PMID- 2994096 TI - Cytomegalovirus (CMV): the agent, its pathogenesis, and its epidemiology. PMID- 2994097 TI - Cytomegalovirus and blood transfusion. PMID- 2994098 TI - Epstein-Barr virus and blood transfusions. PMID- 2994099 TI - Pathobiology of human T-cell leukemia virus. PMID- 2994100 TI - HTLV-I, the prototype human retrovirus: epidemiologic features. PMID- 2994101 TI - Adult T-cell leukemia virus, blood donors and transfusion: experience in Japan. PMID- 2994102 TI - Epidemiologic aspects of acquired immunodeficiency syndrome (AIDS) in the United States: cases associated with transfusions. PMID- 2994103 TI - The cause of acquired immunodeficiency syndrome--is it known? PMID- 2994104 TI - Hemophilia and acquired immune deficiency syndrome. PMID- 2994105 TI - Seroepidemiological evidence for HTLV-III infection as the primary etiologic factor for acquired immunodeficiency syndrome. PMID- 2994106 TI - A new human retrovirus associated with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex. PMID- 2994107 TI - Effects of transfusion on recipient immune status: relationship to transplantation. AB - Blood transfusion therapy confers an immunosuppressive effect on organ transplant recipients. The nonspecific immunosuppressive effect of third-party transfusions is useful for CAD transplantation, while donor transfusions improve the outcome of LRD grafts. Although there is evidence that erythrocytes, modified lymphocytes and/or platelets mediate some effects, the optimal blood product is unknown. Similarly, the mechanism of the effect, be it related to induction of a suppressive immune response, prophylaxis against CMV, active viral infection or a side effect of blood such as iron overload, remains an important issue for investigation. Dissection of the immunosuppressive effect of transfusions may afford insights into host resistance to allografts and new methods to achieve transplantation tolerance. PMID- 2994108 TI - Donor screening and epidemiology. PMID- 2994109 TI - Challenges and opportunities in biotechnology. AB - The biotechnology industry is thriving, and many predicted accomplishments have actually occurred during the last decade. Cloning and expression of genetic information is now simple and routine. Initial commercial products have been realized, but there is much yet to be accomplished in evaluating the clinical significance of many other gene products made available by biotechnology resources. During the next decade, human health care and the pharmaceutical industry should be affected substantially by first- and second-generation recombinant DNA products. Recombinant vaccines, blood coagulation factors, and known biological modulators produced by rDNA technologies should be widely used. Further opportunities will be realized with increasing discoveries of new bioactive molecules and identification of NANB hepatitis and AIDS infectious agents. Full exploitation of health care products will depend on innovative new delivery systems or the ability to reconstruct mammalian and plant genes, providing for in-situ delivery of the necessary gene products. PMID- 2994110 TI - LHRH agonists--mechanism of action and effect on target tissues. PMID- 2994111 TI - The contribution of animal models to the understanding of endocrine management of prostate cancer. Effect of androgen and estrogen. PMID- 2994112 TI - Overview of epidemiology and risk factors associated with colorectal cancer. PMID- 2994113 TI - Strategies and rationale for intervention studies. PMID- 2994114 TI - The response of human carcinoma cell lines to gamma-linolenic acid with special reference to the effects of agents which influence prostaglandin and thromboxane synthesis. AB - Recently, addition of gamma linolenic acid (GLA) which is a precursor of prostaglandin E1 (PGE1) to cell cultures, has been shown to inhibit growth of various carcinoma cells (1,2,3,4). These findings are consistent with Horrobin's proposal that some of the metabolic abnormalities of malignant cells may be due to deficiencies of certain prostanoids. To determine whether the observed effects of GLA are in fact mediated by increasing levels of its metabolites, this study investigated the influence of various inhibitors and stimulants of prostaglandin (PG) synthesis on the effects of GLA on carcinoma cells in vitro. Most of the agents used (aspirin, imidazole, lithium carbonate and ascorbic acid) produced results consistent with the idea that elevation of levels of thromboxane A2 (TxA2) and/or PGE1 may be important as regards the actions of GLA. In sharp contrast was the result obtained with indomethacin. This drug, which could be expected to block conversion of GLA to PGE1 and therefore protect cells against the effects of GLA, actually exaggerated the effects of this fatty acid, thereby causing cell death and desquamation. PMID- 2994115 TI - Dihomo-gamma-linolenic acid increases the metabolism of eicosapentaenoic acid in perfused vascular tissue. AB - The isolated rabbit ear was perfused with 14C-arachidonic acid (AA), with 14C eicosapentaenoic acid (EPA) or with 14C-dihomo-gamma-linolenic acid (DGLA). After incorporation of 14C-AA, the ionophore A 23187 (10 micrograms) stimulated the release of products comigrating with authentic PGI2 (measured as 6-keto-PGF1 alpha), PGE2 and PGD2 on the thin layer chromatography plate. After incorporation of 14C-EPA, A 23187 did not release any trienoic 14C-PGs. After incorporation of 14C-DGLA, A 23187 stimulated the release of labeled products comigrating with 6 keto-PGF1 alpha (but not PGF1 alpha), PGE1 and PGD1. Infusion of unlabeled AA (1 and 10 micrograms/ml) did not influence the metabolism of 14C-EPA or 14C-DGLA. Infusion of unlabeled DGLA (10 micrograms/ml) strongly stimulated the release of trienoic 14C-PGs but did not significantly increase the release of bisenoic 14C PGs. Neither DGLA nor AA influenced the release of any other labeled incorporated PG precursor, indicating that a phospholipase A2 was not affected. The results show that DGLA is able to stimulate the metabolism of incorporated 14C-EPA resulting in an increased release of antiaggregatory trienoic PGs. The mechanism of this effect is unclear but it may be mediated via the formation of a hydroperoxide derivative of DGLA. Thus, an increased generation of antithrombotic trienoic PGs may be expected under special conditions, possibly also in vivo, depending on the supply of unsaturated fatty acids or the level of various hydroperoxide derivatives. PMID- 2994116 TI - Central serotonin neurones in avoidance learning: interactions with noradrenaline and dopamine neurones. AB - The effects of p-chloroamphetamine (PCA), a serotonin (5-HT) releaser, on acquisition and retention were examined in male Sprague-Dawley rats using a one way active avoidance task. PCA was found to impair avoidance acquisition and retention in a time dependent fashion which followed closely the temporal effects of the drug on 5-HT release in the brain. Thus, the avoidance deficit is related to the rate of change and not to the steady-state levels of 5-HT. The 5-HT releasing effect was most pronounced in the forebrain with less effect in the spinal cord. PCA caused time dependent, regional variations in catecholamine content, which was not related to avoidance performance. The avoidance and retention impairment induced by PCA was blocked by the 5-HT synthesis inhibitor p chlorophenylalanine (PCPA) but not by depletion of catecholamines with alpha methyl-p-tyrosine (H44/68) or by the noradrenergic-selective neurotoxin DSP4. Analysis of the time dependent effects of PCA on monoamine content in saline or PCPA-treated rats indicated that the temporal effects of PCA on avoidance performance is not due to a direct or indirect action on catecholamine neurones. The present experiments support the view that the ascending serotonergic pathways play a significant role in aversive learning in the rat. PMID- 2994117 TI - The quasi-morphine withdrawal syndrome: effect of cannabinol, cannabidiol and tetrahydrocannabinol. AB - Delta-9-tetrahydrocannabinol (THC), the main psychoactive principle of cannabis, has been shown to attenuate the exhibition of signs of the quasi-morphine withdrawal syndrome in rats. Cannabinol (CBN) showed the same activity but required a dosage of approximately eight times that of THC to produce an equivalent effect. Cannabidiol was without effect at the dosage levels used. The efficacy of these cannabinoids and the potency differences recorded in this study are in accord with their effects on other behaviours, both in experimental animals and in man. The activity of THC and CBN was not affected by the narcotic antagonist, naloxone. PMID- 2994118 TI - Benzodiazepine-receptor mediated inhibition of isolation-induced aggression in mice. AB - The effects of a benzodiazepine receptor agonist (diazepam), antagonist (Ro 15 1788), and an "active" antagonist [inverse agonist] (3-carboethoxy-beta carboline) were examined in an isolation-induced model of aggression. Diazepam (4 mg/kg) and 3-carboethoxy-beta-carboline (10 mg/kg), but not Ro 15-1788, significantly inhibited aggressive behavior in this model. Ro 15-1788 (10 mg/kg) reduced the anti-aggressive actions of both diazepam and 3-carboethoxy-beta carboline, while mice treated with a combination of diazepam and 3-carboethoxy beta-carboline had aggression scores increased to values not significantly different from vehicle treated mice. These findings suggest that both diazepam and 3-carboethoxy-beta-carboline have anti-aggressive properties in the isolation induced model of aggression which are mediated through CNS benzodiazepine receptors. PMID- 2994119 TI - Blockade of peripheral beta-adrenergic receptors fails to suppress the cerebral spread of an engram in mice. AB - Using bitemporal injections of puromycin, we have reported observations interpreted to indicate that a systemic injection of (-)-propranolol (50 micrograms/kg) drastically suppressed the spread of an engram in mice from the hippocampalentorhinal area to widespread cerebral areas. The present experiments were made with a non-selective, irreversible beta-adrenergic receptor antagonist that fails to cross the blood-brain barrier in order to test the possibility that the propranolol-induced blockade of peripheral beta-receptors might contribute to its effect on engram spread. Prolonged blockade of peripheral receptors by the irreversible antagonist had no effect on engram spread, suggesting that propranolol's effect was centrally mediated. PMID- 2994120 TI - The conditioned place preference is affected by two independent reinforcement processes. AB - The conditioned place preference method for measuring the affective properties of reinforcing events was studied using treatments of known affective value. The size of the place aversion observed increased with dose when the reinforcer was injections of lithium chloride. The size of the place preference observed increased with concentration when the reinforcer was drinking sucrose solutions. However, when the reinforcer was solutions of saccharin (that were consumed in the same amounts as the sucrose solutions) no place preferences were observed. This finding was explained in terms of the dual reinforcement hypothesis [20] which postulates that although sucrose and saccharin both have positive affective properties (based on their tastes) only sucrose has memory improving properties (based on its post-ingestive action). It was therefore proposed that conditioned place preferences depend on the activation of both affective and memory improving processes. This hypothesis was confirmed by the observation of place preferences with a saccharin solution as the reinforcer when the pairing trials were followed by non-contingent, post-pairing injections of glucose or amphetamine (both of which are known to improve memory). Therefore, behavior in the place preference method depends upon both the affective and the memory improving properties of the reinforcers under test. PMID- 2994121 TI - Reduced head-twitch response to quipazine of rats previously treated with methiothepin: possible involvement of dopaminergic system. AB - Methiothepin has been reported to induce an increase of specific binding sites for 3H-5TH 2-3 days following a single administration of a large dose. The present study was intended to ascertain whether methiothepin pretreatment would induce behavioral serotonergic supersensitivity, as assayed by evaluating head twitch response to quipazine and L-5-hydroxytryptophan (L-5HTP). Methiothepin pretreated rats exhibited a significantly reduced response after quipazine but not a significant change after L-5HTP. Such findings could be explained by considering that quipazine stimulates both serotonin and dopamine receptors and by hypothesizing that methiothepin also induced dopaminergic supersensitivity which hampered head-twitch behavior. Such an explanation was supported by the following findings. Rats tested 5 days after a large dose of haloperidol exhibited reduced head-twitch response to quipazine. Moreover, rats which had received a single administration of either haloperidol or methiothepin showed (1) more sustained spontaneous locomotor activity, and (2) enhanced stereotyped response to apomorphine. PMID- 2994122 TI - Prostanoid modulation (mediation?) of certain behavioral effects of ethanol. AB - Prostaglandin E1 (PGE1) and prostanoid precursor fatty acids enhance the acute sedative effects of ethanol in mice, and reduce the intensity of withdrawal after chronic exposure to ethanol. Aspirin, and other inhibitors of prostanoid synthesis, attenuate ethanol's sedative effects, and interfere with the beneficial effects of prostanoid precursors (but not of PGE1 itself) in withdrawal. Neither aspirin nor indomethacin administered alone affect withdrawal behavior. In contrast, ethanol impairment of rotorod behavior is not affected by prostanoid precursors nor by aspirin. These findings support a role for prostanoids as modulators (? mediators) of certain direct effects of ethanol and a role for prostanoid deficiency in the pathogenesis of withdrawal behavior. PMID- 2994123 TI - Opioid activity of lefetamine. AB - In mice lefetamine, at the dose of 50 mg/kg produces motor hyperactivity and at the dose of 60 mg/kg produces analgesia. Both effects are abolished by naloxone. Displacement studies by using [3H]-Naloxone (Nx), [3H]-D-Ala-Met-Enkephalinamide (DAMA) and [3H]-Ethylketocyclazocine (EKC) showed that lefetamine competes with all these opiates with an affinity 50 times lower than that of morphine. The displacing capacity of lefetamine is decreased in the presence of 50 mM Na+. It is concluded that lefetamine is an opioid agonist. PMID- 2994124 TI - Quantitative determination of water in the skin by 1H NMR. AB - Studies were made on determination of the amount of water in rat skin by 1H NMR spectroscopy. The NMR spectrum obtained depended on how the skin was packed into the NMR tube. Consistent results were obtained by packing the skin into a specially designed NMR cell. By this technique the amount of water in the skin could be measured accurately, and results were comparable with those obtained by differential scanning calorimetry. The water content of the skin of rats was consistently about 20% more in females than in males. PMID- 2994125 TI - Rapid modulation of rat hepatocyte HMG-CoA reductase activity by cyclic AMP or cyclic GMP. AB - Rat hepatocytes were used to demonstrate rapid, transient effects on the modulation state (defined as the fraction of the enzyme present in the catalytically active form) of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase, E.C. 1.1.1.34). Insulin elevated, while glucagon, cAMP or cGMP lowered HMG-CoA reductase modulation state within 10 to 15 min. These changes were accompanied by a parallel change in sterol synthesis. Total HMG-CoA reductase activity was not altered. Rapid modulation of HMG-CoA reductase activity therefore constitutes a viable in vivo control mechanism. By contrast to the hormones and second messengers, mevalonolactone lowered both HMG-CoA reductase modulation state and total reductase quantity. PMID- 2994126 TI - The pineal gland and opiate-induced feeding. AB - Opiate systems in the brain are thought to play a major, though not exclusive, role in the regulation of intake. The rough correspondence of feeding and pineal activity rhythms in the rat offers the possibility that the pineal may also modulate ingestive behavior. In these studies we measured the possibility that pineal manipulations would influence feeding responses to opiate agonists and the antagonist naloxone. Male rats received one of four treatments (or a corresponding control treatment): pinealectomy, removal of the superior cervical ganglia (SCG), transection of the optic nerves or chronic melatonin treatment (1 mg/kg daily). Pinealectomy and melatonin treatment reduced intake during the first half of the dark period, and removal of the SCG reduced intake during the second half of the light period. The most striking effect was seen after optic nerve transection, which reduced nocturnal and increased diurnal intake. Pinealectomy, but no other manipulation, caused a slight decrease in sensitivity to the inhibitory effects of naloxone on intake. None of the treatments affected daytime feeding responses to morphine, ketocyclazocine, or butorphanol. These results suggest that the pineal gland has a minimal role in modulating the opioid regulation of food intake. PMID- 2994127 TI - Increase of food intake induced by glucagon in the rat. AB - The effect of intraperitoneal administration of saline, glucose (25 mg/100 g b.w.), insulin (0.025 U/100 g b.w.) and glucagon (50 micrograms/kg b.w.) on glycemia, liver glycogen concentration and food intake was studied on 104 male adult Wistar rats. When saline was injected the amount of food ingested was similar to that expected at the metabolic moment selected for the tests. Glucose administration did not reduce food intake but both insulin and glucagon provoked a threefold increase during the 60 minutes ensuing the injection. The overall ingestion of food during the 24 hours after the injection of the hormones was significantly higher (about 10%) than the control values during the preceding or the succeeding 24 hours. A hyperphagic, rather than a hypophagic effect of glucagon administration is possibly related to the small dose used in the experiments. The mechanisms involved in the increase of food intake due to glucagon are discussed in terms of acceleration of the metabolic reactions that normally prevent large drops of glycemia as glucose utilization proceeds during the inter-meal periods and that in physiological conditions build up until the need for food arises. PMID- 2994128 TI - lacZ gene fusions and insertion mutagenesis in the TL-region of Agrobacterium rhizogenes Ri plasmid. AB - Agrobacterium rhizogenes induces root formation and inserts a fragment of its plasmid into the genome of infected plants. A part of the transferred region (TL region) of the Ri plasmid of A. rhizogenes strain A4 was cloned in pBR322. Insertions of the Escherichia coli lacZ coding region into the hybrid plasmids were made in vivo using mini-Mu-duction. Two mini-Mus were used, one with the Mu A and B transposase genes (MudII1681) and the other without (MudII1734). Two inserts which result in E. coli lacZ expression where shown to be located in the T-DNA region. This indicates that portions of the T-DNA are capable of expression in bacteria. When these two hybrid plasmids were transformed into Agrobacterium only the one harboring MudII1734 insert gave transformants which correspond to homologous recombination. These results indicate that gene fusion and insertion directed mutagenesis can be simultaneously obtained with this mini-Mu and could be used to study Agrobacterium gene expression. PMID- 2994129 TI - Analysis of a region in plasmid R386 containing two functional replicons. AB - A miniplasmid has been obtained from R386 by ligating EcoRI fragments with a fragment carrying a kanamycin-resistance gene. It contains a 6.8-kb Eco fragment of R386 which hybridizes strongly with several IncFI plasmid DNAs but not with the primary or secondary replicons of the F plasmid. This mini-R386 is incompatible with certain IncFI plasmids, and it appears to be one example of a previously unidentified replicon widely distributed in the IncFI group. A region of R386 not closely linked to the 6.8-kb fragment is involved in copy number control of the mini-R386, and a sequence in the same region interacts with mini-F partition functions to cause incompatibility. The 6.8-kb fragment also restricts growth of T7 bacteriophage, and an adjacent fragment restricts phage T4 growth. A further R386 sequence, sharing homology with the F secondary replicon, is capable of autonomous replication. Hence R386, like F, contains at least two functional replicons. PMID- 2994130 TI - Expression of a Rhizobium phaseoli Sym plasmid in R. trifolii and Agrobacterium tumefaciens: incompatibility with a R. trifolii Sym plasmid. AB - Identification of the Sym plasmid in Rhizobium phaseoli strain RCC3622 is described. Introduction of this plasmid into R. trifolii or Agrobacterium tumefaciens strains resulted in bacteria capable of forming characteristic spherical root nodules on beans. This Sym plasmid, designated pSym9, was characterized as 275 MDa and nonconjugative. pSym9 was incompatible with the R. trifolii Sym plasmid pSym5, and carries genes determining a melanin-like black pigment. A second plasmid of 135 MDa, pRph3622a, was also transferred from R. phaseoli to R. trifolii and A. tumefaciens. Transconjugants carrying this plasmid did not form root nodules on beans. In contrast to other Rhizobium plasmids, pRph3622a was unstable in A. tumefaciens. PMID- 2994131 TI - Derivation of a physical map of chloroplast DNA from Nicotiana tabacum by two dimensional gel and computer-aided restriction analysis. AB - A combined approach was used to derive a detailed physical map of Nicotiana tabacum chloroplast DNA for the restriction enzymes SalI, SmaI, KpnI, and BamHI. Complete maps for the restriction enzymes SalI, SmaI, and KpnI were derived by using two-dimensional agarose gel analysis of fragments obtained by reciprocal double digestion of chloroplast DNA. We have characterized a complete cloned library of N. tabacum chloroplast DNA which contains overlapping restriction fragments resulting from partial digestion by BamHI. With these clones and existing data, we used a novel computer-aided analysis to derive a detailed map for the enzyme BamHI. A comparison and compilation of all published N. tabacum chloroplast DNA restriction maps is presented. Differences between ours and a previously published SmaI and BamHI restriction map are discussed. PMID- 2994132 TI - Transposition behavior of IS15 and its progenitor IS15-delta: are cointegrates exclusive end products? AB - We report that the major product of IS15-promoted transposition is a cointegrate. When present in the multicopy plasmid pBR322, IS15 and its progenitor IS15-delta mediate the formation of cointegrates at frequencies of 3.5 X 10(-4) and 2.9 X 10(-5), respectively. We have studied the stability of the cointegrates generated by IS15 and IS15-delta. While these structures are resolved in a rec+ host, they were stable in a rec- host. These observations suggest that neither IS15 nor IS15 delta encode a resolvase and that cointegration is an end product of their transposition process. These properties of IS15-delta and IS15 can explain the transitions from IS15-delta to IS15 and from IS15 to IS15-delta observed in vivo. PMID- 2994133 TI - Preliminary analysis of the incompatibility determinant of a group B miniplasmid. AB - The incompatibility functions of a group B miniplasmid have been studied. The IncB locus has been mapped to within a 360-bp region of the genome, and its primary product shown to be a small RNA molecule. PMID- 2994134 TI - Anomalous electrophoretic mobility of mouse mtDNA restriction fragments. AB - Analysis of the electrophoretic mobilities of mouse mtDNA restriction fragments revealed a high incidence of anomalous migration in polyacrylamide slab gels. Relative to the mobility predicted by the sequence, 6 of 29 200- to 700-bp fragments had deviations of 5-12%. Three of these fragments migrated more slowly than predicted while three were faster. There was little, if any, correlation between electrophoretic mobility and base composition. The anomalous restriction fragments mapped throughout the mitochondrial genome. PMID- 2994135 TI - cAMP levels in reactive tissues around dimethylpolysiloxane solid implants. AB - By the aid of radioimmunologic assay, the authors measured the cAMP content of the reactive capsules around solid dimethylpolysiloxane implants in mice at different times; they also measured the same substance in some human connective tissues (granulation tissue and normal dermis) and compared together all the values they obtained. Different concentrations of cAMP in different tissues seem to reflect correspondent histologic findings, since they vary according to them. These values also seem to indicate a close correspondence between the development of the process of wound healing and of the foreign-body reaction following the implantation of alloplastic materials. On the basis of these data, the authors suggest an experimental therapeutic trial to enhance peripheral cAMP synthesis in order to control the process of reactive capsule constitution. PMID- 2994136 TI - Poland's syndrome: correction of thoracic anomaly through minimal incisions. AB - A method that minimizes residual scarring following Poland's syndrome correction by latissimus dorsi muscle transposition and placement of a submuscular breast implant is described. In order to reduce any resulting unsightly scarring and, in particular, eliminate the anterior thoracic scar, both a dorsal S-shape and an axillary incision were made and the muscle flap was raised. A prosthesis was then inserted and the muscle flap sutured to the anterior chest wall through an anterior incision symmetrical to the inferior border of the contralateral areola. The latter is a previously undescribed approach that produces good cosmetic results. PMID- 2994137 TI - [Cooperation barriers in psychiatric and psychosocial after care: organizational and sociologic aspects]. AB - Community based out-care systems for the long term patient has been a concern ever since deinstitutionalization and rehabilitation became a task in the 60's. Much of our efforts failed. Here we try to make the point of organizational factors and their impact on motivation and cooperative incentives both for the staff in the wards, and in the community based clinics. Exchange theory is offered as a base to outline better planning. PMID- 2994138 TI - Review of the effects of stress on cancer in laboratory animals: importance of time of stress application and type of tumor. PMID- 2994139 TI - Role of the autonomic nervous system in the cytoprotective effect of neurotensin against gastric stress ulcers in rats. AB - Pharmacologic agents were used to study the role of the autonomic nervous system in the cytoprotection produced by intracisternal neurotensin against cold plus restraint stress-induced gastric ulcers in rats. Drugs which stimulated alpha- or beta-adrenergic receptors or blocked muscarinic cholinergic receptors reduced the incidence of ulcers to a similar degree as intracisternal neurotensin; alpha adrenergic or beta-adrenergic blockade as well as cholinergic stimulation prevented neurotensin's beneficial effect. However, pretreatment with indomethacin blocked only the cytoprotective effect of neurotensin or beta adrenergic stimulation, but not that of muscarinic cholinergic blockade. In addition, pretreatment with reserpine or guanethidine also was effective in preventing cytoprotection by intracisternal neurotensin. These data indicate that the mechanism for cytoprotection by centrally administered neurotensin is mediated at least in part through activation of the sympathetic nervous system. This activation by neurotensin appears to produce cytoprotection by stimulation of gastric mucosal prostaglandin synthesis. PMID- 2994140 TI - Influence on memory of posttraining or pre-test injections of ACTH, vasopressin, epinephrine, and beta-endorphin, and their interaction with naloxone. AB - The intraperitoneal (i.p.) injection of ACTH 1-24 (0.2 microgram/kg), lysine- vasopressin (10.0 micrograms/kg) or epinephrine HCl (5.0 micrograms/kg) shortly after training or prior to testing caused memory facilitation of a step-down inhibitory avoidance task in rats, acquired with low intensity training footshocks (0.3 mA, 60 Hz). Naloxone HCl (0.4 mg/kg) potentiated their posttraining effect, but antagonized their pre-test effect. Naloxone on its own caused retrograde memory facilitation but had no effect on the test session. Posttraining human beta-endorphin (1.0 microgram/kg) was amnestic, and its pre test administration enhanced retention. Both effects were naloxone-reversible. Neither the pre-test facilitation caused by beta-endorphin nor those caused by any of the other drugs (which are possible releasers of endogenous beta endorphin) were observed in animals in which the influence of endogenous opioids was prevented at the posttraining period by the administration of naloxone. These results are compatible with, and considerably strengthen, the previously advanced hypothesis that learning of this task, and possibly others, depends on a state induced by beta-endorphin after training, and that it would normally be dissociated because this peptide is normally not released during test sessions. In addition, the posttraining facilitation caused by ACTH, vasopressin, and epinephrine stands out as an effect separate from, and in fact normally hindered by, posttraining beta-endorphin release. PMID- 2994141 TI - Melatonin, cortisol and ACTH in patients with major depressive disorder and healthy humans with special reference to the outcome of the dexamethasone suppression test. AB - The 24 hr profiles of melatonin and cortisol in serum, morning levels of ACTH in plasma, and the dexamethasone suppression test (DST) were investigated in 32 acutely ill patients with a RDC diagnosis of major depressive disorder, 24 patients with a history of longlasting unipolar or bipolar major depressive disorder studied in remission, and 33 healthy subjects. A significant decrease in maximum nocturnal melatonin level (MTmax) was found in the acutely ill depressed patients with abnormal DST compared to both those with normal DSTs and the healthy subjects. The MTmax levels were unaltered when these patients were reinvestigated in remission. A decrease of MTmax was also seen in the group of unipolar and bipolar patients studied in remission. Low nocturnal melatonin is proposed to be a trait marker for major depressive disorder and depressive states with abnormalities in the hypothalamic--pituitary--adrenal (HPA) axis. A significant decrease of ACTH levels at 0800 hr after dexamethasone administration the preceding evening was found in the healthy subjects, the unipolar--bipolar patients in remission, and the acutely ill depressed patients with normal DSTs, but was not found in the acutely ill depressed patients with abnormal DSTs. These findings support the hypothesis that pituitary ACTH regulation is altered in depressed patients with abnormal DST. Morning plasma ACTH before the administration of dexamethasone did not significantly differ between the acutely ill depressed patients with abnormal DSTs, normal DSTs, the patients with unipolar--bipolar disease in remission, or the healthy subjects. Thus, the abnormalities in the HPA axis in depressed patients are proposed to be due to a hypersecretion of corticotrophin releasing factor (CRF) with a subsequent stimulus-induced pituitary desensitization. A significant decrease of melatonin after dexamethasone was seen at 0800 hr in the unipolar--bipolar patients in remission as well as in the healthy subjects, at 1600 hr and 2200 hr in the acutely ill depressed patients in remission, but not at 0800 hr in the acutely ill depressed patients in relapse. A significant regression was found between MTmax levels and the degree of non-suppression of cortisol at 0800 hr in the DST in the acutely ill depressed patients both in relapse and in remission. Melatonin thus is proposed to be an inhibiting factor for CRF during depression. A trend to a phase-advance of cortisol nadir and melatonin peak was seen in the acutely ill depressed patients with abnormal DST, possibly indicating an involvement of the suprachiasmatic nuclei in the hypothalamus. PMID- 2994142 TI - Direct radioimmunoassay of cortisol in saliva and its application to the dexamethasone suppression test in affective disorders. AB - In order to perform the dexamethasone suppression test (DST) with saliva as an alternative to serum, we assayed directly the cortisol concentrations in 25 microliters saliva samples, using a commercial radioimmunoassay kit for serum cortisol with minor modifications. Cortisol in saliva showed a diurnal rhythm parallel to that of cortisol in serum samples collected simultaneously. Saliva cortisol levels increased significantly after ACTH injection, but with a 60 min delay in reaching their peak compared to peak serum cortisol levels. The increase in saliva cortisol was five-fold, while that in serum was two-fold. Saliva cortisol levels continued to increase in some subjects while serum total cortisol levels already had begun to decline. In those subjects, the correlation of saliva with serum cortisol was greater when a quadratic curve was fitted than when calculated for a linear correlation. Considerable variation was observed for within-subject correlations, ranging from + 0.48 to + 0.999. The DST with saliva sample collection was performed on 43 inpatients with affective disorders. Sensitivity, specificity and diagnostic confidence of the DST for major depressive episode with melancholia were 33%, 91%, and 78%, respectively, at the criterion value of 0.3 microgram/100 ml for saliva cortisol, which are similar to those most often reported for the DST with serum cortisol determination. These results indicate that saliva cortisol levels do not always parallel serum cortisol levels and thus are not an unequivocal substitute. The findings for the DST in psychiatric patients, however, support the practical clinical usefulness of saliva cortisol measurements. PMID- 2994143 TI - Hashish extract impairs retention of defeat-induced submissive behavior in mice. AB - The effects of hashish extract on adaptive behavior of male mice were studied in a paradigm which allows the investigation of learning mechanisms in a social context. Mice of the C3H strain, which were not submissive in a confrontation with a nonaggressive DBA mouse on day 1, were defeated on day 2 over 3 min by aggressive, isolated DBA mice, and showed conditioned submissive behavior upon mere contact with a nonaggressive DBA mouse on day 3. A hashish extract containing 38.6-39.4% delta 9-tetrahydrocannabinol (delta 9-THC), 11.6-12.0% cannabinol and 47.7-48.5% cannabidiol was administered orally in all experiments. Hashish extract given 90 min before defeat on day 2, in dosages corresponding to 1, 5, and 10 mg delta 9-THC/kg, impaired retention of defensive upright, defensive sideways and immobility on day 3 (experiment 1). Experiment 2 showed that the drug (5, and 10 mg delta 9-THC/kg) had no antinociceptive potency in mice and did not modify defeat-induced analgesia. Experiment 3, with drug (5 mg delta 9-THC/kg) or solvent administration on day 2 and day 3, showed that the retention deficit was neither due to state-dependent learning, nor to impaired retrieval. It is suggested that hashish extract administered before learning may interfere with memory processing. PMID- 2994144 TI - Motivational properties of kappa and mu opioid receptor agonists studied with place and taste preference conditioning. AB - The reinforcing properties of various opioid agonists acting preferentially on the kappa and mu opioid receptors were assessed using taste and place preference conditioning procedures. Kappa receptor agonists produced conditioned aversions. Taste aversions were produced by all of the drugs used, including racemic mixtures of ethylketazocine, tifluadom, and U50-488, and active isomers (+) tifluadom, (-)-bremazocine, and Mr 2034; corresponding inactive isomers either produced no effect of were less potent. Place aversions were produced by U50-488 and (-)-bremazocine, but not (+)-bremazocine or any of the other kappa receptor agonists tested with the taste procedure. The mu agonists produced predominantly conditioned preferences. Place preferences were produced by morphine, fentanyl and sufentanil. Taste preferences were produced by low doses of these substances; at higher doses the taste preferences were absent or replaced by aversions. Finally, with naloxone and lithium chloride it was shown that the taste procedure was more sensitive to punishing effects than the place procedure. It is concluded that kappa and mu opioid receptor agonists are effective unconditioned stimuli. From the lower portions of the dose response curves it is further concluded that activation of kappa opioid receptors has aversive properties and activation of mu receptors appetitive reinforcing properties. The findings are also discussed with regard to the prevailing notions of taste conditioning with opiates, and the reinforcing properties of activity of the endogenous opioid peptide systems. PMID- 2994145 TI - Effects of valproate on hyponeophagia in rats: competitive antagonism with picrotoxin and non-competitive antagonism with RO 15-1788. AB - The effects of valproate (30-500 mg/kg), alone and in combination with picrotoxin (1.5 mg/kg) or RO 15-1788 (10 mg/kg) were studied in two experiments on hyponeophagia in rats. Valproate reduced eating latency and increased eating time and amount eaten of novel food, except at 500 mg/kg which reduced feeding. Picrotoxin induced generally opposite actions alone and shifted valproate dose/response curves to the right. RO 15-1788 had no detectable intrinsic action, but prevented both the behavioural facilitation and inhibition produced by valproate. These findings are discussed in the context of the GABA hypothesis of benzodiazepine action, with the conclusions that they provide behavioural support for the hypothesis of a receptor complex with GABA and benzodiazepine binding sites, and that an optimal and submaximal level of activity at the benzodiazepine site is a necessary condition for anxiolytic actions of valproate. PMID- 2994146 TI - Behavioural similarities between mother rats and benzodiazepine-treated non maternal animals. AB - Mother rats nursing large litters are hyperphagic, aggressive towards conspecifics, and show less freezing behaviour than non-maternal animals. These naturally occurring adaptations resemble those elicited by benzodiazepine treatment in virgin rats, indicating a common neurochemical change in the brains of mother rats and benzodiazepine-treated virgins. In line with this hypothesis, it was found that three functional benzodiazepine antagonists (FG 7142, pentylenetetrazol, caffeine) decreased food intake, lowered aggression and strengthened freezing in lactating mother rats. These psychopharmacological observations support the idea that GABA neurotransmission is enhanced during motherhood in the rat. PMID- 2994147 TI - Benzodiazepine receptor ligands and the consumption of a highly palatable diet in non-deprived male rats. AB - Non-deprived rats were familiarized with a highly palatable diet until baseline consumption in a 60-min daily access period had stabilised. The benzodiazepine receptor agonist midazolam (1.25-10.0 mg/kg, IP) produced a large, dose-related increase in food consumption during the first 30 min of access. It also produced significant, short-term hyperphagia in animals which had been partially pre satiated on the diet before drug administration, an effect which was reversible by the benzodiazepine receptor antagonist Ro15-1788. Administered alone, Ro15 1788 (1.25-10.0 mg/kg, IP) had no intrinsic activity in the food consumption test. In contrast, CGS 8216 (2.5-40.0 mg/kg, IP) produced a marked dose-related suppression of food intake. This anorectic effect was shared by two benzodiazepine receptor inverse agonists, FG 7142 and DMCM, which also produced dose-dependent reductions in consumption. The effects on feeding produced by FG 7142 (20 mg/kg, IP) and DMCM (1.25 mg/kg, IP) were reversed by either Ro15-1788 (2.5 and 5.0 mg/kg) or midazolam (5.0 and 10.0 mg/kg). A matched anorectic effect produced by CGS 8216 (40 mg/kg) was not, however, reversed by either Ro15-1788 or midazolam. This suggests that at a high dose CGS 8216 may act by a mechanism different from that of the two inverse agonists. The feeding test described in the report proved sensitive to both hyperphagic and anorectic effects of drugs active at benzodiazepine receptors, pointing to a possible bi-directional control of palatable food consumption. PMID- 2994148 TI - Psychotropic effects of enalapril maleate in normal volunteers. AB - In order to establish any psychotropic effects of the angiotensin-converting enzyme inhibitor enalapril, the drug was administered in doses of 20 mg every morning for 14 days to 12 normal subjects, and compared with placebo on a battery of physiological, psychological and subjective tests, before and after the dose on the 1st and 14th days. Diastolic blood pressure was significantly reduced and heart-rate increased by enalapril as compared with placebo; one component (P1-N1) of the auditory evoked EEG response was increased and tapping rate quickened. The commonest side effect was tiredness. It was concluded that enalapril (unlike most other antihypertensive agents) did not lower mood but could enhance attention and alertness. PMID- 2994149 TI - Effects of opiate antagonists and putative kappa agonists on unpunished and punished operant behavior in the rat. AB - The effects of naloxone and diprenorphine, opiate antagonists with different receptor-binding properties, and the putative kappa-receptor agonists, ketocyclazocine and ethylketocyclazocine (EKC), were studied on food-reinforced responding in rats. Behavior was maintained under a multiple-component 1-min variable-interval schedule in which 12-min periods of unpunished responding alternated with 4-min periods in which each response was punished by a brief electric footshock. Daily sessions were 1 h. Naloxone (0.01-10 mg/kg) decreased unpunished responding only slightly; punished responding was decreased significantly to 66% of control by 10 mg/kg. Diprenorphine (0.01-10 mg/kg) did not affect unpunished responding and increased punished responding dose dependently to as much as 190% of control. EKC (0.01-1.0 mg/kg) decreased unpunished and punished responding dose-dependently and comparably, whereas ketocyclazocine (0.01-1.0 mg/kg) decreased unpunished responding but did not significantly affect punished responding. Diprenorphine was more potent than naloxone in blocking the decreases in responding produced by the kappa agonists. Differences in the behavioral effects of naloxone and diprenorphine appear to reflect the different receptor-binding properties of the two opiate antagonists. PMID- 2994151 TI - Implantation of Durapatite ceramic in an osseous defect created by removal of a lateral periodontal cyst--report of a case. PMID- 2994150 TI - Lithium ions inhibit function of low- but not high-affinity muscarinic receptors of murine neuroblastoma cells (clone N1E-115). AB - Murine neuroblastoma cells (clone N1E-115) possess both high- and low-affinity muscarinic receptors. The low-affinity muscarinic receptor, when stimulated, initiates the formation of cyclic GMP by activating the enzyme guanylate cyclase; whereas stimulation of the high-affinity receptor inhibits prostaglanding E1 mediated cyclic AMP formation by inhibiting the enzyme adenylate cyclase. We have reported that lithium ion (Li+) inhibits cyclic GMP formation mediated by the muscarinic receptor agonist, carbachol, in a concentration-dependent manner and that neither ammonium nor sodium ions have such an effect. We extended this study to show that Li+ was an apparently noncompetitive inhibitor of the low-affinity muscarinic receptor with an IC50(+/- SEM) = 13.6 +/- 0.8 mM. In addition, Li+ with a similar IC50 inhibited the cyclic GMP response in intact cells to sodium azide, which is thought to stimulate guanylate cyclase directly. Moreover, though Li+ was found to have a slight inhibitory effect on prostaglandin E1-stimulated cyclic AMP formation (15% inhibition at 10 mM), it had no effect on the function of the high-affinity muscarinic receptor in intact murine neuroblastoma cells. PMID- 2994152 TI - The serotonin/norepinephrine-linked beta-adrenoceptor system and the mode of action of antidepressants. PMID- 2994153 TI - The GABA hypothesis of depression and antidepressant drug action. PMID- 2994154 TI - Disturbances of norepinephrine metabolism and alpha-2 adrenergic receptor activity in anorexia nervosa: relationship to nutritional state. PMID- 2994155 TI - Panic anxiety and lactate metabolism. PMID- 2994156 TI - Regulation of adrenocorticotropin release from the anterior pituitary. PMID- 2994157 TI - Melancholia in rodents: neurobiology and pharmacology. PMID- 2994159 TI - The Ying-Yang hypothesis of opioid peptide immunomodulation. PMID- 2994158 TI - Selectivity of opioid agonists and antagonists for the various opioid receptor types. PMID- 2994160 TI - Yohimbine and clonidine recognize different states of adrenergic receptors on human platelets: reconsideration of studies on platelet alpha-2 receptors in depressive illness. PMID- 2994161 TI - Platelet alpha-adrenergic receptor function in affective disorders and schizophrenia. PMID- 2994162 TI - Studies of beta-adrenergic receptors in leukocytes of patients with affective illness and effects of antidepressant drugs. PMID- 2994163 TI - Delayed biochemical changes following antidepressant treatment. PMID- 2994164 TI - A new postsynaptic serotonin receptor agonist suitable for studies in humans. PMID- 2994165 TI - Actions and interactions of GABA and benzodiazepines in the mouse hippocampal slice. AB - Intracellular recordings have been made from CA1, CA3 and dentate cells of the mouse hippocampal slice. Gamma-aminobutyric acid (GABA) and the water-soluble benzodiazepines, midazolam and flurazepam were applied close to the impaled cell somata by microelectrophoresis. GABA always caused a fall in input resistance, although the associated changes in membrane potential were variable. These were consistent with reports which have defined a hyperpolarizing response to somatic and a depolarizing response to dendritic application of GABA to CA1 and CA3 cells in the rat and guinea-pig. In this study, the phenomenon was seen in CA1, CA3 and dentate cells. The reversal potential for the hyperpolarizing, somatic response to GABA lay between -70 and -75 mV, similar to the reversal potential of the evoked recurrent inhibitory post-synaptic potential (i.p.s.p.). The change in cell input conductance caused by GABA was larger at membrane potentials positive to the resting potential and smaller at hyperpolarized membrane potentials. Extracellular recordings of action potential frequency were made from ten cells in which the application of either midazolam or flurazepam increased the inhibitory potency of GABA. In three of these cells, the benzodiazepine reduced action potential frequency slightly when applied alone. Neither midazolam nor flurazepam had a consistent effect on membrane potential, resting input resistance or the current-voltage (I-V) relations of the cells when ejected alone. In twenty-eight of forty-one cells examined in detail, ejection of either midazolam or flurazepam was found to increase the response to GABA. In six cells, the response to GABA was significantly reduced by the benzodiazepine tested whilst in the remainder, no interaction of the drugs could be demonstrated. Examination of the dose-response relation for GABA alone and in the presence of midazolam or flurazepam showed that the maximal response to GABA was increased by the benzodiazepine in some cells while it was unchanged in others. When intracellular electrodes were filled with potassium chloride, spontaneous depolarizing GABA-mediated post-synaptic potentials (p.s.p.s) were seen. Measurement of the interval and amplitude distributions of these events showed that flurazepam increased their amplitude but not their frequency. PMID- 2994166 TI - The effects of feeding either roughage or concentrate diets on salivary phosphorus secretion, net intestinal phosphorus absorption and urinary phosphorus excretion in the sheep. AB - Mature sheep fitted with rumen and duodenal cannulae were fed either a hay or a concentrate diet and the effects on salivary phosphorus secretion, net intestinal phosphorous absorption and pathway for phosphorous excretion were examined. Route of excretion was markedly affected by diet with urine phosphorus levels being much higher and faecal levels lower when the concentrate diet was fed. This difference was not due to differences in phosphorus intake nor could it be related to differences in either plasma phosphorus levels or net intestinal phosphorus absorption. Salivary phosphorus secretion and renal tubular reabsorptive efficiency for phosphorus were, however, both lower when the concentrate diet was fed. The significance of these effects of diet in relation to the control of phosphorus balance in ruminants is discussed. PMID- 2994167 TI - Mammalian cells are not killed by DNA single-strand breaks caused by hydroxyl radicals from hydrogen peroxide. AB - Cell killing by ionizing radiation has been shown to be caused by hydroxyl free radicals formed by water radiolysis. We have previously suggested that the killing is not caused by individual OH free radicals but by the interaction of volumes of high radical density with DNA to cause locally multiply damaged sites (LMDS) (J. F. Ward, Radiat. Res. 86, 185-195, 1985). Here we test this hypothesis using hydrogen peroxide as an alternate source of OH radicals. The route to OH production from H2O2 is expected to cause singly damaged sites rather than LMDS. Chinese hamster V79-171 cells were treated with H2O2 at varying concentrations for varying times at 0 degree C. DNA damage produced intracellularly was measured by alkaline elution and quantitated in terms of Gray-equivalent damage by comparing the rate of its elution with that of DNA from gamma-irradiated cells. The yield of DNA damage produced increases with increasing concentration of H2O2 and with time of exposure. H2O2 is efficient in producing single-strand breaks; treatment with 50 microM for 30 min produces damage equivalent to that formed by 10 Gy of gamma irradiation. In the presence of a hydroxyl radical scavenger, dimethyl sulfoxide (DMSO), the yield of damage decreases with increasing DMSO concentration consistent with the scavenging of hydroxyl radicals traveling an average of 15 A prior to reacting with the DNA. In contrast to DNA damage production, cell killing by H2O2 treatment at 0 degree C is inefficient. Concentrations of 5 X 10(-2) M H2O2 for 10 min are required to produce significant cell killing; the DNA damage yield from this treatment can be calculated to be equivalent to 6000 Gy of gamma irradiation. The conclusion drawn is that individual DNA damage sites are ineffectual in killing cells. Mechanisms are suggested for killing at 0 degree C at high concentrations and for the efficient cell killing by H2O2 at 37 degrees C at much lower concentrations. PMID- 2994168 TI - Mutation induction by tritiated water and effects of deuterium oxide in cultured mouse leukemia cells. AB - Induction of mutation to 6-thioguanine resistance was studied in L5178Y mouse leukemia cells after exposure to low-dose-rate gamma rays or tritiated water at dose rates of approximately 0.025 to 0.4 Gy/hr for 20 hr in the presence or absence of 45% (v/v) deuterium oxide. The effect of acute gamma-ray exposure was also examined. A higher frequency of induced mutations was observed after tritium beta rays than after gamma rays, both at equivalent doses and cell survival. Deuterium oxide enhanced the mutation induced by gamma rays and tritium beta rays but did not affect the survival-mutation correlation of the two radiations. PMID- 2994169 TI - [Effect of bacterial DNA gyrase inhibitors on the radiosensitivity of thymocytes and on the utilization of exogenous adenine in these cells]. AB - It was established that the postirradiation changes in incorporation of 14C adenine into acid-soluble and acid-insoluble fractions of thymocytes reflected cell death. When added after irradiation, nalidixic and oxolinic acids exerted a radioprotective action. The effect was absent after single washing of thymocytes incubated with these compounds for 60 min before or after irradiation. Both substances inhibited utilization of 14C-adenine and incorporation thereof into the acid-insoluble fraction of nonirradiated thymocytes and did not influence the viability of cells. PMID- 2994170 TI - [Effect of small doses of roentgen radiation on the spontaneous impulse activity of the hippocampus in vitro]. AB - X-Irradiation of rat hippocampus in vitro with low doses accelerated spontaneous impulse passage without concomitant changes in synaptic activity. There was a negative correlation between the original frequency of neuron discharges and the degree of quickening the impulses in response to the effect of radiation. Perfusion of slices by a noncalcium solution blocked the synaptic transmission but did not influence the response to the effect of ionizing radiation. PMID- 2994171 TI - Extremity bone tumors: evaluation by P-31 MR spectroscopy. AB - High-resolution P-31 MR spectra were obtained in four patients with bone tumors of their distal extremities. In one case the tumor, a Ewing sarcoma of the tibia, was investigated during clinical remission after radiation therapy and chemotherapy. The other three cases - one low-grade chondrosarcoma of the tibial head, one malignant fibrous histiocytoma of the tibia, and one chondroblastoma of the medial femoral condyle - showed clinically active tumor growth, with corresponding increased metabolism as demonstrated by bone scintigraphy. The spectra of the three active tumors indicated a comparably high adenosine triphosphate content, similar to previously published spectra from animal tumors or human tumors implanted into animals. There were also high resonances of inorganic phosphate and low resonances of phosphocreatine; there were definite peaks in the phosphodiester and phosphomonoester regions, indicating the existence of these metabolites in the tumors. Slight but definite changes in the metabolite content were observed in one tumor after chemotherapy. The spectra of the unaffected leg did not show any well-resolved P-31 signals, which is typical for healthy bone. These are the first P-31 MR spectra of human bone tumors measured in patients to our knowledge. PMID- 2994172 TI - Intracranial calcification in the infant and neonate: evaluation by sonography and CT. AB - This study reports the sonographic and computed tomography (CT) findings in seven infants and neonates with intracranial calcifications and a spectrum of underlying disorders, including toxoplasmosis, cytomegalic inclusion disease, transverse/straight sinus thrombosis, and probable anoxia. Neurotropic infectious disease usually produced clumped or subependymal calcifications accompanied by sometimes bizarre ventricular configurations and prominent periventricular cystic encephalomalacia. Sonography failed to identify prospectively intracranial calcifications in two of the three patients without infection, although calcifications were visible in retrospect. Overall, CT provided optimum visualization of intracranial calcifications. PMID- 2994173 TI - [Mechanisms of intracellular protein breakdown--distinct role of lysosomal and nonlysosomal route]. PMID- 2994174 TI - Actions of synthetic leukotrienes on platelets and blood vessels in the anesthetised pig: the release of a platelet derived vasodilator. AB - The actions of leukotrienes (LT's) C4, D4, E4 and F4 have been investigated in the perfused hind-limb of the anesthetized pig. In the blood perfused hind limb LTC4, D4 and E4 increased the perfusion pressure in a dose-dependent fashion whereas LTF4 decreased perfusion pressure. In the Tyrode perfused hind limb all LT's increased perfusion pressure (rank order potency LTC4 = LTD4 much greater than LTF4). The actions of LTF4 were not affected by a wide variety of pharmacological treatments, including indomethacin, methysergide and FPL-55712. The LT's aggregated porcine platelets (rank order potency LTC4 greater than LTF4 greater than LTD4) and induced the release of a platelet-derived vasodilatory mediator. The results provide pharmacological evidence of specific leukotriene receptors in vivo and that leukotrienes can independently modulate blood flow. These data suggest that important interactions may occur between platelets, the arachidonate lipoxygenase products and platelet-derived substances in response to inflammatory stimuli in the cardiovascular system. PMID- 2994175 TI - Prostaglandins and their precursors can modify genetic damage-induced by gamma radiation and benzo(a)pyrene. AB - Experiments were performed to study the effect of various prostaglandins (PGs) and their precursors, gamma-linolenic acid (GLA) and arachidonic acid (AA) on gamma-radiation and benzo (a) pyrene (BP)-induced genetic damage to the bone marrow cells of mice, using the sensitive micronucleus (MN) test. Thromboxane B2 prostaglandin E1 and GLA completely prevented BP-induced and reduced to a great degree radiation-induced genetic damage, where as PGE2, PGF2 alpha and AA were without any effect. Since GLA and AA are widely distributed in the cell membranes, and as PGs can be formed virtually in response to any type of stimulus, it is likely that GLA and PGE1 may function as endogenous anti mutagenic chemicals. PMID- 2994176 TI - The effect of colonizing mice with laboratory and wild type strains of E. coli containing tumor virus genomes. AB - Conventional, antibiotic-compromised, and germ-free mice were either fed or subcutaneously inoculated with laboratory or wild type strains of E. coli containing monomeric or dimeric forms of polyoma (PY) virus DNA. Mice were bled at 3 and 6 weeks following administration of E. coli and their sera examined for the presence of PY hemagglutination inhibiting antibodies. None of the mice developed PY infection; despite colonization of the intestinal tract accompanied by prolonged excretion of high titers (10(9) colony-forming units/gram feces) of E. coli harboring potentially infectious PY virus DNA, no evidence of infection could be demonstrated. PMID- 2994177 TI - Pro-opiocortin peptides in rat cerebrospinal fluid. AB - Cerebrospinal fluid (CSF) taken from rats implanted with chronic cisternal cannulae was subjected to gel filtration chromatography on Sephadex G-50. Fractions were monitored using radioimmunoassays for N-terminal pro-opiocortin (N POC), gamma 3-melanotropin (gamma 3-MSH), C-terminal adrenocorticotropin (C ACTH), alpha-endorphin, beta-endorphin, gamma-lipotropin (gamma-LPH) and alpha MSH. Two peaks which corresponded in elution position to rat N-POC (1-74) and gamma 3-MSH were detected. The major C-ACTH-immunoreactive (IR) peak was found to correspond to 14k ACTH. While no alpha-endorphin immunoreactivity was detected in rat CSF, three beta-endorphin-IR peaks were identified in positions expected for beta-LPH, beta-endorphin (1-31) and beta-endorphin (1-27), as well as a major peak of activity with the elution characteristics and cross-reactivity of rat gamma-LPH. HPLC of the alpha-MSH-IR material in rat CSF revealed the presence of a major peak of immunoreactivity whose retention time did not correspond to the known oxidised and reduced forms of alpha-MSH and its desacetylated and diacetylated derivatives. The identity of this peak is unknown. PMID- 2994178 TI - Water and potassium ion absorption by deuterium oxide resistant winter rye seedlings. AB - The concentrations of deuterium oxide (D2O) in the root tissue water of winter rye seedlings equilibrated with the external D2O concentration within 30 min and in the shoot tissue water after 5-6 h. The equilibrated value in the root water was 87% and in the shoot water, 55% of the external concentration. The K+ absorption rate of the seedlings decreased from a value of 39 to 18 mumol g-1 h-1 when the D2O concentration was changed through a range from 0% to 97%. D2O suppressed the absorption of water and K+ by the seedlings. The higher the D2O concentration the greater the suppression, but it was less than with similarly treated rice plants. However, the process of D2O absorption by the seedlings was similar to that of rice seedlings (Envir. exp. Bot., 24, 369). PMID- 2994179 TI - [Chylous ascites in the adult]. PMID- 2994180 TI - [Esophageal giant-cell myoblastoma]. PMID- 2994181 TI - [Immunofixation of the cerebrospinal fluid in post-poliomyelitis progressive muscular atrophy]. PMID- 2994182 TI - [Respiratory effects of naloxone]. AB - The respiratory effects of naloxone (200 micrograms X kg-1) on dogs, was studied measuring the parameters breath rate, inspiratory volume, breath minute volume, inspiratory time, total time of cycle, inspiratory volume/inspiratory time ratio, and inspiratory time/total time of cycle ratio. The statistical study was made by using the analysis of variance. Naloxone increases all the parameters studied as well as the response to CO2 while it decreases inspiratory time. The results suggest that there are opioid neurons in the respiratory centre. The blockade of the opioid receptors by naloxone almost explains the respiratory effects observed. PMID- 2994183 TI - Effect of trazodone on oxidative metabolism of rat brain in vitro. AB - Oxygen uptake of rat brain homogenate was reduced by 1 mM trazodone, a new atypical antidepressant. Na,K-ATPase activity and the associated oxygen consumption of rat brain slices were also reduced. Oxygen consumption of rat brain slices was enhanced by dopamine and this effect was blocked by 0.0001 mM trazodone. This drug uncoupled oxidative phosphorylation. PMID- 2994184 TI - Addition of cAMP to mucosal or serosal medium induces different actions on Necturus gallbladder. AB - Open tip and Cl(-)-selective microelectrodes were used to study the effects of cAMP on apical membrane potential (Va), fractional voltage ratio (fa) and intracellular chloride activity (aicl) in Necturus gallbladder under open-circuit conditions. In the presence of cAMP in the mucosal medium Va depolarized from -68 +/- 5 mV in control conditions to -56 +/- 5 mV and fa decreased from 0.56 +/- 0.15 in control conditions to 0.15 +/- 0.02. Concomitantly aicl fell from 15 +/- 2 mM to 8 +/- 3 mM, a value close to its electrochemical equilibrium activity. These results differ markedly from those obtained when cAMP was added to serosal medium and indicate that cAMP elicits different transport mechanisms whether it is added to the serosal medium or to the mucosal medium. PMID- 2994185 TI - SAR of the effect of endotoxin on serum angiotensin converting enzyme in the mouse. AB - Administration of intact lipopolysaccharide from E. coli and S. minnesota to mice produced a lowering of serum angiotensin converting enzyme (ACE). However, when either lipid A or carbohydrate-free endotoxin from S. minnesota-Re595 was administered, a rise in serum ACE occurred. A possible explanation for these respective effects may be related to an indirect effect on lung perfusion, on the one hand, or a direct toxic effect to endothelial cell membranes, on the other. PMID- 2994186 TI - 63Ni content of renal parenchyma and nuclei from 63NiCl2-treated rats. AB - The time-course and dose-effect relationships for 63Ni uptake in renal parenchyma and nuclei were delineated in 63NiCl2-treated rats, based upon isolation of renal nuclei by sucrose gradient centrifugation and measurements of 63Ni by liquid scintillation counting. In 5 groups of rats killed 2 to 24 hours after 63NiCl2 injection (36 mumol/kg, im), 63Ni content of renal parenchyma (mean +/- SD) diminished from 3.0 +/- 0.3% of the dose at 2 hours to 0.9 +/- 0.3% of the dose at 24 hours; nuclear 63Ni content diminished from 10.5 +/- 4.4 pmol/10(6) nuclei at 2 hours to 3.0 +/- 0.3 pmol/10(6) nuclei at 24 hours. In 8 groups of rats killed 2 hours after injection of 63NiCl2 at dosages from 3 to 250 mumol/kg, 63Ni concentrations in renal nuclei increased progressively from 1.1 +/- 0.3 pmol/10(6) nuclei at 3 mumol/kg to 32.1 +/- 1.7 pmol/10(6) nuclei at 250 mumol/kg. Nuclear 63Ni content generally averaged 1.9 to 2.4% of total renal 63Ni; significantly higher mean values (3.4 +/- 1.0%) were observed in rats killed 48 hours after 5 daily injections of 63NiCl2 (1.1 mumol/kg/day). Combined administration of diethyldithiocarbamate (DDC, 1.33 mmol/kg, im) and 63NiCl2 (36 mumol/kg, im) increased 63Ni uptake in renal nuclei (4.8 +/- 1.4% of renal 63Ni; P less than 0.05 versus corresponding value of 2.0 +/- 0.2% in rats that received 63NiCl2, alone). Enhanced nuclear uptake of 63Ni evidently explains the synergistic effect of DDC and NiCl2 on induction of heme oxygenase activity in rat kidney. PMID- 2994187 TI - Exacerbation of experimental parvoviral enteritis in calves by coccidia and weaning stress. AB - The disease induced in calves following oral dosing with bovine parvovirus (BPV) was found to be substantially exacerbated when the animals had subclinical coccidiosis. Low levels of passive serum antibody appeared to have little influence on the response to BPV. Imposition of weaning stress on coccidia infected calves which had apparently recovered from prior infection with BPV, was found to induce severe diarrhoea with recrudescence of BPV excretion in faeces. This was in marked contrast to the relatively mild diarrhoea which was found following weaning of control animals and animals infected with either agent alone. It was concluded that BPV activity and damage in the intestinal tract was probably enhanced as a consequence of the extra mitotic activity induced in the region by coccidial infection and the local effects of weaning. On the basis of these and previous findings in the field, it is suggested that BPV may play a significant role in the aetiology of post-weaning diarrhoea in calves. PMID- 2994188 TI - Intranasal vaccination of pigs against Aujeszky's disease: comparison with inactivated vaccines in pigs with low maternal antibody titres. AB - Intranasal vaccination with an attenuated Aujeszky's disease virus strain was compared with parenteral vaccination with two inactivated virus vaccines, in 10 week-old pigs with low levels of maternal antibody. Intranasal vaccination conferred a much better protection than parenteral vaccination with the two inactivated vaccines against challenge two months later, as evidenced by shorter periods of growth arrest and fever and a greater reduction of virulent virus shedding after challenge-exposure. PMID- 2994189 TI - Reovirus-induced tenosynovitis: persistence of homologous challenge virus in broiler chicks after vaccination of parents. AB - Broiler chicks from a parent flock previously vaccinated with a commercial tenosynovitis (viral arthritis) vaccine were challenged when one day old with a virulent form of the vaccinal reovirus strain. A group from unvaccinated parents was similarly challenged. At three weeks after infection it was found that, while maternal antibody reduced the incidence of lesions of tenosynovitis by about 50 per cent and also the amount of virus in the gut when compared with the group without maternal antibody, the rates of recovery of virus from the hock joints were very similar. The possible epidemiological importance of persistent virus in the joints of clinically protected chicks is discussed. PMID- 2994190 TI - Effect of parenteral vitamin D3 on plasma 25-hydroxyvitamin D3 concentration in sheep. AB - Vitamin D3 ranging in total amount from 10(5) iu (2.5 mg) to 9 X 10(5) iu (22.5 mg) was given intramuscularly either as a single injection or in three aliquots at three-weekly intervals to housed nonpregnant ewes on a vitamin D deficient diet. The effects on plasma concentrations of 25-hydroxyvitamin D3 (25-OHD3), the major metabolite of vitamin D, were monitored. The increase in plasma 25-OHD3 showed a large variation between animals and was related to, but not proportional to, the dose. None of the treatments produced 25-OHD3 concentrations greater than those in grazing sheep in summer. Repeated dosing provided for more efficient use of the injected vitamin D3. PMID- 2994191 TI - Reaction of goats to infection with infectious bovine rhinotracheitis virus. AB - Intranasal exposure of goats to infectious bovine rhinotracheitis virus resulted in mild respiratory disease and virus reisolation from nasal secretions. No disease was produced in goats exposed to the same virus by the genital or ocular routes. There was serological evidence of contact transmission of infection from infected goats to cattle. Virus recrudescence was not detected in goats treated with dexamethasone two months after virus inoculation. PMID- 2994192 TI - [Implications of anterior ST depression in acute inferior myocardial infarction]. PMID- 2994193 TI - [Acute effect of MK-421 (enalapril) on blood pressure, the renin-angiotensin system and the kallikrein-kinin system in hypertensive patients]. PMID- 2994194 TI - Effects of high K on relaxation produced by drugs in the guinea-pig tracheal muscle. AB - In the guinea-pig tracheal smooth muscle, effects of various relaxants were compared in normal (5.9 mM) and excess (40 mM) K media. The relaxing effect of calcium-channel blockers, nifedipine and verapamil (group I) was potentiated by increasing the external K concentration. The effect of the drugs which are supposed to increase intracellular cyclic AMP, such as isoprenaline, forskolin, isobutylmethylxanthine, theophylline, dibutyryl cyclic AMP (group II) was moderately reduced by excess K. Nitroprusside, 8-bromo-cyclic GMP and sodium nitrite (group III) are generally considered to increase intracellular cyclic GMP and their effect was markedly reduced by excess K. When the tension development was made the same at 5.9 mM K and 40 mM K by adjusting the Ca concentration, the relaxing effect was similar and independent of the K concentration both for group II and group III drugs. It seems that the group II drugs can better overcome a large influx of Ca than group III drugs. PMID- 2994195 TI - CALLA-positive bone marrow B lymphocytes in cytomegalovirus-infectious mononucleosis (case report). PMID- 2994196 TI - Thymopoietin to thymopentin: experimental studies. AB - Thymopoietin is a polypeptide hormone of the thymus consisting of 49 amino acids. The pentapeptide thymopentin (TP-5) Arg-Lys-Asp-Val-Tyr, corresponding to amino acids 32-36 of thymopoietin, appears to represent the active site of thymopoietin in that it has all the biological activities of the native hormone. Thymopoietin is secreted by epithelial cells of the thymus and is pleiotropic in action, affecting neuromuscular transmission, induction of early T cell differentiation and immune regulation. The immuno-regulatory actions of thymopentin on peripheral T cells are mediated by intracellular cyclic GMP elevations in contrast to the intracellular cyclic AMP elevations induced in precursor T cells that trigger their further differentiation to T cells. Thymopoietin and thymopentin have the biological characteristics of being immunonormalizing in a number of animal model systems of immune dysbalance. These include immune dysbalances induced by thymectomy or the thymic involution associated with aging or by other procedures in thymus-intact animals. The normalizing action of thymopentin, whether the immune dysbalance be in the direction of hyper- or hyporesponsiveness, points to its potential utility in human diseases characterized by immune dysbalance. PMID- 2994197 TI - Registry and follow-up systems of trophoblastic disease in Japan. AB - With recent progress in chemotherapy, the prognosis of patients with trophoblastic disease has greatly improved, but the remission rate of patients with choriocarcinoma remains unfavorable. To improve the prognosis of these patients, early detection and early treatment are essential. Under the leadership of the Committee of Trophoblastic Disease of the Japan Society of Obstetrics and Gynecology, regional registries of trophoblastic disease were started in 1974. By 1982 14 prefectures with a total population of 46,893,620 were included in the registry. The early detection and treatment and follow-up made possible by the registry in addition to the introduction of advanced chemotherapy may be responsible for a rapidly decreasing trend in the death rate from this disease. PMID- 2994198 TI - Epidemiology of cytomegaloviral infections: recommendations for prevention and control. AB - Cytomegalovirus (CMV) causes serious illness in immunocompromised patients and congenitally infected neonates. Knowledge of the epidemiologic characteristics of CMV remains limited, but specific and practical measures can help prevent the transmission of infection to persons in high-risk groups. Good personal hygiene, especially hand washing, is the most effective means of preventing the acquisition of CMV by pregnant women and by individuals who care for children and immunocompromised patients. Screening programs for the identification of seronegative pregnant women and female hospital employees or of asymptomatic children who are excreting CMV are not practical or beneficial; educational programs are recommended instead. Since CMV infection is endemic in the community, exclusion of known CMV excretors from schools is not indicated. Exposure of premature infants or of organ transplant recipients and other severely immunocompromised patients to exogenous sources of CMV, such as blood transfusions, should be minimized. These recommendations may need to be revised in the future as more specific knowledge of the epidemiology of CMV is gained. PMID- 2994199 TI - [The vaccine for hepatitis B virus. Definition of criteria for its use]. PMID- 2994200 TI - [Erythrocyte enzyme changes in hemophilia A]. PMID- 2994201 TI - [Prevalence of anti-LAV antibodies in hemophiliacs, correlation with the immunological state]. AB - 49 french haemophiliacs (haemophilia A: 41 patients; haemophilia B: 8 patients) were serologicaly tested for LAV antibodies: 10 patients (20.4%) were seropositive including 9 (21.9%) with haemophilia A and 1 (12,5%) with haemophilia B. Between seronegative and seropositive patients total lymphocyte and T-lymphocyte sub-populations counts were not significantly different. The mean serum IgG level was higher and palpable lymphadenopathy more frequently encountered among seropositive patients. PMID- 2994202 TI - [Acquired immunologic deficiency syndrome. Recommendations of group of French physicians and hemophilia specialists]. PMID- 2994203 TI - [ Methodological evaluation and clinical significance of erythrocyte aggregation]. AB - Red cell aggregation, which occurs in vivo when blood flow slows down, has been studied in 22 healthy subjects and 31 type I diabetics by means of Myrenne MA1 automatic aggregometer. First of all, the most reliable and precise technique has been studied: the best results have been obtained employing 25 microliters of K2 EDTA-anticoagulated blood stored between 0 and 20 degrees C for a maximum of 4 h. The present study has shown statistically significant differences (p = 0.01) between the two groups studied (controls: mean = 11.895 MEA +/- 0.863; diabetics: mean = 15.552 MEA +/- 0.985). Moreover, correlations between erythrocyte aggregability and hematochemical and rheological parameters like fibrinogen, ESR, serum proteins, plasma and blood viscosity, have been evaluated. PMID- 2994204 TI - [Calcitonin, parathyroid hormones, calcium ion, cyclic 3,5-adenosine monophosphate (cAMP) and erythrocyte deformability]. AB - Calcium ion is one of the factors which modulate erythrocyte deformability. It is known that calciotropic hormones such as calcitonin (CT) and parathyroid hormone (PTH) exert hypocalcemizing and hypercalcemizing effects, respectively. Their action is mediated, at the level of their target cells, through adenylcyclase activation with the production of cyclic 3,5-adenosinmonophosphate (cAMP). Modifications of transmembrane calcium fluxes have been described and were attributed to these hormones. Erythrocyte deformability has been evaluated by Dormandy method of red blood cell filtration in hypocalcemic patients affected by hypoparathyroidism, in patients with hypercalcemia due to malignancy or primary hyperparathyroidism and in normal age- and sex-matched subjects. Erythrocyte filtration values resulted to be significantly increased with respect to normal values in hypercalcemic patients and at the lower limits of normality in hypocalcemic subjects. Subsequently, acute studies were performed in normal volunteers in whom venous infusions of synthetic salmon calcitonin determined a significant reduction of erythrocyte filtration values, whereas venous infusions of the 1-34 synthetic human PTH fragment induced a significant increase in filtration values of red blood cells. An infusion of a cAMP analogue, dibutyryl cAMP, determined a slight reduction of erythrocyte filtration values. The calciotropic hormones influence erythrocyte deformability through mechanisms that are yet to be clarified. PMID- 2994205 TI - Toxicity studies on the radioprotective agent WR-2721 in CDF1 mice and beagle dogs. AB - WR-2721, S-2-(3-aminopropylamino)ethylphosphorothioic acid, is used extensively to protect normal cells during the irradiation of neoplastic cells. Dose levels for human radiotherapy are based on results obtained from laboratory animal lethality and toxicity studies. WR-2721 was administered intravenously to CDF1 mice and beagle dogs. Single dose lethality studies in mice showed the average 1/10 of the lethal dose, the median lethal dose and 9/10 the lethal dose to be 508 (1523 mg/m2), 589 (1766 mg/m2), and 682 mg/kg (2047 mg/m2), respectively. The lethal dose for female mice was lower than that for males. The 1/10 lethal dose in mice was slightly toxic to dogs; 1/10 of that dose was nontoxic. The lethal dose for dogs (6000 mg/m2) was higher than that for mice (2000 mg/m2). Clinical signs of toxicosis in the single-dose mouse toxicity study were evident in the 1st week following treatment and declined during the recovery period; signs of toxicosis were transient in dogs. Acute drug-induced pathologic changes included elevated BUN and SGOT levels, lymphoid necrosis, and renal tubular degeneration in mice. These changes were evident in the 1st week following treatment, but had dissipated by study termination. Generalized vascular changes (congestion, hemorrhage, and edema) and renal tubular degeneration occurred in treated dogs that had died or were killed moribund 7 days postinjection. These findings indicate sex-dependent and interspecies variation in the toxicity of WR-2721 with acute, but reversible, pathologic changes. PMID- 2994206 TI - Cyclosporin A inhibits induction but not production of gamma interferon induced by Epstein-Barr virus. AB - Cyclosporin A (CsA) interferes with various T-cell functions in vitro and is a potent inhibitor of T-cell-dependent reactions in vivo, such as graft rejection and control of virus infections. Since human gamma interferon (Hu IFN-gamma) is synthesized by T cells and has a controlling role in regulation of Epstein-Barr virus (EBV) infection, we have studied the effects of CsA on EBV-induced cellular Hu IFN-gamma release. CsA inhibited dose-dependently the EBV-induced Hu IFN-gamma response, studied at the cellular level in human blood lymphocytes. These effects were not due to toxicity of CsA, since at inhibitory levels cellular EBV infection measured as polyclonal IgM production proceeded unaffected. CsA did not affect the number of spontaneous Hu IFN-gamma-secreting cells, nor did it have any inhibitory effect if added after virus exposure. It is concluded that CsA inhibits induction but not production of cellular Hu IFN-gamma. PMID- 2994207 TI - [Pathogenic viruses in activated sludge: problems and quantification]. PMID- 2994208 TI - [Prevalence of antibodies against HTLV-III in various regions in Switzerland]. AB - Acquired immune deficiency syndrome (AIDS) has been etiologically linked with the human T-cell lymphotropic virus HTLV-III. In a study on the prevalence of antibodies to this virus in Switzerland, sera from 941 individuals were collected in 5 major urban areas (Basel, Berne, Geneva, Lausanne and Zurich) in 1983 and 1984. All sera were tested by ELISA and the majority also by Western blot. We found an antibody prevalence of 100% among 22 cases of AIDS, of 94% among 48 cases of AIDS-related complex (ARC), and of 57% among 14 homosexual contacts of AIDS patients. Among 55 sera collected from i.v. drug addicts in 1984, 53% were positive, whereas the rate had been 36% among 103 sera collected in 1983. The rate was 19% among 227 sera collected from asymptomatic active homosexuals in 1984, against 10% among 40 sera collected in 1983. 8% positives were found among 98 patients with various types of viral hepatitis. In addition, 4% of 84 sera of individuals from Equatorial Africa were positive. No positives were found among 15 sera of individuals from other African regions, among 32 sera of persons from various regions of Asia, and among 203 Swiss blood donors. These results demonstrate the high and increasing rate of HTLV-III infection among groups at risk for AIDS, and suggest that HTLV-III-related diseases will be a serious problem in the years ahead. PMID- 2994209 TI - [Paroxysmal phenomena in multiple sclerosis]. AB - Paroxysmal phenomena are rare but relatively typical, and occasionally initial, clinical symptoms of multiple sclerosis. They should be recognized since they can serve as clinical indicators of the disease and can be treated efficiently with carbamazepine. Such shortlived clinical symptoms and signs are probably caused by ephaptic transmission due to myelinoaxonal dissociation within the demyelinated plaque. The clinical picture, pathophysiology and treatment of these disorders are discussed. PMID- 2994210 TI - [Viral hepatitis and AIDS-associated HTLV III/LAV virus infections in drug addicts]. AB - In 1984 intravenous drug users accounted for 25% of all cases of acute hepatitis B and for 32% of all cases of non-A/non-B hepatitis recorded in the Canton of Zurich. Drug addicts also represented an important risk group for hepatitis A, which occurred in small epidemics. Among 141 "healthy" drug users, 10 (7%) individuals showed signs of ongoing hepatitis B virus (HBV-) infection, 26 (19%) individuals had the finding "anti-HBc-alone" (unresolved HBV-infection) and another 69 (49%) individuals were HBV-immune. 30%, 23% and 16% of the three above groups of individuals with HBV-markers were also anti-delta virus positive. Of the 141 "healthy" drug users 23 (16%) showed signs of an ongoing hepatitis A virus (HAV-) infection and 63 (45%) individuals were HAV-immune. Anti-HTLV III/LAV was detected in 69 (49%) of the i.v. drug users, with a higher prevalence (60%) in females than in males and with a higher prevalence in individuals with HBV-markers (50-62%) than in those without such markers (19%). Thus the i.v. drug users are at high risk for infections with hepatitis viruses A, B, D and those responsible for non-A/non-B hepatitis, as well as with the AIDS-associated virus HTLV III/LAV. Preventive measures should include a reduction of needle sharing and of promiscuity, which is abundantly practised by i.v. drug users. PMID- 2994211 TI - DNA damage by benzo[a]pyrene in the liver of mosquito fish Gambusia affinis. AB - Exposure of Gambusia affinis to water containing different concentrations of benzo[a]pyrene (BaP) causes an increase in benzo[a]pyrene monooxygenase (BPMO) activity which reaches a maximum on the second day. Concomitantly, the DNA is altered in such a way that nuclease S1-sensitive sites (SSS) become measurable. The size distribution of liver DNA treated with nuclease S1 in control fish shows two populations of DNA by length, with means of 30 X 10(6) and 60 X 10(6) Daltons, respectively. In fish treated with 100 ppb BaP, the population with longer molecules of DNA disappears and shorter molecules increase in number. This may be explained in terms of the introduction of an additional 0.31-0.46 DNA nicks per control DNA molecule caused by metabolically activated BaP derivatives. PMID- 2994212 TI - Expression in brain of a messenger RNA encoding a novel neuropeptide homologous to calcitonin gene-related peptide. AB - As a consequence of alternative RNA processing events, a single rat gene can generate messenger RNA's (mRNA's) encoding either calcitonin or a neuropeptide referred to as alpha-type calcitonin gene-related peptide (alpha-CGRP). An mRNA product of a related gene has been identified in rat brain and thyroid encoding the protein precursor of a peptide differing from alpha-CGRP by only a single amino acid. The RNA encoding this peptide, which is referred to as beta-CGRP, appears to be the only mature transcript of the beta-CGRP gene. Hybridization histochemistry reveals a similar distribution of alpha- and beta-CGRP mRNA's, but their relative levels of expression vary in different cranial nerve nuclei. Thus beta-CGRP is a new member of a family of related genes with potential functions in regulating the transduction of sensory and motor information. PMID- 2994213 TI - Contribution of promoter to tissue-specific expression of the mouse immunoglobulin kappa gene. AB - The immunoglobulin kappa (kappa) gene promoter was activated by a "neutral" enhancer derived from Harvey murine sarcoma virus (HaMuSV) in immunoglobulin producing myeloma cells, regardless of the enhancer's orientation or position in the vector. In one fibroblast line (3T3) the immunoglobulin kappa gene promoter was completely inactive when linked to the HaMuSV enhancer, whereas in mouse L cells, promoter activity was observed only with the HaMuSV enhancer in tandem with the immunoglobulin kappa gene promoter. The differential behavior of the gene promoter, when activated by a neutral enhancer in these three murine cell lines, suggests that promoter sequences contribute to the tissue-specific expression of this gene. PMID- 2994214 TI - Strategies and applications of in vitro mutagenesis. PMID- 2994215 TI - Genetic engineering of novel genomes of large DNA viruses. AB - Analyses of the function of specific genes and sequences of large DNA viruses such as herpesviruses and poxviruses present special problems because of the size of their genomes (120 to 250 kilobase pairs). Various methods for engineering site-specific insertions or deletions based on the use of selectable markers have been developed and applied for the elucidation of the function of specific DNA sequences, the identification of genes nonessential for virus growth in cell culture, and the expression of foreign genes. These methods should also make possible the construction of viral vectors capable of delivering genes specifying antigens for the prevention of infectious diseases in humans and animals. PMID- 2994216 TI - Benzodiazepine receptor-mediated chemotaxis of human monocytes. AB - Benzodiazepines, which are widely prescribed for their antianxiety effects, are shown to be potent stimulators of human monocyte chemotaxis. The chemotactic effects of benzodiazepine receptor agonists were blocked by the peripheral benzodiazepine receptor antagonist PK-11195, suggesting that these effects are mediated by the peripheral-type benzodiazepine receptor. Diazepam was also active in inducing chemotaxis. Binding studies on purified monocytes revealed high affinity peripheral benzodiazepine receptors, and the displacement potencies of various benzodiazepines correlated with their relative potencies in mediating chemotaxis. The demonstration of functional benzodiazepine receptors on human monocytes, together with recent evidence of receptor-mediated monocyte chemotaxis by other psychoactive peptides (such as opiate peptides), suggests a biochemical substrate for psychosomatic communication. PMID- 2994217 TI - The epidemiology of AIDS: current status and future prospects. AB - The reported incidence of acquired immune deficiency syndrome (AIDS) continues to increase in countries throughout the world. On the basis of a polynomial model for extrapolation, the cumulative number of cases diagnosed and reported since 1981 in the United States is expected to double during the next year with over 12,000 additional cases projected to be diagnosed by July 1986. The annual incidence rates for single (never-married) men in Manhattan and San Francisco, intravenous drug users in New York City and New Jersey, and persons with hemophilia A ranged from 261 to 350 per 100,000 population during 1984. For single men aged 25 to 44 years in Manhattan and San Francisco, AIDS was the leading cause of premature mortality in 1984 as measured by years of potential life lost. Infection with HTLV-III/LAV is considerably more common than reported AIDS in high-risk populations and can persist at least for several years, so the presence of specific antibody should be considered presumptive evidence of current infection. The screening of donated blood and plasma for antibody to HTLV III/LAV and use of safer clotting factor concentrates should greatly reduce HTLV III/LAV transmission through blood and blood products. Most HTLV-III/LAV infections occur through sexual transmission, use of contaminated needles, and as a result of infected mothers passing the virus to newborns. Continued research commitment is needed to develop an HTLV-III/LAV vaccine and therapy for this infection. In the interim, widespread community efforts are needed to minimize transmission. PMID- 2994218 TI - Three-dimensional structure of poliovirus at 2.9 A resolution. AB - The three-dimensional structure of poliovirus has been determined at 2.9 A resolution by x-ray crystallographic methods. Each of the three major capsid proteins (VP1, VP2, and VP3) contains a "core" consisting of an eight-stranded antiparallel beta barrel with two flanking helices. The arrangement of beta strands and helices is structurally similar and topologically identical to the folding pattern of the capsid proteins of several icosahedral plant viruses. In each of the major capsid proteins, the "connecting loops" and NH2- and COOH terminal extensions are structurally dissimilar. The packing of the subunit "cores" to form the virion shell is reminiscent of the packing in the T = 3 plant viruses, but is significantly different in detail. Differences in the orientations of the subunits cause dissimilar contacts at protein-protein interfaces, and are also responsible for two major surface features of the poliovirion: prominent peaks at the fivefold and threefold axes of the particle. The positions and interactions of the NH2- and COOH-terminal strands of the capsid proteins have important implications for virion assembly. Several of the "connecting loops" and COOH-terminal strands form prominent radial projections which are the antigenic sites of the virion. PMID- 2994219 TI - Picornaviruses are no longer black boxes. PMID- 2994220 TI - Transcription of novel open reading frames of AIDS retrovirus during infection of lymphocytes. AB - The retrovirus frequently isolated from patients with the acquired immune deficiency syndrome (AIDS) has two novel open reading frames previously designated "A" and "B." The "A" region was found to be specifically expressed as polyadenylated RNA's of 5.5 and 5.0 kilobases in infected cells. The "B" region was expressed as 1.8- to 2.0-kilobase RNA species. Additional full-length and spliced messenger RNA's of the env region were also identified. PMID- 2994221 TI - Site-specific increased phosphorylation of pp60v-src after treatment of RSV transformed cells with a tumor promoter. AB - When vole cells that had been transformed by Rous sarcoma virus were treated with the tumor-promoting phorbol ester 12-O-tetradecanoyl-13-acetate (TPA), specific phosphorylation of pp60v-src was increased. Partial V8 protease mapping indicated that the increased phosphorylation occurred exclusively on serine residues located in the amino terminus of the molecule. Treatment of cells with dimethyl sulfoxide or 4 alpha-phorbol-12,13-didecanoate did not elicit this response. Two dimensional tryptic phosphopeptide mapping of pp60v-src immunoprecipitated from untreated and TPA-treated cells indicated that a specific tryptic amino-terminal peptide was hyperphosphorylated. PMID- 2994222 TI - Persistent noncytopathic infection of normal human T lymphocytes with AIDS associated retrovirus. AB - Infection of normal peripheral blood T cells by the acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV) was evaluated in long-term cultures of helper-inducer T cells (T4 cells). Cells that were inoculated with ARV and maintained in medium supplemented with interleukin-2 remained productively infected with this virus for more than 4 months in culture, although they showed no cytopathic effects characteristic of acute ARV infection. The presence of replicating virus was demonstrated by reverse transcriptase activity of culture fluids and by viral antigens and budding particles detected on cells by immunofluorescence and electron microscopy. Virus produced in these cultures remained infectious and could induce cytopathic effects and viral antigens in uninfected lymphoid cells. The finding that normal lymphocytes may be productively infected by an AIDS retrovirus in the absence of cell death suggests that a range of biologic effects may occur after infection in vivo. PMID- 2994223 TI - Characterization of gp41 as the transmembrane protein coded by the HTLV-III/LAV envelope gene. AB - Radiolabeled amino acid sequencing was used to characterize gp41, an antigen of HTLV-III/LAV, the virus believed to be the etiological agent of the acquired immune deficiency syndrome. This antigen is the one most commonly detected in immunoblot assays by sera of patients with AIDS or AIDS-related complex (ARC) and other individuals infected with HTLV-III/LAV. A mouse monoclonal antibody that was reactive with gp41 precipitated a 160-kilodalton protein (gp160) in addition to gp41, but did not precipitate a 120-kilodalton protein (gp120) from extracts of metabolically labeled cells producing HTLV-III. Extracts of infected cells that had been labeled with tritiated leucine or isoleucine were immunoprecipitated with the monoclonal antibody. The immunoprecipitates were fractionated by polyacrylamide gel electrophoresis and the p41 was eluted from the gel bands and subjected to amino-terminal radiolabeled amino acid sequencing by the semiautomated Edman degradation. Leucine residues occurred in cycles 7, 9, 12, 26, 33, and 34 among 40 cycles and isoleucine occurred in cycle 4 among 24 cycles analyzed. Comparison of the data with the deduced amino acid sequence of the env gene product of HTLV-III precisely placed gp41 in the COOH-terminal region of the env gene product. Gp160 is thus the primary env gene product and it is processed into gp120 and gp41. PMID- 2994224 TI - Pathways of protein secretion in eukaryotes. AB - Protein secretion from cells can take several forms. Secretion is constitutive if proteins are secreted as fast as they are synthesized. In regulated secretion newly synthesized proteins destined for secretion are stored at high concentration in secretory vesicles until the cell receives an appropriate stimulus. When both constitutive and regulated protein secretion can take place in the same cell a mechanism must exist for sorting the correct secretory protein into the correct secretory vesicle. The secretory vesicle must then be delivered to the appropriate region of plasma membrane. Transfection of DNA encoding foreign secretory proteins into regulated secretory cells has provided insight into the specificity of sorting into secretory vesicles. PMID- 2994225 TI - Human apolipoprotein B: structure of carboxyl-terminal domains, sites of gene expression, and chromosomal localization. AB - Apolipoprotein (apo-) B is the ligand responsible for the receptor-mediated catabolism of low density lipoproteins, the principal cholesterol-transporting lipoproteins in plasma. The primary structure of the carboxyl-terminal 30 percent (1455 amino acids) of human apo-B (apo-B100) has been deduced from the nucleotide sequence of complementary DNA. Portions of the protein structure that may relate to its receptor binding function and lipid binding properties have been identified. The apo-B100 messenger RNA is about 19 kilobases in length. The apo B100 gene is expressed primarily in liver and, to a lesser extent, in small intestine, but in no other tissues. The gene for apo-B100 is located in the p24 region (near the tip of the short arm) of chromosome 2. PMID- 2994226 TI - Intercontinental spread of a new antibiotic resistance gene on an epidemic plasmid. AB - Bacteria of different genera isolated at nine medical centers in different parts of the United States and at one center in Venezuela during the first decade of gentamicin usage carried the gentamicin resistance gene 2"-aminoglycoside nucleotidyltransferase on the same transferable plasmid. Such widespread dissemination of a newly observed resistance gene on one plasmid suggests that a new resistance gene may emerge once on a single plasmid, which then carries it to other centers and other plasmids. The resistance gene might, therefore, be contained if detected early. PMID- 2994227 TI - A potent nonpeptide cholecystokinin antagonist selective for peripheral tissues isolated from Aspergillus alliaceus. AB - A new, competitive, nonpeptide cholecystokinin (CCK) antagonist, asperlicin, was isolated from the fungus Aspergillus alliaceus. The compound has 300 to 400 times the affinity for pancreatic, ileal, and gallbladder CCK receptors than proglumide, a standard agent of this class. Moreover, asperlicin is highly selective for peripheral CCK receptors relative to brain CCK and gastrin receptors. Since asperlicin also exhibits long-lasting CCK antagonist activity in vivo, it should provide a valuable tool for investigating the physiological and pharmacological actions of CCK. PMID- 2994228 TI - Cloning of Shiga-like toxin structural genes from a toxin converting phage of Escherichia coli. AB - The genes controlling high-level production of Shiga-like toxin (SLT) in Escherichia coli were cloned from the SLT converting phage 933J. This phage was isolated from a strain of E. coli that caused a foodborne outbreak of hemorrhagic colitis. The genes that convert normal E. coli to organisms producing high levels of toxin were cloned into the plasmid pBR328 and expressed in E. coli HB101. DNA restriction mapping, subcloning, examination of the cloned gene products by minicell analysis, neutralization, and immunoprecipitation with antibodies to SLT were used to localize the toxin converting genes and identify them as structural genes for SLT. Southern hybridization studies established that the DNA fragment carrying the cloned toxin structural genes had homology with the DNA of Shigella. PMID- 2994229 TI - Externalization of beta-adrenergic receptors promoted by myocardial ischemia. AB - beta-Adrenergic receptors were identified in two fractions of guinea pig myocardium: a purified sarcolemmal fraction and a light vesicle (presumably intracellular) fraction. In the light vesicle fraction, which contained approximately 25 percent of the myocardial receptors under control conditions, the receptors appeared to be segregated from the stimulatory guanine nucleotide binding and catalytic components of adenylate cyclase. During myocardial ischemia, beta-adrenergic receptors were redistributed from the intracellular vesicles to the sarcolemmal fraction, where isoproterenol-stimulated adenylate cyclase activity was increased. These findings should facilitate further studies on cellular and molecular mechanisms that regulate adrenergic receptor traffic in the myocardium and may explain the rapid enhancement in adrenergic receptor expression that occurs with myocardial ischemia. PMID- 2994230 TI - Acquisition by innervated cardiac myocytes of a pertussis toxin-specific regulatory protein linked to the alpha 1-receptor. AB - During development, the chronotropic response of rat ventricular myocardium to alpha 1-adrenergic stimulation changes from positive to negative. The alpha 1 agonist phenylephrine increases the rate of contraction of neonatal rat myocytes cultured alone but decreases the rate of contraction when the myocytes are cultured with functional sympathetic neurons. The developmental induction of the inhibitory myocardial response to alpha 1-adrenergic stimulation in intact ventricle and in cultured myocytes was shown to coincide with the functional acquisition of a substrate for pertussis toxin. A 41-kilodalton protein from myocytes cultured with sympathetic neurons and from adult rat myocardium showed, respectively, 2.2- and 16-fold increases in pertussis toxin-associated ADP ribosylation (ADP, adenosine diphosphate) as compared to controls. In nerve muscle cultures, inhibition of the actions of this protein by pertussis toxin specific ADP-ribosylation reversed the mature inhibitory alpha 1-adrenergic response to an immature stimulatory pattern. The results suggest that innervation is associated with the appearance of a functional pertussis toxin substrate by which the alpha 1-adrenergic response becomes linked to a decrease in automaticity. PMID- 2994231 TI - The synovial lining cell: biology and pathobiology. PMID- 2994232 TI - Radiopharmaceuticals for spleen and bone marrow studies. AB - The spleen and bone marrow have important hematologic functions. Evaluation of bone marrow alone may be incomplete, unless the function of the spleen is studied also. In addition to hematologic functions, each organ has unique characteristics that may need to be evaluated in assessing its activity. The evaluation of the distribution of reticuloendothelial cells, through the phagocytosis of radioactive particles, provides the basis for most of the morphological and functional studies of these organs. During the past three decades, a variety of radioactive agents have been prepared and examined. Among them, colloids of 99mTc, heat-damaged 99mTc labeled RBCs, and the radionuclides of iron and indium, make possible the functional and morphological examination of the spleen and bone marrow by external scintigraphy. PMID- 2994233 TI - The clinical use of radionuclide bone marrow imaging. AB - Bone marrow aspiration and biopsy are excellent techniques for evaluating bone marrow, but this evaluation is limited to a small part of the total blood-forming organ. With the introduction of radionuclide bone marrow imaging, a simple technique became available that overcomes marrow sampling errors by giving a total body view of functioning marrow. Furthermore, the procedure is noninvasive and provides an atraumatic method for evaluating a number of clinical problems including a discrepancy between bone marrow histology and clinical status (possible marrow sampling error), the determination of amount of active marrow after radiation and chemotherapy when further therapy is being considered, detection of sites of extramedullary hematopoiesis, location of the optimal sites for bone marrow biopsy, the diagnosis and staging of diffuse hematologic disorders, detection of metastases, the diagnosis of bone marrow infarcts in hemolytic anemias, and detecting avascular necrosis of the femoral heads. There are two major classes of bone marrow agents: (1) those that are incorporated into the erythroid precursors such as radioiron and (2) colloids that are taken up by the reticuloendothelial system (RES). Indium-111 chloride was originally considered to be an erythropoietic agent but appears to share some properties of RES labels. The best label to use is dependent on the disease being evaluated. PMID- 2994234 TI - Platelet metabolism and activation. AB - Stimulation and execution of platelet responses are intimately coupled to the cells' energy metabolism. The turnover of cytoplasmic ATP is higher than in most other cells; when it is lowered, platelet responses are powerfully inhibited. One third of the total adenine nucleotides are present in the cytoplasm and a small fraction of these, 50% of the ADP, is bound to actin and not available to metabolism. These nucleotides are not synthesized de novo in platelets which readily form them from adenine, adenosine, and hypoxanthine. The remaining two thirds are sequestered in the dense granules together with serotonin and divalent metal ions. NMR studies show that this ATP and ADP are stacked in large aggregates together with the divalent cations. Serotonin is incorporated into the aggregates by being positioned between, and in close proximity to, adjacent adenine rings. In the congenital platelet disorder, "Storage Pool Deficiency," the dense granule content is absent or markedly reduced, which may be caused by an impaired transport of ATP across the dense granule membrane. The content of the dense granules, the beta-thromboglobulin, albumin, fibrinogen, certain coagulation factors, fibronectin of the alpha-granules, and the acid hydrolases of the lysosomes are discharged by exocytosis, but the exact mechanisms are not known. This discharge, platelet secretion, is complete for the dense granule and alpha-granules contents, but is only 30% to 55% for the acid hydrolases. After pretreatment of platelets with certain amines, thrombin causes secretion of all beta-hexosaminidase, indicating that the beta-hexosaminidase usually retained during secretion is bound within the lysosomes at the acid pH but becomes dissociated by raising the pH with the amines. Agonist-induced platelet aggregation and secretion are accompanied by rapid changes in phosphoinositide metabolism which are usually monitored as the radioactivity in phospholipids of platelets preincubated with radioactive arachidonate, glycerol, inositol, or orthophosphate. The mechanisms for precursor incorporation are not well known and interpretations of published results are hampered by much uncertainty. Experimental support for the widely accepted coupling of receptor occupancy to the phosphodiesteratic hydrolysis of phosphatidylinositol or phosphatidylinositol 4,5-biphosphate is nonexistent. These two hydrolytic steps occur concomitantly and together with formation of diglyceride and phosphatidate; all four steps cannot be temporally resolved.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2994235 TI - [Gaviscon]. PMID- 2994236 TI - [What tests and which treatments for liver cancer?]. PMID- 2994237 TI - Isolation and regional mapping of random X sequences from distal human X chromosome. AB - Chromosome-mediated gene transfer (CMGT) lines were shown to be convenient donors of genomic sequences from specific regions of the genome adjacent to selectable markers. Two libraries were prepared from CMGT lines carrying sequences spanning the long arm of the human X chromosome from HPRT (Xq26) to G6PD (Xq28). A series of 22 CMGT lines sharing the same selectable marker (HPRT) were used in conjunction with five standard translocation hybrids to provide fine-resolution regional mapping of the nonrepetitive X specific probes isolated from the libraries. The order of three human recombinant sequences with respect to known X linked markers is: PGK (Xq13), 05-02 (DXS78); HPRT (Xq26), 07-03 (DXS79); surface antigen S11 (Xq27), 07-14 (DXS80); and G6PD (Xq28). PMID- 2994238 TI - Novel approach for restriction mapping repetitive DNA elements using DNA transformation. AB - We demonstrated that DNA transformation can be used to determine the linkage relationship between DNA restriction fragments in mouse genomic DNA. Using this experimental approach, we obtained linkage information which enabled us to construct a restriction map for the multiple thymidine kinase (tk) gene inserts present in a mouse L-cell line. This restriction map included cutting sites for seven restriction enzymes spanning a distance of over 10 kb. It revealed that the tk inserts in this cell line are arranged in a complex array consisting of direct and inverted repeats. In light of these results, we suggest that this approach will be particularly useful for restriction mapping DNA sequences that are repetitive as such DNA may be difficult to characterize by conventional methods alone. PMID- 2994239 TI - Translocation chromosome 7 of A431 cells contains amplification and rearrangement of EGF receptor gene responsible for production of variant mRNA. AB - The epidermal growth factor (EGF) receptor, along with several oncogene protein products, possesses tyrosine-specific protein kinase activity. Furthermore, the EGF receptor has structural similarity to the putitive v-erb-B transforming protein. Because of these closely shared characteristics, it is important to elucidate the possible involvement of the EGF receptor in malignant transformation. The epidermal carcinoma cell line A431 exhibits an abnormally high number of EGF receptors, which is associated with the presence of translocation chromosome M4. Recently, A431 cells have been shown to contain amplified sequences for the EGF receptor gene(s) and also to produce a variant mRNA which diverges from the normal EGF receptor mRNA at the 3' end. Here we report, using the human EGF receptor cDNA probe pE7, that the chromosome M4 has a six- to sevenfold amplification of the EGF receptor gene. Furthermore, the presence of M4 in somatic cell hybrids correlates with the production of the variant 2.9-kb mRNA. This aberrant mRNA is apparently generated by an intrachromosomal rearrangement which was detected using as a probe a fragment of the pE15cDNA encoding the variant mRNA. PMID- 2994240 TI - High spontaneous mutation frequency of BPV shuttle vector. AB - A recombinant shuttle vector containing the entire bovine papillomavirus (BPV) genome, sequences from pBR322, and the Escherichia coli gpt gene was used to transform mouse C127 cells. Plasmid extracted from the transformed mouse cells was used to transform a Gpt- derivative of E. coli HB101, and the relative frequency of plasmids carrying a mutation in the gpt gene was determined. Approximate mutant frequencies of 3-16 X 10(-3) were observed for plasmid molecules which had been passaged through the mammalian cells. Restriction digest analysis indicated that most of the mutant plasmid molecules had gross rearrangements in their DNA structures. PMID- 2994241 TI - Transformation associated p53 protein is encoded by a gene on human chromosome 17. AB - The human gene for the transformation-associated p53 phosphoprotein (P53) was assigned to the short arm of chromosome 17 using human-rodent somatic cell hybrids and Southern filter hybridization of cell hybrid DNA. The filters were hybridized to radiolabeled DNA from a genomic clone which contained P53 nucleotide sequences. Hybridization of the probe to a 2.5-kb human DNA fragment in HindIII-digested DNA was used to identify the human P53 gene. PMID- 2994242 TI - Assignment of human gamma crystallin multigene family to chromosome 2. AB - The multigene family for human gamma-crystallin has been assigned to chromosome 2 using rodent-human somatic cell hybrids and filter hybridization analysis of cell hybrid DNA. Two genomic DNA probes containing human gamma-crystallin gene sequences hybridize to five fragments in human DNA digested with the restriction enzyme EcoRI. By correlating the presence of these fragments in somatic cell hybrid DNA with the human chromosome content of the hybrids, at least six human gamma-crystallin genes can be mapped to chromosome 2. Data obtained with a hybrid clone containing a mouse-human interspecies translocation suggest that these genes may be clustered together on the long arm of human chromosome 2. PMID- 2994243 TI - Defective polymorphonuclear leukocyte migration in AIDS. PMID- 2994244 TI - Nosocomial rotavirus infections in a South African hospital nursery for white infants. AB - Rotavirus infections in a nursery ward for newborn white infants in South Africa do not apparently differ from those encountered in other temperate countries. Neonates began to excrete rotavirus as early as 2 days after birth; a peak incidence of excretion occurred during the winter months. After a period of 4 years the incidence of excretion remained virtually unchanged. Rotaviruses also colonized and spread very rapidly in a newly commissioned nursery ward, possibly via respiratory infections. Passively acquired antibodies had no influence on the prevention of infections but may play a role in the suppression of clinical symptoms since none of the newborn population studied developed gastro-enteritis. The immunosorbent electron microscopy technique used to detect rotaviruses in this study yielded results that compared well with results obtained previously by other techniques. PMID- 2994245 TI - Research agenda for discharge planning. AB - With increased competition over diminishing resources in health care today, discharge planning has become a key element for hospital survival. The sudden high status now attached to this traditional social work function has caused major power struggles for control over discharge planning. Social workers have up to now succeeded in being undisputed leaders in this area because of tradition and demonstrations of clinical competence. Continued success and status in the future will require arguments and organizational design based on scientific research and hard data. This paper describes the main questions that need to be examined and proposes manageable approaches for conducting practice field studies. PMID- 2994246 TI - Occurrence of glioblastoma multiforme in three closely related patients. AB - Three cases of glioblastoma multiforme are presented. These cases have in common the fact that all three patients were relatives but not blood relatives. There had been prolonged intimate contact between them before the development of the neoplastic lesion. PMID- 2994247 TI - Does serum angiotensin converting enzyme reflect intensity of alveolitis in sarcoidosis? AB - Serum angiotensin converting enzyme activity is increased in many patients with pulmonary sarcoidosis and has been proposed as a measure of disease activity. Assay of serum angiotensin converting enzyme, bronchoalveolar lavage, and gallium scans were performed in 27 patients with biopsy proved pulmonary sarcoidosis. There was a positive correlation between serum angiotensin converting enzyme activity and an index of pulmonary gallium uptake assessed by the National Institutes of Health method (r = 0.7, p less than 0.001). There was no significant relationship (r = 0.19) between serum angiotensin converting enzyme activity and bronchoalveolar lavage lymphocytes expressed as a proportion of cells recovered. Increase in the enzyme activity had a sensitivity of 50% as a means of detecting high intensity alveolitis but specificity was only 45%. There was no significant difference in mean angiotensin converting enzyme activity between the following groups: those with positive and those with negative gallium scans; those with bronchoalveolar lavage lymphocyte counts less than or equal to 28% and those with counts greater than 28%. Although there was a significant correlation between the enzyme activity and one component of the alveolitis of sarcoidosis, the data suggest that serum angiotensin converting enzyme activity alone is neither sensitive nor specific enough for high intensity alveolitis. PMID- 2994248 TI - Contiguous granular cell myoblastoma and squamous cell carcinoma in the oesophagus. PMID- 2994249 TI - Lung angiotensin converting enzyme activity in chronically hypoxic rats. AB - A study was carried out to test the hypothesis that the reduced lung angiotensin converting enzyme (ACE) activity which occurs in chronic hypoxia is related to the development of pulmonary hypertension rather than to hypoxia per se. Right ventricular mean systolic pressure (Prvs, mm Hg) and ACE activity (nmol/mg protein/min) in lung tissue homogenates were measured in seven groups of four rats placed in a hypobaric chamber (380 mm Hg; 51 kPa) for two to 24 days. Identical measurements were made on 11 groups of four rats, which were placed in the chamber for 24 days and then allowed to recover in room air for one to 153 days. After two days of hypoxia the mean Prvs (25.5 (SD 3.7] and the mean lung ACE activity (56 (4.6] did not differ significantly from control values. Exposure to hypoxia for four to 24 days caused a progressive increase in mean Prvs to 44.4 (5.9) and a progressive reduction in mean lung ACE activity to 34 (4.0). During recovery lung ACE activity increased and Prvs decreased, so that normal values were achieved by 15 and 56 days respectively. Decreased lung ACE activity may be related to haemodynamic factors associated with pulmonary hypertension rather than to hypoxia. PMID- 2994250 TI - Membrane fluidity and platelet fibrinogen receptor exposure by proteolytic enzymes. AB - Incubation of platelets with pronase or chymotrypsin results in the exposure of fibrinogen receptors. We determined that these enzymes did not affect the membrane fluidity as evaluated by the depolarization of the fluorescence of 1,6 diphenyl-1,3,5 hexatriene (DPH). There was no significant difference in either the depolarization or in its temperature dependence for control, pronase or chymotrypsin-treated platelets. Thus, it can be concluded that the exposure of fibrinogen receptors on the platelet surface by proteolytic enzymes does not depend on the changes of membrane fluidity. We also propose that the proteolytic enzymes do not cause a major alteration in the extent of protein chains embedded in the lipid layers of the platelet membranes. PMID- 2994251 TI - T lymphocyte responses to Coxsackie B4 and mumps virus. I. Influence of HLA-DR restriction elements. AB - The proliferative T lymphocyte responses to Coxsackie B4-, mumps- and varicella zoster viral antigens were characterized. No significant difference in responsiveness was found between healthy individuals and patients with Type 1 (insulin-dependent) diabetes mellitus. Theophyllamine and verapamil decreased antigen-stimulated proliferation, whereas indomethacin in physiologic concentrations (1 microgram/ml) slightly increased proliferation. A major part of the response seemed to be restricted by HLA-DR molecules. Furthermore, for the mumps antigen the DR3- and DR4-determinants which are associated with Type 1 diabetes, seemed to have a different regulatory function on the T lymphocyte response in that an increased frequency of low responders was found among DR3 positive individuals and an increased frequency of high responders among DR4 positives. PMID- 2994252 TI - Delineation of the role of metabolism in the hepatotoxicity of trichloroethylene and perchloroethylene: a dose-effect study. AB - The relationship among dose, metabolism and hepatotoxicity in mice which resulted from subchronic exposure to the chlorinated solvents trichloroethylene (TRI) and perchloroethylene (PER) were examined. Male Swiss-Cox mice received either TRI (0 to 3200 mg/kg/day) of PER (0 to 2000 mg/kg/day) in corn oil by gavage for 6 weeks. Urinary metabolites from individual mice were quantified to estimate the extent to which each compound was metabolized. Four parameters of hepatotoxicity were assessed: liver weight, triglycerides, glucose-6-phosphatase (G6P) activity, and SGPT activity. TRI significantly affected liver weight and G6P activity; PER affected all four parameters. The metabolism of TRI was linearly related to dose through 1600 mg/kg, but then became saturated. The metabolism of PER was saturable. The dose-effect curves of the affected hepatotoxicity parameters of both compounds were nonlinear and resembled the dose-metabolism graph of the corresponding solvent. Plots of the hepatotoxicity data of each compound against total urinary metabolites were linear in all cases, suggesting that the hepatotoxicity of both PER and TRI in mice is directly related to the extent of their metabolism. This pattern is consistent with formation of the toxic intermediate in the primary metabolic pathway of each compound. PMID- 2994253 TI - The relationship between neurological damage and neurotoxic esterase inhibition in rats acutely exposed to tri-ortho-cresyl phosphate. AB - A rodent model of organophosphorus-induced delayed neuropathy (OPIDN) has been developed using Long-Evans adult male rats exposed to tri-ortho-cresyl phosphate (TOCP). In the present study an attempt was made to relate neurochemical with neuropathological changes in rats exposed to single dosages of TOCP ranging from 145 to 3480 mg/kg. The degree of neurotoxic esterase (NTE) inhibition, measured at 20 and 44 hr and at 14 days postexposure was correlated with the appearance of spinal cord pathology 14 days postexposure in a separate group of similarly dosed rats. Those dosages that inhibited mean NTE activity in spinal cord greater than or equal to 72% and brain greater than or equal to 66% of control values within 44 hr postexposure produced marked spinal cord pathology 14 days postexposure in greater than or equal to 90% of similarly dosed animals. In contrast, dosages of TOCP which inhibited mean NTE activity in the spinal cord less than or equal to 65% and in the brain less than or equal to 57% produced spinal cord pathology in less than or equal to 15% of the animals. These data indicate that NTE inhibition may be used as a biochemical predictor for TOCP-induced neurological damage in rats. PMID- 2994254 TI - Toxicity of 3,4,5,3',4',5'-hexabrominated biphenyl and 3,4,3',4'-tetrabrominated biphenyl. AB - Immature male rats were given a single equimolar dose (21.3 mumol/kg body wt) of 3,4,5,3',4',5'-hexabromobiphenyl (HBB) or 3,4,3',4'-tetrabromobiphenyl (TBB) and terminated at various times up to 14 days after treatment. Hepatic microsomal aryl hydrocarbon hydroxylase (AHH) activity for the TBB treatment group was maximal at Day 2 and then steadily decreased, whereas this activity was induced in 1 day and remained high for the HBB treatment group. Tissue concentrations of HBB appeared to be unchanged over time whereas tissue concentrations of TBB decreased in a biphasic manner. Rates of in vitro metabolism of TBB with hepatic microsomes from TBB-treated animals showed a similar time-course relationship to AHH induction. HBB caused moderate to severe hepatic changes while TBB-treated rats had only mild hepatic changes. The relative binding of TBB by the hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was about 10 times that of HBB. The results suggest that even though the receptor-binding affinities imply that TBB should be more toxic than HBB, it is less toxic than HBB because it is metabolized. Studies with the chlorinated analogs of TBB and HBB suggested that PCB behave similarly. These results also suggest that receptor binding and AHH induction do not accurately reflect toxicity for polyhalogenated aromatic hydrocarbons which are metabolized, presumably because continued occupation of the receptor and persistent induction of some enzyme activity are required for toxicity. PMID- 2994255 TI - Studies on the structure-activity relationships for the metabolism of polybrominated biphenyls by rat liver microsomes. AB - The in vitro metabolism of polybrominated biphenyl (PBB) congeners by cytochrome P-450-dependent monooxygenases was investigated using hepatic microsomes isolated from immature male rats pretreated with 3-methylcholanthrene (MC) or phenobarbital (PB). MC pretreatment increased the NADPH-dependent microsomal metabolism of pure PBB congeners which possessed adjacent nonhalogenated ortho and meta carbons on at least one ring. 4,4'-Dibromobiphenyl (-DBB) was metabolized at the fastest rate, followed by 3,4,4'-tribromobiphenyl, 3,4,3',4' tetrabromobiphenyl (-TBB), 2,3,3',4'-TBB, 2,5,3',4'-TBB, and 2,4,2',5'-TBB in decreasing order. It appeared that further bromination prevented metabolism since 2,4,5,3',4'-pentabromobiphenyl (-PBB), 2,3,4,2',4',5'-hexabromobiphenyl (-HBB), and 2,3,4,5,3'.4'-HBB were not metabolized although they possess adjacent nonhalogenated ortho and meta carbons. PB pretreatment increased in vitro rat hepatic microsomal metabolism of PBB congeners which possessed adjacent nonhalogenated meta and para carbons on at least one ring. 2,2'-DBB was metabolized at the fastest rate, followed by 2,4,2',5'-TBB, 2,5,2',5'-TBB, 2,3,3',4'-TBB, 2,5,3',4'-TBB, and 2,4,5,2',5'-PBB in decreasing order. The results suggest that the rates of metabolism of PBB congeners are dependent upon the positions of bromine and the form of cytochrome P-450 induced. In vitro rates of metabolism of 3,4,3',4'-TBB using hepatic microsomes isolated from rats pretreated with either 3,4,5,3',4',5'-HBB or 3,4,3',4'-TBB were also investigated. There was good correlation between the rates of 3,4,3',4'-TBB metabolism, induction of microsomal ethoxyresorufin-O-deethylase activity, and specific content of MC-inducible cytochrome P-450 (P-450 beta NF-B). The results suggest that the isozyme P-450 beta NF-B is responsible for the metabolism of 3,4,3',4'-TBB. PMID- 2994256 TI - Effect of alcohol and aldehyde dehydrogenase inhibitors on the toxicity of 3 nitropropanol in rats. AB - The nitrocompounds 3-nitropropanol (NPOH) and 3-nitropropionic acid (NPA) were shown to be equally toxic when injected intraperitoneally into male Wistar rats. The LD50 for NPOH was 0.58 mmol/kg and for NPA it was 0.56 mmol/kg. NPOH was rapidly metabolized to NPA but this conversion was suppressed by prior administration of ethanol or 4-methylpyrazole to inhibit alcohol dehydrogenase. Administration of ethanol or 4-methylpyrazole before NPOH protected rats from intoxication. However, if the alcohol dehydrogenase inhibitors were given after the nitroalcohol, toxicity still occurred. Administration of the aldehyde dehydrogenase inhibitor diethyldithiocarbamic acid had little effect on the conversion of NPOH to NPA and did not alter the toxicity of NPOH. It was concluded that NPOH and NPA are equally toxic to rats but that NPOH is toxic due to its being rapidly converted to NPA. PMID- 2994258 TI - Interactions of S-(2-haloethyl)-mercapturic acid analogs with plasmid DNA. AB - A series of related S-(2-haloethyl)-L-cysteine analogs were synthesized and their interaction with DNA was studied with plasmid pBR322. Both S-(2-chloroethyl)-L cysteine (CEC) and S-(2-bromoethyl)-L-cysteine (BrEC) rapidly induced relaxation of the supercoiled plasmid as determined by agarose gel electrophoresis and electron microscopy, whereas S-(2-fluoroethyl)-L-cysteine did not interact with DNA. The relaxation was most probably due to strand scission at alkylated labile sites in the DNA. When 35S-labeled CEC or BrEC was used as the substrate, covalent binding of 35S to DNA was obtained; CEF displayed a somewhat higher binding than BrEC. No binding of 35S was obtained with (2-hydroxyethyl)-L [35S]cysteine, [35S]cysteine, or [35S]cystine, substrates which did not induce relaxation of the DNA. Esterification of the carboxyl group resulted in a somewhat lower rate of DNA strand scission, whereas N-acetylation prevented the cysteine analogs from inducing DNA strand breaks. S-(2-Chloroethyl)-glutathione (GSH) did not interact with DNA as determined by lack of effect on the superhelicity of DNA, a finding which is in agreement with the hypothesis that the primary amine groups of CEC or BrEC may participate in the formation of reactive intermediates which can interact with DNA. S-(2-Hydroxyethyl)-GSH and S (2-hydroxyethyl)-L-cysteine were unable to induce DNA strand breaks. Neutral denaturation of supercoiled pBR322 treated with the analogs revealed that compounds which were able to induce DNA strand breaks also interfered with denaturation of double-stranded circular DNA. No such interference was observed when double-stranded linear DNA (obtained by BamH1 restriction digestion) was treated with the analogs prior to denaturation. These data indicate that a marked difference exists between S-(2-chloroethyl)-L-cysteine and S-(2-chloroethyl) glutathione in their reaction with supercoiled plasmid DNA. Either a major difference exists in the reactivity of the corresponding episulfonium ions of these conjugates or a separate mechanism of alkylation based on a free alpha amino of the cysteine conjugate is participating in DNA strand breakage and possible crosslinking. In vivo toxic effects of these S-(2-chloroethyl) conjugates are predicted to be distinctly different. PMID- 2994257 TI - Inhibition of gamma-[3H]aminobutyric acid uptake by organotin compounds in vitro. AB - Trimethyltin, its tetra-, di-, and monomethyl analogs, inorganic tin (Sn II and Sn IV), triethyltin, tripropyltin, tributyltin, and triphenyltin were tested for their ability in inhibiting the uptake of gamma-[3H]aminobutyric acid (GABA) into mouse forebrain synaptosomes in vitro. All organotins containing three carbon-tin bonds were potent inhibitors of [3H]GABA uptake with IC50 values ranging from 10( 4) to 10(-6) M. Various thiol and sulfur compounds, particularly sodium sulfide, were capable of antagonizing the inhibitory effect of triphenyltin and, to a minor extent, of other organotins. All triorganotins also inhibited Na+,K+ ATPase, measured by binding of [3H]ouabain and by hydrolysis of ATP. Although a correlation between inhibition of ouabain binding and GABA uptake by organotins could be found, inhibition of [3H]GABA uptake by the specific inhibitors ouabain and strophantidin was qualitatively and quantitatively different from organotins. These results suggest that all triorganotins are capable of inhibiting synaptosomal [3H]GABA uptake in vitro by a mechanism involving, but not exclusively, inhibition of Na+,K+-ATPase. The role of [3H]GABA uptake inhibition in the neurotoxicity of organotins remains to be determined. PMID- 2994259 TI - Increased susceptibility to mouse hepatitis virus 3 of peritoneal macrophages exposed to dieldrin. AB - Interaction of a single dose (36 mg/kg body wt) of the organochlorine pesticide dieldrin with mouse peritoneal macrophages was examined in C57Bl/6, (C57Bl/6 X A/J)F1, and A/J strains of different genetic resistance to mouse hepatitis virus 3 (MHV3) infection. In vivo studies showed increased susceptibility to MHV3 acute disease of C57Bl/6 and (C57Bl/6 X A/J)F1 animals challenged with the pesticide. Significant decrease of mean time of death in dieldrin-exposed, MHV3-infected susceptible C57Bl/6 mice was observed similarly upon po or ip administration of a single, sublethal dose of dieldrin. In addition, decrease of humoral response to the virus was quantified by determination of anti-MHV3 IgG antibodies in spleen cell supernatant fractions and in blood sera of dieldrin-exposed C57Bl/6 mice. A single dose of dieldrin did not alter the in vivo resistance of A/J animals to acute MHV3 disease. The resistant A/J mice, however, showed increased mortality upon two subsequent exposures to dieldrin followed by infection with high lethal doses of MHV3. Phagocytic activity, cell adherence capacity, and attachment and uptake of 3H-radiolabeled MHV3 by C57Bl/6 peritoneal macrophages were determined by in vitro studies. These affector activities of peritoneal macrophages were slightly decreased or unchanged in cells originating from animals exposed to the pesticide. However, the intrinsic activity of MHV3 restriction appeared to be affected in macrophages derived from dieldrin-treated animals: (i) peritoneal C57Bl/6 macrophages collected from the early phase of acute MHV3 disease contained increased MHV3 antigen and (ii) increased cytolysis was observed after in vitro MHV3 infection of macrophages originating from dieldrin-exposed C57Bl/6 mice. PMID- 2994260 TI - Induction of hepatic metallothionein in mouse liver following administration of chelating agents. AB - Chelating agents commonly used in therapy of heavy metal intoxication alter the levels of essential metals in liver, kidneys, and serum. Induction of metallothionein synthesis in liver occurs following exposure to a variety of chemical and environmental insults and, in some cases, has been attributed to enhanced hepatic uptake of zinc. Therefore, the effect of acute exposure to seven common metal chelators on the concentration of metallothionein in liver was investigated. Adult male Swiss Webster mice were injected intraperitoneally with the chelators and hepatic metallothionein was quantified by the cadmium radioassay. Ethylenediaminetetraacetic acid (EDTA) produced a 5- to 6-fold increase in hepatic metallothionein 24 hr after injection of 0.75 to 3.0 g/kg. No significant increase in hepatic MT was observed until 12 hr following injection of EDTA (1.5 g/kg, ip). Maximal levels were reached between 12 and 48 hr following EDTA injection. Cadmium, a known inducer of hepatic metallothionein, produced a 15-fold increase in the concentration of MT in liver 24 hr following injection. By comparison, 2,3-dimercaptopropanol and diethyldithiocarbamate produced a 9-fold and 13-fold increase in hepatic metallothionein levels, respectively, 24 hr following injection. A 4- to 6-fold increase in metallothionein was observed 24 hr following injection of 2,3-dimercaptosuccinic acid, D,L-penicillamine, diethylenetriaminepentaacetic acid, and EDTA, while nitrilotriacetic acid elevated hepatic metallothionein levels by 2-fold. Alterations in the concentration of hepatic metallothionein by chelators may have implications for their efficacy in the treatment of cadmium intoxication. PMID- 2994261 TI - Impairment of ketocyclazocine antinociception in rats by perinatal lead exposure. AB - The development of ketocyclazocine antinociception has been measured in lead exposed rats as an indirect determinant of kappa-opioid receptor system development. Perinatal lead administration (at 300 and 1000 ppm) in the maternal drinking water from conception to weaning, impaired the antinociceptive activity of ketocyclazocine (using the paw pressure test) in 10-day-old rats. Lead caused a dose-dependent impairment of ketocyclazocine antinociception, the paw pressure threshold for 0.4 mg/kg being reduced from 207 g to 135 g in the 1000 ppm lead dose-group. Ketocyclazocine antinociception was impaired in the high-lead dose group at 21 days, but unaffected at 30 days. Blood lead levels in 10-day-old animals were below 35 micrograms/100 ml in the low-lead dose-group and below 50 micrograms/100 ml in the high-lead dose-group. It is suggested that lead may disrupt the development of kappa-opioid receptor systems in the central nervous system and that this disruption occurs early in development. PMID- 2994262 TI - Effect of slate dust on the rat erythrocyte membrane composition: in vitro and in vivo studies. AB - The biochemical composition of rat erythrocyte ghost membrane obtained by hypotonic lysis and slate-dust-induced lysis were compared in vitro. The phospholipids and glycosamine contents decreased in slate-dust-exposed erythrocyte membrane, whereas there were no changes in protein content. The in vitro and in vivo association of silica, leaching from slate dust, with the components of rat erythrocyte ghost membrane, has also been demonstrated. The interaction of silica with membrane constituents is proposed as a mechanism of action of slate-dust toxicity. PMID- 2994263 TI - Comparison of the DNA-damaging property of photosensitised riboflavin via singlet oxygen (1O2) and superoxide radical O2-. mechanisms. AB - Riboflavin was found to generate singlet oxygen (1O2) and superoxide anion radicals O2-. on exposure to UV-A (320-400 nm) and UV-B (290-320 nm) light. Studies with deoxyguanosine (dGuo) showed that 1O2 was largely responsible for riboflavin-sensitised photodegradation of the guanine base of DNA and RNA. Azide ions (N-3) and 1,4 diazabicyclo-[2.2.2]-octane (DABCO) produced over 90% inhibition of dGuo photo oxidation, whereas superoxide dismutase did not show any noticeable quenching effect under similar conditions. Photo oxidation of dGuo by riboflavin and UV radiation is of significant importance from the point of view of cell-damaging reactions by activated oxygen species produced by the synergistic action of sunlight and chemical agents. It is now known that activated oxygen species are responsible for skin photosensitisation, tumor promotion and carcinogenic properties. PMID- 2994264 TI - Clinical and subclinical nutritional neurological damage in former war prisoners of the Japanese. AB - Five former war prisoners of the Japanese were clinically evaluated and intensively investigated for evidence of neurological disease. On release from imprisonment 36 years previously they were known to have a single neurological problem each, attributable to nutritional damage to the nervous system (four had optic atrophy and one a peripheral neuropathy). The present assessment confirmed these persisting neurological problems; but in addition two patients were found to have extrapyramidal and one pyramidal disease, one was demented, and two had evidence of non-dominant hemisphere damage on psychometric testing. Nutritional damage to the nervous system may be permanent, and more extensive than usually recognized. PMID- 2994265 TI - Disappearance of Chikungunya virus from Bangkok. PMID- 2994266 TI - Multicenter seroepidemiologic study of the impact of cytomegalovirus infection on renal transplantation. AB - The effects of cytomegalovirus (CMV) infection on patient and allograft survival were determined in 1245 renal transplant recipients from 46 transplant centers. When an antilymphocyte preparation was administered to cadaveric allograft recipients, those at risk for primary CMV had a worse outcome than similar patients treated with prednisone and azathioprine (53.1% alive at 6 months with a functioning allograft vs. 70.8%, P = .05) or patients at risk for reactivation CMV (53.1% vs. 71.1%, P = .035). Patients at risk for reactivation CMV had a better outcome if they received an antilymphocyte preparation (71.1% vs. 60.8%, P less than .01). The type of immunosuppression had no effect on patients without CMV. Living-related donor transplantation was not significantly influenced by CMV or type of immunosuppression. We conclude that CMV infection is strongly influenced by the form of immunosuppression employed, and that both are important determinants of the outcome of cadaveric renal transplantation. PMID- 2994267 TI - Natural immunity and graft-vs-host disease. PMID- 2994268 TI - [Phosphorylation of cardiac sarcolemma proteins in isadrine myocarditis]. AB - The incorporation of 33P into 54, 43 and 23 kDalton proteins of rabbit cardiac sarcolemma is demonstrated both in intact muscle and under isadrine myocarditis. The phosphorylation by cAMP-dependent protein kinase is shown only for the 23.5 kDalton protein. The level of this protein phosphorylation decreases under isadrine myocarditis in the presence of 10(-6) M cAMP and absence of "exogenic" protein kinase. In the preparations of the sarcolemma of the cardiac muscle under myocarditis the phosphorylation of the 23.5 kDalton protein is not stimulated by beta-agonists in the medium without "exogenic" protein kinase and cAMP. The latter being present, the phosphorylation reaches the control values. PMID- 2994269 TI - [Various physico-chemical properties of inorganic pyrophosphatase from the mouse spleen]. AB - Manganese, calcium and mercury ions, as well as p-chloromercury benzoate and dithiothreitol are studied for their effect on the activity of inorganic pyrophosphatase (EC 3.6.1.1) of mice spleen. It is shown that Ca2+ and Mn2+ are inhibitors of this enzyme, but Mn2+ in low concentrations may replace Mg2+ in the pyrophosphatase reaction. Hg2+ and p-chloromercury benzoate inhibit the pyrophosphatase activity essentially but not completely. Mice spleen pyrophosphatase is very labile: its preincubation without the substrate for 30 min at 37 degrees C leads to a complete loss of the activity. Neither glycerol, nor glutathione and cysteine but magnesium ions, dithiothreitol and 2 mercaptoethanol protect the enzyme from inactivation. The enzyme is purified by the sulphate ammonium salting-out, gel filtration on Sephadex G-100 as well as by isoelectrofocusing in 5% PAAG. Then pyrophosphatase is eluted from gel and subjected to electrophoresis in the plane layer of the linear gradient of 5-15% PAAG with SDS or 5-25% PAAG without denaturing conditions. One zone corresponding to the molecular mass of 70 kDalton is obtained. It is splitted into two zones in electrophoresis with SDS and 2-mercaptoethanol. PMID- 2994270 TI - [Potential activity of the external pathway of NADH oxidation in mitochondria]. AB - High rate of exogenic NADH oxidation (up to 200 mg-at. oxygen for 1 min per 1 mg of protein and higher) along the rothenone, antimycin-nonsensitive pathway is observed under interaction of mitoplasts with the external membrane and cytochrome c. In the medium with low ionic strength the interaction of external and internal membranes is not a sufficient condition for activating the external pathway of the NADH oxidation: the presence of exogenic cytochrome c is also necessary. With saturated cytochrome c concentrations the addition of outer membranes leads to further stimulation of the NADH oxidation. In the medium with high ionic strength external membranes stimulate oxidation of NADH when exogenic cytochrome c is absent; the subsequent addition of cytochrome c stimulates the NADH oxidation in this medium to a greater extent than in the medium with the low ionic strength. Under the nonlimited interaction of external and internal membranes and cytochrome c the potential activity of the outer pathway of NADH oxidation in the liver mytoplasts of hybernating gophers is lower than in the liver mytoplasts of rats. PMID- 2994271 TI - [The cAMP system in the cerebral cortex of irradiated rats in increased exogenous K+ levels]. AB - Treatment of grey matter of irradiated (0.21 Cl/kg) rats by 60 mM of KCl causing depolarization of cell membranes leads to the increase of the cAMP content and to the decrease of adenylatecyclase activity. Dynamics of the enzyme activity with an increase of the external KCl concentration in control is to a considerable extent similar to that after irradiation, though the latter is more pronounced and a time shift is observed for maximal increase of a cAMP content and cAMP phosphodiesterase activity. PMID- 2994272 TI - [Secondary cicatricial deformities and recurrences after surgical interventions on the hand in congenital syndactylia]. AB - Experience with the treatment of 46 patients with syndactylism of the hand is described. In 28 of them operations for liquidation of syndactylism were repeatedly performed in childhood. The operation technique and results of the treatment are presented. PMID- 2994273 TI - [Detection of cytotoxic antibodies in the serum of cattle with leukemia]. AB - In a set of 55 serum samples obtained from the herd of bulls heavily infested with bovine leukosis we tested the applicability of cytotoxic test for the diagnostics of bovine leukosis we tested the applicability of cytotoxic test for the diagnostics of bovine leukosis and compared the results with immunodiffusion test in agar. The two tests show approximately the same sensitivity because in each examined sample the results of cytotoxic test corresponded at least in two out of three serum dilutions (1:2, 1:5 and 1:7) to the immunodiffusion test. It has been demonstrated that as regards the cytotoxic test, the final serum dilution of 1:5 was satisfying. At the same time it has been checked that the bovine foetal serum, required for the growth of cell cultures, could be replaced by the bovine serum treated with the 40% solution of polyethylene glycol (PEG) 6000 without any influencing the results of cytotoxic test. PMID- 2994274 TI - Chronic hepatitis and hepatocellular carcinoma associated with persistent woodchuck hepatitis virus infection. AB - The livers of 16 woodchucks with naturally acquired chronic infection with woodchuck hepatitis virus were examined both grossly and histologically in 14 biopsy specimens and seven necropsy specimens. Fifteen woodchucks had lesions characteristic of chronic hepatitis; ten of these had chronic active hepatitis, four had chronic persistent hepatitis, and one had cirrhosis with nodular regeneration. In one woodchuck there was massive hepatic necrosis attributed to infection with an unclassified protozoan. Thirteen woodchucks had primary hepatocellular carcinoma. Metastasis to the lung was observed in only one woodchuck. These results were compared to liver lesions in 149 woodchuck hepatitis virus-negative woodchucks. Chronic hepatitis comparable to that associated with woodchuck hepatitis virus infection was not observed in woodchuck hepatitis virus-negative woodchucks although in one, a single, small hepatocellular adenoma was found. PMID- 2994275 TI - Tumors in dwarf galagos (Galagoides demidovii). AB - Three spontaneously occurring tumors are described in dwarf galagos. One tumor was a subcutaneous fibrous histiocytoma in the left inguinal area of an adult male; the other two were bile duct carcinomas in a seven-year-old male and a four year-old female. Both bile duct carcinomas had remarkable invasive and metastasizing capacities. PMID- 2994276 TI - Bluetongue virus-induced encephalopathy in fetal cattle. PMID- 2994277 TI - Pseudorabies virus in dexamethasone-treated pigs. PMID- 2994278 TI - Enzyme-linked immunosorbent assay for the detection of antibodies to bovine viral diarrhea virus in bovine sera. AB - A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was established for the detection of antibodies to bovine viral diarrhea virus (BVDV) in bovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified BVDV was used as test antigen at an optimal amount of 1 microgram/well, whereas the optimal concentration of conjugate was at 1/2000 dilution. The standardized test encountered no non-specific reaction with test sera at a starting dilution of 1/10. A total of 50 bovine serum samples was assayed for the presence of antibodies against BVDV by ELISA and serum neutralization test (SNT). A positive correlation between the 2 tests was found. However, ELISA could be as much as 500-fold more sensitive than SNT in detecting low levels of BVDV antibodies. PMID- 2994279 TI - An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine viral diarrhoea virus (BVDV) in cattle sera. AB - A microtitre ELISA has been established for the quantitation of antibodies to bovine viral diarrhoea virus (BVDV). Single dilutions of sera were assayed and units of antibody were calculated from a standard curve. In order to detect the maximum number of responding animals both IgG1 and IgG2 antibody should be assayed, although detection of IgG1 alone was nearly as effective. The ELISA was as sensitive as the virus neutralization test for detection of antibody; comparison of an ELISA that detected IgG1 plus IgG2 antibody to BVDV with the virus neutralization test gave a correlation coefficient (r) of 0.89 (P less than 0.001 for 95 compared sera). Although similar amounts of IgG1 and IgG2 antibodies were present in sera from both experimentally- and naturally-infected cattle, antibody to BVDV in colostrum and in the sera from young calves was predominantly IgG1. The number of adult cows with antibody was 40 out of 41 while 36 of 44 calves reared in a beef unit were found to have produced antibody by the time they were 31.5 weeks old, an indication of the high prevalence of BVDV in the cattle population. PMID- 2994280 TI - Excretion and reexcretion of thermosensitive and wild-type strains of infectious bovine rhinotracheitis virus after co-infection or two successive infections. AB - Twelve cattle were divided into 2 groups. The first was intranasally co-infected with 2 strains of infectious bovine rhinotracheitis virus (Bovine herpesvirus 1; BHV 1): the thermosensitive vaccine strain IBR/ts RLB106 and a Belgian field isolate IBR/Cu5. Reactivation of BHV 1 was induced by dexamethasone treatment 2 months later and again 5 months later for 3 animals that only reexcreted small quantities of virus during the first dexamethasone treatment. The second group was intranasally infected with IBR/Cu5. Two months later, an attempt to reinfect this group with IBR/ts RLB106 failed. Four months after the primary infection, these cattle were treated with dexamethasone. Except after reinfection and at the beginning or the end of the (re)excretion periods, excreted and reexcreted viruses replicated at 35, 37 and 40 degrees C, indicating the presence of the wild-type virus. Only one isolate, out of 116 cloned from the nasal exudates collected during the excretion and reexcretion periods, expressed the thermosensitive phenotype. This isolate was characterized by its mean plaque size as the IBR/ts RLB106 strain. The epizootiological significance of these findings is discussed, with emphasis on the weak spreading capacity of the ts vaccine strain and the possibility of emergence of recombinant viruses. PMID- 2994281 TI - Humoral and cellular responses in calves experimentally infected with bovine leukemia virus (BLV). AB - In order to elucidate whether infection by Bovine Leukemia Virus (BLV) might induce an immunodeficient state, we inoculated sixteen calves with BLV. The calves were followed up for two years and were tested for humoral and cellular responses using various parameters, namely the appearance of antibodies to the BLV antigens, the changes in the numbers of lymphocytes involved, and the ratio between the two main populations of lymphocytes. Antibodies to the BLV antigens were of both the IgG and the IgM classes of immunoglobulins. The levels of antibodies of the IgM class were higher than those of IgG. There was a temporary decrease of reactive antibodies to the BLV antigens, to below detectable levels, during the 14-24 weeks post infection. A significant decrease in the level of plasma IgM was found in all BLV infected calves exhibiting lymphocytosis, while the level of IgG in the plasma of all experimental calves did not diverge significantly from the initial values, throughout the experiment. BLV infection was followed by lymphocytosis of B-cells in most infected calves, which persisted for the whole course of the experiment, while a decrease in the population of T cells in peripheral blood was observed for a period of several months in all infected calves. PMID- 2994282 TI - The nature of immunity to Snyder-Theilen fibrosarcoma virus induced tumors in cats. AB - Snyder-Theilen fibrosarcoma virus (ST-FeSV) induced tumors evoked a vigorous immune response in adolescent cats. The response was characterized histologically by a lymphoid and histiocytic cell infiltrate beginning around the 9th day post inoculation. Hyperemia edema, hemorrhage, and necrosis of the tumors occurred shortly thereafter. Gross regression of the tumors commenced around the 15th day. Viable fibrosarcoma cells could be recovered as almost pure cultures from tumors biopsied on the 9th day. Biopsies taken between days 9 and 15 contained progressively fewer tumor cells and increasing numbers of lymphoid cells, histiocytes, giant cells, and normal fibroblasts. Tumor cells in such mixed cultures did not replicate as fast as normal and died out within 7 to 14 days. Viable tumor cells were not recovered from biopsies taken after day 15. Fibrosarcoma regression was associated with the appearance of tumor cell specific cytotoxic lymphocytes and antibodies in the blood. Cell mediated immunity, as determined by a chromium release assay, consisted of both antibody dependent and independent mechanisms. Fluorescent and complement dependent cytolytic antibodies were detected in the blood at the same time as cytotoxic lymphocytes, but persisted after regression. In a preliminary experiment, serum from tumor regressor cats was injected into susceptible kittens, and the kittens were then challenged with ST-FeSV transformed fibroblasts or whole FeSV. Immune serum did not prevent the appearance of initial growth of tumors, but did slow their subsequent growth and increased the rate of regression. Immune serum had a much more dramatic inhibitory effect on the accompanying retrovirus infection. PMID- 2994283 TI - Isolation of pigeon herpes encephalomyelitis virus in Saudi Arabia. AB - A virus was isolated from the brains of pigeons suffering from nervous disorders in different localities of the Eastern Province, Kingdom of Saudi Arabia. The new isolate caused a high morbidity, ranging from 33% to 50%, and a mortality rate which reached 40%. The virus produced pinpoint greyish pock lesions on the chorioallantoic membrane of embryonated hens' eggs and induced syncytial formation followed by rounding and lysis of the cells in chicken embryo fibroblast cultures. Virus infectivity was significantly reduced following treatment by 20% ether or chloroform. The isolated virus was identified as pigeon herpes encephalomyelitis virus by serum-neutralization, agar gel diffusion and fluorescent antibody staining techniques. PMID- 2994284 TI - [Trials of peroral vaccination of newborn pigs against transmissible gastroenteritis (TGE)]. AB - Experiments were carried out to apply oral vaccination to newborn pigs against transmissive gastroenteritis on a stationary swine breeding complex at the time when the disease assumed an acute course and on newly infected farms. Used was an attenuated strain an hour prior to allowing the pig to suck. It was found that such vaccination approach was innocuous. It proved effective when applied at the moment of birth both on the infected (stationary) farms and in the new foci of infection--morbidity and mortality were lowered and the body development of pigs was improved. Such vaccination was shown to produce also an antiepizootic effect if systematically used on stationary farms--clinically, there were no new epizootic outbreaks of the diseases. PMID- 2994285 TI - [Isolation and use of a farm-specific hyperimmune serum against transmissible gastroenteritis]. AB - A specific stationary hyperimmune serum was obtained in a pigbreeding complex with a transmissive gastroenteritis infection via trifold (at a fourteen-day interval) i/m injection of sows with 5 cm3 each of undiluted virus (10(6) TCCPE50 per cm3). Tested were hyperimmunization programmes with pigs in the final fattening period. It was found that the use of undiluted virus led to the equation of sows in terms of their immunologic state and to the essential rise of the titer of humoral antibodies. In order to obtain high titer immune serum against transmissive gastroenteritis it is sufficient to proceed with the i/m injection of pigs or adult swine in the final stage of fattening at rising amounts (from 3 to 10 cm3) of attenuated virus of sufficiently high titer (10(6) TCCPE50 per cu. cm). The hyperimmune serum produced a very good prophylactic effect with newborn pigs on the same farm - the twofold oral administration of 5 to 10 cm3 on the day of birth and a couple of days later led to a drop of both morbidity and mortality rate as well as to the improvement of body development. PMID- 2994286 TI - Pathological study of small hepatocellular carcinoma: frequency of their invasion. AB - We studied the pathological findings in 28 small hepatocellular carcinomas (less than 5 cm in diameter), especially the mode of invasion. Seventeen of the 28 lesions were encapsulated. Capsular invasion was present in 14 of the 17 encapsulated tumours. Vascular invasion was found in 12 of the 28 tumours. The small hepatocellular carcinoma, even when encapsulated, frequently invaded blood vessels and adjacent liver tissue, in spite of frequent vascular invasion, distant metastasis was seen in only 1 of the 28 tumours. PMID- 2994287 TI - Ultrastructural studies of adrenal adenoma causing primary aldosteronism. AB - Adrenal adenoma tissue was obtained from 7 patients with the diagnosis of primary aldosteronism and was studied electron microscopically. Spironolactone was administered in 6 of these patients, but not in the remaining patient. Most of the mitochondria of the tumour cells possessed tubular cristae, giving an appearance similar to the mitochondria in the cells of the zona glomerulosa. Spironolactone bodies were seen in the tumour cells of 6 patients who were given spironolactone preoperatively, but were not observed in these cells in the patient not given spironolactone. The literature on the developmental mechanism of this spironolactone body was reviewed. PMID- 2994288 TI - [In vitro effectiveness of new antiviral substances. 1. Results of screening studies against selected picornaviruses]. AB - A number of 3108 new synthetic compounds were subjected to a screening for antiviral activity. The screening resulted in the selection of numerous drugs active against mengovirus in vitro. Some drugs were also active against Coxsackie viruses A9 and B1; most of the drugs efficient against Coxsackie virus A9 also showed activity against mengovirus. Quantitative investigations--such as the determination of a therapeutic index or of the inhibition of infectious virus yields in one-step growth cycle experiments--allowed the selection of the compounds with the highest in vitro efficacy. PMID- 2994289 TI - [In vitro effectiveness of new antiviral substances. 2. studies with enteroviruses and rhinovirus lB]. AB - The antiviral efficacy of several compounds was demonstrated against Coxsackie viruses type A9, B1, B3, B4 and B5, echoviruses type 6, 11, 30, and 33, attenuated polioviruses and rhinovirus 1B. In one-step growth cycle experiments virus multiplication was clearly inhibited by application of the compounds immediately after virus adsorption. PMID- 2994290 TI - Investigation of humoral and cell-mediated immunity aspects in mice with experimental latent herpes infection. AB - An experimental latent herpes infection was established by intradermal inoculation of the mouse ear or of the footpad with a wild strain and a temperature-sensitive (ts) mutant of herpes virus type 2. The wild strain was latency-positive irrespective of the inoculation site; the ts mutant induced latent ganglionic infection only when inoculated in the footpad. Neutralizing antibodies could be detected only after inoculation of the wild strain. The establishment of latency was not prevented by the presence of either a humoral or a cell-mediated immune response. PMID- 2994291 TI - Oligomerization of simian virus 40 tumor antigen may be involved in viral DNA replication. AB - Biological implications of the oligomerization of simian virus 40 (SV40) large T antigen for viral DNA replication were studied by using two temperature-sensitive SV40 A-gene mutants, tsA 58 and tsA 1499. Both mutants are defective at elevated temperature for viral DNA replication whereas tsA 58 is like most other tsA mutants additionally heat sensitive for cell transformation. We found that in contrast to tsA 58 encoded T antigen, tsA 1499 T antigen is thermostable in the ability to bind specifically to the origin of replication of SV40 DNA. Detailed structural analysis of tsA 1499 T antigen by sucrose density gradient centrifugation revealed that it is strictly temperature sensitive for the formation of homologous oligomers but, as we reported previously (M. Montenarh, M. Kohler, and R. Henning, 1984, J. Virol, 49, 658-664), not for the association with the cellular phosphoprotein p53. These observations are compatible with the idea that, in addition to the specific origin-binding ability as well as other functional features, the oligomerization of T antigen may be essential for viral DNA replication. PMID- 2994292 TI - Shope papillomavirus transcription in benign and malignant rabbit tumors. AB - Shope papillomavirus induced benign and malignant tumors from both wild and domestic rabbits were analyzed by the Northern blotting technique for the presence of viral-specific RNAs. Virus-producing benign tumors of wild cottontail rabbits are shown to contain two major RNA species approximately 5000 and 3000 bases in length that originate in the early region and include the majority of the L1 and L2 late open reading frames. Non-productive tumors including wild rabbit carcinomas, and benign warts and primary and metastatic carcinomas of domestic rabbits uniformly are shown to contain two major viral-specific RNA species, 2400 and 1400 bases in length. These transcripts map entirely within the early region of the viral genome. In addition, Southern blot analysis of metastatic tumors indicates that the viral DNA remains exclusively extrachromosomal and apparently unrearranged supporting previous findings with epithelial malignancies. PMID- 2994293 TI - Ablation of humoral immunity in 15I5 x 72 chickens is not predisposing to the formation of subgroup G virus-induced distal sarcomas. AB - Clone (cl.) 85 infection of 15I5 x 72 chickens at 4 weeks posthatch results in a lower frequency of distal sarcomas than does Prague (Pr)-B infection. As higher titers of virus-neutralizing antibody are generated in the cl.85-infected chickens, the possibility was addressed in the present study that the low frequency of cl.85-induced distal sarcomas is a direct consequence of the strong neutralizing response. Our observations indicate that ablation of humoral immunity by embryonic bursectomy does not serve to increase this frequency, implying that the antiviral humoral response is not required to limit formation of cl.85-induced distal sarcomas. PMID- 2994294 TI - Nucleotide sequences responsible for generation of internally deleted Sendai virus defective interfering genomes. AB - The deletion points of four internally deleted defective interfering (DI) RNA species (7a, 7b, 7c, and 7d) that reside in a single Sendai virus strain were defined by nucleotide sequencing. DI RNA 7a (Mr 1.24 x 10(6)) retained the entire NP gene with the complete NP protein-coding sequence, except for the last two U residues of the polyadenylation signal, fused to an 1800-nucleotide sequence comprising 5'-terminal genome and adjacent L gene sequences. DI RNA 7b (Mr, 0.70 x 10(6)) consisted of 100 3'-terminal nucleotides fused to 1900 5'-terminal bases; the deletion point in the NP gene precedes the NP protein initiation codon. DI RNA 7c (Mr 0.55 x 10(6)) retained 420 3'-terminal and 1150 5'-terminal nucleotides. The sequence just downstream of the sequenced deletion site is M gene specific, indicating that 7c arose from at least two deletion events and that it comprises NP, M, and L gene fragments. Transcription of RNA 7c could yield an MRNA encoding a fusion protein with a 14,000 Mr (N-terminal NP sequence fused to out of frame M-specific amino acids). DI RNA 7d (Mr 0.92 x 10(6)) retained 1027 3'-terminal nucleotides fused to 1600 bases from the 5'-terminus. It has an open reading frame for a 33,000 Mr N-terminal NP protein fragment. Nucleotide sequences flanking each deletion and just downstream of the NP gene deletion site suggested that these DI genomes were generated by a copy-choice mechanism, involving polymerase jumping during replication of negative polarity virus genome templates. In this process, the termination and reinitiation of RNA synthesis would involve recognition of sequences that regulate virus genome transcription and replication. PMID- 2994295 TI - Expression of Sendai virus defective-interfering genomes with internal deletions. AB - Sendai virus strain 7 has been shown to contain four defective interfering (DI) RNA species in which both genome termini and various adjacent fragments of the 3' terminal NP gene and 5'-terminal L gene are represented, but most or all internal genes and gene boundaries are deleted. Previous sequence analyses of these mutant RNAs suggested that all four possessed the transcription initiation signal of the NP gene and the transcription termination signal of the L gene. The supposition that these signals should specify transcripts has now been supported by oligo(dT) selection of four DI 7 specific RNA species that had apparent molecular weights slightly lower than each DI genome. DI RNA 7a, which contains the entire NP gene, except for two U residues at the end of the poly(A) initiation signal, appeared to be transcribed solely as a readthrough product. Since DI RNA 7a contains the entire NP protein-coding sequence and DI RNAs 7c and 7d contain fragments of it, whereas DI RNA 7b is devoid of it, only transcripts of RNAs 7c and 7d were expected to specify fusion proteins containing NP gene-specific sequences. A strain 7-induced protein that reacted with monoclonal antibodies against the NP protein had the 33,000 Mr size appropriate for the translation product predicted by the sequence of RNA 7d. Other proteins of lower molecular weight were seen only in cells infected by strain 7, but they did not react with NP-specific antibody and their translation in vitro was not blocked by hybridization to an NP gene-specific oligonucleotide. Therefore, at least some of these proteins may be cellular products induced by DI virus infection. These DI transcripts and translation products may influence interference with replication of the parental helper virus. PMID- 2994296 TI - Detection of the myristylated gag-raf transforming protein with raf-specific antipeptide sera. AB - The post-translational modifications of the gag-raf fusion proteins of the 3611 murine sarcoma virus (MSV) have been examined by inhibiting glycosylation with tunicamycin and by in vivo labeling with [3H]myristic acid. The results show that P75gag-raf is myristylated but not glycosylated and that P90gag-raf is glycosylated but not myristylated (and is now termed gP90gag-raf). gP90gag-raf expression appeared to become lost during passage of the transformed cells, and consequently does not appear to be necessary for the maintenance of transformation. raf-specific sera for detecting gag-raf fusion proteins have been obtained from synthetic peptides made from different regions of the predicted v raf sequence. Immunoprecipitation of P75gag-raf with raf-specific sera directly confirmed the deduced v-raf sequence. The fact that P75gag-raf is both myristylated and precipitated by antiserum to a predicted carboxyl-terminal peptide of the v-raf gene established that the mature protein represents the entire coding region. The gP90gag-raf thus appears to be a glycosylated form of P75gag-raf specified by the gag sequences of the fusion protein, in analogy with Pr65gag and gPr80gag of murine leukemia viruses. Antiserum to the carboxyl terminal P75gag-raf peptide was the most efficient in immunoprecipitation, and will be useful for detecting the product of the c-raf gene. PMID- 2994297 TI - [Platelet alpha2-adrenergic receptors in essential hypertension. Methods of determining specific binding using (3H)-dihydroergocryptine and the effect of physical exertion]. PMID- 2994298 TI - [Platelet alpha2-adrenergic receptors in essential hypertension. Number of binding sites, cyclic adenosine monophosphate and platelet aggregation]. PMID- 2994300 TI - [Cytogenetics of malignant neoplasms]. PMID- 2994299 TI - [Evaluation of the effectiveness of preoperative polychemotherapy of nephroblastomas in children by angiography, ultrasonic tomography and computed tomography]. AB - The results of preoperative polychemotherapy of nephroblastoma in 14 children are presented. Ultrasonic tomography, computed tomography and angiography were performed in all the patients before and after medication. The findings on the efficacy of polychemotherapy were assessed on the basis of surgical evidence and, in particular, resected material examination. A high diagnostic value of all the methods was demonstrated and criteria of the effectiveness of preoperative polychemotherapy as evaluated by application of ultrasonic and computed tomography and angiography were worked out. Optimal terms of carrying out diagnostic procedures in patients with nephroblastoma during preoperative treatment are suggested. PMID- 2994301 TI - [Effect of wheat bran included in an anti-atherosclerotic diet on the lipid metabolic indices of ischemic heart disease patients]. AB - A study was made of the effect of wheat bran contained by the antisclerotic diet on lipid metabolism in patients with chronic coronary heart disease. Eighty-two patients (males) aged 36-59 years with a history of myocardial infarction of 1- to 15 year-standing were entered into the study. It was ascertained that inclusion into the antisclerotic diet of wheat bran in an amount of 70-80 g does not exert any hypolipidemic action. No elevation of the serum alpha-cholesterol content was noticed either. The augmentation of the cholic acid concentration together with a reduction in the cholesterol content in the hepatic bile attests to its reduced lithogenicity. PMID- 2994302 TI - Prevalence of antibodies to lymphadenopathy-AIDS virus in French haemophiliacs. AB - 49 French haemophiliacs (haemophilia A: 41 patients; haemophilia B: 8 patients) were serologically testes for lymphadenopathy-AIDS virus antibodies: 10 patients (20.4%) were seropositive including 9 (21.9%) with haemophilia A and 1 (12.5%) with haemophilia B. Among haemophiliacs A, seropositive patients received significantly larger amounts of factor VIII concentrate during the 2 years preceding the study. PMID- 2994303 TI - [Etiology of acquired immunodeficiency syndrome]. PMID- 2994304 TI - [Importance of echography in diagnosing liver neoplasms and parasitoses]. PMID- 2994305 TI - Squamous carcinoma of the nasopharynx. AB - Nasopharyngeal carcinoma is an unusual neoplasm among squamous cell carcinomas of the head and neck. The tumor is rare in most parts of the world but is strikingly common in several Asian subpopulations, notably Chinese in Hong Kong and Guangdong Province. The Epstein-Barr virus is intimately related to the disease and elicits the formation of antibodies that are useful for diagnosis and follow up study. The virus has not been conclusively shown to cause nasopharyngeal cancer, however.Histologically, nasopharyngeal carcinoma is anaplastic in 75% of cases and better differentiated in 25% of patients. All tumors are treated by high-dose irradiation to the primary site and both sides of the neck. Surgical treatment, in the neck only, is reserved for irradiation failures. The prognosis is better in patients younger than 40 years, in patients without clinical cervical nodal involvement and, unexpectedly, in patients with anaplastic tumors. PMID- 2994306 TI - Studies on the production of endogenous pyrogen by rabbit monocytes: the role of calcium and cyclic nucleotides. AB - Rabbit monocytes stimulated with endotoxin produced endogenous pyrogen, even under conditions of high or low extracellular calcium concentrations. Maximal production occurred when the concentration was in the near-physiological range. Prolonged incubation of cells with a calcium chelator prevented subsequent activation with endotoxin, an effect which was rapidly reversible by re-addition of calcium but not other cations. Addition of small amounts of lanthanum, which acts as a calcium channel blocker, prevented the restoration of pyrogen production, indicating that entry of the added calcium into the monocyte was required. Incorporation of a calcium ionophore into the cell membrane did not stimulate pyrogen production, and no measurable influx or efflux of calcium occurred during stimulation with endotoxin. These observations suggest that a slowly exchangeable calcium pool is necessary for the production of endogenous pyrogen, but that a rise in intracellular calcium is not by itself a necessary or sufficient stimulus. This stands in contrast to other biological systems in which Ca2+ directly couples stimulus and hormone secretion. Incubation of cells with agents shown to increase cyclic 3',5' AMP or cyclic 3',5' GMP levels in monocytes similarly did not stimulate pyrogen production or modulate its production by endotoxin stimulation. Thus, cyclic nucleotides also did not play a detectable role as intracellular messengers in this system. Future work is required to define more clearly the mechanism for the production of endogenous pyrogen, given its marked effects on the immune system through lymphocyte activation and temperature regulation. PMID- 2994307 TI - The poliomyelitis story: a scientific hegira. AB - There is evidence that paralytic poliomyelitis occurred in ancient times, but it was not recognized as a distinct disease until the eighteenth century and did not come into prominence until the late nineteenth century when epidemics began to appear. Outbreaks of increasing size were reported first in the Scandinavian countries, then in the United States and elsewhere, to the surprise and consternation of the medical profession. Poliovirus was first isolated in 1908, but many years of intensive research were required before the epidemiology and pathogenesis of the disease were sufficiently understood to allow preventive measures to be devised. The road to eventual success was complicated by controversies, setbacks, and tragedies, played out and influenced by many powerful personalities. Today there are two effective vaccines. The disease has been virtually eliminated in countries where they have been used extensively, yet in the developing areas of the world recent "lameness surveys" indicate that the incidence of paralytic poliomyelitis is as high as it was during the peak years in the United States in the early 1950s. The challenge now is to use the available vaccines to extend control to the developing countries and eventually to achieve elimination of the disease worldwide. PMID- 2994308 TI - [The importance of radon and its daughter products for housing hygiene]. PMID- 2994309 TI - [Comparison of stationary and person-bound measurements of toluene diisocyanate in workrooms of a polyurethane foam-producing plant]. PMID- 2994310 TI - [Procedure for the determination of angiotensin converting enzyme]. PMID- 2994311 TI - [Mitochondrial enzyme activity of blood neutrophils during the sanatorium-health resort treatment of children with neurodermatitis]. PMID- 2994312 TI - Differential replication of SV40 and polyoma DNAs in Chinese hamster ovary cells. AB - We have investigated the ability of CHO cells to allow growth of papovaviruses by analyzing viral DNA replication after transfection using the calcium-phosphate co precipitation technique. These analyses showed that when SV40-containing plasmids were introduced into CHO cells, viral DNA replicated to a level of approximately 1000 copies per T antigen-expressing cell, and neither late proteins nor virus progeny were produced. When polyoma (Py)-containing plasmids were transfected into CHO cells, a ten-fold higher level of Py DNA was present per T antigen positive cell, and viral capsid proteins and progeny virus were detected, indicating that CHO cells are not equally restricted for all papovaviruses. Infection with intact virions was restricted in both cases. These results indicate that either SV40 or Py DNA introduced into CHO cells are able to express their early viral functions, and that different interactions of cellular proteins involved in the replication machinery with viral nucleic acids and proteins result in different levels of viral DNA synthesis and virus progeny production. We propose that, because of their favorable genetic characteristics, CHO cells should, therefore, provide a valuable experimental system for definition of the cellular contributions to papovavirus replication. PMID- 2994313 TI - Nonpermissive infection of lymphoblastoid cells by vesicular stomatitis virus. II. Effect on viral morphogenesis. AB - The human B-lymphoblastoid cell line Raji is nonpermissive for infection by vesicular stomatitis virus (VSV). The VSV particles released from Raji cells display a more heterogeneous distribution in equilibrium sucrose density gradients than particles released from BHK cells. The particles released from Raji cells contain approximately one-half to one-third as much viral matrix protein, relative to the nucleocapsid protein, as is normal. They also contain a higher proportion of the unglycosylated form of the G protein. The particles released from Raji cells are unstable and many disintegrate in the growth medium. Most of them deform when subjected to ultracentrifugation prior to fixation. The ratio of plaque-forming units to physical particles is much lower for the virions released from Raji cells. PMID- 2994314 TI - The expression of the SV40 early region in the transformed human cell line SV80. AB - The one complete copy of the SV40 early region present in human cells of the transformed line SV80 carries a duplication of the 5' portion of the early region, including its transcriptional control region and splicing signals (W. Gish and M. Botchan, personal communication). Novel SV40-specific RNA species of sufficiently large size, 3.8 and 4.2 kb, to be expressed from the duplicated early transcriptional control region were detected in SV80 cytoplasmic and nuclear RNA preparations by blot hybridization. The results of transcription in a cell-free system of a plasmid, pSV80-04, representing this SV80 cell SV40 DNA integrate (W. Gish and M. Botchan, personal communication) and of nuclease protection experiments with end-labelled pSV80-04 DNA fragments support the conclusion that the duplicated early sequences are transcribed in SV80 cells. It has also been established that the duplicated early splicing signals are functional in SV80 cells. These results are discussed in relation to the large amounts of SV40 early mRNA and T-antigen synthesized in cells of the SV80 line. PMID- 2994315 TI - [Plastic changes of the network properties of neurons during learning in the cat]. AB - Functional connections between neurones of various types in microareas and between microareas of the motor cortex in cats have been studied during elaboration of an electro-defensive reflex to sound. A difference has been shown between neighbouring neurones in formation of their contacts with nearby neurones located within an area of 500 mc. Neurones generating spikes of high amplitude had more active "outputs" to neurones situated at different distances, while neurones generating spikes of low amplitude had more active "inputs" to them. On the other hand, as a result of conditioning "inputs" to distant neurones underwent more significant changes in the first type of neurones, and in the second type--the "outputs" changed more markedly. PMID- 2994316 TI - [Cellular reactions to elaboration of a backward conditioned connection in Helix lucorum]. AB - After 10-15 food stimuli paired with electrical shock in semi-intact snail preparation, responses to strong tactile stimuli identified feeding behaviour neurones were studied. Inhibition evoked by tactile stimulation in these cells before learning procedure disappeared and in some cases noxious stimulus evoked synaptic activation corresponding to feeding reactions in the intact animal. Changes in second-order sensory neurones pre-synaptic to the command neurones of avoidance behaviour are suggested to be the mechanism of forward conditioned connection as well as the mechanism of backward conditioned connection. PMID- 2994317 TI - [Effect of serotonin on cholinergic septo-hippocampal reactions in the rabbit]. AB - In noncurarized and unanaesthetized rabbits, the unit activity and field potentials evoked by testing stimulation of the medial septum were studied before, during (3-10 min), and in different periods (up. to 0.5 h) after microiontophoretic serotonin (5-OT) application or n. Raphe stimulation. In most of the cases, just during 5-OT application or n. Raphe stimulation, cholinergic septo-hippocampal responses decreased. Trace facilitatory effect of native and exogenously applied 5-OT on these responses was found. Increase of efficiency of cholinergic excitatory input was considered as a confirmation of the role of the serotonergic system in hippocampal long-term posttetanic potentiation after the stimulation of the medial septum. On the whole the data obtained indicate a complex modulatory 5-OT influence on the cholinergic septo-hippocampal responses. PMID- 2994318 TI - [Characteristics of the shape of auto- and cross-correlation histograms of the spike trains of polysynaptically connected neurons]. AB - Peculiarities of auto- and cross-correlation histograms of spike trains of polysynaptically (disynaptically) connected neurones were studied by means of mathematical and biomathematical modelling of neuronal interaction (computer controlled experiment with neurones of a mollusc) in wide physiological diapazons of the values of parameters characterizing the properties and conditions of functioning of the neurones and synapses. A comparison was carried out of manifestations of polysynaptic and corresponding monosynaptic connections in auto and cross-correlation histograms. PMID- 2994319 TI - [Tumors of salivary glands]. PMID- 2994320 TI - The advantages of preoperative therapy in Wilms' tumour. A summarised report on clinical trials conducted by the International Society of Paediatric Oncology (SIOP). AB - During 1971-1980, three consecutive clinical studies on nephroblastoma were conducted by the International Society of Paediatric Oncology (SIOP). Besides several questions on different postoperative treatment modalities, the emphasis of these trials was to determine the role of preoperative therapy. In SIOP-1 (1971-1974) we compared preoperative radiotherapy to immediate surgery. There is no difference in survival or recurrence-free survival between both groups. Tumour ruptures, however, did occur significantly less in the pretreated group, and the recurrence-free survival is clearly lower in patients with intraoperative tumour rupture. These results were confirmed by SIOP-2, a non-randomised study conducted between 1974 and 1976. The question tackled by SIOP-5 (1977-1980) was whether preoperative chemotherapy could be a substitute for preoperative radiotherapy. The two preoperative therapies produced equally satisfactory results in terms of recurrence-free survival and 4-year survival rates, and the reduction in the number of operative ruptures. In addition, it was shown that with preoperative chemotherapy about 45% of an unselected population of patients with Wilms' tumour can be treated and cured without any irradiation and its well-known sequelae. The actual need for radiotherapy in stage II tumours with negative lymph node involvement (about 30% of the patients after pretreatment) is under investigation in SIOP-6. Cytohistological grading is equally possible after chemotherapy, and also without pretreatment. If antitumour treatment is begun before histopathological diagnosis, then the risk of overtreating patients with benign conditions, or of administering an inappropriate treatment to patients with malignant tumours of other types, is about 6%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2994321 TI - [incidence of cytomegalovirus infections following transplantation of kidneys from seropositive and seronegative donors]. AB - Serum samples of 110 recipients of kidney transplants and the 100 donors belonging to them taken before the transplantation were examined for complement binding antibodies against cytomegaloviruses. A clear influence of the cytomegalovirus antibody state of donor and recipient on the cytomegalovirus infection appearing after transplantation, serologically ascertained by cytomegalovirus-IgM-antibody proof or seroconversion and fourfold increase of the titre in the complement-binding reaction, respectively, could be made evident. Cytomegalovirus negative recipients of kidneys of cytomegalovirus positive donors had significantly more infections than seronegative recipients of kidneys of seronegative donors (50% vs. 16%, p less than 0.05). According to the infection rate the seropositive recipients stand with about 30% between these two groups. It seems that in these recipients the serological state of the donors has no influence. These findings make evident that seronegative recipients receive the cytomegaloviruses essentially with the transplant, whereas the cytomegalovirus infections in seropositive recipients are endogenous cytomegalovirus reactivations caused by therapy. PMID- 2994322 TI - [Enzyme immunoassay for pregnancy-specific beta 1-glycoprotein (SP-1) in patients with testicular tumors]. AB - The use of a two-side-binding-enzyme-immunoassay for pregnancy-specific beta 1 glycoprotein (SP-1) in tumours of the testicles is described. The lower limit of evidence is with 4 ng/ml near to the physiological region. In 17 of 41 non seminomatous germinal tumours of the testicles (41%) initially increased SP-1 serum concentrations were present, the other measuring values correlated with the course of the disease. In all tumours of the testicles with initially increased SP-1-titres the use of the SP-1-test gives a further possibility of the regulation of therapy and control of the course. PMID- 2994323 TI - [Value of surgery for metastases of non-seminomas in the disseminated tumor stage]. AB - The valency of the surgical treatment of lung metastases in non-seminomatous testicular tumours at the late stage is up to now apparently underestimated. The description and discussion of operative removals of solitary as well as multilocular bilateral pulmonary metastases of teratocarcinomata with regard to stage-related considerations of indication may serve as mental suggestion for a courageous and aggressive position of the treatment as well as in the individual case as therapeutic alternative of an expectation of success which is not to be underestimated. PMID- 2994324 TI - [Diagnosis and surgical therapy of organic hyperinsulinism]. AB - The diagnosis of an insulin producing tumour can be confirmed by a minimum of biochemical investigations. Its preoperative localisation is more difficult. Sonogram, Computertomogram, selective angiography and percutaneous transhepatic collecting of blood samples for insulin analysis from the portal system were preoperative measured to localize the tumours in 32 of 37 patients of our series. In 2 patients intraoperative tumour localisation by measurement of incorporated p32 proved to be effective. In B-cell-carinomas pancreas resection is the adequate therapy. With regard to the therapeutic effects a high risk is involved in the 'blind' left or right sited resection of non-localized tumours. PMID- 2994325 TI - [Skin flap in tracheobronchial reconstructive surgery]. PMID- 2994326 TI - [Galactography in the diagnosis of breast cancer]. AB - Caused by a pathological nipple discharge with a suspect cytological result we made 1818 galactographies. The X-ray results have been classified according to the following points of view: milk duct ectasies, milk duct cysts, ectasies and cysts, intracanalicular proliferations and the so called "normal" duct system. Milk duct carcinomas and carcinomata in situ are found frequently in cases of milk duct abruption and changes in the milk duct borderlines. PMID- 2994327 TI - [Cultivation characteristics of Legionella pneumophila in a liquid medium]. AB - The comparative analysis of liquid media for the cultivation of Legionella pneumophila, sterilized by filtration and autoclaving, has shown that, in contrast to media prepared on the basis of yeast extract, the capacity of media based on proteose peptone for supporting the growth of Legionella is not influenced by the method of their sterilization. The possibility of cultivating this organism in liquid media without ferric pyrophosphate has been demonstrated. PMID- 2994328 TI - [Epidemiological patterns in the spread of hepatitis A and the means for improving the measures for controlling this infection]. PMID- 2994329 TI - [Electron-cytochemical study of microglia in Alzheimer's disease and senile dementia]. AB - Electron cytochemistry was employed to identify microglial cells by nucleoside diphosphatase (NDPase) in the senile plaques of the autopsy brain in Alzheimer's disease and senile dementia. It has been elucidated that processes of microglial cells enter a senile plaque and are closely connected with amyloid accumulation. A positive reaction to NDPase revealed in the extracellular substance suggests that these cells play an important role in the accumulation of amyloid in a senile plaque. PMID- 2994330 TI - [Pituitary adenoma with widespread extrasellar extension (chief topographo anatomic features)]. AB - A group of hypophyseal adenomas with extensive extrasellar spread (179 patients) is distinguished and the main topographo-anatomical characteristics of the tumors are determined. The most important characteristics are a large size of the adenoma and growth in many directions with involvement of several structures of the skull and brain. Frequent (up to 70%) invasive growth is typical of such hypophyseal adenomas irrespective of the histological structure. Multiple anastomoses form between the vessels of the tumor and the vascular networks of all anatomical structures with which the tumor comes in contact. These tumors are always separated from the brain matter by the pia. mater. The invasive character of the growth of hypophyseal adenomas makes their radical removal less possible. Maximum effect of surgery may be expected in demarcated growth of the hypophyseal adenoma, including cases with its extensive extrasellar spread. PMID- 2994331 TI - Kidney scintigraphy in the diagnostics of urological renal diseases. AB - The authors report on their experiences in the diagnostics of urological renal diseases gained by 256 kidney scintigraphies of a total of 227 patients. The more important trends of progress made in the recent years in nuclear medicine are outlined. The sensitivity (93%) and specificity (86%) of the examination based on the surgical and histological findings are determined. The technical importance of the study made from several directions using up-to-date radiopharmaca is emphasized in obtaining the results being favourable even in international comparison. The importance of the coordination between the urologists and experts well-versed in nuclear medicine is pointed out in selecting the examination best suited for the purpose (dynamic, static examination). Based on the literature and on their own experiences, the diagnostic results of the examination in detecting and controlling renal diseases are summarized. PMID- 2994333 TI - Identification of insulin-like growth factor-I and its receptors in the rat testis. AB - As part of a study of the testicular production and action of insulin-like growth factor-I (IGF-I), adult rat testes were extracted with acidified methanol, yielding an immunoreactive IGF-I fraction corresponding in size to human IGF-I. The mean IGF-I content (+/- SEM) of testes weighing approximately 1.1 g was 51.5 +/- 5.6 ng/testis, and was not due to serum contamination. After a 3-day fast testicular IGF-I decreased by 80%, whereas serum IGF-I levels declined by 90%. Testicular homogenates and isolated Leydig cells were shown to contain specific IGF-I receptors, Ka = 2 X 10(9) M-1, with 10% IGF-II cross-reactivity. The concentration of these receptors was 2 pmol binding sites per testis, or 3.3 fmol per 10(6) Leydig cells. However IGF-I at 250 ng/ml had no effect on basal or hCG stimulated testosterone production by isolated Leydig cells, measured over 3 h. Although an effect of IGF-I over longer incubation periods cannot be excluded, it is also possible that testicular IGF-I has a mitogenic role, rather than acting on differentiated testicular functions. PMID- 2994332 TI - Cyproheptadine-mediated inhibition of growth hormone and prolactin release from pituitary adenoma cells of acromegaly and gigantism in culture. AB - The effect of cyproheptadine on growth hormone (GH) and prolactin (Prl) secretion from cultured pituitary adenoma cells of acromegaly and pituitary gigantism was studied. When varying doses of cyproheptadine ranging from 0.01 to 1 microM were added to the incubation media, GH secretion was consistently inhibited and a dose response relationship was observed between the cyproheptadine concentrations and the amounts of GH released into the media. In pituitary adenomas which concurrently produced and secreted Prl, cyproheptadine likewise suppressed Prl release in a dose-related manner. This effect of cyproheptadine was not blocked by coincubation with serotonin. Similarly, coincubation with a dopaminergic antagonist, haloperidol, failed to reverse the inhibitory action produced by cyproheptadine. When coincubated with dopamine, cyproheptadine further inhibited GH and Prl secretion. These results suggest that cyproheptadine possesses a direct action on human somatotroph adenoma cells to inhibit GH and Prl secretion by an unknown mechanism that is different from serotonergic and dopaminergic systems. PMID- 2994334 TI - Beta receptors in peripheral mononuclear cells increase acutely during exercise. AB - Five healthy adult well trained men (averaging 49 miles running per week) were exercised to exhaustion on a treadmill. Plasma epinephrine increased from 168 +/- 37 pg/ml prior to exercise to 633 +/- 155 pg/ml immediately after exercise (P less than 0.025) and plasma norepinephrine increased from 1608 +/- 345 to 8576 +/ 1662 pg/ml (P less than 0.025) in the two respective periods. Beta receptor density increased from 53 +/- 18 fmol/mg protein before exercise, to 223 +/- 63 fmol/mg (P less than 0.05) protein immediately after exercise, and then decreased to 83 +/- 27 fmol/mg protein 1 h later (P less than 0.05 compared to post exercise). The mean Kd value prior to exercise of 1.5 +/- 0.2 X 10(-11) M was unchanged statistically throughout the study. Following correction for haemodilution, serum total and free T4 and T3 concentrations were also unchanged during exercise, although reverse T3 levels did increase from 39 +/- 4 to 45 +/- 0 ng/dl (P less than 0.05). These findings suggest that: 1) plasma epinephrine, norepinephrine and beta receptor density, but not Kd, increase acutely during exercise and 2) total and free T4 and T3, after correction for haemodilution, do not change during acute exercise. Our data indicate that acute exercise represents an unusual condition during which 'down regulation' is not observed, but, rather, there appear to be parallel alterations in beta receptor density and plasma catecholamine levels. PMID- 2994335 TI - Regulation of (Ca2+-Mg2+)-ATPase activity by calcitonin binding to rat liver plasma membranes. AB - The plasma membranes isolated from rat liver bound 125I-labelled ([125I]) synthetic [Asu1,7]eel calcitonin (CT), with increasing concentrations of [125I]CT. This specific binding was completely saturated at a concentration of 0.5 nM CT. A high affinity Ca2+-stimulated, Mg2+-dependent ATPase [(Ca2+-Mg2+) ATPase] activity in the plasma membranes was significantly decreased by the presence of a very low concentration of CT (7.4 pM), although the hormone did not affect the activity of the plasma membrane 5'-nucleotidase. The concentration of CT needed for maximal inhibition of (Ca2+-Mg2+)-ATPase in the plasma membranes was less than 0.74 nM. The plasma membranes washed with 10(-3)% digitonin did not show an inhibitory effect of CT on (Ca2+-Mg2+)-ATPase activity, while the reagent did not have a significant effect on the enzyme. These results suggest that the inhibition of (Ca2+-Mg2+)-ATPase activity may be part of the mechanism by which CT elevates cytosolic Ca2+ in liver cells. PMID- 2994336 TI - Probable ACTH-secreting pituitary tumour in association with Addison's disease. AB - A 61 year old Japanese man with a diagnosis of Addison's disease was admitted to Kyushu University Hospital for further investigation of high ACTH levels and hyperpigmentation which 37.5 mg of cortisone acetate failed to alleviate. The basal level of plasma ACTH was 700-1000 pg/ml, and following 25-37.5 mg cortisone acetate or 1 mg dexamethasone the levels were 300-600 pg/ml. The general pigmentation showed little improvement with such medication. Radiographic studies revealed a double floor of the sella turcica and cisternal herniation. These observations suggested the existence of a pituitary ACTH-secreting tumour. Plasma ACTH showed a circadian rhythm ranging from 440 to 1570 pg/ml and it was not suppressed to a normal range by oral administration of dexamethasone, 8 mg/day or by continuous infusion of dexamethasone, 1.25 mg/h for 2 h. Plasma ACTH responses of 80% above basal level to lysine-vasopressin (LVP), and 12% above basal to synthetic ovine corticotrophin releasing factor (CRF) were observed. FK 33-824, a methionine-enkephalin analogue, suppressed plasma ACTH to 85% of basal level, while bromocriptine (CB-154) caused no significant change. These findings led to a diagnosis of pituitary ACTH-secreting adenoma (corticotropinoma) in association with Addison's disease. The persistent circadian rhythm of plasma ACTH suggested that this adenoma may not be completely free from regulation by the central nervous system. This case may be clinically significant for investigation of the pathogenesis of pituitary adenoma, particularly in Nelson's syndrome. PMID- 2994337 TI - Decrease of LRH receptors in the pituitary gland in streptozotocin-induced diabetic rats. AB - In studies into the mechanism of anovulation in the diabetic condition, the LRH receptor content of the pituitary gland of rats with diabetes mellitus was determined. Normal female rats weighing 180-200 g were injected iv with either streptozotocin (6 mg/100 g body weight) or vehicle in dioestrus of the oestrous cycle. The rats were sacrificed by decapitation 9 days after treatment, and serum LH concentrations and the LRH content of the medial basal hypothalamus (MBH) were measured by radioimmunoassay (RIA), and the LRH receptor content of the pituitary gland was determined. The serum concentration of LH and the LRH content of the MBH in diabetic rats were 34.4 +/- 4.8 ng/ml (mean +/- SEM) and 2.05 +/- 0.04 ng/MBH, respectively, which were similar to the respective values of normal rats in dioestrus. However, the LRH receptor content of diabetic rats (13.2 +/- 3.9 fmol/pituitary) was significantly (P less than 0.05) lower than that of normal rats in dioestrus (68.0 +/- 7.1 fmol/pituitary) and pro-oestrus (59.4 +/- 13.6 fmol/pituitary). These results suggest that anovulation in diabetic rats is at least partly attributable to a low content of LRH receptors in the pituitary gland. PMID- 2994338 TI - Comparison of growth hormone binding and metabolic response in rat adipocytes of epididymal, subcutaneous, and retroperitoneal origin. AB - We undertook a comparison of human growth hormone (hGH) binding and metabolic responses in rat adipocytes of epididymal, subcutaneous, and retroperitoneal origin to determine whether the site of fat depot biopsy might affect the response to hGH stimulation. The results showed highest specific binding in epididymal (3.6%), followed by subcutaneous (2.3%) and retroperitoneal adipocytes (1.5%); half-maximal binding was achieved at 14-18 ng/ml hGH for the three sites. Scatchard analysis of the binding data from each site was linear; there was no significant difference in binding affinities (2.1 to 3.3 X 10(9), M-1), but the number of binding sites was statistically higher in epididymal (9.8 X 10(3) as compared to subcutaneous (7.5 X 10(3), P less than 0.05) and retroperitoneal cells (3.3 X 10(3), P less than 0.01). Stimulation with 5 to 2500 ng pituitary hGH produced a dose-related increase in glucose incorporation, with the largest increase in epididymal fat cells (31%, P less than 0.05) followed by subcutaneous cells (18%, P less than 0.05); no significant increase was seen with retroperitoneal cells. Biosynthetic hGH produced a similar pattern of glucose incorporation in the three sites. Addition of hGH antibodies blocked the glucose incorporation in epididymal adipocytes using both pituitary-derived and biosynthetic hGH. It seems clear that this insulin-like effect is caused by hGH, not an insulin-like impurity. We conclude that the number of binding sites, perhaps related to adipose cell size, differs in adipose tissue from different locations and this influences the metabolic response to hGH stimulation. PMID- 2994339 TI - [Establishment and characterization of B cell line cells (Kei-4 and Kei-5) derived from a patient with adult T-cell leukemia]. PMID- 2994340 TI - [Tac antigen expression on non-T non-B acute lymphocytic leukemia cells]. PMID- 2994341 TI - Trilineage acute leukaemia in combined Ph1-chromosome positivity and monosomy 7. AB - A case of acute leukaemia with Ph1-chromosome and monosomy 7 is reported, in which the peripheral blood contained three types of blast cell as distinguished by light and electron microscopy and immunological phenotyping. The first blast cell type originated from the granulocytic lineage; the cells contained peroxidase-positive granules, and had an HLA-DR+Tdt-CALLA-phenotype. The second blast-cell type was more difficult to define, but had many characteristics of the monocytic series. The phenotype of these blast cells was HLA-DR+Tdt-CALLA-BA-2+ or HLA-DR+/-TA-1+63D3+. Finally, the third type of blast cell was clearly of lymphocytic origin. These cells were peroxidase-negative, and were CALLA+ as studied by electron microscopy using immunogold labelling and fluorescence microscopy. Their phenotype was HLA-DR+Tdt+CALLA+. Cell sorting and double fluorescence assays showed that these three populations were separate; no cells of mixed myeloid/lymphoid phenotype were found. This case suggests the neoplastic transformation of an immature progenitor cell and subsequent differentiation of the neoplastic cells in various directions. PMID- 2994342 TI - Adrenal changes in Niemann-Pick disease: differences between sphingomyelinase deficiency and type C. AB - Structural, chemical, and histochemical analyses of adrenal tissue performed in 8 cases of Niemann-Pick disease (NPD) revealed stark differences of storage between spingomyelinase (SMase) deficiency (6 cases) and type C (2 cases). In all the full-blown cases of the SMase deficiency group, pronounced sphingomyelin (SM) storage was found in all the zones of the cortical epithelium with slightly increasing centripetal gradient. The storage resulted in the reduction or even disappearance of lipofuscinogenesis in the reticular zone, in the reduction of the physiological fat content, in the generalized foamy transformation of the epithelium, and in moderate organomegaly. The storage was expressed in both A and B types and was roughly proportional to the storage in other viscera. The stromal storage was confined to the vascular endothelium, and in particular, to the macrophages. One of the cases showed the presence of typical spirolactone bodies unmodified in fine structure by the lysosomal storage. Their most conspicuous enzymatic activity was that of non-specific esterase and NADH tetrazolium reductase. The adrenals in type C were macroscopically and histologically normal except for a variable population of stromal foam cells. Chemically, there was slight increase in all phospholipids with borderline or moderate percentual increase of SM. There was also slight increase in some of the lower neutral glycosphingolipids. Electron microscopy dislosed rudimentar storage in lower cortical layer epithelium which by its fine structure and according to results of lipid histochemistry was qualitatively different from that in SMase deficiency. The stromal storage was expressed mainly in macrophages in which there was histochemically detectable amount of SM. There was no storage detectable in medullary cells in neither group of NPD complex. The results point not only to striking quantitative differences in storage intensity between the 2 basic groups of NPD showing the cortical epithelium in type C as being remarkably resistant to the metabolic disorder, but also to difference in quality of the storage very much like that found in other tissues, too. PMID- 2994343 TI - Cytochemical study of K+-dependent p-nitrophenyl-phosphatase activity in the urinary bladder of Salamandra salamandra. AB - The ultrastructural Na+-K+-ATPase localization in the salamander urinary bladder has been studied by the histochemical techniques of Ernst (1972) and Mayahara et al. (1980). Reaction products have been specially found on the basal and lateral membranes of granular and mitochondria-rich cells. The variable presence of artifacts, often accompanying Pb-capture phosphatase cytochemistry, is discussed. Additional data on the active ion transport have been obtained by using electrophysiological techniques. PMID- 2994344 TI - Etomidate anesthesia inhibits the cortisol response to surgical stress. AB - Plasma cortisol was measured in 18 patients undergoing gynecological procedures under etomidate or methohexital and nitrous oxide anesthesia. Plasma ACTH was also measured in three patients in each group. The mean plasma cortisol concentration before anesthesia was comparable in both groups. Plasma cortisol increased in patients anesthetized with methohexital, while none of the patients anesthetized with etomidate had an increase in plasma cortisol. The increase in plasma ACTH was equivalent in the two groups. Therefore, etomidate is a potent inhibitor of the adrenal response to surgery. The absence of clinical consequences associated with the blunted response suggests that a major increase in adrenal hormone production may not be necessary during surgery. PMID- 2994345 TI - Inhibition of human leukocyte metabolism and random mobility by local anaesthesia. AB - Hexose-monophosphate shunt (HMS) activity, myelo-peroxidase-(MPO)-mediated iodination and random mobility in human polymorphonuclear leukocytes (PMNs) were studied in the presence of lignocaine. Incubating the PMNs with 0.1% lignocaine during phagocytosis inhibited the 14CO2 produced from glucose-1-14-C via the HMS shunt by 33%. On increasing the concentration of lignocaine, a dose-dependent inhibition was noted. The MPO-mediated iodination was inhibited by 73% in the presence of 0.1% lignocaine, and complete inhibition took place when the concentration was increased to 0.5%. The random mobility of leukocytes was studied by an opto-electronic technique. In the presence of 0.5% lignocaine, all leukocytes examined were completely immobilized; in the presence of 0.1% lignocaine immobilization took place within 45-65 min. PMID- 2994346 TI - Serum levels of cortisol in man during etomidate, fentanyl and air anesthesia, compared with neurolept anesthesia. AB - The effects of etomidate and fentanyl given as a mixture in continuous infusion and of a classical neuroleptanalgesia, with droperidol and fentanyl, on the serum cortisol levels were compared during and after anesthesia in sixteen patients who were to undergo major abdominal surgery. The results showed that the postoperative increase of serum cortisol levels which occurred in the neurolept group was absent in the group receiving etomidate and fentanyl infusion. During etomidate-fentanyl anesthesia there was a more significant decrease of serum cortisol and no rise in serum cortisol levels in response to synthetic ACTH injection. Etomidate and fentanyl, given as a continuous infusion during major abdominal surgery, block the adrenal mechanism to secrete endogenous cortisol in response to stress or after the administration of adreno-corticotrope hormone. PMID- 2994347 TI - Constituents of connective tissue around the capillary of chick pecten oculi. AB - The constituents of the connective tissues around the capillary of the chick pecten oculi were examined electron microscopically by HCl-collagenase and HCl elastase methods. The basal lamina like membrane below the endothelial cell of the pecten capillary was digested by collagenases I, II and IV and elastase, and may be a false basal lamina. The basal lamina of cells with pigment granules which surround the capillary was digested by collagenase IV and elastase, and contained type IV collagen. Fibrils between the basal lamina like membrane of the pecten capillary endothelium and the basal lamina of the cells with pigment granules were digested by collagenases I, II and IV, and elastase. Thus, these fibrils are composed of many kinds of collagen. Elastase may be responsible for the breakdown of most collagens as well as elastin. PMID- 2994348 TI - Percentage of projection neurons and various types of interneurons in the human claustrum. AB - Each neuronal type of the human claustrum is differently pigmented. This has been demonstrated by combining a transparent Golgi technique with the specific staining of lipofuscin deposits. One type of projection cells and four varieties of interneurons have been distinguished. In this study the percentage of these different neuronal types has been evaluated using preparations stained for both Nissl material and pigment deposits. PMID- 2994349 TI - Binding of lincomycin to immunoglobulins has no effect on their antigen-binding capacity. AB - The interaction of lincomycin and immunoglobulins was examined in vitro. While lincomycin bound to the immunoglobulin molecules seemed to decrease the quantity of IgG and IgM detected by radial immunodiffusion and microzone electrophoresis, the level of specific antibodies could not be demonstrated by enzyme-linked immunosorbent assay (ELISA). PMID- 2994350 TI - In vivo model for the acute, latent and reactivated phases of cytomegalovirus infection. AB - Tissue samples from the salivary gland, lung spleen, liver and kidney of Balb/c mice infected with murine cytomegalovirus (MCMV) contained infective MCMV and virus-specific antigens for 6-7 weeks following the infection. After the 8th week no infective virus could be detected in any organ and antigens were found only in the spleen and the mesangial cells of the renal glomerules. As a result of cyclophosphamide treatment applied in the 22nd-24th weeks, the latent viral infection was reactivated in nearly all animals, infective virus production started again in the organs, and lasted for about 3 weeks. During the subsequent latent period the virus was again reactivated by repeated cyclophosphamide treatment. The experimental alternation of the latent and reactivated phases of viral infection renders the model suitable for study of the mechanism and consequences of viral latency and reactivation in vivo. PMID- 2994351 TI - Effect of alpha- and beta-receptor active drugs on corneal thickness. AB - Phenylephrine (10 mg/ml), phentolamine (10 mg/ml), isoprenaline (0.5 mg/ml) and timolol (5 mg/ml) were applied topically to one eye of volunteers with normal corneas, the other eye serving as a control. One drop of each drug was given twice a day for 3 or 4 days. Timolol was found to increase corneal thickness in both the medicated and the control eye. A significantly greater effect was noted on the eye receiving the drug directly. When isoprenaline was given for 3 days, a slight significant decrease in corneal thickness was found in both the medicated and the control eye, with the two eyes exhibiting a similar CCT change. The present results are suggestive of endothelial beta-receptor importance in the regulation of corneal thickness. PMID- 2994352 TI - Histochemical, microchemical (microprobe) and organ culture approaches to the study of auditory development. AB - Cochlear development has been studied by means of biochemistry/histochemistry (Na+/K+-ATPase, adenylate cyclase, 2-deoxy-D-glucose and phospholipids), the energy dispersive X-ray microanalysis technique and organ culture of the mammalian inner ear. A high level of Na+/K+-ATPase and adenylate cyclase occurs in the stria vascularis and has been cytochemically demonstrated at the contraluminal side of marginal cell membranes. An increase in the adenylate cyclase content occurs approximately one day before and in parallel with the rise of the potassium content in endolymph. These processes are preceded by an ultrastructural differentiation of the stria vascularis but appear prior to the rapid increase of the endolymphatic potential. The functional activity of the developing/differentiating cochlea is reflected by increased levels of 2-deoxy-D glucose first during the postnatal morphologic maturation of the organ of Corti and the stria vascularis approximately 1 week after birth, and, later during the maturation of cochlear potentials 10-14 days after birth. Organ culture of the embryonic inner ear is a suitable tool for studies on early (embryonic) morphologic development, neural induction, fate-mapping, epithelio-mesenchymal interactions and neurotrophic interactions. Concerning postnatal inner ear structures, organ culture has focused on development of afferent nerve fibres. The isolated organ is deprived of efferent fibres of central origin. PMID- 2994353 TI - Postnatal development of the mammalian central auditory system and the neural consequences of auditory deprivation. AB - The auditory system of many mammalian species is immature at the time of birth. Peripheral elements, particularly the middle ear and cochlea, account for much of the physiological immaturity observed in central auditory structures. However, there is now considerable evidence that the central pathway undergoes developmental changes that, at least partially, occur in parallel with peripheral development. Auditory nerve fibres and their terminals have been shown to be of smaller diameter and less extensively arborized in neonatal than in adult cats. These factors almost certainly contribute to many of the sluggish physiological properties of neurones in the kitten auditory nerve and cochlear nucleus. At higher levels of the cat auditory system (inferior colliculus, auditory cortex) mechanisms subserving binaural interaction also undergo a period of postnatal development. Recent studies of sound deprivation produced by either deafferentation or a conductive hearing loss have demonstrated that alteration of cochlear output during the immediate postnatal period may change the normal development of the central auditory system. PMID- 2994354 TI - Constraints of intensive care units and follow-up studies in prematures. AB - This paper reviews the main known causes of deafness in newborns at risk. Some of them, like infections, anoxia, prematurity, etc. can be classified as 'clinical factors'. Others, like the ototoxicity of some antibiotics and the noise of the life-supporting equipment, are described in the paragraph on the 'constraints of intensive care'. Finally, the possible potentiating effect that some of the above mentioned factors may have on each other is mentioned in the paragraph entitled 'combined effects'. The need of accurate follow-up studies of neonatal intensive care unit (NICU) babies treated in different ways is stressed in the last paragraph. PMID- 2994355 TI - [Rhinopharyngeal carcinoma and Epstein-Barr virus: study of a sample of Italian patients]. PMID- 2994356 TI - Ultrastructural study of hepatocellular carcinoma with replacing growth pattern. AB - The replacing growth of hepatocellular carcinoma (HCC) has been considered as a basic growth pattern at the tumor-nontumor boundary. In order to clarify the ultrastructural characteristics of HCC with the replacing growth, we have studied 59 surgical cases of HCC. At the tumor-nontumor boundary of HCC with the replacing growth pattern, the tumor cells grow to replace the hepatocytes along the liver cell cord, but oppress and distort the adjacent liver cell cord with varying degrees of aggregation of reticulin fiber. Hepatocytes and cancer cells are in direct contact, but some of the hepatocytes are compressed by cancer cells. The sinusoid in the non-cancerous area is continuous with the blood space in the cancerous tissue, and several layers of endothelial cells are often observed. Accordingly, it is predicted that sinusoidal blood through the portal vein and arterial blood through arterial tumor vessels may mix at the tumor nontumor boundary. PMID- 2994357 TI - Primary signet-ring cell carcinoma of the urinary bladder. Report of two cases with histochemical studies. AB - Two cases of primary signet-ring cell carcinoma of the urinary bladder are presented with histochemical studies. The signet-ring cell carcinoma of the bladder is a rare variant of mucus-producing vesical adenocarcinoma: sixteen cases have been reported in the Occidental and two other cases in Japanese literature. Most of the cases were discovered in the advanced stage and the prognosis was poor: necessity of early detection by cytological examination and treatment with surgery is stressed. The histochemical evidence for the similarity of the neoplasm to colonic epithelium in mucus production is illustrated and the grade of malignancy is discussed from the view of mucus production by the neoplasms. PMID- 2994358 TI - Granular cell tumor of the gallbladder. Report of a case. AB - A rare case of granular cell tumor of the gallbladder in a 58-year-old Japanese man is presented. This is the third report of such a tumor arising in the gallbladder. The immunohistochemical study demonstrated the localization of the nervous-system-specific protein (S-100 protein) in the granular component cells, thereby supporting the Schwann cell origin of this tumor. PMID- 2994359 TI - Malignant giant cell tumor of tendon sheath. Report of a case. AB - In a patient with pigmented villonodular synovitis of the right knee joint, there occurred a malignant giant cell tumor of tendon sheath. There was clinical evidence of metastasis after the second local recurrence and the recurrent tumors were studied enzyme cytochemically and electron microscopically. Ultrastructurally, the malignant tumor consisted of three principal cell types; histiocyte-like cells, fibroblast-like cells, and intermediate cells, with unique attendance of myofibroblasts. This may be the first report of the presence of myofibroblasts in malignant giant cell tumor of tendon sheath. Enzyme cytochemistry revealed various functional properties of histiocytes. PMID- 2994360 TI - Disseminated intravascular coagulation induced by generalized cytomegalic inclusion disease during steroid therapy for polymyositis. AB - A 56-year-old woman came to the hospital with fever and skin eruptions. A rise in myogenic enzyme and the presence of antileucocyte antibody were noticed, along with the gradual appearance of myalgia in both lower extremities, and muscle weakness. Steroid therapy was started under the diagnosis of polymyositis. The steroid was reduced because of mental disturbance but immediately the patient developed high fever. Various forms of treatment were carried out but there was no improvement, and the patient died. At autopsy there were scattered purpura on the skin, and the muscles were atrophic and yellowish-grey in color. Histopathologically, there was inflammatory cell infiltration and muscle fiber degeneration visible in many of the muscles, and the findings showed evidence of polymyositis. There were intranuclear inclusions in the lungs, ovaries, and adrenal glands, and this was diagnosed as generalized cytomegalic inclusion disease. Fibrin thrombi were found in the kidneys, lungs, and adrenal glands and this was pathologically diagnosed as disseminated intravascular coagulation. Endothelial cell damage caused by cytomegalovirus was assumed to be involved to a large extent in triggering the disseminated intravascular coagulation. PMID- 2994361 TI - An immunofluorescent study of generalized Coxsackie virus B3 infection in a newborn infant. AB - An autopsy case of a 10-day-old newborn with generalized infection of Coxsackie virus B3 (CBV3) was reported. CBV3 was isolated from the blood before death. The patient died of cardiac failure. An immunofluorescent study was carried out on autopsy specimens fixed in formalin and embedded in paraffin. CBV3 antigen was detected in the heart, brain, kidney, lungs, spleen, thymus, and pancreas. In the pancreas CBV3 antigen was predominantly seen in the islet cells. No CBV3 antigen was found in the liver and adrenal glands. Electron microscopic examination revealed virion-like particles, 20 nm in diameter, in the endothelial cells of the myocardium. PMID- 2994362 TI - Experimental transmission of Aleutian disease virus (ADV) to different animal species. AB - Two animals from each of 8 different species (mink, Finn raccoon, cat, dog, ferret, blue fox, mouse and rabbit) were inoculated with the highly virulent Utah I strain of ADV. Only the mink developed hypergammaglobulinemia. All animals produced antibodies to ADV antigens, but with different titres. Mink sera had much higher antibody titres than the other animal sera. Antibodies to the ADV coded, non-structural polypeptide (p71) were found in mink, Finn raccoons and dogs only. Presence of this kind of antibodies was taken as evidence of ADV replication, because p71 was not present in the ADV inoculum. The animals were killed 4 weeks after virus inoculation. Homogenates of different organs from mink, Finn raccoons, ferrets, dogs, mice and the cat were shown to infect ADV negative mink, which developed antibodies to ADV following inoculation. We conclude from the present studies that mink and Finn raccoons are potential threats as ADV transmitters. Cats, ferrets, dogs and mice may be considered potential ADV reservoirs, because they possibly harbour ADV for 4 weeks or longer. Blue foxes and rabbits did not seem to be a risk factor for ADV transmission. PMID- 2994363 TI - [Study on the interaction between photoradiation-hematoporphyrin derivative and liposome with spin label and ESR spectrum]. PMID- 2994364 TI - Adenylate cyclase modulation by ammonium ion: GTP-like effect on muscarinic and alpha 2-adrenergic receptors. AB - The stimulatory influence of ammonium sulphate on adenylate cyclase activity has been investigated. By competition binding experiments on the beta-adrenergic stimulatory receptor in rat myocardial membranes, no influence could be detected of ammonium sulphate neither in receptor coupling to the stimulatory guanine nucleotide binding protein nor in the GTP-induced uncoupling. In order to detect an impaired inhibition instead of an increased stimulation of adenylate cyclase activity by ammonium sulphate the investigation was extended to inhibitory receptors. The same type of effect by ammonium sulphate was detected on both the muscarinic cholinergic receptor in rat myocardial membranes as well as on the alpha 2-adrenergic receptor in human platelets. The influence of ammonium sulphate noted in competition binding studies and off-kinetics experiments was GTP-like, i.e. causing a decrease in agonist-receptor affinity leaving all the inhibitory receptors in the low affinity state. In conclusion, this paper indicates that the observed stimulatory effect of ammonium sulphate is exerted by the ammonium ion on the inhibitory guanine nucleotide binding protein, impairing the negative control of adenylate cyclase activity. PMID- 2994365 TI - Contraction and cyclic AMP-related relaxation of the intimal and medial smooth muscle layers of pig thoracic aorta. AB - The contractile responses of an alpha-adrenoceptor agonist, phenylephrine, and of histamine were compared in the intimal and medial smooth muscle layers of the pig aortic arch. Further, the relaxant effects evoked by some compounds influencing the cyclic AMP system were compared in the two muscle layers, as well as their effects on the cyclic AMP content and phosphodiesterase activity. Phenylephrine and histamine induced contraction of the smooth muscle layers. The increase in tension was faster in the intimal than in the medial layer. The alpha adrenoceptor agonist phenylephrine was a more potent contractile agent in the intimal than in the medial smooth muscle. With histamine, no significant difference in the dose-response curves between the two muscle layers was found. Histamine-contracted muscle preparations were relaxed in a dose-dependent manner by the phosphodiesterase-inhibiting compound 3-isobutyl-1-methylaxanthine (MIX) and by 8-bromo-cyclic AMP. The two substances were more potent relaxants in the medial than in the intimal smooth muscle layer. The content of cyclic AMP in the intimal and the medial smooth muscle was increased by MIX. Isoprenaline had no relaxing effect on the muscle preparations and did not change the content of cyclic AMP. There were no differences in the basal levels of cyclic AMP in the intima and media. Vmax of phosphodiesterase activities differed, however, between the two preparations. This study demonstrates that the intimal layer is characterized by a larger contractile responsiveness to phenylephrine and a lower relaxant response to compounds influencing the cyclic AMP-system than those of the medial layer. PMID- 2994366 TI - Effect of one derivative of 2-aminotetralin on the alpha-adrenergic receptors of isolated vessels. AB - Experiments were made to test the effects of originally synthesized piperazine derivative of 2-aminotetralin (P11) on the alpha-adrenergic receptors of isolated vessels. Inhibition of the contracting effects of exogenously introduced noradrenaline into isolated rabbit ear artery was found at concentrations of P11 and phentolamine 1 X 10(-8) to 1 X 10(-4) M. IC50 for P11 is 0.68 microM, for phentolamine -- 0,40 microM. There is a certain shift to the right of the concentration effect curves for noradrenaline in isolated strip of rabbit aorta when P11 and phentolamine are applied in concentrations of 1 X 10(-7) to 1 X 10( 5) M. pA2 for P11 is 7.31, for phentolamine -- 8.04. Potentiation of the maximum effect of noradrenaline is observed after P11 1 X 10(-5) M, which decreases upon preliminary administration of cocaine 1 X 10(-5) M and propranolol 1 X 10(-6) M. The tested compound manifests alpha-adrenergic blocking action in the organs studied. PMID- 2994367 TI - Mode of lipid peroxidation-induced inhibition of Na, K-ATPase. AB - Experimental data are presented concerning inhibition of Na, K-ATPase in rat heart sarcolemmal and rat brain synaptosomal membranes upon generation of activated oxygen species. It is shown that the activity of Na, K-ATPase is inhibited in membranes of both types during incubation with Fe2+ + ascorbate system, which generates O2 and OH- radicals and thus induces lipid peroxidation. The inhibitory effect is linearly dependent on the amount of the lipid peroxidation products (malondialdehyde) accumulated. Exogenous unsaturated phosphatidylethanolamine, when added to partially inactivated enzyme, does not produce reactivation of Na, K-ATPase. Free radical scavenger 4-methyl-2,6-di (tertbutyl) phenol exerts both inhibition of lipid peroxidation and protection of Na, K-ATPase. Mg-ATPase is resistant to the action of lipid peroxidation inducing system. Bubbling of oxygen through membrane suspension results in no malondialdehyde accumulation, but is accompanied by Na, K-ATPase inhibition, which could not be prevented by free radical scavengers. It is suggested that generation of activated oxygen species results in oxidation of one of the essential amino acid residues in the active site of the enzyme. PMID- 2994368 TI - Urinary free cortisol secretion in habitually violent offenders. AB - Male violent offenders (n = 90) and residivious arsonists (n = 10) were investigated by urinary (24 h) free cortisol measurements at mental examination on a psychiatric department. The measurements were made with competitive protein binding radioassay. Only among the habitually violent offenders with antisocial personality were the values low when compared with other violent offenders, antisocial personality without the habitually violent tendency, and male clinic personnel. Poor motivation already in school, truancy, attention deficit and undersocialized aggressive conduct disorder problems seemed to be connected with the low cortisol levels. PMID- 2994369 TI - Radiation dose dependence of the sensitization by oxygen and oxygen mimic sensitizers. AB - New and old evidence is discussed which suggests that the oxygen enhancement ratio (OER) is decreased at lower radiation doses as compared with that of higher radiation doses. In addition, there is evidence that cells irradiated in severe hypoxia have an impaired ability to recover from sublethal damage. However, there is also contrary evidence to these observations. Thus the suggestions that oxygen is not strictly a dose modifying agent have been controversial from the very beginning. It is hoped that new experimental undertakings currently performed in many different laboratories will resolve these issues as well as explain the controversy which has lasted more than 25 years in this field of radiobiologic research. PMID- 2994371 TI - Radiation treatment of laryngeal carcinoma with special reference to CRE values. AB - In a retrospective analysis of 203 cases of laryngeal carcinoma treated with radiation therapy, conventional absorbed dose levels and CRE calculations were compared as regards the prediction of treatment failure, tumor recurrences and major complications. The recurrence rate for T1 and T2 tumors was 14 per cent and for T3-4 tumors 26 per cent. Poorly differentiated (grade 3) tumors had a significantly higher recurrence rate than well and moderately well differentiated (grades 1 and 2) ones. Corrected 10-year survival rates were 89, 82 and 52 per cent, respectively for T1, T2 and T3-4 tumors. There was a significant relationship between the recurrence rate and the CRE level while the total absorbed dose in Gy or the size of the treatment field could not be correlated to treatment failure. Major complications occurred in 8 (3.9%) patients and they had all received treatment giving CRE values of 1920 reu or more. PMID- 2994370 TI - Comparison between diagnoses in the Stockholm Regional Cancer Register and certified underlying causes of death. AB - Data in the Stockholm Regional Cancer Register were compared with death certificates for persons who died of carcinoma in 1978 and were domiciled in Stockholm County. Concordance between cancer register diagnosis and certified underlying cause of death was 87 per cent. Many malignant tumours in organs which often show metastases were unspecified by one of the registers. In many other cases of discordance, differing site codes denoted anatomically close-lying organs. Age distribution did not differ between concordant and discordant diagnostic groups. Coordination of the cancer register and the mortality statistics as regards coding routines and disease classification presumably would enhance inter-register comparability. PMID- 2994372 TI - Management of mediastinal compression due to bronchogenic carcinoma. AB - Fifty-six cases of bronchogenic carcinoma with mediastinal compression were analyzed retrospectively. Microscopic confirmation was obtained in 23 patients and small cell carcinoma was most frequent. Radiation treatment gave complete remission in about 70 per cent when the total tumour dose exceeded 35 Gy fractionated during 3 weeks. Addition of cyclophosphamide marginally increased the remission rate to about 85 per cent. Total tumour doses of less than 35 Gy gave obviously poorer results. The response rate was highest in small cell carcinoma. PMID- 2994373 TI - Postirradiation hemangiosarcoma of the chest wall. Report of a case. AB - An unusual case of cutaneous hemangiosarcoma that developed on a chest wall irradiated after mastectomy for cancer is described. The patient, an elderly woman, had previously received high-dose radiation to the chest wall as well as systemic combination chemotherapy. Sarcoma developed 6 years after mastectomy and progressed rapidly. The time between radiation therapy and occurrence of cutaneous sarcoma was shorter than the median latent period reported for development of radiation-induced sarcoma. Thus, we cannot be certain that radiation was the true or sole etiologic factor. Whether the addition of systemic chemotherapy was a contributory agent is also speculative. PMID- 2994374 TI - Combined transcatheter arterial embolization and regional chemotherapy for locally advanced carcinoma of the breast. A preliminary investigation. AB - Seventeen patients with carcinoma of the breast received a combination of transcatheter arterial embolization and regional chemotherapy before surgery. A gelatin powder mixed with an anticancer agent and blood clotting factors (own technique) was selectively injected into the internal mammary artery, the lateral thoracic artery, and the thoracodorsal artery. Marked regression of both primary tumor and metastatic regional lymph nodes was observed. The potential of this method as presurgical treatment is discussed. PMID- 2994375 TI - Bone mineral content and estrogen receptors in women with breast tumors. AB - The investigation was carried out to elucidate a possible relationship between the amount of estrogen receptors and bone mineral content in patients with breast tumors. Bone mineral content (BMC) was measured by photon absorptiometry in the distal forearm of 54 women with untreated breast carcinoma and 19 with benign breast tumor. The concentration of unoccupied high affinity estrogen receptors was measured in breast tumor biopsy specimens. Higher values of BMC were found in the total group of estrogen receptor-positive patients with breast carcinoma compared with estrogen receptor-negative patients, but not after dividing the patients into smaller groups according to age. No significant correlation could be seen between the amount of estrogen receptors and bone mineral content. In conclusion the present study could not support a relationship between the amount of estrogen receptors in breast cancer tissue and the amount of bone mineral and bone mass in women with breast tumors. PMID- 2994377 TI - Effect of the radioprotector WR 2721 on the response of metastatic Lewis lung carcinoma colonies to alkylating agents. AB - WR 2721 protected 'artificial' lung metastases of Lewis lung carcinoma against the cytotoxic effects of cyclophosphamide and melphalan. When mice were pretreated with WR 2721 30 min before exposure to the alkylating agents a significant increase in the number of lung metastases could be observed. This protection of micrometastases had a significant impact on survival in the case of cyclophosphamide treatment, but not in the case of melphalan treatment. The degree of protection at a standard dose of WR 2721 was dose dependent. PMID- 2994376 TI - DNA distribution, cytosol estrogen receptors and axillary nodes as prognostic predictors in breast carcinoma. AB - One hundred and fifty patients with breast carcinoma were examined to compare axillary node status, estrogen receptor level and cellular DNA content as prognostic indicators. Seventy-four per cent of the patients were postmenopausal and forty per cent had axillary node metastases. Estrogen receptor was measured by isoelectric focusing in polyacrylamide gel. DNA was measured in individual cell nuclei by means of Feulgen-acriflavine-sulphate stained imprints. Fifty-two per cent of the tumors had diploid and/or tetraploid DNA pattern, and the rest aneuploid pattern. Axillary node metastases, aneuploid DNA pattern and low level of estrogen receptor were related to recurrence. When introduced into Cox's proportional hazards procedure, axillary nodes and estrogen receptor level but not DNA pattern remained as significant predictors of recurrence. PMID- 2994378 TI - Relation between number of hemopoietic stem cells in newborn mice and their radiosensitivity. AB - Fractionation of a radiation exposure causes greater damage in newborn mice than a single application since it induces radioresistant foetal hemopoietic stem cells to differentiate prematurely to more radiosensitive adult ones. In the present investigation, it was studied whether other agents that give rise to extensive stem cell destruction also lead to such a change in radiosensitivity. Indeed, treatment with cytostatic drugs which reduces the number of spleen colony forming units (CFU-s) and total cells also diminished the D0 value of the surviving cells 3 days later. Adriamycin was most effective in causing damage to hemopoietic stem cells and in inducing micronuclei in bone marrow; it also had the most marked action on the D0 of the surviving stem cells. PMID- 2994379 TI - Protective effect of selenium against ionizing radiation-induced malformations in mice. AB - A single dose of sodium selenite (0.5 mg Se/kg b.w.) was injected intraperitoneally into mice on day 9 of pregnancy, either 30 min or 2 h before 1.75 Gy whole body irradiation. Administration of selenite 2 h but not 30 min before irradiation resulted in a significant decrease in the number of malformed foetuses (p less than 0.005). The decrease in foetal malformations occurred proportionally for all the major malformations observed, i.e. short or kinked tail, rib and vertebral malformations, coloboma and deformation of retina and iris. In addition, selenium pretreatment also protected against radiation-induced retardation of the sternum of the foetus. PMID- 2994380 TI - Orgotein as a radioprotector in normal tissues. Experiments on mouse skin and a murine adenocarcinoma. AB - The effects of Orgotein (a superoxide dismutase) on the radiation response of normal and malignant murine tissue in vivo were evaluated. The observations were made on the mouse hind leg bearing, in some cases, an adenocarcinoma. The following irradiation protocols were tested: 1) single dose (e.g., 35 and 53 Gy), 2) conventional fractionation (3 Gy/day, 5 days a week) and 3) multiple fractions per day (2 X 3 Gy/day, 3 h fractionation interval, 5 days a week). Radiation was either delivered alone or preceded by a subcutaneous injection of 20, 100 or 400 mg/kg Orgotein administered 1 or 2 h before the beginning of irradiation. No effects of Orgotein on tumor radiation response were detected. A protective effect on normal tissue was observed when radiation was delivered according to aggressive protocols and a relatively high dosage of Orgotein was administered. Furthermore, an accelerated trend of recovery of normal tissue was observed. PMID- 2994381 TI - Comparison between the immediate effects of photon and electron radiation on the ciliary activity of the trachea of the rabbit. An in vitro investigation. AB - The immediate effects of 6 MV roentgen rays and 4 MeV electron rays on the mucociliary activity of the trachea of the rabbit were studied. In spite of the same average LET for the two radiation qualities, there was a clear difference in their effects on the ciliary motility during irradiation. The increase of the mucociliary activity after an absorbed dose of 1.5 Gy roentgen rays reported earlier was confirmed by the present investigation when using 6 MV photons. Electron radiation increased the beat frequency to a corresponding degree already at an administered dose of 0.5 Gy. The differences found in the pattern of the mucociliary activity during irradiation are discussed. The results indicate that the conventional RBE values cannot be used for early physiologic effects of irradiation. PMID- 2994382 TI - Effect of reduced perfusion pressure and acetylcholine on local blood flow in tumors in the rat kidney. AB - The local blood flow (LBF) in a sarcoma and a hepatoma implanted into the rat kidney was examined during renal vasodilation, with and without increased total renal blood flow (RBF). Both tumor types had a control LBF of about 0.7 ml/min per g, i.e. 35 per cent or less as compared with the renal LBF, as simultaneously determined by the H2 gas clearance technique. The RBF averaged 4.6 ml/min per g as recorded electromagnetically. During about 20 mmHg reduction of renal arterial pressure the tumor LBF was maintained relative to the renal LBF, whether the RBF was autoregulated or not. Provided neoplastic vessels account for most of the vascular resistance to tumor blood flow, they must therefore possess autoregulatory ability. The tumor LBF was maintained when the RBF was maximally increased by continuous renal arterial infusion of acetylcholine. This may suggest that a neoplastic vessel vasodilatory response to acetylcholine prevented reduction of the tumor flow. PMID- 2994383 TI - Quantitative changes in the goblet cells of the rat small intestine after irradiation. AB - In order to evaluate the process of cell differentiation in the crypt of the rat small intestine the goblet cells were quantitatively studied in controls and after irradiation of the abdomen. The effect of a single dose, administered at 4 different times of the day, and multiple daily fractionations (MDF) of 6 and 12 Gy with different doses per fraction and different time intervals, were compared. Both regimens caused an initial increase of the goblet cells (both in absolute and relative terms), followed by a decrease and finally return to nearly control levels. After MDF the increase was more marked and the return to a normal level occurred earlier than after the single dose. PMID- 2994384 TI - Correction for the angular dependence of a detector in electron and photon beams. AB - Based on experimentally obtained angular response functions a simple analytic function has been found which can be accurately fitted to such data. Using this function formulas are derived for the variation of the detector response as the angular distribution of the electrons are changing with depth in a phantom both in electron and photon beams. The formulas show that for a flat liquid ionization chamber, a response variation of 2 to 4 per cent is normally obtained in the build-up region of electron and photon beams whereas the response is practically constant beyond dose maximum. PMID- 2994385 TI - Primary retroperitoneal sarcomas. A review of 36 cases. AB - A retrospective study of 36 patients with primary retroperitoneal sarcomas treated at The Norwegian Radium Hospital is presented. The median age at presentation was 57 years. The most common presenting symptom was abdominal pain. Leiomyosarcoma and liposarcoma were the most common histologic subtypes. The median survival in the whole series was 25 months. Patients with completely resected tumors had a longer median survival (59 months) than patients with incomplete resection (16 months) but the difference was not statistically significant. The malignancy grade seemed to be the most important prognostic factor and patients with low grade tumors had a significantly better outlook than those with high grade tumors. PMID- 2994386 TI - Whole brain irradiation for metastases from lung carcinoma. A clinical investigation. AB - Sixty-nine consecutive patients with brain metastases from lung carcinoma were randomly allocated to one of two radiation therapy schedules: 30 Gy/10 fractions/2 weeks or 50 Gy/20 fractions/4 weeks. The improvement rate for neurologic function was similar in the two groups. The median survival times for patients receiving the short course and the long course were 4 months and 3 months, respectively. The half-year survival rate was 42 per cent after the short course and 14 per cent after the long course (p less than 0.05). Performance status and lactate dehydrogenase were other factors which significantly influenced the half-year survival rate. PMID- 2994387 TI - Squamous cell carcinoma of the nasopharynx. An analysis of failure patterns after radiation therapy. AB - Seventy-seven patients with nasopharyngeal squamous cell carcinoma were treated with irradiation, with or without chemotherapy. Sixty-three (82%) developed a relapse in some part of the body; the first relapse appeared at primary, cervical and distant sites in 45 (71%), 30 (48%) and 12 (19%) of the 63 relapsing patients, respectively. In 22 of the 63 relapsing patients, the first relapse occurred simultaneously in two or more sites. Local recurrence-free survival was higher for the T1 + T2 group than for the T3 + T4 group (p less than 0.02). Cervical relapse-free survival was higher for N0 patients than for N+ patients (p less than 0.02). Distant metastases frequently developed as a component of the first relapse. Distant metastases were more common in N+ patients than in N0 patients. Forty-two patients received adjuvant chemotherapy. Although local recurrence-free survival tended to be higher in patients with chemotherapy than without chemotherapy, survival rates and relapse-free survival rates did not differ in the two groups. PMID- 2994388 TI - A therapeutic approach to early vocal cord carcinoma. AB - One hundred and twenty patients with early glottic carcinoma received radiation therapy at the University of Maryland Hospital from 1959 to 1977. The radiation dose ranged from 55 Gy in 4 weeks for small T1a lesions to 65 Gy in 61/2 weeks for T2 lesions. The local control rates by irradiation alone for stages T1a, T1b, and T2 were 92, 91 and 88 per cent, respectively, while 5-year determinate disease-free survival rates were 96 per cent for stage I disease and 88 per cent for stage II disease. Most of the local failures were salvaged by surgery, with a low complication rate. Regional metastases were uncommon, and occurred in 7 per cent in stage I and in 6 per cent in stage II disease. Factors increasing the risk of failures appeared to be bulky tumor, anterior commissure involvement and subglottic extension. PMID- 2994389 TI - Breast retraction assessment. Multiple variable analysis of factors responsible for cosmetic retraction in patients treated conservatively for stage I or II breast carcinoma. AB - A method for objective evaluation of cosmetic outcome of patients treated conservatively for breast carcinoma allowed the location of the nipples on two coordinates. The method was applied in 41 patients, 5 to 41 months following the completion of radiation therapy. Multiple variable analysis revealed that extensiveness of resection of the primary breast tumor was the major factor associated with breast retraction. The only other factor of significance was patient age at diagnosis. Neither the radiation therapy parameters, the use of adjuvant chemotherapy, nor any other analyzed factor was found to be associated with cosmetic breast retraction. PMID- 2994390 TI - Changes in lymphoid cell distribution after intraperitoneal administration of 32P colloids. AB - The effect of intraperitoneal administration of 32P colloids on the distribution of T lymphocyte subpopulations and monocytes was studied using monoclonal antibodies and a flow cytometry technique. Thirty-nine patients with ovarian carcinoma without residual tumor after primary operation were examined either before the administration of 260 to 370 MBq of 32P, 4 to 6 days after therapy, or 4 to 10 months after therapy. A significant reduction of circulating OKT4+ (T helper) cells occurred after therapy, and the reduction lasted throughout the observation period. Monocyte numbers were not significantly changed. It is concluded that intraperitoneal instillation of the 32P isotope may induce the same type of changes in circulating lymphoid cells as those seen after external field irradiation. PMID- 2994391 TI - Comparison between flow cytometry and single cell cytophotometry for DNA content analysis of the uterine cervix. AB - The DNA content of tumor cells in imprints and biopsies from 33 patients with invasive squamous cell carcinoma and 5 patients with adenocarcinoma of the uterine cervix were analyzed by flow cytometric and single cell cytophotometric methods. A correlation of r = 0.83 was found between the modal DNA values obtained by the two methods. Both methods discriminated between diploid and aneuploid tumors and gave comparable but not identical results. Single cell cytophotometric measurements were in this study more reliable than flow cytometric measurements in diploid tumors. Multiple stemlines, small aneuploid peaks and the so called S-phase rate could only be determined by the flow cytometric technique. Flow cytometry seems to be the method of choice in determination of the DNA content in cervical carcinoma cells and cytophotometric single cell measurements are of complementary value. PMID- 2994392 TI - Dynamics of irradiation injury to bone tissue. A vital microscopic investigation. AB - The dynamic changes after a single dose of 15, 25 or 40 Gy 60Co were followed in a titanium vital microscopic bone chamber which permitted observation of the same tissue compartment for over 2 years. The chamber consists of a hollow screw containing 2 glass rods 100 micron apart. The device was inserted into the cortex of the proximal tibial metaphysis of a rabbit. During a healing period of 4 to 6 weeks the space between the glass rods became filled with bone and vessels and in some cases fat. Once a steady state in bone remodelling had been achieved, the animals were irradiated. Vital microscopy was then performed at regular intervals. Mature bone was relatively radioresistant since remodelling continued at a normal rate. In contrast, immature woven bone remained unlamellarized and in some animals tended to increase in amount. The vascular architecture was largely unaltered, even after 40 Gy. Thrombosis or hemorrhage clearly attributable to irradiation was not noted. Initially, the number of fat cells was reduced but repopulation was later seen in several cases. PMID- 2994393 TI - Glucose analogues alter the response of CHO-KI cells to gamma irradiation. AB - Pretreatment of CHO-KI cells with 2-deoxy-D-glucose or L-glucose, two glucose analogues, reduces their survival if subsequently exposed to 60Co irradiation. The reduction in survival is constant irrespective of the time the cells are in contact with the analogues before irradiation and occurred even when cells were irradiated 6 hours after plating, suggesting that cell cycle effects are probably not involved. Interestingly, split-dose recovery was not affected to the expected degree, despite a reduction in the extrapolation number of the primary curve. It is suggested that interference with energy production from glucose is responsible for the reduced capacity of cells to survive the irradiation. PMID- 2994394 TI - Intermittent feeding as a factor enhancing hemopoietic stem cell proliferation and spleen colony formation in irradiated mice. AB - The influence of metabolic stimulation induced by a 3 weeks' adaptation of the animals to intermittent food intake on hemopoietic stem cells was investigated in mice. The methods used included transplantation of bone marrow to lethally irradiated recipients, assay of CFUs number, seeding efficiency, and incorporation of 125iododeoxyuridine into the DNA of spleen cells. A stimulatory effect of the metabolically influenced hemopoietic environment on the proliferative activity in stem cell compartments and on the recovery of hemopoietic organs was demonstrated. These stimulatory effects were most marked when the bone marrow of metabolically influenced donors was transplanted to similarly influenced recipients. PMID- 2994395 TI - Long term effects of ionizing radiation on mouse spermatogenesis. AB - The effects of acute or split dose exposure to radiation on murine stem cell spermatogonia were analysed. Flow cytometry was applied to estimate the percentages of haploid germ cells (round and elongated spermatids) up to 12 months after irradiation. The recovery in the number of haploid germ cells continued gradually during the period under observation. The intervals between the two equal doses in split dose exposures were 0, 4, 8, 24 and 48 hours. Split doses that were 24 h or 48 h apart had more harmful effects on spermatogenesis than split doses with 4 or 8 hours intervals or acute exposures. The repair capacity of the stem cell spermatogonia was remarkably high. PMID- 2994396 TI - Effect of repeat irradiation on the tracheal ciliary cell activity. AB - Rabbit tracheas were initially irradiated with 10 Gy in vitro. A second irradiation of 10 Gy was administered 2 hours later. The initial exposure caused a 25 per cent increase of the ciliary beat frequency during the irradiation--a response which was not found during the second irradiation. Between the exposures, the beat frequency dropped to 20 per cent below the reference level, followed by an 11 per cent overshooting before the second irradiation. Tracheas placed untreated in the experimental chamber for 2 hours and subsequently irradiated with 10 Gy showed a similar pattern of ciliary activity as induced by the initial radiation exposure in the first experiment. The storage during 2 hours did thus not affect the condition of the ciliae. The absence of a great increase of the beat frequency during a second irradiation period may be explained by damages caused by the initial exposure, morphologically seen first a few days later. PMID- 2994397 TI - RNA and protein synthesis of irradiated Ehrlich ascites tumour cells. I. In vivo investigations related to the cell cycle. AB - The effects of roentgen irradiation on the incorporation of 3H-uridine and 14C leucine into RNA and protein and the RNA and protein contents of in vivo growing Ehrlich ascites tumour cells were studied. The results were related to changes in the composition of cells in cell cycle and compared with the synthesis of RNA and protein in cell material from various parts of the cell cycle obtained by means of elutriator centrifuging. The incorporation expressed by the ratio between acid insoluble/acid soluble activity was unchanged for RNA during the observation period up to 24 hours after a dose of 5.0 Gy. The ratio for protein was markedly decreased between 4 and 24 hours. This decrease was partly due to a decrease of the pool size of leucine as studied by changing the amounts of 14C leucine used. From these studies, the existence of at least two pools, an expandable and a non expandable fixed pool can be concluded. There were no differences in the decrease of protein-synthesis between cells from the various parts of the cell cycle. The RNA and protein contents of the irradiated cells from various parts of the cell cycle corresponded to those of non-irradiated cells except for G1/early S-phase cells at 15 and 24 hours after irradiation. Possible reasons for this discrepancy are discussed. PMID- 2994398 TI - [Action of an enzyme complex on the reproduction of para-influenza virus type 1]. PMID- 2994399 TI - European Association of Neurosurgical Societies Fifth European lecture. Barcelona, February 24, 1984. Thresholds of ischaemia applied to aneurysm surgery. PMID- 2994400 TI - [Measurement of relative renal function with Tc 99m 2,3-dimercaptosuccinic acid: a useful method in clinical practice]. PMID- 2994401 TI - [Alpha-adrenergic innervation in the canine ureter]. PMID- 2994402 TI - Seroepidemiological studies on nasopharyngeal carcinoma in China. PMID- 2994403 TI - Retroviruses as chromosomal genes in the mouse. PMID- 2994404 TI - The design and properties of N-carboxyalkyldipeptide inhibitors of angiotensin converting enzyme. AB - Angiotensin-converting enzyme inhibitors promise to make important therapeutic contributions to the control of hypertension and congestive heart failure. The nonapeptide teprotide was the first of these inhibitors to be tested clinically. It was followed by orally active inhibitors, captopril in 1977 and enalapril in 1980. The latter is representative of a new design for the inhibition of metallopeptidases and is the subject of this review. The best of the N carboxyalkyldipeptide inhibitors inhibits angiotensin-converting enzyme with a Ki of 7.6 X 10(-11) M. This compound is the most potent competitive inhibitor of a metallopeptidase yet to have been reported. The basis of this high potency is beginning to be understood and in part is considered to involve precisely arranged multiple interactions within the enzyme active site. X-ray crystallography of a thermolysin-inhibitor complex has been achieved. Assuming that similar interactions within the active site of angiotensin-converting enzyme are mechanistically probable, the authors hypothesize the binding of enalaprilat to converting enzyme as shown in Figure 24. Such interactions are consistent with kinetic studies (Section V) with the understanding that binding to the enzyme is not sensitive to the inhibitor's state of NH protonation. The reason for this surprising conclusion has not been established. Perhaps counterbalancing factors are involved in the energetics of binding or there may be compensating adjustments made in the enzyme which permit NH protonated and nonprotonated inhibitor to bind equally well. Figure 24 also summarizes present understanding of the conformation of enalaprilat when bound to angiotensin-converting enzyme. From studies on conformationally defined analogs of enalaprilat, it seems likely that the Ala-Pro segment of enalaprilat binds in a conformation that is close to a minimum energy conformer. This situation no doubt contributes to the potency of enalaprilat, since little binding energy would be needed to induce conformational changes in this part-structure of enalaprilat when it is bound to the enzyme. The phenethyl group of enalaprilat is believed to be near the alpha-hydrogen of the L Ala residue in the enzyme-inhibitor complex. However, the synthesis of conformationally restricted analogs to establish this point has not yet been reached. The N-carboxyalkylpeptide design was developed from Wolfenden's collected product inhibitors of carboxypeptidase-A. Whether or not N carboxyalkyldipeptides should be classified as collected product or transition state inhibitors is unclear.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2994405 TI - Secretory functions of the mononuclear phagocyte. PMID- 2994406 TI - Dietary factors affecting lipoprotein metabolism. PMID- 2994407 TI - Inhibition of cholesterol absorption by natural products. PMID- 2994408 TI - The role of phosphorylation/dephosphorylation in the regulation of cholesterol 7 alpha-hydroxylase activity. PMID- 2994409 TI - Human NK resistant tumor cell lysis is effected by IL-2 activated killer cells. PMID- 2994410 TI - IgE-dependent platelet cytotoxicity against helminths. PMID- 2994411 TI - The lipoxygenase pathway in the human NK cell system. PMID- 2994412 TI - Mechanism of T-dependent cytotoxicity: role of papain-sensitive non class I MHC target molecules and expression of target antigen for cytotoxicity. PMID- 2994413 TI - DNA fragmentation in targets of CTL: an example of programmed cell death in the immune system. PMID- 2994414 TI - AIDS--an African disease? PMID- 2994415 TI - Oral viral leukoplakia--a new AIDS-associated condition. PMID- 2994416 TI - Thymosin in the staging and treatment of HTLV-III positive homosexuals and hemophiliacs with AIDS-related immune dysfunction. PMID- 2994417 TI - Etiology of AIDS: biological and biochemical characteristics of HTLV-III. AB - The newly identified human HTLV-III virus, the etiologic agent for AIDS, shares many of the biological and physicochemical properties common to a family of retroviruses named human T-cell leukemia (lymphotropic) viruses, or HTLV. Because of the similarities, and because of the uniform nomenclature for human T-cell leukemia (lymphotropic) viruses adopted at the first Cold Spring Harbor Meeting on HTLV (19, 79), this newly discovered virus associated with AIDS as HTLV-III was named HTLV-III. Other investigators making independent isolations of virus have suggested naming the virus lymphadenopathy virus or LAV (3, 16), immunodeficiency associated virus or IADV (48), AIDS-related virus (41). Immunological and nucleic acid comparison has now demonstrated that these viruses are, not surprisingly, very similar to HTLV-III (55, 63, 78). In view of the wide range of disease manifestations caused by the virus, and previous discussions concerning a uniform nomenclature for human T-lymphotropic retroviruses, it would seem ill-advised to restrict the name of this virus to one clinical manifestation of one disease. The frequent isolation of HTLV-III from patients with AIDS and ARC, the detection of antibodies specific for HTLV-III in nearly all patients with these diseases and in a high proportion of individuals at risk, and finally its effect on cells in vitro, leaves little doubt that HTLV-III is causatively involved in the development of these diseases. This etiologic association is further strengthened by the detection of HTLV-III infection in many instances where a direct cause-and-effect association can be made, e.g., hemophiliacs and children with AIDS, and blood from HTLV-III infected donors and the otherwise normal recipients of this blood who subsequently develop AIDS. PMID- 2994418 TI - Lymphadenopathy associated virus (L.A.V.): its association with AIDS or prodromes. PMID- 2994419 TI - Expressions of HTLV-III infection in a pediatric population. PMID- 2994420 TI - Role of Epstein-Barr virus in acquired immune deficiency syndrome. AB - We have reviewed the biologic characteristics, immune responses, and diverse array of diseases occurring from Epstein-Barr virus infections in immune deficient patients. We have summarized possible roles of the virus in the risk groups for AIDS. Data is convincing that EBV is responsible for some of the cases of lymphadenomegaly and Burkitt-like, non-Hodgkin's lymphomas in patients with pre-AIDS and AIDS. A hypothesis has been proposed wherein EBV and other stimulants of B and T cells allow productive infection by the retrovirus and spread of HTLV-III throughout the helper T cell populations. PMID- 2994421 TI - Longitudinal immunological studies on a cohort of initially symptom-free homosexual men in London with respect to HTLV-III serology. PMID- 2994422 TI - Lymphadenopathy: end-point prodrome? Update of a 36-month prospective study. PMID- 2994423 TI - The syndrome of persistent generalized lymphadenopathy: experience with 101 patients. PMID- 2994425 TI - Internalization of insulin: structures involved and significance. AB - The binding of insulin to its receptor is followed by aggregation of hormone receptor complexes and their internalization into the cell. Internalized hormone is concentrated in Golgi-enriched not lysosomal endocytotic structures which, in rat liver, contain lipoprotein particles and can be resolved by centrifugation techniques into three different entities. Recent work has shown that the bulk of endocytotic structures can be resolved from biochemically defined (i.e., galactosyltransferase-containing) Golgi elements. The endosomal apparatus or endosomes appear to function as a sorting center wherein internalized hormone receptor complexes are concentrated and dissociated prior to directing hormone to lysosomes and receptor back to the cell surface for reutilization. Endosomes are heterogeneous and different functions might be subserved by different endosomal structures. Since an insulin stimulable receptor kinase activity can be identified in endosomes certain aspects of insulin action might be initiated herein. PMID- 2994424 TI - Longitudinal assessment of persistent generalized lymphadenopathy (PGL) in homosexual men. PMID- 2994426 TI - Psychosomatic aspects of epilepsy. PMID- 2994427 TI - The effect of drugs upon the assessment of thyroid function. PMID- 2994428 TI - Screening for neutralizing antibodies against hog cholera- and/or bovine viral diarrhea virus in Danish pigs. PMID- 2994430 TI - Are eosinophils the cause of endomyocardial fibrosis in the tropics? PMID- 2994429 TI - Effect of reductones on cyclic 3', 5'-adenosine monophosphate phosphodiesterase. AB - The effect of some reductones such as ascorbic acid (AsA), triose reductone (TR), epinephrine (Ep) and their derivatives on cyclic 3', 5'-adenosine monophosphate phosphodiesterase (cAMP PDE) was studied in the presence or absence of Cu2+. AsA, TR, Ep and the reductones related to them inhibited cAMP PDE activity. Among the reductones, TR showed the highest inhibition. AsA, 5-methyl-3,4-dihydroxytetrone, pyrocatechol, p-hydroxyquinone and resorcinol had a relatively high inhibiting activity. The type of inhibition of AsA, TR and Ep was uncompetitive, competitive and noncompetitive, respectively. Cu2+ enhanced the inhibitory action of the reductones markedly and altered the type of inhibition of the reductones. PMID- 2994431 TI - Sequential hormone measurements after menstrual regulation in normal Nigerian women. AB - The objective of this study was to examine the dynamics of the pituitary hormones (LH, FSH and Prolactin) and Progesterone in normal adult Nigerian women during the first cycle after a menstrual regulation for delayed periods. Hence, eleven healthy volunteers aged between 19 and 35 years were recruited. All the subjects had consulted the Family Planning Clinic for menstrual regulation. Venepuncture was performed immediately before and every other day after the procedure until the onset of the first menstrual period. Serum from the samples were stored at 20 degrees C and later assayed for LH, FSH, Prolactin and Progesterone. The results showed that nine patients (82%) had a demonstrable LH peak whilst seven patients (64%) had a luteal phase serum progesterone in excess of 6 nmol/l. These criteria were used for the diagnosis of ovulation. The combination of two or three indices i.e. LH + FSH peak (64%) LH + Progesterone (64%) LH + FSH + Progesterone (55%) did not seem to confer any special advantages in terms of the diagnosis of ovulation. These results are considered as further evidence for the need to provide contraception in the first cycle after an abortion in women who do not wish to get pregnant immediately. PMID- 2994432 TI - The library in medical education. AB - The process of training medical students must involve the acquisition of knowledge directly from their teachers and from learning resources. The organization and accessibility of materials in a medical library make it an invaluable instructional tool within the Learning Resources System. Most medical schools have omitted the incorporation of library training from their curricula. To correct this anomaly, it is being suggested that a programme of library training be commenced by medical school libraries. Such a programme must comprise instructions in the use of the library, in bibliographic methods and "hands-on' exercises. A practical design is suggested and a method of evaluating the programme should be designed to provide information about its usefulness to the students before it is finally incorporated into the curriculum. PMID- 2994433 TI - Comparative bioavailability studies on brands of diazepam tablets. AB - The chemical and biological equivalence of two brands of diazepam tablets marketed in Nigeria were compared to that of Valium (Roche) tablets. The tablets used were Relanium (Polfa) and Tropium (Biode). Bioequivalence studies of the brands were carried out on nine healthy volunteers by monitoring the cumulative diazepam excreted as oxazepam (both free and conjugated) in urine over 48 h. The bioequivalence of Relanium was found not to be significantly different from that of Valium while that of Tropium was significantly different from that of Valium (P less than 0.05). The results of the investigation showed that the drug products were all chemical equivalents but not biological equivalents. PMID- 2994434 TI - In search of the aetiology of deafness in Nigeria--the probable role of rubella infection. AB - The present study attempts to establish a relationship between maternal rubella and congenital deafness in Nigeria. For the purpose of the survey, the immunological status with respect to rubella infection of 179 healthy but deaf school children (study group) and 248 school children with normal hearing (control group) was determined. The serological technique employed was the haemagglutination inhibition (HI) test. The percentage of immunized persons ranged from 48.6 to 74.2% in the study group and 55.3 to 68.3% in the control group. Furthermore, the HI antibody titres for the study group were not significantly different from those of the control group although both groups were found to exhibit low HI antibody titres. The presence of low titres of HI antibodies in both groups and the absence of any significant correlation between the state of hearing and HI antibody levels seem to indicate that intrauterine rubella is not an important cause of congenital deafness in Nigeria. PMID- 2994435 TI - Outpatient interval female sterilization at the University College Hospital, Ibadan, Nigeria. AB - Between September 1975 and April 1981, 258 patients were sterilized at the University College Hospital (UCH), Ibadan, using minilaparotomy and laparoscopy. Selected sociodemographic data as well as the technical and surgical difficulties encountered have been reviewed in this article. The mean age at sterilization was 38.3 years whilst the mean parity was 7.2. In all, there were 215 minilaparotomy sterilizations and forty-three laparoscopic sterilizations. Surgical difficulties were reported for 12.1% of minilaparotomy and 11.6% for laparoscopic procedures. The most frequently reported difficulties were obesity and pelvic adhesions. The failure rates were 1.4% for minilaparotomy and 2.3% for laparoscopy. PMID- 2994436 TI - Clinical trial of cefoxitin (mefoxin) in parenteral therapy of septicemia (postabortal and postpartum) in Ibadan. AB - Cases of post-abortal sepsis are admitted every day into the University College Hospital (UCH), Ibadan, Nigeria. This derives from the high incidence of induced (illegal) abortions in this environment. The infections are usually caused by mixed bacterial flora, often resistant to the common antibiotics because of the indiscriminate use of these drugs. Any new drug that can be effective in the treatment of these resistant cases will be welcome. The efficacy of Cefoxitin in the treatment of twenty-five cases of postabortal sepsis was therefore compared with the efficiency of other antibiotics in the management of sixty other cases. Response to Cefoxitin was prompt. Temperatures settled within 96 h and no case of pelvic abscess resulted. It was concluded that Cefoxitin could well be a safe and effective alternative antibiotic to replace the common antibiotics to which many hospital organisms have developed resistance. PMID- 2994437 TI - Prospective studies on Hodgkin's disease in Ibadan--a preliminary report. AB - A preliminary report of a prospective study of Hodgkin's disease (HD) is provided, based on clinical and laboratory findings on 21 patients seen between July 1978 and December 1979 at the University College Hospital (UCH), Ibadan. Staging procedure was minimized to relatively simple surgical procedures like lymphangiography and percutaneous biopsy. Staging laparotomy was performed only in one case. Female patients were significantly older than males (P less than 0.05). Patients with lymphadenopathy were significantly older than those without (P less than 0.05), while those with systemic symptoms were significantly younger than those without. Eighteen (86%) of the patients presented with stage IV disease while 12 (57%) had the unfavourable histologic "mixed cellularity' or "lymphocyte depleted' variants. Systemic symptoms were present in 16 (76%) of patients. Fifty-three % of adequately treated patients showed poor response to chemotherapy. The prognosis of HD in Ibadan is on the whole unfavourable. A delineation of the prognostic factors is indicated. PMID- 2994438 TI - Investigations of various extracts of Morinda lucida for antimalarial actions on Plasmodium berghei berghei in mice. AB - Morinda lucida extracts, the stem bark, the root bark and the leaves were screened for antimalarial activity in a "4-day schizontocidal test' against a chloroquine-sensitive strain of P. berghei berghei in mice. Each extract was administered as a single daily dose on days 0, 1, 2 and 3 to mice that had received an intraperitoneal inoculum of 1 X 10(7) infected erythrocytes. Each extract produced a degree of suppression of parasitaemia. The most promising result was obtained with chromatographic fractions of the stem bark extracts, the highest dose producing 96.4% suppression of parasitaemia. PMID- 2994439 TI - Effect of Azadirachta indica on Plasmodium berghei berghei in mice. AB - Azadirachta indica leaf extract has been investigated for antimalarial activity against drug sensitive strain of P. berghei berghei in mice. On administering the extract subcutaneously to infected mice in the "4-day schizontocidal test' 41.2% suppression of parasitaemia was observed. A similar observation was made when the extract was injected for 3 days before the animals were infected with the parasites, 21.7% chemosuppression was obtained. When treatment commenced after the infection had already established, there was no demonstrable suppression of parasitaemia. PMID- 2994440 TI - Lack of schizontocidal activity of three herbal decoctions on Plasmodium berghei berghei in mice. AB - This work reports the effects of three herbal decoctions on Plasmodium berghei berghei in mice. The schizontocidal activity of each decoction was determined on an established infection using chloroquine as a standard and distilled water as control. Also a repository study of the decoction was carried out in another group of mice. The three decoctions neither reduced parasitaemia nor prolonged the life of infected mice that received them. PMID- 2994441 TI - Screening of Morinda lucida leaf extract for antimalarial action on Plasmodium berghei berghei in mice. AB - The leaf extract of Morinda lucida collected in August was administered subcutaneously to albino Swiss mice infected with P. berghei berghei. The schizontocidal activity on early infection was assessed by administering chloroquine (standard) distilled water or Morinda lucida as single daily dose on day 0-3 to infected mice. On day 4 the degree of parasitaemia and percentage was determined in relation to control. Its schizontocidal activity was also observed on an established infection by administering the drugs 72 h after infecting the mice and the degree of parasitaemia was determined daily. The repository action of pyrimethamine was also compared with Morinda lucida. On the early infection, the chloroquine equivalent of Morinda lucida was found to be 1.0 mg/kg. In established infection a daily increase in parasitaemia was observed in control group while the animals that received chloroquine (5 mg/kg) and 1/6 dilution of the stock of Morinda lucida extract showed a sharp fall in parasitaemia from the second day of treatment. For the prophylactic test, 1.2 mg/kg of pyrimethamine and 1/6 dilution of stock of extract produced 80.5% and 70% chemosuppression respectively. It is interesting to note that Morinda lucida leaves extract appears to have schizontocidal and repository effects in mice infected with P. berghei berghei. PMID- 2994442 TI - Parenteral premedication with lorazepam--a dose/response study. AB - The dose/response characteristics of lorazepam when administered parenterally as a premedicant drug were studied. The parameters studied included its' sedative and amnesic effects as well as effects on the cardiovascular and respiratory systems. Intramuscular and intravenous routes using doses 2 mg, 3 mg and 4 mg were compared. The findings suggest that lorazepam's sedative and amnesic effects were more pronounced with the intravenous route, and that in both routes the incidence and degree of sedation and of amnesia were dose dependant. There were minimal changes in arterial blood pressure, pulse rate or quality of respiration. We recommend that due to local logistic factors, the intravenous route might be more suitable, affording a more rapid onset of sedation before induction of anaesthesia, provided that close nursing observation is available. Furthermore, lorazepam 2 mg intravenously is just as effective as the larger doses with less incidence of nausea and vomiting associated with the larger doses. PMID- 2994443 TI - Insulin resistant diabetes mellitus associated with acanthosis nigricans and systemic lupus erythematosus in a Nigerian male. AB - A case is reported of diabetes mellitus associated with insulin resistance, acanthosis nigricans and systemic lupus erythematosus. This will be the first such case described in a Nigerian and most likely the first case in an African, of this recently characterized syndrome. An awareness about this syndrome by physicians in this environment should lead to a prompt referral to where there are adequate facilities for an early diagnosis which could prove to be life saving. PMID- 2994444 TI - Cytopathology and short-term culture of malignant tumours in Ibadan. AB - This report is a retrospective review of early studies (1965-70) on the cytology and short-term tissue culture of fresh specimens from 1643 patients under investigation for cancer at the University College Hospital, Ibadan. A total of 580 specimens were positive for malignant cells. The technical procedures are described in some detail and were found to be particularly useful as aid to laboratory diagnosis of 310 childhood tumours. The relative frequency of Burkitt's lymphoma, retinoblastoma, neuroblastoma and Wilm's tumour in the series was 20:1:1:0.5 respectively. Adoption of the technique as part of routine diagnostic service of teaching hospitals of developing countries is recommended. PMID- 2994445 TI - Blood coagulation activities of the root extracts of Fagara xanthoxyloides plant. AB - The coagulant properties of root bark and root wood extracts of Fagara xanthoxyloides lam plant are reported. Results of an earlier report which showed that the aqueous extract of the root shortened both the PT and PTT of normal and FVIIIC-deficient plasma are confirmed as well as the absence of many such effects on FIXC-deficient plasma. Root bark manifested twice as much potency as an equal concentration of root wood. The activity could still be demonstrated in the residue of root bark after the lyophilized aqueous extract had been successively extracted with methanol and hexane. It is suggested that these results may have clinical implications. PMID- 2994446 TI - Neoplastic diseases of the haemopoietic system in Ibadan: preliminary report of a prospective study. AB - The clinical and epidemiological features of haemopoietic malignancies in Ibadan have been evaluated in this preliminary analysis on findings on 113 patients seen at the University College Hospital, Ibadan, Nigeria, from July 1978 to June 1981. Twenty-seven patients had acute myelogenous (AML), twenty-two acute lymphoblastic (ALL), thirty-one chronic myelocytic (AML), thirty-one chronic lymphocytic leukaemia (CLL), two had polycythaemia rubra vera (PRV) and one myelofibrosis (MF). Incidence rates (IR) of 0.9 X 10(-5) and 1.9 X 10(-5) were estimated for acute leukemia (AL) and all leukemia subtypes respectively. Chloroma occurred frequently in association with AML especially in childhood, and CLL in elderly patients (greater than 50 years) and ALL appeared to manifest unusually aggressive features in spite of apparently adequate chemotherapy. Some of these clinical and epidemiological features suggest deviant biology of some haemopoietic malignancies in Ibadan. PMID- 2994447 TI - Self-administered Entonox (50% nitrous oxide in oxygen) in labour: report of the experience in Ibadan. AB - The effectiveness, safety and acceptability of self-administered Entonox (50% nitrous oxide in oxygen) in 150 Nigerian women during labour was studied. 86.7% of those who received Entonox alone reported satisfactory pain relief while analgesia was also satisfactory in all those who received Entonox plus intramuscular analgesic. Severe drowsiness occurred in two patients and the method was acceptable to 90% of the mothers in the study. To prevent exhaustion of the mothers and marked drowsiness, intramuscular analgesic should be used early in labour followed by Entonox in the second half of the labour. PMID- 2994449 TI - Massive calcium pyrophosphate crystal deposition at the craniovertebral junction. PMID- 2994448 TI - Plasma zinc and copper concentrations in pregnant Nigerian women and newborn. AB - Plasma zinc and copper concentrations were determined in a group of pregnant Nigerian women at various stages of gestation. The levels of these trace elements were also determined in paired maternal and cord blood at delivery. The results showed that the plasma levels rose with increase in gestation from 94.8 (+/- 20.6) micrograms/100 ml in the first trimester to 161.6 (+/- 22.4) micrograms/100 ml at term. This rise is statistically very significant (P less than 0.001). Conversely, there was a fall in the plasma zinc concentrations with increase in gestation--from 77.2 (+/- 14.8) micrograms/100 ml in the first trimester to 65.8 (+/- 15.3) micrograms/100 ml at term. However, this fall is not significant at the 5% level. The mean concentration of copper in the maternal blood at delivery was about three times the mean concentration of 54.6 (+/- 20.8) micrograms/100 ml found in the corresponding paired cord blood. There were no statistically significant differences in zinc concentrations found in maternal and paired cord blood at delivery. Statistically significant correlations were found between copper zinc concentrations and fetal birth weight. PMID- 2994450 TI - Postoperative fractures of lumbar articular facets: occult cause of radiculopathy. AB - Fracture of the inferior lumbar articular facets after laminectomy with facetectomy is a relatively common but unrecognized cause of radiculopathy. Although not all patients may be symptomatic from the fractures, some may have radiculopathy or back pain caused by displacement of the fracture fragment. In a series of 400 postoperative spinal computed tomographic (CT) scans, 25 patients were found who had fractures through the base of the inferior facets. Axial scans revealed no abnormality other than slight widening of the joint on the affected side. Sagittal views demonstrated a lucent defect similar to a pars interarticularis fracture, whereas coronal views showed the fracture at a different location in the base of the facet. Typically patients become symptomatic after a period of postsurgical well-being. A new pain pattern, local tenderness, pain on unusual movements, and relief with recumbency help suggest facet fracture versus recurrent disk herniation. PMID- 2994451 TI - Effect of enalapril on ventricular arrhythmias in congestive heart failure. AB - Twenty-four-hour Holter electrocardiographic recordings were used to measure the effects of a converting-enzyme inhibitor, enalapril, given for 12 weeks, on the frequency of cardiac arrhythmias in 10 patients with congestive heart failure (New York Heart Association functional class II to III) receiving maintenance therapy with digoxin and furosemide. Nine patients were given placebo, and both study groups were conducted in a double-blind, parallel manner. The placebo group had no change in the frequency of arrhythmias, whereas enalapril-treated patients showed a significant decrease in the frequency of premature ventricular complexes, ventricular couplets and ventricular tachycardia. A minor, nonsignificant reduction in atrial premature complexes was seen in patients who received enalapril. Compared with placebo patients, those who received enalapril had an increase in plasma potassium levels of 0.33 mmol/liter, a decrease in plasma digoxin, and decreases in pulmonary artery wedge, mean pulmonary artery and right atrial pressures. However, none of these indexes were correlated with the concomitant decline in cardiac arrhythmias. It is concluded that enalapril reduces the frequency of ventricular arrhythmias in congestive heart failure, although the underlying mechanisms are not known. PMID- 2994452 TI - Food physical factors have different metabolic effects in nondiabetics and diabetics. AB - Physical properties of food may account for differences in glycemic and other metabolic responses to food with similar amounts of carbohydrate, fat and protein. Blending of cooked beans made no difference to plasma glucose, insulin, or GIP (gastric inhibitory polypeptide) responses in nondiabetics, NIDD (noninsulin-dependent diabetics), and IDD (insulin-dependent diabetics). The cooked blended beans gave a greater plasma glucose response and a lesser hormonal response than a cooked flummery (containing cornstarch, protein and fat) in nondiabetics. In NIDD and IDD, however, the reverse applied for plasma glucose. In nondiabetics, cooked flummery gave a lesser glycemic response at some time points than uncooked flummery. In NIDD the opposite occurred. Cooking led to no significant change in insulin response in nondiabetics, but to a lesser insulin response in NIDD. The effect of some physical properties of food on diabetic control cannot be inferred from findings in nondiabetics. PMID- 2994453 TI - Comparison of dietary histories and seven-day food records in a nutritional assessment of older adults. AB - Dietary histories and seven-day food records were obtained for 54 apparently healthy older adults. The two dietary methods correlated for most nutrients, but mean differences were significant for several nutrients. Intakes below recommended levels occurred most frequently for energy, calcium, and zinc. Biochemical evidence of thiamin and riboflavin deficiency was unexpectedly frequent. Using food records, dietary iron correlated with serum ferritin. Using dietary histories, dietary protein correlated with serum albumin, and dietary zinc correlated with plasma zinc. Using either dietary method, plasma ascorbate was associated positively with both dietary ascorbate and ascorbate supplements, and negatively with cigarette smoking. Use of thiamin- or folate-containing supplements was associated with improved biochemical status for the respective vitamin. Though neither dietary histories nor food records give precise intake data for individuals, either method may be useful for epidemiologic studies with appropriate sample sizes. PMID- 2994454 TI - Transcobalamin II: a marker for macrophage/histiocyte proliferation. AB - Increased blood levels of (apo-)transcobalamin II have been observed in several clinical conditions, but persistent inability to find a common denominator for this plasma protein aberration has hampered its introduction as a clinically useful laboratory parameter. Because several observations suggested a relationship between reticuloendothelial cell activity and transcobalamin II, the finding of an extreme transcobalamin II elevation in a patient with malignant histiocytosis was taken seriously. Of 14 consecutive patients with proliferative histiocytosis (8 malignant, 6 reactive), all revealed marked to extreme elevations of transcobalamin II. Macrophage/histiocyte origin of this protein is supported by a close parallelism to increased serum angiotensin-converting enzyme activity. Comparative pre- and postoperative measurements of transcobalamin II and angiotensin-converting enzyme in four patients with histiocytic proliferation who underwent splenectomy, an intervention that led to immediate reduction of the macrophage/histiocyte cell pool, revealed a parallel and impressive drop of both parameters, further corroborating the histiocytic origin of transcobalamin II. It is suggested that transcobalamin II determination provides useful information on activity and size of the macrophage/histiocyte system, and supplements measurements of the traditional acute phase reactants (e.g., C-reactive protein, red blood cell sedimentation rate). PMID- 2994455 TI - AIDS-like immunologic alterations in clinically unaffected drug users. AB - Peripheral blood lymphocyte subpopulations (PBLS) and HLA-DR phenotype have been evaluated in 30 IV drug users who were not affected by acquired immune deficiency syndrome (AIDS). A strongly significant reduction in helper/suppressor ratio was found in these subjects as compared to the control population. When the group under study was subdivided according to the presence or absence of signs of lymphadenopathy syndrome (LAS), the apparently unaffected individuals still had significant modifications in PBLS when compared with controls. These modifications were more marked in subjects within the LAS+ subgroup, who also showed a greater DR5 frequency than those belonging to the LAS- subgroup. The authors concluded that AIDS-like laboratory alterations are present in clinically unaffected IV drug users; the possible role of DR5 in conditioning different individual susceptibility is considered. PMID- 2994456 TI - Hepatitis profile interpretation by microcomputer. AB - A simple BASIC computer program for interpreting two serologic markers of hepatitis A virus infection and five serologic markers of hepatitis B virus infection is described. The program is based on a two-dimensional matrix (table) containing all possible combinations of positive and negative test results with their associated interpretations. Clinically relevant reports are consistent, reliable, and legible. Sample reports are included. PMID- 2994457 TI - Melanocytic neuroectodermal tumor of the foot. Report of a case with multicentric origin. AB - This report describes a case of a two-year-old boy who had a tumor of the right fifth metatarsal. Biopsy revealed an undifferentiated, "small, round cell tumor" initially diagnosed as Ewing's sarcoma. He was treated with intensive chemotherapy, followed by amputation of the right foot when no response was observed. The histology of the amputated specimen was typical of melanocytic neuroectodermal tumor, a diagnosis confirmed by electron microscopy. Apart from documenting origin in a heretofore unrecorded site, this case illustrates the difficulties that arise in making a diagnosis and estimating prognosis of melanocytic neuroectodermal tumor, and provides evidence in favor of the multicentric origin of this uncommon disease. PMID- 2994458 TI - Laryngeal blastoma. A light and electron microscopic study of a novel entity analogous to pulmonary blastoma. AB - A neoplasm morphologically analogous to pulmonary blastoma, but arising in the pyriform fossa of the larynx, is described and illustrated. The patient, a 65 year-old white man, was treated with laryngectomy and is alive and free of disease 13 months later. Grossly polypoid on a broad stalk, the neoplasm consisted of a mixture of blastematous, mesenchymal, and epithelial elements. Ultrastructural examination showed that the spindle and stellate cells were truly mesenchymal. The name "laryngeal blastoma" is proposed for this novel entity. PMID- 2994459 TI - Lymphadenopathy morphologically consistent with Hodgkin's disease associated with Epstein-Barr virus infection. AB - This case report describes a case of chronic Epstein-Barr virus infection resulting in lymphadenopathy morphologically indistinguishable from Hodgkin's disease. Morphologic, immunologic, and virologic studies could be performed on three consecutive lymph nodes. For the first time in the literature, Epstein-Barr Nuclear Antigen (EBNA) could be demonstrated in the nuclei of Sternberg-Reed cells. PMID- 2994460 TI - Cellular inclusions: problems in diagnosis using autoanalyzers. PMID- 2994461 TI - The cefoperazone-sulbactam combination. In vitro qualities including beta lactamase stability, antimicrobial activity, and interpretive criteria for disk diffusion tests. AB - Three concentrations of the penicillanic acid sulfone, sulbactam were tested in combination with cefoperazone against 632 recent clinical bacterial isolates. Cefoperazone was effective alone (less than or equal to 16 micrograms/mL) against 95% of Enterobacteriaceae and combined with 4 micrograms/mL sulbactam inhibited 99.5% of strains. This coverage of enteric bacilli was superior to timentin (99.1%), ceftazidime (98.2%), and tobramycin (90.9%). The minimum inhibitory concentrations (MICs) of cefoperazone-susceptible strains also were markedly decreased by sulbactam (overall MIC90s, 8.0 micrograms/mL for cefoperazone and 1.0 microgram/mL for cefoperazone and 4.0 micrograms/mL for sulbactam). Sulbactam also expanded the spectrum of cefoperazone against Acinetobacter species, some rare Pseudomonas species, and Bacteroides fragilis group species. Sulbactam had direct antimicrobial activity against the acinetobacters and Pseudomonas acidovorans, but the increased activity of cefoperazone-sulbactam against some other Pseudomonas species and anaerobes was attributed to beta-lactamase inhibition. The cefoperazone MICs against beta-lactamase producing Staphylococcus species also were lowered to the level of enzyme-deficient strains. Cefoperazone bactericidal activity was improved by 4.0 micrograms/mL sulbactam, and no antagonism was observed. beta-lactamase hydrolysis studies confirmed a slow hydrolysis of cefoperazone only by TEM beta-lactamases and a high-grade resistance to enzyme breakdown by sulbactam. Differential beta-lactamase affinity studies for cefoperazone and sulbactam showed potential efficacy and applications to plasmid-mediated TEM and OXA enzymes and only marginal effective sulbactam inhibition of Pseudomonas and Klebsiella species enzymes. Disk diffusion studies on 556 strains confirmed the applicability of the cefoperazone 75-micrograms disk to testing routine isolates other than enterococci and methicillin-resistant Staphylococcus aureus. The addition of 4.0 micrograms sulbactam/mL in a fixed concentration to dilution test systems and 15 micrograms sulbactam to the 75 micrograms cefoperazone disk were recommended for in vitro tests. Susceptibility and resistant interpretive criteria for the disk and dilution tests can be applied with confidence. Only 0.4% false-susceptibility errors and a 97.5% absolute interpretive agreement were achieved using the 75 micrograms cefoperazone/15 micrograms sulbactam disk. PMID- 2994462 TI - Epstein-Barr virus infection in a child with acquired immunodeficiency syndrome. AB - A 3-year-old girl, born to an intravenous-drug-dependent mother, had protracted diarrhea, failure to thrive, generalized lymphadenopathy, and recurrent fevers during the first six months of life. At 7 months of age, the Epstein-Barr virus (EBV) genome was detected in her saliva by DNA dot-blot hybridization using a cloned EBV probe. Spontaneous EBV+ lymphoblastoid cell lines had repeatedly developed from her peripheral blood lymphocytes over the subsequent 2 1/2 years. At 11 months of age, persistent tachypnea and a diffuse pulmonary infiltrate developed. Lung biopsy demonstrated a florid, peribronchiolar lymphocytic infiltrate and the EBV genome was identified in the lung tissue. Serum anti-EBV antibodies remained undetectable until 14 months of age. She had a T4+/T8+ ratio of less than 0.8 and serum antibody to human T-cell lymphotropic virus type III. The delayed seroresponse of this patient to symptomatic EBV infection suggests that reliance on EBV serology to diagnose EBV infection in immunocompromised hosts may be inappropriate, and other methods such as DNA probes should be used. PMID- 2994463 TI - Antibody response of infants to two doses of inactivated poliovirus vaccine of enhanced potency. AB - The conventional formulation of injectable poliovirus vaccine (inactivated) contains 20, 2, and 4 D-antigen units of types 1, 2, and 3 polioviruses. Primary immunization requires three doses given at intervals of at least four weeks. A new formulation with 40, 8, and 32 D-antigen units of the three poliovirus types has been prepared to reduce primary immunization to two doses. We evaluated the immunogenic efficacy of this new formulation supplied to us as a liquid vaccine containing diphtheria-pertussis-tetanus vaccines and inactivated poliovirus vaccine. Two doses were administered four weeks apart to 100 infants and eight weeks apart to 114 infants. Antibody titers were determined against the three types of polioviruses before and after immunization. The effects of age, presence of maternal antibody, and interval between doses of the frequency and titers of antibody response were assessed. Irrespective of age or interval between doses, the seroconversion rates to types 1 and 3 antigens were 90% to 100%. To type 2 antigen the rate was below 84% in the 6- to 7-week-old infants, 88% to 95% in 8- to 12-week-old infants, and 90% to 100% in 13- to 45-week-old infants. The seroconversion rates and geometric mean titers of antibody were lower in those infants with maternal antibody than in those without maternal antibody at the time of receiving the first dose. The best results were in infants 8 weeks of age or older, in whom the two doses were given eight weeks apart. We recommend this schedule. PMID- 2994464 TI - Association between hepatitis B surface antigen and hepatocellular carcinoma. PMID- 2994465 TI - Coronaviruslike particles in human gastrointestinal disease. Epidemiologic, clinical, and laboratory observations. AB - Coronaviruslike particles (CVLPs) were visualized by direct electron microscopy (EM) of diarrheal stools in 49 of 126 infants and children between 1 month and 12 years of age during a three-year observation period. The clinical and epidemiologic features of these patients were analyzed and compared with patients with diarrhea who were shedding rotaviruses, or whose stools were negative for enteric viruses by EM. Seasonal and age distributions for CVLP shedding were similar to those for rotaviruses (in most cases less than 1 year of age; peak months were September through January), as were the symptoms and median durations of illness. Prospective studies of three subsequent patients suggest that the duration of shedding in acute illness is five to at least 25 days. Multiple attempts to cultivate the CVLPs were unsuccessful. In addition, partial purification of CVLPs from stool specimens was performed, and immunologic analysis by immunoelectron microscopy and radial immunodiffusion showed no antigenic relatedness to prototype human (OC43 and 229E) or animal (bovine and canine) coronaviruses. These findings suggest that CVLPs may be an important cause of acute gastrointestinal illness in infancy, and may represent a virus antigenically unrelated to known human and animal coronaviruses. PMID- 2994466 TI - Granular cell tumors of the esophagus: a report of two cases and review of the literature. AB - Two patients with granular cell tumors of the esophagus are presented. Both were men, 54 and 51 years old, respectively, and without tumor-related complaints. Diagnosis was made by esophagogram and endoscopic biopsy. The two patients were treated with a left thoracotomy and local excision of the tumors. A review of the world literature was carried out and data of general interest were collected. The symptoms and management of esophageal granular cell tumors, as well as histogenesis, are discussed. PMID- 2994467 TI - Hematochezia and the false negative Meckel's scan: a continued need for barium studies. AB - A patient with hematochezia and a false negative Meckel's scan is presented. A Meckel's diverticulum was subsequently diagnosed on barium small bowel follow through. Meckel's diverticulum is discussed with emphasis on the relationship of barium and radionuclide studies. PMID- 2994468 TI - Malignant fibrous histiocytoma of the liver--a case report. AB - A case of primary malignant fibrous histiocytoma of the liver studied by light and electron microscopy and confirmed at autopsy is presented. Malignant fibrous histiocytoma, the most common adult soft tissue sarcoma, has been reported in most organs but to date has not been described as a primary liver tumor. PMID- 2994469 TI - Thorotrast-associated liver cancer. AB - Thorotrast-related liver cancer is rare. A case of such cancer is presented. Thorotrast had been used intravenously in 1947, 38 years preceding the detection of cancer. A brief history of the use of colloidal solution of thorium dioxide (thorotrast) as a contrast material is discussed, along with a review of literature. Potential hazards of thorotrast in its carriers and a close follow-up are emphasized. PMID- 2994470 TI - Human T-cell leukemia virus (HTLV-I) p24 antibody in New York City blood product recipients. AB - Human T-cell leukemia virus (HTLV-I) is known to be associated with certain hematologic malignancies, and a related virus, HTLV-III/LAV, might be the cause of AIDS. Some persons with AIDS have had evidence of HTLV-I infection. Unrelated to these findings, it has been suggested that HTLV-I is transmitted via blood products. We therefore evaluated the serologic status to the HTLV-I core antigen p24 of 48 persons with hemophilia (Hem A) receiving factor concentrate therapy (a group at risk for AIDS), 49 persons with beta-thalassemia major (Thal) receiving frozen packed red blood cells therapy (FPRC), 26 patients with sickle cell anemia (SCA) receiving FPRC, and 18 persons not receiving any blood products. All participants were clinically well; only one had a risk factor other than hemophilia for AIDS, and all were from New York City, an area with a high incidence of AIDS. No Hem A or nontransfused persons had serum antibody to HTLV core p24 antigen; three with Thal and one with SCA were antibody-positive. These results were confirmed by both radioimmunoprecipitation and Western blot techniques. Positive serology did not correlate with any immune findings or quantity of blood products used. These data support that HTLV-I is preferentially transmitted through cellular blood products and that it is an infection for which cellular blood product recipients in at least some areas of the United States are at risk. Concentrate products appear free of transmission risk relative to cellular blood products, but we cannot be certain that this safety is absolute. The public health implications of blood product transmission of HTLV-I merit active, long-term investigation. PMID- 2994471 TI - Treatment strategies for patients with non-insulin-dependent diabetes mellitus. AB - Strategies for the treatment of patients with non-insulin-dependent diabetes mellitus are discussed. In order to achieve treatment goals, diet and exercise remain important components of an overall treatment program that may include sulfonylurea drugs, especially in cases where patients are of normal weight or only slightly obese, have had the disease less than five years, and are taking little or no insulin. Failure to control blood sugar levels with sulfonylurea drugs may lead to combining this therapy with insulin or administering insulin alone, regardless of patients' weights. PMID- 2994472 TI - Combination cyclophosphamide, Adriamycin, and vincristine rapidly alternating with combination cisplatin and VP-16 in treatment of small cell lung cancer. AB - Forty-four patients with small cell lung cancer were treated with an intensive chemotherapy induction program consisting of combination cyclophosphamide, Adriamycin, and vincristine rapidly alternating with combination cisplatin and VP 16 followed by prophylactic cranial radiotherapy. After chemotherapy induction and cranial radiotherapy, patients with limited disease received multiple-field radiotherapy consolidation to the primary tumor site and mediastinum using thoracic computed tomographic scanning for field planning, and patients with extensive disease received chemotherapy maintenance. Patients with limited disease in complete remission following radiotherapy consolidation received no further treatment unless disease recurred. It was found that cyclophosphamide, Adriamycin, and vincristine could be alternated with cisplatin plus VP-16 at two week intervals in 80 percent of patients on an outpatient basis and that two thirds of patients achieved clinical complete remission after two courses of each regimen. Locoregional radiotherapy delivered via multiple fields was effective in increasing the complete remission rate in patients with limited disease and was well tolerated. The median survival time was 18.5 months in 24 patients with limited disease and 12.2 months in 20 patients with extensive disease. Four patients with limited disease who received chemotherapy induction and radiotherapy consolidation without maintenance chemotherapy and one patient with extensive disease remain alive and disease-free at more than five years. PMID- 2994473 TI - Nasopharyngeal carcinoma. Brief review. AB - Nasopharyngeal carcinoma has a well-defined geographic distribution, primarily affecting persons from southern China and Southeast Asia. Environmental factors are numerous and appear to have a secondary role, mainly in the promotion of the neoplastic process. Relationship with the Epstein-Barr virus is indicated by the identification of viral genome copies within the cells and by a persistent host antibody response with restricted specificity for nasopharyngeal malignancies. The World Health Organization has recently adopted a histologic classification categorized into three subtypes according to the degree of epithelial differentiation, keratinization, and stromal lymphocytic infiltration. The tumor expands locally to contiguous structures, spreads through the cervical lymphatics following the jugular chain, and eventually metastasizes to the skeleton and liver. Primary management consists of radiation therapy to cervicofacial fields and usually offers adequate palliation, with a five-year median survival of 67 percent for stage I and 17 percent for stage IV disease. PMID- 2994475 TI - AIDS. Epidemiology update. PMID- 2994474 TI - Relationship of glucose intolerance and indocyanine green clearance to respiratory enzyme levels in human cirrhotic liver. AB - The relationship of glucose intolerance and indocyanine green clearance to respiratory enzyme levels in liver mitochondria was studied along with standard liver function tests in 40 patients (8 cirrhosis, 19 cirrhosis with hepatoma, 13 non-cirrhotic with hepatoma). There was a negative correlation between cytochrome a(+a3) concentrations and phosphorylative activity per unit of cytochrome a(+a3) (r = -0.75, p less than 0.01), but no correlation between ICG-K and cytochrome a(+a3) concentrations. Cytochrome a(+a3) concentrations in cirrhotic patients with linear oral glucose tolerance pattern, characterized with no return toward normal glucose levels within 120 minutes after an oral glucose load, increased to 1.45 +/- 0.11 (10(-10) mol/mg of protein) compared with 0.90 +/- 0.07 in cirrhotic patients with parabolic OGTT pattern, characterized with a return toward normal glucose levels within 120 minutes (p less than 0.01) (0.82 +/- 0.02 in control patients without liver diseases). The former had high operative mortality regardless of ICG-K value and the latter had virtually uneventful clinical courses. It was suggested that increased cytochrome a(+a3) concentrations and impaired glucose tolerance might be responsible for decreased hepatic functional reserve and poor prognosis in cirrhotics. PMID- 2994476 TI - Controversies in Rh prophylaxis. PMID- 2994477 TI - Genital herpes in pregnancy: risk factors associated with recurrences and asymptomatic viral shedding. AB - One hundred forty-seven women with recurrent symptomatic genital herpes simplex virus acquired prior to the start of pregnancy (group 1) and 15 women whose first symptomatic episode of genital herpes was acquired during pregnancy (group 2) were followed weekly during the course of gestation. Among women with recurrent genital herpes antedating pregnancy, the mean number of recurrences per trimester increased from 0.97 to 1.26 to 1.63 in the first through third trimester, respectively (p less than 0.05 for comparison between each trimester). The median number of symptomatic recurrences of genital herpes during gestation was four in women in group 1 compared to one in women in group 2 (p less than 0.01). Asymptomatic viral excretion from the genital tract was, however, more common in women in group 2 (33%) than in women in group 1 (12.9%) (p less than 0.05). Herpes simplex virus was isolated at 5.5% of routine visits in group 2 women compared to 1% of routine visits among group 1 women. Logistic regression analysis indicated young age also was associated with more frequent asymptomatic viral shedding. Asymptomatic herpes simplex virus excretion was more common from the vulvar area than the cervix, and women in group 2 were more likely to shed virus from both sites simultaneously than women in group 1. Age and recent acquisition of genital herpes are risk factors for asymptomatic excretion of herpes simplex virus during pregnancy. PMID- 2994478 TI - Infection and phagocytosis as possible mechanisms of rupture in premature rupture of the membranes. AB - The concept that premature rupture of the membranes is due to an infectious process is well accepted. However, no definitive data implicating a particular microorganism or a mechanism of action have been advanced. By the use of our recently developed experimental in vitro amnion-chorion reaction vessel model we have studied the effect of the peroxidase-hydrogen peroxide-halide antimicrobial system on these membranes. We have noted that amnion, chorion, decidua, and placental macrophages all possess peroxidase activity. Tissues collected from deliveries following labor (vaginal) are significantly higher in activity than those collected from deliveries with no labor (cesarean section). A mobilization of enzyme from macrophages to amnion appears to occur in the laboring patient. Increased protein hydrolysis is noted in membranes collected from patients without labor subjected to the peroxidase-hydrogen peroxide-halide cytotoxic system when compared with membranes from laboring patients. Bursting pressures of membranes collected from patients without labor are shown to be decreased when the membranes were incubated in the presence of lysolecithin or in the presence of amniotic fluid and phospholipase A2. PMID- 2994479 TI - Serum testosterone concentrations in the evaluation of androgen-producing tumors. AB - During a 5-year study period 18 women with a serum testosterone concentrations of greater than 2 ng/ml were evaluated for a possible androgen-producing tumor. All subjects were hirsute and had menstrual irregularities, with the exception of one postmenopausal woman. The majority of the women were obese and 72% were greater than 50% over ideal body weight. Only two of 11 women undergoing operative and histologic evaluation of the ovaries were found to have an androgen-producing neoplasm. Seven additional women with serum testosterone concentration of greater than 2 ng/ml have been followed for over 1 year with no additional evidence of an androgen-producing neoplasm. The poor predictive value of a serum concentration of greater than 2 ng/ml in identification of an androgen-producing neoplasm is partially explained by the apparent prevalence of high testosterone concentrations in chronically anovulatory, hyperandrogenic obese women and by the large coefficient of variation observed in this study when analyzing testosterone concentrations were analyzed over an 8-hour interval (range, 3% to 42%). In the absence of an adnexal mass or rapidly progressive virilization, it is suggested that the use of venography or operative exploration to diagnose an androgen producing neoplasm be reserved for women with a mean testosterone concentration derived from three daily samples that is at least 2.5 times greater than the upper range of normal in the given laboratory. PMID- 2994480 TI - In vitro binding of insulin and epidermal growth factor to human endometrium and endocervix. AB - The distribution of receptors for insulin and epidermal growth factor along the longitudinal axis of the uterine cavity was studied in 28 uteri obtained from women of reproductive age undergoing hysterectomy for benign conditions. Insulin binding to crude plasma membranes was higher (p less than 0.05) in the secretory than in the proliferative phase of the menstrual cycle in all uterine segments (fundus to cervix). Epidermal growth factor binding did not change during the menstrual cycle but the number of epidermal growth factor binding sites was higher in the cervix than in the fundus (p less than 0.05). Scatchard plots of binding data, obtained with crude plasma membranes from pooled uteri, were curvilinear; the high-affinity sites had dissociation constants of 1 to 4 nmol/L and receptor concentrations of 100 to 300 fmol/mg of protein, for both iodine 125 labeled insulin and 125I-labeled epidermal growth factor. In plasma membranes, obtained from another 15 uteri, mouse nerve growth factor (3.3 micrograms/ml) decreased the binding of insulin by an average of 17% (p less than 0.005); in the decidua of a pregnant uterus at 12 weeks Scatchard analysis showed that nerve growth factor decreased the affinity but not the number of insulin-binding sites. Nerve growth factor had no effect on epidermal growth factor binding. Human prolactin (2 micrograms/ml) also decreased insulin binding by an average of 18% (n = 5, p less than 0.025) but had no effect on epidermal growth factor binding. These "baseline" data will be useful in further studies of the possible interactions between (1) receptors for various peptide growth factors and (2) sex steroid hormones, in normal and neoplastic endometrium and cervix. PMID- 2994481 TI - Pregnancy in a patient with gonadotropin-resistant ovary syndrome. AB - The case of a patient with gonadotropin-resistant ovary syndrome is discussed. Ovulation was successfully induced by the administration of human chorionic gonadotropin and by estrogen replacement therapy. A total of three pregnancies occurred. The first two pregnancies resulted in blighted ova. The third pregnancy resulted in a normal term delivery. PMID- 2994482 TI - Amiodarone-induced lung toxicity. In vitro evidence for the direct toxicity of the drug. AB - Amiodarone, a new antiarrhythmic agent, is associated with serious lung toxicity. This study indicates that in vitro amiodarone can directly induce bovine pulmonary artery endothelial cells to form cytoplasmic lamellar inclusions characteristic of the lung biopsy findings described in vivo. These morphologic changes occur as soon as 4 hours after incubation with the drug and with as little as 1 microgram/ml (within the therapeutic range). Amiodarone-induced endothelial cell injury, monitored by 51Cr release, occurs with as little as 10 20 micrograms/ml. The data suggest that amiodarone toxicity to the lung may be primarily related to its direct toxic effect on lung cells, and that the characteristic morphologic changes of cytoplasmic inclusions may represent an early sign of the drug's effect. PMID- 2994483 TI - Transport properties of the lens. AB - Many studies have shown that the lens is a multicellular syncytial tissue whose electrophysiological properties are the integrated result of membrane transport, low-resistance gap junctions interconnecting the cells, and the restricted extracellular space between cells. There are at least three structurally distinct populations of cells within the lens, and the membrane transport properties of each cell type appear to differ. Indeed, there may be subcellular specialization of membrane transport properties in the surface epithelial cells. We review the physical structure of the lens, its electrical structure, and our present knowledge of the membrane transport properties of the different cell types. Our recent work has focused on radially circulating fluxes generated by the spatial localization of membrane transport in surface cell membranes versus inner fiber cell membranes. We review this work and present some simplified models of the results with some discussion of physiological implications. PMID- 2994484 TI - ATP depletion and loss of cell integrity in anoxic hepatocytes and silica-treated P388D1 macrophages. AB - The relationship between ATP depletion and the loss of cell integrity was examined in the killing of hepatocytes by anoxia and P388D1 macrophages by silica. ATP depletion is a feature of the reaction to either hazard. Treatment of hepatocytes, however, with antimycin, oligomycin, sodium azide, or N,N' dicyclohexylcarbodiimide produced a rate and extent of ATP depletion comparable with anoxia without significant loss of viability. Treatment of P388D1 cells with 2-deoxyglucose plus antimycin, oligomycin, or sodium azide reproduced the loss of ATP accompanying silica particle intoxication. Again, there was no loss of viability. These data dissociate the loss of cellular ATP from the genesis of lethal injury in both cell types. ATP depletion was, however, associated with a loss of lysosomal integrity. With the metabolic inhibitors, loss of lysosomal integrity occurred in the absence of irreversible cell injury over the time course that anoxia and silica intoxication significantly damaged the cells. This implies that neither hazard produces lethal damage through mechanisms dependent on intracellular lysosomal enzyme release. While ATP depletion can cause lysosomal rupture in P388D1 macrophages, phagocytosis of silica particles in the absence of extracellular Ca2+ ions is associated with release of lysosomal contents without depletion of ATP or loss of cell integrity. Silica particles are concluded to interact directly with both the plasma and lysosomal membranes. The former leads to Ca2+ influx with resultant cell death and ATP depletion. The latter leads to release of lysosomal contents that is not followed by irreversible cell injury. PMID- 2994485 TI - Vasopressin stimulates DNA synthesis and ion transport in quiescent epithelial cells. AB - The mitogenic effect of vasopressin was studied in subconfluent quiescent renal epithelial cells (MDCK). Vasopressin stimulated DNA synthesis in the presence of low concentrations of serum. Vasopressin increased the entry of Na into the cells and increased ouabain-sensitive 86Rb uptake, a measure of Na-K pump activity. Because the activity of the Na-K pump in MDCK cells is steeply dependent on intracellular Na, it is likely that stimulation of the Na-K pump by vasopressin was mediated by the increase in Na entry into the cells. Thus both serum and vasopressin stimulate Na uptake and Na-K pump activity in quiescent MDCK cells with a subsequent increase in DNA synthesis. It is concluded that growth regulation in epithelial cells may be mediated in part by changes in monovalent ion transport. PMID- 2994486 TI - The platelet strip. II. Pharmacomechanical coupling in thrombin-activated human platelets. AB - A model of contracted, irreversibly aggregated thrombin-activated human platelets relaxes when treated with ethyleneglycol-bis(beta-aminoethylether-N,N' tetraacetic acid (EGTA) in the presence of Mg2+. Inhibition of the cyclooxygenase or blockade of the thromboxane A2 receptor decreases the tension partially, but EGTA treatment is needed for full relaxation. After a stable relaxation has been achieved (3-4 h), Ca2+ addition in a cumulative manner does not reinduce contraction. Whether in the absence or presence of external Ca2+, the relaxed preparation contracts when stimulated with ADP, epinephrine, thromboxane A2 or its analogues, or thrombin. At supramaximal doses, each of the agonists activates only a partial amount of the total tension capable of being generated. Addition of an agonist of a different class to the partially contracted preparation further increases its force. The contractile responses are reversible on washout, with kinetics dependent on the class of agonist and time of contact with the preparation. The contraction induced by the prolonged simultaneous stimulation with ADP, arachidonate, and thrombin reverts very slowly on washout of the agonists and for all practical purposes reproduces the initial state of irreversible platelet contraction. PMID- 2994487 TI - Role of myosin phosphorylation in contractility of a platelet aggregate. AB - The relationship between tension and myosin 20,000-Da light chain phosphorylation in intact nonmuscle cells was investigated using a preparation of thrombin activated, irreversibly aggregated platelets known as the platelet strip. Steady state levels of tension generated by the platelet strip were found to be linearly related to the level of myosin phosphorylation. This relationship was observed during dose-dependent relaxation induced by the adenylate cyclase activators prostaglandin (PG) E1 and PGI2, and during contraction induced by ADP, epinephrine, and the prostaglandin endoperoxide analogue U-46619, which did not appreciably alter the basal level of adenosine 3',5'-cyclic monophosphate in the preparation. The fully relaxed platelet strip, in the absence of external Ca2+, was associated with a level of 12% light chain phosphorylation, which increased to 72% on maximal contraction. During both relaxation and contraction, changes in myosin phosphorylation were also found to precede or coincide with tension changes. Furthermore, steady-state contraction induced by ADP was associated with a maintained elevation in the level of myosin phosphorylation. These results support the concept that myosin phosphorylation is an important regulatory mechanism for contractility in platelets. PMID- 2994488 TI - Adenosine stimulates sodium transport in kidney A6 epithelia in culture. AB - The effects of adenosine receptor agonists and antagonists were examined in epithelia formed in culture by A6 cells, a continuous cell line derived from Xenopus laevis kidney. A6 epithelia have a high electrical resistance and a short circuit current that is equal to net sodium flux from mucosal to serosal surface. Adenosine, 2-chloroadenosine, 5'-(N-ethyl)carboxamidoadenosine, and N6-(L-2 phenylisopropyl) adenosine produced concentration-dependent increases in short circuit current. Stimulation of short-circuit current by 2-chloroadenosine occurred at concentrations of 0.05 microM and above, with half-maximal stimulation occurring at 0.3 microM. 5'-(N-ethyl)carboxamidoadenosine was more potent than N6-(L-2-phenylisopropyl)adenosine, the usual order of potency for activation of stimulatory adenosine receptors. Theophylline (100 microM), an adenosine receptor antagonist, reduced the short-circuit current response to adenosine and 2-chloroadenosine by 85-90%. Amiloride, an agent that inhibits both basal and adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated short-circuit current in A6 epithelia, completely and reversibly inhibited short-circuit current stimulated by 2-chloroadenosine. Adenosine and 2-chloroadenosine stimulated adenylate cyclase activity in a crude membrane preparation from A6 cells. Stimulation by adenosine was blocked by adenosine deaminase. 2 Chloroadenosine increased cell cAMP accumulation in intact epithelia. The results provide evidence that adenosine and adenosine receptor agonists stimulate adenylate cyclase and active sodium transport in an epithelial cell line of renal origin. PMID- 2994489 TI - Absence of albumin receptor on brain capillaries in vivo or in vitro. AB - Hormones and drugs are known to be available for transport into brain and liver in vivo from the circulating albumin-bound pool. An albumin receptor-mediated mechanism is one possible way in which the transport of ligands from the circulating albumin-bound pool into the tissue may be catalyzed. The albumin receptor model was tested for brain in the present studies using both 125I albumin (labeled by lactoperoxidase) and [3H]albumin (labeled by reductive methylation). The interaction of the labeled albumin with brain capillaries was assessed in vivo with the carotid injection technique in rats and in vitro with isolated bovine brain capillaries. Artifactually high nonspecific binding in both the in vivo and in vitro assays was observed with 125I-albumin. Conversely, the transit time of [3H]albumin through the brain capillaries in vivo was no greater than the transit time of [14C]sucrose. The binding of [3H]albumin to isolated microvessels in vitro was low, less than [3H]inulin and was nonsaturable. In conclusion these studies do not support the albumin receptor model for the transport of albumin-bound ligands into tissues such as brain. PMID- 2994490 TI - Work performance in the iron-deficient rat: improved endurance with exercise training. AB - The effect of an endurance training regimen on muscle oxidative enzymes and work performance was studied in iron-deficient and -sufficient rats. Three-week-old male Sprague-Dawley rats (n = 40) were randomly assigned to diets containing either 6 mg iron/kg (iron deficient) or 50 mg iron/kg (iron sufficient). After 2 wk, each group of rats was further divided into untrained or endurance-trained subgroups. Training consisted of daily treadmill running of gradually increasing duration for a 1-mo period. After the training period, sedentary and endurance trained iron-deficient rats were anemic (Hgb approximately 8 g/dl compared with 16 g/dl in the 2 control groups) and had significantly lower skeletal muscle cytochrome c concentration, cytochrome oxidase activity, and succinic oxidase activity compared with the iron-sufficient groups. In response to training iron deficient rats also generally had a substantial increase in skeletal muscle oxidative enzymes (P less than 0.05), in contrast to iron-sufficient animals, in which there was little or no training effect. Work performance in response to training in the iron-deficient rats improved more than sixfold in an endurance type of exercise (P less than 0.05), but maximal oxygen consumption during a brief, intense type of exercise was not significantly affected. The results suggest that endurance training of iron-deficient rats results in a milder anemia and less drastic reduction of skeletal muscle oxidative enzymes which in turn allows better performance in an endurance type of exercise. PMID- 2994491 TI - Role of H+ transport in ursodeoxycholate-induced biliary HCO-3 secretion in the rat. AB - Biliary bicarbonate secretion may occur by transport of bicarbonate itself or of H+ (or OH-). To distinguish between these two mechanisms, we have studied the effects of bicarbonate deprivation or substitution by weak acids in the perfusate of isolated rat livers on ursodeoxycholate-induced bicarbonate secretion. Livers were perfused with an erythrocyte-free solution containing either the impermeant buffer Tricine (25 mM) or 25 mM Tricine and 13 mM bicarbonate, acetate, or 5,5 dimethyloxazolidine-2,4-dione (DMO), and ursodeoxycholate was infused. Tauroursodeoxycholate, which does not stimulate bicarbonate secretion, served as a control. During ursodeoxycholate infusion 1) the increase in bile flow, in microliter X min-1 X g liver-1 (+/- SE), was significantly higher in livers perfused with Tricine and bicarbonate (1.29 +/- 0.06), Tricine and acetate (1.46 +/- 0.07), and Tricine and DMO (1.30 +/- 0.04) than in livers perfused with Tricine alone (0.99 +/- 0.04); and 2) biliary bicarbonate, acetate, or DMO concentrations and bile pH were significantly higher than the corresponding perfusate values. In contrast, during tauroursodeoxycholate infusion bile flow was the same whatever the perfusate, and bile pH was lower than pH of the perfusate. Therefore, ursodeoxycholate-induced choleresis and bile alkalinization do not depend on bicarbonate as such (which can be replaced by acetate or DMO). This suggests that ursodeoxycholate-induced biliary bicarbonate secretion is the result of H+ (or OH-) transport rather than transport of bicarbonate itself. PMID- 2994492 TI - Influence of luminal pH on rat large bowel epithelial cell cycle. AB - The influence of luminal pH on the colonic epithelial cell cycle was studied by means of dietary modification to produce acidification of colonic contents. Forty male Sprague-Dawley rats were fed one of four diets: defined fiber free, fiber free diluted by either 100 g/kg lactulose or sorbitol, or 25 g/kg of MgSO4. After 2 wk, in vivo pH measurements were recorded at laparotomy under ether anesthesia in the cecum and proximal, mid-, and distal colon. Epithelial cells, isolated from these same regions, were measured for DNA content with flow cytometry in order to determine cell cycle stage. The pH values in all intestinal regions were highest with MgSO4 and then fiber free, sorbitol, and lactulose, the most acidic. In contrast, the percentage of cells in S phase (actively synthesizing DNA) was highest with lactulose and least with MgSO4. There was a significant inverse correlation between luminal pH and percent cells in S phase in the cecum (P less than 0.01), proximal colon (P less than 0.05), and distal colon (P less than 0.01). These results show that acidification of colonic contents by diet modification leads to increased epithelial cell proliferation. PMID- 2994493 TI - Response of serum 1,25(OH)2D3 to variation of ionized calcium during chronic acidosis. AB - Chronic ammonium chloride (NH4Cl) administration causes metabolic acidosis and prevents the normal rise of serum 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] during a low-calcium diet (LCD, 0.002% calcium). The suppression of serum 1,25(OH)2D3 was not due to reduced parathyroid hormone concentration, elevated serum phosphorus, or total calcium concentration. Acidosis increased blood ionized Ca [Ca2+] and proton [H+] concentrations. Serum 1,25(OH)2D3 levels were inversely correlated with both [Ca2+] and [H+]. To determine the independent effects of [Ca2+] on serum 1,25(OH)2D3 we varied [Ca2+] at a constant [H+] by infusing either EGTA or saline for 24 h after 11 days of LCD and NH4Cl. EGTA, preequilibrated with three concentrations of Ca, lowered [Ca2+] and raised 1,25(OH)2D3 but did not alter [H+] or serum phosphorus concentration. The log of serum 1,25(OH)2D3 varied linearly and inversely with arterial blood [Ca2+] during saline (r = -0.884, n = 8, P less than 0.001) and EGTA infusions (r = -0.798, n = 22, P less than 0.001). At all levels of [Ca2+], rats infused with EGTA had a higher serum 1,25(OH)2D3 than those infused with saline. Log serum 1,25(OH)2D3 was correlated neither with [H+] nor pH. Elevated [Ca2+] and not [H+] appears to suppress the serum 1,25(OH)2D3 response to LCD during NH4Cl acidosis in the rat. PMID- 2994494 TI - Relationship among parathyroid hormone, cAMP, and calcium on proximal tubule sodium transport. AB - Parathyroid hormone (PTH), through the generation of cAMP, inhibits the transport of sodium, bicarbonate, and water in the proximal convoluted tubule. The present studies were designed to determine whether the response to PTH or dibutyryl cAMP by proximal renal tubules requires an influx of calcium into the cells or an alteration in the cytosolic concentration of calcium. O2 consumption was determined in an enriched suspension of rabbit proximal convoluted tubules, and the ouabain-sensitive component of O2 consumption was taken as a measure of sodium transport. PTH (1 IU/ml) inhibited the ouabain-sensitive component of O2 consumption from 14.2 +/- 1.6 to 8.9 +/- 1.1 nmol O2 X min-1 X mg protein-1 (P less than 0.005). Dibutyryl cAMP (10(-4) M) inhibited ouabain-sensitive O2 consumption from 12.0 +/- 1.1 to 8.4 +/- 0.8 nmol O2 X min-1 X mg protein-1 (P less than 0.005). In the presence of lanthanum (5-50 microM) or verapamil (20-200 microM), PTH inhibited ouabain-sensitive O2 consumption by 21.3 +/- 3.4% (P less than 0.025) and 33.9 +/- 5.5% (P less than 0.025), respectively. Dibutyryl cAMP inhibited the ouabain-sensitive O2 consumption by 22.6 +/- 7.1% (P less than 0.025) in the presence of lanthanum and 35.4 +/- 3.1% (P less than 0.01) in the presence of verapamil. To more directly assess the cytosolic concentration of calcium, the fluorescent intensity of quin 2-loaded tubules was determined. As compared with timed controls, exposure to PTH resulted in a lower cytosolic concentration of calcium over the 10 min of incubation. Dibutyryl cAMP had a similar effect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2994495 TI - Compartmented pyruvate in perfused working heart. AB - Pyruvate compartmentation and lactate dehydrogenase (LDH) were studied in isolated perfused working guinea pig hearts. The mean intracellular pyruvate (Pyr) contents increased with perfusate Pyr (0-2 mM) but varied only slightly with glucose (0-10 mM) and additional insulin (0.04-5 U/l), respectively. With 5 10 mM glucose plus 5 U/l insulin, but not with Pyr or lactate (Lac) as substrates, a near equilibrium between the LDH and the glycerol-3-phosphate dehydrogenase seemed to exist. Evidence for an inhibitory effect of Pyr on the activity of the LDH system of the perfused hearts was not obtained. With [U 14C]glucose as sole substrate, the specific activity of coronary venous Lac was near half that of precursor glucose. 14CO2 production was thus in quantitative agreement with rates of pyruvate oxidation that were determined as glucose uptake minus (Pyr + Lac) release. In contrast, with 0.2 mM [1-14C]Pyr plus 5 mM glucose, the ratio of 14CO2 production to specific activity of Lac overestimated Pyr oxidation judged from myocardial substrate balances and O2 uptake, respectively; here, at least three pools of [14C]HCO-3 and [14C]lac, respectively, were kinetically demonstrable during washout of trace amounts of 14C-labeled Pyr. Evidently, the specific activity of Lac was equivalent to that of mitochondrial oxidized Pyr provided [14C]glucose was the sole or major precursor of cellular pyruvate. However, exogenously applied [1-14C]Pyr of high specific activity seemed to induce intracellular formation of both a highly and lowly labeled Pyr; the latter Pyr compartment did not seem in ready equilibrium with the cell physiologically prevailing highly labeled Pyr pool. PMID- 2994496 TI - alpha 1-Receptor localization in rat heart and kidney using autoradiography. AB - To study the distribution of alpha 1-adrenergic receptors in rat heart, kidney, and skeletal muscle, we used light-microscopic autoradiography of [3H]prazosin. Scintillation spectrometry of frozen sections demonstrated rapid binding, saturability, stereospecificity, and agonist and antagonist binding characteristic of an alpha-receptor. For autoradiography, sections were incubated, processed, and grain density quantified using a computer-based image analyzer. Specific alpha 1-receptor binding was found over cardiac myocytes in the left and right ventricles but not over skeletal muscle. Scatchard analyses of specific grain densities over cardiac myocytes gave a dissociation constant (Kd) of 0.55 +/- 0.18 nM (SD, n = 4 rats) and a maximum number of binding sites (Bmax) of 448 +/- 90 grains/10(-2) mm2. Renal arterioles had a higher specific grain density than myocardial arterioles at all concentrations of [3H]prazosin (P less than 0.001). Scatchard analyses showed that renal arterioles had a Kd of 0.27 nM and a Bmax of 1,259 grains/10(-2) mm2, whereas myocardial arterioles had a Kd of 1.64 nM and a Bmax of 183 grains/10(-2) mm2. Arterioles in the flexor carpi radialis muscle were not labeled. Renal cortex tubules had the highest grain density of any structure studied, i.e., higher than grain density over glomeruli or tubules in the renal medulla. These observations indicate that significant differences exist in the distribution and affinity of alpha 1-adrenergic receptors in various vascular beds and parenchymal tissues. PMID- 2994497 TI - Effect of hypokinesia-hypodynamia on rat muscle oxidative capacity and glucose uptake. AB - Whole-body hypokinetic-hypodynamic (H/H) suspension, unlike other models of muscle disuse, allows voluntary contractile activity. This study examined the oxidative capacity and insulin sensitivity of rat hindlimb muscles subjected to 7 days of suspension H/H conditions. Oxidative capacity was determined by measuring citrate synthase activity and cytochrome c concentration in soleus and gastrocnemius muscles. A perfused hindquarter preparation was used to measure glucose uptake rates at rest with physiological and supramaximal concentrations of insulin in the perfusate. Citrate synthase activity was 17% lower in soleus and 23% lower in gastrocnemius muscles from H/H rats. Similarly, a 29% decrease in H/H rat gastrocnemius cytochrome c concentration was observed. Rates of glucose uptake were lower in muscles from H/H rats compared with controls at physiological levels of insulin and did not increase in response to a further increase in insulin concentration. Muscles undergoing a significant loss in mass after 7 days suspension were found to have increased glycogen concentrations. In conclusion, data presented in this study suggest that hindlimb muscle disuse, brought about by whole-body suspension, results in a decreased aerobic capacity in load bearing muscles and a lowered insulin sensitivity in perfused rat hindlimb muscles. PMID- 2994499 TI - Opiatergic and dopaminergic function and Lesch-Nyhan syndrome. PMID- 2994498 TI - Mechanisms of antinatriuresis during low-frequency renal nerve stimulation in anesthetized dogs. AB - The influence of 1.0 Hz renal nerve stimulation (RNS) on the renal excretion of sodium and bicarbonate was determined in anesthetized dogs before and during inhibition of renal bicarbonate reabsorption. RNS decreased both urinary sodium and bicarbonate excretion without changing arterial pressure, renal blood flow, or glomerular filtration rate. Pharmacological blockade of bicarbonate reabsorption with acetazolamide prevented RNS-induced decreases in bicarbonate excretion and reduced the antinatriuretic response. Physiological blockade of tubular bicarbonate reabsorption with intrarenal sodium bicarbonate infusion (1 M) abolished both the antinatriuretic response to RNS and the decrease in bicarbonate excretion. This physiological blockade of neurogenic antinatriuresis resulted from alkalinization of the urine and/or peritubular blood rather than an increase in filtered sodium load, because during intrarenal infusion of 1 M sodium chloride RNS concomitantly decreased sodium and urinary bicarbonate excretion. Since antinatriuretic responses and the decrease in bicarbonate excretion response to RNS were significantly decreased by blockade of bicarbonate reabsorption (with acetazolamide and intrarenal sodium bicarbonate infusion), antinatriuresis during RNS is partly mediated by a mechanism dependent on intact bicarbonate reabsorption. The data suggest that renal nerve activity may participate in the normal regulation of acid-base balance via changes in bicarbonate excretion. PMID- 2994500 TI - American cutaneous leishmaniasis: a comparison of three sodium stibogluconate treatment schedules. AB - Thirty-six patients with American cutaneous leishmaniasis were randomized to receive intravenous sodium stibogluconate for 10 days at a dose of 600 mg antimony (Sb) per day by one of three schedules: once daily by rapid infusion (A), by continuous 24 hr infusion (B), or in divided doses every eight hours by rapid infusion (C). Patients not cured after initial treatment were rerandomized to one of the other treatment schedules. An additional 19 patients who were not part of the randomized study received standard (STD) sodium stibogluconate treatment (600 mg Sb once daily by rapid infusion for 10 days, identical with schedule A). In the randomized study, the overall cure rate after the first course of treatment was 64%, but was higher for schedule A (100%) than for B (50%) or C (42%) (P less than 0.01). Considering all courses of treatment, schedule A was more effective (94%) than B (53%) or C (43%) (P less than 0.01). Paradoxically, patients in group A had a higher cure rate than patients in group STD (42% after the first course of treatment and 51% when all courses of treatment were considered). Side effects were mild and well tolerated. Total side effects were more frequent in groups B + C (52%) than A + STD (23%) due to an increased incidence of subjective complaints (26% vs. 10%, P less than 0.05) in patients receiving other than once daily rapid infusion. We conclude that giving the same total amount of sodium stibogluconate in three divided doses or by continuous infusion offers no advantage over standard, once daily treatment of American cutaneous leishmaniasis. PMID- 2994501 TI - Fecal rotaviruses, adenoviruses, coronavirus-like particles, and small round viruses in a cohort of rural Costa Rican children. AB - Excretion patterns of fecal viruses were studied in a cohort of 51 rural Costa Rican children. The presence of rotavirus, adenovirus, coronavirus-like particles, and small round viruses was investigated by electron microscopy (EM) in 2,516 extracts of weekly fecal specimens. Rotavirus was in addition studied with ELISA. The incidence of diarrhea was 0.7 episodes per child-year. Rotavirus was the most common virus (0.53 infection/child-year), followed by adenovirus (0.46 infection/child-year), and coronavirus-like particles (0.24 infection/child year). However, the pathogenicity of rotavirus and adenovirus was low: only 3 of 24 rotavirus infections and 2 of 21 adenovirus infections were associated with diarrheal illness (12.5% and 9.5%, respectively). Small round viruses were detected in 23 specimens, but could not be assigned to a particular group of viruses. Children who excreted coronavirus-like particles and small round viruses were asymptomatic. Typical Norwalk-like viruses, astrovirus or calicivirus were not encountered. Rural conditions, good hygiene and prolonged breast feeding may explain the reduced exposure and pathogenicity of viral enteropathogens in rural Costa Rica. PMID- 2994502 TI - Acute hemorrhagic conjunctivitis caused by enterovirus type 70: an epidemic in American Samoa. AB - From December 1981 to February 1982, an estimated 22,000 cases of acute hemorrhagic conjunctivitis (AHC) caused by enterovirus type 70 (EV 70) occurred among Samoan and non-Samoan residents of American Samoa. The overall attack rate was estimated to be 68%. Samoans of all ages resident in traditional housing and of large family size were at greatest risk of acquiring AHC, while non-Samoan adults resident in western style housing were at lowest risk. Epidemiologic aspects of AHC acquisition were also different for the Samoan and non-Samoan communities; index cases in Samoan households were frequently young adults, whereas index cases in non-Samoan households were commonly school age children, suggesting a role for school transmission in non-Samoans only. In this outbreak, subclinical AHC was rare; of 50 asymptomatic members of affected households, only 3 had neutralizing antibody to EV 70 (all with titers of 1:10). Investigation documented the highly contagious nature of AHC caused by EV 70, and the ease with which epidemic transmission may develop under conditions of crowding and frequent interpersonal contact. PMID- 2994503 TI - Plasma ACTH and aldosterone levels in endotoxin shock in rats. PMID- 2994504 TI - [3 new GABA-ergics: research on their possible site of action]. PMID- 2994505 TI - Primary combined small cell carcinoma of the larynx. AB - The clinical and pathologic findings of six cases of combined small cell carcinoma of the larynx are described. This tumor is a subtype of small cell carcinoma in which there is a definite component of oat cell carcinoma (or intermediate cell type carcinoma) together with squamous cell carcinoma or adenocarcinoma or both. The neoplasm seems to derive from a common cell with subsequent divergent differentiation into the Kulchitsky cells, squamous cells, and/or glandular cells. After histologic diagnosis, adequate evaluation for tumor staging is mandatory. Like pulmonary combined small cell carcinoma, this neoplasm may be best treated with systemic chemotherapy and radiotherapy. The prognosis is similar to that of other subtypes of small cell carcinoma of the larynx. PMID- 2994506 TI - Adenoid cystic carcinoma of the supraglottic larynx: report of a case and review of the literature. AB - Although adenoid cystic carcinoma is the most common malignant tumor of the minor salivary glands, it seldom occurs in the larynx. Less than 0.25 per cent of all laryngeal carcinomas are adenoid cystic, and only 15 such cases of supraglottic origin are recorded in the literature. In only four cases the details of treatment and outcome are described. We report another case and review the literature in order to define the entity of adenoid cystic carcinoma of the supraglottic larynx. This case reveals the aggressive nature of the disease, which is often associated with the solid variant histology and also demonstrates the most rapid course of any cases described in the literature. Other histologic variants of adenoid cystic carcinoma of the supraglottic larynx follow a more prolonged course that can extend many years even with persistent local disease. However, development of distant metastases may eventually occur. Therefore, 5 year statistics are not adequate, and 10 or 20 years' follow-up is required to evaluate the curability of this disorder. Evaluation, treatment, and clinical course of adenoid cystic carcinoma of the supraglottic larynx are discussed. PMID- 2994507 TI - [Possibility of the connatal infection of the upper respiratory tracts with human papilloma virus]. PMID- 2994508 TI - [Possible etiological connection between cancerous diseases of the genitalia and papillomaviruses]. PMID- 2994509 TI - Fatal disseminated adenovirus infection featuring liver necrosis and prolonged viremia in an immunosuppressed child with malignant histiocytosis. AB - Truly disseminated adenovirus disease is rare and appears to be limited to patients with severe combined T and B cell deficiency. This report describes an 18-month-old child with malignant histiocytosis treated with chemotherapy who developed a fatal disseminated type 2 adenovirus infection. Histologic changes in the tissues of multiple organs as well as the presence of typical crystalline structures in sections of the liver seen with electron microscopy provided evidence of viral replication. Adenovirus was recovered from the involved organs and from daily serum samples obtained over a 2-week period prior to death. The possibility that the child may have had virus associated histiocytic disease prior to chemotherapy is discussed. PMID- 2994510 TI - Cytologic diagnosis of the acute nonlymphoid leukemias. I. Morphologic, cytochemical, and ultrastructural features. AB - The cytologic evaluation of a case of acute leukemia proceeds in two stages: 1) assigning the leukemia to one of two major classes, acute lymphocytic leukemia (ALL) or acute nonlymphocytic leukemia (ANLL); and 2) determining the proper subclassification of ALL or ANLL to which the case belongs. Although features such as nuclear morphology, number and nature of nucleoli, and nuclear/cytoplasmic ratio are useful criteria, the major morphologic features which distinguish ANLL cells from ALL cells are the presence of azurophilic granules and/or inclusions derived from fusion of these granules (Auer bodies). In most instances these can be visualized in the conventional blood film or bone marrow preparation. Sometimes, however, granules and Auer bodies are too small or too few to be seen with the light microscope. In such cases histochemical or ultrastructural studies will aid in proper classification. PMID- 2994511 TI - Plasma concentrations of thromboxane and prostacyclin metabolites in patients with bone tumors. PMID- 2994512 TI - [Behavior of the hypophyseal-adrenal cortex system in inhalation and conduction anesthesia. A review with comparative study]. AB - The effects of halothane anaesthesia or epidural analgesia on the per- and postoperative change in blood concentrations of ACTH, beta-lipotropin, cortisol, dehydroepiandrosterone, aldosterone, glucose, lactate and free fatty acids were investigated in connection with elective orthopaedic surgery. Anaesthesia in man with halothane and nitrous oxide was found to be associated with a significant increase in plasma ACTH levels and beta-LPH levels. Changes in plasma dehydroepiandrosterone were similar to those in plasma cortisol. The elevation of plasma aldosterone during major surgery could be explained as the effect of an increased renin secretion. However, simultaneous increase in plasma cortisol and plasma aldosterone during surgery reflect an additional effect of adrenocorticotropin upon aldosterone secretion. PMID- 2994513 TI - [Suppression of the adrenal cortex by infusion of etomidate in general anesthesia]. AB - The effects of anaesthesia with fentanyl and prolonged etomidate infusion (0.8 mg/kg/h) on the peroperative and postoperative change in blood concentrations of aldosterone, cortisol, dehydroepiandrosterone, ACTH, epinephrine, norepinephrine, dopamine, glucose, lactate and free fatty acids (FFA) were investigated in connection with major abdominal surgery. During surgery and anaesthesia with prolonged etomidate infusion no significant alterations in plasma catecholamine concentrations were observed. In contrast, basal plasma levels of ACTH (20.0 +/- 9.5 pg/ml----352.8 +/- 150.8 pg/ml) were elevated in all patients. Despite this endogenous ACTH stimulus, aldosterone (121.8 +/- 19.4 pg/ml----58.4 +/- 10.4 pg/ml) and cortisol (12.3 +/- 2.84 micrograms/dl----5.79 +/- 1.2 micrograms/dl) and dehydroepiandrosterone (2.28 +/- 1.09 ng/ml----1.27 +/- 0.25 ng/ml) levels were markedly depressed in every patient during the perioperative period. Twenty four hours after surgery the basal steroid values were within or above normal limits. PMID- 2994514 TI - [Clinical experiences with the anesthesia and brain monitor (ABM)]. AB - The Anaesthesia and Brain Monitor (ABM) was employed in 83 patients (of 24 to 86 years of age) who were subjected to abdominal surgery, and in 21 intensive-care patients under artificial respiration and sedative treatment (of 45 to 74 years of age), the equipment being used for monitoring anaesthesia or sedation. With this method it became possible to effect a largely integrated surveillance of EEG, EMG, NMT, pCO2, as well as, if desired, external blood pressure measurement by time-synchronous measurement and integration of parameters largely used singly in clinical practice to date. Whereas the EEG parameters (amplitude, frequency), if considered singly, do not yield clear information on the stage of anaesthesia, they can supply additional information especially in very light or very deep anaesthesia, together with the other measured parameters. In intensive care, they afford an estimation of the degree of the sedation. PMID- 2994515 TI - [Familial bilateral carotid and unilateral jugular glomera. Ultrastructural study apropos of 2 clinical cases]. PMID- 2994516 TI - Quantitative in situ gel electrophoretic assay for neomycin phosphotransferase activity in mammalian cell lysates. AB - A method for the assay of neomycin phosphotransferase activity in eucaryotic cell lysates is described. Total cytoplasmic proteins are fractionated in nondenaturing polyacrylamide gels and then allowed to react in situ with [gamma 32P]ATP and kanamycin. The reaction products are detected by blotting to phosphocellulose paper and autoradiography. The assay is linear with protein concentration and sensitive enough to detect expression in transient assays. PMID- 2994517 TI - Rotary dialysis: its application to the preparation of large liposomes and large proteoliposomes (protein-lipid vesicles) with high encapsulation efficiency and efficient reconstitution of membrane proteins. AB - An apparatus for rotary dialysis is introduced and described in detail. The component parts are inexpensive, widely available, and relatively easy to modify and assemble. The apparatus achieves increased mixing of the contents of dialysis bags by constant end-over-end rotation. This technique is particularly useful in systems where maximum contact is desired between substances which would tend to partition under standard dialysis conditions. We have applied rotary dialysis to two liposome production methods. These are (i) the calcium-EDTA-chelation method of Papahadjopoulos et al. (1), which produces large unilamellar liposomes from negatively charged phospholipids, and (ii) a procedure for the reconstitution of membrane proteins into liposomes with a large internal aqueous space, which we have developed using the calcium-EDTA-chelation technique as a point of departure. In both techniques, vesicle formation occurs when a calcium phospholipid precipitate is dissolved by the addition of EDTA. Instead of adding a 150 mM EDTA solution directly, as described in the original method, we have used overnight rotary dialysis against buffer containing 10 mM EDTA at the vesicle formation stage. Materials are encapsulated within the aqueous interior of the vesicles at much higher efficiencies when rotary dialysis is used in either method, compared to efficiencies obtained with direct addition of EDTA (up to 37% of added material vs a maximum published efficiency of 10% for direct addition). Rotary dialysis also promotes the reconstitution of a higher proportion of the membrane proteins present in the dialysis mixture into the bilayer of large liposomes (79 vs 41.6%). It also affects the content of liposomes qualitatively, allowing better reconstitution of the Sendai virus F glycoprotein than does direct addition of EDTA. These effects may be due to the slow time course, the extensive mixing of components, and the low volume-to phospholipid ratios maintained during vesicle formation. PMID- 2994518 TI - A sensitive, specific assay for tissue collagenase using telopeptide-free [3H]acetylated collagen. AB - Collagenase is assayed by incubation with soluble, telopeptide-free collagen extracted from rat skin and labeled with [2-3H]acetic anhydride. Collagen is cleaved by collagenase and the resulting fragments are digested with trypsin and chymotrypsin. Undigested collagen is recovered by precipitation with trichloroacetic acid, collected on glass-fiber filters, and quantitated by liquid scintillation spectrometry. This procedure combines features of the Cawston and Barrett (T.E. Cawston and A.J. Barrett, 1979, Anal. Biochem. 99, 340-345) and the Ryhanen et al. (L. Ryhanen et al., 1982, Collagen Rel. Res. 2, 117-130) methods. The first method provides a simple way to prepare large quantities of uniform substrate, while the second increases the specificity of the assay by removal of the labeled telopeptides. The assay is reproducible and linear with time and enzyme concentration. It is approximately 10X more sensitive than the Cawston and Barrett method and can readily detect 1-8 mU collagenase (1 unit equals 1 microgram collagen cleaved/min at 30 degrees C). The substrate is resistant to elastase, trypsin, and chymotrypsin and is completely degraded by bacterial collagenase. Collagenase is the only tissue metalloprotease found, to date, that cleaves the substrate. PMID- 2994519 TI - Analysis of products of DNA modification by methylases: a procedure for the determination of 5- and N4-methylcytosines in DNA. AB - Although many different methods are used for the identification of methylated heterocyclic bases in DNA not all of them possess the ability to discriminate N4 methylcytosine (m4C) and 5-methylcytosine (m5C). Therefore, some of the methods need additional reexamination. This paper reinvestigates some chromatographic systems (thin-layer chromatography, paper chromatography, electrophoresis) most widely used in the analysis of minor bases occurring in nucleic acids according to their ability to separate m4C and m5C. A simple procedure for the preparation of the sample and a chromatographic system for its analysis was developed. The recommended chromatographic systems may be used for the simultaneous separation of not only of m4C and m5C but also both methylated cytosines together with N6 methyladenine and 7-methylguanine. PMID- 2994520 TI - Separation of inositol phosphates and glycerophosphoinositol phosphates by high performance liquid chromatography. AB - We developed a HPLC method which separates the following nine inositol-containing compounds of biological interest: inositol, inositol 1-monophosphate, inositol 2- or 4-monophosphate, inositol 1,2-cyclic phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate, glycerophosphoinositol, glycerophosphoinositol 4 monophosphate, and glycerophosphoinositol 4,5-bisphosphate. The method shows good resolution and sufficient recovery (70-80%) for each compound. By applying this method to human platelets prelabeled with [3H]inositol and stimulated with thrombin, we found an early increase of inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate. Accumulation of glycerophosphoinositol, inositol 1 monophosphate, and an inositol monophosphate which cochromatographs with inositol 2- and inositol 4-monophosphate occurs later. The method is simple, and--after removal of salts from the incubation buffer--can be directly applied to the measurement of aqueous soluble [3H]inositol-labeled compounds in biological samples. PMID- 2994521 TI - Reversed-phase high-pressure liquid chromatography of arachidonic acid metabolites formed by cyclooxygenase and lipoxygenases. AB - High-pressure liquid chromatography is required to resolve the complex mixtures of arachidonic acid metabolites synthesized by many tissues. We have investigated some of the factors which affect the retention times of these substances in reversed-phase HPLC on columns of 5-micron octadecylsilyl silica. There are considerable differences in selectivity between mobile phases based on methanol and those based on acetonitrile, the latter being much better for cyclooxygenase products. The chromatographic behavior of peptidoleukotrienes (LTC4, LTD4, and LTE4) is quite different from that of other arachidonic acid metabolites which do not contain amino acids. Addition of phosphoric acid to the mobile phase results in very long retention times for peptidoleukotrienes. Very low concentrations of trifluoroacetic acid have effects similar to that of phosphoric acid, but as its concentration is raised, the retention times of peptidoleukotrienes decrease, whereas those of other arachidonic acid metabolites are unaffected. Changing the concentration of acetonitrile in the mobile phase also affects the retention times of peptidoleukotrienes differently from those of other metabolites. This information has been used to devise simple linear gradients which separate most of the major cyclooxygenase and lipoxygenase products of arachidonic acid metabolism. PMID- 2994522 TI - Analysis of urine for 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid using Sep-PAK cartridges for sample cleanup. PMID- 2994523 TI - Molecular biology in viral diagnosis: restriction enzyme analysis of viruses from recurrent genital herpes infections. PMID- 2994525 TI - Platelet alpha-adrenergic receptors are not down-regulated during cardiopulmonary bypass. PMID- 2994524 TI - Modulation of a GABA-ergic inhibitory circuit in the in vitro hippocampus by etomidate isomers. AB - A pulse-paired stimulation technique was used to examine the in vitro effects of the isomers of etomidate on synaptic transmission between Schaffer collaterals and CA1 pyramidal cells in the guinea pig hippocampus. Etomidate produced a dose related, stereospecific, reversible increase in paired-pulse inhibition. Replacement of Cl- by isethionate reversed the inhibition induced by (+) etomidate. Together with earlier biochemical evidence, these results show that in the mammalian CNS (+)-etomidate enhances central inhibition by increasing the effectiveness of gamma-aminobutyric acid (GABA) in a chloride-dependent fashion. PMID- 2994526 TI - A double-protease-resistant variant of transmissible gastroenteritis virus and its ability to induce lactogenic immunity. AB - A virus resistant to 2 major intestinal proteases (trypsin and alpha chymotrypsin) was derived from the attenuated Purdue strain of transmissible gastroenteritis virus. Its enzymatic stability was confirmed, in vitro, by exposure to proteolytic enzymes and to porcine intestinal fluids. Vaccination of 5 seronegative pregnant sows with the variant virus by a series of 2 oral and 1 IM inoculations resulted in high titers of neutralizing antibody in serum and colostrum. The mean antibody titer in milk whey decreased 44-fold within 1 week after parturition. At 3 days of age, the 40 pigs delivered by these sows were challenge exposed orally with virulent transmissible gastroenteritis virus. Pigs nursing the 5 vaccinated sows underwent a relatively mild clinical course of illness. The average mortality of these 40 pigs was 33%. Thirty-six pigs which had been raised by 4 nonvaccinated sows had a more severe illness, greater daily weight loss, and higher mortality (92%). PMID- 2994527 TI - Enzyme-linked immunosorbent assay for the detection of bovine papillomavirus. AB - An enzyme-linked immunosorbent assay has been developed to detect and quantitate bovine papillomavirus in partially purified and in purified viral preparations, using rabbit antiserum against group-specific papillomavirus structural antigens and alkaline phosphatase-labeled affinity purified goat antibody to rabbit immunoglobulin G. Viral detection correlated well with negative-stain electron microscopy of the various preparations and peroxidase-antiperoxidase staining of paraffin sections of the original fibropapillomas. The technique is rapidly done and will detect minute amounts of viral protein (1 ng/ml). PMID- 2994528 TI - Impaired function of bovine alveolar macrophages infected with parainfluenza-3 virus. AB - Bovine alveolar macrophages (BAM) were harvested from nonsedated cattle, adhered to glass or plastic surfaces, and infected with parainfluenza-3 (PI-3) virus at a multiplicity of infection of 10. Control and PI-3 virus-infected BAM were compared at 24-hour intervals up to 168 hours for their ability to phagocytize antibody-coated sheep erythrocytes (EAC) and latex particles, to kill Staphylococcus epidermidis, and to alter intracellular acid phosphatase concentrations. The effect of antiviral serum on phagocytic functions of virus infected cells was also evaluated. Compared with noninfected controls, alveolar macrophages infected with PI-3 virus were 15.3% less adherent to the glass or plastic surfaces at postinoculation hour (PIH) 72 and were 64.0% less adherent at PIH 168. Significant differences (P less than 0.05) between the numbers of control and infected BAM phagocytizing EAC were observed at PIH 24 through 72, with final values differing by approximately 50%. Similar changes were observed in the phagocytic efficiencies of individual cells. The PI-3 virus-infected BAM that were exposed to antiserum or to immunoglobulins against PI-3 virus had approximately a 2-fold greater inhibition in EAC phagocytosis than did infected BAM exposed to serum without PI-3 activity. Significant differences in latex particle phagocytosis were not observed between infected and control BAM. Compared with control BAM, the PI-3 virus-infected BAM contained significantly lower concentrations of acid phosphatase from PIH 48 through 96; at PIH 96, acid phosphatase concentrations were 4-fold less in infected than in control BAM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2994529 TI - Prevention by zinc of rat lung collagen accumulation in carbon tetrachloride injury. AB - Orally administered zinc was studied as a protective antifibrotic agent with respect to experimentally caused lung collagen accumulation in rats. Intraperitoneally injected carbon tetrachloride induced a diffuse alveolar damage with interstitial pulmonary fibrosis, and the morphologic findings suggested a primary toxic effect on the lungs. The carbon tetrachloride induction increased significantly the lung to body weight ratio, lung total protein and collagen content, lung total prolyl hydroxylase and galactosylhydroxylysyl glucosyltransferase activities, and daily urinary hydroxyproline excretion. Treatment with 114 mg/L of zinc in the animals' drinking water inhibited the lung prolyl hydroxylase activity and prevented the increases in lung collagen content and urinary hydroxyproline excretion but did not normalize any of the other above parameters. Enhanced lung prolyl hydroxylase activity was noted when a ferrous ion excess was included in the assay in order to reverse the competitive inhibition of the enzyme activity by zinc. It is suggested that zinc has a direct and selective preventive effect on rat lung collagen accumulation by inhibiting procollagen proline hydroxylation. PMID- 2994530 TI - Neutral metalloendopeptidase in human lung tissue and cultured cells. AB - The distribution of a neutral metalloendopeptidase (NEP), or "enkephalinase," in human lung tissue and cultured cells was compared with that of angiotensin I converting enzyme (ACE). The specific activities of NEP and ACE were measured in homogenates of fetal lung tissue and in isolated airways and pulmonary vessels. NEP activity was highest in airway tissue, and ACE activity was highest in isolated vessels. Human endothelial cells from either umbilical veins or pulmonary arteries had high ACE activity (80 to 90 nmol/h/10(6) cells) but only a trace of NEP activity (0.5 to 0.6 nmol/h/10(6) cells). Fibroblasts cultured from human lungs were low in ACE but richer in NEP than cultured endothelial cells. Fibroblasts from human foreskins or caesarean section skin were the richest source of NEP activity (60 to 80 nmol/h/10(6) cells). Immunohistochemical studies confirmed the biochemical assays. As expected, ACE was localized on the luminal surface of blood vessels, with a distribution similar to that of factor VIII antigen, an endothelial marker. In contrast, NEP was localized within the alveolar septa. Cultured endothelial cells stained only weakly for NEP in contrast to cultured fibroblasts. The location of these 2 enzymes in different cells and the differences in peptide substrate specificity suggests that they act sequentially on circulating peptides or those released within microvascular beds. PMID- 2994531 TI - Thrombocytopenia in homosexual patients. Prognosis, response to therapy, and prevalence of antibody to the retrovirus associated with the acquired immunodeficiency syndrome. AB - Thirty-three homosexual patients with thrombocytopenia (mean [+/- SE] platelet count, 50 000 +/- 7000/mm3; range, 7 to 135 000/mm3) have been followed for a mean period of 20 +/- 2 months. Six patients have developed the acquired immunodeficiency syndrome 1 to 37 months after the diagnosis of thrombocytopenia. Six patients spontaneously reverted to normal platelet counts 5 to 27 months (median, 10 months) after the diagnosis of thrombocytopenia, in the absence of splenectomy and while not receiving corticosteroids. Sixteen of seventeen patients had a moderate to excellent response while on corticosteroid treatment. Ten of ten patients had an excellent response to splenectomy which has persisted. Fifteen patients did not require treatment for their thrombocytopenia. Thirteen of fourteen patients had antibody against the retrovirus associated with the acquired immunodeficiency syndrome, as did 4 of 12 homosexual controls without thrombocytopenia. Thrombocytopenia in homosexuals is part of the complex related to the acquired immunodeficiency syndrome. PMID- 2994532 TI - Transient antibody to lymphadenopathy-associated virus/human T-lymphotropic virus type III and T-lymphocyte abnormalities in the wife of a man who developed the acquired immunodeficiency syndrome. AB - We present evidence of transmission of lymphadenopathy-associated virus (LAV)/human T-lymphotropic virus type III (HTLV-III) from a man to his wife, and a return to a normal number of T-helper lymphocytes and loss of antibody after discontinuing sexual exposure to LAV/HTLV-III. The man had hemophilia A, and developed the lymphadenopathy syndrome, antibody to LAV, and a low number of T helper lymphocytes. His wife, who had no risks for the acquired immunodeficiency syndrome other than sexual contact with him, developed LAV antibody (titer, 1:160) and a mildly decreased number of T-helper cells. The husband subsequently developed the syndrome and lost the LAV antibody. During 10 months of follow-up his wife remained clinically well, discontinued exposure to semen, and then lost the LAV antibody, and regained a normal number of T-helper cells. PMID- 2994533 TI - Prevention and control of influenza. Recommendation of the Immunization Practices Advisory Committee. Centers for Disease Control, Department of Health and Human Services. AB - These recommendations update for 1985-86 the information on the vaccine and antiviral agent available for control of influenza. Changes include addition of statements about the route of vaccine administration; the use of amantadine in medical personnel during influenza A outbreaks; the need to prepare contingency plans to expedite use of amantadine in aborting influenza A outbreaks among residents of institutions; and reduction in the dosage of amantadine for older patients or persons with seizure disorders. PMID- 2994534 TI - Microangiopathic hemolytic anemia and pulmonary small-cell carcinoma. PMID- 2994535 TI - Pancreatitis and diabetes mellitus with metastatic pulmonary oat-cell carcinoma. PMID- 2994536 TI - Methylxanthines as adenosine receptor antagonists. PMID- 2994537 TI - [Tumor markers: new prospects in the diagnosis and clinical monitoring of malignant epithelial neoplasms of the ovary]. PMID- 2994538 TI - [Laryngitis in newborn infants. Apropos of 3 cases]. AB - Laryngoscopic examination of new-born infants with laryngeal dyspnea or dysphonia usually reveals a congenital lesion, but true infections laryngitis, although rare, does still exist. Three cases are reviewed and the literature searched. Functional laryngeal signs are non-pathognomonic, all three levels of the larynx may be affected by inflammation, and pathogenic agents may be viral (herpes), bacterial (Haemophilus Para-Influenzae) or mycotic. In two of the cases reported confirmation of diagnosis was by local swab under laryngoscopic guidance. Recovery occurred after medical treatment alone and intubation was not required in any of the three patients. These findings emphasize the value of laryngoscopy with swab in all neonates with dyspnea or dysphonia in an infectious context. PMID- 2994540 TI - [77 endogalactophoric carcinomas of the breast]. PMID- 2994539 TI - [The role of cytomegalovirus in infantile deafness and deafness of unknown origin]. AB - It is widely accepted that the cause of congenital deafness is genetic in one third of cases roughly, is due to acquired affections during pregnancy or delivery in another third and remains unknown in the last third. It is possible that the cytomegalovirus (CMV) plays an important role in the latter group. The CMV is thought to be involved in 10 to 30% of cases of auditory sequelae from fetal infection, either severe neonatal CMV-induced disease, which is rare, or the frequent subclinical infections affecting an average of 1% of newborn infants. The only certain way to determine the importance of the role of CMV in deafness of unknown etiology is large-scale neonatal biologic screening followed by long-term audiologic surveillance: currently available documented data suggest that this role is very important. PMID- 2994541 TI - Human papilloma virus (HPV) infections related to cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma of the uterine cervix. AB - Human papilloma viruses (HPV) consist of a heterogenic group of viruses (32 different HPV types identified to date) known to induce a variety of squamous cell tumours (papillomas and warts) in the skin, and on mucous membranes of respiratory, gastrointestinal and genitourinary tracts. In the genital tract, the venereal wart (Condyloma acuminatum) has been recognized since ancient times, and known to be a sexually transmitted disease (STD). In 1976, two other morphologically distinct HPV lesions were described in the uterine cervix, known currently as a flat and an inverted condyloma. Subsequently, these new HPV lesions were shown to be frequently associated with concomitant cervical intraepithelial neoplasia (CIN) and carcinoma in situ (CIS) lesions, and occasionally with invasive cervical carcinomas as well. These morphological findings, substantiated by the increasing number of reports of malignant transformation of HPV lesions, as well as data from animal experiments and epidemiological surveys, have lent support to the concept that HPV might be involved in the development of cervical (and other) human squamous cell carcinomas. Further evidence has been provided by the recent discoveries of HPV structural proteins (viral antigens) and HPV type 11 DNA in lesions of CIN, as well as HPV 16 and 18 DNA predominantly in invasive cervical carcinomas. So far, HPV 16 and HPV 18 are the only HPV types with DNA shown to exist integrated in the host cell DNA. At present, cervical (and other) HPV lesions are the subject of intense study utilizing epidemiological, morphological, immunohistochemical, biochemical and molecular biological methods (recombinant gene technology) to provide further evidence of the suggested causal relationship between HPV and cancer. Prospective follow-up studies are also in progress to explore the natural history of cervical HPV lesions as well as the factors (immunological, epidemiological, synergistic actions, etc.) which modify it. Despite the rapid progress made in papilloma virus research in the last few years, many important questions have still to be answered before the final conclusions can be drawn as to the possible role of HPV in cervical carcinogenesis. PMID- 2994542 TI - Immunological findings associated with HTLV-III antibody positivity. AB - In order to evaluate the immunological findings associated with HTLV-III virus infection, T-lymphocyte subpopulations and lymphocyte stimulation responses were studied in a voluntary group of 174 homosexual men living in Finland. Antibodies against HTLV-III were found in 17 individuals. Decreased T-helper/T-suppressor ratio and low responses to mitogens (phytohaemagglutinin, pokeweed mitogen) and a specific antigen (purified protein derivative of tuberculin) were typical for HTLV-III antibody positive individuals. The reason for this decreased Th/Ts ratio was the low level of T-helper cells. In the HTLV-III antibody negative group, where a low Th/Ts ratio was occasionally seen, it was due to elevated T suppressor cells. The low T-helper cell values were in good correlation with the poor clinical status of these persons and with low stimulation responses. PMID- 2994543 TI - The glucagonoma syndrome in a Chinese man. AB - The glucagonoma syndrome is a rare clinical condition. In this syndrome there is a pancreatic islet cell tumour which secretes glucagon, associated with a distinctive skin eruption--migratory necrolytic erythema. We describe a 79 year old Chinese man who presented with the typical skin features of this condition and on investigation had a pancreatic islet cell tumour. Serum glucagon levels were markedly elevated. He had a partial pancreatectomy and the skin rash improved dramatically. However he had liver metastases and responded poorly to streptozotocin. The skin features recurred and the patient died 9 months after diagnosis. As far as we are aware, this is the first report of a Glucagonoma syndrome in a Chinese. PMID- 2994544 TI - The insulin receptor. AB - The insulin receptor is a glycoprotein with a molecular weight in the order of 300,000. There are probably two pairs of subunits joined together by disulphide bonds. The distribution of receptors appears to be tissue-specific. On liver plasma membranes they are found predominantly as singletons, whereas on adipocytes they occur mainly in groups. The groups of receptors are held together by disulphide bonds, but these are different from the bonds holding the subunits together. When insulin binds to the receptor, the hormone-receptor complex is internalised in pinocytotic invaginations in the adipocyte, and in coated pits in fibroblasts. Half the receptors are transported to lysosomes where they are degraded, and the other half are recycled to the cell surface presumably for further re-utilisation. Obese patients and those with type II diabetes have in common both a reduced number of insulin receptors and a post-receptor defect. However the degree of insulin resistance in type II diabetes cannot be accounted for on the basis of obesity alone. Moreover many type II diabetics are not obese. The insulin receptor is also altered in certain physiological states. Fasting and exercise lead to increased binding of insulin to its receptor. Pregnancy, on the other hand, may either increase or reduce binding. The effects of glucocorticoids are heterogeneous, and it is probable that the insulin resistance they induce is post-receptor in nature. Auto-antibodies to the insulin receptor is a rare cause of severe insulin-resistant diabetes, but the condition has given considerable insight into the nature of the insulin receptor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2994545 TI - Beckwith-Wiedemann syndrome. Clinical comparison between patients with and without 11p15 trisomy. AB - Thirteen previously unreported patients with Beckwith-Wiedemann syndrome are reported. They include two unrelated patients having developed nephroblastoma, and three sibs. High resolution banding techniques failed to show any evidence of trisomy 11p15 in any of these 13 patients. The clinical pictures of Beckwith Wiedemann syndrome with and without trisomy 11p15 are compared. PMID- 2994546 TI - Intake of dietary fibre and the age of menarche. AB - The relationship between menarchial age and estimated dietary fibre intake in 46 countries/communities was examined. There was a highly significant positive correlation (r = 0.84) between dietary fibre (g/1000 k calories nutrient energy) and menarcheal age. The nutritional and physiological significance of this relationship is discussed. PMID- 2994547 TI - Nutrition and hypertension. PMID- 2994548 TI - Surface-glycoprotein patterns of established human lung cancer cell lines and primary cultures. AB - The cell surface glycoprotein (GP) profiles of cell lines representing the four major histopathological lung cancer entities, squamous cell (SQC)-, small cell (SCC)-, adeno (ADC)-, and large cell carcinoma (LCC), and two primary cultures of SCC and LCC, respectively, have been examined by the galactose-oxidase tritiated sodium-borohydride cell surface labelling method, The SCC specimens (five cell lines and one biopsy) had a characteristic pattern of major surface GPs, with common GPs at apparent molecular weights (MWs) of 54 -kilo (k) Daltons (D) and 88 kD, which was discriminative from the group of non-SCC (SQC, ADC and LCC). The non-SCC group constantly expressed GPs at apparent MWs of 80 kD and 110 kD, both as established cell lines and in the primary LCC culture. The GP patterns of the SCC cell lines and the LCC cell line were retained in comparison to corresponding primary biopsy material. The propagation of an established SCC cell line without supplementation of serum did not alter the GP expression at the cell surface. Taken together, the surface GP patterns for SCC versus non-SCC appear to be reliable and reproducible markers for these tumor entities, both in biopsy material and in established cell lines. PMID- 2994549 TI - Maturation asynchrony in leukemic cells. An abnormal combination of normal cell markers. AB - Multiple monoclonal antibodies and enzyme assays were used to study maturity markers (myelo-peroxidase) and immaturity markers (terminal transferase, HLA-2) in acute myeloid leukemia cells from 35 patients. In 8 of the patients, indications were found of an expression of maturity and immaturity markers on the same cells, here in called maturation asynchrony. It is suggested that the orderly appearance and disappearance of markers during the maturation of normal cells is disordered in malignant cells, and that single markers should be used with caution for the maturation classification of tumors. The simultaneous expression of maturity and immaturity marker by tumor cells could explain also why such cells can be recognized as abnormal even in the absence of tumor specific antigens. PMID- 2994550 TI - Multiple extractions of hormone receptor from rabbit uteri and human breast tumors. AB - The cytosol hormone receptors were extracted from rabbit uteri and human tumors with tris buffer systems for three consecutive homogenization and centrifugation processes to obtain three cytosols C-1, C-2, and C-3. The assay results evaluated revealed that minimal to tremendous amounts of receptor activities were present in C-2 and/or C-3 fractions. The essential experimental conditions and some speculative mechanisms have been discussed in detail. PMID- 2994551 TI - Prediction of biological behaviour of human breast cancer using multiple parameters. A preliminary study. AB - Prediction of early metastases in individual patients has been attempted by combined evaluation of a battery of recognised parameters such as blood vessel invasion (BVI) of tumor cells, Barr body frequency (BBF), plasminogen activator (PA), collagenase, estradiol receptors (ER), progesterone receptors (PgR), and peroxidase activity (PRA) in 18 malignant and 3 benign (control) breast tumors. Since breast tumor cells spread through the blood vessels, the cases were divided into BVI (+) and BVI (-) groups. Results show that in the former group, all the cases had poor prognosis and two even had distant metastases within one year. In BVI (-) group, 9 out of 12 appeared to have good prognosis. PMID- 2994552 TI - Tissue culture studies of neurofibromatosis: effects of axolemmal fragments and cyclic adenosine 3',5'-monophosphate analogues on proliferation of Schwann-like and fibroblast-like neurofibroma cells. AB - Six dermal neurofibromas obtained from 5 patients with neurofibromatosis were dissociated and the cells were plated on polylysine-coated glass. Two principal cell types were observed in the cultures: elongated and bipolar Schwann-like cells (SLCs), and polymorphic flattened fibroblast-like cells (FLCs). Indirect immunofluorescence demonstrated that SLCs expressed surface laminin but not surface fibronectin; FLCs expressed surface fibronectin but were only weakly positive for surface laminin. Tritiated thymidine autoradiography demonstrated that cultured SLCs proliferated slowly (labeling index, 0.7 to 4.0%), whereas FLCs divided more rapidly (labeling index, 7.5 to 26.4%). Axolemmal fragments prepared from human or rat central nervous system specimens adhered to SLCs derived from each of the 6 neurofibromas, but not to FLCs. Axolemmal fragments induced a marked proliferative response of SLCs from 2 of the 6 neurofibromas but had no effect on proliferation of SLCs from the other 4 neurofibromas or FLCs from any of the 6 neurofibromas. In one patient from whom 2 neurofibromas were obtained, SLCs from one neurofibroma responded to axolemmal fragments, while SLCs from the other did not. Treatment of the cultures with 0.1 mM cyclic adenosine 3'5'-monophosphate (cAMP) analogue, 8-bromo cAMP, caused marked inhibition of proliferation of both SLCs and FLCs derived from all 6 neurofibromas. The same concentration of another cAMP analogue, dibutyryl cAMP, inhibited proliferation of SLCs but not of FLCs. PMID- 2994553 TI - Cytomegalovirus and herpes simplex virus ascending myelitis in a patient with acquired immune deficiency syndrome. AB - Progressive ascending myelitis was the presenting feature of the acquired immune deficiency syndrome (AIDS) in a homosexual man who also had Kaposi's sarcoma, Pneumocystis pneumonia, and disseminated cytomegalovirus (CMV) infection. Neuropathological studies showed profuse cytomegalic cells throughout the brain and spinal cord, but no inflammatory response. At postmortem examination, CMV and herpes simplex virus, type 2 (HSV-2), were recovered from multiple sites throughout the central nervous system (CNS). HSV-2 was isolated from the anus, but from no other extraneural site; in contrast, pathology typical of CMV was also seen in the liver, gastrointestinal tract, adrenals, and lungs. Although histopathological evidence suggesting prior CMV infection has been seen in the brains of AIDS patients, the virus has never been cultured from the CNS in these immunosuppressed hosts, nor has it been known to infect the spinal cord. The absence of an inflammatory response suggests that the pathogenesis of CNS viral infections is altered in AIDS patients. Evidence for CMV infection of the CNS in AIDS patients is no longer circumstantial. PMID- 2994554 TI - Neurotransmitter receptor alterations in Huntington's disease: autoradiographic and homogenate studies with special reference to benzodiazepine receptor complexes. AB - In vitro receptor autoradiography was used to construct semiquantitative maps of subtypes of muscarinic cholinergic (labeled with [3H]N-methylscopolamine), benzodiazepine ([3H]flunitrazepam), gamma-aminobutyric acid ([3H]muscimol), dopamine, and serotonin ([3H]spiperone) receptors in frontal cortex, parietal cortex, caudate, putamen, and globus pallidus in tissue sections from 5 patients with clinically well-evaluated Huntington's disease and 5 controls matched with respect to age, sex, and postmortem delay. Homogenates were prepared from the remaining cortical and striatal tissue and used to characterize pharmacologically these same receptors, as well as histamine, adenosine, and nitrendipine receptors. Neuronal loss and gliosis were assessed in the contralateral formalin fixed caudate and putamen. All binding sites measured (except serotonin) were reduced relative to control values in striatum primarily because of changes in the number of receptors rather than in affinity. Autoradiographic studies generally revealed that these changes were greater in the caudate than the putamen, paralleling the more severe neuropathological changes present in the caudate. In addition, autoradiographic studies demonstrated an increase in gamma aminobutyric acid-related receptors in the globus pallidus. In the cortex, receptor alterations were limited to an increase in the number of benzodiazepine receptors in the frontal cortex which was most prominent in superficial cortical layers. PMID- 2994555 TI - Tetracycline-resistant Mycoplasma hominis strains contain streptococcal tetM sequences. AB - Clinical isolates of Mycoplasma hominis resistant to high levels of tetracycline contained DNA sequences homologous to the streptococcal tetracycline determinant, tetM. In contrast, none of the susceptible M. hominis isolates tested carried this determinant. This is the first description of tetM in an unrelated genus and suggests the spread of tetM from Streptococcus spp. to Mycoplasma spp. PMID- 2994556 TI - Penetration of sulbactam-ampicillin and clavulanic acid-amoxicillin into the pelvic peritoneum. AB - Sixteen patients were given single intravenous injections of ampicillin (0.5 g) with sulbactam (0.5 g), and 15 patients were given amoxicillin (1 g) with clavulanic acid (0.2 g) before elective laparoscopy. At 2 h after dosing, the concentrations of the four compounds in serum and in the peritoneal fluid from the Pouch of Douglas and the ratio of each combination reached levels shown to be effective for antimicrobial activity in vitro. PMID- 2994557 TI - In vitro susceptibility of Campylobacter jejuni to 27 antimicrobial agents and various combinations of beta-lactams with clavulanic acid or sulbactam. AB - The in vitro susceptibility of human isolates of Campylobacter jejuni was investigated with 27 antibiotics and 8 combinations of beta-lactams with clavulanic acid or sulbactam. Ansamycin, the new quinolines, erythromycin, and cefpirome were the most active drugs against C. jejuni; amoxicillin, ampicillin, cefotaxime, and ceftazidime 90% of the isolates, greater than or equal to 50 mg/liter). The activity of various beta-lactams was unchanged by the addition of clavulanic acid or sulbactam. PMID- 2994558 TI - Comparison of three DNA hybridization methods for detection of the aminoglycoside 2"-O-adenylyltransferase gene in clinical bacterial isolates. AB - Two rapid DNA hybridization methods in which whole-cell lysates fixed to nitrocellulose were used were compared with Southern hybridization of purified plasmid or chromosomal DNA for the ability to identify the 2"-O adenylyltransferase [ANT(2")] gene in 42 enzymatically defined isolates of gram negative bacilli. A DNA restriction fragment isolated from an ANT(2") gene cloned into pBR322 and radiolabeled with 32P was used as the probe in all three procedures. Under conditions of high stringency, agreement was obtained between the Southern hybridization method and detection of the ANT(2") enzyme by the phosphocellulose paper binding assay or resistance phenotype in 39 of the 42 strains tested. By using these characterized strains, colony hybridization was shown to be unsatisfactory as a rapid technique for detecting the ANT(2") gene, due to the high number of false-positive and -negative signals obtained. Compared with Southern hybridization, however, spot hybridization (SPH) proved highly reliable for detecting the ANT(2") gene in both members of Enterobacteriaceae and Pseudomonas aeruginosa harboring R factors ranging in size from 23 to 150 kilobases. The relatively low copy number of the 150-kilobase plasmids decreased the sensitivity of SPH, necessitating a minimum cell density of 5 X 10(6) cells per spot. SPH proved to be a very useful method for rapidly screening large numbers of clinical isolates for this resistance determinant. PMID- 2994559 TI - Antiviral and antimetabolic activities of neplanocins. AB - Of a series of carbocyclic analogs of adenosine, in which the ribose moiety was replaced by a cyclopentenyl ring, neplanocin A, or (-)-9-[trans-2, trans-3 dihydroxy-4-(hydroxymethyl)cyclopent-4-enyl]adenine proved particularly effective in inhibiting the multiplication of DNA viruses (i.e., vaccinia), (-)RNA viruses (i.e., parainfluenza, measles, and vesicular stomatitis), and double-stranded RNA viruses (i.e., reo) in vitro in cell culture. Depending on the cells used, the MIC of neplanocin A for these viruses ranged from 0.01 to 4 micrograms/ml, and depending on the parameter used to assess toxicity for the host cell, the specificity index of neplanocin A ranged from 50 to 4,000. As postulated before for other adenosine analogs, neplanocin A may owe its antiviral action to inhibition of S-adenosylhomocysteine hydrolase, hence perturbation of transmethylation reactions. In vivo, neplanocin A afforded only marginal protection against a lethal infection of mice with vesicular stomatitis virus. PMID- 2994561 TI - Metal chelators as potential antiviral agents. AB - Metal-chelating compounds can inhibit virus-induced enzymes in infected cells by coordinating with metal ions at their active sites. Consideration of the coordinating properties of ligands can explain the antiviral activity of these compounds. The antiviral actions of a number of compounds (e.g., thiosemicarbazones, pyrophosphate analogues, beta-diketones, cyclic polyethers and flavanoids) are discussed in the light of their metal-chelating properties. PMID- 2994560 TI - Construction of a gentamicin resistance gene probe for epidemiological studies. AB - A 7.7-kilobase BamHI fragment was cloned from the transconjugant of a clinical isolate of Escherichia coli containing a 120-kilobase multiresistance IncC plasmid. The recombinant plasmid conferred resistance to kanamycin, gentamicin, tobramycin, sulfamethoxazole, and trimethoprim. This clone was used to generate a series of subclones from which a 2.0-kilobase BamHI-HindIII probe containing a gentamicin 2''-O-adenylyltransferase [AAD(2'')] gene was obtained. This probe hybridized specifically in both colony and Southern hybridizations with the AAD(2'') gene but not with other resistance genes, including other aminoglycoside modifying genes, or with a reference IncC plasmid lacking the AAD(2'') gene. The AAD(2'') gene may be part of a transposon, since hybridization occurred with both nonconjugative plasmids and the chromosome in some isolates. PMID- 2994562 TI - Natural antiviral activity of mouse macrophages against encephalomyocarditis virus. AB - Resident mouse peritoneal cells (PC) express a significant antiviral activity against encephalomyocarditis virus (EMCV) in vitro, as judged by decreased virus yield from infected mouse embryo fibroblasts (MEF). This natural antiviral activity of PC was not due either to enhanced lysis of virus-infected cells, as these were protected from lysis rather than destructed by PC, or to interferon (IFN) production, as no direct correlation between IFN and anti-EMCV activity was found. Among PC, macrophages (M phi) appear to be responsible for the anti-EMCV activity, which was indeed attributable to a Thy 1.2-negative, adherent mononuclear cell. Moreover, M phi-defective C3H/HeJ mice showed a significant impairment of anti-EMCV activity, whereas M phi of mice defective for natural killer (NK) activity (bg/bg, SJL/J) or for mature T cells (nu/nu) possessed an intact antiviral capacity. PMID- 2994563 TI - Feline calicivirus subunit vaccine--a prototype. AB - A vaccine was prepared from a subunit component, antigenically similar to the whole feline calicivirus (FCV) particles. Despite the limited number of animals available for this study we were able to demonstrate that the vaccine protected cats when challenged with a virulent strain of the virus while the non-vaccinates kept as controls developed clinical and histopathological symptoms of the calicivirus disease. PMID- 2994564 TI - A paradoxical effect of hydralazine on prolyl and lysyl hydroxylase activities in cultured human skin fibroblasts. AB - The effect of hydralazine on several parameters of collagen biosynthesis has been studied in cultured human skin fibroblasts. Cells treated with hydralazine synthesized procollagen which was severely deficient in hydroxyproline and hydroxylysine, indicating inhibition of prolyl and lysyl hydroxylase reactions in the cell. Assays of prolyl and lysyl hydroxylase activities, however, revealed markedly increased levels in hydralazine-treated cells. The stimulatory effect of hydralazine could not be simulated in cell extracts, demonstrating its requirement for intact cells. The effect occurred slowly over a period of 96 h and was dependent on hydralazine concentration between 10 and 100 microM. This phenomenon was also observed in lysyl hydroxylase-deficient mutants. In both normal and mutant cells the relative magnitude of the hydralazine effect could be modified by ascorbic acid in the culture medium. Ascorbic acid increased the response of prolyl hydroxylase to hydralazine from 1.5- to 2-fold to 3- to 7 fold, whereas it decreased the response of lysyl hydroxylase to hydralazine from 4- to 8-fold to 2- to 3-fold. Total collagen synthesis was substantially reduced in hydralazine-treated cells; the time course and the dose-response relationship were similar to those observed for the hydroxylases. alpha, alpha'-Dipyridyl, an iron chelator, mimicked these effects of hydralazine. The studies suggest the existence in cultured cells of a compensatory mechanism for overproduction of these crucial enzymes in collagen biosynthesis, a mechanism which remains functional in cells derived from patients afflicted with hydroxylysine-deficient collagen disease. PMID- 2994565 TI - Multiple forms of cyclic nucleotide phosphodiesterase in a murine adrenal cortex cell line (Y-1). AB - Murine adrenal cortex tumor Y-1 cells contained both soluble and particulate forms of cyclic nucleotide phosphodiesterase (3',5'-cyclic AMP 5'-nucleotide hydrolase, EC 3.1.4.17). The soluble forms of the enzyme comprised 80% of total cellular phosphodiesterase activity. The soluble enzyme(s) hydrolyzed both cyclic AMP and cyclic GMP, with apparent Km values of 125 and 30 microM, respectively. Soluble cyclic AMP phosphodiesterase showed marked inhibition by the calcium chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), and the anticalmodulin drugs, chlorpromazine, N-(6-aminohexyl)-5-chloro-1 naphthalenesulfonamide (W-7), and calmidazolium. No alteration in soluble cyclic GMP phosphodiesterase activity was observed when cyclic AMP was added to the assay. Resolution of the soluble enzymatic activity by DEAE-cellulose chromatography in the presence of calcium showed two peaks of phosphodiesterase activity. Further purification of one of these peaks on DEAE-cellulose in the presence of EGTA yielded a phosphodiesterase activity peak that was stimulated fivefold by calmodulin. The particulate form of the enzyme hydrolyzed both cyclic AMP anc cyclic GMP; the apparent Km values for these substrates were similar (90 and 100 microM, respectively). Hydrolysis of cyclic GMP by the particulate enzyme was inhibited by cyclic AMP in a concentration-dependent manner with an apparent half-maximal inhibitory concentration of 100 microM. The particulate form of phosphodiesterase was not inhibited by EGTA or anticalmodulin drugs. PMID- 2994566 TI - Rabbit skeletal muscle acylphosphatase: the amino acid sequence of form Ra1. AB - Acylphosphatase was purified from rabbit skeletal muscle by a procedure involving an affinity chromatography step on immunoadsorbent and subsequent ion-exchange chromatography. Three molecular forms with acylphosphatase activity, named Ra1, Ra2, and Ra3, were purified and characterized with respect to molecular weight, amino acid composition, and main kinetic parameters. The amino acid sequence of Ra1 is given in the present paper. The Ra1 form consists of a single polypeptide chain of 98 amino acid residues and contains only one cysteine residue at position 21 that is S-S bound to glutathione. The polypeptide chain has an acetyl group blocking the NH2 terminus. Ra1, Ra2, and Ra3 are compared with the corresponding molecular forms isolated from skeletal muscle of horse and turkey. PMID- 2994567 TI - Isolation and characterization of a cyclic AMP receptor protein from luminous Vibrio harveyi cells. AB - A cAMP receptor protein (CRP) species was purified from the luminous Vibrio harveyi cells to apparent homogeneity. This protein had a dimeric structure with a molecular weight of 23,000 per subunit. Among all eight nucleotides tested, only cAMP (Kd = 3 to 4 microM at 0 degrees C and 52 microM at 23 degrees C) and cGMP (Kd = 6 to 10 microM at 0 degrees C and 67 microM at 23 degrees C) bound to this protein. Its binding to poly(dI-dC), poly(dA-dT), and DNA fragments isolated from V. harveyi cells were all significantly enhanced by the addition of cAMP. Based on patterns of limited proteolysis by trypsin, this CRP assumes different conformations in the absence and presence of cAMP. Also consistent with this conclusion is the finding that the binding of cAMP to CRP induced about 50% quenching of the CRP fluorescence with a concomitant 3-nm blue shift from the original 336-nm emission peak. The binding of cGMP resulted in similar fluorescence changes but had no apparent effect on the pattern of proteolysis by trypsin. Using an in vitro transcription system known to be dependent on cAMP and Escherichia coli CRP, the synthesis of a run-off transcript product was also significantly enhanced by cAMP and this V. harveyi CRP. PMID- 2994568 TI - Response of superoxide anion production by guinea pig eosinophils to various soluble stimuli: comparison to neutrophils. AB - The effect of various soluble stimuli on the superoxide production by guinea pig eosinophils was studied in comparison to neutrophils. Phorbol myristate acetate, A23187, digitonin, NaF, concanavalin A (Con A), and cytochalasin E stimulated eosinophils and neutrophils to release O2-. The O2- production by these active agents, excluding Con A and cytochalasin E, was much greater in eosinophils than in neutrophils. Formyl-Met-Leu-Phe stimulated the O2- production in neutrophils but not in eosinophils. Neither histamine nor Val/Ala-Gly-Ser-Glu stimulated the O2- production in both types of leukocytes. A23187- or Con A-stimulated O2- production was greatly enhanced by cytochalasin B pretreatment in neutrophils but not in eosinophils. Lineweaver-Burk analysis of NADPH oxidase in particulate fractions showed that eosinophils possessed the same Km values as neutrophils and greater Vmax values than neutrophils, suggesting that eosinophils have a similar, but more active, O2- -generating enzyme system than neutrophils. PMID- 2994570 TI - 1H nuclear magnetic resonance studies of the conformations of vitamin D compounds in various solvents. AB - Using proton NMR, the solution conformation of the A ring of vitamin D3 and its analogs has been studied by application of the Karplus relation to the observed coupling constants. The A-ring conformations of vitamins D3, D2, and 25 hydroxyvitamin D3 were found to be solvent dependent, with a clear preference for an equatorial hydroxyl group in polar solvents such as methanol and dimethyl sulfoxide. Conversion of the hydroxyl group to an acetate did not affect solution conformation appreciably, but the corresponding t-butyl-dimethylsilyl ether derivative of vitamin D3 showed a strong preference for the 3 beta-equatorial conformer. The A-ring conformation of the active hormone, 1,25-dihydroxyvitamin D3, which has two hydroxyl groups competing for the equatorial position, was found not to be solvent-dependent. PMID- 2994569 TI - Stimulation of poly(ADP-ribose) synthetase activity in the lungs of mice exposed to a low level of ozone. AB - Toxic effects of O3 are mediated through the formation of free radicals, which can cause DNA strand breaks. Cellular DNA repair is dependent upon the formation of poly(ADP-ribose) (polyADPR) catalyzed by polyADPR synthetase. In order to evaluate whether O3 exposure inflicted DNA damage in lung tissue, we measured the activity of polyADPR synthetase (known to be activated in response to DNA damage) in mouse lungs after exposure to 0.45 ppm (882 micrograms/m3) O3 for up to 7 days. The enzyme activity was stimulated with O3 exposure relative to unexposed controls, showing a 20% (P less than 0.05) increase at Day 5 and 42% (P less than 0.001) at Day 7 of O3 exposure. In addition, the activity of superoxide dismutase (SOD), known to be stimulated in response to production of superoxide anion (.O2 ), was measured as an indicator of free radical involvement. Relative to unexposed controls, the SOD activity in exposed animal lungs increased to the peak level at Day 5 (48%, P less than 0.001) and then declined at Day 7 of O3 exposure but was still higher than controls (17%, P less than 0.05). When animals, after 5 days of O3 exposure, were allowed to recover in filtered room air, the activities of both enzymes declined to their respective control values in 6 days. These results suggest a possible temporal relationship between O3 injury and the activities of polyADPR synthetase and a free radical scavenging enzyme, SOD. The stimulation of polyADPR synthetase activity with O3 exposure, reflecting a response to lung cellular DNA repair, may be a sensitive indicator for assessing DNA damage in oxidant injury. PMID- 2994571 TI - Transient proton inflows during illumination of anaerobic Halobacterium halobium cells. AB - In Halobacterium halobium strain R1 containing both bacteriorhodopsin (bR) and halorhodopsin (hR), the light-driven proton uptake has been experimentally resolved into three transient inflows which are superimposed on the larger proton outflow. Under anaerobic conditions the early proton uptake consists of two components: (i) an inflow which can be blocked using the ATPase inhibitor, Dio-9, and (ii) an inflow which can be abolished by low concentrations (less than 125 nM) of triphenyltin chloride (TPT) with no inhibition of ATP synthesis. At pH 6 these two inflows are approximately equal in magnitude and duration. Measurements of buffering capacity and internal pH indicate that Dio-9 does not alter the passive proton-hydroxyl permeability of the cell membrane and that TPT at these low concentrations slightly decreases it. At later times of illumination (iii) another transient light-driven proton inflow occurs. This inflow is most evident during the first illumination after cells have been stored for extended times in the dark. The internal potassium concentration is not changed by storage, but apparently sodium is taken up, and we attribute the third inflow to sodium extrusion in exchange for protons. These results demonstrate the existence of three distinct triggered secondary proton inflows through the cell membrane. The proton inflow, which can be inhibited by Dio-9, correlates with proton-dependent ATP synthesis. The second inflow, which disappears in the presence of low TPT concentrations, is a passive proton uptake through an otherwise unidentified channel in response to electrogenic chloride pumping by bacteriorhodopsin and/or halorhodopsin. The third system correlates with the Na+/H+ antiporter function that has been demonstrated in H. halobium cell envelope vesicles. In contrast to observations on hR-containing vesicles, which can develop substantial Cl- gradients, the electroneutral OH-/Cl- exchange function can be demonstrated in intact cells only at TPT concentrations greater than 500 nM. PMID- 2994572 TI - Oxidation in H2O and D2O of 6-ethyl-5H-dibenz(c,e)azepine and 1 methylnicotinamide by aldehyde oxidase from rabbit liver. AB - We report the solvent hydrogen isotope effects associated with the oxidation of 6 ethyl-5H-dibenz(c,e)azepine (6-ED) and 1-methylnicotinamide (1-MN) catalyzed by aldehyde oxidase from rabbit liver using two assay methods. The first uses 2,6 dichlorophenolindophenol (DCI) as electron acceptor in an indirect assay in which the bleaching of DCI is measured as the substrate is oxidized. The second uses molecular oxygen as electron acceptor in a direct assay in which the oxidation of 1-MN to its pyridones is accompanied by an increase in absorbance at 300 nm and the oxidation of 6-ED to its lactam product is accompanied by a decrease in absorbance at 335 nm. We have found a solvent hydrogen isotope effect close to unity in the turnover number for each substrate and for each assay method. The solvent hydrogen isotope effects on kcat/Km ranged from 0.4 to 1.1. We conclude that changes in bonding of hydrogen in solvent water, including hydrolysis of or general base attack on an enzyme-intermediate complex, do not play a rate contributing role in the maximal velocity of oxidation of 1-MN and 6-ED catalyzed by aldehyde oxidase from rabbit liver. PMID- 2994573 TI - Further characterization of the peptidyl alpha-amidating enzyme in rat anterior pituitary secretory granules. AB - In previous studies we have demonstrated a secretory granule-associated peptide alpha-amidation activity in rat anterior, intermediate, and posterior pituitary. This activity is capable of converting 125I-labeled synthetic D-Tyr-Val-Gly to labeled D-Tyr-Val-NH2, and requires ascorbic acid, CuSO4, and molecular oxygen for optimal activity. Because of the requirement for peptides with COOH-terminal glycine residues, and cofactor requirements similar to monooxygenases such as dopamine beta-monooxygenase, we have proposed that the alpha-amidating enzyme be named peptidylglycine alpha-amidating monooxygenase, or PAM. The present study focused on (i) verifying that PAM could utilize a physiologically relevant peptide substrate, and (ii) demonstrating the retention of the cofactor requirements with purification of PAM. PAM (Mr = 50,000) was partially purified from rat anterior pituitary secretory granules and was shown to be capable of converting alpha-N-acetyl-ACTH(1-14) to alpha-N-acetyl-ACTH(1-13)NH2 (alpha melanocyte stimulating hormone) and ACTH(9-14) to ACTH(9-13)NH2. The optimal rates for these conversions were dependent on ascorbic acid and CuSO4. Kinetic analyses, using the model compound D-Tyr-Val-Gly as the peptide substrate, demonstrated that, compared to the crude granule extract, the partially purified enzyme displayed increased apparent affinities for both the peptide substrate and ascorbate. These analyses also showed that the Km for D-Tyr-Val-Gly was dependent on the concentration of ascorbate, while the Km for ascorbate was constant over a wide range of D-Tyr-Val-Gly concentrations. The results presented here indicate that PAM can alpha-amidate physiologically relevant peptides related to alpha MSH, and performs the reaction in an ascorbate-dependent fashion. Retention of the ascorbate and copper requirements with purification further support the hypothesis that these cofactors are important requirements for the COOH-terminal alpha-amidation of neuro and endocrine peptides. PMID- 2994574 TI - [Skin cancer and precancerosis]. AB - Both basal cell carcinoma (bcc) and squamous cell carcinoma (SCC) are very common in Japan, followed by Bowen's disease, actinic keratosis and Paget's disease. Bcc has a strong tendency to invade deeply, although distant metastasis is extremely rare. Differentiation from malignant melanoma is sometimes necessary in the case of pigmented bcc. As for scc, the most common predisposing factors in Japan are various kinds of scars. Recently, however, incidence of actinic keratosis-scc is has been increasing. Verrucous carcinoma is a variant with low-grade malignancy, and is considered to be induced by human papilloma virus. In genital Paget's-and Bowen's disease dermal invasion is seen in about 20% of cases various types of internal malignancy occurring in about 15%. Although surgery is the first choice of treatment for skin cancer and precancerous disease, peplomycin is very effective for scc. The latter has generally been used mainly in the preoperative stage. PMID- 2994575 TI - [Cell kinetics of human lung small cell carcinomas transplanted into nude mice]. AB - Three human lung small cell carcinoma (SCC) xenografts serially transplanted into BALB/c nude mice were used for cell kinetic analysis. SCC strains, Lu-24, Lu-130 and Lu-134, were inoculated into the backs of nude mice, and when each tumor reached more than 300 mg, 50 microCi of 3H-thymidine per mouse was administered ip. The percentage labeled mitosis curve was obtained from the autoradiographic specimens which were labeled by the pulse-chase method. Cell cycle phase, growth fraction (GF) and cell loss factor (CL) were assessed by the methods of Quastler, Fujita and Steel, respectively. These cell kinetic parameters were compared with those of six control human gastrocolic and breast carcinoma xenografts which were previously reported by us. It was noticed that the cell cycle times (Tc) of SCC were statistically shorter than those of controls and this short Tc was found to be dependent on their short post-mitotic resting phases. GFs and labeling indices of SCC were observed to be statistically lower than those of controls, suggesting an incomplete adaptation of SCC xenografts to the host nude mice. Whereas some modifications by the host mice on the cell kinetics were supposed, the characteristics of SCC cell kinetics were thought to be essentially preserved in nude mice and these kinetic parameters were observed to be stable throughout the serial transfers. Accordingly, the SCC xenograft-nude mouse system was considered to be useful as an experimental therapeutic model of human lung small cell carcinomas. PMID- 2994576 TI - [A phase II study of oral VP-16 in primary lung cancer]. AB - A clinical trial of a new semi-synthetic podophyllotoxin, VP-16, was undertaken in patients with primary lung cancer; 56 of the 81 evaluable patients had small cell carcinoma, 9 adenocarcinoma, 8 epidermoid carcinoma, 7 large cell carcinoma, and 1 adenosquamous carcinoma. A dose of 200 mg/body/day orally for 5 consecutive days was administered every 3 to 4 weeks. Partial response (PR) was attained in 19 out of 81 (23%) and PR + MR was 35 out of 81 (43%). PR and minor response (MR) were seen as follows; small cell carcinoma, 17 PR (30%), 13 MR; epidermoid carcinoma, 2 PR (25%), 1 MR; adenocarcinoma, 1 MR; adenosquamous carcinoma, 1 MR. The dose-limiting factor was leukopenia, while thrombocytopenia was experienced in 2 cases. Clinical toxicities noted were anorexia, nausea, vomiting, stomatitis, diarrhea and alopecia, but these were well tolerated in all cases. The result indicated that VP-16 has considerable efficacy in small cell carcinoma and epidermoid carcinoma of the lung and hence its usefulness in combination chemotherapy was suggested. PMID- 2994577 TI - [A randomized trial of 3-drug combination chemotherapy in small cell lung cancer- CPA/ACNU/VCR vs ADR/ACNU/VCR]. AB - From April 1981 to February 1983, 116 untreated patients (ECOG PS 0-3) with histologically or cytologically proven small cell lung cancer were randomly allocated to chemotherapy regimen using CPA.ACNU.VCR (CNV, n = 64) or ADR.ACNU.VCR (ANV, n = 52). The objective tumor response was 29.7% (19/53) for the CNV regimen and 48.1% (25/48) for the ANV regimen, but there was no statistically significant difference in these groups. Median survival time was 22.9 w for the CNV regimen (n = 64) and 42.4 w for the ANV (n = 52) regimen. The survival rate was statistically significantly higher for the ANV regimen compared to that of the CNV regimen (P greater than 5%). The toxicity showed no difference between these groups. Addition of ADR to ACNU + VCR was effective, but addition of CPA to these two drugs was not effective. PMID- 2994578 TI - Impetigo herpetiformis and hypoparathyroidism. AB - A 64-year-old woman had recurrent bouts of impetigo herpetiformis triggered by episodes of hypocalcemia secondary to postsurgical hypoparathyroidism. Correction of the hypocalcemia resulted in rapid clearing of her skin disease. Impetigo herpetiformis should be considered a variant of generalized pustular psoriasis. Although best known for its association with pregnancy, this disease should also be considered an important cutaneous manifestation of the postthyroidectomy syndrome. PMID- 2994579 TI - Linear papules on the neck of a child. Syringocystadenoma papilliferum. PMID- 2994580 TI - Firm linear plaque on the lip of a child. Granular cell tumor (granular cell myoblastoma, granular cell schwannoma). PMID- 2994581 TI - [Stromal sarcoma of the breast. 2 cases]. PMID- 2994582 TI - Porcine catabolin stimulates prostaglandin E2 secretion but does not affect intracellular cyclic AMP production in pig synovial fibroblasts. AB - Responses in vitro to partially purified porcine leucocyte catabolin were studied in pig synovial fibroblasts.In serum-free cultures catabolin was found to stimulate secretion of prostaglandin E2 (PGE2) in a time and concentration dependent manner. The initial stimulation of PGE2 secretion occurred only after a latent interval of six hours. In the same cell line catabolin was found to have no effect on the production of cyclic adenosine monophosphate (cAMP) at times ranging from 30 s to 20 min, even at concentrations up to 15 times greater than that required to promote accelerated release of glycosaminoglycans from cultured bovine nasal cartilage. It is therefore concluded that in pig synovial fibroblasts catabolin evokes a delayed secretion of PGE2 but does not alter cyclic AMP production. PMID- 2994583 TI - Proglumide, a gastrin receptor antagonist, inhibits growth of colon cancer and enhances survival in mice. AB - Some tumors are responsive to hormone manipulation. Some gastric and colonic adenocarcinomas from both humans and animals have specific gastrin receptors. A transplantable mouse colon adenocarcinoma cell line (MC-26) contains gastrin receptors; growth of MC-26 colon cancer in vivo is stimulated by pentagastrin (PG). The purpose of this study was to determine whether a gastrin-receptor antagonist, proglumide (PGL), would inhibit growth of MC-26 colon cancer and prolong survival in tumor-bearing mice. Subcutaneous tumors were induced by injecting single-cell suspensions of MC-26 cells into 50 mice divided into 10/group. In Experiment 1, all mice received 1 X 10(5) tumor cells and treatment groups were divided as follows: Group A received intraperitoneal (IP) saline (0.2 ml tid beginning on day 1); B, IP, PGL (250 mg/kg tid) from day of tumor cell inoculation; and C, IP PGL (250 mg/kg tid) from day 7 after tumor implantation. In Experiment 2, mice were inoculated with half the number of tumor cells. Group I mice received saline and Group II received PGL in the same manner starting on day 1. Tumors were measured and all mice were sacrificed on day 23. In Experiment 1, mean tumor area in Group B (PGL-treated) was significantly smaller than Group A on days 11, 14, 17, and 21. Tumors of Group C were significantly smaller than controls on day 21. Survival of PGL-treated mice was significantly prolonged. In Experiment 2, mean tumor area, mean tumor weight, and tumor DNA and RNA content were significantly less in the PGL-treated group than control. It was concluded that growth of a gastrin-responsive colon cancer was inhibited and host survival was enhanced by treatment with a gastrin-receptor antagonist. Hormone manipulation may be a useful treatment for gastrointestinal cancers. PMID- 2994584 TI - Thymic regulation of brain cortex beta-adrenoceptors during development and aging. AB - The influence of the thymus on beta-adrenoceptors has been studied in the brain cortex of mice during developing and aging. Affinity of beta-adrenoceptors shows no statistically significant changes in the various animal models investigated. Receptor density shows a fall in both athymic nude mice and in old normal mice. Receptor density, in particular, decreases progressively with advancing age. It has been demonstrated that thymus exerts a regulatory role in both development and aging, as a neonatal thymic graft is capable of reversing the receptor impairments found in young athymic nude mice and in old normal mice. PMID- 2994585 TI - Angiotensin-converting enzyme activity. A potential marker of tissue hypothyroidism in critical illness. AB - We measured serum angiotensin-converting enzyme (ACE) activity radiometrically as a possible indicator of reduced thyroid function in 57 euthyroid controls, 27 patients in a noncardiac intensive care unit (13 with medical and 14 with surgical disorders), and 29 patients having coronary artery bypass grafting. In the last group, blood was obtained preoperatively and one day and one month after surgery (group 1; n = 18) or preoperatively and six hours and one day after surgery (group 2; n = 11). Patients in group 1 had significant reductions in levels of serum thyroxine (T4), triiodothyronine (T3), and thyrotropin response to protirelin one day postoperatively. The ACE activity fell significantly. Patients in group 2 had low levels of T4, T3, thyrotropin, and ACE six hours postoperatively. All these levels remained low the next day, and free T4 and free T3 levels were also reduced; the reverse T3 level became elevated. Changes in ACE significantly paralleled changes in T3. The 27 patients without coronary artery bypass grafting also had significant reductions in serum T4, T3, and ACE levels. Dilution studies and dialysis of serum with low ACE activity failed to demonstrate an inhibitor to explain the reduced enzyme function. PMID- 2994586 TI - Anomalous glucose and insulin responses in patients with insulinoma. Caveats for diagnosis. AB - Two patients with insulinomas had unusual glucose and insulin-secretory dynamics in response to prolonged fasting. In patient 1, low insulin values persisted throughout three separate supervised fasts without a steady rise in the insulin glucose ratio. In patient 2, a rising insulin-glucose ratio during a fast returned to normal after a documented catecholamine surge following a transient hypoglycemic episode. While patient 1 had clearly elevated proinsulin values of 52% to 57%, patient 2 had a near-normal value of 23%. The diagnosis of an insulinoma can usually be made by obtaining simultaneous glucose and insulin values during a prolonged supervised fast. Rarely, however, anomalous results may be obtained during supervised fasts of patients with insulinoma, and a broader range of diagnostic tests will be required to establish the correct diagnosis. PMID- 2994587 TI - [Topographic diagnosis of true posterior infarction. Comparison of the vectorcardiogram and myocardial scintigraphy]. AB - The aims of this comparative study by vectorcardiography and myocardial scintigraphy in the topographical analysis of primary inferior and/or posterior wall infarction, were: to obtain data confirming the value of identifying true posterior wall infarction; to confirm the diagnostic value of vectorcardiography in this condition. The patients in this retrospective study were admitted to hospital for primary inferior and/or posterior wall infarction and underwent vectorcardiography and myocardial scintigraphy either with Thallium 201 (137 patients) or 99m Technetium (88 patients) in the acute phase. The scintigraphies of all patients included showed hypofixation compatible with inferior and/or posterior infarction as this was used as the topographical reference. The results of vectorcardiography and myocardial scintigraphy were concordant in 164 of the 225 patients (72.8%), 18 with true posterior infarction [%), 110 with inferior wall infarction (48.8%) and 36 with postero-inferior wall infarction (16%). The results were discordant in 61 patients (27.1%); infarcts of the inferior or posterior walls according to one technique, were observed on the posterior or inferior walls with the other. The majority of these cases had postero-inferior wall changes on vectorcardiography and inferior wall infarction alone on scintigraphy (35 patients: 15.5%). The specificity of the vectorcardiographic signs of true posterior wall infarction remained satisfactory.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2994588 TI - Sites of phospholipid biosynthesis during induction of intracytoplasmic membrane formation in Rhodopseudomonas sphaeroides. AB - A rapid, gratuitous and cell-division uncoupled induction of intracytoplasmic photosynthetic membrane formation was demonstrated in low-aeration suspensions of chemotrophically grown Rhodopseudomonas sphaeroides. Despite a nearly 2-fold increase in phospholipid levels, no significant increases were detected in the specific activities of CDP-1,2-diacyl-sn-glycerol:sn-glycerol-3-phosphate phosphatidyltransferase (phosphatidylglycerophosphate synthase, EC 2.7.8.5) and CDP-1,2-diacyl-sn-glycerol:L-serine O-phosphatidyltransferase (phosphatidylserine synthase, EC 2.7.8.8), the first committed enzymes of anionic and zwitterionic phospholipid biosyntheses, respectively. The distribution of phosphatidylglycerophosphate and phosphatidylserine synthase activities after rate-zone sedimentation of cell-free extracts indicated that intracytoplasmic membrane phospholipids were synthesized mainly within distinct domains of the conserved cytoplasmic membrane. Labeling studies with 32Pi and L [3H]phenylalanine suggested that preexisting phospholipid was utilized initially as the matrix for insertion of intracytoplasmic membrane protein that was synthesized and assembled de novo during induction. PMID- 2994589 TI - Evidence for cAMP-mediated control of isoleucyl-tRNA synthetase formation in Escherichia coli K-12. AB - The ability of cAMP to inhibit isoleucyl-tRNA synthetase (IRS) formation has been demonstrated in wild type K-12 Escherichia coli and two adenyl-cyclase (cya) mutants. cAMP appeared not to have any effect on either the valyl- or arginyl tRNA synthetase (VRS and ARS respectively). Addition of cAMP led to a reduction in rate of IRS synthesis but not VRS or ARS. Furthermore, derepression of IRS and VRS by isoleucine limitation was completely prevented by cAMP. PMID- 2994591 TI - Heterogeneity, molecular weight and stability of an oncogenic papovavirus of the Syrian hamster. AB - Sedimentation, isopycnic density gradient and molecular weight studies of an oncogenic papovavirus of the Syrian hamster have been performed. The sedimentation coefficient of 223 S, the buoyant density of 1.340 g/ml, and the molecular weight of 27.5 X 10(6) are in close correspondence with those of SV 40 (simian virus 40) and polyoma viruses. Deviating from these members of the papovavirus group, a sedimentation heterogeneity resulting in the presence of dimers and higher virion oligomers up to pentamers besides heterogeneous material of viral origin was demonstrated by sedimentation velocity. Also, a microheterogeneity of the buoyant density of the virus was demonstrable which is discussed to reflect this virion association. The stability of hamster papovavirus was examined after exposure to alkaline pH and the virus destabilizing agents dithiothreitol and ethyleneglycol-bis-N,N'-tetraacetic acid. In neutral conditions a nucleoprotein complex of 170 S was demonstrated. Further disintegration of this complex with increasing pH resulted in its conversion to nucleoprotein complexes of 125 S and 90 S in analogy to SV 40 and polyoma viruses. PMID- 2994590 TI - Pituitary-adrenal axis rhythm disturbances in psychiatric depression. AB - We studied disturbances in the circadian pattern of plasma corticotropin and cortisol concentrations in 25 depressed patients (eight dexamethasone suppression test [DST] nonsuppressors and 17 suppressors) and 21 normal control subjects. Blood samples were drawn every 20 minutes for 24 hours before the administration of dexamethasone, and for a second 24 hours after the administration of 1 mg of dexamethasone. The corticotropin and cortisol level rhythms were examined using three different statistical methods. Nonsuppressors averaged greater elevations in plasma cortisol and corticotropin levels than did subjects in the other two groups, both before and after administration of the dexamethasone. The cortisol levels of the suppressors were virtually identical to those of the control subjects. However, the suppressors had significant elevations of corticotropin levels compared with normal control subjects, especially on the day before taking dexamethasone. Before taking dexamethasone, the depressed patients reached a daily nadir of cortisol concentration approximately two hours earlier than did the normal control subjects. The DST nonsuppressors also exhibited a blunting in the expected circadian rhythm of the corticotropin level. PMID- 2994592 TI - Adenocarcinoid of the appendix presenting as bilateral Krukenberg's tumor of the ovaries. Immunohistochemical and ultrastructural studies and literature review. AB - A patient with an adenocarcinoid of the appendix presented with bilateral Krukenberg's tumors of the ovaries. Immunohistochemical and ultrastructural studies revealed a selective ability of the mucinous (goblet cell) component of the appendiceal neoplasm to metastasize. A review of the literature has revealed nine previously published cases of appendiceal adenocarcinoid metastatic to the ovaries. All showed involvement of both ovaries, but none provided unequivocal evidence of a metastatic proliferating carcinoid element. As the appendiceal lesion is often grossly inconspicuous, it may be overlooked in cases presenting initially with ovarian tumors. Routine appendectomy is therefore recommended in such patients where no grossly obvious primary tumor is evident. PMID- 2994593 TI - Angiomatoid malignant fibrous histiocytoma. Report of two cases with ultrastructural observations of one case. AB - Two cases of so-called angiomatoid malignant fibrous histiocytoma are reported herein. One of the cases was studied ultrastructurally, representing, to my knowledge, the fourth such set of observations reported in the literature. It is concluded that the histiocytelike cells making up the bulk of the tumor are modified endothelial cells with histiocytic function, so that the term histiocytoid hemangioma may be a valid one. The principal point of difference, however, is that these tumors as described herein are aggressive with a potential both for local recurrence and metastasis. PMID- 2994594 TI - Hydropic cell variant (clear cell variant) of Wilms' tumor. AB - We report an unusual clear cell tumor of the kidney in infancy. The presence in our patient of a discrete mass with foci of primitive nephroblastic cells indicates the neoplastic nature of the clear cells. We discuss its distinction from clear cell sarcomas and carcinomas, and we suggest that this lesion may be inadequately separated from pediatric renal carcinomas in the literature. We recommend the term hydropic cell variant as a less confusing and more accurate descriptive designation. PMID- 2994595 TI - Spinal cord stimulation in patients with a battered root syndrome. AB - Sixteen patients suffering from pain due to a "battered" root syndrome (BRS) [1] were treated with spinal cord stimulation (SCS). All patients had undergone spinal surgery and complained of severe intractable radicular pain. The myelograms revealed an amputated root, the computerized tomograms showed periradicular scar tissues, electromyograms and/or sensory evoked potentials showed pathological changes. Electrical root stimulation via an indwelling, Teflon-coated needle reduced pain significantly within 30 min (80-100 Hz, 0.2-0.3 V). Root blocks with 1 ml bupivacaine (0.5%) abolished the patient's pain completely. No patient revealed major psychosomatic or psychosocial problems. Employing this patient selection schedule, 75% of the patients were pain-free after a mean follow-up period of 16 months range 6-30. PMID- 2994596 TI - Motor and sensory neuropathy secondary to excessive pyridoxine ingestion. AB - This report documents a case of mixed motor and sensory neuropathy that resulted from ingestion of excessive amounts of pyridoxine. An 81-year-old woman was admitted to the hospital because of difficulty in walking and frequent falls. History revealed that she had been taking large doses of pyridoxine daily for several months as treatment for carpal tunnel syndrome. Diagnostic work-up failed to suggest a cause for her symptoms. Nerve conduction studies revealed slowing of motor conduction velocities, prolonged F wave latencies, and prolonged sensory latencies in both lower extremities. We believe the patient's complaints and the results of nerve conduction studies were secondary to pyridoxine neurotoxicity. Since the bases for this neurotoxicity are unknown, we suggest that treatment of carpal tunnel syndrome with oral pyridoxine be carefully monitored and that dosage limits not be exceeded. PMID- 2994597 TI - Pancreatic islet cell carcinoma with gastrin and vasoactive intestinal polypeptide production. AB - The case history of a patient with an islet cell carcinoma, which produced both gastrin and vasoactive intestinal polypeptide (VIP), is presented. Although several examples have been observed of the combined production of these hormones by pancreatic endocrine tumors, few reports have related the clinical details of such cases. Resolution of diarrhea occurred in our patient after institution of nasogastric suction and cimetidine therapy, suggesting that gastric hypersecretion, rather than VIP activity, accounted for this problem. Chemotherapy with streptozotocin and 5-fluorouracil was highly effective in ameliorating clinical symptoms, diminishing serum levels of gastrin and VIP, and greatly reducing the bulk of metastatic disease in this case. PMID- 2994598 TI - The effects of monoclonal antibodies against the hemagglutinin-neuraminidase and fusion protein on the release of Sendai virus from infected cells. AB - Vero cell cultures in Leighton tubes were infected with egg-grown Sendai virus at high multiplicity of infection. Four hours after infection, the cultures were labelled with 35S-methionine, after which various concentrations of fourteen and five mouse monoclonal antibodies directed against different antigenic determinants of the hemagglutinin-neuraminidase (HN) and fusion (F) protein, respectively, were added to the medium. Fourty-eight hours after infection radiolabelled virions released into the medium were collected and purified by discontinuous sucrose gradient centrifugations. The amount of virus-bound radioactivity obtained in the various extracellular materials allowed an estimation of the capacity of the different monoclonal antibodies to inhibit the release of Sendai virus. In addition, the release of virions from infected cells was studied ultrastructurally. Based on their serological reactivity the fourteen anti-HN monoclonal antibodies could be divided into four groups. The first group of clones could not inhibit any biological activity of the virus. These clones were binding proximally, near the base of the HN glycoprotein and could not inhibit the release of the virus. The second group blocked hemolysis, but did not block hemagglutination (HA) or neuraminidase (NA) activity. The third group of clones blocked all biological activities of the HN glycoprotein. The fourth group could only block NA activity. With the exception of one of five monoclonal antibodies belonging to the second group, antibodies of the second, third and fourth group were found to bind more distally on the HN glycoprotein. Except for two monoclonal antibodies of the second group they could all effectively inhibit release of the virus from infected cells. Ultrastructurally, these antibodies caused aggregation of virions in contact with the plasma membrane. The five monoclonal antibodies directed against the F protein reacted with four different antigenic sites. These antibodies could not prevent the release of Sendai virus. PMID- 2994599 TI - Different populations of herpes simplex virus glycoprotein C discriminated by the carbohydrate-binding characteristics of N-acetylgalactosamine specific lectins (soybean and Helix pomatia). Brief report. AB - From the herpes simplex virus specified glycoprotein C two fractions were isolated with affinity either for Helix pomatia lectin (HPA) or soybean lectin (SBA). The data indicated that HPA and SBA, despite their mutual main specificity for N-acetylgalactosamine, recognize structurally different gC populations. PMID- 2994600 TI - Abortive infection with human cytomegalovirus induces an alteration of growth pattern: morphological changes with cytocidal effect in rabbit kidney epithelial cells. Brief report. AB - Rabbit kidney epithelial cells (RK13) exhibited a cytopathic effect (CPE) characterized by cell rounding after infection with human cytomegalovirus (HCMV). Although HCMV-specific immediate early and early antigens were detected by indirect immunofluorescence techniques, neither late antigens nor infectious progeny virus could be observed in virus-infected RK13 cells. HCMV-infected RK13 cells showed a prolonged doubling time and a decreased saturation density in cell growth compared to uninfected control cells. Moreover, colony forming ability (CFA) of virus-infected cells decreased by approximately 70 per cent compared to that of uninfected cells during the first 24 hours after infection. These results indicate that an abortive infection of RK13 cells with HCMV induces an alteration of growth pattern including morphological changes with cytocidal effect. PMID- 2994601 TI - Persistence of virulent canine distemper virus in lymphoblastoid cell lines. AB - Persistent infection with virulent canine distemper virus (CDV-SH) was established in 2 human lymphoblastoid B cell lines (Wi-L2 and Raji), and one human (HSB), one simian (1670) and one canine (CT-45-S) lymphoblastoid T cell line. Cell free virus from persistently infected T cell lines was avirulent for dogs but virulence was maintained during 31 cell passages in persistently infected B cell lines. PMID- 2994603 TI - Machupo virus polypeptides: identification by immunoprecipitation. AB - The most abundant protein in purified Machupo virions (Corvallo strain) labelled with 14C-Protein hydrolysate is a 64 K polypeptide which is associated with virion RNAs. Another structural polypeptide, 37 K, solubilized by nonionic detergent seems to be a major surface glycoprotein. In addition to this, a 78 K polypeptide and a minor 50 K polypeptide have been detected. In Machupo virus infected cells three virus-specific polypeptides similar in size to those described for structural polypeptides were immunoprecipitated with anti-Machupo virus serum. The most abundant virus-specific polypeptide was nonglycosylated (64 K, NP), and the others were glycosylated polypeptides (78 K and 37 K). The synthesis of NP and 78 K polypeptides was recognized at the beginning of a log phase of virus replication. Pulse-chase experiments as well as experiments with an arginine analogue, canavanine (to block proteolytic processing) suggest that 78 K is a precursor for structural glycoproteins of Machupo virions. PMID- 2994602 TI - Thymidine kinase-negative bovine herpesvirus type 1 mutant is stable and highly attenuated in calves. AB - Calves were vaccinated intranasally (IN) or intravenously (IV) with a thymidine kinase-negative (tk-) BHV-1 mutant. Vaccinated calves developed neutralizing antibodies but did not show clinical signs of infectious bovine rhinotracheitis (IBR). Vaccination also prevented clinical signs of IBR disease following IN challenge exposure of the calves to parental Los Angeles (LA) and USDA Cooper strains of tk+ BHV-1. Nasal swabs were collected for 10 days after the vaccination and the challenge exposures to monitor BHV-1 multiplication. At both 91 and 121 days post vaccination (PV), calves were also stressed for 5 days with dexamethasone (DEX) to induce reactivation of BHV-1 and nasal swabs were obtained. tk- BHV-1 multiplied in the nasal mucosa of IN vaccinated calves and was also recovered after DEX treatment. Likewise, tk- BHV-1 was isolated from the buffy coat fraction of IV vaccinated calves, but not from nasal swabs. tk- BHV-1 vaccination reduced the multiplication of tk+ BHV-1 in the nasal mucosa, but did not completely prevent development of a persistent infection by the challenge virus. The phenotypes of viruses isolated from the nasal swabs and buffy coats were analyzed by enzyme assays of extracts from virus-infected cells and by plaque autoradiography. These assays showed that tk- BHV-1 can persist for at least 3 months in vaccinated calves and may also be transmitted from vaccinated to control calves without reverting in vivo to tk+. The results demonstrate that the tk- BHV mutant is attenuated and efficacious as a vaccine. PMID- 2994605 TI - Insulinoma in pregnancy. PMID- 2994604 TI - [Human neutrophil activating factor from a plant seed]. PMID- 2994606 TI - Peripheral neuropathy in cerebrotendinous xanthomatosis. AB - We performed a sural nerve biopsy in a patient with cerebrotendinous xanthomatosis (CTX) because of electrophysiologic evidence of peripheral neuropathy. The sections showed a striking loss of myelinated axons, the distribution of which suggested a compressive and/or ischemic process. Biochemical analysis disclosed large amounts of cholestanol, a cholesterol derivative that characteristically accumulates in CTX. However, the biochemical abnormality was not associated with any obvious structural alterations in the myelin lamellae or with abnormal storage material in Schwann's cells. PMID- 2994607 TI - Severe neonatal centronuclear myopathy with autosomal dominant inheritance. AB - We studied a boy with severe infantile centronuclear myopathy (CNM) and his mother with clinical, electrophysiological, and pathological signs of skeletal muscle, peripheral nerve, and brain-stem disorder, and we believe that her condition represents a variation of her son's disease. His brother had similar symptoms and died at 4 days of age. The occurrence of this syndrome in a symptomatic mother and two severely affected sons suggests an autosomal dominant inheritance with variable expressivity. To our knowledge, this inheritance pattern has not been previously reported in severe (fatal) infantile CNM. The different courses in the mother and her offspring may be manifestations of a single or separate abnormal gene causing alteration of muscle and nerve maturation. PMID- 2994608 TI - Inclusion body myositis and Sjogren's syndrome. PMID- 2994609 TI - Chemical preparation of the eye in ophthalmic surgery. IV. Comparison of povidone iodine on the conjunctiva with a prophylactic antibiotic. AB - We previously found that half-strength (5%) povidone-iodine solution significantly reduced the bacterial flora of the conjunctiva. To compare the antibacterial effect of a topical combination antibiotic (Neosporin ophthalmic solution) given three times daily for three days preoperatively with that of half strength povidone-iodine solution given as part of the preoperative preparation, conjunctival cultures were studied from 35 patients undergoing ocular surgery. When used individually, the antibiotic and povidone-iodine solutions caused a similar and substantial decrease in the number of colonies and species of bacteria cultured. When both drugs were used together, the decrease was even more striking, making 83% of the conjunctivae sterile. To minimize bacterial flora before ocular surgery, we recommend that a broad-spectrum topical antibiotic be given for three days preoperatively and that half-strength povidone-iodine solution be used as part of the preoperative preparation. PMID- 2994610 TI - Cyclic nucleotide and prostaglandin F2 alpha contents of otosclerotic auditory ossicles. AB - We found that the cyclic-adenosine-3',5'-monophosphate (cAMP) contents of otosclerotic human ossicles were 40-50 times greater than basal levels. Cyclic guanosine-3',5'-monophosphate (cGMP) levels were also found to be greater than in physiological conditions, but lower than in cortical bone. These findings suggest the extensive participation of these nucleotide coenzymes in effector cells during the process of bone resorption. This emphasizes the role of bone-resorbing cells in this process as well as the probable osteoclast progenitor role of vascular endothelial cells and their enhanced activity for differentiation. At the same time, the absence of prostaglandin F2 alpha content in the otosclerotic bone analyzed appears to exclude cartilage remnants as a source for inducing the changes in remodelling that occur. PMID- 2994611 TI - A re-examination of otoconia from the Shaker mouse. AB - We have studied saccular and utricular otoconia from Shaker-1 and Shaker-2 mice by X-ray diffraction and scanning electron microscopy. In contrast to previous reports, we found that the crystals were composed of calcite rather than polycrystalline hydroxylapatite. These crystals were indistinguishable mineralogically and morphologically from normal mouse otoconia. The reported occurrence of hydroxylapatite otoconia in the Shaker mouse is probably false. PMID- 2994612 TI - Ross River virus infection of human synovial cells in vitro. AB - Ross River virus (RRV) strains T48 and SC18006 produced a self-limited cytopathic infection of primary and passaged human synovial cell culture. Extracellular (EC) virus titres reached peak levels at 2 days in cell lines and at 4 days in primary cultures, ranging between 10(4.5) and 10(6.6) fluorescent focus-forming units (ffu)/ml. Thereafter titres declined rapidly to undetectable levels at 10-12 days. The proportion of adherent cells showing virus antigen exceeded 60% at 3 days and decreased in all cultures to less than 1/500 after 12 days. Cytopathic effects (CPE) were greatest at 4-8 days and destroyed between 25 and 75% of the cell layer, with subsequent partial regeneration by division of surviving cells. In contrast to rubella virus infection of synovial cells, cultures at 32 degrees and 37 degrees revealed only minor differences and persistent infection was not established. CPE were more extensive at 37 degrees in nearly all synovial cell cultures and in Vero cultures. At 37 degrees synovial cells infected with T48 strain produced higher maximum titres and were more extensively infected than at 32 degrees. PMID- 2994613 TI - Neutralization kinetic analysis of echovirus 30 and coxsackievirus B4 strains revealed little antigenic variation amongst the echovirus strains. AB - An antigenic analysis was made of echovirus 30 and coxsackievirus B4 isolates by determining neutralization rate constants in neutralization kinetic tests. The seven echovirus 30 isolates included the prototype strain and six others isolated in Melbourne, Australia, between 1959 and 1982. Little antigenic heterogeneity was observed in contrast to the evidence of antigenic variation recorded in similar tests on seven coxsackievirus B4 isolates. These isolates also included the prototype strain, as well as six others isolated in Melbourne between 1958 and 1973. The results obtained with the coxsackievirus B4 isolates were in keeping with those observed particularly with the polioviruses and also coxsackievirus B4 by other workers. Those obtained with the echovirus 30 isolates were unexpected, as this virus is also a member of the enterovirus group. PMID- 2994614 TI - Effects of temperature on Epstein-Barr virus replication in epithelial/nasopharyngeal carcinoma hybrid cells. AB - The authors studied the replication of Epstein-Barr virus (EBV) in the epithelial hybrid cells derived from nasopharyngeal carcinoma (NPCKT) caused by the effects of sub-optimal incubation at 34, 32 and 28 degrees C, respectively, using an immunofluorescence technique. The intensity of EBV induction by sub-optimal temperatures was in the order of 32 degrees C----28 degrees C----34 degrees C. In particular, 18-24% positive cells for early antigen and viral capsid antigen were observed at around 10 to 14 days after shift-down of the incubation temperature from 37 to 32 degrees C. PMID- 2994615 TI - Immunofluorescence and electron microscopic investigations of epithelial hybrid cells derived from nasopharyngeal carcinoma (NPC-KT). AB - The present study was made to investigate some characteristics of the epithelial hybrid cells derived from nasopharyngeal carcinoma (NPC-KT cells) both in vivo and in vitro, using immunofluorescence and electron microscopic techniques. Immunofluorescence and electron microscopic studies have shown that the appearance of Epstein-Barr virus (EBV)-related early antigens, EB-viral capsid antigens and virus particles in nude-mouse-grown-tumour cells were rather repressed, in contrast to, in vitro culture of the NPC-KT cells. The tumours after transplantation of the NPC-KT cells to nude mice showed pathological pictures of poorly differentiated carcinoma with EBV-associated nuclear antigen and derived from the NPC-KT cells by means of cytogenetic studies. More importantly, we have detected EBV-related membrane antigens (MA) on the epithelial NPC-KT cells. To our knowledge, the presence of MA on the malignant epithelial cells of the nasopharynx have never been demonstrated. The results reported here show for the first time the presence of MA on nasopharyngeal carcinoma cells. PMID- 2994616 TI - Enzymatic deficiencies of the immune system cells in patients with cancer of the larynx and other malignancies. AB - Activity of N-acetyl-beta-D-glucosaminidase, beta-glucuronidase, and acid phosphatase and myeloperoxidase was determined in neutrophils and lymphocytes of patients with cancer of the larynx and precancerous states of the larynx as well as--for comparative reasons--in patients with malignant tumors of female generation organs, breast carcinoma, cancer of the stomach and endometriosis. The main result of investigations performed was in fact that intracellular deficiency of beta-glucuronidase within the neutrophils characterizes patients with cancer and precancerous states of the larynx. Patients with cancer of the larynx show additionally a deficiency of neutrophil myeloperoxidase. Deficiency of N-acetyl beta-D-glucosaminidase occurs, in contrast, in patients with malignancies of female generation organ. Activity of myeloperoxidase in neutrophils from patients with gastric carcinoma is slightly elevated. PMID- 2994617 TI - Growth factors and cancer. AB - Recent advances in protein chemistry and genetic engineering have revealed new information about the molecular lesions involved in the induction and maintenance of cancer cells. It is now known that a single base change in the DNA of human cells leads to cancer. The normal pathway of proliferation and differentiation is perturbed by changes to molecules involved in the intracellular biochemical pathways controlled by growth factors. Some cancer cells appear to produce their own growth factor, others have higher concentrations of growth factor receptors on their surface and others have mutated versions of the intracellular proteins linked to the growth factor receptors. This increased understanding of growth control in normal and neoplastic cell populations is gradually providing a foundation for new approaches to cancer therapy. PMID- 2994619 TI - Haemagglutinating adenovirus in Victorian chicken flocks 1980-83. PMID- 2994618 TI - Inappropriate luteinizing hormone concentration in human breast cancer. AB - As part of a programme to assess the para-endocrine behaviour of human infiltrating ductal breast carcinoma among Chinese women, this study was designed to compare (Mann-Whitney U test) the immunoreactive mammary tumour tissue concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotrophin (beta-hCG) and prolaction (PRL) at stages I (n = 9), II (n = 12) and III (n = 7) with that of normal breast tissue (n = 8). It was found that with the exception of FSH and beta-hCG in stages I and III significantly elevated concentrations of protein hormones were noted at all stages of malignancy. The possible importance of these findings in multi-stage mammary carcinogenesis is discussed. It is suggested that measurement of LH concentration may provide an endocrine basis to facilitate the diagnosis of tumours to serve as a guide to the biological behaviour of mammary neoplasms. PMID- 2994620 TI - The clinical susceptibility of sheep to four Australian serotypes of bluetongue virus. PMID- 2994621 TI - Gynaecological importance of unusual pituitary tumours. AB - Sixty-eight women with pituitary tumours exhibiting gynaecological symptoms have been reviewed. Fifty-two tumours were prolactinomas while 6 were growth hormone secreting, 6 ACTH secreting, 1 was a large 'inert' adenoma and 3 were late presenting craniopharyngiomas. Several of the prolactinomas exhibited unusual features including excessively high prolactin secretion, a wide spectrum of sensitivity to bromocriptine, rapid expansion in pregnancy, association with the empty sella syndrome and even a secondary malignancy. The behaviour of tumours secreting other hormones but presenting with gynaecological symptoms is described. PMID- 2994622 TI - Neurophysiology. PMID- 2994623 TI - Applied kinesiology and colon health. PMID- 2994624 TI - [Intranasal vaccination of cattle against foot-and-mouth disease]. PMID- 2994625 TI - Polymorphism at the Hor 1 locus of barley (Hordeum vulgare L.). AB - The Hor 1 locus of barley encodes a group of seed storage polypeptides called C hordein. Two-dimensional electrophoretic analysis of C-hordein fractions from six cultivars with different alleles at the Hor 1 locus showed extensive polymorphism. A total of 34 major polypeptides was mapped, with between 4 and 18 present in each cultivar. There was less variation among the same cultivars in the numbers (6 to 10) of restriction fragments of genomic DNA which hybridized to a cDNA clone related to C hordein. The total number of restriction fragments was also lower (22), and most pairs of cultivars had more restriction fragments than polypeptides in common. A total number of about 20-30 C-hordein genes per haploid genome was estimated. The results indicate that cultivars differ mainly in the extent of gene and polypeptide divergence, rather than in the degree of gene reiteration. They are consistent with the proposed origin of the multiple structural genes at the Hor 1 locus by the duplication and divergence of a single ancestral gene. PMID- 2994626 TI - The expression of human glycerol-3-phosphate dehydrogenase in human/rodent somatic-cell hybrids. AB - Our previous studies using rodent/human somatic-cell hybrids suggested that the expression of human mitochondrial glycerol-3-phosphate dehydrogenase (GPDM) is dependent on the presence of human mitochondria. This has now been tested directly by analysis of GPDM activity in a series of nine hybrid-cell lines, four segregating human chromosomes and five losing rodent chromosomes (reverse segregants). The chromosome composition of the hybrids was deduced from analysis of biochemical markers and examination of G- and G11-banded metaphase spreads and the mitochondrial content was determined by Southern blot analysis, using cloned mouse and human mtDNA sequences as probes. We found that the mtDNA species present in these hybrids correlated exactly with the pattern of chromosome segregation such that the conventional hybrids contained rodent mtDNA and the reverse segregants human mtDNA. However, the pattern of GPDM expression was not directly correlated with the species of chromosomes or mitochondria present: all the hybrids showed strong rodent GPDM activity and two from each class of hybrid also showed human GPDM activity but the other hybrids were negative for human GPDM. We conclude that rodent GPDM readily integrates into human mitochondria, that the expression of rodent GPDM is not dependent on the presence of rodent mitochondria, and that GPDM is not coded by mtDNA. Human GPDM either is not capable of being inserted into the rodent mitochondrial membrane or is regulated in some way in the hybrid cells by an unidentified rodent factor. PMID- 2994627 TI - Loss of epidermal growth factor receptors and release of transforming growth factors do not correlate with sarcoma virus-transformation in clonally-related NIH/3T3-derived cell lines. AB - Transformation of NIH/3T3 cells by Kirsten murine sarcoma virus (MSV) caused a dramatic reduction in the number of cell-surface receptors for epidermal growth factor (EGF). However, the number of EGF receptors remained at a very low level in a non-tumourigenic revertant cell line isolated from the virus-transformed cells, indicating that an increase in EGF receptors is not a requirement for the phenotypic reversion of Kirsten MSV-transformed 3T3 cells. Serum-free conditioned medium from normal and virus-transformed cell lines contained similar amounts of cell growth-promoting activity as assayed by the ability to stimulate DNA synthesis in quiescent Swiss 3T3 cell cultures. However, the concentrated conditioned medium from these cell lines showed no evidence of beta-transforming growth factor (TGF) activity as assayed by promotion of anchorage-independent growth of untransformed normal rat kidney (NRK) fibroblasts in agarose. The cellular release of alpha-TGF activity was assayed by measuring the ability of concentrated conditioned medium to inhibit the binding of 125I-EGF to Swiss 3T3 cells. Conditioned medium protein from the virus-transformed cell line inhibited 125I-EGF binding but only to the same extent as conditioned medium protein prepared from the untransformed cell line. The alpha-TGF secretion by these cell lines was estimated to be 30-45-fold lower than the level of alpha-TGF released by a well-characterized alpha-TGF-producing cell line (3B11). These results suggest that the induction of TGF release is not a necessary event in the transformation of NIH/3T3 cells by Kirsten MSV. PMID- 2994628 TI - Differences between collagen hydroxylases and 2-oxoglutarate dehydrogenase in their inhibition by structural analogues of 2-oxoglutarate. AB - Inhibition of lysyl hydroxylase and prolyl 3-hydroxylase was studied with 23 selected aromatic and aliphatic structural analogues of 2-oxoglutarate and the results were compared with those previously reported for prolyl 4-hydroxylase. All the compounds inhibited the hydroxylases competitively with respect to 2 oxoglutarate and noncompetitively with respect to Fe2+ and the peptide substrate. The inhibition patterns for the three collagen hydroxylases were basically similar, but certain differences in detail emerged. One systematic difference was that lysyl hydroxylase had a higher Ki for almost all the compounds than had the two prolyl hydroxylases. Another interesting difference was that pyridine-2,4 dicarboxylate was the most potent inhibitor of lysyl hydroxylase and prolyl 3 hydroxylase, with Ki values of 50 microM and 3 microM respectively, whereas pyridine-2,5-dicarboxylate was the most potent inhibitor of prolyl 4-hydroxylase. These and other data suggest that the three collagen hydroxylases have similar but not identical 2-oxoglutarate-binding sites. Pyridine-2,4-dicarboxylate and pyridine-2,5-dicarboxylate and their corresponding benzene derivatives were also found to inhibit 2-oxoglutarate dehydrogenase, but with this enzyme, unlike the collagen hydroxylases, no distinct difference in the Ki values was found between the corresponding pyridine and benzene derivatives. This demonstrates the importance of the metal ion for the binding of various compounds at the 2 oxoglutarate-binding site of the collagen hydroxylases. 2-Oxoadipate was shown to replace 2-oxoglutarate in the lysyl hydroxylase and 2-oxoglutarate dehydrogenase reactions, as has previously been reported for prolyl 4-hydroxylase, whereas no other 2-oxo acid tested had any co-substrate activity. The 2-oxoglutarate-binding site of these enzymes is thus flexible to a certain degree, as it can accommodate molecules of different shapes and volumes. On the basis of the present data pyridine-2,5-dicarboxylate seems to be a quite specific inhibitor of prolyl 4 hydroxylase, the Ki for 2-oxoglutarate dehydrogenase being about 4000-fold higher. PMID- 2994629 TI - Inactivation of rabbit muscle phosphoglycerate mutase by limited proteolysis with thermolysin. AB - Rabbit muscle phosphoglycerate mutase is inactivated by proteolysis with thermolysin. Inactivation is correlated with the breakage of one (or a few) bond(s) near one end of the polypeptide chain. There is no change in the overall conformation, quaternary structure or binding to Cibacron Blue on proteolysis. The possible analogy with the existence of a flexible tail in the yeast enzyme is discussed. PMID- 2994630 TI - Gluconeogenesis in periportal and perivenous hepatocytes of rat liver, isolated by a new high-yield digitonin/collagenase perfusion technique. AB - A technique is described which allows preparations of hepatocytes, enriched in either periportal or perivenous hepatocytes ('PP-cells' and 'PV-cells' respectively), in a yield of about 30-50% compared with control cell preparations. The liver is first perfused for 40-60s with digitonin (4 mg/ml) to destroy selectively either the periportal or the perivenous part of the microcirculatory unit, and then the remaining hepatocytes are isolated by the ordinary collagenase perfusion technique. In periportal cells the activities of alanine aminotransferase and pyruvate kinase were 29.4 and 18.7 mumol/min per mg of DNA respectively. The rate of gluconeogenesis was 0.402 mumol/min per mg of DNA. In perivenous cells the corresponding values were 9.55, 22.1 and 0.244 mumol/min per mg of DNA respectively. These data support the concept of a zonation of glucose metabolism within the microcirculatory unit of the liver, with the afferent part (periportal zone) having a 2-fold, more active gluconeogenesis than the efferent part (perivenous zone). PMID- 2994631 TI - Hazelhurst-vesicular-stomatitis-virus G and Sindbis-virus E1 glycoproteins undergo similar host-cell-dependent variation in oligosaccharide processing. AB - We have examined and compared the host-cell-dependent glycosylation of the G glycoprotein of vesicular-stomatitis virus (Hazelhurst strain) and the E1 and E2 glycoproteins of Sindbis virus replicated by baby-hamster kidney, chicken-embryo fibroblast and mouse L929 monolayer cell cultures. The results of endo-beta-N acetylglucosaminidase H digestion of viral proteins labelled with [3H]mannose or leucine and Pronase-digested glycopeptides labelled with [3H]mannose indicated that both the G protein and the E1 protein contained a similar mixture of endoglycosidase-resistant oligosaccharides of the complex acidic type and less extensively processed endoglycosidase-sensitive oligosaccharides of the neutral or hybrid type, with a relatively greater content of the endoglycosidase sensitive oligosaccharides for virus replicated in the chicken as against hamster or mouse cells. A large fraction of the G protein and the majority of the E1 proteins from the mammalian host cells contained acidic-type oligosaccharides at both glycosylation sites, whereas most of the G and E1 glycoproteins from the avian host cells and essentially all of the E2 protein from all three host-cell types contained an acidic-type oligosaccharide at one site and neutral- or hybrid type oligosaccharide at the other site. The relative increase in neutral- and hybrid-type oligosaccharides with five-mannose core structures observed for the G and E1 proteins of virus released from the avian host cells suggested that two specific steps in oligosaccharide processing (mediated by alpha-mannoside II and N-acetylglucosaminyltransferase I) were less efficient at one of the glycosylation sites of the vesicular-stomatitis-virus G protein and Sindbis-virus E1 protein in the avian as against mammalian host cells. PMID- 2994632 TI - Preparation of reduced bovine Cu,Zn superoxide dismutase. AB - N.m.r. and e.p.r. were used to measure the oxidation state of copper in Cu,Zn superoxide dismutase treated with reducing agents such as NaBH4, K4Fe(CN)6, Na2S2O4 and H2O2. The activity and the electrophoretic pattern of the treated enzyme were also studied. On the basis of the reducing ability and of the absence of inactivating effects, NaBH4 was the most suitable reducer of those tested. Some characteristics of the reduction of superoxide dismutase by NaBH4 were further investigated. The results obtained indicate that NaBH4 can be used to prepare, in a few minutes, solutions of completely reduced enzyme without any apparent change of the activity and of the structure. PMID- 2994633 TI - Magnesium ion exerts a central role in the regulation of inhibitory adenosine receptors. AB - Guanine nucleotides and Mg2+ differentially regulate agonist binding to adenosine (Ri) receptors in fat-cell plasma membranes. GTP alone decreases binding of the agonist ligand [3H]N6-cyclohexyladenosine (CHA) by increasing the dissociation constant (Kd). Mg2+ alone also decreases [3H]CHA binding, which is associated with a decrease in the number of receptors and in the dissociation constant. In the presence of Mg2+, the effect of GTP is to increase [3H]CHA binding by increasing the total number of receptors. It thus appears that Mg2+ acts specifically at a bivalent-cation site which, with GTP, regulates agonist binding. This putative Mg site is highly sensitive to alkylating agents. Mild treatment with N-ethylmaleimide (NEM) abolishes the characteristic GTP effect on agonist binding in the presence of Mg2+. In addition, the effect of Mg2+ alone is also eliminated. The effect of GTP alone is largely unaltered. Studies of the adenylate cyclase activity indicate that this NEM treatment also abolishes the inhibition of basal activity by adenosine analogues, whereas guanylyl imidodiphosphate inhibition of forskolin-stimulated activity is only slightly impaired at this NEM concentration. These observations indicate that a Mg2+ 'site' or 'component' is required for the integration of receptor (Ri) occupancy with regulation of catalytic activity (C). The regulatory role of Mg2+ is more demonstrable in receptor-GTP-regulatory-protein (Ri-Ni) interactions than in GTP regulatory-protein-catalytic-unit (Ni-C) interactions. PMID- 2994634 TI - Several dehydrogenases and kinases compete for endocytosis from plasma by rat tissues. AB - Plasma contains many enzymes that are probably derived from damaged cells. These enzymes are cleared at characteristic rates. We showed previously that in rats the rapid clearance of alcohol dehydrogenase, lactate dehydrogenase M4 and the mitochondrial and cytosolic isoenzymes of malate dehydrogenase is largely due to endocytosis by macrophages in liver, spleen and bone marrow. We now demonstrate that uptake of each of the enzymes by these tissues is in general decreased by simultaneous injection of a high dose of one of the other dehydrogenases or a high dose of adenylate kinase or creatine kinase. A similar dose of colloidal albumin did not significantly decrease uptake of the four dehydrogenases. Nor was uptake of colloidal albumin, apo-peroxidase from horseradish or multilamellar liposomes influenced by a high dose of mitochondrial malate dehydrogenase. These results indicate that the four dehydrogenases and the two kinases are specifically endocytosed via the same receptor. We suggest that this receptor contains a group, possibly a nucleotide, with affinity for the nucleotide-binding sites of the enzymes. PMID- 2994635 TI - Both acidic-type and neutral-type asparaginyl-oligosaccharides of host-cell glycoproteins are altered in Rous-sarcoma-virus-transformed chick-embryo fibroblasts. AB - In comparisons of [3H]mannose-labelled glycopeptides from chick-embryo fibroblasts infected and transformed with non-defective Prague C Rous-sarcoma virus and from untransformed fibroblasts infected with a transformation-defective derivative of Prague C Rous-sarcoma virus, we have detected transformation dependent alterations in both the acidic-type and the neutral-type asparagine linked oligosaccharides of cellular glycoproteins. Pronase-digested glycopeptides were analysed by the combined techniques of gel filtration, exo- and endo glycosidase digestion and concanavalin A-agarose affinity chromatography. The transformed cell glycoproteins contained more sialic acid and were enriched for more highly branched (versus biantennary) acidic-type structures compared with the untransformed cell glycoproteins, similarly to previously reported transformation-dependent alterations. In addition, the glycopeptides from the virus-transformed cells contained several neutral-type structures that were apparently absent from the untransformed cells: small neutral-type oligosaccharides (Man3GlcNAc2) that were sensitive to endo-beta-N acetylglucosaminidase D but resistant to endo-beta-N-acetylglucosaminidase H, and oligosaccharides with the property of 'truncated' precursor oligosaccharides (endoglycosidase-resistant, alpha-mannosidase-sensitive). Endoglycosidase released oligosaccharides with the properties of hybrid-type structures were derived from the glycoproteins of both transformed and untransformed cells. PMID- 2994636 TI - The control by delta mu H+ of the tonoplast-bound H+-translocating adenosine triphosphatase from rubber-tree (Hevea brasiliensis) latex. AB - The relationship between tonoplast-bound ATPase activity and the magnitude of the electrochemical proton gradient has been investigated on tightly sealed vesicles prepared from rubber-tree (Hevea brasiliensis) latex. A variety of methods have been used to modify, either alone or together, the two components of the electrochemical proton gradient (delta mu H+). When the delta pH component was decreased either by titration with (NH4)2SO4 or by addition of protonophores or nigericin in the presence of K+, ATPase activity was stimulated. On the other hand, when the delta psi component was decreased either by addition of lipophilic cations or by addition of valinomycin in the presence of K+, ATPase activity decreased. It is concluded that activity of the tonoplast-bound ATPase is regulated by changes in the electrochemical proton gradient across the tonoplast, so that, once the maximum proton gradient is established across the tonoplast, any perturbation of the equilibrium state should result in the increased rate of ATP hydrolysis as the enzyme attempts to re-establish the initial gradient. PMID- 2994637 TI - The role of the phosphate group for the structure of phosphopeptide products of adenosine 3',5'-cyclic monophosphate-dependent protein kinase. AB - By c.d. studies it is shown that liver-pyruvate-kinase-related peptide substrates of cyclic AMP-dependent protein kinase have a high tendency towards non-random structures in non-aqueous media. When phosphorylated, the conformation tendencies decrease. This structural change is explained in terms of the formation of strong intrapeptide phosphate-guanidinium salt links. It is proposed that similar events occur at the catalytic site of protein kinase and that such an interaction could facilitate the removal of the phosphorylated products. PMID- 2994639 TI - Complex oscillatory phenomena, including multiple oscillations, in regulated biochemical systems. PMID- 2994638 TI - Metabolism of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate in rat parotid glands. AB - A complete separation of myo-inositol 1,4,5-[4,5-(32)P]trisphosphate prepared from human erythrocytes, and myo-[2-3H]inositol 1,3,4-trisphosphate prepared from carbachol-stimulated rat parotid glands [Irvine, Letcher, Lander & Downes (1984) Biochem. J. 223, 237-243], was achieved by anion-exchange high-performance liquid chromatography. This separation technique was then used to study the metabolism of these two isomers of inositol trisphosphate in carbachol-stimulated rat parotid glands. Fragments of glands were pre-labelled with myo-[2-3H]inositol, washed, and then stimulated with carbachol. At 5s after stimulation a clear increase in inositol 1,4,5-trisphosphate was detected, with no significant increase in inositol 1,3,4-trisphosphate. After this initial lag however, inositol 1,3,4-phosphate rose rapidly; by 15s it predominated over inositol 1,4,5 trisphosphate, and continued to rise so that after 15 min it was at 10-20 times the radiolabelling level of the 1,4,5-isomer. In contrast, after the initial rapid rise (maximal within 15s), inositol 1,4,5-trisphosphate levels declined to near control levels after 1 min and then rose again very gradually over the next 15 min. When a muscarinic blocker (atropine) was added after 15 min of carbachol stimulation, inositol 1,4,5-trisphosphate levels dropped to control levels within 2-3 min, whereas inositol 1,3,4-trisphosphate levels took at least 15 min to fall, consistent with the kinetics observed earlier for total parotid inositol trisphosphates [Downes & Wusteman (1983) Biochem. J. 216, 633-640]. Phosphatidylinositol bisphosphate (PtdInsP2) from stimulated and control cells were degraded chemically to inositol trisphosphate to seek evidence for 3H labelled PtdIns(3,4)P2. No evidence could be obtained that a significant proportion of PtdInsP2 was this isomer; in control tissues it must be less than 5% of the total PtdInsP2 radiolabelled by myo-[2-3H]inositol. These data indicate that, provided that inositol 1,4,5-trisphosphate is studied independently of inositol 1,3,4-trisphosphate, the former shows metabolic characteristics consistent with its proposed role as a second messenger for calcium mobilization. The metabolic profile of inositol 1,3,4-trisphosphate is entirely different, and its function and source remain unclear. PMID- 2994640 TI - Dynamic structures of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle in a reconstituted enzyme system. AB - The dynamics of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle was investigated in an open and homogeneous system reconstituted from purified enzymes. In addition to phosphofructokinase and fructose 1,6-bisphosphatase, pyruvate kinase, adenylate kinase and glucose 6-phosphate isomerases are involved. The time evolution of the metabolite concentrations is governed by a set of differential equations which take into account flow processes and enzymic conversions of metabolites. Depending on the experimental parameters stable attractors, multiple states and sustained oscillations occur. The main source of the nonlinear dynamics is the reciprocal effect of AMP on the activities of phosphofructokinase and fructose 1,6-bisphosphatase. States are characterized by the net flow rates of substrates and by the rate of futile substrate cycling. For efficient glycolytic states high ratios between the influx rates of fructose 6 phosphate and fructose 1,6-bisphosphate and between the maximum activities of phosphofructokinase and fructose 1,6-bisphosphatase must be maintained, while for an efficient gluconeogenic mode the reverse must hold. Fructose 2,6-bisphosphate exerts reciprocal effects on the activities of phosphofructokinase and fructose 1,6-bisphosphatase. In dependence on the experimental conditions fructose 2,6 bisphosphate was found either to generate or to extinguish oscillations. PMID- 2994641 TI - Thermodynamics and control of proton motive free-energy transduction. AB - Theories for (mosaic) non-equilibrium thermodynamics and the control of biological free-energy transduction are briefly recounted. Subsequently they are used to answer questions concerning properties of bacteriorhodopsin and the proton pumps in the mitochondrial respiratory chain, as well as concerning the presence of flux control in the non-irreversible reactions in free-energy transducers. Inhibition of bacteriorhodopsin by the membrane potential, respiratory control by delta muH and the theoretical P/O ratio are central in these discussions. Finally, specific thermodynamic and control properties of systems with localized ('mosaic', 'direct') energy coupling are discussed. PMID- 2994642 TI - Specific photoaffinity labelling of inhibitory adenosine receptors. AB - N6(L-phenylisopropyl)adenosine (L-PIA) and N6(3-iodo-4-azido benzyl)-adenosine (IAzBA) inhibit the adenylate cyclase activity in synaptic membranes of chick cerebellum via Ri adenosine receptors. [3H]L-PIA and [125I]AzBA bind to these membranes with Kd values of approximately 1 nM and Bmax values of approximately 1000 fmol/mg protein. Photolysis of [125I]AzBA bound to synaptic membranes results in the specific incorporation of radioactivity into a protein with Mr = 36,000. This photoincorporation is blocked by simultaneous exposure to L-PIA, theophylline, an adenosine receptor antagonist, or Gpp(NH)p, but not by cytosine, suggesting that the 36,000 dalton protein is the Ri adenosine receptor or a subunit of the receptor that contains the adenosine binding site. PMID- 2994643 TI - Pituitary 1,25-dihydroxyvitamin D3 receptors in hyperthyroid- and hypothyroid rats. AB - The binding of 1 alpha,25-dihydroxy (26,27-methyl-[3H]) cholecalciferol ([3H]1,25 (OH)2D3) to its receptor in cytosol of the anterior pituitary cells was examined in hyperthyroid- and hypothyroid rats, as well as in normal rats. The binding capacity increased by 41% in L-Thyroxine-treated hyperthyroid rats and decreased by 49% in propylthiouracil-ingested hypothyroid rats as compared with normal control rats, whereas the affinity of the receptor for [3H]-1,25(OH)2D3 showed no difference among these 3 animal groups. These findings indicate that the number of 1,25(OH)2D3 receptors in the pituitary may be regulated by thyroid hormone, and further suggest that 1,25-(OH)2D3 may play some role in regulating functions of the anterior pituitary. PMID- 2994644 TI - Regulation of canalicular bile formation by alpha-adrenergic action and by external ATP in the isolated perfused rat liver. AB - In isolated perfused rat liver, addition of adrenaline induced a complex response of bile flow including rapid, reversible stimulation (1/2-2 min), reversible inhibition (2-10 min), and prolonged stimulation. Both the reversible stimulation and the inhibition were mimicked by the alpha-sympathomimetic agonist phenylephrine but not by the beta-agonist isoproterenol. The reversible stimulation was a very early effect being terminated prior to all other alpha adrenergic responses of liver. External ATP considerably lowered bile flow while inducing release of glucose and lactate, inhibition of respiration, and a reversible efflux of Ca2+. Variations of mannitol clearance parallel to those of bile flow indicate a canalicular origin of all changes. PMID- 2994645 TI - Effects of fluoride on retinal rod outer segment cGMP phosphodiesterase and G protein. AB - The effects of fluoride on ROS phosphodiesterase and G-protein have been studied using membrane-free extracts. When G-protein was present NaF, at millimolar concentrations, stimulated PDE activity however, in a G-protein free extract, cGMP hydrolysis was inhibited by high fluoride concentrations. Fluoride was also found to profoundly inhibit the ability of G-protein to bind a GTP analogue, GTP gamma S, both in the presence and absence of rhodopsin. Aluminium greatly modified these effects of fluoride on PDE and G-protein. The possibility that fluoride activates PDE through its effect on G-protein is discussed. PMID- 2994647 TI - Differential susceptibility to GTP formed from added GDP via membrane-associated nucleoside diphosphate kinase of GTP-sensitive adenylate cyclases achieved by hormone and cholera toxin. AB - GTP-sensitive adenylate cyclases in liver membranes achieved by glucagon and by cholera toxin pretreatment displayed similar responses to added GTP in assay with respect to magnitude and sensitivity. However, their susceptibility to GTP formed during incubation from added GDP catalyzed by membrane-associated nucleoside diphosphate kinase(mNDPK) was different. Adenylate cyclase pretreated with cholera toxin was essentially unaffected by added GDP, while further addition of glucagon produced activation. GTP-stimulated adenylate cyclase activity in toxin treated membranes was inhibited by added GDP, whereas glucagon addition reduced the inhibitory action of GDP by two orders of magnitude. Since neither pretreatment with toxin nor glucagon addition altered GTP formation by mNDPK, these observations suggest a possible presence of a mechanism by which hormone makes adenylate cyclase susceptible to the GTP formed via mNDPK for activation. PMID- 2994646 TI - Hormonal regulation of benzo[a]pyrene metabolism in human adrenocortical cell cultures. AB - In cultured fetal human adrenocortical cells, metabolism of the carcinogen benzo[a]pyrene was found to be unresponsive to the xenobiotic inducers 3 methylcholanthrene, benz[a]anthracene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, exposure of cultures to the hormone adrenocorticotropin (ACTH) for 48 hours stimulated benzo[a]pyrene metabolism 3-fold. The major metabolite was the 7,8-diol. Other compounds which stimulate the production of adrenocortical cell cyclic AMP (forskolin and cholera toxin) as well as monobutyryl cyclic AMP also increased benzo[a]pyrene metabolism. Human adrenocortical cells thus provide an unusual example of hormonal regulation of the metabolism of a carcinogen. PMID- 2994648 TI - Amplified, overexpressed and rearranged epidermal growth factor receptor gene in a human astrocytoma cell line. AB - We report here that SK-MG-3, a human astrocytoma cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified and overexpressed EGF receptor gene. Northern blot analysis did not show any abnormal EGF receptor gene related mRNA species. No amplification or rearrangement was noted in 21 other astrocytoma cell lines. In contrast to other cell lines that have EGF receptor gene amplifications, we have not detected inhibition of in vitro proliferation of the SK-MG-3 line by EGF. PMID- 2994649 TI - Specific binding sites for atrial natriuretic peptide (ANP) in cultured mesenchymal nonmyocardial cells from rat heart. AB - Specific binding sites for atrial natriuretic peptide (ANP) were studied in cultured mesenchymal nonmyocardial cells (NMC) from rat heart. Binding study using 125I-labeled synthetic rat (r) ANP revealed the presence of a single class of high-affinity binding sites for rANP in cultured NMCs derived from both atria and ventricles; the apparent dissociation constant (Kd) was approximately 0.2 - 0.3 nM and the number of maximal binding sites was approximately 190,000 - 300,000 sites/cell. rANP significantly stimulated intracellular cGMP formation of cardiac NMCs in a dose-dependent manner (1.6 X 10(-8) M - 3.2 X 10(-7) M). rANP had no effect on synthesis of prostaglandin I2 by cultured cardiac NMCs. The physiological significance of ANP action on cardiac tissue remains to be determined. PMID- 2994650 TI - Conformational microheterogeneity in a DNA double helix: structure of restriction endonuclease Bam H1 recognition site. AB - Structural studies using 500 MHz 1H NMR spectroscopy on Bam H1 recognition site d(GGATCC)2 in solution at 19 degrees is reported. The resonances from the sugar ring and base protons have been assigned from the 2D-COSY and NOESY spectra. Analyses of the NOESY cross-peaks between the base protons H8/H6 and sugar protons H2'/H2", H3' reveal that the nucleotide units G2, A3 and C6 adopt (C3' endo, chi = 200 degrees-220 degrees) conformation while G1, T4 and C5 exhibit (C2'-endo, chi = 240 degrees-260 degrees) conformation. NMR data clearly suggest that the two strands of d(GGATCC)2 are conformationally equivalent and there is a structural two-fold between the two A-T pairs. The above information and the NOESY data are used to generate a structural model of d(GGATCC)2. The important features are: (i) G1-G2 stack, the site of cleavage, shows an alternation in sugar pucker i.e. C2'-endo, C3'-endo as in a B-A junction, (ii) G2-A3 stack adopts a mini A-DNA, both the sugars being C3'-endo, (iii) A3-T4 stack, the site of two-fold, displays an A-B junction with alternation in sugar pucker as C3' endo, C2'-endo, (iv) T4-C5 stack adopts a mini B-DNA both the sugars being C2' endo and (v) C5-C6 stack exhibits a B-A junction with C2'-endo, C3'-endo sugar puckers. Thus, our studies demonstrate that conformational microheterogeneity with a structural two fold, is present in the Bam H1 recognition site. PMID- 2994651 TI - Cysteinyl-cysteine and the microsomal protein from which it is derived act as reducing cofactor for prolyl hydroxylase. AB - Microsomes from L-929 cells contain a reductant which can replace ascorbate as a cofactor for prolyl hydroxylase. The cofactor was extracted with Triton X-100 and exhibited high and low molecular weight forms on S-300 gel columns. Refiltration or trypsin treatment of high molecular weight cofactor produced additional low molecular weight form. The low molecular weight form was purified by P-2 gel filtration, and Dowex-1 and thin layer chromatography. It is ninhydrin reactive, exhibits reduced and oxidized forms with molecular weights of 240 and 460, respectively, and yielded cystine upon acid hydrolysis. The results suggest that it is a dipeptide, cysteinyl-cysteine, derived from a microsomal protein which is the high molecular weight cofactor. PMID- 2994652 TI - Angiotensin II and phorbol ester enhance isoproterenol- and vasoactive intestinal peptide (VIP)-induced cyclic AMP accumulation in vascular smooth muscle cells. AB - The importance of Ca2+ and cAMP in the regulation of cellular functions has been well demonstrated. We studied the effect of angiotensin II (AII), a potent Ca2+ mobilizing hormone, on cAMP accumulation induced by isoproterenol (ISO) and vasoactive intestinal peptide (VIP) in cultured vascular smooth muscle cells (VSMC). Although the addition of AII alone caused little increase of cAMP, it enhanced ISO- and VIP-induced cAMP accumulations in a dose-dependent manner. This enhancement was mimicked by tumor-promoting phorbol ester but not by Ca2+ ionophore. This observation suggested that AII enhanced agonist-induced cAMP accumulation through the activation of protein kinase C in VSMC. PMID- 2994653 TI - Stimulation of high-affinity hormone-sensitive GTPase of human platelets by 1-O alkyl-2-O-acetyl-sn-glyceryl-3-phosphocholine (platelet activating factor). AB - 1-O-Alkyl-2-O-acetyl-sn-glyceryl-3-phosphocholine (platelet activating factor) inhibits human platelet adenylate cyclase via the GTP-dependent mechanism. Inhibition of adenylate cyclase correlates with the stimulation of high affinity hormone-sensitive GTPase. The half-maximal effects of PAF on both enzymes are observed at concentrations about 10(-8) M. Phentolamine, an alpha-adrenergic antagonist, does not abolish the PAF-induced inhibition of adenylate cyclase. The obtained data suggest that PAF receptors are coupled with the GTP-binding inhibitory protein. PMID- 2994654 TI - Combined effect of phorbol ester and, A23187 or dibutyryl cyclic AMP on pepsinogen secretion from isolated gastric glands. AB - In isolated guinea pig gastric glands, pepsinogen secretion was stimulated by the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in a dose dependent manner. Calcium-deprivation from the medium resulted in the decrease in TPA induced pepsinogen secretion. The combination of 0.4 microM Ca2+ionophore A23187 and TPA stimulated pepsinogen secretion slightly higher than the calculated additive value for each agent. This synergistic effect of the agents supports a role of calcium-activated, phospholipid-dependent protein Kinase (protein Kinase C) in gastric pepsinogen secretion. Furthermore, pepsinogen secretion was also stimulated by dibutyryl cyclic AMP (dbc AMP) and dbc AMP slightly enhanced TPA induced pepsinogen secretion. Results suggest that gastric chief cells possess at least two different secretory pathways for pepsinogen which are probably dependent on protein kinase C and cyclic AMP, respectively. PMID- 2994655 TI - Bovine leukemia virus post-envelope gene coded protein: evidence for expression in natural infection. AB - Partial sequence analysis of a 14 kilodalton protein (p14), synthesized by in vitro translation of bovine leukemia virus genomic RNA, showed that it is encoded in the 'X' region of proviral DNA, located between the env gene and the 3' long terminal repeat. The 'X' gene contains a short and a long open reading frame (X SORF and X-LORF) which overlap. BLV p14x is specified by X-SORF and not X-LORF as seen with the related human T-cell leukemia virus which expresses p38-40x. Antibodies in sera from animals with BLV induced tumors were shown to recognize p14x. Expression of this protein in natural infection might be important for virus replication and/or for BLV induced oncogenesis. PMID- 2994656 TI - Native species of helix destabilizing protein-1 in mouse myeloma identified by antibody probing of Western blots. AB - Antibody probing of Western blots is a method for analyzing the apparent Mr of a protein in any given preparation (Renart, J., Reiser, J., and Stark, G., Proc. Natl. Acad. Sci. USA 76: 3116, 1979). We prepared a rabbit antiserum to purified mouse myeloma helix destabilizing protein-1 and used this antiserum in Western blotting experiments with a crude homogenate of mouse myeloma. The results indicated that the native species of helix destabilizing protein-1 can be degraded during purification. This in vitro proteolysis results in complete loss of the native species and accumulation of lower Mr proteins that represent limit digestion products. These findings have identified the true native form of mouse myeloma HDP-1 as a protein of apparent Mr = 36,000 to 38,000, instead of the Mr = 24,000 and 27,000 proteins obtained by routine purification. PMID- 2994657 TI - Calmodulin antagonists elevate the levels of 32P-labeled polyphosphoinositides in human platelets. AB - The calmodulin antagonists trifluoperazine, chlorpromazine, perphenazine, promazine, tamoxifen and the naphthalene sulfonamide derivatives W7 and W13 increased the level of 32P-incorporation into human platelet PIP and PIP2. Various drugs with poor anti-calmodulin activity were ineffective. The increase in 32P-PIP and 32P-PIP2 required micromolar concentrations of trifluoperazine and was time-dependent, reaching half-maximal within two minutes of the addition of the drug. These results indicate that the calmodulin antagonists perturb polyphosphoinositide metabolism, probably at the level of the PI- and PIP-kinases and/or the PIP2- and PIP-phosphomonoesterases. PMID- 2994658 TI - Hormone-dependent phosphorylation of the 1,25-dihydroxyvitamin D3 receptor in mouse fibroblasts. AB - Experimental results, employing several immunologic techniques, suggest that the mouse receptor for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) undergoes hormone dependent phosphorylation in intact cells. Treatment of monolayer cultures of mouse 3T6 fibroblasts with 1,25(OH)2D3 reveals that the occupied 1,25(OH)2D3 receptor displays a minor reduction in electrophoretic mobility as compared to its unoccupied 54,500 dalton counterpart, a change consistent with covalent modification. Similar results were obtained by immunoprecipitation of metabolically-labeled receptors after incubation of 3T6 cells with [35S]methionine. This technique also provided greater insight into the precursor product relationship between the two receptor forms. [32P]Orthophosphate-labeling of 3T6 cells, followed by immunoprecipitation indicated that only the form exhibiting covalent modification was phosphorylated. The temporal correspondence between the binding of 1,25(OH)2D3 to its cellular receptor and its phosphorylation suggests that the biochemical role of 1,25(OH)2D3 may be to induce a conformational change susceptible to phosphorylation and possibly functional activation. PMID- 2994659 TI - Incorporation of GDP-tubulin during elongation of microtubules in vitro. AB - Removal of GDP from tubulin E-site is not obligatory for the in vitro assembly of microtubule protein in 0.5 mM GMPPCP. This assembly, which is significantly enhanced by glycerol, produces microtubules of normal morphology and with normal composition of microtubule-associated proteins (MAPs). [3H]-GDP initially present at the E-site is shown to be incorporated directly into microtubules during assembly; this incorporation, maximally 60% of the assembled polymer, is dependent upon MAPs. These results are consistent with oligomeric species composed principally of GDP-tubulin plus MAPs, being incorporated directly into microtubules. The finding that stoichiometric GTP-tubulin formation is not an essential prerequisite for microtubule assembly may have important implications for the energetics of microtubule formation. PMID- 2994660 TI - Transverse motion of spin-labeled 3,3',5-triiodo-L-thyronine in phospholipid bilayers. AB - Using electron spin resonance stop-flow technique, the transverse motion (flip flop) of 3-([alpha-carboxy-4-(4-hydroxy-3-iodophenoxy)-3,5- diiodophenethyl]carbamoyl)-2,2,5,5-tetramethyl-3-pyrrolin (T3-SL) in dipalmitoyl L-alpha-phosphosphatidylcholine (DPPC) membranes was evaluated. At 22 degrees C, the electron spin resonance spectra of T3-SL in DPPC vesicles were compared before and after the addition of sodium ascorbate, a membrane impermeable reducing agent. The addition of ascorbate reduces the signal amplitude by 67% in 3 min but yields no further reduction for at least 60 min. These results indicate that T3-SL does not flip-flop at any appreciable rate in the membranes. This finding suggests that once partitioned into the membrane, T3 remains in the outer half of the lipid bilayer, thus reducing the possibility that T3 enters the cell by passive diffusion. PMID- 2994661 TI - Desensitization to PAF-induced bronchoconstriction and to activation of alveolar macrophages by repeated inhalations of PAF in the guinea pig. AB - Guinea-pig alveolar macrophages are activated in the presence of PAF-acether (PAF), as shown by O2.- production, suggesting that these cells, abundant in the lungs, are involved in PAF-induced bronchoconstriction. Alveolar macrophages collected after in vivo desensitization to the bronchoconstrictor effect of PAF became refractory to it in vitro, whereas the O2.- production in response to f met-leu-phe persisted, although it was diminished suggesting a partial cross desensitization. A similar desensitization to PAF was also observed in alveolar macrophages in vitro, demonstrating a stimulus-specific process. This study suggests that alveolar macrophages may be involved in bronchoconstriction induced by aerosol of PAF. PMID- 2994662 TI - Purification of the epidermal growth factor receptor by tyrosine-Sepharose affinity chromatography. AB - The EGF receptor has been purified from human epidermoid carcinoma A431 cells by affinity chromatography on wheat germ agglutinin-agarose and tyrosine-Sepharose. The purified EGF receptor was shown to be homogeneous by SDS-polyacrylamide gel electrophoresis and possessed EGF-sensitive tyrosine kinase activity. Kinetic analysis of the autophosphorylation indicated that approximately 1.4 mol of phosphate was incorporated per mol of the EGF receptor. When a synthetic tyrosine containing peptide was used as a phosphorylatable substrate, the specific activity of the EGF-stimulated kinase was 66 nmol/min/mg. PMID- 2994663 TI - Vasopressin induced production of inositol trisphosphate and calcium efflux in a smooth muscle cell line. AB - Phosphatidylinositol metabolism and 45Ca2+ efflux were examined in a vascular smooth muscle cell line (A7r5). [Arg 8]Vasopressin stimulated the rapid formation (measurable at 1 sec) of inositol phosphates in a concentration-dependent manner. The time course for formation of inositol phosphates was similar to that for 45Ca2+ efflux from preloaded cells. The efflux of 45Ca2+ in response to [Arg8]vasopressin could be inhibited by a vasopressin antagonist. This supports the hypothesis that inositol 1,4,5-trisphosphate plays a role in vasopressin stimulated calcium mobilisation from an intracellular source in cultured vascular smooth muscle cells. PMID- 2994664 TI - Phospholipid methyltransferase phosphorylation by intact hepatocytes: effect of glucagon. AB - We have obtained a rabbit antiserum that specifically immunoprecipitates the 50K and 25K proteins of rat liver phospholipid methyltransferase. Exposure of intact rat hepatocytes preincubated with [32P]phosphate to glucagon induces a time dependent phosphorylation of the 50K protein of phospholipid methyltransferase. The incorporation of 32P into the 50K protein was only on phosphoserine. These data support the concept that the activation of rat liver phospholipid methyltransferase by glucagon is mediated by phosphorylation of the enzyme. PMID- 2994665 TI - Synergism between diacylglycerols and calcium ionophore in the induction of human B cell proliferation mimics the inositol lipid polyphosphate breakdown signals induced by crosslinking surface immunoglobulin. AB - Resting human tonsillar B cells were stimulated to divide by heat killed Staphylococcus aureus Cowan strain 1 which was shown to induce hydrolysis of phosphatidylinositol 4, 5-bisphosphate known to give rise to diacylglycerol and an increase in cytosolic free calcium. Addition of the diacylglycerols, 1-oleoyl 2 acetyl glycerol or sn-1, 2-dioctanoylglycerol, together with the calcium ionophore ionomycin to B cell cultures induced marked cell proliferation whereas these agents were ineffective when used alone. Both diacylglycerols were shown to compete with [3H] phorbol 12,13 dibutyrate in binding to protein kinase C. These data support the hypothesis that synergism between cytosolic calcium and endogenous diacylglycerol, which activates protein kinase C, is involved in signal transduction in the proliferation of human B cells. PMID- 2994666 TI - The differential stimulation of brain and heart cyclic-AMP phosphodiesterase by oncomodulin. AB - Ca2+/calmodulin dependent cyclic nucleotide phosphodiesterase, from the bovine heart and brain, purified by monoclonal antibody chromatography were tested with respect to activation by oncomodulin. The heart and brain enzymes which have previously been shown to have slightly different electrophoretic mobilities (1), were found to also differ in the oncomodulin dose-dependent activation of cAMP hydrolysis. Oncomodulin was shown to activate the heart enzyme to the same extent as calmodulin. However, this study indicates that the heart phosphodiesterase has approximately 25-fold higher affinity for oncomodulin than the brain enzyme. The oncomodulin concentration required for the half-maximal activation of the heart phosphodiesterase was estimated to be 2 X 10(-7)M. In addition, the possibility of the observed activation by oncomodulin being due to calmodulin contamination can be ruled out as the oncomodulin activation profiles were unaltered subsequent to chromatography on organomercurial agarose and the activation by oncomodulin could not be reversed by anti-calmodulin IgG. PMID- 2994667 TI - Identification of protein phosphatases dephosphorylating mRNP proteins from cryptobiotic gastrulae of the brine shrimp A. salina. AB - In the cytosol of A. salina cryptobiotic gastrulae at least five protein phosphatases active on phosphorylase a have been detected by ion exchange chromatography on DEAE-cellulose. Only two of these enzymes (PP-X and PP-Y) are active in mRNP dephosphorylation. Both enzymes are insensitive to inhibitor-1 and -2 and stimulation of enzymatic activity (2.5-fold with PP-X and 6.5-fold with PP Y) can be accomplished by ethanol treatment of the native enzymes, or freeze thawing in the presence of 1.7% (v/v) 2-mercaptoethanol. These properties allow PP-X and PP-Y to be classified as type-2A enzymes according to the nomenclature of Cohen. This paper is the first report of protein phosphatases capable of dephosphorylating mRNP proteins. PMID- 2994668 TI - Translational modulation in vitro of a eukaryotic viral mRNA encoding overlapping genes: ribosome scanning and potential roles of conformational changes in the P/C mRNA of Sendai virus. AB - Expression of proteins from three overlapping genes in a single mRNA species of Sendai virus was modulated in a cell-free rabbit reticulocyte translation system. Hybrid-arrested translation by oligodeoxynucleotides complementary to specific regions of the mRNA that specifies the viral P, C, and C' proteins demonstrated that ribosomes scan the RNA from its 5' end to find initiation codons, and suggested that the secondary structure of the mRNA influences the selection of alternative initiation codons. Translational modulation of P, C, and C' proteins by Mg++ and spermidine indicated that RNA folding is involved in this selection process. PMID- 2994669 TI - Metabolism of gammalinolenic acid by human blood platelet microsomes. AB - The microsomal fraction was used to test the ability of human platelets to metabolize gammalinolenic acid. The microsomal delta 6 and delta 5 fatty acid desaturase activities were measured and the incorporation of [14C]malonyl CoA into prostaglandins was also determined. The results indicate that human platelets have the capacity to elongate gammalinolenic acid (18:3 n-6) to dihomogammalinolenic acid (20:3 n-6) precursor of PGE1. Labeled PGE1 could be detected when human platelets microsomes were incubated with [14C]malonyl CoA in the presence of gammalinolenic acid. The results also show that human platelet microsomes have little delta 6 or delta 5 desaturase enzyme activity. PMID- 2994671 TI - Proton release by polymorphonuclear leukocytes during phagocytosis or activation by digitonin or fluoride. AB - The stimulation of polymorphonuclear leukocytes with bacteria or digitonin causes protons to be released into the reaction medium at the same time as the respiratory burst. Although lactic acid is released from the cells due to the stimulated metabolic activity, proton release was not due to lactic acid accumulation as fluoride markedly inhibited lactic acid accumulation in untreated cells within 5 minutes of incubation and a rapid proton release and superoxide anion production occurred after lactate production had ceased. The proton releasing mechanism, however, is closely linked with the activation of the plasma membrane NAD(P)H oxidase since the proton release is depressed only when the activation mechanism and/or the oxidase is inhibited. PMID- 2994670 TI - N-Chloroacetylhydrazone of oxo-GTP irreversibly inhibits the activating function of GTP-binding protein coupled with adenylate cyclase. AB - Guanine nucleotides are successfully used in the studies of regulatory N-proteins coupled with adenylate cyclase. In the present work N-chloroacetylhydrazones of oxo-GTP and oxo-GDP are described. After 4 hr preincubation of these nucleotides with plasma membranes from bovine brain caudate nucleus, the ability of adenylate cyclase to be activated by guanylyl-5'-methylene-diphosphonate is blocked. The degree of inhibition depends on preincubation time and increases in the presence of Mg2+. Guanylyl-5'-methylenediphosphonate protects adenylate cyclase from the action of N-chloroacetylhydrazone of oxo-GTP. These findings suggest that adenylate cyclase activation is diminished as a result of covalent modification of the Ns. N-chloroacetyl-hydrazone of oxo-GDP also causes a loss of the adenylate cyclase sensitivity to the fluoride ion and cholera toxin. PMID- 2994672 TI - Reversible activation of human neutrophil calpain promoted by interaction with plasma membranes. AB - Human neutrophil calpain is a monomer of 85 kDa molecular weight. The proteinase shows an absolute requirement for Ca2+ with maximal catalytic activity at 0.1-0.2 mM Ca2+ and negligible activity at 1-5 microM Ca2+. At this concentration of Ca2+ neutrophil calpain becomes active and reaches 65% of its maximal catalytic activity following interaction with plasma membranes. The activation is fully reversible since the enzyme returns to its native, high Ca2+ requiring form following removal of the membranes. Membrane phospholipids appear to be the physiological compounds responsible for the promotion of such reversible activation. Unlike other Ca2+ dependent proteinases, neutrophil calpain does not undergo conversion to a low Ca2+ requiring form by limited autoproteolysis. PMID- 2994673 TI - Isoproterenol oxidation by tyrosinase: intermediates characterization and kinetic study. AB - The present work deals with isoproterenol oxidation by mushroom tyrosinase and sodium metaperiodate. Intermediates produced at short reaction time were characterized by scanning repetitive spectrophotometry and the stoichiometry of the respective aminochrome appearance was established. The oxidation pathway from isoproterenol to aminochrome is parallel to the previously proposed for L-dopa oxidation by mushroom tyrosinase, whose steps are as follow: Isoproterenol----o quinone-H+----o-quinone----leukoaminochrome---- aminochrome. The stoichiometry for the conversion of o-quinone-H+ into the aminochrome of isoproterenol followed the equation: 2 o-quinone-H+----isoproterenol + aminochrome. The kinetics of chemical reactions that take place from the o-quinone-H+ to aminochrome has been studied as a system of various chemical reactions coupled to an enzymatic reaction (EzCC: Enzymatic-Chemical-Chemical mechanism). PMID- 2994674 TI - Induction of human HL-60 leukemic cell differentiation by immune interferon is accompanied by an increase in NADase activity and by a decrease in DNA-binding proteins. AB - The effects of highly purified human immune interferon (IFN-gamma) on the differentiation of human promyelocytic HL-60 leukemic cells have been studied. The addition of 100 units/ml interferon to HL-60 cells for 5 days results in morphological changes characteristic of macrophages. At the biochemical level, there is a 3-fold increase in the specific activity of the enzyme NADase. Kinetic analysis shows that IFN-gamma causes an increase in the Vmax of NADase without affecting the apparent Km. Pulse labeling experiments with [35S] methionine show a marked change in the de novo synthesis of several proteins in the course of interferon treatment. Chromatography on DNA-agarose show that after treatment with interferon for 24 or 48 h, there is a 60-70% decrease in newly synthesized proteins which bind DNA-agarose and can be subsequently displaced from the column with 2% SDS containing buffer (from 7.7-8.7% bound in control cell extracts to 2.6-3.1% bound in interferon treated cell extract). PMID- 2994675 TI - Nucleotidase activities of human peripheral lymphocytes. AB - Several B lymphoblastic cell lines are known to be relatively resistant to the combination of 2'-deoxyadenosine with an adenosine deaminase inhibitor. These cell lines are believed to have a greater capacity to dephosphorylate 2' deoxyadenosine nucleotides, thus preventing excessive accumulation of potentially toxic metabolites. In this study, the 2'-deoxynucleoside 5'-monophosphate dephosphorylating activities of human peripheral lymphocytes were examined. Peripheral lymphocytes have at least three nucleotide 5'-monophosphate nucleotidases distinguished by different pH optimums, substrate preference, Mg2+ requirement, inhibitors, and molecular weights. Two of the enzymes appeared to be cytosolic, only one of which had significant substrate activity with dAMP. This enzyme had an acidic pH optimum (5.0), no Mg2+ requirement, was inhibited by tartrate, and demonstrated broad substrate specificity. The other cytosolic nucleotidase required Mg2+, had a pH optimum of 5.5 to 6.0, was activated by 2' deoxyinosine, and demonstrated a substrate preference for 3'- and 5' monophosphate 2'-deoxynucleosides of hypoxanthine, guanine, uracil, and thymine. The third enzyme, ecto 5'-nucleotidase, is associated with the cell membrane. Although the ecto 5'-nucleotidase activity was higher in the B lymphocytes, the cytosolic nucleotidases were similar in activity in the T and B lymphocytes. PMID- 2994676 TI - The binding of the radioprotective agent cysteamine with the phospholipidic membrane headgroup-interface region. AB - The interaction of the aminothiol radioprotector cysteamine (beta mercaptoethylamine) (CYST) with dipalmitoylphosphatidylcholine (DPPC) artificial membranes has been studied by differential scanning calorimetry (DSC), turbidimetry and spin labeling. This hydrophilic molecule displays a biphasic, concentration-dependent binding to the phospholipidic head groups at neutral pH. In the CYST/DPPC molar ratio 1:160-1:2 (mole/mole) an increasing ordering effect is observed. At high concentrations (over 3:1 ratio), this ordering effect decreases. With the symmetric disulfide dimer cystamine, the biphasic effect is not shown and the membrane rigidity decrease is obtained only at concentration ratio higher than 1:1. The charge repartition of the cysteamine molecule has been shown to be disymmetric, +0.52 e on the NH3 group and +0.19 e on the SH extremity, [38] whereas the cystamine molecule is electrostatically symmetrical. These properties could be related to their membrane effects. With cysteamine, at a low concentration, an electrostatic bridging between the negatively charged phosphate groups of the polar heads induces the increase in membrane stability: the molecules behave like a divalent cation. At high concentrations a displacement of the slightly charged SH extremity by the amine disrupts the bridges and induces the decrease in rigidity: the drug behaves like a monovalent cation. Due to its symmetric charge and its double length, such an effect is not observed with cystamine. This study could bring further information about the interactions between cysteamine and polyelectrolytic structures (ADN for example) and about the radioprotective properties of this drug. PMID- 2994678 TI - Model for action of local anaesthetics with cytochrome oxidase. PMID- 2994677 TI - Interaction of vinblastine sulfate with artificial phospholipid membranes. A study by differential scanning calorimetry and spin labeling. AB - The effect of the antimitotic drug vinblastine sulfate has been studied on fully hydrated dipalmitoylphosphatidylcholine (DPPC) liposomes in the temperature range 0 degrees to 60 degrees using differential scanning calorimetry and electron spin resonance spectroscopy with two fatty acid spin labels. In the gel phase, vinblastine interacts essentially with the DPPC polar heads and induces an important disorganization of the phospholipidic bilayer. The co-operativity of the main thermal transition is decreased. In the crystal-liquid phase, the drug penetrates inside the artificial membrane and induces the formation of domains which increased thermal stability. These effects are opposite to those observed with the drug isaxonine which is used to reduce the axonal degenerating effects due to vinblastine. PMID- 2994679 TI - Effects of coordinated gold compounds on in vitro and in situ DNA replication. AB - Auranofin, a coordinated gold compound, inhibits in vitro DNA synthesis and displays in vivo antitumor activity. To understand the mechanisms of inhibition of DNA replication, we have examined the effects of auranofin and other gold complexes on the activities of purified cellular and herpesvirus-induced DNA polymerases, and on in situ DNA replication in permeabilized S phase KB cells. Evaluation of the data suggests the following conclusions. (1) The gold compounds varied in their abilities to inhibit DNA polymerase activities. DNA polymerase alpha was more sensitive to inhibition by gold compounds than DNA polymerase beta; (2) Inhibition of purified DNA polymerases by gold (I) compounds was noncompetitive with both DNA template and triphosphate substrates. Inhibition by SKF 101675, a gold (III) complex was competitive with DNA. (3) None of the gold compounds tested preferentially inhibited herpesvirus-induced DNA polymerases. (4) The gold complexes that inhibited in vitro DNA replication also inhibited in situ DNA synthesis. However, the potency and order of potency of the compounds varied between the in vitro and in situ systems. (5) Auranofin and other gold compounds inhibited the clonogenic capacity of KB cells in a concentration dependent manner. The IC50 values measured in the clonogenic assay were significantly lower than those obtained from the in vitro and in situ DNA replication assays. PMID- 2994680 TI - Characterization of histamine H-1 receptors on human peripheral lung. AB - Histamine H-1 receptors in human peripheral lung were characterized by radioligand and biochemical assays employing binding of the H-1 receptor antagonist [3H]pyrilamine to plasma membrane preparations. Simultaneous computerized analyses of the data from fourteen separate equilibrium-binding assays indicated the presence of three distinct classes of binding sites with Kd values of 81 +/- 35 pM, 7 +/- 3 microM, and 320 +/- 167 microM and binding capacities of 23 +/- 3 pmoles, 10 +/- 5 nmoles, and 297 +/- 119 nmoles/mg protein respectively. Dissociation kinetics of [3H]pyrilamine binding also supported the presence of three binding sites or states. Further, competition binding curves for histamine receptor agonists and antagonists also indicated the presence of multiple binding sites for the H-1 receptor. The effect of exogenous stimulation of histamine H-1 receptors on human cyclic nucleotides was also examined. Both histamine and the H-1 agonist 2-methyl histamine caused dose-related increases in the cyclic guanosine monophosphate (GMP) content of human lung. The effects of 2 methyl histamine were selective for cyclic GMP. The histamine-induced increase in cyclic GMP peaked within 1.0 min and was effectively prevented by the H-1 antagonist pyrilamine. Thus, human lung possesses a large number of H-1 receptors which exhibit three binding states and produce cyclic GMP, but not cyclic adenosine monophosphate (AMP), when stimulated. PMID- 2994681 TI - Effect of probenecid on the disposition of captopril and captopril dimer in the rat. AB - The urinary excretion of captopril has been studied in a bladder-cannulated rat model and compared with that obtained after co-administration with probenecid. Probenecid reduced significantly the urinary excretion of captopril from 41% to 21% of the administered dose over a 3-hr period and significantly lowered urine flow rates. In addition, the effect of probenecid on plasma levels of captopril and total captopril (captopril plus disulfides) after oral administration of the disulfide prodrug captopril dimer (10 mg/kg) has been studied in a conscious rat preparation. Co-administration of probenecid (20 mg/kg) given either orally or intravenously increased both the plasma levels of captopril and total captopril (captopril plus captopril disulfides) over a 4-hr period. A prolonged significant inhibition of plasma ACE after co-administration of probenecid and captopril dimer suggests that probenecid may be useful to prolong the action of captopril or the prodrug captopril dimer. PMID- 2994682 TI - Interactions with calmodulin: potential mechanism for some inhibitory actions of tetracyclines and calcium channel blockers. PMID- 2994683 TI - [Effective method of oligonucleotide-controlled mutagenesis of DNA fragments]. AB - A procedure has been designed for changing specific nucleotides in a DNA sequence with efficiency. The method involves the use of the specially constructed cloning vectors pBRS1, pHS1, and pHS2. These plasmids are derivatives of pBR322 in which the EcoRI-HindIII region has been replaced by synthetic duplexes carrying SmaI, HpaI and XhoI sites, in addition to EcoRI and HindIII sites. The DNA fragment to be mutagenized is cloned in pHS1 (or pBRS1, or pHS2) using restriction sites close to the SmaI and HpaI sites. The recombinant plasmid obtained is digested with one of these enzymes to produce double-stranded DNA with blunt ends. This linear DNA is a substrate for exonuclease III (or T4 DNA polymerase). The digestion under controlled conditions produces duplex with protruding single stranded 5'-regions which include the site of the desired mutation. The synthesis of DNA by DNA-polymerase I (Klenow's fragment), primed in part by the synthetic oligonucleotide containing the desired mutation, leads to the linear heteroduplex. The closed circular heteroduplex is formed by ligation. After transformation into E. coli, DNA replication generates homoduplexes, some of which contain the mutation. Colony hybridization with the same 32P-labeled oligonucleotide is used to select mutants. The yield of the mutants is 10-20%. This technique can be extended to replicative form of M13 vectors. It can be also applied to any DNA sequence which has a unique site of restriction endonuclease generating blunt ends. PMID- 2994684 TI - [Hydrolytic stability of a covalent AMP-RNA ligase complex]. AB - Behavior of the covalent [32P]- and [14C]AMP-RNA ligase complex under various conditions has been studied. The covalent structure is shown to be readily cleaved by acid and hydroxylamine and relatively stable to alkali and snake venom phosphodiesterase. Products of degradation of the AMP-RNA ligase and AMP-DNA ligase complexes were compared. The data obtained support the earlier assumption of a phosphoamide bond in the AMP-RNA ligase compound. PMID- 2994685 TI - [Construction of a gene library using partial filling of DNA sticky ends]. AB - To prepare gene libraries, the incomplete filling of protruding ends has been used. DNAs from phages EMBL 3 and EMBL 3a were sequentially digested with SalI and EcoRI, followed by addition of dTTP, dCTP, and DNA polymerase I (Klenow's fragment). Separately, a genomic DNA was partially cleaved with Sau3AI, followed by addition of dATP, dGTP, and Klenow's fragment. The fragmented phage and genomic DNAs were mixed and ligated, and the recombinant DNAs packed in vitro with the phage proteins. The effectiveness of packaging per microgram of genomic DNA was 10(5) to 10(6) (for the wild phage DNA, 10(7)). The proposed procedure is very rapid and needs only microgram quantities of genomic DNA for preparing a representative gene library. It is also useful for other vectors, containing SalI sites. PMID- 2994687 TI - Different defects of T cell regulation of Epstein-Barr virus-induced B cell activation in rheumatoid arthritis. AB - Several reports have shown a defective Epstein-Barr virus (EBV)-specific suppressor T cell function in rheumatoid arthritis (RA), suggesting that EBV may have a role in the pathogenesis of RA. EBV-specific T cell regulation was studied in 47 EBV-immune RA patients and in 14 EBV-immune control subjects by comparing the secretion of IgM into supernatants of 28-day cultures of B cells alone and cocultures of B and autologous T cells. In control subjects, autologous T cells mediated a significant decrease in the secretion of IgM by B cells at 12 and 16 days of culture. Analysis of individual responses demonstrated the existence of 3 subgroups of RA patients: group I (18 patients) had a suppressor T cell function similar to that of controls; group II (21 patients) had a defective T cell function; group III (8 patients) was characterized by a "late help phenomenon." Moreover, in RA group III, IgM secretion in cultures of B cells alone was lower than that seen in controls, RA group I, or RA group II. Differences in the duration or severity of the disease, or in the use of slow-acting therapeutic agents, corticosteroids, and nonsteroidal antiinflammatory drugs could not account for these subdivisions. Thus, our study demonstrates that several immunoregulatory defects exist in subgroups of RA patients. PMID- 2994686 TI - Crystal-induced endogenous pyrogen production. A further look at gouty inflammation. AB - We found previously that crystals of sodium urate and silicon dioxide (silica) can stimulate the production of endogenous pyrogen (EP), now called interleukin-1 (IL-1), the polypeptide mediator of fever and other aspects of inflammation. We have confirmed and extended the work with urate crystals and have examined 2 other crystals associated with joint problems, hydroxyapatite (HA) and calcium pyrophosphate dihydrate (CPPD). The crystals were added to suspensions of human blood leukocytes (2.5 X 10(6) monocytes/dose, with 10% fresh autologous plasma); after 18 hours of incubation, the EP content of the supernatants was assayed in the rabbit pyrogen test. HA and CPPD crystals neither induced EP production nor reduced the amount of staphylococci-induced EP. Presized (10 - 40 micron) urate crystals were pyrogenic, but less so than the unsized and aggregated urate crystals investigated previously and reexamined here. On ultrasonication, the aggregated urate crystals became first more pyrogenic and then less so as the crystals were dispersed and broken down. Ultrasound did not impart pyrogenicity to HA or CPPD crystals: their failure to stimulate EP/IL-1 production from leukocytes in vitro indicates a difference in their phlogistic properties, compared with crystals of urate or silica. The results with urate crystals have pathogenetic implications in a number of areas of gouty inflammation: initiation of the acute attack, other aspects of the acute-phase response, polyarticular involvement, and the inflammatory consequences of chronic stimulation by tophaceous material. PMID- 2994688 TI - Catecholamine regulation of neocortical spindling in DBA/2 mice. AB - The waking EEG of DBA/2 mice is punctuated by conspicuous bursts of high amplitude, 6-7-cps spindles. Catecholamine depletion by 2.0 mg/kg, i.p., reserpine or 120 mg/kg, i.p., alpha-methyl-p-tyrosine increased the occurrence and duration of these brief spindle episodes (BSEs). This effect may reflect noradrenergic depletion because the beta-noradrenergic antagonist propranolol (10 mg/kg, i.p.) powerfully promoted BSE occurrence and increased BSE duration in freely-moving and midpontine-transected mice, whereas the dopamine antagonist haloperidol (2.0 mg/kg, i.p.) neither increased nor decreased BSE occurrence. The alpha-noradrenergic agonist clonidine (0.05 mg/kg, i.p.), which is known to inhibit noradrenergic neuronal firing as well as act at postsynaptic alpha receptors, also promoted BSE occurrence in transected mice. In addition, the dopamine agonist apomorphine (2.0 mg/kg, i.p.) increased BSE occurrence in freely moving mice once the behavioral activation it produced subsided. These effects were blocked by 2.0 mg/kg haloperidol, i.p. The convulsant drug pentylenetetrazol, which is known to promote BSE occurrence at subconvulsant doses in DBA/2 mice, may activate BSEs, in part, by activating dopamine neurons: 2.0 mg/kg haloperidol, i.p., partially blocked the facilitation of BSEs by 20 mg/kg pentylenetetrazol, i.p., in midpontine DBA/2 mice. Thus, noradrenergic neurons may block spindle occurrence in DBA/2 mice whereas dopamine neurons may be one of several systems that can promote spindle occurrence. PMID- 2994689 TI - The painlike effect of gallamine and naloxone differs from sickness induced by lithium chloride. AB - In rats, the conditioned place and taste aversions produced by 31.8 mg/kg lithium chloride were compared with those produced by 10 mg/kg gallamine in Experiment 1 and 20 mg/kg or 2.5 mg/kg naloxone in Experiments 2 and 3, respectively. Lithium produced stronger taste aversions than gallamine or the two doses of naloxone, but gallamine and naloxone each produced stronger place aversions than lithium. These findings support the distinction between two kinds of drug-induced aversive effects, having different associative properties. One effect, called sickness, is more associable with taste than with place cues; the second effect is, like pain, more associable with place than with taste. PMID- 2994690 TI - Fixation of spinal reflex alterations in spinal rats by sensory nerve stimulation. AB - Two preparations in which sensory nerve stimulation was used to obtain peripherally induced spinal fixation in spinal rats are described. In the first preparation, proportionally greater amounts of persisting poststimulation flexor muscle contraction, as measured by a force displacement transducer, were produced as stimulation time was increased from 10 min to 40 min. In the second preparation, sensory nerve stimulation was delivered, and evoked whole-nerve responses were recorded from a flexor motor nerve. Results indicated that 30 min or more of sensory nerve stimulation produced increases in response amplitude and area that persisted for at least 30 min after stimulation. PMID- 2994692 TI - Studies on cytochrome c oxidase, XII. Isolation and primary structure of polypeptide VIb from bovine heart. AB - The isolation and complete sequence analysis of the cytoplasmically synthesized polypeptide VIb from bovine heart cytochrome c oxidase is described. The protein is a stoichiometric constituent of the respiratory complex IV. Its primary structure is deduced from N-terminal sequencing and overlapping peptides obtained from tryptic cleavage and specific cleavage at arginyl and tryptophyl peptide bonds. The polypeptide chain consists of 84 amino acids from which a Mr of 9419 is derived. It has a relatively high content of histidine and proline and contains a single cysteine. A hydrophobic sequence of 20 amino acids points to a membrane-penetrating structure similar to that found in polypeptides I, II, III, IV and VIIIa, VIIIb, VIIIc of the bovine oxidase. The sequence of VIb is tissue specific, it contributes to the formation of nuclear coded isoenzymes of cytochrome c oxidase. The protein thus may be involved in a tissue-specific regulation of cellular respiration. PMID- 2994691 TI - Control of oxygen uptake, microcirculation and glucose release by circulating noradrenaline in perfused rat liver. AB - The effect of noradrenaline on oxygen uptake, on periportal and perivenous oxygen tension at surface acini, on microcirculation and on glucose output were studied in isolated rat livers perfused at constant flow with Krebs-Henseleit-hydrogen carbonate buffer containing 5mM glucose and 2mM lactate. Noradrenaline at 1 microM concentration caused a decrease in oxygen uptake, while at 0.1 microM it led to an increase. Both high and low doses of noradrenaline decreased the tissue surface oxygen tension in periportal and - after a transient rise - in perivenous areas. Noradrenaline at an overall constant flow caused an increase of portal pressure and an alteration of the intrahepatic distribution of the perfusate: at the surface of the liver and in cross sections infused trypan blue led to only a slightly heterogeneous staining after a low dose of noradrenaline but to a clearly heterogeneous staining after a high dose. Both high and low doses of noradrenaline stimulated glucose release. All effects could be inhibited by the alpha-blocking agent phentolamine. In conclusion, control of hepatic oxygen consumption by circulating noradrenaline is a complex result of opposing hemodynamic and metabolic components: the microcirculatory changes inhibit oxygen uptake; they dominate after high catecholamine doses. The metabolic effects include a stimulation of oxygen utilization; they prevail at low catecholamine levels. The noradrenergic control of glucose release is also very complex, involving direct, metabolic and indirect, hemodynamic components. PMID- 2994693 TI - [Sero-epidemiological results in subjects receiving a renal transplant]. AB - In this study a sero-epidemiological investigation on 127 renal allograft recipients was examined. In these patients, treated with Cyclosporine A or conventional drugs, antibody response to various antigens (Cytomegalovirus, Herpes simplex, Varicellae/Zoster and Mycoplasma pneumoniae) was examined. The data were compared to the healthy population and dialyzed subjects. PMID- 2994694 TI - [Radio-hormonal study (radioimmunoassay) in young women with functional hyperprolactinemia. 2) Effects of a dopaminergic agonist, ibopamine, on the regulation of prolactin and gonadotropin secretion (LH, FSH)]. PMID- 2994695 TI - Influence of beta-receptor stimulation on catecholamine and phospholipid concentrations in lungs of fetal rabbits. AB - It was studied whether the beta-receptor stimulating fenoterol had any influence on endogenous catecholamines in the lung tissue of fetuses. On the 23rd, 27th and 31st days of pregnancy, concentrations of noradrenaline and dopamine in the lung homogenate of mature and immature rabbit fetuses were determined by fluorometry, and the basic surfactant components total phospholipids and lecithin by colorimetry, in a group receiving beta-mimetic treatment and in a control group receiving physiological salt solution. The concentration of noradrenaline decreased with the progression of pregnancy but the concentration of dopamine did not change significantly. Application of fenoterol caused an increase in total phospholipids and lecithin in the lungs of 23 and 27 day old rabbit fetuses and decreased the concentration of catecholamines, especially of noradrenaline. The drug had no such effect in mature (31 day) fetuses. PMID- 2994696 TI - [Protective effect of radical scavengers on cerebral infarction--experimental study utilizing the spin trapping method of ESR]. AB - To evaluate the scavenging effect of mannitol, vitamin E and betamethasone in cerebral ischemia, spin trapping technique was applied to the detection of the free radicals generated in ischemic brain homogenate. Thirty Wistar rats were used for this study. In the control group, the brain homogenate prepared immediately after decapitation was preserved at 37 degrees C under N2 gas. Before the preservation and at 30 min, 60 min and 120 min from the start of the preservation, two reaction mixtures containing of spin trapping reagent phenyl-t butyl nitrone (PBN), NADPH, Fe-EDTA and brain homogenate was prepared from each brain sample--one to be incubated for 20 min at 37 degrees C in air and one to be incubated in nitrogen gas under similar condition. Then the free radical adducts of PBN were measured by electron spin resonance (ESR). In pre-treated group, mannitol, vitamin E and betamethasone were administered intravenously 30 min prior to the decapitation and spin adducts of PBN were detected by same procedures as in control group. The ESR spectra, which hyperfine coupling constants were AN = 16.0-16.6 G and AH beta = 3.0-3.8 G, were obtained from the reaction mixtures in each group. Analyses of their relative intensity in control group revealed that the formation of free radical adducts of PBN was increased dependent on the preservation period under aerobic incubation, and increased gradually for 60 min of preservation time followed by a decrease under anaerobic incubation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2994697 TI - Usefulness of plasma renin activity in predicting haemodynamic and clinical responses and survival during long term converting enzyme inhibition in severe chronic heart failure. Experience in 100 consecutive patients. AB - The relation between plasma renin activity before treatment and the haemodynamic and clinical responses to converting enzyme inhibition was determined in 100 consecutive patients with severe chronic heart failure who were treated with captopril or enalapril. Initial doses of captopril produced significant increases in cardiac index and decreases in left ventricular filling pressure, mean arterial pressure, mean right atrial pressure, heart rate, and systemic vascular resistance that varied linearly with the pretreatment value for plasma renin activity. In contrast, there was no relation between the pretreatment activity and the magnitude of haemodynamic improvement after 1-3 months of treatment with the converting enzyme inhibitors, and, consequently, a similar proportion of patients with a high (greater than 6 ng/ml/h; greater than 4.62 mmol/l/h), intermediate (2-6 ng/ml/h; 1.54-4.62 mmol/l/h), and low (less than 2 ng/ml/h; less than 1.54 mmol/l/h) pretreatment value improved clinically during long term treatment (64%, 60%, and 64% respectively). Long term survival after one, two, and three years was similar in the three groups. Estimating the degree of activation of the renin-angiotensin system by measuring pretreatment plasma renin activity fails to predict the long term haemodynamic or clinical responses to converting enzyme inhibitors in patients with severe chronic heart failure, and thus appears to be of limited value in selecting those patients likely to benefit from treatment with these drugs. PMID- 2994699 TI - Effect of trimetazidine on membrane damage induced by oxygen free radicals in human red cells. AB - The effect of trimetazidine, 1-(2, 3, 4 trimethoxybenzyl)piperazine di hydrochloride, on membrane damage induced by oxygen free radicals in red cells was studied in seven healthy volunteers after oral administration. Red cells collected prior to and after a 7 day treatment period with trimetazidine were incubated in the presence of phenazine methosulphate (an intracellular oxygen free radical generator) and diethyldithiocarbamate (a Cu-Zn superoxide dismutase inhibitor). The loss of intracellular K+ induced by oxygen free radicals and the membrane content of peroxidated lipids were significantly reduced in red cells collected after the period of treatment. These results indicate a potent antioxidant activity of trimetazidine which could explain its cardioprotective role during ischaemic and reperfusion phases in which oxygen free radicals are generated and probably implicated in the genesis of cardiac cell injury. PMID- 2994698 TI - Effects of enalapril in heart failure: a double blind study of effects on exercise performance, renal function, hormones, and metabolic state. AB - Several studies have shown symptomatic and haemodynamic improvement after the introduction of angiotensin converting enzyme inhibitors in patients with heart failure treated with diuretics. The concomitant long term effects of the new orally effective long acting angiotensin converting enzyme inhibitor, enalapril, on symptoms, exercise performance, cardiac function, arrhythmias, hormones, electrolytes, body composition, and renal function have been further assessed in a placebo controlled double blind cross over trial with treatment periods of eight weeks. Twenty patients with New York Heart Association functional class II to IV heart failure who were clinically stable on digoxin and diuretic therapy were studied. Apart from the introduction of enalapril, regular treatment was not changed over the study period; no order or period effects were noted. Enalapril treatment significantly improved functional class, symptom score for breathlessness, and exercise tolerance. Systolic blood pressure was significantly lower on enalapril treatment. Echocardiographic assessment indicated a reduction in left ventricular dimensions and an improvement in systolic time intervals. In response to enalapril, the plasma concentration of angiotensin II was reduced and that of active renin rose; plasma concentrations of aldosterone, vasopressin, and noradrenaline fell. There were significant increases in serum potassium and serum magnesium on enalapril. Glomerular filtration rate measured both by isotopic techniques and by creatinine clearance declined on enalapril while serum urea and creatinine rose and effective renal plasma flow increased. Body weight and total body sodium were unchanged indicating that there was no overall diuresis. There was a statistically insignificant rise in total body potassium, though the increase was related directly to pretreatment plasma renin (r = 0.5). On enalapril the improvement in symptoms, exercise performance, fall in plasma noradrenaline, and rise in serum potassium coincided with a decline in the frequency of ventricular extrasystoles recorded during ambulatory monitoring. Adverse effects were few. In patients with heart failure, enalapril had a beneficial effect on symptoms and functional capacity. The decline in glomerular filtration rate on enalapril may not be beneficial in early heart failure. PMID- 2994700 TI - Lipoxygenase products of arachidonic acid in human inflamed skin. AB - Monohydroxy acids (HETEs) and leukotriene B4 (LTB4) metabolites of arachidonic acid were measured in skin of healthy volunteers after ultraviolet B irradiation, and in the uninvolved skin of psoriatics after topical dithranol application. Exudate was collected from suction bullae on control and inflamed abdominal skin, and analysed for 12-HETE and PGE2 by GC-MS and LTB4 by bioassay. 12-HETE and PGE2 were raised at 24 h but not at 72 h after u.v.B irradiation: control and 24 h values were 13.7 and 41.5 ng ml-1 (P less than 0.05, n = 6) for 12-HETE respectively, and 4.5 and 30.2 ng ml-1 (P less than 0.01, n = 6) for PGE2. Dithranol application raised PGE2 levels from 23.1 ng ml-1 in control exudate to 62 ng ml-1 (P less than 0.01, n = 6) at 24 h before declining to base levels at 72 h. However, 12-HETE was raised at 72 h (200 ng ml-1, P less than 0.01, n = 5) but not at 24 h (104 ng ml-1) compared to control levels (50 ng ml-1, n = 5). The levels of the LTB4 were low (less than 100 pg ml-1), and no significant increases were observed. Arachidonic acid in inflamed skin can be metabolised by the cyclo oxygenase and lipoxygenase pathway. It is probable that the lipoxygenase product 12-HETE is involved in these inflammatory reactions. PMID- 2994701 TI - Effects of verapamil and nisoldipine on human platelets: in vivo and in vitro studies. AB - Inhibition of platelet aggregation was observed after 4 days of oral dosing with the calcium antagonists, verapamil (160 mg) or nisoldipine (20 mg) but not following acute dosing. These effects were observed at plasma concentrations that had no effect on platelet aggregation when investigated in vitro. Verapamil added in vitro inhibited adrenaline-induced platelet aggregation at relatively low concentrations (IC50 16 microM) but only inhibited aggregation to adenosine diphosphate at very high concentrations (IC50 700 microM). Nisoldipine, a dihydropyridine, added in vitro had no effect on platelet aggregation induced by adenosine diphosphate but inhibited by 67%, the secondary phase of platelet aggregation induced by adrenaline. Verapamil but not nisoldipine displaced [3H] yohimbine from the specific binding sites on human platelets, suggesting an interaction with alpha 2-adrenoceptors. Inhibition of adrenaline-induced aggregation by verapamil in vitro may be a result of antagonism of alpha 2 adrenoceptors but long term treatment with both verapamil and nisoldipine also inhibits platelet aggregation mechanisms other than by alpha 2-adrenoceptor blockade. PMID- 2994702 TI - The effect of enalapril on baroreceptor mediated reflex function in normotensive subjects. AB - The effects of enalapril, 20 mg orally, on the responses to baroreflex activation and deactivation by respectively phenylephrine and nitroglycerin were investigated in normotensive subjects on a normal sodium diet, with simultaneous measurement of plasma renin activity (PRA), converting enzyme activity (PCEA), aldosterone and catecholamines. Enalapril, 4 h after administration, lowered artificial blood pressure without modifying heart rate and plasma catecholamines. PCEA was abolished, PRA increased and plasma aldosterone decreased. Enalapril (a) displaced to the left the baroreflex set-point, (b) did not affect baroreflex sensitivity since the slopes of the RR-interval/systolic blood pressure regression lines remained unchanged during both activation and deactivation and (c) did not modify baroreflex efficacy since the maximal RR-interval responses as well as the overall RR-interval-time products to identical blood pressure variations were not modified. Thus, enalapril induced a resetting of the baroreflex, which probably accounts for the lack of reflex tachycardia observed during the drug-induced fall in blood pressure. PMID- 2994703 TI - Three months treatment with chemotherapy and radiotherapy for small cell lung cancer. AB - Fifty-five patients with inoperable but limited stage small cell carcinoma of the bronchus and a further 15 patients with contra lateral neck nodes, pleural effusions and marrow involvement were entered into the study and treated. The 3 month treatment regimen comprised 3 courses of etoposide with cyclophosphamide at 2.5 gm-2 followed by methotrexate and radiotherapy, no maintenance treatment was given. The complete response rate in the total patient group was 54% and the partial response rate 21%. The median survival was 11 months for the 70 patients, 15 months for the complete responders, and those patients with a bronchoscopically confirmed complete response survived significantly longer. There was no significant difference between the patients with strictly limited stage disease and those in the broader category. Eight patients are tumour free and alive one year or more after the end of treatment. The median followup is 17 months. Twenty-four patients were delayed 1-2 weeks during treatment because of chemotherapy induced toxicity. Six patients died probably of infection associated with leucopaenia. The majority of the patients' Karnofsky performance improved with the treatment as did their breathlessness assessed on a respiratory score. The short intensive chemotherapy regimen of 3 months produced similar results to those following more prolonged treatment regimens. PMID- 2994704 TI - Evaluation of a radioimmunoassay for neuron specific enolase in small cell lung cancer. AB - A radioimmunoassay for neuron specific enolase (NSE), a marker of neuroendocrine differentiation, has been evaluated in small cell lung cancer (SCLC). In untreated patients 25/38 (68%) with localized SCLC had raised blood levels of NSE (greater than 13 ng ml-1), in extensive disease 34/39 (87%) patients had raised NSE levels. In patients with non-small cell lung cancer (NSCLC) the serum levels were raised in 16/94 (17%). In extensive tumours of non-pulmonary origin NSE levels were increased in 24/116 (20%) patients. Longitudinal studies indicated a good correlation between the response to chemotherapy and fall of NSE levels. Tumour progression was accompanied by a rising NSE in 25/29 patients, with doubling times of 7-90 days. In patients with progression with a normal NSE the recurrence was a NSCLC. Cerebral metastases occurring as the only recurrence during clinical complete remission were not accompanied by a rise of NSE. Serum NSE levels provides a valuable monitor for SCLC during and after chemotherapy. PMID- 2994706 TI - Dietary fibre consumption in Britain: new estimates and their relation to large bowel cancer mortality. PMID- 2994705 TI - The effect of food and concurrent chemotherapy on the bioavailability of oral etoposide. AB - There is no information on the effect of food or concurrent drug administration on the bioavailability of oral etoposide, despite the fact that treatment is frequently administered over several days and most often in combination with other cytotoxic agents. The influence of these factors has been studied in 11 patients, receiving combination cytotoxic therapy for extensive small cell lung carcinoma. Neither food nor concurrent oral or intravenous chemotherapy had a significant effect on the mean plasma concentrations of etoposide, achieved following oral administration. Wide variation in peak plasma concentrations and in area under the concentration time curve (AUC) occurred both between and within patients. It appears unnecessary for patients receiving etoposide (at 100 mg) to fast prior to drug administration. Furthermore, oral etoposide (at 100 mg and at 400 mg) may be given in combination with other cytotoxic agents without compromising its bioavailability. PMID- 2994707 TI - Body reactions during chain saw work. AB - Body reactions during chain saw work were studied in 14 subjects. The subjects divided into three groups (control, sulpiride, and propranolol) successively cut down logs with a chain saw for seven minutes. The start of the sawing led to a pronounced increase in heart rate which persisted during the sawing. The groups taking sulpiride and propranolol showed a smaller increase in heart rate compared with the controls. Hormonal values (adrenocorticotropic hormone (ACTH), cortisol, adrenaline (Ad), noradrenaline (NA), and dopamine) were increased by the operation. A comparison of these values before and after the operation showed that the increase of cortisol, Ad, and NA values was highest in the controls, intermediate in the propranolol group, and lowest in the sulpiride group. The increase in ACTH, however, was greatest in the sulpiride group, intermediate in the controls and correct in the propranolol group. These findings provide some evidence that chain saw work may have an influence on the whole body, including the hypothalamus and the limbic lobe of the brain. PMID- 2994709 TI - A rapid and sensitive culture test for detecting herpes simplex virus from the eye. AB - A rapid and sensitive culture test has been developed for detecting herpes simplex virus (HSV) in ocular infections. The virus is cultured by inoculation and centrifugation of cell monolayers grown on coverslips and the inclusions detected by an indirect immunofluorescence technique. This rapid test takes only two days to complete. By comparison, in our hands the conventional culture test, which depends on the development of cytopathic effect, took between 1 and 20 days with a mean of 4.7 days. Of the 1638 ocular clinical specimens inoculated in parallel by the two methods a total of 188 were positive for HSV. The virus was detected from 184 (97.8%) specimens by the rapid test and from 144 (76.6%) by the conventional test (McNemar's test, U = 5.76, p less than 0.001). PMID- 2994708 TI - Pulmonary response to agate dust in vivo and cytotoxic and haemolytic effects in vitro. AB - The pulmonary response to agate dust in vivo was investigated and also its cytotoxic action on peritoneal macrophages and sheep erythrocytes in vitro. The results were compared with quartz dust as the known fibrogenic dust and emery dust (Corundum) was used as a control dust. Agate increased the wet and dry weight of lungs and induced increased collagen formation and a non-reversible fibrotic reaction in the lungs. The tissue response and lung changes were of milder intensity than seen in rats exposed to quartz. In vitro, the extent of dye uptake and haemolysis yielded results similar to those of the in vivo studies. Release of lactic dehydrogenase into the culture medium was similar in both the agate and emery exposed cells but significantly less when compared with quartz treated cells. PMID- 2994710 TI - Refolding a disulfide dimer of cytochrome c. AB - A covalent dimer of Saccharomyces cerevisiae iso-1 cytochrome c is stabilized by an interchain disulfide bond involving the cysteine residue penultimate to the C terminus. The individual chains in the dimer appear to retain the tertiary structural features characteristic for monomeric cytochrome c albeit with some perturbation. The dimer is reversibly denatured by heat, urea, or guanidine hydrochloride in a single cooperative transition whose midpoint is less than that of the monomeric protein. The kinetic profile observed for the refolding of the denatured dimer is characteristic for monomeric cytochromes except for a markedly enhanced slow-phase amplitude. PMID- 2994711 TI - Kinetic model for the action of the inorganic pyrophosphatase from Streptococcus faecalis. AB - Kinetic studies of the less active form of Streptococcus faecalis inorganic pyrophosphatase (EC 3.6.1.1), together with computational analysis, indicated that cooperativity in ligand binding contributes in a significant way to the behavior of this enzyme. The simplest model applicable to our data was a Monod Wyman-Changeux-type, allosteric model, in which the enzyme is proposed to exist in two states, referred to as R and T states, respectively. In the absence of ligands, 94% of the enzyme was in the T state. MgPPi2- was the only substrate for the enzyme in the R form. This substrate was bound equally well by both enzyme forms, but it was hydrolyzed 5 times more efficiently by the R form than it was by the T form. Mg2PPi was bound exclusively to the T state of the enzyme, and it was hydrolyzed 25% as rapidly as MgPPi2- by the T form. Mg2PPi inhibited the hydrolysis of the more efficient substrate, MgPPi2-, by competing with MgPPi2- for the enzyme in the T form and by shifting the R----T equilibrium in favor of the T form. Mg2+ stabilized the R state, thus activating the hydrolysis of MgPPi2 and inhibiting that of Mg2PPi. PMID- 2994712 TI - Spin-label studies on the origin of the specificity of lipid-protein interactions in Na+,K+-ATPase membranes from Squalus acanthias. AB - The pH dependence and salt dependence of the lipid-protein interactions of phosphatidic acid, phosphatidylserine, and stearic acid with Na+,K+-ATPase membranes from Squalus acanthias have been studied with spin-label electron spin resonance spectroscopy, using lipids with nitroxide labels on the 14-position C atom of the sn-2 chain. For phosphatidic acid and stearic acid, the fraction of motionally restricted spin-label increases with increasing pH, with pKa's of 6.6 and 8.0, respectively. In contrast, the pKa of stearic acid in the bulk lipid environment of the membrane is estimated from spin-label spectroscopy to be approximately equal to 6.6. The fraction of motionally restricted phosphatidylserine spin-label remains constant over the pH range 4.7-9.2. In the fully dissociated state the fractions of motionally restricted spin-labeled phosphatidic and stearic acids decrease with increasing salt concentration, reaching an approximately constant value at [NaCl] = 0.5-1.0 M. For stearic acid the net decrease is comparable to that obtained on protonation, but for phosphatidic acid the decrease is considerably smaller (by approximately 55%) than that obtained on protonating the lipid. The fraction of motionally restricted phosphatidylserine spin-label varies relatively little with salt concentration up to 1 M NaCl. Direct electrostatic effects alone cannot account for the whole of the observed specificity of interaction of the two phospholipids with Na+,K+-ATPase membranes. PMID- 2994713 TI - Axial ligands of chloroplast cytochrome b-559: identification and requirement for a heme-cross-linked polypeptide structure. AB - Optical, resonance Raman, and electron paramagnetic resonance spectroscopies have been used to characterize the ligands and spin state of the chloroplast cytochrome b-559. The protein was isolated from both maize and spinach in a low potential form. The spectroscopic data indicate that the heme iron in both ferric and ferrous cytochrome b-559 is in its low-spin state and ligated in its fifth and sixth coordination positions by histidine nitrogens. Electron paramagnetic resonance data for the purified spinach cytochrome are in good agreement with those determined by Bergstrom and Vanngard [Bergstrom, J., & Vanngard, T. (1982) Biochim. Biophys. Acta 682, 452-456] for a low-potential membrane-bound form of cytochrome b-559. The g values of high-potential cytochrome b-559 are shifted from those of its low-potential forms; this shift is interpreted as arising from a deviation of the planes of the two axial histidine imidazole rings from a parallel orientation. The model is consistent with the physical data and may also account for the facility with which cytochrome b-559 can be converted between low and high-potential forms. Recent biochemical and molecular biological data [Widger, W. R., Cramer, W. A., Hermodson, M., Meyer, D., & Gullifor, M. (1984) J. Biol. Chem. 259, 3870-3876; Herrmann, R. G., Alt, J., Schiller, D., Cramer, W. A., & Widger, W. R. (1984) FEBS Lett. 179, 239-244] have shown that two polypeptides, one with 83 residues and a second with 39 residues, most likely constitute the protein of the cytochrome.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2994714 TI - Interaction of fluorescent 3'-[1,5-(dimethylamino)naphthoyl]adenine nucleotides with the solubilized ADP/ATP carrier. AB - The binding of the 3'-[1,5-(dimethylamino)naphthoyl] (DAN) derivatives of AMP, ADP, and ATP to the solubilized ADP/ATP carrier is studied, evaluating primarily the fluorescence enhancement and 3H-labeled compound binding. DAN nucleotides also fluoresce when adsorbed to Triton X-100 micelles that are used for solubilization of the carrier. The partition of DAN-AMP between water and Triton X-100 micelles is measured, and it is shown to be shifted toward a higher content in Triton micelles with increasing salt concentration. In order to maintain a low level of fluorescence, the Triton content is decreased. The fraction of DAN nucleotide fluorescence due to carrier binding is determined by the suppression with bongkrekate (BKA). In contrast to the membrane-bound carrier, the solubilized preparation shows an increase of total BKA-sensitive fluorescence by 30-60% upon addition of ATP or ADP. In the solubilized atractylate-protein complex, the ADP-stimulated fluorescence amounts even to 80%. The suppression of fluorescence by BKA is independent of the presence of ADP or ATP, while that by carboxyatractylate (CAT) depends on ADP or ATP. The quantitation with [3H]BKA and [3H]CAT of these ligand interactions with DAN-AMP fluorescence shows that DAN-AMP fluorescence reflects the "m"-state carrier population and its redistribution under the influence of ADP or ATP. Thus, besides the "c"/"m" distribution, the kinetics of the c to m transition in the solubilized carrier also can be determined. The m share is increased to 80% when SO4, Pi, or pyrophosphate is present during solubilization. The rate of the ADP- or ATP-stimulated transition to the m state is markedly dependent on pH and on the presence of various anions, whereas the extent is little varied. The affinity decreases 4-fold going from DAN AMP to DAN-ADP and to DAN-ATP (KD = 0.9, 1.6, and 3.2 microM). Comparison with physical binding of [3H]DAN nucleotides shows that the fluorescence yield of bound DAN-AMP is about 1.4 times higher than that of bound DAN-ATP. DAN substitution causes more than a 100-fold affinity increase for AMP and a 50-fold increase for ADP or ATP, probably because of interaction of the DAN group with a hydrophobic niche. A less specific, low-affinity displacement of DAN nucleotides by GDP, ADP, GTP and ATP (Ki = 1-2 mM) probably reflects primarily the ionic interactions at the binding center. PMID- 2994715 TI - Interactions of gelsolin and gelsolin-actin complexes with actin. Effects of calcium on actin nucleation, filament severing, and end blocking. AB - Gelsolin is a calcium binding protein that shortens actin filaments. This effect occurs in the presence but not in the absence of micromolar calcium ion concentrations and is partially reversed following removal of calcium ions. Once two actin molecules have bound to gelsolin in solutions containing Ca2+, one of the actins remains bound following chelation of calcium, so that the reversal of gelsolin's effect cannot be accounted for simply by its dissociation from the ends of the shortened filaments to allow for elongation. In this paper, the interactions with actin of the ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) stable 1:1 gelsolin-actin complexes are compared with those of free gelsolin. The abilities of free or complexed gelsolin to sever actin filaments, nucleate filament assembly, bind to the fast growing (+) filament ends, and lower the filament size distribution in the presence of either Ca2+ or EGTA were examined. The results show that both free gelsolin and gelsolin-actin complexes are highly dependent on Ca2+ concentration when present in a molar ratio to actin less than 1:50. The gelsolin-actin complexes, however, differ from free gelsolin in that they have a higher affinity for (+) filament ends in EGTA and they cannot sever filaments in calcium. The limited reversal of actin-gelsolin binding following removal of calcium and the calcium sensitivity of nucleation by complexes suggest an alternative to reannealing of shortened filaments that involves redistribution of actin monomers and may account for the calcium-sensitive functional reversibility of the solation of actin by gelsolin. PMID- 2994717 TI - The oligosaccharide moiety of the beta 1-adrenergic receptor from turkey erythrocytes has a biantennary, N-acetyllactosamine-containing structure. AB - The turkey erythrocyte beta 1-adrenergic receptor can be purified by affinity chromatography on alprenolol-Sepharose and characterized by photoaffinity labeling with N-(p-azido-m-[125I]iodobenzyl)-carazolol. Through the use of the specific glycosidases neuraminidase and endo-beta-N-acetylglucosaminidase H and affinity chromatography on lectin-Sepharose gels, we show here that the receptor is an N-glycosyl protein that contains complex carbohydrate chains. No high mannose carbohydrate chains appear to be present. The binding of the radiolabeled antagonist dihydroalprenolol to the receptor is affected neither by the enzymic treatments nor by the presence of lectins, suggesting that the carbohydrate moiety is not involved in the catecholamine binding site. PMID- 2994716 TI - Nucleotide sequence of the gene for human factor IX (antihemophilic factor B). AB - Two different human genomic DNA libraries were screened for the gene for blood coagulation factor IX by employing a cDNA for the human protein as a hybridization probe. Five overlapping lambda phages were identified that contained the gene for factor IX. The complete DNA sequence of about 38 kilobases for the gene and the adjacent 5' and 3' flanking regions was established by the dideoxy chain termination and chemical degradation methods. The gene contained about 33.5 kilobases of DNA, including seven introns and eight exons within the coding and 3' noncoding regions of the gene. The eight exons code for a prepro leader sequence and 415 amino acids that make up the mature protein circulating in plasma. The intervening sequences range in size from 188 to 9473 nucleotides and contain four Alu repetitive sequences, including one in intron A and three in intron F. A fifth Alu repetitive sequence was found immediately flanking the 3' end of the gene. A 50 base pair insert in intron A was found in a clone from one of the genomic libraries but was absent in clones from the other library. Intron A as well as the 3' noncoding region of the gene also contained alternating purine-pyrimidine sequences that provide potential left-handed helical DNA or Z DNA structures for the gene. KpnI repetitive sequences were identified in intron D and the region flanking the 5' end of the gene. The 5' flanking region also contained a 1.9-kb HindIII subfamily repeat. The seven introns in the gene for factor IX were located in essentially the same position as the seven introns in the gene for human protein C, while the first three were found in positions identical with those in the gene for human prothrombin. PMID- 2994718 TI - Intrapeptide autophosphorylation of the epidermal growth factor receptor: regulation of kinase catalytic function by receptor dimerization. AB - The epidermal growth factor (EGF) receptor is a transmembrane polypeptide of 170 000 daltons (Da) with a cytoplasmically facing protein kinase domain. The regulation of the tyrosine kinase activity of the EGF receptor by added EGF and by receptor association state was studied in an in vitro system. The rate of autophosphorylation of the solubilized and purified EGF receptor was found to be independent of receptor concentration. To determine whether the zero-order kinetics observed point to intrapeptide phosphorylation, we measured the sedimentation characteristics of the undenatured solubilized receptor. The receptor was found to exist in two association-dissociation states-a monomeric 7.7S form and a dimeric 12S form. The 7.7S form is an active tyrosine kinase; it has high basal activity, and the activity is not further stimulated by EGF; it appears to be an EGF-independent form of the receptor kinase. The 12S form is devoid of catalytic activity, but in the presence of EGF it dissociates into the active monomeric form. Freshly purified receptor preparations contain mainly the monomeric receptor, have high basal kinase activity, and show low EGF stimulatability (less than 1.3-fold). Aging of the receptor results in progressive dimerization and decay of EGF-independent kinase activity (and increase in EGF stimulatability). All of these processes are reversed in the presence of EGF or dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2994719 TI - Studies on the succinate dehydrogenating system. Isolation and properties of the mitochondrial succinate-ubiquinone reductase. AB - A simple procedure for preparation of highly purified soluble succinate ubiquinone reductase from bovine heart mitochondrial particles is described. The enzyme exhibits four major bands on sodium dodecyl sulfate gel electrophoresis and contains (nmol per mg protein): covalently bound flavin, 6; non-heme iron, 53; acid-labile sulfur, 50; cytochrome b-560 heme, 1.2. The enzyme catalyzes thenoyltrifluoroacetone, or carboxin-sensitive (pure non-competitive with Q2) reduction of Q2 by succinate with a turnover number close to that in parent submitochondrial particles. The succinate reduced enzyme exhibits ferredoxin-type iron-sulfur center EPR-signal (g = 1.94 species) and a semiquinone signal (g = 2.00). An oxidized preparation shows a symmetric signal centered around g = 2.01. An unusual dissociation of the enzyme in the absence of a detergent is described. When added to the assay mixture from a concentrated protein-detergent solution, the enzyme does not reduce Q2 being highly reactive towards ferricyanide ('low Km ferricyanide reactive site'; Vinogradov, A.D., Gavrikova, E.V. and Goloveshkina, V.G. (1975) Biochem. Biophys. Res. Commun. 65, 1264-1269). The ubiquinone reductase, not the ferricyanide reductase was observed when the enzyme was added to the assay mixture from the diluted protein-detergent solutions. Thus the dissociation of succinate dehydrogenase from the complex occurs in the absence of a detergent dependent on the concentration of the protein-detergent complex in the stock preparation where the samples for the assay are taken from. An active antimycin-sensitive succinate-cytochrome c reductase was reconstituted by admixing of the soluble succinate-ubiquinone reductase and the cytochrome b-c1 complex, i.e., from the complexes which both contain the ubiquinone reactivity conferring protein (QPs). Cytochrome c reductase was also reconstituted from the succinate-ubiquinone reductase and succinate-cytochrome c reductase containing inactivated succinate dehydrogenase. The reconstitution experiments suggest that there exists a specific protein-protein (or lipid) interaction between QPs and a certain component(s) of the b-c1 complex. PMID- 2994720 TI - Studies of protein-phospholipid interaction in isolated mitochondrial ubiquinone cytochrome c reductase. AB - The interaction between phospholipids, ubiquinone and highly purified ubiquinol cytochrome c reductase was studied using differential scanning calorimetry. The enzyme complex and its delipidated forms undergo thermodenaturation at 337.3 and 322.7 K, respectively. The reduced reductase is more stable toward thermodenaturation than is the oxidized enzyme. While phospholipids restored enzymatic activity to the delipidated enzyme complex and stabilized the enzyme toward thermodenaturation, ubiquinone showed little effect on the thermostability of ubiquinol-cytochrome c reductase. The effect of phospholipids on the thermotropic properties of ubiquinol-cytochrome c reductase is dependent upon the molecular properties of the phospholipid. When ubiquinol-cytochrome c reductase was embedded in closed asolectin vesicles, an exothermic transition peak was observed upon thermodenaturation. When the asolectin concentration in the reconstituted preparation was less than 0.3 mg/mg protein, an amorphous structure was observed in the electron micrograph and the preparation showed an endothermic transition upon thermodenaturation. The thermotropic properties of the enzyme phospholipid vesicles were affected by the phospholipid head groups as well as the fatty-acyl chains, with those phospholipids having the most highly unsaturated fatty-acyl chains having the greatest effect. The energy for the exothermic transition may be derived from the collapse, upon thermodenaturation, of a strained interaction between the unsaturated fatty-acyl groups of phospholipids and protein molecules resulting from vesicle formation. The exothermic transition of the enzyme-phospholipid vesicle was abolished when cholesterol was included in the vesicles and when reductase was treated with a proteolytic enzyme prior to incorporation into the phospholipid vesicles. PMID- 2994722 TI - Studies on the effects of copper deficiency on rat liver mitochondria. I. Changes in mitochondrial composition. AB - As part of an investigation of the lesions of copper (Cu) deficiency a study was undertaken of the copper, iron, cytochrome and fatty acid composition of liver mitochondria from Cu deficient and Cu-adequate control rats. Cu concentrations were significantly decreased in whole liver, liver mitochondria and in blood plasma. Total iron was significantly increased in whole liver but remained at the normal level in mitochondria. Cytochrome c oxidase (EC 1.9.3.1) and its component cytochromes a and a3 were significantly reduced in liver mitochondria from Cu deficient rats, whereas there was no effect on the concentration of cytochromes b, c1 and c. Evidence from comparisons between cytochrome c oxidase activity and the amount of enzyme present, as assessed from the mitochondrial cytochrome a and a3 content, suggests that in addition to an absolute loss of enzyme, Cu deficiency adversely affects the efficiency of the residual enzyme. Severe Cu deficiency had no effect on 'ageing' or 'swelling' properties of liver mitochondria, indicating no marked effects on fatty acid composition. Fatty acid analyses demonstrated a slight but significant increase in docosapentenoic acid (22:5) of Cu-deficient mitochondria, but since this represents a minor component there was no change observed in the 'unsaturation index'. It was concluded that, in contrast to previous reports, Cu deficiency of the severity reported did not have a deleterious effect on the integrity and permeability of the inner mitochondrial membrane as exemplified by any qualitative modification of fatty acid constitution per se. PMID- 2994721 TI - Kinetics of the c-cytochromes in chromatophores from Rhodopseudomonas sphaeroides as a function of the concentration of cytochrome c2. Influence of this concentration on the oscillation of the secondary acceptor of the reaction centers QB. AB - The oxidation kinetics of Cyt c1 and c2 have been measured in normal chromatophores and in chromatophores fused with liposomes in order to increase the internal volume. The kinetics of Cyt c1 oxidation were found to be dependent on Cyt c2 concentration. The initial rate of Cyt c1 oxidation decreased after fusion by a factor of about two, indicating a process dependent on diffusion. The results do not allow a clear distinction between a diffusion of Cyt c2 along the inner membrane surface or through the inner volume of the vesicle; two- and three dimensional models are discussed. In contrast to Cyt c1, the kinetics of oxidation of Cyt c2 were not influenced by changes in concentration. It is concluded that reduced Cyt c2 is preferentially bound to the reaction centers. A binary pattern as a function of flash number from the dark-adapted state was measured in the turn-over of the two-electron gate of the reaction center. In chromatophores with more than 0.5 cytochrome c2 molecules per reaction center, this binary pattern titrated out with a midpoint around 340 mV on reduction of the suspension. In experiments with chromatophores with a low Cyt c2 content, or with spheroplast-derived vesicles which had lost Cyt c2, the binary oscillation in the two-electron gate could be observed at much lower potentials. The results suggest that the binding of reduced cytochrome c2 modifies the behavior of the two-electron gate. A model in which reaction center dimers are stabilized by Cyt c2 is proposed to explain the effect. PMID- 2994723 TI - Monoclonal antibodies to cytochrome c from Paracoccus denitrificans: effects on electron transport reactions. AB - The effect of a monoclonal antibody to a soluble cytochrome c from Paracoccus denitrificans was tested on the membrane-bound electron-transport system of this bacterium. This antibody (F3-10.2) and one previously described (F3-29.4) (Kuo, L.M., Davies, H.C. and Smith, L. (1984) Biochim. Biophys. Acta 766, 472-482) were deduced to bind to the cytochrome c in the area including amino acid residue number 23 on a loop on the side of the heme crevice. In contrast to the observations with the previously tested antibody, the present data show the second antibody to block completely the reaction of the cytochrome c with cytochrome c oxidase but not that with cytochrome c reductase. Neither antibody has an appreciable inhibitory effect on the NADH oxidase of the isolated detergent-treated membranes. The two antibodies bind in different ways, giving insight into the interaction of a soluble protein with membrane-bound enzymes. The data indicate that the reaction sites on the cytochrome c for the oxidase and reductase moieties of P. denitrificans are different. They also argue against the need for a dissociable cytochrome c comparable to that which functions on the mitochondrial inner membrane. PMID- 2994724 TI - Assignment of ESR signals of Escherichia coli terminal oxidase complexes. AB - The ESR signals of all the major components of the aerobic respiratory chain of Escherichia coli were measured and assigned at liquid helium temperature. Cytochrome b-556 gives a weak high-spin signal at g = 6.0. The terminal oxidase cytochrome b-562 . o complex gives signals at g = 6.0, 3.0 and 2.26, and the terminal oxidase cytochrome b-558 . d complex gives signals at g = 6.0, 2.5 and 2.3. A signal derived from cupric ions in the purified cytochrome b-562 . o complex was observed near g = 2.0. It was shown by the effects of KCN or NaN3 on cytochromes under the air-oxidized conditions that cytochrome o has a high-spin heme and cytochrome d has a low-spin heme. The E'm values for cytochromes b-558 and d, respectively, determined by potentiometric titration of the ESR signals were 140 and 240 mV in the membrane preparation, and 30 and 240 mV in the purified preparation. The oxidized cytochrome d gave intense low-spin signals at g = 2.5 and 2.3, while cytochrome d under the air-oxidized conditions gave corresponding signals of only very low intensity. These results suggested that most of the cytochrome d under the air-oxidized conditions contains a diamagnetic iron atom with a bound dioxygen. PMID- 2994725 TI - Identification of PlAl alloantigen domain on a 66 kDa protein derived from glycoprotein IIIa of human platelets. AB - Incubation of platelets with chymotryptin leads to the exposure of fibrinogen receptors and to the appearance of a 66 kDa membrane component on the surface of platelets. Both glycoprotein IIIa (GP IIIa) and a 66 kDa component were precipitated from detergent extracts of solubilized, surface radiolabeled chymotrypsin-treated platelets by human anti-PlAl antisera. Moreover, the presence of the P1A1 antigen was identified on GP IIIa (but not on GP IIb) and on a 66 kDa protein by means of immunoblot procedures using platelet Triton X-114 extracts and these purified proteins. Anti-PlAl antiserum did not recognize GP IIIa on the surface of intact (untreated) platelets nor the 66 kDa protein on the surface of chymotrypsin-treated platelets of PlAl-negative individuals. The present data demonstrate directly that the 66 kDa protein is derived from GP IIIa and contains the PlAl alloantigen. PMID- 2994727 TI - Evidence that the platelet plasma membrane does not contain a (Ca2+ + Mg2+) dependent ATPase. AB - The present study was designed to determine the subcellular distribution of the platelet (Ca2+ + Mg2+)-ATPase. Human platelets were surface labeled by the periodate-boro[3H]hydride method. Plasma membrane vesicles were then isolated to a purity of approx. 90% by a procedure utilizing wheat germ agglutinin affinity chromatography. These membranes were found to be 2.6-fold enriched in surface glycoproteins compared to an unfractionated vesicle fraction and almost 7-fold enriched compared to intact platelets. In contrast, the isolated plasma membranes showed a decreased specific activity of the (Ca2+ + Mg2+)-ATPase compared to the unfractionated vesicle fraction. This decrease in specific activity was found to be similar to that of an endoplasmic reticulum marker, glucose-6-phosphatase, and to that of a platelet inner membrane marker, phospholipase A2. We conclude, therefore, that the (Ca2+ + Mg2+)-ATPase is not located in the platelet plasma membrane but is restricted to membranes of intracellular origin. PMID- 2994726 TI - The mechanism of calcium uptake by liver microsomes: effect of anions and ionophores. AB - The mechanism of calcium uptake by liver microsomes was investigated using various anions and ionophores. Calcium uptake was shown to be specific to microsomes and unlikely to be due to contamination by plasma membranes by correlation of calcium uptake to the marker enzymes specific for these two fractions. Under the conditions employed, phosphates, sulfate, chloride, acetate, nitrate, thiocyanate, maleate, succinate and oxalate all stimulated calcium uptake by microsomes, but to different degrees. The greatest effect (4-6-fold) was observed with phosphate. On the contrary, phosphate is the only anion that stimulates the plasma membrane calcium uptake to any significant degree. Treatment of isolated microsomes with 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) resulted in inhibition of ATP- and anion-dependent calcium uptake. A lipid-permeable organic acid such as maleate retained its ability to promote calcium uptake in DIDS-treated microsomes. However, a lipophilic anion, such as nitrate, stimulated calcium uptake only in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). In addition, 2 microM valinomycin, when added in the absence or presence of 10 to 100 mM K+, had no stimulatory effect on calcium uptake. These results appear to be consistent with a model in which the active uptake of calcium into microsomes involves electroneutral Ca2+-nH+ exchange. PMID- 2994728 TI - Evidence for the asymmetrical binding of p-chloromercuriphenyl sulphonate to the human erythrocyte nucleoside transporter. AB - Nucleosides cross the human erythrocyte membrane by a facilitated-diffusion process which is selectively inhibited by nanomolar concentrations of nitrobenzylthioinosine (NBMPR). The chemical asymmetry of the transporter was investigated by studying the effects of p-chloromercuriphenyl sulphonate (PCMBS) on uridine transport and high-affinity NBMPR binding in inside-out and right-side out membrane vesicles, unsealed erythrocyte ghosts and intact cells. PCMBS was an effective inhibitor of the transporter (50% inhibition at 30 microM), but only when the organomercurial had access to the cytoplasmic membrane surface. PCMBS inhibition of NBMPR binding to ghosts was reversed by incubation with dithiothreitol. Both uridine and NBMPR were able to protect the transporter against PCMBS inhibition. PMID- 2994729 TI - Investigations on the insertion of the mitochondrial precursor protein apocytochrome c into model membranes. AB - Different aspects of the interaction of apocytochrome c and model membranes composed of negatively charged lipids, were studied in order to get insight into the nature of this interaction. The effect of the protein on the lipid packing properties are revealed by DSC, ESR and monolayer techniques. These experiments clearly demonstrate that upon electrostatic interaction with the negatively charged phospholipids, apocytochrome c is able to penetrate into the hydrophobic region of the model membrane. In the case of 1,2-dimyristoyl-sn-glycero-3 phosphoglycerol, this results in a perturbation of 160 lipid molecules per apocytochrome c molecule. Most likely, apocytochrome c disrupts the formation of the gel phase and restricts the lipid chain motion above the gel to liquid crystalline phase transition. Tryptophan fluorescence measurements confirm that at least a part of the protein penetrates into the bilayer, and suggest that after this penetration, the tryptophan (residue no. 59) is located in the glycerol backbone region of the phospholipids. Although the secondary structure of apocytochrome c is predicted to contain about 35% of alpha-helical structure, the CD pattern of an aqueous solution of the protein is featureless. However, negatively charged lipids are able to express this alpha-helical potency in the apocytochrome c, which might be important for the insertion of the protein into lipid membranes. PMID- 2994730 TI - The failure of hydrodynamic analysis to define pore size in cell membranes. AB - The equivalent pore theory predicts that the size of water transporting pores can be calculated from the ratio of osmotic (Pf, cm . s-1) to diffusive (Pd, cm . s 1) water permeability. Determinations of Pf and Pd in human red cells within the last thirty years have increased the ratio of Pf to Pd. According to the equivalent pore theory the pore diameter has increased from 9 A to 25 A. A pore diameter of 25 A is not compatible with the permeability characteristics of the red cell membrane. We conclude that the equivalent pore theory fails to determine pore size in red blood cells. We suggest that water transporting pores in human red cells transport water molecules in a single file fashion. PMID- 2994732 TI - In vitro modification of cholesterol content of rat liver microsomes. Effects upon membrane 'fluidity' and activities of glucose-6-phosphatase and fatty acid desaturation systems. AB - The cholesterol content of rat liver microsomal membranes was modified in vitro by incubating microsomes and cytosol with liposomes prepared by sonication of microsomal lipids and cholesterol. In this way, the cholesterol to phospholipid molar ratio was increased from 0.11-0.13 in untreated microsomes to a maximal of 0.8 in treated ones. Cholesterol incorporation in microsomes produced an increase in the diphenyl-hexatriene steady-state fluorescence anisotropy and a decrease in the efficiency of pyrene-excimer formation which indicated a decrease in the rotational and translational mobility, respectively, of these probes in the membranes lipid phase. Cholesterol incorporation in microsomes did not affect significantly the glucose-6-phosphatase activity in 0.1% Triton X-100 totally disrupted microsomes, but diminished the glucose-6-phosphatase activity of 'intact' microsomes. This indicates that possibly the glucose 6-phosphate translocation across the microsomal membrane is impeded by an increase in the membrane apparent 'microviscosity'. Cholesterol incorporation in microsomes decreased NADH-cytochrome c reductase without affecting NADH-ferricyanide reductase activity. The delta 9 desaturation reaction rate was enhanced by cholesterol incorporation at low but not at high palmitic acid substrate concentration. delta 5 and delta 6 desaturase reaction-rates were increased both at low and high fatty acid substrate concentrations. These results suggest that a mechanism involving fatty acid desaturase enzymes, might exist to self-regulate the microsomal membrane lipid phase 'fluidity' in the rat liver. PMID- 2994731 TI - A rapid isolation procedure of plasma membranes from human neutrophils using self generating Percoll gradients. Importance of pH in avoiding contamination by intracellular membranes. AB - In this study we report an overall procedure for the isolation of both human polymorphonuclear neutrophils and their plasma membrane, by means of self generating Percoll gradients. After efficient purification (40% yield), neutrophils were lysed by nitrogen cavitation and cellular structures quickly isolated in a one-step procedure. Plasma membrane recovery was monitored by [3H]concanavalin A and 5'-nucleotidase (EC 3.1.3.5) activity. We showed the latter activity is indeed present in human neutrophils. The procedure resulted in a good yield of plasma membrane, since 45% and 55% of total 5'-nucleotidase and [3H]concanavalin A activity, respectively, were recovered within two gradient fractions. Depending on the final pH of the Percoll gradient medium, endoplasmic reticulum markers contaminated either the plasma membrane or the granule fractions. At pH 9.05, NADH-ferricyanide reductase activity clearly separated from plasma membrane markers and displayed the same profile as CDPcholine:diacylglycerolcholine phosphotransferase (EC 2.7.8.2), a typical enzyme of endoplasmic reticulum. These results emphasize the need for strict monitoring of the pH of the gradient medium in subcellular fractionation of neutrophils. PMID- 2994733 TI - The effect of general anesthetics on the proton and potassium permeabilities of liposomes. AB - The pump-leak hypothesis of general anesthesia proposes that anesthetics act by increasing the functional proton permeability of membranes, particularly those of synaptic vesicles. Since transmembrane proton gradients are required for neurotransmitter accumulation, decay of such gradients by an uncompensated anesthetic-induced leak would result in loss of neurotransmitter from the vesicles, followed by synaptic block and anesthesia. We have tested this hypothesis by determining the effect of four different general anesthetics on the relative permeabilities of liposome membranes to protons and potassium ions. In all cases, physiologically relevant levels of anesthetics caused a 200 to 500 percent increment in ionic permeability. There was no marked preference for protons, suggesting that the anesthetics did not induce a leak specific for this ionic species. Instead the anesthetics appeared to produce a more general defect available to both protons and potassium ions which resulted in a functional increment in proton permeability. These observations were compared with available data on proton transport rates by synaptic vesicle ATPase enzymes. The magnitude of the anesthetic-induced leak could not be compensated by the ATPase, which is only capable of a 40 percent increase in rate when uncoupled. We consider these results to be consistent with the pump-leak hypothesis. PMID- 2994734 TI - Electron spin resonance studies of lipid fluidity changes in membranes of an uncoupler-resistant mutant of Escherichia coli. AB - The fluidity of the lipids in membrane preparations from a mutant of Escherichia coli resistant to the uncoupler CCCP, grown at different temperatures with and without CCCP, was examined by electron spin resonance using the spin probe 5 doxyl stearic acid. The fluidity of the membrane lipids at the growth temperature, as estimated using electron spin resonance, was less in cells grown at lower temperatures. Precise homeoviscous adaptation was not observed. Growth in the presence of CCCP resulted in a decrease in membrane lipid fluidity, particularly in the inner (cytoplasmic) membrane. There was no change in the proportion of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin in the cell envelope. However, there was an increase in the proportion of unsaturated fatty acids in membranes from cells grown with uncoupler. This was reflected in the increased fluidity of the lipids extracted from these membranes. This result is contrary to that expected from measurements of the fluidity of the lipid in these membranes. The decreased fluidity of the lipid in these membranes may be a consequence of the observed increase in the ratio of protein to phospholipid. PMID- 2994735 TI - Control of free cytoplasmic calcium by intracellular pH in rat lymphocytes. AB - Activation of Na+-H+ exchange in rat thymocytes was found to be followed by an increase in free cytoplasmic Ca2+ concentration [( Ca2+]i). We determined whether the change in [Ca2+]i was secondary to the uptake of Na+, or to the cytoplasmic alkalinization that result from activation of the antiport. Increasing intracellular [Na+] by treating the cells with ouabain or gramicidin failed to affect [Ca2+]i. In contrast, procedures that increased the cytoplasmic pH, such as addition of monensin or NH3, significantly elevated [Ca2+]i. These results suggest an important role of cytoplasmic pH in the control of [Ca2+]i in lymphocytes. PMID- 2994736 TI - DNA sequences homologous to long double-stranded RNA. Transcription of intracisternal A-particle genes and major long repeat of the mouse genome. AB - Long double-stranded RNA (dsRNA-A) (Kramerov, D.A., Ryskov, A.P. and Georgiev, G.P. (1977) Biochim. Biophys. Acta 475, 461-475) from Ehrlich ascites carcinoma cells was used for the search for mobile dispersed genes in the mouse genome. Two kinds of genomic sequences hybridizing to dsRNA-A were cloned. They were designated A1 and A2. The A1 sequence was identified as the gene for intracisternal A particles, while the A2 sequence was found to be the major long repetitive sequence of the mouse genome. Melted dsRNA-A hybridized equally well to both DNA strands of A1 and A2 sequences while total poly(A)+ RNA bound preferentially to one of them. Thus, a partially symmetrical transcription took place in the case of A1 and A2 elements. The analysis of transcripts of A1 elements in Ehrlich carcinoma cells revealed RNAs with sizes of 9.5 kb, 6.8 kb and 6.0 kb. In plasmocytoma MOPC 21 cells, instead of the 6.0 kb RNA, two other kinds of RNA with sizes 5.3 kb and 7.8 kb were found. These transcripts poorly coincided with the four known variants of intracisternal A-particle (IAP) genes. It seemed that at least some of the described RNAs were transcribed from some minor non-identified variants of IAP genes. The A2 transcripts were practically restricted to nuclei, their sizes being heterogeneous. PMID- 2994737 TI - Isolation and construction of mutants of the G4 minus strand origin: analysis of their in vivo activity. AB - An active, rifampicin-resistant primase-dependent bacteriophage G4 origin of complementary DNA strand synthesis has been cloned as a 274 bp fragment into the filamentous phase M13 and its secondary structure altered by deletion and insertion. It has been found that the entire 136 bp G4 intergenic region containing the secondary structure loops I and III is necessary for rifampicin resistant conversion of SS----RF DNA in vivo. The secondary structures, however, can be widely separated by insertion between them of both random DNA sequences, and sequences that form strong additional secondary structure configurations and the origins still retain activity. Primase therefore probably recognises two DNA domains on loops I and III, the physical separation of which is not important. PMID- 2994738 TI - Analysis of the structure and spatial distribution of ultraviolet-induced DNA repair patches in human cells made in the presence of inhibitors of replicative synthesis. AB - The repair of ultraviolet-induced damage in the presence of hydroxyurea or hydroxyurea and arabinosylcytosine was investigated in confluent human fibroblasts at the level of DNA loops attached to the nuclear matrix. Estimation of single-strand break frequencies based on the release of DNA from the DNA nuclear matrix complex after incubation with nuclease S1 revealed the occurrence of multiple incision events per DNA loop in the presence of inhibitors. When both inhibitors were employed, over 90% of the repair-labelled DNA was not ligated within 2 h post-incubation. In the absence of ligation of repair patches, we observed a preferential release of repair-labelled DNA from the nuclear matrix by nuclease S1 compared to prelabelled DNA, regardless of the period of post-UV incubation. The results suggest that repair events are clustered to some extent in a certain area of a DNA loop. However, the position of these clusters relative to the attachment sites of DNA loops at the nuclear matrix is random. The data are discussed in terms of denaturation of a putative repair complex in the presence of hydroxyurea resulting in an excess of incisions over repaired sites. PMID- 2994740 TI - The N-terminal amino acid sequence from alpha 1-antitrypsin isolated from liver inclusion bodies. AB - The alpha 1-antitrypsin from the liver of a subject with alpha i-antitrypsin deficiency was purified and subjected to automated Edman degradation. The N terminal amino acid sequence from position 1 to 12 was identical to that in plasma alpha 1-antitrypsin, type Z. This result precludes that the intrahepatic accumulation of Z alpha 1-antitrypsin is due to a defective removal of a signal peptide. PMID- 2994739 TI - Binding of cyanide to cytochrome c' from Chromatium vinosum. AB - Spectroscopic evidence is presented which demonstrates the binding of cyanide to the ferric cytochrome c' from Chromatium vinosum. The cytochrome was shown to bind one equivalent of cyanide with an equilibrium constant of 2.1 X 10(4) at pH 7.0 and 25 degrees C. This finding represents the first observation of the binding of an anionic ligand to the heme iron in a ferric cytochrome c'. These results suggest that the binding site of the ferric Chromatium cytochrome c' may be significantly more accessible than previously indicated. PMID- 2994741 TI - Purification and characterization of a bone metalloproteinase that degrades gelatin and types IV and V collagen. AB - A third metalloendopeptidase activity, gelatinase, has been completely separated from the collagenase and proteoglycanase activities of rabbit bone culture medium. Although the proteinase could not be purified to homogeneity in large amounts, it was possible to obtain accurate molecular weight values and activity after electrophoresis on non-reduced SDS/polyacrylamide gels. The latent form had an Mr of 65 000 which could be activated with 4-aminophenylmercuric acetate, APMA, to a form of Mr 61 000; under reducing conditions the latent and active forms had Mr of 72 000 and 65 000, respectively. Trypsin was a very poor activator of the latent enzyme. Gelatinase degraded gelatins derived from the interstitial collagens and it also had low activity on native types IV and V collagen and on insoluble elastin. Gelatinase acted synergistically with collagenase in degrading insoluble interstitial collagen. The specific mammalian tissue inhibitor of metalloproteinases inhibited gelatinase by forming a stable inactive complex. Comparison of the properties of gelatinase with those of collagenase and proteoglycanase suggest that the three proteinases form a family which together are capable of degrading all the major macromolecules of connective tissue matrices. PMID- 2994742 TI - Inhibition of smooth muscle 5'-nucleotidase by imidazole and its reversal by magnesium. AB - Imidazole, commonly used as an effective pH-buffering reagent in aqueous media maintained at pH 7-8, was found to depress the 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity of microsomal membrane fraction isolated from rat vas deferens smooth muscle in a dose-dependent manner in the absence of added Mg2+. Such an inhibitory effect of imidazole on the smooth muscle 5' nucleotidase was not dependent upon the purity or integrity of the membrane fractions used and could be fully reversed by the inclusion of 5-10 mM Mg2+ in the assay medium. Of the five different pH-buffering reagents tested, imidazole was specific in exerting inhibitory effect on the 5'-nucleotidase in the absence of Mg2+ and this inhibition could not be accounted for by the impurities present in the imidazole. Differential effects of chelating reagents and other divalent metal ions on the 5'-nucleotidase activity were also observed in imidazole and Tris buffer solutions. The 5'-nucleotidase activity was not affected if the membranes were preincubated and washed with a large volume of 50 mM imidazole and subsequently assayed in 50 mM Tris in the absence of Mg2+. Similar findings were obtained with EDTA treated membrane. These results suggest that imidazole does not act by removal of the activating metal ion but rather interacts directly with 5'-nucleotidase and alters the metal-enzyme interactions. PMID- 2994743 TI - Isolation and partial characterization of rat muscle fructose bisphosphatase. AB - Fructose bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) has been isolated in homogeneous form from rat muscle by a simple and convenient procedure, including adsorption on carboxymethylcellulose and substrate elution. The resultant enzyme preparation has a specific activity comparable to that of the enzymes isolated from rabbit liver, rabbit muscle and rat liver. The native relative molecular mass of the enzyme was estimated by sedimentation equilibrium centrifugation to be approx. 138 000, and the enzyme appears to be a tetramer containing subunits of Mr approx. 34 500. The amino acid composition is distinctly different from that of the rabbit muscle, rabbit liver and rat liver enzymes. The purified enzyme contains no tryptophan and has a blocked amino terminal. PMID- 2994744 TI - Effect of culture in vitro with eicosatetraenoic (20:4(n-6) ) and eicosapentaenoic (20:5(n-3) ) acids on fatty acid composition, prostaglandin synthesis and chemiluminescence of rat peritoneal macrophages. AB - Rat peritoneal macrophages were cultured in either eicosatetraenoic acid (20:4(n 6) ) or eicosapentaenoic acid (20:5(n-3) ) and the effects on phospholipid fatty acids, prostaglandin synthesizing capacity and the ability of the macrophages to show chemiluminescence were examined. Chemiluminescence is an activity resulting from the synthesis of reactive oxygen species. It has been reported that prostaglandins inhibit this activity. The fatty acid profile of the four major phospholipids reflected the fatty acid component of the medium. Macrophages cultured in 20:4(n-6) synthesized twice the prostaglandin produced by controls and those cultured in 20:5(n-3) synthesized 10% that of controls and 5% that of 20:4(n-6)-cultured cells. Macrophages cultured with 20:4(n-6) for 12 h showed half the chemiluminescence of those cultured with 20:5(n-3), while those cultured with 20:4(n-6) for 24 h showed 10% the chemiluminescence of 20:5(n-3)-cultured cells. Addition of the prostaglandin synthase inhibitor, indomethacin, had no effect on chemiluminescence. PMID- 2994745 TI - Opsonized bacteria stimulate leukotriene synthesis in human leukocytes. AB - Incubation of human leukocytes with opsonized bacteria led to leukotriene formation. The main products identified were leukotriene B4, 20-OH leukotriene B4 and 20-COOH leukotriene B4. A lesser amount of leukotriene C4 was formed. In contrast, only minor amounts of leukotrienes were formed by leukocytes challenged with uncoated bacteria. However, both opsonized and unopsonized bacteria stimulated the synthesis of 5S,12S-DHETE and 5S,12S,20-THETE. Opsonized bacteria caused a transient elevation of leukotriene B4 levels, with a maximum after 5 min. After 20 min of incubation the levels of 20-OH leukotriene B4, and 20-COOH leukotriene B4 were 7- and 20-times higher than those of leukotriene B4, showing that the leukocytes effectively degrade leukotriene B4 via omega-oxidation. In the light of the profound biological effects of leukotrienes, the present report indicates that leukotriene formation induced by opsonized bacteria might be important in the host defense against microorganisms. PMID- 2994746 TI - Modification of the carbohydrate in ricin with metaperiodate and cyanoborohydride mixtures: effect on binding, uptake and toxicity to parenchymal and non parenchymal cells of rat liver. AB - The carbohydrate in the toxic glycoprotein ricin was chemically modified by simultaneous treatment with sodium metaperiodate and sodium cyanoborohydride. This treatment causes oxidative cleavage of the sugar residues and reduction of the aldehyde groups which are formed to primary alcohols. The modification markedly decreased the rapid removal of ricin from the blood by hepatic non parenchymal cells with only a relatively small increase in accumulation of the toxin by parenchymal cells. Binding, uptake and toxicity of the modified ricin in primary monolayer cultures of hepatic non-parenchymal cells were all decreased to a much greater extent than in parenchymal cells. The results indicate that native ricin binds to non-parenchymal cells by a dual recognition process which involves both interaction of cell receptors with the mannose-containing oligosaccharides of the toxin and binding of ricin to galactose-containing glycoproteins and glycolipids on the cells. However, uptake and toxicity of native ricin in non parenchymal cells appears to result principally from entry of the toxin through the mannose recognition pathway. By contrast, uptake and toxicity of the expressed essentially through the galactose-recognition route. PMID- 2994747 TI - Purification of 2,3-bisphosphoglycerate synthase-phosphatase from pig skeletal muscle. AB - Two enzymes which possess 2,3-bisphosphoglycerate synthase, 2,3 bisphosphoglycerate phosphatase and phosphoglycerate mutase activities have been purified from pig skeletal muscle. One of the enzymes corresponds to type M phosphoglycerate mutase. The other enzyme shows properties similar to those of the 2,3-bisphosphoglycerate synthase-phosphatase present in mammalian erythrocytes. The erythrocyte and the muscle enzyme possess the same molecular (56 000) and subunit (27 000) weights. The synthase, phosphatase and mutase activity ratio is similar in both enzymes, and they are affected by the same inhibitor (glycerate 3-P) and activators (glycolate 2-P, pyrophosphate, sulfite and bisulfite). PMID- 2994748 TI - The role of thrombin in the regulation of the endothelial prostaglandin production. AB - Prostaglandin synthesis in endothelial cells may be initiated by the addition of exogenous substrate (arachidonic acid) or by addition of thrombin or the CA2+ ionophore A23187, which leads to prostacyclin formation from endogenous substrates. We noticed that endothelial cells produce more than twice the amount of prostacyclin when incubated with thrombin and arachidonic acid together than with arachidonic acid alone. In addition, it was found that the thrombin-induced conversion of endogenous substrates was inhibited by exogenous arachidonic acid. This means that the conversion of exogenous added arachidonic acid to prostacyclin was stimulated by thrombin. This activation of the enzymes involved in prostacyclin synthesis lasted about 5 min and could be inhibited by phospholipase inhibitors such as mepacrine and p-bromophenyl-acylbromide but not by the cAMP analogue dibutyryl cAMP, an inhibitor of arachidonic acid release from cellular phospholipids. These data demonstrate that, in addition to causing release of endogenous substrate, thrombin and the Ca2+-ionophore also activate the enzyme system involved in the further transformation of arachidonic acid. PMID- 2994749 TI - Cultured human epidermis cells produce cell-associated interleukin 1-like prostaglandin E2- and collagenase-stimulating factors. AB - In order to identify factors which may regulate the functions of dermal fibroblasts, cell lysates and conditioned media of cultured human epidermal cells were tested on dermal fibroblasts for the stimulation of prostaglandin E2- and collagenase-production. Both prostaglandin E2- and collagenase-stimulating activities appeared during epidermal cell culture: after 2 d they were detected in the cell lysate, and after 4 d of culture they were found also in the conditioned media. Molecular sieving chromatography of epidermal cell lysates led to the detection of two main peaks showing concomitant prostaglandin E2- and collagenase-stimulating activities at Mr approximately equal to 18 000 and Mr approximately equal to 10 000. A single peak of concomitant prostaglandin E2- and collagenase-stimulating activities were seen at Mr approximately equal to 10 000 in the epidermal cell conditioned media. This suggests that the cell-associated concomitant prostaglandin E2- and collagenase-stimulating activities are processed from a common precursor molecule and released. Collagenase-stimulating activity without accompanying prostaglandin E2 was also detected in the range of Mr approximately equal to 30 000-45 000. PMID- 2994750 TI - Subcellular localization and enzymatic properties of rat liver phosphatidylinositol-4-phosphate kinase. AB - The phosphatidylinositol-4-phosphate kinase activity in rat liver showed a subcellular distribution different from that of phosphatidylinositol kinase. It was preferentially associated with plasma membrane-rich subcellular fractions, while no or minimal activity could be ascribed to mitochondria, lysosomes, Golgi membranes or the endoplasmic reticulum. The plasma membrane enzyme phosphorylated endogenous and exogenously added phosphatidylinositol 4-phosphate at comparable initial rates. The phosphorylation of endogenous substrate was strongly inhibited by Triton X-100, while the phosphorylation of added substrate was enhanced, suggesting that endogenous phosphatidylinositol 4-phosphate was readily available to the enzyme in unperturbed plasma membranes. The total activity of phosphatidylinositol-4-phosphate kinase in rat liver was only 1/20 that of phosphatidylinositol kinase. The enzyme activity showed an unusually broad pH optimum in the neutral range. Mg2+ was the preferred divalent cation and Km towards ATP was about 3-fold higher than the corresponding value for phosphatidylinositol kinase. PMID- 2994751 TI - Cyclic AMP inhibits secretion from bovine adrenal chromaffin cells evoked by carbamylcholine but not by high K+. AB - The role of cAMP in the control of secretion from bovine adrenal chromaffin cells was examined using the adenylate cyclase activator, forskolin. Treatment of chromaffin cells with forskolin resulted in a rise in cAMP levels. Forskolin inhibited catecholamine release elicited by carbamylcholine or nicotine but had no effect on secretion evoked by 55 mM K+. Inhibition of carbamylcholine stimulated release by forskolin was half-maximal at 10 microM forskolin. The inhibition by forskolin of secretion evoked by carbamylcholine was at a step distal to the rise in intracellular free calcium concentration ([Ca2+]i), since this rise was not inhibited by forskolin, which itself produced a small rise in [Ca2+]i. The results suggest that secretion evoked by carbamylcholine is due to the activation of an additional second messenger pathway acting with the rise in [Ca2+]i. This additional pathway may be the target for cAMP action. PMID- 2994752 TI - Cultured endothelial cells do not respond to a platelet-derived growth-factor like protein in an autocrine manner. AB - Cultured endothelial cells produce a growth factor similar or identical to platelet-derived growth factor (PDGF). Endothelial cells are able to proliferate in plasma-supplemented medium, while most nontransformed cells require serum supplemented medium. Since PDGF is a major serum mitogen, we have tested the possibility that endothelial cells interact with and respond to the autologously produced PDGF-like (PDGF-c) protein. We have found that bovine aortic and rat heart endothelial cells express little or no cell surface PDGF receptors as determined by binding of pure 125I-PDGF. Treating these cells under acidic conditions, which release receptor-bound PDGF in control cells without affecting receptor function, did not reveal a population of cryptic receptors. In addition, when rat heart endothelial cells were grown in the presence of an antibody to PDGF, proliferation was unimpaired, though no detectable free PDGF was present in the medium. An equivalent amount of antibody completely blocked the mitogenic response of human fibroblasts that had been preincubated for 1 h at 37 degrees C with an equivalent dose of PDGF. Thus, endothelial cells do not respond mitogenically in a manner that would be expected from the interaction of autologously produced PDGF with its cell surface receptor. Endothelial cells were detergent-solubilized and immobilized on nitrocellulose in an attempt to detect the presence of intracellular PDGF receptors. Specific binding of 125I-PDGF to adsorbed, solubilized bovine aortic or rat heart endothelial cells was undetectable, though significant binding to adsorbed, solubilized fibroblasts, used as a positive control, was observed. We conclude that endothelial cells do not have detectable intracellular PDGF receptors. PMID- 2994753 TI - [Mapping of the different functional domains of (Na+, K+) ATPase]. PMID- 2994754 TI - Distribution of ultraviolet-induced lesions in simian virus 40 DNA. AB - In order to analyze the molecular mechanisms of mutagenesis in mammalian cells, we devised an analytical assay using Simian Virus 40 as biological probe. To study the possible correlations between the distribution of the lesions on the treated DNA and the distribution of mutations, we have located and quantified the lesions induced by ultraviolet light (254 nm) on a SV40 DNA fragment. At a fluence of 2,000 J/m2, our results show that the formation frequency of thymine thymine dimers (TT) is three to four times higher than the formation frequency of the other types of dimers (TC, CT, CC). On the other hand, the formation frequency of a dimer is influenced by the adjacent sequence. In particular, a pyrimidine in the 5' position of a thymine-thymine dimer enhances its formation frequency. At the dose used the formation frequency of the pyrimidine (6-4) pyrimidone photoproducts is twenty times less than the formation frequency of pyrimidine dimers. This paper shows the distribution of the major lesions induced by UV-light on a defined fragment of SV40 genome after UV irradiation. This work is necessary to get an insight into the molecular mechanisms of UV-mutagenesis. PMID- 2994755 TI - The SOS system. AB - In the bacterium Escherichia coli DNA damaging treatments such as ultraviolet or ionizing radiation induce a set of functions called collectively the SOS response, reviewed here. The regulation of the SOS response involves a repressor, the LexA protein, and an inducer, the RecA protein. After DNA damage an effector molecule is produced--possibly single stranded DNA--which activates the RecA protein to a form capable of catalysing proteolytic cleavage of LexA. The repressors of certain temperate prophages are cleaved under the same conditions, resulting in lysogenic induction. SOS functions are involved in DNA repair and mutagenesis, in cell division inhibition, in recovery of normal physiological conditions after the DNA damage is repaired, and possibly in cell death when DNA damage is too extensive. The SOS response also includes several chromosomal genes of unknown function, a number of plasmid encoded genes (bacteriocins, mutagenesis), and lysogenic induction of certain prophages. DNA damaging treatments seem to induce DNA repair and mutagenic activities and proviral development in many species, including mammalian cells. In general, substances which are genotoxic to higher eukaryotes induce the SOS response in bacteria. This correlation is the basis of the numerous bacterial tests for genotoxicity and carcinogenicity. PMID- 2994756 TI - Construction of frameshift mutation hot spots within the tetracycline resistance gene of pBR322. AB - The chemical carcinogen, N-2-acetylaminofluorene (AAF) when bound covalently to DNA induces a majority (greater than 90%) of frameshift mutations. The mutations occur with high frequencies at defined sequences (i.e. mutation hot spots). Two classes of mutation hot spots were found: at repetitive sequences and at specific non-repetitive sequences. Mutations at the repetitive sequences depend upon a functional umuC gene whereas mutations at specific non-repetitive sequences are umuC-independent. The first discovered sequence of this class is the NarI restriction enzyme recognition sequence (5'GGCGCC3'). In an attempt to define a family of such sequences we constructed a related sequence 5'GCGCGC3' within the tetracycline resistance gene of pBR322. This sequence was also found to be an- AAF induced--2 frameshift mutation hot spot in both wild type and umuC strains. PMID- 2994757 TI - Survival and mutagenesis of ultraviolet irradiated simian virus 40 in foetal human fibroblasts. AB - Survival and mutagenesis of UV-irradiated, temperature-sensitive simian virus 40 mutants (SV40) have been studied after infection of human fibroblasts. Survival of the viral progeny obtained after 6,8 or 10 days at permissive temperature decrease as a function of the UV-dose delivered to the virus. In cels which have been pretreated with 10 Jm-2 of UV 24 hours before infection, progeny survival was increased as compared to survival in control cells. The reactivation factor varies from one to ten, depending on the number of lytic cycles carried out at permissive temperature. The level of mutation frequency, as measured by the reversion from a temperature sensitive growth phenotype towards a wild type phenotype, increases with the dose of UV-irradiation given to the virus. Moreover, the mutation frequency is increased in the viral progeny produced in UV irradiated human cells. Similar experiments carried out with SV40-transformed human fibroblasts, which constitutively express SV40 T antigen, gave comparable results. These experiments show that, as in monkey cells, a new error-prone recovery pathway can be induced by pretreating human cells with UV-light before infection. PMID- 2994758 TI - Expression of a bacterial repair gene in mammalian cells. AB - The coding sequence of the uvrA gene from Escherichia coli has been fused to the early promoter, enhancer and origin of replication of the simian virus SV40, and was supplemented with splicing and polyadenylation sites arising from the same virus. Introduction of this hybrid gene into simian cos-1 cells results in the synthesis of a full length UvrA protein (114 kD) which has retained its ability to bind to single-stranded DNA. PMID- 2994759 TI - [Phosphoprotein phosphatases from cell nuclei of the bovine spleen: physico chemical properties]. AB - The physico-chemical properties of phosphoprotein phosphatase (EC 1.3.1.16) from bovine spleen cell nuclei were investigated. The enzyme was shown to possess a wide substrate specificity and to catalyze dephosphorylation of phosphocasein, ATP, ADP and p-nitrophenylphosphate (pNPP). The Km values for ATP, ADP and pNPP are 0.44, 0.43 and 1.25 mM, respectively. The molecular weight of the enzyme as determined by gel filtration on Sephadex G-75 and electrophoresis in polyacrylamide gel of different concentrations is approximately 33 000. SDS polyacrylamide gel electrophoresis revealed two protein bands with Mr 12 000 and 18 000. The enzyme molecule predominantly contains acidic amino acid residues, two free SH-groups and two disulphide bonds. Phosphoprotein phosphatase is a glycoprotein with the carbohydrate content of about 22%, and has an additional absorption maximum at 560 nm. The enzyme is competitively inhibited by ammonium molybdate (Ki = 0.37 microM) and non-competitively by sodium fluoride (Ki = 1.3 mM). Incubation of phosphoprotein phosphatase with 2 mM phenylmethylsulfonylfluoride (PMSF) for 25 hours resulted in a approximately 46% loss of the enzyme activity. Ammonium molybdate, sodium fluoride and PMSF reversibly inhibit the enzyme. Modification of aminoacid SH-groups, NH2-groups and histidine led to a decrease of the enzyme activity. Incubation of phosphoprotein phosphatase with [gamma-33P]ATP resulted in the incorporation of 0.33 mol of 33P per mol of the enzyme. The mechanism of the enzyme-catalyzed hydrolysis of the phosphoester bond is discussed. PMID- 2994760 TI - [The additivity principle in the reaction of angiotensin II and its analogs with antibodies and receptors of glomerular cells from the rat adrenal cortex]. AB - The binding of angiotensin II and its analogues (13) to rabbit antibodies and glomerular cell receptors from rat adrenal cortex was studied, using the radioimmunoassay method and radioreceptor analysis. Double modifications introduced into the angiotensin structure were found to increase in an additive fashion its binding to the antibodies and renal cell receptors. The relative binding activity of the analogues carrying a double modification can be assessed if the activities of the analogues with the appropriate single modifications are known. It was concluded that the testing of modifications in the peptide structure for their additivity may provide some insight into the conformational properties of peptides during their binding to the protein. PMID- 2994761 TI - [Peroxidase activity of succinylated catalase]. AB - The catalase succinylation by succinic anhydride excess results in an almost complete dissociation of the enzyme into subunits possessing no catalase activity. The catalase subunits show the peroxidatic activity on o-dianisidine oxidation. The oxidation kinetics of this substrate by the succinylated enzyme was studied at various temperatures. The activation energy for this process is 10.1 kcal/mole. Within the temperature range of 31-65.5 degrees, the succinylated enzyme thermostability was studied by monitoring the peroxidatic activity decrease upon o-dianisidine oxidation. The activation energy for the succinylated catalase thermoinactivation equals to 15.5 kcal/mole. The peroxidatic activity of catalase subunits obtained by enzyme succinylation and acidic solution treatment was compared to that of horseradish peroxidase in the oxidation of the same substrate, i.e., o-dianisidine. PMID- 2994762 TI - Polymorphonuclear leukocyte function in term and preterm newborn infants. AB - Bacterial infections are a major problem in the care for newborn infants. In search for immunological deficits we investigated phagocytosis and killing of staphylococci using polymorphonuclear leukocytes (PMN) isolated from 2 ml of venous blood. Phagocytosis of PMN from preterm (n = 10) and newborn infants (n = 9; mean birth weights 1,949 and 3,523 g, respectively) was not different from that of adult PMN (n = 14). Killing capacity of PMN from preterm infants was markedly impaired compared to term newborn infants and to adult PMN. We found similar rates of superoxide anion production and similar times for activation in response to phorbolmyristate acetate stimulation. Our study gives further evidence that PMN from term newborn infants have normal phagocytotic and bactericidal capacity. In PMN from preterm newborn infants, however, the bactericidal capacity is diminished similar to newborn infants under stress as described earlier by others. PMID- 2994763 TI - Sequestration of 3H-vitamin D3 by the fetal and neonatal rat liver. AB - 19- to 21-day-old fetuses as well as 3-, 7-, 14-, and 22-day-old pups were used to evaluate the sequestration of a tracer dose of 3H-D3 by the fetal and neonatal rat liver. In fetuses, 3H-D3 was injected directly into the umbilical vein; in neonates, 3H-D3 was injected into the portal vein. In both cases, 14C-sucrose was used as extracellular marker. 3H-D3 sequestration was evaluated at 11.3 +/- 1.1% (means +/- SEM) in fetuses, 3.3 +/- 0.6, 4.9 +/- 0.6, 6.3 +/- 0.5 0.5 and 42.0 +/ 1.9% of the dose administered in 3-, 7-, 14-, and 22-day-old rats, respectively. These results clearly show that, in the rat, the fetal liver can take up 3H-D3. Moreover, when compared to the values obtained in the late fetal period, 3H-D3 uptake decreased significantly during the first 2 weeks post partum (p less than 0.0001) and then dramatically increased (6-fold, p less than 0.0001) between days 14 and 22 post partum, when it reached an uptake capacity similar to that previously observed in adult rats. PMID- 2994764 TI - Differences in plasma ACTH and cortisol between depressed patients and normal controls. AB - Although studies have repeatedly demonstrated that depressed patients average higher baseline and postdexamethasone serum cortisol than normal controls, studies examining similar trends in adrenocorticotrophic hormone (ACTH) have produced conflicting results. The current study uniquely employs 48 hr of every 20-min serum sampling: the first 24 hr prior to dexamethasone administration and the second 24 hr subsequent. The depressed patients showed higher baseline cortisol levels than normal controls, with the greatest differences between 2 AM and 6 AM. After an 11 PM dose of dexamethasone, the difference was greatest between the hours of 8 AM and 4 PM. Among the depressed patients, those who reported recent weight loss had significantly higher plasma ACTH and cortisol levels than those without weight loss. Depressed patients without weight loss had higher baseline plasma ACTH than normal controls, and the differences reached significance during some time periods. PMID- 2994765 TI - Desensitization of mouse Leydig cells in vivo: evidence for the depletion of cellular cholesterol. AB - The study presents a characterization of the refractory state in purified mouse Leydig cells desensitized by a single injection of human chorionic gonadotropin (hCG) in vivo. The treatment of mice with 1 microgram hCG i.p. for 48 h followed by Leydig cell isolation and purification resulted in a decrease in the maxima of hCG-induced cAMP accumulation and testosterone production by approximately 70% and approximately 55%, respectively, when compared to cells of control mice. Despite a 55% reduction in 125I-hCG binding sites, the sensitivity of stimulation was not changed. The refractoriness in testosterone production in vitro was also present when the Leydig cells were stimulated with cholera toxin or dibutyryl cAMP; however, it was not observed when testosterone production was induced by the addition of pregnenolone or 20 alpha- and 22(R)-hydroxycholesterol. Mouse lipoproteins, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) in natural composition, were also able to overcome the steroidogenic block (although not always completely). On the basis of the cholesterol content of the lipoproteins, the two classes were similarly effective. They increased maximal hCG-induced testosterone production not only in desensitized cells, but also in control cells (by 80-100%), whereas their effect on basal testosterone production was negligible. In desensitized cells from hCG-treated mice (2 micrograms i.p., 48 h) cellular unesterified and esterified cholesterol were decreased by 21% and 81%, respectively, when compared to control cells. This loss occurred in the face of unchanged plasma cholesterol levels. In conclusion, our data indicate that the impaired steroidogenesis in mouse Leydig cells desensitized in vivo by a single injection of hCG is the result of a depletion in cellular cholesterol, rather than of an impaired conversion of cholesterol to testosterone. PMID- 2994767 TI - Inhibition of 20 alpha-hydroxysteroid dehydrogenase activity by follicle stimulating hormone and androgens in cultured rat granulosa cells: a search for the mechanism of action. AB - Alterations of progesterone metabolism and especially of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity were studied in cultured rat granulosa cells following various treatments. The cells were incubated for up to 48 h with or without follicle-stimulating hormone (FSH), androgens, hydroxyflutamide, estrogens, chlorea toxin, and dibutyryl cAMP [Bu2 cAMP]. Subsequently, the cells were incubated for 3 h with [4-14 C] progesterone (0.5 microM). The progesterone utilization and accumulation of 20 alpha-reduced and 5 alpha-reduced metabolites were assessed following thin-layer chromatography separation of radiolabeled steroids. Both FSH (1 microgram/ml) and testosterone (0.5 microM) decreased the 20 alpha-HSD activity by decreasing the maximal velocity (by 52% and 37%, respectively) without changing significantly the Km value. The inhibition of 20 alpha-HSD was demonstrable following 12 and 24 h exposure to FSH and following 24 and 48 h exposure to testosterone. Effects comparable to that induced by testosterone were elicited by other androgens (androstenedione and 5 alpha dihydrotestosterone), but not by estrogens (estradiol-17 beta and estrone). Hydroxyflutamide reversed testosterone-induced effects: the increase of endogenous progesterone accumulation and the decrease of 20 alpha-HSD activity. Both cholera toxin (0.001-10 micrograms/ml) and Bu2 cAMP (62.5-1000 micrograms/ml) caused a dose-dependent inhibition of 20 alpha-HSD activity. Present results indicate that: the inhibition of 20 alpha-HSD by both FSH and androgens may be of a noncompetitive nature; androgen action on 20 alpha-HSD may be a true androgenic, receptor-mediated effect; and cAMP may mediate the FSH action on 20 alpha-HSD activity. PMID- 2994766 TI - Effects of aromatizable and nonaromatizable androgen treatments on luteinizing hormone receptors and ovulation induction in immature rats. AB - The effects of androgen pretreatment on follicle-stimulating hormone (FSH) stimulated luteinizing hormone (LH) receptor induction in ovarian granulosa cells was examined. Immature female rats were treated with various doses (0.1-5 mg/rat) of testosterone (T), 5 alpha-dihydrotestosterone (DHT), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol), or 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol). Subsequent follicular development was stimulated by treatment with ovine FSH. LH receptor induction in granulosa cells and ovulatory responses to 10 IU human chorionic gonadotropin (hCG) were examined. Since LH receptor induction requires the synergistic action of both FSH and estradiol, the effects of the androgen pretreatment on FSH-stimulated estradiol production were also examined. Dihydrotestosterone treatment at doses greater than 1 mg inhibited LH receptor induction by approximately 70%, which resulted in absent ovulatory responses. Treatment with 1 mg or more of T or 3 alpha-diol had no effect on LH receptor induction, yet the hCG-stimulated ovulation rate was reduced to 40% of that seen in vehicle-treated controls. 3 beta-Diol, at a dose of 1 mg/rat, did not affect LH receptor induction but did reduce hCG-stimulated ovulation responses. No significant effects of androgen treatment on ovarian or uterine weight or FSH stimulated estradiol production were observed. These results suggest that androgens can act at multiple sites to inhibit ovarian follicular development and function. In addition these studies demonstrate that, although LH receptor induction is necessary, it may not be a sufficient condition to ensure ovulation of ovarian follicles. PMID- 2994769 TI - Mass-analysed ion kinetic energy spectra and B1E-B2 triple sector mass spectrometric analysis of phosphoinositides by fast atom bombardment. AB - Fast atom bombardment is shown to produce useful spectra of the three phosphoinositides and the metabolically related phospholipids, lysophosphatidylinositol and phosphatidic acid. Analysis of the [M-H]- ions for fatty ester composition by mass-analysed ion kinetic energy spectra (MIKES) is shown to be inadequate to resolve fatty acyl daughter ions when the parent ion contains isobaric species. However, analysis on a triple sector instrument with and without collisional activation does provide complete compositional information. Quantitative analysis of the fatty ester content of each lipid molecular species is complicated by dissimilar ion yields from fatty acyl-bearing fragments from compositionally different parent ions. PMID- 2994768 TI - Model-independent electron spin resonance for measuring order of immobile components in a biological assembly. AB - A model-independent description of the angular orientation distribution of elements in an ordered biological assembly is applied to the electron spin resonance (ESR) technique. As in a previous model-independent treatment of fluorescence polarization (Burghardt, T.P., 1984, Biopolymers, 23:2383-2406) the elemental order is described by an angular distribution of molecular frames with one frame fixed in each element of the assembly. The distribution is expanded in a complete orthonormal set of functions. The coefficients of the series expansion (the order parameters) describe the orientation distribution of the elements in the assembly without reference to a model and can be obtained from the observed spectrum. The method establishes the limitations of ESR in detecting order in the assembly by determining which distribution coefficients the technique can detect. A method of determining the order parameters from an ESR spectra, using a set of ESR basis spectra, is developed. We also describe a treatment that incorporates the actual line shape measured from randomly oriented, immobile elements. In this treatment, no model-dependent assumptions about the line shape are required. We have applied the model-independent analysis to ESR spectra from spin-labeled myosin cross-bridges in muscle fibers. The results contain detailed information on the spin-probe angular distribution and differ in interesting ways from previous model-dependent interpretations of the spectra. PMID- 2994770 TI - Separation of human endothelial cells from fibroblasts by centrifugation in Percoll gradients. AB - Human endothelial cells from the umbilical vein and skin fibroblasts can be separated by means of centrifugation in a density gradient of Percoll. Cells show a good recovery in culture. Viability is not impaired. PMID- 2994771 TI - Inhibition by ceruloplasmin of the cardiac sarcolemmal adrenochrome formation. AB - The addition of ceruloplasmin to bovine cardiac sarcolemmal vesicles supplemented with NADPH was able to reduce the formation of adrenochrome from adrenaline. This inhibitory effect appears at 2.5 microM ceruloplasmin and it is almost complete at the level of 20 microM. PMID- 2994772 TI - Interaction of vasoactive intestinal peptide with rat small intestinal epithelial cells after intestinal resection. AB - Specific binding of vasoactive intestinal peptide (VIP) and VIP-stimulated cyclic AMP accumulation were studied in small intestinal epithelial cells (both of crypt and villous levels) 3, 7 and 14 d after a 60% resection of the small intestine. The affinity, but not the binding capacity, of VIP receptors decreased during the adaptive hyperplastic response. Basal cyclic AMP levels were similar in cells of both control and resected rats. Resection induced a decrease of potency, but not of efficiency, of VIP on the stimulation of cyclic AMP accumulation. PMID- 2994773 TI - Sodium butyrate selectively inhibits host cell glycoprotein synthesis in human fibroblasts infected with cytomegalovirus. AB - Host cell as well as viral DNA synthesis in human fibroblasts infected with human cytomegalovirus was found to be largely resistant even to high concentrations of sodium butyrate. Likewise, production of viral progeny was reduced by 1-2 orders of magnitude but not abolished. On the other hand, the drug allowed (modified) glycosylation only of viral polypeptides whereas that of host proteins was suppressed. Immunofluorescence studies on living cells suggested that butyrate may interfere with processing and intracellular transport of virus-specific surface membrane antigens. PMID- 2994774 TI - Viscosities of some triglycerides and ethylester of fatty acids frequently found in cell membranes--a possible effect of viscosity of fatty acids in phospholipids on hemorheology. PMID- 2994775 TI - [Mechanism of the antistressor effect of D-Ala(2)-Leu(5)-Arg(6)-enkephalin]. AB - It has been shown that the stable analog of leu-enkephalin diminishes to a great extent the stress-induced changes in the blood content of ACTH, cortisol, hypophyseal-thyroid hormones, the CAMP level in the adrenal and thymic tissues of white rats. It is assumed that this circumstance may interfere with depletion of the adrenal cortex and suppression of the lymphoid-macrophagal system. PMID- 2994776 TI - [Generation of superoxide radicals by ischemic heart mitochondria]. AB - Tiron (1,2-dihydroxybenzene-3,5-disulfonic acid, disodium salt) was used as a spin trap to detect superoxide radicals produced by rat heart mitochondria. It was shown that ischemia results in the enhancement of the mitochondrial superoxide-forming activity. In the presence of the oxidative phosphorylation uncoupler mesoxalonitrile (3-chlorophenyl)-hydrazone the superoxide production rate in the control mitochondria increases, that in the ischemic mitochondria remains unchanged. PMID- 2994777 TI - [Characteristics of the benzodiazepine receptors in rats with different predispositions to the development of experimental alcoholism]. AB - A study was made of the density and affinity of benzodiazepine receptors in the cortex of the cerebral hemispheres and hippocampus of rats with different predisposition to alcohol consumption. No differences were revealed in the parameters under study in animals with varying duration of ethanol anesthesia and in rats after voluntary consumption of ethanol for 3.5 and 10 months. In a state of abstinence rats with physical dependence manifested a dramatic decrease in the density and affinity of benzodiazepine receptors in the cortex of the cerebral hemispheres. No changes described were detected in the hippocampus. The role of benzodiazepine receptors in the development of abstinence is discussed. PMID- 2994778 TI - [Potentiating action of bi- and trivalent metal salts on the analgesic effect of morphine]. AB - It has been found that the metal salts MnCl2, NiCl2, GdCl3 and LaCl3 in doses up to 30, 20, 5 and 10 micrograms, respectively, potentiate the analgesic effect of intracisternally injected morphine (2 micrograms per mouse). The ability of the metal salts to potentiate the affinity of opiate ligands to the appropriate receptors is effectuated via interaction of metal cation with the specific opiate receptor site. It is suggested that one of the possible mechanisms of the potentiating effect of some metal salts on the morphine-induced analgesia involves the enhancement of morphine affinity for mu-receptors in the brain. PMID- 2994779 TI - Application of molecular genetics to prenatal diagnosis and carrier detection in the hemophilias: some limitations. AB - Prenatal diagnosis and carrier detection in the hemophilias have received much attention in recent years. The error rate in prenatal diagnosis by fetoscopy is less than 1%; fetoscopy is not possible, however, until the second trimester of pregnancy. Carrier detection based on bioassays of plasma has an irreducible error rate (approximately 5%?), because of the "lyonization" phenomenon in heterozygous women, and the final results are always probabilistic. New DNA methods promise to alleviate these difficulties. Prenatal diagnosis can be accomplished in the first trimester. "Lyonization" is bypassed in carrier detection, and the results may sometimes be essentially nonprobabilistic. But the DNA methods have certain limitations of their own which are not widely appreciated. Aside from cost and the necessity to adopt a new technology, there are inherent genetic problems: mothers must be heterozygous for both a disease gene and a marker gene, final results are probabilistic if the marker gene lies outside the disease gene, and multiple marker genes are often in linkage disequilibrium. We have concluded that a clinical unit planning to use the DNA methods must also maintain the conventional methods at a high level of performance. PMID- 2994780 TI - Immunologic and virologic status of multitransfused patients: role of type and origin of blood products. By the AIDS-Hemophilia French Study Group. AB - An immunologic and virologic work-up was undertaken in 425 symptom-free multitransfused patients with hemophilias or hemoglobinopathies living in France. Patients were entered into five groups according to the type of blood product they received: local factor VIII, a mixture of local and imported factor VIII, imported factor IX, local factor IX, washed red blood cells. The overall prevalence of IgG antibodies to the lymphadenopathy-associated virus (LAV) was 45%. The highest rate was observed in hemophiliacs who received factor VIII concentrates prepared from plasma collected mainly on the American continent; intermediary values were found for hemophilic patients treated with local factor VIII or factor IX concentrates; and the lowest values were found for those who were treated with washed red blood cells. Lymphadenopathy, decreased skin hypersensitivity reactions, relative lymphopenia, and altered ratio of T lymphocyte subsets occurred at significantly higher rates in patients positive for LAV antibody, although such abnormalities were also encountered in LAV serologically negative patients. A correlation between treatment intensity and immunologic disturbances was found in patients infused with factor VIII preparations, irrespective of their positive or negative LAV antibody status. This study has shown the prominent role of LAV in the occurrence of immunologic disturbances in multitransfused patients. However, allogenic or altered proteins present in factor VIII but not in factor IX concentrates seem to play a role of immunocompromising agents. The interplay between LAV and additional factors possibly leading to acquired immunodeficiency syndrome remains to be analyzed. PMID- 2994781 TI - Delay in platelet recovery after bone marrow transplantation: impact of cytomegalovirus infection. AB - The effect of cytomegalovirus (CMV) infection on hematopoietic recovery after marrow-ablative chemoradiotherapy followed by autologous bone marrow transplantation (BMT) was studied in patients with non-Hodgkin's lymphoma of high grade malignancy and in patients with acute leukemia. The recovery of platelets after autologous BMT occurred significantly quicker in CMV-negative patients than in CMV-positive patients (platelets greater than 50,000 per cubic millimeter after 21 1/2 v 40 days, respectively). No differences in the recovery of neutrophils were found between those with or without CMV infection. CMV-positive patients required significantly more transfusion support with thrombocyte concentrates than CMV-negative patients (three v six thrombocyte concentrates). In conclusion, CMV infections do not influence neutrophil recovery but do delay platelet recovery. As a consequence, patients with a CMV infection, whether primary, reactivated, or latent, require more thrombocyte concentrates, which increases the risk of transfusion-related infections. PMID- 2994782 TI - Platelet membrane topography: colocalization of thrombospondin and fibrinogen with the glycoprotein IIb-IIIa complex. AB - The distribution of platelet thrombospondin (TSP), fibrinogen, and glycoproteins IIb-IIIa (GPIIb-IIIa) and GPIb were studied in resting and activated human platelets using frozen thin-section immunoelectron microscopy. In resting platelets, TSP and fibrinogen were found within alpha granules and not on the platelet surface. In unstimulated platelets, GPIIb-IIIa and GPIb were distributed diffusely over the platelet membrane as well as within the body of the platelets. Upon thrombin or A23187 stimulation, TSP, fibrinogen, and GPIIb-IIIa colocalized on the platelet membrane and the canalicular system as well as on pseudopodia and between adherent platelets. GPIb distribution was unchanged by platelet activation. The findings support the hypothesis that a macromolecular complex of TSP-fibrinogen and GPIIb-IIIa forms on the activated platelet membrane. PMID- 2994783 TI - Transferrin saturation, plasma iron turnover, and transferrin uptake in normal humans. AB - The relationship between plasma iron, transferrin saturation, and plasma iron turnover was studied in 53 normal subjects whose transferrin saturation varied between 17% and 57%, in 25 normal subjects whose transferrin saturation was increased by iron infusion to between 67% and 100%, and in five subjects with early untreated idiopathic hemochromatosis whose transferrin saturation was continually elevated to between 61% and 86%. The plasma iron turnover of all of these subjects ranged from 0.45 to 1.22 mg/dL whole blood/d. The mean values for the above-mentioned three groups were 0.71 +/- 0.17, 1.01 +/- 0.11, and 1.01 +/- 0.13 mg/dL whole blood/d, respectively. Most of this variation, estimated at 72% by regression analysis, was due to a direct relationship between transferrin saturation and plasma iron turnover. This effect was attributed to a competitive advantage of diferric over monoferric transferrin in delivering iron to tissues. This was confirmed by the demonstration of a more rapid clearance of diferric as compared to monoferric transferrin in an additional group of eight normal subjects. Calculations were made of the amount of transferrin reacting with membrane receptors per unit time. Allowance was made for the noncellular (extravascular) exchange and for the 4.2:1 preference of diferric over monoferric transferrin demonstrated in vitro. The amount of iron-bearing transferrin leaving the plasma to bind to tissue receptors for 53 subjects with a transferrin saturation between 17% and 57% was 71 +/- 13; for 25 subjects with a saturation from 67% to 100%, 72 +/- 12; and for five subjects with early idiopathic hemochromatosis, 82 +/- 11 mumol/L whole blood/d. There were no significant differences among these groups. These studies indicate that while the number of iron atoms delivered to the tissues increases with increasing plasma iron and transferrin saturation, the number of iron-bearing transferrin molecules that leave the plasma per unit time to bind to tissue receptors is relatively constant and within the limits studied, independent of transferrin saturation. PMID- 2994784 TI - A longitudinal study of patients with hemophilia: immunologic correlates of infection with HTLV-III/LAV and other viruses. AB - A comprehensive study was initiated to examine the immunologic status of a sample (n = 47) of the asymptomatic hemophilia A and B populations of metropolitan Atlanta and to determine if any of the abnormalities changed with time or correlated with infection by human T cell leukemia virus type III and lymphadenopathy-associated virus (HTLV-III/LAV) or other viruses either alone or in combination. Patients with hemophilia A (Hem-A) showed a defect in cellular immunity evidenced by a depressed T cell helper/suppressor ratio (P less than .0001), an increased absolute T suppressor cell number (P less than .0001), and a diminished number of T helper cells (P = .003) when compared with health professionals. Lymphocytes from these patients also showed a reduced ability to transform in response to phytohemagglutinin and pokeweed mitogen. No deterioration in immune status was seen during a median ten-month period of follow-up. Sixty-four percent of Hem-A patients had antibodies to HTLV-III/LAV and those who were seropositive had a significantly decreased helper/suppressor cell ratio (P = .018) and a diminished T helper cell number (P = .002); they were also more likely to have had exposure to cytomegalovirus than HTLV-III/LAV negative Hem-A patients (P = .016). Heavy use of factor VIII concentrate was associated with a decreased number of T helper cells (P = .037) and seropositivity for HTLV-III/LAV (P = .011 in 1982). Hemophilia patients had higher IgG, immune complex, and beta 2-microglobulin levels than health professionals (P less than .0001). Although the most prominent abnormality observed in T cell subsets of patients with hemophilia is an increase in T suppressor cells, a finding likely to be associated with immune augmentation in response to multiple stimuli, the T cell abnormality that was predictive of exposure to HTLV-III/LAV, the putative acquired immunodeficiency syndrome agent, was a diminished number of T helper cells. PMID- 2994785 TI - Inhibition of ionophore-stimulated leukotriene B4 production in human leucocytes by monohydroxy fatty acids. AB - Leukotriene B4 (LTB4) release by calcium ionophore-stimulated human leucocytes was measured by use of selective solvent partition of reaction mixtures and an agarose microdroplet chemokinesis assay, and the inhibitory effects of four monohydroxy fatty acids were determined. 15-Hydroxy-eicosatetraenoic acid (15 HETE) was the most effective inhibitor of LTB4 production with an approximate IC50 value of 6 microM and 99% inhibition at 50 microM, whereas 13-hydroxy octadecadienoic acid (13-HODD) and 12-HETE were weaker inhibitors with approximate IC50 values of 32 microM and 23 microM, and 59% and 68% inhibition at 50 microM, respectively. We suggest that 13-HODD and 12-HETE, which are present in large amounts in the lesions of the skin disease psoriasis, may act as endogenous modulators of 5-lipoxygenase activity in skin. PMID- 2994786 TI - Platelet desensitization induced by arachidonic acid is not due to cyclo oxygenase inactivation and involves the endoperoxide receptor. AB - Human platelets pre-exposed to arachidonic acid (AA) (0.1-1 mM) or to the endoperoxide analogue U46619 (1-3 microM) and then washed and resuspended, failed to respond with aggregation or secretion to a second challenge by either agonist. The response to thrombin at low (0.04-0.1 u ml-1) but not at high (2.5 u ml-1) concentrations was also inhibited by pre-exposure to AA and U46619. The ability of platelets to synthesize thromboxane (Tx) B2 from AA or upon challenge with thrombin persisted despite platelet desensitization. In the presence of the reversible cyclo-oxygenase (CO) inhibitors methyl salicylate (MS) or L8027, pre exposure to AA had no effect on subsequent challenge by the same agonist or by U46619, whereas platelet desensitization by pre-exposure to U46619 persisted. However, platelet activation by, and desensitization to AA and U46619, was prevented by trimetoquinol and compound L636499, two thromboxane/endoperoxide receptor antagonists. In contrast to the CO inhibitors, the thromboxane synthetase inhibitor dazoxiben, which in 3 'responders' out of 5 subjects suppressed aggregation, secretion, and Tx formation induced by AA, failed to prevent AA-induced desensitization. Compared to quiescent cells the distances between platelets desensitized after re-exposure to AA were reduced in electron microscopy, but the tight connections associated with aggregated cells were not observed. Degranulation was also not observed and cell morphology resembled that of normal quiescent platelets. In conclusion, (a) AA and U46619 desensitize human platelets at a similar site sensitive to prostaglandin/thromboxane receptor antagonists, and show cross-desensitization; (b) desensitization by AA appears to be mediated by a CO-dependent metabolite, as CO inhibitors prevent desensitization by AA but not to U46619; (c) the failure of dazoxiben to prevent desensitization by AA suggests that a metabolite other than TxA2, possibly the endoperoxides, mediates the phenomenon; (d) desensitization does not involve inactivation of CO or thromboxane synthetase enzymes. PMID- 2994787 TI - The effect of hypoxia on neuroeffector transmission in the bovine retractor penis and rat anococcygeus muscles. AB - The effects of reducing the PO2 of the bathing fluid were studied on non adrenergic non-cholinergic (NANC) transmission in isolated preparations of the bovine retractor penis muscle, the rat anococcygeus muscle, the guinea-pig taenia caeci and the guinea-pig urinary bladder. Hypoxia rapidly and reversibly impaired NANC transmission in the bovine retractor penis and rat anococcygeus muscles but did not affect transmission in the guinea-pig taenia caeci or bladder, suggesting that different NANC mechanisms are involved. Although neurally-evoked relaxation of the bovine retractor penis was impaired by hypoxia, relaxations produced by vasoactive intestinal peptide, prostaglandin E1, sodium nitroprusside or an inhibitory factor isolated from the bovine retractor penis were unaffected. Since the inhibitory factor is similar to, and may actually be the NANC transmitter, the results suggest that the site of action of hypoxia in impairing transmission is prejunctional at the inhibitory nerve endings. PMID- 2994788 TI - Characterization of histamine receptors mediating the stimulation of cyclic AMP accumulation in rabbit cerebral cortical slices. AB - The characteristics of histamine-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in slices of rabbit cerebral cortex have been investigated. The selective H2-receptor antagonists, cimetidine, tiotidine, metiamide and ranitidine appeared to antagonize the stimulation of cyclic AMP accumulation elicited by histamine in a competitive manner consistent with an interaction with histamine H2-receptors. The H1-receptor antagonist mepyramine (0.8 microM) produced only a weak inhibition of the response to histamine. The inhibition appeared to be non-competitive producing a decrease in the maximal response with little effect on the EC50 value. The specific H2-receptor agonist, impromidine, produced a maximum response of only 31 +/- 2% of that obtained with histamine. Studies with histamine and impromidine in combination indicated that impromidine was not acting as a partial agonist. 2-Thiazolylethylamine, a selective H1-agonist, produced only a weak response (EC50 approximately 1mM) yielding a relative potency with respect to histamine (= 100) of 2.5. In the presence of a supramaximal concentration of impromidine, histamine and 2 thiazolylethylamine further elevated the response to impromidine. In these conditions the relative potency of 2-thiazolylethylamine was increased to 59 (histamine = 100), a value which was comparable with that reported for H1 receptor-mediated contractions of guinea-pig ileum. The H1-receptor antagonists mepyramine, promethazine, triprolidine and chlorpheniramine competitively antagonized the potentiation of impromidine-stimulated cyclic AMP accumulation elicited by histamine and 2-thiazolylethylamine in rabbit cerebral cortex without affecting the response to impromidine alone. (+)-Chlorpheniramine was some 150 fold more potent than the (-)-isomer in this respect. Histamine and adenosine in combination had a much greater than additive effect on the accumulation of cyclic AMP in rabbit cerebral cortical slices. The potentiation of the adenosine response could be partially but not completely antagonized by either cimetidine or mepyramine. In the presence of H2-receptor blockade with 0.02 mM tiotidine, histamine elicited a significant potentiation (EC50 44 microM) of the response to adenosine. This response was antagonized competitively by mepyramine yielding a KB value of 0.05 microM similar to that obtained from inhibition of the potentiation of impromidine-stimulated accumulation of cyclic AMP (0.02 microM). These results suggest that there are two components in the response to histamine in rabbit cerebral cortical slices. The first component appears to be mediated by histamine H2-receptors while the second, mepyramine-sensitive, component has some ofthe characteristics ofan H,-receptor mediated response and requires prior stimulation of adenosine- or H2-receptors to produce its effect. PMID- 2994789 TI - An unusual effect of gamma-aminobutyric acid on synaptic transmission of frog tectal neurones in vitro. AB - Bath-applied gamma-aminobutyric acid (GABA) enhanced, in a dose-dependent fashion, the amplitude of optic nerve-evoked monosynaptic excitatory responses of the frog optic tectum superfused in vitro at 7 degrees C. Muscimol was more potent than GABA in eliciting similar effects. GABA-induced responses were antagonized by picrotoxin and were insensitive to bicuculline or strychnine. Raising the bath temperature to 20 degrees C reduced the potency of GABA on these preparations. No significant effect of GABA on the compound action potential of the whole optic nerve was found. These data indicate that GABA can amplify visual inputs to the tectum through bicuculline-insensitive mechanisms. PMID- 2994791 TI - A survey of serum antibodies to eight common viruses in psychiatric patients. AB - Serum antibody titres to eight neurotropic viruses were measured by enzyme immunoassay in 450 psychiatric in-patients and 143 controls. A seasonal variation in schizophrenic births was observed, with a peak incidence between March and April. Both herpes simplex virus and cytomegalovirus antibody titres correlated with age and, when this was controlled for, no significant differences emerged between any patient group and the controls. Mumps antibody titres were significantly lower in patients with mental subnormal and neurosis or personality disorder; measles and rubella antibody titres were lower in male but not female mentally handicapped patients; males had lower antibody titres to mumps, cytomegalovirus and Epstein-Barr virus than females in all groups. A decrease in mumps antibody titres was also found in schizophrenics if the medication factor was excluded. These low antibody titres may indicate an impaired immune response. Thus perinatal or childhood subclinical viral infections of the central nervous system, particularly of mumps, might lead to a range of possible psychiatric outcomes in later life. PMID- 2994792 TI - Serum and CSF antibody titres to seven common viruses in schizophrenic patients. AB - CSF and matched serum antibody titres to seven common viruses were measured in 20 chronic schizophrenic patients, and 17 of these were age and sex-matched with orthopaedic controls. CT scans were carried out in patients and age and sex matched radiological controls. There was a trend for CSF viral antibody titres (except CMV, HSV and VZV) to be decreased in the patients compared to controls, statistically significant for mumps and IgG. The CSF/serum ratios showed a reduction in the patients, compared to controls, statistically significant for measles and rubella as well as mumps and IgG. Cerebral ventricular size was significantly increased in the patients compared to controls, but did not correlate with any of the antibody data. These findings suggest that there is a reduced immune response to certain common viruses in the CNS of schizophrenic patients, but possible effects of institutionalisation or current medication could only be adequately excluded by further prospective studies. PMID- 2994790 TI - Identification of presynaptic beta 2-adrenoceptors on the sympathetic nerve fibres of the human pulmonary artery. AB - Strips of human pulmonary arteries from patients undergoing surgery for lung tumour were incubated with [3H]-noradrenaline. Subsequently, they were superfused with physiological salt solution containing cocaine and corticosterone. Tritium overflow from the strips was stimulated by transmural electrical impulses (2 Hz). The electrically evoked overflow of tritium consisted of 91% unmetabolized [3H] noradrenaline, and this percentage was not altered by isoprenaline. Adrenaline (in the presence of rauwolscine), isoprenaline and the preferential beta 2 adrenoceptor agonist, procaterol, concentration-dependently increased the electrically evoked tritium overflow. Prenalterol, a beta-adrenoceptor agonist with moderate preference for beta 1-adrenoceptors, was considerably less active than the previously mentioned agonists; noradrenaline (in the presence of rauwolscine) was ineffective. The concentration-response curve of procaterol was shifted to the right by the preferential beta 2-adrenoceptor antagonist ICI 118 551 but was not affected by the beta 1-selective antagonist, atenolol. Propranolol, but not atenolol, produced a shift to the right of the concentration response curve of isoprenaline. It is concluded that the sympathetic nerve fibres of the human pulmonary artery are endowed with facilitatory presynaptic beta 2 adrenoceptors. PMID- 2994793 TI - Peculiarities of mucinous colorectal carcinoma. AB - The clinical and pathological features of 54 mucinous carcinomas of the large intestine were compared with those of 576 non-mucinous carcinomas. Tumours were only categorized as mucinous if they contained at least 60 per cent of mucin by volume. Those with a moderate mucin content (60-80 per cent) were indistinguishable in behaviour from 'non-mucinous' tumours. By contrast, those with a high mucin content (greater than 80 per cent) showed several differences from non-mucinous cancers: they had a more proximal distribution through the large intestine, they comprised a greater fraction of cancers in the under 50 age group (24 versus 7 per cent: P less than 0.01), they were more likely to be Dukes' stage 'D' (58 versus 31 per cent: P less than 0.01) and local fixity was commoner (70 versus 37 per cent: P less than 0.001). Consequently the overall resection rate was reduced from 90 to 73 per cent (P less than 0.01), the curative resection rate from 69 to 42 per cent (P less than 0.01) and the 5-year survival rate from 37 to 18 per cent (P less than 0.05). Colorectal carcinomas of high mucin content require wide excision, tend to recur locally and carry a poor prognosis. PMID- 2994794 TI - A retrospective study of male breast cancer in Holland. AB - A retrospective study has been undertaken of 104 men with breast cancer, all of them having a follow-up period of at least 5 years. In 78 cases a histological diagnosis was obtained. The preferred treatment for operable cases was radical mastectomy, in which 60 per cent positive axillary nodes were found. Five-year survival is 54 per cent and the disease-free interval is 42 per cent. Local recurrence occurred in 26 per cent and 16 per cent had developed distant metastases. The overall results are similar to those in the literature with the exception of those for stage III who did better in this series. The generally held beliefs that Klinefelter's syndrome is the strongest predisposing factor to developing male breast cancer and that gynaecomastia is not a premalignant condition are supported by this study. Comparison of results from this series, with those of women of the same age having breast cancer leads to the conclusion that the prognosis in male breast cancer is no worse than for women with comparable disease. PMID- 2994795 TI - Layered versus mass closure of abdominal wounds in infants and children. AB - A prospective trial of layered versus mass closure of laparotomy wounds was performed on 507 infants and children over a 33 month period. All wounds were sutured using polyglycolic acid sutures. There were four wound failures including one disruption in the layered closure group and one wound failure in the mass closure group. The incisional hernias healed spontaneously. Either method of closure yields good results and non-absorbable sutures are unnecessary. PMID- 2994796 TI - Epidermal growth factor receptors. PMID- 2994797 TI - Nervous control of the gut. PMID- 2994798 TI - Experimental intestinal carcinogenesis. PMID- 2994799 TI - Pathophysiology of constipation. PMID- 2994800 TI - Sulpiride and the potentiation of progestogen only contraception. AB - A progestogen (norethisterone) and a dopamine antagonist (sulpiride) were given alone and in combination to volunteers to examine their effects on excretion of ovarian steroids. Compared with non-treatment cycles (n = 15), contraception with a progestogen alone (n = 10) was associated with increased excretion of oestrone and partial suppression of excretion of pregnanediol, suggesting partial luteinization of unruptured follicles. By contrast, the combination of norethisterone and sulpiride (n = 9) suppressed both ovarian steroids to basal values, the suppression being even greater than with sulpiride alone (n = 5). These results suggest that a combination of a progestogen with a dopamine antagonist might have a role in contraception. PMID- 2994802 TI - Haemophilus parainfluenzae and H influenzae respiratory infections: comparison of clinical features. PMID- 2994801 TI - AIDS and haemophilia: morbidity and morality in a well defined population. AB - One hundred and forty-three multitransfused patients with hereditary haemostatic disorders were examined for evidence of disease related to the acquired immune deficiency syndrome (AIDS). Ninety-nine patients with severe haemophilia A were tested for anti-HTLV-III and 76 were found to be positive. All except one of these seropositive patients had received commercial factor VIII concentrates at some time. Eighteen patients with haemophilia B were tested and all were anti HTLV-III negative. Three out of 36 sexual partners of patients with haemophilia A positive for anti-HTLV-III were also seropositive. One, who had recently received blood transfusions, had AIDS with Pneumocystis carinii pneumonia. Three patients with severe haemophilia A died from Aids. A further 30 haemophiliacs had AIDS related complex or lymphadenopathy that could be related to HTLV-III infection. There was a significant correlation between lymphadenopathy and anti-HTLV-III seropositivity. No evidence of casual spread of AIDS was found since all 68 health care staff tested were anti-HTLV-III negative, including three surgeons who regularly worked with patients positive for anti-HTLV-III. The resources devoted to counselling and laboratory support in centres treating people at risk and their families need to be urgently reassessed. PMID- 2994803 TI - IgM and IgG antibodies to HTLV-III in the lymphadenopathy syndrome and subjects at risk for AIDS. PMID- 2994806 TI - HTLV-III infection in spouses of haemophiliacs. PMID- 2994805 TI - Enalapril induced renal impairment in bilateral renal artery stenosis. PMID- 2994804 TI - "AIDS" test a misnomer. PMID- 2994807 TI - Immunocytochemical localization of Na+, K+-ATPase in the canine choroid plexus. AB - To determine the localization of Na+, K+-ATPase in the choroid plexus of the canine fourth ventricle, antisera against canine renal Na+, K+-ATPase holoenzyme and the alpha and beta subunits of the enzyme were employed and the distribution of reaction product examined at the light and electron microscopic level by means of the peroxidase-antiperoxidase immunohistochemical method. In the choroidal epithelial cells, antigenic sites were restricted to the outer surface of the apical plasmalemma, including the membranes of the microvilli. No reaction product was detected in the cytoplasm or along the lateral and basal plasmalemma. These findings strengthen the hypothesis that the apical border of the choroidal epithelial cell plays an important role in cerebrospinal fluid secretion as a result of active transport mediated by Na+, K+-ATPase. PMID- 2994809 TI - The pathology of amiodarone neurotoxicity. II. Peripheral neuropathy in man. AB - Sural nerve changes are described in 2 cases of amiodarone neuropathy. Clinically, 1 patient developed a sensorimotor neuropathy, whereas in the other it was predominantly motor. Examination of sural nerves showed demyelination with only mild axonal loss. Cytoplasmic changes developed in Schwann cells of myelinated and unmyelinated axons, and involved loss of most recognizable organelles. These changes were associated with, and possibly preceded, myelin sheath breakdown. Inclusions, mainly of a lamellated type, were found in all cell types in the nerves. These inclusions, known to be lysosomal in origin, are a characteristic finding in amiodarone-induced neuropathy. The pathogenesis of the neuropathy is discussed, with particular reference to the findings in a parallel experimental study. PMID- 2994808 TI - The pathology of amiodarone neurotoxicity. I. Experimental studies with reference to changes in other tissues. AB - Rats and mice were given amiodarone by mouth at doses of 50 mg/kg/day. The drug induced accumulations of lipids within lysosomes, leading to the formation of cytoplasmic bodies. These were found in many tissues, both nervous and nonnervous, but were excluded from regions with blood-brain or blood-nerve barriers. Of nervous system regions lying outside a vascular barrier, autonomic ganglia were the most affected, with large accumulations of lysosomal bodies in nerve cells and processes, and evidence of degenerative changes. the myenteric plexus was also involved. No significant changes were seen in peripheral nerves. A limited period of increased permeability caused by nerve crush, or due to immaturity of the blood-nerve barrier resulted in the short-lived appearance of drug-induced inclusions. The rate of regeneration of axons following nerve crush was not affected, although there were small defects in myelination. PMID- 2994810 TI - Axonal projections of X-cells to the superior colliculus and to the nucleus of the optic tract in cats. AB - Axonal projections of ganglion cells to the superior colliculus (SC) and to the nucleus of the optic tract (NOT) were re-examined physiologically in the cat's retina. Taking care to minimize current spreads around the stimulating electrodes placed in the NOT or the SC, we reached a conclusion that a substantial proportion of X-cells project to the NOT while a small but definite proportion of them project to the SC. PMID- 2994811 TI - Response of rat paraventricular neurones with central projections to suckling, haemorrhage or osmotic stimuli. AB - In lactating, urethane-anaesthetized female rats extracellular recordings were made from paraventricular nucleus (PVN) neurones that were antidromically activated following electrical stimulation of the neurohypophysis, amygdala or nucleus tractus solitarius/vagal complex (NTS/VC). Of the PVN units, 98 projected to the neurohypophysis but none of these neurosecretory neurones were found to simultaneously project to extrahypothalamic areas. From the firing patterns and the response of these neurons to suckling, haemorrhage or osmotic stimuli both 'vasopressinergic' and 'oxytocinergic' neurones were identified. We found 43 PVN units to project to the NTS/VC and 22% of tested neurones were activated by osmotic or haemorrhage stimuli; no phasic activity was associated with this activation. The suckling stimulus failed to elicit any response from these units. Upon testing the PVN units that projected to the amygdala (n = 35), it was found that haemorrhage and suckling stimuli were without effect, while the osmotic stimulus activated one of 6 units tested. Thus, the extrahypothalamic PVN projections examined in this study were not associated with the suckling reflex response, although there is evidence for their limited involvement in neural response to osmotic or haemorrhage stimuli. PMID- 2994812 TI - Tectospinal neurons in the cat have discharges coding gaze position error. AB - Tectospinal neurons (TSNs) in the caudal superior colliculus of the alert behaving cat have a sustained discharge that depends on the magnitude and direction of the vector between a food target and the visual axis. Each TSN has its 'optimal' vector for which it will be activated at a maximum discharge rate independent of whether the animal's head is free or fixed. The discharge is not due to prolonged stimulation of a TSN's receptive field since the discharge persists even when the target is not visible. PMID- 2994813 TI - Metabolic activity in SmI cortical barrels of adult rats is dependent on patterned sensory stimulation of the mystacial vibrissae. AB - Cytochrome oxidase (CO) histochemistry was used to examine the effect of sensory deprivation on metabolic activity in the somatosensory cortex (SmI) of adult rats. Chronic trimming of one or several rows of mystacial vibrissae resulted in a decrease in CO reactivity in the corresponding barrels in layer IV. Reduced CO staining also was observed in cortical laminae superficial and deep to the affected layer IV barrels, suggesting that patterned deflections of the whiskers are important for maintaining the metabolic activity of neurons at least 3 and perhaps 4 synapses removed from the periphery. PMID- 2994814 TI - Pre- and postsynaptic effects of baclofen in the rat hippocampal slice. AB - CA1 pyramidal cells responded to baclofen with a hyperpolarization. This response was found in the apical and basal dendrites and, like the hyperpolarizing response of the dendrites to GABA, appeared to be Ca2+-dependent since it was blocked or reduced by intracellular injection of EGTA or extracellular application of cadmium. Baclofen also reduced the excitatory and inhibitory postsynaptic potentials produced by stimulation of the Schaffer collaterals. The pre- and postsynaptic effects on the synaptic waveform could be distinguished. PMID- 2994815 TI - Involvement of benzodiazepine recognition sites in the foot shock-induced decrease of low affinity GABA receptors in the rat cerebral cortex. AB - The cerebral cortices of rats habituated to the handling manipulation that precedes sacrifice by guillotine (unstressed rats) have a higher number of low affinity GABA receptors than naive rats (stressed rats). Foot shock stress delivered to handling-habituated rats 5 min before sacrifice decreased the number of low affinity GABA receptors to the level found in naive animals, while leaving almost unchanged the [3H]GABA binding in the latter group. Since benzodiazepine (BZ) recognition sites are the target through which benzodiazepines modulate the emotional states of the animals, we investigated whether these receptors were involved in the action of foot shock stress on GABA binding. The in vitro addition of diazepam (5 X 10(-6) M) to cortical membranes from foot-shocked handling-habituated rats brought back the number of low affinity GABA receptors to the level found in cortical membranes from handling habituated rats. Moreover, the effect of foot shock on low affinity GABA receptors was completely antagonized in vivo by pretreatment with the specific benzodiazepine antagonist Ro15-1788 (30 mg/kg per os). Since the effect of foot shock on [3H]GABA binding is mimicked by the in vitro addition of beta-carbolines to cortical membranes from handling habituated rats, our working hypothesis is that an endogenous ligand for BZ recognition sites, possessing beta-carboline-like properties, is released during foot shock stress. PMID- 2994816 TI - Alpha 1-adrenergic receptors and stimulation of [3H]inositol metabolism in rat brain: regional distribution and parallel inactivation. AB - The relationship between norepinephrine-stimulated phosphatidyl-inositol metabolism and alpha 1-adrenergic receptor density was examined in rat brain. Increases in phosphatidyl-inositol metabolism were determined by accumulation of [3H]inositol phosphates in the presence of lithium in brain slices, while receptor density was determined by specific binding of 125I-BE 2254 (125IBE) in membrane fractions. Treatment of slices of cerebral cortex with increasing concentrations of the irreversible alpha 1-adrenergic receptor antagonist phenoxybenzamine caused a parallel inactivation of specific 125IBE binding sites and norepinephrine-induced [3H]inositol phosphate accumulation, although approximately 20% of the binding sites remained after abolition of the inositol response. Comparison of the density of 125IBE binding sites and the magnitude of norepinephrine-stimulated [3H]inositol phosphate accumulation in 8 different brain regions did not show a particularly good correlation. The thalamus had the highest density of binding sites and an intermediate inositol response, while the hippocampus had the highest inositol response but an intermediate density of binding sites. However, the cerebellum had the lowest density of binding sites and no measurable inositol response. Treatment of slices of each region with 300 nM phenoxybenzamine abolished the inositol response and caused a 59-73% decrease in the density of 125IBE binding sites. The lack of correlation between receptor density and inositol response between brain regions could not be explained on the basis of receptor affinity, spare receptors, protein content, nor differences in slice size.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2994817 TI - Horseradish peroxidase evidence for a spinal projection from the preoptic area of the goldfish, a light and electron microscopic study. AB - Horseradish peroxidase (HRP) was applied to the transected spinal cord of goldfish labeled neurons in the preoptic area. Since leakage of HRP into the blood could produce the labeling of neurosecretory cells, intraperitoneal (i.p.) injections of HRP were made with a wide range of dosages in order to intentionally label preoptic neurosecretory cells. The distribution of preoptic neurons labeled after spinal HRP application was far more restricted than the labeling via uptake of HRP from the blood, even when cells in the spinal cord transected fish were intensely labeled. Furthermore, in HRP electron microscopic material, morphological differences were observed between neurons labeled by the two procedures. Large numbers of dense core vesicles and well-developed stacks of rough endoplasmic reticulum, features typical of cells projecting to the pituitary, were not observed in cells labeled via the spinal cord. These findings indicate that in goldfish a direct projection exists from the preoptic area to the spinal cord which could be homologous to one arising from the paraventricular nucleus of mammals. Both i.p. injection and spinal transection also produced labeling of more caudal periventricular diencephalic cells which resemble preoptic cells in efferent projections as well as ultrastructural features. PMID- 2994818 TI - Inhibitory effect of phenytoin on intracellular cyclic nucleotide and calcium changes during pentylenetetrazole-induced bursting activity in snail neurons. AB - Effects of phenytoin (PHT) on the intracellular calcium reservoir, lysosome-like granules (LLG), and calcium-related intracellular events during pentylenetetrazole (PTZ)-induced bursting activity in the neurons of the Japanese land snail, Euhadra peliomphala, were examined. PTZ-induced morphological change of LLG was inhibited by PHT. Calcium release from LLG was inhibited by PHT. PHT also inhibited the cyclic AMP increase by PTZ. PHT inhibited the increase in calcium-dependent protein kinase activity during PTZ-induced bursting activity. These findings suggest that PHT inhibits, as a first step, cyclic AMP increase which is one of the trigger factors of bursting activity as well as subsequent calcium-related intracellular pathological changes during seizure activity. PMID- 2994819 TI - Conditioning hyperpolarization reveals a property of the light-sensitive current in photoreceptors that is modified by cGMP and EGTA. AB - In the dark, an ionic current flows into the outer segments of retinal rods. The absorption of photons by rhodopsin leads to a graded suppression or inactivation of this inward 'light-sensitive' current. The present study shows that following a conditioning hyperpolarization of the rod membrane in the dark, there was a transient increase in total inward current which could no longer be completely suppressed or inactivated by a bright light. The increased inward current following a hyperpolarization had the same reversal potential as the normal inward current but was apparently less sensitive to light. Thus the chain of events between light stimulation and the subsequent membrane current response can be influenced by voltage. Previously, it has been shown that the light-sensitive current is modulated by cGMP and Ca2+, both of which have been proposed as internal messengers that link the absorption of photons to the change in inward membrane current. In the present study, intracellular injection of cGMP or EGTA produced an additional increase in inward current following a conditioning hyperpolarization. On the other hand, these substances antagonized the effect of voltage by permitting complete inactivation of the total inward current by light after a hyperpolarizing step. These results indicate that cyclic nucleotides and calcium can modify the inactivation of an ionic current following a conditioning hyperpolarization. A simple model suggests that these findings are consistent with the notion that this inward conductance in rods has two open states, one sensitive to light and the other insensitive. cGMP or low Ca2+ appears to favor the open state that can be inactivated by light. PMID- 2994820 TI - Specific bindings of prostaglandin D2, E2 and F2 alpha in postmortem human brain. AB - Binding activities specific for each of [3H]prostaglandin (PG) D2, E2 and F2 alpha were detected in the P2 fraction of the human brain homogenates. The bindings were time-dependent, saturable and of high affinity; Kd values were 30 nM for all the PG bindings. Regional distribution of these binding activities was determined by measuring specific bindings with 10 nM [3H]PG-D2, [3H]PG-E2 and [3H]PG-F2 alpha in the P2 fractions from 17 brain regions. The PG-D2 binding activity was high in the hypothalamus, amygdala and hippocampus followed by cerebellar nuclei, thalamus, nucleus accumbens and cerebral cortex. The PG-E2 binding sites were similarly concentrated in the hypothalamus and the limbic system, but, unlike the PG-D2 binding, no significant binding of [3H]PG-E2 was observed in cerebellar nuclei, cerebellar cortex and putamen. Compared with these two PG bindings, PG-F2 alpha binding activity was low in many areas, but significant binding was detected in the amygdala, cingulate cortex, cerebellar medulla, hippocampus, nucleus accumbens, midbrain and hypothalamus. These results suggest the presence and specific distribution of three distinct types of PG binding activities, i.e. specific binding of PG-D2, PG-E2 and PG-F2 alpha, in the human brain. PMID- 2994821 TI - Dibutyryl cyclic adenosine monophosphate stimulates in vitro luteinizing hormone releasing hormone release only from median eminence derived from ovariectomized, estradiol-primed rats. AB - The present study examined the effect of intermittent infusion of dibutyryl cyclic AMP (dbcAMP; 10(-7) M; 10 min on, 20 min off) on in vitro luteinizing hormone-releasing hormone (LH-RH) release from the rat median eminence (ME) derived from immature rats: intact females, intact males, ovariectomized (OVX) females, castrated (CAST) males, ovariectomized, estradiol primed (OVX + E2) females and castrated, estradiol primed (CAST + E2) males. In intact, OVX and CAST conditions, spontaneous LH-RH release from MEs was not modified by dbcAMP infusion. However, E2 implants in OVX and CAST rats selectively affected the responsiveness of MEs to dbcAMP: ME from OVX + E2 were highly responsive to dbcAMP; contrarily, MEs from CAST + E2 were unresponsive to this nucleotide. Therefore, these differences in MEs responsiveness to dbcAMP-induced LH-RH release appear to be dependent upon a critical effect of E2 priming on this tissue in female but not in male rats. PMID- 2994822 TI - Continuous release of diazepam: electrophysiological, biochemical and behavioral consequences. AB - Neuronal GABAergic sensitivity was assessed using electrophysiological, biochemical and behavioral techniques following the continuous release and maintenance of relatively constant brain levels of diazepam for greater than or equal to 21 days. Our studies indicate that long-term exposure to diazepam results in: (1) a decrease in iontophoretic sensitivity to GABA in the dorsal raphe nucleus, (2) an increase in the affinity of the GABA recognition site in brain tissue and (3) an increase in susceptibility to bicuculline-induced seizures in the intact animal. Since the decrease in GABAergic responsiveness was observed in the presence of measurable levels of diazepam, it was concluded that this subsensitivity phenomenon is associated with tolerance and not with withdrawal effects of the benzodiazepines. PMID- 2994823 TI - Spontaneous epileptiform discharges in hippocampal slices induced by 4 aminopyridine. AB - 4-Aminopyridine (4-AP) induced 2 types of spontaneous field potentials (SFPs) in the hippocampal slice. Type I resembled spontaneous activity induced by other convulsants. They occurred at a rate of approximately 1 Hz, started in the CA2/CA3 region and spread at a velocity of 0.3 m/s to area CA1. Transsection experiments and laminar profiles indicated that they spread synaptically along the Schaffer collateral pathway. Synaptic blockade by low Ca2+/high Mg2+ or kynurenic acid reversibly abolished type I SFPs. Increasing [Ca2+]o lowered the rate and slightly increased the amplitude. Possibly, increased spontaneous transmitter release, and not disinhibition, is responsible for the generation of type I SFPs. Type II occurred at a rate of about 0.15 Hz and travelled in the same direction, but a factor 10 slower. They could not be blocked by separation of the CA1 and CA3 region; coupling remained until stratum moleculare was severed. Type II could not be suppressed by blockade of synaptic transmission. The laminar profile is similar in shape to that of type I but not identical. Increasing [Ca2+]o had the same but stronger effect as on type I. Type II SFPs depressed evoked population spikes up to a second and delayed the next type I SFP. The mechanisms involved remain largely speculative; further analysis is needed to help understand the epileptogenic action of 4-AP. PMID- 2994824 TI - Electrophysiological evidence for a role of the anterolateral quadrant of the spinal cord in the transmission of noxious messages to the thalamic ventrobasal complex in the rat. AB - Responses to noxious mechanical and thermal stimulation applied to the hindpaws were recorded extracellularly from the same neurons of the ventrobasal complex of the rat thalamus (VB) before and after lesions of various areas of the cervical cord in order to determine the pathways carrying the afferent messages. It was demonstrated that lesions of the dorsal and dorsolateral portions of the cord failed to eliminate the VB neuronal responses to noxious stimulation. By contrast, lesion of one anterolateral quadrant eliminated the responses to a noxious stimulation applied to the hindpaw contralateral to the lesion. This occurred whether the lesion was ipsilateral or contralateral to the recording site. From the present study and the data in the literature, it is concluded that the fibers of the spino-thalamic tract which are completely crossed in the spinal cord, travel in the anterolateral quadrant and project directly onto the VB, are involved in the transmission of noxious messages from the cord to the VB neurons. This conclusion indicates that the VB neuronal responses to noxious stimulation of the hindpaw ipsilateral to the recording site depend on the spinothalamic projection to the opposite ventrobasal complex. This therefore suggests that some noxious messages which reach a particular VB neuron are conveyed via the opposite VB and the existence of a thalamo-cortico-thalamic loop is discussed. PMID- 2994825 TI - Correlation of [3H]diazepam binding density with anxiolytic locus in the amygdaloid complex of the rat. AB - Using [3H]diazepam binding, high concentrations of receptors were found in the frontal cortex and lateral amygdala. Infusions of chlordiazepoxide into the lateral amygdala induced a release of responding measured during the component of a conditioned emotional response task previously associated with an aversive stimulus. The lateral amygdala appears to be an important component of the forebrain circuitry involved in the expression of anxiety and sensitive to benzodiazepine drugs. PMID- 2994826 TI - Hypertensive response to saline microinjection in the area of the nucleus tractus solitarii of the rat. AB - We investigated the blood pressure response elicited by microinjection of various hypertonic solutions into the area of the nucleus tractus solitarii (NTS) of the brainstem, an area rich in catecholaminergic neurons. Equiosmolar solutions of NaCl, dextrose, LiCl and KCl were employed. NaCl produced a prolonged blood pressure rise; LiCl and normal saline produced a similar rise of short duration; and KCl produced epileptic-type seizures with postictal hypertension. Dextrose had no effect and neither had NaCl microinjection in areas relatively distant from the NTS. The rise in blood pressure was not reversed by a vasopressin antagonist injected systemically, but was totally abolished by systemic alpha adrenergic blockade with phentolamine. These findings suggest that sodium can cause hypertension by direct stimulation of the central sympathetic nervous system without participation of peripheral mechanisms such as fluid volume expansion or alteration of the vascular wall. PMID- 2994827 TI - A selective increase in phosporylation of protein F1, a protein kinase C substrate, directly related to three day growth of long term synaptic enhancement. AB - Increased in vitro phosphorylation of the 47 kdalton, 4.5 pI protein F1 was observed in dorsal hippocampal tissue from animals exhibiting long term enhancement (LTE) three days after high frequency stimulation of the perforant pathway, as compared to tissue from low frequency stimulated controls or from unoperated animals. The increase in protein F1 phosphorylation was related to LTE rather than simple activation of perforant path-dentate gyrus synapses. This is the first report of a change in brain protein phosphorylation accompanying synaptic enhancement lasting days. The extent of growth of LTE over the three days following stimulation was directly related (r = +0.66, P less than 0.05) to protein F1 phosphorylation. Among the phosphoproteins studied this relationship between LTE and phosphorylation was selective for protein F1. This suggests that protein F1 may regulate growth of synaptic plasticity for at least a three day period. The mechanism for the LTE-related increase in protein F1 phosphorylation has not been established. However, recent evidence from this laboratory indicates: that protein F1 is phosphorylated by the calcium/phospholipid dependent protein kinase C; and that kinase C is activated 1 h after LTE. Therefore, the increase in protein F1 phosphorylation following LTE may result from long term activation of protein C kinase. PMID- 2994828 TI - Ultracytochemical localization of ouabain-sensitive K+-dependent, p-nitrophenyl phosphatase in myelin. AB - Ouabain-sensitive, K+-dependent p-nitrophenyl phosphatase (K-NPPase) activity was demonstrated ultracytochemically in the myelin of nerve fibers in peripheral and central white matter. Enzyme activity was more prominent in paranodal than compact myelin, and it was absent from nodal and interparanodal axolemma. Since K NPPase is part of the Na-KATPase complex, we consider myelin as an important site of the sodium pump and believe that myelin participates in cationic regulation of the nervous tissue. PMID- 2994830 TI - Conformational changes of opioid receptor induced by the electric footshock. AB - The present electric shock (ES) schedule followed in these experiments produced different functional changes in endogenous putative opioid agonist- and antagonist-type receptors, depending on the type of receptor: the amount of antagonist binding was increased by ES application, while the amount of agonist binding was decreased. In order to elucidate the mechanism of these changes, we investigated whether ES application was able to affect sulfhydryl-groups and phospholipids of endogenous opioid receptors. In comparison to the control membrane, the increased antagonist binding sites of the ES membrane were liable to be inactivated by the sulfhydryl-modifying reagents, N-ethylmaleimide (NEM) and iodoacetamide. However, in the presence of 100 mM Na, the antagonist binding sites of both the control and ES membranes were inactivated by NEM in the same manner. On the other hand, the agonist binding sites of both membranes were similarly inactivated by NEM regardless of the absence or presence of 100 mM Na. Another sulfhydryl-modifying reagent, such as p-chloromercuriphenylsulfonic acid, did not produce any difference between the control and ES membranes. The increased antagonist binding sites of the ES membrane were also liable to be inactivated by phospholipase A2. These results suggest that the present ES schedule followed in these experiments produces conformational changes in endogenous opioid receptors in the rat brain. As a result of these conformational changes, the amount of binding in the antagonist sites may be increased, while the amount of binding in the agonist sites may be decreased. However, the increased antagonist binding sites may be liable to be inactivated by NEM, iodoacetamide and phospholipase A2. PMID- 2994829 TI - Developmental and regional differences in the localization of Na,K-ATPase activity in the rabbit hippocampus. AB - Regional differences in Na,K-ATPase activity, and development of Na,K-ATPase activity were examined in rabbit hippocampus using a histochemical marker of enzyme activity. Stratum lucidum of CA3/CA2, corresponding to the mossy fiber terminal field, showed high Na,K-ATPase activity compared to stratum radiatum of CA1. A significant increase in Na,K-ATPase activity was found between 8 and 15 days postnatal. Tissues with limited Na,K-ATPase activity (immature hippocampus, the mature CA1 region) appear particularly prone to seizure-like abnormalities, perhaps reflecting an inability to regulate extracellular potassium. PMID- 2994831 TI - Residual benzodiazepine (BZ) binding in the cortex of pcd mutant cerebella and qualitative BZ binding in the deep cerebellar nuclei of control and mutant mice: an autoradiographic study. AB - In mutant mice 'Purkinje cell degeneration' (pcd), there is an almost complete degeneration of Purkinje cells followed subsequently by a partial degeneration of granule cells. Recent neurochemical studies have revealed a 50% decrease in benzodiazepine (BZ) receptors in 45-day-old pcd mutants after degeneration of the Purkinje cells. At 300 days there is an 80% decrease in BZ receptors concomitant with granule cell losses. To determine the histological localization of these receptor changes this autoradiographic analysis was conducted. An in vitro autoradiographic technique was used to explore [3H]flunitrazepam binding. BZ receptors were found to be more concentrated in the molecular than the granular layer of mutant and control cerebellar cortices. There was, nonetheless, no statistically significant difference in grain counts between control and mutant mice in any layer. Substantial atrophy of cerebellar structures, particularly of the molecular layer, occurred in the mutant mice. It began even before 45 days of age but was extreme at 300 days. When the appropriate mathematical correction factor was introduced for the layer atrophy there was a 60% decrease in grain count in 45-day-old mutants in the molecular layer and a 84% decrease in 300-day old mutants compared to controls. The initial decrease in total BZ receptors in the 45-day-old mutant animals is associated with a selective loss of Purkinje cells. The amount of receptor binding which persists in the 300-day-old mutants in the molecular layer would appear to reflect binding in the remaining parallel fibers from granule cells which remain.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2994832 TI - The effect of light on the spread of signals through the rod network of the salamander retina. AB - Adjacent rods in the amphibian retina are electrically coupled to each other by gap junctions. By injecting current pulses into one rod and recording the voltage change produced in nearby rods, we have studied the extent to which signals spread between rods in the presence and absence of illumination. Light has little effect on the steady potentials produced in nearby rods by the injection of a hyperpolarizing current, but does affect the propagation of transient signals through the rod network. The responses to injection of depolarizing current are increased by light. These effects of light were mimicked by hyperpolarizing the rod network (non-uniformly) by injecting continuous current (on top of which current pulses were superimposed to monitor signal spread). This suggests that the effects of light are due solely to the rod hyperpolarization produced by light. The effects of light are not completely predicted from computer simulations based on a previous characterization of the properties of isolated rods; these experiments thus reveal an inadequacy in the description of the rod membrane currents in that model. Light-induced hyperpolarization of cones has no effect on signal spread between rods. The functional significance of these results is discussed. PMID- 2994833 TI - Specific increase of hypothalamic alpha 1-adrenoceptors in spontaneously hypertensive rats: effect of hypotensive drug treatment. AB - To study potential central adrenoceptor alterations in the hypertension, we have determined alpha 1, alpha 2 and beta-adrenoceptors using [3H]WB4101, [3H]yohimbine and [3H]DHA in the brain regions of spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP) and renal hypertensive rats. There was a significant increase in specific [3H]WB4101 binding only in the hypothalamus of SHR and SHRSP at 16-24 weeks of age compared to that of age-matched Wistar-Kyoto rats (WKY). Scatchard analysis revealed a 28-33% increase in the Bmax value for hypothalamic [3H]WB4101 binding without a change in the Kd value, suggesting a change in the receptor density. An increased density of alpha 1-adrenoceptors was consistently observed in the prehypertensive (5 weeks) and developmental (10 weeks) stages of spontaneous hypertension. In contrast, there was no alpha 1 adrenoceptor alteration in the hypothalamus of rats with renal hypertension. The receptor alteration in the SHRSP hypothalamus was not abolished by a chronic hypotensive treatment which prevented the development of hypertension, thereby suggesting that an increased density of the alpha 1-adrenoceptors in spontaneous hypertension does not occur secondarily to the elevation of blood pressure. The SHRSP hypothalamus showed significantly lowered levels of noradrenaline. There was no change in specific binding of [3H]yohimbine and [3H]DHA in the brain regions of SHRSP, except the brainstem which showed a significant decrease in the [3H]yohimbine binding. Thus, the present study suggests an important role for hypothalamic alpha 1-adrenoceptors in the pathogenesis of spontaneous hypertension. PMID- 2994834 TI - Quantitative localization of [3H]TCP binding in rat brain by light microscopy autoradiography. AB - The anatomical localization of phencyclidine (PCP)/sigma-opiate receptors in rat brain was determined by quantitative light microscopy autoradiography using the new ligand N-(1-[2-thienyl]cyclohexyl) [3H]piperidine ([3H]TCP). TCP is a potent analog of PCP which possesses a higher affinity for PCP/sigma-opiate receptor than does PCP itself. The highest level of [3H]TCP binding was detected in the hippocampus. Intermediate levels were found in frontal cortex, striatum, amygdala and cerebellum. Specific [3H]TCP binding was undetectable in anterior commissure and corpus callosum. The distribution pattern of [3H]TCP binding sites is similar to the pattern obtained with [3H]PCP but more sharply defined. On the basis of its greater potency and specificity, [3H]TCP may prove superior to [3H]PCP as a molecular probe for the study of brain sigma opiate/phencyclidine receptors. PMID- 2994835 TI - Regulation of central alpha-adrenoceptors by serotoninergic denervation. AB - Alpha 1- and alpha 2-adrenoceptors were assessed by binding studies using [3H]prazosin and [3H]p-aminoclonidine as ligands in membrane preparations from the cortex, hippocampus and hypothalamus of rats, 3 weeks after intracerebroventricular injection of the neurotoxin 5,7-dihydroxytryptamine. Cortical alpha 1 and hippocampal alpha 2 adrenoceptors were significantly increased. Treatment also affected the affinity of cortical alpha 2 adrenoceptors. These results suggest a heterologous, region-specific regulation of both subtypes of central alpha-adrenergic receptors by serotonin. PMID- 2994837 TI - Retinal GABA neurons: localization in vertebrate species using an antiserum to rabbit brain glutamate decarboxylase. AB - Retinal gamma-aminobutyric acid (GABA) neurons have been localized immunocytochemically using a new antiserum against rabbit brain glutamate decarboxylase (GAD). The animals examined were: dogfish, ratfish, goldfish, catfish, turtle, chick, mouse, rat, pig, rabbit, cat and New World monkey. GAD containing processes, observed as punctate deposits of immunochemical reaction product, formed discrete bands within the inner plexiform layers of all retinas examined. Immunoreactive, and therefore presumably GABAergic, amacrine cells were observed in all species. Displaced GABAergic amacrine cells were observed in the retinas of goldfish, catfish, turtle and chick, and sparsely in the rabbit as well. GABAergic horizontal cells were detected in catfish, goldfish, chick and turtle. Interplexiform cells in the cat and the rat were clearly immunoreactive for glutamate decarboxylase; this is the first report of GABAergic interplexiform cells in the rat. PMID- 2994838 TI - A differential effect of naloxone on transmission of impulses in primary afferents to ventral roots and ascending spinal tracts. AB - Ventral root reflexes and ascending volleys to stimulation of group I muscle afferents, large diameter cutaneous afferents and unmyelinated primary afferents were examined in barbiturate anaesthetized spinal cats. Intravenous naloxone (0.05-0.10 mg/kg) increased reflexes to stimulation of all primary afferent types but of the ascending volleys, only those to stimulation of unmyelinated primary afferents were increased. Thus it appears that opioid peptides have differential effects on transmission of primary afferent impulses to supraspinal areas, an action possibly relevant to analgesia, in contrast to a non-selective suppression of transmission to motoneurones. PMID- 2994836 TI - The effects of phenoxybenzamine on specific binding and function of central alpha adrenoceptors in the rabbit. AB - We have studied the effects of phenoxybenzamine, an irreversible alpha adrenoceptor antagonist on the binding of the alpha-adrenoceptor ligands [3H]prazosin and [3H]clonidine to rabbit brain membranes. Where possible changes in binding were related to changes in central alpha-adrenoceptor function. Phenoxybenzamine showed a similar alpha 1/alpha 2-adrenoceptor selectivity in the brain to that previously reported in the periphery. Much higher doses were required to reduce specific clonidine binding and to interfere with the hypotensive response to intracisternal clonidine than to reduce specific prazosin binding. Recovery of binding site number of both alpha 1- and alpha 2 adrenoceptor selective ligands was slower than in peripheral tissues (heart and spleen). Recovery was log linear and the half time (t1/2) for recovery of the maximum number of specific prazosin and clonidine binding sites in forebrain was 10.8 +/- 2.6 days and 6.1 +/- 0.1 days and in hindbrain 13.3 +/- 3.1 days and 4.6 +/- 1.8 days, respectively. t1/2 for recovery of the in vivo hypotensive response to intracisternal clonidine was 2.7 +/- 1.0 days. Recovery of this response was attenuated by treatment with the inhibitor of protein synthesis, 5-fluorouracil. This suggests that recovery after phenoxybenzamine in brain, as in the periphery, may depend at least in part on synthesis of new receptor protein. The recovery of brain adrenoceptor number after phenoxybenzamine may be an index of receptor turnover and is much slower in brain than in heart and spleen. PMID- 2994839 TI - Selective increase of R-I subunit of cyclic AMP-dependent protein kinase in glia rich primary cultures upon treatment with dibutyryl cyclic AMP. AB - Levels of cyclic GMP-dependent protein kinase and of the subunits (R-I, R-II and C) of cyclic AMP-dependent protein kinase were determined in two types of neural primary cell cultures that were either treated or not treated with dibutyryl cyclic AMP. Astroglia-rich cell cultures from newborn rat brain responded to exposure to dibutyryl cyclic AMP by a 2-3-fold increase in the level of R-I subunit, as demonstrated by two radioimmunological procedures, while the levels of the other subunits (R-II and C) and of cyclic GMP-dependent protein kinase remained unaffected. In contrast, neuron-rich cell cultures from embryonic rat brain did not display such a change in the level of R-I subunit. Thus, the elevation in the level of R-I elicited by dibutyryl cyclic AMP in normal non malignant neural cells in culture was restricted to glial rather than neuronal cells. PMID- 2994840 TI - Effects of 4-aminopyridine, GABA and bicuculline on cutaneous receptive fields of cat dorsal horn neurons. AB - 4-Aminopyridine (4-AP), bicuculline and GABA were applied locally to dorsal horn cells in the lumbar spinal cord in Nembutal-anaesthetized cats and in spinal cats. 4-AP expanded the cutaneous receptive field of 29 of 33 cells tested. Bicuculline and GABA had little or no effect on receptive field size. PMID- 2994841 TI - Effects of norepinephrine on rat neocortical neurons in dissociated cell culture. AB - Intracellular recordings were made from neurons in dissociated cell culture of neocortex during application of norepinephrine (NE) or other adrenergic agonists. In the population of neurons generally studied, greater than 18 micron in diameter, adrenergic agonists from 1 nM to 50 microM produced no change in membrane potential or input resistance 120 cells). Adrenergic agonists increased synaptic activity impinging on the impaled cell in 25/120 neurons (21%). In neurons in cocultures of locus coeruleus and cerebral cortex, again the same synaptic response to perfusion with NE was noted in 13/93 neurons (14%). In addition, direct effects of NE were noted on 6/93 neurons recorded from in cocultures, all close to the explant. In these cells, NE hyperpolarized the membrane in association with a small decrease in input resistance (11%). These responsive cells may have originated within the explant. A paradigm was used for testing the possibility of a responsive element in the cultures distinct from the impaled soma. 'Hot spots' were found using concentrations of isoproterenol as low as 10 nM. PMID- 2994842 TI - Localization of mu- and delta-opioid receptors in cat respiratory areas: an autoradiographic study. AB - Autoradiography after in vitro binding with selective ligands for either mu ([3H](Tyr-D-Ala-Gly-(NMePhe)-Gly-ol] or delta ([3H](Tyr-D-Thr-Gly-Phe-Leu-Thr)) opioid receptor types revealed the presence of variable amounts of radioactive labeling in the cat brainstem. Areas involved in the respiratory rhythmogenesis were among the most prominently labeled structures. The pneumotaxic center, including the nucleus parabrachialis medialis and the Kolliker-Fuse nucleus, contains a very high density of delta binding sites while the dorsal respiratory nucleus which corresponds to the nucleus tractus solitarius, is more heavily labeled by the mu ligand. The neuroanatomical differences in the distribution of opioid receptors correlates well with the pharmacological responses induced by administration of specific mu- or delta ligands. PMID- 2994843 TI - Opposing effects of oxytocin and of a mu-receptor agonistic opioid peptide on the same class of non-pyramidal neurones in rat hippocampus. AB - A study was made of the effects of opioid peptides on the spontaneous firing of oxytocin-responsive non-pyramidal neurones in hippocampal slices. D-Ala2-Gly-ol5 enkephalin (DAGO), a mu-opiate agonist, decreased or even suppressed the firing of these neurones, an effect reversed by naloxone. In contrast, U-50,488, a kappa opiate agonist, had no effect. When the slices were synaptically uncoupled by elevating the concentration of external magnesium, oxytocin still excited non pyramidal neurones and DAGO still inhibited them. Thus, opiates and oxytocin exerted direct, opposite effects on the same population of neurones, which apparently bear mu-type receptors. An indirect action of opioids on the excitability of pyramidal cells was apparent and is probably mediated by the same interneurones, since the amplitude of the depolarizing component of the synaptic potential elicited by stimulation of Schaffer's collaterals was increased in the presence of DAGO. PMID- 2994845 TI - The effects of temperature on synaptic transmission in hippocampal tissue slices. AB - Fully submerged rat hippocampal tissue slices were exposed to temperature changes, and the effects on CA1 pyramidal cell electrophysiology studied. Raising the temperature from 29 to 33 or 37 degrees C simultaneously increased the focal excitatory postsynaptic potentials and decreased the population spikes. These changes were largely reversible for slices warmed to 33 degrees C, but not for slices warmed to 37 degrees C. During warming transiently increased excitatory transmission was observed; the degree of increased transmission was related to the rate of temperature rise. It is postulated that neuronal membrane hyperpolarization with warming is responsible for several of the effects seen. PMID- 2994844 TI - Pharmacological studies on stress-induced renin and prolactin secretion: effects of benzodiazepines, naloxone, propranolol and diisopropyl fluorophosphate. AB - Stress-induced renin and prolactin secretion was investigated using a conditioned emotional response paradigm. Three minutes after placement in a chamber the rats received an electric shock to their feet via the grid floor, then were immediately returned to their home cage. This procedure was repeated for 3 consecutive days. On the fourth day, instead of receiving an electric shock, they were removed after 3 min and sacrificed by decapitation. Control rats were treated identically with the exception that shock was not administered at any time. There was a significant increase in plasma renin activity and prolactin level in the stressed rats. The administration of the antianxiety drugs chlordiazepoxide (10 mg/kg i.p.) or midazolam (0.125-2 mg/kg i.p.) blocked the stress-induced increase in prolactin levels but not the stress-induced rise in plasma renin activity. Administration of the beta-blocker propranolol (1 mg/kg i.p.) inhibited, but did not completely block, stress-induced rise in plasma renin activity and had no effect on stress-induced prolactin secretion. The opiate antagonist naloxone (0.1-10 mg/kg i.p.) and the acetylcholinesterase inhibitor diisopropyl fluorophosphate (0.5 mg/kg i.p.) did not block stress induced renin or prolactin secretion. It is concluded that stress-induced prolactin secretion is regulated by a benzodiazepine-mediated mechanism and that stress-induced renin but not prolactin secretion is mediated in part via beta receptors. PMID- 2994846 TI - Receptor-mediated inositide hydrolysis is a neuronal response: comparison of primary neuronal and glial cultures. AB - Cholinergic and adrenergic receptor-stimulated inositide hydrolysis was studied in neuronal and glial cells cultured from brains of 1-day-old Wistar-Kyoto rats. Incubation of the cells with [3H]inositol led to the incorporation of radioactivity specifically into inositol phospholipids. Labeling of the membrane lipids reached a maximum in 2-3 days. Receptor-stimulated breakdown of inositides was determined by following the accumulation of inositol phosphates after incubation of the labeled cells for 60 min with carbachol or norepinephrine in the presence of 10 mM lithium. Carbachol (1 mM) stimulated inositol phosphate production in neurons 30 times higher than that seen in glia. The response stimulated by norepinephrine (75 microM) was 6 times higher in neurons than glia. The response to carbachol was blocked by atropine, and the norepinephrine-induced response was inhibited by prazosin suggesting that the receptors mediating the responses were muscarinic and alpha 1-adrenergic, respectively. These results suggest that muscarinic cholinergic and alpha 1-adrenergic stimulated inositide hydrolysis is primarily a neuronal response and that this biochemical event may be important for transmembrane signaling which occurs during neurotransmission. PMID- 2994847 TI - Monoclonal antibodies raised against Lewy bodies in brains from subjects with Parkinson's disease. AB - Monoclonal antibodies which immunocytochemically label Lewy bodies on sections of substantia nigra from subjects with Parkinson's disease were produced by immunization of mice with substantia nigra and locus coeruleus containing Lewy bodies from parkinsonian subjects post-mortem. Tests of specificity indicate that the antibodies do not recognize the same antigen. One of the antibodies (G7) immunocytochemically labels only Lewy bodies, the other (G9) also faintly labels the cell bodies of nigral dopaminergic neurons and cerebellar Purkinje cells in both normal and parkinsonian brains. Absorption experiments show, however, that the G7 antigen is present in normal substantia nigra and the G9 antigen in normal substantia nigra and Purkinje cells. Neither of the antibodies seems to be directed against neurofilament protein. Immunoblots after two-directional electrophoresis indicate that antibody G7 labels a protein with an iso-electric point around 5.6 and a mol. wt. of approximately 40 kdalton, whereas the protein labeled by antibody G9 has an iso-electric point of near 8 and a mol. wt. above 70 kdalton. PMID- 2994848 TI - Autoradiographic visualization of adenosine A1 receptors in the gerbil hippocampus: changes in the receptor density after transient ischemia. AB - N6-cyclohexyl-[3H]adenosine [( 3H]CHA) was used for the in vitro visualization of the hippocampal adenosine A1 receptors in the gerbil. The strata radiatum and oriens of the hippocampus showed particularly high binding activity. Depletion of pyramidal cells and consequent severe decrease in [3H]CHA binding activity in the CA1 subfield were observed after transient ischemic insult. These results suggest that most adenosine receptors in the CA1 region are localized in association with pyramidal cells. PMID- 2994849 TI - Primary cultures from defined brain areas; effects of seeding time on the development of beta-adrenergic- and dopamine-stimulated cAMP-activity during cultivation. AB - The influence of seeding time on the expression of beta-adrenergic- and dopamine stimulated cAMP activity was studied in primary astroglial cultures from various brain areas. In all cultures isoproterenol (10(-7)-10(-5) M) caused an increased cAMP accumulation with time in culture. In brainstem cultures from 17-day-old embryos there was a decreased intracellular cAMP accumulation between 3 and 4 weeks. In all newborn rats and in striatal cultures from 7-day-postnatal rats there was a similar decrease between 2 and 3 weeks. These changes are discussed and interpreted as a differentiation of the cells concerning beta-receptors. The most prominent cAMP accumulation caused by isoproterenol was found in striatal cultures from newborn rat. Although isoproterenol and noradrenaline both caused intracellular cAMP accumulation, isoproterenol was the most effective. There was a cAMP accumulation specific to dopamine (10(-6)-10(-4)M) in 2-week-old striatal cultures from newborn rat, although there was an interaction with beta-adrenergic receptors. In the other cultures studied there was only a very small dopamine stimulation of cAMP. The expression of this heterogeneity among astroglial primary cultures from various brain areas seems to be influenced by the genetic program for cellular maturation and by specific cellular contacts in vivo until seeding and in culture. PMID- 2994850 TI - Effect of retinoic acid on growth and morphological differentiation of mouse NB2a neuroblastoma cells in culture. AB - We have characterized the effects of retinoic acid (RA) on the growth, morphology and biosynthesis of cytoskeletal proteins in NB2a mouse neuroblastoma cells. In addition, the morphological and biochemical changes were compared to those induced by dibutyryl cyclic AMP (db cAMP). Growth inhibition by RA was concentration-dependent and was first detected 24 h after addition of RA. The proliferation of RA-treated NB2a was more dependent on serum than was the proliferation of untreated cultures and RA decreased the saturation density of NB2a cells grown in serum. Morphological changes induced by RA include the formation of an elaborate network of branching neurites in NB2a cells. In contrast, neurites induced by db cAMP or serum deprivation were bipolar and unbranching. Ultrastructural observations of neurites induced by RA revealed dendritic characteristics such as polysomes, spines and absence of intermediate filaments, while neurites induced by db cAMP had axonal characteristics such as filament bundles, absence of ribosomes, and the formation of membrane densities when neurite endings contacted another cell body. These morphological differences were also reflected in a number of changes in the biosynthesis of cytoskeletal proteins. These results suggest that NB2a cells treated with RA and db cAMP are a model system for the study of distinct stages of differentiation. PMID- 2994851 TI - Development of the A1 adenosine receptors in the visual cortex of cats, dark reared and normally reared. AB - The ontogeny of the distribution of the binding sites for [3H]chlorohydroxyladenosine, an A1 adenosine receptor-specific ligand, was visualized autoradiographically within coronal sections of the visual cortical areas of developing cats. In adults, the A1 adenosine receptors were found in all lamina except for lamina IV, and in particularly high concentration within laminas I-III. In brains of kittens 2 months old and younger who were within the critical period for the development of visual neural function, the receptor distribution was less defined and sparser, except that in contrast to adults, it was found in relatively high concentration within lamina VI. Animals dark-reared from birth, so that the critical period was postponed, exhibited an ontogenetic pattern identical to that of the normally reared animals. These results indicate that, at least with respect to ocular dominance determination, A1 adenosine receptors are probably not involved in determining the state of plasticity that is seen during the critical period. PMID- 2994852 TI - The possible modulation of the development of rat dorsal root ganglion cells by the presence of 5-HT-containing neurones of the brainstem in dissociated cell culture. AB - Reliable methods for coculturing dissociated rat brainstem cells together with dorsal root ganglion (DRG) cells have been established. The cells were characterized using autoradiographic, morphological, immunocytochemical and electrophysiological techniques. Light-level microscopy showed the cocultures to be extensively invaded by serotonin (5-HT)-containing neuronal processes and intense clustering of 5-HT-containing varicosities to occur in the vicinity of large round phase-bright neurones thought to be DRGs. Rather extensive fine ramification of the neuronal processes throughout the culture dish were visualized using scanning electron microscopy. Intracellular recording showed the brainstem neurones to be spontaneously active, electrically excitable and sensitive to a variety of transmitter candidates including serotonin (5-HT) and gamma-aminobutyric acid. Several different responses to 5-HT have been observed. These include a depolarization accompanied by an increase in membrane resistance, a depolarization accompanied by a decrease in membrane resistance and a hyperpolarization accompanied by an increase in membrane resistance. As established by others, DRG cells grown in isolation were always quiescent. The application of 5-HT produced no effect on membrane potential, resistance or excitability. In the brainstem-DRG cocultures 52% of DRG cells exhibited synaptic activity and occasional spontaneous spiking, both of which were abolished in the presence of tetrodotoxin or low-Ca2+/high-Mg2+ solution. Transmission electron microscopy confirmed the presence of synapses on the DRG cells. The spontaneously active DRG cells were also found to be sensitive to the application of 5-HT. Thus it appears that a source of 5-HT nerve terminals may regulate the development and pharmacological sensitivity of primary afferent neurones in culture. PMID- 2994853 TI - Modification of neurotransmitter receptor sensitivity in cat visual cortex during the critical period. AB - We have examined the characteristics of various receptors in cat visual cortex during postnatal development. These included beta-adrenergic, GABA, benzodiazepine and acetylcholine receptors. For each population of receptor the number (Bmax) and affinity (Kd) were examined as a function of postnatal age (3 days-adult). For all receptors examined, the Bmax increased during development from low early values to a peak within the critical period. The Kd also changed during development for most receptors. The simultaneous alterations in Bmax and Kd necessitate defining a term which takes both of these receptor properties into consideration. This term, called receptor sensitivity (RS), provides a more comprehensive measure of receptor function than either Bmax or Kd alone. Using this measure, we find that receptor sensitivity is low near birth for the 4 receptor populations studied, rises to a peak within the first two months of life, and then declines to near-neonatal levels for 3 of the 4 receptor populations. PMID- 2994854 TI - Perinatal glucocorticoids alter dentate gyrus electrophysiology. AB - Perinatal glucocorticoid administration produces permanent spatial discrimination learning deficits in rats, presumably referable to changes in the development of neural systems subserving such functions. Because the hippocampal dentate gyrus and its afferent/efferent circuitry appear selectively vulnerable to neonatal steroid treatments, we have examined adult rats treated with neonatally administered glucocorticoids using electrophysiological methods. The techniques were chosen to reveal the topographic and neurophysiologic responsiveness of the major afferent supply to the dentate gyrus. Rats of both sexes received either a high dose (100 mg/kg) or low dose (1 mg/kg) of the synthetic glucocorticoid dexamethasone on postnatal day four, with control subjects receiving an injection of saline. These dosages have been shown to disrupt hippocampal dependent learning [6,38]. Laminar depth profile analyses of entorhinal cortex-dentate gyrus afferents revealed a significant shift in the spatial distribution of evoked extracellular population synaptic potentials (EPSPs) in glucocorticoid treated subjects. Stimulus-response functions also differed between glucocorticoid treated and control subjects. While response amplitudes at threshold stimulus intensities did not differ between groups, at higher stimulus intensities population spike potentials and associated EPSPs differed in glucocorticoid versus control subjects. PMID- 2994855 TI - [Detection of rotavirus by polyacrylamide gel electrophoresis]. PMID- 2994856 TI - [Placental trophoblastic tumor of the uterus: 5 cases]. PMID- 2994858 TI - [7 cases of extramammary Paget's disease]. PMID- 2994857 TI - [Localization of HBsAg and HBcAg by peroxidase-labeled antibody]. PMID- 2994860 TI - Concerning the location of the GTP hydrolysis site on microtubules. AB - The kinetics for GTP hydrolysis associated with microtubule assembly with microtubular protein has been analyzed under reaction conditions where tubulin GDP does not readily assemble into microtubules. The GTPase rate is only slightly faster during the time when net microtubule assembly occurs, as compared with steady state. The slightly slower steady-state GTPase rate apparently results from GDP product inhibition, since the progressive decrease in the rate can be quantitatively accounted for using the previously determined GTP dissociation constant and the Ki value for GDP. Since the GTPase rate is not a function of the rate for net microtubule assembly, it is concluded that GTP hydrolysis is not required for tubulin subunit incorporation into microtubules. The constancy of the rate indicates that the GTPase reaction occurs at a site, the concentration of which does not change during the assembly process. This result is consistent with a reaction scheme in which GTP hydrolysis occurs primarily at microtubule ends. We propose that hydrolysis occurs at microtubule ends, at the interface between a long core of tubulin-GDP subunits and a short cap of tubulin-GTP subunits. PMID- 2994859 TI - Comparison of renal responses to synthetic human PTH(1-34) administration in normal young and elderly male subjects. AB - A parathyroid hormone (PTH) loading test with synthetic human PTH(1-34) was performed in 7 young and 6 elderly normal males. The elderly subjects had significantly higher mean basal levels of serum PTH than the young subjects (0.262 +/- 0.035(SE) vs 0.097 +/- 0.012 ng Eq/ml, P less than 0.001). When human PTH(1-34) at a dose of 100 U was administered to these subjects, the mean increases in urinary excretions of adenosine cyclic 3',5'-monophosphate(cAMP) and inorganic phosphorus (Pi), expressed as increases in absolute amounts per unit time, were significantly lower in the elderly subjects. (3.65 +/- 1.02 vs 7.41 +/ 1.05 mumol/h, P less than 0.05 for cAMP and 14.7 +/- 6.3 vs 41.8 +/- 8.6 mg/2h, P less than 0.05 for Pi) and an inverse correlation was found between the serum PTH levels and the increases in urinary cAMP excretion (mumol/hr; r = -0.63, P less than 0.05). However, when corrected for the glomerular filtration rate (GFR) and the units of PTH administered per kg body weight, the increases were not significantly different in the two groups (elderly 52.1 +/- 7.5 vs young 60.0 +/- 19.2 nmol . kg/100 ml GFR . U . h for cAMP and 0.315 +/- 0.061 vs 0.186 +/- 0.044 mg . kg/100 ml GFR . U . 2 h respectively for Pi).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2994862 TI - Tc1(Hin): a form of the transposable element Tc1 in Caenorhabditis elegans. AB - In this paper we describe the coexistence of two forms of the transposable element Tc1 in the genome of the nematode Caenorhabditis elegans. A copy of the variant form has been isolated from the Bergerac genome and characterized. Restriction mapping and DNA sequencing have shown that a G to T transversion generated a HindIII restriction site to form the variant Tc1(Hin). The presence of this new restriction site makes this variant easily detectable on genomic blot hybridizations. There are approximately 20 copies of Tc1(Hin) amongst the Tc1's present in the Bergerac genome. Bergerac has approximately 250 copies of Tc1 per genome, whereas Bristol has about 30. In the Bristol strain we detected at least one copy Tc1(Hin). The ratio of Tc1(Hin) to total Tc1's is similar in the genomes of Bristol and Bergerac, even though they have markedly different total numbers of Tc1. Our results suggest that a trans-acting change in either the elements or the host genome was responsible for the expansion of Tc1 copy number in the Bergerac genome. PMID- 2994861 TI - Chromaffin cell cytoskeleton: its possible role in secretion. AB - Cytoskeleton proteins (actin, myosin, alpha-actinin, spectrin, tubulin, neurofilament subunits) and their regulatory proteins (calmodulin, gelsolin) have been isolated from adrenal chromaffin cells and characterized. Their physicochemical properties have been studied and their cell localizations have been revealed by biochemical, immunocytochemical, and ultrastructural techniques. alpha-Actinin and spectrin are components of chromaffin granule membranes and some of the cell actin copurifies with these secretory granules. Myosin is not detected in the granules, but is present mainly in the cytosol and close to the cell surface. Trifluoperazine, a calmodulin antagonists, blocks stimulation induced hormone release from chromaffin cells at a step distal from Ca2+ entry. High affinity calmodulin-binding sites have also been found in chromaffin granule membranes and their calmodulin-binding proteins have been characterized. Furthermore, microinjection of calmodulin antibodies into chromaffin cells blocks hormone output in response to stimulation. In view of the above findings, the possible roles of contractile proteins and calmodulin in chromaffin cell functions are discussed. PMID- 2994863 TI - The presence of a heat-stable cytosolic factor in rat submandibular gland which activates Na+,K+-ATPase. AB - A low molecular weight substance is present in the soluble fraction of the rat submandibular gland which activates Na+,K+-ATPase. Varying amounts of cytosolic protein from rat submandibular gland were added to a heavy microsomal preparation of the same tissue. A dose-dependent activation of the Na+,K+-ATPase was observed, with a peak activation of approximately 82% occurring when 9.0 micrograms/mL of cytosolic protein was included in the assay. The activating factor is heat stable and soluble following heat treatment. The factor elutes at a molecular mass of 600 daltons as determined by molecular sieve column chromatography. Ultraviolet-visible scanning of the elute material results in an absorbance at 210 nm, which is characteristic of a low molecular weight peptide. PMID- 2994864 TI - The effect of dietary fibre on feed intake and growth in beagle puppies. AB - We studied the growth, feed intake, feed efficiency and protein efficiency ratio of groups of female Beagle puppies fed 16% or 22% crude protein rations to which 6% or 12% wheat bean was added at the expense of the total diet. The final neutral detergent fibre concentrations were 12%, 16%, 22% and 23% (dry matter basis). The addition of wheat bran to puppy rations, bringing the neutral detergent fibre up to 16% in a 21% crude protein diet had no deleterious effects on feed intake, feed and protein efficiency or growth in Beagle puppies. Over a sufficiently long period of time, the growth of this group did not differ from that of the controls (12% neutral detergent fibre, 23% crude protein) although it was higher at intermediate times. The effects of the high fibre (22 or 23% neutral detergent fibre) diets on growth, feed intake feed efficiency and protein efficiency ratio are consistent with an energy deficit resulting from the animals' inability to adapt fully to the dilution of their rations leading to lower growth, less efficient use of feed and, in the case of group 3 (22% crude protein, 22% neutral detergent fibre), a lower protein efficiency ratio. The protein efficiency ratio of group 4 (16% crude protein, 23% neutral detergent fibre) was higher than that of group 3, most likely the result of a more limiting amount of dietary protein leading to a more efficient use for growth by the animal. We have concluded that intermediate levels of neutral detergent fibre (up to 16%) were not deleterious even in puppy rations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2994865 TI - Experimental inoculation of cats with human coronavirus 229E and subsequent challenge with feline infectious peritonitis virus. AB - Minimal-disease cats exposed to live human coronavirus 229E developed homologous antibody responses that suggested little or no replication of the virus in inoculated animals. Oronasal and subcutaneous inoculation of coronavirus 229E did not elicit an antibody response by heterologous (transmissible gastroenteritis virus, canine coronavirus) neutralization or by heterologous (transmissible gastroenteritis virus) kinetics-based enzyme-linked immunosorbent assay. No clinical signs attributable to coronavirus 229E were seen in inoculated cats. Although the number of animals in each of the five experimental groups was small (n = 2), antibodies produced in response to the virus did not appear to sensitize cats to subsequent feline infectious peritonitis virus challenge, but neither did they cross-protect cats against the challenge dose. PMID- 2994866 TI - Natural transmission of bovine leukemia virus in dairy calves by dehorning. AB - Gouge dehorning was evaluated as a mode of transmitting bovine leukemia virus in Holstein calves at a commercial dairy. Significantly (p less than 0.05) more calves dehorned by the gouge method developed antibodies to bovine leukemia virus, as measured by agar-gel immunodiffusion, three months after dehorning, than calves not dehorned. The field use of a blood-contaminated dehorning device resulted in transmission of bovine leukemia virus. PMID- 2994868 TI - Interaction of 2',3'-di-O-nitro-5'-(N-ethylcarboxamido)adenosine with adenosine receptors in intestinal smooth muscle. AB - The adenosine derivative, 2'3'-di-O-nitro-(5'-N-ethylcarboxamido)adenosine (DINECA), caused relaxation in several isolated smooth muscle preparations including guinea pig taenia caeci, beef coronary arteries, and rabbit small intestine. In rabbit small intestine the response profile of DINECA action differed from that of established adenosine receptor agonists and, in contrast with the latter, its relaxant effect was only partially reversed by the antagonist 8-p-sulfophenyltheophylline. Concentration-response curves to 5'-(N ethylcarboxamido)adenosine (NECA), but not those to DINECA, were significantly shifted to the right by 100 microM of 8-sulfophenyltheophylline. Tissues exposed previously to DINECA became refractory to adenosine, an effect not observed with tissues exposed to NECA, suggesting that DINECA became bound to adenosine receptors. Adenylate cyclase from neuroblastoma cells, containing Ra-type adenosine receptors, was stimulated by 2-chloroadenosine and NECA but not by DINECA. The results suggest that most of the smooth muscle relaxant actions of DINECA are not due to interaction with adenosine receptors but are probably due to its function as a nitrate. However, DINECA appears to interact with adenosine receptors, causing long lasting inhibition of adenosine action in rabbit intestine. Such actions may contribute to the overall response to DINECA application in vivo, although lowering of blood pressure due to the high reactivity of the vasculature to nitrates may be the initial and major effect. PMID- 2994867 TI - The effect of acid-base balance on fatigue of skeletal muscle. AB - H+ ions are generated rapidly when muscles are maximally activated. This results in an intracellular proton load. Typical proton loads in active muscles reach a level of 20-25 mumol X g-1, resulting in a fall in intracellular pH of 0.3-0.5 units in mammalian muscle and 0.6-0.8 units in frog muscle. In isolated frog muscles stimulated to fatigue a proton load of this magnitude is developed, and at the same time maximum isometric force is suppressed by 70-80%. Proton loss is slowed when external pH is kept low. This is paralleled by a slow recovery of contractile tension and seems to support the idea that suppression results from intracellular acidosis. Nonfatigued muscles subjected to similar intracellular proton loads by high CO2 levels show a suppression of maximal tension by only about 30%. This indicates that only a part of the suppression during fatigue is normally due to the direct effect of intracellular acidosis. Further evidence for a component of fatigue that is not due to intracellular acidosis is provided by the fact that some muscle preparations (rat diaphragm) can be fatigued with very little lactate accumulation and very low proton loads. Even under these conditions, a low external pH (6.2) can slow recovery of tension development 10 fold compared with normal pH (7.4). We must conclude that there are at least two components to fatigue. One, due to a direct effect of intracellular acidosis, acting directly on the myofibrils, accounts for a part of the suppression of contractile force. A second, which in many cases may be the major component, is not dependent on intracellular acidosis. This component seems to be due to a change of state in one or more of the steps of the excitation-contraction coupling process. Reversal of this state is sensitive to external pH which suggests that this component is accessible from the outside of the cell. PMID- 2994870 TI - Association of the positive inotropic action of ouabain with a second species of digitalis receptors. AB - Studies were conducted to determine whether Na-K ATPase or a second species of digitalis receptors in canine cardiac sarcolemma membrane preparations is associated with the positive inotropic action of nontoxic concentrations of ouabain. [3H]ouabain association and dissociation experiments using highly enriched sarcolemma preparations from canine ventricle indicate the presence of two species of ouabain binding receptors. Ouabain binding to Na-K ATPase of the sarcolemma preparation requires supporting ligands and is characterized by fast association and very slow dissociation in vitro. The second species of digitalis receptor does not require supporting ligands for ouabain binding and is characterized by slow association and fast dissociation. To determine which species of digitalis receptor is associated with the positive inotropic action of digitalis, ouabain washout experiments were conducted using various isolated canine myocardial preparations. Washout of the positive inotropic effects of 1.2 2.4 X 10(-7) M ouabain gave half-life values of 1.5-2.0 h for the various myocardial preparations. [3H]ouabain dissociation from the second species of digitalis receptors gave half-life values of 1.7-1.8 h, whereas dissociation from the sarcolemma Na-K ATPase gave half-life values of 8.9-9.3 h for the various sarcolemma preparations utilized. Therefore, based on similarities in half-life values between ouabain inotropy and [3H]ouabain dissociation from the second class of digitalis receptors, it is postulated that the positive inotropic action of digitalis glycosides is associated with the second species of digitalis receptors in the sarcolemma and not with the digitalis inhibitory receptor of Na K ATPase for nontoxic concentrations of digitalis. PMID- 2994869 TI - Ca2+ channel antagonist actions in bladder smooth muscle: comparative pharmacologic and [3H]nitrendipine binding studies. AB - The actions of a series of 15 Ca2+ channel antagonists including D-600, nifedipine, and diltiazem were examined against K+ depolarization and muscarinic receptor induced responses in guinea pig bladder smooth muscle. Responses of bladder are very dependent upon extracellular Ca2+ and sensitive to the Ca2+ channel antagonists, the tonic component more than the phasic component of response. Regardless of stimulant, K+ or methylfurmethide (MF), or component of response, the same rank order of antagonist activities is expressed, suggestive of a single structure-activity relationship and the existence of a single category of binding site which may, however, exist in several affinity states. High affinity binding of [3H]nitrendipine (KD = 1.1 X 10(-10) M) occurs in bladder membranes, and similar high affinity binding was found in microsomal preparations from other smooth muscles including guinea pig and rat lung, rat vas deferens, uterus, and stomach. [3H]nitrendipine binding in the bladder was sensitive to displacement by other 1,4-dihydropyridines, paralleling their pharmacologic activities and showing excellent agreement with binding data previously obtained for guinea pig ileal smooth muscle. Comparison of pharmacologic data for inhibition of K+- and MF-induced responses by a common series of Ca2+ channel antagonists in bladder and ileum revealed excellent correlations. Neither pharmacologic nor binding studies suggest significant differences in Ca2+ channel antagonist properties in smooth muscle from bladder and intestine. PMID- 2994871 TI - Role of alpha-adrenergic receptors in the intrinsic inotropic selectivity of dobutamine in anesthetized dogs. AB - The inotropic selectivity of dobutamine was examined in pentobarbital anesthetized, vagotomized dogs pretreated with a ganglion blocker. The purpose was to determine if, in the presence of hexamethonium and vagotomy, the inotropic selectivity of dobutamine could be attributed to an action of dobutamine on alpha adrenoreceptors. Dose-response curves were determined for either isoproterenol or dobutamine 30 min after treatment with hexamethonium (20mg/kg). Analysis of heart rate versus right ventricular contractile force showed that dobutamine produced less tachycardia for a given increase in contractile force than isoproterenol; this was statistically significant when contractile force was increased by either 50 or 100%. In a separate series of experiments, dobutamine (8 micrograms . kg(1 ) . min(-1)) was administered 20 min after propranolol (3 mg/kg). Under these conditions there was a slight increase in contractile force which represented 12% of the dobutamine response prior to propranolol administration. This increase in contractile force in the presence of propranolol was completely prevented by the addition of phentolamine (1 mg/kg). Consequently, in another series of experiments, dose-response curves for dobutamine were performed in the presence of hexamethonium before and 30 min after phentolamine alone (1 mg/kg) or vehicle. Phentolamine did not influence the effect of dobutamine on heart rate or contractile force, but prevented the increase in diastolic blood pressure caused by dobutamine. In addition, analysis of heart rate versus contractile force indicated that there were no statistically significant effects of phentolamine on the inotropic selectivity of dobutamine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2994872 TI - The influence of D2O, perchlorate, and variation in temperature on the potential dependent contractile function of frog skeletal muscle. AB - D2O and perchlorate manifest opposing effects on the contractile function of skeletal muscle (amplitude of twitches and maximum K contractures, potential dependence of contraction activation and inactivation), and when combined the influence of one may effectively antagonize that of the other. The ratio of perchlorate concentrations required to produce effects of equal intensity (e.g., twitch enhancement and restoration of maximum K contractures in media lacking divalent cations or containing a depressant concentration of a cationic amphipath) in H2O and D2O solutions was generally rather constant. These findings are compatible with the view that both agents can influence contractile function by virtue of their effects on solvent structure. In the absence of divalent cations, the effects of reduced temperature resemble those of D2O whereas the effects of increased temperature resemble those of the chaotropic anion. However, in other media, variation in temperature was found to result in additional nonsolvent effects so that low temperature could oppose rather than enhance the effects of D2O. These observations are discussed in terms of a model postulates a role for solvent influences on the kinetics of two separate potential-dependent conformational transitions of membrane proteins which mediate the activation and inactivation of contraction in skeletal muscle. PMID- 2994874 TI - Action of serotonin and norepinephrine on spinal motoneurones following blockade of synaptic transmission. AB - The actions of serotonin and norepinephrine were investigated on spinal motoneurones in isolated, hemisected rat and frog spinal cords. Serotonin and norepinephrine induced slowly developing depolarizations of spinal motoneurones which were frequently preceded by brief, low amplitude hyperpolarizations. Neither the depolarizations nor the hyperpolarizations were attenuated by 20 mM Mg2+ or tetrodotoxin, although synaptic transmission was blocked in both cases. It thus appears unlikely that the action of serotonin and norepinephrine on spinal motoneurone polarization and results from an indirect action via interneurones. PMID- 2994873 TI - Inhibition of ACTH secretion in mouse pituitary tumor cells by activation of muscarinic cholinergic receptors. AB - Hormonally stimulated secretion of ACTH from AtT-20 mouse pituitary tumor cells is a cyclic AMP-mediated process. The presence of inhibitory cholinergic muscarinic receptors on these cells was recently reported, and in this study, the relationship between the activation of these receptors and the consequent inhibition of cyclic AMP formation and ACTH secretion was investigated. The muscarinic agent, oxotremorine, antagonized both cyclic AMP synthesis and ACTH secretion in response to corticotropin-releasing factor (CRF), vasoactive intestinal peptide, a 27-amino acid peptide with an N-terminal histidine and a C terminal isoleucine amide, and forskolin. Other muscarinic agents, carbachol and bethanechol, had similar inhibitory effects. The cholinomimetics reduced basal (unstimulated) ACTH secretion without decreasing basal cyclic AMP levels, and also antagonized hormone release in response to cyclic AMP-independent agonists such as K+, A-23187, and phorbol ester. Scopolamine reversed the inhibitory effects of the muscarinic agents on basal and stimulated ACTH secretion and cyclic AMP formation. Increasing the extracellular calcium concentration reversed the muscarinic antagonism of basal and CRF-stimulated hormone release without affecting the cyclic AMP response. Pertussis toxin pretreatment attenuated the inhibitory effects of the muscarinic agents on forskolin-stimulated cyclic AMP synthesis and ACTH secretion as well as the inhibitory effect of carbachol on basal ACTH release. The data suggest that cyclic AMP is an essential mediator in the ACTH secretory pathway, but that an alternate cyclic AMP-independent ACTH pathway also exists in the clonal cells, and that both pathways may be modulated by a common postcholinergic receptor mechanism. PMID- 2994875 TI - Modulation of inhibition in the hippocampus in vivo. AB - The nature and mechanisms of septohippocampal transmission have been elucidated by taking advantage of an in situ preparation in experiments with Sprague-Dawley rats under urethane. Both extracellular field potentials and intracellular recordings were made in CA1-3 regions of the hippocampus; and the hippocampal commissure and medial septum stimulated to evoke synaptic activity. Using muscarinic and nicotinic agonists and antagonists it was shown that both acetylcholine and medial septal activity can increase the excitability of pyramidal cells, mainly through muscarinic receptors. The effect of septal stimulation was enhanced by local application of physostigmine and reduced by intraventricular injections of hemicholinium. It was also shown that acetylcholine, when applied in the stratum pyramidale, can reduce the voltage and conductance changes observed during evoked inhibitory postsynaptic potentials (IPSP) without affecting the action of gamma-aminobutyric acid on membrane conductance and voltage. It is therefore proposed that acetylcholine can reduce evoked IPSPs through presynaptic inhibition. Evidence is also presented that medial septal stimulation can reduce the efficacy of evoked IPSPs. These observations provide further support for the existence of a cholinergic septohippocampal pathway. PMID- 2994876 TI - Abdominal pain and weight loss in a patient with well controlled diabetes. PMID- 2994879 TI - Cerebrospinal fluid ACTH as a marker of central nervous system metastases from small cell carcinoma of the lung. AB - Adrenocorticotrophic hormone (ACTH) concentrations were measured in the plasma and cerebrospinal fluid (CSF) of 107 consecutive patients with known or suspected central nervous system (CNS) metastases secondary to small cell carcinoma of the lung. The combined results of computerized tomography scans, neurologic examination, and autopsy were used to determine the presence or absence of CNS metastases. On the basis of such an assessment, definitive conclusions were possible in 77 patients. CNS metastases were present in 52 cases and absent in 25. The median CSF ACTH level was 30 ng/ml in both groups. None of five patients with very high CSF ACTH concentrations had elevated ACTH concentrations in plasma. Considering the 95th percentile of patients without CNS metastases as the upper limit of normal, 12 patients with metastases and one without had an elevated CSF ACTH value. Eleven patients with leptomeningeal carcinomatosis (MC) did not constitute a special subgroup in this respect. The median ratio of CSF ACTH and plasma ACTH was 1.0 in patients with CNS metastases and 0.4 in those without (P less than 0.05). Patients with MC had a median ratio of 1.3, which was significantly different from that of both of the other groups (P less than 0.05). Ten patients with CNS metastases (one with MC) and one without exceeded the upper 95th percentile of the CSF/plasma (ACTH) ratio in patients without CNS metastases. The significance levels of these findings disappeared, however, when patients with signs of an elevated ACTH concentration in plasma were excluded. Patients with ectopic ACTH production into CSF do not necessarily have ectopic ACTH production outside the CNS, despite the presence of extracerebral metastases. With the criteria employed in this study, an elevated level of CSF ACTH diagnosed too few patients for the authors to recommend its determination as a single test in diagnosing CNS metastases or MC secondary to small cell carcinoma of the lung. PMID- 2994877 TI - High-dose intracavitary cisplatin with intravenous thiosulfate. Low incidence of serious neurotoxicity. AB - Recent published reports have suggested that cisplatin administered in high doses or in certain combination chemotherapy can cause serious neurotoxicity in a large percentage of patients treated. In several high-dose cisplatin-based intracavitary chemotherapy trials with the simultaneous intravenous administration of sodium thiosulfate, the incidence of clinically relevant neurotoxicity has been extremely low. In addition, several patients with serious preexisting cisplatin-induced neurologic dysfunction were treated without worsening of their clinical condition. It is suggested that thiosulfate might have been responsible for the low incidence of neurotoxicity in this patient population. Further experimental and clinical investigation of the potential of this agent to protect against cisplatin-induced neuropathy appears warranted. PMID- 2994878 TI - Fatal pulmonary toxicity by the association of radiotherapy and medroxyprogesterone acetate. AB - This report describes a fatal pneumonitis occurring in a breast cancer patient while on adjuvant treatment with radiotherapy and medroxyprogesterone acetate. The clinical and radiologic features, as well as the timing of this pneumonitis, make a radiation pneumonitis more than probable. A radiation pneumonitis was also observed in other patients treated in the same way. Medroxyprogesterone acetate thus seems to act as a radiosensitizer since no such effects were seen in patients treated with radiotherapy alone. The radioenhancing effect is not limited to the lungs, since radioesofagitis was also encountered in similarly treated patients. PMID- 2994880 TI - Prognostic factors and outcome in bilateral Wilms' tumor. AB - Twenty-one patients with bilateral Wilms' tumor are reviewed and the details of diagnosis, therapy, and survival presented. All patients had an abdominal mass at the time of diagnosis. Associated findings included hypertension, aniridia, and genitourinary anomalies. Favorable histologic features were found in all simultaneously occurring tumors and in the initial tumor in nonsimultaneous tumors. Eleven of the 18 patients with simultaneously occurring tumors survived for at least 2 years, for an overall 2-year survival rate of 61%, which was similar to the 2-year survival rate of 60% found in a review of 61 other simultaneously occurring bilateral Wilms' tumors reported in the literature since 1971. Two "front-end" factors that affected prognosis were the patient's age and the stage of the most advanced tumor at the time of diagnosis. A significantly better survival was found in children whose tumor was diagnosed before the age of 2 years and in patients who had Stage I or II disease in the most advanced tumor, as compared with those who had Stage III or IV disease. The overall survival rate in this series and in the literature review is much poorer than that reported for bilateral Wilms' tumor in the National Wilm's Tumor Study; some possible reasons for this are given. The authors' current approach to diagnosis and therapy is reviewed. PMID- 2994881 TI - Small cell carcinoma of the urinary bladder with hypercalcemia. AB - This report describes three cases of undifferentiated small cell carcinoma of the urinary bladder. Their light microscopic appearance is closely akin to the small cell carcinoma of lung. The neoplastic cells exhibit few cytoplasmic dense core neurosecretory granules ultrastructurally and immunoreactivity to enolase. Two patients manifested clinically hypercalcemia which is rare in small cell carcinoma in general and, to the best of our knowledge, has not been described in association with bladder small cell carcinoma. PMID- 2994882 TI - Carbamazepine: a neuropsychological and psychiatric profile. PMID- 2994883 TI - The effect of pentoxifylline on the energy metabolism of ischemic gerbil brain. AB - Pentoxifylline decreases the cerebral edema resulting from cortical freezing lesions in cats, produces mitochondrial hypertrophy with preservation of structure in gerbils, and increases survival rate in gerbils rendered ischemic by temporary bilateral carotid occlusion. Since all these findings may be related to energy metabolism, the effect of the drug on postischemic cerebral phosphocreatine and ATP concentrations and on cytochrome oxidase activity has been studied. Brain slices prepared from animals subjected to bilateral carotid occlusion for 30 min and treated with pentoxifylline at release of occlusion, then allowed 3 h for recovery, exhibited higher concentrations of ATP and higher levels of cytochrome oxidase activity than did those of the untreated animals. PMID- 2994884 TI - Cushing's disease: pituitary microadenoma demonstrated by high resolution computed tomography with thin slices. AB - A girl 16 7/12 years of age with Cushing's disease secondary to pituitary microadenoma is described. The tumor was very clearly demonstrated by high resolution contrast-enhanced computed tomography (CT) with thin slices and was removed by transsphenoidal microadenomectomy. In some patients with Cushing's disease levels of ACTH and cortisol are not always high. Therefore, high resolution contrast-enhanced CT with thin slices should be performed when the diagnosis of Cushing's disease is suspected even when the definite diagnosis cannot be established by endocrinological examinations. PMID- 2994885 TI - Implementation of periodic acid-thiosemicarbazide-silver proteinate staining for ultrastructural assessment of muscle glycogen utilization during exercise. AB - Distribution of glycogen particles in semithin and ultrathin sections of biopsy samples from human muscles subjected to either short- or long-term running were investigated using PAS and Periodic Acid-ThioSemiCarbazide-Silver Proteinate (PA TSC-SP) staining methods. Glycogen particles were predominantly found immediately under the sarcolemma or aligned along the myofibrillar I-band. After long-term exhaustive exercise type-1 fibers with a few or no glycogen particles in the core of the fibers were frequently observed. The subsarcolemmal glycogen stores of these "depleted" type-1 fibers were about three times as large as after exhaustive short-time exercise. Another indication of utilization of subsarcolemmal glycogen stores during anaerobic exercise was that many particles displayed a pale, rudimentary shape. This observation suggests fragmental metabolization of glycogen. Thus, depending on type of exercise and type of fiber differential and sequential glycogen utilization patterns can be observed. PMID- 2994886 TI - A two-receptor model for the action of parathyroid hormone on osteoblasts: a role for intracellular free calcium and cAMP. AB - It has been suggested that intracellular Ca2+, in addition to cAMP, plays an important role in PTH-stimulated bone resorption. There is now strong evidence indicating that the osteoblast is the main target cell for PTH action, regulating indirectly, via cell-cell communication, osteoclastic bone resorption. In order to investigate the possible role of free cytosolic calcium in stimulated bone resorption, we studied the effects of the intact hormone (bPTH 1-84) and some of its fragments (bPTH (1-34), bPTH(3-34,) (Nle-8, Nle-18,Tyr-34) bPTH (3-34) amide) on their capacity to modify the cytosolic Ca2+ concentration in rat osteoblast like cells. The experiments were performed using Quin-2, a fluorescent indicator of free calcium. We found an excellent correlation between the ability of PTH and PTH fragments to transiently increase cytosolic Ca2+ concentration in rat osteoblast-like cells and their ability to stimulate bone resorption in embryonic rat calvaria in vitro. On the other hand, no direct correlation was found for the cAMP and bone-resorbing responses. On the ground of these data we propose a two receptor model for PTH action in osteoblasts, in which one receptor is coupled to the production of cAMP, whereas the other is involved in the increase of cytosolic Ca2+. Activation of both receptors by PTH (1-84) or PTH (1-34) leads to the full physiological response in osteoblasts, most probably the release of one or more factors which stimulate the activity of existing osteoclasts and others which stimulate the recruitment of additional osteoclasts. PMID- 2994887 TI - Beta-endorphin and ACTH in plasma during attacks of common and classic migraine. AB - Plasma levels of beta-endorphin and ACTH were measured during and outside migraine attacks in 17 patients with common migraine and 11 patients with classic migraine. Specific radioimmunoassays for beta-endorphin and ACTH were used. The beta-endorphin assay did not cross-react with beta-lipotropin. In common migraine, median plasma beta-endorphin was 3.3 pmol/l (95% confidence limits: 2.5 4.0 pmol/l) during attacks and 2.9 (2.4-3.2) pmol/l in the headache-free period. In classic migraine, plasma beta-endorphin was 3.2 (1.4-4.3) pmol/l during attacks and 2.4 (1.1-3.6) pmol/l outside attacks. ACTH plasma levels were 15 (10.5-20) pmol/l during and 15.7 (13.4-17) pmol/l outside attacks in common migraine. In classic migraine, plasma ACTH was 16 (7-36) pmol/l and 12.3 (8-28) pmol/l respectively. No significant differences were found between attacks and headache-free periods in common or classic migraine. Accordingly, we could not add evidence to the theory of a dysfunction of the endogenous opioid system in migraine. PMID- 2994888 TI - Cytotoxic activity of normal and Herpesvirus saimiri-transformed nonhuman primate cells. AB - Owl monkey mononuclear cells were separated from peripheral blood by centrifugation on Ficoll gradients, removal of adherent cells, and subsequent separation on discontinuous Percoll gradients. Lymphocytes recovered from the various fractions were tested for cytotoxic reactivity immediately after isolation. Low-density cells, enriched in large granular lymphocytes (LGL), demonstrated cytotoxic activity against the human natural killer-susceptible cell lines MOLT 4 and K562. In addition, IL-2-independent T-cell lines which had been obtained by immortalization with the primate herpesvirus Herpesvirus saimiri showed cytotoxicity, even after prolonged culture in vitro, similar to that demonstrated by fresh LGL. Cytotoxic activity of these lines was regulated by IL 2 in a fashion which appeared to be independent of the growth-promoting effects of this lymphokine. These results indicate a function for IL-2 beyond its role in supporting cellular proliferation. Cytotoxic activity could also be demonstrated in culture fluids from one of these cell lines (70N2). In addition, these results indicate the usefulness of immortalized cell lines (like 70N2) as a potential source for studies of the biochemical characterization and purification of supernatants containing cytotoxic factors. PMID- 2994889 TI - Monoclonal antibody that immunoreacts with a subclass of human receptors for epidermal growth factor. AB - Spleen cells from BALBc mice immunized with human epidermoid carcinoma A431 cells were fused with mouse myeloma P3NP cells. One of the isolated hybridoma lines, B4G7, secreted a monoclonal antibody of the IgG class which inhibited the binding of [125I]-EGF to A431 cells and human fibroblasts, but not to mouse 3T3 cells. This inhibition was partial (65-70%) and Scatchard analysis of the EGF binding data suggested that the B4G7 antibody interacts preferentially with a low affinity class of EGF receptors. This monoclonal antibody specifically precipitated EGF receptors (Mr = 170,000 and 155,000) of A431 cells which were directly crosslinked with [125I]-EGF. It also precipitated EGF receptors from cells whose surface proteins were labeled with 125I, from cells grown in the presence of [35S]-methionine or [32P]-orthophosphate, and from membrane fractions phosphorylated in vitro with [32P]-gamma-ATP. Receptors subjected to EGF-induced phosphorylation, both in vivo and in vitro, were also precipitated. The B4G7 antibody blocked approximately 70% of the EGF receptors in human fibroblasts, but did not stimulate DNA synthesis in these cells. However, in the presence of this antibody, cells showed the full mitogenic response to EGF, presumably through the unblocked receptors that are likely to be of the high-affinity type. PMID- 2994890 TI - Myelin formation in dissociated cell cultures of rat embryonic cerebral hemispheres. AB - Developing cultures of dissociated cerebral hemispheres obtained from 18-day-old embryonic rats synthesized, or activated, a myelin-related enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). This increase in activity coincided with the onset of myelination. The presence of CNPase in oligodendrocytes and myelin was demonstrated using immunocytochemical staining techniques. Myelinated axons and myelin sheaths were clearly made visible by electron microscopy of 28-day-old cultures. PMID- 2994891 TI - Effect of monensin on the synthesis, maturation and secretion of vesicular stomatitis virus proteins in a monensin-resistant Chinese hamster ovary cell line. AB - We compared the effects of the cationic ionophore, monensin, on the synthesis, maturation and release of vesicular stomatitis virus (VSV) in cultures of Chinese hamster ovary (CHO) cells and the monensin-resistant clone, MonR-31. Our results depended on the dose and time of the addition of monensin to the infected cells, from 1 h prior to VSV infection to 1 h after infection. VSV production was more resistant in MonR-31 than in CHO cells when the ionophore was added 1 h prior to VSV infection. Monensin added 1 h after VSV infection showed the opposite phenomenon; release of virus particles into the medium was 10- to 10(5)-fold less in MonR-31 cells than in CHO cells, and the intracellular virus number in the resistant cells was one-third to one-fourth of that in the parental CHO cells. Syntheses of all virus-associated G, N and M proteins were inhibited in both cell lines by monensin, but especially so in the MonR-31 cells. There were no marked qualitative changes in the biochemical properties of viral glycoprotein G in virus-infected CHO and MonR-31 cells treated with monensin after virus infection. An endoglycosidase H-resistant G with a molecular weight smaller than that of normal G and attachments of palmitate or fucose on the truncated G protein appeared. Alteration of the secretion of as well as the synthesis of the enveloped virus is discussed in relation to the monensin susceptibility of the resistant MonR-31 clone. PMID- 2994892 TI - Cellular structure and function of mouse adrenocortical tumor cells Y-1 in the post-treatment state of low Ca2+. AB - Under lowered Ca2+ content and in the post-treatment state of low Ca2+, we studied the cellular structure and functioning in mouse adrenocortical tumor cells, Y-1. These cells had been maintained in Ham F12 medium containing 10% fetal calf serum. The Ca2+ present in this complete medium was 0.39 mM. Under a slightly lowered Ca2+ (0.29 mM) produced by EGTA, the cells had many blebs on their surfaces and specific functional activity decreased in steroidogenesis as did ACTH reactivity. In the post-treatment state of a low Ca2+, the cellular surface was covered with many short microvilli and there was greater cellular activity than in the control cells. When the Ca2+ concentration was below 0.17 mM, the cellular structure and functioning were disturbed, and there was no recovery even at the physiological Ca2+ after the removal of EGTA. PMID- 2994893 TI - Distributions of post-proline cleaving enzyme-, converting enzyme-, trypsin- and chymotrypsin-like activities in various nephron segments and in brush-border membranes isolated from rat kidney. PMID- 2994894 TI - Potentiating effect of lysophosphatidylcholine on antibacterial activity of polymyxin antibiotics. PMID- 2994895 TI - [Planting of Lonicera japonica on saline-alkali soil in Yu-Cheng County]. PMID- 2994896 TI - Topological distribution of bovine serum albumin in multilamellar and unilamellar liposomes. AB - The topological distribution of bovine serum albumin (BSA) in multilamellar vesicles (MLV) and unilamellar vesicles (ULV) composed of 1,2-dimyristoyl-sn glycero-3-phosphorylcholine (DML)/cholesterol (molar ratio, 3:1) was studied by ESR using hydrophobic spin-labelled lecithins and hydrophilic Tempocholine. A spin-labelled BSA was also prepared, characterized and used as a probe. Results with hydrophobic spin-labelled lecithin probes showed no significant phospholipid albumin interaction, indicating the virtual absence of albumin from the phospholipid bilayer of MLV and ULV. Reduction with L-ascorbic acid showed that MLV contained about 50% and ULV 90% of spin-labelled albumin on the surface. The distribution of Tempocholine in MLV and ULV was similar to that of entrapped BSA. These findings were confirmed by results using liposomes treated with nickel which broadened the ESR spectra of probes on the surface of vesicles. This study and our previous work suggest that the immunoadjuvant effect of liposomes can be mediated by surface antigens and can be maximized by preferential surface distribution as in ULV-associated BSA. PMID- 2994897 TI - Prevention and control of herpesvirus diseases. Part 2. Epidemiology and immunology. AB - This is the second of two articles summarizing the information currently available on herpes virus diseases. The first described the different members of the herpesvirus group, clinical expression of infection, laboratory diagnosis, and chemotherapy. This article dealing with the epidemiological and immunological aspects of herpesvirus infections also brings up the special problems caused by virus latency and risks associated with congenital infection and immunodeficiency or immunodepressive treatment. The prospects of preventing herpesvirus infections by vaccination and treatment with immune sera are also discussed. PMID- 2994898 TI - Recombinant vaccinia viruses as live virus vectors for vaccine antigens: memorandum from a WHO/USPHS/NIBSC meeting. AB - A scientific workshop sponsored by the World Health Organization, the US Public Health Service, and the National Institute for Biological Standards and Control, London, was held in Bethesda, MD, USA, on 13 and 14 November 1984 to review progress in research relevant to the development of genetically and antigenically modified vaccinia viruses as live vaccines for human and veterinary use. The meeting was followed by an informal consultation convened by WHO to consider the advantages and disadvantages of this approach to vaccine design and production. The needs for further research and the potential role of WHO in coordinating and encouraging international activities in this area were discussed. This report summarizes the proceedings of the scientific workshop and the recommendations made by the WHO consultation. PMID- 2994899 TI - [Rural epidemic of yellow fever with interhuman transmission in the Ivory Coast in 1982]. PMID- 2994900 TI - Lyophilized combination pools of enterovirus equine antisera: new LBM pools prepared from reserves of antisera stored frozen for two decades. AB - This paper describes the preparation and test procedures for a second batch of lyophilized LBM combination antiserum pools, A through H, used for identifying 42 enteroviruses. Each pool is selectively composed of 10 or 11 of 42 individual enterovirus equine sera so that it contains 500 antibody units of each serum component per 0.1 ml. The new pools have been constituted from equine monovalent antisera that were prepared during the period 1962-67 and then evaluated and standardized in a series of collaborative international studies. An essential aspect of preparing the new pools was ensuring that the individual sera had retained high antibody titres through the long period of storage. At the time of retesting, the original stocks of these monovalent sera had been stored frozen at -20 degrees C for periods ranging from 16 to 20 years. PMID- 2994901 TI - On the mechanism of induction of resistance to 6-thioguanine in Chinese hamster V79 cells by 3-methylcholanthrene-diolepoxide. AB - V79 Chinese hamster cells were cultured in the presence of 3-methylcholanthrene diolepoxide (10r,9t-dihydroxy-7,8t-epoxy-tetrahydro-3-methylcholanthrene, MCDE) and mutants were selected in medium containing 6-thioguanine (TG). Of 22 TG resistant mutants examined, 18 were devoid of HPRT (hypoxanthine-guanine phosphoribosyltransferase, EC 2.4.2.8) activity. Two mutants had suffered a total and one a partial gene deletion. The 1.6-kb HPRT mRNA was not detected in these three mutants nor in two others. The remaining mutants did not, however, have a readily demonstrable lesion. PMID- 2994902 TI - c-Ha-ras not c-Ki-ras activation in three colon tumour cell lines. AB - Using the NIH 3T3 transformation assay system, an activated c-Ha-ras transforming gene has been identified in three distinct early passage colon carcinoma cell lines isolated from an invasive, differentiated, adenocarcinoma. The p21 c-Ha-ras gene product from these cell lines displayed an altered electrophorectic mobility and a point mutation in the DNA coding sequence leading to an amino acid substitution at position 12. PMID- 2994904 TI - Characterization of a potentially reversible increase in beta-adrenergic receptors in isolated, neonatal rat cardiac myocytes with impaired energy metabolism. AB - Previous studies have reported that the numbers of beta- and alpha-adrenergic receptors increase in ischemic myocardium. In vivo studies have raised questions regarding the mechanisms involved in the adrenergic receptor alterations and the consequences of these alterations. The purpose of this study was to evaluate potential relationships among beta-adrenergic receptor changes, high energy phosphate reduction, and severity of cell injury in cultured neonatal rat myocytes treated with metabolic inhibitors. The potential for reversal of the receptor changes also was addressed. Binding parameters were measured using [125I]iodocyanopindolol. After 4 hours incubation in potassium cyanide and 2 deoxyglucose, there was a 43% increase in beta-adrenergic receptor number, 41% decrease in adenosine triphosphate, and minimal morphological change in myocytes. Twenty-four hours after removal of the inhibitors, myocytes exhibited a return to normal of the receptor number and adenosine triphosphate level. Iodoacetate treatment for up to 3 hours resulted in marked reduction in adenosine triphosphate and increasing severity of cell injury. The number of beta adrenergic receptors was unchanged at 1.2 hours, increased at 1.5-2 hours, and decreased at 3 hours. Thus: beta-adrenergic receptor density increases during relatively early stages of injury in metabolically impaired myocytes with reduced adenosine triphosphate levels and decreases subsequently, after the myocytes become irreversibly injured; the increased beta-adrenergic receptor density in moderately injured myocytes can be reversed upon removal of the injurious agent and restoration of the cellular adenosine triphosphate level; and changes in catecholamines mediated by an intact nervous system are not required for an increase in beta-adrenergic receptor density in the setting of impaired energy metabolism. PMID- 2994903 TI - George E. Brown memorial lecture. Oxygen radicals in cerebral vascular injury. AB - Acute, severe increases in arterial blood pressure cause sustained cerebral arteriolar dilation, abnormal reactivity to carbon dioxide and to changes in blood pressure, abolition of endothelium-dependent dilation from acetylcholine, discrete morphological lesions of the endothelium and vascular smooth muscle, and breakdown of the blood-brain barrier to plasma proteins. The dilation, abnormal reactivity, and morphological abnormalities are inhibited by pretreatment with cyclooxygenase inhibitors or with free radical scavengers. Superoxide dismutase inhibitable reduction of nitroblue tetrazolium applied to the brain surface was detectable both during hypertension and one hour after hypertension subsided. Nitroblue tetrazolium reduction is also reduced by inhibitors of the anion channel. The abnormalities seen after hypertension are reproduced by topical application of arachidonate. The results are consistent with the view that acute hypertension induces generation of superoxide anion radical in association with accelerated arachidonate metabolism via cyclooxygenase. This radical enters cerebral extracellular space via the anion channel and gives rise to hydrogen peroxide and hydroxyl radical. All three radicals are capable of causing vasodilation by relaxation of cerebral vascular smooth muscle. The hydroxyl radical is the most likely candidate for vascular wall damage. The significance of this mechanism in chronic experimental hypertension or its relevance to human disease is not known. PMID- 2994906 TI - Adenylate kinase: an oncodevelopmental marker in an animal model for human prostatic cancer. AB - Data are presented demonstrating that adenylate kinase (AK; EC 2.7.4.3) is an oncodevelopmental enzyme in the prostate of Copenhagen rats. We selected the Dunning tumor (dorsal rat prostate) as a model system because it most nearly approximates the human pathology. Four sublines of the tumor (R3327-H, R3327-AT, MAT Lu, and MAT LyLu) were studied. The tumor sublines were maintained as solid tumors in syngeneic rats and as monolayers in tissue culture. AK activity appeared in conjunction with malignant transformation of the dorsal prostate. We also determined the normal developmental enzyme pattern: AK was present in prostates of newborns, but was undetectable in prostates of adults. However, AK increased after castration. Therefore, we propose AK as a potential oncofetal tumor marker in prostatic cancer. PMID- 2994905 TI - Alterations of serum carboxypeptidases N and angiotensin-I-converting enzyme in malignant diseases. AB - The activities of serum carboxypeptidases N (E.C. 3.4.17.3) and serum angiotensin I-converting enzyme (E.C. 3.4.15.1) were spectrophotometrically measured in patients with hematological disorders and in histologically proven bronchogenic carcinomas. The enzyme activities have been compared with various laboratory parameters. Controls were healthy subjects. No significant sex differences of carboxypeptidases N and angiotensin-I-converting enzyme were found in the healthy controls or in the whole patients' group. The carboxypeptidases N were significantly elevated in patients with lung cancer (p less than 0.0001) compared to the healthy controls. The angiotensin-I-converting enzyme was significantly reduced (p less than 0.0001) in serum of patients with bronchogenic carcinoma. In responders to chemo- and/or radiotherapy, the carboxypeptidases decreased and the angiotensin-I-converting enzyme increased to normal enzyme values. In non responders to these therapies no change in these enzymes could be observed. PMID- 2994907 TI - Estriol and its conjugates in late pregnancy determined by extraction with Carbopack B and liquid chromatography with fluorometric detection. AB - We report a method for measuring estriol and its intact conjugates in urine, serum, and amniotic fluid. A single assay can be done within about 50 min, eight samples assayed in less than 5 h. A 70-microL urine sample is diluted and the estriol conjugates are adsorbed from it onto graphitized carbon black (Carbopack B, Supelco). After two washings, the analytes are desorbed with chloroform/methanol (60/40, by vol) containing tetrapropylammonium bromide. After solvent evaporation, the residue is redissolved in 100 microL of water/acetonitrile and 20 microL is injected into the chromatograph. Or 1 mL of serum or 0.5 mL of amniotic fluid is deproteinized with cold methanol, then passed through the Carbopack column. After three washings, the estriol and its conjugates are desorbed and treated as for urine. Mean analytical recoveries of the analytes in any of these body fluids were within about 92-98%, except for estriol-3-sulfate-16 alpha-glucuronide in serum (mean recovery 88.3%). The limit of sensitivity is well below the concentrations of clinical interest, and the method is not susceptible to substantial interferences. PMID- 2994908 TI - Single-reagent automated determination of angiotensin converting enzyme in serum. PMID- 2994909 TI - Problems with transporting serum to the laboratory for cryoglobulin assay: a solution. PMID- 2994910 TI - Changes in serum osteocalcin associated with parathyroid hormone infusion in X linked hypophosphatemic rickets. AB - Osteocalcin is a protein unique to bone that can be quantitated in serum by radioimmunoassay. While its function remains unknown, it appears to be a sensitive marker of changes in bone activity. To determine its relationship to parathyroid hormone action, we measured serum osteocalcin in blood samples obtained from patients with vitamin D-resistant rickets before and after administration of exogenous parathyroid hormone. Serum osteocalcin was decreased by 35% at 15 min after infusion and gradually returned toward normal levels by 75 min. We suggest that the acute decline in serum concentrations after infusion is an indication of inhibition of osteoblast activity. Thus, osteocalcin may be a useful means of assessing bone responsiveness to parathyroid hormone. PMID- 2994911 TI - West's syndrome and its treatment. PMID- 2994912 TI - The adrenergic system and the cardiovascular effects of platelet activating factor (1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) in SHR and WKY rats. AB - 1-0-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (1-hexadecyl-2-acetyl-GPC, platelet activating factor, PAF) was previously shown to produce profound hypotension and sympathetic activation in conscious rats. To determine the role of the sympatho-adrenomedullary system in the cardiovascular responses elicited by 1-hexadecyl-2-acetyl-GPC, the vasoactive phospholipid was administered (1 nmol per 300 g) to a) intact, b) bilaterally demedullated, and c) propranolol- (a beta adrenoceptor blocker) treated SHR and WKY rats. The hypotensive response to 1 hexadecyl-2-acetyl-GPC was prolonged in demedullated or propranolol-pretreated WKY rats and in propranolol-treated SHR rats. The extreme tachycardia produced by 1-hexadecyl-2-acetyl-GPC in both the WKY and SHR rats was abolished by propranolol pretreatment. Pressor responses to norepinephrine during the 1 hexadecyl-2-acetyl-GPC-induced hypotension in propranolol-pretreated rats were suppressed in both the normotensive and SHR rats. Plasma acetylhydrolase activity, which inactivates PAF, was higher in hypertensive (SHR) rats or demedullated WKY rats than in the normotensive (WKY) rats. These results show that the tachycardia evoked by 1-hexadecyl-2-acetyl-GPC is mediated solely by sympathetic activation and the beta-adrenergic receptors and further indicate the major role of the sympathetic system and beta-adrenoceptors in recuperation from 1-hexadecyl-2-acetyl-GPC-induced shock. The data also suggest that acetylhydrolase in serum is an important regulatory enzyme for controlling PAF levels in the vascular compartment. PMID- 2994913 TI - Mn++-stimulated 3H-inositol incorporation in cultured vascular smooth muscle cells: deficiency in cells from spontaneously hypertensive rats. AB - Incubation of cultured dissociated aortic smooth muscle cells from Wistar-Kyoto rats (WKY) with Mn++ resulted in a 10-fold stimulation of 3H-myo-inositol incorporation into membrane phospholipid. The stimulation was temperature and energy dependent. The Mn++ EC50 was 1 mM and maximum stimulation occurred at 10 mM Mn++. Other cations were ineffective. In cells from spontaneously hypertensive rats (SHR), the Mn++ EC50 was unchanged whereas the maximum stimulation of 3H inositol incorporation was reduced by 50% compared to cells from parallel WKY cultures. These results suggest that in SHR cultured vascular smooth muscle cells there is a deficiency in an enzymatic process mediating exchange of free inositol with the headgroup inositol of membrane phosphatidylinositol. PMID- 2994914 TI - Effects of enalapril maleate on plasma level of inactive renin in renovascular hypertension. AB - Changes in plasma levels of active and inactive renin after the treatment with enalapril maleate (MK-421), a new angiotensin converting enzyme inhibitor, were studied in five patients with renovascular hypertension (RVH) due to unilateral renal artery stenosis. The dosage was increased when the blood pressure (BP) was not normalized for more than 3 days. Blood sampling was performed before, and 5 hours and 24 hours after the first administration, and on the 3rd day with each dosage. Active and inactive renin concentrations (ARC and IRC) showed a reciprocal change in 4 cases, 5 hours after the first dose. In the chronic treatment, ARC and IRC before the morning dose did not change apparently until the BP was normalized, when both ARC and IRC were evidently increased. It was suspected that a conversion from inactive to active renin may occur in the patients with RVH, when the active renin secretion is stimulated suddenly by the first dose of MK-421. The chronically diminished perfusion pressure in the kidney may stimulate the secretion of inactive renin, but the decrease in endogenous angiotensin II may not. PMID- 2994915 TI - The mechanism of hypertension and bradycardia following lesions of the caudal ventrolateral medulla in the rabbit: the role of sympathetic nerves, circulating adrenaline, vasopressin and renin. AB - Lesions of the ventrolateral medulla of the rabbit, coinciding with the A1 noradrenaline cell bodies (A1 lesions) produced fortyfold increases in the plasma levels of vasopressin and adrenaline, a twofold increase in plasma noradrenaline and a substantial increase in plasma renin activity. These increases accompanied the hypertension and bradycardia that follow A1 lesions. The vasoconstriction and hypertension were completely abolished by phentolamine, an alpha-adrenoceptor antagonist, when it was administered before lesions and were markedly reduced when it was given after lesions. On the other hand, administration of an antagonist to the vasoconstrictor action of vasopressin (d(CH2)5Tyr(Me)AVP) or an angiotensin converting enzyme inhibitor had little effect. Prior removal of the adrenal glands prevented any rise in plasma adrenaline levels but had no effect on the pressure response to subsequent A1 lesions. These results indicate that the vasoconstriction and hypertension were predominantly mediated by alpha adrenoceptor stimulation, acting mainly through sympathetic vasoconstrictor nerves. The fall in heart rate following A1 lesions was approximately halved by pretreatment either with d(CH2)5Tyr(Me)AVP alone, or by blockade of the vagus and sympathetic with scopolamine and propranolol; it was completely abolished by combined pretreatment with all three agents. The experiments show that vasopressin release makes a major contribution to the bradycardia acting at least in part through mechanisms that are independent of cardiac vagal or sympathetic nerves. PMID- 2994916 TI - Alpha- and beta-adrenoceptors in circulating blood cells of essential hypertensive patients: increased receptor density and responsiveness. AB - In 40 male patients with established essential hypertension (P diast greater than 95 mmHg) platelet alpha 2-adrenoceptor density (by 3H-yohimbine binding) and responsiveness (by adrenaline-induced aggregation) as well as lymphocyte beta 2 adrenoceptor density (by (+/-)-125 iodocyanopindolol binding) and -responsiveness (by cyclic AMP responses to isoprenaline) were determined and compared with those in 40 male age-matched normotensives (P diast less than 90 mmHg). In essential hypertensive patients mean platelet alpha 2- and lymphocyte beta 2-adrenoceptor densities were significantly increased. When data from all 80 subjects were combined, significant positive correlations between mean arterial blood pressure and alpha 2- and beta 2-adrenoceptor densities, respectively, were found. The increases in alpha 2- and beta 2-adrenoceptor densities were accompanied by enhanced responsiveness to adrenergic stimulation: in platelets adrenaline induced aggregation--via alpha 2-adrenoceptor stimulation--was exaggerated, and in lymphocytes isoprenaline produced significantly greater increases in the intracellular level of cyclic AMP. It is concluded that the increased density and responsiveness of alpha 2-and beta 2-adrenoceptors in circulating blood cells of essential hypertensive patients may reflect increased sympathetic activity, which might contribute to the elevation of blood pressure. PMID- 2994917 TI - Activities of transport adenosine triphosphatases in erythrocyte membranes of healthy and hypertensive subjects. AB - The Na+, K+-ATPase activity in the erythrocyte membrane was measured in 25 untreated essential hypertensive patients and 25 age-matched healthy normotensive subjects. In addition, the Ca2+, Mg2+-ATPase activity was measured in 20 hypertensive and 25 age-matched normotensive subjects. The Na+, K+-ATPase activity of healthy Chinese measured in this study was similar to the data reported in a Dutch study. We therefore could not support a theory which speculated an ethnic influence on Na+, K+-ATPase activity. Both Na+, K+-ATPase and Ca2+, Mg2+-ATPase activities were slightly lower in hypertensive patients as compared with those in normotensive subjects, but the differences were not statistically significant. We concluded that the transport ATPase activities were not impaired in the erythrocyte membranes of hypertensive patients. PMID- 2994918 TI - Nifedipine as a substitute for converting enzyme inhibitors in the treatment of renovascular hypertension. AB - Nifedipine long acting tablets were substituted for converting enzyme inhibitors in eight patients with renovascular hypertension. A significant drop in blood pressure was observed. Nifedipine was shown to be as effective (in lowering blood pressure) as a drug that acts directly on the renin angiotensin system. These data offer support for previously reported findings that the action of angiotensin II is calcium mediated. PMID- 2994919 TI - A new banding pattern of human chromosomes by in situ nick translation using ECO RI and biotin-dUTP. AB - Here we present first results of an attempt to replace the nicking activity of DNAase I by ECO RI for in situ nick translation of human chromosomes. For the detection of nick translated sites we used biotinylated dUTP. The procedure resulted in a distinct banding pattern of the chromosomes which does not seem to correspond to that reached by any known banding technique. PMID- 2994920 TI - Sarcomas--a clinicopathological guide with particular reference to cutaneous manifestation. III. Angiosarcoma, malignant haemangiopericytoma, fibrosarcoma and synovial sarcoma. PMID- 2994921 TI - Studies on lymphocyte cell surface in ataxia-telangiectasia. AB - Lymphocyte surface proteins of patients with ataxia-telangiectasia were separated by polyacrylamide gel electrophoresis and compared by autoradiography. The patients lacked one of the two main bands (Band I at the origin). The second main band (Band II) was absent in some cases. All patients had one or two additional bands of smaller molecular weight than Band II except one case who had no band detectable. In the patients, alkaline phosphatase, total ATPase and Mg2+. ATPase were increased but 5'-nucleotidase was normal. The results suggest abnormality in the plasma membranes of the patients' lymphocytes. PMID- 2994922 TI - The influence of lithium chloride on experimental autoimmune thyroid disease. AB - Lithium administration is known to be associated with the development of thyroid dysfunction; it also exerts an effect on the immune system. The effect of lithium on experimental autoimmune thyroid disease was studied in female August rats. Following immunization with rat thyroglobulin in Freund's complete adjuvant, lithium chloride was administered i.p. for 30 days to four groups at varying stages of the disease. Control animals received i.p. saline. Anti-thyroglobulin antibody levels (measured by ELISA) were significantly increased in rats given lithium immediately post-immunization (group B) compared to control animals (661 +/- 42 OD vs 448 +/- 68; mean +/- s.e., P less than 0.02). In contrast, animals which received lithium during the spontaneous resolution of the disease (group D) showed a significant fall in anti-TG antibody compared to controls (99 +/- 15 vs 27 +/- 15; P less than 0.001). Anti-TG antibody levels remained undetectable in animals which received lithium but were not immunized. The splenic T cell blastogenic response (as measured following phytohaemagglutinin stimulation) was significantly increased in rats receiving lithium prior to and during immunization (group A) (stimulation index 63.4 +/- 6.9 vs 10.2 +/- 2.4; P less than 0.001). Spontaneous cell proliferation of splenic lymphocytes was decreased in two lithium treated groups (group A P less than 0.005, group C P less than 0.05). There was no alteration in splenic weight or the degree of thyroid lymphocytic infiltration in any of the treated group. Lithium exerted both positive and negative influences on the immune system in rats immunized with thyroglobulin in adjuvant but did not induce autoantibody production in normal rats. PMID- 2994923 TI - Maintenance of cytomegalovirus (CMV) latency and host immune responses of long term renal allograft survivors. II. Secondary CMV infections associated with impaired in vitro proliferative responses to mitogens, allogeneic lymphocytes and CMV infected cells. AB - The relationship between secondary cytomegalovirus (CMV) infections and host general cellular immunocompetence was investigated in 16 renal allograft recipients with minimal immunosuppressive treatment and excellent renal function. Results were compared with 19 CMV seropositive healthy controls. Significantly impaired immune responses were detected in the subgroup of nine recipients who experience at least 2 years before a secondary CMV infection. Their in vitro lymphocyte reactivity (LR) tests to phytohaemagglutinin (PHA, P = 0.01), pokeweed mitogen (PWM, P less than 0.05), microbial antigens (P less than 0.001) and to pooled allogeneic stimulator lymphocytes in the MLC test (P = 0.02) were lower than the controls. The MLC responses, however, increased with graft survival time (r = 0.8810, P = 0.01). This was positively correlated with the virus specific cellular immunity measurable by the LR responses to CMV infected target cells (r = 0.8333, P = 0.02). In contrast, long term renal allograft survivors who maintained their CMV infection in latency after transplantation (n = 7) showed normal responses to PWM, pooled lymphocytes and CMV infected target cells, whereas the responses to PHA and to bacterial antigens were less severely impaired (P less than 0.05 and P less than 0.001, respectively). This study of long term renal allograft survivors shows that a secondary CMV infection has a long lasting negative effect on immunity especially against alloantigens and CMV infected targets. However, in the data presented here it would be as acceptable to suggest that the patients are consistently relapsing with CMV because they initially had poor immune response and not vice versa. PMID- 2994924 TI - Cytomegalovirus (CMV)-specific lysis of CMV-infected target cells can be mediated by both NK-like and virus-specific cytotoxic T lymphocytes. AB - The host response to cytomegalovirus (CMV) involves both humoral and cell mediated immunity. Because virus-specific cytotoxicity appears to be associated with recovery from CMV infection, we have investigated spontaneous (NK) and cytotoxic T cell (Tc) activity against CMV-infected target cells in normal donors and infants with active CMV infection. Fresh mononuclear cells from seropositive donors expressed low-level, non-MHC-restricted cytotoxic activity that preferentially lysed CMV-infected but not uninfected autologous and allogeneic fibroblasts. No cytotoxic activity was observed when mononuclear cells from seronegative donors were studied. Mononuclear cells from infants with active CMV infection and mononuclear cells from seropositive adults that were pre-incubated with cell-free CMV antigen in bulk culture for 6 days had enhanced cytotoxic activity against CMV-infected target cells sharing one or more class I MHC determinants with the effector cell populations. Selective enrichment or depletion experiments were performed to characterize the effector cell populations involved in the augmented cytotoxic response following in vitro induction with CMV antigen. Purified E-rosette-forming cells expressed increased cytotoxic activity against both virus-infected fibroblasts and the NK target cell, K562. Depletion experiments with monoclonal antibodies and complement indicated that the CMV-specific cytotoxic response involves both an E-rosette positive, T8+, M1- T-cell subset that most likely represents CMV-specific Tc, and an NK population that can be induced to differentiate by in vitro stimulation with CMV antigen following quantitative NK-cell depletion with monoclonal antibody, B73.1. PMID- 2994925 TI - Immunotoxicology. PMID- 2994926 TI - Age-dependent alterations of Fc gamma receptor-mediated effector functions of human polymorphonuclear leucocytes. AB - Changes in the effector functions in polymorphonuclear leucocytes (PMNL), harvested from blood of young and aged healthy subjects of both sexes, were studied. FC gamma-receptor (Fc gamma R)-mediated incorporation of IgG coated 51Cr HRBC significantly increased in the aged male group, while the phagocytosis of pre-opsonized fungi (Saccharomyces cerevisiae and Candida albicans) was independent of both the age and sex. However, the intracellular killing capacity of neutrophils obtained from aged male subjects significantly decreased toward 51Cr-labelled c. albicans. The antibody-dependent cellular cytotoxicity (ADCC) was also impaired with ageing in both sexes. The age-dependent decrease in the effector functions of PMNL may be explained, among others, by the fact that during yeast cell incorporation the increased cAMP level does not return to the basic level in the old group. On the other hand, the cGMP level which increased in PMNL of aged subjects does not show any progressive increase as in the young subjects, but remains unchanged. The oxidative metabolism producing free radicals being necessary for the effective intracellular killing and ADCC diminished in PMNL of aged subjects of both sexes. The above findings indicate that the adaptation of cyclic nucleotide system and the oxidative burst to the cell activation becomes impaired with ageing. PMID- 2994928 TI - Polyclonal B cell hyperplasia associated with Epstein-Barr virus causing acute renal allograft failure. AB - Six weeks post cadaver renal transplantation, a patient developed a flu-like illness. Acute renal failure unresponsive to anti-rejection therapy occurred and he died four days later from Pneumocystis carinii pneumonia and Streptococcus viridans septicemia. Autopsy revealed a diffuse polymorphic polyclonal B cell infiltrate occupying most organs, including the allograft. Primary Epstein-Barr Virus (EBV) infection was established by 1) rising anti-EBV antibody titres; 2) the demonstration of EBV nuclear antigen in the infiltrate and 3) the presence of EBV specific DNA sequences in affected tissues. EBV associated polymorphic B cell hyperplasia can mimic rejection and result in acute allograft failure. PMID- 2994927 TI - Altered configuration of Gc on the plasma membrane of transformed and malignant human B lymphocytes. AB - Normal human peripheral blood B cells exhibit strong membrane fluorescence for Gc (vitamin D-binding protein), and this protein can form a close spatial relationship with integral membrane immunoglobulin (mIg) with evidence of codistribution in the lipid bilayer. In contrast, fluorescence for both Gc and mIg has been found in this study to be weak or absent in several B lymphoblastoid cell lines and in chronic lymphocytic leukemia B cells. Moreover, the comobility of these components, where detectable, was also impaired. In abnormal B cells, the intensity of membrane fluorescence for Gc was substantially increased after crosslinking of mIg with antibody, and the latter was also associated with increased specific radioiodination of Gc by lactoperioxidase. These results indicate that Gc can apparently become displaced under certain circumstances within or through the lipid bilayer. The altered content or membrane topography of Gc in such abnormal B cells might be associated with impaired expression and mobility of mIg. PMID- 2994929 TI - Formation of the fibrin clot: the balance of procoagulant and inhibitory factors. AB - Let us now briefly summarize some major known regulating mechanisms, most of which have already been discussed. A general regulating feature of the coagulation system is provided by the cofactors HMW-kininogen, tissue factor, factor V(a), factor VIII:C(a), protein S and thrombomodulin. Tissue factor and thrombomodulin, as cell membrane constituents, and the other cofactors, thanks to their affinity for certain surface sites, localize coagulation reactions and thus avoid generalized intravascular thrombosis when the clotting system is triggered. Thrombin activates factors V and VIII:C and activated protein C inactivates factors Va and VIII:Ca. Thrombin is regulated by AT III, alpha 2M and possibly heparin-cofactor II, whereby endothelial-cell-bound heparin-like molecules enhance thrombin neutralization. Moreover, binding of thrombin to thrombomodulin abolishes its clotting activity, at least in the case of rabbit thrombomodulin. Thrombin is able to cleave PT-fragment 1 from prothrombin, thus generating prethrombin 1, which lacks the gla-region and does not bind to phospholipids. The hypothesis that thrombin may regulate its own formation by this negative feedback, however, must probably be discarded, because no corresponding fragments are found after blood clotting in vitro (Aronson et al, 1977). Factor Xa and factor IXa are inhibited by AT III and endogenous heparin probably enhances their inactivation. However, phospholipid-bound factor Xa in the presence of factor Va (Marciniak, 1973) and phospholipid-bound factor IXa (Varadi and Elodi, 1982) are relatively protected from inhibition. Platelet-bound factor Xa is completely protected from AT III, even in the presence of heparin (Miletich et al, 1978). Thus, specific cell surface sites modulate the inhibition of proteases in situ. Factor XIa is inhibited by several protease inhibitors, the most important being alpha 1-AT. beta-factor XIIa is inhibited mainly by C1-inhibitor and kallikrein by both C1-inhibitor and alpha 2M. No serine protease inhibitor for factor VIIa is as yet known. However, after initial rapid activation by factor Xa, factor VIIa is subsequently proteolytically inactivated by factor Xa, resulting in a transient burst of factor Xa generation by factor VIIa (Morrison and Jesty, 1984). This proteolytic regulation of factor VIIa by factor Xa dampens factor IX or factor X activation via tissue factor-factor VIIa by feedback proteolytic inhibition and this may constitute a major regulatory mechanism for factor VIIa.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2994930 TI - The role of endothelium in the homeostatic balance of haemostasis. AB - As the cells forming the luminal vascular surface, endothelial cells are strategically positioned to play an important role in the regulation of coagulation. They cannot be regarded as an inert surface lining the vessel wall since they possess multiple activities. Anticoagulant properties include provision of a cell surface with heparin-like molecules (which can serve as binding sites for antithrombin III), synthesis of thrombomodulin (which alters the substrate specificity of thrombin), maintenance of a low level of tissue factor and generation of prostacyclin. In addition to these anticoagulant properties, endothelial cells can play a role in procoagulant reactions. Studies which have examined mechanisms underlying the localization of thrombotic processes have suggested the possible involvement of endothelial cells in procoagulant events. Endothelial cells have been found to propagate factor X and prothrombin activation once factor IXa and factor Xa have been formed. Factors regulating the balance of plasminogen activator and inhibitor synthesis are also under study. Perturbation of endothelial cells with induction of tissue factor and production of platelet-activating factor and thromboxane provides a model of the thrombotic state in which endothelium can promote coagulation. The multiple properties of endothelial cells indicate that a continuous blood flow in an unperturbed region of the vessel wall results from a complex interplay of anticoagulant and procoagulant activities. In a perturbed region, endothelial cells might initiate coagulation and the thrombin formed on the surface of endothelial cells might then lead to recruitment of platelets. PMID- 2994931 TI - Rest-exercise radionuclide angiographic assessment of left ventricular function in chronic aortic regurgitation: significance of serial studies in medically versus surgically treated groups. AB - Forty consecutive asymptomatic patients with chronic aortic regurgitation who underwent three serial yearly rest and postexercise radionuclide angiograms were compared with 27 consecutive patients with chronic aortic regurgitation and aortic valve replacement who were studied preoperatively, 3 and 15 months postoperatively. Patients were divided into four subgroups based upon the resting left ventricular ejection fraction and the functional reserve on the initial study. Of the 40 medically treated patients, 19 (47.5%) and 24 (60%) demonstrated a response at least one type lower at 12 months and 24 months, respectively. Initial functional reserve, initial duration of exercise, and the change in exercise duration during the 24 months was not associated with changes in resting or postexercise left ventricular ejection fraction. A seesaw pattern was observed between the resting and the postexercise left ventricular ejection fraction as ventricular function deteriorated. We observed in the surgical groups a reversal of the seesaw interaction between the resting and postexercise ejection fraction seen in the medical patients. In the surgical groups the left ventricular end diastolic pressure, initial functional reserve, initial duration of exercise, and change in exercise duration postoperatively were not predictors of improvement in left ventricular function at 15 months. Comparing medical and surgical serial data, we suggest yearly radionuclide angiographic determination of rest left ventricular ejection fraction in asymptomatic patients with chronic aortic regurgitation. When the rest ejection fraction is less than 50%, exercise angiography should be performed to determine functional reserve. When functional reserve is also abnormal, surgery should be recommended. PMID- 2994932 TI - Effect of enalapril on renin, angiotensin converting enzyme activity, aldosterone and prostaglandins in patients with hypertension. AB - Enalapril (MK-421) was administered orally as a single dose of 2.5, 5.0, 10 and 20 mg to 13 patients with either essential or renovascular hypertension. At these doses, enalapril produced a moderate reduction in both supine and standing blood pressure as well as a significant reduction in angiotensin I-converting enzyme activity, an increase in peripheral plasma renin activity and a decrease in plasma aldosterone concentration 4 to 8 hours after administration of the drug. Plasma levels of prostaglandins E1 and E2 were unchanged. The calculated ratio of urinary Na/K was increased in the patients with renal artery stenosis after enalapril. Creatinine clearance was increased in the patients with essential hypertension and reduced in the patients with renal artery stenosis. No adverse effects occurred in these patients treated with single doses of enalapril. PMID- 2994934 TI - Preoperative scintigraphic detection of cervical metastases from thyroid carcinoma with technetium-99m pertechnetate. AB - A young man with papillary-follicular thyroid carcinoma demonstrated clear visualization of cervical metastases from thyroid cancer on Tc-99m pertechnetate scintigraphy while exhibiting a palpably and scintigraphically normal thyroid gland. This is a very rare occurrence which demonstrates that Tc-99m pertechnetate scintigraphy is capable of detecting cervical metastases from thyroid carcinoma before the appearance of palpable thyroid nodules or defects on scintigraphy. PMID- 2994933 TI - Modulation of right and left ventricular wall thicknesses in experimental hypertension. AB - Enalapril maleate and hydrochlorothiazide were administered over an 8-week period to groups of Dahl salt-sensitive and Dahl salt-resistant rats receiving either a high (8%) or low (0.4%) salt diet. Regional differences of interventricular septal thickness, left ventricular free wall thickness, right ventricular free wall thickness, right ventricular weight/body weight ratio, left ventricular weight/body ratio, and heart weights were determined. For salt-sensitive rats of both diet groups, lowering of blood pressure induced by either drug was associated with a reduction in cardiac weight which was localized to the left ventricle. For salt-resistant rats, irrespective of diet, the associated reduction of cardiac mass induced by enalapril maleate was largely confined to the right ventricle. Regional changes in tissue thickness were, however, not always associated with corresponding changes in tissue mass or blood pressure. These regional modulations appeared to be quite dependent on salt intake and the nature of the antihypertensive drug, for a given animal type, suggesting that pressure afterload is not the only factor in the pathogenesis of left and right ventricular hypertrophy. The responses of the right ventricle were not necessarily the same as those of the left ventricle to identical dietary and drug regimens. In all animal groups, ventricular mass and tissue thickness did not always change in the same sense, suggesting that alterations in the nature and packaging of the cellular constituents may be induced by the combined actions of the dietary salt and antihypertensive medications. PMID- 2994935 TI - Calyceal pertechnetate as a false-positive focus of ectopic gastric mucosa. AB - This case report describes a patient in whom concentration of Tc-99m pertechnetate in a renal calyx created a false diagnostic impression of ectopic gastric mucosa. The administration of furosemide demonstrated the benign cause of the focus. PMID- 2994937 TI - Radionuclide angiography. Parotid hemangioma. PMID- 2994936 TI - Technetium-99m glucoheptonate as a scanning agent in hepatocellular carcinoma. AB - Technetium-99m glucoheptonate (Tc-99m GH) is concentrated in pulmonary and cerebral tumors. The purpose of this study was to assess the uptake of this radionuclide by hepatocellular carcinoma. Its concentration by the primary tumor was compared with that in the non-neoplastic hepatic tissue in 31 patients who showed obvious defects on a colloid scan, and its uptake by pulmonary metastases was examined in six patients with x-ray evidence of this complication. In two patients, the uptake by the tumor was greater than, in six it was equal to, and in ten it was less than that in the non-neoplastic hepatic tissue. In the remaining 13 patients, there was no concentration at all in the tumor. In none of the six patients with multiple pulmonary metastases could uptake of Tc-99m GH by the metastases be demonstrated. It is concluded that Tc-99m GH is of limited value in the diagnosis of primary or metastatic hepatocellular carcinoma. PMID- 2994938 TI - Clinical pharmacokinetics of the angiotensin converting enzyme inhibitors. A review. AB - The angiotensin converting enzyme inhibitors are an important therapeutic advance in the treatment of patients with hypertension and congestive heart failure. In addition, they are useful pharmacological probes to assess the contribution of the renin-angiotensin system to circulatory homeostasis. Captopril was the first angiotensin converting enzyme inhibitor approved for use in patients with hypertension and congestive heart failure. It is rapidly absorbed from the gastrointestinal tract, with detectable plasma concentrations apparent as early as 15 minutes. The extent of absorption is between 60 and 75% of an oral dose and peak plasma concentrations occur after approximately one hour. Captopril is primarily excreted by the kidneys via renal tubular secretion. Renal excretion is rapid, with 90% completed in the first 4 hours. The elimination half-life for unchanged captopril is about 1.7 hours and is markedly increased in the presence of renal insufficiency. Once absorbed, captopril is extensively metabolised to several forms, including a disulphide dimer of captopril, a captopril-cysteine disulphide, and other mixed disulphides with endogenous thiol compounds. It is probable that captopril and its pool of metabolites undergo reversible interconversions. Pharmacokinetic properties of captopril in patients with uncomplicated hypertension appear to be the same as in healthy subjects. However, long term administration of captopril leads to increased concentrations of total captopril, probably from the accumulation of captopril metabolites. Despite the number of potential influences on pharmacokinetic properties in patients with congestive heart failure, due to the many abnormalities in gastrointestinal tract oedema and reductions in splanchnic and renal blood flow, the available data suggest that its pharmacokinetic properties in patients with congestive heart failure resemble those in healthy subjects. However, additional data are necessary to confirm this. Enalapril is the second angiotensin converting enzyme inhibitor to become available. Enalapril is a prodrug that is well absorbed from the gastrointestinal tract, with 60 to 70% of an oral dose being absorbed. However, enalapril must be converted by hepatic esterases to the active form, enalaprilat. After the oral administration of enalapril, the tmax for enalapril is one hour, but for enalaprilat it is 4 hours. There is a prolonged terminal elimination phase with enalaprilat being detectable as late as 96 hours after dosing. Thus, enalapril has a much longer duration of action than captopril. Like captopril, enalapril is primarily excreted by the kidneys.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2994940 TI - (Na+, K+)-ATPase regulation and NaCl intake: effects on circulating inhibitor and sensitivity to noradrenaline. AB - These experiments examined the effects of a high NaCl diet on (Na+,K+)-ATPase in kidney, heart and cerebral cortex, on the level of circulating inhibitor of (Na+,K+)-ATPase in plasma, and on stimulation of (Na+,K+)-ATPase by treatment with dextro (d)-amphetamine. High salt diet increased indices of (Na+,K+)-ATPase activity (K+-activated p-nitrophenyl-phosphatase activity and ouabain binding) in kidney medulla, prevented stimulation by amphetamine in cerebral cortex and reduced amphetamine stimulation in heart. High NaCl feeding increased the plasma level of circulating inhibitor of (Na+,K+)-ATPase. Amphetamine alone had no effect on inhibitor level but amphetamine administration reduced the increase in inhibitor with high NaCl feeding. PMID- 2994939 TI - Adrenergic receptors: molecular mechanisms of clinically relevant regulation. PMID- 2994941 TI - [Adverse effects during chronic treatment with low-dose amiodarone]. PMID- 2994942 TI - Plasmid fingerprinting. A tool for bacterial strain identification and surveillance of nosocomial and community-acquired infections. AB - Plasmid fingerprinting provides a rapid and dependable means of identifying bacterial isolates of the same strain. The stability, wide distribution, and diverse nature and size of extrachromosomal elements make it suitable for virtually all bacterial genera. There are many different procedures available for plasmid screening, and the one chosen depends primarily on the types of organisms to be analyzed. Some procedures are better suited to gram-positive organisms; others are better for visualizing the very large plasmids often seen in Pseudomonas and Rhizobium species. The key to the plasmid fingerprinting technique is agarose gel electrophoresis. In this step of the technique, it is important to differentiate open circular from closed circular forms of plasmids and to recognize the "smile effect." Plasmid fingerprinting can be utilized for epidemiologic studies of both nosocomial and community-acquired infections. The use of restriction endonuclease analysis can greatly enhance the ability of the investigator to differentiate strains that harbor only a single plasmid. Plasmid fingerprinting often provides the only differential characteristic for strains involved in epidemics. PMID- 2994943 TI - Use of nonradioactive DNA probes for the detection of infectious bacteria. AB - Application of the biotinylated DNA probe systems in the detection of Chlamydia trachomatis and penicillin-resistant Neisseria gonorrhoeae is presented. The three formats employed are the dot-blot, in-situ, and Southern hybridization. PMID- 2994944 TI - Microbial diagnosis by nucleic acid sandwich hybridization. AB - Sandwich hybridization is a three-component nucleic acid hybridization method suitable for the identification of microbes. In this method, one specific DNA fragment on solid support acts as catching reagent, and the second reagent is a labeled probe. The labeling of the support is mediated by a specimen nucleic acid homologous to both reagents. Because the specimen is kept in solution, relatively crude specimens not requiring elaborate pretreatments can be tested without background problems. The utility of the method in microbial diagnosis (adenovirus, cytomegalovirus, and Chlamydia trachomatis) has been demonstrated. Increased sensitivity and nonradioactive detection methods will no doubt further extend the applicability of the sandwich hybridization method. PMID- 2994945 TI - Spot hybridization for the detection of adenoviruses and enteroviruses. AB - Nucleic acid spot hybridization was used for detection of adenovirus DNA directly in clinical specimens and enterovirus RNA in infected cells. The test gave results comparable to those obtained with antigen detection assays for adenoviruses. Spot hybridization could also be used for detection of enterovirus growth in cell cultures after a single passage from clinical specimens. PMID- 2994946 TI - Monoclonal antibodies for the diagnosis of sexually transmitted diseases. AB - Monoclonal antibodies are already being used for the diagnosis of human sexually transmitted diseases. These antibodies can be used to detect a wide range of microorganisms, including bacteria, parasites, and viruses. For both culture and direct tests, monoclonal antibodies showed patterns of specificity and reproducibility that exceeded those available with conventionally prepared antisera. The direct tests for these organisms required less than an hour to perform, representing a major advancement in a diagnosis that previously required 2 to 6 days of culture followed by confirmatory testing. Furthermore, rapid differential diagnosis of infection will now be possible. Because some sexually transmitted diseases may be transmitted simultaneously and share similar clinical manifestations (that is, gonorrhea and chlamydia in cervicitis or urethritis, syphilis or herpes in genital ulcers), it will be possible to differentiate a single from a multiple infection by simultaneous testing of direct samples with the appropriate monoclonal antibody reagents. PMID- 2994947 TI - Inflammation in osteoarthritis. PMID- 2994948 TI - Crystal deposition in osteoarthritis: an opportunistic event? PMID- 2994949 TI - Neuromuscular syndromes associated with cancer. PMID- 2994950 TI - Metabolic differentiation of red and white skeletal muscle in vivo and in monolayer culture. AB - Cytochrome oxidase, succinate oxidase and lactate dehydrogenase were compared in: (a) leg and breast muscle from 11-19-day-old chick embryos; and (b) 2, 6, 10 and 14-day-old primary cell cultures established from myoblasts of embryonic leg and breast muscle. Cytochrome oxidase, succinate oxidase and lactate dehydrogenase activities were higher (48.8, 65.4, 277.6%, respectively) in leg muscle after 19 days in ovo. Cytochrome and succinate oxidase activities were higher (111.3, 48.1%, respectively) in leg muscle cell cultures after 14 days in vitro. The data represent evidence for intrinsic developmental patterns for certain enzymes. PMID- 2994951 TI - Purification and properties of the phosphoglycerate mutase isozymes from the mouse. AB - The two homodimeric isozymes of phosphoglycerate mutase have been purified from murine kidney and muscle. No differences were observed in the Michaelis-Menten constant for the substrate 2-phospho-D-glycerate, in molecular weight, temperature and pH optima, when the purified isozymes were compared. The isozymes differ in their inhibition constants for phosphoenolpyruvate, in their Michaelis constants for 3-phospho-D-glycerate and 2,3-bisphospho-D-glycerate, their thermal and pH lability and in their sensitivity towards mercury ions. PMID- 2994952 TI - Metabolism of glucose 1,6-P2--II. Glucose 1,6-P2 phosphatase in pig muscle. AB - Most of the glucose 1,6-P2 phosphatase activity of pig skeletal muscle is present in the cytosolic fraction. Four peaks of glucose 1,6-P2 phosphatase activity are obtained when the cytosolic fraction from pig muscle is subjected to DE-cellulose chromatography. All the peaks hydrolyze other phosphocompounds in addition to glucose 1,6-P2. The glucose 1,6-P2 phosphatase activity of the main peak shows an optimal neutral pH. It is activated by divalent cations, Mg2+ being more effective than Mn2+. The addition of Ca2+ or EGTA does not affect the enzymatic activity. IMP does not possess any effect. It is concluded that this enzyme is different from the glucose 1,6-P2 phosphatases found in mouse brain cytosol and rat skeletal muscle. PMID- 2994953 TI - The importance of membrane sulfhydryl groups to calcium homeostasis in the lens. AB - This study focused on whether changes in lens levels of glutathione and calcium, early events associated with cataract formation, were related or that one might cause the other. The first part of the investigation was concerned with the extent to which an increase in levels of intracellular calcium might alter GSH levels in lens fiber and epithelial cells. The results demonstrate that calcium accumulation, either at 19 degrees C or 37 degrees C, did not diminish the concentration of GSH. More importantly, GSH levels did not decline in opaque regions of a calcium-loaded lens. The reciprocal part of the problem focused on whether a decline in lens thiol might lead to an increase in levels of calcium and subsequent opacification. In particular, it was shown that treatment of lenses with parachloromercuribenzene sulphonic acid (PCMBS), a nonpenetrating sulphydryl probe, resulted in a 10-30% loss of membrane SH groups in the epithelium. Diminished numbers of SH groups was accompanied by chloride fluxes and an increase in membrane permeability to sodium and calcium with an influx of sodium and calcium leading to opacities. It is important to note that these changes occurred in the absence of any change in cellular levels of soluble protein-SH or GSH. Additional experiments suggest that calcium transport was not impaired, as evidenced by lack of inhibition of Ca-ATPase activity in lenses treated with PCMBS. The results suggest that one explanation for opacification is that oxidative insults, which diminish GSH levels, leads to a loss of important membrane SH groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2994954 TI - The BALB/c mouse as a model to study orthopoxviruses. PMID- 2994955 TI - Genetic control of multisystem autoimmune disease in encephalomyocarditis virus infected BALB/cCUM and BALB/cBYJ mice. PMID- 2994956 TI - Genetic differences in BALB/c sublines. PMID- 2994957 TI - Qa2 expression in BALB/c sublines and a BALB/cLAC tumor. PMID- 2994958 TI - The origin and meiotic instability of a polymorphic repetitive sequence PRI family. PMID- 2994959 TI - The genetic stability of endogenous type B and C retroviruses in BALB/c sublines. PMID- 2994960 TI - Sodium bicarbonate during CPR. Does it help or hinder? PMID- 2994961 TI - Reduced beta 2-adrenoceptor responsiveness in exercise-induced asthma. AB - Beta-adrenoceptor responsiveness was studied both in vivo and in vitro in patients with exercise-induced asthma (EIA), asthmatic patients without EIA (NEIA), and control subjects. All subjects were age- and sex-matched and without medication at least one week prior to the tests. In vivo, beta-adrenoceptor responsiveness was evaluated by plasma concentration-effect studies for intravenously infused isoprenaline (0.02-0.1 micrograms X kg-1 X min-1). Mainly beta 2-adrenoceptor mediated responses to isoprenaline, ie, decreases in diastolic blood pressure and increases in plasma cyclic AMP, were reduced in EIA patients but not in NEIA patients. Heart rate and plasma glycerol responses to isoprenaline did not differ between the groups. In vitro, the beta 2-adrenoceptor mediated accumulation of cyclic AMP in lymphocytes stimulated by isoprenaline was attenuated (p less than 0.05) in EIA patients, whereas the beta 2-adrenoceptor responsiveness of lymphocytes from NEIA patients was normal. Thus, beta 2 adrenoceptor mediated responses were reduced both in vivo and in vitro in EIA patients, but not in NEIA patients. This finding that beta 2-adrenoceptor responsiveness was reduced only in a subgroup of asthmatic patients could explain some of the controversies in the literature concerning beta-adrenoceptor function in asthma. PMID- 2994962 TI - Interactions of endothelial cells and smooth muscle cells of arteries. AB - Relaxation of selected isolated arteries by acetylcholine, bradykinin, and certain other vasodilators is mediated by a factor released from endothelial cells. Relaxation by the factor (chemical identity still unknown) is accompanied by an increase of cyclic GMP. The finding that hemoglobin inhibits both endothelium-dependent relaxation and the increase in cyclic GMP may have pathophysiologic relevance. PMID- 2994963 TI - Family-based, in-home services for the severely emotionally disturbed child. AB - FBS was not conceived as a substitute for a residential treatment program. It was instead established to provide a greater array of services in the center's continuum of care and another treatment alternative. Although children were accepted into the FBS program when residential treatment was indicated, it was not because the program was considered an equal to residential treatment. Rather, we felt the child and family could receive greater benefits from an in-home approach [Willner et al. 1972]. The results of intensive family-based services speak for themselves in terms of cost-effectiveness, placement prevention, and family reunification [Bryce and Lloyd 1980a]. It has been the center's experience that the most vulnerable children are those who have been removed for extended periods of time to an institutional setting. Results of table 1 indicate that 68% of the children receiving FBS aftercare remained in their homes, while children who were served before any placement remained in their homes 97% of the time. In spite of major behavioral changes experienced by the child and family during the residential course of treatment, the staff reports the following obstacles to successful reunification: Child "identified" as the problem is often expected to return home as a different human being without needs or problems. Temporary expulsion of the identified child often immediately relieves family stress, yet begins a process of homeostatic adjustment whereby the system closes, excluding the child.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2994964 TI - Lymphocyte subsets and EB virus antibodies in nasopharyngeal carcinoma. AB - Lymphocyte subsets and Epstein-Barr virus (EBV)-associated antibodies were studied in 108 patients with nasopharyngeal carcinoma (NPC) and in 34 normal controls. Lymphocyte subsets were identified with monoclonal antibodies (Ortho Co.) by indirect immunofluorescent antibody (IFA) method. The helper T lymphocytes (OKT4+) in NPC patients comprised 38.2 +/- 10.5% which is significantly different from 45.2 +/- 8.0% in controls. The helper/suppressor ratio in NPC patients was 1.33 +/- 0.65 which is significantly different from 1.64 +/- 0.48 in controls, but the ratio was not correlated with disease extent, sex, age, total lymphocyte counts, WBC counts and EBV-associated antibodies of NPC patients. There were no remarkable differences between NPC patients and controls in B lymphocytes (OKIa+), total T lymphocytes (OKT3+), and suppressor lymphocyte (OKT8+) percentages, total lymphocyte counts and WBC counts. The EBV associated antibodies were titrated by the IFA method using P3HR-1 cells and Raji cells induced by IUdR as target. Mean antibody titers and seropositive rates showed significant increase in NPC patients (1:12-1:502 and 45.4%-68.5%, respectively) compared with controls (1:1-1:87 and 1.0%-5.9%, respectively). The increase in antibodies was positively correlated with NPC disease extent, but was not correlated with the sex, age, total lymphocyte counts, helper/suppressor ratio, and WBC counts of NPC patients. PMID- 2994965 TI - [10 years' experience with the concept of breast-saving therapy in the treatment of breast cancer]. AB - A therapeutic concept dependent on staging of breast carcinoma is presented. In 1974 we started at the 2. Surgical University Clinic Vienna to use the non ablative treatment in patients with breast cancer smaller than 2 cm. Up to 1984 102 patients underwent quadrantectomy axillary dissection, and radiotherapy. With equal therapeutic results the smaller and cosmetically preferable surgical intervention is recommended. PMID- 2994966 TI - Three related centromere proteins are absent from the inactive centromere of a stable isodicentric chromosome. AB - We developed an aqueous spreading procedure that permits simultaneous analysis of human chromosomes by Q-banding and indirect immunofluorescence. Using this methodology and anticentromere antibodies from an autoimmune patient we compared the active and inactive centromeres of an isodicentric X chromosome. We show that a family of structurally related human centromere proteins (CENP-A, CENP-B, and CENP-C) is detectable only at the active centromere. These antigens therefore may be regarded both as morphological and functional markers for active centromeres. PMID- 2994968 TI - [CT diagnosis of primary hepatoma]. PMID- 2994967 TI - [Bronchial adenoma]. PMID- 2994969 TI - [Measurement of bronchial reactivity in asthmatics. III. Relation between bronchial reactivity to methacholine and plasma cyclic nucleotide monophosphate in asthmatic subjects]. PMID- 2994970 TI - Thyroid-dependent maturation of neonatal brain but not lung epidermal growth factor receptors. AB - Although the role of thyroid hormones in enhancing lung and brain maturation during the perinatal period is well established, the cellular mechanisms involved in these processes are incompletely understood. Hypothyroidism retards the development of fetal pulmonary insulin, neonatal pulmonary beta-adrenergic and neonatal brain insulin receptors. In this study, we investigated the effect of hypo- or hyperthyroidism on the development of neonatal brain and lung epidermal growth factor (EGF) receptors. The rabbit pups were rendered hypothyroid by adding 0.05% propylthiouracil to the drinking water starting at 23 days of gestation and thereafter. The neonatal hyperthyroid state was achieved by intramuscular administration of 100 micrograms/kg of synthroid to the rabbit doe on the 29th and 30th day of pregnancy. Neonatal plasma free thyroxine (T4) concentrations were quantitated by a radioimmunoassay. Brain and lung plasma membranes were isolated by differential centrifugation. EGF receptor characteristics were studied using 125I-EGF binding assays and Scatchard analysis. The plasma free T4 concentrations were 0.36 +/- (SEM) 0.02 (n = 6), p less than 0.01 (n = 7) and 1.76 +/- 0.1 (n = 6) ng/dl in the control, hypothyroid and hyperthyroid pups, respectively. The percent specific binding of 125I-EGF to 200 micrograms of brain plasma membrane (BPM) protein was significantly lower in the hypothyroid (0.62 +/- 0.03, n = 7, p less than 0.01), and higher in the thyroxine-treated (1.58 +/- 0.08, n = 6, p less than 0.01) group when compared to control (1.08 +/- 0.06, n = 6) animals. However, the percent specific binding of 125I-EGF to 100 micrograms of lung plasma membrane (LPM) protein was similar in all three groups (2.24 +/- 0.28, control; 2.01 +/- 0.5, hypothyroid, and 2.26 +/- 0.3, hyperthyroid). The number of EGF receptors per milligram of BPM protein (X 10(-10] were lower in the hypothyroid (2.24 +/- 0.03, n = 5) and higher in the hyperthyroid (6.6 +/- 0.02, n = 4) group when compared to control (4.4 +/- 0.05, n = 4) with no apparent difference in Kd. There was no difference in the number of EGF binding sites per milligram of LPM protein (X 10(-10] within the groups (6.6 +/- 0.8, n = 6, control; 7.9 +/- 0.4, n = 4, hypothyroid, and 7.3 +/- 0.3, n = 4, hyperthyroid). Presence of high affinity receptors for EGF in the neonatal brain as well as lung supports the hypothesis that EGF may play an important role in neonatal brain and lung maturation.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2994971 TI - Outpatient management of diabetes mellitus with patient education to increase dietary carbohydrate and fiber. AB - The impact of patient education on dietary fiber intake, diabetes control, and serum lipids was examined in patients with non-insulin-dependent diabetes mellitus. Customary outpatient personnel and procedures were used to teach three diet plans: the American Diabetes Association (ADA) diet, the ADA diet modified to increase high-fiber, high-carbohydrate foods (IF), and the IF diet supplemented with oat bran (IFOB). A control group was instructed on foot care to provide teaching visits. Fifty-two patients were recruited from an outpatient clinic and studied over a 6-wk period. Subjects were of low socioeconomic status and had completed a mean of 8.3 yr of education. Patients instructed to increase their intake of high-fiber foods reported a doubling of fiber intake and tolerated the diets well. Increased fiber and carbohydrate intake and decreased fat intake were associated with reductions in fasting plasma glucose levels. Increased fiber intake was also associated with reductions in total serum cholesterol and high-density lipoprotein cholesterol levels. Changes in fiber, carbohydrate, and fat intake were unrelated to changes in weight, serum insulin levels, or hemoglobin A1c levels over the study period. PMID- 2994972 TI - Studies on cellular tandemization of herpes simplex virus thymidine kinase DNA. AB - The cellular tandemization of the herpes simplex virus (HSV) thymidine kinase (tk) gene was studied in tk- mouse fibroblasts after gene transfer by microinjection into the nucleus or by calcium phosphate-mediated transfection. Three different DNA substrates, designed to yield simple integration patterns, were used: a gel-purified 3.6-kb Bam HI fragment containing the HSV tk gene; the same fragment self-ligated; and the 3.6-kb fragment ligated to a Bam HI-cleaved subset of genomic mouse DNA. The genomic DNA of six independently isolated transformed cell lines was analyzed by Southern blotting and the structure of the tk-specific DNA was studied. The data suggest that modifications (mutations, deletions, recombination events, and recircularization, etc.) of the input DNA fragment occur early after its introduction into the cell. Subsequently these structures are multiplied in a directional manner, generating larger arrays of DNA with distinct and regularly repeated areas. These concatemers can eventually be integrated into the host genome. PMID- 2994973 TI - Retinal degenerations and brain abnormalities in infants and young children. AB - Of 51 infants and children who presented with visual impairment, developmental delays, and suspected brain abnormalities, 28 (55%) had clinical, electroretinographic, and cranial computed tomographic results indicative of cerebroretinal disorders. This report concentrates on the electroretinographic and psychophysical results from 12 patients who had evidence of progression of both brain and retinal disease. We believe these patients represent human cerebroretinal degenerative disorders that have yet to be completely characterized. PMID- 2994974 TI - [Possible mechanisms of the occurrence of new loci of defective endogenous proviruses]. PMID- 2994975 TI - [Amino acid sequence of the 17 kDa fragment from the cytoplasmic region of the alpha-subunit of Na+, K+-ATPase]. PMID- 2994976 TI - [Physiological importance of transferrin]. PMID- 2994977 TI - [LAV/HTLV-III antibodies in blood and blood products]. PMID- 2994978 TI - [Histology of the primary tumor and subsequent metastasis in breast cancer]. AB - The relationship between the histology of the primary tumour and subsequent metastasization was analysed in 343 patients with disseminated carcinoma of the breast. The metastases were subdivided on clinical criteria into osseous, visceral and localized regional ones. It was found that undifferentiated, infiltrating ductal carcinomas without significant productive fibrosis have a special tendency towards visceral metastasization. Purely scirrhous carcinomas, especially without intraductal components, have a marked affinity to the skeletal systems. Intraductally growing and adenomatous carcinomas appear to metastasize ubiquitously. The more components there are to the primary tumour the more frequent are local recurrences and metastases to several organ systems. PMID- 2994979 TI - [Methods and importance of the separation of lipids and water in NMR tomography]. PMID- 2994981 TI - [Initial experiences in the treatment of virus-induced diarrheas in calves with interferon produced by genetic engineering]. PMID- 2994980 TI - [Modification of the Aujeszky's virus neutralization test attributable different test conditions]. PMID- 2994983 TI - A new angiotensin-converting enzyme inhibitor: treatment of hypertension and congestive heart failure. Proceedings of an international symposium on enalapril, sponsored by Merck Sharp & Dohme International, Sao Paulo, Brazil, November 10, 1984. PMID- 2994982 TI - History of the development of inhibitors of angiotensin I conversion. AB - The major steps in the initial development of angiotensin I conversion inhibitors are described, from the discovery of the Bothrops peptides (bradykinin potentiating factor) to the demonstration of the therapeutic potential. It is a history where chance, serendipity and clear scientific reasoning weaved together the work of several scientists. It is also a classical example of drug development for which the initial basic research was made at the university, but the useful product was achieved by industry. PMID- 2994984 TI - Enalapril: a review of human pharmacology. AB - Enalapril, an orally-active, long-acting, nonsulphydryl angiotensin-converting enzyme (ACE) inhibitor, is extensively hydrolysed in vivo to enalaprilat, its bioactive form. Bioactivation probably occurs in the liver. Metabolism beyond activation to enalaprilat is not observed in man. Administration with food does not affect the bioavailability of enalapril; excretion of enalapril and enalaprilat is primarily renal. Peak serum enalaprilat concentrations are reached 4 hours post-dose, and the profile is polyphasic with a prolonged terminal half life (greater than 30 hours) due to the binding of enalaprilat to ACE. Steady state is achieved by the fourth daily dose, with no evidence of accumulation. The effective accumulation half-life following multiple dosing is 11 hours. Higher serum concentrations and delayed urinary excretion occur in patients with severe renal insufficiency. Enalapril reduces blood pressure in hypertensive patients by decreasing systemic vascular resistance. The blood pressure reduction is not accompanied by an increase in heart rate. Furthermore, cardiac output is slightly increased and cardiovascular reflexes are not impaired. Once- and twice-daily dosage regimens reduce blood pressure to a similar extent. Enalapril increases renal blood flow and decreases renal vascular resistance. Enalapril also augments the glomerular filtration rate in patients with a glomerular filtration rate less than 80 ml/min. Enalapril reduces left ventricular mass, and does not affect cardiac function or myocardial perfusion during exercise. There is no rebound hypertension after enalapril therapy is stopped. Enalapril does not produce hypokalaemia, hyperglycaemia, hyperuricaemia or hypercholesterolaemia. When combined with hydrochlorothiazide, enalapril attenuates the undesirable diuretic induced metabolic changes. Therapeutic doses of enalapril do not affect serum prolactin and plasma cortisol in healthy volunteers or T3, rT3, T4 and TSH in hypertensive patients. Enalapril has natriuretic and uricosuric properties. The antihypertensive effect of enalapril is potentiated by hydrochlorothiazide, timolol and methyldopa, but unaffected by indomethacin and sulindac. No interactions occur between enalapril and frusemide, hydrochlorothiazide, digoxin and warfarin. The bioavailability of enalapril is slightly reduced when propranolol is coadministered, but this does not appear to be of any clinical significance. Enalapril increases cardiac output and stroke volume and decreases pulmonary capillary wedge pressure in patients with congestive heart failure refractory to conventional treatment with digitalis and diuretics.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2994985 TI - Cardiovascular pathophysiology of essential hypertension: a clue to therapy. AB - Arterial hypertension is by definition a haemodynamic disorder. At least 3 different subsets of cardiovascular pathophysiological features can be identified in so-called essential hypertension: The young lean patient characterised by an elevated cardiac output and renal blood flow, elevated plasma renin activity and circulating catecholamine levels, as well as symptoms and signs of hyperadrenergic hypertension. The elderly patient characterised by a low cardiac output often with left ventricular hypertrophy, elevated total peripheral resistance, nephrosclerosis, and symptoms and signs of target organ disease. The obese patient (and to a lesser degree the black patient) characterised by expanded fluid volume state, elevated cardiac output, a normal to low total peripheral resistance, and symptoms and signs of volume overload. To initiate antihypertensive therapy, the drug of choice in the young patient is a beta adrenergic receptor blocker; in the elderly it is a haemodynamic vasodilator (anti-adrenergic drug, slow channel calcium blocker, or converting enzyme (ACE) inhibitor), and in black or obese patients it remains a thiazide diuretic. Enalapril, a new ACE inhibitor is indicated as a first-step agent in the great majority of hypertensive patients in whom the elevated arterial pressure should be reduced by a decrease in total peripheral resistance, without compromising systemic or regional blood flow. In contrast to other antihypertensive agents, enalapril will lower preload and afterload to the left ventricle while improving systemic and regional flow in elderly patients with latent or manifest congestive heart failure. PMID- 2994986 TI - Enalapril in essential hypertension. AB - It is now well recognised that the renin-angiotensin system plays a key role in the control of blood pressure not only through circulating angiotensin II but through its interaction with the autonomic and central nervous systems. Angiotensin-converting enzyme (ACE) inhibitors have proved to be effective in lowering blood pressure in different types of hypertension. This study evaluates the antihypertensive effects of enalapril, a new, potent, long acting ACE inhibitor. 50 patients with uncomplicated essential hypertension were included in 4 groups. Group I was used to compare the effects of enalapril and propranolol on blood pressure, renal function, plasma renin activity, aldosterone excretion and plasma lipids in 24 patients after 23 weeks. Group II was used to evaluate long term effects (48 weeks) of these drugs in 13 patients. Group III included 32 patients that received enalapril as monotherapy for 6 to 12 weeks. Group IV was studied to estimate the antihypertensive effect of low doses of hydrochlorothiazide in 18 patients receiving enalapril. The effect on mean blood pressure was similar with enalapril and propranolol (enalapril 117 versus 103 mm Hg and propranolol 115 versus 104 mm Hg); however, the glomerular filtration rate decreased with propranolol (105 versus 87 ml/min; p less than 0.05) and was unaltered with enalapril (102 versus 98 ml/min). Triglycerides rose with propranolol (179 versus 231 mg/dl; p less than 0.05) and did not change with enalapril (157 versus 121 mg/dl). In the long term, antihypertensive effects were similar and no significant side effects were observed. In 14/32 patients blood pressure became normal with enalapril alone. Low doses of hydrochlorothiazide (12.5 to 25 mg) decreased mean blood pressure by 10mm Hg when added to enalapril. The antihypertensive effect of enalapril was similar to that of propranolol; however, the lowering effect on glomerular filtration rate of propranolol did not occur with enalapril. A slight rise in triglycerides occurred only with propranolol. No significant side effects were observed with either propranolol or enalapril. Used as monotherapy, enalapril normalised blood pressure in 44% of cases. Addition of low doses of hydrochlorothiazide significantly increased the antihypertensive effect of enalapril. PMID- 2994988 TI - Enalapril versus triple-drug therapy in the treatment of renovascular hypertension. AB - 18 renovascular hypertensive patients were entered into a randomised, double blind protocol to assess the safety and efficacy of enalapril (5 to 20 mg twice daily) and hydrochlorothiazide (50 to 100 mg/day), versus triple-drug therapy employing hydrochlorothiazide (50 to 100 mg/day), timolol (10 to 30 mg twice daily) and hydralazine (50 to 150 mg twice-daily). Specifically monitored were the effects of each drug regimen on blood pressure, plasma renin activity and angiotensin II, glomerular filtration rate by insulin clearance, and effective renal plasma flow by para-aminohippurate clearance. Results indicate that enalapril/hydrochlorothiazide was more effective than triple-drug therapy in lowering blood pressure. All patients on enalapril/hydrochlorothiazide had excellent control of blood pressure, and there were no adverse effects. In contrast, 50% of the patients on triple-drug therapy had either uncontrolled blood pressure or significant drug-related side effects. Patients who were uncontrolled or intolerant of triple-drug therapy were well controlled on enalapril/hydrochlorothiazide. Patients on enalapril/hydrochlorothiazide demonstrated stimulation of plasma renin activity with inhibition of plasma angiotensin II, indicating adherence with therapy. Therapy for both unilateral and bilateral renovascular hypertension with enalapril/hydrochlorothiazide did not result in reductions in either glomerular filtration rate or effective renal plasma flow, except in 1 patient with a functional solitary stenotic kidney. In contrast, triple-drug therapy was generally associated with modest reductions in glomerular filtration rate and effective renal plasma flow, with a severe reduction in glomerular filtration rate and effective renal plasma flow occurring in 1 patient with bilateral symmetrical renovascular disease. We conclude that the combination of enalapril and hydrochlorothiazide is a safer and more effective regimen, compared with triple-drug therapy, for the treatment of renovascular hypertension. PMID- 2994989 TI - Enalapril maleate versus captopril. A comparison of the hormonal and antihypertensive effects. AB - 24 hypertensive patients were randomised into 2 groups to compare the antihypertensive effects of enalapril and captopril over a 10-week period. In the hydrochlorothiazide run-in period, blood pressure was reduced from 171 +/- 4/109 +/- 1mm Hg to 160 +/- 4/103 +/- 1mm Hg (p less than 0.05). Angiotensin-converting enzyme (ACE) inhibition decreased blood pressure to 132 +/- 3/87 +/- 2mm Hg. Captopril decreased diastolic blood pressure significantly more after 3 hours than enalapril (-24 versus -17mm Hg, p less than 0.05). After 10 weeks of therapy, this antihypertensive response was maintained at 134 +/- 3/83 +/- 1mm Hg. There was no difference between the captopril and enalapril treated groups. Acute and chronic responses of plasma renin activity, plasma aldosterone and ACE were determined. There was an acute positive correlation between the rise in plasma renin activity and the fall in blood pressures with captopril but not with enalapril. With chronic treatment there was no difference in the ability of either of the 2 drugs to reduce blood pressure, inhibit ACE, reduce aldosterone or stimulate plasma renin activity. PMID- 2994987 TI - Natriuretic effect and changes in renal haemodynamics induced by enalapril in essential hypertension. AB - The purpose of this study was to evaluate the natriuretic effect and renal haemodynamic changes induced by enalapril in patients with essential hypertension. In a group of 11 patients with mild to moderate hypertension with normal renal function, and on a controlled sodium intake (80 mmol/day), a decrease in systolic and diastolic blood pressure was observed (p less than 0.001) after 16 weeks of enalapril treatment (20 mg/day), without a change in heart rate. An increase in plasma renin activity (p less than 0.05) without changes in serum aldosterone, and a decrease in exchangeable sodium (p less than 0.001) were present at the end of the treatment period. In 10 hypertensive patients also taking a dietary sodium of 80 mmol/day, the renal haemodynamics, humoral changes, and urinary sodium excretion were measured during 4 days of enalapril treatment (20 mg/day). There was an increase in urinary sodium excretion on the 3rd and 4th days of treatment (p less than 0.01). The effective renal plasma flow and fractional sodium excretion increased 72 hours after the beginning of treatment (p less than 0.01); the glomerular filtration rate did not change, and filtration fraction decreased at 72 hours. Mean blood pressure fell 2 hours after the first dose (p less than 0.01), and the maximum drop in intrarenal vascular resistance occurred after 72 hours of treatment (p less than 0.01). Plasma renin activity increased (p less than 0.05) and serum aldosterone decreased (p less than 0.01) 2 hours after the first dose. Thereafter, serum aldosterone increased progressively until it reached values similar to those with placebo at 48 and 72 hours of treatment. Urinary kallikrein fell during the 2nd and 3rd day of treatment (p less than 0.01). It was concluded that the decrease in exchangeable sodium was due to a natriuretic effect of enalapril. This effect presumably results from renal haemodynamic changes due to the reduction of angiotensin II. Other mechanisms, such as the reduction of aldosterone and accumulation of kinins, could be contributory factors. PMID- 2994991 TI - Massive haemoperitoneum following rupture of hepatoma: report of three autopsy cases. PMID- 2994990 TI - Effects of enalapril on clinical status, biochemistry, exercise performance and haemodynamics in heart failure. AB - The effects of enalapril on clinical well-being, treadmill exercise performance, haemodynamic measurements, hormone levels, and plasma biochemistry in patients with moderate heart failure, were assessed in a 12-week placebo-controlled, double-blind study. Maintenance frusemide and digoxin treatment was continued throughout the study. Compared with placebo, enalapril treatment improved clinical status and increased exercise capacity. The most obvious haemodynamic change was a fall in pulmonary artery wedge pressure and pulmonary artery pressure. Enalapril-induced increases in left-ventricular ejection fraction and cardiac index, and falls in systemic arterial pressure, were small. Of the hormone indices measured, plasma renin activity rose 4-fold, angiotensin II and aldosterone fell slightly, and plasma catecholamines were unaltered by enalapril. Plasma potassium increased on average by 0.3 mmol/L during enalapril therapy. No adverse clinical or biochemical effects were observed. Enalapril has a sustained beneficial action in patients with moderate heart failure. PMID- 2994993 TI - [Changes in rat pleural mesothelium as affected by intrapleural administration of asbestos]. AB - When studying total pleural mesothelium films in rats after intrapleural injection of chrysotile the pathological regeneration of the mesothelium is observed. During 12 months of the experiment rare mitoses, multinuclear cells and symplasts were found. PMID- 2994992 TI - [Human breast cancer markers]. AB - The review contains data concerning breast cancer markers used now in clinical oncology and potential breast cancer markers. Their origin, metabolism, detection methods, potentialities of application for tumour diagnosis, determination of therapy efficiency and prognosis are discussed. PMID- 2994994 TI - [Change in the protein kinase activity of glandular stomach mucosa in rats during malignant transformation]. AB - Changes in the activity of protein kinases in the glandular stomach mucosa of rat were studied in the case of carcinogenesis induced by N-methyl-N'-nitro-N' nitrosoguanidine. No essential changes in the activity of cAMP-dependent protein kinase (histone kinase) were found in the mucosa as well as in tissues of the developed tumours of the rat glandular stomach. The activity of cAMP-independent protein kinase (casein kinase) increased significantly 3 months after the beginning of the carcinogen administration and at the late stages (after 12-15 months) it was decreased considerably both in the glandular stomach tumours and in the stomach mucosa without the characters of the malignant growth. It is supposed that changes in the activity of casein kinase in the stomach mucosa at the late carcinogenesis stages are associated with the neoplastic transformation and precede the appearance of morphological characters of the malignization. PMID- 2994995 TI - [Effect of carcinogenic substances on bone marrow cells in CBA mice]. AB - The effect of some carcinogenic and non-carcinogenic agents on the content of CFUs and frequency of micronuclei (Howell-Jolly corps) in polychromatic erythrocytes of bone marrow cells was studied in CBA mice. Benzene administration decreased the content of CFUs in bone marrow cells, but this effect is considered to be rather a sign of its hematotropic than carcinogenic action. Changes in the CFUs amount induced by chrysotile-asbestos and quartz DQ-12 action were insignificant. All investigated carcinogenic agents (benz(a)pyrene, asbestos and benzene) significantly increased the number of micronuclei in polychromatic erythrocytes of bone marrow in mice. PMID- 2994996 TI - Effect of repeated immobilization stress on central and peripheral adrenoceptors in rats. AB - Repeated forced immobilization stress (40 times for 2.5 h daily) reduced the number of beta-receptors in the heart, hypothalamus and brain stem, but not in the spleen of rats. Repeated immobilization stress has also been found to decrease the number of alpha1-receptors in the heart and increase the number of alpha2-receptors in the spleen and brain stem, as compared to control unstressed rats. The number of heart alpha1-, spleen and brain stem alpha2-receptors was still decreased or increased, respectively, 24 h after the 39th immobilization stress. However, at the same time the number of beta-receptors in the heart and brain stem returned to the control levels. We concluded, that the changes in the number of rat adrenoceptors in the heart, hypothalamus and brain stem correlate with peripheral catecholamines released during repeated immobilization stress. PMID- 2994997 TI - Protein kinase activity and protein kinase inhibitor in mouse kidney: effect of the X-linked Hyp mutation and vitamin D status. AB - cAMP-dependent protein kinase, Ca+2- and phospholipid-dependent protein kinase, and protein kinase inhibitor activity were examined in renal homogenates and 20,000 X g supernatant fractions of normal and Hyp mice. In both genotypes, 70% of total renal cAMP-dependent protein kinase activity was recovered in the soluble fraction in which the activity ratio (without cAMP to with cAMP) of the enzyme was 0.35. The requirement for cAMP was not different for protein kinase of normal and mutant littermates, with an apparent Km for cAMP of 0.05 microM in both genotypes. Furthermore, vitamin D and calcium deficiencies did not significantly affect cAMP-dependent protein kinase activity in normal and Hyp mouse kidney. The concentration of the heat-stable protein kinase inhibitor protein in the 20,000 X g supernatant fraction was identical in normal and Hyp kidney. Whereas protein kinase inhibitor levels were increased 1.8-fold by vitamin D and calcium deficiencies in normal mice (P less than 0.001), no such increase was detectable in Hyp mice. Ca+2- and phospholipid-dependent-protein kinase (protein kinase C) activity in the 20,000 X g supernatant fraction comprised 50% of the total activity of kidney homogenates of both normal and mutant mice. The initial rate of protein kinase C was increased 1.5-fold in kidney supernatants of Hyp mice (P less than 0.001). In contrast, protein kinase C was not significantly different from normal in supernatant fractions of heart, spleen, and liver prepared from Hyp mice. The present demonstration of abnormally high renal protein kinase C activity in Hyp mice may serve to explain the relationship between the previously reported renal defects in brush border membrane phosphate transport and vitamin D metabolism in the mutant strain and elucidate the nature of the primary defect in the Hyp mouse. PMID- 2994999 TI - Studies of nuclear 3,5,3'-triiodothyronine binding in primary cultures of rat brain. AB - Primary cultures of enzymatically dispersed cells from 17-day-old fetal rat cerebral hemispheres were used to detect the presence of nuclear T3 receptors. Cells grown in Minimum Essential Medium supplemented with 10% fetal bovine serum were grown in parallel with cytosine-arabinoside (ARA-C)-treated counterparts which had been exposed to the antimetabolite for 18 h on culture days 3 and 5 or 4 and 6. Five days after the second ARA-C treatment, phase contrast photomicrographs showed substantial loss of the proliferating basal cells, corresponding to an 85% decrease in cell number. Immunocytochemical studies using antiglial fibrillary acidic protein (anti-GFAP) and antineurofilament (anti-NF) antisera demonstrated loss of GFAP-positive cells (astrocytes) and preservation of NF-positive cells (neurons), the latter considered to be a nondividing population under the culture conditions. Nuclei obtained from the brain cell cultures at this time by Triton washing bound T3 with properties similar to those observed in vivo. Scatchard analysis showed a single, high affinity, limited capacity nuclear T3 receptor with a maximal binding capacity (MBC) of 0.53 +/- 0.10 ng T3/mg DNA and a Kd of 0.19 +/- 0.02 nM (+/- SE; n = 4). ARA-C treatment resulted in a mean decrease in DNA per culture dish of 54%, with an accompanying 2-fold enrichment of the MBC, and no change in the Kd. In untreated cultures grown for 20 days, DNA per dish increased until day 14 and subsequently remained stable at approximately 100 micrograms/dish. The MBC also increased from days 0 to 7, and remained stable until day 14. On day 20, the MBC had declined by approximately 60% to 0.21 ng T3/mg DNA, at which time the neuron population was decreased. The extracted nuclear receptor from brain cell cultures had a sedimentation coefficient of 3.6S. The relative binding affinities of the nuclear receptor for T3 and several analogs were identical to those found in vivo, making significant contamination of the nuclei with cytosolic or serum binding proteins very unlikely. Nuclei isolated from long term, neuron-free glial cell cultures failed to show any consistent high affinity saturable T3 binding.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2994998 TI - Structural requirements for the activation of rat anterior pituitary adenylate cyclase by growth hormone-releasing factor (GRF): discovery of (N-Ac-Tyr1, D Arg2)-GRF(1-29)-NH2 as a GRF antagonist on membranes. AB - The efficacy and potency of 14 GH-releasing factor (GRF) analogs, substituted in position 1 to 7, on adenylate cyclase activation in crude homogenates from rat anterior pituitary were related to those of human pancreatic GRF(1-29)-amide and vasoactive intestinal peptide. Among several D-amino acid substitutions, that in position 2 was the only one to yield a super-agonist [with a Kact (concentration required for half-maximal adenylate cyclase activation) 2 times lower than that of GRF(1-29)-NH2]. By contrast, D-isomer substitution in position 1 and 3 was without effect and D-isomer substitution in position 4, 6, or 7 decreased the affinity of the analog. The N-acetylated analog of GRF was as potent and active as the parent peptide, and the identity of the amino acid in position 2 of (N-Ac Tyr1)-GRF(1-29)-NH2 proved to be determining for enzyme activation, with D-Phe2 and D-Trp2 derivatives acting as partial agonists and the (N-Ac-Tyr1,D-Arg2) analog being an efficient competitive antagonist of GRF(1-29)-NH2. With use of this antagonist, it was possible to demonstrate that GRF and vasoactive intestinal peptide receptors represent distinct entities in the rat anterior pituitary. PMID- 2995000 TI - Additive effects of parathyroid hormone and calcitonin on adenosine 3',5' monophosphate release in newly established perfusion system of rat femur. AB - A new perfusion system of isolated rat femora was established, and the effect of PTH or calcitonin (CT) on cAMP release from the adult bone was examined. Stimulation of cAMP release from the isolated perfused bone peaked rapidly between 5 and 10 min after human PTH (hPTH)-(1-34) or eel CT injection, respectively, and declined gradually toward the preinjection level. However CT exhibited a longer lived effect than PTH. Release of cAMP was still significantly greater than control at 60 min after a bolus injection of CT. The rate of cAMP release was directly related to the dose of PTH or CT. H2O2-oxidized PTH (biologically inactive) caused no increase in cAMP release. hPTH-(1-34) was markedly more potent than hPTH-(1-84) in stimulating cAMP release from the perfused bone on an equimolar basis. Simultaneous administration of 5 micrograms PTH and 5 micrograms CT at maximal stimulatory doses produced additive effects, indicating the presence of separate receptor sites for PTH and CT in the bone. In conclusion, this system provides us a means by which hormone actions on adult bone with integrity of the organ can be evaluated and therefore makes possible systemic investigation of the osteoblast-osteoclast function. PMID- 2995001 TI - Binding of intact parathyroid hormone to chicken renal plasma membranes: evidence for a second binding site with carboxyl-terminal specificity. AB - We studied the binding of bovine PTH (bPTH) to chicken renal plasma membranes using an intact hormone radioligand, [125I]bPTH-(1-84). In contrast to previous studies using amino-terminal radioligands, our experiments revealed the presence of two distinct binding sites for intact bPTH. Scatchard analysis of competition curves consistently gave a biphasic curve, and computerized nonlinear regression analysis indicated a high affinity [dissociation constant (Kd) = 1.21 nM] and a low affinity (Kd = 333 nM) site (P less than 0.001). Analysis of binding of [125I] bPTH-(1-34) using identical techniques revealed only one site, similar to the high affinity sites seen with intact hormone tracer. The low affinity site for [125I]bPTH-(1-84) had carboxyl-terminal specificity since analysis of competition curves with unlabeled human PTH-(53-84) gave virtually identical binding parameters to the low affinity site obtained by competition with unlabeled native hormone. Binding to the low affinity site was inhibited in the presence of 10 mM Mg2+ and was reduced after storage of membranes for over 1 month at -70 C. Association of [125I]bPTH-(1-84) to the low affinity site took distinctly longer (approximately 4 h) than to the high affinity site (essentially complete by 2.25 h). Our data suggest that the high affinity site is coupled to adenylate cyclase in these membranes, while the low affinity site is not. The physiological significance of the low affinity carboxyl-terminal PTH binding is not known, and further studies are indicated. PMID- 2995002 TI - Triiodothyronine rapidly decreases transcription of the thyrotropin subunit genes in thyrotropic tumor explants. AB - We have investigated the direct effect of the thyroid hormone T3 on the TSH subunit genes in tissue explants. Minces of TtT 97 thyrotropic tumor were treated with 5 nM T3 for varying periods of time. Nuclei were then isolated from the tumor cells and allowed to continue RNA synthesis in the presence of [alpha 32P]UTP. Newly synthesized RNA sequences were quantified by hybridization to immobilized cloned cDNAs containing sequences specific for either TSH beta or alpha-subunit mRNA. Basal TSH beta and alpha-subunit mRNA synthesis rates were both approximately 300-400 parts/million, the same as in vivo values. After 15 min of T3 treatment, TSH beta mRNA synthesis was significantly decreased by 42% and was maximally decreased by 95% after 1 or more hours of T3. Synthesis of alpha-subunit mRNA was decreased by 38% after 30 min of T3 treatment and by 78% after 1 h or more of T3. The suppressive effects of T3 on transcription correlated with the time course of T3 binding to its nuclear receptor. These changes are quantitatively similar to those observed after in vivo T3 treatment. Decreases in mRNA synthesis preceded significant decreases in tissue steady state mRNA levels or subunit protein levels. The presence of the protein synthesis inhibitor cycloheximide (25 micrograms/ml) during a 4-h incubation with T3 did not change the T3-mediated decreases in TSH beta or alpha-subunit mRNA synthesis or the decreases in cellular mRNA levels. Therefore, T3 can act directly on the thyrotrope to suppress TSH beta and alpha-subunit mRNA synthesis, and protein synthesis is not necessary for the T3-mediated decreases in gene transcription. The data suggest that T3 may act directly at the level of the TSH subunit genes to modulate their expression. PMID- 2995003 TI - Biological activities of synthetic human parathyroid hormone (PTH) 1-84 relative to natural bovine 1-84 PTH in two different in vivo bioassay systems. AB - Synthetic 1-84 human PTH (hPTH) peptides (with either asparagine) or aspartic acid at position 76) were compared with natural bovine PTH (bPTH) in three in vivo bioassays. Surprisingly, in the chick hypercalcemia bioassay, the human 1-84 peptides were approximately 3 times more potent on a molar basis than bPTH. In contrast, in an in vivo mouse kidney cAMP accumulation bioassay, these human peptides were 3-6 times less potent than bPTH. This low potency of synthetic hPTH relative to bPTH in the renal cAMP assay is in accordance with published relative potency estimates for natural extracted hPTH in in vitro rat renal membrane adenylate cyclase assays. The human and bovine 1-84 peptides were weakly active in an in vivo mouse calvaria cAMP accumulation system, producing a shallow dose response curve which was not suitable for any quantitative estimates of potency. In contrast, both human and bovine 1-34 fragments were highly active in stimulating accumulation of cAMP in calvaria thus emphasizing the qualitative differences between 1-84 PTH and the 1-34 fragment of both species of PTH. Despite the homology between human and bovine 1-84 PTH, they have markedly different quantitative biological effects on hypercalcemia in chicks and in vivo renal cAMP accumulation in mice. Any estimate of the biological potency of human 1-84 PTH, relative to bovine 1-84 PTH, will need to be defined in terms of the nature and species of the biological test system. PMID- 2995004 TI - Prolactin stimulates adrenal androgen secretion in infant baboons. AB - It has been suggested that pituitary factors other than ACTH modulate adrenal steroidogenesis during maturation of the pituitary-adrenocortical axis. Therefore, we determined whether hormones other than ACTH influence the production of cortisol (F), dehydroepiandrosterone (DHA), and DHA-sulfate (DHAS) in baboon infants studied between 8 and 24 months of age. Animals (three males, two females) were sedated with ketamine and peripheral blood samples taken 20, 10, and 0 min before a 90-min constant iv infusion of 448 pmol/min of either ACTH, ovine PRL, ovine GH, 2.4 nmol/min human CG (hCG), or normal saline. Serum F, DHA, and DHAS concentrations of blood samples obtained during the infusion (70, 80, 90 min) were averaged and compared with average pretreatment values. Each animal received each of the treatment protocols which included a minimum recovery period of 3-6 weeks. The serum concentrations of F, DHA, and DHAS did not vary with age and averaged (mean +/- SE) 24 +/- 2, 1.9 +/- 0.2, and 36 +/- 5 micrograms/dl, respectively. Compared to pretreatment values, ACTH increased (P less than 0.05) mean serum F concentrations by 155 +/- 20%; PRL, GH, hCG, and saline had no effect. In contrast, serum DHA concentrations were stimulated (P less than 0.05) by both ACTH (131 +/- 20%) and PRL (58 +/- 18%); GH, hCG, and saline had no effect. Similar findings were observed for serum DHAS concentrations. These findings indicate that the majority of serum androgens in young baboons is of adrenal origin. Therefore, we conclude that PRL, in addition to ACTH, may also be an adrenocorticotrophic factor in baboon infants. However, in contrast to ACTH, the action of PRL on the adrenal is apparently specific for androgen production. PMID- 2995005 TI - Insulin and insulin-like growth factor (somatomedin) receptors on cloned rat pituitary tumor cells. AB - Specific receptors for insulin and the somatomedin peptides insulin-like growth factors I and II (IGF-I and IGF-II) have been characterized on three separate cloned strains of rat pituitary tumor cells (GH3, GH1, and GC). Binding of 125I labeled peptides was time, temperature, and pH dependent for all three cell lines. Specific binding of [125I]insulin, which was extremely low in normal rat adenohypophyseal cells, was demonstrable for all three lines, with the Kd for the high affinity receptor ranging from 10(-10) to 4 X 10(-10) M/liter. A specific high affinity IGF-I receptor was also identified, with a Kd of approximately 10( 9) M/liter. IGF-II and insulin were, respectively, 10% and 1% as potent as IGF-I in competing for this receptor. When [125I]insulin and [125I]IGF-I were cross linked to GH3 cells with disuccinimidyl suberate, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both receptors were found to have an apparent mol wt greater than 300,000 in the unreduced state, with subunits of apparent mol wt 125,000 after reduction. A third discrete receptor, which bound [125I]IGF-II, was also identified on all three cell lines. IGF-I was only 10% as potent as IGF-II at displacing [125I]IGF-II, and insulin was virtually unreactive. When [125I]IGF-II was cross-linked to GH3 cells and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two receptors were identified. One had an apparent mol wt of 205,000 unreduced and 250,000 upon reduction, and presumably represents the type II receptor. Additionally, a band was observed at an apparent mol wt greater than 300,000 unreduced and 125,000 upon reduction, probably representing IGF-II binding to the IGF-I or insulin receptor. The presence of specific high affinity receptors for insulin, IGF-I, and IGF-II in these transformed cell lines is consistent with previous observations in normal rat and human pituitary cells and suggests a role for these peptides in the modulation of pituitary function. PMID- 2995006 TI - The prolactin gene hypersensitive sites are present early in development and are not induced by estrogen administration. AB - We have previously shown that two DNase I-hypersensitive sites are present upstream of the PRL gene in pituitary tumors of Fischer 344 rats. In this paper we present a method for examining hypersensitive sites in nontumorous pituitaries where PRL-producing lactotrophs comprise a small percentage of the total cell population. Using this method we are able to show that DNase I-hypersensitive sites in the PRL gene chromatin remain present even when estrogen is withdrawn from animals and PRL synthesis is markedly decreased. Furthermore, the hypersensitive sites appear before sexual development in female rats, and estrogen administration does not affect the appearance of the sites. PMID- 2995007 TI - Subtype-selective down-regulation of rat renal cortical alpha- and beta adrenergic receptors by catecholamines. AB - In the current studies, we have explored agonist-mediated down-regulation of adrenergic receptors in vivo. We infused catecholamines from sc implanted osmotic minipumps and examined the effects of the resultant increases in circulating levels of catecholamines on rat renal cortical alpha- and beta-adrenergic receptor subtypes, as assessed in radioligand binding studies. Infusion of epinephrine or norepinephrine (at 150 micrograms/kg X h) elevated plasma levels of each catecholamine 10- to 20-fold and decreased renal cortical alpha 1 receptor number about 50% without changing alpha 2-receptor number. Isoproterenol infusion (150 micrograms/kg X h) raised plasma levels of this catecholamine, but had no effect on the number of either alpha 1- or alpha 2-receptors. Renal cortical beta-adrenergic receptor number was decreased by infusion of all three catecholamines. However, the beta 1- and beta 2-adrenergic receptors were altered selectively by the different agonists. Infusion of norepinephrine decreased both beta 1- and beta 2-receptor number, but was more effective for the beta 1 receptors; this result was somewhat at variance with that we previously reported for rats bearing transplanted pheochromocytomas. The decrease in beta-receptor number due to epinephrine infusion was largely due to loss of the renal cortical beta 2-receptors. Infusion of isoproterenol decreased the number of both beta 1- and beta 2-receptors (69% and 75%, respectively). Infusion of norepinephrine maximally decreased the number of alpha 1-, beta 1-, and beta 2-receptors within 2 days, and the t 1/2 for receptor loss was about 12 h. beta-Receptors lost in response to isoproterenol infusion could not be recovered in a pellet prepared by high speed centrifugation of the supernatant derived from the preparation of renal cortical membranes. These results indicate that adrenergic receptor subtypes are differentially down-regulated by elevated levels of circulating catecholamines and that this differential loss of receptors depends on the nature of the receptor subtype, the agonist, and perhaps also whether catecholamines are infused rather than increased by pheochromocytoma. PMID- 2995009 TI - Regulation of 1,25-dihydroxyvitamin D3 receptors by vitamin D analogs in cultured mammalian cells. AB - The pig kidney cell line (LLC-PK1) has been shown to possess 1,25 dihydroxyvitamin D3 [1,25-(OH)2D3] receptors and to exhibit functional responses to vitamin D metabolites. Here we report that these receptors appear to undergo homologous up-regulation by 1,25-(OH)2D3 and other vitamin D analogs. This phenomenon was also observed in other cell lines, including human skin fibroblasts and human mammary cancer cells (MCF-7). Treatment with active hormone or vitamin D analogs results in a substantial increase (200-400%) in the number of 1,25-(OH)2D3 receptors without altering the affinity of receptor for hormone. The up-regulated receptor, like the basal receptor, has an apparent Kd of about 0.04 nM and sediments at 3.3S on hypertonic sucrose gradients. In addition, approximately 50% of the total receptors from both control and treated cells bind to DNA-cellulose and elute at 0.18 m KCl. These results indicate that the up regulated receptor is similar to the classical 1,25-(OH)2D3 receptor. While the time necessary to achieve the maximal receptor increment is 16-20 h, there is a rapid component in the rise observed within 5 min. The maximal effect persists for 4-6 h after hormone removal. The increased binding is not a result of differential receptor localization or extractability. 1,25-(OH)2D3, 1,24,25 trihydroxyvitamin D3, 24,25-(OH)2D3, and 25-hydroxyvitamin D3 all increase receptor binding to similar levels, and the dose required closely reflects the affinities of the various metabolites for the receptor. Treatment of cells with the RNA synthesis inhibitor actinomycin D indicates that the increase in receptors is partially dependent on RNA synthesis. Mutant skin fibroblasts from patients with vitamin D-dependent rickets type II, containing nonresponsive 1,25 (OH)2D3 receptors, failed to exhibit the characteristic up-regulation observed in normal cells. Taken together, these results indicate that vitamin D metabolites regulate the number of 1,25-(OH)2D3 receptors in part by receptor occupancy and, more importantly, by a receptor-mediated induction mechanism. PMID- 2995008 TI - Corticotrope response to removal of releasing factors and corticosteroids in vivo. AB - In the intact rat, adrenalectomy (ADX) is known to result in increased ACTH synthesis, content, and secretion from the anterior pituitary compared with those in the sham-adrenalectomized control. Treatment of adrenalectomized, rats with corticosterone prevents or reverses these changes in ACTH. Because corticosterone is known to act both at the corticotrope and at the level of CRF secretion, it is not clear to what extent the ACTH response to ADX is a result of removal of glucocorticoids from the pituitary per se. To test the role of brain input as well as the role of glucocorticoids on the corticotrope response to ADX, we performed the following experiment. Rats were prepared with anterolateral hypothalamic deafferentations (lesion) which severed CRF and arginine vasopressin cell bodies in the hypothalamus from their axonal endings in the median eminence and posterior pituitary. Control rats were subjected to sham lesions. Two days later, half of the rats in each group were subjected to either ADX or sham ADX; a subgroup of the lesioned rats was provided at the time of adrenal surgery with a constant infusion of rat CRF. Five days later, all rats were killed, and anterior pituitary levels of proopiomelanocortin (POMC) mRNA, ACTH, and protein; plasma ACTH and corticosterone, and adrenal and thymus weights were measured. In sham lesioned rats, ADX resulted in increases in POMC mRNA, and plasma ACTH of 2.5- and 12-fold, respectively, compared to sham-adrenalectomized controls. In the absence of hypothalamic drive (lesion only), there were no responses of any of these variables to ADX. In lesioned rats driven with CRF, ADX resulted in increases in POMC mRNA and plasma ACTH of 2.2- and 2.6-fold, respectively, compared to sham ADX. After consideration of the three variables indicating ACTH synthesis, storage, and secretion and comparison of the results of ADX vs. sham ADX within and across the sets of animals, we conclude that 1) there is no autonomous response of the corticotrope to ADX; 2) the removal of corticosterone from the anterior pituitary may account for the majority of the effects of ADX on ACTH synthesis; and 3) the normal response to ADX requires secretion of CRF and increased secretion of another ACTH-releasing factor (possibly arginine vasopressin) that causes increased secretion but little synthesis of ACTH. PMID- 2995010 TI - TSH and cAMP enhance expression of the myc proto-oncogene in cultured thyroid cells. AB - The effect of TSH stimulation on cellular (c)-myc mRNA content in cultured thyroid cells was examined by Northern blot analysis. Polyadenylated mRNA was prepared from quiescent FRTL-5 rat thyroid cells and from cells stimulated by TSH for 8 h. A major 2.1 kilobase (kb) transcript was enhanced 5.5-fold by TSH. (Bu)2cAMP and forskolin each enhanced c-myc expression 2.7-fold. These data indicate that TSH, via cAMP as a second messenger, can regulate expression of a cellular oncogene in a non-neoplastic, specific target cell. PMID- 2995011 TI - Decreased nuclear uptake of 1,25-dihydroxyvitamin D3 by duodenal mucosal cells in the X-linked hypophosphatemic mouse. AB - We have shown that there is a significant decrease in the nuclear uptake of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] by duodenal mucosal cells in the X-linked hypophosphatemic (Hyp) mouse. Duodenal mucosal cells prepared from control and Hyp mice were incubated with 1,25(OH)2[26,27-methyl-3H]D3 ([3H]-1,25(OH)2D3) for 30 min. to evaluate the time-course and perform saturation analysis. The results of time-course studies showed that saturation was attained in 30 min., reaching an average nuclear uptake of 10.4 fmol/tube in the control mice and 6.1 fmol/tube in the Hyp mice. The results of Scatchard analyses were as follows: dissociation constant (Kd) 5.71 X 10(-10) M and maximal binding sites 7.31 X 10(4) sites/cell in the control mice, and Kd 2.92 X 10(-10) M and maximal binding sites 4.88 X 10(4) sites/cell in the Hyp mice, the maximal binding sites of the latter showed a significant decrease (P less than 0.05) by Student's t test. In addition, there was no significant difference in the binding of [3H]-1,25(OH)2D3 to its residual cytosol receptors between the control and Hyp mice. On the basis of these data, we speculate that the reported resistance of Hyp mice to vitamin D may be due to decreased nuclear uptake of 1,25(OH)2D3 by their duodenal mucosal cells. PMID- 2995013 TI - Iodide modulation of Ca2+ efflux from mouse thyroid. AB - In a preceding report, we showed evidence that thyrotropin (TSH) stimulates Ca2+ efflux from mouse thyroid gland and that TSH stimulation of Ca2+ efflux is inhibited by acute administration of excess iodide to mice fed a low iodine diet (Hashizume et al., 1984). The observations suggested that iodide inhibits Ca2+ efflux through an inhibition of TSH-sensitive adenylate cyclase activity. We found further, that iodide inhibits dibutyryl cyclic AMP (DBC)-stimulated Ca2+ efflux. The results suggested that iodide influences the step subsequent to the generation of cyclic AMP. In this report, we studied whether iodide can inhibit Ca2+ efflux by a mechanism which is distinct from adenylate cyclase inhibition. The acute administration of excess iodide to mice fed a regular diet did not decrease the basal Ca2+ efflux rate in the thyroid. TSH-induced stimulation of Ca2+ efflux in thyroids obtained from regular diet-treated mice was not modified by iodide administration. Iodide injection to mice fed a low iodide diet, however, decreased the basal Ca2+ efflux rate though the content of cyclic AMP in the thyroids was not altered by this treatment. The decreased-Ca2+ efflux rate induced by iodide in the low iodine diet-treated thyroids was not modified by TSH in vitro. The results indicate that an acute administration of excess iodide in thyroid inhibits Ca2+ efflux not only by an inhibition of adenylate cyclase but also by an inhibitory action which is distinct from the adenylate cyclase inhibiting action of iodide. PMID- 2995012 TI - Studies on the ontogenesis and the regulatory mechanisms of placental lactogen and insulin binding sites (receptor) in rat liver. AB - The ontogenesis of specific binding of 125I-hPL and 125I-insulin was determined in rat liver cell membranes (10(5) X g pellets), and the regulatory mechanisms of these binding sites were also examined. There were striking differences in the mode of ontogenesis between binding sites of hPL and insulin in rats. HPL binding sites were very few in liver cell membranes from fetal and immature rats. They began to increase after puberty, and markedly increased in late pregnancy. On the other hand, insulin binding sites, which decreased in late pregnancy, were dominant in fetal liver and placenta. Consequently, the lipolytic and glycogenolytic activities of hPL in maternal liver were accentuated, whereas the effects of insulin on maternal liver were suppressed. In contrast, in fetal liver and placenta only the anabolic effects of insulin seemed conspicuous. According to the results of experiments on in vivo administration of estradiol-17 beta, progesterone, hydrocortisone or hPL to intact or hypox-rats, and the measurement of serum rat chorionic mammotropin (rCM), rPRL, estradiol-17 beta, and insulin during pregnancy in rats, the increase in hepatic hPL binding sites observed in late pregnancy might be, at least in part, due to rCM secreted from placenta, and the decrease in insulin binding sites due to the increase in serum insulin itself in rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995014 TI - Long-term treatment of postmenopausal osteoporosis with active vitamin D3, 1 alpha-hydroxycholecalciferol (1 alpha-OHD3) and 1, 24 Dihydroxycholecalciferol (1, 24(OH)2D3). AB - The effects of active vitamin D3 analogues on radial mineral content (RMC) in postmenopausal osteoporosis were examined. Seventy eight subjects with postmenopausal osteoporosis were divided into 5 groups; Group 1 (n = 23) as the control group and Group 2 (n = 27), Group 3 (n = 8), Group 4 (n = 9) and Group 5 (n = 11) which were given 1 microgram of 1, 24(R) (OH)2D3 per day, 1 microgram of 1, 24(S)(OH)2D3 per day, 0.5 and 1 microgram of 1 alpha-OHD3 per day for 6 to 24 months, respectively. After 3-months administration of these drugs, RMC values were significantly increased in Groups 2 (102.8 +/- 1.8%), 4 (103.9 +/- 3.3%) and 5 (114.2 +/- 3.6%), when compared with the controls (97.9 +/- 2.4%). RMC in Group 3 (97.9 +/- 2.4%) was not significantly different from the control value. The administration of 1 alpha-OHD3 caused in increase in RMC in a dose-related manner. A rapid decrease in RMC was observed after withdrawal of the treatment in Groups 2, 4, and 5. A subsequent increase in RMC was observed after re administration of 1 alpha-OHD3 and 1, 24(R)(OH)2D3. Serum Ca levels were increased in the group treated with 1, 24(R)(OH)2D3 and were decreased after the discontinuation of 1 alpha-OHD3 administration. Serum A1-P activity was decreased by treatment with 1 alpha-OHD3 (1 microgram per day) and a subsequent increase was observed in both groups treated with 1, 24(R)(OH)2D3 and 1 alpha-OHD3. Serum PTH levels were decreased by the administration of 1, 24(R)(OH)2D3 and 1 alpha OHD3. In the group treated with 1 microgram of 1 alpha-OHD3 per day, hypercalcemia (2 out of 11 cases and these patients took calcium tablets) and an increase in BUN (1 out of 2 hypercalcemic patients) were observed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995015 TI - Role of extracellular calcium and calcium efflux in the activation of hepatic glycogenolysis by calcium-dependent hormones. AB - The role of extracellular calcium in the glycogenolytic effects of calcium dependent hormones was examined in a rat liver perfusion system. Decreasing the perfusate CaCl2 concentration resulted in a concentration-dependent inhibition of glucose output by maximal concentrations of vasopressin (20 nM) and angiotensin II (10 nM), but not of glucagon (1.4 nM), cyclic AMP (100 microM), dibutyryl cyclic AMP (10 microM) or phenylephrine (5 microM). However, the effect of phenylephrine was inhibited when livers were perfused with CaCl2-free perfusate containing 0.5 mM EGTA in a duration-dependent manner. These effects were exerted through the inhibition of the maximal response of each hormone, and were associated with a parallel decrease in phosphorylase activation but not with changes in tissue cyclic AMP concentrations. When livers were preloaded with 45Ca for 45 min and then washed for either 15 min or 45 min, these hormones elicited a rapid and transient 45Ca efflux regardless of the perfusate calcium concentration. The sequential perfusion of two hormones resulted in the loss of 45Ca efflux by the second hormone. These results suggest that the glycogenolytic effects of vasopressin and angiotensin II depend on the extracellular calcium and that of phenylephrine primarily on the cellular calcium. It was also demonstrated that these calcium-dependent hormones mobilize calcium from the same pools. However, the mobilization of cellular calcium does not necessarily correlate directly with the glycogenolytic actions of vasopressin and angiotensin II. PMID- 2995016 TI - Effect of sodium intake on the excretion of urinary natriuretic factor in essential hypertensives. AB - A simplified method for the determination of natriuretic factor in the urine as measured by digoxin-like substance was studied. Digoxin-like substance in the urine was estimated by RIA using anti-digoxin antibody after being extracted by reversed phase cartridge column but without gel filtration. The values found by radioimmunoassay (RIA) yielded a significant correlation with those of the inhibitory effect of Na-K-ATPase activity which was measured by biochemical assay as described by Hamlyn et al. Using this RIA method, the effect of salt intake on natriuretic factor in urine was studied in patients with essential hypertension. The natriuretic factor on a high sodium diet (NaCl 20 g/day for three days) increased approximately 1.5 times, as compared to those on a low sodium diet (NaCl 3 g/day) (p less than 0.05). The Natriuretic factor showed a positive correlation with urinary Na excretion (P less than 0.050) when the patients were placed on ad. lib. sodium diet. From these results, it is suggested that secretion of natriuretic factor in the urine might be regulated in part by salt intake. PMID- 2995017 TI - Difference in arginine vasopressin responsive cAMP production between apical and basolateral membrane of cultured renal epithelial cells (MDCK). AB - It has been reported that vasopressin (AVP)-sensitive renal epithelial cell line (MDCK) forms morphologically polarized monolayers when cultured on plates. We studied whether the AVP-responsive cAMP production system would be located solely on the basolateral surface of these cells as has already been shown on the renal tubules. We used two methods to overcome the inaccessibility to the basolateral surface of the cultured cell layer and to study the apical and basolateral surfaces separately. One was culture on collagen sheet and the other was on Millipore filters. Our experiments showed that MDCK cell increased adenosine 3':5'-cyclic monophosphate (cAMP) content prominently only when vasopressin was accessible to the basolateral surface. Accordingly, MDCK cells were shown to have the AVP-responsive cAMP production system predominantly on the basolateral surface of the cell membrane. PMID- 2995018 TI - The relation between cyclic nucleotide levels in gastric juice and in blood plasma and blood gastrin in patients with duodenal ulcer following 2-deoxy-D glucose gastric stimulation test. PMID- 2995019 TI - [Peptide hormones of the gastrointestinal tract--a field of endocrinology of growing complexity]. PMID- 2995020 TI - Protein lateral distribution in lipid bilayer membranes. Applications to ESR studies. AB - We analyse recent ESR measurements on Ca2+ ATPase and Myelin proteolipid apoprotein reconstituted in phosphatidylcholine bilayer membranes. Our intention is to discover whether the measurements indicate significant protein-protein repulsive or attractive interactions. In order to do so we have studied a model of a lipid bilayer membrane containing transbilayer proteins. It represents the proteins by hexagons moving on a triangular lattice interacting via an energy E0 which can be attractive, repulsive or zero. The last-named represents that all of the Ca2+ ATPase data is best described either by the "random" model or, possibly, by one in which there is a small repulsive interaction, but not by the "annulus" model or one in which there is always at least one layer of lipid chains between every pair of proteins. We find that all of the Myelin PLA data is best described by a "random" distribution of hexamers and not by an "annulus" model of hexamers. We suggest measurements that can be done in order to unambiguously settle the question of whether these systems are best described by a "random"-type model or an "annulus"-type model. PMID- 2995021 TI - Effect of naloxone on the hormone response to CRF in normal man. AB - Ovine corticotropin releasing factor (CRF) was administered to six normal men and the plasma ACTH and cortisol responses compared with those following the same dose of CRF (200 micrograms) plus the opiate receptor blocker naloxone (20mg). The addition of naloxone was associated with a significant increase in plasma ACTH, cortisol and aldosterone responses. No change was observed in peripheral plasma levels of epinephrine, norepinephrine, arginine vasopressin, angiotensin II or renin activity in response to CRF plus naloxone. It is concluded that endogenous opioid peptides may inhibit the ACTH response to CRF. However the addition of naloxone does not increase the ACTH response to CRF sufficiently to constitute a useful test of pituitary function. PMID- 2995022 TI - Studies on the mechanism of decreased angiotensin I conversion in rat lungs injured with alpha-naphthylthiourea. AB - Lung endothelial cell injury may be an important early event in the pathogenesis of increased permeability pulmonary edema. Since angiotensin converting enzyme (ACE) is located on the luminal surface of the endothelial cell membrane, we sought to determine whether the conversion of angiotensin I (AI) to angiotensin II is decreased after acute lung injury to rats, induced by alpha naphthylthiourea (ANTU), and we investigated the mechanism of the decrease. We found that lungs isolated from rats treated 4 h earlier with ANTU at a dose of 15 mg/kg body weight (BW) had decreased AI conversion when perfused with Krebs Henseleit at a constant flow rate of 30 ml/min/kg BW. When perfusate flow rate was increased from 30 to 50 ml/min/kg BW, lungs isolated from rats treated with 10 mg/kg BW ANTU also had decreased AI conversion when compared to controls treated with a vehicle, Tween 80. Investigating the mechanism of decreased AI conversion, there were no differences among experimental groups in pulmonary arterial pressures or effluent perfusate pH or pO2. There was no correlation between lung wet/dry weight ratios and the extent of AI conversion among control rat lungs. Lung homogenate and serum ACE activity did not differ among control rats and rats pretreated with the two doses of ANTU. Ultrastructural studies revealed an increased percentage of capillaries with blebbing of endothelial cells in lungs injured with ANTU, as compared to controls, but no evidence of increased endothelial cell denudation in injured lungs. We conclude that angiotensin I conversion is decreased after ANTU lung injury and that the extent of decrease is related to the dose of ANTU and to perfusate flow rate. Although we cannot exclude decreased vascular surface area perfused as a cause of decreased conversion, we speculate that subtle changes in the luminal endothelial cell membrane may have caused decreased AI conversion after ANTU lung injury. PMID- 2995023 TI - The ultrastructural study of glycogen and lamellar bodies in the development of fetal monkey lung. AB - Developing fetal monkey lung tissues were studied at the gestational period of 135-145 days. In general, glycogen is distributed throughout the cytoplasm of the type II pneumocytes at this gestational period. It is of interest that we found a structural relationship between glycogen particles and lamellar body formation in the type II pneumocytes. The glycogen particles were found inside the electron dense pre-granules and the immature lamellar bodies. Some of the fully matured lamellar bodies also contained glycogen. This phenomenon has not been observed previously, and it supports the evidence that there is a close and direct structural relationship between glycogen and the development of lamellar bodies at this stage of lung development in the type II pneumocytes. PMID- 2995024 TI - Clinical and electrophysiologic evaluation of peripheral nerve function in chronic phenytoin therapy. AB - Analysis of covariance was employed to explore possible correlations among duration of phenytoin therapy, phenytoin and folate serum concentrations, and clinical and electrophysiologic findings in 32 patients receiving single phenytoin therapy. None of our patients volunteered peripheral nervous system complaints. On examination, 9 of 32 (28%) were found to have signs suggestive of neuropathy (absent knee and/or ankle jerks). Neither phenytoin serum concentration nor duration of phenytoin therapy was found to significantly (p greater than 0.05) affect nerve function after an adjustment for age differences. A test for overall effect of folate serum concentration (grouped greater than or equal to 5 or less than 5 ng/ml) also failed to reach statistical significance (p greater than 0.05). Our findings suggest that phenytoin and folate serum concentrations and duration of phenytoin therapy do not have an important role in the development of clinical neuropathy and electrophysiologic abnormalities. PMID- 2995025 TI - Reduced ACTH content in cerebrospinal fluid of children affected by cryptogenic infantile spasms with hypsarrhythmia. AB - In view of the therapeutic efficacy of adrenocorticotropic hormone (ACTH) in the treatment of infantile spasms (IS) with hypsarrhythmia, we studied the cerebrospinal fluid (CSF) levels of ACTH in 15 children (4-10 months) affected by IS with hypsarrhythmia (eight cryptogenic forms, seven secondary to perinatal distress) and in age-matched controls. Lumbar puncture was performed in all but one case before any kind of treatment. In another case, CSF was collected 3 weeks after a spontaneous remission. Both ACTH and beta-endorphin (beta-EP), the other peptide related to the same precursor (proopiomelanocortin), were measured by specific radioimmunoassay after gel chromatography. While beta-EP levels were unchanged in the two groups of patients, ACTH concentrations of cryptogenic (3.75 +/- 2.40 fmol/ml, Mean +/- SD p less than 0.05) and secondary (6.36 +/- 3.70, NS) forms were lower than in controls (10.90 +/- 5.79). On the other hand, ACTH was higher in the case studied after therapy (9.0) and in the case presenting a spontaneous clinical and EEG remission (15.0). These data indicate that in children affected by IS with hypsarrhythmia (mainly of cryptogenic type), CSF levels of ACTH are lower, while levels of beta-EP remain normal. It would therefore appear that central ACTH content may play a possible role in the pathogenesis of IS with hypsarrhythmia. PMID- 2995026 TI - Chronic carbamazepine treatment increases brain adenosine receptors. AB - The effect of carbamazepine on adenosine receptors in vitro has been well documented, with findings from several groups showing that therapeutic doses of this drug are sufficient to inhibit binding to the major portion of adenosine receptors in brain. In this study, we describe the effects of chronic carbamazepine on central adenosine receptors from several areas of rat brain using [3H]diethylphenylxanthine [( 3H]DPX) and [3H]cyclohexyladenosine [( 3H]CHA) as ligands. Carbamazepine was administered to rats orally in the diet at doses of 2.25 g/kg of diet and 5.0 g/kg of diet for periods of 3 and 11 days, respectively. Carbamazepine-treated animals displayed higher levels of adenosine receptors in virtually all brain areas tested, most of which reached significance in the 11-day treatment group. Scatchard analysis revealed increases in the number of receptors. There was no change in peripheral and central type benzodiazepine receptors or beta-adrenergic receptors in the carbamazepine treated animals. Therefore, carbamazepine treatment in vivo appears to upregulate adenosine receptors, suggesting that this drug may act as an adenosine antagonist. PMID- 2995027 TI - Autoradiographic detection of diphtheria toxin resistant mutants in human diploid fibroblasts. AB - An autoradiographic procedure for the detection of diphtheria toxin (DT) resistant (DipR) mutants in human diploid fibroblast (HDF) cells has been developed. The assay is based on the observation that when HDFs from confluent cultures are seeded in medium containing 0.01 flocculating units/ml or higher concentration of DT, protein synthesis in sensitive cells is severely inhibited by 4-6 hr. If at this or later time, a radiolabeled protein precursor (eg, 3H leucine) is added to the culture, it is almost exclusively incorporated into the resistant cells, which are then readily identified by autoradiography. The DipR cells can also be identified by labeling in the presence of 3H-thymidine, although a higher background is observed in these experiments. Reconstruction experiments using DipS and DipR HDFs show that the frequency of heavily labeled cells that are detected by autoradiography show an excellent correlation with the number of DipR cells added and to the number of DipR cells as detected by conventional colony forming assay. These studies provide strong evidence that the labeled cells identified by autoradiography are bona fide DipR mutants. The detection of DipR cells by autoradiography is apparently not affected by the presence of the sensitive cells in the mixtures. The spontaneous frequency of DipR cells in HDFs has been found to be in the range of 1-5 X 10(-6), and this increases in a dose dependent manner upon treatment with the mutagen ethyl methanesulfonate. These results indicate that the autoradiographic assay could be used for quantitative mutagenesis. Since the autoradiographic assay does not depend on cell division, it may prove useful in estimating the incidence of pre existing mutations in cell populations that either do not divide or have very limited growth potential (eg, lymphocytes, muscle cells, neurons, senescent fibroblasts, etc). PMID- 2995028 TI - Specificity of the collagenase from the insect Hypoderma lineatum. AB - Specificity of the collagenase from the larvae Hypoderma lineatum, a serine protease related to trypsin, has been investigated by using native collagen and non-collagenous substrates. At 25 degrees C and neutral pH the degradation of collagen by the larval enzyme in solution results in a 52% loss of specific viscosity, without loss of helicity. Electron microscopy of segment-long-spacing crystallites of the digest shows the occurrence of one cleavage region between bands 41 and 44 whereas Edman degradation indicates several cleavage loci in this region. Hypoderma collagenase differs from proteinases I and II from the crab Uca pugilator, which catalyse cleavages in multiple regions of the collagen molecule, and also from vertebrate collagenases, which cleave collagen only between residues 775 and 776. Apart of specific action on collagen, Hypoderma collagenase degrades the oxidized chain B of insulin; the major cleavage occurs at the Leu15 Tyr16 bond followed by two minor cleavages at the Arg22-Gly23 and Lys29-Ala30 bonds. The larval enzyme has no action on synthetic peptide substrates of trypsin or chymotrypsin. PMID- 2995029 TI - Cloning of DNA segments of phage 2C, which allows autonomous plasmid replication in Bacillus subtilis. AB - The chromosome of Bacillus subtilis phage 2C is a 100-MDa double-stranded DNA molecule, containing hydroxymethyluracil in place of thymine and carrying redundant ends each encompassing 10% of the genome. 2C DNA was cleaved with EcoRI and HindIII, and cloned in the shuttle plasmids pSC 540 and pCP 115, both containing segments originating from B. subtilis and Escherichia coli plasmids. These chimaerical plasmids, carrying the chloramphenicol resistance gene, were unable to replicate in B. subtilis; this ability was restored, however, after the insertion of viral DNA segments. Physical maps of the recombinant plasmids were made; a large deletion of the E. coli-derived segment of pSC 540 was observed (which paralleled a loss of replication in this host), whereas addition of 2C DNA segments in pCP 115 was not accompanied by deletion (replication in E. coli was conserved in this case). Cloned viral segments mapped mostly, but not exclusively, within the redundant ends of 2C DNA. It is suggested that the thirteen recombinant clones carried the replication origin region of phage 2C DNA, and that these sequences originated within or close to the redundant extremities of the viral chromosome. PMID- 2995030 TI - Events in glucocorticoid hormone action. A correlation of histone H1 variant pattern changes, hormone binding to cell nuclei, and induction of mouse mammary tumor virus RNA. AB - To approach experimentally changes of chromatin structure introduced by glucocorticoids, the histone H1 compositions of hormone-treated and non-treated mouse mammary tumor cells of the GR line [Ringold, G., Lasfargues, E. Y., Bishop, J. M. and Varmus, H. E. (1975) Virology 65, 135-147] were compared. To define the biologically important hormone concentration range, the cells were exposed to different concentrations of triamcinolone, a synthetic glucocorticoid. The induction of mouse mammary tumor virus (MMTV) RNA was measured by cDNA excess hybridization, and the amount of hormone bound to nuclei was determined by a filter-binding assay. Between 0.3 nM and 30 nM triamcinolone the relative increase in nuclear bound hormone corresponded well with the relative induction of MMTV RNA. The half-life of triamcinolone in nuclei of growing cells was 1 h, as measured by a pulse-chase experiment. Reversed-phase high-performance liquid chromatography of histone H1 resulted in its separation into four subfractions. The treatment of cells with biologically active glucocorticoid, 3 nM or 30 nM triamcinolone or 1 microM dexamethasone, resulted in changes in the relative amounts of two subfractions and to a positional shift of two subfractions as compared to untreated cells. No changes were observed after exposure to 3 nM dexamethasone, a concentration which does not induce MMTV RNA [Ringold, G. M., Yamamoto, K. R., Tomkins, G. M., Bishop, J. M. and Varmus, H. E. (1975) Cell 6, 299-305]. PMID- 2995031 TI - Detection and distribution of myosin isozymes in vertebrate smooth muscle. AB - Crude extracts of taenia coli (guinea-pig), gizzard (chicken), stomach, colon, ureter, bladder, mesenteric vein, mesenteric artery, uterus and vas deferens (dog) were electrophoresed under conditions which do not denature myosin (pyrophosphate gels). Two isozymes (G1 and G2) were observed in all cases. Their mobilities are the same in all organs, but there are some variations in their relative proportions. They have an ATPase activity. Based on electrophoretic mobility the light chains (L20 and L17) seem to be the same for both isozymes whilst the heavy chains are different. Isozyme G2 contains one type of heavy chain of an apparent molecular mass of 230 kDa, whilst isozyme G1 contains two types of heavy chains: one of apparent molecular mass of 230 kDa, and the other of apparent molecular mass of 200 kDa. PMID- 2995032 TI - Synthesis of pyrophosphate by chromatophores of Rhodospirillum rubrum in the light and by soluble yeast inorganic pyrophosphatase in water-organic solvent mixtures. AB - Chromatophores of Rhodospirillum rubrum contain a membrane-bound pyrophosphatase that synthesizes pyrophosphate when an electrochemical H+ gradient is formed across the chromatophore membrane upon illumination. In this report it is shown that MgCl2 and Pi have different effects on the synthesis of pyrophosphate in the light depending on whether initial velocities or steady-state levels are examined. When the water activity of the medium is reduced by the addition of organic solvents, soluble yeast inorganic pyrophosphatase (no H+ gradient present) synthesizes pyrophosphate in amounts similar to those synthesized by the chromatophores in totally aqueous medium during illumination, (H+ gradient present). The pH, MgCl2 and Pi dependence for the synthesis of pyrophosphate by the chromatophores at steady-state is similar to that observed at equilibrium with the soluble enzyme in the presence of organic solvents. The possibility is raised that a decrease in water activity may play a role in the mechanism by which the energy derived from the electrochemical H+ gradient is used for the synthesis of pyrophosphate in chromatophores of R. rubrum. PMID- 2995033 TI - Partial purification and characterization of a new phosphoprotein kinase from cells infected with pseudorabies virus. AB - Cytoplasmic fractions from normal baby hamster kidney fibroblasts and from fibroblasts infected with pseudorabies virus were fractionated by DEAE-cellulose chromatography and fractions assayed for protein kinase activity. In preparations from uninfected and infected cells protein kinase activities identified as casein kinase I and II, the two isoforms of the cyclic-AMP-dependent protein kinase, protein kinase C, and a presumed proteolytic fragment of protein kinase C were present in comparable amounts. However in infected cells a new protein kinase activity was detected, appearing about 4 h after infection and increasing during the following 6 h at least. This new protein kinase was purified 100-fold by high performance gel-permeation and ion-exchange chromatography, and characterized. It has an apparent relative molecular mass of 68 000 on the basis of gel-permeation chromatography, and a sedimentation coefficient of 4.3 S. It catalysed the phosphorylation of serine residues of basic proteins in vitro, with protamine a better substrate than mixed histones; and used ATP (apparent Km = 60 microM), but not GTP, as phosphoryl donor. Molecules that can serve as effectors for other protein kinases (cyclic AMP, cyclic GMP, Ca2+ + calmodulin, Ca2+ + phospholipid, double-stranded RNA, and heparin) did not significantly alter the activity of this enzyme. A distinguishing characteristic of the protein kinase was a high KCl concentration optimum with the persistence of activity up to 800 mM KCl, at least. PMID- 2995034 TI - Immuno-isolation of vesicles using antigenic sites either located on the cytoplasmic or the exoplasmic domain of an implanted viral protein. A quantitative analysis. AB - In this study, we present a new general approach for immuno-isolation: a foreign integral membrane protein, the G-protein of vesicular stomatitis virus (VSV), is implanted into the plasma membrane for subsequent immuno-isolation. A quantitative analysis was accomplished using the erythrocyte plasma membrane as a model system. The virus was artificially bound to the membrane via a lectin and subsequently fused at low pH. Vesicles of two opposite orientations were prepared from erythrocytes with fused G-protein. Right-side-out and inside-out vesicles expose the exoplasmic and the cytoplasmic domains of the G-protein on their surfaces respectively. In immuno-isolation experiments antibodies against each of the domains of the G-protein were used. Vesicles were presented to an immunoadsorbent (ImAd) consisting of a solid support with appropriate antibodies bound to its surface. Two commonly used immunoadsorbents prepared from either polyacrylamide beads or fixed Staphylococcus aureus cells were compared and found to have identical immuno-isolation efficiencies. It was possible to control and quantitate the amount of implanted antigen. Therefore, we were able to show that the critical antigen density required for immuno-isolation is 50 G molecules/micron2 plasma membrane surface area for both types of vesicle/antibody couples. This analysis showed that vesicles presenting either the cytoplasmic or the exoplasmic domain of the G-protein are immuno-isolated with the same efficiency. PMID- 2995035 TI - Significance of a positive exercise ECG in middle-aged and old athletes as judged by echocardiographic, radionuclide and follow-up findings. AB - To study the pathologic and prognostic significance of--and possible underlying mechanisms for--a pathological exercise ECG in athletes, two age-matched groups were selected from a total population of 117 middle-aged and old endurance athletes: Group A: 21 with a pathological exercise-ECG, and group B: 21 with normal exercise-ECGs. Data from 201-thallium perfusion scintigraphy, 99 m technetium multiple gated acquisition ventriculography (MUGA), resting echocardiography and 3 years follow-up are as follows: None had thallium findings indicating reversible myocardial ischaemia, but one from group A had a probable old myocardial infarction. All had normal resting MUGA, but group A men slightly more often presented a subnormal increase in ejection fraction according to exercise MUGA than group B men (9/20 vs 4/21). The former also more often had ventricular hypertrophy (LVH) (19/21 vs 14/21). However, apart from slightly longer ventricular filling time among group A men the echocardiograms revealed no group differences e.g. in cardiac dimensions or in indices of systolic or diastolic function. Regardless of exercise-ECG response, 18/42 athletes had one or more value of left ventricular dimensions or diameter exceeding the 95th percentile of the normal range. Since one patient from group A had asymmetric septal hypertrophy, one developed cardiomyopathy during the 3 years follow-up and one had a previous myocardial infarction, only 3/21 had cardiac disease which might explain the pathological exercise-ECG. Thus, pathological exercise-ECG rarely signifies heart disease in athletes, and very rarely coronary heart disease. Rather, the pathological exercise-ECG may be related to LVH and various subtle alterations in cardiac physiology following long-term endurance training. PMID- 2995036 TI - A giant tumor thrombus in the right atrium clearly detected by 111In-oxine labeled platelet scintigraphy. AB - A 54-year-old man was admitted to hospital with a 3-month history of progressive dyspnea with coughing. A giant right atrial mass, originating from a hepatocellular carcinoma, was visualized by computed tomography, and digital subtraction angiography. The volume of the right atrial mass was increasing rapidly. It was therefore essential to determine whether this giant mass was a tumor thrombus or a multiplication of the hepatocellular carcinoma. 111In-oxine labeled platelet scintigraphy revealed active accumulation in the right atrium caused by the presence of active platelet deposition, and slight accumulation in the lung fields probably due to embolic showers originating from the tumor thrombus in the right atrium. This is the first case report showing that 111In oxine labeled platelet scintigraphy can aid in confirming the nature of a giant tumor thrombus in the right atrium and can clarify the pathogenesis of the respiratory symptoms. PMID- 2995037 TI - Hemoptysis and pulmonary artery agenesis: case report. AB - The combination of a pulmonary scintigram using radioactive labeled albumin macroaggregates (MAA) and a study of the circulation in the bronchial artery was performed in one patient. This noninvasive methodology showed that there was increased circulation to the vascular territory of the lung in which the pulmonary artery was missing. This could have resulted from abnormal communications between the bronchial artery and the pulmonary vessels or an increased blood supply to the right lung from bronchial arteries arising from the aorta. The absence of pulmonary circulation in the right lung was proved by the absence of radioactivity in the right lung after an intravenous injection of labeled albumin MAA. PMID- 2995039 TI - Recovery of vaccinal poliovirus type 1 from conjunctiva of an infant with transient, unilateral conjunctivitis. PMID- 2995038 TI - Fructose-1,6-diphosphatase deficiency: glycerol excretion during fasting test. AB - A Turkish boy had suffered since the age of 10 months from recurrent attacks of severe metabolic acidosis and hypoglycaemia precipitated by moderate respiratory tract infections. A liver biopsy showed lack of fructose 1,6-diphosphatase and absence of phosphorylase. The patient died in shock following fructose ingestion. Upon fasting, acidosis with increased lactate and glycerol excretion was found. Findings indicate that, in this inherited disorder of gluconeogenesis, lactic acidosis combined with increased glycerol excretion upon fasting are of diagnostic importance. PMID- 2995040 TI - Signet ring cell adenocarcinoma of the urachus. AB - Signet ring cell adenocarcinoma of the bladder is rare. Only 16 cases are recorded in the literature, although two meet the requirements for being considered of urachal origin. We present the third case of signet cell adenocarcinoma of urachal origin. PMID- 2995041 TI - Genetic instability of cell lines derived from a single human small cell carcinoma of the lung. AB - Specimens from a human small cell carcinoma of the lung were established as a cell line in vitro. Flow cytometric DNA analysis demonstrated only one tumor cell population in the parent tumor as well as in the early passages in vitro. After six passages in vitro, two new subpopulations with different DNA content appeared. By cloning, permanent cell lines were established from the new subpopulations, whereas the original population stopped growing. The cloned cell lines were characterized by morphology, chromosomes analysis, electron microscopy and plating efficiency; the stability of the DNA content was examined regularly by flow cytometric DNA analysis and instability was found in one of the cloned cell lines. Chromosome analysis showed that the cloned cell lines consisted of more than one population after 17 in vitro passages. Both cloned cell lines produced tumors in nude mice. Genetic instability was demonstrated in these mouse grown tumors as well. Development of resistance to antineoplastic treatment may be due to heterogeneity in sensitivity among subpopulations in a tumor. Isolation of populations with different DNA contents allows the study of interaction between subpopulations and the observations provide evidence in support of the hypothesis of clonal evolution. PMID- 2995043 TI - Carcinogenesis of 4-aminobiphenyl in BALB/cStCrlfC3Hf/Nctr mice. AB - Male and female (840 each) BALB/cStCrlfC3Hf/Nctr mice were given 0, 7, 14, 28, 55, 110 and 220, and 0, 7, 19, 38, 75, 150 and 300 ppm, respectively, of 4 aminobiphenyl in their drinking water. Necropsies on killed animals were performed at 13, 26, 39, 52 and 96 weeks on dose. Dose-related neoplasms were angiosarcomas, bladder urothelial carcinomas and hepatocellular neoplasms. The non-neoplastic dose-related lesions were left atrial thrombosis, bladder urothelial hyperplasia, splenic hemosiderosis and splenic erythropoiesis. The incidences of bladder carcinoma and atrial thrombosis were higher in the males and the incidences of hepatocellular neoplasms and angiosarcomas were higher in the females. PMID- 2995042 TI - A comparison of established human lymphoma lines by flow cytometry: quantitation of Ricinus communis agglutinin binding and the effect of specific glycosidases. AB - Two established cell lines of human B-cell lymphomas derived from Burkitt lymphomas and their Epstein-Barr virus-transformed counterparts were analyzed with respect to their ability to bind the beta-galactoside-specific lectin Ricinus communis agglutinin (RCA). Native and sialidase- as well as sialidase beta-galactosidase-treated cells were compared. The method for the quantitative determination of average numbers of binding sites and of apparent affinity constants was flow cytometry with fluorescence-labeled lectin. Although with native cells there was no significant deviation of the values for virus transformed cells from those for the parent cells, some differences could be detected after glycosidase treatment. The general procedure of the combined application of specific glycosidases and the quantitation of sugar-specific lectin binding is recommended as a general strategy for the differentiation of cells with known or putative differences in biological functions. PMID- 2995044 TI - How much does liver disease affect the pharmacokinetics of adriamycin? PMID- 2995046 TI - Relation between 201Tl to 67Ga uptake ratio and histological type in primary lung cancer. AB - Quantitative 201Tl and 67Ga scans were performed on 44 patients with primary lung cancer to ascertain the differences in 201Tl and 67Ga uptake by tumors; especially the crude uptake ratio (CUR) of 201Tl to 67Ga was evaluated in relation to histological type. Adenocarcinomas showed a CUR of 2.00 +/- 1.55, the greatest value among all histological types. Epidermoid carcinomas and oat cell carcinomas had CURs of 0.47 +/- 0.30 and 0.37 +/- 0.05, respectively, both showing significantly lower values than adenocarcinomas (P less than 0.01). On the other hand, adenosquamous carcinomas indicated a CUR of 0.91 +/- 0.15, an intermediate value between adenocarcinomas and epidermoid carcinomas (P less than 0.05). These results indicated that 201Tl and 67Ga uptake by lung cancer was closely correlated with histological type, and also suggested that 201Tl and 67Ga scans enabled various histogeneses of lung cancer to be visualized. PMID- 2995045 TI - Receptors for retinoic acid and retinol in human mammary carcinomas. AB - The cellular content of receptors for retinol (CRBP) and retinoic acid (CRABP) was measured in 148 human mammary carcinomas. High levels of CRABP were found in lobular carcinomas while those of the papillary subgroup had low levels of the receptor. Intermediate values for CRABP were observed for ductal, colloid and medullar carcinomas. The cellular levels of CRBP were high in ductal carcinomas and low in papillar carcinomas. A positive correlation was observed between the receptor for estradiol and CRABP. However, no significant correlation was found between the tumor cell DNA pattern and the content of CRABP and CRBP respectively. Recurrence of disease could be predicted by the nodal status and the level of estradiol receptor while the DNA pattern and the content of receptors for retinoic acid and retinol failed. PMID- 2995047 TI - Decreased activity of liver glutathione peroxidase in human hepatocellular carcinoma. AB - Glutathione peroxidase (GSH-Px) activity, one of the scavenger enzymes of oxygen active radicals, has been measured in hepatocellular carcinoma (HCC) of 17 patients and the values compared with the activity of adjacent tumor-free tissue and with those of 30 histologically normal livers. The results demonstrate a reduced GSH-Px activity in neoplastic tissue (21.19 vs 33.74 U/g prot.; P less than 0.001). However, the adjacent tumor-free liver also had a reduced activity when compared with normal tissue (23.15 vs 33.74 U/g prot.; P less than 0.01), but this value did not differ from that of HCC tissue. These data suggest that HCC might develop in a GSH-Px-deficient condition. PMID- 2995048 TI - Effect of sympathetic stimulation and intrarenal alpha-blockade on the secretion of renin by the human kidney. AB - This study was initiated to explore the possible involvement of renal alpha adrenoceptors in the regulation of active and inactive renin. In fifteen hypertensive patients who proved not to have vascular abnormalities on diagnostic renal arteriography, blood samples were collected simultaneously from the renal artery and vein before and during an intrarenal infusion of either saline (n = 5), or the alpha-1 blocker doxazosin (n = 5) or the non-selective alpha-1 blocker doxazosin (n = 5) or the non-selective alpha-blocker phentolamine (n = 5). Subsequently, responses of renal blood flow and renin secretion were assessed following 3 min of handgrip exercise. In none of the experiments secretion of inactive renin could be detected. Release of active renin increased from 580 (SEM 170) to 650 (SEM 220) microU min-1 (100 g)-1 during infusion of doxazosin (P less than 0.05) and from 760 (SEM 100) to 1000 (SEM 340) microU min-1 (100 g)-1 during infusion of phentolamine (P less than 0.01). Saline infusion had no effect on secretion of active renin. While handgrip exercise had no significant effect on active renin secretion in the saline and in the doxazosin group, it enhanced secretion from 1000 (SEM 340) to 1280 (SEM 390) microU min-1 (100 g)-1 in the phentolamine group (P less than 0.01). The results indicate that mainly alpha-2 adrenoceptors exert an inhibitory effect on release of active renin, although alpha-1 receptors participate to some degree. There is no evidence that the kidney secretes inactive renin. PMID- 2995049 TI - Age related changes of platelet prostacyclin receptors in humans. AB - Platelet receptors for PGI2 were investigated in eighteen healthy volunteers divided in two classes of age: nine were 18-40 years old and nine were 41-65 years old. PGI2 platelet receptors were found to decrease significantly with age; the young subjects had 106 +/- 16 (mean +/- SD) high affinity receptors/platelet and 3509 +/- 529 low affinity receptors/platelet whereas the old subjects had 86 +/- 14 high affinity receptors/platelet (P less than 0.02) and 2471 +/- 640 low affinity receptors/platelet (P less than 0.005). The cumulated data from all the subjects yielded a significant negative correlation with age (r = -0.71, P less than 0.001 for the high affinity class and r = -0.65, P less than 0.01 for the low affinity class). The affinity of receptor sites for the ligand did not statistically change with aging (P less than 0.30 and P less than 0.90 respectively for high and low affinity receptors). PMID- 2995050 TI - Evidence for a Na-K-ATPase-inhibitor in erythrocytes of patients with essential hypertension. AB - ATPase activities were determined in haemolysed and dialysed erythrocytes and in haemoglobin-free membranes of twenty patients with essential hypertension and twenty normotensive controls. Ouabain-sensitive ATPase (Na-K-ATPase) activity of haemolysate but not that of membranes was decreased in hypertensives whereas ouabain-insensitive ATPase (Mg-ATPase + some residual Ca-ATPase) activity was increased in both enzyme preparations when measurements were preformed in the absence of Ca2+-chelating substances. In haemolysed erythrocytes ouabain sensitivity as a percentage of total ATPase activity was a good discriminator between both groups and may be a possible marker for essential hypertension. The decreased activity of Na-K-ATPase in haemolysate is apparently due to a non dialysable inhibitor of Na-K-ATPase which is either tightly bound to the erythrocyte membrane or dissolved in the cytoplasm. Following haemolysis with subsequent centrifugation the Na-K-ATPase inhibitor is removed, at least in part, and thus differences in Na-K-ATPase activity demonstrable in haemolysed and dialysed erythrocytes are no longer apparent in haemoglobin-free membranes. PMID- 2995051 TI - Human lymphocytes induce a differential release of prostaglandin E2 and interleukin 1-like mononuclear cell factor from normal blood monocytes. AB - Lymphocytes influence the production of both prostaglandin E2 (PGE2) and interleukin 1 (IL 1) by monocytes. We have examined whether these two products are released concomitantly or not under identical culture conditions using monocyte- and lymphocyte-enriched populations obtained on Percoll gradient. IL 1 was measured as mononuclear cell factor (IL 1/MCF). When incubated with concanavalin A, the monocyte-enriched fraction (MF; 80-91% monocytes), but not the lymphocyte-enriched fraction (LF; 95% lymphocytes) produced increasing amounts of PGE2 and MCF. However, when LF cells were added to MF cells in culture, a 40% to 60% decrease of PGE2 secretion was observed whereas the MCF production remained unchanged or increased up to 26-fold. Such a dissociation between IL 1 and PGE2 production by monocytes indicates independent regulation mechanisms in controlling the immune response under the influence of lymphocytes. PMID- 2995052 TI - Delayed onset of the amnestic effect of posttraining beta-endorphin: effects of propranolol administered prior to retention testing. AB - Mice were trained in a 1-trial inhibitory avoidance task (0.7 mA FS) and tested for retention at 1, 3, or 6 h following training. Posttraining beta-endorphin (0.1 micrograms/mouse i.p.) administration impaired retention at 6 h, but not 1 or 3 h after training. Propranolol (0.3 mg/mouse i.p.), but not naloxone (0.1 mg/mouse i.p.) administered prior to retention testing at 1 or 3 h accelerated the onset of amnesia in mice given posttraining beta-endorphin. Neither propranolol nor naloxone affected retention when given alone. These findings suggest that the delayed onset of the amnesia produced by posttraining beta endorphin is due to the activation of a beta-adrenergic system. PMID- 2995053 TI - Stereospecific modulation of tumorigenicity by opioid antagonists. AB - The effects of (+) and (-) isomers of naloxone in altering tumor response in A/Jax mice inoculated with S20Y neuroblastoma were determined. Mice given daily subcutaneous injections of 15 mg/kg (-)naloxone had a 71% tumor incidence, a 33% delay in the time before tumor appearance, and a 28% increase in survival time. Inoculation of neuroblastoma cells in control subjects resulted in 100% tumor incidence within 13 days. Animals given 15 mg/kg (+)naloxone were comparable to control subjects in all aspects of neural neoplasia. These results show that opioid antagonists exert a stereospecific action on neural cancers, and provide further evidence that endogenous opioid systems play an important role in neuro oncogenic expression. PMID- 2995054 TI - Chronic haloperidol causes increase in salivary response and alpha 1 adrenoceptors in submandibular gland of the rat. AB - The effect of chronic haloperidol on the receptor-secretion coupling of the submandibular glands of the rat was studied. After injection of 2 mg/kg haloperidol daily for 7 days, the dose-response curve to L-noradrenaline was displaced to the left, with lowering of the threshold and enhancement of the maximal response. This was accompanied by a 73% increase in alpha 1-adrenoceptors in the glands. The effect was selective, since no changes were observed in alpha 2- and beta-adrenoceptors. PMID- 2995055 TI - Differential localization of cholecystokinin-8 binding sites in the rat vs. the guinea pig brain. PMID- 2995056 TI - Benzodiazepine receptor binding with a new ligand, Ro 22-8515. PMID- 2995058 TI - Diazepam antagonizes GABAmimetics in rats with spontaneous petit mal-like epilepsy. AB - Wistar rats in our laboratory breeding colony spontaneously present petit mal like, non-convulsive, epileptic seizures. In these rats, as in other animal petit mal models, GABAmimetics, agonists of GABA-A receptors such as 4, 5, 6, 7 tetrahydroisooxazolo (5,4-c) pyridin-3-ol (THIP), or inhibitors of GABA catabolism such as gamma-vinyl GABA (GVG) or L-cycloserine (CYC), aggravated the seizures. Diazepam not only abolished the spontaneous seizures but also completely blocked the effects of the GABAmimetics, totally suppressing seizures in rats given THIP, GVG or CYC. These findings show that the mode of action of benzodiazepines is not comparable to a non-specific potentiation of GABA transmission, and suggest that the anti-absence effects of the benzodiazepines could depend on interactions with neurotransmitter systems other than GABA. PMID- 2995057 TI - Clonidine pretreatment enhances the sensitivity of the beta-noradrenergic receptor coupled adenylate cyclase system in astrocytes. PMID- 2995059 TI - Discrepancy between alpha 1-adrenoceptor-mediated contraction and the occupation theory in rat vas deferens--possible existence of a 'silent' receptor. AB - The relation of the amount of alpha 1-adrenoceptors (alpha 1-R) with contraction to norepinephrine (NE) through alpha 1-R in rat vas deferens was examined by means of radiobinding assays. Treatment with dibenamine decreased the maximal contraction to NE with a decrease in the amount of alpha 1-R but the relation was not linear. The contractile response disappeared completely when 20% of the alpha 1-R still remained. Moreover, culture of dibenamine-pretreated muscle restored the contraction to NE without a significant increase in the amount of alpha 1-R in the muscle. These findings suggest that some alpha 1-R are 'silent' in the contraction of rat vas deferens in response to NE under physiological conditions. PMID- 2995061 TI - Short-term lithium administration enhances serotonergic neurotransmission: electrophysiological evidence in the rat CNS. AB - Rats received lithium-containing chow for 48 h. Brain and plasma lithium concentrations ranged from 0.4 to 1.0 mEq/l. A first series of experiments served to assess the responsiveness of CA3 hippocampal pyramidal neurons to microiontophoretically applied serotonin (5-HT) and norepinephrine (NE). The response of the same neurons to electrical stimulation of the ventro-medial 5-HT pathway was measured after lithium treatment. The responsiveness to 5-HT and NE was not modified whereas the effect of activation of the ascending 5-HT pathway was increased two-fold by the lithium treatment. These neurons were activated to their physiological firing rate by means of small ejection currents of acetylcholine. A pretreatment with the 5-HT neurotoxin 5,7-dihydroxytryptamine abolished the response to the electrical stimulation in lithium-treated rats. In a second series of experiments, unitary recordings of 5-HT neurons were obtained from the dorsal raphe nucleus. The lithium treatment modified neither the number of spontaneously active 5-HT neurons nor their mean firing rate. These results provide direct electrophysiological evidence for the enhancement of 5-HT neurotransmission by short-term lithium treatment through its presynaptic action on 5-HT terminals. PMID- 2995060 TI - Effects of 4-aminopyridine on cat blood pressure in relation to the sympathetic nervous system. AB - The pressor effect of 4-aminopyridine (4-AP) was studied in anesthetized cats and in isolated cat aortic ring preparations. A significant increase in blood pressure (38.9 +/- 11.4 mmHg) was observed following intravenous administration of 4-AP (0.3 mg/kg). The elevated blood pressure lasted for 1.3 h and returned to the control level after 1.5 h. The pressor effect of 4-AP was not blocked by prior administration of phentolamine (2 mg/kg i.v.) or by pretreatment with reserpine (2 mg/kg i.m., once a day for two consecutive days). In vitro, a dose dependent tonic contraction of aortic ring was noted after addition of 4-AP into the bathing fluid. The ED50 of 4-AP was significantly greater than that of norepinephrine (NE) (8 X 10(-4) M vs. 10(-7) M). Phentolamine (10(-6) M) antagonized the contractile response to NE but not that to 4-AP, whereas verapamil (10(-5) M) attenuated the 4-AP induced contractile response. A diminished contractile effect was also obtained when aortic ring preparations were bathed in calcium-free solution. These data suggest that the sympathetic nervous system plays a minor role in the pressor effect of 4-AP and that the increased calcium influx may account, at least partially, for the vasoconstricting effect of 4-AP. PMID- 2995062 TI - Aging and digitalis sensitivity of cardiac muscle in rats. AB - The cause of the reduced tolerance of aged rats to the arrhythmogenic effect of ouabain was studied in left atrial and left ventricular muscle preparations obtained from 3-, 8- or 26-month old Fischer 344 rats. Under 1.5-Hz stimulation, the concentration-response curves for the positive inotropic effect of ouabain showed a tendency to shift to the left with age. The toxic effects occurred at lower ouabain or digoxin concentrations in the ventricular muscle obtained from senescent rats especially when the muscle was stimulated at a higher frequency (4 Hz), although differences in tolerance to the toxic effect of ouabain were not apparent in atrial muscle preparations stimulated at 1.5 Hz. No age-related difference in intracellular sodium or potassium concentration was observed in perfused ventricular muscle preparations paced at 1.5 Hz. There was no significant age effect on Na+-induced activation of Na+,K+-ATPase. In aged myocardium, however, the upstroke velocity of the action potential was reduced especially under high frequency stimulations, and the ability to follow high frequency stimulation was compromised. These results suggest that reserve capacity of the sodium pump is reduced in aged ventricular muscle, thereby elevating its sensitivity to the cardiac glycoside. PMID- 2995063 TI - Autoradiographic localization of benzomorphan binding sites in rat brain. AB - The benzomorphan subpopulation of opiate binding sites was labeled by [3H]diprenorphine in the presence of unlabeled ligands selected to quench mu and delta opiate binding sites. The distribution of benzomorphan binding sites was then localized autoradiographically. Areas particularly enriched in these sites were nucleus solitarius, nucleus ambiguus, substantia gelantinosa of the trigeminal nerve, the habenula, and the medial nucleus of the amygdala. Within hippocampal formation, binding was relatively enhanced in the pyramidal and granule cell layers. Within the basal ganglia, binding was greatest in the dorsomedial caudate nucleus and least in the globus pallidus. No 'patches' of increased binding were present in the striatum. The interstitial nucleus of the stria terminalis and the medial preoptic nucleus also showed significant binding. This distribution differs from the distributions of mu, delta and kappa opiate binding and is quite similar to the distribution of beta-endorphin immunoreactivity. These observations support the hypothesis, based on biochemical studies in brain membranes, that benzomorphan binding sites may represent the ligand recognition sites of putative epsilon receptors. PMID- 2995064 TI - Effects of methyl beta-carboline-3-carboxylate, Ro 15-1788 and CGS 8216 on muscle tone in genetically spastic rats. AB - Genetically spastic rats were used for studying the effect on muscle tone of beta carboline-3-carboxylic acid methylester (beta-CCM), an inverse benzodiazepine (BDZ) agonist, and that of Ro 15-1788 and CGS 8216, both putative antagonists of pharmacological actions of BDZs. These animals exhibit pathologically increased muscle tone, which can be recorded and quantified in the electromyogram (EMG) of the gastrocnemius (GS) muscle. beta-CCM, 2.5 and 3.0 mg/kg i.p., augmented the tonic activity in the EMG of GS muscle in spastic rats while it did not modify muscle tone at doses of 1.0 and 2.0 mg/kg. Diazepam, 0.4 mg/kg i.p., and Ro 15 1788, 5 mg/kg i.p., but not CGS 8216, 5 mg/kg i.p., antagonised the effect of the beta-carboline on muscle tone. Ro 15-1788, at doses of 0.1-5 mg/kg i.p., did not affect muscle tone in the genetically spastic rats, whilst at doses of 25-200 mg/kg the imidazodiazepine dose dependently increased the tonic activity in the EMG. The action of Ro 15-1788, 50 mg/kg i.p., was reversed by diazepam, 0.4 mg/kg i.p., and beta-CCM, 2 mg/kg i.p., while CGS 8216, 5 mg/kg i.p., facilitated the effect of Ro 15-1788 on muscle tone. CGS 8216 at doses of 5 and 25 mg/kg i.p. was devoid of any effect on muscle tone, whilst at doses of 50 and 200 mg/kg the pyrazoloquinoline increased the tonic activity recorded in the EMG from the GS muscle of the spastic rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995065 TI - Effects of converting enzyme inhibitors: ramipril and enalapril on peptide action and sympathetic neurotransmission in the isolated heart. AB - The role of converting enzyme (CE) in heart function was studied by assessing the inhibition of CE activity in rat heart tissue and in isolated perfused hearts from rat, guinea-pig and rabbit (Langendorff technique). Angiotensin I (ANG I) added to the perfusate reduced coronary flow (FLO) in all species, increased force of contraction (CON) in rats, decreased CON in guinea-pigs and had no effect on CON in rabbits. In contrast, bradykinin (BK) increased FLO in all species, decreased CON in rats, increased CON in guinea-pigs and had no effect on CON in rabbits. The CE inhibitors ramipril (HOE498) 1 mg/kg and enalapril (MK421) 30 mg/kg given orally 1 h prior to killing of the animals inhibited the ANG I effects and potentiated the BK effects but had no effects on the action of ANG II. The same doses of the two CE inhibitors produced up to 24 h inhibition of CE activity measured biochemically in the heart after single oral doses. Electrical sympathetic stimulation of the cardiac nerves (SNS) in isolated rabbit heart resulted in an increase of HR and CON and in an initial decrease and subsequent increase of FLO. The effects of SNS on HR and FLO were significantly reduced following HOE498 (1 mg/kg) pretreatment. The results suggest that local CE is involved in the regulation of peptide effects in the heart, including the ANG II mediated facilitation of neurotransmission. PMID- 2995066 TI - Actions and interactions of benzodiazepine agonists, antagonists and inverse agonists on suprahyoid muscle twitching. AB - Amphetamine-induced twitching of the suprahyoid muscle in nialamide-pretreated, urethane-anaesthetised rats was inhibited by the benzodiazepine agonists RU 32007, diazepam and chlordiazepoxide and the GABAA agonist muscimol. Ro15-1788 and CGS 8216 induced slight, or no reduction in twitching, respectively, but reversed the effect of RU 32007 and diazepam. Ro15-1788 did not reverse the effect of muscimol. Picrotoxin reversed both muscimol and RU 32007 effects at a dose which induced only a small increase in twitching alone. Higher doses of picrotoxin markedly increased amphetamine twitching and induced twitching in the absence of amphetamine. Ethyl-beta-carboline-3-carboxylate (BCE) weakly increased twitching and reversed the effect of RU 32007. CL 218872 weakly reduced twitching but also weakly antagonised RU 32007. In rats pretreated with a higher dose of nialamide methyl beta-carboline-3-carboxylate (BCM) and BCE induced twitching. BCE-induced twitching was antagonised by RU 32007, Ro15-1788, CGS 8216, muscimol and weakly by CL 218872. Thus, benzodiazepine agonists inhibit twitching and inverse agonists induce twitching, both being blocked by antagonists. Partial agonism (CL 218872) or partial inverse agonism (CGS 8216) may be detected by differential effects against different inducers of twitching. PMID- 2995068 TI - K+-dependent 3-O-methylfluorescein phosphatase activity in crude homogenate of rodent heart ventricle: effect of K+ depletion and changes in thyroid status. AB - The total Na+-K+-ATPase activity in rodent heart ventricle was quantified by determination of K+-dependent, ouabain suppressible 3-O-methylfluorescein phosphatase activity. A K+-dependent phosphatase activity of 0.80 mumol/min per g wet wt was obtained from crude homogenate of heart ventricle from 4-week-old guinea pigs. The anticipated association between K+-dependent phosphatase activity and Na+-K+-activated ATP hydrolysis could be clearly demonstrated in the crude homogenate. Based upon a molecular activity of 550/min the corresponding cardiac glycoside receptor concentration in the crude homogenate was 1450 pmol/g wet wt. In crude homogenate of heart ventricle from 3-month-old rats the K+ dependent phosphatase activity was 1.16 mumol/min per g wet wt. Following 4 weeks of K+ depletion of the rats, a decrease of 13% in total K+ content of the heart ventricle was seen. This was associated with a 14% decrease in K+-dependent phosphatase activity. The induction of hypo- and hyperthyroidism for 3 weeks in rats was followed by a decrease of 27% and an increase of 13% in K+-dependent phosphatase activity, respectively. PMID- 2995067 TI - Alpha 2-adrenoceptors modulate kainic acid-induced limbic seizures. AB - We have tested several compounds interfering with the brain monoamine (noradrenaline, dopamine, serotonin) and acetylcholine systems for their effects on limbic seizures produced by systemically (s.c.) injected kainic acid as well as on neurochemical changes in amygdala/pyriform cortex resulting from the kainic acid treatment. The characteristic neurochemical changes induced by s.c. kainic acid were a decrease in noradrenaline and an increase in 5-hydroxyindoleacetic acid in the acute (3 h after kainic acid injection) suggesting strongly increased neurotransmitter turnover in noradrenergic and serotonergic neurons. This was followed by a reduction of glutamic acid decarboxylase and choline acetyltransferase activities during the chronic phase (3 days) of the kainic acid action, indicating destruction of GABAergic and cholinergic neurons. The compounds tested in this model of limbic epilepsy included 1-propranolol, prazosin, clonidine, yohimbine, metergoline, atropine and haloperidol. Among these compounds the alpha 2-adrenergic agonist clonidine (0.1 mg/kg, i.p.) exhibited a powerful protective action on kainic acid-induced limbic seizures as well as on the neurochemical changes in the amygdala and pyriform cortex. In addition, the adrenoceptor antagonists prazosin (alpha 1) and propranolol (beta) as well as the dopamine receptor antagonist haloperidol had significant but less potent - protective actions upon kainic acid-induced seizures and subsequent neurochemical changes. On the other hand, yohimbine (alpha 2-antagonist) and metergoline (serotonin-antagonist) potentiated the limbic seizure syndrome and no effect was found with atropine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995069 TI - The influence of CGP 28392, a 1,4-dihydropyridine, on human platelet calcium and cyclic AMP metabolism. AB - Use of Ca2+ ionophores (e.g. ionophore A 23187) and Ca2+ channel antagonists (e.g. dihydropyridines, phenylalkamines) has provided some insight into the molecular events involved in platelet activation. Recent structural modification of dihydropyridines has generated a novel class of compounds (e.g. CGP 28393) which apparently exert opposing functional effects. We report that CGP 28392 induced an elevation in free intracellular calcium concentrations [( Ca2+]i) via both calcium influx and mobilization of intracellular stores. Treatment of washed human platelets with this Ca2+ agonist resulted in typical Ca2+-linked activation phenomena such as shape change and phosphorylation of Mr 47 000 and Mr 20 000 proteins. CGP 28392 also negatively influenced platelet cyclic AMP metabolism, and appears to exert this effect as a consequence of elevated [Ca2+]i. The data suggest that the mechanisms of action of CGP 28392 involve dihydropyridine susceptible structures on the cell membrane and intracellular Ca2+ binding sites. PMID- 2995070 TI - Further evidence for involvement of adenosine-5'-triphosphate in non-adrenergic non-cholinergic relaxation of the isolated rat duodenum. AB - The nature of the inhbitory non-adrenergic non-cholinergic (NANC) neurotransmitter responsible for neurogenic relaxation of rat duodenum was studied with in vitro techniques. Adenosine 5'-triphosphate (ATP)(1 mM), gamma aminobutyric acid (GABA, 1 mM), dimethylphenylpiperazinium (DMPP, 0.1 mM) and field stimulation (60 V, 2 ms, 0.1 Hz) produced transient relaxation followed by rebound contraction. In contrast vasoactive intestinal polypeptide (VIP) (0.3 microM) and noradrenaline (1 microM) induced relaxation which set in more slowly and lasted longer. Tetrodotoxin (0.85 microM) abolished field stimulation-induced relaxation but not ATP-, VIP- or noradrenaline-induced relaxation. Nucleotide pyrophosphatase (0.25 U/ml), but not the proteolytic enzyme alpha-chymotrypsin (2 U/ml), selectively antagonized NANC relaxation. The rank order of potency of various adenine derivatives for inducing relaxation was adenosine-5'-triphosphate greater than adenosine-5'-diphosphate much greater than adenosine greater than adenosine-5'-monophosphate. ATP-induced relaxation was selectively antagonized by the putative P2 purinoceptor antagonist reactive blue 2, but unaffected by the selective P1 purinoceptor antagonist 8-phenyltheophylline. The duration of ATP- as well as beta-gamma-methylene adenosine-5'-triphosphate (a stable analogue of ATP)-induced relaxation was similar and was unaffected by indomethacin 10 microM (which abolished the rebound contraction). In those preparations whose contractile tone was increased by using a high-K+ medium the ability of ATP to elicit relaxation was markedly reduced, while GABA- and DMPP-induced relaxation was abolished. On the other hand, ATP-, GABA- and DMPP-induced relaxation of the tonic component of 5-hydroxytryptamine (5-HT)(0.1 mM)-induced contraction was similar to that observed in control conditions. These findings add further weight to the proposal that endogenous ATP is involved in determining NANC relaxation of rat duodenum. PMID- 2995071 TI - Dual effects of acetaldehyde on electrical activity in the isolated frog spinal cord. AB - Acetaldehyde (1.4 X 10(-7)-1.4 X 10(-5) M) produced a transient depolarization followed by a distinct hyperpolarization of the ventral and dorsal roots as recorded by sucrose-gap in the isolated spinal cord. This effect occurred more frequently at lower concentrations than at higher concentrations of acetaldehyde, and the depolarization increased with doses increasing. The ventral root reflex potentials evoked by the dorsal root stimulation had decreased amplitudes during depolarization and increased during hyperpolarization. In the presence of Mg2+ or tetrodotoxin, acetaldehyde produced only hyperpolarization. Acetaldehyde slightly prolonged glutamate-induced depolarization of both roots. These results suggest that acetaldehyde acts not only on the spinal motoneuron resulting in membrane hyperpolarization but also on the presynaptic sites, which causes postsynaptic membrane depolarization probably through an increased release and decreased uptake or inactivation of the excitatory transmitter. PMID- 2995072 TI - An anxiogenic benzodiazepine receptor ligand induces learned helplessness. AB - Rats treated with the anxiogenic beta-carboline, N-methyl-beta-carboline-3 carboxamide (FG-7142), failed to acquire an escape response 24 h after treatment. Administration of FG-7142 resulted in a behavioral effect equivalent to a session of inescapable tailshock in this paradigm of learned helplessness. Pretreatment of rats with the selective benzodiazepine receptor antagonist Ro15-1788 blocked the development of learned helplessness elicited by FG-7142. These findings suggest that 'anxiety' may be a major factor in the development of learned helplessness. PMID- 2995073 TI - Phencyclidine and sigma opiate receptors in brain: biochemical and autoradiographical differentiation. PMID- 2995074 TI - Adrenergic modulation of vascular prostacyclin (PGI2) secretion. AB - An in vitro model for the study of adrenoreceptor-prostacyclin (PGI2) relationships in the rat aorta is described. PGI2 synthesis was stimulated by adrenergic agonists (rank order of potency: epinephrine greater than norepinephrine greater than phenylephrine greater than methoxamine). Isoproterenol, UK 14304, clonidine and salbutamol were without effect. Epinephrine (3 X 10(-7) M)-stimulated PGI2 synthesis was inhibited by adrenoreceptor antagonists (rank order of potency: yohimbine greater than prazosin greater than phentolamine greater than corynanthine much greater than propranolol). The absence of calcium in incubation media abolished epinephrine stimulated PGI2 synthesis as did the calcium channel blocker, verapamil, in a dose-dependent manner. Calcium ionophore A23187 (10(-5) M)-stimulated PGI2 synthesis was inhibited by verapamil (in a dose-dependent manner), but not by prazosin, phentolamine or yohimbine. It is concluded that epinephrine-mediated rat aortic PGI2 synthesis is alpha-adrenoceptor- and not beta-adrenoceptor mediated, calcium-dependent, and that the alpha-adrenoceptor antagonists evaluated do not have verapamil-like calcium channel blocking activities. These findings may be relevant to contraction-relaxation cycles of vascular tissue. PMID- 2995075 TI - Alpha 1-adrenoceptor-mediated inositol phospholipid hydrolysis in rat cerebral cortex: relationship between receptor occupancy and response and effects of denervation. AB - The characteristics of catecholamine-mediated breakdown of inositol phospholipids in rat cerebral cortex slices have been examined using a direct assay involving prelabeling with [3H]inositol and examining the production of labelled inositol phosphates in the presence of lithium. Noradrenaline produced a marked stimulation of inositol phosphate accumulation and this response could be potently and competitively antagonised by the alpha 1-adrenoceptor antagonist prazosin. The alpha 2-antagonist yohimbine was almost 1000-fold less potent at antagonising noradrenaline inositol phospholipid response. Noradrenaline and adrenaline were full agonists at alpha 1-adrenoceptors but phenylephrine and methoxamine were only partial agonists in their ability to stimulate inositol phospholipid metabolism. There was a significant correlation between the ability of a variety of agonists and antagonists to activate or inhibit [3H]inositol phosphate accumulation and their ability to displace the alpha 1-adrenoceptor selective ligand [3H]prazosin from specific binding sites when assays were performed on rat cerebral cortical slices under identical conditions. The similarity of EC50 values of agonists stimulating inositol phosphate accumulation and their IC50 values in [3H]prazosin binding experiments suggested a close relationship between receptor occupancy and alpha 1-mediated inositol phosphate accumulation. Further experiments were performed to examine this directly by inactivating alpha 1-adrenoceptors with the alkylating antagonist phenoxybenzamine. After washing out unbound antagonist, [3H]prazosin binding was reduced to a very similar proportion to that observed on the maximal noradrenaline-stimulated accumulation of [3H]inositol phosphates in the slices. The EC50 values for noradrenaline-stimulated inositol phosphate accumulation was unaltered and the affinity of [3H]prazosin for the remaining sites was equally unaffected. In rats treated 14 days previously with i.c.v. 6-hydroxydopamine (2 X 250 micrograms) there was a small increase in alpha 1-adrenoceptor binding sites but a parallel shift to the left in the noradrenaline [3H]inositol phosphate accumulation dose-response curve. On the other hand, the partial agonist phenylephrine induced a larger maximal response in denervated animals without a change in the EC50 values. When slices from 6-hydroxydopamine treated animals were preincubated with phenoxybenzamine, the loss in alpha 1-adrenoceptor binding sites was greater than the reduction in the maximal response to noradrenaline. This may indicate the development of a small receptor reserve after denervation.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2995076 TI - Differential regulation of the cyclooxygenase pathway in starch elicited rat peritoneal macrophages by prostaglandin E2, U-44069, a stable endoperoxide analogue and dibutyryl-cyclic AMP. AB - The basal and carrageenin-stimulated release of thromboxane (TX) B2, the stable product of TXA2, 6-ketoPGF1 alpha, the stable breakdown product of prostacyclin (PGI2) and prostaglandin (PG) E2 from 24 h starch elicited rat peritoneal macrophages was inhibited by dibutyryl-cyclic AMP (db-cAMP). PGE2 also inhibited the release of TXA2 and 6-keto-PGF1 alpha whereas the stable endoperoxide analogue, U-44069, stimulated PGE2 and 6-keto PGF1 alpha release but inhibited TXB2 release. The effects of all three mediators tested were related to an increase of Mo intracellular cAMP content. PMID- 2995077 TI - Action of pinaverium bromide on calmodulin-regulated functions. AB - Pinaverium bromide at concentrations below 10(-5) M did not inhibit calmodulin dependent enzymes such as phosphodiesterase and the Ca transport ATPase of the plasma membrane. At higher concentrations the compound interacted with the stimulation of those enzymes by calmodulin and also inhibited the calmodulin independent activity. A similar inhibitory action was observed for the NaK ATPase. It is concluded that the inhibitory action of pinaverium bromide on smooth muscle concentration at concentrations below 10(-5) M was due to its interaction with the voltage-dependent Ca channels and not to its interference with the calmodulin-dependent activation of the contractile proteins. PMID- 2995079 TI - Involvement of different opioid receptor subtypes in electric shock-induced analgesia and motor suppression in the rat. AB - We have investigated the correlation of electric shock-induced behavioral changes and functional alterations of endogenous opioid receptor subtypes. The degree of electric shock-induced behavioral changes, such as analgesia and motor suppression, was dependent on the duration of and time after electric shock application. The electric shock-induced behavioral changes were completely antagonized by naloxone. The apparent development of tolerance to both behavioral effects as a result of successive daily electric shock was different: Tolerance to electric shock-induced analgesia developed after 2 days' successive electric shock application, while tolerance to motor suppression was not observed even after 7 days' successive electric shock application. There was a decrease of [3H][D-Ala2, Met5]enkephalinamide ([3H]DAMEA, delta agonist) binding and an increase of [3H]naloxone (mu antagonist) binding when potent electric shock induced analgesia developed. On the other hand, the binding of [3H]DAMEA and [3H]ethylketocyclazocine (kappa agonist) was significantly changed when locomotion was suppressed. These results suggest strongly that different opioid systems may participate in electric shock-induced analgesia and motor suppression: electric shock-induced analgesia and motor suppression may be mediated by mu/delta and kappa/delta receptors, respectively. PMID- 2995078 TI - Inhibition of intrathecally administered picrotoxin- and bicuculline-induced convulsions in mice by pipecolic acid or GABA. AB - Pipecolic acid (PA) is an alicyclic amino acid and putative neurotransmitter which may modulate GABAergic transmission in the central nervous system. The present study was designed to investigate the anticonvulsant effect of intrathecally (i.t.) injected PA on picrotoxin- and bicuculline-induced convulsions which are thought to be produced by interactions with GABAergic systems. Intrathecal injections of picrotoxin and bicuculline in mice produced convulsions which were characterized by a rapid onset and short duration. Coadministration of GABA with either bicuculline or picrotoxin, but not strychnine, attenuated the severity of the convulsions. Coadministration of PA also protected against bicuculline- and picrotoxin-induced convulsions. Intrathecal injections of PA produced a dose-related increase in the latency to the onset of these convulsions as well as a decrease in their duration, however PA failed to inhibit the duration of strychnine-induced seizures. The D isomer of PA was found to be more effective than the L isomer as an anticonvulsant in this study. When administered in a high dose (500 micrograms i.t.), the D isomer produced flaccid paralysis while injection of high doses (100-500 micrograms i.t.) of the L isomer actually elicited convulsions. These results further support an interaction between PA and GABAergic activity. PMID- 2995080 TI - Electroencephalographic study of SR 95103, a GABAA antagonist: interaction with inhibitory amino acids and muscimol. AB - SR 95103 has recently been described as a selective GABAA antagonist. In this study, the electroencephalographic (EEG) effects of SR 95103 were investigated as well as its central interaction with inhibitory amino acids and muscimol. Slow intravenous infusions of SR 95103 in rats induced epileptiform EEG activities which were antagonized by intracerebroventricularly injected muscimol, GABA and taurine whereas glycine did not modify and even facilitated the effects of SR 95103. These results suggest that the EEG effects of SR 95103 are due to the specific GABAA antagonistic properties of this compound. PMID- 2995081 TI - Changes in adrenoreceptor density in brown adipose tissue from hyperthyroid rats. AB - Treatment of rats with triiodothyronine (T3 10 micrograms/100 g per day, 20 days) increased the mass of brown adipose tissue (BAT) and heart ventricle and stimulated beta-adrenoreceptor number (assessed from radioligand binding) by 43% in isolated BAT and heart membranes. alpha-Receptor number was slightly increased in BAT membranes (42%) but reduced in heart from T3-treated rats. The ratio of alpha-receptors/beta-receptors was unaffected by T3 treatment in BAT membranes (control and hyperthyroid = 1.54) but reduced in heart (control = 2.8, T3 = 1.04). PMID- 2995082 TI - Effects of GABA receptor agonists on [3H]dopamine release from median eminence and pituitary neurointermediate lobe. AB - The effects of GABAA and GABAB receptor agonists were examined on stimulus induced release of [3H]dopamine ([3H]DA) from a synaptosomal preparation of median eminence (ME) and pituitary neurointermediate lobe (NI). Muscimol was found to inhibit [3H]DA release from ME in a bicuculline-sensitive, strychnine insensitive manner, but had no effect in NI. Homocarnosine also inhibited [3H]DA release from ME. Baclofen had no effect in either area. These results demonstrate an interaction between two putative prolactin release inhibitory factors, DA and GABA, at the site of secretion into hypophysial portal blood. PMID- 2995083 TI - Postsynaptic alpha 2-noradrenergic receptors mediate feeding induced by paraventricular nucleus injection of norepinephrine and clonidine. AB - This study examines the feeding response induced by hypothalamic noradrenergic stimulation, in terms of the type and synaptic position of its mediating receptor. Tests with norepinephrine or the alpha 2 receptor agonist clonidine, injected into the area of the paraventricular nucleus (PVN), revealed a potent feeding response in satiated animals. This response by either agonist was blocked, in a dose-dependent fashion, by local injection of the alpha 2 noradrenergic antagonists, rauwolscine and yohimbine. It was also blocked by the general antagonist, phentolamine. In contrast, it was unaffected by hypothalamic injection of the alpha 1-noradrenergic antagonists, prazosin and corynanthine. These results indicate that feeding elicited by noradrenergic stimulation in the region of the PVN is mediated through alpha 2-type receptors. These alpha 2 receptors appear to be located postsynaptically, since the effectiveness of clonidine in eliciting eating was undisturbed by prior injection of the catecholamine synthesis inhibitor, alpha-methyl-p-tyrosine. PMID- 2995084 TI - Autoradiographic study of the alpha-adrenoceptors of rat aorta and tail artery. PMID- 2995085 TI - Comparison of angiotensin receptors in isolated smooth muscle tissues by photoaffinity labelling. AB - The isolated rat uterus and rabbit aorta were photolabelled with [Azidobenzoic acid 1, isoleucine8]angiotensin II ([AB', Ile8]ANG II) and dose-response curves to angiotensins II and III were determined before and after the irreversible labelling procedure. The data obtained were used to calculate affinities, efficacies and 'spare' receptors for angiotensins II and III. In the rat uterus angiotension II was the higher affinity ligand, whereas in the rabbit aorta angiotensins II and III had similar affinities. 'Spare' receptors were present for both ligands in both tissues. In the rat uterus ANG II and ANG III had similar efficacies, whereas in the rabbit aorta ANG II was the more efficacious ligand. Angiotensin receptors in the rabbit aorta, rat uterus, and rat portal vein appear to be substantially different from one another, and point to the possibility of different functional roles for angiotensins II and III in different smooth muscle tissues. For the vascular tissues, tachyphylaxis to angiotensin was more marked after irreversible labelling than before, suggesting that the mechanism of angiotensin tachyphylaxis is receptor deactivation. PMID- 2995086 TI - Localization of beta-adrenoceptors in the rabbit ear by light microscopic autoradiography. AB - The beta-adrenoceptor antagonist [125I]cyanopindolol (CYP) was used to localize beta-adrenoceptors in sections of rabbit ear. Biochemical studies demonstrated that the binding was stereoselective, and that the beta-adrenoceptors are predominantly of the beta 2-subtype. Autoradiographic studies using 3H-Ultrofilm or nuclear emulsion coated coverslips showed high concentrations of beta 2 adrenoceptors present in the central ear artery, hyaline cartilage, nerve trunks, epithelium and sebaceous glands. PMID- 2995087 TI - The lack of effect of electrochemical stimulation of the hypothalamus and median eminence on the plasma corticosterone level after lesion of the paraventricular nuclei. AB - Electrochemical stimulation of the median eminence of intact rats causes a significant increase in plasma corticosterone level, while the same stimulation within the middle part of the medial basal hypothalamus outside the median eminence is ineffective. Partial removal of the paraventricular nuclei greatly reduces, while complete lesion abolishes the above effect of electrochemical stimulation of the median eminence upon plasma corticosterone level. PMID- 2995088 TI - Effect of the antiserotonin drug, cyproheptadine, on plasma beta-endorphin and beta-lipotropin in patients with Itsenko-Cushing's disease. AB - In the present paper evidences are provided for the changes occurring in blood opioid neuropeptides in patients with Itsenko-Cushing's disease), who were treated with an antiserotonin drug, cyproheptadine (peritol). An increase in the plasma contents of beta-endorphin and beta-lipotrophin in the above patients has been found to be one of the factors conditioning an enhanced activity of the hypothalamic-pituitary-adrenal system. Treatment of the patients suffering from Itsenko-Cushing's disease with cyproheptadine (peritol) resulted in a decrease of the plasma beta-endorphin and beta-lipotropin, as well as a display in 60% of cases of clinical and laboratory remission. Absence of the clinical remission in some patients treated with peritol indicated a need for intervention in the metabolism of other monoamines involved in the modulation of the action of opioid neuropeptides on the activity of the hypothalamic-pituitary system. PMID- 2995089 TI - Prolonged influence of a single neonatal steroid (dexamethasone) treatment on thymocytic steroid binding. AB - Treatment of newborn rats with a single low dose of dexamethasone gave rise to lasting alteration of thymocytic receptor binding capacity. The receptors decreased markedly in number, while their affinity for the hormone was slightly increased. It appears that imprinting with a steroid changes the receptor- hormone relationship in the adult organism. PMID- 2995090 TI - Diacytosis of human asialotransferrin type 3 in the rat liver is due to the sequential engagement of two receptors. AB - The possible role of transferrin receptors in the diacytosis of human asialotransferrin type 3 (HAsTf-3) by the rat liver was studied in vivo. A trace dose of the ligand was allowed to compete for hepatic binding sites against diferric transferrin, the concentration of which was varied between 5 400- and 18 000-fold. Binding of HAsTf-3 was insensitive to the presence of 2Fe-transferrin in this range, and the liver bound the ligand equally efficiently, regardless of whether it was presented in the holo or apo form. In contrast, pretreating the animals with desialylated bovine submaxillary mucin (2 mg/100 g, 2 min before the dose) prevented the asialotransferrin-liver interaction. These findings indicate that endocytosis of HAsTf-3 is mediated by the Gal/GalNAc-specific lectin and not by transferrin receptors. Although 2Fe-transferrin did not affect binding, it did reduce the half-life of the ligand in the liver, thus suggesting that transferrin receptors play an important role in the exocytic leg of the diacytic cycle. Based on our present and earlier data, a model is proposed in which the engagement of lectin and transferrin receptor in the diacytic cycle is envisaged sequentially so that HAsTf-3 switches receptors at an acidified subcellular site. PMID- 2995091 TI - Cell cycle-dependent inhibition of human vascular smooth muscle cell proliferation by prostaglandin E1. AB - We examined the influence of prostaglandins on the initiation of proliferation of growth-arrested human adult aortic and fetal smooth muscle cells. Prostaglandins of the E series (25 nM) exerted a significant (p less than or equal to 0.05) inhibitory effect on DNA synthesis. Inhibition was observed when PGE1 was added in the G1 phase of the cell cycle. PGE1 had no effect when added once DNA synthesis had started. Thus prostaglandins of the E series may inhibit the responsiveness of smooth muscle cells to the mitogenic action of critical growth factors, such as PGDF. This inhibitory response is cell-cycle dependent. Once smooth muscle cells have entered S phase, PGE1 is no longer effective. Our data also suggest that cAMP is involved in the PGE1-induced growth inhibition, since concomitant with PGE1 addition, cAMP levels rose rapidly; addition of the cAMP analogue db-cAMP resulted in a cell-cycle-dependent inhibition pattern comparable to that observed with PGE1. PMID- 2995092 TI - Mobilization and aggregation of integral membrane proteins in erythrocytes induced by interaction with influenza virus at acidic pH. AB - Effect of influenza virus on erythrocyte membranes was investigated by electron microscopy and fluorescence photobleaching recovery measurements. The virus induced mobilization of integral proteins in erythrocyte membrane at acidic pH, where it fused with the cell membrane to cause hemolysis and also cell fusions but not at neutral pH. At lower temperatures (e.g., 4 degrees C), the proteins aggregated in the membrane and, consequently, large protein-free lipid bilayer area was produced. At higher temperatures (e.g., 37 degrees C) the protein distribution became randomized. Spectrin meshwork underneath the erythrocyte membrane was also markedly modified by the virus at acidic pH. Diffuse fibril structure was converted into dense spots and the membrane area lacking the fibril structure was produced. Isolated hemagglutinin rosettes also caused mobilization and aggregation of the integral proteins at acidic pH but to smaller extent than that induced by virus. The membrane perturbation detected as the protein mobilization by the action of hemagglutinin was assigned to be the cause for envelope fusion. PMID- 2995093 TI - Association of phosphorylated simian virus 40 T-antigen with subnuclear fractions of infected and transformed cells. AB - To define the roles of subnuclear structure in SV40 infection, the relative distribution of T-antigen (T-ag) in various subnuclear fractions obtained from both lytically infected and transformed African green monkey kidney cells was determined. Depending on the differential sensitivity of nuclear T-ag to extraction by salt and detergent, nuclear T-ag could be separated into nucleoplasmic T-ag, salt-sensitive T-ag and matrix-bound T-ag subclasses. At least fivefold less matrix-bound T-ag was found in transformed cells than in lytically infected cells. While a cAMP-independent protein kinase was detected in the nuclear matrix, the matrix-bound T-ag (94K) could not be phosphorylated in vitro. The removal of cellular chromosomes by DNase caused changes in the interaction of T-ag with nuclear components. The results suggest that the compartmentalization of nuclear T-ag may be determined by its interaction with host chromosomes. PMID- 2995094 TI - Further characterization of the phenotype of ts20, a DNAts mutant of BALB/3T3 cells. AB - Ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells that is blocked at a step in DNA synthesis involving chain elongation. Following a shift from 33 degrees to 39 degrees C, mutant cells lost ability to grow or form colonies. When mutant cells were infected with polyomavirus, both cell and virus DNA synthesis were inhibited at the restrictive temperature of 39 degrees C. When cell extracts from wild-type cells were added in vitro to lysed infected mutant cells that had been incubated in vivo at 39 degrees C for expression of the mutation, cell DNA synthesis was increased 3-fold (similar to the effect in uninfected mutant cells), whereas virus DNA synthesis was increased only 60%. With harsher lysis conditions, the effect of added extract on virus DNA synthesis was greater, although baseline DNA synthesis (prior to addition of extracts) was much lower. Analysis by alkaline sucrose gradients showed that the addition of cell extract converted small cellular DNA molecules into larger ones, while it increased the synthesis of small virus DNA molecules rather than completed genomes. Analysis of cytosol extracts (in which the activity stimulating DNA synthesis resides) showed that DNA topo-isomerase I activity was more heat-labile when assayed in mutant extracts compared to wild-type extracts. In contrast, cytosol DNA polymerase activity was equally heat-labile in mutant and wild-type extract. This suggested the factor in extract was likely associated with the activity of DNA topo-isomerase I. Analysis of virus DNA synthesized in vitro in restricted mutant cells by gel electrophoresis and fluorography showed an accumulation of topo-isomers migrating between form I and II. These topo-isomers, thought to be a manifestation of the ts defect, did not disappear when extract from wild-type cells was added back in vitro or when mutant cells were shifted back to permissive temperature prior to lysis for in vitro synthesis. The results indicate that polyoma DNA synthesis and cell DNA synthesis differ in their response to the mutant gene product in ts20, although both are inhibited at a step early in DNA chain elongation that may involve DNA topo-isomerase I. PMID- 2995095 TI - Tumor-promoting phorbol esters and vanadate alter the sensitivity of HeLa S3 cells to poliovirus type 1. AB - Treatment of HeLa S3 cells with tumor-promoting phorbol esters and vanadate increased their sensitivity to type 1 poliovirus. Since the sensitization could not be accounted for by increased virus binding or virus production, it appears that virus entry was facilitated by the treatments. When HeLa S3 cells were incubated with TPA for prolonged periods of time, they became resistant to poliovirus due to reduced ability to bind the virus. PMID- 2995097 TI - Effects of cytochalasin B on cell movements and chemoattractant-elicited actin changes of Dictyostelium. AB - The actin-binding drug cytochalasin B (CB) was employed to study the stability and role of cytoskeletal actin following chemotactic stimulation of Dictyostelium discoideum. Intact amoebae were found to be impermeable to this drug, as shown by lack of inhibition of chemotactic movement in its presence and failure of [3H]CB to bind to intact amoebae. However, there were approx. 150 000 high affinity CB binding sites per cell detectable after cell breakage and preparation of Triton insoluble cytoskeletons. The effect of CB on cytoskeletons was to destabilize the second (25-45 sec) and third (60 sec) chemotactically-induced peaks of cytoskeletal actin accumulation and to reduce the actin levels to the low prestimulus amount. In contrast, the drug had no such action on the rapid (3-5 sec) actin peak. This peak appeared to be stable in the presence of CB added before or simultaneously with lysis of the cell. It was also observed that the instability of the second and third peaks to CB gradually decreased after cell lysis (as did the number of CB binding sites) such that if CB was added 5 min after lysis of the chemotactically stimulated amoebae it had no destabilizing effect. Evidence was obtained from experiments employing centrifugation of cytoskeletons at 100 000 g and from the use of the DNase I inhibition assay for G actin, that the first (3-5 sec) actin peak of accumulation involved polymerization rather than just cross-linking of short filamentous actin fragments. The significance of these actin accumulation peaks is discussed and their timing correlated with events involved in chemotaxis. PMID- 2995096 TI - Epithelial HBL-100 cell line derived from milk of an apparently healthy woman harbours SV40 genetic information. AB - The epithelial HBL-100 cell line was established in vitro from milk of an apparently healthy woman. It exhibits characteristics of transformation from the very beginning and evolves during in vitro maintenance, until becoming tumorigenic in nude mice. This immortal cell line represents a useful model for studying the progression of human epithelial cells toward malignancy. In the course of our investigations we detected a 94K protein in HBL-100 cells obtained from four different sources. This protein is shown to be indistinguishable from the SV40 large T-antigen on the basis of: Recognition by polyclonal and different monoclonal antibodies. Partial peptide map analysis. Specific binding capacity to the SV40 DNA origin of replication. The presence of a tandemly integrated SV40 genome is demonstrated by Southern blotting. Successful rescue of SV40 DNA by fusion with permissive COS-7, but not CV-1 cells, indicates that the SV40 T antigen from HBL-100 cells is defective in a function(s) essential to the replication of the viral DNA. The possible origin of the SV40 genetic information that we have detected in HBL-100 cells and the implications of this finding on studies involving this cell line are discussed. PMID- 2995098 TI - Cytoskeletal proteins associated with intracytoplasmic human adenovirus at an early stage of infection. AB - Taking advantage of the sedimentation properties of adenovirus particles, adenovirus-infected baby hamster kidney (BHK21) cells were reversibly fixed with cleavable diimidoester dimethyl 3,3'-dithiobispropionimidate (DTBP) at early times of infection (30 min). Cytoskeletal proteins associated with/or in close vicinity to virions were isolated as a complex cross-linked with carrier virus. Four major cellular proteins were thus found to co-purify with adenovirus particles. They were characterized by their coordinates on 2D maps and immunological reactivity. Two of them were identified as alpha-tubulin (58 kD), and vimentin subunits (56 kD). The two other species 68 and 66 kD might correspond to stress proteins. Affinity blotting on gels showed that both alpha tubulin and vimentin were capable of binding with intact and penton-less adenovirions. Adenovirus components involved in the binding seemed to be mainly core proteins V and VII, and to a lesser extent, hexon. Analysis of neighbor relationships among proteins of the adenovirus-cytoskeletal protein cross-linked complex suggested that some capsid alterations occurred upon/or after entry of the virus into the cell, and that these structural modifications preferentially concerned the vertex components penton and IIIa, and the core protein V. PMID- 2995099 TI - Absence of extensive recombination between inter- and intraspecies mitochondrial DNA in mammalian cells. AB - Recombination of mammalian mitochondrial DNA (mtDNA) was examined using mouse X rat somatic cell hybrid clones and rat cybrid clones. The mouse X rat hybrids were isolated by fusion of chloramphenicol-sensitive (CAPs) mouse and CAP resistant (CAPr) rat cells. The rat cybrids were isolated by fusion of rat cells with type B mtDNA and enucleated cells with type A mtDNA. Genetic and physical analyses showed that the mtDNAs of the hybrids and cybrids were simple mixtures of the two parental mtDNAs except in the following two cases: One was subclone H2 9 of mouse X rat hybrids, which was CAPr even though mtDNA from the CAPs mouse parent was predominantly retained. The other was rat cybrid subclones, Y12-24 and -61, which showed specific loss of one Hinf I fragment of type B mtDNA, B10. These observations suggest that, in contrast to the case with plant mtDNA, recombination of mammalian mtDNA occurs rarely, if at all. PMID- 2995100 TI - Sendai virus envelope glycoproteins become laterally mobile on the surface of human erythrocytes following fusion. AB - Fluorescence photobleaching recovery has been employed to study the lateral mobility of the Sendai virus envelope glycoproteins (HN, neuraminidase/hemagglutinin protein (HN) fusion protein (F) on the surface of human erythrocytes. Our results indicate that the two viral glycoproteins are laterally immobile on the cell surface prior to fusion, and become mobile during the fusion process. The two fused glycoproteins are mobilized to the same extent (diffusion coefficients of 3.1-3.3 X 10(-10) cm2/sec with mobile fractions of 0.53-0.57 for both HN and F). Their mobilization is blocked under conditions that allow virus adsorption and hemagglutination, but not virus-cell or cell-cell fusion. These findings suggest a possible role for the lateral diffusion of the viral glycoproteins in the mechanism of cell-cell fusion, enabling them to perturb the membranes of adjacent cells and lead to cell-cell fusion. PMID- 2995101 TI - Cytotoxic T-cell capacity after autologous bone marrow transplantation. AB - A total of 19 patients, treated for aggressive tumors with high-dose chemo/radiotherapy and autologous bone marrow transplantation (BMT), were studied for concanavalin-A (Con A)-induced proliferation and Con-A-induced cytotoxicity. Ten patients with cytomegalovirus (CMV) antibodies before BMT showed increased Con-A-induced cytotoxicity before and from 100 days after BMT, while Con-A induced proliferation decreased to less than 10% of control values after BMT and remained so. Nine CMV-negative patients showed normal cytotoxic capacity before and after BMT, while Con-A-induced proliferation recovered slowly from day +30 after BMT. Con-A-induced cytotoxicity was not significantly different between CMV positive and CMV-negative patients, while Con-A-induced proliferation showed significant differences from day +100 onward. PMID- 2995102 TI - Identification of ceruloplasmin receptors on the surface of human blood monocytes, granulocytes, and lymphocytes. AB - Membrane receptors for ceruloplasmin (CP) were identified on all human blood leukocytes (granulocytes, monocytes, and lymphocytes) by a visual probe and 125I CP binding. To synthesize the visual probe, amide-modified submicron-sized polystyrene latex minibeads, activated with glutaraldehyde, were covalently bound to CP. Incubation of this probe with human leukocytes, either fractionated or unfractionated, led to its binding, which was visualized on individual cells by using electron microscopy. At 4 degrees C, only surface binding occurred, but internalization also occurred at 37 degrees C. The binding was completely inhibited in the presence of excess nonderived CP, indicating the specificity of the binding. Incubation of fractionated leukocytes with 125I-CP also led to its specific binding to all three fractions. Scatchard analysis indicated the highest number of receptors for granulocytes and the lowest for lymphocytes. The binding affinity was lowest, however, for granulocytes, with monocytes showing the highest affinity. These data, indicating active uptake of CP by blood leukocytes, may reflect the requirement of leukocytes for copper that can be derived from CP. CP may also serve other functions within the cells. PMID- 2995103 TI - Establishment and characterization of a new human cloned myelomonocytic cell line (ZC-1.6). AB - A human cell line, ZC, derived from the blood of a patient with acute myelomonocytic leukemia, was established and cloned. One of the clones, ZC-1.6, was subsequently characterized. As for its morphology and cytochemistry, ZC-1.6 clone shares a number of features with immature cells of the monocytic or myelocytic lineages. Surface marker analysis shows positivity for 4F2 (100% of the cells), and OKM1 (38%) monoclonal antibodies, and presence of surface HLA D/DR antigens (100%) and Fc (11%) and complement (C3b) receptors (100%). Functional capabilities of ZC-1.6 cells include adherence, spreading, and phagocytosis of latex and opsonized zymosan particles. Despite its morphological immaturity, the ZC-1.6 clone produces relevant amounts of O2- in the presence of different stimuli (phorbol myristate acetate, opsonized zymosan, or latex particles). The production of O2- by ZC-1.6 cells is the first evidence that reactive oxygen intermediate production may represent an early feature of cells of the myelomonocytic lineage. PMID- 2995104 TI - Analysis of the binding of a hemopoietic growth factor, P-cell-stimulating factor, to a cell surface receptor using quantitative absorption of bioactivity. AB - Quantitative absorption of biological activity was used to study the interaction of persisting (P)-cell-stimulating factor (PSF), a T-cell-derived lymphokine, with PSF-dependent lines of hemopoietic cells (P cells). It was shown that suspension of P cells in medium containing PSF at 4 degrees C resulted in a diminution of PSF activity in the medium. Similar results were obtained with homogeneous, pure PSF or crude supernatants from cells secreting PSF. This diminution was specific and involved saturable, reversible binding of PSF to the cells rather than degradation of PSF or the release of an inhibitor. Calculations based on the measurement of PSF activity remaining after absorption and estimates of the specific activity of PSF indicated that there were approximately 1000 receptors/cell and that PSF bound with an equilibrium dissociation constant (Kd) of 5 X 10(-12) M. Increased amounts of PSF were absorbed at 37 degrees C; however, in the presence of metabolic inhibitors, the amount of PSF activity removed was equivalent to that seen at 4 degrees C. PMID- 2995105 TI - Effect of smoking on peripheral blood leukocytes and serum antiproteases. AB - Cigarette smokers have an increased risk of chronic obstructive airways disease which has been attributed to a protease-antiprotease imbalance in the lung. The neutrophil is an important source of proteases as well as of myeloperoxidase, which oxidatively inactivates alpha-1-proteinase inhibitor (alpha-1-PI). The purpose of this study is to evaluate the protease-antiprotease imbalance hypothesis by measuring changes in peripheral blood components in a group of 110 young, male, asymptomatic smokers and an equal number of age-matched non-smokers. Significant (p = 0.001), but modest impairment of pulmonary function was observed in the smokers as measured by both forced expiratory spirometry and the single breath nitrogen test. A 35% increase (p = 0.0001) in peripheral blood leukocytes in smokers was attributable to increases in neutrophils (44%), lymphocytes (31%) and monocytes (23%). This increase in leukocyte count correlated significantly (p less than or equal to 0.01) with some of the more sensitive indicators of airway obstruction (FEV1/FVC, CV/VC, CC/TLC, and delta N2/L). Myeloperoxidase activity of neutrophils isolated from peripheral blood of smokers was 13% higher than in non-smokers, while elastase activity per neutrophil was apparently unaffected by smoking. In 50 subject pairs, elevations in serum alpha-1-PI concentrations in smokers (13.7%) were comparable to similar increases in trypsin (9.9%) and elastase (12.4%) inhibitory capacities. Expressed as nanomoles protease inhibited per nanomole of alpha-1-PI, the apparent functional activity of alpha-1-PI was unaltered by smoking. However, a lower, apparent functional activity of alpha-1 PI against trypsin and elastase was observed in both smokers and non-smokers with higher serum alpha-1-PI concentrations. Thus, in a population of young smokers, changes in leukocyte count, neutrophil lysosomal enzyme activities, and functional serum antiprotease activity appear to be consistent with the establishment of a protease-antiprotease imbalance. This imbalance may predispose these smokers to obstructive lung disease. PMID- 2995106 TI - Oxidants, antioxidants and the pathogenesis of emphysema. AB - Emphysema is a chronic pulmonary disorder characterized by a permanent enlargement of the air spaces distal to the terminal bronchioles consequent to destruction of the alveolar walls, including the epithelial and endothelial cells and the connective tissue matrix. There is increasing evidence that an imbalance of oxidants and antioxidants in the lower respiratory tract contributes to this process. Oxidants such as O2-., H2O2, OH, OCl- are generated in the lower respiratory tract as a result of normal biochemical processes, activation of inflammatory cells and inhaled toxic gases. Under normal circumstances, the parenchymal cells are protected by intracellular antioxidants and membrane radical scavengers. In addition, the fluid lining the epithelial surface contains a catalase-like antioxidant that protects the epithelial cells from oxidants. If the oxidant burden overcomes these defenses, the parenchymal cells may be injured, the connective tissue matrix may be partially degraded, the antiprotease screen that protects the lower respiratory tract from attack by neutrophil elastase may be rendered impotent. The alveolar wall then becomes highly vulnerable to elastolytic attack, with a complete destruction of the interstitial connective tissue matrix. In this regard, it is reasonable to hypothesize that reestablishment of the oxidant-antioxidant balance in favor of the antioxidants would be useful as a therapeutic strategy to suppress the emphysematous process. PMID- 2995107 TI - Initial agonist burst duration changes with movement amplitude in a deafferented patient. AB - Changes in the duration of the initial agonist burst were studied in a deafferented human. The patient had been functionally deafferented for five years, having no touch, vibration, pressure or kinesthetic sensation nor any tendon reflexes in the four limbs. Pain and temperature sensation were intact and motor fibres were unaffected. The subject made visually guided step-tracking movements using flexion/extension movements about the elbow. Initial agonist burst duration increased with movement amplitude. Burst duration was approximately 65 ms in small movements (6-12 deg) increasing to 136 ms in intermediate (36 deg) and 200 ms in large (54 and 60 deg) movements. Similar changes in initial burst duration with movement amplitude were seen when the subject made non-visually guided movements. It is concluded that the duration of the initial agonist burst is centrally determined. PMID- 2995108 TI - Neurohypophysial opioids and oxytocin secretion: source of inhibitory opioids. AB - When electrical stimuli are applied to the neural stalk of the pituitary, oxytocin, vasopressin, and probably several opioid peptides also contained in nerve terminals in the gland are released: one action of the released opioids appears to be to inhibit oxytocin release by an action that has been likened to pre-synaptic inhibition. Thus, when Clarke et al. (1979) stimulated the neural stalk following intravenous injection of the opioid antagonist naloxone, they observed that the evoked oxytocin release was potentiated. In the present study we confirm this result and show that oxytocin release evoked by stimulation of the supraoptic nucleus is similarly potentiated by naloxone. This finding is consistent with the hypothesis that the opioid responsible for inhibition of oxytocin release coexists with either oxytocin or vasopressin. We further report that the specific delta-receptor antagonist ICI 174864 does not potentiate oxytocin release either in vivo or in vitro. Thus, it seems unlikely that the enkephalins, putative delta-receptor agonists present in neurohypophysial fibres, are the opioids responsible for the observed inhibition of oxytocin release. PMID- 2995109 TI - Active membrane properties of rat neostriatal neurons in an in vitro slice preparation. AB - The active membrane properties of rat neostriatal neurons have been studied in an in vitro slice preparation. All the neurons examined had resting membrane potentials of more than 50 mV and generated action potentials with amplitudes exceeding 70 mV. The morphological characteristics of the neurons identified by intracellular labeling with HRP indicated that they were medium spiny neurons. Depolarizing current injection through the recording microelectrode generated slow depolarizing potentials and repetitive action potentials with frequencies ranging from less than 10 Hz to over 300 Hz. Adaptation of action potentials was observed when long duration depolarizing current was injected. Depolarizing current injections revealed that the membrane of the striatal neuron had an anomalous rectification when the membrane potential was depolarized to the resting potential. A possible bases for the anomalous rectification might involve inactivation of K-conductance and slow inward Ca- and/or Na-currents. Local electrical stimulation evoked depolarizing postsynaptic potentials (DPSPs) followed by long-lasting small depolarizations. In a double stimulation test, a potentiation of the test DPSP was observed at interstimulus time interval of up to 80 ms. Post-tetanic potentiation of DPSPs was also seen in these neurons. Tests utilizing depolarizing current injection, intracellular Cl- injection, and Cl-conductance blocking drugs indicated that the DPSPs were composed of EPSPs and overlapping IPSPs. The nature of the long-lasting small depolarization succeeding the DPSPs could not be conclusively determined. However, available data suggest that the slow inward Ca-current may be responsible for this response.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995110 TI - Down-regulation of alpha 2- and beta-adrenoceptor binding sites in rat cortex caused by amygdalar kindling. AB - The role of central noradrenergic neurons in kindled seizures was assessed by comparison of alpha 2- and beta-adrenoceptor binding in the cerebral cortex from kindled and control rats. To minimize handling, which may modify kindling-induced changes in binding, the kindling protocol involved stimulation of the amygdala every hour for a maximum of 26 h. Twenty-four hours after kindling, down regulation of beta-adrenoceptors was found in both olfactory cortex and the remaining neocortex, whereas alpha 2 down-regulation was confined to the olfactory cortex. At 21 days after kindling, the only change found was a down regulation of beta-adrenoceptors in the neocortex. The results support the view that functional changes in central noradrenergic transmission are associated with the reduction in seizure threshold induced by kindling. PMID- 2995111 TI - Changes in responsiveness to mu and kappa opiates following a series of convulsions. AB - After a series of seven electroconvulsive shocks, mice (C57BL/6J) showed a marked change in their response to opiates. Although very large doses of mu agonists induce convulsions in normal control mice, our evidence indicated that this was accomplished through nonopiate mechanisms: they could not be blocked by naltrexone and the pattern of drug potencies (codeine greater than morphine greater than levorphanol) was not consistent with an opiate response. In contrast, after electroconvulsive shock small doses of mu agonists induced convulsions that could be blocked by naltrexone and the pattern of drug potency (levorphanol greater than morphine greater than codeine) was consistent with an opiate mechanism. Kappa drugs, on the other hand, produced convulsions in both control and ECS animals, although there was an enhanced responsiveness in the latter. Furthermore, the convulsions produced by kappa drugs were blocked by naltrexone and showed stereoselectivity in both control and ECS animals. The changes in responsiveness to mu and kappa opiates cannot be explained on the basis of a general increase in seizure susceptibility, as sensitivity to the nonopiate convulsant, strychnine, was not enhanced after electroconvulsive shock. The results point to a qualitative change in response to mu agonists after electroconvulsive shock, but only a change in sensitivity to kappa agonists. PMID- 2995113 TI - Inhibition of sarcolemmal Na, K, Mg ATPase from the guinea pig heart is not compatible with a homogeneous population of non-interacting ouabain receptors. AB - The inhibition of sarcolemmal Na, K, Mg ATPase from the guinea pig heart by ouabain was evaluated with a coupled enzyme assay. Models of negative cooperativity and of two independent receptors fitted the inhibition data equally well. The analysis was not compatible with a homogeneous population of non interacting ouabain receptors. PMID- 2995112 TI - Autophagy and lysosomal proteolysis in the liver. AB - Autophagy is defined as any process whereby cellular macromolecules destined for degradation gain access to the lysosomes. A review is presented on the physiological significance, mechanisms and regulation of autophagy in hepatocytes, concentrating on the issue of regulation. The article ends by discussing techniques available for future research. PMID- 2995114 TI - Immobilization of chicken liver fructose 1,6-bisphosphatase on CNBr-activated Sepharose. AB - Chicken liver fructose 1,6-bisphosphatase is readily immobilized on CNBr activated Sepharose. The immobilization alters some enzymatic properties. They include broader pH activity curve, loss of activation by K+ or NH+4, increased resistance to inactivation by trypsin, decreased sensitivity to AMP inhibition, and loss of cooperative interaction among AMP-binding sites. The immobilized enzyme retains about 38% or 19% of the specific activity of the native enzyme when the activity is measured in the absence or presence of K+, respectively. PMID- 2995115 TI - Molecular cloning of the fibroin light chain complementary DNA and its use in the study of the expression of the light chain gene in the posterior silk gland of Bombyx mori. AB - Fibroin light chain (L-chain) mRNA (mol. wt 4.0 X 10(5) daltons) was purified from the posterior silk gland of the silkworm, Bombyx mori (J-131 strain). Double stranded complementary DNA was synthesized and inserted into the PstI site of pBR322 employing the oligo(dC)-oligo(dG) tailing method. Several recombinant plasmids containing the inserts of about 800 base pairs were isolated. Hybridization-translation assay demonstrated that these clones hybridized specifically with the fibroin L-chain mRNA. One of these clones (pLA23) was used as a probe to investigate relative concentrations of the fibroin L-chain gene and mRNA in the posterior silk glands at different stages of late larval development. PMID- 2995116 TI - Hexose monophosphate shunt and cholesterol synthesis in the diabetic and fasting states. AB - The livers of streptozotocin-induced diabetic and fasted rats showed a decreased cholesterol synthesis measured by in vitro incorporation of [2-14C]acetate. A significant decrease of glucose-6-phosphate dehydrogenase (G-6-PD), 6 phosphogluconate dehydrogenase (6-PGD), and pyruvate kinase (PK) was also observed 7 days after administration of streptozotocin. These enzymatic activities were also low in livers of 72 hr fasted animals. An increase of glucose-6-phosphatase (G-6-Pase) was observed consistently in diabetic as well as in fasted rats. Suitable amounts of insulin and refeeding normalized the alterated enzymatic activities in diabetic and in fasted animals, respectively. PMID- 2995117 TI - [Effect of haloperidol on the analgesic activity of opiate agonists administered intracisternally and intrathecally]. AB - The mu-agonist morphine more actively inhibited the complex reflex manifestations of pain responses (the Hafner test and hot plate) in mice as compared to the spinal pain reflexes (tail flick). The delta-agonist D-Ala2-D-Leu5-enkephalin (DADL) abolished the responses of both types in the same doses. Haloperidol potentiated the ability of morphine and DADL to inhibit nociceptive responses at the cerebral level. The potentiating effect of this neuroleptic on inhibition of spinal nociceptive responses by opiate agonists was only observed at a dose of 5 mg/kg. Haloperidol could also in some of the tests potentiate the antinociceptive effect of the kappa-agonist bremazocine and the sigma-agonist SKF 10.047, however their activity was significantly lower than that of morphine and DADL. PMID- 2995118 TI - Synthesis and binding studies of 1-arylpyrazolo[4,5-c]- and 2-arylpyrazolo[4,3 c]quinolin-4-ones. I. AB - The syntheses of some 2-aryl-3-methyl-4,5-dihydro-2H-pyrazolo [4,3-c] quinolin-4 ones and 1-aryl-3-methyl-4,5-dihydro-1H-pyrazolo-[4,5-c] quinolin-4-ones are reported. Some of the latter have shown a high activity in displacing specific [3H]-flunitrazepam binding from bovine brain membranes. PMID- 2995119 TI - Imidazolyl derivatives of enalapril as potential angiotensin converting enzyme inhibitors. AB - The synthesis of a few derivatives of the angiotensin converting enzyme inhibitor enalapril is described. In these molecules the chelating carbethoxy group is substituted with an imidazolyl one. The compounds were tested both in vitro and in vivo. None of them showed in vivo activity but only a marginal in vitro inhibitory activity. PMID- 2995121 TI - Active oxygen forms in photoreaction between DNA and furanochromones khellin and visnagin. AB - The two furanochromones khellin and visnagin react with DNA under irradiation by 365 nm light, forming photoadducts. Recently, the use of khellin as therapeutic agent for skin diseases has been proposed. It is well known that during the formation of photoadducts toxic active oxygen forms are produced. We studied therefore the behaviour of the two furanochromones as producers of 1O-2 and O-2. Our results indicate that visnagin is a strong generator of both superoxide radicals and singlet oxygen, while khellin does not exhibit strong production of OO-2, which is promptly quenched by superoxide dismutase. PMID- 2995120 TI - A novel S = 3/2 EPR signal associated with native Fe-proteins of nitrogenase. AB - In addition to their g = 1.94 EPR signal, nitrogenase Fe-proteins from Azotobacter vinelandii, Azotobacter chroococcum and Klebsiella pneumoniae exhibit a weak EPR signal with g approximately equal to 5. Temperature dependence of the signal was consistent with an S = 3/2 system with negative zero-field splitting, D = -5 +/- 0.7 cm-1. The ms = +/- 3/2 ground state doublet gives rise to a transition with geff = 5.90 and the transition within the excited ms = +/- 1/2 doublet has a split geff = 4.8, 3.4. Quantitation gave 0.6 to 0.8 spin . mol-1 which summed with the spin intensity of the S = 1/2 g = 1.94 line to roughly 1 spin/mol. MgATP and MgADP decreased the intensity of the S = 3/2 signal with no concomitant changes in intensity of the S = 1/2 signal. PMID- 2995123 TI - Locus-specific detection of HLA-DQ and -DR antigens by antibodies against synthetic N-terminal octapeptides of the beta chain. AB - Antibodies against synthetic peptides representing the class-II antigen HLA-DR and -DQ beta chain N-terminal sequences were prepared in rabbits. The two octapeptides only share two amino acids and enzyme-linked immuno-assays showed the antisera only to bind to its own antigen. Both peptide antisera detected a 29 kDa component in immunoblots of Raji and AL-34 cell plasma membrane proteins separated by SDS gel electrophoresis. The binding of either N-terminal peptide antiserum was selectively inhibited only by the peptide used as antigen. Indirect immunofluorescence analysis by flow cytofluorometry showed specific surface immunofluorescence in 1:100-1:1000 dilutions in lymphoblastoid and blood mononucleated cells. In the latter the binding was primarily confined to monocytes and a subpopulation of lymphocytes. It is concluded that locus-specific immunological reagents to distinguish between beta chains of HLA-DR and -DQ have been prepared by the preparation by the production of antibodies against the N terminal sequences of each polypeptide. PMID- 2995124 TI - Redox-dependent activation of 5'-nucleotidase in rat liver plasma membranes. AB - Addition of NADH, but not NAD+ or NADPH, to rat liver plasma membranes resulted in the increase of their 5'-nucleotidase activity. NADH-dependent activation of 5'-nucleotidase was significantly suppressed by atebrine, an inhibitor of NADH dehydrogenase of plasma membranes, and completely abolished by 2,4-dinitrophenol (2 X 10(-4)M) and Triton X-100 (2%). Inhibitors of electron transfer in the mitochondrial respiratory chain, rotenone and potassium cyanide, failed to affect 5'-nucleotidase activity in both the presence and absence of NADH. The data obtained give reasons to suggest a redox-dependent mechanism of 5'-nucleotidase activation in rat liver plasma membranes. PMID- 2995122 TI - E. coli Ada regulatory protein repairs the SP diastereoisomer of alkylated DNA. AB - Using HPLC and 31P NMR spectroscopy on a chemically synthesized asymmetric mixture of the diastereoisomers of thymidyl(3'----5')thymidyl-O-methyl phosphate absolute configuration has been correlated with chromatographic mobility. The methyl phosphotriester system in alkylated DNA which is repaired by the Ada regulatory protein of E. coli has consequently been established to possess the Sp configuration. PMID- 2995125 TI - Exclusive leukotriene C4 synthesis by purified human eosinophils induced by opsonized zymosan. AB - Purified human eosinophils were challenged with N-formyl-methionyl-leucyl phenylalanine, leukotriene B4, platelet-activating-factor, valyl-glycyl-seryl glutamic acid, phorbol myristate acetate, zymosan, opsonized zymosan and the calcium ionophore A23187 to induce leukotriene synthesis. Reversed-phase high performance liquid chromatography analysis demonstrated the almost exclusive synthesis of leukotriene C4 by eosinophils of 11 healthy donors after challenge with opsonized zymosan [(22 +/- 4) X 10(6) molecules LTC4/cell, mean +/- SE] or the calcium ionophore A23187 [(54 +/- 7) X 10(6) molecules LTC4/cell, mean +/- SE]. The other agents were not capable of inducing leukotriene formation. When in addition to opsonized zymosan N-formyl-methionyl-leucyl-phenylalanine or platelet activating factor were added a significant increase of the leukotriene C4 synthesis by eosinophils was observed. These results suggest that eosinophils might be triggered to produce considerable amounts of the spasmogenic leukotriene C4 in vivo by C3b- and/or IgG-mediated mechanisms e.g. phagocytosis. PMID- 2995126 TI - Neurokinin B is hydrolysed by synaptic membranes and by endopeptidase-24.11 (enkephalinase) but not by angiotensin converting enzyme. AB - The major site of hydrolysis was the Gly8-Leu9 bond. Angiotensin converting enzyme (peptidyl dipeptidase A, EC 3.4.15.1) from pig kidney hydrolysed substance P releasing the C-terminal tripeptide Gly-Leu-MetNH2 but failed to hydrolyse neurokinin B. Pig brain striatal synaptic membranes hydrolysed neurokinin B producing a similar pattern of products as did endopeptidase-24.11. Substantial inhibition of this activity was achieved with the selective inhibitor phosphoramidon. A combination of phosphoramidon and bestatin abolished the hydrolysis of neurokinin B by synaptic membranes. Thus, a bestatin-sensitive aminopeptidase may play a role in the synaptic metabolism of neurokinin B in addition to endopeptidase-24.11. This aminopeptidase appears to be distinct from aminopeptidase N (EC 3.4.11.2). PMID- 2995127 TI - Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases. AB - Human thyroid peroxidase (TPO) has been purified from thyroid microsomes by immunoaffinity chromatography using a monoclonal antibody (mAb) to TPO. The eluted material had a specific activity of 381 U/mg and exhibited a peak in the Soret region. The ratio of A411 to A280 ranged from 0.20 to 0.25. Upon SDS polyacrylamide gel electrophoresis, the purified enzyme gave two contiguous bands in the 100 kDa region. Further, it has been demonstrated that sera with anti microsomal autoantibodies from patients presenting Graves' or Hashimoto's thyroiditis diseases were able to bind to purified TPO and to inhibit in a dose dependent manner the mAb binding to purified TPO. This suggests that TPO is the thyroid antigen termed to date the microsomal antigen. PMID- 2995129 TI - A temperature dependent transition in the Pribnow box of the trp promoter. AB - Proton NMR spectra of the trp operator-promoter (sequence CGTACTAGTT.AACTAGTACG) show selective changes in chemical shift and relaxation rates over the range of temperature 0-45 degrees C for the non-exchangeable protons of A11 and A12 only. These bases are in the centre of the Pribnow box. The changes imply that at least three conformational states become significantly populated in this range of temperature, and probably involve a change in the propellor twists of A11 and A12 for one transition, and changes in the helical twist and local pitch for the other. As (1) mutations in the Pribnow box that destroy the TAA sequence impair promoter activity, and (2) the abortive initiation assay for RNA polymerase shows a transition near 20 degrees C, we propose that the observed conformational transitions in the trp promoter are an essential feature of good promoters. PMID- 2995128 TI - Biosynthesis of abnormally glycosylated hepatoma secretory proteins in cell cultures. AB - We studied, by electrophoretic techniques, the physiochemical properties of 4 glycoproteins, alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein and transferrin synthesized by three different human hepatoma cell lines. A common feature was the export of glycoproteins with retarded electrophoretic mobility, indicating incomplete sialylation, and a predominance of atypical, highly branched carbohydrate chains. The abnormal glycosylation pattern may be specific for malignant transformation of hepatocytes and possibly related to the intracellular accumulation of some of these proteins in malignant cells. PMID- 2995130 TI - Three-dimensional structure of (Na+ + K+)-ATPase revealed by electron microscopy of two-dimensional crystals. AB - Prolonged incubation of membrane fragments containing homogeneous (Na+ + K+) ATPase with Mg2+, K+ and VO-3 at 4 degrees C resulted in formation of two dimensional crystals of this enzyme with unit cell parameters: a = 66 A, b = 118 A, gamma = 108 degrees. The crystals correspond to the two-sided plane group p21. By combining tilted electron microscopic views of the crystals, a three dimensional structure of (Na+ + K+)- ATPase was calculated at approximately 20 A resolution. The unit cell is formed by two (alpha beta)-promoters which are in contact in their central parts. The structure was compared with chemical modification and immunochemical data; the arrangement of intra- and extramembrane domains was proposed. PMID- 2995131 TI - Lack of nucleotide cleavage on the binding of G-actin-ATP to plasma gelsolin. AB - G-Actin-ATP bound to plasma gelsolin to form a 2:1 complex. The complex contained approximately equivalent amounts of nucleotide and actin. More than 84% of this nucleotide was ATP. Half of the bound nucleotide was displaced by cold chase and the remainder did not exchange, implying that the two actins in the complex are not equivalent. PMID- 2995132 TI - Isolation and amino acid sequence of the smallest subunit of beef heart bc1 complex. AB - The 11 subunits of beef heart bc1 complex can be separated either by a new SDS PAGE system or by a series of chromatographic steps involving dissociation of the complex by salt treatment. The amino acid sequence of the smallest subunit was determined by complete solid-phase Edman degradation and was confirmed by sequencing the N-terminal part and the C-terminal tryptic fragment by liquid phase Edman degradation. The protein consists of 56 amino acid residues; the Mr was calculated to be 6363. The protein ('ISP binding factor') might be entangled in the reassembly of the iron-sulfur protein with the bc1 subcomplex. PMID- 2995133 TI - The prosthetic groups of succinate dehydrogenase: 30 years from discovery to identification. AB - Recent studies using magnetic circular dichroism at cryogenic temperatures, electron paramagnetic resonance (EPR) and linear electric field effect-EPR (LEFE) of succinate dehydrogenase in membranes and in soluble, homogeneous preparations demonstrated the presence of 3 different Fe-S clusters in the mammalian enzyme, as well as in a similar bacterial enzyme, fumarate reductase from Escherichia coli. There is one each of the 2Fe, 3Fe, and 4Fe clusters. Thus, succinate dehydrogenase is the first enzyme which has been shown to contain all 3 of these Fe-S clusters. The enzyme also contains 1 mol 8 alpha-[N(3)-histidyl]-FAD. It has taken the combined expertise of many laboratories and 15 years of effort to identify the flavin component, and nearly 3 decades to identify the Fe-S clusters. The data from physical methods appear to be internally consistent, in harmony with the results of chemical analysis, and provide a rational explanation for earlier results by the cluster extrusion method. There remains, however, a number of interesting and substantive questions for future investigations. This review traces the tortuous path, the many pitfalls and false leads, which have led us from the discovery of nonheme iron and 'bound' flavin in the enzyme to elucidation of their structures. PMID- 2995134 TI - Resonance Raman spectroscopy shows different temperature-dependent coordination equilibria for native horseradish and cytochrome c peroxidase. AB - Resonance Raman spectra are reported for native horseradish peroxidase (HRP) and cytochrome c peroxidase (CCP) at 290, 77 and 9 K, using 406.7 nm excitation, in resonance with the Soret electronic transition. The spectra reveal temperature dependent equilibria involving changes in coordination or spin state. At 290 K and pH 6.5, CCP contains a mixture of 5- and 6-coordinate high-spin FeIII heme while at 9 K the equilibrium is shifted entirely to the 6-coordinate species. The spectra indicate weak binding of H2O to the heme Fe, consistent with the long distance, 2.4 A, seen in the crystal structure. At 290 K HRP also contains a mixture of high-spin FeIII hemes with the 5-coordinate form predominant. At low temperature, a small 6-coordinate high-spin component remains but the 5 coordinate high-spin spectrum is replaced by another which is characteristic either of 6-coordinate low-spin or 5-coordinate intermediate spin heme. The latter species is definitely indicated by previous EPR studies at low temperature. This behavior implies that, in contrast to CCP, the distal coordination site of HRP is only partially occupied by H2O at any temperature and that lowering the temperature significantly weakens the Fe-proximal imidazole bond. Consistent with this inference, the 77 K spectrum of reduced HRP shows an appreciable fraction of molecules having an Fe-imidazole stretching frequency of 222 cm-1, a value indicating weakened H-bonding of the proximal imidazole. PMID- 2995135 TI - The oxygen reaction of the cytochrome d-terminated respiratory chain of Escherichia coli at sub-zero temperatures. Kinetic resolution by EPR spectroscopy of two high-spin cytochromes. AB - The oxygen reaction of the fully reduced respiratory chain in membranes from oxygen-limited Escherichia coli was studied at sub-zero temperatures using EPR spectroscopy. Laser photolysis of CO-liganded cytochrome oxidase d precedes oxidation of at least 2 kinetically separable high-spin cytochromes. At -120 to 100 degrees C, a rhombic signal appears, attributable to cytochrome d, followed at above -100 degrees C, by appearance of a second, axial signal near g = 6, here assigned to cytochrome(s) b, and changes in the redox state of iron-sulphur clusters. The data kinetically resolve the 2 high-spin signals attributed to the oxidase complex and suggest schemes for electron flow to oxygen. PMID- 2995136 TI - beta-Endorphin and ACTH related peptides in primary cultures of rat anterior pituitary cells: evidence for different intracellular pools. AB - Acid extracts of rat anterior pituitary cells and cell-derived culture media were shown to contain three forms of beta-endorphin immunoreactive peptides, corresponding in molecular size to the prohormone pro-opiomelanocortin (POMC), beta-lipotropin and 3.5 kDa beta-endorphin, and essentially two forms of adrenocorticotropin (ACTH) immunoreactivity, representing a 20 kDa intermediate fragment and 4.5 kDa ACTH. Under basal conditions the intracellular peptides contained a high proportion of the bioactive forms of beta-endorphin and ACTH whereas the extracellular peptides contained a higher proportion of the inactive precursors. When the cells were incubated for 3 h in the presence of 10(-8) M CRF, the levels of intracellular beta-endorphin and ACTH immunoreactivity were reduced by 15-30% and there was a 4-5 fold increase in the level of the secreted peptides; furthermore, unlike the peptides released under basal conditions, the peptides secreted under the influence of CRF contained much higher proportions of 4.5 kDa ACTH and 3.5 kDa beta-endorphin, reflecting the intracellular patterns of these peptides. Similar results were obtained when secretion was stimulated by 10(-7) M epinephrine, which produced a 2-fold increase in peptide release. In the presence of 10(-6) M dexamethasone the basal secretion of ACTH and beta-endorphin related peptides, and the intracellular levels of these peptides, remained unaltered. The results point to the existence of different intracellular compartments from which peptides at different states of maturation can be released selectively. PMID- 2995137 TI - Regulation of pantothenate kinase from various tissues of the rat. AB - The relative tissue activities of pantothenate kinase range from 1 to 5 nmol . min-1 . g-1 wet wt for heart, brain, kidney, and liver. The enzyme partially purified from each tissue is inhibited by CoA, but there is a 10-fold greater potency of inhibition of the heart enzyme compared to those from the other tissues. With the heart and liver enzyme, this difference in potency of CoA inhibition may be a reflection of the differing cytosolic CoA concentrations in these tissues. L-Carnitine specifically reversed the inhibition of each enzyme by CoA. It is concluded that L-carnitine may be a regulator of CoA synthesis in each tissue. PMID- 2995138 TI - Action of urokinase and trypsin on angiotensin, ACTH and the oxidized B chain of insulin. AB - N-terminal analysis of the products of hydrolysis of angiotensin, ACTH and the oxidized B chain of insulin after 4 h incubation with trypsin and urokinase reveals a great qualitative similarity in the action of the two enzymes. As expected, the rates of hydrolysis differ significantly and are much higher in the case of trypsin catalysis than in the case of urokinase catalysis. Unexpectedly, however, a decrease in the difference between the catalytic activity of the two enzymes, by increasing the number of Arg and Lys residues present in the substrate, has been observed. PMID- 2995139 TI - Differentiation of [3H]phencyclidine and (+)-[3H]SKF-10,047 binding sites in rat cerebral cortex. AB - The potency of a series of opioid and non-opioid psychotomimetic drugs to inhibit the specific binding of [3H]PCP and (+)-[3H]SKF-10,047 to rat cerebral cortical membranes was examined. (+)-PCMP, the 3-methylpiperidino analog of PCP, was a potent inhibitor of the specific binding of both ligands. All of the other 12 compounds examined, however, displayed a 3-277-fold selectivity for either [3H]PCP or (+)-[3H]SKF-10,047 binding. These results suggest that although these opioid and non-opioid psychotomimetics bind to both sites, most have significantly different affinities. The binding sites for [3H]PCP appear to be distinct from the 'sigma' binding sites labeled with (+)-[3H]SKF-10,047. PMID- 2995140 TI - Mechanism of coupling periodate-oxidized nucleosides to proteins. AB - This paper describes a mechanism of coupling periodate-oxidized nucleosides to proteins. Each of the dialdehyde groups of a periodate-oxidized nucleoside is shown to couple to lysine residues on different protein molecules through Schiff bases, thereby cross-linking different protein molecules, forming a polymer. This is in contrast to the previous model in which nucleosides were suggested to couple to proteins through a morpholine structure. The cross-linked structure of the nucleoside-antigen, significantly different when compared to the native protein, may affect the specificity and the efficiency of antibody production. PMID- 2995141 TI - Neural regulation of renal tubular sodium reabsorption and renin secretion. AB - The entire mammalian nephron, including the juxtaglomerular apparatus, receives an exclusive noradrenergic innervation. Renal tubular alpha 1 adrenoceptors mediate the alterations in tubular segmental sodium, chloride, and water reabsorption that occur in response to direct or reflex changes in efferent renal sympathetic nerve activity. Specific tubular segments so identified are the proximal convoluted tubule, the loop of Henle (thick ascending limb), and the collecting duct. Alterations in efferent renal sympathetic nerve activity represent an important physiological contribution to the overall role of the kidney in the regulation of external sodium balance in conscious animals during both dietary sodium restriction and acute and chronic increases in total-body sodium. Progressively more intense activation of the renal nerves recruits a series of adrenergically mediated influences on renin secretion that are additive, ranging from subtle (modulation of nonneural mechanisms without directly causing renin secretion) to marked (renal vasoconstriction, antinatriuresis, high renin secretion rates). Juxtaglomerular granular cell beta 1 adrenoceptors mediate renin secretion responses to frequencies of renal nerve stimulation that do not cause renal vasoconstriction; at higher frequencies of renal nerve stimulation where renal vasoconstriction is present, renal vascular alpha 1 adrenoceptors mediate a portion of the renin secretion response. PMID- 2995142 TI - Renal responses to stressful environmental stimuli. AB - The effects of stressful environmental stimuli on urinary sodium excretion in conscious dogs, rats, and humans are reviewed. Environmental stress can increase sympathetic neural outflow and decrease sodium excretion. The antinatriuretic response to environmental stress is accompanied by an unchanged renal blood flow and glomerular filtration rate, which indicates mediation via an increased renal tubular sodium reabsorption. The antinatriuresis resulting from environmental stress is associated with increased renal sympathetic nerve activity, and is abolished by surgical renal denervation. In the central nervous system, but not in the kidney, beta adrenoceptors mediate the increased renal sympathetic nerve activity and antinatriuretic responses to environmental stress. The increased renal sympathetic nerve activity and antinatriuretic responses to environmental stress are greater in spontaneously hypertensive rats (SHR) than in normotensive Wistar-Kyoto (WKY) rats. In SHR, but not WKY rats, the increased renal sympathetic nerve activity and antinatriuretic responses are enhanced by a high sodium diet. Similarly, stressful competition in human young adult males results in an antinatriuresis only if a positive family history of hypertension is present. Thus, environmental stress can increase renal tubular sodium reabsorption via a central beta-adrenoceptor mechanism with activation of the renal sympathetic nerves in both conscious dogs and SHR. The antinatriuretic response to environmental stress is greater in rats and humans with a genetic predisposition to develop hypertension. PMID- 2995144 TI - Multiple opiate receptors: functional implications. PMID- 2995143 TI - Reflex responses to reductions in functioning renal mass. AB - Both acute unilateral nephrectomy (AUN) and unilateral ureteral obstruction (UUO) result in an acute increase in cation excretion from the contralateral kidney. AUN results in reflex changes in systemic hemodynamics owing to an acute and transient increase in arterial pressure that activates carotid sinus baroreceptors and constitutes an afferent limb in the reflex; hemodynamic adjustments and increased cation excretion result. The reflex involves participation of the endogenous opioid system, with receptors located primarily in the central nervous system, and requires intact pituitary function because both hypophysectomy and pretreatment with large doses of dexamethasone prevent the postnephrectomy natriuresis. The natriuresis is closely correlated with an increase in the plasma concentration of the NH2-terminal fragment of the pituitary peptide precursor molecule proopiomelanocortin, which suggests that such a peptide could participate directly or indirectly in the postnephrectomy natriuresis. Surgical denervation of either the ipsilateral or the contralateral kidney markedly alters the response to AUN, which prevents the natriuresis and blunts the kaliuresis, and indicates a role for renal neural reflexes. Renorenal reflex pathways also mediate the response of the contralateral kidney to UUO, because denervation of either the ipsilateral (obstructed) or the contralateral kidney abolishes both the natriuresis and kaliuresis usually seen after UUO. This reflex also involves the endogenous opioid system, for it does not occur in rats receiving an i.v. infusion of the opiate receptor antagonist naloxone. PMID- 2995145 TI - [Reactions of neurons of the suprasylvian gyrus of the cerebral cortex of the cat to acoustic stimulation]. PMID- 2995146 TI - [Electrophysiological study of functional interrelations between the ventral posterolateral nucleus and lateral segments of the reticular thalamic nucleus]. PMID- 2995147 TI - [Effect of ACTH and hydrocortisone on sodium, potassium, calcium and magnesium levels and Ca2-ATPase activity in skeletal muscles]. PMID- 2995148 TI - [Effect of Sr2+ and Ba2+ ions on synaptic transmission in smooth muscles of the gastrointestinal tract of guinea pig]. PMID- 2995149 TI - [Role of alpha- and beta-adrenoreceptors of the hypothalamus in the hyperthermic effect of prostaglandin F2 alpha]. AB - In unanesthetized rabbits, intrahypothalamic injection of prostaglandin F2L (PgF2L) induces a hyperthermic effect, actualised via brain noradrenergic structures and their adrenoreceptors. Blockade of thelad renoreceptors eliminate the hyperthermic effect of PgF2L. PMID- 2995150 TI - [Effect of the autonomic nervous system on kininergic reactions]. AB - Sympathetic activation results in activation of kallikrein-kinin system and in suppression of the sensitivity of the cardiovascular system to bradykinin in animals. Prevalence of cholinergic effects over adrenergic ones suppresses formation of kinin and increases its inactivation. Sympathectomy enhances the sensitivity of cardiovascular system to bradykinin. PMID- 2995151 TI - [Vasomotor reactions in the abdominal skin and muscles of the cat in response to thermal and nociceptive stimuli]. AB - The alterations of blood supply in restricted regions of the abdominal skin and the external oblique muscle were studied in anesthetized cats with photoplethysmographic technique. Local cooling of the abdominal skin decreased blood supply in skin and muscle whereas heating exerted an opposite effect. Vasomotor responses of skin and muscle abdominal blood vessels to somatic and visceral afferent fibre stimulation and injections of alpha- and beta-adrenergic agonists were mostly active in character. The abdominal wall vasomotor responses seem to be a proper experimental model for studying segmental vasomotor control. PMID- 2995152 TI - [Nature of a mediator participating in reflex inhibition of the activity of the lymphatic center of the frog]. AB - Picrotoxin, bicucullin, ammonia and furosemide reversibly blocked the inhibition induced by electrical stimulation of the 9-10th dorsal roots in the frog spinal cord. The data obtained suggest existence of GABA-ergic neurons regulating the lymphatic centre activity in the 9-11th segments of the frog spinal cord. PMID- 2995153 TI - [Functional interaction between alpha- and beta-adrenoreceptors of the small intestine]. AB - Effect of desensitization of alpha-adrenoreceptors on the functional state of beta-receptors and vice versa was studied in isolated sections of the rat small intestine. Neither blockade, nor desensitization of alpha-receptors produced any effect on the sensitivity of beta-receptors to isopropyl-noradrenaline (IPN) whereas desensitization of beta-receptors to IPN resulted in the increase of alpha-receptors sensitivity to phenylephrine with a decreased maximal reaction. In the presence of GTP (0.02 mM) and propranolol (2.00 x 10(-7) M) IPN acts as a competitive reversible auto-inhibitor, GTP fully removing the inhibitory effect of IPN on both alpha- and beta-adrenoreceptors. Existence of functional interrelationship between alpha- and beta-adrenoreceptors is suggested. PMID- 2995154 TI - [Adrenocorticotrophic function of the pituitary and immune response in the rat]. AB - The effect of hypophysis adrenocorticotropic function on the immune response to the ram erythrocytes in rats was studied after hypophysectomy or centragenous dexametasone blockade; the humoral immune response was more obviously activated in animals fed with dexametasone whereas indices of the cellular immunity: the delayed hypersensitivity response (DHR)--were more obvious in hypophysectomised animals. The T-cell populations are normally much more suppressed with glycocorticoids than the cells participating in humoral immune response. PMID- 2995155 TI - Age-dependent complementation of the B (MHC) alleles and a non-B allele controlling resistance to progression of Rous sarcomas, and genetic independence of resistance to induction of tumours in the model of inbred lines of chickens. AB - The effect of age on the resistance to progression of Rous sarcomas was investigated in chickens of different inbred lines and their hybrids. This genetic resistance depends on complementation of B-linked genes and complementation of genes outside the B complex. The reduction of tumour inducibility is controlled by genes outside the B complex, and this type of resistance is independent of the resistance to tumour progression that is primarily controlled by B-linked genes. PMID- 2995156 TI - Comparison of avian MC29 and MC31 viral onc proteins. AB - Lysates of [35S]methionine-labelled quail cells transformed by MC31 virus were immunoprecipitated with anti-myc or anti-gag serum and analysed in SDS-PAGE. A protein with a molecular weight 110K was found in both immunoprecipitates. Thus, the product of the MC31 genome is a gag-myc fusion protein with the molecular weight as the product of the MC29 genome. Comparison by tryptic peptide mapping and p15 cleavage analysis showed no differences between both P110 proteins. PMID- 2995157 TI - Effect of phenoxybenzamine and clonidine on the adrenergic receptor in the rat brain; histofluorescence and ultrastructure examinations. PMID- 2995158 TI - Results of investigations of the cell surface membranes composition of cells transformed by td-mutant of RSV. PMID- 2995159 TI - Effects of various brans on energy intake and glucose metabolism in alloxan diabetic rats. AB - The purpose of the present study was, firstly, to determine whether untreated rye bran and untreated wheat bran differ in their ability to affect energy intake and glucose metabolism in alloxan diabetic rats; and secondly, to find out whether wheat brans processed by extrusion-cooking or acid-treatment differ in this respect. A low-fiber high-starch diet was used as reference. There was a positive correlation between energy intake and urinary glucose excretion. No correlation between these parameters and weight increase was found. The mean urinary glucose excretion was lowest in the animals fed untreated rye bran (68 +/- 3 mmoles/24 h). No difference in urinary glucose excretion was found in the three different wheat bran groups (range of means: 102 +/- 10 - 105 +/- 7 mmoles/24 h). The highest urinary glucose excretion was found in the group fed the low-fiber high starch diet (160 +/- 12 mmoles/24 h). It is thus concluded that untreated rye bran lowered urine glucose more than did untreated wheat bran, and that extrusion cooking or acid-treatment did not change the urine glucose lowering effect of wheat bran. PMID- 2995160 TI - Inhibition of dimethyl sulfoxide-induced Friend erythroleukemia cell differentiation in vitro by lithium chloride. AB - This report describes the ability of ultra-pure lithium chloride (LiCl) to influence the growth kinetics and differentiation of Friend erythroleukemia cells in vitro. LiCl (0.2-50 mEq/l) was effective in reducing the ability of Friend cells to grow in liquid suspension culture (p less than or equal to 0.001). In addition, the capacity of these erythroleukemic cells to respond to the inducing agent dimethyl sulfoxide (DMSO) was also significantly reduced (p less than or equal to 0.001). These results demonstrate that LiCl can influence not only the proliferation of erythroleukemia cells but also their subsequent differentiation after exposure to such chemical inducers. PMID- 2995161 TI - A mechanism for the in vitro stimulation of adrenal cortisol biosynthesis by epidermal growth factor. AB - EGF stimulates adrenal steroidogenesis in ewes and in ovine adrenal slices. In vitro, The stimulation is blocked by the cholesterol synthesis inhibitors compactin and AY 9944. EGF stimulates the incorporation of [14C]acetate into cholesterol. EGF increases the activity of the rate limiting enzyme in cholesterol biosynthesis, HMG CoA reductase. EGF has no effect on the levels of any intermediates involved in the conversion of pregnenolone to cortisol, although ACTH produced changes consistent with 17 alpha-hydroxylase activation. We propose that EGF increases adrenal cortisol synthesis in vitro by a stimulation of cholesterol precursor biosynthesis mediated through activation of HMG CoA reductase. PMID- 2995162 TI - Occurrence of dinucleosidetriphosphatase in the cytosol and particulate fractions from rat liver. AB - Dinucleosidetriphosphatase (EC 3.6.1.29) is present in both the 37,000 g rat liver supernatant and precipitate (50 mU/g each fraction). These two activities show matching molecular weights, isoelectric points, substrate specificities, Km values, bivalent cation requirements and inhibition by zinc (II). The particulate triphosphatase and a residual dinucleosidetetraphosphatase (EC 3.6.1.17) are solubilized by freeze-thawing or by Triton X-100. Detergent treatment also extracts an unspecific phosphodiesterase I activity (EC 3.1.4.1) which also splits dinucleoside polyphosphates. The above findings suggest the occurrence of cytosolic and particulate degradative pathways for dinucleoside polyphosphates. PMID- 2995163 TI - Neurotensin stimulates polyphosphoinositide breakdown and prolactin release in anterior pituitary cells in culture. AB - Biochemical events underlying neurotensin action at the pituitary were investigated in primary culture of anterior pituitary cells prelabeled with [3H]inositol. Incubation with the tridecapeptide produced a dose-dependent increase in the content of total [3H]inositol phosphates. The time-course showed that the effect was rapid and significant within 1 min. Fractionation of [3H]inositol phosphates revealed that inositol triphosphate (IP3) and inositol diphosphate (IP2) increased earlier than inositol monophosphate (IP1). Structure/activity correlation studies demonstrated the specificity of neurotensin effect, showing that acetylneurotensin(8-13) displayed an action similar to the natural peptide, while neurotensin(1-6) hexapeptide did not exhibit any effect. The neurotensin analog [D-Trp11]-neurotensin antagonized in a concentration-dependent manner the effect of neurotensin both on prolactin release and on [3H]inositol phosphate production. The loss of prelabeled phosphoinositides was also investigated. Phosphatidylinositol-4,5-bisphosphate (PtdIns-4,5-P2) and phosphatidylinositol-4-phosphate (PtdIns-4-P) decreased significantly within 15 s, while a slight decline in phosphatidylinositol (PtdIns) level appeared only 1 min after neurotensin addition. These results suggest that neurotensin action at the pituitary is mediated by the early hydrolysis of polyphosphoinositides, leading to the production of 1,2 diacylglycerol and inositol phosphates which may initiate intracellular processes responsible for hormonal release. PMID- 2995164 TI - Concanavalin A stimulates neuron-substratum adhesion and nerve fiber outgrowth in culture. AB - Dorsal root ganglion neurons in culture proceed through a series of shape changes before growing nerve fibers. These shape changes involve: attachment to the substratum, extension of filopodia, and spreading of part of the cell to form broad lamellipodia. With the formation of lamellipodia, neurons adhere firmly to the substratum and retrogradely transport lectins (concanavalin A, wheat germ agglutinin) on their surfaces. In unspread neurons concanavalin A, but not wheat germ agglutinin, rapidly stimulates lamellipodium formation and neuron-substratum adhesion. Neurons treated with concanavalin A also have more, branched nerve fibers than untreated neurons, but otherwise appear similar. These effects of concanavalin A are concentration dependent, blocked by alpha-methyl-D-mannoside (100 mM), and are accompanied by receptor redistribution. Stimulation of lamellipodium extension by concanavalin A is inhibited by low temperature (4 degrees C), 2,4-dinitrophenol (0.2 mM), cytochalasin D (4 microM), or trifluoperazine (10 microM), but not by cycloheximide (360 microM) or colchicine (12.5 microM). Attachment of neurons to the culture substratum was affected little by these treatments. These results indicate differences in the neuron's metabolic requirements for simple attachment to the substratum and the early phases of nerve fiber growth. Moreover, they suggest a convenient system in which to study the cellular and biochemical events of rapid nerve fiber outgrowth in primary neuronal cultures. PMID- 2995165 TI - Expression of nidogen and laminin in basement membranes during mouse embryogenesis and in teratocarcinoma cells. AB - Nidogen and laminin were localized at preimplantation stages of mouse development by immunofluorescence. Laminin was already present on the cell surface at the 2 cell stage, while nidogen was first detectable on compacted 8- to 16-cell stage morulae. Nidogen and laminin colocalized at the blastocyst stage and in postimplantation basement membranes. Immunoblot analyses of tissue extracts and cell culture media indicated the 150-kDa form of nidogen as the largest and predominant form in all tissues examined. Radiolabeled nidogen and laminin synthesized by Reichert's membrane were coprecipitated by antibodies against each antigen, indicating complex formation in situ. Equimolar amounts of laminin and nidogen were determined in 6 M guanidine X HCl extracts of tissues by radioimmunoassays, further indicating stoichiometric complexes. However, lower levels of nidogen than laminin were found in tissue and cell culture media. A less than 2-fold increase in nidogen was found when F9 cells were stimulated to differentiate with retinoic acid and dibutyryl cAMP, compared to a 30-fold increase in laminin secretion. PMID- 2995166 TI - Isoelectric forms of alpha-L-fucosidase in mouse teratocarcinoma-derived cell lines. AB - The alpha-L-fucosidase isoenzyme pattern of mouse teratocarcinoma-derived cell lines was analyzed by isoelectric focusing and compared with the pattern of a mammary carcinoma as an example of a malignant somatic cell line. In addition, these isoenzyme patterns were compared with those of normal fetal and adult mouse tissues from an earlier study. In the normal early fetal and placental tissues as well as in embryonal carcinoma and yolk sac carcinoma cells the alpha-L fucosidase activity is predominantly associated with basic forms of the enzyme. This embryonic pattern of alpha-L-fucosidase is characterized by one to three isoelectric forms of the enzyme with pI values ranging from 7 to 9.5 accounting for more than two-thirds of the total activity. In contrast, the mammary carcinoma pattern resembles adult somatic tissues and primarily expresses acidic enzymatic forms (which comprise approximately 80% of total activity). The somatic cell malignancies arising in retransplantable teratocarcinomas show varying isoenzyme patterns. Thus, a malignant fibrous histiocytoma expresses predominantly basic forms of the enzyme, whereas a leiomyosarcoma expresses approximately equal amounts of acidic and basic forms of the enzyme resembling in this respect late fetal or immature neonatal tissues. These findings show that the embryonal carcinoma and yolk sac carcinoma cells of the mouse express the embryonic isoenzyme pattern of alpha-L-fucosidase in contrast to malignant cells originating in somatic tissue, like mammary carcinoma, which express the adult pattern. Malignancies arising in somatic tissues of teratocarcinomas may retain the embryonic alpha-L-fucosidase phenotype or show a phenotype corresponding to late fetal or neonatal tissues in normal ontogeny. PMID- 2995167 TI - The immortalization of human lymphocytes by spheroplast fusion. AB - A method for immortalizing animal cells based on the spheroplast fusion technique of Schaffner is being developed. Human lymphocytes have been fused with E. coli spheroplasts containing the plasmid pTSV3, which represents the entire SV40 genome cloned into the EcoRI restriction enzyme site of the plasmid pAT153. The efficiency of transformation was examined using pTSV3 and derivatives which have deletions in parts of the late gene sequences. Although there was a marked increase in the survival time of the lymphocyte cultures after fusion compared with cells which had been mitogen-stimulated but not fused, the cells did not continue to proliferate indefinitely. Attempts are now being made to extend the survival time by culturing the transformed lymphocytes in the presence of feeder cells. PMID- 2995168 TI - Cloning, expression and biological activity of a new variant of human interferon alpha identified in virus induced lymphoblastoid cells. AB - A synthetic oligonucleotide complementary to a highly conserved sequence in the IFN-alpha gene family, was used to screen a Namalva cDNA library. Among the cDNA clones having typical IFN-alpha traits, one was distinct from previously characterized IFN-alpha cDNAs. E. coli cells carrying this recombinant cDNA plasmid express an alpha-interferon activity. The sequence of this IFN-cDNA is extremely homologous (99.5%) to that of the IFN-alpha J gene and is designated IFN-alpha J1. Several E. coli trp expression plasmids were constructed for efficient transcription and translation of the mature IFN-alpha J1. The maximal level of expression (5 X 10(3) molecules/cells) was obtained from plasmid pJ1-4. A synthetic consensus translation initiation sequence coupled to the trp p/o region (in pJ1-5) proved to be 10 times less effective in promoting metIFN production in bacteria, than the in-vitro mutated trpL initiation sequence carried on pJ1-4. The bacterial IFN-alpha J1 was purified (to over 90% purity) to a specific activity of 1.3 X 10(8) units/mg. The antiviral activity of the purified IFN-alpha J1 was compared with other highly purified IFN-alpha species (bacterial IFN alpha A and alpha C, leukocyte IFN-alpha 1, leukocyte IFN mixture and Namalva IFN preparation) on a large panel of mammalian cell cultures. IFN alpha J1 exhibits a distinct antiviral activity. PMID- 2995169 TI - The use of BHK suspension cells for the commercial production of foot and mouth disease vaccines over a twenty year period. AB - The Wellcome Foundation Ltd first used BHK suspension cells for the commercial production of foot and mouth disease vaccine in 1964. Since that time both the scale of operation and the volume of production has increased dramatically and in 1983 the Wellcome Group produced almost 350 million monovalent equivalent doses of vaccine from just over 2 million litres of antigen. Experience gained during this period has shown that with careful attention to plant design and operation this large industrial scale tissue culture process can be managed reliably and efficiently. Data will be presented to show the level of production and success rate achieved in our Foot and Mouth Vaccine production units together with an analysis of antigen yields and the comparatively few failures experienced. PMID- 2995170 TI - Flow cytometric analysis of hepatoma tissue and HeLa cells grown on various types of microbeads using hydroxyurea, nocodazole and aphidicolin in succession. AB - Applying Hydroxyurea, Nocodazole and Aphidicolin in succession to obtain parasynchronous growth, the progression of HTC and HeLa cells through the cell cycle has been monitored by laser flow cytometry. The experimental results show that HTC cells behave identically whether grown in monolayer or attached to dextran-based microbeads but that the chemical nature of the micro-support itself plays an important role especially on the speed with which the cells pass from mitosis into G1, polyacrylamide-based microbeads being superior in this respect. PMID- 2995171 TI - A multiplate settling vessel to increase the separation speed of cells from growth medium. AB - The author describes a multiplate settling vessel in which it is possible to separate the cells from the growth medium even if in large scale (up to 1.800 1) in less than 24 hrs, discarding completely the supernatant. PMID- 2995172 TI - Conditions for proper formaldehyde inactivation of foot and mouse disease alhydrogel vaccines. AB - The inactivation of FMDV by formaldehyde (FA) was studied under different conditions, both as free virus and (as in routine vaccine production) after adsorption of the virus to aluminium hydroxide gel (alhydrogel). In the latter case infectivity was monitored after elution of the virus from the gel by isopycnic ultracentrifugation of the virus-alhydrogel mixture in CsCl. By this method good virus recoveries were obtained. Adsorption of the virus to alhydrogel (without formaldehyde) did not reduce infectivity significantly. Both adsorbed and non-absorbed virus lost infectivity at a rate of about one log10 per day (at pH 8.5, 25 degrees C - no formaldehyde). Kinetics of FA inactivation of adsorbed and non-adsorbed virus were also identical, with a fast reduction in the initial phase. After this initial phase inactivation became linear and rather slow. No 'tailing-off' was observed. Some additives (f.i. LAH and especially Tris) reduced the inactivation rate. These findings might explain some data of others who observed 'tailing-off'. PMID- 2995174 TI - Serum free cultivation of lymphoid cells for virus and interferon production. AB - Minor modifications of standard media have given a defined serum free medium for the production of polio virus and measles virus in lymphoblastoid cells and of Namalva interferon in lymphoma cells. By usage of slightly modified standard roller equipment in the order of 2 X 10 12TCID-50 of polio virus and 4 X 10(9) IU of interferon can be produced on one roller rack. The produced poliovirus does comply with requirements for inactivated poliovaccine. PMID- 2995173 TI - Antigenic modification of attenuated Sabin type 1 poliovirus by in vitro passages at supraoptimal temperatures. AB - Mutants were selected from Sabin type 1 attenuated poliovirus (LSc2ab strain), capable of growing at a high temperature (39.5 degrees C). They proved to be neurovirulent for monkeys. No correlation was found between neurovirulence and antigenic structure in Sabin type 1 virus as demonstrated by the analysis of the neutralization epitope formulae of thermo-resistant, neurovirulent mutants derived from LSs2ab strain. The lack of correlation between the antigenic pattern of the virus and the virulence was also confirmed by a mutant resistant to neutralization with monoclonal antibodies derived from the wild Mahoney parent of the Sabin type 1 virus. This mutant continued to be neurovirulent in spite of the complete conversion of its neutralization epitope formula to the Sabin virus pattern. PMID- 2995175 TI - Long term cultivation of Namalva cells for interferon production: stable cytogenetic markers for identification of cells in spite of drastic chromosomal variation. AB - Namalva cells were propagated continuously over a period of up to 18 months. During this period the chromosomal status of the cell populations were investigated cytogenetically. The ability of the cells to produce interferons after induction with Sendai virus was monitored. In contrast to the drastic chromosomal variation observed, interferon production was remarkably stable. Comparison of the various cytogenetic data revealed the presence of marker chromosomes and chromosomal constellations which were excluded from the drift. Some of these are useful for unequivocal identification of Namalva cells during long term cultivation. PMID- 2995176 TI - Production of herpesvirus of turkeys in microcarrier culturing system--a new method for production of vaccine against Marek's disease. AB - A microcarrier (MC) culturing system for production of a vaccine against Marek's disease virus is presented. Cytodex-3 beads (Pharmacia) and DE-53 microgranular (Whatman) are the recommended microcarriers. Primary cells from chick embryo as well as cells from a quail cell line (QT-6, established from a Japanese quail) were found to be good hosts for growth of herpesvirus of turkeys (HVT). HVT, the vaccine strain against Marek's disease, was successfully grown and harvested on the above mentioned MCs and cells. The findings of this study, open new possibilities in the production of HVT for various purposes such as basic research and vaccine preparation. PMID- 2995177 TI - A serum-free growth of a human hepatoma cell line as a tool for hepatocarcinogenesis and virus gene expression. AB - The PLC/PRF/5 human hepatoma cells were grown in a serum-free, hormone-free, chemically defined medium; we recently characterized cell growth and hepatitis B surface antigen (HBsAg) production by these cells under these culture conditions, in the absence of interfering substances. Because of the biochemical and the antigenic properties of PLC/PRF/5 cell line showing a marked similarity to in vivo hepatocellular carcinoma, this cell line provides an in vitro system for the study both of hepatitis B virus (HBV) infection and the relationship between this infection and hepatocarcinogenesis. In this paper we describe the effect of several hormones, growth factors and drugs on growth and HBsAg production of PLC/PRF/5 human hepatoma cells. The results obtained indicate that a serum-free growth of these cells may be useful and facilitate studies either on hormone binding or on the effect of growth factors and drugs. PMID- 2995178 TI - Production of herpes simplex virus from MRC-5 cells grown in a glass bead culture system. AB - The failure of microcarrier systems to provide significant quantities of infectious Herpes simplex virus (HSV) for vaccine production purposes led to an assessment of glass bead column cultures for this requirement. Glass bead columns giving approximately 12000 cm2 culture surface areas have been inoculated at various densities with MRC-5 cells, grown to confluency (as determined by steady state glucose utilization) and infected with HSV. The culture system proved at least as effective as comparable roller bottle cultures. PMID- 2995179 TI - Effect of insulin on an insulin receptor of PLC/PRF/5 human hepatoma cell line. AB - A human hepatoma cell line (PLC/PRF/5) contains several copies of hepatitis B virus (HBV) DNA in integrated form and releases hepatitis B surface antigen (HBsAg) in the form of 22 nm particles in culture medium; studies of the supernatant fluid failed to provide evidence of the morphologically intact virion (Dane particle) or hepatitis B infectivity. In this paper we describe the effect of insulin on cell growth and HBsAg production by these cells; moreover we describe the insulin binding to specific cell membrane receptors. Data in our hands point to a different insulin binding related to different incubation conditions. PMID- 2995180 TI - Reduced glomerular sodium/potassium adenosine triphosphatase activity in acute streptozocin diabetes and its prevention by oral sorbinil. AB - To explore metabolic changes associated with the sorbitol accumulation and myo inositol depletion observed in glomeruli of rats with experimental diabetes, we examined total and ouabain-inhibited adenosine triphosphatase (ATPase) activity in glomeruli isolated from control and streptozocin (STZ)-diabetic rats. Glomerular Na/K-ATPase activity (ouabain-inhibited) was significantly reduced in diabetic animals, while total (composite) ATPase activity remained unchanged. Treatment with insulin partially restored the Na/K-ATPase activity. Administration of the aldose reductase inhibitor, sorbinil, which normalizes glomerular contents of both sorbitol and myo-inositol in diabetes, completely prevented the diminution of Na/K-ATPase activity. These results establish that glomerular Na/K-ATPase activity is reduced in acute experimental diabetes. The ability of sorbinil to prevent this decrease suggests that it is related to polyol accumulation and/or myoinositol depletion, although an effect of the drug unrelated to its aldose reductase inhibiting property has not been excluded. Since increased polyol pathway flux, decreased myo-inositol, and reduced Na/K ATPase activity have also been described in peripheral nerve, another tissue in which typical diabetic complications characteristically occur, the consequences of these metabolic changes may be implicated in the pathogenesis of diabetic nephropathy. PMID- 2995181 TI - New polymorphisms at the insulin locus increase its usefulness as a genetic marker. AB - Polymorphic sites adjacent to known genes can be used to examine the segregation of a disease relative to that gene in families, or to map the gene of interest relative to other loci. The polymorphic region 5' to the human insulin gene (5' FP) permits such analysis, but the three size classes previously identified are insufficient for many studies. More alleles are identified with restriction enzymes that generate small fragments (Pvu II). Nonetheless, sufficient polymorphism for informative family analyses is often not present. To facilitate such analyses, we searched for other polymorphisms in over 20 KB of DNA at the insulin locus in Pima Indians, American Blacks, and Caucasians. The previously described allelic variant at a Pst I site in the 3'-untranslated portion of the gene was not polymorphic in any race. An upstream Hinc II site (-62 BP) was present in only 48% of Black alleles, but was not polymorphic in Pima Indians or Caucasians. New polymorphisms were found at a Taq I site (-11,000 BP) and a Rsa I site (-13,000 BP). The Taq I site was present in 89% of Black alleles, 87% of Pima Indian alleles, and 84% of Caucasian alleles. In contrast, the Rsa I site was present in 60% of Black and Caucasian alleles, but in only 47% of Pima Indian alleles. The Hinc II, Rsa I, and Taq I sites show no obvious linkage with each other or the 5' FP. A fourth polymorphism, previously identified with Sac I, was found to be the creation of a new Sac I site at +2500 BP in 10% of Black alleles.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995182 TI - Genetic influences on the immunologic pathogenesis of encephalomyocarditis (EMC) virus-induced diabetes mellitus. AB - DBA/2 and Balb/cBY mice were infected with approximately 30 plaque-forming units of the M-variant of encephalomyocarditis (EMC-M) virus. Seven days after inoculation the majority of the animals of both strains were hyperglycemic. A significant correlation between increased concentrations of virus in the pancreas and hyperglycemia was found among individual DBA/2 animals, but not among Balb/cBY mice. T-lymphocyte depletion of DBA/2 mice before infection failed to alter the incidence or severity of hyperglycemia in comparison to intact animals. Conversely, hyperglycemia in T-lymphocyte-depleted Balb/cBY mice was reduced substantially in comparison to infected immunocompetent animals. There appears to be at least two genetically influenced pathogenic mechanisms of diabetes in EMC-M virus-infected mice. In some strains of animals, hyperglycemia results exclusively from viral infection and the consequent injury to the beta cells, whereas in other animals, viral damage to the islets is compounded by immunologic events. PMID- 2995183 TI - HLA-DR3 and -DR4 control T-lymphocyte responses to mumps and Coxsackie B4 virus: studies on patients with type 1 (insulin-dependent) diabetes and healthy subjects. AB - To study the relationships between the responses to viral antigens and the HLA DR3 and -DR4 associations in Type 1 (insulin-dependent) diabetes mellitus, the frequency of T-lymphocyte proliferating in response to mumps, Coxsackie B4 and varicella-zoster antigens was determined. A decreased frequency was found in T lymphocytes able to respond to mumps or Coxsackie B4 when presented together with DR3, as compared with the frequency of T lymphocytes able to respond to these viruses together with other DR determinants. This was not found for varicella zoster or purified protein derivative of tuberculin. In contrast, an increased frequency was found in T lymphocytes responding to mumps or Coxsackie B4 together with DR4, compared with other DR determinants. The results were similar in Type 1 diabetic and healthy individuals. The results suggest that elements on the DR3 and DR4 molecules may control T-lymphocyte responses to mumps and Coxsackie B4 viruses. PMID- 2995184 TI - Hyperinsulinaemia and insulin insensitivity: studies in subjects with insulinoma. AB - Hepatic glucose turnover, peripheral insulin sensitivity and insulin receptor binding were measured in four subjects with insulinoma before and 3 months after surgical resection of the insulinoma. Basal hepatic glucose production, quantitated employing a primed constant infusion of tritiated glucose, was low pre-operatively (5.2 +/- 1.7 mumol X kg-1 X min-1) but returned to normal post operatively (14.9 +/- 2.8; normal subjects 13.9 +/- 0.8 mumol X kg-1 X min-1). Paired euglycaemic dose-response curves were developed for each subject. Insulin sensitivity, expressed as a right shift of the dose-response curve (ED50), was low pre- and post-operatively. However, insulin responsiveness (Vmax) remained normal (pre-operatively 13.9 +/- 2.2, post-operatively 13.8 +/- 0.8, normal subjects 16.7 +/- 0.8 ml X kg-1 X min-1). There was no consistent pattern in monocyte or erythrocyte receptor binding before or after surgery. These data suggest that the chronic hyperinsulinaemia causes suppression of hepatic glucose production, and a state of insulin insensitivity which appears to be due to a post-receptor defect. PMID- 2995185 TI - Biochemical and functional changes in hearts from rabbits with diabetes. AB - Biochemical and myocardial functional changes were determined in rabbits made diabetic with alloxan (100 mg/kg, intravenously, two injections 24 h apart). Alloxan-induced diabetes was characterized by a state of hypoinsulinaemia and hyperglycaemia. After 10 weeks of diabetes, significant decreases in heart and left ventricular weights as well as increased serum and heart triglycerides and cholesterol were observed in the diabetic animals (p less than 0.05). In addition, left ventricular pressure, heart rate and rate of left ventricular pressure development were all decreased in the animals. The diabetic state was also associated with a slight elevation in myocardial calcium and a significant decrease in magnesium levels (p less than 0.05). Subcellular fractionation of diabetic hearts indicated the presence of alterations in myofibrillar and sarcoplasmic reticulum marker enzymes (p less than 0.05). Among the lysosomal enzymes, measured, N-acetyl-beta-glucosaminidase activity was significantly increased in the homogenates of diabetic left ventricles (p less than 0.05). These alterations in hearts of diabetic rabbits may be responsible for some aspects of diabetic cardiomyopathy. PMID- 2995186 TI - Mineralization in vitro of matrix formed by osteoblasts isolated by collagenase digestion. AB - Osteoblasts from calvaria of 18-day-old fetal Sprague-Dawley rats were isolated using a dissecting procedure followed by collagenase digestion. Freshly isolated or previously frozen cells were cultured for up to 4 weeks in a Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 micrograms/ml ascorbic acid, with or without 10 mM beta-glycerophosphate. Most of the cells were alkaline phosphatase positive throughout the culture period and expressed a type-I collagen as assessed by immunofluorescence. Cells cultured in the presence of beta-glycerophosphate formed a matrix with type-I collagen in 7 days. The matrix underwent mineralization in less than 2 weeks. In the absence of beta-glycerophosphate, only the formation of a nonmineralized matrix was observed. Electron-microscopic examination revealed osteoblasts embedded in a dense network of collagen fibers, with a well-defined mineralization process in association with matrix vesicles. Scanning electron-microscopy showed that the matrix composed of layers of irregularly shaped spread cells with smooth surfaces trapped in a fiber matrix. No mineralization process was observed when rat skin fibroblasts were cultured under similar conditions. These data demonstrate the ability of enzymatically isolated osteoblasts cultured in the presence of beta glycerophosphate to form bone in vitro, and that this process is similar to bone formation in vivo. PMID- 2995187 TI - Clinical application of the measurement of serum asialoglycoproteins to estimate residual liver function in patients with chronic liver diseases with or without hepatocellular carcinoma. AB - The correlation between the amount of asialoglycoproteins and results of conventional liver function tests was studied in patients with chronic liver diseases, with or without hepatocellular carcinoma. The objective was to determine the clinical significance of the measurement of levels of serum asialoglycoproteins. The levels were elevated in accordance with the progress of liver diseases, and correlated with the decrease in albumin content, cholinesterase activity, the ratio of esterified cholesterol to total cholesterol and to the increase of indocyanine green retention at 15 min (p less than 0.001). There was no correlation with values of glutamic oxaloacetic and pyruvic transaminases. The amount of serum asialoglycoproteins also correlated with survival time in fatal cases of cirrhosis and/or hepatocellular carcinoma. Bilirubin and bile acids did not interfere with the measurement of serum asialoglycoproteins in cases of hyperbilirubinemia. Serum asialoglycoprotein levels are a good indicator of hepatic functional reserve in patients with chronic liver diseases, with or without hepatocellular carcinoma. PMID- 2995188 TI - Immunoglobulin A antibody against hepatitis B core antigen in the acute and persistent infection with hepatitis B virus. AB - Antibody to hepatitis B core antigen of immunoglobulin A class was determined in the serum of patients infected with hepatitis B virus by a sandwich-type solid phase radioimmunoassay with monoclonal antibodies. The antibody, as defined by a sample to normal ratio greater than 2.1, was detected in all of 39 patients with acute hepatitis, with titers varying widely depending on the time of blood sampling. In persons with persistent infection, the antibody was detected in only 2 (4%) of 46 asymptomatic carriers of the virus, contrasting with the positivity in as many as 15 (41%) of 37 patients with chronic persistent hepatitis, in 45 (94%) of 48 patients with chronic active hepatitis, and in 40 (87%) of 46 patients with liver cirrhosis with or without hepatocellular carcinoma. The mean +/- SE titer of antibody in chronic persistent hepatitis (3.8 +/- 0.9) was significantly lower than those in chronic active hepatitis (13.8 +/- 3.2) and cirrhosis with or without carcinoma (25.6 +/- 6.1) (p less than 0.001). Based on the results obtained, the antibody may reflect hepatic injury in the persistent hepatitis B virus infection. PMID- 2995190 TI - Systemic corticosteroid therapy of ulcerative colitis. PMID- 2995189 TI - Oxygen-derived free radicals promote hepatic injury in the rat. AB - We have investigated the possible protective effect of superoxide dismutase and allopurinol in a rat model of mild and severe hepatic necrosis produced by Corynebacterium parvum with or without endotoxin. Histology showed a sinusoidal mononuclear cell infiltrate with multiple granulomata but variable degrees of hepatic necrosis. In the severe hepatic injury model there was a reduction in mortality, associated with a decrease in histologic and biochemical evidence of hepatic necrosis, after treatment with superoxide dismutase. This protective effect was not demonstrated with partially heat-inactivated superoxide dismutase. In the mild hepatic injury model similar trends in reduction of serum levels of hepatic enzymes were observed after treatment with both superoxide dismutase and allopurinol. These results indicate that oxygen-derived free radicals may play an important role in the pathogenesis of hepatic injury in the rat. PMID- 2995191 TI - Comparison of cholinergic inhibition and beta-adrenergic stimulation of adenylate cyclase from rat and guinea-pig hearts: effects of guanine nucleotides and monovalent cations. AB - Adenylate cyclase activities and the effects of isoproterenol (a beta-adrenergic receptor agonist), carbachol (a cholinergic receptor agonist) and forskolin (a plant diterpene) were determined in homogenates and washed particulate fractions prepared from rat and guinea-pig hearts. Many interesting differences were noted in the stimulatory and inhibitory effects of isoproterenol and carbachol respectively on the heart enzyme. The likely significance of these results is presented. PMID- 2995192 TI - Formamidine-mediated inhibition of rat platelet aggregation. AB - The formamidine pesticide chlordimeform (CDF) was a strong inhibitor of aggregation of rat platelets induced by collagen and arachidonic acid and was a weak inhibitor of that induced by ADP. With the exception of 4-chloro-o formotoluidide which was an inhibitor of arachidonic acid-induced aggregation only, the CDF metabolites were without discernible effect on aggregation induced by these agents. Amitraz, another formamidine pesticide, inhibited arachidonic acid-induced aggregation but was without effect on that induced by collagen or ADP. Inhibition of collagen- and/or arachidonic acid-induced aggregation by formamidines was concentration-dependent. Although platelets underwent shape change, primary aggregation was markedly inhibited, and secondary aggregation was abolished in some cases. CDF, its two N-desmethyl metabolites, and octopamine, but not amitraz, caused significantly elevated levels of cyclic AMP in platelet rich plasma as compared to controls; however, this effect did not fully account for the action of formamidines on aggregation. PMID- 2995193 TI - Inhibition of the hydro-osmotic response to vasopressin and hypertonicity by phenothiazines and W7, calmodulin antagonists. AB - Phenothiazines and W7, calmodulin antagonists, inhibit the water flow response produced by ADH, exogenous cyclic AMP, phosphodiesterase inhibition and serosal hypertonicity. Calmodulin antagonists had no effect on osmotic water movement in the absence of hormone. Calmodulin was isolated and localized by immunofluorescence in bladder epithelial cells. Phenothiazines inhibited toad bladder calmodulin activation of phosphodiesterase and prevented fluorescent antibody recognition. The results suggest that the calcium-calmodulin process plays a role in the hydro-osmotic response to ADH and that produced by serosal hypertonicity. The calmodulin common site occurs subsequent to cyclic AMP formation, perhaps on the microtubule-microfilament system. PMID- 2995194 TI - Cirazoline, an alpha 2-adrenoceptor antagonist in guinea-pig ileum. AB - Prejunctional effects of cirazoline have been investigated in guinea pig ileum. At high concentrations cirazoline has been shown to have antimuscarinic activity, pA2 5.25 +/- 0.29. At concentrations below those producing blockade of acetylcholine, cirazoline blocks the prejunctional alpha 2-adrenoceptor activity of clonidine, pA2 6.81 +/- 0.22, and alpha-methylnoradrenaline. Results are discussed in the light of controversial evidence for the activity of cirazoline on alpha-adrenoceptors. PMID- 2995195 TI - Interactions of codeine-7,8-oxide (codeine-epoxide), as a new metabolite of codeine, with multiple opiate receptors. AB - Interactions of codeine-epoxide, a new metabolite of codeine, with multiple opiate receptors were studied using radioligand binding assay. The ability of codeine-epoxide to displace [3H]dihydromorphine binding was the most effective among three labeled ligands, that is, [3H]dihydromorphine, [3H]-D-Ala2-D-Leu5 enkephalin and [3H]ethylketocyclazocine, suggesting that codeine-epoxide had a selectively high affinity to mu-receptors, despite the fact the affinities of codeine-epoxide to opiate receptors were slightly less potent than its parent compound, codeine. Since "sodium ratio" and "GTP ratio" on codeine-epoxide binding lay between those of codeine and naloxone, the interaction of codeine epoxide with opiate receptors may be slightly different from that of codeine. PMID- 2995197 TI - Corticosterone and aldosterone dose-dependent responses to ACTH and angiotensin II in the duck (Anas platyrhynchos). AB - The effects of [1-24]ACTH, [5-Val]angiotensin II, and potassium (K+) on the secretion of corticosterone and aldosterone by tissue slices from the subcapsular (SCZ) and inner (IZ) zones of the duck adrenal gland were determined using incubation and superfusion systems. Both methods demonstrated that the release of corticosterone and aldosterone from IZ and SCZ cells was dependent on the ACTH dose concentration. The IZ cells produced more corticosterone than the SCZ cells in response to stimulation with 1-1000 ng ACTH/ml. The dose response by aldosterone from the cells of the SCZ and the IZ were comparable, but the proportion of aldosterone per total amount of steroid released was greater by cells of the SCZ (11.3%), than by cells of the IZ (3.6%). Elevating the K+ concentration in the incubation medium from 4.0 to 6.5 and 11.2 mM did not directly stimulate corticosteroid release or potentiate the stimulatory effect of ACTH. Superfusion with 10(-12) to 10(-5) M AII stimulated the release of aldosterone from the SCZ cells but had no detectable effect on the IZ and failed to stimulate corticosterone release from either the SCZ or IZ cells. The results presented here demonstrate that in the bird stimulation for the release of corticosterone and aldosterone are different. Methodology for superfusion of adrenal tissue and for the direct radioimmunoassay of aldosterone and corticosterone in the superfusate are described. PMID- 2995196 TI - Effects of glucose feeding on renal electrolyte and H+ transport. AB - Changes of urinary pH, titratable acidity, ammonium, bicarbonate and PCO2, as well as plasma insulin and various plasma and urine electrolytes were studied in 15 normal individuals during a 5-hr glucose tolerance test. After glucose ingestion, urine pH and bicarbonate excretion rose while urine titratable and total acidity, ammonium, Na, K, Cl, phosphorus and PCO2 fell significantly. Serum bicarbonate rose while serum potassium, phosphorus, and anion gap decreased following oral glucose administration. Glucose feeding and the resultant rise in insulin are, therefore, associated with a considerable reduction in urine acidity and Na, K, Cl, and phosphorus excretion. While a significant fall in urinary Na, K and phosphorus has been shown to occur following glucose and insulin administration, their effect on renal H+ excretion in man has not been investigated previously. The mechanism of the observed fall in urine acidity is not clear and requires further investigation. PMID- 2995198 TI - Effects of continuous treatment with synthetic ACTH1-24 or corticosterone on immature Gallus domesticus. AB - Immature chickens were implanted with osmotic pumps filled with ACTH1-24 or pellets consisting of mixtures of cholesterol with corticosterone (0, 10, 20 or 40% by weight). Continuous infusion of ACTH1-24 (2.2 micrograms/hr:120 micrograms/kg body wt/day) caused increases in plasma concentrations of corticosterone, glucose, cholesterol, triglyceride, and uric acid during the first week, and a reduction in weight gain, an increase in the relative weights of the adrenals and liver, and a decrease in the weights of the bursa and spleen. Treatment with pellets containing corticosterone caused dose-related increases in the plasma concentrations of corticosterone, glucose, triglyceride, and cholesterol, an increase in liver size, and a decrease in the size of the bursa and spleen. Thus the effects of ACTH1-24 are probably almost entirely mediated by corticosterone. During the second week of treatment with ACTH or corticosterone the plasma corticosterone concentration was lower than during the first week. Replacing corticosterone implants at Day 7 did not cause plasma corticosterone concentration to return to that observed in the first week suggesting increased removal of the hormone from the circulation. This response resembles the stress induced change in circulating corticosterone and may be part of the process of adaptation. PMID- 2995199 TI - The effects of cortisol and actinomycin D injections on chloride cells and branchial Na+ -K+ -ATPase in rainbow trout (Salmo gairdneri). AB - Injections of cortisol, actinomycin D, or combined administration of the hormone and the antibiotic did not effect rainbow trout (Salmo gairdneri) branchial Na+ K+ -ATPase activity. Numbers of chloride cells also did not change following cortisol and actinomycin D treatment. These results are discussed in light of a similar report concerning Atlantic salmon (Salmo salar). PMID- 2995200 TI - Changes in plasma cortisol during stress and smoltification in coho salmon, Oncorhynchus kisutch. AB - The cortisol stress response in juvenile coho salmon, Oncorhynchus kisutch, considered as the difference between resting and peak poststress cortisol levels, increased from 80 ng/ml in March to 166 ng/ml in July, the period when smoltification normally occurs. Resting plasma cortisol levels also continually increased from 4 ng/ml in March to a maximum of 39 ng/ml in May, but then declined again to 3 ng/ml in July. The results indicate that there is an increased interrenal responsiveness to stress during the period of smoltification in coho salmon. PMID- 2995201 TI - [Complex regulation of the activity of ilv genes in Escherichia coli K-12 cells]. AB - The modern data on Escherichia coli K-12 ilv genes expression are reviewed. The problems of regulation of the ilv genes activity and of their possible role in the process of cell adaptation to changeable environment are discussed. PMID- 2995202 TI - The Streptomyces plasmid SCP2*: its functional analysis and development into useful cloning vectors. AB - Detailed restriction maps of the plasmid SCP2* and its deletion derivative pSCP103 were constructed. DNA fragments carrying hygromycin (Hyg), thiostrepton (Thio) or viomycin-resistance (VioR) determinants were inserted into pSCP103, and various segments were deleted from the resulting plasmids. Changes in plasmid phenotypes associated with these insertions and deletions allowed the localisation and characterisation of plasmid replication, stability, transfer and fertility functions. Several useful cloning vectors were constructed. They are able to maintain large (greater than 30 kb) DNA inserts, with stable inheritance at a low copy number (1-2 per chromosome) and without structural rearrangements, in Streptomyces hosts. The vectors have a broad host range in the genus Streptomyces. One of them (pIJ903) is a shuttle vector for Streptomyces and Escherichia coli. PMID- 2995203 TI - [Experimental study of the effect of potassium sulfate fertilizers on the body in animals]. PMID- 2995204 TI - [Hygienic aspects of environmental protection in areas of the location of oil shale chemistry and power enterprises]. PMID- 2995205 TI - [Hygienic aspects of using sewage treatment wastes in the national economy]. PMID- 2995206 TI - [Dynamics of the response of animals to exposure to energy-producing and coking coal]. PMID- 2995207 TI - [Status and dynamics of morbidity with temporary disability among workers of the artificial leather industry on the basis of PVC]. PMID- 2995208 TI - [Surgery of malignant tumors of the lung in patients with diabetes mellitus]. PMID- 2995209 TI - [Removal of a cancer metastasis from a solitary lung after pneumonectomy, reamputation of the stump of a main-stem bronchus and thoracoplasty for a bronchial fistula]. PMID- 2995210 TI - ABO blood group in patients with malignant trophoblastic disease. AB - ABO blood group was analyzed in 70 patients with hydatidiform mole and 53 patients with malignant trophoblastic disease. 319 healthy pregnant women served as controls. The distribution of ABO blood groups in patients with hydatidiform mole did not deviate significantly from the distribution in the controls. There was a slight excess in group AB and a slight deficiency in group O in patients with hydatidiform mole. However, there was a significant decrease in blood group A and a slight excess in groups O, B and AB in patients with malignant trophoblastic disease compared with the distribution in healthy pregnant women. A higher mortality rate was observed in group B in patients with malignant trophoblastic disease. In conclusion, ABO blood group of patients with malignant trophoblastic disease can be an important prognostic factor in the diagnosis and treatment of the trophoblastic disease, if a large number of cases are compiled by many investigators. PMID- 2995211 TI - Cyclic 3':5'-nucleotide phosphodiesterase in human myometrium at the end of pregnancy: partial purification and characterization of the different soluble isoenzymes. AB - Most of the cyclic nucleotide phosphodiesterase (PDE) activity of pregnant human myometrium was found in the soluble fraction. Chromatography of this fraction on DEAE-cellulose resolved two peaks of PDE activities. Peak I hydrolyzed both cAMP and cGMP and was activated by the Ca2+-calmodulin complex. Peak II, insensitive to this complex, hydrolyzed specifically cAMP. Sucrose gradient centrifugation of the soluble fraction resolved three peaks (A, B, C) of cAMP PDE activities, and only the first two peaks (A, B) were active towards cGMP. A subsequent sucrose gradient centrifugation of peak I, previously determined by DEAE-cellulose, allowed us to restore two peaks A and B identical to those directly obtained from the soluble fraction: peak A hydrolyzes both substrates, while peak B is specific to cGMP hydrolysis. For peak II, a single large cAMP PDE activity peak is generated. Considering Ca2+ and cAMP as intracellular messengers in the control of uterine motility, the characterization of the different forms of PDE in pregnant human myometrium will be of importance in developing improved tocolytic therapy. PMID- 2995212 TI - Luteinizing hormone receptor binding inhibitor in aqueous extract of human corpus luteum. AB - Aqueous extracts of human luteal tissue significantly inhibited binding of 125I labelled human luteinizing hormone (LH) to the 2,000-g subcellular fraction of human corpora lutea. In contrast, aqueous extract of nonluteal tissue of the human ovary did not show a comparable activity to inhibit LH binding. Extracts of human corpus luteum had little or no ability to inhibit LH binding to porcine luteal homogenates. The inhibitory effect of the aqueous extract on LH binding to human luteal homogenate was demonstrated to be dose-related. The inhibitory effect of aqueous extracts of human corpora lutea on LH binding increased from the early to mid and late luteal phase of the menstrual cycle. The results of the present study suggest that there is an LH receptor binding inhibitor (LHRBI) in the 30,000-g aqueous extract of the human corpus luteum and the increase in LHRBI in the luteal tissue as the human corpus luteum ages may explain the means whereby the human corpus luteum regulates its own life span. PMID- 2995213 TI - [Bacterial toxins that affect host cAMP metabolism]. PMID- 2995214 TI - [Nasopharyngeal carcinoma and the Epstein-Barr virus]. PMID- 2995215 TI - [Studies of phosphorylation in rat mast cells (third report)--isolation of calcium-activated, phospholipid, diacylglycerol-dependent protein kinase from rat basophilic leukemia cells (RBL-1 cells)]. AB - Evidence is presented that RBL-1 cells, which are similar to normal rat mast cells in morphology and contain IgE receptors and histamine, contain a calcium activated, phospholipid, diacylglycerol-dependent protein kinase. This enzyme is very similar in its activation requirements to the calcium-dependent enzyme termed protein kinase C in other tissues. The enzyme is activated by Ca2+. Diolein, but not other di, mono or triglycerides, substantially increases the enzyme activity. Among various phospholipids, phosphatidylserine is the most reactive activator; phosphatidylinositol, phosphatidic acid and phosphatidylethanolamine are less effective; and phosphatidylcholine is practically inactive. The enzyme is inhibited by chlorpromazine and local anesthetics such as dibucaine, tetracaine and procaine. PMID- 2995216 TI - [Studies of phosphorylation in rat mast cells (second report)- phosphatidylinositol kinase in rat mast cell granule membranes]. AB - Granules were isolated from sonicated purified rat mast cells on a Percoll gradient. The granules were shown to contain a highly active phosphatidylinositol kinase which catalyzes the formation of diphosphoinositide (DPI) from endogenous phosphatidylinositol in the granule membrane. The enzyme requires ATP and Mg2+ or Mn2+ for activity. DPI formation is almost completely dependent on MgCl2 or MnCl2, and maximal response was observed at 20 mM or 2 mM, respectively. The effects of the divalent cations are competitive. Ca2+, fluoride and cyclic AMP are inhibitory. The Km for ATP is 25 microM. The initial reaction is rapid, but the response ceases within a few min. The maximal response is seen at 28 degrees C. PMID- 2995217 TI - [Hepatic cell carcinoma and the patients with yusho]. PMID- 2995218 TI - [The nature of the endocrine-gastrointestinal hormones]. PMID- 2995219 TI - Plasma growth hormone responses to microinfusion of noradrenergic agents into or electrical stimulation of the hypothalamus and amygdala in baboons. AB - In nine baboons (Papio papio) guide cannulae and electrodes were stereotaxically implanted into the medial basal or lateral hypothalamus, the anterior hypothalamus or the dorsal amygdala. Plasma GH responses were measured after microinfusion (1 microliter) of the alpha2 adrenergic agonist, clonidine, or the beta adrenergic antagonist, propranolol, or electrical stimulation, in each of these sites. Clonidine, 100 nmol/microliter, infused into the medial basal or lateral hypothalamus elevated plasma GH levels by 5-30 ng/ml, 30-45 min post infusion. Plasma GH responses to clonidine infused into the anterior hypothalamus or the dorsal amygdala were all less than 10 ng/ml. The prior, intravenous, administration of piperoxane, 1.0 mg/kg prevented GH responses to clonidine. Propranolol, 50 nmol/microliter, infused into the dorsal amygdala consistently increased plasma GH levels by 5-15 ng/ml. Electrical stimulation of the medial basal or lateral hypothalamus elevated plasma GH levels by 7-35 ng/ml, 15-45 min post stimulation. Electrical stimulation of anterior hypothalamus or dorsal amygdala did not alter plasma GH levels. The stimulation of alpha 2 adrenergic receptors in the medial basal or lateral hypothalamus of the baboons appears to facilitate GH release. PMID- 2995220 TI - Stimulatory effect of epidermal growth factor on prostaglandin E2 production in mouse fibrosarcoma cell line (HSDM1 C1). AB - Using HSDM1 C1 cell line derived from the mouse fibrosarcoma which synthesizes and secretes prostaglandin (PG) E2, specific binding sites for epidermal growth factor (EGF), a potent growth stimulator of many tissues, and its effect on PGE2 production by cultured tumor cells were studied. HSDM1 C1 cell line possessed specific, high-affinity receptors for EGF: Kd (5.5 X 10(-10 M) and binding capacity (17,650 sites/cell). EGF significantly stimulated PGE2 production in HSDM1 C1 line cultured in serum-free medium for 24 h in a dose-dependent manner; a 2.5-fold increase over control was induced by as little as 0.1 ng/ml and the maximal effect (3.5-fold increase) by 1 ng/ml. Its stimulatory effect on PGE2 production was completely blocked by indomethacin, an inhibitor of PG biosynthesis. These data suggest that EGF may be involved in modulation of synthesis and/or secretion of PGE2, a potent bone-resorbing factor, by the tumors which may partly contribute to hypercalcemia in certain types of neoplasms. PMID- 2995222 TI - Bone marrow examination in small cell carcinoma of the bronchus: an unnecessary procedure? AB - One hundred and thirty-seven patients with small cell carcinoma of the bronchus underwent bone marrow trephine and/or aspirate examination as part of their clinical staging. Twenty-four patients (17.5 per cent) were found to have malignant marrow infiltration. In no case was this an isolated finding of metastatic disease, indeed most patients had gross metastatic spread to liver and/or bone. Marrow infiltration has not been shown to be a major prognostic factor in response to chemotherapy or survival in previous studies. We recommend that this staging procedure be discontinued in routine clinical practice, and instead be confined to specific indications in clinical trials. PMID- 2995221 TI - Ovine prolactin potentiates the action of adrenocorticotropic hormone on the secretion of dehydroepiandrosterone sulfate and dehydroepiandrosterone from cultured bovine adrenal cells. AB - To determine the direct effect of prolactin on adrenal androgen secretion, the daily secretions of dehydroepiandrosterone sulfate (DHEA-S), dehydroepiandrosterone (DHEA), androstenedione and cortisol were determined in monolayer culture of bovine adrenal cells in the presence or absence of adrenocorticotropic hormone (ACTH) and/or prolactin. In the absence of ACTH ovine prolactin alone had no effect on steroid secretion during seven-day culture. Ovine prolactin, when administered in combination with ACTH, significantly potentiated the stimulatory effect of ACTH on DHEA-S and DHEA but not androstenedione secretion on the seventh day in culture. On the first day in culture prolactin showed no synergistic effect with ACTH on DHEA and DHEA-S secretion, although ACTH significantly increased DHEA and cortisol secretion. DHEA-S secretion increased as a function of prolactin concentration in the presence of ACTH. These results indicated that long-term treatment by ovine prolactin with ACTH caused the increase in adrenal androgen secretion from bovine adrenal cells. The site of action of prolactin was suggested to be the partial inhibition of adrenal 3 beta-hydroxysteroid dehydrogenase by the result of increases in DHEA-S and DHEA but not androstenedione secretion. PMID- 2995223 TI - Adenosine deaminase in plasma of patients with adult T-cell leukemia (ATL): correlation between enzyme activity and the ATL subtypes. AB - Adenosine deaminase (ADA) was assayed in plasma from 14 patients with adult T cell leukemia (ATL) (eight with acute ATL and six with smoldering or chronic ATL), 20 male family members (ten were anti-ATLA antibody positive and the other ten negative), and 25 normal individuals. ADA activity was uniformly higher in plasma from patients with ATL than normal controls. This enzyme activity significantly increased in acute ATL in comparison to smoldering or chronic ATL. In families of ATL patients, no statistical difference in ADA activity between the anti-ATLA antibody-positive group and -negative group could be discerned. The enzyme activity in a patient with acute ATL, after a bone-marrow transplant, rapidly increased as leukemic cells increased in peripheral blood. These findings indicate that the levels of ADA activities in plasma from ATL patients reflect the condition of this disease. Thus, measurement of this enzyme activity offers a further parameter to distinguish subtypes of ATL, and is of prognostic and therapeutic value. PMID- 2995224 TI - Molecular comparison of retroviruses associated with human and simian AIDS. AB - Infectious retrovirus(es) associated with the human (LAV, HTLV-III, ARV) and simian (SAIDS-1) acquired immune deficiency syndrome were compared by electron microscopy, immunofluorescence and immunoblotting techniques and by restriction endonuclease mapping of the viral genomes. The extracellular virus particles had similar type D morphology, but intracytoplasmic type A nucleoids were found only in SAIDS virus infected cells. Although the antigens of the three prototype AIDS viruses were similar, no cross-reactivity with the SAIDS virus was detected. Molecular hybridization and restriction enzyme analysis also revealed that the SAIDS and AIDS viruses were genetically unrelated. However, only minor differences, consistent with strain polymorphism, were found between the three AIDS virus isolates. Thus, the retroviruses associated with AIDS in macaques and humans are unique to each species. PMID- 2995225 TI - The distribution of epithelial membrane antigen in the kidney and its tumours. AB - The distribution of epithelial membrane antigen (EMA) in the kidney and its tumours has been studied using a polyclonal anti-EMA antiserum and the immunoperoxidase-antiperoxidase technique (PAP). Twelve fetal and 10 adult kidneys examined showed EMA to be confined to the distal tubular or collecting duct epithelium or their embryological precursors. The examination of 55 primary renal tumours showed EMA to be present on the epithelial tumours, on tubular epithelium in nephroblastoma, but not on mesenchymal tumour cells nor on undifferentiated areas of nephroblastoma. PMID- 2995227 TI - Koilocytotic lesions of the cervix: the interrelation of morphometric features, the presence of papilloma-virus antigens, and the degree of koilocytosis. AB - We studied the interrelation of morphometric features, the presence of human papilloma virus antigens (localized by the immunoperoxidase method), and the degree of koilocytosis in koilocytotic cervical intraepithelial neoplasia. We determined the morphometric features of the cells from the deep, the middle, and the superficial layers of the affected koilocytotic epithelium and in non koilocytotic immature metaplasia and squamous cervical epithelium. This approach allows quantification of disturbances of maturation in cervical epithelium. Our quantitative findings support the contention of other authors that human papilloma virus infection is associated with a morphologically distinct lesion, which forms a morphological continuum with neoplasia. It can be argued that, in addition to the degree of koilocytosis, nuclear enlargement and excessive cellular enlargement in the middle layer of the affected epithelium are viral related effects. With increasing immaturity of the cervical intraepithelial neoplasia all investigated viral-related changes are less prominent. These findings suggest that in neoplastic transformation the morphological and antigen expression of human papilloma virus infection is suppressed. PMID- 2995226 TI - Mesoblastic nephromas: a study of 29 tumours from the SIOP nephroblastoma file. AB - In a series of 889 Wilms' tumours we found 29 pure mesoblastic nephromas. The age of the patients varied from newborn to 22 months, but only five were older than four months. Two histologic types were recognized--leiomyomatous (9) and cellular (20) with a fibrohistiocytic appearance. The leiomyomatous type was almost invariably in stage I, smaller and present in younger infants. At operation half of the cellular type has ruptured or infiltrated the renal pelvis or perirenal tissue. Two patients died of postoperative complications. All others are alive after four years and free from disease. PMID- 2995228 TI - Endocrine liver tumour differential diagnosis from hepatocellular carcinoma. AB - A liver tumour, initially diagnosed by light microscopy as a hepatocellular carcinoma, was later shown to be endocrine by argyrophilia and electron microscopy. It was tested by immunohistochemistry for insulin, glucagon, gastrin, VIP, pancreatic polypeptide, glicentin, C-peptide and somatostatin. A few cells were shown to contain somatostatin, but the secretion product in most of the cells was not identified. The patient is well, without any sign of endocrine disturbances, 18 months after the operation. PMID- 2995230 TI - Chemical cholecystitis associated with hepatic arterial chemotherapy delivered by a permanently implanted pump. AB - The introduction of chemotherapeutic agents directly into the proper hepatic artery via an indwelling catheter results in perfusion of the gallbladder, because the cystic artery is usually a branch of the right hepatic artery. Five gallbladders, removed two to 16 months after insertion of permanently implanted Infusaid model 400 pumps, were examined. All of the gallbladders had significant arteritis, with narrowing or occlusion of lumina or necrosis of vessel walls. Fibrosis of the gallbladder wall was also a constant finding. Nuclear atypia of mucosal epithelium and connective tissue was common. Varying degrees of acute and chronic inflammation were present. These abnormalities may have a radiomimetic and direct irritant pathogenesis. PMID- 2995229 TI - Polymorphonuclear leukocyte-mediated cell and tissue injury: oxygen metabolites and their relations to human disease. AB - Reactive oxygen metabolic products derived from an activated NADPH oxidase present in the cell membrane of PMNs and mononuclear phagocytic cells play a critical role in the host's defense against bacterial infection. Recent studies have also demonstrated the ability of these toxic products to initiate eukaryotic cell injury and promote the development of the acute inflammatory responses. Experimental studies suggest that neutrophil-derived oxygen metabolites contribute to the development of the tissue injury associated with a variety of disease states, including emphysema, myocardial infarction, adult respiratory distress syndrome, immune complex-mediated vasculitis, and rheumatoid arthritis. Future studies to define further the mechanisms by which reactive oxygen-derived metabolic products mediate tissue injury will provide insight into the development of new therapeutic strategies for the modulation of disease states that are mediated by the recruitment and activation of PMNs. PMID- 2995231 TI - Segregation analysis of a marker localised Xp21.2-Xp21.3 in Duchenne and Becker muscular dystrophy families. AB - A DNA marker C7, localised Xp21.1-Xp21.3, has been studied in kindreds segregating for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). In DMD families four crossovers were observed in 38 informative meioses between C7 and the DMD locus (theta = 0.12, z max = +2.72). In BMD families no recombinants were observed in the 16 informative meioses studied. These data are consistent with the localisation of the mutations in these disorders being in the same region of Xp21. Studies in families also segregating for the DNA marker 754 support the previously reported physical order of these loci as X centromere-754 DMD-BMD-C7-X telomere. A recombination fraction of 0.11 (z max = +5.58) was found between DMD-754 by combining our previously published data with the data presented here. C7 and 754 thus provide good bridging markers for the diagnosis of DMD and BMD. PMID- 2995233 TI - Heterogeneity of the alpha-globin gene defects in German alpha-thalassemia affected families. AB - Analysis of alpha-thalassemia syndromes in several German families revealed DNA deletion as well as non-deletion forms as the molecular basis for the defects. Thus, the alpha-thalassemia haplotype was identified as the (-alpha)3.7 rightward deletion form, and the region of the putative recombination process generating such a deletion was further characterized. In addition three different alpha(0) thalassemia haplotypes, (--)MED, (--) > 26, and (alpha alpha)T, could be detected using alpha- and zeta-globin gene-specific probes. PMID- 2995232 TI - Evidence against close linkage of the loci for fraXq of Martin-Bell syndrome and for factor IX. AB - Linkage between the loci for fraXq of Martin-Bell syndrome and factor IX was studied in nine families exhibiting this syndrome by means of a restriction fragment length polymorphism at the factor IX locus. Computer analysis of the data indicates there to be no evidence for close linkage between the syndrome and the factor IX locus. PMID- 2995234 TI - Regional localization of the phosphoglycerate kinase gene and pseudogene on the human X chromosome and assignment of a related DNA sequence to chromosome 19. AB - We have used a cDNA clone for human phosphoglycerate kinase (PGK) to examine the chromosomal localization of three members of the human PGK gene family. Using somatic cell hybrids segregating portions of several X-autosome translocations as well as a clone panel of hybrids segregating radiation-induced fragments of the human X chromosome, we assign a PGK pseudogene to the region Xq11-Xq13, proximal to the functional X-linked PGK gene located in Xq13. In addition, using a panel of 24 somatic cell hybrids, we assign an autosomal PGK-related DNA sequence to human chromosome 19. PMID- 2995235 TI - Localization of genes encoding apolipoproteins CI, CII, and E to the p13----cen region of human chromosome 19. AB - The genes encoding apolipoproteins CI, CII, and E have been previously localized to chromosome 19. By use of rodent-human hybrid cell lines containing translocations of chromosome 19 we have now mapped these three genes to the region 19p13-19q13 and most probably 19p13-19cen. The clustering of APOC1, APOC2, and APOE must reflect their common evolutionary background and suggests that they may be coordinately regulated. Polymorphisms detected for any one gene will be useful for inheritance studies of all three. PMID- 2995236 TI - Frequency and types of deletional alpha+-thalassemia in northern Sardinia. AB - We determined by restriction mapping the frequency of the -alpha 3.7 determinant in a random sample of 48 adults in Northern Sardinia. We found a frequency of 0.18 +/- 0.04 and demonstrated that only type I crossover as determined by Apa I digestion (Higgs et al. 1984) is present. Moreover, we showed that this haplotype is not associated with an Rsa I polymorphism 5' to the alpha 2-globin gene. These data support the hypothesis of a unique origin of this deletion in Sardinia. PMID- 2995237 TI - X-linked retinitis pigmentosa: linkage with the centromere and a cloned DNA sequence from the proximal short arm of the X chromosome. AB - A large Danish pedigree segregating for X-linked retinitis pigmentosa (RPX) (Warburg and Simonsen 1968) was restudied for linkage analysis. Using two markers, i.e. the DNA base sequence polymorphism presented by the probe L1.28 defining the chromosomal segment DXS7, and the C-banding heteromorphism (Xcen) (Friedrich 1982), we were able to localize the RPX gene in Xp close to the centromere rather precisely. The gene order could be deduced by three-point linkage analysis, and the gene distances were determined by pairwise analysis using the LIPED program (Ott 1974). Together with previously published data concerning the RPX:DXS7 linkage (Bhattacharya et al. 1984) a regional gene map is constructed. Xcen-11 cM-RPX-6 cM-DXS7. PMID- 2995238 TI - The value of haematological screening for AIDS in an at risk population. AB - The haematological variables measured by automated full blood count in matched homosexual and heterosexual men attending a clinic for sexually transmitted diseases (STD) were compared with those of normal controls and patients infected with the human T lymphotropic virus type III (HTLV-III). Homosexual and heterosexual men were statistically identical for all variables, but both differed noticeably from patients with clinical diagnoses of the acquired immune deficiency syndrome (AIDS) or AIDS related disease. A full blood count as a screening test for AIDS is only interpretable in the context of clinical assessment. PMID- 2995240 TI - AK and ADA polymorphisms in South Sardinia. AB - A sample of the South Sardinia population was studied with respect to adenylate kinase (AK) and adenosine deaminase (ADA) enzymes. The gene frequencies were: AK1 0.975 and ADA1 = 0.933. The results were compared with those of other Italian populations. PMID- 2995239 TI - Pilot study of cervical cytology screening in a sexually transmitted diseases clinic. AB - A pilot study of cervical cytology was carried out on 500 new patients at the women's sexually transmitted disease (STD) clinic at this hospital. The aim was to discover the incidence of abnormal smears in order to gauge the worth of cervical cytology as a routine clinic procedure. Information was also gathered on each patient's age, sexual history, method of contraception used, previous smears, and genital infection. Smears showing carcinoma in situ, dysplasia, or warty atypia were regarded as abnormal, and the relevant patients were referred for colposcopy. Seventy-three (14.6%) had abnormal smears. Eight women (1.6%), average age 29.7 years, had cervical intraepithelial neoplasia grade III (CIN III) confirmed by histology. One third of the patients with abnormal smears had genital warts, and the incidence of abnormal smears was greater in patients with genital warts than in those without warts. We concluded that STD clinics are useful places in which to carry out cervical cytology screening, and we noted a positive association between infection with genital warts and abnormal smears. PMID- 2995241 TI - Structure, sequence and polymorphism in the HLA-D region. AB - Molecular analysis of the HLA-D region has uncovered a complex array of related genes encompassing a minimum of 6 alpha and 7 beta chain sequences. A high level of polymorphism is characteristic of the DQ alpha and beta genes, as well as DR beta. The DP genes, both alpha and beta, are also polymorphic, though to a lesser extent. The genes fit into the previously established loci: DP, DQ and DR, except for a newly-discovered sequence, DZ alpha, which is approximately equally related to all of the other alpha chain genes. Analysis of the polymorphism and evolution of the HLA-D region, by examination of the sequences, calls for several independent duplication events in the generation of this family of genes. PMID- 2995242 TI - Monosomy 6 in a human lymphoma line induced by selection with a monoclonal antibody. AB - The human Epstein Barr Virus-superinfected B lymphoma cell line BJAB-B95.8.6 was mutagenized by gamma irradiation, and HLA mutants were selected with the HLA-Bw6 specific monoclonal antibody SFR8-B6. One of the mutants obtained, BM19, had lost one of the chromosomes 6 present in the wild type cells. Electrophoretic analysis of phosphoglucomutase isozyme PGM3 and erythrocyte glyoxalase 1 from both cells supports this conclusion. The HLA antigens expressed on BM19 were HLA-A2, B13, Bw4, C-, DR2 (questionable), DRw52 (weak) and DQw1. This constitutes one of the haplotypes of the wild type cells, the other (lost from BM19 cells) being HLA-A1, B35, Bw6, Cw4, DR5, DRw52 (strong) and DQw3. Possibilities to employ BM19 cells for the analysis of the major histocompatibility complex and other chromosome 6 encoded genes as well as their products are discussed. PMID- 2995243 TI - Characterization of pulmonary macrophages and bronchus-associated lymphoid tissue (BALT) macrophages in the rat. An enzyme-cytochemical and immunocytochemical study. AB - Three subpopulations of macrophages present in the lung, viz. pulmonary alveolar macrophages (PAM), pulmonary tissue macrophages (PTM), and macrophages in the bronchus-associated lymphoid tissue (BALT), were compared using enzyme cytochemical and immunocytochemical methods. BALT macrophages can be distinguished from PAM and PTM on the basis of their enzyme reaction products. With three monoclonal antibodies, ED1, ED2, and ED3, that recognize different antigen determinants of rat macrophages, the three subpopulations could be clearly distinguished. PAM are ED1-positive and PTM are ED2-positive. The macrophage subpopulation scattered throughout BALT is ED1-positive; macrophages situated at the peripheral site of BALT, near artery and bronchus, are ED2 positive. No ED3-positive macrophages were observed either in the lung or in BALT. These results are discussed with respect to the possible relationship between the subpopulations of pulmonary macrophages. PMID- 2995244 TI - [Our experience with iruxol ointment in the treatment of skin ulcerations of various etiologies]. PMID- 2995245 TI - Interferon modulates the leukotriene C4-induced non-adherence properties of leukocytes: acquisition of an asthmatic phenotype. AB - We have previously shown that peripheral blood leukocytes (PBL) of asthmatic patients acquire non-adherence properties after challenge with leukotriene C4 (LTC4), whereas PBL of normal individuals do not. Hence the use of the LTC4 induced leukocyte-adherence-inhibition (LAI) assay enables one to recognise an asthmatic phenotype on the basis of the ability of PBL to respond in vitro to LTC4. To examine the possibility that alpha interferon (IFN alpha) may have relevance to the pathogenesis of bronchial asthma, various concentrations of IFN were incubated with normal PBL and the acquisition of non-adhering properties was measured. We found that following 24 h incubation with 500 U/ml IFN, normal PBL were induced to respond to a standard dose of LTC4, and this reaction was abrogated by FPL 55712 and cyclohexamide. PMID- 2995247 TI - HLA-DR genotyping by restriction fragment length polymorphism analyses. AB - We have established unique restriction fragment length polymorphism (RFLP) patterns characteristic of homozygous typing cells (HTCs) for HLA-DR-1 through HLA-DR-8 haplotypes. These RFLP patterns were found to segregate in family members and correlate 100% with HLA-DR antibody phenotyping. The RFLP patterns were used to type chronic myelocytic leukemic cells which have a Philadelphia translocation from 23 randomly selected Caucasoid patients. The results show an alternative method for the determination of the HLA-DR types without using live cells and to study disease association with the HLA-DR region. PMID- 2995246 TI - Evidence for methylation as a regulatory mechanism in HLA-DR alpha gene expression. AB - We examined the possibility that one mechanism for controlling HLA-DR alpha gene expression occurs through DNA hypomethylation. We employed the restriction enzyme Hpa II, which recognizes the sequence 5'CCGG3' but not 5'CmCGG3', to study DNA methylation. We first compared a DR-positive B lymphoblastoid cell line (LCL) with an isogenic DR-negative T-LCL. Using a genomic probe for the DR alpha gene, we showed that an Hpa II digestion of DNA from the B-LCL resulted in bands of lower molecular weight than that of the T-LCL. This indicates that the B-LCL DR gene is hypomethylated relative to the T-LCL gene. Demethylation of the gene from the B-LCL is incomplete, suggesting that complete demethylation is not required for its expression. We also examined somatic cell hybrids of T-LCL and B-LCL since the DR alpha gene, which is inactive in the T-LCL, is expressed in the hybrids, providing a system to study DR alpha gene induction. We examined the hybrid line 174 X CEM.T1, which contains and expresses solely the DR alpha gene from the T-LCL parent since both copies of the DR alpha gene from the B-LCL parent, 174, are deleted. The expressed DR alpha gene from the hybrid was compared with the unexpressed gene from the T-LCL parental line, and again an association between DR alpha gene expression and DNA hypomethylation was observed. In contrast to the DR alpha gene from B-LCL, which is not completely demethylated, the DR alpha gene in this hybrid line is not methylated at either of the Msp I sites covered by our probe. This suggests that different regulatory mechanisms operating through DNA methylation may be involved in the expression of DR alpha genes from T-LCL and B-LCL. Examination of another hybrid line which has DR alpha genes from both parental lines supports this contention. The implications of these findings are discussed. PMID- 2995248 TI - IgE heavy chain constant region genes in atopic dermatitis and senile erythroderma patients. AB - By Southern hybridization using a genomic DNA fragment carrying a human IgE heavy chain constant region gene (C epsilon) as a probe, we analyzed the organization of human C epsilon genes and their flanking regions in 23 atopic dermatitis and 6 senile erythroderma patients with elevated serum IgE levels, and 6 atopic dermatitis patients with normal IgE levels. On Bam HI, Hind III, and Eco RI digestions, we detected three hybridizable fragments containing three human C epsilon genes, C epsilon 1, C epsilon 2, and C epsilon 3, respectively, in all leukocyte DNAs. These fragments were almost identical in size among patients and healthy donors. Pst I digestion generated a genetic polymorphism. We, however, could find no correlation between this polymorphism and the disorders. It was concluded that among the patients and healthy donors, there was no marked difference in the organization of the functional C epsilon gene and its flanking region containing a class switch region. Our conclusion cannot rule out the presence of genetic abnormalities of this region in some atopic dermatitis patients which are not resolvable by our method. In the course of this study, we found a novel C epsilon-like gene in placenta DNA which differs from the three C epsilon genes commonly present in normal human DNA. PMID- 2995249 TI - Syrian hamster DNA shows limited polymorphism at class I-like loci. AB - The class I gene products of the Syrian hamster major histocompatibility complex are unique in that they lack functionally detectable polymorphism. Mouse cDNA and hamster genomic probes were used to analyze the hamster class I gene family using genomic Southern hybridization. These studies revealed that the hamster possesses a complex class I multigene family and that it shares extensive sequence homology with the corresponding mouse sequences. Unlike the mouse, however, the Syrian hamster demonstrates only limited restriction endonuclease polymorphism in these genes. These results suggest that the lack of detectable polymorphism in this species is directly related to limited DNA polymorphism. The data presented here support the hypothesis that this species has undergone an evolutionary bottleneck, i.e., that all surviving members of the species arose from a limited number of progenitors. PMID- 2995250 TI - Localization of CT beta and C kappa on mouse chromosome 6. AB - In the mouse three lymphocyte gene families have been positioned on the proximal region of chromosome 6. Originally the immunoglobulin kappa light chain (Igk) and the thymocyte surface antigens Lyt-2 and Lyt-3 were assigned to chromosome 6, and recently the beta chain of the T-cell receptor for antigen was positioned proximal to Igk. Molecular clones which recognize the constant (C) region of the beta chain of the T-cell receptor for antigen (CT beta) and the constant region of the immunoglobulin kappa (C kappa) chain were used to determine recombination frequencies with respect to the morphological marker hypodactyly (Hd). SJL/JL W pi mice were mated with C.B6.C3-Hd/+ mice, and the progeny expressing the Hd phenotype were mated with SJL/JL W pi mice. Backcross progeny which expressed the Hd phenotype were nephrectomized, and kidney DNA was examined by Southern hybridization for the polymorphic restriction endonuclease fragment (REF) patterns of the parental mice. Of the 88 progeny tested in this three-point cross, 3 CT beta and 4 C kappa homozygote REF patterns were detected. These homozygotes were mutually exclusive. This implies the following gene order: centromere-CT beta-Hd-Igk and CT beta 1 would be 7.95 +/- 2.88 centimorgans from C kappa. PMID- 2995251 TI - Linkage of the locus encoding the A chain of alpha-crystallin (Acry-1) to the major histocompatibility complex in the rat. PMID- 2995252 TI - Specificity of the Crithidia luciliae method for detecting anti-DNA antibodies. Effect of absorption for lipoproteins. AB - Using the immunofluorescent (IF) assay with Crithidia luciliae smears, anti native (n) DNA antibodies were detected in the sera of 12 of 20 systemic lupus erythematosus (SLE) patients, in 1 of 6 mixed connective tissue disease cases, in 2 of 38 patients with systemic sclerosis but in none of the sera from 96 normal subjects. All anti-nDNA antibodies were associated with antinuclear antibodies (ANA). However, occasionally sera were encountered in routine screening which appear to be positive for anti-DNA antibodies but negative for ANA. Studies of such sera indicate that this is a nonspecific reaction which can be abolished by treating sera with dextran sulfate or heparin. Treatment of SLE sera with these agents had no effect on their anti-nDNA antibody activity. Absorption of sera with Aerosil eliminated the false positive reactions with C. luciliae; however, this treatment also removed immunoglobulins, ANA and anti-nDNA antibodies. Evidence is reviewed which points to a role of complexes of low density lipoprotein and IgG in the nonspecific binding reactions with C. luciliae which is seen as false positive reactions for anti-nDNA antibodies. PMID- 2995253 TI - Inhibition of neutrophil superoxide production by fanetizole. AB - The effects on neutrophil function of the new immunomodulatory agent fanetizole mesylate were studied. Fanetizole did not affect random or stimulated migration, phagocytosis, or degranulation by normal human neutrophils. Production of superoxide in response to the chemotactic factor formyl-methionyl-leucyl phenylalanine (f-Met-Leu-Phe) was markedly inhibited (41.3 +/- 3.9%) by 250 microM fanetizole. This inhibition was not due to scavenging of superoxide by fanetizole, as there was no impairment of superoxide detection in a cell-free xanthine-xanthine oxidase system. Inhibition was dose dependent (no effect seen with 1 or 10 microM fanetizole) and stimulus specific (no impairment of superoxide production in response to phorbol myristate acetate). Washing the cells after fanetizole treatment partially restored their superoxide response to f-Met-Leu-Phe. Suppression of neutrophil production of toxic oxygen metabolites may partially explain the antiarthritic effect of fanetizole, and study of such selective inhibitors may be useful in probing the contribution of neutrophils to inflammatory tissue damage. PMID- 2995254 TI - Chemiluminescence and superoxide generation by leukocytes stimulated by polyelectrolyte-opsonized bacteria. Role of histones, polyarginine, polylysine, polyhistidine, cytochalasins, and inflammatory exudates as modulators of oxygen burst. AB - Human blood leukocytes generate intense luminol-dependent chemiluminescence (LDCL) following stimulation by streptococci and by Gram negative rods which had been preopsonized by cationic polyelectrolytes (histone, poly L-arginine-PARG, poly L-histidine-PHSTD). Streptococci but not Gram negative rods or hyaluronic acid-rich streptococci (group C) also induced intense LDCL following opsonization with the anionic polyelectrolytes-dextran sulfate or polyanethole sulfonate (liquoid) suggesting that the outer surfaces of different bacteria bound anionic polyelectrolytes to different extents. Both normal and immune serum, synovial fluids and pooled human saliva inhibited the LDCL responses induced by streptococci preopsonized with poly cations. On the other hand, bacteria which had been first preopsonized by the various body fluids and then subjected to a second opsonization by cationic ligands ("sandwiches"), induced a very intense LDCL response in leukocytes. Streptococci which had been preopsonized by PARG, histone or by PHSTD also triggered superoxide generation by blood leukocytes, which was markedly enhanced by a series of cytochalasins. PHSTD alone induced the formation of very large amounts of superoxide. Paradoxically, the same concentrations of cytochalasins B or C which markedly boosted the generation of superoxide following stimulation of leukocytes with soluble or particulate ligands, had a strong inhibitory effect on the generation of LDCL. On the other hand cycochalasins failed to inhibit LDCL which had been induced by phorbol myristate acetate (PMA). Peritoneal macrophages which had been harvested from C. parvum-stimulated mice, generated more LDCL and superoxide following stimulation by PARG than macrophages obtained from proteose peptone-stimulated mice. Macrophages which had been activated either by proteose peptone or by C. parvum and cultivated for 2 hours on teflon surfaces, generated much more LDCL than macrophages which had been cultivated for 24 hours on teflon surfaces. Both cationic and anionic polyelectrolytes mimic the effects of antibodies as activators of the oxygen burst in blood leukocytes and in macrophages. Such polyelectrolytes can serve as models to further study leukocyte-bacteria interactions in infectious and inflammatory sites. PMID- 2995255 TI - Biphasic contraction of isolated guinea pig tracheal chains by superoxide radical. AB - The effects of free radicals on the isolated tracheal chains from guinea pigs were examined in in vitro experiments. When isolated tracheal chains were exposed to the hypoxanthine plus xanthine oxidase system, biphasic contraction was observed. This contraction was prevented by adding superoxide dismutase. However, catalase, mannitol, and indomethacin were ineffective in preventing the contraction due to free radicals. The uric acid plus uricase system had no effect on tracheal muscle tension. These results suggest that superoxide radical contracts tracheal chains. PMID- 2995257 TI - A computerized approach towards the estimation of binding parameters to characterize cooperative and non-cooperative multicomponent ligand-receptor interactions. AB - A mathematical model has been developed to analyse multicomponent ligand-receptor interaction data directly from binding experiments without resorting to approximations such as linearization. This approach may be applied to analyze binding data for radioreceptor systems involving thyroid and steroid hormones, and drugs on neurotransmitters. Affinity constants, maximal binding capacity, cooperativity and nonspecific binding may be calculated for multiple binding systems. PMID- 2995256 TI - Kinetic and chemical analyses of the biologic significance of lipoteichoic acids in mediating adherence of serotype III group B streptococci. AB - The mechanism(s) involved in the binding of lipoteichoic acid (LTA), isolated from virulent, asymptomatic, or avirulent serotype III strains of group B streptococci, to human embryonic epithelial cells (HEC), human fetal epithelial cells (HFC), and human adult buccal epithelial cells was investigated. It was determined that the binding of purified [3H]LTA to human adult buccal epithelial cells differed from the binding to HEC and HFC. LTA from all group B streptococcus strains bound to human adult buccal epithelial cells in a similar manner and was enhanced by the lipid portion of the polymer; in contrast, [3H]LTA binding to HEC and HFC was mediated by hydrophobic as well as specific interactions due to the glycerolphosphate backbone of LTA. Binding avidity of the LTAs to HEC and HFC varied depending on the bacterial strain. Polymers from asymptomatic and avirulent strains were easily dissociated from cell surfaces with unlabeled virulent LTA through competitive interactions; however, 10-fold greater levels of the same material were required to displace virulent [3H]LTA from HEC and HFC surfaces. These observed differences in binding avidity were shown to be due to longer LTA chains (30 to 35 glycerolphosphate units) in virulent strains when compared with LTA chains (10 to 12 glycerolphosphate units) of asymptomatic and avirulent strains. Thus, LTA appears to enhance the ability of virulent group B streptococci to bind to HEC and HFC with stronger avidity by virtue of the increased length of the cell-associated polymers synthesized by these strains. Mild enzymatic treatment of HEC and HFC with trypsin or periodate abolished LTA binding, which suggests the presence of a certain glycoprotein receptor(s) for LTA which does not appear to be present on human adult buccal epithelial cells. These data may therefore partially explain the increased susceptibility of newborn infants to group B streptococcal infections. PMID- 2995258 TI - Detection of sialylated LewisX antigen in cancer sera using a sandwich radioimmunoassay. AB - A monoclonal antibody directed against sialylated LewisX (SLEX) was tested with the serum of 615 cancer patients, 166 patients with non-malignant diseases, and 136 normal persons. The SLEX antibody reacted with the sera of the cancer patients in the following percentages: 29% lung, 19% breast, 12% ovary, 25% colorectal, 13% head and neck, and 13% miscellaneous. SLEX was positive for 22% of stages III and IV (late-stage) cancers as compared with 5% early-stage tumors. Among 80 patients with adenocarcinoma of the lung in the late stages, 45% were positive for SLEX. However, among 54 patients having squamous-cell lung cancer in the late stages, 15% were positive. In 25 cases of small-cell lung cancer, 24% were positive. Among patients who had measurable lung cancer, 4/7 with adenocarcinoma over 3 cm in diameter were positive whereas 0/16 patients with tumors under 3 cm were positive. Four patients who had tumor regression showed a more than 50% decrease in SLEX values whereas in 7 patients with progressive tumors, a more than 50% increase in SLEX levels was found. When tested simultaneously with CEA, SLEX produced positive reactions with the sera of some patients who were negative for CEA. The reaction pattern was distinct, indicating that another antigen was being detected. When used in combination, the percentage of sera that were positive increased. We conclude that the use of SLEX is useful for monitoring of cancer patients. PMID- 2995259 TI - Selective cytotoxicity of AIDS virus infection towards HTLV-I-transformed cell lines. AB - Previously, we reported that cells of the human T-cell lymphotropic virus type I (HTLV-I)-transformed lines MT-2 and MT-4 were extensively killed by infection with AIDS retrovirus HTLV-III. We have investigated this phenomenon more systematically using light and electron microscopy as well as immunofluorescence. The cell lines used in the present studies included 14 of those carrying not only human HTLV-I but also related simian agents and 6 HTLV-I-negative T- and B-cell lines. The results showed that the cytocidal effects occurred in the HTLV-I transformed cell lines exclusively and were not present in further subcultures. In these cell lines the cytotoxic response was closely correlated with the induction of HTLV-III antigens after virus infection. However, cells of 6 HTLV-I free lines were not killed to a marked extent by HTLV-III and were passaged as continuous producers of AIDS virus. Only 2 cell lines were resistant to the cytocidal effect of HTLV-III among 14 HTLV-I carrying cell lines. They were also resistant to the replication of infected HTLV-III. This AIDS virus-specific cytotoxic effect observed in HTLV-I-transformed cell lines did not appear to be associated with gene expression of the gag and pXs region of HTLV-I genomes. This result may indicate that HTLV-III specifically interferes with some steps of HTLV I transformation. PMID- 2995260 TI - Cholera-toxin-enhanced growth of human breast cancer cell lines in vitro and in vivo: interaction with estrogen. AB - Cholera toxin (which increases intracellular cAMP levels) significantly (p less than 0.05) increased the growth of MCF-7, T47-D and Hs578T human breast carcinoma cells in vitro. The effect of cholera toxin on growth of MCF-7 and T47-D cells was more pronounced in the presence of 17 beta-estradiol (p less than 0.05), indicating a synergism between cAMP and estradiol in growth control of estrogen receptor-positive breast carcinoma cells. This interaction was not observed in the estrogen-receptor-negative cell line Hs578T. Daily injections of cholera toxin into female athymic nude mice bearing MCF-7 or Hs578T tumors resulted in significantly (p less than 0.05) increased growth of the tumors. Cholera toxin treatments, in addition, significantly (p less than 0.05) increased cAMP levels in tumor cells and tumor tissue, in vitro and in vivo, respectively. The results of this study clearly demonstrated that an increase in cAMP levels via cholera toxin treatment causes enhanced growth (in vitro and in vivo) of estrogen receptor-positive and -negative human breast carcinoma cells and, although estrogen alone was not mitogenic to the estrogen-receptor-positive breast carcinoma cells in vitro, the steroid was mitogenic to these cells in the presence of elevated cellular cAMP levels. PMID- 2995261 TI - Structure-activity relationship in the induction of Epstein-Barr virus by teleocidin derivatives. AB - New derivatives of (-)-indolactam V (Fig. 1), which have the basic ring-structure of teleocidins without the monoterpenoid moiety, were prepared and their Epstein Barr virus early antigen (EBV-EA)-inducing activity was tested. (-)-14-O-Alkyl indolactam Vs (2 and 3) showed little induction of EBV-EA, while (-)-14 dehydroxyindolactam V (4) and (-)-14-chloroindolactam V (5) proved to be potent EBV-EA inducers, though their activities (EC50) were about 10 times weaker than that of (-)-indolactam V (1). These results indicate that the hydroxyl group at C 14 is not indispensable for EBV-EA induction and can be replaced. The activities (EC50) of (-)-1-N-methyl, (-)-1-N-ethyl, and (-)-1-N-butyl indolactam V (10, 11, and 12) were about 5 times weaker than that of (-)-indolactam V (1), while (-)-1 N-hexyl and (-)-1-N-octyl indolactam V (13 and 14) were even less active, suggesting that the free imino group of the indole ring in (-)-indolactam V (1) plays an important role in the activity, and that the activity cannot be enhanced by alkylation at the N-1 position of (-)-indolactam V (1). PMID- 2995262 TI - Therapeutic issues of marijuana and THC (tetrahydrocannabinol). AB - This article summarizes current knowledge about the medicinal value of cannabis and its principal psychoactive ingredient, delta 9-tetrahydrocannabinol (THC), particularly in the control of nausea and vomiting, in glaucoma, and in reduction of spasticity in multiple sclerosis. The major issues in the controversy about marijuana and medicine, primarily moral and ethical, are discussed. PMID- 2995263 TI - The generation and regulation of human T lymphocytes by Imuthiol. Evidence from an in vitro differentiation induction system. AB - In vitro long term induction of human peripheral blood lymphocytes by sodium diethyldithiocarbamate, DTC (Imuthiol) enhanced the expression of OKT and HLA-DR phenotypic determinants. A population of T-B-DR+ cells was recruited from the null cell population. The increase in OKT+ cells, but not the enhanced HLA-DR expression appeared to be macrophage dependent. The surface markers of NK cells, monocytes or the pokeweed mitogen-induced IgM production were not modified. Collectively, the findings indicate that Imuthiol is not a B cell inducer, but is specifically active on the T-cell population in the way it induces the maturation of null cells into OKT+ DR+ lymphocytes. PMID- 2995264 TI - In vitro modulation of purine enzyme metabolism and lymphocyte surface marker expression by thymosin fraction 5 in homosexual males. AB - Thymosin fraction 5 (Thymosin) has numerous immunoregulatory activities including modulation of enzymes involved in lymphocyte maturation. The effect of Thymosin on the purine metabolic enzymes adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and 5' nucleotidase (5'NT) in null and T-enriched peripheral blood lymphocytes from sexually active asymptomatic homosexual males (AS), patients with the AIDS-related symptom complex (ARC), and those with acquired immune deficiency syndrome (AIDS) was examined and compared to its effect on lymphocytes from healthy heterosexual controls. Mean ADA activity was significantly higher in null cells from fourteen AIDS patients than in five asymptomatic homosexuals, ten ARC patients, or 27 controls. Mean PNP activity was significantly elevated in null-enriched lymphocytes from ten ARC and fourteen AIDS patients compared to controls. No differences in these enzymes were found in T-enriched cells from any group. 5'NT was markedly decreased in both null and T lymphocytes in all homosexual groups relative to controls. Homosexuals had significantly elevated percentages of OKT10 positive and Ia positive lymphocytes compared to controls. Thymosin at an optimal concentration of 150 micrograms/ml caused significant decreases in mean ADA and PNP activity in null lymphocytes from ARC + AIDS patients along with a significant decrease in the percentage of OKT10 positive lymphocytes. No phenotypic changes were seen in AS or control lymphocytes. The data suggest that Thymosin has a maturational effect in vitro on immature T cells from symptomatic homosexuals. PMID- 2995265 TI - Inhibition of leukotriene C4 and B4 generation by human eosinophils and neutrophils with the lipoxygenase pathway inhibitors U60257 and BW755C. AB - Human eosinophils and neutrophils have the capacity to generate leukotriene C4 (LTC4) and leukotriene B4 (LTB4) respectively when stimulated by calcium ionophore A23187. Leukotriene production by mixtures of these cell types was measured by radioimmunoassay for LTC4 and LTB4, and the specificities of the assays determined by assessing cross-reactivities with a number of other arachidonic acid metabolites. The IC50S for LTC4 and LTB4 in their respective assays were 1.76 +/- 0.04 nmol and 3.00 +/- 0.08 nmol. Cross-reactivity for anti LTC4 was shown by leukotriene D4 (LTD4) (70%) and leukotriene E4 (LTE4) (8%), when compared to LTC4, whereas in the radioimmunoassay for LTB4, only the 5(S), 12(R) 6-trans isomer of LTB4 showed appreciable interaction (12%). LTC4 production by eosinophil enriched cell fractions obtained from metrizamide gradients was inhibited in a dose-dependent fashion by the prostacyclin analogue, 6,9-deepoxy-6,9-phenylimino-delta 6,8-prostaglandin I, (U60257) and by 3-amino-1 (3-trifluoromethyl phenyl)-2-pyrazole (BW755C). The ID50 values for U60257 and BW755C were 2 X 10(-6) and 5 X 10(-6) M respectively. This demonstration of LTC4 production by human eosinophils, which are known to be important cells in clinical asthma, provides an in vitro model to assess 5-lipoxygenase inhibitors in human tissue. PMID- 2995266 TI - Long-lived radicals in irradiated apatites of biological interest: an e.s.r. study of apatite samples treated with 13CO2. AB - Hydroxyapatite is used as a model for studying radical formation in the mineral compartment of irradiated calcified tissues. Treating this material with 13C enriched CO2 confirms that radiogenic long-lived radicals correspond to carbon centred species. It is shown, however, that these radicals are not located on the carbonate ions which substitute either the phosphate or the hydroxyl groups. The conditions which allow the formation and the trapping of these radicals are investigated (role of humidity, CO2 and temperature) and this paramagnetic species is identified as CO-2 adsorbed at the surface of apatite crystallites. PMID- 2995268 TI - 99mTc bone scanning agents--I. Influence of experimental conditions on the formation and gel chromatography of 99mTc(Sn)pyrophosphate complexes. AB - The conversion of 99mTcO4- to 99mTc(Sn)pyrophosphate complexes was investigated under various experimental conditions. An increase of the Sn(II) concentration had a beneficial effect, whereas the ligand concentration had little effect. The pH had only a small influence over the range 2-8. Raising the pH to 10 resulted in the partial decomposition of the complexes, which could be reversed by lowering the pH. Furthermore, the occurrence of various complexes was investigated by means of gel chromatography on Biogel P-4 as a function of pH and of the Sn(II) and pyrophosphate concentrations. Four major fractions were found. A single preparation contained, however, no more than two major fractions. The formation of the different complexes was mainly governed by the pH and the ligand concentration. The influence of the eluent on the decomposition and interconversion of the complexes during chromatography was also studied. It appeared to be necessary that the eluent should have the same composition (except for 99mTcO4-) as the reaction mixture. PMID- 2995267 TI - The detection of abscesses with diffusible tracers. AB - In six patients with suspected infection, scintigraphy with diffusible tracers outlined the margins and central portion of an abscess. An animal model was developed to study this process quantitatively. Small inflammatory lesions yielding volumes of pus of 0.1-0.2 mL were shown to have increased blood volume of 0.7 +/- 0.5 mL, increased blood flow by a factor of three and increased extracellular fluid volume of 10 +/- 6 mL. This supports the patient data and indicates that the pathophysiologic features characterising the clinical studies are hyperemia and oedema associated with an abscess. The slow, central area of tracer diffusion corresponded to the presence of pus. Scintigraphy with 99mTc DTPA is a convenient way of detecting suspected inflammatory lesions and localising collections of pus. PMID- 2995269 TI - A dispassionate scientific analysis of Keyes' technique. PMID- 2995270 TI - Biology of rat cytomegalovirus infection. AB - Cytomegalovirus infection of Brown Norway rats was studied after intraperitoneal or subcutaneous inoculation of virus. No clinical illness was apparent during the 1st month postinfection (p.i.). Low titers of virus were detected in many organs at day 4 p.i. for the intraperitoneally inoculated animals and at day 11 p.i. for those inoculated subcutaneously. Thereafter, the virus disappeared from all tested organs except the salivary glands, where it appeared on day 11 p.i. and reached high levels by 4 weeks p.i. Histologically, no abnormalities were observed. The virus had an immunosuppressive effect during the 1st week p.i., as indicated by the immune response to sheep red blood cells. PMID- 2995271 TI - Immunoblotting and enzyme-linked immunosorbent assay analysis of serological responses in patients infected with herpes simplex virus types 1 and 2. AB - Serological responses to soluble membrane and cytoplasmic antigens specified by herpes simplex type 1 (HSV1) and 2 (HSV2) were studied by immunoblotting and by enzyme-linked immunosorbent assay (ELISA). In the immunoblotting test, polypeptides migrating like the HSV1-specified glycoprotein C showed type specific reactivity but could not always be detected. The immunoblotting and ELISA results were in agreement when antibody responses to HSV1- and HSV2 specified antigens were compared, and they allowed the identification of patients with HSV2 and/or HSV1 antibodies. PMID- 2995272 TI - Restrictive events in the replication of hepatitis A virus in vitro. AB - Hepatitis A virus was purified from the feces of 2 patients with unrelated, naturally acquired infections and was inoculated into FRhK-4 cells. Analysis of protein synthesis by double-label coelectrophoresis and subtraction allowed the resolution of virus-specific proteins synthesized during infection. In FRhK-4 cells the two strains of virus studied produced markedly different profiles of virus-specified proteins, with an accumulation of high-molecular-weight proteins for strain HM790 relative to strain HM175, suggesting a level of restriction in the processing of the viral polyprotein. PMID- 2995274 TI - DNA homology relationships between Spodoptera littoralis nuclear polyhedrosis virus and other baculoviruses. AB - The DNA sequence homology relationships between Spodoptera littoralis nuclear polyhedrosis virus (NPV) and five other lepidopteran NPVs were studied by hybridization of 32P-labeled NPV DNAs to Southern blots of restriction endonuclease-digested NPV DNA. The S. littoralis NPV (SlMNPV) shows extensive DNA sequence homology through most of the genome with S. frugiperda NPV and S. exigua NPV. Heliothis armigera NPV, H. zea NPV, and Autographa californica NPV (AcMNPV) share fewer regions of homology with SlMNPV. Using the cloned HindIII fragment V of AcMNPV DNA, fragments with sequence homology to the polyhedrin gene of AcMNPV were identified for the viruses studied. PMID- 2995273 TI - HSV1-specific thymidylate kinase activity in infected cells. AB - Several 5-methoxymethyldeoxyuridine (MMdU)-resistant mutants of herpes simplex virus type 1 (HSV1) were classified by measuring their sensitivities to the deoxythymidine kinase (dTK)-dependent antiviral drugs 9-(2-hydroxyethoxymethyl) guanine (acyclovir, ACV), 1-beta-D-arabinofuranosylthymine (araT), and E-(2)-5 bromovinyldeoxyuridine (BVdU) and to the dTK-independent antiviral drug phosphonoacetate (PAA). Compared to wild-type (WT) virus, all five of the dTK- mutants were highly resistant (greater than or equal to 500-fold) to BVdU and MMdU, moderately resistant to ACV (50- to 100-fold) and araT (10- to 20-fold), but not resistant to PAA. The dTK of the mutant MMdUr-20 (dTK+) appeared to phosphorylate dTMP less well than that of the WT virus, while its affinity for deoxythymidine was not altered. Two other drug-resistant HSV mutants-S1 (isolated against ACV) and B3 (isolated against BVdU)--also showed reduced phosphorylation of dTMP. This suggests that alterations in both dTK and thymidylate kinase activities may determine sensitivity to antiviral drugs. PMID- 2995276 TI - Inflammatory fibroid polyp of the cecum, associated with adenomatous polyp and ovarian thecoma. PMID- 2995275 TI - The artificial endocrine pancreas in the surgical treatment of insulinoma. Usefulness and limits. AB - The usefulness and the limits of the artificial endocrine pancreas in the surgical management of insulinoma has been evaluated in three male patients who underwent pancreatic resection because of previously detected adenoma. In particular, blood glucose and contemporary levels of insulin and C-peptide were continuously monitored before, during and after surgery, to record the temporal relationship between the removal of insulinomas and the variations of these parameters. In the pre-resection phase, only two cases revealed hypoglycemia and required dextrose infusion to correct hypoglycemia and reach euglycemic levels, whereas all the patients showed elevated insulin and C-peptide levels. After anesthesia and surgical incision, the pancreas was observed and manipulated in search of adenoma. In all patients this manoeuvre caused an increase of insulin and C-peptide levels and in two cases a slight decrease of blood glucose levels. After adenoma resection, a prompt increase of glycemia was observed only in one patient, in the other two the time which elapsed before significant blood glucose changes was more prolonged (55 and 80 min. respectively). On the contrary, a rapid fall in insulin and C-peptide levels was observed in all cases. We conclude that artificial endocrine pancreas has the advantage of maintaining the normoglycemia before and during surgery, preventing the risk of dangerous hypoglycemia in basal conditions and following manipulation of pancreas while localizing adenoma. However, the prolonged interval elapsed before significant blood glucose variations limits the usefulness of the artificial endocrine pancreas in localizing intraoperatively previously undetected adenomas. PMID- 2995277 TI - [Bowenoid papulosis and carcinoma in situ of the cervix uteri in sex partners. An example of the transmissibility of HPV-16 infection]. AB - The development of bowenoid papules in a 20-year-old man and a carcinoma in situ of the portio uteri of the 22-year-old female sexual partner is reported. In both lesions HPV-16 DNA could be detected by molecular biological means. This observation led us to the conclusion that HPV 16 had been transmitted sexually. The same seems to be true for HPV-11-induced condylomata acuminata, which appeared on the external genitalia of both patients after recuperation from the HPV-16-induced lesions and conisation treatment. The clinical significance of this observation and its consequences for dermatologists and gynecologists are discussed. PMID- 2995279 TI - Constipation during pregnancy: dietary fibre intake and the effect of fibre supplementation. AB - Forty women who complained of constipation during the third trimester of pregnancy completed 14-day weighed diet records and bowel function charts over a 4-week period. After 2 weeks of baseline observation the women were randomly allocated into three groups which were asked to take 10 g dietary fibre supplements per day in the form of either a corn-based biscuit (Group A), or as wheat bran (Gp B), or to continue without intervention (Gp C). Mean (+/- s.e.m.) daily dietary fibre intake in the first 2 weeks was similar to that in the general population, at 20.4 +/- 1.2 g, for the whole group, and 21.1 +/- 1.6 g for the 26 women who said they had already increased their dietary fibre intakes in attempts to relieve their symptoms. In the final 2 weeks changes in fibre intakes were: Gp A, mean increase 7.2 +/- 1.0 g per day (P less than 0.001); Gp B, mean increase 9.1 +/- 1.6 g per day (P less than 0.001); Gp C mean decrease 3.50 +/- 1.6 g per day (P less than 0.005). These changes were accompanied by an increase in the number of bowel movements and a change to a softer stool consistency in Gps A and B, with no changes in number of bowel movements or stool consistency in Gp C. PMID- 2995278 TI - The effect of an anionic detergent on complex carbohydrates and enzyme activities in the epidermis of the catfish Heteropneustes fossilis (Bloch). AB - The histochemistry of various oxidative enzymes and complex carbohydrates in the epidermis of the catfish Heteropneustes fossils was investigated after exposure to sublethal concentrations of the detergent sodium alkylbenzenesulphonate. It was found that the detergent treatment was accompanied by a marked increase in the number of mucous cells which produce histochemically detectable amounts of acidic glycoproteins with a shift towards the production of O-acetylated sialic acids. The activities of mitochondrial enzymes were lost in the superficial cell layers. In contrast the activities of glucose-6-phosphate and lactate dehydrogenase increased considerably. The rise in glucose-6-phosphate dehydrogenase was correlated with the metabolic requirements for the enhanced production of mucus under stress. The changes in both enzyme activities and in the chemical composition of mucus may provide a suitable experimental model for histochemical investigations of the effects of stress induced by pollutants on aquatic organisms. PMID- 2995280 TI - Increased cardiovascular beta-adrenoceptor responsiveness in underweight subjects. AB - A comparison of cardiovascular responses to increasing bolus doses of the beta agonist, Isoproterenol, was made between 11 normal weight (body mass index 19 22.5) and 9 underweight (body mass index 15.5-19) healthy, euthyroid male subjects from the same socio-economic background and on similar energy intakes. The positive chronotropic response and the fall in diastolic pressure observed during the dose response to Isoproterenol was significantly greater (P less than 0.05) in the underweights suggesting an enhanced beta-adrenoceptor responsiveness in this group. The cardiovascular responses to head-up tilt of underweight subjects were similar to those of the normal weight group. The study suggests that lean, underweight individuals may have a generalized increase in beta adrenoceptor responsiveness. PMID- 2995281 TI - Localisation of receptors using a dimeric ligand and electron immunocytochemistry. AB - The role of regulatory peptides and the existence of specific peptide receptors are becoming established. However, techniques for the ultrastructural localisation of these receptors are fraught with difficulties. We propose here a novel technique for receptor localisation using a dimeric peptide ligand and electron microscopical immunocytochemistry. The dimeric ligand is used as a bridge between the receptor and a specific anti-ligand antibody. By this method we have localised receptors for bombesin in cells of small cell lung carcinoma in culture. The results support previous biochemical evidence for the existence of said receptors and the technique should be applicable for the localisation of other receptors recognising small ligands. PMID- 2995282 TI - Elemental composition of the human atherosclerotic artery wall. AB - The elemental composition of the human atherosclerotic popliteal artery was examined using the proton-induced X-ray-emission (PIXE) method. The application of a narrow proton beam (3 X 10 micron 2) enabled us to determine not only the concentrations of Cl, K, Ca, Fe, Cu, Zn, Br and Pb, but also their localization in different artery-wall regions. The highest mean concentrations of Cl, K, Zn and Br were found in the tunica media. In the investigated sections the distribution of Ca and Fe varied: sometimes, these elements were prevalent in the tunica intima, whereas in other cases, the highest concentrations were observed in the tunica media or tunica adventitia. The concentration profiles of each element were characterized by many sharp, narrow peaks. The highest concentrations of Ca and Fe showed such high levels that only one explanation is possible, i.e. the presence of crystals. The correlation of Ca peaks with those of Zn and Fe is discussed. The usefulness of the micro-PIXE method for the investigation of biomedical materials is also considered. PMID- 2995283 TI - Nucleoside phosphatase and nucleotide tetrazolium reductase as markers of arteriolar differentiation in fetal pig tissue. AB - Nucleoside phosphatase and nucleotide tetrazolium reductase reactions were studied as potential markers of arteriolar differentiation. Arteriolar systems were analyzed cytochemically and morphologically in a variety of fetal pig tissues at 70 and 110 days of gestation. In skeletal muscle and subcutaneous adipose tissue there were age dependent changes in phosphatase and reductase reactivity in arteriolar vessels. These changes were temporally associated with the morphological differentiation of the tunica medial arteriolar layer. There were no age dependent changes in the cytochemistry of arterioles in liver and cardiac muscle. In the youngest fetuses, arterioles in liver and cardiac muscle displayed a typical tunica medial layer (normal morphology). The cytochemical reactions (phosphatase, reductase) of arterioles in cardiac muscle and liver from 70 (and 110) days old fetuses were identical to cytochemical reactions of arterioles in muscle and adipose tissue from 110 days old fetuses. In the skin, there were age dependent increases in phosphatase reactive arterioles and capillaries. Capillary staining (phosphatase) and capillary bed size were inversely correlated in the skin. Capillaries in skeletal muscle, cardiac muscle, adipose tissue and liver were not phosphatase reactive. These results indicate that the morphological and cytochemical differentiation of arterioles is dependent on tissue and age in the fetal pig. Furthermore, several histochemical techniques (phosphatase, reductases) are validated as simple means to analyze arteriolar differentiation in general. PMID- 2995284 TI - Histochemical studies on metabolic zonation of the liver in the trout (Salmo gairdneri). AB - The livers of 26 adult male and female trout were studied histochemically. G6Pase activity was always found to be heterotopically distributed with a constant maximum in the periportal area. In many cases the glycogen content and the activity of phosphorylase predominated in the periportal zone as well. Maximum activity of glucose-6-phosphate-dehydrogenase and malic enzyme, however, could be demonstrated preferentially in the perivenous area. Lactate dehydrogenase, succinate dehydrogenase, alcohol dehydrogenase, acid phosphatase and beta glucuronidase were found equally in all liver cells. 3-Hydroxybutyrate dehydrogenase was absent. Thus, the principles of metabolic zonation have been established in trout liver, the architecture of which differs essentially from that of mammals. The course of the terminal afferent and efferent vessels is the decisive factor for the heterotopic localization of functional units rather than the tubular or plate-forming arrangement of the hepatocytes. PMID- 2995285 TI - Ultracytochemical localization of acetylcholine-like cations in excited motor end plates by means of ionic fixation. AB - Using rapid ionic fixation with molybdic or tungstic heteropolyanions (strong precipitating agents of quaternary ammonium cations such as choline and acetylcholine), acetylcholine-like cations were localized as point-like precipitates in the synaptic vesicles of resting (electrically nonstimulated) motor nerve terminals. When performed at low temperature, the same procedure revealed spot-like precipitates (presumed to be exocytotically released acetylcholine-like cations) in the synaptic cleft in the vicinity of the active zone. These precipitates were often seen in paired forms. Unlike resting motor nerve terminals, excited terminals (electrical stimulation with occasional 4 aminopyridine pretreatment) after ionic fixation exhibited, at first, laminar precipitates both in the vicinity of the active zone inside the nerve terminals and in the synaptic space. In the vicinity of the active zone, the laminar precipitates were directed towards the synaptic membrane, while those in the synaptic space showed no orientation. Ionic fixation also revealed diffused precipitates both around the synaptic vesicles and on the axoplasmic side of the presynaptic membrane. Finally, the same fixation procedure demonstrated the presence of empty synaptic vesicles (without point-like precipitates) in close contact with the presynaptic membrane. The laminar and diffused precipitates are presumed to be two different forms of the same salts of acetylcholine-like cations that are insolubilized by ionic fixation in both the nerve terminals and the synaptic space of excited motor end-plates. PMID- 2995286 TI - [Rare tumors of the base of the tongue and their therapy]. AB - Five rare tumours of the base of the tongue are reported. Diagnosis and therapy of a tuberculoma, an amyloid tumour, a mucoepidermoid carcinoma, a pleomorphic adenoma, and an angioleiomyoma are illustrated. The frequency and other possible sites of these tumours are discussed based on a review of the literature. The standard surgical approaches to the base of the tongue are described, favouring lateral pharyngotomy. The need for careful planning of the operation is stressed including difficulty in intubation and control of bleeding after operation. PMID- 2995287 TI - Virilizing adrenocortical adenoma with a paradoxic response to dexamethasone administration: report of a case. PMID- 2995288 TI - Glomus tumors of the upper extremity: experience with 12 cases. PMID- 2995289 TI - Hyperproduction and gene amplification of the epidermal growth factor receptor in squamous cell carcinomas. AB - Human squamous cell carcinoma tissue was screened to determine the epidermal growth factor (EGF) receptor level. In 9 out of 15 cases, increased EGF binding was observed relative to normal adjacent tissue. In two tissue samples (SCE1 and SCL1), amplification of the EGF receptor gene and increased mRNA level were also observed. In two other cases (SCE2 and SCL3), EGF receptor gene amplification did not occur. We propose that there is an alternative mechanism for EGF receptor hyperproduction, independent of gene content, active in these tissues. A possible role of EGF receptor hyperproduction is envisioned in human neoplasia. PMID- 2995290 TI - Occurrence of human papillomavirus types 16 and 18 DNA in cervical carcinomas from Japan: age of patients and histological type of carcinomas. AB - We have detected by DNA hybridization human papillomavirus (HPV) types 16 and 18 DNA sequences in 19 and 3, respectively, out of 56 cervical carcinomas from Japan. Eighteen out of 19 HPV 16-positive specimens were from squamous cell carcinomas, whereas the three HPV 18-positive specimens were from a squamous cell carcinoma (1/50), an adenosquamous carcinoma (1/3), and an adenocarcinoma (1/3). The occurrence of HPV 16 DNA decreased in patients over 60 years old (less than or equal to 60 years, 15/34 (44%); 60 years less than, 4/22 (18%)) (P less than 0.05). PMID- 2995291 TI - Stimulation of protein glycosylation in cultured hepatoma cells by polyprenyl phosphates. AB - The aim of this work was to selectively stimulate N-glycosylation of proteins in cultured AH 70Btc hepatoma cells. Dolichyl phosphate, alpha-dihydrodecaprenyl phosphate and solanesyl phosphate were entrapped in egg lecithin liposomes and supplied to the cells. After treatment with 0.6-15 nmol of the polyprenyl phosphates/3 ml/dish for 48 hr, the incorporations of [14C]glucosamine into N linked saccharide chains of glycoproteins and into lipid intermediates were increased to various extents. The stimulation by alpha-dihydrodecaprenyl phosphate was most significant and 15 nmol of the lipid/dish increased the incorporation into N-linked saccharide chains of glycoproteins by about 80%. The incorporations of [14C]glucosamine into O-linked saccharide chains and of [14C]leucine into proteins were not changed significantly by alpha dihydrodecaprenyl phosphate. When the N-linked glycopeptides prepared from the cells and those from the cells treated with the polyprenyl phosphate were compared by Sephadex G-50 column chromatography, no significant difference was observed. Analysis of reduced cellular glycoproteins by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis showed that alpha-dihydrodecaprenyl phosphate increased the incorporation of [14C]glucosamine into N-linked saccharide chains of certain high-molecular-weight glycoproteins. In addition, the polyprenyl phosphate enhanced the adhesiveness of the cells to the substratum, probably as a result of the stimulation of N-glycosylation of proteins. PMID- 2995293 TI - Studies on antiviral agents. II. Synthesis and in vitro antiviral activity on new kanamycin A derivatives having higher acyl group at N-1 position. AB - The synthesis and antiviral activity of 3''-N-trifluoroacetylkanamycin A derivatives (6) having higher acyl group at the N-1 position are described. On the basis of the structure-activity relationships between antiviral activity and alkyl chain length in an acyl group at the N-1 position, analogs (6f approximately I) having higher alkylcarbonyl group exhibited antiviral activity against not only HSV-I but also influenza virus. Analogs (6q approximately v) having higher alkyloxycarbonyl group showed antiviral activity against HSV-I. In addition, kanamycin A derivatives (6n, o, y, z) possessing higher alkylcarbonyl group with a functional group, higher alkylaminocarbonyl group, and higher alkylthiocarbonyl group had antiviral activity against HSV-I. The analog (6h) showed a broad antiviral spectrum against both DNA (HSV-I, HSV-II, VZV) and RNA (influenza) viruses. PMID- 2995292 TI - Isolation of A58365A and A58365B, angiotensin converting enzyme inhibitors produced by Streptomyces chromofuscus. AB - A58365A and A58365B, angiotensin converting enzyme inhibitors, were isolated from the culture filtrate of Streptomyces chromofuscus NRRL 15098. A58365A and A58365B are homologous nitrogen-containing bicyclic structures of molecular formulae C12H13NO6 and C13H15NO6. PMID- 2995294 TI - A37812: N-methylstreptothricin F. AB - A37812, a new member of the streptothricin class of antibiotics, has been isolated and characterized as N-methylstreptothricin F. The structure elucidation of A37812 is based on results from 13C and 1H NMR spectroscopies. PMID- 2995295 TI - Methods for the detection and quantitation of angiotensin converting enzyme inhibitors in fermentation broths. AB - Two procedures are described for the detection of inhibitors of angiotensin converting enzyme (ACE). The first is a new agar-plate method useful as a screening tool. Two ACE inhibitors produced by culture A58365 were discovered using this plate test. The second method is a modification of a previously reported spectrophotometric procedure. Both procedures utilize p-nitrobenzyl oxycarbonylglycyl-(S-4-nitrobenzo-2-oxa-1,3- diazole)-L-cysteinyl-glycine as substrate. PMID- 2995296 TI - Angiotensin converting enzyme inhibitors produced by Streptomyces chromofuscus. Discovery, taxonomy and fermentation. AB - Culture A58365.1, NRRL 15098, identified as a new strain of Streptomyces chromofuscus, was found to produce two novel angiotensin converting enzyme (ACE) inhibitors, A58365A and A58365B. Fermentation medium studies afforded an increase in ACE inhibitor titers from less than 1 microgram/ml to greater than 20 micrograms/ml. Proline was the obligatory supplement for ACE inhibitor biosynthesis. PMID- 2995297 TI - Reversible contraction of isolated mammalian cochlear hair cells. AB - Outer hair cells were isolated from the guinea pig cochlea using a micromechanical non-enzymatic procedure. Depolarization of outer hair cells in the presence of 25-125 mM K+ was accompanied by a longitudinal contraction of the isolated cells. A decrease of [K+] to 5.4 mM interrupted contraction and induced a relaxation. Individual hair cells were able to undergo as many as 5 cycles of contraction and relaxation. External Ca2+ was required for relaxation of the contracted hair cells. The contractile event led to the production of a visible cytoplasmic network between the supranuclear area and the cuticular plate. PMID- 2995299 TI - Growth of suppression in the cochlear potentials. AB - Measurement of two-tone effects in the cochlear microphonic and summating potential indicates that the growth of suppression is different for these two cochlear potentials. Whereas the CM response to the fundamental is reduced 10 dB for each 10 dB increase in suppressor level, the SP decreases at a faster rate; approximately 20 dB per 10 dB increase. Slopes of functions for the CM response to the second harmonic are similar to those for the dc component. Since these results are consistent with the notion that suppression operates by attenuating the input to the CM generator, they are consonant with a mechanical origin of suppression. PMID- 2995298 TI - Characteristics of tone-pip response patterns in relationship to spontaneous rate in cat auditory nerve fibers. AB - The responses of single auditory nerve (AN) fibers in the cat were recorded in response to 25 ms tone pips. Peristimulus time histograms (PSTH) of discharge patterns recorded from fibers with high spontaneous rates (high SRs), show that the discharge rate rapidly adapts to a much lower steady-state level over a 15 ms period with shorter times for units with best frequencies (CFs) greater than 5 kHz. The PSTHs of auditory nerve fibers with low SRs do not show this pattern of rapid adaptation. Differences between the high and low SR populations include higher thresholds, better tuning, and longer latency in the low SR population. The peak-to-steady-state discharge ratio is an increasing function of SR and CF; it varies from 1.0 for fibers with SR = 0 to over 8 for fibers with high SRs and CFs near 10 kHz. This ratio increases with increasing stimulus intensity and stimulus recovery time. The high SR population shows a number of responses to transients which are weak or absent in the low SR population. Increasing the recovery time shortened the latency of both high and low SR AN fibers by as much as 1 ms. A number of other response properties of AN fibers are also reported that are important when interpreting the responses of cochlear nucleus neurons to tone pips. PMID- 2995301 TI - Effect of roughage particle size on ruminal, digestive and metabolic characteristics of early-weaned lambs fed pelleted corncob-concentrate diets. AB - Two experiments were conducted to determine effects of feeding corncobs of various mean particle size (MPS) on ruminal, digestive and metabolic characteristics of early-weaned lambs fed pelleted 74.9% concentrate:25.1% corncob diets. The MPS of corncobs in diets was 6.5, 5.4, 1.4 and .8 mm, respectively. As particle size decreased, percentage starch decreased and percentage neutral detergent fiber (NDF), acid detergent fiber (ADF) and cellulose increased. In Exp. 1, 28 crossbred rams (seven/treatment, avg initial wt, 15.3 kg) were used in a randomized complete-block design. In Exp. 2, lambs from Exp. 1 were re-weighed (avg initial wt, 16.8 kg) and fed the same diets as in Exp. 1. In Exp. 1 and 2, lambs ingested dry matter (DM) equal to 2.68 and 3.74% of body weight, respectively. In Exp. 1, apparent DM digestibility was unaffected by corncob MPS; however, in Exp. 2, DM digestibility was highest (68.8%) for lambs fed the 6.5-mm diet and lowest (63.8%) for those fed the .8-mm diet. Apparent starch digestibility was high (greater than 98.8%) in both experiments. Neutral detergent fiber and ADF digestibilities were highest for lambs fed the 1.4-mm diet (50.5 and 43.6%, Exp. 1; 39.6 and 28.9%, Exp. 2). A dramatic increase (6.8 to 39.1%) in acid detergent lignin (ADL) digestibility was observed in Exp. 1 as corncob MPS decreased. In Exp. 2, ADL digestibilities were similar for lambs fed the 6.5-, 5.4- or 1.4-mm diets (avg value, 5.9%) and highest for those fed the .8-mm diet (29.7%). Nitrogen metabolism was unaffected by corncob MPS. In Exp. 1, digestible energy intake, corrected for urinary losses, did not differ among treatments but in Exp. 2, lambs consuming the 6.5-mm diet had higher corrected digestibility energy intakes (1,926.6 kcal/d) than did those fed other diets (avg, 1,832.4 kcal/d). Ruminal pH sampled 4 h post-feeding was highest for lambs consuming the 6.5-mm diet (6.25) in Exp. 1 and the 1.4-mm diet (5.89) in Exp. 2. Lowest ruminal pH (5.30 and 5.36, respectively) was for lambs consuming the .8-mm diet in Exp. 1 and the 5.4-mm diet in Exp. 2. Ruminal lactate concentrations were variable within and among treatments. Total ruminal volatile fatty acid (VFA) concentrations were similar across treatments but in Exp. 2, there was a shift in molar proportions from acetate to propionate as corncob MPS decreased.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2995300 TI - Effects of roughage particle size on site of nutrient digestion and digesta flow through the gastrointestinal tract of sheep fed corncob-concentrate diets. AB - An experiment examining nutritional effects of dietary corncob particle size was conducted using ruminal-, duodenal- and ileal-cannulated sheep in a 4 X 4 Latin square design. Site of nutrient digestion and digesta flow were the principal criteria evaluated. Analyses of dry matter (DM), N, starch and neutral detergent fiber (NDF) were performed on feed, feces and digesta samples. Chromic oxide impregnated paper was used as an external marker to estimate digestibilities at different sites along the gastrointestinal tract. Ruminal pH and volatile fatty acid molar proportions were also determined. All diets (74.9% concentrates: 25.1% corncobs) were pelleted and were similar in ingredient composition but varied in corncob particle size (corncob mean particle sizes were 6.5, 5.4, 1.4 or .8 mm). Dietary crude protein levels differed little among treatments. Starch concentration was higher in diets containing the larger corncob particles while NDF, acid detergent fiber (ADF), cellulose and hemicellulose concentrations were lower in diets containing larger particles, suggesting a reaction between starch and fiber moieties during the pelleting process. Starch flow past the duodenum decreased (P less than .05) as dietary corncob particle size decreased. Apparent NDF digestion before the duodenum was highest for sheep fed diets containing 1.4 mm corncobs (P less than .05). Apparent starch digestion in the small intestine decreased (P less than .05) as dietary corncob particle size decreased. A considerable amount of NDF was apparently digested in the small intestine of sheep consuming diets containing 5.4- and .8-mm corncobs. Likewise, a substantial amount of NDF was apparently digested in the large intestine. Few differences in apparent total tract digestibilities were noted.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995302 TI - The cytochrome c oxidase test for the rapid detection of psychrotrophic bacteria in milk. AB - A new, rapid, simple and cheap method for the detection of the predominant psychrotrophic bacteria in raw and pasteurized milks is described, using tetramethyl-p-phenylene-diamine dihydrochloride to detect cytochrome c oxidase which in general is not present in the non-psychrotrophic bacterial milk flora. The test is sensitive to samples containing over 10(4) organisms/ml. Correlation coefficients of 0.92 and 0.84 between dye oxidation and viable counts for pasteurized and raw milk samples, respectively, were found. PMID- 2995303 TI - Penetration of imidazoles and triazoles into cerebrospinal fluid of rabbits. AB - We studied the penetration of two imidazoles (ketoconazole and vibunazole) and two triazoles (itraconazole and UK-49,858) into cerebrospinal fluid (CSF) of rabbits with and without meningitis. There were wide differences in degree of penetration of these drugs into CSF, from less than 3% to 66% of simultaneous serum concentrations. UK-49,858, which has little protein-binding, penetrated freely while itraconazole, which is highly protein-bound, could not be detected in CSF. Intermediate concentrations of vibunazole and ketoconazole were found in CSF. Presence of meningeal inflammation modestly increased CSF concentrations of ketoconazole but had no significant effect on penetration of the other three drugs. The excellent penetration of UK-49,858 indicates that it has promise for treatment of CNS fungal infections. PMID- 2995304 TI - Isolation of rat hepatocytes with EDTA and their metabolic functions in primary culture. AB - Isolated hepatocytes from adult rat liver were prepared after dissociation of the liver with EDTA. The morphological appearance, viability (94.5%) and yield (1.76.10(7) cells/g liver) compare well with those of previously described methods using collagenase. Differentiated functions of the hepatocytes in primary culture such as albumin secretion (10.9 micrograms/mg cell protein/d) and triglyceride synthesis and secretion are maintained. Induction of triglyceride synthesis and secretion by oleic acid takes place to an extent similar to that observed in vivo and liver perfusion. Particles with a lipid composition resembling circulating very low density lipoproteins are secreted into the medium. These characteristics demonstrate the ability of hepatocytes isolated with EDTA and subsequently used in primary culture to retain complex and highly differentiated functions of the intact liver. PMID- 2995305 TI - Treatment of visceral leishmaniasis. PMID- 2995306 TI - Isolation and characterization of Escherichia coli pantothenate permease (panF) mutants. AB - Mutants of Escherichia coli K-12 defective in the pantothenate permease (panF) were isolated and characterized. The panF mutation resulted in the complete loss of pantothenate uptake and of the ability to use extracellular vitamin for growth. The growth phenotypes of panF panD, panF panB, and panF panC double mutants showed that the cytoplasmic membrane was impermeable to external pantothenate. Analysis of the intracellular and extracellular metabolites from strain DV1 (panF panD) labeled with beta-[3-3H]alanine demonstrated that a carrier-mediated mechanism for efficient pantothenate efflux remained in the panF mutant. Genetic mapping of this nonselectable allele was facilitated by the isolation of three independent Tn10 insertions close to panF. Two- and three factor crosses located panF at minute 72 of the E. coli chromosome and established the gene order fabE panF aroE. PMID- 2995307 TI - Isolation and characterization of Tn5 insertion mutants of Pseudomonas syringae pv. syringae altered in the production of the peptide phytotoxin syringotoxin. AB - A syringotoxin-producing strain of Pseudomonas syringae pv. syringae (B457) was subjected to Tn5 mutagenesis by the transposon vector pSUP1011. Analyses of auxotrophs obtained suggested simple random insertions of Tn5. Syringotoxin negative mutants arose at a frequency of about 0.28%. In a Southern blot analysis, the loss of toxin production was associated with Tn5 insertions into chromosomal EcoRI fragments of about 10.5, 17.8, and 19.3 kilobases. Data from a Southern blot analysis of SstI-digested DNA from these mutants suggest that the 10.5- and 17.8-kilobase EcoRI fragments may be adjacent to or near each other. Mutants that produced only 3 to 4% wild-type toxin levels also were identified. PMID- 2995308 TI - Induction by folate and folate analogs of extracellular and membrane-bound phosphodiesterase from Dictyostelium discoideum. AB - Folate stimulation is known to enhance Dictyostelium discoideum differentiation. During early differentiation, D. discoideum cells possess two classes of folate receptors which can be distinguished by their difference in specificity (R. J. W. de Wit, FEBS Lett. 150, 445-448, 1982). We investigated the type of receptor by which folate affects cell differentiation. Two independently regulated developmental markers were used: the extracellular phosphodiesterase-inhibitor system and cell-surface phosphodiesterase activity. Our results indicate that the major effect of folate on development is mediated by the folate-specific receptor. The nonspecific folate receptor was only involved in a minor, transient enhancement of the extracellular phosphodiesterase activity very early in development. PMID- 2995309 TI - Delineation of two distinct regulatory domains in the 5' region of the nar operon of Escherichia coli. AB - A detailed restriction site map was determined for an 8.4-kilobase DNA fragment containing the 5' regulatory and promoter region of the nar operon of Escherichia coli. The 5' end of the nar operon was subcloned as a 2.5-kilobase fragment, and an intact nar operon was constructed from this subcloned fragment and an EcoRI fragment containing the remainder of the nar operon. A set of Bal 31 deletions extending into the 5' region of the intact operon was selected, mapped, and characterized. Based on the synthesis of the alpha and beta subunits of nitrate reductase in a nar::Tn5 mutant, three categories of deletions were found: (i) those which permitted normal expression, (ii) those which completely prevented expression, and (iii) those which permitted anaerobic expression of the operon but prevented any additional induction by nitrate. The nucleotide sequence was determined for a segment of the nar promoter region starting at one of the latter deletion end points and extending into the first structural gene of the operon. The position of the deletion end point relative to the translation start site for the first structural gene, narG, was defined by identifying the nucleotide sequence for the first 20 N-terminal amino acid residues of the alpha subunit of nitrate reductase. Deletions terminating 161 base pairs (bp) and approximately 200 bp upstream from the narG translation start site permitted anaerobic formation of nitrate reductase but interfered with the stimulation of nar operon expression by nitrate. A maximum size for the regulatory region was defined by two Tn5 insertions, which mapped approximately 550 bp 5' from the translation start site and did not interfere with the normal expression of nitrate reductase under anaerobic conditions with or without nitrate. We conclude that the nar operon 5' regulatory region is divided into two distinct regions: the 100 to 150 bp immediately 5' to the narG gene include a transcriptional start site and the signals necessary for anaerobic expression of the operon, and an adjacent region of 50 to 400 bp is required for the stimulation of operon expression by nitrate. PMID- 2995311 TI - Analysis of the ruv locus of Escherichia coli K-12 and identification of the gene product. AB - The ruv gene of Escherichia coli, which is associated with inducible mechanisms of DNA repair and recombination, has been cloned into the low-copy plasmid vector pHSG415. The recombinant plasmid pPVA101 fully complements the DNA repair deficient phenotype of ruv mutants. Restriction endonuclease analysis of this plasmid revealed a 10.6-kilobase (kb) HindIII DNA insert which contained a 7.7-kb PstI fragment identified as being from the chromosomal ruv region. Deletion analysis and Tn1000 insertional inactivation of ruv function located the ruv coding region to a 2.2-kb section of the cloned DNA fragment. A comparison of the proteins encoded by ruv wild-type and mutant plasmids identified the gene product as a protein of molecular weight 41,000. PMID- 2995310 TI - Novel one-step cloning vector with a transposable element: application to the Myxococcus xanthus genome. AB - A new strategy was developed for rapid cloning of genes with a transposon mutation library. We constructed a transposon designated TnV that was derived from Tn5 and consists of the gene coding for neomycin phosphotransferase II as well as the replication origin of an Escherichia coli plasmid, pSC101, flanked by Tn5 inverted repeats (IS50L and IS50R). TnV can transpose to many different sites of DNA in E. coli and Myxococcus xanthus and confers kanamycin resistance (Kmr) to the cells. From the Kmr cells, one-step cloning of a gene which is mutated as a result of TnV insertion can be achieved as follows. Chromosomal DNA isolated from TnV-mutagenized cells is digested with an appropriate restriction enzyme, ligated, and transformed into E. coli cells with selection for Kmr. The plasmids isolated contain TnV in the target gene. The plasmid DNA can then be used as a probe for characterization of the gene and screening of clones from a genomic library. We used this vector to clone DNA fragments containing genes involved in the development of M. xanthus. PMID- 2995312 TI - Mechanism of bacteriophage conversion of lipase activity in Staphylococcus aureus. AB - Staphylococcus aureus PS54 harbors two temperate bacteriophages and manifests no lipase activity on egg yolk agar. Curing of one of the resident prophages (L54a) restores lipase activity. To study the mechanism of bacteriophage conversion, the prophage was cured, and the gene encoding lipase activity was cloned into pBR322 in Escherichia coli on a 2.9-kilobase DNA fragment of the chromosome. The fragment was subcloned into a shuttle vector and subsequently transformed into S. aureus and Bacillus subtilis. Lipase activity was expressed in all three genetic backgrounds. Transformation and transductional data indicated that conversion is due to insertional inactivation of the lipase gene. Hybridization analysis with probes made from converting-phage DNA and from the cloned fragment confirmed that the phage insertion site resides within the terminal 0.8 kilobase of the insert. PMID- 2995313 TI - Development and use of cloning systems for Bacteroides fragilis: cloning of a plasmid-encoded clindamycin resistance determinant. AB - Chimeric plasmids able to replicate in Bacteroides fragilis or in B. fragilis and Escherichia coli were constructed and used as molecular cloning vectors. The 2.7 kilobase pair (kb) cryptic Bacteroides plasmid pBI143 and the E. coli cloning vector pUC19 were the two replicons used for these constructions. Selection of the plasmid vectors in B. fragilis was made possible by ligation to a restriction fragment bearing the clindamycin resistance (Ccr) determinant from a Bacteroides R plasmid, pBF4;Ccr was not expressed in E. coli. The chimeric plasmids ranged from 5.3 to 7.3 kb in size and contained at least 10 unique restriction enzyme recognition sites suitable for cloning. Transformation of B. fragilis with the chimeric plasmids was dependent upon the source of the DNA; generally 10(5) transformants micrograms-1 of DNA were recovered when plasmid purified from B. fragilis was used. When the source of DNA was E. coli, there was a 1,000-fold decrease in the number of transformants obtained. Two of the shuttle plasmids not containing the pBF4 Ccr determinant were used in an analysis of the transposon like structure encoding Ccr in the R plasmid pBI136. This gene encoding Ccr was located on a 0.85-kb EcoRI-HaeII fragment and cloned nonselectively in E. coli. Recombinants containing the gene inserted in both orientations at the unique ClaI site within the pBI143 portion of the shuttle plasmids could transform B. fragilis to clindamycin resistance. These results together with previous structural data show that the gene encoding Ccr lies directly adjacent to one of the repeated sequences of the pBI136 transposon-like structure. PMID- 2995314 TI - Regulation of the glucose phosphotransferase system in Brochothrix thermosphacta by membrane energization. AB - Uptake of 2-deoxyglucose, alpha-methylglucopyranoside, and glucose into intact cells of Brochothrix thermosphacta (formerly Microbacterium thermosphactum, ATCC 11509) was stimulated by KCN or CCCP. The glucose analogs were recovered almost totally as the sugar phosphates. Membrane vesicles were isolated from protoplasts and shown to be right side out by freeze fracturing and by using ATPase as a marker for the cytoplasmic membrane surface. Uptake of glucose into vesicles was dependent on the presence of phosphoenolpyruvate. NADH oxidation, K+ -diffusion gradients, and externally directed lactate gradients (pH greater than 7 initially) were used to generate transmembrane potentials across membrane vesicles. Above a threshold value of about -50 mV, uptake of glucose into membrane vesicles was reduced. Likewise, the maximum uptake of glucose and its two analogs into cells occurred when the protonmotive force was less than about 50 mV. PMID- 2995315 TI - Participation of cytochromes in some oxidation-reduction systems in Campylobacter fetus. AB - Campylobacter species are rich in c-type cytochromes, including forms which bind carbon monoxide. The role of the various forms of cytochromes in Campylobacter fetus has been examined in cell-free preparations by using physiological electron donor and acceptor systems. Under anaerobic conditions, NADPH reduced essentially all of the cytochrome c in crude cell extracts, whereas the reduction level with succinate was 50 to 60%. The carbon monoxide spectrum with NADPH was predominated by the cytochrome c complex; evidence of a cytochrome o type was seen in the succinate-reduced extracts and in membrane fractions. Succinate-reduced cytochrome c was oxidized by oxygen via a cyanide-sensitive, membrane-associated system. NADPH-reduced cytochrome c was oxidized by a cyanide-insensitive system. Partially purified carbon monoxide-binding cytochrome c, isolated from the cytoplasm, could serve as electron acceptor for NADPH-cytochrome c oxidoreductase; the reduced cytochrome was oxidized by oxygen by a cyanide insensitive system present in the cytoplasmic fraction. Horse heart cytochrome c was also reducible by NADPH and by succinate; the reduced cytochrome was oxidized by a cyanide-sensitive system in the membrane fraction. NADPH and NADH oxidase activities were observed aerobically and under anaerobic conditions with fumarate. NADPH was more active than NADH. NADP was also more effective than NAD as an electron acceptor for the coenzyme A-dependent pyruvate and alpha ketoglutarate dehydrogenase activities found in crude extracts. These dehydrogenases used methyl viologen and metronidazole as electron acceptors; they could be loci for oxygen inhibition of growth. It is proposed that energy provision via the high-potential cytochrome c oxidase system in the cytoplasmic membrane is limited by oxygen-sensitive primary dehydrogenases and that the carbon monoxide-binding cytochrome c may have a role as an oxygen scavenger. PMID- 2995316 TI - Cryptic plasmid and rifampin resistance in Rhizobium meliloti influencing nodulation competitiveness. AB - An assessment was made of the relative contributions of a spontaneous mutation to rifampin resistance and a cryptic plasmid, pTA2, to competitive nodulation of Medicago sativa by a strain of Rhizobium meliloti. This was facilitated by use of rifampin-resistant derivatives of this strain in which pTA2 was originally present, cured, or reintroduced. Both curing of pTA2 and spontaneous mutation to rifampin resistance significantly influenced nodulating competitiveness, but the effect of rifampin resistance was greater and such that the contribution of pTA2 was evident only in cases in which paired competitors had the common rifampin resistance background. The data suggest that rifampin-resistant derivatives contain an altered RNA polymerase insensitive to the action of rifampin. All R. meliloti derivatives had symbiotic characteristics and phage susceptibility patterns similar to those of the wild type. Plasmid pTA2 transfer or other genetic interchange was not detected in nodules of M. sativa inoculated with paired competitors. PMID- 2995317 TI - Restoration of bacterioopsin gene expression in a revertant of Halobacterium halobium. AB - Restoration of bacterioopsin (bop) gene expression in a revertant of Halobacterium halobium was investigated. The phenotype of the revertant is the result of a translocation of the 588-base-pair (bp) sequence "ISH25", adjacent to an ISH24 insertion found in the parental mutant IV-4. These insertions are located about 1,400 bp upstream of the bop gene within the coding region of the putative brp (bacterioopsin-related protein) gene. The level at which the brp gene affects bop gene expression is unknown. Analysis of bop and brp gene transcription in the wild type, mutant IV-4, and the revertant supports the hypothesis that transcription of the putative brp gene is necessary for bop gene expression in the revertant. Eight insertion mutants of the Bop revertant were analyzed to further elucidate restoration of bop gene expression in the revertant. Bop mutants of the revertant were recovered with a frequency of about 10(-4) and were found to contain insertion elements in addition to ISH24 and "ISH25". Six-eighths of these mutants have the insertion element ISH2, and two mutants have previously uncharacterized insertion elements (ISH27 [1,400 bp] and ISH28 [1,000 bp]). ISH27 and ISH28 are confined to the more A + T-rich fraction of the H. halobium genome, as are most copies of other halobacterial insertion elements. The insertion sites in the Bop mutants of the revertant mapped within the coding region of the bop gene (three mutants), immediately upstream of the bop gene presumably in the bop promoter region (two mutants), or within a region from 241 to 449 bp upstream of the bop gene (three mutants). This distribution of insertion sites suggests that the integrity of the 526-bp region between the bop and the brp genes is important for bop gene expression in the revertant. PMID- 2995318 TI - Identification of osmoresponsive genes in Escherichia coli: evidence for participation of potassium and proline transport systems in osmoregulation. AB - Mu d1(Ap lac)-generated operon fusions were used in the identification of genes in Escherichia coli whose transcriptional expression is altered by changes in the osmolarity of the growth medium. One such osmoresponsive gene, designated osrA, was induced 400-fold when the osmolarity of the medium was increased with the addition of either ionic or neutral impermeable solutes but was not induced with glycerol, which is freely permeable across the cell membrane. osrA was mapped to 57.5 min and was shown to be transcribed clockwise on the E. coli chromosome. The ability of small concentrations of L-proline to promote the growth of E. coli in high-osmolar medium was shown to have been specifically lost in osrA mutants; other lines of evidence were also obtained to support the notion that osrA codes for an osmoresponsive L-proline transport system and is homologus to proU in Salmonella typhimurium. A second osmoresponsive operon identified was kdp, which codes for an inducible K+-transport system in E. coli. kdp expression was elevated 12-fold when the osmolarity of the growth medium was increased with the addition of impermeable ionic solutes but not neutral solutes; furthermore, osmoresponsivity of kdp expression was demonstrable only in K+-limiting media. kdp mutants were able to grow normally in high-osmolar media, but strains defective in both kdp and trkA (a gene for a second major K+-transport system) displayed an osmosensitive phenotype. The results suggest that transport systems for L-proline and K+, specified by osrA (proU) and kdp, respectively, play independent and important roles in osmoregulation in E. coli. A third osmoresponsive gene that was identified was lamB, which codes for an outer membrane protein for maltodextrin transport and lambda phage adsorption; its expression was reduced fourfold with increase in the osmolarity of the growth medium. PMID- 2995319 TI - Control of Vibrio fischeri luminescence gene expression in Escherichia coli by cyclic AMP and cyclic AMP receptor protein. AB - Under certain conditions glucose represses the autoinducible synthesis of luminescence enzymes in Vibrio fischeri. To examine the genetic regulation of luminescence more closely, Escherichia coli catabolite repression mutants were transformed with a plasmid (pJE202) that contains V. fischeri genes specifying the luminescence enzymes and encoding regulatory functions for luminescence (the lux genes) or with plasmids (pJE413 and pJE455) containing transcriptional fusions between the lacZ gene on transposon mini-Mu and specific genes in each of the two lux operons. Unless cyclic AMP (cAMP) was added to the growth medium, an adenylate cyclase deletion mutant containing pJE202 produced very little light and low levels of the light-emitting enzyme luciferase. When grown in the presence or absence of cAMP, a cAMP receptor protein (CRP) deletion mutant produced low levels of light and luciferase. A mutant that does not make cAMP but does make an altered CRP which does not require cAMP for activity produced induced levels of luminescence after transformation with pJE202. To test the effects of cAMP and CRP on each of the two lux operons separately rather than on both together, the E. coli catabolite repression mutants were transformed with pJE413 and pJE455. From measurements of beta-galactosidase and luciferase activities it appeared that cAMP and CRP affected transcription of both lux operons. In the presence of autoinducer and its receptor, transcription of the operon encoding all of the luminescence genes except the receptor gene appeared to be activated by cAMP and CRP, whereas in the absence of the receptor, cAMP and CRP appeared to decrease transcription of this operon. Transcription of the operon encoding the autoinducer receptor appeared to be stimulated by cAMP and CRP in the absence of the receptor itself. These results demonstrate that cAMP and CRP are required for proper control of the V. fischeri luminescence system and suggest that lux gene transcription is required by a complex mechanism. PMID- 2995321 TI - Regulation of cyclic AMP synthesis by enzyme IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system in crp strains of Salmonella typhimurium. AB - We investigated the claim (J. Daniel, J. Bacteriol. 157:940-941, 1984) that nonphosphorylated enzyme IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system is required for full synthesis of bacterial cyclic AMP (cAMP). In crp strains of Salmonella typhimurium, cAMP synthesis by intact cells was regulated by the phosphorylation state of enzyme IIIGlc. Introduction of either a pstHI deletion mutation or a crr::Tn10 mutation resulted in a low level of cAMP synthesis. In contrast, crp strains containing a leaky pstI mutation exhibited a high level of cAMP synthesis which was inhibited by phosphotransferase system carbohydrates. From these results, we conclude that phosphorylated enzyme IIIGlc rather than nonphosphorylated enzyme IIIGlc is required for full cAMP synthesis. PMID- 2995320 TI - Effects of a mutation that eliminates UDP glucose-pyrophosphorylase on the pathogenicity of Erwinia carotovora subsp. carotovora. AB - A nonpathogenic mutant of Erwinia carotovora obtained by Mu d1 mutagenesis was defective in the ability to utilize several carbon sources. The basis of the mutation was analyzed biochemically and shown to be a defect in the ability to form UDP glucose-pyrophosphorylase. The nonpathogenic phenotype of the mutant was caused by its sensitivity to galactose. PMID- 2995322 TI - Cell length in a wee dnaA mutant of Escherichia coli. AB - The cell length of the short siblings of dividing pairs formed in the absence of replication by two strains of Escherichia coli, OV-25-9 [dnaA46 wee(Am)] and OV 25-10 [dnaA46 wee(AM) supF] was measured. In the presence of Wee, the length of these cells increased to those values expected for newborn wild-type cells growing under similar conditions. In its absence, cell length remained at values near the minimum unit length possible for newborn cells. Our results show that both cell elongation and the action of Wee are independent of DNA replication, being compatible with the role proposed for Wee in coordination between cell elongation and division. PMID- 2995323 TI - Evidence that adenine methylation influences DNA-protein interactions in Escherichia coli. PMID- 2995324 TI - Marker-exchange mutagenesis of a pectate lyase isozyme gene in Erwinia chrysanthemi. AB - The phytopathogenic enterobacterium Erwinia chrysanthemi contains pel genes encoding several different isozymes of the plant-tissue-disintegrating enzyme pectate lyase (PL). The pelC gene, encoding an isozyme with an approximate isoelectric point of 8.0, was mutagenized by a three-step procedure involving (i) insertional inactivation of the cloned gene by ligation of a kan-containing BamHI fragment from pUC4K with a partial Sau3A digest of E. chrysanthemi pelC DNA in pBR322; (ii) mobilization of the pBR322 derivative from Escherichia coli to E. chrysanthemi by the helper plasmids R64drd11 and pLVC9; and (iii) exchange recombination of the pelC::kan mutation into the E. chrysanthemi chromosome by selection for kanamycin resistance in transconjugants cultured in phosphate limited medium (which renders pBR322 unstable). The resulting E. chrysanthemi mutant was Kanr Amps, lacked pBR322 sequences, and was deficient in only one of the four major PL isozymes, PLc, as determined by activity-stained isoelectric focusing polyacrylamide gels. The rates of PL induction and cell growth in a medium containing polygalacturonic acid as the sole carbon source were not significantly reduced in the mutant. No difference was detected in the ability of the mutant to macerate potato tuber tissue. The evidence suggests that this isozyme is not necessary for soft-rot pathogenesis. PMID- 2995325 TI - Molecular cloning of the Escherichia coli gene for diadenosine 5',5'''-P1,P4 tetraphosphate pyrophosphohydrolase. AB - A clone overproducing diadenosine tetraphosphatase (diadenosine 5', 5'''-P1, P4 tetraphosphate pyrophosphohydrolase) activity was isolated from an Escherichia coli cosmid library. Localization of the DNA region responsible for stimulation of this activity was achieved by deletion mapping and subcloning in various vectors. Maxicell experiments and immunological assays demonstrated that a 3.5 kilobase-pair DNA fragment carried the structural gene apaH encoding the E. coli diadenosine tetraphosphatase. The DNA coding strand was determined by cloning this fragment in both orientations in pUC plasmids. It was also shown that the overproduction of diadenosine tetraphosphatase decreased the dinucleoside tetraphosphate concentration in E. coli by a factor of 10. PMID- 2995326 TI - Self-cloning in Streptomyces griseus of an str gene cluster for streptomycin biosynthesis and streptomycin resistance. AB - An str gene cluster containing at least four genes (strR, strA, strB, and strC) involved in streptomycin biosynthesis or streptomycin resistance or both was self cloned in Streptomyces griseus by using plasmid pOA154. The strA gene was verified to encode streptomycin 6-phosphotransferase, a streptomycin resistance factor in S. griseus, by examining the gene product expressed in Escherichia coli. The other three genes were determined by complementation tests with streptomycin-nonproducing mutants whose biochemical lesions were clearly identified. strR complemented streptomycin-sensitive mutant SM196 which exhibited impaired activity of both streptomycin 6-phosphotransferase and amidinotransferase (one of the streptomycin biosynthetic enzymes) due to a regulatory mutation; strB complemented strain SD141, which was specifically deficient in amidinotransferase; and strC complemented strain SD245, which was deficient in linkage between streptidine 6-phosphate and dihydrostreptose. By deletion analysis of plasmids with appropriate restriction endonucleases, the order of the four genes was determined to be strR-strA-strB-strC. Transformation of S. griseus with plasmids carrying both strR and strB genes enhanced amidinotransferase activity in the transformed cells. Based on the gene dosage effect and the biological characteristics of the mutants complemented by strR and strB, it was concluded that strB encodes amidinotransferase and strR encodes a positive effector required for the full expression of strA and strB genes. Furthermore, it was found that amplification of a specific 0.7-kilobase region of the cloned DNA on a plasmid inhibited streptomycin biosynthesis of the transformants. This DNA region might contain a regulatory apparatus that participates in the control of streptomycin biosynthesis. PMID- 2995327 TI - The new generation antidepressants: promising innovations or disappointments? AB - "New generation" antidepressants are generally considered to include all agents introduced in recent years which are neither tricyclic in structure nor monoamine oxidase inhibitors. They include the tetracyclics (mianserin and maprotiline), the bicyclic serotonergic compounds (fluoxetine and citalopram), and the unicyclics (bupropion), as well as the triazolobenzodiazepine derivatives (alprazolam), the triazolopyridines (trazodone) and the tetrahydroisoquinolines (nomifensine). Methodologic and economic considerations have hampered attempts to develop agents with significantly greater specificity or safety than traditional agents. Traditional agents, while lacking specificity, do have extensive records of efficacy and long-term safety and are usually less expensive than new agents. Patients not responding to traditional agents often have medical or characterologic problems that exclude them from participating in controlled studies of new agents. These problems are discussed and potential approaches to the development of new agents are presented. PMID- 2995328 TI - A cruciform in the direct repeats of the yeast 2 micron DNA: Selective S1 nuclease cleavage at one of the three homologous palindromes. AB - An S1-hypersensitive site was found at the 60 bp direct repeats of the cis acting, stability and/or copy number control region of the yeast 2 micron DNA in the supercoiled hybrid plasmid pDB248'. It was retained in a different plasmid, pYK2121, consisting of pBR322 and the 300 bp long repeated DNA. Analyses of 5' end-labeled fragments and nucleotide sequence determination showed that the S1 cleavage site was at the central part of an AT-rich 19 bp palindrome present in the repeats. Two other homologous palindromes (21 and 15 bp) containing the 12 bp consensus sequences were not cleaved. The nucleotide sequences at the base of the stem and/or loop may determine the efficiency of the cruciform extrusion. PMID- 2995329 TI - Association of Fe-S center(s) with the large subunit(s) of photosystem I particles. AB - Treatment of photosystem I particles from spinach (Spinacia oleracea) with dodecyl sulfate destroyed the protein-bound Fe-S centers and converted some of the acid-labile sulfide to zero-valence sulfur which remained covalently bound to the proteins. When the proteins were resolved by gel-permeation chromatography or by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate, a considerable amount of zero-valence sulfur was associated with the large molecular weight polypeptide(s) (63,000 and 59,000). The results strongly suggest that an intact two-peptide P700 chlorophyll a-protein is an Fe-S protein. PMID- 2995330 TI - Structure of ferricytochrome c' from Rhodospirillum rubrum at 6 A resolution. AB - The structure of a ferricytochrome c' extracted from Rhodospirillum rubrum has been determined at 6 A resolution by the X-ray crystallographic method. The crystals, obtained by dialyzing the protein solution against polyethylene glycol 4000, belong to the hexagonal space group P6(1). Two heavy atom derivatives were obtained by soaking the native crystals in K2PtCl6 and CH3HgCl solution. The phases calculated by the multiple isomorphous replacement method gave an overall figure of merit of 0.90 at 6 A resolution. The resulting electron density map showed the molecular boundary clearly, and gave molecular dimensions of 50 X 25 X 30 A for a monomer molecule. From visual examination of this map, the cytochrome c' from Rhodospirillum rubrum has a similar chain-folding pattern to the cytochrome c' from Rhodospirillum molischianum, the structure determination of which has already been carried out. PMID- 2995331 TI - Reconstitution of a bacterial Na+/H+ antiporter. AB - Membrane proteins from alkalophilic Bacillus firmus RAB were extracted with octylglucoside, reconstituted into liposomes made from alkalophile lipids. The proteoliposomes were loaded with 22Na+. Imposition of a valinomycin-mediated potassium diffusion potential, positive out, resulted in very rapid efflux of radioactive Na+ against its electrochemical gradient. That the Na+ efflux was mediated by the electrogenic Na+/H+ antiporter is indicated by the following characteristics that had been established for the porter in previous studies: dependence upon an electrical potential; pH sensitivity, with activity dependent upon an alkaline pH; inhibition by Li+; and an apparent concentration dependence upon Na+ that correlated well with measurements in cells and membrane vesicles. PMID- 2995332 TI - Characterization of digitalis-like factors in human plasma. Interactions with NaK ATPase and cross-reactivity with cardiac glycoside-specific antibodies. AB - Much of the evidence for a physiologically important endogenous inhibitor of the sodium pump has been either contradictory or indirect. We have identified three discrete fractions in desalted deproteinized plasma from normal humans that resemble the digitalis glycosides in that they: are of low molecular weight; are resistant to acid and enzymatic proteolysis; inhibit NaK-ATPase activity; inhibit Na+ pump activity in human erythrocytes; displace [3H]ouabain bound to the enzyme; and cross-react with high-affinity polyclonal and monoclonal digoxin specific antibodies but not with anti-ouabain or anti-digitoxin antibodies. An additional fraction cross-reacted with digoxin-specific antibodies but had no detectable activity against NaK-ATPase. The three inhibitory fractions differed from cardiac glycosides in that their concentration-effect curves in a NaK-ATPase inhibition and [3H]ouabain radioreceptor assays were steeper than unlabeled ouabain. This suggests that these inhibitors are not simple competitive ligands for binding to NaK-ATPase. In the presence of sodium, no fraction required ATP for binding to NaK-ATPase, and in the presence of potassium, only one fraction had the reduced affinity for the enzyme that is characteristic of cardiac glycosides. Unlike digitalis, all three NaK-ATPase inhibitory fractions stimulated the activity of skeletal muscle sarcoplasmic reticulum Ca-ATPase. The presence of at least three fractions in human plasma that inhibit NaK-ATPase and cross-react to a variable degree with different digoxin-specific antibody populations could explain much of the conflicting evidence for the existence of endogenous digitalis-like compounds in plasma. PMID- 2995333 TI - Monoclonal antibodies against rabbit mammary prolactin receptors. Specific antibodies to the hormone binding domain. AB - Three monoclonal antibodies (M110, A82, and A917) were obtained by fusing myeloma cells and spleen cells from mice immunized with partially purified rabbit mammary gland prolactin (PRL) receptors. All 3 antibodies were capable of complete inhibition of 125I-ovine prolactin (oPRL) binding to rabbit mammary PRL receptors in either particulate or soluble form. M110 showed slightly greater potency than oPRL in competing for 125I-oPRL binding. These antibodies also inhibited PRL binding to microsomal fractions from rabbit liver, kidney, adrenal, ovary, and pig mammary gland, although A82 showed poor inhibition in pig mammary gland. There was no cross-reaction of any of the 3 monoclonal antibodies (mAbs) for the other species tested: human (T-47D breast cancer cells) and rat (liver, ovary). In order to confirm that these antibodies are specific to the binding domain, antibodies were purified, iodinated, and binding characteristics were investigated. 125I-M110 and 125I-A82 binding was completely inhibited by lactogenic hormones, whereas nonlactogenic hormones did not cross-react. Competition of 125I-M110 by oPRL (ID50 = 0.44 nM) was comparable to that of 125I oPRL by unlabeled oPRL (ID50 = 0.35 nM), while 125I-A917 binding was only partially competed (30-60%) by lactogenic hormones. Tissue and species specificity of labeled antibody binding paralleled results of binding inhibition experiments using 125I-oPRL. In addition, A82 and A917 completely inhibited 125I M110 binding. In contrast, 125I-A82 binding was stimulated by A917 and 125I-A917 binding was stimulated by A82. These findings indicate that monoclonal antibodies can be readily prepared from partially purified PRL receptors from rabbit mammary gland; two antibodies (M110 and A82) are hormone binding site specific while the other (A917) binds a domain partially but not entirely distinct from the hormone binding site, and that all three antibodies have strong species specificity. PMID- 2995335 TI - Formation of 4-aminophenoxyl free radical from the acetaminophen metabolite N acetyl-p-benzoquinone imine. AB - N-Acetyl-p-benzoquinone imine, a hepatic metabolite of acetaminophen, and its analogue, N-acetyl-3,5-dimethyl-p-benzoquinone imine, were metabolized by rat liver microsomes and NADPH to their corresponding 4-aminophenoxyl free radicals. ESR spectra were recorded and unambiguously identified. As indicated by the purple color and confirmed by UV and mass spectroscopy, indophenols were formed as final products. The 4-aminophenoxyl free radical formation could be suppressed by the deacetylase inhibitors, sodium fluoride and paraoxon. Microsomal incubations of N-acetyl-2,6-dimethyl-p-benzoquinone imine and NADPH do not result in a detectable radical concentration; in addition, no indophenol was found. Substitution of NADPH-cytochrome P-450 reductase for rat liver microsomes eliminates the deacetylase activity and results in direct reduction of N-acetyl 3,5-dimethyl-p-benzoquinone imine to the N-acetyl-2,6-dimethyl-4-aminophenoxyl free radical. Neither the incubation of N-acetyl-p-benzoquinone imine nor that of N-acetyl-2,6-dimethyl-p-benzoquinone imine with NADPH-cytochrome P-450 reductase yielded a detectable concentration of the corresponding phenoxyl free radical. When starting material that had been exposed to the atmosphere was used, a previously reported free radical with a splitting constant of approximately 2 G was formed. This spectrum is identical with that of the 2,6-dimethyl-p benzosemiquinone free radical, implying hydrolysis of the starting material. Neither the N-acetyl-4-aminophenoxyl nor the N-acetyl-2,6-dimethyl-4 aminophenoxyl radical reduces oxygen to form superoxide or react with oxygen in any other detectable way. PMID- 2995334 TI - Biological activities of binding site specific monoclonal antibodies to prolactin receptors of rabbit mammary gland. AB - The biological activity of three monoclonal antibodies (mAbs) against the rabbit mammary prolactin (PRL) receptor (M110, A82, and A917) were investigated using explants of rabbit mammary gland. The three mAbs which were all able to inhibit the binding of 125I-ovine prolactin to its receptor had different biological activities. Two mAbs (M110 and A82) were able to prevent the stimulating effect of PRL on casein synthesis when the molar ratio between the mAb and PRL was 100. At a lower concentration, M110 moved the PRL dose-response curve to the right by a factor of 2.4. This mAb was also effective in vivo, reducing milk production in a lactating rabbit, in a similar fashion to the prolactin lowering drug, CB-154. One mAb (A917) was able to mimic the action of PRL on both casein and DNA ([3H]thymidine incorporation) synthesis, whereas the other two mAbs were without any stimulatory effect. For this stimulatory effect to be observed, bivalency of the antibody was essential, since monovalent fragments, which were able to inhibit PRL binding, had no agonistic activity. The ability of the mAbs to induce a down-regulation of receptors was also studied. M110, which was equipotent to PRL in occupation of receptors, induced no down-regulation, while A917, which had full biological activity, induced only a small degree of down-regulation. These studies suggest that the binding domain of the receptor might be relatively complex, since only a part of this domain recognized by the antibody with PRL like activity was able to induce hormonal action. Alternatively, only those antibodies able to microaggregate the receptors may possess PRL-like activity. PMID- 2995336 TI - Specific labeling and partial inactivation of cytochrome oxidase by fluorescein mercuric acetate. AB - Addition of 1 eq of fluorescein mercuric acetate (FMA) to beef heart cytochrome oxidase was found to inhibit the steady-state electron transfer activity by 50%, but further additions up to 10 eq had no additional effect on activity. The partial inhibition caused by FMA is thus similar to that observed with other mercury compounds (Mann, A. J., and Auer, H. E. (1980) J. Biol. Chem. 255, 454 458). The fluorescence of FMA was quenched by a factor of 10 upon binding to cytochrome oxidase, consistent with the involvement of a sulfhydryl group. However, addition of mercuric chloride to FMA-cytochrome oxidase resulted in an increase in fluorescence, suggesting that FMA was displaced from the high affinity binding site. Cytochrome c binding to FMA-cytochrome oxidase resulted in a 10% decrease in the fluorescence, possibly caused by Forster energy transfer from FMA to the cytochrome c heme. The binding site for FMA in cytochrome oxidase was investigated by carrying out sodium dodecyl sulfate gel electrophoresis under progressively milder dissociation conditions. When FMA-cytochrome oxidase was dissociated with 3% sodium dodecyl sulfate and 6 M urea, FMA was predominantly bound to subunit II following electrophoresis. However, when the dissociation was carried out at 4 degrees C in the absence of urea with progressively smaller amounts of lithium dodecyl sulfate, the labeling of subunit II decreased and that of subunit I increased. These experiments demonstrate that mercury compounds bind to a high affinity site on cytochrome oxidase, possibly located in subunit I, but then migrate to subunit II under the normal sodium dodecyl sulfate gel electrophoresis conditions. A definitive assignment of the high affinity binding site in the native enzyme cannot be made, however, because it is possible that mercury compounds can migrate from one sulfhydryl to another under even the mildest electrophoresis conditions. PMID- 2995337 TI - Signal transduction and ligand-receptor dynamics in the human neutrophil. Transient responses and occupancy-response relations at the formyl peptide receptor. AB - The responses of neutrophils to formyl peptides are initiated and in many cases achieve a maximal level prior to equilibrium receptor occupancy. In order to begin to understand the linkage between receptor occupancy and cell response we have used a pulsed binding procedure to analyze: 1) the number of receptors contributing to three potential signalling events and six functional responses and 2) the evolution of these responses once ligand binding is interrupted. We find that the half-optimal elevations of the potential signals are produced by less than 1% occupancy (Ca2+) or 1-3% occupancy (cAMP, membrane depolarization). In contrast, actin polymerization and a rapid light scatter response are elicited by less than 0.1% occupancy. Half-optimal elastase release and degranulation require approximately 3% occupancy. While half-optimal O2- production and aggregation require approximately 30% occupancy, the half-optimal rate of O2- production requires less than 10% occupancy. To resolve the apparent lack of correlation between the responses and the signals we examined their time courses following the pulse of stimulation. At least four responses and one signal are transient and decay while occupied receptors remain on the membrane surface. These include the Quin 2-Ca2+ signal, actin polymerization, the light scatter response, O2- generation, and aggregation. Ca2+ elevation is correlated with the responses in that: 1) each of these responses is transient unless new receptors are occupied; 2) occupancy of nearly all of the receptors contributes to the time course of these responses; 3) when binding is interrupted, the responses decay with a half-time of 15 s, following a latency of approximately 10 s or less (except for disaggregation where latency is 30-40 s). We discuss evidence in support of the hypothesis that transient cell responses arise from transient receptor activation. PMID- 2995338 TI - Mechanism of inhibition of transducin GTPase activity by fluoride and aluminum. AB - Transducin, the guanyl nucleotide-binding protein of the retinal light-activated cGMP phosphodiesterase system, is structurally and functionally similar to the inhibitory and stimulatory guanyl nucleotide-binding proteins, Gi and Gs, of the adenylate cyclase complex. All are heterotrimers composed of alpha, beta, and gamma subunits. Gs and Gi can be activated by NaF with AlCl3 as well as by agonists acting through specific receptors. The effects of NaF and AlCl3 on transducin were investigated in a reconstituted system consisting of the purified subunits of transducin (T alpha, T beta, gamma) and rhodopsin. NaF noncompetitively inhibited the GTPase activity of T alpha in a concentration- and time-dependent manner. Inhibition by NaF was enhanced synergistically by AlCl3 which alone only slightly inhibited GTPase activity. None of the other anions tested reproduced the effect of fluoride. Fluoride inhibited [3H]guanosine 5' (beta, gamma-imido)triphosphate binding to T alpha and release of bound GDP. The ADP-ribosylation of T alpha by pertussis toxin and binding of T alpha to rhodopsin, both of which are enhanced in the presence of T beta gamma, were inhibited by NaF and AlCl3. These findings are consistent with the hypothesis that fluoride enhances the dissociation of T alpha from T beta gamma, resulting in the inhibition of GTP-GDP exchange, and therefore, GTP hydrolysis. PMID- 2995340 TI - Prostaglandin A1 metabolism and inhibition of cyclic AMP extrusion by avian erythrocytes. AB - Prostaglandins (PG) inhibit active cyclic AMP export from pigeon red cells, PGA1 and PGA2 most potently (Brunton, L.L., and Mayer, S.E. (1979) J. Biol. Chem. 254, 9714-9720). To probe the mechanism of this action of PGA1, we have studied the interaction of [3H]PGA1 with suspensions of pigeon red cells. The interaction of PGA1 with pigeon red cells is a multistep process of uptake, metabolism, and secretion. [3H] PGA1 rapidly enters red cells and is promptly metabolized (Vmax greater than or equal to 1 nmol/min/10(7) cells) to a compound(s) that remains in the aqueous layer after ethylacetate extraction. The glutathione-depleting agent, diamide, inhibits formation of the PGA1 metabolite. In agreement with the order of potency of other prostaglandins to inhibit cAMP efflux (A much greater than E congruent to B greater than F), PGA2 forms a polar adduct whereas prostaglandins E2, B1, and F2 alpha do not. The red cells secrete the polar metabolite of PGA1 by a saturable mechanism (at 37 degrees C, Km congruent to 0.6 microM, Vmax congruent to 0.5 pmol/min/10(7) cells) that lowered temperatures inhibit (Eact congruent to 21 kcal/mol). Because uptake and metabolism progress with much greater rates than metabolite secretion, red cells transiently concentrate the polar compound intracellularly. Onset and reversal of inhibition of cyclic AMP export by PGA1 coincide with accumulation and secretion of PGA1 metabolite, suggesting that the polar metabolite acts at an intracellular site to inhibit cyclic AMP efflux. In the accompanying Appendix, we present chromatographic and amino acid analyses demonstrating that the polar metabolite is a glutathione adduct of PGA1. PMID- 2995339 TI - Enzymatic properties of separated isozymes of the Na,K-ATPase. Substrate affinities, kinetic cooperativity, and ion transport stoichiometry. AB - There are two isozymes of the Na,K-ATPase, which can be purified separately from rat renal medulla and brainstem axolemma. Here the basic kinetic properties of the two Na,K-ATPases have been compared in conditions permitting enzyme turnover. The two isozymes are half-maximally activated at different concentrations of ATP, the axolemma Na,K-ATPase having the higher affinity. They are half-maximally activated by Na+ and K+ at very similar concentrations but show differences in cooperativity toward Na+. The affinities of both isozymes for ATP and Na+ are affected in a qualitatively similar way by variations in the concentration of K+. Both isozymes transport 22Na+ and 42K+ in a ratio close to 3:2 in artificial lipid vesicles. The two isozymes differ most strikingly in the inhibition of ATPase activity by ouabain. The axolemma Na,K-ATPase has a high affinity for ouabain with positive cooperativity, while the renal medulla Na,K-ATPase has a lower affinity with negative cooperativity. It is likely that the cooperativity differences are due to kinetic effects, reflecting different rates of conformation transitions during enzyme turnover. The functional result of the contrasting cooperativities is that the difference in sensitivity to ouabain is amplified. PMID- 2995342 TI - Characterization of an electrogenic ATP and chloride-dependent proton translocating pump from rat renal medulla. AB - To study acidification mechanisms in the distal nephron, microsomes were prepared from rat renal medulla by differential centrifugation. Microsomes were enriched in the enzyme marker gamma-glutamyl transferase and contained an ATP-dependent proton pump, as evidenced by ATP-dependent, 3,3',4',5-tetrachlorosalicylanilide reversible quenching of acridine orange fluorescence. Acidification was vanadate insensitive, but was completely inhibited by micromolar N-ethylmaleimide. Maximal acidification was achieved in the presence of halide (Cl-, Br-) only and was not attainable with potassium-valinomycin diffusion potentials without halide ion. Microsomal ATPase activity was neither chloride- nor N-ethylmaleimide-sensitive. A chloride conductance was observed only with vesicles which had undergone ATP dependent acidification. An ATP-dependent, N-ethylmaleimide-inhibitable, 3,3',4',5-tetrachlorosalicylanilide-reversible, and chloride-attenuated quench of bis(1,3-dibutylbarbituric acid-(5] pentamethinoxonol fluorescence was seen, consistent with net transfer of positive charge into the vesicles. Nonetheless, positive intravesicular potentials increased the ATP-dependent initial acidification rate, perhaps by increasing availability of chloride ion to the transport site. Our results are consistent with an electrogenic, ATP-dependent proton pump regulated by a voltage-sensitive chloride site. PMID- 2995341 TI - Putative inhibitor of cyclic AMP efflux: chromatography, amino acid composition, and identification as a prostaglandin A1-glutathione adduct. AB - Avian red cells rapidly and extensively metabolize prostaglandin A1 (PGA1) to a polar form that extracts into 80% ethanol and is quantitatively desalted and recovered over a Waters C18 Sep-Pak. This material co-chromatographs with synthetic PGA1-GSH and with the human red cell PGA1 metabolite on cellulose and silica gel thin layer plates and on normal phase high performance liquid chromatography. Quantitation of the amino acid composition by [35S]cysteine incorporation and by fluorometric amino acid analysis of the material eluting from high performance liquid chromatography indicates the presence of PGA1, cysteine, glutamate, and glycine in equimolar ratios. Base sensitivity of the metabolite indicates that it exists largely (80%) in the 9-hydroxyl form. We conclude that the polar metabolite of PGA1 that acts intracellularly to inhibit cyclic AMP efflux by avian red cells is a glutathione conjugate of PGA1. PMID- 2995343 TI - Synergistic stimulation of the Ca2+ influx in rat hepatocytes by glucagon and the Ca2+-linked hormones vasopressin and angiotensin II. AB - Glucagon was added to isolated rat hepatocytes, either alone or together with vasopressin or angiotensin II, and the effects on the initial 45Ca2+ uptake rate were investigated. Addition of glucagon alone which increased cyclic AMP content of the cells slightly increased the initial 45Ca2+ uptake rate. When glucagon was added together with vasopressin or angiotensin II--both of which when added separately increase the initial 45Ca2+ uptake rate but did not affect the cellular content of cyclic AMP--the measured initial 45Ca2+ uptake rate was larger than the sum of that seen with each hormone alone. This indicates that glucagon and Ca2+-linked hormones synergistically enhanced the Ca2+ influx in rat hepatocytes. These effects of glucagon can be mimicked by dibutyryl cyclic AMP or forskolin, suggesting that cyclic AMP augments both the resting Ca2+ and the vasopressin- or angiotensin II-stimulated influx. Measurement of the initial 45Ca2+ uptake rate as a function of the extracellular Ca2+ concentration indicated that the increase in the Ca2+ influx resulting from single or combined glucagon and vasopressin administration occurred through a homogeneous population of Ca2+ gates. These hormones were found to raise both the apparent Km for external Ca2+ and the apparent Vmax of the Ca2+ influx. The maximal increase in these two parameters was observed when the two hormones were added together. This suggests that glucagon and vasopressin synergistically stimulate the same Ca2+ gating mechanism. The dose-response curves for the action of glucagon or vasopressin applied in the presence of increasing concentrations of vasopressin or glucagon, respectively, showed that each hormone increases the maximal response to the other without affecting its ED50. It is proposed that glucagon and the Ca2+-linked hormones control the cellular concentration of two intermediates which are both necessary to allow Ca2+ entry into the cells. PMID- 2995344 TI - Resonance Raman spectra of the pyridoxal coenzyme in aspartate aminotransferase. Evidence for pyridine protonation and a novel photochemical H/D exchange at the imine carbon atom. AB - Resonance Raman (RR) spectra are reported for aspartate aminotransferase from pig heart cytosol, and for inhibitor complexes. They are interpreted with reference to the previously analyzed spectra of pyridoxal phosphate (PLP) Schiff base adducts. This comparison shows that, as expected, the pyridine N atom is protonated in the native enzyme at pH 5, and in the glutarate complexes at pH 8.5, and that it is also protonated in the alpha-methylaspartate complex; the stabilization of the pyridine proton at high pH must be due to the interaction with aspartate 222 seen in the x-ray crystal structure. RR spectra of the erythro beta-hydroxy-DL-aspartate complex, representing the p-quinoid enzyme intermediate, as well as of AlIII complexes of PLP Schiff bases with phenylalanine and tyrosine ethyl ester have been obtained via the coherent anti Stokes Raman scattering technique, and partially assigned. A novel H/D exchange at the coenzyme C4' atom has been observed for the native enzyme in D2O, and has been determined, by a combination of NMR and RR measurements, to be due to the Raman laser irradiation. This photoprocess, which is not observed for PLP Schiff bases in aqueous solution, is attributed to a photoexcited p-quinoid intermediate, similar to that implicated in the enzyme mechanism. It is suggested that this intermediate is stabilized by protein interactions which localize charge on the phenolate O atom, plausibly a hydrogen bond from the nearby tyrosine 225. H/D exchange would then follow via the aldimine-ketimine interconversion known to take place in the enzyme reaction. PMID- 2995345 TI - Spectroscopic, ligand binding, and enzymatic properties of the spleen green hemeprotein. A comparison with myeloperoxidase. AB - The bovine spleen green hemeprotein, a peroxidase which exhibits spectrophotometric properties similar to those of granulocyte myeloperoxidase, was purified using an improved method. The ligand affinity of the ferric enzyme was spectroscopically determined using chloride and cyanide as exogenous ligands. The pH dependence of the apparent dissociation constant of the enzyme-chloride complex showed the presence of a proton dissociable group with a pKa value of 4 on the enzyme; chloride binds to the enzyme when this group is protonated with a dissociation constant of 60 microM. The cyanide affinity of the enzyme is also regulated by the group with a pKa value of 4, but in this case cyanide binds to the unprotonated enzyme with a dissociation constant of 0.6 microM; only the protonated, uncharged form of cyanide reacts with the enzyme. Cyanide binding was competitively inhibited by chloride, and chloride binding was also competitively inhibited by cyanide. The EPR spectrum of the resting enzyme exhibited a rhombic high spin signal at g = 6.65, 5.28, and 1.97 with a low spin signal at g = 2.55, 2.32, and 1.82. Upon formation of the chloride complex, the spectrum was replaced with a new high spin EPR signal with g-values of 6.81, 5.04, and 1.95. The cyanide complex showed a low spin EPR signal with g-values of 2.83, 2.25, and 1.66. Examination of the enzymatic activity of the spleen green hemeprotein by following the chlorination of monochlorodimedon has indicated that the enzyme has the same chlorinating activity as myeloperoxidase; the spleen green peroxidase can catalyze the formation of hypochlorous acid from hydrogen peroxide and chloride ion. Comparison of the present data with those of myeloperoxidase has led to the conclusion that the structure of the iron center and its vicinity in spleen green hemeprotein is very similar, if not identical, to that of myeloperoxidase. The spleen enzyme can thus be used as a model to study the active center, and its environment, in myeloperoxidase. PMID- 2995346 TI - Purification and characterization of the aspartate chemoreceptor. AB - The chemoreceptor for aspartate in Salmonella typhimurium was purified from an Escherichia coli strain containing a plasmid bearing the receptor's structural gene (tar). The receptor was solubilized from salt-washed membranes with the nonionic detergent octyl-beta-D-glucopyranoside and purified by a combination of ion exchange, molecular sieve and hydroxyapatite-agarose chromatography. The inclusion of glycerol and 1,10-phenanthroline in all buffers used prior to ion exchange chromatography prevented scission of the receptor by an endogenous proteolytic activity. The solubilized receptor was estimated to have a molecular weight of 248,000 from its behavior on Sephacryl S-300, suggesting that the receptor may be organized as a multimer containing 4 +/- 1 identical subunits. Circular dichroic measurements of the purified protein indicate that 78% of its residues are arranged in helical secondary structures. PMID- 2995347 TI - Proteolytic fragments identified with domains of the aspartate chemoreceptor. AB - Two proteolytic fragments generated during the preparation of the aspartate receptor from Salmonella typhimurium have been purified. These fragments are the products of a single cleavage by an endogenous protease after amino acid 259 in the sequence of the intact receptor. Proteolytic fragment 1 (PF1) represents amino acids 1-259 (Mr = 29,000); this unit retains the aspartate-binding function of the intact receptor. The second fragment (PF2) includes residues 260-552 (Mr = 31,000) and has the normal sites of reversible methylation for the receptor. Like the purified intact receptor, this fragment can be methylated in vitro, although at a much slower rate. Circular dichroic measurements suggest that both proteolytic fragments contain substantial alpha-helical structure, approximately 95 and 53% for PF1 and PF2, respectively. No beta-structure could be detected in either fragment. Molecular sieve chromatography in the presence of detergent suggests that PF1 occurs as a stable multimer of an order equivalent to that observed for the detergent-solubilized aspartate receptor, i.e. a tetramer (+/- 1). PF2 is found to have a multimeric form which is sensitive to the removal of detergent. It is proposed that these fragments represent structural and functional domains of the aspartate receptor. PMID- 2995348 TI - 5'-Flanking DNA of the rat growth hormone gene mediates regulated expression by thyroid hormone. AB - Thyroid hormone has been shown to rapidly stimulate the rate of rat growth hormone gene transcription which parallels the kinetics of binding of 3,5,3' triiodo-L-thyronine (L-T3) to its nuclear receptor (Yaffe, B. M., and Samuels, H. H. (1984) J. Biol. Chem. 259, 6284-6291). We have constructed a chimeric gene to explore whether the 5'-flanking region of the rat growth hormone gene contains a DNA element which could mediate thyroid hormone control of growth hormone gene expression. The construct consists of 1.8 kilobase pairs of the 5'-flanking region extending 11 nucleotides downstream from the transcription initiation (cap) site ligated to Escherichia coli DNA containing the structural gene for the enzyme xanthine-guanine phosphoribosyltransferase. GC cells, a growth hormone producing rat pituitary cell line, were transfected with this chimeric gene and stable transformants in which the enzyme is regulated by L-T3 were isolated by positive selection using mycophenolic acid and xanthine. These stable transformants develop with relatively high frequency and show marked L-T3 stimulation of xanthine-guanine phosphoribosyltransferase mRNA which is initiated at the cap site of the growth hormone gene. This study provides the first evidence that the 5'-flanking region of the rat growth hormone gene contains a DNA regulatory element which can mediate control of gene expression by thyroid hormone. PMID- 2995349 TI - Production of antibodies directed against Escherichia coli helicase III and the molecular cloning of the helicase III gene. AB - Helicase III from Escherichia coli has been purified to near homogeneity using single-stranded DNA-dependent adenosine nucleoside 5'-triphosphatase activity as an assay to monitor the purification. The denatured form of this 18.5-kilodalton polypeptide, isolated on a preparative polyacrylamide gel run in the presence of sodium dodecyl sulfate, has been used as an antigen to direct the production of rabbit anti-helicase III antibodies. The antibodies obtained fail to inhibit directly either the helicase activity or the DNA-dependent adenosine nucleoside 5'-triphosphatase activity of helicase III. However, when the antigen-antibody complex is removed from solution by binding to Staphylococcus aureus cells with subsequent sedimentation, there is excellent correlation between the loss of both enzymatic activities and the loss of the helicase III polypeptide. The anti helicase III antibodies have been used as a reagent to probe immunologically a library of E. coli DNA fragments inserted into the plasmid pBR322 for expression of the helicase III antigen. The gene encoding helicase III has been localized on a 2.0-kilobase pair PvuII-EcoRI fragment. Bacterial cells harboring a multicopy plasmid containing this fragment overproduce the helicase III antigen approximately 100-fold. PMID- 2995350 TI - The tetrapeptide analogue of the cell attachment site of fibronectin inhibits platelet aggregation and fibrinogen binding to activated platelets. AB - Fibrinogen binding to receptors on activated platelets is a prerequisite for platelet aggregation. However, the regions of fibrinogen interacting with these receptors have not been completely characterized. Fibronectin also binds to platelet fibrinogen receptors. Moreover, the amino acid sequence Arg-Gly-Asp-Ser, corresponding to the cell attachment site of fibronectin, is located near the carboxyl-terminal region of the alpha-chain of fibrinogen. We have examined the ability of this tetrapeptide to inhibit platelet aggregation and fibrinogen binding to activated platelets. Arg-Gly-Asp-Ser, but not the peptide Arg-Gly-Tyr Ser-Leu-Gly, inhibited platelet aggregation stimulated by ADP, collagen, and gamma-thrombin without inhibiting platelet shape change or secretion. At a concentration of 60-80 microM, Arg-Gly-Asp-Ser inhibited the aggregation of ADP stimulated gel-filtered platelets approximately equal to 50%. Arg-Gly-Asp-Ser, but not Arg-Gly-Tyr-Ser-Leu-Gly, also inhibited fibrinogen binding to ADP stimulated platelets. This inhibition was competitive with a Ki of approximately equal to 25 microM but was incomplete even at higher tetrapeptide concentrations, indicating that Arg-Gly-Asp-Ser is a partial competitive inhibitor of fibrinogen binding. These data suggest that a region near the carboxyl-terminus of the alpha chain of fibrinogen interacts with the fibrinogen receptor on activated platelets. The data also support the concept that the sequence Arg-Gly-Asp-Ser has been conserved for use in a variety of cellular adhesive processes. PMID- 2995351 TI - Adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum. Determining ligand dissociation constants of binary and ternary complexes from the kinetics of enzyme inactivation. AB - Adenosine 5-phosphosulfate (APS) kinase from Penicillium chrysogenum is irreversibly inactivated by trinitrobenzene sulfonate in a pseudo-first order process. Under standard assay conditions kapp was 1.9 X 10(-3) s-1. Saturating MgATP or MgADP decreased Kapp to a limit of 4.1 X 10(-4) s-1. There are several explanations for the partial protection, including the presence of two essential lysyl side chains, only one of which is at the active site. Analysis of the inactivation kinetics by means of linear plots derived for partial protection yielded dissociation constants for E X MgATP (Kia) and E X MgADP (Kiq) of 2.9 mM and 1.8 mM, respectively. Low concentrations of APS alone provided no protection against trinitrobenzene sulfonate inactivation, but in the presence of 1 mM MgADP, as little as 2 microM APS provided additional protection while 100 microM APS reduced kapp to the limit of 4.1 X 10(-4) s-1. The results confirm the formation of a dead end E X MgADP X APS proposed earlier as the cause of the potent substrate inhibition by APS. Linear plots of 1/delta k versus 1/[MgADP] at different fixed [APS] and of 1/delta k versus 1/[APS] at different fixed [MgADP] were characteristic of the ordered binding of MgADP before APS (or the highly synergistic random binding of the two ligands). The true APS dissociation constant of the dead end E X MgADP X APS complex (K'ib) was determined to be 1.9 microM. From the value of K'ib and the previously reported value of KIB (apparent inhibition constant of APS as a substrate inhibitor of the catalytic reaction at saturating MgATP), the ratio of the MgADP and PAPS release rate constants (k4/k3) was calculated to be 11. Inactivation kinetics was used to study the effects of Mg2+ and high salt on ADP and APS binding. The results indicated that free ADP binds to the enzyme more tightly than does MgADP at low ionic strength. High salt decreased free ADP binding, but had little effect on MgADP binding. APS binds more tightly to E X MgADP in the absence or presence of salt than to E X ADP. PMID- 2995352 TI - The complete primary structure of HLA-Bw58. AB - Serological studies indicate that HLA-B17 molecules are unusually cross-reactive with products of the HLA-A locus. In particular, a mouse monoclonal antibody MA2.1 defines an epitope that is shared by HLA-A2 and the two subtypes (Bw57 and Bw58) of B17. To investigate these relationships at the structural level, we have isolated a gene coding for Bw58 from the WT49 B cell line. The gene was transfected into mouse L cells and its protein product was characterized with a panel of monoclonal anti-HLA antibodies. The nucleotide sequence of 3520 base pairs of DNA encompassing the seven exons coding for Bw58 and associated introns was determined. The deduced protein sequences for Bw58 and eight other HLA-A,B,C molecules were compared. In the first polymorphic domain (alpha 1), Bw58 is unusual in that it is as homologous to HLA-A locus products as to HLA-B locus products. In the second polymorphic domain (alpha 2), Bw58 has greater homology to B locus products. In the alpha 1 domain of Bw58, small segments of amino acid and nucleotide sequence homology with A2 (residues 62-65) and with Aw24 (residues 75-83) are found in the major region of polymorphic diversity (residues 62-83). These similarities provide structural correlates for the serological relationships between Bw58 and A locus molecules, with residues 62-65 possibly being involved in the MA2.1 epitope. From comparisons of four HLA-A and four HLA B sequences, there is a difference in the patterns of variation for A and B locus molecules. For B locus molecules there is greater variation in the alpha 1 domain than in the alpha 2 domain. For A locus molecules, variation in the two domains is similar and like that for B locus alpha 2 domains. In comparison to other HLA A,B,C genes, novel inverted repeat sequences were found in the nucleotide sequence of HLA-Bw58. These sequences flank the putative RNA splicing sites at the 3' end of the exons encoding the alpha 2 and alpha 3 protein domains. PMID- 2995353 TI - Obligatory role of cholesterol and apolipoprotein E in the formation of large cholesterol-enriched and receptor-active high density lipoproteins. AB - The formation of large cholesterol-enriched high density lipoproteins (HDL1/HDLc) from typical HDL3 requires lecithin:cholesterol acyltransferase activity, additional cholesterol, and a source of apolipoprotein (apo-) E. The present study explores the role of apo-E in promoting HDL1/HDLc formation and in imparting to these lipoprotein particles the ability to interact with the apo B,E(low density lipoprotein (LDL] receptor. Incubation of normal canine serum with cholesterol-loaded mouse peritoneal macrophages resulted in the formation of HDL1/HDLc that competed with 125I-LDL for binding to the apo-B,E(LDL) receptors on cultured human fibroblasts. Cholesterol efflux from macrophages was necessary because incubation of normal canine serum with nonloaded macrophages did not cause HDL1/HDLc formation. However, cholesterol delivery to the serum was not sufficient to result in HDL1/HDLc formation. Apolipoprotein E had to be available. Incubation of apo-E-depleted canine serum with cholesterol-loaded J774 cells, a macrophage cell line that does not synthesize apo-E, demonstrated that no HDL1/HDLc formation was detected even in the presence of significant cholesterol efflux. However, addition of exogenous apo-E to the serum during the incubation with cholesterol-loaded J744 cells promoted the formation of large receptor-active HDL1/HDLc. The receptor binding activity of these particles produced in vitro correlated with the amount of apo-E incorporated into the HDL1/HDLc. Apolipoproteins A-I and C-III were ineffective in promoting HDL1/HDLc formation; thus, apo-E was unique in allowing HDL1/HDLc formation. These results demonstrate that when lecithin:cholesterol acyltransferase activity, cholesterol, and apo-E are present in serum, typical HDL can be transformed in vitro into large cholesterol-rich HDL1/HDLc that are capable of binding to lipoprotein receptors. PMID- 2995354 TI - The oligosaccharide moieties of the epidermal growth factor receptor in A-431 cells. Presence of complex-type N-linked chains that contain terminal N acetylgalactosamine residues. AB - The receptor for epidermal growth factor (EGF) in the human epidermoid carcinoma cell line A-431 is a glycoprotein of apparent molecular weight = 170,000. During biosynthesis, the receptor is first detected as a precursor of apparent Mr = 160,000. In this report we describe our studies on the structures of the oligosaccharide moieties of the mature receptor and its precursor. A-431 cells were grown in medium containing radioactive sugars and the radiolabeled receptors were purified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled glycopeptides were prepared from the purified receptor by proteolysis, and their structures were examined by a variety of techniques. The mature EGF receptor contains both complex-type and high mannose type Asn-linked oligosaccharides in the approximate ratio of 2 to 1, while the precursor contains only high mannose-type chains. A number of experimental results demonstrate that the mature receptor does not contain oligosaccharides in O-linkage through N-acetylgalactosamine to either serine or threonine. The high mannose-type oligosaccharides in both precursor and mature receptor can be cleaved by endo-beta-N-acetylglucosaminidase H and occur in the mature receptor as Man9GlcNAc2 (6%), Man8GlcNAc2 (49%), Man7GlcNAc2 (25%), and Man6GlcNAc2 (20%), whereas, in the receptor precursor the high mannose chains occur primarily as Man8GlcNAc2 (70%). The complex-type oligosaccharides in the mature receptor are predominantly tri- or tetraantennary species and are unusual in several respects. (i) Many of the chains do not contain sialic acid, while the remaining chains contain 1-2 sialic acid residues. (ii) Half of the [3H] mannose-derived radioactivity was recovered as [3H] fucose and the remaining half as [3H] mannose, indicating that there may be an average of 3 fucose residues/chain. (iii) About one-third of the [3H] glucosamine-derived radioactivity in these glycopeptides was recovered as N-acetylgalactosamine and these residues are all alpha-linked and occur at the nonreducing termini. These data demonstrate that the complex-type Asn-linked oligosaccharides in the EGF receptor from A-431 cells contain sugar residues related to human blood type A. In light of other recent studies, these results suggest that in A-431 cells blood group determinants in surface glycoproteins are contained in Asn-linked but not O-linked oligosaccharides. PMID- 2995355 TI - A soluble ATP-dependent system for protein degradation from murine erythroleukemia cells. Evidence for a protease which requires ATP hydrolysis but not ubiquitin. AB - A soluble ATP-dependent system for protein degradation has been demonstrated in reticulocyte lysates, but not in extracts of nucleated cells. We report that extracts of undifferentiated murine erythroleukemia (MEL) cells contain a labile ATP-stimulated proteolytic system. The addition of ATP to MEL cell extracts at alkaline pH enhances degradation of endogenous cell proteins and various radiolabeled exogenous polypeptides from 2-15-fold. Nonhydrolyzable ATP analogs had no effect. In reticulocytes, one role of ATP in proteolysis is for ubiquitin conjugation to protein substrates. MEL cells also contain ubiquitin and extracts can conjugate 125I-ubiquitin to cell proteins; however, this process in MEL cells seems unrelated to protein breakdown. After removal of ubiquitin from these extracts by DEAE- or gel chromatography, the stimulation of proteolysis by ATP was maintained and readdition of purified ubiquitin had no further effect. In addition, these extracts degraded in an ATP-dependent fashion casein whose amino groups were blocked and could not be conjugated to ubiquitin. After gel filtration or DEAE-chromatography of the MEL cell extracts (unlike those from reticulocytes), we isolated a high molecular weight (600,000) ATP-dependent proteolytic activity, which exhibits many of the properties of energy-dependent proteolysis seen in crude cell extracts. For example, both the protease and crude extracts are inhibited by hemin and N-ethylmaleimide and both hydrolyze casein, globin, and lysozyme rapidly and denatured albumin relatively slowly. The protease, like the crude extracts, is also stimulated by UTP, CTP, and GTP, although not as effectively as ATP. Also, nonhydrolyzable ATP analogs and pyrophosphate do not stimulate the protease. Thus, some mammalian cells contain a cytosolic proteolytic pathway that appears independent of ubiquitin and involves and ATP-dependent protease, probably similar to that found in Escherichia coli or mitochondria. PMID- 2995357 TI - Fusion of Sendai virions or reconstituted Sendai virus envelopes with liposomes or erythrocyte membranes lacking virus receptors. AB - Incubation of intact Sendai virions or reconstituted Sendai virus envelopes with phosphatidylcholine/cholesterol liposomes at 37 degrees C results in virus liposome fusion. Neither the liposome nor the virus content was released from the fusion product, indicating a nonleaky fusion process. Only liposomes possessing virus receptors, namely sialoglycolipids or sialoglycoproteins, became leaky upon interaction with Sendai virions. Fusion between the virus envelopes and phosphatidylcholine/cholesterol liposomes was absolutely dependent upon the presence of intact and active hemagglutinin/neuraminidase and fusion viral envelope glycoproteins. Fusion between Sendai virus envelopes and phosphatidylcholine/cholesterol liposomes lacking virus receptors was evident from the following results. Anti-Sendai virus antibody precipitated radiolabeled liposomes only after they had been incubated with fusogenic Sendai virions. Incubation of N-4-nitrobenzo-2-oxa-1,3-diazole-labeled fusogenic reconstituted Sendai virus particles with phosphatidylcholine/cholesterol liposomes resulted in fluorescence dequenching. Incubation of Tb3+-containing virus envelopes with phosphatidylcholine/cholesterol liposomes loaded with sodium dipicolinate resulted in the formation of the chelation complex Tb3+-dipicolinic acid, as was evident from fluorescence studies. Virus envelopes fuse efficiently also with neuraminidase/Pronase-treated erythrocyte membranes, i.e. virus receptor-depleted erythrocyte membranes, although fusion occurred only under hypotonic conditions. PMID- 2995356 TI - Purification and initial characterization of ubiquitin from the higher plant, Avena sativa. AB - Ubiquitin is a highly conserved, 76-amino acid polypeptide recently demonstrated to be involved in ATP-dependent protein degradation in mammalian cells. From immunoblot analyses with anti-human-ubiquitin antibodies we have detected the presence of free ubiquitin in green leaves, etiolated shoots, and dry seeds of the higher plant, oats (Avena sativa L.). We also find that crude oat extracts contain protease(s) that rapidly degrade both oat and human ubiquitin (t1/2 approximately 10 min at 27 degrees C). This proteolysis apparently cleaves ubiquitin at the carboxyl-terminal glycine dipeptide and results in inactivation of the molecule with respect to ligation but does not affect its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using homogenization conditions that preclude this proteolysis (low pH and the addition of the protease inhibitor p-chloromercuribenzoate) and immunoblotting as an assay for the protein, a procedure for the purification of ubiquitin from etiolated oat shoots was developed. Characterization of purified oat ubiquitin by absorption spectra, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, radioimmunoassay with anti-human-ubiquitin antibodies, and kinetic analyses using the ubiquitin activating enzyme isolated from rabbit liver indicates that this protein is remarkably similar to the mammalian form. Small differences between the oat and human proteins have been observed by amino acid compositional analyses indicating that the two forms are not totally homologous. Immunoblotting of crude oat extracts has revealed the presence of high molecular weight proteins recognized by anti-ubiquitin antibodies that represent ubiquitin protein conjugates formed in vivo. Taken together, these data provide evidence that higher plants contain a ubiquitin-dependent proteolytic pathway that is mechanistically identical to that present in animals. PMID- 2995358 TI - Molecular cloning and sequencing of the gene for CDP-diglyceride synthetase of Escherichia coli. AB - The cds gene of Escherichia coli codes for the enzyme CDP-diglyceride synthetase. We now report the construction of plasmids which carry cds. Using these plasmids, we have sequenced 1274 base pairs of DNA, including a 750-base pair open reading frame which is the coding region of the cds gene. This DNA sequence allows the deduction of the primary peptide sequence for CDP-diglyceride synthetase. The protein is very hydrophobic, and, assuming no processing or modification, has a molecular weight of 27,570. Furthermore, there is a second open reading frame immediately after cds, implying that cds may be part of an operon. We have also constructed a runaway replication cds-plasmid that directs approximately 50-fold overproduction of CDP-diglyceride synthetase. This overproduction has been utilized in the purification of the enzyme to homogeneity, as described in the accompanying paper (Sparrow, C.P., and Raetz, C.R.H., J. Biol. Chem. 260, 12084 12091). Finally, the molecular cloning work reported herein allows the exact placement of the cds gene on the E. coli genetic map. PMID- 2995359 TI - Purification and properties of the membrane-bound CDP-diglyceride synthetase from Escherichia coli. AB - The enzyme CDP-diglyceride synthetase (CTP: phosphatidate cytidylyltransferase; EC 2.7.7.41) has been purified to 90% homogeneity from Escherichia coli cells that overproduce the enzyme 50-fold through the use of recombinant DNA technology. The purification required the use of different detergents at each step, illustrating the refractory hydrophobic nature of this protein. Apparent physical effects of EDTA on the enzyme were also utilized in the purification. The enzyme has an apparent minimum subunit mass of 27,000 daltons, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The amino acid composition of the protein was determined, and it correlates well with the theoretical protein product of the cds gene, the sequence of which is reported in the accompanying paper (Icho, T., Sparrow, C. P., and Raetz, C. R. H. (1985) J. Biol. Chem. 260, 12078-12083). The pure enzyme displays surface dilution kinetics when assayed in the presence of Triton X-100. As previously suggested on the basis of studies using partially purified preparations, the enzyme mechanism is sequential, and computer-calculated kinetic constants are reported herein. The substrate specificity of the enzyme is also investigated. This is the first time this enzyme has been purified to homogeneity from any source, despite the fact that it is essential for phospholipid biosynthesis in all organisms. PMID- 2995360 TI - Molecular cloning and sequencing of the gene for CDP-diglyceride hydrolase of Escherichia coli. AB - Previous work from this laboratory had demonstrated that CDP-diglyceride hydrolase of Escherichia coli is encoded by the cdh gene that maps near minute 88 (Bulawa, C. E., and Raetz, C. R. H. (1984) J. Biol. Chem. 259, 11257-11264). We now report the construction of hybrid plasmids and the sequencing of a 1,243-base pair insert carrying cdh. The further construction of BAL31 deletions of this insert, in conjunction with maxicell experiments and in vitro enzyme assay, has led to the identification of a 756-base pair coding sequence for the cdh polypeptide. The molecular weight of the primary translation product deduced from the DNA sequence of the cdh gene is 28,450, in agreement with maxicell experiments. Parallel purification of the enzyme from extracts of wild-type and overproducing strains confirms the presence of a 27-kDa polypeptide in the overproducer, as judged by polyacrylamide gel electrophoresis of the most purified fractions. Inspection of the DNA sequence reveals a very hydrophobic N terminal domain that may be either a signal peptide or a special region, anchoring the hydrolase to the membrane. In contrast to the CDP-diglyceride synthetase, the overall amino acid composition of the CDP-diglyceride hydrolase is not extraordinarily hydrophobic. Although both CDP-diglyceride synthetase and CDP-diglyceride hydrolase can transfer the CMP moiety of CDP-diglyceride to a suitable acceptor, the primary structures and mechanisms of action of these two enzymes are very different. PMID- 2995361 TI - The bacteriophage P22 arc and mnt repressors. Overproduction, purification, and properties. AB - The arc and mnt genes of bacteriophage P22 encode small repressor proteins. We have cloned these genes onto plasmids that overproduce Arc and Mnt to greater than 1% of the soluble cellular protein. Both proteins were purified to greater than 95% homogeneity, and N-terminal sequences and amino acid compositions were determined. These data, in combination with previously determined gene sequences, establish the complete protein sequences for Arc (53 residues) and Mnt (82 residues). Both proteins have melting temperatures between 45 and 55 degrees C and can be renatured to a fully active species. Arc is a dimer in solution and Mnt is a tetramer. PMID- 2995362 TI - Structure and exon to protein domain relationships of the mouse carbonic anhydrase II gene. AB - We have isolated a cosmid clone containing the entire mouse (YBR strain) carbonic anhydrase (CA) II gene in 38 kilobase pairs of genomic DNA. The gene was found to be composed of seven exons and six introns. A TATA box (TATAAAA) and a possible CCAAT box (CCACT) have been located beginning 92 and 142 base pairs, respectively, upstream from the initiation codon ATG. When the regions encoded by exons and protein domains are examined, all but 1 of the 30 putative active site residues are encoded by four exons: exons 2 and 3 mainly code for hydrophilic residues and exons 4 and 6 mostly hydrophobic residues. Two intron splice positions, one between the codons for Glu-116 and Leu-117 and the other interrupting the codon for Gly-143, are located at the bottom of the active site cavity, and the former separates two of the three histidine residues forming ligands to the active site zinc ion. The other four splice sites map to the exterior of the molecule. Thus, except for the possible association of the 29 active site residues encoded by four exons, no obvious correspondence is seen between the regions coded by exons and the functional or secondary structural domains of the mouse CA II molecule. During this study, the possible basis for the two electrophoretic types, CA IIa and CA IIb, of inbred mouse strains was detected as a Gln/His interchange at position 38. PMID- 2995363 TI - Isolation and identification of 24-dehydroprovitamin D3 and its photolysis to 24 dehydroprevitamin D3 in mammalian skin. AB - Until now it had been assumed that mammalian skin contains only one provitamin D, 7-dehydrocholesterol, that is eventually converted to vitamin D3 after the skin is exposed to sunlight. Examination by reverse phase high performance liquid chromatography of lipid extracts from young rat skin, however, led to the observation that 7-dehydrocholesterol is not the only provitamin D in rat skin. Another provitamin D, accounting for 22 +/- 3% of the total provitamin content of the skin, was resolved from 7-dehydrocholesterol, and, on the basis of ultraviolet spectrophotometry, mass spectrometry, and nuclear magnetic resonance spectrometry, was identified as 24-dehydroprovitamin D3 (cholesta-5,7,24-trien-3 beta-ol). This new cutaneous provitamin D is not unique to the rat because it was also detected in the skin of reptiles, amphibians, birds, aquatic mammals, and humans. To be certain that the cutaneous 24-dehydroprovitamin D3 was as susceptible as 7-dehydrocholesterol to ultraviolet photolysis, rat skin was exposed to ultraviolet radiation. A reverse phase high performance liquid chromatographic analysis of a lipid extract of rat skin previously exposed to ultraviolet radiation demonstrated the presence of both previtamin D3 and 24 dehydroprevitamin D3. Therefore, these observations demonstrate for the first time that mammalian skin has the capacity to produce not one but at least two different vitamin Ds. PMID- 2995364 TI - Transient Raman study of horseradish peroxidase. Evidence for a lack of extensive heme pocket relaxation subsequent to carbon monoxide photolysis. AB - Time-resolved resonance Raman spectroscopy is a valuable tool for the study of the dynamics of heme-protein interactions. In particular, the photolysis of a ligand by short laser pulses allows for the examination of the dynamic evolution of heme-protein interactions subsequent to ligand dissociation. To date, such studies have been confined largely to hemoglobins and myoglobins. Here we present the results of the first transient Raman study of a peroxidase. Resonance Raman spectra of horseradish peroxidase were obtained within 10 ns of ligand (CO) photolysis at a variety of pH values. We find that there is only minimal relaxation of the heme pocket of horseradish peroxidase in response to ligand photolysis. This relaxation is pH-dependent and most probably involves the heme vinyl substituents. Such behavior is in sharp contrast to the transient behavior of most hemoglobins and beef heart cytochrome oxidase. PMID- 2995365 TI - Heme binding to murine erythroleukemia cells. Evidence for a heme receptor. AB - Friend virus transformed murine erythroleukemia (MEL) cells are known to take up heme from the surrounding medium and to incorporate it into newly synthesized hemoglobin (Granick, J. L., and Sassa, S. (1978) J. Biol. Chem. 253, 5402-5406), but the mechanism of its uptake is unknown. We hypothesized the existence of a specific receptor for heme in the plasma membrane. Using [55Fe]heme, we examined the characteristics of its interaction with MEL cells at 4 degrees C. [55Fe]heme binding reached equilibrium within 4 h, was 80% dissociable by 16 h, and was independent of pH over the range 7.0-8.2. Specific heme binding was linear with cell number, and competitive binding studies with various heme analogues, such as free protoporphyrin IX, metal-substituted protoporphyrin IX, Fe-mesoporphyrin IX, and Fe-deuteroporphyrin IX, revealed significant stereospecificity for Fe protoporphyrin IX. The dissociation constant of the interaction was 0.03 nM-1 with no evidence of cooperativity or multiple classes of sites. The average number of sites/cell was approximately 10,300. Reduction of binding following preincubation with trypsin, in conjunction with the above data, suggests that this cell type may display a receptor for heme which is comprised, as least in part, of protein. PMID- 2995366 TI - Characterization of specific high affinity receptors for human tumor necrosis factor on mouse fibroblasts. AB - Mouse L-929 fibroblasts, an established line of cells, are very sensitive to lysis by human lymphotoxin (hTNF-beta). Specific binding of a highly purified preparation of hTNF-beta to these cells was examined. Recombinant DNA-derived hTNF-beta was radiolabeled with [3H]propionyl succinimidate at the lysine residues of the molecule to a specific activity of 200 microCi/nmol of protein. [3H]hTNF-beta was purified by high performance gel permeation chromatography and the major fraction was found to be monomeric by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The labeled hTNF-beta was fully active in causing lysis of L-929 fibroblasts and bound specifically to high affinity binding sites on these cells. Scatchard analysis of the binding data revealed the presence of a single class of high affinity receptors with an apparent Kd of 6.7 X 10(-11) M and a capacity of 3200 binding sites/cell. Unlabeled recombinant DNA derived hTNF-beta was found to be approximately 5-fold more effective competitive inhibitor of binding than the natural hTNF-beta. The binding of hTNF-beta to these mouse fibroblasts was also correlated with the ultimate cell lysis. Neutralizing polyclonal antibodies to hTNF-beta efficiently inhibited the binding of [3H]hTNF-beta to the cells. We conclude that the specific high affinity binding site is the receptor for hTNF-beta and may be involved in lysis of cells. PMID- 2995367 TI - Structure of the human prealbumin gene. AB - Using cloned human prealbumin cDNA as a probe, Southern blot hybridization of human genomic DNA revealed that the prealbumin gene consists of an unique, single copy DNA. The nucleotide sequences of the entire human prealbumin gene, including both 581 base pairs of the 5'- and 95 base pairs of the 3'-flanking sequences, were determined. The gene spans about 7.0 kilobase pairs and consists of four exons and three introns. As in most eukaryotic genes, the consensus TATA and CAAT sequences are found 30 and 101 nucleotides, respectively, upstream from the putative cap site, and a polyadenylation signal sequence AA-TAAA is found in the 3'-untranslated region. Unexpectedly, two independent open reading frames provided with respective regulatory sequences were found within the gene: one in the first intron and the other in the third intron. PMID- 2995368 TI - Hormonal regulation of the bovine prolactin promoter in rat pituitary tumor cells. AB - A 1-kilobase DNA fragment containing the promoter of the bovine prolactin gene was fused to the chloramphenicol acetyltransferase gene and the activity of the promoter was assayed by transfection of the fusion gene into COS-1, HeLa, and GH3 cells. Transcription of the chloramphenicol acetyltransferase gene driven by the prolactin promoter was detected only in GH3 cells, a rat pituitary tumor cell line. Epidermal growth factor and thyroid releasing hormone produced a stimulation of transcription, and the synthetic glucocorticoid hormone dexamethasone effected an inhibition of transcription from the prolactin promoter. None of these factors significantly affected transcription of the chloramphenicol acetyltransferase gene fused to the Rous sarcoma virus promoter. Deletion of all but 250 base pairs of bovine prolactin 5'-flanking DNA had no effect, indicating that the signals sufficient for both stimulation and inhibition of transcription reside in close proximity to the promoter. PMID- 2995369 TI - Synthesis and processing of mitochondrial steroid hydroxylases. In vivo maturation of the precursor forms of cytochrome P-450scc, cytochrome P 450(11)beta, and adrenodoxin. AB - The synthesis and maturation of the precursor forms of three mitochondrial enzymes involved in steroid hormone biosynthesis have been studied in vivo. Primary cultures of bovine adrenocortical cells were radiolabeled with [35S] methionine and newly synthesized cholesterol side-chain cleavage cytochrome P-450 (P-450scc), 11 beta-hydroxylase cytochrome P-450 (P-450(11)beta), and adrenodoxin immunoisolated using specific antibodies. Both the precursor and mature forms of P-450scc and P-450(11)beta were detected during short periods of pulse labeling; however, the precursor forms were transitory in nature while their corresponding mature forms accumulated. Pulse-chase experiments showed that the precursor form of each cytochrome P-450 had an apparent half-life of 3.5 min. In contrast, the precursor form of adrenodoxin was not readily detected in pulse-labeling experiments until a substantial amount of its mature form had accumulated. When the cultured cells were treated with a chelator of divalent cations (o phenanthroline) or a mitochondrial uncoupler (dinitrophenol), the maturation of all three precursors was inhibited. The synthesis of the P-450scc and P 450(11)beta precursors was induced in cells maintained in the presence of adrenocorticotropin, and the rates of appearance of their processed forms were also increased. The mature forms of all three proteins were immunoisolated from a trypsinized mitochondrial fraction prepared from the radiolabeled cells, demonstrating that the mature proteins were localized within the organelle. These studies establish that the maturation of the precursor forms of the mitochondrial steroidogenic enzymes are characterized by steps similar to those reported for other mitochondrial precursor proteins. PMID- 2995370 TI - The FLP protein of the 2-micron plasmid of yeast. Purification of the protein from Escherichia coli cells expressing the cloned FLP gene. AB - Most laboratory strains of the yeast Saccharomyces cerevisiae contain many copies of an autonomously replicating plasmid called 2-micron circle DNA. This plasmid codes for a site-specific recombinase, the FLP protein which promotes recombination across two 599-base pair inverted repeats of the plasmid DNA. We have cloned the FLP gene under the control of a strong Escherichia coli promoter and have hyperproduced the protein in that organism. Cell-free extracts from this source promote highly efficient site-specific recombination in vitro and we have used this activity to purify the FLP protein substantially. The enzyme acts efficiently on circular and linear substrates and requires only monovalent or divalent cations for activity. PMID- 2995371 TI - Determination of DNA sequences essential for FLP-mediated recombination by a novel method. AB - The yeast 2-micron circle plasmid encodes a protein, FLP, that mediates site specific recombination across the two FLP-binding sites of the plasmid. We have used a novel technique, "exonuclease-treated substrate analysis," to determine the minimal duplex DNA sequence needed for this recombination event. A linear DNA containing two FLP sites in a direct orientation was treated with the double strand specific 3'-exonuclease, exonuclease III, to generate molecules with a nested set of single-strand deletions that extended into one of the FLP sites. The DNA was then end-labeled at the sites of the deletions and used as a substrate for recombination in vitro. FLP-mediated recombination between two FLP sites excised a restriction endonuclease cleavage site from the DNA. Comparison of the fragments produced by restriction enzyme digestion of untreated and FLP treated DNA showed to the nucleotide the duplex DNA sequence required for FLP mediated recombination. To examine essential sequences in the opposite DNA strand, similar experiments were done using the 5'-exonuclease encoded by phage T7. The minimal essential duplex DNA sequence lies within the region of the FLP site that was previously shown to be protected from nuclease digestion in the presence of FLP. A modified form of this technique can be used to study the minimal sequence requirements of site-specific DNA binding proteins. PMID- 2995372 TI - Internalization of functional epidermal growth factor:receptor/kinase complexes in A-431 cells. AB - We have isolated and partially purified an intracellular vesicle fraction from A 431 cells that contains both epidermal growth factor (EGF) and enzymatically active EGF:receptor/kinase. Exposure of intact A-431 cells to EGF leads to an accumulation of both EGF and kinase activity in this vesicle fraction. The accumulation is time- and temperature-dependent and is blocked by inhibitors of energy production. The EGF receptor in internalized vesicles is capable of autophosphorylation and, in the presence of Ca2+, of phosphorylation of the previously isolated 35-kDa protein (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). The demonstration of an EGF-induced increase in kinase activity of an internalized vesicle fraction lends credence to the hypothesis that EGF-induced endocytosis of the receptor is of physiological significance in the response of cells to this ligand. In addition, these results are consistent with the suggestion that the phosphorylation of the 35-kDa protein is associated with internalization of the EGF:receptor/kinase complex. PMID- 2995373 TI - Purification and characterization of a smooth muscle myosin phosphatase from turkey gizzards. AB - A phosphoprotein phosphatase that dephosphorylates smooth muscle myosin has been purified to apparent homogeneity from turkey gizzards. Smooth muscle phosphatase (SMP) IV has a molecular weight of 150,000 as determined by gel filtration on a Sephadex G-200 column and is composed of two subunits (Mr = 58,000 and 40,000). Although it is active toward a number of proteins, its activities toward the contractile proteins, intact myosin, heavy meromyosin, and isolated myosin light chains are higher than its activities toward phosphorylase alpha, histone IIA, and phosphorylase kinase. SMP-IV preferentially dephosphorylates the beta-subunit of phosphorylase kinase. The properties of the enzyme have been studied using heavy meromyosin, a soluble chymotryptic fragment of myosin, and isolated myosin light chains as substrates. SMP-IV has high affinity for both substrates and is optimally active at neutral pH. Divalent cations, Ca2+ and Mg2+, activate the dephosphorylation of heavy meromyosin but inhibit the activity toward myosin light chains. Low concentrations of ATP (1-5 mM) activate SMP-IV but concentrations higher than 5 mM are inhibitory. Inhibition of 50% of the activity of the enzyme by NaF and PPi requires concentrations higher than 10 mM. Rabbit skeletal muscle heat stable inhibitor-2 has no effect on the activity of SMP-IV toward heavy meromyosin, myosin light chains, and phosphorylase alpha. PMID- 2995374 TI - Stromelysin, a connective tissue-degrading metalloendopeptidase secreted by stimulated rabbit synovial fibroblasts in parallel with collagenase. Biosynthesis, isolation, characterization, and substrates. AB - Rabbit synovial fibroblasts induced to undergo a specific switch in gene expression by agents that alter cell morphology secreted the neutral proteinase precursor procollagenase (apparent Mr of 53,000 and 57,000). A major Mr = 51,000 polypeptide that was always induced coordinately with procollagenase has now been identified as the proenzyme form of a metal-dependent proteinase active at neutral pH. We have named this proteinase stromelysin. Prostromelysin and procollagenase were the most prominent [35S]methionine-labeled secreted proteins of the induced fibroblasts. By the use of casein degradation as an assay for enzyme activity, stromelysin was isolated with high yield from the conditioned culture medium of 12-O-tetradecanoylphorbol 13-acetate-treated fibroblasts and migrated as an active form of Mr = 21,000 that was immunologically identical to the proteoglycan-degrading proteinase purified from rabbit bone. Immunoglobulin G from antiserum raised to purified rabbit bone proteoglycanase immunoprecipitated the Mr = 51,000 proenzyme form from conditioned medium of induced rabbit cells and also immunoprecipitated an Mr = 55,000 polypeptide from induced human fibroblasts. When rabbit prostromelysin was activated by trypsin or 4 aminophenylmercuric acetate, the proenzyme was converted to an active form of Mr = 41,000. During the course of the purification, prostromelysin was converted to an additional activatable form of Mr = 35,000 and additional active forms of Mr = 21,000-25,000, which had related peptide maps distinct from collagenase. All of these forms were immunologically cross-reactive. Purified stromelysin degraded casein, cartilage proteoglycans, fibronectin, alpha 1-proteinase inhibitor, and immunoglobulin G2a and had limited activity on laminin, elastin, type IV collagen, and gelatin, but did not degrade type I collagen. Stromelysin was inhibited by EDTA, 1,10-phenanthroline, and the specific glycoprotein tissue inhibitor of metalloproteinases isolated from human amniotic fluid and was therefore classified as a metalloproteinase. PMID- 2995375 TI - Sequence-specific interaction of histones with the simian virus 40 enhancer region in vitro. AB - DNA fragments containing either one or both of the 72-base pair (bp) elements which constitute the SV40 enhancer and the three adjacent 21-bp repeats were associated with histone octomers from chicken erythrocytes in vitro. Both fragments formed complexes with electrophoretic mobilities of nucleosomes containing the appropriate length of DNA. Analysis of DNase I cutting of uniquely end-labeled complexes suggests that the fragment containing a single 72-bp element forms a positioned core particle. Control experiments show that positioning is not due to the 21-bp repeats or to end effects. The fragment with a tandem repeat of the 72-bp element also does not associate randomly with histones. The data are consistent with formation of a core particle on one or the other of the repeated enhancer sequences. We discuss possible functional consequences of such nucleosome positioning. PMID- 2995376 TI - Insulin receptor kinase activity in rat liver. Regulation by fasting and high carbohydrate feeding. AB - Insulin receptor kinase activity was measured in partially purified receptor preparations from livers of rats fed a standard diet or subjected to either prolonged fasting or a high carbohydrate (CHO) diet, conditions known to decrease (fasting) and increase (CHO) insulin action. Basal and insulin-stimulated phosphorylation of the beta subunit of the insulin receptor was comparable in all groups with a half-maximal effect at approximately 2.0 ng/ml free insulin and a 10-12-fold maximal effect. The kinase activity of insulin receptors from the three groups was further examined using the synthetic polypeptide Glu 4:Tyr 1. Basal and insulin-stimulated rates of Glu 4:Tyr 1 phosphorylation were highest in the CHO-fed and lowest in the fasted group. The magnitude of these differences was the same in the absence or presence of insulin; thus, the alterations in receptor kinase activity in fasting and CHO feeding were entirely expressed in the basal rate of peptide phosphorylation. Antireceptor antibody immunoprecipitated 70-80% of the basal Glu 4:Tyr 1 kinase activity in each group; the remaining 20-30% showed minor group differences when normalized for the amount of protein present in the receptor preparations. These results indicate that the group differences in basal kinase were intrinsic to the insulin receptor. Insulin increased the Vmax of Glu 4:Tyr 1 phosphorylation by approximately 30 fmol of phosphorus/fmol of binding activity/30 min in all three groups; however, the absolute Vmax was highest in the CHO-fed and lowest in the fasted group. The Km of Glu 4:Tyr 1 phosphorylation was unaffected by insulin and was comparable (approximately 0.25 mg/ml) in the three groups. These findings indicate that fasting and CHO feeding produce changes in receptor kinase activity which are regulated by mechanisms independent of insulin and that the alterations show substrate specificity so that differences are detected with one substrate (Glu 4:Tyr 1) but not another (the beta subunit). PMID- 2995377 TI - The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates. AB - ATP, ubiquitin-dependent proteolysis proceeds through covalent intermediates between target proteins destined for degradation and the 8,600-Da polypeptide ubiquitin. The ubiquitin moiety therefore represents a sensitive immunological marker for the specificity and function of this novel post-translational modification. Methods are described for the immunochemical detection of ubiquitin conjugates immobilized on nitrocellulose filters following electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. A further modification allows quantitation of conjugated ubiquitin to the exclusion of free polypeptide. Comparisons of conjugate pools in rabbit reticulocytes and erythrocytes demonstrate that 83 +/- 3% and 31 +/- 0.2%, respectively, of total intracellular ubiquitin exists covalently bound to target proteins. Similar large proportions of conjugated ubiquitin were found in three tissue culture cell lines. Subcellular fractionation revealed that 25% of total ubiquitin conjugates of reticulocytes sediment with the 22,000 X g stromal fraction with the remainder found in the 100,000 X g supernatant. In contrast, significant levels of erythrocyte ubiquitin conjugates occur only in the 100,000 X g supernatant, suggesting ubiquitin-mediated proteolysis actively degrades stromal components lost during terminal maturation. Reticulocytes retain their full complement of active ubiquitin during maturation indicating the concomitant decline in energy dependent proteolysis does not result from ubiquitin inactivation. That the lower level of ubiquitin conjugates and the accompanying rate of energy-dependent proteolysis in erythrocytes is a consequence of limited substrate availability is suggested by observed increases in conjugate pools and induction of specific ubiquitin-protein adducts on incubation with either phenylhydrazine or sodium nitrite. PMID- 2995378 TI - Interleukin 2 induces a rapid increase in intracellular pH through activation of a Na+/H+ antiport. Cytoplasmic alkalinization is not required for lymphocyte proliferation. AB - In several cell types, proliferation initiated by growth factors is associated with a rapid increase in cytoplasmic pH (pHi). This cytoplasmic alkalinization is due to the activation of an amiloride-sensitive Na+/H+ antiport. It is unclear whether growth factor-induced activation of the antiport or the resultant increase in pHi is the trigger for proliferation, an obligatory requirement for proliferation, or simply an associated phenomenon. Interleukin 2 (IL 2) acts as a growth factor for mitogen or antigen-stimulated thymus-derived (T) lymphocytes. In this study, we established that IL 2 produces an increase in pHi and determined whether this increase in pHi plays a role in the proliferative response to IL 2. Monitoring pHi with an intracellularly trapped, pH-sensitive, fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, we demonstrated that IL 2 rapidly (less than 90 s) initiates an increase in pHi in IL 2-sensitive human and murine T cells. Because intracellular alkalinization requires extracellular Na+ and is amiloride-sensitive, it likely occurs through activation of the Na+/H+ antiport. Using partitioning of a weak acid, 5,5-dimethyl-2,4 oxazolidinedione, we confirmed that the IL 2-dependent increase in pHi is sustained for several hours and returns to near base-line levels by 18 h. We also investigated the consequence of preventing Na+/H+ exchange on the proliferative response induced by IL 2. IL 2-driven proliferation occurred in nominally bicarbonate-free medium in the presence of concentrations of amiloride analogs sufficient to inhibit the Na+/H+ antiport and prevent intracellular alkalinization. These data suggest that although the antiport is activated by binding of IL 2 to its receptor, intracellular alkalinization is not essential for IL 2-dependent proliferation. It seems unlikely that either cytoplasmic alkalinization or activation of the Na+/H+ antiport are triggers for T cell proliferation. PMID- 2995380 TI - A comparison of catecholamine-induced internalization of beta-adrenergic receptors and receptor-mediated endocytosis of epidermal growth factor in human astrocytoma cells. Inhibition by phenylarsine oxide. AB - The ligand-induced internalization of beta-adrenergic receptors and the receptor mediated internalization of epidermal growth factor were blocked, under similar conditions, by phenylarsine oxide (PAO) in human astrocytoma cells (1321N1). The inhibition was not prevented or reversed by monofunctional sulfhydryl agents such as 2-mercaptoethanol or glutathione; however, the inhibitory action of PAO was blocked and reversed by bifunctional thiols such as 2,3-dimercaptoethanol or dithiothreitol. The results are consistent with the interaction of PAO with vicinal sulfhydryl groups to form a stabile ring structure. PAO did not prevent isoproterenol-induced uncoupling (desensitization) of beta-adrenergic receptors even though receptor internalization was completely blocked. The effects of PAO on receptor internalization could not be explained by any action of the trivalent arsenical to lower ATP levels. Ligand binding to both receptors was not detectably altered by PAO under conditions selective for inhibition for endocytosis. The results suggest a common mechanism for internalization of beta adrenergic receptors and epidermal growth factor by a process that involves vicinal sulfhydryl groups. PMID- 2995379 TI - Characterization of the gene encoding the major rat liver asialoglycoprotein receptor. AB - A cloned cDNA encoding the major rat liver asialoglycoprotein receptor has been used to analyze the gene for this protein. Genomic Southern blot analysis reveals that the gene is contained on a single EcoRI restriction fragment and is unique. A clone containing the gene (isolated from a rat liver genomic library) has been characterized by sequence analysis. The mRNA for the receptor is encoded by nine exons separated by eight introns. The first exon is confined to the 5' untranslated region of the mRNA, the second exon encodes most of the cytoplasmic NH2-terminal domain of the receptor polypeptide, the third exon corresponds to the hydrophobic transmembrane portion of the polypeptide, and the remaining exons encode the extracellular parts of the receptor. Some, but not all, of the divisions between exons correspond to boundaries between functional domains of the polypeptide. PMID- 2995381 TI - Interrelationships among quinidine, amiloride, and lithium as inhibitors of the renal Na+-H+ exchanger. AB - We examined the effects of quinidine, amiloride and Li+ on the kinetics of Na+-H+ exchange in microvillus membrane vesicles isolated from the rabbit renal cortex. Quinidine reversibly inhibited the initial rate of Na+-H+ exchange (I50 200 microM). The plot of 1/V versus [quinidine] was curvilinear, with Hill coefficient greater than 1.0, indicating that the drug interacts at two or more inhibitory sites or at a single site on at least two different conformations of the transporter. Quinidine decreased the Vmax for Na+-H+ exchange and increased the Km for Na+, indicating a mixed-type mechanism of inhibition. In contrast, plots of 1/V versus [amiloride] and 1/V versus [Li+] were linear, indicating single inhibitory sites; amiloride and Li+ each increased the Km for Na+ with no effect on Vmax, indicating a competitive mechanism of inhibition. Addition of Li+ increased the intercept with no change in slope of the 1/V versus [amiloride] plot, indicating that Li+ and amiloride are mutually exclusive inhibitors of Na+ H+ exchange. Addition of quinidine increased the slopes of the plots of 1/V versus [amiloride] and 1/V versus [Li+], indicating that the binding of quinidine is not mutually exclusive with the binding of amiloride and Li+. Results from this and previous studies are consistent with the concept that the inhibitor amiloride and the transportable substrates Na+, H+, Li+, and NH+4 all mutually compete for binding to a single site, the external transport site of the renal Na+-H+ exchanger. However, our findings indicate that quinidine interacts with the Na+-H+ exchanger on at least one additional site that is not shared by Na+, Li+, or amiloride. PMID- 2995382 TI - Internalization, recycling, and redistribution of vasopressin receptors in rat hepatocytes. AB - Three hours after isolation, cultured hepatocytes have approximately 150,000 surface vasopressin receptors/cell, and these exhibit a Kd for 125I-vasopressin of 6 nM based on calculation of Koff/Kon, or a Kd of 9.5 nM based on Scatchard plot analysis. After the binding of 125I-vasopressin to its receptor on the hepatocyte surface, this complex is internalized with a t1/2 of 3-6 min. Following this internalization, the number of vasopressin receptors on the cell surface is restored both in vitro and in the isolated perfused liver with a t1/2 of 8-10 min. This restoration is blocked in vitro by incubation of the hepatocytes at 18 degrees C, but not by cycloheximide, suggesting that internalized vasopressin receptors recycle back to the cell surface. Prolonged incubation of hepatocytes with vasopressin results in the loss of greater than 75% of the vasopressin surface binding at concentrations of vasopressin approximately equivalent to its Kd. The binding of vasopressin to cultured hepatocytes 3-5 h after isolation resembles binding to the isolated perfused whole liver with respect to receptor dynamics. During culture for 48 h, however, we observe a progressive loss of hepatocyte surface vasopressin receptors. Concomitant with this reduction in surface receptors with time in culture, there appears to be a marked elevation in intracellular receptors. PMID- 2995384 TI - Prevention of the agonist binding to gamma-aminobutyric acid B receptors by guanine nucleotides and islet-activating protein, pertussis toxin, in bovine cerebral cortex. Possible coupling of the toxin-sensitive GTP-binding proteins to receptors. AB - Using the membranes treated with Triton X-100, we studied the interaction between gamma-aminobutyric acid (GABA)B receptors and the GTP-binding proteins which are the substrates for ADP-ribosylation by the islet-activating protein (IAP), pertussis toxin. The addition of guanine nucleotides to the membranes markedly decreased the binding of GABA to GABAB receptors. Preincubation of the membranes with IAP plus NAD caused ADP-ribosylation of the 41,000- and 39,000-Da proteins selectively and decreased GABA binding to GABAB receptors in a time- and dose dependent manner. This decrease of binding appeared to be due to the reduction of receptor affinity for agonist. The GTP-binding proteins which are ADP-ribosylated by IAP were purified from the membrane fraction of bovine cerebral cortex. The addition of the purified GTP-binding proteins to IAP-treated membranes restored the high affinity binding of GABA to GABAB receptor. The two GTP-binding proteins which were resolved by octyl-Sepharose column chromatography showed similar efficacy in restoring GABA binding. Thus, GABAB receptors are coupled to GTP binding proteins, IAP-specific substrates, in the brain membranes. PMID- 2995383 TI - Catecholamine and vasopressin stimulation of gluconeogenesis from dihydroxyacetone in the presence of atractyloside. AB - Atractyloside inhibited gluconeogenesis from dihydroxyacetone in hepatocytes from fasted rats and increased lactate synthesis. In the presence of atractyloside, lactate/pyruvate and beta-hydroxybutyrate/aceto-acetate ratios were increased and the accumulation of Fru-2,6-P2 was prevented. In the absence of atractyloside, gluconeogenesis from dihydroxyacetone was stimulated by dibutyryl-cAMP and, to a much lesser extent, by norepinephrine and vasopressin. Omission of Ca2+ increased the stimulation by norepinephrine but prevented that by vasopressin. High concentrations (greater than or equal to 40 microM) of atractyloside abolished the stimulation of gluconeogenesis by dibutyryl-cAMP but not that by norepinephrine or vasopressin. Exogenous Ca2+ was not required for hormonal stimulation in the presence of atractyloside. The stimulation by norepinephrine was inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N-tetraacetic acid or prazosin but not by propranolol. Atractyloside caused decreases of all glycolytic intermediates and an activation of pyruvate kinase. Norepinephrine partially reversed these effects. The mitochondrial and cytosolic ATP/ADP ratios were determined by digitonin fractionation of hepatocytes. Norepinephrine or vasopressin increased the cytosolic ATP/ADP in the presence of atractyloside. We suggest that the increased availability of cytosolic ATP could be responsible for the stimulation of gluconeogenesis by these hormones. PMID- 2995385 TI - Multisubstrate analogs for deoxynucleoside kinases. Use in novel affinity media applied to resolution of Lactobacillus enzymes. AB - New multisubstrate-type inhibitors of the deoxynucleoside kinases have been synthesized, tested for their specificity as soluble inhibitors of enzymes from Lactobacillus acidophilus, and used to construct media for affinity chromatography. Each inhibitor was a deoxynucleoside 5'-adenosine 5"'-P1,P4 tetraphosphate (abbreviated dNp4A, where dN represents a dAdo, dCyd, dGuo, or dThd moiety linked through its 5'-hydroxyl to the terminal phosphate of adenosine tetraphosphate). At micromolar concentrations, each inhibitor strongly and specifically inhibited the corresponding deoxynucleoside kinase. Each of the four Lactobacillus deoxynucleoside kinase activities was selectively retained on its homologous dNp4A-Sepharose affinity medium. The activity was eluted on addition of the respective dNp4A with up to 70% recovery and 300-500-fold purification (relative to an ammonium sulfate fraction). Whereas dThd kinase was retained only by the dTp4A column, a portion of the dAdo kinase activity was retained, along with all the dCyd kinase or dGuo kinase, on dCp4A- or dGp4A-Sepharose, respectively, and coeluted with these activities. Conversely, all three activities were quantitatively retained on dAp4A-Sepharose, without competition from either dCyd or dGuo, and were eluted simultaneously upon addition of dAp4A. These observations further confirm the understanding that this organism employs paired, and presumably bifunctional, kinases, namely dCyd/dAdo kinase and dGuo/dAdo kinase, along with a separate thymidine kinase. PMID- 2995387 TI - Influence of supramolecular structure on the enzyme mechanisms of rat liver lysyl tRNA synthetase-catalyzed reactions. Synthesis of P1,P4-bis(5' adenosyl)tetraphosphate. AB - Lysyl-tRNA synthetase, dissociated from the multienzyme complexes of aminoacyl tRNA synthetases from rat liver, was previously found to be 6-fold more active than the synthetase complex in the enzymatic synthesis of P1,P4-bis(5' adenosyl)tetraphosphate. The bi-substrate and product inhibition kinetics of the reaction are analyzed. Free lysyl-tRNA synthetase exhibits distinctly different kinetic patterns from those of an 18 S synthetase complex containing lysyl-tRNA synthetase. The 18 S synthetase complex shows kinetic patterns which are consistent with an ordered Bi Uni Uni Bi ping-pong mechanism. Free lysyl-tRNA synthetase shows kinetic patterns consistent with a random mechanism. The differences in the enzymatic properties are attributed to the organization of the supramolecular structure of the synthetase complex. The results suggest that association of the synthetases may affect the mechanisms of the synthesis of AppppA. PMID- 2995386 TI - Involvement of protein kinase C in pulmonary surfactant secretion from alveolar type II cells. AB - The purpose of this study is to clarify the involvement of protein kinase C in pulmonary surfactant secretion from adult rat alveolar type II cells in primary culture. Surfactant secretion in vitro is stimulated by at least two classes of compounds. One class, (e.g. terbutaline) increases intracellular cyclic AMP, whereas the other class (e.g. 12-O-tetradecanoylphorbol 13-acetate (TPA] does not. TPA has been shown to activate protein kinase C in other cell systems. In our studies, 1-oleoyl-2-acetyl-sn-glycerol (OAG), which is a direct activator of protein kinase C, stimulated [3H] phosphatidylcholine secretion by alveolar type II cells in a dose- and time-dependent manner. Tetracaine, which is an inhibitor of protein kinase C, inhibited the TPA-induced secretion of [3H]phosphatidylcholine from alveolar type II cells in a dose-dependent manner. However, tetracaine had no effect on terbutaline-induced secretion. The effects of terbutaline and OAG upon surfactant secretion were significantly more than additive, but those of TPA and OAG were less than additive. The specific activity of protein kinase C was 6-fold higher than cyclic AMP-dependent protein kinase found in type II cells when both kinases were assayed using lysine-rich histone as a common phosphate acceptor. Ninety-four per cent of protein kinase C activity was recovered in the cytosolic fraction of unstimulated type II cells, and 40% of activity in cytosolic fraction was translocated to particulate fraction upon treatment with TPA. As observed in other tissues, protein kinase C of alveolar type II cells was highly activated by 1,2-dioleoyl-sn-glycerol or TPA in the presence of Ca2+ and phosphatidylserine. These results suggest that pulmonary surfactant secretion in vitro is stimulated by both protein kinase C and cyclic AMP-dependent protein kinase. PMID- 2995389 TI - Sensitivity of the response of cytosolic calcium in Quin-2-loaded rat hepatocytes to glucagon, adenine nucleosides, and adenine nucleotides. AB - Hepatocytes from fasted rats were used to study the effect of glucagon on intracellular free cytosolic Ca2+ ([Ca2+]i) indicated by the use of Quin-2 calcium fluorescence. It was found that, in both male and female rats, glucagon increased [Ca2+]i at a half-maximally effective concentration (Kact) of 0.3 nM, a concentration known to be half-maximal for affecting several hepatic functions. Acute chelation of extracellular Ca2+ did not obliterate the hormone effect but shortened its duration. Cyclic AMP, 5'-AMP, ADP, and ATP also increased [Ca2+]i, while adenosine 2':3'-monophosphate and 3'-AMP did not. The rise in [Ca2+]i brought about by glucagon at near physiological concentrations may be responsible for the stimulation of glutamate metabolism produced acutely by glucagon. PMID- 2995388 TI - Steroids, intracellular sodium levels, and Na+/K+-ATPase regulation. AB - In outer medullary kidney tubules, both specific mineralocorticoid, and specific glucocorticoid Na+/K+-ATPase activation in vitro were inhibitable by amiloride, an inhibitor of a number of Na+-transporting mechanisms (Bentley, P.J. (1968) J. Physiol. (Lond.) 195, 317-330; Kinsella, J. L., and Aronson, P. S. (1980) Am. J. Physiol. 238, F461-F469). In addition, dexamethasone raised, whereas amiloride reduced, intracellular Na+ levels. These observations are consistent with the possibility that the steroidal responses are mediated by changes in intracellular Na+ ion activity. However, when intracellular Na+ levels were increased by the incubation of tubule segments in medium containing ouabain (10(-4) M), no Na+/K+ ATPase activation was observed, over incubation periods of up to 6 h. As mineralocorticoid and glucocorticoid effects are maximal within 2 h (Rayson, B.M., and Lowther, S.O. (1984) Am. J. Physiol. 246, F656-F662), these results suggest that the Na+ ion per se does not mediate the steroidal effects observed, directly. Incubation of tubule segments in medium containing 10(-4) M ouabain, at 37 degrees C, for longer periods (18 h), however, did indeed increase Na+/K+ ATPase activity, markedly. Thus, a potential homeostatic mechanism was demonstrable, where a chronic increase in intracellular Na+ level, measured after 2-4 h of treatment, resulted in an increase in Na+/K+-ATPase activity, such that the intracellular Na+ level was restored after 18-20 h of incubation to one not significantly different from the control value. This mechanism, however, appears to be clearly distinguishable from that which mediates steroidal Na+/K+-ATPase activation. PMID- 2995390 TI - Functional alterations in pancreatic beta cells as a factor in virus-induced hyperglycemia in mice. AB - Alterations in the functional capacity of pancreatic beta cells appear to contribute to coxsackievirus B4-induced, long-term hyperglycemia in mice. Mice infected with prototype B4 or its diabetogenic E2 variant were monitored for abnormalities in sugar metabolism (by the glucose tolerance test), for total protein and insulin synthesis in intact beta cells, for alterations in beta cell proteins, and for virus replication. The infected mice were hypoglycemic at 72 h postinfection and hyperglycemic at 6 weeks. At 8 weeks postinfection, few of the prototype- but most of the E2-infected mice remained hyperglycemic. Total protein and synthesis of immunoprecipitable insulin decreased during early infection. At 8 weeks postinfection, insulin synthesis in the prototype-infected mice increased almost to the level of control mice. Although insulin synthesis increased likewise in the E2-infected mice, it remained well below the control level. Two dimensional gel electrophoresis revealed the disappearance of many cellular proteins in beta cells from E2-infected mice but of very few in cells from prototype-infected mice at 72 h postinfection. Many of the disappearing proteins reappeared gradually in the E2-infected group. Infectious virus was recovered from the infected beta cells only at 72 h postinfection. Functional impairment in these cells appears to be a factor in virus-induced hyperglycemia. PMID- 2995391 TI - Polyoma virus major capsid protein, VP1. Purification after high level expression in Escherichia coli. AB - We have expression-cloned in Escherichia coli the major polyoma virus capsid protein, VP1. Under the inducible control of the hybrid tac promoter, VP1 constituted between 2 and 3% of the total host cell protein. The expressed VP1 was purified to near homogeneity with initial yields to 10%. Optimal expression was temperature-dependent, and significant intracellular degradation could be demonstrated. The final product was obtained as one predominant isoelectric focusing species, without the pattern of post-translational modification seen in virus-infected eukaryotic cells. The purified VP1 from E. coli will be useful as a substrate for the purification of VP1 modification enzymes and in the study of inter-VP1 oligomerization. PMID- 2995392 TI - Tangier disease. The complete mRNA sequence encoding for preproapo-A-I. AB - A cDNA library of Tangier liver mRNA has been established, and two apo-A-I containing clones were identified. The complete derived amino acid sequence of preproapo-A-I has been established by nucleic acid sequence analysis of cloned apo-A-I cDNA and specific primer extensions on Tangier liver RNA. Sequence analysis of the longest cDNA clone, pMDB136T, established the derived amino acid sequence of residues 116-243 of plasma apo-A-I. The remaining portion of the sequence of Tangier preproapo-A-I mRNA was established by sequence analysis of specific primer extensions of synthetic oligonucleotides on Tangier liver mRNA. This latter technique provided the derived amino acid sequence of residues -24 to 116, thus completing the entire preproapo-A-I structure. The structure of Tangier preproapo-A-I was identical to normal preproapo-A-I except for a single base substitution (G----T) which resulted in the isosteric replacement of a glutamic acid residue at position 120 to aspartic acid. These results are interpreted as indicating that there is no major structural defect in Tangier apo-A-I, and the rapid rate of catabolism of apo-A-I in Tangier disease is due to a post translational defect in apo-A-I metabolism. PMID- 2995393 TI - Enzymatic hydration of leukotriene A4. Purification and characterization of a novel epoxide hydrolase from human erythrocytes. AB - Human erythrocytes contained a soluble cytosolic epoxide hydrolase for stereospecific enzymatic hydration of leukotriene A4 into leukotriene B4. The enzyme was purified 1100-fold, to apparent electrophoretic homogeneity, by conventional DEAE-Sephacel fractionation followed by high performance anion exchange and chromatofocusing procedures. Its characteristics include a molecular weight of 54,000 +/- 1,000, an isoelectric point 4.9 +/- 0.2, a Km apparent from 7 to 36 microM for enzymatic hydration of leukotriene A4, and a pH optimum ranging from 7 to 8. The enzyme was partially inactivated by its initial exposure to leukotriene A4. There was slow but detectable enzymatic hydration (pmol/min/mg) of certain arachidonic acid epoxides including (+/-)-14,15-oxido 5,8-11-eicosatrienoic acid and (+/-)-11,12-oxido-5,8,14-eicosatrienoic acid, but not others, including 5,6-oxido-8,11,14-eicosatrienoic acid. Human erythrocyte epoxide hydrolase did not hydrate either styrene oxide or trans-stilbene oxide. In terms of its physical properties and substrate preference for leukotriene A4, the erythrocyte enzyme differs from previously described versions of epoxide hydrolase. Human erythrocytes represent a novel source for an extrahepatic, cytosolic epoxide hydrolase with a potential physiological role. PMID- 2995394 TI - Bacteriophage T4 DNA replication protein 41. Cloning of the gene and purification of the expressed protein. AB - The bacteriophage T4 primase, composed of the T4 proteins 41 and 61, synthesizes pentaribonucleotides used to prime DNA synthesis on single-stranded DNA in vitro. 41 protein is also a DNA helicase that opens DNA in the same direction as the growing replication fork. Previously, Mattson et al. (Mattson, T., Van Houwe, G., Bolle, A., Selzer, G., and Epstein, R. (1977) Mol. Gen. Genet. 154, 319-326) located part of gene 41 on a 3400-base pair EcoRI fragment of T4 DNA (map units 24.3 to 21.15). In this paper, we report the cloning of T4 DNA representing map units 24.3 to 20.06 in a multicopy plasmid vector. Extracts of cells containing this plasmid complement gene 41- extracts in a DNA synthesis assay, indicating that this region contains all the information necessary for the expression of active 41 protein. We located gene 41 more precisely between T4 map units 22.01 to 20.06 since our cloning of this region downstream of the strong lambda promoter PL results in the production of active 41 protein at a level 100-fold greater than after T4 infection. We have purified 133 mg of homogeneous 41 protein from 27 g of these cells. Like the 41 protein from T4 infected cells, the purified 41 protein in conjunction with the T4 gene 61 priming protein catalyzes primer formation (assayed by RNA primer-dependent DNA synthesis with T4 polymerase, the genes 44/62 and 45 polymerase accessory proteins, and the gene 32 helix-destabilizing protein) and is a helicase whose activity is stimulated by T4 61 protein. PMID- 2995395 TI - Bacteriophage T4 DNA replication protein 61. Cloning of the gene and purification of the expressed protein. AB - In vitro, a bacteriophage T4 primase composed of T4 61 and 41 proteins, catalyzes the formation of pentaribonucleotides used to initiate DNA synthesis on single stranded DNA. We have determined that cells containing a plasmid with the T4 DNA from 18.68 to 15.05 map units express an activity that substitutes for authentic 61 protein in vitro in catalyzing primer-dependent DNA synthesis with six other T4 DNA replication proteins. This result establishes that this region, genetically assigned to gene 61, is the structural gene for the priming protein. Cells containing a plasmid with gene 61 downstream of the strong phage lambda promoter PL and the antitermination site nutL produce 100-fold more 61 protein than T4-infected cells. We have developed an improved purification procedure which yields 100 mg of homogeneous, active protein from 178 g of these cells. In the plasmid, the T4 DNA downstream of gene 61 expresses a protein of 30,000 daltons. This protein may be the T4 DNA adenine methylase (dam) gene product, since Schlagman and Hattman (Schlagman, S. L. and Hattman, S. (1983) Gene 22, 139 156) have shown that this activity is expressed by plasmids containing T4 DNA from this region. In the PL, nutL vector, the expression of both the 30,000 dalton and 61 proteins is enhanced up to 20-fold by the presence of the phage lambda N protein, a transcription antitermination protein, suggesting that expression of the T4 DNA in the plasmid may be regulated transcriptionally. In addition, in both N+ and N- cells, the level of 61 protein, whose gene is proximal to PL on the plasmid, is lower than that of the product of the promoter distal 30,000-dalton protein gene. This result suggests that, at least in the plasmid construction, the expression of 61 protein may also be regulated after transcription. PMID- 2995396 TI - Radiographic differences between two subtypes of bronchioloalveolar carcinoma. AB - Bronchioloalveolar carcinoma has two light-microscopic, morphologic types, the alveolar type which has cuboidal cells resembling Type II pneumocytes, and the bronchiolar type in which these cells are of the tall columnar variety. To determine if these two different cellular patterns are associated with different clinical or radiologic patterns of disease, we compared the anthropometric, demographic and past medical history, the presenting symptoms, signs, radiographic changes and survival of patients with these two diseases. Clinical records, chest radiographs and pathologic specimens were reviewed by individuals blinded to the hypothesis. Of 30 patients reviewed, we found only one purely alveolar pattern, one predominantly alveolar, 13 mixed, 12 predominantly bronchiolar, and three purely bronchiolar. For analysis we combined the alveolar and the mixed groups and compared them to the purely and predominantly bronchiolar groups. Anthropometric and historical data were similar. The radiographs were different; the most striking difference was the presence of air bronchograms only in the bronchiolar group (p less than 0.0001). Of those who had previous chest films, 80% in the alveolar-mixed group were abnormal, whereas none of those in the bronchiolar group were (p = 0.02). All the initial films in the bronchiolar group had a lesion with definable borders, whereas only two-thirds of the mixed alveolar group did (p = 0.02). Some of the radiographic changes of bronchioloalveolar carcinoma depend on the histologic subtype. PMID- 2995397 TI - Influence of tissue heterogeneity on the determination of steroid receptors in breast cancer. AB - Biochemical determination of hormone receptors in carcinomas is influenced by the potential heterogeneity of the tissue samples. In order to check this, samples of 16 breast cancers were divided into 6 segments. These segments were alternately examined for their ratio of tumor tissue to connective tissue ("percentage of carcinoma") or for the content of estrogen and progesterone receptors. Only 3 of the tumors were homogeneous, and 9 of the heterogeneous tissues had hormone receptors. The segment with the maximum "percentage of carcinoma" was adjacent to that with the peak value of hormone receptors in all but 1 tissue. The same applied to the minima. The maximum and minimum values of estrogen and progesterone receptors were located within the same segment in all but one tissue sample. The results demonstrated that biochemical assay of hormone receptors is reliable. A reduction of the sample volume does not enhance the precision of the receptor assay, since it increases the possibility of a false negative result. PMID- 2995398 TI - Prostaglandin E2 and cyclic nucleotides in plasma and urine of colonic cancer patients. AB - Prostaglandin E2 and cyclic nucleotide levels were measured in plasma and urine of 14 patients with colonic cancer. The measurements were performed 1 day before and 8 days after the removal of the tumor by operation. There was no difference between the plasma PGE2 levels (in form of the 13, 14-dihydro-15-keto metabolite) before and after the operation, but they were significantly higher than the level of a control group. No differences before and after operation were found between plasma cyclic AMP (cAMP) levels, plasma cyclic GMP (cGMP) levels, urinary cAMP levels and urinary cGMP levels. All the cyclic nucleotide concentrations were within the normal range. No correlation could be found between the stage of the tumor spread and any of the substances analyzed. The conclusions are that plasma PGE2 and plasma and urinary cyclic nucleotides do not originate from the colonic tumor tissue, and that these substances cannot be used as tumor markers. PMID- 2995400 TI - Application of restriction endonucleases as a tool for studying the action of 4-S ethanolsulfido-cyclophosphamide on DNA with known sequences. AB - Lambda-DNA and plasmid pBR 322-DNA, respectively, were treated in vitro with increasing amounts of 4-S-ethanolsulfido-cyclophosphamide (CPA-P1). Subsequent digestion with restriction endonuclease Pvu II or Mbo II revealed discrete alterations in the cleavage patterns as compared to the controls, indicating subtle changes in DNA structure due to CPA-P1 interaction. In the case of pBR 322 DNA the CPA-P1 treatment of supercoiled circular DNA was more inhibitory to subsequent digestion as compared to the treatment of linearized plasmid DNA molecules. PMID- 2995399 TI - Epstein-Barr virus serology in nasopharyngeal carcinomas and other head and neck neoplasms in Italy. AB - This paper reports the results of the EBV-specific antibody response in 17 Italian nasopharyngeal carcinoma (NPC) patients, 15 other head and neck tumor patients and 15 normal controls. Nucleic acid hybridization has been performed on the biopsy tissue from 4 of the NPC patients, and EBV-DNA was present in two undifferentiated (WHO 3) tumors, and absent in two samples of the keratinizing (WHO 1) type. EBV serology appears to be specifically related to NPC, more evidently for VCA-IgA and EA-IgG antibodies, and useful as an aid in diagnosis of NPC. However, in order to assess a prognostic value of the above markers, a greater number of patients followed for a longer period of time (at least 5 years) is needed, and is currently being pursued. PMID- 2995401 TI - Cell proliferation and cell loss in intramucosal signet ring cell carcinoma of canine stomachs induced by N-ethyl-N'-nitro-N-nitrosoguanidine. AB - Signet ring cell carcinoma was induced in canine stomachs by N-ethyl-N'-nitro-N nitrosoguanidine, and modes of cell proliferation and turnover in the carcinoma were studied by 3H-thymidine autoradiography in conjunction with morphometric analysis. From 2 to 15 months after the cessation of 8 months carcinogen treatment, carcinomas in an early stage were obtained. Most of the cancer tissues confined to the lamina propria showed a layered structure. This comprised three layers; the superficial and the deep layer were composed of signet ring cells, and the middle layer was composed of small round cells. The dogs were labeled with 3H-thymidine by s.c. injection and by local infusion of the celiac artery. Flash-labeled autoradiographs revealed that most 3H-thymidine incorporating cancer cells were located around the middle layer, with a small amount of mucin. Using a pulse labeling experiment, those labeled carcinoma cells were shown to migrate from the middle layer towards the surface. Morphometric analysis of the autoradiographs showed that the small cells in the middle layer migrated upwards and produced mucin to become full-blown signet ring cells by 5.5 days. In 15 days, most labeled cancer cells in the superficial layer had disappeared. This mode of cellular turnover appeared to mimic a cell renewal system of the normal gastric mucosa. If the cancer cells turn over in this way, the tumor must grow slowly, remaining as an intramucosal cancer for a relatively long period. PMID- 2995402 TI - Phosphorylase a activity as an indicator of neutrophil activation by chemotactic peptide. AB - The activity of glycogen phosphorylase, an enzyme that is activated by both cAMP and calcium, was used as an indicator of the state of the cytoplasm after chemotactic stimulation of polymorphonuclear leukocytes (neutrophils). The activity of the enzyme showed a clear dependence on cytoplasmic calcium. Addition of the calcium ionophore A23187 caused a 4-5-fold increase in activity of phosphorylase a. In the absence of external Ca2+, A23187 caused only brief transient activation of phosphorylase; probably reflecting release of sequestered intracellular Ca2+. Addition of the chemotactic peptide N formylnorleucylleucylphenylalanine (FNLLP) caused a transient 2-3-fold activation of the enzyme. The dose-dependence of activation by FNLLP showed a peak at 10(-8) M, near the Kd of the receptor for FNLLP. The phosphorylase activity peaks by 90 s and then declines, returning to basal levels by 20 min after stimulation with 10(-8) M peptide and by 60 min with 10(-7) M peptide. This finding suggests that the cells do not need to maintain elevated cytoplasmic calcium levels to exhibit stimulated locomotion. Thus, if calcium continues to modulate the motility, there either must be highly localized changes that are not detected in measures of the total cytoplasm, or the sensitivity to calcium must be variable such that basal levels are sufficient to maintain locomotion. Cells loaded with the fluorescence calcium probe quin2 (0.6 mM) in the presence or absence of external Ca2+ had elevated phosphorylase levels before addition of FNLLP. Thus, the presence of quin2 may alter the cytoplasmic Ca2+ level, and it clearly alters some aspects of the neutrophil physiology. Phosphorylase a appears to be a sensitive, nonperturbing indicator of the cytoplasmic calcium levels. PMID- 2995403 TI - Kinetic analysis of F-actin depolymerization in the presence of platelet gelsolin and gelsolin-actin complexes. AB - Platelet gelsolin (G), a 90,000-mol-wt protein, binds tightly to actin (A) and calcium at low ionic strength to form a 1:2:2 complex, GA2Ca2 (Bryan, J., and M. Kurth, 1984, J. Biol. Chem. 259:7480-7487). Chromatography of actin and gelsolin mixtures in EGTA-containing solutions isolates a stable binary complex, GA1Ca1 (Kurth, M., and J. Bryan, 1984, J. Biol. Chem. 259:7473-7479). The effects of platelet gelsolin and the binary gelsolin-actin complex on the depolymerization kinetics of rabbit skeletal muscle actin were studied by diluting pyrenyl F-actin into gelsolin or complex-containing buffers; a decrease in fluorescence represents disassembly of filaments. Dilution of F-actin to below the critical concentration required for filament assembly gave a biphasic depolymerization curve with both fast and slow components. Dilution into buffers containing gelsolin, as GCa2, increased the rate of depolymerization and gave a first order decay. The rate of decrease in fluorescence was found to be gelsolin concentration dependent. Electron microscopy of samples taken shortly after dilution into GCa2 showed a marked reduction in filament length consistent with filament severing and an increase in the number of ends. Conversely, occupancy of the EGTA-stable actin-binding site by an actin monomer eliminated the severing activity. Dilution of F-actin into the gelsolin-actin complex, either as GA1Ca1 or GA1Ca2, resulted in a decrease in the rate of depolymerization that was consistent with filament end capping. This result indicates that the EGTA-stable binding site is required and must be unoccupied for filament severing to occur. The effectiveness of gelsolin, GCa2, in causing filament depolymerization was dependent upon the ionic conditions: in KCI, actin filaments appeared to be more stable and less susceptible to gelsolin, whereas in Mg2+, actin filaments were more easily fragmented. Finally, a comparison of the number of kinetically active ends generated when filaments were diluted into gelsolin versus the number formed when gelsolin can function as a nucleation site suggests that gelsolin may sever more than once. The data are consistent with a mechanism where gelsolin, with both actin-binding sites unoccupied, can sever but not cap F-actin. Occupancy of the EGTA-stable binding site yields a gelsolin-actin complex that can no longer sever filaments, but can cap filament ends. PMID- 2995405 TI - Dissociation of Madin-Darby canine kidney epithelial cells by the monoclonal antibody anti-arc-1: mechanistic aspects and identification of the antigen as a component related to uvomorulin. AB - It has previously been shown that the monoclonal antibody anti-Arc-1 dissociates Madin-Darby canine kidney (MDCK) epithelial cells and changes their morphology in vitro (Imhof, B.A., H.P. Vollmers, S.L. Goodman, and W. Birchmeier, 1983, Cell, 35:667-675). In this article we demonstrate that the anti-Arc-1 antibody recognizes an uvomorulin-like molecule on MDCK cells, i.e., it immunoprecipitates an 84-kD protein fragment from a tryptic digest of cell surfaces in the presence of Ca2+ (as does anti-uvomorulin antiserum). Furthermore, anti-uvomorulin antiserum prevents the binding of anti-Arc-1 to MDCK cells. The distribution of the Arc-1 antigen is also quite similar to that of uvomorulin: it is enriched at the cell-cell contacts both of MDCK cells and of cells in various canine tissues. In the intestinal epithelium the antigen could be further localized in the region of the junctional complex. To study the mechanism of action of the dissociating antibody, MDCK cells grown on Nuclepore filters in Boyden chambers were exposed to anti-Arc-1 from either the upper or lower compartment. It could be shown that the antibody interfered with cell adhesion only from the basolateral but not from the apical cell surface. Antibody action was inhibited in the presence of colchicine but not cytochalasin B. Furthermore, cell dissociation was prevented when the cellular cAMP level was raised. These findings indicate that the anti Arc-1 antibody acts on a target below the tight junctions (possibly on the antigen located in the junctional complex), and they confirm that cytoskeleton and metabolic factors are actively involved in the maintenance of junctional integrity. PMID- 2995404 TI - Processing of the rough endoplasmic reticulum membrane glycoproteins of rotavirus SA11. AB - The synthesis and oligosaccharide processing of the glycoproteins of SA11 rotavirus in infected Ma104 cells was examined. Rotavirus assembles in the rough endoplasmic reticulum (RER) and encodes two glycoproteins: VP7, a component of the outer viral capsid, and NCVP5, a nonstructural protein. A variety of evidence suggests the molecules are limited to the ER, a location consistent with the high mannose N-linked oligosaccharides modifying these proteins. VP7 and NCVP5 were shown to be integral membrane proteins. In an in vitro translation system supplemented with dog pancreas microsomes, they remained membrane associated after high salt treatment and sodium carbonate-mediated release of microsomal contents. In infected cells, the oligosaccharide processing of these molecules proceeded in a time-dependent manner. For VP7, Man8GlcNAc2 and Man6GlcNAc2 were the predominant intracellular species after a 5-min pulse with [3H]mannose and a 90 min chase, while in contrast, trimming of NCVP5 halted at Man8GlcNAc2. VP7 on mature virus was processed to Man5GlcNAc2. It is suggested that the alpha mannosidase activities responsible for the formation of these structures reside in the ER. In the presence of the energy inhibitor carbonyl cyanide m chlorophenylhydrazone (CCCP), processing of VP7 and the vesicular stomatitis virus G protein was blocked at Man8GlcNAc2. After a 20-min chase of [3H]mannose labeled molecules followed by addition of CCCP, trimming of VP7 could continue while processing of G protein remained blocked. Thus, an energy-sensitive translocation step within the ER may mark the divergence of the processing pathways of these glycoproteins. PMID- 2995406 TI - Biosynthesis and intracellular sorting of growth hormone-viral envelope glycoprotein hybrids. AB - Various aspects of the biogenetic mechanisms that are involved in the insertion of nascent plasma membrane proteins into the endoplasmic reticulum (ER) membrane and their subsequent distribution through the cell have been investigated. For these studies chimeric genes that encode hybrid proteins containing carboxy terminal portions of the influenza virus hemagglutinin (154 amino acids) or the vesicular stomatitis virus envelope glycoprotein (G) (60 amino acids) linked to the carboxy terminus of a nearly complete secretory polypeptide, growth hormone (GH), were used. In in vitro transcription-translation experiments, it was found that the insertion signal in the GH portion of the chimeras led to incorporation of the membrane protein segments into the ER membrane. Effectively, GH became part of the luminal segment of membrane proteins of which only very small segments, corresponding to the cytoplasmic portions of the G or HA proteins, remained exposed on the surface of the microsomes. When the chimeric genes were expressed in transfected cells, the products, as expected, failed to be secreted and remained cell-associated. These results support the assignment of a halt transfer role to segments of the membrane polypeptides that include their transmembrane portions. The hybrid polypeptide containing the carboxy-terminal portion of HA linked to GH accumulated in a juxtanuclear region of the cytoplasm within modified ER cisternae, closely apposed to the Golgi apparatus. The location and appearance of these cisternae suggested that they represent overdeveloped transitional ER elements and thus may correspond to a natural way station between the ER and the Golgi apparatus, in which further transfer of the artificial molecules is halted. The GH-G hybrid could only be detected in transfected cells treated with chloroquine, a drug that led to its accumulation in the membranes of endosome or lysosome-like cytoplasmic vesicles. Although the possibility that the chimeric protein entered such vesicles directly from the Golgi apparatus cannot be ruled out, it appears more likely that it was first transferred to the cell surface and was then internalized by endocytosis. PMID- 2995407 TI - Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine. AB - We applied the quick-freezing technique to investigate the precise temporal coincidence between the onset of quantal secretion and the appearance of fusions of synaptic vesicles with the prejunctional membrane. Frog cutaneous pectoris nerve-muscle preparations were soaked in modified Ringer's solution with 1 mM 4 aminopyridine, 10 mM Ca2+, and 10(-4) M d-Tubocurarine and quick-frozen 1-10 ms after a single supramaximal shock. The frozen muscles were then either freeze fractured or cryosubstituted in acetone with 13% OsO4 and processed for thin section electron microscopy. Temporal resolution of less than 1 ms can be achieved using a quick-freeze device that increases the rate of freezing of the muscle after it strikes the chilled copper block (15 degrees K) and that minimizes the precooling of the muscle during its descent toward the block. We minimized variations in transmission time by examining thin sections taken only from the medial edge of the muscle, which was at a fixed distance from the point of stimulation of the nerve. The ultrastructure of the cryosubstituted preparations was well preserved to a depth of 5 - 10 micron, and within this narrow band vesicles were found fused with the axolemma after a minimum delay of 2.5 ms after stimulation of the nerve. Since the total transmission time to this edge of the muscle was approximately 3 ms, these results indicate that the vesicles fuse with the axolemma precisely at the same time the quanta are released. Freeze-fracture does not seem to be an adequate experimental technique for this work because in the well-preserved band of the muscle the fracture plane crosses, but does not cleave, the inner hydrophobic domain of the plasmalemma. Fracture faces may form in deeper regions of the muscle where tissue preservation is unsatisfactory and freezing is delayed. PMID- 2995410 TI - Reduction of DNA synthesis in aging but still proliferating cells. AB - It is well known that cell proliferation (and hence, DNA synthesis) declines in human diploid fibroblast-like cells with increasing passage number. It is not clear whether DNA synthesis declines in the remaining cells that are still actively proliferating. Estimations of cell kinetic parameters permitted extrapolations to be made that reflected the declining numbers of cells still capable of DNA replication. DNA synthesis declined with culture age in intact cells, permeabilized cells, and in the isolated nuclear matrix even when corrected for declining numbers of proliferating cells. With age, DNA polymerase alpha and beta activity in cell lysates declined, but when corrected for the remaining proliferating cells, only polymerase alpha activity declined; DNA polymerase alpha and beta activity bound to the nuclear matrix declined, but when corrected for declining proliferation, no decline was apparent for either enzyme. There was an increase in the number of S1-nuclease sensitive sites and breaks in the parental DNA of the dividing cells in older cultures. It is suggested that in aging cultures, not only does overall DNA synthesis decline owing to decreasing cell proliferation, but also that DNA synthesis declines in the remaining proliferating cells, that this decline is not due to decreasing amounts of DNA polymerase bound to the nuclear matrix, and that alterations in DNA structure occur. PMID- 2995408 TI - Receptor function of mouse sperm surface galactosyltransferase during fertilization. AB - Past studies from this laboratory have suggested that mouse sperm binding to the egg zona pellucida is mediated by a sperm galactosyltransferase (GalTase), which recognizes and binds to terminal N-acetylglucosamine (GlcNAc) residues in the zona pellucida (Shur, B. D., and N. G. Hall, 1982, J. Cell Biol. 95:567-573; 95:574-579). We now present evidence that directly supports this mechanism for gamete binding. GalTase was purified to homogeneity by sequential affinity chromatography on GlcNAc-agarose and alpha-lactalbumin-agarose columns. The purified enzyme produced a dose-dependent inhibition of sperm binding to the zona pellucida, relative to controls. To inhibit sperm/zona binding, GalTase had to retain its native conformation, since neither heat-inactivated nor Mn++-deficient GalTase inhibited sperm binding. GalTase inhibition of sperm/zona binding was not due to steric blocking of an adjacent sperm receptor on the zona, since GalTase could be released from the zona pellucida by forced galactosylation with UDPGal, and the resulting galactosylated zona was still incapable of binding sperm. In control experiments, when UDPGal was replaced with the inappropriate sugar nucleotide, UDPglucose, sperm binding to the zona pellucida remained normal after the adsorbed GalTase was washed away. The addition of UDPGal produced a dose dependent inhibition of sperm/zona binding, and also dissociated preformed sperm/zona adhesions by catalyzing the release of the sperm GalTase from its GlcNAc substrate in the zona pellucida. Under identical conditions, UDP-glucose had no effect on sperm binding to the zona pellucida. The ability of UDPGal to dissociate sperm/zona adhesions was both time- and temperature-dependent. UDPGal produced nearly total inhibition of sperm/zona binding when the zonae pellucidae were first galactosylated to reduce the number of GalTase binding sites. Finally, monospecific anti-GalTase IgG and its Fab fragments produced a dose-dependent inhibition of sperm/zona binding and concomitantly blocked sperm GalTase catalytic activity. Preimmune IgG or anti-mouse brain IgG, which also binds to the sperm surface, had no effect. The sperm GalTase was localized by indirect immunofluorescence to a discrete plasma membrane domain on the dorsal surface of the anterior head overlying the intact acrosome. These results, along with earlier studies, show clearly that sperm GalTase serves as a principal gamete receptor during fertilization. PMID- 2995409 TI - A unique family of endothelial cell polypeptide mitogens: the antigenic and receptor cross-reactivity of bovine endothelial cell growth factor, brain-derived acidic fibroblast growth factor, and eye-derived growth factor-II. AB - Bovine brain, hypothalamus, pituitary, and retina contain potent anionic polypeptide mitogens for endothelial cells. Immunological assays using murine monoclonal antibodies against bovine endothelial cell growth factor (ECGF) and radioreceptor assays using [125I]ECGF were performed to determine the cross reactivity of ECGF with bovine acidic pI brain-derived fibroblast growth factor (acidic FGF) and bovine eye-derived growth factor-II [EDGF-II). We observed that acidic FGF and EDGF-II are recognized by anti-ECGF monoclonal antibodies and compete with [125I] ECGF for receptor occupancy. Furthermore, the biological activity of ECGF, acidic FGF, and EDGF-II is potentiated by the glycosaminoglycan, heparin. These results argue that ECGF, acidic FGF, and EDGF II belong to a common family of polypeptide growth factors. PMID- 2995411 TI - Differential expression of two linked selection genes (HSVI-tk and Eco.gpt) in transformed teratocarcinoma and in L cells. AB - Upon transfection of (TK-)F9 teratocarcinoma stem-cells and (TK-)L fibroblasts with a plasmid carrying two selection genes, Eco.gpt and HSVI-tk, selection for gpt gene yielded ten times fewer colonies than selection for tk. Only the transformed clones selected for gpt had measurable xanthine guanine phosphoribosyltransferase (XGPRT) activity (Jami et al., 1983). Eco.gpt coding for XGPRT was under the control of simian virus 40 (SV40) early genes' regulating sequences (SV-gpt). In the present study, it was verified that the low efficiency of gpt selection in mouse cells was not due to the eucaryotic controlling sequences added to the bacterial gene. The transformed clones selected for tk that had no XGPRT activity possessed at least one uninterrupted copy of the composite SV-gpt gene and as many copies of the transforming plasmid as the cells selected for gpt expression. In a further test, the gpt gene was placed under the control of tk-regulating sequences and inserted with the tk gene in the same vector. Under these conditions, expression of XGPRT in the transformed clones selected for tk was improved, even though relative selection for gpt remained low. PMID- 2995412 TI - Regulation of deoxyglucose uptake by adrenocorticotropic hormone in cultured neurons. AB - The influence of ACTH and some of its N-terminal related peptides was investigated on the uptake of (3H)-2-deoxy-D-glucose in pure cultures of neurons from chick embryo cerebral hemispheres. ACTH influences deoxyglucose uptake in a time and dose-dependent fashion. The stimulation of deoxyglucose uptake is observed after a delay of 6-8 h and requires active protein synthesis. ACTH does not affect deoxyglucose in non-neuronal cells (astroglial cells, hepatocytes, myoblasts, fibroblasts). The effect of various peptide hormones, neuropeptides and growth factors, active in the central nervous system or other tissues, has also been examined. None of these were able to stimulate deoxyglucose uptake, suggesting that the regulation of hexose uptake in neurons is specific for the ACTH-related peptides. PMID- 2995413 TI - Diacylglycerol treatment rapidly decreases the affinity of the epidermal growth factor receptors of Swiss 3T3 cells. AB - The synthetic diacylglycerol 1-oleoyl-2-acetyl glycerol (OAG) and phorbol esters activate protein kinase C in intact cells. We report here that OAG inhibits the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The inhibition was detected as early as 1 min after treatment at 37 degrees C and persisted for at least 120 min. The effect of OAG was reversed upon removal of this diacylglycerol. Detailed Scatchard analysis of 125I-EGF binding to Swiss 3T3 cells at 4 degrees C after a 1 h incubation with a saturating dose of OAG at 37 degrees C, demonstrates that this OAG pretreatment does not change the apparent number of EGF receptors but causes a marked decrease in their apparent affinity for the ligand. Prolonged treatment (40 h) of the cells with phorbol dibutyrate (PBt2) which causes a marked decrease in the number of phorbol ester binding sites and in the activity of protein kinase C, prevented the inhibition of 125I EGF binding by both PBt2 and OAG. The results support the possibility that protein kinase C plays a role in the transmodulation of the EGF receptor in intact cells. PMID- 2995414 TI - Relationship of ornithine decarboxylase activity and cAMP metabolism to proliferation of normal human bronchial epithelial cells. AB - The mechanisms of action of extracellular mitogens for normal human bronchial epithelial cells (NHBE) were investigated by observing their effects on selected biochemical pathways when the cells were incubated in serum-free media. We find that (a) epidermal growth factor (EGF) stimulates ornithine decarboxylase (ODC) activity and the rate of cell division without stimulating cAMP; (b) alone, pituitary extract (PEX) does not stimulate ODC activity, cAMP levels, or cell division; (c) when PEX is added to medium containing EGF there is a further increase in both ODC activity and the rate of cell division, again with no increase in cAMP levels; (d) in contrast, alone, L-epinephrine (EPI) stimulates an increase in both ODC and cAMP but does not stimulate cell division; (e) when EPI is added to medium containing both EGF and PEX a further increase in the rate of cell division is noted; (f) the specific inhibitor of ODC, alpha (difluoromethyl)-ornithine (DMFO), also inhibits NHBE cell proliferation; and (g) the beta-adrenergic receptor antagonist propranolol inhibits the mitogenic action and ODC induction by EPI observed under condition e. We conclude that an increase in ODC activity is necessary but not sufficient for an increase in proliferation of NHBE cells. In contrast, cAMP stimulation is not necessary for an increase in NHBE cell division. However, in the presence of undefined factors in PEX, increases in cAMP levels result in a synergistic increase in the rate of EGF stimulated clonal growth. By correlating the biochemical pathways invoked by EGF, PEX, EPI, and combinations thereof with their mitogenic actions, we have better defined the role each of these different mitogens plays in stimulating epithelial cell division. PMID- 2995416 TI - Receptor-mediated endocytosis of human transferrin and its cell surface receptor. AB - We have studied the process of transferrin endocytosis in human erythromyeloid cell line K562 using fluorescein (FL) and rhodamine (RD) labeled iron-saturated transferrin (FeTF), and a fluorescein labeled monoclonal antibody to the transferrin receptor (FL-mAB). Because the antireceptor antibody and FeTF bind to different sites on the TF receptor molecule, it is possible to simultaneously and independently follow receptor and ligand. We have measured the relative amounts of transferrin or antireceptor antibody bound in the presence or absence of proteolytic enzymes using a cell sorter. At 4 degrees C almost all of the FL-TF and the FL-mAB is surface bound in a diffuse pattern. Within minutes of elevating the temperature to 37 degrees C surface aggregates form and the FL-TF is internalized. At this time about one sixth of the transferrin is still surface bound and accessible to papain digestion. The remainder localizes in a perinuclear cluster of vesicles. Monoclonal antibody binds to the cell surface transferrin receptor but is not internalized at 4 degrees C or 37 degrees C. When unlabeled diferric transferrin is added, it promotes the uptake of the Fl-mAB. The addition of goat anti-mouse immunoglobulin also promotes FL-mAB uptake. These studies support the concept that a specific trigger is required for transferrin receptor endocytosis. PMID- 2995417 TI - F.9 embryonal carcinoma cell calcitonin autocrine system: correlation between immunoreactive calcitonin secretion and calcitonin receptor number. AB - Mouse teratocarcinoma cells in culture offer an in vitro system to study the initial steps of embryogenesis. It has been suggested that, at such early stages, cell functions are regulated by an autocrine process in which embryonic cells produce factors that in turn act on themselves. F.9 cells possess specific membrane receptors for calcitonin (CT) (120 fmol/mg of protein, Ka, = 3.5 X 10(8) M-1). These cells produce CT detected by heterologous radioimmunoassay in serum free culture-conditioned medium (75 pg/10(7) cells/12 h). When F.9 cells are incubated in serum-free medium, CT binding and secretion concomitantly drop by 50% within the first 2 h, then increase progressively to an upper plateau after the sixth hour. Preincubation with 10(-6) M CT leads to disappearance of CT receptors and CT secretion in the culture medium up to 6 h. Avoiding accumulation of CT in the medium by a continuous flow rate for 6 h leads to a progressive decrease of CT receptors. In addition, retinoic acid treatment of cells induces a parallel progressive decrease of CT receptor number and of total CT synthesis. These results suggest a reciprocal regulation of CT receptors and CT secretion, or a close relationship between their regulations. PMID- 2995415 TI - A collagen:glucosyltransferase at the surface of malignant fibroblasts. AB - 3T12 fibroblasts possess glucosyltransferases that catalyze the transfer of glucose from UDP-Glucose to galactosylhydroxylysyl residues on collagenous acceptors. The presence of the enzyme activity at the cell surface is indicated by the following findings: a) suspensions of intact cells, as well as intact cell monolayers, glucosylate gelatinized collagen b) glucose transfer is not due to UDP-Glucose hydrolysis and subsequent intracellular utilization of the free glucose c) experiments using cell suspensions with known proportions of broken cells indicate that the glucosyltransferase activity is attributable to intact cells and not to contamination by intracellular enzymes from broken cells. The Km value for UDP-Glucose is about 20 microM. The enzyme has a pronounced requirement for manganese, and shows highest activity between 2 and 10 mM. The optimal Mn2+ concentration for the intracellular gelatin:glucosyltransferase activity is more restricted (5 to 10 mM). Glucosyltransferase activity is strongly inhibited by diamide and N-ethylmaleimide (5 mM), suggesting that intact sulfhydryl residues present in the enzyme are essential. PMID- 2995418 TI - Platelet-derived growth factor stimulates rapid polyphosphoinositide breakdown in fetal human fibroblasts. AB - The addition of human platelet-derived growth factor (PDGF) to confluent, quiescent cultures of human diploid fibroblasts induced the rapid breakdown of cellular polyphosphoinositides. The levels of 32P-labeled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol (PI) decreased by 30 to 40% within 1 min after exposure of the cells to PDGF. The levels of PIP and PIP2 returned to their initial values within 3 and 10 min, respectively, after PDGF addition. The level of PI continued to increase after it had returned to control values and was up threefold within 30 min after PDGF addition. In cells prelabeled with myo-[3H]inositol PDGF caused an eightfold increase in the levels of inositol trisphosphate (IP3) within 2 min. Lesser increases, twofold and 1.3-fold, respectively, were seen in levels of inositol bisphosphate (IP2) and inositol monophosphate (IP). Within 10 min after PDGF addition the levels of all three inositol phosphates had decreased to control values. The levels of IP3 measured 2 min after PDGF addition depended on the PDGF concentration and were maximal at 5-10 ng/ml of PDGF. Similar concentrations of PDGF stimulate maximal cell growth and DNA synthesis in these cells. PMID- 2995419 TI - The possible cyclic AMP-dependence of an early prereplicative event that determines mitosis in regenerating rat liver. AB - The beta-adrenergic blocker dl-propranolol prevented a large proportion of regenerating rat liver cells from entering the mitotic phase of their first cell division cycle without affecting their ability to initiate or complete DNA replication. The drug, at a dose of 20 or 50 mg/kg of body weight, was most effective in reducing mitosis when injected between 1 and 2 hours after the proliferatively activating partial hepatectomy, which was 22 to 23 hours before the peak of DNA-synthetic activity. Propranolol also inhibited the early prereplicative surge of total liver cyclic AMP, which occurs shortly after partial hepatectomy, but this effect was not correlated to the mitosis-inhibiting activity. However, cyclic AMP or dibutyryl cyclic AMP completely reversed propranolol's mitosis-inhibiting action when injected between 1.5 and 2 hours (but not sooner or later) after partial hepatectomy, which was just before the total liver cyclic AMP content began to rise. Thus, there appears to be a transient, propranolol-inhibitable, probably cyclic AMP-initiated event in the early prereplicative development of rat hepatocytes that determines entry into mitosis rather than the initiation of DNA replication. PMID- 2995420 TI - Adenosine receptor-mediated changes in cyclic AMP production and DNA synthesis in cultured arterial smooth muscle cells. AB - The effects of adenosine and two analogs, L-phenylisopropyladenosine (L-PIA) and 5'-N-ethylcarboxamidoadenosine (NECA), on cAMP production and on platelet-derived growth factor (PDGF)-stimulated initiation of DNA synthesis in growth-arrested cultures of rat arterial smooth muscle cells (SMC) were studied. The intracellular cAMP concentration was dose-dependently enhanced by micromolar concentrations of adenosine and its analogs, with the potency order NECA greater than adenosine greater than L-PIA. The effect was antagonized, in a competitive manner, by the adenosine receptor antagonist 8-phenyltheophylline (8-PT). The stimulatory effect of adenosine was enhanced by 3 microM dipyridamole an adenosine-uptake blocker. DNA synthesis was inhibited in a parallel manner, showing the same potency order. The inhibition was antagonized by 8-PT. Forskolin, a diterpene with the ability to stimulate the catalytic unit of adenylate cyclase and thereby cAMP formation, potentiated the effects of micromolar concentrations of NECA and L-PIA. Forskolin, by itself, stimulated cAMP production and inhibited DNA synthesis. The forskolin-stimulated increase in cAMP was inhibited by L-PIA at nanomolar concentrations. L-PIA in the nanomolar concentration range also stimulated DNA synthesis when initiation was stimulated with suboptimal concentrations of PDGF. These findings suggest the presence of adenosine receptors of both the A1- and A2-subtype on SM-mediating bidirectional changes of cAMP and DNA synthesis. PMID- 2995421 TI - Wheat germ agglutinin and concanavalin A inhibit the response of human fibroblasts to peptide growth factors by a post-receptor mechanism. AB - The effect of plant lectins on amino acid uptake and DNA synthesis in cultured human skin fibroblasts stimulated by various peptide mitogens was studied. Wheat germ agglutinin (WGA), at a concentration of 5 micrograms/ml, which by itself had little effect on 3H-aminoisobutyric acid (AIB) uptake, markedly inhibited stimulation of 3H-AIB uptake by somatomedin-C, insulin, epidermal growth factor (EGF) and platelet-derived growth factor. This inhibition could not be overcome by increasing the concentration of peptide added. Neither WGA nor concanavalin A (Con A) significantly affected basal 3H-thymidine incorporation. However both lectins, at concentrations of 5-20 micrograms/ml, decreased EGF- and insulin stimulated DNA synthesis while succinyl Con A, a divalent lectin derivative, did not. The inhibitory effects of lectins on mitogenic stimulation were reversed by alpha-methyl mannose (Con A) or N-acetylglucosamine (WGA), and were not due to a reduction in the binding of growth factors to their receptors. It is concluded that certain lectins noncompetitively inhibit the response of human fibroblasts to multiple peptide mitogens at the post-receptor level, possibly by interfering with lateral mobility and aggregation of mitogen-receptor complexes. PMID- 2995422 TI - The metabolism of cobalamin bound to transcobalamin II and to glycoproteins that bind Cbl in HepG2 cells (human hepatoma). AB - The binding, internalization, processing and release of labeled cyanocobalamin (CN[57Co]Cbl) bound to human transcobalamin II (TC II) were studied in HepG2 cells, a line of hepatocytes derived from a human hepatoma. The cells bound the TC II-Cbl by specific, high affinity receptors. Within the cell, the CN-Cbl was promptly freed from TC II and the CN-Cbl converted to more active forms including adenosyl Cbl (AdoCbl) and methyl Cbl (MeCbl). Whereas free labeled Cbl was still present at 72 hours after entry, the cells also bound Cbl to an intracellular binder (ICB) presumed to represent the holo enzymes dependent on Cbl. At levels of TC II that saturated the receptors for TC II-Cbl, much of the Cbl entering the cells remained free and was converted to AdoCbl. Under these circumstances the cells released free Cbl, mostly AdoCbl. Human R type binders of Cbl, which are glycoproteins and some having a terminal galactose, were bound by the HepG2 cells. The binding was characteristic of the receptor system responsive to a terminal galactose, or asialoglycoproteins, but was inconsistent and of low affinity. Cbl bound to R binder was internalized and converted to coenzyme forms of Cbl, but the process was much less effective than when the Cbl entered via the TC II receptor system. It was concluded that the receptors for R-Cbl were unlikely to contribute to the physiologic transport of Cbl in man, but may function in some yet unknown way. PMID- 2995423 TI - Reinitiation of cellular DNA synthesis in BrdU-selected nondividing senescent WI 38 cells by simian virus 40 infection. AB - Bromodeoxyuridine-selected nondividing senescent WI-38 cells were stimulated to synthesize DNA, as evidenced by incorporation of [3H]thymidine into nuclei of senescent cells, after infection with simian virus 40 (SV40). Cellular DNA synthesis was confirmed by DNA-DNA hybridization experiments and the use of temperature-sensitive A gene mutants. The DNA synthesis was, at least in part, semiconservative, as microdensitometry of Feulgen-stained nuclei revealed increased DNA content in a large fraction of the cells in the infected population. Thus, senescent cells retain the capacity to replicate their DNA, despite their intrinsic inability to initiate DNA synthesis. PMID- 2995424 TI - Cell growth and differentiation in vitro in mouse macrophages transformed by a tsA mutant of simian virus 40. III. Large T antigen level and cell proliferation and survival in an SV40 tsA640-transformed macrophage line. AB - The levels of simian virus 40 (SV40) large T antigen in a tsA-transformed mouse macrophage line at the permissive (33 degrees C) and the nonpermissive (39 degrees C) temperature were examined by immunofluorescence, sodium dodecylsulfate polyacrylamide gel electrophoresis, complement fixation, and enzyme-linked immunosorbent assay. When the cells were confluent and rested at 33 degrees C, and then were shifted to 39 degrees C, the amount of large T antigen per cell decreased, and most cells survived and remained phagocytic. When the cells were proliferating at 33 degrees C, and then were shifted to 39 degrees C, the cells died with only a small reduction in the amount of large T antigen. Therefore, the physiological state of the cells may determine the survival of cells by affecting the level of large T antigen after exposure to 39 degrees. The confluent cells may be rested with a concomitant decrease of large T antigen. The proliferating cells may not survive in the presence of a relatively high level of functionally defective large T antigen at 39 degrees C. PMID- 2995425 TI - Dibutyryl cyclic AMP resistant MDCK cells in serum free medium have reduced cyclic AMP dependent protein kinase activity and a diminished effect of PGE1 on differentiated function. AB - Prostaglandin E1 (PGE1) has a stimulatory effect both on the growth and the expression of differentiated function of Madin Darby Canine Kidney (MDCK) cells in a hormonally defined medium (Medium K-1). While the stimulatory effect of PGE1 on MDCK cell growth is observed in subconfluent cultures, the effect of PGE1 on differentiated function (i.e., dome formation) is observed at confluency. PGE1 may possibly affect growth and such differentiated functions by separate mechanisms. In order to examine this possibility, dibutyryl cyclic AMP resistant variants of MDCK were selected. All of the variants were partially resistant to the growth inhibitory effects of dibutyryl cyclic AMP and theophylline. The cyclic AMP dependent protein kinase activity of four of the five variant clones studied was significantly reduced as compared with normal MDCK cells. The dependence of the kinase activity of several of the dibutyryl cyclic AMP resistant variants (DBr2 and DBr3) on the cyclic AMP concentration in the reaction mixture was compared with that of normal MDCK cells. At all of the cyclic AMP concentrations tested DBr2 and DBr3 cells had reduced protein kinase activity as compared with normal MDCK cells. This reduced activity could be attributed to a decrease in the Vmax for kinase in the two variants, rather than to a change in the Km of kinase for cyclic AMP. The cyclic AMP phosphodiesterase activity of dibutyryl cyclic AMP resistant variants was also studied. Unlike PGE1 independent clone 1, DBr2 and DBr3 cells did not differ significantly from normal MDCK cells with regard to their ability to degrade cyclic AMP. The growth and functional responsiveness of DBr2 and DBr3 cells to PGE1 was also examined. DBr2 and DBr3 cells were shown to retain a normal growth response to PGE1. However the capacity of DBr2 and DBr3 cells to form domes in response to PGE1 was dramatically reduced as compared with normal MDCK cells. Nevertheless DBr3 cells were shown to still retain the capacity to form domes in response to other inducers. The effect of PGE1 on one of the functional parameters involved in dome formation (the activity of the Na+/K+ATPase) was examined. The rate of ouabain sensitive Rb+ uptake was observed to be elevated in confluent monolayers of normal MDCK cells maintained in Medium K-1, as compared with monolayers maintained in Medium K-1 minus PGE1.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2995426 TI - SV40-transformed human fibroblasts: evidence for cellular aging in pre-crisis cells. AB - Pre-crisis SV40-transformed human diploid fibroblast (HDF) cultures have a finite proliferative lifespan, but they do not enter a viable senescent state at end of lifespan. Little is known about either the mechanism for this finite lifespan in SV40-transformed HDF or its relationship to finite lifespan in normal HDF. Recently we proposed that in normal HDF the phenomena of finite lifespan and arrest in a viable senescent state depend on two separate processes: 1) an age related decrease in the ability of the cells to recognize or respond to serum and/or other mitogens such that the cells become functionally mitogen-deprived at the end of lifespan; and 2) the ability of the cells to enter a viable, G1 arrested state whenever they experience mitogen deprivation. In this paper, data are presented that suggest that pre-crisis SV40-transformed HDF retain the first process described above, but lack the second process. It is shown that SV40 transformed HDF have a progressively decreasing ability to respond to serum as they age, but they continue to traverse the cell cycle at the end of lifespan. Concomitantly, the rate of cell death increases steadily toward the end of lifespan, thereby causing the total population to cease growing and ultimately to decline. Previous studies have shown that when SV40-transformed HDF are environmentally serum deprived, they likewise exhibit continued cell cycle traverse coupled with increased cell death. Thus, these results support the hypothesis that pre-crisis SV40-transformed HDF still undergo the same aging process as do normal HDF, but they end their lifespan in crisis rather than in the normal G1-arrested senescent state because they have lost their ability to enter a viable, G1-arrested state in response to mitogen deprivation. PMID- 2995427 TI - The selective induction of a small number of proteins during G1 transit results from the mitogenic action of pp60v-src in tsASV-infected rat cells. AB - Since the mitogenic/oncogenic pp60v-src product of the avian sarcoma virus (ASV) mutant, tsLA23, is abnormally thermolabile, tsLA23-NRK cells were phenotypically nontransformed at 40 degrees C and were consequently rendered quiescent by serum deprivation at this temperature. These serum-deprived cells were stimulated to transit G1 either as transformed cells by simply dropping the temperature to a pp60v-src -activating 36 degrees C, or as nontransformed cells by adding serum at 40 degrees C. Serum stimulation rapidly increased total protein synthesis in these cells and over 100 changes in cellular proteins (resolved by two dimensional gel electrophoresis) occurred during G1 transit. By contrast, pp60v src-activation did not increase total protein synthesis and only six proteins (18.5-44 kD) were clearly seen to appear or increase when quiescent cells were stimulated to transit G1 by activating pp60v-src. Three of these six pp60v-src- induced proteins also appeared or accumulated during the G1 transit of serum stimulated cells. The appearance and/or accumulation of the six proteins and the subsequent initiation of DNA replication may have resulted from pp60v-src stimulating only a small number of critical cellular genes because both the protein changes and DNA replication were completely suppressed by the transcription inhibitor actinomycin D. PMID- 2995428 TI - [Total duodenopancreatectomy for non-secreting pancreatic nesidioblastoma]. AB - Non-secreting tumors of pancreatic islets of Langerhans are now rarely encountered as a result of the increasing performance of techniques for detecting the different hormones of insular origin or their precursors. Histologically, these tumors, that can be termed nesidioblastomas of the pancreas, have a poorly defined potential course, but there is a definite risk of malignancy. A case is reported of a pancreatic nesidioblastoma located in two regions, treatment being by total duodenopancreatectomy with a good result at 4-year follow up review. A bibliographic list of 13 published reports in the international literature is provided. PMID- 2995430 TI - The in vitro regulation of human thyrocyte HLA-DR antigen expression. AB - The ability of endocrine organs to express human immune response-associated antigens (Ia), such as HLA-DR, is a subject of intense current interest. In this study, the effects of various potential modulators of thyroid follicular cell HLA DR expression were examined using in vitro cultures. A culture supernatant containing T-cell-derived lymphokines caused DR antigen expression on 13-18% of thyroid cells; more consistent effects were produced by recombinant gamma interferon, which led to 46-100% of the thyroid cells becoming HLA-DR positive after 3 days in culture. This effect was both time and concentration dependent and occurred in thyroid cells derived from patients with Graves' disease (n = 7) and Hashimoto's thyroiditis (n = 2) as well as from three subjects with no autoimmune thyroid disease. Thyroid cells stained with the monoclonal antibodies 4F2 and 5E9, which recognize cell activation antigens, regardless of whether they were treated with gamma-interferon. The lectin phytohemagglutinin also induced HLA-DR antigen expression (21-91% of cells positive). This response was dependent on T cell contamination of thyroid cell suspensions, since the effect was inhibited by cyclosporin A. HLA-DQ antigen expression, identified by the Leu-10 monoclonal antibody, was also induced on thyroid cells by gamma-interferon and phytohemagglutinin. In contrast, neither recombinant alpha-interferon nor interleukin-2 induced HLA-DR antigens. Irradiation reduced the response of thyroid cells to gamma-interferon, but two of the known inhibitors of macrophage Ia expression, prostaglandin E2 and (Bu)2cAMP, did not affect gamma-interferon induced thyroid cell HLA-DR expression. We were unable to detect interleukin-1 production by thyroid cells. These results suggest that 1) under normal circumstances, thyroid cells are 4F2 and 5E9 positive, but are incapable of expressing Ia antigens and, thus, of activating T cells to initiate autoimmune thyroiditis; and 2) once activated, for example by a virus, T cells could release gamma-interferon and induce thyroid cell HLA-DR and -DQ antigen expression; these Ia-positive thyroid cells could then have a role in maintaining or enhancing the autoimmune response. PMID- 2995429 TI - Anti-thyroid peroxidase antibody in patients with autoimmune thyroid disease: possible identity with anti-microsomal antibody. AB - Thyroid microsomal antigen and peroxidase (TPO) have a close intracellular anatomical relationship, especially in exocytotic vesicles. We considered that antibodies to microsomal antigen might react with TPO and therefore looked for the presence of antibodies against TPO in the serum of patients with autoimmune thyroid disease (AITD). TPO was prepared from Graves' thyroid glands, solubilized by n-octyl glucoside, and its activity was assayed by the guaiacol method. Control sera and sera with a positive microsomal hemagglutination test (MCHA(+) ) were assayed for their ability to precipitate TPO activity by incubation of sera with TPO and protein A. We identified MCHA(+) sera which caused precipitation of TPO activity, and the extent of precipitation was related to the amount of serum added. A significant correlation was present between this anti-peroxidase activity and microsomal antibodies titers, measured by a micro-ELISA method. Affinity columns prepared from immunoglobulins of MCHA(+) sera, coupled to Reacti Gel (6X), bound TPO activity, whereas using control IgG the recovery in the unbound fraction was high. These data provide evidence of antibodies against thyroid peroxidase in the serum of patients with AITD and suggest a close link between microsomal antigen and thyroid peroxidase. PMID- 2995431 TI - Direct action of opiates on bromocriptine-inhibited prolactin release by human prolactinoma cells in primary culture. AB - The present study was undertaken in order to examine the existence of opioid binding sites on cell membranes of human PRL-secreting tumors. Determination of opioid binding sites using different opiate ligands revealed one class of high affinity (KD, 1.3 nM) binding sites. Pharmacological characterization revealed kappa-1 selectivity (high affinity ethylketocyclazocine (EKC) binding, insensitive to 5 microM (D-Ala2, D-Leu5]enkephalin). Subsequently EKC was added to hPRL-secreting tumor cells in primary culture, alone or in combination with the dopaminergic agonist bromocriptine, and PRL release was measured. Opiates had no direct effect on PRL release by prolactinoma cells. When cells were preincubated with bromocriptine [6.6 +/- 4.8 (SD) X 10(-11) M], EKC (10(-11) to 10(-9) M) antagonized, in a dose-dependent manner, the dopaminergic inhibition of PRL release. The opiate effect was reversed by the opiate antagonist diprenorphine (10(-7) M). Cross-competition studies indicated that this effect was not due to the interaction of opiates with the dopaminergic receptor. In conclusion, opioid binding sites are found on prolactinoma cells. The binding of kappa-1 type opioid ligands modulates the inhibitory effect of dopamine upon PRL release. PMID- 2995432 TI - Circulating activated B cells in primary biliary cirrhosis. AB - Since patients with primary biliary cirrhosis (PBC) have evidence of abnormal function of the humoral immune system, we determined if B cells from patients with this disease show evidence of activation and can be stimulated by polyclonal activators. Using a reverse hemolytic plaque assay, it was found that patients with PBC had a significant increase in the number of circulating immunoglobulin secreting cells, compared to normal controls and patients with chronic type B hepatitis virus (HBV) infection. However, the total number of activated cells was less than 1% of the total circulating B-cell population. Furthermore, we were unable to detect an increase in the expression of transferrin receptors, a membrane receptor associated with B-cell activation, in the majority of B cells in patients with PBC. In other studies, immunoglobulin production by lymphocytes from patients with PBC, when stimulated with the polyclonal activators pokeweed mitogen and Epstein-Barr virus (EBV), was reduced. This hyporesponsiveness was not due to a decrease in the number of B cells, as determined by staining with the monoclonal antibody anti-Leu 12. Furthermore, the decreased response to B cells to polyclonal activation in PBC patients was not due increased suppressor T cell function, since EBV-simulated cultures of lymphocytes from patients with PBC demonstrated diminished suppression of immunoglobulin-secreting cells after 14 days of culture compared to controls. These findings suggest that the humoral abnormalities in PBC are due to the activation of a small subpopulation of B cells rather than to generalized B-cell hyperactivity. PMID- 2995433 TI - Immunological and virological investigation in patients with lymphoadenopathy syndrome and in a population at risk for acquired immunodeficiency syndrome (AIDS), with particular focus on the detection of antibodies to human T lymphotropic retroviruses (HTLV III). AB - Thirteen patients affected with unexplained lymphoadenopathy, fever, weight loss, and diarrhea (lymphoadenopathy syndrome; LAS) were evaluated for the possible appearance of acquired immunodeficiency syndrome (AIDS) and for immunological and virological characterization. The patients belonged to categories of individuals at risk for AIDS and were homosexual and/or drug abusers or hemophiliacs. Lymph node biopsy showed the histological picture of a follicular hyperplasia. The study of cell-mediated immunity (CMI), humoral immune response, and natural killer (NK) activity demonstrated a significant decrease in T cells with the helper/inducer phenotype (OKT4+ cells) and a relatively increased number of lymphocytes with the suppressor/cytotoxic phenotype (OKT8+ cells). NK activity was significantly lower than in normal controls. The in vitro response to polyclonal activators (phytohemagglutinin; PHA) and the cutaneous responsiveness to recall skin tests were impaired, whereas immunoglobulin production was increased, mainly in the IgG fraction. Virological studies showed high serum antibody titers to cytomegalovirus (CMV) but a lack of specific CMI as assayed by the leukocyte migration inhibition test (LMIT). CMV was also isolated from the urine specimen of one patient. The antibody pattern to Epstein-Barr virus (EBV) showed the uncommon contemporary presence of both Epstein-Barr nuclear antigen (EBNA) and early antigen (EA) antibodies. Antibodies to human T-lymphotropic retroviruses (HTLV III) were positive in 10 patients and the virus was isolated in 3 of them. In some patients the presence of serum antibodies to HTLV III was not associated with an impairment of the immune function. A group of individuals at risk for AIDS without LAS was also evaluated for the presence of HTLV III antibodies; the percentage of positive sera was 11.4.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995434 TI - Specificity repertoire of lymphocytes from multiple myeloma patients. I. High frequency of B cells specific for idiotypic and F(ab')2-region determinants on immunoglobulin. AB - The specificity repertoire of B lymphocytes from 14 multiple myeloma patients has been studied using the technique of Epstein-Barr virus (EBV) transformation of peripheral blood lymphocytes (PBL) coupled with clonal analysis by limiting dilution. We find that up to 100% of the B cells from myeloma patients undergoing EBV transformation secrete IgM specific for determinants on the F(ab')2 region of autologous and/or heterologous monoclonal immunoglobulin. In normal individuals 0.02-0.73% of the transformed B cells secrete IgM specific for F(ab')2 determinants. Two patients with monoclonal gammopathy of undetermined significance had only a weak reactivity to F(ab')2 fragments. The number of anti F(ab')2 B cells was up to 145-fold greater in patients than in normal donors. The majority of antibodies from patient clones recognized determinants shared among 3 12 different F(ab')2 fragments, whereas those originating from normal donor B cells saw determinants expressed on only one or two of the panel of test F(ab')2 fragments. There was a preference for autologous M components and a high proportion of antiidiotypic reactivity in five of eight patients so analyzed. We speculate that these findings indicate the existence of an anti-F(ab')2 immunoregulatory network mediating patient immunodeficiency network mediating patient immunodeficiency, thereby creating an abnormality that may enable the progression of multiple myeloma. PMID- 2995436 TI - Pathogenesis of herpes simplex labialis: experimental induction of lesions with UV light. AB - To develop a model system of herpes simplex labialis which would enable the study of patients before lesion onset, five patients were exposed to various doses of UV light from a sunlamp at their usual site of lesions. Six of 10 treatments resulted in the development of herpes labialis. Three of four treatments with the highest exposure levels led to large, vesicular, virus culture-positive sores. Side effects from sunlamp exposure were minimal. PMID- 2995435 TI - Resistance of leishmanial phosphatases to inactivation by oxygen metabolites. AB - Leishmania donovani promastigotes produce large quantities of two distinct acid phosphatases; a tartrate-resistant enzyme is localized to the external surface of the plasma membrane, and a tartrate-sensitive enzyme is secreted into the growth medium. It was shown previously that preincubation of human neutrophils and macrophages with the tartrate-resistant phosphatase markedly reduced the ability of these host cells to produce superoxide anions in response to stimulation with the activator formyl-methionyl-leucyl-phenylalanine. The possibility that the cell surface acid phosphatase or the phosphatase that is secreted into the extracellular fluid might compromise other host cell functions, especially intracellular ones, depends on the ability of the enzyme to resist exposure to toxic oxygen metabolites (e.g., superoxide anion, hydrogen peroxide, hypochlorite) generated by phagocytic cells. In the present report, we show that both leishmanial acid phosphatases were relatively resistant to inactivation by oxygen metabolites. At pH 5.5, the activity of the tartrate-resistant phosphatase was reduced 50% by incubation for 1 h with each of the following: 30 mM O2-, 500 mM hydrogen peroxide, and 6 mM hypochlorite ion. These concentrations are many fold greater than the concentrations of these substances that are generated by stimulated polymorphonuclear phagocytes. The tartrate-sensitive acid phosphatase differed markedly from the tartrate-resistant phosphatase in that the former was essentially insensitive to even very high concentrations of superoxide anion and hydrogen peroxide. Furthermore, 50% inactivation of the tartrate-sensitive leishmanial phosphatase required exposure to 35 mM hypochlorite for 30 min. These results indicate that the catalytic potential of these two leishmanial acid phosphatases probably survives exposure to toxic oxygen metabolites generated by neutrophils and macrophages. PMID- 2995437 TI - Competitive enzyme immunoassays for the rapid detection of antibodies to feline infectious peritonitis virus polypeptides. AB - Monoclonal antibodies specific for the envelope (E1), peplomer (E2), and nucleocapsid (N) polypeptides of feline infectious peritonitis virus (FIPV) were used in rapid, competitive enzyme-linked immunosorbent assays (ELISA) to study the humoral immune response of cats to FIPV infection. Results from the competitive ELISAs were correlated with those from immunofluorescent antibody assays (IFAs) on 203 samples obtained from 64 individual cats. The IFA results correlated best with those obtained with the anti-E1 specific competitive ELISA (85.7%). In contrast, anti-N and anti-E2 competitive ELISA results correlated with IFA results only 65.5 and 2.4% of the time, respectively. The results of the anti-E1 specific competitive ELISA were not influenced by the total immunoglobulin concentration or the possible presence of free viral antigens in the serum. These results suggest that a competitive ELISA involving the use of enzyme-conjugated monoclonal antibody to the E1 glycoprotein of FIPV is a simple and rapid replacement for the more cumbersome IFA. PMID- 2995438 TI - New medium for the production of cholera toxin by Vibrio cholerae O1 biotype El Tor. AB - A new medium that stimulates in vitro production of cholera toxin by Vibrio cholerae O1 El Tor (El Tor vibrios) was developed. The medium contains 0.5% NaCl, 0.3% NaHCO3, 0.4% yeast extract, and 1.5% Bacto-Peptone. El Tor vibrios were cultured in a stationary test tube at 37 degrees C for 20 h. The culture supernatant was assayed for cholera toxin by a reversed passive latex agglutination method. Most vibrios grown in this medium produced 10 to 20 times more toxin than in traditional syncase medium. The number of live vibrios at the end of culture was about 10(8)/ml in the new medium (AKI medium) and about 10(10)/ml in the syncase medium. As a result, each individual organism in the new medium should have produced as much as 1,000 times more toxin than in syncase medium. Sodium bicarbonate was found to be the most important factor in toxin production by El Tor vibrios in the new medium. We recommend this new medium because of its high yield of cholera toxin and its technical simplicity. PMID- 2995439 TI - Fluoroimmunoassay for detection of rubella-specific immunoglobulin M: comparison with indirect enzyme immunoassay and mu-chain capture. AB - The performance of a commercially-available method of fluoroimmunoassay (Rubella M FIAX, International Diagnostic Technology, Santa Clara, Calif.), designed for the detection of rubella-specific immunoglobulin M, was tested with 137 selected sera, including 52 from cases of primary rubella, 29 from healthy pregnant women, 21 containing rheumatoid factor, and 35 from cases of infectious mononucleosis (IM) caused by Epstein-Barr virus. The results were compared with those obtained by commercial indirect enzyme immunoassay (EIA) and EIA anti-mu chain capture tests. The fluoroimmunoassay technique showed a satisfactory level of sensitivity, but low values had to be interpreted with caution as false-positive results were detected with sera with rheumatoid factor and from IM cases, even after preliminary treatment of sera with the anti-human immunoglobulin G antisera provided in the kit. On the other hand, no false-positive results in the analysis of IM sera were seen in the EIA anti-mu chain capture method. Because of its sensitivity and specificity, we recommend the use of the latter technique for the diagnosis of primary rubella. PMID- 2995440 TI - Comparative susceptibilities of strain MRC-5 human embryonic lung fibroblast cells and the Cooney strain of human fetal tonsil cells for isolation of rhinoviruses from clinical specimens. AB - The comparative susceptibilities for rhinovirus isolation were determined for strain MRC-5 human embryonic lung fibroblast and human fetal tonsil cells. Samples from nasopharyngeal and throat swab clinical specimens were inoculated onto duplicate monolayers of each cell type. Of 105 rhinovirus-positive specimens, 78 (74%) were positive in MRC-5 cells, and 102 (97%) were positive in fetal tonsil cells (P less than 0.001). For 75 specimens positive in both, the mean time to cytopathic effect development with standard deviation was 1 +/- 2 days shorter in fetal tonsil cells (P less than 0.01). The use of a second virus culture increased rhinovirus isolations by 10% compared with a single culture. PMID- 2995441 TI - Examination of the Rotazyme II enzyme immunoassay for the diagnosis of rotavirus gastroenteritis. AB - Rotazyme II, which is a shorter version of Rotazyme (less than 3 h), was compared with electron microscopy and Rotazyme for sensitivity and specificity on 229 human stool specimens. Compared with electron microscopy, the newer assay was found to be 99.4% sensitive and 97.3% specific for an overall agreement of 98.7%. After resolution of discordant results by blocking tests, the Rotazyme II enzyme immunoassay was shown to be more sensitive than electron microscopy and, therefore, 100% specific. PMID- 2995442 TI - Renal adaptation to potassium in the adrenalectomized rabbit. Role of distal tubular sodium-potassium adenosine triphosphatase. AB - Potassium secretion and sodium-potassium adenosine triphosphatase (Na-K-ATPase) activity in the distal nephron segments are known to be influenced by the dietary intake of K+. This has been attributed to a change in the plasma aldosterone level, which also influences K+ secretion and Na-K-ATPase activity in the distal nephron. To investigate whether or not dietary K+ can modulate Na-K-ATPase activity in the distal nephron independently of aldosterone, we determined Na-K ATPase activity in four distinct nephron segments of adrenalectomized (adx) rabbits given four specific diets for 1 wk before experimentation. Na-K-ATPase activity was determined by a fluorometric microassay in which ATP hydrolysis is coupled to NADH oxidation. The nephron segments examined were the distal convoluted tubule (DCT), the connecting tubule (CNT), the cortical collecting duct (CCD), and the outer medullary collecting duct (MCD). All diets were similar in composition except for their K+ contents, which were 100, 300, 500, and 700 meq/kg in groups 1-4, respectively. In these adx animals, Na-K-ATPase activity increased greater than 200% in the CCD as the dietary intake of K+ increased. There was a linear relationship between K+ excretion and the enzyme activity in this segment. There was a 50% increase in Na-K-ATPase activity in the CNT as the dietary intake of K+ increased in adx animals. However, there were no significant differences in Na-K-ATPase activities in the DCT and MCD among the four treatment groups. It is concluded that dietary K+ intake can influence Na-K-ATPase activity in the CCD and CNT independently of plasma aldosterone levels. PMID- 2995443 TI - Inhibition of vasopressin-stimulated water flow in toad bladder by phorbol myristate acetate, dioctanoylglycerol, and RHC-80267. Evidence for modulation of action of vasopressin by protein kinase C. AB - The action of vasopressin (AVP) in transporting epithelia is mediated by cyclic AMP(cAMP), whereas its effects in hepatocytes are mediated by calcium and phosphoinositides. Based on our recent observation that AVP stimulates phosphoinositide turnover in toad bladder, we examined the role of calcium phospholipid-dependent kinase (protein kinase C) as a modulator of AVP's hydroosmotic effect. Phorbol myristate acetate (PMA), which can substitute for diglyceride as an activator of protein kinase C, the diglyceride dioctanoylglycerol, and RHC-80267, a glyceride lipase inhibitor that should increase diglyceride levels, inhibited AVP-stimulated water flow, but not water flow stimulated by cAMP, suggesting inhibition of cyclic AMP production. Both the dioctanoylglycerol and RHC-80267, but not PMA, also decreased water flow in response to 8-bromo cAMP indicating a potential inhibition at post-cAMP events as well. PMA increased prostaglandin synthesis; however, inhibition of water flow persisted even when prostaglandin synthesis was completely blocked by incubation with naproxen. Furthermore, water flow was not inhibited by incubation with the inactive diglyceride substitute phorbol didecanoate, supporting the specificity of the PMA inhibition. Consistent with the site of action at adenylate cyclase suggested by the transport experiments, PMA and RHC-80237 decreased both cell cAMP content and the cyclic AMP-dependent kinase ratio (-cAMP/+cAMP), an index of intracellular cyclic AMP effect. Assay for protein kinase C activity in toad bladder epithelial cell supernatant demonstrated that the toad bladder indeed contains a kinase stimulable by phospholipid, calcium, and PMA. As an apparently independent effect, we found that addition of PMA, but not dioctanoylglycerol or RHC-80267, to the mucosal bath increased both water permeability and the frequency of granular cell luminal membrane aggregates in the absence of vasopressin, consistent with stimulation of fusion events at the luminal membrane. Our data suggest that protein kinase C can modulate AVP-stimulated water flow in toad bladder by inhibiting cAMP generation, and perhaps post-cAMP steps as well, and support the hypothesis that AVP-stimulated turnover of membrane phosphoinositides antagonize the effects of AVP via changes in diglyceride, calcium, and protein kinase C. PMID- 2995444 TI - Intracellular pH modulates the generation of superoxide radicals by human neutrophils. AB - The relationship of intracellular pH (pHi) to superoxide radical (O2-) generation was investigated in chemotactic factor-stimulated human neutrophils. Exposure of cells to 100 nM N-formylmethionyl-leucyl-phenylalanine (FMLP) caused activation of Na/H exchange which, in 140 mM Na medium (pH0 7.40), led to a rise in pHi from 7.22 to 7.80. This pHi change was sensitive to amiloride (apparent Ki 78 microM), an inhibitor of Na/H countertransport. The time course of the alkalinization was similar to that of FMLP-stimulated O2- production, which was complete by 5 min. In the presence of 1 mM amiloride, which nearly blocked the pHi transient elicited by FMLP, or in the absence of external Na, where intracellular acidification was observed in FMLP-stimulated cells, O2- release was still roughly 25-45% of normal. Thus, an alkalinization cannot be an obligatory requirement for O2- generation. By independently varying either pH0, pHi, or the internal or external concentrations of Na, both the direction and magnitude of the FMLP-induced pHi transients could be altered. In each instance, the amount of O2- release correlated directly with pHi and was enhanced by intracellular alkalinization. In the absence of FMLP, a rise in pHi to 7.7-7.8 by exposure of cells to 30 mM NH4Cl, 10 microM monensin (a Na/H exchanging ionophore), or after a prepulse with 18% CO2 did not result in O2- generation. Thus, these results imply that an alkalinization per se is not a sufficient trigger. Neutrophils exposed to 4 nM FMLP exhibited a threefold slower rate of alkalinization (reaching pHi approximately 7.80 by 20-30 min) as compared to that obtained with 100 nM FMLP and did not release significant amounts of O2- under normal incubation conditions. However, these cells could be induced to generate O2- when the degree of alkalinization was enhanced by internal Na depletion or by pretreatment with 18% CO2. Together, these results indicate a modulating effect of pHi on O2- production and suggest that other functional responses of neutrophils may be regulated by their pHi. PMID- 2995445 TI - Deoxyribonucleic acid polymorphism in the apolipoprotein A-1-C-III gene cluster. Association with hypertriglyceridemia. AB - A DNA sequence polymorphism, revealed by digestion of human DNA with the restriction endonuclease Sst-1 and hybridization with an apolipoprotein A-I complementary DNA clone, has been shown to be located in or close to the 3' noncoding region of the apolipoprotein C-III gene. This polymorphism is found in significantly increased prevalence (P less than 0.001) in Caucasian hypertriglyceridemic subjects compared with race-matched controls, and its distribution in normal individuals of differing racial origins is reported. Furthermore, no alteration of high density lipoprotein or apolipoprotein A-I and apolipoprotein C-III phenotypes was observed in individuals with or without the polymorphism. PMID- 2995446 TI - Terbutaline-induced desensitization of human lymphocyte beta 2-adrenoceptors. Accelerated restoration of beta-adrenoceptor responsiveness by prednisone and ketotifen. AB - We investigated, in 36 healthy volunteers, the effects of prednisone and ketotifen on recovery of lymphocyte beta 2-adrenoceptor density (determined by ( )-125iodocyanopindolol binding) and responsiveness (assessed by lymphocyte cyclic AMP [cAMP] responses to 10 microM (-)-isoprenaline) after desensitization by the beta 2-agonist terbutaline. Terbutaline (3 X 5 mg/d) decreased lymphocyte beta 2 adrenoceptor density by approximately 40-50%; concomitantly, lymphocyte cAMP responses to 10 microM (-)-isoprenaline were significantly reduced. After withdrawal of terbutaline beta 2-adrenoceptor, density and responsiveness gradually increased, reaching predrug levels after 4 d. Prednisone (1 X 100 mg orally) accelerated beta 2-adrenoceptor recovery; only 8-10 h after administration of the steroid beta 2-adrenoceptor density and cAMP responses to ( )-isoprenaline had reached values not significantly different from pretreatment levels. Similar effects were obtained with ketotifen (2 mg; thereafter 2 X 1 mg/d for 4 d): 24 h after application of the drug beta 2-adrenoceptor density and cAMP responses to (-)-isoprenaline had reached pretreatment levels. Furthermore, ketotifen simultaneously applied with terbutaline completely prevented terbutaline-induced decrease in lymphocyte beta 2-adrenoceptor density and responsiveness. Prednisone (1 X 100 mg orally) or ketotifen (2 mg; thereafter 2 X 1 mg/d for 2 d) had no significant influence on lymphocyte beta 2-adrenoceptor density in healthy volunteers not pretreated with terbutaline, but shifted the ratio high-to-low affinity state of the lymphocyte beta 2-adrenoceptor toward high affinity state. We conclude that glucocorticoids as well as ketotifen can accelerate recovery of density and responsiveness of lymphocyte beta 2 adrenoceptors desensitized by long-term treatment with beta 2-agonists. Such an effect may have clinical implications for preventing tachyphylaxis of asthmatic patients against therapy with beta 2-agonists. PMID- 2995447 TI - Receptor-independent low density lipoprotein transport in the rat in vivo. Quantitation, characterization, and metabolic consequences. AB - Receptor-independent low density lipoprotein (LDL) transport plays a critical role in the regulation of plasma cholesterol levels; hence, these studies were done to characterize this process in the tissues of the rat. High rates of receptor-independent clearance were found in the spleen, but other organs, like liver, gastrointestinal tract, and endocrine glands manifested lower clearance rates that varied from 3 to 9 microliter/h per g, while the rates in nervous tissue, muscle, and adipose tissue were less than 1 microliter/h per g. Receptor dependent uptake was much higher in liver (85 microliter/h per g) and adrenal gland (219 microliter/h per g), but was also low in most other tissues. At normal plasma LDL concentrations, 67% of the receptor-dependent transport in the whole animal was accounted for by LDL uptake in the liver. In contrast, the receptor independent uptake found in the whole animal took place in many organs, including skeletal muscle (20%), liver (16%), small bowel (15%), skin (10%), and spleen (7%). Furthermore, in liver, the rate of cholesterol synthesis could be varied 11 fold, yet the rate of receptor-independent LDL clearance remained constant at approximately 8 microliter/h per g. When the circulating levels of LDL were systematically increased, receptor-independent LDL clearance also remained constant, so that hepatic LDL-cholesterol uptake by this mechanism increased linearly, from 1 to 20 micrograms/h per g, as the plasma LDL-cholesterol level was increased from 10 to 250 mg/dl. Finally, when equal amounts of LDL cholesterol were delivered into the liver by either the receptor-dependent or receptor-independent mechanism, there was significant suppression of cholesterol synthesis and an increase in cholesteryl esters. Thus, in any situation in which receptor-dependent LDL degradation is lost, cholesterol balance in the whole animal and across individual organs is maintained by receptor-independent mechanisms, although when the new steady state is achieved, circulating levels of LDL must necessarily be very much increased. PMID- 2995449 TI - Detection of a frequent restriction fragment length polymorphism in the human T cell antigen receptor beta chain locus. A potential diagnostic tool. AB - Abnormal T cell function is a feature of a spectrum of inherited and acquired diseases. We have detected a frequent restriction fragment length polymorphism in the human T cell antigen receptor beta-chain locus that may aid in the analysis of these disorders. A study of a panel of 18 normal individuals, testing for the presence of the polymorphism, showed it to account for 36% of the alleles in that group. In view of the fact that the T cell receptor beta-chain locus has been mapped to chromosome 7, and that the disease ataxia telangiectasia (AT) is associated both with abnormal T cell function and with chromosomal abnormalities of the same region of chromosome 7, we investigated the possibility that the polymorphism could demonstrate linkage of the T cell receptor locus to the gene for that disease. We demonstrated that the mutation causing AT did not lie within the beta-chain locus itself, and that there was preliminary evidence that the two loci were not closely linked. This polymorphism may provide a useful tool for the study of other genetic disorders associated with abnormalities of T cell function, as well as disorders associated with inherited or acquired abnormalities of chromosome 7. PMID- 2995448 TI - Experimental pulmonary inflammatory injury in the monkey. AB - Inflammatory pulmonary injury was induced in Macaca mulatta rhesus monkeys by the intrabronchial instillation of the formylated peptide norleu-leu-phe (FNLP) or phorbol myristate acetate (PMA). Indicators of pulmonary injury included an increase in mean protein content of bronchoalveolar lavage (BAL) fluid from 0.51 mg/ml in untreated animals to 3.74 mg/ml and 6.64 mg/ml in FNLP- and PMA-treated animals, respectively, the appearance of a diffuse pulmonary infiltrate in chest roentgenograms, and histologic evidence of a predominantly neutrophilic leukocytic infiltration. Concomitant with the appearance of pulmonary injury was the generation of proteases and oxidants in the BAL fluids. Neutrophil elastase, bound to alpha 1-protease inhibitor (alpha 1-PI), was found to increase from 0.47 micrograms/ml in untreated monkeys to 0.99 micrograms/ml in FNLP-treated animals and 1.23 micrograms/ml in monkeys receiving PMA. Radioiodinated human prekallikrein, instilled for 2 min into the inflammatory site and retrieved by lavaging, was found to have undergone proteolytic cleavage; this cleavage was not consistently inhibitable with the inclusion of antibody to elastase. BAL fluids were shown to contain an amidolytic activity when tested on the synthetic substrate H-D-pro-phe-arg-pNA. This activity was partially inhibitable with known inhibitors of active Hageman factor and kallikrein. beta-Glucuronidase levels in the BAL fluids increased from 0.85 U/ml to 4.36 U/ml and 8.25 U/ml in FNLP- and PMA-treated animals, respectively. Myeloperoxidase (MPO) levels also increased from 1.37 OD U/ml X min to 16.59 and 30.47 OD U/ml X min in the same groups of animals. Oxidant generation was also assessed in several different ways. The specific activity of the oxidant-sensitive inhibitor alpha 1-PI recovered in the BAL fluid decreased from 0.80 in control samples to 0.57 and 0.65 in FNLP- and PMA-treated animals. That this inactivation was due to oxidant injury of the molecule was confirmed by the return to full activity of four out of five BAL samples after their incubation with the reducing agent dithiothreitol in the presence of methionine sulfoxide peptide reductase. The specific activity of catalase in the BAL fluids of animals given 3-amino, 1,2,4 triazole (AT) 1 h before lavaging showed drops from 0.97 in untreated monkeys to 0.04 in FNLP treated and 0.49 in PMA-treated monkeys. MPO levels also fell in the AT-treated injured animals from 16.59 to 0.85 delta OD/min X ml in FNLP animals in the absence and presence of AT, and 30.47 to 0.60 delta OD/min X ml in PMA-treated animals. Inhibition of MPO by AT was shown in vitro to be H2O2 dependent. Total glutathione levels in the BAL fluids did not change appreciably after FNLP or PMA treatment. These studies present substantial evidence of the generation of both proteases and oxidants during the establishment of acute pulmonary inflammatory injury in an experimental primate model. PMID- 2995450 TI - Discordance between transferrin receptor expression and susceptibility to lysis by natural killer cells. AB - Expression of the transferrin receptor on target cell lines has recently been implicated as a determinant of susceptibility to cytolysis by natural killer (NK) lymphocytes. We have examined this proposed relationship in several ways. First, K562 (a cell line highly vulnerable to NK lysis) cells were grown for 24 h in the iron chelator desferrioxamine. Under these conditions, the cells doubled their surface transferrin receptor expression as determined both by radioligand binding and surface binding of the OK-T9 monoclonal anti-transferrin receptor antibody. In contrast, cells grown for the same period of time in hemin halved their receptor expression. This fourfold change in transferrin receptor expression between the desferrioxamine-treated and hemin-treated cells produced no change in susceptibility to NK cytolysis. Second, HeLa (a cell line which in its native state is very resistant to NK cytolysis) cells were compared with K562 cells with respect to surface transferrin receptor expression. The difference in NK susceptibility of the two cell lines was not reflected in differences in transferrin receptor expression: the K562 cells expressed approximately 1.5 X 10(5) receptors per cell while HeLa cells expressed 2.0 X 10(5) receptors/cell. Third, infection of HeLa cells by measles virus greatly increased their susceptibility to NK lysis but produced no change in surface transferrin receptor expression. Furthermore, when measles-infected HeLa cells were grown for 6 d in medium supplemented with iron-saturated human transferrin they underwent a 50% reduction in receptor expression but no change in NK susceptibility. Finally, possible alterations in the surface expression of NK target antigens on modified cells were further assayed by their ability to serve as cold-target inhibitors of cytolysis of NK-sensitive target cells. We examined two groups of cells in which transferrin receptor expression was reduced. These were the transferrin-treated, measles-infected HeLa cells with the 50% receptor reduction, and K562 cells grown in medium containing hemin and iron salts where the reduction was five- to sixfold relative to control. In neither case was there a change in the apparent expression of NK target antigen(s). We conclude that there is a discordance between transferrin receptor expression and susceptibility to NK cytolysis in the model systems examined. Therefore, it is unlikely that the transferrin receptor per se is the target recognition structure for human NK cells, although a role in concert with other, as yet undefined molecules, cannot be excluded. PMID- 2995451 TI - Monoclonal anti-human factor VII antibodies. Detection in plasma of a second protein antigenically and genetically related to factor VII. AB - Several murine monoclonal anti-human Factor VII antibodies were produced using hybridoma technology. Two noncompetitive monoclonal antibodies were used to examine by Western blotting the Factor VII cross-reactive material (CRM) in normal human plasma and three commercially available congenitally Factor VII deficient plasmas, and to construct a facile "sandwich" immunoassay for plasma Factor VII. A second, previously undescribed, form of Factor VII CRM was detected in human plasma, which on Western blotting stained with an apparent intensity 5 8% that of Factor VII. This glycoprotein, tentatively called VII*, has a molecular weight 4,500 D less than Factor VII, lacks detectable Factor VII functional activity, does not bind to barium citrate, and is not recognized by a monoclonal antibody that recognizes Factor VII but not alpha-chymotrypsin-treated Factor VII. VII* was not proteolytically produced from Factor VII during in vitro coagulation or after infusion of human Factor VII into rabbits. As determined by Western blotting, the human hepatoma cell line, HepG2, cultured in the presence of vitamin K, secreted relatively greater levels of VII* in proportion to VII (75%) than that found in human plasma. Warfarin treatment of HepG2 cells decreased the quantity of VII secreted by 77%, whereas it only inhibited the secretion of VII* by 14%. Immunologic studies of the plasmas from a patient on chronic warfarin therapy and an individual given a short course of high dose warfarin therapy corroborated the in vitro synthetic studies obtained with HepG2 cells. The data are consistent with the production of VII* by posttranslational, proteolytic, modification of VII, that, at least in the HepG2 cells studied, occurs intracellularly. However, other mechanisms for the production of VII*, in particular, alternative RNA splicing of the transcript from a single gene, cannot be excluded. PMID- 2995452 TI - Oxygen radical-induced erythrocyte hemolysis by neutrophils. Critical role of iron and lactoferrin. AB - Human neutrophils (PMN), when stimulated with such chemotaxins as phorbol myristate acetate (PMA), destroy erythrocytes and other targets. Cytotoxicity depends on PMN-generated reactive oxygen metabolites, yet the exact toxic specie and its mode of production is a matter of some dispute. Using 51Cr-labeled erythrocytes as targets, we compared various reactive-O2 generating systems for their abilities to lyse erythrocytes as well as to oxidize hemoglobin to methemoglobin. PMA-activated PMNs or xanthine oxidase plus acetaldehyde were added to target erythrocytes in amounts that provided similar levels of superoxide. PMNs lysed 68.3 +/- 2.9% (SEM) of targets, whereas the xanthine oxidase system was virtually impotent (2.3 +/- 0.8%). In contrast, methemoglobin formation by xanthine oxidase plus acetaldehyde was significantly greater than that caused by stimulated PMNs (P less than 0.001). A similar dichotomy was noted with added reagent H2O2 or the H2O2-generating system, glucose plus glucose oxidase; neither of these caused 51Cr release, but induced 10-70% methemoglobin formation. Thus, although O2- and H2O2 can cross the erythrocyte membrane and rapidly oxidize hemoglobin, they do so evidently without damaging the cell membrane. That a granule constituent of PMNs is required to promote target cell lysis was suggested by the fact that agranular PMN cytoplasts (neutroplasts), although added to generate equal amounts of O2- as intact PMNs, were significantly less lytic to target erythrocytes (P less than 0.01). Iron was shown to be directly involved in lytic efficiency by supplementation studies with 2 microM iron citrate; such supplementation increased PMN cytotoxicity by approximately 30%, but had much less effect on erythrocyte lysis by neutroplasts (approximately 3% increase), and no effect on lysis in the enzymatic oxygen radical-generating systems. These results suggest a critical role for an iron liganding moiety that is abundantly present in PMN, marginally so in neutroplasts, and not at all in purified enzymatic systems--a moiety that we presume catalyzes very toxic O2 specie generation in the vicinity of juxtaposed erythrocyte targets. The obvious candidate is lactoferrin (LF), and indeed, antilactoferrin IgG, but not nonspecific IgG, reduced PMN cytotoxicity by greater than 85%. Re-adding 10(-8) M pure LF to neutroplasts increased their ability to promote hemolysis by 48.4 +/- 0.9%--to a level near that of intact PMNs. We conclude that O-2 and H2O2 are not sufficient to mediate target cell lysis, but require iron bound to LF, which, in turn, probably generates and focuses toxic O2 radicals, such as OH, to target membrane sites. PMID- 2995454 TI - Screening tests for antibodies to cytomegalovirus: an evaluation of five commercial products. AB - Four hundred and ninety two samples of serum from blood donors were screened for the presence of antibodies specific to cytomegalovirus using radioimmunoassay, a modified complement fixation test, and five commercially available tests: the Cetus CMV IHA, Abbott CMV total AB EIA, Cytomegalisa Stat EIA, Enzygnost EIA, and Virenz G-CMV EIA. A wide variation in results was found, with only 53.5% of the sera giving total concordance by all methods. Rates of seropositivity in the different tests ranged from 34.9% to 59.3%, with sensitivities ranging from 75.2% to 99.1% compared with the radioimmunoassay. Of 211 sera which gave positive results with four or more of the tests, none was negative by the radioimmunoassay and Abbott EIA, three were negative in Cetus IHA and Enzygnost EIA, and 11 were negative in the modified complement fixation test. Virenz G and Cytomegalisa Stat EIAs, however, gave 40 (19%) and 49 (23.2%), respectively, as negative. The results confirmed the reliability of the radioimmunoassay for the detection of the antibody status to CMV, but this test is too elaborate for a screening procedure. The Abbott EIA and Cetus IHA were found to be the most suitable for this purpose in spite of high false positive rates. PMID- 2995455 TI - Organization of cerebral cortical afferent systems in the rat. II. Hypothalamocortical projections. AB - The organization of hypothalamic projections to the cerebral cortex in the rat has been studied using retrograde and anterograde tracer methods. Four separate populations of hypothalamic neurons, which constitute a major source of diffuse cortical innervation, were identified: Tuberal lateral hypothalamic (LHAt) neurons which innervate the cerebral cortex tend to cluster in the perifornical region, in the zona incerta, and along the medial edge of the cerebral peduncle, at levels roughly coextensive with the ventromedial hypothalamic nucleus. Most of these neurons project to the ipsilateral cortex; a small percentage innervate the contralateral cortex, but this varies among cortical terminal fields. The perifornical neurons are organized in a roughly topographic medial-to-lateral relationship with respect to their cortical terminal fields. Field of Forel (FF) neurons, which project primarily to the frontal cortex of the ipsilateral hemisphere, are located just ventral to the medial edge of the medial lemniscus, at the level of the ventromedial basal thalamic nucleus. The more laterally placed neurons innervate the lateral frontal, insular and perirhinal cortex; the more medial neurons, around the mammillothalamic tract, innervate the medial frontopolar, prelimbic, infralimbic, and anterior cingulate cortex. Posterior lateral hypothalamic (LHAp) neurons form a dense cluster spanning the lateral hypothalamus, from the cerebral peduncle to the posterior hypothalamic area at premammillary levels, and extending into the supramammillary nucleus and the adjacent ventral tegmental area. LHAp neurons innervate the entire cerebral cortex, predominantly on the ipsilateral side. Populations of LHAp neurons projecting to different cortical target areas show considerable spatial overlap, but computer plots of the centers of these populations demonstrate a strict topographic relationship with respect to the cerebral cortex. Tuberomammillary (TMN) neurons form a sheet along the ventrolateral surface of the premammillary hypothalamus. About twice as many TMN neurons innervate the ipsilateral, as compared to the contralateral hemisphere; it is not known whether single neurons project to both hemispheres. No topographic organization of the TMN cortical projection is apparent. Injections of different-colored fluorescent dyes into various cortical areas demonstrate that hypothalamic neurons in general have rather restricted cortical terminal fields. Only occasional neurons are found, primarily in LHAt, which are double labeled by injections into different cytoarchitectonic areas.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2995453 TI - Bradykinin-induced changes in phosphatidyl inositol turnover in cultured rabbit papillary collecting tubule cells. AB - Rabbit renal papillary collecting tubule cells were isolated as a homogeneous population and grown in primary culture. These cells were maintained in fully defined medium to inhibit fibroblast overgrowth and to facilitate labeling of endogenous inositol phospholipids with myo-[2-3H]inositol with high specific activity. These cells demonstrated the morphology, cyclic AMP responsiveness, and prostaglandin E2 (PGE2) elaboration, consistent with previous published characterizations. When cells labeled with myo-[2-3H]inositol were stimulated by bradykinin at 10(-7) M, time-dependent and reversible changes in the distribution of inositol polyphosphates were observed. Inositol 1,4,5-triphosphate and inositol 1,4-diphosphate showed time-dependent and dose-dependent increases to maximal levels of 225 and 223% of control, respectively. These data indicate that the elaboration of inositol polyphosphates is a biochemical correlate to bradykinin stimulation and may play a role in PGE2 release in renal papillary collecting tubule cells. PMID- 2995456 TI - Replacement of damaged cortical projections by homotypic transplants of entorhinal cortex. AB - The extent to which transplants of embryonic cortical tissue can be used to replace damaged cortical projections has been examined. Embryonic entorhinal cortex was implanted into the entorhinal region of young adult rats that had previously received a lesion through the angular bundle. Projections between transplant and host were examined by using WGA-HRP and the fluorescent dye Fast Blue. Implants selectively innervated areas of the host hippocampus and amygdala which normally receive entorhinal afferents. Implants were innervated by cells in the host diagonal band and, in one case, by cells in the contralateral entorhinal and/or presubicular cortex. In most cases, host fibers were differentially distributed within transplants, possibly reflecting an ability of host fibers to recognize and selectively innervate their appropriate targets even though the cellular organization of the implant is different from that present during normal development. These data suggest that homotypic implants of embryonic entorhinal cortex can, in some ways, replace severed cortical projections and may eventually be able to reconstitute normal cortical circuitry. PMID- 2995457 TI - Brainstem origins and projections of the cervical and abdominal vagus in the golden hamster: a horseradish peroxidase study. AB - Vagal afferent projections, and preganglionic parasympathetic neurones contributing to the vagus nerve in golden hamsters were traced following application of horseradish peroxidase (HRP) to the proximal end of the cervical or abdominal nerve stump. Efferents in the cervical vagus were traced to their perikarya or origin in the dorsal motor nucleus (DMN) of the vagus, the commissural gray of the cervical spinal cord, the nucleus ambiguus, the nucleus of the accessory spinal nerve (NASN), and in the ventral horn dorsolateral to the NASN. Perikarya in the NASN and the region dorsolateral to it did not contribute efferent fibres to the abdominal vagus. In the remaining cell groups, fewer labelled perikarya were labelled in the abdominal cases than in the cervical cases. Extraperikaryal labelling (presumptive terminals) in the cervical cases was seen primarily in the nucleus of the solitary tract. A modest distribution of extraperikaryal grains was also noted along the inner rim of the area postrema and the ventral border of the DMN. Anterograde labelling was sparser and had a more restricted distribution in the abdominal cases. A detailed description of brainstem pathways of vagal efferent and afferent fibres is provided, as is a comparison of the present observations with those in similar studies of other species. PMID- 2995458 TI - The projection of three extrathalamic cell groups to the cerebral cortex of the turtle Pseudemys. AB - Three extrathalamic subcortical inputs to the part of the cerebral cortex that is known to receive thalamic fibers in the turtle were examined in the present study. Direct projections from the locus coeruleus, the superior medial raphe nucleus, and a wide area of the basal telencephalon that lies ventromedial to the globus pallidus were demonstrated with the horseradish peroxidase method. Fluorescence histochemistry confirmed the presence of catecholamine-containing fibers in the rostral half of dorsal cortex and also demonstrated a dense network of serotoninergic fibers. Biochemical analysis showed the concentration of both monoamines to be relatively high; the norepinephrine concentration was 709 ng/g and the serotonin concentration was 1,750 ng/g. No evidence was found to suggest the existence of either a dopamine fiber projection to cortex comparable to that of mammalian neocortex or the presence of an epinephrine pathway to turtle cortex equivalent to the epinephrine-containing fibers in the pallium of amphibians. The coexistence of the projections from the thalamus with noradrenergic projections from the locus coeruleus, serotoninergic projections from the superior medial raphe nucleus, and presumably cholinergic projections from the basal telencephalon provide at least four distinct subcortical inputs to the reptilian dorsal cortex. Neither thalamic nor similar extrathalamic inputs have been demonstrated in the dorsal pallium of amphibia. Mammalian neocortex, in contrast, has even more elaborately differentiated inputs of both types. These results support the idea that thalamic and extrathalamic inputs to cortex appear at the same time in vertebrate evolution, and that both types of inputs are required for the normal development and function of neocortex. PMID- 2995459 TI - Projections from the cochlear nuclei in the mustache bat, Pteronotus parnellii. AB - Ascending projections of the cochlear nuclei in the mustache bat were analyzed by anterograde transport of [3H]-leucine and by retrograde transport of HRP. We were particularly interested in pathways to two parts of the system: (1) to the medial superior olive, because this nucleus is missing in most echolocating bats, but appears to be present in the mustache bat, and (2) to the intermediate and ventral nuclei of the lateral lemniscus, because these nuclei are hypertrophied and highly differentiated in all echolocating bats that we have examined. The results show a highly systematic projection from the anteroventral cochlear nucleus to all of the auditory nuclei in the brain stem. After an injection of [3H]-leucine in the anterior and dorsal part of the anteroventral cochlear nucleus, presumably in a region sensitive to low frequencies, label is seen in the following locations: ipsilateral to the injection in the lateral part of the lateral superior olive; bilaterally in the dorsal part of the medial superior olive; contralateral to the injection in the dorsal parts of the intermediate and ventral nuclei of the lateral lemniscus; and in the anterolateral part of the central nucleus of the inferior colliculus. After an injection of [3H]-leucine in a posterior part of the anteroventral cochlear nucleus, presumably in a region sensitive to high frequencies, labeling is in the same set of nuclei, but within each nucleus the label is now in a different location: medially in the lateral superior olive, ventrally in the medial superior olive, ventrally in each division of the ventral and intermediate nuclei of the lateral lemniscus, and medially in the central nucleus of the inferior colliculus. Projections from the entire anteroventral cochlear nucleus to the inferior colliculus are confined to the ventral two-thirds of the central nucleus. The dorsal one-third of the central nucleus of the inferior colliculus is the principal target of the dorsal cochlear nucleus and may be a target of the posteroventral cochlear nucleus. Both of these nuclei appear to project sparsely to the ventral parts of the inferior colliculus. We conclude first that the bilateral input to the medial superior olive in the mustache bat is similar to the input seen in other mammals. Thus this bat has a neural structure which is associated with the analysis of binaural time differences and which usually is seen only in animals with heads large enough to create interaural time differences greater than those available to Pteronotus.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2995460 TI - Anatomical connections of the nucleus prepositus of the cat. AB - The afferent and efferent connections of the nucleus prepositus hypoglossi with brainstem nuclei were studied using anterograde and retrograde axonal transport techniques, and by intracellular recordings and injections of horseradish peroxidase into prepositus hypoglossi neurons. The results of experiments in which horseradish peroxidase was injected into the prepositus hypoglossi suggest that the major inputs to the prepositus hypoglossi arise from the ipsi- and contralateral perihypoglossal nuclei (particularly the prepositus hypoglossi and intercalatus), vestibular nuclei (particularly the medial, inferior, and ventrolateral nuclei), the paramedian medullary and pontine reticular formation, and from the cerebellar cortex (flocculus, paraflocculus, and crus I; the nodulus was not available for study). Regions containing fewer labeled cells included the interstitial n. of Cajal, the rostral interstitial n. of the medial longitudinal fasciculus, the n. of the posterior commissure, the superior colliculus, the n. of the optic tract, the extraocular motor nuclei, the spinal trigeminal n., and the central cervical n. The efferent connections of the prepositus hypoglossi were studied by injecting 3H-leucine into the prepositus hypoglossi, and by following the axons of intracellularly injected prepositus hypoglossi neurons. The results suggest that in addition to the cerebellar cortex, the most important extrinsic targets of prepositus hypoglossi efferents are the vestibular nuclei (particularly the medial, inferior, and ventrolateral nuclei, and the area X), the inferior olive (contralateral dorsal cap of Kooy and ipsilateral subnucleus b of the medial accessory olive), the paramedian medullary and pontine reticular formation, the reticular formation surrounding the parabigeminal n., the contralateral superior colliculus and pretectum, the extraocular motor nuclei (particularly the contralateral abducens nucleus and the ipsilateral medial rectus subdivision of the oculomotor nucleus), the ventral lateral geniculate n., and the central lateral thalamic nucleus. Other areas which were lightly labeled in the autoradiographic experiments were the contralateral spinal trigeminal n., the n. raphe pontis, the Edinger Westphal n., the zona incerta, and the paracentral thalamic n. Many of the efferent connections of the prepositus hypoglossi appear to arise from principal prepositus hypoglossi neurons whose axons collateralize extensively in the brainstem. On the other hand, small prepositus hypoglossi neurons project to the inferior olive, and multidendritic neurons project to the cerebellar flocculus, apparently without collateralizing in the brainstem.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2995461 TI - The effects of monocular deprivation on the size of GAD+ neurons in the cat's dorsal lateral geniculate nucleus. AB - Previous studies have shown that suturing one eyelid closed in a newborn kitten results in profound changes in the development of the visual system. Among these is a retardation in the growth of neurons in the layers of the dorsal lateral geniculate nucleus receiving retinal input from the closed eye. Moreover, the greatest effect appears to be in the largest neurons. The present study examines the effects of monocular deprivation on the perikaryal size of a select group of small lateral geniculate neurons. GABAergic neurons, that may be interneurons. These cells were selectively labeled by an antiserum to glutamic acid decarboxylase (GAD) and immunocytochemical methods. The results demonstrate that GAD+ neurons are among the smallest in the lateral geniculate nucleus and that they are insensitive to the effects of monocular deprivation. That is, GAD+ neurons in deprived laminae A and A1 are similar in size to those in the corresponding, nondeprived laminae. These findings are consistent with the hypothesis that GAD+ neurons are interneurons and therefore not subject to binocular competition in the visual cortex. This interpretation, however, is complicated by additional studies of the postnatal development of GAD+ neurons which reveal that GAD+ neurons grow to their adult size relatively early, before the onset of the critical period. Thus the insensitivity of the perikarya of GAD+ neurons to monocular deprivation may be attributable to their precocious growth. PMID- 2995462 TI - Nigral inhibitory termination on efferent neurons of the superior colliculus: an intracellular horseradish peroxidase study in the cat. AB - Intracellularly recorded responses of deeper tectal neurons to stimulation of the substantia nigra and the cerebral peduncle were obtained to demonstrate the monosynaptic inhibitory nature of the nigrotectal pathway in the cat. We also employed antidromic stimulation (contralateral predorsal bundle and superior colliculus) and intracellular labeling with HRP to demonstrate which types of tectal efferent neurons are nigrorecipient. The response to nigral stimulation in 61% of the cells studied was a monosynaptic IPSP of short duration. Recovered HRP labeled nigrorecipient neurons include X1 (large multipolar radiating), X2 (tufted), X4 (medium-size vertical), X5 (medium-size horizontal), T1 (medium-size trapezoid radiating), T2 (small ovoid vertical), I (small sparsely ramified), and A (small horizontal) neurons. Nigrorecipient cells participate in all four of the major efferent axonal systems of the deeper tectal layers: crossed descending (X and T neurons), ipsilateral descending (I and T neurons), ascending (A, X, and T neurons), and commissural (T neurons). EPSPs accompanied by long-lasting hyperpolarizing potentials were recorded from the remaining tectal neurons in response to stimulation of the substantia nigra, cerebral peduncle, and pericruciate cortex. Collision experiments indicate that at least part of the excitatory responses of tectal neurons to nigral and penduncular stimulation are mediated by corticotectal fibers traversing the cerebral peduncle and the substantia nigra. Excitatory effects of nigral, peduncular, and cortical stimulation were disclosed in X neurons including the non-nigrorecipient large vertical neurons of the X3 subgroup. Cortical excitatory and nigral inhibitory inputs converge only on X neurons (X1, X2, X4, X5). In this case, nigrally evoked IPSPs were preceded by a brief EPSP. Collectively, these results demonstrate the inhibitory termination of the nigrotectal pathway on a wide variety of deeper tectal efferent neurons. Such findings imply the versatility of the nigral involvement in tectal mechanisms of gaze control. We suggest that the substantia nigra pars reticulata contacts tectal neurons differing as to their response properties and shapes the signal carried by all the major tectofugal bundles. PMID- 2995463 TI - The natural history of streptococcal skin infection: prevention with topical antibiotics. AB - An investigation on the natural history of streptococcal skin infection was done in fifty-nine children in a rural day care setting. A double-blind study for prevention of streptococcal pyoderma was done during the peak season for skin infection. Triple antibiotic ointment, containing bacitracin, polysporin, and neomycin, was compared to placebo ointment. Ointments were applied thrice daily for minor skin trauma; mosquito bites and abrasions were predominant. Cultures of normal skin surfaces were taken for group A streptococci each week of the 15-week study period. Skin lesions were cultured whenever present. Eighty-one percent of the fifty-nine patients had positive normal skin cultures on one or more occasions. Nineteen children (32%) developed streptococcal pyoderma. Infection occurred significantly more often in children using placebo ointment than in those using topical antibiotic (47% vs 15%; p = 0.01). The infecting strain was first recovered from normal skin surfaces in 67% of placebo patients and in two of the four patients using antibiotic ointment. This study further confirms the importance of skin carriage of group A streptococci as a precursor to pyoderma and demonstrates the importance of minor skin trauma as a predisposing factor. Topical antibiotics may be useful in preventing streptococcal pyoderma, especially in children known to be at increased risk for such infection. PMID- 2995464 TI - Cutaneous manifestations of adult T cell leukemia/lymphoma. Report of three different forms. AB - The clinical and pathologic features of cutaneous lesions observed in three adult T cell leukemia/lymphoma (ATL) patients identified in Taiwan are described. They represent one classical case of ATL and two "smoldering" variants. The classical ATL patient when first seen had numerous erythematous or purpuric papules, nodules, and plaques with or without ulceration. The two "smoldering" cases developed encrusted purpuric plaques with subcutaneous erythematous nodules in one patient and pompholyx-like vesicular eruptions with tumor masses in another patient. The pompholyx-like eruptions have not been described before. Pruritus was the major complaint in two patients. Histopathologic studies revealed pleomorphic infiltration in the classical case and monomorphic infiltration with medium-sized cells in the two "smoldering" variants. Therefore, the cutaneous lesions of ATL are diverse and not pathognomonic. The diagnosis requires a high index of suspicion, detection of circulating characteristic multilobated lymphoid cells with T helper/inducer cell marker, and demonstration of serum antibody against the adult T cell leukemia virus-associated antigen. PMID- 2995465 TI - Effects of dietary vitamin D3 on concentrations of vitamin D and its metabolites in blood plasma and milk of dairy cows. AB - To determine the effect of supplemental dietary vitamin D3 on concentration of vitamin D and its metabolites in milk, 20 Holstein cows were assigned to four groups and fed either 0, 10,000, 50,000, or 250,000 IU of vitamin D3/d beginning approximately 2 wk prepartum and continuing through wk 12 of lactation. Samples of blood plasma and milk were assayed for concentrations of vitamin D, 25 hydroxyvitamin D, 1,25-dihydroxyvitamin D. Only the daily dosage of 250,000 IU caused significant increases of concentrations of vitamin D or 25-hydroxyvitamin D in plasma. Concentrations of vitamin D and 25-hydroxyvitamin D in milk were approximately equal and averaged .2 ng/ml. Little 1,25-dihydroxyvitamin D and no 24,25-dihydroxyvitamin D could be detected in milk from any of the four treatment groups. Cows fed 250,000 IU of vitamin D3/d produced milk containing 54 IU of vitamin D activity per liter, whereas unsupplemented cows produced milk containing 17 IU/L. Oral supplementation with up to 250,000 IU of vitamin D3/d does not increase effectively vitamin D activity of milk. PMID- 2995467 TI - Tumor conference #57. Extramammary Paget's disease. PMID- 2995466 TI - Nevus sebaceus, syringocystadenoma papilliferum, and basal cell epithelioma. AB - This case report illustrates the pluripotential nature of the hair matrix cells in nevus sebaceus. Syringocystadenoma papilliferum and basal cell epithelioma both developed in the same scalp lesion. The staged surgical excision is illustrated. PMID- 2995468 TI - Long-term effects of microbiologically modulated periodontal therapy on advanced adult periodontitis. PMID- 2995469 TI - Survival of herpes simplex virus and other selected microorganisms on patient charts: potential source of infection. AB - This study indicated that when inoculated onto dental charts, both viruses and bacteria were capable of survival allowing the potential for transmission of infection within the dental office. The conscientious dental practitioner can take steps to reduce this possible mode of infection by removing contaminated surgical gloves or washing hands before handling the chart. An additional method of reducing this potential would be to wipe the chart with an antiseptic solution. Although this study has shown that there is a potential for the spread of infection with the organisms tested, the actual extent of dental chart contamination and resultant illnesses contracted are the basis for further study. Additional studies are needed to follow the pattern of chart distribution from person to person within the dental office, determine the types and quantities of pathogens present in the mouth that would contaminate the charts, and sample the charts under actual clinical conditions to determine the types and viability of the organisms present. PMID- 2995470 TI - A nonsteroid anti-inflammatory drug exacerbates Coxsackie B3 murine myocarditis. AB - Nonsteroid anti-inflammatory drugs are often used to treat myalgias and arthralgias in enteroviral infections, but their effects on acute viral myocarditis are unknown. The effect of the nonsteroidal anti-inflammatory drug, ibuprofen, on acute viral myocarditis was studied in 75 four week old male BALB/c mice infected with 1.75 X 10(7) plaque-forming units of Coxsackie virus B3 on day 0. Ibuprofen was given intraperitoneally at a dose of 15 mg/kg body weight daily. The mice were assigned to four groups--Group I, 18 uninfected mice given ibuprofen on days 1 to 14; Group II, 18 infected, untreated mice; Group III, 20 infected mice given ibuprofen on days 1 to 14; and Group IV, 17 infected mice given ibuprofen on days 7 to 14. Nine animals in Group I, eight in Group II and seven in Group III were killed on day 7; the remaining mice were killed on day 14. Heart viral cultures and histologic analysis were done. Cultures at days 7 and 14 were all negative. Inflammation and necrosis analyzed in each animal were graded 0 to 4, with grade 4 representing widespread inflammation and necrosis. The heart was histologically normal in all 18 uninfected mice (Group I) given ibuprofen only. Inflammation and necrosis were not significantly different in Group II (infected, untreated) and Group III (infected, treated beginning day 1) mice killed at day 7. Inflammation scores of mice killed on day 14 were 2.1 +/- 0.6 (Group II), 3.1 +/- 0.7 (Group III) and 2.9 +/- 1.0 (Group IV infected, treated days 7 to 14).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995471 TI - Diet and diabetes: a common approach. PMID- 2995473 TI - Synaptic activity of myenteric neurons in tissue culture. AB - Intracellular recordings were made from myenteric neurons of the guinea-pig caecum grown in tissue culture. The experiments were carried out on cultures older than 15 days, in which the neurons formed ganglion-like aggregates with many interconnecting nerve fibres. Intracellular injection of horseradish peroxidase demonstrated that the neurons project processes both within and outside the aggregates. Spontaneous activity was recorded in about 50% of the cells. This activity was composed of depolarizing potentials 2-20 mV in amplitude, and in most cases 10-20 ms in duration. In some instances, the potentials occurred in short bursts. The spontaneous potentials appear to be nicotinic cholinergic excitatory postsynaptic potentials (EPSPs). Electrical stimulation of the connecting fibers elicited in the neurons nicotinic cholinergic EPSPs, since they were blocked by hexamethonium. By varying the stimulus intensity and by stimulating several fibers, it was shown that the neurons are multiple-innervated. During repetitive presynaptic stimulation at frequencies above 10 Hz, there was a progressive reduction of the EPSP amplitude. It is concluded that the cultured myenteric neurons retain many of the characteristics found in the freshly isolated preparation and may be useful for studying the synaptic properties of these cells. PMID- 2995474 TI - Decreased beta-adrenergic receptor binding in obese female Zucker rats. PMID- 2995472 TI - Dietary studies of children from a biracial population: intakes of carbohydrate and fiber in 10- and 13-year-olds. AB - Dietary intakes of carbohydrate (CHO) and fiber were examined in children randomly selected from a biracial community-Bogalusa, LA. Intakes of CHO per 1,000 kcal were similar for both sexes and both races at ages 10 and 13 years. No group or race differences were found for nine components assessed in two cohorts of 10-year-old children examined three years apart. There were sex differences in sucrose (boys less than girls) and lactose (boys greater than girls) intakes. Comparison of 10- and 13-year-olds examined in 1976 showed a racial difference in fiber and starch intakes (black greater than white). Longitudinal comparisons of a cohort of 148 children examined at both 10 and 13 years showed lower lactose intakes over time. At both ages starch, fiber, and glucose intakes per 1,000 kcal were higher in black children, with higher sucrose/starch ratios in white children. The percent of calories from CHO and sugars was higher in Bogalusa children than values for US adults, but starch intakes were lower. None of the children's intakes was compatible with prudent dietary recommendations. Dietary CHO patterns of Bogalusa children reflect food market trends of increased use of simple CHO and decreased use of complex CHO. PMID- 2995476 TI - Plasma ACTH, cortisol and aldosterone concentrations in chronically cannulated ovine fetuses and in lambs injected with ovine corticotropin releasing factor. AB - Synthetic oCRF was intravenously injected into 3 groups of 5 chronically cannulated ovine fetuses in utero on days 120, 130 and 137 of gestation (10 micrograms/fetus). The respective twin fetuses were used as controls. Ovine CRF was also intravenously injected into 4 groups of 6 lambs on days 1, 3, 7 and 20 after birth (5 micrograms/kg bw). Fetal plasma ACTH and cortisol concentrations increased significantly following oCRF as early as 120 days of gestation without changing maternal plasma cortisol concentrations. The ACTH and cortisol response to CRF increased gradually on stages 130 and 137 of gestation, but on the other hand, plasma aldosterone did not change. In newborns, after oCRF, the pituitary response gave peak values at 10 min for plasma ACTH and adrenal response gave peak values at 15 min for plasma cortisol. Between 1 and 20 days, plasma ACTH and cortisol changes after oCRF decreased in older animals while aldosterone level remained unchanged. In animals receiving both treatments on days 1 and 20, plasma cortisol levels were increased for longer than in animals treated once. PMID- 2995477 TI - Tissue expansion in reconstructive hand surgery: case report. AB - A 22-year-old musician requested the correction of his digital syndactyly deformity without the use of skin grafts. Failure of the original correction to heal primarily resulted in the formation of dense scar tissue. A small, custom designed tissue expander was inserted within the web space and was slowly expanded. The additional expanded tissue permitted reconstruction of the web space and the sides of the fingers without the use of skin grafts. PMID- 2995475 TI - Selected soft tissue malignancies of the foot: an in-depth study with case reports. AB - Soft tissue malignancies represent a diagnostic and therapeutic challenge to the podiatric physician. In order to improve prognosis and increase survival rates of affected patients, prompt clinical and histologic recognition of suspect lesions is necessary. Here we present information and case reports on selected soft tissue malignancies including Kaposi's sarcoma, malignant melanoma, synovial sarcoma, and epithelioid sarcoma to aid the practitioner in recognizing these serious lesions that occur in the foot. PMID- 2995478 TI - Pancreatic pseudocyst with hemorrhage into the gastrointestinal tract through the duct of Santorini. AB - Hemorrhage into a pancreatic pseudocyst frequently goes unrecognized. This catastrophic event can be heralded by intermittent bleeding, or may present as massive gastrointestinal hemorrhage. A high index of suspicion, proper diagnostic workup, and prompt surgical management afford the patient the best chance for survival. We report a patient with massive pseudocyst bleeding into the gastrointestinal tract via the duct of Santorini and discuss the current diagnostic and therapeutic approach. PMID- 2995479 TI - Chronic postcolectomy pericholangitis and cholangiocarcinoma. AB - Two patients with long-standing pericholangitis developed unresectable cholangiocarcinoma after colectomy for chronic ulcerative colitis. The natural history of their illnesses suggest that chronic inflammation of small bile ducts in ulcerative colitis may be a precursor of biliary cancer and may also account for the increased incidence of this tumor in the colitis population. PMID- 2995480 TI - Captopril in the hepatorenal syndrome. AB - Five patients with hepatorenal syndrome were treated with the orally active angiotensin-converting enzyme inhibitor captopril (25 or 50 mg 6 hourly) for up to 48 hours. Only one patient showed a significant increase in urinary sodium concentration (from less than 10 to 70 mmol/liter), but without associated diuresis; renal function continued to deteriorate in all patients with persistent oliguria and rising serum creatinine. The outcome was uniformly fatal. These results suggest that in the hepatorenal syndrome, captopril in standard dosage is without benefit, and provide further evidence that the changes in the renin angiotensin system are probably secondary to reduced renal perfusion from some other cause. PMID- 2995481 TI - Comparative usefulness of tissue fixatives for in situ viral nucleic acid hybridization. AB - Traditionally tissues for in situ hybridization of viral nucleic acid have been small pieces obtained from laboratory rodents, and fixatives that are designed for electron microscopy, such as periodate-lysine-paraformaldehyde (PLP) can handle them adequately. However, these fixatives have limited penetrating ability and may produce no appreciable hardening, so alternative fixation methods were evaluated. The intention was to determine whether fixatives adequate for bulky tissues such as whole or halved pig and cow brains would also be compatible with in situ hybridization. Various fixatives were evaluated using a system of intracranial inoculation of BALB/c mice with pseudorabies virus (PRV) followed by in situ hybridization of brain tissue sections with a 35S-labeled PRV DNA probe. Loss of tissue sections was a major problem, particularly with PLP and formalin, but positive results were obtained with five fixatives tested. Cellular morphology was especially good with PLP and with a modification of Carnoy's fluid, MOCA fixative. An incidental but important observation was that formalin is compatible with in situ hybridization. Retroactive studies of viral diseases using routinely processed blocks of tissue (formalin-fixed, paraffin-embedded) are therefore conceivable. PMID- 2995482 TI - Cytochemical localization of 5'-nucleotidase in the frog (Rana pipiens) retina. A histochemical and cytochemical study. AB - Localization of 5'-nucleotidase in the frog retina was investigated using histochemical and cytochemical techniques. Light-microscopic observations revealed the presence of this enzyme in the inner retinal layers (the nerve fiber layer, ganglion cell layer, and inner plexiform layer). Ultrastructural investigations revealed that the enzyme activity is associated with the plasma membranes of the Muller cell processes, whereas the Muller cell processes present in the outer retinal layers did not demonstrate any detectable enzyme activity. This observation would appear to confirm our previous findings, that 5' nucleotidase is an ectoenzyme, but its distribution in frog retina differs from that in rodents and it is only present in the inner layers of the retina. The prominent localization of 5'-nucleotidase on the glial plasma membrane may be viewed in the context of the widely accepted interaction between neurones and glial cells. Since nucleotides do not penetrate the plasma membrane, a mechanism to produce membrane-permeable adenosine, important for neuronal function, is postulated. It is known that 5'-nucleotidase produces adenosine by hydrolyzing adenosine 5'-monophosphate (5'-AMP). Therefore one would expect that the glial membrane-bound enzyme can accomplish the final step in this mechanism by producing the adenosine in the extracellular spaces. PMID- 2995483 TI - Localization and properties of angiotensin receptors. PMID- 2995484 TI - Effects of converting enzyme inhibition on blood pressure, plasma renin activity (PRA) and plasma aldosterone in hypertensive diabetics compared to patients with essential hypertension. AB - Hypertension with diabetes mellitus has been associated with suppression of the renin-angiotensin-aldosterone system. We have studied the effects of the converting enzyme inhibitor, captopril, on blood pressure, plasma renin activity (PRA) and plasma aldosterone in 10 stable hypertensive diabetic subjects and 10 age-matched patients with essential hypertension. There was no clinical evidence of complication in the diabetic subjects and their diabetic treatment remained unchanged throughout the study. Mean captopril doses used were similar in both groups. In the diabetics and the essential hypertensives, treatment resulted in a significant and similar decrease in blood pressure. Pre-treatment basal and stimulated PRA and the change of PRA with captopril were also similar. Pre treatment stimulated plasma aldosterone and the response of aldosterone to postural stress was significantly lower in the diabetic group, suggesting an impaired adrenal responsiveness to stress. Despite this, our findings indicate that the hypotensive action of captopril is at least as effective in hypertension associated with otherwise uncomplicated diabetes mellitus as in essential hypertension. PMID- 2995485 TI - Binding of monoclonal antibody to the Epstein Barr virus (EBV)/CR2 receptor induces activation and differentiation of human B lymphocytes. AB - A panel of B cell-specific monoclonal antibodies that identify the CR2/EBV receptor were examined for their ability to mimic the T-independent mitogenic agent, EBV, and thus activate human peripheral blood B lymphocytes. Two of four different anti-CR2/EBV monoclonal antibodies, OKB7 and AB-1, produced a 50-fold to 200-fold dose-dependent stimulation of DNA synthesis of peripheral blood mononuclear cells. One of the other monoclonal antibodies, anti-B2, had slight activity, and the other, HB-5, was completely inactive. One of the mitogenic antibodies, OKB7, which directly inhibits binding and infection of B cells by EBV in the absence of a second anti-immunoglobulin antibody, was examined in further detail. Both the intact antibody in soluble form and its pepsin-derived F(ab')2 fragment stimulated DNA synthesis of unseparated B and T lymphocytes. Peak stimulation of DNA synthesis in peripheral blood mononuclear cells occurred between 4 to 6 days. B cells were responsible for incorporation of [3H]thymidine. However, T cells were required for activation of peripheral blood mononuclear cells by OKB7. OKB7, as well as the other mitogenic monoclonal anti-EBV/CR2 receptor antibody, also induced B cells to differentiate after 6 to 10 days of culture as indicated by polyclonal Ig secretion. IgM was the predominate immunoglobulin secreted. These studies thus indicate that certain epitopes on the EBV/CR2 receptor trigger B cells to divide and differentiate. This pathway of B cell activation, in contrast to that produced by EBV, is T cell dependent. PMID- 2995486 TI - Characterization of the requirements for human T cell mitogenesis by using suboptimal concentrations of phytohemagglutinin. AB - To characterize the requirements for T cell proliferation, we have studied the response of purified populations of human T cells to varying concentrations of the mitogen phytohemagglutinin (PHA). Concentrations of PHA which induce optimal proliferative responses induce increases in cytosolic free calcium ([Ca2+]i), expression of interleukin 2 (IL 2) receptors, and production of IL 2. As the concentration of PHA is decreased, each of these processes decreases in parallel. At suboptimal concentrations of PHA, the addition of exogenous IL 2 reconstitutes both the proliferative response and the expression of the IL 2 receptor, as measured by immunofluorescence with antibodies directed against the TAC/IL 2 receptor molecule, but without reconstituting the increase in [Ca2+]i. Therefore, the concentration dependence of responses to PHA appears to be secondary to an absence of IL 2 production due to a failure to induce an increase in [Ca2+]i. The addition of the calcium ionophores A23187 and ionomycin or of accessory cells to low concentrations of PHA induces increases in [Ca2+]i and subsequent proliferative responses, suggesting that the two events are linked. The proliferative response can be inhibited by antibodies directed towards IL 2 or the IL 2 receptor, indicating that the proliferative response was at least partially dependent on the production and action of IL 2. This suggests that, although increases in [Ca2+]i are an integral event in the induction of proliferation by PHA, the increase in [Ca2+]i is required for the production but not the action of IL 2. In addition, low concentrations of PHA deliver an additional signal to cells, independent of an increase in [Ca2+]i, which induces IL 2 receptor expression and allows a proliferative response in the presence of exogenous IL 2. PMID- 2995488 TI - IL 1-like activity in antigen-presenting human B cell lines. AB - The secretion of interleukin 1 (IL 1) by an antigen-presenting cell (APC) may be essential to its function in the stimulation of T cell responses. However, the relevance of IL 1 is less clear in cases where the APC are from continuous B cell lines. We have shown that IL 1-like activity can be demonstrated in human B cell lines by using a cellular co-culture assay for IL 1. Significant IL 1 activity could not be detected in the supernatants of these B cell lines produced either constitutively or after stimulation with various mitogens. The failure to detect IL 1 activity in B cell supernatants was not due to secretion of a detectable inhibitor of IL 1. B cell supernatants or a co-culture assay with B cells failed to demonstrate any IL 2 activity. Co-culture experiments, in which B cells were added to known concentrations of IL 1, showed distinct patterns of stimulation and may suggest that the B cell activity is distinct from conventional IL 1. Not all B cell lines had equivalent levels of IL 1-like activity. However, all B cell lines tested were able to act as effective APC. Thus, B cells that function as APC may utilize a mediator with properties similar to IL 1. PMID- 2995487 TI - Cellular tropism of the human retrovirus HTLV-III/LAV. I. Role of T cell activation and expression of the T4 antigen. AB - In cultures of normal human lymphocytes infected with the human retrovirus HTLV III/LAV, detectable cytoplasmic virus appears and then disappears in a proportion (1 to 10%) of cells, followed by release of virus detected by particulate reverse transcriptase activity, virus antigen assay, and infectivity titer. Virus infection is associated with loss of detectable T4 antigen on infected cells and, ultimately, complete loss of T4+ cells from the culture. Residual non-T4+ cells are not susceptible to a second infection with HTLV-III/LAV, and in cultures of separated cell populations, substantial virus replication occurred in T4+ T cells and minimally, if at all, in non-T4+ cells. We could not detect a disproportionate loss of cell surface phenotype (other than T4) in comparison of infected and noninfected cultures of lymphocytes or purified T4+ T cells when these cultures were monitored with a panel of monoclonal antibodies that detect the major mononuclear cell types (alpha-T11, alpha-T3, alpha-Mo2, alpha-B1), functional T cell subsets (alpha-T8, alpha-Leu-8, alpha-T17), or activated/proliferating cells (alpha-T10, alpha-Ia, alpha-T9, alpha-4F2, alpha Tac). HTLV-III/LAV replication was quantitatively greatest in lymphocytes stimulated with phytohemagglutinin (PHA) and cultured in the presence of interleukin 2 (IL 2). Once activated by PHA, virus production in nondividing (irradiated) cells was similar to that in nonirradiated cells, but was substantially reduced if radiation was performed before PHA stimulation. Omission of PHA, IL 2, or both resulted in progressively lower amounts of virus replication. However, virus replication was detected and T4+ T cell depletion occurred in all cultures, regardless of medium supplement or radiation. T4+ T cells absorb infectious virus, and the binding of HTLV-III/LAV to the surface of T4+ T cells, but not to non-T4+ cells, was directly demonstrated. Binding is equivalent in activated and nonactivated cells and at 4 degrees and 37 degrees C. Reciprocal inhibition of binding was observed with alpha-T4a monoclonal antibody and virus. Exposure of cells to alpha-T4a before and during HTLV-III/LAV inoculation inhibited subsequent virus replication. We conclude that T4+ T cells are the major target for HTLV-III/LAV replication, that this tropism is related to expression of the T4 antigen that serves as a binding site for virus, that infection is inexorable in T4+ T cells regardless of subset or activation state, and that the activation/proliferative state of the cells is not a necessary determinant of infectivity, but rather, determines the amount of replication that will ensue. PMID- 2995489 TI - Synergy between B cell differentiation factors and interleukin 2, using a monoclonal system. AB - By using monoclonal B cell targets, cells derived from patients with chronic lymphocytic leukemia, and B cell differentiation factors (BCDF) derived from monoclonal human T cell hybridomas, we have demonstrated marked synergy for differentiation between interleukin 2 (IL 2) and BCDF. IL 2 alone had no effect on the proliferation of differentiation to immunoglobulin secretion in these cell populations; however, in conjunction with a variety of BCDF, differentiation to plaque-forming cells (PFC) was augmented 10- to 100-fold. There was no increase in proliferation as measured by [3H]thymidine incorporation. These effects could be demonstrated with concentrations of IL 2 as low as 5 U/culture, well within the physiologic range, by using either commercially available or recombinant IL 2. The addition of IL 2 to the B cell and BCDF cultures resulted in almost 100% expression of the IL 2 receptor, Tac, on the surface of these cells, and the augmented PFC response could be inhibited 70 to 80% by the addition of anti-Tac to the culture. Kinetic studies revealed that the addition of IL 2 to the B cell cultures could be delayed for up to 72 hr without a change in the PFC response, suggesting that IL 2 was acting as a secondary or synergistic signal for differentiation. Thus, it appears that IL 2 does have a role in B cell maturation mediated, in part, by IL 2 binding to the IL 2 receptor present on certain B cells. PMID- 2995490 TI - The B10.A mouse B cell response to pigeon cytochrome c is directed against the same area of the protein that is recognized by B10.A T cells in association with the Ek beta:Ek alpha Ia molecule. AB - An analysis of the fine specificities of the primary and hyperimmune antibody responses of B10.A mice to pigeon cytochrome c showed that both were qualitatively very similar. Small amounts of antibody appeared to be directed against the regions of serine 15 and/or glutamic acid 44. The remaining antibodies (greater than 70%) bound to the same complex topographic determinant (including residues 3, 103, and 104) on the back surface of pigeon cytochrome c which had been found to dominate the rabbit antibody response to this protein, and to be involved in Ia-restricted T cell stimulation. The mouse antibodies reacted very poorly with fragmented forms of the immunogen or with tobacco hornworm moth cytochrome c, even though both of these antigens had been shown previously to strongly stimulate pigeon cytochrome c-primed T cells. The specificities of the primary IgG responses of seven other mouse strains were found to be very similar, but not identical, to that of B10.A mice. The cytochrome c-specific antibodies in the hyperimmune serum were shown to bind to determinants involving residues that vary between pigeon and mouse cytochromes c. Comparison of the binding of the antibodies to the immunogen and to the corresponding host protein enabled the calculation of the proportion of the overall binding energy contributed by the variant residues. This was as low as 19 to 35% for the primary response, rose to 25 to 46% for the hyperimmune mouse antibodies, and reached 40 to 63% for hyperimmune rabbit antibodies. The remaining energy of interaction (37 to 81%) was necessarily contributed by the surface of the protein surrounding the variant residues, which is the same for the immunogen and the host protein. These results illustrate the relatively subtle differences in binding affinities which can distinguish self from non-self recognition by antibody molecules. PMID- 2995492 TI - Redistribution of protein kinase C activity in human monocytes: correlation with activation of the respiratory burst. AB - Protein kinase C (PKC) was found to be present in purified human monocytes and lymphocytes isolated by countercurrent centrifugal elutriation. In unstimulated monocytes and lymphocytes, approximately 90% of the PKC activity was cytosolic when the cells were disrupted in the presence of EGTA. The role of this kinase in the stimulation of the respiratory burst in monocytes was investigated. Phorbol esters capable of triggering the release of O2- caused a loss of PKC activity from the cytosol and the appearance of the kinase activity in the particulate cell fraction. Kinase activity was partially extractable from the particulate fraction by 0.1% Triton X-100, whereupon it demonstrated calcium and lipid dependence. The EC50 for the phorbols in initiating the respiratory burst correlated well with their EC50 for stimulating the appearance of PKC activity in the particulate fraction (R = 0.998). Redistribution of PKC activity in monocytes by phorbol myristate acetate (PMA) was rapid and appeared to precede the release of O2-. PMA also shifted PKC activity from the cytosol to the extractable particulate fraction of lymphocytes. We conclude that redistribution of PKC activity by active phorbols or other cell stimulants could provide substrate specificity for phosphorylation reactions. By shifting PKC activity to the monocyte particulate fraction, active phorbols may initiate the phosphorylation of a substrate required for stimulation of the respiratory burst. PMID- 2995491 TI - Expression of polypeptide segments of the human complement component C3 in E. coli: genetic and immunological characterization of cDNA clones specific for the alpha-chain of C3. AB - The third component of complement C3 and its fragments have a central role in a variety of host defense mechanisms. The identification of functionally relevant C3 domains is important because of the marked functional versatility of the C3 molecule. Several human C3 cDNA clones from a human liver cDNA library were isolated and characterized. A bacterial expression vector system was used to express cDNA clones that were identified by an immunological screening procedure. The C3 cDNA clones produced in E. coli the hybrid proteins consisting of cro-beta galactosidase and polypeptide segments of human C3, as revealed by Western blotting with antisera to human C3. The C3 moiety of the hybrid proteins had a m.w. of up to 46.000. Polyclonal antibodies against the C3 segments expressed by one of the C3 cDNA clones (ReC3-1) have been raised in mice and rabbit, and in addition, a monoclonal antibody was produced. The antisera and the monoclonal antibody reacted in Western blotting analysis selectively with the alpha-chain, but not the beta-chain of human C3. Restriction mapping of the different cDNA clones was performed, and revealed that the different clones were partially overlapping. The ReC3-1 cDNA clone included a 0.7 kb noncoding region at the 3' terminal end of the C3 cDNA. One of the restriction sites (Hind III) identified in the ReC3-1 cDNA clone was not present in the recently published sequence of human C3 cDNA. This difference in nucleotide sequence provides direct evidence for C3 polymorphism at the DNA level. The combination of immunologic procedures with recombinant DNA methodology should facilitate additional analysis of the structure-function relationship of the C3 molecule. PMID- 2995493 TI - Relationship between membrane potential changes and superoxide-releasing capacity in resident and activated mouse peritoneal macrophages. AB - In an attempt to understand better the molecular basis for the enhanced respiratory burst of activated macrophages (M phi), we investigated the relationship between stimulus-induced changes in membrane potential and release of superoxide anion (O2-) in mouse peritoneal M phi. Resident M phi and M phi elicited by injection of lipopolysaccharide (LPS-M phi) or obtained from animals infected with bacille Calmette-Guerin (BCG-M phi) were used. LPS-M phi and BCG-M phi showed more pronounced changes in membrane potential (depolarization) and greater release of O2- on contact with phorbol myristate acetate (PMA) than did resident macrophages. The lag time between addition of stimulus and onset of release of O2- was reduced in activated compared with resident cells. Membrane potential changes began 60 to 90 sec before release of O2- could be detected in each cell type. The dose-response curves for triggering of membrane potential changes and O2- release by PMA were identical. The magnitude of membrane potential changes and of O2- release in LPS-M phi and BCG-M phi declined progressively during in vitro culture, and values on day 3 approached those in resident macrophages ("deactivation"). Extracellular glucose was required for effective stimulated change in membrane potential and O2- release. These findings indicate that membrane potential changes are closely associated with O2- releasing capacity in macrophages, and that the systems that mediate membrane potential changes and production of O2- develop or decline concomitantly during activation or deactivation of the cells. Although the plasma membrane was highly depolarized by high extracellular K+ or by the sodium ionophore gramicidin, O2- release was not induced by these maneuvers, indicating that changes in membrane potential by themselves are not sufficient to trigger the respiratory burst in macrophages. Release of O2- was not impaired in buffers in which Na+ was completely replaced with equimolar concentrations of K+ or choline+; thus, induction or maintenance of the respiratory burst in M phi does not require an influx of Na+. PMID- 2995494 TI - Action of novel eicosanoids lipoxin A and B on human natural killer cell cytotoxicity: effects on intracellular cAMP and target cell binding. AB - Lipoxin A (5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic acid) and lipoxin B (5D,14,15-trihydroxy-6,8,10,12-eicosatetraenoic acid), two newly isolated compounds derived from the oxygenation of arachidonic acid in human leukocytes, inhibit the cytotoxic activity of human natural killer (NK) cells. Dose-response studies showed that both lipoxin A and lipoxin B inhibit, at submicromolar concentrations (ID50 10(-7) M), NK cell activity assayed against K562 target cells. Prostaglandin E2 (PGE2) also inhibited cytotoxicity, whereas both 15-HETE (5(S)-hydroxy-5,8,11,13-eicosatetraenoic acid) and leukotriene B4 (synthetic and biologically derived) were ineffective. PGE2 stimulated a time- and dose dependent increase in intracellular cAMP, which was accompanied by a decrease in NK target cell binding. Lipoxin A and lipoxin B did not elevate intracellular cAMP, nor did they inhibit target cell binding. Together these findings suggest that lipoxin A and lipoxin B abrogate NK cell cytotoxicity at a step distal to target effector cell recognition. In contrast, PGE2 appears to exert its effect, at least in part, on cytotoxicity indirectly by decreasing the binding between target and effector cells (in vitro). Moreover, they suggest that novel oxygenated derivatives of arachidonic acid (i.e., lipoxin A, lipoxin B) may regulate the activities of NK cells. PMID- 2995495 TI - Complex regulation of class II gene expression: analysis with class II mutant cell lines. AB - Several Ia-negative variants of a homozygous Iad-expressing antigen-presenting B lymphoma cell line, M12, have been obtained by repeated cycles of negative immunoselection after mutagenesis with ethylmethane sulfonate or gamma irradiation. Two such Iad-negative cell lines, selected with a mixture of alpha I Ad and alpha I-Ed monoclonal antibodies, failed to present antigen to all cloned Iad-restricted T cells tested, whereas the third cell line, selected with alpha I Ad reagents only, stimulated I-Ed but not I-Ad-restricted T cells. The mutations in all three cell lines resulted in the absence of RNA specific for the A beta d gene. In addition, two-dimensional gel electrophoresis of immunoprecipitates from one of the I-Ed-negative cell lines demonstrated the presence of intracytoplasmic Ed polypeptides that exhibited significantly decreased amounts of oligosaccharide induced heterogeneity. The introduction of class II A beta b and A alpha b genes by DNA-mediated transfection resulted in the serologic and functional expression of a class II I-Ab molecule but not the reexpression of the endogenous class II molecules; thus a transacting regulatory element is unlikely to be the target of the mutagenic event. The analysis of these and other Ia variant cell lines may prove useful in understanding the molecular mechanisms that control the expression of class II molecules in B cells. PMID- 2995496 TI - Effects of certain peptides upon protein kinase and phosphorylation patterns of Molt 4b lymphoblasts. PMID- 2995497 TI - A comparative study of neutrophil purification and function. AB - Several different methods are currently used by numerous laboratories for the isolation of human neutrophils. These methods include partial purification by dextran sedimentation followed by water lysis, and more complete purification procedures utilizing discontinuous density gradients coupled with dextran sedimentation and in some cases hypotonic lysis. Some investigators refrain from using certain purification schemes because certain steps or reagents used in a particular method might adversely affect the functional parameter of the neutrophil they wish to measure. In spite of these concerns there has been no systematic comparison of the functional status of neutrophils prepared by the various methodologies. In this study we have compared 4 commonly used methods of isolation with neutrophil function. The results of this study indicate that while neutrophil yield and purity were determined by isolation procedure, all cells were equivalent with regard to chemotactic performance, ability to degranulate, and ability to produce superoxide and hypochlorous acid. PMID- 2995498 TI - Effects of retinoids on the cyclic AMP system of pig skin epidermis. AB - Although retinoids reveal various biologic and biochemical activities on epidermal keratinocytes, their effects on the epidermal cyclic AMP (cAMP) system has been less well characterized. In order to elucidate the relation between them, an in vitro pig skin-slice incubation system was employed. After a long term (up to 24 h) incubation in vitro, control skin responded to epinephrine only slightly. The addition of Ro 10-1670, an active derivative of Ro 10-9359 (etretinate) in the incubation medium, resulted in an increase of the beta adrenergic adenylate cyclase response of epidermis. On the other hand, histamine induced cAMP accumulation was decreased by the retinoid treatment after long-term incubation. The augmentation of the beta-adrenergic response was observed at 1 microM concentration and the maximal effect was observed at 10 microM. There was no significant difference in cAMP phosphodiesterase activities between the control and retinoid-treated skin. The effect was also observed by the addition of all-trans-retinoic acid, retinol, and Ro 10-9359; the latter two compounds revealed much lesser effects. The addition of combinations of various drugs (Ro 10-1670 and hydrocortisone; Ro 10-1670 and colchicine) resulted in more marked (additive or synergistic) effects than the single addition of each chemical. On the other hand, the addition of Ro 10-1670 and all-trans-retinoic acid resulted in neither additive nor synergistic effect, suggesting that they probably work on the same site. Our data indicate that the epidermal beta-adrenergic adenylate cyclase response is modulated by retinoids probably as an independent mechanism stimulated by glucocorticoids or colchicine. PMID- 2995499 TI - Leukotriene B4 and 12-hydroxyeicosatetraenoic acid stimulate epidermal proliferation in vivo in the guinea pig. AB - Epidermal proliferation was studied in the guinea-pig ear in vivo using the incorporation of [3H]thymidine into DNA as a measure of cell proliferation. Twenty-four-hour pretreatment of the skin topically with nanomolar amounts of either leukotriene B4 (LTB4), 12-hydroxyeicosatetraenoic acid, ionophore A23187, or the epidermal mitogen 12-O-tetradecanoylphorbol-13-acetate induced dose dependent increases in epidermal proliferation. The stimulatory effect of LTB4 was stereospecific since a number of biologically inactive LTB4 stereoisomers had no effect. PMID- 2995500 TI - The role of exoenzyme S in infections with Pseudomonas aeruginosa. AB - To define the contribution of exoenzyme S to the pathogenesis of infections with Pseudomonas aeruginosa, we have compared the ability of an exoenzyme S-deficient mutant, 388 exs1::Tn1, and that of its exoenzyme S-producing parent to colonize and disseminate in burned mice infected with this organism. Both the exoenzyme S deficient mutant and the parent strain proliferated in burned skin, but only the parent strain was able to effectively disseminate to blood and other tissues. The reduced ability of the mutant to disseminate was not due to alterations in serum sensitivity, lipopolysaccharide composition, or motility. The exoenzyme S deficient mutant was able to disseminate in the presence of the exoenzyme S producing parent. Antibody to purified exoenzyme S was able to greatly reduce dissemination of the exoenzyme S-producing parent strain but did not prevent colonization in the burned skin. These data suggest that exoenzyme S does not contribute to the initial colonization but does contribute to the establishment of disseminated infection. PMID- 2995501 TI - Rapid determination of molecular relatedness of isolates of human cytomegalovirus. AB - Molecular comparisons of isolates of human cytomegalovirus (CMV) with restriction enzyme digests have helped to identify patterns of CMV transmission. Current techniques, however, require extensive tissue culture passage of the virus, which limits use of these analyses. In this report, we describe a rapid, less expensive, and equally sensitive method of comparing CMV genomes. This procedure, which we have called "junctional hybridization," uses the cloned junction fragments of CMV strain AD169 as probes that hybridize to CMV DNA restriction digests previously separated by electrophoresis and transferred to nitrocellulose filters. The procedure eliminates the need for tissue culture passage of virus beyond the primary isolation and can be used directly with DNA extracted from CMV infected tissue. In all cases, junctional hybridization was as sensitive as currently used methods of restriction enzyme digestion analyses in identifying identical or different CMV isolates. Use of junctional hybridization should facilitate molecular analyses for study of the epidemiology of CMV infections. PMID- 2995502 TI - The molecular epidemiology of cytomegalovirus transmission among children attending a day care center. AB - Cytomegalovirus (CMV) viruria was detected in 16 (25%) of 66 children attending a day care center. A significantly lower prevalence (6.9%) of viruria occurred among an age-matched control group of 1,457 hospitalized children. CMV DNA was compared by restriction endonuclease digestion of cell-associated CMV DNA, prepared by the Hirt procedure. CMV DNA fragments were detected directly on agarose gels by a 2-hr in situ hybridization with 32P-labeled DNA plasmids containing XbaI fragments of the Towne strain. EcoRI digestion of DNA isolated from 11 hospitalized children revealed 11 unique strains. A similar analysis, with EcoRI and several other endonucleases, of DNA isolated from the urine of 16 children attending the day care center revealed that one group of seven children and another group of four children were excreting identical strains of CMV. All seven children in the first group were less than 29 months old; six of these children shared the same classroom. All four children in the second group were greater than 36 months old; three were assigned to the same room. These results prove that CMV was frequently transmitted among children attending the day care center. PMID- 2995503 TI - Epidemiology of rotavirus diarrhea in a prospectively monitored American Indian population. AB - Rotaviral diarrhea is endemic in most areas of the world, yet community-wide epidemics have not been reported in prospectively monitored populations. This prospective study of the etiology of diarrhea included biweekly visits to the homes of 10% of the population of the White Mountain Apache Indians and began in April 1981. During a three-week period beginning 21 October, 1981, 342 new cases of diarrhea were identified on different parts of the reservation. Rotaviral antigen, detected by an enzyme-linked immunosorbent assay, was identified in 169 (73%) of the 233 stool samples that were tested. Rotavirus was not detected in any of the stool samples taken six months before or after the epidemic. During the epidemic, respiratory symptoms were present in 44 (33%) of 135 rotavirus positive patients compared with 17 (17%) of 98 rotavirus-negative patients (P less than .05). This rapidly spreading epidemic involving all areas of the reservation, in the absence of a common source of exposure of ill persons, suggests the possibility of respiratory transmission. PMID- 2995504 TI - Enzyme immunoassay with monoclonal antibodies for the detection of rotavirus in stool specimens. PMID- 2995505 TI - Sensitivity of clinical isolates of human cytomegalovirus to 9-(1,3-dihydroxy-2 propoxymethyl)guanine. PMID- 2995506 TI - Site specificity of epithelial irritants in the reactivation of herpes simplex virus. PMID- 2995507 TI - Antibody to lymphadenopathy-associated virus/human T lymphotropic virus type III in various groups of illicit drug abusers in New York City. PMID- 2995508 TI - Classification of HTLV-III/LAV-related diseases. PMID- 2995509 TI - Live attenuated varicella vaccine. PMID- 2995510 TI - Viral replication and immunologic responses in children naturally infected with varicella-zoster virus and in varicella vaccine recipients. AB - Replication of varicella-zoster virus (VZV) and immunologic responses to VZV were examined by a sensitive culture technique for viral isolation and standard immunologic assays in children after close exposure to wild-type VZV or after inoculation with strain Oka varicella vaccine. Naturally infected children who developed clinical varicella had viremia between five days before and one day after clinical onset of disease, with the highest isolation rate one and two days before onset, and seroconversion followed two days later. Virus was not isolated from blood 12 and 13 days after contact in subclinically infected children. In the vaccine recipients a positive skin test reaction to VZV was observed as early as four to five days after immunization, and antibody appeared later. Virus was not isolated from blood and throat of the vaccinees from three to 14 days after immunization. PMID- 2995511 TI - Determination of immunity to varicella-zoster virus by means of an intradermal skin test. AB - An intradermal varicella skin test, utilizing heat-inactivated noninfectious viral antigen, was evaluated in 16 adults known to be immune or susceptible to varicella and in 109 adults with no history of varicella. The skin test was well tolerated, compared favorably with established methods of determining immunity to varicella, and accurately predicted which subjects would develop clinical varicella after close exposure. PMID- 2995513 TI - Isolation of components of Brucella abortus responsible for inhibition of function in bovine neutrophils. AB - The effects of fractions of Brucella abortus strain 2308 on functions of bovine polymorphonuclear neutrophils (PMNs) were examined in vitro. Ingestion of Staphylococcus aureus and reduction of nitroblue tetrazolium dye by bovine PMNs were not inhibited by heat-killed B. abortus. The ability of PMNs to iodinate proteins was significantly inhibited by live or heat-killed B. abortus and supernatant from heat-killed cells but not by washed heat-killed cells. Two inhibitory components isolated from the supernatant by high-performance liquid chromatography were characterized as nucleotide-like substances with molecular weights of less than 1,000. Inhibition of iodination by these components was concentration dependent. These results indicate that one of the mechanisms by which B. abortus may escape intracellular killing by PMNs is through the production of low-molecular-weight components that inhibit the myeloperoxidase hydrogen peroxide-halide antibacterial system of bovine PMNs. PMID- 2995514 TI - [Effects of chemo-immunotherapy of malignant ovarian tumors. Collaborated study with affiliated hospitals]. PMID- 2995515 TI - [Experimental chemotherapy of ovarian cancers heterotransplanted in nude mice]. PMID- 2995512 TI - Epstein-Barr virus infections and DNA hybridization studies in posttransplantation lymphoma and lymphoproliferative lesions: the role of primary infection. AB - Fourteen patients who developed B cell lymphomas or lymphoproliferative lesions after kidney, liver, heart, or heart-lung transplantation in Pittsburgh during 1981-1983 had active infection with Epstein-Barr virus (EBV) of the primary (six patients), reactivated (seven patients), or chronic (one patient) type. In transplant patients without tumors, the incidence of EBV infection was 30% (39 of 128). Only three of these patients had primary infections. Thus the frequency of active infection was significantly higher in patients with tumors, and patients with primary infections were at greater risk of developing tumors. Five of 13 tumors tested contained EBV nuclear antigen (EBNA) and nine of 11 contained EBV genomes detected by DNA-DNA hybridization with BamHI K, BamHI W, or EcoRI B cloned probes. All EBNA-positive tumors, except one, were also positive by hybridization. Only one tumor was negative for both EBNA and EBV DNA. These data suggest that EBV plays an etiologic role in the development of these lesions. PMID- 2995516 TI - [Therapeutic methods and prognostic factors in common epithelial ovarian cancer]. PMID- 2995517 TI - Evidence for the activity of rifampin on the neuropathy of foot pad-inoculated mice with Mycobacterium leprae. PMID- 2995518 TI - [A case of IgD (lambda)-multiple myeloma presenting as a tonsillar tumor, characterized by various kinds of inclusions in the tumor cells as an initial manifestation]. PMID- 2995519 TI - [Cases of familial vitamin D resistant rickets in mother and child --pathogenesis of ankylosing spinal hyperostosis]. PMID- 2995520 TI - [A case of familial amyloid polyneuropathy demonstrating marked myocardial uptake of technetium-99m-pyrophosphate]. PMID- 2995521 TI - [A case of malignant thymoma associated with massive pericardial effusion, hypogammaglobulinemia and disseminated cytomegalovirus infection]. PMID- 2995522 TI - [Squamous cell carcinoma and cancer genes]. PMID- 2995523 TI - Characteristics of high-affinity and low-affinity adenosine binding sites in human cerebral cortex. AB - The binding characteristics of human brain cortical membrane fractions were evaluated to test the hypothesis that there are A1 and A2 adenosine binding sites. The ligands used were 2-chloro[8-3H]adenosine and N6-[adenine-2,8 3H]cyclohexyladenosine. Binding of chloroadenosine to human brain cortical membranes was time dependent, reversible, and concentration dependent. The dissociation constant (Kd) calculated for chloroadenosine by Scatchard analysis of equilibrium data was 280 nmol/L, with a maximum binding (Bmax) of 1.6 pmol/mg protein, suggesting a single class of binding sites. The specificity of chloroadenosine binding was assessed by the ability of adenosine analogues to compete for binding sites. With this approach, the apparent Kd was estimated to be 0.74 mumol/L for 5'-N-ethylcarboxamideadenosine, 1 mumol/L for cyclohexyladenosine, and 13 mumol/L for N6-(L-2-phenylisopropyl)adenosine. Isobutylmethylxanthine and theophylline, receptor antagonists, had apparent Kd values of 84 mumol/L and 105 mumol/L, respectively. Hill slope factors ranged from 0.3 to 0.6. Chloroadenosine binding to human brain cortical membranes approached equilibrium at 90 minutes, with a t 1/2 of 10 minutes. The kob was 0.080/min, and the k1 was 7.5 X 10(4)/min/mol/L. Reversibility of chloroadenosine binding at equilibrium was completed at approximately 10 minutes, with a k2 value of 0.074/min. The Kd calculated from the rate constants was 990 nmol/L. Cyclohexyladenosine binding was concentration dependent. The Kd calculated for cyclohexyladenosine via Scatchard analysis of equilibrium data was 5 nmol/L with a Bmax of 0.35 pmol/mg protein. Cyclohexyladenosine binding was displaced by three known receptor agonists: N6-(L-2-phenylisopropyl)adenosine (Kd 4 nmol/L), 2 chloroadenosine (Kd 10 nmol/L) and 5'-N-ethylcarboxamideadenosine (Kd 6 nmol/L).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995524 TI - Metastatic tumours of the temporal bone. A histopathological report. AB - Metastatic tumours of the temporal bone seem to be more common than is recognized. Most of these tumours are microscopic and asymptomatic in nature. Microscopic examination of 22 temporal bones belonging to 13 cases of metastatic tumours is reported. The commonest site of involvement in the temporal bone was the petrous apex followed by the tegmen tympani, mastoid bone and internal auditory canal. Primary tumours were most commonly located in the breast. Other sites of primary tumours included the thyroid gland, brain, lungs, prostate and blood (leukaemia). Two cases had undetermined sites of origin. Full neurotologic evaluation is indicated in every case suspected of having a temporal bone metastasis. All three modalities (of surgery, radiotherapy and chemotherapy) are used in combination for the treatment of these tumours. PMID- 2995525 TI - Modulation of neutrophil-reduced pyridine nucleotide content following stimulation with phorbol myristate acetate and chemotactic factors. AB - Modulation of NAD(P)H in human neutrophils (PMN) following stimulation with phorbol myristate acetate (PMA) or chemotactic factors was determined by flow cytometry. Stimulation of PMN with 1 microgram/ml of PMA results in a time dependent decrease in fluorescence, attributable to the oxidation of NAD(P)H. The decrease in fluorescence did not occur with PMN from a patient with chronic granulomatous disease (CGD) and was observed in only half of PMN from the mother of the patient. Loss of fluorescence in normal PMN was maximal following 7-15 min of stimulation with PMA. Simultaneous measurement of PMA-stimulated NAD(P)H oxidation and H2O2 production showed that NAD(P)H oxidation occurred as an all-or none response while H2O2 production showed a graded response. These data suggest that with PMA stimulation, a threshold exists beyond which constitutive NAD(P)+ reduction is suppressed and complete oxidation of NAD(P)H occurs, while H2O2 production is proportional to the concentration of PMA. PMA-stimulated oxidation of NAD(P)H was reversible, and fluorescence returned to the initial level or higher after 40-60 min. Oxidation of NAD(P)H also occurred when cytochalasin B treated PMN were stimulated with 25 nM C5a or 100 nM formyl-methionyl-leucyl phenylalanine (f-MLP), but occurred more rapidly, peaking at 1 to 3 min. Fluorescence also returned by 5-6 min. This response to C5a and f-MLP was graded and proportional to the concentration of chemotactic factor used. Comparative studies showed that the cytochalasin-B treatment was essential for measurement of NAD(P)H oxidation, in response to C5a and F-MLP. PMID- 2995526 TI - Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by reversible phosphorylation-dephosphorylation. PMID- 2995528 TI - Nature of the inhibitory effect of collagenase on phosphodiesterase activity. AB - The level of phosphodiesterase (PDE) activity is lower in collagenase-isolated human fat cells than in adipose tissue fragments. The inhibition is not species specific since collagenase also inhibits PDE in rat adipose tissue and bovine heart. In subcellular fractions from isolated fat cells, the PDE activities were lowest in the plasma membrane-enriched fractions and highest in the cytosolic fractions. This is opposite to PDE in subcellular fractions obtained from adipose tissue fragments. In dose-response experiments, collagenase inhibited particulate PDE to a much larger extent than it inhibited soluble PDE. The extracellular activities of PDE were completely eliminated by collagenase. Repeated washings or reincubation of the isolated fat cells did not restore the PDE activity. A purified collagenase with low specific protease activity reduced the PDE activity in isolated fat cells to a lesser extent than did a collagenase with high specific protease activities. Collagen and several protease inhibitors were ineffective in preventing the reduction of PDE after exposure to collagenase. It is concluded that nonspecific proteases in the collagenase preparations used for fat cell isolation interact with particulate and soluble PDE causing an irreversible inhibition of PDE activity in isolated fat cells. Of the various forms of PDE, plasma membrane-associated PDE seems most sensitive to collagenase. PMID- 2995527 TI - Cholesterol metabolism: use of D2O for determination of synthesis rate in cell culture. AB - Cholesterol synthesis in cell culture in the presence of D2O yields a spectrum of enriched molecules having a relative abundance that indicates random substitution of deuterium for hydrogen. Quantitation of the absolute rate of cholesterol synthesis is obtained by isotope ratio mass spectrometry. Mevinolin and 26 hydroxycholesterol both decrease cholesterol synthesis rate but have a discordant effect on HMG-CoA reductase activity. PMID- 2995529 TI - Antenatal Rh iso-immunization prophylaxis: implications for blood bank assessment. PMID- 2995530 TI - Calmodulin regulation of cellular cyclic AMP production in response to arginine vasopressin, prostaglandin E2 and forskolin in rat renal papillary collecting tubule cells in culture. AB - The effect of calmodulin on the stimulation of cyclic AMP production by arginine vasopressin (AVP), prostaglandin E2 (PGE2) and forskolin was examined in cultured renal papillary collecting tubule cells of the rat. In the presence of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine submaximal concentrations of AVP (1 nmol/l), PGE2 (20 nmol/l) and forskolin (240 nmol/l) significantly increased cellular cyclic AMP accumulation by 2.3-, 6.0- and 8.4-fold respectively. Two chemically dissimilar inhibitors of calmodulin, namely trifluoperazine and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulphonamide (W-7), attenuated the AVP-, PGE2- and forskolin-stimulated cellular production of cyclic AMP in a dose-related manner. Cellular production of cyclic AMP was inhibited by 50% (ID50) by doses ranging from 16 to 28 mumol trifluoperazine/l and 35 to 44 mumol W-7/1. Basal accumulation of cellular cyclic AMP was also decreased by treatment with either trifluoperazine or W-7, but the effective dose was higher than that which inhibited cellular cyclic AMP production stimulated by AVP, PGE2 and forskolin. Since forskolin directly activates adenylate cyclase at a site of the catalytic unit and the cellular action of AVP to activate adenylate cyclase is mediated through receptor-guanine nucleotide regulatory-catalytic units, the present study indicates calmodulin regulation of basal, AVP-, PGE2- and forskolin activated adenylate cyclase in the papillary collecting tubule cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995532 TI - Pituitary gonadotrophin-releasing hormone receptor up-regulation in vitro: dependence on calcium and microtubule function. AB - Exogenous cyclic adenosine nucleotides increase gonadotrophin-releasing hormone (GnRH) receptors in intact cultured rat pituitary cells in a similar manner to that observed with GnRH itself. In this study the calcium and microtubule dependency of GnRH receptor up-regulation was examined in vitro. Treatment of pituitary cells in Ca2+ and serum-containing media with either GnRH (1 nmol/l), K+ (58 mmol/l) or dibutyryl cyclic AMP (dbcAMP; 1 mmol/l) for 7-10 h routinely resulted in a 50-100% increase in GnRH receptors. Incubation of pituitary cells with the calcium channel blocker verapamil, for 7 h, or the calcium chelator EGTA, for 10 h, had no effect on basal receptor levels but prevented the increase in GnRH receptors stimulated by either GnRH, K+ or dbcAMP. Luteinizing hormone release measured with the same stimulators over a 3-h period was prevented by both verapamil and EGTA. Calcium ionophore (A23187) increased GnRH receptors by 40-60% at low concentrations (10 and 100 nmol/l) while higher concentrations (10 and 100 mumol/l) reduced receptor levels. Luteinizing hormone release was not increased by receptor-stimulating concentrations of A23187, but was by higher concentrations (10 mumol/l). None of these pretreatments, for up to 10 h, impaired the subsequent LH response of the cells to increasing doses of GnRH. Vinblastine (1 mumol/l did not affect basal receptor levels but markedly reduced the increase in GnRH receptors stimulated by GnRH, K+ and dbcAMP. This concentration of vinblastine had no effect on LH release.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995531 TI - Homologous ligand regulation of gonadotrophin-releasing hormone receptors in vivo: relationship to gonadotrophin secretion and gonadal steroids. AB - A single injection of gonadotrophin-releasing hormone (GnRH) (60 ng s.c., 42.9 nmol) induced biphasic GnRH receptor regulation in normal intact adult female mice. A transient 22% receptor decrease occurred 30-60 min after injection of GnRH when peak serum decapeptide concentrations were reached (137 +/- 41 (S.E.M.) ng/l). This GnRH receptor decrease occurred shortly after the peak serum LH values at 15-30 min. The subsequent rapid (within 1 h) return of GnRH receptor levels to normal suggested transient receptor occupancy by GnRH rather than true receptor loss. At 8 h after injection of GnRH a significant 35% increase in GnRH receptors was consistently observed, when serum GnRH levels were undetectable and serum LH had returned to basal levels. This receptor increase was not due to increased receptor affinity, and was prevented by a non-specific protein synthesis inhibitor. Ovariectomy, which caused a 50% fall in GnRH receptors (59.4 +/- 4.9 fmol/pituitary gland in intact controls; 26.9 +/- 2.6 in ovariectomized mice) abolished the induction by GnRH of its own receptors, although the initial transient decrease occurred over the period of the acute serum LH and FSH rise. Despite a 50% reduction in GnRH receptors in ovariectomized mice, increased serum gonadotrophin levels and responsiveness to GnRH were maintained, indicating dissociation between receptor changes and gonadotrophin levels. No GnRH receptor up-regulation was observed 8 h after a single GnRH injection (60 ng s.c.) in either intact or orchidectomized normal male mice. However, the same treatment doubled GnRH receptors in GnRH-deficient (hpg) female mice.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995534 TI - Iodine deficiency, hypothyroidism, and endemic goitre in southern Tanzania. A survey showing the positive effects of iodised oil injections by TSH determination in dried blood spots. AB - The Ukinga and Uwanji regions, located in the southern highlands of Tanzania, were studied for the degree of iodine deficiency and the incidence of goitre and hypothyroidism, respectively. A urinary iodine excretion as low as 17.6 +/- 9.3 micrograms/g creatinine was observed in Wangama village. The mean goitre prevalence in 27 villages in Uwanji ranged between 65 and 96% (n = 3031 schoolchildren). Of 681 pregnant women from Ukinga 79.6% had goitre. The prevalence of cretinism as estimated on clinical criteria was 3% in Magoye (Uwanji). A normal serum TSH (below 2.1 mU/l) was observed in only 12 out of 66 school children before iodine prophylaxis, whereas the T4/TBG ratio was decreased in 36 of 63 cases. Blood spot TSH levels in newborn infants (n = 219) from mothers without iodine supplementation were above 12 mU/l in 45%. In contrast, only 20.3% of the newborn (n = 118) had elevated blood spot TSH (p less than 0.002) when the mothers had received an iodised oil injection during pregnancy. Most of the newborn (n = 18; 75%) of the latter group with elevated TSH (n = 24) came from mothers who had received the iodine injection only 1-25 days before delivery. Maternal iodine prophylaxis in late pregnancy does not increase the rate of neonatal hypothyroidism. CONCLUSIONS: It has been confirmed that severe iodine deficiency resulting in endemic goitre, cretinism, and hypothyroidism is prevalent in the regions studied. Dried blood spot TSH determinations may serve as an index for the efficiency of iodine prophylaxis programmes. Such a programme was carried out with relatively little expenditure and effort on a large scale basis. PMID- 2995535 TI - Pig interleukin 1. Purification of two immunologically different leukocyte proteins that cause cartilage resorption, lymphocyte activation, and fever. AB - Two forms of interleukin 1 (IL-1) were purified to homogeneity from the culture supernatants of pig buffy coat leukocytes stimulated with concanavalin A. The two proteins had identical Mr of 21,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but one, which had previously been purified as a cartilage resorbing protein, had pI 5 (IL-1/5) and the other, pI 8.3 (IL-1/8). After initial gel filtration and separation by chromatofocusing IL-1/5 was purified by chromatography on hydroxyapatite and the ion exchangers, Mono S and Mono Q; IL 1/8 was purified by chromatography at pH 4.0 and pH 6.4 on Mono S. Purification was monitored by a cartilage proteoglycan release assay and both proteins had a final specific activity approximately 10(5) times that of the leukocyte culture medium. Medium conditioned by cells from 200 L of blood yielded approximately 15 micrograms of IL-1/5 and 50 micrograms IL-1/8. IL-1/8 augmented mouse thymocyte proliferation, stimulated synovial fibroblasts to produce prostaglandin E and latent collagenase, and was pyrogenic upon intracerebroventricular injection into rabbits. IL-1/5 has previously been shown to possess all these activities. An antiserum to each IL-1 was raised in rabbits. Each antiserum inhibited its respective IL-1 in a cartilage bioassay and stained it upon Western blotting. Neither antiserum inhibited or stained the other IL-1. We conclude that pig leukocytes make two immunologically distinct forms of IL-1 that have identical Mr, demonstrate the same range of biological activity, but differ in isoelectric point. PMID- 2995533 TI - Pituitary receptors for LH-releasing hormone (LHRH) and responsiveness to LHRH in adult female rats after neonatal monosodium L-glutamate treatment. AB - The effects of neonatal monosodium L-glutamate (MSG) treatment on pituitary responsiveness to LH-releasing hormone (LHRH) and on pituitary LHRH receptors have been investigated in the intact adult female rat. Three- to four-month-old rats treated with MSG (4 mg/g body wt) on days 2, 4, 6, 8 and 10 after birth had significantly reduced ovarian and pituitary weights, showed an absence or disruption of ovarian cyclicity after puberty, and had significantly higher concentrations of serum prolactin despite normal levels of LH. In-vitro pituitary LH responses to LHRH were in the normal range for one group of treated animals whilst in a second group the LH responses were markedly enhanced. In contrast, the total number of pituitary LHRH receptors were significantly reduced in all MSG-treated animals showing that the increased pituitary responsiveness of MSG treated animals is not attributable to an increase in pituitary LHRH receptors. PMID- 2995538 TI - Simplification of a commercially available serum angiotensin-converting enzyme determination. AB - The spectrophotometric method for the determination of angiotensin-converting enzyme in serum using p-hydroxyhippuryl-L-histidyl-L-leucine as substrate is commercially available as a test kit. It shows excellent linearity over the whole range of catalytic concentration found in serum. We describe several modifications of this method to simplify and economize the procedure. PMID- 2995537 TI - Pancreatic B-cell peptides as parameters for diagnosis and localisation of hormone secreting tumours. AB - Insulin, C-peptide and proinsulin were measured in peripheral blood of 11 patients suffering from different types of hormone-producing pancreatic B-cell tumours. While proinsulin was elevated in 10/11 patients during prolonged fasting, C-peptide and insulin levels were found within the reference range in 5/11 and 3/5 cases respectively. A rapid insulin assay performed during surgery was helpful for localisation and identification of the respective tumours. PMID- 2995536 TI - Helper T cells induced by an immunopurified herpes simplex virus type I (HSV-I) 115 kilodalton glycoprotein (gB) protect mice against HSV-I infection. AB - Three herpes simplex virus type I (HSV-I) glycoproteins of apparent molecular masses 103, 63, and 115 kD have been purified using virus-specific monoclonal antibodies (mAb) G8D1, C2D2, and T157, respectively. Both G8D1 and C2D2 neutralize HSV-I in vitro and passively protect CBA mice against HSV-I infection in vivo, whereas T157 is neither neutralizing nor passively protective. However, mice given a single subcutaneous injection of 30 micrograms 115 kD glycoprotein in saline were completely protected against lethal challenges of HSV-I administered intraperitoneally or in the footpad 7 d after immunization. In contrast, mice similarly immunized with 103 or 63 kD glycoproteins were only partially protected. The prophylactic immunity was correlated with an early induction of specific antibody, which became even more evident 3 d after virus challenge. There was a remarkable similarity in antibody isotype distribution between the responses to 115 kD glycoprotein and to heat-inactivated intact HSV I. However, the prechallenge sera from 115 kD glycoprotein hyperimmunized mice were again neither virus-neutralizing nor passively protective. All three glycoproteins induced only low levels of delayed-type hypersensitivity (DTH). Pretreatment of mice with cyclophosphamide significantly enhanced DTH to 115 kD and 103 kD glycoproteins in the absence of antibody, but failed to confer significant immunity, indicating that DTH alone is insufficient for protection. Splenic and lymph node Ig- (B cell-depleted) cells from mice protectively immunized with 115 kD glycoprotein could adoptively transfer effective protection and enhance a virus neutralizing antibody response in normal recipients challenged with a lethal dose of HSV-I. Both the protection and the ability to enhance neutralizing antibody were diminished when the cells were treated with mAb GK 1.5 and complement. These results therefore demonstrate that the 115 kD glycoprotein, though not apparently containing accessible epitopes for the induction of virus-neutralizing antibody, possesses determinants capable of activating helper T cells. These L3T4+ cells confer strong protective immunity by enhancing protective antibody upon challenge infection, probably through associative help. PMID- 2995539 TI - Fatal nonmetastatic neurological complications of pulmonary carcinoma. PMID- 2995540 TI - Tongue metastasis from undifferentiated small cell (oat cell) lung cancer. PMID- 2995541 TI - Intrinsic characteristics of the proton pump in the luminal membrane of a tight urinary epithelium. The relation between transport rate and delta mu H. AB - A number of tight urinary epithelia, as exemplified by the turtle bladder, acidify the luminal solution by active transport of H+ across the luminal cell membrane. The rate of active H+ transport (JH) decreases as the electrochemical potential difference for H+ [delta mu H = mu H(lumen) - mu H(serosa)] across the epithelium is increased. The luminal cell membrane has a low permeability for H+ equivalents and a high electrical resistance compared with the basolateral cell membrane. Changes in JH thus reflect changes in active H+ transport across the luminal membrane. To examine the control of JH by delta mu H in the turtle bladder, transepithelial electrical potential differences (delta psi) were imposed at constant acid-base conditions or the luminal pH was varied at delta psi = 0 and constant serosal PCO2 and pH. When the luminal compartment was acidified from pH 7 to 4 or was made electrically positive, JH decreased as a linear function of delta mu H as previously described. When the luminal compartment was made alkaline from pH 7 to 9 or was made electrically negative, JH reached a maximal value, which was the same whether the delta mu H was imposed as a delta pH or a delta psi. The nonlinear JH vs. delta mu H relation does not result from changes in the number of pumps in the luminal membrane or from changes in the intracellular pH, but is a characteristic of the H+ pumps themselves. We propose a general scheme, which, because of its structural features, can account for the nonlinearity of the JH vs. delta mu H relations and, more specifically, for the kinetic equivalence of the effects of the chemical and electrical components of delta mu H. According to this model, the pump complex consists of two components: a catalytic unit at the cytoplasmic side of the luminal membrane, which mediates the ATP-driven H+ translocation, and a transmembrane channel, which mediates the transfer of H+ from the catalytic unit to the luminal solution. These two components may be linked through a buffer compartment for H+ (an antechamber). PMID- 2995542 TI - Biochemical basis of resistance to hygromycin B in Streptomyces hygroscopicus- the producing organism. AB - Hygromycin B, an aminocyclitol antibiotic that strongly inhibits both 70S and 80S ribosomes, is synthesized by Streptomyces hygroscopicus. Ribosomes from this Gram positive mycelial bacterium are inhibited in vitro by the antibiotic. In contrast, the streptomycete is highly resistant to the drug in vivo since it possesses hygromycin B phosphotransferase activity. This enzyme has been shown by gel filtration to have a molecular weight of 42000, and to modify its antibiotic substrate to produce 7"-O-phosphoryl-hygromycin B which totally lacks biological activity both in vivo and in vitro. PMID- 2995543 TI - Temperature-sensitive mutants of the Streptomyces plasmid pIJ702. AB - DNA from the Streptomyces plasmid pIJ702 was mutagenized in vitro using hydroxylamine and transformed into Streptomyces lividans. One plasmid with temperature-sensitive replication (pMT660) and one plasmid with a temperature sensitive tyrosinase (pMT661) were isolated. The plasmid pMT661 contains a novel PstI restriction endonuclease site within the tyrosinase gene. PMID- 2995544 TI - The respirative breakdown of glucose by Saccharomyces cerevisiae: an assessment of a physiological state. AB - Cells of Saccharomyces cerevisiae exhibiting respirative glucose metabolism in continuous culture were able to use ethanol as a co-substrate. The ethanol uptake rate was dependent on the residual respirative capacity of the cells. The activities of gluconeogenic enzymes and of malate dehydrogenase were higher in cells degrading glucose respiratively than in cells metabolizing glucose respiro fermentatively, but were lower than in cells growing on ethanol only. The pattern of distribution of the mitochondrial cytochromes was similar but the differences were less distinct. In synchronously growing cells, the activities of gluconeogenic enzymes and of malate dehydrogenase oscillated, with activities increasing during the budding phase. The increase was preceded by the appearance of ethanol in the culture medium. PMID- 2995545 TI - Regulation by ammonium of glutamate dehydrogenase (NADP+) from Saccharomyces cerevisiae. AB - The activity of glutamate dehydrogenase (NADP+) (EC 1.4.1.4; NADP-GDH) of Saccharomyces cerevisiae is decreased under conditions in which intracellular ammonia concentrations increases. A high internal ammonia concentration can be obtained (a) by increasing the ammonium sulphate concentration in the culture medium, and (b) by growing the yeast either in acetate + ammonia media, where the pH of the medium rises during growth, or in heavily buffered glucose + ammonia media at pH 7.5. Under these conditions cellular oxoglutarate concentrations do not vary and changes in NADP-GDH activity appear to provide a constant rate of oxoglutarate utilization. The following results suggest that the decrease in NADP GDH activity in ammonia-accumulating yeast cells is brought about by repression of synthesis: (i) after a shift to high ammonium sulphate concentrations, the number of units of activity per cell decreased as the inverse of cell doubling; and (ii) the rate of degradation of labelled NADP-GDH was essentially the same in ammonia-accumulating yeast cells and in controls, whereas the synthesis constant was much lower in the ammonia-accumulating cells than in the controls. PMID- 2995546 TI - Molecular homology and incompatibility relationships between F and IncH1 plasmids. AB - IncH1 plasmids and the F plasmid of Escherichia coli display one-way compatibility. An entering IncH1 plasmid is incompatible with a resident F plasmid, but is compatible when it is the resident plasmid. There is little molecular homology between IncH1 plasmids and the F plasmid. A single 5 MDal EcoRI restriction enzyme fragment from digests of several IncH1 plasmids hybridizes with probes constructed from the primary replication region of F. Homology can be demonstrated only with the gene for the essential replication protein of F (gene E), but the expression of incompatibility behaviour appears to be associated with the presence of the secondary replicon of the F plasmid. Thus R27 and F are compatible under growth conditions allowing replication and maintenance of F by the secondary replicon. However, a mutant F plasmid which lacks the secondary replicon of F is incompatible with R27 in both directions, irrespective of the growth conditions used. PMID- 2995547 TI - Genomic analysis of phase I and II Coxiella burnetii with restriction endonucleases. AB - Restriction endonuclease-digested DNAs from several isolates of phase I and phase II Coxiella burnetii were compared using agarose gel electrophoresis and soft laser scanning densitometry. Our results demonstrate that the two phases are, as previously assumed, alternative phases of the same organism. Although the restriction endonuclease digestion revealed genetic differences between clonal isolates of phase I and phase II C. burnetii Nine Mile strain, these differences do not appear to be related to antigenic phase variation. However, analyses of the fragment patterns generated by restriction enzyme digestion suggest potential grouping of the different isolates. PMID- 2995548 TI - Cloning of a DNA fragment from Streptomyces griseus which directs streptomycin phosphotransferase activity. AB - DNA from Streptomyces griseus ATCC 12475 was partially digested with Sau3A and fragments were ligated into BglII-cleaved pIJ702. When the ligation mixture was used to transform protoplasts of Streptomyces lividans TK54, two transformants resistant to both thiostrepton and streptomycin were isolated. The hybrid plasmids pBV3 and pBV4 which they contained, carrying inserts of sizes 4.45 and 11.55 kbp respectively, each retransformed S. lividans to streptomycin resistance at high efficiency. Both plasmids hybridized to restriction digests of S. griseus chromosomal DNA in Southern blot experiments. In vitro deletion and sub-cloning experiments showed the sequence conferring streptomycin resistance to lie within a segment of 1.95 kbp. Extracts of TK54(pBV3) and TK54(pBV4) contained a streptomycin phosphotransferase similar to that in extracts of S. griseus. Streptomycin phosphotransferase activity appeared in extracts of S. griseus, TK54(pBV3) and TK54(pBV4) within 2 d of inoculation. When pBV3 and pBV4 were retransformed into S. griseus with selection for thiostrepton resistance, plasmid DNA of sizes corresponding to the incoming plasmids was found in the transformants. In these transformants the phosphotransferase appeared at 1.5 rather than 2 d, and reached a level over twice that of the original S. griseus strain. PMID- 2995549 TI - Evidence for related virulence sequences in plasmids of Salmonella dublin and Salmonella typhimurium. AB - Transposon-insertion mutants were prepared from virulent field isolates of Salmonella dublin and Salmonella typhimurium. Detailed restriction-enzyme mapping of the single sites of TnA insertion in two mutants (M51 and M173) of S. dublin that showed diminished virulence in a mouse assay indicated that these sites were about 5 kbp apart on the approximately 70 kbp plasmid harboured by the isolate. A Tn10-insertion mutant (M242) of S. typhimurium that showed diminished virulence was also identified. A single copy of Tn10 was inserted into the approximately 90 kbp plasmid harboured by this isolate. Hybridization studies indicated that homology existed between the region encompassing the sites of TnA insertion in M51 and M173 and that encompassing the site of Tn10 insertion in M242. Restriction mapping indicated that the two regions were very similar and could even be identical and, if so, the Tn10 insertion in M242 could be mapped to a point 1.5 kbp from the TnA insertion in M51 and 6.5 kbp from that in M173. It appeared that the maximal extent of the putative similarity/identity was between 13 and 23 kbp. It is proposed that this stretch of high homology could represent a virulence sequence that has been conserved during the evolutionary divergence of the two Salmonella serotypes. PMID- 2995550 TI - Paramyxovirus antigens in osteoclasts from Paget's bone tissue detected by monoclonal antibodies. AB - The fluorescent antibody technique using both monoclonal and specific polyclonal virus antibodies was applied to investigate the nature of the inclusions seen in the abnormal osteoclasts associated with Paget's bone disease. The results show that antigens of measles virus, simian virus 5 (SV5) and human parainfluenza virus type 3 (PF3) could be detected in the osteoclasts but not in control bone cells. Measles and SV5 nucleoprotein (NP) and haemagglutinin-neuraminidase (HN) antigens were apparently present in all the cases of Paget's disease examined, whereas PF3 NP and HN antigens were present only in some of the cases. These investigations suggest that paramyxoviruses may play a role in the aetiology of the bone disease. PMID- 2995551 TI - A cAMP-independent serine/threonine kinase activity is associated with the mos sequences of ts110 Moloney murine sarcoma virus-encoded P85gag-mos. AB - Two proteins, termed P85gag-mos and P58gag, are encoded by the temperature sensitive transformation mutant, ts110 Moloney murine sarcoma virus (MuSV). Based on temperature-shift studies, P85gag-mos is believed to be important for the transforming potential of ts110 MuSV and has been found to be associated with a thermolabile kinase activity that phosphorylates both P85gag-mos and P58gag in immune complexes. Modifications of the original kinase assay conditions are reported here that have allowed a 30-fold increase in the specific activity of P85gag-mos phosphorylated in vitro. The in vitro P85gag-mos-phosphorylating activity was found to be unresponsive to 10 microM-cAMP or 10 microM-cGMP. Addition of 1 mM-pyrophosphate, a known phosphatase inhibitor, to the reaction mixture resulted in an increased yield of phosphorylated P85gag-mos and P58gag; the molar phosphate incorporation per mole of P85gag-mos increased from 0.032 to 0.9, whereas the specific activity of in vitro-phosphorylated P58gag increased 18 fold, from 0.013 to 0.234. pH curves of the in vitro kinase reaction further confirmed the presence of phosphatase activity; in the absence of pyrophosphate, a sharp optimum at pH 4 to 5 was observed, whereas it shifted broadly to pH 7.0 in the presence of pyrophosphate. Under the latter conditions, several experiments were performed in order to determine if the kinase was associated with either gag or mos sequences of P85gag-mos. Antisera directed against p15, p12 and p30 sequences of the gag protein region of P85gag-mos yielded immune complexes that allowed phosphorylation in vitro of P85gag-mos. No phosphorylating activity was detected in immune complexes containing MuSV-124-encoded P62gag. An anti-mos serum generated against a synthetic peptide representing the predicted v mos amino acid residues 37 to 55 recognizes P85gag-mos and allowed phosphorylation of P85gag-mos in vitro in the absence of P58gag. Peptide mapping of both phosphorylated P85gag-mos and P58gag, by using a combination of Cleveland and Western/immunoperoxidase techniques, demonstrated that P85gag-mos became phosphorylated not only on gag sequences, but also at the N-terminal portion of v mos. Phosphoamino acid analyses of P85gag-mos and P58gag phosphorylated in vitro under these modified conditions yielded predominantly phosphoserine and lesser amounts of phosphothreonine. Metabolically 32P-labelled P85gag-mos and P58gag were also found to contain phosphoserine and phosphothreonine. Based on these results, we conclude that a cAMP-independent, serine/threonine protein kinase activity is associated with the mos sequences of P85gag-mos. PMID- 2995552 TI - Hormonal regulation of a polyoma virus middle-size T-antigen gene linked to growth hormone control sequences. AB - We constructed a recombinant DNA gene containing sequences from the 5' flanking region of a dexamethasone-inducible rat growth hormone gene linked to the coding region of the polyoma virus transforming protein, the middle-size T (MT) antigen. We used this gene to derive cell lines in which the expression of the MT antigen could be regulated by dexamethasone. We transfected mouse NIH 3T3 cells and isolated transformed foci from cultures grown in the presence or absence of dexamethasone. The frequency of focus formation and the size of the transformed foci were increased in the presence of dexamethasone. Several transformants showed regulated expression of the MT antigen: the levels of polyoma-specific RNA, MT protein and MT-associated kinase activity were increased two- to fivefold in cells grown in the presence of dexamethasone. These results show that 248 base pairs of rat growth hormone DNA, including the first 237 base pairs upstream from the major transcription initiation site, contain promoter activity and a regulatory element required for glucocorticoid induction. This region of the rat growth hormone can be used to regulate expression of the polyoma MT antigen gene. In some cell lines regulated expression of the MT antigen was accompanied by regulated expression of transformed cell growth properties. The minimum level of the MT antigen required for expression of transformation was considerably less than the level found in a polyoma MT-transformed cell line. Increasing the level of the MT antigen led to increased expression of transformation, assayed by morphology, focus formation and growth in agar. PMID- 2995553 TI - The regulation of translation in reovirus-infected cells. AB - The regulation of translation in reovirus-infected cells has been investigated by double-infection experiments. Different cell lines were able to translate uncapped encephalomyocarditis (EMC) virus mRNA and capped vesicular stomatitis virus (VSV) mRNAs both early and late during reovirus infection. These results are not fully in agreement with a previously suggested model in which, in the early phase of reovirus infection, only capped mRNAs are translated, whereas in the late phase the cells are modified to translate exclusively uncapped mRNAs. The observations that EMC virus and VSV shut down host protein synthesis more efficiently than reovirus translation in the late phase in double-infections indicate that competition for a message-discriminatory factor may not be involved in the shut-off of host protein synthesis in these animal virus-infected cells. PMID- 2995554 TI - Demonstration of the colinearity of human cytomegalovirus genomes and construction of restriction maps of unknown isolates using cloned subgenomic fragments. AB - In this study, we have established the colinearity of human cytomegalovirus (HCMV) genomes using stringent conditions of DNA-DNA filter hybridization of HCMV HindIII fragments and cosmid-cloned AD169 strain HCMV DNA fragments. Large cosmid cloned fragments of AD169 DNA were used for the preparation of radioactive probes by nick translation. These probes were hybridized to HindIII digests of DNA from three fresh isolates of HCMV and to that of the Davis strain. Using published HindIII restriction maps for the AD169 strain as a reference, the results obtained by hybridization allowed us to construct HindIII restriction maps for the genomes of the three fresh isolates. Confirmation of our methodology was found in the correspondence between the HindIII map we constructed for the Davis strain and that published previously. Furthermore, it is shown that variation in the restriction profiles of the unique regions of the genome are due to the absence or gain of restriction sites, and not to a rearrangement of fragments. This technique allows rapid construction of physical maps of the DNA of any fresh isolate for a given restriction enzyme provided the corresponding restriction map of a strain to be used as reference is available. PMID- 2995555 TI - Asynchronous expression of the immediate-early protein of herpesvirus saimiri in populations of productively infected cells. AB - The time course of virus replication in cultures of permissive cells infected with high multiplicities of herpesvirus saimiri (HVS), a gammaherpesvirus, is protracted relative to the replication of herpes simplex virus (HSV), an alphaherpesvirus, under similar conditions. The basis for this difference was investigated by quantitative immunofluorescence microscopy exploiting monoclonal antibodies specific to the HVS 52 000 mol. wt. immediate-early polypeptide (IE 52K) and to delayed-early (DE 51K, DE 110K) and late (130K and capsid proteins) gene products to measure the timing of gene expression in individual cells of infected cultures. The timing of the transitions from IE to DE and from DE to late protein synthesis occurred at proportionately different intervals in the growth cycle of HVS, relative to that of HSV. In particular, the DE to late transition occurred relatively later in HVS infections. However, asynchrony in the events leading to the expression of the first class of virus proteins (IE 52K) was the main source of the extended course of HVS replication in populations of infected cells. This asynchrony was not modified significantly by infection at different stages of the host cell-cycle and was reduced, but not overcome, by very high applied multiplicities of infection. Double-antibody staining revealed a positive correlation between the accumulation of high concentrations of parental virus particles at perinuclear sites and early detection of HVS IE 52K gene expression. Both of these events remained sensitive to a microtubule poison (colcemid) for many hours after infection with HVS, whereas the rapid and synchronous expression of the IE 175K protein (ICP4) in HSV-1-infected cells was insensitive to post-infection exposures to this drug. We conclude that significant differences in early stages of virus entry and intracellular processing which precede immediate-early gene expression are largely responsible for differences between the replicative cycles of these representatives of gamma- and alphaherpesviruses in cultures of permissive cells. PMID- 2995557 TI - Inhibition of herpes simplex virus replication in vitro by human cytotoxic T cell clones and natural killer cell clones. AB - The abilities of human cytotoxic T cell (CTL) clones and natural killer (NK) cell clones to inhibit the replication of herpes simplex virus (HSV) in vitro were shown. The specificities of clones inhibiting HSV replication were the same as those of cytotoxicity in HSV type specificity and HLA restriction, i.e. HSV type 1 (HSV-1) and HSV type 2 (HSV-2) common CTL clones inhibited the replication of both HSV-1 and HSV-2 in autologous cells, but not in allogeneic cells. HSV-1 specific CTL clones inhibited the replication of HSV-1 in autologous cells; however, the replication of HSV-2 was not inhibited. NK cell clones inhibited the replication of HSV-1 and HSV-2 in autologous and allogeneic cells. This is the first demonstration that human virus-specific CTL clones and NK cell clones directly limit virus replication in vitro. PMID- 2995556 TI - Differences in humoral immunogenicity between herpes simplex virus types 1 and 2. AB - Infection of NMRI mice with increasing doses of six different strains of herpes simplex virus type 1 (HSV-1) induced increasing levels of neutralizing antibodies. In contrast, three strains of HSV-2, irrespective of the dose, induced only marginal antibody responses. Only strain HG 52 (HSV-2) at high doses of infection stimulated antibody formation. The virus content of some organs in 6 to 8-week-old mice appeared to be lower after HSV-2 than after HSV-1 infection. Application of immune-modulating drugs [silica or poly(I) X poly(C) coupled via L lysine to CM-cellulose] resulted in little augmentation of antibody formation if compared to HSV-1 infection. Secondary infections with HSV-1 or HSV-2 after a primary dose of HSV-1 were followed by a marked booster response. In contrast, a primary infection with HSV-2 suppressed secondary responses of HSV-1 and HSV-2, thus indicating fundamental differences between the antibody-stimulating potency of HSV-1 and HSV-2 strains. PMID- 2995558 TI - Identification of the products of a varicella-zoster virus glycoprotein gene. AB - Two of the genes identified from the previously published DNA sequence of the Us component of the varicella-zoster virus (VZV) genome were predicted to encode membrane proteins with polypeptide molecular weights of 39000 (39K) and 70K. A rabbit antiserum directed against a unique peptide containing the seven amino acid residues at the carboxy terminus of the 39K gene product specifically precipitated glycoproteins with apparent molecular weights of 55K and 45K from VZV-infected cells labelled with [3H]mannose. The complete inhibition of precipitation of gp55 by free peptide and the partial inhibition of precipitation of gp45 support the conclusion that the 39K gene encodes gp55 and perhaps gp45. The number of VZV genes currently thought to encode glycoproteins is discussed in view of this finding. PMID- 2995559 TI - A comparison of the acid-soluble polypeptides of five herpesviruses. AB - The polypeptides soluble in 0.25 M-HCl were extracted from the nuclei of BHK cells infected with herpes simplex virus type 1 or type 2 and separated by SDS PAGE. Seventeen polypeptides were detectable in each extract of which 10 type 1 and nine type 2 polypeptides were reproducibly effectively extracted. In cells infected with bovine mammillitis virus, pseudorabies virus or equine herpesvirus type 1, at least 12, 13 and eight polypeptides respectively were acid-soluble. In addition to histones, three other cellular polypeptides were present in sizeable quantities in the acid extracts and could obscure other acid-soluble viral polypeptides. Possible relationships between some polypeptides of the five herpesviruses are discussed. PMID- 2995560 TI - Sequencing of coronavirus IBV genomic RNA: three open reading frames in the 5' 'unique' region of mRNA D. AB - The nucleotide sequence of a genomic cDNA clone corresponding to the 5' terminal domain of mRNA D of the Beaudette strain of infectious bronchitis virus (IBV) has been determined. This region contains three open reading frames which predict polypeptides of molecular weights 6700 (6.7K), 7.4K and 12.4K. The predicted 12.4K polypeptide has a codon usage very similar to that predicted for the products of the IBV nucleocapsid, membrane and spike genes. The sequence also predicts a hydrophobic, potentially membrane-anchoring, region in the N terminal half of the 12.4K polypeptide, and a hydrophilic C terminus. PMID- 2995561 TI - The genome structure of a new chicken virus identifies it as a parvovirus. AB - The nucleic acid of chicken parvovirus-like particles showed sensitivity to DNase and S1 nuclease treatment and resistance to digestion with RNase. Viral DNA readily served as a template for self-primed conversion in vitro into a double stranded form of about 5200 base pairs. There was no evidence for encapsidation of strands of opposite polarities. These findings confirm the taxonomic classification of chicken parvovirus-like particles as fowl parvovirus type 1 within the Parvovirus genus of the Parvoviridae. PMID- 2995562 TI - Synthesis of proviral DNA in inbred mouse-derived clones of cells expressing different Fv-1 phenotypes. AB - Formation of proviral DNAs by B-tropic murine leukaemia viruses (MLVs) was examined in N-type and dually permissive mutant cells derived from two inbred mouse strains, DDD and G, both of which are N-type. In the N-type cells, formation of circular proviral DNA was strongly suppressed relative to that of linear DNA. Mutation resulting in loss of the N-type Fv-1 restriction resulted in efficient formation of circular DNA by the previously restricted B-tropic MLV. This showed that Fv-1 restriction and inhibition of closed circular DNA formation were controlled by the same gene. The efficiency of formation of circular proviral DNA by the defective Kirsten murine sarcoma virus was determined by the tropism of the helper virus. PMID- 2995563 TI - A hypothesis accounting for the effect of the host cell on neutralization resistant virus. AB - Evidence is presented showing that a monkey anti-enterovirus 71 immune serum contains several antibody populations which differ in their mode of function. One population reduces infectivity, although inefficiently, by interactions at exposed antigenic sites and can be detected by measuring residual virus infectivity after mixtures of virus and antibody have been allowed to interact. Another antibody population, which is unaffected by the immunosorbent Staphylococcus aureus (Cowan I strain), appears to attach to its antigenic site(s) only after interactions between enterovirus 71 and host cells have already begun. In view of the transience of (presumed) conformational changes in the invading viruses, demonstration of this type of antibody activity requires a particular host cell system. This second type of antibody neutralization could be detected on RD cells but not on green monkey kidney cells. PMID- 2995564 TI - Phosphorylation of the N and M1 proteins of rabies virus. AB - Phosphorylation of rabies virus proteins was followed in vivo and in vitro. The N and M1 proteins were both found to be phosphorylated. The M1 protein was present in the virion in two phosphorylated states, but only the hypophosphorylated form of M1 was found in infected cells. The hypothesis that some of the M1 molecules become hyperphosphorylated during the maturation process by a membrane-bound kinase was examined. The phosphorylation of the viral proteins by the kinase present in purified rabies virions was studied using an in vitro transcriptase assay: under the conditions of the assay, additional phosphate groups were rapidly attached to the N protein. The M1 protein was similarly hyperphosphorylated although more slowly. Whether the hyperphosphorylation of the N protein is responsible for the poor efficiency of the in vitro transcriptase reaction is not clear. No detectable change in the phosphorylation of cellular proteins was observed in the course of rabies virus infection. PMID- 2995565 TI - Conditions for haemolysis by flaviviruses and characterization of the haemolysin. AB - The 17D vaccine strain of yellow fever virus (YF 17D) was used to establish the optimal conditions for lysis of chick erythrocytes. Tissue culture-grown, polyethylene glycol-concentrated virus showed peak activity at pH 5.4 in citrate buffer when incubated at 37 degrees C. A further two- to fourfold increase in titre was obtained by pretreatment of the chick erythrocytes with 250 micrograms/ml trypsin. These conditions were also shown to be optimal for Japanese encephalitis (JE), West Nile (WN) and dengue-2 (den2) viruses. The ratio of haemagglutination (HA) titre to haemolysis (HL) titre approximated to unity, suggesting that the two functions are associated with the same molecule although as separable entities since selective inactivation of the HL activity of the virus was accomplished using 60 micrograms/ml trypsin. HL could be demonstrated at neutral pH if the chick erythrocytes were first subjected to treatment with acidic pH buffer. The effect on the virus envelope is thus not the sole contribution of a low pH environment to optimal HL. Hyperimmune rabbit antiserum prepared against purified YF 17D virions inhibited HA and HL if added before agglutination had occurred by the virus but when added after agglutination had taken place it showed specific anti-HL activity. Monoclonal antibodies that inhibited HA (HAI) by YF 17D did not inhibit HL (HLI) activity when applied after agglutination had taken place. Moreover, monoclonal antibodies specific for the 54K glycoprotein of YF virus but without HAI activity also had no effect on HL when added either before or after agglutination. As yet, we have been unable to identify a monoclonal antibody displaying specific anti-HL activity but all those directed against the 54K envelope glycoprotein possessing HAI activity showed HA to be a prerequisite for HL. PMID- 2995566 TI - Direct detection of the human papovavirus BK in urine of bone marrow transplant recipients: comparison of DNA hybridization with ELISA. AB - Urine specimens from bone marrow transplant (BMT) recipients and from controls were directly tested for BK virus (BKV) DNA sequences by dot hybridization and for BKV antigen by a double-antibody indirect ELISA. A total of 158 specimens from 55 BMT patients (57 collected prior to or at the time of transplantation and 101 in the posttransplant period) and single urines from 125 control subjects were examined by both methods. A molecularly cloned, 32P-labelled BKV probe was hybridized with urine sediments that were spotted directly on nitrocellulose filters and denatured in situ. BKV DNA sequences were detected in 1 (1.8%) pretransplant and 22 (21.8%) posttransplant urines of BMT patients, and in none of control urines. In ELISA of urine supernatants, BKV antigen was detected in 1 (1.8%) pretransplant and 21 (20.8%) posttransplant urines of BMT patients and in 1 (0.8%) of the control urines. The results of the two tests correlated as follows: 16 urines were positive and 253 urines negative by both methods; seven specimens were positive by DNA hybridization only and seven were positive by ELISA alone. Virus excretion in urine was demonstrated in 20 (36.4%) patients by DNA hybridization, in 19 (34.5%) patients by ELISA, in 15 (27.3%) patients by both methods, and in 24 (44%) patients by at least one of the two tests. PMID- 2995567 TI - Detection of integration during active replication of hepatitis B virus in the liver. AB - A method is described that enables the unequivocal detection of the integration of hepatitis B virus DNA (HBV-DNA) into the genomic DNA of the host cell, while at the same time the virus exhibits active replication. This detection was achieved by separating the low molecular weight replicating HBV-DNA from the high molecular weight genomic DNA of the host by electrophoresis of the total undigested cellular DNA through low melting agarose. The high molecular weight DNA was isolated from this gel and electrophoresed after digestion with restriction enzyme(s) on a second agarose gel. Transfer of the DNA content of both gels and hybridization of these blots with 32P-labelled HBV-DNA will reveal whether any integrated and/or actively replicating DNA is present. By using this method the presence of a single copy of HBV-DNA integrated in host DNA was demonstrated in hepatocellular carcinoma tissue of a patient with active HBV replication. PMID- 2995568 TI - The prevalence of antibodies to reovirus type 3 in adults with idiopathic cholestatic liver disease. AB - Recent evidence suggests that neonatal infection with Reovirus type 3 (Reo-3) may play an important role in the pathogenesis of certain idiopathic cholestatic liver diseases of children. Whether this virus is of pathogenetic importance in similar diseases seen in adults is unclear. In this study, sera from 22 adults with idiopathic cholestatic liver disease (16 primary biliary cirrhosis (PBC) and six primary sclerosing cholangitis (PSC] were tested for antibody to Reo-3 virus by a standard hemagglutination inhibition assay. The results were compared to antibody prevalence in ten adults with various other causes of chronic liver disease and 11 healthy volunteers. Although patients with idiopathic cholestatic liver disease had a high prevalence of anti Reo-3 antibodies (20/22, 91%), similar prevalence rates were found in the two control populations (7/10, 70% in chronic liver disease controls and 11/11, 100% in healthy volunteers). Moreover, the geometric mean titres of antibody to Reo-3 virus were similar in all study groups. While these results do not exclude a pathogenetic role for Reo-3 viral infection, they do imply that some genetic predisposition must exist if Reo-3 infection is truly of pathogenetic importance in idiopathic cholestatic liver disease of adults. PMID- 2995569 TI - Human-enteric-coronaviruslike particles (CVLP) with different epidemiological characteristics. AB - One hundred fifty-six diarrheic and 115 control stools collected throughout a year from nonhospitalized children were examined by electron microscopy in Haut Ogooue, Gabon; 65.2% of the controls and 38.5% of the diarrheics were found to contain coronavirus particles (CVLP). In both diarrheic and control groups the CVLP prevalences showed a seasonal variation whereas significant variation of prevalence with age was observed only in the controls. Thus, the CVLP in controls were significantly more abundant in children over 2 years old (76% vs 48%, P less than .01) and more frequently observed during the months of rainy seasons (75% vs 54%, P less than .02). On the other hand, the higher prevalence of CVLP in diarrheics over 2 years old was not significant (48% vs 36%, P = .20), whereas a significantly lower prevalence of CVLP during the months of rainy seasons was observed in this group (27% vs 50%, P less than .01). Studies of the climatological factors in this equatorial climate showed a parallel cyclical variation of parameters representing rainfall, temperature, as well as relative humidity. We were not able to distinguish which of these factors was influencing more directly the prevalence of CVLP. PMID- 2995570 TI - Relative frequency of human rotavirus subgroups 1 and 2 in Japanese children with acute gastroenteritis. AB - Recently developed monoclonal antibodies against the 42,000-dalton major inner capsid protein were used in an enzyme immunoassay to subgroup a total of 156 rotavirus specimens obtained from Japanese infants and young children with acute gastroenteritis during the period between December 1981 and April 1983. Only 2 specimens (1.3%) were identified as subgroup 1, whereas 154 specimens (98.7%) were identified as subgroup 2. The percentage of subgroup 2 rotaviruses obtained in this study was the highest among similar studies thus far performed in different areas of the world. One of the subgroup 1 isolates had fast-moving 10th and 11th gene segments (the "long" pattern) typical of the subgroup 2 human rotaviruses. PMID- 2995571 TI - Human antibody response to immunization with 17D yellow fever and inactivated TBE vaccine. AB - The antibody response against flaviviruses tick-borne encephalitis (TBE), Kyasanur Forest disease (KFD), Murray Valley encephalitis (MVE), West Nile fever (WNF), Japanese B encephalitis (JE), dengue 2 (DEN-2), and yellow fever (YF) was studied in humans after administration of an inactivated TBE virus vaccine. Individuals were either prevaccinated with 17D yellow fever (experimental group) or without any previous exposure to flaviviruses (control group). The appearance of serum titres of homologous and heterologous haemagglutination inhibition (HI) antibodies, heterotypic DEN-2 neutralizing antibodies, and TBE enzyme-linked immunosorbent assay (ELISA) antibodies were examined. Individuals prevaccinated with the 17D yellow fever developed an antibody pattern that contrasted with that of the control group. This pattern was characterized as follows: (1) Predominantly anti-TBE IgG antibodies appeared earlier and in higher titres than in the control group, (2) heterologous HI antibodies cross-reacting with the WN flavivirus subgroup preceded the appearance of homologous HI antibodies, (3) a broad spectrum HI response was observed against all flaviviruses tested, and (4) low titre heterotypic DEN-2 neutralizing antibodies were formed in about half of the cases. These observations are discussed in the context of cross-reactivity, cross-protection and virus infection enhancement. PMID- 2995572 TI - Antibody to Epstein-Barr virus-specific DNase as a marker for the early detection of nasopharyngeal carcinoma. AB - We have previously shown that high levels of antibody to Epstein-Barr virus (EBV) specific DNase may be a useful marker for the early diagnosis of nasopharyngeal carcinoma (NPC). Sera from 3,368 males in an area with a high risk for the development of NPC were examined for the presence of antibody to EBV-specific DNase activity. Significant levels of the antibody were found in 430 of these sera. As a result, 32 individuals have attended the out-patient department of otolaryngology for clinical examination. Among them, one individual was found to have a stage II nasopharyngeal carcinoma. This result supports the value of determination of antibody to EBV-specific DNase as a marker for the early detection of NPC. PMID- 2995573 TI - Acute and latent herpes simplex virus neurological disease in mice immunized with purified virus-specific glycoproteins gB or gD. AB - Groups of 5-week-old BALB/c mice were immunized intraperitoneally with approximately 10 micrograms of purified alum-precipitated glycoprotein gB or gD of either herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) origin. Control mice received injections of alum-precipitated 1% bovine serum albumin (BSA). Following a second immunization 4 weeks later, seroconversion was confirmed by demonstrating the presence of glycoprotein-specific antibody by immune precipitation. All animals were challenged with lethal doses of either HSV-1 or HSV-2 by footpad inoculation and assessed for acute virus-induced neurological disease and the development of ganglionic latency. Whereas 70% of control (BSA immunized) HSV-1-infected animals developed ascending myelitis and died, 100% of mice immunized with either gB-1, gB-2, gD-1, or gD-2 antigens remained free of clinical illness and survived HSV-1 challenge. In contrast, gB-1-or gB-2 immunized mice were not protected against acute HSV-2-induced neurological disease and showed a mortality rate of 60-90% (equivalent to that seen in controls), although mean survival times were prolonged. However, significant protection against HSV-2 challenge was observed with gD-1 or gD-2 immunization. When sacral ganglia were removed from surviving mice 9-12 months after virus challenge, latent virus was detected in all gB- or gD-immunized animals, although the extent of latent infection was restricted. These results provide evidence that glycoprotein gD might be superior to glycoprotein gB as an immunogen for the control of acute HSV-1 and HSV-2 neurological disease in mice. However, neither glycoprotein prevents ganglionic latency, the source of virus for recurrent herpesvirus infections. PMID- 2995574 TI - Pharmacological and neurochemical aspects of kindling. AB - Rats implanted with amygdaloid stimulating and cortical recording electrodes were kindled by daily low-intensity electrical stimulation. In one experiment amino acid concentrations were measured in amygdala, cortex and hippocampus at behavioural stages 1, 2 and 4 (Racine). Control groups consisted of unstimulated rats. Only alanine showed a significant enhancement of concentration in the kindled rats (stage 4 of Racine). In a second experiment, a group of rats was treated daily with 10mg/kg p.o. of diazepam. Diazepam significantly inhibited kindling and no changes in amino acid concentrations were observed in this group. Increased alanine levels are seen after various seizure types; since pentetrazole, isoniazid and beta-vinyllactic acid seizures were associated with alanine level increases only after and never before seizure occurrence, it is suggested that the alanine increases are a consequence rather than a cause of convulsions. In 3H-flunitrazepam binding studies, no change in affinity or receptor number could be demonstrated during kindling. PMID- 2995575 TI - Effect of early malnutrition on dynamic properties of axodendritic synapses in the rat prefrontal cortex. AB - The influence of early protein-calorie malnutrition on synaptic transmission in the rat prefrontal cortex was studied by evoking direct cortical responses with different trains of threshold electrical pulses. Malnutrition resulted in a requirement for increased pulse train stimulating current, reflecting a diminished release of neurotransmitter from the preterminal endings. In addition there was an increase in the time constant of pyramidal cells, indicating that axodendritic synapses have a disability for integrating high frequency inputs at the post synaptic level. It is concluded that early malnutrition alters dynamic properties of axodentritic synapses at both the pre- and the postsynaptic levels. PMID- 2995576 TI - An electron spin resonance investigation of the iron-catalyzed reaction of metronidazole with cysteine. AB - Metronidazole is used in the treatment of protozoal and bacterial infections, and has been used as a radiation sensitizer in experimental research and clinical trials. This drug is rapidly decomposed by cysteine in the presence of ferric or ferrous iron. Electron spin resonance spectroscopy demonstrates the formation of two complexes composed of iron, cysteine, and NO. The nitric oxide is probably formed by the reduction of inorganic nitrite formed by the cleavage of the metronidazole nitro group from the imidazole ring. No such reaction occurred with the 2-nitroimidazole drug, misonidazole. PMID- 2995577 TI - Models for coordination site of cytochrome P-450, characterization of hemin thiolato complexes with S, O, and N donor ligands by electronic absorption and electron spin resonance spectra. AB - Bisthiolato-hemin complexes exhibiting "two split Soret bands" at 370 and 460 nm, classified into "hyperporphyrin spectrum" was prepared with naturally occurring porphyrins (Fe(III)protoporphyrin IX and its dimethyl ester), thioglycolate esters, and tetramethylammonium hydroxide in organic solvents. The structure of the complexes was characterized by electronic absorption and electron spin resonance (ESR) spectrometries. These complexes were stable under air at room temperature, their apparent half-lives being about 30 min monitored by the intensities of the two Soret bands. Thus the bisthiolato-hemin complex containing thioglycolate ester was shown to be a model for the cytochrome P450(P450) thiolato binding complex. Ligand exchange reactions of the bisthiolato-hemin complex with imidazole or methanol indicated that the intermediate species are stabilized as thiolato-hemin-imidazole or -methanol complexes. The latter intermediate complex was suggested to be a good model for low-spin ferric P450 as characterized by distinct beta- and alpha-bands at 530 and 560 nm, respectively, as well as a single Soret peak at approximately 410 nm. The result of the analysis on ESR g values and crystal field parameters for the bisthiolato-hemin, thiolato-hemin-imidazole, and thiolato-hemin-oxygen ligand complexes comparing with those for P450 itself and the ligand binding complexes revealed that the sixth ligand trans to the fifth thiolato ligand of the low-spin ferric P450 can be an oxygen atom of water molecule. PMID- 2995578 TI - The functional role of zinc in angiotensin converting enzyme: implications for the enzyme mechanism. AB - Zinc is essential to the catalytic activity of angiotensin converting enzyme. The enzyme contains one g-atom of zinc per mole of protein. Chelating agents abolish activity by removing the metal ion to yield the inactive, metal-free apoenzyme. Zinc does not stabilize protein structure since the native and apoenzymes are equally susceptible to heat denaturation. Addition of either Zn2+, Co2+, or Mn2+ to the apoenzyme generates an active metalloenzyme; Fe2+, Ni2+, Cu2+, Cd2+, and Hg2+ fail to restore activity. The activities of the metalloenzymes follow the order Zn greater than Co greater than Mn. The protein binds Zn2+ more firmly than it does Co2+ or Mn2+. Hydrolysis of the chromophoric substrate, furanacryloyl-Phe Gly-Gly, by the active metalloenzymes is subject to chloride activation; the activation constant is not metal dependent. Metal replacement mainly affects Kcat with very little change in Km, indicating that the role of zinc is to catalyze peptide hydrolysis. PMID- 2995580 TI - Stimulation of cyclic AMP production in chick renal tissue by cadmium and manganese. AB - The effects of cadmium on production of cyclic AMP by partially purified chick renal plasma membrane preparations and binding of 125I-parathyrin to the membranes have been investigated. At certain concentrations Cd2+ ions (and Mn2+ ions) markedly stimulated the production of cyclic AMP by the tissue. It was found that concentrations of Cd2+ roughly in the same range were also capable of stimulating binding of 125I-parathyrin to the membrane preparations. PMID- 2995581 TI - Kinetic studies on the diaqua form of cis-platin and various nucleobases. AB - Kinetic studies on cis-[Pt(NH3)2(OH2)2]2+ and various nucleobases show that this ion reacts more quickly with guanosine than with adenosine, cytidine, and thymidine, and that a monophosphoric acid unit considerably enhances the rate of reaction of guanosine; the kinetic preference of 5'-GMP over 5'-AMP may point to a greater thermodynamic selectivity. PMID- 2995579 TI - A spectroscopic and electrochemical study of the interaction between copper (II) glycylglycyl-L-histidine-N-methylamide and iron (III) tetra-p sulfophenylporphine. AB - A binuclear complex has been produced by the reaction of an iron porphyrin (sodium tetra-p-sulfophenylporphine iron (III)-FeTPPS) with a copper metallo tripeptide (copper (II) glycylglycyl-L-histidine-N-methylamide-CuGGH) in aqueous solution. The system has been characterized by electron spin resonance (ESR) spectroscopy, optical absorption spectroscopy, and electrochemical methods. Room temperature ESR spectra of the copper complex and low-temperature ESR spectra of the iron porphine provide evidence for the formation of a binuclear complex. These findings are supported by absorption spectroscopy and electrochemical studies, and lead to a value of ca. 2 X 10(-3) M-1 (at room temperature) for the equilibrium constant for complex formation. The relevance of this system to the enzymic active site of mammalian cytochrome c oxidase is discussed. PMID- 2995582 TI - The binding of copper(II) and zinc(II) to oxidized glutathione. AB - 1H and 13C NMR studies of Zn(II) binding to oxidized glutathione (GSSG) in aqueous solution over the pH range 4-11 show that it forms a complex with a 1:1 Zn:GSSG stoichiometry. At pH values between 6 and 11 the metal ligands are the COO- and NH2 groups of the glutamate residues. Below pH 5 the glycine end of the molecule also binds to the metal ions. EPR and visible absorption spectra of Cu(II) GSSG solutions suggest that similar complexes are formed with Cu(II). The solid products obtained from these solutions are shown by analysis and EPR to be primarily binuclear with Cu2GSSG stoichiometry, although the structures depend on the pH and stoichiometry of the solution from which they were obtained. PMID- 2995583 TI - Dithiocarbamates in heavy metal poisoning: complexes of N,N-di(2 hidroxyethyl)dithiocarbamate with Zn(II), Cd(II), Hg(II), CH3Hg(II), and C6H5Hg(II). AB - The complexes M(DHDC)2, CH3Hg(DHDC), and C6H5Hg(DHDC) (M = Zn, Cd, Hg; DHDC = N,N di(2-hydroxyethyl)dithiocarbamate) were prepared and investigated in solution and in the solid state by using 1H and 13C NMR, ir, and Raman spectroscopy. The dithiocarbamate group is anisobidentate and the complexes are associated in solution and the solid state via hydrogen bonding. The possible relation of these structural properties to the behavior of DHDC in the treatment of cadmium poisoning is discussed. PMID- 2995584 TI - Palmitoyl-CoA elongation in brain microsomes: dependence on cytochrome b5 and NADH-cytochrome b5 reductase. AB - Experiments were performed to demonstrate the involvement of electron transport system in fatty acid elongation in rat brain microsomes. Mercuric chloride and p chloromercuriphenylsulfonate, inhibitors on NADH-cytochrome b5 reductase, at 32 microM inhibited NADH-supported palmitoyl-CoA elongation to 30 and 60% of control activity, respectively, whereas NADPH-supported palmitoyl-CoA elongation was unaffected by these mercurials. An antibody to rat liver NADH-cytochrome b5 reductase inhibited brain microsomal NADH-cytochrome b5 reductase activity and NADH-dependent palmitoyl-CoA elongation. Treatment of brain microsomes with trypsin diminished the cytochrome b5 content; NADH- and NADPH-cytochrome c reductase activities were significantly decreased, but the decrease in NADH cytochrome b5 reductase activity was relatively small. Whereas essentially no incorporation of malonyl-CoA into palmitoyl-CoA was observed with trypsin-treated microsomes, addition of detergent-solubilized cytochrome b5 resulted in a recovery of fatty acid elongation. These results indicate the presence of an electron transport system, NADH-NADH-cytochrome b5 reductase-cytochrome b5-fatty acid elongation, in brain microsomes. PMID- 2995585 TI - Gamma-aminobutyric acid and benzodiazepine receptor changes induced by unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. AB - Quantitative autoradiography was used to ascertain alterations in [3H]muscimol, [3H]flunitrazepam (FLU), [3H]naloxone, [3H]D-alanine-D-leucine-enkephalin (DADL), and [3H]spiroperidol binding in basal ganglia 1 week, 4 weeks, and 5 months after unilateral 6-hydroxydopamine lesions of the medial forebrain bundle (MFB) in the rat. At 1 and 4 weeks following lesions, [3H]spiroperidol binding increased 33% in striatum. At 5 months, [3H]spiroperidol was only nonsignificantly increased above control. At 1 week, [3H]muscimol binding decreased 39% in ipsilateral globus pallidus (GP), but increased 41% and 11% in entopeduncular nucleus (EPN) and substantia nigra pars reticulata (SNr), respectively. At 4 weeks, [3H]muscimol binding was reduced 19% in striatum and 44% in GP and remained enhanced by 32% in both EPN and SNr. These changes in [3H]muscimol binding persisted at 5 months. [3H]FLU binding was altered in the same direction as [3H]muscimol binding; however, changes were slower in onset and became significant (and remained so) only at 4 weeks after lesions. Decreases in [3H]naloxone and [3H]DADL binding were seen in striatum, GP, EPN, and SNr. Scatchard analyses revealed that only receptor numbers were altered. This study provides biochemical evidence for differential regulation of striatal GABAergic output to GP and EPN/SNr. PMID- 2995586 TI - Thyrotropin-releasing hormone receptor binding sites: autoradiographic distribution in the rat and guinea pig brain. AB - Thyrotropin-releasing hormone (TRH) binding sites were labeled in vitro in mounted brain tissue sections from rat and guinea pig brains with [3H]methyl TRH and localized autoradiographically using 3H-sensitive film. Regional densities of TRH binding sites were measured by computer-assisted microdensitometry. The distribution of sites in both species was highly heterogeneous. In both guinea pig and rat brains, the highest densities of binding sites were seen in the amygdaloid nuclei and the perirhinal cortex. In contrast, in other brain areas, a clear difference between the distribution of sites in rat and guinea pig was found. The temporal cortex, pontine nuclei, and interpeduncular nucleus, which contained high densities of binding in the guinea pig, were scarcely labeled in the rat. The accessory olfactory bulb and the septohippocampal area presented in the rat higher concentrations of binding sites than in the guinea pig. Other brain areas showing intermediate to low densities in both species were accumbens nucleus, bed nucleus of the stria terminalis, dentate gyrus, facial and hypoglossal nuclei, and gelatinosus subnucleus of the trigeminal nerve, among others. The anterior pituitary also presented low to intermediate concentrations of receptors. The distribution of TRH sites here described does not completely correlate with that of endogenous TRH, but is in good agreement with previous biochemical data. The results are discussed in correlation to the physiological effects that appear to be mediated by TRH. PMID- 2995587 TI - Manganese stimulates the incorporation of [3H]inositol into a pool of phosphatidylinositol in brain that is not coupled to agonist-induced hydrolysis. AB - Mn2+ greatly increases the incorporation of myo-[3H]inositol into phosphatidylinositol (PI) of brain and other tissues by stimulating the activity of a PI-myo-inositol exchange enzyme. This study examined the ability of norepinephrine (NE) and carbachol to stimulate the hydrolysis of [3H]PI formed in the absence and presence of Mn2+-stimulated [3H]inositol exchange. Rat cerebral cortical slices were incubated with myo-[3H]inositol for 60 min in an N-2 hydroxyethyl piperazine-N'-2-ethanesulfonic acid (HEPES) buffer without or with MnCl2 (1 mM). The tissue was washed and further incubated with unlabeled myo inositol and LiCl (10 mM). Prelabeled slices were then incubated with NE (0.1 mM) or carbachol (1 mM) to induce agonist-stimulated [3H]PI hydrolysis. Mn2+ treatment resulted in eight- and sixfold increases in control levels of [3H]PI and [3H]inositol monophosphate [( 3H]IP), respectively. Both NE and carbachol stimulated [3H]IP formation in tissue prelabeled without or with manganese. However, the degree of stimulation (percentage of control values) was greatly attenuated in the presence of Mn2+. In the absence of Mn2+ treatment, NE decreased [3H]PI radioactivity in the tissue to 80% of control values. However, NE did not decrease [3H]PI radioactivity in the Mn2+-treated tissue. These data demonstrate that Mn2+ stimulates incorporation of myo-[3H]inositol into a pool of PI in brain that has a rapid turnover but is not coupled to agonist-induced hydrolysis. PMID- 2995588 TI - Possible involvement of inhibitory GTP binding regulatory protein in alpha 2 adrenoceptor-mediated inhibition of adenylate cyclase activity in cerebral cortical membranes of rats. AB - Influences of alpha 2-adrenoceptor stimulation on adenylate cyclase activity were investigated in cerebral cortical membranes of rats. Pretreatment of the membranes with islet-activating protein and NAD resulted in a significant increase in basal activity as well as in GTP- or forskolin/GTP-induced elevation of adenylate cyclase activity. Strong activation of adenylate cyclase was also caused in membranes pretreated with cholera toxin together with NAD in comparison to that in control membranes, suggesting that adenylate cyclase activity is perhaps regulated by stimulatory and inhibitory GTP binding regulatory protein existing in synaptic membranes. In addition, adrenaline (with propranolol) or clonidine significantly reduced adenylate cyclase activity stimulated by pretreatment with forskolin and GTP. The inhibitory effects of adrenaline were also observed in membranes pretreated with cholera toxin and NAD. Moreover, the inhibition by adrenaline or clonidine was completely abolished by treatment with (a) yohimbine or (b) islet-activating protein and NAD. It is suggested that alpha 2-receptor stimulation causes inhibitory influences on adenylate cyclase activity mediated by the inhibitory GTP binding regulatory protein in synaptic membranes of rat cerebral cortex. PMID- 2995589 TI - Activation of muscarinic and of alpha 1-adrenergic receptors on astrocytes results in the accumulation of inositol phosphates. AB - Astrocyte-enriched cultures prepared from the newborn rat cortex incorporated [3H]myo-inositol into intracellular free inositol and inositol lipid pools. Noradrenaline and carbachol stimulated the turnover of these pools resulting in an increased accumulation of intracellular [3H]inositol phosphates. The effects of noradrenaline and carbachol were dose-dependent and blocked by specific alpha 1-adrenergic and muscarinic cholinergic receptor antagonists, respectively. The increase in [3H]inositol phosphate accumulation caused by these receptor antagonists was virtually unchanged when cultures were incubated in Ca2+-free medium, but was abolished when EGTA was also present in the Ca2+-free medium. Cultures of meningeal fibroblasts, the major cell type contaminating the astrocyte cultures, also accumulated [3H]myo-inositol, but no increased accumulation of [3H]inositol phosphates was found in response to either noradrenaline or carbachol. PMID- 2995590 TI - Glucose transport in astrocytes: regulation by thyroid hormone. AB - Primary cultures of astrocytes from newborn rat brain showed evidence of a substrate-saturable process for glucose transport. The system shows a relatively high affinity for the substrate, with an apparent Km of approximately 1 mM. Maintenance of the cells in medium containing thyroid-hormone-free serum for 3, 6, or 9 days resulted in significantly reduced rates of hexose transport. Addition of exogenous triiodothyronine to the transport incubation medium of these "hypothyroid" cells markedly increased the net rate of 2-deoxyglucose uptake within 60 s to values equal to or above those of control cultures (cells maintained in normal serum). These findings support a key role for thyroid hormone in the transport of glucose across plasma membranes of brain cells and demonstrate the presence of this regulatory system in astrocytes. PMID- 2995591 TI - Tubular aggregates: their association with myalgia. AB - Three thousand consecutive muscle biopsies were reviewed for the presence of tubular aggregates and their association with clinical symptomatology. Tubular aggregates were detected in 19 patients (0.6%). Twelve of these nineteen patients had severe myalgia, and the most abundant tubular aggregates were found in biopsies of patients with myalgia. Seven patients had only myalgia as their clinical symptomatology with normal physical examination. An additional five patients with tubular aggregates and myalgia had concomitant amyotrophic lateral sclerosis (2) or neuropathy (3). The high incidence of myalgia associated with tubular aggregates in our patients and the fact that tubular aggregates originate from sarcoplasmic reticulum suggest a role played by this structure in the pathogenesis of myalgia. PMID- 2995592 TI - A clinical and electrophysiological study of patients with polychlorinated biphenyl poisoning. AB - Neurological examination of 28 patients, 4 years after serious poisoning by polychlorinated biphenyl contaminated cooking oil, are compared with similar examinations of the same patients two years earlier (in 1980). Clinical peripheral sensory neuropathy was found in 54%, headache in 36% and dizziness in 46% of the patients; these findings did not differ (p greater than 0.1) from those in 1980. Although the mean blood polychlorinated biphenyl concentration (19.2 ppb) in the patients was lower (p less than 0.001) than that in 1980 (35.9 ppb), it was still higher than the normal value (less than 4 ppb). There was no difference in the blood polychlorinated biphenyl concentration of patients with neurological manifestation from those without. Although the mean motor and sensory nerve conduction velocities (MNCV and SNCV) were still slower (p less than 0.06) than the mean normal NCV, the mean MNCV of tibial nerve and SNCV of sural nerve were improved (p less than 0.06) as compared with those in 1980. EEGs were normal except in two cases showing nonspecific slow wave changes. In addition, evoked potentials (somatosensory, visual and brain-stem auditory) were measured in this study and found to be normal in all 12 cases examined. PMID- 2995593 TI - A study of the effects of isaxonine on vincristine-induced peripheral neuropathy in man and regeneration following peripheral nerve crush in the rat. AB - Administration of isaxonine (6 mg/kg powdered diet) had no effect on regeneration following sciatic nerve crush in the rat. In 10 patients undergoing treatment with vincristine (1.4 mg/m2 twice monthly) development of peripheral neuropathy was quantitated by neurological symptoms, signs and electrophysiological tests. Five also received isaxonine (1.5 g daily). All patients developed evidence of neuropathy, but in none was it severe. The three lowest disability scores were obtained in isaxonine treated patients, but the highest score was also in an isaxonine treated patient. The equivocal findings in this small study could not be amplified because the drug was withdrawn from clinical use on account of its hepatotoxicity. PMID- 2995594 TI - [3H]Ouabain binding in normal and dystrophic mouse skeletal muscles and the effect of age. AB - The numbers of Na+-K+ ATPase sites in skeletal muscles of normal and dystrophic mice between 3 and 17 months of age have been estimated using [3H]ouabain binding assays. In normal mice, at all ages, slow twitch muscle, soleus (SOL), bound significantly more [3H]ouabain than fast-twitch muscle, extensor digitorum longus (EDL). [3H]Ouabain binding did not alter in either SOL or EDL from normal mice over the age range studied. The numbers of Na+-K+ ATPase sites did alter in muscles taken from dystrophic mice (C57BL/6J dy2J/dy2J). In EDL there was an increase and in SOL a decrease in [3H]ouabain binding. This may be related to a change in muscle fibre metabolism from glycolytic to oxidative or to an altered activity pattern. Increasing age resulted in a progressive reduction in [3H]ouabain binding of both SOL and EDL from dystrophic mice. Part of this reduction may be only apparent and due to an increase in connective tissue composition of dystrophic muscles. A limited study of muscles from neonate dystrophic mice indicated that abnormal [3H]ouabain binding was not present in EDL before two weeks of age. PMID- 2995595 TI - Partial cytochrome oxidase deficiency without subsarcolemmal accumulation of mitochondria in chronic progressive external ophthalmoplegia. AB - Chronic progressive external ophthalmoplegia (CPEO) associated with proximal myopathy and/or craniosomatic abnormalities is a rare syndrome in which morphological mitochondrial changes have been found in some fibres (subsarcolemmal accumulation of mitochondria or "ragged red" fibres). We report a 14-year-old boy with CPEO and a mild proximal myopathy without these characteristic "ragged red" fibres. Histochemistry of skeletal muscle showed a mosaic of fibres without detectable cytochrome oxidase activity, while other mitochondrial enzymes were normal. The total cytochrome oxidase activity and cytochrome aa3 concentration in muscle mitochondrial fractions were only 40% of normal. This case is unique in that a biochemical defect was not accompanied by morphological abnormalities and may represent an early stage of CPEO before the development of morphological changes, or alternatively, a new variant of the disease. PMID- 2995597 TI - Intraductal carcinoma of the breast: results of treatment with excisional biopsy and irradiation. AB - Between 1976 and 1983, 40 women with intraductal carcinoma of the breast without invasion underwent excisional biopsy and irradiation as an alternative to mastectomy. The median age was 53 years (range, 28 to 77 years) and the median follow-up time since initiation of radiation was 44 months (range, 14 to 97 months). Twenty-seven patients presented with a palpable mass; in 13 patients the tumor was detected only by mammography. A limited axillary dissection was performed in 13 patients, and all lymph nodes removed were negative. Treatment was administered to the breast and adjacent chest wall to a dose of 4,600 to 5,000 rad, with 26 patients also receiving a boost dose of 1,000 to 2,000 rad to the site of the primary. Four patients have developed a recurrence in the treated breast, at 17, 19, 35, and 63 months after the beginning of radiation therapy. The 5-year actuarial rate of local recurrence is 10%. Three of the recurrences were in those four patients who presented with a nipple discharge and a central primary. In two cases, the recurrence consisted of only intraductal carcinoma; in the other two, both intraductal and invasive cancer were found. All four patients with recurrence underwent mastectomy and are well without evidence of distant metastases at 1, 12, 15, and 15 months since mastectomy. Cosmetic results were excellent. No patient has developed distant metastases. Since the number of patients treated is small and the period of follow-up is short, one must be cautious in the interpretation of these results. Nonetheless, the treatment of intraductal carcinoma of the breast by excision and irradiation appears to give acceptable local control and excellent survival when suitable precautions of patient selection and evaluation are taken. PMID- 2995596 TI - Polyneuropathy in paraproteinaemia. AB - Paraproteinaemias are frequently associated with peripheral neuropathies. "Benign" paraproteinaemia, myeloma and Waldenstroms macroglobulinaemia may present clinically as polyneuropathy. Therefore immunoelectrophoresis is strongly recommended in the routine diagnosis of polyneuropathies of unknown origin. Peripheral neuropathies associated with paraproteinaemia are clinically, electrophysiologically, pathologically and probably also pathogenetically heterogeneous. There are subgroups such as demyelinating neuropathy associated with IgM paraproteinaemia, which show quite distinctive features. This survey describes the different types of paraproteinaemia and their associated peripheral neuropathies. The incidence, pathogenesis and therapy of peripheral neuropathy associated with monoclonal gammopathies are discussed. PMID- 2995598 TI - Carcinoembryonic antigen: a useful prognostic marker in small-cell lung cancer. AB - Plasma carcinoembryonic antigen (CEA) was determined in 180 patients with small cell lung cancer (SCLC) before treatment. An abnormal level (greater than or equal to 6 ng/mL) was found in 34% of patients tested. Patients with extensive disease (39/83) had a significantly higher frequency of abnormal CEA (P = .001) than those with limited disease (22/97). There was a strong correlation between obtaining an objective response--particularly a complete response (P = .00003)- and the absence of an elevated CEA. Patients with an abnormal CEA also had a shorter survival time (P = .0007) and the difference remained statistically significant after logrank adjustment for extent of disease and ECOG (Eastern Cooperative Oncology Group) performance status. There was also a negative correlation between survival time and the quantitative level of CEA. In this series, only the group of patients with normal initial CEA levels included all survivors beyond 2.5 years. We conclude that CEA is a useful prognostic factor in SCLC. PMID- 2995599 TI - Creatine kinase BB and beta-2-microglobulin as markers of CNS metastases in patients with small-cell lung cancer. AB - Creatine kinase (CK) and its BB isoenzyme (CK-BB) were measured in CSF in 65 evaluable patients suspected of CNS metastases secondary to small-cell lung cancer (SCLC). In addition, CSF and plasma levels of beta-2-microglobulin (beta-2 m) were measured in a group of 73 evaluable patients. Of the 65 patients analysed for CK-BB, 17 had meningeal carcinomatosis (MC), 26 had parenchymal metastases only, and 22 had no CNS disease. Patients with MC had a significantly higher CK BB concentration in CSF than did patients belonging to the other two groups (P less than .01). Taking 0.4 U/L (upper limit in patients without CNS disease) as a cut-off point, 15 patients (88%) with MC had elevated CSF concentrations of CK BB. Patients without CNS metastases had no CSF levels exceeding this value, whereas five patients with multiple CNS metastases did. Receiver operating characteristic (ROC) analysis suggests that CK-BB may be useful in distinguishing MC among patients suspected of having CNS metastases, and CK-BB appears superior to total CK, CSF protein, and CSF lactic dehydrogenase (LDH). In 12 patients with MC at autopsy, CK-BB was, with the above cut-off point, elevated in six patients with a false negative cytology. Of the 73 patients examined for beta-2-m, 18 had MC, 30 had parenchymatous metastases only, and 25 patients had no CNS metastases. The CSF concentrations in the three groups were not significantly different. The median concentrations in the groups were 133 nmol/L, 125 nmol/L, and 107 nmol/L, respectively. The ratios between beta-2-m in CSF and plasma were also not significantly different between the three groups. Thus, the data on CK-BB are promising, and further studies are warranted to see if the usefulness of CK-BB can be more firmly established. By contrast, beta-2-m has no role as a marker of CNS disease secondary to SCLC. PMID- 2995600 TI - High-dose cisplatin administration without hypertonic saline: observation of disabling neurotoxicity. AB - Nephrotoxicity of cisplatin can be ameliorated with intravenous (IV) hydration and forced diuresis with mannitol. Cisplatin has recently been used with hypertonic saline which allows administration of higher doses amounting to 40 mg/m2/d for 5 days, without significant nephrotoxicity. In this report we describe our experience with administration of cisplatin in a dose range of 30 to 40 mg/m2/d for 5 days, administered with IV hydration alone. Thirteen patients with recurrent carcinoma of the head and neck region were treated with high-dose cisplatin along with 5-fluorouracil (5-FU) used as a continuous infusion. Eight patients received a total of 21 courses of cisplatin with the higher dose range (40 mg/m2 for 5 days) and the remainder received 11 courses with the lower dose range. The renal toxicity was minimal but the myelo-suppression was intense, frequently requiring hospitalization for the treatment of infections associated with neutropenia. Furthermore, we encountered severe peripheral neuropathy in five patients, four of whom developed major difficulties with ambulation. Six patients achieved objective regression of their tumor, two had minor response, and five failed to respond to chemotherapy. The study was terminated because of serious nonrenal toxicity from the high-dose cisplatin. Based on our limited experience, we believe that IV hydration alone, without the use of hypertonic saline, allows administration of high-dose cisplatin without significant nephrotoxicity. However, cisplatin used in a dose schedule of 40 mg/m2 for 5 days for more than three courses resulted in a disabling form of peripheral neuropathy. PMID- 2995601 TI - Evidence for shifting connections during development of the chick retinotectal projection. AB - The pattern in which optic axons invade the tectum and begin synaptogenesis was studied in the chick. The anterogradely transported marker, horseradish peroxidase, was injected into one eye of embryos between 5 and 16 days of development (E5 to E16). This labeled the optic axons in the brain. The first retinal axons arrived in the most superficial lamina of the tectum on E6. They entered the tectum at the rostroventral margin. During the next 6 days of development the axons grew over the tectal surface. First they filled the rostral tectum, the oldest portion of the tectum, and then they spread to the caudal pole. Shortly after the first axons entered the tectum on E6, labeled retinal axons were found penetrating from the surface into deeper tectal layers. In any given area of the tectum, optic axons were seen penetrating deeper layers shortly after arriving in that area. Electron microscopic examination showed that at least some of the labeled axons in rostral tectum formed synapses with tectal cells by E7. These results show two things which contrast with results from previous studies. First, there is no delay between the time the retinal axons enter the tectum and the time they penetrate into synaptic layers of the tectum. Second, the first retinotectal connections are formed in rostral tectum and not central tectum. Retrograde tracing showed the first optic axons that arrived in the tectum were from ganglion cells in central retina. Previous studies have shown that the ganglion cells of central retina project to the central tectum in the mature chick. This opens the possibility that the optic axons from central retina, which connect to rostral tectum in the young embryo, shift their connections to central tectum during subsequent development. As they enter the tectum the growth cones of retinal axons appear to be associated with the external limiting membrane. During the time that connections would begin to shift in the tectum a second population of axons appears at the bottom of stratum opticum, some with characteristics of growth cones. This late-appearing population may represent axons shifting their connections. These results have implications for theories on how the retinotopic pattern of retinotectal connections develops. PMID- 2995602 TI - Localization of sodium/potassium adenosine triphosphatase in multiple cell types of the murine nervous system with antibodies raised against the enzyme from kidney. AB - This report describes the development and characterization of a battery of highly specific antibodies to sodium/potassium (Na+ + K+)-ATPase and their use in localizing this enzyme in nervous tissue. The immunolabeling characteristics of polyclonal antibodies and monoclonal antibodies (Schenk, D. B., and H. L. Leffert (1983) Proc. Natl. Acad. Sci. U. S. A. 80: 5281-5285) raised against rat renal (Na+ + K+)-ATPase were compared. The interspecies cross-reactivity of the polyclonal anti-rat antibodies was examined by determining their binding to purified rat, eel, or dog enzyme. The immunostaining characteristics of the IgG fraction of the polyclonal antibody preparations, their affinity-purified derivatives, and the monoclonal antibodies were compared. The results obtained with each of these were similar, providing information about where focal concentrations of the enzyme exist within nervous tissue. The IgG fraction of the polyclonal antibody preparations provided the most sensitive probe, facilitating localization of the (Na+ + K+)-ATPase in the tissue sections from various regions of the nervous system. (Na+ + K+)-ATPase-like immunoreactivity was observed along the plasmalemma of alpha-motor neurons and at the nodal axolemma of myelinated axons from the central or peripheral nervous system. It was determined that the absence of labeling for the enzyme along the paranodal or internodal regions of the axolemma was not an artifact due to a limited accessibility of antibody to these regions. Some central nervous system glial cells demonstrated abundant amounts of plasmalemmal and intracellular (Na+ + K+)-ATPase-like immunoreactivity. These cells were identified as astroglia by positive labeling of cells in serial sections for glial fibrillary acid protein immunoreactivity in the soma and radial processes in optic nerve, or velous processes in the cerebellum. Astrocyte processes overlying the nodal axolemma also stained positively for the enzyme. (Na+ + K+)-ATPase-like immunoreactivity was not observed in association with oligodendroglia cell bodies or their processes forming myelin sheaths. In contrast, the plasmalemma of myelinating Schwann cells showed greatest immunoreactivity in the region of the node of Ranvier. Although a focal concentration of immunoreactivity was observed along node- and paranode associated regions of Schwann cells, a lower level of uniform staining was noted along the entire Schwann cell surface membrane.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2995603 TI - Decreased cerebellar 3',5'-cyclic guanosine monophosphate levels and insensitivity to harmaline in the genetically dystonic rat (dt). AB - The dystonic rat (dt) is an autosomal recessive mutant displaying a complex motor syndrome that includes sustained axial twisting movements. The syndrome is correlated with increased glutamic acid decarboxylase activity in the deep cerebellar nuclei and increased cerebellar norepinephrine levels in comparison with phenotypically normal littermates. Biochemical, behavioral, and anatomical techniques were used to investigate the possibility that the abnormalities noted in the cerebellum of the dt rat were indicative of altered function of the major projection neurons of the cerebellar cortex, the Purkinje cells. Phenotypically normal rats showed tremor in response to harmaline, a drug that acts on the inferior olive to produce bursting in the climbing fiber pathway. Dystonic rats were insensitive to the effects of harmaline but did respond to oxotremorine. Levels of the cyclic nucleotide 3',5'-cyclic guanosine monophosphate, a biochemical marker for Purkinje cells, increased in response to harmaline in normal rats but were significantly lower in dystonic rats under both basal and harmaline-stimulated conditions. Purkinje cell soma size was reduced in the dystonic rats but no other morphological correlates of the behavioral or biochemical deficits were noted. Taken together with other observations on this mutant, the results suggest an impairment in the cerebellum or in its connections with lower brainstem and spinal cord sites. PMID- 2995604 TI - Abnormal development of the retinogeniculate projection in Siamese cats. AB - In the visual system of Siamese cats, the lateral geniculate nucleus (LGN) receives an abnormally large projection from the contralateral eye and a correspondingly reduced projection from the ipsilateral eye. In order to determine how this abnormal pattern of retinal input arises, the prenatal development of the retinogeniculate projection was studied in Siamese cats using the anterograde transport of intraocularly injected [3H]leucine and horseradish peroxidase. Labeled axons from the ipsilateral eye can be detected in the optic tract by embryonic day 30 (E30; gestation is 65 days), several days later than found in normally pigmented animals. The ipsilateral projection is not only apparently delayed but also is reduced in size as compared with normal animals, and this reduction persists throughout development, indicating that the Siamese mutation acts to misdirect growing optic axons to the contralateral side of the brain as originally proposed (Guillery, R. W. (1969) Brain Res. 14: 739-741). The effect of an altered retinal projection on the ingrowth and segregation of retinal fibers to the LGN was also examined. In Siamese fetuses, not until E41 can significant label be seen within the ipsilateral LGN as compared to E35 in normally pigmented fetuses. As in normal animals, in Siamese fetuses, also, the labeled retinogeniculate afferents from the two eyes initially overlap within regions of the LGN before segregating into layers. However, measurements of the area occupied by labeled afferents from the ipsilateral and contralateral eyes indicate that maximum overlap of the two sets of afferents, although close to normal in amount, does not occur until about E51--again several days later than in normally pigmented animals (E47). The time course of segregation is also altered in Siamese cats. The onset of segregation, as signaled by the removal of contralateral eye afferents from territory destined for the ipsilateral eye and by the restriction of ipsilateral eye afferents, does not occur until about E51 in Siamese cats as compared with E47 in normally pigmented animals. Despite this delay in onset, the final segregation of the two sets of afferents in Siamese cats reaches adult-like levels at about the normal time. Thus, the misrouting of axons at the optic chiasm in Siamese cats not only alters the final pattern of innervation from the two eyes within the LGN, but also delays the onset and shortens the total duration of segregation itself. PMID- 2995605 TI - Synaptic plasticity in the molluscan peripheral nervous system: physiology and role for peptides. AB - The plasticity of a synapse in the molluscan peripheral nervous system was examined under a variety of experimental, physiological, and pharmacological conditions. These studies employed the isolated salivary glands and attached buccal ganglia of the freshwater snail Helisoma. Action potentials evoked in buccal neuron 4 normally evoke a large excitatory postsynaptic potential (EPSP) which drives an action potential in gland secretory cells. In order to measure modulation of the EPSP, action potential generation in gland cells was prevented by bathing the preparation in low calcium, high magnesium salines. The relationship between the gland EPSP amplitude and specific physiological properties of neuron 4 was analyzed. In common with some central molluscan synapses, the EPSP was found to be strongly influenced by the membrane potential of neuron 4. Specifically, its amplitude was reduced by hyperpolarization of the neuron 4 soma. The relationship between EPSP amplitude and somatic potential of neuron 4 was linear in the range from resting potential (-47 +/- 6mV) to -100 mV. Furthermore, the EPSP amplitude was directly proportional to the action potential half-width of neuron 4. In order to evaluate the possible physiological role of this action potential/EPSP relationship, we examined whether gland EPSPs are modulated during the spike broadening that occurs in both spontaneous burst activity and imposed impulse trains. The preceding action potential/EPSP relationship was maintained under both of these conditions, i.e., EPSP magnitude increased as spikes broadened during bursts or trains. The peptidergic modulation of neuroglandular transmission was also examined. The molluscan peptide SCPB was found to depolarize neuron 4 and an increase in EPSP amplitude was concomitantly observed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995606 TI - Evidence for a presynaptic adenylate cyclase system facilitating [3H]norepinephrine release from rat brain neocortex slices and synaptosomes. AB - The effects of drugs known to enhance intracellular cyclic AMP levels on depolarization-induced [3H]norepinephrine release from superfused rat neocortical slices and synaptosomes were investigated. The adenylate cyclase activator forskolin, the membrane-permeating cyclic AMP analogues 8-bromo-cyclic AMP and dibutyryl cyclic AMP, as well as the phosphodiesterase inhibitors isobutylmethylxanthine and 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrolidone (ZK 62771) enhanced the electrically evoked release of [3H]norepinephrine from superfused rat brain neocortex slices. 8-Bromo-cyclic GMP was without effect on the electrically evoked release. When [3H]norepinephrine release was enhanced by prolonging the electrical pulse duration from 2 msec to 10 msec, the relative inhibitory effect of the Ca2+ channel blocker Cd2+ and the relative facilitatory effect of the K+ channel blocker 4-aminopyridine remained unaffected. In striking contrast, the relative facilitatory effects of forskolin and 8-bromo-cyclic AMP were strongly reduced, whereas the effect of ZK 62771 was almost doubled. When veratrine-induced release of [3H]norepinephrine from cortex synaptosomes was examined, the facilitatory effects of forskolin, 8-bromo-cyclic AMP, and ZK 62771 were even more pronounced than in brain slices. The data strongly support the hypothesis that a presynaptic adenylate cyclase system plays a facilitatory role in the stimulus-secretion coupling process in central noradrenergic nerve terminals. PMID- 2995607 TI - gamma-Aminobutyric acid receptors on chick ciliary ganglion neurons in vivo and in cell culture. AB - In the chick ciliary ganglion, preganglionic terminals maintain cholinergic synapses on the choroid neurons and both cholinergic and electrical synapses on the ciliary neurons. The preganglionic terminals also contain enkephalin- and substance P-like immunoreactivity, suggesting that transmission through the ganglion is more complicated than is indicated by the known synaptic connections. We report here that embryonic chick ciliary ganglion neurons also have gamma aminobutyric acid (GABA) receptors and that GABA applied to the ganglion can block transmission elicited by preganglionic stimulation. Studies on the neurons in cell culture indicate that the GABA response is mediated by GABAA receptors: GABA activates a Cl- conductance, and the response can be mimicked by muscimol and blocked by bicuculline or picrotoxin. The GABA receptors are regulated independently from acetylcholine (ACh) receptors on the neurons since the levels of ACh and GABA sensitivity are influenced differently by culture age and by chronic exposure to GABA or elevated K+ concentrations. Application of GABA to intact ciliary ganglia increases the membrane conductance of ganglionic neurons (as in culture), reduces to subthreshold the amplitude of excitatory postsynaptic potentials in the neurons elicited by preganglionic stimulation and completely blocks transmission through the ganglion. A native source of ligand for the receptors in vivo has yet to be identified. PMID- 2995608 TI - gamma-Aminobutyric acid and benzodiazepine receptors in the kindling model of epilepsy: a quantitative radiohistochemical study. AB - Quantitative radiohistochemistry was utilized to study alterations of gamma aminobutyric acid (GABA) and benzodiazepine receptors in the kindling model of epilepsy. The radioligands used for GABA and benzodiazepine receptors were [3H] muscimol and [3H]flunitrazepam, respectively. GABA receptor binding was increased by 22% in fascia dentata of the hippocampal formation but not in neocortex or substantia nigra of kindled rats. Within fascia dentata, GABA receptor binding was increased to an equivalent extent in stratum granulosum and throughout stratum moleculare; no increase was found in dentate hilus or stratum lacunosummoleculare or stratum radiatum of CA1. The increased binding was present at 24 hr but not at 28 days after the last kindled seizure. The direction, anatomic distribution, and time course of the increased GABA receptor binding were paralleled by increased benzodiazepine receptor binding. Unexpectedly, GABA receptor-mediated enhancement of benzodiazepine receptor binding was slightly attenuated in fascia dentata of kindled compared to control rats. The anatomic distribution of the increased GABA receptor binding is consistent with a localization to somata and dendritic trees of dentate granule cells. We suggest that increased GABA and benzodiazepine receptor binding may contribute to enhanced inhibition of dentate granule cells demonstrated electrophysiologically in kindled animals. PMID- 2995609 TI - Morphology and physiology of single neurons in the medial interlaminar nucleus of the cat's lateral geniculate nucleus. AB - Physiological studies have shown that the cat's retinogeniculocortical system is comprised of at least three parallel and independent pathways, the W-, X-, and Y cell pathways. The morphological correlates of the constituent W-, X-, and Y cells have been determined both in the retina and in the A and C laminae of the lateral geniculate nucleus. The aim of this study was to extend these structure/function relationships to neurons in laminae 1 and 2 of the medial interlaminar nucleus (MIN), which is a division of the cat's dorsal lateral geniculate nucleus. We used intracellular injection of horseradish peroxidase (HRP) into individual, physiologically identified MIN neurons. Since this procedure may yield an unrepresentative sample of MIN neurons, two controls were performed. First Nissl staining showed that the soma sizes of intracellularly labeled cells were representative of those of all MIN cells. Second, retrograde labeling following HRP injections into the optic radiations or specific visual cortical areas showed that the intracellularly labeled MIN cells were representative of MIN relay neurons. Many of the retrogradely labeled cells were so well filled that their entire dendritic arbors were revealed. Of 70 MIN neurons recorded physiologically, 22 were injected with HRP and successfully recovered. We also completely labeled the somata and dendrites of 114 MIN neurons from HRP injections into the optic radiations and retrogradely labeled 165 MIN neurons by injection of HRP into visual cortical areas. Our sample of intracellularly injected neurons, which were all Y-cells, were morphologically representative of all MIN relay cells. We thus conclude that laminae 1 and 2 of the MIN contain a nearly homogeneous population of Y-cells with properties essentially identical to those of Y-cells in the A and C laminae of the lateral geniculate nucleus. When viewed in the coronal plane, MIN projection neurons typically exhibited oval or elongated somata. In the medial and ventral parts of the MIN, these somata were smaller and more flattened. MIN soma sizes extended over the full range of those seen in the A laminae. Dendritic arbors of most MIN relay neurons radiated in a fairly spherical fashion. In the medial and ventral parts of the MIN, however, dendrites were oriented in a more bipolar fashion, but intermediate forms between spherical and bipolar arbors were also common. Dendrites of MIN projection neurons were typically smooth; most primary dendrites were straight, but secondary dendrites were more variable in structure.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2995611 TI - Topography of cytochrome oxidase activity in owl monkey cortex. AB - In primate cortical tissue which has been stained for the mitochondrial enzyme cytochrome oxidase, a topographical pattern of regularly spaced blobs has been demonstrated in primary visual cortex (Hendrickson, A. E., S. P. Hunt, and J. -Y. Wu (1981) Nature 292: 605-607; Horton, J. C., and D. H. Hubel (1981) Nature 292: 762-764), and a pattern of stripes has been shown in secondary visual cortex (V2) as well (Livingstone, M. S., and D. H. Hubel (1982) Proc. Natl. Acad. Sci. U. S. A. 79: 6098-6101; Tootell, R. B. H., M. S. Silverman, E. Switkes, and R. L. De Valois (1982) Soc. Neurosci. Abstr. 8: 707). These regular cytoarchitectonic landmarks have proven extremely useful in parsing the functional and anatomical architecture of these two cortical areas. In order to look for similar landmarks in other cortical areas of a primate, we completely unfolded the cortical gray matter in the owl monkey (Aotus trivirgatus), sectioned it parallel with the flattened cortical surface, and stained the tissue for cytochrome oxidase. Distinctive cytochrome oxidase topographies were found in about seven different cortical areas. As in other primates, area V1 is characterized by blobs and area V2 is characterized by strips. In the owl monkey, area MT is characterized by an elaborate topography of dark staining in layers 1 to 4, interspersed with light blob-shaped regions, and partially surrounded by a dark ring. Many of these topographic inhomogeneities are also reflected in the lower layer myelination topography in MT. Visual area(s) VP/VA is characterized by an irregular or strip like topography. In some animals, a distinctive topography can be seen in area DX, which is presumably equivalent to either area DM or DI. Primary auditory cortex stains very darkly, but the overall shape of area A is quite variable and the borders are indistinct. Somatosensory area 3B stains quite darkly with sharp borders, but again the overall shape of area 3B is different from that previously described. PMID- 2995610 TI - Association of nerve growth factor receptors with the triton X-100 cytoskeleton of PC12 cells. AB - Triton X-100 solubilizes membranes of PC12 cells and leaves behind a nucleus and an array of cytoskeletal filaments. Nerve growth factor (NGF) receptors (10% of those found in intact cells) are associated with this Triton X-100-insoluble residue. Two classes of NGF receptors are found on PC12 cells which display rapid and slow dissociating kinetics. Although rapidly dissociating binding is predominant (greater than 75%) in intact cells, the majority of binding to the Triton X-100 cytoskeleton is slowly dissociating (greater than 75%). Rapidly dissociating NGF binding on intact cells can be converted to a slowly dissociating form by the plant lectin wheat germ agglutinin (WGA). This lectin also increases the number of receptors which associate with the Triton X-100 cytoskeleton by more than 10-fold. 125I-NGF bound to receptors can be visualized by light microscopy autoradiography in Triton X-100-insoluble residues of cell bodies, as well as growth cones and neurites. The WGA-induced association with the cytoskeleton, however, is not specific for the NGF receptor, since greater than 90% of cell surface glycoprotein receptors for WGA become associated with Triton X-100-insoluble material at lectin concentrations greater than 33 micrograms/ml. Concentrations of WGA which change the Triton X-100 solubility of membrane glycoproteins are similar to those required to alter the kinetic state of the NGF receptor. Both events may be related to the crossbridging of cell surface proteins induced by this multivalent lectin. PMID- 2995612 TI - The faculty work plan and appraisal: its potential for faculty role development. AB - This exploratory study was designed to begin analysis on the "Faculty Work Plan" and its potential for faculty role development in a university setting. The specific goals were: (1) to identify the degree of congruence between faculty work plans and the school goals; and (2) to determine the relationship between faculty work plan/school goal congruence, faculty productivity, potency, and job satisfaction. The sample was composed of 26 respondents, 26 to 50 years old, from a pool of 48 full-time faculty members affiliated with a school of nursing in a university setting. Likert-type rating scores were used to measure Faculty Work Plan-School Goal Congruence (FWP-SG), job satisfaction, productivity, and potency. Data analyses were focused on frequency distribution, group means, and Pearson Correlation Coefficients on selected pairs of variables. Interesting findings were revealed. A strong relationship was found between FWP-SG congruence and faculty productivity (r = .85; p = .000) and potency (r = .84; p = .000). Several recommendations were suggested. PMID- 2995613 TI - A validation study of a self-directed learning readiness scale. AB - In a study that investigated the predictive validity of a self-directed learning readiness scale (SDLRS), scores received by 63 first year nursing students on this scale were compared to alternate measures of self-directedness. These measures included nominations as self-directed learners by peers and faculty; entry grades from high school; and grades received at the end of the first year nursing program in five subjects. Results showed a significant correlation between SDLRS scores and peer nomination scores and between SDLRS scores and first year final subject grades. While these correlations were statistically significant, they explained only 7% and 8% of the variance thus not reaching an educationally meaningful level. Limitations of the alternate measures as a substitution for a "gold standard" are discussed. PMID- 2995614 TI - Nursing's self-image--nursing education's responsibility. AB - This article is concerned primarily with the development of a professional image of nurses and the significant role played by nursing educators. What do nursing educators do or should they do to enhance positive self-image among their students? After many years of dealing with students at various stages along the way toward RN, and seeing their reluctance to enter the nursing world due to uncertainty and fear, it seemed to the authors that the root of the problem may be in the development of professional self-image. The authors believe that even though curricula are sound and faculty well prepared for their responsibilities, students still develop these feelings of doubt and inadequacy. Perhaps nursing educators need to do a self-examination by answering the questions posed throughout the article. It is the belief of the authors that nursing educators sincerely subscribe to their role in self-image development. But, at the same time, they may unconsciously contribute to some aspects of negative image development. Is it something that they "do or don't do"? The article is intended to be thought provoking; to stimulate thinking and self-examination. It is not meant to introduce new and innovative material. It reiterates some facts, enumerates a variety of situations, provides examples, and poses many questions. It is divided into three areas of discussion. Self-concept is briefly reviewed as foundation material for the examples and situations enumerated. Further discussion centers on the importance of the individual (nurse) and the importance of the work (nursing).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995615 TI - How nursing students explain their success and failure in clinical experiences. AB - Student motivation is a faculty concern at all strata of the educational process. A major issue in motivation is the student's perception of the situation and explanation of the cause of success or failure. The education literature indicates little research on how student nurses actually perceive and explain their success and failure. This paper addresses success and failure issues by reviewing attribution theory and describing a study of attribution made by student nurses following completion of clinical nursing courses in one school. Explanations were obtained for success and failure both of nursing care provided and for mastery of theory. The data obtained reveal some interesting patterns and indicate the need for further research in this area. PMID- 2995616 TI - Patterns of practice: Master's prepared nurse practitioners. AB - Graduates of the Primary Care-Family Nurse Clinician Program at the University of Pennsylvania School of Nursing were surveyed as part of an overall, ongoing evaluation of graduate level primary care education in the school. The main focus of the survey was to discover the scope of practice characteristic of the program graduates. The survey revealed that the majority of graduates were providing direct patient care, while some were teaching primary care or embarking on related forms of practice. The graduates were found to be involved in assessment, education and counseling, and were working with physicians and others to provide comprehensive patient care. PMID- 2995617 TI - A theoretical model for developing students' communication skills. PMID- 2995618 TI - Hypothetical dilemmas--a teaching strategy for moral development. PMID- 2995619 TI - Students in transition: imitators of role models. PMID- 2995620 TI - New faculty in nursing: socialization and the role of the mentor. PMID- 2995621 TI - RN students in generic programs: what do we do with them? PMID- 2995622 TI - Ocular infection with herpes simplex virus (HSV-1) in vitamin A-deficient and control rats. AB - An experimental model was developed for studying ocular infections with herpes simplex virus (HSV) type 1 in vitamin A-deficient (-A) and pair-fed control (+A) rats. The severity and course of the disease was evaluated by clinical examination, slit lamp biomicroscopy and histopathologic observations. Experimental animals were in good health and were infected in the early stages of vitamin deficiency (either prior to or at the beginning of the weight plateau). In all trials the onset of herpetic keratitis was more rapid and the clinical disease more severe in -A rats compared to +A controls. Mean slit lamp scores (which assessed the severity of the corneal disease) increased from 3 to 10 d after infection and were higher (P less than 0.002) in -A rats at all time points and doses of virus tested. The inflammatory response in the cornea and uveal tract of -A rats was significantly higher than that of +A animals. Since ocular HSV disease is a common cause of blindness, the availability of a rat model should be valuable in studies of the role of nutritional factors in host susceptibility and response to viral challenge. Mild vitamin A deficiency increased the severity of experimental corneal HSV infections and resulted in a high incidence of epithelial ulceration and necrosis. PMID- 2995623 TI - Localization of lipofuscin in the duodenums of vitamin E-deficient rats. AB - In human malabsorption syndromes, lipofuscin accumulation has been reported to occur exclusively within the muscle layers of the intestine. It has been widely speculated that this lipofuscin deposition is related to vitamin E deficiency. To determine whether vitamin E deficiency leads to the same pattern of intestinal lipofuscin accumulation as that seen in many human malabsorption syndromes, the duodenums of rats that had been fed a vitamin E-deficient diet for 17 or 34 wk were examined for the presence of lipofuscin. Lipofuscin did not appear in the muscle layers of the duodenum until 34 wk, at which time occasional fibers containing large amounts of lipofuscin were present. An earlier and more pronounced deposition of lipofuscin occurred within connective tissue cells of the intestinal villi. After 17 wk, many fibroblastlike cells in the lamina propria of the villi contained large amounts of lipofuscin. By 34 wk, the numbers of these lipofuscin-containing cells in the lamina propria had increased substantially, and scattered cells containing lipofuscin were also seen in the submucosa. The difference in intestinal lipofuscin distribution between vitamin E deficient rats and humans with malabsorption syndromes suggests that other factors, in addition to vitamin E, probably play important roles in regulating lipofuscin accumulation in the intestine. PMID- 2995624 TI - A prospective study of complications related to mandibular third molar surgery. AB - A prospective study that evaluated the surgical and postsurgical problems of 9,574 patients of a wide range of ages who had had 16,127 third molars removed was performed. It was concluded that removal of mandibular third molar teeth during the teenage years resulted in decreased operative and postoperative morbidity. PMID- 2995625 TI - Diphenylhydantoin inhibits parathyroid hormone and prostaglandin E2-stimulated bone resorption in mouse calvaria without affecting cyclic AMP formation. AB - The effect of diphenylhydantoin (DPH) on mouse calvarial bone metabolism was studied in vitro. DPH caused a dose-dependent, reversible inhibition of PTH and PGE2-stimulated bone resorption at concentrations above 20-30 micrograms/ml without affecting cyclic AMP formation. The inhibition was observed already after 60 min and was accompanied by a reduced release of the lysosomal enzymes beta glucuronidase and beta-N-acetylglucosaminidase. The calcium antagonist Verapamil had similar effects on bone resorption and lysosomal enzyme release and it is suggested that DPH influences bone resorption by interfering with calcium fluxes across osteoclastic cell membranes resulting in low intracellular calcium levels and reduced exocytotic processes. PMID- 2995626 TI - Synovial sarcoma: an immunohistochemical study. AB - The immunohistochemical staining pattern of 18 cases of synovial sarcoma with two epithelial-specific monoclonal antibodies is described. This is compared with normal synovium, cases of giant cell tumour of tendon sheath (benign synovioma) and a variety of spindle celled sarcomas. Sixteen cases of synovial sarcoma showed staining of the epithelial component with at least one antibody. No staining was seen in normal synovium or in giant cell tumours of tendon sheath. A small number of malignant schwannomas contained groups of cells which stained positively whilst other spindle cell sarcomas either did not stain or showed 'cross-reaction' type staining only. These results add weight to the proposition that synovial sarcomas do not arise from normal synovium, despite their morphological similarities, but from mesenchymal connective tissue. It is also shown that immunohistochemical staining with anti-epithelial antibodies will emphasize the biphasic pattern of synovial sarcomas allowing their distinction from other sarcomas. PMID- 2995627 TI - Distribution of dense core granules in normal, benign and malignant breast tissue. AB - In this study breast tissue from 114 patients has been examined ultrastructurally for dense core granules (DCG). The tissue included examples of normal 'resting', pregnant and lactating breast plus various benign and malignant lesions. DCG were observed in low numbers in the apical cytoplasm in a proportion of the examples of 'resting' and pregnant breast tissue but were absent in the lactating patients. The incidence appeared to relate to hormonal changes. They were present in 50 per cent of the benign lesions examined. DCG were also observed in a high proportion of the ductal, lobular and tubular carcinomas examined and were associated with luminal differentiation. In the mucoid carcinomas over half the tumours possessed some DCG with large numbers of DCG present within certain of the malignant cells in two cases. It is possible that the granules could be related to mucin secretion. Therefore, in normal, benign and malignant (with the exception of mucoid carcinoma) breast tissue the presence of DCG would appear to be related to hormonal changes and represent prelactational differentiation rather than providing evidence of neuroendocrine differentiation. We emphasize the need for a comprehensive knowledge of the normal morphological variations within a tissue before attempting to interpret its tumours. PMID- 2995628 TI - Diarrheal illness among infants and toddlers in day care centers. I. Epidemiology and pathogens. AB - We conducted a 2-year prospective study of diarrheal illness in children ages 0 to 36 months in 22 day care centers in Maricopa County, Arizona. In 7464 child months of observation, 465 sporadic cases and 170 outbreak-associated cases of diarrhea were identified. Enteric pathogens were identified in 20% of diarrhea episodes. Giardia lamblia, rotavirus, and Campylobacter jejuni were the most common pathogens. Giardia was significantly more common in toddlers than in infants and was found in 19% of asymptomatic child contacts of symptomatic infected children. Rotavirus was significantly more common in infants than in toddlers. In outbreaks, shorter duration of child enrollment was associated with illness. Comparison of day care center characteristics revealed that only a lower score in standardized observations of hygiene and child-handling practices was associated with greater risk of diarrhea. Infectious diarrhea appears to be common in diaper-age children in day care centers, but the patterns of disease differ for different pathogens and for the infant and toddler age groups. PMID- 2995629 TI - Diarrheal illness among infants and toddlers in day care centers. II. Comparison with day care homes and households. AB - During the second year of a prospective study of diarrheal illness among 0- to 36 month-old children in day care centers in Maricopa County, Arizona, we concurrently studied children of the same age in 30 day care homes and 102 households not using day care. The seasonal pattern of diarrhea, frequency of pathogen isolation, and relative frequency of individual pathogens were similar in the three settings. Giardia lamblia and rotavirus were the most common enteropathogens. Asymptomatic infection was identified in 14% to 21% of infant toddler contacts of pathogen-positive cases of diarrhea. We compared rates of diarrhea in the three settings using five serial biweekly family-based surveys during the period of highest diarrhea rates. The incidence in infants and toddlers in DCCs (42 cases per 100 child months) was significantly higher than in DCHs (23 cases per 100 child-months) and in households not using day care (27 cases per 100 child-months); the DCH rate did not differ significantly from that in households not using day care. Among household sample children who began using day care during the survey period, the incidence of diarrhea was significantly higher than in household sample children not using day care. PMID- 2995630 TI - Fecal adenoviruses from a longitudinal study of families in metropolitan Washington, D.C.: laboratory, clinical, and epidemiologic observations. AB - During a 29-month period, we studied enteric infection in 70 families from a pediatric practice in suburban Washington, D.C. Fecal adenoviruses were detected in stools of 18 patients by tissue culture and electron microscopic procedures. From 6 through 11 months of age, the incidence of fecal adenoviruses associated with enteritis was seven per 100, and of confirmed enteric adenoviruses (EAds), three per 100 individuals per year. All EAds belonged to subgenus G (type 41). All three patients with EAds had diarrhea; two had vomiting and one had fever, but none required hospitalization. Ten of the 15 patients with non-EAds were younger than 2 years, and 60% had diarrhea, 40% had vomiting, and 20% had fever. Combined gastrointestinal and respiratory symptoms occurred more often in those who shed non-EAds (three of 11) than in matched controls (two of 48, P = 0.04). An adenovirus was detected in approximately 6% of gastroenteritis episodes, and confirmed EAds were present in approximately 2% of episodes of gastroenteritis in children younger than 2 years of age. None of the contacts of patients with non EAds shed such virus in their stools. None of nine family contacts of those with EAd appeared to shed adenovirus in stool. In contrast, rotavirus spread readily to exposed adults (25% of 65) and children (56% of 62) when a child in similar families had rotavirus infection. PMID- 2995631 TI - Immune thrombocytopenia following actinomycin-D therapy. PMID- 2995632 TI - Nephrolithiasis in childhood inflammatory bowel disease. AB - Six children with inflammatory bowel disease and nephrolithiasis are reported. Their mean age at the passage of the first stone was 12.5 years and the mean duration of active inflammatory bowel disease was 34.5 months. Four had ulcerative colitis and two had Crohn's disease. In three patients, the onset of stone disease was associated with a flare in the bowel disease. Stone passage in four patients was accompanied by an increase in abdominal pain; three experienced gross hematuria. Stones from four of the patients were composed primarily of calcium phosphate; stones from the remaining patients contained uric acid and/or calcium oxalate. The pathogenesis of nephrolithiasis as it relates to inflammatory bowel disease is considered and an approach to therapy offered. PMID- 2995633 TI - Cyclopeptine synthetase activity in surface cultures of Penicillium cyclopium. AB - Cyclopeptine synthetase, the key enzyme of benzodiazepine alkaloid biosynthesis in Penicillium cyclopium forms cyclo-(anthranoyl-phenylalanyl) from anthranilic acid, L-phenylalanine, the methyl group of L-methionine and ATP. The following in vitro measurable partial activities of the enzyme system were followed during the development of P. cyclopium: anthranilic acid and L-phenylalanine adenylyltransferase activities, and the ability for thioester-binding of L phenylalanine to the enzyme protein. These activities became measurable at the beginning of the idiophase and reached a maximum 6 days after inoculation, i.e., the pattern of activity was similar to that of the other enzymes participating in the biosynthesis of the benzodiazepine alkaloids indicating that the activities of all enzymes of the pathway were coordinatedly expressed. Inhibitor experiments indicated that 48-55 h after inoculation a preprotein of anthranilic acid adenylyltransferase was formed, which later on became activated by a hitherto unknown mechanism. PMID- 2995634 TI - Malnutrition and cancer. AB - In relation to cancer, malnutrition needs to be defined comprehensively as the carcinogenic effects produced by nutritional variables through multiple endogenous involvements. Nutritional factors act as primary effectors in four situations: carcinogens in food articles; affected bio-availability of nutrients; non-nutritive dietary items; harmful contaminants. The second situation resulting in malnutrition, often detected by metabolic pathology in high risk groups, is examined. Nutritional carcinogenesis is discussed in relevance to (1) ingestion of toxins; (2) dietary promoters; (3) type, relative proportions, interactions and bioavailability of micronutrients; (4) anti-carcinogenic or protective factors; and (5) the mixed-function oxidases. The etiological role of malnutrition preceding clinical cancer is firmly established. A host of unknown factors presently preclude but do not obliterate association of a specific type of malnutrition to a specific cancer type. PMID- 2995636 TI - Relation of food ingestion to intestinal gas production and gas related symptoms. PMID- 2995635 TI - The effect of dietary fiber and other factors on insulin response: role in obesity. AB - Epidemiologic evidence favors the hypothesis that obesity may result from the fiber-depleted diet of industrialized societies. Since hyperinsulinemia is a universal characteristic and perhaps causal of obesity, the possibility is considered that dietary factors causing excess insulin secretion might lead to obesity. Dietary glucose causes a slightly greater insulin rise than cooked starch containing an equal amount of carbohydrate, and high fiber starchy foods cause a much lesser insulin response than does glucose in solution. Doubling the dose of carbohydrate in a meal causes only a small increase in glucose response but a large increase in insulin response. Dietary fiber could act by displacing some of the carbohydrate that would normally be absorbable in the small intestine, or could translocate the carbohydrate to a point lower in the intestinal tract where less effect on insulin secretion would be observed. Evidence is presented that a higher fiber diet is associated with a higher concentration of fasting circulating free fatty acids, a lesser post-cibal decrease in circulating free fatty acids and triglycerides and less chronic increase in fasting triglycerides than a low fiber diet. These differences are associated with a lesser insulin response to high fiber meals. The extreme fluctuations between the fed and fasted states seen with low fiber diets are thus dampened by high fiber diets. The less complete inhibition of lipolysis during the fed state, and more intense lipolysis during fasting, suggested by the above data, might tend to prevent obesity. The mechanisms of the lesser insulin response to high rather than low fiber meals are not known, but the possibility that dietary fiber decreases the GIP response is considered. PMID- 2995637 TI - Breakdown of polysaccharides by human intestinal bacteria. PMID- 2995638 TI - Fiber, foods, and gastrointestinal microecology. PMID- 2995639 TI - Role of diet in cancer prevention. PMID- 2995640 TI - Pallesthesiometric studies and their use as diagnostic and prognostic indicators. PMID- 2995642 TI - Postsynaptic alpha adrenoceptors in baboon cerebral and mesenteric arteries. AB - Postsynaptic alpha adrenergic mechanisms were compared in cerebral and mesenteric arteries isolated from the baboon. The contractile response to norepinephrine (NE) of the cerebral artery was potent and similar to that of the mesenteric artery. The EC50 was 3.1 (2.0-5.0) X 10(-7) M for the cerebral artery and 2.6 (1.5-4.8) X 10(-7) M for the mesenteric artery. The maximum contraction expressed as a force developed/cross-sectional area did not differ between the two arteries, whereas that expressed as percentage of 30 mM KCl-induced contraction in the cerebral artery (118 +/- 9%) was less than in the mesenteric artery (145 +/- 6%). Phenylephrine produced a contraction in a manner similar to NE, although the EC50 values in both arteries were 2 to 3 times as large as those for NE. Clonidine produced a moderate contraction in the mesenteric artery (35 +/- 8% of the KCl-induced contraction) but no contraction in the cerebral artery. NE induced contraction in the cerebral artery was inhibited more prominently by prazosin, a selective alpha-1 adrenoceptor antagonist, than that in the mesenteric artery; the pA2 value for prazosin in the cerebral artery was higher (9.70) than that in the mesenteric artery (8.95). In contrast, there was no difference in pA2 values for either phentolamine or yohimbine between the two arteries. Clonidine-induced contraction in the mesenteric artery was attenuated by prazosin rather than yohimbine at the same concentration used. Thus, it may be concluded that contractile processes related to postsynaptic alpha-1 adrenoceptor stimulation are predominantly operative in baboon cerebral and mesenteric arteries, the cerebral artery being more susceptible to the inhibitory effect of prazosin. PMID- 2995641 TI - Labeling in vivo of beta adrenergic receptors in the central nervous system of the rat after administration of [125I] iodopindolol. AB - The amount of radioactivity in vivo in the central nervous system (CNS) of the rat has been studied after tail-vein injections of (-)- [125I] iodopindolol (IPIN). The content of radioactivity in cortex and cerebellum 1 to 4 hr after IPIN administration was significantly reduced in rats pretreated with I propranolol (1 mg/kg) given i.v. 5 min before IPIN; only a small effect of I propranolol was seen in brainstem and spinal cord. The maximum reduction in radioactivity caused by I-propranolol was approximately the same in cortex and cerebellum (about 60-65%) and occurred 2 hr after IPIN administration. I Propranolol was approximately 1500-fold more potent than d-propranolol in reducing radioactivity. Pretreatment of rats with other lipophilic drugs that act at beta receptors was able to reduce the binding of IPIN in vivo; in contrast, pretreatment of rats with drugs which do not have direct agonist or antagonist activity at beta adrenergic receptors (desmethylimipramine, metergoline, diazepam, fluoxetine, phentolamine and haloperidol) had no effect. Experiments using ICI 118, 551, a beta-2 antagonist and betaxolol, a beta-1 antagonist, indicated that the majority of radioactivity in the cortex in vivo was bound specifically to the beta-1 subtype of the receptor whereas in the cerebellum the majority of specific binding was to the beta-2-subtype. When the specific binding of IPIN to beta adrenergic receptors was measured in vitro in seven regions of the CNS, at a ligand concentration of 30 pM, a high correlation was found with the I-propranolol displaceable radioactivity measured in vivo (r = 0.97, P less than .001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995643 TI - Differential inhibition of alpha adrenoceptor-mediated pressor responses by rat atrial natriuretic peptide in the pithed rat. AB - Previous investigations have shown that rat atrial natriuretic peptide (r-ANP,5 28, atriopeptin III), antagonizes the effects of various pressor hormones (angiotensin II, vasopressin and norepinephrine) but is ineffective against pressor responses to acute spinal cord stimulation. Because the latter are believed to be mediated by intrajunctional alpha-1 adrenoceptors, whereas the others are thought to involve mainly extrajunctional receptors, we explored the possible specificity of r-ANP for alpha adrenoceptor subtypes, by comparing r ANP, the calcium channel blocker nifedipine and the vasodilator sodium nitroprusside in their ability to inhibit pressor responses to the alpha-2 and alpha-1 adrenoceptor agonists, clonidine and phenylephrine, in pithed, vagotomized rats. Acute pressor responses to bolus-injected clonidine were dose dependently attenuated by both r-ANP (up to 21%) and nifedipine (up to 37%), but acute pressor responses to phenylephrine were unaffected. Sodium nitroprusside inhibited pressor responses to clonidine (up to 67%) and phenylephrine equally (up to 66%). Pressor responses during constant infusions of clonidine and phenylephrine were attenuated similarly by r-ANP and nifedipine. This pattern of results, alpha-2 adrenoceptor specificity during immediate pressor responses but not during sustained pressor responses, suggests that r-ANP, like nifedipine, attenuates those adrenoceptor-mediated pressor responses which depend on slow transmembrane calcium fluxes. PMID- 2995644 TI - Operant acquisition of marihuana by women. AB - Marihuana acquisition and use patterns were studied in 21 women on a clinical research ward. Women could earn one 1-g marihuana cigarette or 50 cents in 30 min of performance on a second-order Fixed-Ratio 300 (Fixed-Interval 1 sec:S) schedule of reinforcement. A 7-day drug-free base line was followed by 21 days of marihuana availability and a postmarihuana drug-free period of 7 days. Five heavy marihuana users smoked an average of 6.1 (+/- 1.45) marihuana cigarettes per day and increased marihuana use significantly through time (P less than .001). Seven moderate marihuana users smoked an average of 2.72 (+/- 0.16) marihuana cigarettes per day and used significantly less marihuana through time (P less than .01). Nine occasional marihuana users smoked less than one cigarette per day (0.90 +/- 0.22) and maintained stable patterns of marihuana use. Women who increased marihuana use during the premenstruum reported significantly greater premenstrual dysphoria on the Premenstrual Assessment Form than women whose marihuana use decreased or remained the same (P less than .05 to .01). There were no marihuana dose-related effects on operant performance. The heavy, moderate and occasional marihuana smokers did not differ in operant purchase points earned, hours worked or money earned. Each marihuana dose-group earned an equivalent number of purchase points during the drug-free periods and the period of marihuana availability. Some subjects continued to work for money when smoking 15 to 20 marihuana cigarettes per day and periods of maximal operant work coincided with periods of maximal marihuana smoking (noon-midnight).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995645 TI - Beta adrenergic receptor-stimulated prostaglandin synthesis in the isolated rabbit heart: relationship to extra- and intracellular calcium. AB - We have investigated the contribution of extra- and intracellular Ca++ and calmodulin to beta adrenergic receptor-stimulated prostaglandin synthesis in the isolated rabbit heart perfused with Krebs-Henseleit buffer. Administration of isoproterenol (100 ng) increased the output of immunoreactive 6-keto prostaglandin F1 alpha and prostaglandin E2 as well as heart rate and developed tension; the coronary perfusion pressure was reduced. Isoproterenol-induced output of prostaglandins was positively correlated with the extracellular Ca++ concentration (0-5 mM). Infusion of the Ca++ channel blockers diltiazem (22 microM) or nifedipine (0.27 microM) inhibited isoproterenol-stimulated output of prostaglandins and the positive inotropic but not the positive chronotropic effect of the amine. Administration of the intracellular Ca++ antagonists 8 (diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (23 microM) or ryanodine (1.6 microM) reduced the outflow of prostaglandins and the positive chronotropic and inotropic effect elicited by isoproterenol. The calmodulin inhibitors trifluoperazine (50 microM) or calmidazolium (1 microM) failed to alter isoproterenol-induced output of prostaglandins; trifluoperazine but not calmidazolium reduced the developed tension and coronary perfusion pressure without altering heart rate. The prostaglandin synthesis elicited by arachidonic acid (3 micrograms) was inhibited by indomethacin but not by alterations in extracellular Ca++, Ca++ channel blockers, intracellular Ca++ antagonists or calmodulin inhibitors. These data suggest that activation of beta adrenergic receptors promotes cardiac prostaglandin synthesis and myocardial contractility by increasing the trans-sarcolemmal flux of Ca++, which releases intracellular Ca++.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995646 TI - Upregulation of gamma-aminobutyric acid (GABA) B binding sites in rat frontal cortex: a common action of repeated administration of different classes of antidepressants and electroshock. AB - The action of different classes of clinically effective antidepressants and electroshock on gamma-aminobutyric acid (GABA) B recognition sites in the frontal cortex was compared to that of other psychotropic agents. After either prolonged (6-18 days) s.c. infusion via osmotic minipumps or repeated i.p. injections of different antidepressants, or a series of electroshocks, treatment was halted and 72 hr later the animals were sacrificed, the brain was dissected and frozen. All major antidepressants (desipramine, amitryptyline or maprotiline), several newer compounds with reported antidepressant activity (viloxazine, zimelidine, fluoxetine, citalopram, progabide, fengabine, sodium valproate, mianserin, trazodone or nomifensine) as well as pargyline and repeated electroshocks, up regulated GABA B binding in the rat frontal cortex but not hippocampus. This appeared to be a maximum binding effect, but in some instance the kinetics were more complex. Reserpine, diphenylhydantoin and phenobarbital down-regulated GABA B binding in the frontal cortex, whereas this was unaltered by haloperidol, chlorpromazine or diazepam administration. Desipramine up-regulated GABA B binding in a dose- and time-dependent manner (minimum effective dose, 1.25 mg/kg/day s.c. for 18 days; onset of action, 6 days at 5 mg/kg/day s.c.). Together with the rather sparse data in the literature on GABA in depression and antidepressant drug action, these findings support a common GABAergic mechanism of action of antidepressant drugs and electroshock, mediated via GABA B synapses. PMID- 2995647 TI - 7-Bromo-1,5-dihydro-3,6-dimethylimidazo[2,1-b]quinazolin-2(3H)- one (Ro 15-2041), a potent antithrombotic agent that selectively inhibits platelet cyclic AMP phosphodiesterase. AB - This study with the new analog Ro 15-2041 (7-bromo-1,5-dihydro-3,6 dimethylimidazo[2,1-b]quinazolin-2(3H)-on e) confirms and substantially extends the activity spectrum of imidazoquinazolinones as potent platelet function inhibitors. Ro 15-2041 inhibited platelet aggregation induced by all common platelet agonists in platelet-rich plasma obtained from various species including man (IC50 = 1-3 microM). The compound potentiated platelet inhibition by prostacyclin, the prostacyclin-induced increase of intraplatelet cyclic (c) AMP levels and inhibited the collagen-induced release of serotonin and beta thromboglobulin. Ro 15-2041 reduced the increase and accelerated the normalization of cytosolic free Ca++ in thrombin-stimulated human platelets. Ro 15-2041 is a potent (IC50 = 70 nM) and selective inhibitor of platelet cAMP phosphodiesterase activity. Whereas Ro 15-2041 caused complete inhibition of cAMP phosphodiesterase activity in human platelet supernatants, breakdown of cAMP in cardiac homogenates was depressed to maximally 50%. In human brain and rabbit uterus Ro 15-2041 was at least 1000 times less potent. By comparison, papaverine fully inhibited phosphodiesterase activity in all four tissues with similar IC50 values of about 5 microM. Furthermore, Ro 15-2041 selectively inhibited cAMP phosphodiesterase activity of a bovine calmodulin-independent but not of a calmodulin-dependent enzyme preparation. The compound exhibited significant p.o. activity in various ex vivo and in vivo platelet function tests.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995648 TI - Effects of beta adrenoceptor down-regulation on the cardiovascular responses to the stereoisomers of dobutamine. AB - Effects of prolonged in vivo infusion of either saline (control) or isoproterenol (beta adrenoceptor desensitization) on acute cardiovascular responses to (+) (beta agonist)-, (-) (alpha agonist)- and (+/-)-dobutamine were studied in pithed rats. Each form of dobutamine resulted in comparable dose-dependent increases in maximum left ventricular dP/dt (LVdP/dtmax) in control animals. Effects of (+) dobutamine were blocked by propranolol whereas those of l-dobutamine were sensitive to prazosin; both alpha and beta antagonists were required to block the inotropic effects of the racemic mixture. Contractile responses to (+)- and (+/-) dobutamine were accompanied by tachycardia (characteristic of beta adrenoceptor stimulation) whereas (-)-dobutamine enhanced LVdP/dtmax without altering heart rate (characteristic of alpha adrenoceptor stimulation). Isoproterenol infusion resulted in a pronounced desensitization to the inotropic effects (LVdP/dtmax) of (+/-)- and (+)-dobutamine. Ed30 values for (+/-)- and (+)-dobutamine were increased by approximately 15- and 50-fold, respectively, and maximal responses to both drugs were severely attenuated. Prazosin further blunted remaining inotropic responses to (+/-)-dobutamine and propranolol resulted in a complete block. Responses to (+)-dobutamine were only sensitive to propranolol. Attenuation of heart rate responses paralleled those observed for LVdP/dtmax. By contrast, the inotropic effects of (-)-dobutamine in either control or desensitized rats were both qualitatively and quantitatively comparable; responses were blocked by the alpha-1 antagonist, prazosin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995649 TI - Prostaglandins and cannabis XIV. Tolerance to the stimulatory actions of cannabinoids on arachidonate metabolism. AB - The stimulation of prostaglandin E2 synthesis by delta 1-tetrahydrocannabinol in cultured cells is rapidly diminished by successive exposures to the drug at 24-hr intervals. Cannabidiol and cannabicyclol, two other constituents of cannabis, also displayed this in vitro tolerance effect. The phenomenon could, in addition, be observed by measuring the release of arachidonic acid from these cells, suggesting that the site of action of the cannabinoids is at one or more of the lipases that are believed to control prostaglandin synthesis under most conditions. Tolerance to cannabinoid action has been reported for a variety of in vivo parameters; thus, this in vitro system exhibits similar behavior and may, therefore, be a good model for studies on the molecular mechanisms involved in tetrahydrocannabinol action. PMID- 2995651 TI - Primary hepatocellular carcinoma (a review of 74 cases). PMID- 2995650 TI - CGS 9896: agonist-antagonist benzodiazepine receptor activity revealed by anxiolytic, anticonvulsant and muscle relaxation assessment in rodents. AB - CGS 9896, a pyrazoloquinoline that potently binds to benzodiazepine receptors, has been reported to have anticonflict activity in conventional footshock paradigms and to antagonize pentylenetetrazol-induced seizures. In the present experiments, the pentylenetetrazol discriminative cue was blocked by CGS 9896 with a potency comparable to that of diazepam. CGS 9896 also selectively lengthened the latency to terminate self-initiated brain stimulation reward. These procedures extend the anxiolytic activity of CGS 9896 to models that do not rely upon footshock-induced conflict. CGS 9896 did not impair the traction reflex in mice, did not impair rotorod performance in rats, did not reduce unpunished operant responding and decreased motor activity only slightly, indicating no distinguishable sedation or muscle relaxation in rodent models. In fact, diazepam induced rotorod impairment was blocked by CGS 9896. The anticonvulsant effects of CGS 9896, as indicated by audiogenic seizure and pentylenetetrazol-induced seizure studies, were substantial but were weaker than those of diazepam, possibly because of the muscle relaxant component of diazepam. Ethanol-induced motor impairment was potentiated more markedly by diazepam than by CGS 9896. Mixed agonist-antagonist properties of CGS 9896 therefore emerge when a comprehensive battery of behavioral assessments is utilized. CGS 9896 may have clinical anxiolytic activity without sedation or muscle relaxation. PMID- 2995652 TI - Updating rDNA restriction enzyme maps of Tetrahymena reveals four new intron containing species. AB - The extrachromosomal rDNA molecules from a number of Tetrahymena strains were characterized by restriction enzyme mapping using three different restriction enzymes combined with gel blotting and hybridization analysis. Strains from four out of six recently described species were found to contain an intron in the 26s rRNA coding region. The evolutionary relationship among the species of the T. pyriformis complex was examined on the basis of the rDNA maps with emphasis on similarities between two of the new species and the widely studied T. thermophila and T. pigmentosa. Examination of a large number of T. pigmentosa strains showed this species to exhibit an unusual polymorphism with respect to its rDNA. It is suggested that recombinational cross-over events play a role in the formation of new rDNA alleles in this species. PMID- 2995653 TI - [Gallbladder pathology in the Ivory Coast. Contribution of ultrasonics]. AB - Ultrasound imaging was first used in the two hospital and university centers of Abidjan in 1983, and findings since then have demonstrated the previously unrecognized frequency and variability of gallbladder affections. Particular conditions of functioning of an ultrasonography department in tropical surroundings are analyzed and ultrasound features of bile stones, cholecystitis and gallbladder cancer described. PMID- 2995654 TI - [Comparative study of Hexabrix and Lipiodol UF in sialography]. AB - Many new lipid- and water-soluble contrast media can be used in sialography, the three main criteria of choice being the cost, the quality of filling, particularly of the parenchyma, and its evacuation and clinical tolerance. Duroliopaque possesses very similar physicochemical properties to those of Lipiodol UF, but a sufficiently satisfactory filling of the parenchyma is not obtained. Comparison of Lipiodol UF and Hexabrix in parotid and submaxillary contrast imaging showed good clinical tolerance for both products, but improved parenchymatous filling with Lipiodol UF. However, the more effective glandular evacuation with Hexabrix makes this the product most suitable at the present time for morphologic exploration of salivary glands. PMID- 2995655 TI - Cyclic nucleotide phosphodiesterases in somatic and germ cells of mouse seminiferous tubules. AB - The distribution of phosphodiesterase forms in somatic and germ cells, and their variations during testicular development and germ cell differentiation have been investigated. Seminiferous tubules from immature mice and Sertoli cells in culture possessed two enzyme activities which were comparable to forms described for different tissues and species: (a) a calcium-calmodulin-dependent enzyme with high affinity for guanosine 3',5'-(cyclic)-monophosphate (cGMP), and (b) a calcium-calmodulin-independent enzyme with high affinity for adenosine 3',5' (cyclic)-monophosphate (cAMP) the activity of which increased in cultured Sertoli cells after treatment with FSH or dibutyryl cAMP. Seminiferous tubules from adult animals and germ cells at the meiotic and post-meiotic stage of differentiation possessed two enzyme forms that could be distinguished from those present in somatic cells of the seminiferous tubules: (a) a calcium-calmodulin-dependent form with high affinity for both cAMP and cGMP, similar to forms described in other tissues from different species, and (b) a calcium-calmodulin-independent phosphodiesterase with high affinity for cAMP and present only in post-meiotic cells, previously identified also in germ cells of the rat. PMID- 2995656 TI - Follicular fluid modulation of functional LH receptor induction in pig granulosa cells. AB - Follicular fluid was collected from small (1-2 mm), medium (3-5 mm) and large (6 12 mm) follicles of pigs, treated with charcoal to remove steroids, and tested for effects on the induction of functional LH/hCG receptors in cultures of granulosa cells from small antral pig follicles. Granulosa cells were cultured for 2, 4 or 6 days in Medium 199 + 10% pig serum. Granulosa cells cultured in the presence of purified human FSH (0.1 microgram/ml, LER 8/117), insulin (1 mU/ml), cortisol (0.01 microgram/ml) and thyroxine (10(-7) M) accumulated a 4- to 8-fold increase in LH/hCG receptors compared to control cultures. The amounts of cyclic AMP and progesterone secreted after exposure to ovine LH (1 microgram/ml: NIH S19) were also increased 2-3-fold and 80-100-fold, respectively. Exposure to FSH alone resulted in lower amounts of LH/hCG receptors with a concomitant decrease in optimum LH responses. Addition of 12.5-50% follicular fluid obtained from small (1-2 mm) follicles led to a dose-dependent inhibition of the FSH plus insulin, cortisol and thyroxine induction of LH/hCG receptors after 4 days of culture. Fluid from medium follicles showed reduced ability to inhibit LH/hCG receptor induction, and fluid from large follicles exerted only a slight inhibition or no inhibition of receptor induction. Fluid from medium-sized and large follicles exerted a progressive dose-dependent stimulation of progesterone secretion by the granulosa cell cultures. The inhibitory activity was precipitated primarily with 70% ethanol and to a lesser degree by 36 and 90% ethanol. These studies demonstrate that induction of functional LH/hCG receptors in cultures of pig granulosa cells from immature follicles is enhanced by including insulin, cortisol and thyroxine, in addition to FSH, in the culture medium, and that follicular fluid modulates both receptor induction and progesterone secretion as a function of follicular maturation. PMID- 2995657 TI - Role of GnRH in the regulation of pituitary GnRH receptors in female mice. AB - The fall in pituitary GnRH receptors in female mice after ovariectomy (Ovx) was further decreased (greater than 50%), rather than prevented, by treatment with a GnRH antiserum, despite suppression of the post-gonadectomy increase in serum gonadotrophins, suggesting that increased endogenous GnRH secretion is not the mediator of GnRH receptor fall after ovariectomy in mice. Furthermore, GnRH antiserum reduced GnRH receptors by 30-50% in intact normal females, without altering receptor affinity, and rendered serum LH and FSH undetectable but did not reduce receptors in GnRH-deficient, hpg mice. When GnRH was administered to ovariectomized mice this failed to restore receptor values (fmol/pituitary) (intact = 55.3 +/- 2.4; Ovx = 30.1 +/- 2; Ovx + GnRH = 31.6 +/- 2.8), but serum LH was reduced from high post-ovariectomy values (231 +/- 42 ng/ml) to values normal for intact females (24 +/- 2 ng/ml). In contrast, multiple GnRH injections to intact female mice increased GnRH receptor by 35%, while serum LH was reduced to just detectable levels. A marked dissociation between GnRH receptor and serum gonadotrophin concentrations was observed. Administration of oestrogen (E2) plus progesterone (P) to ovariectomized mice in which endogenous GnRH had been immunoneutralized reversed the inhibitory effect of GnRH antiserum on GnRH receptors and increased values above those of ovariectomized controls, although no increase in serum or pituitary gonadotrophin levels was seen in ovariectomized mice treated with E2 + P + GnRH antiserum. Treatment with E2 and P of intact females receiving GnRH antiserum did not prevent the inhibitory effect of antiserum on receptors, while E2 + P treatment alone of intact female mice reduced GnRH receptors by 30%. These data suggest that the gonadal steroids reduce GnRH receptors in intact female mice by inhibiting hypothalamic GnRH secretion, and that a certain degree of pituitary exposure to GnRH is required for maintenance of a normal receptor complement. These results suggest that (1) the fall in GnRH receptors after ovariectomy is primarily attributable to removal of gonadal factors. The fall is not a reflection of alteration in endogenous GnRH interaction with the gonadotroph; (2) homologous ligand 'up-regulation' of GnRH receptors in female mice depends upon the presence of the ovaries; (3) endogenous GnRH is also required for GnRH receptor maintenance in intact female mice; and (4) GnRH receptor and serum gonadotrophin responses to hormonal changes can be dissociated and their relationship is complex. PMID- 2995658 TI - Evidence for a pituitary site of gonadal steroid stimulation of GnRH receptors in female mice. AB - Treatment of GnRH-deficient (hpg) female mice with oestradiol-17 beta (E2) for 7 days increased GnRH receptors from 4.1 +/- 0.4 fmol/pituitary (control) to 7.2 +/ 0.7 fmol/pituitary (GnRH-treated), and consistently increased pituitary FSH content. Treatment of hpg female mice with E2 plus progesterone (P) for 14 days stimulated GnRH receptors more than did E2 alone, although values still remained lower than those of normal intact female mice. In contrast, GnRH treatment of intact hpg female mice alone, or combined with E2 + P, increased GnRH receptors to values similar to those of intact normal female mice. In contrast, the receptor rise after GnRH treatment alone of ovariectomized hpg mice was significantly less than in intact hpg mice similarly treated. However, the combination of GnRH + E2 + P treatment of ovariectomized hpg mice increased GnRH receptors to normal intact female values, indicating the synergistic actions of these hormones on GnRH receptor up-regulation at the pituitary. Oestradiol treatment of ovariectomized normal female mice prevented the receptor fall after ovariectomy, and when combined with exogenous GnRH further increased receptors to values identical to those of intact female mice receiving GnRH alone. Ovariectomy of hpg mice had no effect on GnRH receptor, serum or pituitary LH and FSH values. There was no change in serum LH concentration after GnRH treatment of hpg female mice, but serum FSH increased and this was accentuated by ovariectomy, indicating that in intact mice an ovarian factor(s) normally inhibits GnRH-stimulated FSH release. This factor did not appear to be an ovarian steroid since serum FSH was not suppressed in intact or ovariectomized GnRH-treated hpg mice concurrently receiving E2 + P treatment. These results suggest that: (1) gonadal steroids alone have a major direct stimulatory action on the pituitary to increase GnRH receptors; (2) the oestrogen-induced increase in GnRH receptors is enhanced in the presence of GnRH; (3) steroids exert inhibitory feedback on gonadotrophin secretion that is mediated at some cellular regulatory locus other than the GnRH receptor complex. PMID- 2995660 TI - Inclusion body myositis and systemic lupus erythematosus. AB - Inclusion body myositis (IBM) has been recognized as a distinct type of idiopathic inflammatory myopathy. Previous reports have emphasized the absence of immunologic abnormalities and lack of response to corticosteroid therapy in patients with IBM. We report a case of IBM associated with systemic lupus erythematosus and modest response of the myopathy to corticosteroid therapy. The clinical and immunologic features of IBM are more diverse than previously appreciated. PMID- 2995659 TI - Effects of photoperiod and hCG on the regulation of testicular LH/hCG receptors in Syrian hamsters (Mesocricetus auratus). AB - The regulation of testicular LH/hCG receptors was studied in Syrian (golden) hamsters with testicular atrophy induced by exposure to short photoperiod (5L:19D) and in gonadally active hamsters kept in a long photoperiod (14L:10D). By 24 h after injection of hCG, long-photoperiod hamsters showed a dose-related decrease in the number of testicular LH/hCG receptors. At 48 and 72 h, there was a recovery from this 'down-regulation'. The recovery was much faster than has been reported for the rat and mouse, and it resulted in elevation of testicular LH/hCG receptor concentrations above basal values. Hamsters with short photoperiod-induced testicular atrophy showed an increase in testicular LH/hCG receptors after injection of hCG, except for animals injected with a very high dose. The hCG-induced increase in testicular LH/hCG binding in these animals was associated with reappearance of testosterone responses to subsequent hCG stimulation. Response of testicular LH/hCG receptors to hCG in prepubertal hamsters resembled that measured in animals with short photoperiod-induced gonadal atrophy. PMID- 2995661 TI - Antibody to cytomegalovirus in various connective tissue disorders. PMID- 2995662 TI - Non-chromaffin paraganglioma presenting as a pelvic metastasis. PMID- 2995663 TI - N6-cycloalkyladenosines. Potent, A1-selective adenosine agonists. PMID- 2995664 TI - N alpha-(diphenoxyphosphoryl)-L-alanyl-L-proline, N alpha-[bis (4 nitrophenoxy)phosphoryl]-L-alanyl-L-proline, and N alpha-[ (2 phenylethyl)phenoxyphosphoryl]-L-alanyl-L-proline: releasers of potent inhibitors of angiotensin converting enzyme at physiological pH and temperature. AB - The rate of loss of phenol or 4-nitrophenol from N alpha-(diphenoxyphosphoryl)-L alanyl-L-proline (2), N alpha-[bis(4-nitrophenoxy)phosphoryl]-L-alanyl-L-proline (5), and N alpha-[(2-phenylethyl)phenoxyphosphoryl]-L-alanyl-L-proline (12) was determined spectrophotometrically at pH 7.5 and 37 degrees C in both Tris and phosphate buffers. These moderately potent inhibitors of angiotensin converting enzyme (Ki greater than 0.8 microM) all hydrolyze, losing 1 mol of phenol to yield highly potent inhibitors (Ki = 0.5-18 nM). The half-times for loss of 1 mol of phenol in Tris buffer are 22 days (2), 3.4 h (5), and 21 days (12). The half times in phosphate buffer were not significantly different. The mono(4 nitrophenoxy) ester 6 (Ki = 18 nM) loses its 1 mol of nitrophenol with a half time of 35 h to yield N alpha-phosphoryl-L-alanyl-L-proline 16 (Ki = 1.4 nM), which hydrolyzes at the P-N bond with a half-time of 2.2 h. Hydrolysis of the P-N bond in 2 and 12 was not observed during the time course of the kinetic experiments. The two phosphoramidate diesters 2 and 5 and the phosphonamidate monoester 12 thus release powerful inhibitors of angiotensin converting enzyme with a known time course at physiological pH and temperature in vitro. A time dependent increase in inhibitory potency against converting enzyme that paralleled the kinetics of phenyl ester hydrolysis was confirmed in vitro. PMID- 2995665 TI - Synthesis and activity of 5-(aminomethylene)-1,3-cyclohexanediones: enolic analogues of gamma-aminobutyric acid. AB - Eight 1,3-cyclohexanediones with an aminoalkyl side chain in the 5-position were synthesized as rigid enolic analogues of GABA (gamma-aminobutyric acid). Biochemical investigations about their abilities to displace [3H]GABA and [3H]baclofen [beta-(p-chlorophenyl)-gamma-aminobutyric acid] in binding studies or to inhibit the high-affinity sodium-dependent GABA uptake showed that these compounds were generally devoid of affinity for the two GABA receptors and for the GABA carrier. Only compound 1 exhibited a weak affinity in the GABA-A binding experiments (IC50 = 6.5 X 10(-5) M). Graphic computer modeling was applied in an attempt to explain this activity in comparison to some reference GABA agonists. Electrophysiological studies on dorsal root ganglia (DRG) also excluded agonistic or antagonistic properties on GABA-A or GABA-B receptor models but pointed out an atypical prolongation of Ca2+-dependent action potential for compound 1. PMID- 2995666 TI - Synthesis and biological activity of certain 6-substituted and 2,6-disubstituted 2'-deoxytubercidins prepared via the stereospecific sodium salt glycosylation procedure. AB - A number of 6-substituted and 2,6-disubstituted pyrrolo[2,3-d]pyrimidine 2' deoxyribonucleosides were prepared by the direct stereospecific sodium salt glycosylation procedure. Reaction of the sodium salt of 4-chloro-6-methyl-2 (methylthio)pyrrolo[2,3-d]pyrimidine (6a) or 4,6-dichloro-2 (methylthio)pyrrolo[2,3-d]pyrimidine (6b) with 1-chloro-2-deoxy-3,5-di-O-p toluoyl-alpha-D-erythro-pentofuranose (9) provided the corresponding N7 2'-deoxy beta-D-ribofuranosyl blocked derivatives (8a and 8c) which, on ammonolysis, gave 4-amino-6-methyl-2-(methylthio)-7-(2-deoxy-beta-D-erythro-pentofuranosyl )pyrrolo[2,3-d]pyrimidine (11a) and 4-amino-6-chloro-2-(methylthio)-7-(2-deoxy beta-D-erythro-pentofuranosyl )pyrrolo[2,3-d]pyrimidine (11b), respectively. Dethiation of 11a and 11b afforded 6-methyl-2'-deoxytubercidin (10a) and 6-chloro 2'-deoxytubercidin (10b), respectively. Dehalogenation of 10b provided an alternate route to the reported 2'-deoxytubercidin (3a). Application of this glycosylation procedure to 4,6-dichloro and 4,6-dichloro-2-methyl derivatives of pyrrolo[2,3-d]pyrimidine (15a and 15b) gave the corresponding blocked 2' deoxyribonucleosides (18a and 18b), which on ammonolysis furnished 10b and 4 amino-6-chloro-2-methyl-7-(2-deoxy-beta-D-erythro- pentofuranosyl)pyrrolo[2,3 d]pyrimidine (17), respectively. This stereospecific attachment of the 2-deoxy beta-D-ribofuranosyl moiety appears to be due to a Walden inversion at the C1 carbon by the anionic heterocyclic nitrogen. Controlled deacylation of 4-chloro-7 (2-deoxy-3,5-di-O-p-toluoyl-beta-D-erythro-pentofuranosyl) pyrrolo[2,3 d]pyrimidine (20a) gave 4-chloro-7-(2-deoxy-beta-D-erythro pentofuranosyl)pyrrolo[2,3-d] pyrimidine (20b). Dehalogenation of 20b gave the 2' deoxynebularin analogue 7-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrrolo[2,3 d]pyrimidine (19), and reaction of 20b with thiourea gave 7-(2-deoxy-beta-D erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidine-4(3H)- thione (21). All of these compounds were tested in vitro against certain viruses and tumor cells. Only compounds 12a, 20b, and 21 showed significant activity against measles in vitro, and the activity is comparable to that of ribavirin. Although compounds 3a and 12b are slightly more active than ribavirin against HSV-2 in vitro, they are relatively more toxic to Vero cells. Compounds 3a and 20b exhibited moderate cytostatic activity against L1210 and P388 leukemia in vitro but are considerably less active than 2-chloro-2'-deoxyadenosine (1). PMID- 2995667 TI - (+/-)-2-Amino-3,4-dihydro-7-[2,3-dihydroxy-4-(hydroxymethyl)-1- cyclopentyl]-7H pyrrolo[2,3-d]pyrimidin-4-ones: new carbocyclic analogues of 7-deazaguanosine with antiviral activity. AB - 5-Allyl-2-amino-4,6-dihydroxypyrimidine (3) was chlorinated and ozonized to yield (2-amino-4,6-dichloro-pyrimidin-5-yl)acetaldehyde (5). Acetalization of 5 with ethanol afforded a new pyrimidine intermediate 6 which can lead to 2-amino-3,4 dihydro-7-alkyl-7H-pyrrolo[2,3-d]pyrimidin-4-ones and therefore to carbocyclic analogues of 7-deazaguanosine. The 7-substituent was a cyclopentyl analogue of the arabinofuranosyl moiety in 10a, lyxofuranosyl moiety in 10b, and ribofuranosyl moiety in 10c. Compounds 10a and 10b exhibited selective inhibitory activities against the multiplication of HSV1 and HSV2 in cell culture. Repeated administration of compound 10a at 10mg/kg ip to mice infected with HSV2 increased the number of survivors and lengthened significantly the mean survival time. PMID- 2995668 TI - Synthesis and receptor-binding affinity of fluorotamoxifen, a possible estrogen receptor imaging agent. AB - Aminotamoxifen was totally synthesized from p-nitrobenzoyl chloride via a Friedel Crafts acylation. Then, by means of a Balz-Schiemann reaction, aminotamoxifen was converted into fluorotamoxifen. The triazene variation of this conversion, with a 25% yield, enables a rapid, one-step diazotization, incorporating a fluorine atom into the phenyl ring of the tamoxifen. This reaction may be useful for the preparation of low specific activity 18F-labeled tamoxifen, for distribution, and for estrogen-receptor studies. For these in vivo and in vitro studies, fluorotamoxifen was also synthesized from p-fluorobenzoyl chloride, and its chemical intermediates were compared with estradiol and hexestrol, for their receptor binding and competition, as well as for their uterotropic activity. It is demonstrated that tamoxifen and fluorotamoxifen are strong estradiol agonists and partial hexestrol agonists, while aminotamoxifen is a weak estradiol and hexestrol agonist. PMID- 2995669 TI - Synthesis and biological properties of (carboxyalkyl)amino-substituted bicyclic lactam inhibitors of angiotensin converting enzyme. AB - Syntheses of the potent angiotensin converting enzyme inhibitor (3S)-1 (carboxymethyl)-3-[[(1S)-1-carboxy-3-phenylpropyl]amino]- 2,3,4,5-tetrahydro-1H [1]benzazepin-2-one (4b; CGS 14831) and the related monoester prodrug (17a; CGS 14824A) are described together with preparative details for six- and eight membered ring analogues. Inhibitory potencies and in vivo biological activity of the compounds are discussed. The data indicate that 17a has a biological profile comparable to that of enalapril. PMID- 2995671 TI - Seasonal survival and expectation of infective life of Culicoides spp. (Diptera: Ceratopogonidae) in Israel, with implications for bluetongue virus transmission and a comparison of the parous rate in C. imicola from Israel and Zimbabwe. PMID- 2995670 TI - Angiotensin converting enzyme inhibitors: 1,5-benzothiazepine derivatives. AB - The synthesis of chiral 1,5-benzothiazepines 2a-c, 14a-c, 15c, and 16a prepared from cysteine is described. In vitro inhibition of angiotensin converting enzyme (ACE) is reported for each compound. Compound 2c was the most potent in vitro having an IC50 of 2.95 nM. The ester of 2c, i.e. 14c, was found to inhibit the AI pressor response by 75% at a dose of 0.05 mg/kg iv and by 39% at 1.0 mg/kg po. Additionally, 14c lowered blood pressure in the spontaneous hypertensive rat (SHR) by 35 mmHg, at a dose of 10 mg/kg po. PMID- 2995672 TI - Gene mapping and medical genetics. PMID- 2995674 TI - Zygosity determination in newborn twins using DNA variants. AB - A prerequisite for the optimal use of the twin method in human genetics is an accurate determination of the zygosity at birth. This diagnosis is sometimes hampered by the lack of available specific markers. We report here the use of DNA variants (restriction fragment length polymorphisms) as genetic markers for zygosity determination. We have analysed the placental DNA of 22 twin pairs with known zygosity on Southern blots by hybridisation with polymorphic human DNA probes. We looked at six different polymorphic sites using four restriction enzymes and six DNA probes. Among 10 dizygotic (DZ) pairs, only one was not demonstrably different and seven had at least two discordances. Within each of the 12 monozygotic (MZ) pairs there was complete concordance. Thus, nine of 10 dizygotic and 12 of 12 monozygotic twins were assigned their correct zygosity solely by comparison of six DNA variants. The use of these highly polymorphic DNA probes may have practical importance for antenatal diagnosis and paternity testing. PMID- 2995673 TI - Molecular genetics of the human X chromosome. AB - The human X chromosome will soon be mapped at 10 cM intervals. This will permit the localisation of any X linked disorder provided that informative families are available for linkage analysis. The location of RFLPs currently in use for clinical diagnosis is summarised. The next decade should witness the elucidation of the molecular basis of some of the more common defects, such as the muscular dystrophies and X linked mental retardation. PMID- 2995675 TI - Susceptibility to antimicrobial agents and analysis of plasmids in gentamicin- and methicillin-resistant Staphylococcus aureus from Dublin hospitals. AB - Methicillin- and gentamicin-resistant Staphylococcus aureus (MGRSA) strains isolated from Dublin Hospitals were classified into two groups (phenotypes). Phenotype-I strains expressed high level resistance to gentamicin and were susceptible to fusidic acid; strains resistant to tetracycline harboured a 3 X 10(6)-mol. wt plasmid. Strains in phenotype II usually expressed low level resistance to gentamicin, were resistant to fusidic acid and often harboured a (22-24) X 10(6)-mol. wt plasmid that specified resistance to ethidium bromide, tetracycline, kanamycin, neomycin and trimethoprim, or to combinations of these markers. A few phenotype-II strains expressed higher levels of resistance to gentamicin and other aminoglycosides. All MGRSA strains carried a 21 X 10(6)-mol. wt plasmid conferring resistance to penicillin, ethidium bromide, cadmium and mercury. Gentamicin resistance was invariably chromosomal and all strains carried chromosomal resistance to methicillin, erythromycin, streptomycin and spectinomycin. Several methicillin-resistant S. aureus (MRSA) strains isolated before the emergence of gentamicin resistance harboured a 21 X 10(6)-mol. wt penicillinase plasmid with the same restriction endonuclease profile as that from some MGRSA strains. Some MRSA strains carried other plasmids related to those found in MGRSA strains. PMID- 2995676 TI - Transferable resistance and aminoglycoside-modifying enzymes in enterococci. AB - Ten isolates of Streptococcus faecalis and two isolates of S. faecium were studied together with an NCTC strain of each species. Antibiotic susceptibility tests showed different patterns of high-level resistance to aminoglycosides. Three S. faecalis strains were highly resistant to gentamicin and other aminoglycosides. By means of a filter membrane technique, transfer of high-level resistance to aminoglycosides was demonstrated from S. faecalis to S. faecalis, from S. faecium to S. faecalis and from S. faecalis to S. faecium, the last of which has not previously been described. Aminoglycoside-modifying enzymes were assayed with radiolabelled cofactors. High-level resistance to gentamicin and other aminoglycosides could be attributed to the production of 2''-O phosphotransferases, 3'-O-phosphotransferases, 6-O-adenylyltransferases and 6'-N acetyltransferases. Other resistance mechanisms accounted for resistance in two strains of S. faecalis and one strain of S. faecium that were highly resistant only to streptomycin and one S. faecalis strain that was moderately resistant to all aminoglycosides. A low level of 6'-N-acetyltransferases was detected in the three strains of S. faecium but this did not confer high-level resistance to aminoglycosides and this trait could not be transferred. PMID- 2995677 TI - Enrichment of dog leukocyte subpopulations using density gradients. AB - Density gradient methods have been used for the enrichment of leukocyte subpopulations from human blood. We have adapted a three step method utilizing centrifugation on Hypaque-Ficoll (HF), double density HF (ddHF) and Percoll density gradients to separate lymphocytes, monocytes and basophils from dog blood. After HF separation, both Basenji-Greyhound (BG) and mongrel dogs had similar percentages of lymphocytes and monocytes, but BG dogs had significantly greater (p greater than 0.025) numbers of granulocytes. When cells from HF gradients were spun directly on Percoll, the presence of granulocytes decreased the purification of leukocyte subpopulations. An intervening separation on a ddHF gradient removed granulocytes without a selective loss of lymphocytes or monocytes. When cells from ddHF gradients were further separated on Percoll, 2 distinct cell layers resulted. Layer 1 was monocyte rich with 72.4 +/- 6.5% monocytes, 26.6 +/- 6.2% lymphocytes, and 0.8 +/- 1.8% granulocytes. Layer 2 was lymphocyte rich with 74.3 +/- 11.1% lymphocytes, 21.4 +/- 7.8% monocytes and 3.9 +/- 4.2% granulocytes. Viability as determined by Trypan blue exclusion was above 90%. Using cAMP-Phosphodiesterase (PDE) as a marker, higher PDE activity was present in the monocyte rich layer. This was similar to that reported in humans. Basophil numbers were enhanced by the use of a 1-step separation on a ddHF gradient. The percentage of basophils was increased 10-fold over baseline levels, with a viability of 90%. These techniques can be used to purify various canine leukocyte subpopulations and provide cells for biochemical analysis. PMID- 2995678 TI - Proliferative responses to human cytomegalovirus in lymphocyte cultures of male homosexuals. AB - 29 healthy adults and 32 male homosexuals were investigated for their humoral and cellular immunity against human cytomegalovirus (HCMV). In contrast to 30% of the controls, all homosexuals had HCMV serum antibodies. In addition, 84% of the homosexuals reacted positively in HCMV induced in vitro lymphocyte proliferation as compared to 66% of the controls. The study group also showed significantly higher mean reactivities in the lymphoproliferative response towards HCMV. In the group of homosexuals, a correlation could be established between the humoral and cellular immune reactivity to HCMV. PMID- 2995679 TI - Surface charge of mammalian neurones as revealed by microelectrophoresis. AB - The surface charge of isolated rat dorsal root ganglion neurones was studied by microelectrophoresis technique. The increase of Ca concentration caused greater reduction of the electrophoretic mobility compared to that produced by an equivalent amount of divalent organic cations, dimethonium or hexamethonium. No charge reversal for Ca concentrations up to 80 mM was observed. These data fit the suggestion that two anion groups of the outer membrane surface can bind one Ca ion with apparent binding constant of about 50 M-1. In solutions of low pH the electrophoretic mobility of cells decreased corresponding to titration of acidic groups with apparent pK = 4.2. Trypsin treatment in mild conditions markedly reduced the surface charge; however, neuraminidase and hyaluronidase did not change it. N-bromosuccinimide (a specific reagent for carboxylic groups of proteins) decreased the electrophoretic mobility about 60%. However, no increase of the surface charge after the action of specific reagents for amino groups (2,4,6-trinitrobenzene-sulfonic acid and maleic anhydride) was observed. It was shown that the surface charge depends also on the intracellular metabolism. If 1 mM dibutyryl cAMP or theophilline was added to the culture medium (thus, raising the concentration of cAMP inside the cell) the surface charge increased. This effect developed slowly and reached its maximum on the third day of incubation. Treatment of cells by 5 mM tolbutamide (an inhibitor of some protein kinases) did not change cell mobility. Addition of 5 mM N-ethylmaleimide (an inhibitor of adenylate cyclase) to the culture medium produced some decrease of the surface charge.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995680 TI - Structure and expression of the cell division genes ftsQ, ftsA and ftsZ. AB - The essential cell division genes ftsQ, ftsA and ftsZ map in a cluster of cell envelope genes located at two minutes on the Escherichia coli genetic map and appear to constitute an atypical operon. These closely clustered genes are all transcribed in the same direction and yet each of these genes was independently cloned on a multicopy plasmid and found to be expressed. Tn5 insertion mutagenesis of this region confirmed that these genes were independently expressed, indicating that each of these genes has its own promoter. However, maximum expression of the distal ftsZ gene required the promoter for the proximal ftsQ gene. The proximal ftsZ promoter was located within the ftsA structural gene and the proximal ftsA promoter was located within the ftsQ structural gene. No transcription terminators were evident from the DNA sequence analysis of this region. The sequence analysis also revealed that the termination codon for ftsQ overlapped the initiation codon of the ftsA gene and that the ftsA and ftsZ genes were separated by 60 base-pairs. The ftsQ gene product has a calculated Mr of 31,432 and the ftsA gene product has a calculated Mr of 45,327. The structure and expression of these genes are discussed. PMID- 2995682 TI - Comparative study of the L1 family in the genus Mus. Possible role of retroposition and conversion events in its concerted evolution. AB - The long interspersed repetitive family L1 was analysed in different species belonging to the genus Mus. It is shown to be highly conserved even in M.n. setulosus, which diverged from the other species around ten million years ago. The study of the linkage between diagnostic restriction sites in the various species and the sequence variations of different regions of the L1Md repeat shows that the L1 family undergoes concerted changes involving subsets of repeats. The rate at which this homogenization process occurs does not appear to be the same for all the subfamilies detected. The L1Md repeat in the twelfth intron of the serum albumin gene of Balb/c mice is shown to be a recent insertion. The role retroposon- and gene conversion-like events may play in the concerted evolution of the L1 family is discussed. PMID- 2995681 TI - Sites of dnaA protein-binding in the replication origin of the Escherichia coli K 12 chromosome. AB - On the basis of the observation that dnaA protein binds preferentially to DNA fragments carrying the Escherichia coli chromosomal replication origin (oriC), the binding sites were investigated by DNase I footprinting. As a result, three strong binding sites were identified in the minimal oriC sequence. The respective binding sites were 16 to 17 base-pairs long, and contained a common sequence (5') T-G-T-G-(G/T)-A-T-A-A-C (3') in the middle, although their polarities were not the same. Since mutants defective in function for autonomous replication have been isolated in the corresponding positions of the common sequence at each binding site, dnaA protein-binding at these sites seems to be significant for replication initiation. PMID- 2995683 TI - Promoter mutations affecting divergent transcription in the Tn10 tetracycline resistance determinant. AB - The tetracycline resistance determinant in transposon Tn10 consists of two genes, the tetA resistance gene and the tetR repressor gene, that are transcribed from divergent overlapping promoters. We determined the levels of pulse-labeled tet messenger RNA in Escherichia coli strains with the Tn10 tet genes on a multicopy plasmid. Addition of the inducer 5a,6-anhydrotetracycline results in a 270- to 430-fold increase in tetA mRNA and a 35- to 65-fold increase in tetR mRNA. As judged by the relative molar amounts of tetA and tetR mRNA synthesized under maximally inducing conditions, the tetA promoter (tetPA) is 7 to 11 times more active than the two tetR promoters (tetPR1 and tetPR2) combined. We characterized ten mutations in tetPA, including nine single-base-pair substitutions and a 30 base-pair deletion. All of the single-base-pair changes reduce the agreement with the consensus sequence for promoters recognized by E. coli RNA polymerase. Mutations in highly conserved nucleotides result in a 200- to 600-fold reduction in tetPA activity in vivo. Unexpectedly, tetPA mutations reduce by two- to fourfold the combined activity in vivo of tetPR1 and tetPR2, in spite of their locations outside the -35 and -10 regions of tetPR1 and tetPR2. For two tetPA mutations, the negative effect on tetPR activity was also demonstrated in tetR- tetPR-lacZ operon fusion strains, thus eliminating the possibility that it is due to variations in either plasmid copy-number or induction efficiency. The pleiotropic effects of tetPA mutations are discussed in terms of the expectation that the overlapping tet promoters compete for RNA polymerase. PMID- 2995684 TI - Neurospora crassa and S1 nuclease cleavage in hsp 83 gene chromatin. AB - We have examined the distribution of Neurospora crassa and S1 nuclease cleavage products in the chromatin of the hsp 83 heat shock gene from the Drosophila melanogaster cytogenetic locus 63 BC. Both of these nucleases generate double strand breaks in chromatin at specific sites close to the 5' end of the hsp 83 gene. With N. crassa nuclease we observe one major 5' fragment which is derived from nuclease cleavage in a DNA segment mapping approximately 120 base-pairs from the beginning of the transcription unit. With S1 nuclease we observe one major fragment which overlaps the transcription start site. In addition to the major hypersensitive sites at the beginning of the gene, the hsp 83 transcription unit is also sensitive to attack by these nucleases both before and after heat shock; however, the yield of cleavage products from within the gene is considerably greater after heat induction. PMID- 2995685 TI - Crystallization and preliminary diffraction data for iso-1-cytochrome c from yeast. AB - Deep red crystals of the electron transfer protein, iso-1-cytochrome c from yeast (Saccharomyces cerevisiae), have been obtained from a 90% saturated solution of (NH4)2SO4 containing 2 mg protein/ml, 0.1 M-sodium phosphate and adjusted to pH 6.7. The space group is P4(1)2(1)2 (or P4(3)2(1)2) with a = b = 36.4 A, c = 136.8 A and Z = 8. Crystals are stable for at least ten days in the X-ray beam and diffract to better than 2.0 A resolution. Comparable and morphologically similar crystal forms of three iso-1-cytochrome c mutants at Phe87, a pivotal residue in the electron transport chain, have also been obtained. PMID- 2995686 TI - Refinement of the solution structure of the B DNA hexamer 5'd(C-G-T-A-C-G)2 on the basis of inter-proton distance data. AB - A restrained least-squares refinement of the solution structure of the self complementary B DNA hexamer 5'd(C-G-T-A-C-G)2 is presented. The structure is refined on the basis of 190 inter-proton distances determined by pre-steady-state nuclear Overhauser enhancement measurements. Two refinements were carried out starting from two initial B DNA structures differing by an overall root-mean square (r.m.s.) difference of 0.32 A. In both cases, the final r.m.s. difference between the experimental and calculated inter-proton distances was 0.12 A compared to 0.61 A and 0.58 A for the two initial structures. The difference between the two refined structures is small, with an overall r.m.s. difference of 0.16 A, and represents the error in the refined co-ordinates. The refined structures have a B-type conformation with local structural variations in backbone and glycosidic bond torsion angles, and base-pair propellor twist, base roll, base tilt and local helical twist angles. PMID- 2995687 TI - DNA topoisomerase I mutants. Increased heterogeneity in linking number and other replicon-dependent changes in DNA supercoiling. AB - Plasmid pBR322 DNA isolated from Salmonella typhimurium supX (topoisomerase I) mutants exhibits a novel supercoiling distribution characterized by extreme heterogeneity in linking number and the presence of highly negatively supercoiled topoisomers. The most negatively supercoiled topoisomers isolated from one supX mutant have more than twice the wild-type level of supercoiling; the distribution as a whole has a median superhelix density about 1.3 times that of wild type. Surprisingly, the supercoiling distribution of plasmid pUC9 DNA isolated from supX mutants differs from that of pBR322. Escherichia coli topoisomerase I mutants have been shown to acquire compensatory mutations that reduce bacterial chromosome supercoiling to below the wild-type level even in the absence of topoisomerase I. We find that such a compensatory mutation in an E. coli topoisomerase I deletion mutant does not reduce pBR322 DNA supercoiling to a level below that of wild type. Thus, the effects of topoisomerase mutations on supercoiling depend on the replicon. PMID- 2995688 TI - Phosphorylation of myosin light chain kinase from vascular smooth muscle by cAMP- and cGMP-dependent protein kinases. AB - The enzyme, myosin light chain kinase, has been purified to homogeneity from bovine aortic vascular smooth muscle. Approximately 10 mg of enzyme could be obtained from 1 kg of fresh aortas with an overall yield of 26% of the original activity. The vascular myosin light chain kinase has a molecular weight of 160 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Antiserum raised to the aortic myosin light chain kinase in rabbits strongly inhibited phosphotransferase activity. In addition, the antiserum was used to identify myosin kinase in a crude homogenate of vascular smooth muscle by radioimmunoblotting. A single species of the enzyme (Mr = 160 000) was identified. The bovine aortic myosin kinase could be phosphorylated by both cyclic AMP- and GMP-dependent protein kinases. Approximately 2 mols PO4/mole of enzyme could be incorporated by the cyclic AMP-dependent protein kinase in the absence of calmodulin. If Ca2+ and calmodulin were included in the reaction mixture, phosphate incorporation by the cyclic AMP-dependent protein kinase was reduced to 1 mol and phosphorylation by cyclic GMP-dependent protein kinase was completely inhibited. These results were confirmed by tryptic peptide mapping. Two distinct phosphopeptides were identified: site-1 and site-2. Both could be phosphorylated by the cyclic AMP-dependent protein kinase but only site-1 was phosphorylated by the cyclic GMP-dependent enzyme. In the presence of Ca2+ and calmodulin, phosphorylation by cAMP-dependent protein kinase was restricted to site-1. The effect of phosphorylation on myosin light chain kinase activity was determined. Only phosphorylation by cyclic AMP-dependent protein kinase was found to alter the requirement of myosin kinase for calmodulin. The K0.5 (i.e. the concentration of calmodulin required for half-maximal enzyme activation) for calmodulin was 5 nM for the unphosphorylated myosin kinase. With 2 mol PO4/mol myosin kinase incorporated, the K0.5 for calmodulin was increased to 82 nM. When only 1 mol PO4/mol myosin kinase was incorporated, no effect on calmodulin requirement was observed. Moreover, single site phosphorylation had no effect on other activity parameters, including Km for ATP and for light chains. Our studies suggest that cyclic AMP-dependent protein kinase may play an important role in the regulation of vascular myosin kinase activity. Moreover, our results indicate that cyclic GMP-dependent protein kinase does not affect calmodulin-activation of myosin kinase or several other activity parameters. PMID- 2995689 TI - Identification of a neuroregulated phosphoprotein in skeletal muscle as the regulatory subunit of cyclic AMP-dependent protein kinase II. AB - When soluble proteins in cytosolic fractions of rat soleus muscles are 32P phosphorylated in vitro by an ATP:protein phosphotransferase reaction, the major substrate is a 56-kilodalton (56K) protein. As we have also reported previously, the onset and development of increased 32P-phosphorylation of this 56K protein, which are observed after the soleus is denervated, temporally correlate with the denervation period and length of the distal nerve stump [Held et al, 1983]. Conclusive evidence which identifies this neuroregulated muscle protein as the regulatory subunit of cyclic AMP-dependent protein kinase type II (R-II) is presented in this paper. The 56K soleus protein and purified bovine heart R-II were 32P-phosphorylated and subjected to limited proteolysis with bovine pancreas trypsin. After resolution of the generated 32P-phosphopeptides by SDS slab PAGE and visualization by autoradiography, no tryptic products were observed from the 56K soleus protein which were not also produced by proteolysis of the purified R II. These tryptic phosphopeptides included 39, 16.5, and 12K fragments which retained the autophosphorylation site of R-II. After denervation, the 32P phosphorylation of the 56K soleus protein and of the 39K tryptic peptide product were comparably increased. The identification of the neuroregulated 56K soleus protein as R-II was also confirmed by Western blotting with a specific anti-R-II sera. Taken together, our results demonstrate that the previously observed neuroregulation of the 32P-phosphorylation of the 56K soleus protein is identifiable with some alteration which affects the intramolecular 32P autophosphorylation of R-II. PMID- 2995691 TI - Epstein-Barr virus serology and malaria exposure in a small group of Liberian children with splenomegaly. PMID- 2995690 TI - Phosphorylative neuromodulation of the regulatory subunit of cyclic AMP-dependent protein kinase type II in skeletal muscle. AB - The phosphorylative neuromodulation of the regulatory subunit of protein kinase type II (R-II) in cytosolic fractions from denervated and sham-operated, contralateral soleus muscles of the rat was evaluated. The denervation-induced increase in the 32P-phosphorylation of R-II is not related to an increased dephosphorylation by cation-dependent or cation-independent protein phosphatases in the cytosolic fractions. The level of 32P-phosphorylation of an exogenous heptapeptide substrate (Kemptide) by dissociated catalytic subunits of cyclic AMP dependent protein kinase in cytosolic fractions from denervated and sham-operated solei did not differ. Also, no change in the concentration of cytosolic R-II assessed by competitive enzyme-linked immunosorbent assays (ELISA) was found after denervation. However, the in vitro 32P-phosphorylation of R-II in these samples was increased. Taken together, our results suggest that the increased availability of autophosphorylatable sites reflects an in vivo modulation of R-II phosphorylation rather than a significant change in total R-II content. PMID- 2995692 TI - Retrovirus and malignant lymphoma in homosexual men. AB - We report malignant lymphoma in 27 homosexual men, of whom 22 had high-grade lymphomas (B-cell immunoblastic sarcoma or small non-cleaved lymphoma) and five had low-grade disease. Antibody to human T-cell lymphotropic virus type III (HTLV III) was present in 13 (87%) of 15 with high-grade lymphoma and in two (40%) of five with low-grade disease. In contrast, only one (9%) of 11 "control" heterosexual patients with high-grade lymphoma had antibody to HTLV-III, while such antibody was found in none of 40 asymptomatic heterosexual controls and in 17 (55%) of 31 asymptomatic homosexual men. Of the homosexual lymphoma patients, 85% presented with disease in extranodal sites, including the central nervous system and rectum, and 81% had reversed T-helper/suppressor ratios. Median survival, despite treatment, is eight months. The acquired immunodeficiency syndrome-related lymphomas in homosexual men are extranodal, high-grade, B lymphoid tumors, associated with exposure to HTLV-III and unusual clinical characteristics. PMID- 2995693 TI - Crystalline inclusions in multiple myeloma. PMID- 2995694 TI - HTLV-III infection among health care workers. Association with needle-stick injuries. AB - Health care workers are caring for an increasing number of persons infected with human T-cell lymphotropic virus type III (HTLV-III), the primary etiologic agent of the acquired immunodeficiency syndrome (AIDS). We studied 361 health care and clinical laboratory personnel from institutions in several metropolitan areas with both high and moderate levels of HTLV-III infection among high-risk group members to evaluate routes of exposure to and seropositivity for HTLV-III. Protection of the privacy of subjects and prospective determination of risk factors were integral components of the study design. Six (26%) of 23 health care workers with recognized risk factors for AIDS had HTLV-III antibodies. Thirty nine (14%) of 278 workers at one institution as well as a total of five workers from other institutions reported possible percutaneous exposure to HTLV-III, usually injuries with needles that had been used on AIDS patients. There were three HTLV-III seropositive subjects who reported possible parenteral exposure to HTLV-III but no recognized AIDS risk factors. One was a symptomatic female, subject A, and her apparent sources of HTLV-III exposure were two puncture wounds, without injection of blood, made with needles used on AIDS patients. Human T-cell lymphotropic virus type III was cultured from her asymptomatic, seronegative long-term sexual partner, apparently representing female-to-male transmission. For the two other seropositive workers (subjects B and C) with nosocomial parenteral exposure, we could not rule out heterosexual transmission as a possible source of HTLV-III exposure. These latter two cases as well as the identification of seropositive health care providers from known risk groups point to the need for thorough case investigation to identify routes of exposure in health care workers. The risk of nosocomial HTLV-III transmission appears to be low and related to percutaneous exposure. Medical personnel should be trained systematically in the proper techniques and handling of instruments for phlebotomy and similar procedures to decrease occupational exposure to HTLV-III. PMID- 2995696 TI - HTLV-III transmission. PMID- 2995695 TI - Heterosexually acquired HTLV-III/LAV disease (AIDS-related complex and AIDS). Epidemiologic evidence for female-to-male transmission. AB - Thirty-seven percent (15/41) of patients with human T-cell lymphotropic virus type III (HTLV-III) disease (acquired immunodeficiency syndrome [AIDS] or AIDS related complex) sequentially evaluated at Walter Reed Army Medical Center, Washington, DC, acquired this infection from a partner(s) of the opposite sex. Demographic features of these 15 patients (ten males and five females) differed substantially from those for patients reported to the Centers for Disease Control. Heterosexual contact with partners who developed AIDS or who were at risk for AIDS was confirmed in six patients. The remaining nine patients had multiple (greater than 50) heterosexual partners and/or sexual contact with prostitutes. The method of sexual activity did not appear to be related to disease acquisition; however, this study clearly demonstrated that receptive anal intercourse was not a requirement. The observations reported herein provide further epidemiologic evidence to support the occurrence of bidirectional heterosexual transmission (both male to female and female to male) of HTLV-III infection and disease. PMID- 2995697 TI - Live Oka/Merck varicella vaccine in healthy children. Further clinical and laboratory assessment. AB - A clinical trial among 137 healthy children, ages 1 to 12 years, was conducted with four different doses (4,350, 870, 435, and 43 plaque-forming units [pfu]) of live Oka/Merck varicella vaccine to evaluate clinical reactions and selected laboratory parameters and to determine the minimum effective dose and induction time of antibody. The vaccine was well tolerated with no significant difference in the rate of reported symptoms by dose. The frequency of varicellalike rash was 3% (4/137); all rashes were mild. Serum aminotransferase values were essentially unchanged after vaccination. Minor variations found in platelet counts after vaccination were not associated with any bleeding, bruising, or clotting. Among initially seronegative children who received doses of 435 pfu or greater, 94% assayed at two weeks and 100% assayed at four or six weeks seroconverted. The geometric mean titers were similar for all four doses at six weeks. IgG and IgA responses were demonstrated with no relation to the vaccine dose. PMID- 2995698 TI - Activity of endogenous inhibitor(s) of platelet aggregation in plasma of normal males, females and pregnant women. PMID- 2995700 TI - [A patient with immunoblastic lymphadenopathy: possible relation to Epstein-Barr virus infection]. PMID- 2995699 TI - [Interstitial pneumonitis and smoldering adult T-cell leukemia. Study of anti ATLA antibody in patients with chronic pulmonary diseases in ATL endemic area]. PMID- 2995701 TI - [A case of adult T-cell leukemia who developed systemic swelling of lymph nodes 4 years after disappearance of dermal symptoms]. PMID- 2995702 TI - [Transition of toxic hepatitis. 2. Drug-induced liver neoplasms]. PMID- 2995703 TI - [Microquantitation of immunoglobulin]. PMID- 2995704 TI - [Antireceptor antibody assay--methods and clinical significance]. PMID- 2995706 TI - Antibody to lymphadenopathy-associated virus in two Japanese hemophiliacs. PMID- 2995705 TI - [Structure and function of thyroid follicular cells--biochemical and histochemical studies on the enzymes related to thyroid hormone synthesis]. PMID- 2995707 TI - [Gastric carcinoma in the aged--a comparison between Japan and Germany]. PMID- 2995708 TI - [Effects of calcium hopantenate on GABA receptors in the aged rat brain cortex]. PMID- 2995709 TI - [Diseases caused by abnormal hormone receptors]. PMID- 2995710 TI - [TAE-TPE combination for the treatment of liver cell carcinoma]. PMID- 2995711 TI - Dynamic topography of visual evoked potential in children: a study of the development of the visual system. AB - To study the development of the visual system, the dynamic topography of the visual evoked potential (VDT) was investigated in 30 normal children aged from two weeks to four years. Twenty-three of the 30 children (77%) showed reliable VDT. The results showed three steps in the development of the visual system in these young children. The first step was seen during the first 4 months after birth, and it was represented by a shortening of duration and an increasing of amplitude in N70 in the occipital region. The second step was seen between 9 months and 2 years of age, and it was represented by a shortening of duration and an increasing of amplitude in P100 and P150 over a widespread area in the occipito-parieto-temporal region. The third step was not clearly seen until 3 years of age, and was represented by the appearance of P200, the so called "vertex potential", around the occipitoparietal region. These three steps in the development of the visual system seemed to be closely related to the time-course of the development in the myelination and maturation of the neuron synapses in the visual pathway and visual cortex. With the aid of VDT, it was possible to clarify that the visual system in children develops step by step from a low level to a high level of the visual function. PMID- 2995712 TI - Proton-coupled transport of organic solutes in animal cell membranes and its relation to Na+ transport. AB - Recent studies on proton-coupled transport of organic solutes in animal cell membranes were reviewed. In the intestinal and renal brush border membranes, transport of intact small peptides (di- or tri-peptides) has been established to be cotransported with H+. The peptide transport is Na+-independent, dependent on a pH gradient, electrogenic as revealed by transport-associated membrane depolarization and conductance increase, and reveals a marked overshoot uptake when a sufficiently large proton gradient is imposed across the membrane. Similar properties are found for L-lysine transport by the brush border membrane vesicles from mullet kidney and for L-leucine transport in some cultured cells. Partial involvement of H+ in Na+-dependent transport has also been reported for some organic acids, L-glutamate, and citrate. The physiological meanings of these purely H+-dependent and partially H+-dependent transports have been discussed based on available data. PMID- 2995713 TI - [A case of adenoid cystic carcinoma of the trachea treated by circumferential resection and anastomosis]. PMID- 2995714 TI - Characterization of canine rotavirus RS 15 strain and comparison with other rotaviruses. PMID- 2995715 TI - Pathogenic characteristics of highly virulent avian reovirus, strain 58-132, isolated from a chicken with tenosynovitis. PMID- 2995716 TI - Prevalence of Hong Kong (H3N2) influenza virus-antibody in swine. PMID- 2995717 TI - Epizootiologic survey of feline leukemia virus in South-Kyushu area. PMID- 2995718 TI - Neurogenic tumors in coho salmon (Oncorhynchus kisutch) reared in well water in Japan. AB - Neurogenic tumors were found protruding from various parts of the body of 23 coho salmon. The tumor-bearing fish were first- or second-generation fish derived from eggs imported at the eyed stage to Japan from the United States. Twenty-two of the tumors were in young adults and varied from 14 to 80 mm in maximum diameter. Histologically, the tumors were composed of spindle-shaped cells with abundant fibrous stroma. One tumor showed typical nuclear palisading. All tumors in young adults invaded locally muscle and adipose tissues. These tumors were similar in histologic appearance to malignant schwannomas in humans. One tumor found in a fingerling coho salmon was identified as an ependymoblastoma. The Vectstain avidin-biotin-peroxidase complex immunoperoxidase staining procedure for S-100 protein revealed that the S-100 protein existed in an ependymoblastoma and in areas of typical nuclear palisading in a malignant schwannoma in coho salmon. The occurrence of soft tissue tumors in coho salmon was first recorded in Japan. The morphology and etiology of the present cases were compared with those of the tumors in salmon reported from the United States. Judging from the conditions in which the fish were reared, the development of these tumors was not related to halogenated compounds formed during water chlorination, as suggested previously. The environmental factor(s) responsible for their development has not yet been identified, and genetic influences may have been a contributory factor. PMID- 2995719 TI - [Histochemical studies of neutrophils in patients with myocardial infarction]. PMID- 2995720 TI - [Cellular mechanisms of the disorders of cardiac rhythm. I. Circulating excitation]. PMID- 2995722 TI - [Virus-staphylococcal destructive pneumonia in children]. PMID- 2995724 TI - [Do-in: in search of equilibrium]. PMID- 2995725 TI - [What to think about those new body therapies. Interview by Christophe Baroni]. PMID- 2995723 TI - [Growth factors. A new dimension in understanding oncogenesis]. AB - The current understanding in biology and function of 4 growth factors is reviewed. PDGF suggests functions for proto-oncogens in normal cells, which may interact in tightly linked hierachies to induce malignant growth. PDGF requirement of normal fibroblast cell-lines is lost when the cells are infected with tumor viruses. TGF is able to stimulate growth of normally anchorage dependent cells in an anchorage independent manner in soft agar. This ability is thought to be the best in-vitro correlate of neoplastic transformation. The peptide hormones bombesin/gastrin releasing factor and EGF can act as autocrine growth factors in various lung cancer cell-lines and stimulate clonal tumor cell growth in-vitro. The potential clinical application of these types of growth factors may enable the in-vitro growth from any lung cancer patient and allow individual drug testing. TCGF produced by T-cells to activate T-cells, is central to immune stimulation and immune response. Models for potential indirect anticancer effects either by in-vivo administration or by in-vivo incubation plus passive transfer of T-cells are presented to be initiated in future clinical trials. PMID- 2995726 TI - Suppressed neutrophil oxidative activity in sepsis: a receptor-mediated regulatory response. AB - We have reported that neutrophils from patients with intraabdominal sepsis show suppressed superoxide production in response to soluble chemoattractants. To determine whether this could be a result of altered receptor function, neutrophil function studies and characterization of receptors were performed upon 22 patients recently operated upon for intraabdominal sepsis. Patient cells showed an absence of subagarose migratory response to activation serum (C5) but intact FMLP response. Superoxide production to FMLP was 5.3 +/- 0.1 nmole FcC compared to 11.2 +/- 0.1 nmole for laboratory normals. Addition of cytochalasin B reversed this. Scatchard analyses of patients' cells showed affinity of 55.5 +/- 0.6 as compared to 36.5 +/- 1.8 for controls. The number of receptors of cell was approximately 55,000 for both controls and patients. Internalization studies revealed significant increase in tritiated-FMLP uptake at room temperature. These results support the hypothesis that superoxide suppression is due to enhanced clearance of stimulant by enhanced internalization of the receptor-ligand complex. PMID- 2995727 TI - Multiple calcified hepatic lesions. PMID- 2995721 TI - [Humoral and cellular immunity in perimyocarditis and congestive cardiomyopathy]. AB - In coxsackie B-, influenza and mumps-induced perimyocarditis the characteristic immunological features were cross-reacting cytolytic antimyolemmal antibodies, circulating immune complexes and no NK- or K-cell activity. Cytolytic antimyolemmal antibodies are markers of a secondary immunopathogenesis in perimyocarditis. In cytomegalovirus myocarditis antiinterfibrillary antibodies could be found and immune complexes were rarely observed. Postmyocarditic cardiomyopathy could be identified by circulating antimyolemmal and antisarcolemmal antibodies which also bind to the endomyocardial autopsy specimens. In these patients circulating immune complexes are more frequent than in normals and in dilated cardiomyopathy. In dilated cardiomyopathy antifibrillary and antiinterfibrillary antibodies were observed, the incidence of which correlated with the severity (NYHA) of the disease. A group of patients with dilated cardiomyopathy could be distinguished who demonstrated regardless of the severity lymphocytotoxic reactions against vital cardiocytes. PMID- 2995728 TI - Pituitary-testicular axis abnormalities in immature male hypothyroid rats. AB - The pituitary-testicular disturbances which follow the onset of hypothyroidism were studied in immature male Wistar rats rendered hypothyroid by treatment with methimazole (MMI) given in drinking water, starting at 40 days of age. Half of the animals continued on MMI (MMI group) up to 140 days of age; the remaining rats were withdrawn MMI at 100 days and injected thereafter s.c. with 3 micrograms of T3 daily, during the last 40 days (MMI + T3 group). Ten rats were used as controls (C group). Hypothyroidism induced in immature animals significantly decreased serum T4, T3, LH, PRL, and testosterone levels, and also impaired the normal growth of body and sex accessory glands. T3 replacement therapy helped to normalize serum hormonal levels, but the body and sex accessory gland weights were not fully corrected. Hypothyroidism also reduced the [125I]LH/hCG binding sites of testicular homogenates. T3 replacement was not able to improve the binding; nonetheless, the hormone-receptor affinity constant remained unaltered among the groups. Leydig cell responsiveness to hCG stimulation in vitro (0-82 nM) showed impaired testosterone production in the MMI group (25% of that found in the C group) and also in the MMI + T3 group (80% of that found in the C group). These data demonstrate that induction of hypothyroidism in the immature male rat leads to alterations in serum LH, PRL and testosterone levels, and suggest that thyroid hormones have a modulating action on the testis as far as LH-mediated testosterone secretion is concerned. PMID- 2995729 TI - Metabolism of adrenal androgens by human endometrium and adrenal cortex. AB - The enzyme 17 beta-hydroxysteroid dehydrogenase (17OHSD) was studied in human endometrium and adrenal cortex with respect to the metabolism of 5-androstene-3 beta,17 beta-diol (androstenediol) and dehydroepiandrosterone (DHA). The aim was to provide further information concerning the origin and biological significance of these androgens in endometrium, particularly the increased concentrations of the secretory phase and to compare the characteristics of the enzyme in the two tissues. In both endometrium and adrenal cortex the metabolism of androstenediol to DHA was linear with time and increasing enzyme concentration. The preferred cofactor was NAD and the apparent Km values were 3.4 +/- 0.2 (SD) microM (n = 3) for endometrium and 30.5 +/- 6.1 microM (n = 3) for adrenal cortex. In endometrium DHA was not metabolised to androstenediol in the presence of either NADH or NADPH whereas in the adrenal cortex both cofactors were utilised. However, the concentration of NADH required to achieve maximum enzyme activity was 10-fold higher (1 mM) than for NADPH (0.1 mM) and maximum activity with NADH was only 30% of that using NADPH. The apparent Km was 125 microM DHA (n = 2). The study indicates that androstenediol in endometrium does not arise from DHA metabolism but that its presence could be due to a binding protein particularly during the secretory phase. Our findings also suggest that the enzyme of endometrium differs from that of the adrenal cortex and that the kinetic properties may be related to the physiological requirements of the two tissues. PMID- 2995730 TI - Inactivation of soluble 17 beta-hydroxysteroid dehydrogenase of human placenta by fatty acids. AB - The sensitivity of soluble, 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) of human placenta to inactivation by fatty acids was examined. Exposure to the unsaturated fatty acids oleic, arachidonic, linoleic and linolenic acid resulted in the loss of activity. Methyl and ethyl esters of oleic acid, the saturated fatty acid, stearic acid and prostaglandins E2 and F2 alpha were without effect. Inactivation by oleic acid required the fatty acid at levels above its critical micelle concentration, 50 microM, as estimated by light-scattering. Steroid substrates and inhibitors did not protect against inactivation. NAD+, NADH, NADP+ and NADPH did protect. The concentrations of NADP+, 50 microM, and NAD, 1.5 mM, necessary for complete protection were significantly greater than their respective Michaelis constants, 0.16 microM and 15.2 microM. The data suggest that soluble 17 beta-HSD can bind to fatty acid micelles and that the binding site(s) on the enzyme are at or near pyridine nucleotide binding sites. PMID- 2995731 TI - Effect of high and low density lipoproteins on corticotropin-mediated cortisol synthesis by bovine zona fasciculata cells. AB - The role of bovine HDL and LDL in supporting corticotropin-stimulated steroidogenesis has been investigated, using acutely dispersed zona fasciculata cells. Using a dose of corticotropin sufficient to maximally stimulate steroidogenesis (in the absence of lipoproteins) both HDL and LDL increased steroidogenesis in a dose-dependent manner. At higher concentrations of lipoprotein, HDL caused approx 3-fold greater increase in steroid production than did LDL. Taken with the knowledge that HDL is the major cholesterol carrier in bovine serum, these findings suggest that in cattle HDL is a major source of cholesterol for steroidogenesis. PMID- 2995732 TI - Comparison of the DNA sequence and secondary structure of the herpes simplex virus L/S junction and the adeno-associated virus terminal repeat. AB - The defective parvovirus Adeno-associated virus (AAV) is absolutely dependent upon coinfection with either Adenovirus or Herpes Simplex Virus (HSV) for its multiplication. We have compared the terminal repeats of HSV-1F strain DNA with the terminal 200 nucleotides of AAV DNA. Our findings demonstrate similarities between portions of the HSV inverted repeats found at the L/S junction and the termini of AAV. By computer analysis we have determined potential secondary folding patterns for both genomes. The following points can be made about the a, b, and c repeats in HSV: (1) Regions b and c are complementary over a significant portion of their length. (2) The ends of a can fold back on themselves to form large secondary structures. Moreover, when the b and c homology is used to align the ends of a, the b/a and c/a junctions are within 1 base of each other. (3) The short direct repeats within a are essentially a large loop with little secondary structure. The potential implications of this structure are discussed and a model for HSV DNA replication is presented. PMID- 2995733 TI - Transfer of reactivity with in vitro produced transfer factor in rhesus and owl monkeys. AB - Transfer factors against two heterologous antigens, Herpesvirus saimiri and owl monkey kidney cells, were replicated in vitro in a human lymphoblastoid cell line (LDV/7) and injected into rhesus and owl monkeys. Transfer of immunity was demonstrated by the leukocyte migration inhibition assay. This study suggests that heterologous transfer factor, replicated in vitro, can transfer cellular immunity against membrane antigens in rhesus and owl monkeys. PMID- 2995734 TI - In vitro studies on the relative potency of bronchodilator agents. PMID- 2995735 TI - Primary hypomagnesemia with secondary hypocalcemia. Report of a case and review of the world literature. AB - Primary hypomagnesemia with secondary hypocalcemia (PHSH) is a rare type of hypocalcemic disorder which occurs in early infancy and is clinically characterized by recurrent tetany and/or convulsion. In this paper, a male infant with PHSH who had frequent seizures at the age of 9 days is described. Besides PHSH, several illnesses in infancy are manifested by hypomagnesemia and hypocalcemia, i.e. transient neonatal hypomagnesemic hypocalcemia, congenital renal or hepatic insufficiencies, magnesium-losing nephropathy, combined impairments of intestinal absorption and renal reabsorption of magnesium. PHSH is to be differentiated from these illnesses by the demonstration of a combination of the following findings; hypocalcemia refractory to calcium but responsive to magnesium, continuous requirement for magnesium supplementation to maintain normocalcemia, lack of hypermagnesiuria and/or impaired intestinal absorption of magnesium. Twenty cases from the literature were found to exhibit these characteristics. The clinical, biochemical, and endocrine features of PHSH are summarized on the basis of a review of the data of these and the present case. No associated illness was known in the afflicted infants or mothers. Both male and female infants were afflicted at a male to female ratio of 15:6. Some siblings were afflicted but none of the parents or relatives. The onset of tetany and/or convulsion was between the 9th day and 4th month, which is later than that of other neonatal hypocalcemic illnesses. Hypocalcemia was more pronounced than other infantile hypocalcemic illnesses. The role of the parathyroid hormone in the pathogenesis of hypocalcemia has been studied in several studies but no unifying concepts have yet been established. PMID- 2995736 TI - [Angiokeratoma corporis diffusum (Fabry's disease). Update. Apropos of 2 cases]. AB - Fabry's disease (angiokeratoma corporis diffusum) is an X-linked recessive inherited metabolic defect due to the lack of the enzyme alpha-galactosidase A. We reviewed the Argentine literature on the subject, the main features of the disease and its differential diagnosis. Two patients aged ten and fifteen are described showing the characteristic clinical picture of the disease since ages four and nine respectively. Skin and conjunctival ultrastructural studies showed intracytoplasmatic granules with a lamellar appearance in the endothelial cells, pericytes and fibroblasts. Plasma levels of alpha-galactosidase activity were sharply decreased in the two patients studied and partially decreased in their heterozygous mothers. PMID- 2995738 TI - [Laboratory diagnosis in sexually transmitted viral diseases. Its importance in medical practice]. AB - We describe the most popular current laboratory techniques used to demonstrate poxvirus, papovavirus and herpes simplex. In the latter case, the microbiological diagnosis was done. PMID- 2995737 TI - [Sclerodermiform porphyria cutanea tarda. Ultrastructural study]. AB - Approximately 30% of patients affected of PCT present scleroderma-like lesion of the skin. Two cases of PCT, presenting scleroderma-like lesions are reported. The patients were diabetic but not alcoholic and tolerate relatively well sunshine. Porphyrin elimination diminished with urine alkalinization and phlebotomies, and the scleroderma-like lesions improved with N-acetyl-hydroxy-proline administration. PMID- 2995739 TI - [Kaposi sarcoma in acquired immune deficiency syndrome (AIDS). I: Clinical findings and laboratory diagnosis]. AB - In addition to the classical type of Kaposi's sarcoma, a new, aggressive and prognostically ominous variant occurs since 1980: the AIDS-associated Kaposi's sarcoma. This aggressive variant is associated with viral infection by the lymphotropic retrovirus HTLV-III, identical to LAV. In the U.S.A. and in Europe, this virus is transmitted mainly by homosexual contact, whereas in Central Africa the characteristic mode of transmission is heterosexual promiscuity. The authors present for the benefit of the ENT-specialist, case-reports of an American/European and of an African AIDS-associated Kaposi's sarcoma of the palate illustrating the major symptoms of generalized lymphadenopathy, opportunistic infections, and the relevant immunological and serological features. PMID- 2995741 TI - Differential effects of morphine on the rates of dopamine turnover in the neural and intermediate lobes of the rat pituitary gland. AB - The acute administration of morphine to male rats decreased the rate of dopamine turnover in the median eminence and in the neural lobe of the pituitary, but was without effect in the intermediate lobe of the pituitary. Pretreatment with the opiate antagonist, naltrexone, reduced the effects of morphine. These results indicate that morphine, by acting on opiate receptors, inhibits the activity of tuberoinfundibular dopaminergic neurons that terminate in the median eminence and those tuberohypophysial dopaminergic neurons that terminate in the neural lobe of the pituitary. PMID- 2995740 TI - Synergistic interaction between alpha- and beta-adrenergic receptors in rat brain slices: possible site for antidepressant drug action. AB - Experiments were undertaken to examine the characteristics of the adrenergic receptor-coupled cAMP system in rat brain slices. It was found that the potentiation of isoproterenol-stimulated cAMP accumulation by 6 fluoronorepinephrine, an alpha-adrenergic agonist, is largely dependent upon the degree of beta-receptor occupancy, with prazosin-sensitive alpha-adrenergic receptors contributing less to this interaction. Chronic administration of a variety of antidepressants decreased the potentiating interaction between 6 fluoronorepinephrine and isoproterenol even under conditions where there were no obvious effects on the alpha- or beta- adrenergic components themselves. Chronic administration of imipramine had no effect on the interaction between 6 fluoronorepinephrine and adenosine, suggesting that the drug selectively modifies the coupling between the alpha- and beta-adrenergic systems. The results suggest that antidepressants influence the coupling between 6-fluoronorepinephrine and isoproterenol receptors independent of any effect on the individual recognition sites. PMID- 2995742 TI - Inhibition of [3H]nitrendipine binding by phospholipase A2. AB - Phospholipase A2 from several sources inhibited [3H]nitrendipine binding to membranes from brain, heart and ileal longitudinal muscle. The enzymes from bee venom and Russell's viper venom were most potent, having IC50 values of approximately 5 and 14 ng/ml, respectively, in all three membrane preparations. Inhibition of binding by bee venom phospholipase A2 was time- and dose-dependent. Mastoparan, a known facilitator of phospholipase A2 enzymatic activity, shifted the bee venom phospholipase A2 dose-response curve to the left. Pretreatment of brain membranes with bee venom phospholipase A2 (10 ng/ml) for 15 min caused a 2 fold increase in the Kd without changing the Bmax compared with untreated membranes. Extension of the preincubation period to 30 min caused no further increase in the Kd but significantly decreased the Bmax to 71% the value for untreated membranes. [3H]Nitrendipine, preincubated with bee venom phospholipase A2, was recovered and found to be fully active, indicating that the phospholipase A2 did not modify the ligand. It is concluded that phospholipase A2 acts on the membrane at or near the [3H]nitrendipine binding site and that phospholipids play a key role in the interactions of 1,4 dihydropyridine calcium channel antagonists with the dihydropyridine binding site. PMID- 2995743 TI - Solubilization of quisqualate-sensitive L-[3H]glutamate binding sites from guinea pig brain membranes using a zwitterionic detergent. AB - L-[3H]Glutamate binding sites were solubilized with a zwitterionic detergent 3 [(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) plus ammonium thiocyanate from guinea pig synaptosomal membranes. The binding of L [3H]glutamate to the solubilized binding sites was saturable and reversible. Scatchard analysis suggested the existence of two different classes of binding sites with KDs of 63.8 and 644 nM. The L-[3H]glutamate binding was displaced by excitatory amino acids with such an order of potency that L-glutamate much greater than D-glutamate congruent to L-aspartate greater than D-aspartate. Quisqualate effectively displaced the glutamate binding in biphasic manner. L Glutamic acid diethyl ester, the quisqualate receptor antagonist, also showed a moderate displacing ability. Other neuroactive amino acid analogues displaced the glutamate binding only weakly, except for L- and D-homocysteic acids which had moderate potency. It is very likely from these results that the glutamate binding sites solubilized in this study are relevant to the physiological glutamate receptors especially of quisqualate-type. PMID- 2995744 TI - Naloxone inhibits superoxide release from human neutrophils. AB - Using the superoxide dismutase inhibitable reduction of cytochrome c assay, we studied, the effect of (-) naloxone on N-formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated superoxide (O2-) release from human neutrophils. Neutrophils were pre-incubated with the range of concentrations of (-) naloxone that is administered in models of experimental sepsis (10(-6) - 10(-4.5) M). (-) Naloxone inhibited O2- release in a dose dependent manner. 02- produced by a cell-free xanthine-xanthine oxidase system was not inhibited by (-) naloxone, indicating that (-) naloxone was not scavanging O2-. There was no difference between the effect of (-) and (+) naloxone suggesting that the inhibition of O2- was not specific for an opiate receptor. Another opiate antagonist, nalorphine, as well as the opiate agonist, morphine, also inhibited O2- release in the same concentration range. There was no difference between the effect of naloxone and morphine. PMID- 2995745 TI - Effects of delta 9-tetrahydrocannabinol on testosterone production in vitro: influence of Ca++, Mg++ or glucose. AB - The major psychoactive component of marihuana, delta 9-tetrahydrocannabinol (THC), influences testicular function. In the present experiments, the addition of THC to incubations of whole decapsulated mouse testes altered testosterone (T) production differentially, depending on the specific gonadotropin used, the dose of THC and/or the amount of divalent cation present in the media. In the presence of luteinizing hormone (LH; 10 ng/ml), and a dose of 25 micrograms THC/ml, T production was significantly decreased, compared to that by testes incubated with LH and vehicle at all Ca++ levels, except at 0.127 or 1.0 mM Ca++. The production of T by these paired testes exposed to either THC or vehicle (ethanol; ETOH), increased as Ca++ concentration approached physiological levels. In contrast, in the presence of follicle-stimulating hormone (FSH; 1 microgram/ml), THC-induced suppression of T production was significant in the absence of Ca++ from the media, and at 12.7 mM Ca++. However, it appeared that the levels of Ca++ did not differentially affect T production in the presence of FSH, whether or not THC was also added. In the presence of human chorionic gonadotropin (hCG; 12.5 mIU/ml), a lower dose of THC (25 ng/ml), stimulated T production at 0.25 to 1 mM Ca++, but had no effect as Ca++ reached 2.5 mM. Without additional Ca++ in the media, this dose of THC significantly reduced T secretion. In contrast, in the presence of hCG, a higher THC dose (25 micrograms/ml), suppressed T accumulation at 0.127, and from 1.0 to 12.7, but had no effect at 0.25 mM, or in the absence of Ca++. In the presence of hCG, the high 25 micrograms/ml dose of THC stimulated T production, in the absence of additional Mg++, and at 0.01 mM Mg++, but THC had no effect at 0.1 mM Mg++, but inhibited T production at 1.1 mM Mg++. In the presence of hCG, 25 micrograms THC/ml produced a consistent suppression of T production across glucose concentrations examined. These findings suggest that the mechanisms by which THC effects testicular steroidogenesis may involve Ca++- and/or Mg++-dependent processes. Differential requirements for these divalent cations by the gonadotropins may explain the interactive effects of THC with LH, hCG or FSH. PMID- 2995746 TI - Central alpha-activation by clonidine reduces plasma level of beta-endorphin in patients with essential hypertension. AB - Whether peripheral beta-endorphin contributes to the antihypertensive action of clonidine was examined by measuring plasma levels of beta-endorphin-like immunoreactivity (beta EpLI) after acute administration of clonidine in patients with essential hypertension. Administration of clonidine (0.225 mg) in one dose significantly lowered blood pressure, decreased heart rate and reduced the plasma level of beta EpLI and ACTH, while the placebo had no effect on blood pressure, heart rate or plasma level of beta EpLI suggesting that peripheral beta-endorphin does not play a major role in the antihypertensive action of acute clonidine administration. PMID- 2995747 TI - Protection from cerebral ischemia by U-50,488E, a specific kappa opioid analgesic agent. AB - U-50,488E is a novel analgesic agent with a specific agonist property on the kappa opioid receptor. It is found to protect against the lethal effect of temporary bilateral carotid occlusion (BCO) in Mongolian gerbils and rats of the Fischer strain. Pretreatment with U-50,488E in gerbils before 7 min of BCO reduced the development of behavioral hyperactivity and preserved the hippocampal neurons from ischemic death. This protective effect of U-50,488E resided predominantly in the levo-enantiomer which is also more potent as a kappa analgesic. Two other kappa opioid analgesics, ethylketocyclazocine and bremazocine, shared the effects of U-50,488E in the gerbils. Naloxone and dynorphin 1-13, on the other hand, were without protective effects in the same ischemic model. The ischemic protective effects of U-50,488E may involve the kappa opioid receptor. PMID- 2995748 TI - In vitro study of immunoreactive corticotropin-releasing factor release from the rat hypothalamus. AB - Immunoreactive corticotropin-releasing factor (I-CRF) release from rat hypothalami was studied in vitro utilizing a perifusion of rat hypothalami and a rat CRF RIA. Basal release of I-CRF from the hypothalamus of adrenalectomized or hypophysectomized rats was higher than in that of normal rats. K+-induced I-CRF release was completely suppressed by omission of Ca++ from the medium. Dexamethasone suppressed I-CRF release from hypothalami, but not from median eminence (ME). C-AMP and angiotensin II had mild stimulatory effects on I-CRF release. These results suggest that 1) the feedback mechanism acts mainly on a higher level than ME, and 2) c-AMP and angiotensin II may be involved in CRF releasing mechanism(s). PMID- 2995749 TI - Plasma cortisol and corticosterone concentrations in the golden hamster, (Mesocricetus auratus). AB - Because some recent studies of hamster adrenocortical function have depended on older studies that may have been inadequate or misinterpreted, the present study re-examined plasma corticosterone and cortisol concentrations in hamsters under several conditions to determine which plasma glucocorticoid predominated in this animal. Sensitive radioimmunoassays were used to measure separately the two glucocorticoids in the basal condition, after adrenocorticotropin (ACTH) treatment, after acute stress, and after chronic stress. In the basal condition, corticosterone concentrations were 3-4 times higher than those of cortisol. After stimulation, this difference disappeared, but rarely were any hamster's cortisol levels higher than their corticosterone levels. Both ACTH and acute stress elevated plasma corticosterone and cortisol concentrations, but only plasma cortisol concentrations were elevated following chronic stress. The dissociation between cortisol and corticosterone concentrations after chronic stress suggests that the two glucocorticoid hormones in the hamster may be regulated independently. The data also indicate that both corticosterone and cortisol should be measured when assessing adrenocortical function in the hamster. PMID- 2995750 TI - Gamma linolenic acid attenuates cardiovascular responses to stress in borderline hypertensive rats. AB - The purpose of the present study was to investigate the effects of gamma linolenic acid (GLA) on cardiovascular responses to psychosocial stress (isolation) and to pressor hormones in the genetically borderline hypertensive rat (SHR X WKY). Adult male SHR X WKY were divided into two groups following five weeks of group housing. One group (GLA) received eight weeks constant flow osmotic pumps releasing 0.04 mg GLA in olive oil/kg-hr, while the second group received dummy pumps (DUM). One week following pump implantation, each group was divided into two subgroups and exposed to a four-week experimental period of either continued group housing (no stress) or isolation (stress). A two-week recovery period of group housing followed the experimental period. Blood pressure and heart rate were determined weekly by the tail cuff technique. At the end of the recovery period, animals in the no stress condition were anesthetized and received an arterial cannula for NOR and ANG infusion and direct BP recording. Then the responses to an ED50 of NOR and ANG were determined. All animals were then killed for determination of heart weight and adrenal weight. All groups had mean control period systolic BP values ranging from 143-146 mm Hg. In the no stress condition, neither GLA nor DUM altered BP over the course of the study. However, BP increased in the DUM group during all four weeks of the isolation period vs the control period (p less than 0.01), whereas BP increased only in week 1 in the GLA group (p less than 0.05). Heart rate increased during stress in the DUM group (p less than 0.05), but not in the GLA group. Vascular reactivity to NOR was unaffected by GLA administration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995751 TI - Liver disease and its effect on haemostasis during liver transplantation. AB - In a prospective study involving 25 consecutive adult orthotopic liver transplantation (OLT) patients, of whom 23 had cirrhosis, we have related pretransplantation recipient parameters to blood loss during transplantation. In phase 1 (explantation of diseased liver) blood loss was 0.1-7.2 1, in phase 3 (following restoration of the portal blood flow after implantation) 0.1-39.7 1, and total blood loss was 1.6-47.2, median 9.2 1. Five patients (20%) died from causes directly related to defective haemostasis during the operation. Pretransplantation cholinesterase, antithrombin III and albumin correlated most strongly with blood loss in phase 1; a history of ascites, antithrombin III and cholinesterase levels correlated with blood loss in phase 3, and a history of ascites, urinary sodium and antithrombin III with total blood loss. Cholestasis did not influence blood loss. Portal hypertension per se presumably played only a restricted role. A pretransplant 24-h urinary sodium excretion of 10 mmol or less and a serum sodium of 132 mmol/l or less were highly predictive of blood loss exceeding 10 1 during OLT. Urinary sodium determination under test conditions and serum sodium measurement should already be part of the assessment of potential OLT candidates by the referring hospital. PMID- 2995753 TI - Acquired immune deficiency syndrome (AIDS). PMID- 2995752 TI - DNA content of hepatocytes in various stages of liver cirrhosis. AB - In order to search for some parameters that would make it possible to predict a potentially high risk of evolution from liver cirrhosis to hepatocellular carcinoma (HCC), the DNA content of hepatocytes in patients with liver cirrhosis with or without development to HCC was investigated by means of microspectrophotometry in Feulgen-stained specimens. In patients without development of HCC in more than 5 years of follow-up, 90% or more of tested hepatocytes were diploid, whereas in patients with liver cirrhosis who developed HCC within 3 years the frequency histogram of nuclear DNA content was widely spread from diploid to hyperpolyploid with a small peak of triploid. Furthermore, in the latter group of patients, many of the binucleate cells had different DNA contents in each paired nucleus. In non-cancerous portions of HCC, the ploidy histogram of nuclear DNA content was widely spread from diploid to polyploid with different DNA contents of each paired nucleus in binucleate cells, without the small peak of triploid. These results suggest that the deranged cell kinetics of hepatocytes in cirrhosis may be considered to be a state of potentially high risk for the evolution of HCC, and that a patient with liver cirrhosis with such an abnormal hepatocyte DNA content should be followed up carefully for the early diagnosis of HCC. PMID- 2995754 TI - Prevention of human cytomegalovirus infection. PMID- 2995755 TI - [Preoperative irradiation of laryngeal cancer patients under different oxygen regimens]. AB - Radiation therapy under the conditions of hyperbaric oxygenation (HBO) using the method of mean fractionation (3.3 Gy 3 times a week up to the summary dose of 33 Gy) was employed to overcome tumor hypoxia and to raise selectively radiosensitivity of laryngeal cancer. A randomized study of 120 patients has shown that the use of radiation therapy under HBO conditions makes it possible to reduce the frequency of radiation reactions, to increase tumor resorption and the degree of radiation damage, to reduce the frequency of postoperative purulent complications, and to prevent the occurrence of early and late recurrences and metastases. PMID- 2995756 TI - [Hyperthermia and hyperglycemia in the radiation and combined treatment of locally disseminated and recurrent rectal cancer]. AB - The results of the use of short-term induced hyperglycemia and local UHF hyperthermia in radiation therapy of 27 rectal cancer patients aged 17 to 65 were analysed. A primary inoperable locally disseminated tumor process was noted in 8 patients, recurring cancer in 19. A noticeable tumor regression (over 50%) was observed in 7 out of 8 patients making a radical operation possible. With computerized tomography tumor regression was recorded by over 50% in 9 out of 19 nonoperated patients. The data obtained illustrate some potentialities of raising the efficacy of radiotherapy using radiomodifiers. PMID- 2995757 TI - [Use of metronidazole in the preoperative irradiation of stomach cancer patients]. AB - Altogether 70 patients received combined therapy for stomach cancer. A course of gamma-beam therapy (a summary focal dose of 20 Gy) combined with metronidazole was given at the first stage. Three-five days after the discontinuation of radiation therapy the patients were operated upon. Forty-nine (70%) patients were subjected to radical operation. The area of surgical intervention ranged from subtotal resection of the stomach to combined gastrectomy. Lethality after radical operations was 7.5%. Pronounced and extensive dystrophic changes were found in tumors at microscopic examination of resected specimens. PMID- 2995758 TI - [Modern contrast preparations for lymphography and their prospective performance]. PMID- 2995759 TI - Pattern recognition in diffuse lung disease. A review of theory and practice. PMID- 2995760 TI - Transferrin receptor as a target for antibody-drug conjugates. PMID- 2995761 TI - Autogenous regulation of synthesis of the replication protein in plasmid pSC101. AB - A 1.3-kb segment of plasmid pSC101 includes the replication origin (ori) and the gene (rep) encoding the 37 kilodalton (K) protein required for autonomous replication of the plasmid. The present work describes the regulation of the rep gene expression. The gamma promoters PR and PL fail to promote rep gene expression when located upstream of a sequence with dyad symmetry overlapping the rep promoter, whereas elimination of this sequence allows expression and results in over-production of the rep protein. When expression of trpA'-lacZ is controlled under the rep promoter, beta-galactosidase is produced without the lac inducer. However, this enzyme synthesis is effectively reduced when the complete rep sequence is provided in trans. A partial disruption of the sequence with dyad symmetry relieves the repression. These results suggest that expression of the rep gene is negatively regulated by its own product and that the sequence with dyad symmetry plays the role of a receptor site for the rep protein. PMID- 2995763 TI - Analysis of the flanking regions from different haemolysin determinants of Escherichia coli. AB - The haemolysin (hly) determinant of the plasmid pHly152 contains an IS2 element at 469 bp upstream of the hlyC gene. The sequence at the other (right-hand) end (RS) also shows multiple hybridization with the plasmid pHly152 and the chromosome of some Escherichia coli strains but the nucleotide sequence of this region does not reveal the typical properties of an IS element. Similar arrangements in the regions flanking the hly determinant are also found on various Hly plasmids from uropathogenic E. coli strains. Chromosomal hly determinants lack both flanking sequences (IS2 and RS) in the immediate vicinity of the hly genes. The sequences immediately upstream of the hlyC gene have been determined from several chromosomal hly determinants and compared with the corresponding sequence of the hly determinant of the plasmid pHly152. We show that these sequences, which contain one promoter (left promoter, phlyL) in all hly determinants tested, vary considerably although common sequence elements can still be identified. In contrast, only relatively few nucleotide exchanges have been detected in the adjacent structural hlyC genes. The A + T content of the 200 bp sequence upstream of hlyC is very high (72 mol% A + T) but even the structural hly genes show a considerably higher A + T content (about 60 mol%) than the E. coli chromosome on average (50 mol% A + T) suggesting that the hly determinant may not have originated in E. coli. PMID- 2995762 TI - Certain chromosomal regions in Streptomyces glaucescens tend to carry amplifications and deletions. AB - Streptomycetes are subject to a high degree of genetic instability. One manifestation of this phenomenon is the occurrence of tandemly reiterated DNA stretches within the chromosome. We describe the analysis of ten reiterated sequences observed in various ethidium bromide-treated streptomycin-sensitive and melanin-negative mutant strains of Streptomyces glaucescens. The repeated DNA units were 2.9 to 35 kb in length. No two sequences were identical. The amplified sequences occupied up to 45% of the total genomic DNA. Structural analysis of the cloned repeated DNA stretches by means of restriction enzymes and by cross hybridization revealed the presence of two chromosomal areas rich in DNA reiterations. In some cases reiterated regions were accompanied by nearby rearrangements. PMID- 2995764 TI - The expression of the MET25 gene of Saccharomyces cerevisiae is regulated transcriptionally. AB - The MET25 gene of Saccharomyces cerevisiae was cloned by functional complementation after transformation of a yeast met25 mutant. Subcloning of the DNA fragment bearing MET25 located the gene on a 2.3 kb region. The gene was formally identified by integration at the chromosomal MET25 locus. The cloned MET25 gene was used as a probe to measure the MET25 messenger RNA in a wild-type strain grown under conditions which promoted or failed to promote repression of MET25 expression. It was found that, under repression conditions, MET25 messenger RNA was reduced tenfold when compared with non-repression conditions. This suggests that the expression of MET25 is regulated transcriptionally. The direction of transcription, the size of the transcript and the position of the transcribed part of the gene were determined. Deletion mapping of the regulatory region was carried out. Deleted plasmids were introduced back into yeast cells and tested for their ability to complement met25 mutations and to promote regulation of expression of the MET25 gene by exogenous methionine. By this method the regulatory region was found to be confined to a 130 bp region. PMID- 2995765 TI - Cloning and expression of the genetically unstable tyrosinase structural gene from Streptomyces glaucescens. AB - The gene from Streptomyces glaucescens coding for an inducible tyrosinase was cloned using the low copy vector pIJ41 and the melanin-negative strain Streptomyces lividans TK23 as host. Hybridisation experiments as well as complementation studies showed that melC mutant strains carry large deletions of more than 10.5 kb, comprising the structural gene for tyrosinase, while melA and melB strains carry mutations in genes involved in the expression of tyrosinase activity. Strong DNA homology was found between the Streptomyces antibioticus and the S. glaucescens tyrosinase structural genes and both genes showed a similar regulation when introduced into melanin-negative hosts. While both tyrosinases exhibited clear induction in S. glaucescens, constitutive expression was observed in S. lividans. Northern blot experiments showed that tyrosinase expression is regulated at the transcriptional level and that the gene (822 bp) is part of a 2.3 kb transcript. The main start of the mRNA at about 475 bp upstream from the tyrosinase N-terminus was located by S1-mapping experiments. PMID- 2995766 TI - Autoregulation of the dnaA gene of Escherichia coli K12. AB - Regulation of the dnaA gene, which codes for an essential factor for the initiation of replication from the chromosomal origin, was studied in vivo using transcriptional and translational gene fusions. We found that the dnaA gene was autoregulated over a 30-fold range by the activity of dnaA protein. Expression from the dnaA promoter region of a dnaA"lacZ fusion was inhibited up to sevenfold by surplus dnaA protein and was stimulated up to fivefold upon thermoinactivation of the mutant protein in five different dnaA(Ts) strains. The autoregulation was found to be exerted at transcription from the major dnaA promoter and was eliminated by deletion of sequences around position -65 of this promoter where a 9-bp sequence, which is also found four times in the chromosomal origin, is located. PMID- 2995768 TI - The temperate B. subtilis phage phi 105 genome contains at least two distinct regions encoding superinfection immunity. AB - Two different PstI fragments of temperate phage phi 105 DNA are shown to confer superinfection immunity upon Bacillus subtilis when inserted into the multicopy cloning vector pE194 cop-6. The 2.3 kb PstI fragment I is located almost entirely within EcoRI fragment F and encompasses a region previously known to encode a repressor. The other fragment, PstI-E (4.3 kb) maps inside the EcoRI-B fragment, and allows an explanation of the clear-plaque phenotype of the deletion mutant phi 105DII:6c. The two regions can be distinguished functionally, since only the PstI fragment I product interacts with a specific phi 105 promoter-operator site. PMID- 2995769 TI - A restriction endonuclease map of Streptomyces phage VWB. AB - A physical map of the actinophage VWB has been constructed using the restriction endonucleases BglII, ClaI, EcoRI, EcoRV, HindIII, KpnI and SphI. Phage VWB, genome size 47.3 kb, propagates on Streptomyces venezuelae, and it can also lysogenise this species. The three BglII-generated fragments of VWB DNA were cloned in pBR322, and subsequently mapped. In this manner the restriction map of the VWB phage genome was constructed. PMID- 2995767 TI - The hexB mismatch repair gene of Streptococcus pneumoniae: characterisation, cloning and identification of the product. AB - A second gene involved in mismatch repair in Streptococcus pneumoniae, the hexB gene, has been characterised. The gene was cloned into a multicopy plasmid vector. The cloned hexB gene is expressed as judged by its ability to complement a chromosomal hexB- allele. Its direction of transcription and its functional limits were defined. Comparison of the proteins encoded by recombinant plasmids and by restriction fragments allowed us to identify an Mr 83,000 protein as the probable product of the hexB gene. We offer evidence that this gene together with the hexA gene is essential for repair of transition as well as frameshift mismatches. PMID- 2995770 TI - Malignant hyperthermia in a Saudi child. AB - This is the first report of a case of malignant hyperthermia (MH) from Saudi Arabia. MH was probably triggered by response to Halothane. The diagnosis was suspected by the clinical signs of tachycardia, severe rigidity, cyanosis and rising temperature. The case was successfully managed by vigorous cooling, dantrolene sodium and diuresis. PMID- 2995771 TI - Varicella-zoster virus (VZV)-specific polypeptides detected in cells treated with metabolic inhibitors. PMID- 2995772 TI - The polypeptides of human parainfluenza 1 (HA-2) virus and their antigenic relationships to those of Sendai virus (HVJ). PMID- 2995773 TI - RISH IV. Immunoglobulin diversity. AB - RISH considers that cell surface components involved in like cell identification are not involved in the structure of the plasma membrane per se and are attached to a part of their mRNA. the mRNA then acts as a template for the synthesis of DNA. Thus the component at the cell surface is attached to an RNA/DNA receptor. If there is a conformational change in the component (antigen) this will cause a distortion in its RNA/DNA receptor. This distortion is then detected by a tissue specific T lymphocyte which removes all or part of the RNA/DNA receptor from the aberrant cell and the lymphocyte then undergoes replication. During this process receptor RNA/DNA is incorporated into the daughter lymphocyte which becomes a B lymphocyte/plasma cell producing immunoglobulin. The initial tissue specific T lymphocyte becomes a dual functional helper/suppressor cell. The B lymphocytes use the RNA from the RNA/DNA receptor to synthesize the variable region of the first antibody, IgM1. This antibody (IgM1) does not react with the antigen, ie. the distorted component, or the receptor RNA, but with receptor DNA. The DNA of the receptor base pairs with its complementary strand in the B lymphocyte, and the complementary DNA acts as a template for mRNA synthesis. This results in the production of IgM2 and IgG that can bind the antigen and receptor RNA. These antibodies (IgM1, IgM2 and IgG) when endocytosed by the stimulating cell will also complex cytoplasmic mRNA and nuclear DNA and prevent the synthesis of the antigen that initiated the immune response. If other classes of antibodies are to be produced they will follow a similar pattern (IgM1, IgA and IgG or IgM1, IgE and IgG). From the codons of the known amino acids, the codons for amino acids from translation of the complementary DNA strand have been calculated. The amino acids derived from the complementary codons are considered to represent sequences of amino acids in the antigen as represented by the DNA of an RNA/DNA receptor. For these sequences of amino acids, each has a complementary amino acid as defined by the normal codon. These complementary amino acids are then used in the synthesis of the variable region of the antibody. PMID- 2995774 TI - Hypoglycaemia and insulin resistance in uraemia associated with insulin fragments. AB - The pathogenetic role of insulin fragments in spontaneous hypoglycaemia and insulin resistance in chronic renal failure is discussed. Four different evidences are presented. In our studies, a dialysable 'toxin' with insulin-like actions was found in uraemia. Indeed small polypeptide fragments of hormones with potent activities have been recently identified in uraemic plasma. The varying bioactivities of different insulin fragments can explain the coexistence of insulin resistance and hypoglycaemia in these patients. As shown by our studies, removal of these fragments by haemodialysis results in normalisation of metabolic disturbances. However actual proof is difficult with the present technological problems. PMID- 2995775 TI - The use of drugs for the inhibition of prostaglandin or leukotriene synthesis from arachidonic acid is theoretically hazardous. AB - It would appear that it has become almost common practice to regard arachidonic acid (AA) as the sole precursor of eicosanoids. The fact that both dihomogamma linolenic acid (DGLA) and eicosapentaenoic acid (EPA) give rise to distinct families of eicosanoids is commonly almost completely ignored. Elevated tissue levels of AA eicosanoids have been found in and have been implicated in the etiology of a number of diseases. Drugs which selectively block AA mobilization or its eicosanoid metabolism have therefore been developed for therapeutic use in these conditions. The fact that such drugs will also simultaneously block the eicosanoid metabolism from DGLA as well as from EPA is also commonly ignored. It is suggested that the profoundly adverse side-effects displayed by some of these drugs, resulting in some instances in their withdrawal from use, could be the direct result of their concomitant action of interfering with the eicosanoid metabolism of DGLA and EPA. It is further suggested that, before the interactions between the eicosanoids derived from AA and those derived from DGLA and EPA are understood, the use of drugs for the manipulation of AA eicosanoid metabolism in isolation, could be hazardous. This implies that all such drugs currently in use are to be regarded as experimental and provisionally toxic in terms of their effects on the whole system of eicosanoid metabolism. Thus even drugs which have been passed by the FDA and similar Drug Control Councils require total re evaluation especially in view of the fact that the non-steroidal anti inflammatory drugs are often prescribed for chronic conditions which require therapy for several years.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995776 TI - Entropic DNA. AB - The presence of mobile genetic elements without apparent biological function within genomes requires explanation. It is argued that consideration of DNA as an open thermodynamic system leads to the simple hypothesis that mobile genetic material increases the internal entropy thereby lowering the free energy of the DNA relative to DNA of the same size. This phenomenon enhances the survival of such sequences by simple structural stabilization. The consequences of this idea are discussed in terms of the proposed Bekenstein limit to the entropy to energy ratio of thermodynamic systems and biological entropy/information flow. PMID- 2995777 TI - False-positive anti-HTLV-III. PMID- 2995778 TI - Adrenal corticosteroid replacement therapy. PMID- 2995779 TI - Signal strength on a 0.15-T magnetic resonance imager. AB - Signal strength in magnetic resonance (MR) images made on a 0.15-T imager was calculated for spin-echo (SE) and inversion-recovery with echo (ISE) pulse sequences and was compared to measurements of signal strength for aqueous MnCl2 solutions whose T1 and T2 relaxation times encompassed the range of values commonly found for tissues. Although measured signal strength generally agreed with calculated signal strength (correlation coefficient = 0.996 for both SE and ISE), significant reductions in measured strength (greater than 15%) were sometimes observed. Diffusion alone could not account for this discrepancy. Further experiments showed that imaging gradients reduced the effective T2 estimated from the imager. In addition, SE pulse sequences with short repetition times exhibited a reduction in signal strength. PMID- 2995781 TI - [Use of calcium hydroxyapatite in oromaxillofacial surgery. II. The treatment of mandibular atrophy. The surgical technic and case presentation]. PMID- 2995782 TI - Electrodiagnosis of radiculopathies. AB - Electrophysiology assesses function and should be considered complementary to, not as competing with, myelography, which assesses structure. Electromyography, being devoid of morbidity and significant side effects, should precede invasive radiology: myelography and contrast-enhanced computerized tomography (CT). Needle electromyography is the most useful electrophysiologic test when evaluating radiculopathies. Presence of denervation (fibrillation and positive sharp waves) indicates axonal interruption, a major determinant to the completeness and speed of recovery. It occurs first in the paraspinal muscles. In the first few days before denervation can develop, abnormalities of motor unit recruitment are most helpful in differentiating weakness due to impaired conduction as opposed to pain or voluntary lack of effort. An abnormal paraspinal EMG is electrophysiologic proof of a lesion at or proximal to the spinal root. Such abnormalities persist for long periods following surgery, limiting the use of EMG in the postoperative period. Conventional conduction studies are usually normal in radiculopathies. An abnormal or unrecordable sensory nerve action potential should suggest a "ganglionopathy" or double crush syndrome. F wave, H reflex, and somatosensory evoked potential studies, although having a lower diagnostic yield than needle EMG, can be helpful in confirming some root lesions early in the disease. The diagnostic value of these tests might be increased by consideration of characteristics other than latency such as their amplitude and dispersion. PMID- 2995784 TI - Electrodiagnostic evaluation of patients with myasthenia gravis and related disorders. AB - The technique of repetitive nerve stimulation was developed as a means of confirming a diagnosis of MG. The application of this test in the clinical evaluation of patients with muscle weakness has identified other disorders of neuromuscular transmission such as LEMS, infantile botulism, and several forms of congenital myasthenia. Repetitive nerve stimulation to several different nerves, before and after exercising the muscles, usually permits identification of disorders of neuromuscular transmission more readily than very detailed study of one muscle. There are other useful electrodiagnostic tests, but in most cases, repetitive nerve stimulation of weak muscles will permit the identification of a disorder of neuromuscular transmission. PMID- 2995783 TI - Electrodiagnosis of plexopathies. AB - The pathophysiologic processes underlying plexus lesions and their recognition by electromyographic examination are detailed in this article. The nerve conduction studies most helpful for localization of plexopathies are discussed, and the electromyographic findings of the most commonly encountered plexopathies are described. PMID- 2995785 TI - [Diagnostic value of angiography in differentiating the degree of the depressed type of gastric cancer]. AB - Endoscopy and superselective angiography has been performed in 68 preoperative patients with the depressed type gastric cancer. The results are summarized as follows. Differentiation of the cancers into the early and the advanced was correct in 91%, and classification of the cancers into 3 groups (1 ; m, 2 ; sm, 3 ; advanced) was correct in 72% by endoscopy. Differentiation of the cancers into the early and the advanced was correct in 90%, and classification of the cancers into 4 groups (1 ; m X sm, 2 ; pm, 3 ; ss X se, 4 ; sei) was correct in 81% by the angiography. The rate of misdiagnosis, taking the early simulating advanced gastric cancer as the early, was 20% by the endoscopy, as compared to 8% by the angiography. Classification of the early simulating advanced gastric cancers into 3 groups (1 ; pm, 2 ; ss X se, 3 ; sei) was correct in 68% by the angiography. Excavated type cancer was likely to be overestimated as to the depth of invasion by the angiography. Poorly differentiated type, scirrhous type, and infiltrative growth with an ill defined border were found difficult to be correctly evaluated by both endoscopy and angiography. Preoperative angiography was useful for appropriate surgical therapy of the gastric cancer. PMID- 2995787 TI - [Reevaluation of the histological classification of breast cancer--a presentation of a new histological classification considering the relationship between scirrhous tendency and postoperative prognosis]. AB - The present study was designed to evaluate the correlation between the histology and the prognosis of the patients with breast cancer. In this study, 1271 cases with breast cancer were retrospectively studied and an attempt of a new histological classification was presented, which was intended to reflect the prognosis more exactly than the ordinarily used classification by Japan Mammary Cancer Society. Japan Mammary Cancer Society classifies infiltrating carcinoma into 3 types; papillotubular, medullary tubular and scirrhous carcinoma. In this study, when scirrhous interstitial infiltration was observed in papillotubular or medullary tubular carcinoma, even if scirrhous tendency was observed only in small part, the patients' prognosis was as poor as scirrhous carcinoma. The prognosis of common type of infiltrating carcinoma with scirrhous tendency corresponded to that of one grade advanced stage without scirrhous tendency. Considering these results and clinical usefulness, common type of infiltrating carcinoma should be classified to 2 types, namely with or without scirrhous tendency in order to reflect the prognosis and to correspond to WHO classification. PMID- 2995786 TI - [Morphological study of carcinoma of the gallbladder: its differences between calculous and acalculous carcinoma]. AB - The most characteristic phenomenon in patients with carcinoma of the gallbladder is frequent coexistence of gallstones. In this study, morphological differences of carcinoma of the gallbladder in between calculous and acalculous cases is investigated. This study involved fifty-three cases (eleven early carcinoma and forty-two advanced) that had been surgically treated at the Hamamatsu Medical Center Hospital during the past ten years. Nine of 11 early carcinomas were calculous and two, acalculous. All calculous carcinomas showed grossly superficial type and two acalculous, polypoid type. All of them were histologically differentiated adenocarcinoma. In advanced carcinoma, thirty-one of 42 cases were calculous and eleven, acalculous. Fifteen of 31 calculous cases showed polypoid type and sixteen, diffuse infiltrative type. In all cases with the latter type, the cystic duct was completely obstructed by impacted gallstones. All calculous carcinomas were histologically belonged to differentiated adenocarcinoma, mucinous carcinoma or adenosquamous carcinoma. On the other hand, six of 11 acalculous carcinomas revealed grossly polypoid type, and histologically differentiated adenocarcinoma or mucinous carcinoma. The remaining five were of diffuse infiltrative type, and of poorly-differentiated adenocarcinoma or signet-ring cell carcinoma. From these data, it may be highly suggestive that differentiated adenocarcinoma or adenosquamous carcinoma of histologic type irrespective of it's gross type is characteristic of calculous carcinoma, and poorly-differentiated adenocarcinoma or signet-ring cell carcinoma possessing primary diffuse infiltrative growth, of acalculous. The majority of the superficial type in early carcinoma and the diffuse infiltrative type in advanced are considered as a secondary modified gross appearance by direct or indirect effects of coexistent gallstones onto the main tumor. PMID- 2995780 TI - Genetic map of Saccharomyces cerevisiae, edition 9. PMID- 2995788 TI - Guanine nucleotides modulate the function of chemotactic cyclic AMP receptors in Dictyostelium discoideum. AB - Guanosine di- and triphosphates specifically decrease the affinity of chemotactic cAMP receptors in isolated Dictyostelium discoideum membranes. The K0.5 was increased from 50 nM to 150 nM. Receptors were shown to be heterogeneous in dissociation kinetics. In the absence of guanine nucleotides three dissociation processes could be resolved, having first order rate constants of 8.7 X 10(-4), 1.3 X 10(-2), and higher than 0.1 s-1. Guanine nucleotides decreased the affinity for cAMP by transforming the slowest dissociating receptor form (KD is 8 nM) to forms dissociating more rapidly. Our data indicate that a guanine nucleotide binding protein (G-protein) is involved in the transduction of the cAMP signal in D. discoideum. PMID- 2995790 TI - [Interaction (integration and excision) of the pRP19.6 plasmid, a derivative of the RP1 plasmid containing duplicated sequence IS21, with the chromosome of Escherichia coli K12]. AB - A pRP19.6 plasmid is the derivative of the temperature sensitive RPlts12 plasmid and contains a duplicated IS21 (IS8) element. Using temperature sensitive pRP19.6 replication, Hfr strains have been obtained by integration of the plasmid into the chromosome of E. coli rec+ and recA- cells and their properties were studied. According to the results obtained, pRP19.6 insertion into the genome of the rec+ bacteria IS reversible, and its integration into the chromosome of the recA- bacteria produced the stable Hfr strains. To elucidate the mechanism of pRP19.6 excision from the bacterial chromosome, plasmids of R+ transconjugates generated with a low frequency in the crosses between the stable Hfr strains and the rec+ recipients were analyzed. It was shown that the stable Hfr clones might produce stable R1 plasmids as well as a family of deletion KmsTra- derivatives of the pRP19.6. The structure of the KmsTra- was investigated and the mechanism of their formation was proposed. In the light of the data obtained, prospects of pRP19.6 practical application are discussed. PMID- 2995789 TI - Cyclic AMP and theophylline enhance DNA synthesis in L cells stimulated with anti actin IgG and [(IgG)2 protein A]2 complex by recruiting cells into S-phase. AB - Surface binding of anti-actin IgG alone or in a Mr = 716 000 [(IgG)2 Protein A]2 complex results in a stimulation of DNA synthesis and cell growth in L cells. Cyclic-AMP (0.01-1.0 mM) added to such cell cultures augmented DNA synthesis as measured by incorporation of [3H]thymidine into DNA. Theophylline (0.1-1.0 mM), a phosphodiesterase inhibitor which prevents enzymatic breakdown of cAMP, had similar effects, but cGMP (0.01-1.0 microM) reversed the effects of cAMP and theophylline upon DNA synthesis. Analysis of the cell cycle by flow cytometry revealed that antibody produced a shift (7%) of cells from the G1-phase to the S phase (DNA-synthetic) of the cell cycle at 72 hr of incubation. Addition of cAMP (0.5 mM) to cell cultures, however, produced significant shifts of antibody stimulated cells from G1-phase to S-phase at all time points measured, i.e., 24 (12%), 48 (22%), 72 hr (23%). Thus, antibody recruited cells into S-phase of the cell cycle and cAMP greatly augmented the effect. These observations suggest that the mechanism of activation of L cell growth by antibody to surface antigens involves a recruitment of cells into the DNA-synthetic phase and that the effect may be mediated by cAMP. PMID- 2995791 TI - [Molecular cloning of MuLV proviruses integrated into the genome of mouse erythroleukemia cells. I. Characteristics of endogenous proviruses]. AB - The library of genes was obtained from erythroleukemic AKR cells (C-1), that were maintained as suspension culture. Thirty four clones that had homology with 60 70S RNA of Rauscher Leukemia virus (RLV) were separated from this library. The restriction mapping was carried out with 14 clones, that contained most extensive proviral sequences. One clone (107) contains proviral sequences that are derived from one of the components of the RLV complex. The other 13 clones contain sequences of endogenous xenotropic viruses. The endogenous retroviral sequences obtained differ in restrictive maps from proviruses of ecotropic and xenotropic infectious endogenous MuLV and, apparently, might be attributed as non-inducible infectious xenotropic MuLV of class III. Some of the cloned retroviral sequences had symmetrical structure, that is typical for integrated proviruses, i. e. these sequences were separated from flanking cellular ones by long terminal repeats. All investigated retroviral sequences are deletion mutants of MuLV proviruses. It was shown that the inner regions of proviruses diverged more than the long terminal repeats. The expression of the main inner MuLV polypeptide (p30) was detected in NIH 3T3 cells, transfected with DNA of some clones. PMID- 2995792 TI - [Structuro-functional organization of the SV40 virus chromosome. IV. Specific organization of regulatory sequences of the SV40 genome in the mini-chromosome]. AB - Using previously described technique of hybridization end-labeling, we analysed nucleosomal organization of the regulatory region of SV40 minichromosome. We showed that DNAase II, in spite of certain specificity observed on the naked DNA, cut the minichromosome in a highly specific manner with the major hypersensitive site inside the enhancer. This hypersensitivity and that to micrococcal nuclease were not found when the chromosome of mature SV40 virions was tested. PMID- 2995794 TI - [Molecular organization of plasmid R906 (Inc P-1)]. AB - Genetic and restriction (for enzymes EcoRI, BamHI and HindIII) maps of the relatively broad host range plasmid R906 are constructed. There are two non essential regions on the R906 DNA which can be deleted and cloned. Non-essential regions confer a resistance to different agents and restriction sites are clustered in these regions. Essential and conjugativity genes are located in two other DNA regions approximately at 0-23 and 29-44 kb of the R906 map. These large regions share a high level of homology with Inc-1 group plasmids R751 and RP4 according to Southern-blot hybridization and heteroduplex analyses. A transposon like structure is found on the R751 DNA among R751/R906 heteroduplex molecules. This transposon of total length 5.1 kb has 1.4 kb inverted repeats at the ends. Bla genes of R906 and RP4 plasmids do not have homologous sequences. Data evidence that IncP-1 group plasmids irrespective to their original bacterial source and range of coded antibiotic resistance have very similar molecular organization. The role of possible factors which are responsible for the broad host range property of the IncP-1 group plasmids is discussed. PMID- 2995793 TI - [Interaction of the peptide antibiotic bacitracin with DNA and synthetic polynucleotides]. AB - The aim of the present paper was to study the specific character of interaction of peptide antibiotic bacitracin with DNA and to estimate the interaction constant. The influence of bacitracin on bacteriophage DNA restriction by HindIII and SmaI endonucleases was studied. The possibility of arranging the polynucleotide template by small ligands was shown. PMID- 2995796 TI - Reverse relationship between lysosomal-enzyme release and active-oxygen generation in stimulated human neutrophils. AB - The relationship between the generation of active species of oxygen (O-2, H2O2 and OH.), chemiluminescence, and the release of lysosomal enzymes (lysozyme, alpha-mannosidase and beta-glucuronidase) was examined in human neutrophils stimulated with opsonized zymosan in the presence or absence of active-oxygen scavengers. In the absence of scavengers, increasing zymosan concn stimulated a marked increase in active-oxygen production in a concn-dependent manner and a less rigorously dose-dependent increase in enzyme secretion. Addition of OH. and/or 1O2 scavengers (benzoate, 1,4-diazo-bicyclo-2,2,2-octane or xanthine) caused a marked increase in enzyme release and a decrease in the generation of active-oxygen species except O-2 and H2O2. These findings suggest that exocytosis of lysosomal enzymes by stimulated neutrophils might be attenuated by the active generation of OH. and chemiluminescence. Superoxide dismutase (SOD) at low concns inhibited lysosomal enzyme release while promoting OH formation; and SOD at high concns decreased OH. and O-2 formation and chemiluminescence, accompanied by higher levels of lysosomal enzyme release. Catalase showed an effect similar to that of SOD. Our data suggest that the reduction by scavengers of active-oxygen levels, particularly of the species detected in the OH. and chemiluminescence assays, results in an increase in lysosomal enzyme release. PMID- 2995795 TI - Quantitation of specific antibodies bound to feline leukemia virus in the plasma of pet cats. AB - A method is described for determining levels of circulating immune complexes (CIC) composed of feline leukemia virus (FeLV) antigens and corresponding antibodies in plasma of persistently-infected pet cats. The procedure is based on the ability of high-titered heterologous anti-FeLV serum to chase cat anti-FeLV IgG from dissociated CIC by successfully competing for binding of free antigen. The eluted cat antibody is then collected and quantitated. In a study of cats in the process of clearing persistent FeLV infections, measured levels of FeLV specific CIC correlated well with fluctuating levels of free FeLV antigen and antibody. The Raji cell assay for CIC in those cats was of comparatively little value in following the clearance of the virus, presumably because that assay does not distinguish between CIC containing viral and those containing non-viral antigens. The method described can be adapted to studies of specific immune complexes associated with a variety of syndromes, provided that the antigen eliciting the immune response is known. PMID- 2995797 TI - [Treatment of juvenile rheumatoid arthritis]. AB - Recently interest in pediatric rheumatology has increased. This review tries to summarize our knowledge about pharmacological mechanisms, as well as practical application and adverse of antirheumatic drugs. In addition, forms of treatment which sofar lack any scientific backing are mentioned and discussed. Various data about efficacy and side effects in children are poor or lacking, and current treatment in children is based on analogy conclusions derived from experiences with adult RA. The therapeutic concept presented in this article offers a good chance to improve the functional status of the rheumatic child. However, one should be aware that several controlled trials with antirheumatic drugs in children still have to be performed. Uncertainties have to be replaced by knowledge. This is true also for the surgical treatment mentioned. PMID- 2995798 TI - A leukocyte subset bearing HLA-DR antigens is responsible for in vitro alpha interferon production in response to viruses. AB - In this report, we characterize the major human peripheral blood nonadherent mononuclear cell subset that is responsible for the production in vitro of alpha interferon (alpha IFN) in response to influenza, Sendai, and Newcastle disease viruses. Using a panel of anti-monocyte, anti-B, anti-T and anti-natural killer cell monoclonal antibodies to purify and recover both positive and negative cell populations, we show that the major alpha IFN-producing cells are HLA-DR(+) cells with no other surface markers characteristic of either lymphocytes or myelomonocytic cells. These cells copurify, on Percoll density gradients, with cells mediating NK activity, but the cell population responsible for alpha IFN production can be distinguished unambiguously from NK cells based on the former's reactivity with an anti-HLA-DR monoclonal antibody, the lack of reactivity with antibodies that detect the low-affinity receptor for IgG on human natural killer cells and granulocytes, and the inability to mediate spontaneous cytotoxicity. Double immunofluorescence assays indicate that cells of this subset, the lineage of which is as yet undetermined but which might be related to dendritic cells, constitute a minor proportion (approximately 1-1.5%) of the nonadherent mononuclear cells from healthy donors. PMID- 2995799 TI - The chromosome-breaking activity of the restriction endonuclease Alu I in CHO cells is independent of the S-phase of the cell cycle. AB - The restriction endonuclease Alu I induces chromosomal aberrations in living Chinese hamster ovary (CHO) cells. Multiple fixation times reveal that the chromosome-breaking activity of Alu I is similar to that of ionizing radiation in that it is independent of the S-phase of the cell cycle. These results indicate that DNA double-strand breaks are the ultimate lesions for the production of chromosomal aberrations in all stages of the cell cycle. PMID- 2995800 TI - Metabolic epidemiology of colon cancer: fecal mutagens in healthy subjects from rural Kuopio and urban Helsinki, Finland. AB - Fecal mutagenic activity and dietary pattern of rural and urban Finnish population groups with distinct risk for the development of colon cancer were studied in a low-risk population in rural Kuopio and an intermediate-risk population in urban Helsinki. The average daily intake of protein and fat was the same in the two groups but the frequency of consumption of whole-grain cereals and whole-grain bread, as well as the amount of fiber from the bread were higher in Kuopio as compared to Helsinki. Fecal samples collected for 2 days were incubated under anaerobic conditions at 37 degrees C for 96 h, extracted with hexane: peroxide-free diethyl ether, partially purified on a silica Sep-Pak cartridge, and assayed for mutagenic activity using the Salmonella typhimurium/mammalian microsome system. The fecal mutagenic activity was observed with the tester strains TA98 and TA100 with and without microsomal activation in both the population groups. The percentage of samples showing fecal mutagenic ratio greater than 3 with TA98 and TA100 with microsomal activation, was higher in volunteers from Helsinki than in Kuopio. PMID- 2995801 TI - The mutagenicity of bile acids using a fluctuation test. AB - The mutagenicity of bile acids was detected by a fluctuation test using Salmonella typhimurium TA100 and TA98 as tester strains. Cholic acid, chenodeoxycholic acid, deoxycholic acid and ursodeoxycholic acid were mutagenic in this test while lithocholic acid was not. The mutagenicity of the bile acids on a molar basis was roughly one-fourth that of methyl methanesulfonate, a moderately potent mutagen. Epidemiological studies have shown a high correlation between levels of bile acids excreted and colon cancer. However, no evidence has previously been reported showing that bile acids are mutagenic. Our results suggest that bile acids may be important in the etiology of colon cancer. PMID- 2995803 TI - In vitro assays of in vivo exposure to cyclophosphamide: induction of sister chromatid exchanges in peripheral lymphocytes, bone-marrow cells and in cultured cells exposed to plasma. AB - The in vivo genotoxic potential of cyclophosphamide (CY) was assessed by sister chromatid exchange (SCE) induction after removal and in vitro culture of circulating peripheral lymphocytes and bone marrow from CY exposed male, Fischer 344 rats. Plasma was simultaneously obtained and assessed for genotoxic activity by incubation with 4 cultured mammalian cell lines: HepG2, H4-II-E (H4), V79 and IMR-90. These 4 cell types were used to help discriminate the role of metabolism in generating SCE-inducing factors in plasma. HepG2 and H4 have been shown to metabolize certain xenobiotics while V79 and IMR-90 do not. An in vivo dose response to CY at doses of 0, 5, 10, 20, 30 and 50 mg CY/kg assayed 1 h post-i.p. injection was performed. Phytohemagglutinin (PHA)-stimulated lymphocytes showed a dose-related increase in SCE up to 66.7 SCE/cell at 20 mg/kg (30 mg/kg was cytotoxic). Bone marrow also showed an SCE increase to 34.8 SCE/cell at 10 mg/kg (higher doses were cytotoxic). Plasma induced a dose-dependent SCE increase in the 4 cultured cell lines at all tested doses indicating the presence of direct acting SCE-inducing metabolites of CY. A time course study using 20 mg CY/kg indicated peak plasma levels of CY genotoxic activity at approximately 0.5-1 h post-injection. By 3 h, the level of genotoxic activity in plasma was considerably reduced. Lymphocytes, however, showed a cumulative increase in SCE to 74.7 SCE/cell after 3 h of exposure. These in vivo exposure--in vitro assay techniques may be useful and facile systems with which to develop an integrative testing system for assessing the in vivo genotoxicity of a chemical. PMID- 2995802 TI - Fecal mutagens and Bacteroides fragilis levels in the feces of dimethylhydrazine treated rats: influence of diet. AB - In a study designed to investigate the effects of dietary synergisms on 1,2 dimethylhydrazine (DMH)-induced rat colon carcinogenesis, fecal pellets were examined for the presence of direct-acting fecal mutagens and levels of Bacteroides fragilis group organisms. Intraperitoneal injections of DMH at 10 mg/kg were given for 16 weeks (weeks 3-18) to 160 male F344 rats consuming 4 supplemental dietary factors in all possible combinations. The dietary factors examined were wheat bran (15%), cholesterol (1%), beef tallow (18%) and indole-3 carbinol (IC) (0.1%). Feces were collected 3, 10, 17, 24 and 31 weeks after commencing the dietary treatments and dichloromethane extracts were assayed using the Salmonella typhimurium TA100 without metabolic activation. The numbers of B. fragilis group organisms were enumerated in feces collected at the same time. Most feces samples were negative for mutagens but extracts from weeks 17-31 showed a significant mutagenic response from the IC factor in the diet. The fecal levels of B. fragilis were significantly increased by the inclusion of cholesterol in the diets. The B. fragilis counts and fecal mutagen production were not correlated (r = 0.09), although species of the B. fragilis group have been implicated in the production on human fecal mutagens. PMID- 2995804 TI - Electrophysiology of the facial nerve in hemifacial spasm: ectopic/ephaptic excitation. AB - Pathologic and pathophysiologic findings in hemifacial spasm are reviewed in connection with recent theoretical and experimental studies of ectopic/ephaptic excitation. The intracranial segment of the normal facial nerve is ensheathed by an arachnoid membrane only and shows no fascicular organization. In hemifacial spasm, this segment shows signs of demyelination. Several electrical phenomena relating to ectopic excitation, ephaptic transmission between facial nerve fibers, and autoexcitation can be reproduced in clinical electrophysiologic studies of hemifacial spasm. These abnormalities gradually disappear after facial nerve decompression in the cerebellopontine recess. It is concluded that the normal facial nerve is vulnerable to minor compression, the primary pathophysiologic mechanism in hemifacial spasm is ectopic/ephaptic excitation due to compression and demyelination of the intracranial segment of the facial nerve, and the facial nerve in hemifacial spasm is a useful model for the study of ephaptic transmission, which has provided new information about the resolution of abnormal electrical events after decompression. PMID- 2995805 TI - Electrophysiologic study in benign human botulism type B. AB - Conventional electromyography (EMG) and single fiber EMG (SFEMG) were performed in a 64-year-old diabetic woman with mild type B botulism. The main clinical signs were autonomic dysfunction and cranial nerves paresis. Conventional EMG was normal, except for small changes that were consistent with mild mixed peripheral neuropathy in the lower limbs and were related to diabetes. Repetitive stimulation and results of single stimulus before and after full effort were normal. SFEMG showed increased jitter and impulse blocking in clinically normal muscles. The jitter was frequency dependent and improved at a higher innervation rate. Impulse blocking in potentials with only slightly increased jitter was found. The follow-up showed improvement of the jitter in agreement with clinical recovery. Jitter abnormalities were recorded after 16 weeks, when clinical signs of botulism had been reversed to normal. Motor unit fiber density increased progressively, and on examination at 8 weeks, some potentials showed very high jitter values. Both findings might suggest new endplate formation, perhaps due to ultraterminal sprouting development. PMID- 2995806 TI - Accelerated conversion of androgen to estrogen in plasma-cell dyscrasia associated with polyneuropathy, anasarca, and skin pigmentation. PMID- 2995807 TI - Risk of localized and widespread endometrial cancer in relation to recent and discontinued use of conjugated estrogens. AB - In a case-control study of the risk of adenocarcinoma of the endometrium in relation to conjugated-estrogen use, we found that 31 per cent of 425 women with endometrial cancer and 15 per cent of 792 controls reported having used conjugated estrogens; the rate-ratio estimate was 3.5 with a 95 per cent confidence interval of 2.6 to 4.7. For use that lasted at least one year, the rate-ratio estimate for Stage I or II cancer was 5.2 (95 per cent confidence interval, 3.7 to 7.2), and for Stages III and IV combined it was 3.1 (1.5 to 6.4). Among women who had used estrogen for at least one year and then discontinued it, the risk of endometrial cancer remained significantly elevated even after estrogen-free intervals of over 10 years. The findings suggest that long-term use of conjugated estrogen increases the risk of both localized and widespread endometrial cancer. The data also suggest that women who have taken conjugated estrogen for one or more years remain at increased risk for at least 10 years after they discontinue use. Such women should be considered for long term gynecologic surveillance. PMID- 2995808 TI - Postmenopausal estrogen use, cigarette smoking, and cardiovascular morbidity in women over 50. The Framingham Study. AB - We studied the effect of estrogen use on morbidity from cardiovascular disease in 1234 postmenopausal women, aged 50 to 83 years, participating in the Framingham Heart Study's 12th biennial examination (index examination). The medication history recorded at biennial examinations 8 through 12 was used to classify the degree of estrogen exposure before eight years of observation for cardiovascular morbidity and mortality. Despite a favorable cardiovascular risk profile and control for the major known risk factors for heart disease, women reporting postmenopausal estrogen use at one or more examinations had over a 50 per cent elevated risk of cardiovascular morbidity (P less than 0.01) and more than a twofold risk for cerebrovascular disease (P less than 0.01) after the index examination. Increased rates for myocardial infarction (P less than 0.05) were observed particularly among the estrogen users who smoked cigarettes. Conversely, among nonsmokers estrogen use was associated only with an increased incidence of stroke (P less than 0.05). No benefits from estrogen use were observed in the study group; in particular, mortality from all causes and from cardiovascular disease did not differ for estrogen users and nonusers. PMID- 2995809 TI - When research results are in conflict. PMID- 2995811 TI - Alternative sites for screening blood for antibodies to AIDS virus. PMID- 2995810 TI - Sensitivity of the parathyroid hormone-1,25-dihydroxyvitamin D axis to variations in calcium intake in patients with primary hyperparathyroidism. AB - Previous studies have demonstrated a spectrum of parathyroid responsivity to alterations in the extracellular calcium concentration in patients with primary hyperparathyroidism, but studies employing physiologic amounts of calcium have not, to our knowledge, been reported. We studied 18 unselected patients with primary hyperparathyroidism at the lower (400 mg) and upper (1000 mg) limits of a normal dietary intake of calcium. The diet containing high-normal amounts of calcium induced only a slight increase in 24-hour calcium excretion (from 281 to 337 mg per day) yet was associated with significant reductions in fasting serum levels of immunoreactive parathyroid hormone (from 60 to 50 nleq per milliliter; P less than 0.001), nephrogenous cyclic AMP (from 3.52 to 2.63 nmol per deciliter of glomerular filtrate; P less than 0.001), and plasma levels of 1,25 dihydroxyvitamin D (from 74 to 58 pg per milliliter; P less than 0.001). A wide spectrum of responses was observed, with some patients appearing to have essentially autonomous parathyroid function and others having marked suppressibility (up to 50 per cent) of the parathyroid hormone-vitamin D axis. We conclude that parathyroid function may be suppressed by dietary calcium in some patients with primary hyperparathyroidism. PMID- 2995812 TI - Radon and lung cancer in Swedish miners. PMID- 2995813 TI - Association of Epstein-Barr virus with lethal midline granuloma. PMID- 2995814 TI - Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. AB - A 'rescuable' plasmid containing globin gene sequences allowing recombination with homologous chromosomal sequences has enabled us to produce, score and clone mammalian cells with the plasmid integrated into the human beta-globin locus. The planned modification was achieved in about one per thousand transformed cells whether or not the target gene was expressed. PMID- 2995815 TI - Expression of human HPRT in the central nervous system of transgenic mice. AB - Severe deficiency of hypoxanthine phosphoribosyltransferase (HPRT) in man results in the Lesch-Nyhan syndrome, an X-linked neurological disorder characterized by mental retardation, choreoathetosis and a compulsive tendency towards self mutilation. Although the HPRT gene is normally constitutively expressed in all tissues at low levels, expression is elevated approximately fourfold in several regions of the central nervous system, particularly in the basal ganglia. The relationships between HPRT deficiency, tissue-specific alterations of nucleotide metabolism and the neuropathology of the Lesch-Nyhan syndrome remain unclear. Here we have microinjected recombinant molecules containing human HPRT (hHPRT) complementary DNA, the mouse metallothionein-I (MT-I) promoter and the 3' untranslated portion of the human growth hormone (hGH) gene into mouse embryos to produce transgenic animals that express hHPRT on induction by cadmium. The hHPRT cDNA in these experiments contained 88 base pairs (bp) of 5'-untranslated and 190 bp of 3'-untranslated sequences, and the full-length coding sequence. We studied the in vivo expression of this MT-hHPRT fusion gene and observed preferential hHPRT expression in tissues of the central nervous system (CNS). This study suggests that sequences within the hHPRT transcript (cDNA) influence CNS expression via increased synthesis or stability of messenger RNA. PMID- 2995816 TI - Light-suppressible, cyclic GMP-sensitive conductance in the plasma membrane of a truncated rod outer segment. AB - Recent experiments by Fesenko et al and ourselves have shown that excised membrane patches from retinal rod outer segments contain a cyclic GMP-sensitive conductance which has electrical properties similar to those of the light sensitive conductance. This finding supports the notion that cGMP mediates phototransduction (see ref. 3) by directly modulating the light-sensitive conductance. However, some uncertainty remained about whether the patch experiments had discriminated completely between plasma and intracellular disk membranes; thus the cGMP response in an excised membrane could have resulted from contaminating disk membrane fragments, which are known to contain a cGMP regulated conductance. Furthermore, the patch conductance has not yet been shown to be light-suppressible, an ultimate criterion for identity with the light sensitive conductance. We now report experiments on a truncated rod outer segment preparation which resolved these issues. The results demonstrated that the cGMP sensitive conductance was present in the plasma membrane of the outer segment, and that in the presence of GTP the conductance could be suppressed by a light flash. With added ATP, the effectiveness of the light flash was reduced and the suppression was more transient. The effects of both GTP and ATP were consistent with the known biochemistry. From the maximum current inducible by cGMP, we estimate that approximately 1% of the light-sensitive conductance is normally open in the dark; this would give an effective free cGMP concentration of a few micromolar in the intact outer segment in the dark. PMID- 2995817 TI - Expression of insulin-like growth factor-II transcripts in Wilms' tumour. AB - Wilms' tumour probably arises from embryonal kidney cells and occurs in both hereditary and sporadic forms. Knudson and Strong have suggested that both forms of the disease are initiated by two mutational events. In the case of the inherited form, cytogenetic evidence indicates that a germline deletion of chromosome band 11p13 may correspond to one of the two mutations. DNA mapping evidence is consistent with the notion that the tumour susceptibility gene (Wg) on chromosome 11 is actually recessive. Comings has proposed that the dominantly inherited tumours may arise by the inactivation or loss of a diploid pair of regulatory genes which normally suppress the expression of a structural transforming gene (Tg). It has recently been suggested that the N-myc oncogene may serve as a transforming gene in retinoblastoma, although no such gene has yet been identified in Wilms' tumour. We now report that in four cases of Wilms' tumour, insulin-like growth factor-II (IGF-II) transcripts are highly elevated compared with the adjacent normal kidney. In addition, we have mapped the gene for IGF-II to chromosome band 11p14.1, which is in the immediate vicinity of Wg. These findings suggest that IGF-II may be involved in the aetiology of Wilms' tumour. PMID- 2995818 TI - Insulin-like growth factor-II gene expression in Wilms' tumour and embryonic tissues. AB - Wilms' tumour (nephroblastoma) is an embryonal neoplasm occurring in hereditary and spontaneous forms. Both types show rearrangements of the short arm of chromosome 11. The germ line of children with the rare inherited triad of aniridia, genito-urinary abnormality and mental retardation carry a chromosome 11 that has a deletion in its short arm (band 11p13) and these children are at increased risk of developing Wilms' tumour. Neonates with the Beckwith-Wiedemann syndrome, in which there may be duplication of the 11p13-11p15 region, are similarly predisposed. In the spontaneous form of the tumour a deletion of the 11p14 band in tumour cells, but not in normal cells, has been reported, and the development of homozygosity for recessive mutations in the 11p region is implicated in the aetiology of Wilms' tumour. In view of these chromosomal rearrangements and because Wilms' tumour is histologically indistinguishable from the early stages of kidney development, we have now examined the expression of genes localized to 11p in Wilms' tumour and human embryonic tissue. In 12 sporadic tumours examined, the expression of the gene coding for insulin-like growth factor-II (IGF-II), localized to the 11p15 region, was markedly increased relative to adult tissues, but was comparable to the level of expression in several fetal tissues including kidney, liver, adrenals and striated muscle. This may reflect the stage of tumour differentiation, but could also contribute to the malignant process, as IGF-II is an embryonal mitogen. PMID- 2995819 TI - A single genetic locus encoded by Yersinia pseudotuberculosis permits invasion of cultured animal cells by Escherichia coli K-12. AB - For many species of pathogenic bacteria, invasion and survival within animal cells is central to establishing a successful host-parasite relationship. Localization within host cells protects the microorganism from host defences, or permits it to cross epithelial barriers and subsequently become systemically distributed. The precise mechanisms that permit entry of bacteria into host tissues are unclear, therefore we have been studying the invasion of epithelial cells by Yersinia pseudotuberculosis. As a first step towards identifying the factors required for this process, we report here the identification of a single genetic locus from this organism that is sufficient to convert the innocuous Escherichia coli K-12 strain into an organism capable of invading cultured animal cells. PMID- 2995820 TI - Primary structure of the precursor to the three major surface antigens of Plasmodium falciparum merozoites. AB - Recently, a class of protein antigens of high relative molecular mass (Mt) which can induce protective immunity against blood-stage malaria has been identified. In Plasmodium falciparum the protein has a Mr of approximately 195,000 (P195). It is the precursor of three proteins of Mr 83,000 (83K), 42K and 19K which are the major surface antigens of merozoites; thus it may also be useful for immunization against P. falciparum. Three studies describing the isolation of single short complementary DNA clones for part of the P195 gene sequence have been reported. Here we describe the complete structure of the P195 gene determined from further DNA clones, its organization within genomic DNA and the location of the specific processing fragments within the primary amino-acid sequence. PMID- 2995821 TI - Isolation and characterization of the human pulmonary surfactant apoprotein gene. AB - Pulmonary surfactant is a phospholipid-protein complex which serves to lower the surface tension at the air-liquid interface in the alveoli of the mammalian lung and is essential for normal respiration. Inadequate levels of surfactant at birth, a frequent situation in premature infants, results in respiratory failure. In all species examined, surfactant is composed primarily of dipalmitoylphosphatidylcholine and two major protein species of relative molecular mass (Mr) 32,000 (32K) and 10K (refs 2-5). Reconstitution in vitro of purified 32K pulmonary surfactant apoprotein (PSAP) with synthetic lipids forms a lipoprotein complex that lowers surface tension by spreading to create a thin interfacial film. Here we describe the cloning of the human PSAP gene and complementary DNA, and discuss features of the unusual encoded protein. PMID- 2995822 TI - Nucleotide sequence evidence for relationship of AIDS retrovirus to lentiviruses. AB - Lentiviruses are a subfamily of retroviruses which have been aetiologically linked to the induction of arthritis, encephalitis, progressive pneumonia and slow neurological diseases in certain species. Relatively little is known about their genome structure, mechanisms of pathogenesis or evolutionary relationships with other retroviral subfamilies. In an effort to understand better the mechanisms by which these viruses induce such a variety of chronic diseases, we have molecularly cloned and physically characterized the genomes of caprine arthritis-encephalitis virus (CAEV) and equine infectious anaemia virus (EIAV) (A.Y. et al., in preparation). The latter, which bears some morphological similarity to the lentiviruses, has yet to be classified definitively as one. Here, we have determined the nucleotide sequence of a highly conserved region within the CAEV and EIAV pol genes. We demonstrate a much closer relationship of their predicted pol gene products to that of the presumed aetiological agent of human acquired immune deficiency syndrome (AIDS) than to those of other retroviruses. Additional pairwise comparisons allowed us to generate an evolutionary tree showing that the pol genes of lentiviruses and oncoviruses have evolved from a common progenitor. PMID- 2995823 TI - AIDS. US and French institutes in patents struggle. PMID- 2995825 TI - First comparison of two animal viruses in three dimensions. PMID- 2995824 TI - Events at the surface of the cell. PMID- 2995826 TI - Oncogenes. Growth factors short-circuited. PMID- 2995827 TI - Variability and repertoire size of T-cell receptor V alpha gene segments. AB - The immune system of higher organisms is composed largely of two distinct cell types, B lymphocytes and T lymphocytes, each of which is independently capable of recognizing an enormous number of distinct entities through their antigen receptors; surface immunoglobulin in the case of the former, and the T-cell receptor (TCR) in the case of the latter. In both cell types, the genes encoding the antigen receptors consist of multiple gene segments which recombine during maturation to produce many possible peptides. One striking difference between B- and T-cell recognition that has not yet been resolved by the structural data is the fact that T cells generally require a major histocompatibility determinant together with an antigen whereas, in most cases, antibodies recognize antigen alone. Recently, we and others have found that a series of TCR V beta gene sequences show conservation of many of the same residues that are conserved between heavy- and light-chain immunoglobulin V regions, and these V beta sequences are predicted to have an immunoglobulin-like secondary structure. To extend these studies, we have isolated and sequenced eight additional alpha-chain complementary cDNA clones and compared them with published sequences. Analyses of these sequences, reported here, indicate that V alpha regions have many of the characteristics of V beta gene segments but differ in that they almost always occur as cross-hybridizing gene families. We conclude that there may be very different selective pressures operating on V alpha and V beta sequences and that the V alpha repertoire may be considerably larger than that of V beta. PMID- 2995828 TI - Antibodies against platelet-derived growth factor inhibit acute transformation by simian sarcoma virus. AB - A clue to the molecular mechanism of neoplastic transformation was provided by the finding of a near identity in amino-acid sequence between the platelet derived growth factor (PDGF) B-chain and a region in the transforming protein, p28sis, of simian sarcoma virus (SSV), an agent that causes sarcomas and gliomas in experimental animals. This finding infers a direct link between the molecular biology of normal mitogenesis and oncogenesis since it suggests that the transforming activity of SSV is caused by a growth factor. Although PDGF agonist activity has been isolated from conditioned medium of SSV-transformed cells, it is not clear whether infection of responsive cells by SSV leads solely to autocrine stimulation of growth by a secreted PDGF-like factor or whether other, possibly intracellular, activities of p28sis or its processed products contribute to the transformation. To distinguish between these possibilities, we have studied the effect of anti-PDGF antibodies on acute SSV-transformation, and report here that these antibodies inhibit both proliferation and SSV-induced morphological changes in human diploid fibroblasts. PMID- 2995830 TI - Identification of talin as a major cytoplasmic protein implicated in platelet activation. AB - During platelet activation there is a major reorganization in the platelet cytoskeleton that accompanies a rapid change in platelet shape. Many of the events associated with activation are attributed to a rise in calcium concentration within the platelet cytoplasm. One direct consequence of the elevated calcium is the activation of a calcium-dependent protease that cleaves a major platelet protein of relative molecular mass (Mr) approximately 235,000 (235K) to 200K. This protein, P235, has been purified and reported to interact with actin, but the significance of the proteolytic cleavage is unknown. Talin, a cytoskeletal protein in smooth muscle and fibroblasts, binds vinculin and, together with vinculin, is localized in fibroblasts at sites of actin-membrane attachment. Talin and P235 have similar purification procedures, sedimentation coefficients and Stokes' radii (ref. 6 and Molony et al., unpublished observations). Of particular significance, talin is readily cleaved by proteases from approximately 215K to a fragment of approximately 190K. Given these similarities we have investigated the possible relationship between these proteins. Here we demonstrate that platelet P235 is recognized by anti-talin antibody and that it binds vinculin. Both proteins are cleaved in vitro by the calcium-activated protease to yield similar fragments. We conclude that P235 corresponds to the platelet form of talin. PMID- 2995831 TI - Sequence-induced DNA curvature at the bacteriophage lambda origin of replication. AB - DNA replication in bacteriophage lambda begins at a unique origin between residues 39,000 and 39,200 of the lambda genome. This segment of DNA serves a dual function since it also lies within the coding sequence of the lambda replication initiator protein O which binds origin DNA. The lambda origin sequence contains four 19-base-pair (bp) segments (iterons) which have dyad symmetry, followed by a 40-bp A + T-rich zone of highly asymmetrical base composition. It was noted earlier that lambda origin DNA exhibits an anomalous electrophoretic mobility on gels; that is, the length of DNA as determined by DNA sequencing is approximately 20% less than is predicted from electrophoretic mobility. Recent studies of kinetoplast minicircle DNA (K-DNA) from the protozoan Leishmania tarentolae have led to the proposal that sequence-induced DNA curvature could account for such electrophoretic anomalies by alteration of the shape of the DNA molecule. We now present evidence that the lambda origin contains a static curve. PMID- 2995829 TI - Amplification and altered expression of the c-myc oncogene in A-MuLV-transformed fibroblasts. AB - Chromosomal rearrangements involving the c-myc oncogene are a prevalent feature of plasmacytomas that arise after inoculating BALB/c mice with pristane and Abelson murine leukaemia virus (A-MuLV). With this observation in mind, we decided to determine if any genetic alterations of the c-myc locus could be observed in cells of a different type, when transformed in vitro by A-MuLV. Here we have analysed three independent A-MuLV-transformed NIH 3T3 lines (ANN-I, 54c12 and N25), and found that the c-myc locus is amplified 8-19-fold in each transformant. Quantitative S1 nuclease mapping performed on ANN-I and 54c12 RNAs demonstrated that: (1) c-myc messenger RNAs accumulated to double the levels found in NIH 3T3 cells; and (2) a shift in the use of the two normal c-myc transcription initiation sites (P1 and P2) occurred in favour of the 3' site, P2. Analysis of c-myc chromatin by DNase I treatment of 54c12 nuclei revealed that most, if not all, of the c-myc gene copies were transcriptionally competent. We present alternative ideas to explain why amplification of the c-myc gene occurs repeatedly in A-MuLV-transformed fibroblasts. Finally, we discuss our results in relation to the hypothesis linking the phenomenon of tumour progression with the amplification of oncogenes. PMID- 2995832 TI - DNA sequence at the end of IS1 required for transposition. AB - The insertion sequence IS1 belongs to a class of bacterial transposable genetic elements that can form compound transposons in which two copies of IS1 flank an otherwise non-transposable segment of DNA. IS1 differs from other known elements of this class (such as IS10, IS50 and IS903) in several respects. It is one of the smallest known insertion elements, exhibits a relatively complex array of open reading frames, is present in the chromosomes of various Enterobacteria, in some cases in many copies, and its insertion can result in the duplication of either 8 or 9 base pairs (bp) in the target DNA. Furthermore, although, like other members of the compound class, it seems to undergo direct transposition, IS1 also promotes replicon fusion (co-integrate formation) at a relatively high frequency. Like all other elements studied to date, the integrity of the extremities of IS1 are essential for efficient transposition. We have constructed a test system to determine the minimal DNA sequences at the extremities of IS1 required for transposition. Sequential deletions of the end sequences reveal that 21-25 bp of an isolated extremity are sufficient for transposition. A specific sequence 13-23 bp from the ends, defining the edge of the minimal sequence, is implicated as an essential site. The sites, symmetrically arrayed at both ends of IS1, correspond to the apparent consensus sequence of the known binding sites for the Escherichia coli DNA-binding protein (called integration host factor or IHF) which is required for the site-specific recombination that leads to integration of bacteriophage lambda into the bacterial genome. The sites at the ends of IS1 may thus bind a host protein, such as JHF or a related protein, that is involved in regulating the transposition apparatus. PMID- 2995833 TI - What turns on heat shock genes? PMID- 2995834 TI - Human lymphocytopathic retroviruses (HLRV)? PMID- 2995836 TI - A highly polymorphic DNA marker linked to adult polycystic kidney disease on chromosome 16. AB - Adult polycystic kidney disease (APCKD) is a common and often lethal multi-organ disease with an autosomal dominant pattern of inheritance; approximately 1 in 1,000 people carry the mutant gene. The major pathological abnormality is the development and progressive enlargement of cysts in several organs including the liver, pancreas and spleen as well as the kidneys. The basic biochemical defect which leads to the formation of cysts remains unknown. Cyst development, which is not retarded by any known therapy, leads to irreversible renal failure and death at a mean age of 51 unless dialysis or transplantation are used. Patients with the disease account for 9% of chronic dialysis requirement. The first symptoms tend to occur in the fourth decade, after most patients have reproduced. Presymptomatic diagnosis depends on the ultrasonographic detection of cysts, but exclusion cannot be achieved by this means; 34% of at-risk patients in the second decade and 14% in the third will go on to develop cysts after negative diagnosis. The low sensitivity of diagnostic techniques in this critical age-range imposes severe limitations on genetic counselling and the condition cannot be identified prenatally. Hence we have searched for a linkage marker for APCKD; we show here that the APCKD locus is closely linked to the alpha-globin locus on the short arm of chromosome 16 (zeta = 25.85, theta = 0.05). PMID- 2995835 TI - The hepatitis B virus. AB - DNA recombinant technology has radically changed hepatitis B virus (HBV) virology. The genetic organization, transcription and replication of the virus are basically understood, structures of integrated HBV sequences in hepatocellular carcinoma have been characterized, and new vaccines produced by recombinant DNA technique are being developed. PMID- 2995838 TI - Oncogene chromosome breakpoints and alu sequences. PMID- 2995837 TI - Role of intracellular calcium mobilization in the regulation of protein kinase C mediated membrane processes. AB - Phorbol esters are potent tumour-promoting agents that exert pleiotropic effects on cells. Among these are the control of growth, stimulation of release of stored bioactive constituents and regulation of growth-factor surface receptors. Phorbol esters bind to and activate protein kinase C, leading to the phosphorylation of specific protein substrates presumed to be necessary for eliciting the full response. Strong evidence exists that specific binding of tumour promoter occurs at the membrane level in intact cells, resulting in activation of protein kinase C. Recent evidence concerning the release of bioactive constituents from platelets and neutrophils has linked agonist-induced protein kinase C activation and Ca2+ mobilization in a synergistic mechanism. Here we present a novel model of synergism between Ca2+ and phorbol esters that leads to transferrin receptor phosphorylation and down-regulation in HL-60 human leukaemic cells. Raising intracellular Ca2+, although ineffective by itself, increases the potency and rate of action of phorbol ester for activating protein kinase C and mediating transferrin receptor phosphorylation and down-regulation. We propose a molecular model in which increased intracellular Ca2+ recruits protein kinase C to the plasma membrane, thus "priming' the system for activation by phorbol ester. PMID- 2995839 TI - Autoradiographic localisation of 3H-apomorphine binding sites in rat brain. AB - The best experimental conditions for a selective binding of 3H-apomorphine to dopamine receptors on cryostat sections were first selected by liquid scintillation quantification of the bound radioactivity. In the corpus striatum, a specific binding occurred with a half-maximal saturation concentration of about 1 nM and a maximal capacity of 180 fmol/mg of slice protein, both values in agreement with previous binding data on either membranes or slices incubated in a physiological medium. Inhibition with domperidone was clearly biphasic, indicating two classes of sites corresponding to the D-2 and D-3 sites as previously defined on membranes. When 3H-apomorphine was used at low concentrations (0.8-1.5 nM), a condition ensuring a preferential labelling of D-2 sites, rather well contrasted autoradiographic pictures were generated. The major dopaminergic projection fields in telencephalon (caudate-putamen, nucleus accumbens, olfactory tubercles) were visualised as well as other catecholaminergic regions such as the superficial gray layer of superior colliculi. Within the striatum, differences in density of these sites were observed in three perpendicular planes and confirmed by a computer densitometric image analysis. Labelling of areas of origin of the cerebral dopaminergic neurons in substantia nigra or ventral tegmental area were also observed. When a higher concentration of 3H-apomorphine (3.5 nM) was used in the presence of domperidone, another, but autoradiographically less distinct subclass of sites (D-3 sites) was demonstrated. PMID- 2995840 TI - Calcium entry blockers inhibit vasoconstrictor responses to sympathetic nerve stimulation mediated by alpha 1-adrenoceptors. AB - Stimulation of the sympathetic outflow (spinal cord segments T 7-9) in pithed rats resulted in an increase in mean arterial pressure, heart rate, total peripheral vascular resistance and cardiac output. The increase in blood pressure and peripheral resistance was markedly depressed by prazosin and to a lesser extent by yohimbine, suggesting that these responses were mediated primarily by postjunctional alpha 1-adrenoceptors. The calcium entry blockers nifedipine, tiapamil and verapamil also depressed pressor responses and the increase in total peripheral resistance to electrical stimulation of the sympathetic outflow in these rats. This depression resulted primarily from an effect on peripheral vascular resistance components, as cardiac output remained unaffected by the calcium entry blockers. This conclusion was supported by studies on isolated, perfused rat renal arteries. Vasoconstrictor responses of this in vitro preparation to perivascular nerve stimulation were depressed by 1,000-fold lower concentrations of prazosin than rauwolscine, demonstrating the predominantly alpha 1-adrenoceptor nature of these effects. Likewise, these vasoconstrictor responses were depressed by nifedipine, tiapamil and verapamil in a concentration dependent manner. The results of this study suggest that vasoconstrictor responses of rat resistance vessels to sympathetic nerve stimulation are mediated primarily by postjunctional alpha 1-adrenoceptors and can be inhibited by calcium entry blockers. This implies that contractile responses of these resistance vessels to alpha 1-adrenoceptor stimulation are not independent of the availability of extracellular calcium. PMID- 2995841 TI - Cyclic AMP facilitates the electrically evoked release of radiolabelled noradrenaline, dopamine and 5-hydroxytryptamine from rat brain slices. AB - The adenylate cyclase activator forskolin as well as 8-bromo-cyclic AMP enhanced the electrically evoked release of 3H-noradrenaline and 3H-5-hydroxytryptamine from superfused rat neocortical slices and that of 3H-dopamine from neostriatal slices with comparable EC50's of about 0.5 and 50 microM, respectively, without affecting spontaneous tritium efflux. The phosphodiesterase inhibitor ZK 62771 (3 100 microM) also enhanced 3H-noradrenaline and 3H-dopamine release but slightly reduced 3H-5-hydroxytryptamine release. However, this drug profoundly enhanced spontaneous tritium release in the latter case. The facilitatory effect of forskolin (0.3 microM) on the release of the amine neurotransmitters was potentiated in the presence of ZK 62771 (30 microM). Therefore, cyclic AMP appears to exert a general facilitatory effect on the release of these biogenic amines from central nerve terminals. PMID- 2995842 TI - Interaction between presynaptic facilitatory angiotensin II receptors and inhibitory muscarinic cholinoceptors on 3H-noradrenaline release in the rabbit heart. AB - The existence of a functional interaction between presynaptic receptors modulating the release of noradrenaline was studied in the rabbit heart. Isolated right atria were prelabelled with 3H-noradrenaline and the overflow of tritium was induced by field stimulation (2 Hz, 0.1 ms duration, supramaximal voltage for a total of 180 pulses). In atria superfused with Krebs' solution containing 10 mumol/l cocaine and 30 mumol/l corticosterone, angiotensin II (10 nmol/l) increased the stimulation-evoked overflow of 3H-transmitter by 2.8-fold. The addition of atropine (0.3 mumol/l) to the perfusion medium, either in the presence or in the absence of uptake inhibitors, further enhanced the facilitatory effect of angiotensin II (3H-transmitter release increased by 3.5 fold). Exposure to 1 mumol/l carbachol decreased by 65% the stimulation-evoked release of 3H-transmitter while the facilitatory effect of angiotensin II determined in the presence of the muscarinic cholinoceptor agonist was enhanced (3H-transmitter release increased by 6.6-fold). Conversely, during sustained activation of presynaptic angiotensin receptors producing a 2.5-fold increase in the release of 3H-transmitter, the inhibitory effect of carbachol remained unchanged. These results suggest a functional interaction between presynaptic inhibitory muscarinic cholinoceptors and the presynaptic facilitatory angiotensin receptor which modulate the release of noradrenaline from cardiac noradrenergic nerves. PMID- 2995845 TI - [AIDS: how do we continue in the interim?]. PMID- 2995844 TI - Leukotrienes C4, D4 and E4 cause widespread and extensive plasma extravasation in the guinea pig. AB - Intravenous injection of leukotriene C4 (1 nmol X kg-1) caused substantial plasma exudation in anesthetized guinea pigs, as evidenced by marked hemoconcentration 15% in 5 min) and significant extravasation of Evans blue. Fluorometric quantitation of Evans blue content in 38 selected tissues documented that leukotriene C4 caused significant plasma extravasation throughout the body, except for the brain, stomach, duodenum, colon and gonads. In particular, the respiratory and the uro-genital tracts, but also the conjunctiva, the esophagus, the bile ducts and the umbilical ligaments, were very sensitive to the edema promoting effect of leukotriene C4. Intravenous injection of leukotriene D4 (1 nmol- X kg-1) or E4 (5 nmol X kg-1) evoked plasma extravasation with a distribution and magnitude that was similar to that induced by leukotriene C4. It is concluded that the three major constituents of slow reacting substance of anaphylaxis (SRS-A), leukotrienes C4, D4 and E4, cause a generalized and extensive plasma exudation that is consistent with the proposal that these leukotrienes are important mediators of inflammation. PMID- 2995846 TI - [Mammographically occult breast carcinoma]. PMID- 2995843 TI - Dopaminergic modulation of hippocampal noradrenaline release. Evidence for alpha 2-antagonistic effects of some dopamine receptor agonists and antagonists. AB - 3H-Noradrenaline release in the rabbit hippocampus and its possible modulation via presynaptic dopamine receptors was studied. Hippocampal slices were preincubated with 3H-noradrenaline, continuously superfused in the presence of cocaine (30 mumol/l) and subjected to electrical field stimulation. The electrically evoked tritium overflow from the slices was reduced by 0.1 and 1 mumol/l dopamine and apomorphine, but significantly enhanced by 10 mumol/l apomorphine or by 0.1 and 1 mumol/l bromocriptine. If the alpha 2-adrenoceptor antagonist yohimbine (0.1 mumol/l) was present throughout superfusion, the inhibitory effects of dopamine and apomorphine were more pronounced and even 10 mumol/l apomorphine and 1 mumol/l bromocriptine inhibited noradrenaline release. Qualitatively similar observations were made in the presence of another alpha 2 antagonist, idazoxane (0.1 mumol/l). In the presence of the D2-receptor antagonist domperidone (0.1 mumol/l) the inhibitory effects of dopamine were almost abolished, whereas both apomorphine (greater than 1 mumol/l) and bromocriptine (greater than 0.01 mumol/l) greatly facilitated noradrenaline release. The D2-receptor agonist LY 171555 (0.1 and 1 mumol/l) significantly reduced the evoked noradrenaline release whereas the D1-selective agonist SK & F 38393 was ineffective at similar concentrations. The effects of LY 171555 were abolished in the presence of domperidone (0.1 mumol/l) but remained unchanged in the presence of yohimbine or idazoxane (0.1 mumol/l, each). At 1 mumol/l the D2 receptor antagonists domperidone and (-)sulpiride significantly increased the evoked noradrenaline release by about 10%. However, at this concentration, domperidone (but not (-)sulpiride) affected also basal tritium outflow. Bulbocapnine and the preferential D1-receptor antagonists SCH 23390 enhanced the evoked noradrenaline release already at 0.1 mumol/l.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995848 TI - [Reactions of neurons of the auditory cortex of unanesthetized cats to tones of a characteristic frequency]. AB - Extra- and intracellular responses of primary auditory cortex (AI) neurons were studied in acute experiments on non-anaesthetized cats. It was found that auditory cortex neurons having similar best frequencies revealed various forms of responses to corresponding frequency tones. Neurons responded to the tone by on reactions constituted about 40% of nerve cells studied. 27% of neurons revealed responses of on-off and off types. 27% of cortical neurons responded by steady excitation or by inhibition of background activity. About 6% of neurons did not respond to the tone. During intracellular recordings about 85% of neurons studied responded to switching on and/or off of the tone by a spike-IPSP sequence. 96% of cortical neurons generated IPSP as a constant component of the response to tone. Tonic responses of auditory cortical neurons were the result of powerful and lasting depolarization of the postsynaptic membranes. The conclusion is made that interaction of excitatory and inhibitory processes is the most significant in any kind of responses of the auditory cortical neurons to tones. PMID- 2995847 TI - [Ionic mechanisms of the effects of phenibut and GABA unrelated to changes in the function of chloride channels]. AB - It is established that beta-phenyl-GABA (phenibut) and partly GABA elicit direct depolarization of the isolated spinal cord motoneurons. The depolarizing effect of phenibut and a depolarizing component of GABA action do not alter in the presence of picrotoxin (10(-5) mol/l) and in the chloride-deficient medium. This depolarizing phenibut effect which is not bound with activation of GABAA receptors and chloride channels coupled with them does not alter in Na+-deficient medium, enhances in the medium with excess of K+ ions (10 mol/l) and in presence of imidazol (5 . 10(-4) mol/l) and is completely abolished in the Ca2+-deficient medium with 2 mmol/l of Mn2+ or in the presence of 10(-4) mol/l theophylline. It is supposed that phenibut and partly GABA diminish intracellular concentration of cAMP via GABAB-receptor activation and decrease functional activity of voltage dependent Ca2+-ionic channels and Ca2+-activated outward K+-currents. PMID- 2995849 TI - [Desensitization of cholinoreceptors of the somatic membrane of command neurons of Helix lucorum taurica L]. AB - Desensitization of receptors by the rhythmic application of acetylcholine (AC) was studied in Helix lucorum command neurons PPa2, PPa3, LPa2, LPa3. Application of AC induced in them monophasic depolarization wave. Repetitive AC stimulation with intervals less than 1-2 min induced a decrement of the response which gradually reached plato. Rate and degree of desensitization were constant in every following series and did not depend on preceding periods of depression. Response restoration was unchanged if intervals between series were equal. Stimulation with higher frequencies induced more considerable desensitization. Desensitization was specific to the locus of the membrane, on which AC was applied. Extrastimulation (intracellular stimulation and application of AC) did not lead to restoration of the response. It is supposed that desensitization is a short-term process and results from changes in receptors. The obtained data are explained by means of the Katz-Thesleff model of desensitization. As certain characteristics of synaptic depression and desensitization are similar, a suggestion is put forward that postsynaptic receptors can participate in the processes of synaptic effectiveness decrement during habituation. PMID- 2995850 TI - [Reactions of neurons of the motor cortex of the cat to acoustic stimulation and their role in the performance of an instrumental alimentary reflex]. AB - Neuronal reactions evoked in the motor cortex by single and series of sound clicks were studied in trained and untrained cats in chronic experiments. Presentation of conditional stimuli (single sound click) led to appearance of instrumental conditional movement for 7s after the conditioning. At the same time neuronal reaction to conditional sound stimulus did not influence neuronal activity directly connected with instrumental movements. Neuronal reactions to conditional stimuli did not prevent instrumental reflex. They carried out motor function during unconditional defense reaction which appeared in response to acoustic stimuli. Presence or absence of reactions to acoustic conditional stimuli depended on signal significance of stimuli, their physical parameters and level of excitability of the animal. PMID- 2995852 TI - [Preventive prosthetics. The value of patient instruction and after-care for edentulous patients]. PMID- 2995851 TI - [Presynaptic mechanisms of synchronizing fluctuations in the amplitude of synaptic potentials in the central nervous system of Helix lucorum taurica L]. AB - The amplitude of EPSP arising in different command neurons (LPa2, LPa3 and PPa3) of the snail Helix lucorum after stimulation of presynaptic element LPa7 fluctuated synchronously. Synchronous application of acetylcholine on two command neurons showed that synchronous EPSP fluctuations were connected not with postsynaptic but with presynaptic mechanisms. The existence of a special modulating neuron is suggested. PMID- 2995853 TI - Effect of picrotoxinin on benzodiazepine receptor binding. AB - The effect of picrotoxinin on [3H]flunitrazepam binding to benzodiazepine receptors was investigated. In mouse forebrain membranes, picrotoxinin inhibited basal, GABA- and pentobarbital-stimulated [3H]flunitrazepam binding; this inhibitory activity was temperature- and chloride ion-dependent. Scatchard analysis of the data indicates that picrotoxinin decreases the number of binding sites without altering binding affinity. In cerebellar membranes, picrotoxinin did not alter [3H]flunitrazepam receptor binding. PMID- 2995854 TI - Differential expression of 2':3'-cyclic nucleotide 3'-phosphodiesterase in cultured central, peripheral, and extraneural cells. AB - The relative levels of the central nervous system myelin marker enzyme 2':3' cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37, CNPase) were determined in neuroblastoma, astrocyte, oligodendrocyte and Schwann cell cultures and in freshly isolated human lymphocytes and platelets. The highest specific activities were associated with the cells that elaborate myelin membrane in the central and peripheral nervous system, oligodendrocytes and Schwann cells, respectively. Antiserum to bovine CNPase recognized both CNP1 and CNP2 in CNS myelin and human oligodendroglioma. In addition, a 53,000 dalton protein was evident on autoradiographs of immunoblotted PNS myelin and human oligodendroglioma proteins. Cultured rat oligodendrocyte, C6 and mouse NA neuroblastoma CNPase appear to share common determinants with the corresponding normal rat CNS enzyme. PMID- 2995855 TI - Occurrence of phenolsulfotransferase in primary glial culture cells of rat. AB - Phenolsulfotransferase (PST) activity towards phenol and monoamines was determined in rat brain and in primary cultures of rat astrocytes. The pH requirement, Km values and the proportion of PST activity with respect to phenol and dopamine as substrates were similar between PST from the glial cells and the rat cortex. The enzyme activity increased with age in the brain of older animals, and also with increasing incubation time in the primary culture of astroglia. The specific PST activity of the astroglia appeared to be higher than that of the brain enzyme. In glial cultures treated with 0.25 mM dibutyryl cyclic AMP in the same culture conditions, PST activity is suppressed to about 25% of its untreated counterpart, even though dibutyryl cyclic AMP at concentrations of ImM only slightly inhibited PST activity in vitro. PMID- 2995856 TI - [Adherence of peripheral blood neutrophils in patients with multiple sclerosis treated with ACTH. Preliminary report]. AB - The authors studied the adherence of peripheral blood neutrophils in 15 patients with multiple sclerosis treated with ACTH. Increased adherence was found only in the whole population of white blood cells and immature precursors of granulocytes and polymorphonuclears but not in lymphocytes and eosinophils. Administration of ACTH caused a statistically not significant decrease of the adherence of various forms of white blood cells. PMID- 2995857 TI - Sex steroids modulate prolactin response to naloxone in postmenopausal women. AB - To evaluate whether ovarian steroids modify the prolactin (PRL) response to opioid receptor blockade, the effects of naloxone infusion (1.6 mg/h for 4 h) on PRL secretion were studied in 5 postmenopausal women. Naloxone infusion was performed in basal conditions and after chronic oral treatment with conjugated estrogens (CE) (1.25 mg/day, for 20 days) or CE plus medroxyprogesterone acetate (MPA) (10 mg/day, for 20 days). Under basal conditions, 17 beta-estradiol, estrone, gonadotropin, and PRL plasma levels were in the normal range for postmenopausal women, and naloxone failed to affect PRL secretion. Naloxone induced a significant PRL increase after CE treatment alone (p less than 0.001) or in combination with MPA (p less than 0.001). The increase was significantly higher (p less than 0.05) after CE + MPA treatment than after CE treatment alone. These data suggest that steroids modulate the stimulatory effect of naloxone on PRL secretion in postmenopausal women. PMID- 2995858 TI - Estradiol-induced LH release in juvenile female rats: nembutal blocks both the decline and the exposure of nonavailable pituitary LHRH receptor sites. AB - We have previously shown that, as seen in the adult rat, pituitary LHRH receptor content declines during the hours encompassing the first preovulatory LH surge. This decrease in receptor number was prevented, however, when pituitary membranes were treated with MgCl2 to dissociate endogenously bound ligand(s). The present study investigates: whether the first proestrous reduction in available LHRH receptors can be reversed by subjecting the pituitary membranes to a less drastic, dilution-washing procedure previously reported to be highly effective in dissociating bound LHRH, and whether Nembutal blockade of the proestrous decline in LHRH receptors also prevents the dissociation-induced exposure of additional LHRH binding sites. A premature LH surge was induced by exposing juvenile female rats to proestrous-type levels of plasma estradiol (E2) via Silastic capsules. A decline in available LHRH receptors was found around the time of this steroid induced LH surge. A marked increase in receptor number was, however, observed upon dilution-washing of the membranes. The increase in receptor number was not accompanied by any changes in receptor affinity (ka). Nembutal administration blocked both the LH surge and the decline in available LHRH receptors. Moreover, dilution-washing of pituitary membranes from Nembutal-treated rats failed to uncover additional LHRH binding sites. The results suggest that a significant portion of the proestrous decline in pituitary LHRH receptors is due to a reduced availability of the receptor to binding. Whether such a phenomenon is due to true occupancy by endogenous ligand(s) or to binding-dependent localization of the receptor within the cell membrane is unclear.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995859 TI - Beta-2-adrenergic receptors in pituitary. Identification, characterization, and autoradiographic localization. AB - In the present study, we have used 125I-cyanopindolol (125ICYP) to identify, characterize, and localize beta-adrenergic receptors in bovine, rat, and human pituitary gland by in vitro labeling light microscopic autoradiography. The binding of 125ICYP to slide-mounted bovine pituitary sections was saturable and of high affinity with an apparent Kd of 0.2 nM. The pharmacological profile of 125ICYP binding obtained from competition studies demonstrates that the beta adrenergic receptors in the pituitary gland are predominantly of the beta 2 subtype. Rat pituitary autoradiograms show specific binding sites for 125ICYP in anterior, intermediate, and posterior lobes with highest concentrations found in the intermediate lobe and progressively lower concentrations in posterior and anterior lobes, respectively. Autoradiograms of 125ICYP binding in human pituitary show a significantly higher concentration of beta 2-adrenergic receptors in posterior than in anterior lobe of the pituitary. There is a homogeneous distribution of beta 2-adrenergic receptors within each lobe of both rat and human pituitary glands. The results of the present study provide the first visualization of beta 2-adrenergic receptors in rat and human pituitary and demonstrate the presence of significant concentrations of beta 2-adrenergic receptors in the posterior lobe. The data support a role for epinephrine and norepinephrine in modulating pituitary function. PMID- 2995860 TI - Ovarian LHRH receptors increase following lesions of the major LHRH structures in the rat brain: involvement of a direct neural pathway. AB - Specific lesions of different brain structures known to contain or to be coursed by LHRH neurons have been carried out in intact cycling female rats in order to investigate the role played by central LHRH and neuronal systems in the regulation of ovarian and hypophyseal LHRH receptor (LHRH-R) levels, using the stable LHRH analog [D-Ser (TBU)6] Des-Gly10, LHRH ethylamide, buserelin. Radiofrequency lesions were placed bilaterally or unilaterally into female rats showing at least two consecutive estrous cycles, under pentobarbital anesthesia, while groups of animals were sham-operated. Bilateral lesions placed into the septal area and into the nucleus and tract of the diagonal band of Broca (DBB) resulted in a nearly 25-40% stimulation of LHRH binding activity within the ovaries and a 30% inhibition of LHRH analog binding to pituitary homogenate, while lesions placed into the medial preoptic area (MPO), where the majority of LHRH neurons are found, produced a doubling of ovarian LHRH-R accompanied by a 60% decrease of pituitary binding sites. Lesions involving the median eminence and arcuate hypothalamic nucleus, where the most conspicuous convergence of LHRH fibers occurs, produced a twofold increase in ovarian LHRH-R levels and an almost complete loss of pituitary LHRH-R binding capacity, with no change in affinity. In order to clarify the importance of direct neural signals modulating the ovarian LHRH-R concentration, lesions were placed in the right or left MPO and DBB, and the content of LHRH-R measured in right and left ovaries. Lesions placed unilaterally (right or left) produced a significant increase of LHRH-R binding activity within the ovary ipsilateral to the lesion, while a reduction or no effect was observed in the contralateral gland. Data show a marked stimulation of ovarian LHRH-R number following bilateral lesions placed in the major LHRH containing structures, while pituitary LHRH binding sites are significantly inhibited, indicating impairment in the rate and/or amplitude of endogenous hypothalamic LHRH release. Furthermore results obtained following unilateral lesions indicate that a neural mechanism is involved in ovarian LHRH receptor induction and further reinforce our view that a direct neural connection links the brain and the ovaries. PMID- 2995861 TI - Delta-sleep-inducing peptide reduces CRF-induced corticosterone release. AB - It has been reported that delta-sleep-inducing peptide (DSIP) can affect several activities other than sleep, including reduction of stress. We studied the effects of this nonapeptide on corticotropin releasing factor (CRF)-stimulated release of corticosterone in rats treated with chlorpromazine-morphine pentobarbital. Significant reduction of corticosterone levels were observed after intravenous injection of 5-30 micrograms/kg DSIP. No effect of DSIP was found on the corticosterone release elicited by injection of adrenocorticotropic hormone. The results suggest that DSIP attenuates the effects of CRF at the level of the pituitary. PMID- 2995862 TI - Self-injurious behavior in acquired sensory neuropathy. AB - Self-abusive behavior, noted frequently in congenital sensory neuropathy, was observed in two children with acquired peripheral nerve dysfunction. In one case a laceration over the median nerve was followed by self-induced trauma to the fingers distal to the cut, while the other patient developed self-mutilation in all the extremities following insecticide poisoning and presented with signs of diffuse peripheral neuropathy. Improvement of the self-injurious behavior in each case seemed temporally related to the use of anticonvulsant medications, a treatment that is often suggested for older patients with paresthesias related to peripheral neuropathy. The apparent improvement in these two patients suggests that a trial of these drugs in additional patients with self-abusive behavior associated with peripheral neuropathy would be justified. PMID- 2995863 TI - Sulfonic acid enkephalin: binding specificity to rat brain opiate receptors. AB - We tested sulfonic-acid enkephalin, a SO3H-Tyr derivative of the Leu-enkephalin, for its binding capacity to rat brain opiate receptors, by competition against several tritiated enkephalins. Using [3H]DADLE as the competitor, we demonstrated that sulfonic-acid enkephalin binds to rat brain opiate receptors, and using [3H]DSLET and [3H]DAGO as the competitors, we demonstrated that sulfonic-acid enkephalin binds preferentially to delta-opiate receptors. The affinity ratio of sulfonic-acid enkephalin for delta-opiate receptors versus mu-opiate receptors, is considerably higher than that of the parent compound, Leu-enkephalin, and of DADLE and is similar to the affinity ratio of DSLET for the same receptors. PMID- 2995864 TI - Autoradiographic localization of mu, delta and kappa opioid receptor binding sites in rat and guinea pig spinal cord. AB - The autoradiographic distribution of mu, delta and kappa opioid binding sites was evaluated in various segments of the rat and guinea pig spinal cord. Mu opioid receptor binding sites are highly concentrated in the superficial layers of the dorsal horn (laminae II and III) in both species, without any marked gradient along the cord. Delta binding sites are somewhat concentrated in the superficial layers of the dorsal horn. However, delta binding sites are also present and evenly distributed in other areas of the gray matter. The highest density of delta sites is found in the cervical segment with only low levels in the lumbo sacral region of the rat and guinea pig spinal cord. Kappa opioid binding sites are highly concentrated in the superficial layers of the dorsal horn of the spinal cord. Lower levels are seen in the rest of the gray matter with some enrichment in lamina X. Moreover, the lumbo-sacral portion of the spinal cord is enriched in kappa sites as compared to the cervical and thoracic segments. These data demonstrate the differential laminar distribution of mu, delta and kappa opioid binding sites in rat and guinea pig spinal cord. PMID- 2995865 TI - Leucine enkephalin noncompetitively inhibits the binding of [3H]naloxone to the opiate mu-recognition site: evidence for delta----mu binding site interactions in vitro. AB - Using quantitative methods, this study examined the hypothesis that delta-ligands are noncompetitive inhibitors at a population of mu-binding sites. Evidence is presented that with the defined set of in vitro assay conditions utilized, [3H]naloxone labels two binding sites: the mu binding site and a second site tentatively identified as a kappa binding site, and that leucine enkephalin is a noncompetitive inhibitor at the mu recognition site. PMID- 2995866 TI - Microwave hyperthermia for brain tumors. AB - Twelve patients with malignant brain tumors who had failed to respond to conventional therapies were treated with thermotherapy. Hyperthermic temperatures (approximately 43 degrees C) were induced in the tumors using microwaves at a frequency of 2450 MHz that were guided into the tumors by one or more semirigid coaxial applicators. These applicators fit into 16 gauge tubes or needles and can be inserted into the brain with minimal damage to healthy tissues. During each treatment, the tumors were maintained at hyperthermic temperatures for 1 hour. Several treatments spaced a few days apart were usually administered. The procedure used for producing hyperthermia in brain tumors with microwaves proved to be safe and could be repeated several times without producing toxic effects. Objective tumor responses were obtained in 75% of the patients (decrease in tumor size, 3 patients; slowing of tumor growth, 2 patients; necrosis of tumor tissues verified by pathological examination, 4 patients). Favorable clinical responses were observed in 75% of the patients (rapid decrease in intractable headaches, 5 patients; improvements in clinical deficits, 4 patients). Also, in all patients, the microwave power required to heat for a given time or a given volume decreased during most of the thermotherapy sessions, possibly because of heat damage to the tumor vasculature. Our results, taken together with the results of other investigators, indicate that thermotherapy is a promising modality for treating malignant brain tumors, either as the sole therapy or in combination with radiotherapy and chemotherapy. The next logical steps would be Phase I/II type trials of subjects whose disease is less advanced than the disease of patients treated in the current series of investigations. PMID- 2995867 TI - Radiation-associated gliomas: a report of four cases and analysis of postradiation tumors of the central nervous system. AB - Four cases of radiation-associated gliomas are described. All patients were white men, irradiated in childhood for craniopharyngioma, anaplastic ependymoma, retinoblastoma of the orbit, and Burkitt's lymphoma, respectively. The dose ranged from 1800 to 5900 rads, and the latency period was 5 to 25 years. All primary and secondary tumors were verified histologically, and no evidence of persistence of the primary tumors was found. All secondary tumors arose in the fields of irradiation. Ninety-six cases of radiation-induced tumors of the central nervous system have been reported in the literature to date. Twenty-four were gliomas and occurred mainly in young men. PMID- 2995868 TI - Ontogeny and regional distribution of proenkephalin- and prodynorphin-derived peptides and opioid receptors in rat hippocampus. AB - Levels of prodynorphin- and proenkephalin-derived peptides were determined in whole hippocampus of prenatal and early postnatal rats and in five regions of the hippocampus of the adult rat. Using autoradiography, opioid receptor subtypes were localized in coronal sections of adult hippocampus. The opioid peptides are present in very low concentrations in prenatal hippocampus, with only dynorphin B and alpha-neo-endorphin being present in significant amounts. The main increase in concentrations of the opioid peptides occur between day 7 and 14 postnatally, when dynorphin A, dynorphin A-(1-8), dynorphin B and alpha-neo-endorphin reach their adult levels. beta-Neo-endorphin and [Met]enkephalyl-Arg-Gly-Leu do not reach their maximal level until later in development. There is a distinct differential distribution of the opioid peptides in the subregions of the hippocampus; the subiculum and CA1 are relatively poor in prodynorphin-derived peptides but do contain significant amounts of [Met]enkephalin and [Leu]enkephalin. Very high concentrations of dynorphin B and alpha-neo-endorphin are present in region CA4. Dynorphin A-(1-8) and [Met]enkephalin have their highest concentrations in the dentate gyrus. There is a 5-fold higher concentration of [Met]enkephalin in the ventral hippocampus compared to the dorsal hippocampus. A similar trend is seen with dynorphin A-(1-8) but not with the other opioid peptides. The most abundant opioid receptor population in the hippocampus is of the mu type and it is densest in and around stratum pyramidale of the region CA3. There are relatively few kappa opioid receptors in the rat hippocampus. These results indicate the presence of at least two independent opioid neuronal systems (enkephalin and dynorphin) in rat hippocampus and the presence of mu-, delta- and kappa-opioid receptor subtypes. PMID- 2995870 TI - [Biochemical diagnosis of insulinoma: correlations between blood glucose, blood insulin and the titer of plasma non-esterified fatty acids during fasting and during various diagnostic tests]. AB - The difficulty of clinical and biochemical diagnosis of insulinoma lies in the extreme variability of both clinical symptoms and glycaemia/insulinaemia levels, so that neither parameter is a totally reliable diagnostic indicator. The possibility of introducing a third parameter, the non-esterified fatty acid (FFA) level, to enhance the reliability of insulinoma diagnosis was therefore investigated. The behaviour of glucose, insulin and plasmatic FFA levels and the correlation of the three parameters were studied in 4 patients whose suspected insulinomas were later surgically confirmed. The tests applied were as follows: 24 hour fast followed by endovenous glucose, oral glucose administration, tolbutamide stimulus test, adrenaline and glucagon tests. The results revealed anomalies in insulinaemia and glycaemia behaviour such as have already, in part, been described in organic hyperinsulinism, e.g. the discrepancy between insulin and glucose levels after prolonged fasting. It was also clear that the parameters examined still leave much room for uncertainly in the biochemical diagnosis of insulinomas. Numerous anomalies were also observed in the behaviour of plasmatic FFA. For example lipid mobilisation was often reduced in conditions that normally stimulate it (fasting, the administration of catecholamines). Equally the prolonged blockage of lipid mobilisation was encountered in the presence of factors that normally reduce lipolysis (endovenous glucose). On the basis of these results it is suggested that a combined assessment of glucose and plasmatic FFA levels may be a more sensitive diagnostic indicator of insulinoma than the insulin/glucose ratio commonly reported in the literature. The mechanism behind the lipid mobilisation anomalies of insulinomas is also discussed in the light of its apparent connection with the glucose/insulin interaction characteristic of the condition. PMID- 2995869 TI - Affinity partitioning and centrifugal counter-current distribution of membrane bound opiate receptors using naloxone-poly(ethylene glycol). AB - Crude synaptic membranes isolated from calf brain cortex were subjected to an aqueous two-phase system and the partition of the various membrane constituents and activities between the phases were studied. These constituents were phosphate, cholesterol and protein. The activities measured were acetyl cholinesterase, succinate dehydrogenase, 2',3'-cyclicnucleotide-3' phosphohydrolase and stereospecific opiate-binding. The successful fractionation of the membranes was achieved by the use of an aqueous two-phase system in a counter-current distribution process. A ligand bound to poly(ethylene glycol) with an affinity for opiate receptors was synthesized by reacting 6-aminonaloxone with tresylpoly(ethylene glycol). The ligand-polymer was used to extract membrane bound opiate receptors into the upper, poly(ethylene glycol)-rich phase. This use of affinity partitioning resulted in membrane fractions with a 3-4 fold higher ability to bind stereospecifically etorphine than the original preparations of synaptic membranes. PMID- 2995871 TI - [Etiologic determination of chronic liver diseases]. AB - Sixty patients with chronic liver conditions (mostly cirrhotic) were examined for HBV markers. The percentage of markers identified, far higher than in samples of healthy subjects, suggests that HBV either alone or in association with other causes may be implicated in the pathogenesis of these conditions. PMID- 2995872 TI - Excitatory amino acids in the chick retina: possible involvement of cyclic guanosine monophosphate. AB - An investigation has been made of the ability of excitatory amino acids to alter the levels of chick retinal cyclic guanosine monophosphate (GMP). Kainic acid, N methyl-D-aspartic acid and quisqualic acid each produced dose-related increases in cyclic GMP, and these could be attenuated by the addition of excitatory amino acid antagonists. It appears likely that in addition to its proposed role in visual transduction, this nucleotide may be involved in excitatory amino acid function in the chick retina. PMID- 2995874 TI - The functional projections of the anterior ventralis nucleus, as revealed by autoradiography. AB - The effect of stimulation of ventral anterior nuclei of the thalamus (VA) was studied in different areas of cat cortex (sensory, motor and association) with [3H]glycine autoradiography. One-hour VA stimulation resulted in the most intensive [3H]glycine incorporation in the second, third and fifth layers of the anterior suprasylvian gyrus and of the motor cortex. The labelling was less intensive in the sensory (auditory) and association (posterior middle suprasylvian association area) cortices, but the effect of VA stimulation reached these regions as well. The VA stimulation resulted in evoked potentials over the whole area examined, with highest amplitude and shortest latency in the anterior suprasylvian gyrus. It is suggested that the [3H]glycine autoradiography is suitable to study the functional projection of non-specific thalamocortical systems. PMID- 2995873 TI - The use of lectin transport in the mouse central nervous system as an anterograde axonal marker for electron microscopy. AB - Lesion-induced axonal degeneration and autoradiography-electron microscopy have been the only reliable anterograde axonal markers available for electron microscopic examination of neuronal circuitry. However, these methods have their limitations. Recently, Phaseolus vulgaris-leucoagglutinin (PHA-L) has been used as an anterograde axonal marker for light microscopy. This report describes the use of this lectin as an anterograde marker for electron microscopy. PHA-L was injected into mouse SmI cortex or ventrobasal thalamus. Using standard immunohistochemical techniques, the transported lectin was tagged with antibody, which was then visualized with avidin-biotin-horseradish peroxidase binding. Light microscopy demonstrated anterograde transport to predicted cortical regions. With the electron microscope, labeled axon terminals were seen forming asymmetric synapses with spines, dendrites and cell bodies. PMID- 2995875 TI - Synaptic actions of the ventral root afferents on cat hindlimb motoneurons. AB - Postsynaptic potentials (PSPs) with long latencies were evoked in cat hindlimb motoneurons by stimulation of the distal stump of a cut ventral root. Measurements of their latency and the threshold in the responsible afferent fibers showed that they were produced mainly by the activity of the unmyelinated fibers in the ventral root that enter the spinal cord through the dorsal root. Patterns of PSPs evoked in flexor and extensor motoneurons by ventral root stimulation were similar to those observed in the flexion reflex. PMID- 2995876 TI - Perivascular peptides relax cerebral arteries concomitant with stimulation of cyclic adenosine monophosphate accumulation or release of an endothelium-derived relaxing factor in the cat. AB - Calcitonin gene-related peptide (CGRP), substance P (SP) and vasoactive intestinal polypeptide (VIP) have been proposed to be neurotransmitters/neuromodulators in cerebral perivascular nerve fibers. Here, we present pharmacological and biochemical evidence showing that these peptides have different modes of relaxing cerebral blood vessels in the cat. CGRP causes pronounced relaxation, this occurs simultaneously with stimulation of cyclic adenosine monophosphate (cAMP) accumulation. The strong VIP-induced dilatation is parallelled by cAMP accumulation, albeit of a lower magnitude than with CGRP. The SP-induced relaxation was much weaker than that of CGRP and VIP, and it was not associated with cAMP accumulation. Only at concentrations of SP where maximum relaxation had occurred, was a nonsignificant cAMP accumulation seen. The responses to SP and acetylcholine were absent in arteries where the endothelium had been removed, whereas the relaxations induced by CGRP and VIP persisted. PMID- 2995877 TI - Brain gamma-aminobutyric acid and benzodiazepine receptor binding in dialysis encephalopathy. AB - We measured gamma-aminobutyric acid (GABA) and benzodiazepine binding in autopsied frontal cortex of 8 patients dying with dialysis encephalopathy (DE). No alteration in [3H] GABA binding was observed. However, a mild reduction (-23%, P less than 0.05) of [3H] flunitrazepam-binding density was found in DE cortex. The magnitude of this reduction was similar to that observed in frontal cortex of amygdala-kindled rats [10]. We suggest that a reduction in benzodiazepine receptor number, in combination with markedly reduced GABA concentration in DE cerebral cortex may contribute to some of the clinical features (especially seizures) characteristically observed in this syndrome. PMID- 2995878 TI - Forebrain serotonergic involvement in avoidance learning. AB - The one-way active avoidance deficit caused by the serotonergic (5-HT) releasing compound p-chloroamphetamine (PCA) was examined in rats after degeneration of 5 HT neurons in the forebrain. Injection of 5,7-dihydroxytryptamine (5,7-DHT) into the forebrain in desipramine (20 mg/kg)-pretreated rats resulted in a 65-70% decrease in 5-HT concentrations in the prefrontal cortex and hippocampus without any significant effect on striatal 5-HT. Slight reductions in noradrenaline (NA) (25%) and dopamine (DA) (34%) concentrations were observed in the prefrontal cortex only. The 5,7-DHT lesions markedly attenuated the impaired avoidance performance induced by PCA (2.5 mg/kg), suggesting that the avoidance deficit depends on intact 5-HT terminals in cortex and/or hippocampus. The 5,7-DHT lesion alone caused a slight but significant impairment of acquisition. The results suggest that 5-HT terminal systems in the forebrain play an important role in avoidance learning. PMID- 2995879 TI - Physiological doses of estradiol can increase lingual dyskinesia and cerebrospinal fluid homovanillic acid in monkeys. AB - In 4 ovariectomized monkeys bearing a dopamine-sensitive lingual dyskinesia due to a previous mid-brain lesion, 30 ng of 17 beta-estradiol injected subcutaneously, caused a 4-fold increase in the dyskinesia in the hour following the injection. In a separate experiment done on 7 anesthetized monkeys, the same dose of estradiol caused a significant increase of homovanillic acid in the cerebrospinal fluid obtained by cisternal puncture. The above findings suggest that very small doses of estradiol in the physiological range can increase dopaminergic transmission in the striatum via increased release. PMID- 2995880 TI - Demyelination-induced plasticity in the axon membrane: an ultrastructural cytochemical study of lead neuropathy in the rat. AB - We examined the distribution of ferric ion-ferrocyanide stain (a marker for excitable regions of myelinated fibers) in the lead-induced demyelinating neuropathy of the rat. By electron microscopy, we found that paranodal degeneration resulted in spreading of the reaction product from nodal to internodal axolemma. During repair, nodal-like stained areas formed at the contact zones between preremyelinating Schwann cells. These data suggest that the location and extent of excitable axonal regions are influenced by axoglial relationships. Additionally, some fibers displayed staining at paranodal axolemma adjacent to demyelinated segments, suggesting it might be an alternative site for impulse generation in demyelinated fibers. PMID- 2995881 TI - Inhibitory effect of adenosine on electrically evoked contractions in the rat vas deferens: pharmacological characterization. AB - The inhibitory effects of adenosine as well as its related analogues on the contractile response of the rat vas deferens to field stimulation were compared in the absence and in the presence of nitrobenzylthioguanosine (NBTGR), a potent adenosine uptake inhibitor. In the presence of NBTGR, the order of potency was N6 cyclohexyladenosine (CHA) greater than or equal to L-N6-phenylisopropyladenosine (L-PIA) greater than 2-chloroadenosine greater than D-N6-phenylisopropyladenosine (D-PIA) greater than or equal to adenosine greater than 2'-deoxyadenosine. The inhibitory effect of adenosine but not that of clonidine, beta-endorphin and somatostatin was blocked by 1,3-diethyl-8-phenylxanthine (DPX, pA2 = 7.2), a potent P1-purinergic antagonist. The results suggest that adenosine inhibited the electrically evoked contractions of the rat vas deferens via the activation of the A1 subtype of P1-purinergic receptors. PMID- 2995882 TI - Effects of L-lysine and its metabolites on pentylenetetrazol-induced seizures. AB - Lysine and its metabolic intermediates were studied for their effect on pentylenetetrazol (PTZ)-induced seizures in mice. L-Lysine at dosages above 2 mmol/kg given i.p. significantly increased seizure protection and seizure latency (the time required to develop seizures after PTZ injection) with a peak effect dose at 10 mmol/kg. A pretreatment time of 15 min was required to significantly prolong seizure latency with a peak effect time of 45 min. D-Lysine at 10 mmol/kg i.p. afforded some seizure protection and significantly prolonged seizure latency but has a peak effect time of 15 min. When administered intracerebroventricularly, both L-lysine and piperidine at 0.1 mmol/kg prolonged seizure latency significantly, and increased seizure protection slightly. L Pipecolic acid at the same dose given through the same route, however, shortened seizure latency significantly. L-alpha-Aminoadipic acid, on the other hand, had no significant effect. Lysine metabolites that prolonged seizure latency also increased seizure protection and decreased seizure death, and one that shortened seizure latency had the opposite effect. The anticonvulsant activity of lysine and its metabolites was explained on the basis of their connection with the GABAergic transmission. PMID- 2995883 TI - Effects of formaldehyde on the isolated phrenic nerve and the phrenic nerve diaphragm preparation of the rat, in vitro. AB - Formaldehyde, 0.5-4.5 mM, increased the threshold for electrical excitation of the nerve, and led to a partial and reversible inhibition of the compound action potential (cAP). The depression was not enhanced by high frequency stimulation. At 8.9 mM or higher, the depression of the nerve excitability could not be reversed. The inhibition of the nerve developed more slowly than that of the muscle, and the nerve was unaffected after 10 min exposure to 2.2 mM. Formaldehyde, 2.2 mM, caused an immediate depression of the indirectly (through the nerve) and directory (at the muscle) elicited twitch tension. After 10 min the tensions were reduced to respectively, 56% and 49% of control. However, the electromyogram was not changed, indicating that the effect was localized to the excitation-contraction coupling. Tetanic tension (100 Hz in 5 sec) was inhibited more than twitch tension during indirect stimulation, whereas the opposite was found during direct stimulation of the muscle. Thus, during high frequency stimulation, formaldehyde must have an additional effect on the neuromuscular transmission. This effect was localized presynaptically since a fall out of endplate potentials was observed in the formaldehyde-treated diaphragm. In 6.7 mM or higher concentrations the directly or indirectly induced contractions were irreversibly blocked. The resting membrane potential of the muscle cells was unchanged after exposure to formaldehyde. Formaldehyde caused myotonia-like contractions of the diaphragm, occasionally after exposure to low concentrations (2.2 mM), and always after exposure to higher concentrations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2995884 TI - Chick brain hypomyelination produced by 2,4-dichlorophenoxyacetic butyl ester treatment. AB - Fertilized hens' eggs were externally treated with 2,4-dichlorophenoxyacetic (2,4 D) butyl ester (2.4 mg/egg) before starting incubation. The specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) was diminished in the whole brain of animals born from treated eggs, as compared with controls. Myelin was isolated by ultracentrifugation from the central nervous system. Treatment with 2,4-D butyl ester produced a 65% reduction. There were no statistically significant differences in myelin phospholipids, cerebrosides, sulphatides and gangliosides between control and treated animals. Cholesterol was diminished by 12% in the treated group, while cholesterol esters were not detectable in either myelin fraction. Total myelin proteins were also decreased by 40%, without variations in their pattern. There were changes in the following ratios in the myelin of the treated group: lipid/protein, cholesterol/phospholipids, cholesterol/protein, phospholipids/protein. Collectively these findings indicate that 2,4-D butyl ester produced hypomyelination. PMID- 2995885 TI - Vitamin A-binding proteins. PMID- 2995886 TI - Effect of vitamin A status on retinoid-binding protein levels in rat tissues. PMID- 2995888 TI - Cipollone v Liggett Group, Inc. The application of theories of liability in current cigarette litigation. PMID- 2995887 TI - Vitamin D hormone regulates myc-oncogene expression in tissue culture. PMID- 2995889 TI - The relationship between renal parenchymal mass and absolute quantitation of 99Tcm-DMSA uptake. An experimental study in the growing pig. AB - Five Gottingen minipigs from the same litter aged 1 month entered the study and right nephrectomies were performed at staggered intervals between weeks 1 and 5. Absolute renal uptake of 99Tcm-DMSA, expressed as a percentage of the administered dose, was recorded in each animal weekly before right nephrectomy and at weekly intervals for 5 weeks post-nephrectomy. Before nephrectomy absolute renal uptake of 99Tcm-DMSA ranged between 16.8 and 21.5% (mean 18.6%) per kidney with no difference between right and left kidneys. After right nephrectomy, uptake in the left kidney approximately doubled within 1 week. No correlation was shown between renal parenchymal mass and absolute 99Tcm-DMSA uptake in paired or solitary normal kidneys undergoing growth or compensatory growth. PMID- 2995890 TI - Menopausal hot flush. PMID- 2995892 TI - Pruritic vulvar squamous papillomatosis: evidence for human papillomavirus etiology. AB - The origin of squamous papillae of the vulvar vestibule is controversial. Although some are considered as asymptomatic normal variants of pelvic anatomy, a review of 12 cases of vulvar squamous papillae in patients visiting the Infectious Diseases Clinic at UCLA reveals a distinctly symptomatic variety. A syndrome complex of premonitory vulvar vestibular pruritus, pain or burning, dyspareunia, and progressive development of squamous papillae was noted. Microscopic examination of tissue specimens of the areas of squamous papillae reveals the presence of koilocytic change suggestive of viral infection with human papillomavirus. Furthermore, immunoperoxidase stain revealed human papillomavirus capsid antigen in two cases, which has heretofore not been reported in the literature to the authors' knowledge. Evidence of partner infection on physical examination of sexual partners of these women revealed changes consistent with human papillomavirus in four of six partners who were available for examination. Treatment with podophyllin, cryotherapy, laser, or a combination seems to give predictable resolution of the condition and associated symptoms. PMID- 2995891 TI - New criteria for the diagnosis of gestational trophoblastic disease. AB - The purpose of the study was to test the hypothesis of whether the combined use of ultrasound and human chorionic gonadotropin (hCG) determinations could increase the diagnostic accuracy of sonography in the diagnosis of hydatidiform mole. The criteria used were the absence of fetal heart movement by ultrasound when the hCG level was above 82,350 mIU/mL and the presence of an hCG level in excess of 2 SD above the mean for the biometrically derived gestational age for suspected partial moles. The threshold of 82,350 mIU/mL was derived by probit analysis of the hCG serum levels of a population of normal intrauterine pregnancies prospectively examined to determine the level of hCG at which fetal heart activity would be visible by sonography. The diagnostic accuracy of these criteria was compared with the preoperative sonographic examination in 36 hydatidiform moles. When sonography was used alone, 15 of 36 cases (41.6%) did not have a definitive diagnosis on the first examination. The combination of hCG and ultrasound would have correctly identified 32 of the 36 cases (88.8%). This improvement was statistically significant (P less than .005). PMID- 2995894 TI - [AIDS--from the clinical viewpoint]. PMID- 2995893 TI - [Epidemiology of AIDS]. PMID- 2995895 TI - [Elements of the genome regulating structural gene transcription in eukaryotes]. AB - A constant fine regulation of gene expression is needed for the normal development to proceed and for the physiological homeostasis to be maintained. In many cases such a regulation in eukaryotes is realized at the level of transcription, involving various cis- and trans-regulatory genomic elements. The review provides the data on the structure of elements determining the level of gene transcription in response to the action of various environmental factors and effectors, responsible for coordinated expression of the genes which provide for tissue-specific transcription and self-regulation of gene transcription. The data were considered on regulation of gene activity by mobile genetic elements and a relationship between the mechanisms of regulation of gene expression and evolution has been formulated. PMID- 2995896 TI - Buffalo in the northern Natal game parks show no serological evidence of infection with foot-and-mouth disease virus. AB - A total of 594 sera collected from buffalo (Syncerus caffer) in the Hluhluwe/Umfolozi Game Reserve complex, Ndumu Game Reserve and the eastern shores of Lake St Lucia were examined for antibody to SAT 1, 2 and 3 types of foot-and mouth disease (FMD) virus in neutralization tests. No neutralization of SAT 2 or 3 viruses was exhibited by any of the sera tested at final dilutions greater than 10. A small proportion (2,9%) of sera neutralized SAT 1 virus at dilutions up to 10, but these were considered to be due to non-specific reactions. This, together with the absence of clinical FMD in both cattle and game in this region over at least a 45-year period and the failure to isolate FMD virus from pharyngeal scrapings of buffalo sampled in the area, leads to the conclusion that FMD does not occur in these buffalo populations. PMID- 2995897 TI - [Antibodies specific to members of the Herpesvirus group in membranous, membranoproliferative and IgA glomerulonephritis]. PMID- 2995898 TI - Head and neck oncology--1985. Reflections on changing times. AB - The field of head and neck oncology is keeping pace with the dramatic shift in cancer treatment modalities occurring during this period of medical history. Having achieved a level of surgical elegance, the head and neck oncologist has the future to rely upon for more biological methods of management of this disease. Biochemistry, immunology, and virology occupy much of the research interest pertaining to malignancies of these body systems and it is within this research that the answers will probably be found. PMID- 2995899 TI - Nasopharyngeal carcinoma. Clinical presentation, diagnosis, treatment, and prognosis. AB - Serologic testing is a useful diagnostic aid for patients with NPC, particularly those in whom the tumors are small and submucosal (difficult to see or occult). If a metastatic tumor is found in the neck but its primary source is occult, positive titers provide reason for a detailed investigation of the nasopharynx, including a thorough examination with the patient under anesthesia and a random biopsy procedure. This approach can spare the patient a biopsy of neck nodes. Dickson compared two groups of patients with NPC metastatic lesions in the neck- the only difference between the groups was that the patients in one group had undergone a neck biopsy before radiation treatment--and found a somewhat poorer survival rate in the biopsied group. A large body of clinical evidence, histopathologic data, and, more recently, immunologic studies support the concept that carcinomas of the nasopharynx constitute two distinct diseases. Today, these are classified as WHO type 1 tumors (according to previous terminology, the "keratinizing, squamous cell carcinomas") and combined WHO type 2 and 3 tumors (the "combined grade 4 undifferentiated carcinomas," which are mostly the lymphoepitheliomas and transitional cell carcinomas in previous terminology). Clearly, the anti-EBV serologic findings separate the WHO type 1 tumors from the WHO type 2 and 3 tumors. The serologic findings in the former group are essentially the same as those in control groups, and the WHO type 1 tumors can be considered the "common garden variety" of squamous cell carcinomas found in other areas of the head and neck region. Furthermore, the WHO type 2 and 3 tumors occur at an earlier age; disease-free periods after treatment are longer; survival after treatment is better; and early and advanced neck metastasis is more common. In addition, primary WHO type 2 and 3 tumors in the nasopharynx are more often small, submucosal, and sometimes difficult to detect; indeed, they may be clinically occult. The tumors seem to be more radiation-sensitive than the WHO type 1 carcinomas, which are more likely to recur or persist in the nasopharynx after treatment. PMID- 2995900 TI - Squamous cell carcinoma. Metastasis to the neck from an unknown or undiscovered primary. AB - With an unknown or occult primary cancer, the clinical presentation can be either undiagnosed lump or lumps in the neck that has been biopsied and diagnosed as metastatic cancer on the basis of the histopathologic findings. PMID- 2995901 TI - Interferon sensitivity of fibroblast cell cultures derived from patients with neoplasms of the head and neck. AB - Interferon (IFN) is a protein with antiviral activity that has been shown to inhibit the growth of many different types of cells. We have measured the IFN sensitivity of nine cell cultures isolated from patients with squamous cell carcinoma and one with malignant melanoma of the head and neck. Normal-appearing fibroblast cultures isolated from these tissues appear quite sensitive to the antiviral effects of IFN. When the encephalomyocarditis virus yield reduction assay is used, these diploid cells are as sensitive to IFN-alpha as are newborn foreskin fibroblast cultures. A similar antiviral effect is seen with IFN-gamma. These cells are relatively insensitive to the antigrowth effect of both IFN preparations as measured by 3H thymidine incorporation and direct observations of cell growth. This is the same relative sensitivity as fibroblasts derived from normal patients. Since these cells are at least 100 times less sensitive to the antigrowth action of the IFNs, it appears unlikely that the IFNs play a significant role in the control of normal fibroblast growth, in contrast to the sensitivity of malignant cell lines. PMID- 2995903 TI - Cryoanalgesia for painful peripheral nerve lesions. AB - Twelve patients with chronically painful peripheral nerve lesions were treated with cryoanalgesia. The pain was relieved in 6 patients for 1-12 months. Although the pain eventually recurred, the patients resumed normal activities during remission. It is necessary to improve the techniques of nerve localization and to determine the proper mode of nerve freezing. PMID- 2995902 TI - Effects of human interferon on cell proliferation and antigens induced by Epstein Barr virus in epithelial hybrid cells derived from nasopharyngeal carcinoma. AB - The effects of human leukocyte interferon (HulFN-alpha) and fibroblast interferon (HulFN-beta) on cell growth and induction of antigens related to Epstein-Barr virus (EBV) in a nasopharyngeal carcinoma hybrid cell line (NPC-KT) were studied. HulFN-alpha showed little antiproliferative and anti-EBV effect on NPC-KT cells. On the other hand, HulFN-beta showed modest antiproliferative effect on NPC-KT cells; the effect was dependent on time and HulFN-beta concentration. HulFN-beta alone was ineffective in inhibiting EBV-related antigens, but NPC-KT cells treated for more than 1 week with HulFN-beta inhibited the action of the chemical inducer of EBV, n-butyric acid, and the appearance of EBV-induced early antigen and EB viral capsid antigen was inhibited more than 50% compared with NPC-KT cells treated with n-butyric acid alone. PMID- 2995904 TI - [Evaluation of 17-beta-hydroxysteroid dehydrogenase activity as a marker of the hormone dependence of breast cancers]. AB - Intratumoral activity of the enzyme 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) was measured in 55 patients with breast cancer (17 pre- and 38 post menopausal) before and/or after 8 days of a progestin treatment (lynestrenol 10 mg/day). In 12 patients the 17 beta-HSD ability to be stimulated was compared to estradiol and progesterone receptor (ER and PR) levels. In premenopausal patients 17 beta-HSD was higher when tumorectomy was performed in the luteal phase than in the follicular phase. In post-menopausal patients, 17 beta-HSD is higher after progestin treatment. However 17 beta-HSD stimulation by lynestrenol depends on receptor levels. It is most after markedly stimulated in ER+ PR+ tumors. It remains low in ER- PR- tumors. In conclusion, intratumoral measurement of the progesterone dependent enzyme (17 beta-HSD) in breast cancer after progestin treatment provides a fine and reliable index of the presence and functional character of PR and hormone dependence of the tumor. PMID- 2995905 TI - The pathological spectrum of alcoholic liver disease. PMID- 2995906 TI - S-100 protein as a marker for melanocytic and other tumours. AB - The majority of melanocytic tumours are easily diagnosed but they become a problem when they are amelanotic and the tumour cells resemble those of other tumours. This applies particularly to secondary melanoma. Detection of S100 protein is a useful identifying marker. S100 protein, so named for its solubility in saturated ammonium sulphate, is derived from brain tissue. It is a dimer and belongs to a calcium binding group of proteins. The protein was first thought to be in neural or neural crest derived tissues but has been found in chondrocytes, adipocytes, myoepithelial cells, dendritic cells of lymphoid tissue, Langerhans cells and T lymphocytes. The protein is present in a high proportion of malignant melanomas and nevocytic nevi of skin, but is less positive in eye melanomas. It is present in gliomas, Schwannomas and neurofibromas but not in neurone derived tumours such as neuroblastomas. Chondromas, chondrosarcomas, liposarcomas, some osteogenic sarcomas and some histiocytic tumours are positive. The tumours that do not contain S100 protein are listed. Pending development of melanoma-directed monoclonal antibodies, the use of anti-serum to S100 protein plus anti-keratin and anti-leukocyte reagents is useful in the identification of tumours of doubtful histogenesis. PMID- 2995907 TI - [An introduction to histopathology of germ cell neoplasms of the ovary]. PMID- 2995908 TI - [Malignant gonocytic neoplasms of the ovary in girls]. PMID- 2995909 TI - [Serum 25-hydroxyvitamin D3 levels in 3- and 6-month-old Warsaw infants receiving daily prophylactically vitamin D3 "Polfa"]. PMID- 2995911 TI - [Clinico-diagnostic value of blood oxyproline in children with rickets and rickets-like diseases]. PMID- 2995910 TI - [Idiopathic hypercalcemia as a syndrome of hypersensitivity to vitamin D3 in 19 infants]. PMID- 2995912 TI - [Preoperative chemotherapy of nephroblastoma including adriamycin]. PMID- 2995913 TI - [The significance of prognostic score in the follow-up study of the patients with postoperative breast cancer with special reference to bone metastasis]. PMID- 2995914 TI - [Diagnostic accuracy of medical imagings and an integrated statistical approach to diagnosis--with special attention to hepatocellular carcinoma]. PMID- 2995915 TI - Cloning and the nucleotide sequence of rat glutathione S-transferase P cDNA. AB - A cDNA library prepared from poly(A)+ RNA of 2-acetylaminofluorene (AAF) induced rat hepatocellular carcinoma was screened by synthetic DNA probes deduced from a partial amino acid sequence of glutathione S-transferase P subunit that had been isolated from the tumor by two-dimensional gel electrophoresis. One of the four clones analyzed contained an mRNA region encoding the total amino acid sequence of this enzyme subunit and the complete 3'-noncoding region. The nucleotide sequence indicates that this enzyme subunit has 209 amino acids (calculated Mr=23,307) distinct from other glutathione S-transferase subunits such as Ya and Yc. Comparison of the amino acid sequences between these proteins indicates that glutathione S-transferase P subunit gene has been evolved from the ancestral gene at an earlier stage than the separation of Ya and Yc and that there are at least three domains having a considerable homology with each other in these enzymes. The very large increase of this mRNA in chemically induced hepatocellular carcinoma suggests a characteristic derepression of this gene during hepatocarcinogenesis. PMID- 2995917 TI - Altered DNA conformations detected by mung bean nuclease occur in promoter and terminator regions of supercoiled pBR322 DNA. AB - Mung bean nuclease was used to probe for recognizable DNA unwinding and unpairing in the plasmid pBR322. In negatively supercoiled DNA, but not relaxed DNA, cleavages occurred preferentially in non-coding regions of the genome. The types of nucleotide sequences cleaved and which non-coding regions were cleaved depended upon environmental conditions. At 37 degrees C, cleavages occurred in an 84 bp A+T-rich sequence in the terminator region of the ampicillin-resistance gene. Recognition is likely based on a novel DNA conformation which occurs in the longest, most dA+dT-rich region of pBR322. In the presence of 1 mM Mg2+, cleavages occurred in inverted repeated sequences in the promoter regions of the RNA primer for DNA replication and ampicillin- and tetracycline-resistance genes as well as the terminator of RNA-1. Potential loops of hairpin (cruciform) structures were cleaved. At 27 degrees C, cleavages occurred near a promoter activated by cAMP receptor protein in vitro and in the 3' non-coding region of the tetracycline-resistance gene. Thus, in supercoiled pBR322 DNA, recognizable DNA unwinding and unpairing occurs preferentially in regulatory regions for transcription and DNA replication. PMID- 2995918 TI - Cloning and characterization of the gene for the yeast cytoplasmic threonyl-tRNA synthetase. AB - A fragment of DNA from the yeast nuclear gene MST1 that codes for the mitochondrial tRNAThr1 synthetase was used as a probe to screen for other yeast threonyl-tRNA synthetase genes. At low stringency, the MST1 probe hybridizes strongly to a 6.6 kb EcoRI fragment of yeast genomic DNA with the homologous gene and in addition hybridizes more weakly to a smaller 3.6 kb EcoRI fragment with a second threonyl-tRNA synthetase gene (THS1). To clone THS1, a library was constructed by ligation to pUC18 of size selected (3-4.5 kb) EcoRI fragments of genomic DNA. Several clones containing the 3.6 kb EcoRI fragment were isolated. A 2,202 nucleotide long open reading frame corresponding to THS1 has been identified in the cloned fragment of DNA. The predicted protein encoded by THS1 is 38% identical to the E. coli threonyl-tRNA synthetase over the latter's length (642 amino acids) and is 42% identical to the predicted MST1 product over its 462 residues. In situ disruption of the chromosomal copy of THS1 is lethal to the cell, indicating that this gene codes for the cytoplasmic threonyl-tRNA synthetase. PMID- 2995916 TI - Tissue specific transcription of the human epsilon-globin gene following transfection into the embryonic erythroid cell line K562. AB - We have introduced a plasmid containing the human epsilon-globin gene either stably or transiently into a number of erythroid or non-erythroid cell lines, and analysed the accuracy and efficiency of transcription. In non-erythroid cells (or in mouse erythroleukaemia (MEL) cells in which adult but not embryonic globin genes are expressed) transcription of the epsilon-globin gene occurs mainly from a site 200 bp upstream of the major cap site (the -200 cap site). In the human K562 cell line, in which the endogenous epsilon-globin gene is transcribed at high levels, transcription initiation from the introduced gene occurs mainly from the major cap site. Transcriptional activity of the epsilon-globin gene introduced into K562 cell is quantitatively similar to that of the endogenous gene. This suggests the presence (or absence) in K562 cells of factor(s) which activate (or repress) the epsilon-globin gene in a tissue specific manner. PMID- 2995919 TI - Transient alterations of the chromatin structure of sea urchin early histone genes during embryogenesis. AB - We describe features of the chromatin structure of the early histone gene family of Strongylocentrotus purpuratus during development. Before and after the histone genes are transcriptionally active, chromatin structure is quite similar with well-defined spaced nucleosomes and no major 5'-flanking sites hypersensitive to nucleases. During the period when the genes are active, marked changes in chromatin structure occur. Micrococcal nuclease digestion generates monomer nucleosomes and only trace amounts of higher multimers. Regions hypersensitive to an endogenous nuclease and DNAase I appear in the 5'-flanking regions of genes for H2A, H2B and H3. Each region consists of four sites spanning a DNA length of 200-250 base pairs. In each case, one major cutting site is near the TATA box; the bulk of the sensitive region is in the nontranscribed spacer. Other sites, in 3'-flanking regions of the genes, are sensitive to nucleases only when the histone genes are no longer transcribed. PMID- 2995921 TI - A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure. AB - The in vitro synthesis of extraneous RNA sequences by SP6 and T7 RNA polymerases from specific DNA templates is described. Transcription of templates prepared by digestion with restriction enzymes that leave 3' protruding ends resulted in the production of significant amounts of long, template-sized RNA transcripts which hybridized to vector DNA. Sequences copied from the noncoding template strand were among the extraneous transcripts. The presence of these sequences in probe preparations were detected in Southern and RNase protection hybridization assays. In contrast, transcription of DNA templates with blunt or 5' protruding ends yielded few RNA products as extraneous sequences. PMID- 2995920 TI - Site-specific nicking at the avian retrovirus LTR circle junction by the viral pp32 DNA endonuclease. AB - The avian retrovirus pp32 DNA endonuclease prefers to nick supercoiled DNA containing long terminal repeat (LTR) circle junction sequences at one or the other of two sites, each which mapped two nucleotides back from the circle junction. The sequence at the sites of nicking was (sequence: see text) where increases indicates the positions of the two alternative nicked sites. This site specific nicking was observed when the circle junction LTR DNA was present in supercoiled form, the divalent metal ion was Mg2+ and the molar ratio of protein to DNA was low. The majority of other LTR DNA sites nicked by pp32 in the presence of Mg2+ were adjacent to or within the dinucleotide CA. PMID- 2995922 TI - The ribosomal RNA promoter of Acanthamoeba castellanii determined by transcription in a cell-free system. AB - The DNA sequences required for faithful initiation of ribosomal RNA transcription were determined. BAL-31 digestion was used to modify the rDNA template by introducing deletions from its 3'- and 5'-ends. The resulting mutant DNAs were tested for template activity individually or in competition with wild type utilizing an in vitro transcription system from Acanthamoeba castellanii. The results identify the sequence extending from -31 to +8 to be absolutely required for transcription. In addition; when the region between -47 and -32 is left intact, transcription is augmented. PMID- 2995923 TI - The Ty transposon of Saccharomyces cerevisiae determines the synthesis of at least three proteins. AB - Two new Ty determined proteins have been identified by placing a Ty transcriptional unit under the control of a high efficiency yeast expression vector. One of these proteins is the product of a post-translational processing event and it binds nucleic acids. A previously identified protein, pl (Tyl-15), has also been shown to bind nucleic acids and to be modified by phosphorylation. PMID- 2995924 TI - Suppression of a nonsense mutation in mammalian cells in vivo by the aminoglycoside antibiotics G-418 and paromomycin. AB - Aminoglycoside antibiotics in Escherichia coli and yeast can cause ribosomes to read through stop codons during translation. This can result in the phenotypic suppression of nonsense mutations. We show here for the first time that the aminoglycosides G-418 and paromomycin have similar effects in monkey (COS-7) cells in vivo. Suppression of an amber mutation (TAG) by aminoglycosides can restore the activity of a mutant gene transfected into COS-7 cells to almost 20% of wild type levels. PMID- 2995925 TI - Nucleotide sequence of Candida pelliculosa beta-glucosidase gene. AB - The nucleotide sequence of the DNA fragment containing the beta-glucosidase gene of Candida pelliculosa was determined. Analysis of the sequence revealed three open reading frames which could encode 65,825, and 412 amino acid residues. The presence of the second frame was found to be sufficient for the expression of the beta-glucosidase gene in a heterologous host Saccharomyces cerevisiae. Putative protein encoded by this gene had hydrophobic amino acids, resembling a signal peptide, at its N-terminal region and 19 potential glycosylation sites. Codon usage of Candida genes had the similar pattern shown in S.cerevisiae. Codon bias of the beta-glucosidase gene of Candida was relatively low, compared with that of the highly expressed genes of S. cerevisiae. PMID- 2995926 TI - Splicing of the adenovirus-2 E1A 13S mRNA requires a minimal intron length and specific intron signals. AB - The adenovirus E1A region encodes three overlapping mRNAs, designated 9S, 12S and 13S. They differ from each other with regard to the length of the intron which is removed by RNA splicing. We have constructed E1A genes with deletions and insertions in the intervening sequence that is common to all three E1A mRNAs, in a search for signals which influence splicing of the 13S mRNA. Mutant plasmids were transfected into HeLa cells and the transiently expressed E1A mRNAs characterized by the S1 protection assay. The results show that five upstream and 20 downstream nucleotides are sufficient to allow for a correct utilization of the 5'-splice junction for the E1A 13S mRNA. Moreover, we show that a minimal intron length of 78 nucleotides is required for efficient 13S mRNA splicing. The ability of mutants with large intron deletions to maturate a 13S mRNA could partially be restored by expanding the intron length with phage lambda sequences. However, in no case was the normal splicing efficiency obtained with these mutants. In contrast, one mutant in which sequences from the authentic 13S mRNA intron were used to expand the intron expressed almost normal levels of 13S mRNA, thus suggesting that signals which specifically promote 13S mRNA splicing exist. PMID- 2995929 TI - Clinical. Positive pain relief. PMID- 2995928 TI - The human apolipoprotein AII gene: structural organization and sites of expression. AB - The complete nucleotide sequence of the human apolipoprotein All gene together with 911 bases of 5' flanking sequence and 687 bases of 3' flanking sequence have been determined. The mRNA coding region is interrupted by three introns of 169, 293 and 395bp. The Intro-exon structure of the apo All gene is similar to that of the apo AI, apo CIII and apo E genes: three introns separate 4 coding sequences specifying the 5' untranslated region, pre-peptide, a short N-terminal domain and a C-terminal domain composed of a variable number of lipid-binding amphipathic helices. Intron II carries a 33bp dG-dT repetitive element adjacent to the 3' splice junction which has the potential to adopt the Z-DNA conformation. The 5' and 3' terminuses of the mRNA have been identified by primer extension and S1 nuclease mapping. A number of short direct repeats are found in the 5' flanking region and an inverted repeat occurs between the CAAT and TATA boxes. Downstream of the the gene is an Alu family repeat containing a polymorphic MspI site, the deletion of which is associated with increased circulating levels of apoAII. ApoAII gene expression was demonstrated in adult human liver and HepG2 cells but not in human small intestine. Of ten Rhesus monkey tissues examined apo All mRNA was detected only in liver. PMID- 2995927 TI - Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosomal localization. AB - Human Tumor Necrosis Factor and Lymphotoxin are cytotoxic proteins which have similar biological activities and share 30 percent amino acid homology. The single copy genes which encode these proteins share several structural features: each gene is approximately three kilobase pairs in length and is interrupted by three introns. In addition, these genes are closely linked and have been mapped to human chromosome 6. However, only the last exons of both genes, which code for more than 80 percent of each secreted protein, are significantly homologous (56 percent). PMID- 2995930 TI - Pharmacology. Counteracting side-effects. PMID- 2995931 TI - [Correlation between in vitro degranulation of basophils by polymyxin B and immunoglobulin E level in the blood of patients with asthma]. PMID- 2995932 TI - [Small-cell cancer of the lung, Gdansk, 15 September 1984]. PMID- 2995933 TI - Evidence against transmission of human T-lymphotropic virus/lymphadenopathy associated virus (HTLV-III/LAV) in families of children with the acquired immunodeficiency syndrome. AB - Six children with the acquired immunodeficiency syndrome (AIDS) and 12 of their household contacts were investigated serologically for evidence of infection with human T-lymphotropic virus/lymphadenopathy-associated virus (HTLV-III/LAV), the presumed etiologic agent of AIDS. All six children had antibody against HTLV III/LAV, as measured by enzyme-linked immunosorbent assay, in each specimen tested. Of the two mothers studied both were seropositive; one was diagnosed with and died from AIDS. Four of the remaining 10 household members were seropositive, including three adults in groups at high risk for the development of AIDS and one sibling who was younger than the child with AIDS. Among the seronegative household contacts were four foster mothers or grandmothers of the children with AIDS, three of whom had cared for the children since infancy. Household contact with children with AIDS may include persons in groups at high risk for AIDS who have been infected with HTLV-III/LAV. However, the negative findings in household contacts without risk factors for AIDS suggest that horizontal transmission of the virus within households by means other than sexual contact must be infrequent. PMID- 2995934 TI - Clinical manifestations of herpesvirus infections in pediatric renal transplant recipients. AB - We report our experience with 29 symptomatic herpesvirus infections occurring during the course of 87 pediatric transplant procedures performed over the 10 year period, 1973 to 1982. The yearly attack rate ranged from 0.05 to 0.40 case per cumulative patient years at risk. A greater proportion (9 of 14) of children who received more than 10 units of whole blood or packed red blood cells prior to transplantation developed a viral infection compared with those given 10 transfusions or fewer (8 of 25) (P = 0.10). Fever occurred in 22 (76%) children, pulmonary disease in 8 (28%), hepatitis in 11 (35%), leukopenia in 7 (24%), thrombocytopenia in 9 (31%) and central nervous system disease in 3 (10%). Herpesvirus infections were responsible for allograft loss in 7 (24%) patients. However, no differences in the actuarial graft survival curves were noted for transplants performed since 1979 in children with and without viral infection. The etiologic viral agents were cytomegalovirus in 19 (65%) episodes, herpes simplex virus in 8 (28%), Epstein-Barr virus in 2 (7%) and varicella-zoster virus in 2 (7%). Cytomegalovirus-infected patients were younger and more commonly developed primary infection compared with children with herpes simplex virus disease who were more likely to have secondary infection and to manifest a mucocutaneous vesicular rash. We conclude that the etiologic agents and clinical features of herpesvirus infections are similar in pediatric and adult renal allograft recipients. Moreover except for distinctive syndromes such as mucocutaneous vesicular eruption or a central nervous system lymphoma, the various herpes-viruses cause clinically indistinguishable illnesses in pediatric transplant patients with similar end organ involvement and untoward renal consequences. PMID- 2995935 TI - Premortem isolation of Saint Louis encephalitis virus: case report and implications for hospital and laboratory personnel. PMID- 2995937 TI - Stimulated secretion of pro-opiomelanocortin-related peptides in hypothalamic cells. AB - Although it has been suggested pro-opiomelanocortin (POMC) related peptides in brain may be neurotransmitters or neuromodulators, little is known about their secretion from neurons because it is difficult to study neurosecretion with an in vivo model. To demonstrate the possibility that POMC peptides may be neuroregulators which can be secreted in response to specific stimuli, we studied the secretion of immunoreactive (IR-) adrenocorticotropin (ACTH) and IR-beta endorphin from dissociated hypothalamic cells during potassium-induced depolarization. Significant increments (p less than 0.025) in secretion of IR ACTH (267%) and IR-beta-endorphin (88-172%) over basal secretion were stimulated by 60 mM KCl in the presence of calcium. CONCLUSION: Stimulated secretion of POMC peptides from hypothalamic cells by potassium and calcium follows classical neurosecretory mechanisms and suggests these neuropeptides could be neuroregulators in brain. PMID- 2995938 TI - Properties and distribution of vasoactive intestinal polypeptide receptors in the rat brain. AB - Vasoactive intestinal polypeptide (VIP) interaction with the rat brain synaptic membrane was examined using 125I-labeled VIP as a tracer molecule. Ion, pH and incubation time significantly influenced VIP receptor binding. Scatchard analysis suggested that the rat brain membrane had a single binding site with an apparent dissociation constant (Kd) of 9.8 X 10(-9) M. The receptor activity was sensitive to trypsin and phospholipase A digestion, and heating at 100 degrees C for 5 min completely destroyed the binding activity. This indicates that protein and phospholipid moieties are essential for VIP binding. Thiol reagents reduced receptor binding activity, suggesting that an intrachain disulfide bond might be an important constituent of the VIP binding site. High levels of binding were found in the hippocampus, striatum and cerebral cortex, and binding was very low in the medulla/pons and cerebellum. The receptor density did not always parallel the brain distribution of immunoreactive VIP (IR-VIP) concentration. The cerebral cortex had the highest ratio of IR-VIP-to-receptor, and the striatum had the lowest ratio of IR-VIP-to receptor. Although intra-nigral or intra-striatal injection of 6-hydroxydopamine had no effect on striatal VIP-binding, an intra striatal injection of kainic acid resulted in a substantial lowering of striatal VIP receptors. The neurotoxic effects of kainic acid have been shown to be dependent on the corticostriatal tract, and this suggests that the striatum receives the VIPergic innervation from the cerebral cortex. Our findings indicate that endogenous VIP and VIP receptors might act as a neurotransmission modulator of extrapyramidal function in the striatum. PMID- 2995936 TI - Cytomegalovirus pyloric obstruction in a child with acquired immunodeficiency syndrome. PMID- 2995939 TI - Central effects of mu, delta, and kappa receptor agonists in hemorrhagic shock. AB - We have recently shown that selective mu-opiate receptor agonists increase blood pressure and heart rate when injected into the anteroventral hypothalamus (AV3V) of the conscious rat, and that lesions of this area may worsen the effects of hypovolemia. These experiments suggested the possibility that mu-agonists might enhance cardiovascular recuperation following acute hemorrhagic shock. Sprague Dawley rats (250-300 g) were prepared with indwelling polyethylene catheters (under halothane anesthesia) in both femoral arteries, and a guide cannula to allow intrahypothalamic injections. Twenty-four hours after surgery, animals were made hypovolemic by withdrawing arterial blood (8.5 ml/300 g) over a 5 min period. After the bleeding, 0.9% NaCl or D-Ala2-D-Leu5-enkephalin (DADL; delta agonist), D-Ala2-MePhe4-Gly-ol-enkephalin (DAGO; mu-agonist) and U-50488H (trans(+)-dichloro-N-methyl-N-(2-[1-pryodynyl])-benzene-acetami ne, methane sulfate hydrate) (kappa-agonist) were injected in 1 microliter volume into the AV3V. Hemodynamic parameters were followed for 2 hr. Neither DADL nor U-50488H affected the blood pressure and heart rate responses to hemorrhage. In contrast, the mu-opiate receptor agonist, DAGO, enhanced the recovery of blood pressure and stimulated the heart rate. These data suggest that specific mu-opiate receptor agonists might possess beneficial effects in shock and trauma by enhancing cardiovascular recuperation through centrally-mediated mechanisms. PMID- 2995940 TI - Receptors for GRP/bombesin-like peptides in the rat forebrain. AB - Binding sites in the rat forebrain were characterized using (125I-Tyr4)bombesin as a receptor probe. Pharmacology experiments indicate that gastrin releasing peptide (GRP) and the GRP fragments GRP as well as Ac-GRP inhibited radiolabeled (Tyr4)bombesin binding with high affinity. Biochemistry experiments indicated that heat, N-ethyl maleimide or trypsin greatly reduced radiolabeled (Tyr4)bombesin binding. Also, autoradiographic studies indicated that highest grain densities were present in the stria terminalis, periventricular and suprachiasmatic nucleus of the hypothalamus, dorsomedial and rhomboid thalamus, dentate gyrus, hippocampus and medial amygdaloid nucleus. The data suggest that CNS protein receptors, which are discretely distributed in the rat forebrain, may mediate the action of endogenous GRP/bombesin-like peptides. PMID- 2995941 TI - Motor dysfunction after spinal cord injury is mediated by opiate receptors. AB - Naloxone, an opiate receptor antagonist, improves neurological outcome following traumatic cervical spinal cord injury in the cat and ischemic lumbar spinal cord injury in the rabbit. However, the doses of naloxone required have been quite high (greater than 1 mg/kg), suggesting that its beneficial actions are either not opiate receptor mediated or mediated by non-mu opiate receptors (which are less naloxone-sensitive). The kappa receptor appears to be the predominant opiate receptor in the spinal cord in a variety of species. For these reasons we evaluated the effects of a somewhat kappa selective opiate receptor antagonist WIN44,441-3 [WIN(-)] on neurological recovery following traumatic spinal injury in the cat and ischemic spinal injury in the rabbit. Animals treated with this more selective antagonist showed improved motor recovery as compared with animals treated with either physiological saline or with the dextroisomer of the WIN compound [WIN44,441-2; WIN(+)]. These findings support the hypothesis that motor dysfunction after spinal cord injury is in part mediated by opiate receptors and indicate that kappa selective opiate receptor antagonists may have particular therapeutic utility in spinal cord injury. PMID- 2995942 TI - Effects of guanine nucleotides, transition metals and temperature on enkephalin receptors of rat brain membranes. AB - The interactions among guanine nucleotides, transition metals and enkephalin (ENK) receptors were investigated by assaying the [3H]ENK binding to brain synaptic membranes. In the presence of 100 mM NaCl, GTP and Gpp(NH)p reduced the binding of [3H]ENK at 0 degree C and 25 degrees C, reflecting a decrease mainly in the affinity of the binding sites. In the absence of sodium, however, guanine nucleotides only slightly lowered the ENK binding at 25 degrees C, and at 0 degree C they strongly increased the ENK binding dose-dependently. This nucleotide-induced increase of specific ENK binding reflects an increase in the affinity and the number of the binding sites. In micromolar concentrations, zinc and cupric ions inhibited the ENK binding to opioid receptors by respectively reducing the affinity and the number of binding sites. In the absence of sodium at 0 degree C, cupric ions completely antagonized the nucleotide-induced increase in the ENK binding, although zinc ions did not affect it. The results suggest complex interactions of transition metals and guanine nucleotides with ENK binding sites. PMID- 2995944 TI - ACTH/MSH 4-10 improves motor unit reorganization during peripheral nerve regeneration in the rat. AB - ACTH/MSH 4-10 administration (10 micrograms/48 hr IP for 7 days) enhances neuromuscular activity following crush denervation of rat extensor digitorum longus muscle. The number of regenerated functional motor units is greater in peptide treated rats than in saline treated denervated controls. Selective activation of responding motor units indicates that ACTH/MSH 4-10 preferentially accelerates the reformation and stabilization of small size motor units. These observed effects may be beneficial since they contribute to the early reestablishment of more organized motor units, thereby restoring fine control of motor functions, in contrast to the disorderly reorganization of untreated regenerating systems. Possible mechanisms of peptide action (neurotransmitter synthesis and release, excitability changes, etc.) are discussed. PMID- 2995943 TI - Combined autoradiographic-immunocytochemical analysis of opioid receptors and opioid peptide neuronal systems in brain. AB - Using adjacent section autoradiography-immunocytochemistry, the distribution of [3H]naloxone binding sites was studied in relation to neuronal systems containing [Leu]enkephalin, dynorphin A, or beta-endorphin immunoreactivity in rat brain. Brain sections from formaldehyde-perfused rats show robust specific binding of [3H]naloxone, the pharmacological (mu-like) properties of which appear unaltered. In contrast, specific binding of the delta ligand [3H]D-Ala2,D-Leu5-enkephalin was virtually totally eliminated as a result of formaldehyde perfusion. Using adjacent section analysis, we have noted associations between [3H]naloxone binding sites and one, two, or all three opioid systems in different brain regions; however, in some areas, no apparent relationship could be observed. Within regions, the relationship was complex; for example, in caudate-putamen, patches of opioid receptors did not correspond to the distribution of enkephalin immunoreactivity, but there was a correspondence between subcallosal streaks of binding sites and enkephalin. The complexity of the association between [3H]naloxone binding sites and the multiple opioid systems, and previous reports of colocalization of mu and kappa receptors in rat brain, are inconsistent with a simple-one-to-one relationship between a given opioid precursor and opioid receptor subtype. Instead, since differential processing of the three precursors gives rise to peptides of varying receptor subtype potencies and selectivities, the multiple peptide-receptor relationships may point to a key role of post translational processing in determining the physiological consequences of opioid neurotransmission. PMID- 2995945 TI - Vasoactive intestinal peptide: expression of the prohormone in bacterial cells. AB - A complementary DNA (cDNA) to the messenger RNA for the preprohormone of human vasoactive intestinal peptide (VIP) has been isolated and characterized. This cDNA extends from 65 bases 5' of the AUG translation start codon through the entire 3' untranslated region. Using this cDNA we have constructed expression plasmids which allow the synthesis of 120 out of the 150 amino acids of the prohormone in E. coli. This portion of the prohormone gene was either fused to a segment of a bacteriophage structural gene or expressed alone. When expression was induced the fusion protein constituted 15% of the total bacterial cell protein while the prohormone alone was 5%. Both proteins are recognized by antiserum raised against porcine VIP. They provide protein to study the precursor product relationship of the hormone plus the possibility of identifying cryptic regulatory peptides contained within the prohormone. PMID- 2995946 TI - [Effect of naloxone on plasma renin activity and aldosterone level in the blood in patients with acute renal failure]. PMID- 2995947 TI - [Difficulties in the diagnosis of leprosy]. PMID- 2995948 TI - [Basic concepts of genetic engineering]. PMID- 2995949 TI - [A rare case of psychotic reaction following non-conventional methods of treatment]. PMID- 2995950 TI - [Cancer of the lung in women]. PMID- 2995951 TI - [Effect of the status of fitness and extent of pathological changes on prognosis in small-cell lung cancer]. PMID- 2995952 TI - Involvement of opioid and other systems in ethanol abstinence audiogenic seizures in the rat? AB - The action of opiate receptor agonists: (D-Ala2)-methionine enkephalinamide (D MEA), morphine, heroin, etorphine, and antagonists: naloxone and diprenorphine on audiogenic seizures was tested during ethanol abstinence. The action of diazepam and clonidine was also tested Morphine (5 and 20 mg/kg), but not heroin and etorphine, given intraperitoneally inhibited the seizures, similarly as intraventricularly administered D-MEA did. However, morphine given by this route was ineffective. Diazepam and clonidine inhibited audiogenic seizures: the action of clonidine was counteracted by yohimbine, but not by prazosin. The results may be considered as supporting the hypothesis on the participation of opioid system in ethanol abstinence. However, the participation of gabergic and noradrenergic systems cannot be ruled out: these systems may possibly interact with the opioid system in evoking the symptoms of ethanol abstinence. PMID- 2995953 TI - Effects of electroconvulsive shock on central GABA-ergic mechanisms. AB - A single electroconvulsive shock (ECS) has no influence on seizures induced by picrotoxin and bicuculline, although it decreases the level of GABA in the cortex. A repeated ECS (once daily for 7 days) does not change the level of GABA in the cortex, brain stem, and cerebellum, but depresses seizures induced by both compounds. It prolongs the time of their occurrence and decreases their intensity. This effect is stronger in the case of bicuculline. It manifests itself in the increase of the number of animals protected from seizures and decrease of their lethality. Baclofen does not change ECS action on seizures induced by picrotoxin and bicuculline whereas it enhances catalepsy caused by ECS. Bicuculline does not change the time-course of catalepsy. The obtained results suggest that repeated ECS reduces the seizures induced by GABA antagonists probably y increasing GABA-ergic transmission or/and by increasing dopaminergic and serotoninergic transmission which significantly modify the activity of GABA-ergic neurons. PMID- 2995955 TI - [5'-nucleotidase in various organs and the blood]. PMID- 2995956 TI - [Osmotic and non-osmotic regulation of vasopressin secretion]. PMID- 2995954 TI - Synthesis, receptor binding affinities and alpha-MSH releasing activities of TRH analogues. AB - New syntheses of three thyrotropin releasing hormone (TRH) analogues ([Dopa2]THR, [Nic1]TRH, and [Tyr(30NO2)2]TRH) have been reported (Dopa stands for L-3,4 dihydroxyphenylalanine, Nic--for nicotinic acid and Tyr(3-NO2)--for L-3 nitrotyrosine). These three TRH analogues and five already known ones ([Aad1Tca3]TRH, [D-His2]TRH, [D-Pro3]TRH, [Pro-NH-NH2(3)]TRH and [Tyr2]TRH), were studied in vitro for their binding activity to rat pituitary TRH receptors and a MSH releasing activity in the neuro-intermediate lobe of frogs. Competition of analogues for 3H-TRH binding to rat anterior pituitary membrane fraction was used. One of ten tested analogues ([Aad1, Tca]3 TRH) was as potent as TRH in competing for high-affinity binding sites (Kd = 8.5 nM). The binding activity of diastereoisomers ([D-His2]TRH and [D-Pro3]TRH) was reduced as well as that of analogue [Pro-NH-NH2(3)]TRH. The rest of the analogues were inactive. The binding activities were in good accordance with alpha-MSH releasing activities. PMID- 2995957 TI - Estimates of heritability of response to Rous sarcomas of chickens. AB - The stocks used for this investigation consisted of 1039 F3 generation progeny from the cross of two highly inbred lines and 355 and 462 offspring from subpopulations UNH 105A and UNH 105B, respectively, of a noninbred line of New Hampshires. Matings were such that B complex alleles were segregated in the three experimental populations with minor exceptions. Each chicken was inoculated at 6 weeks of age with a subgroup of Rous sarcoma virus (RSV). Resulting tumors were subjectively scored on a scale from 0 (no tumor) to 6 (massive tumor) six times during a 10-week experimental period. Based upon the six tumor scores, each chicken was then assigned a tumor profile index (TPI), a criterion of antitumor response. The TPI were analyzed by least squares analysis of variance and corrected for hatch, sex, and virus prior to obtaining components of variance and estimates of heritability from a nested analysis of variance. Estimates of heritability from the sire component ranged from 0 to .41 +/- .27 and from the dam component .18 +/- .09 to .26 +/- .14, which are rather low estimates in general. PMID- 2995959 TI - Effects of cellulase from Trichoderma viride on nutrient utilization by broilers. AB - Studies were conducted to evaluate the effects of cellulase from Trichoderma viride in a diet for broilers containing high levels of wheat bran. Broiler-type, mixed-sex chicks were fed from 3 to 8 weeks of age. Wheat bran was added at 0, 10, and 20% levels. A fourth group received the 20% wheat bran plus the cellulase enzyme added at the level of .008%. A portion of the chicks was used in a digestibility study with chromic oxide as an indicator. The summarized data showed that cellulose treatment had a significant effect on reducing feed consumption (P less than .01) and an apparent effect in improving feed-to-gain ratio. Cellulase supplementation significantly improved the digestibility of cell wall components (P less than .01). Calcium, phosphorus, iron, zinc, and copper associated with cell walls were solubilized by cellulase. Iron balance was negative in the groups without cellulase; however, iron, which is bound by the bran, apparently was made available for absorption by cellulase. PMID- 2995958 TI - Induction of urolithiasis in single comb white Leghorn pullets: effect on glomerular number. AB - Urolithiasis was induced in an experimental group of Single Comb White Leghorn pullets by feeding them layer ration and exposing them to nephrotrophic Gray strain infectious bronchitis virus (IBV). Gray strain IBV was recovered from kidney and cloacal swabs for up to 26 days after exposure to the virus. Control pullets fed pullet grower ration and not exposed to Gray strain IBV did not develop urolithiasis. The experimental design did not allow differentiation between the roles of layer ration and IBV in triggering urolithiasis. Urolithiasis was associated with asymmetry in left vs. right kidney weight comparisons for individual pullets. Pullets from the urolithiasis group had 43,800 +/- 4,500 glomeruli/gram kidney weight, whereas control pullets had 68,770 +/- 3,500 glomeruli/gram kidney weight. This difference was significant (P less than .01). Total kidney weights did not differ significantly when the experimental and control pullets were compared. Comparisons of glomeruli size distributions indicated that the number of intermediate sized glomeruli (.15 to .22 mm in circumference) was significantly reduced in pullets from the urolithiasis treatment group. These observations indicate that a significant reduction in nephron number can be masked by compensatory hypertrophy of the remaining kidney tissue in pullets with urolithiasis. PMID- 2995960 TI - [Polypoid gangliocytic paraganglioma of the duodenum]. PMID- 2995961 TI - [Mucosecreting tumor of the appendix with hepatic metastases]. PMID- 2995962 TI - [Acute polyarthritis associated with Sweet's syndrome]. AB - A 43-year old man presents with acute febrile neutrophilic dermatosis (Sweet's syndrome) associated with acute seronegative polyarthritis. Although no definite diagnosis of viral infection could be made, the patient had raised serum antibodies against cytomegalovirus (1/1280; 1/2560). Histological examination showed typical lesions of Sweet's syndrome as well as the presence of extra- and intracellular unidentified particles in histiocytes. PMID- 2995963 TI - [Recurring anguilluliasis and pleomorphic T lymphoma associated with anti-HTLV-I antibodies]. PMID- 2995964 TI - [Effect of the cultivation conditions and ultrasonic rupture of the biomass of Streptomyces achromogenes ATCC 12767 on the yield of restriction endonucleases]. PMID- 2995965 TI - Binding of protein kinase C to neutrophil membranes in the presence of Ca2+ and its activation by a Ca2+-requiring proteinase. AB - In the presence of micromolar concentrations of Ca2+, both protein kinase C and a cytosolic Ca2+-requiring neutral proteinase of human neutrophils become associated with the neutrophil membrane. Binding to the membrane results in activation of the proteinase, which then catalyzes limited proteolysis of the kinase to produce a form that is fully active in the absence of Ca2+ and phospholipid. This irreversibly activated protein kinase is released from the membrane and may thus have access, in the intact cell, to intracellular protein substrates. In the absence of the proteinase, Ca2+ promotes the binding of protein kinase C, but conversion to the Ca2+/phospholipid-independent form does not occur and the kinase remains associated with the membrane fraction. PMID- 2995966 TI - Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures. AB - Formaldehyde (HCHO) produces DNA-protein crosslinks both in vitro and in vivo. Simian virus 40 (SV40) chromosomes that have been fixed by prolonged incubation with HCHO either in vitro or in vivo (within SV40-infected cells) can be converted to nearly protein-free DNA by limit-digestion with Pronase in the presence of NaDodSO4. The remaining Pronase-resistant DNA-peptide adducts retard the DNA upon gel electrophoresis, allowing resolution of free and crosslink containing DNA. Though efficiently crosslinking histones to DNA within nucleosomes both in vitro and in vivo, HCHO does not crosslink either purified lac repressor to lac operator-containing DNA or an (A + T)-DNA-binding protein (alpha-protein) to its cognate DNA in vitro. Furthermore, a protein that does not bind to DNA, such as serum albumin, is not crosslinked to DNA by HCHO even at extremely high protein concentrations. These properties of HCHO as a DNA-protein crosslinker are used to probe the distribution of nucleosomes in vivo. We show that there are no HCHO-crosslinkable DNA-protein contacts in a subset of SV40 chromosomes in vivo within a 325-base-pair stretch that spans the "exposed" (nuclease-hypersensitive) region of the SV40 chromosome. This replication origin proximal region has been found previously to lack nucleosomes in a subset of isolated SV40 chromosomes. We discuss other applications of the HCHO technique, including the possibility of obtaining base-resolution in vivo nucleosome "footprints." PMID- 2995967 TI - A v-erbB-related protooncogene, c-erbB-2, is distinct from the c-erbB-1/epidermal growth factor-receptor gene and is amplified in a human salivary gland adenocarcinoma. AB - From a human genomic library, we obtained six v-erbB-related DNA clones. A DNA probe prepared from one of the clones, lambda 107, hybridized to EcoRI fragments of 6.4 and 13 kilobase pairs of human DNA. Neither of these fragments was amplified in A431 vulva carcinoma cells, in which the gene encoding the epidermal growth factor receptor is amplified. In addition, the probe from lambda 107 hybridized with a single, 4.8-kilobase poly(A)+ RNA species and did not react with EGF receptor mRNA. Thus, we conclude that clone lambda 107 represents a v erbB-related gene (c-erbB-2) that is distinct from the EGF receptor gene. In contrast, the other five clones were shown to represent the EGF receptor gene (c erbB-1). Partial nucleotide sequence analysis of the lambda 107 insert showed that this clone contained at least seven putative exons and that six of them could encode the kinase domain characteristic of protein products of the src oncogene family. Southern blot analysis showed close similarity of the restriction patterns of the rat c-erbB-2 gene and the rat neu oncogene, suggesting possible involvement of c-erbB-2 in human cancer. In fact, approximately 30-fold amplification of c-erbB-2 was observed in a human adenocarcinoma of the salivary gland. PMID- 2995968 TI - Location of cis-acting regulatory sequences in the human T-cell leukemia virus type I long terminal repeat. AB - The location of cis-acting regulatory regions within the long terminal repeat (LTR) of the human T-cell leukemia virus type I (HTLV-1) was determined. The sequences present between nucleotides -350 and -55 (cap site +1) contain an enhancer element that is active in lymphoid and nonlymphoid cell lines. The sequences located near the "TATA" and RNA initiation sites contain a promoter, the activity of which can be augmented by homologous and heterologous enhancer elements. A region responsive to trans-acting transcription factors present in HTLV-I- and HTLV type II-infected cells is located between nucleotides -159 and +315. HTLV-I LTR deletion mutants respond in a similar manner both to the trans acting factors present in infected cells and to the tat protein encoded by the x lor region of the genome, thus providing further evidence that the tat protein mediates transcriptional trans-activation of the LTR in HTLV-infected cells. PMID- 2995970 TI - Active auxin uptake by zucchini membrane vesicles: quantitation using ESR volume and delta pH determinations. AB - Closed and pH-tight membrane vesicles prepared from hypocotyls of 5-day-old dark grown seedlings of Cucurbita pepo accumulate the plant growth hormone indole-3 acetic acid along an imposed proton gradient (pH low outside, high inside). The use of electron paramagnetic spin probes permitted quantitation both of apparent vesicle volume and magnitude of the pH gradient. Under the experimental conditions used, hormone accumulation was at minimum 20-fold, a value 4 times larger than what one would predict if accumulation reflected only diffusional equilibrium at the measured pH gradient. It is concluded that hormone uptake is an active process, with each protonated molecule of hormone accompanied by an additional proton. Experiments with ionophores confirm that it is the pH gradient itself which drives the uptake. PMID- 2995969 TI - The oncofetal domain of fibronectin defined by monoclonal antibody FDC-6: its presence in fibronectins from fetal and tumor tissues and its absence in those from normal adult tissues and plasma. AB - An IgG1 monoclonal antibody, FDC-6, was established, which defines a unique fibronectin (FN) domain, located between the "Hep-2" and the "Fib-2" domains, in the COOH-terminal region of FNs isolated from hepatoma, sarcoma, and fetal fibroblasts. A systematic study with this antibody indicates the presence of two classes of human FNs. (i) FN from fetal connective tissue, placenta, amniotic fluid, hepatoma, and colon carcinoma as well as cell lines from fetal tissues (WI 38), hepatomas (HuH-6 and HuH-7), and sarcoma (VA13) was characterized by the presence of the FDC-6-defined domain and by a high molecular weight (subunit Mr, 310,000-335,000). (ii) In contrast, FN from normal adult tissues and plasma was characterized by a lower molecular weight (subunit Mr, 285,000-295,000) and lack of reactivity with FDC-6 and is therefore devoid of the FDC-6-defined domain. The FDC-6-defined domain is therefore called the "oncofetal" domain, and FN containing this domain is hereby called "oncofetal FN" (onf FN). The onf FN is similar to the previously known "cellular-form" FN. FN from normal adult tissues and plasma, lacking the oncofetal domain, is hereby called "normal FN" (nor FN). The nor FN is similar to the previously known "plasma-form" FN. Development of FN from fetal to adult form is associated with loss of the oncofetal domain defined by the FDC-6 antibody, and oncogenic transformation is associated with activation in synthesis of the oncofetal domain defined by the FDC-6 antibody. PMID- 2995971 TI - Peroxisomal organization in normal and cerebrohepatorenal (Zellweger) syndrome fibroblasts. AB - The reported absence of morphologically detectable peroxisomes in liver and kidney tissue cells from patients affected by the autosomic recessive, inherited metabolic disease known as cerebrohepatorenal, or Zellweger, syndrome was studied in fibroblasts, assuming it to be a generalized defect. Normal cultured fibroblasts were shown to contain peroxisomes according to morphological, biochemical, and subcellular fractionation criteria: particle-bound catalase and fatty acyl-CoA oxidase copurify in subcellular fractionation by differential centrifugation or isopycnic equilibrium in continuous density gradients and peroxidase-positive organelles of approximately equal to 0.1 micron in diameter are detected in the cytoplasm. In Zellweger cultured fibroblasts, these peroxisomal enzymes are present; however, they behave as cytosolic enzymes in the different subcellular fractionation procedures employed and peroxisomes are not detected cytochemically. These findings support the hypothesis that the lack of peroxisomes in this genetic disease is the consequence of a defect in the assembly of the peroxisomal constituents. Furthermore, the value of fibroblasts for subcellular analysis of peroxisomal defects is illustrated. PMID- 2995972 TI - Isolation and chromosomal localization of the human fgr protooncogene, a distinct member of the tyrosine kinase gene family. AB - The cell-derived domain of Gardner-Rasheed feline sarcoma virus (GR-FeSV) consists of a gamma-actin- and a tyrosine-specific protein kinase-encoding sequence designated v-fgr. By utilizing a v-fgr probe, it was possible to detect related sequences present at low copy number in DNAs of a variety of mammalian species and to isolate a human fgr homologue. Comparative studies revealed that this human DNA clone represented all but 200 base pairs of v-fgr. Analysis of human genomic DNA demonstrated that the fgr protooncogene was distinct from the cellular homologues of other retrovirus onc genes. In addition, the fgr protooncogene was localized to the distal portion of the short arm of human chromosome 1 at p36.1-36.2 by in situ hybridization. Taken together, our findings establish that the fgr protooncogene is a unique member of the tyrosine kinase gene family. PMID- 2995974 TI - UV-induced mutagenesis of phage S13 can occur in the absence of the RecA and UmuC proteins of Escherichia coli. AB - The UV-induced mutagenesis of phage S13 that accompanies Weigle repair is known to require the products of the recA and umuDC genes, as does the UV-induced mutagenesis of the Escherichia coli chromosome. I found that UV-induced mutagenesis of phage S13 occurred in the absence of both the RecA and UmuC functions when the irradiated phage was photoreactivated. Furthermore, UV-induced phage mutations were produced in a recA- umuC- cell even without photoreactivation and in the absence of any other known UV repair mechanism, at a frequency 29% of that found after photoreactivation and 7% of that found after Weigle repair, implying that DNA synthesis can proceed past a dimer at an unexpectedly high frequency even when unaided by the UmuC-RecA SOS repair functions. The unaided DNA synthesis appears capable of producing mutations in the vicinity of a pyrimidine dimer; by aiding synthesis past a dimer, a repair mechanism may disclose a mutation without having any active role in producing it. PMID- 2995973 TI - Retroviral insertional mutagenesis of a target allele in a heterozygous murine cell line. AB - A clonal murine cell line that is heterozygous at the beta 2-microglobulin locus (B2m) was obtained by Abelson murine leukemia virus (Ab-MuLV) transformation of liver cells from (C57BL/6 X BALB/c) F1 fetuses. To obtain proviral insertional mutants, we superinfected a subclone of these cells, which does not express the env surface protein of the Moloney leukemia virus (Mo-MuLV, the helper virus that was used to transmit the defective Ab-MuLV genome during transformation), with Mo MuLV. Mutant clones that fail to express the C57BL/6 allele of B2m (B2mb) were then immunoselected by using a monoclonal antibody that specifically recognizes the B2mb gene product and not that of the B2ma allele. Of 22 independent clones obtained, one contains a proviral insertion that is near or in the first exon of the B2mb gene. Surprisingly, the insertion is of the Ab-MuLV genome and not of replication-competent Mo-MuLV. This indicates that superinfection with Mo-MuLV "rescued" the defective Ab-MuLV genome, which then inserted into the B2mb gene. We conclude that when an allele-specific selection procedure exists, proviral insertion is a potential method for obtaining mutations in heterozygous mammalian cells. This approach may thereby provide a method for molecular cloning of such selectable genes, using a retroviral hybridization probe. PMID- 2995975 TI - One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells. AB - We have developed a host cell reactivation assay of DNA repair utilizing UV treated plasmid vectors. The assay primarily reflects cellular repair of transcriptional activity of damaged DNA measured indirectly as enzyme activity of the transfected genes. We studied three plasmids (pSV2cat, 5020 base pairs; pSV2catSVgpt, 7268 base pairs; and pRSVcat, 5027 base pairs) with different sizes and promoters carrying the bacterial cat gene (CAT, chloramphenicol acetyltransferase) in a construction that permits cat expression in human cells. All human simian virus 40-transformed cells studied expressed high levels of the transfected cat gene. UV treatment of the plasmids prior to transfection resulted in differential decrease in CAT activity in different cell lines. With pSV2catSVgpt, UV inactivation of CAT expression was greater in the xeroderma pigmentosum group A and D lines (D0 = 56 J X m-2) than in the other human cell lines tested (normal, ataxia-telangiectasia, Lesch-Nyhan, retinoblastoma)(D0 = 680 J X m-2)(D0 is the dose that reduces the percentage of CAT activity by 63% along the exponential portion of the dose-response curve). The D0 of the CAT inactivation curve was 50 J X m-2 for pSV2cat and for pRSVcat in the xeroderma pigmentosum group A cells. The similarity of the D0 data in the xeroderma pigmentosum group A cells for three plasmids of different size and promoters implies they all have similar UV-inactivation target size. UV-induced pyrimidine dimer formation in the plasmids was quantified by assay of the number of UV induced T4 endonuclease V-sensitive sites. In the most sensitive xeroderma pigmentosum cells, with all three plasmids, one UV-induced pyrimidine dimer inactivates a target of about 2 kilobases, close to the size of the putative CAT mRNA. PMID- 2995976 TI - Leukotriene C4 production by murine mast cells: evidence of a role for extracellular leukotriene A4. AB - The glutathione-containing leukotriene C4 (LTC4) is a major mediator of smooth muscle contraction and is released by mast cells when antigen interacts with cell bound IgE. Antigen-stimulated mast cells undergo phospholipase activation. We report a pathway of LTC4 production by mast cells that does not require phospholipase activation but depends on the interaction of activated neutrophils with unstimulated mast cells, using as an intermediate extracellular leukotriene A4 (LTA4). The epoxide LTA4 is released by neutrophils and, together with leukotriene B4 and 5-hydroxyeicosatetraenoic acid, constitutes the major lipoxygenase metabolites found in supernatants of stimulated neutrophils. Five minutes after activation of neutrophils by calcium ionophore A23187 we measured 136 pmol of extracellular LTA4 per 10(7) neutrophils (range 40-300, n = 7) by trapping the epoxide with alcohols. Therefore, we conclude that LTA4 is not just an intracellular leukotriene precursor but is released as a lipoxygenase metabolite. LTA4 is known to be stabilized by albumin and is efficiently converted by mast cells into LTC4 even at low LTA4 concentrations. The LTA4 complexed to albumin is converted into LTC4 rapidly and completely within 10-15 min. More than 50% of the LTA4 presented to mast cells is metabolized to LTC4 at concentrations of LTA4 between 0.2 and 2 nmol of LTA4 per 10(7) mast cells. This observation establishes a potential physiologic role for extracellular LTA4. Therefore, interactions between various cell types that release or utilize LTA4 may provide an important metabolic pathway for the production of leukotrienes. PMID- 2995978 TI - Visualization of mu1 opiate receptors in rat brain by using a computerized autoradiographic subtraction technique. AB - We have developed a quantitative computerized subtraction technique to demonstrate in rat brain the regional distribution of mu1 sites, a common very high-affinity binding site for both morphine and the enkephalins. Low concentrations of [D-Ala2, D-Leu5]enkephalin selectively inhibit the mu1 binding of [3H]dihydromorphine, leaving mu2 sites, while low morphine concentrations eliminate the mu1 binding of [3H][D-Ala2, D-Leu5]enkephalin, leaving delta sites. Thus, quantitative differences between images of sections incubated in the presence and absence of these low concentrations of unlabeled opioid represent mu1 binding sites. The regional distributions of mu1 sites labeled with [3H]dihydromorphine were quite similar to those determined by using [3H][D-Ala2, D-Leu5]enkephalin. High levels of mu1 binding were observed in the periaqueductal gray, medial thalamus, and median raphe, consistent with the previously described role of mu1 sites in analgesia. Other regions with high levels of mu1 binding include the nucleus accumbens, the clusters and subcallosal streak of the striatum, hypothalamus, medial habenula, and the medial septum/diagonal band region. The proportion of total specific binding corresponding to mu1 sites varied among the regions, ranging from 14% to 75% for [3H][D-Ala2, D Leu5]enkephalin and 20% to 52% for [3H]dihydromorphine. PMID- 2995977 TI - Glial cells metabolically cooperate: a potential requirement for gene replacement therapy. AB - Immunofluorescently labeled glial cells are shown by radioautography to metabolically cooperate with hypoxanthine phosphoribosyltransferase-deficient fibroblasts. The observations of cooperation without cell contact and of incorporation gradients around the glial cells suggest that cooperation occurs through extracellular transport of radiolabeled purine compounds. The transfer of radiolabeled adenine, adenosine, or methylthioadeninosine is supported by the quantitative loss of cooperation when the recipient cell is also deficient in enzymes required for adenine or adenosine salvage. The demonstration of glial cell cooperation provides impetus for current research toward gene replacement therapy for the neurologic symptoms of the Lesch-Nyhan syndrome. PMID- 2995979 TI - Correlation of cell-surface phenotype with the establishment of interleukin 3 dependent cell lines from wild-mouse murine leukemia virus-induced neoplasms. AB - The wild mouse ecotropic virus, Cas-Br-M murine leukemia virus, induces myeloid and erythroid leukemias as well as T-cell and B-cell lymphomas in NFS mice. The ability to establish long-term cell lines from these tumors in the presence or absence of the T-cell-derived lymphokine interleukin 3 (IL-3) was examined. IL-3 dependent cell lines were readily obtained from the majority of the myeloid or erythroid leukemias and immunoblastic lymphomas. In the absence of IL-3, only one long-term factor-independent cell line was obtained from a myelogenous leukemia. The majority of the thymic T-cell lymphomas or B-lineage lymphomas could not be cultured in the presence or absence of IL-3. The results suggest that transformation of hematopoietic lineages does not necessarily obviate the requirement for normal growth factors. The acquisition of independence from growth factors may require additional transforming events. PMID- 2995980 TI - Benzodiazepine/gamma-aminobutyric acid receptor deficit in the midbrain of the seizure-susceptible gerbil. AB - The density of benzodiazepine/gamma-aminobutyric acid receptor binding sites was lower in the midbrain of seizure-susceptible gerbils compared to control seizure resistant gerbils. Binding of [3H]diazepam to high-affinity brain-specific sites in membrane homogenates of gerbil brain showed a 20-30% lower binding in midbrain (but not other regions) in adult seizure-susceptible gerbils than in controls. This binding deficit was localized by tissue slice autoradiography with [3H]flunitrazepam to the substantia nigra and mesencephalic periaqueductal gray regions, while higher binding was observed in the interpeduncular nucleus. These differences were also seen in animals sacrificed immediately after a seizure. A parallel deficit of [3H]bicuculline methochloride binding to low-affinity gamma aminobutyric acid receptors also was seen in the same midbrain regions. Scatchard plot analysis showed that the benzodiazepine binding deficit in the nigra was due to a lower number of binding sites with not significant difference in affinity. Lower [3H]flunitrazepam binding was likewise seen in younger animals (29% lower at 30 days of age, 38% at 60 days, and 21% at 90 days), indicating that the midbrain receptor deficit is present in the seizure-susceptible gerbil prior to the age of onset of seizures at 50-100 days. Therefore, these changes are not likely to result from seizures but reflect genetically determined biochemical differences that could play a role in the expression of seizure susceptibility. The deficit in midbrain benzodiazepine/gamma-aminobutyric acid receptors in the seizure-susceptible gerbil would be consistent with the hypothesis that a deficit of gamma-aminobutyric acid-mediated inhibition might contribute to some kinds of epilepsy. PMID- 2995981 TI - Adrenergic regulation of gluconeogenesis: possible involvement of two mechanisms of signal transduction in alpha 1-adrenergic action. AB - We have previously suggested that the effects of alpha 1-adrenergic agents on hepatocyte metabolism involve two mechanisms: (i) a calcium-independent insulin sensitive process that is modulated by glucocorticoids and (ii) a calcium dependent insulin-insensitive process that is modulated by thyroid hormones. We have studied the effect of epinephrine (plus propranolol) on gluconeogenesis from lactate and dihydroxyacetone. It was observed that the adrenergic stimulation of gluconeogenesis from lactate seemed to occur through both mechanisms, whereas when the substrate was dihydroxyacetone the action took place exclusively through the calcium-independent insulin-sensitive process. This effect was absent in hepatocytes from adrenalectomized rats, suggesting that it is modulated by glucocorticoids. PMID- 2995982 TI - Use of a protein-blotting procedure and a specific DNA probe to identify nuclear proteins that recognize the promoter region of the transferrin receptor gene. AB - We describe a procedure for detecting high-affinity, sequence-specific DNA binding proteins from crude nuclear extracts. The technique utilizes electrophoretic transfer of NaDodSO4/PAGE-fractionated proteins onto nitrocellulose filters. Incubation of the filters with a 5% (wt/vol) solution of nonfat dry milk effectively blocks nonspecific and low-affinity DNA-binding sites. Incubation of the blocked filters with radiolabeled DNA under optimal binding conditions and subsequent autoradiography reveals high-affinity DNA protein interactions. We have used this procedure to identify proteins that bind specifically to the promoter region of the transferrin receptor gene. PMID- 2995983 TI - Methionine-sensitive glycolysis in transformed cells. AB - Glycolysis in several tumor cell lines grown in tissue culture was inhibited by methionine. Kirsten murine sarcoma virus-transformed rat kidney cells (K-NRK) were inhibited 60-75% by 10 mM methionine, whereas normal rat kidney (NRK-49F) cells showed little or no inhibition. Inhibition of glycolysis in K-NRK cells was manifest 2-4 hr after exposure to the amino acid. Glycolysis in a chemically transformed cell line of Madin-Darby canine kidney cells was also sensitive to methionine, but maximal inhibition (75%) required 18-24 hr of incubation with the amino acid. Under the same conditions glycolysis in the nontransformed canine cells was less than 20% inhibited by methionine. In Ehrlich ascites tumor cells grown in tissue culture, 10 mM methionine inhibited glycolysis by about 50%. Inhibition of glycolysis, even by 50 mM methionine, was rapidly reversible. Within 2 hr after removal of methionine the rate of glycolytic activity was restored to that observed in control cells. Furthermore, inhibition by methionine required a minimum level (7%) of serum in the growth medium and inhibition was not sensitive to cycloheximide. Only amino acids that are transported by system A (including the nonmetabolized analogue methylaminoisobutyric acid) specifically inhibited glycolysis in tumor cells. The only exception was phenylalanine, which was toxic to both transformed and normal cell lines. PMID- 2995984 TI - Bacteriophage T7 DNA polymerase: cloning and high-level expression. AB - Phage T7 DNA polymerase consists of a 1:1 complex of the viral T7 gene 5 protein and the host cell thioredoxin. A 3.25-kilobase T7 DNA fragment containing the complete coding sequence of gene 5, and the nearby genes 4.7 and 5.3, was cloned in the BamHI site of the plasmid pBR322. Transformation of the thioredoxin negative (trxA-) Escherichia coli strain BH215 with the recombinant plasmid pRS101 resulted in large overproduction of gene 5 protein corresponding to a level about 60-fold higher than in T7-infected cells. Transcription of gene 5 probably originates from a previously unknown E. coli RNA polymerase promoter located immediately upstream of the structural gene. Contrary to expectation, pRS101 could be maintained also in E. coli trxA+ cells despite the in vivo formation of active T7 DNA polymerase. However, the expression of gene 5 was lower by a factor of 5-10 than in trxA- cells. Since the plasmid copy number in the two strains was the same, a gene dosage effect can be excluded. The observed difference suggests an autoregulatory interaction of T7 DNA polymerase holoenzyme on the expression of T7 gene 5. The trxA- strain BH215/pRS101 is an excellent source of gene 5 protein and T7 DNA polymerase. After in vitro reconstitution of holoenzyme by addition of excess thioredoxin, highly active T7 DNA polymerase was purified to homogeneity by a simple antithioredoxin immunoadsorbent chromatography technique. PMID- 2995985 TI - Oligomeric structure of the multifunctional protein CAD that initiates pyrimidine biosynthesis in mammalian cells. AB - The first three steps in mammalian de novo pyrimidine biosynthesis are catalyzed by the multifunctional protein designated CAD. Regions of the single 240-kDa poly peptide chain are folded into separate structural domains that have discrete functions. Previous studies suggested that CAD forms predominantly trimers. The trimers are found to be in slow equilibrium with hexamers and higher oligomers composed of multiples of three copies of the CAD polypeptide chain. However, quantitative chemical crosslinking studies of CAD with dimethyl suberimidate were used here to show a progressive conversion of monomer to crosslinked hexamer. High levels of the hexamer accumulate in the reaction mixture, suggesting that the major oligomeric form is hexameric, although residual amounts of smaller oligomers remain present. Larger oligomers may form by association of hexamers and are seen after longer crosslinking times. Sucrose gradient centrifugation shows a 20.8S species to be the slowest sedimenting peak, while the larger species sediments at 27.9S. Electron microscopic studies of rotary-shadowed preparations of CAD have confirmed that, while small amounts of other oligomeric forms are present, the CAD monomer is primarily associated into cyclic hexamers with an open planar appearance. PMID- 2995986 TI - Evidence that an iron-nickel-carbon complex is formed by reaction of CO with the CO dehydrogenase from Clostridium thermoaceticum. AB - The interaction between carbon monoxide and the CO dehydrogenase from Clostridium thermoaceticum was studied by electron spin resonance (ESR) techniques. When the enzyme reacts with CO, a paramagnetic complex is formed which previously was shown, by isotope substitution, to be due to a nickel-carbon species. In this paper, we demonstrate that iron is also a component of this ESR-detectable complex. When the iron in the enzyme is replaced with 57Fe, a broadening of 18 G in the g parallel and 7 G in the g perpendicular region is seen. This hyperfine interaction is probably due to more than one iron atom in the complex. Coenzyme A influences this ESR spectrum. In the absence of CoA, the ESR spectrum consists of two superimposed signals, which were simulated using the following ESR parameters: signal 1, with g = 2.074 and g = 2.028, and signal 2 with gx = 2.062, gy = 2.047, and gz = 2.028. CoA converts signal 2 into signal 1. Since iron, nickel, and carbon all are part of this ESR-detectable complex, we propose that these atoms exist in a spin-coupled complex with net spin = 1/2, analogous to other iron-sulfur centers in which the metals are bridged by acid-labile sulfide. PMID- 2995987 TI - Locating a nucleotide-binding site in the thymidine kinase of vaccinia virus and of herpes simplex virus by scoring triply aligned protein sequences. AB - Computer techniques were used to locate related segments of amino acid sequences in the thymidine kinases of vaccinia virus and of herpes simplex virus type 1 and in porcine adenylate kinase. As determined by a procedure that evaluates triply aligned sequences, the probability that the similarities among the segments described here arose by chance was no greater than 0.001. Because the sequence in porcine adenylate kinase is a nucleotide phosphate-binding site it is concluded that the segments in the vaccinia virus and herpes simplex virus thymidine kinases perform similar functions. The segments are residues 16-23 in porcine adenylate kinase, 11-19 in vaccinia virus thymidine kinase, and 56-64 in herpes simplex virus thymidine kinase. PMID- 2995988 TI - The production of DNA strand breaks in human leukocytes by superoxide anion may involve a metabolic process. AB - H2O2 is known to be capable of inducing strand-break damage in intracellular DNA, but whether O2- also can do so in the absence of H2O2 is uncertain. The difficulty in distinguishing the effects of the two is that, under physiological conditions, dismutation of O2- to H2O2 can readily occur. When human leukocytes are stimulated with phorbol 12-myristate 13-acetate (PMA), they release O2-, and within a few minutes strand breakage in intracellular DNA can be observed. We have attempted to determine whether the O2- produced is itself capable of causing DNA damage or whether H2O2 alone, or in combination with O2-, is responsible for the observed damage. Addition of catalase (up to 250 micrograms/ml) to remove H2O2 prevented no more than about 50% of the DNA damage. The majority of the remaining damage could be blocked, in a dose-dependent manner, by superoxide dismutase (SOD) or a SOD-mimetic copper complex, identifying a fraction of damage to intracellular DNA dependent upon extracellular O2-. We studied this O2(-) specific fraction through the use of three metabolic poisons (fluoride, 2 deoxyglucose, and A23187). These agents largely blocked DNA damage, while affecting extracellular O2- levels only slightly. For comparison, H2O2-induced DNA damage was studied with glucose oxidase to generate a flux of H2O2. The first two metabolic poisons had little effect, whereas A23187 did inhibit H2O2-induced DNA damage. We conclude that O2(-)-induced damage occurs through a mechanism that differs, at least in part, from the H2O2 damage pathway and that the former may involve one or more metabolic steps. PMID- 2995989 TI - Human apolipoprotein B cDNA clone isolation and demonstration that liver apolipoprotein B mRNA is 22 kilobases in length. AB - An expression library made in plasmids pUC8 and pUC9 with mRNA derived from the human hepatoma cell line HepG2 was screened with a rabbit antiserum to human low density lipoprotein (LDL). Approximately 12,000 clones were screened and five positives were identified. The cDNA inserts were all 1500-1600 base pairs in length. The insert from one clone, pB8, was isolated, labeled by nicktranslation, and found to cross-hybridize strongly with the other four cDNA clones. The pB8 clone produces a fusion protein of approximately equal to 37.5 kDa that reacts in electrophoretic transfer blot analysis with rabbit anti-human LDL. This reactivity can be abolished by pretreatment of the antiserum with purified human LDL, p = 1.025 - 1.050 g/ml. A pB8-derived probe was used to demonstrate that apolipoprotein B (apo B) mRNA is present in HepG2 cells and liver extracts but not in HeLa cells or extracts from small intestine, heart, aorta, spleen, brain, skeletal muscle, lung, kidney, or ovary. RNA transfer blot analysis revealed that HepG2 cell apo B mRNA was approximately equal to 22 kilobases in length. These cDNA clones should allow the isolation of the apo B gene and ultimately the elucidation of the primary structure of this protein. PMID- 2995990 TI - Identification of distinct receptor complexes that account for high-and low affinity glucagon binding to hepatic plasma membranes. AB - We have analyzed ligand-receptor complexes resulting from (i) the incubation of canine hepatic plasma membranes with [125I]iodoglucagon and (ii) subsequent gentle solubilization of receptor-bound ligand with digitonin. The complexes (molecular weight approximately equal to 500,000) retain the radiolabeled ligand during gel filtration and subsequent manipulation at 4 degrees C in the absence of covalent crosslinking. Affinity chromatography of the glucagon-receptor complexes on columns of wheat germ lectin linked to agarose resulted in two fractions, one of which was not bound by the column and the other of which was specifically eluted by N-acetylglucosamine. The presence of GTP during the incubation of plasma membranes with [125I]iodoglucagon caused about a 50% decrease in total ligand binding but affected only the ligand-receptor complexes that bound to wheat germ lectin. Moreover, it was found that the proportion of the two forms of ligand-receptor complexes identified by chromatography on wheat germ lectin depended on the degree of saturation of the membrane receptor. Thus, both the inhibition by glucagon of radiolabeled glucagon binding to membranes and the concomitantly decreased extent of association of the radiolabeled ligand with solubilized receptor complexes could be modeled in terms of two noninteracting receptor populations (having dissociation constants of about 0.35 and 4.94 X 10( 9) M). We conclude that (i) glucagon-receptor complexes formed on canine hepatic plasma membranes exist in two forms that differ after solubilization by digitonin in their avidities for wheat germ lectin, (ii) the high-and low-affinity binding of glucagon characteristic of hepatic plasma membranes arises from distinct receptor populations that probably differ in glycosylation, and (iii) the effect of GTP to decrease binding of glucagon to membranes arises from interactions of the nucleotide with the receptor complex that binds to wheat germ lectin. PMID- 2995991 TI - The replication initiator protein of plasmid pT181 has sequence-specific endonuclease and topoisomerase-like activities. AB - Initiation of pT181 DNA replication specifically requires the plasmid-encoded RepC protein. Here we demonstrate that highly purified RepC protein has sequence specific endonuclease and topoisomerase-like activities. A maximum sequence of 127 base pairs containing the pT181 origin of replication is required for nicking closing by RepC protein. RepC introduces a single strand break within the pT181 origin. The nick site has been shown by DNA sequencing to lie between nucleotides 70 and 71 in the bottom strand of the DNA within the origin sequence. This nick site probably corresponds to the start site of pT181 replication. The results presented here suggest that, unlike most other plasmids, pT181 replicates by a rolling circle mechanism. PMID- 2995992 TI - Induction of the early stages of Friend erythroleukemia with helper-free Friend spleen focus-forming virus. AB - The polycythemia-inducing strain of Friend virus (FV-P) causes a multistage erythroleukemia in susceptible mice. FV-P is a complex of two viruses, a replication-competent virus [Friend murine leukemia virus (F-MuLV)] and a replication-defective spleen focus-forming virus (SFFVp). We have addressed directly the role of SFFVp in the induction of the early stages of Friend disease by constructing stocks of SFFVp free of detectable F-MuLV, using a recently described retroviral helper-cell line. These preparations are capable of inducing erythroid bursts (vBFU-E) whose inducibility, kinetics, and responsiveness to erythropoietin suggest that they are very similar, if not identical, to the vBFU E induced by FV-P. Single injections of helper-free SFFVp had no apparent effects in vivo, although the addition of exogenous helper virus to the inoculum resulted in the induction of classic Friend disease. Increasing the effective titer by giving mice five daily virus injections resulted in the induction of splenomegaly and a large increase in the number of erythroid colony-forming units that were independent of erythropoietin. When the injections were discontinued, the spleens regressed and all the mice survived. When the injections were continued, all the mice died within 25 days of the first injection. These results demonstrate that SFFVp alone can alter the growth characteristics of erythroid progenitors and is directly responsible for the induction of vBFU-E in vitro and the erythroid hyperplasia in vivo. They also demonstrate that the initial polyclonal stage of Friend disease is reversible and can be reproduced by using preparations of SFFVp free of detectable F-MuLV. PMID- 2995993 TI - Alteration of leukotriene release by macrophages ingesting Toxoplasma gondii. AB - Mouse resident peritoneal macrophages incubated with ionophore A23187 or opsonized zymosan released leukotrienes (LT) B4 and C4 (LTB4 and LTC4) and LTC4 and LTD4, respectively. In contrast, incubation with Toxoplasma gondii, an obligate intracellular protozoan, led to the formation of 11-, 12-, and 15 hydroxyicosatetraenoic acids (HETEs), together with an unidentified compound, designated compound X. Each of these compounds incorporated [3H]arachidonic acid from the macrophage during phagocytosis of T. gondii. Compound X migrated immediately prior to 15-HETE by reverse-phase HPLC and was distinct from authentic monoHETE, monohydroperoxyicosatetraenoic acid (mono-HPETE), and dihydroxyicosatetraenoic acid (diHETE) standards. The generation of compound X by macrophages correlated with the extent of phagocytosis of T. gondii and with intracellular survival of the organisms. Prior antibody-coating of T. gondii or activation of macrophages, either of which inhibited survival and replication of ingested organisms, was associated with production of LTD4 but not compound X. Killed organisms also stimulated LTD4 release only. Although T. gondii concentrated arachidonic acid, they did not metabolize the compound to identifiable lipoxygenase products. Preincubation of macrophages with the relative lipoxygenase inhibitors nordihydroguaiaretic acid or 5,8,11,14 icosatetraynoic acid inhibited the formation of compound X. The absence of leukotriene production by macrophages ingesting T. gondii may explain the relative lack of a neutrophil inflammatory response in diseases due to obligate intracellular organisms. Alternatively, compound X may have functional activities that might mediate some of the host responses to cellular parasitism. PMID- 2995994 TI - Chloroplast DNA diversity is low in a wild plant, Lupinus texensis. AB - Chloroplast DNA diversity was measured in an annual flowering plant, Lupinus texensis. Individual plants were collected from 21 local populations throughout the range of the species in Texas. Chloroplast DNA was isolated separately from each plant and digested with seven restriction enzymes. The most common form of the 150-kilobase-pair genome was cut at 134 sites, so that about 0.5% of the base pairs in the genome were sampled. Of the 100 plants examined, 88 had identical restriction fragment patterns. Three variant forms were found in different local populations. Two, represented in single plants, differed from wild type in the presence or absence of single restriction sites. The third variant was fixed in one of the local populations; it had lost a restriction site and also had a deletion of approximately equal to 100 base pairs. The data suggest that chloroplast DNA in this plant is much less polymorphic than mitochondrial DNA from animals and is probably less polymorphic than nuclear genes in the same plant or in animals. PMID- 2995995 TI - Structure of the human phosphoglycerate kinase gene and the intron-mediated evolution and dispersal of the nucleotide-binding domain. AB - The human X-linked phosphoglycerate kinase (PGK) gene, which is expressed in all somatic cells, was cloned and its structure was determined. The gene is interrupted by 10 introns and spans 23 kilobases. When projected on the three dimensional structure of the PGK protein molecule, splice junctions are located between established peptide domains. In particular, an intron separates the two mononucleotide subdomains of the ATP-binding region, and additional introns divide each of these subdomains between their characteristic beta-strands. Similar correlations are found in the bipartite NAD-binding domains of alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Furthermore, in each case the nucleotide-binding domain is separated from the catalytic domain by at least one intron. The homology of the exon organization in structurally similar regions of these three enzymes suggests that a nucleotide-binding domain evolved by gene duplication and was subsequently dispersed to different proteins through a process of intron-mediated recombination. PMID- 2995996 TI - Use of pooled DNA samples to detect linkage disequilibrium of polymorphic restriction fragments and human disease: studies of the HLA class II loci. AB - A rapid method has been developed and used to search for restriction fragment length polymorphisms (RFLPs) that are in linkage disequilibrium with disease associated loci. By using genomic blot-hybridization analysis with DQ beta-chain and DR beta-chain cDNA probes, we examined DNA polymorphisms within the HLA class II loci associated with susceptibility to insulin-dependent mellitus (IDDM). To facilitate the search for informative RFLPs, we compared pooled DNA samples from IDDM patients with pooled DNA samples from randomly selected control individuals, instead of using the conventional approach of examining DNA samples from individuals in two groups. (The conditions under which this approach is useful are treated theoretically in the Appendix.) Several specific polymorphic restriction fragments associated with IDDM were revealed by using this economical and rapid approach. The restriction enzymes and probes identified as informative in this screening were then used to analyze HLA-DR-typed IDDM families, homozygous typing cells, and unrelated individuals to determine the association of the specific restriction fragments with HLA-DR serological type and the frequency in control and IDDM populations. Some individual polymorphic fragments for which the IDDM population was enriched correlated strongly with HLA-DR3, whereas others correlated strongly with HLA-DR4. Some fragments (e.g., a 10 kilobase Taq I fragment detected with the DR beta probe) that were more prevalent in the IDDM population subdivided the serologically defined HLA-DR type and may be informative markers for IDDM susceptibility. PMID- 2995997 TI - Gene localization on sorted chromosomes: definitive evidence on the relative positioning of genes participating in the mouse plasmacytoma-associated typical translocation. AB - Experiments have been carried out to establish the relative position of the mouse Ig heavy chain locus and the c-myc oncogene. In mouse plasmacytoma with the typical rcpt(12;15) chromosome translocation the c-myc oncogene is juxtaposed to one of the heavy chain genes in a head-to-head orientation. Since the relative orientations of the c-myc locus and the Ig heavy chain gene cluster on the corresponding mouse chromosomes had not been settled, it was not known whether the rearranged c-myc gene is transposed to chromosome 12 or remains on chromosome 15. To decide which of the two alternatives is correct, we separated the translocation chromosomes by fluorescence-activated chromosome sorting. The separated chromosomal fractions were hybridized with myc-specific DNA probes corresponding to the first or second/third exons in a chromosome spot assay. The results presented here indicate that the c-myc gene in mouse plasmacytoma carrying the typical translocation, as in the human Burkitt lymphoma analogous translocation, transposes to the chromosome carrying the Ig heavy chain locus. These results also establish the orientations of the Ig heavy chain locus and the c-myc locus on their normal chromosomes. PMID- 2995998 TI - Translocation of c-myc in the hereditary renal cell carcinoma associated with a t(3;8)(p14.2;q24.13) chromosomal translocation. AB - A translocation between chromosomes 3 and 8, t(3;8)(p14.2;q24.13), has been reported in a family with hereditary renal cell carcinoma. Using somatic cell hybrids, we have isolated, separately, both derivative chromosomes. We find that the c-myc oncogene (8q24.1) has been translocated to the derivative 3 [der(3)]. We have not detected a rearrangement within an approximately equal to 21-kilobase region around the c-myc gene using restriction enzyme digestion and Southern blot hybridization analysis. The translocated c-myc gene should provide a probe to the chromosome 3p14 region, which appears to be important not only in renal cell carcinoma but also in small cell carcinoma of the lung. These hybrids have also been useful for the regional mapping of the Chinese hamster ovary cell Gly-B defect to 8q22.1----q24.13 and support the regional assignment of acylase I to 3p21. PMID- 2995999 TI - Generation of single base-pair deletions, insertions, and substitutions by a site specific recombination system. AB - The sequence analysis of both products of individual phi 80 site-specific recombination events in vivo shows that recombination with a secondary attachment (att) site generates several different novel joints at the mismatched position: one recombination event resulted in a single base-pair deletion and two other recombination events resulted in two different single base-pair substitutions. The characterized products of recombination can be straightforwardly interpreted as the outcome of strand exchange involving staggered nicks bracketing the heterology within an overlap region of five to nine base pairs. In comparison, more complex segregation patterns have been observed in previous studies of lambda recombination between nonidentical att sites; the nature of the overlap region heterology may have a significant effect on the segregation patterns. To recover both products of a single recombination event, we used a plasmid that carries the phi 80 int and xis genes and both att sites. Because the two att sites are situated in opposite orientation, intramolecular recombination between them inverts rather than deletes the intervening segment of DNA. Although subsequent reinversion restores the original gross genetic arrangement, single base-pair insertions, deletions, and substitutions are introduced at the sites of recombination. One of the mutations improves the recombination efficiency of the secondary att site and thereby converts a formerly "stable" sequence to an efficient target for rearrangement, and other mutations are predicted to alter the specificity of recombination. These pathways may also provide useful models for the efficient generation of localized sequence diversity on a development (as well as evolutionary) time scale. PMID- 2996000 TI - Identification and order of sequential mutations in beta-actin genes isolated from increasingly tumorigenic human fibroblast strains. AB - We have sequenced the mutant beta-actin gene of a tumorigenic human fibroblast cell line (HuT-14T) and found that it carries three mutations that alter the amino acids at positions 36, 83, and 244 as well as a 22-base-pair "insertion" sequence, in the 5' intron, not present in a wild-type gene. The less tumorigenic cell line HuT-14, a progenitor of HuT-14T, has the same codon-244 mutation and the insertion sequence but not the other two mutations. A nontumorigenic cell line that is related to HuT-14 but that has no beta-actin mutations does carry the intron-length polymorphism. We conclude that the mutation at codon 244 occurred first in a beta-actin allele already bearing the 22-base-pair intron insert and that mutations at codons 36 and 83 arose subsequently during the selection for the HuT-14T phenotype. Rat-2 cells synthesize the appropriate charge-variant species of mutant actin protein when transfected with either the singly or the triply mutated beta-actin gene. PMID- 2996001 TI - Sex- and cell-specific regulation of yolk polypeptide genes introduced into Drosophila by P-element-mediated gene transfer. AB - To find whether cis-acting regulatory sequences necessary for sex- and cell specific expression of two yolk polypeptide genes (Yps) reside near the structural genes themselves, we introduced a 5.0-kilobase genomic DNA segment containing a 3' truncated Yp1 and a complete Yp2 into five different autosomal locations by P-element-mediated gene transfer. Transcripts from the introduced Yp1 were not found in male flies but appeared on a normal developmental schedule in adult females, accumulating in their body walls and ovarian follicles but not in guts or malpighian tubules. Protein from the introduced Yp2 allele was present in female hemolymph and vitellogenic ovaries but was lacking from male hemolymph. We conclude that sequences necessary for the correct stage-, cell-, and sex specific expression of the Yp1 and Yp2 genes are included in this genomic fragment. These results combined with published work suggest that two tissue specific, cis-acting, bidirectional, positive regulatory elements placed on either side of a centrally located HindIII site govern expression of both Yp genes--one element specific for fat body and the other specific for ovarian follicle cells. PMID- 2996002 TI - In vitro growth characteristics of simian T-lymphotropic virus type III. AB - The type C retrovirus simian T-lymphotropic virus type III (STLV-III) has been isolated recently from immunodeficient macaque monkeys at the New England Regional Primate Research Center. The present studies were done to define the in vitro growth characteristics of this agent. STLV-III replicates efficiently in interleukin 2-dependent T-cell cultures of macaque peripheral blood lymphocytes (PBL), less efficiently in such cultures of human and gibbon PBL, and inefficiently in baboon PBL. No replication, as assessed by measuring reverse transcriptase activity in these culture supernatants, could be detected in similarly maintained cultures of chimpanzee, squirrel monkey, and cotton-top tamarin PBL. Like the human acquired immunodeficiency syndrome (AIDS) virus, human T-cell lymphotropic virus III/lymphadenopathy-associated virus (HTLV III/LAV), STLV-III replicates in T4+ but not T8+ lymphocytes and its infection of macaque and human lymphocytes can be blocked with monoclonal anti-T4 antibodies. STLV-III differs from the human AIDS virus, however, in its apparent inability to grow in the Epstein-Barr virus-transformed B lymphocytes tested, the differing range of nonhuman primate T-cell populations that support its growth, and its less striking toxicity for T lymphocytes. These studies provide further characterization of an agent that will be extremely important in facilitating the development of vaccines and antiviral therapy for AIDS. PMID- 2996003 TI - Presence of an allelic EcoRI restriction fragment of the c-mos locus in leukocyte and tumor cell DNAs of breast cancer patients. AB - Structure of the human c-mos protooncogene in DNAs from breast tumors, leukemic cells, and lymphocytes from normal individuals was analyzed by restriction enzyme digestion and Southern blot. In 6 of 75 breast tumor DNAs, we found an EcoRI 5 kilobase extra band hybridizing with a human c-mos probe containing all of the sequences homologous to v-mos oncogene. This band was also found in lymphocyte DNA from 3 of these patients, indicating a restriction fragment length polymorphism. This polymorphism was not found in a series of 69 lymphocyte DNAs from the unaffected population. Moreover, 1 of 73 leukemic cell DNAs exhibited the 5-kilobase band. These results indicate that this rare polymorphism is significantly more frequently found in patients with breast cancer than in the rest of the population (P less than 0.05, by a chi 2 test with Yates correction. PMID- 2996005 TI - Light-activated guanosinetriphosphatase in Musca eye membranes resembles the prolonged depolarizing afterpotential in photoreceptor cells. AB - Measurement of light-dependent GTPase (EC 3.1.5.1) activity in a paradigm guided by electrophysiological experiments was used to examine the involvement of a guanine nucleotide binding protein in fly phototransduction. Cell-free membrane preparations of Musca eyes responded to blue light by a 10- to 20-fold increase in GTP-hydrolyzing activity. This light-dependent GTPase had a low Km for GTP (0.5 microM) and was effectively inhibited by guanosine (5'----O3)-1 thiotriphosphate and guanosine 5'-[beta-gamma-imino]triphosphate but not by adenosine 5'-[beta-gamma-imino]triphosphate and ATP. The action spectrum of GTPase activity measured with intense light resembled closely the photoequilibrium spectrum of metarhodopsin. After illumination with blue (less than 480 nm) light, which converted rhodopsin to metarhodopsin, the GTPase remained highly active for at least 60 min in the dark. Similarly, rhodopsin-to metarhodopsin conversion in intact cells induced a prolonged excitation in the dark, known as the prolonged depolarizing afterpotential (PDA). The persistent GTPase activity (like the PDA) was suppressed to the low basal activity of the unilluminated membranes after conversion of metarhodopsin to rhodopsin with red light (greater than 570 nm), whereas during illumination with red light, some GTPase activity was maintained. The magnitude of the persistent GTPase activity in the dark, like the PDA, depended in a supralinear manner on the amount of pigment conversion. Thus, the dependence of GTPase activity of Musca membrane preparations on photopigment conversion resembles the induction and suppression of the PDA measured in intact photoreceptors of Musca. These findings indicate that a guanine nucleotide binding protein is part of the chain of events leading to both the generation of the receptor potential and the PDA. PMID- 2996004 TI - Slow, persistent replication of lentiviruses: role of tissue macrophages and macrophage precursors in bone marrow. AB - Lentiviruses, as exemplified by visna virus of sheep, are nononcogenic retroviruses that cause slowly progressive diseases after prolonged periods of incubation. Earlier studies on visna have shown that the long incubation period of the disease is associated with constant production of minimal quantities of virus in tissues, whereas virus could be obtained by culturing monocytes and macrophages from explants of lymphatic tissues and inflamed organs. In this study the role of macrophages in lentivirus infection was explored using two sheep that were intrabronchially inoculated with virus. When sections of paraffin-embedded tissue, processed by a recently described technique which combines immunocytochemistry for the identification of macrophages and in situ hybridization for identification of viral nucleic acid, were examined, we found that virus replication is associated almost exclusively with infection in selected populations of macrophages in the interalveolar region of the alveoli, in inflammatory exudate cells in the lung, in lymph nodes, and in the spleen. Although large numbers of alveolar macrophages had viral RNA, few of these cells produced virus. While this minimally productive type of viral replication provides an explanation for the slow pace of the infection, restricted replication in terminally differentiated, short-lived macrophages does not explain persistent virus replication in the animal. With the discovery of clusters of infected macrophage precursors in the bone marrow, a mechanism for persistence was found. The macrophage precursor cells provide an important missing link in the virus-target-cell circuit and may be the reservoir of latently infected cells which perpetuate lentivirus infections in both animals and humans. PMID- 2996007 TI - Inositol phospholipid and phosphatidic acid metabolism in response to membrane receptor activation. PMID- 2996008 TI - The effects of dietary intake of essential fatty acids on prostaglandin and leukotriene synthesis. PMID- 2996006 TI - Increases in muscle Ca2+ mediate changes in acetylcholinesterase and acetylcholine receptors caused by muscle contraction. AB - The synthesis of acetylcholinesterase (AcChoE; acetylcholine acetylhydrolase, EC 3.1.1.7) and of acetylcholine receptors (AcChoR) by cultured rat muscle fibers is influenced strongly by the level of muscle contractile activity. If fibers are grown in the presence of tetrodotoxin (TTX) to block spontaneous contraction, the total amount of AcChoE decreases markedly, as does the percentage of AcChoE assembled as the collagen-tailed presumed synaptic form of the enzyme. Under these conditions, however, the number of AcChoR increases. We demonstrate here that each effect of TTX can be prevented by treating the muscle cells with the calcium ionophore A23187. Thus, cells treated with A23187 and TTX have 30- to 40 fold higher levels of collagen-tailed AcChoE and lower levels of AcChoR by a factor of 4-5 than do cells grown in TTX alone. These results suggest that an increase in muscle cytoplasmic Ca2+ mediates the known effects of muscle contraction on these cholinergic macromolecules. PMID- 2996009 TI - Alterations in membrane function, organization and composition in the obese ob/ob mouse. PMID- 2996010 TI - The physiological determination of meal size in pigs. PMID- 2996011 TI - Pulmonary compartmentalization of interferon and natural killer cell activity. AB - Studies were performed to determine if natural killer (NK) activity in the mononuclear cells harvested from infected lungs was dependent on local or systemic factors. Mice were inoculated by intratracheal (it), intraperitoneal (ip), or intravenous (iv) routes with (a LD50 dose of) influenza virus A PR/8/34. At various days postinoculation cells from lungs, spleens, and peripheral blood were assayed for NK activity, and lung wash, lung homogenates, and serum were assayed for interferon. After it inoculation there was three- to fourfold increase of NK activity in the lung with little or no increase in NK activity in spleens or peripheral blood. The local augmentation of NK activity in the lung correlated with an increase in interferon (IFN) titer in the lung wash and lung homogenate of PR8 inoculated mice. The virus failed to induce IFN or augment NK activity when it was inoculated systemically. The observed local augmentation of NK activity and local induction of interferon production following it inoculation suggests that the NK population in the lung is capable of responding to locally derived regulatory factors. PMID- 2996012 TI - Blockade of thromboxane and the prevention of eicosanoid-induced sudden death in mice. AB - We studied the effects of thromboxane-receptor antagonism and thromboxane synthetase inhibition in a thrombotic model of sudden death in mice. Intravenous injection of arachidonic acid (AA; 80 mg/kg) or the prostaglandin-endoperoxide analog U-46,619 (2.3 mg/kg) results in sudden death in approximately 90% of the animals. Pretreatment with the thromboxane receptor antagonist SQ-29,548 (0.3-10 mg/kg) protects dose-dependently against AA and U-46,619-induced sudden death. In contrast, CGS-13,080, a thromboxane synthetase inhibitor, shows a dose-dependent beneficial effect in AA-induced sudden death only. Although PTA2 has partial thromboxane agonistic properties in the rabbit, it protected the mice against AA induced sudden death, thus demonstrating TxA2 antagonistic properties in this species. These data emphasize the importance of thromboxane A2 as a major mediator of arachidonic acid-induced sudden death and the effectiveness of thromboxane-receptor antagonists in endoperoxide-induced sudden death. PMID- 2996014 TI - Action of purified colony stimulating factor on hemopoietic cells. PMID- 2996013 TI - Effects of acute and subchronic delta 9-tetrahydrocannabinol administration on the plasma catecholamine, beta-endorphin, and corticosterone levels and splenic natural killer cell activity in rats. AB - The effect of acute (1 day) or subchronic (25 days) treatment with delta 9 tetrahydrocannabinol (THC), the major psychoactive constituent of marihuana, on plasma norepinephrine (NE), epinephrine (E), corticosterone, beta-endorphin (beta end), and splenic natural killer (NK) cell activity of the rat was studied. Groups of animals received subcutaneously, either THC in corn oil + saline (3 mg THC/kg); oil + saline; or THC + naloxone (2 mg naloxone/kg and 3 mg THC/kg). Acute injection of THC with or without naloxone did not significantly change plasma levels of NE, E corticosterone, beta-end, or the NK cell activity. However, subchronic treatment with THC significantly reduced plasma levels of NE, E, corticosterone, and NK cell activity, compared to controls. The plasma beta end levels were significantly elevated in the THC-treated animals. In the THC + naloxone group of animals, the plasma hormone levels (corticosterone and beta end) were similar to control levels and the NK cell activity was significantly higher than in THC-treated animals. These results indicate that subchronic exposure to THC results in suppression of splenic NK cell activity. The interaction of THC with the endogenous opiate system appears to be a contributing factor leading to the NK cell suppression in rats. A direct suppressive action of THC or its metabolites on the NK cell is not ruled out by this study. PMID- 2996015 TI - The central role of the macrophage in hemopoietic microenvironmental regulation. AB - Based on the macrophage-specific release using crystalline silica, and the production and secretion of at least three hemopoietic regulatory factors (erythropoietin, colony stimulating factor and a multipotential stem cell enhancing and maintaining factor) into the extracellular fluid of bone marrow derived macrophages grown on hydrophobic teflon foils, a hypothesis for the regulation of hemopoiesis is proposed. The macrophage is viewed as that component in the hemopoietic microenvironment responsible for hemopoietic regulation, by acting as an "intermediary" between extracellular signals and the controlled release of regulatory factors acting on responsive cells at different levels in the hemopoietic hierarchy. PMID- 2996016 TI - The role of ion fluxes in hematopoietic cell differentiation. PMID- 2996017 TI - Mechanisms of iron uptake in reticulocytes. PMID- 2996018 TI - T cell malignancies and human T cell leukemia (lymphotropic) retroviruses (HTLV). PMID- 2996019 TI - The action of erythropoietin as the inducer of erythroid differentiation. PMID- 2996020 TI - A specific cyclic GMP phosphodiesterase of the retinal interphotoreceptor matrix. PMID- 2996021 TI - DNA sequences required for regulated expression of the human beta-globin gene. PMID- 2996023 TI - Distant sequences which regulate globin genes. AB - Besides the major cap site, transcription of the human epsilon-globin gene initiates at several upstream sites, the furthest 4.5 kb away. The upstream initiation sites occur in regions of hypersensitivity to DNaseI. There is also a very prominent DNaseI hypersensitive site 6.5 kb upstream which corresponds to an unusual nucleotide sequence. Upstream promoters, particularly one 200 bp upstream can be regulated independently of the major cap site. This site behaves as if it were a unidirectional enhancer. A fragment upstream of the mouse beta-globin promoter acts as a negative regulator in cis; it contains a long stretch of alternating purine and pyrimidine bases. The significance of upstream regulatory sequences adjacent to globin genes is discussed. PMID- 2996022 TI - Regulation of human globin gene expression. PMID- 2996024 TI - Tissue- and stage-specific expression of a cloned adult beta globin gene in transgenic mice. PMID- 2996025 TI - Characterization of recombinant monkey and human erythropoietin. AB - We have isolated the human EPO gene from a human genomic lambda library and a monkey EPO cDNA from an anemic monkey kidney cDNA library. These sequences have been expressed in both COS1 and Chinese hamster ovary cells. The expressed EPO has been shown to be fully biologically active both in vitro and in vivo and immunologically identical to the native hormone. In addition, administration of recombinant EPO to animals causes a dramatic increase in their hematocrit. PMID- 2996027 TI - Formation of inositol phosphates and calcium mobilization in Swiss 3T3 cells in response to prostaglandin F2 alpha. AB - Signal transduction in the mitogenic action of prostaglandin F2 alpha on Swiss 3T3 cells has been studied. Confluent and quiescent Swiss 3T3 cells prelabeled with myo-[2-3H]inositol were stimulated with PGF2 alpha for 15 min at 37 degrees C in the presence of 5 mM LiCl, and the amount of total [3H]inositol phosphates, a sum of inositol tris-, bis-, and mono-phosphates, accumulated in the cells was determined. Addition of PGF2 alpha to the cells at 0.2 to 10 microM induced a 1.7 to 2.4-fold increase in [3H]-inositol phosphates. The accumulation was dose dependent. Since assay of the agonist-dependent accumulation of inositol phosphates in the presence of LiCl has been used as a sensitive method for identifying those receptors that are coupled to the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns (4,5)P2], these results indicate that PGF2 alpha induces in Swiss 3T3 cells hydrolysis of inositol lipids by a phospholipase C. The receptor-stimulated hydrolysis of PtdIns(4,5)P2 is usually coupled with a rise in cytoplasmic free Ca2+ concentration ([Ca2+]i). The effect of PGF2 alpha on [Ca2+]i was studied in quin-2 loaded Swiss 3T3 cells. On addition of 0.1 microM and 1 microM PGF2 alpha, there was an immediate increase in quin-2 fluorescence by 16 to 19% indicating a 1.5 to 1.8-fold increase in [Ca2+]i. These results therefore indicate that PGF2 alpha at 0.1 to 1 microM induces in Swiss 3T3 cells the hydrolysis of inositol lipids and a rise in [Ca2+]i. PMID- 2996026 TI - Dietary sodium deficiency potentiates the effect of prostaglandin E2 on in vitro renin release in the rat. AB - Using a renal cortical slice system from sodium loaded (SL) or sodium deficient (SD) rats, this study investigated whether the effect of prostaglandin E2 (PGE2) on renin release (RR) is mediated by tissue cyclic AMP content (TcAMPc) changes, and if it can be modified by dietary sodium manipulation. At 10(-5) M, PGE2 significantly stimulated RR and TcAMPc in both SL and SD groups of slices. PGE2 doses of 10(-9) M and 10(-7) M were ineffective, although RR, but not TcAMPc, was significantly greater in the SD group in response to 10(-7) M PGE2 than RR in the SL group. Addition of the phosphodiesterase inhibitor theophylline (10(-4) M) together with the same three PGE2 doses maintained the stimulatory effect of 10( 5)M PGE2 alone on RR and TcAMPc in both groups of slices, and reversed the effect of 10(-7) M PGE2 alone on RR and TcAMPc in the SD group of slices only. Added by itself, theophylline was ineffective. These data indicate that: PGE2 can stimulate RR by a direct effect on the juxtaglomerular cells; the RR responses to PGE2 and theophylline administration are enhanced in the SD state; and the possibility of cAMP mediation of the effect of PGE2 on RR is discussed. PMID- 2996028 TI - Effects of an ACTH 4-9 analog on auditory evoked brainstem responses and middle latency responses. AB - Early and middle latency auditory evoked potentials (EAEPs and MAEPs) were recorded from thirteen male volunteers after oral administration of either 40 mg of an ACTH 4-9 analog (ORG 2766) or placebo. Main results indicate slightly longer latencies of the later components of the EAEPs after ACTH 4-9 analog. Effects of differences in treatment were clearest with very high stimulus rates. Therefore, these effects do not lend themselves for the explanation of ACTH 4-9 analog-induced changes in long latency auditory evoked potentials of cortical origin obtained with comparatively slow stimulus rates in earlier studies. In addition, the ACTH 4-9 analog inhibited a decrease in amplitudes of the Na component of the MAEP across the session. This latter result may be in line with dishabituating actions of the peptide. PMID- 2996029 TI - Comparison of abstinence syndromes following chronic administration of mu and kappa opioid agonists in the rat. AB - Abstinent states were compared following chronic administration of mu and kappa opioid agonists, morphine and ethylketocyclazocine, respectively. Rats were prepared with chronic EEG and EMG electrodes and indwelling IV cannulae. One group of rats was chronically administered IV morphine, while a second group received chronic injections of IV ethylketocyclazocine. Morphine abstinence was associated with suppression of REM sleep occurrences, increases in number of wet dog shakes, and a decline in EEG spectral power during slow-wave sleep episodes. In contrast, the ethylketocyclazocine abstinence syndrome included minor abstinence signs. Differences in abstinent states between morphine and ethylketocyclazocine indicate the involvement of separate receptor populations in the process of dependence on morphine and ethylketocyclazocine. PMID- 2996030 TI - Serotonin uptake and imipramine binding in rat platelets after chronic dexamethasone and amitriptyline treatment. AB - Chronic adrenocorticotrophin treatment was shown to decrease platelet 5HT uptake, increase Km and decrease Vmax. Similarly, chronic dexamethasone treatment decreased 5HT uptake, but had no effect on the Kd and Bmax of high affinity imipramine binding sites in the platelets. Chronic amitriptyline treatment induced similar changes, with the exception that the drug induced a decrease in the number of high affinity imipramine binding sites. Chronic dexamethasone treatment potentiated the effects of amitriptyline and decreased the IC50 of imipramine and desipramine as 5HT uptake inhibitors in vitro. The results suggest that whereas 5HT uptake is related to high affinity imipramine binding sites, it can be modified independently of the high affinity imipramine binding. PMID- 2996031 TI - Noradrenergic modulation of 4-aminopyridine-induced acetylcholine release from rat cerebral cortex. AB - The noradrenergic influence on cortical acetylcholine (ACh) release was investigated by the cortical cup technique in urethane anaestetized rats treated with 4-aminopyridine (4-AP). The following results were obtained: 1) The increase in ACh release induced by 4-AP (3 mg/kg i.p.) was strongly potentiated by pretreatment with -methyl-p-tyrosine (alpha-MPT) which inhibits catecholamine biosynthesis or by N-(2-chloroethyl)-N-ethyl-bromobenzylamine (DSP4) bringing about a selective degeneration of noradrenergic fibres. Neither pretreatment enhanced the spontaneous ACh output. 2) Pretreatment with p-chlorophenylalanine (PCPA), an inhibitor of serotonin synthesis, did not modify 4-AP effect on ACh output. 3) The alpha blockers, yohimbine (1 mg/kg i.p.) and prazosin (4 mg/kg i.p.), did not enhance the 4-AP effect on ACh release but only delayed its onset. 4) Yohimbine (7 mg/kg i.p.) completely reversed 4-AP effect on ACh release which was significantly decreased. It is concluded therefore that pretreatments with alpha-MPT and DSP4 remove an inhibitory noradrenergic control on cortical ACh release. On the other hand, the alpha blockers might interfere with the ionic mechanisms underlaying the 4-AP effect thus, masking the removal of the noradrenergic control, due to an alpha blockade. PMID- 2996032 TI - Effect of morphine and naloxone on the levels of ACTH and beta-endorphin in plasma, brain and pituitary of rats. AB - The effect of i.p. administration of morphine (25 mg/kg) and of naloxone (50 mg/kg) on the levels of ACTH and beta-endorphin in plasma, in brain and in pituitary has been studied in rats. An opposite effect morphine and naloxone is seen in plasma ACTH with an increase elicited by morphine and a decrease produced by naloxone. Small changes are seen in plasma levels of beta-endorphin as well as in ACTH and in beta-endorphin levels in brain and in pituitary. To every change in ACTH levels in plasma an opposite change in ACTH brain levels is observed. PMID- 2996033 TI - Influence of yohimbine on the ACTH-induced behavioural syndrome, in rats. AB - In adult male rats, yohimbine at low doses (0.1, 0.5 and 1 mg/kg i.p.) potentiated, and at high doses (30.0 mg/Kg) antagonized, the behavioural syndrome induced by the intracerebroventricular injection of ACTH 1-24 (3 micrograms/rat) (stretching-yawning syndrome and penile erections). These results support the hypothesis that brain catecholaminergic systems play a positive role in the ACTH induced behavioural syndrome. PMID- 2996035 TI - An investigation on the mechanism of anticonvulsant action of ketamine and phencyclidine on convulsions due to cortical application of penicillin in rabbits. AB - The intracortical injection of 500 units of penicillin in rabbits elicited intermittent bilateral spikes or spike-wave complexes followed by generalization of the epileptiform activity characterized by repeated ictal high-voltage, high frequency spikes paralleled by generalized convulsions. Administration of phencyclidine (PCP) (0.7-1.0 mg/Kg i.v.), ketamine (KT) (20-40 mg/Kg i.v.), pentobarbital (PB) (10 mg/Kg i.v.) and diazepam (3 mg/Kg i.v.) inhibited the generalization of the epileptiform activity induced by penicillin (500 units) counteracting the EEG and motor patterns of the ictal events, while did not influence the interictal spike-and-wave complexes. Physostigmine (0.1 mg/Kg i.v.), clonidine (0.1 mg/Kg i.v.), haloperidol (1 mg/Kg i.v.) and naloxone (10 mg/Kg i.v.) did not affect the inhibitory influence of PCP on epileptiform activity due to cortical application of penicillin. Thus, the mechanism of this anticonvulsant action of PCP seems not to depend on the neurotransmitter system related to the reported drugs. The mechanism of action of PCP and KT is discussed in connexion with the similarities of the effects of this drugs in respect to sigma opiate agonists and pentobarbital. PMID- 2996034 TI - Neurochemical studies on GABAergic and aminergic systems in the rat brain following acute and chronic piracetam administration. AB - In view of the metabolic and behavioural effects of piracetam (P), a cyclic derivative of gamma-aminobutyric acid (GABA), in experimental animals and in man, it was of interest to investigate the effect of acute or chronic administration of the compound on the function of different brain neurotransmitter systems. P was ineffective in modifying either synthesis release, uptake or post- synaptic binding sites for GABA. Acute P injection decreased dose-dependently cGMP levels in the rat cerebellum. Moreover, this effect was not mediated through a GABAergic mechanism. An acute challenge with Piracetam 15 days after chronic treatment with the compound increased DOPAC levels in the striatum and counteracted haloperidol induced PRL rise. Furthermore, chronic P administration increased normetanephrine levels in the cerebral cortex, an index of the release of norepinephrine at the synaptic level, and induced a desensitization of beta-adrenoceptors in this same brain area. In conclusion, besides the well documented effect of P on cholinergic neurons, P seems to exert its biological and behavioural effects through activation of catecholaminergic mechanisms. PMID- 2996036 TI - Differential sensitivity to prazosin and yohimbine blockade of tyramine and noradrenaline. AB - Previous researches demonstrated that in the rat vas deferens the effects of noradrenaline released by tyramine were more easily affected than those induced by exogenous noradrenaline by the non selective alpha-blockers, phentolamine and dihydroergocristine. The investigation has now been extended to the effects of selective alpha-blockers, prazosin and yohimbine with the aim to see whether the major sensitivity of tyramine to alpha-blockade correlates with the type of receptor activated. The results obtained with the two antagonists which selectively block alpha 1 and alpha 2 receptors strongly resemble each other and those previously obtained with the non selective alpha-adrenoceptor blockers. Thus, the peculiar sensitivity to alpha-blockade of noradrenaline released by tyramine with respect to exogenous noradrenaline does not seem to be dependent on the type (alpha 1 or alpha 2) of receptor involved. PMID- 2996037 TI - [Inhibiton of the mitotic activity of thymocytes in mice in vitro by diethyldithiocarbamate]. PMID- 2996038 TI - Neuronal dopamine receptors on autonomic ganglia and sympathetic nerves and dopamine receptors in the gastrointestinal system. PMID- 2996039 TI - Pharmacological correction of the energy metabolism of the ischemic myocardium. PMID- 2996040 TI - Amitriptylinoxide: receptor-binding profile compared with other antidepressant drugs. AB - The interactions of amitriptylinoxide and various antidepressant drugs with different neurotransmitter and drug receptors were investigated by receptor binding studies. Amitriptylinoxide had less affinity than amitriptyline for most of the receptors studied. Half maximal inhibition of acetylcholine receptor binding occurred for amitriptylinoxide at 18 mumol/l (amitriptyline: 0.32 mumol/l). Comparing all studied antidepressants for muscarinic acetylcholine receptor binding, amitriptylinoxide had the weakest affinity of all tested tricyclic compounds. Also the affinity of amitriptylinoxide for alpha-receptor binding was about 60 fold less than that of amitriptyline. For all antidepressants investigated, the lowest affinities were found for benzodiazepine, opiate and beta-receptor binding. The weak affinities of amitriptylinoxide for various receptors may be responsible for its reduced side effects, while it still retains potent antidepressant properties by stabilising the amitriptyline-level in the brain. PMID- 2996041 TI - Photosensitized production of superoxide anion by monochromatic (290-405 nm) ultraviolet irradiation of NADH and NADPH coenzymes. PMID- 2996043 TI - Restriction endonuclease mapping of the hepatitis B viral genome isolated from Taiwan. AB - The Eco RI fragment of hepatitis B virus (HBV) DNA isolated from human blood plasma Dane particles were inserted into plasmid pUC8 Eco RI site and transformed into E. coli JM103 host. Two recombinants pTWL1 and pTWL2 were found to carry 3.2 kbp fragment and proved to have HBV genome by Southern hybridization method. The 1.4 kbp Bam HI fragment which carried the hepatitis B viral surface antigen (HBsAg) gene, obtained via Bam HI digestion of Dane particles DNA which was made fully double stranded by endogenous DNA polymerase reaction, was also inserted into plasmid pUC8 Bam HI site. Four recombinant clones, pTWS1, pTWS2, pTWS3, and pTWS4 were found. Only one of the clones pTWS1 carried the HBsAg gene in a correct orientation with respect to the lac promoter sequence. The physical mapping of HBV DNA was performed with several restriction endonucleases. Our results indicated that the HBV DNA insert contains unique XbaI and HpaI cleavage sites and lacks the cleavage sites for the HindIII, SmaI, KpnI, SalI, and SstI endonucleases. The locations of Bam HI, BglII, and HincII endonucleases cleavage sites within the cloned HBV DNA of the pTWL1 plasmid were similar to that HBV DNA of adw and adw2 subtypes. PMID- 2996042 TI - Analgesic consultation. PMID- 2996044 TI - Neurotoxins with phospholipase A2 activity in snake venoms. PMID- 2996046 TI - GABAergic effects of reserpine following chronic treatment. AB - Reserpine-induced depression has been used as an animal model to screen for antidepressant agents. Chronic (14-day) treatment with reserpine resulted in a significant increase in beta-adrenergic receptor binding in the cerebral cortex and the hippocampus, which was partially prevented by chronic treatment with either the antidepressant imipramine, the GABA-A agonist THIP or, the GABA-B agonist baclofen. Chronic treatment with reserpine also significantly increased 3H-GABA receptor binding, which was partially prevented by chronic treatment with either imipramine or THIP. Both subchronic and chronic administration of reserpine resulted in a decrease in GABA concentrations in the cerebral cortex and hippocampus. These data demonstrate the effect of reserpine treatment on the GABA-ergic system, and add further support for a functional coupling between the noradrenergic and GABA-ergic systems, which may be important for the mechanism of action of antidepressant agents. PMID- 2996047 TI - [Correction of maxillary anterior resorption defects with calcium hydroxylapatite implants]. PMID- 2996045 TI - Comparative effects of thyrotropin releasing hormone, MK-771 and DN-1417 on morphine abstinence syndrome. AB - The effects of thyrotropin releasing hormone (TRH) were compared with two of its analogs, L-N-(2-oxopiperidine-6-yl-carbonyl)-L-histidyl-L-thiazolidine-4-carbo xam ide (MK-771) and gamma-butyrolactone-4-carboxyl-histidyl-prolineamide (DN 1417) on the abrupt and naloxone-precipitated abstinence symptoms in morphine dependent male Swiss-Wester mice. Mice were made physically dependent on morphine by subcutaneous implantation for 3 days of a pellet containing 75 mg morphine free base. Control mice were implanted with placebo pellets. Intracerebral administration of TRH (10 ng-10 micrograms per mouse) immediately after removal of placebo pellets had no effect on the basal temperature of mice. Mice implanted with morphine pellets exhibited a characteristic hypothermic response following the removal of the pellets. TRH at all doses employed prevented the hypothermia observed during abrupt withdrawal of morphine (pellet removal). DN-1417 and MK 771 (10 ng-10 micrograms per mouse) on the other hand produced a short lived hyperthermic response in mice from which placebo pellets had been removed. However, both TRH analogs produced long-lasting antagonism of withdrawal hypothermia in mice from which morphine pellets had been removed. TRH and its analogs had no effect on the body weight loss observed during abrupt withdrawal of morphine. Intracerebral administration of 10 micrograms TRH and its analogs inhibited the naloxone-induced jumping response as evidenced by increases in naloxone ED50 values to elicit this response. It is concluded that TRH and its analogs may be useful in combating some of the withdrawal symptoms in opiate dependent subjects. PMID- 2996049 TI - [Results of perfusion scintigraphic follow-up studies of patients with occlusions of the internal carotid artery and the middle cerebral artery]. PMID- 2996048 TI - [Dynamic computed tomography--an aid in the differential diagnosis of liver masses]. PMID- 2996050 TI - [Brain perfusion studies during carotid compression]. PMID- 2996051 TI - Gallbladder bile: an experimental study in dogs using MR imaging and proton MR spectroscopy. AB - Magnetic resonance (MR) imaging, proton MR spectroscopy, and biochemical analysis were performed to investigate MR signal intensity (SI) differences between concentrated and dilute gallbladder bile of seven fasting and five sincalide treated dogs. MR images revealed high SI from bile of fasting dogs and low to medium SI in sincalide-treated dogs when spin-echo (SE) pulse sequences with repetition rates of 0.5 and 2.0 sec were used. Proton MR spectra were similar for fasting and sincalide-treated dogs. In fasting dogs, water content in the bile was slightly lower, and cholesterol, phospholipid, and bile acid concentrations were higher. More than 90% of proton signals in all Fourier transform free induction decay spectra emanated from water molecules, and no lipid proton resonances were detected in Fourier transform SE spectra after tau delays of 7 msec. These results indicate that the differences in SI are caused by alterations in relaxation times of water protons, possibly resulting from the interactions of water protons and macromolecules. PMID- 2996052 TI - Esophageal source measurement of Tc-99m attenuation coefficients for use in left ventricular volume determinations. AB - A linear attenuation coefficient for water (mu = .15 cm-1) at 140 keV has been used in the determination of left ventricular volumes (LVV) by attenuation corrected equilibrium methods. This theoretical value ignores the effect of Compton scatter and thus may be too high for human LVV determinations. The effective attenuation coefficient, mu', of the human chest was determined in ten normal volunteers using a Tc-99m esophageal source imaged with a gamma camera. Values for mu' at 30 degrees LAO in end-expiration, quiet breathing, and end inspiration were .125 +/- .006 cm-1, .125 +/- .005 cm-1, and .113 +/- .007 cm-1, respectively (95% confidence interval). Values of mu' at 45 degrees LAO were .122 +/- .006 cm-1, .119 +/- .007 cm-1, and .099 +/- .009 cm-1, respectively, for the same conditions. The measured value of mu' for the source in a water phantom was .127 +/- .001 cm-1. This suggests that a value of mu' of .125 cm-1 may be appropriate for use in determining LVV in patients. PMID- 2996054 TI - Placental vascular responses to leukotriene receptor antagonist FPL 55712. AB - We have previously shown that FPL 55712, a specific leukotriene receptor antagonist, potentiates estrogen induced uterine hyperemia in nonpregnant rabbits. We therefore chose to investigate the vascular responses of pregnant rabbits to leukotriene blockade. Nine unanesthetized animals carrying 46 viable fetuses were used in this study. Regional blood flows were measured by the radioactive microsphere technique. In 5 rabbits control blood flows were measured after vehicle administration and FPL 55712, 1 mg/kg bolus, followed by infusion of 100 micrograms/kg/min was given via the jugular vein. Regional blood flows were measured again after 10 minutes of infusion. The procedural order was reversed in the remaining 4 animals. Resistance was calculated as mean arterial pressure divided by total flow to an organ. FPL 55712 decreased the blood pressure from 83 +/- 2 to 76 +/- 3 mmHg (P less than .001). Uterine resistance was not significantly changed (387 +/- 44 to 362 +/- 42 mmHg X ml-1 X min X gm), but renal resistance fell from 18.5 +/- 1.1 to 15.1 +/- 1.2 mmHg X ml-1 X min X gm (P less than .01). FPL 55712 induced maternal placental vasodilatation with resistance decreasing from 291 +/- 33 to 261 +/- 31 mmHg X ml-1 X min X gm (P less than .04). Vehicle administration did not cause dilation in any vascular bed. FPL 55712 appears to be a placental vasodilator whose action is most likely due to receptor blockade of the vasoconstrictive endogenous leukotrienes. PMID- 2996053 TI - The role of calcium in eicosanoid production induced by ricinoleic acid or the calcium ionophore A23187. AB - Rat isolated intestine incubated in Krebs solution converted exogenous [14C] arachidonic acid into products that chromatographed with prostaglandins, leukotriene B4 and 5-hydroxy-eicosatetraenoic acid. Accumulation of these products was increased by the laxative ricinoleic acid (0.34 mM) or the calcium ionophore A23187 (7.6 microM). In the presence of the calcium antagonists TMB-8 (0.43 microM) or verapamil (0.2 microM) the mean effects of ricinoleic acid or the calcium ionophore were smaller. Stimulation of arachidonic acid metabolism by ricinoleic acid therefore seems likely to involve a calcium-dependent mechanism. PMID- 2996056 TI - In vitro and in vivo chemotaxis of guinea pig leukocytes toward leukotriene B4 and its w-oxidation products. AB - Leukotriene B4 (LTB4), 20-OH-LTB4, and 20-COOH-LTB4 were studied for their relative activities towards guinea pig peritoneal eosinophils and neutrophils during in vitro chemotaxis in modified Boyden chambers. The leukotrienes were also injected into guinea pig skin, and the cellular infiltrate in 4 hour biopsies was evaluated histologically. Eosinophils migrated more actively than neutrophils towards LTB4 in vitro, while in vivo, more neutrophils were observed. 20-OH-LTB4 was markedly less active than LTB4 in vivo and in vitro, and 20-COOH LTB was barely active at all. Crude ionophore-stimulated neutrophil supernatants (ECF) were more active towards eosinophils than towards neutrophils, both in vivo and in vitro, compared to the pure leukotrienes. The data confirm the potent chemotactic properties of LTB4 for eosinophils and neutrophils, with less activity of its w-metabolites. PMID- 2996055 TI - Correlation between the myotropic activity of leukotriene A4 on guinea-pig lung, trachea and ileum and its biotransformation in situ. AB - Leukotrienes A4 and D4 displayed equivalent myotropic activity on guinea pig lung parenchyma strips. However, on the trachea, the activity of LTD4 was much higher than that of LTA4. The potencies of these two leukotrienes were also different on strips of longitudinal muscles of the ileum where LTD4 was very active whereas LTA4 was inactive. Since the activities of both leukotrienes were blocked by FPL 55712, our results suggested that the transformation of LTA4 by the smooth muscle preparations was a prerequisite to its biological activity. LTA4 was then incubated for 10 min with homogenates of guinea pig lung parenchyma, trachea and longitudinal muscles of ileum, and the metabolites were analysed by bioassay using strips of guinea pig ileum and lung parenchyma in a cascade superfusion system and also by reversed phase high performance liquid chromatography (RP HPLC). Homogenates of lung parenchyma rapidly transformed LTA4 to LTB4, LTC4, LTD4 and LTE4. Incubation of LTA4 with homogenates of trachea or of the longitudinal muscles of ileum showed the formation of LTB4 and its isomers but no significant amount of peptido-leukotrienes were detected. These findings reveal that LTA4 undergoes distinctly different metabolic transformations in these tissues which correspond to the biological activities of the products recovered. These results strongly suggest that the myotropic activity and potency of LTA4 is related to the tissue levels of enzymes which catalyse its biotransformation. PMID- 2996057 TI - The effects of a diet rich in fish oil on human neutrophils: identification of leukotriene B5 as a metabolite. AB - Diets that are enriched with fish oil have been shown to alter arachidonic acid metabolism via the cyclooxygenase pathway. Recently it has been shown that one of the major component fatty acids of fish oil, eicosapentaenoate (EPA), is a substrate for the leukotriene B (LTB) pathway when added exogenously to human neutrophils in vitro. We fed a diet that contained 8-10gm/day of EPA to four human subjects for three weeks and compared the arachidonate metabolism of their neutrophils to the same functions while the subjects were on their usual diet. The fish oil-supplementation increased neutrophil EPA content from undetectable levels to 7.4 +/- 2.4% (p less than 0.01, expressed as % of total fatty acid), and decreased arachidonate from 15.4 +/- 2.3% to 12.8 +/- 2.3% (p less than 0.05). Leukotriene B5 was identified as a metabolite during the fish oil-diet by its chromatographic profile and mass spectrum. During the experimental diet LTB4, decreased from 160 +/- 37 ng/10(7) neutrophils to 120 +/- 12 (p less than 0.05), and LTB5 increased from 0 to 39 +/- 9 ng/10(7) neutrophils (p less than 0.005). The diet had no effect on neutrophil aggregation or adherence to nylon fibers. PMID- 2996058 TI - Prostaglandins, not cyclic GMP, inhibit oxytocinase isoenzymes from human amniotic fluid in vitro. AB - Two isoenzymes of oxytocinase (EC 3.4.11.3) activity were fractionated from human amniotic fluid samples between the 14th and 22nd weeks of gestation by Ultrogel acrylamide-agarose gel filtration and partially characterized. The isoenzymes were competitively inhibited by PGE1, PGE2 and PGF2 alpha more at pH 6.2 than at pH 6.8, whereas cyclic GMP (cGMP) and its 8-bromo derivative had no effect at either pH. The implications of these findings are discussed and it is suggested that since the activity of amniotic fluid oxytocinases is very low or minimal at or near term, inhibition of these by prostaglandins may not have physiological significance in the initiation of human parturition. PMID- 2996059 TI - [Clinical picture and etiopathogenesis of AIDS]. PMID- 2996061 TI - [Significance of pulmonary DSA for assessing the operability of lung tumors]. AB - In lung tumours histological findings, exclusion of metastatic disease and different diagnostic procedures such as bronchoscopy, mediastinoscopy and thoracic CT play an important role for tumour staging to plan the correct therapeutic approach. Selection of patients for thoracotomy depends on results of these examinations. This study was carried out to assess the value of an additionally applied pulmonary DSA in 17 patients with lung tumours (bronchogenic carcinoma n = 14). Our results demonstrate that in 4 cases angiography could prove inoperability of tumours (compression of sup. v. cava, occlusion of a main pulmonary artery): one patient had a negative mediastinoscopy, one patient had a small cell bronchogenic carcinoma; in two patients an additional mediastinoscopy could be omitted. In two other patients with bronchogenic carcinoma who appeared operable in mediastinoscopy and DSA, explorative thoracotomy only could ensure inoperability due to an infiltration of the thoracic wall. As a minimal invasive method applicable in outpatients pulmonary DSA seems to be useful in giving additional important diagnostic information particularly in central bronchogenic carcinomas. PMID- 2996060 TI - Recent prevention and treatment possibilities of viral infections in dialytic and transplanted patients. PMID- 2996062 TI - [Morphometric and densitometric assessment of left ventricular function in the digital subtraction angiocardiogram]. AB - The paper deals with the early clinical experience, using a new DSA installation with a specially programmed cardiological module. DSA provides very good contrast and special resolution and morphometric estimation of the ejection fraction and regional movement of the left ventricular myocardium derived from DSA. This is as good as the results of the left cardiogram. The significant advantage of DSA ist the ability to make a rapid and simple diagnosis. In addition, it is possible with densitometric methods to calculate ventricular volume curves and to provide phasic indications of the amplitude to contraction of the left ventricle. These investigations have so far proved valuable and provide new scope for the radiological evaluation of left ventricular function. PMID- 2996063 TI - [Bronchogenic cysts of the upper mediastinum]. AB - A case report of a congenital bronchogenic cyst in the upper mediastinum on the level of the thoracic aperture, which very likely communicates with the oesophagus, is presented. The embryogenesis, aetiopathogeny, diagnostics, location and histological structure of this rare case are discussed. PMID- 2996064 TI - [Computed tomographic findings in diffuse malignant pleural mesothelioma]. AB - Malignant pleural mesothelioma is a rare tumour and often difficult to diagnose. In this report, the CT findings of 50 patients with proven malignant pleural mesothelioma are described and tabulated. In 31 cases the CT findings could be compared with the results of thoracotomy and thoracoscopy. CT proved reliable in evaluating the exact extent of the disease. Additionally, a combination of findings can be detected by CT, suggesting the diagnosis of malignant pleural mesothelioma. The limitations of CT in malignant pleural mesothelioma are discussed. PMID- 2996066 TI - [Computed tomography in esophageal carcinoma. A prospective study]. AB - In a prospective study, 83 patients with esophageal carcinoma were examined by computed tomography. The CT data were compared with intraoperative, postmortem and microscopical findings in 74 patients. The T stage as determined by computed tomography was confirmed by surgery or autopsy in 88% of the cases and by microscopical examination in 82%. Infiltration of surrounding structures could be proved with a sensitivity of 87%, a specificity of 91% and an accuracy of 89%. Suspected lymph nodes were detected by computed tomography in 68% of the patients with lymph node metastases. Computed tomography enabled additional information on localisation, length and diameter of the tumors and the existence of metastases in the liver, lung and pleura. Due to the very good correlation between pre- and postoperative staging, computed tomography gives important information on local tumor resectability. In this manner, operations and therapeutic strategy can be planned with greater accuracy, and diagnostic thoracotomy can be reduced to a minimum. PMID- 2996067 TI - Small-bowel radiography. A prospective comparative study of three techniques in 200 patients. AB - The authors report on a prospective study on small-bowel radiography, comparing conventional barium follow-through, single-contrast enteroclysis (Sellink's method) and double-contrast enteroclysis. Enteroclysis appeared to be superior even when the follow-through was done with frequent fluoroscopy and large amounts of contrast medium. Single and double-contrast techniques did not differ essentially in image quality. Single-contrast technique being less time-consuming is to be preferred. PMID- 2996065 TI - [Comparative scintigraphic analysis of 99mTc aerosol inhalation and 81m Kr ventilation in chronic obstructive bronchial disease]. AB - The usefulness of 99mTc-labelled serum albumin aerosol as a ventilation agent was compared to that of 81mKr gas in normal subjects and in patients with a wide range of pathological pulmonary function. In addition to visual analysis of the scintigrams, lung activity distribution was quantified by an index. In healthy subjects aerosol distribution was very similar to that of 81mKr gas. In patients with persistent obstruction of bronchial air flow, the aerosol tended to penetrate less well than 81mKr gas to the lung periphery and to show a more uneven pulmonary tracer distribution. Consequently the 81mKr gas technique resulted in underestimating bronchial obstructive defects compared with the radioaerosol method. The results indicate that unlike 81mKr gas imaging, small particle aerosol scintigraphy provides a useful diagnostic technique for the detection of even mild air flow obstructions. PMID- 2996068 TI - [Echographic findings in gallbladder empyema]. AB - Retrospectively the sonographic findings of 11 patients with proven empyema of the gallbladder were analysed. In one patient sonography only showed signs of cholelithiasis, whereas the other ten patients showed one or more signs of cholecystitis. Echogenic bile was established nine times, always connected with layering. PMID- 2996069 TI - [Value of MR tomography vs. CT in the diagnosis of rectal carcinoma and its recurrence]. AB - The value of NMR tomography for the diagnosis of carcinoma of the rectum and of recurrences has been studied, using a 1.5 Tesla NMR apparatus and comparing the results with high resolution CT. There were five patients with a histologically proven primary tumour, eighteen patients with a recurrence and five patients who had had a rectal carcinoma removed, where there was no evidence of recurrence. By obtaining images in three planes, NMR showed the true tumour extent in all cases and was superior to CT in the diagnosis of the primary tumour (in four patients out of five) and in showing recurrences (in five out of eighteen patients). NMR also had advantages in demonstrating lymph node enlargement in the pelvis, where difficulties are often encountered using CT. Early experience with tissue characterisation indicates that it is possible to diagnose rectal tumours confined to that organ. These are usually missed by CT. CT is superior to NMR in demonstrating destructive lesions in bone. Early clinical experience suggests that NMR is a further advance in the early diagnosis of carcinoma of the rectum and of recurrences. PMID- 2996070 TI - [High-resolution coronal computed tomography in endocrine-active pituitary microadenomas with special reference to their contrast medium dynamics]. AB - Coronal dynamic high-resolution CT (DHRCT) proved a highly accurate and sensitive diagnostic tool for detecting microadenomas as small as two millimetres in 179 patients having clinically and endocrinologically proven pituitary adenomas. Their different patterns of contrast enhancement were analysed versus time. It is important to stress that the diagnosis of a pituitary microadenoma must be strictly correlated with clinical, endocrinological and histo-immunocytochemical findings and cannot be made exclusively on the basis of CT-morphological criteria. PMID- 2996071 TI - [Value of high-resolution CT in diagnosing acquired cholesteatoma of the middle ear]. AB - In thirty patients with cholesteatomas of the middle ear, high resolution CT of the petrous bone was performed in conjunction with the clinical examination. The extent of the soft tissue process can be demonstrated by CT, but differentiation of the cholesteatoma from the accompanying inflammatory changes is not possible. Typical complications such as destruction of the auditory ossicles, the bony labyrinth, the facial canal, the lateral wall of the attic and the superior and inferior walls of the tympanic cavity are clearly demonstrated. Changes in the mastoids are better demonstrated than by conventional radiography. Using the high contrast and special resolution of modern high resolution CT, this has become the method of choice in the investigation of cholesteatomas of the middle ear. PMID- 2996072 TI - [Temporary preoperative balloon catheter blockage of a traumatic giant aneurysm and of an a.v. fistula of the vertebral artery]. AB - A new method of embolisation with a dilatation catheter is described as an example in a case with traumatic aneurysm and an a.v. fistula of the vertebral artery. The lesion was temporarily occluded with a conventional catheter for angioplasty and operated upon. The method is available for e.g. rapid management in case of imminent rupture of traumatic aneurysms, multiple lesions of vessels and for operative reconstruction of traumatised arteries. PMID- 2996073 TI - [Pre- and postoperative diagnosis of an aortic aneurysm by dynamic computed tomography with omission of angiography]. AB - A study which was prospective with regard to the method, showed 41 cases of aortic aneurysms in a collective of 3597 body-CT-examinations (1.14%). By means of CT and its possibility of reconstruction all morphological details of an aneurysm become visible. The course of the vessels to the bowel and to the kidneys are shown with the same excellence as in angiography. However, additional information which angiography cannot supply, is also given, like non-perfused lumen, changed wall structure and perivascular changes. Measurement of flow can be performed in the vessel and in the organ, the arterial supply of which is involved in aneurysmatic disease. The method is non-invasive. This is important for control after operation. Morphological changes of the vessel prosthesis are detected, as well as bleeding and infection. Functional parameters are determined semiquantitatively. PMID- 2996074 TI - [Selective arteriography of the internal pudendal artery in posttraumatic erectile dysfunction]. AB - 102 patients with erectile dysfunction were evaluated by a multidisciplinary approach including measurement of the bulbocavernous reflex (BCR)-latency, cystometry, dynamic cavernosography and bilateral selective arteriography of the pudendal arteries. Seven patients had a posttraumatic source of their erectile failure. Three patients showed a mixed neurogenic-arteriogenic aetiology of erectile dysfunction, two patients had evidence of isolated arterial damage, one patient had a venous and one an isolated neurogenic cause of erectile dysfunction. The passage of pudendal-penile vessels and nerves through the urogenital diaphragm is the most vulnerable portion of its way through the pelvis. Ruptures of the prostatomembranous urethra are frequently accompanied by injury to the pudendal-penile vessels and nerves, thus causing erectile dysfunction. PMID- 2996075 TI - [Peripheral venous DSA of the calf arteries in arterial obstructive disease patients. A comparison with conventional angiography]. AB - The ability of venous DSA to demonstrate the calf vessels distal to the trifurcation in patients with proximal blocks was compared with conventional angiography in 33 patients (44 limbs) in a study carried out by four observers. In 11.1% cases, the vessels were demonstrated better by DSA and in 2.5% by conventional angiography. In 86.4% the quality was the same. The reasons for this are analysed. PMID- 2996076 TI - [Injuries of the medial epicondyle of the humerus in children. Conservative or surgical therapy?]. AB - 18 patients with avulsions of the medial epicondyle 1 10/12 to 16 2/12 years after trauma are presented. Non-union occurs very often after conservative treatment involving avulsion with or without displacement. Although non-union of the medial epicondyle gives no or only little disability, the fixation with one or two Kirschner wires is recommended to avoid non-union. Open reposition is necessary in cases with injury of the n. ulnaris and in cases with incarceration of the epicondyle in the elbow joint. PMID- 2996077 TI - [Menkes syndrome with excessive skeletal changes]. AB - An infant was seen for multiple fractures at the age of 10 weeks. He developed marked cortical thickening of many bones, which raised the suspicion of a battered child syndrome. Unusual progression of bone thickening and hitherto undescribed excessive bone remodeling led to the diagnosis of Menkes'kinky hair disease, a disorder of the connective tissue caused by a decreased copper bioavailability, to which disease the infant finally succumbed. PMID- 2996078 TI - [Muscle tissue relaxation times after experimental injuries]. AB - The proton relaxation times of muscles in the limbs of rats were measured after various types of experimental injury. Abnormalities of perfusion, the effect of histamine and of a non-specific myositis resulted in a significant prolongation of T1 and T2 in in-vitro experiments. It must be remembered that these were serious types of injury. It may be possible to demonstrate less extensive muscle changes by means of MR. PMID- 2996079 TI - [Osteochondrosis dissecans of the acetabulum. A very rare site]. PMID- 2996080 TI - [Psoas abscess after a sub partu pelvic girdle rupture]. PMID- 2996081 TI - MR imaging of a cystic lesion of the bone. A case report. PMID- 2996082 TI - [Imaging of a truncus arteriosus by nuclear resonance tomography]. PMID- 2996083 TI - Perinephric abscess causing subcutaneous emphysema. A case report. PMID- 2996084 TI - Massive haemorrhage from a caecal diverticulum. PMID- 2996085 TI - [Uptake of technetium-99m pyrophosphate in cardiac amyloidosis]. PMID- 2996086 TI - Trophic stimulation of steroidogenesis: in search of the elusive trigger. PMID- 2996087 TI - Regulation of luteal function in domestic ruminants: new concepts. PMID- 2996089 TI - The defective glucocorticoid receptor in man and nonhuman primates. PMID- 2996088 TI - Regulation of microsomal cytochrome P-450 enzymes and testosterone production in Leydig cells. PMID- 2996090 TI - Regulation of hormone receptors and adenylyl cyclases by guanine nucleotide binding N proteins. PMID- 2996091 TI - Progesterone action on the LHRH and the nigrostriatal dopamine neuronal systems: in vitro and in vivo studies. PMID- 2996092 TI - Neurohormones from fish tails. II: Actions of urotensin I in mammals and fishes. PMID- 2996093 TI - Thyrotropin-releasing hormone action: mechanism of calcium-mediated stimulation of prolactin secretion. PMID- 2996094 TI - Effect of wheat bran and pectin on the absorption and retention of phosphorus, calcium, magnesium and zinc by the growing pig. AB - Two separate balance experiments of P, Ca, Mg and Zn were carried out on 5 lots of 4 growing pigs each (35-40 kg) adapted for 3 weeks to one of the diets studied. In the first experiment, the control diet was compared with a diet containing 20% of coarse wheat bran, thus richer in minerals, the quantities ingested not being equalized. In the second experiment, three diets were compared: a control diet, a diet with 2.5% of high-methoxylated (HM) apple pectin, and a diet with 2.5% low-methoxylated (LM) apple pectin. The supplement of P and Mg provided by the wheat bran was well absorbed (apparent absorption) and retained by the pigs. On the contrary, in spite of higher intake of Ca and Zn with bran diet, the absorption of these minerals was not improved. The action of wheat bran phytase and the possible absorption of P and Mg (but not of Ca and Zn) in the large intestine could explain these results. Compared to HM pectin that had relatively little effect on mineral utilization, LM pectin drastically diminished the absorption and retention of the minerals studied and resulted in negative Ca, Mg and Zn balances. The degree of pectin esterification would thus be the main factor determining the effect of pectin on mineral availability. In conclusion, wheat bran is a source of available P and Mg for the pig but it might have an unfavorable effect on the utilization of Ca and Zn. LM pectin produces a deleterious influence on mineral balances. PMID- 2996095 TI - [Absence of effect of vitamin D on intestinal phytase and alkaline phosphatase: relation with phytic phosphorus in the pig]. AB - At least two-thirds of the phosphorus ingested by pigs is in the form of phytates. Two intestinal vitamin D-sensitive enzymes, alkaline phosphatase and phytase, might be involved in phytate-P digestion. The effects of dietary vitamin D upon the two intestinal phosphatases and P utilization in pigs fed a high phytate-P diet are reported here. Fourteen vit. D-depleted pigs (25-hydroxy vit. D: about 2 ng/ml plasma) were divided into two groups fed the same basal diet containing 0.6% P (of which 80% was phytic) and 0.6% Ca. One group (+D) was supplemented for 5 weeks with vit. D (1 000 IU D3/kg diet) and the other (-D) received none. P absorption and retention was two times higher in +D pigs than in -D animals (balance technique), and tibia X-ray pictures showed a lower bone density with the -D diet than with the +D diet. Surprisingly, vit. D supplementation had no effect on either of the mucosal enzymes (phytase and alkaline phosphatase). It may be concluded that vit. D improves phytate-P absorption via a mechanism which does not involve an increase in the activity of the intestinal phosphatases. PMID- 2996096 TI - Oxygen consumption and carbon dioxide production following open heart surgery. AB - The VO2, VCO2 and RQ were measured in 24 patients after open heart surgery using a CO2 analyzer and O2 Consumption Calculator (Siemen Elma, Sweden). With prolonged ventilatory care, shivering was noticed in only one patient and VO2, VCO2 and RQ were rather stable and constant in patients in the Intensive Care Unit (ICU). With dopamine infusion the 3-5 kg body wt/min cardiovascular status was stable and the oxygen extraction ratio was a mean of 0.264. From those data we suggest that ventilatory support would be desirable for about 2 days for those patients in the post-operative period. PMID- 2996097 TI - Hemofiltration in severe high microvascular permeability pulmonary edema secondary to rickettsial spotted fever. AB - Two patients, affected by spotted fever, developed low pulmonary capillary wedge pressure (PCWP) pulmonary edema with severe hypoxemia. Conventional specific and supportive therapy, including mechanical ventilation, failed to induce significant respiratory and hemodynamic improvement which was dramatically reached by means of hemofiltration. Removal of circulating middle molecular weight peptides by the convective mass transfer, characteristic of hemofiltration, offers a new and effective therapeutic approach for the adult respiratory distress syndrome secondary to rickettsial diseases. PMID- 2996098 TI - Perspectives in resuscitation. PMID- 2996099 TI - The effects of volume loading during epidural analgesia. AB - We have determined the effects of volume loading on the cardiovascular changes during epidural analgesia in 37 patients, who underwent various kinds of surgery. The patients were placed in 4 groups, depending upon the level of analgesia and utilization of volume loading with colloidal solutions. If the analgesia extended above Th4 we grouped them as "high epidural" and lower than Th5 level they were grouped as "low epidural". The cardiac output was measured through a Swan-Ganz catheter with thermo-dilution methods and cardiovascular variables were calculated by standard formulas. Under epidural block the most significant changes were a fall in blood pressure with decrease in cardiac output which were more pronounced during high epidural analgesia. Volume loading during the induction period with colloidal solutions would prevent the marked fall of blood pressure in half of cases studied, but in the other half the infusion was not effective for the prevention of fall in blood pressure. On every occasion over loading effects on the right side of the heart were observed with the infusion of colloidal solutions. Also, a marked fall in systemic vascular resistance was observed with the infusion. In consequence the volume loading did not prevent the fall in arterial pressure. To manage the latter which was observed during epidural block, some sympathomimetic agents would be necessary with the volume loading. This approach would be much more important in patients with dehydration and high level of epidural analgesia. PMID- 2996100 TI - Five years experience of cardiopulmonary resuscitation in a children's hospital. AB - The emergency call for resuscitation in a children's hospital is reported. Forty seven resuscitation attempts via the emergency call were made in 43 patients over the past 5 years; 24 out of 43 patients had heart diseases. Cardiac patients were more likely to die within a few days after their resuscitation. The patients, who had previously been in cardiac arrest, lost their lives more often. These results implied that it was very crucial to prevent cardiac arrest especially for cardiac patients in order to save lives. Among the causes of emergency calls, complications with the endotracheal tubes were most common (13) and the airway obstructions followed (11). Summation of two causes, the airway problems, accounted for more than half of the series. Sixteen out of all emergency calls occurred on weekends, and all endotracheal tube troubles broke out during a night shift or on a holiday, that is, the lives of those patients were threatened especially when fewer staff were on duty. Those accidents were embarrassing for us because we believe that all patients must have been under our surveillance all day. Some hospitals have a successful resuscitation rate, organizing a cardiopulmonary resuscitation (CPR) team. However, we obtained better results (about 30%) without a team. It should be borne in mind that it is more desirable to prevent cardiac or respiratory arrests initially rather than save the patients in emergencies. PMID- 2996103 TI - Two aspects of intensive therapy in reanimatology. PMID- 2996101 TI - Morphology of blood cells and the surface of charcoal by scanning electron microscopy during haemoperfusion in patients with hepatorenal syndrome. AB - The surface structure of non-spherical natural and spherical polymeric charcoal as well as the composition of blood cells in peripheral blood was studied in 52 patients with hepatorenal syndrome using the scanning electron microscope. Our data have allowed us to conclude that the optimal varieties of charcoal in respect of haemocompatibility are non-coated polymeric charcoals with spherical granules. Moreover, these varieties are characterised by an increased ability to absorb deformed erythrocytes. The possible mechanisms of blood-cell and fibrin adsorption on the surface of charcoal together with a specific approach to the choice of charcoal depending on the patients initial thrombocyte count is given. PMID- 2996102 TI - Cerebral blood flow during open-chest cardiac massage with occlusion of the descending aorta in dogs. AB - Cerebral blood flow (CBF) and carotid blood flow (CAF) during open-chest cardiac massage with and without occlusion of the descending aorta was examined in 10 dogs to assess whether they were augmented by occlusion. In control measurements with a normally beating heart, CBF with and without occlusion of the descending aorta were 17.8 +/- 2.3 and 13.8 +/- 2.2 ml/min (+/- S.E.M.), which were not significantly different. Cerebral blood flow during open-chest cardiac massage were 6.1 +/- 1.0 with occlusion and 5.7 +/- 1.0 ml/min without occlusion of the descending aorta, which were also not significantly different. By contrast, CAF increased significantly with occlusion of the descending aorta both during control measurement, with mean increases of 61.1% and 92.2% during open-chest cardiac massage (P = 0.05). While occlusion generally failed to augment CBF; in two dogs resuscitation was successful by manual cardiac massage. With the restoration of cardiac activity it increased immediately to twice that of control blood flow, and then gradually returned to the control level. Based on these observations, it is the author's opinion that every effort should be directed toward the restoration of cardiac activity as quickly as possible during circulatory arrest, and to increase CBF which is essential for neurological recovery. PMID- 2996105 TI - [Delayed neuropathy caused by amiodarone hydrochloride]. PMID- 2996104 TI - Enzyme-assisted vitrectomy with bacterial collagenase. Pilot human studies. AB - Clostridiopeptidase A, a bacterial collagenase, was used to assist vitrectomy with membrane stripping in six patients with dense intravitreal fibroproliferative tissue associated with retinopathy of prematurity, diabetic retinopathy, or proliferative vitreoretinopathy. When it was injected intra operatively, allowed to incubate for 15 minutes, and then removed by irrigation/aspiration, no side effects of lens opacity, lens dislocation, or retinal hemorrhage were observed. Use of this enzyme may facilitate removal of fibroproliferative tissue in certain difficult vitrectomy cases. PMID- 2996106 TI - [Gynecologic skin diseases of the perineum and perianal region]. PMID- 2996107 TI - Ultrastructural characterization of pulmonary neoplasms. II. The role of electron microscopy in characterization of uncommon epithelial pulmonary neoplasms, metastatic neoplasms to and from lung, and other tumors, including mesenchymal neoplasms. AB - Ultrastructural analysis through better resolution adds significant information to the evaluation and classification of primary pulmonary neoplasms. Light microscopy is limited in the evaluation of lung neoplasms. In some cases the light microscopic appearance may be entirely misleading, whereas in others it is inconclusive. Immunocytochemistry provides information on cytoplasmic differentiation of various tumors and hence more data on their corresponding phenotypes. The data from immunocytochemistry without corresponding objective electron microscopic evaluation may be very difficult to interpret. Correlation of historical, gross, light, electron microscopic, and immunocytochemical data is essential for a final accurate diagnosis (fig. 20). Fine needle aspiration of pulmonary neoplasms is becoming very fashionable and a diagnosis, including type of neoplasm, is expected on the basis of examination of a limited number of cells which further emphasizes the importance of ultrastructural characterization in helping to establish an accurate diagnosis [63-69]. The current classification of pulmonary neoplasms may need to be modified in the near future to incorporate the newly created data [70-72]. At the present time, there appears to be, at least, a need for a 'double standard', as Sobin [73] has suggested, which would permit the evaluation of the biologic significance of the ultrastructural and immunocytochemical findings (as applied to classification of neoplasms) in an effort to derive meaningful clinicopathologic correlations. Figure 20 emphasizes the additive role which should be played by the various diagnostic modalities to enable a morphologic assessment which would be an accurate predictor of biologic behavior. With an accurate assessment of biologic behavior, a more appropriate and rational approach for therapy is possible. There is also an important role for ultrastructural analysis in metastatic pleural and pulmonary neoplasms, primarily adenocarcinomas, as well as in the differential diagnosis of pulmonary neoplasms versus other tumors that may be similar in histological appearance. The role of ultrastructure in mesenchymal neoplasms is also crucial in defining specific neoplastic cell populations and in some cases in the differentiation from other non-mesenchymal tumors. It seems that routine electron microscopic examination of pulmonary neoplasms provides additional information that may be of great value in the management of patients and in understanding the differentiation, and perhaps histogenesis, of pulmonary neoplasms.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2996108 TI - Structural and functional properties of ganglioside antigens on human tumors of neuroectodermal origin. PMID- 2996109 TI - [Results of muscular x-ray computed tomography in 145 cases of neuromuscular disease]. AB - CT Scan examination in 145 cases of neuromuscular diseases yielded the following results: Diagnosis between myogenic and neurogenic process is inconstant and cannot be considered as absolute. In myogenic diseases the X ray density of muscle is early decreased with a preservation of muscle outline. In neurogenic diseases muscle volume is early decreased. Coexistence of atrophic and hypertrophic muscles indicates primarily muscle disease. Some patterns of involvement appear to be frequent. In Duchenne's dystrophy a contrast exists between atrophic "empty" or hypertrophic muscles during the ambulatory period and "ghostly" muscles during the terminal period. In facio-scapulo-humeral muscular dystrophy, tibialis anterior and hamstring muscles have often a decreased density and psoas muscles are normal or hypertrophic. In myotonic dystrophy a hypodense perifemoral crescent is frequently observed. Diagnosis between limb-girdle myopathy ("empty" muscles with preserved limits, hypertrophic muscles, hypodense gastrocnemius medialis muscles) and chronic spinal amyotrophy (irregular and atrophic muscles without selective involvement and hypertrophic muscles) is tentatively proposed but is not considered to be clear-cut. Muscle involvement has an asymmetric distribution in amyotrophic lateral sclerosis and is rather symmetric in peripheral neuropathies. PMID- 2996110 TI - [Progabide treatment of a case of the syndrome of painful legs and moving toes]. AB - There is presently no recognised therapy for the painful legs and moving toes syndrome. Treatment by progabide, a GABA-agonist, was successful in one case: a gradual improvement was observed during four weeks and the movements have now disappeared after six months of progabide therapy. PMID- 2996112 TI - [Evaluation of the content and composition of dietary fiber in daily meals of children]. PMID- 2996111 TI - [Chemoluminescence of the human neutrophilic granulocyte, II. Origin, normal values, hereditary deficiencies]. PMID- 2996114 TI - Effect of corticotherapy on CSF and serum immunoglobulins in patients with acute polyradiculoneuritis. PMID- 2996113 TI - Sodium-potassium stimulated adenosine triphosphatase and postnatal ontogenetic development of epileptic reactivity in rabbit motor cortex. PMID- 2996115 TI - [Muscular ultrasonic diagnosis. Etiological approach to various backaches]. AB - Certain cases of mechanical back pain present unusual clinical and ultrasonographic features involving the posterior lumbar muscle groups, which can be used to distinguish various types. The clinical examination may isolate a juxta-vertebral territory in which pressure triggers or exacerbates the back pain; anaesthesia of this territory is accompanied by pain relief. Ultrasonography may reveal a hypoechogenic area in this muscle territory in which histomorphometric and ultrastructural studies, performed in three cases, revealed endomysial and perimysial adiposis associated with atrophy probably due to peripheral neuropathy. PMID- 2996116 TI - [Disk metastasis simulating disk hernia]. PMID- 2996117 TI - [The effect of in vitro administration of isoprenaline on Na+ K+ and Mg++ dependent ATPase in the brain of rats of various ages]. PMID- 2996118 TI - Intragastric bile acid concentrations in healthy subjects and in patients with gastric and duodenal ulcer and the influence of fiber-enriched wheat bran in patients with gastric ulcer. AB - Fasting and postprandial intragastric bile acid concentrations were determined in healthy subjects and in patients with gastric and duodenal ulcer. The results showed great interindividual variation and wide overlapping between groups. Mean bile acid concentrations, fasting and postprandially, were significantly higher in patients with gastric ulcer than in healthy subjects. The differences between gastric and duodenal ulcer patients and between duodenal ulcer patients and healthy subjects were not significant. Fiber-enriched wheat bran reduced bile acid concentrations significantly in patients with gastric ulcer disease. PMID- 2996119 TI - Chronic 'cryptogenic' liver disease and malignant hepatoma in intermediate alpha 1-antitrypsin deficiency identified by a Pi Z-specific monoclonal antibody. AB - Using a monoclonal antibody against the Pi Z genetic variant of alpha 1 antitrypsin in an enzyme-linked immunosorbent assay, we have screened plasma samples from 857 consecutive patients with liver disease for the presence of Pi Z alpha 1-antitrypsin. Intermediate alpha 1-antitrypsin deficiency (Pi MZ and SZ) was found in 64 cases, or 7.6%, compared with an expected 4.8% (p less than 0.001). The plasma alpha 1-antitrypsin level was subnormal in only 50% of them. Forty-three of the 64 heterozygotes were men, compared with 494 of 857 (58%) in the total study population (p less than 0.001). At least 14 heterozygotes had cryptogenic liver disease, compared with 3 of 128 sex- and age-matched controls from the same study population (p less than 0.001). Malignant hepatoma occurred in 6 heterozygotes compared with 1 control (p less than 0.01), and in 13 of all 793 non-Pi Z patients (p less than 0.001). PMID- 2996120 TI - Bulk laxatives: their dietary fibre composition, degradation, and faecal bulking capacity in the rat. AB - The intestinal dietary fibre degradation and faecal bulking capacity of various bulk laxatives were investigated by means of balance experiments on rats. Nitrogen, fat, and mineral excretion in faeces was also studied. The dietary fibre content of the various bulk laxatives was quite different (in g/kg dry matter): ACO fibre tablets (barley and citrus pulp), 451; Fiberform (wheat bran based), 817; Inolaxol (sterkulia gum), 696; and Vi-Siblin (ispaghula husk), 533. The increase in faecal dry matter per 1 g dietary fibre was similar with ACO fibre tablets, Fiberform, and Vi-Siblin. Inolaxol gave a significantly (p less than 0.001) higher faecal dry-weight increment, mainly due to an increased mineral excretion. Of the dry-weight increment, 59-82% constituted undegraded dietary fibre. Thus, 68-97% of the fibre passed through the gastrointestinal tract without being degraded. All the bulk laxatives caused a similar increase in the faecal N content, whereas the increase in faecal lipids was most pronounced with Vi-Siblin. The water-holding capacity of faeces was more pronounced with Inolaxol and Vi-Siblin than with ACO fibre tablets and Fiberform. PMID- 2996121 TI - The dynamic morphology of the transferrin-transferrin receptor system in human leukaemia/lymphoma cell lines and its relation to iron metabolism and cell proliferation. AB - Some functional and morphological aspects of the transferrin-transferrin-receptor (Tf-TfR) system were investigated in 12 human haematopoietic tumour cell lines. The iron uptake ability, studied by 59Fe labelling, varied considerably between individual tumour cell lines. The studies of the morphology of the Tf-TfR system by fluorescence based methods using labelling by FITC-Tf, and by indirect immunofluorescence (OKT9 anti-TfR monoclonal antibody) demonstrated that the iron uptake was the result of receptor-mediated endocytosis (RME) of the Tf-TfR complex. The RME was better developed in the haemoglobin synthesizing K-562 cells than in the monocytic U-937 cells, suggesting that the intracellular iron processing capacity differs from cell type to cell type. The efficiency of iron uptake, transport and storage was related to cell growth. Inhibition of growth by increasing cell densities or by drug treatments (phorbol ester TPA and difluoromethylornithine DFMO) was thus accompanied by a decrease in surface Tf binding, in cellular Tf handling capacity and in iron accumulation. TPA induced a redistribution of the TfR-pool to the intracellular space as demonstrated by morphology. PMID- 2996122 TI - Myeloperoxidase-deficient polymorphonuclear leucocytes. (IV): Relation to FAB classification in acute myeloid leukaemia. AB - In 148 consecutive cases of untreated acute myeloid leukaemia (AML) the relation between an increased number of myeloperoxidase (MPO)-deficient polymorphonuclear leucocytes (PMN) and a classification modified after the proposals outlined by the French-American-British Co-operative Group, the FAB classification, was investigated. In 22 cases with minimal granulocytic component, 8 M5, 13 M6, 1 M7, no one (0%) showed an increased number of MPO-deficient PMN. In 3 (13%) of 23 cases with slight granulocytic component, 3 M0, 20 M1, and in 49 (57%) of 86 cases with granulocytic differentiation (53 M2, 1 M3, 32 M4), an increased number was demonstrated. This difference was statistically significant at a high level (P less than 0.0001). The number of MPO-deficient PMN could not be estimated in 15 cases of AML and 2 additional cases were mixed leukaemias. It is concluded that an increased number of MPO-deficient PMN in acute leukaemia speaks in favour not only of AML, but suggests the diagnosis of subtypes with some granulocytic component, most likely M1, M2, M3 or M4. Furthermore, the results support the concept that in AML at least some of the mature myeloid cells may be involved in the leukaemic process. PMID- 2996123 TI - Myeloperoxidase-deficient polymorphonuclear leucocytes. (V): Relation to FAB classification and neutrophil alkaline phosphatase activity in primary myelodysplastic syndromes. AB - In 45 cases of primary myelodysplastic syndrome; 16 refractory anaemia (RA), 11 RA with ring sideroblasts (RA+), 13 RA with excess of blasts (RAEB), 5 chronic myelomonocytic leukaemia (CMML), the relations between myeloperoxidase (MPO) activity in polymorphonuclear leucocytes (PMN), neutrophil alkaline phosphatase (NAP) activity, absolute number of PMN and thrombocytopenia were investigated. 11 patients (26%) showed abnormal numbers (greater than 4%) of MPO-deficient PMN and 27 (75%) showed abnormal NAP activity (NAP score; greater than 134.0, less than 15.0), mostly decreased. No significant correlations between MPO activity and NAP activity were demonstrated, nor were any significant correlations found with the other parameters investigated. The FAB-subtypes, RAEB and CMML, showed a significant correlation to thrombocytopenia (p = 0.028) and to pancytopenia (p = 0.024). The findings may support the view that at least some of the myelodysplastic syndromes may be fundamentally the same disease as acute myeloid leukaemia. PMID- 2996124 TI - Sickle gene in western Arabia is mostly associated with the 13.0 kb Hpa I fragment. PMID- 2996126 TI - The effect of non-inflamed synovial fluid on neutrophil function in vitro. AB - In neutrophils preincubated with non-inflamed synovial fluid, opsonized zymosan induced O2- production was increased by 232.7 +/- 19.1% and degranulation induced by zymosan or phorbol myristate acetate was increased by 152.8 +/- 21.8% and 217.4 +/- 26.3 respectively. Unstimulated neutrophils were not directly activated by the fluid, nor did it affect their chemotactic response. The activity was not abolished by heat (56 degrees C, 30 min, or 100 degrees C, 3 min) or by treatment with trypsin or hyaluronidase. The activity was not present in the serum albumin bound lipid extract of the synovial fluid. Thus, this activity may represent a heat- and trypsin-resistant factor which is neither a complement component nor hyaluronic acid. It is proposed that this synovial fluid factor plays a role in increasing the neutrophil killing potential during the inflammatory process. PMID- 2996125 TI - Second messenger function of arachidonic acid lipoxygenation products in human natural killer cell lysis? AB - Human natural killer (NK) cells were tested in the presence of several fatty acid oxygenase inhibitors such as nordihydroguaiaretic acid (NDGA), 5,8,11,14 eicosatetraynoic acid, 3-amino-1-m-(trifluoromethyl)-phenyl-2-pyrazoline (BW 755C), and indomethacin. All drugs inhibited NK lysis at the post-target cell binding level at concentrations that also suppressed lipoxygenation of arachidonic acid, suggesting that such reactivity may be required for effector cell triggering. NDGA gave a 50% NK cell inhibition in the range of 10-30 microM and also suppressed antibody-dependent and lectin-dependent systems. Further evidence of the involvement of arachidonic acid lipoxygenation was found as NK activity could be reconstituted to NDGA-suppressed effector cells with several metabolites such as LTB4, LTB4 analogues, and hydroxyeicosatetraenoic acids lipoxygenated at C-5, C-12, and C-15. Cyclic nucleotides such as cGMP and cAMP could also reconstitute activity with optimal effects at approximately 10(-8) M. The combined evidence is compatible with a model for triggering lysis in which lipoxygenation products have a second messenger function. Whether arachidonic acid lipoxygenation is necessary for effector cells at all different activation/differentiation stages and whether the lipoxygenation products activate guanylic cyclase, protein kinase C, or some other target molecule remain to be further investigated. PMID- 2996127 TI - Identification of patients at risk of symptomatic cytomegalovirus infection after open-heart surgery. AB - In a prospective study, 58 consecutive patients submitted to open-heart surgery were followed from the preoperative period up to 3 months postoperatively with regard to clinical status, cytomegalovirus (CMV) antibodies and signs of enzymatic liver damage. Forty-eight patients (83%) were seropositive prior to operation. Of the ten (17%) preoperatively seronegative patients, five became CMV seropositive during follow-up. Of the 48 initially seropositive patients, nine showed heightened CMV-antibody titers after the operation. Significant symptomatic CMV illness with protracted fever developed in four patients, all from the group of five with seroconversion. All five of these patients had enzymatic liver damage. The incidence of symptomatic CMV infection after open heart surgery in CMV-negative patients probably is higher than previously assumed, while CMV-positive patients seem to have complete protection against CMV morbidity. PMID- 2996128 TI - Ohmefentanyl--a new agonist for mu-opiate receptor. AB - Ohmefentanyl (F 7302, N[1-(beta-hydroxy-beta-phenylethyl)-3-methyl-4-piperidyl]-N -phenylpropionamide) is a potent synthetic analgesic agent. The analgesic activity of ohmefentanyl in mice is 6300 times more potent than that of morphine. The potency of ohmefentanyl in competing with specific binding of 3H-naloxone is reduced by Na+ (100 mM) and GTP (50 microM), thus suggesting the agonist properties of this compound. Binding characteristics of 3H-ohmefentanyl with mice brain P2 fraction are studied. An important saturable, specific and reversible binding is demonstrated. Scatchard analysis indicates the existence of two classes of binding sites (KD1 = 0.32nM, KD2 = 3.91nM). Various opiate drugs strongly inhibit the binding of 3H-ohmefentanyl, but nonopiate drugs have negligible affinity. Comparison of the relative potencies of morphine, DSTLE(Tyr D-Ser-Gly-Phe-Leu-Thr, a specific ligand for the delta-opiate receptor) and ohmefentanyl in competing with 3H-dihydromorphine (mu) and 3H-[D-Ala2, D-Leu5] enkephalin (delta) binding to crude synaptic plasma membranes of mice brain shows that the inhibitory potencies of morphine, DSTLE and ohmefentanyl on 3H-DHM binding are 79, 0.11 and 81.5 times those on 3H-DADLE binding respectively. This suggests that ohmefentanyl has potent mu-agonist properties. PMID- 2996129 TI - Molecular cloning of a complementary DNA encoding human macrophage-specific colony-stimulating factor (CSF-1). AB - Complementary DNA (cDNA) clones encoding human macrophage-specific specific colony-stimulating factor (CSF-1) were isolated. One cDNA clone codes for a mature polypeptide of 224 amino acids and a putative leader of 32 amino acids. This cDNA, which was cloned in the Okayama-Berg expression vector, specifies the synthesis of biologically active CSF-1 in COS cells, as determined by a specific radioreceptor assay, macrophage bone marrow colony formation, and antibody neutralization. Most of the cDNA isolates contain part of an intron sequence that changes the reading frame, resulting in an abrupt termination of translation; these cDNA's were inactive in COS cells. The CSF-1 appears to be encoded by a single-copy gene, but its expression results in the synthesis of several messenger RNA species, ranging in size from about 1.5 to 4.5 kilobases. PMID- 2996130 TI - Dispute over access to Reye's study data. PMID- 2996131 TI - Deletion in chromosome 11p associated with a hepatitis B integration site in hepatocellular carcinoma. AB - Hepatitis B virus (HBV), a virus with known carcinogenic potential, integrates into cellular DNA during long-term persistent infection in man. Hepatocellular carcinomas isolated from viral carriers often contain clonally propagated viral DNA integrations. As small chromosomal deletions are associated with several types of carcinomas, the occurrence of chromosomal deletions in association with HBV integration in hepatocellular carcinoma was studied. HBV integration was accompanied by a deletion of at least 13.5 kilobases of cellular sequences in a human hepatocellular carcinoma. The viral DNA integration and deletion of cellular sequences occurred on the short arm of chromosome 11 at location 11p13 11p14. The cellular sequences that were deleted at the site of HBV integration were lost from the tumor cells, leaving only a single copy of the remaining cellular allele. PMID- 2996132 TI - A strong influence of serotonin axons on beta-adrenergic receptors in rat brain. AB - The role of serotonin axons in modulating the norepinephrine neurotransmission system in rat brain was investigated. Selective lesions of the forebrain serotonergic system were made by injecting 5,7-dihydroxytryptamine into the midbrain raphe nuclei. Four to six weeks after the lesion, the uptake of 3H labeled serotonin in the frontal cortex and the hippocampus was reduced by more than 90 percent, while neither the uptake of 3H-labeled norepinephrine nor the content of norepinephrine was affected in either tissue. The number of beta adrenergic receptors, as measured by radioligand binding with 3H-labeled dihydroalprenolol, was increased in the frontal cortex and hippocampus of rats with lesions. Similarly, specific lesions of central serotonin axons produced by systemically administered p-chloramphetamine resulted in an increase in the binding of 3H-labeled dihydroalprenolol to beta-adrenergic receptors and in the production of adenosine 3',5'-monophosphate in response to isoproterenol. These results indicate that serotonin axons may regulate beta-adrenergic receptor number and function in brain. PMID- 2996133 TI - Evidence that the v-sis gene product transforms by interaction with the receptor for platelet-derived growth factor. AB - A scheme for partial purification of biologically active v-sis-coded protein from cells transformed with simian sarcoma virus (SSV) has made possible a functional comparison of the transforming protein with platelet-derived growth factor (PDGF). The SSV-transforming gene product is capable of specifically binding PDGF receptors, stimulating tyrosine phosphorylation of PDGF receptors, and inducing DNA synthesis in quiescent fibroblasts. Each of these activities was specifically inhibited by antibodies to different regions of the v-sis gene product. Moreover, viral infection of a variety of cell types revealed a strict correlation between those cells possessing PDGF receptors and those susceptible to transformation by SSV. These findings provide evidence that SSV-transforming activity is mediated by the interaction of a virus-coded mitogen with PDGF receptors. PMID- 2996134 TI - Identification of the protein encoded by the E6 transforming gene of bovine papillomavirus. AB - Papillomaviruses (PV) contain several conserved genes that may encode nonstructural proteins; however, none of these predicted gene products have been identified. Papillomavirus E6 genes are retained and expressed as RNA in PV associated human and animal carcinomas and cell lines. This suggests that the E6 gene product may be important in the maintenance of the malignant phenotype. The E6 open reading frame of the bovine papillomavirus (BPV) genome has been identified as one of two BPV genes that can independently transform mouse cells in vitro. A polypeptide encoded by this region of BPV was produced in a bacterial expression vector and used to raise antisera. The antisera specifically immunoprecipitated the predicted 15.5-kilodalton BPV E6 protein from cells transformed by the E6 gene. The E6 protein was identified in both the nuclear and membrane fractions of these transformed cells. PMID- 2996135 TI - Transcription of class III genes activated by viral immediate early proteins. AB - The adenovirus EIA and pseudorabies virus immediate early (IE) proteins induce transcription from transfected viral and nonviral genes transcribed by RNA polymerase II (class II genes). These proteins have now been shown also to activate transcription of transfected genes transcribed by RNA polymerase III (class III genes). As previously observed for class II genes, this stimulation of class III gene transcription was much greater for transfected genes than for the major endogenous cellular class III genes. Extracts made from cell lines stably expressing a transfected pseudorabies virus IE gene were 10 to 20 times more active in the in vitro transcription of exogenously added class III genes than extracts of the parental cell line. These results indicate that the E1A and IE proteins stimulate the expression of class III genes by a mechanism similar to the mechanism for stimulation of class II gene transcription by these proteins. PMID- 2996137 TI - Bidirectional SV40 transcription mediated by tandem Sp1 binding interactions. AB - The 21-base pair repeat elements of the SV40 promoter contain six tandem copies of the GGGCGG hexanucleotide (GC-box), each of which can bind, with varying affinity, to the cellular transcription factor, Sp1. In vitro SV40 early RNA synthesis is mediated by interaction of Sp1 with GC-boxes I, II, and III, whereas transcription in the late direction is mediated by binding to GC-boxes III, V, and VI. PMID- 2996136 TI - Inhibition of lymphocyte proliferation by a synthetic peptide homologous to retroviral envelope proteins. AB - The retroviral transmembrane envelope protein p15E is immunosuppressive in that it inhibits immune responses of lymphocytes, monocytes, and macrophages. A region of p15E has been conserved among murine and feline retroviruses; a homologous region is also found in the transmembrane envelope proteins of the human retroviruses HTLV-I and HTLV-II and in a putative envelope protein encoded by an endogenous C-type human retroviral DNA. A peptide (CKS-17) was synthesized to correspond to this region of homology and was examined for its effects on lymphocyte proliferation. CKS-17 inhibited the proliferation of an interleukin-2 dependent murine cytotoxic T-cell line as well as alloantigen-stimulated proliferation of murine and human lymphocytes. Four other peptides, representing different regions of virus proteins, were inactive. These results suggest that the immunosuppressive portion of retroviral transmembrane envelope proteins may reside, at least in part, in a-conserved sequence represented by the CKS-17 peptide. PMID- 2996138 TI - Transposon tagging to detect a latent virus in Myxococcus xanthus. AB - Transposon mutagenesis of the bacterium Myxococcus xanthus with the transposon Tn5 revealed a special class of bacterial mutants that transduced the transposon through culture supernatant fluids. Virus-like particles copurified with transducing activity. Transposon tagging for detecting these virus-like particles may be generally useful in isolating endogenous viral agents capable of transferring genetic information between cells. PMID- 2996139 TI - The 36-kilodalton substrate of pp60v-src is myristylated in a transformation sensitive manner. AB - A primary intracellular substrate for pp60v-src kinase in a variety of avian and mammalian cells is a protein of 34 to 39 kilodaltons (kD). After incubation of chicken embryo fibroblasts (CEF) with [3H]myristic acid for 4 hours, the 36-kD protein contained covalently bound myristic acid by several criteria: (i) the radioactively labeled material comigrated with the 36-kD protein on sodium dodecyl sulfate-polyacrylamide gels in one and two dimensions, (ii) the labeled material was insoluble in chloroform-methanol, and (iii) radioactively labeled myristate could be recovered from the purified 36-kD protein. The resistance of the acyl fatty acid moiety to hydrolysis by hydroxylamine suggested that the covalent linkage to the 36-kD protein may be through an amide linkage. The [3H]myristic-acid labeling of the 36-kD protein in Rous sarcoma virus-transformed CEF showed a reduction of up to 45 percent when compared to an identical amount of 36-kD protein derived from normal cells; this reduction was not due to general changes in myristic acid metabolism in transformed cells. PMID- 2996140 TI - Functional relation between HTLV-II x and adenovirus E1A proteins in transcriptional activation. AB - The mechanism of cellular transformation by the human T-cell leukemia viruses (HTLV) is thought to involve a novel gene known as the x gene. This gene is essential for HTLV replication and acts by enhancing transcription from the HTLV long terminal repeat. The HTLV x gene product may also cause aberrant transcription of normal cellular genes, resulting in transformation of the infected cells. Although there is no evidence as yet for such a mechanism, it was shown that the HTLV-II x gene product can activate transcription from adenovirus E1A-dependent early promoters and therefore has the potential to activate cellular genes. It was also shown that the adenovirus and herpes pseudorabies immediate early proteins activate expression from the HTLV-I and HTLV-II long terminal repeats, though at lower levels than with the x gene product. These findings indicate possible common mechanisms of action for transcription regulatory genes of distinct viruses. PMID- 2996142 TI - Patent dispute divides AIDS researchers. PMID- 2996143 TI - HTLV-III and LAV: similar, or identical? PMID- 2996141 TI - Structure of the human interleukin-2 receptor gene. AB - The gene encoding the human interleukin-2 (IL-2) receptor consists of 8 exons spanning more than 25 kilobases on chromosome 10. Exons 2 and 4 were derived from a gene duplication event and unexpectedly also are homologous to the recognition domain of human complement factor B. Alternative messenger RNA (mRNA) splicing may delete exon 4 sequences, resulting in a mRNA that does not encode a functional IL-2 receptor. Leukemic T cells infected with HTLV-I and normal activated T cells express IL-2 receptors with identical deduced protein sequences. Receptor gene transcription is initiated at two principal sites in normal activated T cells. Adult T cell leukemia cells infected with HTLV-I show activity at both of these sites, but also at a third transcription initiation site. PMID- 2996144 TI - Wounding and its role in RSV-mediated tumor formation. AB - Tumors induced in chickens by Rous sarcoma virus remain localized at the site of injection even though the animals become viremic. Tumors have now been shown to be inducible at other sites if a wound is inflicted or if the tissue is injured by administration of tumor promoters. These findings indicate that local wounding plays a role in the spread of tumorigenicity of Rous sarcoma virus. PMID- 2996145 TI - Iron(II) EDTA used to measure the helical twist along any DNA molecule. AB - A new method has been devised to measure the number of base pairs per helical turn along any DNA molecule in solution. A DNA restriction fragment is adsorbed onto crystalline calcium phosphate, fragmented by reaction with iron(II) EDTA, and subjected to electrophoresis on a denaturing polyacrylamide gel. A modulated cutting pattern results, which gives directly the helical periodicity of the DNA molecule. A 150-base pair sequence directly upstream of the thymidine kinase gene of the type 1 herpes simplex virus was found to have an overall helical twist of 10.5 base pairs per turn, which is characteristic of the B conformation of DNA. In addition, purines 3' to pyrimidines showed lower than expected reactivity toward the iron cutting reagent, which is evidence for sequence-dependent variability in DNA conformation. PMID- 2996146 TI - Hypoglycemia-induced neuronal damage prevented by an N-methyl-D-aspartate antagonist. AB - The possibility that neuronal damage due to hypoglycemia is induced by agonists acting on the N-methyl-D-aspartate (NMDA) receptor was investigated in the rat caudate nucleus. Local injections of an NMDA receptor antagonist, 2-amino-7 phosphonoheptanoic acid, were performed before induction of 30 minutes of reversible, insulin-induced, hypoglycemic coma. Neuronal necrosis in these animals after 1 week of recovery was reduced 90 percent compared to that in saline-injected animals. The results suggest that hypoglycemic neuronal damage is induced by NMDA receptor agonists, such as the excitatory amino acids or related compounds. PMID- 2996147 TI - Heterogeneity of childhood acute lymphoblastic leukemia--impact on prognosis and therapy. PMID- 2996149 TI - The platelet fibrinogen receptor. PMID- 2996148 TI - Biological heterogeneity of small cell lung cancer. AB - Over the past 20 years considerable advances have been made in the characterization of the biologic properties of small cell lung cancer. The recognition that this histologic type of lung cancer, in contrast to the other major types of lung cancer, is highly sensitive to both chemotherapy and radiation therapy lead to significant improvements in the overall survival of patients with this disease. However, in spite of the initial major advances in therapy, overall results and survival have remained unchanged over the past 5 years. The majority of patients, although they will demonstrate an initial response to cytotoxic therapy, will ultimately die of their disease due to the development of drug resistance. Whether this development of drug resistance within a tumor cell represents the emergence of resistant clones of cells present at diagnosis, or a change within the cells exposed to cytotoxic therapy that renders them resistant to further therapy remains to be determined. The development of laboratory techniques that facilitate the culture and establishment of SCLC cell lines has greatly improved our understanding of the biology of SCLC, in addition to providing useful models for studies of mechanisms of drug resistance and metabolism. The ease of establishment of SCLC cell lines in defined medium suggests that these cells secrete "autocrine growth factors" essential for their growth in vitro. The characterization of these factors may provide an alternative means for treating this tumor in vivo. Moreover, by developing specific culture media for other types of lung cancer, similar advances in our knowledge of these tumors will occur. Although the growth of SCLC cells in a clonogenic assay may be of value in chemotherapy selection, the established cell lines provide a model for studying mechanisms of drug and radiation sensitivity; and drug metabolism. The recognition that SCLC cell lines have elevated levels of L-dopa decarboxylase has lead to the development of specific agents that may alter the growth of these cells through inhibition of polyamine synthesis. Such specifically designed therapy may become important in the future therapy of these patients. The establishment of cell lines has clearly indicated the considerable heterogeneity that exists in SCLC.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2996151 TI - Problems in the management of insulinoma. PMID- 2996150 TI - Association of proteins with the platelet cytoskeleton. PMID- 2996152 TI - Hypophosphatemic rickets: still misdiagnosed and inadequately treated. AB - We studied the presentation and results of medical therapy in 25 children with sex-linked dominant hypophosphatemic rickets. The average age at diagnosis was 3.8 years. Reasons for the delay included misdiagnosis and failure to recognize the normal range of serum phosphorus levels in children. Early diagnosis and treatment (before age 1) was associated with normal alignment of the lower limbs. Combination therapy with phosphate and vitamin D2 improved growth and bone mineralization, but did not change the height percentile or limb alignment. Limited use of calcitriol (1,25-dihydroxyvitamin D3) was not helpful in the adolescent but was associated with limited height increase in two younger children. Early diagnosis and medical therapy should prevent bowing of the legs. PMID- 2996153 TI - Surgical aspects of limb deformity in hypophosphatemic rickets. AB - Our assessment of the surgical correction of bowleg deformity in eight patients with sex-linked dominant hypophosphatemic rickets showed the best results in patients having staged, proximal tibial osteotomies at completion of growth. Lack of medical therapy was associated with recurrent genu varum or markedly delayed union. Undercorrection may be prevented by using the hip-knee-ankle axis to plan the realignment procedure. PMID- 2996154 TI - Fatal coxsackievirus B4 infection in a neonate. AB - We reported a fatal coxsackievirus B4 infection in a neonate. The CSF WBC was elevated, with polymorphonuclear leukocytes predominating initially and lymphocytes predominating later in the illness. Autopsy findings included inflammation of the heart and liver. Coxsackievirus B4 was isolated from the heart and quantitated as 10(3) 50% tissue culture infectious doses (TCID50) per gram of myocardium. PMID- 2996155 TI - [Problems in the diagnosis of tumors of the pancreas]. PMID- 2996156 TI - Spinal movements in ankylosing spondylitis and the effect of treatment. AB - Fourteen male patients with ankylosing spondylitis, admitted for a 2-week period of inpatient treatment, had their spinal mobility assessed on admission and at the end of treatment by clinical measures and a three-dimensional radiographic technique. The patients were given injections of low-dose corticotrophin (ACTH) or placebo under a double-blind protocol. Initially all the patients had restricted movements compared with normal. After treatment all showed some improvement of mobility but no additional benefit accrued from ACTH. Clinical measures of mobility must be interpreted with care as the changes in these measurements were not closely reflected in the lumbar movements measured radiographically. Changes seen in plain radiographs were of little predictive value for improvements in mobility. PMID- 2996157 TI - [Monoclonal antibodies and cancer]. PMID- 2996159 TI - Limitations of enzyme immunoassay tests for detection of HTLV-III/LAV antibodies. AB - A commercial enzyme immunoassay (EIA) kit was compared with an immunofluorescence test for the detection of antibodies directed against human T-cell lymphotropic virus type III (HTLV-III). The EIA kit is recommended for the screening of blood and plasma products only and is not suitable for diagnostic purposes because of the problem of nonspecific reactions. It is also recommended that the EIA kit not be used for screening immunoglobulin preparations for anti-HTLV-III antibodies because of the problem of false-positive reactions. PMID- 2996158 TI - [Monoclonal antibodies in diseases of viral origin]. PMID- 2996160 TI - AIDS--report of an epidemiological seminar. PMID- 2996161 TI - The role of Pseudomonas species in patients treated with ampicillin and Sulbactam for gangrenous and perforated appendicitis. AB - A prospective, randomized, double-blinded comparison of Sulbactam and ampicillin and clindamycin and gentamicin is described. The combination of ampicillin and Sulbactam was not as effective in the management of perforated appendicitis and gangrenous appendicitis as was clindamycin and gentamicin. While both combinations of antibiotics had good anaerobic activity and failures were not associated with the recovery of Bacteroides fragilis group organisms, infectious complications were seen in patients from whom Pseudomonas were isolated. These pseudomonads were not nosocomially acquired and were found especially in patients with perforated appendicitis. We concluded that the combination of clindamycin and gentamicin, although less convenient to administer to the patient, remains the adjunctive antibiotic management of choice for perforated or gangrenous appendicitis. The epidemiologic factors of Pseudomonas species as a primary pathogen in peritonitis deserves further attention. PMID- 2996162 TI - Ultrasonically guided subsegmentectomy. AB - A new operative procedure for systematic subsegmentectomy guided by ultrasound has been described. This operation consists of operative sonography, ultrasonically guided puncture and injection of dye and hemihepatic blood occlusion. Systematic subsegmentectomy was performed upon 57 patients without operative mortality. The cumulative one year survival rate of 35 patients with hepatocellular carcinoma who underwent operation at our hospital was 80.3 per cent. The two and three year survival rates were 63.3 and 52.6 per cent, respectively. PMID- 2996163 TI - Historical article: Glioblastomas: by K.G. McKenzie. PMID- 2996164 TI - Bendectin and interventricular septal defects. PMID- 2996165 TI - Comments on "Alcohol enhancement of marihuana-induced fetotoxicity". PMID- 2996166 TI - [Hypertension treatment 1985: significance of calcium antagonists and angiotensin converting enzyme inhibitors]. PMID- 2996167 TI - Epinephrine-induced aggregation of rabbit platelets refractory to ADP. AB - The mechanisms involved in platelet aggregation induced by epinephrine are unclear. Although epinephrine does not aggregate washed rabbit platelets, platelets made refractory to ADP will aggregate in response to epinephrine in the presence of ADP. We have examined whether the mechanism(s) by which epinephrine induces aggregation of refractory platelets involves fibrinogen binding and Ca2+ association. With normal platelets, ADP causes aggregation, fibrinogen binding and Ca2+ association in a medium containing 0.2 mM 45Ca2+. After 3 min of incubation with ADP, fibrinogen dissociates from platelets, but 45Ca2+ does not. Epinephrine alone does not cause aggregation, fibrinogen binding or 45Ca2+ association. Platelets that are refractory to ADP do not aggregate and bind fibrinogen upon addition of ADP, but aggregate and bind fibrinogen in response to epinephrine, provided ADP is still present. These effects of epinephrine are mediated by the alpha-adrenergic receptor since they are blocked by phentolamine or verapamil and potentiated by propranolol. However, epinephrine-induced aggregation of platelets refractory to ADP does not involve further detectable increase in the amount of 45Ca2+ associated with the platelets. PMID- 2996168 TI - Studies of suloctidil in experimental thrombosis in baboons. AB - Suloctidil has been evaluated in the baboon for its antithrombotic efficacy using models of both acute and chronic arterial thrombogenesis. Acute thrombus formation was initiated by Dacron vascular grafts inserted as extension segments into chronic arteriovenous shunts. 111In-platelet deposition was measured by scintillation camera imaging for one hour. The results after oral administration of suloctidil (100 mg/kg/d in two divided doses) were not different from control studies. Moreover, concurrent heparin anticoagulation did not affect 111In platelet deposition compared with control data. In contrast, ticlopidine (20 mg/kg/d) significantly decreased platelet deposition that was reduced further by the addition of heparin. Chronic arterial-thromboembolism was initiated by segments of polyurethane (Biomer) cannula introduced into chronic arteriovenous shunts. Thrombus formation by the polyurethane cannula was measured as 111In platelet turnover (corrected for removal of senescent platelets). Cannula platelet consumption was unaffected by suloctidil (20 mg/kg/d given in two divided doses for two days preceding and throughout the period of platelet survival measurement). In contrast, dipyridamole (10 mg/kg/d) and sulfinpyrazone (100 mg/kg/d) completely interrupted cannula platelet consumption. We conclude that suloctidil probably has little or no effect on platelet-dependent thrombus formation. PMID- 2996169 TI - Enhancing effect of dietary supplementation with omega-3 fatty acids on plasma fibrinolysis in normal subjects. AB - A supplement of MaxEPA oil containing 5 g of omega-3 polyunsaturated fatty acids was given for two weeks to nine normal volunteers. The vascular plasminogen activator (VPA) level increased and there was a fall in the levels of inhibitors of vascular plasminogen activator (IPA) and of plasmin, alpha 2-antiplasmin (alpha 2-AP). No significant changes occur in serum cholesterol, triglycerides, HDL or LDL levels. PMID- 2996170 TI - Inhibitory effect of sodium salicylate on ADP-induced platelet aggregation and on 45Ca2+ uptake into platelets. AB - The effects of sodium salicylate (SS) on ADP-induced platelet aggregation and on a metabolism of calcium in platelets were studied, using gel-filtrated platelets (GFP). SS inhibited dose-responsively ADP-induced aggregation in the presence of fibrinogen and Ca2+. It was found that extracellular 45Ca2+ was rapidly taken up into platelets after stimulation by ADP, while SS significantly inhibited this activity. On the other hand, SS had no effect on platelet aggregation induced by 0.11-1.0 microM ionophore A23187. Therefore, it was found that the inhibitory effect of SS on ADP-induced platelet aggregation may be due to the inhibition of the active influx of extracellular Ca2+ into platelets during aggregation. PMID- 2996171 TI - HLA typing of Epstein-Barr virus transformed lymphoblastoid cell lines (LCL). AB - The application of standard tissue typing techniques to cells other than peripheral blood lymphocytes has been accompanied by the problem of extra reactions. This applies as well to Epstein-Barr virus transformed lymphoblastoid cell lines (LCL) as to leukemic cells and human spleen cells. These extra reactions are attributable to additional antibodies in the typing sera which are not apparent under standard conditions with PBLs. Two types are described: Type 1 extras, which becomes apparent after longer incubation times and are attributed to weak antibodies and type 2 extras which are apparent after shorter incubation times and are attributed to subpopulation specific or differentiation antigens. Technical modifications are proposed by which these extras can be circumvented. They include: Only start typing when cells have been cultured for 2 to 3 days. Remove dead cells by spinning over standard ficoll-hypaque or 11% triosil. Use shorter incubation times. Avoid using sera that give too many type 2 extras. In this way phenotypes can be accurately identified on LCL's obtained from kidney transplant donors and recipients. When LCL's were compared with their matching PBL, HLA phenotypes were concordant in 87% of cases for HLA-A, 90% for HLA-B, 81% for HLA-C and 70% for HLA-DR. PMID- 2996172 TI - Effects of dobutamine on cyclic AMP content in canine upper urinary tract tissue. AB - There was no regional difference in tissue cAMP content throughout the upper urinary tract specimens obtained from dogs immobilized by pancuronium bromide (0.1 mg/kg, i.v.). The cAMP content was increased in all regions examined 5 min after administration of dobutamine (1 mg/kg, i.v.) and pancuronium bromide. The increase was much higher in the pelvicalyceal region than in the other upper urinary tract tissue. The responsiveness of the pelvicalyceal region where pacemaker cells for ureteral peristalsis exist is considered to be higher than that in the other upper urinary tract tissue in respect to dobutamine-induced cAMP increase. PMID- 2996173 TI - Oxidation of N-methylthiobenzamide and N-methylthiobenzamide S-oxide by liver and lung microsomes. AB - The in vitro oxidation of N-[14C]methylthiobenzamide (NMTB) and N [14C]methylthiobenzamide S-oxide (NMTBSO) by rat lung and liver microsomes was studied. In the presence of an NADPH-generating system, NMTB was rapidly converted to NMTBSO and small amounts of N-methylbenzamide (NMBA) and covalently bound metabolites (CVB). Under similar conditions, NMTBSO was converted to NMBA and CVB. Studies with metabolic inhibitors indicate that both the S-oxidation of NMTB and its further conversion to NMBA and CVB, probably via enzymatic oxidation to the S,S-dioxide, are catalyzed by both cytochromes P-450 and the FAD containing monooxygenase (MFMO). Based on differential effects of inhibitors with lung vs liver microsomes, it would appear that in lung microsomes the MFMO plays a significantly greater role than cytochrome P-450 in the oxidation of NMTB and NMTBSO, whereas in the liver the contribution of these two pathways is more nearly equal. 1-Methyl-1-phenyl-3-benzoylthiourea, which blocks the in vivo pneumotoxicity of both NMTB and NMTBSO, also inhibited their in vitro microsomal metabolism and CVB, suggesting that these oxidations are obligatory steps in the expression of toxicity by these compounds. PMID- 2996174 TI - Acrylamide neurotoxicity: altered spinal monosynaptic responses to quipazine, a serotonin agonist, in cats. AB - The effects of the serotonin agonist quipazine on the spinal monosynaptic reflex (MSR) were investigated in acrylamide-treated cats. Cats were administered 30 mg/kg acrylamide (ACR) for 10 days, and the MSR and spontaneous ventral root discharge (VRD) were recorded on the tenth day (ACR 10) or 10 days subsequent to the last injection (ACR 20). Cumulative doses of quipazine were administered to increase the MSR and VRD. It was found that the MSR in ACR 10 cats was significantly depressed by ACR while the ACR 20 cats showed increased MSRs with double peaks. Dose-response relationships between quipazine and the area-under the-MSR showed a significant shift to the right in the ACR 10 from control and no significant change in the ACR 20 from control. Dose-response curves of quipazine vs VRD showed no significant difference in either group from control. These data suggest that the alpha-motoneuron in ACR-treated cats was responding normally to quipazine while the primary afferent terminal was not. PMID- 2996175 TI - Evidence for direct action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thymic epithelium. AB - 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) acts on selected targets within the immune system to produce a characteristic profile of pathologic responses typified by thymic atrophy, suppressed cellular immunity, and inhibition of antibody production to T-lymphocyte-dependent antigens. Studies in inbred mice differing in sensitivity to TCDD indicate that TCDD-induced thymic atrophy is mediated by a receptor protein (designated the Ah receptor). To study the cellular and molecular basis for TCDD-induced thymic atrophy, primary cultures of thymic epithelial (TE) cells were established from C57BL/6 mice, a strain sensitive to TCDD. Treatment of TE monolayers with TCDD (0.1 to 10 nM) resulted in the altered maturation of cocultured syngeneic thymocytes as judged by suppression (40% of control at 10 nM TCDD) of TE-dependent responsiveness of thymocytes to the mitogens concanavalin A and phytohemagglutinin. TE-conditioned medium enhanced the mitogen responsiveness of thymocytes three- to four-fold; however, the enhanced mitogen response mediated by the TE-conditioned medium was not suppressed in thymocytes incubated in medium collected from TCDD-treated cultures or in TE-conditioned medium to which TCDD (10 nM) had been added directly. The suppression of TE-dependent maturation of thymocytes was concentration dependent (EC50 approximately 1 nM) and stereospecific, suggesting involvement of the Ah receptor. The Ah receptor in cytosol fractions from cultured TE cells was measured directly and was found to be present at a concentration 3 and 3.5 times greater than that measured in whole thymus and thymocytes, respectively. The results of this study indicate that TCDD can act directly on epithelial target cells in the thymus: one consequence of this action appears to be the altered thymus-dependent maturation of T-lymphocyte precursors, mediated through direct cell-cell contact between thymocytes and TE cells. PMID- 2996176 TI - Failure to induce mutations in Chinese hamster V79 cells and WB rat liver cells by the polybrominated biphenyls, Firemaster BP-6, 2,2',4,4',5,5' hexabromobiphenyl, 3,3',4,4',5,5'-hexabromobiphenyl, and 3,3',4,4' tetrabromobiphenyl. AB - Firemaster BP-6 (FM), a mixture of polybrominated biphenyls (PBB), and the congeners 2,2',4,4',5,5'-hexabromobiphenyl (2,4,5-HBB), 3,3',4,4',5,5' hexabromobiphenyl (3,4,5-HBB), and 3,3',4,4'-tetrabromobiphenyl (3,4-TBB) were tested for their ability to induce mutations in mammalian cells in culture. A rat liver microsome-mediated (S 15) Chinese hamster V79 cell mutation assay was used to test the mutagenicity of PBB and 3,4-TBB. V79 cells and WB rat liver cells were used to detect the mutagenicity of 2,4,5-HBB and 3,4,5-HBB. No mutagenic effects were detected at the dose levels tested. The possibility that these compounds promote liver neoplasms via a nongenotoxic mechanism is discussed. PMID- 2996177 TI - Acute myelotoxic responses in mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AB - Blood cells are derived from a multitiered system of progenitor stem cells that lose their capacity for proliferation and self-renewal as they continue along pathways of differentiation. Since these hematopoietic events can be readily monitored in vivo and in vitro in the mouse, we have utilized this system to examine altered cellular differentiation associated with 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) toxicity. Progenitor cells were suppressed following acute exposure of mice to TCDD at doses as low as 1.0 micrograms/kg body wt. In vitro studies demonstrated that myelotoxicity occurs by a direct inhibition of proliferating stem cells. Genetic studies indicated that the myelotoxic responses to TCDD, both in vivo and in vitro, segregate with the Ah locus. In addition, the in vitro myelotoxicity of various polyhalogenated aromatic hydrocarbon congeners correlated with their previously reported ability to induce hepatic microsomal enzyme activity and to bind to an intracellular receptor for TCDD. TCDD was also found to bind specifically to bone marrow cells from Ah-responsive, but not nonresponsive mice, indicating that bone marrow cells possess a specific receptor for TCDD. These data indicate that the myelotoxic response to TCDD is regulated by the Ah receptor present in the target tissue and demonstrates the utility of this system for examining the cellular and molecular events associated with the toxicity of polyhalogenated aromatic hydrocarbons, the prototype for which is TCDD. PMID- 2996178 TI - Effects of heavy metals on corticosteroid production in cultured rat adrenocortical cells. AB - Application of 10(-4) M Zn, 10(-5) M Ba, 10(-6) M Se, 10(-6) M Cd, 10(-6) M Hg, and 10(-6) M Mn did not affect the adrenocorticotrophic hormone (ACTH)-induced steroid production in cultured adrenocortical cells. The application of 10(-5) M Pb significantly reduced the ACTH-induced steroid production in cultured cells. However, Pb did not reduce the steroidogenesis induced by dibutyryl cyclic AMP (Dbc-AMP), suggesting that the plasma membrane is the site where Pb interferes with steroid production. The morphological changes induced by the addition of ACTH or Dbc-AMP plus the test metals were similar to those induced by ACTH or Dbc AMP alone. PMID- 2996179 TI - Acute polybrominated biphenyl toxicosis alters vitamin A homeostasis and enhances degradation of vitamin A. AB - A single oral dose of HBB (3,3',4,4',5,5',-hexabromobiphenyl), a congener of the polybrominated biphenyls (PBBs), and a dioxin-type cytochrome P-450 enzyme inducer rapidly and dramatically altered the steady-state metabolism of vitamin A and caused an abnormal twofold enhancement in the metabolic output of degraded vitamin A in urine and feces of rats. The effects are most likely explained by an increased metabolism of vitamin A in kidney and deregulation of vitamin A metabolism in liver; this may lead to an increased dietary vitamin A requirement. PMID- 2996180 TI - Probicyclophosphates: monocyclophosphates as potential prodrugs for bicyclophosphate GABA antagonists. AB - 4-Alkylbicyclophosphates, R1C(CH2O)3P(O), with suitable R1 substituents (e.g., t butyl, isopropyl, or cyclohexyl) are highly toxic compounds [mouse intraperitoneal (ip) LD50 values 0.036-0.52 mg/kg] and are potent noncompetitive gamma-aminobutyric acid (GABA) antagonists. 4-Alkylmonocyclophosphates of the type R1(HOCH2)C(CH2O)2P(O)R2 (where R2 is O-p-nitrophenyl, O-phenyl, S-ethyl, or S-n-propyl) are potential prodrugs by virtue of their cyclization to bicyclophosphates; thus, they may be considered as probicyclophosphates. Both the O-aryl and S-alkyl compounds cyclize via intramolecular transesterification in aqueous medium at pH 7.4 and the S-alkyl derivatives also on oxidation with m chloroperoxybenzoic acid in chloroform and possibly with microsomal oxidases. For the isomeric O-nitrophenyl, 4-isopropyl compounds, one is equitoxic with the corresponding bicyclophosphate on ip administration to mice while the other is of very low toxicity, a result paralleled by their relative cyclization rates [k(min 1) 4 X 10(-2) and less than 4 X 10(-5), respectively]. The S-alkyl compounds are of intermediate toxicity and the O-phenyl compound is of low toxicity to mice, apparently due to less efficient conversion to the bicyclophosphates. The probable mode of action of these monocyclophosphates as potential prodrugs for bicyclophosphate GABA antagonists is further supported by receptor inhibition studies and identification of the bicyclophosphate products in enzyme systems and in the urine of treated rats. PMID- 2996182 TI - [Rare case of a melanotic progonoma of the maxillary alveolar process in a child]. PMID- 2996181 TI - Effect of ochratoxin A on rat liver mitochondrial respiration and oxidative phosphorylation. AB - The in vitro effects of ochratoxin A on the membrane structure and bioenergetic functions of rat liver mitochondria were studied. It was found that when the toxin was added to the assay medium the respiratory control of the isolated mitochondria was decreased as the concentration of the toxin increased. The mitochondrial respiration was gradually uncoupled by the toxin when its concentration was raised above 1.2 X 10(-6) M, and became fully uncoupled at 6.2 X 10(-4) M. The oxidative phosphorylation was not damaged until the toxin concentration was higher than 9.3 X 10(-5) M. On the other hand, ochratoxin A inhibited the electron transfer functions of the mitochondria. At the concentration above 1.0 X 10(-4) M, ochratoxin A inhibited the succinate dehydrogenase, succinate-cytochrome c reductase, and succinate oxidase activities of the respiratory chain. Fifty percent of succinate-cytochrome c reductase and succinate oxidase activity was lost in the presence of 8.0 X 10(-4) and 6.2 X 10( 4) M ochratoxin A, respectively. The inhibition kinetic studies revealed that ochratoxin A is an uncompetitive inhibitor of both succinate-cytochrome c reductase and succinate dehydrogenase, and the inhibition constants for the 2 enzyme activities were estimated to be 4.4 X 10(-4) and 2.2 X 10(-4) M, respectively. However, the toxin did not inhibit either cytochrome oxidase or NADH dehydrogenase activity of the mitochondrial respiratory chain. It is thus concluded that ochratoxin A exerts its effect on the mitochondrial respiration and oxidative phosphorylation through the impairment of the mitochondrial membrane and inhibition of the succinate-supported electron transfer activities of the respiratory chain. PMID- 2996183 TI - [Maxillofacial actinomycosis with dissemination to the spine]. PMID- 2996184 TI - [Comparative diagnosis of recurrent herpetic stomatitis by the methods of immunofluorescence and direct electron microscopy]. PMID- 2996185 TI - Cerebral platelet thromboembolism and thromboxane synthetase inhibition. AB - Platelet aggregating sodium arachidonate was slowly infused into the internal carotid artery (1 mg, 100 microliters, 1 microliter/s) of nitrous oxide anesthetized rats. The electroencephalographic activity recorded by a Cerebral Function Monitor from the injected hemisphere was reduced within minutes. The somatosensory evoked responses to contralateral electrical stimulation of the whisker area were eliminated on the same side in most cases when measured five and fifteen minutes after the infusion. The brain was frozen in situ with liquid nitrogen after fifteen minutes. Regional tissue analysis showed ipsilateral derangement of the cerebral energy state and increased lactate levels. Pretreatment with the platelet antiaggregating thromboxane synthetase inhibitor OKY-1581 (Sodium-3-4-(3-pyridylmethyl)phenyl-2-methyl-acrylate), 30 mg/kg i.v., fifteen minutes before the sodium arachidonate infusion prevented cerebral energy failure and elimination of the sensory evoked responses. PMID- 2996187 TI - An improved method for isolation of mouse pancreatic islets. PMID- 2996186 TI - The biologic significance of the mixed lymphocyte kidney culture in humans. AB - The mixed lymphocyte kidney culture (MLKC) in humans has been studied in normal and abnormal clinical conditions. Human renal cortical cells were extracted by collagenase treatment from the kidneys of "normal" heart-beating cadaver organ donors (n = 13), patients with end-stage renal disease (ESRD) at pretransplant bilateral nephrectomy and splenectomy (n = 13), and from irreversibly rejected renal allografts at the time of graft nephrectomy (n = 5). Proliferation of peripheral blood T lymphocytes of 2-DR-mismatched volunteers occurred in response to kidney cortical cells extracted from each of the 3 donor categories in a reaction termed the allogeneic mixed lymphocyte kidney culture. Additionally, splenic T cells from cadavers and patients with ESRD were seen to react to their autologous kidney cells. The renal cortical cells extracted from ESRD kidneys were more stimulatory in the allogeneic and autologous MLKC responses than those extracted from "normal" cadaver kidneys even when the ESRD kidneys were 99% depleted of passenger T and B lymphocytes by treatment with monoclonal antibodies T11 and B1. In order to help define the antigens operative in the MLKC, we pretreated stimulating lymphocytes and renal cortical cells with anti-class II monoclonal antibodies. The allogeneic mixed lymphocyte reaction and MLKC were inhibited ca. 80% and 30%, respectively. The autologous MLKC was unaffected by this treatment. To further support that tissue-specific immune mechanisms were operative in the reaction, experiments were performed with infiltrating lymphocytes isolated from the ESRD kidneys, which were seen to generate a proliferative response when stimulated with autologous cortical cells. However, the response of these same infiltrating lymphocytes when stimulated with allogeneic lymphocytes (MLR), was markedly weaker than the response of the patients' autologous spleen cells. In addition, two kidneys were obtained at rejection from recipients that had received grafts from HLA-MLR-identical sibling donors. A lymphoproliferative reaction of recipient peripheral blood T lymphocytes occurred in response to (donor) renal cortical cells, but not to donor peripheral blood lymphocytes. In contrast, infiltrating (recipient) kidney lymphocytes responded to the kidney cortical cells and to donor peripheral blood lymphocytes. Moreover, peripheral blood T lymphocytes of the HLA-identical donor responded to his own kidney cortical cells, which were isolated from the rejected recipient kidney, and did not respond to recipient peripheral blood lymphocytes. Finally, a "normal" cadaveric kidney was fortuitously available at the same time that a rejected transplant (cadaver) PMID- 2996188 TI - [Specific binding of organic acids by a fraction of the basolateral membrane of the rat kidney cortex isolated by osmotic shock]. AB - Non-vesiculated membrane fragments of the basolateral membrane of the rat kidney cortex were isolated by the osmotic shock method and fractionated by means of differentional centrifugation. Formation and purity of membrane fragments were tested morphologically (contact luminescent, phase-contrast and electron microscopy) and biochemically (determination of the activity of marker enzymes- Na+, K+-dependent ATPase and alkaline phosphatase). The activities of Na+, K+ ATPase and alkaline phosphatase in the purified fraction of the basolateral membrane were 21 and 0.2%, respectively, of those in the kidney cortex homogenate. The binding of 14C-hyppuric and 14C-uric acids with basolateral membrane fragments was studied by means of filtration through the millipore filters. The existence of competitive inhibition and substrate saturation of the binding testify to the presence of organic acid carrier in the basolateral membrane. The affinity of the carrier to hyppurate in membrane preparations was proved to be the same as in the intact proximal tubules (the apparent Michaelis constant is equal to 0.7 mM). The equilibrium constant (Kf) for the carrier hyppurate complex does not exceed 10 M-1. That means that the complex of the carrier with hyppurate is not strong. PMID- 2996189 TI - [Genetic transformation of somatic cells. VII. The loss of the transformant phenotype is accompanied by both stable and unstable changes in DNA]. AB - From 6 clones of Chinese hamster cells varying in the rate of the loss of transformant phenotype and containing a thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1), 25 subclones negative in thymidine kinase (TK-) were isolated on a medium with 50 micrograms/ml 5-bromodeoxyuridine (BrdU). A study was made of the frequency of spontaneous reversions to the TK+ phenotype in cell populations of BrdU-resistant subclones, and of the transforming activity (upon transformation of TK- cells of A238 clone to the TK+ phenotype) of DNA preparations from a row BrdU-resistant subclones. In 7 of 11 BrdU-resistant subclones the TK- phenotype is associated with changes reducing significantly the transforming activity of DNA. Some of these alterations are stable and undergo no spontaneous reversion, while the other ones are unstable, being reversed or suppressed at a high frequency. BrdU-resistant subclones produced from clones more stable in transformant phenotype are on the whole more stable in the TK- phenotype than BrdU-resistant subclones from the clones with the high rate of the loss of the TK+ phenotype. PMID- 2996190 TI - [Genetic transformation of somatic cells. IX. The loss of the transformant phenotype is accompanied by rearrangements in the plasmid DNA containing the thymidine kinase gene of the herpes virus]. AB - As demonstrated by dot-hybridization, the cells of HT-subclones isolated from the cells of transformant clones cultured on a non-selective medium differ significantly in the number of copies of thymidine kinase gene (tk-gene) of Herpes simplex virus (HSV1). Since the cells of transformant clones lose thymidine kinase-positive (TK+) phenotype during cultivation, this data are indicative of high frequence rearrangements in the region of transforming DNA as responsible for the transformant phenotype nonstability. These rearrangements, among other things, induce alterations in the number of copies of tk gene of HSV1. The analysis of cells of subclones isolated on a medium containing 5 bromodeoxyuridine (BrdU) shows that the number of copies of tk gene of HSV1 decreases as compared to the cells of parental clones. The decrease in the number of copies of tk gene of HSV1 in a row of BrdU-resistant subclones is accompanied by simultaneous increase in the number of sequences of pBR325 plasmide DNA to which tk gene of HSV1 is linked covalently in the pST826 plasmide introduced into cells of transformant clones. This evidence implies a most complex nature of transforming DNA rearrangements reducing the number of copies of tk gene of HSV1 due possibly to a genetic correction. The analysis of results permits a hypothesis that instability of cells in transformant phenotype may be determined by the genetic instability of insertion type. The rate of the loss of transformant phenotype depends on the frequency of rearrangements in the transforming DNA locus. PMID- 2996192 TI - [DNA analysis. A new diagnostic tool in clinical genetics]. PMID- 2996191 TI - Bone marrow necrosis: an unusual presenting feature of small cell lung carcinoma. AB - The authors describe a case of bone-marrow necrosis which represented the initial clinical feature of a "small cell" lung carcinoma with bone metastases. PMID- 2996193 TI - [Polymorphism of the DNA restriction fragment length flanking the insulin gene. A genetic marker for diabetic mellitus or atherosclerosis?]. PMID- 2996194 TI - Frequency-dependent attenuation in breast tissue characterization. AB - The aim of this study is to establish whether an index derived from the slope of the frequency-dependent ultrasonic attenuation can provide quantitative information on normal and pathological breast tissue. Ultrasonic measurements were performed by using pulsed transmitted ultrasound in the frequency range 2-8 MHz. Thirty-three specimens were selected for their probable pathologic classification, by macroscopic observation, before ultrasonic study, and subsequently histologically classified. Ultrasonic results suggest the possibility that the examined specimens fall into four groups: (1) fat, fibroadenoma, giant fibroadenoma, infiltrating ductal carcinoma, medullary carcinoma; (2) infiltrating lobular carcinoma, tubular carcinoma, scirrhous carcinoma; (3) fibrosis; (4) fibrofatty tissue, fibrocystic disease. Correlative morphological studies indicate that the employed index can classify breast tissues on the basis of their cellular and fibrous composition and the inhomogeneity of their structure. PMID- 2996196 TI - [Arterial occlusion in tumors of the kidneys]. PMID- 2996195 TI - Effect of sodium bicarbonate and Tris (THAM) infusion on intra- and extracellular acid-base status during experimental uremia in the rat. AB - The intra- and extracellular acid-base status of nephrectomized rats was determined following infusion of 10 mmol/kg body mass Tris (THAM) or sodium bicarbonate. The 'mean whole body pHi', an overall estimate of the intracellular pH (complementary to the in vivo determined arterial plasma pH) was calculated from the distribution of 14C-labelled 5,5-dimethyl-2,4-oxazolidinedione. For evaluation of buffer effect of Tris or sodium bicarbonate, extra- and intracellular bicarbonate concentration was calculated from the Henderson Hasselbalch equation. Intracellular and extracellular pH and bicarbonate concentration were significantly more increased when sodium bicarbonate was infused, while PCO2 increased significantly more when Tris was administered. On the basis of these results it is concluded that treatment of both in intracellular and extracellular uremic acidosis can be performed more efficiently with bicarbonate than with Tris buffer. PMID- 2996197 TI - Scrotal malignancies: the University of Iowa experience and a review of the literature. AB - Cancer of the scrotum is of special interest, despite its relative infrequency, because of its surprising virulence and also by virtue of its historical importance. This was the first known occupational cancer and showed the need for research involving potential environmental carcinogens. No one individual has sufficient experience to randomize patients to different methods of treatment, and the disease is becoming even less common. Therefore, recommendations are often based on compilations from the literature, many reports being fifty to one hundred fifty years old. This report reviews the clinical features of scrotal malignancies and the forty-five-year experience with this disease at the University of Iowa. PMID- 2996199 TI - [Experience with the administration and the effectiveness of systematic preventive otolaryngological services in a compound chemical fertilizer industry]. PMID- 2996198 TI - [Primary multiple chemodectoma]. PMID- 2996201 TI - [Clinical aspects and laboratory methods of diagnosis of acute viral non perforative otitis media]. PMID- 2996200 TI - [Angiofibroma of nasal cavity in a woman]. PMID- 2996202 TI - [Linitis plastica]. PMID- 2996203 TI - Powassan viral encephalitis: a review and experimental studies in the horse and rabbit. AB - Powassan virus strain M794, a member of the Flavivirus genus known to infect man and animals in Canada, was inoculated intracerebrally into rabbits and horses. No clinical signs were observed in rabbits, but widespread encephalitis resulted, characterized by lymphoid perivascular cuffing, lymphocytic meningitis, and lymphocytic choroiditis. In horses, eight days after inoculation, prominent neurological signs occurred and lesions were those of non-suppurative encephalomyelitis, neuronal necrosis, and focal parenchymal necrosis. The virus could not be reisolated from the rabbit or horse brains. Pathologic features, useful in separating some of the common North American equine neurological diseases, are discussed. PMID- 2996204 TI - Mucinous adenocarcinoma of the tongue in a great horned owl. PMID- 2996205 TI - Response of merino sheep to inoculation with a caprine retrovirus. AB - Maedi-visna has never been recorded in Australia. However, caprine retroviruses have been isolated which cause clinical disease similar to caprine arthritis encephalitis and produce antibodies in goats similar to those caused by maedi visna virus. Merino lambs in close contact for up to 109 weeks with three goats experimentally infected with a caprine retrovirus did not seroconvert or show any significant pathological lesions. No viruses were isolated from any of these sheep though the goats seroconverted and two out of the three developed a non suppurative bursitis and arthritis. Merino lambs inoculated with a caprine retrovirus showed no significant clinical signs or pathological lesions over a period of 140 weeks though nine out of the 12 seroconverted and viraemia was demonstrated in seven. PMID- 2996206 TI - Serological survey of lentivirus (maedi-visna/caprine arthritis-encephalitis) infection in British goat herds. AB - A serological survey for lentivirus infection was undertaken on a volunteer sample of 331 British goat herds. The prevalence of infected herds was 10.3 per cent and the prevalence of seropositive individual animals was 4.3 per cent. There was evidence of a geographical variation in the distribution of infection. Herds consuming pooled colostrum appeared to be at greater risk from infection. Approximately half the herds sampled had been established within the past six years. There was evidence of recently introduced infection in a relatively high proportion of infected herds. PMID- 2996207 TI - New agent causing hepatitis. PMID- 2996208 TI - Parvovirus vaccination. PMID- 2996209 TI - Parvovirus vaccination. PMID- 2996210 TI - Vaccines against Aujeszky's disease: evaluation of their efficacy under standardized laboratory conditions. AB - A standardized test was developed to compare the efficacy of Aujeszky's disease virus (ADV) vaccines under laboratory conditions. Per test 3 groups of 6 to 8 sero-negative pigs were used. The first vaccination was done at 10 weeks of age. One group was vaccinated once, another was vaccinated twice and the 3rd served as control. Pigs were challenge exposed to the virulent NIA-3 strain of ADV 12 weeks after the first vaccination. Apart from mortality, average periods of growth arrest, fever and virus shedding after challenge were used as parameters to evaluate vaccine efficacy. Two inactivated and 4 attenuated vaccines were tested. Two attenuated vaccine viruses were excreted after vaccination. Despite maximal standardization, a considerable variation still existed between the experiments in mortality and growth arrest periods of control pigs after challenge. However, the controls were always more severely affected than the vaccinated pigs. All vaccines except one were effective in preventing death after challenge, but none conferred complete protection. Most vaccinated pigs still lost weight, developed fever and shed virus after challenge. Revaccination after 3 or 4 weeks had little effect, particularly with the attenuated vaccines. The results of the present study indicate that 2 of the attenuated vaccines conferred the best protection, 1 attenuated vaccine appeared to be as effective as the 2 inactivated ones, and the 4th attenuated vaccine was least effective. PMID- 2996211 TI - Enzymes of isolated brush border membrane of Cotugnia digonopora, and their insensitivity to anthelmintics in vitro. AB - Purified brush border membrane of Cotugnia digonopora showed the presence of a number of phosphohydrolases. Among these, alkaline phosphatase was extremely active. Other enzymes such as glucose-6-phosphatase, fructose-1,6-diphosphatase, cAMP-phosphodiesterase, 5'-nucleotidase and adenosine-triphosphatase were also active. Observations were made on the activities of various ATPases; whereas the enzyme was activated by Ca++ and Mg++ in an additive manner, its sensitivity to ouabain was negligible. Furthermore, in the presence of EDTA the enzyme activity was quite significant. The treatment of isolated brush border membrane with mebendazole, niclosamide and praziquantel in vitro did not alter the activity of these enzymes. However, treatment of intact worms drastically affected the integrity of the membrane. PMID- 2996212 TI - Intranasal vaccination of pigs against Aujeszky's disease. 4. Comparison with one or two doses of an inactivated vaccine in pigs with moderate maternal antibody titres. AB - Intranasal (IN) vaccination of pigs with low levels of maternally-derived antibody (MDA) has previously been shown to confer good protection against challenge with virulent Aujeszky's disease virus (ADV). The objective of the present study was to determine the efficacy of IN vaccination with an attenuated ADV, in comparison with that of an inactivated vaccine given parenterally, in pigs with higher MDA titres at the time of vaccination. In one experiment, vaccinations were done at 6 weeks of age, and in another experiment pigs were vaccinated at 4 and/or 9 weeks of age. Two months after (the last) vaccination pigs were challenged intranasally with a virulent ADV. Protection was evaluated on the basis of mortality, periods of growth arrest, fever and virus shedding after challenge. The presence of MDA markedly depressed the serum-neutralizing antibody response after vaccination. Sensitisation occurred after parenteral vaccination with an inactivated vaccine despite high MDA levels. Although the intranasally-vaccinated pigs had lower levels of neutralizing antibody at the time of challenge, they were significantly better protected than pigs given 1 or 2 doses of the inactivated vaccine. Comparing the present results with those of a previous study, it appears that the efficacy of parenteral as well as intranasal ADV vaccination decreases with increasing levels of MDA at the time of vaccination. PMID- 2996213 TI - Restriction endonuclease analysis of Brucella ovis and other Brucella species. AB - Brucella ovis DNA was analysed by using 11 different restriction endonucleases. The most clearly resolved DNA fragment patterns were obtained after digestion with the enzyme Hind III. When DNA preparations from 35 strains of B. ovis were digested with this enzyme, the fragment patterns appeared to be identical. The patterns obtained after Hind III digestion of DNA from one strain each of B. abortus, B. canis and B. melitensis were more similar to each other than to the B. ovis pattern. PMID- 2996214 TI - The rapid detection of Aujeszky's disease virus in pigs by direct immunoperoxidase labelling. AB - Direct immunoperoxidase labelling on impression smears of brain and pharynx was compared with virus isolation and direct immunofluorescence for the detection of Aujeszky's disease virus in experimentally-infected pigs. Immunoperoxidase labelling was as sensitive as immunofluorescence and more sensitive than virus isolation for tissue that had been stored at room temperature (approximately 20 degrees C) for up to 144 h. PMID- 2996215 TI - Differentiation between metaplastic carcinomas and sarcomas of the human female breast by fibronectin. AB - The distribution pattern of fibronectin in metaplastic carcinomas, stromal sarcomas, malignant cystosarcoma phyllodes tumours and histiocytic type lymphomas of the human female breast has been studied using the indirect immunoperoxidase technique on formalin fixed paraffin embedded tissue. Fibronectin was demonstrated as intensely stained strands between tumour cells forming an irregular network in metaplastic carcinomas and lymphomas. Stromal sarcomas and the malignant stromal component of the phyllodes tumours exhibited, in contrast, a uniform staining throughout tumour cells and stroma which was weaker than in adjacent normal-looking connective tissue. We suggest that the intense staining reaction of metaplastic carcinomas is due to the scirrhous reaction generally associated with invasive human breast carcinomas. The advantage of using fibronectin as a diagnostic tool in the differentiation of carcinoma/lymphoma versus sarcoma is the fact that the antigen is a stromal marker and its staining intensity is not influenced by the morphology or degree of differentiation of non mesenchymal tumours. PMID- 2996216 TI - Effect of dimethyl sulfoxide on interaction of human cytomegalovirus with host cell: conversion of a nonproductive state of cell to a productive state for virus replication. AB - The effect of dimethyl sulfoxide (DMSO) on the interaction of human cytomegalovirus (HCMV) with host cell was studied. Confluent state of a human rhabdomyosarcoma cell line (A204) showed a much lower susceptibility to HCMV infection when compared to that in subconfluent actively growing cell cultures. Treatment of confluent cultures with DMSO, however, converted many nonproductive cells in these cultures to a productive state for virus replication. Infectious center assay revealed that approximately 100-fold more cells in the compound treated cultures are able to produce infectious virus. The amount of infectious virus produced in DMSO-treated confluent cultures was enhanced by approximately 10,000-fold over production in untreated cultures and recovered to the level of that produced in subconfluent cultures productive state for virus replication. This cell physiology-dependent inhibition of HCMV replication and enhancement of virus growth by DMSO did not occur with herpes simplex virus type 2. Immunofluorescence staining, gel electrophoresis, and DNA analyses indicate that block of HCMV replication in confluent cultures probably occurs at the level of early transcription or translation of the viral genome. In contrast, in DMSO treated confluent cultures appreciable amounts of HCMV DNA polymerase (an early virus function), viral DNA, and late antigens were synthesized. Pretreatment of confluent cultures with DMSO enabled the cells to support HCMV replication. In addition, the most effective enhancement by DMSO was found in cultures that had been treated with the compound up to 5 hr after infection. These results suggest that the enhancing effect by DMSO is primarily expressed through some host cellular function(s) and the early stages in virus growth cycle are most likely under control by DMSO action. PMID- 2996217 TI - Identification and biochemical analysis of DNA replication-defective large T antigens from SV40-transformed cells. AB - Nine commonly studied Simian virus 40 (SV40)-transformed rodent cell lines were screened for tumor (T) antigens defective in SV40 DNA replication using a simple polyethylene glycol-mediated cell fusion assay. Each line contained a functional origin of SV40 DNA replication, as shown by fusion with Cos 1 cells. Fusion with uninfected monkey cells revealed that T antigens from two lines lacked detectable replicative activity, while T antigens from five other lines exhibited only very weak replicative activity. One line, and a tumor cell line derived from it, expressed T antigen with wild-type replication activity. Biochemical analysis of these proteins revealed defects in DNA binding activity and ATPase activity. One line expressed large T antigen defective in both activities. All of the lines contained complexes of T antigen with the cellular protein p53 and all of the T antigens exhibited nucleotide-binding activity. The results indicate that some of these lines may constitute a useful source of new replication-defective T antigens. PMID- 2996218 TI - Malignant transformation of rat cells by the polyomavirus middle T gene. AB - To gain an insight into the molecular mechanism of cooperation between the polyomavirus middle T gene and cellular genes in the tumorigenic process, we have examined various properties of rat cell lines transformed by middle T alone. Middle T transformants display a phenotype ranging from nontumorigenic (flat) to fully transformed (tumorigenic) and the phenotype of a given cell line correlates very well with its cellular level of middle T antigen. Highly transformed, tumorigenic variants arise spontaneously in the flat cells during their growth with a mutation rate of 2.2 X 10(-5) per cell per generation. These variants contain elevated levels of both middle T antigen and middle T transcripts, suggesting that fully transformed cells arise as a consequence of an efficient mode of viral gene expression. PMID- 2996219 TI - Differential requirement for SV40 early genes in immortalization and transformation of primary rat and human embryonic cells. AB - A series of recombinant plasmids carrying various DNA fragments of SV40 early region were used to test for their ability to immortalize primary cultures of rat embryo (RE) and human embryonic kidney (HEK) cells. When primary RE cells were transfected with plasmids containing an entire early region of wild-type SV40- or a deletion mutant in the small tumor (t) antigen, dl 1410-DNA, they were all immortalized. The immortalized cells could grow in soft-agar medium and produced large tumor (T)-antigen. Cultured RE cells transfected with pW2-t, which contains a deletion in the large-T-specific coding region, also gave rise to continuous cell lines. Interestingly, two of nine RE lines immortalized by pW2-t could also grow in soft-agar medium. The plasmid pW-t8 carrying a similar fragment of SV40 DNA as pW2-t, but lacking the processing and polyadenylation signal sequences, also immortalized RE cells. Surprisingly, the plasmid pD-t1 which contains neither the intact large-T nor the small-t function also immortalized RE cells. However, the RE lines immortalized by pW-t8 or pD-t1 were unable to grow in soft agar medium and displayed a wide range of growth phenotypes. On the contrary, when primary HEK cells were used for immortalization experiments, only those SV40 plasmids carrying the intact large-T function were able to generate immortalized lines. The growth properties of these immortalized HEK lines can be categorized into two groups. Those HEK lines immortalized by the large T alone grew slightly denser and rounder than their parental normal HEK cells, while those immortalized by both the large-T and small-t antigens grew extremely fast, reached higher density, piled up on each other, and were anchorage independent. In addition, when these SV40 plasmids were used to directly transform primary HEK cells by the focus assay, the large-T clone, pD3-05, only transformed HEK cells to form light foci. Transfection by the large-T plus the small-t sequences either in cis or in trans, did increase the frequency of focus formation, and gave rise to dense foci which could grow in soft-agar medium. PMID- 2996220 TI - Selection and characterisation of acyclovir-resistant herpes simplex virus type 1 mutants inducing altered DNA polymerase activities. AB - A collection of TK+, ACV-resistant mutants of herpes simplex virus type 1 (HSV-1) has been derived using a selection system based on biochemically transformed cells. Evidence is presented suggesting that most of these mutants induce resistant DNA polymerase activities and are thus likely to express variant DNA polymerases. Preliminary data on the pathogenesis of these mutants show that most are similar to wild type virus in the majority of their characteristics, although they may be reduced in their ability to kill mice. PMID- 2996221 TI - Molecular cloning and physical mapping of the genome of fish lymphocystis disease virus. AB - A defined and complete gene library of the fish lymphocystis disease virus (FLDV) genome was established. FLDV DNA was cleaved with EcoRI, BamHI, EcoRI/BamHI and EcoRI/HindIII and the resulting fragments were inserted into the corresponding sites of the pACYC184 or pAT153 plasmid vectors using T4 DNA ligase. Since FLDV DNA is highly methylated at CpG sequences (Darai et al., 1983; Wagner et al., 1985), an Escherichia coli GC-3 strain was required to amplify the recombinant plasmids harboring the FLDV DNA fragments. Bacterial colonies harboring recombinant plasmids were selected. All cloned fragments were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridization of recombinant plasmid DNA to viral DNA. This analysis revealed that sequences representing 100% of the viral genome were cloned. Using these recombinant plasmids, the physical maps of the genome were constructed for BamHI, EcoRI, BestEII, and PstI restriction endonucleases. Although the FLDV genome is linear, due to circular permutation the restriction maps are circular. PMID- 2996222 TI - Defining the borders of the chicken proto-fps gene, a precursor of Fujinami sarcoma virus. AB - The transforming (onc) genes of retroviruses contain specific sequences, derived from as yet poorly defined, normal cellular genes, termed proto-onc genes. Proto onc genes must be defined to explain their docility compared to the oncogenicity of the viral derivatives. Here we set out to determine the borders of the chicken proto-fps gene from which the onc genes of avian Fujinami (FSV) and PRC sarcoma viruses (PRCSV) are derived. These onc genes are hybrids of an element from the gag gene of retroviruses (delta gag) linked to a 2.8-kb domain from proto-fps. To identify the 5' border of proto-fps we have sequenced 1.5 kb beyond the 5' border of overlap with viral fps utilizing a proto-fps clone derived previously. A possible promoter was identified that maps 736 nucleotides from this border. The 736 nucleotides contain two possible exons with 121 codons, and short regions of homology with the delta gag termini of FSV and PRCII. A translation stop codon and an adjacent polyadenylation signal were identified just prior to the 3' border of overlap with viral fps within a 1.15-kb sequence of a newly isolated proto-fps clone. Comparing four exons within this 1.15 kb proto-fps sequence with known fps equivalents of FSV and PRCSV, we have detected strain-specific, but no common point mutations in each viral genome. A 3.3-kb polyadenylated proto-fps mRNA was detected in chicken liver RNA by gel electrophoresis and hybridization with proto-fps DNA. We conclude that the coding capacity of proto-fps is just over 3 kb, consistent with the size of the putative proto-fps protein of 98 kDa and hence slightly larger than that of viral fps. Thus proto-fps and the viral delta gag-fps genes each contain distinct 5' regulatory and coding sequences and share the 3' terminal fps domains. It is suggested that this difference, rather than scattered point mutations, is responsible for the oncogenic function of the viral genes and the unknown cellular function of proto-fps. PMID- 2996223 TI - [Changes in the cyclic nucleotide system in experimental anaphylaxis and approaches to their correction]. AB - Components of the cyclic nucleotide system--cAMP, cGMP, activities of adenylate cyclase (AC) and phosphodiesterase (PDase) were studied in brain, lung, adrenal gland, liver tissues, in blood plasma (cAMP, cGMP) and in leukocytes (AC and PDase) of guinea pigs at the periods of sensibilization and development of anaphylaxis. Dibutyryl cAMP was preadministered in a number of the animals in order to correct possible alterations in the system of cyclic nucleotides and to stimulate unspecific resistance of the organism. Distinct alterations were observed in the patterns studied after sensibilization of the animals, especially pronounced in anaphylactic shock. Preadministration of dibutyryl cAMP prevented development of anaphylactic shock in all the animals treated with the antigen. Even after repeated administration of the antigen the antigen. Even after repeated administration of the antigen the anaphylactic shock was observed only in 50% of these animals. The data obtained suggest that synthetic analogues of cAMP may be used for treatment of patients with allergic disorders. PMID- 2996225 TI - [Degree of differentiation of ovarian cancer and its clinical significance]. AB - The study was concerned with morphological criteria for assessing the degree of differentiation of ovarian cancer and establishing their relationship with clinical course in 323 patients. The degree of ovarian cancer differentiation was found to influence the course and prognosis considerably. PMID- 2996224 TI - [Cyclic AMP level, dissociation of membrane-bound calmodulin and regulation of calcium transport in the heart sarcoplasmatic reticulum in circulatory hypoxia]. AB - Slight alterations in calmodulin-stimulated and distinct alterations in cAMP dependent regulation of calcium transport were found in dog heart microsomes under conditions of circulatory hypoxia as compared with myocardium of healthy animals. After addition into the incubation mixture of exogenous protein kinase simultaneously with calmodulin and cAMP the rate of 45Ca accumulation became similar both in microsomes of intact and impaired myocardium. In the hypoxia membrane-bound calmodulin was dissociated in cardiomyocytes and its content was increased in cytosol, whereas content of cAMP tended to decrease; under these conditions activity of Ca2+-ATPase in sarcoplasmic reticulum was similar to the control values. PMID- 2996226 TI - [Role of spleen macrophage activation in inhibiting the metastasis of transplantable tumors]. AB - The role of macrophages in the development of the inhibitory effect of subcutaneously transplanted tumor on the formation of experimental metastases in the lung was studied. Four murine lines of tumor were investigated in a syngeneic system. The treatment of tumor-bearing mice with antimacrophage agents (carrageenan or silica) proved to stimulate experimental metastasis formation which correlated with the absence of antitumor activity of splenocytes tested in a transfer system (by counting lung metastases). Antitumor activity of the following types of splenocytes of tumor bearers was studied in this system: intact, those treated in vitro with different doses of carrageenan, separated from macrophages by carbon iron and, vice versa, enriched with macrophages. It was shown that (I) antitumor activity of tumor-bearers' splenocytes correlates with the ability of relevant tumors to inhibit experimental lung metastasis formation, (2) the key role in this effect is played by activated macrophages of the spleen, and (3) activation of macrophages is not tumor-specific. PMID- 2996227 TI - A new approach to the diagnosis of active Rh0(D) immunization in passively immunized pregnant women. AB - An assay has been developed to distinguish active from passive Rh0(D) immunization in a patient who had recently received hyperimmune anti-Rh0(D) immunoglobulin therapy. Isolated peripheral B lymphocytes from a pregnant woman at 32 weeks gestation were co-cultured with Epstein-Barr virus in a liquid growth medium. After 7 days, anti-Rh0(D) antibodies produced in vitro by the transformed lymphocytes were detected in culture supernatants, thereby proving active immunization and indicating the potential of hemolytic disease of the newborn in the current pregnancy. This assay was also performed with peripheral B lymphocytes from three groups of individuals: mothers known to be Rh0(D) immunized and who recently delivered Rh-positive infants, women with longstanding Rh0(D) immunization, and women who were treated with anti-Rh0(D) globulin. In the first group, anti-Rh0(D) antibodies were again detected after in vitro viral stimulation. In the latter two groups, essentially no anti-Rh0(D) activity was detected. PMID- 2996228 TI - Antibodies to HTLV-III and the lymphadenopathy syndrome in multitransfused beta thalassemia patients. AB - Antibodies to HTLV-III have been investigated in 118 multitransfused beta thalassemia patients. Thirteen patients (11.01%) were found to be positive; 3 of these 13 showed clinical and immunological signs of the lymphadenopathy syndrome. A retrospective study carried out on 65 sera has shown that at least 6 patients were negative 3 years before the present investigation. This is the first extensive study on HTLV-III infection in multitransfused beta-thalassemics. It suggests that these patients are at risk for the acquired immune deficiency syndrome and related diseases. PMID- 2996229 TI - How should we handle the ethical questions regarding information to donors and patients and the practical implications regarding deferral of donors and handling of donated blood in the event of introducing a screening test for HTLV-III as in order to prevent transmission of AIDS by blood transfusion? PMID- 2996230 TI - [Alcoholic polyneuropathies]. PMID- 2996232 TI - [Stomach cancer with multiple metastases under the guise of Moschcowitz syndrome]. PMID- 2996231 TI - [Clinico-morphological characteristics of viral-bacterial meningoencephalitis in adults]. PMID- 2996233 TI - [Levels of corticotropin, cortisol, gonadotropic hormones, prolactin and sex steroids in women suffering from Itsenko-Cushing disease]. PMID- 2996234 TI - [Cytochrome C in the treatment of patients in the acute stage of ischemic strokes]. PMID- 2996235 TI - [Diagnosis and classification of diseases of the peripheral nervous system]. PMID- 2996236 TI - [The virus causing human T-cell lymphomas-leukemias]. PMID- 2996237 TI - [Virological and immunological research on patients with influenza and acute respiratory diseases with central nervous system involvement]. AB - Virological examinations of the cerebrospinal fluid and nasopharyngeal washings from 96 patients with influenza and other acute respiratory viral diseases and of the autopsy material from 14 patients who had had symptoms of the involvement of the central nervous system permitted isolation of respiratory viruses and detection of viral antigens in the cerebrospinal fluid and brain tissue. The level of immunoglobulins in the cerebrospinal fluid was found to be increased, however, their concentration did not exceed that in the blood serum which only indicates the possibility of their penetration through the hematoencephalic barrier. This factor, as well as the pattern of pathomorphological changes in the brain tissues indicate the toxicoallergic genesis of meningoencephalitis in acute respiratory diseases. PMID- 2996238 TI - [Isolation of the causative agent of Karelian fever from Aedes sp. mosquitoes]. AB - The results of isolation of an Alphavirus strain from Aedes sp. mosquitoes collected in the focus of Karelian fever are presented. According to the results of serological studies, the isolated strain LEIV-9298 Karelia belongs to the antigenic complex Sindbis-Western equine encephalomyelitis. The appurtenance of the isolated agent to the Alphavirus genus has been confirmed by electron microscopic examinations. A rise of antibody titres to LEIV-9298 Karelia virus in paired sera of subjects with a history of Karelian fever allows it to be considered the causative agent of this disease. PMID- 2996239 TI - [Ultrastructural organization of liposomes and isolated glycoproteins of enveloped viruses obtained by using the nonionic detergent MESK]. AB - Materials on comparative ultrastructural analysis of isolated glycoproteins (GP) of influenza, parainfluenza, and Venezuelan equine encephalomyelitis viruses as well as liposomes reconstructed on their basis prepared with the use of MECK, a nonionic detergent, are presented. Original virions, subvirion particles, and isolated GP were characterized by typical morphology and remained intact in highly purified and concentrated suspensions owing to the use of the sparing and effective method of treatment with MECK. After removal of the detergent typical micellar aggregates were shown to form from GP monomers for all three viruses under study. The forming rosettes morphologically and biologically are similar to those isolated from ortho-, paramyxo-, and togaviruses using the Triton X-100 detergent. Stages of liposome formation and interaction with cell membrane were studied electron microscopically. A common feature for liposomes from GP of all the three viruses consisted in the formation of two morphological variants of structural complexes: filaments of various lengths and composition in them of GP and vesicles, the arrays of GP in which were similar or identical to those of viral membrane. Liposomes from Sendai virus GP possessed hemolytic activity, however, their interaction with the cell studied by means of the immunoferritine label occurred not by fusion but apparently as a result of destruction of the cell membrane at the sites of protein F contact with plasmalemma and formation in the latter of defects leading to erythrocyte lysis. PMID- 2996240 TI - [Characteristics of the etiological structure of viral hepatitis in children determined by radioimmunological analysis]. AB - Determination of the specific markers of acute viral hepatitides A and B (HA and HB) by radioimmunoassay demonstrated that in the structure of acute viral hepatitides in children HA prevails in the interepidemic period (49.4%) followed by HB (19.3%), then by a polyetiological group of acute viral hepatitides of obscure etiology (17.2%), then by HA in the presence of chronic HB (9.7%) and combined acute forms of HA and HB (4.3%). PMID- 2996241 TI - [Evaluation of the size of the continuous poly(G) site necessary for the biological activity of the poly(G).poly(C) complex]. AB - On the basis of synthesis of a series of poly(G, A).poly(C) copolymers with changing G:A ratio from 15:1 to 90:1 and trials of their biological activity in comparison with poly(G).poly(C), the size of poly(G) in it was evaluated within the range of a continuous double-stranded area necessary for the activity. The antiviral activity close to that of poly(G).poly(C) in experimental tick-borne encephalitis of mice and vesicular stomatitis virus infection of chick embryo cells was found only in poly(G,A).poly(C) complexes with a G:A ratio equal to or higher than 90:1. Consequently, the high activity of poly(G).poly(C) is present at an average length of poly(G) equal to 90-100 nucleotides within the limits of the continuous double-stranded area. PMID- 2996242 TI - [Indices of alpha-interferon and of lymphocyte natural killer activity in genital herpes and the effect on them of specific vaccinal therapy and interferon therapy]. AB - Fifty-six patients with recurrent genital herpes (RGH) and 53 normal subjects were examined. No interferon was found in the blood sera of the patients either in the period of the disease relapse or during remission. The leukocyte capacity to produce interferon in vitro (leukocyte interferon reaction, LIR) in the patients was found to be 4-5 times lower than in normal subjects. Study of the normal killer activity of lymphocytes in patients with RGH as compared with that in normal subjects revealed its decrease in 37.5% of the patients and only in 5.1% of normal subjects. Interferon therapy with purified human leukocyte interferon given to 36 patients with RGH resulted in clinical improvement in 88.9% of the patients that was accompanied by increasing normal killer activity of lymphocytes. Vaccine therapy given to 35 patients resulted in a stable clinical effect in 91.4% of the patients, however, without activation of LIR in them. It is concluded that the therapeutic effects of interferon therapy and vaccine therapy have different mechanisms. PMID- 2996243 TI - [Patterns in the biosynthesis and action of human gamma-interferon]. AB - The highest yields of gamma-interferon activity were obtained by using a fraction of mononuclears recovered from freshly collected donor blood in ficoll-verografin density gradient without using hemolysis. Unification of mononuclears from individual donors into a common pool stimulated interferon production. Staphylococcal enterotoxins A and B, concanavalin A, and lentyl-lectin were found to be the most effective inducers. Immobilization of inducers on neutral carriers reduced their effectiveness. Upon induction with lectin the synthesis was complete within 24 hours, and with enterotoxin in 3 days. In the latter instance the synthesis dynamics was of a two-phase nature. Gamma-interferon produced the antiviral condition later (in 10 hours) than alpha-interferon. PMID- 2996244 TI - [Significance of the radial hemolysis reaction for the diagnosis of herpetic and adenovirus eye infections]. PMID- 2996246 TI - [Immunoglobulin A serum antibodies against the capsid antigen of Epstein-Barr virus in the differential diagnosis and follow-up of nasopharyngeal cancer]. AB - IgA antibodies to Epstein-Barr virus capsid antigen (IgA anti-VCA) can be detected in sera of patients with certain types of nasopharyngeal carcinoma (NPC). IgA anti-VCA titres were determined by the indirect immunofluorescence technique. 17 control patients with benign diseases or carcinomas of the head and neck other than NPC had negative IgA anti-VCA titres less than or equal to 1:16. NPC was diagnosed histologically according to the Cologne modification of the WHO classification. Among 16 cases of untreated or recurrent NPC, a rare disease in Europe, seen over the past three years, those with undifferentiated carcinomas with and without lymphoid stroma and the non-keratinizing carcinomas with lymphoid stroma were IgA anti-VCA positive (1:32 to 1:512), whereas patients with squamous cell carcinomas were negative. In four cases the primary tumour had not been diagnosed by other ENT doctors in spite of known regional or distant metastases consisting of undifferentiated carcinomas with or without lymphoid stroma. IgA anti-VCA antibodies in the sera of these patients indicated the probable site of the primary tumour. NPC was verified by biopsy in all these cases. In 2 serologically negative patients the original diagnosis of undifferentiated NPC with lymphoid stroma had to be revised to malignant Non Hodgkin lymphoma. In the follow-up of 6 NPC patients the trend of changes in IgA anti-VCA titres correlated with the course of the disease, but the minute tumour related changes could be detected only when at least two previous sera of the same patient were included in every test.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996245 TI - Na+-H+ exchange, oncogenes and growth regulation in normal and tumor cells. PMID- 2996247 TI - [Effect of a new tube-feeding formula on metabolism, urinary electrolytes and gastrointestinal tract of healthy adults with and without a supplement of ballast material from soy bran]. AB - Over a period of three weeks eight apparently healthy subjects received a new formula diet. From the second week on 30 grams of dietary fibers were administrated. This dietary fiber was produced from soya-bran and the eight subjects accepted this diet very well. No change of blood chemistry was measured. The stool weight without this dietary fiber amounted to 57 +/- + 15 g/day, where as by giving the fibre stoolweight rose up to 86 + 4 g/day. Transit time without fiber was 83 +/- 29 h and with fiber it dropped to 74 +/- 11 h (n.s.). The renal excretion of potassium, sodium, magnesium and calcium remained constant. PMID- 2996248 TI - [Causes of labor initiation in man: role of oxytocin and prostaglandins]. AB - Questions related to the mechanism of human labor are of central clinical importance nowadays, since the majority of perinatal mortality and morbidity is due to disregulation of uterine contractility mainly premature onset of labor. During delivery of spontaneous or induced onset endogenous prostaglandin F synthesis increases dramatically and reaches a maximum at the time of placental separation. These increased amounts of prostaglandin F and E lead to myometrial contractions and to a reduction of the cervical resistance. Post partum, prostaglandins lead to the contracture of the myometrium necessary for separation and expulsion of the placenta. The precise causes for initiation of parturition at term however have not been fully elucidated. The present review presents a theory in which oxytocin acts as central trigger of labor. At the end of human pregnancy a marked rise in the concentration of oxytocin receptors in the myometrium can be observed, thereby leading to an increased sensitivity of the myometrium towards oxytocin. Therefore, a small increase of the circulating oxytocin concentration in the maternal peripheral blood (for example through addition of fetal oxytocin) is sufficient to induce contractions. Apart from inducing contractions, oxytocin also leads to a stimulation of prostaglandin synthesis through receptors in the decidua. Prostaglandins themselves lead to further contractions, soften the cervix, induce gap-junctions and sensitize the myometrium further for oxytocin, thereby leading to a progressive cervical dilatation. At the end of the first stage of labor, the membranes usually rupture leading to a further increase in prostaglandin synthesis, so that the mechanism can no longer be interrupted exogenously.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996249 TI - [Effect of a cyclic analog of enkephalin on learning and memory in the mouse]. AB - In experiments on mice, the effect of cyclic analogue of enkephalin (CAE) on the processes of learning and memory was studied in control animals and in animals with changed functional state of monoaminergic brain systems. Administration of the peptide to intact animals significantly accelerated the acquisition of conditioned reflexes of two-way avoidance and did not significantly affect the retention of these reflexes and their subsequent reproduction. Retention and reproduction of conditioned reflexes elaborated in one combination, was disturbed. Administration of iprazid did not eliminate the "accelerating effect" of CAE on the formation of conditioned reflexes of the two-way avoidance but sharply disturbed their retention. In such conditions, the amnesing iprazid effect increased still more. Besides, under CAE effect, the activity of acetylcholinesterase in the motor and especially in the visual cortex of the mice increased. The obtained data testify to an important role of the monoaminergic and cholinergic brain mechanisms in realization of CAE effects on the processes of learning and memory. PMID- 2996250 TI - [Frequency modulation of theta volleys of septal neurons during rhythmic oligosynaptic stimulation in the rabbit]. AB - Activity of the neurones with stable theta-bursts was recorded extracellularly in intact and hippocampectomized septum of unanaesthetized chronic rabbits during low-frequency (3-17 Hz) stimulation of horizontal limb of the diagonal band or the lateral septal nucleus. Gradual entrainment and phase-locking of the spontaneous theta-cycles occurred. Two types of entrainment were observed: "entrainment by pause", where interburst interval was reset by the stimuli; and "entrainment by burst", where bursts were time-locked to the stimuli. Such reorganization of the spontaneous bursts occurred in a narrow frequency range of stimulation (from 4 Hz up to 9-12 Hz), with the best resonance following in the range of "basic" theta frequencies of the awake rabbit (5-6 Hz). With stimulation beyond the theta-range three phenomena occurred: shift of the burst frequencies to higher or lower harmonics of stimulation frequencies; complex interactions of basic background frequency with the rhythm of stimulation ("beating"); escape from the influence of the stimuli with return to background theta-burst frequency. PMID- 2996252 TI - [Methods for determination of insoluble and soluble dietary fiber in food stuff]. AB - A comparative evaluation of two enzymatic methods (method after Asp and the Berlin Method) and a detergent method (modified NDF-Method) for the determination of dietary fiber in food was carried out. The procedure of the Berlin Method is described in detail. The methods were used to analyse the dietary fiber content of cereal-based samples. In comparison with the other methods, the NDF-Method consistently gave lower dietary fiber values. This was also the case when only the insoluble dietary fiber was determined. With the enzymatic methods, the method after Asp gives higher values for the insoluble and lower values for the soluble dietary fiber than the Berlin Method. PMID- 2996251 TI - [Formation and extinction of drinking conditioned reflexes after unilateral intercollicular sectioning of the brain in the rat]. PMID- 2996253 TI - Effect of sulfite or nitrite on the ATP content and the carbohydrate metabolism in yeast. AB - Low concentrations of sulfite or nitrite (about 0.5 mmol) when applied at pH 3.6, caused a rapid and drastic decrease of the concentration of ATP in yeast cells. Under these conditions, alcoholic fermentation was inhibited by sulfite and to a lesser extent by nitrite. Ethanol consumption under aerobic conditions was shown to be more sensitive to nitrite than to sulfite. This indicates a higher sensitivity of respiratory processes to nitrite than to sulfite. Among 15 enzyme activities assayed in extracts from yeast cells after incubation with sulfite or nitrite, glyceraldehyde-3-phosphate dehydrogenase was shown to be the most sensitive. Analysis of the steady-state concentrations of intermediates of alcoholic fermentation in intact yeast cells also implies inhibition by sulfite or nitrite of the glyceraldehyde-3-phosphate dehydrogenase step of fermentation. In contrast to nitrite, sulfite had an additional effect by accumulating the intracellular steady state concentration of glyceraldehyde-3-phosphate 10 to 100 fold over the concentration in the absence of sulfite. In vitro studies on the equilibrium catalyzed by triosephosphate isomerase or aldolase confirmed the postulated shift of equilibrium concentrations by a formation of complex of glyceraldehyde-3-phosphate with sulfite. PMID- 2996254 TI - [Effect of TRH and its analog DN-1417 on anoxia-induced amnesia in mice]. AB - The effect of neuropeptides and their analogs on anoxia-induced amnesia was examined using one-trial passive avoidance task in mice. Anoxia, produced by the exposure to CO2 immediately after the acquisition of avoidance response, induced amnesia which is shown by a short latency to enter from the safety compartment into the shocked compartment in the retention test conducted 24 hr later. In these anoxia-treated animals, thyrotropin-releasing hormone (TRH: 10-20 mg/kg), its analog DN-1417 (10-20 mg/kg) and ACTH 4-10 (66 micrograms/body), which were given sc 15-60 min before the retention test, markedly prolonged the latency in a dose-dependent manner, indicating a reversal of the amnesia. Arginine- and lysine vasopressin also reversed the amnesia at a dose of 100 micrograms/body. These results suggest that TRH and DN-1417, known to reverse the amnesia produced by the protein synthesis inhibitor cycloheximide, have ameliorating effects on the retrieval process of memory. PMID- 2996255 TI - [Immunohistochemistry: theoretical potentials and practical application]. AB - Immunhistochemical methods are increasingly used and their application in surgical pathology is obvious. Nine different immunohistochemical techniques are compared in this review article. The peroxidase anti-peroxidase method of Sternberger et al. (1970), the avidin biotin complex method of Hsu et al. (1981) and the labeled avidin biotin technique of Guesdon et al. (1979) are to be preferred. Two step methods using labeled antibodies such as alkaline phosphatase or peroxidase-labeled second antibodies are less sensitive. However, two-step methods using alkaline phosphatase-labeled antibodies offer the advantage of double staining procedures since alkaline phosphatase reacts with different azo dyes and results in a wide spectrum of colours in the final reaction product. At present, the major problems of immunohistochemistry in daily work are the selection of the fixative and of appropriate controls. Though both problems remain unresolved, immunohistochemical methods should be used for special problems in surgical pathology. PMID- 2996256 TI - [Frequency of perilymphadenitis in regional lymph nodes in carcinoma]. AB - Intensity, frequency and extension of an inflammatory reaction of the lymph node capsule (perilymphadenitis) were investigated in nodes draining invasive ductal breast cancer and infiltrating adenocarcinoma of the large bowel. Occurrence of chronic perilymphadenitis was significantly higher in paracolic lymph nodes than in the axillary ones (p less than 0.05). Both paracolic and axillary lymph nodes developed perilymphadenitis more frequently when they were infiltrated by carcinoma (p less than 0.001). Frequency and extension of the inflammatory reaction of the capsule depended on the degree of carcinomatous infiltration. PMID- 2996257 TI - [Report of experiences with primary liver tumors in childhood]. PMID- 2996258 TI - [Free serum amino acids in patients with ovarian cancer]. AB - Free serum amino acids were measured in patients with untreated ovarian cancer, stage T1-T3 and compared to amino acid concentration in normal patients. Of 10 measured amino acids, 6 were significantly elevated in the cancer group. We didn't observe a decrease of any amino acid level in serum. PMID- 2996260 TI - [Pox in zoo and wild birds. Light and electron microscopic studies]. PMID- 2996259 TI - [Estrogen metabolism and hormone substitution in the climacteric]. AB - Report about the metabolism of estrogens in the postmenopausal period and the hormone replacement therapy. The indications and contraindications for the estrogen- and estrogen-progesterone-therapy were discussed. Osteoporosis in the aging women constitutes a major public health and socio-economic importance than the climacteric syndrome. PMID- 2996261 TI - Canine parvovirus infection demonstrated by immunofluorescence. PMID- 2996262 TI - Persistent infection of the genital tract and excretion of the vaccine strain after live virus immunization with bovine herpesvirus 1 (IBR/IPV virus). PMID- 2996263 TI - Electron microscopic characterization of a viral agent isolated from arthritis in chicken. PMID- 2996264 TI - Comparison of interferon production in cattle after intranasal infection with parainfluenza-3 live vaccine and avirulent IBR/IPV-herpesvirus. PMID- 2996265 TI - [Treatment of patients with disorders of spinal circulation using physical factors]. AB - The main clinical syndromes were identified in 136 patients with impaired circulation in the cervical, thoracic and lumbosacral segments of the spinal cord. Using EMG, ENMG and examination of the monosynaptic H-reflex, the authors give a detailed clinico-electromyographic presentation of these syndromes in the process of physiotherapy, aimed at improving the blood circulation in the involved area and at compensation for motor disorders. The study made it possible to determine the effectiveness of the employed therapeutic complexes and to discuss some neurophysiological mechanisms underlying the restoration of motor function under the impact of physiotherapy. PMID- 2996267 TI - Anal abscess imaged with 99mTc-labeled leukocytes. Case report. AB - Abscess imaging with leukocyte scintigraphy is described in a case report. Autologous leukocytes were separated from whole blood in a patient with localized inflammation in the perineal region and labeled with 99mTc, an ideal radionuclide for clinical examination with gamma camera. Scintigraphic investigations demonstrated a deep-lying anal abscess. The procedure may prove useful for detection of occult infections and may provide a new diagnostic approach in fever of unknown origin. PMID- 2996266 TI - [Efferent innervation of intracranial arteries]. PMID- 2996268 TI - Association of human papillomavirus and Chlamydia infections with incidence cervical neoplasia. AB - Incidence cervical neoplasia is defined as disease that becomes manifest during a given period of observation. Association with preceding genital infections having characteristic cytologic findings would seem to be more likely for incidence than for prevalence cases since the usual long latency period of carcinoma in situ (CIS) could allow resolution of infectious processes. For this reason, it was elected to examine the preceding Papanicolaou smears from patients with tissue confirmed incidence CIS or invasive epidermoid carcinoma. There were 67 women with biopsy-proven CIS or invasive carcinoma of the uterine cervix identified in the files of the University of New Mexico Cytopathology Laboratory from 1966 to 1982 who had two initial negative smears as well as smears at intervals of three years or less. All cytologic smears prior to tissue diagnosis were rescreened for confirmation of cytologic atypia or its absence as well as for morphologic evidence of human papillomavirus (HPV) or chlamydial infections. Control cases matched for age, gravidity, ethnicity and number of smears were reviewed in an identical manner. Koilocytes indicative of HPV infection were found in 17 index cases (25%) and 5 controls (7%) (p = 0.005). Chlamydial infections were identified in 18 index cases (27%) and in 4 controls (6%) (p = 0.001). The times required for conversion from smear negativity to malignancy were determined for each incidence case. The results showed great variability but suggest that the progression to malignancy is not hastened in women with antecedent HPV or chlamydial infections. Our results indicate that the presence of koilocytes and/or chlamydial inclusions in cervical smears serves to identify a group of women with a significantly increased risk of developing cervical carcinoma, even in the absence of concurrent dysplasia. PMID- 2996269 TI - Reproducibility of the cytologic diagnosis of human papillomavirus infection. AB - As part of a larger epidemiologic investigation of the association between human papillomavirus (HPV) infection and cervical intraepithelial neoplasia, the reliability of the cytologic diagnosis of HPV infection was examined. A random sample of cervicovaginal specimens with cytologic changes characteristic of HPV infection were matched with a second set of slides, with regard to the date and severity of the smear and the age of the woman from whom the smear was obtained. The kappa statistic for interobserver agreement was 0.38 (p less than 0.0005), increasing to 0.68 (p less than 0.0005) when uncertain diagnoses were excluded. Intraobserver agreement ranged from kappa = 0.40 to 1.00. Although this agreement is within the range of reliability found for the diagnosis of other atypical cytologic changes, considerable variation is present. The effect of this variability on the validity of estimating the risk of cervical cancer associated with HPV infection may be considerable. PMID- 2996270 TI - Immunoperoxidase staining for the detection of herpes simplex virus antigen in cervicovaginal smears. AB - Destained cervicovaginal smears from eight patients with herpes simplex virus (HSV) infections were stained by means of the peroxidase-antiperoxidase (PAP) technique to demonstrate the presence of the HSV type 2 (HSV-2) antigen. Positive results were obtained in six of the eight cases, with intense staining for the HSV-2-specific antigen throughout the cytoplasm and nuclei of cells having a ground-glass nuclear appearance as well as in multinucleated giant cells. Virus isolation was successfully performed for the HSV-2-positive case that also had a histologically confirmed squamous-cell carcinoma of the cervix. The combined use of cytology and the PAP staining technique was of great value in the demonstration of cervical HSV infections. PMID- 2996271 TI - Cytodiagnosis of herpes simplex keratitis by means of an immunoperoxidase technique. A case report. AB - Typical herpes simplex keratitis that developed in a 5-year-old boy was initially diagnosed cytologically in Papanicolaou-stained samples. Subsequently, an immunoperoxidase staining technique was used to identify the specific type of herpes simplex virus (HSV) in the destained cellular samples. The positive staining helped to establish the diagnosis of a type 1 HSV infection, permitting early treatment with acyclovir and subsequent complete recovery from the ocular herpetic infection. Emphasis is placed on the value of the immunoperoxidase technique for the rapid and specific diagnosis of cases of suspected HSV infection. PMID- 2996272 TI - Congenital herpes simplex virus infection diagnosed by cytology of aspirated tracheobronchial material. AB - Aspirated tracheal secretions from a ten-day-old newborn having signs of sepsis showed small clusters of cells with cytopathic changes consistent with herpes simplex virus (HSV) infection. The presence of type 2 HSV was confirmed by an immunoperoxidase procedure on the aspirated bronchial mucus and at necropsy in most of the viscera. Since prompt antiviral chemotherapy may favorably affect the outcome of HSV infections, early cytologic studies of tracheobronchial secretions may prove useful for rapid diagnosis. PMID- 2996274 TI - Fine needle aspiration cytology of fibrolamellar hepatocellular carcinoma. AB - The major cytologic features seen in fine needle aspirates from two cases of fibrolamellar hepatocellular carcinoma were: liver-like tumor cells, characterized by plump, polygonal forms with eosinophilic, granular cytoplasm; large oval nuclei with extremely prominent solitary nucleoli; and parallel bands of fibrous tissue and fibrocytes seen within the tumor fragments. Other helpful features included intracytoplasmic hyaline globules and well-delineated pale bodies. Clinically, the tumors occurred in young patients with noncirrhotic livers and ran a more favorable course than do other types of hepatocellular carcinoma. PMID- 2996275 TI - Fine needle aspiration cytology of bronchioloalveolar-cell carcinoma of the lung. AB - The fine needle aspiration cytology features of twelve peripherally located bronchioloalveolar cell carcinomas of the lung diagnosed by fine needle aspiration biopsy are described. A spectrum of cytomorphologic changes was appreciated, including classic groups showing uniform malignant cells having prominent depth of focus with a lack of significant nuclear molding. Other cells showed features of atypical alveolar macrophages and bronchial-lining cells. The smears demonstrated malignant cells arranged along alveolar septae and possessing hobnail-shaped nuclei. Two cases had associated psammoma bodies, and one case demonstrated optically clear nuclei in the malignant cells. This series stresses the fine needle aspiration features that aid in the recognition of this specific lung neoplasm and its differentiation from benign reactive pulmonary lesions, other primary lung cancers and metastatic tumors. PMID- 2996273 TI - Identification of types and primary sites of malignant tumors by examination of exfoliated tumor cells in serous fluids. Comparison with the diagnostic accuracy on small histologic biopsies. AB - The accuracy of identification of tumor type and primary site of malignant tumors by examination of exfoliated tumor cells was cytologically studied in 448 malignant effusions from 366 patients for whom the primary tumor site had been confirmed by histology. Ninety-seven corresponding small biopsies from metastases were separately reviewed histopathologically. In four fluids, the cells were too scanty or too poorly preserved for tumor typing. The cytologic tumor typing was performed with nearly 100% accuracy in the remaining 444 fluids, except for those of intermediate-cell anaplastic carcinomas (0 of 3) and poorly differentiated squamous (epidermoid) carcinomas (1 of 5). Adenocarcinoma was correctly identified in 98% of 285 fluids, large-cell carcinoma in 97% of 108 fluids, oat cell carcinoma in 94% of 16 fluids, well-differentiated (keratinizing) squamous carcinoma in 100% of 3 fluids, malignant lymphoma in 100% of 22 fluids and sarcoma in 100% of 2 fluids. The criteria and the failures are discussed at length. In the investigation of the accuracy of cytologic and histologic diagnoses with respect to the primary tumor site, tumors with variable sites of origin (sarcomas and lymphomas) and those with usually singular sites of origin (e.g., small-cell anaplastic carcinoma of the lung) were excluded, leaving 387 cytologic and 83 histologic specimens available for review. The breast as a primary site was correctly identified in 70% of both the cytologic and histologic specimens; the primary cytodiagnostic criteria included a uniform cell pattern, finely granular chromatin, dense cytoplasm and cell balls with smooth borders. Ovarian primaries were correctly identified in 70% of the fluids and 83% of the biopsy samples on the basis of very irregular clusters of large pleomorphic tumor cells, large nucleoli and psammoma bodies. Lung primaries, identified in 50% of the fluids and 29% of the biopsy samples, showed quite variable cell patterns, most often including large pleomorphic cells with or without mucus formation and prominent multinucleation. Gastric cancers of the diffuse type were accurately identified in 52% of the corresponding fluids, which showed mainly isolated cells with dense cytoplasmic rims, occasional signet-ring cells, "embryo-shaped" nuclei, marked hyperchromasia and densely granular chromatin.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2996276 TI - Human papillomavirus-related neoplasia of the lower female genital tract. PMID- 2996277 TI - Cytopathology of clear-cell hepatocellular carcinoma in ascitic fluid. PMID- 2996278 TI - Effect of estrogen on the affinity of malachite green for staining cardiac lipid inclusions in mice. AB - The effect of estradiol-17-beta on lipids of the ventricular myocardium of mice has been studied with a cytochemical technique in which malachite green was added to glutaraldehyde. This malachite green-glutaraldehyde fixative enhances the visualization of certain phospholipid-related elements. Estrogen induces an affinity of ventricular cardiac lipid inclusions for the cationic dye malachite green. The staining affinity is evidenced only in the estrous female, not in diestrus. In oophorectomized animals, malachite green staining is seen only following estradiol injection, but this effect is blocked by progesterone. In the male, ventricular lipids do not stain, nor do they develop malachite green affinity with estrogen stimulation. These results imply a blockade of the estradiol-mediated dye affinity by progesterone and testosterone. This reinforces the concept of the heart as a target organ for sex steroids and expands the previously described estrogen effects on myocardium. PMID- 2996280 TI - Autonomic dysfunction in experimental allergic neuritis. AB - Beat-to-beat variation (R-R variation) in the electrocardiogram was studied in experimental allergic neuritis in the Sprague-Dawley rat. Reduced R-R variations were found in 2 of 10 animals, probably as a sign of autonomic dysfunction. The vagal nerves from these two animals, studied in vitro, showed disturbed conduction. In one animal prolonged conduction latencies to supramaximal electrical stimuli were found. Vagal nerves from controls and from animals without clinical symptoms showed normal conduction. Histologically, the vagal nerves from affected animals showed a slight inflammatory cell infiltration and signs of demyelination but there was no evidence of involvement of the brainstem vasomotor nuclei. Thus, we suggest that the autonomic dysfunction in experimental allergic neuritis, measured as reduced R-R variations, is caused by a peripheral vagal neuropathy. PMID- 2996279 TI - Metabolism of coronary vasculature in euthyroid, hyperthyroid and recovering hyperthyroid rats: a histochemical study. AB - Coronary arteries and arterioles from normal rats, from rats made hyperthyroid by administration of desiccated thyroid for 10 weeks, and from hyperthyroid rats which were then fed normal control diets for 10 weeks, were examined histochemically to determine the activity of key metabolic pathways. The primary aims of this study were to determine if the alterations in particular enzyme and substrate activities that occur in thyrotoxic rat myocardium, arteries and arterioles were reversible and would return to normal levels following cessation of the hyperthyroid state. Our results suggest that hyperthyroid rats, even after 10 weeks on the normal diet, still show some compromise in arteriolar aerobic metabolism in favor of anaerobic pathways, while coronary arteries still demonstrate little glucose-6-phosphate dehydrogenase activity. Myocardial metabolic activity approximates that of normal control animals by the end of the 10th week on the normal diet. PMID- 2996281 TI - Neurological manifestations in a phase 2 study of 13 patients treated with doxyfluridine. AB - Doxyfluridine is a new cytostatic drug of the fluoropyrimidine group, which may prove to have a high antitumor activity with less toxic side effects. Thirteen patients with advanced colorectal cancer were given doxyfluridine 2 g/m2 as a one hour infusion for five days every three weeks and examined neurologically and neurophysiologically. One patient experienced an acute, reversible cerebellopathy after the second treatment course and developed a progressive generalized encephalopathy during the subsequent third and fourth cycle. The frequency of cerebellopathy may depend on the dosage and type of administration of the drug. Five patients developed a mild, mainly sensory polyneuropathy. High age, weight loss and decreasing general condition seem to be important factors in the development of polyneuropathy during doxyfluridine treatment, and this stresses the importance of optimal nutrition during the treatment period. PMID- 2996282 TI - Endoneurial fibrosis following nerve transection. An immunohistological study of collagen types and fibronectin in the rat. AB - Indirect immunofluorescent techniques with antibodies to type I, III, and V collagens and fibronectin were used to study rat sciatic nerve tributaries after transection with intact contralateral nerves as controls. Codistribution of type I and III collagens characterized the epineurium of normal nerve. In the perineurium, however, type I collagen was absent, but type III and V collagens and fibronectin were detected. Type I and III collagens were codistributed in the endoneurial stroma where a homogeneous staining by antibodies to fibronectin was also observed. During the 4-week observation period after transection the perineurium reacted by slight thickening which was most clearly demonstrated by staining with antibodies to fibronectin and to type V collagen. A widening of the type I-negative cleft also occurred. Endoneurial, type V collagen-positive cuffs around the nerve fibers became disorganized, and a concomitant increase of the stroma containing type I and III collagens and fibronectin was observed. The codistribution of the fibrous collagen types appeared similar in normal epineurium and endoneurium. Type V collagen was located in the perineurium and in endoneurial cuffs surrounding the nerve fibers. The present data indicate that collagen accumulation takes place in the perineurium and endoneurium of transected nerve. The cell type responsible for the synthesis of the connective tissue material is discussed. PMID- 2996283 TI - Immunocytochemical studies of serum proteins and immunoglobulins in human sural nerve biopsies. AB - Post-embedding immunocytochemical studies on immunoglobulins (Ig) and other serum proteins were carried out on 38 human sural nerve biopsies using the PAP method. In addition to toxic, hereditary, metabolic, dysproteinemic, and vasculitic neuritic neuropathies, morphologically normal sural nerves were included as controls. The intensity of the immunocytochemical reactions was strong for proteins, such as IgG, the light chains of Igs, and albumin, but weak or absent for others like complement component C3, IgA, ceruloplasmin, and alpha-1 antitrypsin (AAT) in normal nerve biopsies and in all pathologic groups. IgG, the light chains of immunoglobulins, and albumin could readily be detected in perineurium, endoneurial interstitium, and blood vessel walls. IgM, C3, and beta lipoprotein (BLP) were largely confined to the walls of blood vessels and perineurium, thus indicating that they do not penetrate the blood nerve barrier. Only in a few cases, in vasculitic-neuritic and dysproteinemic neuropathies, staining of the endoneurial interstitium for IgM and C3 was observed. Increased staining for the corresponding heavy or light chains was not detected in the endoneurium in any of the neuropathies associated with gammopathy. The results stress that PAP immunocytochemistry is suitable for studying the blood-nerve barrier (BNB) and provides new aspects to the concept of the BNB with respect to the steady state of serum proteins between endoneurial and vascular spaces. It is suggested that, in addition to serum concentration and molecular weight of serum proteins, the permeability of the BNB is influenced by other yet undefined factors. PMID- 2996284 TI - The rostral mesencephalon in Parkinson's disease and Alzheimer's disease. AB - The rostal mesencephalon at the level of the posterior commissure was studied by light microscopy in two patients with idiopathic Parkinson's disease, one patient with Alzheimer's disease, and one patient with senile dementia of Alzheimer's type. In the Parkinsonian cases, the rostral part of the nucleus of Edinger Westphal disclosed Lewy bodies in 3% of the neurons, neurofibrillary degeneration in 2% of the neurons, and a 54% neuronal loss. In Alzheimer's disease, 2% of Edinger Westphal neurons contained neurofibrillary degeneration, whereas in senile dementia of Alzheimer's type only rare neurofibrillary degeneration was evident in this nucleus. Neuronal loss was not apparent in the nucleus of Edinger Westphal in either of the Alzheimer's cases. The pathologic changes observed in this presumably cholinergic nucleus resemble in some respects changes reported in the cholinergic centers of the basal forebrain in these diseases. In addition, the central gray matter and pretectal region in Parkinson's disease contained a patchy increase in astroglia, some with scant reactive cell bodies; however, Lewy bodies were limited to that part of the central gray matter corresponding to the nucleus of Darkschewitsch. A few neurofibrillary tangles were present in the nucleus of Darkschewitsch in both diseases. PMID- 2996285 TI - Vitreous surgery in patients with primary neuropathic amyloidosis. AB - Pars plana vitrectomy was performed in 9 patients from the north of Sweden because of amyloid deposits of the vitreous. Operative technique and post operative results are presented. PMID- 2996286 TI - Stimulation of adenylate cyclase of ciliary processes in response to decreased inflow of aqueous humour. AB - Intravitreal injection of ouabain was used to induce unilateral hypotony and to study the relationship of adenylate cyclase (AC) and sodium-potassium activated adenosine triphosphatase (NaK-ATPase) both of which are involved in the production of aqueous humour. After preliminary experiments, days 3 and 5 were chosen as representative times when IOP was maximally reduced and stable following ouabain injection. NaK-ATPase and adenylate cyclase activities were measured biochemically in the same homogenates of isolated ciliary processes (CP). Biochemical measurements showed that 46% of NaK-ATPase activity was inhibited after 3 days, and about 78% of NaK-ATPase was inhibited 5 days after ouabain injection. At the beginning of NaK-ATPase inhibition there was a significant stimulation of adenylate cyclase activity of the CP. The highest activities were seen 2 and 3 days after ouabain injection. The suggestion is made that these 2 enzymes are interdependent. PMID- 2996287 TI - Cataract surgery. Outcome assessments and epidemiologic aspects. AB - Part one defines the topics and study purposes. The 12 studies reviewed in this survey focuses on following 3 topics of which the current knowledge is very limited: Assessment of outcome of cataract surgery (effectiveness studies). Pre operative prediction of outcome of cataract surgery. Epidemiologic aspects of cataract surgery. These three topics were chosen as study objectives because more accurate knowledge of these points is necessary to assure a better cataract treatment in future, and as a basis for securing sufficient resource allocation to cataract surgery in the public health care. Each topic will be treated separately in the following 3 parts. Part two concerns the effectiveness studies on cataract surgery (publ. I-VI). Primary, a visual functioning index is presented and evaluated. The index is meant as a new supplementary tool for evaluation of the rehabilitation effect. This index, together with visual acuity and other outcome evaluators, was then used for assessment of the effectiveness of cataract extraction generally and in different sub-groups, supplemented with quantitative assessment of rehabilitation problems and analysis of outcome predictors. In a consecutive series of patients, in which intraocular lenses were not used, it was found, that 82% of the patients obtained a final visual acuity of 0.5 or better and 74% of the patients had normal or near normal basic visual functioning at one year follow-up. In spite of these good results, the rehabilitation of these aphakics were difficult. The difference between the two figures above represents some of the 'aphakic vision cripples' with severe difficulties of adaptation to aphakic spectacles. 26% of cataract extracted patients in a normal consecutive group were found to be dissatisfied with the outcome. Two main reasons for dissatisfaction were found: macular disease and low quality of vision with aphakic spectacles, especially in monaphakics, of which 75% had complaints about vision. Although monaphakics nearly reach the same level in basic functioning as the biaphakics, the latter are much more satisfied. It seems likely that basic visual functioning is gained by first eye surgery and more delicate visual functioning and a subjective 'visual comfort' is obtained by second eye surgery. Adaptation to aphakic spectacles seem to be far less difficult for the biaphakics. In series of patients rehabilitated with intraocular lenses or extended-wear contact lenses, the monaphakics appear to have a generally better visual functioning and they are much more satisfied with outcome than spectacles corrected monaphakics.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2996288 TI - Epstein-Barr virus infection and serological profile in Greenland Eskimo children. AB - The Epstein-Barr virus (EBV)-specific antibody profile of 101 Greenland Eskimo children was determined. The proportion of children with serological evidence of recent or past primary EBV infections rose from 22% at 6 months of age to 79% at 24 months of age. All but 2 of 49 children more than 4 years of age proved seropositive. The geometric mean titre (GMT) of antibodies to the viral capsid antigen (VCA) was highest during the first 3 years of life and declined sharply to a lower, nearly constant level in older children. The GMT of antibodies to the nuclear antigen (EBNA), rose slowly during the first 4 years of life to its persistent level. None of the children had a history of illnesses comparable to infectious mononucleosis. The results have shown that in this population with an enhanced risk of nasopharyngeal carcinoma, primary EBV infection occurs at a very early age. PMID- 2996289 TI - Combined [99mTc]DMSA kidney scintigraphy and [131I]hippuran renography in children with urinary tract infections. AB - Combined [99mTc]DMSA kidney scintigraphy and [131I]hippuran renography were performed consecutively in 87 children with recurrent urinary tract infections in a retrospective study. This procedure allows a description of renal cortical morphology, split function determination and run-off evaluation. Signs of cortical scarring were found in 41 of 172 kidneys (24%) and were significantly associated with vesico-ureteral reflux (p less than 0.001) and with delayed urinary run-off (p less than 0.01). Split renal function was significantly reduced in kidneys with unilateral scarring (p less than 0.001). The radio isotope investigations and intravenous urography were performed within 3 months of each other in 56 patients (110 kidneys). Good agreement between the findings was found except for 13 kidneys, where cortical activity defects were revealed by scintiscan despite normal urography. The extended scintigraphic procedure described is considered useful for urological screening of children with urinary tract infections and may thus replace urography as a first-line investigation. It should be followed by micturition cysto-urethrography when evaluation for vesico ureteral reflux is indicated. PMID- 2996290 TI - Rheumatoid arthritis cells in experimental pleuritis in mice. AB - In mice immunized with bovine fibrin, the same antigen was applied to the pleural cavity. A granulomatous pleuritis appeared affecting both the visceral and the parietal pleura, especially located around the antigen particle. Rheumatoid arthritis (RA) cells were constantly found in the pleural cavity when pleural lesions were present. This immunological, granulomatous pleuritis is the first experimental model for the study of RA cell-positive types of pleurisy in humans. PMID- 2996291 TI - Effect of general anaesthetics and organic solvents on alpha 1-adrenoceptors in the myometrium. AB - The alpha 1 selective radioligand 3H-prazosin was used to assay alpha 1 receptors in membranes prepared from the rabbit myometrium. 3H-Prazosin was found to bind to a single high affinity site in these membranes which was the presumed alpha 1 receptor. A series of general anaesthetics and organic solvents were tested for their ability to inhibit 3H-prazosin binding. The order of potency of the tested agents to inhibit the binding was: chloroform=halothane=trichloroethylene greater than carbon tetrachloride greater than dichloromethane. The depression of 3H prazosin binding seemed to be induced on the alpha 1 receptor since non-specific radioligand binding was not affected as revealed by a saturation experiment with 3H-prazosin where halothane was used as inhibiting agent. Computer analysis of the latter experiment also showed that halothane depressed mainly the affinity of 3H-prazosin for the alpha 1 receptor. The ability of the general anaesthetics and organic solvents to inhibit contractions elicited by alpha 1 stimulation with phenylephrine in the rabbit uterus was also investigated. In these tests the order of potency for the inhibition of the contractile response was: carbon tetrachloride greater than or equal to halothane=chloroform greater than trichloroethylene greater than dichloromethane. The mechanism of action for alpha 1 receptor and myometrial depression is discussed. PMID- 2996292 TI - Effect of halothane on calf cortex alpha 1-adrenoceptors. PMID- 2996293 TI - Opioid receptors in human placenta may be affected by methadone and propoxyphene treatment. PMID- 2996294 TI - 5-Hydroxy-2-methyl-2-(di-n-propylamino)tetralin: synthesis and central pharmacological effects. PMID- 2996295 TI - Influence of receptor formation and receptor movement inhibitors on hormonal imprinting in cell culture. AB - Hormonal imprinting takes place at the first interaction of the cell with the adequate hormone, and exerts a lasting influence on cellular binding capacity and functional response over many subsequent cell generations. Hormonal imprinting can also be induced in cell lines. In a Chinese hamster ovary (CHO K1) cell line, inhibitor of endocytosis and cellular protein synthesis inhibited hormone binding in themselves, and in cultures preexposed to TSH they inhibited imprinting by TSH in a dose-dependent manner. The protein synthesis inhibitor cycloheximide and the microfilament de-organizing agent cytochalasin-B inhibited imprinting by TSH to a greater degree than all other inhibitors tested, indicating that apart from cellular binding capacity, unimpaired cellular protein synthesis and microfilament activity are essential prerequisites of hormonal imprinting. PMID- 2996296 TI - Changes in the adrenergic reactivity of spontaneously hypertensive rats after abrupt withdrawal of chronic clonidine treatment. AB - An experimental model of the "withdrawal syndrome" in chronic clonidine treatment is reproduced in spontaneously hypertensive rats (SHR). Medication is withdrawn abruptly after daily oral administration of 100 micrograms/kg clonidine for 12 days or after it has been received with the drinking water in a dose of approximately 250 micrograms/kg daily for 14 days. In the course of the treatment, as well as on the 24th and 48th hour after withdrawal, studies are carried out of the changes in the arterial pressure, heart rate, vascular resistance, the contractions of electrically stimulated m.anococcygeus and vas deferens, as well as some biochemical parameters (noradrenaline content in the brain and myocardium, the level of glucose and free fatty acids in the blood, the activity of phosphorylase "a" in the myocardium). In the course of clonidine treatment there is potentiation of the peripheral noradrenaline effects on the arterial pressure and vascular resistance, increasing hyperglycaemia, reduced noradrenaline content in the brain and myocardium, as well as potentiation of the inhibitory effect of clonidine on the contractions of electrically stimulated m.anococcygeus, as well as potentiation of the isoprenaline responses on the arterial pressure. The isoprenaline effects on electrically stimulated vas deferens, as well as the activity of the stimulated phosphorylase "a", decrease. No sharp increase in the arterial blood pressure was observed upon withdrawal of the clonidine treatment. The reactivity of the peripheral pre- and postsynaptic alpha-adrenergic receptors is intensified (the effects of noradrenaline on the arterial pressure and vascular resistance become even stronger, the noradrenaline content in the brain and myocardium and hyperglycaemia increase, and there is potentiation of the inhibitory effect of clonidine on electrically stimulated m.anococcygeus.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996297 TI - Memory effects of GABA-ergic antagonists in rats trained with two-way active avoidance tasks. AB - In experiments on male Wistar rats, trained and tested for memory after 24 h in a shuttle box, it was found that bicuculline applied intraperitoneally 10 min before the training in a dose of 0.5 mg/kg, improves learning and retention; bicuculline applied immediately after training in a dose of 1.0 mg/kg, improves retention. Picrotoxin, administered i.p. 20 min before training in doses of 0.25 and 0.50 mg/kg, improves learning, while in doses of 0.10-2.0 mg/kg it facilitates retention; picrotoxin, applied immediately after training in doses of 1.0 and 2.0 mg/kg, improves retention in the experimental animals. It is assumed that the GABA-ergic receptor antagonists, administered intraperitoneally, facilitate the processes of memory formation, whereby the decrease in the inhibitory effect of GABA is essential. PMID- 2996298 TI - Further investigation on the antiinflammatory properties of adolapin--bee venom polypeptide. AB - Adolapin is a basic polypeptide (M. W. 11500) isolated from bee venom. It showed marked antiinflammatory and analgetic properties and inhibited cyclooxygenase. It was found that adolapin inhibited also the activity of bee venom phospholipase A2 (7 nmole/ml producing about 80% inhibition of 2.5 nmole/ml phospholipase). In addition it inhibited the lipoxygenase from human platelets (4.5 nmole/ml inhibited about 80% of the activity of 0.8 mg protein/ml). Adolapin (20 micrograms/kg) caused an elevation of c-GMP level in rat spleen and brain as well as a decrease of c-AMP in rat spleen. Adolapin was tested by the "tail flick" method which allowed the demonstration of its analgetic action. The partial inhibition of the analgetic effect of adolapin induced by naloxon, proved the participation of a central mechanism of action. Similar to other nonsteroid analgetics, adolapin displayed antipyretic effect (40 micrograms/kg caused an inhibition of the mean temperature rise about 62%. PMID- 2996299 TI - Neuropeptide Y is a potent inhibitor of cyclic AMP accumulation in feline cerebral blood vessels. PMID- 2996300 TI - Distribution of carbonic anhydrase in cells and membranes isolated from pig gastric mucosa. AB - Mucosal cells were isolated from pig stomachs and then fractionated on linear density gradients of Percoll. Different types of cells were identified by their typical staining and morphology. In disrupted cell fractions, hydration of CO2 by carbonic anhydrase was measured by means of pH-stat technique. Localization of carbonic anhydrase to certain cell fractions was also studied by histochemical staining. Both parietal cells and carbonic anhydrase were confined to the low and intermediate density fractions of the gradients. Purified membranes from pig gastric mucosa, which contained the acid pump of the stomach, the H,K-ATPase, also contained a firmly bound carbonic anhydrase of high activity. The enzyme activity in the membranes was inhibited by acetazolamide, furosemide and KSCN. The molecular mass of the carbonic anhydrase was 33 kDA as estimated by its binding of [14C]furosemide followed by polyacrylamide gel electrophoresis. Previous suggestions of a role of carbonic anhydrase as a supplier of H+ in the secretion of acid are supported by its high activity and its localization to the same membrane as the acid pump of the stomach. PMID- 2996301 TI - Studies on the oxygen radical mechanism involved in the small intestinal reperfusion damage. AB - Characteristic mucosal lesions develop in the small intestine during ischaemia and hypotension. This tissue damage can be further aggravated in the immediate reperfusion phase, presumably secondary to the generation of oxygen free radicals which have been proposed to be generated in this situation through the hypoxanthine-xanthine oxidase system. This was further investigated in the cat small intestine using a standardized regional intestinal hypotension model in which the effects of allopurinol (a xanthine oxidase inhibitor) were compared to those of an exogenous supply of inosine. The grade of mucosal damage, the nucleotide levels, the concentrations of hypoxanthine, total and oxidized glutathione, and of conjugated dienes were measured in the intestinal tissue. The results indicate that oxygen radicals generated by xanthine oxidase are very important, but not the only significant factor in the small intestinal reperfusion damage. PMID- 2996302 TI - Effects of fasting and refeeding on the activity of hepatic glucose-6-phosphatase in rats. AB - The activities of glucose-6-phosphate hydrolase and glucose-6-phosphate translocase were determined in rats fasted for 1-3 days and in animals fasted for one day and then either refed with mixed pellet or given oral or intraperitoneal glucose. The assay was based on the colorimetric measurement of the released inorganic phosphate. Fasting over 24 h significantly increased both the translocase and the hydrolase activity of glucose-6-phosphatase. These parameters showed a further increase when rats were fasted for another 24 h. In animals fasted for 24 h and then refed with standardized pellet diet, a progressive fall of enzyme activity was noticed. However, even 72 h of refeeding did not lead to complete normalization. Glucose given orally or intraperitoneally also suppressed the enzyme activity, although the effect was somewhat delayed. As expected, in fasting rats glucose and insulin levels were significantly decreased. Normoglycaemia was established after just 24 h, regardless of refeeding with pellets or with glucose. The former group exhibited hyper- and the latter hypo insulinaemic pattern. We speculate that augmented activity of hepatic glucose-6 phosphatase during fasting stimulates the metabolism of glucose through the glucose cycle and is thereby at least partially responsible for insulin resistance accompanying the fasting state. PMID- 2996303 TI - Facilitation of hippocampal long-lasting potentiation by GABA antagonists. AB - Long-lasting potentiation (LLP) of synaptic transmission in the CAI region of the hippocampal slice preparation has been examined. The effects of reduced postsynaptic inhibition given by application of gamma-aminobutyric acid (GABA) antagonists (mainly picrotoxin) on the generation of LLP were investigated. It was first demonstrated that picrotoxin had little effect on excitatory synaptic transmission itself as judged by the rising phase of the field EPSP. Moreover, there were largely no actions on short-lasting synaptic effects such as paired pulse facilitation and frequency potentiation. On the other hand, following drug application, much fewer afferent volleys were needed to generate a given amount of LLP. Long-lasting potentiation could be produced by trains containing as few as 2-5 impulses, trains that normally give rise to only short-lasting effects. There was no apparent difference in the maximal amount of LLP that could be produced for a given input, suggesting that the GABA antagonists do not operate by enhancing the capacity for LLP production but by facilitating its induction. As in normal solution, the LLP in the presence of the drugs was confined to the tetanized pathway. Tetanization in the treated slices was associated with enhanced somatic firing as well as an increase of the negative extracellular potential recorded in the dendritic layer. It is proposed that part of this increased negativity represents current through synaptically opened N-methyl-D aspartate (NMDA) receptor channels. Furthermore, it is suggested that the facilitated induction of LLP in the presence of GABA antagonists is related to a facilitated activation of these NMDA receptor channels which is secondary to the higher levels of dendritic depolarization attained during tetanization under conditions of reduced postsynaptic inhibition. PMID- 2996304 TI - Effects of ganglioside GM1 treatment on striatal glucose metabolism, blood flow, and protein phosphorylation of the rat. AB - Effects of ganglioside GM1 administration have been studied in unilaterally partially hemitransected rats on striatal energy metabolism, using the radioactive deoxyglucose (DG) technique, on striatal blood flow, using radiolabelled iodoantipyrine (IAP) as tracer, and on cyclic AMP (cAMP) and Ca2+ induced protein phosphorylation in striatal membranes (P2 fraction). Ganglioside GM1 treatment counteracted the imbalance in striatal energy metabolism, in striatal blood flow, as well as in protein phosphorylation found between the striata of the lesioned and unlesioned side, possibly due to excitatory effects on the lesioned side and inhibitory effects on the unlesioned side. In intact animals, GM1 treatment produced a reduction in cAMP and Ca2+ induced striatal protein phosphorylation. Facilitatory actions of the ganglioside GM1 dominate following a lesion, probably due to its possible function as a modulator of receptors for neuronotrophic factors, leading to restoration of metabolic rate and of cAMP and Ca2+ induced protein phosphorylation in the striatum of the lesioned side. The results emphasize that ganglioside GM1 treatment can restore the metabolism of a partially innervated striatum towards normal, as evaluated both at the level of the entire striatal structure by means of the DG and IAP techniques and at the molecular level by means of studies on the cAMP and Ca2+ induced protein phosphorylation. PMID- 2996305 TI - Adrenoreceptor interactions of the enantiomers and metabolites of mianserin: are they responsible for the antidepressant effect? AB - Mianserin is a tetracyclic antidepressant whose postulated mechanism of action involves release of noradrenaline mediated via cortical alpha 2-adrenergic autoreceptor blockade. This property resides stereoselectively in the S(+) enantiomer of mianserin, which is also more potent in behavioural tests indicative of antidepressant activity, and in the reversal of clonidine-induced effects. Cortical receptor binding studies have indicated that although a similar stereoselectivity prevails for the inhibition of both alpha 2-binding (clonidine) and alpha 1-binding (prazosin), mianserin and its enantiomers are more potent antagonists at alpha 2- than at alpha 1-binding sites. However, no stereoselectivity is apparent for the antagonism of cortical alpha 2 heteroreceptors controlling serotonin release. Following chronic administration, (+/-)- and S(+)-mianserin, but not the R(-)-enantiomer, produce functional supersensitivity at alpha 2-autoreceptors which is unaccompanied by changes in clonidine binding. Neither mianserin nor its enantiomers alter the sensitivity of alpha 2-heteroreceptors following chronic administration. Like mianserin and its S(+)-enantiomer, but unlike R(-)-mianserin and the 8-hydroxy metabolite, the desmethyl metabolite inhibits noradrenaline uptake in vitro. 8-hydroxymianserin and, to a lesser extent, desmethylmianserin release noradrenaline from cortical slices via alpha 2-autoreceptor antagonism, but only the 8-hydroxy metabolite blocks alpha 2-autoreceptors and alpha 2-heteroreceptors in synaptosomal preparations. It is likely that S(+)-mianserin, desmethylmianserin, and 8 hydroxymianserin contribute substantially to the overall facilitating effect of mianserin on noradrenergic transmission in vivo. As yet it is unclear whether this effect is exclusively responsible for the antidepressant activity of mianserin or whether the stereoselectivity also shown by the mianserin enantiomers towards serotonin receptors plays a complementary role. PMID- 2996307 TI - Neuropeptides and their receptors in aged-rat brain. AB - Age-associated changes in methionine-enkephalin (ENK) and thyrotropin releasing hormone (TRH) concentrations, and their receptors were examined in discrete regions of the rat brain. The ENK and TRH concentrations in aged rats were nearly identical to those in young adult rats, except for a slightly lower TRH value in the hypothalamus of the aged rats. On the other hand, the ENK and TRH receptor levels in the cerebral cortex of aged rats was markedly lower than that of young adults rats. The results suggest that determinations of both neuropeptide and receptor levels are indispensable for evaluation of peptide-mediated neural systems in the central nervous system. PMID- 2996306 TI - The transplanted kidney. Diagnostic and interventional radiology. AB - Following kidney allotransplantation a great number of complications threaten the patient and his graft, e.g. acute tubular necrosis, acute and chronic rejection, urologic and vascular complications and complications due to the immunosuppressive treatment. During the last decade a number of technical developments in radionuclide, ultrasonographic and radiographic imaging and intervention has significantly improved the possibility of early recognition and handling of such complications. Knowledge of the capability and limitations of the various techniques is of vital importance for their rational use. The aim of this review article is to give a short description of the various imaging modalities, the rational monitoring of the posttransplant patient, and possible handling of complications by the aid of imaging techniques. PMID- 2996308 TI - Red cell abnormalities in a kindred with an uncommon form of hereditary spherocytosis. PMID- 2996309 TI - Systemic manifestations and enzyme studies in sarcoidosis with neurologic involvement. AB - The dissemination and activity of systemic disease was evaluated retrospectively in 50 patients with neurosarcoidosis, 24 of whom presented with neurologic symptoms. During follow-up, five patients never developed detectable systemic disease. In 26 patients, sarcoidosis had previously been diagnosed, but in 11 (42%) of them the neurologic symptoms were initially not connected with this disease. During follow-up, extraneural features were those of sarcoidosis in general. However, 23 patients (46%) had normal chest X-ray on admission to neurologic examinations. Fourteen (35%) of 40 examined patients had ocular changes, 13 (33%) of 39 hypercalciuria and 13 (26%) of 50 skin manifestations. Serum angiotensin converting enzyme (ACE) was elevated in only 31% of the patients. Measureable amounts of ACE were recorded in the cerebrospinal fluid from 13 of 17 examined patients. During follow-up the activity of neurosarcoidosis seemed to be linked to the course of systemic disease in general. PMID- 2996310 TI - Effect of lipid lowering diet on low density lipoprotein receptor activity in freshly isolated peripheral blood mononuclear cells. AB - The effect of lipid lowering diet on low density lipoprotein (LDL) receptor activity has been studied in freshly isolated peripheral blood mononuclear cells (PBMCs) from 16 hypercholesterolemic male subjects during a three weeks' dietary intervention trial. The participants were randomized to an intervention group or to a control group. The subjects in the intervention group had a non significantly larger increase in LDL receptor activity, determined as degradation of 125I-LDL at 37 degrees C, than the control group (49.7 +/- 18.1 and 35.4 +/- 21.2 ng/mg, respectively). Irrespective of assignment to the intervention group or control group, the eight subjects with the largest reduction in total serum cholesterol had significantly larger absolute and percentage increases in LDL receptor activity than the eight subjects with the smallest reduction in total serum cholesterol (p less than 0.05). Thus, it appears that the cholesterol reduction that can be achieved in humans by dietary intervention results in a small increase in LDL receptor activity in freshly isolated PBMCs. PMID- 2996311 TI - The fatty acid composition of 12 North-European fish species. AB - Cardiovascular diseases among Greenland Eskimos are rare because their diet is rich in fatty fish and marine mammals. The beneficial effect of the fish diet appears to be mediated, at least in part, by the high amount of eicosapentaenoic acid in fish. We investigated the total lipid amount and fatty acid composition of 12 commonly eaten North-European fish species. Most of the detected fatty acids were unsaturated, and the content of eicosapentaenoic acid varied usually between 6 and 16%. The amount of total lipid varied between 3.5 and 216 mg/g wet tissue. The total amount of lipid in different fish species seems to be more important than the respective fatty acid composition when considering which fish should be especially beneficial in the diet. Herring, salmon, Baltic herring, turbot and trout seem to contain most abundantly eicosapentaenoic acid. PMID- 2996312 TI - Effects of synthetic corticotropin-releasing factor in normal individuals and in patients with hypothalamic-pituitary-adrenocortical disorders. AB - Plasma adrenocortical hormone (ACTH) and cortisol response to four dose levels (25, 50, 100 and 300 micrograms) corticotropin-releasing factor (CRF) were studied in 5 healthy men, and the response to 100 micrograms CRF in 12 patients with various disorders of the hypothalamic-pituitary-adrenocortical function. In normals, mean plasma ACTH and cortisol concentration rose at all dose levels of CRF and peaked at 30 and 60 min respectively. The increment in plasma cortisol at 60 and 90 min was significantly higher on 100 and 300 micrograms CRF than on 25 micrograms, but the total cortisol concentration was not. Seven patients had Cushing's syndrome. In 2 patients with adrenocortical carcinoma the basal plasma ACTH was suppressed. After CRF a small increase was seen in plasma ACTH and cortisol in one patient successfully treated with mitotane, while the other patient did not respond. In 1 patient with ectopic ACTH syndrome an increase in plasma ACTH 15 min after CRF was not accompanied by any increase in plasma cortisol. One patient with bilateral multinodular adrenocortical hyperplasia did not respond to CRF. The plasma ACTH and cortisol response to CRF was supernormal in 2 patients with Cushing's disease, while a third patient responded in the normal range. In 2 patients with Nelson's syndrome the plasma ACTH response was excessive. Two out of three hypophysectomized patients did not respond to CRF, while one patient with a slightly positive response to hypoglycemia also responded (subnormally) to CRF. Our data indicate that CRF in doses of 50-100 micrograms will be a valuable substance in the differential diagnosis of Cushing's syndrome. Some overlap in the response is, however, seen between patients with Cushing's disease and other patients with Cushing' syndrome. CRF will possibly be of value also for the diagnosis of secondary adrenocortical failure. PMID- 2996313 TI - In vivo regulation of B lymphocyte production in the bone marrow: effects and mechanism of action of exogenous stimuli on pre-B cell proliferation and lymphocyte turnover. AB - The present studies demonstrate that a single administration of an extrinsic agent (SRBC) can stimulate increased production of B lymphocytes in mouse bone marrow as revealed by 2 in vivo assays which quantitate pre-B cell proliferation and small lymphocyte renewal, respectively. The mechanisms mediating this stimulatory effect are sensitive to silica in vivo and require the presence of the spleen. Early events are both silica-sensitive and spleen-dependent, while a subsequent stage appears still to be spleen-dependent but not silica-sensitive. Sustained exogenous stimulation by multiple SRBC injections for 4 wk in young mice produces an expanded population size and increased production of pre-B cells and B lymphocytes in the bone marrow, apparently an elevated kinetic steady state of B lymphocyte production. (Formula: see text). As depicted schematically in Figure 1, the results suggest that the magnitude of bone marrow B lymphocyte production in vivo may reflect a basal level, putatively regulated by microenvironmental and other endogenous factors, which is amplified by exogenous environmental stimuli mediated by the action of macrophages located in the spleen. Further questions about such an environmental amplification (Fig. 1) concern the nature of later events in the spleen, the identity of putative stimulatory factors or cells circulating from the spleen to the bone marrow, the receptive target cell stages in the bone marrow and the consequences of this process with respect to the size and diversity of B lymphocyte clones and of primary humoral immune responses in vivo. PMID- 2996314 TI - Human lymphocyte-high endothelial venule interaction: functional and molecular characterization. PMID- 2996315 TI - T cell clones: a model of a non-recirculating phase of T cell differentiation. PMID- 2996316 TI - Evidence for local neuromodulation of T cell migration in vivo. PMID- 2996317 TI - Interaction mechanism of clustering lymphocytes: specificity molecular properties and its relation to migration. PMID- 2996318 TI - Protective effects of monoclonal antibodies and immunopurified glycoproteins in primary HSV-1 infection. PMID- 2996319 TI - Follicular dendritic cells isolated from human tonsils. PMID- 2996320 TI - Stimulation of neutrophil oxidative metabolism by indomethacin. AB - Human neutrophils exposed to indomethacin demonstrate an enhanced capacity for superoxide ion (O2-) generation when stimulated with opsonized zymosan. Enhancement is not seen with indomethacin-treated cells exposed to soluble oxidative stimuli. To further investigate this phenomenon, O2- generation, chemiluminescence, and phagocytosis were assessed in human neutrophils preincubated with indomethacin. Zymosan-stimulated O2- release was increased from 150 to 300% of controls in neutrophils exposed to 400 micrograms/ml indomethacin. Enhancement was not reversed by removal of indomethacin from the medium prior to addition of the stimulus and was dose-dependent at drug concentrations of 5 to 400 micrograms/ml. Neutrophils exposed to methacin alone also generated more O2- than control cells, although this increment was not sufficient to account for the degree of enhancement seen when indomethacin-treated cells were exposed to zymosan. Neutrophil chemiluminescence induced by zymosan was also increased by exposure to indomethacin, and at a drug concentration of 400 micrograms/ml (1.1 mM) enhancement ranged from 253 to 333% of controls. As was observed with O2- generation, chemiluminescence of neutrophils was increased in the presence of indomethacin alone, although, to a degree far less than was seen when drug treated cells were stimulated with zymosan. Phagocytosis of radiolabeled S. aureus by neutrophils incubated with indomethacin was increased 13 +/- 5% over controls (P less than 0.01, n = 5), but was unaltered by incubation of cells with the buffer used to solubilize the drug. The modest degree of enhancement of phagocytosis suggests that increased particle uptake is not the sole mechanism of oxidative enhancement.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996322 TI - Structure and alpha-adrenergic activity of pyrazinimidazolines. AB - The effects of newly synthetized pyrazinimidazolines on the contraction of isolated rat tail artery and on the chronotropic action of rat atria as well as the effects on blood pressure in anaesthetized rats were determined. The structure-activity relationships were studied, including standard imidazoline drugs. Starting from practically inactive derivatives the step-by-step structural modifications have been made resulting in markedly active chemical congeners, which were designed, synthetized and tested pharmacologically. PMID- 2996321 TI - Effect of benzydamine on exocytosis and respiratory burst in human neutrophils and mononuclear phagocytes. AB - The effect of benzydamine on stimulus-dependent respiratory burst activity and enzyme release was tested in human neutrophils, monocytes and monocyte-derived macrophages. Established anti-inflammatory compounds, indomethacin, phenylbutazone and bufexamac, were tested for comparison. Care was taken to avoid cytotoxic or cytolytic concentrations of the test compounds, and their effect on release of lactate dehydrogenase was also tested. Release of specific and azurophil granules contents were induced in human neutrophils by A23187, PMA and fMLP with and without cytochalasin B pretreatment. Benzydamine inhibited stimulus dependent release of vitamin B12-binding proteins, a marker for the specific granules, in a concentration-dependent fashion. By contrast, phenylbutazone and bufexamac were practically inactive. The effect of benzydamine on exocytosis of azurophil granules was tested in cytochalasin B-pretreated neutrophils. Benzydamine, again in contrast to the two reference anti-inflammatory compounds, inhibited release concentration-dependently also under these conditions. The concentration of the compound which inhibited exocytosis by 50% was 30-100 microM in normal and 3-10 microM in cytochalasin B-treated neutrophils. The effect of benzydamine and reference compounds on the respiratory burst was tested by assaying for superoxide formation in neutrophils and H2O2 formation in mononuclear phagocytes. Benzydamine was inactive on neutrophils and inhibited slightly the burst response of monocytes and macrophages. Two reference compounds, bufexamac and phenylbutazone, were generally more active. The strongest inhibitory effect was that of phenylbutazone on fMLP-stimulated cells. Benzydamine lacked activity under these conditions, indicating that it does not bind to the receptor of formylated chemotactic peptides.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996323 TI - Diagnosing infectious mononucleosis. AB - Infectious mononucleosis can be diagnosed with certainty only when suggestive clinical findings are corroborated by relative and absolute lymphocytosis, lymphocyte atypia of more than 20 percent and a positive serologic test. A serologic test is not absolutely necessary when atypical lymphocytes exceed 40 percent since this is specific to infectious mononucleosis. The diagnosis is difficult when clinical findings are scanty and the blood picture is unhelpful. PMID- 2996324 TI - Low glycemic index carbohydrate foods in the management of hyperlipidemia. AB - Reduction in the mean glycemic index (GI) of diets of 12 hyperlipidemic patients from 82 +/- 1 to 69 +/- 2 units (p less than 0.001) for a 1 mo period resulted in a significant reduction in total and LDL serum cholesterol and serum triglyceride by comparison with the mean lipid values for the preceding and following control months. The change in GI of the diet was achieved largely through manipulation of the cereal products and was not related to large differences in the amount of dietary fiber. In addition, apart from a small mean increase in unsaturated fat and calorie intake during the control periods, no difference was seen between the proportion of macronutrients on either treatment as determined by 1 wk diet histories recorded on alternate weeks throughout the 3 mo study. Selection of low glycemic index foods may therefore be a useful adjunct to the management of hyperlipidemia. PMID- 2996325 TI - The effect of raw wheat bran, alfalfa meal and alpha-cellulose on iron ascorbate chelate and ferric chloride in three binding solutions. AB - Iron ascorbate chelate was prepared and purified with iron and ascorbate combined in a 1:1 molar ratio. Some iron dissociated from the chelate and bound to raw wheat bran and alfalfa meal, indicating something in these fiber sources produced the dissociation. The chelate bound intact to alpha-cellulose in bicarbonate and phosphate buffer. More iron as ferric chloride bound to the three fiber sources than did iron as chelate. Iron became more soluble in bicarbonate buffer in the presence of raw wheat bran and alfalfa meal, but became less soluble in phosphate buffer in the presence of these fiber sources. PMID- 2996327 TI - Prophylactic versus no brain irradiation in regional small cell lung carcinoma. AB - Among 104 complete responders entered in a randomized prospective trial of treatments for regional small cell undifferentiated carcinoma of the lung, 52 received prophylactic irradiation of the brain, 3,000 rad in 10 fractions, and 52 did not. The median survivals were 53 and 52 weeks respectively, and the incidences of brain metastases were 5% and 20%. Prophylactic brain irradiation was not associated with significant long-term toxicity. PMID- 2996326 TI - Effects of dietary fibers and cholestyramine on the activity of pancreatic lipase in vitro. AB - Most experiments were conducted in the presence of human gallbladder bile; colipase and pancreatic lipase were purified using porcine pancreas. The adsorption of bile salts, phospholipids and cholesterol from the bile, together with that of pancreatic lipase was measured on wheat bran, cellulose, hemicellulose (xylan), slightly methylated pectin (42%) and cholestyramine. In contrast to cholestyramine which intensively binds biliary lipids (61.7-81.7%) and pancreatic lipase (47.5%), the fibers studied only had a low adsorbent power. The direct influence of these fibers and of cholestyramine at concentrations ranging from 0-5% on lipase activity was measured at constant pH, using two conventional assay systems, long chain triglycerides and tributyrin. In the presence of human bile and colipase, a drastic reduction in triglyceride hydrolysis by lipase was observed with cholestyramine (loss of 66-82%) and wheat bran (loss of 77-94%) at 1% concentration. The other fibers did not have any marked effects on enzyme activity. The use of a radio labeled lipase made it possible to demonstrate that the inhibitory effect of bran on enzyme activity was independent of adsorption phenomena on bran. The fraction of bran that can be solubilized in the aqueous phase, in fact, induced this reduction in activity. The presence of protein inhibitor in bran may be responsible for the reduction in pancreatic lipase activity. PMID- 2996329 TI - Watery diarrhea in a patient with myasthenia gravis, thymoma, and immunodeficiency. AB - An elderly man with thymoma, myasthenia gravis, and hypogammaglobulinemia developed profuse watery diarrhea. Infusions of gamma-globulin caused the diarrhea to resolve. The patient succumbed to fulminant bronchopneumonia. At necropsy he was found to have widespread cytomegalovirus infection with duodenal and ileal ulceration, subtotal villous atrophy, marked nonspecific inflammation of the small intestine and bronchopneumonia. In addition Herpes simplex infection and invasive candidiasis were present. Patients with immunodeficiency are susceptible to a variety of gastrointestinal pathogens, particularly viral. PMID- 2996328 TI - Cobalamin-specific R binder in pernicious anemia gastric juice: production by digestive enzyme action on saliva R binder. AB - We found in four of five pernicious anemia gastric juices a partly degraded R binder which was cobalamin specific and has an apparent molecular weight of 60 70,000 daltons. Twenty-nine to 74% (4.8-27.0 ng/ml) of the corrinoid binding capacity could not be blocked by cobinamide (a noncobalamin corrinoid). The fifth pernicious anemia gastric juice and five nonpernicious anemia gastric juices had minimal amounts of this binder (2.5 and 2.2 +/- 1.4 ng/ml). Scatchard analysis revealed that cobalamin-specific R binder has 1000-fold lower affinity for cobinamide than cobalamin. Increasing quantities of trypsin and/or chymotrypsin digested increasing amounts of saliva R binder and an increasing percentage of the remaining digest-resistant R binder acquired cobalamin specificity. Partly degraded R binder in pernicious anemia gastric juice was resistant to further proteolysis. Cobalamin-specific R binder, perhaps produced in vivo by the action of refluxed pancreatic enzymes on swallowed R would preferentially bind ingested and/or biliary cobalamin rather than analogue and thereby could play a role in hastening the development of cobalamin deficiency in pernicious anemia. PMID- 2996330 TI - Rhinoviruses in Seattle families, 1975-1979. AB - Rhinovirus infections in Seattle families with schoolchildren (1975-1979) and in selected outpatients were revealed by virus shedding or antibody rise. These observations extend those in the Seattle Virus Watch (1965-1969). Analysis of rhinovirus serotype prevalence again revealed certain "common" persisting serotypes but provided no further evidence that new serotypes are continuing to emerge. Two seasonal peaks, spring higher than fall, were again evident. Infection rates, again inversely related to age, were lower overall than in the Virus Watch (0.42 vs. 0.64 per person-year), probably because there were fewer young children. Frequencies of antibody response by virus shedders again varied widely by serotype but differed greatly from those in the Virus Watch in rank order of response rate, suggesting that immunogenicity is not a stable serotype characteristic. The frequency and magnitude of antibody response of virus shedders increased with age. Antibody-related protection against infection was evident only in persons age greater than or equal to 10 years. Observations in 7 families during successive homotypic infection episodes indicate that postinfection immunity to natural challenge requires persistence of antibody. Of all reported respiratory illness, 11.9% (0.31 per person-year) were due to rhinoviruses and 6.9% to influenza viruses. Of viruses recovered from family members, rhinoviruses, herpes simplex, and influenza comprised 56%, 12.6%, and 12.4%, respectively. Although households often experienced greater than or equal to 2 concurrent or closely consecutive episodes of infection with different viruses, only 29 individuals were shown to shed 2 viruses at the same time. Most of the second viruses, include 3 rhinoviruses and 18 other nonhemadsorbing viruses, appeared when 582 rhinovirus-positive specimens were retested after treatment with homotypic antibody. These results suggest that rhinoviruses interfere with nonhemadsorbing viruses in cell culture but mostly with other rhinoviruses in humans. PMID- 2996331 TI - Enzootic vesicular stomatitis New Jersey type in an insular feral swine population. AB - Free-ranging feral swine from Ossabaw Island, Chatham County, Georgia, were serially bled and tested for vesicular stomatitis New Jersey type serum neutralizing antibody to determine the intensity and progression of annual vesicular stomatitis activity. From November 21, 1981 to October 11, 1982, and from March 15, 1983 to October 14, 1983, 307 and 340 swine were sampled, respectively. Seroconversions were initially detected during the first week of June and continued into September in both 1982 and 1983. Serologic results indicate a seroconversion incidence during 1982 and 1983 of approximately 12% and 60%, respectively. Similar patterns in timing and affected geographic area were observed during both years, with the earliest viral activity and highest incidence restricted to the southern portion of the island. Clinical vesicular stomatitis was not seen during 1982. However, during 1983, vesicular stomatitis New Jersey type virus was isolated from vesicular lesions on two swine. PMID- 2996332 TI - Effects of enalapril alone, and in combination with hydrochlorothiazide, on renin angiotensin-aldosterone, renal function, salt and water excretion, and body fluid composition. AB - Enalapril is a new, oral, long-acting nonsulfhydral angiotensin converting enzyme inhibitor. Thirty-nine patients with primary hypertension were entered into a randomized, double-blind protocol to assess the efficacy of enalapril (10 to 20 mg bid), hydrochlorothiazide (25 to 50 mg bid), or combined drug therapy. Enalapril, either alone or in combination with hydrochlorothiazide, effectively controlled blood pressure. Enalapril monotherapy was associated with an increase in plasma renin activity and a decrease in angiotensin II concentration; in patients with an initial inulin clearance less than or equal to 80 mL/min/1.73 m2, inulin and para-aminohippurate clearances were markedly improved, without producing adverse effects on salt and water excretion or body fluid composition. Combination therapy was associated with a marked increase in plasma renin activity; however, only those patients with an initial inulin clearance less than or equal to 80 mL/min/1.73 m2 demonstrated suppression of angiotensin II concentration and marked improvement in inulin and para-aminohippurate clearances. These observations suggest that enalapril, either alone or in combination with a diuretic, has the potential to reverse renal function abnormalities encountered in the hypertensive state. PMID- 2996333 TI - Isolation and subregional mapping of an arbitrary cloned probe detecting a common RFLP on human chromosome 2. AB - In the course of studies on restriction fragment length polymorphisms (RFLPs) being conducted in our laboratory, several single-copy cloned probes have been generated from specific human chromosomes using murine-human hybrid cell libraries. The following describes the isolation and subregional localization of an arbitrary single-copy cloned probe for human chromosome 2. This probe, designated pXG-18, has detected a common TaqI polymorphism in addition to two other RFLPs using the restriction enzymes MspI and HindIII. This sequence maps to the interval q32-q36 of chromosome 2, a region of the human genome to which very few markers have been assigned. PMID- 2996334 TI - Deletion mapping of human chromosome 5 using chromosome-specific DNA probes. AB - A complete genomic DNA library was prepared from a Chinese hamster-human cell hybrid that contains human chromosome 5 as its only human DNA. Unique or low-copy DNA fragments, isolated form recombinant bacteriophage that contained human DNA inserts, were regionally mapped on chromosome 5 using Southern blot analysis of genomic DNA from a series of hybrid cell lines that were selected as having deletions of various portions of 5q. The chromosome 5-specific DNA library, together with a genetic selective procedure allowing the isolation of hybrid cell lines with deletions of virtually any portion of 5q, will provide a means to construct very accurate physical and recombinational maps of this human chromosome. This system represents an excellent opportunity to examine very precisely the relationship between physical and genetic distances for many loci along the length of this autosome. PMID- 2996335 TI - The E7-associated cell-surface antigen: a marker for the 11p13 chromosomal deletion associated with aniridia-Wilms tumor. AB - Unbalanced interstitial deletions of the p13 region of human chromosome 11 have been associated with congenital hypoplasia or aplasia of the iris, mental retardation, ambiguous genitalia, and predisposition to Wilms tumor of the kidney. Utilizing somatic cell hybrids containing either the normal or abnormal chromosome 11 from a child with Wilms tumor and aniridia, we previously mapped the E7 cell-surface antigen to the 11p1300-to-11p15.1 region. To localize even further the site of this antigen on chromosome arm 11p, we have produced somatic cell hybrids from the fibroblasts of a second child with Wilms tumor and aniridia and a different deletion of 11p [46,XY, del (11)(pter----p14.1::p11.2----qter)]. Furthermore, the normal and deleted chromosome 11 could also be distinguished on the basis of a restriction fragment length polymorphism for the beta-globin gene. Hybrid cells containing the deleted chromosome were not killed in the presence of complement and the E7 monoclonal antibody (which recognizes E7 cell surface antigen), while hybrid cells containing the patient's normal chromosome 11 were killed. Thus, expression of the E7-associated cell-surface antigen can be mapped to the 11p13 region, and it appears to be a potential marker of the chromosome abnormality associated with aniridia-Wilms tumor. PMID- 2996336 TI - Determination of the parental origin of sex-chromosome monosomy using restriction fragment length polymorphisms. AB - The parental origin of the single X chromosome in sex-chromosome monosomy was evaluated by comparing restriction fragment length polymorphisms (RFLPs) of 10 spontaneous aborted 45,X conceptions with those of their parents. Seven X-linked marker loci were used, and we were able to specify the origin of the X in nine cases, with six being maternally and three paternally derived. These results demonstrate the efficiency of the technique and show that the single X chromosome in 45,X spontaneous abortions can be derived from either parent. PMID- 2996337 TI - A strategy for using multiple linked markers for genetic counseling. AB - A strategy for using multiple linked markers for genetic counseling is to test sequentially individual markers until a diagnosis can be made. We show that in order to minimize the number of tests performed per case while diagnosing all informative cases the order in which the markers are to be tested is critical. We describe an algorithm to obtain this order using the parameter "I," the frequency of informative cases. The I value for a specific locus used depends on the marker frequency, association with the disease locus, and also on the informativeness of the marker loci already tested. Realizing that a direct assay for the beta S gene already exists, and that most cases of beta-thalassemia in Mediterraneans can be directly diagnosed using synthetic oligonucleotide probes, we illustrate the above technique by examining nine DNA polymorphisms in the human beta-globin cluster for their ability to diagnose sickle-cell anemia in American blacks and beta-thalassemia in Mediterraneans. This analysis shows that 95.39% of all sickle cell pregnancies can be diagnosed by testing a subset of only six markers chosen by our algorithm. Furthermore, six markers can also diagnose 88.03% of beta thalassemia in Greeks and 83.56% of beta-thalassemia in Italians. The test set is different from that suggested by the individual informative frequencies due to nonrandom associations between the restriction sites. PMID- 2996338 TI - Silicosis in slate pencil workers: I. An environmental and medical study. AB - An environmental and medical survey was undertaken in the slate-pencil industry in the central part of India. The industrial hygiene survey revealed that concentrations of free silica dust were very high. The medical survey, involving 593 workers, revealed that the prevalence of silicosis in this industry was 54.6%. Of these, 17.7% of workers had conglomerate silicosis (progressive massive fibrosis, PMF). The radiologic appearance of simple and conglomerate silicosis resembled closely the simple pneumoconiosis and progressive massive fibrosis (PMF) among other occupational groups exposed to free silica and also found in coal workers. The pulmonary lesions were detectable after a relatively short duration of exposure. The short latent period of development and the high prevalence of silicosis observed among these workers are related to exposure to high concentrations of siliceous dust in the work environment. PMID- 2996339 TI - Rapid progression of silicosis in slate pencil workers: II. A follow-up study. AB - Findings of the follow-up examination of slate-pencil workers after 16 months are described. The progression of silicosis with this short duration was very rapid with high mortality among those who had conglomerate silicosis at the initial examination. Twenty-three workers had died during this period at a mean age of only 34.7 years, with a mean duration of exposure of 11.9 years. This high mortality is attributed to exposure to high concentrations of silica dust leading to early onset of PMF at a relatively young age. The progression of silicosis within this period was related to the intensity and duration of dust exposure, and also to the severity of silicosis found at the initial examination. Smoking habits had an adverse, though statistically nonsignificant, effect on the evolution of silicosis. PMID- 2996340 TI - Advances in therapeutics: converting enzyme inhibition and the kidney. PMID- 2996341 TI - Hemodynamic and renal function in essential hypertension during treatment with enalapril. AB - Enalapril (at a mean dose of 25 mg), a potent, long-acting angiotensin converting enzyme inhibitor, was prescribed in combination with hydrochlorothiazide (at a mean dose of 64 mg) for 96 weeks in 11 patients with essential hypertension who had pretreatment (placebo) glomerular filtration rates of less than 80 ml/minute/1.73 m2. Blood pressure was well controlled. After 56 weeks of therapy, glomerular filtration rate (assessed by inulin clearance) increased 55 percent and effective renal plasma flow (assessed by para-amino-hippurate clearance) increased by 32 percent; these increases were sustained through the 96 weeks of therapy. Furthermore, gains in renal function were sustained without adversely affecting 24-hour urinary protein excretion, sodium excretion, or body fluid composition. These results suggest that enalapril, in combination with hydrochlorothiazide, has the pharmacologic capability to favorably modify a primary pathophysiologic characteristic of essential hypertension by decreasing renal vascular and mesangial tone. PMID- 2996342 TI - Comparison of effects of enalapril plus hydrochlorothiazide versus standard triple therapy on renal function in renovascular hypertension. AB - Renal function and antihypertensive drug efficacy were determined in a prospective, double-blind, multicenter study comparing enalapril plus hydrochlorothiazide with standard triple therapy (hydrochlorothiazide, timolol, and hydralazine) in 75 patients with documented renovascular hypertension. Both groups had significant mean decreases in systolic and diastolic blood pressures. Effective control of diastolic hypertension occurred in 96 percent of patients receiving enalapril compared with 82 percent of patients receiving the triple drug regimen. Effective renal plasma flow was significantly increased by enalapril therapy. In contrast, the glomerular filtration rate had a bimodal response. In 80 percent of enalapril-treated patients, there was no significant change in the inulin clearance, although in 20 percent of patients (10), there was a 28 percent decrease in the inulin clearance with a concomitant 12 percent increase in renal plasma flow. Seven of the 10 patients had unilateral renal artery stenosis, but in all 10, it was high-grade stenosis (more than 80 to 90 percent stenosis). Although a significant rise in the serum creatinine level occurred in one patient in association with diuretic therapy, volume repletion reversed this azotemia. No oliguric acute renal failure occurred in the enalapril treated group. The cause of the decrease in glomerular filtration rate induced by enalapril plus hydrochlorothiazide in a minority of patients with renal artery stenosis appears to be quite complex. Although the abolishment of the autoregulation of glomerular filtration secondary to blockage of angiotensin II appears to be a primary cause, the roles of decreased arterial pressure, renal counterbalance, concurrent diuretic therapy, and other hemodynamic factors that may maintain glomerular ultrafiltration pressure must also be considered. The results of this study show that enalapril plus hydrochlorothiazide is effective in treating renovascular hypertension. Special care is needed for a small group of patients with renovascular hypertension in whom there is a decrease in the glomerular filtration rate with this therapy. This may identify a subset of patients with unilateral or bilateral high-grade renal artery stenosis in whom alternative therapy--percutaneous angioplasty or surgical intervention--may be considered. PMID- 2996343 TI - Are non-modulating patients with essential hypertension a distinct subgroup? Implications for therapy. AB - In 40 to 50 percent of the essential hypertensive population, a high intake of sodium does not increase renal blood flow. These patients have been called "non modulators" since their adrenal and renal vascular responses to angiotensin II are not modified by changes in sodium intake. To determine if these patients form a distinct subgroup, the frequency distribution of four characteristics that have been reported to be abnormal in non-modulators were analyzed: aldosterone secretory response to acute volume depletion, plasma aldosterone response to angiotensin II infusion, plasma renin activity response to saline infusion, and renal blood flow response to salt loading. All four characteristics had a bimodal distribution in patients with hypertension. The effect of angiotensin converting enzyme inhibition on two of these abnormalities was also reviewed. In both cases- aldosterone secretory response to angiotensin II and renal blood flow response to salt loading--converting enzyme inhibition restored the abnormal responses towards normal values in non-modulators without altering the responses in normotensives or modulators. Indeed, the correction of the abnormal renal blood flow response to salt loading through converting enzyme inhibition may explain how converting enzyme inhibitors normalize blood pressure in 50 percent of the patients in whom the renin-angiotensin system is suppressed by an unrestricted, typically high, intake of salt. In summary, non-modulators are a distinct subset of the hypertensive population. Converting enzyme inhibition corrects the abnormalities that may be responsible for their hypertensive condition and, therefore, may be a specific form of therapy for these patients. PMID- 2996344 TI - Converting enzyme inhibitor therapy limits progressive glomerular injury in rats with renal insufficiency. AB - Sustained increases in glomerular capillary pressure and flow accompany systemic hypertension in rats that have undergone extensive ablation of the renal mass. These intrarenal hemodynamic changes are, in turn, associated with the progressive development of proteinuria and glomerular sclerosis, leading ultimately to failure of remnant nephron units. The efficacy of antihypertensive therapy with enalapril was evaluated in this animal model of chronic renal insufficiency. A dose of enalapril sufficient to prevent systemic hypertension normalized the glomerular capillary pressure without reducing the glomerular filtration rate in the remnant kidney. Maintenance of normal capillary pressure markedly reduced the development of proteinuria and sclerotic lesions in remnant glomeruli. These results suggest that antihypertensive therapy directed at reducing the glomerular capillary pressure could retard the progressive loss of renal function in patients whose functional renal mass has been reduced by disease. PMID- 2996345 TI - Therapeutic implications of hypertension-induced glomerular injury. Comparison of enalapril and a combination of hydralazine, reserpine, and hydrochlorothiazide in an experimental model. AB - Systemic hypertension does not always reflect concomitant glomerular hypertension. At similar levels of systemic hypertension, glomerular injury occurs only in kidneys that lack protective preglomerular vasoconstriction, which results in glomerular hypertension. indeed, glomerular hypertension and glomerular injury do not develop in rats with spontaneous hypertension that have effective preglomerular vasoconstriction. In the experiments reported herein, the normal adaptive response (afferent arteriolar dilation) to a reduction of one and five-sixths of the renal mass in rats with spontaneous hypertension was examined to ascertain whether that response would expose the remaining nephrons to the injurious effects of high perfusion pressure. In addition, the efficacies of two different antihypertensive regimens were compared. Rats with spontaneous hypertension received either no therapy, or a combination of hydralazine, reserpine, and hydrochlorothiazide, or the angiotensin converting enzyme inhibitor enalapril. Three weeks after ablation of one and five-sixths of the renal mass, blood pressure, glomerular filtration rate, urinary protein excretion, and histologic injury scores for mesangial expansion and glomerulosclerosis were determined. Untreated rats with hypertension had severe glomerulosclerosis and mesangial expansion. Both antihypertensive regimens normalized systemic blood pressure and reduced glomerulosclerosis. However, enalapril was more effective than the combination of hydralazine, reserpine, and hydrochlorothiazide in reducing the exaggerated glomerular filtration rate (0.52 +/- 0.40 versus 0.82 +/- 0.10 ml per minute; p less than 0.05), the injury score for mesangial expansion (79 versus 103; p less than 0.05), and the degree of proteinuria (32 +/- 4 versus 42 +/- 3 mg per 24 hours; p less than 0.05). Persistence of hyperfiltration accompanied by increased mesangial expansion, may lead to progression of glomerular damage despite "adequate" control of systemic hypertension, as observed in rats treated with a combination of hydralazine, reserpine, and hydrochlorothiazide. PMID- 2996346 TI - Infectious complications in heart-lung transplant recipients. AB - Infectious complications were studied in 14 patients who received heart-lung transplants at Stanford University Medical Center from March 1981 to November 1983. Twenty-nine infections occurred in 12 patients: 18 bacterial, nine viral, and two fungal. Sixteen (89 percent) of the bacterial infections occurred in the lung. Because of frequent colonization of the lower respiratory tract, the specificity of transtracheal aspiration and bronchoscopy was low. Empiric broad spectrum antibiotic therapy was usually successful, and no patient died of bacterial infection. Cytomegalovirus infection occurred in six and herpes simplex virus infection in three patients. Two patients had invasive candidiasis at postmortem examination. This series emphasizes the importance of infection, particularly of the lung, in causing morbidity and mortality in heart-lung transplant recipients. PMID- 2996347 TI - Giant cell arteritis associated with mononeuritis multiplex and complement activating 19S IgM rheumatoid factor. AB - Giant cell arteritis is a necrotizing granulomatous arteritis of large arteries, especially the aorta and its branches. Mononeuritis multiplex is a peripheral sensorimotor neuropathy usually producing foot or wrist drop, commonly associated with necrotizing arteritis of small and medium-sized arteries. Rheumatoid vasculitis is an example of the latter type of arteritis associated with high titer 19S IgM rheumatoid factor typically occurring in patients with long standing erosive rheumatoid arthritis. This report describes a 71-year-old man with biopsy-proved giant cell arteritis, mononeuritis (foot drop) multiplex, and high-titer complement-activating rheumatoid factor without rheumatoid arthritis. Possible pathogenic relationships are discussed. PMID- 2996349 TI - The insidious development of symptomatic secondary hormone syndromes in patients with malignant endocrine tumors. AB - Endocrine tumors may produce secondary or "ectopic" hormones that cause paraneoplastic syndromes. Such syndromes may be confused with more common complications related to a patient's tumor, and thus escape detection and appropriate treatment. The secondary hormone secretion responsible for these syndromes often occurs late in the course of such diseases and presents in an insidious manner. Two patients are presented that illustrate these points. The first, a woman with medullary carcinoma of the thyroid (MCT), developed a syndrome secondary to ACTH secretion that was confused initially with the changes caused by the massive diarrhea that accompanies MCT. The second, a man with malignant glucagonoma, is the first with this disease to have developed symptomatic hyperinsulinemia as a late complication. We stress the clinical courses of these patients and note that treatment of these syndromes may improve the quality of patients' lives. PMID- 2996348 TI - Lactic acidosis with small cell carcinoma. Rapid response to chemotherapy. AB - A patient presented with widespread small cell carcinoma, abnormalities on liver function tests, and rapidly progressive lactic acidosis. The acidosis was totally corrected following chemotherapy. Prompt treatment in tumor-associated lactic acidosis may significantly prolong survival. PMID- 2996350 TI - Minute chromosomes replacing the Y chromosome carry Y-specific sequences by restriction fragment analysis and in situ hybridization. AB - Two unrelated males, a 43-year-old man with azoospermia and a 4-year-old boy with stature at the 10th centile, had similar karyotypes: 46,X,min. The minutes, present in all cells analyzed, stained weakly with G-, C-, and Q-banding methods. To elucidate their origin we used molecular techniques: In HaeIII digests of total genomic DNA from both individuals, no Y-specific reiterated sequences were detected. However, restriction fragment analysis with probe pDP31 demonstrated that the patients' DNA contained the Y-specific fragment. In situ hybridization with the same probe showed that these sequences were present on the minute chromosomes and have not been translocated elsewhere. PMID- 2996351 TI - Paradoxical excitement to sedative-hypnotics in mentally retarded clients. AB - The relationships among "paradoxical" excitement to sedative--hypnotic medication, self-injurious behavior, and perinatal trauma were evaluated. Mentally retarded patients were classified as either paradoxical or normal responders to sedative-hypnotics. Paradoxical responders to these medications have a lower MA, a history of perinatal trauma, self-injurious behavior (SIB), and aggressive behavior when compared to normal responders. These findings confirmed and extended previous reports that a type of SIB may be indexed by paradoxical response to sedative-hypnotics. Results also suggested that perinatal trauma may be of etiological importance in the development of SIB. Because perinatal trauma or fetal distress results in excessive levels of B-endorphin, in utero, an impaired endogenous opiate system may be a critical factor maintaining a syndrome of SIB. Thus, these data may indicate psychopharmacological markers of SIB that may have both treatment and etiological significance. PMID- 2996352 TI - Inhibition of human platelet aggregation by parathyroid hormone. Is cyclic AMP implicated? AB - Parathyroid hormone (PTH) is a polypeptide which in different in vitro systems raises intracellular cyclic AMP (cAMP) levels via adenyl cyclase activation and stimulates Ca2+ transport across cell membranes. We tested whether, on the basis of this mechanism, PTH would inhibit human platelet aggregation. The latter was tested in vitro by a photometric technique. Platelet aggregation induced by the calcium ionophore A 23187 was inhibited by PTH at concentrations (0.5-3 USP U/ml) similar to those effective in other in vitro systems. Higher concentrations of PTH were required to prevent aggregation initiated by adenosine-5'-diphosphate, arachidonic acid, or platelet-aggregating factor. The terminal synthetic fragment 1-34 b PTH was ineffective against all aggregation stimuli. The antiaggregating effect of PTH was potentiated by verapamil and theophylline and was additive to that of PGI2. However, PTH did not appear to increase platelet cAMP levels and was not counteracted by an inhibitor of platelet adenyl cyclase. It is therefore unlikely that PTH inhibits platelet aggregation through an adenyl cyclase stimulated increase of cAMP. Since PTH levels are markedly increased in uremic plasma, it might contribute to the defective platelet function and the bleeding tendency frequently occurring in uremic patients. PMID- 2996353 TI - HTLV-III AIDS link. PMID- 2996354 TI - Risk factors for gestational trophoblastic neoplasia. AB - A case-control study to determine the gynecologic and reproductive risk factors for gestational trophoblastic neoplasia was conducted in the Baltimore Metropolitan Area. All cases (N = 190) that were pathologically diagnosed from 1975 to 1982 as hydatidiform mole, invasive mole, or choriocarcinoma were ascertained. Slides were independently reviewed by two pathologists. Cases were matched by age, race, and last menstrual period to controls who were delivered of normal pregnancies at term. In the analysis of medical record and interview data, factors found to be positively associated with gestational trophoblastic neoplasia included professional occupations (odds ratio = 2.56, p less than 0.0001), prior spontaneous abortions (odds ratio = 2.32, p = 0.02), and the mean number of months from the last pregnancy to the index pregnancy (cases = 35.9, controls = 28.2; p = 0.03). Factors found not to be associated with disease included contraceptive history, irradiation, ABO blood group, and smoking factors of the male partner. The findings suggest that gestational trophoblastic neoplasia may be part of a continuum of early (first-trimester) reproductive abnormalities. PMID- 2996355 TI - Propranolol inhibits the maturational effect of adrenocorticotropin in the fetal sheep lung. AB - We reported previously that metyrapone inhibited the maturational effect of adrenocorticotropin in the fetal sheep lung, even in the presence of exogenous glucocorticoids. To examine the role of beta-adrenergic input in this response we examined lung maturation in fetal sheep treated for 100 hours in vivo with adrenocorticotropin (66 ng/min for 15 minutes every 2 hours, n = 5); adrenocorticotropin plus propranolol (40 micrograms/min, n = 4), or saline solution (n = 8). Pulmonary maturation was assessed by pressure-volume curves, phospholipid content, and morphologic features. The basal cortisol level rose from less than 5 to 32.0 +/- 12.1 and 83.5 +/- 8.0 ng/ml in the adrenocorticotropin and adrenocorticotropin plus propranolol groups, respectively. The adrenal:body weight ratio (X 10(-4)) rose from 1.43 +/- 0.12 in the saline solution group to 2.90 +/- 0.16 and 2.51 +/- 0.14 in the adrenocorticotropin and adrenocorticotropin plus propranolol groups, respectively. Lung distensibility (milliliters of air per gram of lung) rose from 1.10 +/- 0.14 to 1.90 +/- 0.20 in the adrenocorticotropin group but was unchanged (0.98 +/- 0.24) in the adrenocorticotropin plus propranolol group. Phosphatidylcholine (milligrams per gram of lung) in the lung lavage rose from 0.07 +/- 0.02 to 0.23 +/- 0.11 in the adrenocorticotropin group but was not significantly changed (0.12 +/- 0.06) in the adrenocorticotropin plus propranolol group. We conclude that propranolol inhibits the maturational effects of adrenocorticotropin on the fetal lung, which implies that the mechanism of pulmonary maturation is not solely dependent on endogenous cortisol and must be mediated, at least in part, through adrenergic responses. PMID- 2996356 TI - Human T-cell leukemia/lymphotropic virus type III in the conjunctival epithelium of a patient with AIDS. AB - The human T-cell leukemia/lymphotropic virus type III (HTLV-III), the causative agent of the acquired immunodeficiency syndrome (AIDS), has been isolated from the conjunctival epithelium of a 33-year-old woman with AIDS, suggesting that an important reservoir of the virus may be the ocular surface epithelial cells. The tears and conjunctival epithelium from normal controls were negative for HTLV III. The finding of HTLV-III in the tears and conjunctival epithelium indicated that HTLV-III may be ubiquitous in bodily cells and fluids. Repeated contact with the tears and ocular surface epithelium of patients with AIDS may possibly facilitate transmission of HTLV-III, and precautions are advisable during routine ophthalmologic procedures such as glaucoma testing and contact-lens fitting. PMID- 2996357 TI - The impact of the AIDS epidemic on corneal transplantation. PMID- 2996358 TI - Production of a human monoclonal antibody to HLA by human-human hybridoma technology. A preliminary report. AB - An Epstein-Barr-virus-transformed lymphoblastoid cell line (ECEBV) was derived from a multiply transfused renal dialysis patient. ECEBV was shown to secrete specific antibody in a cellular enzyme-linked immunosorbent assay (CELISA) and was hybridized with the mutagenized human fusion partner G M1500 resistant to 6 thioguanine and ouabain. Hybridomas surviving hypoxanthine-aminopterin-thymidine (HAT) and ouabain selection were cloned by limiting dilution. The hybridomas continue to secrete antibody which reacts with some human cells but not with others after 14 months in culture. None reacts with K562 (no HLA-A, -B, -C or DR) or with Daudi (no HLA-A, -B, or -C). This is a preliminary report of the production of a human monoclonal antibody to HLA. Application of this technique could result in the large-scale production of human monoclonal antibodies for HLA typing, the production of anti-idiotype antibodies for use in transplant patients to prevent acute rejection, and for the study of the structure and function of HLA in man. PMID- 2996359 TI - Aortic endothelial cell death and replication in normal and lipopolysaccharide treated rats. AB - Endothelial cell death and replication was analyzed in the rat aorta with the use of combined IgG immunocytochemistry and 3H-thymidine autoradiography. Dead and replicating cells were clustered in the aortic endothelium of normal rats with a low level of spontaneous cell death and replication. Death and replication were also clustered in rats treated with endotoxin (Escherichia coli lipopolysaccharide [LPS]), a substance which is cytotoxic to endothelial cells in culture. LPS caused a dramatic increase both in endothelial cell death and replication, while no endothelial denudation could be discerned with the use of the 111In-labeled platelet technique. We conclude that endothelial cell death and replication are topographically clustered phenomena, which may imply that cell death leads to a local stimulation of cell replication. Furthermore, LPS induces a massive cell death, which is paralleled by cell replication, yet does not cause any significant increase in denudation. This suggests that the primary effect of LPS on the vessel wall is its cytotoxicity for endothelial cells. Endothelial cell death appears to provide the stimulus for cell replication both in normal and in LPS-treated rats. PMID- 2996361 TI - Thermogenesis and BAT activity in hypophysectomized rats with and without corticotropin replacement. AB - Surgical hypophysectomy (Hypox) suppressed body weight, body energy gain, and net energetic efficiency when compared with pair-fed, sham-operated controls but caused marked increases in the thermic response to food, brown adipose tissue (BAT) mass, protein content, and BAT thermogenic activity (estimated from mitochondrial purine nucleotide binding). Chronic replacement of Hypox rats with ACTH restored adrenal weight, plasma corticosterone levels, body energy gain, energetic efficiency, and the thermic response to food to normal. However, the Hypox-replaced animals were apparently fatter (greater carcass energy density) and retained a greater mass of brown fat than controls. Purine nucleotide binding to BAT mitochondria was lower in the ACTH-replaced group than in Hypox rats but remained above the level in controls. These data indicate that hypophysectomy stimulates thermogenesis, probably as a result of decreased adrenal steroid release. However, ACTH does not totally prevent the effects of Hypox, and an involvement of other pituitary hormones and/or hypothalamic-releasing factors cannot be excluded. PMID- 2996363 TI - By 94 days of gestation plasma cortisol increases block ACTH response to hypotension in lamb fetuses. AB - By use of a crossover design, we studied the effects of increasing plasma cortisol concentration on ACTH responses to a standardized stress in 14 lamb fetuses between 94 and 108 days gestation. On a random basis we assigned the animals into two groups of seven. Animals in groups I and II received infusions of cortisol (5 and 1 microgram/min, respectively) or saline for 4 h. After the cortisol or saline pretreatment, we reduced arterial pressure approximately 40 50% in both groups of animals with nitroprusside. After saline pretreatment, hypotension in the group I animals produced an increase in the fetal plasma ACTH from 15 +/- 3 to 200 +/- 20 pg/ml (P less than 0.001), and in the group II animals pretreated with saline plasma ACTH increased from 21 +/- 4 to 141 +/- 19 pg/ml (P less than 0.001) with hypotension. Cortisol pretreatment elevated fetal plasma cortisol levels from 7 +/- 3 to 36 +/- 5 ng/ml in group I and from 8 +/- 4 to 20 +/- 2 ng/ml in group II. The ACTH response to hypotension in both groups was abolished by the cortisol pretreatment. We conclude that by 94 days gestation increases in plasma cortisol within a physiological range block ACTH responses to hypotension in lamb fetuses. PMID- 2996360 TI - Collagenase, elastase, and nonspecific protease production by vigorous or immunomodulated liver granulomas and granuloma macrophages/eosinophils of S mansoni-infected mice. AB - Schistosomiasis mansoni is characterized by granulomatous inflammations, deposition of collagen, and irreversible liver fibrosis. In chronic infections fibrosis is cumulative, while collagen synthesis and degradation are diminished. In the present study, we compared collagenase, elastase, and nonspecific neutral protease (NP) activities in isolated vigorous (8-week infection) and immunomodulated (18-20-week infection) liver granulomas. Enzyme activity was localized in the adherent macrophage (greater than 90% purity) and nonadherent eosinophil-rich (greater than 70% purity) cell fractions of the disaggregated granulomas. Collagenase levels were approximately two times higher in granuloma extracts and explant culture supernates of the vigorous as compared with the immunoregulated lesions. However, macrophages and eosinophil-rich cells derived from either type of granuloma secreted similar enzyme levels. Elastase and NP levels in granuloma extracts and secretions of adherent macrophage and eosinophil rich cell populations were the same in vigorous or immunomodulated lesions but were significantly greater in vigorous granuloma culture supernates. In addition to active collagenase, trypsin activatable latent collagenase was also present in both types of granuloma extracts, explants and eosinophil-rich cell culture supernates. Latent elastase was also detected in granuloma extracts or explant supernates but was absent from secretions of granuloma cells. These results suggest that the presence of active neutral proteases within granulomas may play an important role in the regulation of tissue repair and remodeling during the fibrotic process. PMID- 2996362 TI - Calcium, cAMP, and dopamine affect single mammotrophs assayed by hemolytic plaques. AB - We investigated the effects of dopamine, forskolin, and maitotoxin on prolactin release from individual rat and monkey mammotrophs. Forskolin increases cAMP levels, whereas maitotoxin amplifies the influx of extracellular calcium. Prolactin secretion from single mammotrophs was visualized by the reverse hemolytic plaque assay and then quantified by measuring the proportion of plaque forming cells and the mean plaque area. In the presence of dopamine alone both the plaque proportion and mean area of the plaques formed by rat mammotrophs decreased by 50 and 40%, respectively. This inhibition of secretion was blocked by the dopaminergic antagonist spiperone as well as forskolin and maitotoxin. Forskolin and maitotoxin alone significantly elevated both the proportion (+30%) and area (+180% for forskolin and +250% for maitotoxin) of the plaques. These actions of maitotoxin were neutralized by D-600, a calcium channel blocker. All of these agents induced similar trends with monkey prolactin cells. We conclude that single mammotrophs in culture respond to perturbations in a differential manner and in a way predicted by earlier results based on macropopulation measurements. PMID- 2996364 TI - Differential effects of forskolin on LH and GH release. AB - The effects of forskolin, an agent which increases intracellular levels of cAMP, on basal luteinizing hormone (LH) and growth hormone (GH) release and on gonadotropin-releasing hormone (GnRH)-stimulated LH release were documented. Continuously perifused dispersed anterior pituitary cells from female rats at random stages of the estrous cycle were used. Secretory rates of both LH and GH increased in a concentration-dependent manner in response to a 1-h challenge with 0.03, 0.1, 0.3, 1, or 3 microM forskolin. In response to 0.3 microM forskolin, maximum GH release was achieved within 15-20 min, after which secretion decreased. In contrast, LH release increased gradually, became maximal at 1.5-2 h, and remained constant until the forskolin was withdrawn. Cells exposed to 10 nM GnRH for 4 h exhibited a biphasic release of LH with the interphase nadir occurring at 30 min. The second phase of LH release was enhanced by simultaneous addition of forskolin with the GnRH. Whereas second phase release did not increase further, exposure of the cells to forskolin for 60 or 120 min before GnRH resulted in increased first-phase LH release. We suggest that, whereas our data are consistent with a role for cAMP in mediating the acute release of GH, cAMP may be involved in the process through which nonimmediately releasable LH becomes available for release. PMID- 2996365 TI - Contractile properties of cultured glomerular mesangial cells. AB - The glomerular mesangium is composed of matrix material and at least two cell types. One is a bone marrow-derived phagocyte and the other is a smooth muscle like cell. The phagocytic cell represents approximately 3-7% of the total mesangial cell population. The other, more abundant, cell type appears to be contractile and therefore has been proposed to play a role in regulating the surface area for filtration, one component of the ultrafiltration coefficient, Kf. In this review we discuss the contractile properties of cultured mesangial cells as well as the phenotypic alterations that lead to loss of isotonic contraction after prolonged culture. PMID- 2996366 TI - Alpha and beta types of carbonic anhydrase-rich cells in turtle bladder. AB - The carbonic anhydrase-rich (CA) cell population of the turtle urinary bladder, which is responsible for the secretion of H+ and probably of HCO-3, was studied by freeze-fracture and thin-section electron microscopy. The apical membrane of the major CA cell type (alpha type) was characterized by microplicae and by a coat of studs on its cytoplasmic side; on freeze-fracture, it contained a dense population of rod-shaped intra-membrane particles. When fixed at low CO2 tension, the apical membrane area of the alpha cell was reduced; its surface displayed microplicae as well as microvilli, and the apical cytoplasm contained many vesicles with rod-shaped particles and studs. The apical membrane of the other (beta type) CA cell was characterized by numerous individual microvilli without microplicae and by a relative absence of rod-shaped particles and studs. Instead, the beta cell contained studs and rod-shaped particles in its basolateral membrane. The ultrastructure and frequency of the beta CA cell were not affected by changes in CO2 tension. We suggest that the alpha cell is responsible for H+ secretion. The reversal of the polarity of the membrane elements in the beta cell and failure to respond to CO2 with amplification of its apical membrane are consistent with a role in HCO-3 secretion. PMID- 2996367 TI - Effect of metabolic acidosis on the PTH receptor-adenylate cyclase system of canine kidney. AB - The phosphaturic action of parathyroid hormone (PTH) is blunted during metabolic acidosis. Previous studies suggest that the activation of renal cortical adenylate cyclase by PTH is decreased under this condition. However, the mechanisms underlying the defect are not completely defined. The present studies were designed to examine the interaction of PTH with its receptor-adenylate cyclase system in basolateral cortical membranes from dogs with metabolic acidosis. Chronic metabolic acidosis was induced in seven normal dogs. Venous blood pH decreased to 7.21 +/- 0.01 and serum bicarbonate to 12.58 +/- 0.32 meq/liter. In seven control dogs blood pH was 7.38 +/- 0.002 and serum bicarbonate was 20.14 +/- 0.26 meq/liter. The kidneys were surgically removed and basolateral membranes were prepared by differential centrifugation and ultracentrifugation in discontinuous sucrose density gradients for studies of adenylate cyclase activity and hormone-receptor binding. Metabolic acidosis resulted in a significant decrease in PTH-dependent adenylate cyclase activity (Vmax 2,119 +/- 150 pmol cAMP X mg prot-1 .30 min-1 vs. 3,548 +/- 116 in the controls). The PTH concentration giving half-maximal activation of adenylate cyclase was unchanged. However, PTH-receptor binding showed similar affinity and binding capacity in both groups of membranes. Basal enzyme activity was also similar. In the presence of the GTP analogue 5'-guanylylimidodiphosphate, PTH dependent adenylate cyclase activity remained markedly decreased in the acidotic dog membranes compared with the controls. The ability of NaF to stimulate enzyme activity was also depressed in the membrane of acidotic dogs. Enzyme activity in the presence of Mn2+ was similar in the two groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996368 TI - Transport of citrate across renal brush border membrane: effects of dietary acid and alkali loading. AB - Dietary acid or alkali loading was given to rats by providing 150 mM NH4Cl or 150 mM NaHCO3 in place of drinking water for 6 days; control animals received 150 mM NaCl. After 6 days, the citrate clearance was 0.04 +/- 0.01 ml/min (mean +/- SE) in the acid-loaded group, 0.9 +/- 0.1 ml/min in the control group, and 2.5 +/- 0.2 ml/min in the alkali-loaded group. At the end of the experiment, the rats were killed, and the Na+ gradient-dependent (Nao+ greater than Nai+) citrate uptake (pmol/mg protein) was measured in brush border membrane (BBM) vesicles prepared from each group. At 0.3 min, the [14C]citrate uptake was 198 +/- 8 pmol/mg protein (mean +/- SE) in the acid-loaded group, 94 +/- 16 pmol/mg protein in the control group, and 94 +/- 13 pmol/mg protein in the alkali-loaded group. The rate of Na+-independent (NaCl in medium replaced by KCl) [14C]-citrate uptake by BBM vesicles was the same for acid-loaded, control, and alkali-loaded animals. Thus, the increased capacity of the proximal tubular BBM to transport citrate from the tubular lumen into the cell interior may be an important factor that contributes to decreased urinary citrate in the presence of metabolic acidosis induced by chronic dietary acid loading. PMID- 2996369 TI - Effect of insulin on renal phosphorus handling in the rat: interaction with PTH and nicotinamide. AB - Previous studies suggested an antiphosphaturic action of insulin. However, effects of parathyroid hormone (PTH), anti-natriuresis, or other variables were not vigorously controlled. Recently it has been suggested that nicotinamide restores phosphaturia in several antiphosphaturic states. Clearance studies were therefore performed in acutely parathyroidectomized rats to test the hypothesis that insulin abolishes the phosphaturic action of PTH and that this effect is prevented by nicotinamide. Superimposition of euglycemic hyperinsulinemia on PTH infusion (plasma insulin 19.1 +/- 2.7 vs. 9.8 +/- 1.1 microU/ml, P less than 0.05) during steady-state Pi excretion decreased fractional excretion (FE) of Pi compared with PTH-infused controls (3.16 +/- 0.61 vs. 18.02 +/- 0.81%, P less than 0.001). Renal cortical NAD+ was lower in the former than the latter group (455 +/- 22 vs. 689 +/- 30 pmol/mg, P less than 0.01). Nicotinamide pretreatment prevented the antiphosphaturic action of insulin and the decrease in cortical NAD+ in both the presence and absence of exogenous PTH. These studies offer direct evidence that in acutely parathyroidectomized rats insulin can abolish the phosphaturic action of PTH, independent of glomerular filtration rate, the filtered loads of Pi and glucose, FENa+, and cAMP excretion, an effect that is prevented by nicotinamide pretreatment. In the absence of nicotinamide pretreatment, superimposition of insulin on PTH infusion was associated with a decrease in renal cortical NAD+. A role for intracellular NAD+, probably indirect, in the antiphosphaturic action of insulin is suggested. PMID- 2996371 TI - Renal hypertension impairs inotropic isoproterenol effect without beta-receptor changes. AB - Experiments were performed in male Wistar rats with renovascular hypertension (167 +/- 4.2 mmHg) produced by clipping the renal artery for a 3-wk period (2 kidney, 1-clip Goldblatt). The results were compared with those obtained in age matched normotensive controls. Hypertension of 3-wk duration elicited a significant increase in ventricular weight (1.01 +/- 0.02 g) with respect to the controls (0.82 +/- 0.01 g) but had no significant effect on body weight. The inotropic responsiveness to beta-adrenergic stimulation was diminished in papillary muscles from renal hypertensive rats: the maximum increase in the maximal rate of rise of tension produced by isoproterenol was 27.39 +/- 5.4 and 11.77 +/- 2.91 g X mm-2 X s-1 (P less than 0.05) in control and hypertensive animals, respectively. Similar results were obtained when the estimated maximal velocity of shortening of the contractile element (Vmax) was used to assess myocardial contractility. The inotropic response to CaCl2 was also significantly depressed in the 2-kidney, 1-clip rats. However, the relaxant and the chronotropic responses to isoproterenol were not significantly modified in the Goldblatt rats. Assays of beta-adrenergic receptors to l-[3H]dihydroalprenolol binding, showed no significant changes in the number (expressed per mg of membrane protein) or in the affinity of the beta-receptors. These results suggest that at an early stage of the renal hypertensive model the impaired inotropic response to isoproterenol is not mediated by an alteration of the beta-receptors and should be searched at a postreceptor adenyl cyclase level. PMID- 2996370 TI - Positive inotropic effect of acetylcholine in canine cardiac Purkinje fibers. AB - The purpose of this study was to investigate possible mechanisms to explain the positive inotropic effects of acetylcholine in canine cardiac Purkinje fibers. Action potentials and tension were recorded from Purkinje fibers in vitro using microelectrodes and a force transducer. Acetylcholine (10(-9) to 10(-4) M) produced a dose-dependent increase in tension that was blocked by atropine but not by propranolol, phentolamine, hexamethonium, or verapamil. At 10(-5) and 10( 4) M, acetylcholine increased action potential duration at 50% of repolarization (APD50) but did not affect resting membrane potential, action potential amplitude, Vmax, or action potential duration at 90% of repolarization (APD90). Isoproterenol (10(-7) M) shortened APD50 and APD90 and increased developed tension. Subsequent addition of acetylcholine (10(-5) M) prolonged APD50 and APD90 and decreased tension. Increasing extracellular Ca2+ concentration [( Ca2+]o) from 2.0 to 3.0 mM increased tension and shortened APD50. Addition of acetylcholine (10(-5) M) increased tension further and prolonged APD50. In K+ depolarized fibers high concentrations of acetylcholine (10(-4) M) restored excitability, but lower concentrations (10(-6) M) suppressed slow responses induced by isoproterenol. Thus acetylcholine alone or with elevated [Ca2+]o increased APD50 and tension and facilitated the induction of slow responses, yet in the presence of isoproterenol acetylcholine increased APD50, decreased tension, and suppressed slow responses. These effects were mediated by muscarinic receptors and were independent of catecholamine release. PMID- 2996373 TI - Signet-ring-cell thyroid tumors. Follicle cell tumors with arrest of folliculogenesis. AB - This study describes three carcinomas and three benign follicle-cell lesions that consisted of vacuolated thyrocytes. In five of these tumors the neoplastic cells, wholly or in part, had the same unique signet-ring-cell feature as recently described for a mucinous microfollicular thyroid adenoma case. On electron microscopy, however, intracellular lumina that were bordered by microvilli were shown. Serial sections of the epon-embedded material and the results of mucin stains and thyroglobulin-immunostaining excluded primary mucin-producing tumors of the thyroid and in each of our six thyroid lesions gave evidence of an arrest of folliculogenesis. PMID- 2996372 TI - Age-related changes of action potential plateau shape in isolated human atrial fibers. AB - Because the frequency of atrial arrhythmia increases dramatically with age in humans, we investigated age-related changes of cellular electrical activity in human atrial fibers, utilizing standard microelectrode techniques. Twenty-four atrial samples, uniformly exhibiting fast responses, were selected. Patients were in two different age groups: 10.0 +/- 7.0 mo and 54.4 +/- 9.7 yr. Although mean maximum diastolic electrical potential did not significantly differ, the two groups showed marked dissimilarities in action potential (AP) shape. Adult-type APs always exhibited an initial notch followed by a low-level prolonged plateau, whereas APs of the young were more triangular and had a short plateau approaching zero potential. 4-Aminopyridine (0.5 mM) markedly increased the level of the adult AP plateau without suppressing the initial notch, which disappeared after further addition of caffeine (10 mM). The simultaneous action of the two drugs changed an adult-type AP into a young type. The drug effects were much less marked in young atria. Our results suggest that an age-related increase in transient outward currents can account for the differences in plateau shape described. Moreover, age-related differences in plateau configuration in response to a period of rest suggest a faster repriming kinetic of these currents in adults. PMID- 2996375 TI - Syringocystadenoma papilliferum. A plasmacytotropic tumor. AB - Seven cases of syringocystadenoma papilliferum were studied by immunohistochemical methods for the presence of IgG, IgA, IgM, and secretory component in tumor epithelial cells and IgG, IgA, and IgM in plasma cells underlying the tumor epithelium. Six of seven cases showed IgA positivity within epithelial cells, one case showed faint intraepithelial IgG staining, and none stained for IgM. Four of five cases were positive for secretory component. The plasma cells were predominantly of the IgG and IgA class. These findings suggest that the association of plasma cells with this tumor is a consequence of epithelial attraction via a mechanism similar to that utilized by glands of the normal secretory immune system. PMID- 2996374 TI - Cellular differentiation in ovarian sex-cord-stromal and germ-cell tumors studied with antibodies to intermediate-filament proteins. AB - Seventy ovarian sex-cord-stromal and germ-cell tumors were immunohistochemically studied for the presence of intermediate-filament proteins of different types used as markers for cellular differentiation. Cells of ovarian granulosa-cell tumors constantly expressed vimentin and appeared to lack cytokeratin. Two tumors previously classified as granulosa-cell tumors were reclassified as poorly differentiated "common" epithelial tumors based on their cytokeratin positivity, vimentin negativity, and morphologic features. Dysgerminomas and Leydig-cell tumors showed only vimentin positivity. Tubular structures in androblastomas, which are considered to represent Sertoli-cell differentiation, were cytokeratin positive, and thus differed from the majority of normal Sertoli cells that are known to express vimentin and not cytokeratin. Embryonal carcinomas, choriocarcinomas, and endodermal sinus tumors showed cytokeratin positivity in the neoplastic cells whereas vimentin was observed in the stromal cells. In immature teratomas, epithelial differentiation was demonstrated with cytokeratin antibodies, and neural and glial differentiation was also frequently demonstrated by immunostaining with antibodies to neurofilaments and glial fibrillary acidic protein. The results show that antibodies to intermediate filaments can be used in the differential diagnosis between ovarian epithelial and nonepithelial tumors, and they provide a very accurate additional method to characterize the cellular differentiation of ovarian neoplasms. PMID- 2996376 TI - Signet-ring cell lymphoma of T-cell origin. An immunocytochemical and ultrastructural study relating giant vacuole formation to cytoplasmic sequestration of surface membrane. AB - In contrast to previous accounts of signet-ring lymphoma as a B-cell neoplasm, we report a case of signet-ring, large-cell lymphoma of T-cell lineage. Immunologic and ultrastructural studies were performed on a subcutaneous mass noted initially, as well as on an enlarged lymph node that developed later, in a 69 year-old man. Immunologic assessment indicated strong expression of T-helper antigen (Leu 3a + b), universal T-antigens (Leu 1, 5), and Ia. There was an absence of T-suppressor/cytotoxic antigen (Leu 2a), universal T-antigens (Leu 4, 9), and immunoglobulin light and heavy chains. Collectively, these findings indicate a mature T-cell lymphoma of T-helper type in an activated (Ia+) state. In contrast to previous reports of T-cell and Ia occurring solely as surface antigens, we demonstrated pools of cytoplasmic Leu 1, 3, 5 and Ia that displaced the nucleus. The ultrastructure of the giant cytoplasmic vacuoles was identical to the microvesicle-containing vacuoles reported in signet-ring cell lymphomas of B-cell lineage. In our case of T-cell lineage, we found substantial evidence of endocytosis by the neoplastic cells and numerous giant multivesicular bodies. The pools of cytoplasmic T and Ia antigens may result from abnormal internalization of surface T-antigens or the sequestration of T-antigen-containing Golgi-derived vesicles. Our combined immunologic and ultrastructural findings suggest that aberrant membrane recycling may be the common denominator of signet-ring formation in both B- and T-cell signet-ring lymphomas. PMID- 2996377 TI - Cancer of the minor salivary glands of the larynx. AB - Minor salivary gland carcinomas of the larynx are rare and few large series have been reported from a single institution. Eighteen patients were treated for this disease at M.D. Anderson Hospital between 1944 and 1982. Of these, 8 patients had adenoid cystic carcinoma and 10 had poorly differentiated adenocarcinoma. Characteristically these tumors presented as predominantly submucosal masses in the supraglottic or subglottic regions. Surgery was the primary treatment modality used in most cases. The average 2 and 5 year survival rates for patients with this disease were 70.6 percent and 42.8 percent, respectively. Although the 5 year survival rates were comparable between the adenocarcinoma and cystic carcinoma groups, adenocarcinoma was a more rapidly lethal disease than adenoid cystic carcinoma. Salvage after recurrence was seldom possible, although local and regional control could usually be achieved. Distant metastases remain the principal cause of treatment failure. PMID- 2996378 TI - gamma-Linolenic acid fails to prevent the effects of prenatal ethanol exposure on brain and behavioral development in B6D2F2 mice. AB - The purpose of this study was to obtain a quantitative assessment of the behavioral retardation caused by prenatal ethanol exposure in mice and to test the hypothesis that gamma-linolenic acid (GLA) supplementation would prevent such effects. Pregnant B6D2F1 mice were fed liquid diets containing 25% ethanol derived calories from days 7-17 of gestation. The experimental groups were given GLA via subcutaneous injection; the control was administered vehicle only. All groups were pair fed to this ethanol control, including a second control group which received sucrose substituted isocalorically for ethanol. Additional control groups included one fed lab chow ad libitum and two further ethanol groups, one treated with coconut oil, the other with arachidonic acid (AA). Behavioral development of the pups was measured on day 32 postconception and open field behavior was measured on day 50. Body and brain weight were also measured. The results indicated that reproductive outcome, as measured by animals which produced live pups, was worse in the GLA- and AA-treated groups. Ethanol produced significant behavioral retardation of the order of 1.7 days. Body and brain weight were lower in ethanol-treated pups. Covariance analysis indicated that the effect on brain weight was independent of the effect on body weight. Open field scores suggested that ethanol-treated males were more active then sucrose controls. The data did not support the hypothesis that GLA would prevent the deleterious effects of prenatal ethanol exposure; in no instance was a GLA treated group different from the ethanol control. PMID- 2996379 TI - Production of leukotrienes in human skin and conjunctival mucosa after specific allergen challenge. AB - The production of leukotrienes has been monitored in tear fluids from subjects following a conjunctival provocation test, and skin blister fluids following initiation of a Prausnitz-Kustner reaction. In tear fluids elevated levels of leukotriene C4 (LTC4)-immunoreactive material were measured following allergen challenge as compared to control tear fluid obtained by mechanical or reflex stimulation. Analysis by high performance liquid chromatography indicated the presence of LTC4, LTD4 and LTE4. In the skin, significantly elevated levels of LTC4-immunoreactive material were measured following allergen challenge in the Prausnitz-Kustner reaction. HPLC analysis indicated the presence of both LTC4 and LTD4. LTB4 immunoreactive material was detected both in the tear fluid and the skin tissue fluid. However, no significant increase occurred in either tissue after the allergic reactions. These results indicate that the SRS-A leukotrienes are released in vivo in man following allergen challenge, and indicate these mediators may be important in human allergic diseases. PMID- 2996381 TI - The effect of zinc, arginine, fructose and seminal supernatant of normal semen on the triple adenosine triphosphatase activities of the spermatozoa from males with oligoasthenozoospermia. AB - When the spermatozoa of oligoasthenozoospermic patients were suspended with the supernatant of normal semen, an increase in triple ATPase enzyme activities besides enhancement of spermatozoa motilities were observed. This suggests that factor or factors present in the supernatant of normal semen that effects spermatozoa motility also have a positive effect on triple ATPase enzyme activities. In an attempt to produce such an effect, zinc, arginine and fructose were added to the incubation media where the spermatozoal ATPase enzyme activities were determined. Zinc increased Ca2+-Mg2+ ATPase enzyme activity without affecting Na+/K+-Mg2+ and Mg2+ ATPase activities. Triple ATPase enzyme activities remained unchanged after arginine and fructose additions. As a result zinc is thought to be one of the factors that affect spermatozoa motility in seminal plasma. PMID- 2996380 TI - The fasciculus longitudinalis medialis in the lizard Varanus exanthematicus. 2. Vestibular and internuclear components. AB - In the present study the vestibular components of the fasciculus longitudinalis medialis (flm) were investigated in the lizard Varanus exanthematicus with various tracing techniques: anterograde transport of horseradish peroxidase to study vestibulo-oculomotor and vestibulospinal projections, the multiple retrograde fluorescent tracer technique for the cells of origin of such projections. Internuclear projections between the oculomotor and abducens nuclei could also be studied in this way. Rather extensive vestibulo-ocular projections passing via the flm were demonstrated. Mainly ipsilateral ascending projections arise in the dorsolateral vestibular nucleus, mainly contralateral ascending projections in the ventromedial vestibular nucleus and adjacent parts of the ventrolateral and descending vestibular nuclei. Furthermore, distinct bilateral ascending projections of the nucleus prepositus hypoglossi were demonstrated. Extensive vestibulospinal projections pass via the flm and form the medial vestibulospinal tract. This largely contralateral descending pathway arises predominantly in the ventromedial and descending vestibular nuclei. Terminal structures presumably arising in the ventromedial and descending vestibular nuclei were found on contralateral neurons, probably motoneurons innervating neck muscles. Vestibular neurons with both ascending (presumably to extra-ocular motoneurons) and descending projections to the spinal cord are present in all vestibular nuclei, although preferentially in the ventromedial vestibular nucleus and adjacent parts of the ventrolateral and descending vestibular nuclei. However, also in the dorsolateral vestibular nucleus a substantial number of double labeled neurons were found. These vestibular neurons with both vestibulomesencephalic and vestibulospinal projections are probably involved in combined movements of eyes and head. Evidence for reciprocal internuclear connections between the oculomotor and abducens nuclei was found. Neurons in the dorsal part of the oculomotor nucleus probably project to the ipsilateral abducens nucleus, while neurons in the abducens nucleus most likely project to the contralateral oculomotor nucleus. These reciprocal internuclear connections between the oculomotor and abducens nuclei probably play an important role in conjugate horizontal eye movements. PMID- 2996382 TI - Spermatogenesis of rat: effect of central norepinephrine synthesis inhibition by diethyldithiocarbamate. AB - Spermatogenesis and steroidogenesis of male Wistar rats under the influence of a decreased central NE level was studied. Inhibition of NE synthesis was produced by chronic injection (7 days) of DDC, a Dopamine hydroxylase blocker, which decreased brain NE, increased brain DA without significantly affecting brain 5 HT. Rats were sacrificed on the day after cessation of treatment (8th day) and after an interval of 13 days following the cessation of treatment (21st day). The time interval of 13 days is equivalent to one cycle of the seminiferous epithelium in Wistar strain rats. A degenerative change (reduced spermatid count) in the spermatogenic pattern at stage-VII of the cycle of the seminiferous epithelium was observed in the rats sacrificed on the 8th day, the degeneration being much greater in the rats sacrificed on the 21st day. Rats sacrificed on the day after cessation of DDC treatment revealed an inhibition of plasma testosterone level. Such change was not observed in the rats sacrificed after the interval of 13 days following DDC treatment. Human chorionic gonadotropin supplementation in the rats sacrificed on the 21st day partially restored the spermatogenesis towards the vehicle treated control. The inhibition of spermatogenesis and steroidogenesis reflects a decrease in the pituitary gonadotropin secretion under the influence of a decreased NE synthesis in brain, following chronic treatment of DDC. PMID- 2996383 TI - Production of angiotensin-converting enzyme in rat epididymis. AB - Unilateral ductuli efferentia testis of immature rat were severed in order to interrupt the continuity between testis and epididymis and angiotensin-converting enzyme (ACE) activities were measured 5 weeks after the operation. The epididymal ACE on the operated side increased to nearly the same level as that on the contralateral side, but the testicular ACE activity on the operated side was markedly lower than that on the contralateral side. On immunofluorescent study using antiserum against purified rat lung ACE, ACE could be found in the tubular epithelial cells of the operated epididymis. These results suggest that epididymis itself synthesize ACE without connection to testis. PMID- 2996384 TI - Hepatic necrosis associated with herpes virus after isoflurane anesthesia. PMID- 2996385 TI - Adrenocortical function following multiple anesthesia inductions with etomidate. PMID- 2996386 TI - [Immunological aspects of surgical stress]. PMID- 2996387 TI - Influences of Ca2+: antagonist and cyclic AMP on vascular smooth muscle cells in culture. AB - When cultured with a Ca2+ antagonist diltiazem, fetal bovine aortic smooth muscle cells showed a cobblestone-like monolayer and a positive factor VIII-related antigen in culture, which are supposed to be specific to the endothelial cells in general. Similar change was produced by addition of a cyclic AMP analogue, dibutyryl cyclic AMP or a phosphodiesterase inhibitor, theophylline. All of these changes were reversible. Both smooth muscle cells and endothelial cells are derived from mesenchymal cells embryologically and it is of great interest to presume the differentiation process of both cells. PMID- 2996388 TI - Observations on transplacental infection with bluetongue virus in sheep. AB - Twenty-four ewes were inoculated with 1 of 2 strains of bluetongue virus type 4 at 40, 60, or 80 days of gestation. Two ewes aborted, 2 ewes died, and 1 was killed during the experiment, but their fetuses were recovered. At term, 2 mummified fetuses, 4 dead lambs, and 17 clinically healthy lambs were produced by 12 sheep, and the remaining 7 sheep were barren. Porencephaly and cerebellar dysgenesis were found in term lambs born to sheep inoculated at 40 and 60 days of gestation. Radiographic examination of 12 fetuses showed developmental ages far less than their chronologic age; 8 fetuses had skeletal growth-retardation lines, which were also observed in the dead lambs. A systemic lymphoreticular hyperplasia was observed in the dead lambs and in all lambs at 12 weeks of age; in 4 of the latter, granulomatous reactions were present in the liver and kidney. Lungs of the full-term lambs were reduced in weight and showed poor alveolar development and mononuclear cell infiltration, which persisted in the 12-week-old lambs. It was concluded that bluetongue virus is capable of causing not only gross abnormalities of the CNS, but also generalized growth retardation and fetal lymphoreticular hyperplasia. PMID- 2996389 TI - Binding characteristics of swine erythrocyte insulin receptors. AB - Crossbred gilts (controls; n = 7) had 8.8 +/- 1.1% (mean +/- SEM) maximum binding of [125I]insulin to insulin receptors on erythrocytes. The number of insulin binding sites per cell was 137 +/- 19, with a binding affinity ranging from 7.4 X 10(7)M-1 to 11.2 X 10(7)M-1 and mean of 8.8 X 10(7)M-1. Pregnant sows (n = 5) had a significant increase (P less than 0.01) in maximum binding due to an increase in number of receptor sites per cell. Lactating sows fed a high-fiber diet (n = 3) and a low-fiber diet (n = 4) did not develop a significant difference in maximum binding of insulin. Sows fed the low-fiber diet had a significantly higher number of binding sites and a significantly lower binding affinity than did sows fed a high-fiber diet. Receptor-binding affinity was lower in the low fiber diet group than in cycling gilts, whereas data from sows fed the high-fiber diet did not differ from data for cycling gilts. Data from this study indicated that insulin receptors of swine erythrocytes have binding characteristics similar to those in other species. Pregnancy and diet will alter insulin receptor binding in swine. PMID- 2996390 TI - Viral matrix inclusion bodies in myocardium of lymphoid leukosis virus-infected chickens. AB - Chicken embryos and healthy adult chickens naturally infected with lymphoid leukosis virus were used to investigate viral inclusion bodies in myocardial cells by light and electron microscopies and by immunocytochemical technique. Intracytoplasmic viral matrix inclusion bodies frequently appeared in the myocardium of adult chickens, but not in that of embryos. In light microscopic preparations, inclusions were irregularly distributed, were basophilic, and contained ribonucleic acid. Ultrastructurally, inclusions in myocardial cells were in areas containing numerous interstitial C-type particles. Early inclusions were composed of clusters of ribosomes associated with sarcoplasmic tubules; spherical bodies developed among these ribosomes. Mature inclusions were composed of numerous spherical bodies (50 to 75 nm) with interspersed ribosomes and of ribosomes clustered at the periphery. Inclusions were not membrane-enclosed. Occasionally, spherical bodies were in paracrystalline arrays. Multiple budding occurred on cell membranes adjacent to matrix inclusions. The viral group specific protein, p27, was demonstrated by the peroxidase-antiperoxidase method and by the protein A-gold method in the spherical bodies, in nucleoids of mature virus particles, and among ribosomes of inclusions. The results indicate that the matrix inclusions were the result of lymphoid leukosis virus infection and were the product of viral protein synthesis on ribosomes. PMID- 2996391 TI - Association of blood cholesterol with occurrence of fat necrosis in cows and tall fescue summer toxicosis in steers. AB - Factors associated with fat necrosis in cows and tall-fescue summer toxicosis in steers were studied. In the cow study, fescue pastures were fertilized, using 3 rates of N: high N (703 to 483 kg and 0 kg of N/ha/year from broiler litter in 1972 to 1974 and 1975, respectively), moderate N, and low N (224 and 74 kg of N/ha/yr from NH4NO3, 1972 to 1975, respectively). Bermuda grass pastures were fertilized at 2 rates of N: moderate N and low N (280 and 20 kg of N/ha/year from NH4NO3, 1972 to 1975, respectively). Fat necrosis developed only in cows grazing tall fescue, with an occurrence of 60%, 8%, and 3% for high-N, moderate-N, and low-N pastures, respectively. Cows grazing the high-N fescue, and to some extent those grazing the moderate-N fescue, had clinical signs of summer fescue toxicosis. Plasma cholesterol concentrations were lowest in cattle grazing the high-N fescue, averaging 114 mg/dl, followed by 134 and 127 mg/dl in cattle grazing the moderate-N and low-N fescue, respectively. In the steer grazing study, 24 paddocks of 0.49 ha each were seeded with tall-fescue lines G1-307 or G1-306 or with tall-fescue cultivars, KY-31 or Kenhy. All paddocks were fertilized with 170 kg of N/ha/year. Serum cholesterol concentrations were lower in steers grazing on G1-307 than in steers grazing on G1-306 or cultivars. Serum total lipids followed a similar trend, with a positive correlation (r = 0.49) between cholesterol and total lipids.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996392 TI - Ovarian lesions induced in heifers by intravenous inoculation with modified-live infectious bovine rhinotracheitis virus on the day after breeding. AB - Four commercially available vaccine strains of modified-live infectious bovine rhinotracheitis virus were passaged once in embryonic bovine kidney cells. Heifers were inoculated IV on the day after breeding with 5.0 ml of nondiluted cell culture fluid of each of the 4 strains. Virus was reisolated from nasal swabs and blood collected during the week after inoculation. The heifers were killed 9 to 14 days after inoculation. Mild-to-marked inflammatory and necrotic lesions were seen in the corpora lutea and ovaries of the heifers. The lesions were similar to, and almost as severe as, those resulting from the inoculation of virulent strains of infectious bovine rhinotracheitis virus. Adrenal lesions were also found in all heifers examined. Virus was reisolated from the ovaries of only 4 of the 8 heifers. However, virus was confirmed in the ovaries of all 8 heifers, using immunofluorescent or ultrastructural studies. Heifers with severe luteal damage had abnormally low plasma progesterone concentrations. PMID- 2996393 TI - [Cutis marmorata telangiectatica congenita. Associated abnormalities]. AB - Eight cases of cutis marmorata telangiectatica congenita are presented and an analysis of associated alterations on seven is done. These abnormalities are mainly asymmetrical body development (hypotrophy of the leg in two cases, hemihypertrophy of body in five), hemihypertrophy of brain with psychomotor retardation in three patients, Nevus flameus on lumbar region in one and syndactylies on hands and feet in one. Inclusion of the disease into neurocutaneous syndromes is suggested and is emphasized the importance of the pediatric and neurological follow-up of these patients. PMID- 2996394 TI - The challenge of the acquired immunodeficiency syndrome. PMID- 2996396 TI - The acquired immunodeficiency syndrome in gay men. AB - The acquired immunodeficiency syndrome (AIDS) is a major health problem for gay men in the United States. About three fourths of all reported cases have occurred in this population, and the number is projected to double in the next year. In Manhattan and San Francisco, AIDS is now the leading cause of premature mortality in men aged 25 to 44 years who have never married. In a sample of a cohort of gay men enrolled in a San Francisco clinic, 2.7% of the men had the syndrome and 26% had related conditions in 1984. Antibody to human T-lymphotropic virus, type III/lymphadenopathy-associated virus was found in sera from 67% of the men, including 58% of asymptomatic men. Behavioral factors associated with an increased risk of AIDS include large numbers of sexual partners, receptive anal intercourse, and "fisting." The adoption of safer lifestyles is currently the basis of attempts to control the syndrome in gay men. PMID- 2996395 TI - The epidemiology and prevention of the acquired immunodeficiency syndrome. AB - The incidence of the acquired immunodeficiency syndrome (AIDS) has continued to increase worldwide. From June 1981 to September 1985, 12 932 cases have been diagnosed and reported in the United States; this number is expected to double during the next year. The incubation period is long and few persons infected with human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV III/LAV) have AIDS diagnosed within 2 to 5 years of infection. The widespread use of HTLV-III/LAV serologic tests to screen donated blood and plasma, the continued deferral of donors from groups with an increased incidence of AIDS, and the use of heat-treated clotting factor concentrates should help prevent HTLV-III/LAV transmission through blood and blood products. Preventing transmission among sexual partners, among intravenous drug users, and from infected mothers to newborns will continue to be difficult without a vaccine, specific antiviral therapy, or both. Risk reduction strategies should involve community groups to provide accurate information and influence behaviors that lead to transmission of the virus. PMID- 2996397 TI - Epidemiology of human T-lymphotropic virus type III and the risk of the acquired immunodeficiency syndrome. AB - The discovery of human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) has opened a window to the understanding of the spectrum of the acquired immunodeficiency syndrome (AIDS) and related clinical syndromes. Analysis of risk factors for seropositivity has shown that HTLV-III is transmitted most efficiently via routes that involve close personal contact or parenteral exposure. Longitudinal studies have shown that HTLV-III infection has a long latent period. The prevalence of AIDS in different geographic areas and among different risk groups appears to depend in part on duration of exposure. Co factors for AIDS outcome such as manner and route of exposure, underlying immune status, and host susceptibility are also likely to play a role in risk. PMID- 2996399 TI - A human T-lymphotropic retrovirus (HTLV-III) as the cause of the acquired immunodeficiency syndrome. AB - Three human T-lymphotropic viruses have been isolated and characterized in the past 5 years. The ability to culture target cells with T-cell growth factor and sensitive detection systems for the virally encoded polymerase reverse transcriptase permitted isolation of HTLV-I, which is strongly linked to the cause of adult T-cell leukemia and associated with other lymphoid malignancies in endemic areas. The same techniques, using a permissive human tumor cell line, allowed the isolation and characterization of HTLV-III/lymphadenopathy-associated virus, which is implicated as the primary cause of the acquired immunodeficiency syndrome (AIDS). This virus shares some features with HTLV-I and HTLV-II, such as additional genes not found in most retroviruses. One gene codes for a transcriptional activator protein and may be a feature of a larger group of related retroviruses. The clear identification of the primary cause of AIDS has resulted in the development of specific immunologic reagents, preventive and therapeutic proposals, and comprehensive identification of the clinical diseases associated with this virus. PMID- 2996400 TI - Lymphadenopathy-associated virus: from molecular biology to pathogenicity. AB - Recent data indicate that the lymphadenopathy-associated virus (LAV) is morphologically similar to animal lentiviruses, such as equine infectious anemia and visna viruses. This finding, together with the cross-reactivity of the core proteins of LAV with those of the equine infectious anemia virus and a similarity in genome structure and biological properties, allows LAV to be placed in the retroviral subfamily of Lentivirinae. Molecular data indicate a high degree of genetic variation of the virus, especially in the envelope gene, which have important implications for the origin of the virus (the T4 lymphotropism may be a recently acquired property) and for future immunization. Another problem is the role of viral infection in the induction of irreversible immunodeficiency. This syndrome occurs in a minority of infected persons, who generally have in common a past of antigenic stimulation and of immune depression before LAV infection. PMID- 2996398 TI - The international occurrence of the acquired immunodeficiency syndrome. AB - Through December 1984, 9932 cases of the acquired immunodeficiency syndrome have been reported, mainly from North and South America and Europe; 85% of these cases occurred in the United States. Haiti and the United States have the highest incidence rates, 59 and 36 per million population, respectively. Rates in the United States range from 0.3 (beginning of 1981) to 10.4 (end of 1984). Brazil, Canada, Denmark, Switzerland, France, West Germany, the United Kingdom, and the Netherlands show a slower increase. Homosexual men and intravenous drug users are still the main risk groups in the United States and Europe. The disease is prevalent in heterosexual Haitians and Africans whether they live in their own countries or abroad. Cases of the syndrome have been identified in Zaire, Rwanda, Zambia, and Uganda, but its full extent is not yet known. Consistent with the general history of epidemics, the appearance of geographically separated sites of incidence of the syndrome could be linked to population migrations; however no evidence has been found to identify an index location. PMID- 2996401 TI - Infection by the retrovirus associated with the acquired immunodeficiency syndrome. Clinical, biological, and molecular features. AB - Peripheral mononuclear cells from more than 160 persons from groups at risk for the acquired immunodeficiency syndrome (AIDS) have yielded AIDS-associated retroviruses (ARV). Antibodies to ARV can also be found in these risk groups. Antibody-negative, virus-positive persons have been identified with early infection or possible viremia with immune complex formation. Established lines of human T and B cells, monocytes, and promyelocytes have been infected with ARV. Moreover, infectious virus has been recovered from macrophages cultured from the blood of some persons with AIDS. The cytopathic effects of ARV in T cells is associated with the accumulation of unintegrated viral forms in the infected cells. The ARV has also been isolated from plasma, serum, saliva, semen, urine, cerebrospinal fluid, and brain tissue. All these results reflect the wide host range of ARV and support its role in neurologic abnormalities seen in some patients. Molecular studies of independent ARV isolates indicate a polymorphism of nucleotide sequences, particularly in the viral envelope region. All these features place ARV in the lentivirus subfamily of human retroviruses. PMID- 2996402 TI - Antigens of human T-lymphotropic virus type III/lymphadenopathy-associated virus. AB - Antigens encoded by the gag and env genes of the human T-lymphotropic virus type III/lymphadenopathy associated virus (HTLV-III/LAV) include a p55 gag polyprotein that yields p24 as the major virus core protein, and an env gene polyprotein, gp 160, that produces gp 120, the most immunogenic protein in humans, at the amino terminus. Although its use is limited to research laboratories due to the cost and specialized procedures involved, the analysis of sera by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is the test providing the optimal balance of specificity and sensitivity. Because the gp 120 represents the external virus protein, it would be the most appropriate antigen for vaccine development. Also viruses serologically related to HTLV-III/LAV were detected recently in two species of Old World monkeys. Because about half the healthy African green monkeys appear to have been exposed to simian T-lymphotropic virus type III (STLV-III), a related agent of the species, a characterization of the STLV-III gp 120 and immune response of the host may provide additional information for vaccine development. PMID- 2996404 TI - Mechanisms of T-cell functional deficiency in the acquired immunodeficiency syndrome. AB - Cell-mediated immune responses to cytomegalovirus infections were studied to define mechanisms for deficient effector T-cell responses in patients with the acquired immunodeficiency syndrome (AIDS). Progressive cytomegalovirus infection is common in such patients and is accompanied by a failure to develop HLA restricted cytotoxic T-cell responses. The mechanism for the cytotoxic T-cell deficiency is presumably the basis for susceptibility to opportunistic infections. Precursors to these effector cells circulate in the peripheral blood of patients, but maturation of these cells into cytotoxic T cells is arrested during an interleukin-2-dependent phase. Production of interleukin-2 by lymphocytes from patients with AIDS is deficient because sera from these patients contain an inhibitor of interleukin-2 production. Excess suppressor cell activity does not appear to account for this deficient production. Studies of the source of the serum inhibitor should provide insights into the pathogenesis of AIDS and possible leads for effective treatment. PMID- 2996403 TI - Immunopathogenesis of the acquired immunodeficiency syndrome. AB - Many abnormalities of humoral and cellular immunity associated with the acquired immunodeficiency syndrome can be explained by the preferential infection of the T4 lymphocyte subset with the etiologic retrovirus. Severe alterations in specific T4 functions, such as inadequate immune responsiveness to specific antigen, result in devastating morbidity and mortality. On the basis of accumulated scientific knowledge, we outline the immunopathogenesis of this syndrome. PMID- 2996405 TI - Immunologic reconstitution in the acquired immunodeficiency syndrome. AB - Although effective therapies are available for many of the infections and tumors that occur in patients with the acquired immunodeficiency syndrome (AIDS), no therapy exists for the underlying immunodeficiency. Three approaches used to treat this immunodeficiency have included wholescale immune replacement through lymphocyte transfers, bone marrow transplantation, and thymic implantation; immunologic enhancement with biologic-response modifiers, such as gamma interferon, interleukin-2, and some drugs; and antiretroviral therapy. Because of the persistence of the etiologic virus, both immune replacement and enhancement will probably be ineffective unless effective strategies are developed against the virus. Agents that have shown antiviral activity in vitro which are currently in clinical trials include suramin, heteropolyanion-23 (HPA-23), ribavirin, and alpha interferon. Foscarnet and monoclonal antibodies have yet to enter clinical trials in the United States. It is still too early to tell if any of the antiviral agents will prove of value in the management of patients with AIDS. PMID- 2996406 TI - The natural history of infection with the lymphadenopathy-associated virus/human T-lymphotropic virus type III. AB - Over half of the persons infected with the lymphadenopathy-associated virus/human T-lymphotropic virus type III (LAV/HTLV-III), the retrovirus that causes the acquired immunodeficiency syndrome (AIDS), become persistently infected with the virus. These "carriers" serve as the major reservoir of infection for others. Virus from their vascular and lymphatic spaces infects others through direct blood or mucous membrane exposure. After months to years, a high proportion of those infected will develop clinical manifestations of infection. For infected homosexual men, approximately 25% have developed AIDS-related conditions, mainly lymphadenopathy, and approximately 10% have developed AIDS. Because of the large number of infected persons in the United States, increasing rates of disease can be expected. PMID- 2996407 TI - The acquired immunodeficiency syndrome in persons with hemophilia. AB - The widespread use of coagulation factor concentrates prepared from the blood of numerous donors has doubled the life expectancy of persons with hemophilia, but parenteral exposure to alloantigens and infectious agents is not free of risk. The prevalence of the acquired immunodeficiency syndrome (AIDS) now approaches 1% in patients with hemophilia, and laboratory evidence of abnormal immunoregulation is found in at least 50% of treated patients with severe hemophilia. The immune defect is multifactorial. The attack rate of AIDS among patients with severe hemophilia appears to have reached a peak; further evidence suggests that only a minority of those infected with human T-lymphotropic virus type III will develop AIDS. The advent of improved donor screening methods and the application of heat treatment of coagulation factor concentrates should further reduce the risk of AIDS in persons with hemophilia. PMID- 2996408 TI - Licensed tests for antibody to human T-lymphotropic virus type III. Sensitivity and specificity. AB - Before the discovery of the etiologic agent of the acquired immunodeficiency syndrome (AIDS), substantial epidemiologic evidence suggested that the responsible agent could be transmitted by blood and blood products such as factor VIII. The Public Health Service and the Food and Drug Administration therefore took general steps to increase the safety of the blood supply. With the discovery of the human T-lymphotropic virus type III (HTLV-III) it became possible to test blood for evidence of infection using an antibody detection assay. Currently licensed tests to detect antibody to HTLV-III range in sensitivity between 93% and 99%, and all are above 99% in specificity. The Public Health Service has issued provisional recommendations regarding the voluntary use of the antibody tests to screen blood and plasma donations. PMID- 2996409 TI - The acquired immunodeficiency syndrome in infants and children. AB - The classification of the pediatric acquired immunodeficiency syndrome (AIDS) is based on epidemiologic, immunologic, and virologic data. Persons at risk include mothers who use intravenous drugs, infants who have received blood transfusions from subjects with risk factors, patients receiving factor VIII therapy, and infants born to heterosexual mothers with bisexual husbands. A distinct immunologic phenotype, rarely seen in other immunodeficiency disorders, is associated with pediatric AIDS consisting of polyclonal hypergammaglobulinemia and T-cell immunodeficiency. Detection of antibody to the AIDS retrovirus or isolation of virus are essential in establishing a diagnosis. During early infancy, viral isolation is essential as passive transfer of material IgG may occur. Primary immunodeficiency diseases, in particular adenosine deaminase and purine nucleoside phosphorylase deficiency, should be excluded. A diagnosis of pediatric AIDS may be established in a patient who has a risk factor associated with AIDS, polyclonal hypergammaglobulinemia, T-cell immunodeficiency, and antibody to the AIDS retrovirus or isolation of virus. PMID- 2996410 TI - Treatment of infections in patients with the acquired immunodeficiency syndrome. AB - The microorganisms that regularly infect patients with the acquired immunodeficiency syndrome (AIDS) have become well recognized. Most take advantage of defects in T-lymphocyte function, but others, such as Streptococcus pneumoniae and Haemophilus influenzae, take advantage of B-cell defects. Still others, such as Staphylococcus aureus and Shigella species, occur or persist for reasons that are unclear. Infections with organisms associated with hospitalization and medical procedures are also seen and should be anticipated. Among the infections taking advantage of T-cell defects, Pneumocystis carinii pneumonia is the most commonly diagnosed, but cytomegalovirus infection may be equally common. Disseminated Mycobacterium avium-intracellulare infection has been found in one half of our patients at postmortem examination. The retrovirus responsible for AIDS commonly infects the central nervous system, as does Toxoplasma gondii. Although candida infections are common, dissemination is uncommon. Many of the infections respond to appropriate therapy but tend to recur when treatment is stopped. Often treatment courses must be prolonged even beyond those used in other immunocompromised hosts. PMID- 2996411 TI - Myoepithelioma. AB - Myoepithelial cells are integral components of normal salivary glands. Their active or passive participation in the histogenesis of several salivary gland tumors is a debated issue. This debate notwithstanding, a rare form of salivary tumor composed entirely of myoepithelial cells exists and probably represents a monomorphic form of mixed tumor. This tumor, the myoepithelioma, occurs in most major and minor salivary tissues and is generally a biologically benign lesion. PMID- 2996413 TI - DNA modification in Neisseria gonorrhoeae: resistance of DNA of 19 strains to cleavage by restriction enzymes. PMID- 2996412 TI - Effect of soybean crude fiber on the concentrations of serum lipids and apolipoproteins in hyperlipemic subjects. AB - Effect of soybean crude fiber on the concentrations of serum lipids and apolipoproteins in hyperlipemic subjects was examined. The concentrations of serum triglycerides, VLDL triglycerides and VLDL cholesterol were decreased significantly following the administration of soybean crude fiber for 2 months. Neither were significant changes found in total cholesterol, LDL cholesterol, HDL cholesterol, apo A-I, apo A-II, apo C-II and apo B levels, nor in body weight before and after the administration of soybean crude fiber. PMID- 2996415 TI - Brachial plexus neuropathy in the population of Rochester, Minnesota, 1970-1981. AB - Brachial plexus neuropathy (BPN) is a clinical entity of unknown cause characterized by the acute or subacute onset of pain and weakness, with occasional atrophy of the arm muscles. Information on the incidence of the disease in a delineated population is lacking, as the data available on BPN have come essentially from case reports or selected series. Using the Mayo Clinic records-linkage system as the source of data, 579 clinical records were reviewed of Rochester, Minnesota, residents in which a diagnosis suggestive of BPN was reported for the period 1970 through 1981. Eleven cases fulfilled all criteria, providing an overall annual incidence rate of 1.64 cases per 100,000 population. An infectious disease and/or tetanus toxoid immunization preceded the onset of BPN in 4 cases. The upper brachial plexus was involved in 6 cases, the lower brachial plexus in 2, and the whole plexus in 3; in 1 case there was bilateral BPN. The neuropathy ran a mild to moderate course in 10 cases, and complete recovery was recorded in 6, with slight residua in the others. The occurrence of antecedent events and the features of the disease are supportive of the concept of an immune-mediated process. PMID- 2996414 TI - Susceptibility of Neisseria gonorrhoeae DNA to cleavage by restriction endonuclease KpnI. AB - We compared the susceptibility of Escherichia coli and Neisseria gonorrhoeae DNA to cleavage by KpnI and found that KpnI has a lower affinity for gonococcal DNA. Site-specific methylation is suggested as the cause of this altered affinity. PMID- 2996416 TI - Detection of picornavirus sequences in nervous tissue of amyotrophic lateral sclerosis and control patients. AB - We used in situ hybridization to look for picornavirus ribonucleic acid (RNA) sequences in frozen sections of central nervous system (CNS) tissue of amyotrophic lateral sclerosis (ALS) and control patients. Using reconstruction experiments, we concluded that 30 copies of viral RNA per cell could be detected with the assay. RNA which hybridized to DNAs complementary (cDNAs) to both poliovirus and Theiler's virus was found at several levels in the CNS of 2 patients, 1 ALS patient, and 1 control. In transverse sections of the spinal cord, these sequences predominated in cells of the anterior horns. We assessed the specificity of hybridization by several criteria: no hybridization was observed with heterologous visna virus cDNA probes; hybridization was abolished by pretreatment of the sections with ribonuclease; chemography artifacts were ruled out; and the results were reproduced in three independent experiments. We concluded that RNA molecules, possibly belonging to a picornavirus having sequences in common with poliovirus and Theiler's virus, were present in the CNS of these 2 patients. On the other hand, 14 cases of classic ALS, 2 cases of Guamanian parkinsonian dementia, and 5 controls had negative results. However, the presence of picornavirus sequences in our series could be underestimated because in many cases autolysis times were 10 hours or longer. PMID- 2996417 TI - Leukotrienes increase blood-brain barrier permeability following intraparenchymal injections in rats. AB - To examine whether leukotrienes could increase blood-brain barrier permeability, rats were anesthetized and injected intravenously with Evans blue. Ten microliters of vehicle, of leukotrienes B4, C4, or E4, or of arachidonic acid was injected over 1 hour directly into the brain parenchyma. The percentage of the total surface area of Evans blue extravasation in a coronal section of brain centered on the injection site was then determined as an estimate of blood-brain barrier permeability. Leukotrienes B4, C4, and E4, and arachidonic acid all increased blood-brain barrier permeability, but this effect was lost when the total dose was reduced to 20 ng. Increased blood-brain barrier permeability induced by arachidonic acid could be prevented by pretreatment with the lipoxygenase inhibitor BW755C, but not with indomethacin. Leukotrienes may play a role in the development of increased blood-brain barrier permeability after cerebral injury. PMID- 2996418 TI - Azathioprine and steroids are not more effective in decreasing multiple sclerosis intra-blood-brain-barrier IgG synthesis than steroids alone. AB - Intra-blood-brain-barrier IgG synthesis rates and oligoclonal IgG banding patterns were examined in 9 patients with multiple sclerosis who were treated with azathioprine and steroids for 2 to 4.5 years. The IgG synthesis rates of 5 patients were significantly decreased from the pretreatment mean values 1 month after treatment, and their synthesis rates remained at the decreased levels throughout treatment. However, among the remaining 4 patients, the rates exceeded the pretreatment means. This continuous suppressive effect of the combined azathioprine and steroids upon the IgG synthesis rate was similar to that of steroids, suggesting that azathioprine and steroids in combination were not more effective in reducing intra-blood-brain-barrier IgG synthesis than steroids alone. Oligoclonal IgG patterns in all cerebrospinal fluid samples were not significantly altered during the study. PMID- 2996419 TI - The genetics of GBS. PMID- 2996420 TI - [Genetic and physicochemical analysis of the resistance of Pseudomonas aeruginosa strains to gentamycin and other antibiotics]. AB - The genetic and physicochemical mechanisms of antibiotic resistance were studied in 50 clinical strains of P. aeruginosa resistant to gentamicin. The serotypes and pyocinotypes of the bacteria were determined. The spectra and levels of antibiotic resistance and the plasmid profiles of the strains were estimated. It was shown that 16 multiresistant isolates had identical spectra and levels of antibiotic resistance, belonged to the same serotype and pyocinotype and were characterized by the absence of the extrachromosomal DNA, which indicated the circulation of the same polyresistant strain in hospital. Plasmids with identical molecular weights and antibiotic resistance spectra were detected in 14 strains belonging to different serotypes and pyocinotypes. These plasmids determined synthesis of the same aminoglycoside inactivating enzymes: APH (3') and AAC (3). Epidemiologic distribution of the same high molecular R plasmid among the clinical strains of P. aeruginosa is suggested. PMID- 2996421 TI - Inactivation of Norwalk virus in drinking water by chlorine. AB - Norwalk virus in water was found to be more resistant to chlorine inactivation than poliovirus type 1 (LSc2Ab), human rotavirus (Wa), simian rotavirus (SA11), or f2 bacteriophage. A 3.75 mg/liter dose of chlorine was found to be effective against other viruses but failed to inactivate Norwalk virus. The Norwalk virus inoculum remained infectious for five of eight volunteers, despite the initial presence of free residual chlorine. Infectivity in volunteers was demonstrated by seroconversion to Norwalk virus. Fourteen of 16 subjects receiving untreated inoculum seroconverted to Norwalk virus. Illness was produced in four of the eight volunteers and in 11 of 16 control subjects. A similar Norwalk virus inoculum treated with a 10 mg/liter dose of chlorine produced illness in only one and failed to induce seroconversion in any of eight volunteers. Free chlorine (5 to 6 mg/liter) was measured in the reaction vessel after a 30-minute contact period. Norwalk virus appears to be very resistant to chlorine which may explain its importance in outbreaks of waterborne disease. PMID- 2996422 TI - Enteroviruses in sludge: multiyear experience with four wastewater treatment plants. AB - We describe our experience with the isolation of viruses from four treatment plants located in different geographic areas. Over a period of 3 years, 297 enteroviruses were isolated from 307 sludge samples. The highest frequency of viral isolation (92%), including multiple isolates from single samples, was obtained from a treatment plant serving the smallest population. Excluding the polioviruses, 22 different enterovirus serotypes were isolated. The methods used to isolate the viruses were relatively simple and included an elution procedure in which beef extract was used and a disinfection step. No concentration procedure was used. Of three cell culture systems used, the RD line of human rhabdomyosarcoma cells was by far the most useful for the isolation of echoviruses; BGM and HeLa cells were particularly useful for the isolation of group B coxsackieviruses. A seasonal effect on viral isolation rates from sludge was observed. PMID- 2996423 TI - Recombinant plasmid conferring proline overproduction and osmotic tolerance. AB - A recombinant plasmid carrying the proBA (pro-74) mutant allele which governs osmotic tolerance and proline overproduction was constructed by using the broad host-range plasmid vector pQSR49. The physiological, biochemical, and genetic properties of strains carrying the pQSR49 derivatives pMJ101 and pMJ1, mutant and wild type, respectively, were investigated. pMJ101 conferred enhanced osmotolerance compared with strains carrying the wild type, pMJ1. These results are in contrast to those obtained previously with strains carrying recombinant plasmids based on pBR322 that failed to confer the osmotic tolerance phenotype. gamma-Glutamyl kinase (first step in proline biosynthesis) from strains carrying pMJ101 was 200-fold less sensitive to feedback inhibition than was the wild-type enzyme. As expected, the intracellular proline levels of strains carrying pMJ101 were more than an order of magnitude higher than those of the wild type. An analysis of copy number revealed that the pQSR49 constructs were present in the cell at a level six- to eightfold lower than those of the pBR322 recombinants, which may account for the difference in phenotype. We found that the genetic stability of the pQSR49 derivative in a variety of gram-negative bacteria was dependent on the insert orientation and the presence of foreign DNA on the plasmid. These factors may be significant in future studies aimed at expanding the osmotolerance phenotype to a broad range of gram-negative bacteria. PMID- 2996424 TI - Membrane-associated viral complexes observed in stools and cell culture. AB - Viral complexes observed to be membrane associated rather than clumped by antibody were detected in a rotavirus-containing stool specimen by negative-stain electron microscopy. These "viral packets" were also observed in cell culture fluids after repeated passaging and contained up to 100 virions. Other stool specimens have been observed to contain similar packets of parvovirus-like particles. Such complexes must be expected in fecally contaminated water. PMID- 2996425 TI - Construction of cloning, promoter-screening, and terminator-screening shuttle vectors for Bacillus subtilis and Streptococcus lactis. AB - Shuttle vectors have been constructed which are suitable both for the selection of regulatory sequences and for gene cloning in Bacillus subtilis and Streptococcus lactis. The promoter screening vectors contain a promoterless chloramphenicol acetyltransferase gene; the insertion of suitable DNA fragments upstream of the gene restored the enzyme activity. With a related set of vectors, transcription termination signals can be selected. PMID- 2996427 TI - Calcium-activated neutral protease purified from beef lung: properties and use in defining structure of epidermal growth factor receptors. AB - Ca2+-Requiring proteases degrade cytosolic and integral membrane proteins as well as alter, by limited proteolysis, the activity of certain protein kinases. When cells are lysed, a Ca2+-requiring protease degrades the epidermal growth factor (EGF) receptor, an integral membrane protein with an intrinsic kinase activity, from its 170-kDa form to a 150-kDa form. This Ca2+-requiring protease has all of the characteristics of calcium-activated neutral protease (CANP). To show that CANP is the protease uniquely responsible for the degradation of the native EGF receptor in vitro, CANP was highly purified from beef lung. This affinity purified CANP had properties previously described for other CANPs: heterodimer of 80 and 30 kDa; neutral pH optimum; activation by millimolar Ca2+; and inhibition by an endogenous, heat-stable proteinaceous inhibitor, by leupeptin, and by sulfhydryl alkylating agents. Using the EGF receptor labeled by covalent attachment to 125I-EGF, this purified CANP quantitatively generated the 150-kDa form from the native receptor in A-431 cell membranes. As with the native receptor, the 150-kDa receptor forms produced by the endogenous Ca2+-requiring protease, by CANP, by chymotrypsin, and by elastase were all capable of EGF stimulated autophosphorylation. When the 150-kDa receptor forms were generated by the three exogenously added proteases, autophosphorylation with [gamma-32P]ATP followed by trypsinization produced 32P-labeled peptides that were not the same. However, the tryptic 32P-labeled peptides from the autophosphorylated 150-kDa receptor form produced by CANP or by the endogenous Ca2+-requiring protease were identical. These data indicate that CANP is identical to the endogenous Ca2+ requiring protease responsible for producing the autophosphorylating 150-kDa receptor form from the native EGF receptor when cells are lysed. PMID- 2996426 TI - Components of purified sarcolemma from porcine skeletal muscle. AB - Sarcolemmal membranes were isolated from porcine skeletal muscle by modifications of a LiBr-extraction technique. Latency determinations of acetylcholinesterase, ouabain-sensitive p-nitrophenylphosphatase, [3H]ouabain binding, and (Na+ + K+) ATPase activities indicated that 65-76% of the membranes were sealed inside-out vesicles. The preparations were enriched in cholesterol and phospholipid, and demonstrated adenylate cyclase activity and both cAMP and cGMP phosphodiesterase activities. An indication of the purity of this fraction was that the Ca2+-ATPase activity (0.13 mumol Pi mg-1 min-1 at 37 degrees C) was 3.8% of that of porcine skeletal muscle sarcoplasmic reticulum preparations. Pertussis toxin specifically catalyzed the ADP-ribosylation of a Mr 41,000 sarcolemmal protein, indicating the presence of the inhibitory guanine nucleotide regulatory protein of adenylate cyclase, Ni. An endogenous ADP-ribosyltransferase activity, with several membrane protein substrates, was also demonstrated. The addition of exogenous cAMP dependent protein kinase or calmodulin promoted the phosphorylation of a number of sarcolemmal proteins. The calmodulin-dependent phosphorylation exhibited an approximate K 1/2 for Ca2+ of 0.5 microM, and an approximate K 1/2 for calmodulin of 0.1 microM. 125I-Calmodulin affinity labeling of the sarcolemma, using dithiobis(succinimidyl propionate), demonstrated the presence of Mr 160,000 and 280,000 calmodulin-binding components in these membranes. These results demonstrate that this porcine preparation will be valuable in the study of skeletal muscle sarcolemmal ion transport, protein and hormonal receptors, and protein kinase-catalyzed phosphorylation. PMID- 2996428 TI - Characterization of a new repetitive sequence in the Dictyostelium discoideum genome. AB - A repetitive DNA sequence was isolated from a Dictyostelium discoideum genomic plasmid library of BglII-digested DNA ligated to the BamHI site in pBR322. This clone, called pBS582, hybridized to a large number of phage lambda Dictyostelium genomic clones. Southern blot analysis indicated that pBS582 DNA hybridized to many differently sized genomic DNA fragments generated by digestion with Eco RI, AvaI, or HindIII. Restriction maps of pBS582 and five genomic clones showed that the flanking regions of each of the genomic clones were different. These findings indicate that the sequence specific to pBS582 is scattered throughout the Dictyostelium genome and is reiterated approximately 100 times in the haploid genome. Northern blot analysis revealed that RNA which hybridized to pBS582 DNA was present during all stages of growth and development and did not seem to be developmentally regulated. Southern blot analysis of DNAs from other slime molds (D. giganteum, D. purpureum, and Polysphondylium violaceum) were performed to determine whether the pBS582 sequence was present in other species of slime molds. Hybridization of pBS582 was observed to DNA from the two Dictyostelium species but not to Polysphondylium. It may thus be possible to use hybridization of specific sequences as a biochemical tool to study the relatedness of different slime mold species and their molecular taxonomy. PMID- 2996429 TI - Microsomal interactions between iron, paraquat, and menadione: effect on hydroxyl radical production and alcohol oxidation. AB - The addition of menadione or paraquat to rat liver microsomes resulted in about a threefold increase in the production of hydroxyl radical (.OH) as reflected by the increased oxidation of 2-keto-4-thiomethylbutyric acid (KMBA) to ethylene. This increase was not sensitive to superoxide dismutase but was blocked by catalase. The increase occurred in the absence of added iron and was not affected by the potent iron chelating agent, desferrioxamine, which suggests the possibility that .OH was produced from an interaction between H2O2 and the paraquat or menadione radical. Menadione and paraquat were especially effective in stimulating the oxidation of KMBA in the presence of certain iron chelates such as ferric-ADP, -ATP, or -EDTA, but not ferric-desferrioxamine, -citrate, or histidine, or unchelated iron. In fact, ferric-ADP or -ATP only stimulated .OH production in the presence of menadione or paraquat. In the presence of ferric EDTA, the greater than additive increase of .OH production was sensitive to catalase, but not to superoxide dismutase, suggesting the possibility of reduction of ferric-EDTA by paraquat or menadione radical. The interactions with ferric adenine nucleotides may increase the catalytic effectiveness of menadione or paraquat in producing potent oxidants such as the hydroxyl radical, and thus play a role in the toxicity associated with these agents. Paraquat and menadione had little effect on the overall oxidation of ethanol by microsomes. Microsomal drug metabolism was decreased by menadione or paraquat. As a consequence, the effect of these agents on the microsomal oxidation of ethanol was complex since it appeared that paraquat and menadione stimulated the oxidation of ethanol by a .OH-dependent mechanism, but inhibited the oxidation of ethanol by a cytochrome P 450-dependent oxidation pathway. Experiments with carbon monoxide, ferric-EDTA, and 2-butanol plus catalase tended to verify that microsomal oxidation of alcohols was increased by a .OH-dependent pathway when menadione or paraquat were added to microsomes. PMID- 2996430 TI - Electron spin relaxation of synthetic melanin and melanin-containing human tissues as studied by electron spin echo and electron spin resonance. AB - Electron spin lattice relaxation times (T1) and the phase memory times (Tm) were obtained for the synthetic melanin system from 3-hydroxytyrosine (dopa) by means of electron spin echo spectroscopy at 77 degrees K. Saturation behavior of the ESR spectra of melanins in melanin-containing tissue and of the synthetic melanin was also determined at the same temperature. The spin lattice relaxation time and the spectral diffusion time of the synthetic melanin are very long (4.3 ms and 101 microseconds, respectively, in the solid state), and the ESR signal saturates readily at low microwave powers. On the other hand, ESR spectra of natural melanins from the tissues chosen for this study, as well as those of synthetic melanins which contain Fe3+ of g = 4.3 and Mn2+ of g = 2, are relatively difficult to saturate compared with samples without such metal ions. These results show clearly that a large part of those two metal ions in sites responsible for the ESR spectral components with these particular g values are coordinated to melanin in melanin-containing tissue, and modify the magnetic relaxation behavior of the melanin. Accumulations of these metal ions in melanins are different from system to system, and they increase in the order: hair (black), retina and choroid (brown), malignant melanoma of eye and skin, and lentigo and nevus of skin. PMID- 2996431 TI - Structural requirements for the biological activity of thymopentin analogs. AB - Thymopentin, the synthetic pentapeptide Arg-Lys-Asp-Val-Tyr, corresponds to residues 32-36 of the thymic hormone thymopoietin. Thymopentin, like thymopoietin, induces intracellular cGMP elevations in the human T-cell line, CEM. Thymopentin also displaces radiolabeled thymopoietin from a receptor glycoprotein prepared from CEM cells, provided that a nonapeptide corresponding to thymopoietin is added to block thymopoietin binding to an additional binding site. Twenty nine analogs with single position substitutions were synthesized by solid-phase or classical solution synthesis, and are evaluated in these assays. All analogs that were active gave positive effects in both assays. A number of substitutions were tolerated at positions 2, 4, and 5, but there was an absolute requirement for L- or D-Arg at position 1 and L- or D-Asp at position 3 to maintain biological activity. PMID- 2996432 TI - Chemical and kinetic characterization of tadpole back skin collagenase. AB - The purified collagenase from tadpole (Rana catesbiana) back skin was studied with respect to its activation energy using soluble and fibrillar type I collagen, as well as a synthetic peptide substrate, DNP-Pro-Gln-Gly-Ile-Ala-Gly Gln-D-Arg. The activation energy appeared to be independent of the nature of the substrate, ranging between 28 and 35 kcal/mol. The peptide was cleaved at the Gly Ile bond and proved to be a poor substrate (kcat/Km, 1.21 h-1 microM-1) when compared with native type I collagen in solution (kcat/Km, 40.6 h-1 microM-1), consistent with the enzyme's low activity versus gelatin [T. A. Bicsak and E. Harper (1984) J. Biol. Chem. 259, 13145]. The amino acid composition of the collagenase was shown to be high in glycine and glutamic acid, and the preparation was shown not to be contaminated with collagen by digestion with bacterial collagenase. The enzyme was not inhibited by iodoacetic acid or 2 hydroxy-5-nitrobenzyl bromide, suggesting the lack of essential cysteinyl and tryptophanyl residues, but was inhibited by micromolar concentrations of ZnCl2, consistent with the presence of essential histidine(s). Ethoxyformic anhydride irreversibly inhibited the collagenase suggesting the presence of essential lysyl residues. PMID- 2996433 TI - Studies on the dark/light regulation of maize leaf pyruvate, orthophosphate dikinase by reversible phosphorylation. AB - In maize leaves, pyruvate, orthophosphate dikinase (PPDK) is deactivated in the dark and reactivated in the light. Studies in vitro using purified PPDK and a partially purified regulatory protein from maize confirmed previous reports correlating deactivation/reactivation with the reversible phosphorylation/dephosphorylation of a threonyl residue. By monitoring the stability of the exogenous 32P-labeled adenylate substrates during deactivation, we have firmly established ADP as the specific phosphate donor. In isolated maize leaf mesophyll protoplasts preilluminated with 32Pi, we observed a three- to fivefold higher PPDK activity in situ in the light, and a corresponding three- to fivefold higher level of phosphorylation of the 94-kDa PPDK protomer in the dark. HPLC-based phosphoamino acid analysis of PPDK purified from maize leaves of both light- and dark-adapted plants revealed the presence of P-serine. The inactive enzyme from dark-adapted plants (inactivated in vivo) also contained P-threonine. Total phosphate content of PPDK purified from leaves of light-adapted plants was approximately 0.5 mol/mol protomer, and 1.5 mol/mol protomer from leaves of dark adapted plants. Since the difference between enzyme purified from light-adapted (active PPDK) and dark-adapted (inactive PPDK) plants is the presence of P threonine in the latter, this suggests an inactivation stoichiometry in vivo of 1 mol P-threonine/mol 94-kDa protomer. These complementary studies with maize leaf PPDK in vitro, in situ, and in vivo provide convincing evidence for the dark/light regulation of this key C4-photosynthesis enzyme by reversible phosphorylation. PMID- 2996434 TI - Identification of two populations of cardiac microsomes with nitrendipine receptors: correlation of the distribution of dihydropyridine receptors with organelle specific markers. AB - Conventional sarcolemma and microsome preparations from rabbit and cat ventricular muscle were fractionated on continuous linear sucrose gradients. The distribution of nitrendipine receptors was compared with the distribution of organelle specific markers. For the conventional sarcolemma preparation, the dihydropyridine receptor distribution matched the pattern for external membrane markers in position and shape. The number of nitrendipine receptors was three times the number of muscarine binding sites (approximately 1.0 pmol/mg protein) at the isopycnic point of the vesicles. In contrast, two populations of vesicles with nitrendipine receptors were found in the microsome preparations. One population banded with the external membrane vesicles at a mean buoyant density of 24% (w/w) sucrose. The specific content of dihydropyridine receptors (0.2 pmol/mg) was 1/5 that for the muscarine receptors. The second and major population followed the distribution of an Mr 300K polypeptide, a marker for the junctional cisternae of the sarcoplasmic reticulum (SR). Muscarine receptors, however, were also present throughout that band, albeit at a reduced specific content (approximately 0.1 pmol/mg) compared to the light vesicles. The nitrendipine specific content increased over threefold from that of the light vesicles such that the relative content (nitrendipine/muscarine) was twice that determined for the conventional sarcolemma preparation. Nitrendipine receptors were not associated with nonjunctional SR or mitochondria. The light and heavy microsome populations were incubated with 0.2 mg digitonin/mg protein, a treatment which preferentially perturbs the isopycnic point of external membrane vesicles. For the light vesicles, the membranes with muscarine and nitrendipine receptors became heavier than the bulk of the SR. In contrast, after digitonin treatment of the heavy vesicle population, the nitrendipine and muscarine receptors and the SR marker appeared to comigrate into a sharpened band at 39% sucrose. The possibility that the dihydropyridine binding sites in the heavy microsome population are on external membrane vesicles physically linked to the junctional SR is discussed. PMID- 2996435 TI - Spectral characteristics and catalytic properties of thyroid peroxidase-H2O2 compounds in the iodination and coupling reactions. AB - Hog thyroid peroxidase (TPO) was highly purified in order to study the spectral properties and catalytic specificities of its H2O2 compounds in iodothyronine biosynthesis. Purified TPO exhibited a Soret spectrum with an absorption maximum at 410 nm and had an A410/A280 value of 0.55. Protein iodination was only catalyzed under conditions which allowed formation of the transient TPO compound I (Fe(IV)-pi o+). On addition of an equimolar amount of H2O2, TPO formed a stable compound with an absorption maximum at 417 nm. This compound efficiently catalyzed the coupling reaction, but was unable to iodinate proteins. It catalyzed the formation of 1 mol iodothyronines/mol TPO, and therefore retained two oxidizing equivalents per molecule. It is proposed that this compound constitutes a second form of compound I whose structure might be Fe(IV)-Ro, analogous to that of cytochrome c peroxidase compound I. In the presence of an excess of H2O2, it formed TPO-compound III with an absorption maximum at 420 nm. TPO-compound III catalyzed neither the iodination nor the coupling reaction. PMID- 2996436 TI - Phospholipid methyltransferase activity in pancreatic islets: activation by calcium. AB - Pancreatic islet homogenates contain a Mg2+-requiring phospholipid methyltransferase activity, the activity of which was doubled by calcium (K0.5 less than 5 microM). Other divalent metal ions stimulated the activity from 11 to 35%, but zinc and strontium were inhibitory. Cyclic AMP had no effect on the enzyme activity and cyclic GMP inhibited it slightly. Calcium increased the Vmax of the enzyme without affecting its Km with respect to S-adenosylmethionine (6 microM). Chlorpromazine, trifluoperazine, and dibucaine inhibited the calcium stimulatable activity without affecting the activity in the absence of calcium. Phosphatidylserine stimulated, and arachidonic acid and palmitic acid inhibited, the basal enzyme activity. The methylated products were found to be primarily mono- and dimethylphosphatidylethanolamine (30%) and phosphatidylcholine (43%) and an, as yet unidentified, nonpolar lipid fraction (27%), as judged by thin layer chromatography. In the presence of calcium, incorporation of methyl groups into phosphatidylcholine, mono- and dimethylphosphatidylethanolamine, and nonpolar lipids was increased by 131, 60, and 46%, respectively. Based on the localization of the enzyme activity in the insulin secretory granule fraction, it is proposed that phospholipid methylation plays a role in coupling the stimulus to the initial events in insulin secretion, leading to the exocytosis of insulin. PMID- 2996437 TI - The stereochemical configuration of the natural 1 alpha,25-dihydroxyvitamin D3 26,23-lactone. AB - Four possible diastereoisomers of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone were chemically synthesized and compared with the natural metabolite by high pressure liquid chromatography. The four synthetic diastereoisomers of 1 alpha,25 dihydroxyvitamin D3-26,23-lactone could be separated into three peaks by high pressure liquid chromatography. The naturally occurring 1 alpha,25 dihydroxyvitamin D3-26,23-lactone isolated from dog serum and in vitro incubation of chick kidney homogenates comigrated with 23(S)25(R)-1 alpha,25 dihydroxyvitamin D3-26,23-lactone. The four diastereoisomers of 1 alpha,25 dihydroxyvitamin D3-26,23-lactone were tested against naturally occurring 1 alpha,25-dihydroxyvitamin D3-26,23-lactone to determine their relative competition in the 1 alpha,25-dihydroxyvitamin D3-specific cytosol receptor binding assay for 1 alpha,25-dihydroxyvitamin D3. 23(S)25(S)-1 alpha,25 Dihydroxyvitamin D3-26,23-lactone was the best competitor followed by 23(R)25(R) 1 alpha,25-dihydroxyvitamin D3-26,23-lactone and 23(R)25(S)-1 alpha,25 dihydroxyvitamin D3-26,23-lactone, and 23(S)25(R)-1 alpha,25-dihydroxyvitamin D3 26,23-lactone was the poorest competitor. Natural 1 alpha,25-dihydroxyvitamin D3 26,23-lactone isolated from dog serum had almost the same binding affinity as that of 23(S)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone. These data unequivocally demonstrate that the stereochemistry of the natural 1 alpha,25 dihydroxyvitamin D3-26,23-lactone has the 23(S) and 25(R) configuration. PMID- 2996438 TI - [Low-dose intermittent intra-arterial infusion chemotherapy]. AB - Arterial infusion chemotherapy is commonly-used modality for controlling cancers located in specific regions. Previously we described a new method of intra hepatic arterial catheterization through the left subclavian artery using a subcutaneously-implanted silicone reservoir. In the present paper, we report our experience using a low dose-intermittent intraarterial (i.a.) infusion chemotherapy. Since February, 1982, 70 patients including 44 cases of metastatic liver cancer, 16 cases of primary hepatocellular carcinoma and 10 cases of other gastrointestinal malignancies, have been treated with this low dose-intermittent i.a. infusion chemotherapy, the drugs used being as follows. 1) MMC 4 mg, 5-FU 500 mg, AraC 40 mg/2w, 2) MMC 4 mg/w, 3) 5-FU 500 mg/w, MMC 4 mg/2w, ADM 30 mg/4w. Here, we briefly review the effectiveness of this modality for controlling regional diseases including liver metastases. The average hospital-free interval was 156 days and partial responses were observed in 43% (21/49) of cases. Side effects during the therapy were only mild bone marrow suppression and anorexia, which were tolerable in out-hospital care. We also studied the pharmacokinetics of i.a. infusion into the liver in comparison with i.v. infusion using 99mTc-RBC, and found that the ratio of i.a. to i.v. with regard to trans-arterial drug delivery to the liver was 10.0. From the viewpoints of first pass effect and increased local concentration theory, this ratio suggests that the effectiveness of a low-dose anti-tumor agent administered intraarterially is not so low. Accordingly, we believe that low dose-intermittent i.a. infusion chemotherapy is beneficial as an induction and maintenance chemotherapy for patients with regionally located cancers because it is effective, safer and prolongs the hospital-free interval. PMID- 2996439 TI - [One-shot therapy and transcatheter arterial embolization (TAE) therapy of unresectable hepatocellular carcinoma]. AB - 407 cases of unresectable hepatocellular carcinoma (HCC) occurring from 1970 to March 1985, including 107 cases receiving conservative therapy, 176 cases receiving one-shot therapy and 124 cases receiving transcatheter arterial embolization (TAE) therapy, were studied and the efficacy of chemotherapy was compared with that of TAE therapy. The results were as follows; One-year survival rate was 2.8% with a median survival time of 1.3 months in conservative therapy. In the 176 cases of one-shot therapy, one-year survival rate was 21.0%, two-year 6.8% and three-year 2.3% and the median survival time was 4.8% months. In 120 cases of one-shot therapy which were compatible with criteria for one-shot injection of anticancer drugs via the hepatic artery for HCC, one-year survival rate was 30%. However the rate was 1.8% in 56 cases which were not compatible with the criteria. In 37 cases in which Mitomycin C (MMC) and Adriamycin (ADR) were administered alternately, one-year survival rate was 41.7%, two-year 16.1% and three-year 4.3%. The highest survival rate was obtained by TAE therapy. One year survival rate was 66.9%, two-year 33.8% and three-year 28.9%. Decrease of AFP after therapy was noted in 42.4% of cases given one-shot therapy and in 95.2% of cases given TEA therapy. The results suggest that alternate administration of anticancer agents produces good chemotherapeutic effects and that the best life prolongation is obtained by TAE therapy. PMID- 2996440 TI - [Basic and clinical study of liver cancer using intra-arterial infusion of an emulsion of the oily contrast medium lipiodol with 5-FU]. AB - An emulsion of Lipiodol and the anti-cancer agent 5-FU has been made using the microtip of an ultrasonic homogenizer, and we have been performing super selective intra-arterial infusions of the emulsion by the Seldinger method on patients with liver tumor. In our basic study, the emulsion was in a water-in-oil state in which 5-FU granules 5-45 mu in diameter were enclosed in the Lipiodol. In an in vivo experiment, the concentration of 5-FU in the tumor tissue remained as high as that existing 30 minutes after infusion of the emulsion, while almost no activity was detected 30 minutes after infusion of 5-FU only. Therefore, the use of Lipiodol as a carrier enables the anti-cancer agent to reach the affected site and makes it possible to obtain a high concentration of the drug within the tumor itself in cases of liver tumor with abundant vasculature significantly involving the hepatic artery. In addition, with this method, it is possible to select different drugs according to the drug-sensitivity of the particular tumor, and the side-effects are extremely slight. In conclusion, we believe that this method can be an effective therapy not only for unresectable primary liver tumor, but for metastatic liver tumor as well. PMID- 2996441 TI - [Transcatheter hepatic arterial embolization therapy using degradable polylactic acid microspheres]. AB - Transcatheter hepatic arterial embolization therapy (TAE) is an established method for the treatment of hepatocellular carcinoma (HCC). We have developed a new embolus consisting of microspheres with a mean diameter of about 200 microns which are composed of biodegradable polymolecular polylactic and the anti-cancer agent, aclarubicin -HCl, in a ratio of 12% w/w. These microspheres were applied to 28 patients with inoperable HCC using the following method microspheres containing 50 mg of aclarubicin-HCl were injected through a catheter placed in the hepatic artery or one of its branches, 2 or 3 times at intervals of 2 to 4 weeks. The effectiveness was confirmed by decreased levels of serum AFP and regression of the tumor size, and the accumulated survival rate for 6 months was 83.3%. PMID- 2996443 TI - Malignant transformation of eccrine spiradenoma. AB - Two patients are described in whom malignant transformation occurred within an eccrine spiradenoma. Their findings were compared with those of six similar patients from the literature, and a characteristic clinicopathologic picture was evident. The typical history was one of rapid enlargement of a cutaneous nodule of long standing. The neoplasms were composed, at least in part, of large, markedly atypical basaloid cells with numerous mitotic figures. Adjacent benign spiradenoma was observed microscopically in each case, and some of the carcinomas retained architectural features reminiscent of spiradenoma. One patient died of disseminated tumor, but follow-up was short or lacking in several cases. Malignant transformation of eccrine spiradenoma shares many clinical and pathologic features with malignant transformation of dermal cylindroma. PMID- 2996442 TI - [A phase II study of NK171 (etoposide)]. AB - A phase II study of NK171 (etoposide), a semisynthetic derivative of podophyllotoxin, was carried out in patients with primary lung cancer at 46 institutes throughout Japan. Out of 198 patients registered for the study, 130 were judged eligible by the committee for extramural review and the response rate was 13.8% (18/130). The response rates were 17.4% (15/86) by i.v. administration and 6.8% (3/44) by oral administration. In the case of i.v. administration, the response was observed only for small cell carcinoma and the response rate was 32.6% (15/46). In the case of oral administration, the response rates were 16.7% (1/6) for squamous cell carcinoma, 12.5% (1/8) for adenocarcinoma and 3.4% (1/29) for small carcinoma. As a hematological toxicity, leukopenia (less than 3,000/mm3) was observed in 66.7% and 35.3% of cases treated, for i.v. and oral administration, respectively. With regard to the clinical picture, alopecia was observed at high incidences of 73-82% in the cases of both i.v. and oral administration. PMID- 2996444 TI - Can potassium citrate replace sodium bicarbonate and potassium chloride of oral rehydration solution? AB - Ninety four children aged less than 5 years with diarrhoeal dehydration and acidosis were treated randomly with either World Health Organisation (WHO) oral rehydration solution containing sodium chloride, potassium chloride, sodium bicarbonate and glucose or an oral solution with tripotassium citrate monohydrate replacing the sodium bicarbonate and potassium chloride in the WHO solution. Fifty five children (58%) were hypokalaemic (potassium less than 3.5 mmol/l) on admission. All but two in the citrate group were successfully treated. There were no significant differences in rehydration solution intake, stool output, gain in body weight, and fall in plasma specific gravity and haematocrit between the two treatment groups after 48 hours' treatment. Significant improvement in the serum potassium concentration was observed in the hypokalaemic children receiving potassium citrate solution compared with children receiving WHO solution after 24 and 48 hours' treatment. None developed hyperkalaemia. Although children receiving potassium citrate solution corrected their acidosis at a slower rate than the WHO solution group during the first 24 hours, by 48 hours satisfactory correction was observed in all. Tripotassium citrate can safely replace sodium bicarbonate and potassium chloride and may be the most useful and beneficial treatment for diarrhoea and associated hypokalaemia. PMID- 2996446 TI - Cyclic AMP and cyclic AMP-dependent protein kinase in mouse skin. I. Systemic effects of retinoids and PUVA. PMID- 2996445 TI - Inhibitory effects of dapsone on enzymatic activities of membrane phospholipids in human blood cells. AB - In order to elucidate the action mechanism of dapsone on blood cell membranes, we assessed the dose-dependent effect of dapsone on the activities of choline phosphotransferase (which mediates the production of the structural phospholipid, phosphatidylcholine) and methyltransferase (which produces phosphatidylcholine from phosphatidylethanolamine, representing the dynamics of the cells) in the membranes of red cells, lymphocytes, and neutrophils obtained from 16 healthy human subjects. The methyltransferase activity of lymphocyte and neutrophil cell membranes was slightly inhibited by dapsone, although only at a high concentration (1 mM), while that of red cells was not affected. On the other hand, dapsone significantly decreased the choline-phosphotransferase activity of red-cell membranes in a dose-dependent fashion, but did not significantly inhibit that of lymphocytes or neutrophils. The mechanisms of the hemolytic side effect of dapsone on erythrocytes and its anti-inflammatory effect on neutrophils are discussed in connection with its inhibitory effect on the enzymatic activities of membrane phospholipids. PMID- 2996447 TI - Retention of lead in the rat. PMID- 2996448 TI - Serum levels of vitamins A and E in women with ovarian cancer. AB - Serum concentrations of the vitamins A and E were measured in 38 postmenopausal women with either malignant, borderline, or benign epithelial ovarian tumors or with a normal ovary. No statistically significant differences were found in the levels of the vitamins between the various groups. The levels of both vitamins in the ovarian vein were also measured in malignant and benign ovarian tumors. The concentration of vitamin A in the ovarian vein of a patient with a malignant tumor was lower than in the peripheral vein, although the difference was not significant. The findings indicate that the vitamin A and E are not of great importance to the metabolism of epithelial ovarian cancer. PMID- 2996450 TI - Hyperparathyroidism associated with the enlargement of two or three parathyroid glands. AB - Eighty-five (23%) of 375 patients undergoing surgery for primary hyperparathyroidism were found to have enlargement (greater than 50 mg) of two or three parathyroid glands. Of 76 patients followed from 12 to 140 months after surgery, eight (10.5%) developed hypercalcemia at 1, 4, 45, 64, 74, 79, 84, and 133 months. In a comparison of pertinent preoperative biochemical and pathologic data between 55 patients with two- or three-gland hyperparathyroidism and 55 age- and sex-matched patients with single-gland hyperparathyroidism, only the preoperative serum phosphate differed significantly, being lower in the patients with single-gland disease (2.4 +/- 0.1 vs. 2.6 +/- 0.1; p less than 0.04). In the eight patients with two- or three-gland hyperparathyroidism who developed postoperative hypercalcemia, the preoperative concentrations of serum calcium were lower (10.8 +/- 0.2 vs. 11.5 +/- 0.2; p less than 0.019), the preoperative concentrations of serum phosphate were higher (3.1 +/- 0.2 vs. 2.5 +/- 0.1; p less than 0.020), and the weights of the excised parathyroid tissues were less (356 +/- 72 mg vs. 1354 +/- 215 mg; p less than 0.02) than those of patients with two- or three-gland disease who did not develop postoperative hypercalcemia, indicating a milder form of hyperparathyroidism. In the 68 patients without recurrent hypercalcemia, there was no tendency for the serum calcium concentration to increase with time. Patients with primary hyperparathyroidism associated with two or three enlarged parathyroid glands have an appreciable incidence of persistent or recurrent hypercalcemia, which may increase even further with longer observation. PMID- 2996449 TI - Role of liver transplantation in cancer therapy. AB - Fifty-four patients underwent total hepatectomy and liver replacement in the presence of a primary liver malignancy. In 13 recipients in whom the hepatic tumors were incidental to some other endstage liver disease, recurrence was not seen and 12 of the 13 patients are alive after 4 months to 15 1/2 years. In contrast, tumors recurred in 3 of every 4 patients who received liver replacement primarily because of hepatic malignancies that could not be resected by conventional techniques of subtotal hepatectomy and who lived for at least 2 months after transplantation. The most encouraging results were in patients with the fibrolamellar hepatocellular carcinomas that grow slowly and metastasize late, but even with this lesion, the recurrence rate was 57%. In future trials, additional effective anticancer therapy will be needed to improve the results of liver transplantation for primary liver malignancy, but what an improved strategy should be has not yet been defined. PMID- 2996451 TI - The etiology of the Klippel-Trenaunay syndrome. AB - The etiology of the Klippel-Trenaunay syndrome (KTS) remains obscure. Although venous hypertension secondary to deep venous obstruction has been suggested as a cause, recent studies have demonstrated that most patients have unimpeded venous drainage. Calf blood flows have been measured in 33 patients with KTS using venous occlusion plethysmography. Although all flow rates were within normal limits, flow in affected limbs was invariably greater than in normal limbs (p less than 0.001), and this is related to the presence of the nevus. Biopsies of subcutaneous veins demonstrate the histological features of a response to chronically raised flow. The authors suggest that KTS is caused by mesodermal abnormality during fetal development, leading to the maintenance of microscopic arteriovenous communications in the limb bud, as a result of which the triad of nevus, hypertrophy, and superficial varices is produced. Deep venous abnormalities occur pari passu with the triad and are not responsible for its development. PMID- 2996452 TI - In vivo and in vitro actions of low-molecular-weight gonadal peptides. AB - Two low-molecular-weight gonadal peptides, IRRP3 and IRRP5, have been isolated from sheep ovaries having different biological properties in male rats. The peptide IRRP3 has specific effect on FSH secretion as well as release at the pituitary level, in vitro binding of labeled FSH to testicular receptor, and in vivo uptake of labeled FSH by the testis, whereas peptide IRRP5 has specific effect on LH secretion at the pituitary level, in vitro binding of labeled LH to testicular receptor, and in vivo uptake of labeled LH by the testis. PMID- 2996453 TI - Pharmacological analysis of the postjunctional alpha-adrenoceptors of the rat anococcygeus muscle and vas deferens. AB - The alpha-adrenoceptor sub-type of the rat vas deferens was characterized with noradrenaline (alpha-1, alpha-2-agonist), phenylephrine (alpha-1-agonist), prazosin (alpha-1-antagonist) and yohimbine (alpha-2-antagonist) and compared with the results obtained in the rat anococcygeus muscle. In the vas deferens, both prazosin and phentolamine were competitive antagonists of noradrenaline and phenylephrine whereas yohimbine was a non-competitive antagonist irrespective of the concentrations of these antagonists. On the other hand, in the anococcygeus muscle, at low concentrations, prazosin was non-competitive against noradrenaline whilst it was competitive against phenylephrine. Yohimbine behaved as a competitive antagonist of noradrenaline at both low and high concentrations whilst at low concentrations it was non-competitive against phenylephrine. These results suggest a predominance of alpha-1-adrenoceptors in the vas deferens located postjunctionally with a small population of alpha-2-subtype adrenoceptors, whilst the anococcygeus muscle seems to contain relatively more alpha 2-adrenoceptors postjunctionally than the vas deferens. PMID- 2996454 TI - Effect of lithium treatment on blood pressure and angiotensin-converting enzyme activity in normotensive Wistar-Kyoto and spontaneously hypertensive rats. AB - Short-term administration of LiCl (4 mEq/kg) reduced blood pressure of SH rats but had no effect on WKY animals. Lower doses of lithium, however, did not alter blood pressure in the SH or WKY rats. Acute and chronic administration of lithium had no appreciable effect on heart rate. Although all rats gained weight during the treatment period, the per cent increase in body weight of LiCl (4 mEq/kg) treated SH and WKY rats was significantly lower than the corresponding vehicle treated animals. Angiotensin converting enzyme (ACE) activity of anterior pituitary of WKY rats was significantly higher than that of SH rats. ACE activity of neurohypophysis, instead was lower in WKY rats than in SH rats. ACE activities in plasma, striatum and hypothalamus of SH and WKY rats did not differ. After short-term lithium treatment (4 mEq/kg), plasma ACE activity was significantly reduced only in the SH animals although ACE activities in pituitary, striatum or hypothalamus remained unaffected in the SH animals. The results suggest that the antihypertensive effect of lithium in SH animals may be related to decreased plasma ACE activity. PMID- 2996455 TI - Pharmacodynamic properties of adrenal cortical extracts in comparison to synthetic corticosteroid mixture in the rat. AB - The effects of a total extract of adrenal cortex on corticosterone and ACTH plasma levels have been studied in the rat, either during circadian rhythms or in conditions of stress, in comparison to the effects of a synthetic corticosteroid mixture. A total extract of adrenal cortex, acutely and chronically administered at low and high doses, showed an inhibitory effect on plasma steroids and ACTH, whereas a synthetic corticosteroid mixture was more effective in producing such an inhibition. On the other hand, rats treated with a total extract of adrenal cortex responded better to stress than animals treated with a synthetic corticosteroid mixture. This difference may be due to the presence in the total extract of the adrenal cortex of certain substances having stimulatory properties on the steroidogenesis and/or on the activity of the hypothalamus-hypophysis adrenal axis. PMID- 2996456 TI - [Determination of virus-specific antibodies against the infectious bovine rhinotracheitis/infectious pustular vulvovaginitis virus in cattle sera using ELISA and the serum neutralization test]. PMID- 2996457 TI - [Intraerythrocytic acid-base status in decompensated metabolic acidosis and with parenteral buffer administration]. PMID- 2996458 TI - Ataxia telangiectasia with hepatocellular carcinoma in a 15-year-old girl and studies of her kindred. AB - We studied a case of familial ataxia-telangiectasia in a 15-year-old girl who had a clinical history of cerebellar ataxia and recurrent pulmonary infections. She was found at autopsy to have a hepatocellular carcinoma, which has been described twice previously in the literature as occurring with this disorder. Family studies on the majority of her seven siblings (the product of one father and two mothers who were identical twins) showed one brother to have classic features of cerebellar ataxia, IgA deficiency, markedly elevated alpha-fetoprotein levels, and characteristic chromosomal abnormalities. This boy also died later of hepatocellular carcinoma in 1984. An affected sister had previously died of a respiratory tract infection. PMID- 2996459 TI - Myeloperoxidase deficiency. Increased sensitivity for immunocytochemical compared to cytochemical detection of enzyme. AB - The discovery of hereditary deficiency of myeloperoxidase (MPO) in neutrophils and monocytes of affected individuals has been based on the absence of cytochemical staining in these peripheral blood cells. We report that an immunocytochemical method shows more sensitivity than either the benzidine or 4 chloro-1-naphthol cytochemical methods. In MPO-deficient subjects, immunocytochemistry detects a marked decrease, but not absence, of MPO. PMID- 2996460 TI - Lymph node immunohistology in intravenous drug abusers with persistent generalized lymphadenopathy. AB - Lymph node biopsy specimens from 16 intravenous drug abusers with persistent generalized lymphadenopathy were evaluated by immunohistochemical methods using a panel of antisera to detect different cell populations. The 11 cases that we tested on cryostat sections showed an increased number of Leu-2a-positive cells (cytotoxic-suppressor phenotype) in the follicular centers and a significantly reduced helper-to-suppressor T-cell mean ratio when compared with control tissues. In these 11 patients the peripheral helper-suppressor ratio was at the lower normal limit or inverted. Ten cases tested for anti-human T-cell lymphotropic virus type III antibodies were positive. In all 16 cases, immunohistology of paraffin-embedded sections demonstrated a polyclonal B population; 12 of 15 patients tested had polyclonal hypergammaglobulinemia, mostly IgG. The mixed-cell population of the lymph node sinuses was composed mostly of Leu-M1-positive and lysozyme-positive cells and, to a lesser extent, by alpha 1-antichymotrypsin-positive and S100 protein-positive cells. It seems that many of the immunologic dysfunctions found in these patients appear to be reflected in a fairly repetitive immunohistologic pattern. PMID- 2996461 TI - Lymphadenopathy during cytomegalovirus-induced mononucleosis in guinea pigs. AB - Hartley guinea pigs infected with guinea pig cytomegalovirus (GP-CMV) develop a mononucleosis syndrome during primary infection that is similar to that seen in immunocompetent humans. In the present study this animal model of human disease was used to investigate the sequential changes in the lymph nodes during the course of primary GP-CMV infection. Infectious virus could be isolated from the nodes up to eight weeks after inoculation. When compared with nodes of control animals, the nodes of GP-CMV-infected animals were found to be enlarged up to a year after infection. During the first ten days of infection, histologic changes due to virus proliferation and immune stimulation of the paracortical areas of the lymph node, in a diffuse hyperplasia pattern, were noted. Although typical cytomegalovirus inclusions were seen only rarely, many cells demonstrated intranuclear staining using an avidin-biotin immunoglucose-oxidase histochemical reaction for GP-CMV. During late acute and chronic infection, the lymph nodes showed immune stimulation of the germinal centers, in the pattern of follicular hyperplasia. Specific histologic changes related to active virus proliferation were not seen after the early acute phase. PMID- 2996463 TI - Investigations in the thiazolidine series. PMID- 2996462 TI - Primary lymph node pathology in AIDS and AIDS-related lymphadenopathy. AB - Lymph nodes of patients symptomatically infected with the acquired immunodeficiency syndrome (AIDS) virus show a spectrum of morphologic changes ranging from marked lymphoid hyperplasia to marked lymphocytic depletion. These changes can be grouped into three distinct patterns. The type I pattern features follicular and paracortical hyperplasia, and is associated with chronic lymphadenopathy. The type II pattern, which shows diffuse lymphoid hyperplasia but loss of germinal centers, signifies evolution of chronic lymphadenopathy to AIDS. The type III pattern shows marked lymphocytic depletion and represents the end-stage lymph node seen in fatal AIDS. These histologic patterns are closely correlated with the clinical and immunologic status of patients infected with the AIDS virus. PMID- 2996464 TI - Lumbosacral radiculopathy. PMID- 2996465 TI - Evidence for a captopril-sensitive angiotensin converting enzyme in the hindquarter vasculature of SHR and WKY. AB - Vascular angiotensin converting enzyme could contribute to the elevated vascular resistance found in hypertension. The purpose of this study was to determine if angiotensin converting enzyme activity was present in the hindquarter vasculature of one model of hypertension, the spontaneously hypertensive rat (SHR) and its normotensive control, the Wistar-Kyoto rat (WKY). We evaluated the effect of a maximal blocking dose of captopril (0.5 mg) on the angiotensin I pressor response during the infusion of the hindquarter with an artificial perfusate. Angiotensin I (1000 ng/ml) produced a significant increase in peripheral vascular resistance (PVR) in both SHR and WKY, but the increase was greater in SHR. Captopril inhibited the elevation in PVR in both. A lower concentration of angiotensin I (250 ng/ml) produced a significant and similar pressor response in SHR (less than the pressor response to 1000 ng/ml) and WKY (same as the pressor response to 1000 ng/ml). Again, captopril prevented the elevation in PVR to A1 in both SHR and WKY. Because these studies were performed using an artificial perfusate, angiotensin converting enzyme must be present in the SHR and WKY hindquarter vasculature including resistance vessels. PMID- 2996467 TI - [Changes in skeletal muscles in experimental thyrotoxic myopathy]. AB - In an experimental model of thyrotoxic myopathy in mice certain decrease in average diameter of muscle fibers (MF) has been revealed (by 16%). In 1-20% of the MF various types of focal pathologic reactions (loss of cross and longitudinal striation, glial, glomerular and adipose degeneration, Zenker's necrosis) increasing number, structural changes and position of nuclei are observed. Degree of atrophy and part of the altered fibers depend on duration and severity of thyrotoxicosis. Morphologic disorders localize focally and are not so vast as to be the cause of muscle weakness. The main cause of the latter and of the structural disorders in the skeletal muscle at thyrotoxic myopathy is, evidently, slacken of the hormonal control in cyclic adenosine monophosphate dependent processes. PMID- 2996466 TI - [Manifestation of the neurotoxic effect of the organochlorine pesticide hexachlorobutadiene in the postnatal period of ontogeny in the rat]. AB - Daily peroral administration of chlororganic pesticide hexachlorobutadiene in doses 8.1 mg/kg (1/20 LD50) to pregnant rats results in certain ultrastructural changes of neurocytes and myelin fibers of the spinal cord both in the animals and their offspring (newborns and 1-2-month-old rats). By means of electron paramagnetic resonance (EPR) method, changes in intensity of the EPR-signals of free radicals in the spinal cord, ceruloplasmin of blood serum have been revealed in the experimental pregnant animals, as well as in 1-month-old rats (in the latter--in the brain, too). Gas-liquid chromatography reveals the preparation contents in the adrenals, heart, brain and spinal cord, in the uterus of the pregnant animals, as well as in corresponding organs of their offspring. Certain retardation in growth and decrease in body mass are noted in the offspring. PMID- 2996469 TI - [Granular-cell tumor of the rectum]. AB - Histological and ultrastructural description of a granular-cell tumor of the rectum in a male of 33 is presented. PMID- 2996468 TI - [Correlation between the electron microscopic picture of small cell cancer of the lung and treatment results]. AB - Electronmicroscopic study of 35 small-cell lung carcinomas showed these to be a group of lung carcinomas having different histogenesis (cytogenesis). Apart from carcinoma without signs of differentiation, this group also includes squamous cell carcinoma, adenocarcinoma, apudoma and mixed carcinoma with various differentiation of tumour cells. A certain correlation between the ultrastructural features of the tumour and its susceptibility to the treatment is established. Small-cell carcinomas consisting of undifferentiated cells are most susceptible. A relatively good prognosis can be expected when a variant with the endocrine differentiation of tumour cells is treated. Tumours interpreted light microscopically as small-cell carcinoma but ultrastructurally identified as having squamous-cell differentiation, or as adenocarcinoma and mixed carcinoma have the least favourable prognosis after treatment. PMID- 2996471 TI - [Ultrastructural changes in the skin of patients with Fabry's angiokeratoma]. AB - The skin of a patient with Fabry's diffuse angiokeratoma accompanied by a severe decrease of leucocyte alpha-galactosidase (0,7-1,2 nmol/mg protein/h) was studied by a method of semithin and ultrathin sections. Cytoplasmic inclusions having lamellar structure in the form of alternating electron-dense and light strips with a period about 6 nm were found in the endotheliocytes of dilated vessels, lymphoid cells, neutrophil leucocytes, axons and leucocytes of nerve trunks. The presence of these specific inclusions together with the decrease of leucocytic alpha-galactosidase allows the differential diagnosis with other types of angiokeratomas and some skin angiomas. PMID- 2996470 TI - [Histogenesis of malignant fibrous histiocytoma and giant-cell tumor of bone]. AB - Ten tumours: 5 malignant fibrous histiocytomas (2 with a primary site in the bone and 3 in the soft tissue), 3 giant cell bone tumours (one of them was malignant with metastasis to the lung) and 2 osteogenic sarcomas were studied in organ culture. The blood was added into some tumour pieces with a subsequent examination of blood phagocytosis by means of checking the hemosiderin granule accumulation in the cell cytoplasm. Malignant fibrous histiocytoma and giant cell bone tumour were very similar by the pattern of their growth and phagocytic activity of their cells; both differed strikingly from the osteogenic sarcoma. This implies the histogenetic similarity of malignant fibrous histiocytoma and giant cell tumour of the bone. PMID- 2996472 TI - The effect of delta-9-tetrahydrocannabinol on rat cerebrospinal fluid. AB - The effect of delta-9-tetrahydrocannabinol (THC) on cerebrospinal fluid (CSF) flow in rats was studied. The ventricular system of rats anesthetized with ketamine sulfate was cannulated via cisternal puncture, and CSF production was recorded. When administered in doses of 25 mg/kg to 45 mg/kg intraperitoneally, THC caused inhibition of CSF flow; in larger doses a smaller response was noted. In response to THC, CSF flow showed an initial drop, a return toward baseline, and a secondary decrease. It is postulated that this biphasic effect is due to a combination of THC's sympathomimetic effects on the CNS plus the local action that this drug has on choroidal synaptosomal neurotransmitters. PMID- 2996473 TI - Swine influenza vaccine and Guillain-Barre syndrome. Epidemic or artifact? PMID- 2996474 TI - Juvenile multisystem degeneration with motor neuron involvement and eosinophilic intracytoplasmic inclusions. AB - A case of juvenile multisystem degeneration with motor neuron involvement, possibly of familial type, showing many unusual clinical and pathologic features is reported. Eosinophilic intracytoplasmic inclusions were present in some remaining anterior horn cells and motor nerve nuclei of the brain stem as well as in a few neurons of the reticular activating system, the dorsal vagus nuclei, and the intermediolateral cell column. Smaller eosinophilic inclusions were seen in large neurons of the caudate nucleus and putamen, substantia nigra, and subthalamic nucleus. PMID- 2996476 TI - Magnetic resonance imaging vs computed tomography. Comparison in imaging oral cavity and pharyngeal carcinomas. AB - Twenty-one patients with oral cavity and pharyngeal carcinomas were enrolled in a prospective protocol to study the diagnostic efficacy of magnetic resonance imaging vs computed tomography. Magnetic resonance imaging was found to be superior to computed tomography in the area of tumor contrast (conspicuity) and equal or inferior in edge definition, delineation of regional disease, and lymph node metastasis. These findings are consistent with the current applications and limitations of magnetic resonance imaging. PMID- 2996475 TI - Verrucous carcinoma of the larynx. Possible human papillomavirus etiology. AB - Verrucous carcinoma of the larynx is a distinct and uncommon variant of well differentiated squamous cell carcinoma. By DNA hybridization techniques, we clearly demonstrated human papillomavirus (HPV-16-related) sequences in five patients with this neoplasm. In addition, HPV-16-related sequences were found in adjacent normal tissues. The DNAs from squamous cell carcinomas of the larynx were negative when hybridized to HPV-6, -11, or -16. Postirradiation anaplastic transformation of verrucous carcinoma has been described. We believe that radiotherapy should not be given unless the potential consequences are fully explained because of its potential to activate or alter HPV-16-related sequences. PMID- 2996477 TI - Tumors of the parapharyngeal space. AB - We retrospectively studied tumors of the parapharyngeal space treated at the Baylor College of Medicine Affiliated Hospital System, Houston, from 1972 to 1985. Of the 42 lesions, 30 (71.4%) were benign and 12 (28.6%) were malignant. Tumors of neurogenic origin were present in 17 (40.5%). Tumors of salivary gland origin were present in 16 (38.1%): ten were benign, six were malignant. Nine (21.4%) of the patients presented with miscellaneous lesions, six of which proved to be malignant. We have found that a preoperative arteriogram is no longer routinely indicated. High-resolution computed tomography is now the best initial diagnostic study because it helps determine the size and extent of the tumor, differentiate tumors of parotid and extraparotid origin, demonstrate degree of tumor vascularity, and separate benign from malignant lesions. PMID- 2996478 TI - The Kendall oration. The changing scene of veterinary administration. PMID- 2996480 TI - Role of ethanol in conditioning aversion to alcoholic beverages in rats. PMID- 2996479 TI - Another look at initiation of human parturition. AB - Despite our moderate understanding of the physiology of the initiation of labour in various animal species, notably ruminants and rodents, the mechanism in man remains enigmatic. In the initiation process in the human the major role is apparently taken by the placental and fetal membranes, while the fetus may modulate the timing of labour. This review sets out to examine current knowledge of the process of initiating and maintaining parturition in the human. PMID- 2996482 TI - [Effect of nonoxynol (Patentex) on forensic sperm detection]. PMID- 2996481 TI - Myocardial catecholamine responsiveness of spontaneously hypertensive rats as influenced by swimming training. AB - Alterations of myocardial mechanical catecholamine responsiveness by swimming training (2 X 90 min/day, 4 weeks) were examined in 13-week-old spontaneously hypertensive males rats (SHR). The relationships between myocardial mechanical catecholamine responsiveness and ventricular beta-adrenoceptors as well as myosin isoenzyme pattern were also examined. Compared with sedentary controls, trained rats showed a greater responsiveness to isoproterenol (10(-6) mol/l) on isometric tension (T) and its first derivative (dT/dt) (delta T: 0.45 +/- 0.55 vs. -0.15 +/ 0.11 10(-2) N/mm2, p less than 0.01, delta dT/dt: 17.1 +/- 10.1 vs. 8.3 +/- 3.6 10(-2) N/mm2 X s, p less than 0.05). In sedentary SHR, dT/dtmax increased significantly, whereas developed tension decreased slightly, coupled with a decrease of time to peak tension by high dose (10(-6) mol/l) isoproterenol. Therefore, it can be stated that dT/dt is a better indicator for catecholamine sensitivity than isometric tension. beta-adrenoceptor density [( 3H] dihydroalprenolol binding) decreased significantly in trained rats (68.7 +/- 7.62 vs. 102.4 +/- 4.37 fmol/mg protein, p less than 0.01) with no significant difference in KD values (4.61 +/- 2.26 vs. 6.11 +/- 1.94 nM, ns). In addition, myosin isoenzyme pattern revealed by pyrophosphate gel electrophoresis shifted towards VM-1 after swimming training. The increased catecholamine sensitivity of fast contracting myocardium is, in principle, compatible with the assumption of cAMP-dependent regulation of myofibrillar ATPase activity (21) or cross bridge kinetics (9), although other postreceptor processes should also be taken into consideration for the increased catecholamine sensitivity. PMID- 2996483 TI - [Comparative serological studies of methods for demonstrating antibodies in infectious laryngotracheitis of chickens]. PMID- 2996484 TI - Fructose bisphosphatase isozymes of the mouse. I. Inheritance. AB - Electrophoretically detectable polymorphisms of fructose bisphosphatase (EC 3.1.3.11) have been found in the mouse. One polymorphism, found among inbred strains of Mus musculus and feral animals, affects the isozymes found in the muscle and in most other tissues examined but is not expressed in kidney, liver, or testis. These tissues have other electrophoretically distinct isozymes which are monomorphic in Mus musculus but are present as a different electromorph in the sympatric species Mus spretus. Breeding data have established that the genetic control of the muscle enzyme is expressed by an autosomal structural locus Fbp-1 which is distinct from that expressing the liver, kidney, and testis enzyme, Fbp-2. The organ-specific expression of the two loci suggests possible functional differences between the two products. PMID- 2996485 TI - An electrometric method for the determination of tyrosinase activity. AB - The pathway of dopachrome formation from L-dopa involves the net release of one proton for each molecule of dopachrome formed. The protons produced as a consequence of the enzymic step catalysed by tyrosinase can be measured by an electrometric device able to monitor changes in H+ concentration below 1 microM. This electrometric recording can be used as a simple, sensitive and continuous method for determining tyrosinase activity. The electrometric method can also be used in the presence of ascorbate by the spontaneous coupling of ascorbate oxidation to dopaquinone reduction, but measuring proton uptake instead of proton release. PMID- 2996486 TI - Elicitor-induced prolyl hydroxylase from French bean (Phaseolus vulgaris). Localization, purification and properties. AB - The enzyme prolyl hydroxylase (proline: 2-oxoglutarate dioxygenase, EC 1.14.11.12), induced in suspension-cultured cells of Phaseolus vulgaris L. (French bean) by treatment with an elicitor preparation from the phytopathogenic fungus Colletotrichum lindemuthianum, has been investigated. The enzyme, which catalyses the hydroxylation of poly-L-proline with the stoichiometric decarboxylation of 2-oxoglutarate, has been shown to be localized mainly in smooth endoplasmic reticulum. After solubilization from microsomal membranes, the hydroxylase was purified by ion-exchange chromatography and affinity chromatography on poly-L-proline-Sepharose 4B. The subunit Mr, as assessed by sodium dodecyl sulphate/poly-acrylamide-gel electrophoresis, was 65 000, the subunit apparently being recovered as a doublet: the subunits associate under non denaturing conditions to give at least a tetramer. The bean hydroxylase has kinetic properties and cofactor requirements similar to those previously reported for the enzyme from other plants. Elicitor treatment of suspension-cultured bean cells leads to a rapid induction of prolyl hydroxylase activity concomitant with induction of a protein: arabinosyl-transferase and increased levels of an arabinosylated hydroxyproline-rich protein. PMID- 2996487 TI - Tumourigenicity, cell-surface glycoprotein changes and ornithine decarboxylase gene pattern in Ehrlich ascites-carcinoma cells. AB - We selected a 2-difluoromethylornithine-resistant Ehrlich ascites-carcinoma cell line that grows in the presence of 20 mM-difluoromethylornithine. These cells contain 10-20 times the normal amount of hybridizable sequences for ornithine decarboxylase (EC 4.1.1.17) in their genomic DNA. We used these gene-amplified cells, their revertant counterparts (grown in the absence of the drug after an established gene amplification) and tumour cells grown in the presence of putrescine to investigate the changes of ornithine decarboxylase gene pattern and simultaneously occurring phenotypic changes, such as tumourigenicity and the expression of cell-surface glycoproteins. In the tumour cells reverted back to the normal gene frequency, not only did the amplified sequences disappear, but there were also signs of gene re-arrangements seen as a "gene jump', when a signal evidently moved to a heavier restriction fragment. Similar gene re arrangement likewise occurred in cells exposed to putrescine. Although the wild type tumour cells and the gene-amplified cells readily grew in the peritoneal cavity of mice, the revertant cells and the putrescine-treated cells had lost their tumourigenicity in mice. Gene-amplified tumour cells and the revertant cells showed distinct changes in their surface glycoprotein pattern in comparison with the parental cell line. These findings indicate that alterations of ornithine decarboxylase gene pattern/dosage may be associated with phenotypic changes possibly related to the tumourigenicity of these carcinoma cells. PMID- 2996489 TI - On the role of the membrane proton conductance in the relationship between rate of respiration and protonmotive force. AB - The rate of respiration in mitochondria is not a unique function of the protonmotive force, depending on whether the protonmotive force is varied by addition of ADP or uncouplers. This result has been generally considered to contradict the chemiosmotic theory. Recently, O'Shea & Chappell [Biochem. J. (1984) 219, 401-404] claimed that this observation can be reconciled with the chemiosmotic theory, provided only that the proton conductance of the membrane is different in the presence of ADP or uncouplers. This hypothesis is shown here to be necessary but not sufficient to account for the experimental data and the reason for the contradiction between this recent interpretation and earlier interpretations is pointed out. PMID- 2996488 TI - Adenosine formation and release from neonatal-rat heart cells in culture. AB - The incorporation of [3H]adenosine (10 microM) into neonatal-rat heart cell nucleotides was inhibited in a concentration-dependent manner, such that 50% inhibition was obtained with 0.75 microM-dipyridamole, 0.26 microM-hexobendine or 0.22 microM-dilazep. Adenosine formation was accelerated 2.5-fold to 2.1 +/- 0.3 nmol/10(7) cells in 10 min when cells were incubated with a combination of 30 mM 2-deoxyglucose and 2 micrograms of oligomycin/ml. Of the newly formed adenosine, 6 +/- 2% was in the cells. Dipyridamole, hexobendine or dilazep (10 microM) increased the amount of adenosine in the cells and decreased that in the medium such that 45-50% of the newly formed adenosine was in the cells. Antibodies which inhibited ecto-5'-nucleotidase by 98.7 +/- 0.3% did not alter the rate of adenosine formation or its distribution between cells and medium. We conclude that adenosine was formed in the cytoplasm during catabolism of cellular ATP and was released via the dipyridamole-sensitive symmetric nucleoside transporter. PMID- 2996491 TI - Adenosine potentiates lutropin-stimulated cyclic AMP production and inhibits lutropin-induced desensitization of adenylate cyclase in rat Leydig tumour cells. AB - The action of adenosine on lutropin (LH)-stimulated cyclic AMP production and LH induced desensitization of adenylate cyclase in rat Leydig tumour cells was investigated. Adenosine and N6-(phenylisopropyl)adenosine caused a dose-dependent potentiation of LH-stimulated cyclic AMP production at concentrations (0.01-10 microM) which alone did not produce an increase in cyclic AMP production. However, 2-deoxyadenosine had no effect either alone or in combination with LH on cyclic AMP production. The potentiation produced by adenosine was unaffected by concentrations of the specific nucleoside-transport inhibitor dipyridamole, which inhibited [3H]adenosine uptake by up to 90%. The phosphodiesterase inhibitor 3 isobutyl-l-methylxanthine, but not RO-10-1724, inhibited the adenosine-induced potentiation. In the presence of adenosine, the kinetics of LH-stimulated cyclic AMP production were linear with time up to 2h, compared with those with LH alone, which showed a characteristic decrease in rate of cyclic AMP production after the first 15-20 min. Consistent with the altered kinetics, adenosine also inhibited the LH-induced desensitization of adenylate cyclase. These results suggest that adenosine has effects on rat tumour Leydig cells through receptors on the external surface of the plasma membrane. This receptor has characteristics similar to those of the R-type receptors, which have been shown either to stimulate or to inhibit adenylate cyclase. However, the effects of adenosine in the present studies does not involve a direct inhibition or activation of adenylate cyclase, but may involve an as yet undefined receptor-mediated modulation of adenylate cyclase. PMID- 2996490 TI - Eukaryotic nuclear ADP-ribosylation reactions. PMID- 2996492 TI - Redox-linked spin-state changes in the di-haem cytochrome c-551 peroxidase from Pseudomonas aeruginosa. AB - Magnetic-c.d., e.p.r. and optical-absorption spectra are reported for the half reduced form of Pseudomonas aeruginosa cytochrome c-551 peroxidase, a di-haem protein, and its fluoride derivative. Comparison of this enzyme species with oxidized peroxidase shows the occurrence of spin-state changes at both haem sites. The high-potential haem changes its state from partially high-spin to low spin upon reduction. This is linked to a structural alteration at the ferric low potential haem group, causing it to change from low-spin to high-spin. Low temperature spectra demonstrate photolysis of an endogenous ligand of the high potential haem. In addition, an inactive form of enzyme is examined in which the structural change at the ferric low-potential haem does not occur on reduction of the high-potential haem. PMID- 2996493 TI - Enzymic determination of inorganic phosphates, organic phosphates and phosphate liberating enzymes by use of nucleoside phosphorylase-xanthine oxidase (dehydrogenase)-coupled reactions. AB - Coupled enzyme assays are described for measuring inorganic phosphates, organic phosphates and phosphate-liberating enzymes in biological material. The assays all determine Pi by its reaction with inosine, catalysed by nucleoside phosphorylase; this yields ribose 1-phosphate and hypoxanthine. The hypoxanthine is oxidized to uric acid by xanthine oxidase, and may be measured either by the absorbance of the uric acid, or by the formazan formed when a tetrazolium salt is used as the oxidant. The coupled enzyme assays are characterized by high sensitivity, quantitative utilization of phosphates and stoichiometric formation of the measurable products, measurement at pH 6.0-8.5, determination of phosphates within a single analytical step, and continuous measurement of phosphohydrolase activity in a corresponding rate assay. Examples include determinations of substrates such as Pi, PPi and AMP, and of enzymes such as 5' nucleotidase, inorganic pyrophosphatase and glucose-6-phosphatase. Directions for further examples are given. PMID- 2996494 TI - Seminalplasmin. An endogenous calmodulin antagonist. AB - Seminalplasmin, a strongly basic protein isolated from bull semen, was found to antagonize with high potency and extraordinary specificity the function of calmodulin. Calmodulin antagonism is the result of an interaction between the two proteins, which is mainly determined by electrostatic forces. The stimulation of Ca2+-transporting ATPase and phosphodiesterase by calmodulin was half-maximally inhibited at approx. 0.1 microM-seminalplasmin. However, the basal activity of calmodulin-dependent enzymes was not significantly altered by seminalplasmin over the concentration range investigated. PMID- 2996496 TI - The inhibition of diacylglycerol-stimulated intracellular phospholipases by phospholipids with a phosphocholine-containing polar group. A possible physiological role for sphingomyelin. AB - Phosphatidylinositol phosphodiesterase activated by diacylglycerol is substantially inhibited by all phospholipids containing a phosphocholine head group, including phosphatidylcholine, hydrogenated phosphatidylcholine, choline plasmalogen, lysophosphatidylcholine, lysocholine plasmalogen, sphingomyelin and sphingosylphosphocholine. The sphingosine-containing phospholipids are the most inhibitory. Phosphatidic acid does not inhibit, and phosphatidylethanolamine activates the hydrolysis still further. Sphingomyelin is highly inhibitory to a diacylglycerol-stimulated intestinal mucosal phospholipase A2, or a liver lysosomal phospholipase A1 + A2, both hydrolysing a phosphatidylcholine substrate. Sphingomyelin [20% molar (20 mol of sphingomyelin/80 mol of phosphatidylethanolamine)] activates phosphatidylethanolamine hydrolysis by intestinal mucosal phospholipase A2, and then at higher concentrations (40% molar) substantially inhibits the activity. The results are discussed in relation to possible molecular reorganizations brought about in the hydrated phospholipid substrate complex, and in particular the possible stabilizing role of sphingomyelin in the maintenance of membrane structure, and hence in the modulation of phospholipase activity. PMID- 2996495 TI - The purification and properties of human liver ketohexokinase. A role for ketohexokinase and fructose-bisphosphate aldolase in the metabolic production of oxalate from xylitol. AB - Ketohexokinase (EC 2.7.1.3) was purified to homogeneity from human liver, and fructose-bisphosphate aldolase (EC 4.1.2.13) was partially purified from the same source. Ketohexokinase was shown, by column chromatography and polyacrylamide-gel electrophoresis, to be a dimer of Mr 75000. Inhibition studies with p chloromercuribenzoate and N-ethylmaleimide indicate that ketohexokinase contains thiol groups, which are required for full activity. With D-xylulose as substrate, ketohexokinase and aldolase can catalyse a reaction sequence which forms glycolaldehyde, a known precursor of oxalate. The distribution of both enzymes in human tissues indicates that this reaction sequence occurs mainly in the liver, to a lesser extent in the kidney, and very little in heart, brain and muscle. The kinetic properties of ketohexokinase show that this enzyme can phosphorylate D xylulose as readily as D-fructose, except that higher concentrations of D xylulose are required. The kinetic properties of aldolase show that the enzyme has a higher affinity for D-xylulose 1-phosphate than for D-fructose 1-phosphate. These findings support a role for ketohexokinase and aldolase in the formation of glycolaldehyde. The effect of various metabolites on the activity of the two enzymes was tested to determine the conditions that favour the formation of glycolaldehyde from xylitol. The results indicate that few of these metabolites affect the activity of ketohexokinase, but that aldolase can be inhibited by several phosphorylated compounds. This work suggests that, although the formation of oxalate from xylitol is normally a minor pathway, under certain conditions of increased xylitol metabolism oxalate production can become significant and may result in oxalosis. PMID- 2996497 TI - Mast-cell products and heparin stimulate the production of mononuclear-cell factor by cultured human monocyte/macrophages. AB - Purified mast cells derived from rat peritoneal fluids and dog mastocytomas were extracted with 1 M-NaCl and sonication techniques. The mast-cell products increased the production of mononuclear cell factor from human peripheral blood mononuclear cells in culture, as judged by the enhanced stimulation of prostaglandin E (2-5 fold) and collagenase (3-11-fold) production by cultured adherent synovial cells. Heparin alone (1-10 micrograms/ml) induced a similar stimulation of mononuclear-cell-factor production by monocyte cultures, whereas histamine (1-10 micrograms/ml) had no effect. The stimulatory effect of mast-cell products and heparin represented a direct effect on mononuclear cells; they did not potentiate the effect of monokine on the synovial cells. These results suggest that mast-cell-macrophage interactions may play a significant role in the pathogenesis of inflammation and connective-tissue degradation. PMID- 2996498 TI - Polyamine-stimulated phosphorylation of prostatic spermine-binding protein is mediated only by cyclic AMP-independent protein kinases. AB - Spermine-binding protein (a rat ventral prostatic protein with high affinity for spermine) was phosphorylated in situ through the action of intrinsic cellular protein kinase(s), suggesting it to be a phosphoprotein in vivo. The purified protein served as a substrate in a number of cyclic AMP-independent protein kinase reactions in vitro, but not for cyclic AMP-dependent, Ca2+ + calmodulin dependent or Ca2+ + phospholipid-dependent protein kinases. Available data indicate that at least one of the cyclic AMP-independent protein kinases (cytosolic protein kinase C2) may be physiologically relevant in mediating the phosphorylation of this protein. The phosphorylation reaction was stimulated several-fold in the presence of spermine. Spermidine was somewhat less effective, whereas putrescine, cadaverine and 1,6-hexanediamine were minimally active. Phospho amino acid analysis of 32P-labelled spermine-binding protein indicated that phosphoserine was the only labelled phospho amino acid. Spermine-binding protein did not undergo autophosphorylation, or modify the stimulative effect of spermine on the phosphorylation of other substrates such as non-histone proteins. In situ the phosphorylation of spermine-binding protein in tissue from castrated rats was markedly diminished as compared with the normal. Since the phosphorylation of spermine-binding protein appears to be mediated by cyclic AMP independent protein kinase(s) whose activity in the prostate is under androgenic control, it is suggested that androgen-dependent modulation of the protein kinase(s) exerts a regulatory control (via phosphorylation-dephosphorylation) on the spermine-binding activity and stability of this protein in vivo. Further, since this protein is a substrate for only the cyclic AMP-independent protein kinases, it could serve as a tool for the investigation of such kinases. PMID- 2996499 TI - Enzymic basis of deranged foetal flavin-nucleotide metabolism consequent on immunoneutralization of maternal riboflavin carrier protein in the pregnant rat. AB - A comparison of the kinetic and other parameters of enzymes of flavin-nucleotide metabolism in the whole foetus vis-a-vis the maternal liver in the pregnant rat revealed relatively lower activities of foetal flavokinase and FAD pyrophosphorylase. Passive immunoneutralization of the maternal riboflavin carrier protein suppresses foetal FAD pyrophosphorylase rather selectively. Additionally, although the activities of foetal nucleotide pyrophosphatase and FMN phosphatase were unchanged owing to immunoneutralization, higher activities of these enzymes in the whole foetus as compared with the maternal liver may be responsible for the drastic depletion of FAD levels that precipitates foetal degeneration. PMID- 2996500 TI - Effects of 1 alpha,25-dihydroxycholecalciferol on sodium-ion translocation across chick intestinal brush-border membrane. AB - By utilizing isolated brush-border vesicles, Na+ transport across the luminal membrane of chick small intestine was found to be a composite of (i) a saturable (Km 10mM-Na+) amiloride-sensitive Na+/H+ antiport and (ii) a potential-sensitive conductive pathway. No evidence was obtained for the existence of a Na+/Cl- symport system. With the exception of the duodenum, luminal Na+ transfer in the entire small intestine was subject to regulation by vitamin D. Repletion of vitamin D-deficient chicks with 1 alpha,25-dihydroxycholecalciferol [1 alpha,25(OH)2D3] significantly decreased net Na+ uptake by isolated membrane vesicles (by approximately 30%). The sterol suppresses the conductive pathway (25 45% inhibition) as well as the Na+/H+ antiport system. Kinetic analysis of the latter revealed that 1 alpha,25(OH)2D3 altered Vmax (from 12.9 to 4.8 nmol of Na+/20s per mg of protein), but did not change Km. Diminution of Na+ transfer, entailing an increase in the electrochemical transmembrane Na+ gradient, provides an explanation of the simultaneously observed stimulatory action of 1 alpha,25(OH)2D3 on Na+-gradient-driven solute transport in chick small intestine. Indirect evidence was obtained that the luminal plasma membrane of chick small intestine displays a definite H+ permeability that is positively affected by 1 alpha,25(OH)2D3. PMID- 2996502 TI - Characterization of ectonucleotidases on vascular smooth-muscle cells. AB - We compared the properties of the ectonucleotidases (nucleoside triphosphatase, EC 3.6.1.15; nucleoside diphosphatase, EC 3.6.1.6; 5'-nucleotidase, EC 3.1.3.5) in intact pig aortic smooth-muscle cells in culture with the properties that we previously investigated for ectonucleotidases of aortic endothelial cells [Cusack, Pearson & Gordon (1983) Biochem. J. 214, 975-981]. In experiments with nucleotide phosphorothioate diastereoisomers, stereoselective catabolism of adenosine 5'-[beta-thio]triphosphate, but not of adenosine 5'-[alpha thio]triphosphate, by the triphosphatase and stereoselective catabolism of adenosine 5'-[alpha-thio]diphosphate by the diphosphatase were found, as occurs in endothelial cells. In contrast with endothelial ecto-5'-nucleotidase, the smooth-muscle-cell enzyme catabolized adenosine 5'-monophosphorothioate (AMPS) to adenosine: the affinity of the enzyme for AMPS was greater than for AMP, and Vmax for AMPS was about one-sixth that for AMP. In both cell types AMPS was an apparently competitive inhibitor of AMP catabolism by 5'-nucleotidase. The relative rates of catabolism of nucleotide enantiomers in which the natural D ribofuranosyl moiety is replaced by an L-ribofuranosyl moiety were similar to those in endothelial cells. No ectopyrophosphatase activity was detected in smooth-muscle cells, in contrast with endothelial cells, where modest activity is present. PMID- 2996501 TI - Stabilization of glucose-6-phosphatase activity by a 21 000-dalton hepatic microsomal protein. AB - Hepatic microsomal glucose-6-phosphatase activity was rendered extremely unstable by a variety of techniques: (a) incubation at pH 5.0; (b) extraction of the microsomal fraction in the presence of 1% Lubrol; (c) various purification procedures. These techniques all result in the removal of a 21 kDa polypeptide from the fraction containing glucose-6-phosphatase activity. The 21 kDa protein was purified to apparent homogeneity by solubilization in the detergent Lubrol 12A-9 and chromatography on Fractogel TSK DEAE-650(S) and centrifugation at 105 000 g. The 21 kDa protein stabilizes glucose-6-phosphatase activity, whereas other purified hepatic microsomal proteins do not. The 21 kDa protein appears to be a potential regulator of glucose-6-phosphatase activity. PMID- 2996503 TI - Calpain inhibition by peptide epoxides. AB - A Ca2+-activated cysteine proteinase (calpain II) was purified from chicken gizzard smooth muscle by use of isoelectric precipitation, (NH4)2SO4 fractionation, chromatography on DEAE-Sepharose CL-6B, Reactive-Red 120-agarose and Mono Q. The apparent second-order rate constants for the inactivation of calpain by a series of structural analogues of L-3-carboxy-trans-2, 3 epoxypropionyl-leucylamido-(4-guanidino)butane (E-64) were determined. The fastest rate of inactivation was observed with L-3-carboxy-trans-2, 3 epoxypropionyl-leucylamido-(4-benzyloxy-carbonylamino)buta ne. It was possible to determine the active-site molarity of solutions of calpain by titration with E 64. When incubated with Ca2+, calpain underwent several steps of intermolecular limited proteolysis, via multiple pathways, followed by a slower loss of enzymic activity. The proteolytic steps preceding the loss of activity did not affect the rates of reaction of calpain with E-64 analogues. PMID- 2996504 TI - Interactions of insulin, glucagon and dexamethasone in controlling the activity of glycerol phosphate acyltransferase and the activity and subcellular distribution of phosphatidate phosphohydrolase in cultured rat hepatocytes. AB - Rat hepatocytes were incubated in monolayer culture for 8 h. Glucagon (10nM) increased the total phosphatidate phosphohydrolase activity by 1.7-fold. This effect was abolished by adding cycloheximide, actinomycin D or 500 pM-insulin to the incubations. The glucagon-induced increase was synergistic with that produced by an optimum concentration of 100 nM-dexamethasone. Theophylline (1mM) potentiated the effect of glucagon, but it did not affect the dexamethasone induced increase in the phosphohydrolase activity. The relative proportion of the phosphohydrolase activity associated with membranes was decreased by glucagon when 0.15 mM-oleate was added 15 min before the end of the incubations to translocate the phosphohydrolase from the cytosol. This glucagon effect was not seen at 0.5 mM-oleate. Since glucagon also increased the total phosphohydrolase activity, the membrane-associated activity was maintained at 0.15 mM-oleate and was increased at 0.5 mM-oleate. This activity at both oleate concentrations was also increased in incubations that contained dexamethasone, particularly in the presence of glucagon. Insulin increased the relative proportion of phosphatidate phosphohydrolase that was associated with membranes at 0.15 mM-oleate, but not at 0.5 mM-oleate. It also decreased the absolute phosphohydrolase activity on the membranes at both oleate concentrations in incubations that also contained glucagon and dexamethasone. None of the hormonal combinations significantly altered the total glycerol phosphate acyltransferase activity. However, glucagon significantly increased the microsomal activities, and insulin had the opposite effect. Glucagon also decreased the mitochondrial acyltransferase activity. There was a highly significant correlation between the total phosphatidate phosphohydrolase activity and the synthesis of neutral lipids from glycerol phosphate and 0.5 mM-oleate in homogenates of cells from all of the hormonal combinations. Phosphatidate phosphohydrolase activity is increased in the long term by glucocorticoids and also by glucagon through cyclic AMP. In the short term, glucagon increases the concentration of fatty acid required to translocate the cytosolic reservoir of activity to the membranes on which phosphatidate is synthesized. Insulin opposes the combined actions of glucagon and glucocorticoids. The long-term events explain the large increases in the phosphohydrolase activity that occur in vivo in a variety of stress conditions. The expression of this activity depends on increases in the net availability of fatty acids and their CoA esters in the liver. PMID- 2996505 TI - The phosphonomethyl analogue of 3-phosphoglycerate is a potent competitive inhibitor of phosphoglycerate mutases. AB - The phosphonomethyl analogue of 3-phosphoglycerate (2-hydroxy-4 phosphonobutanoate) is a potent competitive inhibitor of cofactor-dependent phosphoglycerate mutase from yeast and of cofactor-independent phosphoglycerate mutase from wheat germ. For the yeast enzyme Ki is 1.3 mM (Km for substrate is 0.71 mM); for the wheatgerm enzyme Ki is 18 mM (Km for substrate is 0.86 mM). This analogue should be a useful tool for n.m.r. spectroscopic studies on the mechanism of action of the two mutases. The arsonomethyl analogue of 3 phosphoglycerate (4-arsono-2-hydroxybutanoate) was a relatively poor inhibitor. PMID- 2996507 TI - Catecholamine-stimulated lipolysis in adipocytes of spontaneously hypertensive rats. AB - We investigated the catecholamine-stimulated lipolytic response of perirenal adipocytes isolated from spontaneously hypertensive (SHR) and normotensive (C) rats of the Sprague-Dawley (SD) and Wistar-Kyoto (WKY) strain. Younger rats (10 17 weeks) were matched with respect to age and body weight. Age-matched SHR rats were smaller than their C counterparts, had equal-size adipocytes, and demonstrated lipolytic responses equal to C cells. Weight-matched SHR rats were older than normotensive controls, had larger adipocytes, and showed depressed norepinephrine (NE)-stimulated responses with a rightward shift in the dose response curve. Rates of lipolysis of SHR and C cells were not different in the simultaneous presence of norepinephrine and theophylline. Nine- to ten-month-old rats were of comparable body weight and adipocyte size regardless of blood pressure status; however, SHR cells still showed a significantly blunted response to catecholamine stimulation. We conclude that (1) the NE-stimulated lipolytic response of adipocytes of SHR rats is significantly less than that elicited from C cells; (2) this function difference seems unrelated to a size difference between cells of younger, SHR and C rats, thus implicating the adrenergic system; and (3) whole body growth (as reflected by body weight) and perirenal adipocyte growth do not proceed in parallel in actively growing SHR rats. PMID- 2996506 TI - A critical appraisal of evidence for localized energy coupling. Kinetic studies on liposomes containing bacteriorhodopsin and ATP synthase. AB - In intact systems (chloroplasts, mitochondria and bacteria) many experiments have been reported which are indicative of localized coupling between ATP synthase and electron transfer complexes. We have carried out similar experiments with a system in which we may assume that specific interactions between the proton pumps are absent: reconstituted vesicles containing bacteriorhodopsin and yeast mitochondrial ATP synthase. The only experiment that gives results which differ from those previously published for intact systems concerns the effect of uncouplers on the rate of ATP synthesis at different levels of inhibition of the ATP synthase. We propose that this type of experiment may discriminate between localized and delocalized coupling. PMID- 2996509 TI - Aromatic L-amino acid decarboxylase activities in human lung tissues: comparison between normal lung and lung carcinomas. AB - We measured the activity of aromatic L-amino acid decarboxylase with L dihydroxyphenylalanine as a substrate (DOPA decarboxylase) in normal lung tissues and lung tumors obtained fresh at surgery. The activity in control human lung tissues was low and variable: 3.50 +/- 0.42 pmole/min/mg protein (n = 56, mean +/ SE, range 0.01-15), indicating the wide individual variations. Most of small cell carcinoma specimens showed very high activity, as compared with both control lung tissues and with other types of non-SCC lung cancers. Similar results were also obtained in the athymic mice heterotransplants of SCC. High activity was also observed using 5-L-hydroxytryptophan as a substrate (5-HTP decarboxylase) in nine SCC samples. Serotonin was not detected in any control lung tissues, but was detected in all the nine SCC samples, but dopamine was detected only in three out of nine SCC samples. PMID- 2996508 TI - Substrate-induced spectral changes in human normal and chronic myeloid leukemic granulocytes. AB - Several drugs/chemicals were allowed to interact with the cytochrome P-450 dependent mixed function oxidase system in the postmitochrondrial supernatant fractions of Ficoll-Hypaque-separated granulocytes from human normal subjects and patients with chronic myeloid leukemia. The substrate-induced spectral changes were followed by recording the difference spectra. Compounds conventionally classified as type I and type II substrates, on addition to S1 fractions of both normal and leukemic granulocytes, caused spectral changes that were reverse to those reported for the rat liver microsomes. Aminopyrine, phenobarbital, and Tween 80 evoked a reverse type I spectral change with a peak at 420-430 nm and a trough at 380-400 nm, whereas aniline and pyridine induced a modified type I (a reverse type II) spectral change characterized by a peak at 408 nm and a trough at 421 nm. These changes were found to be quantitatively proportional to the amounts of substrate added. However, the magnitude of the peaks and troughs was considerably less in the S1 fraction of the leukemic granulocytes. Correspondingly, total heme content was significantly decreased in S1 fractions of CML granulocytes as compared to similar fractions of normal granulocytes. PMID- 2996511 TI - Human spleen dihydroorotate dehydrogenase: properties and partial purification. AB - Human spleen dihydroorotate dehydrogenase is associated with the mitochondrial membrane and is linked to the respiratory chain via ubiquinone. The enzyme activity was unaffected by pyridine nucleotides. The product of the reaction, orotate, was a potent inhibitor. However, a range of other naturally occurring pyrimidines or purines had no significant effect on the activity. No evidence for the involvement of a complexed metal ion or for an active sulfhydryl group was obtained. Purification of the enzyme was achieved by preparation of an acetone powder and extraction with Triton X-100, followed by preparative polyacrylamide gel electrophoresis. Activity was observed by the addition of the artificial electron acceptors, ubiquinone 50 or PMS. Purification resulted in alteration of the pH optimum and of other kinetic characteristics. Two molecular-weight species, of molecular weight 88,000 and 98,000, were consistently observed. The properties of the human spleen enzyme were similar in principle to those for the rat liver enzyme. Differences in the mode of linkage to the respiratory chain for the mitochondrially bound enzyme, and in the characteristics of the purified enzyme, were observed. PMID- 2996510 TI - Human spleen dihydroorotate dehydrogenase: a study of inhibition of the enzyme. AB - Seventy-one pyrimidine analogs have been tested as possible inhibitors of human spleen mitochondrial dihydroorotate dehydrogenase. Of these nine were demonstrated to be effective inhibitors of the enzymic activity. Two compounds, dihydro-5-azaorotate and 6-thiobarbiturate appeared to be specific inhibitors of the DHO-DHase. In addition, three compounds, 5-azaorotate, 5-bromoorotate, and barbiturate were also inhibitory against the two subsequent enzymes of the pathway, orotate phosphoribosyltransferase and orotidylate decarboxylase, so that they could act against three enzymes of the mammalian pyrimidine de novo biosynthetic pathway. PMID- 2996512 TI - The insulin-mimetic action of Mn2+: involvement of cyclic nucleotides and insulin in the regulation of hepatic hexokinase and glucokinase. AB - Manganese causes a significant rise in hepatic glucokinase and hexokinase in 16 day-old suckling rats, and has an insulinomimetic effect in producing a precocious emergence of glucokinase (EC 2.7.1.2) and a rise in the low Km, hexokinases (EC 2.7.1.1) activities. These enzyme changes occur within 6 hr of manganese administration and there are accompanying increases in plasma insulin and hepatic cyclic GMP. That the effect of manganese is at a site other than, or in addition to, insulin secretion is suggested by the significant increases in glucokinase and hexokinase in 16-day-old streptozotocin-diabetic rats; in this group there is also an increase in hepatic cGMP similar in time scale to that of the normal-manganese-treated group. The effects of manganese and insulin were not additive. It is proposed that one site of action of manganese may be at the level of cyclic GMP systems. The results are also discussed in relation to the known action of manganese at the level of the protein phosphatases. PMID- 2996513 TI - Calcitonin and calcitonin gene-related peptide interact with the same receptor in cultured LLC-PK1 kidney cells. AB - Calcitonin and calcitonin gene-related peptide stimulate adenylate cyclase activity and plasminogen activator production in cultured renal tubular LLC-PK1 cells. Salmon [125I]calcitonin and human [125I]calcitonin gene-related peptide bound specifically to the cells. Salmon [125I]calcitonin binding was reduced at lower concentrations of non-radioactive salmon calcitonin than of human calcitonin gene-related peptide. For the stimulation of adenylate cyclase activity and plasminogen activator production, the potency of salmon calcitonin was higher than that of human calcitonin and calcitonin gene-related peptide. In a subclone of LLC-PK cells lacking salmon calcitonin binding sites, no specific binding of [125I]CGRP occurred, and adenylate cyclase activity and plasminogen activator production was not increased by the peptides. Thus, in LLC-PK1 cells the stimulation of adenylate cyclase activity and plasminogen activator production by calcitonin gene-related peptide is probably mediated by the calcitonin receptor. PMID- 2996514 TI - Stimulation of human myelopoiesis by leukotriene B4. AB - Cultivation of human mononuclear bone marrow cells for 10 days in the presence of leukotriene B4 (8 X 10(-8) - 3 X 10(-6)M) led to an increase in the formation of granulocyte-macrophage colonies. The increase varied between 19 and 122% when compared to control cells. 5S, 12S-Dihydroxy-6, 8, 10, 14-eicosatetraenoic acid (5S, 12S-DHETE), an isomer of leukotriene B4, did not stimulate colony formation. Preincubation of the cells with 5S, 12S-DHETE inhibited the stimulatory action of leukotriene B4 on the proliferation of bone marrow cells. The present study indicates that leukotriene B4 amplifies the stimulation caused by the colony stimulating factor(s) and may play a role in modulating granulocyte and macrophage poiesis by a positive feedback mechanism. PMID- 2996515 TI - Comparison of heme environments and proximal ligands in peroxidases and other hemoproteins through carbon-13 nuclear magnetic resonance spectroscopy of carbon monoxide complexes. AB - Carbon-13 nuclear magnetic resonance signals for the carbon monoxide ligand in ferrous complexes of horseradish peroxidase, lactoperoxidase, and chloroperoxidase are located respectively at 209.1, 208.3, and 200.8 parts per million from the tetramethylsilane reference. On the basis of previous hemoprotein and model compound studies these resonance positions are consistent with coordination of a proximal histidine ligand in horseradish peroxidase and lactoperoxidase, and coordination of a cysteinyl mercaptide ligand in chloroperoxidase. Carbonyl chemical shift values for acidic and basic horseradish peroxidase isoenzymes are very similar. PMID- 2996516 TI - A revised structure of the antitumor drug elliptinium--amino(acid) adducts. AB - Using simple organic reactions and spectrometric techniques (nuclear magnetic resonance and mass spectrometry), we propose a revised structure of the adducts obtained through the H2O2-peroxidase oxidation of the antitumor drug ellipticinium and aliphatic amino(acid) compounds. These derivatives display an oxazole ring structure between the O(9)- and N(10)- atoms. PMID- 2996517 TI - ATP depletion in human platelets caused by permeabilization with saponin does not prevent serotonin secretion induced by collagen. AB - Saponin (5 to 25 micrograms/ml) produced a concentration-dependent decrease in the cellular content of total ATP and [32P]ATP in 32P-labeled human platelets. In platelets whose ATP had been profoundly decreased by saponin, Ca2+ produced phosphomonoesteratic cleavage of the polyphosphoinositides with a concomitant accumulation of phosphatidylinositol. Collagen still induced secretion of serotonin in platelets that had been treated with saponin in the presence or absence of Ca2+. This effect of collagen occurred in the absence of the formation of cyclooxygenase metabolites. In platelet permeabilized with saponin, agonist induced secretion and aggregation seems to be unrelated to protein phosphorylation, breakdown of the inositol phospholipids by phospholipase C and formation of cyclooxygenase metabolites. PMID- 2996518 TI - Purification of opioid-binding materials from rat brain. AB - Opiate receptors were solubilized from rat brain with digitonin and treated with an affinity resin, AH-Sepharose coupled with [D-Ala2, Leu5]enkephalin. The eluted materials from the resin with the opioid peptide had opioid-binding activity. Based on the protein content of the purified materials, 450-fold purification over the solubilized receptors was achieved. Analyses by gel electrophoresis revealed that the purified materials were rich in one polypeptide with a molecular weight of 62,000. PMID- 2996519 TI - Oxidation of reduced cucumber ascorbate oxidase. AB - Reoxidation process of reduced cucumber ascorbate oxidase (1.10.3.3) with dioxygen was investigated in detail through absorption, circular dichroic (CD) and electron paramagnetic resonance (EPR) spectra. One of the three type I coppers and the type II copper were reoxidized more rapidly than other type I coppers. The principal active site of ascorbate oxidase was considered to be comprised of one type I, one type II and a pair of type III coppers similarly to the active sites in laccase and ceruloplasmin. Remaining two type I and a pair of type III coppers were also disclosed to contribute to the oxidation of L ascorbate. PMID- 2996520 TI - In vivo detection of a three iron cluster in fumarate reductase from Escherichia coli. AB - Escherichia coli with plasmid amplified expression of fumarate reductase was grown anaerobically on a medium containing fumarate and glycerol and investigated by electron paramagnetic resonance spectroscopy. Anaerobically harvested cells exhibit an EPR signal characteristic of a reduced [2Fe-2S] cluster. Anaerobic addition of fumarate results in diminution of the reduced [2Fe-2S] signal and the appearance of the EPR signal associated with the oxidized 3Fe cluster. The results provide the first evidence for a trinuclear iron-sulfur cluster that exists in vivo, and suggest that the 3Fe cluster in purified fumarate reductase samples is not an artifact of the isolation procedure. The significance of this observation is discussed in relation to the physiological relevance of trinuclear iron-sulfur clusters. PMID- 2996521 TI - Mechanism of dismutation of superoxide produced during autoxidation of melanin pigments. AB - The hydrogen peroxide produced during the autoxidation of melanin pigments has been measured using an oxidase electrode. The autoxidation has been shown to occur via the superoxide intermediate. The melanin pigment competes with superoxide dismutase for the scavenging of superoxide radicals. However, superoxide dismutase at high concentrations caused a substantial increase in the production of hydrogen peroxide, formed during melanin autoxidation. The implications of this finding are discussed in light of melanin's ability to function as a pseudo-dismutase. PMID- 2996522 TI - Detection of a catalytic intermediate of peroxidase in hog thyroid microsomes. AB - A catalytic intermediate, Compound II of peroxidase was detected spectrophotometrically in thyroid microsomes. From comparison with the spectral data on purified thyroid peroxidase, the content of the peroxidase was estimated to be 0.019 nmol per mg of the microsomal protein, being about one-eighth of the amount of cytochrome b5. It was concluded that thyroid peroxidase exhibits the same peroxidase activity for guaiacol or ascorbate in the free and the microsome bound forms. PMID- 2996523 TI - Preparation and reconstitution of a phospholipid deficient cytochrome b6-f complex from spinach chloroplasts. AB - A simple, rapid procedure suitable for large scale preparation of a lipid deficient cytochrome b6-f complex from spinach chloroplasts has been developed. The procedure involves solubilization with a mixture of sodium cholate and octylglucoside, ammonium sulfate fractionation and calcium phosphate column chromatography. The purified complex contains, in nanomoles per milligram protein, 20.6 cytochrome b, 10.8 cytochrome f and 54 phospholipids. The purified complex has little plastoquinol-cytochrome c reductase activity in the absence of added lipid. Full reductase activity was reconstituted by the addition of plastoquinone prior to the addition of lipid. PMID- 2996524 TI - Wheat germ phosphoglycerate mutase: purification, polymorphism, and inhibition. AB - An improved method for purifying the bisphosphoglycerate-independent phosphoglycerate mutase from wheat germ has been devised. The method yields enzyme with a specific activity of 2,300 units/mg in 0.1 M Tris-C1 at pH 8.7 and 30 degrees C. Electrophoresis on electrofocusing and analytical polyacrylamide gels reveals only one protein band (pI = 7.3); however, under denaturing conditions (sodium dodecyl sulfate polyacrylamide gel electrophoresis), two prominent enzyme forms, with molecular masses of 63 and 74 kDa, manifest themselves along with several minor, high molecular mass components (126-141 kDa). Non-denaturing exclusion chromatography shows that both major species are catalytically active, and suggests that each species is capable of participating in reversible monomer/dimer association. Wheat germ mutase is inhibited by time dependent reactions involving either polydentate chelators or sulfhydryl reagents. PMID- 2996525 TI - Detection of a tetranuclear iron-sulfur center in fumarate reductase from Escherichia coli by electron paramagnetic resonance spectroscopy. AB - Soluble fumarate reductase and fumarate reductase complex from Escherichia coli have been investigated by electron paramagnetic resonance spectroscopy. Both succinate- and dithionite-reduced samples show signals associated with a [2Fe 2S]1+ cluster that account maximally for slightly more than one spin/molecule. In addition, at temperatures below 20 K, dithionite-reduced samples exhibit broad and complex features, to high and low field of the [2Fe-2S]1+ signal, that are attributable to a spin coupled [4Fe-4S]1+ cluster. Preliminary attempts to quantify the signals indicate that the [4Fe-4S] cluster is present in an approximate 1:1 stoichiometry with the [2Fe-2S] cluster. The observed enhancement of the spin relaxation of the [2Fe-2S]1+ cluster on dithionite reduction is attributed to spin-spin interaction between the S = 1/2, reduced tetranuclear and binuclear clusters. PMID- 2996526 TI - Regulation of growth hormone and epidermal growth factor receptors by progestins in breast cancer cells. AB - A 24 hr incubation of T-47D human breast cancer cells with R5020, a synthetic progestin, resulted in a 200-250% increase in the specific binding of human growth hormone (hGH) and epidermal growth factor (EGF) by these cells. This effect was specific for progestins in that similar responses were observed with progesterone, medroxyprogesterone acetate and ORG 2058 but no significant increases in hGH or EGF binding were observed in cells incubated with testosterone, estradiol or hydrocortisone. Increased binding was due to an increase in the concentration of receptors (hGH, control = 6,490 +/- 500, progestin treated = 13,180 +/- 3,270 sites/cell; EGF, control = 33,380 +/- 7,410, progestin treated = 67,460 +/- 20,330 sites/cell) while the affinity constants for the hormone-receptor interactions were unchanged by progestin treatment. The specific binding of insulin, calcitonin, transferrin and concanavalin A was unaffected by these treatments. It is concluded that expression of hGH and EGF receptors in this breast cancer cell line is regulated by progestins. PMID- 2996527 TI - Possible mechanism of the allosteric activation of cAMP receptor protein. AB - Secondary structure of cAMP receptor protein of E. coli was predicted and compared to its crystal structure in the complex with cAMP solved by McKay and Steitz. The two conformations coincide in the DNA binding domain but strikingly differ in the other domain which binds cAMP and causes protein dimerization. The comparison indicates that cAMP destabilizes a very long helix instead of which sheets are formed creating a hydrophobic pocket where cAMP binds. Consequently, the helix-sheets isomerization and a resulting change in the relative monomer disposition in the dimer appears to be the origin of cAMP-induced allosteric activation of the protein. Extremely long helices were also predicted in the regions of the regulatory subunit of cAMP-dependent protein kinase from bovine cardiac muscle where cAMP binds. It is thus likely that the proposed mechanism of the effect of cAMP on protein structure has wider implications. PMID- 2996528 TI - Leukotriene A5 is a substrate and an inhibitor of rat and human neutrophil LTA4 hydrolase. AB - The epoxide 5(S) trans-5,6 oxido, 7,9 trans-11,14,17 cis eicosatetraenoic acid (leukotriene A5) was chemically synthesized and demonstrated to be both a substrate and an inhibitor of partially purified rat and human LTA4 hydrolase. Both rat and human LTA4 hydrolase utilized leukotriene A5 less effectively as a substrate than leukotriene A4. Incubation of leukotriene A5 (10 microM) or leukotriene A4 (10 microM) with rat neutrophils demonstrated formation of 123 pmol LTB5/min/10(7) cells and 408 pmol LTB4/min/10(7) cells respectively. Purified rat neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 22 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 50 nmol LTB4/min/mg protein. Human neutrophil LTA4 hydrolase incubated with 100 microM leukotriene A5 produced 24 nmol LTB5/min/mg protein and when incubated with 100 microM leukotriene A4 produced 52 nmol LTB4/min/mg protein. Leukotriene A5 was an inhibitor of the formation of leukotriene B4 from leukotriene A4 by both the rat and human neutrophil LTA4 hydrolase. Excess leukotriene A5 prevented covalent coupling of [3H] leukotriene A4 to LTA4 hydrolase suggesting inhibition may involve covalent coupling of leukotriene A5 to the LTA4 hydrolase. PMID- 2996529 TI - In vitro metabolism of [3H]-peptide leukotrienes in human and ferret lung: a comparison with the guinea pig. AB - Fragmented lung tissue prepared from human, ferret and guinea pig converted [3H] leukotriene C4 ([3H]-LTC4) to [3H]-LTD4 and [3H]-LTE4. [3H]-LTD4 was the major product recovered from incubations with human and guinea pig lung, whereas [3H] LTE4 was the predominant metabolite in ferret. Kinetic analysis in human lung yielded a much faster rate for the conversion of [3H]-LTC4 to [3H]-LTD4+E4 than for the conversion of [3H]-LTD4 to [3H]-LTE4. In all three species serine-borate complex blocked the metabolism of [3H]-LTC4, whereas L-cysteine blocked the metabolism of [3H]-LTD4. These studies demonstrate that guinea-pig and human lungs have a similar metabolic pattern and capacity which is dissimilar to the ferret. PMID- 2996530 TI - Quasi-stationary concentrations of fructose-2,6-bisphosphate in the phosphofructokinase-2/fructose-2,6-bisphosphatase cycle. AB - The cooperation of phosphofructokinase-2 and fructose-2,6-bisphosphatase is investigated. Experimentally derived rate laws of the kinase and bisphosphatase activities introduced into the respective differential equations permitted to describe the time evolution of fructose-2,6-bisphosphate to quasi-stationary levels. The two enzyme activities were found to exert strong temperature dependence. The quasi-stationary levels of fructose-2,6-bisphosphate, however, are independent on temperature. PMID- 2996531 TI - Preparation of rat gastric heavy and light microsomal membranes enriched in (H+ K+)-ATPase using 2H2O and Percoll gradients. AB - Gastric heavy microsomal membranes highly enriched in (H+-K+)-ATPase were obtained from cimetidine- or carbachol-treated rats through 2H2O and Percoll gradient centrifugations. Both the resting (cimetidine-treated) and the stimulated (carbachol-treated) heavy membranes which presumably represent the apical membrane of gastric parietal cells were enriched with the polypeptides of 81,000 and 45,000 besides that of 93,000 representing (H+-K+)-ATPase. No apparent differences could be detected between the resting and the stimulated heavy membranes in their polypeptide profiles or their specific activity of (H+-K+) ATPase. Nevertheless, the level of 86RbCl uptake was greater in the stimulated than the resting heavy microsomal membrane vesicles. The light gastric microsomes which abound in intracellular tubulovesicles containing reserve (H+-K+)-ATPase as isolated from cimetidine-treated rats were similarly purified with respect to (H+ K+)-ATPase. The purified light gastric membranes were largely devoid of the polypeptides of 81,000 and 45,000 found in the heavy gastric membranes. These observations further support the current hypothesis that secretagogues bring about changes in the environment of (H+-K+)-ATPase and induce KCl permeability in the apical membrane of the parietal cells, although at present we have been unable to identify the polypeptide(s) responsible for the KCl pathway. PMID- 2996532 TI - Formation of benzo(alpha)pyrene metabolites and DNA adducts catalyzed by a rat liver mitochondrial monooxygenase system. AB - Sonic disrupted mitoplasts from 3-methylcholanthrene (MCA) treated rats can catalyze the formation of benzo(a)pyrene (BaP) adducts with calf thymus DNA in the presence of an NADPH generating system. The mitoplasts used in this study contained less than 1% microsomal marker enzymes: rotenone insensitive NADPH cytochrome c reductase and glucose-6-phosphatase. The rates of BaP metabolism and DNA adduct formation per nanomole cytochrome P-450 were different for MCA induced mitochondrial and microsomal enzymes. The major B(a)P DNA adducts formed in incubations with lysed mitoplasts were derived from reaction of 9-OH-B(a)P-4,5 oxide with deoxyguanosine. The results suggest a potential role of mitochondrial monooxygenase activity in the covalent binding of B(a)P to mitochondrial DNA. PMID- 2996533 TI - Effects of acetylcholine, TSH and other stimulators on intracellular calcium concentration in dog thyroid cells. AB - The intracellular free calcium concentration, [Ca2+]i, has been measured in dog thyroid cells using the fluorescent Ca2+-indicator, quin2. Acetylcholine or its non-hydrolyzable analog, carbamylcholine rapidly increased [Ca2+]i by 40 +/- 4% (mean +/- SE) over the basal level of 81 +/- 2 nM. This increase was totally abolished by atropine, a muscarinic cholinergic receptor blocker, but was not influenced by verapamil, a voltage dependent-calcium channel blocker. Depletion of extracellular Ca2+ by the addition of EGTA, diminished but did not abolish the response to carbamylcholine. These data suggest that cholinergic effectors increase [Ca2+]i by mobilization of Ca2+ from intracellular stores rather than from an influx of Ca2+. Addition of TSH, isoproterenol, phorbol ester, dibutyryl cyclic GMP or cyclic AMP did not elicit any change in [Ca2+]i suggesting that their action may not involve any mobilization of intracellular Ca2+. These data provide direct evidence that in the thyroid cell, cholinergic agents act via their receptors to cause a rapid increase in [Ca2+]i, which may mediate their metabolic effects. PMID- 2996534 TI - Synthesis and biological activity of a linear fragment of the atrial natriuretic factor (ANF). AB - A linear fragment of the atrial natriuretic factor, ANF(106-125), unable to form an intramolecular cystine bridge, was synthesized by the solid-phase method. The fragment showed smooth muscle relaxant activity in the rabbit aorta and chick rectum assays, an inhibitory effect on aldosterone secretion from bovine adrenal zona glomerulosa cells, and had affinity for specific ANF receptors located in zona glomerulosa cell membranes. The potency of ANF(106-125) in these four assay systems was about two to three orders of magnitude lower than that of ANF(103 125) which contains the intact cyclic structure. The obtained results indicate that the disulfide linkage stabilizes the bioactive conformation of ANF peptides but is not an absolute requirement for biological activity. PMID- 2996535 TI - Bryostatin, a non-phorbol macrocyclic lactone, activates intact human polymorphonuclear leukocytes and binds to the phorbol ester receptor. AB - Bryostatin, is an antineoplastic agent with activity in both solid and liquid tumors. When added to tissue culture cells this agent shares a number of similarities with phorbol esters. In this report, we evaluate Bryostatin's effect on human polymorphonuclear leukocytes. Bryostatin stimulates the release of specific granules with a parallel dose response curve to phorbol 12-myristate 13 acetate (PMA), but induces release of superoxide at a significantly slower rate than PMA. Competition experiments demonstrate that Bryostatin, although sharing little structural similarity with PMA, can bind to the PMA receptor. In addition, both Bryostatin and PMA stimulate the phosphorylation of almost identical proteins in intact PMNs. These experiments suggest that Bryostatin may activate PMNs by binding to the PMA receptor, which is currently felt to be the calcium, phospholipid-dependent protein kinase. PMID- 2996536 TI - Regional heterogeneity of rat brain phencyclidine (PCP) receptors revealed by photoaffinity labeling with [3H] azido phencyclidine. AB - Photoaffinity labeling of rat brain phencyclidine (PCP) receptors with [3H] azido phencyclidine ([3H]AZ-PCP) reveals the existence of five polypeptides which are specifically labeled by the affinity probe (Mr's 90,000, 62,000, 49,000, 40,000 and 33,000). These labeled components are unevenly distributed in rat brain. In the frontal cortex, thalamus and olfactory bulb, the major bands labeled are the Mr's 90 K and 62 K polypeptides; in the cerebellum most of the labeling is in the 90 K and 33 K bands; and in the hippocampus all but the Mr 40 K band are heavily labeled. Together with dexoxadrol/[3H]PCP competition binding data, which indicated the existence of high and low affinity dexoxadrol/PCP binding sites, these results suggest regional heterogeneity of PCP receptors. The regional distribution of the high affinity dexoxadrol binding sites correlates best with that of the Mr 90 K polypeptide. PMID- 2996537 TI - The effect of plasma gelsolin on actin filaments. Ca2+-dependency of the capping and severing activities. AB - The Ca2+-dependency of plasma gelsolin capping and severing properties was investigated using viscometry, fluorescence of N-(1-pyrenyliodoacetamide) labelled actin and Triton X-100 cell models. The two properties of plasma gelsolin appear to be expressed differently, severing being fully Ca2+-dependent, whereas capping seems to take place, even in the absence of Ca2+, although less efficiently. PMID- 2996538 TI - Muramyl peptides and serotonin interact at specific binding sites on macrophages and enhance superoxide release. AB - Serotonin at low micromolar concentrations inhibited binding of two [125I] labeled muramyl peptides to resident mouse peritoneal cells and to a macrophage derived cell line, PU5-1.8-F7. Binding of [3H]serotonin was inhibited in parallel fashion. Overnight incubation with serotonin or muramyl peptide enhanced the release of superoxide by both types of cells when later stimulated with phorbol myristate acetate. Serotonin antagonists decreased binding of muramyl peptide and serotonin and diminished the subsequent enhancement of superoxide release. A cell line variant lacking detectable binding sites for muramyl peptide was far less responsive (superoxide release) than the parent line, to either drug. The data are consistent with sharing of a common set of receptors on the macrophage by muramyl peptide and serotonin and with involvement of these receptors in enhancing superoxide release. PMID- 2996539 TI - Evidence for a functionally active inhibitory guanine nucleotide-binding regulatory protein in the swine ovary. AB - Incubation of particulate fractions of swine granulosa cells or luteal minces with purified pertussis toxin (islet-activating protein) and [32P]-NAD catalyzed the (32P)-ADP ribosylation of a 41,000 dalton membrane protein. ADP-ribosylation was markedly reduced by prior incubation of intact cells with toxin. The functional relevance of this presumptive inhibitory guanine nucleotide-binding protein in pig granulosa cells was indicated by the ability of prior treatment with pertussis toxin to increase cyclic AMP generation and progesterone production significantly in response to follicle stimulating hormone. Prior cellular intoxication also enhanced cyclic AMP production stimulated by luteinizing hormone and choleratoxin, but not basally or after forskolin. These results demonstrate the presence of an inhibitory guanine nucleotide-binding protein in both the follicular (granulosa cell) and luteal compartments of the mammalian ovary, and indicate its functional relevance in cyclic AMP generation and progesterone secretion. PMID- 2996540 TI - Glycerol-3-phosphate dehydrogenase mRNA content in cultured 3T3-L1 adipocytes: regulation by dibutyryl cyclic AMP. AB - Incubation of differentiated 3T3-L1 adipocytes with 0.5 mM dibutyryl cAMP plus 0.5 mM theophylline for 2 hours results in an 85% decrease in glycerol-3 phosphate dehydrogenase (G3PD) mRNA content. Incubation of the adipocytes with insulin (1 microgram/ml) up to 24 hrs yielded no significant change in the G3PD mRNA content compared with control. PMID- 2996542 TI - Endogenous dephosphorylation of synaptosomal calmodulin-dependent protein kinase type II. AB - Calmodulin-dependent protein kinase Type II autophosphorylation in synaptosomes is localized to the cytoskeleton (synaptic junction), while a potent dephosphorylating activity is present in the lipid bilayer. The dephosphorylating activity is operative in intact synaptosomes and in a reconstitution system comprised of the cytoskeletal and Triton X-100 - soluble fractions. Dephosphorylation is inhibited by EDTA and pyrophosphate, but not by EGTA or NaF. The present characterization of endogenous synaptosomal dephosphorylating activity completes the regulatory cycle operating on this enzyme in which phosphorylation of calmodulin-dependent protein kinase type II inhibits its response to Ca+2 and calmodulin. PMID- 2996541 TI - Glucose-6-phosphate modification of bovine renal Na,K-ATPase: a model for changes occurring in the human renal medulla in diabetes. AB - The kinetics of hydrolysis of ATP were determined for the renal Na,K-ATPase, in the K+ conformation, modified with glucose-6-phosphate. There was a shift in the ATP hydrolysis kinetics from negative kinetic co-operativity for the control enzyme preparations to substrate inhibition kinetics for the modified enzyme preparations. The effect was reversible and stabilized after NaBH4 reduction. Approximately 4 moles of glucose-6-phosphate were incorporated per mole of Na,K ATPase (based on MW of 150,000 daltons). Similar substrate inhibition kinetics were observed for the renal Na,K-ATPase isolated from several human subjects with mature onset diabetes. PMID- 2996543 TI - High yield-purification of a urinary Na+-pump inhibitor. AB - A Na+-pump inhibitor was purified from 140 liters of human urine to an apparent homogeneity. Tracing of the inhibitor during the different steps of purification was achieved by simultaneous determination of its capacity to inhibit the activity of Na+,K+-ATPase and ouabain binding, and to cross-react with antidigoxin antibodies. The final purification achieved a 400,000 fold. The purification steps included flash chromatography, anionic exchange chromatography, and reversed-phase HPLC on RP18, diphenyl and phenyl packings. NMR studies indicated that the final product was a non-peptidic, possibly steroidal compound. Its molecular weight as determined by mass spectrometry was 431. PMID- 2996544 TI - Dephosphorylation of cytoplasmic non-polysomal messenger ribonucleoproteins from cryptobiotic gastrulae of Artemia salina. AB - Cytoplasmic non-polysomal mRNP from cryptobiotic gastrulae of the brine shrimp Artemia salina do not contain endogeneous protein phosphatase activity. However, both non-polysomal mRNP and purified mRNP proteins, phosphorylated by mRNP associated protein kinase, can be dephosphorylated by protein phosphatases purified from A. salina cytosol and rabbit skeletal muscle. The 38 kDa and 23.5 kDa poly(A) binding proteins (P38 and P23.5) and a 65 kDa protein are the major substrates of each protein phosphatase used. The reversible phosphorylation dephosphorylation of mRNP may be involved in the regulation of mRNP metabolism, by altering the poly(A) binding capacities of the mRNP proteins. PMID- 2996545 TI - Full, reversible copper removal from ascorbate oxidase. AB - Anaerobic treatment with cyanide of reduced ascorbate oxidase causes total depletion of copper. No significant amount of the metal is reincorporated when the apo-enzyme is incubated with cupric ions, but it is upon incubation with a stoichiometric amount (eight mol per mol of native enzyme) of a Cu(I) complex stable in air [Cu(I)(thiourea)3]Cl. The yield in reconstituted protein is higher under anaerobic conditions (85-90%) than in air (70-75%). By treatment with less than stoichiometric amounts of [Cu(I)(thiourea)3]Cl the apo-protein binds copper preferentially at the blue copper site. As a consequence the recovery of enzymatic activity is percentually lower than copper reincorporation. PMID- 2996546 TI - A catalyst function for MPTP in superoxide formation. AB - We demonstrate that 1-methyl-4-phenyl-1,2-dihydropyridine (MPDP) can be generated, in an alternate pathway, from the catalyst action of 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) upon the iron redox equilibrium reaction. Superoxide and ferric iron are instantaneously produced after addition of MPTP to a solution of ferrous iron. This reaction is oxygen and pH dependent. Superoxide, through a iron dependent Haber-Weiss reaction with peroxide, can generate the cytotoxic hydroxyl radical. A small portion of the superoxide reacts with MPTP to produce the reactive species X. which, in the presence of Fe+3 can also generate MPDP. PMID- 2996547 TI - Beta-adrenergic receptor subtypes and subcellular compartmentation of cyclic AMP and cyclic AMP-dependent protein kinase in rabbit cardiomyocytes. AB - In purified ventricular myocytes from adult rabbit, beta-adrenergic stimulation causes cyclic AMP accumulation and cyclic AMP-protein kinase activation in both particulate and soluble fractions of the cell, whereas prostaglandin E1 elevates cyclic AMP and cyclic AMP-protein kinase activity in the soluble fraction exclusively. Only activation of particulate cyclic AMP-protein kinase activity results in phosphorylase b----a conversion. Using radioligand binding technics, we have determined whether beta 1- and beta 2-receptor subtypes mediate beta adrenergic effects in particulate and soluble subcellular compartments, respectively. The non-selective antagonist [125I]iodocyanopindolol binds to intact ventricular myocytes with KD of 25 pM and a Bmax of 2.6 X 10(5) receptors/myocyte. Competition for [125I]iodocyanopindolol binding to intact myocytes by the beta-receptor subtype-specific antagonists practolol (beta 1) and zinterol (beta 2) results in monophasic curves with antagonist KD values of 1 microM and 1.5 microM, respectively. We conclude that adult rabbit cardiac myocytes do not possess detectable beta 2 receptors. Further, the ability of isoproterenol to cause elevation of cyclic AMP in two functionally distinct regions within the myocyte must pertain to the actions of a single subtype of beta-receptor, the beta 1-receptor. PMID- 2996548 TI - Desensitization of testicular ornithine decarboxylase to gonadotropin releasing hormone and gonadotropic hormones. AB - Prior exposure of the testis to gonadotropin releasing hormone, luteinizing hormone or follicle stimulating hormone caused the testis refractory to these hormones in terms of ornithine decarboxylase activity at 24 h. Luteinizing hormone caused desensitization in the Leydig cells while the levels of ornithine decarboxylase in the seminiferous tubules were unaltered. In gonadotropin releasing hormone desensitized testis all the other treated compounds namely, luteinizing hormone, follicle stimulating hormone, prostaglandin F2 alpha, norepinephrine and cyclic AMP caused stimulation of ornithine decarboxylase activity. The testis desensitized with LH responded to cyclic AMP and norepinephrine whereas prostaglandin E2 or gonadotropin releasing hormone caused less stimulation of ornithine decarboxylase activity. These results indicate that testicular desensitization to gonadotropin releasing hormone and luteinizing hormone is not due to a post cyclic AMP block. PMID- 2996549 TI - Altered synaptosomal ATPase activity in rat brain following prolonged in vivo treatment with nicotine. AB - Effects of prolonged in vivo treatment with nicotine on synaptosomal ATPase activity in rat brain were examined by employing doses of nicotine (0.02 and 0.04 mg/ml in the drinking water) which simulated intake by moderate and heavy smokers. Under these conditions the "low" dose of nicotine resulted in increased body weight whereas "high" dose of nicotine inhibited weight gain. Examination of synaptosomal ATPase activities revealed a dose and time dependent stimulatory/inhibitory effect. With "low" dose of nicotine, maximum stimulatory effect on ATPase activity was seen at the end of the 3rd week, while "high" dose stimulated the enzyme activities maximally by the 2nd week itself; further treatment up to the 4th week caused inhibition of the ATPase activities (10% to 43% decrease). PMID- 2996550 TI - Aldehyde dehydrogenase activity as the basis for the relative insensitivity of murine pluripotent hematopoietic stem cells to oxazaphosphorines. AB - The ex vivo sensitivity of murine pluripotent hematopoietic stem cells (CFU-S) and myeloid progenitor cells (CFU-GM) to 4-hydroperoxycyclophosphamide, ASTA Z 7557, phosphoramide mustard, acrolein, melphalan, and cis-platinum was determined in the absence and presence of known (disulfiram, diethyldithiocarbamate, cyanamide) or suspected [ethylphenyl(2-formylethyl)phosphinate] inhibitors of aldehyde dehydrogenase activity. As compared to CFU-GM, CFU-S were less sensitive to the oxazaphosphorine agents, 4-hydroperoxycyclophosphamide and ASTA Z 7557. The two cell populations were approximately equisensitive to acrolein as well as to the non-oxazaphosphorine cross-linking agents, phosphoramide mustard, melphalan and cis-platinum. All four inhibitors of aldehyde dehydrogenase activity potentiated the cytotoxic action of the oxazaphosphorines toward CFU-S; they did not potentiate the cytotoxic action of acrolein or the non oxazaphosphorines toward these cells. The inhibitors did not potentiate the cytotoxic action of the oxazaphosphorines, non-oxazaphosphorines, or acrolein toward CFU-GM. Pyridoxal, a substrate for aldehyde oxidase, did not potentiate the cytotoxic action of oxazaphosphorines toward CFU-S. Cellular NAD-linked aldehyde dehydrogenases are known to catalyze the oxidation of the major transport form of cyclophosphamide, 4-hydroxycyclophosphamide/aldophosphamide, to an inactive metabolite, carboxyphosphamide. Our observations suggest that (1) aldehyde dehydrogenase activity is an important determinant of the sensitivity of a cell population to the oxazaphosphorines, (2) CFU-GM lack the relevant aldehyde dehydrogenase activity, and (3) the phenotypic basis for the relative insensitivity of CFU-S to oxazaphosphorines is the aldehyde dehydrogenase activity contained by these cells. PMID- 2996551 TI - Disappearance and metabolism of leukotriene B4 during carrageenan-induced pleurisy. AB - Leukotriene B4 (LTB4) has been implicated as a mediator in the inflammatory process by virtue of its potent chemotactic activity. At present, very little is known of the stability of this compound in vivo; therefore, the present study was designed to determine the half-life and metabolic fate of radiolabeled LTB4 during a 2-hr intrapleural incubation in rats with acute carrageenan pleurisy. After injection of 0.2 ml of 1% sodium carrageenan (Viscarin), inflammation was allowed to develop for 4 hr. A small polyethylene cannula was then inserted into the chest, and 0.1 microCi of [14C]LTB4 was injected into the chest. Samples for radioactivity determination were taken at 0, 1, 2, 3, 4, 5, 7, 10, 15, 20, 30, 45, 60, 90 and 120 min via the cannula, and at 120 min the entire content of the chest was collected. The half-life for the disappearance of radioactivity from the chest was 45.8 +/- 3.5 min. The 120-min samples were treated with acetone to precipitate protein and extracted with Sep-Paks. The extracts were analyzed by reversed phase high performance liquid chromatography using an ultraviolet detector set at 269 nm and a radioactivity detector. An additional experiment was run using multi-[3H]LTB4, and the only major metabolites detected were omega hydroxylated compounds. It can be concluded from these results that LTB4 is relatively stable in vivo and could be present for long enough at the inflammatory site to have an influence upon inflammatory cell migration. PMID- 2996552 TI - A double inhibition kinetic analysis of [3H]-muscimol binding to the gamma aminobutyric acid receptor on calf brain synaptic membranes. Further studies on the mechanism of homocysteine-induced seizures. AB - The simultaneous action of pyridoxal 5'-phosphate (PLP) and L-homocysteine on specific [3H]muscimol binding to the gamma-aminobutyric acid receptor on freeze thawed, Triton-treated calf-brain synaptic membranes was examined kinetically by double inhibition analysis. PLP was found to be a pure inhibitor and L homocysteine, a partial inhibitor, with respect to [3H]muscimol. Diagnostic analysis of the experimental data showed that the interaction constant (alpha) of the two inhibitors for the free receptor is between 0 and 1, confirming that the two inhibitors act synergistically. Further double inhibition analysis showed that no quaternary receptor-[3H]muscimol-homocysteine-PLP complex is formed although the ternary receptor-homocysteine-PLP complex is present. The localization and relationship of binding groups for both inhibitors is discussed as in their association with the ligand binding site. PMID- 2996553 TI - Role of uridine phosphorylase in the anabolism of 5-fluorouracil. AB - The activities of enzymes responsible for activating 5-fluorouracil (FUra) to 5 fluorouridine-5'-monophosphate (FUMP) were compared in normal and tumor tissues of rodents to assess the potential capacity of uridine phosphorylase to anabolize FUra to the nucleoside in the presence of ribose-1-phosphate (R-1-P). The activity of the alternative pathway to FUMP with a pyrimidine phosphoribosyltransferase [FUra + 1-pyrophosphoribosyl-5-phosphate (PPRP)] was approximately 15 to 17 nmoles/mg protein/hr in bone marrow from mice and rats and ranged from 28 to 47 nmoles/mg protein/hr in tumor tissues. Uridine phosphorylase [measured as the formation of 5-fluorouridine (FUrd) from FUra and R-1-P] was 35 230 nmoles/mg/hr in bone marrow and in two FUra-sensitive solid tumors, colon tumor No. 38 in mice and RPMI colon tumor in rats; the activity of uridine phosphorylase from L5178Y ascites leukemic cells was notably lower, 8 nmoles/mg/hr. Levels of uridine kinase ranged from 55 to 187 nmoles/mg protein/hr. Thus, the activities of the enzymes of the two-step FUra activating pathway were high compared to the PPRP-dependent activity in all tissues except L5178Y; also, the FUra-sensitive tumors yielded extracts with 1.5 to 6.5 times greater enzyme activity than the corresponding activity in bone marrow. Uridine phosphorylase was partially purified from rat liver, RPMI rat tumor and colon tumor No. 38; the apparent Km of FUra averaged 50 microM, almost 9-fold lower than that of uracil, and the apparent Km of R-1-P for condensation with FUra was 33 microM. The tissue concentration of R-1-P was greater than 70 microM in kidney and liver of rodents and somewhat less in spleen. Colon tumor No. 38 and RPMI colon tumor had 12 and 20 microM R-1-P, respectively, but these low values may reflect low tumor viability. The high levels of uridine phosphorylase and uridine kinase activities in normal tissues and even higher levels in tissues from FUra sensitive tumors, as well as the sufficient concentration of R-1-P relative to its kinetic constant, suggest that FUra metabolism by the two-step pathway to FUMP may be a significant factor in the activity and selectivity of FUra. PMID- 2996554 TI - Protection against alloxan-induced diabetes in mice by the free radical scavenger butylated hydroxyanisole. PMID- 2996555 TI - Inhibition by ethanol of forskolin-stimulated adenylate cyclase in a murine neuroblastoma clone (N1E-115). AB - Forskolin, a diterpene activator of adenylate cyclase, stimulated the formation of cyclic AMP in intact murine neuroblastoma clone N1E-115 cells and stimulated adenylate cyclase activity in a membranal preparation from these cells. Ethanol caused a concentration-dependent inhibition of the forskolin-stimulated responses in both preparations. In intact cells, the inhibition appeared to be noncompetitive. However, in the membranal preparation the inhibition was more of a competitive nature. In addition, there was also a large difference in the amount of inhibition in the two systems. Thus, the inhibition by ethanol was nearly twice as much with intact cells as with membranes. Sucrose appeared to mimic these effects of ethanol, suggesting that with intact cells the effect of this alcohol may be due, in part, to changes in cellular osmotic pressure. PMID- 2996556 TI - Effect of allopurinol on neutrophil superoxide production, chemotaxis, or degranulation. AB - Recent studies examining the effect of allopurinol on bacterial killing by leukocytes [Tubaro et al., Biochem. Pharmac. 29, 3018 (1980); Tritsch and Neiswander, Life Sci. 32, 1359 (1983)] have been interpreted by those authors as proof that xanthine oxidase is the major superoxide producing enzyme in activated leukocytes. To test the assertion that xanthine oxidase is involved in the production of superoxide by activated human neutrophils, the xanthine oxidase content of neutrophils was measured, and the effect of allopurinol on neutrophil functions, including superoxide production, was studied. Neutrophils were found to contain a level of xanthine oxidase insufficient to account for the flux of superoxide associated with neutrophil activation. Allopurinol did not inhibit superoxide production induced by opsonized zymosan, phorbol myristic acetate, or formylmethionylleucylphenylalanine. Furthermore, neither chemotaxis nor degranulation was affected by allopurinol. Allopurinol was also found ineffective in blocking superoxide-mediated carrageenan-induced foot edema in the rat. These studies are interpreted as evidence that xanthine oxidase is not a major superoxide-generating system in activated neutrophils as has been suggested by others. PMID- 2996557 TI - The influence of omeprazole on the synthesis and secretion of pepsinogen in isolated rabbit gastric glands. AB - Regulation mechanisms of pepsinogen (EC 3.4.23.) synthesis and secretion were studied by following newly synthesized [14C]-labeled pepsinogen during culture of isolated rabbit gastric glands. Omeprazole, a substituted benzimidazole, while almost completely abolishing acid production at 10(-4) M, strongly stimulated secretion of preformed and newly synthesized pepsinogen. Although the pepsinogen synthesis at this concentration of omeprazole was reduced to about 55% of the control rate, a two-fold absolute increase of total secreted pepsinogen was found. This increase was not due to a non specific leakage through disruption of chief cell membranes, as no increase of lactate dehydrogenase in the culture medium could be demonstrated. The stimulated secretion was influenced neither by 10(-3) M cimetidine, 10(-3) sodium thiocyanate nor 10(-4) M atropine. No additivity was found between the carbachol (10(-4) M) or dibutyryl cyclic AMP (10(-3) M) and the omeprazole induced pepsinogen secretion. PMID- 2996558 TI - Comparison of ouabain receptors in sheep myocardium and Purkinje fibres. AB - The conducting system of the heart has been reported to be more sensitive to the toxic effects of digitalis than the working myocardium. To investigate the molecular basis of these observations, we have characterized the ouabain receptor in Purkinje fibres and ventricular muscle of the digitalis-sensitive sheep heart using cell membrane preparations, crude homogenates and contracting heart tissues. [3H]-Ouabain binding has the following characteristics: in sheep left ventricular cell membranes, specific binding was of high affinity (KD 1.9 X 10( 9) M at 37 degrees); was co-incident with an inhibition of (Na+ + K+)-ATPase activity; and was inhibited by K+ and unlabelled cardiotonic steroids; in crude homogenates, the maximal binding capacity but not the affinity for ouabain varied in different parts of the sheep heart with Purkinje fibres containing markedly fewer binding sites (0.33 X 10(14)/g wet weight; left ventricle, 1.3 X 10(14)/g wet weight) and in isolated, contracting Purkinje fibres and right ventricular moderator band strips, concentration-response curves for [3H]-ouabain binding, increase in force of contraction and inhibition of [86Rb+]-uptake were co incident. In both contracting tissues, a ouabain concentration of 3 X 10(-7) M occupied about 50% of the specific binding sites, gave the maximal inotropic effect without toxicity and inhibited [86Rb+]-uptake by about 50%. The maximal binding capacity was lower in contracting Purkinje fibres (2 X 10(14) binding sites/g wet weight) than in contracting moderator band strips (3.9 X 10(14) binding sites/g wet weight). The maximal inotropic effects were reached slightly faster in Purkinje fibres but toxicity also occurred faster in these fibers. We conclude that the specific ouabain binding site is the receptor mediating positive inotropy and inhibition of (Na+ + K+)-ATPase in the sheep heart. Further, this receptor is identical in both the conducting system and working myocardium but the conducting system contains many fewer receptors. This change in receptor number, rather than affinity, may underlie the increased ouabain toxicity observed in Purkinje fibres. PMID- 2996559 TI - Effect of plasma on human erythrocyte beta-adrenergic receptors. PMID- 2996560 TI - [Substrate specificity of restriction endonucleases Eco471 and Eco521]. AB - Cleavage sites for Eco47I and Eco52I restriction endonucleases, which are isoschizomers of Ava II and Xma III, respectively, have been structurally elucidated. PMID- 2996561 TI - Failure to demonstrate (cross-reacting) antibodies to human T lymphotropic viruses in patients with rheumatic diseases. PMID- 2996562 TI - In vitro and in vivo inhibitory effects of propentofylline on cyclic AMP phosphodiesterase activity. AB - 1-(5'-Oxohexyl)-3-methyl-7-propylxanthine (propentofylline), a vasoactive agent, was investigated for its in vitro and in vivo inhibitory effects on cyclic AMP phosphodiesterases activity. Soluble cyclic AMP phosphodiesterases from the cerebral cortex, heart muscle, descending aorta and platelet showed two Km values, high and low. The low Km values were in the range of 2.1-3.0 mumol/l and the high Km values were 111, 28.7, 30.2 and 18.7 mumol/l, respectively. Propentofylline inhibited the enzyme from the 4 tissues noncompetitively. Ki values for the low and high Km cyclic AMP phosphodiesterases were 83.4-135 and 107-188 mumol/l, respectively. In the four tissues, the enzyme inhibitory effect of propentofylline was 1/4 to 1/10 times that of 3-isobutyl-1-methylxanthine (IBMX) but 3-9 and 6-17 times as potent as those of theophylline and caffeine, respectively. The in vivo cyclic AMP phosphodiesterase inhibitory effect of propentofylline was determined in mice using an elevation of plasma cyclic AMP levels as a measure. When given both singly and in combination with isoprenaline (isoproterenol), propentofylline was a potent inhibitor of cyclic AMP phosphodiesterase in the case of intravenous administration. PMID- 2996563 TI - The cytotoxic action of diethyldithiocarbamate in vitro. Different inhibition of scheduled and unscheduled DNA synthesis of rat thymic and splenic cells. AB - Within a concentration range of 1-10 micrograms/ml, an addition of diethyldithiocarbamate (DDC) to splenic and thymic rat lymphocytes (2 X 10(6)-4 X 10(6) cells/ml) resulted in a complete inhibition of scheduled (semiconservative) DNA synthesis. Lower and higher concentrations were less effective. Under the same conditions, a strictly dose-dependent inhibition of unscheduled (excision repair) DNA synthesis, a decrease of the sedimentation rate of nucleoids, as well as changes of the thymidine pool were observed. The results suggest that the cytotoxic action of DDC in vitro may be mediated a) by the chelating properties of the drug with inhibitory effects on a variety of cellular functions, including nucleic acid precursor metabolism, b) by an immediate radiomimetic attack of the SH-group of DDC on the DNA. PMID- 2996564 TI - [Sciatica and chemonucleolysis]. PMID- 2996565 TI - The use of ROC curves in histopathologic decision making. AB - The applicability of receiver operator characteristic (ROC) curves to histopathology is presented in three examples, along with a basic discussion of some properties of ROC curves. One major application is in the attempt to define stages in diseases that show a continuous spectrum of histologic patterns, in which the uncertainty of boundary points and the overlap of features makes such definition difficult. Construction and analysis of ROC curves may help to identify the features with the greatest utility, as, for example, in the grading of mucinous carcinomas of the ovary. ROC curves can also be used to assess diagnostic differences between histopathologists, whether they are using different criteria or the same criteria but with different weightings, as, for example, in cervical premalignancy or borderline ovarian tumors. PMID- 2996566 TI - Medical applications of image analysis with the Magiscan 2. AB - The previous generation of image analysis machines were capable of processing and analyzing binary, i.e., black and white images, and making measurements and decisions thereon. The Magiscan 2, one of the new generation computers, is capable of analyzing gray-level images in a variety of sophisticated ways. Its uses in the medical application of image analysis are presented, as are the techniques used to analyze images in general. The two principal current medical uses are the automatic karyotyping of chromosomes and the automatic screening of cervical smears. Other applications discussed include three-dimensional reconstruction of structures from two-dimensional sections and the possibility of developing expert systems for medical diagnostics. PMID- 2996567 TI - Early detection of precancerous lesions in dysplasias of the lung by rapid DNA image cytometry. AB - Hematoxylin-and-eosin-stained cytologic smears of sputum from 28 patients with dysplastic and suspicious cell findings were subjected to DNA image cytometry after Feulgen restaining. The nuclear DNA contents were measured with a TV-based image-analysis system, the Leitz TAS plus, combined with an automatic microscope. Computation of DNA data was performed according to an algorithm for the diagnosis and grading of malignancy. Of the 19 cases that were proven to be malignant in the follow-up, either by histologic examination, sputum cytology, fine needle aspiration biopsy or autopsy, the algorithm identified 17 as malignant in a stage (dysplasia) in which cytology was not yet able to present a definite diagnosis of malignancy. Only two cases of bronchial carcinoma were not detected in the state of dysplasia by this procedure. The periods between the DNA diagnosis of malignancy in dysplasia and the morphologic evidence of cancer varied from three days up to six months. Of the 11 cases that had been classified as benign by the algorithm, 9 were confirmed as benign during the clinical follow-up. Rapid DNA image cytometry appears able to separate squamous dysplasias of the lung into precancerous and nonprecancerous lesions. PMID- 2996568 TI - [Fabry's disease. Apropos of a family]. PMID- 2996569 TI - Genetic engineering of human interferons from lymphoblastoid cells: I. Cloning of interferon species expressed in Sendai-induced Namalva cells. PMID- 2996570 TI - In vitro effects of verapamil and methoxy-verapamil [D 600] on transmembrane sodium and potassium transport in human erythrocytes. PMID- 2996571 TI - Evaluation of ouabain-insensitive red blood cell cation transport in obese children. PMID- 2996572 TI - [Hormonal correlations during calcitonin therapy]. PMID- 2996573 TI - Subclone heterogeneity in a clonally-derived osteoblast-like cell line. AB - To analyze the phenotypic diversity of a clonal rat osteosarcoma cell line (ROS 17/2) we have subcloned the cell line and characterized four subclones, ROS 17/2 A.II, A.III, A.V, and A.XIV. The subclones retained many of the characteristics of the parent clone that are considered typical of normal osteoblast-like cells; they responded to parathyroid hormone and isoproterenol, and had a negligible response to prostaglandin E2 as measured by their respective changes in cyclic AMP concentration. In addition up to a 75% decrease in 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) binding was observed over a four-fold increase in cell density. The morphologies of the subclones varied from spindle-shaped, fibroblast-like to cuboidal. Doubling times varied from 24 to 48 hours, and basal alkaline phosphatase (AP) levels differed by as much as 10 times over the initial 3 months in culture. After 6 months (approximately 100 PDL), the population doubling time of subclone A.XIV decreased from approximately 48 to approximately 20 hrs and there was a 2.5 to 3-fold increase in saturation density. This cell line was designated A.XIV.1 and was compared to a thawed sample from frozen stock of the original A.XIV isolate, designated A.XIV.2. These two populations, the parent cell line (ROS 17/2) and subclone A.V had similar growth properties, but differed with respect to changes in their alkaline phosphatase activity (AP) with time in culture: that is, all clones increased AP with time but there was a three to five fold difference in their respective AP levels at various times in culture. All clones except A.V exhibited decreased AP activity upon reaching their saturation densities.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996574 TI - A viral antigen-bearing cell line derived from culture of Paget's bone cells. AB - This study documents the characteristics of a viral antigen-bearing cell line derived from co-culture of the bone from a 71-year-old woman with Paget's disease of bone and HEp-2 cells. The cell line has survived in continuous culture for 3 1/2 years and 185 subcultures. The cells are epithelioid in appearance, produce alkaline and acid phosphatase, increase alkaline phosphatase activity in response to 1,25-(OH)2-D3 and contain receptors for 1,25-(OH)2-D3. Immunofluorescent studies utilizing antisera to respiratory syncytial virus and measles virus reveal antigens of both viruses in the cells. These cells do not produce bone in culture and the adenylate cyclase activity found in their plasma membrane does not increase significantly in response to parathyroid hormone or calcitonin. The cells are not contact inhibited and form spherical colonies in agarose. They are aneuploid and have a modal number of 62-74 as well as HeLa markers. When injected into athymic mice, osteosarcomas are produced. These tumors continue to bear viral antigens. The availability of this cell line should aid in further studies of the viral antigens associated with Paget's disease of bone. PMID- 2996575 TI - Controlled trial of enalapril in congestive cardiac failure. AB - Twenty five patients with chronic congestive cardiac failure had enalapril (n = 13) or placebo (n = 12) added to their existing regimen of digoxin and frusemide in a randomised double blind trial. Four hours after the first 5 mg dose, the enalapril group showed significant falls in blood pressure, heart rate, and concentrations of plasma angiotensin II, angiotensin converting enzyme, and noradrenaline. During the 12 week trial heart failure became worse in one enalapril treated patient (8%) and in seven placebo treated patients (58%). There were no significant changes in cardiac ejection fraction or exercise duration in either group. Plasma noradrenaline response to graded exercise and maximum exercise rate-pressure product were significantly reduced after four and 12 weeks of active treatment but unchanged with placebo treatment. There was a sustained increase in plasma potassium and a slight rise in plasma creatinine in the enalapril group. Plasma concentrations of the active drug, enalaprilate, were dose related and log enalaprilate correlated significantly with percentage of plasma angiotensin converting enzyme activity (r = -0.66). Enalapril was well tolerated and produced no adverse effects. The drug appears to be superior to placebo and offers considerable promise for the treatment of this condition. PMID- 2996576 TI - Quantification of intracardiac shunts by gold-195m, a new radionuclide with a short half life. AB - Gold-195m, a radionuclide with a short half life (30.5 s) was used to quantify left to right intracardiac shunts. The results of this method were compared with those obtained with technetium-99m, a method that was validated against oximetry. In five patients the pulmonary to systemic flow ratio (greater than 3:1) obtained by both radionuclides indicated that the level of shunting was too high to be measured accurately. In one patient fragmentation of the bolus meant that no satisfactory gamma fit could be obtained. In the remaining 16 patients there was no significant difference between two successive 195mAu studies. The agreement between 99mTc results and 195mAu results was excellent. Oxygen administration, straight leg raising exercise, and the use of oblique projections did not affect the values of the pulmonary to systemic flow ratio. The technique of quantification of intracardiac shunts by 195mAu gives reproducible and accurate results and the low radiation dose means that it is suitable for use in children with suspected left to right shunts. PMID- 2996577 TI - Effect of meptazinol on neuromuscular transmission in the isolated rat phrenic nerve-diaphragm preparation. AB - Meptazinol has been shown to have significant effects on neuromuscular transmission in the isolated rat phrenic nerve-diaphragm preparation. The response of the preparation to indirect electrical stimulation was increased in a concentration-dependent manner by meptazinol hydrochloride 2-32 micrograms ml-1. Meptazinol 0.5-2 micrograms ml-1 antagonized the effects the tubocurarine on this preparation, and in concentrations of 1 microgram ml-1 and greater, potentiated suxamethonium. These effects were similar to those obtained with neostigmine and it was demonstrated that meptazinol had significant anti-cholinesterase activity in the concentrations used. Inhibition of cholinesterase with ecothiopate revealed a neuromuscular blocking activity of meptazinol in concentrations as low as 0.25 micrograms ml-1. PMID- 2996578 TI - Calcium channel antagonists: pharmacological considerations. PMID- 2996579 TI - Possible role of calcium in neurotransmission to the lower airways. PMID- 2996580 TI - Nedocromil, a mucosal and connective tissue mast cell stabilizer, inhibits exercise-induced asthma. AB - Nedocromil, the disodium salt of a pyranoquinoline dicarboxylic acid, has a similar profile of activity to disodium cromoglycate (DSCG) but appears to be more active in stabilizing mucosal-type mast cells. In a double-blind placebo controlled trial nedocromil (2 mg) was given by inhalation to eight asthmatic patients prior to a treadmill exercise task. There was a significant reduction in the fall in FEV1 (P less than 0.01) and FVC (P less than 0.05) after nedocromil when compared to placebo. This study indicates that nedocromil might be an effective anti-asthma agent and in addition may help define the role of the mucosal mast cell. PMID- 2996581 TI - Differential metabolism of deoxyribonucleosides by leukaemic T cells of immature and mature phenotype. AB - Experimental evidence has indicated that T lymphoblasts are more sensitive to deoxynucleoside toxicity than are B lymphoblasts. These data have led to the use of purine enzyme inhibitors as selective chemotherapeutic drugs in the treatment of T cell malignancies ranging from T cell acute lymphoblastic leukaemia to cutaneous T cell lymphomas. We have compared the toxicities of 2'-deoxyadenosine, 2'-deoxyguanosine, and thymidine for T cell lines derived from patients with T cell acute lymphoblastic leukaemia with those for mature T cell lines derived from patients with cutaneous T cell leukaemia/lymphoma. We have found that both deoxynucleosides are far less toxic to the mature T cell lies than to T lymphoblasts and that the mature cells accumulate much lower amounts of dATP and dGTP when exposed to deoxyadenosine and deoxyguanosine, respectively. Similar studies performed on peripheral blood cells from patients with T cell leukaemias of mature phenotype and on peripheral blood T cells demonstrate similar low amounts of deoxynucleotide accumulation. Measurements of the activities of several purine metabolizing enzymes that participate in deoxynucleoside phosphorylation or degradation do not reveal differences which would explain the toxicity of deoxynucleosides for immature, as compared to mature, T cells. We conclude that deoxynucleoside metabolism in leukaemic T cells varies with their degree of differentiation. These observations may be relevant to the design of chemotherapeutic regimes for T cell malignancies. PMID- 2996582 TI - Production of human active BPA from TCGF independent T cell lines that do not excrete HTLV: proof of direct action of MLA-144 derived BPA using purified BFU-E. AB - Burst promoting activity (BPA) and colony stimulating activity (CSA) were measured in the supernatants from six T-cell lines which are IL-2 independent and do not excrete human T-cell leukaemia virus. Two cell lines were shown to produce high levels of BPA, namely MLA-144 and the Jurkat cell line. MLA-144 is a gibbon T-cell line which can proliferate and produce BPA in serum free HB101 medium. The BPA from this line was shown to act directly on BFU-E by studies using highly purified peripheral blood derived BFU-E. The Jurkat CM had far less activity against purified progenitors. These cells lines will be of value in the study of erythropoietic regulation. PMID- 2996583 TI - ADP/ATP carrier protein from beef heart mitochondria has high amounts of tightly bound cardiolipin, as revealed by 31P nuclear magnetic resonance. AB - An unusual binding of cardiolipin to the ADP/ATP carrier has been found, which is distinguished by the relatively large amount and by the tightness of binding. High-resolution 31P NMR studies on the detergent-solubilized ADP/ATP carrier from beef heart mitochondria revealed narrow signals from phosphatidylcholine and phosphatidylethanolamine and a broadened signal of 30-40-Hz line width, suggestive of cardiolipin. Line broadening of this magnitude is to be expected when tumbling of the whole protein-detergent micelle is the only source of phosphorus spin-spin relaxation. Thus a strong immobilization of the protein bound cardiolipin is inferred. By sucrose density gradient centrifugation phosphatidylcholine and phosphatidylethanolamine were removed, while approximately six +/- one molecules of cardiolipin remained tightly bound in the dimeric protein molecule. The cardiolipin binding was stable against treatment with sodium dodecyl sulfate although release of the inhibitor carboxyatractyloside revealed at least partial protein denaturation. Ca2+ ions did not readily interact either with the bound cardiolipin. Complete detachment of the bound phospholipid was achieved by a short heat pulse in the presence of sodium dodecyl sulfate. Denaturation of the carrier protein by guanidinium chloride or NaClO4 also led to release of the bound phospholipid. Thus different stages of protein denaturation must be envisaged. PMID- 2996584 TI - Effect of alkyl side chain variation on the electron-transfer activity of ubiquinone derivatives. AB - The effect of the alkyl side chain of the ubiquinone molecule on the electron transfer activity of ubiquinone in mitochondrial succinate-cytochrome c reductase is studied by using synthetic ubiquinone derivatives that possess the basic ubiquinone structure of 2,3-dimethoxy-5-methyl-1,4-benzoquinone with different alkyl side chains at the 6-position. The alkyl side chains vary in chain length, degree of saturation, and location of double bonds. When a ubiquinone derivative is used as an electron acceptor for succinate-ubiquinone reductase, an alkyl side chain of six carbons is needed to obtain the maximum activity. However, when it serves as an electron donor for ubiquinol-cytochrome c reductase or as a mediator in succinate-cytochrome c reductase, an alkyl side chain of 10 carbons gives maximal efficiency. Introduction of one or two isolated double bonds into the alkyl side chain of the ubiquinone molecule has little effect on electron transfer activity. However, a conjugated double bond system in the alkyl side chain drastically reduces electron-transfer efficiency. The effect of the conjugated double bond system on the electron-transferring efficiency of ubiquinone depends on its location in the alkyl side chain. When location is far from the benzoquinone ring, the effect is minimal. These observations together with the results obtained from photoaffinity-labeling studies lead us to conclude that flexibility in the portion of the alkyl side chain immediately adjacent to the benzoquinone ring is required for the electron-transfer activity of ubiquinone. PMID- 2996585 TI - Reaction of electron-transfer flavoprotein with electron-transfer flavoprotein ubiquinone oxidoreductase. AB - The oxidative half-reaction of electron-transfer flavoprotein (ETF), electron transfer from ETF to electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), is dependent on complementary surface charges on the two proteins. ETF is the positively charged member of the redox pair. The evidence is based on the pH and ionic strength dependencies of the comproportionation of oxidized ETF and ETF hydroquinone catalyzed by ETF-QO and on the effects of chemical modification of ETF on the comproportionation reaction. Acetylation of one and five epsilon amino groups of lysyl residues results in 3- and 13-fold increases, respectively, in the Km of ETF-QO for ETF but no change in Vmax. Amidination, which maintains positive charge at modified loci, has no effect on steady-state kinetic constants. These chemical modifications have no effect on the equilibrium constant for equilibration of ETF redox states. The Km of ETF-QO for ETF is pH dependent above pH 8.5, suggesting titration of lysyl residues as previously observed in studies of the reductive half-reaction of ETF [Beckmann, J. D., & Frerman, F. E. (1983) J. Biol. Chem. 258, 7563-7569]. The ionic strength dependence of TN/KmETF for the reaction follows the limiting Bronsted equation ln (TN/Km) = ln k0 + 2 alpha Z1Z2I1/2, and Z1Z2, the product of charges on the reacting proteins, is similar to the value of Z1Z2 for the reductive half reaction of ETF by the general acyl-CoA dehydrogenase. The ETF-QO-catalyzed comproportionation reaction exhibits a primary deuterium isotope effect in D2O, perhaps indicating the participation of solvent water in the electron-transfer reaction. PMID- 2996587 TI - Chemical modification of the CuA center in cytochrome c oxidase by sodium p (hydroxymercuri)benzoate. AB - Cytochrome c oxidase contains a copper ion electron-transfer site, CuA, which has previously been found to be unreactive with externally added reagents under conditions in which the protein remains structurally intact. We have studied the reaction of cytochrome oxidase with sodium p-(hydroxymercuri) benzoate (pHMB) and found that the reaction proceeds, under appropriate conditions, to give an excellent yield of a particular derivative of the CuA center that has electron paramagnetic resonance and near-infrared absorption spectroscopic properties which are distinctly different from those of the unmodified center. Spectroscopic and chemical characterization of the other metal ion sites of the enzyme reveals little or no effect of the pHMB modification on the structures of and reactions at those sites. Of particular interest is the observation that the modified enzyme still displays a substantial fraction of the native steady-state activity of electron transfer from ferrocytochrome c to O2. Although the modified copper center retains the ability to receive electrons from the powerful reductant Na2S2O4 and to transfer electrons to O2, it is not significantly reduced when the enzyme is treated with milder (higher potential) reductants such as NADH/phenazine methosulfate or the physiological substrate ferrocytochrome c. CuA exhibits many spectroscopic and chemical properties which make it highly atypical of cuproprotein active sites; the singular nature of this site has prompted speculation about the importance of the structural peculiarities of this metal ion center in the catalytic cycle of the enzyme. In this work, we demonstrate that the unusual features of this site are not prerequisites for competent catalysis of electron transfer and O2 reduction by the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996586 TI - Symmetry and asymmetry in mandelate racemase catalysis. AB - Kinetic properties of mandelate racemase catalysis (Vmax, Km, deuterium isotope effects, and pH profiles) were all measured in both directions by the circular dichroic assay of Sharp et al. [Sharp, T. R., Hegeman, G. D., & Kenyon, G. L. (1979) Anal. Biochem. 94, 329]. These results, along with those of studying interactions of mandelate racemase with resolved, enantiomeric competitive inhibitors [(R)- and (S)-alpha-phenylglycerates], indicate a high degree of symmetry in both binding and catalysis. Racemization of either enantiomer of mandelate in D2O did not show an overshoot region of molecular ellipticity in circular dichroic measurements upon approach to equilibrium. Both the absence of such an overshoot region and the high degree of kinetic symmetry are consistent with a one-base acceptor mechanism for mandelate racemase. On the other hand, results of irreversible inhibition with partially resolved, enantiomeric affinity labels [(R)- and (S)-alpha-phenylglycidates] reveal a "functional asymmetry" at the active site. Mechanistic proposals, consistent with these results, are presented. PMID- 2996588 TI - Change of conformation and internal dynamics of supercoiled DNA upon binding of Escherichia coli single-strand binding protein. AB - The influence of Escherichia coli single-strand binding (SSB) protein on the conformation and internal dynamics of pBR322 and pUC8 supercoiled DNAs has been investigated by using dynamic light scattering at 632.8 and 351.1 nm and time resolved fluorescence polarization anisotropy of intercalated ethidium. SSB protein binds to both DNAs up to a stoichiometry that is sufficient to almost completely relax the superhelical turns. Upon saturation binding, the translational diffusion coefficients (D0) of both DNAs decrease by approximately 20%. Apparent diffusion coefficients (Dapp) obtained from dynamic light scattering display the well-known increase with K2 (K = scattering vector), leveling off toward a plateau value (Dplat) at high K2. For both DNAs, the difference Dplat - D0 increases upon relaxation of supercoils by SSB protein, which indicates a corresponding enhancement of the subunit mobilities in internal motions. Fluorescence polarization anisotropy measurements on free and complexed pBR322 DNA indicate a (predominantly) uniform torsional rigidity for the saturated DNA/SSB protein complex that is significantly reduced compared to the free DNA. These observations are all consistent with the notion that binding of SSB protein is accompanied by a gradual loss of supercoils and saturates when the superhelical twist is largely removed. PMID- 2996589 TI - Detection of neocarzinostatin chromophore-deoxyribose adducts as exonuclease resistant sites in defined-sequence DNA. AB - A 5'-end-labeled DNA restriction fragment was treated with the nonprotein chromophore of neocarzinostatin under anoxia in the presence of dithiothreitol, conditions known to maximize formation of chromophore-deoxyribose adducts. Under conditions where unmodified DNA was digested to completion, chromophore-treated DNA was highly resistant to digestion by exonuclease III plus the 3'----5' exonucleolytic activity of T4 DNA polymerase and partially resistant to digestion by exonuclease III plus snake venom exonuclease. The electrophoretic mobilities of the products of exonucleolytic digestion suggested that (i) digestion by exonuclease III or T4 polymerase terminated one nucleotide before the nucleotide containing the adduct, (ii) the remaining nucleotide directly adjacent to the adduct (3' side) could be removed by snake venom phosphodiesterase, but at a slow rate, (iii) the covalently linked chromophore decreased the electrophoretic mobilities of the digestion products by the equivalent of approximately three nucleotides, and (iv) adducts formed under anaerobic conditions occurred at the same nucleotide positions as the strand breaks formed under aerobic conditions (primarily at T and, to a lesser extent, A residues). The close similarity in sequence specificity of adducts and strand breaks suggests that a common form of nascent DNA damage may be a precursor to both lesions. A chromophore-induced free radical on C-5' of deoxyribose, subject to competitive fixation by addition reactions with either oxygen or chromophore, is the most likely candidate for such a precursor. The base specificity of adduct formation does not reflect the reported base specificity of neocarzinostatin-induced mutagenesis, suggesting that lesions other than adducts may be responsible for at least some neocarzinostatin-induced mutations, particularly those occurring at G X C base pairs. PMID- 2996590 TI - Reconstitution of membrane proteins. Spontaneous association of integral membrane proteins with preformed unilamellar lipid bilayers. AB - We have developed a simple method for reconstituting pure, integral membrane proteins into phospholipid-protein vesicles. The method does not depend on use of detergents or sonication. It has been used successfully with three different types of integral membrane proteins: UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, cytochrome oxidase (EC 1.9.3.1) from pig heart, and bacteriorhodopsin from Halobacterium halobium. The method depends on preparing unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) that contain a small amount of myristate as fusogen. Under conditions that the vesicles of DMPC have the property of fusing, all of the above proteins incorporated into bilayers. Two events appear to be involved in forming the phospholipid-protein complexes. The first is a rapid insertion of all proteins into a small percentage of total vesicles. The second is slower but continued fusion of the remaining phospholipid-protein vesicles, or proteoliposomes, with small unilamellar vesicles of DMPC. This latter process was inhibited by conditions under which vesicles of DMPC themselves would not fuse. On the basis of proton pumping by bacteriorhodopsin and negative staining, the vesicles were unilamellar and large. The data suggest that insertion of the above integral membrane proteins into vesicles occurred independently of fusion between vesicles. PMID- 2996591 TI - Role of superoxide in the N-oxidation of N-(2-methyl-1-phenyl-2 propyl)hydroxylamine by the rat liver cytochrome P-450 system. AB - The N-oxidation of N-(2-methyl-1-phenyl-2-propyl)hydroxylamine (N hydroxyphentermine, MPPNHOH) and the N-hydroxylation of 2-methyl-1-phenyl-2 propylamine (phentermine) by reconstituted systems that contained cytochromes P 450 purified from rat liver microsomes were demonstrated. The oxidation of MPPNHOH, but not of phentermine, could also be mediated by a superoxide and hydrogen peroxide generating system that contained xanthine and xanthine oxidase. Superoxide dismutase completely inhibited the oxidation of MPPNHOH by the xanthine/xanthine oxidase system and inhibited by 70% the oxidation mediated by a reconstituted cytochrome P-450 oxidase system. The majority of the microsomal oxidation was inhibited by an antibody raised against the major isozyme of cytochrome P-450 purified from livers of phenobarbital-pretreated rats. 2-Methyl 2-nitroso-1-phenylpropane (MPPNO) was found to be an intermediate in the overall oxidation of MPPNHOH to 2-methyl-2-nitro-1-phenylpropane (MPPNO2). Superoxide dismutase appeared to inhibit the first step, the conversion of MPPNHOH to MPPNO. These observations are accounted for by a sequence of two mechanistically distinct P-450-mediated oxidations. In the first reaction, N-hydroxylation of phentermine occurs by a normal cytochrome P-450 pathway. The formed hydroxylamine then uncouples the cytochrome P-450 system to generate superoxide and hydrogen peroxide. The superoxide oxidizes MPPNHOH to MPPNO which is then oxidized to MPPNO2, the ultimate product. This superoxide-mediated oxidation represents another pathway for N-oxidation by cytochrome P-450. PMID- 2996592 TI - Mechanism of oxidation of N-hydroxyphentermine by superoxide. AB - Cytochrome P-450 oxidizes N-hydroxyphentermine (MPPNHOH) by an indirect pathway involving superoxide. The chemical details of this oxidation, in which N hydroxyphentermine is converted to 2-methyl-2-nitro-1-phenylpropane (MPPNO2), have been elucidated by examining the interaction of MPPNHOH with superoxide in aqueous and organic solvents. The role of peroxide, hydroperoxy radicals, and oxygen in the reaction was also examined. The results indicate that superoxide itself is oxidizing MPPNHOH to a nitroxide that disproportionates to MPPNHOH and 2-methyl-2-nitroso-1-phenylpropane (MPPNO). MPPNO is then oxidized to MPPNO2 by O2 or hydroperoxide. Two possible mechanisms for the superoxide oxidation were considered, a proton abstraction and a hydrogen atom abstraction. Stoichiometric and oxygen evolution studies favor the hydrogen abstraction pathway. PMID- 2996593 TI - Construction and characterization of cDNA encoding the alpha 2 chain of chicken type IX collagen. AB - We have isolated and characterized a cDNA encoding the carboxy-terminal half of one of the polypeptide subunits of a novel disulfide-bonded collagen found in hyaline cartilage. This collagen has been given the type assignment type IX, and it has several unusual characteristics. First, the polypeptide subunits are shorter than alpha-chains of the fibrillar collagens types I, II, and III. Second, type IX molecules are heterotrimers of three genetically distinct polypeptide subunits. Third, type IX molecules contain three triple-helical collagenous domains interspersed with noncollagenous domains. When chicken cartilage collagens are extracted with pepsin, type IX collagen is cleaved and gives rise to the triple-helical fragments HMW and LMW. The identification of the cDNA reported here is based on a comparison of the amino acid composition of tryptic peptides derived from LMW with the composition of tryptic peptides predicted from the nucleotide sequence of the cDNA. We also show that the amino terminal sequence of one of the subunits of LMW is identical with the sequence predicted from the nucleotide sequence of the cDNA. Finally, we demonstrate that the amino-terminal amino acid sequence of a tryptic peptide isolated from one of the subunits of HMW is identical with a sequence predicted from the cDNA. We have given the polypeptide chain encoded by the cDNA reported here the name alpha 2(IX), and we show that it is homologous to the alpha 1(IX) chain previously characterized by us. PMID- 2996594 TI - Amplified DNA of the Novikoff hepatoma nucleolus is arranged in a 7.3-kilobase tandem repeat. AB - We have reported previously the cloning and characterization of a nucleolar localized 5.8-kilobase (kb) EcoRI fragment that is approximately 50-fold more highly reiterated in Novikoff hepatoma tumor cells than in normal rat liver [Parker, D. L., Busch, H., & Rothblum, L. I. (1981) Biochemistry 20, 762]. In the present study, the arrangement of these 5.8-kb EcoRI segments within the Novikoff hepatoma genome was investigated. Through the use of "indirect" restriction site mapping, partial restriction enzyme digestions, and molecular cloning, we have determined that the 5.8-kb EcoRI fragment and a 1.5-kb EcoRI fragment together constitute a 7.3-kb unit. The 7.3-kb unit is present in the hepatoma genome as a tandem repeat and constitutes the unit of the DNA that has been amplified. Studies on the arrangement of homologous sequences in the normal rat genome indicate that the amplified DNA may have been derived by a rearrangement and amplification of the nontranscribed spacer of the ribosomal DNA (rDNA) repeat. PMID- 2996595 TI - Kinetics of DNA renaturation catalyzed by the RecA protein of Escherichia coli. AB - The recA enzyme of Escherichia coli catalyzes renaturation of DNA coupled to hydrolysis of ATP. The rate of enzymatic renaturation is linearly dependent on recA protein concentration and shows saturation kinetics with respect to DNA concentration. The kinetic analysis of the reaction indicates that the Km for DNA is 65 microM while the kcat is approximately 48 pmol of duplex formed (pmol of recA)-1 (20 min)-1. RecA protein catalyzed renaturation has been characterized with respect to salt sensitivity, Mg2+ ion and pH optima, requirements for nucleoside triphosphates, and inhibition by nonhydrolyzable nucleoside triphosphates and analogues. These results are consistent with a Michaelis-Menten mechanism for DNA renaturation catalyzed by recA protein. A model is described in which oligomers of recA protein bind rapidly to single-stranded DNA, and in the presence of ATP, these nucleoprotein intermediates aggregate to bring complementary sequences into close proximity for homologous pairing. As with other DNA pairing reactions catalyzed by recA protein, ongoing DNA hydrolysis is required for renaturation. However, unlike the strand assimilation or transfer reaction, renaturation is inhibited by E. coli helix-destabilizing protein. PMID- 2996596 TI - Electron spin resonance study of phospholipid membranes employing a comprehensive line-shape model. AB - The electron spin resonance spectra of the 1-myristoyl-2-[6-(4,4 dimethyloxazolidine-N-oxyl)myristoyl]-sn-glycero- 3-phosphocholine spin-label in highly oriented, fully hydrated bilayers of 1,2-dimyristoyl-sn-glycero-3 phosphocholine have been studied as a function of temperature and magnetic field orientation. The oriented spectra show clear indications of slow motional components (rotational correlation times greater than 3 ns) even in the fluid phase (T greater than 23 degrees C), indicating that motional narrowing theory is not applicable to the spectral analysis. The spectra have been simulated by a comprehensive line-shape model that incorporates trans-gauche isomerization in addition to restricted anisotropic motion of the lipid long molecular axis and that is valid in all motional regimes. In the gel (L beta') phase the spin-label chains are found to be tilted at 28 degrees with respect to the normal of the orienting plane. In the intermediate (P beta') phase there is a continuous distribution of tilt angles between 0 degrees and 25 degrees. In fluid (L alpha) phase there is no net tilt of the lipid chains. The chains rotate at an intermediate rate about their long axis in the fluid phase (tau R,parallel = 1.4 6.6 ns for T = 50-25 degrees C), but the reorientation of the chain axis is much slower (tau R, perpendicular= 13-61 ns for T = 50-25 degrees C), whereas trans gauche isomerization (at the C-6 position) is rapid (tau J less than or equal to 0.2 ns). Below the chain melting transition both chain reorientation and chain rotation are at the ESR rigid limit (tau R greater than or equal to 100 ns), and trans-gauche isomerization is in the slow-motion regime (tau J = 3.7-9.5 ns for T = 22-2 degrees C). The chain order parameter increases continuously with decreasing temperature in the fluid phase (SZZ = 0.47-0.61 for T = 50-25 degrees C), increases abruptly on going below the chain melting transition, and then increases continuously in the intermediate phase (SZZ = 0.79-0.85 for T = 22-14 degrees C) to an approximately constant value in the gel phase (SZZ congruent to 0.86 for T = 10-2 degrees C).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2996597 TI - The interaction of Sendai virus with negatively charged liposomes: virus-induced lysis of carboxyfluorescein-loaded small unilamellar vesicles. AB - The interaction of Sendai virus with small, unilamellar vesicles, lacking virus receptors and loaded with self-quenched 6-carboxyfluorescein, was studied. Sendai virions induced release of carboxyfluorescein from vesicles composed of negative charged phospholipids, despite the fact that they did not contain virus receptors. Preliminary experiments indicate that the carboxyfluorescein release is accompanied by mixing of the virus and liposome lipids and their entrapped contents, suggesting liposome-virus fusion. No release of carboxyfluorescein was observed with vesicles containing only phosphatidylcholine. The rate of virus induced carboxyfluorescein release was temperature dependent; the lytic activity of the virus was greatly enhanced above 25 degrees C. This effect was not due to a thermal phase transition of the lipids in either the lipid vesicles or the virions. Virus-induced carboxyfluorescein release was inhibited by the presence of calcium ions in the medium and of cholesterol in the lipid vesicles. It increased with increasing concentrations of either the lipid vesicles or the virions. pretreatment of virions with increasing concentrations of three different proteolytic enzymes (trypsin, chymotrypsin and proteinase) inhibited the virus' ability to cause release of carboxyfluorescein from negatively charged liposomes. Inhibition of the viral lytic activity was also observed after virions were incubated above 56 degrees C. PMID- 2996598 TI - Interaction of the polyene antibiotic etruscomycin with large unilamellar lipid vesicles: binding and proton permeability inducement. AB - The effect of the polyene antibiotic etruscomycin on the permeability of large unilamellar lipid vesicles was investigated. Proton leakage was induced in egg yolk phosphatidylcholine (EPC) vesicles only when sterol was present in the membrane; the extent of leakage was limited. High etruscomycin/lipid ratios (R) were necessary (R greater than 0.1). Higher percentages of sterol increased the permeability, slightly more strongly for ergosterol than for cholesterol. Dipalmitoylphosphatidylcholine (DPPC) vesicles were more sensitive to permeability inducement, even in the absence of sterol in the bilayer (inducement for R greater than 0.06). The interactions of etruscomycin with the vesicles were examined by circular dichroism, fluorescence and 31P-NMR. In the range of antibiotic concentration where permeability was induced, R greater than 0.1 for EPC vesicles, R greater than 0.06 for DPPC vesicles, etruscomycin exhibited characteristic circular dichroism spectra independent of the presence of sterol. Under the same conditions, 31P-NMR and fluorescence studies indicated a destruction or a fusion of the vesicle bilayer. At lower etruscomycin concentrations (R less than 0.03), the etruscomycin circular dichroism spectra were different, indicating that the interaction with membranes containing ergosterol differed from that with membranes containing cholesterol. From correlating the increase in fluorescence intensity with this interaction, as well as from exchange experiments, it was inferred that etruscomycin at a low antibiotic/lipid ratio is more strongly bound to ergosterol-containing vesicles than to cholesterol-containing vesicles. These results and their comparison with the results obtained with other polyene antibiotics indicate that at low R etruscomycin resembles amphotericin rather than filipin in its preferential binding to ergosterol-containing vesicles. At higher R, that is in conditions where permeability is induced, the selectivity is different. The corresponding mechanism seems not to involve the formation of an etruscomycin-sterol channel, since the hydrophobic chain of the complex would be too short to form a channel. PMID- 2996599 TI - ESR spectral changes induced by chlorpromazine in spin-labeled erythrocyte ghost membranes. AB - Chlorpromazine interacted preferentially with membrane proteins rather than membrane lipids in the initial incorporation into human erythrocyte ghosts, as demonstrated by means of the fluorescence quenching and a maleimide spin label. In this state the membrane fluidity increased. At higher concentrations of chlorpromazine, the membrane fluidity decreased and a motionally restricted signal from fatty acid spin labels appeared predominantly. However, no such signal appeared in protein-free vesicles. The temperature and pH dependences of the outer hyperfine splitting of this restricted signal were very similar to those of bovine serum albumin. On the basis of sodium dodecyl sulfate polyacrylamide gel electrophoresis of chlorpromazine-treated and -untreated ghosts, it was found that there was no significant difference in membrane proteins between both samples except for the changes of a few bands which were not directly concerned with the occurrence of this restricted signal. These results suggest that the fatty acid spin labels bind preferably to membrane proteins as the lipid domain becomes packed with chlorpromazine. PMID- 2996601 TI - Interaction of cytoplasmic L-glycerol-3-phosphate dehydrogenase with liposomes. AB - NAD-linked L-glycerol-3-phosphate dehydrogenase binds to phosphatidylcholine liposomes as shown by the changes in the properties of both the enzyme and the membrane. The surface potential and the fluidity of the liposome membrane (monitored at the 5th C atom depth) change due to the presence of the enzyme, whereas the enzyme is activated by the liposomes. These findings suggest the occurrence of peripheral protein-lipid interactions. PMID- 2996600 TI - Distribution of Ca2+-ATPase, ATP-dependent Ca2+-transport, calmodulin and vitamin D-dependent Ca2+-binding protein along the villus-crypt axis in rat duodenum. AB - The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Ca2+-ATPase activity and ATP-dependent Ca2+-transport rates in basolateral membranes from rat duodenum were measured during migration along the crypt-villus axis. In addition, vitamin D-dependent calcium-binding protein and calmodulin content were measured in homogenates of six cell populations which were sequentially derived from villus tip to crypt base. Alkaline phosphatase activity was highest at the tip of the villus (fraction I) and decreased more than 20-fold towards the crypt base (fraction VI). (Na+ + K+)-ATPase activity also decreased along the villus-crypt axis but in a less pronounced manner than alkaline phosphatase. ATP-dependent Ca2+-transport in basolateral membranes was highest in fraction II (8.2 +/- 0.3 nmol Ca2+/min per mg protein) and decreased slightly towards the villus tip and base (fraction V). The youngest cells in the crypt had the lowest Ca2+-transport activity (0.9 +/- 0.1 nmol Ca2+/min per mg protein). The distribution of high affinity Ca2+-ATPase activity in basolateral membranes correlated with the distribution of ATP-dependent Ca2+-transport. The activity of Na+/Ca2+ exchange was equal in villus and crypt basolateral membranes. Compared to the ATP dependent Ca2+-transport system, the Na+/Ca2+ exchanger is of minor importance in villus cells but may play a more significant role in crypt cells. Calcium-binding protein decreased from mid-villus towards the villus base and was undetectable in crypt cells. Calmodulin levels were equal along the villus-crypt axis. It is concluded that vitamin D-dependent calcium absorption takes primarily place in villus cells of rat duodenum. PMID- 2996603 TI - DNA methyltransferases in normal and avian sarcoma virus-transformed rat cells. Quantitation of 5-methyldeoxycytidine in DNA and enzyme kinetics study. AB - In rat kidney cells transformed by avian sarcoma virus (B77 strain) DNA is hypomethylated (2.61 +/- 0.07%) when compared to DNA extracted from normal cells (3.33 +/- 0.11%) as revealed by high-performance liquid chromatography analysis. Kinetics studies showed that no significant differences could be detected between DNA methyltransferase activities from normal and transformed cells with regard to apparent Vmax, apparent Km for S-adenosylmethionine (2.32 X 10(-6) M and 6.64 X 10(-6) M respectively) and apparent Ki for S-adenosylhomocysteine (9.2 X 10(-7) M and 7.8 X 10(-7) M respectively), when unmethylated duplex DNA was used as second substrate. Equivalent ratios of S-adenosylmethionine over S-adenosylhomocysteine were measured in each cell type and DNA methyltransferase activities from both sources were found to be strictly additive. These results show that the hypomethylation of DNA detected in transformed cells is related neither to alterations of enzymatic activities extracted from nuclei nor to unbalanced S adenosylmethionine/S-adenosylhomocysteine ratios. PMID- 2996602 TI - Activation of the abl oncogene in murine and human leukemias. PMID- 2996604 TI - P31, a mammalian housekeeping protein encoded by a multigene family containing a high proportion of pseudogenes. AB - The primary structure of P31, a 13.300 kDa mammalian 'housekeeping' protein, was deduced from the nucleotide sequence of a rat recombinant cDNA clone. The P31 mRNA has 950 nucleotides and codes for a protein of 121 amino acids. This mRNA is present in several mouse tissues and in other mammals. Nuclei of rapidly growing cells contain mature P31 mRNA and a distinctive set of larger transcripts. P31 is encoded by a multigene family. Five members of the family were isolated from a mouse genomic library and restriction mapping and S1 nuclease analysis of four of them (P31-4, P31-12, P31-13 and P31-14) suggest that they are processed genes. The very low homology of the fifth (P31-8) with respect to the corresponding cDNA sequence indicates that it is a pseudogene. Although the features of the mRNA and the genes encoding P31 exhibit extensive similarity with those of ribosomal proteins, neither the identity nor the biological function of the protein has yet been determined. PMID- 2996605 TI - Effect of spermine on cleavage of plasmid DNA by nucleases S1 and Bal 31. AB - S1, a single-strand-specific nuclease, cleaves both strands of supercoiled DNA mostly once at unpaired sites. However, all the sites are not cleaved with the same frequency by the enzyme, there being sites of preferential cleavage and infrequent ones. Spermine was found to reduce this cleavage specificity of S1 with supercoiled plasmid DNA. A similar effect of spermine was observed with Bal 31 nuclease. Bal 31 contains in addition to S1-like activity a quasi-processive exonuclease activity that simultaneously degrades both 3'- and 5'-termini of linearized duplex DNA. Spermine stimulated the exonuclease activity. The above results were obtained within the concentration range of spermine that did not induce DNA aggregation. PMID- 2996606 TI - Identification of heme axial ligands of cytochrome c' from Alcaligenes sp. N.C.I.B. 11015. AB - The spectral properties of both ferric and ferrous cytochromes c' from Alcaligenes sp. N.C.I.B. 11015 are reported. The EPR spectra at 77 K and the electronic, resonance Raman, CD and MCD spectra at room temperature have been compared with those of the other cytochromes c' and various hemoproteins. In the ferrous form, all the spectral results at physiological pH strongly indicated that the heme iron(II) is in a high-spin state. In the ferric form, the EPR and electronic absorption spectra were markedly dependent upon pH. EPR and electronic spectral results suggested that the ground state of heme iron(III) at physiological pH consists of a quantum mechanical admixture of an intermediate spin and a high-spin state. Under highly alkaline conditions, identification of the axial ligands of heme iron(III) was attempted by crystal field analysis of the low-spin EPR g values. Upon the addition of sodium dodecyl sulfate to ferric and ferrous cytochrome c', the low-spin type spectra were induced. The heme environment of this low-spin species is also discussed. PMID- 2996607 TI - Following association to the membrane, human erythrocyte procalpain is converted and released as fully active calpain. AB - When exposed to inside-out human erythrocyte vesicles, in the presence of micromolar Ca2+, the 80 kDa catalytic subunit of procalpain is processed through three successive and sequential steps. These include binding to the cytosolic surface of the membrane, followed by a very rapid conversion into the 75 kDa active subunit, and ultimately by spontaneous and complete release of this active proteinase form. Binding to the membranes is competitively inhibited by the endogenous natural inhibitor through the formation of the proteinase-inhibitor complex, in which form the 80 kDa subunit can no longer be associated to the membranes. Calcium ions and the natural endogenous inhibitor appear to be crucially involved in the modulation of this novel membrane-bound mediated activation of human red cell procalpain. PMID- 2996608 TI - Proteolysis of poly(ADPribose) polymerase by a pyrophosphate- and nucleotide stimulated system dependent on two different classes of proteinase. AB - We have identified a system in human lymphocytes which proteolytically cleaves poly(ADPribose) polymerase to specific fragments of molecular weight 96 000, 79 000 and 62 000-60 000. This proteolytic processing is dependent on two different classes of proteinase. One of these proteinases is a serine proteinase, since the processing is inhibited by phenylmethylsulfonyl fluoride, antipain, soybean trypsin inhibitor and diisopropylfluorophosphate, the other is a cathepsin D-like proteinase, since processing is also inhibited by pepstatin A. The processing that occurs in permeabilized cells can be simulated in vitro by treating purified poly(ADPribose) polymerase with trypsin, but not by treating the polymerase with cathepsin D. Since processing at the cellular level is blocked by inhibitors of either of the two proteinases, but only trypsin could cleave the purified polymerase, this suggests that in the cell the action of the cathepsin D-like proteinase is a prerequisite for cleavage of poly(ADPribose) polymerase by the serine proteinase. Thus, a pathway involving sequential action of these proteinases may exist. Proteolysis in permeabilized human lymphocytes is stimulated by nucleotides containing a pyrophosphate group, such as 5',5'''-P1,P4 tetraphosphate and ATP, or by pyrophosphate itself. In contrast, nucleotides containing only a single phosphate, such as AMP and cyclic AMP, or inorganic sodium phosphate, do not show this stimulation of proteolysis. These results suggest that a pyrophosphate linkage is the minimum molecular requirement for stimulation of proteolytic processing of poly(ADPribose) polymerase. Proteolytic processing of poly(ADPribose) polymerase is independent of ADPribosylation. Following proteolysis, specific fragments of the polymerase, particularly the 62 000-60 000 molecular weight fragment(s), are still capable of being ADPribosylated. PMID- 2996609 TI - The acid-alkaline transition of a sea turtle myoglobin: coexistence at high pH of high- and low-spin forms. AB - The microenvironment of the iron in a sea turtle Dermochelys coriacea myoglobin is studied using the spectroscopic techniques EPR and optical absorption. Optical absorption spectra in the visible region suggest a great homology between turtle Mb and other myoglobins, such as those from whale, human and elephant. The pK of the acid-alkaline transition is 8.4 slightly lower than the pK of whale and equal to that of elephant myoglobin. The EPR spectrum at pH 7.0 is characteristic of a high-spin configuration with axial symmetry (gx = gy = 5.95). At higher pH, this signal changes in a way different from that observed for whale myoglobin. We observe for turtle Mb both the formation of a low-spin configuration with rhombic symmetry (gx = 2.56, gy = 2.20, gz = 1.90) and of a high-spin species with rhombic distortion (gx = 6.79, gy = 5.18, gz = 2.12). This suggests a lowering of symmetry at the haem, so that now the x and y directions are no more equivalent. This can be explained by amino acid substitution at the distal positions of haem or to off-axial positioning of distal residues. The coexistence at high pH (pH 11.0) of these two spin forms could be explained by the existence of two protein conformations, in which the crystal field splitting factor, delta, and the electron exchange energy are of the same order, allowing the presence of different configurations simultaneously. The presence of different kinds of haem is ruled out by the experiments with nitrosyl turtle Mb and turtle Mb-F showing spectra very similar to those of whale myoglobin. The pk of the acid-alkaline transition, 8.5, obtained from EPR spectra, agrees very well with results from optical absorption. PMID- 2996610 TI - Metabolic profile of linoleic acid in porcine leukocytes through the lipoxygenase pathway. AB - Porcine neutrophilic leukocytes were found to contain a lipoxygenase which converted linoleic acid into 13-hydroxy-9,11-octadecadienoic acid (n-6 specificity), arachidonic acid into 12-hydroxy-5,8,10,14-eicosatetraenoic acid (n - 9 specificity) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid into 5,12 dihydroxy-6,8,10,14-eicosatetraenoic acid. This lipoxygenase was partially purified and it appeared that its substrate specificity and other properties were quite different from the 12-lipoxygenase of blood platelets. Incubations of intact or broken porcine leukocytes with added linoleic acid revealed the formation of not only 13-hydroxy-9,11-octadecadienoic acid but also of substantial amounts of epoxyhydroxy and trihydroxy isomers. These products from linoleate, collectively described by the name 'octadecanoids' were characterized in detail by a combination of chemical, chromatographic and mass spectrometric techniques. The phospholipids of porcine leukocytes contain more than twice as much linoleate than arachidonate (22 vs. 8%). In accordance with this fatty acid composition we found that in the stimulated neutrophil the endogenous production of octadecanoids often surpassed that of the eicosanoids. Lipoxygenation of endogenously liberated linoleic acid was especially pronounced when a suspension of leukocytes in citrated plasma was recalcified and allowed to clot. PMID- 2996611 TI - Iron uptake and heme synthesis by isolated rat liver mitochondria. Diferric transferrin as iron donor and the effect of pyrophosphate. AB - Isolated rat liver mitochondria accumulate iron from fully saturated transferrin at neutral pH. With 5 microM iron as diferric transferrin, accumulation at 30 degrees C amounts to approx. 40 pmol/mg protein per h. With access to a suitable porphyrin substrate, 70-80% of the amount of iron accumulated is recovered in heme. Mobilization of iron and synthesis of heme both depend on a functioning respiratory chain. Vacant iron-binding sites on mono- and apotransferrin compete with the mitochondria for iron mobilized from transferrin. Pyrophosphate at concentrations in the range 10-50 microM enhances mobilization of iron, counterbalances the inhibitory effect of mono- and apotransferrin and enhances metallochelatase activity. The results emphasize the putative suitability of pyrophosphate as an intracellular iron-transport ligand in situ. PMID- 2996613 TI - Ribosomal protein S6 phosphorylation and morphological changes in response to the tumour promoter 12-O-tetradecanoylphorbol 13-acetate in primary human tumour cells, established and transformed cell lines. AB - The phosphorylation of ribosomal protein S6 in fibroblasts, primary human tumour cells, established and SV40-transformed human cell lines was compared after the addition of 12-O-tetradecanoylphorbol 13-acetate (TPA). In fibroblasts and primary tumour cell cultures, stimulation of S6 phosphorylation was about 4-6 fold. Established and transformed cell lines showed enhanced S6 phosphorylation which was not further stimulated by the addition of TPA. These findings indicated that the influence of TPA on the metabolic pathway, that finally leads to the phosphorylation of protein S6 in cells with a limited lifespan (fibroblasts, primary human tumour cells) can be mimicked by unknown steps also associated with immortalization (establishment function) and the transformed state of the tumour cells. Another interesting observation were morphological changes of the established and SV40-transformed cells which were visible as early as 20 min after the addition of TPA. In fibroblasts and primary tumour cells no changes in morphology were observed, even after 8h incubation. PMID- 2996612 TI - Galactose-specific receptors on liver cells. I. Hepatocyte and liver macrophage receptors differ in their membrane anchorage. AB - Colloidal gold particles coated with asialoglycoproteins are bound by hepatocytes as well as by liver macrophages. Binding by both cell types is inhibited by N acetylgalactosamine and related saccharides and is dependent on the presence of Ca2+. We have now performed an electron microscopic study on receptor anchorage in the plasma membranes. Cells with prebound ligand were treated with 20 mM EDTA at 4 degrees C, washed free of chelator and tested for residual galactose specific receptor activity. Whereas hepatocytes preserve binding activity (73% of untreated control), liver macrophages lose galactose-specific receptor activity (12% of untreated control). Liver macrophages regain binding activity after a 2 min incubation at 37 degrees C allowing for receptor recycling. If the macrophages were fixed with low glutaraldehyde concentration prior to EDTA treatment they fully retained their receptor activity (74% of control). Ligands were also removed from both cell types by incubation with 80 mM N acetylgalactosamine. After washing the cells free of the competing monosaccharide, both the hepatocytes as well as the macrophages show full binding activity (120% and 85% of untreated controls). Therefore, membrane anchorage sites of the macrophage receptors are not identical to ligand-binding sites. These results suggest a Ca2+-Mg2+-dependent receptor anchorage on the macrophage plasma membrane. As shown in the accompanying paper (Roos, P.H., Hartmann, H.J., Schlepper-Schafer, J., Kolb, H. and Kolb-Bachofen, V. (1985) Biochim. Biophys. Acta 847, 115-121), EDTA-induced dissociation from the membrane can be used for isolation of the galactose-specific receptors of liver macrophages. PMID- 2996615 TI - Identification of ecto-nucleoside triphosphate pyrophosphatase in human articular chondrocytes in monolayer culture. AB - In cultured monolayers of human articular chondrocytes we have observed an enzyme activity which catalyzes the extracellular conversion of ATP to AMP and PPi. The enzyme was active at very low concentrations of ATP (microM) and exhibited optimal activity at concentrations of ATP of approx. 100 microM. The enzyme was active in intact cells as judged by measurement of the release of the cytoplasmic marker enzyme lactate dehydrogenase. No increase in production of PPi from ATP was observed on mechanically disrupting the cells and no activity was shed into the medium by intact cells. Activity was stable between days 4 and 8 after subculturing the cells and was not affected by the timing of the final medium change prior to assay. Activity was also observed with other nucleoside triphosphates (GTP, CTP and UTP). We suggest that this activity is attributable to ecto-nucleoside triphosphate pyrophosphatase. This observation may be important in relation to the pathogenesis of the human disease of chondrocalcinosis in which crystals of calcium pyrophosphate dihydrate deposit in articular cartilage. PMID- 2996614 TI - Differential effects of hyperoxia and hydrogen peroxide on thymidine kinase and adenosine kinase activities of cultured endothelial cells. AB - To compare the respective sensitivity of two nucleoside kinases, adenosine kinase and thymidine kinase, to oxidative stress, we measured these enzyme activities in cultured aortic endothelial cells exposed for 48 h to various O2 concentrations, and in cell extracts treated with H2O2 or the enzyme system hypoxanthine-xanthine oxidase. Adenosine kinase activity was not significantly influenced by the exposure to hyperoxia, nor by treatment with the enzyme system hypoxanthine xanthine oxidase or with H2O2. On the other hand, there was a dose-dependent inhibitory effect on thymidine kinase activity resulting from exposure to various O2 concentrations or from treatment with various amounts of xanthine oxidase. Incubation of cell extracts in the presence of H2O2 also resulted in a significant reduction of thymidine kinase activity. These results indicate that thymidine kinase exhibits a selective sensitivity to the toxic effect of O2 concentrations and of O2 intermediates such as H2O2. PMID- 2996616 TI - Protection of mammalian cells by o-phenanthroline from lethal and DNA-damaging effects produced by active oxygen species. AB - Active oxygen species are suspected as being a cause of the cellular damage that occurs at the site of inflammation. Phagocytic cells accumulate at these sites and produce superoxide ion, hydrogen peroxide and hydroxyl radical. The ultimate killing species, the cellular target and the mechanism whereby the lethal injury is produced are unknown. We exposed mouse fibroblasts to xanthine oxidase and acetaldehyde, a system which mimics the membrane of phagocytic cells in terms of production of oxygen species. We observed that the generation of these species produced DNA strand breaks and cellular death. The metal chelator o phenanthroline completely abolished the former effect, and at the same time it effectively protected the cells from lethal injuries. Because complexing iron o phenanthroline prevents the formation of hydroxyl radical by the Fendon reaction (Fe(II) + H2O2----Fe(III) + OH- + OH.), it is proposed that most of the cell death and DNA damage are brought about by OH radical, produced from other species by iron-mediated reactions. PMID- 2996617 TI - [Study of cytochrome c refolding kinetics by the "stopped flow" method at acid pH]. AB - It is shown that refolding of horse cytochrome c at pH 2.0 induced by an increase of ionic strength occurs in three kinetic phases with the rate constants similar to those observed for the refolding of this protein into the native state. The rate constants and amplitudes of these phases do not depend on the final ionic strength and phase amplitudes are not temperature-dependent. Reasons for the complex kinetics of the cytochrome c refolding at acid pH are discussed. A description of a "stopped-flow" attachment compatible with different commercial optical devices is provided. PMID- 2996619 TI - [Effect of hyperbaric oxygenation on paramagnetic centers in the rabbit myocardium during experimental myocardial infarction]. AB - Changes in paramagnetic centres (PC) concentration in rabbit's myocardial muscle were studied under experimental myocardial infarction during 168 hours by means of ESR-spectroscopy. PC characterized by ESR signal with g = 3.0 was found. It was shown that hyperbaric oxygenation did not affect PC content in the infarction zone, and resulted in an increase of PC concentration with g = 1.94 and g = 2.0 in the intact myocardial zone. PMID- 2996618 TI - [The use of fluorescence probes in the study of the beta-adrenoreceptor function of human erythrocytes]. AB - Possibility of making use of fluorescent probe 4-(p-dimethyl aminostyril)-1 methyl pyridinium p-toluene-sulphonate (DSM) for observing the interaction of beta-adrenoactive substances (adrenaline and propranolole) and specific receptors of human blood erythrocytes has been studied. Two types of DSM bindings with erythrocyte membranes have been revealed--specific and non-specific. It has been found that DSM reacts selectively to preliminary influence of beta-adrenoactive substances: antagonist propranolole diminishes both specific and nonspecific bindings and agonist adrenaline increases specific binding with the membrane erythrocytes. The conclusion is made as to possible use of DSM probe for investigating beta-adrenoreceptive function of the cell membranes. PMID- 2996620 TI - [Formation of the left-handed helix during simultaneous treatment of poly[d(GC)] with bis-netropsin and Zn(II) ions]. AB - It has been found that the effect of AT-specific ligand and Zn2+ on GC alternating polymer brings about transfer of the latter into Z-conformation. PMID- 2996623 TI - [Detection of superoxide radicals in intact heart mitochondria by spin trapping]. AB - Tiron (4,5-dihydroxybenzene-1,3-disulfonic acid, disodium salt) has been used as a spin trap to detect superoxide radicals produced by the rat heart mitochondria. In the presence of the oxidative phosphorylation uncoupler carbonyl cyanide (3 chlorophenyl)-hydrazone the superoxide production rate increases. PMID- 2996621 TI - [Formation of intermediate products in the reaction of hemoglobin with cyanide]. AB - Interaction between methemoglobin and cyanide was studied by low temperature inhibition method in combination with ESR. A new, not earlier described in literature ESR signal of low spin cyanide complex of this protein was recorded. PMID- 2996624 TI - [Study of the flexibility of serum albumin molecules using the spin label method]. AB - In is shown that the description of the motion character of N.- O-group of spin label conjugated with human serum albumin molecule on the basis of the jump--way isotropic diffusion model is correct. The structural flexibility of serum albumin molecule was revealed. It is the result of the ability of three domains that form the molecule for relative thermal reorientations. General tendency of elevation of protein correlation time from 20 to 45 ns with rising temperature from 5 to 44 degrees reflects the strengthening of hydrophobic interactions between domains. The presence in solution of 5 and 15% D2O essentially increases the amplitude of protein flexibility changes in the range of two thermoinduced transitions of serum albumin: 15-20 degrees and 32-35 degrees C. It reflects the important role of water in this process. PMID- 2996622 TI - [Effect of cross-linking agents on electron transport and proton transfer in the mitochondrial respiratory chain]. AB - The effect of protein cross-linkage on proton translocation and electron transport in mitochondria respiratory chain was studied. Dimethylsuberimidate (1 mM) or dicyclohexyl carbodiimide (50 g/ml) inhibit proton translocation with concomitant stimulation of respiration. It is concluded that the definite level of dynamic mobility of proteins is needed for proton translocation. PMID- 2996625 TI - [Spin label study of structural changes at different depths of phospholipid membranes after their peroxidation]. AB - Changes of structural organization of liposomal phospholipid membranes, after their Fe2+-induced peroxidation were studied at different depth using nitroxyl derivatives of stearic acid. It was established that during Fe2+-induced lipid peroxidation a strong rise in lipid polarity accompanied with immobilization of acyl chains of phospholipids at the depth of 0.6-0.8 nm from the surface was measured. At the same time no significant changes in the structural organization were seen at the depth of 20-22 nm. PMID- 2996626 TI - [Determination of the surface charge of lipoproteins and its changes during lipid peroxidation]. AB - Surface potential of human plasma lipoproteins was studied by the use of positively charged spin probe. The calculated values of surface potential of high and low density lipoproteins appeared to be -29 +/- 1 and -16 +/- 1 respectively. It was shown that lipid peroxidation process induces an increase of surface potential of both high and low density lipoproteins. Probably, it is connected with the increase of the negative charge density on their surface. This fact can play an important role in pathogenesis of diseases with lipid metabolism and lipid peroxidation level disorders in plasma (atherosclerosis, ischemic heart disease etc.). PMID- 2996627 TI - [Generation of active forms of oxygen by human peripheral blood neutrophils and lymphocytes during adhesion to glass]. AB - Oxygen activation by neutrophils and human blood lymphocytes at adhesion to glass have been studied by luminol--dependent chemiluminescence. It has been established that the cell interaction with glass leads to the formation of O2-., O2', .OH and H2O2. Chemiluminescence kinetics and the excited--oxygen forms ratio at adhesion were different for neutrophils and lymphocytes. The desorption of cells resulted in a decrease of the chemiluminescent response to neutrophils and lymphocytes when they again adhered to glass and in practically complete inhibition of chemiluminescence induced by adding concanavalin A. It has been determined that at adhesion of neutrophils and lymphocytes to glass different mechanisms of oxygen activation take place. PMID- 2996628 TI - [Initiation of the mouse sarcoma virus oncogene v-K-ras during the chemical treatment of proto-oncogene C-ras]. AB - On the basis of the results obtained in the laboratories of Weinberg, Barbacid et al. describing the responsibility of one or another mutation in one of coding triplets of the ras family gene both for chemical and viral cancerogenesis, we draw the conclusion that it is impossible to distinguish these two causes of cancer appearance. It is shown that the cause of mice viral sarcoma v-K ras development from the cellular protooncogene C-ras may consist in the chemical reaction of one of coding triplet nucleotides with cancerogenic substance. PMID- 2996630 TI - Purification and properties of glycogen synthase I from bovine polymorphonuclear leucocytes. AB - Glycogen synthase I was purified from bovine polymorphonuclear leucocytes (PMNs) by a procedure involving concanavalin A-Sepharose affinity chromatography. The purified glycogen-bound glycogen synthase I had a specific activity of 9.83 U/mg protein and the glycogen free enzyme 21 U/mg protein. Molecular ratio of the native enzyme and the subunit were 340 K and 85 K respectively. After phosphorylation by the catalytic subunit of cAMP-dependent protein kinase the phosphorylated sites were studied using high-performance liquid chromatography (HPLC) of the tryptic 32P-peptides. The enzyme was phosphorylated at three different sites with retention times identical to site 1a, site 1b, and site 2 from rabbit skeletal muscle glycogen synthase. PMID- 2996629 TI - Sequence analysis of a Dictyostelium discoideum gene coding for an active dihydroorotate dehydrogenase in yeast. AB - A Dictyostelium discoideum DNA fragment isolated on the basis of its ability to complement the ural mutation of yeast, codes for a dihydroorotate dehydrogenase activity. The complete nucleotide sequence of this 1898 bp fragment has been determined and reveals an open reading frame capable of coding for a 369 amino acid polypeptide of molecular mass 47.000. The gene shows preferential use of codons with weak pairing forces. Eleven codons, mainly those with a G in the third position, are absent. The flanking sequences are unusually rich in A + T (80%). Several direct and inverted repeats exist in the 5' flanking sequence. PMID- 2996631 TI - Overproduction and purification of initiation factor IF2 and pNUSA proteins from a recombinant plasmid bearing strain. AB - The genes for translational initiation factor, IF2 and pNusA have been cloned into a plasmid vector where they are placed under the control of the inducible lambdapL promoter and the c1857 thermosensitive repressor. When a strain carrying this plasmid is heat induced, IF2 alpha, IF2 beta and pNusA are overproduced 15 to 20 fold. This has allowed us to purify the IF2 and NusA proteins in large amounts. PMID- 2996632 TI - A new statistical method for identifying interconnections between neuronal network elements. AB - A new method is proposed to analyse dependencies in point processes, which takes into account specific character of neuronal activity. Simulation modelling of neuronal network revealed that the estimated weight of connection depends monotonically on the value of the model synaptic strength. In contrast to the crosscorrelation, the method allows for nonlinear interconnections and does not require point processes to be stationary and samples to be large. Examples are presented of the method's application to neurophysiological data analysis. PMID- 2996634 TI - Sustained oscillations generated by mutually inhibiting neurons with adaptation. AB - Autonomic oscillatory activities exist in almost every living thing and most of them are produced by rhythmic activities of the corresponding neural systems (locomotion, respiration, heart beat, etc.). This paper mathematically discusses sustained oscillations generated by mutual inhibition of the neurons which are represented by a continuous-variable model with a kind of fatigue or adaptation effect. If the neural network has no stable stationary state for constant input stimuli, it will generate and sustain some oscillation for any initial state and for any disturbance. Some sufficient conditions for that are given to three types of neural networks: lateral inhibition networks of linearly arrayed neurons, symmetric inhibition networks and cyclic inhibition networks. The result suggests that the adaptation of the neurons plays a very important role for the appearance of the oscillations. Some computer simulations of rhythmic activities are also presented for cyclic inhibition networks consisting of a few neurons. PMID- 2996633 TI - A maximum entropy criterion of filtering and coding for stationary autoregressive signals: its physical interpretations and suggestions for its application to neural information transmission. AB - The operations of encoding and decoding in communication agree with filtering operations of convolution and deconvolution for Gaussian signal processing. In an analogy with power transmission in thermodynamics, an autoregressive model of information transmission is proposed for representing a continuous communication system which requires a pair of an internal noise source and a signal source to encode or decode a message. In this model transinformation (informational entropy) equals the increase in stationary nonequilibrium organization formed through the amplification of white noise by a positive feedback system. The channel capacity is finite due to the existence of inherent noise in the system. The maximum entropy criterion in information dynamics corresponds to the 2nd law of thermodynamics. If the process is stationary, the communication system is invertible, and has the maximum efficiency of transformation. The total variation in informational entropy is zero in the cycle of the invertible system, while in the noninvertible system the entropy of decoding is less than that of encoding. A noisy autoregressive coding which maximizes transinformation is optimum, but is also ideal. PMID- 2996635 TI - [Generation of the superoxide radical and lipid peroxidation in skeletal muscles]. AB - The production of O2-, H2O2 and ADP X Fe3+-dependent lipid peroxidation (LPO) in skeletal muscle sarcoplasmic reticulum and mitochondria was studied. It was shown that in the muscle cell the O2- sources and LPO substrates are spatially separated, i. e., the former are localized in mitochondrial membranes, while the latter are predominantly located in sarcoplasmic reticulum membranes. It is concluded that in isolated subcellular fractions of the muscle LPO is generated via direct reduction of ADP-Fe3+ by corresponding redox chains without the involvement of free O2-. The feasibility of intermembrane diffusion of O2- in vivo is postulated, which results in the initiation by mitochondrial O2- of LPO in sarcoplasmic reticulum membranes. PMID- 2996636 TI - [Biosynthesis of thiamine triphosphate and identification of thiamine diphosphate binding proteins in the rat liver hyaloplasm]. AB - The nature of the thiamine diphosphate binding proteins from rat liver hyaloplasm was studied. When [14C]thiamine was used as a marker, a [14C]thiamine diphosphate containing electrophoretically homogeneous protein preparation was isolated from the liver soluble fraction and classified as transketolase. No other non enzymatic proteins which bind thiamine diphosphate and can serve as substrates in the reaction of thiamine diphosphate synthesis in the hyaloplasm were found. It was shown that the phosphate group is transferred by rat liver thiamine diphosphate kinase to the free (but not to the protein-bound) thiamine diphosphate as it was believed earlier. PMID- 2996637 TI - [Chromatographic properties of human leukocyte alpha-interferon A on sorbents with sepharose and silochrome matrices]. AB - Chromatography on immobilized monoclonal antibodies NK-2 from a bacterial strain producer resulted in a pure human leukocyte alpha-interferon A (alpha-INF-A) homogeneous upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reverse phase high performance liquid chromatography. The chromatographic properties of partially purified alpha-INF-A on synthetic and commercial sorbents containing immobilized dyes, aromatic dipeptides, chelating and hydrophobic ligands as well as on porous glass have been investigated. In most cases, [125I]alpha-INF-A was used as an inner standard. The chromatographic behaviour of native and [125I]-labeled alpha-INF-A was practically the same. alpha-INF-A was most effectively chromatographed on porous glass, L-Trp-L-Trp Sepharose 4B and Cu2+-chelate sorbents. In the latter case, the feasibility of substitution of the Sepharose matrix for the silochrome one has been demonstrated. It has been proposed that alpha-INF-A has a hydrophobic "pocket" with exposed aromatic amino acid residues which are capable of selective binding to aromatic dipeptides. PMID- 2996638 TI - [Functional changes in the properties of beta-adrenoreceptors of pigeon erythrocytes after treatment with a catalytic subunit of cAMP-dependent protein kinase]. AB - beta-Adrenoreceptors were solubilized by deoxycholate from pigeon erythrocyte plasma membranes treated with N-ethylmaleimide. Removal of the detergent resulted in the incorporation of receptors into phospholipid vesicles as well as in the reconstitution of their biological activity. After fusion of vesicles containing reconstituted receptors to vesicles containing the Ns protein and a catalytic component, the hormonal activation of the enzyme was restored. When prior to fusion the beta-adrenoreceptor-containing vesicles were preincubated with the catalytic subunit of cAMP dependent protein kinase, the hormone-induced activation of the enzyme diminished by 45-50%. The decrease of activation is due to the increase in the lag phase of the enzyme activation in the presence of isoproterenol and Gpp(NH)p as well as to the loss of activity in the steady-state phase of activation. Phosphorylation of beta-adrenoreceptors decreased the concentration of the ternary isoproterenol-receptor-Ns protein complex involved in the activation of adenylate cyclase. Thus, the phosphorylation of receptors is responsible for the disturbances in the mechanism of hormonal signal transmission that are similar to those observed in adenylate cyclase desensitization. PMID- 2996639 TI - [Submitochondrial distribution of cAMP during incubation with rat liver mitochondria]. AB - Labeled cAMP incubated with rat liver mitochondria penetrates not only through outer mitochondrial membranes, but also into mitoplasts, where it is accumulated mainly in the matrix. Damage of mitochondrial membranes caused by single freezing thawing treatment promotes no influx, but efflux of cAMP from mitoplasts. cAMP molecules penetrate inside mitochondria largely in an unchanged state in all submitochondrial fractions, as was demonstrated by the TLC method. cAMP transport into mitochondria can serve as a reason for: 1) stimulation of mitochondrial function by hormones whose effects are realized through activation of cytoplasmic adenylate cyclase and by extramitochondrial (cytosolic) cAMP; 2) existence of cAMP-dependent protein kinase and cAMP-phosphodiesterase in mitochondria. PMID- 2996640 TI - Association between amino acid alterations and hallucinations in alcoholic patients. AB - Because available evidence suggests that alterations in the serotonergic as well as dopaminergic tones underlie hallucinatory activity, we decided to investigate whether serotonin and dopamine pathways are modified in alcoholics with a history of hallucinosis. Brain serotonin has been shown to depend on the plasma ratio of its precursor tryptophan over other amino acids competing with it for brain entry. Similarly, brain dopamine depends on the plasma ratio of its precursors phenylalanine and dopamine over their competitors. Amino acid abnormalities are common in alcoholics. For this reason, we assessed whether alcoholics who had experienced hallucinations have alterations in amino acids believed to be associated with neurotransmitter modifications. Patients with a history of hallucinations were found to have a tryptophan ratio significantly lower than that of patients without such a history, and a tyrosine + phenylalanine ratio significantly higher. These data suggest that amino acid abnormalities believed to result in decreased brain serotonin and in increased brain dopamine render certain individuals more vulnerable to hallucinatory experiences. PMID- 2996641 TI - High thresholds for movement perception in schizophrenia may indicate abnormal extraneous noise levels of central vestibular activity. AB - A theoretical argument proposes that thresholds for visual perception of movement should be abnormally high in schizophrenia. This may reflect a central vestibular dysfunction, consisting of abnormally high levels of extraneous noise within the neural activity of the central vestibulo-cerebellar complex. Two experiments are reported with results that support the hypothesis. To some extent, the disorder may explain the smooth pursuit eye movement dysfunction in schizophrenia. Relations to the dopamine hypothesis in schizophrenia are discussed. PMID- 2996643 TI - Regulation of baboon fetal adrenal androgen production by adrenocorticotropic hormone, prolactin, and growth hormone. AB - Factors other than adrenocorticotropic hormone (ACTH) are thought to influence fetal adrenal steroidogenesis during primate pregnancy. Therefore, we determined the effects of prolactin (Prl), growth hormone (GH), and human chorionic gonadotropin (hCG) as well as ACTH on steroid secretion by collagenase-dispersed baboon fetal adrenal cells. Adrenal glands were obtained from seven baboon (Papio anubis) fetuses following cesarean section at Day 100-107 of gestation (term = Day 184). Tissue was minced with a fine scissors and cells were dispersed with 0.2% collagenase, then washed with Medium 199 containing penicillin/streptomycin. Cells (0.5 X 10(4)) were placed in 4 ml Medium 199 with or without 10 nmol ovine Prl, ovine GH, or ACTH, or 50 nmol hCG. After 18 h incubation (37 degrees C), cells were separated by centrifugation and the quantities of cortisol (F), dehydroepiandrosterone (DHA), and DHA-sulfate (DHAS) secreted into the medium were determined. In controls, DHA secretion [224 +/- 96 ng/(24 h X 10(5) cells] was greater (P less than 0.05) than that of DHAS (20 +/- 12) and F (14 +/- 12). Adrenocorticotropic hormone, Prl, and GH stimulated (P less than 0.05) DHA secretion by 370% +/- 71%, 215% +/- 61%, and 292% +/- 73%, respectively; hCG was not effective. Due primarily to the relatively low secretion rates, DHAS and F secretion were not altered by hormonal treatment. Moreover, addition of 20 nmol progesterone to the medium in the presence or absence of ACTH did not influence F production. These findings indicate that the baboon fetal adrenal at midgestation does not utilize placental progesterone for F synthesis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996642 TI - Dopamine and the action of opiates: a reevaluation of the dopamine hypothesis of schizophrenia. With special consideration of the role of endogenous opioids in the pathogenesis of schizophrenia. AB - It is suggested that the antipsychotic efficacy of opioids in patients suffering from schizophrenia may result from an interaction of opioids with the dopaminergic system. The modulatory effect of opioids on dopaminergic functions has already been demonstrated in basic experiments: Anatomical and biochemical data reveal an interaction between opioid receptors and dopamine (DA) actions on dopaminergic nerve terminals, cell bodies, and afferent nerve endings. Endogenous enkephalin levels correlate well with the endogenous dopamine content in various brain areas. Systemic or iontophoretic administration of morphine alters the spontaneous activity of ventral tegmental dopaminergic neurons. Morphine and enkephalin effectively enhance pituitary prolactin release, whereas dopamine inhibits it. Opioid agonists effectively alter DA release, DA reuptake, and DA metabolism in the striatum and substantia nigra. In reverse, chronic neuroleptic treatment enhances the synthesis and release of pituitary beta-endorphin. Opioids affect contralateral rotation elicited by dopamine agonists in animals with unilateral lesions of the nigrostriatal pathway. Phencyclidine, a psychotropic drug that shares certain pharmacological characteristics with the putative sigma opioid receptor ligand SKF 10,047, indirectly mimics the effects of dopamine agonists on prolactin release, release of acetylcholine, etc. It is suggested that an imbalance of opiate-DA interaction might be involved in the pathogenesis of schizophrenia. Consequently, clinical studies on the effects of opioids on psychotic symptoms should also examine opioid influence on dopaminergic functions in these patients. PMID- 2996644 TI - The postpartum induced corpus luteum: functional differences from that of cycling cows and the effects of progesterone pretreatment. AB - Anestrous postpartum (PP) Hereford cows (n = 41) were used to compare corpora lutea (CL) from gonadotropin-releasing hormone (GnRH)-induced ovulation with CL from cycling cows. Postpartum cows were injected i.m. daily with 100 mg progesterone (P4) or oil on Days 25 through 28 PP and then given 200 micrograms GnRH i.m. on Day 30 PP. Corpora lutea were removed from one-half of the PP cows in the oil- and P4-treated groups 6.5 days after GnRH injection, and from the cycling cows 7 days after estrus. Intact PP cows were used to evaluate cycle length. Blood was collected daily from all PP cows from Day 25 PP through luteectomy and on Days 9, 11, and 13 post-GnRH from the oil- and P4-intact cows to determine short (SHORT) versus normal (NORM) luteal phases. Cycling cows were bled daily from estrus until CL removal NORM PP cows had higher (P less than 0.001) P4 levels than did SHORT PP cows from Day 7 through Day 13 post-GnRH, and more (P less than 0.05) P4-intact cows were NORM compared with oil-intact cows (45.5% vs. 14.3%, respectively). Corpora lutea from cycling cows were heavier (P less than 0.05) and had a higher luteinizing hormone (LH) receptor concentration (P less than 0.05), but CL P4 concentration did not differ from PP cows. Corpora lutea weight, LH receptor and P4 concentration, and in vitro P4 production were similar in the oil-and P4-treated PP cows. NORM cows had heavier CL (P less than 0.05) than SHORT cows, although P4 content and LH receptor concentration did not differ.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996645 TI - Regulation of mouse oocyte maturation: effect of elevating cumulus cell cAMP on oocyte cAMP levels. AB - We have reexamined the possibility that cumulus cell cAMP can enter the oocyte via the gap junctions connecting the two cell types (Schultz et al., 1983a). Since our recent results indicate that the mouse oocyte possesses a very active cyclic nucleotide phosphodiesterase (PDE) (Bornslaeger et al., 1984), we have altered our experimental protocol to ensure that mouse oocyte PDE activity is inhibited throughout the duration of an experiment. Our results demonstrate the apparent transfer of cAMP from cumulus cells to the oocyte; these results are discussed in terms of current models for regulation of mammalian oocyte maturation. PMID- 2996646 TI - Bioreactions at the tissue/hydroxyapatite interface. AB - The events at the hydroxyapatite implant material/tissue interface in the rat middle ear were studied by light microscopy, autoradiography, morphometry, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and X ray microanalysis. Deposition of calcium, partially in the form of calcium phosphate, was found at the interface. Resorption of the implant material occurred as the result of mono- and multinuclear phagocyte activity. Resorption decreased 6 mnth after the operation, possibly due to the decreasing number of phagocytes at the interface and the increasing amount of bone in the macropores. PMID- 2996647 TI - The use of principal component analysis to resolve the spectra and kinetics of cytochrome c oxidase reduction by 5,10-dihydro-5-methyl phenazine. AB - The method of principal component analysis (PCA) was applied to the absorption wavelength-time surfaces generated by rapid scanning stopped-flow spectrophotometry (RSSFS). The method was used to resolve the absorption surfaces generated during the reduction of cytochrome c oxidase by 5,10-dihydro-5-methyl phenazine (MPH) into the individual spectral shapes and time courses of the component chromophores. Two forms of resting cytochrome oxidase were used in these analyses: one that has its maximum absorption in the Soret region at 418 nm (418-nm species) and the other has its absorption maximum at 424 nm (424-nm species). A weighting scheme suitable for RSSFS data was developed. The optical absorption spectra obtained by W.H. Vanneste (1966, Biochemistry, 5:838-848) for the oxidase components were found to fit adequately as components of the experimental surfaces. Among these spectra were the oxidized forms of cytochromes a and a3 in the wavelength region 330-520 nm for the 418-nm species. Vanneste's spectral shape for the oxidized cytochrome a3 did not fit as a component in the spectrum of the 424-nm species. After accounting for the spectral shape of all components present, PCA provided a straightforward method for determining the separate time courses of each chromophore. We have found for both forms used that cytochrome a is reduced by MPH in the initial stages of the reaction, while cytochrome a3 is reduced in subsequent, slow phases. An important aspect of PCA is that it provided confirmation of the spectra of the various oxidase components without requiring the use of inhibitors or the use of simplifying mechanistic assumptions. The resolution of time profiles of strongly overlapping chromophores is also demonstrated. PMID- 2996649 TI - Thromboxane (TX)A3 and prostaglandin (PG)I3 are formed in man after dietary eicosapentaenoic acid: identification and quantification by capillary gas chromatography-electron impact mass spectrometry. AB - The low incidence of myocardial infarction in Greenland Eskimos has been related to their traditional marine diet rich in eicosapentaenoic acid. However, whether dietary eicosapentaenoic acid is indeed transformed in man to antiaggregatory PGI3 and weakly proaggregatory TXA3 has not been clarified. In our studies we ingested either cod liver oil or mackerel both rich in eicosapentaenoic acid. Formation of TXB3, the hydrolysis product of TXA3, in platelet-rich plasma stimulated ex vivo with collagen was traced by capillary GC/EIMS. Via external standard, TXB3 formation in platelets was estimated to be 5-15% of TXB2 formation. From urine we extracted dinor metabolites of PGI according to a selective method. We utilized delta 17-2,3-dinor-6-keto-PGF1 alpha (PGI3-M) as an index of total body production of PGI3 in analogy to 2,3-dinor-6-keto-PGF1 alpha (PGI2-M), the major urinary metabolite of PGI2. We separated PGI2-M and PGI3-M as the Me, MO, Me3Si derivatives by capillary gas chromatography and identified PGI3 M by EI mass spectrometry. Excretion of PGI3-M, which was not detectable under control conditions, was 83 +/- 25 ng/24 h (SD) after ingestion of cod liver oil and 134 +/- 38 ng/24 h after mackerel ingestion, while excretion of PGI2-M was 162 +/- 52 ng/24 h and 236 +/- 32 ng/24 h, respectively. Our findings with diets rich in EPA show that it is possible in man to change in vivo the spectrum of biologically active prostanoids by nutritional means and alter it in a favourable direction. PMID- 2996648 TI - Electron spin resonance and electron-spin-echo study of oriented multilayers of L alpha-dipalmitoylphosphatidylcholine water systems. AB - A detailed electron spin resonance (ESR) study of spin-labeled-oriented multilayers of L alpha-dipalmitoylphosphatidylcholine (DPPC) water systems for low water content (2-10% by weight) is reported with the purpose of characterizing the dynamical and structural properties of model membrane systems. Emphasis is placed on the value of combining such experiments with detailed simulations based on current slow-motional theories. Information is obtained regarding ordering and anisotropic rotational diffusion rates via ESR lineshape analysis over the entire motional range, from the fast motional region through the moderately slow and slow to the rigid limit. This includes the low temperature gel phase, the liquid crystalline L alpha (1) phase and what appears to be a third high-temperature phase above the L alpha phase. Cholestane (CSL) and spin-labeled DPPC (5-PC, 8-PC, and 16-PC) have been used to probe different depths of the bilayer. While CSL and 5-PC both reflect the high ordering of the bilayer close to the lipid-water interface, CSL appears to be located close enough to the water for the nitroxide to be involved in hydrogen bonding with water molecules. 16-PC reflects the relatively low ordering near the tail of the hydrocarbon chain in the bilayer. Quantitative estimates of ordering and motion are obtained for these cases. The results from CSL indicate that close to the lipid-water interface the DPPC molecule is oriented approximately perpendicular to the bilayer in these low water-content systems. However, all three labeled lipid probes indicate that the hydrocarbon chain of DPPC may be bent away from the bilayer normal by as much as 30 degrees and this evidence is stronger at low temperatures. When cholesterol is added to the DPPC-water system at a concentration greater than or equal to 2.5 mol %, the ordering is greatly increased although the rotational diffusion rate remains almost unaffected in the gel phase. Electron spin echoes (ESE) are observed for the first time from oriented lipid-water multilayers. Results obtained from cw ESR lineshape analysis are correlated with data from ESE experiments, which give a more direct measurement of relaxation times. These results indicate that for detection of very slow motions (close to the rigid limit) ESE experiments are more sensitive to dynamics than continuous wave ESR for which inhomogeneous broadening becomes a major problem. PMID- 2996651 TI - [Effect of dopamine and dopamine mimetics on Na, K-ATPase of corpus striatum synaptosomes]. AB - Dopamine (DA) and DA-mimetics (apomorphine, midantan, piribedil) have a dual effect on Na, K-ATPase of the rat brain striate synaptosomes: activating at micromolar concentrations and inhibitory at higher concentrations (less than or equal to 30 microM). In the presence of Ca2+ (1 mM EGTA + 2.5 mM Ca2+) DA activating effect completely disappears and the inhibitory effect becomes even more pronounced. In the presence of cAMP (50 microM) which has no effect of its own on Na, K-ATPase, DA activation maximum is shifted towards lower concentrations, and the inhibitory effect remains unchanged. The above mentioned effects of DA persist in the presence of ouabain (1 mM), i.e. during measuring of Na, K-ATPase activity by an "ouabain" method, with DA activation maximum shifted towards higher concentrations. PMID- 2996650 TI - The differentiation of isomeric biological compounds using collision-induced dissociation of ions generated by fast atom bombardment. AB - Though fast atom bombardment ionization makes possible the ionization and molecular weight determination of polar or thermally labile biological compounds, the resulting mass spectra commonly give few or no fragment ions which would allow detailed structural analysis. In particular, isomeric compounds often give identical spectra. Collision-induced dissociation of ions resulting from fast atom bombardment ionization is shown to be a powerful combination which can differentiate isomeric substances. The technique is applied to isomeric bile acid salts and steroid conjugates and is capable of differentiating structural isomers which have similar fast atom bombardment mass spectra. A range of isomeric cyclic nucleotides is also shown to be amenable to the method. Sensitivity limits are examined and the unequivocal identification of two 3',5'-cyclic nucleotides isolated from living systems is demonstrated. PMID- 2996652 TI - [Adenylate cyclase activity and cyclic AMP content in brain tissue of dogs during clinical death and in the post-resuscitation period]. AB - The cAMP levels and adenylate cyclase activity have been studied in the grey brain substance and striatum system of dogs during circulation arrest due to electrotrauma of different duration (1-2, 15, 45 min) and in postresuscitation period in animals recovered after 15-min clinical death. Adenylate cyclase is strongly activated and cAMP levels are increased in the brain areas under study during complete brain ischemia. The cAMP levels in the grey substance and in striatum system are reduced considerably compared to the control, accounting for 12-20 on days 2-5 of postresuscitation period in animals with neurologic deficit. Adenylate cyclase and phosphodiesterase enzyme activity is twice higher in the striatum system and 50% lower in the grey brain substance than the baseline. The disturbances in cyclic nucleotide exchange, along with other factors seem to play an important role in the pathogenesis of postresuscitation encephalopathy. PMID- 2996654 TI - [Effect of 4-aminopyridine on the development of experimental botulism]. AB - The studies of survival and the life span of mice and the toxin in LD50 have shown that 4-aminopyridine (4-AP) at doses of 1, 2 and 5 mg/kg has a therapeutic effect on type C botulinic intoxication. Maximum protective effect is observed when 4-AP at a dose of 2 mg/kg is injected twice daily. Therefore, 4-AP, enhancing the transmitter release into cholinergic synapses, weakens the toxic effect of botulinic toxin. PMID- 2996653 TI - [Possibilities of pharmacologic correction of Na, K-ATPase activity in the spinal cord in botulism]. AB - The experiments on white rats have shown that gutimin is capable of reactivating Na, K-ATPase of the synaptosomes of the jugular spinal cord in type C botulinic intoxication. Serotonin prevented Na, K-ATPase activity inhibition only in preclinical period of intoxication. Parmidin injection did not prevent suppression of Na, K-ATPase activity either in preclinical period or in skeletal muscle paresis. PMID- 2996655 TI - [Genetic regulation of the function of E. coli plasmid pAP42 transfer genes]. AB - Sensitivity of the genetic transfer system of F-like plasmid pAP42 marked with the transposons Tn1 and TN9 to fertility inhibitors of six reference Fin-groups was studied. It was shown that transfer function and donor-specific piliformation of the plasmid under study were inhibited by reference plasmids of FinU and FinV groups, surface exclusion by plasmids of FinU and FinQ groups. The different influence of the FinOP group plasmid on transfer functions of the marked plasmids pAP42::Tn1 and pAP42::Tn9 that is likely to be connected with the effect of incorporated transposons was determined. PMID- 2996656 TI - [Inheritance and expression of the restrictive activity of plasmids coding EcoRI restrictase in Vibrio cholerae cells]. AB - Recombinant E. coli plasmids are known to be obtained from E. coli cells using the plasmids coding EcoR1 restriction endonuclease. These plasmids were shown to possess various chromosomal or plasmid genes. The paper presents data on the construction of conjugative recombinant plasmid pSA1002, capable of conjugate transfer into V. cholerae cells. The stable maintenance and inheritance of the plasmid in V. cholerae cells have been demonstrated as well as phenotypic expression of its genes, including EcoR1 restriction endonuclease genes. The possibility of recombinant plasmids formation in V. cholerae cells dependent on EcoR1 restriction endonuclease, coded by pSA1002, is discussed. PMID- 2996658 TI - [Differentiation of human stromal bone marrow cell precursors in monolayer culture]. AB - The colonies of human bone marrow fibroblasts in monolayer culture have been studied. It has been shown that there are two types of colonies in the cultures: monolayer and multilayer ones, both having alkaline phosphatase-positive cells. In monolayer colonies one can observe calcium deposition indicative of osteogenic differentiation of human bone marrow stromal cells. PMID- 2996657 TI - [Effect of treatments of different modalities on the nucleoside phosphate kinase activity of normal and neoplastic cells]. AB - The activity of nucleoside phosphate kinases was studied in experimental transplantable tumours under chemotherapy, in the liver of normal rats treated with hepatocarcinogens, or in human lung tumours after irradiation. The above factors have been found to increase the activity of uridine phosphate kinase, reducing at the same time the activity of thymidine phosphate kinase. The data suggest the existence of an unknown regulatory mechanism responsible for the normal levels of uridylates and thymidylates, thymidine kinase and uridine kinase shunt. PMID- 2996659 TI - Incidence of involvement of the B and T lymphocyte lineages in chronic myelogenous leukemia. AB - Peripheral blood specimens were obtained from 22 patients with Philadelphia chromosome (Ph1) positive chronic myelogenous leukemia (CML) (16 in chronic phase, 2 in an accelerated phase, and 4 in blast crisis). Studies were performed to determine the frequency of the presence of the Ph1 chromosome in cells of lymphoid lineages. Rosetted (E+) lymphocytes (T lymphocytes) from nine patients in chronic phase and one patient in blast crisis were stimulated with T cell growth factor interleukin 2 (IL-2) and/or phytohemagglutinin (PHA). All ten patients had sufficient T lymphocyte metaphases for analysis and of a total of 461 metaphases examined, only one contained the Ph1 chromosome. Nucleated cells of density less than 1.077 g/mL were infected with Epstein-Barr virus (EBV). Following infection, cell lines were established from individual colonies attached to egg albumin-coated Lab-Tek slide chambers (clonal cell lines) or from suspension culture in 96-well tissue culture cluster dishes (nonclonal cell lines). Cell surface and intracellular marker analysis confirmed the B lymphocyte phenotype of all the cell lines examined. B lymphoblastoid cell lines were established from 16 of the 22 patients. All lines from 12 patients were Ph1 negative. From two chronic phase patients, both Ph1-positive and Ph1-negative lines were established. From one patient in an accelerated phase, only Ph1 positive lines were established. From another patient in blast crisis (of myeloblastic phenotype), only Ph1-positive lines were established initially; however, five months later, after the patient had been treated with mitoxantrone, only Ph1-negative lines were derived from this patient. Based on these results, it appears that most B cells and mature T cells in most CML patients are Ph1 negative, but that about 25% of patients have predominantly Ph1-positive B cells or a mixture of Ph1-positive and Ph1-negative B cells that are capable of growing as established cell lines after transformation with EBV. PMID- 2996660 TI - Quantitation of adenosine-5'-triphosphate used for phosphoinositide metabolism in human erythrocytes. AB - The human erythrocyte actively phosphorylates and dephosphorylates phosphatidylinositol present in the membrane in an apparent "futile cycle." Recent reports have proposed that this phosphorylation/dephosphorylation cycle is a significant consumer of adenosine-5'-triphosphate (ATP) in the erythrocyte. This study details two independent techniques for quantitating the ATP consumed by this phosphoinositide futile cycle. With the first technique a quasi-steady state labeling of erythrocyte ATP with 32P-phosphate was obtained, and the rate of synthesis of 32P-phosphoinositides was then monitored. The second technique used a novel labeling strategy that allowed only ATP to be labeled with 32P; the transfer of 32P from ATP to phosphoinositides was then an independent measure of the ATP consumed for phosphoinositide synthesis. These two techniques documented that 0.5% to 1.0% of net ATP produced by the erythrocyte is used for phosphoinositide synthesis. PMID- 2996661 TI - Purification of eosinophil peroxidase and studies of biosynthesis and processing in human marrow cells. AB - Human eosinophil peroxidase (EPO) was purified from leukocytes obtained from a patient with hypereosinophilia. EPO was extracted from the granule fraction using 0.2 mol/L sodium acetate pH 4.0, and the extract was subjected to gel chromatography on Sephadex G-75 and ion exchange chromatography on Biorex 70. The mol wt calculated from gel chromatography was approximately 50,000. However, under reducing and denaturing conditions, polyacrylamide gel electrophoresis revealed two subunits with mol wt of 50,000 and 15,000. The biosynthesis of EPO was studied in marrow cells from patients with eosinophilia using labeling with (14C)-leucine, followed by immunoprecipitation with anti-EPO, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography for visualization of labeled EPO. Biosynthesis of an Mr 53,000 subunit was demonstrated. Biosynthetic labeling of the Mr 15,000 subunit was not demonstrated. A labeled Mr 25,000 chain was detected and may represent a degradation product or a chain that, after further modification, produces the Mr 15,000 subunit. Labeling was also detected in two polypeptides with mol wt of 78,000 and 72,000. These forms of EPO seem to represent precursor polypeptides subjected to proteolytic processing in a similar manner as has been reported for myeloperoxidase (MPO). However, Monensin, a proton ionophore, which blocks the processing of MPO, did not inhibit processing of EPO, indicating separate mechanisms by which MPO and EPO are directed to granules. PMID- 2996663 TI - Hypomethylation of DNA derived from purified human erythroid cells correlates with gene activity of the beta-globin cluster. AB - Analysis of methylation at the beta-globin gene cluster was carried out on DNA derived from nucleated RBCs (orthochromatic normoblasts) isolated from peripheral blood of patients with beta-thalassemia major or other congenital hemolytic anemia after splenectomy. A procedure to separate these normoblasts from the other nucleated cells of the peripheral blood was developed, providing us with a convenient source of DNA for investigating parameters related to human erythroid differentiation. Blood samples were obtained from six adult patients who express their gamma-globin genes at different levels. Inverse correlation between methylation and gene activity was consistently observed for five of the eight sites analyzed. A site 3' to the beta gene was always unmethylated, two sites flanking the epsilon gene were always found to be methylated, and two sites 5' to the two gamma genes, G gamma and A gamma, were hypomethylated in correlation with gamma gene activity of the individual patients. A site 5' to the delta gene was unmethylated in normoblasts as well as in WBC. No apparent relation between hypomethylation and gene activity was observed for two additional sites. The results suggest that methylation at specific chromosomal locations participate in genetic regulation of the beta-like globin genes in humans. PMID- 2996662 TI - Graded responses of human neutrophils induced by serum-treated zymosan. AB - A modified zymosan preparation was used to probe the interaction of particulate stimuli with human neutrophils (PMNs). After extraction with alkali and detergent, the zymosan particles retained their ability to be opsonized in serum and to stimulate PMNs. Serum-treated zymosan (STZ) induced dose-dependent superoxide (O2-) production and membrane potential depolarization in the range of 1 to 10 mg/mL of STZ. The rate and extent of secretion of lysozyme and beta glucuronidase were also dose-dependent in the range of 1 to 10 mg/mL of STZ. Cytochemical studies using nitroblue tetrazolium, however, showed that 92% of PMNs were stimulated to produce O2- at 0.1 mg/mL of STZ. The dose response of O2- production induced by STZ is therefore due to increasing O2- production by individual PMNs and not to the stimulation of more PMNs to produce O2-. Evidence for O2- production was found only in the area of PMN-zymosan contact, suggesting a mechanism for the graded responses of PMNs treated with particulate stimuli. In order to determine the nature of the dose dependence of depolarization (a measure of PMN activation), PMNs equilibrated with the fluorescent probe 3,3' dipentyloxacarbocyanine were analyzed by flow cytometry. The results demonstrate that STZ induces a dose-dependent depolarization of the membrane potential of individual PMNs. These results also demonstrate that increasing concentrations of STZ can induce increasing PMN responses even when all of the PMNs have been activated. These results are consistent with the hypothesis that receptor mediated particulate stimulation of PMNs is a phenomenon that results in graded PMN responses. PMID- 2996664 TI - Prevalence of antibodies to human T-lymphotropic virus-III (HTLV-III) in hemophiliacs and other patients chronically substituted with blood products. AB - The prevalence of antibodies to human T-lymphotropic virus III (HTLV-III) was determined in a total of 140 hemophiliacs and 36 polytransfused patients from three medical centers by an enzyme linked immunosorbent assay (ELISA) and confirmatory tests. 58 hemophiliacs (41.4%) were seropositive. In all instances where the origin of the coagulation factors given to these patients could be determined, blood products came from the United States. In addition, 2 of 36 polytransfused patients, mostly with acute leukemias, who were transfused with blood products from local donors were positive for HTLV-III antibodies. No HTLV III antibodies were detected in 237 blood donors selected in part from the donor pool of the polytransfused patients. PMID- 2996665 TI - Tumor-associated antigen TAG-72: correlation of expression in primary and metastatic breast carcinoma lesions. AB - Variability of tumor-associated antigens among and within human tumor cell groups presents a potential problem in the development and optimization of immunodiagnostic and therapeutic procedures for cancer. We determined the degree of expression of a tumor-associated antigen in the primary and metastatic lesions of 23 patients with infiltrating ductal carcinoma; this was accomplished using monoclonal antibody B72.3, an IgG1 generated against membrane-enriched fractions of human metastatic breast carcinomas and reactive with a 220,000-400,000 d glycoprotein complex, termed TAG-72, and the avidin-biotin complex immunoperoxidase method on fixed tissue sections. Sixteen of the 23 breast carcinomas (70%) demonstrated MAb B72.3 reactivity (range 5% to 100% of tumor cells staining). Reactivity of lymph node metastases was present in 14 of 21 patients (67%). MAb reactivity in metastases to distant sites, including bone, adrenals, liver, skin and effusions, was present in 10 of 18 patients (56%). In one patient, neither the primary carcinoma nor the metastasis to the lymph node demonstrated reactivity. There was a statistically significant positive correlation between MAb B72.3 reactivity in both primary and lymph node metastases (Kendall's Correlation Coefficient = 0.60, p = 0.0006) and between lymph node and distant metastases (Kendall's Correlation Coefficient = 0.48, p = 0.02) of the same patient. No correlation existed between antibody reactivity seen in the primary and that found in the distant lesions of that patient. These studies thus demonstrate that monoclonal antibody B72.3 can detect expression of a tumor-associated antigen in both primary and metastatic infiltrating ductal carcinoma lesions, and may prove valuable in the understanding of tumor biology of metastases and as a means for diagnosing occult disease. PMID- 2996667 TI - Human papillomavirus. AB - Whilst papillomaviruses mainly induce epithelial proliferations in man and in different animal species, the association of this group of viruses with malignant tumours has become increasingly evident. Despite the heterogeneity of human papillomavirus (HPV), only a small number are identified within carcinomas, implying a different, oncogenic potential for different HPV types. PMID- 2996668 TI - The management of cardiac arrest in infants and children. AB - The management of cardiac arrest in adults is a well known and, in most hospitals, a well practised procedure. Similarly the resuscitation of the newborn is usually well provided for. However, cardiac arrests occurring in older children can cause difficulties. PMID- 2996669 TI - The role of the atria in fluid volume control. AB - Neural reflexes from the heart, in particular the atria, are well documented and are involved in sodium and water excretion. Current work also indicates that the atria have an important endocrine function. Atrial peptides, which have been recently characterized and synthesized, may play a significant role in the homeostasis of body fluids. PMID- 2996666 TI - Cellular retinol-binding protein in normal and neoplastic human mammary gland. AB - The concentration of cellular retinol-binding protein (CRBP) was determined in samples of normal and neoplastic mammary gland, using a specific and sensitive radioimmunoassay. The CRBP concentration was significantly higher in neoplastic tissue, but detectable levels were also present in all samples of normal gland. Tubulo-ductal cancers had significantly lower CRBP levels than other cancer types. The CRBP concentration of the neoplastic tissue showed no correlation with the concentration of progesterone or estrogen receptor. PMID- 2996670 TI - Metastatic tumours of the nasal tip. AB - Two cases of metastatic tumour at the nasal tip are described. Both originated from a primary tumour of the bronchus. In each case the lesion was the first sign of an otherwise silent neoplasm. PMID- 2996671 TI - Small cell carcinoma of skin: a report of two cases. AB - We present two cases of small cell carcinoma of skin and review the evidence for the origin of these tumours from Merkel cells situated in the basal layers of the epidermis. The aggressive behaviour of these tumours makes their initial histological diagnosis important if careful follow-up is to be instituted and we suggest that more radical primary treatment might improve results. We emphasise the need to exclude an origin from other sites apart from skin and the role of electron microscopy and immunohistochemistry in identifying these tumours as being distinct from other poorly differentiated carcinomas. PMID- 2996672 TI - The effects of sodium nitroprusside and 8-bromo-cyclic GMP on electrical and mechanical activities of the rat tail artery. AB - The effects of sodium nitroprusside and 8-bromo-guanosine 3':5'-cyclic monophosphate (8-bromocyclic GMP) on the electrical and mechanical activities of the rat tail artery were compared. The inhibitory effects of sodium nitroprusside on the contractions induced by noradrenaline, phenylephrine, KC1 and clonidine were mimicked by 8-bromo-cyclic GMP. Sodium nitroprusside and 8-bromo-cyclic GMP increased the resting membrane potential only in preparations with low initial resting membrane potentials. In tissues previously contracted and depolarized with noradrenaline, KC1 and clonidine, both sodium nitroprusside and 8-bromo cyclic GMP caused relaxation without significantly affecting the membrane potential. Both sodium nitroprusside and 8-bromo-cyclic GMP abolished neurally mediated contractions without any significant effect on the electrical responses. These results suggest that the actions of sodium nitroprusside and 8-bromo-cyclic GMP are not related to membrane hyperpolarization or inhibition of membrane excitability. PMID- 2996673 TI - Analysis of the alpha-adrenoceptor-mediated, and other, components in the sympathetic vasopressor responses of the pithed rat. AB - The vascular receptors activated following sympatho-adrenal stimulation were determined by analysing the effects of 'selective' antagonists on the vasopressor response to spinal sympathetic nerve activation in the pithed rat. The net vascular response to adrenal stimulation was a balance between alpha-adrenoceptor mediated vasoconstriction and beta-adrenoceptor-mediated vasodepression. Part of the alpha-adrenoceptor-mediated response was 'prazosin-sensitive' (alpha 1) and the remainder was abolished by rauwolscine (alpha 2). As with adrenal stimulation, direct sympathetic nerve stimulation of the vasculature evoked pressor responses which were partly resistant to prazosin. Rauwolscine only partly blocked the prazosin-sensitive component. Reserpine pretreatment led to smaller responses than prazosin plus rauwolscine. Thus, the response resistant to alpha-adrenoceptor antagonists could be mediated, in part, by adrenoceptors distinct from alpha-adrenoceptors, as currently defined. alpha, beta-Methylene ATP reduced the nerve-mediated pressor response after alpha-adrenoceptor blockade or reserpine pretreatment but not in drug-free controls. The results suggest that stimulation of the adrenal medulla can produce a vasopressor response which consists of summating alpha 1- and alpha 2-adrenoceptor-mediated components, and is identical to the effect of injected adrenaline. In contrast, the response to vasopressor nerve stimulation appears to be essentially mediated by alpha 1 adrenoceptors, with a facilitatory influence from alpha 2-adrenoceptors. A further response obtained after alpha-adrenoceptor blockade may contain a purinergic component and another which is adrenergic but not mediated by stimulation of alpha-adrenoceptors. PMID- 2996674 TI - Modulation of 5-hydroxytryptamine-induced head-twitch response by drugs acting at GABA and related receptors. AB - The effects of drugs acting at the gamma-aminobutyric acid (GABA) receptors and other chloride ionophore-related sites have been studied for their ability to modulate the head-twitch induced by 1-5-hydroxytryptophan (5-HTP) in the mouse. The GABAa receptor agonists, muscimol, imidazoleacetic acid and 3 aminopropanesulphonic acid, produced a dose-related potentiation, while bicuculline inhibited the head-twitch. The GABAb receptor agonist, baclofen, produced dose-related inhibition. Diazepam potentiated the head-twitch while the 'inverse' benzodiazepine receptor agonist ethyl-beta-carboline-3-carboxylate inhibited the head-twitch. The antagonist Ro15-1788 also produced inhibition. Ro05-4864, a ligand for the benzodiazepine 'acceptor' site, potentiated the head twitch. Pentobarbitone and pentylenetetrazol potentiated the 5-HTP-induced head twitch at low doses, changing to inhibition as the dose was increased. Picrotoxin in subconvulsant doses, produced only potentiation. More than one site may be involved in the action of these substances. GABA, amino-oxyacetic acid and 1-2-4 diaminobutyric acid inhibited the head-twitch, while the GABA-depletor, 3 mercaptopropionic acid potentiated it. Of all the agents tested, only muscimol produced head-twitching when given alone. It was concluded that both GABAa and GABAb receptors modulate the head-twitch response to 5-HTP. PMID- 2996676 TI - The influence of blood gases on alpha 1- and alpha 2- adrenoceptor-mediated pressor responses in the pithed rat. AB - The influence of blood gases on alpha 1- and alpha 2-adrenoceptor-mediated pressor responses was studied in the pithed rat by varying the inspired gas mixture or the ventilation stroke volume. Acidosis favoured the peak responses to the alpha 2-adrenoceptor agonist, xylazine, while alkalosis favoured the peak responses to the alpha 1-adrenoceptor agonist, phenylephrine. A combination of hypoxia and hypercapnia greatly depressed the alpha 1 response to phenylephrine whereas the alpha 2 response to xylazine remained relatively unaffected. When Pao2 was varied in either acidotic or alkalotic conditions the response to the phenylephrine increased as Pao2 increased. To prevent hypoxia in air ventilated rats, large stroke volumes were required. This caused alkalosis and hence decreased responsiveness to xylazine. Consequently, air ventilated pithed rats gave poorer responses to xylazine than did those ventilated on 100% O2. The results show that alpha 1- and alpha 2-adrenoceptor-mediated pressor responses can be differentially affected by blood gases. The relative contribution of alpha 1- and alpha 2-adrenoceptors to vascular tone may be either under- or over estimated depending on the arterial blood gases. PMID- 2996675 TI - Differential release of eicosanoids by bradykinin, arachidonic acid and calcium ionophore A23187 in guinea-pig isolated perfused lung. AB - The effects of infusions of bradykinin (0.2 microM), calcium ionophore A23187 (0.5 microM) and arachidonic acid (13 microM) on the release of eicosanoids from the guinea-pig isolated perfused lung were investigated using radioimmunoassay for thromboxane B2 (TXB2), 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha), PGE2, leukotriene B4 (LTB4) and LTC4 and bioassay using the superfusion cascade. Bradykinin released more 6-oxo-PGF1 alpha than TXB2, whereas arachidonic acid and ionophore released more TXB2 than 6-oxo-PGF1 alpha. The time course of eicosanoid release varied with the stimulus: bradykinin and arachidonic acid produced an immediate release, whereas the ionophore showed a slower onset of release. Although the amounts of LTB4 and LTC4 released by the ionophore were very low according to radioimmunoassays, there was evidence from the bioassay of release of a leukotriene-like substance, thought to be LTD4. The leukotriene antagonist FPL 55712 lacks specificity in the guinea-pig trachea; at the concentration used (2 microM) it antagonized contractions of the tracheal strip to PGE2 as well as to LTC4. Our results show that in the guinea-pig perfused lung the metabolism of exogenous arachidonic acid is both qualitatively and quantitatively different from the metabolism of endogenous arachidonic acid; furthermore, the profile of eicosanoid production is stimulus-dependent. PMID- 2996677 TI - Theophylline-sensitive modulation of non-cholinergic excitatory neurotransmission in the guinea-pig ileum. AB - Atropine-resistant longitudinal contractions of the guinea-pig ileum due to field stimulation were greatly augmented by theophylline (150 microM). Adenosine exerted an opposite effect. Theophylline did not potentiate contractions evoked by exogenous substance P. It is suggested that a theophylline-sensitive inhibitory mechanism (possibly mediated by adenosine or a related substance) controls transmitter release from enteric non-cholinergic excitatory neurones. PMID- 2996679 TI - Effects of tyramine on noradrenaline outflow and electrical responses induced by field stimulation in the perfused rabbit ear artery. AB - In the perfused rabbit ear artery the basal outflows of noradrenaline (NA) and 3,4-dihydroxyphenylglycol (DOPEG) were less than 1 ng g-1 and 1-2 ng g-1 wet weight of tissue respectively. Field stimulation increased outflows of NA and DOPEG in a frequency-dependent manner, and they reached the maximum value at frequencies over 5 Hz. Tyramine (1 X 10(-6) -1 X 10(-4) M) increased basal outflow of NA and DOPEG, in a dose-dependent manner. This effect was not blocked by tetrodotoxin (TTX, 3 X 10(-7) M), but was prevented by pretreatment with 6 hydroxydopamine (6-OHDA). Tyramine increased the field stimulation-induced outflow of NA but not that of DOPEG in a dose-dependent manner. Cocaine (1 X 10( 5) M) reduced the increased outflow of NA induced by tyramine at rest and during field stimulation, without modifying DOPEG-outflow. Guanethidine (5 X 10(-6) M), increased outflows of NA and DOPEG at rest, and reduced the NA outflow induced by field stimulation. Pretreatment with guanethidine (5 X 10(-6) M) did not block the action of tyramine on NA and DOPEG basal outflows. Additional application of guanethidine during the presence of tyramine did reduce the outflow of NA induced by field stimulation, but did not modify the outflow of NA and DOPEG at rest. Tyramine at concentrations over 1 X 10(-5) M depolarized the smooth muscle membrane of the rabbit ear artery. After chemical denervation with 6 hydroxydopamine (6-OHDA) the depolarizing action of tyramine was reduced. Tyramine-induced depolarization was attenuated by prazosin (5 X 10(-6) M) or phentolamine (5 X 10(-6) M), but not by guanethidine (5 X 10(-6) M). In 6-OHDA denervated tissues, tyramine-induced depolarization was attenuated by phentolamine but not by prazosin. Field stimulation evoked excitatory junction potential (e.j.p.), slow depolarization and spike potential in the rabbit ear artery. Tyramine reduced, while guanethidine blocked these electrical responses. Tyramine did not alter the facilitation process of e.j.ps. In tissues pretreated with guanethidine, tyramine evoked either no electrical response or a slow depolarization during field stimulation. The slow depolarization was blocked by prazosin. Tyramine reduced the NA content of tissues in a dose-dependent manner (by 31% at 10(-4) M). Guanethidine (5 X 10(-6) M) reduced the NA content by 20%. 10 We conclude that in the rabbit ear artery, tyramine depolarizes the smooth muscle membrane indirectly by releasing neuronal NA which acts on alpha adrenoceptors, and directly by an action on the smooth muscle cells. Two NA compartments (guanethidine-sensitive and tyramine-sensitive NA) could be identified. Field stimulation releases the former with associated generation of ej.p. and slow depolarization whilst the release of the latter is not accompanied by ej.p. generation. PMID- 2996678 TI - The mechanism of action of maitotoxin in relation to Ca2+ movements in guinea-pig and rat cardiac muscles. AB - Maitotoxin (MTX), the most potent marine toxin known, produced a dose-dependent positive inotropic effect on guinea-pig isolated left atria and rat ventricle strips at concentrations of 0.1 ng to 4 ng ml-1. MTX (4 ng ml-1) also exhibited a positive chronotropic effect on guinea-pig right atria. The MTX-induced inotropic effect was almost abolished by Co2+ or verapamil, but was little affected by propranolol, reserpine or tetrodotoxin. The tissue Ca content of guinea-pig left atria was increased by MTX (2-30 ng ml-1) in a dose-dependent manner, and this increase was markedly inhibited by Co2+ or verapamil. Furthermore, on the rat isolated cardiac myocytes MTX (0.01-10 ng ml-1) caused an arrhythmogenic effect which was followed by their transformation into irreversibly rounded cells. The effects of MTX on the isolated cells were inhibited by verapamil or Ca2+-free solution. These results suggest that the excitatory effects of MTX on heart muscle are caused by a direct action on the cardiac muscle membrane mainly due to an increase in Ca2+ permeability. PMID- 2996680 TI - Treatment with N-ethylmaleimide selectively reduces adenosine receptor-mediated decreases in cyclic AMP accumulation in rat hippocampal slices. AB - N-ethylmaleimide (NEM) has been reported to interact with the GTP-binding Ni protein; we have examined its effect on adenosine receptor binding in feline cortical membranes and on adenosine-receptor mediated effects on cyclic AMP accumulation in rat hippocampal slices. Treatment of cortical membranes with NEM (100 microM for 5 min) altered the binding of [3H]-phenylisopropyladenosine (PIA) from being almost exclusively to a single class of high affinity sites (KD = 1.65 nM) to binding at two classes of sites (KDH = 2.1 nM, KDL = 102 nM). The total number of binding sites was similar (825-845 fmol mg-1 in control membranes, 944 1428 fmol mg-1 in NEM-treated membranes). In rat hippocampal slices treated with forskolin (0.3 microM) L-PIA produced a biphasic effect on cyclic AMP accumulation: an inhibition at 0.03 to 1 microM and at higher concentrations, a stimulation. Treatment with 50 microM NEM selectively inhibited the inhibitory phase, causing stimulation at lower concentrations of L-PIA. At 50 microM, NEM did not alter basal or forskolin-stimulated cyclic AMP accumulation but at higher concentrations inhibition was observed. It is concluded that NEM can, in certain doses, selectively block adenosine A1-receptor-mediated effects without affecting A2-receptor-mediated actions in the same tissue. It is suggested that this is due to NEM affecting the Ni guanine nucleotide binding protein. PMID- 2996681 TI - A history of the discovery and clinical application of antiviral drugs. PMID- 2996682 TI - Rapid diagnosis of virus diseases. PMID- 2996683 TI - The laboratory selection of antiviral agents. PMID- 2996685 TI - Resistance and latency. PMID- 2996684 TI - Herpes viruses: a background. PMID- 2996686 TI - Antiviral therapy in the immunocompromised patient. PMID- 2996687 TI - Rhinovirus colds. PMID- 2996688 TI - Persistence of antibrain antibodies in cerebrospinal fluid during plasmapheresis for multiple sclerosis. AB - A man with chronic progressive multiple sclerosis received a 10 day course of treatment with adrenocorticotrophic hormone without beneficial effect. He then received six sessions of plasmapheresis, again without improvement. Treatment with adrenocorticotrophic hormone had no effect on serum antibrain antibody titres, but plasmapheresis virtually eliminated the antibodies from serum and caused a fall in serum IgG concentrations; neither treatment had any effect on the IgG concentration and antibody titre in the cerebrospinal fluid. Treatment with plasmapheresis may fail in patients with multiple sclerosis because it does not remove antibrain antibodies from the intrathecal space. PMID- 2996689 TI - Large hepatocellular cancers: hepatic resection or liver transplantation? AB - Thirteen patients with primary hepatocellular cancer were studied to outline criteria for resectability in patients with large liver tumours. The mean age was 34 and the mean tumour diameter 13 cm (range 7-18 cm). Five of the tumours had a diameter of 15 cm or more. Extensive radiological investigations showed that seven of the patients had tumours of both right and left lobes of the liver, 10 had evidence of vascular invasion, and three had evidence of extrahepatic spread. Only two of the patients underwent a classically described formal hepatic resection, the rest having extensive resections crossing major anatomical planes. In no instance did the vascular invasion preclude resection, and extrahepatic spread could be verified in only one patient. The traditional criteria of resectability--that is, tumours located to one main lobe of the liver without vascular invasion and extrahepatic spread--can and should be extended. Resection may be preferable to transplantation even in patients with large primary liver tumours. PMID- 2996690 TI - Nabilone and prochlorperazine: a useful combination for emesis induced by cytotoxic drugs. PMID- 2996691 TI - Variation in human T lymphotropic virus III (HTLV-III) antibodies in homosexual men: decline before onset of illness related to acquired immune deficiency syndrome (AIDS). AB - Western blot analysis was used to document the development and changes in human T lymphotropic virus III (HTLV-III) antibody among Danish homosexual men followed longitudinally over three years. Reactivity against p15, p24, and p55 appeared earliest. After seroconversion the antibody concentration fluctuated, but in one instance a steady decline in banding intensity was seen during the 18 months before onset of the acquired immune deficiency syndrome (AIDS) and throughout the remaining eight months of his life. PMID- 2996692 TI - Large hepatocellular cancers: hepatic resection or liver transplantation? PMID- 2996693 TI - Alfacalcidol as a modulator of growth of low grade non-Hodgkin's lymphomas. AB - Ten patients with low grade non-Hodgkin's lymphoma (seven follicular small cleaved and three small lymphocytic) were treated with 1 microgram oral alfacalcidol (1 alpha-hydroxycholecalciferol) daily. Of the seven patients with lymphomas of follicular small cleaved subtype, one achieved complete and three partial remission, whereas none of three patients with small lymphocytic lymphomas responded. In seven of the 10 patients, 1,25(OH)2D3 receptors were measured in tissue from lymph nodes, and a positive correlation between the presence and amount of receptor and response to alfacalcidol was found. These preliminary data suggest that alfacalcidol has appreciable antitumour activity in low grade non-Hodgkin's lymphomas. PMID- 2996695 TI - Effects of intrathecally administered methysergide and yohimbine on microstimulation-produced antinociception in the rat. AB - This study examined whether intrathecal (i.t.) administration of the serotonergic antagonist methysergide, of the alpha 2 noradrenergic antagonist yohimbine, or of both drugs antagonized stimulation-produced antinociception (SPA) evoked from the nucleus raphe magnus (NRM) and the nucleus reticularis paragigantocellularis (NRPG) of lightly anesthetized rats. The increase in tail flick latency (TFL), but not the increase in paw pinch withdrawal threshold (PWT), evoked from NRM sites was antagonized by i.t. administration of methysergide. Intrathecal administration of yohimbine antagonized both the increase in TFL and the increase in PWT produced by stimulation of NRM sites. Stimulation of sites in the NRPG also increased TFL and PWT; these increases were not antagonized by i.t. administration of methysergide. Although i.t. administration of yohimbine antagonized the increase in TFL evoked from the NRPG, the increase in PWT was not antagonized. When coadministered intrathecally, methysergide and yohimbine antagonized the increases in TFL and PWT produced by stimulation of NRM and of NRPG sites. In contrast, i.v. administration of the same doses of methysergide and yohimbine did not antagonize either the increase in TFL or the increase in PWT evoked from either set of sites. These results support the concept that activation of serotonergic and noradrenergic bulbospinal neurons mediates SPA and additionally suggest that the noradrenergic component involves an alpha 2 noradrenergic receptor. PMID- 2996694 TI - Calcitonin receptors in the rat mesencephalon mediate its analgesic actions: autoradiographic and behavioral analyses. AB - Autoradiographic analyses of salmon calcitonin (sCT) binding in the rat mesencephalon revealed an exceptionally high concentration of receptors in the ventral and ventrolateral segments of the periaqueductal gray matter (PAG) extending along the entire rostral-caudal axis. Relatively heavy labeling was also seen along a band extending ventrolaterally through the mesencephalic reticular formation. Other receptor-rich areas include the nucleus linearis, pars compacta and lateralis of the substantia nigra, locus coeruleus, parabrachial nuclei and nucleus raphe pontis of the pontine reticular formation. Injections of sCT into the PAG induced a dose-dependent increase in hot-plate latencies. All rostral-caudal levels of these brain regions appeared to be equally responsive. Injections into the midline pontine reticular formation were also effective in increasing response latencies. Unilateral injections into the hypothalamus, medial thalamus, ventral thalamus and mesencephalic reticular formation proved to be ineffective. Human calcitonin (hCT) was considerably less potent. These biological effects are consistent with the potencies of both peptides in displacing 125I-sCT from slide-mounted sections of rat PAG. Naloxone failed to antagonize sCT-induced analgesia, suggesting an opiate independent mechanism for this peptide in eliciting analgesia. PMID- 2996696 TI - Synaptic delay in the lateral geniculate nucleus of the cat. AB - Unitary, presynaptic spike potentials were observed in single cell recordings from the dorsal lateral geniculate nucleus of the cat. In 11 cells, spontaneous S potentials (extracellularly recorded excitatory postsynaptic potentials) were preceded at a fixed interval by a small wave (the 'T' potential). In another 14 cells, a T potential, although not detected in single traces, was revealed by averaging 20-100 samples synchronized to the peak of the S potential. Provided the field response was not too large a T potential could also be detected in the response to a stimulus to the optic nerve. The T potential would appear to be the spike potential of the afferent optic axon which is excitatory to the geniculate cell because it precedes the S potential at a very exact interval and also follows the corresponding retinal ganglion cell spike at a very exact interval and because the interval between T potential and S potential is reversibly decreased by cooling with a temperature coefficient characteristic of synapses. T potentials ranged in amplitude from 8 to 134 microV and were all predominantly positive-going suggesting a failure of the nerve impulse to invade fully the terminals of the optic nerve. The time from the positive peak of the T potential to the start of the S potentials was taken as a good measure of the synaptic delay. The T-S interval averaged 0.29 ms (+/- 0.045 ms S.D.). PMID- 2996697 TI - Aging: effect on the interaction of ethanol and pentobarbital with the benzodiazepine-GABA receptor-ionophore complex. AB - Benzodiazepine receptor binding and modulation by pentobarbital and ethanol was studied using the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1 propanesulfonate to solubilize the gamma-aminobutyric acid (GABA)-benzodiazepine receptor-ionophore complex from the brains of Fischer 344 rats of 3-4, 12-15 and more than 28 months of age. The affinity of the benzodiazepine binding site was significantly lower in the young rats compared to either the mature or senescent animals. However, no age-related changes in the maximum number of benzodiazepine binding sites or GABA concentrations occurred in the detergent extract. Pentobarbital produced practically identical dose-dependent enhancement of [3H]flunitrazepam specific binding in all 3 age groups. In contrast, ethanol between 0.1 and 200 microM failed to produce a dose-dependent effect on [3H]flunitrazepam specific binding in any age group. The effect of pentobarbital and ethanol on [35S]t-butyl-bicyclophosphorothionate [( 35S]TBPS) specific binding to the picrotoxinin binding site was examined in the above solubilized receptor/ionophore complex under the same binding conditions. Both sedative hypnotics produced a dose-dependent decrease in [35S]TBPS specific binding. However, pentobarbital was over 10,000 times more potent. It appears that ethanol may not enhance [3H]flunitrazepam specific binding in this solubilized preparation because of its weak action at the picrotoxinin binding site. PMID- 2996698 TI - Opposite actions of forskolin at pre- and postsynaptic sites in rat sympathetic ganglia. AB - In isolated rat superior cervical ganglia, forskolin, a powerful activator of adenylate cyclase, augmented the amplitude of fast excitatory postsynaptic potentials. Quantal analysis showed that forskolin acts presynaptically to facilitate the release of the transmitter. The time course of the presynaptic action of forskolin paralleled that of the increase in cyclic AMP level in the ganglia. In addition, forskolin exerted a postsynaptic action on the nicotinic acetylcholine receptor so that the acetylcholine-induced depolarization was depressed. The action of forskolin on the nicotinic acetylcholine receptor seems to be unrelated to the cyclic AMP system. PMID- 2996699 TI - Glutamine can enhance synaptic transmission in hippocampal slices. PMID- 2996700 TI - Stress enhances electrical synapse formation between regenerating neurons. AB - A pair of adult molluscan neurons (L5 and R5) are known to sprout and form a new electrical synapse following axotomy. Additionally, certain forms of stress can elicit restricted sprouting of neurons L5 and R5 within the intact nervous system. This study found that the strength of 5-5 electrical coupling was increased during sprouting in ganglia from previously stressed animals. The increase of electrical coupling was due to a decrease of coupling resistance without any observable change in the degree of sprouting. Thus prior stress can increase the strength of an electrical synapse formed between sprouting neurons. PMID- 2996701 TI - Do antagonists of pancreatic cholecystokinin receptors interact with central nervous system cholecystokinin receptors? AB - The abilities of the pancreatic cholecystokinin (CCK) receptor antagonists dibutyryl cyclic GMP, proglumide, benzotript, CBZ-tryptophan, CBZ-cysteine and CCK-27-32-amide to inhibit CCK binding to its receptor in the pancreas and brain of mice and guinea pigs was examined. In both species, the same relative potencies of the antagonists in brain and pancreas was seen except that dibutyryl cyclic GMP was considerably more potent on pancreas than on cerebral cortex CCK receptors. CCK-27-32-amide was the most potent inhibitor for both brain and pancreas but was more potent in the guinea pig than in the mouse. Proglumide, a relatively weak antagonist, was a more potent inhibitor of the guinea pig than of the mouse pancreas receptor. Thus, these data suggest that there are both tissue specific and species-specific differences in CCK antagonist interactions with the CCK receptor. PMID- 2996702 TI - 4-Aminopyridine induces expansion of cutaneous receptive fields of dorsal horn cells. AB - Systemic administration of 4-aminopyridine (4-AP) increased the size of the cutaneous receptive fields of 9 of the 15 dorsal horn cells tested. These receptive fields were on the feet and toes of the hind limbs of cats. Receptive field sizes increased with increasing doses of 4-AP. However, 4-AP administration did not change the responses of dorsal horn cells to graded mechanical stimuli administered near the centers of their receptive fields. PMID- 2996704 TI - Repeated electroconvulsive shock downregulates the opioid receptors in rat brain. AB - Ten consecutive daily electroconvulsive shocks (ECSs), which produce maximal tonic and clonic convulsions, caused reductions of mu- and delta-opioid receptor binding in the hypothalamus, hippocampus and caudate nucleus, but not in the frontal cortex and brainstem. These changes of opioid receptor binding were not observed in rats receiving a single ECS. Scatchard analysis revealed that ECS induced reduction of mu- and delta-receptor binding was due to a decrease in the binding sites but not to a change in the binding affinity. Time course studies showed that 7 days after the end of 10 consecutive daily ECSs, both mu- and delta receptor binding remained lower than those of sham controls. However, the effects of ECS on the opioid receptor binding disappeared in 2-3 weeks. These observations are consistent with the hypothesis that ECS treatments increase the release of opioid peptides in certain brain regions which in turn down-regulate the opioid receptors. PMID- 2996703 TI - Modulation of dopaminergic activity in the nucleus accumbens following facilitation or blockade of the dopaminergic transmission in the amygdala: a study by in vivo differential pulse voltammetry. AB - Using in vivo voltammetry, dopaminergic (DAergic) activity in the nucleus accumbens (ACC) was assessed following microinjection of DAergic agonists and antagonists in the amygdala (AMY). It was found that DAergic activity in the ACC was under the inhibitory influence of DAergic transmission in the AMY. Therefore, it can be suggested that there is a functional interdependence between the activity of these two DAergic pathways originating from the ventral mesencephalic region. PMID- 2996705 TI - The anorectic effect of FG 7142, a partial inverse agonist at benzodiazepine recognition sites, is reversed by CGS 8216 and clonazepam but not food deprivation. AB - The benzodiazepine partial inverse agonist N'-methyl-beta-carboline-3-carboxamide (FG 7142; 5.0 and 10.0 mg/kg, i.p.) produced a dose-dependent reduction in the consumption of a familiar, highly palatable diet by non-food-deprived male rats. At dose levels which exhibited no significant intrinsic effects, the benzodiazepine receptor antagonist 2-phenylpyrazolo-[4,3-c]-quinoline-3(5H)-one (CGS 8216; 1.25-5.0 mg/kg, i.p.) reversed the anorectic effect of FG 7142. When clonazepam and FG 7142 were given in combination, mutual cancelling of their opposite effects occurred. These results are consistent with an action of FG 7142 at benzodiazepine recognition sites to reduce the level of palatable food consumption, and imply that a bidirectional control of food intake via benzodiazepine recognition sites can be achieved. The anorectic effect of FG 7142 was not reversed by 24-h food deprivation, indicating a possible separation from the effects of hunger mechanisms. PMID- 2996706 TI - Folic acid has a disinhibitory action in the rat hippocampal slice preparation. AB - The effects of folic acid on synaptic transmission in the hippocampal slice have been studied. Application of folic acid (0.1-1 mM) increased the size of population spikes recorded extracellularly in the CAl pyramidal cell layer and caused the appearance of multiple population spikes. Intracellular recording revealed that folic acid had no consistent effect on the membrane potential, but greatly reduced the rapid chloride-mediated phase of the inhibitory postsynaptic potential (IPSP) evoked by ortho- and antidromic stimulation. The slower, potassium-mediated phase of the IPSP was usually enhanced. Furthermore, folic acid abolished spontaneous IPSPs recorded with potassium chloride-filled microelectrodes. All of these effects were quickly reversible when the drug was washed from the chamber. Finally bath-applied folic acid reduced the hyperpolarization produced by iontophoretically applied GABA. Based on these results, we conclude that folic acid produces its excitatory effects on hippocampal pyramidal cells by a disinhibitory action which involves a postsynaptic blockade of GABA responses. PMID- 2996707 TI - Modulation of the endogenous acetylcholine release from rat striatal slices. AB - The dopaminergic modulation of the acetylcholine release from rat striatal slices has been investigated using a chemiluminescent method. Dopamine, more efficiently than apomorphine, decreased the potassium-evoked release of acetylcholine. The effect of dopamine antagonists, haloperidol and sulpiride, has been studied, and haloperidol was a better antagonist than sulpiride to the dopamine effect. Haloperidol elicited an acetylcholine release from striatal slices at 0.1 nM, probably by removing endogenous dopamine from dopaminergic receptors. PMID- 2996709 TI - Neural elements subserving pulmonary stretch receptor-mediated facilitation of phrenic motoneurons. AB - The neural elements responsible for facilitation of phrenic nerve activity by lung inflation were investigated in cats by the simultaneous recording of individual pulmonary stretch receptor afferents, respiratory neurons of the ventrolateral nucleus of the tractus solitarius and phrenic nerve activity. Monosynaptic excitation of I beta neurons by slowly adapting pulmonary stretch receptors was demonstrated by cross-correlational analysis. It was also demonstrated that the majority of these same I beta neurons projected to the contralateral C5 phrenic motoneuron pool. Thus, this study has shown that I beta neurons can act as central neural elements to mediate the facilitatory effect of lung inflation upon phrenic nerve activity. PMID- 2996708 TI - Maitotoxin activates quantal transmitter release at the neuromuscular junction: evidence for elevated intraterminal Ca2+ in the motor nerve terminal. AB - Maitotoxin (MTX), applied in vitro to the mouse neuromuscular junction, briskly activates the spontaneous release of acetylcholine quanta, manifest as miniature end-plate potentials (MEPPs). This effect requires external Ca2+ and is accompanied by a steady post-junctional depolarization. After the peak activation of the spontaneous release process, the quantal discharge gradually declines with eventual abolishment of MEPPs. In contrast to the striking increase in MEPP frequency, the quantum content of the nerve-evoked end-plate potentials (EPPs) is increased only moderately by MTX. These effects are attributable to the ability of the toxin to elevate the level of free intracellular Ca2+ in the motor nerve terminal. With further characterization of its presynaptic site of action, maitotoxin may become a useful tool in studying synaptic physiology. PMID- 2996710 TI - Interfering with inhibition may improve motor function. AB - Motor behavior not seen in newborn cats can be revealed by spinal transection and is therefore normally suppressed. Motor performance of these spinal kittens after reaching adulthood surpasses that of chronic adult-operated spinal cats but the latter display a significant improvement when GABAergic inhibition is blocked pharmacologically. Thus, inhibitory processes influence normal motor development as well as recovery of function after neurological damage. PMID- 2996711 TI - Neuropeptide Y reduces orthodromically evoked population spike in rat hippocampal CA1 by a possibly presynaptic mechanism. AB - Application of the brain neuropeptide Y (NPY) to rat hippocampus in vitro reversibly reduced the amplitude of the CA1 population spike evoked by stratum radiatum stimulation. Threshold for the effect was 10(-8) M. NPY had similar effects on single pulse- and paired pulse-evoked population spikes. Antidromic population spikes, evoked from the alveus, were unaffected by NPY. Thus, NPY appears to modulate excitatory transmission in the hippocampus by a presynaptic mechanism. PMID- 2996712 TI - Projection neurons of the nucleus accumbens: an intracellular labeling study. AB - Projection neurons of nucleus accumbens (NAC) of the rat were identified by either antidromic activation from stimulation of midbrain ventral tegmental area substantia nigra (VTA-SN) regions, or by tracing axons of intracellularly labeled NAC neurons into the ventral pallidum. The morphology of these NAC projection neurons were determined to be medium spiny neurons similar to those identified in the caudate-putamen. PMID- 2996713 TI - Effects of chronic ouabain treatment on [3H]ouabain binding sites and electrogenic component of membrane potential in cultured rat myotubes. AB - The effects of incubation of cultured rat skeletal myotubes in ouabain were studied on the number of [3H]ouabain binding sites and electrogenic component of membrane potential. Ouabain treatment for 2-6 days increased the number of binding sites, resting membrane potential (Em) and the ouabain-sensitive component of Em in the muscle cells. The findings strengthen the idea that Na,K ATPase has an important role in regulation of Em in cultured skeletal muscle and suggest that Na-K pump inhibition during development may be a regulatory mechanism for cellular synthesis of Na,K-ATPase. PMID- 2996715 TI - Quantitative autoradiographic determination of angiotensin-converting enzyme (kininase II) binding in individual rat brain nuclei with 125I-351A, a specific enzyme inhibitor. AB - We report the localization and characterization of angiotensin-converting enzyme (kininase II) in discrete nuclei from individual rat brains by a quantitative autoradiographic technique coupled to computerized microdensitometry. The enzyme was quantitated by incubation of 16-micron-thick brain sections with 0.07-2 nM of the converting enzyme inhibitor 125I-351A and comparison to 125I-standards. This technique can be applied to the study of other enzymes in single rat brain nuclei. PMID- 2996714 TI - Ibotenic acid decreases thyrotropin-releasing hormone receptor binding in the rat amygdala. AB - Thyrotropin-releasing hormone (TRH) receptor densities in the amygdala were examined with quantitative autoradiography in rats treated with the cellular neurotoxin ibotenic acid (IBO). Microinjections of IBO (10 micrograms) into the right basolateral amygdaloid nucleus (BsA) reduced the concentration of TRH receptors in this nucleus by over 50%, when compared to the contralateral BsA and to vehicle-injected control rats. IBO lesions left amygdaloid terminals intact, as shown by normal levels of presynaptic choline uptake sites. Our results strongly suggest that TRH receptors in the amygdala are predominantly located on cell bodies. PMID- 2996716 TI - Contribution of electrogenic sodium-potassium ATPase to resting membrane potential of cultured rat skeletal myotubes. AB - The contribution of electrogenic Na+ -K+ ATPase to resting membrane potential (Em) of mature and developing rat skeletal myotubes in culture was determined by examining effects of inhibition of this enzyme on Em. Ouabain, a specific Na+-K+ ATPase inhibitor, caused resting Em to decrease within 30 s by 5-8 mV and reach a minimum value of about -60 mV after 5 min. The decrease in Em was not accompanied by a decrease in input resistance for up to 15 min after application. Resting Em was found to be dependent on the temperature of the recording medium with maximum values of Em ranging from -85 to -90 mV at a temperature of 35-37 degrees C and minimum values about -60 mV at 10-15 degrees C. Ouabain (1 mM), added to cultures at low temperature (10-15 degrees C) did not further decrease Em but did prevent the increase in Em that occurs with increasing temperature up to 37 degrees C. Resting Em of cultured myotubes was reduced to about -60 mV by reducing the supply of ATP either with 2,4 dinitrophenol (DNP), which inhibits oxidative phosphorylation or with fluorodinitrobenzene (FDNB), which inhibits creatine phosphokinase. Neither of these compounds, when added to cultures in the presence of ouabain, reduced resting Em to a value lower than that obtained with ouabain alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996717 TI - Altered postnatal ontogeny of alpha 1- and alpha 2-adrenoceptor binding sites in the brain of a convulsive mutant mouse (quaking). AB - Binding assays of [3H]dihydroalprenolol ([3H]DHA), [3H]prazosin and [3H]clonidine have been performed on whole brain (minus cerebellum) homogenates of the convulsive mutant mice quaking (qk) and the controls of the same strain (C57BL/6J:B6). In 70-day-old mutants (which fully exhibit the qk convulsive phenotype), the binding of [3H]DHA to beta-adrenoceptor binding sites was not different from the controls, whereas the binding capacities of [3H]prazosin and [3H]clonidine to alpha 1-and alpha 2-adrenoceptor sites, respectively, were greatly enhanced. The biphasic ontogenic pattern of alpha 2-adrenoceptors had a greater amplitude in the brain of 30- to 90-day-old mutants than in the corresponding B6 controls. In mutants younger than 30 days or older than 90 days, the number of alpha 2-adrenoceptor sites was not modified. The number of alpha 1 adrenoceptor binding sites was increased in the brain of the mutants, only in animals older than 70 days. In younger mice, the postnatal modulation of alpha 1 adrenoceptor sites was identical to the controls. Regional studies were performed in 70-day-old mice. [3H]clonidine binding was increased in the brainstem of the mutants, and to a lesser extent in the cerebral cortex, while it was slightly diminished in the hypothalamic area. [3H]prazosin binding was also increased in the brainstem of the mutants, and decreased in the olfactory bulbs. Our results suggest that the convulsions of the qk mutants are selectively associated with modifications of alpha- and not beta-adrenoceptor binding sites.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996718 TI - Electrophysiological and morphological correlates in the development of visual cortical circuitry in infant kittens. AB - Geniculate axons in cat visual cortex establish excitatory connections with cortical cells in supragranular and granular layers at birth. They are localized to the granular layer for the first month after birth. Retraction of geniculocortical synapses parallels axonal extension of spiny stellate cells to the supragranular layer and establishment of synapses for intracortical transmission. PMID- 2996719 TI - Beneficial effect of chronic treatment with Org 2766 and alpha-MSH on impaired reversal learning of rats with bilateral lesions of the parafascicular area. AB - The effects of chronic treatment with the ACTH-(4-9) analogue Org 2766, alpha MSH, and gamma 2-MSH were studied on T-maze reversal learning and on behavior assessed on the basis of open-field and other gross behavioral activities, grasping responses, inspection of various reflexes and electrical footshock sensitivity of rats with parafascicular lesions or sham-lesions. Repeated administration of Org 2766 and alpha-MSH to parafascicular area-lesioned rats resulted in functional recovery of impaired T-maze reversal learning. The structurally related neuropeptide gamma 2-MSH was without any effect. The alpha MSH effect did not depend on time after lesioning as treatments during the first or second post-operative week were equally effective. Chronic peptide treatments did not change disturbed motor functions of parafascicular-lesioned rats, as measured by open-field activity, other gross behavioral activities and grasping responses. Since acute peptide treatments did not affect the impaired reversal learning performance of lesioned rats, the beneficial effect of Org 2766 and alpha-MSH could not be explained as a short-term effect on attention and motivation. It was more likely to be an accelerated recovery of cognitive function as a result of long-term neurotropic influences. PMID- 2996720 TI - The effect of ACTH4-10 on protein synthesis, actin and tubulin during regeneration. AB - The effects of ACTH4-10, a peptide fragment of corticotropin, on rat dorsal root ganglia (DRG), spinal cord and sciatic nerve were studied following a crush lesion of the sciatic nerve. The in vitro total protein synthesis rate of DRG L4, L5 and L6, measured one and three days after ipsilateral nerve crush, were not altered by various ACTH4-10 treatment regimes. Likewise, neither ACTH4-10 treatment of sham-operated rats nor in vitro exposure of control ganglia to peptide, resulted in changes in synthesis rate. Four days after crush lesion, the amounts of actin and tubulin in the ventral horn L2-L5 region of the spinal cord and of actin in DRG L5 were estimated following 2-dimensional separation. No significant effect of ACTH treatment was found. Degeneration-associated changes in the protein profiles of segments of sciatic nerve were not altered by ACTH4-10 treatment. The data are discussed in relation to the possible site of action of neurotrophic ACTH-like peptides. PMID- 2996721 TI - Direct effects of androgens on lateral hypothalamic neuronal activity in the male rat: I. A microiontophoretic study. AB - Unit neuronal activity in the lateral hypothalamic-medial forebrain bundle region (LHA-MFB) of the male rat is modified following an increase of plasma testosterone level. In order to determine possible direct action of hormones on LHA-MFB neurons, unit discharge frequencies were recorded during local microiontophoresis of testosterone and estradiol, and electroosmotic application of cholesterol. Thirteen cells did not respond, 9 were nonspecifically depressed by all the steroids tested, 13 were excited by both sex-steroids, and 11 were specifically activated by testosterone. The short latencies of the responses suggest a direct effect of steroids on the plasma membrane sites. PMID- 2996722 TI - The effect of in vitro and in vivo ethanol administration on [35S]t butylbicyclophosphorothionate binding in C57 mice. AB - Previous studies have shown that ethanol may produce some of its effects by facilitation of GABAergic transmission. One of the potential sites of drug action at the GABA receptor complex is the picrotoxin site, which can be studied with [35S]t-butylbicyclophosphorothionate (TBPS). Ethanol inhibited the binding of [35S]TBPS to C57 mice brain regions in vitro. This inhibition appears to be noncompetitive since ethanol decreased the Bmax and not the KD value of [35S]TBPS. C57 mice were chronically treated with ethanol in liquid diet to determine if the sensitivity of TBPS binding is altered following chronic treatment or during withdrawal. Chronic treatment with ethanol and during withdrawal did not alter the KD or Bmax values of [35S]TBPS binding in C57 mice brain regions. It is suggested that the sensitivity of picrotoxin site on the oligomeric GABA receptor complex is not altered during ethanol tolerance or withdrawal. The effects of ethanol on GABA system may be mediated by its interaction with the coupling mechanism(s) or a direct effect on the chloride channels. PMID- 2996724 TI - Heart mitochondrial function in chronic experimental diabetes in rats. AB - Diabetes was introduced in rats by an intravenous injection of streptozotocin (65 mg/kg). Animals were sacrificed 8 weeks later and mitochondria were isolated from the ventricular tissue by differential centrifugation. The state 3 respiration, oxidative phosphorylation rate and Mg2+-dependent ATPase activities were depressed in mitochondria from diabetic hearts. These changes were partially reversible upon 2 weeks of insulin and fully reversible after 4 weeks of insulin therapy. Mitochondrial calcium uptake but not calcium binding, was decreased in diabetes and this change was fully reversible by 2 weeks of insulin administration. The observed alterations in mitochondrial function could not be explained on the basis of any changes in mitochondrial lipid and protein composition or subcellular contamination. These results indicate the presence of a generalized depression in mitochondrial function in chronic diabetes and such a defect is suggested to contribute in the development of cardiomyopathy at late stages of diabetes. PMID- 2996723 TI - 5',5'''-P1, P4 diadenosine tetraphosphate (Ap4A): a putative initiator of DNA replication. AB - The proposal that Ap4A acts as an inducer of DNA replication is based primarily on two pieces of evidence (7). The intracellular levels of Ap4A increase ten- to 1000-fold as cells progress into S phase and the introduction of Ap4A into nonproliferating cells stimulated DNA synthesis. There is also some additional suggestive evidence such as the binding of Ap4A to a protein that is associated with multiprotein forms of the replicative DNA polymerase alpha and the ability of this enzyme to use Ap4A as a primer for DNA synthesis in vitro with single stranded DNA templates. These observations have stimulated interest in the cellular metabolism of Ap4A. This is well since there is a great need for additional experimentation in order to clearly establish Ap4A as an inducer of DNA replication. Microinjection experiments of Ap4A into quiescent cells are needed in order to ascertain if Ap4A will stimulate DNA replication and possibly cell division in intact cells. Studies of the effects of nonhydrolyzable analogs of Ap4A on DNA replication in intact quiescent cells could also prove valuable. Although Ap4A can function as a primer for in vitro DNA synthesis by DNA polymerase alpha this may not be relevant in regard to its in vivo role in DNA replication. Ap4A in vivo could interact with key protein(s) in DNA replication and in this way act as an effector molecule in the initiation of DNA replication. In this regard the interaction of Ap4A with a protein associated with a multiprotein form of DNA polymerase alpha isolated from S-phase cells is of interest. More experiments are required to determine if there is a specific target protein(s) for Ap4A in vivo and what its role in DNA replication is. The cofractionation of tryptophanyl-tRNA synthetase with the replicative DNA polymerase alpha from animal and plant cells is of interest. The DNA polymerase alpha from synchronized animal cells also interacted with Ap4A. Although the plant cell alpha-like DNA polymerase did not interact with Ap4A this DNA polymerase was not a multiprotein form of polymerase alpha and the synchrony of the wheat germ embryos was not known. A possible tie between protein-synthesizing systems and the regulation of proteins involved in DNA replication may exist. The requirement of protein synthesis for the initiation of DNA replication has long been known. Also, it is well established that many temperature-sensitive mutants for tRNA synthetases are also DNA-synthesizing mutants. More investigation in this area may be warranted.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2996725 TI - Modification of cardiac sarcolemmal Na+-Ca2+ exchange by diltiazem and verapamil. AB - Cardiac sarcolemmal membranes were isolated from the rat heart and their ability for Na+-Ca2+ exchange in the absence or presence of diltiazem and verapamil was examined. Maximal Ca2+ influx activity of membranes due to Na+-dependent reaction occurred within 3 min and was about 5 nmol Ca2+/mg protein. Diltiazem (0.1 to 10 microM) depressed the Ca2+ influx activity significantly whereas verapamil (0.1 to 10 microM) had no effect at initial stages of the reaction (10 to 20 sec). The inhibitory effect of diltiazem on Ca2+ influx was found to be of an uncompetitive nature. Sodium was found to cause a rapid Ca2+ efflux from the calcium loaded membrane vesicles; about 70% of the Ca2+ efflux activity was increased by 0.1 to 10 microM of verapamil and 10 microM of diltiazem significantly. The stimulatory effect of these agents on Ca2+ efflux was associated with a change in Ka value from 16 to 5 mM Na+. Both diltiazem (0.1-3 microM) and verapamil (0.1-10 microM) did not affect the membrane Na+-K+ ATPase activity, but diltiazem in high concentrations (10-30 microM) had an inhibitory action. Specific calcium channel blocking agents, nitrendipine and nifedipine, depressed sodium-dependent Ca2+ efflux activity. A beta-adrenoreceptor antagonist, propranolol, unlike acebutolol, increased sodium-induced Ca2+-influx at high concentrations (10-100 microM).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996726 TI - The influence of intrinsic sympathomimetic activity on regional left ventricular function; use of regional ejection fraction to demonstrate a beneficial action by pindolol over propranolol on hypokinetic segments. AB - In a previous report from our laboratory, visual assessment of wall motion in patients with coronary artery disease demonstrated no advantage for pindolol, a beta blocking agent with intrinsic sympathomimetic activity (ISA), over propranolol on impaired regional left ventricular (LV) function. In this study, we reanalyzed the radionuclide ventriculograms using a computer-assisted method of deriving regional ejection fraction. Use of normalized values allowed hypokinetic and normokinetic segments to be identified and examined separately. Pindolol (5-10 mg twice a day) was compared to propranolol (40-80 mg 4 times a day) in 23 patients using a randomized, crossover study design. Supine resting heart rate was reduced less (70 +/- 12 beats/min vs 63 +/- 10 beats/min, p less than 0.01) by pindolol; exercise heart rate was reduced equally by both agents. Derivation of normalized regional LV ejection fractions showed that 17 segments were hypokinetic at rest. Function of these segments increased (p less than 0.02) with pindolol. This improvement was not detected by visual assessment of regional wall motion. Thirty-seven segments were found to be hypokinetic during exercise and a significant (p less than 0.05) improvement in function occurred with pindolol and propranolol. In summary, derivation of normalized regional LV ejection fraction values allows the demonstration of significant improvement in resting LV function with pindolol, but not with propranolol in patients with regional dysfunction due to coronary artery disease. This advantage may provide a rationale for further evaluation of this agent in patients with more widespread ventricular dysfunction. PMID- 2996727 TI - Heart sarcolemmal ATPase and calcium binding activities in rats fed a high cholesterol diet. AB - ATPase and calcium binding activities were studied in sarcolemmal membranes from hearts of male rats fed either a control or 2% cholesterol diet for different time periods. Studies with isolated membrane revealed a significant increase in Na+-K+ ATPase activity, sialic acid content and ATP-independent calcium binding capacity in the presence of 1.25 mM CaCl2 in the 6 week cholesterol fed group. By 12 weeks, Na+-K+ ATPase, Mg2+-ATPase and Ca2+-ATPase activities as well as ATP independent calcium binding in the presence of 0.05 mM CaCl2 were increased in membranes from cholesterol fed rats. A significant increase (P less than 0.05) in the sarcolemmal cholesterol/phospholipid molar ratio, which is an indicator of a decrease in membrane fluidity, was also noted in the 12 week cholesterol fed group. Concanavalin A, which is believed to decrease membrane fluidity, stimulated both Mg2+ and Ca2+-dependent ATPase activities and increased ATP independent calcium binding in control sarcolemmal preparations and these changes resembled those observed in the sarcolemma from cholesterol fed rats. Since concanavalin A did not alter the activity of Na+-K+ ATPase, it appears that some of the observed differences in sarcolemmal activities upon cholesterol feeding did not correlate well with changes in membrane order. At 24 weeks, there was a generalized depression in the sarcolemmal ATPase activities of the cholesterol group; both Mg2+ ATPase and Ca2+ ATPase were significantly less than in control.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996729 TI - Evaluation of the alpha and beta blocking effects of intravenous labetalol on left ventricular function in coronary artery disease. AB - Intravenous labetalol was evaluated in 10 patients with stable angina without heart failure. Mean dose was 1.75 mg/kg (range 1.5-2 mg/kg). Measurements were taken within one minute after the injection, and at 1, 5 and 15 minutes thereafter. Labetalol significantly decreased blood pressure and increased heart rate. Peak aortic flow velocity increased only significantly at 1 minute; dP/dt+ max. was significantly decreased during all the measurements. Left ventricular end diastolic pressure did not change. Thus in patients without failure left ventricular function remained stable despite the negative inotropic effects of labetalol. PMID- 2996730 TI - Temporal desensitization of rat uteri for the decidual cell reaction is abolished by cholera toxin acting by a mechanism apparently not involving adenosine 3':5' cyclic monophosphate. AB - To determine if increased endometrial vascular permeability (a response which precedes decidualization) could be obtained in temporally nonsensitized uteri by treatments designed to increase endometrial adenosine 3':5'-cyclic monophosphate (cAMP) concentrations, cholera toxin (an activator of adenylate cyclase) was injected into the uterine lumen of immature rats treated to be at the equivalent of day 4, 5, or 6 of pseudopregnancy. In all experiments, the rats were pretreated with indomethacin to inhibit endogenous prostaglandin (PG) synthesis. Endometrial vascular permeability, determined using 125I-labeled bovine serum albumin, was assessed 8 h later. Cholera toxin increased endometrial vascular permeability to the same level in all groups. As determined by uterine weights 5 days after the intrauterine administration of cholera toxin or its vehicle, the toxin produced the same extent of decidualization in all groups. Cholera toxin had no detectable effect on uterine cAMP concentrations in rats sacrificed 15 min after intrauterine treatment. In contrast, intrauterine administration of PGE2 increased uterine cAMP concentrations at 15 min in all groups. These data suggest that the effects of cholera toxin and of PGE2 on endometrial vascular permeability and decidualization are not mediated by cAMP. PMID- 2996728 TI - Vascular and contractile responses to extracellular ATP: studies in the isolated rat heart. AB - The dose-response characteristics for the effect of ATP upon cardiac function and vascular tone have been investigated in the isolated perfused rat heart. Vasodilation was observed with low ATP concentrations (0.01-0.1 mM) whereas severe vasoconstriction occurred with high concentrations (1.0-10.0 mM). At all doses studied, heart rate and pressure-rate product were reduced in a dose dependent manner, with 10 mM ATP almost complete cardiac arrest was observed. Analysis of epicardial electrograms revealed that ATP induced arrhythmias, prolonged the P-R interval and induced partial blockade of S-A nodal activity and A-V conduction. Investigating possible mechanisms for the vascular and contractile effects of ATP, it was possible to exclude the calcium chelating properties of ATP and the effects of coincident ischemia arising as a consequence of ATP-induced vasoconstriction. Pharmacological studies revealed the ATP-induced vasoconstriction to be unresponsive to a range of coronary vasodilators and also allowed exclusion of prostaglandins, catecholamines and adrenergic receptors in the mediation of ATP effects. Investigations with acetylcholine revealed remarkably similar effects upon both contractile and vascular activity but studies with atropine suggested that the muscarinic receptor was not involved. Studies with theophylline allowed a dissociation of the vascular and contractile effects of ATP and indicated a possible involvement of the adenosine receptor in the cellular response to both high and low concentrations of ATP. PMID- 2996731 TI - The in vitro metabolism of benzo[a]pyrene by polychlorinated and polybrominated biphenyl induced rat hepatic microsomal monooxygenases. AB - The metabolism of benzo[a]pyrene by halogenated biphenyl-induced rat hepatic microsomal monooxygenases was determined using a high pressure liquid chromatographic assay system. Incubation of benzo[a]pyrene with microsomes from rats pretreated with phenobarbitone or phenobarbitone-type inducers (2,2',4,4',5,5'-hexachlorobiphenyl, 2,2',4,4',6,6'-hexachlorobiphenyl, 2,2',5,5' tetrachlorobiphenyl, 2,2',4,4',5,5'-hexabromobiphenyl, and 2,2',5,5' tetrabromobiphenyl) resulted in increased overall metabolism of the hydrocarbon (less than fourfold) into phenolic, quinone, and diol metabolites, with the most striking increase observed in the formation of 4,5-dihydro-4,5 dihydroxybenzo[a]pyrene. In contrast, the metabolism of benzo[a]pyrene by microsomes from rats induced with 3-methylcholanthrene or 3,3',4,4' tetrachlorobiphenyl resulted in a greater than 10-fold increase in overall benzo[a]pyrene metabolism, with the largest increases observed in the formation of the trans-7,8- and -9,10-dihydrodiol metabolites of benzo[a]pyrene. However, in comparison to control and phenobarbitone-induced microsomes, the oxidative conversion of benzo[a]pyrene by microsomes induced with 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl into the 6,12-quinone was substantially inhibited. Previous reports have shown that the commercial halogenated biphenyl mixtures, fireMaster BP-6, and Aroclor 1254 are mixed-type inducers and that microsomes from rats pretreated with these mixtures markedly enhance the overall metabolism of benzo[a]pyrene. Not surprisingly, the metabolism of benzo[a]pyrene by microsomes from rats pretreated with the mixed-type inducers, 2,3,3',4,4'-penta ,2,3,3',4,4',5-hexa-, and 2',3,3',4,4',5-hexa- chlorobiphenyl was also increased and the metabolic profile was similar to that observed with fireMaster BP-6 and Aroclor 1254 induced microsomes. PMID- 2996732 TI - Effect of lanthanum on the inotropic response of isoproterenol: role of the superficially bound calcium. AB - Mammalian myocardial contractility is believed to be related to the amount of calcium contained in a highly labile superficial calcium pool. The purpose of this study was to determine the role of such sites in the positive inotropic effect of isoproterenol. Lanthanum, an ion that is restricted to the extracellular space and that displaces the superficially bound calcium, was selected as a tool for this investigation. In Langendorff preparations of the guinea pig heart, lanthanum decreased the basal contractility index (+dP/dtmax) in a concentration-dependent fashion (0.05-3 microM) and blocked the inotropic response of isoproterenol in a noncompetitive manner (0.25-3 microM). Three micromolar lanthanum (i) reduced basal contractility and the maximum response to isoproterenol by 97 and 95%, respectively, (ii) had no significant effect (p greater than 0.05) on basal and isoproterenol-induced cyclic AMP levels, and (iii) had no effect on the Kd of [3H]nitrendipine binding, but reduced the Bmax by 31%. While 1 microM lanthanum reduced basal contractility and the maximum response to isoproterenol by 90 and 70%, respectively, it had no effect on [3H]nitrendipine binding. These results suggest that the effects of such low concentrations of lanthanum (less than or equal to 3 microM) are not related to a direct action on the calcium channels and are not mediated by an inhibition of isoproterenol stimulation of the enzyme adenylate cyclase. Therefore, one interpretation of these results suggests that superficially bound calcium is required for the inotropic response of isoproterenol. PMID- 2996733 TI - Noradrenergic alpha 2 binding sites in vagal dorsal motor nucleus and nucleus tractus solitarius: autoradiographic localization. AB - The distribution in the canine medulla oblongata of binding sites for p [3H]aminoclonidine, a ligand specific for alpha 2-adrenergic receptors, was studied with light microscopic autoradiographic methods. Specific labelling was determined using unlabelled phentolamine as a displacer. The greatest density of sites was localized in the dorsal motor nucleus of the vagus nerve, the area postrema, and in several regions of the nucleus tractus solitarius. Less dense binding of the radioligand was also seen in the inferior olivary nucleus. Dorsomedial regions of the nucleus tractus solitarius were the most densely labelled in this nucleus, and dorsolateral and ventral regions were much less densely labelled. The region of the nucleus tractus solitarius shown in this study to be heavily labelled with alpha 2-adrenergic binding sites has been implicated in the autonomic control of blood pressure. The dorsal motor nucleus of the vagus, together with the nucleus tractus solitarius, may thus represent the site of the antihypertensive action of the drug clonidine, an alpha 2 adrenoreceptor agonist. PMID- 2996735 TI - The medical treatment of the hypersecreting pituitary gland. AB - Pituitary adenomas may produce local endocrine and neurological effects, as well as systemic metabolic complications due to hormonal hypersecretion. Medical therapy with pharmacological agents has been developed and is based on the neurotransmitter regulation of normal pituitary hormonal secretion. 189 patients with secretory pituitary adenomas underwent medical therapy for the hypersecretory state. 156 of these were prolactin-secreting adenomas, 16 of which were in males. The response of bromocriptine was almost universal with lowering of serum prolactin and reversal of the clinical symptoms, as well as tumor shrinkage of most large adenomas with suprasellar extension. 23 patients with acromegaly were treated with bromocriptine, with 11 noting clinical improvement, and decreased tumor size in two. Five patients with Cushing's disease were treated with cyproheptadine, with only one showing a biochemical and clinical improvement. Two patients with Nelson's syndrome each had progressive tumor growth stabilized with cyproheptadine and bromocriptine in one, and sodium valproate in the other. There appears to be a role for medical therapy in the majority of prolactin-secreting pituitary tumors, some growth hormone secreting pituitary tumors, and selected adrenocorticotropin secreting-pituitary tumors. PMID- 2996734 TI - Antagonism by theophylline of the adenosine receptor agonist 5'-N ethylcarboxamidoadenosine at the guinea pig ileum. AB - The inhibitory effect of the putative adenosine A2 receptor agonist 5'-N ethylcarboxamidoadenosine (NECA) on acetylcholine release from the stimulated guinea pig ileum preparation and the nature of its antagonism by theophylline were investigated. NECA was shown to inhibit the response of the ileum preparation in a dose-dependent fashion, and an EC50 value of 1.62 X 10(-8) M was determined. This value was comparable with that determined for the A1 receptor agonist N6-R-phenylisopropyladenosine (R-PIA) (2.57 X 10(-8) M) using the same preparation. Competitive antagonism of the inhibitory effect of NECA by theophylline was quantitated and a pA2 value of 5.04 for the methylxanthine was obtained. This value was similar to those obtained previously for R-PIA and adenosine itself and suggests that these nucleosides may be interacting with the same receptor site on myenteric nerve endings. These findings do not permit the designation of the receptor as an A1 or A2 subtype according to current criteria. PMID- 2996736 TI - Refined Romberg test. PMID- 2996737 TI - The fate of "outstanding postdischarge data". PMID- 2996739 TI - Classification of patients with AIDS in Canada. PMID- 2996740 TI - Non-small cell lung cancer. Neuroresection of the solitary intracranial metastasis followed by radiochemotherapy. AB - Fifteen selected patients with advanced intrathoracic non-small cell lung cancer and solitary metastasis were treated by a combined program including craniotomy, brain and chest irradiation, and systemic chemotherapy. One patient died because of cerebral hemorrhage after the operation. Five patients failed to achieve neurologic benefit. Nine patients improved their neurologic grading, and the median duration of improvement was 10 months (range, 1-26 months). The responses to systemic treatment were: one complete response, three partial responses, six stable disease responses, and four progressive disease responses. The overall median survival was 6 months from craniotomy and 12 months from diagnosis. Five patients became long survivors; they had a survival time ranging between 12 and 26 months after craniotomy. In conclusion, one third of patients had a satisfactory response to treatment; this outlines the value of the combined aggressive therapeutic approach also performed in patients who had a highly unfavorable prognoses. PMID- 2996738 TI - Compliance with postpartum Rh isoimmunization prophylaxis in Alberta. AB - A retrospective review of obstetric records for 1979 in two major Calgary hospitals was undertaken to determine the rate of compliance with postpartum Rh isoimmunization prophylaxis in Alberta. The charts of 4528 women ranging in age from 13 to 46 years were reviewed. The prevalence rate of Rh negativity was found to be 16%. Of the 710 Rh-negative women 490 (69%) were eligible to receive Rh immune globulin (RhIG); that is, they had no anti-D antibodies, and the baby/fetus was Rh-positive or Rh-unknown. RhIG had been administered to 93.6% of the eligible women; the compliance rate ranged from 66.7% for obstetric emergencies (i.e., spontaneous abortion, antepartum or early-pregnancy hemorrhage, or ectopic pregnancy) to 98.2% for postpartum diagnoses. In more than half (54.7%) of the women who underwent amniocentesis Rh type was not determined; the implications of this finding are discussed. Although poor compliance with postpartum RhIG administration is not a reason for withholding antepartum administration of RhIG, maximum compliance with the more cost-effective programs should be attained before antepartum programs are fully implemented. PMID- 2996742 TI - Long-term survival with tumor regression in androgen-induced liver tumors. AB - Two patients with androgen-induced liver tumors, one of whom had been partially treated by a liver resection, are reported. Hepatocellular carcinoma was diagnosed on histologic grounds. The patients had been receiving androgen therapy for primary diagnoses of either hypopituitarism or paroxysmal nocturnal hemoglobinuria. After androgen withdrawal, both are alive and well with no evidence of residual tumor 10 and 14 years after diagnosis, respectively. PMID- 2996741 TI - Altered vitamin A-binding proteins in carcinoma of the head and neck. AB - Since vitamin A inhibits the terminal differentiation of keratinocytes, elevated levels of cytoplasmic vitamin A-binding proteins could explain the reduced tendency of squamous cell carcinomas to undergo terminal differentiation. The levels of cellular retinol-binding protein (CRBP) and cellular retinoic acid binding protein (CRABP) were determined in 37 squamous cell carcinomas of the head and neck and were compared with the corresponding levels in adjacent normal tissue. The CRBP levels expressed per milligram of DNA were increased threefold (P = 0.0005) in the tumor as compared with normal tissue. In contrast, CRABP levels expressed per milligram were decreased 2.5-fold (P = 0.0007) in the tumor as compared with normal tissue. Despite significant differences in retinoid binding protein levels between tumor and adjacent normal tissue, there was no significant correlation of these differences with any of the patient clinical parameters or any of the tumor growth characteristics. The increased CRBP levels may explain why terminal differentiation, an important mechanism for limiting growth in normal keratinocytes, is less readily induced in squamous cell carcinomas. PMID- 2996743 TI - Mucin in carcinomas of the thyroid. AB - One hundred forty-two cases of thyroid carcinomas were studied to assess the frequency with which mucinous deposits occur within the neoplastic cells. In contrast to the literature, which contains only a few scattered reports on this topic, the production of mucin by carcinomas of the thyroid was relatively frequently observed in our material. Almost one half of the cases displayed mucinous substances, which occurred in about 50% of the papillary and the medullary carcinomas, 35% of the follicular types, and about 21% of the anaplastic varieties. The amount of mucinous substances varied considerably: 28 cases were weakly positive, 30 cases were moderately positive, and 6 cases were strongly positive. These findings are discussed with respect to diagnostic and differential diagnostic problems, especially with respect to the interpretation of mucinous deposits that occur in metastatic carcinomata. PMID- 2996744 TI - Hepatocellular carcinoma. Relationship to wine and pork consumption. AB - The authors investigated the possibility that dietary fat, meat, beef, and pork consumption might be factors that would, in addition to alcohol, correlate with mortality from hepatocellular carcinoma (HCC) in different countries. The authors also relate the consumption of different alcoholic beverages (wine, beer, spirits) to HCC mortality. The significant relationships were between HCC mortality and alcohol consumption (r = 0.40, P less than 0.05), wine consumption (r = 0.46, P less than 0.05), and pork consumption (r = 0.40, P less than 0.05). The intake of alcohol, wine, and pork was also significantly higher in the countries with HCC mortality of greater than 3/100,000, compared with countries with HCC mortality of less than 3/100,000. PMID- 2996745 TI - Primary signet-ring carcinoma of the large bowel. Report of nine cases. AB - Nine cases of signet-ring carcinoma have been observed from among 800 consecutive histologic cases diagnosed as adenocarcinoma of the colon during a period of 10 years (0.9%). This group of nine patients (Group A) has been matched for sex, age, and stage with a group of 45 patients affected by ordinary carcinoma of the colon (Group B). Clinical and histologic parameters, including symptoms, primary tumor site, free interval from primary surgery, histochemical investigation of intracytoplasmic mucins, and survival, were evaluated. The results of this investigation showed no clinical differences between signet-ring carcinoma and ordinary carcinoma, and no statistically significant results were observed regarding the frequency of local recurrence and actuarial survival. PMID- 2996747 TI - Increased survival with high-dose multifield radiotherapy and intensive chemotherapy in limited small cell carcinoma of the lung. AB - From June 1979 through April 1982, we treated 35 patients with limited small cell carcinoma on an intensive chemo-radio-immunotherapy regimen, consisting of induction with cyclophosphamide, doxorubicin, and vincristine, alternately cycled with VP-16 and cisplatin. Patients were stratified by performance status and randomized to thymosin, fraction V, or no thymosin. Induction was followed by consolidation, consisting of prophylactic whole-brain radiotherapy and multifield radiotherapy to the primary and mediastinum with cyclophosphamide and vincristine. Patients who were complete responders (CRs) postconsolidation resumed maintenance immediately. Patients were followed from 1 to 3.8 years (median, 2.2 years) at the time of analysis. After induction, 35% (12/34) had become CRs; after consolidation radiotherapy, an additional 10/34 became CRs for a total CR rate of 65% (22/34). There were only 9/34 local failures (26%), of which all but one were impatients who had not become CRs. A prolonged median survival (21 months) has been obtained in patients with limited small cell carcinoma of lung treated with an intensive combined modality regimen. At 1 year, survival is 83%; at 2 years, 46%. There is a 33% long-term survival (greater than 3 years). There is no difference in survival or recurrence rate between patients treated with or without thymosin. PMID- 2996746 TI - Treatment of malignant nondysgerminomatous germ cell tumors of the ovary with vincristine, dactinomycin, and cyclophosphamide. AB - Eighty patients with malignant nondysgerminomatous germ cell tumors of the ovary were treated with the combination of vincristine, dactinomycin, and cyclophosphamide (VAC) at The University of Texas M.D. Anderson Hospital and Tumor Institute. All patients underwent initial surgery: biopsy alone in 3 patients, unilateral salpingo-oophorectomy in 48 patients, and bilateral salpingo oophorectomy with or without hysterectomy in 29 patients. Sixty-six patients received VAC as primary postoperative therapy; 46 patients (70%) achieved a sustained remission. VAC produced sustained remission in 86% of patients with Stage I, 57% of patients with Stage II, 50% of patients with Stage III, and no patients with Stage IV disease. For patients with Stage I disease, survival rates did not differ among histologic groups, but in advanced disease, patients with immature teratoma did significantly better than the others. Four of the 20 patients who failed primary VAC therapy were salvaged with other therapies, and 8 of 14 treated with VAC after relapse or failure of other treatments were salvaged. Although VAC produces excellent results with very acceptable toxicity in patients with Stage I disease and advanced immature teratoma, survival of patients with other advanced histologic types has been disappointing. The authors are therefore treating this latter group with alternative therapy such as vinblastine, bleomycin, and cisplatin with the goal of achieving improved efficacy. PMID- 2996748 TI - A study to analyze the origin of tumor cells in malignant fibrous histiocytomas. A multiparametric characterization. AB - To study the derivation of tumor cells of malignant fibrous histiocytomas (MFH), their phenotypical marker profile was investigated and compared with those of malignant histiocytosis (MH) and of different types of soft tissue tumors (STT). The presence of the following markers was investigated: on paraffin sections, alpha-1-antichymotrypsin (ACT); on frozen sections antigens associated with lymphocytes, macrophages and fibroblasts, the enzymes acid phosphatase, nonspecific esterase, and beta-glucuronidase; and, on isolated and cultured cells, the receptors for EA-gamma and complement. Furthermore, the capacity to phagocytose sensitized erythrocytes and carbon particles was studied in vitro. MFH tumor cells and a part of other types of STT shared the expression of ACT and lysosomal enzymes with MH. They differed, however, from MH by the absence of monocyte/macrophage-associated antigens and by the expression of fibroblast associated antigens, which property they had in common with other STT. MFH tumor cells were not able to form rosettes or to phagocytose Ig-sensitized erythrocytes, but they showed phagocytosis of carbon particles. The results strongly indicate that MFH tumor cells originate from (primitive) fixed mesenchymal cells and are not related to monocyte-derived histiocytes. PMID- 2996750 TI - Liver iron stores and hepatitis B antigen status. AB - Higher serum iron and ferritin levels noted in hepatitis B antigen (HBAg) carriers than in noncarriers suggests that virus might actively replicate in hepatocytes, stimulate ferritin synthesis, and result in increased liver iron stores. A comparative semiquantitative study of immunohistochemical ferritin (0 12) and hemosiderin (0-9) was performed on 54 normal, 13 cirrhotic, and 70 nonneoplastic livers from patients with hepatocellular carcinoma, in each group, comparing amounts in HBAg-positive and HBAg-negative patients. Mean scores for ferritin and hemosiderin were high in all three groups, normal livers averaging 8.3 and 6, respectively, cirrhotic livers, 8.5 and 7.4, respectively, and carcinoma livers, 5.6 and 6.1, respectively. In each group, there was no significant difference in ferritin and hemosiderin mean scores in HBAg-positive and HBAg-negative patients. The large liver iron stores do not appear to be a consequence of hepatitis B virus infection alone. Their role in the development of hepatocellular carcinoma is still to be elucidated. PMID- 2996749 TI - Palliation of pelvic recurrence of colorectal cancer with intra-arterial 5 fluorouracil and mitomycin. AB - Twenty-one patients with inoperable colon cancer in the pelvis were treated with intra-arterial 5-fluorouracil (5-FU) and mitomycin C, given bilaterally into the internal iliac arteries. Seventeen of the 21 patients had failed previous radiation therapy and 15 had also failed systemic intravenous chemotherapy. Eighteen of the 21 patients received intra-arterial treatments because of pelvic pain. Effect of this treatment on the pain could be evaluated in 16 patients. A measurable decrease in pain medication occurred in 8 of 16, whereas a subjective feeling of pain relief was observed in 12 of 16 patients for a mean period of 3.5 months. However, objective tumor response was considered definite only if associated with a greater than 50% decline of an elevated plasma carcinoembryonic antigen level; this was observed in 5 of 11 patients (45%). Reduction in tumor mass as measured by imaging techniques was observed in two of ten patients in whom it was evaluable. Improvement in hydronephrosis was observed in five of seven evaluable patients. Hematuria was present in 12 patients and improved in 10 of those patients. The most significant side effect of chemotherapy was perineal and gluteal skin erythema, which was observed in 36% of the patients after the first course and in 24% during the second course. This frequently escalated to cutaneous vesiculation and desquamation. This side effect was prevented by concurrent administration of steroids. Pelvic arterial infusion of 5-FU and mitomycin C can offer temporary pain relief to patients who have failed other means of therapy. Objective antitumor effects may have also resulted but were much harder to assess in this group of patients. PMID- 2996751 TI - Correlation of steroid receptors with histologic differentiation in mammary carcinoma. A Singapore experience. AB - Cancer of the breast is the most common tumor in females in Singapore, with the rate of 20.7 per 100,000 per year (1977 estimate), which is predicted to increase to 29.8 per 100,000 women per year by 1995. A detailed histopathologic review of 50 primary breast cancer tumors analyzed for estrogen receptor (ER) level was carried out and a variety of morphologic features correlated with ER results to identify any factors that will improve the management and prognosis for breast cancer. Cytosol was incubated with 3H-estradiol in the presence and absence of cold diethylstilbestrol, and bound and free hormone were separated by Dextran coated charcoal method. Tumors binding more than 5 fmol/mg cytosol protein were classified as ER-positive. Progesterone receptor (PR) level was analyzed in some specimens with the use of a similar method. Most of the patients were Chinese (90%). Three patients were Malays, one was Indian, and one was European in this series. Results indicated that there was strong correlation between ER level, age, and histologic grade of the tumors. No correlation existed between absence or presence of lymph node metastases and ER. Although there was a trend for ER positive tumors to have a low-grade lymphocytic infiltration, the difference was not statistically significant. PMID- 2996752 TI - Smoking as a risk factor in hepatocellular carcinoma. A case-control study in southern African blacks. AB - Two hundred forty southern African black patients with hepatocellular carcinoma and control subjects matched for race, sex, age, and urban or rural background were questioned about their smoking habits. Patients with hepatocellular carcinoma were not more likely to smoke or to smoke heavily than the control subjects. This was also true of the subgroups: men and women, and urban and rural background. There was a slightly increased relative risk associated with smoking in all patients who showed no serum markers of current or past hepatitis-B virus infection and in patients older than 50 years who did not have markers of current or past hepatitis-B virus infection. However, this was not statistically significant, and was not supported by a linear trend, the risk in heavy smokers being less than 1. Rural black patients, who have a higher incidence of hepatocellular carcinoma than urban black patients, smoked less than their urban counterparts. We conclude that smoking is not an unqualified risk factor for hepatocellular carcinoma in southern African black patients. There may, however, be a trend toward smoking playing an etiologic role in patients without hepatitis B virus infection, especially in older patients. PMID- 2996753 TI - Flow cytometric DNA and 5'-nucleotide phosphodiesterase in endometrium. AB - One hundred endometrium specimens have been studied with flow cytometry for DNA analysis (FCDA) and a proliferative enzyme marker, 5'-nucleotide phosphodiesterase (5'-NPD). FCDA data showed that aneuploidy was present in only 5 of 40 cancer specimens. However, with corrected histograms, a higher DNA value was observed in the G2/M (6%) of all cancer compared with noncancer specimens (4%). Thus, FCDA can be a useful diagnostic aid for endometrial cancer. The determination of 5'-NPD was done with a quenching method based on the use of 5' (5-iodo-3-indoxyl)-thymidine phosphodiester as a substrate and 4',6-diamidino-2 phenylindole for DNA. This method could qualitatively define which population of the cell cycle had a higher enzyme level and also quantitatively gave the enzyme units per cell. It was found that 12.5% of all cancer specimens had 5'-NPD activity in the G0/G1 cells and 87.5% in the S and/or G2/M cells, whereas in the noncancer specimens 5'-NPD was found in 28.5% of the G0/G1 cells and 71.5% of the specimens had 5'-NPD in the S and/or G2/M cells. Furthermore, the concentration of 5'-NPD was found to be five times higher in the G2/M cells of the cancer specimens than that in the noncancer specimens. However, in the hyperplasia specimens, the activity was only two times higher in the same cell cycle fraction than in the normal specimens. The results of this investigation provided for the first time evidence that this exonuclease activity alters in the cell cycle fractions and that a decrease in the enzyme activity in G0/G1 cells and an increase in G2/M cells may be a useful marker for neoplastic development in human endometrial cancer. PMID- 2996754 TI - A human c-src gene resides on the proximal long arm of chromosome 20 (cen--- q131). AB - A molecular clone of v-src, the oncogene of Rous sarcoma virus, was used to detect and regionally localize a human c-src proto-oncogene on chromosome #20. The human c-src gene, detected as either a 28-kbp EcoRI DNA fragment or as a 15.4 kbp BglII DNA fragment, was localized to 20 cen----q131 by filter hybridization analysis of DNAs from human-rodent somatic cell hybrids. The results indicate that c-src is on the same chromosome arm as aberrations associated with myeloproliferative disease, although the possible involvement of c-src in these aberrations is unknown. PMID- 2996755 TI - Epstein-Barr virus-inducing activity of Euphorbiaceae plants commonly grown in Cameroon. AB - Epstein-Barr virus (EBV) activating potency of 12 species of plants belonging to the Euphorbiaceae family, that are commonly grown in Cameroon, one of the endemic areas of Burkitt's lymphoma (BL), was investigated. The EBV-inducing activity was found in most of the plants tested and in the soil around the plants whose root extracts were active. These findings support the notion that such EBV-activating principles are one of the environmental co-factors causing BL. PMID- 2996757 TI - The biological role of oncogenes--insights from platelet-derived growth factor: Rhoads Memorial Award lecture. AB - No one in tumor biology can now be unaware of the overlap between growth factors and oncogenes. Many if not all oncogenes are now perceived as functional components of a mitogenic cascade which is normally controlled by growth factors. Some oncogenes function at the onset of this cascade by directing the synthesis of an automitogenic growth factor. Others function in the interior of the cascade by directing synthesis of a growth factor receptor or a structurally altered receptor derivative. Still other oncogenes appear to be mutated or rearranged homologues of genes the expression of which is normally induced by growth factors. Those of us working with platelet-derived growth factor (PDGF) take particular satisfaction in this new conceptual framework. It is within the molecular biology of PDGF that the overlap between growth factors and oncogenes is illustrated to its fullest and most tangible extent. An oncogene termed c-sis directs synthesis of a functional PDGF subunit. The PDGF receptor protein is in all probability encoded by a member of the src family of oncogenes. Formation of the PDGF:receptor complex stimulates expression of the c-myc and c-fos protooncogenes. My associates and I have devoted the past 10 years to the molecular biology of PDGF. Our studies on the control of the 3T3 cell cycle by PDGF contributed a pair of new jargon terms to the oncology literature- "competence" and "progression." We also had some input into the bottom end of the "oncogene hierarchy" displayed in Chart 1. The effort that we invested paid a pleasant dividend for me when, in the spring of 1984, the American Association for Cancer Research honored me with the Rhoads Memorial Award. What follows is an overview of the PDGF literature which is more anecdotal than comprehensive. My object is to show how the PDGF field moved from the level of whole animal biology, through biochemistry, down to molecular genetics in just 10 years time. PMID- 2996756 TI - Epstein-Barr virus activation by human semen principle: synergistic effect of culture fluids of bacteria isolated from patients with carcinoma of uterine cervix. AB - Epstein-Barr virus early antigens (EBV EA) were induced in a Raji cell system in order to assay the activity of the EA inducer in human semen. In semen from 53 Chinese, 45.3% induced EBV EA in Raji cells. Such positive semen and EBV-inducing positive culture fluids of bacteria isolated from the uterine cervix of patients with cervical carcinoma had a synergistic effect on the induction of EBV EA. This synergistic effect as related to the cause of cervical carcinoma is discussed. PMID- 2996758 TI - Modulation of cytotoxicity of menadione sodium bisulfite versus leukemia L1210 by the acid-soluble thiol pool. AB - We investigated the mechanism of antitumor activity of the water-soluble derivative of menadione, menadione sodium bisulfite (vitamin K3), versus murine leukemia L1210. Vitamin K3, in concentrations greater than 27 microM, caused time and concentration-dependent depletion of the acid-soluble thiol (GSH) pool. Maximal GSH depletion to 15% of control occurred at 45 microM vitamin K3. Vitamin K3-mediated GSH depletion and vitamin K3-mediated growth inhibition were abrogated by coincubation with 1 mM cysteine or 1 mM reduced glutathione but not by 1 mM ascorbic acid or 180 microM alpha-tocopherol. Low concentrations of vitamin K3 (9-27 microM) elevated both the GSH pool and the total glutathione pool, the latter to a greater degree. Vitamin K3 also caused an increased rate of superoxide anion generation by L1210, maximal at 45 microM vitamin K3 (300% of control), and a concentration-dependent depletion of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) and total nicotinamide adenine dinucleotide phosphate (NADP) pools. Forty-fifty % depletion of the NADPH pool occurred after exposure to 27 microM vitamin K3 and 100% occurred at 36 microM vitamin K3; 27 microM vitamin K3 is a nontoxic concentration of vitamin K3. Loss of NADPH and total NADP was prevented by coincubation with 1 mM cysteine but not by coincubation with ascorbic acid or alpha-tocopherol. We conclude that tumor cell growth inhibition by vitamin K3 is modulated by acid-soluble thiols and may be caused by GSH pool and/or NADPH depletion. Toleration of partial NADPH depletion by L1210 cells may indicate that a threshold level of NADPH loss of greater than 50% is necessary for toxicity. NADPH depletion may be a toxic effect common to quinone drugs. Equitoxic concentrations of vitamin K3, phylloquinone, lapachol, dichlorolapachol, and doxorubicin caused L1210 NADPH pools to deplete to 30 +/- 10 (SD), 60 +/- 10, 60 +/- 11, and 80 +/- 12% of control, respectively. In contrast, GSH depletion may not be a common mechanism of toxicity. Of these quinones, only vitamin K3 caused significant GSH depletion when studied in equitoxic concentrations. PMID- 2996759 TI - Activation of the parathyroid hormone receptor-adenylate cyclase system in osteosarcoma cells by a human renal carcinoma factor. AB - Human renal carcinoma cell line 786-0 elaborates a protein that is structurally and immunochemically distinct from parathyroid hormone (PTH) and that activates renal cortical adenylate cyclase via an interaction with the PTH receptor. Because of the high frequency of excessive bone resorption and resultant hypercalcemia in patients with malignant disease we evaluated the ability of this 786-0 cell factor to reproduce PTH action in bone-derived cells. The 786-0 factor as well as bovine PTH (BPTH) (1-34) and prostaglandin E1 produced marked increases in cyclic adenosine 3':5'-monophosphate (cAMP) accumulation in the clonal rat osteosarcoma cell line UMR-106. A competitive antagonist of PTH action, [norleucine8, norleucine18, tyrosine34] BPTH(3-34)amide, blocked the cAMP stimulation produced by 786-0 factor and BPTH(1-34) but not that produced by prostaglandin E1. In the presence of forskolin (0.1 microM) UMR-106 cells were extremely sensitive to 786-0 factor, showing significant increases in cAMP production at a concentration 10-fold less than that required to activate adenylate cyclase in renal membranes. In contrast UMR-106 cells were less sensitive to BPTH(1-34) than were renal membranes. This preferential increase in sensitivity to 786-0 factor was not seen in membranes prepared from UMR-106 cells suggesting the importance of cytosolic components. Six additional human genitourinary carcinoma cell lines were found to produce factors that increased cAMP levels in UMR-106 cells. We conclude that 786-0 factor is a potent activator of the PTH receptor-adenylate cyclase system in these bone-derived cells. These findings are consistent with the view that cancer-associated hypercalcemia may frequently be attributable to tumor secretion of proteins (such as 786-0 factor) that are distinct from PTH but are capable of activating skeletal PTH receptors. PMID- 2996760 TI - Cytotoxicity of human beta-interferon for differentiating leukemic HL-60 cells. AB - We examined the effect of human interferon (HuIFN) -alpha and -beta on the proliferation and differentiation induced by dimethyl sulfoxide (DMSO) of HL-60 human promyelocytic leukemia cells into mature granulocytes. Neither HuIFN-alpha nor -beta, alone, from 1 to 1000 IU/ml, nor the homologous mock HuIFN preparations affected HL-60 cell differentiation or proliferation. Whereas the combination of HuIFN-alpha (10 to 1000 IU/ml) with DMSO also did not affect the proliferation or differentiation of HL-60 cells, the addition of HuIFN-beta (1000 IU/ml) and DMSO (1.25%) to growing cultures reduced cell viability as much as 14% of that observed for cells treated with DMSO alone or to 4% of that observed for either untreated cells or those treated with HuIFN-beta alone. The cytotoxic effect declined with decreasing concentrations of HuIFN-beta. The cytotoxic effect of DMSO and HuIFN-beta was exerted only as cells began to differentiate. Removal of HuIFN-beta at Day 2 did not reverse the cytotoxic effect, and addition of HuIFN-beta at Day 2 did not inhibit cell proliferation. Addition of HuIFN-beta to postmitotic cells on Day 4 after DMSO treatment did not affect proliferation but did slow differentiation. The cytotoxic and antidifferentiative effects of naturally produced HuIFN-beta were confirmed with highly purified recombinant HuIFN-beta. Undifferentiated HL-60 cells were resistant to the antiviral effects of HuIFN-beta, requiring 4 to 6 times the concentration to protect 50% of the cells against vesicular stomatitis virus as that needed to produce a cytotoxic or antidifferentiative effect. The profoundly cytotoxic effects of HuIFN-beta reported here may provide a model to study this interferon in combination with inducers of leukemic cell differentiation as a possible strategy in cancer therapy. PMID- 2996761 TI - Differential expression of transforming growth factor-alpha during prenatal development of the mouse. AB - Transforming growth factors (TGFs) are differentially expressed in the mouse during neonatal development. Highest levels are seen early at Day 7, and lower levels, at Day 13. Both small- and large-molecular-weight forms of TGF are found; they share many biochemical properties with rat TGF-alpha, including a similar high-pressure liquid chromatography elution profile. Although the embryo-derived activities compete with epidermal growth factor for binding to epidermal growth factor membrane receptors, they are immunologically distinct from epidermal growth factor. These embryonic polypeptides, however, do cross-react in a competitive radioimmunoassay developed using a synthetic peptide corresponding to the carboxy-17 amino acids of rat TGF-alpha as the immunogen. The highly conserved TGF-alpha family of peptides produced by some tumor cells may therefore represent derepressed forms of these embryonic growth factors. A functional role in neonatal development is proposed. PMID- 2996762 TI - Effect of retinoic acid on cellular content and human parathyroid hormone activation of cyclic adenosine 3':5'-monophosphate-dependent protein kinase isoenzymes in clonal rat osteogenic sarcoma cells. AB - Pretreatment with 10(-8) M retinoic acid for 4 days caused changes in three distinct components of the parathyroid hormone (PTH)-stimulated cyclic adenosine 3':5'-monophosphate response in a clonal rat osteogenic sarcoma cell line, UMR 106-06: the amplitude of the cyclic adenosine 3':5'-monophosphate response to PTH was moderately increased after pretreatment with retinoic acid; while the cellular content of the two isoenzymes of the cyclic adenosine 3':5' monophosphate-dependent protein kinase was approximately equal in control cells, retinoic acid pretreatment was associated with a marked increase in the ratio of type II to type I holoenzyme activity. This change might be due to a decrease in the type I holoenzyme as suggested by immunofluorescence detection of decreased type I regulatory subunit in fixed cells together with the relative decrease in type I holoenzyme determined biochemically; there was a marked alteration of the pattern of PTH-stimulated protein kinase isoenzyme activation from predominantly type I isoenzyme in control cells to almost exclusively type II isoenzyme in retinoic acid-treated cells. Growth inhibition by submaximal amounts of PTH and retinoic acid when added together was greater than that for either agent alone. PMID- 2996763 TI - Identification of DNA topoisomerase II as an intracellular target of antitumor epipodophyllotoxins in simian virus 40-infected monkey cells. AB - The effect of antitumor epipodophyllotoxins, etoposide (VP-16) and teniposide (VM 26), on chromosomal DNA in mammalian cells was studied using SV40 virus-infected monkey cells as a model system. Treatment of SV40 virus-infected monkey cells with these drugs results in DNA breaks on intracellular SV40 DNA. The broken DNA strands are sensitive to phenol extraction, suggesting that they are associated with tightly linked protein(s). Several pieces of evidence suggest that DNA topoisomerase II is covalently linked to the broken SV40 DNA strands following drug treatment. ovobiocin, an inhibitor of topoisomerase II, blocks the epipodophyllotoxin-induced SV40 DNA breaks in vivo and in vitro. Epipodophyllotoxin-induced cleavage sites on intracellular SV40 DNA are strikingly similar to those produced on purified SV40 DNA by purified calf thymus DNA topoisomerase II. The protein-linked SV40 DNA is specifically immunoprecipitated by antisera against topoisomerase II. We thus conclude that epipodophyllotoxins induce chromosomal DNA breakage via DNA topoisomerase II. The physiological effects of epipodophyllotoxins on cell death, chromosomal DNA breakage, sister chromatid exchanges, and chromosomal aberrations may be the consequence of drug interaction with DNA topoisomerase II. Our present results are also consistent with the proposal that epipodophyllotoxins interfere with the breakage-reunion reaction of DNA topoisomerase II by stabilizing an enzyme-DNA complex in its putative cleavable state. PMID- 2996765 TI - Multistage neoplastic transformation of normal and preneoplastic Syrian hamster embryo cells by viral oncogenes. PMID- 2996764 TI - Genetic targets in multistage carcinogenesis. PMID- 2996766 TI - Human epithelial cell carcinogenesis: combined action of DNA and RNA tumor viruses produces malignant transformation of primary human epidermal keratinocytes. PMID- 2996767 TI - Direct hypotensive action of intravascular bradykinin in man. AB - To investigate the cardiovascular effects of bradykinin (BK), the peptide was injected into 6 normotensive volunteers in the supine position. BK was given intravenously as a bolus in a dose of 0.001-7.5 microgram BK/kg body weight. Intraarterial blood pressure decreased dose related in a range of 0.25-1.0 microgram BK/kg body weight. Pretreatment with 2 X 50 mg indomethacin or 80 mg propranolol as well as changes in oral salt intake (from 10 to 300 mmol Na+/day) had no effect on the blood pressure-lowering effect of BK. Captopril (25 mg) potentiated the effect of BK 20- to 50-fold. In primary hyperaldosteronism, renal kallikrein activity and absolute vascular reaction to BK was increased. The results showed clearly that in man, similarly as in rats, BK lowers blood pressure by direct vasodilation and acts independently of prostaglandins, beta receptors or salt intake. PMID- 2996769 TI - Na-K pump activity in erythrocytes of patients with endogenous and exogenous glucocorticoid excess. AB - The mechanism of glucocorticoid-induced hypertension is not clarified. Recent data suggest an alteration of active electrolyte transport systems by glucocorticoids. We therefore studied the activities of the Na-K pump by measuring the Na-K-ATPase activity in erythrocyte ghosts and the ouabain sensitive uptake of rubidium in erythrocytes of patients with Cushing's syndrome and of patients with exogenous glucocorticoid excess after treatment with fluocortolone or ACTH. Na-K-ATPase activity was significantly increased in patients with Cushing's syndrome and in patients treated with ACTH compared to the controls. Similarly, total uptake of 86Rb and the ouabain-sensitive uptake in erythrocytes were found to be above the control range both in patients with Cushing's syndrome and in patients treated with fluocortolone. There was no difference in furosemide-sensitive uptake of 86Rb. The results demonstrate an increased maximal activity of the Na-K pump in patients with glucocorticoid excess. PMID- 2996768 TI - Adrenal scintigraphy in the morphological and functional evaluation of Cushing's syndrome. AB - In recent years, cholesterol adrenal scintigraphy has been employed in the morphofunctional study of adrenal hypercorticism. Particularly in Cushing's syndrome, this noninvasive procedure can give a determinant contribution to distinguish ACTH-dependent from ACTH-independent forms. In our Institute, adrenal scintigraphy was performed in 77 patients with Cushing's syndrome diagnosed on clinical grounds confirmed by laboratory investigations and functional tests (17 with cortisol-secreting adenoma, 54 with pituitary ACTH-dependent bilateral adrenal hyperplasia, 2 with ectopic ACTH-dependent bilateral hyperplasia and 4 with bilateral nodular hyperplasia). Three distinct scintigraphic patterns have been identified. The 56 patients with ACTH-dependent Cushing's syndrome showed bilateral symmetric or slightly asymmetric visualization of the adrenal glands; in the 17 patients with ACTH-independent Cushing's syndrome the adrenal scintigraphy only visualized the gland harboring the adenoma; finally, a marked asymmetric visualization of the glands was observed in the 4 patients with adrenal nodular hyperplasia. These data confirm that adrenal scintigraphy is able to distinguish between ACTH-dependent and ACTH-independent Cushing's syndrome and reliably lateralizes adenomas when they are present. Moreover, the morphofunctional information obtained by this procedure, together with the high resolution morphological data by computerized tomography, allows to recognize the presence of bilateral nodular hyperplasia, an uncommon cause of Cushing's syndrome. PMID- 2996771 TI - Beta-adrenoceptor density and relative number of beta-adrenoceptor subtypes in biopsies from human right atrial, left ventricular, and right ventricular myocard. AB - In the present study we estimated the relative number of beta-adrenoceptors in membrane preparations from right atrial and left ventricular biopsies from 8 patients, as well as from right ventricular biopsy from one patient. Determinations of relative number of beta adrenoceptor subtypes (beta1, beta2) were also performed. Estimations were performed in a radioligand assay, measuring specific binding of [125I]-cyanopindolol (CYP). Inhibition of specific [125I] CYP binding was studied by adding propranolol (non-selective antagonist), atenolol (beta1 selective antagonist) and ICI 118551 (beta2 selective antagonist) to the preparations. Some individual variance in total number of receptors was found. Per mg protein, the number of binding sites were higher in atrial preparations (mean 71.4 +/- 14.4 fmol X mg-1) than in left ventricular preparations (mean 30.2 +/- 7.1 fmol X mg-1). Receptor number in the right ventricular preparation was 49.5 +/- 2.6 fmol X mg-1. Approximately 25% of beta adrenoceptors were of beta2 subtype in preparations from both right atrial and left ventricular biopsies, as well as from the right ventricular biopsy. Thus, there are regional differences in the quantity of beta adrenoceptors in human myocard whereas the relative proportion of beta1 and beta2 receptors appears to be fairly constant. PMID- 2996770 TI - Mineralocorticoid and glucocorticoid receptors in circulating mononuclear leukocytes of patients with primary hyperaldosteronism. AB - Mineralocorticoid and glucocorticoid receptors were measured in circulating mononuclear leukocytes in 5 patients affected by Conn's syndrome (3 cases of bilateral adrenal hyperplasia and 2 cases of adenoma plus unilateral hyperplasia). The number of the binding sites per cell resulted significantly lower (189 +/- 114, mean +/- SD), as compared with the normal controls (298 +/- 105). The affinity of aldosterone for the receptor was found to be not different than that of healthy control subjects. The capacity and the affinity of dexamethasone for glucocorticoid receptors ranged in the normal values. These data suggest a possible down-regulation of mineralocorticoid receptors in humans. PMID- 2996772 TI - [Excretion of cyclic adenosine 3',5'-monophosphate in patients with type I diabetes mellitus]. PMID- 2996773 TI - Assembly and propagation of repressed and depressed chromosomal states. PMID- 2996774 TI - Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for pre-mRNA splicing in vitro. AB - We have identified six distinct factors necessary for pre-mRNA splicing in vitro by selective inactivation and complementation studies, and by fractionation procedures. Splicing factor 1 (SF1) is sensitive to micrococcal nuclease, and appears to consist of at least U1 and U2 snRNPs, since splicing is inhibited when the 5' termini of U1 and U2 snRNAs are removed by site-directed cleavage with RNAase H. SF2 is a micrococcal nuclease-resistant factor present in the nuclear extract but absent from an S100 extract. SF3 is a factor that can be preferentially inactivated by moderate heat treatment. Two additional factors (SF4A and SF4B) were identified by fractionation of the nuclear extract using spermine-agarose and CM-sepharose chromatography. SF1, SF2, and SF4B appear to be required for cleavage of the pre-mRNA at the 5' splice site and lariat formation, whereas SF3 and SF4A are only required for cleavage at the 3' splice site and exon ligation. PMID- 2996775 TI - U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing. AB - Two different experimental approaches have provided evidence that both U2 and U1 snRNPs function in pre-mRNA splicing. When the U2 snRNPs in a nuclear extract are selectively degraded using ribonuclease H and either of two deoxyoligonucleotides complementary to U2 RNA, splicing activity is abolished. Mixing an extract in which U2 has been degraded with one in which U1 has been degraded recovers activity. Use of anti-(U2)RNP autoantibodies demonstrates that U2 snRNPs associate with the precursor RNA during in vitro splicing. At 60 min, but not at 0 min, into the reaction intron fragments that include the branch-point sequence are immunoprecipitated by anti-(U2)RNP. At all times, U1 snRNPs bind the 5' splice site of the pre-mRNA. Possible interactions of the U2 snRNP with the U1 snRNP and with the pre-mRNA during splicing are considered. PMID- 2996776 TI - Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones: a model system for study of higher order structure. AB - We describe a model system for study of chromatin structure at levels above that of the nucleosome. A series of fragments with lengths ranging from 172 to 207 bp tandemly repeated three to greater than 50 times was prepared; each repeat contains the region important in forming a positioned core particle on a sea urchin 5S rRNA gene upon in vitro association with histones. The tandemly repeated sequences can be studied as linear DNA fragments or as relaxed or supercoiled circular molecules. A number of criteria indicate that nucleosomes position correctly on all the tandemly repeated elements. Measurement of the change in linking number per core particle led to a value of -1.0. Both length and repeat number dependent changes in conformation of the nucleoproteins are observed. We discuss the possibility that some ordered higher level chromatin structure can form with DNA and core histones alone. PMID- 2996777 TI - Recombination within the human embryonic xi-globin locus: a common xi-xi chromosome produced by gene conversion of the psi xi gene. AB - The duplicated human embryonic alpha-like globin genes encode a 5' functional zeta (xi 2) gene and a highly homologous pseudogene (psi xi 1). We have identified chromosomes with a xi 2-xi 1 rather than a xi 2-psi xi 1 arrangement by genomic mapping and oligonucleotide analysis. The DNA sequence of a cloned downstream xi-like gene provides direct evidence for the conversion of a psi xi 1 ---xi 1 gene, by a xi 2 gene. We present data suggesting that this gene conversion, which removed the only identifiable inactivating mutation in the psi xi 1 gene, was an interchromosomal event. The xi 2-xi 1 arrangement is common in all eight populations studied representing a previously undescribed type of polymorphism between individuals. Stable mRNA transcripts from the converted gene are absent at 16-20 weeks of gestation when transcripts from the xi 2 gene are readily detectable. PMID- 2996778 TI - The nucleotide sequence and the tissue-specific expression of Drosophila c-src. AB - We have examined the coding capability and expression of the Drosophila homolog of the vertebrate proto-oncogene c-src. Sequence analysis of a cDNA clone representing the Drosophila c-src locus suggests that the gene encodes a 62 kd protein that is remarkably similar to the protein product of chicken c-src. The Drosophila c-src locus is transcribed into three mRNAs that are each regulated independently during development. Drosophila c-src RNA is abundant in embryos and pupae but rare in larvae and adults. In situ hybridization reveals that after the first 8 hr of development, c-src RNA accumulates almost exclusively in neural tissues such as the brain, ventral nerve chord, and eye-antennal discs, and in differentiating smooth muscle. We conclude that c-src may not be a mitotic signal but instead may play a role in the development of neural tissue and smooth muscle. PMID- 2996779 TI - Microinjection of the ras oncogene protein into PC12 cells induces morphological differentiation. AB - To investigate the possible role of ras proteins in the differentiation process signaled by nerve growth factor, we have microinjected the proto-oncogenic and oncogenic (T24) forms of the human H-ras protein into living rat pheochromocytoma cells (PC12). PC12 cells, which have the phenotype of replicating chromaffin-like cells under normal growth conditions, respond to nerve growth factor by differentiating into nonreplicating sympathetic neuron-like cells. Microinjection of the ras oncogene protein promoted the morphological differentiation of PC12 cells into neuron-like cells. In contrast, microinjection of similar amounts of the proto-oncogene form of the ras protein had no apparent effect on PC12 cells. The induction of morphological differentiation by the ras oncogene protein occurred in the absence of nerve growth factor, was dependent on protein synthesis, and was accompanied by cessation of cell division. Treatment of PC12 cells with nerve growth factor or cAMP analogue prior to injection did not alter the phenotypic changes induced by the ras oncogene protein. PMID- 2996781 TI - Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region. AB - Latently infected B lymphocytes continuously express an Epstein-Barr Virus nuclear antigen (EBNA-1) required in trans for maintenance of the plasmid state of the EBV genome. Filter binding assays and DNAase I footprinting analyses revealed that the carboxy-terminal domain of EBNA-1 protects binding sites at three different loci in the 172,000 bp EBV genome. Two of these loci correspond to essential elements within an 1800 bp segment defined as the minimal region required for plasmid maintenance (ori-P). Binding to each of 20 X 30 bp tandem repeats in the "sink" locus protects 25 bp centered over a 12 bp palindromic consensus sequence TAGCATATGCTA. The nearby dyad symmetry "origin" locus contains two 46 bp protected regions each encompassing two paired core binding sites. The demonstration of sequence-specific binding at multiple loci suggests that EBNA-1 has pleiotropic functions, which may include control of copy number and segregation of the EBV plasmids as well as initiation of replication. PMID- 2996780 TI - Protein kinase C phosphorylates pp60src at a novel site. AB - The transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homolog (pp60c-src) are demonstrated to be phosphorylated at serine 12 in vivo under certain conditions. We propose that protein kinase C is responsible for this modification based on the following evidence. First, the tumor promoters, 12-O-tetradecanoylphorbol-13-acetate and teleocidin, and synthetic diacylglycerol, known activators of protein kinase C in vivo, cause nearly complete phosphorylation of pp60src at serine 12. Second, among five purified serine/threonine-specific protein kinases tested, only protein kinase C phosphorylates pp60c-src and pp60v-src in vitro at serine 12. Third, purified protein kinase C phosphorylates a synthetic peptide corresponding to the N terminal 20 amino acids of pp60c-src at serine 12. The physiological significance of this novel phosphorylation is discussed. PMID- 2996782 TI - Molecular organization of the cut locus of Drosophila melanogaster. AB - Mutations of the cut locus (ct) of Drosophila can be divided into four groups based on their phenotypes and complementation patterns. Each group alters the phenotype of a different set of tissues. Two hundred kilobases of ct DNA, located in 7B1-2, have been cloned by chromosomal walking, and the cloned sequences have been used to analyze more than 40 mutants. Based on the location of transposable element mutations and the extent of deficiencies and an inversion, four cut locus regions can be defined. Mutations in each region affect the phenotype of a different set of tissues. The most centromere proximal region contains mutations that are null for cut locus function. Within individual regions, a higher level of organization can be detected. PMID- 2996784 TI - Overproduction of FtsZ induces minicell formation in E. coli. AB - The ftsZ gene in E. coli K-12 is an essential cell division gene. We report that a two to sevenfold increase in the level of the FtsZ protein resulted in induction of the minicell phenotype. An increase in the level of FtsZ beyond this range resulted in an inhibition of all cell division. Unlike the classical minicell mutant, the formation of minicells induced by increased levels of FtsZ did not occur at the expense of normal divisions, indicating that increasing FtsZ resulted in additional division events per cell cycle. In addition, increased FtsZ caused cell division to be initiated earlier in the cell cycle. These results are consistent with the level or activity of FtsZ controlling the frequency of cell division in E. coli. PMID- 2996783 TI - Functional selection and analysis of yeast centromeric DNA. AB - A direct selection procedure has been used to isolate 11 distinct yeast genomic DNA fragments that eliminate the extreme segregation bias characteristic of autonomously replicating yeast plasmids. The selection scheme takes advantage of the fact that the cloned ochre suppressing tRNA gene, SUP11, is lethal at high copy number and therefore causes cell death when present on an ARS plasmid that lacks a cis-acting partition function. Each of the cloned DNA sequences was mapped to specific yeast chromosomes by hybridization to chromosome-sized DNA molecules separated by alternating field electrophoresis. Ten of the cloned fragments correspond to chromosomal centromeres; one fragment corresponds to the cis-acting locus required for endogenous 2 mu plasmid stability. Nucleotide sequence comparison of the ten centromere DNAs gives a new picture of conserved centromere DNA elements. PMID- 2996785 TI - In vitro replication of human mitochondrial DNA: accurate initiation at the origin of light-strand synthesis. AB - Synthesis of human light-strand mitochondrial DNA was accomplished in vitro using DNA primase, DNA polymerase, and other accessory proteins isolated from human mitochondria. Replication begins with the synthesis of primer RNA on a T-rich sequence in the origin stem-loop structure of the template DNA and absolutely requires ATP. A transition from RNA synthesis to DNA synthesis occurs near the base of the stem-loop structure and a potential recognition site for signaling that transition has been identified. The start sites of the in vitro products were mapped at the nucleotide level and were found to be in excellent agreement with those of in vivo nascent light-strand DNA. Isolated human mitochondrial enzymes recognize and utilize the bovine, but not the mouse, origin of light strand replication. PMID- 2996786 TI - The effect of prostaglandins on the cyclic AMP content of limb mesenchymal cells. AB - We have been investigating the hypothesis that prostaglandins including prostaglandin E2 (PGE2) produced during the critical condensation phase of limb chondrogenesis are involved in the regulation of cartilage differentiation by acting as local modulators of cyclic AMP (cAMP) accumulation. The purpose of the present study was to determine directly whether PGE2 and other prostanoids which had previously been shown to stimulate in vitro chondrogenic differentiation do indeed elevate the cAMP content of limb mesenchymal cells, and to determine whether the ability of various prostanoids to increase cAMP production by these cells directly reflects the potencies of these same molecules in stimulating chondrogenesis. We have found that PGE2 does indeed elicit a striking elevation in the cAMP content of subridge mesenchymal cells, indicating that the cells possess adenylate cyclase-coupled receptors for this molecule. The effect of PGE2 on cAMP accumulation is potentiated by a phosphodiesterase inhibitor, thus paralleling the potentiating effect phosphodiesterase inhibitors have on PGE2 stimulated in vitro chondrogenesis. The effect of PGE2 on cAMP content is dose dependent with a 3-fold increase seen at 10(-8)M, which is the lowest concentration at which PGE2 effectively stimulates chondrogenesis. PGE1, which is just as effective as PGE2 in stimulating chondrogenesis, is just as effective as PGE2 in stimulating cAMP accumulation. PGA1, which is a much less effective stimulator of chondrogenesis than PGE2 or PGE1, is less than half as potent as these molecules in elevating cAMP levels. PGF1 alpha, 6-keto PGF1 alpha, and thromboxane B2, which have little or no effect on chondrogenesis, have little or no effect on cAMP content.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996788 TI - [Monitoring the possibilities of the importation of virus diseases. Virologic serologic study]. PMID- 2996787 TI - Modulation of oocyte maturation by cyclic adenosine 3', 5'-pyrophosphate. AB - The germinal vesicle (GV) of follicle-enclosed oocytes in mammals remains arrested at the dictyate state of meiosis. Upon releasing the oocytes from the follicles, the meiotic process resumes, leading to dissolution of the GV (GVBD), suggesting that factors in the follicular fluid sustain the meiotic arrest of oocytes. In the present study the spontaneous resumption of meiosis was blocked by the addition of cyclic adenosine 3', 5'-pyrophosphate (cAPP) plus dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP), at final concentrations of 25 and 50 microM, respectively. These compounds were ineffective when added separately at these concentrations. None of the other related compounds tested with dbcAMP blocked GVBD. Bovine follicular fluid (BFF) was analyzed for inhibitors of GVBD. BFF was extracted with 70% ethanol and the ethanolic extract chromatographed on Dowex 1-X8 column. The fraction eluted with 0.1 N HCl markedly inhibited GVBD of isolated mouse oocytes in combination with dbcAMP. The active BFF substance and cAPP block spontaneous GVBD of mouse oocytes and may be related substances. The present study supports the thesis that meiotic arrest at the dictyate stage in oocytes is sustained by factors present in follicular fluid and may act in association with cAMP. PMID- 2996789 TI - [Cytomegaloviruses in sperm and the risk of sexual transmission]. PMID- 2996790 TI - The lipid peroxidation model for halogenated hydrocarbon toxicity. Kinetics of peroxyl radical processes involving fatty acids and Fe(III) porphyrins. AB - The toxicity of halogenated alkanes originates from their metabolism by cytochrome P-450 which leads to the formation of reactive intermediates. In particular, peroxyl radicals derived from the halogenated compounds are believed to induce peroxidative chain degradation of lipids. To examine this hypothesis, radical reactions in a system involving FeIII-deuteroporphyrin as a model of cytochrome P-450, fatty acids or cholesterol, and carbon tetrachloride or the anesthetic agent halothane are studied by means of pulse radiolysis. It is shown that haloperoxyl radicals react with the fatty acids in competition with their reaction with the ferriporphyrin. Moreover, the secondary fatty acid peroxyl radicals also react efficiently with the porphyrin. A model for halogenated alkane toxicity is discussed in terms of these new findings. The importance of local oxygen concentration and structural arrangement of fatty acids around cytochrome P-450 are emphasized. PMID- 2996792 TI - 9-Anilinoacridines: novel compounds active against herpes simplex virus. PMID- 2996791 TI - The induction of cytochrome P-450 by isosafrole and related methylenedioxyphenyl compounds. AB - Using sucrose gradients, the Ah receptor and a 3-4S binding peak were measured in hepatic cytosol from Dub: ICR, C57BL/6, and DBA/2 male mice. Isosafrole, piperonyl butoxide, and 5-t-butyl-1,3-benzodioxole were unable to displace 2,3,7,8-tetrachlorodibenzo-p-dioxin or 3-methylcholanthrene from either the Ah receptor or the 3-4S binding peak, in vitro. In in vivo experiments, treatment of C57BL/6 mice with 3-methylcholanthrene caused a 4-fold reduction in Ah receptor binding 2 h after i.p. injection; whereas, isosafrole caused a 2-fold enhancement of the Ah receptor after 24 h. This increase in the Ah receptor binding following isosafrole treatment may be due to induction. 3-Methylcholanthrene treatment of C57BL/6 mice also caused a 3-fold reduction in the 3-4S binding peak 2 h after i.p. injection; isosafrole treatment had little or no effect on the 3-4S peak in C57BL/6 or DBA/2 mice. Both in vivo and in vitro data appear to demonstrate that there is no direct role for the Ah receptor or the 3-4S protein in the regulation of cytochrome P-450 by methylenedioxyphenyl compounds. Using Sephadex G-100 chromatography, a cytosolic protein fraction was obtained from C57BL/6 and Dub:ICR mice which was previously implicated by others as a carrier in the metabolism of benzo[a]pyrene (B[a]P). This fraction was applied to sucrose gradients and sedimented in the 3-4S region. Hence it appears that the 3-4S binding peak may be the carrier described by these workers. PMID- 2996793 TI - Studies on angiotensin converting enzyme inhibitors. III. 2-Carboxyethylcarbamoyl 1,2,3,4-tetrahydroisoquinoline- 3-carboxylic acid derivatives. PMID- 2996794 TI - [The treatment of epidemic pharyngoconjunctivitis fever by traditional Chinese medicine]. PMID- 2996795 TI - Phase behaviour of a diglyceride prodrug: spontaneous formation of unilamellar vesicles. AB - A prodrug (Fig. 1(IV)) is synthesized consisting of the beta-blocker bupranolol which is covalently linked to 1, 3-dipalmitoyl-2-succinyl-glycerol. The resulting lipid-like prodrug is amphipathic and surface active. It disperses readily in H2O above 30 degrees C forming a smectic lamellar phase. This prodrug bears one positive charge at neutral pH and hence the swelling behaviour of dispersions in H2O is similar to that of charged phospholipids: the dispersions show continuous swelling with increasing water content and consequently in the excess H2O region of the phase diagram the thermodynamically most stable structure is the unilamellar vesicle. This includes oligomeric vesicles which may be defined as unilamellar vesicles containing smaller, also unilamellar vesicles entrapped in their internal aqueous compartment. The prodrug dispersions in H2O are polydisperse with vesicle sizes ranging from 0.1 micron to several micron. Sonication of these dispersions produce small unilamellar vesicles of an average size and size distribution similar to sonicated egg phosphatidylcholine dispersions. Unsonicated dispersions of the prodrug in H2O undergo reversibly sharp order-disorder transitions at 32 degrees C with an enthalpy change of delta H = 10 kcal/mol. In sonicated aqueous dispersions this phase transition is asymmetric and significantly broadened indicating that the cooperativity is markedly reduced. The peak temperature and enthalpy change of this broad transition are reduced compared to the transition observed with unsonicated dispersions. The temperature dependence of the electron spin resonance (ESR) hyperfine splitting and order parameter also reflects the order-disorder transition. From ESR spin labeling it is concluded that in sonicated dispersions the prodrug molecule is more mobile and its anisotropy of motion is reduced compared to unsonicated dispersions. This result indicates that the molecular packing in the highly curved bilayers of small unilamellar prodrug vesicles is significantly perturbed compared to bilayers of unsonicated dispersions. PMID- 2996796 TI - [The story of oxygen. (3)]. AB - This last part of the oxygen story seems to stray from the line followed till now, and contradict itself, by electing ATP instead of oxygen as its protagonist. Yet, of course, this deviation is only apparent: in reality the hypoxanthine, which is at the basis of the production of superoxide, during the post-ischemic perfusion, is just a catabolic product of the intercellular reserves of ATP, pillaged by the ischemia itself. The attempts to resolve the ischemia and cellular anoxia through the supply, added to the volemic and respiratory reintegration, of performed packets of energy, such as ATP with MgCl2, FDP, etc., cannot be unimportant for traumatologist surgeon, accustomed to face the multiform physiopathologic facets of the shock. Although the flattering results tend to increase, some doubts remain about the effectiveness of such measures, especially ATP, and someone also suggests possible negative effects, partly framed in the well known and complex consequences of the "drug" on the cardiovascular dynamics, partly put forth by its not enough defined metabolic outline. Evidently, the way of the straight energetic supply, which, undoubtedly, represents the fulfillment of a "mirage" of release, at least partially, and in critical situations, from oxygen, although still long and burdened with problems, is also, decidedly, suggestive with promises. It seems surgeon, the true protagonist of such investigations, wants to seek a new dimension in them, being aware, and transplantations supplied an exemplary lesson in such sense, the biologic language is congenial to him, as it is high time. PMID- 2996797 TI - New frontiers in the treatment of lung cancer. AB - Surgical resection still is the only significant curative approach in nonsmall cell lung cancer. Recent surgical experience indicates that a modest decrease in the death rate from bronchogenic carcinoma may occur in three general areas: (1) the detection and treatment of radiographically occult squamous cell carcinoma; (2) the combination of adjuvant chemotherapy and surgical excision in selected patients with small cell carcinoma; and (3) surgical resection and postop irradiation of patients with hilar and mediastinal lymph node metastases. At the time of diagnosis, 80 to 85% of the patients present with unresectable lung cancer. These patients may benefit from other modalities of therapy, i.e., radiotherapy, chemotherapy, or immunotherapy. Failures following radiotherapy in unresectable nonsmall cell lung cancer are due to (1) distant metastasis, (2) local region failure, and (3) local and distant failure. To increase the local control, new methods of treatment have been tried, such as hyperfractionation of radiotherapy and the use of 131I antiferritin immunoglobulin. The development of effective systemic chemotherapy is necessary to treat metastatic bronchogenic carcinoma. The response rate to chemotherapeutic agents is substantially lower in nonsmall cell carcinoma than in small cell carcinoma. Investigation is ongoing to assess the effectiveness of new antitumor drugs used alone, in combination with other drugs, or combined with other modalities for the treatment of bronchogenic carcinoma. PMID- 2996798 TI - On the search for new anticancer drugs 14: the plasma pharmacokinetics and tissue distribution of spin-labeled thio-TEPA (SL-O-TT). AB - We defined the plasma and tissue concentrations and pharmacokinetics of SL-O-TT, a spin-labeled analog of thio-TEPA, in 35-44-g male Swiss Webster mice that had received spin-labeled thio-TEPA at a dosage of 10 mg/kg. Concentrations of spin labeled thio-TEPA in ethyl acetate extracts of tissue and plasma were determined by gas-liquid chromatography and electron spin resonance spectroscopy. Plasma concentrations of spin-labeled thio-TEPA declined in a biexponential fashion that was well described by the equation: Ct = 21.5e-0.276t + 2.30e-0.026t indicating a half-life alpha of 2.5 min and a half-life beta of 26.6 min. After 2 h there was still spin-labeled thio-TE-PA in plasma, but not in tissues. In tissues, no spin labeled thio-TEPA was detected with gas-liquid chromatography 15 min after injection, but with electron-spin resonance label was found in lung and skeletal muscle. The main metabolite of spin-labeled thio-TEPA is spin-labeled TEPA, where oxidative desulfurization is invoked as the main metabolic mechanism. Reduction of the spin label to the hydroxylamine was also observed with time. PMID- 2996800 TI - Differential renal tumor response to N-ethylnitrosourea and dimethylnitrosamine in the Nb rat: basis for a new rodent model of nephroblastoma. AB - Previous studies have indicated that the Nb rat has a higher predisposition to the spontaneous development of nephroblastoma than most other strains of laboratory rat. In order to determine whether this inherent susceptibility could be exploited as a model for Wilms' tumor, Nb rats were treated with various chemicals in regimens known to be associated with renal tumor induction in the young rat. Nb rats were either exposed in utero to various dose levels of N ethylnitrosourea (ENU) on day 18 +/- 1 of gestation, injected s.c. with one 25 mg/kg dose of dimethylnitrosamine (DMN) as neonates or dosed twice orally with 7,12-dimethylbenz[a]anthracene (DMBA) shortly after ovariectomy. Transplancental ENU at a dose of 60 mg/kg resulted in an average of 50% frequency of unequivocal nephroblastomas in both sexes with no renal mesenchymal tumors. In contrast, DMN administered to neonates produced a similar incidence of renal mesenchymal tumors but no nephroblastomas. DMBA in the ovariectomized female was not a successful strategy for inducing primary renal tumors in the Nb rat (0 nephroblastomas, 1 renal mesenchymal tumor in 16 effective survivors), although the kidney was sometimes the site for metastatic invasion by tumors originating at other locations. The induction of nephroblastomas and renal mesenchymal tumors by ENU and DMN in parallel experiments emphasized the many differences which establish them as distinct and unrelated tumor entities. The relatively high incidence of nephroblastoma in the Nb rat using transplacentally administered ENU appears to represent a suitable basis for developing a rodent model of human nephroblastoma or Wilms' tumor. PMID- 2996799 TI - High-dose cyclophosphamide and VP 16 as late dosage intensification therapy for small cell carcinoma of lung. AB - This study investigated the use of late dose intensification therapy (LDIT) with cyclophosphamide (180 mg/kg) and VP 16 (1 g/m2) plus autologous bone marrow rescue in 22 patients with small cell lung cancer (SCLC). These patients were selected from a group of 95 patients who received three courses of a five-drug induction regimen comprising cyclophosphamide (750-1000 mg/m2), adriamycin (40 mg/m2), VP 16 (100 mg/m2) for 3 days, methotrexate (50 mg/m2) and vincristine (2 mg) (CAVMO). There were 16 patients with limited disease, 8 of whom were in complete remission (CR) and 8 in partial remission (PR) after the induction therapy. The other 6 patients had extensive disease; 3 of these achieved CR and 3 PR after induction therapy. Of the 11 patients in PR, 5 responded to LDIT; 3 had a further PR, and 2 CR. Subsequent to LDIT radiotherapy 4000 cGy was given to the primary site in 10 of the 22 patients. Since the start of the study, 19 of the 22 patients have relapsed and died (median survival 11 months), while 3 remain alive and in remission at 11, 11, and 24 months. Comparison of the survival of patients receiving LDIT with that of an equivalent group (with respect to staging and response to induction chemotherapy) of patients who received induction chemotherapy alone showed no significant difference. In this study, LDIT following conventional induction therapy in patients with chemosensitive tumours did not improve survival. PMID- 2996801 TI - Nalbuphine's reversal of hypovolemic shock in the anesthetized rat. AB - Nalbuphine is an agonist-antagonist analgesic chemically related to the opiate agonist oxymorphone and to the opiate antagonist naloxone. Because naloxone has been shown to be beneficial in hemorrhagic shock, this study was undertaken to investigate the hemodynamic effects of nalbuphine in anesthetized rats. The animals were bled 1% of body weight, which caused a significant decrease in mean arterial pressure (MAP) and heart rate (HR). Results indicate an immediate increase in MAP and HR above posthemorrhage values in the rats treated with 1 and 5 mg/kg nalbuphine compared with no improvement for the rats in the saline treated group. The increases in MAP were sustained for periods up to 120 min postadministration of nalbuphine. Measurements of cardiac output and regional distribution of blood flow indicate that nalbuphine caused an increase in cardiac output, heart rate, stroke volume, and dP/dt with no change in total peripheral resistance. PMID- 2996802 TI - Hemodynamic effects of naloxone in early canine hypovolemic shock. AB - The contribution of endorphins (endogenous opiates) to the pathophysiology of shock was evaluated by the administration of naloxone (NAL) at different time intervals after inducing hypovolemia. Forty-four dogs were bled into a reservoir to a mean arterial pressure (MAP) of 40-45 mmHg and maintained at this pressure for 30, 45, or 60 minutes. The animals were then given either intravenous 0.9% NaCl (S) or NAL. Animals treated after 30 minutes of hypovolemia at a fixed MAP had similar hemodynamic responses to both NAL or S. After 45 minutes, the NAL treated animals showed significant improvement in MAP, cardiac output (CO), and survival (P less than 0.05) when compared with S-treated animals. Animals treated with NAL after 60 minutes showed little significant hemodynamic difference from animals treated with S but all S-treated animals died during the 60-minute treatment period. Plasma endorphinlike activity (PELA) rose with hypovolemia and then returned toward control levels in all NAL treated animals before reinfusion of shed blood while it remained elevated in S-treated animals until after reinfusion. PMID- 2996803 TI - Alpha 1- and alpha 2-adrenoreceptor relationships in SHRs during hypotension. AB - Previous studies have suggested that spontaneously hypertensive rats (SHRs) are less able to tolerate the cardiovascular stress of hemorrhagic hypotension than normotensive WKYs. The present studies were designed to determine whether or not this deficiency on the part of the SHRs is related to a genetically derived inappropriate balance between the alpha 1- and alpha 2-adrenoreceptors in the peripheral vasculature. To answer this question, SHRs were divided into untreated controls and experimental rats, which were pretreated with either the relatively specific alpha 1-antagonist prazosin or the alpha 2-antagonist yohimbine. In another series, the adrenal medullae were removed 2-3 days before hemorrhage. The data indicate that alpha 1-receptor blockade might offer some degree of protection to SHRs subjected to hemorrhagic hypotension, whereas inhibition of the alpha 2-adrenoreceptors proved detrimental. We conclude that the innervated alpha 1-adrenoreceptors play a more dominant role in SHR blood pressure regulation than the extrasynaptic alpha 2-adrenoreceptors but that these alpha 1 receptors are unable to maintain the compensatory vasoconstrictor response as effectively as the alpha 2-adrenoreceptors. PMID- 2996804 TI - The genesis of arrhythmias during myocardial ischemia. Dissociation between changes in cyclic adenosine monophosphate and electrical instability in the rat. AB - It has been proposed that increases in tissue cyclic adenosine monophosphate during ischemia may be responsible for the induction of arrhythmias that occur during the early minutes of ischemia. We have tested this hypothesis using the isolated perfused rat heart with coronary artery occlusion for 30 minutes. In control hearts, after a transient small rise, cyclic adenosine monophosphate content remained close to its preischemic value (3.0 +/- 0.1 nM/g dry weight) throughout the period of occlusion. Eight percent (1/12) of the hearts fibrillated. Ninety-two percent (11/12) of the hearts exhibited ventricular tachycardia, and the mean total number of premature ventricular complexes was 528 +/- 121. Inclusion of epinephrine (1.0 microM) in the perfusion fluid elevated cyclic adenosine monophosphate prior to coronary occlusion (to 10.7 +/- 0.6 nM/g dry weight) and also throughout the ischemic period. It also increased arrhythmias such that 83% (20/24) of hearts fibrillated, 100% exhibited ventricular tachycardia, and the mean number of premature ventricular complexes increased to 747 +/- 86. Inclusion of forskolin (0.2 microM), which stimulates adenyl cyclase independently of the beta-receptor, increased cyclic adenosine monophosphate content to a greater extent than epinephrine, to 14.1 +/- 0.9 nM/g dry weight before the onset of ischemia and to 8.2 +/- 0.4 nM/g dry weight after 30 minutes of ischemia. Despite the large increase in cyclic adenosine monophosphate, there was no increase in rhythm disturbances which were less than those seen in controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996807 TI - Copper dependent control of the enzymic and phagocyte induced degradation of some biopolymers, a possible link to systemic inflammation. AB - The role of copper during inflammation is unknown. An attempt was made to examine the reactivity of copper on the oxygen free radical induced depolymerization of hyaluronic acid and synovial fluid. Thionein-copper and CuSO4 at 2 mumol/l concentrations inhibited the degradation of this biopolymer successfully. Translation of the enzymically generated excited oxygen species onto a cellular level was performed. Activated PMN cells were used to decompose hyaluronic acid in the presence of CuSO4, Cu-thionein and ceruloplasmin not exceeding physiological levels. All employed copper compounds inhibited the depolymerizing process. Furthermore, PMN cell induced bleaching of cytochrome c was also affected in the presence of both CuSO4 and thionein-copper. PMID- 2996806 TI - Use of proton nuclear magnetic resonance spectroscopy in detection and study of organic acidurias. AB - We used high-resolution proton nuclear magnetic resonance spectroscopy to detect, identify, and study the major normal and abnormal organic acid metabolites in urine from patients with propionic acidemia, methylmalonic aciduria, branched chain ketoaciduria, isovaleric acidemia, and glutaric aciduria type I. Characteristic and diagnostic spectra were obtained at 400 MHz for each disorder in all the patients studied and neutral and basic compounds, including amino acids and acylcarnitines, were also detected. The technique is rapid (10 min) and requires small samples (0.5 mL) and no preliminary extraction or derivative preparation. We believe that it is particularly suitable for the rapid and acute diagnosis of inborn errors of metabolism, especially the organic acidurias, and for acute pediatric clinical care, when rapid monitoring of major metabolic alterations is required in a time scale suitable to influence directly and immediately the therapy of the patients concerned. PMID- 2996805 TI - Afferent renal nerve-dependent hypertension following acute renal artery stenosis in the conscious rat. AB - Anatomical and electrophysiological evidence indicates that the kidneys contain both mechano- and chemoreceptor nerve endings. We conducted the present study to determine whether conditions of reduced renal blood flow elicit cardiovascular alterations that are dependent on afferent renal nerves. Removal of the renin angiotensin system with the angiotensin I-converting enzyme inhibitor, captopril, and/or reduction in baroreflex gain by sinoaortic denervation, were combined in conscious rats with acute renal artery stenosis to prevent these systems from potentially obscuring any afferent renal nerve-dependent effects. One week after sinoaortic denervation or sham sinoaortic denervation, each rat was chronically instrumented with Doppler flow probes on the lower abdominal aorta and superior mesenteric and right renal arteries, as well as with intravascular catheters, and a perivascular balloon occluder on the right renal artery. After surgical recovery, sham sinoaortic-denervated animals responded to a 60-minute period of stenosis (50% reduction in renal blood flow) with increases in arterial pressure, regional resistance, and plasma renin activity. Captopril abolished the increases in arterial pressure, hindquarters, and left renal resistance, but both bradycardia and increased mesenteric resistance persisted, indicating that baroreflex activation might be buffering a non-renin-angiotensin system pro hypertensive mechanism. In support of this, sinoaortic-denervated animals during captopril administration responded to stenosis with substantial increases in arterial pressure (25-30 mm Hg) and regional resistance (30-50%) that were unrelated to the renin-angiotensin system, but which were abolished after denervation of the stenotic kidney. The data suggest that acute reductions in renal blood flow activate an afferent renal nerve-dependent cardiovascular response that is strongly expressed under conditions of reduced gain of the renin angiotensin and baroreflex systems. We speculate that this reflex may assume particular importance in chronic renal hypertension when baroreflexes become impaired and activation of the renin-angiotensin system is reduced. PMID- 2996808 TI - Unaltered stimulation of pituitary adrenocorticotrophin secretion by corticotrophin-releasing factor following sodium valproate administration in a patient with Nelson's syndrome. AB - A 53-year-old woman with Nelson's syndrome was treated with 600 mg sodium valproate daily. Her plasma ACTH was effectively decreased, whilst the response of plasma ACTH to corticotrophin-releasing factor (CRF) was unaltered. The result of the CRF test suggested that sodium valproate, at least in the present patient, acted at the hypothalamic level. PMID- 2996809 TI - Thyroid stimulation by (Fab)2 and Fab fragments of TSH receptor antibody. AB - The TSH receptor binding and thyroid stimulating properties of (Fab)2 and Fab fragments of Graves' IgG have been investigated. (Fab)2 fragments were prepared by pepsin digestion of IgG and Fab fragments by reduction of (Fab)2 or papain digestion of IgG. (Fab)2 and Fab were effective in inhibiting TSH binding to its receptor with all five patients' sera studied and both preparations stimulated cyclic AMP release from isolated thyroid cells. However Fab fragments were less active thyroid stimulators than their parent (Fab)2 in all five cases. These studies indicate that antibody divalency is not essential for thyroid stimulation by TSH receptor antibodies. PMID- 2996811 TI - Adenoid cystic carcinoma: the results of radical surgery. AB - A personal series of 267 salivary tumours seen in a 20-year period was reviewed. Thirty-six patients with a previously untreated histologically proven adenoid cystic carcinoma of the major and minor salivary glands were submitted to radical surgery. The 5-year survival was 73%; no patient died of disease or suffered a recurrence of disease beyond the 5-year mark, suggesting that radical surgery achieves good results and largely pre-empts the notorious pattern of repeated recurrence. Only 8% of the patients died solely of primary recurrence, suggesting that the place of supraradical surgery is likely to be very limited. PMID- 2996810 TI - Calcium calmodulin and hormone secretion. AB - As long ago as 1970, it was proposed that Ca2+ can act as a 'second messenger' like cAMP (Rasmussen & Nagata, 1979). The recognition that calmodulin is a major Ca2+ binding protein in non-muscle cells has prompted the suggestion that calmodulin may serve an analogous role for Ca2+ to that served by protein kinase for cAMP (Wang & Waisman, 1979), or at least to the regulatory subunit of the cyclic nucleotide-dependent kinases. It is becoming clear that calmodulin probably does play a role in stimulus secretion coupling in endocrine cells. Nevertheless, some of the experimental approaches which have led to this rather tentative conclusion do induce some doubts, as we have attempted to indicate. Many of the pharmacological agents used in the studies cited in this review are not specific in their interaction with calmodulin. For example, the phenothiazines also inhibit phospholipid-sensitive protein kinase. The introduction of more specific drugs, such as the naphthalene sulphonamides, may lead to a clearer picture of the role of calmodulin in hormone secretion. Relationships probably exist between cyclic nucleotides, calcium, calmodulin, phosphatidylinositol (PI) turnover and phospholipids in the overall control of the secretory process (see Fig. 1). There is considerable evidence that calcium is the primary internal signal initiating exocytosis of hormone from many glands. However, it appears that cyclic nucleotides can modulate the calcium signal either positively or negatively and it is possible that cAMP and calcium can separately activate secretion. The presence of both calmodulin-activated adenylate cyclase and cyclic nucleotide phosphodiesterase in the same tissue would appear to suggest either spatial or temporal control mechanisms or that (diagram; see text) the calcium requirement for calmodulin activation differs between the two enzymes. The true explanation is probably far more complex and involves perhaps as yet unknown factors that can differentially influence the activity of calmodulin itself in membranes and in cytosol. Berridge (1982) and Rasmussen (1980) give detailed accounts and review current hypotheses regarding relationships between the cyclic nucleotide and calcium second messenger systems. The various possible interrelationships of the putative messengers have been encompassed by the term 'Synarchic regulation' (Rasmussen, 1980). These concepts and the elucidation of the mechanisms by which cyclic AMP and calcium are involved in the control of secretion from particular cell types will make fascinating reading over the next few years.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2996812 TI - Plasma exchange in the treatment of acute systemic lupus erythematosus without circulating immune complexes. AB - One patient affected by systemic lupus erythematosus with recurrent phlebothrombosis and peripheral neuropathy is described. False positive VDRL test, anticardiolipin antibodies, and high titres of antinuclear antibodies were present. Circulating immune complexes were not found. The effect of plasma exchange on clinical symptoms and on serological abnormalities was rapid and striking. PMID- 2996813 TI - [A case of perineurioma showing right radial nerve palsy--special reference to histological findings of myelinated fiber degeneration due to perineurioma]. PMID- 2996814 TI - [Enzyme-linked immunoadsorbent assay (ELISA) of cerebrospinal fluid antibody in the diagnosis of herpes simplex encephalitis]. PMID- 2996815 TI - [A case report of Crow-Fukase syndrome with markedly retroperitoneal lymphadenopathy]. PMID- 2996816 TI - Long-term survival and recurrence-free interval in combined surgical, radio- and chemotherapy of malignant brain gliomas. AB - A prospective randomized study has been conducted since 1978 to judge the value of combined radio- and chemotherapy (CCNU) in the postoperative treatment of grade III and IV supratentorial gliomas. Inoperable or recurrent tumors were excluded from these series, but evaluated in addition. Increase in median survival time (18 months) for the entire series (62.2% glioblastomas) was only small compared to other series mentioned in literature, but there was a significant prolongation of the recurrence-free interval (16.5 months) and in the total survival time from the onset of symptoms in 46.4% of cases living more than 18 months. If 'useful recovery' is the major goal of present treatment of cancer, the recurrence-free interval and the fraction of long-term survivors may be more important statistical parameters than median survival time. To improve the 'quality of life' in a number of patients (so-called long-term survivors) and the length of recurrence-free intervals should be the therapeutic goal, even if overall survival time remains essentially unaffected. We conclude that combined therapy, including radio- and chemotherapy, following surgical resection is of value in most cases of malignant glioma. PMID- 2996817 TI - Eczema herpeticum of the child. An unusual manifestation of herpes simplex virus infection. AB - Two children aged 7 months with eczema herpeticum received treatment consisting of intravenous acyclovir and human plasma with a high titer of herpes simplex virus antibodies. One recovered following two recurrences, but the other died rapidly, suffering both septicemia due to Pseudomonas aeruginosa and herpetic encephalitis. In both cases, the virus involved was a herpes simplex virus type 1 (HSV 1). The various isolates obtained before, during and after treatment remained equally sensitive to acyclovir. These observations highlight three points: the unpredictable and sometimes dramatic development of eczema herpeticum in the young child; the urgency of early diagnosis and treatment; the role of environment in viral contamination. PMID- 2996818 TI - Evaluation and application of the linear variable differential transformer technique for the assessment of human dorsal hand vein alpha-receptor activity. AB - Diurnal, day-to-day, intrasubject, and intersubject variability of responsiveness of dorsal hand veins to norepinephrine has been investigated in healthy young subjects through the use of a novel technique in which a linear variable differential transformer (LVDT) is placed directly over the vein. Under constant operating conditions, control vein diameter remained consistent. There is a log dose responsiveness to norepinephrine infused directly into the hand vein. There was little diurnal, day-to-day, or intrasubject variability in the dose of norepinephrine required to induce 50% constriction of hand vein diameter. The responsiveness to norepinephrine of different veins in either hand was also consistent. However, there was wide intersubject variability, apparently unrelated to age, gender, or other subject characteristics. We conclude that the LVDT method is reproducible and reliable and offers a relatively noninvasive means of assessing the effects of disease and drugs on the human dorsal hand vein in vivo. The LVDT technique has been applied to study the rate of onset, magnitude of effect, dose responsiveness, and duration of action of intravenous dihydroergotamine, 0.1, 0.2, and 0.4 mg, on human dorsal hand veins. Despite systemic intravenous administration, there was an average delay in maximum response of 30 minutes to 1 hour. Venoconstriction was incomplete, with a maximum reduction of approximately 50% of vein diameter after each of the larger doses. There was no significant difference between the effects produced by 0.2 or 0.4 mg, which persisted for 6 hours after dosing. PMID- 2996820 TI - Effect of naloxone on the actions of captopril. AB - Captopril inhibits the metabolism of endogenous opioids in vitro and potentiates their effects in vivo. We examined the hypothesis that endogenous opioids contribute to the actions of captopril in man. The acute cardiovascular and autonomic effects of oral captopril, intravenous naloxone, and their combination were examined in eight healthy men with normotension in a double-blind, placebo controlled study of a Latin squares design. Naloxone altered neither blood pressure nor heart rate. There were significant falls in systolic blood pressure during captopril dosing alone, but there was no fall in blood pressure during combination therapy. Heart rates were higher during the combination than during captopril alone. The combination caused sedation, but neither captopril nor naloxone alone had any behavioral effects. Modification of the acute circulatory effects of captopril by naloxone suggests a role for endogenous opioids in the responses to converting enzyme inhibition. The sedation caused by the combination raises the possibility that captopril may exert central nervous actions in man. PMID- 2996819 TI - Pharmacologic reduction of sympathetic drive increases platelet alpha-2-receptor number. AB - Several lines of evidence implicate sympathetic nervous system involvement in the pathophysiology of essential hypertension in man. Extrapolations are frequently made from in vitro measurements of plasma catecholamine levels to the physiologic role of the sympathetic system in hypertension. We assessed the utility and validity of such extrapolation from in vitro to in vivo measures of adrenergic function. Addition of guanadrel to diuretic therapy in 11 patients with essential hypertension reduced supine intra-arterial blood pressure from 135 +/- 14/76 +/- 9 to 127 +/- 13/67 +/- 5 mm Hg (P less than 0.02). Supine heart rate was also reduced, from 77 +/- 14 to 63 +/- 13 bpm (P less than 0.001). Plasma norepinephrine levels fell from 303 +/- 107 to 170 +/- 46 pg/ml (P less than 0.01). Platelet alpha 2-receptor number ([3H]yohimbine maximal binding) increased from 204 +/- 77 to 301 +/- 150 fmol/mg (P less than 0.02). The pupillary mydriatic response to phenylephrine and the forearm arterial vasoconstrictor response to intra-arterial norepinephrine did not change. Thus guanadrel reduced blood pressure by decreasing sympathetic tone. In this milieu of low sympathetic activity the platelet alpha 2-receptor number increased, but physiologic responses to exogenous alpha-agonists did not change. Caution is therefore advised when extrapolating from in vitro measurement of plasma catecholamine levels and platelet alpha 2-receptor number to the in vivo physiologic significance. PMID- 2996821 TI - Impairment of cellular immunity and OKT4 lymphocytes in symptom-free hemophiliacs with antibodies to human T leukemia virus III (HTLV III). AB - Antibodies against the human T-leukemia virus III (HTLV III) were detected by immunofluorescence in the sera of 17 out of 48 hemophiliacs (35.4%) without AIDS or ARC, frozen in 1983-84. Immunological data collected at that time were re evaluated by separating HTLV III-positive and negative subjects. HTLV III positive patients had significantly reduced OKT4 cells (both in %: 26.1 +/- 10.9 vs 41.2 +/- 15.2; P less than 0.01; and in absolute numbers: 469 +/- 291 vs 1,038 +/- 541; P less than 0.005) and OKT3 lymphocytes (in absolute numbers: 1,234 +/- 550 vs 2,050 +/- 1,067; P less than 0.01). Subpopulations identified by other monoclonal reagents (OKT8, Leu 7, OKM1, anti-Tac) showed no significant differences between the two groups. Patients subsequently found to be seropositive had significantly more frequent anergy to skin tests to recall antigens and often an impairment of in vitro response to phytohemagglutinin A. Despite these relevant defects of some tests of cell-mediated immunity, in HTLV III-positive cases no clinical progression toward AIDS-related complex was observed in a mean period of follow-up of more than 1.5 years. PMID- 2996822 TI - [T2-analysis of normal and pathological structures of the head]. AB - Analyses of T2 values (spin-spin relaxation time constant) in magnetic resonance tomography were carried out in 29 patients with brain tumours. 21 of these had tumours of the glioma group (17 astrocytomas WHO I-III and oligoastrocytomas, 4 glioblastomas). Measurements were effected both pixel by pixel and according to relevant ROI (regions of interest). Although the measurements yielded a T2 value which was typical of the disease, it was individually difficult to effect proper grading on account of the scatter occurring from case to case. Markedly more relevant information was obtained by the introduction of profile measurements in T2 images. The relation between T2 profile and histology of the gliomas permits rough grading between more or less differentiated gliomas. PMID- 2996823 TI - [Value of nuclear magnetic resonance tomography in lung tumors]. AB - A total of 93 patients with pulmonary diseases were examined by means of magnetic resonance tomography. Of these, 39 had a bronchial neoplasm of varying histological structure and varying hilar and mediastinal extension. Compared with other imaging methods, magnetic resonance tomography offers advantages in the imaging of a malignant process and accurate definition of its spatial extension. MR and CT seem to be essentially of equal value in respect of the diagnostic problem of deciding whether lymph node metastases are present in the mediastinum and in the hilus. On the other hand, CT is presently still superior to MR with regard to verifying small-scale processes which are in the process of undergoing endobronchial growth, since CT provides better spatial resolution. Hence, in our opinion the special value of magnetic resonance tomography is the exact definition of the extension of an already identified neoplastic process. This method can supply accurate pointers towards the operability of a bronchial neoplasm. In this regard it is an ideal complement to mediastinoscopy. However, MR will not be an important tool--even in the foreseeable future--for effecting early diagnosis of a neoplasm. The value of diagnostic information supplied by this method may improve by the future use of breath triggering, and additional diagnostic advantages are envisaged by the use of contrast media and the future possibility of producing thinner layers. That is why the catalogue of indications prepared at this time should be seen as a kind of momentary snapshot which may have to be supplemented or extended after the prevailing technical and equipment parameters have undergone further optimisation. PMID- 2996824 TI - [Recurrent extensive pleomorphic adenoma (mixed tumor) of the submandibular gland -diagnosis and therapy]. AB - The authors report an extensive recurrent pleomorphic adenoma of the submandibular gland with destruction of the ramus of the lower jaw. The advantages of computed tomography are the possibility of assessing the osseous structures, the extension of the soft parts, and the relation to the adjacent neck structures. Despite the unusual bone infiltration, histological examination did not point to malignancy. PMID- 2996825 TI - Vitamin D-dependent rickets type I in pigs. AB - We have bred a strain of pigs with an inherited condition of hypocalcaemic rickets, transmitted by an autosomal-recessive mechanism. Homozygous (affected) piglets grew at half the rate of their heterozygous (clinically normal) littermates, and developed profound hypocalcaemia with severe secondary hyperparathyroidism and hypophosphataemia by 8 weeks of age. In the hypocalcaemic piglets, plasma 1,25-dihydroxyvitamin D levels were low or undetectable, and 24,25-dihydroxyvitamin D3 levels were also reduced despite 25-hydroxyvitamin D3 levels being 2-fold higher. There was no detectable 25-hydroxyvitamin D3-1- or 24-hydroxylase enzyme activity in renal homogenates prepared from affected animals. Plasma and intestinal calcium-binding protein levels were reduced in the hypocalcaemic piglets. Sucrose density gradient analysis of intestinal cytosol, prepared in high-salt buffer, revealed the presence of a similar amount of a specific less than 4.2S 1,25-dihydroxyvitamin D3 binder in both groups of piglets. Administration of pharmacological doses of vitamin D3 to affected animals reversed the hypocalcaemia. We conclude that this strain of pigs has vitamin D-dependent rickets type I. PMID- 2996826 TI - Effect of 1,25-dihydroxyvitamin D deficiency on the metabolic clearance rate of 1,25-dihydroxyvitamin D3: studies using pigs with vitamin D-dependent rickets type 1. AB - We have used pigs with inherited vitamin D-dependent rickets type 1 to study the effect of 1,25-dihydroxyvitamin D deficiency on the metabolic clearance rate of 3H-1,25-dihydroxyvitamin D3 infused to steady-state levels in plasma. Plasma levels of 1,25-dihydroxyvitamin D were 24 +/- 1 (SEM) pmol/l in the hypocalcaemic, homozygous piglets and 196 +/- 27 pmol/l in their normocalcaemic, heterozygous siblings. The metabolic clearance rate of 1,25-dihydroxyvitamin D3 was the same in both normal heterozygous (0.90 +/- 0.02) and hypocalcaemic, homozygous piglets (0.90 +/- 0.01 ml-1 min-1 kg-1 metabolic body size). We conclude that a deficiency of circulating 1,25-dihydroxyvitamin D does not influence the clearance of 1,25-dihydroxyvitamin D3 from the circulation of pigs. PMID- 2996827 TI - Metabolism of vitamin D in patients with primary biliary cirrhosis and alcoholic liver disease. AB - The metabolism of isotopically labelled vitamin D2 and D3 has been investigated in eight patients with primary biliary cirrhosis and in five controls. The concentration of labelled vitamin D2 was lower than that of vitamin D3 in serum of patients with primary biliary cirrhosis on days 1 and 2 after intravenous injection (P less than 0.005 and P less than 0.05, respectively) but no difference was seen in controls. Similar amounts of labelled 25-hydroxyvitamin D2 and D3 were seen in serum of the control group; the same pattern was observed in the primary biliary cirrhosis group, and no significant differences were observed between the two groups. In both control and primary biliary cirrhosis groups, the serum concentration of labelled 24,25-dihydroxyvitamin D2 exceeded that of 24,25 dihydroxyvitamin D3 (significant for controls on day 2, P less than 0.02) but concentrations in the two groups were not different. Concentrations of labelled 25,26-dihydroxyvitamin D3 were significantly higher than those of 25,26 dihydroxyvitamin D2 in the primary biliary cirrhosis group at all times and in the control group on days 2 and 3. Both 25,26-dihydroxyvitamin D2 and D3 were higher in the serum of patients with primary biliary cirrhosis than in controls (significant on day 1; P less than 0.05). Urinary excretion over days 0-3 of radioactivity from both vitamins D2 and D3 was significantly higher in the primary biliary cirrhosis group than in controls: 12.03 vs 1.80% for vitamin D2 and 8.98 vs 1.76% for vitamin D3 (P less than 0.005). Vitamin D2-derived urinary radioactivity in primary biliary cirrhosis correlated strongly with serum bilirubin (P = 0.005). The metabolism of labelled vitamin D3 was studied in seven patients with alcoholic liver disease, three of whom showed low serum concentrations of labelled 25-hydroxyvitamin D3 suggesting impaired hepatic synthesis. The 25-hydroxylation response was quantified as the relative index of 25-hydroxylation and was significantly related to two other indices of liver function. It is concluded that impaired 25-hydroxylation of vitamin D may occur in alcoholic liver disease and results from hepatocellular dysfunction. Less than the predicted amounts of 1,25-dihydroxyvitamin D3 were produced in four of the seven patients with alcoholic liver disease; this defect may be attributable in part to decreased precursor 25-hydroxyvitamin D and to poor renal function. PMID- 2996828 TI - The opioid-noradrenergic link in Gilles de la Tourette's syndrome. PMID- 2996829 TI - Treatment of infectious complications of acquired immunodeficiency syndrome. AB - The infectious complications of the acquired immunodeficiency syndrome (AIDS) are discussed, and the conventional and nonconventional therapies used for these infections are reviewed. The infections most commonly encountered in patients with AIDS are Pneumocystis carinii pneumonia (58%), Candida esophagitis (31%), toxoplasmosis (21%), cytomegalovirus infections (15%), and herpes-simplex virus infections (12%). Pneumocystis carinii pneumonia is the most common life threatening process in these patients. Trimethoprim-sulfamethoxazole (TMP-SMZ) is considered the drug of choice for its treatment. Oral candidiasis often indicates the progression to AIDS in the high-risk populations of homosexual or bisexual men, intravenous drug abusers, and individuals with hemophilia. Nystatin suspension is commonly used to treat oral candidiasis, while Candida esophagitis demands systemic therapy with ketoconazole. Toxoplasmosis most commonly manifests itself in patients with AIDS as a cerebral mass lesion. The recommended therapy includes sulfadiazine and pyrimethamine. AIDS patients frequently experience protozoal invasion of the intestinal tract with Giardia lamblia, Isospora belli, and Cryptosporidium muris. Various drugs have been tried for these infections, including quinacrine hydrochloride, metronidazole, TMP-SMZ, and spiramycin. Cytomegalovirus (CMV) infections commonly involve the lungs, gastrointestinal tract, eyes, brain, and nervous system. Attempts to treat these disseminated CMV infections with antiviral agents, including acyclovir, have not been successful. However, acyclovir has been found beneficial in the treatment of herpes-simplex virus infections. Multiple infectious complications may occur in patients with AIDS as a result of the cellular-immune deficiency associated with this disease. Until more research is done with AIDS patients, therapy must be based on the data available from the treatment of these infections in immunosuppressed patients without AIDS. PMID- 2996830 TI - Haemodynamic effects of low and high doses of insulin during beta receptor blockade in dogs. AB - Haemodynamic effects of small and high doses of insulin during beta receptor blockade were studied in nine dogs. Beta receptor blockade was induced by 0.5 mg/kg propranolol and caused depression of cardiac performance with a significant increase in left ventricular end-diastolic pressure (LVEDP) and a significant decrease in heart rate; maximum rate of left ventricular (LV) pressure rise (LVdP/dtmax), stroke volume and cardiac output. At 15 min, after beta receptor blockade, a bolus injection of 0.5 IU/kg of insulin, free of glucagon and calcium, was given followed by a continuous infusion of 0.5 IU/kg/h. After 30 min another bolus dose of 300 IU insulin was injected. Glucose and potassium were given to maintain physiological levels of these factors. Five minutes after a low dose of insulin there was a significant decrease in LVEDP (P less than 0.01), and a significant increase in LVdP/dtmax (P less than 0.01), in stroke volume (P less than 0.01) and in cardiac output (P less than 0.01). The other haemodynamic variables were not significantly changed. Administration of a high dose of insulin further, significantly, improved performance of the beta receptor blocked heart and caused a significant reduction in total peripheral resistance. In conclusion, insulin exerts inotropic and vasodilator effects which are dose dependent and not related to adrenergic mechanisms. PMID- 2996831 TI - Metabolic effects of low and high doses of insulin during beta-receptor blockade in dogs. AB - Metabolic effects of low and high doses of insulin during beta-receptor blockade were studied in eight dogs. Beta-receptor blockade was induced by 0.5 mg/kg propranolol which caused depression of heart performance. This was accompanied by a significant reduction in myocardial blood-flow and oxygen consumption. There was also a significant reduction in arterial concentrations and myocardial uptake of free fatty acids, while arterial concentrations and myocardial uptake of glucose and lactate were not significantly changed. Fifteen minutes after beta receptor blockade, an intravenous (i.v.) bolus injection of 0.5 IU/kg, of insulin, free of glucagon and calcium, was given followed by a continuous infusion of 0.5 IU/kg/h. Glucose and potassium were given to maintain constant levels of these factors. After 30 min another bolus dose of 300 IU insulin was injected. Thirty minutes after a low dose of insulin, a significant increase in heart performance was recorded at unaltered myocardial oxygen consumption. Arterial concentrations of free fatty acids were significantly reduced while levels of glucose and lactate were unchanged. Myocardial uptake of glucose increased significantly while uptake of lactate and free fatty acids was unchanged. After a high dose of insulin there was a considerable improvement in heart performance. Myocardial blood-flow and oxygen consumption were not changed, nor were there alterations in arterial concentrations and myocardial uptake of glucose, lactate and free fatty acids. It is concluded that, during beta-receptor blockade high doses of insulin improve the mechanical performance of the heart through mechanisms that are independent of insulin's effects on substrate metabolism. PMID- 2996833 TI - Attenuation may regulate gene expression in animal viruses and cells. AB - In eukaryotes, an abundant population of promoter-proximal RNA chains have been observed and studied, mainly in whole nuclear RNA, in denovirus type 2, and in SV40. On the basis of these results it has been suggested that a premature termination process resembling attenuation in prokaryotes occurs in eukaryotes. Moreover, these studies have shown that the adenosine analog 5,6-dichloro-1-beta D-ribofuranosylbenzimidazole (DRB) enhances premature termination, but its mode of action is not understood. The determination of the nucleotide sequences of SV40 and other viruses and cellular genes provide means for elucidating the nucleotide sequences involved in the attenuation mechanism. A model has recently been described in which attenuation and mRNA modulation in a feedback control system quantitatively regulate SV40 gene expression. The suggested mechanism described in this model opens up approaches to the investigation of attenuation and mRNA modulation as a possible mechanism whereby eukaryotes may regulate transcription in a variety of different circumstances. PMID- 2996832 TI - A computer-assisted rapid-scanning spectrophotometer with applications to tissues in vitro and in vivo. AB - A flexible approach is described to acquire, store, retrieve, and manipulate the large volume of data provided by a rapid-scanning spectrophotometer designed for biological preparations. Data can be displayed in spectral and kinetic formats. This spectrophotometer provides a means to simultaneously measure the concentrations of components of light scattering preparations and to record chemical reactions as they occur. Although application of this instrument for the study of reduction/oxidation shifts of mitochondrial respiratory chain components is emphasized, the design is suitable for many spectrophotometric applications. PMID- 2996834 TI - Metastatic mediastinal neoplasm masquerading as aortic dissection: a skip sign on computed tomography for their distinction. AB - Four years after undergoing a left lower lobe lobectomy for squamous cell carcinoma, a 77-year-old man presented with a mass lesion in the left upper mediastinum associated with chest pain. Computed tomography revealed a homogeneous density immediately adjacent to the aortic arch and thoracic aorta consistent with aortic dissection. Upon thoracotomy, however, the lesion was found to be an oat cell carcinoma. Retrospective review of the computed tomography scans detected a normal segment of descending aorta, indicating the interruption of the paraaortic lesion. Such a skip sign can be used to rule out aortic dissection. PMID- 2996836 TI - Genetic basis of iron assimilation in pathogenic Escherichia coli. PMID- 2996835 TI - Infantile spasms. PMID- 2996837 TI - Nonbronchoscopic bronchoalveolar lavage for the diagnosis for Pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome. AB - We compared conventional bronchoscopic transbronchial biopsy (TBB) and bronchoalveolar lavage (BAL) with non-bronchoscopic bronchoalveolar lavage (NB BAL) in nine patients with acquired immunodeficiency syndrome (AIDS) and bilateral lung infiltrates. NB-BAL was carried out with a control-tipped reusable catheter. In each patient, bronchoscopic procedures were performed in the right lung, followed immediately by NB-BAL in the left lung. The specimens obtained by NB-BAL confirmed the presence of P carinii pneumonia in seven of eight patients in whom the diagnosis was established by TBB or BAL. Viral cultures of NB-BAL specimens yielded cytomegalovirus (CMV) in four of five subjects with evidence of CMV via bronchoscopic technique, including two instances in which CMV was not detected by BAL. Complications were limited to right-sided pneumothorax attributable to TBB. Accuracy of NB-BAL appears to be comparable to that of conventional bronchoscopic approaches in the diagnosis of AIDS-related pulmonary infection with P carinii or CMV. NB-BAL may be a safer and more economical alternative to TBB and BAL in the diagnosis of pulmonary opportunistic infections. PMID- 2996838 TI - Spontaneous pneumothorax. A complication of lung cancer? AB - Among 338 adults (258 men and 80 women) presenting with spontaneous pneumothorax, there were six men with lung cancer: five squamous cell carcinoma and one oat cell carcinoma. Pneumothorax led to the diagnosis in five cases and the remaining occurred as a complication of known neoplastic disease. The average age was 67 years. We analyze these six cases, along with 46 others from the literature. In patients less than 40 years old with normal chest x-ray film findings after lung expansion, further investigation for neoplastic disease is not justified. However, heavy smoking, chronic bronchitis, bullous emphysema and incomplete lung expansion after chest drainage in patients over 40 years old are indications for cancer screening through sputum cytologic study, bronchoscopic examination and surgical exploration. The occurrence of a pneumothorax neither alters the treatment of the underlying disease nor modifies the one-year prognosis. Five year survival is nil, suggesting that lung cancers present as pneumothorax at an advanced stage of disease. PMID- 2996839 TI - Cobalt-induced bronchial asthma in diamond polishers. AB - Three diamond workers had occupational asthma attributed to the inhalation of cobalt powder. The exposure originated from high speed polishing disks with an abrasive consisting of microdiamonds cemented in extra fine cobalt not alloyed to tungsten carbide. The bronchoconstriction progressed towards the end of working days; it was especially pronounced in the absence of an adequate exhaust ventilation; and it could be accompanied by rhinitis and chest tightness. Cobalt inhalation challenge tests were positive in all three patients, and exposure to cobalt temporarily increased nonspecific hyperreactivity. PMID- 2996840 TI - Virologic and immunologic study on acquired immune deficiency syndrome. PMID- 2996842 TI - Determination of cyclic AMP and cyclic GMP in normal human epidermis and dermis. PMID- 2996841 TI - Hemophiliacs and acquired immune deficiency syndrome. PMID- 2996843 TI - Vaginal metastasis of choriocarcinoma and invasive mole. Clinical and pathological characteristics, diagnosis and treatment. PMID- 2996844 TI - [Malignant ovarian tumors stage I: a clinico-pathological analysis of 105 cases]. PMID- 2996845 TI - A cloned sequence, p82H, of the alphoid repeated DNA family found at the centromeres of all human chromosomes. AB - Clone p82H is a human DNA sequence which hybridises in situ exclusively to the centromeric regions of all human chromosomes. It is composed of approximately 14 tandemly repeated variants of a basic 172 bp sequence, and is related to the alphoid family. The organisation of the family of cross-hybridising sequences, detected by the clone p82H, is described both in the human genome and on certain chromosomes, and its relationship to known sequence families is discussed. PMID- 2996847 TI - [CT study of hepatocellular carcinoma]. PMID- 2996848 TI - [A radiologic-pathologic study of 119 cases of peripheral lung cancer]. PMID- 2996846 TI - T-antigen is the only detectable protein on the nucleosome-free origin region of isolated simian virus 40 minichromosomes. AB - A nucleosome-free region or nucleosome gap, containing the origin of replication and the transcriptional promoter elements, is observed on 20%-25% of the SV40 minichromosomes isolated at physiological ionic strength at late time during the infectious cycle. We found that this subpopulation of gapped minichromosomes was more sensitive to digestion with a variety of single-cut restriction enzymes than the rest of the minichromosomes. This increased digestibility of gapped minichromosomes allowed us to excise the gap region by concomitant digestion with Bgl I and Msp I. T-antigen was the only detectable protein bound to this isolated chromatin fragment. In particular no histones could be detected. The presence of T-antigen on the gap region was confirmed by immunoelectron microscopy. Most of the T-antigen appeared to be located on the late side of the Bgl I restriction enzyme site. PMID- 2996849 TI - [X-ray diagnosis of tumors of the trachea and major bronchi]. PMID- 2996851 TI - [Myoclonus epilepsy, Lafora body form--a case report]. PMID- 2996850 TI - [The relationship between Epstein-Barr virus and rheumatoid arthritis]. PMID- 2996852 TI - [Present status and trends in drug dependence]. PMID- 2996853 TI - [A sero-epidemiological study of viral hepatitis A in a village]. PMID- 2996854 TI - [Effect of aluminium citrate on electrokinetic potential on the surface of SiO2 and TiO2 particles]. PMID- 2996855 TI - [Toxicity of organic sulfur fungicide-urbacid and its residues in food]. PMID- 2996857 TI - [Glomus tumors of the finger]. PMID- 2996856 TI - [Toluene diisocyanate induced asthma]. PMID- 2996859 TI - [Paget's disease of the scrotum: report of 13 cases]. PMID- 2996858 TI - [Changes in trace elements (zinc, copper and manganese) in the hair before and after severe fractures by proton-induced X-ray emission spectrometry (PIXE)]. PMID- 2996860 TI - Bowel function in an urban black African population. AB - Despite a low daily dietary fiber intake, an urbanized black African population in southern Africa has a low incidence of those diseases associated with fiber deficiency. Stool weights, defecation frequencies, and transit times in this group are much closer to those of westernized whites than to rural blacks. PMID- 2996862 TI - Efficacy of a simplified lower gastrointestinal flexible endoscope cleaning method. AB - Published guidelines from the Center for Disease Control (CDC) "strongly recommended" gas sterilization or 30 minutes of high-level disinfection with either 2 percent glutaraldehyde or 6 percent hydrogen peroxide following each flexible endoscope cleansing for proper care. The guidelines were proposed on the basis of previous CDC studies performed on glutaraldehyde disinfection of respiratory equipment. A prospective study was performed culturing flexible endoscopes following cannulation of the lower gastrointestinal tract and cleansing. A uniform endoscope cleansing method without gas sterilization or high level disinfection was used between patients. Thirty aerobic and 30 anaerobic RODAC bacterial culturings revealed no obligate anaerobic organism growth and only sparse, aerobic, environmental and cutaneous organism growth. There were no instances of documented or suspected postendoscopy infectious complications. Our results indicate that high-level disinfection and gas sterilization of flexible endoscopes are not necessary to prevent bacterial disease transmission from patient to patient. PMID- 2996861 TI - Retrorectal tumors. Mayo Clinic experience, 1960-1979. AB - One hundred twenty patients with primary retrorectal tumors (79 congenital, 14 neurogenic, 13 osseous, and 14 miscellaneous) had their initial treatment at the Mayo Clinic from 1960 to 1979. The mean age was 43 years (100 patients were adults). Female predominance was associated with congenital cysts (15:1) and male predominance with chordomas (5:1). Forty-three percent of the patients had malignant lesions. No dermoid cysts were found in this series. Diagnosis was made by digital examination or sacral radiographs in all patients. Computed tomography scan was the most important diagnostic method; the rate of positive findings was 100 percent in 20 patients. Approach to the tumor was posterior in 79 of 102 patients in whom resection was possible. Ten of 66 patients with benign tumors had recurrence. The five-year survival rate for patients with chordomas was 75 percent and for patients with other malignant lesions was 17 percent. Because preoperative biopsy can cause tumor spread, abscess, fecal fistula, or meningitis, it should not be performed if tumors are potentially resectable. Whenever possible, total resection should be done. PMID- 2996864 TI - Peritoneovenous shunts in malignant ascites. AB - A 51-year-old woman with malignant ascites secondary to hepatocellular carcinoma had a peritoneovenous (LeVeen) shunt inserted with effective control of ascites and amelioration of symptoms. The results of 12 recent series evaluating the efficacy of peritoneovenous shunts in the treatment of 198 patients with malignant ascites were reviewed. Peritoneovenous shunts effectively controlled malignant ascites in 77% of patients. Complications occurred in 25%, although the majority of these were related to shunt occlusion and transient congestive heart failure. PMID- 2996865 TI - Perspectives on the role of viruses in insulin-dependent diabetes. AB - Insulin-dependent diabetes mellitus (IDDM) results from the destruction of pancreatic beta cells. Viruses have been suggested as one of the possible causes. The evidence for viruses comes largely from experiments in animals, but several studies in humans also point to viruses as a trigger of this disease in some cases. Encephalomyocarditis (EMC) virus, Mengovirus (2T), and Coxsackie B4 virus infect and destroy pancreatic beta cells when inoculated into mice. This results in hypoinsulinemia and hyperglycemia. The development of EMC virus-induced diabetes is dependent on the genetic background of the host and genetic makeup of the virus. Animals with diabetes for several months show some long-term complications, including glomerulosclerosis, ocular changes, and decreased bone formation and mineralization in addition to acute metabolic changes. EMC virus induced diabetes can be prevented by a live-attenuated vaccine. The capacity of Coxsackie B4 virus to induce diabetes is also influenced by the genetic background of the host. However, Mengovirus-induced diabetes is not dependent on the genetic background of the host. In contrast to the EMC, Mengo, and Coxsackie B4 viruses, reovirus type 1 seems to be somehow associated with an autoimmune response producing a diabetes-like syndrome in suckling mice. This virus produces an autoimmune polyendocrinopathy that results in very mild and transient glucose intolerance. Several common human viruses including mumps, Coxsackie B3 and B4 viruses, and reovirus type 3 can infect human beta cells in culture and destroy them. A variant of Coxsackie B4 virus has been isolated from the pancreas of a child who died of acute-onset IDDM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2996863 TI - Serological diagnosis of acute viral hepatitis. AB - Fifty cases of symptomatic acute viral hepatitis presenting at the Washington, D.C., Veterans Administration Medical Center between 1976 and 1978 were tested for serological markers of hepatitis virus infection. The etiology of the acute hepatitis appeared to be hepatitis A virus in 20%, hepatitis B virus in 52%, non A, non-B agents in 22%, delta hepatitis in 4%, and infectious mononucleosis in 2%. The diagnosis of type B hepatitis was difficult to verify because 10% of cases were seronegative for HBsAg and another 10% were seronegative by conventional testing for IgM antibody to hepatitis B core antigen (a putative marker of acute hepatitis B virus infection). Accurate serodiagnosis of acute viral hepatitis depends upon the correct application of testing for IgM antibody to hepatitis A virus, IgM antibody to hepatitis B core antigen, HBsAg, and tests for syphilis and mononucleosis. PMID- 2996866 TI - [Effect of the antioxidant butylated hydroxytoluene (dibunol) on hormonal regulation in rats of various ages]. PMID- 2996867 TI - [Identification of 2 types of DNA-matrix contacts in the cells of human fibrosarcoma HT1080 with replicative and post-replicative complexes]. PMID- 2996869 TI - Sciatic nerve damage associated with pentamidine. PMID- 2996868 TI - A study of the mediator spectrum of delta-9 tetrahydrocanabinol effects on cerebral bioelectric activity. AB - Defense conditioning in cats with simultaneous recording of bioelectric activity was used to show that serotonin-negative and dopamine-positive substances normalize delta-9 THC-elicited dissimilarity of amplitudinal and temporal parameters of evoked potentials in visual associative and somatosensoric zones II of the cerebral cortex. It was also found that serotoninergic substances affect both primary and secondary waves of evoked potentials while dopaminergic substances influence only late waves. PMID- 2996870 TI - [Vaccination of geese (Anser anser dom.) and Muscovy ducks (Cairina moschata dom.) against parvovirus hepatitis (s. Derzsy's disease)-- report of a field trial with the attenuated live virus vaccine Palmivax]. PMID- 2996871 TI - Graduated potentials of the median nerve and their usefulness in diagnosis of the type of lesion of the peripheral nervous system. PMID- 2996872 TI - Ephapse between two motor units in chronically denervated muscle. PMID- 2996873 TI - Management of life-threatening dermatoses. AB - The notion of life-threatening dermatoses may seem to be a contradiction in terms, but in fact there are a number of serious dermatologic conditions that require prompt attention to prevent fatal consequences. This article will review four such diseases, for which prompt recognition and treatment will be emphasized. PMID- 2996874 TI - A selective screen for transposable element mobilization in Drosophila melanogaster. AB - A selective system is described that provides a simple and sensitive assay for the detection and analysis of induced mobile element transpositions in Drosophila melanogaster. The system will detect a single event in samples greater than 10(6) and thus provides a eucaryotic assay system for monitoring the induction of transposition by a variety of agents including, but not limited to, chemical carcinogens and toxins, ionizing radiation, and various environmental pollutants. The experimental system focuses on an X-linked rosy+ transposon and a conditional lethal system that permits the detection of a single transposition event in very large samples. The results of a pilot experiment utilizing this system are presented. PMID- 2996875 TI - Mutagenesis at the ouabain-resistance locus of 3.7.2C L5178Y cells by chromosomal mutagens. AB - Chemical mutagens including methyl methanesulfonate, N-methyl-N'-nitro-N nitrosoguanidine, iodomethane, and epichlorohydrin have been classified as "chromosomal mutagens" in the L5178Y/thymidine kinase (TK) gene mutation assay by Clive and coworkers [Mutat Res 59:61-108, 1979; and "The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation." Amsterdam: Elsevier/North Holland, pp 103-123, 1980] who observed mutagen-dependent increases in small TK-deficient mutant colonies with detectable damage to the chromosome (11) that carries the TK locus. In this study, we tested these four chemicals for the induction of gene mutations at the ouabain-resistance (ouares) locus of 3.7.2C L5178Y cells to determine if presumptive chromosomal mutagens would go undetected at a gene locus that is unresponsive to chromosomal damage. A final concentration of 375 micrograms/ml ouabain in soft-agar medium selected against the ouabain-sensitive phenotype without loss of the mutagen-induced ouabain-resistant phenotype. Verification of the mutant phenotype was completed for six individual soft-agar ouares colonies derived from mutagen-treated cultures via growth for 10-11 days in nonselective medium followed by retesting for colony formation in selective soft-agar medium. Dose-related reproducible increases in the frequency of ouabain-resistant mutants were observed for 3.7.2C L5178Y cells that had been exposed for 3 hr to 24-46 micrograms/ml epichlorohydrin, 1.9-3.6 micrograms/ml iodomethane, 0.006-0.011 micrograms/ml N methyl-N'-nitro-N-nitrosoguanidine and and 2.0-5.4 micrograms/ml methyl methanesulfonate. Also, treatments with EMS, which induced sufficient numbers of ouares colonies to permit analysis of colony size distribution, showed the existence of a bimodal size distribution similar to those reported for TK deficient mutants. This discovery suggests that mutant colony size in this cell line may be independent of specific gene locus effects. We conclude that (1) chemicals that induce a high proportion of chromosomal mutants, as detected at the TK locus in earlier studies, also induce single gene mutations at the ouabain resistance locus and (2) a bimodal distribution of mutant colony sizes in soft agar medium after short expression periods may be a distinctive characteristic of the 3.7.2C L5178Y cell line and is not confined to the TK locus. PMID- 2996877 TI - Equid herpesvirus 1 (EHV 1) latency: more questions than answers. PMID- 2996876 TI - Validation of an in vivo alkaline elution assay to detect DNA damage in rat testicular cells. AB - An alkaline elution assay was used to measure DNA damage in the rat testis after in vivo treatment with chemicals. All of the chemicals reported to induce heritable mutations in a mammalian assay (mouse specific locus test) were positive in the rat alkaline elution assay. Chemicals that are negative or inconclusive in the specific locus test did not cause detectable DNA damage in rat testes. DNA damage was detected by alkaline elution after either intraperitoneal or oral administration of chemical mutagens. Data from these validation studies were used to establish criteria to be used for evaluation of alkaline elution results with materials of unknown potential for genotoxic effects in germinal tissue. PMID- 2996878 TI - Activities of key enzymes of aerobic and anaerobic metabolism in middle gluteal muscle from trained and untrained horses. AB - The effect of physical training on the in vitro activities of key enzymes that provide quantitative information on the maximum capacities of anaerobic and aerobic metabolism has been investigated in the gluteal muscle of the horse. Training had no effect on the activities of 6-phosphofructokinase or creatine kinase, suggesting that there was no effect on the capacity of anaerobic metabolism in this muscle. However, the activities of hexokinase and citrate synthase were increased, indicating that training increased the capacity of aerobic metabolism. For comparative purposes, muscle fibre composition and enzyme activities were also determined in a group of foals and a group of broodmares. PMID- 2996879 TI - Experimental reactivation of equid herpesvirus 1 (EHV 1) following the administration of corticosteroids. AB - Eight ponies were experimentally infected with equid herpesvirus 1 (EHV 1) (subtype 1). All animals showed clinical and serological evidence of infection and virus was isolated from nasal swabs and leucocytes. These ponies were kept in isolation for a further three months during which time complement fixing antibody decreased at least four-fold. Following immunosuppression with dexamethasone and prednisolone subtype 1 virus was recovered from six of the eight animals within 14 days. Five of these six ponies were viraemic and three of them shed virus in nasal secretions; only four displayed significant rises in complement fixing antibody and only two in neutralising antibody. Clinical abnormalities were not detected during reactivation. PMID- 2996880 TI - Cell surface influenza haemagglutinin can mediate infection by other animal viruses. AB - We have used filter-grown Madin-Darby canine kidney (MDCK) cells to explore the mechanism by which influenza virus facilitates secondary virus infection. Vesicular stomatitis virus (VSV) and Semliki Forest virus (SFV) infect only through the basolateral surface of these polarized epithelial cells and not through the apical surface. Prior infection with influenza virus rendered the cell susceptible to infection by VSV or SFV through either surface. The presence of both a permissive and a restrictive surface for virus entry in the same cell allowed us to determine how the influenza infection enhanced the subsequent infection of a second virus. Biochemical and morphological evidence showed that influenza haemagglutinin on the apical surface serves as a receptor for the superinfecting virus by binding to its sialic acid-bearing envelope proteins. Influenza virus also facilitates secondary virus infection in non-epithelial cells; baby hamster kidney cells (BHK-21), which are normally resistant to infection by the coronavirus (mouse hepatitis virus MHV-A59), could be infected via the haemagglutinin-sialic acid interaction. Facilitation of secondary virus infection requires only the sialic acid-binding properties of the haemagglutinin since the uncleaved haemagglutinin could also mediate virus entry. PMID- 2996881 TI - Deletion of complement C4 and steroid 21-hydroxylase genes in the HLA class III region. AB - Molecular maps have been prepared of the HLA region on human chromosome 6 that includes the complement C4 and steroid 21-hydroxylase genes (21-OH), using DNA of individuals deficient (QO) in either of the two forms C4A or C4B. In all, 18 haplotypes with C4A QO were examined by Southern analysis and two had deletions of 28-30 kb that included both the C4A and 21-OHA genes. Of six C4B QO haplotypes, one had a deletion that included both the C4B and 21-OHA genes. Thus, some of the C4 null alleles are due to deletion of the gene but the majority in this sample are not. Deletion occurred in two common haplotypes suggesting that in the population as a whole, C4A deficiency is due to deletion in about one-half the C4A QO haplotypes. As duplication of C4A or C4B genes does occur, the possibility that unequal cross-over could explain the C4 deletion was examined by preparing cosmid clones from the DNA of an individual typed C4A QO. A cloned genomic fragment containing the single C4B gene was isolated and found to be similar to the homologous region of a cosmid from a normal individual carrying a C4A gene. This suggests that if a cross-over has occurred it is in a region where the two genes are identical. The biological significance of the rather frequent occurrence in the population of haplotypes with C4A or C4B deletion together with the accompanying deletion of the 21-OHA gene is discussed. PMID- 2996882 TI - Identification of proteins involved in the regulation of yeast iso- 1-cytochrome C expression by oxygen. AB - On the basis of a gel electrophoresis retardation assay, protein(s) which interact specifically with the upstream activating site (UASc) of the yeast iso-1 cytochrome C (CYC1) gene were identified and separated by heparin ultrogel chromatography. DNase I protection experiments indicate that these factors protect a 23-bp sequence overlapping the UASc site previously defined. The specific binding activity is strongly reduced in extracts prepared from a wild type strain grown anaerobically. It is absent in a mutant strain blocked in the biosynthesis of heme but it is restored upon the addition of the missing precursor, delta amino levulinic acid (dALA) to the growth medium. In contrast, the binding activity does not differ significantly in extracts form a wild-type strain grown in either glucose or glycerol as carbon source. These data strongly argue that the CYC1 UAS binding protein(s) that we have identified mediate the oxygen and heme control of cytochrome C biosynthesis. PMID- 2996883 TI - Yeast cdc35 mutants are defective in adenylate cyclase and are allelic with cyr1 mutants while CAS1, a new gene, is involved in the regulation of adenylate cyclase. AB - Newly isolated temperature-sensitive cdc35 mutants of Saccharomyces cerevisiae have been characterized. They show the morphology, growth and conjugation characteristics typical of class-A or class-II start mutants. The cdc35 mutation induces a significant decrease of the intracellular cAMP level and produces a thermolabile adenylate cyclase. By classical genetic criteria the CDC35 gene is identical with the structural gene of adenylate cyclase, CYR1. The results of the mutant selection, the kinetics of macromolecule accumulation and the cell-density change of cdc35 mutants at the restrictive temperature, indicate that CDC35 function may not be cell cycle-specific. A new mutation, cas1, was isolated and partially characterized. It mediates the suppression by external cAMP of the unlinked cdc35 mutation. It causes a slight increase of the intracellular cAMP level and has strong effects on the adenylate cyclase activities, especially on the Mg2+ dependent activity. The data suggest that the CAS1 protein is a controlling element of adenylated cyclase. The CAS1 locus is different from the RAS1 and RAS2 loci. PMID- 2996884 TI - A transcription enhancer in the Herpesvirus saimiri genome. AB - Herpesvirus saimiri, an oncogenic agent of New World primates, has a linear double-stranded DNA genome of approximately 155 kb. To test its genome for the presence of a transcription enhancer, we have mixed randomly fragmented H. saimiri DNA with non-infectious, linear SV40 DNA lacking the 72-bp repeat enhancer region (the so-called SV40 enhancer trap) and co-transfected this DNA mixture into monkey CV-1 cells. Viable SV40-like viruses were generated by intracellular ligation/repair processes with short H. saimiri DNA fragments. One recombinant, SVHS-2, had integrated a 377-bp enhancer segment from the righthand region of the H. saimiri genome, 7 kb upstream of DNA sequences encoding an immediate-early mRNA. This enhancer sequence is contained within the non repetitive portions of the viral genome known to be preserved episomally in all lymphoid tumor cell lines. Further recombinant viruses (SVHS-14, SVHS-7, and SVHS 8) essentially contain subsets of the 377-bp insert. Unlike in the previous enhancer trap experiments, where heterologous enhancers were incorporated without any sequence alterations, SVHS-14 and SVHS-7 have suffered short internal deletions of a very similar segment of the H. saimiri insert. This renders the enhancer more active, implying that the deleted segment, while it may have a role in the herpesvirus infection cycle, exerts a negative effect within the isolated enhancer. PMID- 2996886 TI - T4 polynucleotide kinase; cloning of the gene (pseT) and amplification of its product. AB - The T4 gene (pseT) for polynucleotide kinase (pnk) has been cloned in lambda. Induction of a lambda E-W-S-cI857 prophage in which the pseT gene can be transcribed from the late lambda promoter, PR1, leads to greater than 100-fold amplification of pnk activity; pnk comprises approximately 7% of the total soluble cell protein. The purified enzyme, as expected, is both a 5'-kinase and a 3'-phosphatase. The amino acid sequence deduced from an open reading frame identified as the pseT gene contains a sequence which corresponds particularly well with that part of the adenine nucleotide binding site of adenylate kinase shown to form a flexible loop. A deletion mutant that lacks 5'-kinase activity, and possibly also 3'-phosphatase activity, has lost two amino acids from within the proposed loop structure. A second region of the pnk sequence shares homology with phosphoglycerate kinase, yeast inorganic pyrophosphatase and histone 2b from various organisms. PMID- 2996885 TI - Specific interaction of cellular factors with the B enhancer of polyoma virus. AB - Specific interactions between proteins from mouse 3T6 cells and the enhancer sequence of polyoma virus were detected using the method of band shifting on polyacrylamide gels. Proteins eluted from 3T6 nuclei using a buffer containing 0.55 M NaCl, formed a stable complex with the B enhancer of polyoma virus. At least two different factors are involved in this interaction. The contact sites which were mapped on the DNA sequence using DNase I footprinting correspond to a GC-rich palindrome surrounded by two sequences homologous respectively to the immunoglobulin and to the immunoglobulin and SV40 enhancers. Moreover Bal31 deletion analysis confirmed that similar sequences are required for the formation of the complex. In spite of a common function and partial sequence homology among some enhancers, neither the polyoma A enhancer, the mouse immunoglobulin heavy chain gene enhancer, nor the origin-promoter-enhancer region of SV40 efficiently competed with the polyoma B enhancer for the binding of these molecules. PMID- 2996888 TI - The EcoDXX1 restriction and modification system of Escherichia coli ET7. Purification, subunit structure and properties of the restriction endonuclease. AB - The Escherichia coli plasmid pDXX1 codes for a new restriction-modification system. The specific restriction endonuclease coded by this system has been purified by a procedure that includes phosphocellulose and heparin-agarose chromatography. Sedimentation on glycerol gradients showed one peak of activity with a value of about 12 S. The highly purified enzyme require ATP and Mg2+ for activity as well as S-adenosylmethionine, although some S-adenosylmethionine molecules are probably bound to the enzyme. The enzyme does not cleave lambda DNA at well-defined sites and has a strong non-modified DNA-dependent ATPase activity. The enzyme has also methylase activity acting against non-modified DNA. PMID- 2996889 TI - NMR studies of the trp repressor from Escherichia coli. Characterisation and assignments of residue types. AB - High-resolution proton nuclear magnetic resonance spectra of the trp repressor of Escherichia coli under various conditions are reported and analysed. The spectrum of the denatured state agrees with that predicted from the amino acid composition, with the exception of the two histidine residues, which have different chemical shifts although they titrate normally. The spectrum of the native protein shows the presence of extensive secondary and tertiary structure. Using information from chemical shifts, numbers of protons, titration behaviour, homonuclear chemical-shift-correlated spectroscopy and nuclear Overhauser enhancement correlated spectroscopy, most of the aromatic protons have been assigned to residue type. Further, about 30% of the aliphatic protons have been assigned to residue type by two-dimensional spectroscopy. Nuclear Overhauser enhancements establish that high-field methyl groups belonging to a valine residue lie directly over an aromatic ring. PMID- 2996887 TI - Antagonistic effects of thyrotropin and epidermal growth factor on thyroglobulin mRNA level in cultured thyroid cells. AB - Both thyrotropin (TSH) and epidermal growth factor (EGF) are potent mitogenic agents when added to dog thyroid cells in primary culture [Roger, P. P. and Dumont, J. E. (1984) Mol. Cell. Endocrinol. 36, 79-93]. The concomitant effect of these agents on the differentiation state of the cells was appreciated using cell morphology, iodide trapping, thyroglobulin synthesis and cytoplasmic thyroglobulin mRNA content as markers. Together with previous results [Mol. Cell. Endocrinol. 36, 79-93 (1984)] it is shown that cells cultured in the continuous presence of TSH maintain all the parameters at a near normal level. In the absence of TSH, thyroglobulin mRNA decreased to very low, though still detectable levels. Addition of TSH restored subnormal mRNA levels. Culture of cells in the presence of EGF for 4-6 days affected profoundly their morphology, abolished iodide trapping and decreased thyroglobulin synthesis and cytoplasmic mRNA content to undetectable levels. Addition of TSH to cells previously exposed to EGF reversed the growth factor effect on all four indexes. The redifferentiating effect of TSH was well observed within 3-4 days and was mimicked by the adenylate cyclase activators, forskolin and cholera toxin. When administered simultaneously, TSH and EGF achieved an intermediate situation, EGF antagonizing partially the effect of TSH on the expression of thyroglobulin gene. Another growth factor, fibroblast growth factor, while promoting thyroid cell proliferation also, did not interfere at all with TSH effects on cytoplasmic thyroglobulin mRNA content. Our results make the dog thyroid cell in primary culture an appropriate model to study the mechanisms involved in gene regulation by cyclic AMP and growth factors. PMID- 2996890 TI - Preliminary identification of the secondary structure of the trp repressor from Escherichia coli. AB - The probable secondary structure content of the trp repressor from Escherichia coli has been inferred from NMR and circular dichroic measurements; the results are compared with those of prediction algorithms. 70% of the amide protons have exchange rate constants orders of magnitude smaller than the intrinsic rate constants, identifying them as participating in hydrogen bonds. The exchange rate constants fall into two distinct classes, one having half-lives of 20 min and the other more than 24 h. The latter class, consisting of 50% of all amide protons, indicates a stable core. The exchange data are consistent with circular dichroism and predictions that suggest that about 55% of the peptides from alpha helix, and 20% form beta sheets and turns. The NMR spectrum further indicates that there is little beta sheet, suggesting that the secondary structure class is alpha. PMID- 2996891 TI - Identification of surface residues in the trp repressor of Escherichia coli. AB - A subset of the spin systems assigned in the 1H NMR spectrum of the trp repressor in the first paper in this series (our penultimate preceding paper in this journal) can be identified as surface or buried residues on the basis of four independent types of measurement: selective spin-lattice relaxation times; the dependence of line widths on temperature and the concentration of manganous ion; fluorescence quenching; and titration behaviour. Criteria are developed for distinguishing surface and buried residues. The significance for the function of DNA binding proteins is discussed. PMID- 2996892 TI - Chemical modification and nuclear magnetic resonance studies on human plasminogen kringle 4. Assignment of tyrosine and histidine resonances to specific residues in the sequence. AB - Modification of kringle 4 with tetranitromethane leads to the selective nitration of tyrosine 40 but on prolonged incubation with reagent, reaction of tyrosine 49 is also observed. Nitration of tyrosines 40 and 49 had no influence on the lysine Sepharose affinity of kringle 4, indicating that these residues are not important for the functional integrity of the ligand-binding site. Comparison of the NMR spectra of native kringle 4 with those of kringle 4 in which tyrosine 40 or tyrosines 40 and 49 are nitrated permitted the identification of the resonances of these residues. These NMR studies also showed that the chemical modifications caused little perturbation of the three-dimensional structure of the protein. Cross-linking of lysine 35 and tyrosine 40 with 1,3-difluoro-4,6-dinitrobenzene demonstrates that in the kringle-fold the reactive epsilon-amino and phenolic groups of these residues can approach each other to a distance of 0.5 nm. NMR spectra of this kringle 4 species also confirmed the assignment of the resonances to tyrosine 40. NMR spectra of a kringle 4 derivative in which the disulphide bridge between cysteines 1 and 79 has been broken by selective reduction and alkylation showed that the core structure of the kringle-fold and the lysine binding site are unaltered by this modification. This observation is in agreement with earlier results which showed that the lysine-Sepharose affinity of kringle 4 is not affected by reduction and alkylation of this disulphide bridge. Comparison of the NMR spectra of native and disulphide-cleaved kringle 4 aided in the assignment of resonances to residues adjacent to the site of modification (tyrosine 2 and histidine 3) and permitted the tentative assignment of the resonances of tyrosines 9 and 73. PMID- 2996893 TI - Two-dimensional 1H NMR investigation of ribonuclease A and ribonuclease-A- pyrimidine-nucleotide complexes. AB - Ribonuclease A was studied by two-dimensional 1H NMR spectroscopy. 10 out of 12 alanine and 9 out of 10 threonine spin systems as well as all valine [9], leucine [2] and isoleucine [3] spin systems were identified from the correlated spectroscopy (COSY) and relayed coherence transfer spectroscopy (RCT). Sequence specific assignments were obtained from nuclear Overhauser effect spectra for proton resonances of 21 amino acid moieties. 2' and 3'-pyrimidine-nucleotide RNase-A complexes were also investigated by two-dimensional NMR. We were able to monitor structural changes in the active center, the vicinity of the active center and in regions far from the catalytic region. Chemical shift changes of resonances of protons near Thr-45 reflected the binding of the same moiety. This in turn is also dependent on the position of the nucleotide phosphate group. Binding of 2' nucleotides led to characteristic changes in protein regions not affected by the binding of 3' nucleotides. These results are interpreted in terms of structural differences between the 2' and 3'-nucleotide-RNase-A complexes; the structure of the complex of the native 3' nucleotide inhibitor being more closely related to that of the free protein. PMID- 2996894 TI - Equilibrium binding of thioredoxin fB to chloroplastic fructose bisphosphatase. Evidence for a thioredoxin site distinct from the active site. AB - Thioredoxin fB, the protein activator of chloroplastic fructose 1,6 bisphosphatase, strongly binds its target enzyme with a stoichiometry of one protein dimer per enzyme tetramer. The thioredoxin binding site is distinct from the active site and the dissociation constant of the protein-enzyme complex has the extremely small value of 769 nM at pH 7.5. This interaction involves both ionic and hydrophobic contributions and is enhanced by a pH increase from 7 to 8. These results suggest that the above molecular properties may be involved in the light activation of chloroplastic fructose bisphosphatase. PMID- 2996895 TI - Photoaffinity labelling of the renal V2 vasopressin receptor. Identification and enrichment of a vasopressin-binding subunit. AB - To identify renal vasopressin receptor proteins, analogues of 1-deamino vasopressin i.e. ([1-(2-mercapto)propionic acid]vasopressin, [Mpa1]VP) with photoreactive aryl-azido groups in position 4 and 8 of the vasopressin sequence were prepared. In the absence of ultraviolet light, these ligands exhibit a high binding affinity for the V2 vasopressin receptor in plasma membranes from bovine and rat kidney medulla (apparent dissociation constants 1.8 X 10(-9) M to 1.7 X 10(-8)M); the photoreactive analogues stimulate the renal vasopressin-sensitive adenylate cyclase. In photoaffinity labelling experiments with tritium-labelled ligands (34-50 Ci/mmol), a membrane protein from bovine kidney or rat kidney medulla with an apparent relative molecular mass (Mr) of 30 000 was preferentially and specifically labelled. The labelling of the 30 000-Mr protein was completely inhibited by a 10-100-fold molar excess of vasopressin; in contrast, angiotensin II, bradykinin or low-affinity analogues of vasopressin did not suppress the incorporation of the reactive ligands into this protein. The highest specific labelling yield and only a low amount of unspecific labelling was obtained with the analogue [Mpa1,Lys(N6-4-azidobenzoyl)8]VP. Preparative sodium dodecyl sulfate gel electrophoresis of bovine kidney membranes photolabelled with this analogue resulted in a 20-30-fold enrichment of the 30 000-Mr vasopressin-binding protein. Our results suggest that this photoreactive analogue of [1-deamino, 8-lysine]vasopressin is a suitable tool for further purification of the renal V2 vasopressin receptor binding subunit. PMID- 2996896 TI - Analysis of secondary structures in M13mp8 (+) single-stranded DNA by the pausing of DNA polymerase alpha. AB - The pausing of DNA replication has been used as a tool for analyzing secondary structures in a single-stranded DNA. M13mp8 (+) single-stranded DNA was replicated in vitro by the DNA polymerase alpha from calf thymus. The positions of pausing were determined from DNA sequencing gels. All experimentally observed pausing sites could be correlated with computer-predicted secondary structures of the M13 single-stranded DNA. In the computer calculations of the secondary structures, long-range base-pairing, G.T mispairs and loop-out of bases were allowed. By using six different primers, the pausing site pattern and the corresponding secondary structure map of a region comprising 1400 nucleotides of the M13 genome has been established. Our experiments indicate that the M13 DNA is highly structured. Most of the stable structures are clustered around the origin of replication. With fragments of the M13 DNA, we show that long-range base pairing exists in the M13 single-stranded genome and we present evidence for tertiary structure interactions. Furthermore we observe structures that form newly during the course of replication. The Escherichia coli single-stranded DNA binding protein facilitates replication through the barriers. PMID- 2996897 TI - Analysis of modification-dependent structural alterations in the anticodon loop of Escherichia coli tRNAArg and their effects on the translation of MS2 RNA. AB - The conformation of the anticodon loop of Escherichia coli tRNAArg was investigated. It is shown that the structure of the anticodon loop is influenced by the base composition of the anticodon stem, and the natural modification of the nucleoside residue 32 in the anticodon loop. The structural effects detected by analysis of the accessibility of the anticodon loop to nuclease S1 could be correlated with the ability of different Arg-tRNAArg species to suppress frame shifting during translation of MS2 RNA. PMID- 2996899 TI - Chlamydia trachomatis infection monitoring with cytomorphology, direct immunofluorescence and "in vitro" growth method. AB - The increased occurrence of genital infections from Chlamydia trachomatis (Ct) suggested the need for a simple, rapid, sensitive method for detection of Ct. The purpose of the present study was to select symptomatic or asymptomatic women through two fast screenings: Pap-test and direct immunofluorescence (IF) test with monoclonal antibody. From 1,816 cervical cytology samples, 32 (1.76%) were selected for intracytoplasmic inclusions pathognomonic of Ct infection. Only 19 women underwent a check-up. The direct IF gave positive results in ten cases out of 19 (52.63%), and culture in eleven (57.89%). A correlation was made between the direct IF test and culture and also between cytologic and colposcipc findings. We thus conclude that direct IF, for its specificity, sensitivity, easy execution and low cost, could be currently utilized when clinical signs or Pap smears are suggestive of Ct infections. PMID- 2996898 TI - Demonstration of an Mg2+-induced conformational change by photoaffinity labelling of the high-affinity ATP-binding site of (Na+ + K+)-ATPase with 8-azido-ATP. AB - 8-Azido-ATP (8-N3ATP) is a substrate of (Na+ + K+)-ATPase from pork kidney and photoinactivates it by binding to the Mr = 100 000 alpha-subunit. The photoinactivation requires the presence of Mg2+ even though 8-azido-ATP is recognized by the high-affinity ATP binding site (Kd = 3.1 microM). K+ ions protect the enzyme against photoinactivation as does excess ATP. To see whether the Mg2+-requirement of the photoinactivation is due to the action of free Mg2+ or to the existence of an Mg X 8-azido-ATP complex, the action of the stable Mg X ATP complex analogue, chromium X 8-N3ATP (Cr X 8-N3ATP), was studied. Cr X 8 N3ATP photoinactivates (Na+ + K+)-ATPase in the absence of Mg2+, but the photoinactivation is enhanced by Mg2+, indicating that the formation of a Mg X ATP complex is an absolute requirement for photoinactivation. However, the interaction of Mg2+ with a low-affinity site also enhances the photoinactivation. It is therefore concluded that interactions with MgATP and free Mg induce conformational changes in the purine subsite of the high-affinity ATP binding site. Controlled trypsinolysis of the [alpha-32P]8-N3ATP-photolabelled enzyme in the presence of K+ results in the formation of an Mr = 56 000 radioactive peptide, whereas trypsinolysis of a [gamma-32P]Cr X ATP-labelled enzyme under identical conditions forms an Mr = 41 000 radioactive peptide. Extensive trypsinolysis of the [alpha-32P] 8-N3ATP-photolabelled alpha-subunit leads to the formation of a radioactive peptide of Mr = 1800. PMID- 2996900 TI - Association of human papilloma virus and Chlamydia trachomatis infections with incidence cervical neoplasia. AB - Out of a sample of 2,470 smears screened in order to identify of cervical intraepithelial neoplasia (CIN) 67 (2.712%) smears were selected. 39 (58.21%) of these showed evidence of human papilloma virus infection (HPV), 28 (41.79%) showed changes which suggested chlamydial infections (Ct). Results were verified by means of direct immunofluorescence tests (IF) and confronted with colposcopic and cytologic findings. The cytologic findings suggest a possible association between HPV and Ct infections with the incidence of the cervical neoplasia. PMID- 2996901 TI - Progress in gynecological oncology. PMID- 2996902 TI - A critical approach to gamma variate fit of radionuclide-angiography pulmonary histograms. AB - Radionuclide-angiography (RNA) left-to-right intracardiac-shunt quantification algorithms, based on the part-by-part fit technique and the use of a so-called gamma variate model function (GVF), were tested via simulation analysis using data obtained from normal subjects. A good bolus of radioindicator was obtained by administering it directly into the vena subclavia. Normal subjects were defined as those having pulmonary histograms (PH) with no visible distortion caused by a shunt. Pure, non-superimposed data on the downslope of the PH curves, which are lost in presence of a shunt, proved to be appropriate reference values for testing the accuracy of results of standard shunt quantification algorithms. A generalized four-parameter GVF was introduced in order to extend the flexibility of the model function. The use of the three-parametric GVF to reconstruct the downslope of the PH curve out of the upslope data proved to be inadequate. This reveals an evident source of error in algorithms that calculate the shunt contribution by fitting GVF parameters to so-called difference-curve data. It is concluded that the inherent restricted statistical weight of RNA data prevents accurate results being obtained from standard RNA-shunt-assessment algorithms. PMID- 2996903 TI - Preferential blood flow increase to the brain stem in moderate neonatal hypoxia: reversal by naloxone. AB - Previous studies have shown that severe neonatal asphyxia and hypoxia lead to a redistribution of cerebral blood flow (CBF) with a preferential perfusion of the brain stem. The present study shows that this mechanism is operative also in moderately hypoxic newborn lambs (oxygen saturation 32.7-65.2) with a threshold of about 25% reduction in oxygen saturation. In hypoxia, the mean increase in total CBF, brain stem and telencephalic blood flow was 44%, 68% and 43%, respectively (five lambs). We found that naloxone reverses this redistribution, and that the effects of naloxone on telencephalic perfusion and cerebral metabolic rate of oxygen (CMRO2) were proportional. In hypoxia + naloxone (1 mg/kg), a further increase in total CBF, brain stem, and telencephalic blood flow of 30%, 7% and 31% was noted. We therefore suggest that the redistribution of CBF is an important opioid-mediated homeostatic mechanism, which diminishes the metabolic requirements of the newer part of the brain in hypoxia and allows a preferential perfusion of the vital structures of the brain stem. PMID- 2996904 TI - Peripheral neuropathy in meningococcal septicemia. AB - We report a case of a mixed sensorimotor, predominantly axonal mononeuritis multiplex that developed after a severe meningococcal septicemia and disseminated intravascular coagulation (DIC) with associated distal limb necrosis. Ischemia resulting from the DIC-induced multiple vascular occlusions is suggested as the leading cause of this neuropathy. PMID- 2996905 TI - Haemodynamic effects of enalapril, a new converting enzyme inhibitor, in hypertensive patients. AB - The haemodynamic effects of enalapril (EN), a new, long-acting, nonsulphhydryl converting enzyme inhibitor, were evaluated by non-invasive methods in 10 adult patients with mild to moderate essential hypertension (EH). Patients were randomly assigned, double blind to 2 treatment groups (EN 20 mg o.d. or 10 mg b.d.) for 4 weeks, and were crossed over to the other dosage regimen after a 2 week washout period. Measurements included mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), limb blood flow (LBF), plasma aldosterone (ALD), plasma renin activity (PRA) and systolic time intervals (STI). Both regimens (b.d. and o.d.) significantly reduced MAP (15.3% and 16.3%, respectively), total peripheral resistance (20.3% and 21.8%, respectively), limb vascular resistance (24.1% and 24.9%) and ALD (33.5% and 36.9%) and increased CO (7.8% and 8.7%), LBF (10.9% and 11.6%) and PRA (10.4% and 9.5%). No significant change was observed in HR or STI. EN 20 mg o.d. or 10 mg b.d. reduced arterial pressure to a similar extent through a fall in total peripheral resistance. An increase in CO was also observed. PMID- 2996907 TI - Blood pressure response to enalaprilic acid in essential hypertension: dose response and effect of pre-treatment with furosemide. AB - Enalaprilic acid (MK 422), the active metabolite of enalapril, has recently become available for intravenous administration. In order to establish the proper dose for rapid blood pressure reduction, 9 patients with moderate to severe essential hypertension on a constant sodium intake of 100 mmol/24h were studied. They received four single doses of MK 422 according to an up-and-down titration schedule. Doses between 5 and 80 mg resulted in effective blood pressure reduction with an onset of action of about 10 minutes. Within this dose range the response was flat. No symptomatic hypotension was observed. The fall in blood pressure was less pronounced in patients with low initial plasma renin activity (PRA). Accordingly, a study was done to show whether the blood pressure response could be augmented by preceding stimulation of PRA by injection of 40 mg furosemide 15 minutes before the administration of MK 422. PRA increased after furosemide, but the blood pressure response to MK 422 was not augmented. PMID- 2996906 TI - The effect of pentoxifylline on filterability of normal red blood cells and their adhesiveness to cultured endothelial cells. AB - The effects of pentoxifylline on filterability of normal red blood cells (RBCs) and their adhesiveness to cultured endothelial cells were investigated. In a balanced randomized and double blind trial, six healthy volunteers received 400 mg pentoxifylline or matching placebo 2 h before blood samples were taken. Filterability of RBCs of the subject while on pentoxifylline was significantly increased at 25 degrees C and 18 degrees C. Lowering of the filteration temperature to 18 degrees C significantly decreased filterability of RBCs. In vitro studies showed that 12 micrograms/ml pentoxifylline significantly increased RBC filterability and also partially prevented the effect of decreasing temperature on RBC filterability. 12 micrograms/ml pentoxifylline significantly decreased the adherence of normal RBCs to cultured endothelial cells. Our results suggest that in addition to increasing filterability of RBCs, pentoxifylline also decreased the adherence of RBCs to endothelial cells and this may contribute to its therapeutic effect. PMID- 2996908 TI - Antibody density on rat red cells determines the rate of activation of the complement component C1. AB - It is a common observation that there is variability in the rate of activation of C1, the first component of complement, when bound to immune complexes. The cause of this variation has been investigated with experiments designed to assess separately the effect of antibody, antigen and C1 density. Using 125I-labeled C1 and a rat monoclonal antibody specific for the class I antigen, it has been found that the rate of activation is primarily dependent on antibody density on the cell surface and not on antigen or C1 density. This finding supports the suggestion that direct contact between the C1r2C1s2 subcomponent of C1 and antibody may be required for potentiation of C1 activation. PMID- 2996909 TI - Phencyclidine binding sites in the nucleus accumbens and phencyclidine-induced hyperactivity are decreased following lesions of the mesolimbic dopamine system. AB - [3H]Phencyclidine [( 3H]PCP) binding to rat nucleus accumbens, hippocampal and striatal membranes, and PCP-induced locomotor hyperactivity were assessed following selective lesions of the mesolimbic dopaminergic system. 6 Hydroxydopamine (6-OHDA) injections into the A10 region of the ventral tegmental area or into the accumbens itself resulted in a blockade of PCP's stimulatory effects and a highly significant reduction in the number of [3H]PCP binding sites and dopamine content of the nucleus accumbens. However, destruction of the dopaminergic mesolimbic fibers did not significantly alter hippocampal or striatal [3H]PCP binding. The data suggest that PCP elicits its locomotor stimulating effects via an interaction with PCP binding sites located mostly on mesolimbic dopaminergic terminals within the nucleus accumbens. PMID- 2996911 TI - Alpha 2-adrenoceptor coupling to adenylate cyclase in adrenaline insensitive human platelets. AB - Coupling of the alpha 2-adrenoceptor to adenylate cyclase was assessed in platelets from 3 groups of subjects: normal controls and patients with myeloproliferative disorders whose platelets were either sensitive or specifically insensitive to the aggregatory effects of adrenaline. The ability of adrenaline to induce the formation of a complex between the alpha 2-adrenoceptor and the inhibitory guanine nucleotide binding protein was examined in all three groups by assessment of the effect of mono and divalent cations and Gpp(NH)p on the displacement of [3H]yohimbine binding to platelet membranes by adrenaline. Coupling to adenylate cyclase was also assessed in separate studies of the inhibition by adrenaline of PGE1 stimulated cyclic AMP accumulation in whole platelets. Both the formation of the ternary complex and the inhibition by adrenaline of cyclic AMP accumulation was not significantly different in platelets sensitive or insensitive to adrenaline. These results suggest that inhibition of cyclic AMP alone does not explain the mechanisms of adrenaline induced platelet aggregation. PMID- 2996910 TI - Evidence for purinergic transmission in mouse bladder and for modulation of responses to electrical stimulation by 5-hydroxytryptamine. AB - Electrical stimulation (ES) contracted superfused mouse bladder, and 10(-7) M tetrodotoxin (TTX) abolished the twitches without impairing responses to acetylcholine (ACh) or beta,gamma-methylene ATP. ES acted largely through nerves which were not cholinergic, adrenergic or histaminergic. They may be purinergic because the bladder was contracted by stable analogues of ATP, and after desensitisation by a high concentration of alpha,beta-methylene ATP the response to ES was selectively reduced. 5-Hydroxytryptamine (5-HT) at 0.03-3 X 10(-6) M and tetraethylammonium (TEA) at 0.1-10 X 10(-3) M potentiated responses to ES, on average by 64% and 182%. Pempidine had no effect on responses to ES. The action of TEA was different from that of 5-HT; potentiation of responses was greater than could be produced by 5-HT, and whereas 5-HT did not increase responses to ACh, TEA markedly increased twitch tensions. The mode of action of 5-HT is not clear. PMID- 2996912 TI - Effect of mu and kappa opioid receptor agonists on rat plasma corticosterone levels. AB - The effect of several mu and kappa opioid receptor agonists on rat plasma corticosterone levels, measured using radioimmunoassay, was investigated. The mu agonists, morphine and fentanyl, and the kappa agonists, U-50,488, tifluadom and bremazocine, all produced dose-related increases in rat plasma corticosterone levels. The effects of both fentanyl and U-50,488 were reversed by naloxone, indicating an action at opioid receptors. Pretreatment of the rats with the irreversible, mu-selective antagonist, beta-funaltrexamine, reduced the effect of fentanyl, but not that of U-50,488, indicating that both mu and kappa opioid receptors are involved in mediating this effect. PMID- 2996913 TI - Effects of hycanthone on the neuromuscular transmission. AB - The effects of the antischistosomal drug, hycanthone, on the synaptic transmission at the frog neuromuscular junction were studied. The mean quantal content increased in the presence of 20 microM hycanthone. The amplitude of the miniature end-plate current was unaffected by 20 microM hycanthone, while 2 microM hycanthone decreased the ionophoretic ACh response (ACh induced current). The decay time constants of the evoked end-plate current and the miniature end plate current were increased with 1-5 microM hycanthone, but were decreased at concentrations over 20 microM. Analysis of the ACh induced noise revealed that 1 microM hycanthone slightly increased the channel lifetime whereas the single channel conductance was not affected. It was concluded that the primary site of action of hycanthone is the 'transient state' or ACh bound but closed conformation of the ACh receptor ion channel, but this drug also has other sites of action (presynaptic nerve terminal and open conformation of ACh receptor-ion channel complex). PMID- 2996914 TI - [Abstracts of the 2d symposium on Current Problems in Pediatric Endocrinology. Reinhardsbrunn, 9-12 April 1984]. PMID- 2996915 TI - Analysis of a tyrosine-specific protein kinase activity associated with the retroviral erbB oncogene product. AB - The transforming protein erbB of avian erythroblastosis virus (AEV) has considerable sequence homology with the epidermal growth factor (EGF) and appears to represent a truncated form of this receptor. The sequence of the erbB gene is furthermore related to that of other viral transforming genes such as src, fps, yes or abl. The transforming proteins of these src-related oncogenes as well as receptors for EGF, platelet-derived growth factor (PDGF), and insulin are associated with tyrosine-specific protein kinases. It has been difficult to demonstrate this activity for the erbB protein. To analyze the erbB gene product, we prepared polyclonal antibodies against a bacterially expressed erbB DNA restriction fragment (BamHI/BamHI). The antiserum is shown to immunoprecipitate the erbB protein from AEV-transformed chicken fibroblasts and also recognizes the EGF receptor protein. Both proteins become phosphorylated in vitro on tyrosine residues upon the addition of [gamma-32P]ATP. The protein kinase activity is low compared to other oncogene-specific kinases. This is not due to kinase blocking by the serum, because erbB carboxyterminal synthetic peptide antibodies give rise to low levels of protein kinase activity as well indicating that this may be a characteristic property of erbB in vitro. PMID- 2996916 TI - Polarized plasma membrane domains in cultured endothelial cells. AB - To determine whether distinct plasma membrane domains exist in endothelial cells, we infected monolayer cultures of macro- and microvascular endothelial cells with enveloped RNA viruses known to bud selectively from either the apical or basal surface in polarized epithelial cells. We found that vesicular stomatitis (VSV) and Sendai virus emerge asymmetrically from cultured endothelial cells. This provides direct evidence for the existence of polarized plasma membrane domains in vascular endothelial cells. PMID- 2996917 TI - Epidemiology of genital herpes simplex virus infection. PMID- 2996918 TI - Comments on the role of epidemiology in the investigation of hepatitis B virus. PMID- 2996919 TI - Noncontraceptive estrogen use and cardiovascular disease. AB - To summarize, estrogens have powerful effects on certain biologic parameters, the alteration of which could influence cardiovascular disease risk. Estrogens have long been known to influence lipid and lipoprotein levels by decreasing LDL (the atherogenic lipoprotein) and by increasing HDL (the protective lipoprotein). THese lipid alterations could favorably influence the risk of cardiovascular disease. Estrogens also temporarily increase glucose intolerance and lower fasting glucose levels; although the former event could adversely affect cardiovascular disease risk, the clinical significance of increased glucose intolerance with low fasting levels has not been determined. There is no consistent evidence that menopausal estrogens adversely affect coagulation or blood pressure levels, although both of these parameters could be affected in selected individuals. Overall, the estrogenic effects on lipid/lipoprotein levels appear to be the most consistent and the most powerful; given this assumption, estrogens should protect against cardiovascular disease. There is another interpretation of the biologic effect of estrogens on cardiovascular risk. It is possible that estrogens may increase the risk of a thromboembolic event due to an adverse influence on coagulation parameters and, at the same time, decrease the risk of an atherogenic event (via favorably altered lipid/lipoprotein levels). This proposed dual action of estrogens may explain the apparently conflicting results of increased risks of thromboembolism and decreased risks of atherosclerosis or myocardial infarction in men treated with high doses of estrogen (36, 196, 197). In women, there is some suggestion that (low-dose) postmenopausal estrogens may increase the risk of thromboembolism (79, 204), although the majority of studies report no such increase. The difference in risk of thromboembolism between men and women using estrogens may be due to several factors. First, the dose (potency) of the estrogen used by men is usually higher than that used by postmenopausal women, and the risk of estrogen-induced thrombus formation may be dose-dependent. Second, men tend to have more atherosclerotic lesions than women and thus would have more substrate available for thrombus formation. (This hypothesis is supported by the observation that the increased risk of thromboembolism was evident in men using estrogens for the secondary prevention of cardiovascular disease.) Indirect evidence supporting the hypothesis that endogenous estrogen levels are protective against cardiovascular disease is most consistent for women.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2996920 TI - An analysis of factors predisposing to chronic graft-versus-host disease. AB - Among 75 consecutive allogeneic bone marrow transplant recipients, 24 developed chronic graft-versus-host disease (GVHD), which was diagnosed from day 31 to day 368 after transplantation. Eight (33%) patients had more extensive chronic GVHD, and five patients died. The actuarial incidence of chronic GVHD was 48% at 400 days. A number of factors were analyzed for their association with chronic GVHD. Recipients of marrow from donors over 17 years of age had an actuarial incidence of chronic GVHD at 400 days of 74%, compared with 27% if the donors were under 17 years of age (log-rank test on survival curves, p less than 0.001). Other factors that appeared to predispose for chronic GVHD in bivariate analysis were recipient age above 17 years (p less than 0.02), treatment with donor unirradiated buffy coat cells (p less than 0.01), grade-II-IV acute GVHD (p less than 0.01), and a preceding cytomegalovirus (CMV) infection (p less than 0.01). In multi-variate analysis, however, only donor age above 17 years, treatment with donor buffy-coat cells, and grade-II-IV acute GVHD were significantly associated with chronic GVHD. The possible role of CMV infection in the development of chronic GVHD is discussed. PMID- 2996921 TI - Bronchiolo-alveolar carcinoma after inhalation of vegetable oil through a tracheal cannula. AB - Ten cases of carcinoma of the lung, mostly alveolar cell carcinomata, arising in patients after continuous use of (mostly mineral) oils as nose drops or laxatives, have till now been reported in the literature. We describe another such case, a man who used sesame oil to lubricate his tracheal cannula. PMID- 2996922 TI - Myeloperoxidase activity in smokers; an additional factor in the development of pulmonary emphysema. PMID- 2996923 TI - Increased neutrophil myeloperoxidase activity associated with cigarette smoking. AB - Neutrophils from 49 young male smokers contained significantly higher myeloperoxidase (MPO) activity than those from 49 age-matched, nonsmoking controls, while the elastase-like activity was not different in the two populations. MPO activity was increased in some smokers, but did not correlate significantly with the increased number of peripheral blood neutrophils, cigarette usage (present or cumulative), or the mild pulmonary dysfunction detected by forced expiratory spirometry and the single breath nitrogen test. This increased MPO activity in smokers' neutrophils may contribute to the greater risk of obstructive pulmonary disease in some smokers by an exacerbation of the protease-antiprotease imbalance in the lung. This hypothesis is supported by the prior observations that neutrophils are recruited in greater numbers into the lungs of smokers and that MPO (in the presence of H2O2 and chloride ion) oxidatively inactivates antiproteases of both the alveoli and the mucus-lined airways. PMID- 2996924 TI - An electrophysiological characterization of inhibitions and postsynaptic potentials in rat hippocampal CA3 neurones in vitro. AB - Inhibitory processes in the CA3 region of the rat hippocampal slice were studied extracellularly using paired stimuli and with intracellular impalements of pyramidal neurones. As with mossy fibre (MF) or commissural (COMM) conditioning stimuli (Kehl and McLennan 1983), activation of the perforant path (PP) input caused a long-lasting inhibition of test orthodromic population spikes (PSs) evoked by shocks delivered to the fimbria. That at least a portion of this orthodromically-evoked inhibition reflected postsynaptic events was shown by the reduction both of the amplitude of antidromic PSs and the firing rate of spontaneously active single units. Experiments in which the extracellular concentration of chloride was reduced indicated that only an early component of the inhibition was due to a conductance for that anion. The existence of two inhibitory mechanisms distinguishable extracellularly by their sensitivity to bicuculline and manipulation of extracellular ion concentrations was correlated intracellularly with two hyperpolarising peaks occurring approximately 20 and 150 ms following MF, COMM or PP stimuli. The later hyperpolarisation had an equilibrium potential 20-25 mV more negative than the early IPSP, was unaffected by manipulations of extra- or intracellular concentrations of chloride and was associated with a decrease of membrane resistance suggesting that a potassium conductance was involved in its generation. The fact that it was recorded in the absence of any preceding depolarisation, was blocked by drugs acting presynaptically to cause disinhibition (Kehl and McLennan 1985) and, like the early inhibition, was reversibly reduced by hypoxia suggested that the late inhibition/hyperpolarisation was a synaptic phenomenon rather than an intrinsic membrane event. Because the late inhibition/IPSP could be shown to have a lower threshold for activation vis-a-vis the chloride- dependent early inhibition, it is possible that two distinct populations of interneurones mediate these two synaptic events. PMID- 2996925 TI - Quantitative method for detection of blood-nerve barrier alterations in experimental animal models of neuropathy. AB - A solid phase radioimmunoassay was developed for measuring albumin concentrations in both endoneurium and serum, which were normalized to total endoneurium and serum protein to obtain a blood-nerve barrier index (BNB-index). The BNB-index in experimental lead neuropathy demonstrated barrier dysfunction beginning at 6 weeks of 4% lead carbonate ingestion and at 14 weeks was 5.2 times that of pair fed controls. These data, therefore, confirmed investigations that indicated a gradual alteration of the BNB beginning at 6 weeks and were based on (i) direct measurement of endoneurial albumin concentration by densitometry after sodium dodecyl sulfate-pore gradient electrophoresis and (ii) intravenous injection of 125I-albumin (J. F. Poduslo, P. A. Low, A. J. Windebank, P. J. Dyck, C. T. Berg, and J. D. Schmeltzer, 1982. J. Neurosci. 2: 1507-1514). The BNB-index after crush injury was 2.2 times that of control nerves at 24 h and gradually decreased toward normal values but was still 1.6 times that of controls at 70 days, a value consistent with the prolonged time course for complete repair. The BNB-index, therefore, can be used to evaluate BNB alterations quantitatively in animal models of neuropathy. Furthermore, we suggest that the BNB-index can also be used on biopsied, neuropathic, human sural nerve for evaluation of blood-nerve barrier abnormality. PMID- 2996926 TI - Aging of motoneurons and synaptic processes in the cat. AB - The aging of spinal cord alpha motoneurons was explored in old cats with intracellular recording techniques to determine the basic membrane properties of these neurons and their monosynaptic response following activation of group Ia afferent fibers. The conduction velocity of the motoneurons' axons decreased in old animals (14 to 15 years of age) compared with adult controls (1 to 3 years of age). The input resistance of the motoneurons increased in the old cats; no change occurred in the resting membrane potential or spike amplitude. There was a reduction in the delay between the initial segment and the somadendritic components of the antidromic spike. The half-width duration of the monosynaptic EPSP in the old cats increased, but its amplitude did not change. These data indicate that a host of different membrane properties of spinal cord motoneurons and their Ia-monosynaptic input are affected by the aging process. Analysis of the results suggests that the degradation of neuronal processes occurs in all motoneurons rather than preferentially affecting a specific population of motoneurons. PMID- 2996927 TI - Biogenesis of liver delta-aminolevulinate synthase. The role of cAMP in the induction. AB - In primary cultures of chick embryo hepatocytes pulse labeled with [35S]methionine, immunochemical analyses indicated that adenosine 3':5'-cyclic monophosphate (cAMP) did not affect either the rate of production or the maturation of delta-aminolevulinate synthase (ALA synthase). In addition, allylisopropylacetamide caused a slight drop in intracellular cAMP while testosterone caused the levels of cAMP to rise to 260% of the basal levels measured in hepatocytes in culture. Thus the results of this study did not indicate a direct short-term role for cAMP in the regulation of production of ALA synthase. PMID- 2996928 TI - Isolation and amino acid sequence of the 8 kDa DCCD-binding protein of beef heart ubiquinol:cytochrome c reductase. AB - The 8 kDa protein of beef heart ubiquinol:cytochrome c reductase was detected by means of a new SDS-PAGE [(1985) FEBS Lett. 190, 89-94] system and was isolated by a series of chromatographic steps involving dissociation of the complex by salt treatment. The amino acid sequence was determined by solid-phase Edman degradation of both the N-terminal part of the whole protein and proteolytic cleavage fragments of the protein. The protein consists of 78 amino acid residues: its Mr was calculated to be 7998. Structure predictions have been made from average and sided hydropathy profiles. The suggested structure encompasses an alpha-helix and a beta-strand, the latter comprising a glutamic acid residue situated in a relatively hydrophobic neighbourhood. This residue may be responsible for the fact that the 8 kDa protein is the first subunit of the whole reductase (consisting of 11 subunits) to be labelled by DCCD when the reductase is in free form or inlaid in phospholipid vesicles. PMID- 2996929 TI - Stimulation of procollagenase synthesis in human rheumatoid synovial fibroblasts by mononuclear cell factor/interleukin 1. AB - In order to define mechanisms regulating the synthesis of procollagenase in human rheumatoid synovial fibroblasts, the proteins synthesized by cultured cells were labeled with [35S]methionine. Labeled medium proteins were analyzed by SDS-PAGE directly and after immunocomplexing with a specific antibody to human fibroblast collagenase. Labeling of both the predominant form of the enzyme (Mr approximately 55 000) as well as a minor species (Mr approximately 61 000) was increased following incubation with the monokine, mononuclear cell factor/interleukin 1. The approximately 61 kDa form of the procollagenase appears to be a glycosylated form of the approximately 55 kDa precursor based on binding to Con A-Sepharose and decrease in the approximately 61 kDa form after culture in the presence of tunicamycin. Thus, mononuclear cell factor, homologous with interleukin 1, partially purified from monocyte conditioned medium increased incorporation of [35S]methionine into several medium proteins, including those complexed by the anticollagenase antibody. In the presence of mononuclear cell factor/interleukin 1, labeling of the procollagenase was increased 12-14-fold over control cultures incubated with medium alone. Therefore, one of the mechanisms involved in increase of collagenase activity in the medium of cultured synovial fibroblasts in the presence of mononuclear cell factor/interleukin 1 is a stimulation of enzyme protein synthesis. PMID- 2996930 TI - 1-N6-Etheno-ADP-ribosylation of elongation factor-2 by diphtheria toxin. AB - Diphtheria toxin fragment A is able to inhibit protein synthesis in the eukaryotic cell by ADP-ribosylating the diphthamide residue of elongation factor 2 (EF-2) [(1980) J. Biol. Chem. 255, 10710-10720]. The reaction requires NAD as ADP-ribose donor. This work reports on the capacity of an NAD analog, the nicotinamide 1-N6-ethenoadenine dinucleotide (epsilon NAD), to be a substrate of diphtheria toxin fragment A in the transferring reaction of the fluorescent moiety, the epsilon ADP-ribose, to the EF-2. As a consequence of the transfer of the epsilon ADP-ribosyl moiety to the EF-2, there is an increase in the emission intensity of the fluorophore and a blue shift in its emission maximum. The epsilon ADP-ribosylated EF-2, like ADP-ribosylated EF-2, retains the capacity to bind GTP and ribosome. The utility of introducing a fluorescent probe in a well defined point of the EF-2 molecule for conformational or binding studies is discussed. PMID- 2996931 TI - Induction of protein kinase C activation and Ca2+ mobilization by fibroblast growth factor in Swiss 3T3 cells. AB - Addition of fibroblast growth factor (FGF) to quiescent cultures of Swiss 3T3 cells rapidly induced diacylglycerol formation, protein kinase C activation and Ca2+ mobilization. Protein kinase C-activating agents such as 12-O tetradecanoylphorbol-13-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG) mimicked the action of FGF and stimulated DNA synthesis in the presence of insulin. Prolonged treatment of the cells with phorbol-12,13-dibutyrate (PDBu) led to the down-regulation and complete disappearance of protein kinase C. In these cells, TPA and OAG did not induce DNA synthesis any more. FGF still elicited Ca2+ mobilization and DNA synthesis, but the magnitude of DNA synthesis was reduced to almost half as compared with that in the control cells. These results clearly indicate that both diacylglycerol and Ca2+ may serve as second messengers for FGF and suggest that these messengers may be involved in the mitogenic action of this growth factor. PMID- 2996932 TI - Involvement of lysine residues in the binding of ovine prolactin and human growth hormone to lactogenic receptors. AB - The lactogenic activity (L.A.) of oPRL and hGH derivatives obtained by chemical modifications of lysine residues was studied by radioreceptor assay. Control treatment with borohydride had a slight effect on the L.A. of hGH but drastically reduced the oPRL activity; this latter was preserved in the presence of iodoacetamide. Methylation, ethylation, guanidination and acetimidination affected the L.A. of both hormones as a function of the degree of modification. The structure-binding relationships to the lactogenic receptors are discussed, suggesting that the lysine or arginine residues in homologous positions 42, 51, 73, 128, 146 of oPRL and 47, 50, 73, 128, 147 of hGH might be particularly involved. PMID- 2996934 TI - An opsonised microelectrode. Observation of the respiratory burst of a single human neutrophil. AB - A 10 micron diameter gold microvoltammetric electrode, opsonised with human IgG, was used to study the respiratory burst of a single human neutrophil. The electrode oxidised superoxide produced near its surface by the neutrophil back to dioxygen. It is suggested that the current so detected is proportional to the rate of superoxide production by the NADPH oxidase of a single cell. In all cases the response consisted of a relatively rapid rise in current after cell addition, followed by a 2-phase decay. It is further suggested that this complex decay results from the production of superoxide being rate-limited initially by the NADPH concentration and later by the coupled metabolism of the hexose monophosphate shunt. PMID- 2996933 TI - A novel side-chain-linked antiparallel cyclic dimer of enkephalin. AB - The dimeric cyclic enkephalin analog, (H-Tyr-D-Lys-Gly-Phe-Glu-NH2)2, was isolated as a second major component from the crude product obtained in a solid phase synthesis of the corresponding cyclic monomer, H-Tyr-D-Lys-Gly-Phe-Glu-NH2. In comparison with [Leu5]enkephalin the cyclic dimer is about equipotent in assays representative for mu-opioid receptor interactions and 1/10 as potent at the delta-receptor. The fact that the enkephalin dimer shows a receptor selectivity pattern distinct from that of the cyclic monomer and of the corresponding linear analog suggests that cyclodimerization via side-chain linkages might be generally useful as a means to produce shifts in the activity profiles of peptide hormones and neurotransmitters. PMID- 2996936 TI - Further comments on the logic of the application of uncoupler- inhibitor titrations for the elucidation of the mechanisms of energy coupling. AB - Following the appearance of two papers in this journal commenting on the logic of the application of uncoupler-inhibitor titrations as a means of discriminating between 'delocalized' and 'localized' chemiosmotic mechanisms [(1984) FEBS Lett. 176,79-82; (1985) FEBS Lett. 186, 8-10], and in contrast with the arguments presented there and elsewhere, we show that in a linear model the increase in delta mu H which accompanies partial inhibition of the ATPases always leads to a relatively higher decrease of the rate of ATP synthesis by a given concentration of uncoupler in the presence of an ATPase inhibitor than in its absence. This is due to the fact that the same titre of uncoupler leads to a higher dissipative H+ flow in the presence of inhibitor, since the driving force delta mu H is higher. PMID- 2996935 TI - Enhancement of collagen-induced phosphoinositide turnover by thromboxane A2 analogue through Ca2+ mobilization in human platelets. AB - In human washed platelets, collagen-induced phosphoinositide turnover was inhibited by indomethacin, an inhibitor of thromboxane A2 (TXA2) formation, particularly at lower doses of collagen. This inhibition was counteracted by the addition of 9,11-epithio-11,12-methano-TXA2 (STA2), a stable analogue of TXA2 as well as by the Ca2+ ionophore A23187. STA2 and A23187 did not stimulate phosphoinositide turnover markedly, but significantly increased cytoplasmic free Ca2+ concentrations. The actions of STA2 were blocked by 13-azaprostanoic acid, a TXA2 receptor antagonist. The results suggest that TXA2 is generated during the action of collagen and increases cytoplasmic free Ca2+ which then stimulates phosphoinositide turnover in cooperation with collagen. PMID- 2996937 TI - Calcium-activated, phospholipid-dependent protein kinase in cultured rat mesangial cells. AB - The analysis of the 100 000 X g supernatant fraction of cultured rat glomerular mesangial cells with DEAE-cellulose ion-exchange chromatography revealed a large peak showing the activity of a protein kinase (protein kinase C) which depended on phospholipid and diolein as well as Ca2+. Furthermore, it was shown that angiotensin II (AII) (10(-6)M) induced rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate, leading to production of diacylglycerol rich in arachidonic acid, in the cultured rat mesangial cells. These results suggest that activation of protein kinase C resulting from enhancement of phosphoinositide metabolism may be important as an intracellular regulatory mechanism of AII upon cultured mesangial cells. PMID- 2996938 TI - Atrial natriuretic factor inhibits the early pathway of steroid biosynthesis in bovine adrenal cortex. AB - We have previously determined that atrial natriuretic factor (ANF) is a potent inhibitor of steroid secretion in cultured bovine zona glomerulosa and fasciculata cells. The present report describes a comparison of the effect produced by ANF on aldosterone, deoxycorticosterone and progesterone secretions by zona glomerulosa cells and on cortisol, corticosterone and progesterone secretions by zona fasciculata cells. The equipotent inhibitory action of ANF on the stimulated secretion of these steroids in both cell types indicates a common site of action prior to progesterone synthesis at which ANF inhibits the steroidogenic pathway. PMID- 2996940 TI - Structural analysis of a new GC-specific insertion element IS186. AB - A new insertion sequence, IS186, has been identified from Escherichia coli and sequenced. It contains 1336 base pairs with a terminal inverted repeat of 22 nucleotides. A long open reading frame, from an ATG codon at position 55 to a TAG termination codon at 1327, could code for a polypeptide of 424 amino acids. This element recognizes GC-rich regions as target sites for insertion. PMID- 2996939 TI - Myeloperoxidase oxidation of sulfur-centered and benzoic acid hydroxyl radical scavengers. AB - Myeloperoxidase (MPO) oxidizes sulfur-centered and benzoate hydroxyl radical scavengers through formation of HOCl. Sulfur-centered hydroxyl radical scavengers compete with benzoate as antioxidants of HOCl. We conclude from these observations that competition experiments between benzoate and sulfur-centered hydroxyl radical scavengers are not sufficiently specific to infer participation of hydroxyl radicals in oxidative reactions mediated by neutrophils because of the unique action of MPO in affecting oxidation of the test radical scavengers. PMID- 2996941 TI - Specific transcription of orthopox virus DNA by HeLa cell RNA polymerase II. AB - A HeLa cell extract was used to transcribe DNA isolated from cowpox virus. Truncated templates generate accurately initiated run-off transcripts of discrete sizes and whose sensitivity to inhibition by alpha-amanitin indicates synthesis by cell RNA polymerase II. A mapped restriction fragment of wild-type cowpox DNA contains specific sites of initiation which are not detected in the geographically equivalent fragment from a cowpox mutant having a defined sequence rearrangement in this region. PMID- 2996942 TI - A restriction endonuclease SuaI from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. AB - A type II restriction endonuclease (SuaI) has been isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. The enzyme is an isoschizomer of BspRI. It does not cut S. acidocaldarius DNA, as the recognition sequence GGCC in this DNA contains modified nucleotide(s). The enzyme is most active at 60-70 degrees C and is highly thermostable. PMID- 2996943 TI - Activation of yeast plasma membrane ATPase by phorbol ester. AB - Addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) to yeast cells produces a 2-fold activation of the plasma membrane ATPase. The activation is reversible and time-and dose-dependent. The activated enzyme shows an increased affinity for its substrate, ATP, and its optimum pH is shifted to a more alkaline range. These changes are similar to those observed in the reported activation by glucose. Upon incubation of yeast cells with 32Pi incorporation of radioactivity in a membrane polypeptide of 105 kDa is observed after addition of either glucose or TPA. PMID- 2996944 TI - Physiological and metabolic effects of dietary fiber. AB - William Beaumont noted the gastric effects of vegetable fiber and suggested that dietary fiber may provide health benefits. In the last decade investigators documented the physiological effects of fiber on gastric emptying, intestinal nutrient absorption rates, and colon function. Further clinical investigation and much more of the type of repetitive observations pioneered by Beaumont are required to definitively establish the physiological effects of fiber on gastrointestinal physiology. High-fiber intake provides well-established benefits for persons with diabetes: it lowers insulin requirements, provides better control of blood glucose, and reduces serum lipids. Foods rich in soluble fiber, such as oat or bean products, lower cholesterol significantly for persons with hypercholesterolemia and for healthy young subjects. High-fiber foods also lower serum triglycerides and blood pressure. Several studies indicate that high intake of fiber protects against coronary heart disease. PMID- 2996945 TI - Proteolytic enzymes, past and present. AB - William Beaumont's pioneering research on gastric secretion has been germinal in the discovery of proteolytic enzymes and the elucidation of their chemical structure, physiological roles, and biochemical evolution. Although the mammalian digestive enzymes, notably those of gastric and pancreatic origin, have been among the best characterized, of even greater interest and complexity are those that fulfill regulatory functions by limiting their action on specific peptide bonds in target protein substrates. The difference between digestive and regulatory proteases can best be understood by considering their evolutionary relationships on the basis of the organization of both their genes and the proteins themselves. An analysis of representative members of protease families, notably the mammalian serine proteases, suggests that they are the products of processes of recombination of gene segments that give rise to functionally and structurally distinct domains. The evolutionary variability introduced by combinations of domains appears to be far more restricted than if each protein molecule were the product of a single and unique evolutionary event. PMID- 2996946 TI - Macrophages as effector cells. Introduction. PMID- 2996947 TI - Molecular basis for the enhanced respiratory burst of activated macrophages. AB - Macrophages elicited by injection of agents that produce inflammation or obtained from animals infected with intracellular parasites are primed so that they respond to phagocytosis or exposure to phorbol myristate acetate with a marked increase in the respiratory burst. This capacity to respond to stimulation with increased release of reactive oxygen metabolites appears to play an essential role in the increased microbicidal capability of activated macrophages. Macrophages can be primed for this capacity by incubation in vitro with bacterial products, proteases, or gamma interferon. The molecular basis for this priming is presently under investigation. An increase in the number or affinity of plasma membrane receptors does not appear to explain priming. Changes in one or more of the transduction events responsible for stimulus-response coupling might lead to more efficient stimulation or function of the enzyme responsible for the respiratory burst; these events are just beginning to be studied in macrophages. Priming can be explained at least in part by a modification of the respiratory burst enzyme such that it binds its substrate NADPH, the source of electrons for reduction of oxygen to superoxide anion, more efficiently. Understanding the molecular basis for priming of the respiratory burst might permit its eventual therapeutic manipulation. PMID- 2996950 TI - A simple improvement in the technique of hysterosalpingography achieving optimal imaging and avoiding possible complications. PMID- 2996948 TI - Lipoxygenase pathways of macrophages. AB - Resident mouse peritoneal macrophages when exposed to zymosan during the first day of cell culture synthesize and secrete large amounts of prostaglandin E2 and leukotriene (LT) C4, the respective products of cyclooxygenase- and 5 lipoxygenase-catalyzed oxygenations of arachidonic acid. Under these conditions of cell stimulation only small amounts of hydroxyeicosatetraenoic acids (HETEs) are concomitantly produced. However, exogenously added arachidonic acid is metabolized to large amounts of 12- and 15-HETE. No LTC4 is formed under these conditions. Inasmuch as 12- and 15-HETE have been shown to modulate certain lymphocyte responses, further study of the regulation of their production by macrophages is warranted. PMID- 2996949 TI - Effect of gestrinone in endometriosis tissue and endometrium. AB - The effects of gestrinone (R 2323) on endometrial and endometriosis tissue concentrations of cytosol estrogen and progestin receptors and the activity of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) were investigated in 11 patients operated on because of suspected external endometriosis. Serum concentrations of luteinizing hormone, follicle-stimulating hormone, estradiol (E2), progesterone, testosterone (T), and sex-hormone-binding globulin (SHBG) were also investigated. After one control cycle, the patients received 2.5 mg of oral gestrinone twice weekly from the fifth day of the first treatment cycle until the eighth day of the second treatment cycle, the day of operation being day 10. Treatment with gestrinone decreased serum concentrations of T during the second treatment cycle and effected a major decrease in SHBG during both treatment cycles, resulting in highly increased free T and free E2 indices. The effects of gestrinone on the endometrium, a decrease in estrogen and progestin receptors, and induction of 17 beta-HSD are characteristic progestin actions. These parameters remained unchanged in endometriosis tissue. Our data indicate that gestrinone has effects that are typical of androgens and progestins in patients with endometriosis. PMID- 2996951 TI - The effects of spironolactone on adrenal steroidogenesis in hirsute women. AB - The effect of spironolactone on adrenal androgen and cortisol production was studied in six hirsute women. Hirsute women were evaluated before and 1 month after receiving 200 mg of spironolactone daily. Basal levels of serum androgens, 17-hydroxyprogesterone (17-OHP), cortisol (F), corticosteroid-binding globulin, and plasma adrenocorticotropic hormone (ACTH) were normal and did not change with therapy. The delta maximum (delta max) responses after dexamethasone suppression and ACTH administration of dehydroepiandrosterone (DHEA), androstenedione (delta 4A), 17-hydroxypregnenolone, and 17-OHP were similar in hirsute women and ovulatory control subjects. After spironolactone administration, the delta max DHEA response was unchanged, whereas the delta max delta 4A response was decreased (P less than 0.05). The delta max ratios of DHEA/delta 4A and 17 OHP/delta 4A were significantly increased after spironolactone in hirsute women, which suggested inhibitions of 3 beta-ol-dehydrogenase-isomerase and delta 4 17,20 desmolase activities. A significant reduction in delta max F occurred after spironolactone administration (P less than 0.05). Although baseline 11 desoxycortisol (S) and the plasma S/ACTH ratio were unaltered, the delta max S/F ratio increased after treatment (P less than 0.01), suggesting an inhibition of 11 beta-hydroxylase activity. Inhibition of adrenal androgen production occurs with spironolactone, but only serum levels of delta 4A are decreased, whereas DHEA and its sulfate (DHEA-S) levels remain unchanged. PMID- 2996953 TI - [Possible mechanisms of the spread of inhibition in the cerebral cortex]. PMID- 2996952 TI - Down-regulation of testicular follicle-stimulating hormone receptors by human menopausal gonadotropin in infertile men. AB - We measured testicular FSH receptors in 27 infertile men before, or 1, 3, 5, 7, or 14 days after a single administration of 150 IU of hMG. The administration of hMG reduced Bmax for FSH receptors to about 50% of that in the preadministration testes for 5 days. From the seventh day, Bmax of FSH receptors began to increase and returned to the preadministration level 14 days after the administration. PMID- 2996954 TI - [Image diagnosis of adrenal disorders. III. "Nonfunctioning" adrenal adenoma, weak mineralocorticoids producing adrenal carcinoma, congenital adrenogenital syndrome due to 21-hydroxylase deficiency--simple virilizing form, and pheochromocytoma]. AB - The image diagnoses of a case of so-called "nonfunctioning" adrenal adenoma, weak mineralocorticoids producing adrenal carcinoma, congenital adrenogenital syndrome due to 21-hydroxylase deficiency--simple virilizing form--, and 5 cases of pheochromocytoma were studied. In a patient with so-called "nonfunctioning" adrenal adenoma (2.3 X 3.0 X 3.3 cm), in which steroids biosynthesis was confirmed, computed tomography (CT) delineated the tumor shadow with extremely low density, and ultrasonography (US) demonstrated the round tumor echo with homogenous and low echogenicity at the superior region of the right renal pole. Adrenal scintigraphy also showed the tumor image. A weak mineralocorticoids producing left adrenal carcinoma (3.5 X 3.5 X 3.0 cm) was shown as a heterogenous round tumor at the left lateral portion of the vertebra by CT. On adrenal scintigraphy under dexamethasone pretreatment, there was good uptake in the tumor and disappearance of the contralateral. Both bilateral adrenal images on CT in a patient with congenital adrenogenital syndrome were linear-shaped and markedly enlarged. The enlarged right adrenal was clearly demonstrated by US with an electronic sector scanner but not with an electronic linear scanner, although the left one was hardly shown by either US instruments. Three of 4 patients with pheochromocytomas examined by US were correctly detected, while in the remaining one the tumor image was judged to be a retroperitoneal tumor. CT also correctly demonstrated the former 3 pheochromocytomas, but misjudged the latter one as a pancreatic cancer. Good uptake of Adosterol by bilateral adrenals was shown in a case of extra-adrenal pheochromocytoma. Three of 4 cases of adrenal pheochromocytoma showed the isotope uptake of the contralateral normal adrenal alone. In another case of right adrenal pheochromocytoma, isotope accumulation in the colon obscured whether the isotope uptake in the right adrenal was shown or not. PMID- 2996955 TI - Malignant mixed mesodermal tumor of ovary. AB - A case of primary malignant mixed mesodermal tumor (MMMT) of the ovary arising de novo in a post-menopausal multiparous female is reported. The tumor contained heterologous elements in the form of cartilage and fat in addition to carcinosarcomatous areas. Post-operatively, single-agent chemotherapy was administered and up to the time of reporting the patient has been alive and well. Because of the rarity of these tumors the proper treatment remains a dilemma. The differential diagnosis of MMMT of ovary and teratocarcinoma is considered, as the two are confused frequently. PMID- 2996956 TI - Sensitivity of 5'-nucleotidase and phospholipase A2 towards liver plasma membranes modifications. AB - The changes in the phospholipid and fatty acid composition of liver plasma membranes isolated from rats, fed two different diets, containing either saturated or unsaturated fatty acids, were investigated. We established that dietary treatment can considerably modify the fatty acid as well as the phospholipid composition of liver plasma membranes. Lipid transfer proteins were used for enrichment of liver plasma membranes with sphingomyelin, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and phosphatidylinositol. A marked sphingomyelin and membrane fluidity dependence of the membrane-bound 5'-nucleotidase and phospholipase A2 was observed. PMID- 2996957 TI - Purification and preliminary characterization of phosphoglycerate mutase from Schizosaccharomyces pombe. AB - Phosphoglycerate mutase could be purified to over 95% homogeneity by a single step procedure involving elution from Cibacron Blue-Sepharose by a pulse of cofactor 2,3-bisphosphoglycerate. Although the enzyme has been isolated in only small quantities (c. 100 micrograms), gel filtration and sodium dodecylsulphate polyacrylamide gel electrophoresis indicated that it is monomeric with Mr approximately 23,000, an extremely low value for this enzyme. Preliminary investigations of the kinetic characteristics and the nature of important amino acid side chains have been undertaken. PMID- 2996958 TI - Appearance of nerve growth factor receptors on cultured neural crest cells. AB - Light microscopic radioautography of differentiating quail neural crest cultures (1 to 2 weeks after explanation) incubated with Iodine-125-labeled nerve growth factor (125I-NGF) revealed that approximately 35% of the cells bound NGF. The binding was specific and saturable; it was blocked by an excess of nonradioactive NGF, and was not detected following incubation with biologically inactive 125I NGF. In addition, the binding did not appear to be blocked or diminished by insulin. Cell cultures prepared from somites or notochord showed no specific binding of 125I-NGF. Melanocytes comprised approximately 10% of the cell population in these cultures and appeared to be unlabeled. The subpopulation of cells with NGF receptors that were morphologically similar to other non melanocyte unlabeled cells present in the neural crest cultures are probably the targets of the factor during differentiation and development. In contrast, there was no evidence of 125I-NGF binding by premigratory neural crest (adherent to the isolated neural tube) or by early migratory neural crest cells (24 hr after explantation). Both of these types of neural crest cells are relatively undifferentiated. The cells of the neural tube were also unlabeled. The binding of 125I-NGF to differentiating neural crest cells was not noticeably diminished by a brief pretreatment with trypsin or Dispase, enzymes used in the isolation of neural tubes. Hence, the absence of NGF receptors on premigratory neural crest and early migratory neural crest cultures was not due to enzymatic alterations of the receptor. It seems, therefore, that receptors for NGF appear on neural crest cells during the time when these cells are acquiring their phenotypic characteristics. PMID- 2996959 TI - Placodal sensory neurons in culture: nodose ganglion neurons are unresponsive to NGF, lack NGF receptors but are supported by a liver-derived neurotrophic factor. AB - Explant and dissociated neuron-enriched cultures of nodose ganglia (inferior or distal sensory ganglion of the Xth cranial nerve) were established from chick embryos taken between embryonic Day 4 (E4) and Day 16 (E16). The response of each type of culture to nerve growth factor (NGF) was examined over this developmental range. At the earliest ages taken (E4-E6), NGF elicited modest neurite outgrowth from ganglion explants cultured in collagen gel for 24 hr, although the effect of NGF on ganglia taken from E4 chicks was only marginally greater than spontaneous neurite extension from control ganglia of the same developmental age. The response of nodose explants to NGF was maximal at E6-E7, but declined to a negligible level in ganglia taken from E9-E10 or older chick embryos. In dissociated neuron-enriched cultures, nodose ganglion neurons were unresponsive to NGF throughtout the entire developmental age range between E5 and E12. In contrast to the lack of effect of NGF, up to 50% of nodose ganglion neurons survived and produced extensive neurites in dissociated cultures, on either collagen- or polylysine-coated substrates, in the presence of extracts of late embryonic or early posthatched chick liver (E18-P7). Antiserum to mouse NGF did not block the neurotrophic activity of chick (or rat or bovine) liver extracts. Whether cultured with chick liver extract alone or with chick liver extract plus NGF, nodose ganglion neurons taken from E6-E12 chick embryos and maintained in culture for 2 days were devoid of NGF receptors, as assessed by autoradiography of cultures incubated with 125I-NGF. Under similar conditions 70-95% of spinal sensory neurons (dorsal root ganglion--DRG) were heavily labeled. 2+ PMID- 2996960 TI - Glucokinase is not the pancreatic B-cell glucoreceptor. AB - A series of recent experimental findings are reviewed to indicate that glucokinase does not represent the pancreatic B-cell glucoreceptor. Whether in liver, pancreatic islet or insulin-producing tumoral cell homogenates, glucokinase fails to yield a higher reaction velocity with alpha-than beta-D glucose. At a high glucose concentration (40 mmol/l), when the phosphorylation of glucose by glucokinase is indeed higher with beta- than alpha-D-glucose, no preference for beta-D-glucose is observed in intact islets, as judged from the utilization of D-[5-3H]glucose, production of lactic acid, oxidation of D-[U 14C]glucose, net uptake of 45Ca or release of insulin. The glucose 6-phosphate content of intact islets is higher in the presence of beta- than alpha-D-glucose. At a low glucose concentration (3.3 mmol/l), when the participation of glucokinase to hexose phosphorylation is minimal, alpha-D-glucose is still better metabolized and stimulates both 45Ca net uptake and insulin release more efficiently than beta-D-glucose, despite the fact that hexokinase yields a higher reaction velocity with beta- than alpha-D-glucose. In intact islets, beta-D glucose is used preferentially to alpha-D-glucose in the pentose cycle pathway as judged from the oxidation of alpha- or beta-D-[1-14C]glucose relative to that of alpha- or beta-D-[6-14C]glucose. In islets removed from fasted rats, the rate of glycolysis is more severely decreased than expected from the repression of glucokinase. The metabolism of glucose in tumoral insulin-producing cells differs, in several respects, from that in normal pancreatic islets, although the pattern of hexokinase and glucokinase activities is similar in these two types of cells. All these observations point to the participation of regulatory sites distal to glucose phosphorylation in the control of glucose metabolism in islet cells. PMID- 2996962 TI - Safety of hepatitis B vaccine confirmed. PMID- 2996963 TI - Progress on AIDS. PMID- 2996961 TI - Insulin-gene flanking sequences, diabetes mellitus and atherosclerosis: a review. AB - A highly polymorphic locus flanking the human insulin gene contains two major size classes of DNA restriction fragments, which segregate in families as stable genetic elements. The L-allele, i.e. fragments with an average size of about 600 base-pairs seems to be a weak genetic marker for Type 1 (insulin-dependent) diabetes mellitus, whereas the U-allele, i.e. fragments of an average size of about 2500 base-pairs hitherto has been associated with Type 2 (non-insulin dependent) diabetes mellitus and diabetic hypertriglyceridaemia. The most recent reports on this subject do not confirm an association between the U-allele and Type 2 diabetes. Our own studies indicate that the U-allele is a fairly strong marker for the development of atherosclerosis (relative risk for U-carriers 3.36). The putative functions of the polymorphic region in atherogenesis and the relation of this region to other genetic markers for atherosclerosis are not known. PMID- 2996964 TI - Pig gastric mucus: a one-way barrier for H+. AB - Gastric mucus is thought to protect the underlying mucosal cells from mechanical hazards and back-diffusion of luminal H+. In health, a pH gradient exists across the mucus layer from the variable low pH of the lumen to a pH approaching neutrality at the epithelial cell surface. By current hypotheses this gradient is maintained by the combined effects of an unstirred layer, restricted or slowed diffusion of H+ in the mucus, and the epithelial cell secretion of bicarbonate, which is confined to the cell surface by the mucus layer. These mechanisms do not explain how H+ is secreted through mucus in the first place. Using a modified diffusion chamber we have shown that pig gastric mucus facilitates a low efficiency Na+/H+ exchange--a property that helps to clarify some previously unexplained components of H+ secretion. When a solution containing Na+ was separated by a layer of fresh pig gastric mucus from a solution of similar pH containing a much lower concentration of sodium, the sodium-rich solution was electrically negative relative to the sodium-poor solution and its pH decreased significantly with time. A similar pH gradient developed when the barrier was a synthetic cation-exchange membrane, and one of opposite sign when it was an anion exchanger; no pH gradient developed across neutral barriers. It is suggested that similar electrical coupling of H+ diffusion to active Na+ transport might in vivo ensure that secreted H+ moves into the gastric lumen. PMID- 2996965 TI - Opisthorchis viverrini infection and cholangiocarcinoma. PMID- 2996966 TI - [Current trends in the development of the science of coagulation abroad]. PMID- 2996967 TI - [Sorbamine--a new infusion medium for correcting metabolic alkalosis]. PMID- 2996968 TI - Is there a basis for distinguishing two types of P2-purinoceptor? AB - It is suggested that the P2-purinoceptor may be separated into two subtypes largely on the basis of the rank order of agonist potency of structural analogues of ATP and also on the activity of antagonists at the P2-purinoceptor: Subtype 1 (designated P2X), potency order: alpha, beta-methyleneATP, beta, gamma methyleneATP greater than ATP = 2 methylthioATP; antagonism by ANAPP3 and selectively desensitisation following administration of alpha, beta-methyleneATP; present in the vas deferens and urinary bladder of guinea-pig and rat, frog and rat ventricle, and also in the smooth muscle of the rat femoral artery and rabbit central ear artery, where they mediate excitation. Subtype 2 (designated P2Y), potency order: 2-methylthioATP much greater than ATP greater than alpha, beta methyleneATP, beta, gamma-methyleneATP; weak antagonism by ANAPP3 and desensitisation following administration of alpha, beta-methyleneATP; present in the guinea-pig taenia coli and the longitudinal muscle layer of the rabbit portal vein, where they mediate relaxation and also on the vascular endothelial cells of the rat femoral artery and pig aorta (where occupation leads to the production of endothelium-derived relaxing factor). Differences in the structure of the P2 purinoceptor in various tissues may be useful in the development of drugs for the treatment of vascular, gastrointestinal and urinoglenital disorders. PMID- 2996969 TI - Prolonged treatment with (+/-) propranolol induces supersensitivity to (L)noradrenaline in mesenteric arteries in the rat. AB - In the isolated perfused mesenteric arteries of the rat, neither (+/-) propranolol (0.1 microM) nor (+/-)isoproterenol (0.05 microM) modified the overflow of DL-[3H]noradrenaline (DL-[3H]NA) induced by sympathetic nerve stimulation at either 5 or 10 Hz. The blockade of alpha presynaptic receptors with phentolamine (4.7 microM) increased the 3H-transmitter overflow at 5 and 10 Hz. (+/-)Propranolol (0.1 microM) failed to modify this effect. Vasoconstrictor responses to exogenous NA or sympathetic nerve stimulation were not modified by (+/-)propranolol (0.1 microM). Prolonged treatment with (+/-)propranolol (7 mg/kg) for 15 days potentiates responses to both exogenous NA and sympathetic nerve stimulation; however, the fractional release per pulse of DL-[3H]NA was not modified at either 5 or 10 Hz. These results provide no evidence to support the hypothesis that the release of NA is regulated by presynaptic beta adrenoreceptors in the mesenteric arteries of the rat. The enhancement of vascular responses after prolonged treatment with propranolol could be caused by postsynaptic supersensitivity. PMID- 2996970 TI - VIP binding to epithelial cell membranes of rat ventral prostate: effect of guanine nucleotides. AB - Specific binding of vasoactive intestinal peptide (VIP) to epithelial cell membranes of rat ventral prostate was reversible, saturable and dependent on time and temperature. The data suggested the presence of two classes of VIP receptors: a class with high affinity (Kd = 1.7 nM) and low binding capacity (0.5 pmol VIP/mg protein), and another class with low affinity (Kd = 36.2 nM) and high binding capacity (7.5 pmol VIP/mg protein). Chicken VIP and porcine secretin recognized VIP receptors but exhibited a 10-fold higher and a 40-fold lower affinity than porcine VIP, respectively. However, glucagon, somatostatin, Met enkephalin and cholecystokinin were ineffective. GTP inhibited markedly the interaction of VIP with membranes by increasing the rate of dissociation of peptide bound to its receptors. GDP and Gpp(NH)p behaved as GTP but other purine nucleotides and nucleosides did not show any effect. PMID- 2996971 TI - Some pharmacological effects of tizanidine on smooth muscle organs and alpha 2 adrenoceptor. AB - Microsomal and crude synaptosomal fractions were prepared from the longitudinal muscle of guinea-pig ileum. A specific binding of [3H]yohimbine (10 nM) to alpha 2-adrenoceptor in the crude synaptosomal fraction was inhibited by tizanidine and clonidine. Tizanidine is about one-third as potent as clonidine. A specific binding of [3H]QNB (0.3 nM) to muscarine receptor in the microsomal fraction was inhibited by atropine (10(-8)-10(-7) M) but not by tizanidine (up to 10(-4) M). Tizanidine inhibited spontaneous movements of guinea pig ileum and rat stomach (in situ) and intestinal transit in mice, and induced mydriasis in mice. These effects induced by tizanidine might be due to activation of alpha 2-adrenoceptor but not to atropine like action. PMID- 2996972 TI - Involvement of bicuculline-insensitive receptors in the hypothermic effect of GABA and its agonists. AB - GABA, delta-aminovaleric acid (DAVA) and sodium valproate (VPA) decrease core temperature in conscious rats. Bicuculline increases GABA-induced hypothermia, does not modify DAVA (250 mg/kg) and VPA (100 and 400 mg/kg) hypothermia and antagonizes initial hypothermia by DAVA (1000 mg/kg) and VPA (200 mg/kg) and late hypothermia by DAVA (500 mg/kg) and VPA (200 mg/kg). Picrotoxin increases late hypothermia by GABA (250 mg/kg) and VPA (400 mg/kg), but decreases initial hypothermia by VPA (200 mg/kg). Pentylenetetrazol increases variably GABA-induced hypothermia, inhibits early early hypothermia by DAVA and increases hypothermia induced by VPA (400 mg/kg). We conclude that GABA and GABA-agonists (DAVA and VPA) may induce hypothermia partly mediated by activation of bicuculline insensitive GABA-receptors. PMID- 2996973 TI - Depressant effect of forskolin on spontaneous locomotor activity in mice. AB - Forskolin, an activator of adenylate cyclase, depressed spontaneous locomotor activity when injected intracerebroventricularly into mice in doses of 10 and 100 micrograms. The findings suggest that increases in brain cAMP levels may be associated with a reduction in excitability. PMID- 2996974 TI - Effects of permanent deafferentation of the anterior preoptic area on serum aldosterone levels in the crested newt (Triturus cristatus carnifex Laur.). AB - By cutting off the fiber systems running along the medial forebrain bundle of the urodele amphibian Triturus cristatus, a wide deafferentation of the preoptic area was evoked. This operation elicited a decrease in aldosterone serum level, probably through a reduction of ACTH secretion. At present we are not able to ascertain whether such reduction was prompted by changes in the hypothalamic production of the neurohypophysial hormones or the corticotropin-releasing factor (CRF). PMID- 2996976 TI - Joint distribution of insertion elements IS4 and IS5 in natural isolates of Escherichia coli. AB - A reference collection of natural isolates of Escherichia coli has been studied in order to determine the distribution, abundance and joint occurrence of DNA insertion elements IS4 and IS5. Among these isolates, 36% were found to contain IS4 and 30% were found to contain IS5. Among strains containing IS4 the mean number of copies per strain was 4.4 +/- 0.8; the comparable figure for IS5 was 3.7 +/- 1.0. Although the presence of the elements among the isolates was independent, among those isolates containing both IS4 and IS5, there was a significant negative correlation in the number of copies of the elements. The reference collection was also studied for the presence of the DNA sequences flanking the single copy of IS4 in the chromosome of E. coli K12. Homologous sequences were found in only 26% of the isolates. The sequences flanking the IS4 invariably occur together, and their presence is significantly correlated with the presence of IS4. In eight of the strains that carry these flanking sequences, an IS4 is located between them, and the sequences are present at the homologous position as in the K12 strain. We suggest that IS4 and its flanking sequences share a common mechanism of dissemination, such as plasmids, and we present evidence that they are included in a much larger transposable element. PMID- 2996975 TI - Characterization of hepatic growth hormone binding sites in two fish species, Gillichthys mirabilis (Teleostei) and Acipenser transmontanus (Chondrostei). AB - To obtain information on the presence of growth hormone (GH) receptors in liver of nonmammalian vertebrates the specific binding of 125I-bovine growth hormone (bGH) to liver membranes of seven species representing the major groups was studied by radioreceptor assay. A substantial degree of specific binding was detected with sturgeon (Acipenser transmontanus) liver membranes and a much lower amount was detected on hepatic membranes of Gillichthys mirabilis. No significant specific binding was detected on liver membranes of pigeon, turtle, bullfrog, tilapia, or leopard shark. Gillichthys and sturgeon liver membranes were further characterized and compared with hepatic membranes from male rabbits. The sturgeon and Gillichthys membranes showed binding that was dependent upon time, temperature, pH, and membrane concentration. Scatchard analysis of the binding of 125I-bGH to sturgeon and rabbit membranes revealed both high and low affinity binding sites. The high affinity sites had KA values of 3.1 X 10(11) and 1.0 X 10(11) M-1, and capacities of 12 and 50 fmol/mg protein, respectively. Membranes from Gillichthys liver contained only a single class of binding sites with a KA of 6.7 X 10(9) M-1 and a binding capacity of 49 fmol/mg. Hormonal specificity of the sturgeon and Gillichthys hepatic binding sites was studied using methionyl human GH (met-hGH), ovine prolactin (oPRL), and a crude preparation of sturgeon (st)GH. The met-hGH and stGH inhibited the binding of 125I-bGH to sturgeon liver membranes while only met-hGH displaced labeled bGH from Gillichthys liver membranes. One microgram of oPRL did not significantly inhibit 125I-bGH binding in either membrane assay. Based on these studies, sturgeon hepatic GH receptors seem to be more like those of nonprimate mammals than those of teleosts. Our results, in conjunction with the data of J. N. Fryer (Gen. Comp. Endocrinol. 39, 123-130 (1979)), indicate that considerable evolutionary divergence has occurred among teleost hepatic GH receptors. Thus, vertebrate GH receptors seem to have undergone at least as much evolution as has the hormone itself. PMID- 2996978 TI - Hybrid dysgenesis in Drosophila melanogaster: nature and inheritance of P element regulation. AB - The genetic determination of the control of resistance or susceptibility to germ line changes mediated by P elements was studied in two strains and in derivatives of crosses between them. One strain, characterized as true M, completely lacked P elements. The second strain, pseudo-M (M'), carried a number of P elements, but these did not have the potential to induce the gonadal sterility that is associated with P-M hybrid dysgenesis. Individuals from the true M strain were invariably unable to suppress P factor activity (i.e., all daughters of outcrosses of M females and P males were sterile). In contrast, individuals from the M' strain showed variable degrees of suppression that were manifested in a wide range of gonadal sterility frequencies in standard tests. This continuous distribution pattern was reproducible for more than 25 generations.--The results of the genetic analysis indicate that a strain with a variable degree of suppression of gonadal dysgenesis is not necessarily in a transient state between the extreme conditions of P and M cytotype. A large variance in the ability to suppress gonadal dysgenesis with a mean value intermediate between the extremes of P and M cytotype may be a relatively stable strain characteristic. No reciprocal cross effect was observed in the suppression of sterility of F1 females from M X M' matings. Thus, the existence of M' strains indicates a Mendelian component in P element regulation and suggests that cytotype, which has an extrachromosomal aspect, may be only one of perhaps several mechanisms involved in regulation. Analysis of the effects of individual chromosomes from the M' strain showed that each chromosome contributed to the reduction of gonadal dysgenesis in the progeny of test matings. The results are consistent with a one component titration model for P element regulation. PMID- 2996977 TI - Both nucleolar organizers are replicated in Dipteran polyploid tissues: a study at the level of individual nuclei. AB - Working with the Dipteran Calliphora erythrocephala, we have tested the hypothesis that only one nucleolar organizer region (NO) is replicated during polyploidization. NO replication was examined in two very different highly polyploid nuclear types: salivary gland nuclei and nurse cell nuclei. Two strains of the organism containing NO regions with highly diagnostic nontranscribed spacer (NTS) polymorphisms were prepared and reciprocal single pair-matings between members of the strains were performed. The representation of the two distinguishable NOs in diploid and polyploid DNAs of individual F1 progeny from each cross was then examined. DNA from a total polyploid nuclear DNA preparation and from individual polyploid nuclei of both tissue types was analyzed. Our results show conclusively that both genomic NOs are replicated in individual polyploid nuclei of both types. Further, evidence for variation in the relative replication of cistrons from the two NOs by individual nuclei was obtained. The cistron types present in the NOs of both strains showed differential replication upon polyploidization. In general, the patterns of differential cistron replication seen in salivary gland and nurse cell nuclei were similar. PMID- 2996979 TI - Transposable element-induced response to artificial selection in Drosophila melanogaster. AB - The P family of transposable elements in Drosophila melanogaster transpose with exceptionally high frequency when males from P strains carrying multiple copies of these elements are crossed to females from M strains that lack P elements, but with substantially lower frequency in the reciprocal cross. Transposition is associated with enhanced mutation rates, caused by insertion and deletion of P elements, and chromosome rearrangements. If P element mutagenesis creates additional variation for quantitative traits, accelerated response to artificial selection of progeny of M female female X P male male strain crosses is expected, compared with that from progeny of P female female X M male male strain crosses.- Divergent artificial selection for number of bristles on the last abdominal tergite was carried out for 16 generations among the progeny of P-strain males (Harwich) and M-strain females (Canton-S) and also of M-strain males (Canton-S) and P-strain females (Harwich). Each cross was replicated four times. Average realized heritability of abdominal bristle score for the crosses in which P transposition was expected was 0.244 +/- 0.017, 1.5 times greater than average heritability estimated from crosses in which transposition was expected to be rare (0.163 +/- 0.010). Phenotypic variance of abdominal bristle score increased by a factor of four in lines selected from M female female X P male male crosses when compared with those selected from P female female X M male male hybrids. Not all quantitative genetic variation induced by P elements is additive. A substantial fraction of nonadditive genetic variation is implicated by chromosomal analysis, which demonstrates deleterious fitness effects of the mutations when homozygous.--Several putative "quantitative" mutations were identified from chromosomes extracted from the selected lines; these will form the basis for further investigation at the molecular level of the genes controlling quantitative inheritance. PMID- 2996980 TI - Homologous recombination between autonomously replicating plasmids in mammalian cells. AB - The ability of autonomously replicating plasmids to recombine in mammalian cells was investigated. Two deletion plasmids of the eukaryotic-prokaryotic shuttle vector pSV2neo were cotransfected into transformed monkey COS cells. Examination of the low molecular weight DNA isolated after 48 hr of incubation revealed that recombination between the plasmids had occurred. The DNA was also used to transform recA- E. coli. Yield of neoR colonies signified homologous recombination. Examination of the plasmid DNA from these colonies confirmed this view. Double-strand breaks in one or both of the input plasmids at the sites of deletion resulted in an enhancement of recombination frequency. The recombination process yielded monomeric and dimeric molecules. Examination of these molecules revealed that reciprocal recombination as well as gene conversion events were involved in the generation of plasmids bearing an intact neo gene. The COS cell system we describe is analogous to study of bacteriophage recombination and yeast random-spore analysis. PMID- 2996981 TI - Elevated levels of petite formation in strains of Saccharomyces cerevisiae restored to respiratory competence. II. Organization of mitochondrial genomes in strains having high and moderate frequencies of petite mutant formation. AB - Restriction enzyme analysis of aberrant mtDNA molecules in restored strains of Saccharomyces cerevisiae that displays an elevated level of petite formation has shown the occurrence of novel junction fragments and nonstoichiometric amounts for some unaltered bands. Five aberrant mitochondrial genomes from high-frequency petite-forming (hfp) strains (greater than 60% petites per generation) contain like-oriented duplications and single copy regions. High-frequency petite formation is postulated to arise from increased intramolecular recombination between duplicated segments. Mitochondrial DNA structures in two other hfp strains cannot be easily interpreted and might arise from intramolecular recombination. Mitochondria DNA from moderate-frequency petite-forming (mfp) strains (5-16% petites per generation) contains inverted duplications in two cases. The elevated petite formation is postulated to arise from homologous recombination between directly repeated sequences. In mtDNA from one mfp strain, deletion end-points have been shown to overlap. Such deletion endpoint overlap is postulated to be required for the maintenance of the tandem duplication in hfp strains. Two regions of the wild-type mtDNA (between cyb and oli2 and between SrRNA and oxi2) appear to be dispensable for mitochondrial function. PMID- 2996983 TI - Nucleotide sequence of the tetracycline resistance gene of pTHT15, a thermophilic Bacillus plasmid: comparison with staphylococcal TcR controls. AB - The nucleotide (nt) sequence of the tetracycline resistance (TcR) region (1628 bp) of the Bacillus plasmid pTHT15 was determined. A single open reading frame (ORF), encoding a 458 amino acid (aa) 50-kDal protein (TET), is present after a GTG initiation codon preceded by a ribosome-binding site (RBS-2). The transcriptional start point, at a position 120 nt upstream from the GTG codon was determined by S1 mapping. This upstream region contains a short ORF (30 aa) which is preceded by RBS-1. The presence of three inverted repeats, which can form two different conformations of the mRNA very similar to those of the control region of the macrolide-lincosamide streptogramine B resistance gene of pE194 [Horinouchi and Weisblum, Proc. Natl. Acad. Sci. USA 77 (1980) 7079-7083; Gryczan et al., Nucl. Acids Res. 8 (1980) 6081-6097; Shivakumar et al., Proc. Natl. Acad. Sci. USA 77 (1980) 3903-3907], suggests that the TcR gene is regulated by a translational attenuation mechanism. A Rho-independent transcriptional terminator structure is present immediately after the translational stop codon (TAA) of the TET protein. Comparison of the TET protein with the staphylococcal TcR proteins of pT181 revealed considerable homology. PMID- 2996985 TI - A powerful method for the preparation of cDNA libraries: isolation of cDNA encoding a 100-kDal nucleolar protein. AB - An easy and quick method to synthesize large cDNA molecules and to clone them with very high efficiency in the expression vector lambda gt11 is described. The technique employs RNase H and Escherichia coli DNA ligase treatment during second strand synthesis, followed by repair of the ds cDNA extremities by S1 nuclease and PolIk (Klenow fragment) treatment. This treatment allows efficient addition of suitable linkers and results in a 100-fold increase in the yield of cloned cDNA, when compared with other published techniques. Using 75 ng of poly(A)+ RNA from CHO cells, we have prepared a library of 1.1 X 10(7) clones. This library was screened with polyclonal antibodies raised against a 100-kDal nucleolar protein of CHO cells. Five recombinants were isolated with inserts of 500-2500 bp. The average size of cDNA obtained by this method is considerable: the 2500-bp cDNA represents 90% of the mRNA coding for the 100-kDal protein. PMID- 2996982 TI - Cellular genes in the mouse regulate in trans the expression of endogenous mouse mammary tumor viruses. AB - The transcriptional activities of the eleven mouse mammary tumor virus (MMTV) proviruses endogenous to two sets of recombinant inbred (RI) mouse strains, BXD and BXH, were characterized. Comparison of the levels of virus-specific RNA quantitated in each strain showed no direct relationship between the presence of a particular endogenous provirus or with increasing numbers of proviruses. Association of specific genetic markers with the level of MMTV-specific RNA was examined by using multiple regression analysis. Several cellular loci as well as proviral loci were identified that were significantly associated with viral expression. Importantly, these cellular loci associated with MMTV expression segregated independently of viral sequences. PMID- 2996984 TI - Expression of amplified human beta interferon genes using heavy metal induction in Chinese hamster ovary cells. AB - An expression cassette consisting of the human beta-interferon (beta-IFN) cDNA fused to the human metallothionein (MeT)IIA promoter has been linked to a selectable mouse dihydrofolate reductase gene (dhfr) and used to transform dhfr deficient Chinese hamster ovary (CHO) cells. Transformants resistant to increasing concentrations of methotrexate (Mtx) were isolated and found to secrete beta-IFN either constitutively or upon induction with cadmium (up to 325 000 units beta-IFN/10(6) cells/24 h). Molecular analysis demonstrates a large increase in beta-IFN-specific DNA sequences and beta-IFN mRNA levels in amplified cell lines, with initiation of transcription occurring at the authentic start point for the MeT promoter. PMID- 2996986 TI - High-level production of the EcoRI endonuclease under the control of the pL promoter of bacteriophage lambda. AB - Escherichia coli strains overproducing the EcoRI restriction endonuclease have been constructed, using lambda pL promoter expression vectors. In a first step we constructed endRI::lacZ gene fusions by fusing the N-terminal part of the endRI gene with a lacZ gene fragment, whereafter the hybrid gene was positioned randomly under the control of the pL promoter to optimize the level of expression. These plasmids direct the synthesis of large amounts of fusion protein approaching 30% of the total cellular protein content. In most cases the overproduced protein forms enzymatically inactive intracellular aggregates. The position of the promoter in front of the hybrid gene had little effect on the level of expression, except in fusions directly affecting the ribosome-binding site (RBS). In a second step, several of these promoter-gene configurations were used to reconstruct the intact endRI gene in appropriate hosts producing EcoRI methylase and cI-coded repressor. The levels of EcoRI endonuclease overproduction were similar to that obtained for the corresponding fusion protein, despite the fourfold difference in protein size. Intracellular precipitation was also observed with the overproduced EcoRI endonuclease. PMID- 2996987 TI - Solubility of the EcoRI restriction endonuclease and its purification from an over-producing strain. AB - The solubility of the EcoRI restriction endonuclease was measured in solutions of varying NaCl concentrations, at different temperatures and in the presence of DNA. The precipitation of the protein was enhanced by low NaCl concentrations, by elevated temperatures, and by the addition of DNA. These observations are discussed in relationship to the interaction of this protein with DNA. The purification of the EcoRI restriction enzyme from a strain of Escherichia coli that over-produces this enzyme was hampered by the insolubility of the protein, and hence the purification procedure was modified to optimize the recovery of active enzyme. PMID- 2996988 TI - Nuclear localization of poliovirus capsid polypeptide VP1 expressed as a fusion protein with SV40-VP1. AB - The poliovirus cDNA fragment coding for capsid polypeptide VP1 was inserted between the EcoRI and BamHI sites of SV40 DNA, generating a chimaeric gene in which the sequence of the 302 amino acids (aa) of poliovirus capsid polypeptide VP1 was placed downstream from that of the 94 N-terminal aa of SV40 capsid polypeptide VP1. The resulting defective, hybrid virus, SV40-delta 1 polio, was propagated in CV1 cells using an early SV40 mutant, am404, as a helper. Cells doubly infected by SV40-delta 1 polio and am404 expressed a 50-kDal fusion protein which was specifically immunoprecipitated by polyclonal and/or monoclonal antibodies raised against poliovirus capsids or against poliovirus polypeptide VP1. Examination of the infected cells by immunofluorescence after staining with anti-poliovirus VP1 immune sera revealed that the fusion protein was mostly located in the intra- and perinuclear space of the cells, in contrast to the exclusively intracytoplasmic location of genuine poliovirus VP1 polypeptide that was observed in poliovirus-infected cells. This suggests that the N-terminal part of the SV40-VP1 polypeptide could contain an important sequence element acting as a migration signal for the transport of proteins from the cytoplasm to the nucleus. PMID- 2996989 TI - Effect of aging on the response of the cerebral cortex to noradrenergic denervation. AB - Cyclic AMP generation was measured in isolated minces of the ipsilateral and contralateral cortex of young (3-4 months) and aged (28-29 months) Fischer-344 rats, 2 weeks after unilateral lesion of the nucleus locus ceruleus (LC). Measurements were conducted under basal conditions and in the presence of 0.1 mM isoproterenol. Despite marked norepinephrine (NE) depletion of about 90% in the ipsilateral cerebral cortex in both age-groups, cyclic AMP generation under basal conditions was unaffected. However, whereas isoproterenol-stimulated cyclic AMP generation was significantly higher in the NE-depleted ipsilateral cerebral cortex of young rats, such 'denervation supersensitivity' was not apparent in aged rats. Also, endogenous levels of NE were 31% higher, and isoproterenol stimulated cyclic AMP generation was 33% lower in the contralateral 'control' cortex of aged rats than in young rats. These results suggest an age-related defect in the postsynaptic expression of noradrenergic mechanisms in the rat cerebral cortex. Further research using intermediate ages is needed to establish whether these age-related findings are caused by 'senescence' or are 'developmental' phenomena. PMID- 2996990 TI - Elution of cytomegalovirus antibodies from adenocarcinoma of the colon. AB - Eluates were prepared by high salt extraction from normal colonic mucosa and adenocarcinomatous tissue from 28 patients, eight more from unmatched colonic tissue and five from patients with other gastrointestinal disease. Immunoglobulins were detected by ELISA: IgG was present in 24% eluates from normal colon and 21% from carcinomas; IgA in 55% eluates from normal colon and 39% from carcinomas; IgM in 55% from normal colon and 37% from carcinomas. Cytomegalovirus-specific antibody was found in 15% eluates from normal colon and in 18% carcinomas. Out of the 28 matched specimens, cytomegalovirus-specific IgG was detected in one normal and four tumour eluates, specific IgA in two normal and four tumour eluates, and specific IgM in two normal and two tumour eluates. In two instances cytomegalovirus-specific antibody was present in the eluates prepared from the normal and tumour tissue of the same patient. Of those eluates which contained cytomegalovirus-specific antibodies by ELISA, two were positive by anti-complement immunofluorescence of human embryo fibroblasts infected with cytomegalovirus strain AD-169. It seems possible, therefore, that cytomegalovirus antigens on colonic cells may be masked by complexing with anti-cytomegalovirus antibodies, and may not therefore be detected by techniques such as immunofluorescence. PMID- 2996991 TI - Low residue or normal diet in Crohn's disease: a prospective controlled study in Italian patients. AB - Seventy patients with non-stenosing Crohn's disease were randomly assigned to follow a low residue diet or a normal Italian diet for a mean of 29 months. The two groups were comparable at the onset in various measures of disease severity and diet. Patients complied well with the diet prescriptions, the low residue group eating a mean of 8.1 portions a week of fibre containing foods and the liberalized group a mean of 26.6 portions (p less than 0.005). There was no difference in outcome between the two groups, including symptoms, need for hospitalisation, need for surgery, new complications, nutritional status, or postoperative recurrence. Eighty six per cent of patients eating ad libitum and 65% of patients who avoided roughage eliminated one or more permitted foods because of subjective intolerance. Lifting of dietary restrictions, which results in a more appetizing and nutritious diet, does not cause symptomatic deterioration or precipitate intestinal obstruction in Crohn's disease. PMID- 2996992 TI - Management of the mildly abnormal Pap smear: a conservative approach. AB - Follow-up and management of the mildly abnormal Pap smear has been the subject of controversy in the medical literature. At Fitzsimons Army Medical Center, patients with initial Pap smears reported as "inflammatory atypia" or mild dysplasia were treated with specific therapy for vaginitis and/or cervicitis. Follow-up smears were performed 6 to 8 weeks later. Of the above groups, 80 and 58%, respectively, were negative on repeat smear. A third group of patients with Pap smears reported as consistent with human papillomavirus (HPV) were not treated but had repeat smears performed at 6 to 8 weeks. Of these, 76.4% reverted to normal. The results of the colposcopically directed cervical biopsies obtained on patients with persistently abnormal smears are reported. These findings support a conservative plan for follow-up of mildly abnormal Pap smears. PMID- 2996994 TI - Cervical adenoid cystic carcinoma associated with ascites. AB - Two new cases of cervical adenoid cystic carcinoma are reported. The addition of these two cases brings the total number of reported cases in the literature to 88 making this the most common gynecologic site of occurrence for this unusual tumor. Ascites developed in both of these cases after primary radiation therapy. The significance of this unique association is discussed. PMID- 2996993 TI - Adenoid cystic carcinoma of the cervix: a report of 17 cases. AB - Seventeen cases of adenoid cystic carcinoma of the cervix are presented. The long term survival was in early stage IB-IIA. Patients with more than stage IIB had no survival rate over 5 years. One thing of interest was the fact that lung metastasis was 29.4%. PMID- 2996996 TI - A case of verrucous carcinoma of the uterine cervix: clinical, light, and electron microscopic, and immunohistological observations. AB - A case of verrucous carcinoma of the uterine cervix in a 67-year-old patient is reported. On transmission electron microscopy, virus-like particles with a diameter of 45 to 55 nm were found in the nuclei of koilocytotic cells. However, human papilloma virus antigen could not be detected by immunohistochemistry, using the peroxidase-antiperoxidase technique. PMID- 2996995 TI - Long-dormant invasive mole associated with multiple malignancies. AB - A 65-year-old previously healthy housewife, gravida 3, para 3, was first diagnosed as Stage Ib carcinoma of the uterine cervix (poorly differentiated squamous cell carcinoma) and admitted. The external radiation of 5400 rad by telecobalt source was performed. No intracavitary radiation was added. After about 7 1/2 years the patient noticed a tumor of fist size on her buttocks, but she did not present in our clinic regularly. Because of enlarging tumor and general malaise she was readmitted a year later. On the fifth hospital day she died with ileus. Autopsy revealed osteosarcoma of buttocks in the radiation field, stomach cancer (tubular adenocarcinoma) with perforated peritonitis, and invasive mole of the uterine corpus. The patient's last pregnancy terminated as a full-term delivery at 26 years of age and she was 43 years at her menopause. The dormant period of invasive mole was 47 years after her last pregnancy, 30 years after her menopause, and at least 8 years after pelvic radiation. PMID- 2996997 TI - Adenoid cystic carcinoma of the cervix: a case report with implications for chemotherapeutic treatment. AB - There have been 59 other cases of adenoid cystic carcinoma of the cervix reported in the literature. Therefore, little is known of the true biological behavior or therapeutic responsiveness of these tumors. We are reporting a case of adenoid cystic carcinoma of the cervix, FIGO Stage I-B, initially treated with radical hysterectomy and bilateral pelvic lymphadenectomy. Two years after surgery, the patient presented with recurrent disease in the pelvis and multiple lung metastasis. She subsequently was treated with chemotherapy consisting of cytoxan, Adriamycin, and cis-platinum with a sustained complete clinical response. PMID- 2996998 TI - [Aftercare of gynecologic malignancies. Continuing education meeting of the Austrian Society for Gynecology and Obstetrics. Bad Mitterndorf, 21 June 1984]. PMID- 2996999 TI - [Cytomegalovirus infection--significance in gynecology and obstetrics]. PMID- 2997000 TI - [Condylomas--evidence for the involvement of papillomaviruses in the development of cervical cancer]. PMID- 2997001 TI - [Herpes genitalis]. PMID- 2997002 TI - [AIDS--a new threat]. PMID- 2997003 TI - Effects of dibutyryl cyclic AMP and gonadotropin-releasing hormone added prior to filter sterilization of the media and variation in oxygen partial pressure on output of human chorionic gonadotropin from term placental explants. AB - The effect of 2 mM dibutyryl cyclic AMP (dbcAMP) and 10 and 100 micrograms/ml gonadotropin-releasing hormone (GnRH) added prior to filter sterilization and of varying oxygen partial pressure (5, 20, and 95% oxygen) were evaluated using explants from each of 4 placentas on each of 6 days of culture. There were significant differences between placentas on days 1 and 2 of culture (both p less than 0.025), and day 4 (p less than 0.05), but there were no effects of GnRH or varying oxygen partial pressure on output of human chorionic gonadotropin under the conditions evaluated. PMID- 2997004 TI - Cell-mediated immune responses to cytomegalovirus in patients with dysplasia of the uterine cervix. AB - Peripheral blood lymphocytes from 172 women with various degrees of cervical dysplasia were assayed for responsiveness to cytomegalovirus-induced early and late antigens using lymphocyte stimulation tests. No evidence was found to suggest that any group of patients was specifically sensitized to either of the antigens tested. Neither was there any difference between the patient groups in respect of mitogen-induced stimulation. The concept that cytomegalovirus might be involved in cervical cell transformation leading to carcinoma has therefore not been strengthened as a result of this study. PMID- 2997005 TI - [Studies of phosphorylation in rat mast cells (sixth report). Cyclic AMP dependent protein phosphorylation in rat mast cell granule membranes]. AB - Granules with broken membranes were isolated from sonicated, purified rat serosal mast cells on a Percoll gradient. When the granules were incubated with [gamma 32P]ATP and Mg2+ in the absence of exogenous protein kinases and substrates, one major radioactive band was recovered in the 44 K area after SDS/PAG electrophoresis. The phosphorylation reaction with ATP requires Mg2+ and 1 mM Mg2+ produces the maximal response. The initial reaction is rapid and the maximal response is seen at 30 degrees C. The reaction is enhanced by 0.05-0.5 microM cyclic AMP and is inhibited by Ca2+ (0.45 mM and higher). There is no obvious effect of phosphatidylserine and phorbol ester TPA on the reaction. The 44 K phosphorylated protein is made up of several components ranging in isoelectric point from approximately 7.6 to 6.6. This cyclic AMP-dependent phosphorylating activity in the granules may provide a mechanism by which the granules respond rapidly to cyclic AMP during mediator release. PMID- 2997007 TI - New replication mutant pNH602 and its relationship to plasmid pAS3, another deletion derivative of plasmid R6K. AB - A stable deletion derivative pNH602 was obtained when the recently described higher-copy-number point mutant pNH601 of plasmid R6K was introduced to a minicells-producing strain of Escherichia coli. The size of plasmid pNH602 is 18.8 Mg/mol as determined by electron microscopy. The 7.2 Mg/mol fragment of R6K genome missing in pNH602 carries the Smr-determinant and the region finO and, according to the results of restriction analysis, it includes one EcoRI site. With its radioisotopically determined 33 copies of pNH602 per E. coli K-12 chromosome (npc), representing a 23% increase of the point mutant pNH601 and 150% enhancement of R6K npc, plasmid pNH602 differs from another closely related R6K deletion derivative pAS3 of the same size which exhibits only 20 npc. Both pNH602 and pAS3 plasmids are conjugative. PMID- 2997006 TI - Isolation and characterization of mutants of the RP4 plasmid coding for increased resistance to ampicillin. AB - E. coli strain J53(RP4) was mutagenized with ethyl methanesulfonate and N-methyl N'-nitro-N-nitrosoguanidine. Clones showing a two-to threefold increase in resistance to ampicillin were produced. This increase was not due to an increased number of RP4 copies per chromosome. The level of penicillinase activity was twice higher in comparison with the parental strain. No detectable changes were found in the region coding for the resistance to ampicillin on the plasmid by restriction analysis. PMID- 2997008 TI - Sensory and autonomic polyneuropathy associated with hypergammaglobulinemia. AB - Sensory and autonomic polyneuropathy is a rare disease characterized by a sensory nerve disorder and postganglionic autonomic dysfunction. The etiology of this disease is unknown. We described a 51-year-old woman who had a chronic sensory dominant polyneuropathy and dysautonomia associated with hypergammaglobulinemia. In the previous reports of sensory and autonomic polyneuropathy, not much attention was given to coexisting hypergammaglobulinemia. By reviewing the literatures, hypergammaglobulinemia was frequently present in these case reports. This fact leads us to consider that an immunological mechanism may be playing a role in the pathogenesis of this disorder. PMID- 2997009 TI - Epstein-Barr virus antibodies in neurological diseases. AB - Antibody titers to the Epstein-Barr virus (EBV) in the serum and cerebrospinal fluid (CSF) were determined in 57 cases of acute or subacute neurological diseases. As a result, sera from 11 cases (7 Bell's palsy, 2 encephalitis and 2 acute cerebellar ataxia) were found to be positive for antibodies to early antigen. Seven of these 11 cases either seroconverted for IgG antibodies to viral capsid antigen (VCA) or were proven positive for anti-VCA-IgM antibodies in the serum. While 4 were found positive for IgG antibodies to VCA in CSF, 3 tested by the anti-complement immunofluorescence method were all negative for EBV associated nuclear antigen (EBNA) in CSF. Three of the 11 cases were considered to be of a primary infection with EBV because of a negative serologic test for antibodies to EBNA initially; a reinfection with the virus or a reactivation of the latent infection was suspected in some of the remaining cases. PMID- 2997010 TI - [Structural analysis of Borrmann type 4 of gastric scirrhous carcinoma in terms of tumor cell aggregation patterns]. PMID- 2997011 TI - [Study on stromal component of mastopathy--content and type of collagen]. AB - Mastopathy is one of most common diseases involving adult female. Hormonal disturbance is implicated to play one role promoting the disease; however, the role of stromal component, i. e., collagen has not been examined to discuss its pathogenesis. The present study is to investigate the difference of content and type of collagen between mastopathy and normal mammary tissue. In addition, other mammary tumors were also studied. Materials were consisted of 8 cases of normal control, 23 mastopathy, 11 fibroadenoma, and 26 carcinoma. The content of collagen was analysed using a Woessner's method. The typing of collagen was determined by amino acid analysis, densitometry, and electron microscopy. The results were as follows; 1. Normal. The content was 59.8 +/- 4.0% (mean +/- SE). The collagen type was classified as type I. 2. Mastopathy. 58.9 +/- 4.0 The collagen was composed of both type I and type III. 3. Fibroadenoma. 56.3 +/- 3.5, type I 4. Carcinoma. Except for the scirrhous carcinoma, its content was 50.7 +/- 2.2. Type I 5. The amino acid composition of the mastopathy except for the fibrosing type demonstrated high levels of hydroxyproline and glutamic acid and low levels of hydroxylysine, histidine, and arginine. That of the carcinoma showed only increased level of histidine. 6. The rate of lysyl hydroxylation of collagen in the normal control was 39.2% and the carcinoma 26.1%. With regard to the content of collagen, there was no significant difference between the cases of mastopathy and normal control. However, in the mastopathy, an aberrant type of collagen, i.e., type III was detected. The amino acid composition and rate of lysyl hydroxylation of the mastopathy differed from those of the carcinoma. This indicates that there is a maturation disturbance of collagen in the development of mastopathy. In conclusion, it is considered that analysing these two factors may be one diagnostic aid to predict the malignant transformation of mastopathy. PMID- 2997012 TI - Effect of ovine corticotropin releasing factor and arginine vasopressin on ACTH and aldosterone secretion in sheep. PMID- 2997014 TI - Prolactin receptor: identification of the binding unit by affinity labelling and characterization of poly- and monoclonal antibodies. AB - The prolactin receptor localized in rabbit mammary gland membranes has been identified by affinity labelling using covalent cross-linking agents such as a unique protein chain of approximately 32,000 daltons. After partial purification (5,000-fold) of these receptors from mammary gland homogenate, polyclonal antibodies, which specifically and completely inhibit prolactin binding in all organs and in all species studied, were raised. These antibodies possessed prolactin-like biological activity (casein synthesis) on rabbit mammary gland explants. Monoclonal antibodies specifically directed against the binding domain of the receptor were also obtained. These antibodies were more species-specific than the polyclonal antibodies. The most potent (M110) possessed higher affinity than prolactin for the receptor and could be a very effective tool to elucidate the structure of the receptor and its immunological detection. PMID- 2997013 TI - Effect of dopamine on ACTH-induced glucocorticoid secretion in rat adrenal suspended cells. PMID- 2997015 TI - Histological and fine structural features of pancreatic ductal adenocarcinomas in relation to growth and prognosis: studies in xenografted tumours and clinico histopathological correlation in a series of 75 cases. AB - Histology and fine structure of pancreatic ductal adenocarcinomas were assessed with respect to their significance for tumour growth and prognosis. The histological parameters included glandular differentiation, nuclear anaplasia, nuclear size, and mitotic activity (number of mitoses per high power field). Using these criteria three grades of malignancy were distinguished. They correlated well with the growth kinetics of seven human pancreatic ductal adenocarcinomas transplanted into nude mice. The tumour doubling time of a G 3 carcinoma was about half that of a G 1 carcinoma. On electron microscopy the tumour grade was reflected in the degree of functional differentiation of the neoplastic duct cells. In an additional clinicopathological evaluation of 75 patients operated upon for ductal adenocarcinoma of the pancreatic head, a positive relationship was found between grade and duration of symptoms until diagnosis. Moreover, the G 1 tumours showed generally a lower stage symptoms until diagnosis. Moreover, the G 1 tumours showed generally a lower stage at the time of surgery than G 2 and G 3 carcinomas. Finally, the median survival times correlated significantly with the tumour grade. From the various parameters used nuclear grade proved to be the most significant prognostic criterion, since a separate morphometric study revealed a very close correlation between median nuclear size of the tumours and survival time. PMID- 2997016 TI - Glycogen-rich, clear cell breast cancer: with comments concerning other clear cell variants. PMID- 2997017 TI - Herpes simplex viral infection in human neonates: an immunohistochemical and electron microscopic study. AB - Specimens obtained at autopsy from six neonates with herpes simplex virus (HSV) infections were examined microscopically, electron microscopically, and immunohistochemically. Coagulative necrosis with inclusions was found in the livers and adrenal glands in all cases, as well as in various other organs, including the spleen, bone marrow, lungs, esophagus, tongue, and thymus, in some cases. Distinct hemorrhagic diathesis was found in three cases. No characteristic clinical findings, such as skin rashes or elevated titers of the antibody to HSV, were found, and clinical diagnosis was therefore difficult. In three cases isolation and typing of the causative virus were performed virologically, and type 1 HSV (HSV-1) was identified as the causative virus. Immunohistochemically, the type and distribution of the virus were evaluated in all cases with type specific antisera to types 1 and 2 (HSV-2) antigens by the peroxidase antiperoxidase method. In five cases the infections were found to be due to HSV-1 and in only one case to HSV-2. In the placenta in one case of HSV-2 infection, HSV antigen was demonstrated in the chorionic villi. Electron microscopic study confirmed the existence of viral particles in the placenta in that case and, thus, the possibility of a transplacental route of infection. PMID- 2997018 TI - The ultrastructure of small cell lung carcinoma in bronchial biopsy specimens. AB - Forty-three bronchial biopsy specimens from patients with small cell lung cancer (SCLC) were studied by electron microscopy. In 38 specimens the diagnosis was based on the light microscopic examination of Epon-embedded tissue; 36 of these specimens contained dense-core granules on electron microscopic examination. In five cases the light microscopic diagnosis was either different from the electron microscopic diagnosis or in doubt. Electron microscopy revealed dense-core granules as the only sign of differentiation, and the diagnosis was changed to SCLC. The tumor cell populations in the biopsy specimens were quite heterogeneous. Cells of the oat cell type were always present and, on electron microscopic examination, always showed degenerative changes. It was therefore decided that this cell type represents an artifact. The true SCLC tumor cell, which constitutes only a small portion of the tumor in biopsy specimens, is characterized by a regular oval or rounded cell with pale cytoplasm and a ground glass nucleus with finely dispersed chromatin. Nucleolated cells, similar to those seen in large cell cancer, are often present but are not ultrastructurally different from nonnucleolated tumor cells. PMID- 2997019 TI - Congenital cytomegalovirus infection presenting as massive ascites with secondary pulmonary hypoplasia. AB - A unique presentation of congenital cytomegalovirus infection occurred in a premature infant who had massive ascites of undetermined etiology. The ascites, apparently present since early gestation, had compressed the thoracic cavity by elevating the diaphragm, resulting in severe pulmonary hypoplasia. PMID- 2997020 TI - Splenic histiocytosis in idiopathic thrombocytopenic purpura: a relative sphingomyelinase deficiency? AB - The case of a patient in whom idiopathic thrombocytopenic purpura (ITP) was associated with diffuse splenic histiocytosis is described; the patient's subsequent sphingomyelinase level was at the lower limits of the normal range. The patient's splenic lecithin:sphingomyelin ratio was not significantly different from that of 11 age-matched control subjects. It is postulated that the sporadic cases of splenic histiocytosis in patients with ITP are due to a relative, acquired sphingomyelinase deficiency. PMID- 2997021 TI - "Fibrinothrombotic glomerulopathy" in patients with placental site trophoblastic tumor and nephrotic proteinuria. PMID- 2997022 TI - Correlation between alimentary mycotoxin contamination and specific diseases. AB - Several pathological cases including primitive hepatomas, Reye's syndrome, alimentary toxic aleukaemia, were encountered in two different Tunisian Sahel hospitals. Contamination of some nutriments of the patients by mycotoxins (aflatoxins, trichothecenes, ochratoxin A, citrinin) are most likely involved in the origin of these diseases. PMID- 2997023 TI - Molecular genetics of the fourth component of human complement and steroid 21 hydroxylase. PMID- 2997025 TI - Decreased membrane potential of T lymphocytes in ageing mice: flow cytometric studies with a carbocyanine dye. AB - The membrane potential of lymphocytes from young (1-4-month-old) and old (25-37 month-old) CBA/Ca mice was studied with the aid of the fluorescent dye 3,3' dihexyloxacarbocyanine iodide (DiOC6(3)). In young mice, most B and T lymphocytes showed a high, and equal, degree of polarization. In old animals most, and in the older individuals almost all, T lymphocytes were found to be depolarized; both Lyt-2+ and Lyt-2- subsets were affected. B cells were largely unaffected. Since changes in transmembrane potential, including temporary hyperpolarization, are known to accompany lymphocyte activation, the depolarized state of T cells in old mice may be related to the decline of T-cell function that occurs during senescence. PMID- 2997024 TI - Molecular cloning and characterization of complementary and genomic DNA clones for mouse C4 and Slp. PMID- 2997027 TI - Mode of action of Agrobacterium tumefaciens lipopolysaccharide (LPS) in mice hepatic tissue: a comparative study with Salmonella typhimurium LPS. PMID- 2997026 TI - Autocrine models of B-lymphocyte growth. I. Role of cell contact and soluble factors in T-independent B-cell responses. AB - The requirements for triggering human B cells to DNA synthesis by T-independent polyclonal activators were examined. Optimal S phase entry of purified resting B cells infected with Epstein-Barr virus (EBV) or confronted with killed particles of Staphylococcus aureus Cowan Strain I (SAC) required a high density of cells in culture. Experiments varying culture vessel geometry and culture volumes revealed that the initial limiting quantity was a soluble activity generated in the B-cell cultures. A parallel observation was noted in the requirements for the sustained growth of EBV-transformed lymphoblasts. Autostimulatory soluble factors harvested from such cultures were able to augment DNA synthesis in low density cultures of resting cells triggered by EBV or SAC. Below a critical cell number, however, soluble factors by themselves, were not sufficient either for supporting primary B-cell responses or for maintaining the proliferation of transformed lymphoblasts. By employing conditions which encouraged cell contact it was found that a second, non-harvestable factor requiring cell proximity for its action was also necessary to promote B-cell growth. The implications of these findings for autocrine and paracrine models of B-cell activation are discussed. PMID- 2997028 TI - DNA and nuclear matrix relations: ultrastructural and cytochemical study of nuclei treated with high molarity LiCl-urea. PMID- 2997029 TI - MHC and non-MHC genetic influences on Rous sarcoma metastasis in chickens. AB - The B5/B5 genotype, in Leghorns, was associated with a high degree of metastasis of Rous sarcoma virus-induced tumors, but in combination with a Leghorn-New Hampshire background markedly less metastasis occurred. Initially, four mating types were used: B5/B5 X B5/B5 chickens from the F5 generation of the cross of Leghorn lines 6(1) and 15(1), B24/B24 X B24/B24 chickens from line UNH 105 (New Hampshires), and reciprocal crosses of B5/B5 X B24/B24 chickens. Subsequently, F2 generation progeny of the cross of B5/B5 and B24/B24 breeders, as well as B24/B24 line UNH 105 and B5/B5 (6(1) X 15(1))F2 chickens, were used. Six-week-old chickens were inoculated in the wingweb with Rous sarcoma virus. Chickens dying during a 10-week period after inoculation were necropsied and suspect metastatic lesions examined histologically. Among 234 terminal chickens from the initial four mating types the incidence of metastasis associated with B5/B5 Leghorns (66%) was substantially higher than for B24/B24 New Hampshires (12%) and B5/B24 progeny of reciprocal Leghorn-New Hampshire crosses (19 and 24%). Subsequently, among 524 terminal hosts in the Leghorn-New Hampshire F2 population, B genotype significantly influenced tumor dissemination. However, among 52 concurrently challenged B5/B5 hosts from the (6(1) X 15(1))F2 population the incidence of metastasis (60%) was significantly higher than among 122 B5/B5 hosts from the Leghorn-New Hampshire F2 population (31%), indicating a non-major histocompatibility complex genetic effect on metastasis. PMID- 2997030 TI - Analysis of the sheep MHC using HLA class I, II, and C4 cDNA probes. AB - Four cDNA probes for the human major histocompatibility complex (MHC) were used to investigate the sheep MHC, in conjunction with serological typing for ovine lymphocyte antigen (OLA). Lymphocytes from a family (two parents and five offspring) of Romanov sheep were subjected to genomic DNA digestion by the restriction endonuclease Eco RI, followed by gel electrophoresis. A single Southern blot representing all seven individuals was then consecutively hybridized with the class I, alpha-DC, beta-DR, and C4 probes, which were originally designed to identify HLA class I, class II (DC and DR), and C4 products, respectively. Using each of the three class I/class II probes, several bands showing DNA polymorphism were detected. The segregation of these bands in the five offspring exactly paralleled the OLA haplotype segregation established by serological typing. A further eight individuals carrying haplotypes which were phenotypically identical to those in the above-mentioned family showed bands in the corresponding positions when tested with the same three probes. Using the C4 probe, no polymorphism was detected in these fifteen individuals. PMID- 2997033 TI - Current status of liver transplantation. PMID- 2997034 TI - Impairment of blood-CSF barrier in patients with neurological complications following acute haemorrhagic conjunctivitis. PMID- 2997032 TI - Molecular cloning and partial nucleotide sequence of a 3.5 kb HLA-B27-associated fragment of genomic DNA. PMID- 2997031 TI - DNA polymorphism of the C2 locus. AB - The extent of the C2 locus in the HLA class III region has been determined by Southern blotting techniques and by DNA sequence analysis. The gene is 18 kb in length and therefore provides a marked contrast to the adjacent factor B gene of 6 kb. A novel restriction fragment length polymorphism (RFLP) has been identified using the endonuclease Sst I and a genomic probe derived from the 5' region of the C2 gene. Four variants have been detected in a sample of unrelated individuals with haplotypes carrying the C2C allele. Further analysis using C2 and factor B cDNA probes has determined the relationship between this and the other RFLPs previously identified in this region of the genome. Together, the three polymorphisms identified so far make the subdivision of previously indistinguishable haplotypes possible. They therefore constitute a series of markers which increase the resolution of genetic variation in the C2 locus and they may be important in studies of diseases associated with this region of the major histocompatibility complex. PMID- 2997035 TI - Rotavirus & bacterial enteropathogens in acute diarrhoeas of young children in Bangalore. PMID- 2997037 TI - Modulation of 5-hydroxytryptamine-evoked responses of the isolated rat uterus by imidazole, a phosphodiesterase stimulator. AB - Imidazole, a phosphodiesterase stimulator potentiated the responses of rat uterus to 5-HT, without increasing the maximal response. Aminophylline, papaverine and diazoxide significantly inhibited the responses to 5-HT including the maximal response. Imidazole did not affect the inhibitory effect of aminophylline, papaverine and diazoxide. The effect of imidazole on myometrium may be due to its direct effect on membrane permeability resulting in an increased influx of calcium. Phosphodiesterase stimulation if at all seems to play only a minor role. PMID- 2997036 TI - Adenosine triphosphatase systems in genital tract of testosterone treated male adult monkeys. AB - Studies on the distribution of Na+, K+-dependent, Mg2+ dependent and Ca2+ dependent Adenosine Triphosphatase (ATP-ases) in the testes, epididymis, seminal vesicles and prostate glands of mature bonnet monkeys were carried out with and without Testosterone propionate (TP) treatment. Comparatively, the Ca2+ dependent ATP-ase was very active in the testes, caput and cauda epididymis and prostate of control animals. However, the Mg2+-dependent ATP-ase activity was predominant in the seminal vesicles. In all the genital tissues the Na+, K+ dependent ATP-ase exhibited low activity compared to other ATP-ase systems. On TP treatment at 1 mg/kg body wt. dose for 30 consecutive days to the second group of animals, all classes of ATP-ases drastically decreased in the testes, cauda epididymis, seminal vesicles and prostate. While in caput epididymis the Mg2+-dependent ATP ase was stimulated, the Na+, K+-dependent ATP-ase was decreased both in the caput and corpus epididymis by the hormone treatment. The present study reveals the general inhibitory influence on the ATP-ase systems and thereby ionic transport after long term TP administration. PMID- 2997038 TI - Modification of interactions between neutrophils and staphylococci by lysosomotropic weak bases. AB - Weak bases that alkalinize the pH within neutrophil lysosomes inhibit in vitro cell functions, including lysosomal enzyme release and superoxide production. To determine the relevance of this inhibition to microbicidal activity, the effect of lysosomotropic weak bases on interactions between human neutrophils and Staphylococcus aureus 502a was studied. After treatment with 1 mM chloroquine, neutrophils showed significantly impaired phagocytosis of 14C-labeled S. aureus. However, 50 mM ammonium chloride had no effect on phagocytosis, although we have previously shown that this concentration raises lysosomal pH and inhibits degranulation and superoxide production. This base was therefore used to study effects on intracellular microbicidal activity. Incubation of neutrophils with 50 mM ammonium chloride diminished killing of S. aureus (22.9 +/- 6.3% of bacteria surviving versus 8.2 +/- 1.3% in suspensions without ammonium chloride). At 1 mM, ammonium chloride had no significant effect. The inhibition of cellular function could be neither explained as a function of neutrophil death, as measured by trypan blue dye exclusion, nor attributed to direct promotion of bacterial growth (in the absence of neutrophils, colony counts were similar in the presence or absence of ammonium chloride) or enhanced resistance to neutrophil microbicidal mechanisms (bacteria treated with ammonium chloride and washed before neutrophil exposure showed no improvement in survival). Ammonium chloride at 50 mM also impaired neutrophil killing of S. aureus in an anaerobic chamber, but microbicidal activity against Escherichia coli S15 was not affected. These findings suggest that optimal neutrophil killing of staphylococci requires a highly acid intralysosomal compartment, but ingestion of bacteria does not. This may reflect primary failure of acidification of the phagocytic vacuole or differential pH requirements for fusion of the plasma membrane with itself and with lysosome membranes. The difference between effects on killing of S. aureus and E. coli is probably a result of the relative importance of the components of neutrophil microbicidal activity against the two organisms. PMID- 2997040 TI - Myelopoiesis in experimentally contaminated specific-pathogen-free and germfree mice during oral administration of polymyxin. AB - Oral administration of polymyxin to specific-pathogen-free C3H/Law mice which with previously contaminated with gram-negative bacteria resulted in complete suppression of cecal gram-negative bacteria. Suppression of cecal gram-negative bacteria was accompanied by reduction of the cecal endotoxin concentration from 10 to 1 microgram/g of cecal content as measured with a microtechnique for the Limulus amebocyte lysate assay. Endotoxin determination by this assay appeared to be unaffected by the amount of polymyxin present in cecal preparations after oral administration of this antibiotic. In experimentally contaminated specific pathogen-free mice, the femoral concentration of progenitor cells forming granulocyte-macrophage colonies in vitro (CFU-GM) decreased significantly (P less than 0.001) to 66% of the initial control after 4 days of polymyxin treatment. However, the femoral CFU-GM concentration in germfree mice and splenic CFU-GM concentration in experimentally contaminated specific-pathogen-free and germfree mice was not affected by polymyxin treatment. The kinetic behavior of femoral and splenic CFU-GM in experimentally contaminated specific-pathogen-free and germfree mice was expressed as the in vivo sensitivity to the S-phase-specific cytostatic drug hydroxyurea, i.e., the hydroxyurea kill. Administration of polymyxin to experimentally contaminated specific-pathogen-free mice significantly diminished the hydroxyurea kill of femoral CFU-GM from 29 to 13% (P less than 0.02) and of splenic CFU-GM from 53 to 27% (P less than 0.005). The hydroxyurea kill of femoral CFU-GM in germfree mice was not significantly affected by polymyxin treatment. On basis of these results we conclude that the effect of polymyxin treatment on myelopoiesis is most likely due to elimination of intestinal gram negative bacteria and may indicate a significant role of intestinal gram-negative bacteria in the regulation of myelopoiesis. PMID- 2997039 TI - Plasmid-mediated virulence in Salmonella dublin demonstrated by use of a Tn5-oriT construct. AB - Salmonella dublin, a serotype which causes invasive disease in cattle and humans, carries a characteristic 80-kilobase plasmid (pSDL2). We were able to cure the plasmid from a strain of S. dublin. The cured strain was avirulent for mice by either the oral or intraperitoneal route of infection. A derivative of Tn5 which contains the transfer origin of the broad-host-range plasmid RK2 (Tn5-oriT) was transposed onto pSDL2, allowing mobilization of the plasmid by an RK2 helper plasmid. Reintroduction of the pSDL2 derivative plasmid into the cured strain restored virulence, demonstrating that the plasmid is necessary for virulence. These studies also demonstrate the usefulness of the Tn5-oriT construct for genetic manipulations. PMID- 2997041 TI - Interleukin 2 therapy in infectious diseases: rationale and prospects. AB - Clinical trials using interleukin 2 as a therapeutic immunomodulating agent in patients with acquired immunodeficiency syndrome have recently begun. In this article we present data from studies which indicate the ability of interleukin 2 in vitro to augment clinically important cytotoxic immune responses in lymphocytes from these patients. These studies provide both a rationale for the current trials and a model for evaluating the potential for use of interleukin 2 in other infectious diseases. We outline the types of infectious diseases in which interleukin 2 may prove to be useful and the therapeutic strategies in which it may play a role. PMID- 2997043 TI - HTLV-I is endemic in southern Italy: detection of the first infectious cluster in a white population. AB - Human T-cell leukemia virus (HTLV-I) infection is observed among black and Japanese populations in well-delimited endemic spots in association with a high incidence of adult T-cell leukemia (ATL). We present evidence of HTLV-I infection in two ATL patients from southeastern Italy who have not travelled and who have no known relations abroad, and in 8% of non-leukemic controls from the same area. Thus, populations exhibiting HTLV-I infection appear more widespread than supposed up to now. PMID- 2997044 TI - Papillomavirus DNA in human tongue carcinomas. AB - Seven biopsies from different carcinomas of the tongue were analyzed for human papilloma virus (HPV) sequences by Southern blot analysis. After hybridization with various types of human papilloma viruses, 3 tumors were found to be positive. Whereas DNA from one tumor hybridized with HPV 2 DNA under conditions of high stringency, the 2 other positive biopsies contained HPV 16 DNA. All positive tumors revealed high copy numbers of the respective DNA. The cleavage pattern of the HPV-2-positive tumor differed from the established HPV 2 prototypes. Minor differences from the HPV 16 prototype were also noted in one of the HPV-16-positive tongue carcinomas. PMID- 2997042 TI - A glycoprotein antigen detected with new monoclonal antibodies on the surface of human lymphocytes infected with human T-cell leukemia virus type-I (HTLV-I). AB - We have prepared two new mouse monoclonal antibodies (MAbs) named TARM-34 (IgM) and TAG-34 (IgG1), that react with surface antigens of lines of human lymphocytes bearing a human T-cell leukemia virus type-I (HTLV-I). The characters of these antibodies are compared with those of anti-HTLV-1 gp21 MAb (TA-21, IgG1), anti HTLV-I p19 MAb (GIN-14, IgG1) and human antibodies from patients with adult T cell leukemia (ATL). An indirect membrane immunofluorescence assay showed that TARM-34, TAG-34 and TA-21 all reacted specifically with cell-surface antigens of HTLV-I-positive T- and B-cell lines and cultured peripheral blood lymphocytes from HTLV-I-infected adults. Radioimmunoassay showed that serum antibodies from the ATL patients interfered with the binding of TA-21 antibody to cells of the HTLV-I-positive T-cell line MT-2, but not with the bindings of TARM-34 and TAG-34 antibodies. TARM-34 and TAG-34 both precipitated a 34-kd glycoprotein (gP34), while TA-21 precipitated gp21 from a lysate of 3H-glucosamine-labelled MT-2 cells. TARM-34 and TAG-34 also precipitated the 34-kd protein from lysates of MT 2 and HUT 102 cells labelled with 125I- or 35S-cysteine. Interestingly, TARM-34 and TAG-34 also precipitated 35-kd protein from a lysate of other HTLV-I-positive cells (F-Taj cell line) derived from an ATL patient. TA-21 precipitated the 21-kd protein from the lysates of 35S-cysteine-labelled HTLV-IMT-2 virions, but TARM-34 and TAG-34 did not precipitate any protein from this lysate. TARM-34 lysed HTLV-I bearing cells in the presence of rabbit complement. These results indicate that TARM-34 and TAG-34 both recognize a glycoprotein antigen that is expressed on the surface of HTLV-I-infected cells. PMID- 2997045 TI - Antibodies reacting with human T-lymphotropic retrovirus (HTLV-I) or related antigens in lymphomatous and healthy hamadryas baboons. AB - The sera of lymphomatous and healthy hamadryas baboons of the main, lymphoma prone Sukhumi stock were tested for antibodies reacting with HTLV-I antigens in the indirect immunofluorescence test. Antibodies of this specificity were found in all but one of 58 lymphomatous baboons and in 45% of 177 healthy ones. The prevalence of HTLV-I reactive antibodies in lymphoma-free baboon populations (including 118 Sukhumi "forest" stock animals and 195 baboons imported in 3 groups from Ethiopia) was consistently lower (5-8%). The specificity of baboon antibodies reacting with HTLV-I or a related agent is supported by the following evidence: Concordant reactivity pattern of baboon sera with several HTLV-I positive and-negative human cell lines; elimination of baboon sera anti-HTLV reactivity by absorption with purified HTLV-I, but not by other retroviruses; significant correlation between immunofluorescence titers of baboon sera and their reactivity in enzyme-linked immunosorbent assay with purified HTLV-I; competition of baboon anti-HTLV with monoclonal antibodies GIN-14 for binding of the epitope on p19HTLV-I. The prevalence of anti-HTLV positives in the main Sukhumi stock increased by age, reaching its maximum (approx. 80%) at 5-15 years, and showed no significant sex-related variation. The level of anti-HTLV antibodies in lymphomatous baboons and in age-, sex- and population-matched healthy ones did not differ. However, in pre-lymphoma sera these antibodies reached significantly higher levels than in sera of lymphomatous baboons (obtained in the terminal stage) or of matched, healthy controls. PMID- 2997046 TI - Evaluation of a new bronchodilator, Formoterol, using biochemical parameters. AB - The authors administered orally 40, 60 and 80 micrograms of Formoterol which was developed as a new bronchodilator and 4 mg of Salbutamol which is considered to be a beta 2-selective drug, to healthy adults. Pulse rate, blood pressure and blood levels of cyclic GMP, free fatty acid, cyclic AMP, glucose and lactic acid were measured in order to evaluate efficacy and dosage of Formoterol and to compare efficacy of this drug with that of Salbutamol. As a result, it is considered that Formoterol has a strong dose dependent beta 2-effect with 40 micrograms of Formoterol almost as high as 4 mg of Salbutamol in beta 2-effect. In addition, Formoterol has almost no alpha-effect and its beta 1-effect can be considered negligible in the doses of 60 micrograms and 80 micrograms. PMID- 2997047 TI - Effects of L-carnitine administration on mitochondrial electron transport activity present in human muscle during circulatory shock. AB - Carnitine was administered to a group of patients in shock, and the activities of cytochrome oxidase and succinate cytochrome c reductase in muscle needle biopsies from these patients were compared to those activities present in a non-carnitine treated control group of patients. Carnitine seemingly exerted a significant protective action on cytochrome oxidase activity during the initial phases of shock, but not to such an extent on succinate cytochrome c reductase activities. PMID- 2997048 TI - Cannabinoids block release of serotonin from platelets induced by plasma from migraine patients. AB - The effects were assessed of delta'THC (the psychoactive component of cannabis) and CBD and DMHP-CBD (the non-psychomimetic components of marijuana derivatives) on 14C labelled serotonin release from normal platelets, when incubated with patient's plasma obtained during migraine attack. A statistically significant inhibitory effect (p greater than 0.005) of 14C serotonin release was found at 10(-5)M, 10(-6)M, 10(-7)M delta'THC concentrations. Plasma of migraine patients obtained in attack-free periods revealed no significant inhibitory effect on 14C serotonin release from normal platelets using the same delta'THC concentration. CBD and DMHP-CBD had no significant inhibitory effect on 14C serotonin release from normal platelets when tested either at migraine-free period plasma or plasma obtained during migraine attack. PMID- 2997050 TI - Metabolic and hormonal changes in moderate and intense long-term running exercises. AB - The changes in selected muscle and blood metabolites - blood catecholamines, cyclic AMP, glucagon, growth hormone, insulin, and C-peptide--were measured in five male long-distance runners after moderate 90 min (4.3 min/km) and intense 45 min (3.3 min/km) of continuous running exercises. After the 45-min exercise, the mean concentrations of blood lactate and glucose were 2.8 and 8.8 mmol/l, respectively, and the muscle glycogen concentration had decreased by 31%. No changes could be found after the 90 min of running. The increase in blood glycerol concentration was greater after the 45-min exercise (5.8-fold) than after the 90-min exercise (3.9-fold), although the serum free fatty acid concentration increased significantly only during the 90-min exercise (1.6-fold). Plasma noradrenaline increases were 2.7-fold and 7.9-fold during the 90- and 45 min exercises, respectively, whereas the plasma adrenaline concentration was significantly elevated only after the 45-min exercise (4.7-fold). The respective changes of plasma cyclic AMP were 2.5- and 2.7-fold, and the increase in the concentration of plasma growth hormone was 5.8-fold after the 45-min exercise. The plasma insulin level decreased during the 90-min exercise, but was elevated after the 45-min exercise. The increase was greater than could be expected on the basis of the plasma C-peptide concentration. It is concluded that the hormonal and metabolic changes during submaximal long-term exercises are highly dependent on the intensity of exercise, even when the intensity of exercise is below the level of the anaerobic threshold.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2997049 TI - Adrenergic regulation of adaptation to muscular activity. PMID- 2997051 TI - Acute sodium bicarbonate does not affect renal hemodynamics in splenectomized dogs. AB - The glomerular filtration rate and the renal plasma flow were measured in conscious splenectomized dogs. The rapid intravenous administration of sodium bicarbonate (0.21 g . kg-1) did not affect either the glomerular filtration rate or the renal plasma flow. PMID- 2997052 TI - The demographic characteristics of pregnant women infected with cytomegalovirus. AB - An analysis was made of the demographic characteristics of 1000 women who were screened for serological evidence of cytomegalovirus (CMV) infection while receiving antenatal care in central London. The prevalence of antibodies against CMV was shown, by multiple discriminant analysis, to be significantly associated with non-Caucasian race (p less than 0.001), increasing maternal age (p less than 0.001) and poor social class (p less than 0.05). These results are interpreted as reflecting increased childhood exposure to CMV as a result of poor social environments. When data from 48 women who acquired primary CMV during pregnancy were analysed, infection was also related to non-Caucasian race (p less than 0.01), but in contrast, there was no demonstrable effect of social class, maternal age or marital status. We conclude that pregnant women acquiring the form of CMV infection with the greatest pathological potential for the fetus (primary infection) belong primarily to the middle-class sections of communities. Since middle-class women traditionally avail themselves of prophylactic measures, this result provides some optimism for the ultimate control of this common disease by immunization. PMID- 2997053 TI - The prognostic value of morphometry in ovarian epithelial tumors of borderline malignancy. AB - A fully "blind" morphometric analysis was made of 20 ovarian epithelial tumors; 18 of these were of borderline malignancy, one was a well-differentiated adenocarcinoma and one had been categorized as "borderline? malignant?" Morphometry correctly identified the adenocarcinoma, which had proved fatal, and the two tumors of borderline malignancy that had directly led to the patients' deaths, as having a "poor" prognosis. One tumor thought to have a "poor" prognosis was associated with long-term survival, but the patient had received chemotherapy. All the patients whose tumors were morphometrically graded as having a "good" prognosis were alive and tumor-free at intervals ranging from 4 to 14 years. It is concluded that the predictive prognostic power of morphometry, when applied to ovarian epithelial tumors of borderline malignancy, is greater than that of unaided light microscopy. PMID- 2997054 TI - Specific identification of human papillomavirus type in cervical smears and paraffin sections by in situ hybridization with radioactive probes: a preliminary communication. AB - Cervical Papanicolaou smears and paraffin sections of biopsy specimens obtained from women attending dysplasia clinics were examined for viral DNA sequences by in situ hybridization technique using 35S-labeled cloned recombinant DNA probes of human papillomavirus (HPV) types 6, 11, and 16. These and one unrelated DNA probe complementary to measles virus RNA were labeled by nick translation using either one or two 35S-labeled nucleotides. The radiolabeled probes were reduced in size with DNase to 60-160 nucleotides. Paraffin sections and cervical smears were collected on pretreated slides, hybridized with the probes under stringent or nonstringent conditions for 50 h, and autoradiographed. Additional cervical specimens from the same women were examined for the presence of genus-specific papillomavirus capsid antigen by the immunoperoxidase technique. Preliminary results may be summarized as follows. The infecting virus could be identified in smears as well as in sections. Viral DNA sequences were detected only when there were condylomatous cells in the specimen and in only a proportion of the condylomatous cells. Even under stringent conditions, some specimens reacted with both HPV-6 and HPV-11. None of the specimens hybridized with HPV-16 or with the unrelated probe. In some instances, the cells did not hybridize with any of the three probes even when duplicate specimens contained frankly condylomatous, capsid antigen-positive cells. In situ hybridization of Papanicolaou smears or of tissue sections is a practical method for diagnosis and follow-up of specific papillomavirus infection using routinely collected material. PMID- 2997055 TI - Disseminated intravascular coagulation syndrome. AB - This paper reports a patient with malignant fibrous histiocytoma of the maxilla who developed DIC during the 12-month observation of the hemostatic course, and a case of squamous cell cancer of the tongue associated with post-operative DIC. The triggers in these 2 cases were malignant tumor, infection, shock and operation. Heparin and aprotinin were administered in both cases. Hemostatic improvement was obtained in case 2, but neither cases were cured. The etiology, diagnosis and treatment of DIC are discussed. PMID- 2997056 TI - 1-Deoxyglycitolation of protein amino groups and their regeneration by periodate oxidation. AB - Reductive alkylation of lysyl epsilon-amino groups with sugars (1 deoxyglycitolation) using pyridine borane as the reducing agent has been recently described [Wong, W.S.D., Osuga, D.T. & Feeney, R.E. (1984) Anal. Biochem. 139, 58 67]. The regeneration of the lysines has now been achieved by oxidation with approximately 10 mM periodate. In experiments with glycitolated bovine serum albumin, reactions using 5, 20, and 80 mM periodate were essentially complete in the first 10 min. Oxidations of methionine to the sulfoxide were evident even at this lower concentration of periodate, while higher concentrations and prolonged reaction times only caused unnecessary destruction of amino acids. The removal was shown to occur in a wide pH range with little variation in the recovery of the lysines. The degree of glycitolation, or the nature of the attached carbohydrate residues, had no effect on the yield of products. Reductive 1 deoxygalactitolation of approximately 55% of the lysyl epsilon-amino groups of lysozyme caused no loss in enzymatic activity; when 5 mM periodate was used to remove the 1-deoxygalactitol moiety, approximately 95% of the amino groups were regenerated, concomitant with destruction of approximately 16% of the activity. On reductive 1-deoxygalactitolation of the amino groups of turkey ovomucoid, 65% of the lysyl epsilon-amino groups were modified with 70% loss of the inhibitory activity against trypsin. On treatment with 5 mM periodate, approximately 80% of the amino groups were regained with a similar recovery of the inhibitory activity. PMID- 2997057 TI - Photoreactive enkephalin analogue: [D-Ala2, p-N3-Phe4-Met5]-enkephalin. Synthesis, purification by high performance liquid chromatography and characterization. AB - A photoreactive [D-Ala2, p-N3-Phe4-Met5]enkephalin was synthesized by classical solution peptide synthetic methods. The hydroxysuccinimide ester was used in all the coupling steps in the presence of a weak base, triethylamine. The deprotected enkephalin analogue was purified on high performance liquid chromatography using a Waters, C18 muBondapak reverse phase column and its purity was assessed by thin layer chromatography. The composition of the analogue was determined and confirmed by elemental analysis and amino acid analysis. Its photoreactivity was demonstrated by the time dependent ultraviolet spectral changes on exposure to light. Competition receptor binding showed that [D-Ala2, p-N3-Phe4 Met5]enkephalin was 4-fold more potent than [D-Ala2, Met5]-enkephalin in competing for binding to the enkephalin binding site. The data presented suggest that this photoreactive enkephalin analogue may be suitable for use in the in situ photoaffinity labeling of the enkephalin receptor. PMID- 2997058 TI - Comparison of hydrolysis of atriopeptin II stand-in substrate by atrial dipeptidyl carboxyhydrolase and angiotension I-converting enzyme. AB - We recently found and partially purified a new membrane-bound metallo dipeptidyl dipeptidase from bovine atrial tissue homogenates (Harris, R.B. & Wilson, I.B. (1984) Arch. Biochem. Biophys. 233, 667-675). We suggested that this enzyme was capable of cleaving the dipeptide, phenylalanyl-arginine from the C-terminus of atriopeptin II to give atriopeptin I. The atriopeptins are two atrial natriuretic peptides and the existence of the atrial peptide system has implicated the mammalian heart as an endocrine organ. The tetrapeptide benzoyl-glycyl-seryl phenylalanyl-arginine was synthesized because it contains the C-terminal tripeptide sequence of atriopeptin II and should be useful to test the roles of the atrial enzyme and angiotensin I-converting enzyme in processing the atrial peptides. We found that for the atrial enzyme, Vmax was 13-fold higher and Km 7 fold-lower for this stand-in substrate than for benzoyl-glycyl-histidyl-leucine, a standard substrate used to measure converting enzyme activity. The ratio of Vmax/Km as a measure of substrate specificity indicates that the stand-in substrate is 86-fold better than benzoyl-glycyl-histidyl-leucine. In contrast, the stand-in substrate is a 20-fold poorer substrate for the converting enzyme than benzoyl-glycyl-histidyl-leucine. With the stand-in substrate, the converting enzyme showed pronounced substrate inhibition. An effective Vmax and Km were calculated using only concentrations of S below the optimum substrate concentration. These results confirm that the atrial enzyme is distinct from the converting enzyme. They also suggest that the conversion of atriopeptin II to atriopeptin I is a physiological process that is mediated by this enzyme. PMID- 2997059 TI - Synthesis of Z-CCK-27-32-NH2, Z-Tyr(SO-3)-Met-Gly-Trp-Met-Asp-NH2, a cholecystokinin receptor antagonist and an inhibitor of gastrin-induced acid secretion. AB - The synthesis of the hexapeptide Z-Tyr(SO-3)-Met-Gly-Trp-Met-Asp-NH2, representing the C-terminal sequence of cholecystokinin minus the C-terminal phenylalanyl residue is described. This peptide was shown to be the most potent cholecystokinin receptor antagonist in vitro described to date. It is also able to inhibit gastrin-induced acid secretion in vivo, in the rat and was proved to antagonize the action of the C-terminal octapeptide of cholecystokinin in the central nervous system. PMID- 2997060 TI - Chemical synthesis of insulin-like growth factor II. AB - Human insulin-like growth factor II (IGF-II) with 67 amino acids and three disulfide bridges has been synthesized by the solid-phase method. The synthetic hormone is shown to be homogeneous in high performance liquid chromatography (HPLC), high performance partition chromatography (HPPC), and chromatofocusing. It is indistinguishable from natural hormone in HPLC, peptide map of thermolysin digests, amino acid composition and radioreceptor binding assay. Thus, synthetic and natural IGF-II are identical. PMID- 2997061 TI - The significance of increased echogenicity in the detection and differentiation of pediatric disease. AB - Increased renal echogenicity, by ultrasonography, is seen in various pathological conditions. Correlation of renal size with the pattern and distribution of the increased echogenicity, and the patient's age and clinical data will limit the differential diagnosis and suggest the appropriate radiographic follow-up studies, obviating the need for unnecessary procedures. PMID- 2997062 TI - Depression of the radioprotective effect of isoproterenol on mammalian cells in vitro after desensitization of the cAMP system. AB - A short (5 min) incubation of cultured Chinese hamster fibroblasts with the specific beta-agonist isoproterenol (1 microM) leads to an increase in the intracellular content of cAMP and a decrease in radiosensitivity of the cells. Prolonged (up to 1 h) incubation induces a desensitization of the cAMP system to isoproterenol and causes a decrease both in the cAMP-stimulating and radioprotective effect of isoproterenol. There were no detectable changes in the beta-adrenoreceptor number or binding affinity of beta-receptors to the radiolabelled beta-antagonist dihydroalprenolol in desensitized cells; cAMP phosphodiesterase activity was also the same as in intact cells. It is proposed that a 1 h incubation of the cells with isoproterenol induces the first step of desensitization, i.e. the functional "uncoupling' of beta-receptors from adenylate cyclase. Thus, the presence of beta-receptors in cells is not enough for the realization of the radioprotective potency of isoproterenol; an intact, non-desensitized, state of the cAMP system is obligatory. PMID- 2997064 TI - Photochemical generation of superoxide radical and the cytotoxicity of phthalocyanines. AB - The effect of the central metal atom on the photodynamic activity of phthalocyanine dyes has been estimated by cytotoxicity to cultured Chinese hamster cells. Chloroaluminium phthalocyanine,, followed by the Zn- derivate, were found to be the only active dyes. In parallel it was found that visible light (615 +/- 10 nm) excitation of phthalocyanines dissolved in dimethylsulphoxide in the presence of oxygen generates superoxide radical anion. O2- radicals were spin--trapped with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and identified by electron spin resonance. The quantum yields for O2- generation range from 10(-5) (Zn-phthalocyanine) to 4.2 X 10(-4) (Ga-phthalocyanine). The efficiency of generating O2- was apparently uncorrelated with the phototoxicity of the same dyes. Furthermore, the biological photodamage could not be inhibited by the addition of superoxide dismutase. It is concluded that O2- is involved very little, if at all, in the phthalocyanine-induced photo-killing of mammalian cells. PMID- 2997065 TI - The isolation and characterization of kadsurenone from haifenteng (Piper futokadsura) as an orally active specific receptor antagonist of platelet activating factor. AB - A natural product, kadsurenone, was isolated from the Chinese herbal preparation haifenteng (Caulis piperis futokadsurae) and characterized as an orally-active specific antagonist of the platelet-activating factor (PAF). Kadsurenone inhibits the specific binding of 3H-PAF to a receptor preparation from rabbit platelet membrane in a competitive and reversible manner, its Ki being 3.88 X 10(-8) M. It inhibits the aggregation of rabbit platelets in plasma induced by PAF with a pA2 of 6.28, but not those induced by arachidonic acid, epinephrine, ADP or A-23187. It inhibits the aggregation of isolated human neutrophils with a pA2 of 6.32. It also inhibits PAF-induced degranulation and release of beta-D-glucuronidase at 2 24 microM in vitro. In the rat, kadsurenone at 8-40 mg/kg i.p. inhibits the increases of plasma lysosomal enzymes and haematocrit induced by intravenous PAF. In the guinea pig, kadsurenone at 25-50 mg/kg p.o. reduces the increase of cutaneous vascular permeability induced by PAF. These results indicate that kadsurenone is a specific and effective receptor antagonist of PAF in several in vitro and in vivo systems. PMID- 2997063 TI - Uptake of 59Fe and 239Pu by rat liver cells and human hepatoma cells. AB - The accumulation of 59Fe and 239Pu was studied in rat hepatocytes in primary culture and in human hepatoma cells (Hep-G2 cells) and was compared with the uptake in isolated perfused rat liver and in rat liver in vivo. With respect to iron uptake from citrate both cell types react similarly: time and concentration dependence as well as the influence of temperature point to a non-specific but energy-dependent uptake mechanism. Pu shows similar behaviour except that a relatively large fraction remains bound to the cell membrane and uptake is lower. This is much more pronounced in Hep-G2 cells than in hepatocytes. If the metals are bound to transferrin, uptake into both cell lines, as well as into isolated perfused rat liver, is very low. After intravenous injection of 239Pu-citrate, accumulation in the rat liver is very rapid during the first 10 min and much slower after that. The role of citrate in metal uptake is discussed. PMID- 2997066 TI - Synthesis of platelet-activating factor from human polymorphonuclear leukocytes: regulation and pharmacological approaches. AB - Human polymorphonuclears release a platelet-activating factor (PAF-acether) when challenged with various stimuli. PAF-acether is a mediator that is synthesized during cell activation in a process in which a phospholipase A2 and an acetyltransferase take part. These enzymes are finely regulated and accordingly PAF-acether release may be modulated. The authors have studied some of the transductory mechanisms which are triggered during cell stimulation and the effect of their pharmacological modulation on PAF-acether release. Theophylline, methylisobutylxanthine and dipyridamole, which block phosphodiesterase of cyclic nucleotides, induce a dose-dependent inhibition of PAF-acether release without affecting phagocytic uptake. Polyamines (dansylcadaverine, rimantadine and amantadine) reduced PAF-acether release and the phagocytic process in an order of potency similar to their ability to inhibit phospholipid methylation and the cholinephosphotransferase pathway. The calmodulin antagonist trifluoperazine induced a dose-dependent inhibition of PAF-acether release and acetyltransferase at concentrations from 10(-4) to 10(-5) M. Hence it appears that modulation of PAF-acether release can be obtained by different pharmacological blockades: phosphodiesterase of cyclic nucleotides, phospholipid metabolism and calcium calmodulin. PMID- 2997067 TI - Platelet-dependent granulocyte activation in vitro: effect of ticlopidine. AB - The presence of platelets or platelet release products is known to augment the injury of endothelial cells caused by stimulated neutrophils (PMN leukocytes). In our in vitro studies, platelet-rich or platelet-poor plasma (PRP or PPP) was placed in one compartment and a PMN suspension in the other of a modified Boyden chamber divided by a dialysis membrane. The addition of the aggregating substance (ADP, collagen) to PRP but not to PPP was followed by PMN activation as shown by enzyme release and O-2 generation. The in vivo treatment with ASA completely prevented the platelets from triggering PMN activation. The in vitro addition of a thromboxane synthetase inhibitor (imidazole 10(-3) M) or a lipoxygenase inhibitor (NDGA 10(-6) M) did not show any effect on platelet-dependent PMN activation, thus suggesting that neither TxA2 nor lipoxygenase by-products are involved. Finally, in vitro and in vivo treatment with ticlopidine blunted the stimulating activity of the platelets on PMN. Our data further support the hypothesis that a sequential platelet-PMN interaction may occur, and that the therapeutic effect of some antiplatelet drugs may be partly due to a protective effect against platelet-dependent PMN-mediated vascular damage. PMID- 2997068 TI - Activation of latent polymorphonuclear leucocyte collagenase by the glutathione cycle. AB - The intrapleural injection of carrageenin into rats caused accumulation of leucocytes in the pleural cavity and changes in blood leucocyte populations. An increased number of polymorphonuclear (PMN) leucocytes was observed. The peripheral blood PMN leucocytes of untreated rats contained 33.2 nM reduced glutathione/10(7) cells and collagenase mainly in the latent form. During experimental pleurisy in blood leucocytes as well as in the leucocytes obtained from the pleural exudate, the level of reduced glutathione decreased significantly by about 33% (22.4 nM/10(7) cells) and parallel collagenase activation up to 80-100% was observed. Two tested antiinflammatory drugs, Timegadine and piroxicam, which significantly inhibited activation of latent collagenase during the course of inflammation and partially protected reduced glutathione against oxidation. These results suggest that the activation of latent leucocyte collagenase in vivo via the disulphide-thiol interchange reaction may be coupled to the glutathione cycle and regulated by variation in its redox potential. PMID- 2997070 TI - The unicellular Tetrahymena as a model cell for receptor research. PMID- 2997069 TI - An outbreak of invasive aspergillosis among allogeneic bone marrow transplants: a case-control study. AB - Between April 1982 and March 1983, 10 of 26 (38.4%) allogeneic bone marrow transplant recipients housed on a newly opened bone marrow transplant unit developed invasive aspergillosis. By contrast, between September 1977 and March 1982, only 3 of 46 (6%) transplant recipients developed invasive aspergillosis. A case-control study to identify host factors related to Aspergillus infection found that aspergillosis was more common in patients with chronic myelogenous leukemia and aplastic anemia, older patients, patients having cytomegalovirus disease, patients who experienced prolonged granulocytopenia, patients conditioned with ara-C (100-200 mg/day), and patients who received longer duration of antimicrobial therapy. A series of logistic regression analyses revealed that underlying disease was the single best predictor of Aspergillus infection. This study demonstrates that underlying disease is an important risk factor for aspergillosis and that special measures may be warranted when transplanting certain patients. PMID- 2997071 TI - Target cell prolactin, II. AB - Is the entry hypothesis compatible with all the existing data about "the" second messenger for prolactin listed in Section II? All of these messengers, in some way either participate in, or modify, prolactin's actions or, in an end point dependent manner, may actually mimic prolactin. There remains considerable uncertainty as to whether these findings reflect phenomena, some independent of and others quite dependent upon entry, on the one hand, or merely portions of a relatively large number of molecular cascades, some (but not necessarily all) begun initially at the plasmalemma and many (if not all) orchestrated toward completion by intracellular prolactin or agonist-receptor complex. PMID- 2997072 TI - Interferon production and sensitivity of rabbit corneal epithelial and stromal cells. AB - The induction of interferon and the ability of interferon to induce the antiviral state were studied using rabbit corneal epithelial and stromal cells which were cultured for fewer than five passages. Interferon titers in the range of 7000 units/ml were induced in epithelial cell cultures and 76,000 units/ml in stromal cell cultures treated with UV-inactivated bluetongue virus. The interferon induced was stable to pH 2.0 treatment and heating to 56 degrees C for 16 hr. Infection of epithelial and stromal cell cultures with various strains of herpes simplex virus type 1 showed that all strains tested replicated to equivalent titers in the respective cell types, and that no detectable interferon was induced in stromal cells and only trace amounts in epithelial cells. Exogenously supplied rabbit interferon induced the antiviral state in cultures of both cell types restricting the replication of not only encephalomyocarditis virus but also herpes simplex virus. Sixty to ninety units of rabbit interferon reduced HSV-1 virus replication by 50%. Human interferons had less than 27% of the antiviral activity in rabbit cells than they had in a human cell line. The data indicate that exogenously supplied interferon may act to reduce the severity of herpetic keratitis by directly inducing the antiviral state in corneal epithelial and stromal cells. However, interferon endogenously produced by rabbit corneal cells in response to HSV-1 infection probably plays a minor role in the pathogenesis of ocular HSV-1 infections. PMID- 2997073 TI - Resolution of HSV corneal infection in the absence of delayed-type hypersensitivity. AB - The role of delayed-type hypersensitivity (DTH) in the resolution of herpes simplex virus type 1 (HSV-1) ocular infection was examined. Infection of Balb/c mice on the sacrificed cornea with HSV-1 resulted in sensitization for DTH. This response, demonstrable by swelling of the ear following inoculation with ultraviolet-irradiated virus, was optimal 7 days postinfection. The reaction was immunologically specific and characterized histologically by a predominately mononuclear cell infiltrate. DTH responsiveness could be completely abrogated if the mice were inoculated intravenously with an attenuated strain of HSV-17 days before corneal infection. DTH-unresponsive mice were, nevertheless, resistant to corneal challenge with sublethal or lethal doses of HSV-1. Resistance was accompanied by a greater than 30-fold reduction in infectious virus in the eye 24 hr post challenge. A cellular infiltrate characteristic of a DTH response was not observed within the cornea during virus clearance. Tolerance was restricted to DTH, as antibodies to HSV antigens could be readily demonstrated 6-7 days after intravenous virus immunization. These antibodies may have contributed to the resistance observed. The results establish that neither a systemic nor local DTH response is required by the host to resist HSV-1 ocular infection. PMID- 2997074 TI - Distribution patterns of photoreceptors, protein, and cyclic nucleotides in the human retina. AB - The concentration of cGMP, cAMP, protein and the number of cone and rod photoreceptors have been measured in parallel arrays of punches, 3 mm in diameter, taken from each quadrant of normal human retinas. A separate punch containing the fovea and parafoveal region was also analyzed. Eyes were obtained from four male donors ranging in age from 35 to 67 yr. The retina thins considerably from the center to the periphery, and consequently the protein content forms a gradient in the same direction. Similar gradients were observed for cAMP and cGMP concentrations. In all eyes studied, the foveal-parafoveal region had higher levels of cAMP than cGMP. The data was analyzed with the aid of a computer in order to obtain three-dimensional maps of the patterns of distribution of the different parameters. A strong correlation between the areas of higher cone density, non-photoreceptor neurons, and cAMP, and an equally strong correlation between rod distribution and that of cGMP was observed. These maps will serve as baseline data in studies of pathological conditions such as retinitis pigmentosa. PMID- 2997076 TI - A cutaneous fibropapilloma from a red deer (Cervus elaphus) associated with a papillomavirus. AB - A cutaneous fibropapilloma was found on a Scottish red deer (Cervus elaphus), and a papillomavirus was isolated from it. The virus appeared to be related to bovine papillomavirus type 1 (BPV1) or type 2 (BPV2) because: (i) it cross-reacted in peroxidase-antiperoxidase tests with antisera raised against these virions; (ii) BPV1 and BPV2 DNAs cross-hybridized to the red deer papillomavirus in situ; and (iii) BPV1 and/or BPV2 DNA cross-hybridized to the red deer papillomavirus DNA on Southern blots under conditions of high stringency. These tests also revealed a unique restriction enzyme cleavage pattern for the red deer papillomavirus DNA. PMID- 2997075 TI - Phosphodiesterase-probes show distinct defects in rd mice and Irish setter dog disorders. AB - The phosphodiesterase from the visual cells of rd mice and affected Irish setter dogs has been analyzed, using biochemical, biophysical, and immunological techniques. The authors' findings demonstrate that the mechanisms that cause a deficiency in phosphodiesterase activity in rd mice and Irish setter dogs are distinctly different. Apparently, the phosphodiesterase complex is normal in affected Irish setter dogs but is abnormal in rd mice. The criteria used for determining the normalcy of the phosphodiesterase complex were sedimentation characteristics, immuno-cross-reactivity, and histone-activation, which is shown to be a unique characteristic of the visual cell enzyme. According to these criteria, the phosphodiesterase complex in the visual cells of rd mice is either absent or abnormal from the onset of visual cell differentiation until degeneration, because it exhibits no cross-reactivity with antibody to phosphodiesterase; it is not activated by histone; and if present, it exhibits abnormal sedimentation characteristics and perhaps subunit structure. On the other hand, phosphodiesterase from the visual cells of affected Irish setter dogs is normal by the same criteria, because it cross-reacts with antibody against phosphodiesterase; it is activated by histone; and it exhibits normal sedimentation and electrophoretic patterns. It is proposed that depressed levels of phosphodiesterase activity in affected setter photoreceptors are due, perhaps, to a defect in the light-initiated cascade which activates the enzyme normally, in situ. PMID- 2997077 TI - Interaction between polymorphonuclear leukocytes and varicella-zoster virus infected cells. AB - The addition of polymorphonuclear leukocytes (PMNL) to human fibroblasts infected with varicella-zoster virus (VZV) resulted in a reduced virus yield. The reduction was greater when antibodies specific for VZV were added to the system. Addition of VZV-specific antibodies without PMNL also reduced virus yield, but a 10-fold greater concentration of antibodies was required to effect the same reduction. PMNL adhered to and formed rosettes around VZV-infected cells. By electron microscopy, it was possible to observe activation of PMNL incubated with VZV-infected fibroblasts. Activated PMNL extended cytoplasmic projections toward virions and had vacuoles containing virions in various stages of digestion. PMID- 2997078 TI - Isolation and adaptation characteristics of hepatitis A virus in primary African green monkey kidney cells: production of antigen useful for ELISA serology. AB - Four strains of hepatitis A virus (HAV) were isolated from four fecal samples of patients with type A hepatitis by using primary African green monkey kidney (PAGMK) cells or FRhK-4 cells. In all four samples viral antigen became detectable in PAGMK cells at the 3rd passage level after 9 weeks of incubation; detectable levels of antigen were reached earlier in FRhK-4 cells. An enzyme linked immunosorbent assay (ELISA) was used to detect HAV antigen (HAV-Ag). Blocking experiments with negative and positive human sera and with paired marmoset sera established the identity of the virus. Infectious virus appeared to be both intracellular and extracellular. Although HAV-Ag could not be detected in culture medium by ELISA, the HAV infectivity titers of culture media were as high as those of cell-associated viruses (greater than 10(6) TCID50/0.2 ml). The passage procedure was simplified by using only virus isolated from cell-free medium as seed material, and the HAV strains were successfully propagated for 12 consecutive passages through PAGMK cells at 2-week intervals. The tissue-culture produced HAV-Ag proved to be useful as a source of antigen in ELISA for detection of human anti-HAVIgG and IgM. The HAV strains adapted to PAGMK cells lost or decreased their ability to grow in FRhK-4 cells, while one strain adapted to FRhK 4 cells grew equally well in both cell systems. PMID- 2997080 TI - Inhibitory effect of superoxide radicals on cardiac myofibrillar ATPase activity. AB - The superoxide radicals generated by the xanthine oxidase reaction reduced the myofibrillar Ca2+-ATPase activity. This negative effect was prevented by superoxide dismutase or by dithiothreitol, a protective thiol compound. Partial protection was achieved by catalase, while mannitol was ineffective. The myofibrillar Ca2+-ATPase exposed to O2-. radicals did not modify the affinity for Ca2+ while it showed a remarkable reduction of Vmax measured at the saturating level of Ca2+. The O2-. inhibited myofibrillar ATPase showed a higher value of Km for the cofactor associated to a reduced value of Vmax when studied in the presence of increasing concentration of ATP. Thus, circumstances that enhance the production of cardiac O2- radicals can be considered a negative metabolic event capable of depressing the myofibrillar Ca2+-ATPase activity. PMID- 2997079 TI - Anterior cervical decompression and fusion for cervical degenerative disc diseases. PMID- 2997081 TI - Metabolism of exogenous nucleosides in Bacillus cereus and Escherichia coli. PMID- 2997082 TI - The restoration of impaired macrophage functions using as immunomodulator the Corynebacterium granulosum-derived P40 fraction. AB - Many microorganisms and compounds of microbial origin exhibit immunomodulatory activities and have been extensively used in immunotherapy of experimental animal tumors and in patients with neoplasia. In this paper we describe the effect of the C. granulosum-derived P40 fraction on the growth and metastatization of the transplantable epithelioma T8 of Guerin. Moreover, we evaluated the effect of P40 treatment on several depressed macrophage functions of tumor-bearing rats. In particular, the phagocytic and chemotactic activities of such cells were studied, as well as the antiviral intrinsic and extrinsic activities against HSV-1 and the anti-Toxoplasma gondii activity. All these functions were depressed in untreated tumor-bearing rats. Administration of a single intravenous injection of P40 fraction led to the restoration of all depressed macrophage activities to normal values. In particular, the possibility of restoring the antimicrobial activity of macrophages from tumor-bearing rats by treatment with this immunomodulator is of great concern when one considers the increasing incidence of opportunistic infections in immunocompromised hosts. Results are discussed in terms of both the possible mechanism of action of P40 and of its possible target cells. PMID- 2997084 TI - The epidemiological investigation of AIDS. PMID- 2997083 TI - Effects of anti-inflammatory and anti-rheumatic drugs on the activities of purified and membrane-bound Na+/K+ adenosine triphosphatase. AB - We have examined the effects of anti-inflammatory and anti-rheumatic drugs on membrane-bound and purified Na+/K+-ATPase activity in vitro. Only the gold containing compounds (gold sodium thiomalate and auranofin) were found to inhibit the enzyme activity in a dose-dependent manner. Sodium thiomalate and triethylphosphine, the ligand compounds for gold sodium thiomalate and auranofin, respectively, had no effect on ATPase activity. The antagonistic properties was abolished by preincubation of the gold compounds with dithiothreitol. Lineweaver Burke analysis of the inhibitions of purified ATPase by the gold compounds was found to follow uncompetitive kinetics. Inhibition of ATPase by gold may cause disruption of transmembrane cation transport and thus result in impairment of several metabolic processes and cellular functions. PMID- 2997085 TI - AIDS: The challenge to science and medicine. PMID- 2997086 TI - Screening blood: public health and medical uncertainty. PMID- 2997087 TI - Colorectal cancer in Hawaii Japanese. PMID- 2997088 TI - Molecular determination of T-cell receptor alpha and beta chain genotypes in human families. AB - Polymorphism in genes encoding the alpha and beta chain of the human T cell receptor has been detected by Southern blot analysis. Genomic DNA samples were isolated from B lymphoblastoid cell lines derived from members of families, each family including at least one individual with a recombinant HLA haplotype. T cell receptor alpha and beta chain haplotypes could be assigned in the families on the basis of observed restriction fragment length polymorphism (RFLP). Polymorphism in the alpha chain gene was detected in BglII digests using an alpha chain probe that included the V, J, C, and 3' untranslated sequences. A probe consisting of only the constant region (C alpha) revealed no polymorphism indicating that the polymorphic fragment hybridized to V, J, or 3' untranslated sequences of the alpha chain. Polymorphism in beta chain genes was observed in BglII digested DNA samples using a probe that corresponds to the constant region (C beta). Polymorphic C beta restriction fragments of 10.0 and 9.2 kilobase segregated in six of the eight families studied. Recent structural data for the C beta region suggest that the polymorphic BglII site lies in the region 5' to the C beta 2 gene. These polymorphisms should serve as markers for alpha and beta chain complexes allowing genetic studies of these immunologically important gene families. PMID- 2997089 TI - Primary spinal cord tumors treated with surgery and postoperative irradiation. AB - Between 1954 and 1979, 37 patients with a primary spinal cord tumor received postoperative irradiation after laminectomy. There were 26 intramedullary and 11 tumors of the conus/cauda equina. The 26 intramedullary tumors were divided as follows: 14 astrocytomas, eight ependymomas, three unbiopsied tumors, and one diffuse histiocytic lymphoma. Of the cauda equina tumors, 10 were ependymomas and one was an astrocytoma. Patients were followed until death or for a minimum of 4 years. The 5- and 10-year actuarial survivals for the entire group were 70 and 58%, respectively. Anatomic location of the tumor was the most important predictor of both survival and neurologic function. Patients with tumors of the cauda equina had superior neurologic function and a significantly better survival than those with tumors at other sites. Recurrent tumor was the cause of death in 82% of the patients dead at the time of analysis. Of the patients alive at the conclusion of the study, 10 were completely normal or had only mild neurologic deficit; the remaining 10 patients were severely disabled. Increasing radiation dose correlated with an increase in tumor control and survival. Of those receiving less than 40 Gy, 77% died of recurrent tumor, while 83% of those who received greater than 40 Gy are alive 4.1 to 28.9 years after treatment. PMID- 2997090 TI - A phase I study of intravenous iododeoxyuridine as a clinical radiosensitizer. AB - Twenty-four patients with locally advanced (19 patients) or metastatic (5 patients) tumors were treated in a Phase I study combining constant intravenous infusions of iododeoxyuridine (IUdR) and hyperfractionated radiation therapy. IUdR was given as a constant infusion for 12 hours/day for two separate 14-day infusion periods in most patients. The dose of IUdR was escalated from 250 to 1200 mg/m2/12-hour infusion in this study. The initial tumor volume was treated to 45 Gy/1.5 Gy BID/3 weeks followed by a cone-down boost to 20-25 Gy/1.25 Gy BID/2 weeks after a planned 2-week break. THe IUdR infusion preceded the initial and cone-down irradiation by 1 week. Local acute toxicity (within the radiation volume) was uncommon and few patients required an alteration of the planned treatment schedule. Two patients developed late local toxicity with one patient showing clinical signs of radiation hepatitis and another patient developing a large bowel obstruction that required surgical bypass. Dose-limiting systemic toxicity was confined to the bone marrow with moderate to severe thrombocytopenia developing on Day 10-14 of infusions at 1200 mg/m2/12 hours. Mild stomatitis and partial alopecia occurred in some patients at this dose level. No systemic skin toxicity was seen. Pharmacology studies revealed steady-state arterial plasma levels of IUdR of 1 to 8 X 10(-6) M over the dose range used. In vivo IUdR incorporation into tumors was studied in three patients with high-grade sarcomas using an anti-IUdR monoclonal antibody and immunohistochemistry and demonstrated incorporation in up to 50-70% of tumor cells. The preliminary treatment results, particularly in patients with unresectable sarcomas, are encouraging. In comparison to our previous experience with intravenous bromodeoxyuridine, this Phase I study of IUdR shows less systemic toxicity (especially to skin), higher (2-3X) steady-state arterial levels, and comparable in vivo tumor cell incorporation. PMID- 2997091 TI - The relationship between endothelial dysfunction and collagen accumulation in irradiated rat lung. AB - Male rats were killed 2 months (early fibrosis) or 6 months (peak fibrosis) after a range of single doses of 60Co gamma rays to the right hemithorax. Pulmonary arterial perfusion scans were performed at 2 months on animals scheduled for autopsy at 6 months. Lung angiotensin converting enzyme (ACE) activity was used to monitor endothelial function, and hydroxyproline (HP) concentration served as an index of interstitial collagen accumulation (fibrosis). ACE activity also was measured in right lung bronchoalveolar lavage (BAL) fluid and blood serum, to determine whether information obtained from a minimally invasive procedure might serve as an index or predictor of the severity of lung damage. Linear dose response curves (r = 0.92-0.99) were obtained for right lung arterial perfusion, ACE activity and HP concentration. At 2 months, perfusion decreased 2.7% per Gy, ACE activity (per lung, per mg wet weight, or per mg protein) decreased 3.0-4.2% per Gy, and HP concentration (per g dry weight) increased 1.7% per Gy. At 6 months, the slopes of the response curves were virtually identical to those at 2 months; the Y intercept of the response curve for ACE activity was unchanged, whereas that for HP concentration was 50% higher at 6 than at 2 months. ACE activity and protein concentration in the BAL increased with increasing dose, but the variation within groups was too large, and the sensitivity was too low to serve as a reliable index of lung status. Serum ACE activity was independent of radiation dose at both autopsy times. Thus in rat lung, arterial perfusion, endothelial dysfunction and interstitial fibrosis exhibit similar but not identical radiosensitivities. The dose-effect curves for these three responses of the lung in situ change less than 5% per Gy over the dose range of 10-30 Gy, a smaller variation than would be predicted from endothelial cell survival data based on clonogenic assays in vitro or in vivo. PMID- 2997093 TI - Prenatal and preweaning deaths caused by pseudorabies virus and porcine parvovirus in a swine herd. AB - Sequential outbreaks of pseudorabies virus and porcine parvovirus infections were documented at a swine farm in southern Minnesota. Data for the prevalence of mummified fetuses born and the preweaning mortality were recorded over a 3-year period. The farm was a farrow-to-finish facility, with breeding females housed in 4 groups according to their stage of pregnancy. The herd consisted of approximately 130 breeding females in December 1981, and expanded to 220 females during the 12 months of 1982. Excluding the outbreaks, the mean preweaning mortality was 20.43% (SE 1.59) and the number of mummified fetuses per litter was 0.19 (SE 0.01). An outbreak of porcine parvovirus infection caused the preweaning mortality and number of mummified fetuses to increase to 50% and 4.10 per litter, respectively. Two outbreaks of pseudorabies 27 months apart, caused the preweaning mortality to increase to 95% and 82%, and the number of mummified fetuses to increase to 0.96 and 1.25 mummified fetuses per litter, respectively. The increase in mummification was observed 1 month after the increase in preweaning mortality caused by pseudorabies virus infections, whereas the increase in mummification and preweaning mortality was simultaneous with porcine parvovirus infections. PMID- 2997092 TI - Humoral immune response in infectious mononucleosis. Late emergence of anti-EA(R) and the effects of corticosteroid therapy. AB - The antibody response to Epstein-Barr virus (EBV) antigens in patients with infectious mononucleosis (IM) was studied to assess antibody appearance to the restricted (R) component of the early antigen (EA) complex and to determine the effect of corticosteroids on all aspects of the humoral immune response. Sixty college students with heterophil-positive clinical IM, confirmed by EBV-specific serology, were followed for a period of 4-26 weeks, Half received prednisone for six days, and the remainder received no corticosteroid therapy. Regardless of therapy, 48% of the patients developed anti-EA(R) antibodies. The response to other antigens was similar in both groups with the exception that antibodies to the EB-associated nuclear antigen (EBNA) developed later during convalescence and at lower titers in the corticosteroid-treated group. We conclude that 1) anti EA(R) antibodies develop with considerable frequency following IM and are not a marker, as previously proposed, of unusually severe disease, and 2) corticosteroid therapy may retard the formation of anti-EBNA antibodies but it does not otherwise influence the humoral immune response to EBV. PMID- 2997094 TI - Isolation of equine coital exanthema virus (equine herpesvirus 3) from the nostril of a foal. AB - The virus causing equine coital exanthema (equine herpesvirus 3) was isolated from a lesion on the nostril of a 2-month-old foal. One week after the mare had returned from a stallion station, vesicular lesions developed on her vulva. They were diagnosed clinically as coital exanthema, and 5 days later a lesion developed on the nostril of her foal. This case is an example of horse-to-horse transmission of coital exanthema virus without coitus. A laboratory diagnosis is necessary to differentiate viruses that cause vesicular lesions about the oral and nasal cavities of horses. PMID- 2997095 TI - Idiopathic hyperaldosteronism in a dog. AB - Idiopathic hyperaldosteronism was diagnosed in an 8-year-old castrated male Yorkshire Terrier, based on increased concentration of plasma aldosterone, hypertension, hypernatremia, decreased natriuresis, hypokalemia, and hyperkaluria. Unilateral adrenalectomy was performed after visualization of a nodule on the right adrenal gland. Hyperplasia of the zona glomerulosa and increased postoperative aldosterone concentrations supported the diagnosis of idiopathic hyperaldosteronism. PMID- 2997096 TI - Correlation between high susceptibility to AIDS virus and surface expression of OKT-4 antigen in HTLV-I-positive cell lines. AB - Most human T-cell lymphotropic virus type I (HTLV-I)-carrying cell lines possess high susceptibility to AIDS retrovirus. This high permissiveness was clearly correlated with the amount of OKT-4 molecules, the possible receptor for AIDS retrovirus, expressed on the cell surface of HTLV-I-bearing cell lines. However, no correlation was noted in HTLV-I-negative cell lines. PMID- 2997097 TI - Phase II study of vindesine in patients with non-small cell lung cancer. AB - A phase II study of vindesine (VDS) was carried out in 21 patients with non-small cell lung cancer (NSCLC). There were 13 and 8 patients with and without prior chemotherapy, respectively. VDS was administered at a weekly iv dose of 3 mg/m2. Partial response was observed in two of 15 adenocarcinomas and one of 2 adenosquamous cell carcinomas, and the overall response rate was 14.3% (3/21). Myelosuppression, especially leukopenia, was the most common dose-limiting side effect. Neurotoxicity was also a common side effect but the degree was mild. It was concluded that VDS at a dose of 3 mg/m2 every week seems to be active against NSCLC. PMID- 2997098 TI - Culture of ciliated and nonciliated cells from rat ductuli efferentes. AB - The isolation and culture of ciliated and nonciliated cells from rat ductuli efferentes is described. Fragments of epithelium obtained after two collagenase digestions attached to plastic and to extracellular matrix and could be maintained in culture for at least 2 weeks. Ciliary beating in cells grown on epididymal extracellular matrix-coated plastic could be observed for up to 7 days in culture. Although cells maintained on this substrate retained organelles characteristic of cells in vivo, they assumed a flattened, squamous appearance. In contrast, cells growing on the surface of permeable supports impregnated with extracellular matrix were polarized and exhibited a cuboidal/columnar appearance. Androgen binding protein conjugated to colloidal gold was taken up by these cells via coated pits and was found sequentially in uncoated endosomes, multivesicular bodies and lysosomes. PMID- 2997099 TI - A bacteriological investigation of the effectiveness of cleaning and disinfection procedures for toilet hygiene. AB - The bacterial contamination of hospital and institutional toilets and toilet areas which were cleaned daily was investigated. The effect of daily disinfection with hypochlorite or a quaternary ammonium product, or with a continuous-release hypochlorite disinfectant system, based on the chlorine-releasing agent trichloroisocyanuric acid, was determined. The continuous release system produced substantial and sustained reduction in contamination of the toilet itself (water, toilet bowl and rim) and some reduction in contamination of sites surrounding the toilet (seat, floor, and air). By contrast, although daily disinfection produced some reduction in contamination compared with daily cleaning, the reductions were less than that associated with the continuous release system and indicated the inadequacy of daily disinfection and/or cleaning for toilets where effective procedures are required. PMID- 2997100 TI - The in-vitro activity of a novel penem FCE 22101 compared to other beta-lactam antibiotics. AB - FCE 22101 is a penem antibiotic which inhibits the majority of Enterobacteriaceae, Haemophilus influenzae, and Neisseria gonorrhoeae at concentrations of 0.5-4 mg/l. It inhibits staphylococci, haemolytic streptococci and Streptococcus pneumoniae at less than or equal to 0.25 mg/l. Pseudomonas aeruginosa and other Pseudomonas species are resistant. Bacteroides fragilis and Clostridium species are inhibited by less than or equal to 1 mg/l. FCE 22101 is not hydrolyzed by the common plasmid and chrosmosomal beta-lactamases. It shows minimal discrepancy between MIC and MBC values and there is minimal effect of inoculum size. Although FCE 22101 is generally less active against Enterobacteriaceae than are cefotaxime and ceftazidime, it does inhibit some Enterobacter spp. resistant to these agents. FCE 22101 and imipenem are similar in activity against Gram-positive and anaerobic species. PMID- 2997101 TI - Once-daily intravenous acyclovir for prophylaxis of herpes simplex virus reactivation after marrow transplantation. AB - To determine the most convenient and least expensive regimen for prevention of recurrent herpes simplex virus (HSV) infection after marrow transplantation, we conducted a randomized, double-blind comparison of intravenous acyclovir 250 mg/m2 and placebo given once daily for four weeks. Six of 14 acyclovir and nine of 13 placebo recipients shed HSV during prophylaxis. All nine culture-positive placebo recipients developed associated lesions during prophylaxis compared to four of six acyclovir recipients. Median time to first culture-positive lesion was significantly delayed by acyclovir compared to placebo (33 days after transplant vs. 10; P = 0.05). Acyclovir-resistant HSV was recovered from one acyclovir recipient while receiving prophylactic acyclovir, and from two placebo recipients during subsequent administration of therapeutic acyclovir. Once-daily intravenous acyclovir can significantly delay time to appearance of culture positive HSV lesions after marrow transplant, but virological and clinical breakthrough may occur and optimal prevention will require administration of intravenous acyclovir more than once daily. PMID- 2997102 TI - A unique aminoglycoside-O-phosphorylating activity mediating resistance to aminoglycosides in Escherichia coli. PMID- 2997103 TI - Simple procedure for removal of AMP from NADP preparation. AB - A simple and reliable procedure for removal of AMP from NADP preparation is described. In this procedure, a mixture of AMP and NADP solution is first incubated with 5'-nucleotidase to hydrolyze AMP to adenosine and inorganic phosphate (Pi). The reaction mixture is then applied to a Dowex 1 (formate) column. Adenosine and 5'-nucleotidase are removed by washing the column with 20 mM HCOOH. NADP is finally eluted with 3.5 M HCOOH followed by precipitation and washing with acetone. The yield of salt-free NADP is about 80%. Although Pi is coeluted with NADP in the acid form (H3PO4), it is removed during the precipitation and repeated washing with acetone. A slight modification of this procedure for simultaneous removal of AMP, ADP, and ATP from NADP preparation has also been discussed. PMID- 2997104 TI - Beta-adrenergic mediators increase pulmonary retention of instilled phospholipids. AB - Retention of radiolabeled phospholipid vesicles instilled into the alveolar space was studied with the isolated perfused rat lung and quantitated by measuring the percent of instilled radioactivity remaining in lung after five lavages. With synthetic [14C]dipalmitoyl phosphatidylcholine (PC):egg PC: phosphatidylglycerol:cholesterol (10:5:2:3) vesicles, there was a rapid (within 5 min) base-line retention of 10.3 +/- 0.25% (n = 11) followed by a slower phase of linear retention over the next 4 h. Retention at 2 h with perfused lungs was 18.6 +/- 0.60% (n = 9) and was similar to values obtained with lungs in vivo. Net retention (total minus base line) was stimulated 93% by isoproterenol, 173% by 8 bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), and 39% by 8 bromoguanosine 3',5'-cyclic monophosphate; propranolol blocked the effect of isoproterenol. The retention of natural (biosynthesized) surfactant [14C]PC was stimulated 92% by 8-Br-cAMP. The results suggest that the retention of exogenous phospholipid by the isolated perfused lung represents phospholipid uptake and that this process is under beta-adrenergic control. Secretion and uptake may be physiologically linked to regulate the concentration of surfactant on the alveolar surface. PMID- 2997105 TI - Regulation of ventilation and oxygen consumption by delta- and mu-opioid receptor agonists. AB - To study the effect of endorphins on metabolic rate and on the relationship between O2 consumption (VO2) and ventilation, we administered enkephalin analogues (relatively selective delta-receptor agonists) and a morphiceptin analogue (a highly selective mu-receptor agonist) intracisternally in nine unanesthetized chronically instrumented adult dogs. Both delta- and mu-agonists decreased VO2 by 40-60%. delta-Agonists induced a dose-dependent decrease in mean instantaneous minute ventilation (VT/TT) associated with periodic breathing. The decrease in VT/TT started and resolved prior to the decrease and returned to baseline of VO2, respectively. In contrast, the mu-agonists induced an increase in VT/TT associated with rapid shallow breathing. Arterial PCO2 increased and arterial PO2 decreased after both delta- and mu-agonists. Low doses of intracisternal naloxone (0.002-2.0 micrograms/kg) reversed the opioid effect on VT/TT but not on VO2; higher doses of naloxone (5-25 micrograms/kg) reversed both. Naloxone administered alone had no effect on VT/TT or VO2. These data suggest that 1) both delta- and mu-agonists induce alveolar hypoventilation despite a decrease in VO2, 2) this hypoventilation results from a decrease in VT/TT after delta-agonists but an increase in dead space ventilation after mu agonists, and 3) endorphins do not modulate ventilation and metabolic rate tonically, but we speculate that they may do so in response to stressful stimulation. PMID- 2997106 TI - Tonic beta-sympathetic activity in the lung periphery in anesthetized dogs. AB - The present study was undertaken to determine whether beta-adrenoceptors could be physiologically detected in the lung periphery and whether they were under tonic stimulation in the resting state in anesthetized dogs. A fiberoptic bronchoscope was wedged in a sublobar segment of lung in anesthetized male mongrel dogs for measurement of resistance through the collateral system (Rcs). beta-Agents were delivered locally as aerosols through the bronchoscope, and the response was evaluated by changes in Rcs. Distilled water alone produced a mean increase of 8.5 +/- 2.43% (SE) in Rcs at 2 min in six dogs, whereas dl-isoproterenol produced a mean decrease of 8.9 +/- 2.10% (P less than 0.03), thus demonstrating the presence of submaximally stimulated beta-receptors. To test whether the beta receptors were under tonic stimulation, we compared the effect of aerosolized d- and dl-propranolol in 5 dogs. d-Propranolol that lacks significant beta-blocking activity and dl-propranolol both produced large transient increases in Rcs. However, with d-propranolol, Rcs had returned to base line at 15 min, whereas with dl-propranolol Rcs remained elevated at a mean of 20% above base line for greater than 2 h (P less than 0.01). Local timolol aerosol also produced a sustained increase in Rcs. After pretreatment with reserpine or after bilateral adrenalectomy, both d- and dl-propranolol still produced large transient increases in Rcs, but dl-propranolol no longer produced a sustained increase. Neither isoproterenol nor atropine affected Rcs in the presence of dl propranolol, nor did pretreatment with atropine affect the response of Rcs to dl propranolol.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2997107 TI - Elevated diaphragm electromyogram during neonatal hypoxic ventilatory depression. AB - Diaphragmatic electromyogram (EMG) was obtained in eight 48-h-old unanesthetized monkeys while breathing air and then either of two different hypoxic gas mixtures (12 or 8% O2 in N2) for 5 min. Minute ventilation (VI) rose significantly above control levels by 1 min of hypoxemia while animals were breathing either of the hypoxic gas mixtures as tidal volume (VT) and slope and rate moving average EMG increased. The relative gains in VI were associated with comparable increases in diaphragmatic neural activity per minute (EMG/min = peak EMG X frequency) during this early phase of hypoxemia. VI subsequently fell to control levels (inspired O2 fraction = 12%, arterial PO2 = 23 +/- 3 Torr) or significantly below (inspired O2 fraction = 8%, arterial PO2 = 18 +/- 0.4 Torr) by 5 min of hypoxemia, secondary to changes in VT. Despite the decline in VI, slope and rate moving average EMG, and EMG/min remained statistically above control values by 5 min of hypoxemia, although there was a trend for EMG/min to decrease slightly from the 1 min peak response. These findings demonstrate that hypoxic-induced depression of neural input to the diaphragm is not independently responsible for the biphasic nature of the newborn ventilatory response, although it cannot be ruled out as a contributor. The fall in inspiratory volumes despite constant elevated EMG activity suggests the presence of a change in respiratory mechanics and/or an impairment in diaphragmatic contractile function without offsetting neural compensatory activity. PMID- 2997109 TI - Detection of low numbers of poliovirus 1 in oysters: collaborative study. AB - A collaborative study was performed to evaluate a method for determining numbers of poliovirus 1 in oysters. Commonly available laboratory equipment and materials were used. Raw oysters in the shell were shipped to each investigator along with 12 tubes of unknown concentrations of virus. Six 100 g duplicate oyster samples were analyzed by 5 collaborating laboratories. Each analyst used a prescribed procedure for diluting the inocula. Two samples contained approximately 100 plaque-forming units (pfu)/100 g, 2 samples contained approximately 50 pfu/100 g, and the other 2 contained approximately 25 pfu/100 g. Recoveries varied from 35 to 55% at inocula levels of 30-100 pfu/100 g. The limit of detectability was hypothesized to be 14 pfu in a 100 g sample when the recovery was 35%. The method has been adopted official first action. PMID- 2997108 TI - Mechanical damage to murine neuronal-enriched cultures during harvesting: effects on free fatty acids, diglycerides, Na+,K+-ATPase, and lipid peroxidation. AB - The most commonly used procedure to harvest cultured cells from petri dishes is to scrape the cells off the plates with a rubber or Teflon policeman. However, the results reported herein demonstrate that this technique, with its associated mechanical trauma, significantly perturbed cell membranes in neuronal-enriched cultures derived from the ventral half of fetal murine spinal cords. This is evidenced by liberation of free fatty acids and diglycerides, partial inhibition of Na+,K+-ATPase activity, and increased malondialdehyde production. Harvesting the cells by freezing, either on liquid nitrogen or dry ice, significantly attenuated these effects. This important observation indicates that mechanical manipulation of cultured cells during harvesting significantly affects subsequent biochemical analyses, particularly those associated with the cell membrane (e.g., membrane lipid metabolism and assay of intrinsic membrane enzymes). PMID- 2997111 TI - Moebius-Poland syndrome. PMID- 2997110 TI - Digestion of food samples for total mercury determination. AB - A method of digestion by using a mixture of hydrochloric, nitric, and sulfuric acids has been developed for the determination of total mercury in a wide range of food samples. Good recoveries of mercury were obtained from NBS (National Bureau of Standards) Albacore Tuna and from food samples spiked with inorganic mercury. A detection limit of 0.01 microgram mercury/g can be obtained. PMID- 2997112 TI - A case of West Nile (WN) virus encephalitis. PMID- 2997113 TI - Cloning of a restriction-modification system from Proteus vulgaris and its use in analyzing a methylase-sensitive phenotype in Escherichia coli. AB - A 4.84-kilobase-pair plasmid was isolated from Proteus vulgaris (ATCC 13315) and cloned into the plasmid vector pBR322. Plasmid pBR322 contains substrate sites for the restriction endonucleases PvuI and PvuII. The recombinant plasmids were resistant to in vitro cleavage by PvuII but not PvuI endonuclease and were found to cause production of PvuII endonuclease or methylase activity or both in Escherichia coli HB101. The approximate endonuclease and methylase gene boundaries were determined through subcloning, Bal 31 resection, insertional inactivation, DNA-dependent translation, and partial DNA sequencing. The two genes are adjacent and appear to be divergently transcribed. Most E. coli strains tested were poorly transformed by the recombinant plasmids, and this was shown by subcloning and insertional inactivation to be due to the PvuII methylase gene. At a low frequency, stable methylase-producing transformants of a methylase sensitive strain were obtained, and efficiently transformed cell mutants were isolated from them. PMID- 2997114 TI - Cloning and expression in Escherichia coli of a gene coding for a chondroitin lyase from Bacteroides thetaiotaomicron. AB - We cloned the gene for one of the two chondroitin lyases of Bacteroides thetaiotaomicron into the cosmid vector pHC79 and subcloned it into pBR328. No proteins the size of B. thetaiotaomicron chondroitin lyase I or II (104 to 108 kilodaltons) were detectable in maxicell or in vitro transcription-translation preparations. However, partial purification of the chondroitin lyase activity from the Escherichia coli subclone showed that its properties were similar to those of the B. thetaiotaomicron chondroitin lyases. Antibodies to the chondroitin lyase that was produced in E. coli cross-reacted with the B. thetaiotaomicron chondroitin lyase II but not with chondroitin lyase I. The molecular weight of the enzyme produced in E. coli, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration, was slightly lower than those of the two chondroitin lyases from B. thetaiotaomicron; the enzyme had a higher affinity for bacterial membranes and for heparin-agarose, and cyanogen bromide digestion products of the chondroitin lyase produced in E. coli differed slightly from those of B. thetaiotaomicron chondroitin lyase II. gamma delta mutagenesis was used to locate the chondroitin lyase gene on the subcloned 7.8-kilobase EcoRI fragment. The size of the gene was approximately 3.3 kilobases, as expected for a protein with a molecular weight of 104,000. PMID- 2997115 TI - Mutations affecting gyrase in Haemophilus influenzae. AB - Mutants separately resistant to novobiocin, coumermycin, nalidixic acid, and oxolinic acid contained gyrase activity as measured in vitro that was resistant to the antibiotics, indicating that the mutations represented structural alterations of the enzyme. One Novr mutant contained an altered B subunit of the enzyme, as judged by the ability of a plasmid, pNov1, containing the mutation to complement a temperature-sensitive gyrase B mutation in Escherichia coli and to cause novobiocin resistance in that strain. Three other Novr mutations did not confer antibiotic resistance to the gyrase but appeared to increase the amount of active enzyme in the cell. One of these, novB1, could only act in cis, whereas a new mutation, novC, could act in trans. An RNA polymerase mutation partially substituted for the novB1 mutation, suggesting that novB1 may be a mutation in a promoter region for the B subunit gene. Growth responses of strains containing various combinations of mutations on plasmids or on the chromosome indicated that low-level resistance to novobiocin or coumermycin may have resulted from multiple copies of wild-type genes coding for the gyrase B subunit, whereas high-level resistance required a structural change in the gyrase B gene and was also dependent on alteration in a regulatory region. When there was mismatch at the novB locus, with the novB1 mutation either on a plasmid or the chromosome, and the corresponding wild-type gene present in trans, chromosome to plasmid recombination during transformation was much higher than when the genes matched, probably because plasmid to chromosome recombination, eliminating the plasmid, was inhibited by the mismatch. PMID- 2997116 TI - Gyrase activity and number of copies of the gyrase B subunit gene in Haemophilus influenzae. AB - Gyrase activities in extracts of various strains of Haemophilus influenzae can differ by more than an order of magnitude (J. K. Setlow, E. Cabrera-Juarez, W. L. Albritton, D. Spikes, and A. Mutschler, J. Bacteriol. 164:525-534, 1985). Measurements of in vitro activity and copy number indicated that most of these differences arose from variations in the number of copies of the gene for the gyrase B subunit, with some strains containing multicopy plasmids coding for that subunit. The quantitative relationship between gyrase and copy number depended on the mutations in the plasmids and in the host. The gyrase and copy number were considerably lower in plasmid-bearing strains carrying the prophage HP1c1. Two mutations affecting gyrase that are apparently regulatory caused an increase in gyrase without a concomitant increase in copy number. The possibility that the in vivo gyrase activity did not reflect the in vitro data was explored by measurement of alkaline phosphatase and ATPase activity in the extracts. Alkaline phosphatase activity increased with increasing gyrase activity measured in vitro, but ATPase activity did not. We conclude that extra supercoiling enhanced transcription of the alkaline phosphatase gene but not the ATPase gene and that it is unlikely that there is much discrepancy between gyrase activity assayed in vitro and the activity in the cell. PMID- 2997117 TI - Translational coupling in Bacillus subtilis of a heterologous Bacillus subtilis Escherichia coli gene fusion. AB - Translational coupling was demonstrated in a gene fusion in which the promoter and the N-terminal region of the Bacillus subtilis subtilisin (aprA) gene were fused to a promoterless Tn9-derived chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) gene. Expression of this gene fusion results in the production of a native-sized CAT product, whereas the Tn9-derived CAT gene is usually not translated from its own ribosome binding site in B. subtilis (D. S. Goldfarb, R. L. Rodriguez, and R. H. Doi, Proc. Natl. Acad. Sci. USA 79:5886-5890, 1982). A 178-base-pair deletion, which removed part of the signal peptide and the propeptide of the aprA gene and created a translational stop codon 230 base pairs upstream of the CAT gene ribosome binding site, reduced expression of the CAT gene. A BamHI 10-mer linker insertion into this deletion site, which restored the reading frame and simultaneously removed the translation stop codon, restored CAT gene expression. The data indicate that expression of the CAT gene was dependent on translation of the truncated aprA gene into the ribosome binding site of the CAT gene. PMID- 2997118 TI - Isolation and analysis of genes involved in siderophore biosynthesis in plant growth-stimulating Pseudomonas putida WCS358. AB - The plant-growth-stimulating Pseudomonas putida WCS358 was mutagenized with transposon Tn5. The resulting mutant colony bank was screened for mutants defective in the biosynthesis of the fluorescent siderophore. A total of 28 mutants, divided into six different classes, were isolated that were nonfluorescent or defective in iron acquisition or both. These different types of mutants together with the probable overall structure of the siderophore, i.e., a small peptide chain attached to a fluorescing group, suggest a biosynthetic pathway in which the synthesis of the fluorescing group is preceded by the synthesis of the peptide part. A gene colony bank of P. putida WCS358 was constructed with the broad-host-range cosmid vector pLAFR1. This genomic library, established in Escherichia coli, was mobilized into the 28 individual mutants, screening for transconjugants restored in fluorescence or growth under iron limiting conditions or both. A total of 13 cosmids were found to complement 13 distinct mutants. The complementation analysis revealed that at least five gene clusters, with a minimum of seven genes, are needed for siderophore biosynthesis. Some of these genes seem to be arranged in an operon-like structure. PMID- 2997119 TI - Comparative studies of the amino acid and nucleotide sequences of pilin derived from Pseudomonas aeruginosa PAK and PAO. AB - The entire amino acid sequence for Pseudomonas aeruginosa PAO pilin was determined through peptide sequencing and from the complete nucleotide sequence encoding the pilin gene. The precursor PAO pilin is 149 amino acids in length which includes a 6-amino-acid positively charged leader sequence. Comparison of the amino acid sequences of pilin produced by P. aeruginosa PAO and PAK reveals a region of high homology corresponding to the leader peptide and residues 1 to 54 of the mature pilin. The amino acid sequence of the peptide encompassing the major antigenic determinant of PAK differs greatly from that of the equivalent region in PAO. The C-terminal regions of these proteins are semiconserved. Few major differences were found when the predicted secondary structures for PAO and PAK pilins were compared. Major nucleotide sequence variation between the equivalent restriction fragments from PAO and PAK occurred within the areas coding for the peptides containing the immunodominant site for PAK pilin and the C termini. PMID- 2997121 TI - Rhizobium meliloti nodulation genes: identification of nodDABC gene products, purification of nodA protein, and expression of nodA in Rhizobium meliloti. AB - A set of conserved, or common, bacterial nodulation (nod) loci is required for host plant infection by Rhizobium meliloti and other Rhizobium species. Four such genes, nodDABC, have been indicated in R. meliloti 1021 by genetic analysis and DNA sequencing. An essential step toward understanding the function of these genes is to characterize their protein products. We used in vitro and maxicell Escherichia coli expression systems, together with gel electrophoresis and autoradiography, to detect proteins encoded by nodDABC. We facilitated expression of genes on these DNA fragments by inserting them downstream of the Salmonella typhimurium trp promoter, both in colE1 and incP plasmid-based vectors. Use of the incP trp promoter plasmid allowed overexpression of a nodABC gene fragment in R. meliloti. We found that nodA encodes a protein of 21 kilodaltons (kDa), and nodB encodes one of 28 kDa; the nodC product appears as two polypeptide bands at 44 and 45 kDa. Expression of the divergently read nodD yields a single polypeptide of 33 kDa. Whether these represent true Rhizobium gene products must be demonstrated by correlating these proteins with genetically defined Rhizobium loci. We purified the 21-kDa putative nodA protein product by gel electrophoresis, selective precipitation, and ion-exchange chromatography and generated antiserum to the purified gene product. This permitted the immunological demonstration that the 21-kDa protein is present in wild-type cells and in nodB- or nodC-defective strains, but is absent from nodA::Tn5 mutants, which confirms that the product expressed in E. coli is identical to that produced by R. meliloti nodA. Using antisera detection, we found that the level of nodA protein is increased by exposure of R. meliloti cells to plant exudate, indicating regulation of the bacterial nod genes by the plant host. PMID- 2997120 TI - Primary characterization of the protein products of the Escherichia coli ompB locus: structure and regulation of synthesis of the OmpR and EnvZ proteins. AB - The ompB operon of Escherichia coli contains the structural genes for two proteins, OmpR and EnvZ, which control the osmoregulated biosynthesis of the porin proteins OmpF and OmpC. By inserting XbaI octamer linkers into the cloned ompB locus, four distinct frameshift mutants were isolated and subsequently characterized for their OmpR and EnvZ protein products and their outer membrane porin phenotype. In a minicell expression system, the wild-type products of the ompR and envZ genes were found to be approximately 28 and 50 kilodaltons in size, respectively, whereas the mutant proteins were either truncated or extended due to the frame shift. The identity of the envZ gene product was confirmed by immunoprecipitation. M13 dideoxy sequencing of the DNA around the wild-type ompR envZ junction revealed an error in the sequence published for this operon; the complete corrected sequence is presented. A sequence, ATGA, was found that forms the termination codon for the OmpR reading frame and a possible initiation codon for the EnvZ protein; these sequences are consistent with the sizes of the proteins observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The translational activity of this ATG codon was confirmed by fusing the lacZ gene in frame with the putative EnvZ coding sequence. The implications of these results are discussed with respect to the regulation of synthesis of the ompB gene products. PMID- 2997122 TI - Integration of bacteriophage SP24 into the chromosome of group A streptococci. AB - The group A streptococcal bacteriophage SP24 contains a unique phage att site and integrates into a common chromosomal locus in two unrelated group A streptococcal strains, CS24 and CS112. Southern blot analysis suggested that the terminally redundant phage DNA recombines to form the unit-length genome observed in the prophage state. Phage DNA integration appears to be required for stable lysogen formation and conversion to the M+ state; however, the precise role of the bacteriophage and the relationship of phage integration to increased M protein synthesis are unclear. PMID- 2997123 TI - Streptococcus faecalis R plasmid pJH1 contains a pAM alpha 1 delta 1-like replicon. AB - Streptococcus faecalis R plasmid pJH1 did not transform competent strains of Streptococcus sanguis. A hybrid plasmid, pDL310, consisting of virtually all of the S. faecalis hemolysin-bacteriocin plasmid pJH2 and a segment of pJH1 DNA that included the tetracycline resistance determinant, yielded tetracycline-resistant transformants at a frequency of less than 10(-8) transformants per CFU, when it was added to a competent culture of S. sanguis Wicky. Four of the transformants contained a 4.7-kilobase plasmid (pDL316) that transformed strain Wicky at a frequency of 8.6 X 10(-8). Restriction endonuclease digests, agarose gel electrophoresis, and Southern blot hybridizations indicated that pDL316 consisted entirely of pJH1-derived DNA. Additional restriction studies, Southern blot hybridizations, and heteroduplex analyses indicated that pDL316 was very closely related to 4.6-kilobase tetracycline resistance plasmid pAM alpha 1 delta 1, a derivative of 9.0-kilobase S. faecalis plasmid pAM alpha 1. PMID- 2997124 TI - Identification of the Escherichia coli recN gene product as a major SOS protein. AB - The recA+ lexA+-dependent induction of four Escherichia coli SOS proteins was readily observed by two-dimensional gel analysis. In addition to the 38 kilodalton (kDa) RecA protein, which was induced in the greatest amounts and was readily identified, three other proteins of 115, 62, and 12 kDa were seen. The 115-kDa protein is the product of the uvrA gene, which is required for nucleotide excision repair and has previously been shown to be induced in the SOS response. The 62-kDa protein, which was induced to high intracellular levels, is the product of recN, a gene required for recBC-independent recombination. The recA and recN genes were partially derepressed in a recBC sbcB genetic background, a phenomenon which might account for the recombination proficiency of such strains. The 12-kDa protein has yet to be identified. PMID- 2997125 TI - Comparative stability and catalytic and chemical properties of the sulfate activating enzymes from Penicillium chrysogenum (mesophile) and Penicillium duponti (thermophile). AB - ATP sulfurylases from Penicillium chrysogenum (a mesophile) and from Penicillium duponti (a thermophile) had a native molecular weight of about 440,000 and a subunit molecular weight of about 69,000. (The P. duponti subunit appeared to be a little smaller than the P. chrysogenum subunit.) The P. duponti enzyme was about 100 times more heat stable than the P. chrysogenum enzyme; k inact (the first-order rate constant for inactivation) at 65 degrees C = 3.3 X 10(-4) s-1 for P. duponti and 3.0 X 10(-2) s-1 for P. chrysogenum. The P. duponti enzyme was also more stable to low pH and urea at 30 degrees C. Rabbit serum antibodies to each enzyme showed heterologous cross-reaction. Amino acid analyses disclosed no major compositional differences between the two enzymes. The analogous Km and Ki values of the forward and reverse reactions were also essentially identical at 30 degrees C. At 30 degrees C, the physiologically important adenosine 5' phosphosulfate (APS) synthesis activity of the P. duponti enzyme was 4 U mg of protein-1, which is about half that of the P. chrysogenum enzyme. The molybdolysis and ATP synthesis activities of the P. duponti enzyme at 30 degrees C were similar to those of the P. chrysogenum enzyme. At 50 degrees C, the APS synthesis activity of the P. duponti enzyme was 12 to 19 U mg of protein-1, which was higher than that of the P. chrysogenum enzyme at 30 degrees C (8 +/- 1 U mg of protein-1). Treatment of the P. chrysogenum enzyme with 5,5'-dithiobis(2 nitrobenzoate) (DTNB) at 30 degrees C under nondenaturing conditions modified one free sulfhydryl group per subunit. Vmax was not significantly altered, but the catalytic activity at low magnesium-ATP or SO4(2-) (or MoO4(2-)) was markedly reduced. Chemical modification with tetranitromethane had the same results on the kinetics. The native P. duponti enzyme was relatively unreactive toward DTNB or tetranitromethane at 30 degrees C and pH 8.0 or pH 9.0, but at 50 degrees C and pH 8.0, DTNB rapidly modified one SH group per subunit. APS kinase (the second sulfate-activating enzyme) of P. chrysogenum dissociated into inactive subunits at 42 degrees C. The P. duponti enzyme remained intact and active at 42 degrees C. PMID- 2997126 TI - Isolation of insertion, deletion, and nonsense mutations of the uracil-DNA glycosylase (ung) gene of Escherichia coli K-12. AB - Two uracil-DNA glycosylase (ung) mutation selection procedures based upon the ability of uracil glycosylase to degrade the chromosomes of organisms containing uracil-DNA were devised to obtain a collection of well-defined ung alleles. In an enrichment procedure, lysogens were selected from Escherichia coli cultures infected with lambda pKanr phage containing uracil in their DNA. (These uracil DNA phage were prepared by growth on host cells deficient in both dUTPase and uracil-DNA glycosylase.) The lysogenic Kanr population was enriched for uracil glycosylase-deficient mutants by a factor of 10(4). In a phage suicide selection procedure, lambda pung+ phage were unable to form plaques on dut ung cells containing uracil-DNA in their chromosomes, and all of the progeny were lambda pung-. Deletion, insertion (ung::Mu and ung::Tn10), nonsense, and missense mutants were isolated by using these procedures. Extracts of three insertion mutants contained no detectable enzyme activity. All of the other mutant isolates had less than 1% of the normal uracil glycosylase specific activity. The previously studied ung-1 allele, which was derived by N-methyl-N'-nitro-N nitrosoguanidine mutagenesis, produced about 0.02% of the normal amount of uracil glycosylase activity. No significant phenotypic differences between ung-1 and ung::Tn10 alleles were observed. Variations of the lysogen selection procedure may be helpful for isolating other DNA glycosylase mutations in E. coli and other organisms. PMID- 2997127 TI - Isolation and characterization of Ca2+-blind mutants of Yersinia pestis. AB - The plasmid pCD1 is required for expression of the low-calcium response (LCR), virulence, and production of V antigen in Yersinia pestis KIM. Five independent mutants constitutive for the LCR at 37 degrees C (Lcrc) were obtained through ethyl methanesulfonate mutagenesis followed by ampicillin enrichment. A sixth, spontaneous mutant was obtained directly through ampicillin enrichment. These mutants failed to grow at 37 degrees C regardless of calcium concentration and produced V antigen constitutively at this temperature. All six mutations were located on pCD1. One mutation was mapped to a 1-kilobase region of lcrA. Based on complementation mapping of this mutation, the lcrA locus was divided into two new loci, lcrD and lcrE. This mutation, lcrE1, did not alter the transcription of other genes in the LCR region and was cis-recessive to lcr mutations. Several lower-molecular-weight outer membrane proteins which were observed in the parent strain grown at 37 degrees C in the presence of 2.5 mM calcium were reduced in quantity or absent from the mutant strain. PMID- 2997128 TI - Export of alpha-amylase by Bacillus amyloliquefaciens requires proton motive force. AB - The secretion of protein directly into the extracellular medium by Bacillus amyloliquefaciens, a gram-positive bacterium, was shown to be dependent on proton motive force. When the electrochemical membrane potential gradient of protons was dissipated either by uncouplers or by valinomycin in combination with K+, a precursor form of alpha-amylase accumulated on the cellular membrane. The proton motive force could be dissipated without altering the intracellular level of ATP, indicating that the observed inhibition of export was not the result of decreased ATP concentration. PMID- 2997129 TI - Inhibition of growth of Rhizobium japonicum by cyclic GMP. AB - Exogenous cyclic guanosine-3',5'-monophosphate (cGMP) inhibited the growth of Rhizobium japonicum at less than 100 microM. Other nucleotides, including cyclic AMP, cyclic IMP, and cyclic CMP, had no inhibitory effect even at higher concentrations nor was the inhibition by cGMP reversed by cyclic AMP. The inhibitory effect was independent of the carbon and nitrogen source(s) used. cGMP did not inhibit the growth of any other species of bacterium tested, including several fast-growing Rhizobium species. The kinetics of growth inhibition are multiphasic, with no apparent effect for several hours after addition, followed by a period of total inhibition. Subsequently, growth resumed at a slower rate. Resumption of growth was not due to destruction of the nucleotide. Studies of the intracellular cGMP concentration did not reveal significant changes in cells grown under aerobic or microaerobic conditions. No effect of cGMP on the derepression of respiratory nitrate reductase was observed. PMID- 2997131 TI - Construction of a series of ompF-ompC chimeric genes by in vivo homologous recombination in Escherichia coli and characterization of the translational products. AB - OmpF and OmpC are major outer membrane proteins. Although they are homologous proteins, they function differently in several respects. As an approach to elucidate the submolecular structures that determine the difference, a method was developed to construct a series of ompF-ompC chimeric genes by in vivo homologous recombination between these two genes, which are adjacent on a plasmid. The genomic structures of these chimeric genes were determined by restriction endonuclease analysis and nucleotide sequence determination. In almost all cases, recombination took place between the corresponding homologous regions of the ompF and ompC genes. Many of the chimeric genes produced proteins that migrated to various positions between the OmpF and OmpC proteins on polyacrylamide gel. On the basis of the results, a domain contributing to the mobility difference the OmpF and OmpC proteins was identified. Some chimeric genes did not accumulate outer membrane proteins, despite the fact that the fusion of the ompF and ompC genes was in frame. Bacterial cells possessing the chimeric proteins were also tested as to their sensitivity to phages which require either OmpF or OmpC as a receptor component. The chimeric proteins were either of the OmpF or OmpC type with respect to receptor activity. Based on the observations, the roles of submolecular domains in the structure, function, and biogenesis of the OmpF and OmpC proteins are discussed. PMID- 2997130 TI - Complete nucleotide sequence of macrolide-lincosamide-streptogramin B-resistance transposon Tn917 in Streptococcus faecalis. AB - Streptococcus faecalis transposon Tn917 was cloned in Escherichia coli on plasmid vector pBR325. The erythromycin resistance determinant of Tn917 was not expressed in the E. coli background. The nucleotide sequence of Tn917 was determined and found to be 5,257 base pairs in length. Six open reading frames (ORFs) were identified and designated 1 through 6 (5' to 3'); all were on the same DNA strand. A region exhibiting strong homology with known promoters was identified upstream from ORF1. ORFs 1 to 3 were virtually identical to the previously sequenced erythromycin resistance determinant on Streptococcus sanguis plasmid pAM77. At the 3' point, where the homology between Tn917 and pAM77 ends, was a 20 base-pair region about 80% homologous with a component of the res site of Tn3. The amino acid sequence of ORF4 showed homology with other site-specific recombination enzymes, including approximately 30% homology with the resolvase of Tn3. Contained within Tn917 was a directly oriented 73-base-pair duplication of the left terminus. The Tn917 sequence revealed that antibiotic-enhanced transposition might be due to extension of transcription from the resistance related genes (in ORFs 1 to 3) into transposition genes (in ORFs 4 to 6). Transcription analyses resulted in data consistent with this interpretation. PMID- 2997132 TI - Enzymatic activities in cell fractions of mycoplasmalike organisms purified from aster yellows-infected plants. AB - Mycoplasmalike organisms (MLOs), purified from aster yellows-infected plants were osmotically lysed, and the membranes were separated from the cytoplasmic fraction through differential centrifugation. Electron microscopic examinations of sections of the purified MLOs and the isolated membranes showed pleomorphic bodies and unit membranous empty vesicles, respectively. Cell fractions were tested for NADH oxidase, NADPH oxidase, ATPase, RNase, DNase, and p-nitrophenyl phosphatase activity. NADH oxidase and ATPase were confined to the membrane fraction and NADPH oxidase to the cytoplasmic fraction of the MLOs. para Nitrophenyl phosphatase, RNase, and DNase activities were detected in both membrane and cytoplasmic fractions, but p-nitrophenyl phosphatase and RNase appeared to be associated with membranes and DNase with the cytoplasmic fraction. Glucose-6-phosphate dehydrogenase was found in the cytoplasmic fraction of the MLO cells. Our findings on the distribution of enzymes in MLO cells and cell fractions are the first basic documentation on nonhelical, nonculturable microbes parasitic to plants. PMID- 2997133 TI - Nitrous oxide reduction by members of the family Rhodospirillaceae and the nitrous oxide reductase of Rhodopseudomonas capsulata. AB - After growth in the absence of nitrogenous oxides under anaerobic phototrophic conditions, several strains of Rhodopseudomonas capsulata were shown to possess a nitrous oxide reductase activity. The enzyme responsible for this activity had a periplasmic location and resembled a nitrous oxide reductase purified from Pseudomonas perfectomarinus. Electron flow to nitrous oxide reductase was coupled to generation of a membrane potential and inhibited by rotenone but not antimycin. It is suggested that electron flow to nitrous oxide reductase branches at the level of ubiquinone from the previously characterized electron transfer components of R. capsulata. This pathway of electron transport could include cytochrome c', a component hitherto without a recognized function. R. capsulata grew under dark anaerobic conditions in the presence of malate as carbon source and nitrous oxide as electron acceptor. This confirms that nitrous oxide respiration is linked to ATP synthesis. Phototrophically and anaerobically grown cultures of nondenitrifying strains of Rhodopseudomonas sphaeroides, Rhodopseudomonas palustris, and Rhodospirillum rubrum also possessed nitrous oxide reductase activity. PMID- 2997134 TI - Evidence that polygalacturonase is a virulence determinant in Erwinia carotovora. AB - Polygalacturonase (PG) was purified from Erwinia carotovora EC. A hybrid cosmid, pSH711, that encodes PG activity but not pectate lyase activity was identified from an E. carotovora genomic library by an immunological screening method. A cell extract of Escherichia coli cells containing pSH711 was able to produce plant tissue maceration when spotted on carrot, potato, or turnip slices. In addition, the E. coli strain containing this plasmid was able to macerate carrot, potato, and turnip slices. Our results suggest that PG plays an important role in soft-rot disease. PMID- 2997135 TI - Negative regulation of adenylate cyclase gene (cya) expression by cyclic AMP cyclic AMP receptor protein in Escherichia coli: studies with cya-lac protein and operon fusion plasmids. AB - We constructed cya-lac protein and operon fusion plasmids in vitro. The effect of cyclic AMP (cAMP) on cya expression was examined by measuring the synthesis of beta-galactosidase in Escherichia coli cells containing fused plasmids. In the cya-lacZ fused protein system, cya expression was strongly repressed by exogenous cAMP. Functional cAMP receptor protein (CRP) was necessary for this effect. On the other hand, in a tet-lacZ fused protein as a control system, tet expression was not affected by cAMP. The inhibition of cya expression by cAMP was also observed in the cya-lac fused operon system, although it was necessary to increase the amount of cAMP or CRP in the cells to detect the effect. The results indicate that cAMP-CRP is a negative regulator of cya expression at the level of transcription. PMID- 2997136 TI - Evolutionary conservation of genes coding for meta pathway enzymes within TOL plasmids pWW0 and pWW53. AB - Pseudomonas putida MT53 contains a TOL plasmid, pWW53, that encodes toluene xylene catabolism. pWW53 is nonconjugative, is about 105 to 110 kilobase pairs (kbp) in size, and differs significantly in its restriction endonuclease digestion pattern and incompatibility group from the archetypal TOL plasmid pWW0. An RP4::pWW53 cointegrate plasmid, pWW53-4, containing about 35 kbp of pWW53 DNA, including the entire catabolic pathway genes, was formed, and a restriction map for KpnI, HindIII, and BamHI was derived. The entire regulated meta pathway genes for the catabolism of m-toluate were cloned into pKT230 from pWW53 on a 17.5-kbp HindIII fragment. The recombinant plasmid supported growth on m-toluate when mobilized into plasmid-free P. putida PaW130. A restriction map of the insert for 10 restriction enzymes was derived, and the locations of xylD, xylL, xylE, xylG, and xylF were determined by subcloning and assaying for their gene products in both Escherichia coli and P. putida hosts. Good induction of the enzymes by m toluate and m-methylbenzyl alcohol but not by m-xylene was measured in P. putida, but little or no regulation was found in E. coli. The restriction map and the gene order showed strong similarities with published maps of the DNA encoding both the entire meta pathway operon (xylDLEGFJIH) and the regulatory genes xylS and xylR on the archetype TOL plasmid pWW0, suggesting a high degree of conservation in DNA structure for the catabolic operon on the two different plasmids. PMID- 2997138 TI - Initiation of chromosome replication in Escherichia coli after induction of dnaA gene expression from a lac promoter. AB - Escherichia coli HB282 carries a dnaA46(Ts) allele on the chromosome, a wild-type dnaA allele under the control of the lacUV5 promoter on the multicopy plasmid pBC32, and an overproducing lac repressor allele on an F' factor. When the plasmid dnaA gene is repressed, the strain is thermosensitive. After a temporary deficiency in active dnaA protein at nonpermissive temperature, the addition of isopropyl-beta-D-thiogalactopyranoside to the culture was found to produce a burst of initiations within 5 to 10 min at 30% of the origins in 90% of the cells. Initiations then continued at a rate slightly faster than the mass doubling time such that after 2 h the origin-to-mass ratio of the control culture was restored. PMID- 2997137 TI - Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. AB - We constructed the broad-host-range plasmid pUCD800 containing the sacB gene of Bacillus subtilis for use in the positive selection and isolation of insertion sequence (IS) elements in gram-negative bacteria. Cells containing pUCD800 do not grow on medium containing 5% sucrose unless the sacB gene is inactivated. By using pUCD800, we isolated a 1.4-kilobase putative IS element from Agrobacterium tumefaciens NT1RE by selection for growth on sucrose medium. This putative IS element appears to be unique to Agrobacterium strains. PMID- 2997139 TI - Measurement of the proton motive force in Rhizobium meliloti with the Escherichia coli lacY gene product. AB - An Escherichia coli lac operon constitutive for lacY was subcloned into the EcoRI site of a wide-host-range plasmid of the Q incompatibility group, and the resulting recombinant plasmid was introduced into Tn5-generated Lac- mutants of Rhizobium meliloti. The R. meliloti transconjugants accumulated lactose about 1,000-fold, equivalent to a proton motive force of -170 to -180 mV, not significantly different from the values calculated from the distributions of weak acids and lipophilic cations. PMID- 2997140 TI - DNA supercoiling and suppression of the leu-500 promoter mutation. AB - top mutations (formerly supX) eliminate DNA topoisomerase I activity and suppress the leu-500 promoter mutation in Salmonella typhimurium (K. M. Overbye, S. K. Basu, and P. Margolin, Cold Spring Harbor Symp. Quant. Biol. 47:785-791, 1983). Sublethal doses of coumermycin which reduce intracellular levels of supercoiling activity in a top mutant eliminated suppression of the leu-500 mutation. This result provides evidence that increased DNA supercoiling suppresses the leu-500 promoter mutation in top mutants. PMID- 2997141 TI - Chromosomal and plasmid locations for phosphoribulokinase genes in Alcaligenes eutrophus. AB - Genes coding for phosphoribulokinase (PRK), a key enzyme of the Calvin cycle, were localized in the genome of the chemoautotroph Alcaligenes eutrophus. The NH2 terminal sequence of the PRK subunit was determined. With a synthetic oligodeoxynucleotide probe complementary to a portion of this sequence, hybridization analysis revealed PRK genes to be located on both the chromosome and the megaplasmid pHG1 of A. eutrophus H16. PMID- 2997142 TI - IS186: an Escherichia coli insertion element isolated from a cDNA library. AB - Escherichia coli IS186 was isolated from cDNA libraries made from rainbow trout RNA and maintained in E. coli RR1. The element was 1,347 base pairs in length, had a perfect inverted repeat of 25 base pairs, and had an open reading frame of 375 amino acids. The hypothetical protein sequence of IS186 had limited homology to the E. coli IS4 hypothetical protein I sequence. There were three copies of IS186 in E. coli RR1. PMID- 2997143 TI - The kinetics of the flash-induced P515 response in relation to the H+ permeability of the membrane bound ATPase in spinach chloroplasts. AB - The effect of dicyclohexylcarbodiimide (DCCD) on the kinetics of the flash induced P515 response and on the activity of the ATPase was investigated in isolated spinach chloroplasts. It was found that after the addition of 5 X 10( 8)mol DCCD the rate of ATP hydrolysis induced by a period of 60 sec illumination was decreased to less than 5% of its original value. At this concentration, hardly any effect, if at all, could be detected on the kinetics of the flash induced P515 response, neither in dark-adapted nor in light-activated chloroplasts. It was concluded that the presence of concentrations of DCCD, sufficiently high to affect the ATPase activity, does not affect the kinetics of the flash-induced P515 response. Since DCCD decreases the H+ permeability of the membrane-bound ATPase, it was concluded that this permeability coefficient for protons is not an important factor in the regulation of the flash-induced membrane potential and, therefore, does not affect the kinetics of the flash induced P515 response. PMID- 2997145 TI - Stoichiometry of proton uptake in isolated pea chloroplasts under different light intensities. AB - The number of protons released inside the chloroplast thylakoids per electron which is transferred through the electron transport chain (H+/e- ratio) was measured in isolated pea chloroplasts at pH 6.0 under continuous illumination and with methyl viologen as an electron acceptor. At saturating light intensity (200 W X m-2) ("strong" light) the H+/e- ratio was 3. At low intensity (0.9 W X m-2) ("weak" light) the H+/e- ratio was 2 with dark-adapted chloroplasts, but it was close to 3 with chloroplasts that were preilluminated with strong light. It is shown that the presence of azide in the reaction mixture leads to errors in the determination of the H+/e- ratio due to underestimation of the initial rate of H+ efflux on switching off the light. To explain the above data, we assume that transformation of the electron transport chain occurs during illumination with strong light, namely, the Q cycle becomes operative. PMID- 2997144 TI - Anion (and cation) requirements of the coupled electron flow in spinach thylakoids. AB - Proton uptake as well as coupled electron flow of chloroplasts swollen in dilute impermeable buffers became dependent upon the addition of exogenous permeable anions. This dependence was observed with both cyclic and noncyclic electron acceptors, suggesting that this anion requirement is associated with the electrogenic proton uptake step rather than with the oxygen-evolving reactions of photosystem II. PMID- 2997147 TI - mRNA and apolipoprotein E synthesis abnormalities in peripheral blood monocyte macrophages in familial apolipoprotein E deficiency. AB - We have studied synthesis of apolipoprotein E (apo-E) and apo-E mRNA in cultures of peripheral blood human monocyte macrophages (M-M cultures) obtained from a patient with familial apolipoprotein E deficiency. We have found that the M-M cultures of the apo-E-deficient patients contained two apo-E mRNA species with slightly different molecular weight as compared to normal apo-E mRNA. The apo-E mRNA concentration of the apo-E-deficient cultures was approximately 50-fold reduced as compared to the normal cultures, whereas the actin mRNA concentrations were identical in both M-M cultures. Genomic blotting analysis using a full length apo-E cDNA clone as hybridization probe did not show gross differences between the restriction patterns of the DNA obtained from the apo-E-deficient patient and two normal controls. When normal M-M cultures were grown in media containing [35S]methionine they synthesized and secreted apo-E into the culture media. In contrast we could not detect any intracellular or extracellular apo-E in the patient's M-M cultures grown under identical conditions. These observations are consistent with the hypothesis that familial apo-E deficiency results from structural apo-E gene mutation(s). The putative mutation(s) affect either the transcription of the apo-E gene or the processing of the primary apo-E mRNA transcript. These abnormalities are associated with low levels of synthesis of aberrant apo-E mRNA forms which are either very unstable or cannot be translated into protein. PMID- 2997146 TI - Further studies on the binding of DCCD to cytochrome B and subunit VIII of complex III isolated from beef heart mitochondria. AB - Complex III (the cytochrome b-c1 complex) from beef heart mitochondria was incubated with [14C]DCCD for various periods of time. The polypeptide profile of the complex was compared in both stained gels and their autoradiograms when three different methods were used to terminate the reaction. Precipitation with ammonium sulfate resulted in the formation of a new band with an apparent molecular weight of 39,000 in both incubated samples and the zero time controls. Reisolation of the complex by centrifugation through 10% sucrose or by precipitation with trichloroacetic acid did not result in any changes in the appearance of the subunit peptides of the complex. Subunit III (cytochrome b) and subunit VIII were the only bands labeled after termination of the reaction by centrifugation through sucrose, while both ammonium sulfate and trichloroacetic precipitation resulted in nonspecific labeling of several other subunits of the complex and increased labeling of subunit VIII relative to subunit III. Preincubation of the complex with antimycin prior to treatment with [14C]DCCD resulted in a 50% decrease in the binding of DCCD to both cytochrome b and subunit VIII. Furthermore, treatment of the complex III with DCCD resulted in a change in the red shift observed after antimycin or myxothiazol addition to the dithionite-reduced complex resulting in a broad peak with no sharp maximum. These results provide further confirmation that DCCD binds preferentially to cytochrome b and subunit VIII of complex III from beef heart mitochondria and suggest that cytochrome b may play a role in proton translocation. PMID- 2997148 TI - Inhibitors of Na+/H+ exchange block stimulus-provoked arachidonic acid release in human platelets. Selective effects on platelet activation by epinephrine, ADP, and lower concentrations of thrombin. AB - We have observed previously that removal of extraplatelet Na+ blocks platelet secretion of dense granule contents in response to epinephrine, ADP, and 0.004 unit/ml thrombin, all agents which must mobilize arachidonic acid for its subsequent conversion to cyclooxygenase products in order to elicit platelet secretion. The present studies demonstrate that removal of extraplatelet Na+ blocks arachidonic acid mobilization in response to epinephrine, ADP, and 0.004 unit/ml thrombin without altering arachidonic acid conversion to thromboxane A2. The data also provide several lines of evidence which suggest that the blockade of arachidonic acid release due to removal of extraplatelet Na+ is a manifestation of blockade of Na+/H+ exchange system. 1) There is a concentration dependent effect of extraplatelet Na+ (EC50 congruent to 55 mM) on [3H]arachidonic acid release such that mobilization is observed when [Na+]o greater than [Na+]i. 2) Increasing extraplatelet [H+] (i.e. decreasing extraplatelet pH from pH 7.35 to 6.8) causes a concentration-dependent decline in stimulus-provoked [3H]arachidonic acid release. 3) Ethylisopropylamiloride and other potent 5-amino analogs of amiloride block [3H]arachidonic acid release with a potency that parallels their effects on Na+/H+ exchange in other cellular systems. None of the above manipulations alter primary aggregation induced by epinephrine, ADP, or 0.004 unit/ml thrombin, indicating that stimulus-receptor binding, subsequent exposure of fibrinogen receptors, and fibrinogen-mediated platelet-platelet cross-linking are not significantly inhibited by [3H]arachidonic acid release in response to greater than 0.1 unit/ml thrombin, a stimulus that can elicit platelet secretion in the absence of products of the cyclooxygenase pathway. Therefore, Na+/H+ exchange may selectively modulate arachidonic acid mobilization in response to the so-called "weak agonists," agonists that require this mobilization to effect vigorous platelet aggregation and dense granule secretion. PMID- 2997149 TI - Evidence for a phosphoenzyme intermediate in the reaction pathway of rat hepatic fructose-2,6-bisphosphatase. AB - We re-examined the kinetics of the bisphosphatase reaction of rat hepatic 6 phosphofructo-2-kinase/fructose-2,6-bisphosphatase after depleting the enzyme of bound fructose 6-phosphate and found a hyperbolic dependence on fructose 2,6 bisphosphate at concentrations below 100 nM. The Michaelis constant was 4 nM, the Vmax was about 12 nmol X mg-1 X min-1 at 22 degrees C but the substrate inhibited at concentrations above 100 nM. Both phosphate and alpha-glycerol phosphate strongly inhibited phosphoenzyme formation and hydrolytic rate below 100 nM, but relieved the inhibition by substrate at higher concentrations probably by antagonizing substrate binding. A number of observations support the proposition that the phosphoenzyme is a necessary participant in catalysis. 1) The amount of phosphoenzyme measured during steady-state hydrolysis as a function of substrate concentration correlated with the velocity profile. 2) Rapid mixing experiments demonstrated that over a broad range of substrate concentrations phosphoenzyme formation was faster than the net rate of hydrolysis. 3) Both phosphate and alpha glycerol phosphate inhibited the rate of phosphoenzyme formation and, at low substrate concentrations, reduced the steady-state phosphoenzyme levels. The latter correlated with inhibition of substrate hydrolysis. 4) Both phosphate and alpha-glycerol phosphate stimulate the rate of phosphoenzyme breakdown, consistent with their stimulation of substrate hydrolysis at high substrate concentrations. 5) The fractional rate of phosphoenzyme breakdown, which was pH and substrate dependent, multiplied by the amount of phosphoenzyme obtained in the steady state at that pH and substrate concentration approximated the observed rate of hydrolysis. We conclude that the phosphoenzyme is a reaction intermediate in the hepatic fructose-2,6-bisphosphatase reaction. PMID- 2997150 TI - Regulation of the manganese-containing superoxide dismutase is independent of the inducible DNA repair system in Escherichia coli. AB - Studies on the induction of the manganese-containing superoxide dismutase in several strains of Escherichia coli with different mutations in recA and lexA revealed that the inductions of the Mn-isozyme and of the SOS system by oxygen free radicals are not coregulated. We also studied the synthesis of the manganese superoxide dismutase in the temperature-dependent, protease-constitutive strain recA441(tif-1) that also contained a lac fusion in an SOS gene. A shift to the temperature at which recA441 has constitutive protease activity did not induce Mn superoxide dismutase but did induce beta-galactosidase. The data clearly demonstrate that induction of the Mn-superoxide dismutase is independent of the SOS system. PMID- 2997151 TI - The polymerase subunit of DNA polymerase III of Escherichia coli. II. Purification of the alpha subunit, devoid of nuclease activities. AB - The alpha subunit (140 kDa) of DNA polymerase III (pol III) holoenzyme has been purified to near-homogeneity from a plasmid-carrying Escherichia coli strain which overproduced the alpha subunit about 20-fold. Pol III core (containing only the alpha, epsilon, and theta subunits), produced at twice the normal level, was also purified in good yield. The isolated alpha subunit has DNA polymerase activity, which is completely inhibited by 10 mM N-ethylmaleimide or 150 mM KCl as observed in the pol III core or holoenzyme. The alpha subunit has an apparent turnover number of 7.7 nucleotides polymerized per s, compared to 20 for pol III core, and is more thermolabile. The alpha subunit lacks the 3'----5' exonuclease (proofreading) activity of pol III core; neither alpha subunit nor core (nor holoenzyme) possesses any of the previously reported 5'----3' exonuclease activity. Thus, the alpha polypeptide is the polymerase subunit and epsilon (27 kDa) is the proofreading subunit (Scheuermann, R. H., and Echols, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7747-7751). Together with the theta polypeptide (10 kDa), of unknown function, they form a pol III core with greater stability and catalytic efficiency. PMID- 2997152 TI - The lipoxins. Stereochemical identification and determination of their biosynthesis. AB - The stereochemistry and double bond geometry of a novel series of leukocyte derived arachidonic acid metabolites, the lipoxins, was determined by comparison to pure unambiguous synthetic standards. The lipoxins were found to be a mixture of four lipoxin A isomers and two lipoxin B isomers. In determining the biosynthesis of these compounds, they were shown to be formed via a tetraene epoxide. In addition, it was shown that all of the lipoxin isomers formed by the incubation of 15-hydroperoxyeicosatetraenoic acid with human leukocytes were also formed by nonenzymatic hydrolysis of this tetraene epoxide. PMID- 2997153 TI - Exons of the human pancreatic polypeptide gene define functional domains of the precursor. AB - Pancreatic polypeptide is a 36-amino acid peptide which inhibits pancreatic exocrine function. We have previously determined from the nucleotide sequence of a cDNA that pancreatic polypeptide is derived from a 95-amino acid precursor, prepropancreatic polypeptide. Pulse-chase studies have suggested that the precursor is cleaved to produce three peptides: pancreatic polypeptide, an icosapeptide, and a smaller peptide. In the present study, we have used the cloned cDNA as a hybridization probe to isolate the pancreatic polypeptide gene from a human bacteriophage genomic library. The nucleotide sequence of 2.8 kilobases of DNA representing the entire human pancreatic polypeptide gene was determined. The gene contains four exons and three introns. Exon 1 encodes the 5' untranslated region of the mRNA, exon 2 encodes the signal sequence and the sequence of pancreatic polypeptide, exon 3 encodes the icosapeptide, and exon 4 encodes a carboxyl-terminal heptapeptide and the 3'-untranslated region of the mRNA. By Southern blot analysis, the gene detected in a pancreatic polypeptide producing islet cell tumor was indistinguishable from that in normal human leukocytes. The structure of the human pancreatic polypeptide gene is consistent with the hypothesis that prepropancreatic polypeptide generates three distinct peptides, each encoded by a separate exon. Increased expression of pancreatic polypeptide in the islet cell tumor does not appear to be correlated with major alterations in pancreatic polypeptide gene structure. PMID- 2997154 TI - Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase. AB - Rat liver L-type pyruvate kinase was phosphorylated in vitro by a Ca2+/calmodulin dependent protein kinase purified from rabbit liver. The calmodulin (CaM) dependent kinase catalyzed incorporation of up to 1.7 mol of 32P/mol of pyruvate kinase subunit; maximum phosphorylation was associated with a 3.0-fold increase in the K0.5 for P-enolpyruvate. This compares to incorporation of 0.7 to 1.0 mol of 32P/mol catalyzed by the cAMP-dependent protein kinase with a 2-fold increase in K0.5 for P-enolpyruvate. When [32P]pyruvate kinase, phosphorylated by the CaM dependent protein kinase, was subsequently incubated with 5 mM ADP and cAMP dependent protein kinase (kinase reversal conditions), 50-60% of the 32PO4 was removed from pyruvate kinase, but the K0.5 for P-enolpyruvate decreased only 20 30%. Identification of 32P-amino acids after partial acid hydrolysis showed that the CaM-dependent protein kinase phosphorylated both threonyl and seryl residues (ratio of 1:2, respectively) whereas the cAMP-dependent protein kinase phosphorylated only seryl groups. The two phosphorylation sites were present in the same 3-4-kDa CNBr fragment located near the amino terminus of the enzyme subunit. These results indicate that the CaM-dependent protein kinase catalyzed phosphorylation of L-type pyruvate kinase at two discrete sites. One site is apparently the same serine which is phosphorylated by the cAMP-dependent protein kinase. The second site is a unique threonine residue whose phosphorylation also inactivates pyruvate kinase by elevating the K0.5 for P-enolpyruvate. These results may account for the Ca2+-dependent phosphorylation of pyruvate kinase observed in isolated hepatocytes. PMID- 2997155 TI - Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Suicidal inactivation by acetylenic fatty acids. AB - Human polymorphonuclear leukocytes (PMN) not only generate and respond to leukotriene B4 (LTB4), but also catabolize this mediator of inflammation rapidly and specifically by omega-oxidation (probably due to the action of a cytochrome P 450 enzyme). To develop pharmacologically useful inhibitors of the LTB4 omega hydroxylase in human PMN, we devised a general scheme for synthesizing terminal acetylenic fatty acids based on the "acetylenic zipper" reaction. We found that the LTB4 omega-hydroxylase in intact PMN and in PMN sonicates is inactivated in a concentration-dependent fashion by terminal acetylenic analogues of lauric, palmitic, and stearic acids (i.e. 11-dodecynoic, 15-hexadecynoic, and 17 octadecynoic acids). Consistent with a suicidal process, inactivation of the LTB4 omega-hydroxylase requires molecular oxygen and NADPH, is time-dependent, and follows pseudo-first-order kinetics. Inactivation of the omega-hydroxylase by acetylenic fatty acids also is dependent on the terminal acetylenic moiety and the carbon chain length. Saturated fatty acids lacking a terminal acetylenic moiety do not inactivate the omega-hydroxylase. In addition, the two long-chain (C16, C18) acetylenic fatty acids inactivate the omega-hydroxylase at much lower concentrations (less than 5.0 microM) than those required for inactivation by the short-chain (C12) terminal acetylenic fatty acid (100 microM). Potent suicidal inhibitors of the LTB4 omega-hydroxylase in human PMN will help elucidate the roles played by LTB4 and its omega-oxidation products in regulating PMN function and in mediating inflammation. PMID- 2997156 TI - Inhibition of transferrin receptor expression by interferon-alpha in human lymphoblastoid cells and mitogen-induced lymphocytes. AB - 125I-Transferrin binding to lymphoblastoid K562 and Daudi cells markedly increased after exposure of the cells to culture conditions that stimulated proliferation. Treatment of these cells with interferon-alpha (IFN-alpha) resulted in concurrent inhibition of cell growth and of the rise in transferrin binding. Scatchard analyses revealed that IFN reduced the number of transferrin receptors without altering the binding constant. When 125I-transferrin binding was measured using permeabilized cells, the IFN-induced reduction of binding was comparable to that observed with intact cells, indicating that IFN diminished the total number of cellular transferrin receptors. We also found that addition of IFN-alpha to phytohemagglutinin-stimulated human lymphocytes inhibited the mitogen-induced enhancement of [3H]thymidine incorporation as well as surface binding of 125I-transferrin. Our findings suggest that the decrease in transferrin receptor expression on IFN-alpha-treated cells may be one of the mechanisms responsible for the antiproliferative action of IFN. PMID- 2997157 TI - Facilitated diffusion during catalysis by EcoRI endonuclease. Nonspecific interactions in EcoRI catalysis. AB - The potential for processive EcoRI endonuclease hydrolysis has been examined on several DNA substrates containing two EcoRI sites which were embedded in identical sequence environments. With a 388-base pair circular DNA, in which the two recognition sites are separated by 51 base pairs (shorter distance) or 337 base pairs (longer distance), 77 and 34% of all events involved processive hydrolysis at ionic strengths of 0.059 and 0.13, respectively. However, the frequency of processive action on linear substrates, in which the two sites were separated by 51 base pairs, was only 42 and 17% at these ionic strengths, values half those observed with the circular DNA. Processive action was not detectable on circular or linear substrates at an ionic strength of 0.23. These findings indicate that DNA search by the endonuclease occurs by facilitated diffusion, a mechanism in which the protein locates and leaves its recognition sequence by interacting with nonspecific DNA sites. We suggest that processivity on linear substrates is limited to values half that for small circles due to partitioning of the enzyme between the two products generated by cleavage of a linear molecule. Given such topological effects, measured processivity values imply that the endonuclease can diffuse within a DNA domain to locate and recognize an EcoRI site 50 to 300 base pairs distant from an initial binding site, with minimum search efficiencies being 80 and 30% at ionic strengths of 0.059 and 0.13, respectively. The high efficiency of processive action indicates that a positionally correlated mode of search plays a major role in facilitated diffusion in this system under such conditions. Also consistent with this view was the identification of a striking position effect when two closely spaced EcoRI sites were asymmetrically positioned near the end of a linear DNA. The endonuclease displays a substantial preference for the more centrally located recognition sequence. This preference does not reflect differential sensitivity of the two sites to cleavage per se, but can be simply explained by preferential entry of the enzyme via the larger nonspecific target available to the more centrally positioned recognition sequence. These conclusions differ from those of a previous qualitative analysis of endonuclease processivity over short distances (Langowski, J., Alves, J., Pingoud, A., and Maass, G. (1983) Nucleic Acids Res. 11, 501-513). PMID- 2997158 TI - Substitutions of proline 76 in yeast iso-1-cytochrome c. Analysis of residues compatible and incompatible with folding requirements. AB - Fine-structure genetic mapping previously revealed numerous nonfunctional cyc1 mutations having alterations at or near the site corresponding to amino acid position 76 of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae. DNA sequencing of the alterations in four of these cyc1 mutations indicated that the normal Pro-76 was replaced by Leu-76. Revertants containing at least partially functional iso-1-cytochromes c were isolated, and the alterations were analyzed by DNA sequencing and protein analysis. Specific activities of the altered iso-1 cytochromes c were estimated in vivo by growth of the strains in lactate medium; compared to normal iso-1-cytochrome c with Pro-76, the following activities were associated with the following replacements: approximately 90% for Val-76, approximately 60% for Thr-76, approximately 30% for Ser-76, approximately 20% for Ile-76, and 0% for Leu-76. In order to develop an understanding of the factors that determine whether or not an altered iso-1-cytochrome c will function, we undertook a theoretical analysis which led to the conclusion that the activity of the proteins was dependent on both short- and long-range interactions. Short range interactions were estimated from studies on known protein structures which gave the likelihood that various amino acids would be found in a local backbone configuration similar to the native protein; long-range interactions with the rest of the molecule were analyzed by considering the size of the side chain. We believe this approach can be used to analyze a wide variety of mutant proteins. PMID- 2997159 TI - Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils. I. Sodium fluxes. AB - The nature of Na+ fluxes in resting and in chemotactic factor-activated human neutrophils was investigated. In resting cells, ouabain-insensitive unidirectional 22Na+ in- and effluxes represented passive electrodiffusional fluxes through ion channels: they were nonsaturable and voltage-dependent (PNa = 4.3 X 10(-9) cm/s). Amiloride (1 mM) had little effect on resting 22Na+ influx (approximately 0.8 meq/liter X min), thereby suggesting a minor contribution of Na+/H+ exchange and a lack of amiloride-sensitive Na+ channels. When neutrophils were exposed to the chemotactic tripeptide N-formyl-methionyl-leucyl phenylalanine (FMLP, 0.1 microM), 22Na+ influx was stimulated approximately 30 fold (initial rate approximately 22 meq/liter X min). The FMLP-induced 22Na+ influx was saturable with respect to external Na+ (Km 26-35 mM, Vmax approximately 28 meq/liter X min), was electroneutral, and could be competitively inhibited by amiloride (Ki 10.6 microM). From a resting value of approximately 30 meq/liter of cell water, internal Na+ in FMLP-stimulated cells rose exponentially to reach a concentration of approximately 60 meq/liter by 10-15 min. This uptake was blocked by amiloride. FMLP also stimulated the efflux of 22Na+ which followed a single exponential time course (rate coefficient approximately 0.16 min-1). The FMLP-induced 22Na+ fluxes were similar to those observed with 10 microM monensin, a known Na+/H+ exchanging ionophore. The data indicate that FMLP activates an otherwise quiescent, amiloride-sensitive Na+/H+ exchange. Furthermore, all of the FMLP-induced 22Na+ fluxes can be satisfactorily accounted for by transport through the exchanger, leaving little room for an appreciable increase in Na+ conductance. PMID- 2997160 TI - Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils. II. Intracellular pH changes. AB - The intracellular pH (pHi) changes resulting from chemotactic factor-induced activation of Na+/H+ exchange in isolated human neutrophils were characterized. Intracellular pH was measured from the equilibrium distribution of [14C]-5,5 dimethyloxazolidine-2,4-dione and from the fluorescence of 6-carboxyfluorescein. Exposure of cells to 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (FMLP) in 140 mM Na+ medium at extracellular pH (pHo) 7.40 led to a rise in pHi along an exponential time course (rate coefficient approximately 0.55 min-1). By 10 min, a new steady-state pHi was reached (7.75-7.80) that was 0.55-0.60 units higher than the resting pHi of control cells (7.20-7.25). The initial rate of H+ efflux from the cells (approximately 15 meq/liter X min), calculated from the intrinsic intracellular buffering power of approximately 50 mM/pH, was comparable to the rate of net Na+ influx (approximately 17 meq/liter X min), an observation consistent with a 1:1 stoichiometry for Na+/H+ exchange. This counter-transport could be inhibited by amiloride (apparent Ki approximately 75 microM). When either the external ([Na+]o) or internal Na ([Na+]i) concentrations, pHo, or pHi were varied independently, the new steady-state [Na+]i and pHi values in FMLP stimulated cells were those corresponding to a chemical equilibrium distribution of Na+ and H+ across the cell membrane. By analogy to other activated cells, these results indicate that an alkalinization of pHi in human neutrophils is mediated by a chemotactic factor-induced exchange of internal H+ for external Na+. PMID- 2997161 TI - The sources of thymidine nucleotides for virus DNA synthesis in herpes simplex virus type 2-infected cells. AB - The thymidine nucleotide sources present during herpes simplex virus type 2 (HSV 2) infection were examined. It was concluded that the source of dTTP in HSV-2 infected cells is not only derived from the ribonucleotide reductase-catalyzed de novo pathway, but also from host DNA. When the de novo pathway was inhibited by the addition of hydroxyurea, an inhibitor of ribonucleotide reductase, the dTTP levels were maintained by a compensatory increase in dTTP derived from host DNA. The utilization of host DNA-derived dTTP for viral DNA synthesis was demonstrated. In spite of an increased contribution of dTTP from host DNA in the presence of hydroxyurea, the level of utilization of host DNA-derived dTTP appeared to remain constant. More than one dTTP pool in virus-infected cells is implicated. PMID- 2997162 TI - Effect of actin filament length and filament number concentration on the actin activated ATPase activity of Acanthamoeba myosin I. AB - The actin-activated Mg2+-ATPase activities of phosphorylated Acanthamoeba myosins IA and IB were previously found to have a highly cooperative dependence on myosin concentration (Albanesi, J. P., Fujisaki, H., and Korn, E. D. (1985) J. Biol. Chem. 260, 11174-11179). This behavior is reflected in the requirement for a higher concentration of F-actin for half-maximal activation of the myosin Mg2+ ATPase at low ratios of myosin:actin (noncooperative phase) than at high ratios of myosin:actin (cooperative phase). These phenomena could be explained by a model in which each molecule of the nonfilamentous myosins IA and IB contains two F-actin-binding sites of different affinities with binding of the lower affinity site being required for expression of actin-activated ATPase activity. Thus, enzymatic activity would coincide with cross-linking of actin filaments by myosin. This theoretical model predicts that shortening the actin filaments and increasing their number concentration at constant total F-actin should increase the myosin concentration required to obtain the cooperative increase in activity and should decrease the F-actin concentration required to reach half-maximal activity at low myosin:actin ratios. These predictions have been experimentally confirmed by shortening actin filaments by addition of plasma gelsolin, an F actin capping/severing protein. In addition, we have found that actin "filaments" as short as the 1:2 gelsolin-actin complex can significantly activate Acanthamoeba myosin I. PMID- 2997163 TI - Solubilization and reconstitution of the Na+/Ca2+ exchanger of cardiac sarcolemma. Properties of the reconstituted system and tentative identification of the protein(s) responsible for the exchange activity. AB - The cardiac sarcolemma Na+/Ca2+ exchanger has been solubilized in Triton X-100 and reconstituted into asolectin liposomes by removing the detergent with Amberlite XAD-2 beads. The specific Na+/Ca2+ exchange activity of the proteoliposomes was increased about 10 times with respect to the original sarcolemma. The exchange activity was strongly dependent on the amount of acidic phospholipids present in the liposomes. Phosphatidylserine and phosphatidylinositol increased the initial rate of the reconstituted exchanger. Fractionation of the solubilized sarcolemma by rate zonal centrifugation produced fractions in which the specific activity of the exchanger was enriched up to 130 fold with respect to the original membranes. The fractionation scheme used has shown that the exchange activity correlated best with a 33-kDa protein present in the fractionated extract. No correlation has been found between exchange activity and other proteins of higher molecular mass (70 or 82 kDa) which have previously been suggested to correspond to the Na+/Ca2+ exchanger. Thus, the 33-kDa protein may represent the Na+/Ca2+ exchanger of cardiac sarcolemma. PMID- 2997164 TI - Coelution of the type II holoenzyme form of cAMP-dependent protein kinase with regulatory subunits of the type I form of cAMP-dependent protein kinase. AB - The types and subunit composition of cAMP-dependent protein kinases in soluble rat ovarian extracts were investigated. Results demonstrated that three peaks of cAMP-dependent kinase activity could be resolved using DEAE-cellulose chromatography. Based on the sedimentation of cAMP-dependent protein kinase and regulatory subunits using sucrose density gradient centrifugation, identification of 8-N3[32P]cAMP labeled RI and RII in DEAE-cellulose column and sucrose gradient fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Scatchard analysis of the cAMP-stimulated activation of the eluted peaks of kinase activity, the following conclusions were drawn regarding the composition of the three peaks of cAMP-dependent protein kinase activity: peak 1, eluting with less than or equal to 0.05 M potassium phosphate, consisted of the type I form of cAMP-dependent protein kinase; peak 2, eluting with 0.065-0.11 M potassium phosphate, consisted of free RI and a type II tetrameric holoenzyme; peak 3, eluting with 0.125 M potassium phosphate, consisted of an apparent RIIC trimer, followed by the elution with 0.15 M potassium phosphate of free RII. The regulatory subunits were confirmed as authentic RI and RII based upon their molecular weights and autophosphorylation characteristics. The more basic elution of the type II holoenzyme with free RI was not attributable to the ionic properties of the regulatory subunits, based upon the isoelectric points of photolabeled RI and RII and upon the elution location from DEAE-cellulose of RI and RII on dissociation from their respective holoenzymes by cAMP. This is the first report of a type II holoenzyme eluting in low salt fractions with free RI, and of the presence of an apparent RIIC trimer in a soluble tissue extract. PMID- 2997165 TI - Genomic DNA sequence for human C-reactive protein. AB - The gene for the prototype acute phase reactant, C-reactive protein, has been isolated from two lambda phage libraries containing inserted human DNA fragments using synthetic oligonucleotide probes. Nucleotide sequence analysis indicates that after coding for a signal peptide of 18 amino acids and the first two amino acids of the mature protein, there is an intron of 278 base pairs followed by the nucleotide sequence for the remaining 204 amino acids. The intron is unusual in that it contains on the positive strand a poly(A) stretch 16 nucleotides long and a poly(GT) region 30 nucleotides long which could adopt the Z-form of DNA. The nucleotide sequence reported here confirms the amino acid sequence of mature C reactive protein as originally reported except that it codes for an additional 19 amino acids beginning at position 62. Thus DNA sequence analysis predicts that the mature protein consists of 206 amino acids rather than 187 as originally reported. The mRNA cap site is located 104 nucleotides from the start of the signal peptide and there is a 3' noncoding region 1.2 kilobase pairs in length. The gene has a typical promoter containing the sequences TATAAAT and CAAT 29 and 81 base pairs upstream, respectively, of the cap site. PMID- 2997166 TI - Binding of human tumor necrosis factor to high affinity receptors on HeLa and lymphoblastoid cells sensitive to growth inhibition. AB - Purified human tumor necrosis factor (TNF) was iodinated to high specific activity with good retention of its biological activity, as determined by the cytotoxic titer on murine L929 cells. The binding of 125I-TNF to L929 and human HeLa S2 cells grown in monolayer was initially measured, but high levels of nonspecific binding were observed. Specific binding to high affinity receptors of HeLa S2 cells grown in suspension culture was demonstrated by competitive displacement experiments and analysis of the binding data with the LIGAND program. A KD of 2 X 10(-10) M and 6000 receptors/cell were calculated in this way. These observations provide the first direct evidence for a cellular receptor for TNF. The cell-bound 125I-TNF was internalized at 37 degrees C, presumably by receptor-mediated endocytosis, and subsequently degraded to acid-soluble products. Three lines of human lymphoblastoid cells were examined for sensitivity to the cytostatic effect of TNF and for the presence of high affinity receptors. Daudi and Raji cells were insensitive to TNF and showed very few specific binding sites when incubated with 125I-TNF. Jurkat cells were growth-inhibited by TNF and showed a significantly greater number of specific binding sites than the other lymphoblastoid cells. These findings suggest that the sensitivity of some cell lines to the biological effects of TNF may be correlated with the presence of a relatively high number of receptors for this factor. PMID- 2997167 TI - Isolation and characterization of the inositol cyclic phosphate products of polyphosphoinositide cleavage by phospholipase C. Physiological effects in permeabilized platelets and Limulus photoreceptor cells. AB - Cleavage of the polyphosphoinositides, catalyzed by phospholipase C purified from ram seminal vesicles, produces phosphorylated inositols containing cyclic phosphate esters (Wilson, D. B., Bross, T. E., Sherman, W. R., Berger, R. A., and Majerus, P. W. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4013-4017). In the present study we describe the isolation and characterization of inositol 1:2 cyclic 4-bisphosphate and inositol 1:2-cyclic 4,5-trisphosphate, the two cyclic phosphate products of phospholipase C catalyzed cleavage of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, respectively. We established the structures of these two cyclic compounds through 18O labeling of phosphate moieties, phosphomonoesterase digestion, and fast atom bombardment-mass spectrometry. We examined the physiological effects of these compounds in two systems: saponin-permeabilized platelets loaded with 45Ca2+ and intact Limulus photoreceptors. Both inositol 1:2-cyclic 4,5-trisphosphate and the noncyclic inositol 1,4,5-trisphosphate, but not inositol 1:2-cyclic 4-bisphosphate, release 45Ca2+ from permeabilized platelets in a concentration-dependent manner. Injection of inositol 1:2-cyclic 4,5-trisphosphate into Limulus ventral photoreceptor cells induces both a change in membrane conductance and a transient increase in intracellular calcium ion concentration similar to those induced by light. We injected inositol 1,4,5-trisphosphate and inositol 1:2-cyclic 4,5 trisphosphate into the same photoreceptor cell and found that the cyclic compound is approximately five times more potent than the noncyclic compound in stimulating a conductance change. We speculate that inositol 1:2-cyclic 4,5 trisphosphate may function as a second messenger in stimulated cells. PMID- 2997168 TI - Activation of NADPH-dependent superoxide production in a cell-free system by sodium dodecyl sulfate. AB - Sodium dodecyl sulfate (SDS) elicits the production of superoxide (O2-) by a cell free system represented by sonically disrupted guinea pig peritoneal macrophages. O2- generation requires NADPH and a heat-sensitive cellular component, is proportional to the amount of macrophage protein, and exhibits a pH optimum of 6.5-7. The kinetic parameters of the SDS-stimulated enzyme are: Km (+/- S.E.) = 0.0367 +/- 0.003 mM NADPH and Vmax (+/- S.E.) = 73.46 +/- 9.09 nmol O2-/mg of protein/min. O2- production is dependent on the cooperation between a particulate subcellular component sedimentable at 48,000 X g and a cytosolic factor present in the 48,000 X g supernatant. The activity of both components is destroyed by heating at 80 degrees C. Pretreatment of intact macrophages with phorbol myristate acetate results in the partial removal of the requirement for cytosolic factor; SDS is now capable of activating the isolated 48,000 X g pellet. Among a large number of anionic, cationic, and nonionic detergents tested, only the anionic detergents SDS and sodium dodecyl sulfonate are capable of eliciting O2- production in the cell-free system, SDS being the more potent stimulant. It is proposed that the structural requirements that make these compounds capable of activating the O2- forming NADPH oxidase in a cell-free system are the presence of an anionic polar head and a long hydrophobic alkyl tail. We suggest that sodium salts of long chain unsaturated fatty acids that were found by us to be capable of stimulating O2- production in a cell-free system (Bromberg, Y., and Pick, E. (1984) Cell. Immunol. 88, 213-221) owe their activity to the fact that they function as anionic detergents. PMID- 2997169 TI - Thyroid peroxidase selects the mechanism of either 1- or 2-electron oxidation of phenols, depending on their substituents. AB - Unlike lactoperoxidase and horseradish peroxidase, thyroid peroxidase catalyzed the oxidation of hydroquinone mostly by way of 2-electron transfer. This conclusion could be derived from three independent experiments: ESR measurements of p-benzosemiquinone, trapping the unpaired electron by cytochrome c, and spectrophotometric analysis of catalytic intermediates of the enzymes. The 1 electron flux for hydroquinone oxidation was found to be 15-19% in the reaction of thyroid peroxidase, while it was nearly 100% in the reactions of lactoperoxidase and horseradish peroxidase. From the spectrophotometric analysis of the catalytic intermediates of enzyme, it was suggested that the mechanism of oxidation catalyzed by thyroid peroxidase changes from a 2-electron to a 1 electron type as the substituents at 2- and 6-positions of phenol become bulky or heavy. On the other hand, the mechanism was invariably a 1-electron type when the oxidation of phenols was catalyzed by lactoperoxidase or horseradish peroxidase. These three peroxidases all catalyzed 1-electron oxidation of ascorbate. PMID- 2997170 TI - Des-1-25-fructose-1,6-bisphosphatase, a nonallosteric derivative produced by trypsin treatment of the native protein. AB - Limited tryptic digestion of pig kidney fructose-1,6-bisphosphatase in the presence of magnesium ions results in the formation of an active enzyme derivative which is no longer inhibited by the allosteric effector AMP. The presence of AMP during incubation of fructose-1,6-bisphosphatase with trypsin protects against the loss of AMP inhibition. By contrast, the presence of the nonhydrolyzable substrate analog fructose 2,6-bisphosphate accelerates the rate of formation of that form of fructose-1,6-bisphosphatase which is insensitive to AMP inhibition. Sodium dodecyl sulfate-polyacrylamide electrophoresis of samples taken during trypsin treatment shows that the loss of AMP inhibition parallels the conversion of the native 36,500 molecular weight fructose-1,6-bisphosphatase subunit into a 34,000 molecular weight species. Automated Edman degradation of trypsin-treated fructose-1,6-bisphosphatase following gel filtration shows a single sequence beginning at Gly-26 in the original enzyme, but no changes in the COOH-terminal region of fructose-1,6-bisphosphatase. Thus, the proteolytic product has been characterized as "des-1-25-fructose-1,6-bisphosphatase." A comparison of the kinetic properties of control enzyme and des-1-25-fructose-1,6 bisphosphatase reveals some differences in properties (pH optimum, Ka for Mg2+, K+ activation, inhibition by fructose 2,6-bisphosphate) between the two enzymes, but none is so striking as the complete loss of AMP sensitivity shown by des-1-25 fructose-1,6-bisphosphatase. The loss of AMP inhibition is due to the loss of AMP binding capacity, but it is not known at this stage whether residues of the AMP site are present in the 25-amino acid NH2-terminal region or the removal of this region leads to a conformational change that abolishes the function of an AMP site located elsewhere in the molecule. PMID- 2997171 TI - Hepatic retinol metabolism. Distribution of retinoids, enzymes, and binding proteins in isolated rat liver cells. AB - The main retinoids and some binding proteins and enzymes involved in retinol metabolism have been quantified in different types of rat liver cells. Hepatic perisinusoidal stellate cells contained 28-34 nmol of retinoids/10(6) cells, and parenchymal liver cells contained 0.5-0.8 nmol of retinoids/10(6) cells, suggesting that as much as 80% of more of total liver retinoids might be stored in stellate cells with the rest stored in parenchymal cells. Isolated endothelial cells and Kupffer cells contained very low levels of retinoids. More than 98% of the retinoids recovered in stellate cells were retinyl esters. Isolated parenchymal and stellate cell preparations both contained considerable retinyl palmitate hydrolase and acyl-CoA:retinol acyltransferase activities. Parenchymal cells accounted for about 75-80% of the total hepatic content of these two enzyme activities, with the rest located in stellate cells. On a cell protein basis, the concentrations of both of these activities were much greater in stellate cells than in parenchymal cells. In contrast, cholesteryl oleate and triolein hydrolase activities were fairly evenly distributed in all types of liver cells. Large amounts of cellular retinol binding proteins were also found in parenchymal and stellate cells. Although parenchymal cells accounted for more than 90% of hepatic cellular retinol binding protein, the concentration of the protein in stellate cells (per unit protein) was 22 X greater than that in parenchymal cells. Stellate cells were also enriched in cellular retinoic acid binding protein. Thus, both parenchymal and stellate cells contain substantial amounts of retinoids and of the enzymes and intracellular binding proteins involved in retinol metabolism. Stellate cells are particularly enriched in these several components. PMID- 2997172 TI - Control of insulin gene expression in pancreatic beta-cells and in an insulin producing cell line, RIN-5F cells. I. Effects of glucose and cyclic AMP on the transcription of insulin mRNA. AB - To define the mechanism whereby glucose regulates islet insulin mRNA content, insulin gene transcription rates were determined in islets labeled with [3H]uridine at low (3.3) or high (17 mM) glucose. Glucose stimulated the transcription of total RNA almost 2-fold and insulin mRNA 5.6-fold. Addition of dibutyryl cAMP to islets in vitro could partially mimic the effect of glucose on insulin gene-specific transcription. In the insulin-producing RIN-5F cell line, glucose did not affect transcription, while cholera toxin acted as a secretagogue and increased total RNA and insulin gene-specific transcription as well. We conclude that glucose exerts a specific stimulatory effect on the transcription of the insulin gene(s) in normal islets and that this effect may be mediated in part by cAMP. PMID- 2997173 TI - Characterization and partial purification of the sodium-potassium-ATPase inhibitor released from cultured rat hypothalamic cells. AB - An inhibitor of sodium-potassium-ATPase has been partially purified from the culture medium obtained from hypothalamic cells maintained in a capillary membrane perfusion system, and some of the properties of this inhibitory factor have been investigated. Gel filtration (Sephadex G-25 Superfine) of heat-treated medium (80 degrees C for 10 min) resulted in elution of inhibitory activity in the post-salt fraction. These fractions inhibited active (i.e. sodium-potassium ATPase-mediated) sodium transport in intact human erythrocytes, displaced [3H]ouabain from its binding site, and directly inhibited canine kidney sodium potassium-ATPase as measured by NADH oxidation. High-performance liquid chromatography (on Hypersil ODS) of these fractions after desalting yielded one region which showed inhibitory activity on all three assays. Inhibition of sodium potassium-ATPase was dose-related and filtered through an Amicon UM10 membrane. Incubation of this material with dispase, carboxypeptidase A, chymotrypsin, and prolidase destroyed inhibitory activity, whereas trypsin and leucine aminopeptidase were ineffective. These studies show that hypothalamic neurones release a low molecular weight heat-stable peptide which inhibits active sodium transport, ouabain binding, and sodium-potassium-ATPase. PMID- 2997174 TI - The gelatinolytic activity of rat uterus collagenase. AB - The collagenase produced by rat uterine cells in culture has been examined for its ability to degrade denatured collagen. Acting as a gelatinase, rat uterus collagenase was able to successfully degrade the denatured chains of collagen types I through V. In addition, the enzyme produced multiple cleavages in these chains and displayed values for Km of 4-5 microM, compared to values of 1-2 microM when native collagen was used as substrate. Furthermore, rat uterus collagenase degraded the alpha 2 chain of denatured type I collagen at a significantly faster rate than the alpha 1 chain, as previously observed for human skin fibroblast collagenase. In contrast to the action of human skin collagenase, however, the rat uterus enzyme was found to be a markedly better gelatinase than a collagenase, degrading the alpha chains of denatured type I guinea pig skin collagen at rates some 7-15-fold greater than native collagen. Human skin collagenase degrades the same denatured chains at rates ranging from 13-44% of its rate on native collagen. Rat uterus collagenase, then, is approximately 50 times better a gelatinase than is human skin collagenase. In addition to its ability to cleave denatured collagen chains at greater rates than native collagen, the rat uterus collagenase also attacked a wider spectrum of peptide bonds in gelatin than does human skin collagenase. In addition to cleaving the Gly-Leu and Gly-Ile bonds characteristic of its action on native collagen, rat uterus collagenase readily catalyzed the cleavage of Gly-Phe bonds in gelatin. The rat enzyme was also capable of cleaving Gly-Ala and Gly-Val bonds, although these bonds were somewhat less preferred by the enzyme. The cleavage of peptide bonds other than Gly-Leu and Gly-Ile appears to be a property of the collagenase itself and not a contaminating protease. Thus, it appears that the collagenase responsible for the degradation of collagen during the massive involution of the uterus might also act as a gelatinase to further degrade the initial products of collagenolysis to small peptides suitable for further metabolism. PMID- 2997175 TI - Metabolism and pharmacokinetics of 24,25-dihydroxyvitamin D3 in the vitamin D3 replete rat. AB - The time course of in vivo metabolism of 24,25-dihydroxyvitamin D3 in rats has been examined. Several tissues were surveyed in an effort to discover new metabolites of 24,25-dihydroxyvitamin D3 and to estimate the concentrations of previously identified metabolites. Rapidly growing male rats were dosed with 24,25-dihydroxyvitamin D3 orally until plasma concentrations of 24,25 dihydroxyvitamin D3 were at steady state. 24,25-Dihydroxyvitamin [3-3H]D3 was then administered. At 10 min and 1, 6, 15, 24, 96, and 192 h after dosing, the animals were killed, and plasma, liver, intestine, and bones were analyzed with a newly developed gradient straight-phase high performance liquid chromatography system. The high performance liquid chromatography system is capable of base-line resolution of most of the major vitamin D metabolites. 24,25-Dihydroxyvitamin D3 clearance from plasma, liver, and kidney but not intestine followed a two compartment model. 24,25-Dihydroxyvitamin D3 disappeared from plasma with a half life of 0.55 h (fast phase) and 73.8 h (slow phase). Only two lipid-soluble metabolites of 24,25-dihydroxyvitamin D3 were detected: 24-oxo-25-hydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3. These compounds circulate at very low concentrations in the plasma (50 pg/ml of plasma). PMID- 2997176 TI - The high potential iron-sulfur center in Escherichia coli fumarate reductase is a three-iron cluster. AB - The fumarate reductase complex and soluble enzyme from Escherichia coli have been investigated by low temperature magnetic circular dichroism and electron paramagnetic resonance spectroscopies. The results confirm the presence of one [2Fe-2S] cluster and show that the high potential iron-sulfur center is a 3Fe cluster of the type found in bacterial ferredoxins. Since the 3Fe cluster is present in catalytically competent enzyme and does not appear to be involved in any type of cluster conversion under reducing conditions, we conclude that it is an intrinsic component of the functional enzyme. The significance of the results is discussed in relation to the published amino acid sequence and the iron-sulfur cluster composition of bacterial fumarate reductases. PMID- 2997177 TI - Isolation of novel microbial 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid dehydrogenases. Purification, characterization, and analytical applications of a 17 beta-hydroxysteroid dehydrogenase from an Alcaligenes sp. AB - By selecting for growth on testosterone or estradiol-17 beta as the only source of organic carbon, we have isolated a number of soil microorganisms which contain highly active and novel, inducible, NAD-linked 3 alpha-, 3 beta-, and 17 beta hydroxysteroid dehydrogenases. Such enzymes are suitable for the microanalysis of steroids and of steroid-transforming enzymes, as well as for performing stereoselective oxidations and reductions of steroids. Of particular interest among these organisms is a new species of Alcaligenes containing 17 beta hydroxysteroid dehydrogenase, easily separable from 3 beta-hydroxysteroid dehydrogenase. Unlike any of the other isolated organisms, this Alcaligenes sp. contained no 3 alpha-hydroxysteroid dehydrogenase activity. A large-scale purification (763-fold) to homogeneity of the major induced 17 beta hydroxysteroid dehydrogenase was achieved by ion-exchange, hydrophobic, and affinity chromatographies. The enzyme has high specific activity for the oxidation of testosterone (Vmax = 303 mumol/min/mg of protein; Km = 3.6 microM) and reacts almost equally well with estradiol-17 beta (Vmax = 356 mumol/min/mg; Km = 6.4 microM). It consists of apparently identical subunits (Mr = 32,000) and exists in polymeric form under nondenaturing conditions (Mr = 68,000 by gel filtration and 86,000 by polyacrylamide gel electrophoresis). The isoelectric point is pH 5.1. The enzyme is almost completely specific for 17 beta hydroxysteroids which may be delta 5-olefins or ring A phenols or have cis or trans A/B ring fusions. Substituents at other positions are tolerated, although the presence of a 16 alpha- or 16 beta-hydroxyl group blocks the oxidation of the 17 beta-hydroxyl function. 3 beta-Hydroxysteroids (A/B ring fusion trans, but not cis, or delta 5-olefins) are very poor substrates. The application of this highly active, specific, and stable 17 beta-hydroxysteroid dehydrogenase to the microestimation of steroids by enzymatic cycling of nicotinamide nucleotides and for the stereospecific oxidation of steroids is demonstrated. PMID- 2997178 TI - The relationship between gastric acid secretion and gastric H+,K+-ATPase activity. AB - The H+,K+-ATPase has been postulated to be the enzyme responsible for H+ secretion by the parietal cell. Omeprazole has been shown to be an inhibitor of acid secretion in vivo, but also in in vitro test models for acid secretion, including partly purified H+,K+-ATPase, the inhibitory action of omeprazole has been demonstrated (Wallmark, B., Jaresten, B. M., Larsson, H., Ryberg, B., Brandstrom, A., and Fellenius, E. (1983) Am. J. Physiol. 245, G64-G71). It was thus possible to use this compound to demonstrate a correlation between H+,K+ ATPase activity in rat oxyntic mucosa and in vivo H+ secretion. Two results were found. (a) Increasing oral doses of omeprazole progressively inhibited acid secretion, H+,K+-ATPase activity, and phosphoenzyme formation of a microsomal fraction isolated from the inhibited rat mucosa. Furthermore, a Mg2+-stimulated ATPase activity, associated with the H+,K+-ATPase membrane fraction, was not affected by the omeprazole treatment. (b) Recovery of H+,K+-ATPase activity following complete omeprazole inhibition was correlated with the appearance of acid secretion. The results indicate a strict relationship between the activity of the gastric H+,K+-ATPase in the microsomal fraction and gastric acid secretion. PMID- 2997179 TI - Effect of 1,25-dihydroxyvitamin D3 on phospholipid metabolism in a clonal osteoblast-like rat osteogenic sarcoma cell line. AB - The effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on phospholipid metabolism was examined in clonal rat osteogenic sarcoma cells, UMR 106, of osteoblastic phenotype. Treatment of UMR 106 cells with 10(-8)M 1,25-(OH)2D3 for 48 h caused an increase in [14C]serine incorporation into phosphatidylserine (PS) and a decrease in [3H]ethanolamine, [3H]linositol, and [14C]choline incorporation into phosphatidylethanolamine (PE), phosphatidylinositol, and phosphatidylcholine, respectively; the decrease in [3H]ethanolamine incorporation into PE was the largest. The total contents of phospholipids were similarly affected by 10(-8)M 1,25-(OH)2D3 treatment, suggesting that the effects of 1,25-(OH)2D3 are due largely to alterations in the synthesis of these phospholipids. The effects of 1,25-(OH)2D3 were evident at 10(-10) M 1,25-(OH)2D3, and 10(-8)M 1,25-(OH)2D3 caused a maximal stimulation of [14C]PS synthesis (167% of control) and a maximal reduction in the [3H]PE synthesis (41% of control). The [14C]PS/[3H]PE ratio increased gradually and reached a maximum after 70 h of treatment with 10(-8)M 1,25-(OH)2D3. When the cells were cultured in calcium-free medium containing 0.5 mM EGTA or when 5 microM cycloheximide was added to the medium, the effect of 1,25-(OH)2D3 on phospholipid metabolism was almost completely inhibited. Neither 25-hydroxyvitamin D3 nor 24,25-dihydroxyvitamin D3 caused significant changes in phospholipid metabolism. These results suggest that 1,25-(OH)2D3 alters phospholipid metabolism by enhancing PS synthesis through a calcium-dependent stimulation of the base exchange reaction of serine with other phospholipids and that the effect of 1,25-(OH)2D3 requires the synthesis of new proteins. Because PS is thought to be important for apatite formation and bone mineralization by binding calcium and phosphate to form calcium-PS-phosphate complexes, the present data suggest that 1,25-(OH)2D3 may stimulate bone mineralization by a direct effect on osteoblasts, stimulating PS synthesis. PMID- 2997182 TI - Phosphorylation and inactivation of yeast fructose-1,6-bisphosphatase by cyclic AMP-dependent protein kinase from yeast. AB - Purified fructose-1,6-bisphosphatase from Saccharomyces cerevisiae was phosphorylated in vitro by purified yeast cAMP-dependent protein kinase. Maximal phosphorylation was accompanied by an inactivation of the enzyme by about 60%. In vitro phosphorylation caused changes in the kinetic properties of fructose-1,6 bisphosphatase: 1) the ratio R(Mg2+/Mn2+) of the enzyme activities measured at 10 mM Mg2+ and 2 mM Mn2+, respectively, decreased from 2.6 to 1.2; 2) the ratio R(pH 7/9) of the activities measured at pH 7.0 and pH 9.0, respectively, decreased from 0.62 to 0.38, indicating a shift of the pH optimum to the alkaline range. However, the affinity of the enzyme for its inhibitors fructose-2,6-bisphosphate (Fru-2,6-P2) and AMP, expressed as the concentration required for 50% inhibition, was not changed. The maximum amount of phosphate incorporated into fructose-1,6 bisphosphatase was 0.6-0.75 mol/mol of the 40-kDa subunit. Serine was identified as the phosphate-labeled amino acid. The initial rate of in vitro phosphorylation of fructose-1,6-bisphosphatase, obtained with a maximally cAMP-activated protein kinase, increased when Fru-2,6-P2 and AMP, both potent inhibitors of the enzyme, were added. As Fru-2,6-P2 and AMP did not affect the phosphorylation of histone by cAMP-dependent protein kinase, the inhibitors must bind to fructose-1,6 bisphosphatase in such a way that the enzyme becomes a better substrate for phosphorylation. Nevertheless, Fru-2,6-P2 and AMP did not increase the maximum amount of phosphate incorporated into fructose-1,6-bisphosphatase beyond that observed in the presence of cAMP alone. PMID- 2997180 TI - Dolichyl phosphate metabolism in brain. Developmental increase in polyisoprenyl phosphate phosphatase activity. AB - The enzymatic dephosphorylation of endogenous and exogenous dolichyl phosphate (Dol-P) by membrane preparations from calf brain has been reported previously (Burton, W.A., Scher, M.G., and Waechter, C. J. (1981) Arch. Biochem. Biophys. 208, 409-417). The study presented here shows that polyisoprenyl phosphate phosphatase activity increases with age in crude microsomal fractions from white (glial-enriched) and grey (neuronal-enriched) matter of pig brain. A kinetic analysis of grey matter phosphatase activity revealed that the apparent Km values for Dol-P are similar for membranes from adults (0.13 mM) and prenatal subjects (0.12 mM), but the Vmax is significantly higher in adults. In direct contrast to this result, the specific activity of dolichol kinase is nearly 2-fold higher in membranes from pig brain grey matter of prenatal subjects as compared to identical preparations from animals 90 days of age. While the changes in kinase and phosphatase activity are approximately 2-fold, reciprocal changes in these activities would enhance shifts in the metabolic balance of a phosphorylation dephosphorylation process. In another aspect of this study on polyisoprenyl phosphate phosphatase in nervous tissue, membrane preparations from calf brain are shown to dephosphorylate S- and R-Dol-P. Similar apparent Km values were obtained for S-Dol-P and R-Dol-P, but the Vmax for the dephosphorylation reaction was found to be 1.8-fold greater for S-Dol-P. These results extend the description of polyisoprenyl phosphate phosphatase activity in brain and provide information on developmental changes in phosphatase and kinase activities involved in Dol-P metabolism in nervous tissue. PMID- 2997183 TI - The identification and partial characterization of the fibroblast growth factor receptor of baby hamster kidney cells. AB - The binding of biologically active, 125I-labeled basic fibroblast growth factor (FGF) to baby hamster kidney-derived cell line cells (BHK-21) was studied at 4 degrees C. Unlabeled FGF displaced cell surface bound 125I-FGF, but platelet derived growth factor, epidermal growth factor, insulin, or transferrin did not. Binding was saturable both as a function of time and as a function of increasing 125I-FGF concentrations. Scatchard analysis of the binding data revealed the presence of about 1.2 X 10(5) binding sites/cell with an apparent KD of 270 pM. The number of the binding sites was down-regulated following preincubation of the cells with FGF. The density of binding sites/cell also decreased as an inverse function of cell density. When 125I-FGF binding was studied in a BHK-21 cell membrane preparation, it was found that the membranal binding site displayed a lower KD of 21 pM. 125I-FGF was covalently cross-linked to its cell surface receptor on intact BHK-21 cells using the homobifunctional agent disuccinimidyl suberate. Two macromolecular species with an apparent molecular weight of 145,000 and 125,000, respectively, were labeled under both reducing and nonreducing conditions. Unlabeled FGF competed with 125I-FGF for binding to both macromolecular species. The labeling of the macromolecules was also inhibited by heparin. No labeling was observed in the absence of the cross-linkers or when heat-inactivated 125I-FGF was used instead of radiolabeled, biologically active FGF. PMID- 2997181 TI - Structural characterization of cardiac protein phosphatase with a monoclonal antibody. Evidence that the Mr = 38,000 phosphatase is the catalytic subunit of the native enzyme(s). AB - The native structures of protein phosphatases have not been clearly established. Several tissues contain high molecular weight enzymes which are converted to active species of Mr approximately 35,000 by denaturing treatments or partial proteolysis. We have used a monoclonal antibody directed against purified bovine cardiac Mr = 38,000 protein phosphatase to determine whether this species is the native catalytic subunit or a proteolytic product of a larger polypeptide. Monoclonal antibody was obtained from a cloned hybrid cell line produced by the fusion of Sp2 myeloma cells with spleen cells from a mouse immunized with phosphatase coupled to hemocyanin. This antibody was specific for the Mr = 38,000 phosphatase as determined by immunoblot analysis of purified enzyme or cardiac tissue extracts after native or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single immunoreactive protein of Mr = 38,000 was present in cardiac tissue extracts including extracts prepared from freeze-clamped rat heart rapidly denatured in hot sodium dodecyl sulfate buffer. Precipitation of cardiac extract with 80% ethanol did not alter the Mr of the phosphatase nor did it liberate new immunoreactive material not observed in the extract. Ethanol precipitation caused the dissociation of both phosphatase activity and immunoreactivity from a high Mr form to a form of Mr between 30,000 and 40,000. An immunoreactive protein of Mr = 38,000 was identified in several bovine and rat tissues as well as tissues from rabbits, mice and chickens and human HT-29 cells. From these data we conclude that the Mr = 38,000 cardiac phosphatase is a native catalytic subunit of higher molecular complexes which are dissociated by ethanol precipitation. A very similar, or identical, protein is present in several tissues and species suggesting that this catalytic subunit is a ubiquitous enzyme important in many dephosphorylation reactions. PMID- 2997184 TI - Identification of an endogenous protein kinase C activity and its intrinsic 15 kilodalton substrate in purified canine cardiac sarcolemmal vesicles. AB - The cardiac sarcolemmal 15-kDa protein, previously shown to be the principal sarcolemmal substrate phosphorylated in intact heart in response to beta adrenergic stimulation (Presti, C. F., Jones, L. R., and Lindemann J. P. (1985) J. Biol. Chem. 260, 3860-3867), was demonstrated to be the major substrate phosphorylated in purified canine cardiac sarcolemmal vesicles by an intrinsic protein kinase C activity. The intrinsic protein kinase C, detected by its ability to phosphorylate H1 histones, was most concentrated in cardiac sarcolemmal vesicles and absent from sarcoplasmic reticulum membranes. Unmasking techniques localized the intrinsic protein kinase activity and its principal endogenous substrate, the 15-kDa protein, to the cytoplasmic surfaces of sarcolemmal vesicles; phospholamban contaminating the sarcolemmal preparation was not significantly phosphorylated. The intrinsic protein kinase C required micromolar Ca2+ for activity, but not calmodulin. Half-maximal phosphorylation of the 15-kDa protein occurred at 10 microM Ca2+; optimal phosphorylation of the 15 kDa protein by protein kinase C and Ca2+ was additive to that produced by cAMP dependent protein kinase. Exogenous phospholipids were not required to activate endogenous protein kinase C. However, heat-treated sarcolemmal vesicles, in which intrinsic protein kinase activities were inactivated, were sufficient to maximally activate soluble protein kinase C prepared from rat brain, suggesting that all the necessary phospholipid cofactors were already present in sarcolemmal vesicles. Of the many proteins present in sarcolemmal vesicles, only the 15-kDa protein was phosphorylated significantly in heat-inactivated sarcolemmal vesicles by soluble protein kinase C, confirming that the 15-kDa protein was a preferential substrate for this enzyme. Consistent with a protein kinase C activity in sarcolemmal vesicles, the tumor-promoting phorbol ester 12-O tetradecanoylphorbol 13-acetate stimulated 15-kDa protein phosphorylation severalfold, producing approximately 70% of the maximal phosphorylation even in the absence of significant ionized Ca2+. The results are compatible with an intrinsic protein kinase C activity in sarcolemmal vesicles whose major substrate is the 15-kDa protein. PMID- 2997185 TI - An increase in prolyl-4-hydroxylase activity occurs during the retinoic acid induced differentiation of mouse teratocarcinoma stem cell lines F9 and P19. AB - Monolayer cultures of F9 teratocarcinoma stem cells and P19 stem cells differentiate into endoderm, and fibroblast-like cells, respectively, when treated with retinoic acid. We demonstrate that this differentiation is associated with a large increase (greater than 40-fold) in the activity of an enzyme, prolyl-4-hydroxylase, involved in the posttranslational modification of collagens. This large increase in prolyl-4-hydroxylase activity occurs between 42 and 72 h after retinoic acid addition, and is associated with an increased amount of immunoprecipitable prolyl hydroxylase enzyme. This enzyme should be a useful marker for certain differentiated cell types produced during differentiation of teratocarcinoma stem cell lines. PMID- 2997186 TI - The interaction of phosphorothioate analogues of ATP with phosphomevalonate kinase. Kinetic and 31P NMR studies. AB - The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S), adenosine 5'-O-(2-thiotriphosphate) (ATP beta S), and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) could act as substrates for phosphomevalonate kinase in the presence of Mg2+ and Cd2+ as activating divalent metal cations. The Sp diastereomer of ATP alpha S was the preferred substrate regardless of the metal ion used, consistent with the metal ion not binding to the alpha-phosphate. With ATP beta S, the Sp diastereomer was the preferred substrate with Mg2+, and the Rp diastereomer was the preferred substrate with Cd2+. The reversal of specificity establishes that the metal is chelated through the beta-phosphate in the active site of the phosphomevalonate kinase reaction. A comparison of the Vmax values as a function of substitution of oxygen by sulfur showed the order for Mg2+ to be: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Sp) greater than ATP gamma S greater than ATP beta S(Rp). With Cd2+ as the activating metal ion, the order was: ATP greater than ATP alpha S(Sp) greater than ATP alpha S(Rp) greater than ATP beta S(Rp) greater than ATP gamma S greater than ATP beta S(Sp). It is concluded that the chelate structure of metal ATP substrate in the phosphomevalonate kinase reaction is the delta, beta, gamma bidentate complex. 31P NMR measurements and radioassay with [2-14C] phosphomevalonate were used to measure the equilibrium of the reaction catalyzed by phosphomevalonate kinase with ATP and phosphorothioate analogues of ATP as the phosphoryl group donor. The order as a phosphate donor as determined by both methods in the phosphomevalonate kinase reaction is ATP beta S greater than ATP alpha S greater than ATP greater than ATP gamma S. Except for ATP gamma S, the equilibrium is shifted in the direction of formation of ADP alpha S and ADP beta S relative to ADP formation. Thus, ATP beta S rather than ATP would be effective for the synthesis of diphosphomevalonate. The phosphomevalonate kinase reaction could also be used to synthesize mevalonate 5-(2-thiodiphosphate) using ATP gamma S as the phosphoryl group donor. PMID- 2997187 TI - Characterization of a cyclic AMP-resistant Chinese hamster ovary cell mutant containing both wild-type and mutant species of type I regulatory subunit of cyclic AMP-dependent protein kinase. AB - We have characterized a cyclic AMP-resistant Chinese hamster ovary (CHO) cell mutant in which one of two major species of type I regulatory subunit (RI) of cyclic AMP-dependent protein kinase is altered. Wild-type CHO cell extracts contain two cyclic AMP-dependent protein kinase activities. As shown by DEAE cellulose chromatography, there is a peak of type I protein kinase activity in mutant extracts, but the type II protein kinase activity is considerably reduced even though free type II regulatory subunit (RII) is present. The type I kinase from the mutant has an altered RI (RI*) whose KD for the binding of 8-N3[32P] cAMP (KD = 1.3 X 10(-5) M) is increased by more than 200-fold compared to RI from the wild-type enzyme (KD = 5.5 X 10(-8) M). No differences were found between the catalytic subunits from the wild-type and mutant type I kinases. A large portion of RI in mutant and wild-type extracts is present in the free form. The RI* derived from mutant type I protein kinase shows altered labeling by 8-N3[32P]cAMP (KD = 1.3 X 10(-5) M) whereas the free RI from the mutant is labeled normally by the photoaffinity label (KD = 7.2 X 10(-8) M), suggesting that the RI* which binds to the catalytic subunit is functionally different from the free form of RI. The decreased amount of type II kinase activity in the mutant appears to be due to competition of RI* with RII for binding to the catalytic subunit. Translation of mRNA from wild-type CHO cells results in the synthesis of two different charge forms of RI, providing biochemical confirmation of two different species of RI in CHO cells. Additional biochemical evidence based on isoelectric focusing behavior of 8-N3[32P]cAMP-labeled RI species and [35S]methionine-labeled RI from mutant and wild-type extracts confirms the charge heterogeneity of RI species in CHO cells. These genetic and biochemical data taken together are consistent with the conclusion that there are at least two different species of RI present in CHO cells and that one of these species is altered in the mutant analyzed in this work. PMID- 2997189 TI - Effect of modification of lysine residues of fructose-6-phosphate 2 kinase:fructose-2,6-bisphosphatase with pyridoxal 5'-phosphate. AB - Inactivation of a bifunctional enzyme, fructose-6-P,2-kinase:fructose-2,6 bisphosphatase by pyridoxal 5'-P followed by reduction with NaBH4 was studied. Fructose-6-P,2-kinase is over 80% inactivated by 2 mM pyridoxal 5'-P. The stoichiometry of the pyridoxyl-P incorporation and the inactivation of the kinase follows a biphasic curve. The first P-pyridoxyl residue incorporated per protomer does not affect fructose-6-P,2-kinase, but the next two P-pyridoxyl incorporation/protomer results in 80% inactivation. The Km values for ATP and fructose-6-P of the enzymes containing varying amounts of P-pyridoxyl groups at intermediate levels of inactivation are not altered, but Vmax is decreased. Among the metabolites tested, only fructose-2,6-P2 and Mg-ATP are competitive with pyridoxal-P and protect the enzyme against the inactivation. Neither the activity nor the fructose-6-P inhibition of fructose-2,6-bisphosphatase is affected by the modification. The acid hydrolysate of the inactive P-[3H]pyridoxyl enzyme contained only [3H]pyridoxyl lysine. High performance liquid chromatography of tryptic peptides of phospho[3H]pyridoxyl enzymes reveals two peptides which were missing in the enzyme protected by fructose-2,6-P2 or ATP during the modification reaction. These peptides have been isolated, and their amino acid sequences have been determined as Asp-Gln-Asp-Lys-Tyr-Arg and Asp-Val-His-Lys-Tyr. Pyridoxal-P reacts specifically with two lysine residues at the fructose-2,6-P2-binding site of fructose-6-P,2-kinase but not that of fructose-2,6-bisphosphatase. The site may also overlap with the ATP-binding site. PMID- 2997188 TI - DNA-mediated gene transfer of a mutant regulatory subunit of cAMP-dependent protein kinase. AB - We have used DNA-mediated gene transfer of genomic DNA to introduce into wild type Chinese hamster ovary (CHO) cells a mutant gene that confers resistance to the growth inhibitory effect of cAMP. This dominant mutation in CHO cell line 10248 is responsible for an alteration in the RI subunit (RI*) of the type I cAMP dependent protein kinase (Singh, T. J., Hochman, J., Verna, R., Chapman, M., Abraham, I., Pastan, I.H., and Gottesman, M.M. (1985) J. Biol. Chem. 260, 13927 13933). The transformant 11564 which was studied in detail, has the same characteristics as the original mutant 10248 including continued growth in medium containing 8-Br-cAMP, an increase in the Ka for cAMP activation of the kinase, a greatly reduced amount of type II protein kinase activity, an altered incorporation of the photoaffinity label 8-N3[32P]cAMP into the RI* subunit of PKI, and an absence of cAMP-dependent phosphorylation of a Mr = 52,000 protein in intact cells. In addition, analysis of the DNA of the transformant indicates the presence of an increased amount of DNA for the RI gene. These results are consistent with the transfer of a mutant gene for the RI* subunit of the cAMP dependent protein kinase and its phenotypic expression in the transformant and also support the hypothesis that the mutation responsible for the defect in cell line 10248 is due to an alteration in the gene for RI. PMID- 2997190 TI - [17O]Water and nitric oxide binding by protocatechuate 4,5-dioxygenase and catechol 2,3-dioxygenase. Evidence for binding of exogenous ligands to the active site Fe2+ of extradiol dioxygenases. AB - Pseudomonas testosteroni protocatechuate 4,5-dioxygenase and Pseudomonas putida catechol 2,3-dioxygenase (metapyrocatechase) catalyze extradiol-type oxygenolytic cleavage of the aromatic ring of their substrates. The essential active site Fe2+ of each enzyme binds nitric oxide (NO) to produce an EPR active complex with an electronic spin of S = 3/2. Hyperfine broadening of the EPR resonances of the nitrosyl complexes by 17O-enriched H2O shows that water is bound directly to the Fe2+ in the native enzymes, but is apparently displaced in substrate complexes. NO is not displaced by either substrates or inhibitors. The EPR spectra of several enzyme-inhibitor-NO complexes are different from those of enzyme-NO or enzyme-substrate-NO complexes and are found to be broadened by 17O-enriched water. The data show that at least 2 and perhaps 3 sites in the Fe ligation can be occupied by exogenous ligands. Furthermore, it is likely that substrates and inhibitors displace water by binding either at or near to the Fe in the nitrosyl complex. Nitric oxide binding is found to be substrate-dependent for each enzyme. Native catechol 2,3-dioxygenase exhibits KD values of 190 microM and 2.0 mM for NO binding in two types of independent sites. Only one type of site is observed in the catechol complex which exhibits a KD for NO of 3.4 microM. One type of NO binding site is observed for both the native and substrate complexed protocatechuate 4,5-dioxygenase with KD values of 360 and 3 microM, respectively. The presence of a specific site in the Fe coordination for NO which is modified in the substrate complex, suggests that O2 binding by the extradiol dioxygenases may also occur at the Fe. PMID- 2997191 TI - Stimulation of the T3-T cell receptor-associated Ca2+ influx enhances the activity of the Na+/H+ exchanger in a leukemic human T cell line. AB - Three monoclonal antibodies reactive with different structural domains of the T3 T cell receptor complex of the human T cell leukemia line, HPB-ALL, were previously shown to activate a membrane potential-sensitive, La3+-inhibitable Ca2+ influx (Oettgen, H. C., Terhorst, C., Cantley, L. C., and Rosoff, P. M. (1985) Cell 40, 583-590). OKT3 (anti-T3), WT-31 (anti-receptor constant region), and T40/25 (anti-receptor variable region) also enhance the activity of the Na+/H+ exchanger in these cells. The associated rise in pHi was dependent on the presence of external Ca2+ and Na+, was inhibited by dimethylamiloride and La3+, and was maintained for at least 20 min. Phorbol esters, which are co-mitogenic in T cells and activate protein kinase C, also stimulated the exchanger, but by a mechanism not requiring an elevation in cytoplasmic Ca2+; the rise in pHi rapidly peaked and returned to baseline levels within 20 min. Pretreatment with phorbols prevented an increase in pHi by OKT3 although a transient additive effect was observed when the two were added simultaneously. Receptor function was maintained in the presence of phorbol esters as OKT3 still stimulated a Ca2+ influx. These data demonstrate the existence of two interdependent pathways to activate Na+/H+ exchange in T lymphocytes and suggest a pathway of internal regulation of antigen activated signal transduction. PMID- 2997192 TI - 7Li, 31P, and 1H NMR studies of interactions between ATP, monovalent cations, and divalent cation sites on rabbit muscle pyruvate kinase. AB - The interactions between ATP, monovalent cations, and divalent cations on rabbit muscle pyruvate kinase have been examined using 7Li, 31P, and 1H nuclear magnetic resonance. Water proton nuclear relaxation studies are consistent with the binding of Li+ to the K+ site on pyruvate kinase with an affinity of 120 mM in the absence of substrates and 16 mM in the presence of P-enolpyruvate. Titrations with pyruvate demonstrate that pyruvate binds to the enzyme with an affinity of 0.65 mM in the presence of Li+ and 0.4 mM in the presence of K+. 7Li+ nuclear relaxation rates in solutions of pyruvate kinase are increased upon titration with the metal-nucleotide analogue, Cr(H2O)4ATP. Mn2+ EPR spectra were used to determined the distribution of the enzyme between the so-called isotropic and anisotropic conformations of the enzyme (Ash, D. E., Kayne, F., and Reed, G.H. Arch. Biochem. Biophys. (1978) 190, 571-577). Li-Cr distances of 5.6 and 11.0 A were calculated for the anisotropic and isotropic forms, respectively, in the absence or presence of pyruvate. When the divalent cation site on the enzyme was saturated with Mg2+, these distances increased to 6.7 and 9.5 A, respectively, regardless of the presence or absence of pyruvate. 31P nuclear relaxation studies with the diamagnetic metal-nucleotide analogue, Co(NH3)4ATP, indicated that addition of Mn2+ ion to the divalent cation site on the enzyme increased the longitudinal relaxation rates of all three phosphorus nuclei of the analogue. The 31P data indicate that the presence of pyruvate at the active site effects a decrease in the Mn-P distances, bringing Mn2+ and Co(NH3)4ATP closer together at the active site. The data also permit an evaluation of the role of the metal coordinated to the beta-P and gamma-P of ATP at the active site. PMID- 2997193 TI - Sequence of gene and cDNA encoding murine major histocompatibility complex class II gene A beta 2. AB - The murine major histocompatibility complex is known to express two class II molecules, A and E. They are composed of one alpha- and one beta-polypeptide chain. Recently, we reported the finding of an additional beta-chain second domain exon tentatively designated A beta 2. We describe here the nucleotide sequence of the A beta 2 gene and an A beta 2 cDNA clone. A beta 2 displays the same exon organization as the known expressed beta-chain genes, and the predicted A beta 2 polypeptide shows all the characteristic features of the expressed beta chains. The predicted A beta 2 polypeptide shows 49-56% amino acid sequence identity to A beta and E beta chains and to the human DP, DQ, and DR beta-chains. These are 63-79% homologous to each other, indicating that A beta 2 is the most divergent member of the beta-chain family. The A beta 2 gene seems to be transcribed in the same types of cells as other class II genes. Detection of incompletely spliced A beta 2 mRNA and the finding of a cDNA clone containing an intron segment suggest that A beta 2 transcripts are processed slowly. Hybridizations with A beta 2 probes to restriction enzyme-digested genomic DNA indicate that the A beta 2 gene displays lower allelic polymorphism than the A beta gene. PMID- 2997194 TI - [3H]Ethylpropylamiloride, a ligand to analyze the properties of the Na+/H+ exchange system in the membranes of normal and hypertrophied kidneys. AB - [3H]Ethylpropylamiloride is a useful radioactive label to identify the Na+/H+ exchange system (Vigne, P., Frelin, C., Audinot, M., Borsotto, M., Cragoe, E. J., and Lazdunski, M. (1984) EMBO J. 3, 2647-2651). This paper extends the analysis of the properties of interaction of [3H]ethylpropylamiloride with the exchanger and describes its use with hypertrophied kidneys. [3H]Ethylpropylamiloride binding sites copurify with the luminal membrane marker alkaline phosphatase but not with the basolateral membrane marker (Na+,K+)ATPase, thus indicating an asymmetric distribution of the Na+/H+ exchanger. Specific [3H]ethylpropylamiloride binding is dependent on pH. The pH dependency indicates that an ionizable function with a pKapp of 7.0 is essential in the association of the amiloride derivative. H+ acts competitively on [3H]ethylpropylamiloride binding; Na+, Li+, or cholinium ions have no effect on the association. Compensatory adaptation of the kidney to chronic reduction of renal mass is accompanied by a 1.7-fold increase in the activity of the Na+/H+ exchange system. Properties of interaction of internal and external pH with the Na+/H+ exchanger of normal and hypertrophied kidneys are identical. Titration of [3H]ethylpropylamiloride-binding sites in normal and hypertrophied kidneys suggests that the increased activity of the Na+/H+ exchange system is not accompanied by an increased concentration of exchangers. PMID- 2997196 TI - Functional activation of beta-adrenergic receptors by thiols in the presence or absence of agonists. AB - Treatment of beta-adrenergic receptor with dithiothreitol (DTT) or other thiol compounds caused its functional activation in the presence or absence of agonist ligands. Such activation was observed in reconstituted unilamellar phospholipid vesicles that contained beta-adrenergic receptors, purified to greater than or equal to 95% homogeneity from turkey erythrocyte plasma membranes, and the stimulatory GTP-binding protein of the adenylate cyclase system (Gs) purified from rabbit liver. Incubation of the vesicles with 2-10 mM DTT at 0 degrees C for 1 h increased the rate (4-5-fold) and the extent (3-4-fold) of activation of Gs by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding, an effect about equivalent to the addition of beta-adrenergic agonists. Treatment with DTT also markedly potentiated the ability of agonists to stimulate GTP gamma S binding, increasing the initial rate about 10-fold. DTT treatment was as effective as agonist in stimulating GTPase activity, and maximal stimulation was obtained when DTT-treated vesicles were assayed in the presence of agonist. Other thiol compounds produced effects similar to those of DTT but were at least 10-fold less potent. Stimulation of GTP gamma S binding or GTPase activity required active receptor, and treatment of the receptor with DTT prior to reconstitution also increased its efficacy. There was no effect of DTT on Gs alone. Thus, the site of action of DTT appears to be on the beta-adrenergic receptor itself, and the reduction of disulfides and the binding of agonist act synergistically to activate the receptor. DTT treatment made the receptor more labile to thermal denaturation. Inclusion of cholesterol or cholesteryl-hemisuccinate (5-25%) in the vesicles protected the reduced receptor against such denaturation and enhanced its recovery during reconstitution. No effect of cholesterol or cholesteryl-hemisuccinate was observed on the stability of the nonreduced receptor, which was comparable to that observed in native membranes. PMID- 2997195 TI - Production of 10-oxo-19-nor-25-hydroxyvitamin D3 by solubilized kidney mitochondria from chick and rat. AB - Cholate-solubilized chick kidney mitochondria that 1-hydroxylated 25 hydroxyvitamin-D3 (25-OH-D3) upon reconstitution also produced 10-oxo-19-nor-25 OH-D3, which co-eluted with 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) on normal phase high performance liquid chromatography (HPLC) with hexane:propanol-2 (9:1), the traditional chromatographic system for isolating 1,25-(OH)2-D3. The 10-oxo derivative was separated from 1,25-(OH)2-D3 by normal phase HPLC with dichloromethane:propanol-2 (19:1) or by reverse phase HPLC with methanol:water (4:1). Unlike 1,25-(OH)2-D3 production, formation of 10-oxo-19-nor-25-OH-D3 did not require a source of reducing equivalents and was blocked by the antioxidants, diphenyl-rho-phenylenediamine, and butylated hydroxytoluene, implicating a free radical or peroxidative synthetic mechanism. Rat kidney mitochondria solubilized with cholate or with cholate and Emulgen 911 produced 10-oxo-19-nor-25-OH-D3 but no detectable 1 alpha,25-(OH)2-D3. These results stress the importance of careful identification of vitamin D metabolites produced in vitro and suggest the use of alternate chromatographic conditions for isolating 1,25-(OH)2-D3 or inclusion of antioxidants in the assay of solubilized 1 alpha-hydroxylase to eliminate contamination of 1,25-dihydroxyvitamin D3 with 10-oxo-19-nor-25-OH-D3. PMID- 2997197 TI - Phorbol ester and 1-oleoyl-2-acetylglycerol inhibit angiotensin activation of phospholipase C in cultured vascular smooth muscle cells. AB - Angiotensin II acts on cultured rat aortic vascular smooth muscle cells (VSMC) to induce the rapid, phospholipase C-mediated generation of inositol trisphosphate from phosphatidylinositol 4,5-bisphosphate and mobilization of intracellular Ca2+. sn-1,2-Diacylglycerol, the other major product of inositol phospholipid breakdown, is known to activate protein kinase C, but its role in angiotensin II action on VSMC has not been defined. We report herein that, in cultured VSMC prelabeled with [3H]myoinositol, brief incubations (2-5 min) with 4 beta-phorbol 12-myristate 13-acetate (PMA) (1-100 nM) or 1-oleoyl-2-acetylglycerol (10-100 microM), two potent activators of protein kinase C, inhibit subsequent angiotensin II (100 nM)-induced increases in phosphatidylinositol 4,5 bisphosphate breakdown and inositol trisphosphate formation. In addition, pretreatment of VSMC with either PMA (IC50 approximately 1 nM) or 1-oleoyl-2 acetylglycerol (IC50 approximately 7.5 microM) also markedly inhibits angiotensin II (1 nM)-stimulated increases in cytosolic free Ca2+, as measured with the calcium-sensitive fluorescent indicator quin 2, or 45Ca2+ efflux. Neither PMA nor 1-oleoyl-2-acetylglycerol initiated phosphatidylinositol 4,5-bisphosphate breakdown or Ca2+ flux by itself. PMA treatment (10 nM, 5 min) did not influence the number or affinity of 125I-angiotensin II-binding sites in intact cells. These data suggest that one function of angiotensin II-generated sn-1,2 diacylglycerol in vascular smooth muscle may be to modulate, by protein kinase C mediated mechanisms, angiotensin II receptor coupling to phospholipase C. PMID- 2997198 TI - Cyclic AMP and Ca2+-activated K+ transport in a human colonic epithelial cell line. AB - Addition of either vasoactive intestinal peptide (VIP) or the Ca2+ ionophore, A23187, to confluent monolayers of the T84 epithelial cell line derived from a human colon carcinoma increased the rate of 86Rb+ or 42K+ efflux from preloaded cells. Stimulation of the rate of efflux by VIP and A23187 still occurred in the presence of ouabain and bumetanide, inhibitors of the Na+,K+-ATPase and Na+,K+,Cl cotransport, respectively. The effect of A23187 required extracellular Ca2+, while that of VIP correlated with its known effect on cyclic AMP production. Other agents which increased cyclic AMP production or mimicked its effect also increased 86Rb+ efflux. VIP- or A23187-stimulated efflux was inhibited by 5 mM Ba2+ or 1 mM quinidine, but not by 20 mM tetraethylammonium, 4 mM 4 aminopyridine, or 1 microM apamin. Under appropriate conditions, VIP and A23187 also increased the rate of 86Rb+ or 42K+ uptake. Stimulation of the initial rate of uptake by either agent required high intracellular K+ and was not markedly affected by the imposition of transcellular pH gradients. The effect of A23187, but not VIP or dibutyryl cyclic AMP, was refractory to depletion of cellular energy stores. A23187-stimulated uptake was not significantly affected by anion substitution, however, stimulation of uptake by VIP required the presence of a permeant anion. This result may be due to the simultaneous activation of a cyclic AMP-dependent Cl- transport system. The kinetics of both VIP- and A23187 stimulated uptake and efflux were consistent with a channel-rather than a carrier mediated K+ transport mechanism. The results also suggest that cyclic AMP and Ca2+ may activate two different kinds of K+ transport systems. Finally, both transport systems have been localized to the basolateral membrane of T84 monolayers, a result compatible with their possible regulatory role in hormone activated electrogenic Cl- secretion. PMID- 2997199 TI - Fructose-1,6-bisphosphatase from rat liver. A comparison of the kinetics of the unphosphorylated enzyme and the enzyme phosphorylated by cyclic AMP-dependent protein kinase. AB - A purification procedure for rat hepatic fructose-1,6-bisphosphatase, described earlier, has been improved, resulting in an enzyme preparation with a neutral pH optimum and with both phosphorylatable serine residues present. The subunit Mr was 40,000. Phosphorylation in vitro with cyclic AMP-dependent protein kinase resulted in the incorporation of 1.4 mol of phosphate/mol of subunit and led to an almost 2-fold decrease in apparent Km for fructose-1,6-bisphosphate. In contrast to yeast fructose-1,6-bisphosphatase, fructose-2,6-bisphosphate had no effect on the rate of phosphorylation or dephosphorylation of the intact enzyme. The effects of the composition of the assay medium, with regard to buffering substance and Mg2+ concentration, on the apparent Km values of phosphorylated and unphosphorylated enzyme were investigated. The kinetics of phosphorylated and unphosphorylated fructose-1,6-bisphosphatase were studied with special reference to the inhibitory effects of adenine nucleotides and fructose-2,6-bisphosphate. Unphosphorylated fructose-1,6-bisphosphatase was more susceptible to inhibition by both AMP and fructose 2,6-bisphosphate than phosphorylated enzyme, at high and low substrate concentrations. Both ATP and ADP had a similar effect on the two enzyme forms, ADP being the more potent inhibitor. Finally, the combined effect of several inhibitors at physiological concentrations was studied. Under conditions resembling the gluconeogenic state, phosphorylated fructose-1,6 bisphosphatase was found to have twice the activity of the unphosphorylated enzyme. PMID- 2997200 TI - Appearance of specific leukotriene B4 binding sites in myeloid differentiated HL 60 cells. AB - Exposure of HL-60 cells for 6 days to a combination of 1.25% (v/v) dimethyl sulfoxide and 10 microM dexamethasone induces myeloid differentiation which results in a cell with many of the characteristics of a mature granulocyte. At 4 degrees C myeloid differentiated, but not undifferentiated, monocytic differentiated or eosinophilic differentiated HL-60 cells display marked specific leukotriene B4 binding. Leukotriene B4 binding at 4 degrees C reaches a maximum within 10 min, is readily reversed by unlabeled leukotriene B4, and is stereospecific. Only molecules with structural and biological similarity to leukotriene B4 can competitively inhibit leukotriene B4 binding. Scatchard analysis at 4 degrees C in differentiated cells shows two classes of binding sites. The high affinity sites have a Kd of 0.27 nM and a Bmax of 14.8 fmol/10(7) cells; the low affinity sites have a Kd of 0.58 microM and a Bmax of 2453 fmol/10(7) cells. The appearance of specific leukotriene B4 binding sites in the myeloid differentiated cells correlates with their ability to chemotax in response to leukotriene B4. Undifferentiated cells do not chemotax to leukotriene B4. At 37 degrees C leukotriene B4 is incorporated into phospholipid and triglyceride species in both undifferentiated and myeloid differentiated HL-60 cells making binding studies at 37 degrees C in intact cells impossible. No evidence of omega-hydroxylase activity was found in HL-60 cells. These data suggest that the HL-60 cell may be an excellent model system for the study of leukotriene B4 receptor binding, processing, and gene expression. PMID- 2997201 TI - The 1,4-dihydropyridine receptor associated with the skeletal muscle voltage dependent Ca2+ channel. Purification and subunit composition. AB - The dihydropyridine receptor associated with the voltage-dependent Ca2+ channel from rabbit skeletal muscle has been purified using the tritiated derivative of (+)-PN 200-110. The drug was used not only as a marker associated with the solubilized receptor but also in direct binding experiments performed after each purification step. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilization of a microsomal preparation resulted in an extract with a specific binding activity of 10 pmol/mg of protein. A combination of chromatographic steps utilizing anion exchange, lectin affinity, and gel filtration resulted in an 80 fold purification to a specific binding activity of 800 pmol/mg of protein. The affinity of (+)-[3H]PN 200-110 for the solubilized receptor was only slightly altered after the purification procedure. The KD values were 0.7 and 1.8 nM on the starting material and the most purified fractions, respectively. The subunit composition of the dihydropyridine receptor was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was consistent with three polypeptides of Mr 142,000, 33,000, and 32,000. The last two small components were not covalently associated with the larger one. In spite of a careful investigation of the conditions which improved the stability of the dihydropyridine receptor, a partial denaturation could not be prevented during purification. This resulted in an underestimation of receptor purity when calculated from the maximal specific binding activity as compared to the enrichment in the three polypeptides observed after polyacrylamide gel electrophoresis. Finally, application of the same purification procedure to solubilized microsomal preparations of chick and frog skeletal muscle demonstrated the presence of a large polypeptide component of Mr 135,000-141,000 associated with the Ca2+ channel from these sources. The doublet of small molecular weight was not found with the frog muscle. PMID- 2997202 TI - The subunit structure of the follitropin (FSH) receptor. Photoaffinity labeling of the membrane-bound receptor follitropin complex in situ. AB - Human follicle-stimulating hormone (hFSH) was acylated with N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) and radioiodinated (55 microCi/micrograms) for use as a photoaffinity probe to investigate the subunit structure of the FSH receptor in calf testis. After incubation with the photoaffinity probe and photolysis with UV light, the cross-linked hormone-receptor complex was solubilized from the membrane and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Autoradiography of the polyacrylamide gels revealed two major bands, 64 kDa and 84 kDa. These were equivalent in molecular mass to those observed in a previous study (Branca, A. A., Sluss, P. M., Smith, A. A., and Reichert, L. E., Jr. (1985) J. Biol. Chem. 260, 9988-9993) in which performed hormone-receptor complexes were solubilized with detergent prior to formation of covalent cross-linkages through the use of homobifunctional cross-linking reagents. Reduction with dithiothreitol resulted in the loss of radioactivity from the 84-kDa band with a concomitant increase in the intensity of the 64-kDa band. Since dithiothreitol increases the dissociation of intact radioiodinated azidobenzoyl-FSH into subunits, it is suggested that the conversion of the 84-kDa band to the 64-kDa band by dithiothreitol is due to the loss of non-cross-linked hFSH subunit from the 84 kDa band and that the two bands observed after photoaffinity labeling arise from covalent bond formation between hFSH and a receptor subunit having a relative molecular weight (Mr) of 48,000. In addition to the predominant photolabeling of the receptor to yield the 64-kDa and 84-kDa bands, several other, less intense bands (54 kDa, 76 kDa, 97 kDa, and 116 kDa) were also consistently observed on autoradiographs. The appearance of all bands, however, was inhibited by the inclusion of unlabeled hFSH in the initial binding incubation mixtures. The results of this study indicate that the calf testis FSH receptor has a multimeric structure containing at least one 48-kDa subunit and suggest the presence of other nonidentical receptor subunit proteins. PMID- 2997203 TI - D1 protein of Drosophila melanogaster. Purification and AT-DNA binding properties. AB - D1 protein of Drosophila melanogaster is a sequence-specific DNA-binding protein which recognizes AT-rich DNA sequences. AT-rich DNA sequences in eukaryotic organisms are distributed in two characteristic ways: flanking transcriptional units and in constitutive heterochromatin. D1 could play a role in regulation of gene expression and in geographical localization of DNA sequences within the nucleus. D1 has been partially purified by ion exchange chromatography. DNA binding activity was investigated by nucleoprotein gel electrophoresis, using end labeled restriction fragments varying in AT sequence content. D1 binds most tightly to the satellite sequence -AATAT-, with intermediate strength to the complex satellite (359-base pair repeat) and another AT-rich (68% AT) mixed sequence DNA, and least to the simple satellite sequence -AAGAG-. PMID- 2997204 TI - Purification and characterization of a high molecular weight phosphoprotein phosphatase from rabbit liver. AB - A high molecular weight phosphoprotein phosphatase was purified from rabbit liver using high speed centrifugation, acid precipitation, ammonium sulfate fractionation, chromatography on DEAE-cellulose, Sepharose-histone, and Bio-Gel A 0.5m. The purified enzyme showed a single band on a nondenaturing polyacrylamide anionic disc gel which was associated with the enzyme activity. The enzyme was made up of equimolar concentrations of two subunits whose molecular weights were 58,000 (range 58,000-62,000) and 35,000 (range 35,000-38,000). Two other polypeptides (Mr 76,000 and 27,000) were also closely associated with our enzyme preparation, but their roles, if any, in phosphatase activity are not known. The optimum pH for the reaction was 7.5-8.0. Km value of phosphoprotein phosphatase for phosphorylase a was 0.10-0.12 mg/ml. Freezing and thawing of the enzyme in the presence of 0.2 M beta-mercaptoethanol caused an activation (100-140%) of phosphatase activity with a concomitant partial dissociation of the enzyme into a Mr 35,000 catalytic subunit. Divalent cations (Mg2+, Mn2+, and Co2+) and EDTA were inhibitory at concentrations higher than 1 mM. Spermine and spermidine were also found to be inhibitory at 1 mM concentrations. The enzyme was inhibited by nucleotides (ATP, ADP, AMP), PPi, Pi, and NaF; the degree of inhibition was different with each compound and was dependent on their concentrations employed in the assay. Among various types of histones examined, maximum activation of phosphoprotein phosphatase activity was observed with type III and type V histone (Sigma). Further studies with type III histone indicated that it increased both the Km for phosphorylase a and the Vmax of the dephosphorylation reaction. Purified liver phosphatase, in addition to the dephosphorylation of phosphorylase a, also catalyzed the dephosphorylation of 32P-labeled phosphorylase kinase, myosin light chain, myosin, histone III-S, and myelin basic protein. The effects of Mn2+, KCl, and histone III-S on phosphatase activity were variable depending on the substrate used. PMID- 2997205 TI - Interactions between spinach ferredoxin-nitrite reductase and its substrates. Evidence for the specificity of ferredoxin. AB - Reduced ferredoxin can serve as electron donor in the 6-electron reduction of nitrite to ammonia catalyzed by spinach nitrite reductase. We have examined interactions between nitrite reductase and its substrates, ferredoxin and nitrite, with emphasis upon protein-protein interactions between ferredoxin and nitrite reductase. Ferredoxin, of the proteins tested, is the most effective in retarding low ionic strength inactivation of nitrite reductase. The interaction appears to be electrostatic, and the apparent Kd, calculated from the concentration dependence of ferredoxin protection, is about 1 microM in 2 mM Tris. Chemical modification of carboxyl residues of ferredoxin resulting in a change of charge reduces its reactivity with both ferredoxin:NADP+ oxidoreductase and nitrite reductase, indicating the importance of charge-charge interactions. Cross-linking studies provided no evidence for a ternary complex containing the oxidoreductase and nitrite reductase but indicated that the two enzymes will compete for ferredoxin, possibly using the same site (or overlapping sites) on the ferredoxin. A complex containing ferredoxin:NADP+ oxidoreductase, ferredoxin, and cytochrome c was detected, indicating that ferredoxin has different binding sites for cytochrome c and ferredoxin:NADP+ oxidoreductase. Active cross-linked complexes of ferredoxin and nitrite reductase were obtained and were less sensitive to low ionic strength inactivation than free reductase and had decreased ferredoxin-supported nitrite reductase activity. The evidence presented of protein-protein interactions between ferredoxin and nitrite reductase indicates that ferredoxin is indeed the specific physiological electron donor to the reductase. PMID- 2997206 TI - Transient expression of human neutral alpha-glucosidase AB (glucosidase II) in enzyme-deficient mouse lymphoma cells. AB - To define new methods for gene isolation exploiting mutant mammalian cells we transformed a mutant mouse cell line deficient in glucosidase II with total human genomic DNA and detected transient expression of the human glucosidase II gene. Maximum gene expression was detected 48 h after addition of DNA as a 2.5-fold increase in neutral alpha-glucosidase activity (2.47 +/- 0.15, n = 4). When mutant mouse DNA was used for transformation, no increase in enzyme activity was seen. The increased enzyme activity was due to expression of the human gene product. Thus, by rocket immunoelectrophoresis, cells transformed with human DNA yielded a "rocket" which reacted with antibody to human but not to mouse glucosidase II and which hydrolyzed substrate in situ. Specific DNA sequences were required for expression of the enzyme activity, since digestion of DNA with EcoRI and SstI rendered the DNA ineffective for eliciting expression of the enzyme, while digestion of DNA with BamHI and XhoI did not affect the increase. Transfection with intact phage from a human genomic DNA library also resulted in transient expression of the human gene. These results demonstrate the feasibility of detecting, by enzymatic assay, transient expression of a human gene for an intracellular enzyme following DNA-mediated transformation both with total human DNA and with intact phage from a human recombinant library. This system could be used as an assay for isolation of a gene from a genomic library by sibling selection. PMID- 2997207 TI - Sequence-specific BamHI endonuclease. The proposed role of arginine residues in substrate binding and recognition. AB - Arginyl residues in BamHI endonuclease were examined because of their alleged role in proteins that contain nucleotide- or phosphate-binding sites. Butanedione, an arginine-specific reagent, inhibited the endonuclease in the presence of sodium borate. The inhibition was decreased by preliminary incubation of the enzyme with DNA or competitive inhibitors which were the 5'-phosphoryl deoxydinucleotide subsets of the BamHI recognition sequence. The dinucleotide pdGpdG protected the enzyme most efficiently against the butanedione modification. Dinucleotides that were unrelated to the recognition sequence failed to protect the enzyme from inactivation. These studies indicate that arginine residues may reside in the enzyme's active site and might function in the sequence-specific recognition of the BamHI palindrome. PMID- 2997208 TI - Vesicular stomatitis virus produced from infected LSTRA lymphoma cells bear tyrosine protein kinase activity (p56). AB - Murine LSTRA lymphoma cells contain a very active tyrosine protein kinase of 56 kDa (p56) which is not related to any of the other known tyrosine kinases. In the past the purification and characterization of the p56 have been hampered because of the low amount of this protein in LSTRA membranes. In this study, we have utilized a different approach for purification which consisted of trapping the protein in the membrane of vesicular stomatitis virus. Incubation of the virions with [gamma-32P]ATP resulted in the phosphorylation of p56 on tyrosine residues. Moreover, the phosphopeptide digest profile of vesicular stomatitis virus-p56 was identical to that observed with authentic LSTRA-p56. The p56 from such virions could be resolved from other proteins by two-dimensional gels, and furthermore, such virions have been used to prepare several antisera directed against the p56. PMID- 2997209 TI - Role of a guanine nucleotide-binding regulatory protein in the hydrolysis of hepatocyte phosphatidylinositol 4,5-bisphosphate by calcium-mobilizing hormones and the control of cell calcium. Studies utilizing aluminum fluoride. AB - Treatment of isolated hepatocytes with NaF produced a concentration-dependent activation of phosphorylase, inactivation of glycogen synthase, efflux of Ca2+, rise in cytosolic free Ca2+ ([Ca2+]i), increase in myo-inositol-1,4,5,-P3 levels, decrease in phosphatidylinositol-4,5-P2 levels, and increase in 1,2 diacylglycerol levels. These changes were evident within 1 min and maximum at 2-5 min. Maximum effects on Ca2+ efflux, [Ca2+]i, glycogen synthase, and phosphorylase were observed with 15 mM NaF, whereas myo-inositol-1,4,5-P3 and 1,2 diacylglycerol levels were maximally stimulated by 50 mM NaF. The levels of intracellular cAMP were decreased by NaF (up to 10 mM) in the absence or presence of glucagon (0.1-1 nM) or forskolin (2 microM). The effects of low doses of NaF (2-15 mM) to inhibit basal or glucagon-stimulated cAMP accumulation, mobilize Ca2+, activate phosphorylase, and inactivate glycogen synthase were all potentiated by AlCl3. This potentiation was abolished by the Al3+ chelator deferoxamine. These results illustrate that AlF4- can mimic the effects of Ca2+ mobilizing hormones in hepatocytes and suggest that the coupling of the receptors for these hormones to the hydrolysis of phosphatidylinositol-4,5-P2 to myo inositol 1,4,5-P3 is through a guanine nucleotide-binding regulatory protein. This is because AlF4- is known to modulate the activity of other guanine nucleotide regulatory proteins (Ni, Ns, and transducin). PMID- 2997210 TI - Mechanisms of inhibition of (Na,K)-ATPase by hydrostatic pressure studied with fluorescent probes. AB - To investigate the mechanisms by which hydrostatic pressure inhibits (Na,K) ATPase, we measured enzyme activity, as a function of pressure and temperature, of purified (Na,K)-ATPase from dog kidney and eel electroplax, and we monitored protein conformation, possible subunit interactions, and the fluidity of the membrane with fluorescent probes. The (Na,K)-ATPase and p-nitrophenylphosphatase activities were inhibited reversibly by pressures below 1.5 kilobars (eel enzyme) and 2.5 kilobars (dog kidney enzyme). Above these pressures, the enzymes were inactivated irreversibly. The plots of 1n(activity) versus pressure were curvilinear; this indicates that the reversible inhibition by pressure involves two or more rate-limiting steps. The calculated activation volumes varied with temperature and pressure and were larger for the (Na,K)-ATPase activity compared to the p-nitrophenylphosphatase activity. The fluorescence polarization of three hydrophobic probes decreased with increasing temperature (10-36 degrees C) and increased with increasing pressure (10(-3)-1.5 kilobars), reversibly, without any evidence of a lipid phase transition. Plots of enzyme activity versus fluorescence polarization of the lipid probes showed an inverse relationship; this indicates that enzyme activity was directly related to the fluidity of the membrane as measured by the lipid probes. Pressure had no effect on the fluorescence polarization of two cardiac glycoside probes nor on the efficiency of resonance energy transfer between either donor and acceptor cardiac glycosides specifically bound to the ouabain sites of different alpha-subunits, or tryptophan and the bound cardiac glycoside probe. These results suggest that dissociation of dimeric alpha-subunits is not related to the inhibition by pressure, and that the cardiac glycoside-complexed enzyme conformation is stabilized by pressure. It is concluded that increased pressure decreases the membrane fluidity which hinders conformational transitions associated with rate limiting steps of the (Na,K)-ATPase reaction. It is proposed that ion-bound or occluded forms of (Na,K)-ATPase are stabilized by pressure because they occupy a smaller volume. PMID- 2997211 TI - Characterization of a potent catenation activity of HeLa cell nuclei. AB - Using an assay which measures catenation of a supercoiled DNA template, we have characterized and quantitated a potent activity identified in crude fractions of HeLa cell nuclei. Catenation requires Mg-ATP and a DNA-condensing agent, polyvinyl alcohol. A filter-binding or agarose gel assay can be used to quantitate activity. In this reaction, DNA topoisomerase I relaxes the input supercoiled DNA to provide DNA topoisomerase II, a strongly favored template for catenation. DNA topoisomerase II preferentially catenates relaxed DNA over supercoiled DNA by a factor of 100. One molecule of DNA topoisomerase II is able to catenate about 20 circles of relaxed DNA/min at 30 degrees C but only 0.16 circle of supercoiled DNA/min at 30 degrees C. The purified HeLa topoisomerase I can also catenate DNA under these assay conditions, yet in an ATP-independent fashion. It is much less efficient than topoisomerase II; one molecule of topoisomerase I catenates only about 3.8 X 10(-3) molecules of supercoiled DNA/min at 30 degrees C with a DNA template containing 5% nicked circles. This remarkable difference between the two enzymes allows quantitation of DNA topoisomerase II activity seen in the presence of excess topoisomerase I. Unlike Escherichia coli topoisomerase I (omega), catenation by the HeLa topoisomerase I is not stimulated by gapped circles. PMID- 2997212 TI - The structure of two fast-white myosin heavy chain promoters. A comparative study. AB - Two complete myosin heavy chain genes were isolated from chicken genomic libraries, and shown to code for fast-white isoforms. Isoform specific probes were developed from the 5' nontranslated regions of the two genes and used to identify the developmental stages at which each of the genes are expressed. One of the genes is transcribed in the embryo and the other only in the adult. The 5' flanking regions of the two genes were sequenced along with the first three exons. The 5' untranslated sequences in both genes are not contiguous, one intron is present in the adult gene while the embryonic gene contains two. The promoters of both genes contain the conserved CAAT and TATA box elements observed in other eucaryotic genes. A computer assisted comparison was performed on the two genes at the nucleotide and amino acid levels. No homology could be detected in the 5' flanking regions of the genes except in and around the CAAT and TATA elements, however, structural sequences at the 5' ends were highly conserved as well as the position of the first three introns. The amino acids in and around the ATP binding site are completely conserved between the two isoforms. PMID- 2997213 TI - Autophosphorylation and protein kinase C phosphorylation of the epidermal growth factor receptor. Effect on tyrosine kinase activity and ligand binding affinity. AB - The effect of autophosphorylation and protein kinase C-catalyzed phosphorylation on the tyrosine-protein kinase activity and ligand binding affinity of the epidermal growth factor (EGF) receptor has been studied. Kinetic parameters for the phosphorylation by the receptor kinase of synthetic peptide substrates having sequences related to the 3 in vitro receptor autophosphorylation sites (tyrosine residues 1173 (P1), 1148 (P2), and 1068 (P3)) were measured. The Km of peptide P1 (residues 1164-1176) was significantly lower than that for peptides P2 (residues 1141-1151) or P3 (residues 1059-1072). The tyrosine residue 1173 was also the most rapidly autophosphorylated in purified receptor preparations, consistent with previous observations for the receptor in intact cells (Downward, J., Parker, P., and Waterfield, M. D. (1984) Nature 311, 483-485). Variation in the extent of receptor autophosphorylation from 0.1 to 2.8 mol of phosphate/mol of receptor did not influence kinase activity or EGF binding affinity either for purified receptor or receptor in membrane preparations. Phosphorylation of the EGF receptor by protein kinase C was shown to cause a 3-fold decrease in the affinity of purified EGF receptor for EGF and to reduce the receptor kinase activity. In membrane preparations, phosphorylation of the EGF receptor by protein kinase C resulted in conversion of high affinity EGF binding sites to a low affinity state. This suggests that activation of protein kinase C by certain growth promoting agents and tumor promoters is directly responsible for modulation of the affinity of the EGF receptor for its ligand EGF. The regulation of the EGF receptor function by protein kinase C is discussed. PMID- 2997214 TI - Properties of beta-adrenergic receptors of cultured mammalian cells. Interaction of receptors with a guanine nucleotide-binding protein in membranes prepared from L6 myoblasts and from wild-type and cyc- S49 lymphoma cells. AB - The radiolabeled agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) and antagonist [125I]iodopindolol ([125I]IPIN) were used to investigate the properties of beta adrenergic receptors on membranes prepared from L6 myoblasts and S49 lymphoma cells. The high affinity binding of (-)-[3H]HBI to membranes prepared from L6 myoblasts was stereoselectively inhibited by the active isomers of isoproterenol and propranolol. The density of receptors determined with (-)-[3H]HBI was less than that determined with [125I]IPIN. The binding of (-)-[3H]HBI was inhibited by guanine nucleotides, suggesting an agonist-mediated association of the receptor with a guanine nucleotide-binding protein, presumably the stimulatory guanine nucleotide-binding protein (Ns) of adenylate cyclase. Results obtained in studies with membranes prepared from wild-type S49 lymphoma cells and the adenylate cyclase-deficient variant (cyc-) were similar to those obtained in experiments carried out with membranes prepared from L6 myoblasts. Thus, the high affinity binding of (-)-[3H]HBI to membranes prepared from wild-type and cyc- S49 lymphoma cells was stereoselectively inhibited by the active isomers of isoproterenol and propranolol, and was inhibited by GTP. Moreover, the density of sites determined with (-)-[3H]HBI was less than that determined with [125I]IPIN. These results suggest either that cyc- cells contain a partially functional Ns, or alternatively, that the inhibitory guanine nucleotide-binding protein (Ni) is capable of interacting with beta-adrenergic receptors. PMID- 2997215 TI - An inducible periplasmic blue copper protein from Paracoccus denitrificans. Purification, properties, and physiological role. AB - When grown on methylamine as a sole carbon source, Paracoccus denitrificans synthesizes a Type I blue copper protein which mediates electron transfer between methylamine dehydrogenase and cytochrome c. This blue copper protein does not serve as an electron acceptor for methanol dehydrogenase and is not synthesized by cells grown on methanol or succinate. The blue copper protein and methylamine dehydrogenase were localized in the periplasm of P. denitrificans, whereas formate dehydrogenase was cytoplasmic. The copper protein can be purified to high yield in a single step from the periplasmic subcellular fraction prepared from P. denitrificans. The purified protein contains a single 15,000-Da polypeptide chain and one copper atom/molecule and exhibits a pI of 4.8. The oxidized form of the protein absorbs strongly at 595 nm and weakly at 464 nm. The physical and physiological properties of this protein indicate that it is not an azurin, but representative of another class of blue copper proteins. PMID- 2997216 TI - Crystallographic characterization of phthalate oxygenase reductase, an iron sulfur flavoprotein from Pseudomonas cepacia. AB - Pseudomonads grown on phthalate synthesize a series of enzymes that metabolize this aromatic substrate. Among the inducible enzymes is a reductase which transfers electrons from NADH to the terminal dioxygenase that converts phthalate to the corresponding cis-1,2-dihydrodiol (Keyser, P. (1976) Ph. D. thesis, University of Miami; Ribbons, D. W., and Evans, W. C. (1960) Biochem. J. 76, 310 318). The phthalate oxygenase reductase induced in Pseudomonas cepacia is a single polypeptide chain (Mr approximately equal to 33,000) with two prosthetic groups, FMN and [2Fe-2S]. This oxidoreductase has been crystallized at pH 6.7 from polyethylene glycol 6000 in space group R3 with a = b = 113.4 A and c = 77.7 A (hexagonal indexing). PMID- 2997217 TI - Self-phosphorylation enhances the protein-tyrosine kinase activity of the epidermal growth factor receptor. AB - The effect of self-phosphorylation on the protein-tyrosine kinase activity of the epidermal growth factor receptor has been investigated using immunoaffinity purified protein. Enzyme was first incubated for various times with excess ATP to phosphorylate it to differing extents; the ability of the enzyme to phosphorylate exogenous peptide substrates was then measured as a function of its self phosphorylation state. Increasing self-phosphorylation to 1.3-1.8 mol of phosphate mol-1 of epidermal growth factor receptor enhanced protein-tyrosine kinase activity 2-3-fold. Comparison of the kinetics of protein-tyrosine kinase activity at different ATP concentrations revealed significant differences between unphosphorylated and phosphorylated enzyme. At low levels of ATP, a double reciprocal plot of the protein-tyrosine kinase activity of the unphosphorylated enzyme was hyperbolic, suggesting that ATP may act as an activator of the enzyme. At higher ATP concentrations, where greater levels of self-phosphorylation occurred during the reaction, the kinetics appeared linear and similar to those of the phosphorylated enzyme. Dose-response studies using three different peptide substrates (angiotensin II, gastrin, and a synthetic peptide corresponding to the self-phosphorylation site in p60v-src) showed that exogenous substrates inhibit receptor self-phosphorylation. In each case, half-maximal inhibition was observed at a peptide concentration approximately equal to the substrate's Km. A kinetic analysis comparing peptide phosphorylation using unphosphorylated and prephosphorylated enzyme indicated that the self-phosphorylation site can act as a competitive inhibitor (alternate substrate) versus peptide substrates. These results suggest that self-phosphorylation of the epidermal growth factor receptor removes a competitive constraint so that exogenous substrates can be more readily phosphorylated. PMID- 2997218 TI - Photoaffinity labeling of A1-adenosine receptors. AB - The ligand-binding subunit of the A1-adenosine receptor has been identified by photoaffinity labeling. A photolabile derivative of R-N6 phenylisopropyladenosine, R-2-azido-N6-p-hydroxyphenylisopropyladenosine (R AHPIA), has been synthesized as a covalent specific ligand for A1-adenosine receptors. In adenylate cyclase studies with membranes of rat fat cells and human platelets, R-AHPIA has adenosine receptor agonist activity with a more than 60 fold selectivity for the A1-subtype. It competes for [3H]N6 phenylisopropyladenosine binding to A1-receptors of rat brain membranes with a Ki value of 1.6 nM. After UV irradiation, R-AHPIA binds irreversibly to the receptor, as indicated by a loss of [3H]N6-phenylisopropyladenosine binding after extensive washing; the Ki value for this photoinactivation is 1.3 nM. The p hydroxyphenyl substituent of R-AHPIA can be directly radioiodinated to give a photoaffinity label of high specific radioactivity (125I-AHPIA). This compound has a KD value of about 1.5 nM as assessed from saturation and kinetic experiments. Adenosine analogues compete for 125I-AHPIA binding to rat brain membranes with an order of potency characteristic for A1-adenosine receptors. Dissociation curves following UV irradiation at equilibrium demonstrate 30-40% irreversible specific binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the probe is photoincorporated into a single peptide of Mr = 35,000. Labeling of this peptide can be blocked specifically and stereoselectively by adenosine receptor agonists and antagonists in a manner which is typical for the A1-subtype. The results indicate that 125I-AHPIA identifies the ligand-binding subunit of the A1-adenosine receptor, which is a peptide with Mr = 35,000. PMID- 2997219 TI - Disruption of phosphatidylserine translocation to the mitochondria in baby hamster kidney cells. AB - Phosphatidylserine synthase is found predominantly in the microsomal fraction, and phosphatidylserine decarboxylase is found predominantly in the mitochondrial fraction of baby hamster kidney (BHK-21) cells. This segregation of enzymes of phosphatidylserine metabolism allows serine metabolism to phosphatidylserine and phosphatidylethanolamine to be used as an indicator of the intracellular movement of phosphatidylserine. After BHK-21 cells were pulse-labeled with [3H]serine, phosphatidylserine was efficiently labeled, and subsequently 40-50% of this radiolabeled lipid turned over to form phosphatidylethanolamine during a 7.5-h chase. Treatment of cells with NaN3 plus NaF or cycloheximide at the end of the pulse labeling period markedly inhibited the rate and extent of phosphatidylserine turnover during the chase period. The inhibition of phosphatidylserine turnover could not be attributed to inhibition of either phosphatidylserine decarboxylase or phosphatidylserine exchange protein activity. Subcellular fractionation of the BHK-21 cells demonstrated that cells poisoned with NaN3 plus NaF accumulated phosphatidylserine in the microsomal fraction relative to unpoisoned cells. The results indicate that metabolic energy is required for the transport of phosphatidylserine to the mitochondria. PMID- 2997220 TI - Inhibition of 3-O-methylglucose transport in human erythrocytes by forskolin. AB - The effect of forskolin, an activator of adenylate cyclase, was investigated on glucose transport in human erythrocytes. Forskolin was found to be a potent inhibitor of 3-O-methylglucose (3-O-MG) influx in human erythrocytes. The inhibition of 3-O-MG transport was instantaneous and reversible. The inhibitory effect of forskolin was concentration-dependent, having an IC50 value of 7.5 microM. Forskolin caused a decrease in Vmax of carrier-mediated 3-O-MG transport from 35.32 to 1.56 mumol/ml of cell X min in the presence of 50 microM forskolin. Inhibition of influx was not reversed at high concentrations of 3-O-MG. In addition, forskolin inhibited the influx of other carbohydrates including galactose, ribose, and fructose. In contrast, forskolin was without effect on adenosine transport. To unravel the underlying mechanism responsible for the inhibitory action of forskolin, the possible involvement of cyclic AMP in controlling glucose transport was examined. Erythrocytes treated with 50 microM forskolin exhibited an increase in cyclic AMP content from the basal levels of 258 fmol/ml of cell to 334 fmol/ml of cell within 10 s after forskolin exposure. However, erythrocytes in which cyclic AMP was allowed to accumulate in excess of 10,000 times the basal level, by means of preincubation with exogenous cyclic AMP, displayed 3-O-MG transport indistinguishable from that of cyclic AMP-poor control cells. In view of the finding that cyclic AMP plays no discernible role in the erythrocyte 3-O-MG transport, it is suggested that the forskolin inhibition is mediated by a mechanism other than by stimulating adenylate cyclase activity. Moreover, forskolin appears to directly inactivate the 3-O-MG transport system since glucose-sensitive cytochalasin B binding to erythrocyte membranes is virtually abolished by 50 microM forskolin. PMID- 2997221 TI - Determination of the anomeric specificity of the Escherichia coli CTP:CMP-3-deoxy D-manno-octulosonate cytidylyltransferase by 13C NMR spectroscopy. AB - [99%, 1-13C]- and [90%, 2-13C]3-deoxy-D-manno-octulosonic acid (KDO) were prepared enzymatically and used to determine the anomeric specificity of the CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl transferase (CMP-KDO synthetase) by 13C NMR spectroscopy. Addition of CMP-KDO synthetase to reaction mixtures containing either 1-13C- or 2-13C-labeled KDO resulted in rapid CMP-KDO formation which was accompanied by a substantial decrease in the 13C-enriched resonances of the beta-pyranose form of KDO relative to the resonances of other KDO species in solution, demonstrating that the beta-pyranose is the preferred substrate. Concomitant with the production of CMP-KDO was the appearance of peaks at 174.3 and 101.4 ppm when [1-13C]- and [2-13C]KDO, respectively, were used as substrates. The correspondence of these resonances to the enriched carbons in CMP KDO was confirmed by the expected 3-bond (3JP,C-1 = 6.9 Hz) and 2-bond coupling (2JP,C-2 = 8.3 Hz) between the labeled carbons and the ketosidically linked phosphoryl group. A large coupling (3J = 5.7 Hz) was observed in proton-coupled spectra of CMP-[1-13C]KDO between carbon 1 and the axial proton at carbon 3 of KDO. The magnitude of this coupling constant supports a diaxial relationship between these two groups and, along with chemical shift data, indicates that KDO retains the beta-configuration when linked in CMP-KDO. PMID- 2997222 TI - Glucocorticoids regulate the transcription of glycerol phosphate dehydrogenase in cultured glial cells. AB - Utilizing a glycerol phosphate dehydrogenase cDNA clone from the rat glioma C6 cell line, we report that the glucocorticoid regulation of glycerol phosphate dehydrogenase gene expression in glial cells occurs at the transcriptional level. Furthermore, our transcription results in vitro, as well as Northern blot analysis, show that the short-chain fatty acid, sodium butyrate, totally blocks this hydrocortisone-induced transcription of glycerol phosphate dehydrogenase, demonstrating that the site of action of this fatty acid resides in the cell nucleus. The C6 glycerol phosphate dehydrogenase cDNA clone (pGPDH-1,1800 base pairs) used in these studies was selected from a C6 library generated from polysomal poly(A)+ RNA. pGPDH-1 hybridized to a 4.7-kilobase RNA from rat muscle and brain, mouse liver, and C6 cells; this mRNA is at least four times larger than the required coding sequence for glycerol phosphate dehydrogenase. Northern blot analysis of developing rat brain reveals a striking increase in glycerol phosphate dehydrogenase transcripts during the period most associated with peak myelination. PMID- 2997223 TI - Interaction of ribonuclease H from Drosophila melanogaster embryos with DNA polymerase-primase. AB - An RNase H was purified 2,500-fold to near homogeneity from early embryos of Drosophila melanogaster. The purified enzyme has an approximate molecular weight of 180,000 and appears to consist of two 49,000- and two 39,000-dalton polypeptides. The enzyme specifically hydrolyzes RNA.DNA hybrids and releases oligoribonucleotides ranging in size from 2-9 residues. The RNase H can also remove RNA primers that are synthesized and subsequently elongated by the Drosophila polymerase-primase. Preincubation of the RNase H from D. melanogaster embryos with the homologous DNA polymerase-primase results in an increased rate of DNA synthesis. The DNA chains synthesized under these conditions are shorter than those synthesized in the absence of the RNase H, and the rate of primer synthesis is increased significantly. These findings suggest that the RNase H forms a complex with the polymerase-primase, increasing its recycling capacity and thereby increasing the frequency of chain initiation. PMID- 2997224 TI - Yeast mRNA splicing in vitro. AB - Synthetic actin and CYH2 pre-mRNAs containing a single intron are accurately spliced in a soluble whole cell extract of yeast. Splicing in vitro requires ATP. The excised intron is released as a lariat in which an RNA branch connects the 5' end of the molecule to the last A in the "intron conserved sequence" UACUAAC. Two other discrete RNA species produced during splicing in vitro may represent reaction intermediates: free, linear exon 1 and a form of the intron lariat extending beyond the 3' splice site to include exon 2. Both lariat forms correspond to molecules previously shown to be produced during yeast pre-mRNA splicing in vivo. PMID- 2997225 TI - Effect of pertussis toxin treatment on the down-regulation of opiate receptors in neuroblastoma X glioma NG108-15 hybrid cells. AB - Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with 10 nM [D Ala2,D-Leu5] enkephalin (DADLE) results in a reduction of cell-surface opiate delta receptors. Whether opiate receptor internalization requires the activation of the guanine nucleotide-binding protein (Ni) is unclear. Hence, activation of Ni was attenuated by treating hybrid cells with 100 ng/ml pertussis toxin (PT) for 3 h, which resulted in a decrease in DADLE's ability to inhibit adenylate cyclase activity. Despite this prior treatment with PT, chronic exposure of these cells to 10 nM DADLE resulted in a time-dependent decrease in both [3H]diprenorphine and [3H]DADLE binding. This reduction in 3H-ligand binding in cells previously treated with PT represented internalization of the receptors because translocation was observed of bound [3H]DADLE from plasma membrane fractions to the lysosomal fractions in the Percoll gradients. Thus, opiate receptors internalize without activation of Ni. The internalization of opiate receptors was not accompanied by Ni. By measuring the amount of the 41-kDa alpha subunit being labeled by PT with [32P]NAD+, it was determined that plasma membrane preparations, of both the control cells and cells treated with 10 nM of DADLE for 4 h, contained equal concentrations of Ni, 2 pmol of Ni/mg of protein. Additionally, there was no measurable alteration in the amount of PT substrate in the lysosomal fractions of the DADLE-treated cells as compared to that of control cells. Chronic DADLE treatment resulted in a decrease in Km value of NAD+ in the ADP-ribosylation of 41-kDa subunit of Ni. In summary, opiate receptors internalize as agonist-receptor complexes without the guanine nucleotide-binding component. PMID- 2997226 TI - Hemin, chelatable iron, and the regulation of transferrin receptor biosynthesis. AB - We have examined the mechanism by which hemin regulates the expression of the human transferrin receptor. Previous work led to the suggestion that the regulatory signal is provided by heme (Ward J. H., Jordan, I., Kushner, J. P., and Kaplan, J. (1984) J. Biol. Chem. 259, 13235-13240). We demonstrated that hemin regulates the expression of the receptor via alterations in the rate of receptor biosynthesis. However, this effect can be completely abolished by addition of desferrioxamine, an intracellular iron chelator. Competition curves demonstrate that desferrioxamine and hemin affect the same intracellular iron pool. Since the chelator cannot remove iron from heme, we propose that hemin acts simply by delivering iron to a chelatable iron pool and that levels of chelatable iron provide the regulatory signal for expression of the transferrin receptor gene. PMID- 2997227 TI - Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. AB - Camptothecin, a cytotoxic drug, is a strong inhibitor of nucleic acid synthesis in mammalian cells and a potent inducer of strand breaks in chromosomal DNA. Neither the equilibrium dialysis nor the unwinding measurement indicates any interaction between camptothecin and purified DNA. However, camptothecin induces extensive single strand DNA breaks in reactions containing purified mammalian DNA topoisomerase I. DNA breakage in vitro is immediate and reversible. Analyses of camptothecin-induced DNA breaks show that topoisomerase I is covalently linked to the 3' end of the broken DNA. In addition, camptothecin inhibits the catalytic activity of mammalian DNA topoisomerase I. We propose that camptothecin blocks the rejoining step of the breakage-reunion reaction of mammalian DNA topoisomerase I. This blockage results in the accumulation of a cleavable complex which resembles the transient intermediate proposed for eukaryotic DNA topoisomerase I. The inhibition of nucleic acid synthesis and the induction of DNA strand breaks observed in vivo may be related to the formation of this drug induced cleavable complex. PMID- 2997228 TI - An international collaborative study on foot and mouth disease virus assay methods. 2. Quantification of 146S particles. AB - Workers in 11 laboratories in Europe and one in North America participated in a collaborative study to assess the variability of a sucrose gradient procedure used for the quantification of foot and mouth disease virus (FMDV). To this end, a range of standards was distributed from one of the participating laboratories. A series of adenine preparations were used to assess the various spectrophotometers/UV monitors and it showed most to be accurate and linear in their responses. The FMDV and MS2 ribophage standards were used to assess the sucrose gradient procedure and indicated low levels of within laboratory variation whereas between laboratory variation was greater. Recalculation of results from the unprocessed data in the coordinating laboratory permitted the identification and reduction of one source of between laboratory variation. Despite the observed variations, the results indicated that meaningful comparisons could be made between the data of different laboratories provided that a procedure similar to the one described in this report is used. PMID- 2997229 TI - Effects of isoxuprine and nylidrin on adrenoreceptors in rat vas deferens. AB - The interaction of isoxuprine and nylidrin with alpha 1- and beta 2 adrenoreceptors in rat vas deferens was examined using radioligand binding assays and physiological studies in vitro. Isoxuprine and nylidrin have a greater affinity for binding to alpha 1 (isoxuprine KD = 59 +/- 15 nM; nylidrin KD = 41 +/- 3 nM) than beta 2-(isoxuprine KD = 3,900 +/- 500 nM; nylidrin KD = 900 +/- 50 nM) adrenoreceptors in rat vas deferens. Vas deferens from rats pretreated for 16 24 h with reserpine (3 mg/kg i.p.) were exposed to 10 microM phenoxybenzamine for 15 min to inactivate alpha-adrenoreceptors. Under these conditions high concentrations of both isoxuprine and nylidrin relaxed vas deferens contracted with 55 mM K+, however the relaxation was not blocked by the beta-adrenoreceptor antagonist propranolol (10 microM). Both isoxuprine and nylidrin were potent competitive antagonists of alpha 1-adrenoreceptor mediated contraction of vas deferens. pA2 values for isoxuprine (6.9 +/- .05) and nylidrin (7.1 +/- .08) agreed well with KD values for binding to alpha 1-adrenoreceptors in vas deferens. The greater potency of isoxuprine and nylidrin in inhibiting alpha 1 adrenoreceptors than binding to beta 2-adrenoreceptors or causing nonspecific relaxation suggest that alpha-adrenoreceptor antagonist actions of these drugs may be important in their ability to inhibit smooth muscle tone. PMID- 2997230 TI - The use of forskolin to investigate the site of cardiac beta-adrenoreceptor supersensitivity. AB - The positive inotropic responses of left atria and papillary muscles and the positive chronotropic responses of right atria of guinea-pigs to isoprenaline and forskolin were examined. An increase in sensitivity of the three preparations to isoprenaline was observed by lowering the bath temperature from 38 to 30 degrees C as demonstrated by a leftwards shift of the concentration-response curves. A similar degree of supersensitivity was observed for forskolin. Since forskolin is reputed to stimulate adenylate cyclase directly, whereas isoprenaline stimulates via the regulatory nucleotide Ns protein, this would suggest a common site for the supersensitivity at adenylate cyclase. However, the possibility that forskolin also stimulates via the Ns protein in producing cardiac stimulation and that this is the site of hypothermia-induced supersensitivity is discussed. Supersensitivity to isoprenaline was also observed in left atria and papillary muscles from guinea-pigs chronically pretreated with reserpine for 3 days (5 mg/kg at 72 h, 3 mg/kg at 48 and 24 h) or 7 days (0.1 mg/kg daily). In the same tissues, there was no change in the sensitivity to forskolin. The site of the supersensitivity can therefore be concluded to occur before the level of adenylate cyclase activation either directly or via the regulatory Ns protein; possibly at the beta-adrenoreceptor itself. PMID- 2997231 TI - Effect of drugs interacting with adrenoreceptors and muscarinic receptors in the epididymal and prostatic parts of the human isolated vas deferens. AB - Electrical field stimulation of ring preparations of the epididymal (Ve) and prostatic (Vp) parts of the human isolated vas deferens produced contractions with similar frequency-dependence and appearance. The contractions of Ve, but not of Vp preparations were abolished by tetrodotoxin (10(-6)M). Noradrenaline (NA), phenylephrine, and methoxamine, but not clonidine induced repetitive, phasic contractions in both Ve and Vp preparations, and increased the amplitude of electrically induced responses. Clonidine concentration-dependently decreased electrically induced contractions in Ve preparations, but had no significant effects in Vp preparations. Phentolamine and prazosin abolished electrically induced contractions in Ve but not in Vp preparations. In Ve rings the contractions were increased by rauwolscine; no such effect was observed in Vp preparations. Isoprenaline, propranolol, acetylcholine and carbachol had no effects in the Ve or Vp preparations. Scopolamine and atropine reduced electrically induced responses. Clonidine decreased and rauwolscine increased the electrically induced release of 3H in both Ve and Vp preparations pre-loaded with 3H-NA. Phenylephrine, prazosin, isoprenaline, propranolol, carbachol and scopolamine had minor or no effects on the 3H release. Radioligand receptor binding experiments using 3H-prazosin and 3H-rauwolscine as ligands revealed similar densities of alpha 1- and alpha 2-adrenoreceptors in the human vas deferens. There seemed to be no differences in their distribution between the epididymal, middle and prostatic part of the organ. It is concluded that the neurotransmission in the human vas deferens is noradrenergic and mediated via alpha 1-adrenoreceptors. The prazosin and tetrodotoxin resistant part of the electrically induced contraction in Vp preparations may be caused by direct smooth muscle stimulation. PMID- 2997232 TI - Lower-extremity sensibility testing in patients with herniated lumbar intervertebral discs. AB - The significance of sensory changes determined by pinprick and light touch in individuals with a herniated lumbar disc has been questioned. Discrepancies may be related to the subjectiveness of the test, failure to use dermatome-specific testing sites, overlap of areas that are innervated by different nerve roots, anatomical variations, or lack of sensitivity of the testing technique. For this study, we assessed the results of sensory examinations of twenty-five patients with documented herniation of a lumbar disc. The examinations were done using Semmes-Weinstein monofilaments, vibrometry, pinprick, and light touch in the autonomous skin areas supplied by the fourth and fifth lumbar and first sacral nerve roots. Right-left differences in Semmes-Weinstein pressure thresholds of more than fifteen milligrams per square millimeter enabled us to localize disc lesions to a specific root in 100 per cent of patients and differences in vibratory thresholds of more than 3.5 micrometers, to localize the correct level in 88 per cent. Lesser differences in thresholds did not help to identify the involved root. The mean sensory threshold on the side of the disc lesion was found to be significantly greater than that on the opposite side by both vibrometry and pressure aesthesiometry (p less than 0.005). These findings were not duplicated using light touch or pinprick testing. Even with the most sophisticated sensibility-testing techniques, correct identification of the nerve root that was compressed by a herniated lumbar disc was accurate in only 50 per cent of patients. PMID- 2997233 TI - Large-scale purification of presynaptic plasma membranes from Torpedo marmorata electric organ. AB - The presynaptic plasma membrane (PSPM) of cholinergic nerve terminals was purified from Torpedo electric organ using a large-scale procedure. Up to 500 g of frozen electric organ were fractioned in a single run, leading to the isolation of greater than 100 mg of PSPM proteins. The purity of the fraction is similar to that of the synaptosomal plasma membrane obtained after subfractionation of Torpedo synaptosomes as judged by its membrane-bound acetylcholinesterase activity, the number of Glycera convoluta neurotoxin binding sites, and the binding of two monoclonal antibodies directed against PSPM. The specificity of these antibodies for the PSPM is demonstrated by immunofluorescence microscopy. PMID- 2997234 TI - Secretory protein targeting in a pituitary cell line: differential transport of foreign secretory proteins to distinct secretory pathways. AB - The mouse pituitary cell line, AtT-20, packages the adrenocorticotropic hormone (ACTH) in secretory vesicles and releases it when the cell is stimulated with secretagogues. These cells have the capacity, after transfection with the appropriate DNA, to package heterologous peptide hormones into the regulated secretory vesicles (Moore, H. P. H., M. D. Walker, F. Lee, and R. B. Kelly, 1983, Cell, 35:531-538). To test if other secreted proteins prefer a different route to the surface, we have transfected AtT-20 cells with DNAs coding for a fragment of a membrane protein, the vesicular stomatitis virus G protein from which the membrane spanning domain has been deleted (Rose, J. K., and J. E. Bergmann, 1982, Cell, 17:813-819). We found that the secreted vesicular stomatitis virus G proteins were not transported to the regulated secretory vesicles. Instead they preferentially exited the cell by the constitutive pathway previously found in these cells (Gumbiner, B., and R. B. Kelly, 1982, Cell, 28:51-59). In contrast, human growth hormone transfected into the cells by the same procedure was transported to the regulated pathway with a similar efficiency as the endogenous hormone ACTH. Transport of the secreted G protein to the regulated pathway, if it occurs at all, is at least 30-fold less efficient than peptide hormones. We conclude that the transport machinery in AtT-20 cells must selectively recognize different secreted proteins and sort them into distinct secretory pathways. PMID- 2997235 TI - The extracellular matrix of normal chick embryo fibroblasts: its effect on transformed chick fibroblasts and its proteolytic degradation by the transformants. AB - Extracellular matrix (ECM), prepared from chick embryo fibroblasts, contains fibronectin as the major structural protein along with collagen and other polypeptides as less abundant protein components. When Rous sarcoma virus transformed chick embryo fibroblasts are cultured on the ECM in the presence of the tumor promoter tetradecanoyl phorbol acetate, the transformed cells lose their characteristic rounded morphology and align on and within the ECM fibrillar network. This restrictive aspect of ECM is only temporary, however, and with time (24-72 h) the transformed cells progressively degrade the ECM fibers and resume their rounded appearance. The matrix degradation can be monitored by employing biosynthetically radiolabeled ECM. The addition of purified chicken plasminogen to the Rous sarcoma virus-transformed chick embryo fibroblast cultures enhances the rate and extent of ECM degradation, due to the elevated levels in the transformed cultures of plasminogen activator. Plasminogen-dependent and independent degradation of ECM has been characterized with regard to sensitivity to various natural and synthetic protease inhibitors and to the requirement of cell/ECM contact. Plasminogen-dependent degradation of ECM occurs rapidly when ECM and cells are in contact or separated, whereas plasminogen-independent degradation is greatly reduced when ECM and cells are separated, which suggests that cell surface-associated proteolytic enzymes are involved. A possible role in ECM degradation has been indicated for cysteine proteases, metallo enzymes, and plasminogen activator, the latter as both a zymogen activator and a direct catalytic mediator. PMID- 2997237 TI - Interactions between cyclic AMP- and phorbol ester-dependent phosphorylation systems in S49 mouse lymphoma cells. AB - High-resolution two-dimensional gel electrophoresis of proteins labeled with either 32Pi or [35S]methionine was used to study interactions between cyclic AMP and tetradecanoyl phorbol acetate (TPA) at the level of intracellular protein phosphorylation. Cultured S49 mouse lymphoma cells were used as a model system, and mutant sublines lacking either the catalytic subunit of cyclic AMP-dependent protein kinase or the guanyl nucleotide-binding "Ns" factor of adenylate cyclase provided tools to probe mechanisms underlying the interactions observed. Three sets of phosphoproteins responded differently to TPA treatment of wild-type and mutant cells: Phosphorylations shown previously to be responsive to activation of intracellular cyclic AMP-dependent protein kinase were stimulated by TPA in wild type cells but not in mutant cells, a subset of phosphorylations stimulated strongly by TPA in mutant cells was inhibited in wild-type cells, and two novel phosphoprotein species appeared in response to TPA only in wild-type cells. The latter two classes of TPA-mediated responses specific to wild-type cells could be evoked in adenylate cyclase-deficient cells by treating concomitantly with TPA and either forskolin or an analog of cyclic AMP. Three conclusions are drawn from our results: 1) TPA stimulates adenylate cyclase in wild-type cells causing increased phosphorylation of endogenous substrates by cyclic AMP-dependent protein kinase, 2) activated cyclic AMP-dependent protein kinase inhibits phosphorylation (or enhances dephosphorylation) of a specific subset of TPA dependent phosphoproteins, and 3) cyclic AMP-dependent events facilitate TPA dependent phosphorylation of some substrate proteins. PMID- 2997238 TI - Human spreading factor: synthesis and response by HepG2 hepatoma cells in culture. AB - Human serum spreading factor (SF) is a cell adhesion and spreading-promoting glycoprotein purified from serum or plasma that mediates effects in a wide variety of animal cell culture systems. HepG2 human hepatoma cells were found to synthesize and secrete SF into culture medium. Quantitative immunoassay of the protein indicated a concentration of about 1 microgram/ml in 48 hr-conditioned medium from confluent cultures. Although fibronectin also was synthesized and secreted into the culture medium, HepG2 cell spreading was observed in response to human serum SF, but not in response to human plasma fibronectin. Immunoprecipitation of SF from culture medium of cells metabolically-labeled with leucine, fucose or glucosamine identified a single form of the molecule of approximately 70,000 daltons. Treatment of cultures with tunicamycin inhibited incorporation of fucose and glucosamine into immunoprecipitated SF, but did not prevent synthesis and secretion of the protein. Electrophoretic analysis and cell spreading assays showed that SF secreted by tunicamycin-treated HepG2 cells was of molecular weight (mw) approximately 60,000, and was biologically active. PMID- 2997236 TI - Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors. AB - Nerve growth factor (NGF) regulates the microtubule-dependent extension and maintenance of axons by some peripheral neurons. We show here that one effect of NGF is to promote microtubule assembly during neurite outgrowth in PC12 cells. Though NGF causes an increase in total tubulin levels, the formation of neurites and the assembly of microtubules follow a time course completely distinct from that of the tubulin induction. The increases in microtubule mass and neurite extension closely parallel 10- and 20-fold inductions of tau and MAP1, proteins shown previously to promote microtubule assembly in vitro. When NGF is removed from PC12 cells, neurites disappear, microtubule mass decreases, and both microtubule-associated proteins return to undifferentiated levels. These data suggest that the induction of tau and MAP1 in response to NGF promotes microtubule assembly and that these factors are therefore key regulators of neurite outgrowth. PMID- 2997239 TI - Reversible effects of dibutyryl cAMP on laminin and type IV collagen secretion from retinoic acid treated F9 cells. AB - The effects of dibutyryl cAMP on the differentiation of embryonal carcinoma F9 cells were studied mainly using the secretion of laminin and type IV collagen as the marker. For this purpose, F9 cells were labeled with 35S-methionine and radioactive proteins in the medium were analyzed by SDS-polyacrylamide gel electrophoresis. Treatment of F9 cells with retinoic acid alone induced differentiation into cells secreting type IV collagen. The combination of retinoic acid and dibutyryl cAMP stimulated laminin secretion in addition to type IV collagen secretion. This effect of dibutyryl cAMP was observed only 16 h after adding dibutyryl cAMP. Immunofluorescence staining demonstrated that the majority of the cells in culture were converted into cells secreting laminin under these conditions. In contrast to the irreversible effect of retinoic acid, the effect of dibutyryl cAMP on laminin and type IV collagen secretion was reversible at least during the first 5 days of maintaining cells in the medium containing retinoic acid plus dibutyryl cAMP. Removal of dibutyryl cAMP from the culture medium decreased the protein secretion to the basal levels within 2 days. This reversibility was not due to a change in cell number. An in vitro translation assay also suggested the reversible effect of dibutyryl cAMP on the levels of laminin mRNA. Coinciding with variations of the protein secretion, a reversible and homogeneous change in the morphology of retinoic acid generated F9 cells was observed by dibutyryl cAMP. PMID- 2997240 TI - Neurite formation by neuroblastoma-glioma hybrid cells (NG108-15) in defined medium: stochastic initiation with persistence of differentiated functions. AB - Neurite formation and proliferation by NG108-15 cells were studied in short-term serum-free N2 medium. Neuritogenesis by individual cells was observed at widely differing times, suggesting a stochastic component to initiation of differentiation. Cells with and without neurites could also enter one or more rounds of proliferation at varying times. The initial choice between these divergent behaviors influenced subsequent growth. Cells initially extending neurites had a high probability of continuing neuritic elongation. Cells initially dividing had a high probability of yielding further progeny. Addition of dibutyryl cyclic AMP to cells cultured in N2 medium rapidly increased the probability of differentiating and decreased the probability of proliferating. To test whether or not cells with highly differentiated morphologies had irreversibly lost the capacity for proliferation, induced cultures were washed and challenged by the addition of serum-containing medium. The length of time required for individual cells to divide increased with increasing time of preincubation in the induction medium. However, few cells appeared to be permanently removed from the proliferative pool. These observations suggest that differentiating cells exhibit persistence, a tendency to continue on the differentiation pathway. Persistence is extinguished following one round of proliferation in serum-containing medium. PMID- 2997242 TI - Transforming growth factor (TGF) activity in human urine: synergism between TGF beta and urogastrone. AB - Human urine contains an acid and heat stable peptide with an apparent molecular weight of 8,000-10,000 that, in the presence of urogastrone (EGF), induces the anchorage-independent growth of nontransformed cells in semisolid media. This nonmitogenic growth-modulating activity does not compete with EGF for binding to EGF membrane receptor sites and can be resolved from EGF by high-performance liquid chromatography. The urine-derived growth factor has been purified more than 10,000-fold and shares many biochemical properties with and is functionally related to the B class of TGFs isolated from transformed cells and non-neoplastic tissues. The low molecular weight anchorage-independent growth-stimulating activity universally present in human urine is a result of the synergistic interaction of this urine-derived TGF-beta and urogastrone. PMID- 2997241 TI - Evidence for p15 cleavage site in myc-specific proteins of avian MC29 and OK10 viruses. AB - Myc-related proteins were precipitated from MC29 virus-transformed cells (PR-2) and from OK10 virus-transformed cells (9C) by anti-gag and anti-myc sera. Immunoprecipitates were cleaved with the avian retroviral protease p15 and the cleavage products analyzed in SDS-PAGE. Cleavage fragments of p110gag-myc (product of MC29 virus) and p58myc (product of OK10 virus) showed the presence of a p15 cleavage site within the myc-specific region. The site is missing in deletion mutants of MC29 virus. PMID- 2997243 TI - Differential effects of polyamines on rat thyroid protein kinase activities. AB - Ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis, has been shown to be regulated in thyroid by thyrotropin both in vivo and in vitro. Little, however, is known of the role of polyamines in thyroid cell function. Since studies in other tissues suggest that polyamines may influence protein phosphorylation, we studied the effect of the polyamines on various protein kinase activities in rat thyroid. Putrescine, spermidine, and spermine inhibit cyclic-AMP-dependent histone H1 kinase activity when measured in the cytosol fraction of rat thyroid; this effect is largely reproduced by NaCl concentrations of equivalent ionic strength. Both spermidine and spermine effect a 1.6-2.4-fold increase in cytosolic cyclic-AMP-independent (messenger-independent) casein kinase activity; stimulation by both polyamines is maximal at 5mM. A similar profile of stimulation is observed for messenger-independent casein kinase activity in crude nuclear preparations. Sodium chloride fails to stimulate both cytosolic and nuclear messenger-independent casein kinase activities at ionic strength equivalent to the spermine concentrations used. Spermine, but not putrescine, spermidine, or sodium chloride, inhibits calcium/phospholipid dependent protein kinase C activity in cytosol extracts partially purified by DEAE chromatography. These findings suggest that regulation of protein kinase(s) by polyamines may represent a proximal locus (i) of action of thyrotropin regulated ornithine decarboxylase activity in thyroid. PMID- 2997244 TI - Effect of nimodipine on cerebral metabolism during ischemia and recirculation in the mongolian gerbil. AB - The effect of nimodipine on cerebral metabolism during ischemia and reflow was studied in female mongolian gerbils. Animals were divided into three experimental groups. Group 1 received 1 mg/kg nimodipine i.p. 1 h prior to ischemia. Group 2 received an injection of the vehicle, 5% polyethylene glycol 400. Group 3 received an equal volume of normal saline. Cerebral ischemia was induced by bilateral common carotid artery occlusion for 1, 2, or 5 min. Recirculation was established for 0, 1, or 5 min. Sham-operated animals served as nonischemic controls. Gerbils were killed by microwave irradiation. Regional levels of ATP, phosphocreatine, glucose, glycogen, cyclic AMP, and cyclic GMP were measured in brain extracts using standard assay techniques. Levels of metabolites in sham operated animals did not differ among Groups 1, 2, and 3. At 1 min of ischemia, cortical and striatal ATP levels were highest in Group 1 (p less than 0.05 and p less than 0.01, respectively). After 5 min of recirculation, cortical and striatal glucose levels were highest in Group 1 (p less than 0.005). Regional levels of the metabolites measured at other times did not differ significantly among the three groups. Pretreatment with nimodipine thus retards the fall in ATP and facilitates the recovery of glucose in mongolian gerbils subjected to common carotid artery occlusion. A regional variability of this effect was observed. PMID- 2997245 TI - Thermography of pain: instrumentation and uses. PMID- 2997246 TI - The status of bone marrow transplantation for leukemia. PMID- 2997247 TI - A woman with refractory diarrhea for five months. PMID- 2997248 TI - A depressed nurse with hypoglycemia. PMID- 2997249 TI - Rash and hyperglycemia. That wasn't diabetic. PMID- 2997250 TI - Diagnosis when the gene locus is unknown. PMID- 2997251 TI - Effects of solutions used for storage of size-exclusion columns on subsequent chromatography of peptides and proteins. AB - The effects of storage of size-exclusion column packing materials in methanolic or azide-water solutions on subsequent separations were tested. Three commercially available columns were used in these studies; the Toyo-Soda Bio-Sil TSK 125, Bio-Sil TSK 250 and the DuPont Bio-Series GF-250. Upon initial chromatography, all three columns bound up to 760 micrograms of cytochrome c tryptic peptides. Sample binding to packing material is probably a function of the positively charged basic groups on peptides or proteins interacting with silanol groups. The larger the peptide, the less the opportunity for silanol charged group interaction, hence, less binding. Initial samples introduced to a new column occupy the binding sites. Equilibration with neat methanol removes the bound protein revealing sites which bind sample. After absorption of peptides to binding sites on the packing material, storage in neat methanol regenerates the binding sites. Storage in 10% methanol diminished the binding phenomenon, but storage in azide-water reduced binding to a range below detection at the microgram level. Our recommendation to users of size-exclusion chromatographic columns is that one satisfy the absorption capacity of a new column by injecting a sufficient quantity of a basic peptide standard or other convenient sample to reduce available binding sites before using the column for important separations. Store columns in azide-water or 10% methanol to prevent the regeneration of exposed silanol groups. PMID- 2997252 TI - Antibody-mediated extraction of the main tetrahydrocannabinol metabolite, 11-nor delta 9-tetrahydrocannabinol-9-carboxylic acid, from human urine and its identification by gas chromatography-mass spectrometry in the sub-nanogram range. PMID- 2997253 TI - Use and regeneration of amicon ultrafiltration cones for deproteination of microsomal solutions before chromatographic analysis. PMID- 2997254 TI - Detection and quantitation of rotavirus using monoclonal antibody coupled red blood cells: comparison with ELISA. AB - A total of 125 faecal extracts from infants were tested by reverse passive haemagglutination (RPH) using red cells coated with a monoclonal antibody against the major group-specific rotavirus antigen (VP 6). Results were compared with those obtained using a rabbit anti-rotavirus capture, guinea pig anti-rotavirus detector-based ELISA. The specificity of the assay was confirmed by use of 'normal' immunoglobulin coupled red cells and by inhibition with rabbit antiserum. The antibody-coated red cells could be stabilised by treatment with glutaraldehyde and subsequent freeze-drying with no detectable loss of activity even after storage at 45 degrees C for 4 wk. Good correlation was obtained between RPH and ELISA. Purified bovine rotavirus could be detected by RPH down to approximately 10(5) particles in a 25 microliters vol. Similar results were obtained with polyclonal antibody coupled cells and an ELISA using monoclonal antibody. Experiments using subgroup-specific monoclonal antibodies indicated the feasibility of rapid subgroup determination. PMID- 2997255 TI - An enzyme-linked immunosorbent assay, ELISA, for SV40 antigen detection. AB - The exclusion of SV40 contamination in poliovaccine produced in Cynomolgus monkey kidney cell cultures is routinely done by inoculation of the inactivated vaccine into Cercopithecus monkey kidney cell cultures where a cytopathic effect reveals the presence of the virus. An ELISA is described for the detection of SV40 antigen and the efficiency of antigen detection was compared with the development of cytopathic effect in Cercopithecus tissue cultures. The assay shortened considerably the time for production control and was in full agreement with the conventional test method. PMID- 2997256 TI - Dermorphin reduces the metyrapone-evoked release of adrenocorticotropin, beta endorphin, and beta-lipotropin in man. AB - The aim of this study was to investigate further the influence of dermorphin (D), a new potent opioid peptide (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), on the functional activity of the pituitary-adrenocortical system in man. Six normal men were treated with oral metyrapone to stimulate the secretion of ACTH, beta lipotropin, and beta-endorphin. In these subjects, significant suppression of metyrapone-evoked release of ACTH and related peptides occurred during D infusion (5.5 micrograms/kg X min for 30 min) compared with that during saline infusion. These results indicate that D can induce a significant decline in plasma levels of ACTH, beta-lipotropin, and beta-endorphin, the major circulating peptides from the C-terminal part of proopiocortin, and suggest that opioid peptides may be involved in the control of the functional activity of pituitary-adrenocortical activity in man. PMID- 2997257 TI - Platelet alpha 2-adrenoceptor and adenylate cyclase in patients with anorexia nervosa and bulimia. AB - Platelets alpha 2-adrenoceptors were studied in 24 patients with anorexia nervosa shortly after admission to the hospital and after 10% weight gain. Twenty patients with bulimia and 24 healthy age- and sex-matched normal subjects also were studied. Receptor number was significantly increased in patients with bulimia and anorexia nervosa. After 10% weight gain, the receptor number almost normalized in anorexia nervosa patients. Kd values were increased in all patients groups at all times of study. In patients with bulimia or anorexia nervosa, both initially and after weight gain, the maximal effect of prostaglandin E1 (PGE1) on platelet cAMP production was greatly increased, while the half-maximally effective dose was unchanged. Also, the maximal inhibitory effects of epinephrine and clonidine on PGE1-stimulated platelet cAMP production were greater, while the half-maximal dose of both alpha 2-agonists was unchanged. Metabolic and endocrine indicators of starvation were present in both bulimic and anorexia nervosa patients initially. Blood beta-hydroxybutyric acid was elevated, and plasma T3 values and the orthostatic response of plasma norepinephrine (delta NA) were reduced, while cortisol was elevated (only in anorexia nervosa patients). Among these parameters, only delta NA significantly correlated with the actions of PGE1 and epinephrine on cAMP production. In conclusion, the activity of the sympathetic nervous system was reduced in patients with anorexia nervosa and bulimia. This reduction was accompanied by an increased capacity and a decreased affinity of platelet alpha 2-receptors and an increased PGE1 stimulatory and epinephrine inhibitory effects on cAMP production. PMID- 2997258 TI - Characterization of insulin-like growth factor I receptor on human erythrocytes. AB - [125I]Insulin-like growth factor I (IGF-I) specifically bound to erythrocytes; the binding was saturable, and time and temperature dependent. Steady state binding was reached at 16 h at 4 C, and specific binding averaged 14.3 +/- 0.7% (+/- SEM) at a concentration of 3.6 X 10(9) cells/ml in seven normal subjects. [125I]IGF-I binding to the cells was displaced by unlabeled IGF-I in a dose dependent manner. Scatchard analysis indicated a linear plot, and Ka and number of binding sites/cell were 1.43 +/- 0.07 X 10(9) M-1 and 20.7 +/- 2.2, respectively. Compared to IGF-I, the relative potencies of multiplication stimulating activity and insulin for displacing [125I]IGF-I binding were 20% and 1%, respectively. [125I]IGF-I binding to erythrocytes from patients with acromegaly was lower than binding to cells from pituitary dwarfs. An inverse correlation between plasma IGF-I level and the number of IGF-I-binding sites per cell was found (r = -0.75; P less than 0.005). These results demonstrate that [125I]IGF-I binding to erythrocytes can be used for clinical measurement of the IGF-I receptor. PMID- 2997259 TI - The effectiveness of oral iodized oil in the treatment and prophylaxis of endemic goiter. AB - In a longitudinal study carried out for 2 yr in the Darfur region, western Sudan, 2316 school children received a single dose of 2 capsules of iodized oil (400 mg iodine) orally, and 1161 school children received 1 ml of the same preparation im (475 mg iodine); 2393 school children served as controls. One year after treatment, goiter prevalence was reduced from 67.0% to 36.0% among the children who had received oral iodized oil and from 71.0% to 42.0% in those who received it im. The prevalence in the control group did not change. The prevalences in each group were approximately the same 2 yr after treatment. Urinary iodine excretion increased after treatment and remained significantly higher than the initial value during the trial. In subjects from rural Darfur, serum T4 levels were increased 1 yr after treatment with oral iodized oil (P less than 0.001) and im iodized oil (P less than 0.01), and remained high in the former (P less than 0.05) but not in the latter. This increase was accompanied by reduction of serum T3 and TSH levels. Sialadenitis occurred in 3.7% of the children who received oral iodized oil. Thyroid antibodies were not detected before treatment, but microsomal antibodies were detected in 2 of the 128 subjects studied who received iodized oil orally. Comparable results occurred when oral and im iodized oil were given to 841 individuals covering a wider age range. It is concluded that a single oral dose of iodized oil is effective in the correction of iodine deficiency, reducing the goiter size and preventing the recurrence of goiter for at least 2 yr. PMID- 2997260 TI - Gonadotropin-releasing hormone receptors in human pituitary: ligand structural requirements, molecular size, and cationic effects. AB - A specific, high affinity receptor for GnRH in human pituitaries obtained post mortem is described. The human pituitary GnRH receptor bound GnRH, a GnRH agonist [(D-Ala6,N alpha-MeLeu7,Pro9NEt)-GnRH], and a GnRH antagonist [Ac-D-Nal(2)1,D alpha-Me-4-ClPhe2,D-3-Pal3,D-Arg6,D-Ala10 )-GnRH] with similar affinities (KdS of 4.81 nM, 0.32 nM, and 0.32 nM) to those of the rat pituitary (KdS of 4.71 nM, 0.31 nM, and 0.40 nM). A second GnRH antagonist [(D-pGlu1,D-Phe2,D-Trp3,6)-GnRH], however, was bound with a much lower affinity by the human receptor (Kd of 4.21 nM) than that of the rat (Kd of 0.09 nM). Monovalent and divalent cations affected [125I]GnRH agonist binding to rat and human pituitary receptors differently. In the presence of Mg2+ or Ca2+, binding to the human receptor was significantly lower than in the rat. At near physiological concentrations, Na+ and K+ (100 mM and 10 mM, respectively) inhibited [125I]GnRH agonist binding to the receptors to a similar extent in both species. At unphysiological concentrations (10 mM Na+ and 100 mM K+) these ions decreased binding to the human pituitary receptor to a greater extent than to the rat receptor. Using a ligand-immunoblotting technique, the human receptor or binding component of the receptor complex was found to be of greater molecular size (64,000 daltons) than that of the rat (60,000 daltons). The results show that the human and rat pituitary GnRH receptors have similar binding affinities for GnRH and certain GnRH analogs but differ in their binding of an antagonist, their sensitivity to cationic effects on GnRH agonist binding, and in the molecular size of the receptor GnRH-binding protein. PMID- 2997262 TI - Rapid increase in plasma and urinary cyclic GMP after bolus injection of atrial natriuretic factor in man. AB - We studied the effects of a bolus injection of 50/micrograms synthetic human atrial natriuretic factor (ANF) on the cyclic GMP and cyclic AMP levels in plasma and urine of eight normal men. Administration of the hormone increased basal immunoreactive (IR-) ANF levels in plasma 2.8-fold to 110 pM three minutes after injection. Thereafter, IR-ANF levels rapidly declined to basal levels. Plasma cyclic GMP levels increased 2.6-fold to 16.6 nM within 6 minutes after ANF and decreased to near basal values within 30 minutes. Urinary cyclic GMP excretion increased 2.8-fold, whereas urinary volume and sodium excretion increased less than two-fold in the 30 minutes after ANF. Plasma cyclic AMP levels did not change. The data indicate that changes in plasma IR-ANF levels are followed by changes in plasma cyclic GMP and in urinary cyclic GMP excretion and suggest that cyclic GMP is a biological marker for circulating ANF in man. PMID- 2997261 TI - Low dose adrenocorticotropin infusion in continuous ambulatory peritoneal dialysis patients. AB - The adrenocorticoid responses to low doses of ACTH (0.03-10 ng/min) in sodium deplete normal subjects and end-stage renal disease patients maintained on continuous ambulator peritoneal dialysis (CAPD) were compared. All subjects were pretreated with dexamethasone. ACTH was administered by graded iv infusions in doses of 0.03, 0.3, 1.0, 3.0, and 10 ng ACTH/min. Each rate of infusion was maintained for 30 min. Plasma aldosterone, 18-hydroxycorticosterone, corticosterone, 18-hydroxy-11-deoxycorticosterone, and cortisol were measured in plasma sampled at the end of each rate of infusion in both groups. Plasma 11 deoxycorticosterone was measured in CAPD patients. The plasma steroid levels in the CAPD patients after each infusion rate were equal to or greater than the levels in normal subjects. The slopes of the cumulative increases above baseline in plasma steroid levels in the CAPD patients were equal to or greater than those in the normal subjects. In both groups, plasma corticosterone increased the most and aldosterone the least. Kinetic analyses indicated that the adrenal responses to low dose ACTH were not linear. A distinct threshold for ACTH-stimulated increase in plasma adrenocorticoid levels, if present, is very low. The responses of plasma adrenocorticoids to low dose ACTH are normal in CAPD patients. PMID- 2997263 TI - Adrenal steroids in maternal and cord blood after dexamethasone administration at midterm. AB - We undertook a study designed to evaluate whether it is feasible to suppress fetal adrenal secretion of androgens at mid-pregnancy by giving dexamethasone (DX) to the mother. Levels of DX and adrenal steroids were measured in maternal and cord plasma of 13 DX-treated and 16 untreated mothers undergoing abortion at 18-20 weeks of pregnancy. Maternal adrenal suppression was evidenced by a sharp fall of plasma cortisol (F), cortisone (E), corticosterone (B), and dehydroepiandrosterone sulfate (DHEA-S). However, in cord blood no fall of DHEA-S or corticosterone sulfate (BS) was found up to 20 hours after DX administration, and cord plasma ACTH remained detectable. The failure of DX to suppress the fetal adrenal at mid-pregnancy suggests that this drug would not be effective in the intrauterine treatment of congenital adrenal hyperplasia (C.A.H.). PMID- 2997265 TI - [Growth appearance of cancer cells derived from human gastric carcinoma with special reference to morphological features in vitro]. PMID- 2997264 TI - Human T-cell leukemia/lymphoma virus I and/or Epstein-Barr virus-infected B-cell lines spontaneously produce acid-labile alpha-interferon. AB - B-cell lines were established as spontaneous outgrowths of cell cultures from patients with adult T-cell leukemia (ATL). Three such lines were shown to have integrated human T-cell leukemia/lymphoma virus 1 (HTLV-I) proviral sequences as well as Epstein-Barr virus (EBV) infection. Supernatant fluids from these cultured cells were assayed for interferon (IFN) production. Acid-stable alpha IFN was found to be produced by one cell line (CF), and acid-labile alpha-IFN by the other two (HS, MJB). In contrast, HTLV-I-infected T-cell lines did not produce IFN. Some EBV-infected B-cell lines produce acid-labile alpha-IFN, while others do not. Since alpha-IFN, both acid stable and acid labile, is found in sera from patients with the polyclonal activation of B cells as a constitutive part of the disease, the above observations suggest a possible role of polyclonal B-cell activation in alpha-IFN production in these diseases. PMID- 2997266 TI - [The fine structure of intracranial neoplasms induced in dogs with Rous sarcoma virus]. PMID- 2997267 TI - Comparative efficiency of commercial immunoassays for the diagnosis of rotavirus gastroenteritis during the course of infection. AB - We evaluated the performance characteristics of three commercially available immunoassays for the detection of rotavirus antigens in stool samples obtained from infants during the course of rotavirus gastroenteritis. Two of the assays, Bio-EnzaBead (Litton Bionetics, Charleston, S.C.) and Rotazyme (Abbott Laboratories, North Chicago, Ill.), are enzyme immunoassays, while the third, Rotalex (Medical Technology Corporation, Somerset, N.J.), is a latex agglutination assay. We tested a total of 122 samples obtained from 26 children with gastroenteritis; 56 samples, obtained from 21 children, were found to contain rotavirus antigen by a reference microplate enzyme immunoassay. Rotavirus antigen was found by the Bio-EnzaBead, Rotazyme, and Rotalex assays in 53, 42, and 29 samples, respectively. The true positivity of samples which were positive by the reference microplate assay but negative by the other assays was confirmed by a specific neutralization assay or by the visualization of bands of double stranded RNA by polyacrylamide gel electrophoresis or both. No false-positive assay results were noted with any of the commercial assays. The sensitivity of the assays was determined to a great extent by the time after the onset of illness at which the specimen was collected. Thus, the sensitivity of commercial assays with specimens collected early in the course of illness did not differ significantly from that of the microplate assay. However, significantly lower degrees of sensitivity were noted later in the course of illness. Results of our studies indicate that all three commercial assays can accurately detect rotavirus in stools from children with rotavirus gastroenteritis. However, the choice of assay systems for use in the clinical laboratory will depend on the conditions in which stool specimens are collected and tested in the laboratory. PMID- 2997268 TI - Enzyme immunoassay inhibition assay for the detection of rat rotavirus-like agent in intestinal and fecal specimens obtained from diarrheic rats and humans. AB - An enzyme immunoassay inhibition assay was developed to detect rat rotavirus-like agent (RVLA) or antigenically related viruses in intestinal washings and homogenates obtained from diarrheic rats and in fecal specimens obtained from diarrheic humans. In this assay, RVLA antigens in a test sample inhibited the binding of a biotin-labeled anti-rat RVLA antibody preparation to rat RVLA antigens bound to the solid phase. Intestinal washings and homogenates obtained 1 day after RVLA infection of suckling rats inhibited the binding of the biotin labeled antibody to the solid-phase rat RVLA antigens by 76 to 100%. The inhibition was blocked by RVLA immune rat serum but not by nonimmune rat serum. Of 27 fecal specimens obtained from diarrheic humans, 6 produced disease characteristic of rat RVLA infection when inoculated into suckling rats. Four of these six specimens produced greater than 50% inhibition in the enzyme immunoassay. Fecal specimens obtained from diarrheic humans that were determined to be negative for RVLA produced an average inhibition of 9.2%. This enzyme immunoassay appears to be a useful diagnostic and research tool for the study of infections with at least one of the antigenically distinct rotaviruses. PMID- 2997269 TI - Comparison of the detection of herpes simplex virus in direct clinical specimens with herpes simplex virus-specific DNA probes and monoclonal antibodies. AB - A comparison of two commercially available kits for rapid herpes simplex virus (HSV) detection directly in patient specimens was performed. The immunofluorescence assay (IFA) utilized monoclonal antibodies to HSV, and the DNA probe assay utilized three HSV sequences cloned into pBR322. A sample of 243 specimens received in viral transport medium were inoculated into MRC-5 tissue cultures. The remainder of the specimen was centrifuged, and the cellular pellet was examined by IFA and DNA probes. One hundred and sixty-two (66.7%) specimens were considered satisfactory for IFA and DNA probe testing, based on a criterion of observing greater than or equal to 2 intact cells per high-power field. Of the 162 specimens, 35 (21.6%) yielded HSV by culture. By IFA, the sensitivity of detecting HSV culture-positive specimens was 77.1%; specificity was 100%, positive predictive value was 100%, and negative predictive value was 93.3%. DNA probe sensitivity was 71.4%; specificity was 90.6%; positive predictive value was 67.6%; and negative predictive value was 92%. Forty-four (27.2%) of the 162 specimens exhibited nonspecific cytoplasmic staining with the DNA probe. IFA and DNA probe assays can be completed in 2 to 3 h, whereas the average time to culture positivity in this series was 2.2 days. Rapid HSV diagnosis can aid in timely and appropriate patient management. PMID- 2997271 TI - Cytomegalovirus antigenic heterogeneity can cause false-negative results in indirect hemagglutination and complement fixation antibody assays. AB - Cord sera and antepartum maternal sera from three congenitally cytomegalovirus (CMV)-infected infants and their mothers were CMV seronegative (titer, less than 8) in a complement fixation (CF) assay with a glycine-extracted CMV AD169 antigen; sera from two of the infants and mothers were also seronegative in a commercial indirect hemagglutination (IHA) assay with AD169 antigen. In tests with their own CMV isolates propagated and made into glycine-extracted CF antigen, all were seropositive. When 108 random cord sera were assayed for CF antibody with AD169, Davis, and A antigens (A is a locally derived antigen from one of the above infants), 44 were seropositive and 54 were seronegative for all three antigens. Of the remaining 10 sera, 4 were positive for A only, 3 were positive for A and Davis only, 2 were positive for Davis and AD169 only, and 1 was positive for AD169 only. All 10 were positive when a mixture of all three antigens was used. The IHA assay with AD169 antigen was positive with only 4 of these 10 sera. These results suggest that up to 6% of sera may be misclassified as seronegative in the CF and IHA assays if only a single antigen is used. PMID- 2997270 TI - Rapid viral diagnosis of acute respiratory infections: comparison of enzyme linked immunosorbent assay and the immunofluorescence technique for detection of viral antigens in nasopharyngeal secretions. AB - Nasopharyngeal secretions from adults and children were obtained in Stockholm, Sweden, for routine diagnosis of influenza A virus, influenza B virus, respiratory syncytial (RS) virus, parainfluenza type 3 virus, and adenovirus infections by demonstration of viral antigens directly in the specimens. The cells in nasopharyngeal secretions were pelleted by centrifugation for preparation of cell deposits for diagnosis by the immunofluorescence technique (IF) in London, England, and in Stockholm, whereas the supernatants were used to diagnose infection by the enzyme-linked immunosorbent assay (ELISA) in Stockholm. Titrations of the various purified viruses showed that ELISA could detect viral antigens in amounts corresponding to 1 to 10 ng of virus protein per test well. In a series of 73 specimens tested for influenza A, RS, and parainfluenza type 3 viruses by IF in London and by ELISA in Stockholm, 15 of 18 RS, 14 of 15 influenza A, and 2 of 2 parainfluenza type 3 viral infections were diagnosed by ELISA as compared with IF, giving sensitivities for RS and influenza A viral diagnosis of 83 and 93%, respectively, and a specificity of 100%. In another series of specimens from 35 patients tested for influenza B virus and adenovirus, five influenza B virus and four adenovirus infections were diagnosed by both methods; one additional influenza B infection was detected only by IF and another only by ELISA. Comparisons of diagnostic results between the two methods performed in Stockholm gave nonagreement of results for 37 of 1,593 tests (2.5%) for the five viruses. The conclusion reached was that the described ELISA, although a satisfactory test, had somewhat less sensitivity than did IF for the detection of respiratory viral infections. This could possibly be explained by unnecessary dilutions of specimens at the time of collection; transportation, processing, and storage of specimens were less complicated than for IF. PMID- 2997272 TI - Rapid diagnosis of rotavirus gastroenteritis by a commercial latex agglutination test. AB - The Rotalex test, a commercial latex agglutination test for rotavirus, was compared with direct electron microscopy (EM) and the Rotazyme test I, a commercial enzyme immunoassay, for detection of rotavirus in stools of children and neonates. For initial stool specimens from 265 children (less than 3 years old) with diarrhea, the Rotalex test had a sensitivity of 81.7% and specificity of 99.5% compared with EM results. Positive and negative predictive values were 98 and 94.9%, respectively. The Rotalex test was slightly more sensitive and specific than the Rotazyme test. When daily stool specimens from patients with rotavirus gastroenteritis were examined, the sensitivity of the Rotalex test varied depending on the time of stool collection relative to the onset of symptoms. Sensitivity was 100 (20/20), 96 (23/24), and 54% (7/13) during 1 to 4, 5 to 7, and 8 to 18 days, respectively, after the onset of symptoms. The sensitivity of the Rotazyme test varied similarly with days from onset. We also examined 214 EM-negative stool specimens from asymptomatic newborns. False positivity by the Rotalex test was only 3.3% (7/214) compared with 4.2% (9/215) for the Rotazyme test. The Rotalex test was as sensitive and specific as EM for detection of rotavirus during the acute stage of illness and much faster and cheaper than EM or the Rotazyme test. The test appears to be suitable for routine use in small hospitals, emergency wards, or even the physician's office for rapid diagnosis of rotavirus gastroenteritis. PMID- 2997273 TI - Comparison of immunofluorescence with commercial monoclonal antibodies to biochemical and biological techniques for typing clinical herpes simplex virus isolates. AB - Immunofluorescence with monoclonal antibody reagents from two commercial sources for differentiating herpes simplex viruses types 1 and 2 demonstrated 100% agreement with cell culture selectivity (chicken embryo and guinea pig embryo cells) and (E)-5-(2-bromovinyl)-2'-deoxyuridine sensitivity for typing a total of 94 clinical herpes simplex virus isolates. PMID- 2997274 TI - Cefoperazone and cefoperazone-sulbactam susceptibility tests with anaerobic bacteria by the thioglycolate disk elution method. AB - Tests were performed with 104 anaerobic microorganisms to evaluate the thioglycolate disk elution technique for the detection of resistance to cefoperazone and cefoperazone-sulbactam. An unacceptably high false-resistance rate and a poor reproducibility record make the disk elution procedure unsatisfactory for routine testing of this drug or combination of drugs. PMID- 2997275 TI - Degradation of heparan sulfate in the subendothelial extracellular matrix by a readily released heparanase from human neutrophils. Possible role in invasion through basement membranes. AB - Freshly isolated human neutrophils were investigated for their ability to degrade heparan sulfate proteoglycans in the subendothelial extracellular matrix (ECM) produced by cultured corneal and vascular endothelial cells. The ECM was metabolically labeled with Na2(35S)O4 and labeled degradation products were analyzed by gel filtration over Sepharose 6B. More than 90% of the released radioactivity consisted of heparan sulfate fragments 5-6 times smaller than intact heparan sulfate side chains released from the ECM by either papain or alkaline borohydride. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of either heparin or serine protease inhibitors. In contrast, degradation of soluble high molecular weight heparan sulfate proteoglycan, which was first released from the ECM, was inhibited by heparin but there was no effect of protease inhibitors. These results indicate that interaction of human neutrophils with the subendothelial ECM is associated with degradation of its heparan sulfate by means of a specific, newly identified, heparanase activity and that this degradation is facilitated to a large extent by serine proteases. The neutrophil heparanase was readily and preferentially released (15-25% of the cellular content in 60 min) by simply incubating the cells at 4 degrees C in the absence of added stimuli. Under these conditions, less than 5% of the cellular content of lactate dehydrogenase, lysozyme, and globin degrading proteases was released. Further purification of the neutrophil heparanase was achieved by its binding to heparin-Sepharose and elution at 1 M NaCl. It is suggested that heparanase activity is involved in the early events of extravasation and diapedesis of neutrophils in response to a threshold signal from an extravascular inflamed organ. PMID- 2997276 TI - Low expression of human histocompatibility leukocyte antigen-DR is associated with hypermethylation of human histocompatibility leukocyte antigen-DR alpha gene regions in B cells from patients with systemic lupus erythematosus. AB - The relationship between the expression of HLA-DR antigens and the HLA-DR alpha gene methylation was examined in systemic lupus erythematosus (SLE). Using permanent B cell lines, we found reduced DR expression in SLE. The low DR expression was correlated with high anti-DNA antibody titers in patients' sera. The amounts of DR alpha message were lower in SLE cells than in normal controls, suggesting that the low expression of DR antigens is associated with gene functions. The extent of DNA methylation was examined at five CCGG sites in the HLA-DR alpha locus. DNA from both SLE and normal cells showed variable methylation patterns. Since the DR alpha gene is a single-copy gene, such a variability is the result of assaying a mixture of transformed clones containing methylated DR alpha gene, with other clones containing unmethylated DR alpha gene. A distinctive feature of normal cells was a consistent methylation pattern: 12 normal cell lines showed exactly the same pattern. In contrast, 28 SLE cell lines showed a cell-line-specific methylation, and hypermethylation at the DR alpha locus. The hypermethylation is often associated with transcriptionally inactive genes. Thus, our results suggest that (a) B cells with hypermethylated DR genes might express no or few DR antigens; (b) the ratio of cells with differently methylated DR genes is consistent in normal individuals, while, in SLE patients, cells with hypermethylated DR genes predominate, resulting in apparently reduced DR antigen expression; and (c) the aberrant DR expression could be associated directly with immunoregulatory dysfunctions in SLE disease. PMID- 2997277 TI - Possible role of adenosine in the macula densa mechanism of renin release in rabbits. AB - This study was designed to examine: (a) the effects of adenosine and its analogues on renin release in the absence of tubules, glomeruli, and macula densa, and (b) whether adenosine may be involved in a macula densa-mediated renin release mechanism. Rabbit afferent arterioles (Af) alone and afferent arterioles with macula densa attached (Af + MD) were microdissected and incubated for two consecutive 30-min periods. Hourly renin release rate from a single arteriole (or an arteriole with macula densa) was calculated and expressed as ng AI X h-1 X Af 1 (or Af + MD-1)/h (where AI is angiotensin I). Basal renin release rate from Af was 0.69 +/- 0.09 ng AI X h-1 X Af-1/h (means +/- SEM, n = 16) and remained stable for 60 min. Basal renin release rate from Af + MD was 0.20 +/- 0.04 ng AI X h-1 X Af + MD-1/h (n = 6), which was significantly lower (P less than 0.0025) than that from Af. When adenosine (0.1 microM) was added to Af, renin release decreased from 0.72 +/- 0.16 to 0.24 +/- 0.04 ng AI X h-1 X Af-1/h (P less than 0.025; n = 9). However, when adenosine was added to Af + MD, no significant change in renin release was observed. N6-cyclohexyl adenosine (an A1 adenosine receptor agonist) at 0.1 microM decreased renin release from Af from 0.69 +/- 0.14 to 0.39 +/- 0.12 ng AI X h-1 X Af-1/h (n = 5, P less than 0.05). However, 5' N-ethylcarboxamide adenosine (an A2 adenosine receptor agonist) either at 0.1 microM or at 10 microM had no effect. Theophylline, at a concentration (10 microM) that does not block phosphodiesterase but does block adenosine receptors, increased renin release from Af + MD from 0.21 +/- 0.03 to 0.46 +/- 0.08 ng AI X h-1 X Af + MD-1/h (P less than 0.05; n = 8). The results are consistent with the hypotheses that adenosine decreases renin release via the activation of A1 adenosine receptors, and that adenosine may be an inhibitory signal from the macula densa to juxtaglomerular cells. PMID- 2997279 TI - Alterations in adenosine triphosphate and energy charge in cultured endothelial and P388D1 cells after oxidant injury. AB - To investigate mechanisms whereby oxidant injury of cells results in cell dysfunction and death, cultured endothelial cells or P388D1 murine macrophage like cells were exposed to oxidants including H2O2, O2-. (generated by the enzymatic oxidation of xanthine), or to stimulated polymorphonuclear leukocytes (PMN). Although Trypan Blue exclusion was not diminished before 30 min, cellular ATP was found to fall to less than 30% of control values within 3 min of exposure to 5 mM H2O2. Stimulated PMN plus P388D1 caused a 50% fall in cellular ATP levels. During the first minutes of oxidant injury, total adenylate content of cells fell by 85%. Cellular ADP increased 170%, AMP increased 900%, and an 83% loss of ATP was accompanied by a stoichiometric increase in IMP and inosine. Calculated energy charge [(ATP + 1/2 AMP)/(ATP + ADP + AMP)] fell from 0.95 to 0.66. Exposure of P388D1 to oligomycin plus 2-deoxyglucose (which inhibit oxidative and glycolytic generation of ATP, respectively) resulted in a rate of ATP fall similar to that induced by H2O2. In addition, nucleotide alterations induced by exposure to oligomycin plus 2-deoxyglucose were qualitatively similar to those induced by the oxidant. Loss of cell adenylates could not be explained by arrest of de novo purine synthesis or increased ATP consumption by the Na+-K+ ATPase or the mitochondrial F0-ATPase. These results indicate that H2O2 causes a rapid and profound fall in cellular ATP levels similar to that seen when ATP production is arrested by metabolic inhibitors. PMID- 2997278 TI - Defensins. Natural peptide antibiotics of human neutrophils. AB - We extracted a granule-rich sediment from normal human neutrophils and subjected it to chromatographic, electrophoretic, and functional analysis. The extract contained three small (molecular weight less than 3,500) antibiotic peptides that were named human neutrophil peptide (HNP)-1, HNP-2, and HNP-3, and which will be referred to as "defensins." HNP 1-3, a mixture of the three defensins, killed Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli effectively in vitro when tested in 10 mM phosphate buffer containing certain nutrients, but it had little or no bactericidal activity in nutrient-free buffer. In contrast, the nutrient-free buffer supported a high degree of activity by HNP 1-3 against Cryptococcus neoformans. In addition to its antibacterial and antifungal properties, HNP 1-3 directly inactivated herpes simplex virus, Type 1. Two of the individual purified defensins, HNP-1 and HNP-2, were as microbicidal as the mixture HNP 1-3. HNP-3 was less active than the other defensins against most but not all of the microbes tested. Immunoperoxidase stains revealed HNP 1-3 to have a granular localization in the neutrophil's cytoplasm by light microscopy. Frozen thin section immunogold transmission electron microscopy showed HNP 1-3 to be localized in azurophil granules. These studies define a broad-spectrum antimicrobial system in human neutrophils. The defensin system may operate in conjunction with or independently from oxygen-dependent microbicidal processes to enable human neutrophils to inactivate and destroy potential pathogens. PMID- 2997280 TI - Central serotonergic stimulation of aldosterone secretion. AB - Serotonin stimulates aldosterone secretion both in vitro and in vivo, and serotonin antagonism decreases plasma aldosterone levels in patients with idiopathic aldosteronism. This study was designed to assess the effects of the serotonin precursor, 5-hydroxytryptophan (5HTP), upon aldosterone secretion in man, and to determine whether stimulatory effects of 5HTP are mediated through the central nervous system. Oral 5HTP, administered as a single 200-mg dose, increased plasma aldosterone levels from 4.7 +/- 0.6 to 13.3 +/- 2.8 ng/dl in dexamethasone-pretreated, normal volunteers. Peripheral inhibition of decarboxylation of 5HTP, achieved by pretreatment with carboxydopa, 25 mg three times daily for 3 d, significantly increased the stimulatory effects of 5HTP on aldosterone levels (P less than 0.001). No change in aldosterone levels occurred in subjects who received placebo after pretreatment with dexamethasone and carboxydopa. Increased aldosterone was not accompanied by increases in plasma levels of renin activity, potassium, or ACTH. Plasma levels of 5HTP were markedly increased by carboxydopa pretreatment, but peak plasma levels of serotonin were not significantly altered. Four patients with idiopathic aldosteronism all had an increase in plasma aldosterone levels after 5HTP administration, whereas the response in four patients with aldosterone-producing adenoma was variable. Incubation of isolated human and rat adrenal glomerulosa cells with serotonin resulted in increased aldosterone secretion by both sets of cells, whereas 5HTP was ineffective in stimulating aldosterone secretion in vitro. We conclude that central serotonergic pathways are involved in the stimulation of aldosterone induced by administration of 5HTP. This mechanism may be an important etiologic factor in the hypersecretion of aldosterone that occurs in patients with idiopathic aldosteronism. PMID- 2997281 TI - Effects of vasopressin antagonist on vasopressin binding, adenylate cyclase activation, and water flux. AB - We studied the effect of an arginine vasopressin (AVP) analogue, (1-[beta mercapto-beta, beta-cyclopentamethylenepropionic acid],2-O-ethyltyrosine, 4 valine)AVP(d[CH2]5Tyr[Et]VAVP), on the stimulation of adenylate cyclase by various hormones in the isolated nephron segments and 3H-AVP binding to renal papillary membranes from the rat. The net water flux across the renal cortical collecting tubules of the rabbit was also examined. We found that d(CH2)5Tyr(Et)VAVP significantly inhibited adenylate cyclase activation by AVP in cortical, medullary, and papillary collecting tubules and in the medullary thick ascending limb. In contrast, the AVP analogue did not alter the stimulation of adenylate cyclase by parathyroid hormone in the cortical thick ascending limb, by glucagon in the medullary thick ascending limb, and by calcitonin in cortical collecting tubules. In addition, d(CH2)5Tyr(Et)VAVP blocked [3H]AVP binding to renal papillary membranes. The enhanced net water transport induced by AVP in isolated, perfused rabbit cortical collecting tubules also was completely blocked by this AVP analogue. These results indicate that d(CH2)5Tyr(Et)VAVP specifically antagonizes the cellular action of AVP on the medullary thick ascending limb and on the cortical, medullary, and papillary collecting tubules. Evidence is also presented for competitive antagonism as the cellular mechanism of action. PMID- 2997282 TI - Aging decreases the capacity of human skin to produce vitamin D3. AB - An evaluation of surgically obtained skin (age range, 8-92 yr) revealed that there is an age-dependent decrease in the epidermal concentrations of provitamin D3 (7-dehydrocholesterol). To ascertain that aging indeed decreased the capacity of human skin to produce vitamin D3, some of the skin samples were exposed to ultraviolet radiation and the content of previtamin D3 was determined in the epidermis and dermis. The epidermis in the young and older subjects was the major site for the formation of previtamin D3, accounting for greater than 80% of the total previtamin D3 that was produced in the skin. A comparison of the amount of previtamin D3 produced in the skin from the 8- and 18-yr-old subjects with the amount produced in the skin from the 77- and 82-yr-old subjects revealed that aging can decrease by greater than twofold the capacity of the skin to produce previtamin D3. Recognition of this difference may be extremely important for the elderly, who infrequently expose a small area of skin to sunlight and who depend on this exposure for their vitamin D nutritional needs. PMID- 2997283 TI - A distant gene deletion affects beta-globin gene function in an atypical gamma delta beta-thalassemia. AB - We describe an English family with an atypical gamma delta beta-thalassemia syndrome. Heterozygosity results in a beta-thalassemia phenotype with normal hemoglobin A2. However, unlike previously described cases, no history of neonatal hemolytic anemia requiring blood transfusion was obtained. Gene mapping showed a deletion that extended from the third exon of the G gamma-globin gene upstream for approximately 100 kilobases (kb). The A gamma-globin, psi beta-, delta-, and beta-globin genes in cis remained intact. The malfunction of the beta-globin gene on a chromosome in which the deletion is located 25 kb away suggests that chromatin structure and conformation are important for globin gene expression. PMID- 2997284 TI - Human fat cell lipolysis is primarily regulated by inhibitory modulators acting through distinct mechanisms. AB - The effects of adenosine deaminase and of pertussis toxin on hormonal regulation of lipolysis were investigated in isolated human fat cells. Adenosine deaminase (1.6 micrograms/ml) caused a two-to threefold increase in cyclic AMP, which was associated with an increase in glycerol release averaging 150-200% above basal levels. Clonidine, N6-phenylisopropyladenosine, prostaglandin E2, and insulin caused a dose-dependent inhibition of glycerol release in the presence of adenosine deaminase. Pretreatment of adipocytes with pertussis toxin (5 micrograms/ml) for 180 min resulted in a five- to sevenfold increase in cyclic AMP. Glycerol release was almost maximal and isoproterenol caused either no further increase or only a marginal additional increase of lipolysis after pretreatment with pertussis toxin, whereas cyclic AMP levels were 500 times higher than in controls. The effects of antilipolytic agents known to affect lipolysis by inhibition of adenylate cyclase activity, i.e., clonidine, N6 phenylisopropyladenosine, and prostaglandin E2, were impaired. In contrast, the antilipolytic action of insulin was preserved in adipocytes pretreated with pertussis toxin. As in controls, the peptide hormone had no detectable effect on cyclic AMP after pertussis toxin treatment. The findings support the view that the antilipolytic effect of insulin does not require adenylate cyclase or phosphodiesterase action. In addition, the results demonstrate that, upon relief of endogenous inhibition, human fat cell lipolysis proceeds at considerable (adenosine deaminase) or almost maximal (pertussis toxin) rates. A certain degree of inhibition, therefore, appears to be necessary for human fat cell lipolysis to be susceptible for hormonal activation. PMID- 2997285 TI - Presence of epidermal-derived thymocyte activating factor/interleukin 1 in normal human stratum corneum. AB - Keratinocytes produce a molecule, epidermal-derived thymocyte activating factor (ETAF), which is biologically and physiochemically similar to the polypeptide hormone interleukin 1 (IL-1). Because the stratum corneum (SC) is composed of terminally differentiated keratinocytes, we questioned whether ETAF/IL-1 could be isolated from this tissue. The extraction of normal human SC with a physiologic saline solution yielded a large amount of ETAF/IL-1 activity, as measured by the in vitro thymocyte co-stimulator assay. SC-derived ETAF/IL-1 (scETAF/IL-1) eluted from a sizing column with an approximate molecular weight of 15,000, and demonstrated three isoelectric point forms after separation on a chromatofocusing column. By these physiochemical characteristics, scETAF/IL-1 was found to be similar, if not identical to human keratinocyte- and macrophage-derived ETAF/IL 1. Further, a number of biologic effects known to occur in vivo after the administration of ETAF/IL-1, such as fever, neutrophilia, and an increase in plasma levels of acute-phase proteins, were all induced by the injection of scETAF/IL-1 into endotoxin-nonresponsive mice. scETAF/IL-1 was also found to stimulate collagenase production by human fibroblasts in vitro. In summary, our studies have established that normal human SC contains a large quantity of scETAF/IL-1. Whether scETAF/IL-1 integrates into the earliest afferents phases of local inflammatory responses, or merely represents a means of disposal of excessively produced hormone is currently unresolved. PMID- 2997286 TI - Long-term effects of dietary marine omega-3 fatty acids upon plasma and cellular lipids, platelet function, and eicosanoid formation in humans. AB - We studied the incorporation and metabolism of eicosapentanoic (EPA) and docosahexaenoic acid in six human volunteers who supplemented their normal Western diet for 5 mo daily with 10-40 ml of cod liver oil, rich in omega-3 polyunsaturated fatty acids. EPA and docosahexaenoic acid were incorporated into the total phospholipids of plasma, platelets, and erythrocytes in a dose- and time-dependent manner. During omega-3 fatty acid ingestion serum triacylglycerols were lowered and platelet aggregation upon low doses of collagen was reduced. Concomitantly, formation and excretion of prostanoids showed a characteristic change. As measured in serum from whole clotted blood, thromboxane A3 was formed in small amounts, whereas thromboxane A2 formation was reduced to 50% of control values. Excretion of the main urinary thromboxane A metabolites was unaltered in subjects with low basal excretion rates, but decreased markedly in two subjects with high control values. As determined from the main urinary metabolite, prostaglandin I3 was formed from EPA at rates up to 50% of unaltered prostaglandin I2 formation. The biochemical and functional changes observed lasted for the entire supplementation period of 5 mo and were reversible within 12 wk after cessation of cod liver oil intake. Favorable changes induced by long chain omega-3 fatty acids include a dose-related and sustained shift of the prostaglandin I/thromboxane A balance to a more antiaggregatory and vasodilatory state. PMID- 2997288 TI - Effect of atrial peptides on aldosterone production. AB - This study examines the effects of the synthetic atrial peptides (atriopeptin I, II, and III) on aldosterone and corticosterone production by rat adrenal cell suspensions. Furthermore, we studied the effect of atriopeptin II infusion on the plasma aldosterone response to angiotensin II in the rat in vivo. Atriopeptin I, II, and III decreased aldosterone release from zona glomerulosa cells in a dose dependent fashion. 10 pM atriopeptin II inhibited basal aldosterone release significantly (P less than 0.01), and 10 nM atriopeptin II or III lowered it by 79%. Atriopeptin II decreased the sensitivity of the glomerulosa cells to adrenocorticotropic hormone (ACTH) and angiotensin II. Atriopeptin II had no effect on basal or ACTH-stimulated corticosterone release by fasciculata medullary cells. In vivo infusions of angiotensin II with or without simultaneous infusions of atriopeptin II showed that atriopeptin II significantly inhibited the aldosterone response to angiotensin II. This inhibition by atriopeptin II was independent of any effect on plasma renin activity, serum potassium, or ACTH. These data raise the possibility that the atrial natriuretic peptides may affect sodium excretion by the kidney, not only directly, but also indirectly through the inhibition of aldosterone production. PMID- 2997287 TI - Prospective study of cytotoxic T lymphocyte responses to influenza and antibodies to human T lymphotropic virus-III in homosexual men. Selective loss of an influenza-specific, human leukocyte antigen-restricted cytotoxic T lymphocyte response in human T lymphotropic virus-III positive individuals with symptoms of acquired immunodeficiency syndrome and in a patient with acquired immunodeficiency syndrome. AB - Peripheral blood leukocytes (PBL) from 18 homosexual men who did not have acquired immunodeficiency syndrome (AIDS) and from 9 heterosexual men were repetitively tested for their ability to generate HLA self-restricted cytotoxic T lymphocyte responses to influenza virus (flu-self) over a 2-yr period. The sera of the same donors were tested for antibodies to human T lymphotropic virus-III (HTLV-III). Six of the homosexual and none of the heterosexual donors consistently generated weak cytotoxic T lymphocyte responses to flu-self. Seven of the homosexual and none of the heterosexual donors were seropositive for antibodies to HTLV-III. No obvious correlation was detected between weak flu-self cytotoxic T lymphocyte responses and antibodies to HTLV-III. However, one homosexual donor generated no detectable cytotoxic T lymphocyte activity to flu self, although he was a strong responder to HLA-alloantigens. This donor had an OKT4:OKT8 ratio of 0.4 and was seropositive for HTLV-III antigens; HTLV-III virus was identified in his PBL; and he developed AIDS during the course of this study. A second donor with lymphadenopathy and who was seropositive for HTLV-III antigens exhibited marginal cytotoxic T lymphocyte activity to flu-self which he subsequently lost. PBL from two patients, one with Kaposi's sarcoma and one with generalized lymphadenopathy, were also tested for cytotoxic T lymphocyte responses to flu-self and to alloantigens. Both donors failed to generate cytotoxic T lymphocyte to flu-self, but generated strong cytotoxic T lymphocyte responses to alloantigens. The selective loss of an HLA-restricted cytotoxic T lymphocyte response without loss of HLA alloantigenic cytotoxic T lymphocyte activity may be an important functional immunologic characteristic in the development of AIDS. PMID- 2997289 TI - Bradykinin stimulation of oxidative metabolism in renal cortical tubules from rabbit. Possible role of arachidonic acid. AB - Vasoactive peptides may have direct effects on both renal vasculature and renal tubules. In this study, we examined the direct and immediate effects of bradykinin on oxygen consumption by suspensions of cortical tubules from rabbit kidney. Bradykinin (10(-11) to 10(-7) M) stimulated oxygen consumption rates (QO2) in a dose-dependent manner with a maximal increase of +0.80 +/- 0.13 nmol X mg protein-1 X min-1. This stimulation was prevented by calcium-free media or by the addition of inhibitors of calcium transport, calcium-calmodulin complex formation, Na,K-ATPase activity, mitochondrial respiration, and phospholipase activity. Addition of bradykinin increased the ADP and AMP contents of cortical tubules without changing the ATP content. These data indicate that bradykinin stimulates ATP use and Na,K-ATPase activity. We also examined the effects of exogenous arachidonic acid on QO2 in cortical tubules. Acute additions of arachidonic acid stimulated QO2 at low concentrations (10(-8) to 10(-6) M) and uncoupled mitochondrial respiration at high concentrations (10(-5) M). The effect of arachidonic acid on adenosine nucleotide content was dose-dependent and indicated increased use of ATP. Bradykinin increased QO2 in the presence of low concentrations of arachidonic acid (10(-11) to 10(-9) M), but had no further effect on QO2 in the presence of higher concentrations of arachidonic acid (10( 8) to 10(-6) M). Bradykinin stimulation of QO2 was not prevented by inhibition of cyclooxygenase activity with indomethacin but was prevented by inhibition of lipoxygenase-like activity with nordihydroguariaretic acid. These results suggest that the bradykinin effect on QO2 may be mediated by arachidonic acid release and subsequent metabolism. PMID- 2997290 TI - Chloride secretory mechanism induced by prostaglandin E1 in a colonic epithelial cell line. AB - Confluent T84 monolayers grown on permeable supports and mounted in a modified Ussing chamber secrete chloride (Cl-) in response to prostaglandin E1. The threshold stimulation was observed at 10(-9) M and a maximal effect at 10(-6) M. Unidirectional flux studies showed an increase in both serosal to mucosal and mucosal to serosal Cl- fluxes with 10(-6) M prostaglandin E1; the increase in serosal to mucosal Cl- flux exceeded the increase in mucosal to serosal flux, resulting in net Cl- secretion. Na+ transport was not affected in either direction and the changes in net Cl- flux correlated well with the changes in short circuit current. To identify the electrolyte transport pathways involved in the Cl- secretory process, the effect of prostaglandin E1 on ion fluxes was tested in the presence of putative inhibitors. Bumetanide was used as an inhibitor for the basolaterally localized Na+,K+,Cl- cotransport system whose existence and bumetanide sensitivity have been verified in earlier studies (Dharmsathaphorn et al. 1984. J. Clin. Invest. 75:462-471). Barium was used as an inhibitor for the K+ efflux pathway on the basolateral membrane whose existence and barium sensitivity were demonstrated in this study by preloading the monolayers with 86Rb+ (as a tracer for K+) and simultaneously measuring 86Rb+ efflux into both serosal and mucosal reservoirs. Both bumetanide and barium inhibited the net chloride secretion induced by prostaglandin E1 suggesting the involvement of the Na+,K+,Cl- cotransport and a K+ efflux pathways on the basolateral membrane in the Cl- secretory process. The activation of another Cl- transport pathway on the apical membrane by prostaglandin E1 was suggested by Cl- uptake studies. Our findings indicate that the prostaglandin E1-stimulated Cl- secretion, which is associated with an increase in cyclic AMP level, intimately involves (a) a bumetanide-sensitive Na+,K+,Cl- cotransport pathway that serves as a Cl- uptake step across the basolateral membrane, (b) the stimulation of a barium-sensitive K+ efflux mechanism on the basolateral membrane that most likely acts to recycle K+, and (c) the activation of a Cl- transport pathway on the apical membrane that serves as a Cl- exit pathway. PMID- 2997291 TI - Synergistic action of cyclic adenosine monophosphate- and calcium-mediated chloride secretion in a colonic epithelial cell line. AB - Vasoactive intestinal polypeptide (VIP) and the calcium ionophore A23187 caused dose-dependent changes in the potential difference and the short circuit current (Isc) across confluent T84 cell monolayers mounted in modified Ussing chambers. Both VIP and A23187 stimulated net chloride secretion without altering sodium transport. Net chloride secretion accounted for the increase in Isc. When A23187 was tested in combination with VIP, net chloride secretion was significantly greater than predicted from the calculated sum of their individual responses indicating a synergistic effect. VIP increased cellular cyclic AMP (cAMP) production in a dose-dependent manner, whereas A23187 had no effect on cellular cAMP. We then determined whether VIP and A23187 activated different transport pathways. Earlier studies suggest that VIP activates a basolaterally localized, barium-sensitive potassium channel as well as an apically localized chloride conductance pathway. In this study, stimulation of basolateral membrane potassium efflux by A23187 was documented by preloading the monolayers with 86Rb+. Stimulation of potassium efflux by A23187 was additive to the VIP-stimulated potassium efflux. By itself, 0.3 microM A23187 did not alter transepithelial chloride permeability, and its stimulation of basolateral membrane potassium efflux caused only a relatively small amount of chloride secretion. However, in the presence of an increased transepithelial chloride permeability induced by VIP, the effectiveness of A23187 on chloride secretion was greatly augmented. Our studies suggest that cAMP and calcium each activate basolateral potassium channels, but cAMP also activates an apically localized chloride channel. Synergism results from cooperative interaction of potassium channels and the chloride channel. PMID- 2997293 TI - Characterization of human platelet vasopressin receptors. AB - Using tritiated arginine-8-vasopressin [3H]AVP, vasopressin-specific binding sites were detected on human platelet membranes. One class of high-affinity binding sites was characterized with an equilibrium dissociation constant of 1.01 +/- 0.06 nM and a maximal binding capacity of 100 +/- 10 fmol/mg of protein (n = 12). Highly significant correlations were found between the relative agonistic (r = 0.87, P = 0.002) or antagonistic (r = 0.99, P = 0.007) vasopressor activities of a series of 13 AVP structural analogues and their relative abilities to inhibit [3H]AVP binding to platelet receptors whereas no such relationship existed when antidiuretic activities were considered (r = 0.28, P = 0.47). AVP did not stimulate cyclic AMP production of human platelets; on the contrary, high AVP concentrations (10(-6) M) inhibited cyclic AMP production measured in basal and prostaglandin E1-stimulated conditions. AVP caused intact platelet aggregation with a half-maximal aggregation (EC50) of 28 +/- 2 nM. This effect was more potently reversed by the specific vascular antagonist d(CH2)5Tyr(Me)AVP (pA2 = 8.10 +/- 0.23) than by the specific renal antagonist d(CH2)5IleuAlaAVP (pA2 = 6.67 +/- 0.12). The pA2 values of these two antagonists in platelets are in close agreement with the pKi values obtained in competition experiments (respectively 8.59 and 6.93) and with pA2 values reported in the literature for their in vivo antivasopressor activity (respectively 8.62 and 6.03). The observation that human platelets bear AVP receptors belonging to the vascular class suggests that platelet receptors can be used to further explore the role of vasopressin in cardiovascular homeostasis. PMID- 2997292 TI - Inhibition of parathyroid hormone secretion and parathyroid hormone-independent diminution of tubular calcium reabsorption by WR-2721, a unique hypocalcemic agent. AB - Hypocalcemia has been observed in patients receiving WR-2721 [S-,2-(3 aminopropylamino)-, ethylphosphorothioic acid]. WR-2721 is a compound that, after being dephosphorylated, provides protection of normal tissues against radio- and chemotherapy. The hypocalcemic response was accompanied by a decrease in the plasma level of parathyroid hormone (PTH) and by hypomagnesemia. Our present studies in rats on the mechanism of the hypocalcemic effect of WR-2721 indicate that: (a) The phosphorylated and dephosphorylated form of WR-2721 induced an equal dose-dependent decrement in plasma calcium. (b) In intact rats a maximal hypocalcemic dose of WR-2721 reduced urinary cyclic AMP excretion from 70.5 +/- 6.3 to 38.2 +/- 3.1 pmol/ml glomerular filtration rate (GFR), a level comparable to that observed (35.9 +/- 5.2 pmol/ml GFR) in thyroparathyroidectomized (TPTX) rats. (c) WR-2721 given to TPTX rats did not significantly interfere with the calcemic effect of bovine PTH 1-34 infused at 2.5 IU/h. Likewise, the drug did not impair the PTH actions on the renal Ca and inorganic phosphate (Pi) handling, and on the urine cyclic AMP excretion. (d) In TPTX rats made normocalcemic by low Pi diet, the hypocalcemic effect of WR-2721 was only about 25% of that observed in intact animals. However, it was associated with increased urine Ca per milliliter GFR, indicating a PTH-independent inhibitory effect on tubular Ca reabsorption. (e) In WR-2721-treated intact rats, prevention of hypomagnesemia by infusing magnesium chloride did not reduce hypocalcemia. In conclusion, the hypocalcemic effect of WR-2721 is not dependent upon the presence of a phosphate group in the molecule and is not causally related to hypomagnesemia. WR-2721 appears to be a unique hypocalcemic pharmacologic agent with strong inhibitory activity on PTH secretion and additional PTH-independent action on renal Ca reabsorption. PMID- 2997295 TI - Studies on multiple thyroid cell membrane-directed antibodies in Graves' disease. AB - Immunoglobulin G was obtained from the serum of a woman who had given birth to three children with a delayed onset of hyperthyroidism; the clinical events were due to the coexistence of thyroid-stimulating antibody (TSAb) and an inhibitor of TSAb in the maternal serum. The current studies explore the possible existence of additional thyroid membrane-directed antibodies. Human thyroid slices, cells in monolayer culture, and functioning rat thyroid cells (FRTL5), with measurement of cyclic AMP concentration, were used for TSAb assays. Assays of the inhibition of binding of 125I-thyrotropin (TSH) to its receptor used human thyroid and FRTL5 cells, and human thyroid and guinea pig fat cell membranes as receptors. All activities were associated with IgG kappa. Fractions of IgG kappa obtained by adsorption to and the desorption from human thyroid and guinea pig fat cell preparations and F(ab')2 and Fab fragments of the parent IgG were tested. Results indicated that there were three activities in the IgG, namely, TSAb; an inhibitor of TSH-binding that was active in all species and preparations tested, and was effective as Fab and F(ab')2 on both particulate and solubilized thyroid membranes; and an enhancer of TSH-binding (e.g., approximately equal to 220% increase in binding) that was relatively specific for human thyroid membranes only in particulate form, was not adsorbed by fat, and was active as F(ab')2, but minimally as Fab. The concept is developed that dilution of the total IgG, experimentally in vitro or by metabolic clearance in vivo in neonates, determines the effect on either thyroid stimulation or TSH-binding. The incidence of such multiple antibodies and their interaction remains to be determined. PMID- 2997294 TI - Calcium uptake by intestinal brush border membrane vesicles. Comparison with in vivo calcium transport. AB - In prior studies, we examined kinetics of steady state in vivo transepithelial calcium transport in rat and hamster. The present studies related calcium uptake by the brush border to in vivo transport. We measured calcium uptake by brush border membrane vesicles from the two species. In the rat, our prior in vivo studies had shown that (a) calcium transport was mediated, (b) no nonmediated component was detectable, and (c) Vmax was 2.5 times greater in proximal than distal small intestine. In brush border membrane vesicles from the rat, Vmax for the saturable component of calcium uptake was again 2.5 times greater in proximal than distal intestine. Contrasting with in vivo studies, a major nonsaturable component was present in vesicles from proximal and distal small intestine. In the hamster, our previous in vivo studies had shown (1) both mediated and nonmediated components of calcium transport, (2) greater nonmediated transport in proximal than distal small intestines, and (3) Vmax for calcium transport twice as great in distal as in proximal small intestine. In the present study with brush border membrane vesicles from hamster, Vmax for saturable calcium transport was again twice as great in distal as in proximal small intestine. However, nonsaturable calcium transport rates relative to saturable rates were much greater with vesicles than in in vivo studies, and were greater in vesicles from distal than proximal small intestine. Since rates of saturable calcium uptake by brush border membrane vesicles parallel corresponding in vivo mediated transport rates, we conclude that the segmental rates of calcium transport in rat and hamster could be determined by brush border function. PMID- 2997296 TI - Altered proopiomelanocortin gene expression in adrenocorticotropin-producing nonpituitary tumors. Comparative studies with corticotropic adenomas and normal pituitaries. AB - In order to assess the mechanisms of proopiomelanocortin (POMC) gene expression in human ACTH-producing tumors, we performed the simultaneous evaluation of POMC products and messenger RNA (mRNA) in tissue fragments obtained from two corticotropic adenomas, five nonpituitary tumors, and two normal human pituitaries. The POMC products were examined using a combination of gel exclusion chromatography and four different radioimmunoassays directed against gamma 3 melanocyte stimulating hormone (gamma 3MSH), ACTH, gamma-lipotropin (gamma LPH), and beta-endorphin. The POMCmRNA was detected and analyzed by dot and northern blot hybridization using a single-stranded genomic DNA probe corresponding to the coding region of the human POMC gene. Tissue concentrations of POMC products and mRNA showed parallel distributions. Immunoreactive gamma 3MSH and gamma LPH patterns revealed only 16-kD fragment- and gamma LPH-like peptides in normal and tumoral pituitaries; additional gamma 3MSH- and/or beta MSH-like peptides were found in all five nonpituitary tumors. A single POMCmRNA of approximately 1,200 bases (b) was detected in normal and tumoral pituitaries; a single identical POMCmRNA was also found in four nonpituitary tumors. A thymic carcinoid tumor, in addition to the 1,200-b POMCmRNA, contained equal amounts of a second larger POMCmRNA of approximately 1,450 b. It is concluded that POMC gene expression appears qualitatively unaltered in corticotropic adenomas. In nonpituitary tumors, in contrast, abnormal POMC processing is frequent; in addition, an extra POMCmRNA was detected in a thymic tumor with a greater length than the normal mRNA; the mechanisms and pathophysiological implications of these modifications remain to be elucidated. PMID- 2997297 TI - Activation of the human neutrophil nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase by protein kinase C. AB - A variety of phagocytosable and soluble agonists stimulate the human neutrophil respiratory burst enzyme, NADPH-oxidase, an activity required for normal microbicidal function. Of these agonists, the phorbol esters, which stimulate diverse systems by their ability to substitute for diacylglycerol to activate protein kinase C (the major phorbol ester receptor), have now been shown to directly stimulate NADPH-oxidase through this same receptor. Almost 90% of the specific receptors for phorbol 12,13-dibutyrate (PDBu) were found in the cytosol upon subcellular fractionation. The dissociation constant for [3H]PDBu was 1.2 nM. No significant difference was found in the distribution of the receptor between subcellular fractions from resting as compared with phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils. On the basis of these binding studies, we were able to establish a reconstituted system in which PMA activated dormant NADPH-oxidase in a light membrane fraction when cytosol, NADPH, phosphatidylserine, or phosphatidylinositol and ATP were added. The calcium chelator, EGTA, inhibited the activation, which suggested a requirement for calcium at low concentrations. The half-maximally effective PMA dose was 1.1 nM, as predicted from the receptor content in these preparations. Reconstitution of oxidase activity was rapid, peaking within 1 min of incubation. Purified protein kinase C was able to substitute for the cytosol fraction, and accounted for 80% of the cytosol activity. These studies demonstrate that phorbol esters stimulate the neutrophil respiratory burst through activation of cytosolic protein kinase C, which in turn activates either a regulatory constituent or the NADPH-oxidase directly in the plasma membrane to generate an active O-2-generating system. PMID- 2997298 TI - 3-Hydroxy-3-methylglutaryl coenzyme A reductase in anencephalic and normal human fetal liver. AB - In previous investigations, we have found that the liver appears to be the major source of cholesterol in the human fetus, and, in particular, a principal source of circulating low density lipo-protein-cholesterol (LDL-C). LDL-C plasma levels are low in the normal fetus, most likely due to the rapid uptake and metabolism by the fetal adrenal as precursor for steroid hormone biosynthesis. In contrast, in the anencephalic fetus the adrenals are atrophic, the rate of estrogen and glucocorticoid production is low, and the levels of LDL-C in fetal plasma are high. The purpose of the present investigation was to determine the activity of 3 hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the primary rate limiting enzyme of cholesterol biosynthesis, in anencephalic liver and normal fetal liver. We found that the specific activity of HMG-CoA reductase in normal fetal liver microsomes was 0.428 +/- 0.054 nmol mevalonate formed times mg-1 protein X min-1 (mean +/- SE, n = 9). The rate of HMG-CoA reductase in anencephalic liver microsome preparations was 10-fold less (0.040 +/- 0.003) (mean +/- SE, n = 7) P less than 0.001. Furthermore, we detected HMG-CoA reductase (97,000-mol wt protein) in normal human fetal liver after SDS PAGE and immunoblotting by using a monoclonal antibody directed against HMG-CoA reductase. We were unable to detect any significant quantity of HMG-CoA reductase protein in anencephalic fetal liver, which indicates that low reductase activity was due to low amounts of enzyme protein rather than inactive enzyme. In summary, we conclude that the low levels of cholesterol synthesis observed in anencephalic fetal liver are probably due to both the high levels of LDL-C in fetal plasma as well as the presence of low circulating levels of estrogens and glucocorticoids and that these factors regulate cholesterol synthesis both in vivo and in vitro in fetal liver. This occurs most probably by the modulation of the amount of HMG CoA reductase, a primary rate-limiting and regulatory enzyme of the cholesterol biosynthetic sequence. PMID- 2997300 TI - Defective binding and function of 1,25-dihydroxyvitamin D3 receptors in peripheral mononuclear cells of patients with end-organ resistance to 1,25 dihydroxyvitamin D. AB - Lectin-induced DNA synthesis by peripheral mononuclear cells from 17 normal donors was inhibited (40-60%) by 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) at physiological concentrations (10(-10)-10(-9) M). The lymphocytes acquire specific receptors for 1,25(OH)2D3 upon activation by the lectins. This process precedes the inhibitory effect of 1,25(OH)2D3. We studied lymphocytes from six patients from four different kindreds with the syndrome of hereditary end-organ resistance to 1,25(OH)2D (the so-called vitamin D-dependent rickets type II). In five patients (three kindreds) peripheral blood mononuclear cells did not acquire receptors for 1,25(OH)2D3 upon phytohemagglutinin-induced activation. Moreover, in contrast to normal lymphocytes, the mitogenic stimulation of these patients' lymphocytes by phytohemagglutinin and concanavalin A was not inhibited by 1,25(OH)2D3. Activated lymphocytes of the sixth patient from a fourth kindred exhibited normal binding of [3H]1,25(OH)2D3 but the hormone failed to inhibit the mitogenic stimulation. A similar pattern of the vitamin D effector system was previously observed in fibroblasts cultured from skin biopsies of the same group of patients. The conclusions from these findings are: (a) the inhibition of mitogenic stimulation by 1,25(OH)2D3 is mediated by specific functional receptors to the hormone; and (b) the receptors for 1,25(OH)2D3 in mononuclear cells are probably controlled genetically by the same mechanisms as the effector system in well-characterized target organs of the hormone, such as intestine and kidney. PMID- 2997299 TI - Production of and in vitro response to interleukin 2 in the acquired immunodeficiency syndrome. AB - To test the hypothesis that deficient interleukin 2 (IL-2) secretion may underlie the impaired capacity of T cells from patients with Acquired Immunodeficiency Syndrome (AIDS) and the AIDS-related complex (ARC) to generate the macrophage activating lymphokine, gamma interferon (IFN-gamma), we used five specific microbial antigens to examine IL-2 production. Mononuclear cells from only one of 32 (3%) AIDS patients secreted normal levels of IL-2, and 21 (66%) failed to produce any detectable IL-2. For 36 ARC patients, IL-2 generation was normal in nine (25%) and absent in 11 (31%). Given these results, recombinant (r) IL-2 was tested for its capacity to stimulate or enhance IFN-gamma production. rIL-2 (10 U/ml) alone stimulated cells from controls, ARC, and AIDS patients to secrete 93 +/- 25, 99 +/- 33, and 7 +/- 3 U/ml of IFN-gamma, respectively. rIL 2 (10 U/ml) plus antigen induced no change in mean IFN-gamma levels for controls, a 4.4-fold increase for 17 AIDS patients (16 +/- 16 vs. 71 +/- 21 U/ml), and a 7.2-fold increase (18 +/- 5 vs. 130 +/- 27 U/ml) for 19 ARC patients with abnormal IFN gamma generation to antigen alone. Individual responses indicated that six of the 17 (35%) AIDS patients with opportunistic infections and 12 of the 19 (63%) with ARC were apparent responders to 10-100 U/ml of rIL-2. These results (a) document profound impairment in antigen-induced IL-2 secretion by AIDS and ARC T cells, (b) indicate that, in vitro, mononuclear cells from certain patients can respond to rIL-2 with enhanced IFN-gamma production, and thus (c) suggest that in selected patients rIL-2 might have a potentially beneficial therapeutic (AIDS) or prophylactic (ARC) effect against opportunistic infections. PMID- 2997302 TI - Acoustic intensity histogram pattern diagnosis of liver diseases. AB - Display information on the histogram pattern includes echo intensity and echo frequency in the region of interest (ROI). The level and frequency of the center of gravity and the width of the echo level at 0%, 10%, and 50% of normalized frequency are calculated from the histogram. The width of the echo from the liver is significantly elongated on the X axis (P less than 0.001) but that of the center of gravity is not. Fatty liver shows the same attenuation rate curve as normal liver, although echo intensity is greatly increased. The curve of the attenuation rate derived from the histogram can be used to distinguish between liver cirrhosis and the tissue of normal or fatty liver. This study clearly demonstrates the clinical usefulness of the histogram as a diagnostic tool. PMID- 2997303 TI - Prenatal appearance of a mesoblastic nephroma associated with polyhydramnios. PMID- 2997301 TI - Immunoreactive corticotropin-releasing factor in human plasma. AB - Plasma immunoreactive corticotropin-releasing factor (I-CRF) levels were determined by using a human CRF radioimmunoassay and an immunoaffinity procedure. The basal plasma I-CRF level in normal subjects was 6 +/- 0.5 pg/ml (mean +/- SD). We found that most plasma I-CRF levels were affected by stress, negative feedback, and circadian rhythm. Basal I-CRF levels were high in patients with Addison's disease, Nelson's syndrome, hypopituitarism stemming from pituitary macroadenoma, and CRF- and adrenocorticotropic hormone-producing tumors. A very low, but significant, amount of I-CRF was detected (1-3 pg/ml) in patients with Cushing's syndrome, in corticosteroid-treated patients, and in a patient with hypothalamic hypopituitarism. These results suggest that a major component of plasma I-CRF is of hypothalamic origin, however, other extrahypothalamic tissues cannot be ruled out as a minor source of plasma I-CRF. PMID- 2997304 TI - Congenital mesoblastic nephroma: prenatal ultrasonic findings and surgical excision in a very-low-birth-weight infant. PMID- 2997305 TI - Comparison of an avidin-biotin immunoassay with three commercially available immunofluorescence kits for typing of herpes simplex virus. AB - An avidin-biotin complex system was compared with three commercially available immunofluorescence kits for serotyping herpes simplex virus isolates from clinical specimens. Sensitivity values showed that the Electro-Nucleonics and Immulok reagents were useful in detecting the presence of virus, whereas the predictive values showed that the Syva and Immulok reagents possessed adequate discrimination between the herpes simplex virus serotypes. The avidin-biotin complex system was equal or superior to the immunofluorescence reagents tested both in detecting herpes simplex virus antigens in cell culture and in serotyping herpes simplex virus isolates. PMID- 2997306 TI - Effect of food on the bioavailability of pentopril, an angiotensin-converting enzyme inhibitor, in healthy subjects. AB - Pentopril (CGS 13945) was administered in 125-mg capsules to eight healthy men on two occasions according to a randomized schedule; on one occasion in the fasting state and on the other occasion immediately following the ingestion of a standardized meal. Unlike captopril, a prototype angiotensin-converting-enzyme inhibitor, there was no significant difference in the peak plasma concentration for either the drug or its active metabolite (CGS 13934) between the fasting and the fed states. There was also no appreciable change in the area under the plasma curve for the drug and its metabolite after administration of drug in the presence of food compared with a fasting state. There was, however, a lag time in drug absorption after ingestion of food, which resulted in a significant increase in peak time for the active metabolite in plasma. Food delays the body's absorption of the drug and hence the appearance of its active metabolite in plasma without any significant effect on the relative bioavailability. Because relative bioavailability is not affected in the presence of food, such a delay may not have any therapeutic importance on chronic administration. PMID- 2997307 TI - Adrenergic beta receptors are not uniformly distributed in the cerebellar cortex. AB - The noradrenergic (NE) innervation of the cerebellar cortex is sparse, forming a broad plexus of radially oriented axons distributing throughout the granular and molecular layers. Autoradiographic studies of beta-adrenergic receptor distribution in the rat show the greatest density of silver grains in the molecular layer (Palacios and Kuhar, '82). In the course of studies of NE hyperinnervated structures, we found that beta receptors are nonhomogeneously distributed in the Purkinje cell layer, where they occur in "patches" overlying small groups of Purkinje cell somata. Tissue sections were incubated in 10 pM 125iodocyanopindolol (ICYP), which binds equally to beta1 and beta2 adrenergic receptors. Nonspecific binding was determined in sections incubated in 125ICYP and 1 microM dl-propranolol. Beta-adrenergic receptor patches are of irregular size and are most prominent in the vermis of lobules I-IX, although the medial cerebellar hemispheres also show areas of increased silver grains over Purkinje cells. In order to determine the subtype of beta receptors, adjacent sections were incubated with either 125ICYP and the beta 2-selective antagonist IPS-339, or 125ICYP and the beta 1-selective antagonist practolol. Patches were observed after each incubation procedure, indicating that they are composed of both beta1 and beta2 receptors. Patches are observed in normal animals and also in rats in which cerebellar NE content was increased 165% by neonatal treatment with 6 hydroxydopamine. This treatment does not alter the density of beta receptors. The cerebellar elements in which the beta receptors are located is not known. While silver grains accumulate over small groups of Purkinje cell somata, they are not coextensive with these cell bodies. The distribution of beta-adrenergic receptors does not parallel the arrangement of noradrenergic varicosities in the rat cerebellar cortex. PMID- 2997309 TI - Eccrine syringofibroadenoma (Mascaro). Report of two cases. AB - We report two cases of eccrine syringofibroadenoma. Both patients had a large, solitary hyperkeratotic nodular lesion over the extremities. Histologic sections showed spongelike masses of small cuboidal acrosyringeal cells. Fibrovascular connective tissue stroma resembling premalignant fibroepithelial tumor of Pinkus filled the spaces between the masses. PMID- 2997308 TI - Selective retrograde labeling with D-[3H]-aspartate in afferents to the mammalian superior colliculus. AB - The selectivity previously reported for retrograde labeling patterns obtained following D-[3H]-aspartate injections and proposed to be related to the transmitter specificity of the labeled pathways was tested in afferents to the superior colliculus (SC) of rats and rabbits. In rats selectivity was assessed by comparing retrograde perikaryal labeling patterns observed in D-[3H]-aspartate experiments with those found after administration of a nonselective tracer, horseradish-peroxidase-labeled wheat germ agglutinin (HRP-WGA), and of the tritiated neurotrasmitter gamma-aminobutyric acid (GABA). Following D-[3H] aspartate injections into the SC labeling was intense in a large number of cortical and hypothalamic neurons. Other afferents to the SC, however, such as those originating from the ventrolateral geniculate nucleus, the pars reticulata of the substantia nigra, the locus coeruleus, the pontine nuclei, or the retinal ganglion cells, were not labeled. Similar results were obtained in rabbits. In cats, the analysis was focused on the cerebral cortex, since in an earlier investigation no retrograde labeling had been detected in the visual cortex following D-[3H]-aspartate injections in the SC. In the present work, however, retrogradely labeled neurons were observed in various cortical areas including a few in visual cortex. This report shows selective retrograde labeling for a part of the afferents to the SC. This selectivity does not display major differences among the mammalian species studied. Moreover, according to the information available about the distribution of neurotransmitters in the brain, the findings reported here favour the idea that D-[3H]-aspartate is a retrograde tracer selective for glutamatergic and/or aspartatergic pathways. PMID- 2997310 TI - Eccrine gland clear reticulated cytoplasm. AB - This report relates how a normal variation of the eccrine secretory coil was mistaken for pathologic change. Electron microscopic studies of this variation, which to the best of our knowledge have not previously been reported, are presented. PMID- 2997311 TI - CT diagnosis of mediastinal lymphocele. AB - A mediastinal lymphocele occurring after esophagogastrectomy is demonstrated with CT following lymphography. This technique allows the definitive diagnosis of lymphocele, permitting differentiation from other post-operative mediastinal masses. PMID- 2997313 TI - Feelings and attitudes during early convalescence following vascular surgery. AB - This descriptive study examined patients' attitudes and feelings towards convalescence following vascular surgery. A convenience sample of 25 subjects consisted of patients who had initial (18) and repeat (7) surgery. A self administered questionnaire and a semi-structured interview guide were utilized to examine patients' perceptions concerning health, uncertainty about the outcome of surgery and course of disease, and their degree of adherence to discharge instructions. The findings suggested that the early convalescent period was difficult for all patients, but particularly for those in the repeat surgery group. Although data analysis revealed no statistically significant differences in perceptions between groups, repeat surgery patients tended to experience less favourable perceptions of health, a higher level of uncertainty, and a lower rate of adherence to discharge instructions. PMID- 2997312 TI - Calcification of pseudomyxoma peritonei following intraperitoneal chemotherapy: CT demonstration. AB - Pseudomyxoma peritonei is essentially mucinous ascites, resulting from the intraperitoneal rupture of a malignant mucinous lesion with seeding of peritoneal surfaces. We report a case of pseudomyxoma peritonei from a perforated mucinous adenocarcinoma of the appendix in which radiographically demonstrable punctate calcifications developed during the course of intraperitoneal chemotherapy. PMID- 2997314 TI - Effect of dietary fiber in insulin-dependent diabetics: insulin requirements and serum lipids. AB - Four young adult (18 to 26 years old), nonobese human subjects (two men and two women) with insulin-dependent diabetes mellitus volunteered to consume a series of three diets: baseline (normal daily intake), wheat bran (normal daily intake + 78 gm wheat bran per day), and cellulose (normal daily intake + 30 gm cellulose per day). Wheat bran and cellulose diets both contained 60 gm dietary fiber, with 50% of the dietary fiber from wheat bran or cellulose, respectively. Each patient served as his or her own control. Randomized diets were of 6 weeks' duration, separated by a 4-week "recovery" period. At the conclusion of each diet, subjects were hospitalized and underwent 12 hours of computer-controlled, insulin-glucose infusions. Significant decreases were seen in fasting cholesterol (p less than .05), but the decreases seemed to result largely from the significant reductions in high-density lipoprotein cholesterol. A large reduction in triglycerides was noted with cellulose feeding but not with wheat bran. The mean daily insulin dose decreased (p less than .05) in response to fiber addition (8% and 10% decrease for wheat bran and cellulose feeding, respectively). Mean biostator insulin requirements decreased 11% with wheat bran (p less than .05) but not with cellulose. During biostator monitoring, subjects experienced delayed postprandial blood glucose and insulin-infusion rate peaks with both wheat bran and cellulose feeding. The wheat bran diet reduced peak blood glucose concentration and peak insulin infusion rate in comparison with baseline and cellulose diets. The data suggest that high levels of cellulose or wheat bran are of marginal benefit to insulin-dependent diabetic subjects. PMID- 2997315 TI - Effects of soybean polysaccharide on plasma lipids. AB - Free-living volunteers with mild to moderate hypercholesteremia added 25 gm soybean polysaccharide or starch placebo in crouton or cookie form to their normal, daily diets. A total of 31 persons completed the blind, crossover design, 8-week, experimental protocol. Subjects ingesting soybean polysaccharide prior to placebo showed an 11% decrease (from 252 to 224 mg/dl) in total plasma cholesterol; those who followed placebo with fiber showed a 5% decrease (from 241 to 230 mg/dl). Starch placebo was associated with a 2% decrease in total cholesterol when consumed first and a 4% increase when consumed following the fiber consumption period. High-density-lipoprotein (HDL) cholesterol decreased 8% and 6% from initial values during the first period for the fiber and starch groups, respectively. HDL cholesterol increased 2% but decreased 1% during the second period for starch and fiber, respectively. No significant changes in triglyceride levels occurred. The data indicate that soybean polysaccharide fiber promotes a significant decrease in plasma cholesterol in persons with mild to moderate hypercholesteremia. The addition of fiber may represent an important adjunct to traditional fat- and cholesterol-controlled diets for such persons. PMID- 2997316 TI - Regional specification of cell-specific gene expression during craniofacial development. AB - The core problem in craniofacial development is regional specification of cell specific gene expression. Regional specification is also referred to as pattern formation or spatial organization. During early embryogenesis, regional specification is possibly operant following blastula, and is apparent during gastrulation and thereafter during embryonic and fetal stages of development. Current interdisciplinary approaches toward understanding embryonic development incorporate a number of scientific approaches including those of recombinant DNA technology. Three major advances have significantly enhanced the utility of so called "genetic engineering," including the discovery of restriction nuclease enzymes that cleave DNA sequences at specific sites; the discovery of DNA ligase enzymes that facilitate ligation and annealing of DNA sequences to one another, so as to facilitate the joining of foreign DNA sequences with host DNA; and the discovery of effective techniques for the introduction of foreign DNA sequences into previously refractory organisms. The present discussion analyzes the problem of regional specification of ameloblast-specific gene expression as a paradigm for utilizing recombinant DNA technology in studies of normal and abnormal craniofacial development. PMID- 2997317 TI - Catecholamine metabolites and cyclic nucleotides in cerebrospinal fluid in dementia of Alzheimer type. AB - Three, 4-dihydroxyphenylacetic acid (DOPAC); 3-methoxy, 4-hydroxyphenylacetic acid (HVA); 3-methoxy; 4-hydroxyphenylglycol (MHPG); adenosine 3', 5'-cyclic monophosphate (cyclic AMP); and guanosine 3', 5'-cyclic monophosphate (cyclic GMP) were measured in cerebrospinal fluid (CSF) of patients with dementia of Alzheimer type (DAT) and controls. DOPAC levels were lower and HVA levels were higher in DAT patients than in controls. In the most rostral fractions of CSF from DAT patients there was a negative correlation between DOPAC and cyclic AMP levels. In addition, patients with onset of DAT symptoms before the age of 65 had lower DOPAC levels, a higher HVA/DOPAC ratio, and higher cyclic nucleotide levels than patients with late onset of DAT. By combining DOPAC and cyclic AMP levels, we could clearly distinguish these two groups. PMID- 2997318 TI - Development of the hypothalamic-pituitary-axis in the ovine fetus: ontogeny of action of ovine corticotropin-releasing factor. AB - In samples from twenty chronically cannulated ovine fetuses the plasma immunoreactive adrenocorticotrophin (ACTH) concentrations were 12.5 +/- 3.2(8), 15.2 +/- 4.1(9) and 21.2 +/- 5.6(8) pg/ml at periods, prior to parturition, of 30 to -35, -25 to -29 and -20 to -24 days respectively. Values are mean +/- SEM (number of samples). These values were not significantly different from each other but were significantly lower (P less than 0.02) than values in the next two age groups -36.0 +/- 4.9(7) pg/ml at -19 to -15 days, and 39.6 +/- 6.6(11) pg/ml at -14 to -9 days. A further significant increase (P less than 0.05) occurred in the -8 to -3 day period, ACTH being 53.9 +/- 5.4(12) pg/ml. On day of delivery two samples had values of 325 and 360 pg/ml. A single injection, intravenously of 1.0 microgram ovine corticotrophin-releasing factor (O-CRF), caused a significant increase in fetal plasma ACTH concentrations in fetuses of -6 to -23 days prior to delivery but not in fetuses -24 to -35 days prior to parturition. The maximum values of ACTH after O-CRF were significantly greater in fetuses -2 to 0 days prior to parturition than in younger fetuses (P less than 0.01). In 6 experiments in 4 fetuses (parturition -1 to -13 days) the effect of 1.0 microgram O-CRF persisted for at least 2.5 h. The results support the hypothesis that the pituitary release of ACTH changes sensitivity to hypothalamic O-CRF at least twice during the last fifth of gestation; an increasing sensitivity is seen as term approaches. PMID- 2997319 TI - The value of urine osmolality as an index of stress in the ovine fetus. AB - In ovine fetuses, during 100-130 days of gestation, urine osmolalities less than 175 mosmol/kg water were associated with plasma immunoreactive adrenocorticotrophin (ACTH) concentrations below 40 pg/ml in 40/41 samples. In 18/29 fetuses with urine osmolalities greater than 175 mosmol/kg water plasma ACTH was significantly elevated. In 38 samples of fetal blood there was a significant correlation between plasma ADH and ACTH concentrations. By least squares regression the equation to the line was [ACTH] = 5.06 + 3.70 [ADH] (r = 0.62, P less than 0.001). In 50 samples from fetuses of gestational ages 100-140 days, with urine osmolalities of 302 +/- 86 mosmol/kg (mean +/- SD) the blood pH, pO2 and pCO2 values were not significantly different from those in 50 samples from fetuses with urine osmolalities of 125 +/- 22 mosmol/kg. It is proposed that the measurement of fetal urine osmolality provides a good index of fetal stress. A fetus with a urine osmolality less than 175 mosmol/kg is almost invariably in the optimum, unstressed condition. PMID- 2997320 TI - Sympathetic nervous activity in cirrhosis. A survey of plasma catecholamine studies. AB - This review summarizes recent progress in the knowledge of catecholamines in cirrhosis. Compensated patients have normal plasma concentration of noradrenaline. Highly elevated plasma noradrenaline concentration in decompensated patients indicates that the sympathetic nervous system is enhanced in this condition. This may especially apply to the sympathetic tone in the kidney, as evaluated by regional measurements of noradrenaline overflow. Hepatic elimination of catecholamines is only slightly reduced. Activation of the sympathetic nervous system seems to play an important role in the avid sodium water retention and decreased kidney perfusion observed in decompensated cirrhosis. Volume- en baro-receptors are likely to elicit this overactivity. The decreased systemic arterial blood pressure may be a primary event which is in part counteracted by enhanced sympathetic nervous activity and activated renin angiotensin system. The role of a non-volume-dependent hepatic baro-receptor, false neurotransmitters, postsynaptic receptors, and autonomous neuropathy are yet unknown issues of further research. PMID- 2997321 TI - Detection of serum HBV-DNA by molecular hybridisation. Correlation with HBeAg/anti-HBe status, racial origin, liver histology and hepatocellular carcinoma. AB - The relationship of the presence of hepatitis B virus (HBV) DNA in serum, a measure of HBV-replication, to HBeAg/anti-HBe status has been examined. In Northern Europe, there is a strong positive correlation between the presence of HBV-DNA and HBe antigenaemia and a negative correlation with the presence of anti HBe. These associations are less marked in patients from Southern Europe, Africa, the Middle and Far East. When HBV-DNA is present in the serum of anti-HBe carriers, it is usually associated with the presence of severe liver disease or carcinoma. Forty percent of patients with hepatocellular carcinoma had evidence of continuing HBV replication. PMID- 2997322 TI - Mitozantrone as single agent therapy in hepatocellular carcinoma. A phase II study. AB - Thirty-five patients with hepatocellular carcinoma (HCC) have been treated with the Anthracenedione derivative, Mitozantrone. One complete and 5 partial responses were seen in 22 evaluable patients and in a further 4 patients tumour size remained static for lengths of time ranging from 13 to 42 weeks. Therapy was well tolerated. Bone marrow suppression was dose-limiting but easily managed by dose reduction. Other acute toxicity was rare, vomiting and hair loss being reported only once each. Cardiac 'events' occurred in 5 patients, 3 of whom had received high total cumulative doses of Mitozantrone. We conclude that Mitozantrone is of clinical benefit in HCC and that its usage is compatible with a good quality of survival. Its cardiotoxic potential requires further evaluation. PMID- 2997323 TI - Porphyria cutanea tarda and hepatocellular carcinoma. Frequency of occurrence and related factors. AB - In order to assess the incidence of hepatocellular carcinoma (HCC) in porphyria cutanea tarda (PCT), 83 patients (77 males, 6 females, mean age 57.4 years) were studied. Thirteen patients (15.7%) had HCC, all of whom were male and cirrhotics with a mean age of 58.5 years. HCC patients showed a statistically significant (P less than 0.0005) longer evolution time (23 years since onset of the cutaneous disease) than patients without HCC (9.4 years), while the age of onset was similar in both groups. Differences in alcohol intake and hepatitis B virus (HBV) markers were non-significant, although high prevalence (54%) of past HBV infection was found in both groups. In HCC development, attributable risks of 100% were found for cirrhosis (P less than 0.001), male sex (P = NS) and for age over 51 (P less than 0.025). Therefore, PCT harbours a high incidence of HCC; evolution time, cirrhosis and age over 51 appear to be the most important contributing factors. PMID- 2997324 TI - Multiple hepatic adenomas after long-term therapy with testosterone enanthate. Review of the literature. AB - We report a case of multiple hepatic adenomas developed in a 32-year-old man with renal allograft after long-term therapy with testosterone enanthate. Histological assessment showed a benign hepatocellular adenoma, which has not regressed over a 6-month follow-up since androgen withdrawal. A review of primary hepatic tumors associated with androgenic-anabolic steroid therapy published in the English literature has been carried out, concentrating on the more relevant recent publications. No previous case of hepatic adenoma with possible relationship to the use of non-17-alpha alkylated compounds has been reported. PMID- 2997325 TI - Opiate receptors and beta-endorphin levels in brain areas of dogs with portal systemic encephalopathy. AB - Clinical observation has indicated a supersensitivity to morphine in patients with hepatic encephalopathy. With the aim of clarifying the issue, radioreceptor binding studies of opiate receptors were performed in frontal cortex and hypothalamus of 6 dogs with mild portal-systemic encephalopathy induced by chronic treatment with dimethylnitrosamine followed by porto-caval shunt end-to side. beta-Endorphin assays were performed in the same areas with radioimmunoassay. Opiate receptors labeled with [3H]naloxone in both areas showed a significant increase in the receptor densities (Bmax) without changes in the dissociation constant (KD). In parallel beta-endorphin levels showed a decline during the development of encephalopathy in both areas. The increased densities of opiate receptors in the mild stage of encephalopathy may explain the supersensitivity to morphine in patients with liver diseases. PMID- 2997326 TI - Maternal inheritance of mitochondrial DNA during backcrossing of two species of mice. AB - As judged by restriction analysis, mitochondrial DNA shows strictly maternal inheritance during 6-8 generations of backcrossing in both directions between Mus domesticus and Mus spretus. The average number of paternal mitochondrial genomes contributed to the next generation is estimated to be no more than one per thousand maternal mitochondrial genomes contributed. Despite the estimated accumulation of over 2000 mutational differences between M. spretus and M. domesticus mtDNAs since their divergence from a common ancestor, each of these mitochondrial DNAs, whether on a M. spretus or a M. domesticus nuclear background, allows mice to develop with seemingly normal viability and fertility. PMID- 2997327 TI - Intracellular transport of transferrin- and asialoorosomucoid-colloidal gold conjugates to lysosomes after receptor-mediated endocytosis. AB - Proteins coupled to colloidal gold particles have been widely used to visualize the uptake and intracellular transport of specific ligands by receptor-mediated endocytosis. The intracellular route of lysosome-directed ligands such as asialoglycoproteins (ASGP) are apparently unaltered by conjugation to gold, but the pathway of transferrin, a ligand that normally recycles to the cell surface, was reported to be altered by conjugation to 15-20 nm gold. In this study, we sought to determine whether a smaller transferrin-gold probe would recycle, and whether it might enter the same endosomal and lysosomal compartments as does a larger, lysosome-directed ASGP gold probe by visualizing their simultaneous uptake in human hepatoma (HepG2) cells. In the same cells, endocytosis of fluid phase protein was followed using the soluble tracer native ferritin; lysosomal compartments were identified by acid phosphatase cytochemistry; and cell surfaces were labeled with ruthenium red or cationized ferritin. During the first 10 min of uptake at 37 degrees C, specific receptor-bound ferrotransferrin (FeTf)-8 nm gold and asialoorosomucoid (ASOR)-20 nm gold were clustered together in coated pits and entered the same coated vesicles, smooth vesicles, and tubules in the peripheral cytoplasm. At later times, however, transferrin-gold did not return to the cell surface; unlike native transferrin, this gold probe accompanied ASOR gold into multivesicular bodies (MVB). The MVBs that contained probes were at first devoid of acid phosphatase activity, but at 30 min, enzyme activity was detected in a few MVBs. Native ferritin was present, along with gold probes, in all compartments of the endocytic pathway. We conclude that the normal intracellular pathway of transferrin is altered by its association with a colloidal gold particle. PMID- 2997328 TI - Polymyxin B use does not ensure endotoxin-free solution. AB - Polymyxin B is often added to in vitro samples to 'ensure' that endotoxin activity is removed. We present data, from the standard rabbit pyrogen test and the Limulus amebocyte lysate assay, that polymyxin B bound to a gel support will bind some, but not all, endotoxin. These data, in conjunction with previously published data by Morrison and Curry (1979), indicate that those studies that have relied on polymyxin B to inactivate endotoxin must be re-evaluated. PMID- 2997329 TI - Immobilization of antibodies on a new solid phase for use in ELISA. AB - Antibodies to myoglobin were immobilized by covalent linkage to polyester film for use in a solid-phase ELISA. The covalent linkage of antibody to this new solid phase was accomplished by partial acid hydrolysis of the film followed by periodate oxidation. About 60 ng of protein could be immobilized per cm2 of film and the binding was stable. Antimyoglobin IgM immobilized on the films was used in the ELISA technique to detect myoglobin within the range 0.25-10 ng. The assay procedure was found to be very accurate and the coefficient of variation of each concentration ranged from 0.63 to 2.1%. Furthermore the immobilized film could be re-used after dissociating the antigen antibody complexes. PMID- 2997330 TI - An objective filter-based, enzymatic method for the in vivo measurement of the migration of human polymorphonuclear leucocytes. AB - Incorporation of control valves into a previously described device enabled us to regulate the formation of 8 suction blisters on the upper surface of the forearm in adult human volunteers. After the removal of the raised epidermis and blister fluid, uniform areas of denuded dermis were obtained by placing hollow adhesive ring reinforcers onto each of the regions of exposed dermis. Single or double nitrocellulose filters were then placed onto each of the areas of moistened, exposed dermis. The chemotactic tripeptide FMLP was incorporated into 1% agarose containing 0.1% bovine serum albumin (BSA) to give a concentration range of 10( 8) M to 10(-6) M FMLP. In control systems the FMLP was omitted. Cylindrical agarose blocks +/- FMLP were then placed onto the filters and encased in individual perspex cups glued firmly onto the skin. The filter(s) and agarose blocks were replaced at 2 h intervals and polymorphonuclear leucocyte (PMNL) migration onto (single filter) and into (double filter) the filters was measured by microscopic enumeration or according to the amount of myeloperoxidase (MPO) and lysozyme in the supernatants of filters immersed in 0.1% Triton-X for 10 min to lyse the PMNL. Microscopic enumeration was found to be unsuitable but the method based on MPO and lysozyme release from filter-associated PMNL was rapid, accurate and reproducible. Detectable PMNL migration (greater than 90%) occurred at 3-4 h and was maximal at 8-10 h. This pattern was observed for both the control and FMLP-containing systems. However, PMNL migration was significantly greater in FMLP-exposed dermis. FMLP at 10(-6) M was found to promote maximal PMNL migration. Significantly greater MPO and lysozyme activities were observed with the double filter system. This method is suitable for the objective quantitation of PMNL migration in vivo. PMID- 2997331 TI - Immunological cross-reactivities among three herpesviruses. AB - Sixty adults were tested for humoral and cell-mediated immunity to varicella zoster virus (VZV), type 1 herpes simplex virus (HSV-1) and the human cytomegalovirus (CMV). Since herpesviruses share common antigens, we compared results in these individuals to assess whether our tests gave false positives due to cross-reactions. Of the 60, IgG antibody to VZV, HSV-1, CMV tested by ELISA was detected in 51 (85%), 34 (57%) and 20 (33%) respectively. All possible permutations of results were obtained and there was no evidence of cross reactivity among the viruses. Lymphocyte transformation performed in 20 was found to be positive in 16 (80%), 13 (65%) and 8 (40%) for VZV, HSV-1 and CMV respectively. Similarly, all possible permutations of results were obtained. In addition, 8 subjects were found to have positive lymphocyte transformation responses for one or other of the viral antigens but no specific antibody was found in their sera. Thus, the antibody and lymphocyte transformation test were not influenced by possible cross-reacting antigens among the 3 herpesviruses we studied. Both tests are necessary to firmly establish whether a subject has had a prior infection. PMID- 2997332 TI - Prevalence of a human retrovirus in native Japanese: evidence for a possible ancient origin. AB - The origin of a human retrovirus (ATLV or HTLV-I) is, at present, unknown although carriers of the virus have been found in Japan, the Caribbean basin and Africa. By means of a sero-epidemiological study, the Ainu people of Hokkaido, located in the northernmost island of Japan, were shown to have antibody to the virus in high frequency. Since the Ainu are regarded as descendants of the pre agriculture native population of northern Japan, this finding appears to indicate that the retrovirus was already present in the aboriginal Japanese of prehistoric times. PMID- 2997333 TI - Sources of research support: overview and discussion. PMID- 2997334 TI - Calmodulin activities are significantly increased in both uninvolved and involved epidermis in psoriasis. AB - In order to elucidate a part of calmodulin actions in the hyperproliferative state in human epidermis, calmodulin activities in the psoriatic and in the normal human epidermis were determined using calmodulin-deficient phosphodiesterase from bovine heart and purified pig skin epidermal calmodulin as a standard. Skin samples were obtained from 11 normal healthy controls and from both the uninvolved and involved regions of 8 nonconsanguineous psoriatic patients. Pure epidermal samples, prepared by the microdissection method, were used for calmodulin assays. Normal human epidermis contained 270 +/- 13 ng/mg dry weight, whereas calmodulin activities were significantly increased in psoriatic epidermis, 412 +/- 29 ng/mg dry weight for the uninvolved epidermis and 747 +/- 46 ng/mg dry weight for the involved epidermis, respectively. These results suggest that calmodulin may play an important role in cell proliferation in human epidermis. PMID- 2997335 TI - Antiviral and antiproliferative activities of human leukocyte interferon potentiated by cimetidine in vitro. AB - The influence of cimetidine on antiviral activity of leukocyte interferon (IFN alpha (Le] was studied in plaque-reduction assays using Utrecht (U) amnion cells challenged with vesicular stomatitis virus (VSV) and in CPE inhibition assays using A549 cells challenged with encephalomyocarditis (EMC) virus and WISH cells challenged with VSV. The IFN-alpha (Le)-induced antiviral activity was slightly enhanced in cells treated with cimetidine, whereas cimetidine treatment alone did not show any antiviral effect. The observed titer (OT) was significantly higher (p less than 0.05) in cells treated with cimetidine together with IFN-alpha (Le) compared with the control without cimetidine. The effect of cimetidine on IFN alpha (Le)-induced cell growth inhibition was studied on Daudi (a Burkitt's lymphoma cell line) and on G361 (a melanoma cell line) cells. The growth of these cells was slightly suppressed by cimetidine alone. When cells were treated with IFN-alpha (Le)/cimetidine, the cell growth inhibition rates were significantly higher (p less than 0.02) than the rates obtained with IFN-alpha (Le) or cimetidine alone. These results indicate that cimetidine can enhance the antiviral as well as the antiproliferative activities of IFN-alpha (Le) in "in vitro" studies. PMID- 2997336 TI - HuIFN-gamma antiviral activity correlates with immunological reactivity in a double-monoclonal antibody radiometric assay. AB - Several lines of evidence have been obtained that correlate biological activity with reactivity in an immunoradiometric assay (IRMA) employing two different monoclonal antibodies. Antiviral activity and immunological reactivity decreased in parallel when freeze-dried human gamma-interferon (HuIFN-gamma) was heated for prolonged periods of time at elevated temperatures. Likewise, aqueous preparations of HuIFN-gamma inactivated by acid pH or heat responded similarly in the biological assay and IRMA. These results indicate that the monoclonal antibodies used in this commercially available IRMA are specific for the epitopes on biologically activity HuIFN-gamma. PMID- 2997337 TI - Cellular mechanisms in in vivo production of gamma interferon induced by lipopolysaccharide in mice infected with Mycobacterium bovis BCG. AB - Infection of mice with Mycobacterium bovis BCG sensitizes them for immune induction of interferon (IFN)-gamma by specific antigen. These mice were found also to respond to lipopolysaccharide (LPS) and concanavalin A (Con A) with high level production of IFN-gamma in the circulation, which was not observed in control mice. In this study, we compared the IFN-gamma response to LPS with that to Con A in an attempt to clarify the cellular mechanisms responsible for in vivo LPS-induced IFN-gamma production. Consequently, (i) the responses to LPS and Con A differed in the kinetics, that to LPS having a longer lag period. (ii) Spleen cells taken from infected mice produced little IFN-gamma in response to LPS, but they showed a higher IFN-gamma response to Con A than those from control mice. (iii) By treating infected mice with immunosuppressive drugs or antibodies to T and natural killer cells before challenge with the inducers, it was indicated that different cellular systems are involved in the IFN-gamma responses to LPS and to Con A. PMID- 2997338 TI - Role of macrophage D-region antigens and T-lymphocyte differentiation antigens in induction of gamma interferon. AB - Macrophage-T-lymphocyte cultures from patients with recent recurrent herpes labialis were stimulated to produce gamma interferon by either herpes simplex antigen or mitogens (PHA and Con A). The ability of monoclonal antibodies to HLA DR and DC/DS, HSV glycoprotein antigens and T-lymphocyte surface antigens to inhibit interferon production and lymphocyte proliferation were studied. Anti-D region antibodies inhibited HSV antigen-induced but not mitogen-induced interferon and proliferation. The extent of inhibition varied mainly according to the determinants recognized by the antibodies and, to a lesser degree, between patients. Inhibition probably resulted from inhibition of antigen presentation, through antibody binding to macrophage D-region antigens. Interferon production was a more sensitive index of inhibition than lymphocyte proliferation. With one antibody (L227) a marked difference in inhibition in the two assays was noted, suggesting that lymphocytes producing gamma interferon may differ from the majority of the proliferating cells in their recognition of D-region determinants. Antibodies to the HSV glycoprotein antigens (gA/B, gC, gD, gE) did not produce consistent significant inhibition of interferon production and had no effect on proliferation. Anti-Leu 4 and -Leu 5 inhibited HSV antigen and mitogen induction of gamma interferon (and proliferation). Anti-Leu 2 and anti-Leu 3, mildly inhibitory alone, produced synergistic inhibition together. Hence, in human systems, the interaction between macrophages and T lymphocytes in producing gamma interferon appears to differ according to mode of induction. Macrophage D region antigens are required for antigen induction probably via presentation whereas other factor(s), probably monokine secretion, are necessary for mitogen induction. Antibodies acting on the T-lymphocyte surface appear to affect a final common pathway of interferon induction, similar to their effects on proliferation and secretion of other lymphokines. PMID- 2997339 TI - Differential regulation of interferon synthesis in lymphoblastoid cells. AB - We have examined the molecular mechanisms involved in the induction and regulation of expression of alpha and beta 1 human interferons (HuIFN) in Namalva cells. Cloned IFN-alpha and -beta 1 cDNAs, and antisera to purified IFN-alpha and -beta 1 were used as specific probes to determine the expression of HuIFN genes both on the RNA and protein levels. The rates of gene transcription were correlated with the relative levels of HuIFN mRNA present in induced cells and with the amounts of HuIFN peptides synthesized by these cells. The comparative rate of transcription of HuIFN-alpha and -beta 1 genes was measured in nuclei isolated from Namalva cells before and after induction. No transcription of HuIFN alpha and -beta 1 genes was detected in nuclei isolated from the uninduced cells. The correspondence in the rate of HuIFN-alpha and -beta 1 genes transcription after virus infection with the relative levels of HuIFN mRNA in the induced cells indicates that the stimulation of HuIFN synthesis by viral infection results from the activation of the transcription of HuIFN genes. The relative levels of alpha and beta 1 induced transcripts were the same in spite of the differences in the number of copies of HuIFN-alpha and -beta 1 genes indicating that the beta 1 gene is transcribed more efficiently than the alpha genes. The steady-state levels of HuIFN-alpha and -beta 1 mRNAs in induced Namalva cells are comparable, however, the overall amount of HuIFN-beta 1 synthesized (as determined by radioimmunoassay and biological activity) is approximately 10-fold lower than that of IFN-alpha. No evidence has been found that would indicate that HuIFN-beta 1 mRNA induced in Namalva cells is different from that induced in human fibroblasts. The data indicate, however, that in Namalva cells, the IFN-beta 1 polypeptide has a higher turnover rate and slower rate of release into medium than the HuIFN-alpha polypeptides, indicating that the observed difference in the overall amounts of these two types of interferons present in the medium is due to regulation on posttranslational level. PMID- 2997340 TI - Molecular cloning of murine interferon gamma (MuIFN-gamma) cDNA and its expression in heterologous mammalian cells. AB - A complete cDNA clone of murine interferon-gamma (MuIFN-gamma) was obtained by recombining two appropriate segments from partial cDNA clones originally identified by colony hybridization with rat IFN-gamma chromosomal gene fragments as probes. An expression vector was constructed in which the cDNA was placed under control of the SV40 early promoter. Transient expression of MuIFN-gamma was obtained by transformation of COS-1 cells. Subsequently, this interferon expression unit was linked to a vector containing a dihydrofolate reductase (DHFR) modular gene and used to transform DHFR(-)-CHO cells. Cell clones were selected that constitutively produce an interferon activity which by several criteria was found to be indistinguishable from natural, splenocyte-derived MuIFN gamma. PMID- 2997341 TI - [Sensitivity of group A coxsackieviruses and ECHO viruses, and isolation from clinical specimens by RD-18S cells derived by cloning from RD cells]. PMID- 2997342 TI - [Comparative study on sultamicillin and bacampicillin in the treatment of respiratory tract infections]. PMID- 2997343 TI - [Examination of pharmacokinetics and intestinal flora following 15 days of sultamicillin or ampicillin administration]. PMID- 2997345 TI - [A case of surgical treatment of triple cancer]. PMID- 2997344 TI - Surgical treatment for tracheal diseases. A report of 72 cases. PMID- 2997346 TI - [Studies on metabolic regulation of liver specific functions and growth control of mature hepatocytes in primary culture]. PMID- 2997347 TI - [The study of opioid peptides in the normal menstrual cycle, pregnancy and labor]. AB - The present investigation was performed for the purpose of measuring serum immuno reactive (IR)-beta-lipotropin (LPH), IR-beta-endorphin (EP) and adrenocorticotropic hormone (ACTH) in normal menstrual cycle, pregnancy and labor of normal human subjects. The normal menstrual cycle was divided to menstrual (n = 7), follicular (n = 18), ovulatory (n = 12) and luteal phases (n = 16). The serum concentration of IR-beta-EP and ACTH was significantly higher in the luteal phase than those in other phases. The values for IR-beta-LPH were also higher in luteal phase, but statistically not significant. Normal pregnancy was divided to three groups; I: first trimester (n = 12-15), II: second trimester (n = 11) and III: third trimester (n = 13-18). Normal labor (n = 7) was also divided into two stages, the period with a cervix dilatation of 5cm and 10cm dilatation. The serum concentration of IR-beta-LPH and IR-beta-EP increased progressively with gestational weeks. During labor these two peptides showed maximum values at the period with cervix dilatation of 10cm. These findings indicate that the basic values for opioid peptides in human reproductive cycle can be applied to further clinical analysis of several gynecological disorders. PMID- 2997348 TI - [Studies of immunological function in patients with ovarian cancer]. AB - Immunological function in patients with ovarian cancer was studied using parameters including peripheral blood lymphocyte counts, lymphocyte subsets, PHA induced lymphoproliferative (PHA-LP) reaction, PPD skin test and immunoglobulin levels. The following results were obtained: The number of total lymphocytes, T cells (OKT 11+) and B cells (OKIa 1+) in peripheral blood were found to be decreased in patients with advanced carcinoma. The positive rate of PPD skin test was also lower in the patients. OKT8+ cells were significantly increased in the disseminated carcinoma (DC) group and the OKT4+/OKT8+ ratio in the DC group was markedly decreased when the results were compared with the control and the patients in the localized carcinoma (LC) group. PHA-LP reaction and serum IgM level in the patients in the LC and DC groups were found to be significantly lower than in the control group. The percentage of T cell (OKT11+), serum IgG and IgA levels were not altered in any clinical stage of the patients. PMID- 2997349 TI - [Diagnostic usefulness of stepwise discriminant analysis employing the values of CA125, TPA, IAP, CEA and ferritin in sera measured simultaneously for gynecological malignant neoplasms]. AB - The values for CA125, TPA, IAP, CEA, and ferritin in sera were measured simultaneously in 68 healthy nonpregnant females and 133 patients with various gynecological diseases, and examined by stepwise discriminant analysis. The usefulness and the limits for diagnosis of various gynecological diseases were investigated for each tumor marker. Also, the diagnostic usefulness of stepwise discriminant analysis employing the values for five tumor markers in sera was studied for gynecological malignancies compared with that of measuring serum CA125 alone. Because the mean values for CA125 in sera were increased specifically in the ovarian cancer patient group compared with those of other tumor markers in sera, the measurement of serum CA125 was considered to be more useful in diagnosing ovarian cancer than that of the other tumor markers. The mean values for CA125 in sera, however, were also increased more significantly in the groups of patients with endometriosis and normal pregnancies than in the group of healthy nonpregnant females (p less than 0.005). In the stepwise discriminant analysis employing the values for CA125 and four other tumor markers in sera, the diagnostic usefulness of each tumor marker was demonstrated in the early diagnosis, the differential diagnosis, and the determination of complete remission after several therapies for ovarian cancers. PMID- 2997350 TI - A controlled study of intravenous doxorubicin versus oral tegafur in patients with hepatocellular carcinoma. PMID- 2997351 TI - [An evaluation of the clinical usefulness of amifostine (YM-08310), radioprotective agent. A double-blind placebo-controlled study. 1. Head and neck tumors]. PMID- 2997352 TI - [An evaluation of the clinical usefulness of amifostine (YM-08310), radioprotective agent. A double-blind placebo-controlled study. 2. Abdominal and pelvic tumors]. PMID- 2997353 TI - Refractoriness of platelets to prostaglandins after infusion in rabbits. AB - Continuous intravenous infusion of prostacyclin (prostaglandin I2, PGI2) in rabbits induced refractoriness to PGI2-induced inhibition of platelet function. Although inhibited during earlier stages, platelet response to adenosine diphosphate and thrombin became normal within 24 hours of PGI2 infusion. Cross mixing experiments with platelets and plasma from infused and control animals suggested that the altered response to PGI2 was caused by a defect intrinsic in the platelets. PGI2-stimulated cyclic adenosine monophosphate (cAMP) production was reduced in platelets from infused rabbits as compared with those from controls. Platelets refractory to PGI2 were refractory to PGE1 and PGD2, as well. Because PGE1 but not PGD2 shares the same platelet receptor as PGI2, the phenomenon could not be ascribed to receptor-specific downregulation, which was also shown by refractoriness of platelets from infused rabbits to the nonprostanoid inhibitor of platelet function adenosine. Either increased concentrations of ineffective inhibitors or their combination with phosphodiesterase inhibitors overcame refractoriness of resistant platelets, which also responded to inhibition by dibutyryl cAMP, indicating residual activity of adenylate cyclase. That at least the catalytic subunit of the enzyme was still working in refractory platelets was shown by inhibition of aggregation induced by forskolin, a non-receptor-mediated activator of adenylate cyclase. Impairment of the adenylate cyclase regulatory subunit, possibly accompanied by multireceptor downregulation, may explain the paradoxical refractoriness of platelets to prolonged infusion of PGI2. Such an effect may limit the benefit of PGI2 in treatment of thromboembolic disease. PMID- 2997356 TI - Dermal type cylindroma of the parotid gland with malignant transformation. PMID- 2997354 TI - Effects of thyroid hormone on Na-K-adenosine triphosphatase activity along the rat nephron. AB - Na-K-adenosine triphosphatase (ATPase) activity was determined in individual nephron segments obtained from the kidneys of euthyroid and hypothyroid rats. One group of animals was made hypothyroid by feeding 0.05% aminotriazole (ATZ) in the diet for 2 weeks. A second group received the same amount of ATZ plus 500 micrograms/kg body weight of L-thyroxine (T4) given subcutaneously each day for 2 weeks. A third group received the same diet without ATZ or T4. There was a 57% (P less than 0.01) decrease in Na-K-ATPase activity in the proximal convoluted tubule (PCT) in ATZ-treated rats that was corrected by the simultaneous administration of T4 with ATZ. A smaller (15% to 25%) and statistically nonsignificant decrease in Na-K-ATPase activity was observed in the cortical portion of the proximal straight tubule and in both the cortical and the medullary portions of the thick ascending limb in ATZ-treated rats. These changes in the enzyme activity were also corrected by simultaneous administration of T4 with ATZ. The results suggest that under the conditions of these experiments the PCT is a major site of action of thyroid hormone in the rat kidney. PMID- 2997355 TI - Aetiology of bilateral sensori-neural hearing loss in young children. PMID- 2997357 TI - Augmentation with hydroxylapatite and vestibuloplasty in the atrophic maxilla with a flabby ridge. AB - Hydroxylapatite is used for maxillary ridge augmentation in combination with a submucous vestibuloplasty. Undermining and internal incision of the flabby ridge provides a tunnel for Hydroxylapatite insertion. After three weeks the augmented maxilla is stable enough for prosthetic treatment. Indications, technique and first year experience with 7 cases are described. PMID- 2997359 TI - Giant mitochondria with filamentous structures in bone marrow macrophages. PMID- 2997358 TI - A modified hydroxylapatite implant material. A preliminary report. AB - Hydroxylapatite implants are becoming widely used, but this new material is not without its drawbacks. These mainly concern its physical characteristics, complicating and limiting its clinical applications. A method of altering these with fibrin adhesives is described and early laboratory and clinical experience is presented. PMID- 2997360 TI - Transformed lymphocytes from Abelson-diseased mice express levels of a B lineage transformation-associated antigen elevated from that found on normal lymphocytes. AB - Animals injected with Abelson murine leukemia virus (A-MuLV) rapidly develop fatal bone marrow-derived lymphosarcomas. In all such diseased animals tested, a subpopulation of bone marrow cells expressed a monoclonal antibody-defined, B lineage transformation-associated antigen (6C3 Ag) at levels increased from that detected on normal lymphocytes. Cells bearing a high level of this antigen were found to be transformed as measured by clonal growth in agar, and they expressed surface antigen markers characteristic of early pre-B cells. High-level antigen expressing cells were found in the bone marrow, lymph nodes, and spleen, but never in the thymus of diseased animals. This distribution agrees with the published pathology of Abelson disease. PMID- 2997362 TI - Isolation and enzymatic characterization of the plasmalemma from bovine spermatozoa. AB - An improved method for the isolation of pure plasma and acrosomal membranes from bull spermatozoa is presented. Plasma membranes were isolated from the spermatozoa of bulls of different breeds, and some enzymatic activity, such as (Na+-K+) ATPase, Ca++ ATPase, Mg++ ATPase, alkaline and acidic phosphatases were assayed. Such enzymatic activity levels differ noticeably from those published by other authors, whose preparations were probably contaminated by other cellular components. Highly statistically significant differences of these activities have been found among the several breeds. PMID- 2997363 TI - Nutrition and cancer: recommendations of the American Cancer Society. PMID- 2997361 TI - Molecular basis of the dm1 mutation in the major histocompatibility complex of the mouse: a D/L hybrid gene. AB - The H-2dm1 mutation is unique among all described H-2 mutations in that two transplantation antigens, the H-2Dd and the H-2Ld, are affected. Here, we show that the mutant gene, Ddm1, is formed by fusion of the 5' part of the Dd gene and the 3' part of the Ld gene, with the region in between deleted. The recombination junction is located in the third exon, which encodes the alpha 2 region of the protein. When the hybrid gene is transfected into mouse L cells, serological and biochemical analyses indicate the Ddm1 antigen expressed in the transformant line is identical to the mutant molecule in dm1 spleen cells. These results demonstrate that the D/L hybrid gene is most likely responsible for the dm1 mutant phenotype. PMID- 2997365 TI - Further studies of the volume-regulatory response of Amphiuma red cells in hypertonic media. Evidence for amiloride-sensitive Na/H exchange. AB - When Amphiuma red cells are shrunken in hypertonic media, they return toward their original volume by gaining Na through an amiloride-sensitive pathway. As cells recover their volume during this volume-regulatory increase (VRI) response, acid is extruded into the medium. Medium acidification is correlated with cell Na uptake. Both medium acidification and cell Na uptake are blocked by 10(-3) M amiloride or by replacing medium Na with K or choline. Perturbations that increase cell Na uptake (such as increasing medium osmolality) also increase medium acidification. As the medium becomes more acidic, the cells become more alkaline. These changes in cell and medium pH are increased if pH equilibration across the cell membrane is prevented by inhibiting the anion exchanger with SITS (4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonic acid). The quantity of acid extruded by SITS-treated cells is the same as the quantity of Na gained, which strongly suggests 1:1 exchange of Na for H. Cell enlargement in SITS-treated cells results from the exchange of osmotically active Na ions for H ions that are not osmotically active when combined with cellular buffers. Previous evidence indicates that the normal VRI response involves an increase in the cellular content of Cl as well as Na. We show that SITS completely blocks net Cl uptake, which suggests that Cl enters via the anion exchanger. SITS also slows Na entry, presumably as a result of the above-mentioned increase in cell pH caused by SITS. We suggest that the initial event in the VRI response is net Na uptake via a Na/H exchanger, and that net Cl uptake results from secondary Cl/HCO3 exchange via the anion exchanger. PMID- 2997364 TI - Large and rapid changes in light scattering accompany secretion by nerve terminals in the mammalian neurohypophysis. AB - Large changes in the opacity of the unstained mouse neurohypophysis follow membrane potential changes known to trigger the release of peptide hormones. These intrinsic optical signals, arising in neurosecretory terminals, reflect variations in light scattering and depend upon both the frequency of stimulation and [Ca2+]o. Their magnitude is decreased in the presence of Ca2+ antagonists and by the replacement of H2O in the medium by D2O. These observations suggest a correspondence between the intrinsic optical changes and secretory activity in these nerve terminals. PMID- 2997366 TI - Plasmids mediating iron uptake in Vibrio anguillarum strains isolated from turbot in Spain. AB - Vibrio strains isolated from diseased turbot in an experimental fish farm on the Atlantic coast of northwest Spain were identified as Vibrio anguillarum. The isolates shared many biochemical characteristics with V. anguillarum strains obtained from other sources, and harboured a plasmid species that showed extensive homology with plasmid pJM1, carried by V. anguillarum strain 775 isolated from an epizootic in North America. Restriction endonuclease analysis showed that the two plasmids were very similar albeit not identical. The presence of the plasmid in the turbot isolates was associated with their ability to cause disease in fish. Plasmid-carrying bacteria could also grow under conditions of iron limitation. Two outer membrane proteins, of 86 and 79 kDal, were induced, and a similar siderophore activity to that produced by V. anguillarum 775 was also detected under these conditions. The 86 kDal outer membrane protein cross reacted immunologically with antiserum raised against the outer membrane protein OM2 produced by strain 775. Nonvirulent plasmidless derivatives were unable to grow under iron-limiting conditions, and were also unable to produce either siderophore activity or the 86 kDal outer membrane protein, suggesting the plasmid-mediated nature of these components. PMID- 2997367 TI - Studies of the 2':3'-cyclic nucleotide phosphodiesterase of Haemophilus influenzae. AB - The 2':3'-cyclic nucleotide phosphodiesterase:3'-nucleotidase of Haemophilus influenzae was purified from a periplasmic preparation by affinity chromatographic techniques. The enzyme-catalysed hydrolysis of 2':3'-cyclic AMP to adenosine without accumulation of the intermediate substrate 3'-AMP was demonstrated by high performance liquid chromatography. Competitive inhibition of the enzyme by a variety of nucleosides and mononucleotides indicated the presence of either purine or pyrimidine bases to be essential for selective interactions with the enzyme, and confirmed the need for a 3'-position phosphate for the functioning of mononucleotides as substrates for the enzyme. The enzyme had a molecular weight of 79 000, was stable at low temperatures and was thermally denatured at temperatures above 50 degrees C. PMID- 2997368 TI - Identification of restriction fragments from two cryptic Clostridium butyricum plasmids that promote the establishment of a replication-defective plasmid in Bacillus subtilis. AB - Clostridium butyricum NCIB 7423 carries two cryptic plasmids, pCB101 (6.05 kbp) and pCB102 (7.8 kbp). Sites for the restriction enzymes EcoRI, EcoRV, HindIII, ClaI and PstI have been found in one or both of these plasmids and their relative positions determined. Restriction fragments from both plasmids have been inserted into a vector plasmid (pJAB1) that is able to replicate in Escherichia coli but not in Bacillus subtilis and the recombinant plasmids have been established in E. coli. A 3.3 kbp Sau3A fragment of pCB101 conferred upon the vector the ability to transform both Rec+ and Rec- strains of B. subtilis. Plasmid pRB1, a representative chimaera carrying only the 3.3 kbp Sau3A fragment of pCB101, was successfully transferred from B. subtilis back to E. coli. Plasmid pRB1 was readily lost from B. subtilis in the absence of selection. This evidence, together with the results of hybridization experiments, suggests that pRB1 is present as a weakly replicating autonomous element in B. subtilis. A recombinant plasmid carrying a 2.0 kbp Sau3A fragment of pCB102 underwent integration into the B. subtilis chromosome. PMID- 2997369 TI - The role of haemagglutinin-neuraminidase glycoprotein cell surface expression in the survival of Sendai virus-infected BHK-21 cells. AB - A temperature-sensitive mutant of Sendai virus with a lesion in the haemagglutinin-neuraminidase protein (HN) (ts 271) was used to study the effect of HN cell surface expression on the fate of infected BHK-21 cells. The total amount of HN was reduced in ts 271 virus-infected cells at the non-permissive temperature (38 degrees C) presumably due to degradation of the protein. At this temperature, neither HN nor a modified form of HN were found expressed at the surface of the infected cells. BHK-21 cells infected with ts 271 were nevertheless killed by the infection at 38 degrees C as well as at 30 degrees C. These results ruled out the hypothesis that the lack of HN cell surface expression could be the unique requirement allowing BHK cell survival. PMID- 2997370 TI - Immunological priming with synthetic peptides of foot-and-mouth disease virus. AB - A sub-immunizing dose of a synthetic peptide corresponding to the amino acids 141 to 160 region of protein VP1 from foot-and-mouth disease virus (FMDV), serotype O1, coupled to keyhole limpet haemocyanin (141-160KLH) has been shown to prime the immune system of guinea-pigs for an FMDV serotype-specific neutralizing antibody response to a second sub-immunizing dose of the same peptide. Optimal priming required an interval of 42 days between the priming dose and the booster dose. No priming was observed in the absence of adjuvant. The secondary response was not restricted by the carrier since animals primed with 141-160KLH could be boosted with uncoupled 141-160 or 141-160 coupled to tetanus toxoid. It has also been shown that uncoupled peptide 141-160 will prime for a neutralizing antibody response when it is incorporated into a relatively non-immunogenic carrier such as small unilamellar liposomes. These results indicate that the 141-160 peptide of FMDV, as well as containing an important neutralizing antibody site, can initiate its own T-helper cell response. PMID- 2997371 TI - The pathogenicity of the A7, M9 and L10 strains of Semliki Forest virus for weanling mice and primary mouse brain cell cultures. AB - The multiplication of the M9, A7 and L10 strains of Semliki Forest virus (SFV), both in weanling mice and primary mouse brain cell cultures, was compared. Following both intraperitoneal (i.p.) and intracerebral (i.c.) injection, the virulent L10 strain multiplied to higher titre in the mouse central nervous system (CNS) than did the less virulent M9 and A7 strains, whereas M9 multiplied to higher titre than A7. By the i.c. route, all three virus strains multiplied to higher titre than following i.p. injection. Multiplication of A7 and M9 in oligodendrocytes, but not neurons, was detected following i.c. injection. All three virus strains showed a tropism for cultured mouse glial cells rather than neurons. The L10 strain multiplied better in neurons than did A7 or M9. It is concluded that the mechanism of acute demyelination induced by the M9 and A7 strains is similar. Based on this and previous studies, it is proposed that infection of glial cells triggers immune-mediated demyelination. The virulence of the L10 strain is due to its ability to exceed a lethal threshold of damage to neurons before immune intervention can occur. PMID- 2997372 TI - Effect of bovine alpha 1 interferon on bovine herpesvirus type 1-induced respiratory disease. AB - Treatment of calves with bovine recombinant alpha 1 interferon prior to challenge with bovine herpesvirus type 1 increased the animals' ability to withstand a subsequent Pasteurella haemolytica challenge. The reduction in viral-bacterial synergy observed following interferon treatment did not appear to be due to a direct effect of the interferon on virus replication in the upper respiratory tract. Thus, even though interferon-treated animals shed slightly less virus from their nasal passages than did untreated animals, this reduction was not statistically significant. Furthermore, there was no difference in the level of intranasal interferon secreted by control or interferon-treated animals. These results suggest that interferon treatment does not affect the production of endogenous interferon. In contrast, a significant difference was observed between the number of days that control animals were sick, the levels of serum fibrinogen and the functional activity of polymorphonuclear neutrophilic granulocytes obtained from infected calves. These results suggest that bovine recombinant alpha 1 interferon may have a greater immunomodulatory effect than a direct antiviral effect in this model. This is further supported by the observation that bovine herpesvirus type 1 is relatively resistant to the direct antiviral effect of bovine recombinant alpha 1 interferon in vitro. PMID- 2997373 TI - Transformation of human 143 tk- cells with plasmids containing the gene encoding the adenovirus DNA-binding protein. AB - Human cell lines that contain and express the gene encoding the adenovirus type 5 DNA-binding protein (Ad5 DBP) are very useful for the isolation of adenovirus mutants with an altered DBP. In order to obtain these cells, human 143 tk- cells were transfected, using the calcium phosphate technique, with plasmids containing the Ad5 DBP gene and the herpes simplex virus thymidine kinase (HSV tk) gene as a selectable marker. Characterization of several tk+ transformants revealed that these cells did contain the HSV tk gene, but in none of these cells could Ad5 DBP DNA sequences be detected. However, when 143 tk- cells were co-transfected with a plasmid containing the Ad5 DBP gene and another plasmid carrying early region E1, integration of the Ad5 DBP gene in chromosomal DNA could be detected. Integration of Ad5 DNA sequences was also observed when transfection was performed with plasmids containing the Ad5 DBP gene and the long terminal repeat of Moloney murine leukaemia virus. By employing a radioimmunoassay it could be shown that DBP-related proteins were synthesized in two of the cell lines containing the Ad5 DBP gene. Since both cell lines support the growth of the temperature-sensitive viral DBP mutant, H5ts125, at the non-permissive temperature, the DBP-related proteins expressed in these cells must be functional. PMID- 2997374 TI - Study of the 78A1 isolate of Moloney murine sarcoma virus. I. Molecular cloning and characterization. AB - The 78A1 isolate of Moloney murine sarcoma virus (78A1 Mo-MuSV) was cloned from a genomic library obtained from virus producer rat cells, in the lambda vector L47. Among the recombinants hybridizing with a probe specific for the v-mos sequences, we recovered a recombinant which contained leukaemia virus (MuLV) sequences and was able to transform both mouse and rat cells in transfection experiments. The cloned provirus could be rescued by both Mo-MuLV ecotropic and amphotropic viruses in mouse cells, but only with the amphotropic helper virus in rat cells. Comparative restriction mapping indicates that the 78A1 provirus is 200 bp longer than the HT1 provirus. The difference lies in the gag-pol junction region of Mo MuSV. Other minor differences were found in the gag region, whereas the restriction patterns of the 3' parts of the proviruses were identical. PMID- 2997375 TI - Study of the 78A1 isolate of Moloney murine sarcoma virus. II. Haematopoietic tropism and tumourigenicity. AB - A new isolate of Moloney murine sarcoma virus (Mo-MuSV), designated 78A1, has been molecularly cloned. The cloned genome, found to be larger than that of other known isolates of the same virus is close in size to that of the myeloproliferative sarcoma virus (MPSV), also a derivative of the original Mo MuSV/Moloney murine leukaemia virus (Mo-MuLV) complex. Until now, MPSV was the only Mo-MuSV isolate known to be capable of inducing a myeloproliferative disease associated with a tumoural syndrome when injected intravenously into sensitive mice. We compared the biological activity of our cloned virus isolate (78A1) and that of another cloned Mo-MuSV virus (HT1) whose genome is slightly smaller than that of 78A1. The helper virus (Mo-MuLV) associated with the Mo-MuSV isolates was also injected alone as control. After injection into sensitive mice only the isolate 78A1, as well as MPSV caused a tumoural syndrome invading spleen, liver and other haematopoietic organs, and the appearance of granulo-macrophage precursors not requiring exogenous stimulating factors for their proliferation and differentiation. The 78A1 virus has a longer latency period (3 months) than MPSV (several days) and does not induce a typical myeloproliferative disease. PMID- 2997376 TI - Baculovirus (MNPV) genomic variants: characterization of Spodoptera exempta MNPV DNAs and comparison with other Autographa californica MNPV DNAs. AB - A nuclear polyhedrosis virus (NPV) strain from Spodoptera exempta (SeMNPV-25 baculovirus) is a restriction endonuclease DNA map variant similar to Autographa californica NPV (AcMNPV baculovirus). Fourteen restriction endonuclease variants were identified and isolated from a SeMNPV baculovirus stock with 12 of the variants found at low frequency (less than 3%). The DNA from each variant was compared to the prototype SeMNPV-25 for insertions, deletions and new restriction sites. Regions of variation were defined on the prototype SeMNPV-25 genome, and the nature of the variation within these regions was determined. These data are discussed and compared with the existing data on other variants of AcMNPV. A comparison of the physical maps revealed that all the SeMNPV variants were different from those reported for AcMNPV. Although the SeMNPV variants were distinctive, they were clearly genomic AcMNPV variants. The regions of the baculovirus genomic variation were identified, and three separate mechanisms are suggested for their generation. Five regions (hr1 to hr5) were associated with intragenic homologous viral sequences, five regions (vI to vIII) may be associated with the insertion of DNA sequences of cellular origin, and two regions (pI and pII) were associated with mutations resulting in the addition or loss of a PstI site. Physical maps were generated for SeMNPV variant regions vI, hr2, vII and vIII. PMID- 2997378 TI - Acid-labile interferon produced in human peripheral leukocytes by induction with Sendai virus. AB - An acid-labile interferon-alpha (IFN-alpha) exists in IFN produced in human peripheral leukocytes with Sendai virus. This activity was found in fractions of molecular weights about 100 000 (100K), 58K and 44K. The IFN activity in the 100K fraction was neutralized with anti-HuIFN-alpha serum alone, but activity in the 58K and 44K fractions was neutralized only by a mixture of anti-HuIFN-alpha and anti-HuIFN-beta sera. PMID- 2997377 TI - Effects of glycosylation inhibitors on the expression of polyalbumin receptors by hepatitis B surface antigen produced in vitro. AB - In this study we examined the role of secondary modifications in the expression of polyalbumin receptors by hepatitis B surface antigen (HBsAg) produced in vitro. Several clones isolated from 3T3 mouse fibroblasts after transfection with the hepatitis B virus genome produced HBsAg with marked variation in the expression of polyalbumin receptors. Treatment of the cells with glycosylation inhibitors (tunicamycin, glucosamine) resulted in loss of the 27 000 mol. wt. HBsAg glycopolypeptide, concomitantly with a marked increase in polyalbumin receptors. These data suggest that glycosylation regulates the activity of polyalbumin receptors associated with native HBsAg. PMID- 2997379 TI - Hit-and-run neutralization of poliovirus. AB - At physiological ionic strength, monoclonal antibody 35-1f4 has previously been shown to neutralize poliovirus type 1 by antibody-mediated polymerization. At low ionic strength, this antibody neutralized the virus by a hit-and-run mechanism: the virions were converted to non-infectious, empty capsids devoid of antibodies. These empty capsids resembled those formed by thermal denaturation of native polio virions in their sedimentation coefficient (80S), antigenicity (H) and isoelectric pH (6.3). PMID- 2997380 TI - Incomplete neutralization of hepatitis A virus in vitro due to lipid-associated virions. AB - Hepatitis A virus (HAV) released from infected BS-C-1 cells was incompletely neutralized when incubated with a variety of convalescent sera (non-neutralizable fraction of 17 to 32%). Chloroform extraction of virus resulted in a substantial reduction of the non-neutralizable fraction (to less than 1%), suggesting that non-neutralizable virions might be associated with lipids. Non-neutralizable HAV recovered from untreated cell culture supernatant fluids sedimented heterogeneously and less rapidly than normal virus in rate-zonal sucrose gradients and also banded at a lower density in CsCl (1.14 to 1.18 g/ml) than normal, neutralizable virus (1.32 g/ml). This bimodal distribution of HAV in CsCl gradients was confirmed by cDNA-RNA hybridization. Together, these observations suggest that a substantial proportion of HAV particles released from infected cells are lipid-associated and imply an important role for cell membranes in the assembly and release of HAV in vitro. PMID- 2997381 TI - A 28000 molecular weight human cytomegalovirus structural polypeptide studied by means of a specific monoclonal antibody. AB - We have produced a monoclonal antibody against human cytomegalovirus (HCMV) which recognizes a structural protein of 28 000 mol. wt. which is present in both the cytoplasm of infected cells during the late phase of the viral replication cycle and in the extracellular viral particles. This antigen was detected in all HCMV strains assayed and reacted with human sera having anti-HCMV antibodies. PMID- 2997382 TI - Detection of Norwalk virus in stools by enzyme immunoassay. AB - The development of a solid-phase microtiter enzyme immunoassay (EIA) for detection of Norwalk virus antigen in stool samples is described. The EIA was compared with a previously developed radioimmunoassay (RIA) for detection of Norwalk virus antigen in stools obtained from 30 volunteers who received Norwalk virus. The EIA detected viral antigen in stools from 17 of the volunteers and the RIA detected viral antigen in 15. Seroconversion was a more sensitive indicator of infection in some patients. However, two samples from volunteers who were clinically ill but did not show seroconversion to Norwalk virus were positive for Norwalk virus antigen by both immunoassays. This indicates that antigen detection may be important for use in epidemiological studies. Neither of the immunoassays gave positive reactions for stools known to contain enteric adenovirus, rotavirus, or Hawaii virus, or in stools from patients with acute diarrhea of unknown cause. The stability of the EIA reagents and ease of use should provide a means for more extensive testing for Norwalk virus in outbreaks of gastroenteritis. PMID- 2997383 TI - Genetic relatedness among human rotaviruses. AB - The genetic relatedness of 81 clinical rotavirus isolates to the human rotavirus prototype strains Wa (subgroup 2, serotype 1) and DS-1 (subgroup 1, serotype 2) was examined by RNA hybridization techniques. Labeled single-stranded (+) transcripts of Wa or DS-1 virus were incubated with denatured genomic rotaviral RNAs, and the resulting hybrids were subjected to gel electrophoresis and autoradiography. Nineteen of the specimens contained subgroup 1 rotavirus with a "short" RNA migration pattern. These viruses were found to be closely related to the DS-1 strain and were associated with illness of short duration. The remaining 62 isolates belonged to subgroup 2 and exhibited a "long" RNA migration pattern. Fifty-four of these isolates exhibited significant hybridization with the Wa strain probe. Four isolates yielded multiple hybrid bands with the Wa probe but also possessed at least one gene segment homologous to the DS-1 strain. The remaining four subgroup 2 rotaviruses did not exhibit significant homology in the form of labeled hybrid bands when tested with either the Wa or DS-1 probe. These findings suggest that most clinical rotavirus isolates belong to one of two human rotavirus "families" defined as Wa-like or DS-1-like. Our observations also suggest that reassortment occurs in vivo between rotaviruses belonging to the two human rotavirus "families" and that there are one or more additional families of human rotavirus. PMID- 2997384 TI - Humoral immune response to HSV-1 and HSV-2 viral proteins in patients with primary genital herpes. AB - The humoral immune response to HSV-1 and HSV-2 proteins was examined in patients with primary first-episode genital herpes. Ten patients had culture-proven HSV-1 infections, 37 had HSV-2 infections, and all were seronegative to HSV proteins before developing their infections. Development of serum antibodies to individual HSV proteins and glycoproteins was determined by immunoprecipitation of radiolabeled HSV-1- and HSV-2-infected cell proteins and subsequent gel electrophoresis. In HSV-1 patients, a sequential development of antibodies to HSV 1 proteins was observed with early appearance of antibodies to the nucleocapsid protein p148 and to glycoproteins gB and gC. Seroconversion to gD and to a polypeptide of 88,000 molecular weight (p88) occurred next, and, finally, seroconversion to gE and to a nonglycosylated 66,000 dalton protein p66. In HSV-2 patients, antibodies to HSV-2 proteins p148, gB, and p88 appeared within 1 week of onset of symptoms. Seroconversion to p66, gD, and to a complex of glycoproteins gC and gE ("g80") occurred later, at a mean time of approximately 3 weeks. Seroconversion to HSV-1 gB, p88, and p66 occurred significantly later than seroconversion to the homologous counterparts. Seroconversion within 21 days of onset to HSV-2 gD, g80, and p66 was associated with a longer time to the first recurrence in HSV-2 patients, suggesting a possible role of these antibodies, alone or in combination, in the maintenance of HSV-2 latency in humans. PMID- 2997385 TI - Acute glandular fever-like illness in a patient with HTLV-III antibody. AB - A lymph node biopsy obtained from a patient with human T-cell lymphocytotropic virus III/lymphadenopathy-associated virus (HTLV-III/LAV) antibody, presenting with an acute glandular fever-like illness, was examined by electron microscopy. Numerous pathological changes were present in the biopsy, including hypertrophy of smooth endoplasmic reticulum, intracytoplasmic rod-like inclusions within the cisternae of endoplasmic reticulum, multivesicular bodies, test-tube and ring shaped forms, and tubulo-reticular structures. Intranuclear and intracytoplasmic viral-like particles measuring 105-120 nm in diameter and small cytoplasmic particles measuring 50-70 nm in diameter were found in some degenerating lymph node cells. These pathological findings may reflect a host cell response to various pathological and viral stimuli resulting from immune deficiency owing to infection with HTLV-III/LAV. PMID- 2997386 TI - Detection of BK virus IgM antibodies by two enzyme-linked immunosorbent assays (ELISA) and a hemagglutination inhibition method. AB - We have used an antigen solid-phase enzyme-linked immunosorbent assay (SP-ELISA) and an IgM antibody capture ELISA (MACELISA) for detecting IgM antibodies to human polyomavirus BK (BKV). These tests were compared with the standard hemagglutination inhibition test (HAI) of IgM serum fractions following sucrose density gradient fractionation. The SP- and MACELISA were not influenced by concomitant BKV-IgG, but high levels of both BKV-IgG and rheumatoid factor could cause false positive results by SPELISA, but not by MACELISA. The MACELISA gave much higher positive to negative ratios than the SPELISA. The sensitivity and specificity of the two tests were high compared to the IgM-HAI method. The sera could be tested in a single dilution (1:160), and thus the ELISA-tests are useful for testing large numbers of sera. PMID- 2997387 TI - Tolerance of one-month intranasal interferon. AB - Under double-blind conditions, groups of volunteers (68 in total) were allocated at random to take intranasal solutions of placebo or one of three doses of highly purified leucocyte interferon by intranasal spray twice a day for 28 days. The highest dose would have been expected to protect against experimental colds. Treatment was discontinued because of upper respiratory symptoms as often in each of the interferon groups as in the placebo group. However, it was possible to distinguish clinically between "colds" on placebo and low-dose interferon and "reactions to treatment" on high-dose interferon. The features of the reactions to treatment were a protracted build-up of local symptoms and minor epistaxis. None of the volunteers on the high-dose interferon were thought to have a definite cold, but viruses were isolated from four out of six volunteers on low dose interferon who had definite colds. Previous experiments had also shown this dose to be insufficient to protect against experimental rhinovirus challenge. The dose of interferon that appeared to protect against virus infection caused significant unwanted effects. It is essential to find interferon preparations with less inflammatory activity before interferon can be considered for use as a long-term prophylactic against the common cold. PMID- 2997388 TI - Cystosarcoma phyllodes: the wide spectrum of clinical behavior. PMID- 2997389 TI - Experimental coxsackievirus B4 meningitis. PMID- 2997390 TI - Reaction of human ceruloplasmin and anion treated ceruloplasmin with diethyldithiocarbamate. AB - The reaction of human ceruloplasmin and anion treated ceruloplasmin with diethyldithiocarbamate was studied at pH 5.5. The analysis of optical and EPR spectra at 9 GHz showed that ceruloplasmin contains five paramagnetic copper ions, two of which, X and Y, not involved in enzymatic activity, are chelated by diethyldithiocarbamate; the complex thus formed is easily removed by high-speed centrifugation. However, the enzyme depleted of these two X and Y copper ions is able to compete with the Cu(II)-diethyldithiocarbamate complex, as time elapses, recovering both Cu(II) atoms. In addition diethyldithiocarbamate acts as a reducing agent for the two type-I copper atoms when added in large excess to the enzyme or the anion treated enzyme. PMID- 2997391 TI - Interaction between bound cupric ion and spin-labeled cysteine beta-93 in human and horse hemoglobins. AB - The location of the various copper binding sites for horse and human hemoglobin was probed using spin labels attached to the beta-93 cysteine residue. Dipole dipole interactions between the spin label and bound copper produce a decrease in the amplitude of the spin label spectrum which was used to estimate the Cu(II) spin label distance. By comparing the results with horse and human hemoglobin at 298 and 77 K four different Cu(II) binding sites were identified. The low affinity horse hemoglobin site with the sulfhydryl blocked (site 1) was found to be located 10-13 A from the sulfhydryl spin label on the surface of the molecule. Only with a free sulfhydryl is the site (site 2) in the pocket between the F and H helices closer to the SH-group and the iron populated. It is site 2 which is responsible for the oxidation. In frozen solutions a Cu-nitroxide distance of about 17 A was determined with human hemoglobin. This distance is consistent with the previously postulated location of the "high affinity" human hemoglobin site near the amino terminus of the beta-chain. At 298 K a much shorter Cu-nitroxide distance of about 7 A was calculated for human hemoglobin. This shorter distance at higher temperature also correlated with a slightly smaller value of g11 and A11 for the Cu(II) ESR spectrum. It is postulated that in solution cross-linking between nitrogenous ligands in the region of the amino terminus of one beta-chain and the carboxyl terminus of the other beta-chain can explain this shorter distance. This cross-link could involve histidine beta-143, which is one of the ligands thought to be also involved in site 1. Binding to the "high-affinity" site in solution thus stabilizes the "low-affinity" site 2 relative to site 1 explaining the reported interaction between the "high-affinity" and "low affinity" sites. PMID- 2997392 TI - Effect of hyperglycemia and its prevention by insulin treatment on the incorporation of 32P into polyphosphoinositides and other phospholipids in peripheral nerve of the streptozotocin diabetic rat. AB - The influence of varying doses of streptozotocin and preventive insulin treatment on phospholipid metabolism in sciatic nerve in vitro from diabetic rats was studied. Animals were given 30, 45, and 60 mg/kg injections of streptozotocin and 10 weeks later nerves were removed and incubated in the presence of [32P] orthophosphate. The quantity of isotope incorporated into phosphatidylinositol 4,5-bisphosphate (PIP2) was progressively greater with increasing drug dosage, whereas uptake of label into other phospholipids was unchanged. Rats were made diabetic and within 72 h were implanted with long-acting, insulin-containing osmotic minipumps and the incorporation of [32P]orthophosphate into phospholipids of intact and epineurium-free nerves was examined 8 weeks later. For whole nerve, increased labeling in nerves from diabetic animals occurred only in PIP2 and phosphatidylinositol-4-phosphate (PIP) and was completely prevented by insulin treatment. Isotope incorporation into polyphosphoinositides was also markedly elevated (greater than or equal to 100%) in desheathed diabetic nerves, but not in nerves from insulin-treated animals. Other phospholipids in epineurium-free nerves displayed some rise in isotope uptake, but the increases were not prevented by insulin treatment and appeared unrelated to hyperglycemia. Morphological examination of nerves extended previous findings that prolonged insulin treatment produces axonal degeneration. These observations indicate that abnormal nerve polyphosphoinositide metabolism is at least in part a consequence of hyperglycemia. The metabolic alterations may be intimately involved in reduced nerve conduction velocity, which is characteristic of diabetic neuropathy. PMID- 2997393 TI - Evidence for [D-Ala2,D-Leu5]enkephalin-induced supersensitivity to 5 hydroxytryptamine in a neurotumor x brain hybrid cell line (NCB-20). AB - A neuroblastoma X Chinese hamster embryonic brain explant hybrid cell line (NCB 20) expressed 5-hydroxytryptamine (5-HT1) receptors, linked to adenylate cyclase, which closely resembled 5-HT1 receptors previously characterized in central nervous tissue. However, the affinity of the receptors for 5-HT was only 150 nM compared to 5 nM in membranes prepared from cerebral cortex. The elevation of cyclic AMP levels in NCB-20 cells produced by 5-HT was found additive to that produced by cholera toxin but synergistic with that produced by either prostaglandin E1 (PGE1) or forskolin, suggesting that these latter two agents elevate cyclic AMP levels by a different mechanism than 5-HT. The elevation of cyclic AMP levels by either 5-HT or PGE1 was reversed by [D-Ala2,D Leu5]enkephalin (DADLE), morphine, clonidine, and 3,4-dihydroxyphenylethylamine (dopamine) on a short (30 min) time scale. However, continued exposure to DADLE resulted in loss of the initial inhibitory effects of DADLE after 6 h and return of cyclic AMP levels to that seen with either 5-HT or PGE1 alone. When the DADLE exposure time was increased to 48 h, 5-HT produced a further twofold increase in cyclic AMP levels, but there was no increase in the responsiveness of the cells to PGE1 unless naloxone was added 1 h prior to treatment with PGE1. Scatchard analysis showed that the increased potency of 5-HT resulted from an increase in receptor affinity for 5-HT (from a KD of 150 +/- 20 nM to one of 20 +/- 7 nM), with a reduction in the number of apparent binding sites. The 5-HT supersensitivity observed in NCB-20 cells may be a good model for neurotransmitter interactions that produce desensitization or facilitation in the intact nervous system. PMID- 2997394 TI - Possible role of gangliosides in regulating an adenylate cyclase-linked 5 hydroxytryptamine (5-HT1) receptor. AB - Cultured NCB-20 hybrid cells express adenylate cyclase-coupled receptors for 5 hydroxytryptamine (5-HT) that correspond biochemically and pharmacologically to 5 HT1 receptors in rodent brain membrane preparations, apart from a much-reduced affinity for 5-HT (160 nM compared to less than 5 nM in brain). Since NCB-20 cells also differ from rodent brain both qualitatively and quantitatively in their ganglioside composition, the effects of exogenously added gangliosides on the affinity of the 5-HT1 receptor for 5-HT were tested. Both GM1 ganglioside (the cholera toxin receptor) and tetrasialoganglioside GQ1b produced a 10-fold increase in receptor affinity for [3H]5-HT, measured by binding studies. All gangliosides, at submicromolar concentrations, resulted in significantly reduced EC50 values for 5-HT-mediated elevation of intracellular cyclic AMP levels. GQ1b had the capacity to most dramatically enhance the potency of 5-HT in mediating increases in cyclic AMP levels. Gangliosides had no effect on the potency of DADLE or 3,4-dihydroxyphenylethylamine (dopamine)-mediated depression of cyclic AMP levels, suggesting some specificity for 5-HT. Our data are interpreted as implying a specific role for polysialogangliosides in modulating the affinity of the 5-HT1 receptor and the coupling of the 5-HT1 receptor-guanine nucleotide binding protein adenylate cyclase complex. PMID- 2997395 TI - Monoclonal antibodies to benzodiazepines. AB - Four hybridoma lines secreting monoclonal antibodies to benzodiazepines were produced after BALB/c mice were immunized with a benzodiazepine-bovine serum albumin conjugate. The monoclonal antibodies were purified from ascites fluids, and their binding affinities for benzodiazepines and other benzodiazepine receptor ligands were determined. These antibodies have very high binding affinities for diazepam, flunitrazepam, Ro5-4864, Ro5-3453, Ro11-6896, and Ro5 3438 (the KD values are in the 10(-9) M range). However, these antibodies have low affinities for the benzodiazepine receptor inverse agonists (beta-carbolines) and antagonists (Ro15-1788 and CGS-8216). PMID- 2997396 TI - Barbiturates are selective antagonists at A1 adenosine receptors. AB - Barbiturates in pharmacologically relevant concentrations inhibit binding of (R) N6-phenylisopropyl[3H]adenosine ([3H]PIA) to solubilized A1 adenosine receptors in a concentration-dependent, stereospecific, and competitive manner. Ki values are similar to those obtained for membrane-bound receptors and are 31 microM for (+/-)-5-(1,3-dimethyl)-5-ethylbarbituric acid [(+/-)-DMBB] and 89 microM for (+/ )-pentobarbital. Kinetic experiments demonstrate that barbiturates compete directly for the binding site of the receptor. The inhibition of rat striatal adenylate cyclase by unlabelled (R)-N6-phenylisopropyladenosine [(R)-PIA] is antagonized by barbiturates in the same concentrations that inhibit radioligand binding. The stimulation of adenylate cyclase via A2 adenosine receptors in membranes from N1E 115 neuroblastoma cells is antagonized only by 10-30 times higher concentrations of barbiturates. It is concluded that barbiturates are selective antagonists at the A1 receptor subtype. In analogy to the excitatory effects of methylxanthines it is suggested that A1 adenosine receptor antagonism may convey excitatory properties to barbiturates. PMID- 2997397 TI - Normal myelin composition and phospholipid synthesis in adrenalectomized rats with reduced brain myelin and glycerol 3-phosphate dehydrogenase activity. AB - The composition of CNS myelin was investigated in rats adrenalectomized at day 14 and killed 7 days later, previously shown to result in a 25% reduction in the amount of bulk-isolated myelin and a 40% decrease in brain glycerol 3-phosphate dehydrogenase activity. The proportions of the major myelin proteins, as well as the specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase, were the same in the myelin from both adrenalectomized and control animals. The amount of total phospholipid and the proportions of individual phospholipids were also normal in myelin from the adrenalectomized animals. The amount of nonmyelin phospholipid in whole brain was unchanged by adrenalectomy. Labeling studies carried out 4 days after adrenalectomy of 14-day-old animals showed no change in the synthesis rates of the major myelin phospholipids as compared with the synthesis rate of nonmyelin phospholipids. Furthermore, incorporation of [1,(3) 14C]glycerol into the glycerol moiety of ethanolamine plasmalogen, which requires glycerol 3-phosphate dehydrogenase, was also normal, showing that the reduced oligodendroglial glycerol 3-phosphate dehydrogenase activity following adrenalectomy was not rate-limiting for myelin phospholipid synthesis. PMID- 2997398 TI - Benzodiazepine binding characteristics of embryonic rat brain neurons grown in culture. AB - The binding of [3H]diazepam to cell homogenates of embryonic rat brain neurons grown in culture was examined. Under the conditions used to prepare and maintain these neurons, only a single, saturable, high-affinity binding site was observed. The binding of [3H]diazepam was potently inhibited by the CNS-specific benzodiazepine clonazepam (Ki = 0.56 +/- 0.08 nM) but was not affected by the peripheral-type receptor ligand Ro5-4864. The KD for [3H]diazepam bound specifically to cell homogenates was 2.64 +/- 0.24 nM, and the Bmax was 952 +/- 43 fmol/mg of protein. [3H]Diazepam binding to cell membranes washed three times was stimulated dose-dependently by gamma-aminobutyric acid (GABA), reaching 112 +/- 7.5% above control values at 10(-4) M. The rank order for potency of drug binding to the benzodiazepine receptor site in cultured neurons was clonazepam greater than diazepam greater than beta-carboline-3-carboxylate ethyl ester greater than Ro15-1788 greater than CL218,872 much greater than Ro5-4864. The binding characteristics of this site are very similar to those of the Type II benzodiazepine receptors present in rat brain. These data demonstrate that part, if not all, of the benzodiazepine-GABA-chloride ionophore receptor complex is being expressed by cultured embryonic rat brain neurons in the absence of accompanying glial cells and suggest that these cultures may serve as a model system for the study of Type II benzodiazepine receptor function. PMID- 2997399 TI - Autonomic nervous system dysfunction in workers exposed to organic solvents. AB - Tests for cardiovascular function were made in 34 workers exposed to organic solvents for several years (mean 16.2 years). The results were compared with those of 52 healthy subjects. All the cardiovascular parameters were lower in the exposed group. The exposed subjects with evidence of peripheral neuropathy had lower values than those without neuropathy. The findings suggest that peripheral parasympathetic nerves may also be involved in mild sensory-motor peripheral neuropathy among subjects with long-term occupational exposure to organic solvents. PMID- 2997400 TI - Differential effects of alpha-adrenoceptor blockade on essential, physiological and isoprenaline-induced tremor: evidence for a central origin of essential tremor. AB - Intravenous thymoxamine reduced the power of essential tremor but increased that of physiological and isoprenaline-induced tremor. These findings indicate that essential and physiological tremor have dissimilar pathophysiological mechanisms. They also suggest that central adrenergic mechanisms are involved in the pathophysiology of essential tremor and that isoprenaline-induced tremor is not a good model of essential tremor. Furthermore, alpha-adrenoceptor blockers may be a useful therapy for essential tremor. PMID- 2997401 TI - Rhabdomyolysis and concomitant neurological lesions after intravenous heroin abuse. AB - Seven cases of rhabdomyolysis in heroin addicts are presented. All patients showed concomitant neurological symptoms suggesting mononeuropathy, incomplete plexus lesions or myelopathy. In most cases rhabdomyolysis occurred without preceding trauma to the muscles (for example tissue compression or coma). Five patients had a history of recently resumed heroin abuse after prolonged abstinence. An allergic or toxic reaction to heroin or adulterants seems to be more likely than trauma in the pathogenesis of these complications. Severe rhabdomyolysis can occur without visible muscular swelling. Routine screening of creatine kinase is recommended in heroin addicts with neurological complications, as rhabdomyolysis may lead to fatal renal failure and may easily fail to be diagnosed. PMID- 2997402 TI - Comparison of in vitro demyelination and cytotoxicity of humoral factors in multiple sclerosis and other neurological diseases. AB - The distribution and nature of serum factors causing in vitro demyelination and glial lysis were investigated in multiple sclerosis (MS), other neurological diseases (OND), ill control and control groups. MS sera were unique in affecting only CNS myelin and glia whereas stroke and Guillain-Barre syndrome (GBS) sera brought changes to both CNS and PNS tissue. Through both visual scoring of myelin damage and the quantitative measurement of radiolabel release from cerebellar cultures, it was evident that the MS and OND groups have similar myelino- and cytotoxic effects. This may reflect MS and OND sera sharing similar humoral factors. 74% MS, 68% OND and 22% of control scores were above a score threshold designed to exclude culture handling trauma effects. When classified by their current disease state MS patients with severe and mild disease yielded higher in vitro scores than did those with moderate disease who comprised an older age group. No other clinical features of MS patients gave any association with in vitro serum effects. The rare demonstration of bound Fab IgG in cultures after MS serum tests indicates that immune mechanisms are unlikely to make a large contribution to serum-induced demyelination and cellular change in vitro. PMID- 2997403 TI - 2,4-Dichlorophenoxyacetic acid (2,4-D) does not cause polyneuropathy in the rat. AB - 2,4-Dichlorophenoxyacetic acid (2,4-D), a component of Agent Orange, was injected intraperitoneally into adult male Fisher rats for 3-12 weeks. During the period of study gait and toe-spreading reflexes remained normal and distal motor latencies, motor and mixed nerve conduction velocities and amplitudes remained similar (P greater than 0.05) in animals receiving 2,4-D or vehicle. This study suggests that 2,4-D is not toxic to peripheral nerves in the rat. PMID- 2997404 TI - Evaluation of renal parathyroid hormone receptor function in myotonic dystrophy. AB - Endocrine abnormalities in myotonic dystrophy (MyD) reflect some of the multi systemic involvement resulting from this disorder. One of these, abnormal insulin secretion, is considered to be caused by receptor dysfunction. Bone abnormalities, cataract and calcium transport defect suggest the abnormal calcium metabolism in MyD. The calcium metabolism is chiefly regulated by parathyroid hormone (PTH). An interest in the similarity between MyD and pseudohypoparathyroidism, which is a disorder of PTH receptor dysfunction, encouraged the authors to evaluate renal PTH receptor function from the responses of urinary adenosine 3',5'-monophosphate (cAMP) and phosphate excretion after administration of human PTH(1-34). The responses of cAMP were high in 3 cases, low in one case, but normal in the 4 other cases. The phosphaturic responses were elevated in 3 cases, reduced in 3 cases, and normal in 2 other cases. Since these abnormal responses closely mimic those in hypoparathyroidism, there may also be renal PTH receptor dysfunction in some cases of MyD. The results of the present study suggest another peptide hormone receptor defect, similar to insulin, which supports the hypothesis of generalised receptor dysfunction in MyD. PMID- 2997406 TI - VP-16 and cisplatin as first-line therapy for small-cell lung cancer. AB - Thirty-one patients with small-cell lung cancer (SCLC) were treated with VP-16 and cisplatin as first-line therapy. In the majority of cases an Adriamycin (Adria Laboratories, Columbus, Ohio) containing regimen was contraindicated because of severe cardiac or hepatic disease. Eight patients who presented with cerebral metastases were also included in the series. Eleven patients had limited disease (LD), and 20 had extensive disease (ED). Of the 28 evaluable patients, 12 (43%) achieved a complete response (CR) and 12 (43%) had a partial response (PR). Four patients (14%) either had no response or progressed on treatment. The median duration of response for patients with LD was 39 weeks and for those with ED, 26 weeks. The median survival time (MST) for the whole group of responding (CR and PR) LD patients was 70 weeks (range, 28 to 181 + weeks), and for responding ED patients, it was 43 weeks (range, 17 to 68 weeks). Gastrointestinal toxicity was mild, but leukopenia and thrombocytopenia were common. There were four febrile episodes during periods of drug-induced neutropenia and this led to one treatment related death. Nephrotoxicity occurred in 15 patients and required discontinuation of cisplatin in two. These results compare favorably with reports of standard induction chemotherapy regimens and provide further evidence of the activity of the VP-16 and cisplatin regimen in patients with SCLC. PMID- 2997405 TI - Severe polyneuropathy in Tangier disease mimicking syringomyelia or leprosy. Clinical, biochemical, electrophysiological, and morphological evaluation, including electron microscopy of nerve, muscle, and skin biopsies. AB - Polyneuropathy in Tangier disease can be divided into three clinical types. The most severe form (type III) with a syringomyelia-like syndrome has been described in three cases only. Here, a fourth case of this type is presented. Because of unusual trophic disturbances even leprosy was suspected. Electrodiagnostic findings, including evoked cerebral potentials in this case, were suggestive of a generalized neuropathy with some degree of primary or secondary demyelination and implied possible impairment of central structures. Sural nerve biopsy, including electron microscopy and quantitative analysis, revealed a predominant reduction of smaller myelinated and unmyelinated fibres. The main morphological feature was the abundance of abnormal non-membrane-bound vacuoles in Schwann cells, mostly of the unmyelinated type, and in some endoneurial fibroblasts, macrophages and perineurial cells. There was no inverse relationship between lipid vacuoles and axons in Schwann cell complexes as suspected by others. An excess of endoneurial collagen as well as an increased fascicular area were obvious. In five skin biopsy specimens of different regions typical vacuoles were noted in Schwann cells, histiocytes, nevus cells, and rarely in perineurial cells. PMID- 2997407 TI - Single agent vincristine by infusion in refractory multiple myeloma. AB - A phase 2 trial of vincristine infusion was conducted in a group of 21 patients with refractory multiple myeloma. Patients were generally heavily pretreated with radiotherapy and chemotherapy. Vincristine was given intravenously (IV) as a 0.5 mg bolus and followed immediately by infusion of 0.25 to 0.50 mg/m2/d for 5 days. Courses were repeated every 3 weeks in the absence of disease progression or prohibitive toxicity. Objective responses (partial) were noted in two patients (10%), both of whom were administered 0.5 mg/m2/d infusions. Response durations were brief (2.2 and 1.2 months). Toxicity consisted of neurotoxicity and myelosuppression. In addition to the occurrence of paresthesias and myalgias, ileus (two cases) and moderately severe loss of motor function (two cases) were observed. The mean lowest WBC count following treatment was 2.67 X 10(3)/microL v 3.96 X 10(3)/microL pretreatment (P = .008). The mean lowest platelet count was 75.0 X 10(3)/microL v 106.8 X 10(3)/microL pretreatment (P = .008). Vincristine infusion appears to have limited activity in the treatment of refractory multiple myeloma. Additionally, response durations were short lived and toxicity, both neurologic and hematologic, was appreciable. PMID- 2997408 TI - 4'Epidoxorubicin (epirubicin): activity in hepatocellular carcinoma. AB - Doxorubicin provides the most consistent response rate in hepatocellular carcinoma. We therefore initiated a trial with its analog 4'epidoxorubicin. Eighteen patients, all without prior treatment, were given the drug as a single agent every 3 weeks with dose escalation whenever possible. Five patients were treated by six-hour infusion and 13 by intravenous (IV) bolus injection, with the median dose being 90 mg/m2. The patients were of diverse ethnic background and included some with underlying cirrhosis and hepatitis B surface antigenemia. Three patients had partial remissions (6, 12, 48 weeks) for a response rate of 17%. Four patients also had prolonged stable disease (14, 26, 27, 38 weeks). Toxicity was mild, although cardiac toxicity developed in three patients at 685, 825, and 1,460 mg/m2 cumulative dose. The response to 4'epidoxorubicin in this study appears to be equivalent to the reported response rates for doxorubicin, with decreased toxicity. PMID- 2997409 TI - Solubilization of peripheral benzodiazepine-binding sites from rat kidney. AB - The ability of a variety of detergents to solubilize peripheral benzodiazepine binding sites from rat kidney was tested. Of all the detergents tested, only digitonin was found to be suitable for solubilization. This detergent solubilized 21% of the binding activity; 47% was inactivated, and 32% remained in the pellet. Specific binding of [3H]Ro 5-4864 to membrane-bound and solubilized peripheral benzodiazepine-binding sites was saturable, yielding a linear Scatchard plot (r = 0.96). KD values obtained for the membrane-bound and solubilized peripheral benzodiazepine binding sites were 3.9 +/- 0.4 nM and 5.4 +/- 0.4 nM, respectively. Respective Bmax values were 4.6 +/- 0.5 and 1.9 +/- 0.2 pmol/mg of protein. The KD value for the solubilized material obtained from kinetic experiments was 5.3 +/- 0.6 nM. The potency of PK 11195, Ro 5-4864, diazepam, flurazepam, chlordiazepoxide, Ro 15-1788, methyl-beta-carboline-3-carboxylate, and clonazepam to displace bound [3H]Ro 5-4864 from peripheral binding sites was similar in the membrane-bound and the soluble states. Most of the binding activity of the solubilized binding sites was destroyed by heating at 60 degrees C for 30 min or by treatment with 2 M guanidinium chloride or 4 M urea. More than 95% of the binding activity of the solubilized binding sites was retained after 18 hr at 4 degrees C, and more than 60% was retained after 4 days at the same temperature. These results indicate that the binding characteristics of peripheral benzodiazepine-binding sites extant in the membrane-bound state are retained after solubilization. PMID- 2997411 TI - Opiate and alpha 2-adrenoceptor responses of rat amygdaloid neurons: co localization and interactions during withdrawal. AB - Interactions between neuronal responses mediated by opiate receptors and by alpha 2-adrenoceptors were characterized in the amygdala. Extracellular single-unit recordings and microiontophoresis were performed using five-barrel microelectrodes in chloral hydrate-anesthetized rats. A subpopulation of amygdaloid cells displayed inhibitory responses to morphine or D-Ala,D-Leu enkephalin; antagonist studies suggested that both mu- and delta-opiate receptor subtypes were present. The same neurons displayed inhibitory responses to norepinephrine or clonidine mediated by alpha 2-adrenoceptors. Responses mediated by opiate receptors and by alpha 2-adrenoceptors were highly co-localized to the same subpopulation of amygdaloid neurons. Such cells responded to microiontophoresis of either morphine or clonidine, whereas other cells in the amygdala generally showed neither response. Responsive cells were characterized by a distinctive, triphasic waveform and a high sensitivity to glutamate. These cells were largely restricted to the nucleus centralis and the posterior portion of the nucleus medialis. Cells outside of this group showed suppressant responses to norepinephrine which appeared not to be mediated by alpha 2-adrenoceptors. After chronic morphine treatment, application of opioid antagonists elicited a withdrawal response, consisting of an increase in firing rate. Clonidine reversed the withdrawal response of these cells. The amygdala may be one of the regions of the nervous system in which clonidine acts to reduce symptoms of opiate withdrawal. PMID- 2997410 TI - Characterization of barbiturate-stimulated chloride efflux from rat brain synaptoneurosomes. AB - Membrane chloride (Cl-) permeability was studied in a novel subcellular brain preparation, the synaptoneurosome. Using a radioactive tracer exchange technique, Cl- transport was determined by measuring 36Cl- efflux from rat cerebral cortical synaptoneurosomes. Barbiturates increased 36Cl- efflux in a dose-dependent manner with the following relative order of potency: 5-(1,3-dimethylbutyl)-5-ethyl barbituric acid ((-)-DMBB) greater than pentobarbital greater than secobarbital greater than (+)-DMBB greater than hexobarbital greater than amobarbital greater than mephobarbital. Phenobarbital and barbital were virtually inactive. A good correlation was observed between the potencies of these barbiturates in stimulating 36Cl- efflux and their anesthetic potencies in mice (r = 0.90, p less than 0.01) and their abilities to enhance [3H] diazepam binding to brain membranes (r = 0.77, p less than 0.05). The effect of pentobarbital in enhancing 36Cl- efflux was reversed by the gamma-aminobutyric acid (GABA) antagonists picrotoxin and bicuculline. Picrotoxin and bicuculline both decreased 36Cl- efflux in the absence of pentobarbital, suggesting the presence of endogenous GABA. Incubation of synaptoneurosomes with 4,4'-di-isothiocyano- or dinitro-2,2' disulfonic acid stilbene, inhibitors of anion transport, also decreased both basal and pentobarbital-induced 36Cl- efflux. Pentobarbital (500 microM) was most effective in inducing 36Cl- efflux in the cerebellum, hippocampus, and cortex (23.7, 23.6, and 22.5%, respectively), and was less effective in stimulating 36Cl efflux in the striatum (15.1%) and pons-medulla (6.2%). The relative efficacy of pentobarbital in enhancing 36Cl- efflux among these various brain regions was highly correlated (r = 0.96, p 0.01) with the relative densities of [35S]-t butylbicyclophosphorothionate-binding sites, a measure of GABA-gated Cl- channel density.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2997412 TI - The role of the polymorphonuclear leukocyte in hyperalgesia. AB - The results of recent studies of the mechanism of leukotriene B4-induced hyperalgesia suggest a dependence on polymorphonuclear leukocytes (PMNLs). In this study, we addressed the contribution of PMNLs to hyperalgesia evoked by the peptide chemotactic factors N-formyl-methionyl-leucyl-phenylalanine (fMLP) and the anaphylatoxin fragment of the fifth component of the complement pathway (C5a). Local injection of glycogen, which attracts but does not activate PMNLs, produced a marked shift to the left (toward lower concentrations) in the concentration dependence curve of fMLP-induced hyperalgesia. In addition, PMNL repletion by transfusion with syngeneic PMNLs reestablished fMLP-induced hyperalgesia in PMNL-depleted rats. Finally, supernatants from rat and human PMNLs, that had been stimulated with fMLP in vitro, produced hyperalgesia in PMNL depleted rats. Preliminary characterization of the hyperalgesia-inducing activity released by stimulated PMNLs indicated that it is lipid in nature. The nonsteroidal anti-inflammatory indomethacin did not attenuate C5a and fMLP induced hyperalgesia. Thus, the hyperalgesia produced by fMLP and C5a is similar to that produced by leukotriene B4 in that it is dependent on PMNLs and independent of the cyclo-oxygenation of arachidonic acid. Taken together, these data suggest that structurally diverse PMNL-chemotactic factors produce hyperalgesia by a novel mechanism, involving PMNL-derived factors. PMID- 2997413 TI - Eicosanoids in the central nervous system. AB - All mammalian tissue investigated to date is capable of eicosanoid biosynthesis in response to various activating stimuli. While the importance of these metabolites as major mediators of many normal physiological processes and some pathophysiological conditions has not been proven, it is evident that these compounds are at least important modulators of many cellular and organ system functions. This review is intended to provide the reader with a brief overview of eicosanoid biology, with specific reference to the neurosciences. The increasing knowledge about the role of the eicosanoids in neurobiology may contribute to the understanding and treatment of many neurological diseases. The eicosanoids comprise several groups of biologically active unsaturated fatty acids: the "primary" prostaglandins, the cyclic endoperoxides, the prostanoids, the leukotrienes, and other acid lipids. This article includes a review of the enzymatic pathways of biosynthesis and metabolism of eicosanoids in man, and the pertinent structural nomenclature. The general basic and clinical pharmacological effects of the more important compounds on vascular perfusion, platelet function, intracellular enzyme activity, and interactions with other mediators of cellular activity are reviewed. A more detailed review of the actions of eicosanoids as mediators or modifiers of central nervous system physiology and pathophysiology is presented. Recent animal and human studies on the use and alterations of the eicosanoid metabolites is summarized, specifically where they relate to several clinical problem areas of interest to the neurosurgeon and neurobiologist. These areas include cerebrovascular circulation physiology, cerebral ischemia, cerebral vasospasm following subarachnoid hemorrhage, migraine headaches, hypothalamic function, neurotransmission, and nociception. A bibliography of 92 articles for further review is also included. PMID- 2997414 TI - Transsphenoidal surgery following unsuccessful prior therapy. An assessment of benefits and risks in 158 patients. AB - The authors report the results of a retrospective study conducted in an effort to define the results and risks of transsphenoidal surgery for patients whose prior therapy had failed. In a series of 1210 patients undergoing transsphenoidal surgery during a 10-year period, 158 had received prior therapy: 127 for pituitary adenoma, 20 for craniopharyngioma, and 11 for other lesions. Prior therapy was considered "direct" when it consisted of craniotomy or transsphenoidal surgery (either open or stereotaxic), and "indirect" when it consisted of radiation therapy, adrenalectomy, or bromocriptine therapy. The current transsphenoidal operation was performed for persistent hyperfunctioning endocrinopathy in 63 patients, for visual loss in 72 patients, and for cerebrospinal fluid (CSF) rhinorrhea in 21 patients. Success rates were as follows: normalization of endocrinopathy was achieved in 35% of cases; improvement or stabilization of vision in 59%; and successful repair of CSF rhinorrhea in 74%. The risks associated with repeat transsphenoidal surgery are significantly greater than the same procedure in a previously untreated patient. PMID- 2997415 TI - The rationale and methodology for intra-arterial chemotherapy with BCNU as treatment for glioblastoma. AB - The rationale for, methodology of, and experience with intra-arterial BCNU infusion therapy of malignant glioma are described. This approach achieves tumor levels of drug four times greater than equal doses infused intravenously, and has been used to treat 79 patients over the course of 4 years. The drug was given in 192 infraophthalmic and 66 supraophthalmic carotid artery infusions. Patients who were treated via infraophthalmic carotid artery infusion following tumor recurrence (after both operation and irradiation) survived 54 additional weeks (92 weeks after initial diagnosis). Patients who were treated with BCNU immediately after initial irradiation therapy survived 64 weeks (infraophthalmic carotid artery infusion) and 49.5 weeks (supraophthalmic carotid artery infusion). The major ocular complications (pain and diminished visual acuity) associated with infraophthalmic carotid artery infusion are avoided by selective balloon-guided supraophthalmic carotid artery administration. However, both approaches were associated with white-matter changes, seen as diminished absorption on computerized tomography scans, in 20% of patients treated following irradiation therapy. This toxicity appears to preclude intra-arterial BCNU treatment in the immediate postirradiation period. Better results are being achieved with our current therapy, which involves four infusions of BCNU (400 mg every 4 weeks) into the infraophthalmic or supraophthalmic carotid artery in advance of irradiation. Cisplatin infusions (60 to 90 mg/sq m every 5 weeks) are offered for recurrent glioblastoma. PMID- 2997417 TI - Technetium-99m NGA functional hepatic imaging: preliminary clinical experience. AB - Technetium-99m galactosyl-neoglycoalbumin ( [Tc]NGA) is a radiolabeled ligand to hepatic binding protein, a receptor which resides at the plasma membrane of hepatocytes. This receptor-binding radiopharmaceutical and its kinetic model provide a noninvasive method for the assessment of liver function. Eighteen patients were studied: seven with hepatoma, eight with liver metastases, four with cirrhosis (two had concurrent hepatoma and one chronic active hepatitis), and one patient with acute fulminant non-A, non-B hepatitis. Technetium-99m NGA liver imaging provided anatomic information of diagnostic quality comparable to that obtained with other routine imaging modalities, including computed tomography, angiography, ultrasound, and [Tc]sulfur colloid scintigraphy. Kinetic modeling of dynamic [Tc]NGA data produced estimates of standardized hepatic blood flow, Q (hepatic blood flow divided by total blood volume), and hepatic binding protein concentration, [HBP]. Clinical correlation was by classical Child Turcotte criteria (CTC). Significant rank correlation was obtained between [HBP] estimates and CTC scores (rs = -0.72, p = 0.001). This correlation supports the hypothesis that [HBP] is a measure of functional hepatocyte mass. The combination of decreased Q and markedly reduced [HBP] may have prognostic significance; all three patients with this combination died of hepatic failure within 6 wk of imaging. PMID- 2997416 TI - Establishment of a brain-tumor model in adult monkeys. AB - A brain-tumor model in adult monkeys may be significant because of the biological similarity to humans as well as the feasibility for surgical manipulation and for sequential computerized tomography (CT) scanning. In the present study, brain tumors were successfully produced in Japanese monkeys (Macaca fuscata), each weighing 2 to 10.8 kg, with an average age of 5.1 years old. Tumor cells were implanted by intracerebral inoculation of 4 X 10(7) chick embryo fibroblasts infected with the Schmidt-Ruppin strain of Rous sarcoma virus (RSV). With a 15- to 67-day latency, brain tumors were induced in 11 (73.3%) of 15 RSV-inoculated monkeys. Contrast-enhanced CT scans delineated all solitary intracerebral tumors greater than 4 to 6 mm in diameter. The CT images were proved at autopsy to be accurate within 2 mm in determining the size of tumor. Five of the 11 monkeys with intracerebral tumors died, with an average survival time of 26.6 days after RSV inoculation. The induced tumors were classified as either glioma or sarcoma by the presence or absence of glial fibrillary acidic protein (GFAP) and S-100 protein. A chromosome analysis of cultured tumor cells showed a diploid number of 42, indicating monkey origin. It is concluded that the reproducible brain tumor in the adult Japanese monkey inoculated with RSV can serve as a good experimental brain-tumor model for the further study of human malignant brain tumors. PMID- 2997418 TI - Mechanism of renal concentration of technetium-99m glucoheptonate. AB - Seventy female Sprague-Dawley rats were studied to determine the mechanism of tubular localization and the effects of commonly encountered changes in hydration and acid-base balance on renal uptake and urinary excretion of technetium-99m glucoheptonate ([99mTc]GHA). The in-vivo protein binding and protein-free plasma clearance of [99mTc]GHA also were quantitated. Twenty additional rats were studied to determine the effects of PAH competition and probenecid blockade on renal uptake of [99mTc]dimercaptosuccinic acid (DMSA) in comparison with their effects on [99mTc]GHA localization. Kidney uptake of [99mTc]GHA averaged 11.17 +/ 0.49 (s.e.)% of the injected dose in control animals. This varied slightly among groups but was significantly reduced by probenecid blockade and para aminohippuric acid (PAH) competition to 4.08 +/- 0.55 (p less than 0.005) and 2.39 +/- 0.14 (p less than 0.005), respectively. Technetium-99m DMSA was not affected in its renal accumulation by these maneuvers. The total plasma clearance of [99mTc]GHA was lower than iodine-125(125I)iothalamate but the clearance of the protein free supernate was higher, raising a possibility of some tubular secretion. Acidification of the urine which has been shown to reduce [99mTc]DMSA uptake appeared to have no effect on [99mTc]GHA. Hepatic uptake was minimal in all groups averaging less than 1% injected dose. These data demonstrate that renal accumulation of [99mTc]GHA is blocked by probenecid and PAH suggesting that it is actively concentrated in the proximal tubule by enzyme systems similar to those involved in PAH and hippuran transport. It appears that [99mTc]GHA uptake measures a different aspect of kidney function than [99mTc]DMSA. PMID- 2997419 TI - Phosphorus-32 therapy of cystic Grade IV astrocytomas: technique and preliminary application. AB - Instillation of [32P]chromic phosphate to cystic brain tumors was performed in six patients. Three patients had craniopharyngioma, two had Grade IV astrocytoma and one had Grade II astrocytoma. The cyst volumes ranged from 2 to 44 cc. A calculated dose of 20,000 rad was delivered to the cyst wall. The [32P]chromic phosphate dose given to achieve this dose ranged from 0.11 mCi to 2.5 mCi. Radionuclide leakage was not detected in either the central nervous system or the reticuloendothelial system by bremsstrahlung scanning. Stereotactic instillation was done in some cases, others had indwelling catheters. The frequency of cyst fluid aspiration in the three patients with craniopharyngioma decreased postinstillation. In the two patients with Grade IV astrocytoma, reductions in both the CT documented cyst size as well as the frequency of cyst aspiration were noted. We conclude that [32P]chromic phosphate installation by stereotactic or indwelling catheter method is a safe and helpful procedure in the management of cystic brain tumors. PMID- 2997420 TI - Iodine-123 IMP uptake in brain metastases from lung cancer. PMID- 2997421 TI - Experimental study of the use of collagen tubes for implantation of particulate hydroxylapatite. AB - A pilot study was conducted in rats to evaluate the role of collagen tubes in subperiosteal implantation of particulate hydroxylapatite. The results showed that the collagen tube shaped the hydroxylapatite, facilitated precise implant placement, and prevented particle migration while not interfering with healing. PMID- 2997423 TI - Malignant mixed salivary tumor presenting as a mandibular metastasis. AB - A rare case of a central mandibular metastatic mixed salivary gland tumor is reported which presented following a 45-year history of recurrent benign mixed salivary gland tumor of the parotid gland on the same side. The mandibular tumor included a predominantly benign mixed salivary gland component as well as frankly invasive adenocarcinoma. The clinicopathologic features of the mandibular and residual parotid tumors suggested that the metastatic event may have occurred prior to the development of frank carcinoma in the parotid tumor. PMID- 2997424 TI - [RI scintigraphy of parotid adenolymphoma]. PMID- 2997422 TI - Composite graft for mandibular alveolar ridge augmentation: a preliminary report. AB - Augmentation of atrophic mandibles with a composite graft system consisting of allogeneic freeze-dried rib, autogenous cancellous bone and marrow, and hydroxylapatite is reported. The six patients subjected to this procedure tolerated the surgery well and showed marked improvement in dental function, with maintenance of 78% of the augmented height one year later and 67% of the height at two years. PMID- 2997425 TI - Prostaglandins and hypokalemia. PMID- 2997426 TI - The use of the portal system for the transplantation of a neonate kidney graft in a child with Wilms' tumor. AB - A single an-encephalus neonate kidney graft was transplanted into the portal system of a 6-year-old recipient who had previously undergone removal of the right kidney and inferior vena cava because of Wilms tumor. The left kidney ceased to function shortly thereafter. The child was supported very poorly on hemodialysis, and showed repeated very high levels of cytotoxic antibodies in her serum. The first cross-negative kidney graft that was available harbored two main arteries and duplicate collecting system with two very thin ureters. These vascular anatomic and pathologic variations of both donor graft and recipient necessitated the use of the portal system for renal graft venous drainage and the aorta for the graft revascularization. The ureters that had pinpoint-like lumen were inserted together into the lumen of the native ureter stump and fixated. One year after the transplantation the serum creatinine level is 1.8 mg/dL. PMID- 2997427 TI - Bilateral multifocal Wilms' tumor. AB - A three-year-old boy who presented with symptoms of peritonitis was found to have four Wilms' tumors affecting both kidneys. Individual enucleation of three tumors in the right kidney plus left lower nephrectomy were performed. Chemotherapy was administered for one year. The diagnosis of Wilms' tumor was confirmed on each specimen by the histologic studies. The child remains asymptomatic and developing normally six years after the initial surgical treatment. Bilateral partial nephrectomies is the most conservative of the surgical treatments available for bilateral Wilms' tumor. PMID- 2997429 TI - Furosemide choleresis in isolated perfused rat liver: partial dependency on perfusate sodium and chloride. AB - The effect of furosemide on hepatic bile formation was studied in isolated perfused rat liver to determine if 1) the observed cholestatic effect at lower dose of furosemide in vivo is a primary effect or a secondary effect due to decreased hepatic blood flow caused by the furosemide-induced volume contraction and if 2) the observed choleretic effect at higher doses can be explained by the osmotic effect of furosemide and its metabolites in bile. A single dose of furosemide (initial perfusate concentration 0.01, 0.1 or 1 mM) produced choleresis, whereas 0.001 mM furosemide did not affect bile flow significantly. Because furosemide failed to produce cholestasis at tested doses, the observed cholestasis in vivo at similar blood concentrations must be a secondary effect. Furosemide choleresis was associated with biliary secretion of furosemide and its metabolites. However, the choleretic effect expressed as microliters per micromole of drug secreted declined with increasing dose and biliary secretion. Furosemide choleresis was also associated with an increase in the net biliary secretion of Na+ and Cl-. The effect of Na+ and Cl- replacement on furosemide choleresis was studied to determine if the choleresis was a result of direct effect of furosemide on hepatic electrolyte transport. Replacement of perfusate Na+ completely by Li+ or partially by choline+ resulted in a 30 to 50% reduction in choleretic effect and furosemide-induced biliary Cl- secretion. A similar decline in choleretic effect and net furosemide-induced biliary Na+ secretion was also observed when perfusate Cl- was replaced by nitrate, acetate or isethionate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2997428 TI - Beneficial effects of a new potent and specific thromboxane receptor antagonist (SQ-29,548) in vitro and in vivo. AB - SQ-29,548, a newly synthetized thromboxane receptor antagonist, was investigated for its effects on platelet and vascular thromboxane receptors in vivo and in vitro. Arachidonic acid (AA)-induced sudden death in rabbits was dose-dependently inhibited by SQ-29,548 at doses ranging from 0.2 to 2 mg/kg. Sudden death was accompanied by a 46 +/- 6% decrease in continuously measured circulating platelet count, which was also dose-dependently inhibited by SQ-29,548. The AA-induced increase in continuously recorded whole blood ATP content was 1.2 +/- 0.4 microM and was significantly diminished by all SQ-29,548 doses used. Platelet aggregation induced by AA, the endoperoxide analog U-46,619 or collagen in platelet-rich plasma was dose-dependently inhibited by SQ-29,548 which exerted an IC50 of 0.8, 0.3 or 2.9 microM, respectively. In contrast, ADP and platelet activating factor acether-induced platelet aggregation were unaffected at concentrations of SQ-29,548 up to 260 microM. Thromboxane B2 formation was not significantly altered by SQ-29,548 (1-100 microM) in platelet-rich plasma stimulated with AA or in spontaneously clotting whole blood. Thromboxane synthetase, cyclooxygenase and lipoxygenase product formation were unaffected by SQ-29,548 when washed rabbit platelets were stimulated with radiolabeled AA and the products were measured after separation by thin-layer chromatography. U 46,619 (500 nM), carbocyclic thromboxane A2 (15 nM) and prostaglandin F2 alpha (3 microM)-induced contractions of rabbit pulmonary artery were antagonized by SQ 29,548 exerting a IC50 value between 120 and 40 nM, whereas norepinephrine induced contractions were unaltered.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2997430 TI - A role for adenylate cyclase in the subsensitivity to isoproterenol during ontogenesis of the embryonic chick ventricle. AB - Isoproterenol (ISO) increases contractility and cyclic AMP content in ventricles of embryonic and hatched (H) chicks. A transient decrease in beta agonist sensitivity for both effects (10-fold shift in ISO EC50) is seen in 18-day embryos (18 E) (Pappano and Biegon, 1982). Beta adrenoceptor-coupled adenylate cyclase (AC) and receptor binding were characterized in 14,000 X g ventricular particulates from 10 to 11E, 17 to 19E and 1-week-old chicks (5-6H). The concentration for half-maximal enzyme activation (Kact) for ISO [+100 microM 5' guanylylimidodiphosphate (Gpp(NH)p)]-stimulated AC is greatest in the 17 to 19E (1.6 microM vs. 0.1-0.2 microM in the 5-6H and 10-11E). Maximal enzyme activity in the presence of ISO + Gpp(NH)p (picomoles of cyclic AMP/20 min X mg of protein), however, is greatest in the 10 to 11E (2018 +/- 80 vs. 1020 +/- 80 and 1120 +/- 60 in the 17 to 18E and 5 to 6H, respectively). The Kact values for NaF and Gpp(NH)p are the same for all three ages (2.4 mM and 8 microM, respectively), whereas maximal activity with these effectors of the regulatory and catalytic protein-dependent AC decreases with increasing age. Neither the Kact nor maximal activity of catalytic AC, assessed with MnCl2 and forskolin, changes with age. The respective Kact values for MnCl2 and forskolin are 0.3 mM and 4 microM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2997431 TI - Sensitivity changes in the smooth muscle of the guinea-pig isolated vas deferens after treatment of animals with 6-hydroxydopamine. AB - Postjunctional supersensitivity of the smooth muscle of the guinea-pig vas deferens induced by denervation, decentralization and treatment of animals with reserpine has been attributed, in part, to a partial membrane depolarization (8 10 mV) resulting from reduced electrogenic Na+,K+-pumping activity. This study was undertaken to characterize sensitivity changes which occur after treatment of animals with 6-hydroxydopamine (100 mg/kg + 250 mg/kg i.v., 1 day apart). Seven days after the second injection, concentration-response curves for isometric contractile responses to norepinephrine, methoxamine, acetylcholine and histamine were shifted 40.6-, 1.7-, 3.6- and 2.7-fold, respectively, to the left of control; however, the sensitivity to KCl was not increased, which contrasts with the results after denervation, decentralization and reserpine treatment. Ouabain (10(-5) M) produced 1.8- and 1.3-fold leftward shifts of the KCl concentration response curves in tissues from control and 6-hydroxydopamine-treated animals, respectively. The pronounced effect of ouabain in tissues from treated animals may be an indication that 6-hydroxydopamine treatment does not result in as much inhibition of electrogenic Na+,K+-pumping, and resultant membrane depolarization, as other methods which induce supersensitivity of the guinea-pig vas deferens. PMID- 2997432 TI - Receptors for opioid peptides in the guinea-pig ileum. AB - Cryptic receptor sites in the guinea-pig ileum preparation have been uncovered by the treatment of the preparation with the highly selective, irreversible mu opioid receptor antagonist, beta-funaltrexamine. These beta-funaltrexamine insensitive sites appear to interact only with opioid peptides ([D-Ala2, D Leu5]enkephalin, [D-Ala2, Met5]enkephalinamide, Tyr-D-Ser-Gly-Phe-Leu-Thr and [D Ala2, MePhe4, Gly-ol5]enkephalin) but not with nonpeptide agonists. These new sites could not be protected by either mu-selective (morphiceptin and [D-Ala2, MePhe4, Gly-ol5]enkephalin) or delta-selective ([D-Ala2, D-Leu5]enkephalin, Tyr-D Ser-Gly-Phe-Leu-Thr, (Allyl)2-Tyr-Gly-Gly-psi-(CH2S)-Phe-Leu, and (Allyl)2-Tyr Aib-Aib-Phe-Leu) peptides against beta-chlornaltrexamine alkylation. However, naloxone afforded full protection of these sites against beta-chlornaltrexamine alkylation. The delta-selective antagonist, (Allyl)2-Tyr-Aib-Aib-Phe-Leu, had no activity at these cryptic sites at concentrations that effectively blocked delta receptors in the mouse vas deferens. The cryptic sites do not appear to be typical mu or delta receptors. The new receptors were termed mu', a mu subtype, and a receptor model that is consonant with our data is presented. PMID- 2997433 TI - Reduced neutrophil superoxide anion release after prolonged infusions of lidocaine. AB - Lidocaine is used extensively in coronary care units, yet the effect of lidocaine infusions on neutrophil function has not been known. Lidocaine and other local anesthetics impair leukocyte antibacterial functions when added in vitro. We found that lidocaine added to human neutrophils in vitro markedly impaired the release of superoxide anion (O2-) and the granule enzymes lysozyme and myeloperoxidase after stimulation by phorbol myristate acetate or opsonized zymosan. We then measured production of O2- during stimulation of neutrophils from eight normal subjects, five coronary artery disease patients not receiving lidocaine and 13 coronary artery disease patients receiving lidocaine infusions for at least 12 hr. Release of O2- by cells from lidocaine-treated patients (14.2 +/- 3.8 nmol/2.5 X 10(6) neutrophils per 15 min) was significantly lower than from cells of the normal subjects (78.4 +/- 7.2 nmol; P less than .001) and the coronary patients not receiving lidocaine (70.6 +/- 4.0 nmol; P less than .001). Bactericidal assays at a high concentration (2 mg/ml) of lidocaine demonstrated slight reductions in 2 hr killing rates for Escherichia coli (70% with lidocaine vs. 95% control). Inhibition by lidocaine of the release of toxic oxygen metabolites from neutrophils could potentially reduce infarct size in patients with acute myocardial infarction; but as there is only a slightly reduced ability to kill bacteria, increased susceptibility to infections is unlikely although it cannot be excluded. PMID- 2997434 TI - Synaptic facilitation by 3-aminopyridine and its antagonism by verapamil and diltiazem. AB - The effects of 3-aminopyridine (3-AP) on synaptic transmission in bullfrog sympathetic ganglion were studied in normal Ringer's solution and during graded reductions in extracellular Ca++ by means of intra- and extracellular recording techniques. 3-AP caused a single orthodromic stimulus to generate a brief burst of repetitive postganglionic discharges (SBR). In the absence of 3-AP, synaptic transmission, measured as the amplitude of the postganglionic compound action potential, failed progressively as Ca++ was reduced from 1.8 to 0.47 mM. This Ca++ dependence curve of synaptic transmission was shifted to the left (lower Ca++) by 3-AP in dose-related fashion, with maximum shift (4- to 5-fold) at 1 mM 3-AP. The magnitude of the maximum shift produced by 3-AP was precisely the same as that produced by 3,4-diaminopyridine and 4-aminopyridine. Although 3-AP could prevent transmission failure at otherwise suboptimal Ca++ levels, its ability to generate SBR failed progressively as Ca++ was reduced from normal (1.8 mM) to 0.5 mM. Thus, there was a wide difference between the Ca++ dependence domains of synaptic transmission and of SBR in the presence of 3-AP. To confirm this difference in Ca++ dependence domains by a method other than reduction of [Ca++]0, we investigated the interactions between 3-AP and two Ca++ entry blockers, verapamil and diltiazem. 3-AP SBR was abolished by verapamil and by diltiazem at concentrations significantly below those required to block synaptic transmission in the presence of 3-AP. The results thus demonstrate a competitive interaction between aminopyridines and Ca++ entry blockers and further confirm the Ca++ dependent nature of the synaptic actions of aminopyridines. PMID- 2997435 TI - Protective effect of a selective leukotriene antagonist in endotoxemia in the rat. AB - The role of lipoxygenase metabolites as inflammatory mediators in endotoxic shock remains uncertain. In the present study, the effects of a selective leukotriene (LT) D4/LTE4 antagonist, LY171883, 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazol-5-YL) butoxy]-phenyl ethanone], on endotoxin-induced sequelae in the rat were assessed. LY171883 was given as a bolus (30 mg/kg i.v.) 10 min before Salmonella enteritidis endotoxin (40 mg/kg) in ketamine-anesthetized (200 mg/kg i.m.) rats, followed by an infusion (10 mg/kg/hr) starting at 30 min postendotoxin. Carotid artery blood pressures were determined at 10 min before and at intervals up to 240 minutes postendotoxin administration. Compared to shocked vehicle controls LY171883 attenuated (analysis of variance, P less than .02) the initial (0-30 min), but not the later, endotoxin-induced hypotension. LY171883 prevented completely (analysis of variance, P less than .001) the neutropenia (0-180 min), but not the thrombocytopenia induced by endotoxin. Hemoconcentration resulting from endotoxemia was reduced by LY171883 compared to the vehicle control group (P less than .02). These data demonstrate that this LTD4/LTE4 antagonist has significant salutary actions in endotoxemia and suggest that LTs may contribute to some endotoxin-induced sequelae. PMID- 2997436 TI - Relaxation of vascular smooth muscle by HA-1004, an inhibitor of cyclic nucleotide-dependent protein kinase. AB - A newly synthesized compound, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), was shown to be a potent inhibitor of two cyclic nucleotide-dependent protein kinases, cyclic GMP-dependent protein kinase and cyclic AMP-dependent protein kinase and the Ki values were 1.4 and 2.3 microM, respectively. HA-1004 relaxed rabbit aortic strips contracted by various agonists and with similar ED50 values. Phenotolamine, propranolol and atropine did not affect this HA-1004 induced relaxation, thereby suggesting that this compound does not act through these membrane receptor associated mechanisms. HA-1004 shifted the dose-response curve for CaCl2 to the right in a competitive manner in depolarized rabbit renal arterial strips. This compound also relaxed the A-23187 and phenylephrine-induced contractions elicited in Ca++-free solution. These findings suggest that HA-1004 exerts its action at the intracellular or submembranal level. This vasodilator has little effect on actomyosin adenosine triphosphatase and Ca++-calmodulin dependent myosin light chain kinase. Studies using its derivatives with various lengths of alkyl chain (C0-C6) indicated that the potencies of these compounds, as vasorelaxants, correlated well with their potential to inhibit cyclic nucleotide-dependent protein kinase. HA-1004 should be a useful tool for investigating in smooth muscle, regulatory mechanism(s) by second messengers, cyclic AMP and cyclic GMP. PMID- 2997437 TI - Effect of altered plasma protein binding on pharmacokinetics and pharmacodynamics of propranolol in rats after surgery: role of alpha-1-acid glycoprotein. AB - The pharmacokinetics and pharmacodynamics of propranolol in rats 2 days after laparotomy were compared to control animals. The apparent volumes of distribution and the systemic clearance of propranolol were decreased to about 20 to 40 and 70% of control values, respectively. The area under the blood concentration-time curve (AUC) of propranolol after p.o. administration showed a marked elevation after surgery and its availability increased about 2-fold at doses of 5.0 and 12.5 mg/kg. These changes were associated with a decreased plasma unbound fraction of propranolol after surgery. Immunological determination of alpha-1 acid glycoprotein (AGP) revealed a marked increase after laparotomy and a linear relationship was found between the plasma AGP concentration and the binding capacity of high-affinity binding site for propranolol in plasma (r = 0.961, P less than .001). AUC of p.o. administered propranolol was also correlated with plasma AGP concentration. The beta-blocking activity of propranolol assessed by the reduction in the isoproterenol-induced tachycardia was decreased in rats after laparotomy when it was evaluated in terms of the total plasma concentration of propranolol. In contrast, its activity evaluated by the unbound plasma concentration showed no difference between control and laparotomized rats, suggesting the dependence of the pharmacological activity of propranolol on its unbound level in plasma. Thus, laparotomy-induced changes in both pharmacokinetics and pharmacodynamics could be considered largely due to an increase in its binding to the increased plasma level of AGP. PMID- 2997438 TI - Dual effects of clonidine on micturition reflex in urethane anesthetized rats. AB - The effects of clonidine (CLO) on reflexly activated bladder motility have been examined in urethane-anesthetized rats and compared to its effects on field stimulation-induced contractions of isolated urinary bladder. Intravenous CLO suppressed micturition reflex transiently in a yohimbine-sensitive manner. The suppressive effect of i.v. CLO was greater after surgical sympathectomy (bilateral section of the hypogastric nerves). Intracisternal CLO was more effective than topical (on the bladder dome) or intracerebroventricular CLO in suppressing micturition. When tested in experimental conditions involving activation of both excitatory and inhibitory reflexes to the bladder CLO produced either inhibitory and/or excitatory effects on bladder motility in dependence of factors such as dose, route of administration and integrity of the sympathetic (inhibitory) innervation to the bladder. CLO suppressed bladder contractions produced by dimethylphenilpiperazinium, a ganglionic stimulant, and reduced those produced by postganglionic nerve stimulation. CLO inhibited, in a yohimbine sensitive manner, amplitude of field stimulation-induced contractions of isolated rat bladder, and its effectiveness was inversely related to the frequency of stimulation. Our findings are suggestive that the inhibitory action of CLO on micturition reflex is counteracted, in normal animals, by a negative feedback on sympathetic inhibitory influences carried out through the hypogastric nerves. PMID- 2997439 TI - The effects of bicarbonate and foreign anions on chloride transport in smooth muscle of the guinea-pig vas deferens. AB - The selectivity of the external site of the Cl transporting mechanism in the guinea-pig vas deferens has been investigated by measurement of 36Cl uptake and efflux and by direct measurement of intracellular pH. Replacing 50% of the Cl in normal Krebs solution inhibited the 15 min uptake of 36Cl in the order NO3 greater than Br greater than SCN greater than F greater than I greater than glucuronate, both in Cl-depleted tissues and tissues pre-incubated in the 50%-Cl solutions (steady-state uptake). After 3 h incubation in these solutions, the total cellular Cl was reduced by the anions in the order Br greater than NO3 greater than I greater than SCN greater than F greater than glucuronate. Br, NO3 and I reduced the cellular Cl to less than 50% of normal, suggesting that they are actively taken up by the cells. The ability of foreign anions to inhibit a 3 min uptake of high specific activity, low concentration Cl (6.5 mM) suggests an apparent affinity series of NO3 greater than Cl = SCN = Br greater than I greater than F at the external site. Addition of NO3, Cl, Br, HCO3, F, SCN or I to a Cl free, nominally HCO3-free bathing solution accelerated 36Cl efflux. The first four mentioned were powerful stimulants, the other three less potent. However, the exact position of HCO3 in the sequence is uncertain. The rapidity with which CO2 crosses the membrane and forms HCO3 intracellularly may allow competition between HCO3 and Cl at the internal site and so distort the result. The action of F is also questionable since this ion drastically reduces the divalent cation activity and is a metabolic inhibitor. Measurement of intracellular pH provided conclusive evidence that Cl, NO3, Br and I can exchange with HCO3 across the cell membrane. This exchange is as rapid with NO3 as with Cl but slower with Br and considerably slower with I. The results also indicate that SCN ions cross the cell membrane. It is concluded that Cl, HCO3, Br and NO3 are all translocated by the exchange carrier. I and perhaps SCN also interact with the transport mechanism, but the translocation rate is then greatly reduced. The precise order of the affinity of these anions remains uncertain but the following sequence: NO3 greater than Cl = SCN = Br greater than I greater than F is considered to be the most likely. PMID- 2997440 TI - Regional differences in responsiveness to octopamine within a locust skeletal muscle. AB - Regional differences in the physiological and biochemical responses to octopamine have been investigated in the extensor tibiae muscle of the hind leg of the locust. Octopamine increases the rate of relaxation of twitch tension generated by the slow motoneurone by different amounts in different regions of the muscle. It also increases the amplitude of twitch tension by the same amount in different regions of the muscle. The relaxation rate of fast twitch tension is increased by the same amount in all regions of the muscle innervated by the fast motoneurone. Octopamine also increases cyclic AMP levels by different amounts in different regions of both the extensor muscle and its antagonistic muscle, the flexor tibiae. In both muscles the maximal responses are obtained in the regions of the muscles containing the highest proportions of slow and intermediate muscle fibres as characterized by their contractile and ultrastructural properties. The results are discussed in terms of their functional significance and compared with examples of differential responses of different muscle fibre types in other invertebrate and vertebrate skeletal muscles. PMID- 2997442 TI - Compressive strength measurement and microstructure studies of hydroxyapatite cones. AB - Compressive strength measurement and microstructure studies of standard, diamond bur-ground, and fracture surfaces of durapatite cones were reported. The compressive strength measurements of durapatite cones were variable and strongly dependent on the test method. The fracture force ranged from 1090 to 3692 N and the calculated compressive strengths ranged from 113 (16,333 psi) to 389 mN/m2 (56,462 psi). The outer surface of durapatite cones was rough, irregular, and dense. Sharp fracture lines occurred along the longitudinal axis of the cones. Isolated porosity was seen in the fracture surface of one specimen. Grinding hydroxyapatite cones using a high-speed diamond bur with water spray produced a surface grinding feature, an irregular surface topography, and microcracks. The microcrack directions were perpendicular to the direction of the diamond bur cutting features. PMID- 2997441 TI - Selective cardiovascular and neuroendocrine effects of a kappa-opioid agonist in the nucleus tractus solitarii of rats. AB - The cardiovascular and neuroendocrine effects of a selective kappa-opiate receptor agonist (U50488H) microinjected into the nucleus tractus solitarii have been investigated in urethane-anaesthetized rats. Comparative experiments were conducted using 8-arginine vasopressin (AVP)-deficient Brattleboro rats and an opiate agonist selective for delta receptors. Unilateral injection of U50488H elicited a significant dose-dependent increase in mean arterial pressure and a small decrease in heart rate in Sprague-Dawley rats. The pressor effect was blocked preferentially by the relatively selective kappa-receptor antagonist MR2266BS compared to naloxone. Bilateral injections of U50488H elicited a relatively greater increase in mean arterial pressure than unilateral injections and a significant decrease in heart rate. U50488H did not elicit a pressor effect in Brattleboro rats, whereas a marked response (associated with a significant increase in AVP secretion) was found in parent strain Long-Evans rats. In contrast, no such differential effects in the response of Brattleboro and Long Evans rats were observed in parallel experiments using equimolar doses of the selective delta-opiate agonist Tyr-D-Ser-Gly-Phe-Leu-Thr which elicited a transient pressor response. An antagonist [1-(beta-mercapto-beta, beta cyclopentamethylene-propionic acid)2-(0-methyl) tyrosine] arginine vasopressin (1,d(CH2)5Tyr(ME)AVP) specific for the vasopressor action of AVP blocked the U50488H-induced pressor response in a dose-dependent manner when administered intravenously 10 min prior to the kappa agonist, but did not significantly attenuate the response to the delta agonist. Conversely, the U50488H-induced response was not modified by pre-treatment with phenoxybenzamine whereas the delta-agonist pressor response was completely blocked by it. The results provide evidence for specific kappa-opiate cardiovascular and neuroendocrine responses in the nucleus tractus solitarii and suggest that a kappa-receptor mechanism, possibly involving a peptide of the dynorphin group as the endogenous ligand, may operate in the central control of blood pressure and AVP secretion. PMID- 2997443 TI - Protective and sensitizing effects of D2O treatment on thermal responses of Chinese hamster cells. PMID- 2997444 TI - Hormonal steroids indirectly influence insulin binding in rat testis. AB - Pituitary LH is a key factor in controlling Leydig cell activity. In order to verify how steroids produced by the male gonads could influence testicular insulin binding, different steroids at varying doses were administered. Testosterone propionate (Tp) as well as estradiol-17 beta (E2-17 beta) and 5 alpha-dihydrotestosterone (DHT) led to a 50% decrease of insulin binding in testis while no effect was noted when HCG was administered concomitantly with DHT. LH receptors were parallely measured: DHT and E2-17 beta significantly depressed testis LH binding. It is concluded that steroids play a role in the regulation of testis membrane insulin receptor via their feedback mechanism on pituitary LH. PMID- 2997445 TI - Presynaptic regulation by ACh of the NE mediated responses in the rat vas deferens. AB - In the isolated electrically stimulated vas deferens preparation the effect of exogenous acetylcholine (ACh) was studied. It was possible to differentiate two separate sites for the action of ACh. A postsynaptic effect (M1) which is revealed as a sudden decrease in the basal tension of the muscular twitch, antagonized competitively by atropine (pA2 = 8.44 +/- 0.79), potentiated at a ratio of 10.24 by neostigmine, and not altered by hexamethonium, yohimbine, clonidine, theophylline or prazosin. Treatment with reserpine or 6-OH-DA induced a supersensitivity of this effect. The second action is a presynaptic effect (M2), which is manifested by a rapid increase in the muscular twitch, is dose dependent to ACh, and is potentiated significantly by reserpine, neostigmine and clonidine. This latter effect was not modified by atropine, hexamethonium, yohimbine, prazosin or theophylline. The normal efficacy of ACh is 185 times greater in presynaptic over postsynaptic receptors. The increase in the twitch is not considered to be by the release of endogenous ACh or ATP. The evidence presented here indicates that the presynaptic effect of ACh (M2) is due to participation of alpha 2-adrenoceptors in enhancement of the release of norepinephrine (NE) from the nerve terminals. PMID- 2997446 TI - Comparative study between histochemical and biochemical estimation of estrogen receptors in tumors. AB - Sucrose gradient analysis (SDGC) was compared with histofluoroassay, using 17 beta-Estradiol-6-carboxymethyloxime-bovine serum albumin-fluorescein isothiocyanate (E2-BSA-FITC) as fluorescent ligand, for the estimation of estrogen receptors (ER) in human breast tumors. No correlation was seen between fluorescent ligand binding capacity by the tumoral tissues on the one hand and ER levels estimated by SDGC on the other hand. The fluorescent ligand had a lower affinity for the receptor than estradiol itself and was contaminated with free estradiol. It was concluded that the absence of correlation between both techniques was for the greatest part due to unspecific binding of E2-BSA-FITC. PMID- 2997447 TI - Demonstration of receptors for insulin-like growth factor-II on human T lymphocytes. AB - Primary human T-lymphocytes that have been mitogen activated in chemically defined medium demonstrate cell surface receptor for insulin-like growth factor II (IGF-II). In contrast resting T-lymphocytes demonstrate little or no IGF-II receptor. Receptors appear within 24 hours of mitogen activation with maximal binding occurring at 72 hours. After this point IGF-II binding declines. Receptor binding of IGF-II to T-lymphocytes does not show a sharp pH dependence but is maximal above pH 7. Insulin does not compete for IGF-II binding sites and proinsulin competes only weakly, suggesting that this is a type 2 IGF receptor and not an insulin receptor. Furthermore, anti-insulin antibodies do not inhibit IGF-II from binding to activated T-lymphocytes indicating divergent binding domains on the two peptide hormones. IGF-II demonstrates stimulating action on T lymphocyte proliferation probably mediated by binding of IGF-II to this receptor. PMID- 2997448 TI - Some nonsteroidal antiinflammatory drugs inhibit the generation of superoxide anions by activated polymorphs by blocking ligand-receptor interactions. AB - Representatives of the major classes of nonsteroidal antiinflammatory drugs (NSAID) were assessed for their effects on superoxide anion (O2-.) production by human polymorphonuclear leukocytes stimulated with phorbol myristate acetate or N formyl methionyl-leucyl phenylalanine (fMLP). Three levels of effects were studied: (1) overall inhibition of O2-. production, (2) the inhibition of interaction between fMLP and specific receptors at the cell surface, and (3) intermediate proenzyme and enzyme effects. Some, but not all drugs inhibited O2-. production. In general, drugs that inhibited O2-. production inhibited fMLP receptor interactions in a consistent dose dependent fashion, showing noncompetitive kinetics. Drugs that failed to inhibit O2-. production showed weak and variable effects on receptor binding and on the intermediate enzymes. Clinical observations suggest that inflammation in diseases such as gout respond differently to NSAID than diseases such as rheumatoid arthritis; studies of drug effects may help to clarify the differences in pathogenesis of these inflammatory diseases. PMID- 2997449 TI - The 87A7 chromomere. Identification of novel chromatin structures flanking the heat shock locus that may define the boundaries of higher order domains. AB - The chromatin fiber of eukaryotic chromosomes is thought to be organized into a series of discrete domains or loops. To learn more about these large-scale structures, we have examined the sequence and chromatin organization of the DNA segments surrounding the two hsp 70 genes at the Drosophila melanogaster cytogenetic locus 87A7. These studies indicate that this heat shock locus is flanked on both the proximal and distal sides by novel chromatin structures, which we have called, respectively, scs and scs' (specialized chromatin structures). Each structure is defined by two sets of closely spaced nuclease hypersensitive sites arranged around a central nuclease-resistant segment. Our findings suggest that these two structures define the proximal and distal boundaries of the 87A7 chromomere and, hence, may be one of the first examples of anchor points for the organization of eukaryotic chromosomes into a series of discrete higher order domains. Moreover, these structures may provide focal points both for the decondensation of the chromomere when the hsp 70 genes are induced by heat shock and for the subsequent rewinding and condensation of the chromomere during recovery from heat shock. PMID- 2997450 TI - Substrate specificity of the DNA unwinding activity of the RecBC enzyme of Escherichia coli. AB - The RecBC enzyme of Escherichia coli promotes genetic recombination of phage or bacterial chromosomes. The purified enzyme travels through duplex DNA, unwinding and rewinding the DNA with the transient production of potentially recombinogenic single-stranded DNA. The studies reported here are aimed at understanding which chromosomal forms allow the entry of RecBC enzyme and hence may undergo RecBC enzyme-mediated recombination. Circular duplex molecules, whether covalently closed, nicked or containing single-stranded gaps of 10 to 774 nucleotides, are not detectably unwound by RecBC enzyme. Linear duplex molecules are readily unwound if they have a nearly flush-ended terminus whose 5' and 3' ends are offset by no more than about 25 nucleotides; molecules with longer single stranded tails are poorly bound by RecBC enzyme and are infrequently unwound. The single-strand endonuclease activity of RecBC enzyme can slowly cleave gapped circles to produce molecules presumably capable of being unwound. These results provide an enzymatic basis for the recombinogenicity of double-stranded DNA ends established from genetic studies of RecBC enzyme and Chi sites, recognition sites for RecBC enzyme-mediated DNA strand cleavage. PMID- 2997451 TI - Facile cruciform formation by an (A-T)34 sequence from a Xenopus globin gene. AB - We have studied the structure adopted by an (A-T)34 sequence from a Xenopus globin gene when present in a negatively supercoiled plasmid. A variety of enzyme and chemical probing experiments and electrophoretic migration shift methods reveal that the sequence adopts cruciform geometry at moderate levels of supercoiling. The structure has the lowest free energy of formation yet observed for a cruciform, and no detectable kinetic barrier preventing rapid interconversion between extruded and unextruded conformations. Analysis of band shift experiments reveals a twist change on cruciform formation of -5.8, slightly smaller than the -6.5 we would predict on the basis of a transition from B DNA. An attractive explanation consistent with this discrepancy is that the (A-T)34 stretch is locally underwound to about 11.7 base-pairs/helical turn at low levels of supercoiling. This calculation is made on the assumption that the cruciform junction is structurally similar to those examined previously, which is supported by the nuclease digestion results. This perturbed helical structure could be of considerable biological significance. PMID- 2997452 TI - Specificity of insertion of IS1. AB - A systematic study of the specificity of insertion of the transposable element IS1 into small defined-sequence plasmids (pBR322 and derivatives) was conducted to determine the features of the DNA sequence that influence target site selection. We have physically mapped several collections of independent insertions of IS1 into these plasmids and have determined: (1) that about 80% of all insertions occur in the DNA segment (about 200 base-pairs) between the unique EcoRI site of pBR322 and the beginning of the beta-lactamase gene, one of the two regions of high A + T density in this plasmid; (2) that there is a strong orientation effect in this region (almost all IS1 insertions are in one orientation) that depends on both the pBR322 sequence and the environment of the transposon in the donor molecule; and (3) that the orientation effect does not depend on the strong transcription that is directed through this region in pBR322. Furthermore, we have found that insertion of a poly(dA X dT) segment into pBR322 creates an artificial hotspot for IS1 insertion, even though it is not as attractive for insertion as the above-mentioned major hotspot. Our observations suggest that an interplay between several properties of the target sequences and the sequence environment of the donor transposon is responsible for the observed specificity of position and orientation. One of the possibilities discussed here is that preferred "entry-sites", or "signal" sequences, for the transposition complex play a major role in determining the positions and orientations of IS1 insertions. PMID- 2997453 TI - Properties of lac P2 in vivo and in vitro. An overlapping RNA polymerase binding site within the lactose promoter. AB - The Escherichia coli lac promoter has been shown to contain an RNA polymerase binding site (P2) that overlaps with, and is shifted 22 base-pairs upstream from the normal lac promoter (P1). In this paper, we provide RNA polymerase protection data obtained in vitro that show that, in the absence of CAP-cAMP, in vitro P2 is the preferred polymerase binding site on the P+ template. In the presence of CAP cAMP, polymerase binding to P2 is reduced and more polymerase is bound at P1. Two lac P1 "-35 region" mutations, L157 and 4, which increase the homology between this region and the consensus "-10 region" sequence, are both shown to have an increased affinity for polymerase binding at P2. CAP-cAMP is also able to decrease the amount of polymerase bound to P2 and to increase the amount bound to P1 on these mutant promoter fragments. P2 does not initiate transcription efficiently in vivo. Nuclease S1 mapping experiments detect only a low level of transcription from one of the P2 "up" mutations, but no beta-galactosidase synthesis is directed by this mutant. Mutations such as L157 and 4, which alter the P2-10 region, also alter lac P sensitivity to CAP-cAMP in vivo, suggesting that the P2 sequence plays a role in CAP-cAMP regulation of lac P. Possible roles for P2 in vivo are discussed. PMID- 2997454 TI - Bacterial DNA topoisomerase I can relax positively supercoiled DNA containing a single-stranded loop. AB - Using heteroduplex molecules formed from a pair of plasmids, one of which contains a small deletion relative to the other, it is shown that bacterial topoisomerase I can relax a positively supercoiled DNA if a short single-stranded loop is placed in the DNA. This result supports the postulate that the specificity of bacterial DNA topoisomerase I for negatively supercoiled DNA in its relaxation reaction derives from the requirement of a short single-stranded DNA segment in the active enzyme-substrate complex. Nucleolytic and chemical probing of complexes between bacterial DNA topoisomerase I and heteroduplex DNA molecules containing single-stranded loops ranging from 13 to 27 nucleotides in length suggests that the enzyme binds specifically to the region containing a single-stranded loop; the site of DNA cleavage by the topoisomerase appears to lie within the single-stranded loop, with the enzyme interacting with nucleotides on both sides of the point of cleavage. PMID- 2997455 TI - Excision-amplification of mitochondrial DNA during senescence in Podospora anserina. DNA sequence analysis of three unique "plasmids". AB - During senescence in the filamentous fungus Podospora anserina, specific regions of the mitochondrial genome, termed senDNA are excised, ligated and amplified. We have cloned in their entirety three such autonomously replicating plasmids, alpha, beta and epsilon senDNA. None of these plasmids displayed cross hybridization nor did we detect any significant DNA homology by computer analysis. The complete DNA sequence of the 2.5 kb alpha, the 5.5 kb epsilon and about 3.4 kb of the 9.8 kb beta senDNA is presented (kb = 10(3) base-pairs). These sequences were analyzed for the presence of consensus sequences common to introns, and it was found that alpha senDNA has the characteristics of a group II intron, epsilon senDNA contains three group I introns, and beta senDNA did not show relevant sequences in the 3.4 kb examined. Comparison of the 5' and 3' flanking sequences of alpha senDNA with oxi 3 (Co I) amino acid sequences from Neurospora crassa and Saccharomyces cerevisiae revealed significant homology and provided strong support that the excised alpha senDNA itself consists entirely of an intron. Upstream from the oxi 3 gene a transfer RNA cysteine sequence was detected. beta senDNA contained four tRNA sequences, aspartic acid, serine, valine and tryptophan, and sequences homologous to URFC (untranslated reading frame C) as well as two new URFs. epsilon senDNA contained sequences homologous to ATPase 8 and URFl; URFl was interrupted by three group I introns. The excision site sequences, as located by S1 nuclease mapping were unique for each senDNA. Analysis for repeated units showed that each plasmid contained elements which could be involved in secondary structure required for the alignment of distal ends preparatory to excision. These results are interpreted in terms of the structural requirements of mobile elements including the possible involvement of reverse transcriptase in the excision-ligation-amplification process. PMID- 2997456 TI - Nuclear magnetic resonance study of the proton exchange rate in the operator promoter DNA sequence of the trp operon of Escherichia coli. AB - The dynamic behavior of a palindromic oligonucleotide (C-G-T-A-C-T-A-G-T-T-A-A-C T-A-G-T-A-C-G) representative of the operator sequence and containing the Pribnow box of the trp operon of Escherichia coli was investigated. The resonances of the imino protons and of the H2 protons of the adenosine residues were all assigned. The opening rate constants of the base-pairs were calculated by monitoring the exchange rate of the observable imino protons (nine out of ten), using selective temperature (T1) measurements, which avoid the complication of cross-relaxation and spin diffusion. These measurements have to be performed in conditions where the exchange process is much faster than the opening and closing of the base pairs, so that the observed exchange rate is equal to the opening rate. It is shown that the catalysis of the exchange process by phosphate dianions is not very efficient (kB approximately equal to 7 X 10(4) M-1 S-1). Hence, in phosphate buffer, the necessary opening-rate limiting condition is met only at high pH values (approximately equal to 9.5) where efficient catalysis by OH- takes place, or at very high buffer concentration. While G X C base-pairs show very little exchange, acting in the sequence as molecular "staples", the A X T base-pairs that are protected from the fraying have very different opening and closing rates, depending on the sequence. Although it seems possible that the opening process could occur at the base-pair level, it is localized at most to two base pairs in that particular sequence. The activation energies for the opening process of all non-fraying base-pairs are very similar (19 +/- 1 kcal mol-1; 1 cal = 4.184 J), and the differences in the opening rates are essentially due to differences in the activation entropies. With regard to the role of this sequence in the promoter, it is observed that the end of the Pribnow box exchanges relatively easily, and that the activation parameters for the "breathing" process and for the isomerization step of the promoter--RNA polymerase are not very different. PMID- 2997458 TI - Oxygen toxicity and protective systems. PMID- 2997459 TI - Unusual neurological symptoms in a case of severe crotalid envenomation. AB - In Sweden bites by non-European venomous snakes are reported to the Poison Information Centre 5-10 times annually. These incidents generally take place in private homes and may result in severe poisoning. We report a recent case of envenomation from a bite by Crotalus durissus terrificus with a prolonged, atypical course. The patient, a 24-year old man, was admitted to hospital approximately eight hours after the snakebite. On admission we noted coma, circulatory failure, hypofibrinogenaemia with bleeding from fang marks on his right arm, melaena and haematemesis. Antishock therapy including intravenous fluids, steroids and epinephrine was instituted immediately and within six hours infusion of polyvalent antivenom was started. Next day, when the initial disturbances were corrected, peripheral neurological features were noted and the patient gradually became comatose. Antivenin therapy was reinstituted. The coma lasted for one week and recovery extended over several months with persisting neurological symptoms. Six months after the bite there were still pathological findings in the electromyogram. PMID- 2997457 TI - Histone H4 and H2B genes in rainbow trout (Salmo gairdnerii). AB - The complete nucleotide sequence of the 3.0-kb BamH I-Sst I restriction fragment contained within the rainbow trout genomic clone lambda TH2 has been determined. This region contains the rainbow trout H4 and H2B histone genes and 5' and 3' flanking and spacer sequences, and represents the 5' half of the histone-gene cluster; the remaining half has been characterized previously. The genes are uninterrupted, and are transcribed from the same strand. The protein sequence of H4, as determined from the nucleic acid sequence, is the same as that derived for other vertebrate H4 proteins, although comparison of nucleotide sequences shows a great deal of sequence divergence, especially in the third base position. The amino acid sequence of H2B, though largely homologous to those of other vertebrate H2B proteins, displays some characteristic differences in primary structure. Consensus sequences noted in many other eukaryotic genes, as well as histone-specific consensus sequences, have been identified. An unusual feature of the spacer region between the H4 and H2B genes is the presence of a duplicated sequence 87 bp in length. The 5' and 3' ends of each repeat are complementary, and each repeat contains smaller repeated sequences internally, as well as a possible cruciform structure. PMID- 2997460 TI - Crystallographical analysis of tooth enamel using milligram samples. AB - An X-ray diffraction microanalytical method, in which sample is loaded onto a silver membrane filter, was applied to assess the crystal content in tooth enamel. Each enamel powder was first examined at room temperature, and then examined again at intervals after heating to 200, 400, 600, 800, and 1000 degrees C. The hydroxyapatite composition weight and crystal weight of the samples were derived from the standard calibration curves. The "crystal content ratio" was defined as the ratio of crystal weight to sample weight. The following results were obtained: (1) beta-tricalcium phosphate (beta-TCP) replaced the hydroxyapatite after heating at the high temperatures; (2) the "crystal content ratio" in the tooth enamel increased with the rise in temperature; and (3) the lattice parameters of the enamel apatite and the beta-TCP were changed by the heating. The X-ray diffraction technique has the potential to analyze the crystal content using milligram samples. PMID- 2997461 TI - Cholangiocarcinoma in pregnancy: the contributions of ultrasound-guided interventional techniques. PMID- 2997462 TI - Central and peripheral serotonergic control of forestomach motility in sheep. AB - Participation of tryptaminergic receptors in the control of forestomach motility was investigated in conscious sheep using strain-gauges and chronically implanted electrodes. Two hours after feeding the sheep, serotonin (5-HT) was infused into the jugular vein (i.v.), or the carotid artery (i.c.), or into the lateral cerebral ventricles (i.c.v.), over a 10-min period. An i.v. dose of 16 micrograms/kg/min abolished the cyclic propagated contractions throughout the forestomach, increased ruminoreticular tone, and induced simultaneous contractions of all the parts of the rumen. A dose of 1.6 micrograms/kg/min i.c. or i.v. 5-HT inhibited phasic contractions. The effects of 5-HT were blocked completely by i.c.v. administration of methysergide (20 micrograms/kg) and imipramine (200 micrograms/kg), and blocked partially by naloxone (25 micrograms/kg), but unaffected by atropine (50 micrograms/kg). The inhibitory effects of i.v. 5-HT were antagonized by methysergide (200 micrograms/kg, i.v.) but unaffected by imipramine (2 mg/kg, i.v.) and atropine (250 micrograms/kg, i.v.). Only the i.v. administration of methysergide blocked the inhibition induced by i.c. infusion (1.6 micrograms/kg/min) of 5-HT. It is suggested that 5 HT exhibits an inhibitory control on forestomach phasic contractions through hypothalamic and bulbar 5-HT receptors, and exerts peripheral excitatory effects on the tone of the rumen wall. PMID- 2997464 TI - Mapping of herpesvirus saimiri proteins on the viral genome: proteins dependent and not dependent on viral DNA synthesis. AB - Hybrid selected translation was used to map the genome of herpesvirus saimiri, a lymphotropic and oncogenic herpesvirus. RNA extracted from virus-infected cells was hybridized to cloned genomic fragments, and the hybrid selected mRNAs were translated in vitro in a rabbit reticulocyte lysate. Forty-five virus-induced polypeptides were identified and correlated to their coding regions on the herpesvirus saimiri genome. Inhibition of the replication of viral DNA with phosphonoacetic acid showed that 22 of these polypeptides belong to the early group of herpesvirus saimiri gene products. PMID- 2997463 TI - Transcription of vaccinia virus early genes by a template-dependent soluble extract of purified virions. AB - An extract capable of selectively transcribing early vaccinia virus genes was prepared by disrupting purified vaccinia virions and passing the soluble material through a DEAE-cellulose column to remove endogenous DNA. Runoff transcripts of predicted size were synthesized by using double-stranded DNA templates that contained truncated early vaccinia virus genes, whereas several late vaccinia virus genes were not transcribed under these conditions. Proper dilution of the enzyme extract was critical, and a threshold concentration of DNA was required. At 30 degrees C, runoff transcripts were detected after 5 min and synthesis slowed appreciably after 30 min. Mg2+ was the preferred divalent cation, and KCl concentrations above 20 mM were inhibitory. Correct initiation of transcription was demonstrated by high-resolution analysis of S1 nuclease-digested hybrids formed by annealing in vitro-synthesized RNA with 5'-end-labeled DNA. A requirement for a 31-base-pair transcriptional regulatory sequence was found by using templates with deletions in an early promoter region. This in vitro system may be useful for mapping early transcriptional initiation sites, measuring the effects of additional promoter mutations, and isolating transcription factors. PMID- 2997465 TI - Effect of cloned human interferons on protein synthesis and morphogenesis of herpes simplex virus. AB - Pretreatment of human fibroblast cells with 100 U of either cloned human alpha-2 or beta interferon per ml for 24 h reduced the release of infectious herpes simplex virus type 1 by more than 99%. This inhibition in infectivity correlated well with the total number of extracellular virus particles released from treated cells as determined by DNA dot blot hybridization analysis. Electron microscopic observations of interferon-treated human fibroblast cells clearly demonstrated typical assembly of nucleocapsids inside the nucleus, even though very few mature extracellular particles were seen. Analysis of virus-specific proteins by the immunoblot technique showed that neither species of interferon had a significant inhibitory effect on the synthesis of major nucleocapsid proteins. However, the synthesis of specific glycoproteins (D and B) was drastically reduced or delayed in beta-interferon-treated cells. The results presented in this communication suggest that cloned human interferons block herpes simplex virus morphogenesis at a late stage and inhibit the release of particles from the treated cells. PMID- 2997466 TI - In vivo and in vitro models of demyelinating disease: JHM virus in the rat central nervous system localized by in situ cDNA hybridization and immunofluorescent microscopy. AB - In situ probing of central nervous system (CNS) tissues has made it possible to associate the presence of JHM virus (JHMV) RNA with individual cells in the rat CNS. The presence of viral RNA was not always associated with antigen expression. The in situ hybridization revealed that cerebellar Purkinje cells and hippocampal neurons were highly susceptible to JHMV infection during either acute or paralytic disease. In the paralytic disease, Purkinje cell neurons frequently contained viral RNA. This observation suggests that these neurons, and perhaps others, may be repositories for JHMV in rats that undergo prolonged infections. PMID- 2997467 TI - Recombination between nonsegmented RNA genomes of murine coronaviruses. AB - We have isolated a recombinant virus between the A59 and JHM strains of mouse hepatitis virus, which contain a single species of nonsegmented RNA genome. This recombinant was derived by mixed infection of DBT cells with temperature sensitive mutants of A59 and JHM at nonpermissive temperature. Viruses recovered at this temperature were screened by oligonucleotide fingerprinting of their genomic RNAs. One recombinant virus, B1, was found to contain mostly A59-derived sequences, but the 3 kilobases at the 5' end of the genomic RNA was derived from JHM. Thus, the crossover point in the B1 genome is located within gene A, which codes for the viral RNA polymerases. The study of the intracellular RNA species of B1 virus revealed that probably all of the virus-specific subgenomic mRNA species contained the body sequences of strain A59 but the leader sequences of JHM. This result indicates that the JHM leader RNA, which differs from the A59 leader RNA, could be fused to the mRNAs of a different virus strain during RNA transcription. Furthermore, B1 virus-infected cells contain an additional subgenomic mRNA species which is transcribed from a new initiation site within gene C, suggesting that the leader RNA could determine the site of initiation for coronavirus mRNAs. These data represent a first report of RNA recombination between viruses, other than picornaviruses, which contain nonsegmented RNA genomes. PMID- 2997469 TI - Analysis of the genomic termini of tupaia herpesvirus DNA by restriction mapping and nucleotide sequencing. AB - A recombinant plasmid harboring both genomic termini of tupaia herpesvirus (THV) DNA was characterized by restriction enzyme analysis and by determination of the nucleotide sequence. A unique NotI cleavage site was found that is located approximately 19 base pairs upstream of the THV terminal junction. THV DNA fragments from virion DNA were analyzed by using the same restriction enzymes as for the recombinant plasmid. The comparative fine mapping of virion THV DNA revealed heterogeneous molecules of variable lengths with the NotI cleavage site conserved. A number of short direct and inverted repeats and palindromes were found surrounding the THV terminal joint. The THV repetitive sequences were compared with the repeats reported for the DNA termini of herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus and are discussed in respect to signals for a site-specific endonuclease required for packaging. PMID- 2997468 TI - Structure of simian virus 40-adeno-associated virus recombinant genomes. AB - The structures of recombinant genomes formed by recombination between simian virus 40 (SV40) and adeno-associated virus 2 (AAV) DNAs after either DNA cotransfection or coinfection by virions were characterized. Two types of structures were found. Group A structures, found after cotransfection and in one of seven recombinants arising from coinfection, represented a simple deletion of SV40 sequences replaced by a slightly shorter AAV sequence. Group B structures were found in six of seven recombinants arising after virion coinfection. All contained either the left or right terminal sequences (approximately 250 to 450 bases) of the AAV genome adjacent to the SV40 origin of DNA replication. Only 350 to 650 bases (including the origin) remained of the SV40 sequence. The joined SV40-AAV sequences were present in the recombinant genome as a tandem repeat of a size that can be packaged into SV40 capsids. PMID- 2997470 TI - Humoral immune response to herpes simplex virus type 2 glycoproteins in patients receiving a glycoprotein subunit vaccine. AB - Serial serum specimens from 22 herpes simplex virus (HSV)-seronegative recipients of an HSV type 2 (HSV-2) glycoprotein subunit vaccine were analyzed by radioimmunoprecipitation and polyacrylamide gel electrophoresis for the development of antibodies to HSV-2 gB, gD, and g80, a complex of gC and gE. Volunteers received 50 (n = 12) or 100 micrograms (n = 10) of vaccine at days 0, 28, and 140; sera were drawn weekly for 8 weeks and again at days 140, 147, and 365. Among seronegative volunteers, antibody to gB was detected 2 weeks after the first dose, while antibodies to g80 and gD were detected after the second dose (day 35). Antibodies to nonglycosylated HSV-specific proteins were not detected. A dose-response effect between recipients of 50- and 100-micrograms doses was observed in the proportion of vaccine recipients seroconverting to g80 and in the proportion of recipients retaining antibodies to both gD and g80 over time. Diminishing complement-independent neutralizing antibody titers occurred after the second dose and were associated with loss or reduction of detectable antibody to gD. Volunteers who were seropositive for HSV-1-specific antibody (n = 11) were also enrolled in the trial and received 50-micrograms doses of vaccine. Vaccination resulted in conversion to HSV-2 complement-independent neutralizing antibody specificity or indeterminant specificity in 10 of 11 volunteers. These shifts were accompanied by changes in the radioimmunoprecipitation and polyacrylamide gel electrophoresis profile. These changes, which were apparent by 14 days after the first vaccine dose, included de novo appearance or increased levels of antibody to g80 and increased levels of antibody to gD and gB. These studies document the immunogenicity of solubilized glycoproteins gB, gD, gC, and, possibly, gE in humans. PMID- 2997471 TI - Specificity of Fc receptors induced by herpes simplex virus type 1: comparison of immunoglobulin G from different animal species. AB - Cells infected with herpes simplex virus type 1 (HSV-1) express a cell surface receptor able to bind the Fc portion of immunoglobulin G (IgG). Of the four human IgG subclasses, the HSV-1 Fc receptor, like staphylococcal protein A, binds to all except IgG3. In this paper, we describe the binding of a number of animal IgG and IgG subclass molecules to HSV-1-infected cells and compare this binding to that of protein A. Although only few representatives from each animal order were tested, we found that IgG from Carnivora and Rodentia did not bind or bound only slightly to the HSV-1 receptor, whereas IgG from Primates, Lagomorpha, and Artiodactyla bound well. This pattern was clearly different from the species spectrum of IgG binding of protein A. Differences between the two receptors were also found when animal IgG subclasses were tested. The pronounced differences in affinity for the HSV-1 Fc receptor between immunoglobulins from, for example, mouse and rabbit may influence the interpretation of animal studies with this virus. PMID- 2997473 TI - Functional implications of oligomerization of simian virus 40 large T antigen during lytic virus infection. AB - The formation of oligomers of simian virus 40 (SV40) large T antigen in SV40 infected and -transformed monkey cells was analyzed by sucrose density gradient centrifugation. The overall distribution of total T antigen during lytic infection showed mainly low-molecular-weight forms (monomers and dimers) in the early phase (10 h postinfection) and an increase in the number of oligomers in the late phase of the lytic cycle (36 h postinfection), indicating an accumulation of these final products. In contrast, studying the conversion of newly synthesized T antigen into oligomers by appropriate pulse-chase radiolabeling of infected cells revealed that this processing decelerates considerably during the late phase of infection. This mechanism can be reaccelerated by blocking DNA replication with aphidicolin. Since none of these results could be obtained by using synchronized SV40-transformed monkey cells (COS-1), these observations are compatible with the idea that the process of T antigen oligomerization may be involved in viral, but not in cellular, DNA synthesis. PMID- 2997472 TI - Utilization of internal AUG codons for initiation of protein synthesis directed by mRNAs from normal and mutant genes encoding herpes simplex virus-specified thymidine kinase. AB - Previous studies (H.S. Marsden, L. Haarr, and C.M. Preston, J. Virol. 46:434-445, 1983) have shown that at least three polypeptides, with molecular weights of 43,000, 39,000, and 38,000, are encoded by the herpes simplex virus type 1 (HSV 1) thymidine kinase (TK) gene. It has been suggested that the 39,000- and 38,000 molecular-weight polypeptides arise from preinitiation complexes bypassing the first and second AUG codons before commencement of translation since, according to previous work (M. Kozak, Nucleic Acids Res. 9:5233-5252, 1981), these codons are not of the most efficient structure for initiation. This possibility was investigated by using specific herpes simplex virus mutants with alterations in the TK gene. Mutant TK4 has an amber mutation between the first and second AUG codons, whereas mutant delta 1 has a deletion which removes the first AUG codon but leaves other AUG codons, as well as transcriptional promoter sequences, intact. Both mutants synthesized only the 39,000- and 38,000-molecular-weight polypeptides, and the amounts produced were normal in TK4-infected cells but increased in delta 1-infected cells. Furthermore, the levels of TK produced after infection with the mutant viruses correlated with the amounts of the 39,000- and 38,000-molecular-weight polypeptides synthesized. The 43,000-, 39,000-, and 38,000-molecular-weight polypeptides were shown to be related by their positive reaction with anti-TK serum in both immunoprecipitation and immunoblotting experiments. The production of the 39,000- and 38,000-molecular-weight polypeptides through bypassing of the first AUG codon was examined by hybrid arrest experiments with a DNA fragment complementary to only 50 bases at the 5' terminus of TK mRNA. This fragment arrested the synthesis of the 30,000- and 38,000-molecular-weight polypeptides when annealed to mRNA from wild-type HSV-1- or TK4-infected cells, showing that those polypeptides arise from an mRNA initiated upstream from the first AUG codon. mRNA from cells infected with mutant delta 1, which lacks DNA sequences upstream from the first AUG, was not affected by the 50-base-pair fragment. The data therefore confirm that three polypeptides encoded by the HSV-1 TK gene arise by differential use of in-phase AUG codons for the initiation of protein synthesis. This mechanism for the production of related but distinct polypeptides has not previously been demonstrated in a eucaryotic system, and the implications for the regulation of TK enzyme activities are discussed. PMID- 2997474 TI - Evidence for transmembrane orientation of acylated simian virus 40 large T antigen. AB - In mKSA cells (a simian virus 40-transformed BALB/c mouse tumor cell line), plasma membrane-associated large T antigen (large T) is found in two subfractions of the plasma membrane; a minor amount of large T is recovered from the Nonidet P 40 (NP-40)-soluble plasma membrane fraction, whereas the majority is tightly bound to a substructure of the plasma membrane, the plasma membrane lamina (PML). Only PML-associated large T is fatty acid acylated (U. Klockmann and W. Deppert, EMBO J. 2:1151-1157, 1983). We have analyzed whether these two forms of plasma membrane-associated large T might differ in features like cell surface expression or metabolic stability. In addition, we have asked whether one of the two large Ts might represent the hypothetic, large T-related protein T* (D. F. Mark and P. Berg, Cold Spring Harbor Symp. Quant. Biol. 44:55-62, 1979). We show that in mKSA cells grown in suspension culture, large T associated with the PML is also exposed on the cell surface. This form of large T, therefore, exhibits properties of a transmembrane protein. Large T in the NP-40-soluble plasma membrane fraction could not be labeled with radioiodine on the cell surface and, for this reason, does not seem to be oriented towards the cell surface. In contrast, when mKSA cells were grown on substratum (culture dish), we found that in these cells both NP-40-soluble large T as well as large T anchored in the PML could be cell surface iodinated. We also have analyzed the plasma membrane association of surface T antigen in mKSA cells grown in a mouse as ascites tumor. In tumor cells, only PML-bound large T is cell surface associated. We conclude that differences in extractibility of cell surface-associated large T most likely depend on cell shape and are not an artifact of cell culture. Both NP-40-soluble and PML-bound large Ts are associated with the plasma membrane in a metabolically stable fashion. Neither of the two large Ts represents T*. PMID- 2997475 TI - Poliovirus metabolism and the cytoskeletal framework: detergent extraction and resinless section electron microscopy. AB - The association of poliovirus metabolism with the cytoskeleton was investigated. Infected cells were extracted by using the nonionic detergent Triton X-100 in the physiological cytoskeleton buffer. The skeletal framework obtained was examined by transmission electron microscopy of resinless sections. The fibers of the framework were grossly distorted in infected cells. No virions or procapsids were seen but many virus-specific spheroidal bodies were associated with the framework. They had a diameter of 40 to 70 nm, were characterized by a dense core and a translucent periphery, and occurred in strings, often near the remnants of flattened vesicles. These spheres may correspond to virus-synthesizing bodies. The metabolism of poliovirus RNA was shown to be associated with the skeletal framework by pulse-labeling cells with [3H]uridine and measuring the RNA retained on the framework. 20S double-stranded RNA, a form of poliovirus RNA found only in the replication complex, was attached to the skeleton throughout a 60-min pulse label. 35S single-stranded viral RNA, a form found in virions, in polyribosomes, and in the replication complex, appeared first on the framework but after a few minutes was also found in the soluble cytoplasmic phase, encapsidated in virions. In contrast to viral RNA, viral proteins exhibited a varied association with the skeletal framework. Viral proteins were pulse-labeled with [35S]methionine and chased with unlabeled methionine. Although all of the virus-specific proteins were found, to some extent, in the skeletal fraction, the derivatives of P2 (P2-X and P2-5) and a derivative of P3 (P3-2) showed a preferential association with the skeletal framework. Virions and procapsids, on the other hand, were not associated with the cytoskeleton; both they and their component proteins (P1-VP0, P1-VP1, P1-VP2, and P1-VP3) were found dominantly in the soluble cytoplasmic phase. The pathway of poliovirus assembly can be inferred from the above data. It is different from that found previously for the enveloped vesicular stomatitis virus and may be representative of encapsidated cytoplasmic virus assembly. PMID- 2997476 TI - Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4. AB - Using Vero cells transformed with the wild-type gene for ICP4 as the permissive host cell, we isolated herpes simplex virus type 1 (HSV-1) mutants containing deletions in both copies of the ICP4 gene. The mutants, d120 and d202, contained deletions of 4.1 and 0.5 kilobases, respectively, in each copy of ICP4. ICP4 mRNA synthesized in d202-infected Vero cells was 0.5 kilobases smaller than that synthesized in cells infected with the wild-type virus. No ICP4 mRNA was detected in d120-infected Vero cells. d120 and d202 specified polypeptides that reacted with ICP4 antiserum and were smaller than the wild-type ICP4 polypeptide by 130 and 30 kilodaltons, respectively. The only other HSV-1 gene products detectable on infection of Vero cells with d120 and d202 were ICP6 (of the early kinetic class of HSV-1 polypeptides), ICP0 (immediate early), ICP22 (immediate early), and ICP27 (immediate early). Immediate-early polypeptides ICP0 and ICP27 were expressed to a higher level in Vero cells infected with an ICP4 temperature sensitive (ts) mutant (tsB32) at 39 degrees C, indicating immediate-early stimulatory activity associated with the ts ICP4 polypeptide. In addition, the patterns of complementation of d120, d202, and tsB32 in ICP4-transformed cells also demonstrated inhibitory activity associated with the ts form of the ICP4 polypeptide. PMID- 2997477 TI - Isolation of a new serotype of simian acquired immune deficiency syndrome type D retrovirus from Celebes black macaques (Macaca nigra) with immune deficiency and retroperitoneal fibromatosis. AB - A new serotype of simian acquired immune deficiency syndrome (SAIDS) retrovirus (type 2) belonging to the D genus of retroviruses is associated with a SAIDS occurring spontaneously in a colony of Celebes macaques (Macaca nigra) and rhesus macaques (Macaca mulatta) at the Oregon Regional Primate Research Center. This syndrome resembles SAIDS in M. mulatta at the California Primate Research Center, which is associated with a similar type D retrovirus (type 1). However, at the Oregon Center, SAIDS is distinguished by the occurrence of retroperitoneal fibromatosis in some of the affected monkeys. Type 2 virus was isolated from seven of seven macaques with SAIDS, retroperitoneal fibromatosis, or both and from one of six healthy macaques. The new strain is closely related to SAIDS retrovirus type 1 and Mason-Pfizer monkey virus but can be distinguished by competitive radioimmunoassay for minor core (p10) antigen and by genomic restriction endonuclease cleavage patterns. Neutralization tests indicate that type 1 and type 2 SAIDS retroviruses are distinct serotypes. Therefore, separate vaccines may be necessary to control these infections in colonies of captive macaques. PMID- 2997478 TI - Isolation and molecular cloning of a fast-growing strain of human hepatitis A virus from its double-stranded replicative form. AB - A fast-growing strain of human hepatitis A virus was selected and characterized. The virus has the unusual property of developing a strong cytopathic effect in tissue culture in 7 to 10 days. Sequences of the viral genome were cloned into recombinant plasmids with the double-stranded replicative form as a template for the reverse transcription of cDNA. Restriction analysis and direct sequencing indicate that this strain is different from that described by Ticehurst et al. (Proc. Natl. Acad. Sci. USA 80:5885-5889, 1983) in the region that presumptively codes for the major capsid protein VP1, but both isolates have conserved large areas of homology in the untranslated 5'-terminal sequences of the genome. PMID- 2997479 TI - Transcription mapping of the varicella-zoster virus genome. AB - RNA was isolated from varicella-zoster virus-infected Flow 5000 cells (diploid fibroblasts) at late times after infection. With the use of overlapping DNA probes representing all regions of the varicella-zoster genome, an extensive Northern blot analysis of the RNA was carried out. The analysis revealed at least 58 discrete transcripts ranging in size from approximately 0.8 to 6.5 kilobases. RNAs were found to be homologous to all probes used except for those mapping at approximately map unit 0.3, where no RNA transcripts could be detected. Comparison of the sizes and locations of RNA transcripts mapping in the right hand ends of the varicella-zoster virus and the herpes simplex virus DNAs shows a number of striking analogies, suggesting their similar genomic organization. PMID- 2997481 TI - Foot-and-mouth disease virus-induced RNA polymerase is associated with Golgi apparatus. AB - Electrophoretic analysis of the Golgi apparatus isolated by differential centrifugation from radiolabeled cells infected with foot-and-mouth disease virus showed about 10 protein bands. The virus-induced RNA polymerase was identified by immunoprecipitation and electron microscope staining procedures. Pulse-chase experiments indicated that the polymerase passed through the Golgi apparatus in less than 1 h. PMID- 2997480 TI - Posttranslational processing of p21 ras proteins involves palmitylation of the C terminal tetrapeptide containing cysteine-186. AB - The p21 proteins of ras oncogenes are synthesized as precursors in the cytosol. After processing, which involves acylation, the products are associated with the plasma membrane in eucaryotic cells. The p21 overproduced in Escherichia coli, however, is not processed by acylation. A synthetic tetrapeptide of the p21 C terminus is used to identify the acylation site in eucaryotic p21 as cysteine 186. The same peptide of bacterial p21 is not acylated. Although p21 of Harvey murine sarcoma virus-transformed NRK cells can be metabolically labeled with either [3H]palmitate or [3H]myristate, the lipid moiety of the hydrophobic peptide is identified as palmitic acid. We suggest that the enzymatic mechanism for p21 palmitylation may be different from N-terminal myristylation of many other membrane proteins. PMID- 2997482 TI - Amplification of a bovine papillomavirus-simian virus 40 chimera. AB - A chimeric plasmid, pBOP, containing bovine papillomavirus (BPV) and the origin of replication from simian virus 40 (SV40) was constructed. The plasmid was established in mouse cells, where it was maintained stably as an autonomous BPV replicon. Lines carrying pBOP were fused to cells of COS-7, a simian line producing SV40 T antigen. Replication dependent on the SV40 origin and having the kinetics and approximate amplitude of an SV40 infection ensued. SV40 replication is therefore dominant over BPV replication, and the SV40 origin can conveniently be used to amplify lower-copy-number plasmids in mammalian cells. PMID- 2997483 TI - In vitro synthesis of an infectious RNA from cDNA clones of human rhinovirus type 14. AB - Development of a novel infectious cDNA assay is described for human rhinovirus type 14. A full-length cDNA clone of the human rhinovirus type 14 genome RNA was assembled and transcribed in vitro by using the SP6 transcription system. Transfection of HeLa cells with the nascent RNA resulted in the production of rhinovirus indistinguishable from the parental virus by both immunological and polyacrylamide gel analysis. PMID- 2997484 TI - Neurotropic Cas-BR-E murine leukemia virus harbors several determinants of leukemogenicity mapping in different regions of the genome. AB - The infectious virus derived from the molecularly cloned genome of the neurotropic ecotropic murine Cas-BR-E retrovirus was previously shown to have retained the ability to induce hind-limb paralysis and leukemia when inoculated into susceptible mice (P. Jolicoeur, N. Nicolaiew, L. DesGroseillers, and E. Rassart, J. Virol. 45:1159-1163, 1983). To map the viral sequences encoding the leukemogenic determinant(s) of this virus, we used chimeric viral genomes constructed in vitro between cloned viral DNAs from the leukemogenic Cas-BR-E murine leukemia virus (MuLV) and from the related nonleukemogenic amphotropic 4070-A MuLV. Infectious chimeric MuLVs, recovered from NIH 3T3 cells microinjected with these DNAs, were inoculated into newborn NIH Swiss, SIM.S, and SWR/J mice to test their leukemogenic potential. We found that each chimeric MuLV, harboring either the long terminal repeat, the gag-pol, or the pol-env region of the Cas-BR-E MuLV genome, was leukemogenic, indicating that this virus harbors several determinants of leukemogenicity mapping in different regions of its genome. This result suggests that the amphotropic 4070-A MuLV has multiple regions along its genome which prevent the expression of its leukemogenic phenotype, and it also shows that substitution of only one of these regions for Cas-BR-E MuLV sequences is sufficient to make it leukemogenic. PMID- 2997485 TI - Restriction endonuclease mapping of adenovirus 35, a type isolated from immunocompromised hosts. AB - The DNA of adenovirus 35 (Ad35), a type recently associated with infections in immunocompromised hosts, was mapped by the use of BamHI, SmaI, PstI, EcoRI, and HpaI restriction endonucleases. In addition to standard mapping procedures, we used the in vitro adenovirus DNA replication system with origins at both physical ends of the linear molecule to determine the terminal fragments. Deletions of single restriction endonuclease sites in a group of closely related adenovirus isolates from patients with acquired immunodeficiency syndrome helped determine the location of some DNA fragments on the genome. PMID- 2997486 TI - Group B coxsackieviruses readily establish persistent infections in human lymphoid cell lines. AB - Exposing human lymphoid cell lines to uncloned or recently cloned group B coxsackieviruses results in the frequent establishment of chronically infected cultures. Persistence is maintained by a carrier culture mechanism involving virus spread through the medium and replication among a minority of cells at any given time. These studies provide a model for persistence by highly cytocidal viruses. PMID- 2997487 TI - Simultaneous occurrence of Wilms tumor and renal calculus. AB - Nephroblastoma and renal stone disease are seen infrequently in children. We report on a 2-year-old girl with bilateral nephroblastoma and a simultaneous staghorn calculus in 1 kidney. PMID- 2997488 TI - Inhibitors of crystal growth of hydroxyapatite: a constant composition approach. AB - Pyrophosphate, citrate and magnesium, inhibitors of hydroxyapatite crystal growth, were studied using a seeded crystal growth system of constant composition at pH 5.80, 6.60 and 7.40. With this technique, crystal growth was studied at constant supersaturation at different pH values without the induction of other calcium phosphate phases. One inhibitor unit (that concentration of material that results in a reduction of 50 per cent in the growth rate from control) was calculated using the Langmuir adsorption isotherm. Pyrophosphate and citrate increased inhibitor activity with decreasing pH, whereas magnesium increased inhibitor activity with increasing pH. These data suggest that, at the urinary concentrations of these inhibitors, pyrophosphate is the most potent inhibitor, citrate less, and magnesium least. Pooled urine collections were studied using the same system and were found to have decreased inhibitor activity as pH decreased. This suggests that other modulators of hydroxyapatite, either promoters or inhibitors, are active in this system at the pH values studied. PMID- 2997489 TI - Renal accumulation of 99mTc-DMSA in the artificially perfused isolated rat kidney. AB - In order to investigate aspects of the renal handling of 99mTc-DMSA, 68 isolated rat kidneys were artificially perfused. The experimental groups were: Group 1 (no. = 32)-oxygenated filtering kidneys; Group 2 (no. = 29)-oxygenated non filtering kidneys; Group 3 (no. = 7)-anaerobic non-filtering kidneys. We conclude that the 99mTc-DMSA complex is strongly bound to albumin, is not filtered and is removed from perfusion fluid through the renal peritubular capillary route and that this occurs by an active process which depends upon aerobic metabolism. This process has a high capacity and is not inhibited by probenecid. PMID- 2997490 TI - Anti-AIDS agents show varying early results in vitro and in vivo. PMID- 2997491 TI - Seroepidemiological studies of HTLV-III antibody prevalence among selected groups of heterosexual Africans. AB - T-lymphocyte subsets and human T-cell lymphotropic virus type III antibody prevalence were studied in African patients with the acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC), and in female prostitutes. African blood donors and healthy Zairian and Rwandese persons matched for age, sex, and annual income served as controls. Seropositivity was noted in 46 (87%) of 53 patients with AIDS, 29 (88%) of 33 patients with ARC, 67 (80%) of 84 prostitutes, and five (12.5%) of 40 and eight (15.5%) of 51 healthy controls and blood donors, respectively. Patients with AIDS and ARC had a significantly lower OKT4/OKT8 ratio than healthy African controls. These studies suggest that human T-cell lymphotropic virus type III infection has already spread extensively into the general African population and that female prostitutes could be an important human reservoir of AIDS virus in the heterosexual population. PMID- 2997492 TI - AIDS-associated virus yields data to intensifying scientific study. PMID- 2997493 TI - Transfusion-associated acquired immunodeficiency syndrome in the United States. AB - By Aug 15, 1985, one hundred ninety-four cases of possible transfusion-associated acquired immunodeficiency syndrome (AIDS) had been reported to the Centers for Disease Control. Cases received their transfusions in 30 states. Infants account for 10% of the cases, suggesting an increased susceptibility to developing AIDS. Investigations one to six years after the transfusions have identified high-risk donors to 47 cases. Of 47 high-risk donors tested, 40 had a reactive serology for human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV III/LAV) antibody, including five with no risk for AIDS by history. The HTLV III/LAV was isolated from 23 of 26 seroreactive high-risk donors, most of whom remained asymptomatic. Blood components that transmitted HTLV-III/LAV included red cells, platelets, plasma, and whole blood. The time from transfusion to diagnosis of AIDS ranged from four to 84 months. The risk of developing AIDS after a blood transfusion has been low and will be lowered further by using both self-deferral and antibody screening. PMID- 2997494 TI - Leads from the MMWR. Summary: Recommendations for preventing transmission of infection with HTLV-III/LAV in the workplace. PMID- 2997495 TI - Night of the living dead: could the mummy strike again? PMID- 2997496 TI - [Changes in Y-protein and ligandin in human liver tumor tissues]. AB - Since the relationship between tissue ligandin and liver tumors has not been studied yet, we investigated the changes of Y protein and ligandin in human hepatoma and cholangioma by gel filtration, BSP-affinity chromatography, and SDS gel electrophoresis. The concentration of Y protein was markedly increased in both hepatoma and cholangioma, 2.8 and 4.8 times that of control, respectively. The content of ligandin was also increased in both conditions. SDS polyacrylamide gel electrophoresis of the increased ligandin confirmed the increment of 2.3 K dalton protein, which coincided with the MW of the ligandin subunit. Although the mechanism of the ligandin increase in hepatoma tissue is not clear, one possible reason might be due to the degree of differentiation of the tumor cells. In our case, the pathological examination revealed that the tumor cell was classified as Edmondoson Type II. PMID- 2997497 TI - [Sequential chemotherapy with low-dose methotrexate and 5-fluorouracil in advanced gastric cancer]. AB - Sequential chemotherapy with low-dose methotrexate (30 mg/body) and 5-FU (750 mg/m2) was undertaken in 16 patients with advanced gastric cancer. Following the chemotherapy, leucovorin (30 mg/body) was injected intravenously. These treatment were repeated weekly for two to eight weeks. Out of 16 patients, two partial remissions and two minor responses were obtained. The overall response rate was 12.5%. There were no cases of renal or hematologic toxicity. The only adverse effect was nausea. We concluded therefore, that sequential chemotherapy with low dose methotrexate and 5-FU is a useful method for advanced gastric cancer. PMID- 2997498 TI - [An autopsy case of triple cancer associated with superior vena cava syndrome]. AB - In recent years, because of advances in diagnostic techniques and therapy for malignant tumors, it is not rare to encounter cases of primary multiple malignant neoplasms. However, triple cancer is still rare. We report a case of triple cancer--gastric cancer, sigmoid colon cancer and malignant fibrous histiocytoma. To our regret, we could not make a diagnosis while the patient was alive. Careful examination of a cancer patient is important because malignant tumors may develop at any time. PMID- 2997499 TI - [Evaluation of the effect of transcatheter arterial embolization as preoperative therapy of hepatocellular carcinoma]. AB - From May 1982 till Nov. 1984, we performed 90 hepatic resection for primary or metastatic liver tumor. Preoperative transcatheter arterial embolization (TAE) was performed on 14 of them. All resected specimens of 14 cases were examined histologically. Effectiveness of TEA was estimated by the ratio of the necrotic area to the whole area of the tumor. Necrosis of the main tumor more than 90% was found in 10 of 14 cases, though necrosis of the daughter nodules was detected in only one of six cases. In hepatectomized patients, two-year-survival of the preoperative TAE cases had not been statistically improved compared with the non TAE cases. PMID- 2997500 TI - [Multidisciplinary treatment of lung cancer]. AB - In our hospital 94 patients with small cell lung cancer were admitted the past 10 years. 21 patient have undergone surgical resection following the chemotherapy with VEMT regimen. The 5 year survival rate of the patients after resection was 32.7%. The MST for 52 non-surgical patient received VEMT regimen was 8 M, but MST for the recent 15 patients received CAV or COMP regimens is prolonged by 17 M. On the treatment for non-small cell lung cancer, our recent study shows a significant improvement in response rates for patients on platinum-containing combination chemotherapy. CAP regimen produces a longer MST in patients with adenocarcinoma or large cell carcinoma compared with MFC regimen (13 M vs 9 M). PMID- 2997501 TI - [Chemo-endocrine therapy of diffuse carcinoma of the stomach and its clinical evaluation]. AB - Based on experimental study that some human gastric cancers do contain measurable estrogen receptor (ER) and the growth of diffuse carcinoma of the stomach on nude mice depended on sex hormone. Chemo-endocrine therapy after gastrectomy for female patients are starting since 1980. RESULTS: 1) The cumulative 2, 3 and 4 year survival rate in 8 patients with chemo-endocrine therapy (TAM+) revealed significant higher (100%, 80% and 53.4%) than those (59.2%, 23.7% and 0%) in 10 patients with chemotherapy alone (TAM-) after curative gastrectomy. Average disease free interval showed two times longer (32.0 m0) in TAM(+) than those (15.3 m0) in TAM(-). There was no significant on survival rate in both groups of noncurativelly resected patients. 2) Patients with higher ER contents (greater than or equal to 3.0 fmols) have a tendency with favorable prognosis and better response to chemo-endocrine therapy. PMID- 2997502 TI - [A case of congenital stomatocytosis with decreased red cell sodium content, increased red cell sodium efflux and pulmonary embolism]. PMID- 2997503 TI - [Superoxide productivity and bactericidal activity of neutrophils in patients with multiple myeloma]. PMID- 2997504 TI - [Memory--7. Serotonin, peptide hormones and memory]. PMID- 2997505 TI - [Sleep--2. Substances in the mechanism of sleep and wakefulness]. PMID- 2997506 TI - [Development of live varicella vaccine]. PMID- 2997507 TI - [AIDS virus]. PMID- 2997508 TI - Lung cancer in chromate workers--analysis of 11 cases. AB - We have experienced 11 cases of lung carcinoma in workers at a chromate factory during the past 14 years. All patients were males. The age of onset ranged from 41 to 68 years. Ten of the 11 were heavy smokers. The time of exposure to chromate was from 17 to 29 years and the average was 23.9 years. Seven patients had perforation of their nasal septa. The primary sites of the cancers were from the lobar to the subsegmental bronchi. There were nine squamous cell carcinomas and three small cell carcinomas. Four squamous cell carcinomas were hilar type early stage cancers and two of them were found in one patient at the same time. The chromium content of the lung tissue in the seven patients tested was from 13.9 to 2,368.4 micrograms/g of dry tissue and was higher than that of lung cancer or non-lung cancer cases without chromate exposure. There was no severe dysplasia of the bronchial epithelium in these 11 patients. PMID- 2997509 TI - Characteristics of gastric cancer invading to the proper muscle layer--with special reference to mortality and cause of death. AB - A total of 2,608 patients with gastric cancer received curative or palliative resection in the surgical department of the National Cancer Center Hospital during the 13-year period from 1968 to 1980. Of these, 261 patients with gastric cancer invading to the proper muscle layer (pm cancer) were studied. The incidence of pm cancer was 10.0% (261/2,608). Annual incidence showed a tendency of a little increase as compared with the pronounced increase in early cancer in recent years. Male patients comprised 67% of the total. Sixty- to 69-yr-old patients constituted 28.7%. This cancer occurred more frequently in the lower portion and the lesser curvature of the stomach. The type looking like early cancer comprised 38.3% (100/261). Histologically, undifferentiated types constituted 50% (131/261) and differentiated types comprised 44% (117/261). There were nine patients with distant metastasis despite the fact that the invasion was limited to the pm layer. Eighty-one (31%) of the 261 patients had died by November 1984. However, the survival period was longer than that for advanced cancer invading to the serosal layer. Concerning the cause of death, 48 (59.2%) of the 81 patients died of recurrent cancer and 27 (33.3%) died of nonmalignant disease. Death caused by hematogenous metastasis, especially liver metastasis, was the most characteristic. A close relationship was not found between the mode of invasion to the pm layer and mortality. PMID- 2997510 TI - Statistical analyses of clinico-pathological, virological and epidemiological data on lymphoid malignancies with special reference to adult T-cell leukemia/lymphoma: a report of the second nationwide study of Japan. The T- and B Cell Malignancy Study Group. AB - In the present nationwide survey, 1,040 new cases of lymphoid malignancy, in most of which expression of cell surface markers had been determined, and 243 controls for a case-control study on adult T-cell leukemia/lymphoma (ATL) and other types of lymphoid malignancies were enrolled during the two years (1982-84) from 24 institutions throughout Japan. Among the 1,040 cases, 197 cases of ATL, 183 cases of T-cell lymphoma and 236 cases of non T-cell lymphoma were used in the detailed analysis of the clinico-pathological and epidemiological findings. Furthermore, 66 cases of ATL and 174 cases of other lymphoid malignancies were used for case control analysis. In order to standardize the clinico-pathological classification throughout Japan, 20 hematologists and 12 pathologists met once a year and made the final consensus diagnosis in each case of lymphoid malignancy. Five hundred cases of non-Hodgkin's lymphoma were examined for antibody to ATL-associated antigen (ATLA). Clinico-pathological and epidemiological features for these cases were compared according to their anti-ATLA antibody status. The new results obtained from this survey are as follows: All of the patients with ATL in Kyushu had anti-ATLA antibody, but several patients with ATL in other districts had no anti-ATLA antibody, suggesting that there was no association with ATL virus (ATLV) infection in these cases. There was a difference in the histopathological patterns in non-Hodgkin's lymphoma between Kyushu and the other districts, which was due to the difference in distribution of the ATLV-associated lymphoid malignancies, namely ATL, in each area. The histopathological distribution in anti-ATLA negative cases in the Kyushu district was almost the same as that in the other districts. PMID- 2997511 TI - Phase II study of co-administration of uracil and tegafur (UFT) in hepatocellular carcinoma. Tokyo Liver Cancer Chemotherapy Study Group. AB - A Phase II study of co-administration of uracil and tegafur (UFT) was performed in 32 patients with unresectable hepatocellular carcinoma. A dose of 400 mg/m2/day of UFT was administered orally, three times a day, for more than 4 weeks. Of 26 patients evaluable for response, one (3.8%) showed a partial response of 9 months' duration. There were no complete responders. A dose limiting toxicity was gastrointestinal tract disturbance. Six patients (18.8%) had to discontinue UFT treatment because of gastrointestinal toxicity. The clinical advantage of tegafur in the treatment of hepatocellular carcinoma was not enhanced by co-administration of uracil. PMID- 2997512 TI - [Recent progress in thyroid function tests--TBII, free T3, thyroglobulin and calcitonin]. PMID- 2997513 TI - [Nephrogenous cyclic AMP, plasma vitamin D metabolites and the Ellsworth-Howard test using human PTH(1-34)]. PMID- 2997515 TI - [Ultrastructure of inclusion bodies in myrmecia]. PMID- 2997514 TI - [Present status of cancer marker research]. PMID- 2997516 TI - [Electron microscopic observations of epidermal cells separated by colloidal silica density gradient centrifugation]. PMID- 2997517 TI - [Tissue and cellular localization of carcinoembryonic antigen (CEA) in gastric cancer and correlation to serum CEA levels and prognosis]. PMID- 2997518 TI - [Prolyl hydroxylase activity and collagenase activity in the liver tissue of non alcoholic chronic liver diseases]. PMID- 2997519 TI - [A new estimation method of nerve conduction velocity distribution by spectrum analysis]. PMID- 2997520 TI - [Evaluation of infantile liver cirrhosis by the RI hepatogram using 99mTcO4-]. PMID- 2997521 TI - [Basic and clinical evaluation of TRAb measurement by using the TRAK kit]. PMID- 2997522 TI - [Fundamental and clinical evaluation on measurement of the TSH receptor antibody (TRAb) by the radioreceptor assay kit]. PMID- 2997523 TI - Thermosensitive neurons in the brain. AB - Since the discovery of thermosensitive neurons in the POAH, numerous papers have been published suggesting the primary importance of these neurons in thermoregulation. The basic properties of the neurons per se were well studied in cultured explants and slice preparations, outside of the influences of anesthesia and extrahypothalamic inputs. Attempts have been made to classify thermosensitive neurons according to firing rate and response pattern and to correlate each neuron group with thermoregulatory responses. In complex thermoregulatory networks, thermosensitive neurons are always under the influences of extrahypothalamic and non-thermal inputs. Most studies on POAH neurons have shown a variety of responses, some contrary to others; for instance, three-fourths of POAH warm-sensitive neurons may be facilitated by scrotal warming whereas the rest are inhibited or uninfluenced. These inconsistencies in responses among thermosensitive neurons, observed also in the effects of chemicals, may reflect different roles of POAH thermosensitive neurons in a variety of thermoregulatory responses. Unit recordings from conscious animals over long periods, together with observation on whole body responses, are expected to throw more light on the physiological significance of POAH thermosensitive neurons. PMID- 2997524 TI - Studies on the affinity and selectivity of denopamine (TA-064), a new cardiotonic agent, for beta-adrenergic receptors. AB - Denopamine is a new orally active cardiotonic agent. The present experiment was carried out to characterize the binding affinity and selectivity of this drug for beta-adrenergic receptor subtypes. Binding studies were performed using 3H dihydroalprenolol as the radioligand. Binding affinities of denopamine and some beta-agonists for rat heart membranes (KiH), which contain predominantly the beta 1-subtype, were in the order of isoproterenol (Iso, 14.1 nM) greater than prenalterol (158) greater than norepinephrine (Nor, 227) greater than or equal to epinephrine (Epi, 248) greater than denopamine (545) greater than or equal to dobutamine (645) greater than procaterol (1440) greater than terbutaline (6420). In rat lung membranes (predominantly beta 2-subtype), the order of potency (KiL) was Iso (20.6 nM) greater than procaterol (70.2) greater than Epi (136) greater than prenalterol (412) greater than dobutamine (735) greater than or equal to Nor (744) greater than denopamine (2205) greater than terbutaline (2500). The beta 1/beta 2-selectivity as judged from the KiL/KiH values was in the order of denopamine (4.1) greater than Nor (3.3) greater than prenalterol (2.6) greater than Iso (1.5) greater than dobutamine (1.1) greater than Epi (0.55) greater than terbutaline (0.39) greater than procaterol (0.05). Practolol, a beta 1 antagonist, showed a high beta 1-selectivity (KiL/KiH = 15.3). In the presence of guanine nucleotide (GTP), the denopamine radioligand competition curve showed a rightward shift, and its Hill coefficient increased like other agonists, although the degree of the shift was less than that observed with full agonists such as Iso. These results essentially correspond with the pharmacological and biochemical properties of denopamine and confirm the beta 1-selectivity and the agonist property of this compound. PMID- 2997525 TI - Autoradiographic studies on benzodiazepine receptor subtypes in the rat brain. AB - The technique of in vitro autoradiography which was developed by Kuhar and others was applied to the rat brain with use of 3H-flunitrazepam (flu) as a radioactive ligand. The cerebral cortex, hippocampus, substantia nigra and cerebellar cortex were rich in 3H-flu binding sites. To differentiate the benzodiazepine receptor (BZR) subtype, the authors used a type 1 specific ligand, either triazolopiridazine (CI 218872) or methyl-beta-carboline-carboxylate (beta-CCM), as an unlabeled displacer. The preparations were exposed on a 3H-sensitive film and then the film was developed. Computer-analysis of thus obtained autoradiographic pictures revealed that type 2 binding sites were distributed evenly within the rat brain, but with slight predominance in the hippocampus. After adding beta-CCM, no silver grains were noticed in the cerebellum and substantia nigra. These data meant that these two structures contained essentially type 1 BZR, while the hippocampus contained both type 1 and type 2 receptors. Autoradiographically, characteristic distribution of BZR represented by 3H-flu binding was considerably lost by adding a type 1 specific ligand, and this treatment caused the silver grains to be evenly distributed. These data suggest that the BZR which is directly associated with characteristic pharmacological actions such as anxiolytic and hypnotic effects is type 1, and type 2 binding sites have a less characteristic distribution pattern and might be pharmacologically less specific. PMID- 2997526 TI - The effects of morphine and morphine-related compounds on the rate of afferent discharges from the pulmonary receptors in bullfrog. AB - We examined the effects of morphine and morphine-related compounds on the rate of afferent discharges from the pulmonary receptors in bullfrog. Morphine (1 X 10( 5) - 1 X 10(-3) M) decreased the rate of spontaneous afferent discharges in a concentration-dependent manner, but hardly affected the rate of afferent discharges synchronized with lung inflation. Dihydrocodeine, naloxone and dextrorphan had the same effects as morphine. Levallophan (1 X 10(-4) M), pentazocine (1 X 10(-4) M) and pethidine (1 X 10(-3) M) clearly decreased both rates of the spontaneous afferent discharges and the afferent discharges synchronized with lung inflation. In the presence of naloxone or levallorphan at the concentration of 1 X 10(-5) M, morphine (1 X 10(-5) and 1 X 10(-4) M) caused an additive decrease in the rate of afferent discharges. Dextromethorphan and apomorphine at the concentration of 1 X 10(-4) M caused an increase in the rate of spontaneous afferent discharges followed by decrease in both rates of the spontaneous afferent discharges and the afferent discharges synchronized with lung inflation. When dextromethorphan or apomorphine was washed out and the rates of afferent discharges were almost restored to the levels before application of each drug, reapplication of these drugs caused no increase in the rate of spontaneous afferent discharges, but the drugs inhibited the generation of afferent discharges. All of these drugs did not affect the flow rate of perfusion solution from the pulmonary vein. PMID- 2997527 TI - Isoproterenol inhibition of potassium release from rat parotid gland. AB - The mechanism of isoproterenol-induced inhibition of potassium release from rat parotid slices has been determined. Spontaneous potassium release from the slices was significantly inhibited by isoproterenol at concentrations above 10(-6) M. This isoproterenol effect was completely abolished in the presence of propranolol (10(-5) M) and ouabain (10(-3) M) and was abolished during Na+-exclusion from the incubation medium. Isoproterenol caused an enhancement of the microsomal Na+, K+ ATPase activity at concentrations above 10(-5) M, and this activity was inhibited by propranolol (10(-5) M). The stimulatory effect of isoproterenol on the Na+, K+ ATPase exhibited a strong correlation with the inhibition of potassium release on each dose of isoproterenol. Moreover, dibutyryl cyclic AMP at concentrations above 10(-4) M inhibited potassium release in a dose-dependent manner and cyclic AMP caused an enhancement of the microsomal Na+, K+-ATPase activity. These results suggest that the inhibitory effect of isoproterenol on potassium release is clearly derived from the elevated Na+, K+-ATPase activity and that it may in part be mediated by cyclic AMP. PMID- 2997528 TI - Equipotency of anti-curare activity of 4-aminopyridine and 3,4-diaminopyridine in anesthetized rats. AB - In the curarized preparation, 3,4-diaminopyridine (3,4-DAP) and 4-aminopyridine (4-AP) were equiactive in their ability to antagonize d-tubocurarine caused complete depression of the indirectly elicited twitches of the sciatic nerve tibialis anterior muscle preparation in anesthetized rats. In the non-curarized preparation, 3,4-DAP showed 2.3 to 4.0 times stronger augmentation of the indirectly elicited twitches than 4-AP, but both the drugs increased equivalently and slightly the maximally elicited twitches of the chronically denervated muscle. The results suggest that the difference of their prejunctional effects is masked by the postjunctional effects of d-tubocurarine in the indirectly elicited twitches. PMID- 2997529 TI - Participation of the nucleus reticularis gigantocellularis in the morphine induced elevation of plasma corticosterone in rats. AB - The effects of morphine, adrenocorticotropic hormone (ACTH) and formalin on plasma corticosterone levels were investigated in the nucleus reticularis gigantocellularis (NRGC)-lesioned rats. ACTH (1.0 U/kg) or formalin (6.4%, 0.2 ml/rat) elevated plasma corticosterone in both sham-lesioned and NRGC-lesioned rats at the same degree, while morphine (10 mg/kg) also elevated plasma corticosterone in sham-lesioned rats, the elevation of which was significantly reduced by NRGC-lesioning. These findings suggest that the NRGC is involved in the corticosterone-increasing effect of morphine, but not involved in the effect of ACTH or formalin. PMID- 2997531 TI - [Interaction of various leukotrienes in guinea pig tracheal and lung parenchymal strips]. PMID- 2997530 TI - Intra-hepato-arterial infusions of cis-diamminedichloroplatinum (II) and 5 fluorouracil clinically effective for malignant liver tumors. AB - Four hepatocellular cancer patients and 11 metastatic liver cancer patients were treated with intra-hepato-arterial infusions of cis-diamminedichloroplatinum (II) plus 5-fluorouracil. Cis-diamminedichloroplatinum (II) (25-35 mg/m2) was given once a week, 5-fluorouracil (150-180 mg/m2) was infused daily. A partial response was obtained in 3 of 4 patients with primary cancer and in 5 of 9 evaluable metastatic cancer patients; the mean response durations were 27+ weeks in the former and 47+ weeks in the latter. However, severe bone marrow suppression occurred in 4 patients; 3 died of septicemia (2 cases) and massive intra-tracheal bleeding, respectively. This combined intra-hepato-arterial chemotherapy exerts a synergistic anticancer effect on malignant liver tumors, however, the related bone marrow suppression remains to be overcome. PMID- 2997532 TI - [A case of lung carcinoma associated with production of human chorionic gonadotropin]. PMID- 2997533 TI - Changes in cyclic AMP and cyclic GMP levels in the contraction-relaxation cycle of isolated smooth muscle cells from guinea-pig taenia caeci. AB - The present study was undertaken to investigate the role of cyclic AMP and cyclic GMP in the regulation of the contraction-relaxation cycle in isolated smooth muscle cells from guinea pig taenia caeci. The contraction of isolated smooth muscle cells induced by both caffeine (4 X 10(-3) M) and carbachol (10(-4) M) consisted of an initial activation phase and a following spontaneous relaxation phase. Isoproterenol (10(-4) M) inhibited the carbachol-induced contraction and raised intracellular cyclic AMP level of isolated smooth muscle cells. Both the inhibition of the carbachol-induced contraction and the rise in cyclic AMP level elicited by isoproterenol were blocked by propranolol (10(-4) M). Caffeine increased intracellular cyclic AMP level significantly and contracted isolated smooth muscle cells. Caffeine and carbachol contracted isolated smooth muscle cells and increased intracellular cyclic GMP level. Cyclic GMP was significantly increased in spontaneous relaxation phase. The data suggest that beta-adrenergic inhibitory effect on cholinergic excitation in the mammalian isolated smooth muscle cells is mediated by the change in intracellular cyclic AMP level and that cyclic GMP related to the relaxation in these single cells. Thus, it appears that caffeine-induced contraction might be due to the stimulation of Ca2+-induced Ca2+ release by cyclic AMP. PMID- 2997534 TI - Osteomas in OF-1 mice: no alteration in biologic behavior during long-term treatment with cyclosporine. AB - During the course of a long-term oral study in OF-1 mice for the assessment of any carcinogenic potential of the immunosuppressive agent cyclosporine (CS-A), a high incidence of osteomas was found in all treatment groups as well as in controls. The incidences of osteoma-bearing mice were 20% in control male and 30% in control female mice; the respective incidences in all treated mice were 14.7% for males and 38% for females. The osteomas were found to occur in multiple sites in 70% of control males, in 73.3% of control females, and from all treated groups in 54.5% of males and 64.9% of females. The pelvis and sacrum were most frequently involved (42%), followed by the hindlimbs (32%), the skull (14.5%), the vertebral column (6.5%), the forelimbs (4%), and the sternum and ribs (1%) in control animals. The distribution of osteomas was similar in treated mice and did not differ significantly from controls. Histologic and ultrastructural analysis confirmed benign osteomas with abundant C-type viral particles. Thus a previously unobserved spontaneous high incidence of osteomas was reported in OF-1 mice. Immunosuppressive treatment with CS-A did not change the incidence or the biologic behavior of these osteomas. PMID- 2997535 TI - Effect of a chemically defined diet in liquid form on colon carcinogenesis in rats. AB - The effects of ad libitum feeding of a chemically defined diet in liquid form on the incidence and histology of colon cancer induced by 10 weekly sc injections of 7.4 mg/kg of azoxymethane [(AOM) CAS: 25843-45-2] were investigated in W-rats. The chemically defined diet was adjusted once every 24 hours from 4 weeks before injection of the carcinogen to the end of the experiment at week 40. Oral administration of the defined diet resulted in significant increase in the incidence of colon cancer at week 40. Histologic examination showed that unlike adenocarcinomas with high mucin-producing activity, which were common in rats on pellet diet, most of the adenocarcinomas that developed in rats fed on defined diet were highly or well differentiated, with a typical glandular pattern. Administration of the chemically defined diet also resulted in marked colon mucosal hypoplasia and reduced gastrin levels in the serum at weeks 4 and 40. PMID- 2997536 TI - Attenuation of exogenous murine mammary tumor virus virulence in the C3H/HeJ mouse substrain bearing the Lps mutation. AB - A major change in mammary tumor incidence (MTI) and latency has occurred in the C3H/HeJ mouse substrain maintained at The Jackson Laboratory. The average time required for 50% of the C3H/HeJ mice to develop a mammary tumor changed from 40 weeks of age to the current 61 weeks of age. This 61-week median MTI in the C3H/HeJ substrain is significantly different from the less than 40-week median MTI seen in other C3H substrains infected with an exogenous milk-transmitted murine mammary tumor virus (MuMTV). The median MTI of over 80 weeks in MuMTV negative C3H substrains also is significantly longer than that seen in C3H/HeJ mice. Although the median MTI for the C3H/HeJ substrain has changed significantly, the continued presence of an exogenous MuMTV in C3H/HeJ mice was confirmed by the presence of high levels of gp52 antigen in the milk of lactating females. Presence of an exogenous MuMTV in C3H/HeJ female mice also was confirmed by their ability to pass their exogenous MuMTV(HeJ) via their milk to exogenous MuMTV-negative BALB/cByJ, C3H, or C3H hybrid mice. These foster-nursed mice exhibited the reduced tumor frequency and the increased median MTI seen in their C3H/HeJ foster mothers. The change in tumor incidence and latency in the C3H/HeJ substrain is not due to the loss of the exogenous MuMTV but to the occurrence of an attenuated MuMTV. Selection of this attenuated MuMTV may be related to the presence of the Lps mutation that occurred during the same time period that the MTI changed in the C3H/HeJ substrain. PMID- 2997537 TI - Isolation of antibodies specific for avian viral and cellular myc proteins. AB - The myc gene has been implicated in the genesis of various neoplasms in birds, mice, and humans and was originally identified as the cellular homologue of the transforming gene (v-myc) of the avian myelocytomatosis virus MC29. For specific antisera to be obtained for the myc gene product, a bacterial expression vector was constructed in which the coding sequences for approximately 20 kd of MC29 p110gag-myc (amino acid residues 502 to 678) were placed between the coding sequences for the amino terminal 13 kd of Rous sarcoma virus pp60src and the coding sequences for 112 kd of beta-galactosidase. Expression of this tripartite gene was driven by a hybrid trp-lac promoter under lac repressor control. Induction of expression resulted in the production of a 145-kd hybrid protein containing src, myc, and beta-galactosidase sequences. The hybrid protein was purified and injected into rabbits to produce antisera. The resultant antisera immunoprecipitated p110gag-myc and p58myc -p60myc from MC29- and MH2-infected nonproducer quail fibroblasts, respectively. In addition, the antisera also immunoprecipitated a 58-kd protein from the bursal lymphoma cell line BK25, which was identified as chicken c (cellular)-myc gene product. PMID- 2997538 TI - [Current views on endocrine neoplasms of the pancreas (review of the literature)]. PMID- 2997541 TI - Reexamination of the efficacy of vaccination against mousepox. AB - Experiments were conducted to evaluate the efficacy of three strains of vaccinia virus, IHD-T, Lister and Wyeth, to immunize the BALB/cByJ mouse against infection with ectromelia virus. Mice vaccinated with any of the strains were protected for at least 12 weeks against clinically apparent disease when challenged with cage mates infected with a virulent stain (NIH-79) of ectromelia virus. However, 4 to 8 weeks after vaccination mice were capable of transmitting virus to non vaccinated cage-mates. The results are discussed within the context of the current practices for preventing and controlling ectromelia epizootics. PMID- 2997539 TI - [Hereditary salt sensitivity as a cause of essential hypertension: studies of membrane transport and intracellular electrolytes]. AB - Based on oscillatory long-term blood pressure recordings and on biochemical findings in 62 normotensive and 54 untreated hypertensive subjects, who were investigated during their usual high sodium diet and after moderate salt restriction, we have developed a concept for the pathogenesis of essential hypertension, which differs from current concept proposed by others: We demonstrated that normotensive subjects with a positive family history of hypertension respond to sodium restriction from 200 to 50 mmol/day over 2 weeks with a minute fall of mean blood pressure of 2.9 +/- 0.7 mmHg (+/- SEM), whereas in subjects with a negative family history of hypertension blood pressure remained unchanged (-0.93 +/- 0.67 mmHg). This difference was only revealed by computing the "basal blood pressure average" from 240 heart beats, but not by conventional sphygmomanometric blood pressure measurements. Normotensives with heredity of hypertension or "salt sensitive" normotensive subjects were not different from subjects with a negative family history in the sodium pump, Na-K cotransport or intracellular sodium and potassium of erythrocytes. In contrast, the former group had an increased sensitivity to infused noradrenaline, which might be responsible for enhanced tubular sodium reabsorption in subjects with a positive family history of hypertension (or "salt sensitive" subjects). We only found an increased K-permeability of red cells in established hypertension, which was compensated for by a an increased activity of the sodium pump. These cell membrane defects were more pronounced in more severe hypertension. In the course of essential hypertension a cell membrane defect may develop as a consequence rather than a cause of the disease. PMID- 2997542 TI - Tumors and polycystic renal disease in two captive woodchucks (Marmota monax). AB - Hepatocellular carcinoma, polycystic renal disease and pneumonia are reported in an aged woodchuck, and a metastatic fibrosarcoma is reported in a relatively young animal born and raised in the laboratory. PMID- 2997543 TI - Cryptic adenosine triphosphatase activities in plasma membranes of CCl4-cirrhotic rats. Its modulation by changes in cholesterol/phospholipid ratios. AB - The activities of Na+,K+-, and Ca2+-ATPases were determined in plasma membranes obtained from livers of rats treated acutely and chronically with CCl4. Twenty four hours after a single oral dose of CCl4 the ATPases decreased below 50% of control values. The activity of Ca2+-ATPase returned to normal after 4 days, and Na+,K+-ATPase activity returned to normal values after 12 days. One week after initiation of the chronic intraperitoneal treatment with CCl4, the Na,K+-ATPase decreased to 40% of control values and continued to decrease further until reaching values below 1%. Ca2+-ATPase followed a pattern similar to that obtained with Na+,K+-ATPase, except that the decrease was not as severe. Colchicine treatment prevented the modifications in ATPases when given simultaneously with CCl4 and reverted the alterations in ATPase activities of the CCl4-cirrhotic animals. Because ATPases are known to be modulated by the lipid composition of the membrane, we also determined the cholesterol to phospholipid ratio in all the isolated membranes. The ratios were increased in membranes with low ATPase activity due to an increase in the total concentration of cholesterol. Plasma membranes of cirrhotic rats treated with colchicine showed a low concentration of cholesterol, a decreased cholesterol to phospholipid ratio, and Na+,K+-ATPase activity was almost normal. When plasma membranes of cirrhotic rats were fused with phosphatidyl serine-containing liposomes, the cholesterol to phospholipid ratio decreased and the ATPase activity increased. The ATPase activity of normal plasma membranes decreased below 20% of control values when enriched with cholesterol. Our results suggest that the decrease in the plasma membrane Na+,K+ ATPase activity of the cirrhotic rat is due in part to an increase in its cholesterol concentration and in the cholesterol to phospholipid ratio. PMID- 2997545 TI - Improved separation of rhesus monkey lymphocytes with Percoll. AB - The total yield and the proportions of Rhesus monkey peripheral blood mononuclear cells (PBMC) with detectable T4, T8, T11 and Ia markers that were purified by density gradient centrifugation through solutions of polyvinyl pyrrolidone (Percoll) were significantly greater than those found in PBMC suspensions obtained by using Ficoll-Hypaque. The percentages of recovery of PBMC and of cells expressing the above markers were lowest among PBMC isolated with a Ficoll Hypaque mixture of density 1.077 and highest among PBMC obtained by using a solution of Percoll of density 1.077. Percoll of density 1.077 was also better than Ficoll-Hypaque in that it yielded PBMC with the lowest levels of erythrocyte (less than 1%) and granulocyte (less than 1%) contamination. The reduced levels of PBMC with detectable markers appeared to result from an effect of either Ficoll or Hypaque on the lymphocytes rather than from loss of lymphocyte subpopulations since higher proportions of cells bearing T4, T8, T11 and Ia were found when PBMC were isolated with a solution of Percoll which yielded a percentage of PBMC as low as that recovered with Ficoll-Hypaque. Altered marker expression by cells obtained with Ficoll-Hypaque was not seen with similarly prepared human PBMC. PMID- 2997544 TI - Vascular sarcomas (probably angiosarcomas) transplanted from suspensions of liver cells from diethylnitrosamine-treated rats. AB - F344 male rats were given 90 ppm diethylnitrosamine in their drinking water ad libitum in two cycles. Livers containing neoplastic nodules, hepatomas, and no sarcomas in the sections sampled were digested in parallel with 0.05% collagenase, 0.1% Pronase, or 0.25% trypsin. Cells were transplanted into 9- to 19-day-old F344 rats. Despite the absence of sarcomas in the sections examined microscopically from each such liver before digestion and the presence of multiple hepatomas in all sections examined, vascular sarcomas, probably angiosarcomas, were observed in a large proportion of animals injected with the suspensions of cells; hepatomas were not observed in these animals. Morphology by light microscopy, immunohistochemical demonstration of factor VIII, histochemical demonstration of alkaline phosphatase, and the presence of Weibel-Palade bodies strongly suggest that these tumors are angiosarcomas. Similar tumors developed from cells obtained in parallel with the aid of Pronase, collagenase, or trypsin. Cell suspensions obtained with Pronase yielded tumors with the shortest latent period between the injection of cells and the death of one-half of the transplant recipients. The procedure that we used provides a consistent method for the production of transplantable sarcomas. The absence of sarcomas in the single sections taken from donor livers and multiple sections of similar livers not used for transplantation suggests that transplantability of these sarcoma cells is acquired very early in this neoplasm. PMID- 2997540 TI - [Angiotensin-converting enzyme inhibition: direct and indirect mechanisms]. AB - The introduction of angiotensin-converting-enzyme (ACE)-inhibitors into the analysis of the renin-angiotensin system (RAS) had broadened our knowledge of the integral role of renin and the kidney in circulatory homeostasis and has provided a pathophysiologically based concept for the treatment of hypertension. When the RAS is activated, as it is when sodium is restricted, the renal blood supply shows the most striking vasodilatation among vascular beds assessed after ACE inhibition. Sodium excretion rises, there is a fall in blood-pressure, and plasma concentrations of angiotensin II (AII) and aldosterone are reduced. Conversely, with sodium loading the hemodynamic and hormonal effects of ACE-inhibitors are small. In 50-60% of normal or high-renin patients with essential hypertension ACE inhibitors induce a potentiated acute renal response: renal blood flow and sodium excretion increase more than they do in the remainder of the hypertensives or in normal subjects. The responders of the hypertensive patients fail to increase renal blood flow or to enhance renal vascular responsiveness to infused AII when they shift from a low to a high sodium intake. The altered renal response of these "sodium-sensitive" hypertensives could be related to local activity of the RAS which is insufficiently suppressed by sodium loading. ACE-inhibition reverses this failure of the renal blood supply to respond to sodium loading. Kidneys of spontaneously hypertensive rats and the renin-rich kidney of Goldblatt hypertensive rats show an increased tubulo glomerular (TG) feedback response as compared to normal kidneys. The change in TG-feedback response might be expected to contribute to the inability of the hypertensive kidney to respond adequately to sodium loading. ACE-inhibition reduces TG-feedback sensitivity. In renal artery stenosis glomerular capillary pressure tends to be maintained by an AII mediated rise in postglomerular resistance. Suppression of AII by ACE-inhibition reduces efferent vascular tone and thus filtration rate. There is a potential for interaction of ACE-inhibitors with the kallikrein and prostaglandin pathways as well as with the sympathetic nervous system and endogenous opioids. This may modify the renal and blood pressure responses to these compounds. PMID- 2997546 TI - Measurement of hepatic blood flow. AB - Measurement of hepatic blood flow is fundamental to understanding hepatic physiology and biochemistry, quantitating drugs metabolized in the liver, and describing pathophysiologic states such as portal hypertension. However, the dual blood supply, altered handling of labels during disease states, and the relative inaccessibility of the portal system have made precise measurements of hepatic blood flow difficult. A variety of techniques can be utilized in both laboratory animals and patients; the more invasive techniques are generally available for animal studies, while semi-invasive and noninvasive methodologies yield information from patients. Each method has its inherent strengths and weaknesses, and may be utilized in a particular circumstance to accurately assess hepatic blood flow. It is therefore critical to select a measurement technique which yields the desired accuracy without interfering with the physiologic conditions under study. The methodology is presently available to achieve this end. PMID- 2997548 TI - Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemic myocardium. AB - The infiltration of neutrophils into ischemic myocardium exacerbates myocardial damage upon reperfusion, whereas drugs that inhibit neutrophil activity or function reduce infarct size. Consequently, it is important to accurately assess the myocardial neutrophil content. Histologic sections and radiolabeled cells have been used, but have major limitations. We have developed a method to measure the neutrophils present in cardiac tissue by utilizing a spectrophotometric assay for the neutrophil-specific myeloperoxidase enzyme (MPO) (Bradley et al., 1982a). Coronary artery occlusion and reperfusion in the anesthetized dog induces neutrophil accumulation into the ischemic heart, which shows a linear relationship with time. An increase in activity from 0.014 +/- 0.001 units (u) MPO/100 mg tissue to 0.091 +/- 0.02 u MPO/100 mg is already apparent at the end of the 90-min occlusion period. This activity increases over 5 hr reperfusion to 0.32 +/- 0.018 u MPO/100 mg tissue. Histologic analyses confirmed the temporal association of neutrophil accumulation. Moreover, there is a correlation between infarct size and tissue MPO activity. Measuring the MPO content in preparations of canine neutrophils, which is directly correlated with cell number, allows units of MPO activity to be converted into a tissue neutrophil content. This assay is simple, sensitive, and provides a quantitative index of myocardial neutrophil accumulation that can be used to study the relationship between leukocyte infiltration and myocardial injury. PMID- 2997547 TI - [In vivo and in vitro study of (N-piperidinomethyl)-2-chromanne on pre- and postsynaptic alpha-adrenoreceptors in the rat]. AB - The alpha-adrenoceptors blocking effect of (N-piperidinomethyl)-2-chromanne was studied in vivo and in vitro in the rat. In the pithed rat, (N-piperidinomethyl) 2-chromanne (1 mg/kg i.v.) antagonized the pressor effects induced by alpha agonists. (N-piperidinomethyl)-2-chromanne competitively antagonized the pressor effects of M7 (alpha 2-agonist) and cirazoline (alpha 1-agonist). (N piperidinomethyl)-2-chromanne antagonized the inhibitory effects of clonidine on presynaptic alpha 2-receptors in stimulated pithed rats. On the vas deferens of the rat (N-piperidinomethyl)-2-chromanne antagonized the inhibitory effects of clonidine on the twitch response produced by electrical stimulation and also the contractions induced by phenylephrine. This derivate appears to be an alpha 1 and alpha 2 blocking agent. PMID- 2997549 TI - The use of 2,2,6,6-tetramethyl-4-maleimido-piperidin-1-oxyl in electron paramagnetic resonance spin label studies of drug interactions with erythrocyte membranes. AB - The electron spin resonance (EPR) spectrum of erythrocyte membranes labeled with 2,2,6,6-tetra-methyl-4-maleimido-piperidin-1-oxyl (MAL-6) indicates both weakly and strongly immobilized labeling sites. Changes in the ratio of the membrane's low field spectral peaks (W/S) have often been used to monitor drug-erythrocyte interactions. We have investigated a number of experimental factors that may influence this ratio even in the absence of drug. Instrumental settings on the EPR spectrometer had no obvious effect. As the weight ratio of label/protein decreased, the W/S ratio increased. Similarly an increase in labeling time and temperature lead to an increase in the ratio. The ratio also increased with time after labeling; this change was most marked in samples kept at 37 degrees C, but was insignificant in samples kept at 4 degrees C. Increasing the viscosity of the sample with low-molecular-weight substances such as sucrose or glycerol, but not with those with high molecular weight such as dextran and PVP, caused a reduction in the ratio. Increasing the pH and changing the buffer concentrations also lead to a small increase in W/S. These results suggest that it is very important that all of these factors be kept constant and at some optimal value if reliable and consistent results are to be obtained using this method to monitor drug erythrocyte interactions. PMID- 2997550 TI - The effects of kidney invigorating Chinese traditional drugs on the functions of the hypothalamus-pituitary-gonads axis. PMID- 2997551 TI - Different mechanisms underlying reduced beta 2-adrenoceptor responsiveness in lymphocytes from neonates and old subjects. AB - To investigate the mechanism underlying age-dependent changes in beta adrenoceptor function we have determined beta 2-adrenoceptor density (by (+/-) 125iodocyanopindolol (ICYP) binding) and beta 2-responsiveness (cyclic AMP responses to isoprenaline stimulation) in lymphocytes derived from 20 neonates, 54 young adults (19-30 years) and 15 old subjects (60-86 years). In young adults the mean number of lymphocyte beta 2-adrenoceptors amounted to 862 +/- 36 (range 500-1560) ICYP binding sites/cell (N = 54); it was slightly higher in old subjects with 1230 +/- 94 (698-1980) ICYP binding sites/cell (N = 15). In contrast, lymphocytes derived from neonatal blood had a significantly lower mean beta 2-adrenoceptor number (385 +/- 35 (130-608) ICYP binding sites/cell, N = 20, P less than 0.01). (-)-Isoprenaline (0.01-100 microM)-induced increases in lymphocyte cyclic AMP content were significantly lower in neonates and old subjects than in young adults. While for neonates and young adults significant positive correlations between beta 2-adrenoceptor density and 10 microM (-) isoprenaline-induced cyclic AMP increases exist, in old subjects cyclic AMP increases were much lower than could be expected from the beta 2-adrenoceptor number. It is concluded that the mechanism underlying reduced beta 2-adrenoceptor responsiveness in neonates and old subjects is different: while in neonates it seems to be due to the reduced beta-adrenoceptor number, in old subjects it is caused by a post-receptor defect--presumably by a decreased activity of the adenylate cyclase. PMID- 2997553 TI - [Epidemiologic, clinicopathologic and developmental analysis of a series of 8 peripheral cholangiocarcinomas]. PMID- 2997552 TI - Kappa casein, lactalbumin and GCDFP 70 localization in human breast carcinomas: an immunohistochemical study using the avidin-biotin-peroxidase complex method. AB - A retrospective study of 67 human breast carcinomas of various types and grades was conducted using the avidin-biotin-peroxidase complex (ABC) to localize casein, lactalbumin, and GCDFP 70 on paraffin sections. Estrogen and progesteron receptors also were evaluated. This study demonstrated the following: (1) Casein positive cells were present in all cases with a variable distribution and degree of staining, whereas lactalbumin and GCDFP 70 were seen in only 40 and 43% of the cases, respectively. (2) No significant relationship was observed between casein, lactalbumin, GCDFP 70 and the histologic types of tumors or the extent of stromal elastosis, with the exception GCDFP 70, which was observed more often in well differentiated ductal carcinomas. (3) No significance was established in the relationship between antigens and steroid receptor content, with the exception of casein; strong casein immunostaining was significantly related to high progestin receptor levels. (4) Lactalbumin and GCDFP 70 were significantly associated with each other, but independently so of the histologic grades and types, the extent of stromal elastosis, and the steroid receptor content of the tumor cells. PMID- 2997554 TI - Acquired immune deficiency syndrome. A perspective for the medical practitioner. AB - AIDS is a new disease process with complications and management problems unlike anything ever seen before. An attempt has been made to summarize the available information about its various clinical presentations and how to manage them. Subjects covered will include associated malignancies, infections, transmissibility, prodromal states, and infection control issues. PMID- 2997555 TI - The immunologic effects, kinetics, and use of glucocorticosteroids. AB - This article describes our current understanding of the immunologic effects of glucocorticosteroids and uses this information in an attempt to place the therapeutic dosing of glucocorticosteroids on a more rational basis. PMID- 2997556 TI - Mechanism of action of thyroid hormones. AB - Thyroid hormones have ubiquitous effects and influence the function of most organs. The influences that thyroid hormones have on these diverse functions are primarily mediated through binding of T3 and T4 to specific nuclear receptor sites. The nuclear action of T3 results in organ-specific increases and decreases of specific mRNAs, leading to alteration in the level of the corresponding proteins. In addition to the well established nuclear action of T3, effects of thyroid hormone on other sites including cell membranes and mitochondria have been documented. PMID- 2997557 TI - Effects of a new 2-piperazinotetralin on alpha-1 and alpha-2 adrenergic receptors in isolated rat vas deferens. AB - The pharmacological action of a newly synthesized piperazine derivative of 2 aminotetralin (P11) on pre- and postsynaptic alpha-adrenoceptors of isolated rat vas deferens has been investigated. P11 shifted noradrenaline concentration effect curves to the right. The pA2 value was 5.88 for P11, 5.38 for phentolamine, and 7.73 for prazosin. The maximum effect of the agonist was enhanced by P11 and phentolamine, but this enhancement was less pronounced in the presence of 1 X 10(-5) M cocaine and 1 X 10(-6) M propranolol. 1 X 10(-6) M to 1 X 10(-5) M P11 increased the contractions of a field-stimulated vas deferens and antagonized the effects of clonidine (1 X 10(-9) M on the same preparation. The IC50 values of P11, phentolamine and prazosin were 0.40 microM, 0.03 microM and greater than 10.0 microM, respectively. The results show that P11 possesses alpha 1-and alpha 2-adrenolytic activity. The effect of P11 on these receptors most likely participates in the mechanisms of its hypotensive action. PMID- 2997558 TI - In vitro inhibition of renal sodium-potassium ATPase activity by progesterone. AB - The effect of various steroids and prostaglandins on the activity of canine kidney Na+, K+-ATPase activity was tested in vitro. Progesterone induced a dose dependent increase in inhibition of the canine kidney Na+, K+-ATPase activity. Aldosterone, cortisol, cortisone as well as prostaglandin E2 and 13,14-dihydro-15 keto-prostaglandin F2 alpha, did not cause an inhibition of canine kidney Na+, K+ ATPase. PMID- 2997559 TI - Delta-9-Tetrahydrocannabinol: effect on mitochondrial swelling of different tissues of rat. AB - In vivo delta-9-tetrahydrocannabinol (delta-9-THC) under short (10 and 50 mg/kg) and long term (10 mg/kg/day for 15 consecutive days) administration (i.p.) to adult male albino rats increased the mitochondrial swelling of hypothalamus, heart and liver. Similarly, in vitro treatment with delta-9-THC (2-8 micrograms/mg protein) produced a dose-dependent stimulation in the mitochondrial swelling of different tissues. Ca2+ ions (0.15 mM - 0.3 mM) increased the mitochondrial swelling. Calcium (0.3 mM)-induced stimulation of mitochondrial swelling was enhanced with delta-9-THC under both in vivo and in vitro conditions. Chaotropic agents e.g. KNO3 (100-200 mM) and KSCN (200-400 mM), decreased the mitochondrial swelling. Chaotropic (200 mM)-induced inhibitory effect in different tissues was reduced with delta-9-THC under in vivo and in vitro treatments. These results suggest that delta-9-THC, like calcium, stimulated the mitochondrial swelling, and that the action of delta-9-THC may be localized on the specific structures of the membrane which are sensitive to chaotropic agents. PMID- 2997560 TI - Histaminergic agents can modulate adrenergic smooth muscle neurotransmission. AB - The effects of histamine and some agonists and antagonists of the two types of histaminergic receptors on adrenergic smooth muscle neurotransmission were studied. The experiments were carried out on electrical stimulated (0.1 Hz, 1 pulse, 1 msec pulse duration, supramaximal voltage) prostatic part of rat vasa deferentia. Histamine, 2-(aminoethyl) thiazole and 2-methylhistamine (H1 agonists) as well as dimaprit (H2-agonist) had a biphasic effect on smooth muscle contractions induced by electrical stimulation (ES): low drug concentrations potentiated and high concentrations markedly inhibited the ES-contractions. The agonist of H1-receptors pyridylethylamine in low concentrations did not affect the smooth muscle contractions and in high concentrations decreased their amplitude. Diphenhydramine and mepyramine (H1-antagonists) strongly potentiated (up to 250%) the ES-contractions, while the H2-antagonist cimetidine did not change them. These effects might be due to the interaction of the drugs tested with different populations of the histaminergic receptors. PMID- 2997561 TI - [Surgical prevention in precancerous conditions and noninvasive cancers of the breast]. AB - The biological significance of precancerous lesions and non-invasive carcinoma of the breast are compared, particularly proliferative mastopathy with cellular atypia (so-called mastopathy grade III), ductal papillomatosis, non-infiltrating intraductal carcinoma (IDC) and lobular in situ carcinoma (CLIS). While biological significance has been described for most tissue pathology choice of therapy is difficult because the clinical course following local excision is not readily assessed. For therapy of individual patients, their pathology, individual situation and risk factors are to be considered. In general a conservative, expectant treatment is recommended for all lesions following local excision. More radical surgery is recommended for IDC, although a number of factors precluding radical procedures may be discussed. PMID- 2997562 TI - Treatment of Bell's palsy. PMID- 2997563 TI - [Mucoepidermoid carcinoma of the stomach]. AB - A case report is given of a 65 year old female patient harboring a rare mucoepidermoid carcinoma of the stomach. On X-ray examination a smooth faced mass with a diameter of 3 cm was found in the corpus region of the stomach. During gastroscopy the tumor was seen to be localized in the lower third of the stomach close to the lesser curvature with an ulceration on top. On microscopic examination a cornified squamous cell carcinoma was found including interspersed mucilaginous adenocarcinoma tissue. Prognosis of mucoepidermoid carcinoma is bad, histogenesis controversial. Discussion of this case as well as of other unusual adenocarcinomas for example with trophoblastic differentiation has to include the possibility of bidirectional development of undifferentiated stem cells. However it still remains unclear, why such tumors of the stomach are almost always adenocarcinomas and not signet ring cell carcinoma. PMID- 2997564 TI - [Cutaneous implantation metastasis following fine-needle puncture of pancreatic cancer]. AB - A case report form is given of a patient who developed a cutaneous implantation metastasis of a punctured pancreatic carcinoma. Needle biopsy of the tumor, located in the head of the pancreas, had been performed before, as well as drainage of the necrotic pancreas tail. Necrosis had been caused by pancreatitis due to stenosis of the pancreatic duct. This is a rare complication which may be due to local changes induced by puncture in the surroundings of the puncture channel. PMID- 2997565 TI - Regulation of anterior pituitary and brain beta-adrenergic receptors by ovarian steroids. AB - Ovariectomy of adult female rats (200-230g) resulted in an increase in beta adrenergic receptors in the cerebral cortex, hypothalamus and anterior pituitary. The anterior pituitary had the largest overall increase as well as the most rapid increase in beta-adrenergic receptor density of the tissues examined. The increase in hypothalamic or cerebral cortical beta-adrenergic receptors became apparent only long after ovariectomy (7-14 days). Fourteen days after ovariectomy, the density of beta-adrenergic receptors was 79%, 40%, and 24% in excess of control values in crude membranes prepared from anterior pituitary, hypothalamus and cerebral cortex, respectively. Over the same interval, the plasma concentration of luteinizing hormone (LH) increased 28-fold, while the concentration of follicle-stimulating hormone (FSH) rose 5-fold compared to control levels. Estradiol replacement (20 micrograms/kg/day) in these animals for four days before sacrifice concomitantly reduced plasma levels of the gonadotropins as well as the density of beta-adrenergic receptors in both the anterior pituitary and the hypothalamus. Long-term steroid replacement during the fifth and sixth week after ovariectomy, with implants of estradiol and progesterone which released the steroids in approximately physiological concentrations, significantly reduced beta-adrenergic density in anterior pituitary, but not in the hypothalamic membranes. This treatment significantly reduced plasma LH, but not FSH. Beta-adrenergic receptor density was also found to fluctuate significantly during the 4-day estrous cycle. The highest values were found on proestrus, and the lowest on diestrus 1. These studies indicate that changes in plasma concentrations of gonadal steroids (e.g. during the estrous cycle) influence the density of beta-adrenergic receptors in tissues involved in the control and release of anterior pituitary gonadotropins. PMID- 2997566 TI - Epinephrine infusion induces hyporesponsiveness of vascular smooth muscle. AB - Exposure to vasoactive drugs may lead to desensitization of vascular smooth muscle responsiveness. We have explored this phenomenon by infusing epinephrine into awake rabbits for 2h and then assessing smooth muscle contraction, both in vivo and ex vivo. Epinephrine was infused at a rate of 1 microgram X min-1 which resulted in a 15-fold increase in the plasma epinephrine concentration. The dose of phenylephrine required to cause a 25 mmHg increase in mean arterial pressure significantly increased from 109 +/- 56 micrograms prior to the infusion to 261 +/- 143 at the end of the 2h infusion (p less than 0.01). The sensitivity to phenylephrine remained decreased when reassessed 2h later. Untreated rabbits displayed no change in alpha-adrenergic responsiveness when assessed at 2 hourly intervals over the time-course of the experiment. Contraction of aortic rings removed from both epinephrine-treated and control rabbits was determined in vitro in tissue baths. The EC50 of norepinephrine-induced contraction increased from 31 +/- 6 to 210 +/- 20 nM while there was also a 30% decrease in the maximal force of contraction (EMax) in treated vessels. The EC50 only partially recovered after 4h of incubation ex vivo, while the EMax was restored to the control value. The EC50 for histamine in the aortic rings from epinephrine-treated rabbits was not different from controls although there was a 25% reduction in the EMax at 2h. We conclude that desensitization of alpha-adrenergic mediated vascular contractility develops rapidly in vivo and is only slowly reversible after removal of the agonist. PMID- 2997567 TI - Combined administration of human corticotropin-releasing factor and lysine vasopressin induces cortisol escape from dexamethasone suppression in healthy subjects. AB - Inadequate suppression of plasma cortisol after 1-2 mg dexamethasone is frequently observed in depressive patients. To further investigate the pathophysiology underlying cortisol nonsuppression after dexamethasone we compared cortisol and corticotropin (ACTH) response to human corticotropin releasing factor (h-CRF), lysine vasopressin (LVP), and a concurrent administration of both peptides after pretreatment with 1.5 mg dexamethasone in six male controls. Neither h-CRF nor LVP were able to produce a marked elevation of dexamethasone suppressed plasma cortisol and ACTH. If both peptides were administered in combination, a substantial escape of plasma cortisol from dexamethasone suppression was observed. ACTH responses changed in concordance with those of cortisol indicating that the LVP-CRF interaction takes place at the pituitary level. Our finding is consistent with a multihormonal control of pituitary-adrenal activity and bears several implications for interpretation of dexamethasone suppression test results in depressive illness. PMID- 2997568 TI - Serotonergic agonists stimulate inositol lipid metabolism in rabbit platelets. AB - The metabolism of inositol phospholipids in response to serotonergic agonists was investigated in rabbit platelets. In platelets prelabelled with [3H]-inositol, in a medium containing 10 mM LiCl which blocks the enzyme inositol-1-phosphatase, 5 hydroxytryptamine (5-HT) caused a dose-dependent accumulation of inositol phosphates (IP). This suggests a phospholipase-C-mediated breakdown of phosphoinositides. Ketanserin, a selective 5-HT2 antagonist, was a potent inhibitor of the 5-HT response, with a Ki of 28 nM, indicating that 5-HT is activating receptors of the 5-HT2 type in the platelet. Lysergic acid diethylamide (LSD) and quipazine also caused dose-related increases in inositol phosphate levels, though these were considerably less than those produced by 5 HT. These results show that relatively small changes in phosphoinositide metabolism induced by serotonergic agonists can be investigated in the rabbit platelet, and this cell may therefore be a useful model for the study of some 5 HT receptors. PMID- 2997569 TI - Effects of cannabinoids on progesterone release in cultures of rat luteal cells. AB - This study investigated the direct effects of tetrahydrocannabinols (THC) on progesterone release by cultured rat luteal cells, as a function of dose and time. During a 24-h incubation, the level of progesterone in the culture medium was decreased by 35% and 60% in the presence of 1 microM 11-OH-delta 9-THC and 8 beta-OH-delta 9-THC, respectively, when compared with control cultures. Dose response analysis revealed that 8 beta-OH-delta 9-THC inhibited progesterone levels at 0.1 microM but not at lower concentrations. The action of 8 beta-OH delta 9-THC was rapid in onset and a significant effect could be observed as early as 2 h following the addition of the cannabinoid. While luteinizing hormone (LH, 1 microgram/ml) significantly enhanced progesterone release in the culture medium over the respective control levels, this action of LH was dramatically suppressed by the concomitant presence of 8 beta-OH-delta 9-THC at 2, 4 and 24 h in separate experiments. Moreover, the increase in the level of progesterone in the culture medium induced by 8-bromo-cyclic AMP was also attenuated by the concomitant presence of 8 beta-OH-delta 9-THC in the cultures. These results further substantiate a direct action of cannabinoids on the steroidogenic function of the corpus luteum, and that it involves at least some step(s) distal to the LH-sensitive adenylate cyclase system. PMID- 2997570 TI - Metabolic profiling of radioreceptor-assayable opioid peptides in a human pituitary ACTH-secreting tumor. AB - The profile of endogenous opioid peptides in the peptide-rich fraction obtained from a homogenate of an ACTH-secreting human pituitary tumor is presented. Gradient RP-HPLC is used to separate the mixture into peptide constituents. A preparation of opioid receptors is used in a radioreceptor assay with ethorphine a relatively non-specific ligand that is used as a screen because it interacts with mu, sigma, and delta receptors - as the HPLC detector to detect a range of peptides that derive from proenkephalin A and POMC. PMID- 2997572 TI - Convulsant component of a depressant benzodiazepine. AB - The convulsant influence of high doses of diazepam, in the presence of the benzodiazepine receptor antagonist Ro 15-1788, was studied in rats. Animals were implanted with permanent cortical screw electrodes for EEG recording. EEG spiking and accompanying clonic activity was observed in rats receiving greater than or equal to 200 mg/kg diazepam, followed 10 minutes later by Ro 15-1788 (20 mg/kg). Pentylenetetrazole and picrotoxin seizure thresholds, measured during constant rate iv infusion, were significantly lowered by pretreatment with diazepam (250 mg/kg) and Ro 15-1788 (20 mg/kg) administered 30 and 20 minutes, respectively, before seizure threshold measurement. It is proposed that this convulsive activity of diazepam is mediated through the picrotoxinin receptor. PMID- 2997571 TI - The effects of the acute administration of buprenorphine hydrochloride on the release of anterior pituitary hormones in the rat: evidence for the involvement of multiple opiate receptors. AB - Multiple opiate receptor agonists and antagonists have been found to produce different patterns of anterior pituitary hormone release. The present studies examined the pattern of anterior pituitary hormone release produced by buprenorphine. The effects of the kappa agonist ethylketocyclazocine on thyroid stimulating hormone release were also examined. Following buprenorphine, serum levels of corticosterone and luteinizing hormone were not changed while growth hormone release was stimulated in a dose-dependent manner. Prolactin release was stimulated after the lowest dose of buprenorphine while the highest dose induced a fall in serum prolactin. Similar biphasic effects on thyroid stimulating hormone were seen after either buprenorphine or ethylketocyclazocine. The results provide support for the role of multiple opiate receptors in opiate-induced changes in anterior pituitary hormone release. PMID- 2997573 TI - Regulation of beta-endorphin and ACTH in brain by estradiol. AB - The effect of estradiol on the brain concentration of immunoreactive beta endorphin (beta-EP) and C-terminal ACTH (CLIP) was studied in ovariectomized rats. Dopamine, a known inhibitor of pituitary intermediate lobe pro opiomelanocortin (POMC), was examined as a possible mediator of the estradiol induced changes in brain POMC. Animals were treated for 1 or 3 weeks with either 1) saline; 2) silastic estradiol implants; or 3) estradiol implants plus haloperidol 1 mg/kg/day. After one week of treatment no significant change in hypothalamic beta-EP content was noted in any group compared to the control level of 4.13 +/- .33 (SEM) pmoles although in the neurointermediate lobe beta-EP increased from 566 +/- 72 to 942 +/- 73 pmoles after haloperidol (p less than .005). After 3 weeks, however, hypothalamic beta-EP decreased from 3.96 +/- .28 to 2.74 +/- .19 pmoles (p less than .005) and C-terminal ACTH decreased from 3.78 +/- .33 to 2.82 +/- .18 pmoles (p less than .02) in the estradiol treated rats. This estradiol induced decrease in the hypothalamic content of beta-EP and C terminal ACTH was not blocked by haloperidol. We conclude that estradiol lowers the hypothalamic content of beta-EP and CLIP and that this effect does not appear to be mediated by dopamine. PMID- 2997574 TI - Phospholipids and sialic acid changes produced by vitamin D3 on intestinal mitochondria. AB - Vitamin D3 administration to vitamin D-deficient chicks produces, 24 hours after treatment, a large increment of phospholipids in mitochondria from intestinal mucosa. Proportion of the different phospholipid classes and fatty acid composition of the organelles were not modified by that treatment. A time course study of the effects of 1,25(OH)2D3 on calcium absorption and sialic acid content of intestinal mitochondria glycoproteins showed that both effects were correlated. The results suggest that either vitamin D or the increase of calcium transfer are involved in the make up of intestinal mitochondria membranes. PMID- 2997575 TI - The effect of Synacthen administration on lipoprotein lipase activity in the epididymal fat pad of the rat. AB - Conflicting data have been reported on the influence of (excess) glucocorticoids on lipoprotein lipase (LPL) activity in adipose tissue. To solve this problem hypercorticism was induced in rats by treatment for varying periods with Synacthen, a synthetic corticotrophin-1-24 preparation, and LPL was measured in the epididymal fat pads using different methods. In extracts of defatted tissue preparations from overnight fasted rats treated for 3 days with Synacthen we observed an increase in LPL activity (acetone-ether powder LPL) to values similar to those found in normally fed controls. In contrast, the heparin-elutable part of LPL activity in the tissue was not influenced by the Synacthen treatment. This activity remained significantly lower in overnight fasted animals, Synacthen treated or not, than in normally fed rats. Adrenalectomy lowered the acetone ether powder LPL activity of the epididymal adipose tissue in fasted as well as in fed rats. In fasted rats it prevented the stimulation of the LPL activity by Synacthen. PMID- 2997576 TI - Increased body fat due to elevated energetic efficiency following chronic administration of inhibitors of sympathetic nervous system activity. AB - The effects on energy balance of drugs known to inhibit the sympathetic nervous system at different sites and with various modes of action were studied in lean mice of the CFLP strain. Metabolizable energy intake was monitored continuously over seven-week experimental periods and energy expenditure was determined by the comparative carcass method as well as by measurements of 24-hour oxygen consumption. Administration of either propranolol, phenoxybenzamine, clonidine, or mecamylamine had no influence on energy balance nor on body composition. On the other hand, administration of alpha-methyl para tyrosine, reserpine, and bethanidine, ie, drugs capable of reducing the levels of noradrenaline at sympathetic terminals, resulted in significant increases in body fat by 38%, 24%, and 46% respectively: this effect was brought about by a reduction in the metabolic rate of these drug-treated groups and the efficiency of energy utilization (both gross and net) was increased by three to nine times. These studies indicate that chronic administration of drugs that act at the receptor level to inhibit sympathetic activity fail to influence metabolic rate, thereby suggesting that thermogenesis under noradrenaline control may involve mechanisms other than the alpha and beta classification of adrenergic receptors. Nonetheless, the ability of some drugs which both reduce noradrenaline levels and decrease metabolic rate would support the concept of an important role of the sympathetic nervous system in the overall control of thermogenesis and in energy balance regulation. PMID- 2997577 TI - Rearrangements between the operators in the bacteriophage lambda. AB - The left operator mutant lambda v2s develops poorly during infection as a result of constitutive expression of the left operon. A revertant of lambda v2s, designated lambda iri, was found to contain an inversion of the cI region with the inversion endpoints to be within the lambda operators OL and OR. Formation of the inversion is facilitated by a translocation of right operator OcR mutant sequence to the left operator in lambda v2s. The inversion in lambda iri positions wild-type OR sequence at OL returning control of the left operon to repression by the lambda cro repressor. PMID- 2997578 TI - A new allele with abnormal cyclic-AMP phosphodiesterase activity in Phycomyces. AB - A mutant of the fungus Phycomyces blakesleeanus (Burgeff), C21 (madA7) that was isolated for its abnormal phototropism (Bergman et al. 1973) carries a secondary mutation pde-1 which is unlinked to the madA gene. The pde-1 allele causes the loss of about 80% of the cAMP phosphodiesterase activity. This allele is not essential for the photoreactions of the mycelium or the sporangiophore, and the bulk activity of the phosphodiesterase appears to play no role in the phototransduction pathway of Phycomyces. PMID- 2997579 TI - Enhancement of genetic transformation frequencies of mammalian cell cultures by damage to the cell DNA. AB - Ultraviolet (UV)-light and 5-fluorodeoxyuridine (FUdR), two known DNA damaging agents, were found to enhance the frequency of stable plasmid transformations in several different animal cell lines. Combined treatment with the two agents was more effective than treatment with either agent alone. A correlation between the transformability of a cell line in the absence of treatment and its response to damaging treatment was also observed. Southern blot analysis of transformed clones indicated that the stimulation in transformation frequency was not due to an increased number of copies of the integrated plasmid in the transformed cells. PMID- 2997581 TI - Conjugation-dependent enhancement of induced and spontaneous mutation in the lacI gene of E. coli. AB - The frequency of lac mutations induced in an F'lacIS plasmid, transferred by conjugation from UV-irradiated, excision-deficient donors to excision-deficient, delta pro lac recipients, is 2-3 fold higher than that typical of nonmating cells which contain the plasmid. These additional induced mutations can probably be ascribed to errors made during the first, or repliconation, synthesis that takes place in the recipient during the course of plasmid transfer. We also find that spontaneous mutation rates are enhanced in conjugating cells, indicating that fewer errors are corrected, or more made, during transfer replication. PMID- 2997580 TI - Untargeted mutagenesis induced by UV in the lacI gene of Escherichia coli. AB - Using a nonselective method, we have estimated the proportion of untargeted mutations in the lacI gene of E. coli by transferring either irradiated or unirradiated F' pro lac plasmids from an excision deficient donor to an excision deficient pro lac deleted recipient that had been irradiated and allowed to induce recA dependent functions for 30 min. We find that about 10 percent of the mutations induced by either 3.5 Jm-2 or 7 Jm-2 UV are untargeted. PMID- 2997582 TI - The catabolite gene activator protein (CAP) is not required for indole-3-acetic acid to activate transcription of the araBAD operon of Escherichia coli K-12. AB - Kline et al. (1980) have reported that indole-3-acetic acid (IAA) and four other indole derivatives are able to substitute for cAMP in activating expression of the ara regulon of E. coli. We have examined this phenomenon in detail, utilizing fusions between the structural gene for beta-galactosidase and the promoters for the araBAD, araE, and araFG operons. We confirm that IAA potently stimulates transcription from the araBAD promoter. The effect is highly specific to araBAD, as IAA has no, or only slight, effects on the araE and araFG operons. However, contrary to the results of Kline et al., we find that the action of IAA does not require CAP. Thus, IAA fully stimulates the transcription of araBAD in a strain which bears a complete deletion of the crp gene. PMID- 2997583 TI - Eliminated sequences with different copy numbers clustered in the micronuclear genome of Tetrahymena thermophila. AB - As the ciliated protozoan Tetrahymena thermophila develops a new macronucleus (MAC) from products of its micronucleus (MIC), several repetitive sequences are eliminated from the MAC genome. Four MIC DNA clones containing repetitive sequences that are eliminated from the MAC were obtained. One clone contains a representative from each of three families of eliminated sequences. One, present in 200-300 copies in the MIC, is almost completely eliminated from the MAC. A second, present in approximately 50 copies in the MIC, is scattered throughout the genome, although up to half of the family members examined could be localized to chromosome 2. Approximately one tenth of the members of this less repetitive family persist in the MAC while the rest are eliminated. The third type of eliminated sequence has three to four members, all of which are eliminated from the MAC. Three of the members are located on three of the five MIC chromosomes, and one could not be mapped. This sequence is clustered with the other two families of sequences in at least three of the four sites. All three types of eliminated sequences are found in similar arrangements in the MIC of several different inbred strains of T. thermophila. PMID- 2997584 TI - Lethal mutations in the structural gene of an outer membrane protein (OmpA) of Escherichia coli K12. AB - The gene ompA encodes a major outer membrane protein of Escherichia coli. Localized mutagenesis of the part of the gene corresponding to the 21-residue signal sequence and the first 45 residues of the protein resulted in alterations which caused cell lysis when expressed. DNA sequence analyses revealed that in one mutant type the last CO2H-terminal residue of the signal sequence, alanine, was replaced by valine. The proteolytic removal of the signal peptide was much delayed and most of the unprocessed precursor protein was fractioned with the outer membrane. However, this precursor was completely soluble in sodium lauryl sarcosinate which does not solubilize the OmpA protein or fragments thereof present in the outer membrane. Synthesis of the mutant protein did not inhibit processing of the OmpA or OmpF proteins. In the other mutant type, multiple mutational alterations had occurred leading to four amino acid substitutions in the signal sequence and two affecting the first two residues of the mature protein. A reduced rate of processing could not be clearly demonstrated. Membrane fractionation suggested that small amounts of this precursor were associated with the plasma membrane but synthesis of this mutant protein also did not inhibit processing of the wild-type OmpA or OmpF proteins. Several lines of evidence left no doubt that the mature mutant protein is stably incorporated into the outer membrane. It is suggested that the presence, in the outer membrane, of the mutant precursor protein in the former case, or of the mutant protein in the latter case perturbs the membrane architecture enough to cause cell death. PMID- 2997588 TI - Maternal mortality: pilot surveillance in seven states. PMID- 2997586 TI - [Physiological activity of Escherichia coli cells studied by the spin exchange method]. AB - The respiration and reducing activity of Escherichia coli M-17 cells was studied at physiological temperatures using the phenomenon of spin exchange between water soluble nitroxyl radicals and molecular oxygen in liquid bacterial media. The peak intensity of the EPR signal from nitroxyl probes was characterized by a two stage kinetics. The first stage was due to the rapid uptake of dissolved O2 whereas the second stage was caused by the anaerobic reduction of free radicals. The maximal rates of respiration and radical reduction were found at 45 to 55 degrees C. The rate of cell respiration changed when lipids underwent a gel liquid crystal structural transition. PMID- 2997587 TI - Recommendations for preventing transmission of infection with human T lymphotropic virus type III/lymphadenopathy-associated virus in the workplace. PMID- 2997585 TI - Genetic mapping of rpoD implicates the major sigma factor of Bacillus subtilis RNA polymerase in sporulation initiation. AB - We have mapped the chromosomal locus of rpoD, which encodes the major sigma factor of Bacillus subtilis RNA polymerase. The rpoD locus lay between aroD and lys, tightly linked to dnaE and inseparable from crsA. Marker order in this region was acf-aroD-dnaE-rpoD(crsA)-spoOG-lys. By transformation using cloned donor DNA from the rpoD region, we identified the gene immediately upstream of rpoD as dnaE, which coded for a 62,000 dalton protein essential for DNA replication. Both dnaE and rpoD were transcribed in the same direction, counterclockwise on the chromosome. The gene functions and organization in the rpoD region are thus similar to those of the E. coli sigma operon. We also used transformation to identify crsA47 as a mutation within the sigma coding region itself. The crsA alteration of sigma renders the sporulation process insensitive to glucose catabolite repression, and also restores sporulation ability to strains carrying early-blocked spoOE, spoOF, and spoOK mutations. Thus the major sigma factor and these spoO gene products directly or indirectly affect the same cellular function. PMID- 2997589 TI - Hepatitis surveillance, 1982-1983. PMID- 2997590 TI - [Clinicopathological studies on pancreatic carcinoma in totally pancreatectomized cases--with special reference to its intrapancreatic development and the pancreatogram]. AB - Histological examination and pancreatography were performed on 31 totally pancreatectomized cases of pancreatic ductal carcinoma, which was divided into four types according to its growth pattern in the pancreas. Pancreatograms of the resected specimens were also classified according to histological findings. Pancreatic carcinoma of Type I (20 cases) was diffusely infiltrating carcinoma with scirrhous stroma which was particularly present between the lobules. The pancreatogram of this type showed constriction of the main pancreatic duct accompanied by dilatation of the distal pancreatic duct. Carcinoma of Type II (6 cases) was diffusely developing in or around the dilated main pancreatic duct or its branches. The pancreatogram of Type II demonstrated the irregularly dilated main pancreatic duct with absence of its branches. Type III (4 cases) was the localized carcinoma with medullary structure. The pancreatogram showed replacement and dilatation of pancreatic ducts without marked stenosis of the main pancreatic duct. Type IV (1 case) was intraductal protruding carcinoma which showed polypoid shadow defects in remarkably dilated pancreatic ducts. The results suggested that a total pancreatectomy should be applied mainly for pancreatic carcinomas of Type II. PMID- 2997591 TI - [Portosystemic A-V fistula and portal hypertension associated with islet-cell tumor of the pancreas]. AB - A 50-year-old woman was admitted on Feb. 25, 1983, complaining of hematemesis and melena. Endoscopic examination revealed the rupture of the esophageal varices. At first she was treated with pressure hemostasis using a S-B tube, but it was failed. Then she underwent esophageal transection under emergency thoracotomy. After the operation, she developed ascites and watery diarrhea, though there was no episode of hematemesis and melena. A bruit was audible at the epigastric region. Abdominal angiograms demonstrated a fist-sized portosystemic A-V fistula (PAVF) locating at the pancreas head, and suggested concomitant existence of portal hypertension. On May 16, 1983, laparotomy was carried out in order to reduce portal pressure, but it was not successful. She died 3 weeks after the second operation. Autopsy and pathological findings revealed that PAVF associated with a hen-egg-sized islet-cell tumor of the pancreas head. Etiology and clinical features of this rare entity may be characterized by the nature of the islet-cell tumor having rich and rapidly growing vascular supply. PMID- 2997593 TI - Differentiation between amine transport and beta-adrenergic receptor-mediated binding in cultured mammalian cells. AB - We have found that several types of cultured mammalian cells, including both normal and transformed human, rat, and mouse cell lines, have an active transport system for a diverse group of structurally related compounds possessing an amine group and various types of aromatic ring structures. Ligands such as the beta adrenergic antagonists (-)-[3H] dihydroalprenolol (DHA), (-)-[3H]propranolol, and (-)-[125I] iodocyanopindolol, and the tricyclic antidepressant [3H]imipramine, which are used to assess cell surface receptors for catecholamines and serotonin, appear to be actively transported into cells rather than simply bound to cell surface sites or accumulated by passive diffusion. DHA transport was competed by many structurally related amines including imipramine and certain alpha-and beta adrenergic ligands, but not by catecholamines or serotonin. Ligand transport in HeLa cells was saturable at micromolar levels, selective, nonstereospecific, temperature- and pH-dependent, and sensitive to the ionophore monensin and the amine transport inhibitor reserpine, thus indicating dependence on a carrier system driven by a transmembrane proton gradient. In C6 glioma cells, amine transport was clearly distinguishable from beta-adrenergic receptor binding which could be measured with the recently developed hydrophilic beta-blocker (+/-)-[3H] 4-(3-tertiarybutylamino-2-hydroxy-propoxy)-benzimidazole-2-on hydrochloride (CGP 12177); binding of this ligand met rigorous pharmacological criteria, was not influenced by monensin or reserpine, and, therefore, did not appear to be transported. Membrane vesicles from HeLa and C6 cells transported DHA but not CGP 12177 via a MgATP-dependent mechanism which was inhibited by N,N' dicyclohexylcarbodiimide, monensin, and reserpine, indicating a carrier system driven by a proton gradient maintained by a proton-pumping ATPase. PMID- 2997592 TI - Phosphatidate and monooleylphosphatidate inhibition of fibroblast adenylate cyclase is mediated by the inhibitory coupling protein, Ni. AB - It has previously been shown that monooleylphosphatidate (MOPA) and phosphatidate inhibit cAMP accumulation in VA13 and WI-38 fibroblasts. In this study we investigated whether this inhibition might be due to a decrease in adenylate cyclase activity. Our results showed that both MOPA and phosphatidate inhibit prostaglandin E1-stimulated adenylate cyclase in WI-38 membranes in a concentration-dependent manner with half-maximal inhibitions at 0.1 and 0.5 microM, respectively, and maximal inhibitions of 35-55%. A 5 microM concentration of structurally similar lipids caused no significant inhibition. The inhibitory effects of MOPA and phosphatidate on adenylate cyclase were GTP dependent, greater at low concentrations of Mg2+, eliminated following treatment of cells with islet-activating protein, nonadditive with carbachol, and noncompetitive with prostaglandin E1. Collectively these data suggested that MOPA and phosphatidate inhibitions of cAMP accumulation were due at least in part to an Ni mediated inhibition of adenylate cyclase. Furthermore, the inhibitions showed the same characteristics normally associated with hormonal inhibition of this enzyme. PMID- 2997594 TI - Interactions of indoles with specific binding sites for 2,3,7,8 tetrachlorodibenzo-p-dioxin in rat liver. AB - In order to identify some of the structural requirements for binding of indoles to the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we have investigated the capacity of various indoles to inhibit specific [1,6-3H]TCDD binding in rat liver cytosol, as analyzed by electrofocusing in polyacrylamide gel. Of these indoles, indolo[3,2-b]carbazole was the most active. The IC50 value for receptor binding of indolo[3,2-b]carbazole as well as for 2,3,7,8 tetrachlorodibenzofuran was 3.6 nM, whereas that of 5,6-benzoflavone was 26 nM. Both indolo[3,2-b]carbazole and 2,3,7,8-tetrachlorodibenzofuran competitively inhibited the binding of [3H]TCDD to the receptor. The well-known microsomal enzyme inducer 3,3'-diindolymethane did not interact significantly with the TCDD receptor. Previous concepts of structure-activity relationships for binding of chlorinated dioxins to the TCDD receptor fail to account for the receptor binding of unhalogenated aryl hydrocarbon hydroxylase inducers such as 5,6-benzoflavone. We have instead considered the true three-dimensional space occupied by some receptor ligands by means of a computer using crystallographic data as inputs. When the atomic van der Waals radii were included, all potent receptor ligands studied could be fitted into a rectangle of 6.8 X 13.7 A. PMID- 2997595 TI - Epinephrine-stimulated maintained rubidium efflux from guinea pig hepatocytes may involve alpha 1- and alpha 2-adrenoceptors. AB - The epinephrine-stimulated maintained increase in 86Rb efflux from guinea pig hepatocytes may consist of both alpha 1- and alpha 2-adrenoceptor-mediated components. Both the alpha 1-selective adrenoceptor agonist phenylephrine and the alpha 2-selective agonist clonidine evoked a maintained increase in 86Rb efflux. Prazosin, an alpha 1-selective antagonist, did not inhibit the maintained increase in 86Rb efflux elicited by epinephrine, whereas yohimbine, an alpha 2 selective antagonist, did. In the absence of external Ca, no maintained increase in 86Rb efflux was observed. The results suggest that there may be two separate alpha-adrenergic sensitive Ca influx pathways into guinea pig hepatocytes, one mediated by alpha 1- and the other by alpha 2-adrenoceptor activation. PMID- 2997596 TI - Inhibition of uridine kinase and the salvage of uridine by modified pyrimidine nucleosides. AB - Uridine kinase can play a crucial role in the provision of pyrimidine nucleotides for cellular nucleic acid synthesis, particularly when de novo synthesis is inhibited by chemotherapeutic agents. Therefore, uridine kinase is an attractive target for drug development. We examined a series of 29 analogs of uridine, most with modifications at the 5'-position, as inhibitors of uridine kinase in vitro and of uridine salvage by intact L1210 cells. Substitution at the 5'-position resulted in decreased efficacy as inhibitors of uridine kinase, particularly if the substituent was large. None of the analogs with 5'-position modifications effectively inhibited salvage of uridine by intact L1210 cells. Four carbocyclic pyrimidine nucleoside analogs (one series) were all effective competitive inhibitors of uridine kinase and of uridine salvage by intact L1210 cells. Cyclopentenyl uracil 19 shows promise for further development as it inhibits uridine salvage at nontoxic concentrations. PMID- 2997597 TI - [Herpes simplex encephalitis in the newborn infant--a review]. AB - Neonatal herpes simplex encephalitis is a rare, but very threatening disease which leads to death or to severe cerebral damage with only a few exceptions. In the past few years antiviral agents have become available which have proved to influence the course of the disease in a positive manner. Therefore the problem is a challenge for neonatologists and pediatric neurologists. The virologic and epidemiologic aspects of neonatal herpes encephalitis and its prophylactic, diagnostic and therapeutic possibilities are reviewed. The efficacy of virustatic therapy is related to the onset of therapy which should be started as soon as possible. The typical EEG- and CT-changes are due to an already advanced stage of the disease. Virologic examinations in the first days are of limited value. Brain biopsy should not be performed in this age group. Therefore the indication to start antiviral chemotherapy depends exclusively on the clinical picture. PMID- 2997598 TI - Detection of environmental effects through anatomic pathology. PMID- 2997599 TI - Human liver carcinogenesis: a complex problem. PMID- 2997600 TI - The role of specific DNA base damages in the X-ray-induced inactivation of bacteriophage PM2. AB - Two types of X-ray-induced base damages, alkali-labile sites and thymine ring saturation products, were quantitated in PM2 DNA irradiated in the phage capsid under oxic and anoxic conditions. The extent of formation of these base damages was compared with the number of single- and double-strand breaks and lethal hits produced under the same conditions. The individual inactivation efficiencies of alkali-labile sites and thymine ring saturation products were determined by selectively inducing each of these damages in isolated PM2 DNA by chemical means in vitro and determining the rate of biological inactivation of the treated DNA by transfection. For each lethal X-ray hit induced in oxic conditions there were 1.06 alkali-labile sites, 0.40 thymine ring saturation products, 2.09 singe strand breaks and 0.11 double-strand breaks in the PM2 genome. In anoxic conditions, the respective number of lesions was 1.00, 0.19, 1.73 and 0.09. The individual inactivation efficiencies of thymine ring saturation products and alkali-labile sites were found to be essentially equal, 7-8 lesions per lethal event in the PM2 genome. Alkali-labile sites and thymine ring saturation products together accounted for 15-20% of the biological inactivation of X-irradiated bacteriophage PM2. The presence or absence of oxygen during irradiation did not affect the contribution to inactivation made by alkali-labile sites, but the contribution by thymine ring saturation products to inactivation was about 2-fold higher in oxic compared with anoxic conditions. With the 4 lesions measured, we have accounted for some 28-34% of the lethal events in X-irradiated PM2 phage, most of the remaining events being caused by as yet unidentified base damages. PMID- 2997601 TI - Use of nitrite and hypochlorite treatments in determination of the contributions of IQ-type and non-IQ-type heterocyclic amines to the mutagenicities in crude pyrolyzed materials. AB - The mutagenic heterocyclic amines Glu-P-2, MeA alpha C and Phe-P-1, which possess a 2-aminopyridine structure in their molecule (non-IQ-type mutagens), were found to be inactivated by nitrite treatment under acidic conditions, as observed previously with Trp-P-1, Trp-P-2, Glu-P-1 and A alpha C. In contrast, MeIQx, 4,8- and 7,8-DiMeIQx, which were originally isolated from fried beef or heated model mixtures of creatinine, amino acids and glucose, and which have a 2 aminoimidazole moiety in their molecules (IQ-type mutagens), were very resistant to nitrite treatment like IQ and MeIQ. Both types of mutagenic heterocyclic amines were completely inactivated by treatment with hypochlorite. This differential inactivation of mutagenic heterocyclic amines by nitrite and hypochlorite was used in determination of the contributions of IQ-type and non-IQ type mutagens to the total mutagenicities of various pyrolyzed materials. The percentage contributions of IQ-type mutagens to the mutagenicities of broiled sardine, fried beef, broiled horse mackerel, cigarette smoke condensate and albumin tar were 88, 75, 48, 6 and 4, respectively. PMID- 2997602 TI - Granular nuclear inclusion body disease: fine structure of tibial muscle and sural nerve. AB - Fine granular (hyaline) intranuclear inclusion bodies were found in perivascular cells of a muscle and a sural nerve biopsy from a 32-year-old woman with slowly progressive motor disturbances. The hyaline nuclear inclusion bodies could be distinguished from other intranuclear hyaline inclusions by their granularity, the size of the granules (approximately 5-15 nm), and the positive iron staining reaction. They were not seen in muscle fibers or Schwann cells. Because of these apparently pathognomonic structural features the patient appears to present a condition that has not been described before. PMID- 2997603 TI - Pseudo-anterior interosseous nerve syndrome. AB - The anterior interosseous nerve syndrome (AINS) is well known. A case is presented that had electromyographic findings limited to the distribution of the anterior interosseous nerve. A small area of sensory loss and slight asymmetry of amplitudes noted on median nerve conduction studies were inconsistent with an AIN syndrome. An antecubital level partial median nerve compromise primarily involving the bundles that form the anterior interosseous nerve was surgically noted. This case illustrates that localization of a peripheral nerve lesion must include consideration of the internal topography of peripheral nerves. PMID- 2997604 TI - Peripheral neuropathies associated with monoclonal proteins: a clinical review. AB - Over the last decade, the increasing use of serum and urine protein electrophoretic screening of patients with idiopathic peripheral neuropathy has led to greater recognition of peripheral neuropathy syndromes that are associated with monoclonal proteins and plasma cell dycrasias. After careful evaluation, most of these patients have benign monoclonal gammopathy, followed in frequency by primary systemic amyloidosis and osteosclerotic myeloma, with occasional cases associated with osteolytic multiple myeloma, Waldenstrom's macroglobulinemia, gamma heavy chain disease, and other rare disorders. Several of these syndromes have distinctive presentations and are recognizable clinically, whereas others (especially multiple myeloma neuropathy) are diverse clinically and are not clearly distinguishable from other chronic neuropathies. The discovery of IgM kappa monoclonal proteins directed at myelin antigens in some patients with benign monoclonal gammopathy and the delineation of the syndrome of neuropathy and multiorgan involvement in osteosclerotic myeloma are important developments which may shed light on the mechanism of the remote effects of malignancies on the nervous system. PMID- 2997605 TI - Passive transfer of the Lambert-Eaton myasthenic syndrome: neuromuscular transmission in mice injected with plasma. AB - Mice were given 1 to 67 daily injections of whole plasma from a control subject and a patient with the Lambert-Eaton myasthenic syndrome (LES), and the parameters of neuromuscular transmission in diaphragm muscles were studied in vitro. In animals that received 41 to 59 injections of LES plasma, mean quantum content of the endplate potentials, as measured under the conditions of low [Ca2+]o (1.2 mM) and high [Mg2+]o (10 mM), was reduced to 30% of the control, or 40% of the value found in normal untreated mice. When [K+]o was elevated from 5 mM to 17.5 mM, the frequency of spontaneous miniature endplate potentials increased by a factor of 98 and 105 respectively in the control and normal mice but only 39 times in the LES plasma recipients. Despite these presynaptic abnormalities, no evidence of postsynaptic deficit, nerve conduction failure, or inability of the muscle fibers to produce normal action potentials was found. These electrophysiologic features in mice thus represent a reproduction of the human condition, supporting the concept of humoral involvement in the pathophysiology of LES. Passively transferred LES in mice is a faithful animal model of the human disease which will be useful in exploring the molecular basis of the presynaptic impairment. PMID- 2997606 TI - Peripheral neuropathy following amitriptyline overdose. PMID- 2997607 TI - Herpesvirus infections of pregnancy. Part I: Cytomegalovirus and Epstein-Barr virus infections. PMID- 2997608 TI - Seroepidemiologic screening for antibodies to LAV/HTLV-III in Sri Lanka, 1980 1982. PMID- 2997610 TI - Diet and colonic cancer: putting the puzzle together. PMID- 2997609 TI - Target platelet antigen in homosexual men with immune thrombocytopenia. AB - A syndrome of isolated immune thrombocytopenic purpura (ITP) has recently been described in homosexual men. We have identified an antiplatelet antibody in the serum of 29 of 30 homosexual men with isolated ITP. The antibody binds to a platelet membrane antigen of 25,000 daltons, and binding is effected by the F(ab)2 portion of the immunoglobulin. Similar antibody activity was not detected in serum from 30 nonhomosexual patients with either ITP or nonimmune thrombocytopenia. The 25,000-dalton antigen was not found on other hematopoietic cells, and it was distinct from the core protein of the AIDS-associated retrovirus. In contrast, serum antibody reacted with a 25,000-dalton antigen associated with cultured herpes simplex virus Types I and II. In these experiments the antigen appeared to be derived from green-monkey kidney cells in which the herpes simplex viruses were grown. Identical antigenic activity was also demonstrated in uninfected human skin fibroblasts. We conclude that ITP in homosexual men is accompanied by a serum antibody directed against a platelet antigen of 25,000 daltons. The nature of the antigen and the relation of the serum antibody to ITP require further study. PMID- 2997611 TI - The T4 lymphocyte in AIDS. PMID- 2997612 TI - Inactivation of human T-cell lymphotropic retrovirus (HTLV-III) by LD. PMID- 2997613 TI - Maternal-fetal transfer of abused substances: pharmacokinetic and pharmacodynamic data. PMID- 2997614 TI - Opioids and development: new lessons from old problems. PMID- 2997615 TI - Epstein-Barr virus-positive Burkitt's lymphoma cells not recognized by virus specific T-cell surveillance. AB - The pathogenesis of Epstein-Barr (EB) virus-positive Burkitt's lymphoma (BL) appears to involve the combined actions of virus-induced B-cell proliferation, and a rare chromosomal translocation juxtaposing c-myc and immunoglobulin gene loci in a single B cell; holoendemic malarial infection in some way facilitates the oncogenic process. Outgrowth of the EB virus-positive tumour suggests either breakdown or evasion of those immune controls, in particular cytotoxic T-cell responses against the virus-induced lymphocyte-detected membrane antigen LYDMA, which limit virus-infected B-cell numbers in healthy virus carriers. Immunosuppression, such as that which malarial infection may induce, cannot itself be a sufficient explanation in this regard since our studies have identified a number of BL patients who retain detectable LYDMA-specific T-cell surveillance. The present work shows that in many cases of virus-associated BL, the emerging malignant clone is insensitive to such surveillance. Several EB virus-positive BL cell lines, recently established in vitro and expressing the class I histocompatibility locus antigens (HLAs) which restrict cytotoxic T-cell function, were not killed by HLA-matched LYDMA-specific effector populations in assays where the EB virus-positive lymphoblastoid cell line (LCL), derived from normal B cells of the same patient, sustained high levels of lysis. PMID- 2997616 TI - Morphological transformation in vivo of human uterine cervix with papillomavirus from condylomata acuminata. AB - Carcinoma of the human uterine cervix has been associated with several infectious agents including papillomavirus. Papillomavirus group-specific antigen (GSA) and viral particles have been demonstrated in human condylomata acuminata (CA) and flat warts of the uterine cervix. Cell alterations consisting of nuclear enlargement, hyperchromasia, irregularity, binucleation and cytoplasmic clearing (koilocytosis) are often interpreted as mild to moderate dysplasia. Present evidence that human papillomavirus (HPV) is responsible for the development of these lesions relies on the association of GSA and virus particles in the affected tissue, fulfilling the first two of Koch's postulates. Direct proof of an aetiological relationship, however, requires induction of the CA change in normal, human uterine cervix after exposure to papillomavirus. Infecting human subjects with HPV is ethically unacceptable and no satisfactory alternative systems have been defined. Also, human cell cultures do not support growth or transformation by HPV. Here we report the first demonstration of the morphological transformation of human tissues with a human papillomavirus under controlled, experimental conditions. 'Transformation' is used here in its literal sense to refer to a heritable morphological alteration in the appearance of the cells. The use of this term does not indicate that the changes described are neoplastic, but they are identical to the dysplastic changes found in biopsies of uterine cervical CA. Our results demonstrate the direct involvement of CA virus in dysplastic change of human cervical tissue and indicate that the experimental system described may be useful in elucidating the contribution of human papillomaviruses to the pathogenesis of human cervical cancer. PMID- 2997617 TI - Porin channel triplets merge into single outlets in Escherichia coli outer membranes. AB - Previous observations on the structural and functional properties of porin, the matrix protein of Escherichia coli, have indicated that the channel-forming trimers span the outer membranes of the bacterial cell, forming a molecular sieve. By using electron microscopy and image reconstruction, we demonstrate here that three channels on the outer surface of the cell merge into a single channel at the periplasmic face. Conductance measurements using conditions under which single activated triplets could be observed led us to conclude that the three individual consecutive closing steps reflect three channels within a single trimeric unit. Statistical analysis of conductance levels revealed that the first relaxation step is distinctly smaller than the two subsequent channel closings. This functional observation can be explained if the channels of porin trimers coalesce. PMID- 2997618 TI - Has morphine a physiological function in the animal kingdom? PMID- 2997619 TI - Hypervariable telomeric sequences from the human sex chromosomes are pseudoautosomal. AB - Pairing of human X and Y chromosomes during meiosis initiates within the so called pairing region at the telomeres or the chromosome short arms. Using DNA from the Y chromosome we found sequence homology in the pairing region of the human X and Y chromosomes. This DNA is telomeric, contains repetitive sequences and is highly polymorphic in the population. The polymorphism has allowed family studies which show the sequences are not inherited as though linked to the sex chromosomes. This 'pseudoautosomal' pattern of inheritance points to an obligate recombination in the pairing region of the sex chromosomes during male meiosis. PMID- 2997620 TI - Pseudoautosomal DNA sequences in the pairing region of the human sex chromosomes. AB - A DNA probe from a human Y chromosome-derived cosmid detects a single-copy genomic DNA fragment which can appear in different allelic forms shared by both sex chromosomes. Variants at this DNA locus show an autosomal pattern of inheritance, undergo recombination with sexual phenotype and can therefore be described as 'pseudoautosomal'. Another probe from the same cosmid detects a sequence repeated 15-20 times per haploid genome. These repeats also appear pseudoautosomal and map exclusively to the short-arm terminal region of each sex chromosome. PMID- 2997621 TI - An inherited limb deformity created by insertional mutagenesis in a transgenic mouse. AB - We have created an insertional mutation that leads to a severe defect in the pattern of limb formation in the developing mouse. The novel recessive mutation is phenotypically identical and non-complementary to two previously encountered limb deformity mutations, and is closely linked to a dominant mutation that gives rise to a related limb dysmorphism. The inserted element thus provides a molecular genetic link with the control of pattern formation in the mammalian embryo. PMID- 2997622 TI - L-myc, a new myc-related gene amplified and expressed in human small cell lung cancer. AB - Altered structure and regulation of the c-myc proto-oncogene have been associated with a variety of human tumours and derivative cell lines, including Burkitt's lymphoma, promyelocytic leukaemia and small cell lung cancer (SCLC). The N-myc gene, first detected by its homology to the second exon of the c-myc gene, is amplified and/or expressed in tumours or cell lines derived from neuroblastoma, retinoblastoma and SCLC. Here we describe a third myc-related gene (L-myc) cloned from SCLC DNA with homology to a small region of both the c-myc and N-myc genes. Human genomic DNA shows an EcoRI restriction fragment length polymorphism (RFLP) of L-myc defined by two alleles (10.0- and 6.6-kilobase (kb) EcoRI fragments), neither associated disproportionately with SCLC. Mouse and hamster DNAs exhibit a 12-kb EcoRI L-myc homologue, which indicates conservation of the gene in mammals. Gene mapping studies assign L-myc to human chromosome region 1p32, a location distinct from that of either c-myc or N-myc but associated with cytogenetic abnormalities in certain human tumours. This L-myc sequence is amplified 10-20 fold in four SCLC cell line DNAs and in one SCLC tumour specimen taken directly from a patient. Either the 10.0- or 6.6-kb allele can be amplified and in heterozygotes only one of the two alleles was amplified in any SCLC genome. SCLC cell lines with amplified L-myc sequences express L-myc-derived transcripts not seen in SCLC with amplified c-myc or N-myc genes. In addition, some SCLCs without amplification also express L-myc-related transcripts. Together, these findings suggest an enlarging role for myc-related genes in human lung cancer and provide evidence for the concept of a myc family of proto-oncogenes. PMID- 2997623 TI - Deletion of Huntington's disease-linked G8 (D4S10) locus in Wolf-Hirschhorn syndrome. AB - Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by progressive involuntary movements and dementia. The symptoms of the disease, although devastating in severity, do not usually appear until the third to fourth decade of life. The gene defect is highly penetrant, and results in the loss of neurones in the basal ganglia, globus pallidus, and more diffusely in the cortex. A DNA marker, G8 (or D4S10), is tightly linked to Huntington's disease and this gene has been localized to chromosome 4 (ref. 3). The discovery of this linkage marker raises the possibility of developing a presymptomatic test for the disorder, and of eventually isolating the disease gene based on its map position. We have now regionally localized the DNA marker G8 to the terminal band of the short arm of the chromosome, a region representing approximately 0.5% of the total human genome. The assignment was made by examining DNA from patients with Wolf-Hirschhorn syndrome, a birth defect resulting from partial heterozygous deletion of the short arm of chromosome 4. PMID- 2997624 TI - Fission yeast Schizosaccharomyces pombe correctly excises a mammalian RNA transcript intervening sequence. AB - Study of heterologous gene expression in the budding yeast Saccharomyces cerevisiae has shown that this organism is incapable of correctly removing intervening sequences from transcripts of higher eukaryotic genes. This is probably due to the stringent requirement for the presence of a TACTAAC box close to the 3' end of the intervening sequence if splicing in S. cerevisiae is to occur. Comparison of the introns found in the fission yeast Schizosaccharomyces pombe has identified conserved sequences similar to those found in higher eukaryotes. Therefore, we have investigated whether Schiz. pombe is capable of accurately excising intervening sequences from the transcripts of higher eukarotic genes. We show here that both the 5' and 3' splice sites of the simian virus 40 (SV40) small-T antigen transcript are accurately utilized when cloned viral DNA is expressed in Schiz. pombe cells. These data suggest that Schiz. pombe may be a better model system than S. cerevisiae for the genetic study of RNA splicing and for expressing higher eukaryotic genes. PMID- 2997625 TI - Origin of AIDS. PMID- 2997626 TI - Multiple sclerosis and viruses. PMID- 2997627 TI - Multiple sclerosis and human T-cell lymphotropic retroviruses. AB - A combination of different types of data suggests that some multiple sclerosis patients respond immunologically to, and have cerebrospinal T cells containing, a retrovirus that is related to, but distinct from, the three types of human T-cell lymphotropic viruses. The role of this virus in multiple sclerosis is uncertain. PMID- 2997629 TI - A caffeine analogue (1,3,7-trimethyl-6-thioxo-2-oxopurine) with a negative inotropic and chronotropic effect. AB - Cardiac effects of thio-xanthine derivatives, S-caffeine and S-theophylline, were studied on isolated guinea-pig atria and on partially purified cardiac cAMP phosphodiesterase enzymes. Theophylline and caffeine were taken as reference compounds. On electrically driven left atria S-caffeine (0.01-1 mmol/l) decreased contractile tension in a concentration dependent manner. On spontaneously beating atria, the same concentrations of S-caffeine showed negative inotropic as well as negative chronotropic effects. On electrically driven left atria, S-theophylline (0.01-1 mmol/l) increased heart contractile tension but, at higher concentrations, a reversal of the stimulating effect was observed. Both S caffeine and S-theophylline inhibited bovine heart cAMP phosphodiesterase activity to a comparable extent. Their inhibitory potencies were about three and nine times higher than those of theophylline or caffeine but consistently lower than that of IBMX. The results show that the replacement of O with S in the methylxanthine molecule drastically modifies the effects induced by the drugs on cardiac function without changing those on cAMP phosphodiesterase. PMID- 2997628 TI - Xanthine derivatives as antagonists at A1 and A2 adenosine receptors. AB - A variety of alkylxanthines has been comparatively examined as antagonists of A1 adenosine receptors in rat fat cells, rat and bovine cerebral cortex and of A2 adenosine receptors in human platelets. With few exceptions all xanthine derivatives with 7-position substituents such as diprophylline, proxyfylline, pentoxifylline and etofylline were less potent antagonists than xanthine itself which had Ki-values of 170 mumol/l (A1) and 93 mumol/l (A2). Theophylline, caffeine and 3-isobutyl-1-methylxanthine were more potent than xanthine but nearly equipotent antagonists at both receptor subtypes. 8-Phenyl substituents considerably increased the antagonist potency at A1 and A2 receptors. 1,3-Diethyl 8-phenylxanthine was the most potent A2 antagonist (Ki 0.2 mumol/l) in human platelets. At A1 receptors 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX) was the most potent antagonist in all three tissues with Ki-values from 0.3 to 8.6 nmol/l. Several 8-phenylxanthine derivatives were remarkably selective antagonists at A1 receptors. 8-Phenyltheophylline was approximately 700 times more potent as antagonist at A1 receptors (bovine brain) than at A2 receptors (human platelets), and PACPX was even 1,600 times more potent as A1 adenosine receptor antagonist. These compounds offer a possibility for a subtype-selective blockade of adenosine receptors. PMID- 2997630 TI - Involvement of cAMP in modulation of noradrenaline release in the human pulmonary artery. AB - After incubation with 3H-noradrenaline, strips of human pulmonary arteries from patients undergoing surgery for lung tumour were superfused with physiological salt solution containing cocaine and corticosterone. Forskolin, AH 21-132 (a cAMP phosphodiesterase inhibitor), 8-Br-cAMP and isoprenaline did not affect the basal tritium efflux from the strips, but produced a concentration-dependent facilitation of the tritium overflow evoked by transmural electrical stimulation (2 Hz). The facilitatory effect of isoprenaline was potentiated by forskolin which produced a shift to the left of the concentration-response curve of isoprenaline. It is concluded that cAMP plays a role in the modulation of noradrenaline release in the human pulmonary artery and that presynaptic beta adrenoceptors appear to be coupled to an adenylate cyclase in the sympathetic nerve terminals. PMID- 2997631 TI - Pharmacological profile of the imidazopyridine zolpidem at benzodiazepine receptors and electrocorticogram in rats. AB - Zolpidem is a novel non-benzodiazepine related hypnotic, which possesses an imidazopyridine structure. This drug has preferential affinity for the 3H diazepam binding site in the rat cerebellum, while it is only weakly active at inhibiting 3H-Ro 5-4864 binding to the rat kidney. The potency of zolpidem at displacing 3H-Ro 15-1788 binding to rat cerebral cortex membranes is enhanced in the presence of GABA. On the sleep pattern of the electrocorticogram in the curarised rat, zolpidem induces a physiological type of slow wave sleep with rapid onset of action. Zolpidem differs from classical benzodiazepine drugs, in possessing an atypical binding profile to 3H-benzodiazepine receptors, and because it does not affect the sleep patterns. PMID- 2997632 TI - [A special, nonidiopathic form of lymphedema]. PMID- 2997633 TI - [Galactosemia; variability in clinical aspects and problems in the diagnosis]. PMID- 2997634 TI - [Progressive multifocal leukoencephalopathy in hemophilia A. Is there a relation to AIDS?]. AB - Progressive multifocal leukencephalopathy (PML) due to Papovavirus is usually combined with a cellular immunodeficiency as a consequence either of a neoproliferative disease or of medical treatment. In the first description of a case of PML in combination with substituted hemophilia A with no known cellular immunodeficiency, possible pathogenetic relations to factor VIII substitution as well as to AIDS, caused by HTLV-III virus, are discussed. PMID- 2997635 TI - Center for Environmental Health. PMID- 2997636 TI - Quantifying sensory loss in peripheral neuropathies. AB - Alterations in cutaneous sensation is the most common presentation of toxic neuropathies. The clinical assessment of cutaneous sensation can be graded only very roughly, and frequently there are wide fluctuations in findings from one examiner to another and from one examination to another in the same patients. Screening populations for sensory impairment has relied mainly upon nerve conduction studies. Limitations in this technique resulted in the development of portable instruments which can quantitate sensory thresholds. Instruments for measuring vibration, thermal and current perception thresholds will be described. These tools are able to quantitate nerve impairment at its most distal extension and also examine axon populations not reflected in nerve conduction studies. PMID- 2997637 TI - Psychophysical testing in human populations exposed to neurotoxicants. AB - Sensory organs can be studied from different points of view: biochemical, morphological, electrophysiological, clinical, and psychophysical. In this paper, the psychophysical and electrophysiological approaches are discussed and compared. Even though close parallels have been drawn between these two methodologies, discrepancies clearly indicate that they do not measure the same phenomenon. Vibration sensitivity is used to illustrate the role sensory assessment plays in neurotoxicology. Several psychophysical techniques are described to measure this sensory modality. Emphasis is placed on the need to quantitatively define stimulus parameters. The validity of vibration sensitivity as an indicator of sensory peripheral nerve dysfunction is established through a review of diseases, physical agents, pharmaceuticals, and other neurotoxicants known to alter the morphology and/or the physiology of the peripheral nervous system or sense organs. A series of factors or conditions affecting vibration detection thresholds, such as age, laterality, gender, temperature and others, are examined. Finally, a review of the psychophysical methods and procedures illustrates the advantages of the forced-choice paradigm and the tracking technique over other procedures and methods of stimulus presentation. The importance of psychophysical assessment of sensory functions in neurotoxicology is emphasized. PMID- 2997638 TI - Conduction studies in peripheral nerve. AB - Conventional nerve conduction studies assess only a small proportion of the fiber population in peripheral nerve. Nerve conduction velocity is a measure of the very fast conducting fibers and varies with temperature and age. The amplitude of the compound nerve action potential is determined by fibers 9-14 microns in diameter and is dependent on the number of active fibers. The amplitude of the normal sural nerve action potential varies by a factor of 3-4 due to the large variation in the number of large myelinated fibers (1700-3300 per nerve, 7-14 microns). Nerve conduction studies performed under adequate conditions are of value in ascertaining whether there is involvement of peripheral nerve. Due to the large variation in normal nerve these studies are, however, not well suited as screening procedures in individuals exposed to toxic substances. PMID- 2997639 TI - Peripheral neurotoxicity testing by pairs of stimuli. AB - At the present time the best electrophysiological test of peripheral nerve function for purposes of evaluating neurotoxicity in humans is the analysis of the response to pairs of stimuli. This test is a more sensitive measure of axonal conduction deficit than is the single-action potential of standard clinical technique. While not as sensitive a measure as a train of stimuli at any given frequency of stimulation, the paired stimulus technique has the following advantages. The interpretation of responses to trains of impulses can be made inaccurate by alternate blocking. Under such conditions the pairs response will already have shown impaired conduction, The method is sufficiently sensitive that the second response of the pair is decreased in normals. Thus the test will show any neurotoxic impairment which is additive to such normal physiological decrement. The method has already been reported in the literature as being sensitive to a number of different peripheral and central neuropathies in humans, including segmental demyelination and axonal degeneration. The equipment required is often already in the standard clinical facility, or can be added at reasonable cost. Stimulation with pairs is more acceptable to the subject since it is not as painful as presentation of stimulus trains. PMID- 2997640 TI - Inhibition of polyphosphoinositide phosphodiesterase by aminoglycoside antibiotics. AB - The calcium-activated phosphodiesteratic hydrolysis of 32P-labeled phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate in prelabeled nerve ending membranes is inhibited by the aminoglycosides neomycin and gentamicin, and to a lesser extent, by streptomycin. The inhibition is overcome by increasing concentrations of Ca2+, indicating that the aminoglycosides exert their effect by displacing Ca2+ from lipid. PMID- 2997641 TI - Potentiation of Na+-dependent uptake of gamma-aminobutyric acid in mouse brain particles by buffer-mediated proton removal. AB - A number of buffers showed a remarkable facilitatory effect on the uptake of GABA into a mouse brain microsomal subfraction (P3) at 0 degrees C and pH 7.3 in the presence of 80 mM NaCl. Complete dose-response curves were obtained for 21 buffers, ranging in pKa values from 6.2 to 9.9. The data are consistent with the interpretation that the unprotonated forms of the buffers are responsible for the enhancement of GABA uptake by P3 particles and that this is a result of the removal of protons from a membrane site (or sites) in such a manner as to allow the GABA transporter to function. However, the enhancing effects of the buffers could not solely be attributable to unhindered interaction of protonated membrane sites with unprotonated forms of the buffer. Additional factors related to structures of the buffers and membrane properties which might be importantly operative in the enhancement are discussed. PMID- 2997642 TI - An opiate receptor-associated aminopeptidase that degrades enkephalins. AB - During the purification of opiate receptor by affinity chromatography on wheat germ agglutinin-agarose, an aminopeptidase is coeluted with the receptor. Virtually all of both the enzyme and the receptor is retained on the hydroxylapatite column. The aminopeptidase functions optimally at neutral pH and is activated by Mn2+. The enzyme is sensitive to dithiothreitol, is inhibited by amastatin and bestatin, and is insensitive to puromycin. The enzyme seems to be linked to the receptor, since its activity is enhanced by D-Ala2-Met enkephalinamide or naltrexone. The properties of this aminopeptidase indicate that it is distinct from neutral arylamidase, leucine-aminopeptidase, aminopeptidases A and B, brain acidic aminopeptidase, and the membrane aminoenkephalinase that we purified recently (4). PMID- 2997643 TI - Effects of hyperphenylalaninemia in the fetal stage on the postnatal development of fetal rat brain. AB - The effect of exposure at different prenatal stages to maternal hyperphenylalaninemia (HyPhe) on the somatic and neurological development of fetuses in rats was studied, with special respect to the change of relevant enzyme activities in the brain. While evident somatic damage was found only in the fetuses exposed to maternal HyPhe at a last stage of gestation, distinct mental retardation seemingly due to some irreversible damage to the brain was observed in all the treated fetuses regardless of the timing of exposure, and a significantly reduced activity of 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), a marker enzyme of myelin, was confirmed in the mantle region of the brain. PMID- 2997645 TI - Changes in the alpha-adrenoceptors in the medulla oblongata including nucleus tractus solitarii of spontaneously hypertensive rats. AB - The nucleus tractus solitarii (NTS) is a brain stem center mediating depression of blood pressure. In order to elucidate a possible mechanism for the central regulation of blood pressure, we studied noradrenergic indices in the medulla oblongata, a region including the NTS, in spontaneously hypertensive rats (SHR) as compared with normotensive controls of the Wistar Kyoto strain (WKY) at 12 weeks of age. The medulla oblongata was the only brain region showing a significantly low noradrenaline level in the SHR as compared with WKY rats; the level is also significantly decreased at 8 weeks of age. The alpha 1-adrenergic binding sites, as measured with 2-(2', 6'-dimethoxy) phenoxyethylamine methylbenzodioxan [3H]WB4101 showed significant increases in KD and Bmax values in medulla oblongata homogenates from rats of both strains from 4-12 weeks after birth, with no significant interstrain difference. On the other hand, the KD and Bmax of the alpha 2-sites, measured by [3H]yohimbine binding, were reduced in SHR as compared to WKY animals, even at 4 weeks after birth when hypertension was not yet apparent. As expected, the relatively selective alpha 2-antagonist, clonidine, was a potent inhibitor of [3H]yohimbine binding but not of [3H]WB4101 binding in these homogenates. The results suggest that some genetic disorder in the alpha 2-adrenergic transmission system in the NTS region may be involved in the development of hypertension in the SHR rats. PMID- 2997644 TI - Immunohistochemical localization of 2',3'-cyclic nucleotide 3'-phosphodiesterase in adult bovine cerebrum and cerebellum. AB - We have previously shown that 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP; EC 3.1.4.37) in rat central nervous tissues can be immunohistochemically stained with anti-bovine CNP serum. However, the anti-bovine CNP serum prepared in our laboratory has only weak cross-reactivity with rat CNP. Sections of bovine nervous tissues were found to be stained effectively with the serum, and the localization of CNP has been revealed in greater detail. We describe here the immunohistochemical localization of CNP in adult bovine cerebrum and cerebellum. CNP stained was localized in myelin sheaths, oligodendrocytes, and the processes of oligodendrocytes; astrocytes and neurons were negative. All myelinated nerve fibers appeared to be stained with the anti-CNP serum. Perineuronal and perivascular oligodendrocytes, and oligodendrocytes extending their processes to isolated myelin fibers were stained. Interfascicular oligodendrocytes, however, did not react or reacted faintly to the anti-CNP serum; only their processes were reactive. Comparison with the stain for S-100 protein was helpful to distinguish oligodendrocytes from astrocytes particularly when both glial cells were situated together at the perineuronal and perivascular positions. PMID- 2997646 TI - Changes in the levels of neural cell specific proteins in the developing rat brain. AB - The levels of S-100 protein (S-100) and neuron-specific enolase (NSE) in the developing rat brain were determined by a sensitive enzyme immunoassay and the results were compared with those obtained by other methods. Changes with development in the levels of S-100, NSE, and 2', 3'-cyclic nucleotide 3' phosphodiesterase (CNPase), biochemical markers for astroglia, neurons and oligodendroglia respectively, were determined in various brain regions including the cerebral hemisphere (CH), brain stem (BS) and cerebellum (Ce). The peak increments of S-100, NSE, and CNPase activity were reached later than that of the brain weight in all of the regions. The ratios of S-100/NSE and CNPase/NSE rose during the 21 days after birth in the CH and BS; the S-100/NSE ratio in the CH began to decrease from the 21st day, whereas the CNPase/NSE ratio continued to rise even after the 30th day, suggesting different maturation periods of the different glial cells. In the Ce, the change of these ratios showed a pattern different from those in the other regions. In the CH of rats with experimental microencephaly induced by methylazoxymethanol (MAM), the ratios were almost normal, in spite of the reduction of the brain weight to about 50% of the control. PMID- 2997647 TI - Stimulative effect of nerve growth factor on alpha-aminoisobutyric acid uptake and Na,K-ATPase activity in superior cervical sympathetic ganglia excised from adult rats. AB - Effects of nerve growth factor (NGF) on the uptake of non-metabolizable alpha aminoisobutyric acid (AIB) and on Na,K-ATPase activity in superior cervical sympathetic ganglia (SCG) excised from adult rats were examined during aerobic incubation in vitro. Active uptake of labelled AIB into isolated SCG during 1 to 5 hours incubation at 37 degrees C was significantly accelerated by the addition of NGF to the incubation medium in a dose-dependent manner. Although the Km value of the AIB uptake by the SCG did not change with the addition of NGF, Vmax was nearly doubled. The NGF-evoked increase in AIB uptake was antagonized by the further addition of its specific antiserum in a dose-dependent fashion, and was largely suppressed in a medium containing ouabain. In SCG, axotomized one week prior to the examination, from which most of the neurons had disappeared and reactive proliferation of satellite glial components was in progress, the NGF induced acceleration of AIB uptake was completely absent. The ganglionic Na,K ATPase activity was greatly stimulated in the presence of NGF, and the effect was completely eliminated in the axotomized SCG. These results strongly suggest that the NGF-induced acceleration of active AIB uptake by the isolated SCG occurs not in glial cells but exclusively in the neuronal components with the apparent coupling of an Na ion extrusion process. PMID- 2997649 TI - Adenoid cystic carcinoma presenting as intracranial tumour. AB - Two women are described in whom adenoid cystic carcinoma of upper respiratory tract origin presented as a large intracranial tumour with involvement of cranial nerves. Smears of the neurosurgical biopsies were distinguished by abundant metachromatic mucin. PMID- 2997648 TI - The stokes radius of the CHAPS-solubilized benzodiazepine receptor complex. AB - The Stokes radii and the apparent molecular weights of the CHAPS or Triton X-100 solubilized benzodiazepine receptors are calculated by gel exclusion chromatography. The results suggest that the molecular receptor complex solubilized by CHAPS is much larger than the complex solubilized by Triton X-100. PMID- 2997650 TI - Serotonergic stimulation of prolactin and corticosterone secretion is mediated by different pathways from the mediobasal hypothalamus. AB - In previous studies we obtained evidence that serotonin release by p chloroamphetamine (PCA) causes an increase in corticosterone secretion but that this effect is not mediated via the raphe nuclei in the midbrain. In contrast, PCA-induced prolactin secretion was abolished by dorsal raphe lesions. In the present study, posterolateral cuts which interrupted caudal inputs to the hypothalamus attenuated the effect of PCA on plasma prolactin but did not block the PCA-induced increase in plasma corticosterone levels. Large lesions of the mediobasal hypothalamus produced a significant reduction of plasma corticosterone concentration but did not completely prevent the effect of PCA on corticosterone secretion. Hypophysectomy performed 24 h before sacrifice caused a marked decrease in plasma corticosterone levels but did not completely abolish the effect of PCA. These results suggest that PCA also stimulates corticosterone secretion via a direct action on the adrenal gland. The lesions in the mediobasal hypothalamus caused an increase in plasma prolactin concentration, and in these rats, PCA suppressed rather than stimulated prolactin secretion. This suggests that the known weak dopamine agonist activity of PCA is exposed when the effects of serotonin release in the brain are eliminated. PMID- 2997651 TI - Distribution of cystatin C (gamma-trace), an inhibitor of lysosomal cysteine proteinases, in the anterior lobe of simian and human pituitary glands. AB - Cystatin C, a protein inhibitor of lysosomal cysteine proteinases, was demonstrated by immunohistochemical techniques to be present in most luteinizing hormone- (LH-)containing cells in simian and human adenohypophyses. Immunoreactivity of cystatin C was also found in simian adrenocorticotrophic hormone- (ACTH-)containing cells localized to an area corresponding to the pars intermedia but not in the ACTH-containing cells of the anterior pituitary lobe of monkey. No immunoreactivity of cystatin C was detected in the growth hormone- (GH ) and prolactin-containing cells of monkey and man. PMID- 2997653 TI - Effects of tifluadom on food consumption compared with chlordiazepoxide and kappa agonists in the rat. AB - Tifluadom (0.625-10.0 mg kg-1) was administered to non-deprived male rats which had been accustomed to eating a highly palatable diet in a 30 min test period. This compound, an opioid benzodiazepine, produced a significant increase in consumption of food when administered by the subcutaneous route, but not after intraperitoneal injection. Both chlordiazepoxide (1.25-20.0 mg kg-1) and the selective kappa opiate receptor agonist U-50,488 (0.3125-2.5 mg kg-1) also produced significant hyperphagic effects in the same feeding situation. In contrast, the two kappa opiate receptor agonists, ethylketocyclazocine (0.1-3.0 mg kg-1) and bremazocine (0.078-1.25 mg kg-1) brought about a dose-related suppression of food intake. Hence, the effects of kappa opiate receptor agonists in the feeding situation described here were not uniform. Furthermore, tifluadom could be likened either to a benzodiazepine or to a selective kappa receptor agonist. The hyperphagia induced by tifluadom was antagonized by naloxone, suggesting that the effect was mediated by an action at opiate receptors. It was not antagonized however by Ro15-1788 (10.0 and 20.0 mg kg-1), a selective benzodiazepine receptor antagonist, ruling out possible mediation by benzodiazepine receptors. The benzodiazepine receptor antagonist, CGS 8216, exhibited intrinsic activity when administered alone, and significantly reduced food consumption in tifluadom-treated and control animals. PMID- 2997654 TI - Are delta-opioid receptors involved in the regulation of food and water intake? AB - The effect of the delta-opioid antagonists ICI 154,129 (1-100 micrograms, i.c.v.) and ICI 174,864 (1-100 micrograms, i.c.v.) and of the delta-agonist D-Ala2-D-Leu5 enkephalin (DADL; 1-10 micrograms, i.c.v.) on the intake of food and water of non deprived rats was investigated. Animals treated with either ICI 154,129 or ICI 174,864 ate and drank significantly less than vehicle-treated controls over a 3 hr test period. The suppressant effects of these peptides on appetite were similar to those observed with the more commonly-used opioid antagonists, naltrexone and Mr 2266. In contrast, the delta-agonist DADL produced an increase in both the consumption of food and water in the 3 hr following administration of drug. The findings presented in this study lend further support to the hypothesis that an endogenous enkephalin/delta-receptor system may play a role in the tonic induction of ingestive behaviour. PMID- 2997652 TI - Luteinizing hormone and prolactin release by cultured pituitary cells and by gonadotrope- and lactotrope-enriched populations in the presence of modulators of adenylate cyclase. AB - The effects of 3 compounds previously known to alter the adenylate cyclase activity in various tissues were tested on rat pituitary. Forskolin increased while RMI 12,330 A and SQ 22,536 decreased enzyme activity of pituitary homogenates. Forskolin (10(-5) M) promoted in cultured cells a massive synthesis of cyclic adenosine monophosphate (cAMP) and intense output of the nucleotide in the medium; at the same concentration, RMI 12,330 A decreased the cell content of cAMP while release was unaffected, and SQ 22,536 exerted no effect on the cyclic nucleotide amount of both cell and medium. The basal release of plated total cells or enriched gonadotropes was slightly stimulated by forskolin (10(-7) - 10( 5) M) though the level of stimulation never attained the one produced by 10(-7) M luteinizing hormone-releasing hormone (LHRH); it was affected neither by RMI 12,330 A (10(-7) - 10(-5) M) nor by SQ 22,536 (10(-7) - 10(-4) M). The LHRH stimulating effect was slightly amplified during the 1st h (sensitivity response of cells) by low concentrations of forskolin (10(-7) and 10(-6) M) but the overall luteinizing hormone (LH) release after 4 h of incubation (capacity response of cells) was unmodified. Higher concentrations of the diterpene inhibited the LHRH effect as seen with total cells and with enriched gonadotropes. RMI 12,330 A at 10(-5) M significantly inhibited the LHRH-promoted LH release from total cells and from gonadotropes but a higher concentration (10( 4) M) promoted a large unspecific release that masked the LHRH effect. SQ 22,536 never modified the cell response to LHRH, whatever the drug concentration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2997656 TI - Beta-endorphin: interaction with specific nonopioid binding sites on EL4 thymoma cells. AB - Binding of 125I-labeled camel beta-endorphin (125I-beta C-endorphin) to cells of several mouse thymoma cell lines was examined and was highest to EL4 cells. 125I beta C-endorphin binding to EL4 cells was temperature-dependent; it was further characterized at 4 degrees C and exhibited saturability, complete reversibility, structural specificity and pH-dependence. 125I-beta C-endorphin binding was not inhibited by the opioid pentapeptides [Leu] enkephalin or [Met] enkephalin (which share common sequences with the N-terminus of beta C-endorphin) or by the N terminal beta C-endorphin fragments beta C-endorphin (1-16) or beta C-endorphin (1-27). In contrast, binding was inhibited by beta C-endorphin (1-31), indicating that beta C-endorphin binding to EL4 cells was with a C-terminal beta C-endorphin segment. We suggest that binding of beta-endorphin to such nonopioid binding sites may precede its apparent effects on the proliferation of T-lymphocytes (5,6). PMID- 2997655 TI - Effects of pyroglutamic acid on corticostriatal glutamatergic transmission. AB - The effects of L-pyroglutamic acid, a molecule structurally derived from L glutamic acid (Glu), were measured on the high affinity of uptake of glutamic acid from striatal synaptosomes of the rat and on the binding of [L-3H]glutamic acid to striatal membranes. The results showed a competitive inhibition of the high affinity transport of glutamic acid by L-pyroglutamic acid in vitro with no effect on the uptake of gamma-aminobutyric acid (GABA). An inhibition of the binding of [L-3H]glutamic acid to striatal membranes was also detected. Significant high affinity uptake of [L-3H]pyroglutamic acid was evident in synaptosomes from the striatum. A regional distribution study of the uptake processes for [L-3H]glutamic acid and [L-3H]pyroglutamic acid in different areas of the brain showed a similar distribution, suggesting that an uptake of [L 3H]pyroglutamic acid, although weak, occurs in glutamatergic nerve terminals. This proposal was further reinforced by measuring the effects of a large cortical lesion involving frontal and parietal areas on the uptake of [L-3H]glutamic acid and [L-3H]pyroglutamic acid in synaptosomes from the striatum. The results showed a large decrease in the uptake processes of both labelled molecules showing that the uptake of [L-3H]pyroglutamic acid, as for glutamic acid mainly occurred in corticostriatal nerve terminals, although other uptake sites are not excluded. PMID- 2997657 TI - Calcium-dependent potassium-stimulated release of neurokinin A and neurokinin B from rat brain regions in vitro. AB - The release of the tachykinins neurokinin A (NKA) and neurokinin B (NKB) from superfused slices of rat brain was studied. For radioimmunoassay of superfusates and tissue extracts an antiserum which reacts with both NKA and NKB but not with substance P was used. The released immunoreactive material, as well as the immunoreactive material in extracts of the tissue slices, was characterized by cation exchange chromatography and reversed phase high performance liquid chromatography (reversed phase-HPLC). Potassium (50 mM) evoked a calcium dependent release of tachykinin-like immunoreactivity from slices of frontal cortex and striatum. Reversed phase-HPLC accumbens, striatum and the ventral part of the mesencephalon revealed two major immunoreactive components which co-eluted with synthetic NKA and NKB. Furthermore, the superfusates also contained three minor unidentified immunoreactive components. PMID- 2997658 TI - Subarachnoid hemorrhage from a peripheral intracranial aneurysm associated with malignant glioma: report of a case. AB - The case of a patient who initially presented with a subarachnoid hemorrhage from an aneurysm of the distal left middle cerebral artery is reported. The aneurysm was later found to have occurred within a malignant glioma. Histological analysis showed tumor infiltrating the wall of the aneurysm. A causal relationship between growth of the tumor and development and rupture of the aneurysm is postulated. PMID- 2997659 TI - Pyridoxine neuropathy in rats: specific degeneration of sensory axons. AB - When rats received pyridoxine in doses large enough to cause neuropathy in humans, the animals developed gait ataxia that subsided after the toxin was withdrawn. By using quantitative histologic techniques, we found axonal degeneration of sensory system fibers and that the fibers derived from the ventral root were spared. Although the degeneration approached the dorsal root ganglion, neurons in the ganglion did not degenerate. We found no early decrease in oxygen consumption of nerve, suggesting that impaired oxidative metabolism was not the primary event. PMID- 2997660 TI - Long-lasting conduction block in hereditary neuropathy with liability to pressure palsies. AB - We detected 29 conduction blocks in 12 patients with hereditary neuropathy and liability to pressure palsies. The blocks occurred at entrapment sites, most often the ulnar nerve at the elbow. Some had lasted for several years. This kind of block seems to be characteristic of the neuropathy, and may be related to the localized thickening of myelin ("tomacula"). In one patient with two permanent and severe blocks, neurolysis was beneficial. PMID- 2997661 TI - Autoradiographic localization of thyrotropin-releasing hormone receptors in amyotrophic lateral sclerosis spinal cord. AB - We used quantitative autoradiography to determine the density of thyrotropin releasing hormone (TRH) receptors in discrete regions of spinal cord from four patients with amyotrophic lateral sclerosis (ALS). The density and distribution of [3H]-3-methyl-histidine-TRH binding to TRH receptors differed from reported values in normal individuals, with fewer TRH receptors in lamina II and lamina IX. The diminished concentration of TRH receptors in lamina IX may reflect the loss of motor neurons in ALS. PMID- 2997662 TI - [A case of hemoperitoneum caused by the spontaneous rupture of a hepatic metastasis of Wilms' tumor]. PMID- 2997663 TI - [A case of giant cutaneous fibrohistiocytoma]. PMID- 2997664 TI - [Recent pharmacotoxicologic and clinical findings on adriamycin]. AB - Anthracycline derivative adriamycin (ADR) is one of the most important anticancer drugs with major clinical application in carcinomas of the brest, endometrium, ovary, testicle, thyroid, lung and in treatment of many sarcomas. It is useful also in haematological cancers including acute leukaemia, multiple myeloma, Hodgkin's disease and the diffuse non-Hodgkin lymphomas. A factor limiting ADR clinical practice is represented by a severe dose-dependent cardiac toxicity, the mechanism of which is still under study, but appears to involve excessive intracellular production of free radicals within myocardium: this is rarely seen at ADR dosage below 500 mg/m2. A series of new anthracycline analogues has recently entered clinical trials: they include 4'-epiadriamycin, 4'-deoxy adriamycin, aclacynomycin A, carminomycin and N-trifluoroacetyladriamycin-14 valerate. These new agents appear to have different spectrum of action and somewhat less toxicity, however antitumor activity in only now being defined, consequently major clinical interest is still concentrated in the use of ADR. The present article reviews the most relevant data from literature concerning the pharmacology, the toxicology and the clinical use of ADR. PMID- 2997665 TI - [The inoperable non-microcytoma lung cancer. Results of chemotherapeutic and chemoradiotherapeutic treatment]. AB - The cases of 36 patients with inoperable non-small cell lung cancers and similar anatomoclinical features were retrospectively analysed on the basis of treatment received (21 combined chemical and radiation therapy, 15 chemotherapy). The results showed 7 PR (partial response) 5 S (stable) 9 P (progression) in the group given combined chemical and radiation treatment; 2 PR, 7 S and 6 P in the group given chemotherapy alone. The patients with the best Performance Status produced the best PR figures (6/9) and the longest mean survival (10 months). Analysis of 9 patients in each group indicated that the length of survival is not affected by the timing or doses of drug treatment. Altogether the data support the view that no treatment has any influence on the prognosis for inoperable lung cancers. PMID- 2997666 TI - Regional differences in mu 1-binding of [3H][D-Ala2,D-Leu5]-enkephalin: comparisons of thalamus and cortex in the rat. AB - Typically, mu 1-sites represent approximately 25-35% of binding in rat brain homogenates. Competition studies indicated that approximately 60% of [3H][D Ala2,D-Leu5]-enkephalin ([3H]DADLE) binding in the thalamus was inhibited by low concentrations of morphine (2-5 nM). This high proportion of mu 1-binding was anticipated based upon the low levels of delta-sites and the high levels of mu 1 sites in this region observed in autoradiography studies. In contrast, morphine lowered [3H]DADLE binding by only approximately 5-15% in the cortex, a region known to possess large amounts of delta- and few mu 1-receptors. These results support previous autoradiography studies and illustrate the advantages of using tissue regions in the characterization of opiate receptor subtypes. PMID- 2997667 TI - Glutamic acid decarboxylase-like immunoreactive neurites in senile plaques. AB - In the neocortex of an aged (26-year-old) rhesus monkey, a small percentage of abnormal neurites within some senile plaques (defined by the presence of amyloid) were immunoreactive for glutamic acid decarboxylase. This suggests that gamma aminobutyric acid-synthesizing neurons may contribute to plaque formation in the aged brain. PMID- 2997668 TI - Reserpine-induced potentiation of the inhibitory action of neuropeptide Y on the rat vas deferens neurotransmission. AB - The inhibitory action of neuropeptide Y (NPY) on the muscular activity of the prostatic end of the rat vas deferens elicited by transmural electrical stimulation was examined in control and in reserpinized rats. Pretreatment with 1 mg/kg reserpine for 48 h induced a 6-fold increase in NPY potency. Likewise, the potency of clonidine to inhibit the electrically induced muscular activity or noradrenaline to contract the ductus musculature was also potentiated. It is hypothesized that reserpine via a denervation super-sensitivity-like process increases the density of the NPY receptors. The functional significance of NPY in the motor activity of the vas deferens is discussed. PMID- 2997669 TI - The effect of opioids and of naloxone on Na+,K+-adenosine triphosphatase activity in frog spinal cord membrane fractions. AB - The effects of opioids and of naloxone on ouabain-sensitive Na+,K+-adenosine triphosphatase (ATPase) activity were studied in vitro on membrane fractions from frog spinal cords. The addition of morphine and of the stable enkephalin analogue, D-Ala2,D-Leu5-enkephalin, in concentrations from 10(-7) to 10(-4) M significantly increased Na+,K+-ATPase activity. No effect was found with methionine enkephalin (Met-Enk). However, the addition of two peptidase inhibitors, captopril and phosphoramidon (10(-5) M each), significantly increased Na+,K+-ATPase activity. A further increase in enzyme activity was found when Met Enk (10(-4) or 10(-7) M) was added simultaneously with peptidase inhibitors. On the other hand, the addition of the opiate antagonist, naloxone, at low concentration (10(-7) M) decreased the activity of Na+,K+-ATPase. These results are discussed with respect to the effect of synthetic and endogenous opioids on the activity of Na+,K+-ATPase. PMID- 2997670 TI - Evidence for the involvement of kainate receptors in synaptic transmission in the avian cochlear nucleus. AB - Previous studies using various excitatory amino acid antagonists have shown that synaptic transmission between the auditory nerve and the cochlear nucleus of chickens (nuc. magnocellularis; NM) is mediated by non-N-methyl-D-aspartate (non NMDA) receptors. In the present study we have attempted to define the subclass of non-NMDA receptor in the NM by examining the effects of various excitatory amino acid agonists on synaptically evoked field potentials in an in vitro preparation of the chicken brain stem. Both quisqualate and DL-alpha-amino-3-hydroxy-5-methyl 4-isoxazolepropionic acid (AMPA), whose actions operationally define the quisqualate receptor class, caused variable and weak depression of evoked responses in the NM, as did L-glutamate. Kainic acid, on the other hand, completely blocked postsynaptic responses at micromolar concentrations. We conclude that kainate-preferring non-NMDA receptors play a predominant role in mediating transmission in the NM. PMID- 2997671 TI - Insect central nervous system gamma-aminobutyric acid. AB - Specific saturable binding of radiolabelled gamma-aminobutyric acid (GABA) has been demonstrated in central nervous system (CNS) extracts of the cockroach, Periplaneta americana. The pharmacological properties of these putative insect CNS GABA receptors differ from those of both the vertebrate GABAA and GABAB receptor sites. Autoradiographical techniques have been used to examine the localization of these [3H]GABA binding sites in a cockroach ganglion and have shown that they are found predominantly in the neuropile, the region where the majority of synaptic connections occur. The results suggest that these insect CNS [3H]GABA binding sites may represent a novel class of GABA receptors. PMID- 2997672 TI - Intracellular demonstration of an N-methyl-D-aspartate receptor mediated component of synaptic transmission in the rat hippocampus. AB - Rat hippocampal CA1 pyramidal neurones were monosynaptically activated via stimulation of the Schaffer collateral-commissural pathway. On changing from a 1 mM Mg2+-containing to a Mg2+-free medium there was a pronounced prolongation of the intracellularly recorded excitatory postsynaptic potential. This effect was reversibly abolished by the selective N-methyl-D-aspartate (NMDA) antagonist, D-2 amino-5-phosphonovalerate (APV). We propose that Mg2+ normally prevents expression of NMDA receptor-mediated responses during low-frequency stimulation. During a period of tetanic stimulation, however, cells may depolarize sufficiently to allow a significant NMDA component of the response to be manifest. This could then initiate long-term potentiation. PMID- 2997673 TI - Captopril and teprotide as discriminators of angiotensin-converting enzyme activity in brain tissue. AB - Titrations of angiotensin-converting enzyme (ACE; E.C. 3.4.15.1) present in human serum, as well as in homogenates prepared from post-mortem human caudate or mouse (C57BL1/6J) whole brain tissue, were performed with the selective ACE inhibitors, captopril (SQ 14225) and teprotide (SQ 20881). ACE activity present in human serum was more sensitive to inhibition by either inhibitor than the activity present in the brain homogenates. The inhibition curves for the titration of the human serum activity by both inhibitors were sigmoidal while the inhibition curves for the ACE activity present in the brain homogenates were more complex. These results suggest that the brain homogenates contained: at least two species of enzyme activity with properties similar to ACE but with differing affinities for the inhibitors, or substances without ACE activity that are capable of competing with ACE for the binding of the inhibitors. Therefore, measurements of captopril or teprotide-sensitive peptidase activity as well as inhibitor-binding activity may not always reflect ACE concentrations in brain tissue. PMID- 2997674 TI - Serotonin modulates photoresponses in Hermissenda type-B photoreceptors. AB - Light responses of Hermissenda type-B photoreceptors evoked by flashes of light were enhanced and prolonged by bath application of serotonin (5-HT). 5-HT appears to act directly on the B photoreceptors and not indirectly through 5-HT-sensitive interneurons since similar effects were observed in the presence of tetrodotoxin and in axotomized preparations which eliminated synaptic input to the B photoreceptors. We also examined the effect of 5-HT on light-evoked membrane currents with voltage-clamp techniques. The increase in the duration of the light response in 5-HT can be explained by the prolonged decay of the light-activated current evoked by constant-intensity test flashes. 5-HT appears to prolong the light response by slowing a late process in the decay of light-activated membrane current. In contrast to the effects of 5-HT an alpha 2-agonist, clonidine, enhanced the peak photoresponse but did not prolong the light response. Serotoninergic modulation of light-dependent processes in the B photoreceptors may contribute to the development of long-term changes produced by conditioning. PMID- 2997676 TI - Norepinephrine depolarizes lateral horn cells of neonatal rat spinal cord in vitro. AB - Superfusion of norepinephrine (NE) (1-50 microM) onto lateral horn cells, including antidromically identified sympathetic preganglionic neurons (SPNs), situated in thin transverse neonatal rat thoracolumbar spinal cord slices caused a membrane depolarization and repetitive cell discharges. The NE depolarization was associated with an increase in membrane resistance, and the response became smaller upon conditioning hyperpolarization; a clear reversal of polarity, however, was not observed. Pretreating the slices with phentolamine and prazosin but not yohimbine or propranolol prevented the depolarizing effect of NE. This finding, in conjunction with the evidence of the presence of noradrenergic fibers in the spinal cord, suggests that NE may serve as an excitatory neurotransmitter to neurons of the lateral horn. PMID- 2997675 TI - Studies on the depression of gamma-aminobutyric acid potentials by phloridzin in cat dorsal root ganglion cells in vitro. AB - Phloridzin has been used as a tool to investigate membrane transport mechanisms. We have demonstrated that phloridzin acts to depress chloride flux resulting from activation of a gamma-aminobutyric acidA (GABAA) receptor on cat dorsal root ganglion cells. This action appears not to involve an inwardly directed chloride pump postulated for these neurons but rather affects a site associated with the GABA receptor-chloride complex. PMID- 2997678 TI - AIDS 1985. PMID- 2997679 TI - False positive anti-HTLV III serology. PMID- 2997677 TI - Antagonism of L-5-hydroxytryptophan-induced head twitching in rats by lisuride: a mixed 5-hydroxytryptamine agonist-antagonist? AB - Lisuride antagonized L-5-hydroxytryptophan (5-HTP)-induced head twitches at doses lower than those sufficient to induce the serotonin (5-HT) syndrome. Among several other 5-HT agonists tested, only LSD and 1-(m-trifluoromethylphenyl) piperazine (TFMPP) shared this paradoxical profile. Assessment of various dopamine (DA) agonists revealed a lack of correlation between DA-mediated stereotyped behavior (indicative of postsynaptic DA agonism) and blockade of 5 HTP-induced head twitches. Lisuride displaced specific ligand binding from putative S1a, S1b and S2 receptors at nanomolar concentrations, and other drugs that blocked 5-HTP-induced head twitches also displaced binding at S2 sites. It is proposed that lisuride may have agonist properties at S1a receptors mediating the 5-HT syndrome but antagonist properties at S2 receptors mediating 5-HTP induced head twitching. PMID- 2997680 TI - Short-term effects of tamoxifen, medroxyprogesterone acetate, and their combination on receptor kinetics and 17 beta-hydroxysteroid dehydrogenase in human endometrium. AB - The interactions of an antiestrogen (tamoxifen) and a progestin (medroxyprogesterone acetate) on endometrial 17 beta-hydroxysteroid dehydrogenase activities were studied in short-term experiments (four to 96 hours) in normally menstruating women at the follicular phase and were related to simultaneously measured concentrations of cytosol and nuclear estrogen and progestin receptors. Tamoxifen effected a decrease in the activity of 17 beta-hydroxysteroid dehydrogenase. This was associated with an apparent translocation to and retention of estrogen receptor in the nucleus without any significant changes in cellular progestin receptor. Medroxyprogesterone acetate administration led to a rapid increase in endometrial 17 beta-hydroxysteroid dehydrogenase activity, and depletion of cytosol and total cellular progestin receptor. Combination of the drugs led to effects that could be addressed to the individual drugs separately, and under the experimental conditions the effects of medroxyprogesterone acetate were uninfluenced by simultaneous tamoxifen administration. Put together with the authors' previous findings on the same parameters during long-term (three-week) medroxyprogesterone acetate administration, it seems possible that potentiation of progestin effects on endometrial carcinoma is not to be expected during long term simultaneous antiestrogen-progestin treatment. It is therefore likely that the favorable effects of combining these two drugs in long-term treatment are due to their different endocrine action mechanisms. PMID- 2997681 TI - Identification of human papilloma virus in cervical swabs. PMID- 2997682 TI - [Lattice degeneration of the peripheral retina: ultrastructural study]. AB - The ultrastructural study of a case of snail track degeneration shows the presence of lipid inclusions in both the glial and the macrophage cells in every layer of the retina, and the existence of intraretinal fibers different from collagen fibers appearing to be glial filaments similar to those found in astrocytic gliomes and to the Rosenthal fibers observed in senile nervous cells. Other features were thinning of the retina and absence of blood vessels in the retina. There are no abnormalities of the vitreo-retinal juncture. All the lesions are in agreement with those observed by Daicker [Ophthalmologica, Basel 165: 360-365, 1972; Klin. Mbl. Augenheilk. 172: 581-583, 1978] with some differences, however. They are different from those found in lattice degeneration. They show that snail track degeneration is a specific form of peripheral retinal degeneration which is quite different from lattice degeneration and must not be considered similar. PMID- 2997683 TI - Rapid diagnosis of herpetic infections by immunoperoxidase method. AB - The PAP-(peroxidase-antiperoxidase)-immunohistochemical method was tested for rapid diagnosis on corneal specimens in clinically suspected herpetic disease. The method proved accurate, easy and useful in rapid diagnosis of corneal specimens from epithelial keratitis. Corneal buttons after keratoplasties for herpetic lesions were similarly tested for the presence of herpetic antigen. In a total of five corneal buttons, antigen was found in two corneas with necrotizing keratitis, whereas it was not noted in the three corneas opacified by disciform edema. PMID- 2997685 TI - Absolute indications for salivary gland scintigraphy with 99mTc-pertechnetate. AB - In recent years salivary gland scintigraphy has gained widespread acceptance as a useful means for evaluating salivary gland disorders. An absolute indication for this procedure exists when the ductal orifice of one or several major salivary glands cannot be found or cannot be cannulated. Clinical conditions in which this problem occurs include technical failure to probe and cannulate the duct, developmental anomalies, obstructive disorders, traumatic lesions and fistulae and the need of postsurgical information after glandular excision or after ligation or repositioning of a major excretory duct. The clinical value of scintigraphy in these conditions is demonstrated by means of case presentations. PMID- 2997684 TI - Cytomegalovirus presence and salivary composition in acquired immunodeficiency syndrome. AB - Parotid and whole saliva was collected from nine patients with acquired immunodeficiency syndrome (AIDS) and nine controls. Cytomegalovirus (CMV) was cultured from both salivary samples in six of the AIDS patients but was not present in any of the controls. In the AIDS samples parotid sodium (p less than 0.05), IgG (p less than 0.01), and albumin (p less than 0.05) were higher than in control samples. Parotid potassium (p less than 0.05) and total protein (p less than 0.05) were lower than control values, whereas flow rate, lactoferrin, lysozyme, IgA, and IgM levels were similar in both sets of samples. AIDS does not appear to affect secretory IgA levels. Sodium (p less than 0.01) and IgA (p less than 0.05) were higher in the whole saliva of AIDS patients. Serum IgG, IgM (p less than 0.01), and IgA (p less than 0.05) were also elevated when compared with the controls. The prevalence of CMV in parotid and whole saliva of AIDS patients is consistent with the known susceptibility of this group to adventitious infection and the predilection of this virus for the salivary glands. The changes in salivary composition suggest a low level of inflammation, which occurs independently of the virus. PMID- 2997686 TI - The sagittal split osteotomy of the mandible. AB - Modifications of the sagittal split osteotomy of the mandible have essentially reduced the major drawbacks of the procedure, such as condyle displacement, short term skeletal relapse, and protracted maxillomandibular fixation and mental nerve dysesthesia. These techniques have proved effective over a period of 4 years in fifty-seven patients treated. PMID- 2997687 TI - [Active immunization of children exposed to varicella infection in hospitals, using subcutaneous and intradermal attenuated live vaccine]. PMID- 2997688 TI - [Neurologic, electrophysiologic and angiologic examination of patients with vibration disease]. PMID- 2997689 TI - [Complications of infectious mononucleosis in childhood]. PMID- 2997690 TI - The analytical sensitivity of Tc99m radionuclide 'milk' scanning in the detection of gastro-oesophageal reflux. AB - The analytical sensitivity of radionuclide 'milk' scans for detecting gastro oesophageal reflux (GOR) has been assessed using an in vitro simulation test. Five factors were found to affect the ability to detect simulated reflux: isotope concentration, absolute gamma camera sensitivity, absorber thickness overlying the 'oesophagus' and volume and duration of reflux. We found that a critical volume-duration product must be exceeded for reflux to be detected. Radionuclide milk scanning appears to be much less sensitive in detecting transient events like GOR than might be expected from previously reported static simulation studies. PMID- 2997691 TI - Glucagon receptors along the nephron: [125I]glucagon binding in rat tubules. AB - Binding of [125I]glucagon was measured in microdissected pieces of tubules from the rat nephron. Specific glucagon binding sites were found only in nephron segments containing a glucagon-sensitive adenylate cyclase activity. At 7.5 nM labelled hormone, higher levels of specific binding (16-27 X 10(-18) mol mm-1) were found in the thick ascending limb of the Henle's loop and in the distal convoluted tubule and lower binding levels (2-5 X 10(-18) mol mm-1) in the collecting tubule whereas specific binding could not be detected in the proximal tubule and in the thin segments of the Henle's loop. In the medullary thick ascending limb, Scatchard analysis of specific [125I]glucagon binding indicated an apparent equilibrium dissociation constant of 2.4 nM. The stereospecificity of binding sites in medullary thick ascending limbs and medullary collecting tubules, was assessed by competition experiments using unlabelled glucagon, enteroglucagon and unrelated hormones (vasopressin, calcitonin, parathyroid hormone and insulin); in both segments, glucagon was more active than enteroglucagon in displacing labelled glucagon from its tubular binding sites, whereas all other hormones tested were inactive. These results indicate that tubule binding sites might be the physiological receptors for glucagon involved in adenylate cyclase activation. PMID- 2997692 TI - Transmission phenomena and early tension recovery in skinned muscle fibres of the frog. AB - Tension responses due to small rapid length changes completed in 50 microseconds were obtained from segments with different length of single fibres of the ileofibularis muscle of the frog. The very early parts of the responses varied with segment length. A simulation of the early parts of the response was carried out by means of a linear model in which the fibre is regarded as a rod of infinitesimally small segments containing undamped elasticity, damped elasticity and mass in series. In the simulation corrections were included for the effects caused by the viscosity and density of the surrounding fluid and for the force transducer characteristics. The results indicate the presence of a very rapid component in the fast recovery with a time constant of 5-15 microseconds. The undamped elasticity of the activated fibres corrected for their passive properties was such that a sudden shortening corresponding to 2.6 nm/half sarcomere would reduce active tension to zero. PMID- 2997693 TI - Tension responses to rapid length changes in skinned muscle fibres of the frog. AB - Tension responses due to rapid length changes completed in 50 and 150 microseconds were obtained from activated skinned single fibres of the ileofibularis muscle of the frog. The natural frequency of the force transducer was about 50 kHz. The length changes ranged between -1% and +0.5% of the fibre segment length. The sarcomere length was adjusted to 2.15 micron. The temperature was maintained at 2.5 degrees C. The transmission velocity estimated from these recordings obtained on fibre segments with different length was 230 m/s in fully activated segments and 112 m/s in relaxed segments. The initial part of the responses during the length changes consisted of an abrupt change in tension reaching an extreme value T1, which depended on the amplitude as well as the duration of the length change. A partial rapid recovery towards a plateau occurred after the length change. The reciprocal half-time of this recovery increased with the amplitude of the displacement both for stretches as well as releases up to about 5 nm/half sarcomere. PMID- 2997694 TI - Sites of thyroid hormone action on Na-K-ATPase along the rabbit nephron. AB - Although Ismail-Beigi and Edelman demonstrated in 1971 that thyroid hormones control the activity of Na-K-ATPase in the mammalian kidney, the actual site of this regulation inside the organ was not located. We therefore decided to study the relationship between thyroid hormones and Na-K-ATPase activity in individual nephron segments obtained by microdissection of collagenase-treated rabbit kidneys. For this purpose, the changes in the activity and number of catalytic sites of Na-K-ATPase in response to thyroidectomy or triiodothyronine administration were examined. Eight to 12 days after thyroidectomy, Na-K-ATPase activity had dropped by 40 to 80% in the convoluted and straight portions of the proximal tubules, and in the cortical and outer medullary collecting tubules, but not in the thick ascending limbs of Henle's loops or distal convoluted tubules. The apparent number of catalytic sites for Na-K-ATPase, as measured by specific binding of 3H-ouabain, decreased in parallel with Na-K-ATPase activity, and therefore this enzyme's specific activity was not altered. Fourty eight hours after injection of thyroidectomized animals with a single dose of either 100 or 500 micrograms/kg triiodothyronine, Na-K-ATPase activity in target segments was restored to the level measured in control animals. These effects of thyroid hormone were specific for Na-K-ATPase, since the activity of adenylate cyclase, another marker of the basolateral membrane, was not altered by thyroidectomy. The results obtained indicate that triiodothyronine controls Na-K-ATPase activity in specific nephron segments, by altering the number of this enzyme's catalytic sites. PMID- 2997695 TI - Membrane potentials of individual cells of isolated gastric glands of rabbit. AB - Individual glands of rabbit gastric mucosa were prepared for measurements of cell membrane potentials. In the first experiments a collagenase isolation technique was used which produced gland fragments that were fixed on agarose. In later experiments a microdissection technique was used which allowed whole glands to be isolated that were held in suction pipettes. Individual parietal or chief cells could be recognized and impaled with microelectrodes, however, the yield of reliable recordings was small and the distinction from artifacts sometimes difficult. In acceptable recordings the membrane potentials of both cell types varied between around -20 and -35 mV or exceptionally -50 mV in both preparations, with mean values being around -26 mV. The significance of the recordings was tested by ion substitution experiments. Substitution of all chloride by sulfate increased the membrane potential to values ranging up to -60 and -80 mV that are commonly observed in other cells. PMID- 2997696 TI - Electrical properties of Madin-Darby canine kidney cells. Effects of extracellular potassium and bicarbonate. AB - To gain some insight into electrogenic transport processes across the plasma membrane of Madin-Darby canine kidney (MDCK)-cells, continuous measurements of the potential difference across the plasma measurements of the potential difference across the plasma membrane (PD) were made during step changes of extracellular ion composition as well as application of barium or valinomycin. During control conditions mimicking in vivo extracellular fluid, PD approaches 51.5 +/- 0.8 mV (n = 62). Step increase of extracellular potassium concentration from 5.4 to 10, to 20 or to 35 mmol/l, depolarizes PD by +5.5 +/- 0.8 mV (n = 7), by +15.8 +/- 0.5 mV (n = 64) and by +23.8 +/- 1.2 mV (n = 12), respectively. 1 mmol/l barium depolarizes PD by +19.8 +/- 0.6 mV (n = 38) and abolishes the effect of increasing extracellular potassium from 5.4 to 10 mmol/l but not to 35 mmol/l. Ten mumol/l valinomycin hyperpolarizes PD to -69.3 +/- 2.9 mV (n = 7). In the presence of valinomycin, increase of extracellular potassium from 5.4 to 20 mmol/l depolarizes PD by +31.0 +/- 1.0 mV (n = 7). Ouabain depolarizes PD and reduces the sensitivity of PD to extracellular potassium concentration. Omission of extracellular bicarbonate and carbon dioxide as well as increase of extracellular bicarbonate at constant carbon dioxide lead to a hyperpolarization and enhanced sensitivity of PD to extracellular potassium. In the presence of barium, the effects of omitted bicarbonate and carbon dioxide of MDCK-cells is highly conductive to potassium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2997699 TI - Biochemical messengers of platelet activation. PMID- 2997697 TI - The effect of acute saline volume expansion on renal Na-K-ATPase. AB - To examine the role of Na-K-ATPase in the natriuresis that occurs after acute extracellular volume expansion, we performed acute clearance experiments and in vitro analysis of renal microsomal ATPase activity in rats receiving intravenous 0.9% sodium chloride (0.1 ml/100 g bw/min). Despite increased absolute reabsorption of filtered sodium (196 +/- 8.1 vs. 165 +/- 11.4 uEq/min, p less than 0.05), renal medullary microsomal Na-K-ATPase activity was decreased from 134 +/- 5.9 to 110 +/- 6.3 pmol Pi/mg protein/hour (p less than 0.02). No changes occurred in cortical or papillary regions and Mg-ATPase was unaffected. Similar results were obtained after adding 4 mEq/l potassium chloride to the infusion to prevent any fall in serum K+. These data suggest that a considerable percentage of sodium reabsorption is suppressed in acutely volume expanded animals and it is proposed that this is mediated by inhibition of medullary Na-K-ATPase. PMID- 2997698 TI - Occurrence of adenoviral pneumoenteritis among sheep kept under extensive conditions in Sweden. PMID- 2997700 TI - Measurement of platelet ionized calcium. AB - We measured ionized cytoplasmic calcium in human blood platelets by two techniques: one based on the fluorescence of intracellular Quin 2 and the other based on the luminescence of the calcium sensitive photoprotein, aequorin. Platelet activation (shape change, aggregation, secretion) induced by thrombin, ADP, epinephrine, collagen, A23187, or phorbol ester was invariably preceded or accompanied by a rise in platelet free calcium indicated by aequorin, but in some cases Quin 2 failed to detect the elevation of calcium concentration. Aequorin appears to be sensitive to localized changes in cytoplasmic calcium levels, while Quin 2 reflects average or diffuse calcium values. Failure of Quin 2 to indicate a rise in free calcium levels in association with a platelet activity does not establish the process as "calcium independent". PMID- 2997701 TI - The interaction of extracellular calcium with the platelet membrane glycoprotein IIb-IIIa complex. AB - Platelet aggregation requires the presence of extracellular Ca2+ and fibrinogen. When platelets are activated, fibrinogen receptors become expressed on the cell surface. These receptors are composed of a heterodimer complex of two integral membrane glycoproteins, IIb and IIIa. The requirement for Ca2+ in platelet aggregation can be explained in part by the interaction of Ca2+ with this complex. Conceptually, this interaction can be divided into three parts. First, Ca2+ holds the membrane IIb-IIIa complex together. Half-maximal dissociation of the complex occurs at 37 degrees in the presence of 0.4 uM extracellular Ca2. Mg2+ ions are unable to serve as a substitute for Ca2+. Second, studies with a complex-specific monoclonal antibody that binds only to activated platelets indicate that Ca2+ also is required for the agonist-induced conversion of the IIb IIIa complex into a functional fibrinogen receptor. The KCa for receptor expression is also 0.4 uM, and Mg2+ is unable to substitute for the Ca2+. Third, Ca2+ is required for the actual binding of fibrinogen to its receptor. In this case, the KCa is 10-100 uM, and Mg2+ is an effective substitute for Ca2+. Thus, Ca2+ holds the IIb-IIIa complex together, is involved in fibrinogen receptor expression and supports fibrinogen binding. These multiple interactions of extracellular Ca2+ with glycoproteins IIb and IIIa help to explain the known requirement for Ca2+ in platelet aggregation. PMID- 2997702 TI - Polyphosphoinositides and cell activation. AB - This review surveys various aspects of the metabolism of inositol containing lipids and focuses upon the role of polyphosphoinositides in the triggering of physiological responses of platelets. Since the latter responses are evoked by a rise in cytosolic Ca2+ concentration the relationship between the phosphoinositide metabolism and the intracellular Ca2+ mobilization is discussed. PMID- 2997703 TI - Relative importance of diacylglycerol, phosphatidate, lysophosphatidate, inositol trisphosphate and arachidonate metabolism in platelet receptor signalling. AB - Activation of platelets is correlated with phospholipase C-induced degradation of phosphatidylinositol 4,5-bisphosphate and the rapid formation of 1,2 diacylglycerol and myo-inositol 1,4,5-trisphosphate. Both products are considered second messengers and they, respectively, stimulate protein kinase C and Ca2+ mobilization. Mobilization of Ca2+ leads to activation of a Ca2+/calmodulin dependent myosin light chain kinase and phospholipases A2 which liberate arachidonic acid from phospholipids. Arachidonate is then immediately converted to active endoperoxides and thromboxanes which are released and activate further platelets again through phospholipase C. The levels of phosphatidic acid and lysophosphatidic acid are also increased following receptor-stimulated hydrolysis of the inositol phospholipids. Lysophosphatidic acid might have a direct action on the opening of Ca2+-channels. PMID- 2997704 TI - The role of phosphorylations in the regulation of ion fluxes. AB - Ions appear as very important second messenger since steep ionic gradients are established between subcellular compartments as well as between the outside and the inside of the cell and ions are very diffusible molecules. Ions have been shown to induce many biological events as exemplified by the triggering of skeletal muscle contraction by calcium ions. There is more and more evidence that the same ions might play as tuning systems for biological functions. Therefore their concentration must be precisely controlled. This paper summarizes evidences that phosphorylation of regulatory proteins modulates the cytoplasmic level of some ions. PMID- 2997705 TI - Calcium mobilization and signal transduction in the neutrophils. AB - Neutrophil responsiveness is initiated by increases in the intracellular concentration of calcium through mechanisms the elucidation of which is of interest to the field of signal transduction in calcium mobilizing systems. Some, but not all, neutrophil chemotactic factors specifically stimulate the hydrolysis of phosphatidylinositol 4,5-bisphosphate. A non-mitochondrial pool of internal calcium has been shown to be released in permeabilized cells by inositol 1,4,5 trisphosphate. Diglyceride is thought to activate protein kinase C producing stimulatory and inhibitory signals for neutrophil activation. The lack of effect of leukotriene B4, on polyphosphoinositide hydrolysis indicates that mechanisms independent of inositol 1,4,5-trisphosphate are also available to the neutrophils. Pertussis toxin inhibits the stimulated mobilization of calcium, hydrolysis of the polyphosphoinositides and activation of protein kinase C. The inhibitory effects of pertussis toxin can be bypassed by phorbol esters and calcium ionophores thus indicating that a guanine nucleotide binding protein is functionally located at a step preceding the activation of phospholipase C. The similarities between the biochemical events activated by chemotactic factors and those described in other hormonally sensitive cells emphasize the generality of the relevance of these concepts. The differences raise the possibility that elements of the excitation-response coupling sequence other than those commonly monitored will still be identified. The later may be more evident in the neutrophils because these cells' predominant function is motility and not secretion. PMID- 2997706 TI - Endogenous and pharmacological mechanisms for the regulation of human platelet cytosolic free Ca2+. AB - Because they inhibit the processes that promote elevation of [Ca2+]i and augment the processes that promote removal of Ca2+ from the cytosol, receptor antagonists, agents that mimic or elevate cAMP, cGMP or 1,2-Diacylglycerol (DG), and both inorganic and organic Ca2+ channel blockers can be considered to act as 'Ca2+ antagonists' on human platelets. Agonist-induced elevation of [Ca2+]i is associated with phosphoinositide hydrolysis. Unlike agents that mimic or elevate cAMP, cGMP or DG, receptor antagonists and organic Ca2+ influx, mobilisation of internal Ca2+ and inositol lipid hydrolysis. Lanthanides apparently inhibit only Ca2+ influx. Thus La3+ but not Verapamil or Diltiazem block receptor-operated Ca2+ channels on human platelets. The endogenous processes that promote extrusion or sequestration of cytosolic Ca2+ may be augmented by cAMP, cGMP, DG and by Ca2+. DG, via activation of protein kinase C, may serve as a bi-directional regulator of platelet reactivity. PMID- 2997707 TI - Molecular membrane organization in normal and pathological platelets: changes in inositide metabolism and membrane fluidity. AB - The role of platelet plasma membrane in mediating cellular response to external stimuli was investigated by studying phosphatidylinositol turnover and structural physical molecular alterations. Phosphoinositide turnover was evaluated by phosphatidylinositol breakdown and phosphatidic acid (PA) synthesis. The membrane structure was investigated by measuring fatty acid chain organization, flexibility and movements using the spin label method. When platelets are stimulated by thrombin, a rapid phosphatidylinositol bisphosphate (PIP2) breakdown is observed, accompanied by an immediate PA synthesis. At the same time a membrane-bound calcium liberation within the cell is revealed by the decrease in chlortetracycline fluorescence, and the dense granule constituent serotonin is released from the cell. These events result in disorganization of the platelet membrane as estimated by the decrease in order parameter. All these thrombin induced effects occur in the presence of EDTA, thus being independent of external calcium and aggregation. By contrast ionophore A 23187 does not induce any PIP2 breakdown nor structural disorganization and the dense granule release measured at 10 seconds is only very weak. These results were confirmed by studies on pathological platelets in which PIP2 breakdown was normal when release was normal, such as in Glanzman thrombasthenia, or did not occur when no granule release was measurable as in Gray-platelet syndrome. It is suggested that membrane-bound calcium has a structural rather than a functional role. PMID- 2997708 TI - Nucleotide sequence of the BsuRI restriction-modification system. AB - The genes of the 5'-GGCC specific BsuRI restriction-modification system of Bacillus subtilis have been cloned and expressed in E. coli and their nucleotide sequence has been determined. The restriction and modification genes code for polypeptides with calculated molecular weights of 66,314 and 49,642, respectively. Both enzymes are coded by the same DNA strand. The restriction gene is upstream of the methylase gene and the coding regions are separated by 780 bp. Analysis of the RNA transcripts by S1-nuclease mapping indicates that the restriction and modification genes are transcribed from different promoters. Comparison of the amino acid sequences revealed no homology between the BsuRI restriction and modification enzymes. There are, however, regions of homology between the BsuRI methylase and two other GGCC specific modification enzymes, the BspRI and SPR methylases. PMID- 2997709 TI - Termination of a transcription unit comprising highly expressed genes in the archaebacterium Methanococcus voltae. AB - The 3'termini of transcripts originating from genes organized in a highly expressed transcription unit were analyzed in the archaebacterium Methanococcus voltae. The putative termination signals were found in an AT-rich intergenic region following the 3'-terminal gene. The two detected signals both contain oligo(T) sequences. A possible stem/loop structure immediately precedes one of the oligo(T) tracts. This secondary structure is considered to have an additional function in stabilizing the transcripts. PMID- 2997710 TI - Nucleotide sequences from phaseolin cDNA clones: the major storage proteins from Phaseolus vulgaris are encoded by two unique gene families. AB - The nucleotide sequences of eight partial and five full-length phaseolin cDNA clones show that phaseolin polypeptides are encoded by two distinct gene families which differ in their coding regions by the presence or absence of two different size direct repeats. The alpha-type phaseolin polypeptides are encoded by genes containing direct repeats which encode 14 additional amino acids. Aside from these differences, the alpha-and beta-type phaseolin genes show a high degree of homology (98%) which is consistent with these genes being derived from a common ancestral gene. Much of the heterogeneity found in the phaseolin polypeptides appears to be due to post-translational processing. Nucleotide sequence analysis demonstrates that the alpha-type genes contain only a few amino acid replacement substitutions and that the beta-type genes appear to contain no amino acid replacement substitutions. S1 nuclease mapping shows a complex pattern for transcriptional initiation of phaseolin mRNA. Hydropathy analysis shows that phaseolin polypeptides are predominately hydrophilic, and that the two N-glycosyl recognition sites are located in different hydropathic environments. PMID- 2997712 TI - Subgroup IV of human immunoglobulin K light chains is encoded by a single germline gene. AB - The series of studies on the human K light chain genes of the various subgroups is concluded by this report on the isolation and nucleotide sequence determination of a functional VKIV gene (abbreviations ref. 1) and its germline counterpart. The rearranged gene which stems from a lymphoid cell line and the germline gene differ in four nucleotides which can be attributed to somatic mutations; three of the mutations are clustered in CDR3. The germline gene regions of two unrelated individuals were identical over a stretch of 1267 bp. By hybridization experiments it is shown that the human K locus contains only one VKIV gene. In 16 lymphoid cell lines studied here, the VKIV gene is frequently deleted or aberrantly rearranged which may be a consequence of peculiarities of its function and/or its structural organization. PMID- 2997713 TI - Detection of a unique human V kappa IV germline gene by a cloned cDNA probe. AB - We have cloned the cDNA encoding the KIV chain of a human antibody with specificity against the major carbohydrate antigen of Streptococcus A. The cDNA has been used as a genetic probe to estimate the number of germline VKIV genes in human DNA. The presence of unique hybridizing bands on digestion of human DNA with several restriction endonucleases and the equivalence of the DNA in a band to a single gene per haploid genome point to the conclusion that there is a unique human VKIV germline gene. The corollary of this conclusion is that the diversity of human VKIV chains must be exclusively due to somatic mutation. This is supported by examination of the sequences of human KIV chain genes and their KIV chain products. Fusion of the unique germline VKIV gene (1) with one of several JK segments, followed by somatic mutations in the V region of the rearranged KIV gene, can account for the known sequences. The restricted germline gene repertoire may account for the small proportion of human KIV chains in the human K chain sequence library (2). PMID- 2997711 TI - Human immunoglobulin kappa light chain genes of subgroups II and III. AB - The first complete sequences of functionally rearranged VK genes (abbreviations ref. 1) of subgroups II and III are reported. The genes have been cloned from lymphoid cell lines synthesizing KII or KIII light chains as evidenced from immunochemical analyses with anti-VK subgroup-specific antisera. These data, together with the sequence of a KIV gene (described in the accompanying paper) and those of previously published KI genes make possible a comparison of genes representative of the four known V region subgroups of human K light chains. The VKII gene is distinguished from the VKI, VKIII, and VKIV genes by a much longer intron within the leader sequence: 426 bp vs ca. 120-220 bp. Blot hybridization experiments with human DNA digests using probes from the KII and KIII genes and from the respective upstream regions help to define subgroup specific probes and hybridization conditions. PMID- 2997714 TI - Developmental modulation of DNA methylation in the fungus Phycomyces blakesleeanus. AB - DNA methylation is a rather sparse event among fungi. Phycomyces blakesleeanus seems to be one of the few exceptions in this context. 5-Methylcytosine represents 2.9% of the total cytosine in spore DNA and is located in approximately the same amount at any of the four CA, CT, CC or CG dinucleotides. A progressive and gradual drop in total 5-methylcytosine parallels the development of the fungus. This demethylation is non random but sequence specific and is not accounted for equally by the four different methylated dinucleotides, CG being much less affected (20% demethylated) than CA, CT and CC (more than 90% demethylated at the same time). "De novo" methylation to restore the initial pattern probably takes place during spore maturation. By using specific hybridization probes we have been able to show that the rRNA genes are not significantly methylated at any stage of development, regardless of their transcription status. PMID- 2997715 TI - A Chinese G gamma + (A gamma delta beta)zero thalassemia deletion: comparison to other deletions in the human beta-globin gene cluster and sequence analysis of the breakpoints. AB - A clone was isolated that contains the deletion junction region from an individual with a deletion associated with Chinese G gamma + (A gamma delta beta)zero thalassemia. A clone containing the normal DNA corresponding to the 3' breakpoint of this deletion was also isolated. Portions of these two clones were sequenced and compared to the region in the A gamma-globin gene where the 5' breakpoint occurs. This comparison reveals that the breakage and reunion event was nonhomologous and that it probably involved the insertion of 36-41 bases of DNA belonging to the L1 (KpnI) family of repetitive DNA. Genomic mapping revealed that the DNA on the 3' side of this deletion is closely linked in normal DNA to the 3' breakpoints of two different large deletions that are associated with hereditary persistence of fetal hemoglobin (HPFH). We cloned and mapped 35 kbp of normal DNA from this region (greater than 45 kbp downstream of the human beta globin gene) that contains the 3' breakpoints of the Chinese thalassemia and the two HPFH deletions. An endogenous retrovirus-like element and several other repetitive sequences are located within this region. We show that the Chinese thalassemia deletion is greater than 80 kbp in length and differs in size from the two HPFH deletions by less than 6%. We also show that the Chinese thalassemia deletion is at least 40 kbp larger than several other deletions associated with a very similar phenotype. PMID- 2997716 TI - Structure of viral DNA in a rat cell line transformed by the cloned EcoRI-C fragment of adenovirus 12. AB - A DNA segment carrying viral DNA was cloned from a rat cell line transformed by the cloned EcoRI-C fragment (0 to 16.4 map units) of human adenovirus type 12(Ad12), and the viral sequence in the clone was analysed. The cloned segment contained the region from nucleotide positions 118 to 3520 of the Ad12 genome in the middle. No unique structure was found at the viral and non-viral DNA junctions. When examined the transforming activity, the conserved viral sequence was able to transform rat 3Y1 cells efficiently. Southern blotting analysis of the viral sequence in five re-transformed cell lines showed that the viral sequence was inserted at different sites of cellular DNA. These results indicate that (I) the Ad12 DNA moiety from the enhancer-promoter region of the E1A gene to the end of the E1B gene contains enough information for efficient transformation of the rat cell, and (II) integration of the viral sequence at unique cellular sites is not prerequisite for transformation. PMID- 2997717 TI - Binding studies of SV40 T-antigen to SV40 binding site II. AB - SV40 T-Antigen binding site II was synthesized, cloned and analyzed for its ability to bind purified SV40 T-antigen. We report the binding constant of T antigen for isolated site II. Using a filter binding assay the calculated binding constant was 6-8 fold less efficient than site I previously reported. Binding constants were calculated using two methods. The first was a direct calculation using a protein titration curve (KD). The second was by the ratio of measured association and dissociation rates. Both methods gave similar constants. Protection studies with SV40 T-antigen on the T-antigen binding sites in the wild type array demonstrated that the binding constants of site I and site II are similar to those calculated for the individual sites. These results demonstrate that SV40 T-antigen does not bind cooperatively to sites one and two as earlier believed and are in agreement with recent observations emanating from several laboratories. PMID- 2997718 TI - Sequence of the D beta 2-J beta 2 region of the human T-cell receptor beta-chain locus. AB - We have sequenced the region encompassing a D beta 2 segment and the J beta 2 segments of the human T-cell antigen receptor beta-chain genes. The D beta 2 element lies about 650 base pairs upstream of a cluster of seven potentially functional J beta 2 sequences and one J beta 2 pseudogene. Examination of human beta-chain cDNA sequences which involve rearranged D beta 2 and J beta 2 elements demonstrates that N-region, as well as junctional, diversity can occur during D-J joining. Further, we present evidence for possible somatic mutation in active J beta 2 segments. PMID- 2997719 TI - Nucleotide sequence characterization of Ty 1-17, a class II transposon from yeast. AB - We have determined the nucleotide sequence of a class II yeast transposon (Ty 1 17) which is found just centromere-distal to the LEU2 structural gene on chromosome III of Saccharomyces cerevisiae. The complete element is 5961 bp long and is bounded by two identical, directly repeated, delta sequences of 332 bp each. The sequence organization indicates that Ty 1-17 is a retrotransposon, like the class I elements characterized previously. It contains two long open reading frames, TyA (439 amino acids) and TyB (1349 amino acids). In this paper, the sequences of the two classes of yeast transposon are compared with one another and with analogous elements, such as retroviral proviruses, cauliflower mosaic virus and copia sequences. Features of the Ty 1-17 sequence which may be important to its mechanism of transposition and its genetic action are discussed. PMID- 2997721 TI - The 5'-flanking regions of three pea legumin genes: comparison of the DNA sequences. AB - Approximately 1200 nucleotides of sequence data from the promoter and 5'-flanking regions of each of three pea (Pisum sativum L.) legumin genes (legA, legB and legC) are presented. The promoter regions of all three genes were found to be identical including the "TATA box", and "CAAT box', and sequences showing homology to the SV40 enhancers. The legA sequence begins to diverge from the others about 300bp from the start codon, whereas the other two genes remain identical for another 550bp. The regions of partial homology exhibit deletions or insertions and some short, comparatively well conserved sequences. The significance of these features is discussed in terms of evolutionary mechanisms and their possible functional roles. The legC gene contains a region that may potentially form either of two mutually exclusive stem-loop structures, one of which has a stem 42bp long, which suggests that it could be fairly stable. We suggest that a mechanism of switching between such alternative structures may play some role in gene control or may represent the insertion of a transposable element. PMID- 2997720 TI - A cDNA clone encoding a glycinin A1a subunit precursor of soybean. AB - A cDNA clone covering the whole coding region for a glycinin subunit precursor containing the A1a acidic subunit, one of the A2 family, has been identified from a library of soybean cotyledonary cDNA clones using a mixed oligonucleotide probe. Analysis of the cDNA insert revealed that it contained 1746 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 54 nucleotides, a signal peptide region corresponding to 19 amino acids, an acidic subunit region (A1a) corresponding to 291 amino acids followed by a basic subunit region corresponding to 185 amino acids, and a 3'-terminal nontranslated region of 207 nucleotides. By comparing the predicted protein sequence of this precursor with that of the legumin A precursor of pea, it was found that glycinin A2 subunit family appeared to be more closely related to the legumin than to the A3 subunit family, and that the evolutional rearrangement of glycinin genes has occurred. PMID- 2997723 TI - A RFLP for A56 (D5S) an anonymous DNA sequence from chromosome 5. PMID- 2997722 TI - A new endonuclease recognizing the deoxynucleotide sequence CCNNGG from the cyanobacterium Synechocystis 6701. AB - A new sequence-specific endonuclease from the cyanobacterium Synechocystis species PCC 6701 has been purified and characterized. This enzyme, SecI, is unique in recognizing the nucleotide sequence: 5' -CCNNGG-3' 3' -GGNNCC-5' and cleaves it at the position indicated by the symbol. Two other restriction endonucleases, SecII and SecIII, found in this organism are isoschizomers of MspI and MstII, respectively. PMID- 2997724 TI - RFLP for the human apolipoprotein B gene: I;BamHI. PMID- 2997725 TI - RFLP for the human apolipoprotein B gene: II;EcoRI. PMID- 2997726 TI - RFLP for the human apolipoprotein B gene: III;EcoRV. PMID- 2997727 TI - RFLP for the human apolipoprotein B gene: V;XbaI. PMID- 2997728 TI - Poliovirus genome RNA hybridizes specifically to higher eukaryotic rRNAs. AB - The RNA genome of poliovirus hybridizes to 28S and 18S rRNAs of higher eukaryotes under stringent conditions. The hybridization detected by Northern blot analyses is specific since little or no signal was detected for yeast or prokaryotic rRNAs or other major cellular RNAs. Southern blot analysis of DNA clones of mouse rRNA genes leads us to conclude that several regions of 28S rRNA, and at least one region in 18S rRNA, are involved in the hybridization to polio RNA, and that G/C regions are not responsible for this phenomenon. We have precisely mapped one of these hybridizing regions in both molecules. Computer analysis confirms that extensive intermolecular base-pairing (81 out of 104 contiguous bases in the rRNA strand) could be responsible for this one particular site of interaction (polio genome, bases 5075-5250; 28S rRNA, bases 1097-1200). We discuss the possible functional and/or evolutionary significance of this novel type of interaction. PMID- 2997729 TI - The nucleotide sequence of a HMW glutenin subunit gene located on chromosome 1A of wheat (Triticum aestivum L.). AB - A cloned 8.2 kb EcoRI fragment has been isolated from a genomic library of DNA derived from Triticum aestivum L. cv. Cheyenne. This fragment contains sequences related to the high molecular weight (HMW) subunits of glutenin, proteins considered to be important in determining the elastic properties of gluten. The cloned HMW subunit gene appears to be derived from chromosome 1A. The nucleotide sequence of this gene has provided new information on the structure and evolution of the HMW subunits. However, hybrid-selection translation experiments suggest that this gene is silent. PMID- 2997730 TI - Structure and transcription of the Drosophila mulleri alcohol dehydrogenase genes. AB - The D. melanogaster Adh gene is transcribed from two different promoters; a proximal (larval) promoter is active during late embryonic and larval stages, and a distal (adult) promoter is active primarily in third instar larvae and in adult flies (1). Genetic analyses suggest that several species of the mulleri subgroup (distant relatives of D. melanogaster) have two closely-linked Adh genes, Adh-1 and Adh-2, each of which expresses a different ADH protein (2). The temporal pattern of expression of Adh-1 and Adh-2 is similar to the expression of D. melanogaster Adh from the proximal and distal promoters (2,3,4). We are interested in the molecular basis for the pattern of Adh expression in the mulleri subgroup species and in the mechanism of the switch in Adh promoter utilization. For these reasons, we have studied the structure and transcription of the Adh locus of D. mulleri, a species of the mulleri subgroup. We show that the ADH-1 and ADH-2 proteins are expressed from two distinct genes separated by 2 kilobase pairs, and that Adh-1 and Adh-2 are transcribed in the expected temporal pattern. In addition, we find a pseudogene 1.2 kb upstream from Adh-2, which is transcribed in a temporal pattern similar to Adh-2. PMID- 2997731 TI - Investigation of the tertiary folding of Escherichia coli 16S RNA by in situ intra-RNA cross-linking within 30S ribosomal subunits. AB - Intra-RNA cross-links were introduced into E. coli 30S ribosomal subunits by mild ultraviolet irradiation. The subunits were partially digested with cobra venom nuclease, followed in some cases by a second partial digestion with ribonuclease H in the presence of the hexanucleotide d-(CTTCCC). The cross-linked RNA complexes were separated by two-dimensional gel electrophoresis and the sites of cross-linking analysed by our published procedures. Tertiary structural cross links in the 16S RNA were identified between positions 31 and 48, between oligonucleotides 1090-1094 and 1161-1164, and between oligonucleotides 1125-1127 and 1280-1281. The first of these imposes a rigid constraint on the relative orientations of helices 3 and 4 of the 16S secondary structure. A further tertiary cross-link (which could not be precisely localised) was found between regions 1-72 and 1020-1095, and secondary structural cross-links were identified between positions 497 and 545-548, and positions 1238-1240 and 1298. PMID- 2997732 TI - Inhibition of HeLa cell DNA topoisomerase I by ATP and phosphate. AB - The relaxation activity of DNA topoisomerase I from HeLa cell nuclei is strongly inhibited by a variety of purine nucleotides in the presence but not absence of 1 mM potassium phosphate. For ATP, 3-4 mM causes nearly complete inhibition. The 2' and 3'-AMP isomer are active as well in the presence of 1 mM phosphate, but the 5'-AMP isomer and adenosine are inert. At 3 mM ATP, the titration curve for phosphate is sigmoidal with inhibition beginning abruptly at about 0.5 mM. The negatively-supercoiled DNA isolated from an "inhibited" reaction is relaxed as well as the standard DNA template in the absence of ATP and phosphate suggesting that inhibition does not result from an alteration of the template which protects against its relaxation. Relaxation of positively-supercoiled DNA is also inhibited. Catalysis by E. coli DNA topoisomerase I and HeLa DNA topoisomerase II is not inhibited at concentrations of ATP and phosphate sufficient to cause 80 90% inhibition of HeLa type 1 enzyme. PMID- 2997733 TI - Interaction of the restriction endonuclease ScaI with its substrates. AB - The kinetic constants of the site-specific endonuclease, ScaI, for various substrates were determined. We estimated Vmax and Km for octa-, deca-, dodeca-, and hexadecanucleotides and for plasmid pBR322 DNA. Vmax for these substrates were close, but Km were quite different (in decreasing order, octa- greater than deca-, dodeca-, hexadeca- greater than pBR322). The results were discussed with respect to the tertiary structure of substrate. PMID- 2997734 TI - DNA sequence and characterization of the Escherichia coli serB gene. AB - We have determined the sequence of a DNA fragment containing the Escherichia coli serB gene. An open reading frame of 966 nucleotides was identified that encodes a polypeptide of 322 amino acids with a molecular weight of 35,002 daltons. The transcription start site was determined by Mung Bean nuclease mapping. The -10 and -35 regions of the serB promoter lack homology to the consensus sequences. In addition, the -35 region of the serB promoter overlaps the -35 region of a second divergent promoter. Frameshift mutations were constructed at three different sites within the serB gene. When plasmids carrying these mutations were used as templates in a minicell system, mutations closer to the proposed transcription and translation start sites resulted in smaller polypeptides than those further away, confirming the proposed direction of transcription and translation. The observed sizes of the truncated and native polypeptides were in agreement with those predicted from the DNA sequence. A very stable stem and loop structure (delta G= -32 kcal/mole) that does not fit the criteria of known transcription terminators was found one nucleotide downstream from the putative UAA translation stop codon. PMID- 2997736 TI - Effect of X-ray induced DNA damage on DNAase I hypersensitivity of SV40 chromatin: relation to elastic torsional strain in DNA. AB - The effect of X-irradiation on DNAase I hypersensitivity of SV40 minichromosomes within nuclei or free in solution was investigated. The susceptibility of the specific DNA sites in the control region of minichromosomes to DNAase I decreased in a dose dependent manner after irradiation of isolated nuclei. On the other hand, the irradiation of minichromosomes extracted from nuclei in 0.1 M NaCl containing buffer almost did not affect the level of their hypersensitivity to DNAase I. This suggests that DNAase I hypersensitivity may be determined by two different mechanisms. One of them may be connected with elastic torsional strain within a fraction of minichromosomes and another seems to be determined by nucleosome free region. The first mechanism may be primarily responsible for the hypersensitivity of minichromosomes within nuclei. After irradiation of the intact cells, DNAase I hypersensitivity tested in nuclei substantially increased. This was connected with activation of endogeneous nucleases by X-irradiation which led to accumulation of single- and double-strand breaks superimposed to DNAase I induced breaks in the control region of SV40 DNA. PMID- 2997735 TI - Dynamic gene interactions in the evolution of rabbit VH genes: a four codon duplication and block homologies provide evidence for intergenic exchange. AB - Two rabbit VHa-negative genes, RVH831 and RVH832, were isolated from a single genomic fragment selected by hybridization with the mouse VHIII gene S107V1. RVH831 is a pseudogene with a frameshift mutation in FR3 and a 19 bp deletion within the VH-D splice site. In contrast, RVH832 has an open reading frame and an intact VH-D splice site and thus may be functional. However, RVH832 displays a unique 4 codon duplication/insertion in FR1 that may be the result of an unequal exchange event between two ancestral VH genes. Sequence comparisons between these and other rabbit VH genes reveal patterns of shared blocks of nucleotide substitutions, suggestive of gene conversion. A high overall homology (greater than or equal to 73%) between the compared VH nucleotide sequences suggests that rabbit VH genes may not be organized in clearly divergent families or subgroups. PMID- 2997737 TI - Nucleotide sequence of the transposon Tn7 gene encoding an aminoglycoside modifying enzyme, 3"(9)-O-nucleotidyltransferase. AB - The nucleotide sequence of a transposon Tn7 DNA fragment encoding a 3"(9)-O nucleotidyltransferase, an aminoglycoside-modifying enzyme, which mediates bacterial resistance to spectinomycin and streptomycin, was determined. The aadA structural gene was 786 bases long and predicted a polypeptide of 262 amino acids with a calculated molecular weight of 29,207. Comparison of the DNA sequences of Tn7 and plasmid R538-1 indicated that their aadA genes were nearly identical. Comparison of the polypeptides predicted by the aadA genes of Tn7 and Tn554 indicated that the genes were related. PMID- 2997738 TI - The majority of minicircle DNA in Crithidia fasciculata strain CF-C1 is of a single class with nearly homogeneous DNA sequence. AB - DNA minicircles found within the kinetoplast of the trypanosomatid Crithidia fasciculata, like those of most other kinetoplastid species, are heterogeneous in sequence. The pattern of minicircle DNA fragments generated by cleavage of kinetoplast DNA with various restriction enzymes has been used to demonstrate this heterogeneity. Here we describe a strain of Crithidia fasciculata in which more than 90% of the DNA minicircles exhibit a common pattern of restriction enzyme cleavage sites. A map of cleavage sites within this major minicircle DNA class is presented for seven restriction enzymes with hexanucleotide recognition sequences. Sequence homogeneity at an even finer level is reflected in minicircle DNA digestion patterns generated by restriction enzymes with tetranucleotide recognition sites. Partial DNA sequence analysis of multiple clones from the major minicircle class shows nearly complete homogeneity at the nucleotide level. The existence of a near homogeneous complement of DNA minicircles in Crithidia should facilitate the study of their replication in this organism. PMID- 2997740 TI - An anonymous single copy chromosome 22 clone, D22S10 (22c1-18) identifies an RFLP with PstI. PMID- 2997741 TI - RFLPS at the D21S19 locus of human chromosome 21. PMID- 2997742 TI - Purification of Mbo II methylase (GAAGmA) from Moraxella bovis: site specific cleavage of DNA at nine and ten base pair sequences. AB - The restriction modification methylase M. Mbo II has been purified using a sensitive oligonucleotide linker assay. The enzyme methylates the Mbo II recognition sequence* GAAGA at adenine to produce GAAGmA. M. Mbo II can be used in conjunction with the methylation dependent restriction endonuclease Dpn I (GmATC) to produce cleavage at the 10 base sequence GAAGATCTTC. When M. Mbo II is used in combination with M. Cla I (ATCGATCGAT), cleavage by Dpn I occurs at the four ten base sequences GAAGATCTTC, GAAGATCGAT, ATCGATCTTC and ATCGATCGAT, which is equivalent to a nine base recognition site. The use of combinations of adenine methylases and Dpn I to generate highly selective DNA cleavages at a variety of sequences up to fourteen base pairs is discussed. PMID- 2997739 TI - Cloning and sequencing of the adenylate kinase gene (adk) of Escherichia coli. AB - Adenylate kinase, the product of the adk locus in Escherichia coli K12, catalyzes the conversion of AMP and ATP to two molecules of ADP. The gene has been cloned by complementation of an adk temperature sensitive mutation. The DNA sequence of the complete coding region and of 5'- and 3'-untranslated regions were determined. The resulting protein sequence was found to contain several regions of high homology with cytosolic adenylate kinase of pig muscle (AK1), whose three dimensional structure has been determined. The most significant of the amino acid exchanges is the replacement of histidine 36 with glutamine. This residue is believed to play a role in catalysis through metal ion binding. The codon usage pattern and the determination of adenylate kinase molecules per cell shows that the enzyme is one of the more abundant soluble proteins of the bacterial cells. PMID- 2997743 TI - Ozonolysis of supercoiled pBR322 DNA resulting in strand scission to open circular DNA. AB - Treatment of supercoiled pBR322 DNA with ozone resulted in the conversion of closed circular DNA to open circular DNA. Restriction analysis of the resulting open circular DNA showed that ozonolysis in the absence of salt caused single strand cleavage at specific sites. PMID- 2997744 TI - Molecular cloning and nucleotide sequence of a developmentally regulated gene from the cyanobacterium Calothrix PCC 7601: a gas vesicle protein gene. AB - Since the gas vesicle protein (GVP) is highly conserved among the different gas vacuolate prokaryotes, a 29-mer oligonucleotide corresponding to a portion of the Anabaena flos-aquae GVP gene was synthesized and used to isolate the GVP structural gene from Calothrix PCC 7601 (= Fremyella diplosiphon). Gas vacuole production in this filamentous cyanobacterium is restricted to hormogonia which occur at a specific stage during the developmental cell cycle. The GVP gene (gvpA) was localized on a 709 bp HindIII-HincII fragment. Nucleotide sequence analysis revealed a 213 bp open reading frame whose deduced amino-acid sequence shows a very high homology with that of the Anabaena flos-aquae GVP. Assuming that the first methionine residue is proteolytically processed, the molecular mass of the Calothrix GVP is 7375 daltons. Sequences resembling the Escherichia coli consensus promoter were found upstream from the gvpA gene. The initiator codon of the gvpA gene is preceded by a polypurine sequence assumed to be the ribosome binding site. Southern hybridizations with a probe specific for the gvpA gene indicated that this gene is not plasmid-borne, and that another homologous gene is present in the Calothrix genome. PMID- 2997746 TI - Autoregulation plus upstream positive and negative control regions associated with transcriptional activation of the mouse P1(450) gene. AB - 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to interact with a cytosolic receptor and, in turn, activate transcription of the mouse P1(450) gene. Various lengths of DNA upstream of the P1(450) gene were inserted into the pSV0-cat expression vector, with and without addition of the Harvey murine sarcoma virus (Ha-MSV) 72-bp repeat enhancer element. The constructs were cotransfected with pSV2-neo into mouse hepatoma wild-type cells and two variant cell lines. One variant is believed to result from a mutation in the P1(450) structural gene and expresses high levels of P1(450) mRNA constitutively; the other variant has a defect in nuclear translocation of the inducer-receptor complex. After selection in G418, the cells were treated with control medium, TCDD, cycloheximide, or TCDD plus cycloheximide and then assayed for chloramphenicol acetyltransferase (CAT) activity. The data are consistent with the presence of several functional regions within the upstream sequence: a promoter region, a region that is negatively autoregulated, possible repressor-binding and inducer-receptor complex-binding sites, and an upstream activation element that is required for transcriptional activation by TCDD. The Ha-MSV enhancer can substitute for this upstream activation element. PMID- 2997745 TI - The nucleotide sequence of the yeast MEL1 gene. AB - The complete nucleotide sequence of the MEL1 gene of the yeast, Saccharomyces cerevisiae, encoding alpha-galactosidase was determined. The nucleotide sequence contains an open reading frame of 1413 bp encoding a protein of 471 amino acids. Comparison with the known N-terminal amino acid sequence of the mature secreted protein indicated that alpha-galactosidase is synthesized as a precursor with an N-terminal signal sequence of 18 amino acids. The general features of this signal peptide resemble those of other yeast signal peptides. Molecular weight of the mature alpha-galactosidase polypeptide deduced from the nucleotide sequence is 50.049 kd. The 5' regulatory region has sequences in common with other yeast genes regulated by the GAL4-protein. PMID- 2997747 TI - A gastrin gene is expressed in both porcine pituitary and antral mucosal tissues. AB - By employing S1 nuclease mapping of total RNA isolated from porcine cerebral cortex, cerebellum, hypothalamus, pituitary, kidney, liver, pancreas, intestine, and antral mucosa, we have investigated gastrin gene expression in these tissues. Our results show that a gastrin gene is expressed only in the antral mucosal and pituitary tissues. Based on the amount of gastrin specific probe protected from S1 nuclease digestion in the presence of a given weight of total RNA, the amount of gastrin mRNA present in pituitary is approximately 330 times lower than in antral mucosa. These findings help establish the tissue distribution of gastrin gene expression. PMID- 2997749 TI - Transfection of mouse ribosomal DNA into rat cells: faithful transcription and processing. AB - Truncated mouse ribosomal DNA (rDNA) genes were stably incorporated into rat HTC 5 cells by DNA-mediated cell transfection techniques. The mouse rDNA genes were accurately transcribed in these rat cells indicating that there is no absolute species specificity of rDNA transcription between mouse and rat. No more than 170 nucleotides of the 5' nontranscribed spacer was required for the accurate initiation of mouse rDNA transcription in rat cells. Further, the mouse transcripts were accurately cleaved at the 5' end of the 18S rRNA sequence, even though these transcripts contained neither the 3' end of mouse 18S rRNA nor any other downstream mouse sequences. Thus, cleavage at the 5' end of 18S rRNA is not dependent on long range interactions involving these downstream sequences. PMID- 2997748 TI - A novel expression selection approach allows precise mapping of the hepatitis B virus enhancer. AB - We have used a novel approach called expression selection to precisely define the hepatitis B virus (HBV) enhancer. Expression selection is based on a shuttle vector containing an enhancerless SV40 T antigen gene, the SV40 origin of replication and a plasmid replicon. This vector is linearized, ligated with the sonicated DNA to be analyzed and transfected into eukaryotic cells, where only plasmids which have incorporated an enhancer can express T antigen and therefore replicate. Vectors amplified by replication are selectively rescued in E. coli and their inserts analyzed. When we performed this protocol with HBV DNA we rescued two overlapping fragments of 166 and 214 bp which in HBV DNA map about 500 bp upstream of the core antigen mRNA initiation site and 1150 bp downstream of the surface antigen mRNA initiation site. These results were confirmed by conventional deletion mapping. When compared to the SV40 enhancer in nonhepatic cell lines, the HBV enhancer is only 5 to 10% as active; nevertheless, it also acts in an orientation-independent manner and in a position downstream of a gene. The HBV enhancer is situated in the coding region of the potential reverse transcriptase, and thus is the first enhancer identified to map in a protein coding region. PMID- 2997751 TI - An anonymous single copy X-chromosome clone DXS91, from Xq11-q13, identifies a moderately frequent RFLP. PMID- 2997752 TI - An anonymous single copy X-chromosome clone DXS92, from Xq26-27, identifies two frequent RFLPs. PMID- 2997753 TI - [99mTc uptake and TSH receptor autoantibodies--comparative study in Basedow's disease and other thyroid diseases]. AB - In 255 patients (normals: group I, n = 30; nontoxic goitres: group II, n = 134; toxic goitres without ophthalmopathy: group III, n = 63; Graves' disease: group IV, n = 28) a TSH-receptor-autoantibody-assay (TRAK assay) for detection of thyrotropin-binding inhibiting antibodies (TBIAb) was tested and 99mTcO4-uptake (TcTU) was measured. Normal TcTU (range: 1.5-5.5%) and normal TRAK values (normal limit: F less than 11%) were only found in group I. An increased TcTU was found in group II in 22.4% (increased TRAK values only in 2.2%). In group III an increased TcTU was measured in 34.9% of the patients (all with normal TRAK titers). The stimulation of the TSH-receptor in immunogenic hyperthyroidism by TBIAb could be demonstrated by increased TRAK values in 71.4% of the patients with Graves' disease. In correlation, TcTU was also increased in 82.1% of the patients in group IV. As the measurement of TcTU can be helpful in differential diagnosis, the functional imaging with gamma camera and computer is today a conditio sine qua non, especially in suspected hyperthyroidism. PMID- 2997750 TI - The genomic organisation and nucleotide sequence of the HLA-SB(DP) alpha gene. AB - We have isolated a unique fragment of the HLA-DR alpha gene and probed human genomic DNA at low stringency to search for homologous sequences. A minimum of six non-polymorphic cross-hybridizing high molecular weight fragments were found in all DNAs examined. In order to obtain molecular clones of these cross hybridizing fragments, we constructed lambda and cosmid libraries of human DNA and screened them at low stringency with the HLA-DR alpha gene specific subclone. We have isolated clones corresponding to each of the six fragments and, in this paper, describe those which contain the gene encoding HLA-SB(DP) alpha. PMID- 2997754 TI - Investigation of the parameters related to the formation of 99mTc-pyrophosphate complexes using gel chromatography column scanning. AB - Using gel chromatography two different 99mTc-PPi complexes (complex I and II) were observed under various pH-values. In neutral medium complex II was the main product while in acidic or alkaline media complexes I and II were formed. Our results related to the organ distribution in mice revealed that complex II is a bone-seeking agent whereas complex I concentrates in the kidneys. PMID- 2997755 TI - Vegetarianism--blowing the myths. PMID- 2997756 TI - News analysis. Just another excuse to persecute? PMID- 2997757 TI - Radionuclide diagnosis of infradiaphragmatic total anomalous pulmonary venous drainage. AB - We hypothesized that infradiaphagmatic total anomalous pulmonary venous drainage (ITAPVD), because of its unique physiology, could be diagnosed with radionuclide angiography. Seven neonates with severe respiratory distress were injected intravenously with 3 mCi technetium-99m pertechnetate. In each of four neonates demonstrated to have ITAPVD by pulmonary angiography, nuclide recirculation through the right atrium occurred 3-6 s after initial passage. In addition, direct visualization of the anomalous common pulmonary trunk with nuclide as a "tail" below the diaphragm was obvious in the third infant studied. This prompted review of the first two infants with ITAPVD; in retrospect the anomalous trunk was also visualized with nuclide in both of these infants. All three were injected via the upper extremity. In the fourth ITAPVD infant, nuclide was injected via the lower extremity. In that infant, preferential streaming of the inferior vena caval flow and nuclide across the foramen ovale into the left heart led to simultaneous opacification of anomalous trunk and descending aorta, obscuring the "tail" sign. PMID- 2997758 TI - [Effect of naloxone on corticotropin and somatotropin secretion in patients with acute renal failure]. PMID- 2997759 TI - [Occurrence of antibodies against Herpes simplex virus type 1 in patients treated by long-term dialysis and in personnel of the dialysis department]. PMID- 2997760 TI - [Serotherapy of leukemia and lymphoma using monoclonal antibodies]. PMID- 2997761 TI - Morphological and biochemical responses of leukocytes in chemokinesis and chemotaxis. AB - Morphological changes associated with leukocyte chemokinesis and chemotaxis are briefly described. Leukocyte stimulation by chemokinetic and chemotactic factors (cytotaxins) elicits various biochemical responses including ligand-receptor interactions, ion fluxes, alterations in phospholipid and arachidonic acid metabolism, cyclic nucleotides, protein phosphorylation and reorganization of the cytoskeleton. These biochemical processes may be related to signal transduction, the effector mechanisms of directional locomotion or possibly other leukocyte responses which occur in parallel. PMID- 2997762 TI - Differential diagnosis of acute allograft rejection and CMV-infection in renal transplantation by urinary cytology. AB - Acute allograft rejection and CMV-infection are the most common complications after renal transplantation. Quick differential diagnosis between these two complications is still difficult but necessary, since both complications demand a different therapy. More than 2500 urinary samples from 33 transplanted patients were prospectively examined and part of them evaluated quantitatively. Urinary samples of patients with acute renal failure, long-term haemodialysis or immunosuppressive therapy served as controls. The following cytomorphological criteria proved to be useful: Tubular epithelial cells, casts, oxalate crystals (sand-glass shaped), dirty background, increasing erythrocyturia, mixed cell clusters, lymphocytes and mitoses. Rejection is going on when the number of renal tubular cells is increased and two or more further criteria are positive. 25 acute allograft rejections without acute renal failure were diagnosed clinically. All 25 rejections were also diagnosed by urinary cytology. Nevertheless, it is not possible to differentiate between acute allograft rejection and acute renal failure of other origin. CMV-infection was serologically detected in 7 patients. In 6 of them viral infected cells were found in the urine shortly after the onset of unspecific clinical symptoms. Besides the typical "owl-eye" cells milkglass nuclei, sometimes with eosinophilic condensation, were seen while criteria for transplant rejection were never observed at the same time. Cytologic examination of voided urine is a simple diagnostic help for the differentiation between allograft rejection and CMV-infection after renal transplantation. PMID- 2997763 TI - [The Dental Institute of Abidjan from its founding to 1985]. PMID- 2997764 TI - Microcrystalline hydroxyapatite compound in prevention of bone loss in corticosteroid-treated patients with chronic active hepatitis. AB - To determine whether microcrystalline hydroxyapatite compound (MCHC) could reduce bone loss or its consequences in patients with chronic active hepatitis (CAH) on corticosteroid therapy, a controlled trial was conducted in 36 such patients over a period of 2 years. Both skeletal symptoms (back pain) and fractures were uncommon during the trial period but both showed non-significant differences in favour of the MCHC group and biochemical investigations were suggestive of a reduction in parathyroid over-activity. Continued reduction in bone mineral content of the radius (photon absorptiometry) was halted in those receiving MCHC and iliac crest bone biopsy showed a non-significant increase in trabecular bone volume. The fall in iliac crest cortical plate thickness was significantly less (P less than 0.025) in the MCHC group and the results overall were consistent with a beneficial effect from MCHC in corticosteroid-induced osteoporosis. PMID- 2997765 TI - On the biopotency and site of action of drugs affecting endocrine tissues with special reference to the anti-steroidogenic effect of anaesthetic agents. AB - Dispersed guinea-pig adrenal cells or mouse Leydig cells were stimulated with a saturating dose of adrenocorticotrophin (ACTH, 50 ng/1) or luteinizing hormone (LH, 5IU/1), respectively. The incubations were performed in the presence of increasing concentrations (10(-9) - 5 X 10(-4)mol/l) of the anaesthetic agents propofol, thiopentone and etomidate. At the end of this stimulation period, cortisol (from the adrenal preparation) or testosterone (from the Leydig cell culture) were assayed by radioimmunoassay. Propofol, thiopentone and etomidate all inhibited ACTH-stimulated cortisol secretion in a dose-related fashion. Similar inhibition of LH-stimulated testosterone output was found with propofol and thiopentone whereas etomidate was without effect at any concentration employed, an observation in accordance with its known site of action, 11 beta hydroxylase, an enzyme which is not involved in the biosynthesis of testosterone. The concentration (mumol/l) of anaesthetics which gave 50% inhibition (ED50) of ACTH-stimulated cortisol secretion was 0.1 +/- 0.002 (n = 7), 160 +/- 18 (n = 3) and 170 +/- 18 (n = 3) (mean +/- s.e.m.) for etomidate, thiopentone and propofol, respectively. The corresponding values for the LH stimulated testosterone output from the Leydig cell preparations were 186 (thiopentone) and 180 (propofol) mumol/l. In a separate series of experiments adrenal cells were stimulated with (a) the cortisol precursor steroids (all at 10(-5)mol/l) pregnenolone, 17 hydroxypregnenolone, progesterone, 17-hydroxyprogesterone and 11-deoxycortisol, (b) dibutyryl cAMP (10(-3)mol/l) or (c) ACTH (100 ng/l) in the presence and absence of either etomidate (5 X 10(-5)mol/l), propofol (2.5 X 10(-4)mol/l) or thiopentone (5 X 10(-4)mol/l). All the stimulators increased cortisol production by > 7-fold over that seen in their absence. Propofol depressed ACTH and dibutyryl cAMP induced cortisol output by > 60% (P < 0.05) but was without effect when the steroid precursors were used, suggestive of an inhibition between the sequence involving ACTH binding -> pregnenolone production. In contrast, etomidate and thiopentone reduced cortisol secretion by > 40% (P < 0.05) regardless of the stimulator used, indicating that at least one site of action was at the level of the final enzymic step of cortisol synthesis, i.e. 11beta hydroxylase. PMID- 2997767 TI - HTLV III infection and AIDS. PMID- 2997766 TI - Monitoring for tardive dyskinesia: a community-based approach. PMID- 2997768 TI - Improvement of a simple method to purify ribonucleotide reductase. AB - The use of an ATP-agarose column to purify ribonucleotide reductase from human D 98 cells was recently reported. The column selectively retains greater than 99.9% of the contaminating nucleoside diphosphate (NDP) kinase from crude preparations of ribonucleotide reductase. It was presently found, however, that extending the length of the column caused the ribonucleotide reductase to dissociate into subunits. One subunit appeared in the low ionic strength buffer wash while the other required 0.5 M KCl for elution. The enzyme could also be recovered intact (non-dissociated) by equilibrating the enzyme preparation and the column with 0.5 M KCl prior to chromatography. Either method greatly improved the overall yield and the specific activity of the ribonucleotide reductase because it prevented the binding and subsequent loss of any of the subunits. In addition, the use of a larger column permitted the gel-filtration properties of the ATP-agarose to separate the bulk of the residual (not bound) NDP kinase from the ribonucleotide reductase. PMID- 2997769 TI - [The role of cytogenetics in malignant proliferations]. PMID- 2997770 TI - [Adsorption of spin-labeled flocculants on E. coli cells]. AB - The interaction of intact E. coli cells with polymeric flocculants polyethylenimine and dextran was being studied by spin-labelling. The nitroxyl groups of spin-labelled polymers are reduced during formation of the adsorption layer on the cell surface. Some peculiarities of redox reactions between polymer macroradicals and units of the bacterial electron-transport chain were studied depending of temperature and flocculation regime. The course of the reduction of spin-labelled polymer radicals and characteristics of EPR spectra of the flocculant give evidence on the formation of a loose polymeric layer on the surface of E. coli cells. PMID- 2997771 TI - [Immunologic indices in Itsenko-Cushing disease]. AB - The status of the immune system was studied in 36 patients with the Itsenko Cushing disease (10 at the initial stage, 12 in the period of recurrence, 14 during remission) and in 20 healthy persons comparing the corticotropin and hydrocortisone content in the plasma. The level of T lymphocytes was lowered at the initial stage of disease (40.6 +/- 3.4%) as well as during recurrence and remission (37.8 +/- 3.9 and 44.5 +/- 4.6%, respectively) as compared to this level in the healthy persons (60.5 +/- 3.6%). The content of B lymphocytes was lowered in all 3 groups (4.3 +/- 0.8,2.9 +/- 0.5 and 4.8 +/- 0.7%, respectively; in the group of healthy persons, it was 19.2 +/- 3.0%). The phagocytic activity of leukocytes was lowered at the initial stage of disease (39.0 +/- 7.6%) and during recurrence (36.7 +/- 4.5%) as compared to that of the healthy persons (44.6 +/- 1.5%). The hydrocortisone sensitive population of T lymphocytes was decreased 1.5-2 times. An increase in the corticotropin level (148.1 +/- 16.9 and 85.2 +/- 19.7 pg/ml, respectively; in health 47.9 +/- 3.8 pg/ml) and in the hydrocortisone level (142.5 +/- 11.1 and 181.3 +/- 20.5 ng/ml, respectively; in health 79 +/- 23 ng/ml) was marked simultaneously in the patients of these 2 groups. During remission (corticotropin and hydrocortisone normal levels), immunological indicators did not completely return to normal, thus suggesting a stable disorder of the cell immunity. PMID- 2997772 TI - [Specific estrogen-binding protein in the rat liver: effect on the accumulation of estradiol-receptor complexes in nuclei in vitro]. PMID- 2997773 TI - [Effect of blood serum hormones and lipoproteins on levels of cAMP and cGMP in viable sections of rat liver]. AB - Cyclic nucleotides are universal intermediary agents of hormones in the target tissues, however mechanisms of the regulation of the cAMP and cGMP content in the cell are rather complex and still obscure. More data have appeared of late on the involvement of blood serum lipoproteins in the regulation of various intracellular processes including adenylate cyclase activity. Therefore the purpose of the study was to investigate the effect of adrenaline, hydrocortisone, glucagon, insulin and blood serum lipoproteins on the cAMP and cGMP content in rat liver surviving sections. An attempt was made to study a cooperative effect of the above hormones and lipoproteins of various classes. The results obtained have shown that adrenaline and glucagon raise the cAMP level in rat liver surviving sections. The effect of adrenaline is mediated by beta-adrenoreceptors. Insulin lowers an increases the level of cAMP in liver sections determined by the effect of glucagon. A decrease of the initial cAMP content in response to insulin occurs after a short lag period. High density lipoproteins (HDLP3) reduce the cAMP content in liver surviving sections. A cooperative effect of lipoproteins of a very low density and adrenaline (or hydrocortisone) in the regulation of the cAMP content in the rat liver was revealed. The cGMP content in rat liver surviving sections does not change under the influence of the above hormones and lipoproteins. PMID- 2997774 TI - [Diagnosis of malignant thyroid tumors by indirect thyroid lymphography]. AB - Indirect thyroidolymphography was used for 38 patients with malignant thyroid tumors. A conclusion has been made that indirect lymphography of the thyroid is a simple and rather informative diagnostic method for malignant thyroid tumors and should be followed by spot biopsy. PMID- 2997775 TI - [Role of phospholipase A2 in regulating lipid peroxidation and the respiratory chain in experimental tuberculosis]. PMID- 2997776 TI - uvrA and recA mutations inhibit a site-specific transition produced by a single O6-methylguanine in gene G of bacteriophage phi X174. AB - Using site-specific mutagenesis, we have examined the mutagenic activity in vivo of O6-methylguanine or O6-n-butylguanine located at a preselected site in gene G of bacteriophage phi X174. The experiments were designed so that the phage mutant produced by a targeted transition from either of these alkylated derivatives would be recognizable by a simple plaque assay. Spheroplasts derived from normal and repair-deficient cells were transfected, and the lysates were screened for mutant virus. In cells with normal repair, DNA carrying the methylguanine produced the expected transition in 15% of the total phage; DNA carrying the butylguanine produced the same mutation in 0.3% of the phage. In cells deficient in excision repair (uvrA) the transition frequency went up by a factor of 8 for O6-butylguanine and down by a factor of 40 for O6-methylguanine. In cells deficient in recombination (recA), the transition frequency increased 1.5-fold for butylguanine and decreased by a factor of 8 for methylguanine. The data show that both methyl- and butylguanine produce site-directed transitions in phi X174; the transition occurs in recA cells; the frequency of the transition is influenced by both recA and uvrA mutations; the recA and uvrA mutations alter the transition frequency for methylguanine and butylguanine in opposite directions. PMID- 2997777 TI - Cloning of yeast TOP1, the gene encoding DNA topoisomerase I, and construction of mutants defective in both DNA topoisomerase I and DNA topoisomerase II. AB - Rabbit antibodies specific to yeast DNA topoisomerase I were used in immunological screening of a Saccharomyces cerevisiae genomic DNA library in Escherichia coli. One of the clones identified by its expression of antigenic determinants of the yeast enzyme is shown to contain the coding sequence of the enzyme: no active DNA topoisomerase I is detectable in cell extracts when insertion or deletion mutations are introduced into a 2-kilobase-pair (kb) region of the sequence in a haploid yeast genome. Blot hybridizations show that there is a single copy of the cloned sequence per haploid and that the sequence is transcribed to give a 2.7-kb poly(A)+ message. Mutants in which 1.7 kb of the sequence is deleted are viable. Temperature-shift experiments using synchronously grown cells of a delta top1 top2 temperature-sensitive (ts) double mutant and its isogenic top2 ts strain show that, whereas mitotic blocks can prevent killing of the top2 ts mutant at a nonpermissive temperature, the same treatments are ineffective in preventing cell death of the delta top1 top2 ts double mutant. These experiments suggest that in yeast DNA topoisomerase I serves a role auxiliary to DNA topoisomerase II. PMID- 2997779 TI - Deduced product of the stage 0 sporulation gene spo0F shares homology with the Spo0A, OmpR, and SfrA proteins. AB - The location of the stage 0 sporulation locus spo0F has been determined on a cloned fragment of Bacillus subtilis DNA. The spo0F gene and surrounding region was sequenced and was shown to code for a protein of Mr 14,286. The amino acid sequence of this deduced protein was 56% homologous to the amino-terminal domain of the spo0A gene product. The molecular weight of the Spo0F protein was approximately half that of the Spo0A protein, and its sequence was homologous to the amino-terminal half of the Spo0A protein. This same portion of the Spo0A protein showed ancestral relationship to the OmpR and SfrA regulatory proteins of Escherichia coli. Mutations in any of the genes encoding these proteins in either organism are highly pleiotropic and result in alterations in the regulation of membrane components, suggesting that they may have related roles in both organisms and that the stage 0 sporulation defect of spo0A and spo0F mutants is an indirect consequence of this regulatory system. PMID- 2997778 TI - Modes of action of aspirin-like drugs. AB - Current dogma holds that nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibition of the synthesis and release of prostaglandins. However, NSAIDs also inhibit the activation of neutrophils, which provoke inflammation by releasing products other than prostaglandins. We now report that NSAIDs (e.g., indomethacin, piroxicam) inhibit activation of neutrophils by inflammatory stimuli, such as C5-derived peptides and leukotriene B4, even when cyclooxygenase products generated in suspensions of stimulated neutrophils (prostaglandin E and thromboxanes) are present. Sodium salicylate (3 mM) greatly inhibited aggregation of neutrophils but had no effect on aggregation of platelets or production of thromboxane induced by arachidonate. Sodium salicylate and other NSAIDs also inhibit calcium movements (45Ca uptake, changes in fluorescence of chlortetracycline and quin-2). Aspirin, sodium salicylate, indomethacin, and piroxicam also enhanced the poststimulation increase in intracellular cyclic AMP. NSAIDs therefore inhibit early steps in neutrophil activation as reflected by their capacity to inhibit movements of Ca and to enhance intracellular levels of cyclic AMP. PMID- 2997781 TI - The ets sequence from the transforming gene of avian erythroblastosis virus, E26, has unique domains on human chromosomes 11 and 21: both loci are transcriptionally active. AB - Human DNA segments homologous to the ets region from the transforming gene of avian erythroblastosis virus, E26, were molecularly cloned and shown to be closely related to the viral equivalent by hybridization and partial sequence analysis. The transforming gene of E26 has a tripartite origin with the structure delta gag [1.2 kilobases (kb) from the viral gag gene]-myb(0.9 kb from the chicken myb gene)-ets (1.6 kb from the chicken ets gene). Human ets DNA is located on two distinct human chromosomes. The human ets-1 locus on chromosome 11 encodes a single mRNA of 6.8 kb; the second locus, ets-2 on chromosome 21, encodes three mRNAs of 4.7, 3.2, and 2.7 kb. The ets-related sequences of human DNA on chromosomes 11 and 21 are discontiguous, except for a small overlap region encoding 14 amino acids, where 12 are conserved between these two loci. By contrast, the chicken homolog has contiguous ets-1 and ets-2 sequences and is primarily expressed in normal chicken cells as a single 7.5-kb mRNA. We conclude that the ets sequence shared by the virus, the chicken, and humans is likely to contain at least two dissociable functional domains, ets-1 and ets-2. Thus, the tripartite transforming gene of E26 includes four distinct domains that may be functionally relevant for the transforming function of the virus (delta gag, myb, ets-1, and ets-2). PMID- 2997780 TI - The FLP recombinase of the yeast 2-micron plasmid: characterization of its recombination site. AB - The minimal size of the recombination site required for efficient FLP recombinase catalyzed recombination in vitro is no more than 28 base pairs, which includes parts of two 13-base-pair inverted repeats and all of an 8-base-pair spacer. The FLP recombinase cleaves the DNA at the boundaries of the spacer, becomes covalently linked to the spacer DNA via a 3' phosphate, and leaves a free 5' hydroxyl at the other end of the 8-base-pair spacer. The efficiency of recombination is reduced if the size of the spacer in a recombinant site is increased or decreased by 1 base pair, while the spacer in the second site is unaltered. Recombination between two sites with identical 1-base-pair additions or deletions in the spacer, however, is relatively unaffected. This result suggests that pairing of sequences in the spacer region is important in FLP promoted recombination events. The sequence asymmetry utilized by the recombinase to determine the orientation of the site is located uniquely within the spacer region. PMID- 2997782 TI - Interaction between two transcriptional control sequences required for tumor antigen-mediated simian virus 40 late gene expression. AB - Transcriptional control signals required for tumor (T)-antigen trans-activation of the simian virus 40 (SV40) late promoter include T-antigen binding sites I and II and the SV40 72-base-pair (bp) repeats. We have used in vivo competition studies to examine how these signals function in relationship to one another. In vivo competition with recombinant plasmids containing the entire SV40 late regulatory region and promoter sequences [map position (mp) 5171-272] results in quantitative removal of limiting trans-acting factor(s) required for late gene expression in COS-1 cells. Deletion of either the T-antigen binding sites (mp 5171-5243) or the 72-bp tandem repeat (mp 128-272) from the competitor plasmid results in markedly less efficient binding of the trans-acting factor, as judged by the loss of competition. Cotransfection of two separate plasmids, one containing the T-antigen binding sites I and II and the other containing the 72 bp repeats, fails to compete for the trans-acting factors. Insertion of increasing lengths of DNA sequences between the T-antigen binding sites and the enhancer sequences also dramatically reduces the efficiency of competition. These results suggest that efficient binding of trans-acting factors requires the presence, in cis, of at least two SV40 regulatory domains. Our studies further suggest that the distance separating these two transcriptional signals is important. PMID- 2997783 TI - Targeting of loaded Sendai virus envelopes by covalently attached insulin molecules to virus receptor-depleted cells: fusion-mediated microinjection of ricin A and simian virus 40 DNA. AB - Insulin molecules were covalently attached to detergent-solubilized Sendai virus envelope glycoproteins (HN and F polypeptides) by the use of the crosslinking reagent succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB). Reconstitution of modified viral glycoproteins (carrying covalently attached insulin) together with unmodified viral glycoproteins resulted in the formation of "fusogenic" viral envelopes bearing insulin molecules. Reconstitution of such fusogenic viral envelopes in the presence of ricin A or simian virus 40 (SV40) DNA resulted in the formation of viral envelopes bearing insulin molecules and loaded with ricin A or SV40 DNA. Such viral envelopes were able to bind to hepatoma tissue culture cells (HTCC) from which Sendai virus receptors were removed by treatment with neuraminidase. Incubation of viral envelopes loaded with ricin A with virus receptor-depleted HTCC resulted in fusion-mediated injection of the toxin, as inferred from inhibition of protein synthesis and decrease in cell viability of the microinjected cells. Fusion-mediated injection of SV40 DNA was inferred from the appearance of SV40 tumor antigen in microinjected cells. Binding and fusion of the loaded viral envelopes to neuraminidase-treated HTCC was mediated solely by the virus-associated insulin molecules. Addition of free insulin molecules inhibited binding of the viral envelopes and, consequently, the microinjection of ricin A and SV40 DNA. PMID- 2997784 TI - Mechanism of insulin action on membrane protein recycling: a selective decrease in the phosphorylation state of insulin-like growth factor II receptors in the cell surface membrane. AB - Insulin action in adipocytes leads to an increase in the steady-state number of cell surface glucose transporters and insulin-like growth factor II (IGF-II) receptors that appear to cycle continuously between the plasma membrane and a low density membrane fraction. The IGF-II receptor could be labeled to constant specific activity by incubating adipocytes with [32P]phosphate for 2 hr. The extent of phosphorylation of IGF-II receptors in plasma membranes and in low density microsomes was compared using 125I-labeled IGF-II binding and immunoblotting to quantitate the receptors present in each fraction. Receptors in the plasma membrane fraction of control cells incorporated approximately 1 molecule of phosphate per IGF-II binding site or 2 to 3 times more phosphate than was incorporated into IGF-II receptors in the low-density microsomes. Addition of insulin to labeled adipocytes did not change the specific activity of the gamma phosphate of ATP but produced a specific and sharp decrease in the 32P-phosphate content of IGF-II receptors in the plasma membrane. No change due to insulin in the phosphorylation of receptors derived from low-density microsomes was observed. The insulin-mediated decrease in the [32P]phosphate content of IGF-II receptors from the plasma membrane was rapid in onset, paralleled the increase in the number of IGF-II receptors on the cell surface, and persisted for at least 30 min in the presence of insulin. Furthermore, when the effect of insulin to increase the number of IGF-II receptors in the cell surface was prevented by cooling cells to 5 degrees C, the decrease in phosphorylation of plasma membrane receptors could still be observed, indicating that this latter effect is not secondary to receptor redistribution. These data indicate that insulin inhibits one or more IGF-II receptor kinases or increases phosphatase activity, or both. Decreased phosphorylation of such insulin-sensitive plasma membrane components as IGF-II receptors may play a role in increasing their steady-state cell surface concentration, perhaps by delaying their internalization. PMID- 2997785 TI - Evidence that intracellular magnesium is present in cells at a regulatory concentration for protein synthesis. AB - When extracellular magnesium is reduced by a factor of 50 (from 1.0 to 0.02 mM), the total intracellular magnesium of a spontaneously transformed clone of 3T3 cells decreases by 30-50%. Protein synthesis rates in these cells were measured as the intracellular magnesium decreased. Protein synthesis rates and magnesium content were found to decrease in parallel with each other. At 3 hr, a decrease to 84% of control values of magnesium content was accompanied by a decrease to 85% of control values of leucine incorporation rates. A larger inhibition had occurred by 12 hr, when the magnesium had decreased to 67% and leucine incorporation rates had decreased to 57%. When magnesium was restored to magnesium-deprived cells, both magnesium content and leucine incorporation increased about 2-fold by 1 hr. In the experiments reported here, initial small changes in magnesium content are associated with changes in protein synthesis rates. This strongly suggests that magnesium is present at a regulatory rather than excess concentration for protein synthesis. The results are consistent with a role for intracellular magnesium in the regulation of protein synthesis and support the hypothesis that magnesium has a central role in the regulation of metabolism and growth. PMID- 2997788 TI - One role for DNA methylation in vertebrate cells is strand discrimination in mismatch repair. AB - Although the occurrence of 5-methylcytosine (m5C) in DNA is widespread, the function of this modified base remains unclear. At some specific sites it apparently has an effect in controlling gene expression, but many sites do not appear to be involved in this regulation. Balanced against its regulatory usefulness at some sites is the mutational risk it imposes upon the cell. Deamination of m5C can lead to its replacement by thymine (T). One possible role for excess methylation is strand discrimination in the repair of mismatches. We constructed the complementary hemimethylated single-base-pair mismatches, G T and A C, at a CG site in simian virus 40 DNA, transfected these into the host African green monkey kidney cells (CV-1), and examined DNA of the progeny for repair at this site. Hemimethylation at two Hha I sites (Gm5CGC) bracketing the mismatch directed repair to occur only on the unmethylated strand. Methylation at the multiple Cm5CATGG and Gm6ATC sites, a pattern normally seen in bacteria, also instructed repair to proceed on the unmethylated strand, although less efficiently. Hemimethylation at only one site, adjacent to the mispaired bases (Hpa II, Cm5CGG) produced repaired molecules in a ratio that may represent random repair of the A C mismatch and strand-directed repair in the complementary G T mismatch. The -mCG- -GT- mismatch could result from deamination of m5C in the most commonly methylated dinucleotide in vertebrates, CpG. Methylation may be able to compensate for the errors it causes by serving as a mechanism for strand discrimination in correcting those errors. In addition, single-strand nicks were also shown to direct repair. PMID- 2997787 TI - Transforming and nontransforming growth factors are present in medium conditioned by fetal rat calvariae. AB - Conditioned medium recovered from fetal rat calvarial cultures contains an autocrine factor termed bone-derived growth factor (BDGF); this factor has been purified by acid extraction, gel-permeation chromatography, and two reversed phase HPLC steps and examined for mitogenicity on normal rat kidney fibroblasts (NRK, clone 49F). HPLC-purified BDGF caused a dose-related increase in cell number, DNA content, and [3H]thymidine incorporation into acid-insoluble material. Since highly purified BDGF appeared less mitogenic than cruder preparations, the latter were tested for additional growth factors, with particular attention to those required for anchorage-independent colony formation in soft agar. BDGF did not displace 125I-labeled epidermal growth factor (EGF) in a radioligand-receptor assay, indicating the absence of EGF and transforming growth factor alpha (TGF-alpha). Without EGF, no BDGF preparation induced NRK cells to form soft agar colonies. However, calvarial conditioned medium contained a factor which, like TGF-beta, induced large soft-agar colonies in the presence of EGF; this TGF-beta-like factor did not copurify with BDGF. Polyclonal antibodies against platelet-derived growth factor did not neutralize the effects of BDGF on NRK cells. BDGF is a potent mitogen for nonskeletal-tissue-derived fibroblasts. Although crude BDGF preparations do contain TGF-beta, BDGF is distinct from this factor and others necessary for NRK cell transformation to anchorage-independent growth. PMID- 2997786 TI - Induction of the proto-oncogene fos by nerve growth factor. AB - Nerve growth factor (NGF) causes the differentiation of PC12 cells to sympathetic neuron-like cells and also induces a rapid but transient expression of fos mRNA and protein. fos mRNA transcripts can be detected 5 min after the addition of NGF, are maximally abundant after 30 min, and then their levels decrease. fos protein synthesis parallels the expression of fos mRNA, and the induced fos proteins are located in the nucleus. cAMP, epidermal growth factor, the phorbol ester phorbol 12-myristate 13-acetate, and K+ depolarization also induce the fos gene. Growth of PC12 cells in the presence of dexamethasone, which induces differentiation into chromaffin-like cells, is not accompanied by fos expression. We propose that while fos gene induction is associated with the differentiation of PC12 cells to sympathetic nerve, its enhanced expression is primarily involved in the anabolic responses induced by NGF and many growth factors. PMID- 2997789 TI - Fabry disease: isolation of a cDNA clone encoding human alpha-galactosidase A. AB - Fabry disease is an X-linked inborn error of metabolism resulting from the deficient activity of the lysosomal hydrolase, alpha-galactosidase A (alpha-Gal A; alpha-D-galactoside galactohydrolase, EC 3.2.1.22). To investigate the structure, organization, and expression of alpha-Gal A, as well as the nature of mutations in Fabry disease, a clone encoding human alpha-Gal A was isolated from a lambda gt11 human liver cDNA expression library. To facilitate screening, an improved affinity purification procedure was used to obtain sufficient homogeneous enzyme for production of monospecific antibodies and for amino terminal and peptide microsequencing. On the basis of an amino-terminal sequence of 24 residues, two sets of oligonucleotide mixtures were synthesized corresponding to adjacent, but not overlapping, amino acid sequences. In addition, an oligonucleotide mixture was synthesized based on a sequence derived from an alpha-Gal A internal tryptic peptide isolated by reversed-phase HPLC. Four positive clones were initially identified by antibody screening of 1.4 X 10(7) plaques. Of these, only one clone (designated lambda AG18) demonstrated both antibody binding specificity by competition studies using homogeneous enzyme and specific hybridization to synthetic oligonucleotide mixtures corresponding to amino-terminal and internal amino acid sequences. Nucleotide sequencing of the 5' end of the 1250-base-pair EcoRI insert of clone lambda AG18 revealed an exact correspondence between the predicted and known amino-terminal amino acid sequence. The insert of clone lambda AG18 appears to contain the full-length coding region of the processed, enzymatically active alpha-Gal A, as well as sequences coding for five amino acids of the amino-terminal propeptide, which is posttranslationally cleaved during enzyme maturation. PMID- 2997790 TI - Identification and expression of a nuclear antigen from the genomic region of the Jijoye strain of Epstein-Barr virus that is missing in its nonimmortalizing deletion mutant, P3HR-1. AB - An Epstein-Barr virus (EBV) deletion mutant, HR-1, cannot immortalize lymphocytes. HR-1 was derived from a virus strain, Jijoye, that is immortalization competent. Using human antiserum from certain patients with chronic active EBV infection, we have identified in Jijoye cells a protein of apparent mass of 78-80 kDa that is missing in cells with the HR-1 genome. A protein of identical size and antigenicity has been stably expressed in mouse LTK cells by gene transfer with cloned Jijoye EBV DNA that encompasses the deletion in the HR-1 genome. The expressed product is a nuclear neoantigen. The polypeptide we have identified is likely to be essential in the immortalization process. PMID- 2997791 TI - Solubilization of a thromboxane A2/prostaglandin H2 antagonist binding site from human platelets. AB - A binding site for 9,11-dimethylmethano-11,12-methano-16-(3-[125I]iodo-4 hydroxyph eny l)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranorthromboxane A2 ([125I]-PTA-OH), a thromboxane A2/prostaglandin H2 antagonist, was solubilized into the 200,000 X g supernatant from human platelet membranes by using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Binding to the solubilized site was saturable, displaceable, and reversible. Displaceable binding was not affected by sodium, potassium, or phosphate concentrations up to 50 mM or by magnesium to 5 mM but was increased 14% (P less than 0.05) by 5 mM calcium. A pH optimum for displaceable binding occurred between pH 7.0 and 7.5. Scatchard analysis of [125I]-PTA-OH binding to the solubilized binding site revealed a single class of sites, having a dissociation constant (Kd) of 66 +/- 16 nM (n = 3) and a Bmax of 750 +/- 80 fmol/mg of protein. The Kd for the membranes prior to solubilization was 47 +/- 11 nM (n = 3) and the Bmax was 700 +/- 90 fmol sites per mg of protein. The association rate constant, k1, was 1.57 X 10(7) M-1 X min-1 and the dissociation rate constant, k 1, was 0.61 +/- 0.04 min-1 (n = 4), yielding a Kd (k-1/k1) of 39 nM. Several thromboxane A2/prostaglandin H2 agonists and antagonists displaced bound [125I] PTA-OH at concentrations similar to those at which they affect platelet aggregation. Collectively, these observations suggest that the solubilized protein is the thromboxane A2/prostaglandin H2 binding site that mediates platelet aggregation. PMID- 2997792 TI - The low affinity taurine-binding protein may be related to the insulin receptor. PMID- 2997793 TI - Taurine scavenges oxidized chlorine in biological systems. PMID- 2997794 TI - The anticonvulsant actions of two taurine derivatives in genetic and chemically induced seizures. PMID- 2997795 TI - The biological oxidation of hypotaurine to taurine: hypotaurine as an antioxidant. PMID- 2997797 TI - Determination of fluorescent oxytetracycline complexes in dental and skeletal hard tissues by rapid and accurate quantitative method. PMID- 2997796 TI - Biochemical characterizations of human osteoblasts in culture. PMID- 2997798 TI - Physiologic root resorption in cats: a process involving cyclic nucleotides and prostaglandins. PMID- 2997799 TI - Bone cells, bone metabolism and cGMP. PMID- 2997800 TI - Polyunsaturated fat diet: the Cleveland Clinic Foundation's experience. PMID- 2997801 TI - Alterations in genome structure and expression in aging human fibroblasts. PMID- 2997802 TI - Hemoglobin oxidation and inter-relationship with lipid peroxidation in the red cell. PMID- 2997803 TI - Prostaglandins, cyclic AMP production and biological activity of alloimmune thymocytes. AB - The effect of alloimmunized non-adherent thymocytes on the spontaneous activity of the mouse atria was studied. BALB/c anti C3H non-adherent thymocytes induced negative inotropic effect on C3H atria. Cell-free supernatant from non-adherent thymocytes induced the same biological activity. This activity was blunted by the inhibition of non-adherent immune thymocytes' cyclo-oxygenase activity. PGE was present in higher amounts in free-cell supernatant from BALB/c anti C3H thymocytes plus C3H atria than in those from non-immune thymocytes. Intracellular levels of immune thymocytes cAMP are raised in comparison with those of normal thymocytes. It is proposed that alloantigen stimulates non-adherent thymocytes, increasing intracellular levels of cAMP and PGE. Immune thymocytes, release PGE upon recognition of the alloantigens expressed in the atria and this triggers a negative inotropic effect. The increment in immune thymocyte cAMP appears to be associated with the activation of thymocytes cyclo-oxygenase activity by alloimmunization. PMID- 2997804 TI - Platelet aggregation and evaluation of the ratio thromboxane B2/6-keto prostaglandin F1 alpha in the plasma of patients on long term cimetidine treatment. AB - It has been reported that a long term treatment with cimetidine may give rise to thrombotic complications and may cause reversible damage to blood cells. In 57 patients on long term cimetidine treatment, platelet aggregates, platelet aggregation in vitro, plasma 6-keto-PGF1 alpha/thromboxane B2 ratio and platelet cyclic AMP levels were assessed. In 52% of the patients, platelet aggregate ratios were abnormal and collagen and ADP-hypersensitive platelets were observed. Such alterations began occurring after the first month of therapy and were shown to worsen progressively during the administration. Four of these patients, who developed unexpected thrombotic compliances after about 7 months of therapy, showed higher than normal plasma thromboxane B2, lower plasma 6-keto-PGF1 alpha and two of them, lower platelet cyclic AMP concentrations. It is suggested that cimetidine, through an unknown mechanism which probably involves activation of endogenous cyclic AMP phosphodiesterase, may favour the action of platelet aggregating agents. PMID- 2997805 TI - Involvement of brain histamine in delta 9-tetrahydrocannabinol tolerance and withdrawal. AB - The involvement of brain histamine (HA) in delta 9-tetrahydrocannabinol (delta 9 THC) tolerance and dependence was studied in rats. Rats treated for 5 days with delta 9-THC (2-6 mg/kg, IV) developed tolerance to the hypothermic effects of the drug. Tolerance also developed over the 5 day period to the decrease in brain regional HA concentrations observed after an acute injection of delta 9-THC. Administration of the tricyclic antidepressant drug clomipramine hydrochloride to tolerant rats induced a withdrawal-like behavioural syndrome. Accompanying this behaviour was a fall in HA concentrations of the midbrain, cortex, medulla oblongata/pons and the cerebellum. Administration of delta 9-THC, but not of the delta 9-THC vehicle, prior to clomipramine challenge attenuated both the intensity of the withdrawal-like syndrome and the reductions in brain regional HA concentration. PMID- 2997806 TI - Gammahydroxybutyric acid: central biochemical and behavioral effects in neonatal rats. AB - Administration of gammahydroxybutyric acid (GHBA) to 4 days old animals caused a dose dependent decrease in locomotor activity. GHBA also induced a marked hypoventilation, irregular breathing and finally apnea, while heart rate was slightly increased. Changes in monoamine neurotransmitter turnover indicated an inhibition of dopamine (DA) neurotransmission. It is concluded that GHBA mechanisms in the neonatal rat brain are biochemically as well as functionally mature at an early age and that the effects on locomotor activity and respiratory regulation at least partly may involve interactions with central DA neurotransmission. PMID- 2997807 TI - Attenuation of cyclophosphamide-induced taste aversions in mice by prochlorperazine, delta 9-tetrahydrocannabinol, nabilone and levonantradol. AB - A series of experiments were performed with adult CD-1 male mice to evaluate the antiemetic effects of several compounds using the conditioned taste aversion procedure. The antiemetics were administered IP immediately prior to a 30-min conditioning trial in which a novel tasting solution (0.3% saccharin) was presented to the subjects. The emetics, apomorphine and the cancer chemotherapeutic drug cyclophosphamide, were given IP immediately after the conditioning trial at doses that induced taste aversions. Three days later the mice received a two bottle preference test (saccharin vs. water) and the percent saccharin consumed of the total fluid intake was calculated. Doses of the phenothiazine antiemetic prochlorperazine (1 and 3 mg/kg) attenuated the aversions produced by 0.3 and 1.0 mg/kg apomorphine. Doses of drugs currently approved or under clinical investigation as antiemetics in conjunction with cancer chemotherapy, i.e., prochlorperazine (1.0 mg/kg), delta 9 tetrahydrocannabinol (0.3 and 1.0 mg/kg) and nabilone (0.01 and 0.03 mg/kg), significantly attenuated the taste aversions induced by cyclophosphamide. Levonantradol at doses of 0.03 and 0.06 mg/kg, however, did not attenuate cyclophosphamide-induced taste aversions. Conditioned taste aversions produced by emetic drugs warrants investigation as a model for evaluating potential antiemetics. PMID- 2997808 TI - A possible role of a GABAergic mechanism in the convulsant action of RO5-4864. AB - The purpose of the present investigation was to determine the possible role of GABAergic mechanism in the convulsant action of RO5-4864. Benzodiazepines (BZ) and other agents which facilitate central GABAergic transmission delayed the onset of facial and forelimb clonus, whereas tonic hind limb extension was blocked in a dose-dependent manner. RO5-4864-induced convulsions were blocked by diazepam, clonazepam, pentobarbital, ethanol and amino-oxyacetic acid (AOAA). RO5 4864-induced convulsions were not blocked by the BZ antagonist RO15-1788. Specifically, RO15-1788 caused a decrease in the onset of severity component of tonic seizures, which tended to become generalized and precipitated in a tonic extension of the hindlimbs. Further, subconvulsive doses of a direct GABA receptor antagonist, bicuculline, enhanced the proconvulsant action of RO5-4864, indicating thereby a potential antagonism of the central GABAergic transmission. These observations strongly suggest that RO5-4864 probably elicits convulsions by selective impairment of the GABAergic transmission. PMID- 2997809 TI - Pyrazoloquinoline benzodiazepine receptor ligands: effects on schedule-controlled behavior in dogs. AB - The effects of diazepam and the pyrazoloquinoline benzodiazepine receptor ligands CGS8216, CGS9896, and CGS9895 on schedule-controlled responding were studied in dogs. Responding was maintained under a multiple fixed-interval (FI) 5-min fixed ratio (FR) 30 response schedule of food presentation. Diazepam (PO) produced dose related decreases in response rates under FR component. Under the FI, rates first increased and then decreased with increasing doses of diazepam. Diazepam also produced a dose-related disruption of the temporal pattern of responding under the FI as measured by decreases in quarter-life values. CGS8216 IV produced dose related decreases in response rates under both components. The highest oral dose of CGS8216 also decreased rates in both components. CGS8216 was approximately 100 times more potent by the IV route as compared to the oral route. CGS9896 IV had no significant effect on responding under either component of the multiple schedule. However, with increasing doses of CGS9896 PO, response rates under both components first decreased and then returned to control values. CGS9895 PO was without significant effect on responding. When CGS8216 was administered concomitantly with graded doses of diazepam, the former drug blocked the rate decreasing effects of diazepam under the FR component, but not the rate increasing effects of diazepam under the FI. The present results demonstrate that although these three pyrazoloquinolines are benzodiazepine receptor ligands, they do not exhibit diazepam-like effects on schedule-controlled behavior. PMID- 2997810 TI - Effect of ACTH, beta-endorphin, morphine and naloxone on the release of cortisol by isolated adrenal glands. AB - The release of cortisol (determined by RIA) from isolated slices of adrenal glands of guinea pigs is stimulated by ACTH, by beta-endorphin, and by morphine in a concentration-dependent way; naloxone gives a small stimulation which is not related to its concentration. Naloxone inhibits the effect of ACTH (1.11 X 10( 11) M) in a competitive manner with an IC50 of about 3.10(-9) M. Also morphine and beta-endorphin inhibit the effect of ACTH, but not in competitive manner. Naloxone (10(-9)-10(-7) M) gives a concentration-related inhibition of the increase of cortisol release produced by morphine (10(-8) M) and by beta endorphin (1.44 X 10(-10) M). These data suggest a similarity in the conformation of ACTH, beta-endorphin, morphine and naloxone towards the binding sites of ACTH of the guinea pig adrenal glands. PMID- 2997811 TI - Role of cyclooxygenase products in the lung action of leukotrienes A4, B4, C4, D4 and E4. AB - Leukotrienes (LT) LTA4, LTB4, LTC4, LTD4 and LTE4 induced marked contractions of guinea pig lung parenchymal strips mounted in organ baths. These contractions were inhibited differentially (40-50% for LTA4, LTC4, LTD4 and LTE4, and 90% for LTB4) by indomethacin (20 micrograms.ml-1; 55.9 microM). Two novel inhibitors of thromboxane synthetase (OKY-1581 and OKY-046) reduced the myotropic activity of the lung strips and the release of prostaglandins and thromboxanes from the perfused guinea pig lungs stimulated by LTB4 and LTD4. The release of cyclooxygenase products prostaglandin F2 alpha, thromboxane B2 and 12 hydroxyheptadecatrienoic acid by guinea pig lungs following stimulation with LTB4 and LTD4 was also measured by gas chromatography-mass spectrometry. The role of prostaglandins and thromboxanes in the lung actions of leukotrienes was confirmed using a cascade superfusion system and classical organ baths. Although prostaglandins and thromboxanes contribute to the contractile effect of LTB4 on the guinea pig lung whereas they may play a lesser role in the action of the peptidoleukotrienes (approx. 40-50%), stimulation of their release by the peptidoleukotrienes is many times more effective than by LTB4. PMID- 2997812 TI - Monoclonal antibodies to the insulin receptor. PMID- 2997813 TI - Evaluation of testing modalities for peripheral neuropathy in lepromatous Hansen's disease. AB - To assess methods for detecting peripheral neuropathy, 28 previously untreated patients with lepromatous Hansen's disease underwent upper extremity manual muscle testing, sensory testing by using monofilaments, and electrophysiological nerve conduction studies (motor and sensory) at their initial examination. All but three patients demonstrated some abnormality identified by at least one of the testing procedures. Sensory testing with monofilaments located the greatest number of abnormalities found in 24 of the 28 patients. Next, electrophysiological testing demonstrated neuropathy in 21 of the 28 patients tested; 20 of these patients had sensory abnormalities and 20 had motor irregularities. The least sensitive method was manual muscle testing, which detected abnormalities in only 12 patients. Furthermore, sensory testing with monofilaments revealed peripheral neuropathy in 5 patients whose electrophysiological studies showed normal sensory patterns, and electrophysiological testing detected abnormalities in 1 patient whose sensory monofilament examination was normal. The results of this study support the usefulness of all three testing modalities. PMID- 2997814 TI - Post-weaning crowding induces corticoadrenal hyperreactivity in male mice. AB - The effect of various population densities on corticoadrenal function was studied in prepuberal male mice. High population densities decreased body weight gain. Neither adrenal weight nor basal serum corticosterone were modified by crowding. However, corticoadrenal response to some acute stresses such as noise and forced swimming was higher in crowded mice. As corticoadrenal response to adrenocorticotropin remained unaffected, it appears that crowding induced pituitary-adrenal hyperreactivity. Neither the defecation rate nor exploratory activity were altered by crowding, suggesting a dissociation between pituitary adrenal responsiveness and behavioral measures presumably related to emotional arousal. These discrepancies may possibly be due to the higher sensitivity of corticoadrenal function to environmental changes. Our results suggest that crowding would be suitable as a model for chronic continuous stress. PMID- 2997815 TI - A retrograde gradient for disruption of a conditioned aversion to drinking cold water by ECS administered during the CS-US interval. AB - Rats were used to examine the effects, upon a conditioned aversion to cold drinking water, of electroconvulsive shock (ECS) delivered during the delay between cue and unconditioned stimulus. An injection of LiCl (US) 30 min after ingestion of novel cold water (CS) produced a reliable aversion to the cold water. ECS given immediately following the ingestion of cold water substantially attenuated this aversion. An orderly decrease in the attenuation of the aversion was observed when ECS was delayed 5, 10 or 20 min after offset of the cold water cue. The results indicate that ingestive cue aversions can be formed without electrochemical neural-transmission-based representation of the cue being maintained during the CS-US interval. The differential effectiveness of ECS suggests that this agent retroactively interferes with processing of the ingestive cue. PMID- 2997816 TI - Influence of regimen (roughage vs. concentrates) on satiety and forestomach motility in sheep. AB - The influence of concentrate intake on the subsequent intake of hay was investigated in sheep fitted with a rumen cannula and electrodes on the reticulum wall to measure volatile fatty acids concentration and reticular motility, respectively. In sheep fasted during night time, a previous meal of concentrates given 30, 60 or 120 min before feeding hay, did not modify significantly (p less than 0.05) the first hour and the first 3 hour intake of hay despite a large increase in ruminal total volatile fatty acid concentration. However, the daily intake of hay was significantly (p less than 0.05) reduced. Similarly, the reticular motility in response to feeding hay was not affected by the previous meals of concentrate. It is concluded that in fasted sheep on a hay ration the short-term satiation of a hay meal is not affected by foregoing intake of concentrates and that the hay intake is not controlled by ruminal volatile fatty acid levels. PMID- 2997817 TI - Dopaminergic behavior in frontal decorticated rats. AB - Decortication of the frontal neocortex in rats enhanced the increased general activity caused by methamphetamine, 0.15 mg/kg, SC. However, the low, 0.1 mg/kg, SC, and high, 0.5 mg/kg, SC, dose effects of apomorphine were not affected by the decortication. The cataleptic effect of haloperidol, 2 mg/kg, SC, was decreased. The data suggest that the frontal cortex may inhibit dopamine release in mesolimbic and nigrostriatal areas. However, the sensitivity of presynaptic or postsynaptic dopamine receptor systems in the nucleus accumbens area appears to be unaltered by frontal decortication. PMID- 2997818 TI - Mapping of contraversive and ipsiversive circling responses to ventral tegmental and substantia nigra electrical stimulation. AB - Circling responses to ventral mesencephalic electrical stimulation were studied over a range of stimulation sites and a range of stimulation frequencies. Contraversive circling was seen with 62% of the sites stimulated; positive sites were found in the ventral tegmental area, the medial lemniscus, and the zona compacta and zona reticulata of the substantia nigra; frequency thresholds were in the range of 15-60 Hz. Ipsiversive circling was seen with 30% of the sites stimulated; these sites tended to be in the region of nigral dopamine cell bodies, but this correlation was not perfect; some ipsiversive circling sites were found in zona reticulata, and some were found dorsal to zona compacta. Ipsiversive circling had high frequency thresholds, in the range of 100-150 Hz, and generally had longer latencies than those for contraversive circling. In one third of the cases where ipsiversive circling was seen with high frequency stimulation, contraversive circling was obtained with lower frequency stimulation at the same site. In these cases contraversive circling was seen first, with short latency at low frequencies. As stimulation frequency was raised, the period of contraversive circling became shorter and the animals then stopped and reversed direction. The dispersion of positive sites rules out the suggestion that there are simple medial-lateral differences in the direction of circling elicited by nigral stimulation, and the dispersion of sites and the frequency response of the effects suggest that neither direction of circling results from direct depolarization of the dopaminergic cells themselves. PMID- 2997820 TI - Circadian changes of gluconeogenic enzymes in irradiated rats. AB - The authors studied the effect of whole body irradiation at different times of day on the circadian rhythms of gluconeogenic enzymes. They found that: 1. liver and kidney enzyme activities were highest in the light part of the day and lowest in the middle of the dark part; 2. 12-h circadian rhythm of glucose-6-phosphatase and fructose-1, 6-bisphosphatase activity in the liver and the renal cortex followed a similar course; 3. a lethal whole body dose of 14.4 Gy X-rays did not affect the circadian oscillation curves of the given enzymes, with the exception of fructose-1, 6-bisphosphatase in the liver of irradiated rats, where the rhythm was lost. PMID- 2997819 TI - The effects of thyrotropin releasing hormone on rats with lesions of the mesolimbic and nigrostriatal dopamine systems. AB - We report the effects of intracerebroventricular (ICV) administration of thyrotropin releasing hormone (TRH), a TRH metabolite histidyl-proline diketopiperazine (DKP) and systemically administered d-amphetamine (AMP) on the locomotor activity of two groups of rats which had previously received bilateral injections of either 6-hydroxydopamine (6-OHDA) or saline into the nucleus accumbens. Both TRH and AMP enhanced locomotor activity in control, but not lesioned animals, whereas DKP had very little effect. In a second experiment, the effects of ICV administration of saline, TRH and DKP were tested on rotational behaviour in rats with unilateral lesions of the substantia nigra. Neither peptide induced significant circling on its own. However, coadministration of TRH or DKP with systemically administered AMP enhanced rotation above that found after injection of AMP alone. These results suggest that TRH can act on mesolimbic and nigrostriatal dopamine systems, either directly or by modulating the effects of other dopaminergic agents. PMID- 2997821 TI - The influence of tissue treatment on brain Na+, K+-ATPase activity and its response to vanadate inhibition. AB - This study has compared the effect of freezing in situ and decapitation without freezing on the Na+,K+-ATPase activity in mouse cerebral cortex homogenates under otherwise comparable conditions. The Na+,K+-ATPase activity was substantially influenced by the sample preparation; a twofold value was obtained for frozen samples as compared to that in fresh samples. Not only basal activity, but also the sensitivity of the enzyme towards vanadate inhibition depended on tissue treatment; lesser inhibition was observed in frozen samples. These findings suggest the possible implication of altered enzyme characteristics due to sample preparation while studying the influence of various other factors on enzyme activity. PMID- 2997823 TI - ACTH, beta-endorphin and met-enkephalin: peripheral modifications during the stress of human labor. AB - We investigated the psychoneuroendocrine and emotional correlates of the natural stress situation of human labor. State anxiety, subjective pain, plasma ACTH, peripheral plasma beta-lipotropin (Beta-LPH), beta-endorphin (Beta-EP), and met enkephalin (Met-Enk) were serially evaluated at six predetermined time points before, and after labor in a sample of 14 women with normal pregnancies. State anxiety and subjective pain showed a progressive increase during labor, with a levelling during the final stage. Plasma Beta-EP and ACTH showed a similar progressive increasing from baseline until the end of labor. Beta-LPH showed no significant modification. Met-Enk remained at nearly baseline values throughout labor, with a marked progressive rise in the postpartum stage. The findings of this study seem to confirm the role of plasma Beta-EP as a stress hormone. Possible relationship between pain and anxiety curves and plasma Beta-EP are discussed in light of psychobiological studies on stress, the opioid system and analgesia. Plasma Met-Enk, according to our findings, should probably not be regarded as a stress hormone. Its rise in the postpartum stage might be as one of the psychoneuroendocrine mechanisms maintaining elevated prolactin levels during lactation. PMID- 2997822 TI - Tritiated imipramine binding to platelets is decreased in patients with agoraphobia. AB - Controversy exists regarding the relationship between anxiety states and major depression. We studied the binding of tritiated imipramine to platelet membranes in order to determine if patients with agoraphobia and panic attacks differed from depressed subjects or healthy volunteers on this biological parameter. Mean (+/- SD) Bmax and Kd values were significantly lower in patients with agoraphobia and panic attacks (787 +/- 276 fmole/mg protein and 0.35 +/- 0.14 nM, respectively) than in healthy volunteers (1237 +/- 201 fmole/mg protein and 0.71 +/- 0.37 nM, respectively). In addition, patients with agoraphobia and panic attacks had binding parameters that were similar to those of patients with bipolar or familial pure depressive disorder, but significantly lower than those of patients with depressive spectrum or sporadic depressive disorder. These findings have implications for both the nosology and pathophysiology of anxiety disorders. PMID- 2997824 TI - Characterization of the discriminative stimulus effects of centrally administered morphine in the rat. AB - The discriminative stimulus effects of centrally administered morphine were characterized in rats trained to discriminate 3.0 mg/kg SC morphine from saline in a two-choice discrete-trial avoidance paradigm. The intracerebroventricular (ICV) administration of 0.3-10 micrograms morphine engendered morphine appropriate responding, morphine administered ICV being nearly 1000 times as potent as morphine administered SC. Cannula implantation itself did not affect the sensitivity of the rats to the discriminative effects of morphine. The onset of the discriminative stimulus effects of ICV morphine was not immediate; stimulus generalization comparable to that produced by 3.0 mg/kg morphine occurred 30-60 min after the injection of 1.0 or 10 micrograms ICV morphine and persisted for 90 and 150 min, respectively. Naltrexone blocked the discriminative stimulus effects of 10 micrograms ICV morphine in a dose-related manner. Complete antagonism of the stimulus effects of this dose of morphine was obtained with 0.01-0.03 mg/kg SC naltrexone. When administered centrally, the relatively lipid insoluble naltrexone methobromide completely antagonized the discriminative effects of 3.0 mg/kg morphine at a median effective dose of 0.3 micrograms. In contrast, when injected systemically at a dose of 1.0 mg/kg (approximately 500 micrograms), naltrexone methobromide failed to block the discriminative stimulus effects of either 10 micrograms ICV morphine or the SC training dose. Thus, periventricular brain sites appear to be involved in mediating the discriminative stimulus effects of morphine in the rat. PMID- 2997826 TI - Sucrose intake unaffected by fenfluramine but suppressed by amphetamine administration. AB - The present study examined the acute effects of the anorectic agents, fenfluramine and amphetamine, on nutrient selection in male Sprague-Dawley rats. One group of animals received continuous access to both granulated sucrose and Purina chow (presented in separate cups), while the other group received only Purina chow. Four doses of fenfluramine (0, 1.5, 3.0, 6.0 mg/kg) and four doses of amphetamine (0, 0.5, 1.0, 2.0 mg/kg) were tested. Injections were given at the beginning of the feeding period, and nutrient intakes were measured at 2, 4, and 8 h postinjection. While both fenfluramine and amphetamine resulted in similar overall decreases in caloric intakes, the two drugs produced different effects on nutrient selection. Following fenfluramine administration, sucrose intakes remained relatively unaffected, while Purina chow intakes showed pronounced suppression. In contrast, amphetamine administration resulted in a more equal suppression of both sucrose and Purina chow intakes. While the overall decreases in food intake seen with fenfluramine and amphetamine may be linked to changes in central neurotransmitter levels, the sparing of sucrose consumption observed with fenfluramine may also involve drug-induced changes in peripheral metabolism. PMID- 2997825 TI - A stimulatory effect of intraaccumbens injections of noradrenaline on the behavior of rats in the forced swim test. AB - Intraaccumbens injections of catecholamines noradrenaline and dopamine, though not of serotonin, stimulated locomotion by rats in an open field, 10-15 min later. Similar effects were observed 5 min after microinjection of apomorphine whereas clonidine only attenuated locomotor activity. On the other hand, intraaccumbens administration of phenylephrine, isoproterenol and quipazine, in doses similar to an effective dose of noradrenaline, did not alter rat open field behavior. The escape-directed activity of rats in the forced swim test (FST) was stimulated 5 min after local administration of noradrenaline, phenylephrine, isoproterenol or apomorphine only. No effects in the FST were observed 15 min after noradrenaline injection or after intracaudate noradrenaline administration. The stimulatory effects of intraaccumbens noradrenaline injection in the FST were antagonized by the local pretreatment of rats with phentolamine, though not with propranolol. Accordingly, it is possible to conclude that both catecholamines, but not serotonin, play complex and probably distinct roles within the nucleus accumbens in the stimulation of activity by rats in the FST and the open field test. PMID- 2997827 TI - Morphine and delta 9-tetrahydrocannabinol: two-way cross tolerance for antinociceptive and heart-rate responses in the rat. AB - Tail-flick analgesic responses and heart-rate changes were measured in male Sprague-Dawley rats challenged with an acute IP morphine sulfate (MS) or delta 9 THC injection after receiving daily injections of delta 9-THC or morphine, respectively. Degree of tolerance development to each agent was determined before the cross-tolerance challenge was administered. Cross tolerance occurred to analgesic and bradycardic effects of a 10 mg/kg THC challenge in rats receiving 50 mg/kg MS injections over a 23-day period. Cross tolerance to the bradycardic effects of a 20 mg/kg MS challenge occurred in rats receiving seven daily 10 mg/kg delta 9-THC injections and to MS tail-flick analgesia after 14 days. Although rapid tolerance occurred during administration of both agents, cross tolerance to THC bradycardia occurred only in groups exhibiting complete tolerance to MS injections; cross tolerance to MS bradycardia was observed in animals that were only partially tolerant to THC injections. The data extend earlier cross tolerance data in the mouse to the rat, and provide new information using heart rate, a response that may mirror aversive internal states induced by drugs. PMID- 2997828 TI - The locomotor-reducing effects of GABAergic drugs do not depend on the GABAA receptor. AB - The locomotion-reducing effect of the GABAB agonist baclofen was compared with that of the GABAA agonists, aminopropanesulfonic acid (APSA) and THIP. It was found that baclofen was more potent than the other drugs. After intraventricular injection, baclofen induced almost complete immobility, whereas APSA did not affect locomotor activity. THIP had an intermediate effect. The GABA transaminase inhibitor gamma-acetylenic GABA (GAG) provoked a dose-dependent reduction of locomotion. Neither the effects of THIP nor those of GAG could be blocked by concurrent administration of bicuculline. The antagonist itself did not affect locomotor activity. It is concluded that the GABAA receptor is not important for the locomotion-reducing effects of GABAergic drugs. PMID- 2997829 TI - Effects of carbamazepine on noradrenergic mechanisms in affectively ill patients. AB - Noradrenergic mechanisms have been postulated to account for the anticonvulsant and psychotropic effects of carbamazepine. In order to assess this possibility in man, cerebrospinal fluid (CSF) was obtained from affectively ill patients before and during treatment with carbamazepine (average duration 29 days) at doses averaging 860 mg/day, achieving blood levels of 8.86 micrograms/ml. Neither plasma nor CSF norepinephrine (NE) nor CSF 3-methoxy-4-hydroxy-phenylglycol (MHPG) was significantly altered by carbamazepine. Baseline medication-free values in 21 depressed patients were not predictive of the degree of subsequent clinical antidepressant response. CSF NE decreased in four manic patients treated with carbamazepine. The many effects of carbamazepine on noradrenergic mechanisms in animals are discussed in relationship to these first studies of carbamazepine in man. PMID- 2997830 TI - Studies on radioprotectors in mammals and their possible use in radiotherapy--a summary. PMID- 2997831 TI - Studies on sulphydryl radioprotectors in mouse spleen and peripheral blood cells against gamma rays. PMID- 2997832 TI - Radioprotection of mouse testis by S-2-(3-aminopropylamino)ethylphosphorothioic acid. PMID- 2997833 TI - Pancreatic imaging. AB - In this article, the pancreas is evaluated with regard to the controversies surrounding this organ from a clinical standpoint, and an approach to imaging modalities is examined. The roles of computed tomography, ultrasound, endoscopic retrograde cholangiopancreatography, and magnetic resonance imaging are put into proper perspective. PMID- 2997834 TI - Imaging approach to the suspected renal mass. AB - The authors present their algorithmic approach to the detection, characterization, and staging of renal masses. Based on classification of urographic findings, the patient may be triaged to the appropriate cross sectional or invasive imaging modality that will result in the most cost effective management. PMID- 2997835 TI - Fibrolamellar hepatocellular carcinoma. AB - Fibrolamellar hepatocellular carcinoma (HCC) has recently been separated as a distinct clinicopathologic entity with a better prognosis than the usual HCC associated with cirrhosis. The mean age of our 17 patients was 20 years. Alpha fetoprotein levels were normal, and none of the risk factors for HCC was present. Distinctive histologic features included deeply eosinophilic polygonal hepatocytes and abundant fibrous stroma. Calcification was present on plain films of five of 13 cases. Sonography usually showed a homogeneous, echogenic mass. Computed tomography (CT) demonstrated small, central calcification in four of ten cases. A central echodensity, hypodense on CT scans, was seen in two cases and corresponded to a central scar. By combining clinical and laboratory data with radiologic tests, a correct diagnosis can often be suggested before biopsy is performed. PMID- 2997836 TI - Blood flow imaging with MR: spin-phase phenomena. AB - Blood flow phenomena occurring when flow is within the magnetic resonance (MR) imaging plane were analyzed. In this situation, the signal intensity of vascular lumina is predominantly determined by spin-phase change phenomena, and section transition effects of moving spins can be neglected. In this paper, we develop the concepts of in-plane flow, with emphasis on the notion that the spatial variations in velocity and acceleration of blood, which mainly occur along vessel walls, are important determinants of intravascular signal loss in MR images. Flow patterns in the large mediastinal arteries were qualitatively and quantitatively analyzed in six healthy subjects and 14 patients with hemodynamic abnormalities using multiple electrocardiograph-gated image acquisition; ungated studies of 30 patients were analyzed for venous flow effects. Intraluminal signal was strongly dependent on the phase of the cardiac cycle and the echo number. Signal loss was found to occur along vessel walls, in vascular bends, and at bifurcations. PMID- 2997837 TI - Fatty infiltration of the liver: evaluation by proton spectroscopic imaging. AB - The reliability of proton spectroscopic imaging in evaluating fatty infiltration of the liver was investigated in 35 subjects (12 healthy volunteers and 23 patients with fatty livers). With this modified spin-echo technique, fatty liver could be separated from normal liver both visually and quantitatively. On the opposed image, normal liver had an intermediate signal intensity, greater than that of muscle, whereas fatty liver had a lower signal intensity, equal to or less than that of muscle. In normal livers, the lipid signal fraction was less than 10%, while in fatty livers it was greater than 10% and usually exceeded 20%. With this technique, nonuniform fatty infiltration of the liver can be differentiated from hepatic metastases, and the technique may prove useful in the differentiation of some hepatic disorders. PMID- 2997839 TI - Thromboxane synthetase inhibitors, thromboxane receptor antagonists, and aspirin in cardiovascular disease. PMID- 2997838 TI - Testicular neoplasms: 29 tumors studied by high-resolution US. AB - High-resolution (10-MHz) ultrasonography produces extremely detailed anatomic images of the testis. The sonographic features most helpful in detecting tumors are mass, bright echogenic foci, and diffuse parenchymal texture change. Of 29 patients with testicular neoplasms, 21 (72%) had one or more masses, 19 (66%) had one or more echogenic foci, and nine (31%) had a diffuse parenchymal texture change. Bright echogenic foci were present in six (86%) of seven testes that had a regressed germ-cell tumor. In an attempt to define the histologic features of bright echogenic foci, we performed needle localization under real-time guidance on four operative specimens. We observed immature bone and cartilage, calcification, tubular atrophy and fibrosis, and focal noncalcific scarring. Discovery of occult testicular neoplasms was common (9/29); four patients were thought to have had "extragonadal" germ-cell tumors before abnormalities were found on the sonograms. PMID- 2997841 TI - [S1 nuclease]. PMID- 2997840 TI - [Protein engineering]. PMID- 2997842 TI - [Cloning of human 20 K growth hormone cDNA and alternative splicing sites]. PMID- 2997843 TI - Effect of bromocriptine on prostacyclin release and cyclic nucleotides on rat aortic and uterine tissues. AB - The effect of bromocriptine mesylate on cyclic nucleotides and PGI2 release by rat aortic and uterine tissues was investigated. Treatment of rats with bromocriptine (10 mg kg-1 I.P. daily for 14 days) increased PGI2 release by the thoracic aorta from 0.67 +/- 0.02 to 1.4 +/- 0.03 ng/mg wet tissue (P less than 0.001; n = 6). This increase was antagonized by treatment with sulpiride (15 mg kg-l). Incubation of the arterial tissue with bromocriptine (50 micrograms ml-1) in vitro also stimulated PGI2 release. Mepacrine (160 micrograms ml-1) significantly decreased both basal and stimulated PGI2 release. Incubation of myometrial tissue from pregnant rats with bromocriptine (50 micrograms ml-1) in vitro significantly decreased PGI2 release from 1.25 +/- 0.07 to 0.60 +/- 0.08 ng/mg wet tissue (P less than 0.05, n = 6). It also elevated uterine cAMP from 40 +/- 2 to 64 +/- 3 pmoles/100 mg wet tissue. Both effects were antagonized by sulpiride. Bromocriptine did not affect uterine cGMP or the cyclic nucleotides in the aorta. It is concluded that the increase in aortic PGI2 was mediated via activation of dopamine D-2 receptors that stimulate phospholipase A2 enzyme. The decrease in myometrial PGI2 release may be related to the increase in uterine cAMP resulting from activation of dopamine D-1 receptors. Previous studies suggested a role for PGI2 in implantation in the rat. The results suggest that the inhibitory effect on uterine PGI2 may underlie the reported inhibition of bromocriptine on implantation. On broad basis, the decrease in uterine PGI2 together with the reported luteolytic effect of bromocriptine point to a potential role for the compound in postcoital contraception. PMID- 2997845 TI - Electrophysiological consequences of leukotrienes applied on isolated rat retina. AB - Scotopic vision is the result of a cascade of light-dependent biochemical events in rod outer segments (ROS) involving mainly a cGMP-modulation of sodium current. This modification of ionic currents induces changes of membrane potential which generates electroretinographic (ERG) waves. As (i) ERG disturbances are commonly recorded in hypoxic and inflammatory retinal diseases (ii) leukotrienes (LTs), a very potent mediators of inflammation, disturb ionic exchanges in several artificial or natural membrane systems, we undertook the investigation of the effects of LTs on ERG record in mammalian isolated retina. LTB4, LTC4 and LTD4, all induced a dose-dependent marked reduction of the b wave amplitude of ERG. This effect is correlated with a significant decrease in the survival time of the retina. The analysis of the modification of ERG indicates that LTs exhibit a real toxic effect since b wave is mainly affected while P III wave is unchanged. Comparatively with other nervous cells, this phenomenon may be attributed to an increase in Na+ permeability of ROS. It is suggested that LTs may be involved in the development of inflammatory or ischemic retinal diseases. PMID- 2997844 TI - Pulmonary microcirculatory responses to leukotrienes B4, C4 and D4 in sheep. AB - The pulmonary microvascular responses to leukotrienes B4, C4, and D4 (total dosage of 4 micrograms/kg i.v.) were examined in acutely-prepared halothane anesthetized and awake sheep prepared with lung lymph fistulas. In anesthetized as well as unanesthetized sheep, LTB4 caused a marked and transient decrease in the circulating leukocyte count. Pulmonary transvascular protein clearance (pulmonary lymph flow X lymph-to-plasma protein concentration ratio) increased transiently in awake sheep, suggesting a small increase in pulmonary vascular permeability. The mean pulmonary artery pressure (Ppa) also increased. In the acutely-prepared sheep, the LTB4-induced pulmonary hemodynamic and lymph flow responses were damped. Leukotriene C4 increased Ppa to a greater extent in awake sheep than in anesthetized sheep, but did not significantly affect the pulmonary lymph flow rate (Qlym) and lymph-to-plasma protein concentration (L/P) ratio in either group. LTD4 increased Ppa and Qlym in both acute and awake sheep; Qlym increased without a significant change in the L/P ratio. The LTD4-induced rise in Ppa occurred in association with an increase in plasma thromboxane B2 (TxB2) concentration. The relatively small increase in Qlym with LTD4 suggests that the increase in the transvascular fluid filtration rate is the result of a rise in the pulmonary capillary hydrostatic pressure. In conclusion, LTB4 induces a marked neutropenia, pulmonary hypertension, and may transiently increase lung vascular permeability. Both LTC4 and LTD4 cause a similar degree of pulmonary hypertension in awake sheep, but had different lymph flow responses which may be due to pulmonary vasoconstriction at different sites, i.e. greater precapillary constriction with LTC4 because Qlym did not change and greater postcapillary constriction with LTD4 because Qlym increased with the same rise in Ppa. PMID- 2997846 TI - Proinflammatory properties of unsaturated fatty acids and their monohydroxy metabolites. AB - The proinflammatory effects of unsaturated fatty acids and, where appropriate, their monohydroxy derivatives, have been investigated both by application to human skin and with respect to human polymorphonuclear leukocyte (PMN) migration. Of the fatty acids applied to the skin only eicosapentaenoic and arachidonic acids (EPA; AA) produced consistent, measurable erythema. The monohydroxy derivatives of the two fatty acids also caused erythema, the 12-hydroxy isomers being the most potent. Chemokinetic activity towards PMNs was observed in the presence of AA, EPA and alpha-linolenic acid using an agarose microdroplet chemokinesis assay. In contrast to their in vivo properties, the 5-hydroxy isomers of AA and EPA were the most potent, being approximately 10 times more chemokinetically active than the other isomers. Quantification of the hydroxyeicosatetraenoic and hydroxyeicosapentaenoic acids (HETEs; HEPEs) in the lesional skin of psoriatic patients demonstrated that, of the metabolites measured, 12-HETE was present in the greatest amounts. Twenty five times more 12 HETE than 12- or 15-HEPE was detected, these being the most abundant of the HEPEs formed. The monohydroxy derivatives of AA and EPA may contribute to the inflammatory changes observed in psoriasis. The HETEs appear to be of greater importance than the HEPEs in view of the relative amounts present. PMID- 2997847 TI - Recent developments in the bioassay of opioids. PMID- 2997848 TI - The influence of atriopeptin on renal function. PMID- 2997849 TI - Natriuretic and hypertensinogenic pro-opiomelanocortin derived peptides. PMID- 2997850 TI - Further characterization of the endogenous natriuretic and digoxin-like immunoreacting activities in human urine. PMID- 2997851 TI - 24-H patterns of erythrocyte membrane-bound Na/K ATPase, plasma renin and aldosterone in normotensives and essential hypertensives. PMID- 2997852 TI - Cellular mechanisms for neurotensin receptor-mediated release of prolactin. PMID- 2997854 TI - The significance of the effects of insulin on Na,K-transport in muscle cells. PMID- 2997853 TI - Insulin sensitivity of human red blood cells cation transport. PMID- 2997855 TI - Atrial natriuretic peptides affect renal metabolism and angiotensin II receptors. PMID- 2997856 TI - Involvement of cytosolic free calcium in the action mechanism of atrial natriuretic factor (ANF). PMID- 2997857 TI - Response of the RAC Risk Assessment Subcommittee to scientific issues raised in an HHS memorandum. PMID- 2997858 TI - Structure-activity studies with ACTH/alpha-MSH fragments on corticosteroid secretion of isolated zona glomerulosa and fasciculata cells. AB - The steroidogenic action of ACTH/alpha-MSH fragments was studied on isolated zona glomerulosa and zona fasciculata cells dispersed by collagenase. ACTH-(4-7), ACTH (6-10), ACTH-(4-10) and ACTH-(11-13) stimulated corticosterone production of the zona fasciculata and aldosterone production of the zona glomerulosa cells. ACTH (7-10) was ineffective. ACTH-(4-7) appeared to be the most potent peptide of the tested fragments. None of the fragments affected the steroidogenic action of ACTH (1-39). It is suggested that similar to the melanotropic effect of alpha-MSH two 'message' sequences for adrenocortical stimulation exist in the alpha-MSH part of the ACTH molecule. PMID- 2997859 TI - High-affinity angiotensin receptors in rat adrenal medulla. AB - Angiotensin II receptors have been quantitated in single rat adrenal medullas by incubation of tissue sections with 125I-[Sar1]-AII, autoradiography with exposure to 3H-sensitive Ultrofilm, computerized densitometry and comparison with 125I labelled standards. Rat adrenal medulla contains a single class of high affinity AII receptors with a Ka of 0.84 +/- 0.02 X 10(9) M-1 and a Bmax of 3259 +/- 502 fmol/mg protein, one of the highest densities in AII receptors found in rat tissues. These observations provide evidence for a local site of action of AII in the release of adrenal medullary catecholamines. PMID- 2997861 TI - [Prevalence of HTLV-III and changes in immunity in heroin addicts]. PMID- 2997860 TI - Adsorption of anionic chloro complexes of 59Fe and 195Au on non-ionic resins of macro-reticular type. AB - Adsorption behavior of 59Fe and 195Au on the non-ionic macro-reticular resin, Amberlite XAD-7 is studied. Distribution coefficients (Kd) for both nuclides in hydrochloric acid or lithium chloride solutions above 6M are particularly high. In nitric acid solution below 2M, 195Au is highly adsorbed on the resin but 59Fe is negligible over any concentrations. This resin did not adsorb other important radionuclides such as 54Mn, 60Co, 65Zn, 90Sr, 106Ru, 137Cs and 144Ce at all. Based on such peculiar adsorption behavior, application to selective separation and determination of radio and stable iron in sea water was also studied. PMID- 2997862 TI - [NMR and CT studies in acute and late-stage cerebral infarct]. AB - MR offers an additional non-invasive means for the investigation of cerebro vascular disease. This new digital imaging method competes with CT. Twenty-four patients with cerebral infarcts were examined by CT and MR; of these, 12 were in the acute stage of necrosis and resorption, and 12 were examined after six weeks following the formation of cysts and glial scars. Because of the increased relaxation time of T1 and T2, there is a large signal difference between infarcted and normal brain. Consequently, cerebral infarcts can be clearly recognised during the first 24 hours. MR also has advantages in demonstrating infarcts in the pons and medulla and if one wishes to avoid contrast enhancement during CT. Problems may arise in differentiating fresh infarcts from haemorrhage and from gliomas. At the present time, the length of the procedure and its high cost justify MR in exceptional circumstances only. PMID- 2997863 TI - [Intraoperative sonographic localization of brain tumors]. AB - Twenty-six patients with mostly small intracranial space-occupying lesions were examined sonographically during surgery. In all patients, accurate localisation and complete delineation of the intra-cerebral lesion proved possible. The extent of the lesion could be determined accurately and surrounding cerebral structures were identified unequivocally. There were no significant differences in the appearances before and after opening of the dura. The significant advantage of the intra-operative use of sonography lies in the accurate localisation of the space-occupying lesion. This results in a reduction of interference with normal cerebral tissue and in a reduction of the duration of the operation. PMID- 2997864 TI - [Clinical importance of emission-computed tomography of the skull in bone scintigraphy]. AB - Sixty-five ECT examinations were carried out in patients with abnormalities of the skull base and paranasal sinuses and the results were compared with ordinary scintigraphy and CT. ECT provides additional information as compared with scintigraphy by its images in three planes, which are free of superimposition. This is of value in the localisation of the lesion and for determining its extent and activity. It is a functional tomographic method which is complementary to the morphological information provided by CT. The indications and value of the method in inflammatory, metastatic and neoplastic diseases are discussed. PMID- 2997865 TI - [Value of computerized tomography in the diagnosis of recurrence in malignant head and neck tumors]. AB - Among 441 CT scans of 303 patients with a malignant tumor of the head or neck 138 scans of 83 patients were performed for evaluation of recurrent tumor. In diagnosing nodal recurrence computed tomography (CT) had a sensitivity of 92% (52 out of 56) and a specificity of 96% (55 out of 57). In diagnosing recurrence at the primary site sensitivity of 72% (56 out of 77) and specificity of 60% (30 out of 50) was lower and clearly inferior to physical examination with 92 and 88% respectively. CT was very useful in delineating advanced recurrencies, especially in regions that were not readily accessible by physical examination or were indurated by flat scarring. In addition to the complete staging of nodal involvement this makes CT the radiologic method of choice in follow-up of patients with malignant tumors of the head and neck. A post-therapeutic baseline scan obtained 6-8 weeks after surgery resp. radiotherapy is expected to further improve results, especially in differentiating scar from recurrent tumor. PMID- 2997866 TI - Density of the caput mandibulae in computed tomography compared with clinical findings related to TMJ dysfunction. AB - Computed tomography (CT) is an excellent method for evaluating the temporomandibular joint, since it shows the bone structures and soft tissues at the same time and constitutes the most practical method for measuring tissue densities in vivo. The aim of this work was to compare densities of the caput mandibulae obtained by CT with the clinical findings in patients with TMJ dysfunction (25) and controls (29). The densities in the patient group were higher than in the controls and seemed to correlate with the clinical symptoms and signs of TMJ dysfunction, especially with muscle pain and deviation in the mouth opening movement. PMID- 2997867 TI - [Differential arthrographic diagnosis of the painful shoulder joint]. AB - 60 arthrograms of the shoulder were explored under morphologic and pathologic anatomic aspects. Differentiation was made between acute and chronic pain. The possible findings in leaking of the contrast material out of the joint capsule are shown and discussed. Signs are quoted of degeneration of soft tissue and bone. The findings in patients with rheumatoid arthritis and a history of shoulder dislocation are summarised. Distension of the joint capsule without shoulder dislocation is described. Filling of the periarticular lymphatics is mentioned in different cases. PMID- 2997868 TI - [NMR tomography of the skeletal muscles in neuromuscular diseases]. AB - MR tomography is a useful procedure for assessing changes in size and structure of skeletal muscles in three dimensions. It provides excellent soft tissue contrast resolution, but spatial resolution requires to be improved. Myatrophic alterations can be shown best by using short echo delay times (TE) and short repetition times (TR). Muscles with fatty degeneration reveal a change in signal intensity and in relaxation times. These reproducible MR findings indicate appropriate areas of EMG diagnosis and biopsy and provide objective follow-ups. PMID- 2997869 TI - [Contribution of digital subtraction angiography to the diagnosis of rejection reactions after kidney transplantation]. AB - The ability to diagnose rejection changes in renal transplants using DSA was evaluated retrospectively taking intrarenal vascular changes into consideration. The findings obtained with DSA were compared to those of scintigraphy in 26 graft recipients, on whom both methods had been performed within a narrow time period for evaluation of hypertensive disease. The scintigraphic diagnosis was based on function and perfusion studies. The intrarenal vascular tree was demonstrated on DSA better by intraarterial than by intravenous contrast media injection, however both techniques delivered useful diagnostic information. The status of the graft was evaluated with comparable results by DSA and scintigraphy in 73% of the cases. In our experience, functional effects produced by morphological changes demonstrated by DSA, can be defined better with the help of scintigraphy. On the other hand, by performing DSA it is possible to clarify morphologically functional findings, which may be detected by scintigraphy but often are diagnostically nonspecific. The availability of both morphological and functional data increases the diagnostic accuracy which can be obtained with these two low invasive procedures in the assessment of vascular changes in renal transplants. PMID- 2997870 TI - [Value of ultrasonography in the diagnosis of bladder tumors]. AB - The authors have compared the value of transabdominal, transrectal and intravesical ultrasonography in 100 patients with carcinoma of the bladder. They have also compared the pathological stage, determined at operation or post mortem, with the "ultrasound" stage. Transabdominal examination resulted in agreement in 61% cases, transrectal 69% and intravesical in 92%. In the authors' opinion, transabdominal ultrasonography is suitable for general orientation. It can determine the size and position of the tumour. Transrectal examination is particularly valuable if, for any reason, it is impossible to perform cystoscopy. It is also valuable for the examination of tumours localised at the bladder base. Intravesical sonography demonstrated the tumour in every case. During this examination, changes in the elasticity and distensibility of the bladder due to tumour and changes in the bladder volume can be determined. Intravesical ultrasonography carried out at the same time as cystoscopy is a rapid and highly practical procedure, which ideally supplements cystoscopy. At present it is the best method for demonstrating infiltration of the bladder wall. PMID- 2997871 TI - [Importance of computed tomography in the diagnosis and differential diagnosis of primary adrenal tumors]. AB - In the present study, 29 patients with primary adrenal tumours and one with an extra-adrenal paraganglioma were examined. With an accuracy of over 90%, computed tomography is the method of choice if there is clinical suspicion of a suprarenal tumour. Pathological changes greater than 8 to 10 mm can be demonstrated. Low absorption values, up to 25 HU, are found predominantly with hyperplasias and adenomas. Phaeochromocytomas, carcinomas and metastases, on the other hand, show significantly higher densities between 30 and 60 HU. CT also demonstrates neighbouring structures. Differentiation between benign and malignant processes is also possible by demonstrating tumour infiltration or metastases. PMID- 2997872 TI - [Hernias in the computed tomogram of the abdomen]. AB - Asymptomatic hernias may be accidental findings in abdominal computed tomography. The characteristic features in our own 47 own cases, and the results of other authors, are discussed. Suspected diaphragmatic hernias, with the exception of hernias, are a special indication for CT. The method may also be useful for the detection of rare abdominal and pelvic wall herniations. PMID- 2997873 TI - [Value of computer tomographic determination of spleen size for the elucidation of splenic involvement within the framework of primary lymph node neoplasms]. AB - The splenic index was determined in 155 persons, without evidence of splenic disease, in order to obtain a simple measure of splenic size. Subsequently 36 patients with malignant lymphomas and who then had their spleens removed were examined by CT. A comparison of the CT and pathological-anatomical findings showed that it is a valuable non-invasive method for diagnosing splenic involvement, having a specificity of 86%, sensitivity of 77% and accuracy of 83%. PMID- 2997874 TI - [The use of Gianturco spirals in percutaneous transhepatic bile duct drainage]. AB - One hundred and thirty percutaneous transhepatic biliary drainage procedures have been carried out so far (catheter drainage in 80, biliary endoprostheses in 50 cases). In 36 patients Gianturco coils were introduced into the fistulous tract in order to control severe bleeding and to stop biliary leakage. Three patients of this group had rectifiable complications. PMID- 2997875 TI - [The obscure primary tumor]. AB - The imaging techniques and their results were analysed in 202 patients in whom a primary tumour was diagnosed only at autopsy. The main cause for failure to find the primary tumour was misinterpretation by the radiologist. Pure description offers no more than can be seen by a skilled clinician. The examination should lead to a diagnosis; even a suspicion should be indicated and one should insist on additional measures for follow-up or for obtaining a diagnosis. Improved diagnosis due to the introduction of sonography and CT has only been partially achieved. Malignant pulmonary tumours are the most commonly overlooked lesions and represent 30% of undiagnosed tumours. They are the most frequent primary tumours giving rise to cerebral and bone metastases. PMID- 2997876 TI - [Experimental studies on the sonographic image of the cruciate ligament]. PMID- 2997877 TI - [Tumor hemorrhage with fluid accumulation in a pituitary adenoma]. PMID- 2997878 TI - [Relation between endocrine ophthalmopathy and sinusitis]. PMID- 2997879 TI - [Fenestration of the median cerebral artery]. PMID- 2997880 TI - [Duplication of the external iliac artery]. PMID- 2997881 TI - Filiform polyposis: a manifestation of histiocytosis X. PMID- 2997883 TI - [Gas in the portal system]. PMID- 2997882 TI - [Cystic lymphangioma of the greater omentum]. PMID- 2997884 TI - Gas dissection into the psoas associated with Knutsson's sign. PMID- 2997885 TI - Leiomyosarcoma of the rectum versus prostatic malignancy. Differentiation by magnetic resonance imaging. PMID- 2997886 TI - Acute effects of the new oral angiotensin converting enzyme inhibitor 1-(D-3 acetylthio-2-methylpropanoyl)-L-prolyl-L-phenylalanine (Alacepril) in essential hypertension. AB - In 15 patients with mild-to-moderate essential hypertension a new orally active angiotensin converting enzyme inhibitor, 1-(D-3-acetylthio-2-methylpropanoyl)-L prolyl-L-phenylalanine (Alacepril), was administered with a single oral dose of 50 mg to evaluate its antihypertensive effect. Following Alacepril plasma angiotensin converting enzyme (ACE) activity was inhibited by approximately 75% within 2 h. Plasma renin activity increased slightly whereas plasma levels of aldosterone decreased significantly. Blood pressure fell markedly not only in patients with high renin levels but also in those with low renin levels. Nevertheless, the magnitude of blood pressure reduction was correlated with the pre-treatment plasma renin values (r = -0.602, p less than 0.05 systolic, r = 0.667, p less than 0.01 diastolic). No relevant changes in pulse rate was observed. These findings demonstrate that in essential hypertension the novel orally active ACE inhibitor Alacepril exerts marked antihypertensive effect, which may offer a new effective approach to treatment of hypertension. PMID- 2997888 TI - Dehydroepiandrosterone sulfate as a digitalis like factor in plasma of healthy human adults. AB - Plasma values for digitalis like factors (DLF) and dehydroepiandrosterone sulfate (DHEA-S) in 11 healthy adults were (mean +/- SD) 44 +/- 6 pmol digoxin equivalents/L and 11 +/- 3 mumol/L respectively. DHEA-S accounted for 62-100% of the total plasma DLF. DHEA-S and plasma DLF displaced [125I]digoxin from digoxin antibody, inhibited hog brain Na,K-ATPase and displaced [3H]ouabain from ATPase in a concentration dependent manner similar to digoxin. Plasma extracts (11 subjects) inhibited Na,K-ATPase and displaced [3H]ouabain from Na,K-ATPase with mean values +/- SD of 1.7 +/- 0.22 and 1.8 +/- 0.25 nmol digoxin equivalents/L plasma respectively. HPLC fractionation of plasma DLF showed several peaks. The major peak was due to DHEA-S, identified by its retention time, radioimmunoassay of DHEA-S and mass spectrometry. PMID- 2997887 TI - N-(3-Fluoropropyl)-N-normetazocine, a potentially useful opiate antagonist for opiate receptor studies with positron emission tomography (PET). AB - A new fluorinated derivative of N-propylnormetazocine, N-(3-fluoropropyl)-N normetazocine (1) was synthesized. 1 was similar to the unfluorinated analog 3 in its ability to compete with (3H)-naltrexone for binding sites in rat brain membranes and its potency in antagonizing morphine analgesia in rats. Competition of both compounds against (3H)-naltrexone was little affected by the presence of sodium chloride, a characteristic frequently exhibited by opiate antagonists. Morphine analgesia in rats was measured by suppression of locomotion and vocalization responses to footshock. The ability of 1 to antagonize morphine analgesia in rats was similar to that of 3. Neither 1 nor 3 showed any evidence of agonist activity in rats at doses as high as 1.0 mg/kg (the highest dose tested). These results suggest that 1, labeled with 18F, may be useful for in vivo studies of the opiate receptor using positron emission tomography (PET). PMID- 2997889 TI - Effects of diethyldithiocarbamate and N-methyl-N-dithiocarboxyglucamine on murine hepatic cadmium-metallothionein in vitro. AB - A study was made of the effects of diethyldithiocarbamate (DDTC) and N-methyl-N dithiocarboxyglucamine (MDCG) on partially purified cadmium-metallothionein (Cd MT) in vitro obtained from livers of mice previously injected with CdCl2 containing 109CdCl2. Analytical Sephadex G-75 gel filtration showed that MDCG effected a time-dependent removal of Cd from MT, and 99% of the Cd was recovered as the soluble Cd(MDCG)2 complex after 24 hr of incubation. Only a portion of Cd of Cd-MT was complexed by DDTC after 24 hr of incubation; 44% remained as Cd-MT, and the net loss following centrifugation prior to application of the samples to the column corresponded to 56% of the Cd originally present. It was proposed that MDCG complexes the 4 g-atoms of Cd in cluster A of MT as well as the 3 g-atoms in cluster B, while DDTC complexes only the 4 g-atoms of Cd in cluster A. PMID- 2997890 TI - A comparison of the relative in vitro and in vivo binding affinities of various benzodiazepines and related compounds for the benzodiazepine receptor and for the peripheral benzodiazepine binding site. PMID- 2997891 TI - Effects of trifluoperazine and verapamil on the hydro-osmotic response to antidiuretic hormone in the urinary bladder of the toad. AB - To investigate the role of the intracellular calcium-calmodulin complex in the hydro-osmotic response to antidiuretic hormone (ADH), the effects of trifluoperazine (TFP), a well-established inhibitor of calmodulin-mediated functions, and of verapamil (V), a calcium entry blocker, were examined in the urinary bladder of the toad, a model for the late distal tubule and the collecting duct of the mammalian nephron. Preincubation of the hemibladders with TFP at serosal concentrations of 10(-5) and 10(-4) M was without effect on basal water flow but markedly reduced the maximal hydroosmotic response to ADH (50 mU/ml) in a dose-dependent manner as compared to control hemibladders (23.60 +/- 1.23 vs. 42.17 +/- 4.18 mg/min per hemibladder (10(-5) M TFP) and 5.43 +/- 0.59 vs. 52.50 +/- 4.67 mg/min per hemibladder (10(-4) M TFP). This inhibitory effect of TFP on the ADH-stimulated osmotic water flow persisted in the presence of naproxen (10(-5) M), a known inhibitor of prostaglandin synthesis. The hydro osmotic response to cyclic adenosine 3',5' monophosphate (cAMP, 10(-3) M) was also significantly reduced in TFP-pretreated tissues (11.68 +/- 1.84 vs. 32.83 +/ 3.14 mg/min per hemibladder), suggesting a post-cAMP inhibitory effect of TFP. V (10(-4) M) had no effect on basal water flow but significantly reduced the hydro osmotic effect of 50 mU/ml ADH (15.17 +/- 1.05 vs. 38.00 +/- 3.39 mg/min per hemibladder). In contrast, cAMP-stimulated osmotic water flow was significantly stimulated in V-treated tissues (48.07 +/- 1.95 vs. 27.13 +/- 1.50 mg/min per hemibladder).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2997892 TI - Gamma camera evaluation of the effects of degradable starch microspheres on arterial liver blood flow in the rat. AB - Degradable starch microspheres injected into an artery causes a temporary reduction of regional blood flow and improves the exposure of the organ to a drug injected simultaneously. The purpose of this study was to quantify this effect when microspheres are injected arterially into the liver of rats. As model substance radioactive pertechnetate ions (99TcmO4-) was used. The results were evaluated from external gamma camera measurements. The amounts of microspheres injected together with pertechnetate was 1.5-12 mg. When compared to injections of pertechnetate only, the integrated exposure of the liver to pertechnetate was increased by a factor of 1.4-2.4 when microspheres were added. PMID- 2997893 TI - [Biochemistry of prostacyclin]. PMID- 2997894 TI - [Pharmacology of prostacyclin]. PMID- 2997895 TI - [Role of prostacyclin in thromboresistance]. PMID- 2997896 TI - Assessment of some risk factors for hepatocellular carcinoma: a case control study. AB - A case-control study of risk for hepatocellular carcinoma (HCC) was carried out in our Department from December 1980 to December 1983. One hundred and twenty consecutive inpatients with HCC were compared with 360 controls pair-matched by sex and age (within years). For each case three different controls were selected from inpatients at the same hospital: one patient with liver cirrhosis; one patient with solid tumor and one patient with chronic illness other than neoplasm or liver disease. We report here the results on alcohol consumption, smoking habit and hepatitis B virus infection. The risk factors investigated are distributed similarly in HCC and cirrhosis. The prevalence of alcohol abuse in HCC is similar to that in cirrhosis and is significantly higher than in other neoplastic or otherwise chronically ill patients (odds ratio 2 X 3 and 3 X 2 respectively). Thus alcohol abuse is probably a risk factor for HCC as a cause of cirrhosis. Smoking habits were similar among the various disease groups and independent of alcohol consumption. The prevalence of heavy smoking was comparable in cases and controls. HbsAg negative-HCC with an ultrasonographic pattern of 'diffuse' alteration was more frequent in heavy smokers. PMID- 2997897 TI - [Value of abdominal sonography and laparoscopy in the diagnosis of primary hepatocellular cancer]. PMID- 2997899 TI - [Etiopathogenetic aspects of juvenile bronchial asthma. Mechanisms and mediators. II]. PMID- 2997898 TI - [Chronic monoarthritis induced by a synovial sarcoma]. PMID- 2997900 TI - [In situ cancer of the cervix and papillomavirus. Study of lll cases of conization]. AB - A series of 111 cervical conization specimens from patients with carcinoma in situ (CIS) have been examined by the authors in order to point out the real incidence of condylomatous lesions and their different aspects. The histological criteria of condyloma and cervical intra-epithelial neoplasia (dysplasia and CIS) are recalled, and relationships between them are discussed. Koilocytes have been observed in 77.5% of CIS examined. Different localisations of condylomatous aspects as regards dysplastic and neoplastic lesions are described and discussed. The histological pattern defined as CIN III with incomplete signs of condylomatous lesion, is significantly associated to flat condyloma (72.7% of cases). Morphological and biological border between condyloma and CIN seems to be not clean; therefore the authors stress on the careful screening, treatment and follow-up of this patients. PMID- 2997903 TI - Recombinant DNA techniques in the study of hepatitis B virus infection. AB - Recombinant DNA techniques have recently contributed a great deal of informations on hepatitis B virus (HBV) infection. Serum HBV-DNA appeared as the most sensitive marker of viral replication activity both in hepatitis B e antigen (HBeAg)-positive and in anti-HBe-positive patients. In the latter group, a significant correlation between serum viral DNA positivity and liver disease activity was present. In our experience, more than 50% of anti-HBe-positive cases with chronic liver disease showed circulating HBV-DNA, while none of healthy HBsAg chronic carriers was found positive for serum HBV-DNA. In type B acute hepatitis, viral nucleic acid sequences were detectable only in a small number of uncomplicated cases, but were observed in all the patients who progressed to chronic hepatitis. HBV-DNA represents therefore an early and useful prognostic parameter in acute infection. Several epidemiological studies have established a striking correlation between HBV infection and development of hepatoma. Using molecular hybridization techniques, viral DNA has been identified in liver cancer cells. Finally, HBV-DNA has also been identified in the pancreas, kidney, skin, bile ducts and in cells of the vascular system. In addition, the presence of viral genome has been recently identified in circulating lymphocytes of patients with acute or chronic HBsAg-positive hepatitis. These findings add further informations to the understanding of viral biology and of virus-host interactions in the natural history of the infection and associated liver disease. PMID- 2997901 TI - [A rare form of benign tumor of the liver possibly related to the use of oral contraceptives: focal pediculated nodular hyperplasia]. AB - Following a recent case, the authors review the literature of hepatic nodular hyperplasia. The incidence of this condition increases regularly with the consumption of oestrogens. They attempt to define the methods of detection for patients at highest risk. Without exaggerating the incidence of this complication of oral contraceptives, the authors believe that it will become increasingly more common than the vascular complications which, although frequently discussed, are relatively rare. PMID- 2997902 TI - [Varicella and pregnancy]. AB - Two recent cases of varicella in pregnant women are reported. A fortuitous review of fetal and neonatal risks is made that serves as a guide to therapy: elective abortion if the infection occurs early on in pregnancy; diagnosis of congenital varicella if it occurs some time after delivery; avoidance of delivery two days before and five days following eruption of rash by reason of the high neonatal mortality. PMID- 2997904 TI - Characterization of growth parameters of a human hepatoma cell line cultured under serum-free conditions. AB - The PLC/PRF/5 human hepatoma cell line has at least four complete series of hepatitis B virus (HBV)-DNA sequences integrated into the host DNA and produces hepatitis B surface antigen (HBsAg) and several serum proteins in the culture medium. In order to study serum proteins and HBsAg released by these cells under controlled conditions, the serum-free growth of several human hepatoma-derived cell lines was recently investigated. In this paper the growth of PLC/PRF/5 human hepatoma cell line in a serum- and hormone-free medium was investigated. The results represent a tool which might be used in pharmacological research, studies on hormone-binding and virus gene expression. PMID- 2997906 TI - [Focal nodular hyperplasia--mixed liver cell and bile duct adenoma]. AB - Although benign tumorous lesions of the liver are rare, they can be found particularly among women in the preclimacteric age group. Usually, the clinical findings are non-characteristic. Differentiation from other space-occupying growths can be difficult due to the variable appearance of focal nodular hyperplasia. The selective angiogram is typical in some cases and no further radiological procedures are required. Nevertheless, histological examinations should be performed for complete clarification. PMID- 2997907 TI - [Peroperative ultrasonics in hepatic surgery]. PMID- 2997905 TI - [Huntington chorea. Review of the most recent aspects of research]. AB - The authors are presenting a review concerning the most visible aspects of the research directed to emphasize that Huntington's Chorea cannot be compared, either clinically or pathologically, to Parkinson's Disease. It is a hereditary disease in which we can discern an alteration of some neurotransmitters, neuromodulators, neurohormones that are not necessarily opposite in respect to pathophysiological findings of Parkinson disease. PMID- 2997909 TI - Detection of rotavirus in faecal specimens by enzyme immunoassay, latex agglutination and electron microscopy. AB - Faecal specimens from 570 patients with gastroenteritis were studied for rotaviruses by enzyme immunoassay (EIA), electron microscopy (EM) and latex agglutination test (LX). Specimens from 127 patients were positive and 379 were negative with all 3 methods. 64 (11%) specimens gave contradictory results in the tests. EIA gave significantly more positive results than EM (168 vs 145). 30 EM negative and EIA-positive specimens were positive in a confirmatory test. LX was positive in 161 specimens and no significant differences to EM or EIA were observed. There were 16 (2.8%) LX-positive samples that were negative both in EM and EIA. The positivity of these samples could not be confirmed and 15 of them were only slightly positive. Our results can be concluded: (1) Enzyme immunoassay may be more sensitive than electron microscopy but requires a confirmatory test and is tedious when only a few specimens per day are to be examined; (2) LX seems to be suitable for primary screening of faecal specimens for rotavirus; for definitely positive results the test is reliable. PMID- 2997908 TI - T-cell prolymphocytic leukaemia: a clinical and immunological study. AB - 2 cases of T-cell prolymphocytic leukaemia (T-PLL) were investigated for their reactivity with a series of monoclonal antibodies (MoAbs) as well as for the cytochemical expression and functional activity of the pathological cells. Both patients showed morphological (large cells with abundant cytoplasm and eccentric and irregularly shaped nucleus with large and prominent nucleoli) and clinical (high leucocyte count and splenomegaly) features typical of T-PLL. The cells from 1 patient expressed a helper/inducer phenotype (T4+, T8-) and were reactive with the anti-Tac (interleukin-2 receptor) MoAb, while the other case co-expressed both the T4 and the T8 antigens. The response to phytohaemagglutinin and the natural killer activity (assessed by 51chromium release) were significantly reduced in both cases, while the helper capacity, tested in a pokeweed mitogen driven system, was maintained only in the 1st case. This latter case which expressed a more mature phenotype (T4+, T8-) responded well to chemotherapy. PMID- 2997910 TI - Outbreak of hepatitis A in a day care center in Finland. AB - An outbreak of hepatitis A occurred in a Helsinki day care center attended by 12 children less than 3 years of age. After return of one of the children from Israel, several cases of hepatitis A appeared. Altogether 9 persons, 3 children and 6 adults, developed hepatitis A. The diagnosis was based on measurement of hepatitis A IgM antibodies in serum. Instructions for hygiene and administration of prophylactic gammaglobulin to the personnel and children of the day care center, and to their family members, prevented further spread of the infection. The important role of small infants for spread of hepatitis A is emphasized. PMID- 2997911 TI - The diagnosis of vesico-ureteral reflux. Radiologic and nuclear medicine methods. AB - Video voiding cystourethrography in 47 patients was compared to 99mTc cystography performed suprapubically in 20 patients and 123I Hippuran renocystography performed in 43 patients. The degrees of reflux were equally distributed through the grades. 99mTc cystography was negative in 4 of 5 grade 2 refluxes and positive in 4 out of 5 grade 3-4 refluxes. Computerized dynamic tracings of the 123I renocystography showed an activity level increase over the kidneys of more than 1% of the bladder activity level in 47 units, 18 were equivocal and 21 negative. Grade 2 and 3 reflux found on the video VCU was refound on the renocystography. 24 false positives were found. It is concluded that 99mTc cystography performed as in our unit adds no further information to the diagnosis of reflux and that 123I renocystography diagnosing the more severe degrees of reflux may be a future method of non-invasive reflux control. PMID- 2997912 TI - Arsenic exposure to smelter workers. Clinical and neurophysiological studies. AB - Forty-seven copper smelter workers, exposed to airborne arsenic for 8-40 years, were examined clinically with electromyography, and the motor and sensory conduction velocities in their arms and legs were determined. Fifty age-matched industrial workers not exposed to arsenic formed a reference group. The level of arsenic in the air at the smeltery was estimated to be below 500 micrograms/m3 before 1975 and approximately 50 micrograms/m3 thereafter. Urine analyses of arsenic showed a mean value of 71 micrograms/l (1 mumol/l) in the exposed group; this value is lower than that found in earlier studies reporting clinically detectable neuropathy. Only minor neurological and electromyographic abnormalities were found. A slightly reduced nerve conduction velocity in two or more peripheral nerves was more common among the arsenic workers than the referents, and a statistically significant correlation between cumulative exposure to arsenic and reduced nerve conduction velocity in three peripheral motor nerves was found. This occurrence was interpreted as a sign of slight subclinical neuropathy. In conclusion the risk of clinically significant neuropathy is small when exposure is kept below 50 micrograms/m3 in workroom air. The subclinical findings may be of interest in relation to the prevention of early adverse health effects from arsenic exposure. PMID- 2997913 TI - AIDS--again. PMID- 2997914 TI - Illness associated with a package holiday in Romania. AB - A study of 370 holidaymakers returning from Romania revealed that 279 (75%) reported illness. Alimentary symptoms predominated and were recorded either alone or along with other symptoms by 71 per cent of the tourists. The highest illness rate (82%) occurred in those under 39 years of age and those over 60 years had least illness (38%). Most of the tourists attributed their illnesses to the supply, handling or preparation of food and drink. Twenty-six (21%) tourists had serological evidence of typhoid immunisation out of 121 from whom blood samples were obtained. Most of the tourists studied (85%) were immune to poliomyelitis. PMID- 2997915 TI - Poliomyelitis in an adult male. AB - Paralytic poliomyelitis is now unusual in developed countries following the success of vaccination programmes. This paper reports a case of poliomyelitis in a patient with incomplete vaccination where the source of infection is unclear. PMID- 2997916 TI - Rapidly progressive renal failure in a child. PMID- 2997917 TI - The action of oncogenes in the cytoplasm and nucleus. AB - As many as 40 distinct oncogenes of viral and cellular origin have been identified to date. Many of these genes can be grouped into functional classes on the basis of their effects on cellular phenotype. These groupings suggest a small number of mechanisms of action of the oncogene-encoded proteins. Some data suggest that, in the cytoplasm, these proteins may regulate levels of critical second messenger molecules; in the nucleus, these proteins may modulate the activity of the cell's transcriptional machinery. Many of the gene products can also be related to a signaling pathway that determines the cell's response to growth-stimulating factors. Because some of these genes are expressed in nongrowing, differentiated cells, the encoded proteins may in certain tissues mediate functions that are unrelated to cellular growth control. PMID- 2997919 TI - Z-DNA: still searching for a function. PMID- 2997918 TI - Histocompatibility antigens on murine tumors. AB - Recent advances in tumor immunology suggest that the expression of the histocompatibility antigens, encoded by the major histocompatibility complex, is important in controlling the metastatic growth of certain murine tumors. The anomalous expression of histocompatibility antigens in many neoplasms appears to be associated with the ability of these cells to evade the immune system and progress to metastasis. This review examines some of the underlying molecular and immunobiological interactions that might determine the metastatic outcome of cellular transformation. PMID- 2997920 TI - Expression of the Escherichia coli lacZ gene on a plasmid vector in a cyanobacterium. AB - A biphasic plasmid vector was used to introduce the Escherichia coli K-12 lac operon into the unicellular cyanobacterium Agmenellum quadruplicatum PR-6. The PR 6 transformants expressed beta-galactosidase at nearly as high a level as did Escherichia coli transformants. In order to accomplish this, it was necessary to obtain PR-6 mutants that could be transformed by plasmids with unmodified recognition sites for the endogenous PR-6 restriction endonuclease Aqu I. These mutants were generated by a variation of the ectopic mutagenesis techniques that have been used in other naturally transforming bacteria. The ability to assay the expression of lacZ in PR-6 paves the way for the construction of gene fusions with various PR-6 promoters and quantitation of their expression under specific in vivo conditions. PMID- 2997921 TI - A new HTLV-III/LAV encoded antigen detected by antibodies from AIDS patients. AB - A newly identified protein from HTLV-III/LAV, the virus implicated as the etiologic agent of the acquired immune deficiency syndrome, was studied. This protein, which has a molecular weight of 27,000 (p27), was shown by amino acid sequencing to have a coding origin 3' to the env gene on the HTLV-III genome. The presence of antibodies to p27 in virus-exposed individuals indicated that this gene is functional in the natural host. PMID- 2997922 TI - Genomic heterogeneity of AIDS retroviral isolates from North America and Zaire. AB - In an analysis of the genomic variation of AIDS retroviral isolates from patients living in New York, Alabama, and Zaire, restriction maps were constructed by using seven enzymes, each known to cleave the proviral DNA more than once, in conjunction with Southern blot analysis. The maps of LAV, HTLV-III, and ARV-2 as deduced from their published nucleotide sequences were included in this analysis. The results demonstrated that (i) several "signature" restriction sites were common to all isolates; (ii) with the exception of LAV and HTLV-III, the North American and European isolates were all different from one another and showed no geographical specificity; (iii) the African isolates as a group were more diverse than those from North America and Europe; and (iv) the genomic variability was concentrated within the env gene. PMID- 2997923 TI - Isolation of T-lymphotropic retrovirus related to HTLV-III/LAV from wild-caught African green monkeys. AB - Present evidence suggests that the acquired immune deficiency syndrome (AIDS) emerged in Central Africa as a new disease in recent decades. This disease has recently approached epidemic proportions in many parts of the world. The etiologic agent of AIDS is believed to be the virus HTLV-III/LAV, which has been proposed as having originated from a recent simian-human transmission in Africa. This report describes the isolation of a designated STLV-IIIAGM retrovirus closely related to HTLV-III/LAV from seven healthy wild-caught African Green monkeys (Cercopithecus aethiops) that showed the presence of antibodies designated STLV-IIIAGM. In vitro growth characteristics, ultrastructural morphology, and major proteins of 160,000 kilodaltons (kD), 120 kD, 55 kD, and 24 kD are similar to and cross-reactive with the analogous antigens of HTLV-III/LAV. The use of these serologic markers in the detection of STLV-IIIAGM-infected monkeys may be important in assuring the continued safety of a variety of biologic reagents that are derived from these primate species. The existence of a retrovirus closely related to HTLV-III/LAV that naturally infects an African nonhuman primate in the apparent absence of disease may provide a unique model for the study of human AIDS and the development of an effective vaccine. PMID- 2997925 TI - Neuroendocrine response to estrogen and sexual orientation. PMID- 2997924 TI - Association of crossover points with topoisomerase I cleavage sites: a model for nonhomologous recombination. AB - Nonhomologous DNA recombination is frequently observed in somatic cells upon the introduction of DNA into cells or in chromosomal events involving sequences already stably carried by the genome. In this report, the DNA sequences at the crossover points for excision of SV40 from chromosomes were shown to be associated with eukaryotic topoisomerase I cleavage sites in vitro. The precise location of the cleavage sites relative to the crossover points has suggested a general model for nonhomologous recombination mediated by topoisomerase I. PMID- 2997926 TI - A disease in many guises. PMID- 2997927 TI - The epidemic's unsung heroes. PMID- 2997928 TI - Indications of a new virus in MS patients. PMID- 2997929 TI - Corticotropin-releasing activity of monokines. AB - Hepatocyte-stimulating factor and interleukin-1 are proteins produced by monocytes in response to inflammatory challenge. Neither of these monokines had direct effects on steroid production by cultured adrenocortical cells. Both monokines stimulated pituitary cells (AtT-20) to release adrenocorticotropic hormone; interleukin-1 was equipotent with a combination of corticotropin releasing factor and arginine vasopressin, and hepatocyte-stimulating factor was at least three times as effective. The synthetic glucocorticoid, dexamethasone, inhibited production of hepatocyte-stimulating factor by cultured monocytes. These results indicate an axis between monocytes and pituitary and adrenocortical cells which may play a role in regulating host defense. PMID- 2997930 TI - Detection of human cytomegalovirus in peripheral blood lymphocytes in a natural infection. AB - In situ hybridization was used to detect human cytomegalovirus (HCMV) in the peripheral blood mononuclear cells of some naturally infected (seropositive) individuals. A subpopulation of cells hybridized specifically to a portion of the HCMV genome that is heavily transcribed during the immediate-early period of infection. The hybridization signal was markedly reduced by base hydrolysis and ribonuclease, and therefore the probe appears to be detecting viral RNA. A fluorescence-activated cell sorter was used to select lymphocytes bearing the OKT4 and OKT8 markers. Hybridization with the HCMV probe revealed a higher proportion of positive cells in the OKT4 than in the OKT8 subset. This observation specifically identifies lymphocytes as a cell population involved in natural HCMV infection and suggests that lymphocytes may be a reservoir for maintaining infection and may also serve as a vehicle for its spread by blood transfusion. PMID- 2997931 TI - Cystic fibrosis locus defined by a genetically linked polymorphic DNA marker. AB - A polymorphic DNA marker has been found genetically linked, in a set of 39 human families, to an autosomal recessive gene that causes cystic fibrosis (CF), a disease affecting one in 2000 Caucasian children. The DNA marker (called D0CRI 917) is also linked to the PON locus, which by independent evidence is linked to the CF locus. The best estimates of the genetic distances are 5 centimorgans between the DNA marker and PON and 15 centimorgans between the DNA marker and the CF locus, meaning that the location of the disease gene has been narrowed to about 1 percent of the human genome (about 30 million base pairs). Although the data are consistent with the interpretation that a single locus causes cystic fibrosis, the possibility of genetic heterogeneity remains. The discovery of a linked DNA polymorphism is the first step in molecular analysis of the CF gene and its causative role in the disease. PMID- 2997932 TI - Scintigraphic evaluation of cerebrospinal fluid diversionary shunt: complications of the proximal limb. PMID- 2997933 TI - Scintigraphic evaluation of cerebrospinal fluid diversionary shunt: complications of the atrial limb. PMID- 2997934 TI - [Acquired immunodeficiency syndrome. A status report. The statement of a World Health Organization meeting]. PMID- 2997935 TI - [Histo-pathological studies of periodontal tissue reactions after furcation perforations treated with various inorganic biomaterials in dog teeth]. PMID- 2997937 TI - "Adult T-cell leukemia/lymphoma" with bone demineralization. AB - Two patients with T-cell malignancy having radiographic manifestations of generalized and localized bone demineralization are reported. One, a 53-year-old man, had marked osteoporosis and severe hypercalcemia, but no clinical evidence of leukemia throughout his illness. At autopsy there was no definite evidence of bone involvement. Histologic proof was obtained from abdominal skin which revealed "adult T-cell leukemia/lymphoma (ATLL)." The second case, a 33-year-old man, complained of arthralgia in his hands and feet; radiographs showed severe localized demineralization and pathologic fractures. Specimens of his peripheral blood, cervical lymph nodes, and bone marrow revealed ATLL cells. PMID- 2997936 TI - A diagnostic approach to lytic lesions of the mandible. AB - Fifty seven patients with histologically proven cyst-like lesions of the mandible are reviewed. The importance of combining radiographic with scintigraphic imaging is stressed. These two imaging modalities are complementary in demonstrating both the biological activity and the extent of the lesion. In osteomyelitis the bone scan, which is more sensitive than the radiograph, is also the method of choice in assessing the result of treatment. PMID- 2997939 TI - [Features of hematopoiesis in patients with small cell carcinoma of the lung]. PMID- 2997938 TI - [Radioisotope scintigraphy in the diagnosis of spinal fractures]. PMID- 2997940 TI - [Clinico-endoscopic diagnosis of recurrences of cancer of the rectum]. PMID- 2997941 TI - [Polygynax]. PMID- 2997942 TI - Extracranial metastasis of glioblastoma with sarcomatous component. AB - A case of glioblastoma with sarcomatous component is presented. Craniotomy was performed with total resection of the left occipital tumor. The patient received radiotherapy postoperatively but developed extracranial metastases only two months after the first surgical procedure. PMID- 2997943 TI - Etiology of intracranial tumors. PMID- 2997944 TI - Partial hepatectomy on cirrhotic liver with a right lateral tumor. AB - A total of 24 patients with cirrhotic liver and solitary, small hepatocellular carcinoma (HCC) located at the lateral part of the right lobe underwent surgery with our technique of hepatic clamping and finger dissection. There were no operative mortality or acute or chronic hepatic failure. Total operating time was 129 +/- 20 minutes; actual resection time was only 22.7 +/- 4.9 minutes. The average amount of blood transfused during this procedure was 1552 +/- 909 ml. The preoperative serum bromsulphalein retention rate proportionately reflected the postoperative peak serum conjugated bilirubin concentration if the weight of the resected specimen was less than 310 gm (p less than 0.001). An evaluation of the enzymes (SGOT, SGPT, and lactate dehydrogenase) released from liver cells on the first postoperative day found that more prominent elevation was observed in the group of patients with hypotension than in those without hypotension (all p less than 0.001). Although all enzyme levels returned to the preoperative level on the fourteenth postoperative day, the excretory capacity of liver cells as measured by serum bromsulphalein retention rate on day 14 time was still abnormally high (p less than 0.001) and took 2 to 3 months to decline to a level that still exceeded preoperative levels (p less than 0.05). In conclusion, partial hepatectomy on cirrhotic liver by hepatic clamping and finger dissection was a simple, rapid technique without any serious side effects. PMID- 2997945 TI - Surgical resection of segment VIII (anterosuperior subsegment of the right lobe) in patients with liver cirrhosis and hepatocellular carcinoma. AB - A limited liver resection was performed in two patients with cirrhosis and a hepatocellular carcinoma situated in segment VIII (anterosuperior subsegment of the right lobe). One of the patient had previously bled from esophageal varices. Resection of segment VIII was performed following the anatomical planes of section after complete mobilization of the right lobe of the liver. Both patients were alive and free of recurrence 14 and 30 months after surgery. Hepatocellular carcinomas are thus treatable by limited anatomic liver resection even when they are situated in the vicinity of the major hepatic veins and the vena cava. PMID- 2997946 TI - Angiotensin converting enzyme and endotoxin induced lung damage in the mouse. AB - Acute pulmonary oedema can be induced by intraperitoneal injection of Escherichia coli endotoxin in the mouse. A fall in serum angiotensin converting enzyme activity is found in mice given endotoxin and in patients with septic adult respiratory distress syndrome, and has been proposed as an indicator of lung microvascular injury. Protein concentration and angiotensin converting enzyme activity in serum, lung, and bronchoalveolar lavage fluid were determined in male mice up to eight hours after injection of endotoxin. By six hours the serum protein concentration had increased and the bronchoalveolar lavage fluid protein concentration had fallen, suggesting fluid shift into the lung. Angiotensin converting enzyme activity fell in serum and lung but increased in bronchoalveolar lavage fluid. As these changes in enzyme activity were not paralleled by changes in protein concentration they are unlikely to be a result of fluid shift or protein leak, and may indicate an active role of the enzyme in the response to sepsis. PMID- 2997947 TI - [Angiotensin-converting enzyme in sarcoidosis]. PMID- 2997949 TI - Influence of mercury on uptake of [3H]dopamine and [3H]norepinephrine by rat brain synaptosomes. AB - Mercuric compounds have been shown to alter several membrane-bound enzymes and associated receptor activities. The present studies were initiated to investigate the in vitro effects of mercuric chloride (HgCl2) and methylmercury chloride (CH3HgCl) on the uptake of [3H]dopamine (3HDA), [3H]norepinephrine (3HNE), and Na+, K+-ATPase in rat brain synaptosomes. Brain synaptosomes were prepared by the ficoll-sucrose gradient method from normal, adult male Sprague-Dawley rats, weighing approx. 200 g. The effect of mercury on Na+, K+-ATPase was determined by using a coupled enzymatic method. Uptake of DA and NE by brain synaptosomes was determined by filtration in the presence and absence of 0-30 microM HgCl2 and 0 100 microM CH3HgCl. A parallel inhibition in the synaptosomal uptake of 3HDA and 3HNE, and the activity of the synaptosomal membrane Na+, K+-ATPase, was observed in both mercuric chloride and methylmercury treatments. The mercury compounds also significantly inhibited the mitochondrial ATPase (Mg2+-oligomycin-sensitive ATPase). The inhibitory influences of the toxins were concentration-dependent. The results suggest that the mercury compound mediated decrease in DA and NE uptake in brain synaptosomes may be related to the inhibition of Na+, K+-ATPase by the same toxins. PMID- 2997948 TI - Antagonism of cyanide poisoning by chlorpromazine and sodium thiosulfate. AB - Anti-cyanide action by sodium thiosulfate (ST) was enhanced by prior administration of chlorpromazine (CPZ). However, CPZ (alone) provided no protection against cyanide lethality. To investigate the possibility that CPZ enhances thiocyanate formation in ST-pretreated mice, the effects of CPZ on rhodanese activity and the time course of plasma thiocyanate concentrations were investigated. CPZ did not alter hepatic rhodanese kinetics nor did it enhance plasma thiocyanate concentrations in ST-pretreated mice. The effect of CPZ and ST on the time course of cytochrome oxidase inhibition and recovery, in vivo, was also investigated. At 4 mg KCN/kg, maximal inhibition of brain (40%) and heart (60%) cytochrome oxidase occurred 10 to 20 min post-challenge in control and CPZ pretreated mice, while no inhibition occurred in ST- and CPZ/ST-pretreated mice. Twenty milligrams KCN/kg caused 100% lethality in control and CPZ-pretreated mice and 6/25 and 4/20 deaths were observed in ST- and CPZ/ST-pretreated mice, respectively. No significant inhibition of brain, heart, and liver cytochrome oxidase activities was observed in surviving ST- and CPZ/ST-pretreated mice challenged with 20 mg KCN/kg. Control and CPZ-pretreated mice died within 5 min of KCN challenge and had almost the same degree of inhibition of brain (35 and 29%, respectively) and heart (60 and 55%, respectively) cytochrome oxidase as did similarly pretreated mice 5 min after challenge with a nonlethal cyanide dose (4 mg/kg). Our results suggest that CPZ does not enhance the formation of thiocyanate in ST-pretreated mice. In addition, the similar degree of cytochrome oxidase inhibition noted after both lethal and nonlethal KCN treatments raises questions as to the ultimate target in cyanide-induced lethality. PMID- 2997950 TI - Surgical treatment for cholangio-carcinoma (inserting silastic transhepatic biliary stents). PMID- 2997951 TI - Cytomegalovirus immunity in allogeneic marrow grafting. AB - IgM and IgG class antibodies to cytomegalovirus (CMV) late antigen were studied in 58 bone marrow transplant (BMT) recipients and their donors using a sensitive enzyme-linked immunosorbent assay (ELISA) and with standard virological and histomorphological techniques. Patients who were CMV-seropositive before BMT had a significantly higher risk for active CMV infection after BMT than seronegative ones (23 of 29 vs. 3 of 26 patients; P less than 1 X 10(-6)). Transplantation of marrow from CMV-seropositive donors was associated with a higher incidence of active CMV infection after BMT than transplantation of marrow from seronegative donors (17 of 28 vs. 9 of 27 patients). Such transplantations also had a significantly higher incidence of grades II-IV acute graft-versus-host disease (23 of 29 vs. 11 of 27 patients; P = 0.007). Following BMT, the evolution of the IgG class CMV antibody response was influenced by the serological status of the marrow donor. First, a fall in IgG class CMV antibody titers during the first month after BMT was seen less often after transplantation of marrow from seropositive donors than after transplantation of marrow from seronegative donors. Second, recipients of marrow from CMV-seropositive donors who developed active CMV infection had an earlier IgG antibody response than those with seronegative marrow donors. These results suggest that the transfer of memory B and T cells occurs with the graft. Failure to mount a sustained IgM or IgG antibody response upon active CMV infection was associated with a fatal outcome. PMID- 2997952 TI - Lymphocyte responses after cytomegalovirus infection in bone marrow transplant recipients--a one-year follow-up. AB - A group of 46 bone marrow transplant (BMT) recipients were studied by lymphocyte stimulation with cytomegalovirus (CMV) antigen before, and repeatedly in the first year after, BMT. Of these, 25 patients developed CMV infection and 68% of these got a positive lymphocyte response to CMV. The recipients with an early response (up to 3 months) to CMV antigen after the CMV infection were less prone to develop chronic graft-versus-host disease (GVHD) than those who responded later or not at all (P = 0.002). After the CMV infection, the increased lymphocyte CMV reactivity remained in recipients without chronic GVHD, but recipients with chronic GVHD usually lost their reactivity. The results suggest that it may be possible to predict patients who are not going to develop chronic GVHD by studying lymphocyte responses to CMV infections. PMID- 2997954 TI - [Uptake of cyclic guanosine-3',5'-monophosphate and its metabolism in 3T6 cells]. AB - Uptake of 3H-cGMP by cultured murine 3T6 cells was studied. The cells were shown to contain radioactivity 1 minute after its addition, with the level of radioactivity increasing during a 3 hour incubation period. By this time, the intracellular non-metabolized cGMP corresponded to 1-5% of the whole intracellular radioactivity. In the presence of theophylline the uptake of 3H cGMP by cells was seen decreasing, however, the portion of non-metabolized cGMP reached 45-50% of the whole intracellular radioactivity. Thus, the presence of theophylline made it possible to maintain the high level of intracellular cGMP. It is concluded that the incubation of cell cultures in the medium with cGMP may be useful for achieving an elevating intracellular cGMP concentration and for studying the biological effect of cyclic nucleotide. PMID- 2997953 TI - [cAMP-dependent protein phosphorylation and its regulatory role in the cell]. AB - Literary data are reviewed on the role of the cAMP-dependent phosphorylation of proteins in the regulation of cell metabolism, membrane permeability, and muscle contraction. It is suggested that a low cAMP level in the tumor and other actively proliferating cells may be associated with a raised exit of cAMP from cells into the surrounding medium because of the increased phosphorylation of the membrane proteins of these cells. PMID- 2997955 TI - [Antibody-dependent cell-mediated cytotoxicity of peripheral blood neutrophils in vitro in acute leukemia]. AB - A study was made of in vitro antibody-dependent cellular cytotoxicity of neutrophils of peripheral blood in 14 cases of acute myeloblastic leukemia and in 20 healthy patients (control group). A decrease in the number of neutrophils, carrying receptors to Fc-IgG and to C3b-component of complement was registered compared to the results obtained for healthy people. Besides, the number of cells restoring Nitro-blue tetrazolium and myeloperoxidase activity decreased. The decrease in antibody-dependent cellular cytotoxicity of neutrophils in the cases of acute myeloblastic leukaemia is explained by the deficiency of the cell receptor apparatus and by the decrease in the intracellular metabolism in them. PMID- 2997956 TI - Retrospective serological study on bluetongue antibody prevalence in a Louisiana herd. PMID- 2997959 TI - Fibronectin, laminin in hybrids of Rous sarcoma virus transformed and normal mouse fibroblasts. AB - Hybrid clones derived from the fusion of normal and Rous sarcoma virus transformed 3T3 fibroblasts were analyzed for fibronectin and laminin pattern of expression in order to find a possible correlation with tumorigenicity. Both organization in the pericellular matrix and secretion into the culture media were investigated by immunofluorescence and ELISA techniques. No significant difference in fibronectin or laminin release was found among hybrid clones exhibiting different levels of tumorigenicity. In contrast, distinctive immunofluorescence patterns of concomitant presence of low levels of fibronectin and high levels of laminin were constantly observed in all the tumorigenic clones. PMID- 2997957 TI - Bluetongue in exotic sheep in Cameroon. AB - Bluetongue virus (BTV) was diagnosed in the Animal Research Institute, Mankon, Bamenda from tissue samples collected from five sheep of exotic breeds which had died of suspected bluetongue in a series of outbreaks between June and October 1982. Five serotypes BTV 1, 4, 5, 12 and 14 were isolated during the period mentioned. Similar disease occurred during June of the following year and BTV type 16 was isolated from a spleen sample from a dead sheep. PMID- 2997958 TI - Prevalence of bluetongue virus and antibodies in ruminants in Cameroon. PMID- 2997960 TI - [Mechanisms of regulation of Ca2+ levels in myometrium cells]. AB - The ways and mechanisms of the Ca2+ concentration regulation in myometrium cells are analyzed. The plasma membrane is thoroughly studied for its role in the calcium control provision for the contractile activity of the uterus. The systems of Mg2+-ATP-dependent transport of Ca2+, sodium-calcium metabolism as well as regularities of the Ca2+ passive transfer in the sarcolemma vesicles are considered. The systems of the Mg2+-ATP- and N+-dependent transport of calcium are discussed for their contribution into regulation of calcium concentration in the myoplasm. Oxytocin and ions of bivalent metals (stimulators of the contractile activity of the uterus) are studied for their effect on the activity of the sarcolemma calcium pump. PMID- 2997962 TI - Ultrastructure of well-differentiated adenocarcinomas of the lung with special reference to bronchioloalveolar carcinoma. AB - The cytologic phenotypes of 20 well-differentiated pulmonary adenocarcinomas were determined by electron microscopy. On examination of more than 100 cells in each case, the tumors were classified according to the predominant cell types. Nine cases (45%) were of mucous cell type, further divided into 7 cases of bronchial surface epithelial cell type, 1 case of bronchial gland cell type, and 1 case of metaplastic bronchiolar goblet cell type. The remainder included 5 cases (25%) of Clara cell type, 2 cases (10%) of type II cell type, and 4 cases (20%) of mixed cell type. The predominant histologic pattern by light microscopy was "typically" bronchioloalveolar (Manning et al.'s type 1) in the metaplastic goblet cell tumor and papillary in most Clara cell-type tumors, while it was glandular in bronchial surface and bronchial gland cell types, although variable in type II cell or mixed cell type. Therefore, bronchioloalveolar carcinomas, when histologically defined inclusive of papillary tumors, present cytologic phenotypes also related to the bronchioloalveolar epithelium, i.e., metaplastic goblet or Clara or type II cell subtypes, which is in accordance with some previous reports. These tumors could be distinguished from the other (glandular) adenocarcinomas that show primarily bronchial mucous cell differentiation. PMID- 2997963 TI - [Muller adenosarcoma of the uterus]. PMID- 2997961 TI - [Biochemical aspects of the action of local anesthetics]. AB - Notions on the molecular mechanisms of anesthesia are presented. The chemical characteristics are given for main representatives of certain groups of local anesthetics with peculiarities of their membrane-tropic action mentioned. The effect of local anesthetics on the synaptic transmission, membrane enzymes, ion transport through the cell membranes is considered simultaneously with the anesthesia phenomenon on the basis of the data available in literature and results of the authors' investigations. PMID- 2997964 TI - [Leukotrienes and inflammation]. PMID- 2997965 TI - [Hormone receptors in cancer of the breast: preliminary analysis of 2000 cases]. PMID- 2997966 TI - [Radiotherapy of 211 cerebral gliomas]. PMID- 2997967 TI - Radionuclide imaging of the urinary tract. AB - This article describes the role of nuclear medicine in the evaluation of the genitourinary tract. The technical aspects of radionuclide imaging (radiopharmaceuticals, radiation dosimetry, instrumentation, and method) are briefly presented, and each of the indications for renal scintigraphy--including the evaluation of differential renal function, hypertension, obstruction, renal transplants, masses, trauma, congenital anomalies, vesicoureteral reflux, and infection--are discussed. The relative advantages and disadvantages of radionuclide imaging with respect to alternative radiographic examinations (such as intravenous urography, ultrasonography, CT, angiography, and magnetic resonance imaging) are emphasized wherever applicable. PMID- 2997969 TI - Renal masses in children. An integrated imaging approach to diagnosis. AB - In view of the continuing technologic advancements in the development and availability of diagnostic imaging modalities, it is appropriate to assess periodically the currently accepted approaches to the evaluation of renal masses in children. The roles, advantages, and disadvantages of plain film, intravenous urography, ultrasonography, radionuclide scintigraphy, computed tomography, angiography, and magnetic resonance imaging in the approach to the evaluation of renal masses in children are discussed. An integrated imaging approach that provides the most accurate and necessary information for diagnosis and treatment is recommended. PMID- 2997968 TI - Computed tomography of the kidney. AB - The development and refinement of computed tomography have revolutionized the diagnostic evaluation of patients in urologic practice. This article reviews the applications and interpretation, as well as the limitations, of renal computed tomography and compares this modality with other imaging studies of the kidney. PMID- 2997970 TI - [Methods of increasing the operability of retroperitoneal metastases of testicular tumors]. PMID- 2997971 TI - [Intraperitoneal paraganglioma simulating a retroperitoneal neoplasm]. PMID- 2997972 TI - [Familial case of duplication of the optic disk associated with syndactyly and oligophrenia]. PMID- 2997973 TI - [Computerized tomography and spatial image filtration in the diagnosis of temporal bone diseases]. PMID- 2997974 TI - [Profuse intra-abdominal hemorrhage from a tumor of the liver]. PMID- 2997975 TI - Survey of antibodies to infectious laryngotracheitis virus in Northern Ireland poultry flocks. PMID- 2997976 TI - Parvovirus vaccination. PMID- 2997977 TI - Metabolism and effects of organic compounds in animals. AB - In recent years, society has become increasingly aware and concerned about protection from chemicals released into the environment. The knowledge of the metabolism and effects of organic compounds in animals, specifically food producing animals, are of paramount importance in assessing potential human health hazards. An intensive effort has been directed at detection of chemicals in the environment, determination of their physiological insult and cellular interaction; in particular their carcinogenic and mutagenic induction capability. The chemical exposure of food-producing animals can be extremely difficult to evaluate and quite devastating. The exposure of food-producing animals to polybrominated biphenyls (PBBs) and polychlorinated biphenyls (PCBs) emphasizes the seriousness of the problem. The polycyclic aromatic hydrocarbons (PAHs), capable of inducing cancer in experimental animals, have been studied extensively in laboratory animals. Current studies deal with the mechanism of metabolism of PAHs and the ultimate carcinogenic form. Although our knowledge concerning the health hazards of organic chemicals is continually increasing, its impact on food producing animals and man's food chain is poorly understood. Awareness of the problem by practicing veterinarians and toxicologists, environmental toxicologists and public health officials is required to evaluate the impact of organic chemicals on the human food chain. PMID- 2997978 TI - Size and map locations of early transcription products on the Autographa californica nuclear polyhedrosis virus genome. AB - The size classes and map locations of early transcripts of Autographa californica nuclear polyhedrosis virus were identified by probing Northern blots with cloned viral DNA fragments. Twelve abundant and 30 minor transcripts were detected by 4 hr postinfection. There was an increase in the abundancy and number of virus specific transcripts between 6 and 8 hr postinfection, coincident with the onset of DNA replication. The change in transcription pattern did not occur in cells infected at the nonpermissive temperature with a DNA-negative mutant indicating that these early transcripts were synthesized prior to the initiation of viral DNA replication. Two early transcripts, 4.6 and 1.1 kb, mapped to the genomic region of the temperature-sensitive lesion, between 60.1 and 62.0%. PMID- 2997979 TI - Thymotropic envelope gene recombinants of Moloney leukemia virus have highly conserved envelope structures. AB - Envelope gp70s were isolated from the thymotropic recombinant viruses related to Moloney murine leukemia virus (RM-M-MuLVs) which were generated by the inoculation of two strains of ecotropic M-MuLV (strain 1869 and temperature sensitive mutant-1) into BALB/c or CFW/D mice. Chymotrypsin oligopeptide maps of parental ecotropic MuLV, RM-M-MuLV, and inducible xenotropic MuLV showed each of the above virus types had a distinctly characteristic peptide map. The majority of RM-M-MuLV gp70 molecules examined showed a high degree of peptide homology. Data from restriction endonuclease mapping demonstrated that the newly acquired sequences in each of the RM-M-MuLVs were very related and encompassed both the polymerase and the envelope genes. The source of the sequences acquired by the RM M-MuLV was from endogenous nonecotropic and nonxenotropic proviruses. This suggested that the family of endogenous proviruses which combined with the parental ecotropic virus was either specifically selected or was much more available than other endogenous proviruses. Although slight variations of envelope-specific sequences and peptides existed among various RM-M-MuLV isolates; within a single thymoma, individual clones of tumor cells yielded RM-M MuLV gp70s which were identical to each other. These findings are discussed within the context of the leukemogenic potential of RM-MuLVs. PMID- 2997980 TI - Human T-cell leukemia virus type II: primary structure analysis of the major internal protein, p24 and the nucleic acid binding protein, p15. AB - The 24,000-molecular-weight major internal protein (p24) and the 15,000-molecular weight nucleic acid binding protein (p15) of human T-cell leukemia virus type II (HTLV-II) were subjected to amino acid composition and amino-terminal amino acid sequence analysis. A comparison of amino acid composition of p24 and p15 of HTLV II with those of the analogous proteins of HTLV-I revealed that these two proteins share overall similarity. Further, alignment of the amino-terminal amino acid sequence for the first 27 residues of p24 and 34 residues of p15 from HTLV II showed extensive sequence homology with analogous proteins of HTLV-I. These data suggest that although disease associated with HTLV-I is malignant T-cell leukemia and that associated with HTLV-II is a relatively benign variant of hairy cell leukemia, HTLV-I and HTLV-II are closely related to each other, at least in their gag-gene-encoded sequences. PMID- 2997981 TI - Structure of the Epstein-Barr virus nuclear antigen as probed with monoclonal antibodies. AB - Five hybridoma cell lines secreting antibodies directed against an Epstein-Barr virus (EBV) nuclear antigen (BamHI K antigen) have been isolated. All five antibodies detect this antigen in a variety of EBV-positive lymphoblastoid cell lines. The reaction of these antibodies with several mutant forms of this protein has allowed the antibody binding site(s) to be predicted. PMID- 2997982 TI - Transcription and replication of eight RNA segments of influenza virus. AB - A novel quantitation system of both plus- and minus-strand RNAs for all eight genome segments of influenza virus was developed using single-strand cDNAs as the probes for hybridization, and employed for the measurement of various RNA species in influenza virus WSN-infected MDBK cells. The synthesis rate and accumulation level of plus-strand RNAs differed considerably among eight RNA segments and were under temporal control. In contrast, eight vRNA molecules of minus polarity were synthesized coordinately at similar rates. Newly synthesized plus-strand RNAs were rapidly transported into the cytoplasm, particularly during the early phase of virus infection, but vRNAs accumulated in the nuclei until the late infection phase. The present data supported the differential regulation of synthesis and the separate transport between plus- and minus-strand RNAs. PMID- 2997983 TI - Cloning and mapping of the SPO1 genome. AB - Many of the XbaI, EcoRI, KpnI, and BglII fragments of bacteriophage SPO1, accounting for about 65% of the genomic sequences, were cloned in Bacillus subtilis. Four of the EcoRI fragments were specifically refractory to cloning in both Escherichia coli and B. subtilis, probably because of expression of deleterious genes carried on the SPO1 fragments. To permit complete identification of the regions cloned, the SPO1 restriction map has been extended to include the XbaI fragments and the previously unmapped KpnI fragments. Markers for 26 of the 39 known genes have been located on specific cloned fragments, permitting more precise determination of the positions of most of the genes. One cloned SPO1 fragment was inhibitory to SPO1 development. PMID- 2997984 TI - Molecular cloning of the Mason-Pfizer monkey virus genome: characterization and cloning of subgenomic fragments. AB - The molecular characterization of the proviral DNA genome of Mason-Pfizer monkey virus (M-PMV), the prototype D-type retrovirus, is described. An analysis of unintegrated viral DNAs present in acutely infected cells revealed open and closed circular molecules and linear species. The size of the M-PMV linear proviral DNA is determined to be 8.1 kbp in length. A preliminary screening of restriction enzymes indicated that many of those commonly used for cloning (EcoRI, SalI, ClaI, XhoI) did not cut the provirus. Digestion of a mixture of linear and circular forms of unintegrated DNA with HindIII produced a set of restriction fragments 2.3-3 kbp in length. These subgenomic fragments where cloned into the bacterial plasmid pAT153, and two classes of M-PMV subgenomic clones isolated. The first of these contained fragments that spanned the ends of the linear genome and presumably were derived from circular proviruses. Six of the seven clones in this class contained a single long terminal repeat (LTR), represented by pMP6, while the seventh, pMP9, contains two LTRs. Digestion of the latter clone with an enzyme that cleaves once within the LTR allowed the length of the M-PMV LTR to be determined as 350 bp. Both the LTR containing clones and the second class of subgenomic clones have been used in developing a detailed restriction map of the M-PMV proviral DNA and in orienting it with regard to transcription of viral RNA. Thus, pMP6/pMP9 contain sequences from the LTR-gag region of the genome and the second class of subclones (represented by pMP1) span the env-coding region. No clones containing the pol-coding region have been isolated. In order to determine the nature of M-PMV-related endogenous sequences in the chromosomal DNA of Old World primates, EcoRI-digested primate DNA was hybridized at low stringency to the subgenomic clones and then washed under conditions of low, moderate, and high stringencies. Multiple sequences closely related to the LTR-gag region of the M-PMV genome, were detected. Sequences more distantly related to the env region were also found in Old World monkeys. Ape and human DNAs were shown to contain sequences related to the LTR-gag region of the M PMV genome, but were only weakly detectable at low stringency. PMID- 2997985 TI - Structure of eight streptococcal bacteriophages. AB - Eight phages from groups A, B, C, E, G, and H streptococci were propagated in their own hosts, purified, and examined for morphology; the size, shape, and structure of their extracted nucleic acids was also examined. Electron microscopy showed three types of phage morphology. All eight phages possess linear double stranded DNAs of molecular weights ranging from 10.5 X 10(6) to 24 X 10(6). Four phages from three different serological groups presented an identical pattern of restriction enzyme fragments. As shown by BAL31 digestion prior to restriction and by reanneling experiments, all but one DNA is circularly permuted. Terminal repetition is also present in six phage DNAs. PMID- 2997986 TI - Synthesis in cell culture of the gapped linear duplex DNA of the slow virus visna. AB - Visna virus is a nontransforming retrovirus that causes slow infections in animals and a rapidly progressive-lytic infection in cell culture. The results of an analysis of the synthesis of viral DNA in cell culture are reported. Region- and strand-specific probes cloned in M13 have been used to define the dynamics of DNA synthesis and the major nucleic acid species formed. It is shown that (i) within the first hours of infection, a full-length copy of the viral RNA genome is synthesized by reverse transcription, (ii) early in infection a major species of DNA is formed that extends from a site near the center of the molecule to the 3' end, (iii) somewhat later a second major species of plus-strand DNA is generated that extends from the 5' end to the middle of the genome. As a consequence, most viral DNA molecules consist of a full-length minus strand, and two plus strands separated by a gap or nick in the center of the molecule (J. D. Harris, J. V. Scott, B. Traynor, M. Brahic, L. Stowring, P. Ventura, A. T. Haase, and R. Peluso (1981). Virology 113, 573-583). The implications of this viral DNA structure for one unusual aspect of the lentivirus life cycle, the production of viral RNA, and virions from extrachromosomal DNA are discussed (J. D. Harris, H. Blum, J. Scott, B. Traynor, P. Ventura, and A. T. Haase (1984). Proc. Natl. Acad. Sci. USA 81, 7212-7215). PMID- 2997987 TI - Characterization and chromosomal distribution of endogenous mouse mammary tumor viruses of European mouse strains STS/A and GR/A. AB - The endogenous mouse mammary tumor virus (MMTV) proviral copies in two genetically dissimilar mouse strains, STS/A, a European mouse strain, and BALB/c, were characterized. STS/A carries the same four MMTV proviral copies as GR.Mtv-2 ; these strains share also most of the isoenzyme markers and are therefore highly related. Cellular DNA of GR.Mtv-2- contains a partial MMTV provirus that is not present in STS/A. GR.Mtv-2- is derived from GR; they differ in the locus Mtv-2 that contains one MMTV provirus. Expression of this Mtv-2 endogenous MMTV provirus is directly linked to mammary tumorigenesis in GR. MMTV proviral loci were studied using restriction enzyme analysis and the Southern transfer procedure using liver DNAs from recombinant inbred strains between BALB/c and STS/A. All segregating MMTV-specific EcoRI fragments were identified to MMTV proviral loci and most of these were localized by studying the cosegregation of the Mtv units and known chromosomal markers. Since STS/A, GR.Mtv-2-, and GR are highly related, the five complete endogenous MMTV proviruses of GR were located on the following chromosomes: Mtv-2 on chromosome 18, Mtv-3 on 11, Mtv-19 on 1, Mtv-20 on 4, whereas Mtv-8 has tentatively been located on chromosome 18 by Callahan et al. (R. Callahan, D. Gallahan, and Ch. Kozak (1984), J. Virol. 49, 1005-1008). GR and GR.Mtv-2 furthermore contain two incomplete MMTV proviral elements, one of which is also present in STS/A. PMID- 2997988 TI - Isolation and characterization of defective-interfering particles of poliovirus Sabin 1 strain. AB - Defective-interfering (DI) particles of the Sabin strain of type 1 poliovirus were generated on serial high m.o.i. passaging. The deletions, measured by agarose gel electrophoresis, appeared to comprise approximately 10% of the total genome. Analysis of the RNAs, after digestion with RNase T1, by two-dimensional polyacrylamide gel electrophoresis revealed that the locations of the deleted genome regions were similar to those of the DI particles of the Mahoney strain of type 1 poliovirus (A. Nomoto, A. Jacobson, Y. F. Lee, J. Dunn, and E. Wimmer, (1979), J. Mol. Biol. 128, 179-196). Taking the known nucleotide sequences of the total genome and large RNase T1-resistant oligonucleotides into account, the deletions of almost all DI RNAs were found to exist between nucleotide positions 1307 and 2630, a genome region encoding capsid polypeptides VP2, VP3, and VP1. In cells coinfected with the purified DI particles and the Sabin strain of type 2 or type 3 poliovirus, particles containing the DI genomes were effectively produced. These results suggest that encapsidation signals are conserved in all three serotypes of polioviruses. However, only a very small amount of similar DI particles appeared to be produced in cells coinfected with coxsackie virus B1, although the genomes of polioviruses and coxsackie viruses have common sequences and therefore these viruses are considered to have arisen from a common ancestor. These data may suggest differences in encapsidation signals between polioviruses and coxsackie viruses. PMID- 2997989 TI - Induction of a deoxyuridine triphosphate nucleotidohydrolase activity in Epstein Barr virus-infected cells. AB - Superinfection of Raji cells with Epstein-Barr virus (EBV) or chemical induction of HR-1 cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) results in the induction of a deoxyuridine triphosphate nucleotidohydrolase (dUTPase) which is not observed in mock-treated cells or TPA-treated EBV genome-negative BJAB cells. The EBV-induced dUTPase could be distinguished from the host dUTPase based upon differences in their migration in polyacrylamide gels and sensitivity to the 5 mercurithioguanosine derivitive of dUTP. The expression of the EBV-specified dUTPase is prevented by phosphonoacetic acid indicating that its expression is dependent upon EBV-DNA replication. PMID- 2997990 TI - The gag and pol genes of bovine leukemia virus: nucleotide sequence and analysis. AB - The DNA sequence of the gag and pol regions of a provirus cloned from a bovine tumor is presented. In order to confirm these results the sequence of portions of a second clone, derived from a virus-producing cell line, was also determined. The gag gene was found to consist of 1179 nucleotides, which probably encode only three proteins: an N-terminal protein of 109 amino acids, a major core protein (p24) of 215 amino acids, and a nucleic acid binding protein (p12) of 69 residues. An open reading frame, whose translated product showed clear homology to the avian and murine proteases, was found beginning immediately upstream of the 3' end of gag. Following this protease region, a third long open reading frame, encoding 852 amino acids, showed clear homology to both avian and murine pol genes. The mechanism of translation of the protease and pol gene products cannot be predicted with certainty. Like Moloney murine leukemia virus (M-MuLV), BLV has a termination signal at the 3' end of gag, but unlike M-MuLV the protease is in a different reading frame. Like Rous sarcoma virus (RSV), BLV has a termination signal at the 3' end of the protease region and the reverse transcriptase is in a different (i.e., the third) reading frame. Possible translation mechanisms are discussed. Finally, the BLV gag and pol gene products are highly related to those of the human T-cell leukemia virus (HTLV); relatedness varied from 37% amino acid identities within the N terminal gag protein to 54% within the nucleic acid binding protein. Highly significant homology with both murine and avian type-C proteins was found within p24, p12, and the putative protease, reverse transcriptase, and endonuclease. Based on this homology, the BLV-HTLV family of viruses appears about equally distantly related to murine and avian type-C viruses. PMID- 2997991 TI - Attenuation of murine coronavirus infection by ammonium chloride. AB - Ammonium chloride at a concentration of 20 mM delayed by 4-5 hr the production of virus progeny in mouse L-2 cells infected at high multiplicity with mouse hepatitis virus (MHV). This delay was seen in the production of both intracellular and extracellular virus. However, the final titers were similar to those produced by MHV-infected cells maintained in normal medium. The manifestation of virus-induced cell fusion was similarly found to be delayed, but not otherwise decreased in severity, when ammonium chloride was present in the culture medium. Ammonium chloride caused similar delays in production of virus specific, positive-sense RNAs and of viral polypeptides. The relative proportions and apparent molecular weights of viral RNAs and polypeptides were similar to those found in MHV-infected cells cultured in normal medium. In vitro translation of endogenously produced viral RNAs in cell extracts, prepared from MHV-infected cells, was not inhibited by ammonium chloride. Thus, ammonium chloride has no specific, inhibitory effect on viral protein synthesis. Ammonium chloride did not reduce the number of virus-infected cells in culture, as monitored by infectious center assay. Analysis of early events in MHV infection showed that ammonium chloride did not affect adsorption or internalization of MHV by L-2 cells. However, the subsequent eclipse phase, as monitored by decline in infectivity of internalized virus inoculum proceeded less efficiently in the presence of ammonium chloride. On the basis of the known inhibitory effects of ammonium chloride on lysosomal/endosomal functions, the results suggest an endosomal mechanism of MHV uncoating. Thus the primary effect of ammonium chloride on MHV infection of L-2 cells is to attenuate virus uncoating, thereby chronologically displacing all subsequent virus-encoded functions. PMID- 2997994 TI - The herpes simplex virus amplicon. IV. Efficient expression of a chimeric chicken ovalbumin gene amplified within defective virus genomes. AB - cDNA sequences of the chicken ovalbumin gene were fused to an alpha (immediate early) promoter of herpes simplex virus and to genomic ovalbumin 3'-flanking sequences. The chimeric alpha-ovalbumin gene was introduced into defective virus genomes which were stably propagated in serially passaged virus stocks in the presence of helper virus. Analyses of polypeptides synthesized in cells infected with the resultant defective virus stocks revealed the abundant expression of the chimeric alpha-ovalbumin gene. The presence of introns was not essential for this expression. PMID- 2997993 TI - Ultrastructural localization of L and NS enzyme subunits on vesicular stomatitis virus RNPs using gold sphere-staphylococcal protein A-monospecific IgG conjugates. AB - Colloidal gold spheres were coated with staphylococcal protein A and were used to determine the location of NS and L proteins on vesicular stomatitis virus (VSV) ribonucleoprotein (RNP) complexes using monospecific anti-NS and anti-L IgG preparations. Conjugates using either anti-NS or anti-L demonstrated that these enzyme subunits were uniformly distributed along the entire length of the RNP complex. Under saturating conditions of IgG concentrations, it was observed that there were at least 60-70 molecules of NS protein and 30-35 molecules of L protein labeled per RNP complex. PMID- 2997995 TI - Characterization of New Jersey vesicular stomatitis virus isolates from horses and black flies during the 1982 outbreak in Colorado. AB - Vesicular stomatitis viruses isolated from horses, afflicted during the recent outbreak in the western United States, and from black flies (Simuliidae) were characterized with respect to the homology of their genomic RNAs and the mobility of their proteins in polyacrylamide gels. All the isolates were very similar, if not identical, with respect to these two parameters. When the black fly isolate was compared to other VSV isolates, this virus appeared to belong in the Hazelhurst subgroup of the New Jersey serotype of VSV. Since the other viruses in this division were obtained from infected swine, the natural host range of this subgroup has been extended to horses. PMID- 2997992 TI - Stable induction of a 51K cellular protein in neuronal cells surviving herpes simplex virus type 1 infection. AB - A series of survivor cell lines derived by infection of B103 rat neuroma cells with active wild-type herpes simplex virus type 1 (HSV-1) (M. Levine, A. L. Goldin, and J. C. Glorioso, J. Virol. 35, 203-210 (1980)) has been isolated. The survivor cells produced no infectious virus, yet they continued to react with HSV 1 antiserum for over 100 cell generations following the initial infection. The reactivity of the survivor cells with HSV-1 antiserum is characterized as being due to expression of a 51K protein. The 51K protein reacted with antiserum prepared against HSV-1 virions and was not detectable in the parental B103 cells. A protein of the same molecular weight was seen in productively infected B103 and HEL cells. The protein detected in the survivor cells comigrated with that seen in the infected cells on two-dimensional gel electrophoresis, indicating that they represent similar proteins. Despite the presence of the 51K protein reactive with HSV-1 antiserum, the survivor cells contain no detectable HSV-1 DNA sequences. They do contain DNA sequences which cross-hybridize with HSV-1 DNA, but similar cross-hybridizing sequences were also present in the parental B103 cells. No hybridizing polysomal, polyadenylated RNA species were present in the survivor cells that were not present in the parental B103 cells when probed with the cross-hybridizing HSV-1 restriction fragments. Therefore, the 51K protein evidently represents a cellular protein induced by the HSV-1 infection. PMID- 2997997 TI - Effect of tunicamycin and monensin on biosynthesis, transport, and maturation of bovine herpesvirus type-1 glycoproteins. AB - The effect of tunicamycin and monensin on the biosynthesis, intracellular transport, and maturation of bovine herpesvirus type-1 (BHV-1) glycoproteins was examined. Tunicamycin completely inhibited the production of infectious virus particles and significantly reduced the incorporation of [3H]glucosamine into viral glycoproteins. In the presence of monensin, reduced amounts of infectious virus particles were produced, which was mainly due to inhibition of virus release, rather than virus production. Monensin only slightly inhibited viral glycoprotein synthesis. The effects of these compounds on infectivity indicated that glycosylation is required for the production of infectious virus, though complete processing of the glycoproteins is not essential. In addition, egress of the virions from infected cells probably requires a functional Golgi complex. In the presence of tunicamycin or monensin various degrees of glycosylation of the major glycoproteins occurred, consequently their rates of migration differed from that of the normal glycoproteins. Tunicamycin completely blocked glycosylation of GVP 6/11a/16 and GVP 7. In contrast, GVP 3/9 and GVP 11b were partially glycosylated in the presence of tunicamycin. These results indicated that GVP 6/11a/16 and GVP 7 are N-linked glycoproteins, but GVP 3/9 and GVP 11b contain both N- and O-linked oligosaccharide side chains. Tunicamycin blocked the transport of all viral glycoproteins to the cell surface, suggesting that glycosylation is required for this process. In the presence of monensin, the viral glycoproteins were transported and expressed on the cell surface indicating that transport does not require complete processing of the glycoproteins and may occur via a Golgi-independent pathway. In addition, monensin-treated BHV-1 infected cells could act as target cells in an antibody-dependent cell cytotoxicity assay. Thus, complete glycosylation may not be essential for maintenance of antigenicity and participation in immune destruction. PMID- 2997996 TI - Molecular characterization of two strains of Shope fibroma virus. AB - An isolate of Shope fibroma virus (SFV), designated Indiana (SFV-I), was previously described to be tumorigenic in vivo as SFV, cytocidal in vitro as the orthopoxviruses (vaccinia, rabbitpox, etc.), and to share antigenic determinants with SFV and vaccinia. The genetic relatedness of SFV-I to SFV and vaccinia was studied by means of Southern blotting and hybridization. The results indicated that SFV-I shares extensive DNA homology with vaccinia, but few common sequences with SFV. By contrast, SFV and vaccinia show no sequence homology. These findings suggest that SFV-I is an orthopoxvirus which carries some genetic information from the leporipoxviruses. Recombinants between two genera of poxviruses have not been reported before. PMID- 2997998 TI - Identification and characterization of a mouse mammary tumor virus protein uniquely expressed on the surface of BALB/cV mammary tumor cells. AB - A unique subline of BALB/c mice, designated BALB/cV, exhibits an intermediate mammary tumor incidence (47%) and harbors a distinct milk-transmitted mouse mammary tumor virus (MMTV). The BALB/cV subline was used to study the molecular basis of potential virus-host interactions involving cell surface-expressed MMTV proteins. Cell surface iodination identified virus-specific proteins expressed on BALB/cV primary mammary tumor cells grown in culture. In contrast to (C3H)MMTV producing cell lines which expressed MMTV gp52, BALB/cV tumor cells lacked gp52 and expressed instead a 68K, env-related protein. The 68Kenv protein was also detected on the surface of metabolically labeled BALB/cV tumor cells by an external immunoprecipitation technique. The expression of 68Kenv was restricted to mammary tissues of BALB/cV mice that also expressed other MMTV proteins. Biochemical analysis established that 68Kenv was not modified by N-linked glycosylation. 125I-labeled 68Kenv was rapidly released into the media of tumor cell cultures and was recovered both in the form of a soluble protein and in a 100,000 g pellet. The biologic function of this cell surface-expressed viral protein remains unknown. PMID- 2997999 TI - Simian sarcoma virus-encoded gag-related protein: in vitro cleavage by Friend leukemia virus-associated proteolytic activity. AB - The simian sarcoma virus (SSV) encodes a gag-related 65,000-Da protein (SSV p65) which is not processed in SSV nonproducer cells (SSV-NP cells) (H.-J. Thiel, T. J. Matthews, E. M. Broughton, K. J. Weinhold, D. P. Bolognesi, T. Graf, and H. Beug (1981a), Virology 114, 124-131). In order to cleave SSV p65, retroviral particles containing this antigen were incubated with extracts from the heterologous helper virus Friend leukemia virus (FLV). Superinfection of SSV-NP cells by FLV has been previously shown to result in processing of SSV p65 in vivo (H.-J. Thiel, F. Weiland, R. Hafenrichter, T. J. Matthews, and K. J. Weinhold (1982), Virology 123, 229-234). In vitro cleavage was most efficient in the presence of a nonionic detergent (greater than 0.1% Nonidet-P40) and a reducing agent (greater than 5 mM dithiothreitol) at a pH of 7.0. The products, termed SSV p55 (p15, p12, p30), SSV p30, SSV p25 (p15, p12), and SSV p10, were characterized by (1) molecular weight, (2) kinetics experiments, (3) incorporation of different radiolabeled amino acids, and (4) comparison with SSAV structural proteins. Kinetics experiments with two amino acids ([3H]leucine, [35S]cysteine) revealed that initial processing of SSV p65 produced SSV p55 and SSV p10, with subsequent processing of SSV p55 occurring thereafter. In contrast to the Moloney system, the major intermediate p40 (p30, p10) could not be clearly demonstrated. A direct comparison of SSAV p10 and the cleavage product SSV p10 by SDS-PAGE suggests that SSAV pr65gag and SSV p65 differ slightly by molecular weight. PMID- 2998000 TI - Correlation of the genetic and physical maps of phage T1. AB - A series of BglII- and Sau3A-derived fragments of phage T1 DNA were cloned into the plasmid vector pLV59 and the clones were tested for their ability to complement and recombine with infecting genomes of am mutants representing all known T1 essential genes. The resulting data were used to correlate the T1 genetic and physical maps. This analysis led to the following conclusions: the large map distances observed at the left (early) end of the genetic map arise partly from increased genetic recombination at the chromosomal left end and partly from increased physical distances between identified genetic markers in this region, the right chromosomal end is also recombinogenic, genes 3.5 to 18, which occupy the right two-thirds of the DNA molecule, are tightly compacted with little space for additional protein-specifying sequences in this region. PMID- 2998001 TI - Complete DNA sequence of lymphotropic papovavirus: prototype of a new species of the polyomavirus genus. AB - Lymphotropic papovavirus (LPV) is a new member of the polyomavirus genus. Its host range in vitro is restricted to transformed cells of B-lymphocyte origin. Here the complete 5270-bp DNA sequence of LPV is presented. The LPV early region can encode a large T and a small t antigen but no middle T antigen and the late region can encode the three structural proteins VP1, VP2, and VP3. Based on sequence conservation of shared proteins LPV is equally related to both mouse polyomavirus (Py) and simian virus 40 (SV40) and represents a new distinct species of the polyomavirus genus. Sequence comparisons of LPV, SV40, and Py point out essential conserved sequence features of the polyomavirus genus more clearly than the comparison of only SV40 and Py. The most conserved proteins are VP1 with 42% and large T antigen with 28% of the amino acids conserved among the three viruses. Although least conserved the noncoding DNA sequences of LPV show significant homologies both to SV40 and Py (origin of viral DNA replication and putative early promoter). A 63-bp tandem repeat at the late side of the replication origin possibly represents a LPV enhancer element. PMID- 2998003 TI - Orthopoxvirus DNA: a comparison of restriction profiles and maps. AB - Characteristic DNA endonuclease digest fragment electropherograms and restriction site maps permitted differentiation and genome structure analysis of 38 orthopoxviruses that included isolates of monkeypox virus from humans and animals, monkeypox white variants, variola, vaccinia, ectromelia, Tatera (gerbil) and raccoon poxviruses, and cowpox and camelpox viruses. HindIII cleavage sites mapped on the 38 virus genome DNAs plus SmaI, BglI, SacI, KpnI, XhoI, and SalI maps for variola (Harvey) and monkeypox (Copenhagen) virus DNAs were derived essentially by cross-hybridizations with monkeypox, vaccinia, and variola virus cloned DNA restriction fragments, thus digest fragments could be assigned homologous regions on previously established genome maps. Salient of our observations, the DNA HindIII maps correlated to a high degree, but variations in middle and especially terminal DNA region cleavage sites provided a basis for discerning species, strains and variants. The extent of the inverted terminal repetitions (ITRs) for 37 DNAs were determined with HindIII, PvuI, SalI, and ClaI, plus nine more restriction enzymes for Bangladesh variola virus DNA by hybridizations with either the terminal tandemly repeated 70-bp segment or an EcoRI-PvuI near hairpin-end 75-bp segment from WR vaccinia virus. The opposite terminal regions of variola DNA were considerably asymmetrical compared to the large symmetrical ITRs of the other species examined. An apparent DNA inversion and concurrent deletion (1 kbp) with subsequent repair of DNA to original structure was suggested from right terminal region maps of four viruses chosen from a variola virus passage series in monkeys. Correlative with virus geographic distribution, two strains of monkeypox virus, each containing two variants, were differentiated by DNA profiles of isolates from smallpox-like disease (SLD) patients of the African rainforest region. The DNAs of five monkeypox viruses isolated from laboratory and zoo animals resembled most DNAs from SLD monkeypox viruses from Sierra Leone. A poxvirus from an American raccoon contained 40% DNA that did not cross-hybridize with orthopoxvirus DNA probes. The DNAs of recent isolates from a gerbil and from a camel each mapped as unique African orthopoxvirus species and differed from variola virus. PMID- 2998002 TI - Amino-terminal amino acid sequences of structural proteins of three flaviviruses. AB - N-terminal amino acid sequences of structural proteins of three flaviviruses, yellow fever, St. Louis encephalitis, and dengue-2 viruses, have been obtained. The glycoproteins of these three viruses are 52-60% conserved in the region sequenced, depending upon which pair of viruses are compared, and 40% of the amino acids are invariant in all three viruses. Thus, flaviviruses are closely related and have in all probability descended from a common ancestor. Furthermore, residues important in the secondary structure of proteins are conserved, suggesting that the overall conformation of the glycoproteins is the same in all three viruses while considerable variation in the primary sequence can be accommodated. The N-terminal regions of the nucleocapsid proteins of yellow fever and St. Louis encephalitis viruses show markedly less homology (25%) and this region is highly basic with one-quarter (yellow fever) or one-third (St. Louis encephalitis) of the residues being lysine or arginine. N-terminal sequences for the M protein of yellow fever and for NV2(GP19) of St. Louis encephalitis viruses are also reported. PMID- 2998004 TI - Processing of virus-specific glycoproteins of varicella zoster virus. AB - Monoclonal antibodies to varicella zoster virus (VZV) glycoproteins were used to study the processing of three glycoproteins with molecular weights of 83K-94K (gp 2), 64K (gp 3), and 55K (gp 5). Immunoprecipitation experiments performed with VZV-infected cells, pulse labeled with [3H]glucosamine in the presence of tunicamycin, suggest that O-linked oligosaccharide is present on the glycoprotein of gp 2. Use of the enzyme endo-beta-N-acetylglucosaminidase H revealed that the fully processed form of gp 3 had high-mannose type and that of gp 5 had only complex type of N-linked oligosaccharides. Experiments with monensin suggest that the precursor form (116K) of gp 3 is cleaved during the processing from Golgi apparatus to cell surface membrane. The extension of O-linked oligosaccharide chain and the complex type of N-linked oligosaccharide chains also occurs during this processing. PMID- 2998005 TI - Phage P22 lysis genes: nucleotide sequences and functional relationships with T4 and lambda genes. AB - Wild-type and amber mutant alleles of the lysis genes of P22 were cloned and sequenced. Gene 13 encodes an 11,520-Da basic hydrophobic protein that has 89% amino acid homology to lambda S protein. Gene 19 encodes a protein that has a small degree of amino acid homology with T4 lysozyme, but we could detect no homology to lambda R or RZ proteins. The protein product of gene 19 was purified; its amino terminal amino acid sequence is as predicted by the DNA sequence. It starts with a single amino terminal methionine residue and is a basic protein with a molecular weight of 15,968. Plasmids expressing P22 gene 19, lambda genes R and RZ, and T4 gene e were constructed. All of these plasmids were able to complement both lambda R- and P22 19-. PMID- 2998006 TI - Molecular cloning of the PRCII sarcoma viral genome and the chicken proto oncogene c-fps. AB - The class II avian sarcoma viruses comprise PRCII, PRCIIp, PRCIV, URI, 16L, and Fujinami. The members of this class are all replication-defective viruses containing various amounts of a transforming sequence called v-fps. PRCII contains the smallest amount of fps-specific sequences, transforms fibroblasts in tissue culture, but is only weakly tumorigenic. As a first step in understanding variations in pathogenicity among the class II avian sarcoma viruses and the mechanism by which the oncogene of these viruses was transduced from a single cellular locus, we have molecularly cloned the viral genome of PRCII, its related helper PRCII-AV, and the chicken proto-oncogene (c-fps) from which v-fps derived. The fps-specific region within the cloned PRCII genome was shown to be 0.8-1.0 kb smaller than that of the Fujinami fps-specific region, in agreement with previous studies. Transfection of the cloned DNAs into primary chicken cells demonstrated that both clones (PRCII and PRCII-AV) are biologically active. The cloned PRCII genome is helper dependent and produces a gag-fusion phosphoprotein (P105) which is phosphorylated on a tyrosine residue. The cloned PRCII-AV genome produces infectious virus and can function as a helper for the cloned PRCII genome in transfection assays. Three overlapping recombinant lambda clones homologous to v fps from a chicken genomic library have been isolated. One of these, lambda-c fps(2), contains all of the cellular sequences homologous to v-fps. In the aggregate, the three molecular clones may represent the entirety of c-fps. PMID- 2998007 TI - Assignment of simian rotavirus SA11 temperature-sensitive mutant groups B and E to genome segments. AB - Recombinant (reassortant) viruses were selected from crosses between temperature sensitive (ts) mutants of simian rotavirus SA11 and wild-type human rotavirus Wa. The double-stranded genome RNAs of the reassortants were examined by electrophoresis in Tris-glycine-buffered polyacrylamide gels and by dot hybridization with a cloned DNA probe for genome segment 2. Analysis of replacements of genome segments in the reassortants allowed construction of a map correlating genome segments providing functions interchangeable between SA11 and Wa. The reassortants revealed a functional correspondence in order of increasing electrophoretic mobility of genome segments. Analysis of the parental origin of genome segments in ts+ SA11/Wa reassortants derived from the crosses SA11 tsB(339) X Wa and SA11 tsE(1400) X Wa revealed that the group B lesion of tsB(339) was located on genome segment 3 and the group E lesion of tsE(1400) was on segment 8. PMID- 2998008 TI - Serine kinase activity associated with Maloney murine sarcoma virus-124-encoded p37mos. AB - An antiserum directed against amino acid residues 37-55 [anti-mos (37-55) serum] of the predicted v-mos sequence was used to precipitate p37mos from Moloney murine sarcoma virus-124 (Mo-MuSV-124) acutely infected 3T3 cells. Proteins with sizes ranging from p37mos to 43 kDa (p43) were found to be phosphorylated when anti-mos (37-55) immune complexes containing p37mos were incubated with [gamma 32P]ATP and Mn2+. The phosphorylation of p37mos and p43 could be specifically blocked when the anti-mos (37-55) serum was incubated with 37-55 cyclic mos peptide prior to immunoprecipitation, but not if the serum was preincubated with an unrelated peptide representing amino acids of the myc protein sequence. Anti mos (37-55) immune complexes from uninfected 3T3 cells did not produce any phosphorylated proteins the size of p37mos or p43. However, a 50-kDa protein (p50) was phosphorylated in both unblocked and mos peptide-blocked anti-mos (37 55) immune complexes from infected 3T3 cells, and in immune complexes from uninfected cells. Quercetin, an inhibitor of some protein kinases, inhibited the kinase phosphorylating p50 but not the kinase phosphorylating p37mos and p43. Preabsorption of the cell extract prior to immunoprecipitation with an excess of formalin-fixed Staphylococcus aureus, complexed with preimmune normal rabbit serum IgG, specifically removed the kinase phosphorylating p50. The amount of in vitro phosphorylated p37mos and p43 in the immune-complex kinase assay reached a maximum in extracts of 3T3 cells 2-3 days postinfection with Mo-MuSV 124 but decreased to trace levels after 5 days. Metabolically and in vitro phosphorylated p37mos generated an identical pattern of phosphopeptides upon partial V8 protease digestion. Based on peptide mapping and a kinetic analysis of the in vitro phosphorylation reaction, p37mos appears to be a precursor to the p43 phosphorylated species. Phosphoamino acid analyses revealed only phosphoserine in in vitro phosphorylated p37mos and p43mos. It was concluded that p37mos is closely associated with a serine kinase activity and that the in vitro phosphorylation of p37mos may lead to formation of a highly modified mos protein (p43) by way of superphosphorylation. PMID- 2998009 TI - Natural variants of the Sabin type 1 vaccine strain of poliovirus and correlation with a poliovirus neutralization site. AB - Independent substitution mutations have been detected in capsid polypeptide VP1 of the type 1 oral poliovirus vaccine isolated from normal infant vaccine recipients. These mutations map at amino acid residues 142 and 147 of VP1, a region only minimally hydrophilic. A synthetic peptide, corresponding to residues 141 to 147 of VP1 was synthesized, conjugated to a carrier polypeptide of bovine serum albumin. The conjugate was found to elicit a weak poliovirus neutralizing antibody response. It was also capable of priming the immune system for the production of IgG-type antibodies able to neutralize greater than 99.999% of infectious type 1 virus. It is suggested that region 141 to 147 of VP1 may be involved in neutralization of the virus and that the mutants may have accumulated by antibody selection. PMID- 2998010 TI - The mannosidase inhibitors 1-deoxymannojirimycin and swainsonine have no effect on the biosynthesis and infectivity of Rous sarcoma virus. AB - The effects of inhibitors, which interfere with oligosaccharide trimming by blocking mannosidases, on the processing and export of the viral glycoproteins of Rous sarcoma virus (RSV), have been studied. 1-Deoxymannojirimycin (DIM) prevents removal of mannose residues from the Man9 (GlcNAc)2 oligosaccharide whereas swainsonine (SW) blocks at a later stage resulting in the formation of so-called hybrid oligosaccharides. Under a regime of these inhibitors, proteolytic cleavage of the viral glycoprotein precursor can still occur to yield aberrant glycoprotein products, gp75DIM/gp30DIM and gp80SW/gp30SW. Virus particles carrying these aberrant viral glycoproteins are released from inhibitor-treated cultures in normal amounts and these virions are fully infectious. Thus blocking oligosaccharide trimming at the stages described here or, using different inhibitors, at different stages as described previously (J. V. Bosch and R. T. Schwarz, Virology 132, 95-109 (1984)), does not have any influence on the infectivity of Rous sarcoma virus. PMID- 2998011 TI - Crossover sites cix for inversion of the invertible DNA segment C on the bacteriophage P7 genome. AB - The bacteriophage P7 genome contains an invertible DNA segment called C which determines its host range. P7 C(+) phages produce plaques on Escherichia coli K12. The C segment consists of a 3-kb unique sequence and 0.62-kb inverted repeats of which one carries an internal 0.2-kb deletion. This deletion has been mapped within the right inverted repeat in the C(+) orientation. The crossover sites cix for inversion of the C segment do not map at the inside boundaries of the inverted repeats, as had been proposed. They are localized at the external ends of these repeats. Thus organization of the C segment in phage P7 is analogous to that in the related phage P1. PMID- 2998012 TI - The characterization and molecular cloning of the double-stranded RNA genome of an Australian strain of infectious bursal disease virus. AB - The genome of infectious bursal disease virus (IBDV) strain 002-73 was found to consist of two segments of double-stranded (ds) RNA which were 3400 bp (MW 2.06 X 10(6)) and 2900 bp (MW 1.76 X 10(6)) long, respectively. The ds IBDV RNA could be translated, in vitro, only after extensive denaturation. The small RNA segment was found to code for a single polypeptide of MW 90K, while the large RNA segment coded for three major polypeptides of MW 52K, 32K, and 28K, and two minor polypeptides of MW 41K and 16K. The large RNA segment could encode proteins of MW 125K while the MW of the translated products was 169K suggesting that a precursor product relationship exists between some of the translation products. A method is described for the synthesis of ds cDNA from large ds RNA molecules. Analyses of recombinant colonies showed that inserts covering the entire IBDV genome had been cloned. PMID- 2998013 TI - Studies on human parainfluenza virus 3: characterization of the structural proteins and in vitro synthesized proteins coded by mRNAs isolated from infected cells. AB - The structural proteins of human parainfluenza virus 3, a member of the paramyxovirus family, were characterized by SDS-polyacrylamide gel electrophoresis of radiolabeled virus. The purified virion contains at least eight structural proteins, with estimated molecular weights of 251K, 90K, 71K, 68K, 65K, 51K, 35K, and 21K, respectively. Three of the polypeptides (71K, 65K, and 51K) were identified as glycoproteins based on their incorporation of [3H]glucosamine. Disruption of the virus by Triton X-100 in the presence of increasing salt concentrations indicated that the polypeptides of molecular weights 251K, 90K, 68K, and 21K were components of the nucleocapsid. In parainfluenza virus 3 infected BS-C-1 cells, seven virus structural polypeptides were identified. Six structural proteins (90K, 71K, 68K, 51K, 35K, and 21K) were detected in the cell lysate at 7 hr after infection, while at 10 hr an additional polypeptide (251K) was also observed. At least two nonstructural polypeptides of molecular weights 30K and 25K were also detected in infected cells. mRNAs isolated from virus-infected cells were translated in a cell-free protein synthesizing system. The in vitro translation products were identical to the authentic virion polypeptides as determined by partial digestion with staphylococcal V8 protease. PMID- 2998014 TI - Isolation of a papovavirus with a bipartite genome containing unlinked SV40 and BKV sequences. AB - Wild-type (wt) BK virus was introduced into permissive BSC-1 cells along with either early or late defective SV40 genomes. The defectives contained all of the late (L-SV40) or all of the early (E-SV40) coding sequences. Persistently infected (PI) BSC-1 cultures were established and contained wt BKV DNA and E- or L-SV40 DNA in Hirt supernatants. Each of the BKV/SV40 combinations could be serially passed in BSC-1 cells. Also, DNase I digestion of virus stocks from BKV/E-SV40 infections did not eliminate E-SV40. This suggested that (1) E-SV40 genomes could be packaged in BKV capsids and (2) BKV T antigen acted to stimulate the growth of L-SV40 genomes. During continuous culture of PI BSC-1 cells containing BKV and L-SV40, wt BKV genomes were lost and replaced by a BKV defective. The BKV defective (E-BKV) contained a deletion in the late region, an intact early region, and a duplication of the origin. This combination represents a new papovavirus with a bipartite genome in which the early region is derived from BKV and the late region from SV40, and both are present in separate molecules. The BKV and SV40 defectives complement each other for infectivity. Infectious virus is formed with the E-BKV genomes packaged in SV40 capsids. It is hypothesized that this kind of recombination (reassortment) is a way in which papovaviruses may generate variation. The host range for the new BKV/SV40 is narrow. It propagates well in BSC-1 cells, relatively poorly in fetal human brain cells, and not at all in green monkey TC-7 or human embryonic kidney cells. However, it transforms fetal human brain cells at a frequency 25-50 times greater than wt BKV does. PMID- 2998015 TI - Genome rearrangements of bovine rotavirus after serial passage at high multiplicity of infection. AB - After serial passage at high multiplicity of infection of standard bovine rotavirus in MA104 cells, different genome rearrangements occurred in which segment 5 was lost from the RNA profile and distinct additional bands of double stranded (ds) RNA were found in positions on gels between segments 1 and 6. It was shown that some of the additional RNA bands contained segment 5-specific sequences. The additional RNA bands were transcribed in vitro to apparent full length. Analysis of the proteins synthesized in cells infected with viruses possessing rearranged genomes showed that in all cases the product of RNA segment 5, VP5, was missing; however, in one case an abnormal protein was observed which corresponded in size to the coding capacity of the mRNA transcribed from the additional genomic RNA band. Viruses with rearranged genomes could be plaque purified, and they grew in the absence of standard virus to titers comparable to those obtained from standard virus. In mixed infections of standard virus and virus possessing genome rearrangements, standard virus overgrew during passage at low multiplicity of infection whereas virus possessing genome rearrangements overgrew during passage at high multiplicity of infection. PMID- 2998016 TI - Nucleotide sequence analysis of the chicken gene c-mil, the progenitor of the retroviral oncogene v-mil. AB - The nucleotide sequence of the chicken gene c-mil was determined within and around all regions homologous to the oncogene v-mil of avian retrovirus MH2. The regions of homology to the previously determined v-mil sequence, ranging in size from 28 to 177 base pairs (bp), are distributed over 14 kilobase pairs (kbp) of the chicken genome and are organized in 11 exons. All exon-intron boundaries of c mil, except the 5' boundary of exon 1 and the 3' boundary of exon 11, were unambiguously defined by the identification of consensus splice donor and acceptor sites precisely at positions where homology to v-mil ceases or resumes. The homology to v-mil starts within the coding sequence of exon 1 and ends within the 3' untranslated region of exon 11, 12 nucleotides downstream from the nonsense codon terminating the large open reading frame shared between c-mil and v-mil. The c-mil and v-mil sequences differ at only 7 out of 1153 nucleotide positions, and the predicted sequences of v-mil and c-mil proteins differ by one conservative and four nonconservative substitutions among 379 amino acid residues. Hence, the carboxy-terminal domains of the MH2 gag-mil hybrid protein and of the putative c-mil protein are very similar. However, the amino-terminal domain of the cellular protein is possibly encoded by additional 5' c-mil sequences not present in the transduced v-mil oncogene, while that of the MH2 hybrid protein is encoded by viral gag sequences. The sequence analysis also revealed that c-mil and c-myc derived sequences are immediately adjacent on the MH2 genome carrying both the v-mil and the v-myc oncogene. Hence, transduction of c-mil into MH2 involved recombination, at the 3' site, with either the c-myc locus or a previously transduced v-myc gene, and, at the 5' site, with gag sequences of the transducing virus. At both sites, no significant homologies were found between the sequence elements involved in the recombination. PMID- 2998017 TI - A late gene product of phage P22 affecting virus infectivity. AB - Gene 14 is a recently discovered late gene of phage P22, mapping between the DNA injection and head completion genes (P. Youderian and M. Susskind (1980), Virology 107, 258-269). The gene 14 product has not been detected in phage particles. We have studied the defective phenotype of amber mutants in gene 14 to determine the role of gp14. The yield of physical particles from 14- infections is normal, but the infectivity of those particles is reduced by 60-80%. The noninfectious particles adsorb to but do not kill the host cell, as if they were defective in DNA injection. No differences in morphology, DNA composition, DNA permutation, or protein composition have been detected between 14- and wild-type particles. Procapsids, the capsid precursor to DNA packaging, exhibit a similar reduction in viability when isolated from 14- infected cells, assayed by in vitro DNA packaging. This is consistent with the gene 14 product functioning in the assembly or maturation of the procapsid. The three DNA injection proteins, encoded by genes 7, 16, and 20, are assembled into the particle at the procapsid stage. The defect in 14- particles may arise from improper organization or modification of one or more of the three proteins needed for DNA injection. PMID- 2998018 TI - Cloning and physical mapping of Yaba monkey tumor virus DNA. AB - The physical map positions for the BamHI, EcoRI, and SalI restriction fragments of Yaba monkey tumor pox virus DNA were determined using cloned virus DNA fragments as probes for hybridization as well as analyzing the secondary digests of larger DNA restriction fragments. Digests of EcoRI A and B fragments and SalI A and B fragments with BamHI allowed for the orientation of most of the BamHI restriction map. These secondary digest products were confirmed and the map positions for the EcoRI fragments were established using cloned BamHI fragments. Yaba monkey tumor virus DNA was cloned using the plasmid vector pBR322. PMID- 2998020 TI - Complementary DNA cloning and expression of the papaya ringspot potyvirus sequences encoding capsid protein and a nuclear inclusion-like protein in Escherichia coli. AB - Three cDNA clones that express viral gene products in Escherichia coli JM83 were derived from a watermelon mosaic virus-1 strain of papaya ringspot virus (PRSV W). DNAs complementary to portions of the viral RNA were inserted into the pUC8 and pUC9 plasmids, and the expressed polypeptides were fusion products with the amino terminus of beta-galactosidase. Clones W1-77 and W2-1 expressed fusion products with apparent molecular weights of 40,000 (40K) and 14K, respectively, which were serologically related to PRSV capsid protein. A 52K product serologically related to a 54K nuclear inclusion protein of tobacco etch virus was produced by clone W1-18. The sequences encoding the capsid and 57K nuclear inclusion-like proteins of PRSV were physically mapped to adjacent positions through Southern blot analyses of clones W1-77 and W1-18. PMID- 2998019 TI - pp60src-dependent protein phosphorylation in membranes from Rous sarcoma virus transformed chicken embryo fibroblasts. AB - The Rous sarcoma virus (RSV)-transforming protein, pp60src, is a plasma membrane associated tyrosine-specific protein kinase. A 36,000-Da cellular polypeptide (p36) which is phosphorylated at tyrosine in RSV-transformed chicken embryo fibroblasts (RSV-CEF) is also plasma membrane associated. To determine if p36 is directly phosphorylation and kinase activity in situ in the plasma membrane, src dependent protein phosphorylation in membranes isolated from RSV-CEF has been characterized. These membrane preparations contained high ATPase and phosphoprotein phosphatase activities; but when sufficient concentrations of [gamma-32P]ATP were used, the phosphorylation of pp60src and the phosphorylation of p36 were linear for 1 min or more, and the initial rates of phosphorylation could therefore be determined. In membranes from RSV-CEF pp60src and p36 became phosphorylated predominantly at tyrosine, while in membranes from uninfected cells p36 was phosphorylated at low levels at serine. When membranes from RSV-CEF were preincubated with tumor-bearing rabbit (TBR) serum, the IgG became phosphorylated while the phosphorylation of p36 was inhibited, suggesting that p36 is directly phosphorylated by pp60src. Phosphorylation of pp60src, p36, and TBR-IgG was dependent on growth temperature in membranes from cells infected by a temperature-sensitive mutant, tsNY68, although some dependence on growth temperature was observed even with membranes from wild-type RSV-infected cells. However, at the nonpermissive temperature, tsNY68 pp60src retained 20-40% of its kinase activity, providing supporting for the proposal (B. M. Sefton, T. Hunter, and K. Beemon (1980, J. Virol, 33, 220-229) that transformation may result from a small quantitative change in pp60src activity. The phosphorylation of pp60src and its kinase activity were not coordinately affected by growth temperature or mutations within src, indicating that different factors affect the phosphoacceptor capacity and kinase activity of the protein. PMID- 2998021 TI - Nucleotide sequences of the 1B and 1C nonstructural protein mRNAs of human respiratory syncytial virus. AB - The genes encoding the 1C and 1B mRNAs of human respiratory syncytial (RS) virus are first in the order of viral transcription and encode nonstructural (NS) proteins of approximate molecular weights 14,000 and 11,000, respectively, estimated by gel electrophoresis. The complete nucleotide sequences of the 1C and 1B mRNAs determined from several full-length cDNA clones are described. The 1C and 1B mRNAs contain 528 and 499 nucleotides, respectively, exclusive of poly(A), and encode proteins of 139 and 124 amino acids. The calculated molecular weights of the predicted NS1C and NS1B proteins are 15,567 and 14,674, respectively. Both mRNA sequences contain the 5'-terminal sequence, 5' GGGGCAAAU . . . , and the 3' terminal sequence, 5' . . . AGUAUA(N)1-4-poly(A), that were identified previously as conserved among six other RS viral mRNAs. In addition, a dicistronic readthrough RNA having the general structure 5' 1C mRNA-intergenic sequence-1B mRNA 3' was identified by dideoxynucleotide sequencing of intracellular poly(A)+ RNA using a DNA primer derived from a 1B-cDNA clone. In the dicistronic RNA, the nucleotide sequences of the 1C and 1B cistrons are separated by, in mRNA sense, four A residues and the intergenic sequence 5' . . . CUUAACAGAAGACAAAAAN . . . 3' (N represents unidentified nucleotide). The significance of these sequences is discussed. PMID- 2998022 TI - Recombinants between attenuated and virulent strains of poliovirus type 1: derivation and characterization of recombinants with centrally located crossover points. AB - Recombinants with a centrally located crossover point were selected from crosses between poliovirus type 1 strains and intertypic (type 3/type 1) recombinants. Two such recombinants were characterized in some detail. In one of them (v1/a1 6), the 5' half of the genome was derived from a virulent type 1 strain, while the 3' half came from an attenuated type 1 strain. The genome of the other recombinant (a1/v1-7) had the reverse organization, with the 5' and 3' halves being derived from the type 1 attenuated and virulent strains, respectively. As deduced from the RNase T1 oligonucleotide maps, the a1/v1-7 genome also had a relatively short centrally located insert of the poliovirus type 3 origin. Both recombinants exhibited ts phenotypes. The RNA phenotypes of the recombinants corresponded to that of the parent donating the 3' half of the genome, v1/a1-6 and a1/v1-7 expressing RNA- and RNA +/- characters, respectively. Despite being a ts RNA- virus, v1/a1-6 proved to be neurovirulent when injected intracerebrally into Cercopithecus aethiops monkeys, although it exhibited a somewhat diminished level of pathogenicity as compared to its virulent type 1 parent. Recombinant a1/v1-7 behaved as an attenuated strain. These data supported our previous conclusion drawn from the experiments with intertypic poliovirus recombinants that the attenuated phenotype of poliovirus depends largely on the structure of the 5' half of its genome, although mutations of the 3' half may alleviate the virulence of the virus to a degree. PMID- 2998023 TI - An identification of a transforming region of Epstein-Barr viral DNA cannot be confirmed. AB - We have analyzed Epstein-Barr viral DNA sequences in two cell lines HI-26-36 and HI-HFX. Stoerker et al. (J. Stoerker, J.E. Holliday, and R. Glaser (1983), Virology 129, 199-206) concluded that these cells were transformed by virus that arose as the result of marker rescue of a transformation-incompetent deletion mutant of EBV. We compared viral DNA sequences in prototype EBV strains with data presented in the Virology paper. Our findings are not consistent with the data of Stoerker et al. and indicate that the cell lines supplied to us did not arise by "marker rescue" but appear to contain the Jijoye viral genome. PMID- 2998024 TI - Effect of tunicamycin on the development of the cytopathic effect in Sindbis virus-infected avian fibroblasts. AB - In Sindbis virus-infected avian cells the development of the cytopathic effect is correlated with the disruption of plasma membrane function. Sindbis virus inhibits the activity of the Na+K+ATPase, a membrane-associated enzyme complex which regulates intracellular monovalent cation levels. Tunicamycin, which blocks envelope protein glycosylation, prevents inhibition of Na+K+ATPase activity and the development of morphological changes in Sindbis virus-infected cells. Although inhibition of Na+K+ATPase activity is not essential for the termination of host protein synthesis, membrane-mediated events may favor the selective translation of viral proteins. The termination of host protein synthesis does not contribute to the development of these cytopathic changes in the time frame examined. In tunicamycin-treated, Sindbis virus-infected cells, unglycosylated E1 is inserted into the plasma membrane but virus release is prevented. In productively infected cells, therefore, the inhibition of Na+K+ATPase activity and the development of the cytopathic effect may result from terminal events in virus assembly and/or virus release. PMID- 2998025 TI - Relation between the levels of mRNA abundance and kinetics of protein synthesis in pseudorabies virus-infected cells. AB - Virus proteins synthesized by pseudorabies virus-infected cells can be classified into five groups on the basis of the kinetics of their synthesis at various stages of the infective process; virus mRNAs can similarly be classified into four groups. To determine whether the kinetics of synthesis of specific proteins are determined solely by the level of abundance in the cells of their mRNAs, we have compared at various times after infection the relative synthesis of these proteins with the relative abundance of their mRNAs. We have focused on two proteins: the 142K major capsid protein, an early-late protein, and the 136K major DNA binding protein, an early protein. The mRNAs encoding these proteins were identified. The relative abundances of these mRNAs in the cytoplasms of the infected cells were found to be the same as those associated with the polysome fractions. The relative amount of the proteins synthesized by the infected cells at a given stage of the infective process closely reflected the relative amount of the mRNA encoding these proteins that was present in the cells at that stage of the infective process. Most virus mRNA species that are present in the cytoplasm of infected cells were represented on polysomes to approximately an equal extent. Some RNA species were, however, significantly underrepresented under certain conditions in the polysomal fractions. We conclude that whereas the amount of many virus proteins synthesized by the infected cells is determined mainly by the level of the abundance of their mRNAs, additional controls operate in the cells that determine the relative rates of synthesis of some other virus proteins. PMID- 2998026 TI - An endogenous virus from Lophortyx quail is the prototype for envelope subgroup 1 of avian retroviruses. PMID- 2998028 TI - Electron microscopic evidence for the cruciform structure in intracellular SV40 DNA. AB - The conformation of intracellular SV40 DNA during lytic infection of CV-1 cells was studied by the psoralen crosslinking technique. Analysis of the crosslinked SV40 DNA in the electron microscope revealed a rare population (0.1%) with a cruciform structure at coordinates 0.62 +/- 0.05 or at 0.37 +/- 0.05 of the SV40 genome. The implication of this observation in relation to SV40 DNA replication is discussed. PMID- 2998027 TI - Sequence homologies between bovine papillomavirus genomes mapped by a novel low stringency heteroduplex method. AB - The bovine papillomaviruses (BPVs) types 1, 2, and 5 cause fibropapillomas whereas BPVs types 3, 4, and 6 cause true papillomas. A novel method of heteroduplex mapping at low stringency of hybridisation has identified the position and relative orientation of distantly related sequences in the genomes of these viruses. The genomes of BPV-1 and BPV-2 are closely related but both show a high degree of sequence divergence from the BPV-5 genome. A 1.25-kb sequence adjacent to the unique BamHI site of the BPV-5 genome hybridised to BPV 1 and to the equivalent region of BPV-2. The hybridising sequence in the BPV-1 genome mapped to the C-terminal region of the E1 open reading frame (ORF) and the N-terminal region of the E2 ORF. The BPV-3, BPV-4, and BPV-6 genomes show moderate homology to each other but minimal homology to the fibropapillomavirus genomes. Low-stringency heteroduplex mapping revealed that overlapping sequences in the BPV-1 E1 and L1 ORFs (or the equivalent regions in BPV-2) hybridised to sequences in BPV-3, BPV-4, and BPV-6. Hybrid regions were less than 1 kb long and were sometimes interrupted by short nonhybridising segments. The hybridising sequences in BPV-3 and BPV-4 are positioned in a way that parallels the spacing of the E1 and L1 ORFs in BPV-1. These data suggest that the bovine fibropapilloma viruses and true papilloma viruses share a similar genomic organization, but have undergone extensive sequence divergence. PMID- 2998029 TI - Frequent generation of new 3'-defective interfering particles of vesicular stomatitis virus. AB - We have isolated and partially characterized a number of different genome types of defective interfering (DI) particles newly generated by a highly heat resistant strain of vesicular stomatitis virus in either Rat(B77) or Vero cells. Northern blot analyses revealed that many of these DI genomes contain N gene sequences and/or sequences of the NS, M, and G genes. One type contains NS sequences without any indication for the presence of either N, M, or G sequences. Another type of DI particle genomes did not contain any detectable sequences of N, NS, M, or G, but contain panhandle-type sequences and, thus, most likely resembles the 5'-panhandle-type DI particles. Unlike previously assumed, these data demonstrate that DI genomes which have the 3'-terminal N, NS, M, and G genes or portions of these genes conserved do frequently arise together with 5'-DI particle genomes after serial undiluted passages of the heat-resistant strain of vesicular stomatitis virus. PMID- 2998030 TI - Determination of a splice acceptor site of pX gene in HTLV-I infected cells. AB - The splice acceptor site of pX gene of HTLV-I has been determined to be at base position 7301 using S1 nuclease protection analysis. This splice acceptor site is used in all HTLV-I immortalized T-cell clones studied despite variation in the abundance levels of pX mRNA. Our results confirmed the proposal by Haseltine et al. (W. A. Haseltine, J. Sodroski, R. Patarca, D. Briggs, D. Perkins, and F. Wong Staal, Science (Washington, D. C. 225, 421-424 (1984); K. Shimotono, W. Wachsman, Y. Takahashi, D. W. Golde, M. Miwa, T. Sigimura, and I. S. Y. Chen, Proc. Natl. Acad. Sci. USA 81, 6657-6661 (1984)) that a pX protein with a molecular weight of at least 38,000 could be synthesized. Generation of a 2.0-kb pX mRNA may involve a double-splicing event. PMID- 2998031 TI - Clonal selection of T lymphocytes infected by cell-free human T-cell leukemia/lymphoma virus type I: parameters of virus integration and expression. AB - We have successfully transmitted cell-free HTLV-I to normal cord blood and peripheral blood lymphocytes and have exploited this system to study the kinetics of infection and transformation of these cells. Transmission was successful in 4 out of 23 attempts. In all 4 cases, the infected cells progressed from an initial stage of polyclonality to predominantly monoclonal cells within 4-6 weeks. Both complete and defective proviruses were transmitted to the recipient cells initially, but cells with a complete provirus were preferentially maintained. The monoclonally infected cells have persisted in culture for more than 6 months and may be considered immortalized. Expression of core antigens as detected by immunoflourescence and the reverse transcriptase activity in the medium at least in one case was not observed until weeks after the cells had become monoclonal, suggesting that expression of virus or viral structural proteins is not necessary for selected growth of the infected cells in vitro. PMID- 2998032 TI - The mature form of the Friend spleen focus-forming virus envelope protein, gp65, is efficiently secreted from cells. AB - The env genes of Friend spleen focus-forming viruses (F-SFFV) have been implicated in the rapid pathogenicity of these agents. Two env-gene products are detected in SFFV-infected cells: the primary translation product, gp52, and a more highly processed form, gp65. In this communication we demonstrate that gp65 is the major end product of the SFFV env gene, and is efficiently secreted from both erythroleukemia cells and infected fibroblasts. Secretion was observed for the mature env-gene products of both polycythemia- and anemia-inducing strains of SFFV. These results suggest that one function of the point mutation near the 3' end of the env gene, which is invariant in the formation of SFFVs, is to allow secretion of gp65, and that secreted gp65 may be the factor mediating the leukemogenic activity of these viruses. PMID- 2998033 TI - Characterization, molecular cloning, and physical mapping of the Shope fibroma virus genome. AB - Five strains of Shope fibroma virus (SFV), a strain of rabbit myxoma virus, and a strain of vaccinia virus were compared by restriction endonuclease digestion of their viral DNAs. Restriction digest patterns revealed that SFV and rabbit myxoma, both members of the Leporipoxvirus genus, were distinct from vaccinia, an Orthopoxvirus. All strains of SFV examined had a high degree of nucleotide sequence homology as shown by conservation of restriction sites within their genomes. However, restriction patterns of SFV and myxoma were quite different from one another suggesting that the genomes from these two viruses of the Leporipoxvirus genus do not share a large, highly conserved region of homology as do the viruses belonging to the Orthopoxvirus genus. Restriction mapping identified inverted terminal repeats of approximately 12 kb in length. Restriction fragments representing all but 400 bp of the termini were cloned in plasmid vectors. PMID- 2998034 TI - Residual transforming activity of PY1178T, a mutant lacking the principal in vitro tyrosine phosphorylation site, is not affected by removal of the secondary tyrosine phosphorylation site at residue 322. AB - Polyoma virus mutants lacking one or both tyrosines at position 315 and 322 of wild-type middle T antigen have been constructed. The effects of the removal of these tyrosines are additive for middle T phosphorylation in immune complexes, with tyrosine 315 being the major acceptor site and 322 a secondary site. Previous studies have shown little or no effect of deletion of tyrosine 322 on transforming ability, whereas a strong effect has been seen by substitution of phenylalanine for tyrosine 315. In contrast to the phosphokinase results, there is no additive effect of combining these mutations on the viruses' transforming ability. Thus the double mutant lacking both tyrosines has the same weak transforming activity as the single mutant containing tyrosine 322 and phenylalanine 315. Phosphorylation of middle T antigen at tyrosine 322 by pp60c src or other tyrosine-specific cellular protein kinase is therefore unimportant for transformation. PMID- 2998035 TI - Isolation of three new avian sarcoma viruses: ASV 9, ASV 17, and ASV 25. AB - The newly isolated avian sarcoma viruses, ASV 9, 17, and 25, cause fibrosarcomas in young chickens and induce foci of transformed cells in chick embryo fibroblast cultures. They are defective in replication and belong to envelope subgroup A. The sizes of their genomes are 6 kb (ASV 9), 5 kb (ASV 17), and 6 kb (ASV 25), respectively. All three contain long terminal repeat (LTR) and gag sequences but lack pol. env is absent from ASV 9 and ASV 25, but some env sequences are detectable in ASV 17. None of the defective viral genomes hybridized to selected onc probes representing src, fps, yes, myc, myb, and erb A. erb B appears absent from ASV 9 and ASV 17, but some hybridization between the erb B probe and the RNA of ASV 25 was detected. ASV 9 codes for a transformation-specific gag-linked protein of 130kDa. Multiple gag-linked transformation-specific proteins are seen in ASV 17 and 25; they require further study. PMID- 2998037 TI - Production of mouse mammary tumor virus upon transfection of a recombinant proviral DNA into cultured cells. AB - We have investigated the intracellular proteins synthesized in rat XC and feline kidney cells transfected with endogenous mouse mammary tumor virus (MMTV) proviral DNA. The endogenous provirus GR40, associated with the Mtv-8 locus, directs the synthesis of gag proteins indistinguishable from those found in MMTV infected cells. The env precursor Pr73env and the mature gp52 proteins could not be detected in these cells. Instead an env-related protein of 68K is synthesized. In contrast to this endogenous provirus, a cloned exogenous proviral variant directs the synthesis of apparently normal env proteins upon transfection into the same cell lines. These results suggest that the env gene of the endogenous MMTV provirus GR40 is defective. The exogenous proviral variant is not expected to synthesize virus particles since it carries a rearrangement in the gag gene. In order to obtain an MMTV provirus capable of correctly expressing both gag and env functions, we have constructed a hybrid endogenous-exogenous provirus containing the 5' long terminal repeat (LTR)-gag of GR40 and the pol-env-3' LTR of the exogenous provirus. Upon transfection into feline kidney cells, this hybrid provirus directed the synthesis of apparently authentic gag and env proteins. Further, virus particles can be detected in the culture medium of the transfected cells by electron microscopy. Viral proteins obtained from viral particles banded in a sucrose gradient were detected by immunoprecipitation. PMID- 2998036 TI - Cloned DNA of defective avian sarcoma virus mutant LA46 encodes the cis-acting temperature-sensitive defect in replication. AB - Avian defective sarcoma virus mutant LA46 carries a temperature-sensitive defect in replication and transformation. To elucidate this defect, we cloned the integrated provirus of LA46. By DNA-mediated transfection, the cloned DNA induced fusiform-transformed foci in chick embryo fibroblasts without helper virus. LA46 encoded transformation-specific protein p105 was expressed in these transformants in the absence of helper virus-encoded proteins. Superinfection of the transformed cells with different helper viruses resulted in the rescue of pseudotypes. All the rescued pseudotypes retained the temperature-sensitive phenotype in virus replication and transformation, suggesting that the defect was due to a cis-acting lesion in the LA46 genome. Restriction enzyme comparison between LA46 and wild-type virus revealed sequence differences near the 5' and 3' termini of the LA46 genome, including the long terminal repeat regions. PMID- 2998038 TI - Primary structure of the cleavage site associated with trypsin enhancement of rotavirus SA11 infectivity. AB - The primary structure of the trypsin cleavage site in the outer layer protein VP3 of rotavirus SA11 was determined. This cleavage enhances the infectivity of rotavirus SA11. Both VP8, one of the polypeptides generated by the cleavage, and VP3 had their alpha-NH2 blocked. Only VP5, the other polypeptide produced by the cleavage, was susceptible to sequential Edman degradation, indicating that it contained the new alpha-NH2 terminus generated by trypsin hydrolysis. The results indicated that purified VP5 is composed of two polypeptides with the following amino acid sequence at their N terminus: (a) ??VYTRAQPNQDAVVSKTS...; (b) AQPNQDAVVSKTS.... Sequencing of the DNA complementary to ds RNA segment 4 revealed a nucleotide sequence encoding the amino acid sequences indicated above, with only one different amino acid. From these results, the amino acid sequence of the site cleaved by trypsin was extended to cover the C termini (present in VP8). The following sequence, which contains two sites (indicated with asterisks) and can be cleaved by trypsin was deduced: ... VPVSIVSR*NIVYTR*AQPNQDIVVSKTS.... PMID- 2998039 TI - Identification and protein analysis of polyomavirus assembly intermediates from infected primary mouse embryo cells. AB - A method is described for the isolation of polyoma virus assembly intermediates from infected mouse embryo cells. Sucrose gradient profiles revealed the presence of 90 S, 200 S, and 240 S intermediates. These intermediates were shown to be sensitive to a number of factors: ionic condition of the isolation buffer, presence of chelating agents and nonionic detergents during isolation, and sonication of nuclei during extraction of intermediates. Pulse-chase experiments demonstrated that the order of formation of the intermediates to be 90 S----240 S, with the 200 S particles as a possible intermediate form linking the 90 S and 240 S particles. Viral structural proteins VP1, VP2, and VP3 were shown to be present on all three intermediates, but the ratio of each protein varied on each intermediate species. Two-dimensional gel electrophoresis demonstrated that the distribution of the VP1 isoelectric focusing species were different among the three intermediates. Histone H1 was found exclusively with the 90 S species. PMID- 2998040 TI - Cell lines derived from avian lymphomas exhibit two distinct phenotypes. AB - Lymphoid cell lines were derived from three avian leukosis virus (ALV)-induced lymphomas. These cell lines contained proviral DNA sequences integrated upstream from the c-myc proto-oncogene, expressed increased levels of c-myc RNA, and were tumorigenic in syngeneic animals. While cell surface immunoglobulin (IgM) was expressed by all three cell lines, only one of the lines secreted IgM into the culture medium. Further, analysis by light microscopy and flow cytometry demonstrated that these cell lines exhibited two distinct morphological and light scattering profiles. The two nonsecreting lines exhibited a lymphoblastoid phenotype, whereas, the secreting line possessed a more differentiated plasmacytoid phenotype. These findings implicate the activation of c-myc in the pathogenesis of tumors representing two distinct stages of B-cell differentiation within a single animal species. PMID- 2998041 TI - Role of a membrane glycoprotein in Friend virus-induced erythroleukemia: studies of mutant and revertant viruses. AB - We previously reported the isolation and characterization of spontaneous, transmissible mutants of Friend spleen focus-forming virus (SFFV) that are nonpathogenic in adult NIH/Swiss mice and that contain abnormalities in nonoverlapping regions of their envelope glycoprotein (env) genes (M. Ruta, R. Bestwick, C. Machida, and D. Kabat, 1983, Proc. Natl. Acad. Sci. USA 80, 4704 4708). In newborn NIH/Swiss mice, these mutant SFFVs form revertants that are pathogenic in mice of all ages. At least two of three studied revertants contain second site env mutations which affect the sizes and proteolytic fragmentation patterns of their encoded glycoproteins. A variety of structural and genetic evidence suggests that the xenotropic- and ecotropic-related regions of the SFFV glycoprotein fold into separate globular domains that are connected by a flexible proline-rich joint. A glutamyl peptide bond within this joint is exceptionally susceptible to cleavage with Staphylococcus aureus V8 protease. Moreover, disulfide bonds occur within the xenotropic-related domain, but not between the globular domains. These results provide strong additional evidence that the env gene is required for SFFV pathogenesis, and they provide a new system for identifying the features of glycoprotein structure and localization which are essential for its leukemogenic activity. PMID- 2998042 TI - Complete nucleotide sequence of the M RNA segment of Rift Valley fever virus. AB - The entire M RNA segment of the phlebovirus Rift Valley fever virus (RVFV) has been molecularly cloned and the complete nucleotide sequence determined. The RNA is 3884 nucleotides in length, corresponding to a molecular weight of 1.38 X 10(6), having a base composition of 27.3% A, 25.4% G, 27.2% U, and 20.1% C. Sequences present at the 3' and 5' termini of the molecule are largely complementary for some 51 residues and can form a stable duplex structure when the potential secondary structure of the entire molecule is considered. A single major open reading frame, capable of encoding 1206 amino acids (131,845 Da), was found in the viral-complementary sequence ("positive" polarity). Amino-terminal amino acid sequencing of the purified viral glycoproteins G1 and G2 allowed for the positioning of the coding sequences for these polypeptides within this major open reading frame in the following orientation with respect to the genomic M RNA: 3'-G2-G1-5'. From the predicted amino acid composition of the two mature viral glycoproteins, both were found to have a high cysteine content (G2, 6%; G1, 5%). Sequences within the open reading frame capable of encoding up to 23,000 Da of polypeptide were found in addition to those required for the viral glycoproteins. The potential contribution of these sequences to the coding capacity of the M RNA, viral protein processing, and intracellular protein distribution is discussed. PMID- 2998043 TI - Complete sequences of the glycoproteins and M RNA of Punta Toro phlebovirus compared to those of Rift Valley fever virus. AB - The complete sequence of Punta Toro virus (Phlebovirus, Bunyaviridae) middle size (M), RNA has been determined. The RNA is 4330 nucleotides long (mol wt 1.46 X 10(6), base composition: 26.7% A, 33.6% U, 18.5% G, 21.2% C) and has 3'- and 5' terminal sequences that, depending on the arrangement, are complementary for some 15 residues. The viral RNA codes in its viral-complementary sequence for a single primary gene product (the viral glycoprotein precursor) that is comprised of 1313 amino acids (146,376 Da) and is abundant in cysteine residues but has few potential asparagine-linked glycosylation sites. The 5'-noncoding region of the Punta Toro M viral-complementary RNA is short (16 nucleotides); the 3'-noncoding sequence is much longer (372 nucleotides). The latter is rich in short stretches of adenylate residues, like the 3'-noncoding regions of the Punta Toro S mRNA species (T. Ihara, H. Akashi, and D. H. L. Bishop, 1984, Virology 136, 293-306). No other large open reading frame has been identified in either the viral, or viral-complementary, M RNA sequences. Limited amino-terminal sequence analyses of the two viral glycoproteins have indicated the gene order and potential cleavage sites in the glycoprotein precursor. The data suggest the existence of a 30 X 10(3)-Da polypeptide (designated NSM) in the glycoprotein precursor that precedes the G1 protein (i.e., gene product order: NSM-G1-G2). Examination of the sequence of the Punta Toro M gene product reveals the presence of multiple hydrophobic sequences including a 19-amino acid, carboxy-proximal, hydrophobic region (G2). This hydrophobic sequence is followed by a 13-amino acid-terminal sequence rich in charged amino acids. The size and constitution of the carboxy-terminal region is consistent with a transmembranal and anchor function for the glycoprotein in the viral envelope. Other regions of the glycoprotein precursor contain sequences of amino acids with a predominantly hydrophobic character (23, 50, and 20 amino acids in length). Their functions are unknown. The amino terminus of the G1 protein is located near the end of the 23-amino acid-long hydrophobic sequence of the presumptive precursor, the hydrophobic 50-amino acid sequence lies within G1, and the amino terminus of G2 is located in the middle of the 20-amino acid-long hydrophobic sequence.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2998044 TI - Phospholipid liposomes enhance the infectivity of purified simian virus 40 virions. AB - When simian virus 40 virions purified after treatment with sodium deoxycholate were incubated with the extract of monkey kidney CV-1 cells, infectivity of the virions was enhanced. The infectivity-enhancing activity was recovered from the phospholipid fraction of CV-1 cells. The constructed liposomes composed of phosphatidylserine were able to enhance the infectivity of the purified virions, but those composed either of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, or phosphatidylinositol could not. The liposomes constructed with a mixture of phosphatidylethanolamine and phosphatidylcholine at a ratio of 1:1 (w:w) also enhanced the infectivity of the purified virions. Pretreatment of cells with liposomes either of phosphatidylserine or of phosphatidylethanolamine did not enhance susceptibility of the cells to infection with the purified virions. These observations suggest that the major phospholipids of the cellular membrane, when associated with virions, play a vital role in activation of purified virions. PMID- 2998045 TI - Human papillomavirus type 17 DNA in skin carcinoma tissue of a patient with epidermodysplasia verruciformis. AB - Epidermodysplasia verruciformis (EV) is a serious skin disease caused by certain types of human papillomavirus (HPV), because the flat wart-like lesions of EV very frequently change to malignant squamous cell carcinoma. The relation between HPV and skin carcinoma was examined by studies on an EV patient who had a squamous cell carcinoma. HPV-17 was isolated from EV lesions of this patient. With HPV-17 DNA as a probe, cellular DNA prepared from the carcinoma tissue was analyzed by Southern blot hybridization. Results showed that cells contained about 100 copies of monomeric and oligomeric extrachromosomal HPV DNA. These results suggest that HPV-17 is involved in skin carcinogenesis in EV. PMID- 2998046 TI - Activation and inhibition of expression of the 72,000-Da early protein of adenovirus type 5 in mouse cells constitutively expressing an immediate early protein of herpes simplex virus type 1. AB - It has been previously reported that immediate early proteins of pseudorabies and cytomegalo viruses can substitute for the products of the human adenovirus type 5 (Ad5) E1A gene in the activation of early Ad5 transcription. In the present report the effect of one of the herpes simplex virus type 1 (HSV-1) immediate early genes, ICP4, on Ad5 early gene expression has been examined using mouse cell lines that constitutively express ICP4. These lines as well as nonproducers were infected with wild-type (wt) Ad5 or with various Ad5 E1A mutants and the levels of expression of the Ad5 E2A 72K DNA binding protein were measured by immunoprecipitation with a monoclonal antibody specific for 72K. With dl 312, which lacks E1A, some 72K expression was seen in nonproducer lines but levels were considerably higher in the producer lines. A similar result was also obtained using dl 312-infected nonproducer cells that were superinfected with HSV 1 virions. These data suggest that HSV-1 ICP4 can substitute for E1A in the activation of expression of early Ad5 proteins. With wt Ad5, 72K was also expressed at high levels in nonproducer mouse cells, however, in the ICP4 producer cell lines, a marked inhibition of 72K expression was observed and this inhibition correlated with the amount of ICP4 present. Using the E1A mutants pm 975 and hr 1, this inhibition was found to be specific for the products of the 1.1-kb E1A mRNA. These data suggest that ICP4 and E1A proteins either directly inhibit each other, or more likely, operate independently and competitively on factors required for viral gene activation. PMID- 2998047 TI - Sequence homology of the simian retrovirus genome with human T-cell leukemia virus type I. AB - A retrovirus highly related to human T-cell leukemia virus type I (HTLV-I) was isolated from a T-cell line established from a seropositive pig-tailed monkey and the provirus genome was molecularly cloned using HTLV-I as a probe. The monkey virus (STLV) had the genomic structure of the LTR-gag-pol-env-pX-LTR. Analysis of the env-pX-LTR region revealed the 90% homology of the nucleotide sequence with that of HTLV-I in each region. This high homology of the sequence indicates that STLV is a member of the HTLV family, but apparently different from HTLV-I. This suggests that the possibility of recent interspecies transmission from monkeys to humans in the endemic area is very small. From its similarity to HTLV, STLV should be useful as an animal model in studies on natural HTLV infection and leukemogenesis of HTLV in humans. PMID- 2998048 TI - Embryonic erythroid cells transformed by avian erythroblastosis virus may proliferate and differentiate. AB - Embryonic chick cells from the primitive streak stage to later stages of the developing embryo were infected with avian erythroblastosis virus (AEV). The data indicate that the greatest number of target cells for AEV was observed in the 12 somite blastoderm and gradually decreased in hemopoietic tissues with the development of the embryo. The target cell for AEV is not in the BFU-E compartment, as it is in the adult bone marrow, but is probably recruited within the CFU-M compartment which precedes the BFU-E compartment. Our studies also show that a significant number of transformed colonies derived from embryonic hemopoietic tissues undergo hemoglobinization in contrast with what is observed in transformed colonies of bone marrow. A complete characterization of the embryonic and adult hemoglobin is at present under study. PMID- 2998049 TI - An immunoaffinity purification procedure for SV40 large T antigen. AB - A rapid purification procedure for SV40 large T antigen has been developed which combines the use of an adenovirus-SV40 hybrid virus which overproduces large T antigen, and immunoaffinity chromatography on an anti-large T monoclonal antibody coupled to protein A Sepharose. The protein exhibits the p53-binding, ATPase, and sequence-specific DNA-binding activities of T antigen. The purification procedure can be completed in 1 day and allows the isolation of milligram amounts of large T in excellent yield. The pure protein is extremely antigenic and is tolerant of iodination to high specific activity, permitting the development of a competition radioimmunoassay for large T that reliably detects nanogram amounts of the protein. PMID- 2998050 TI - In vitro packaging of plasmid DNA oligomers by Salmonella phage P22: independence of the pac site, and evidence for the termination cut in vitro. AB - In vitro packaging experiments with phage P22 using artificially ligated plasmid concatemers have shown that the pac site is not necessary for DNA packaging although in vivo this initiation signal is indispensable. This indicates that the phage-coded protein gp3 also executes other important functions during phage maturation in addition to the recognition of pac, or that its site specificity is lost in vitro. It has been shown previously that gp3 is necessary for in vitro packaging. Further, it was demonstrated that DNA which is only 74% of headful size cannot be packaged. Oversized DNA, however, is cut in vitro to unit length. PMID- 2998051 TI - Structural homologies between RNA gene segments 10 and 11 from UK bovine, simian SA11, and human Wa rotaviruses. AB - The nucleotide sequences of gene segments 10 and 11 from UK bovine rotavirus have been determined. Gene 10 is 751 nucleotides long and contains a single long open reading frame capable of coding for a protein of 175 amino acids. When compared with the published data for gene 10 of the simian rotavirus SA11 and human Wa strains it was found to be more closely related to the SA11 structure (92% nucleotide sequence homology; 97% amino acid sequence homology) than to the human Wa structure (84% nucleotide, 86% amino acid sequence homology). All three strains have two potential N-glycosylation sites in the hydrophobic N terminus of the gene 10 protein. Gene 11 from UK bovine rotavirus is 667 nucleotides long with a single long open reading frame capable of coding for a protein of 198 amino acids. When compared with the published sequence of gene 11 from the human rotavirus Wa, the UK bovine rotavirus gene 11 was found to be one nucleotide longer in the 5'-noncoding region and three nucleotides longer in the coding region. The nucleotide sequence homology was 86%. The predicted proteins coded by segment 11 in UK and Wa rotaviruses are both rich in serine and threonine (23%) and very hydrophilic, but differ appreciably in amino acid sequence (83% homology). PMID- 2998052 TI - Hybridization of herpes simplex virus DNA and human ribosomal DNA and RNA. AB - A small DNA segment from the inverted repeats at the termini of the unique long sequence region of herpes simplex virus DNA was found to hybridize with human 28 S ribosomal DNA and RNA but not 18 S ribosomal DNA and RNA. The hybridization occurred under stringent conditions and was not blocked by nucleic acids high in guanine plus cytosine content. These data strongly suggest that the hybridization represented authentic base sequence homology. PMID- 2998053 TI - Transformation by polyoma ts-a mutants. I. Characterization of the transformed phenotype. AB - Seven clonal lines of Fischer rat cells transformed with ts-a mutants of polyoma virus were studied. Four clones are characterized by a temperature-sensitive (ts) and three clones by a temperature-insensitive-transformed phenotype. Six clones have retained a functional though temperature-sensitive large T antigen, as judged by a 10- to 20-fold amplification of viral sequences in clones grown at low temperature compared to those grown at high temperature. No amplification is observed in one non-ts clone. As analyzed by Southern blotting, no obvious difference appears in the integration pattern of viral sequences in ts and non-ts clones concerning the number of sites of genome integration, the presence or absence of tandem repeats of the viral genome, or the absence of specific viral sequences. In autoradiograms of gel-electrophoresed immunoprecipitates, no correlation can be drawn between the amounts of either large T antigen or middle T antigen and the type of transformed state of the clones under the conditions tested. In assays of the middle T-antigen-associated kinase, no reproducible difference can be observed between the non-ts and ts clones. Finally, no correlation was observed between a temperature-insensitive phenotype and the production of an N-terminal fragment of large T antigen. Thus the molecular basis for the difference between ts-a transformants with ts or non-ts phenotypes remains elusive. PMID- 2998054 TI - Nucleotide sequence of the envelope gene of radiation leukemia virus. AB - The nucleotide sequence and the predicted amino acid sequence of the envelope gene (env) of RadLV/VL3 (T+L+) has been determined. RadLV/VL3 (T+L+) is a highly thymotropic (T+) and leukemogenic (L+) murine recombinant retrovirus continuously produced at high titer by BL/VL3 cells, a line established in culture from a radiation leukemia virus (RadLV)-induced C57BL/Ka mouse thymic lymphoma. The envelope gene is strikingly similar (92% homologous) to that encoded by the ecotropic Akv-MuLV. Among the differences, 134 scattered, although unevenly distributed point mutations (6.4%), two 9-bp deletions as well as a 6-bp insertion were observed (which result in 49 amino acid changes in the env gene of RadLV/VL3 (T+L+)). PMID- 2998055 TI - Presence of circulating antibodies against gag-gene MuLV proteins in patients with autoimmune connective tissue disorders. AB - An immunoblotting procedure using viral proteins from purified murine sarcoma virus or MSV-(MLV) has been developed to characterize antiviral antibodies in sera from patients with autoimmune connective tissue disorders. Fifty-eight sera with anti-Sm, anti-RNP, anti-SS-B (La), and other undefined specificities were found to react with several major viral polypeptide bands. Most of them corresponded to gag-gene-encoded products: pr65gag, p40gag, p30, p15, p12 and p10. Other bands with molecular weights averaging 90K, 60K, 45K, and 28K were recognized by a few sera. Immunological specificity of the reaction was assessed by reproducing the tests with IgG purified from sera and from corresponding F(ab')2 fragments. Moreover, the specificity of the reaction with gag proteins was confirmed by repeating the tests with p30 and p15 prior purified by immunoprecipitation with anti-p30 and anti-p15 goat sera. Furthermore, the gag polypeptides were recognized by human sera by replacing MSV-(MLV) by three other murine retroviruses of different origin. An indirect confirmation of these results was obtained by applying this method to sera of MRL lpr/lpr mice which develop an autoimmune syndrome comparable to that of human systemic lupus erythematosus. In agreement with previously published results (C. Rordorf, C. Gambke, and J. Gordon (1983), J. Immunol. Methods 59, 105-112), we found that anti-gag-gene antibodies were present in the sera of individual mice. Patterns of reactivity were found to vary with the age of the animals. No retroviral polypeptide was significantly detected in the great majority (80%) of sera from normal donors. However, 5 out of 25 sera showed faint bands although to a lesser extent than pathological sera. These five sera also reacted with HeLa cell purified HnRNPs, suggesting that their normal status should be reconsidered. PMID- 2998056 TI - Isolation and characterization of bacteriophage T3/T7 hybrids and their use in studies on molecular basis of DNA-packaging specificity. AB - In vitro DNA-packaging systems of bacteriophages T3 and T7 packaged homologous DNA more efficiently than heterologous DNA. Packaging of phage DNA proceeds by way of concatemeric intermediates (H. Fujisawa, J. Miyazaki, and T. Minagawa (1978), Virology 87, 394-400). The conversion of mature homologous and heterologous DNAs to concatemers was efficient in both the T3- and T7-packaging systems. In vitro complementation experiments indicate that the gene 19 product (gp19) specifies which DNA enters the capsid. To identify DNA regions recognized by the packaging systems, T3/T7 hybrids were constructed and physical maps of the hybrid DNAs were determined by restriction enzyme analysis. By comparing restriction maps and in vitro packaging of hybrid DNAs, it is concluded that the sequence responsible for specificity of DNA packaging is confined within 5% of the ends of the T3 and T7 genomes. PMID- 2998058 TI - Effect of herpes simplex virus type 1 on cellular pools of oligosaccharide-lipid. AB - Incorporation of [3H]mannose into cellular pools of mannosylphosphoryl dolichol (Man-P-Dol), oligosaccharide-lipid, and glycoprotein was measured and compared in herpes simplex virus type 1 (HSV-1)-infected cells and -uninfected cells. While mannose incorporation into the monosaccharide-dolichol fraction was similar in infected or uninfected Vero cells, incorporation into the oligosaccharide-lipid fraction was markedly reduced in HSV-1-infected cells (64% of control levels). In contrast, mannose incorporation into glycoprotein was significantly increased in virus-infected cells versus uninfected cells (194% of control levels). The kinetics of incorporation into the various fractions was examined and it was determined that there was minimal increase in mannose incorporation into oligosaccharide-lipid after 8 hr postinfection in virus-infected cells. This corresponded to the time at which nonglycosylated precursors of the HSV-1 glycoproteins were first detected in association with the nuclear fraction. These data suggest that there is an accelerated turnover of oligosaccharide-lipid in virus-infected Vero cells which is most likely due to extensive glycoprotein synthesis. PMID- 2998059 TI - Early steps in FMDV replication: further analysis on the effects of chloroquine. AB - We have previously demonstrated that chloroquine and NH4Cl, two well-known lysosomotropic drugs inhibit foot-and-mouth disease virus (FMDV) replication. This fact points to the relevance of an acidic environment during FMDV penetration. In the present report, we show that chloroquine prevents the cell mediated disruption of 140 S virions into 12 S particles. This dissociation, which resembles that caused by low pH in vitro, might be an initial uncoating step. Furthermore, we demonstrated that a decrease in the environmental pH counteracts the effect of chloroquine indicating that viral disruption is a low pH cell-mediated process. The fact that it still occurs at low temperature (20 degrees) and shortly after viral adsorption suggests not only that prelysosomal vesicles represent the putative site for uncoating but also cause the virion to uncoat. PMID- 2998057 TI - A comparative study on the translation of cardiovirus RNAs in rabbit reticulocyte lysates. AB - In rabbit reticulocyte lysates the RNAs of encephalomyocarditis (EMC) virus, mengovirus, and Mous-Elberfeld (ME) virus directed the synthesis of similar sets of products. Moreover, the viral protease synthesized from any one of the three viral RNAs could cause cleavage of the viral capsid precursor proteins synthesized from any of the three RNAs. However, the three RNAs differed in their dependence on tRNA supplementation (to the lysates) for effective translation. In the absence of tRNA supplementation, synthesis of 5'-derived proteins of EMC viral RNA proceeded normally, but little synthesis of the proteins coded by the remaining portion of the viral genome occurred. In the case of mengoviral RNA, omission of tRNA supplementation caused mostly a generalized reduction of the synthesis of all viral proteins. In contrast, synthesis of ME viral proteins stopped almost completely in the absence of tRNA supplementation. PMID- 2998060 TI - Cloning and gene assignment of mRNAs of human parainfluenza virus 3. AB - Cytoplasmic poly(A)-containing RNA from parainfluenza virus 3-infected cells was used as template to construct a cDNA library that was cloned into the EcoRV site of the plasmid pMG5. The resulting clones were screened with [32P]-labeled cDNA probes made from infected and mock-infected cell mRNAs. The virus specificity of the clones was confirmed by Northern blot hybridization. The viral clones were grouped into five different families by hybridization with individual size selected reverse transcripts representing the major classes of poly(A)+-RNA from virus-infected cells. The five groups were shown to be unrelated on the basis of cross-colony hybridization and corresponded to five unique classes of intracellular viral poly(A)+-RNAs. Clones representing the NP and P genes of PIV 3 were identified by both hybrid-selected and hybrid-arrested translation. Clones specific for the P gene selected mRNA that directed the synthesis of P protein and another polypeptide of 21 kDa. This additional polypeptide comigrated with protein VP8 previously identified in virions and in infected cell lysates. PMID- 2998061 TI - Spontaneous reiterations of DNA sequences near the ends of adenovirus type 3 genomes. AB - Repeated passage of adenovirus type 3 in HeLa cells has led to a novel stock of variant genomes. Most of the DNA molecules in this stock are characterized by deletions and substitutions of DNA sequences near the left end of the adenovirus type 3 genome map, as reported earlier (C.C. Robinson and C. Tibbetts (1984) Virology 137, 276-286). In this report the characterization of the variant genomes is extended and reveals elongated DNA molecules bearing tandem repetitions of viral DNA sequences near the left and right ends of the viral DNA. Evidence is also presented supporting the cellular DNA origin of short insert sequences found in substitution variants. The elongated variants are of interest because of their novel repeated DNA structures. The locations of these aberrant sequences raise questions about their potential impact on viral gene expression. PMID- 2998063 TI - The antiherpes drug (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) interferes with formation of N-linked and of O-linked oligosaccharides of the herpes simplex virus type 1 glycoprotein C. AB - In HSV-1 infected cell the antiherpes drug (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) exerted at least three different effects on glycosylation of glycoprotein gC. First, an overall decrease of protein glycosylation occurred due to inhibition of synthesis of the lipid-linked oligosaccharides, precursors of N linked oligosaccharides of gC. Second, an inhibition of processing of N-linked oligosaccharides occurred after the acquisition of endo H-resistance, and possibly due to inhibition of galactose incorporation. Third, a small inhibition of incorporation of glucosamine into O-linked oligosaccharides, and, may be associated with this, a change in the proportion of two different classes of O linked oligosaccharides of gC, namely those with terminal N-acetylgalactosamine and those with terminal sialic acid. PMID- 2998062 TI - Anti-complement immunofluorescence establishes nuclear localization of human cytomegalovirus matrix protein. AB - A monospecific, polyclonal antiserum to the 69-kDa matrix protein of human cytomegalovirus (HCMV) was prepared in a guinea pig and used to determine the intracellular distribution of this viral antigen. The resulting antiserum was specific for infected cells as tested by immunofluorescence, and specific for the HCMV matrix protein as determined by "nitrocellulose immunoassay" of electrophoretically separated, infected-cell proteins. Antibodies were reacted with fixed, infected human fibroblasts, and visualized by the anti-complement immunofluorescence procedure to avoid complications arising from the strong IgG Fc binding activity of the infected-cell-specific cytoplasmic inclusion. Results establish that the matrix protein is located in the nucleus, and indicate that it is concentrated in the nucleoplasm rather than within the intranuclear inclusions. PMID- 2998064 TI - The SV40 large T-p53 complex: evidence for the presence of two immunologically distinct forms of p53. AB - The transforming protein of SV40 is the large T antigen. Large T binds a cellular protein, p53, which is potentially oncogenic by virtue of its functional involvement in the control of cell proliferation. This raises the possibility that p53 may mediate, in part, the transforming function of SV40 large T. Two immunologically distinct forms of p53 have been identified in normal cells: the forms are cell-cycle dependent, one being restricted to nondividing cells (p53 Go) and the second to dividing cells (p53-G divided by). We have now dissociated and probed the multimeric complex of SV40 large T-p53 for the presence of immunologically distinct forms of p53. Here we present evidence for the presence of p53-Go and p53-G divided by complexed with SV40 large T. PMID- 2998065 TI - Mouse mammary tumor virus gene expression regulated in trans by Lps locus. AB - Expression of mouse mammary tumor virus (MMTV) in the lactating mammary glands of uninfected mice varies between strains of mice in a manner largely independent of the proviral content. Previous linkage analysis in the mouse suggested that the Lps locus was associated with steady-state levels of MMTV RNA. The Lps locus mediates the mouse's response to the injection of lipopolysaccharide (LPS) in the responder mouse while mice with the deficient allele are incapable of responding. Injecting LPS-responder mice, C3HfB/HeN, and nonresponder mice, C3Hf/HeJ, with LPS resulted in a threefold increase in the level of MMTV RNA in responder mice but had no effect on nonresponders. The increased level was due to only one of the possible MMTV transcripts: the 1.7-kb transcript containing the open reading frame (orf) of the long terminal repeat (LTR). The level of MMTV-specific transcripts, then, is regulated by the Lps locus, a cellular gene which is not linked to any viral coding sequences and therefore must act in trans. PMID- 2998066 TI - Human cytomegalovirus completely helps adeno-associated virus replication. AB - Coinfection of adeno-associated virus (AAV) with human cytomegalovirus (HCMV) strain Towne in human embryonic fibroblasts resulted in accumulation of AAV capsid antigen and production of infectious AAV with a lag of 24 hr compared to AAV replication in AAV-adenovirus coinfections. In contrast to previous observation, these findings demonstrated that HCMV is a competent helper virus for the complete replication of AAV. In addition, HCMV and AAV were synergistic in their cytopathic effects on cells, suggesting the possibility that AAV may play a role in the pathogenicity of HCMV infections. PMID- 2998067 TI - Transmission of human T-cell leukemia virus type I to an S+L- cat kidney cell line. AB - The S+L- cat kidney cell line CCC was cocultivated with lethally irradiated human lymphoid cell lines that were producing human T-cell leukemia virus type I (HTLV I). Eight of nine S+L- CCC sublines that had been cocultivated with nine different HTLV-producing T-cell lines gave positive reactions for HTLV antigens by indirect immunofluorescence assay. One subline CCC/2M was cloned. The percentages of fluorescent cells differed markedly in different sublines and clones. Southern blot hybridization with HTLV probes and electrophoresis of immunoprecipitates indicated that defective HTLVs were often transmitted into S+L cat cells. S+L- CCC cells were permissive for HTLV and the properties of HTLV infected cat cells were heterogeneous. PMID- 2998068 TI - A Trojan Horse mechanism for the spread of visna virus in monocytes. AB - Visna virus is the prototype of the lentivirus subfamily of retroviruses that cause slow infections of sheep and goats. These viruses persist and can be isolated from blood and cerebrospinal fluid for years despite neutralizing antibody. In the studies reported here we have used quantitative in situ hybridization to analyze infected leukocytes. We show that (1) monocytes harbor the visna genome; and (2) virus gene expression is as constrained in this cell as it is in glial and epithelial cells. These results are in accord with a Trojan Horse mechanism of virus dissemination in an immunologically responsive host. PMID- 2998070 TI - Difference in sensitivity to interferon among mouse hepatitis viruses with high and low virulence for mice. AB - Mouse hepatitis viruses (MHV) of different virulence for mice were studied with respect to interferon (IFN) sensitivity. The growth of low-virulent MHV-S and intermediately virulent MHV-JHM was significantly suppressed in IFN-treated L cells compared with untreated cells. However, a comparable suppression of the growth of highly virulent MHV-2 was not observed in IFN-treated cells. This differential effect of IFN treatment could also be demonstrated at the level of viral mRNA and viral proteins. In cells infected with MHV-S or MHV-JHM the amount of viral mRNAs was remarkably reduced by IFN treatment. Also the levels of the major intracellular viral proteins, in particular the E1 protein, were affected by IFN treatment. Similar effects could not be demonstrated in MHV-2-infected cells. These results suggest that during MHV-S or MHV-JHM infection IFN treatment suppresses virus replication at several stages. The significance of these results is discussed in terms of the pathogenecity of these viruses. PMID- 2998069 TI - The expression of viral functions is necessary for recombination of a herpesvirus (pseudorabies). AB - To determine whether viral functions are necessary for recombination of the pseudorabies virus genome in infected cells, we have used as a model system marker rescue at the permissive temperature (PT) and nonpermissive temperature (NPT) of a temperature sensitive mutant (tsG1) deficient in the immediate-early (180K) protein. Two restriction fragments, both of which can rescue tsG1 at the PT but only one of which encompasses the whole immediate-early gene and can complement tsG1, were compared for their ability to rescue the mutant at the NPT. Although both restriction fragments rescued the mutant with equal frequency at the PT, only the fragment which could express the immediate-early 180K protein prior to recombination, i.e. could complement tsG1, rescued the mutant at the NPT. We conclude that the expression of viral functions is necessary for high frequency recombination of the pseudorabies virus genome. PMID- 2998071 TI - Biochemical characterization of an aphthovirus type 0(1) strain campos attenuated for cattle by serial passages in chicken embryos. AB - The biochemical properties of a virulent and an attenuated strain of foot-and mouth disease virus (FMDV) Type 0(1) Campos (0(1)C) were compared in order to establish differences that could account for their altered biological functions. The avirulent strain (0(1)C-O/E) was derived from the virulent strain 0(1)C by serial passages in chicken embryos. Analysis of the RNase T1-generated oligonucleotides of the viral RNA through one- and two-dimensional (2D) gel electrophoresis (fingerprints) revealed a few changes in the genome structure of the 0(1)C-O/E strain compared to the wild type strain. In addition there was a significant decrease in the length of the poly(C) rich tract of the 0(1)C-O/E RNA. All virion structural proteins, except VP4, their precursors, and the viral RNA polymerase (p56a) show charge differences. In addition a significant decrease in the apparent molecular weight of polypeptide p100 (primary translational product from the 3' end region of the genome) of the attenuated strain was observed. PMID- 2998072 TI - Replicative functions of the SV40(cT)-3 mutant defective for nuclear transport of T antigen. AB - The SV40(cT)-3 mutant is defective in transport of SV40 large tumor antigen (T ag) to the nucleus. Several properties of T-ag associated with SV40 lytic infection and attributed to its nuclear localization were examined to determine whether biologically significant levels of the mutant T-ag (cT-ag) that were immunologically undetectable were transported to the nucleus in SV40(cT)-3 infected TC-7 cells. SV40(cT)-3 was defective in regulation of T-ag synthesis and initiation of viral DNA synthesis. These defects were presumably due to the lack of nuclear transport of cT-ag, since cT-ag was capable of interacting with the SV40 origin of viral DNA synthesis in a solution binding assay. The level of fatty acid acylation, a modification specific for the cell surface associated T ag, was not affected by the cT mutation. The cT mutation sufficiently suppressed the nuclear transport of wild-type (WT) T-ag in SV40(cT)-3-infected COS-1 cells to result in the cessation of WT-T-ag-stimulated SV40(cT)-3 viral DNA synthesis. These results are discussed with respect to the recent findings that SV40(cT)-3 is fully competent for the transformation of established cell lines and the induction of cellular DNA synthesis in quiescent cells. PMID- 2998073 TI - The short unique region of the B95-8 Epstein-Barr virus genome. AB - The 12-kbp short unique region of the B95-8 Epstein-Barr virus (EBV) genome has been sequenced and analysed for latent and lytic cycle transcripts. Two latent and three late mRNAs have been detected, the largest of the late transcripts potentially encoding a 143-kDa protein. The region containing oriP, the putative origin of replication of the genome as a plasmid in latently infected B lymphocytes, is shown to contain 21 direct repeats of a 30-bp A+T-rich sequence and a related large inverted repeat. PMID- 2998074 TI - Biosynthesis of reovirus-specified polypeptides. The s1 mRNA synthesized in vivo is structurally and functionally indistinguishable from in vitro-synthesized s1 mRNA and encodes two polypeptides, sigma 1a and sigma 1bNS. AB - The structural and functional properties of the reovirus serotype 1 (Lang strain) s1 mRNA were examined. Reovirus s-class mRNAs, synthesized either in vivo within infected mouse L cells or in vitro by chymotrypsin-derived cores of purified virions, were purified by filter-hybridization using cDNA clones of the S-class genome segments. S1 cDNA-selected mRNA encoded the synthesis of the Mr approximately 12,000 nonstructural polypeptide designated sigma 1bNS in addition to the well-established structural polypeptide sigma 1, now designated sigma 1a. The coding properties of in vivo- and in vitro-synthesized s1 mRNA were equivalent: both encoded sigma 1a and sigma 1bNS. Primer extension analysis of s1 mRNA revealed a single major 5' terminus for both in vivo- and in vitro synthesized s1 mRNA. These results suggest that there is a single transcript of the reovirus S1 genome segment which is functionally dicistronic, and likely encodes both sigma 1a and sigma 1bNS. PMID- 2998075 TI - The BamHI F region of the B95-8 Epstein-Barr virus genome. AB - The BamHI F region of the B95-8 Epstein-Barr virus (EBV) genome has been sequenced and analysed for transcription signals and open reading frames. S1 mapping and northern blotting with probes from M13 recombinants have been used to search for mRNAs. Four rightward-reading frames encoding basic proteins appear to be expressed by 3'-coterminal early mRNAs. Two leftward-reading frames appear to be expressed by 3'-coterminal early mRNAs. PMID- 2998076 TI - [Mechanisms involved in latent herpes simplex virus infection. I. In vivo studies]. PMID- 2998077 TI - [Mechanisms involved in latent herpes simplex virus infection. II. In vitro studies on virus-cell interactions]. PMID- 2998078 TI - [Effect of tunicamycin on human cytomegalovirus replication]. PMID- 2998079 TI - [Comparative study of humic acids of muds and peat by the methods of electron paramagnetic resonance and infrared spectroscopy]. PMID- 2998080 TI - [All-Union registry data on long-term surviving patients with small cell lung cancer]. AB - Data borrowed from the All-Union Cancer Register on 42 small cell lung cancer survivors over 2.5 years are presented. The average age of patients was 56 years; the male/female ratio was 6:1. Seventeen patients survived over 5 years. PMID- 2998081 TI - [Endoscopic diagnosis and evaluation of the effectiveness of combination chemotherapy of small cell lung cancer]. AB - The results of bronchoscopy for small cell lung carcinoma demonstrated endoscopic procedure to provide much reliable information indispensable in making primary diagnosis and evaluating the efficacy of combined chemotherapy. It was shown that assessment of the results of chemotherapy for lung cancer should be carried out on the basis of a wide range of parameters (including endoscopic and roentgenologic findings) as determined by the resolution of relevant diagnostic procedures for different types of tumor growth. PMID- 2998082 TI - [Roentgenological semeiotics of small cell lung cancer]. AB - The study was concerned with description of roentgenologic semeiotics of central and peripheral small cell lung cancer in 141 patients receiving chemoradiation therapy. The frequency of carcinoma metastasis into intrathoracic lymph nodes was high. Small cell lung cancer showed a good response to conservative treatment, which, in particular, manifested itself in regression of metastases into intrathoracic lymph nodes. PMID- 2998083 TI - [Biochemical methods and their place in prognosis and evaluation of the effectiveness of treatment of small cell lung cancer]. AB - The blood plasma levels of ACTH, CEA, calcitonin, parathyrin, hydrocortisone, serotonin, and histamine were measured radioimmunologically in 58 cases of small cell lung cancer prior to treatment. Elevated concentrations of CEA (61%) and ACTH (44%) were relatively frequent. Blood plasma--ACTH level in cases of expanded small cell lung cancer was higher than in healthy subjects and patients with localized tumor. A correlation was found between cancer patients survival time and the basal levels of CEA, ACTH and calcitonin. Polyamines were assayed in diurnal urine of 24 cancer patients prior to treatment and during chemotherapy. The mean level of putrescine before treatment was much in excess of normal value. Responders to treatment revealed a considerable rise in spermidine excretion within the first 10 days after treatment. In non-responders, spermidine excretion remained at the same level. PMID- 2998084 TI - [Present status of the problem of small cell cancer of the lung]. PMID- 2998085 TI - [Cytological diagnosis of small cell lung cancer]. AB - Morphometric studies using the Integral instrumentation complex and the ICP--II pulsed cytophotometer assays of DNA in lung tumor cells of various histological patterns were conducted to obviate difficulties involved in establishing differential cytologic diagnosis of small cell undifferentiated lung cancer. It was shown that morphometric and pulsed cytophotometric measurements may offer considerable advantage when used in conjunction with other procedures of differential diagnosis with a view to improving diagnosis of various forms of lung tumors and small cell undifferentiated lung cancer, in particular. PMID- 2998086 TI - [Electron microscopy in the diagnosis of small cell lung cancer]. AB - The paper deals with an ultrastructural classification of small cell lung cancer distinguishing between wholly-undifferentiated tumors which do not contain cells with organ-, tissue- or cytospecific features (group 1) and tumors which incorporate both undifferentiated and differentiated cells (group 2). Depending on certain cytospecific characteristics, group 2 tumors histologically identifiable as small cell lung cancer may prove to be endocrine cancer, squamous cell cancer, adenocarcinoma or a mixed type tumor incorporating differentiated cells of two or more patterns. There is a correlation between the ultrastructural features of small cell lung cancer and its response to radiation and chemotherapy. PMID- 2998087 TI - [Role of ambulatory care in the diagnosis of small cell lung cancer]. AB - Data on detection of small cell lung cancer in the course of mass screenings are presented. Small cell lung carcinoma was identified in 17.5% of all those who underwent check-ups (stage 1-1,5; stage II--16.7; stage III--30.3 and stage IV- 51.5%). PMID- 2998088 TI - [Surgery in the treatment of patients with small cell lung cancer]. AB - The data on surgical and combined treatment of 52 cases of small cell lung cancer are presented and the end results are evaluated. The findings on survival time make the case for surgery as a component of combined treatment of tumor. The best results were obtained in cases of stage I and II tumor, absence of metastases into intrathoracic lymph nodes, tumor invasion limited to bronchopulmonary lymph nodes or the root of the lung, intermediate cell pattern of small cell lung cancer and surgery followed by chemotherapy. PMID- 2998089 TI - [Place of surgical intervention in the treatment of small cell lung cancer]. AB - On the basis of evaluation of their own findings (162 cases) and data available in the literature the authors suggest that surgery be used as a component of combined treatment for small cell lung cancer. Preoperative assessment of tumor process expansion appeared to be unreliable. Pneumonectomy was found to remain an operation of choice although application of extended pneumonectomy to deal with tumor expansion should not be recommended because the end results do not differ from those of conservative (chemoradiation, polychemotherapy) treatment. PMID- 2998090 TI - [Chemotherapy and combined chemo- and radiotherapy of small cell lung cancer]. AB - Objective response of small cell lung cancer to different modalities of chemo-and radiation chemotherapy was recorded in 68-94% while complete regression of tumor- in 22-50% of a total of 166 cases. Complete regression was registered in approximately 33% of cases of localized tumor process and in 16% of cases of expanded process. The survival median for different treatment modalities was 13.7 22.5 months in cases of complete regression and 8.7-12.4 months for partial regression. The cyclophosphamide + adriamycin + methotrexate treatment scheme proved effective in 87% of localized tumor (complete regression--27%). As a result of treatment with the said drugs plus radiation, objective response was obtained in 95%, with complete regression being registered in 68% of the latter group. Therefore, radiation should be an indispensable component of treatment for small cell lung cancer. A two year relapse- and metastasis--free period was observed in 18% cases of localized process and a five year period--in 9%. PMID- 2998091 TI - [Role of radiation therapy in the treatment of patients with small cell lung cancer]. AB - The paper presents a review of up-to-date literature on the problems of morphological heterogeneity of small cell lung cancer, clinico-morphological parallels, main prognostic factors and rationale for application of radiation therapy in the complex treatment of the disease. The authors also discuss their own findings on 208 cases, with 170 of them having localized tumor. Superfractionated irradiation of a locoregional zone with 1.2 Gy thrice a day was conducted at the initial stage of treatment (total dose--46.8 Gy). As a result, complete regression was obtained in 65.5% and partial regression in 34.5%. All patients with localized tumor survived six months after radiation chemotherapy, 58.5%--12 months, and 57.1%--24 months. PMID- 2998092 TI - [Prophylactic brain irradiation and radiotherapy of brain metastases of small cell lung cancer]. AB - The report presents the results of cranial irradiation of 44 small cell lung cancer patients with clinically-identified intracranial metastases and 40 patients for metastatic spread prevention. Whole brain irradiation was carried out with single doses of 2-4 Gy (total dose--30-40 Gy) in both groups 5 times weekly. Patients irradiated for metastasis prevention revealed a 3.3-fold decrease in intracranial metastasis frequency and a good post-treatment tolerance. In the other group, radiation failed to reach tumor lesions in 20%; treatment produced a poor effect in 30%. There was a correlation between survival time, initial expansion of process and tumor response to primary treatment. No relationship was observed between survival time and procedure and duration of cranial irradiation. Prophylactic irradiation may be beneficial in responders to therapy. However, randomized research into the effectiveness of preventive irradiation and possible radiation injury to cranial and brain tissues is required, particularly, in patients responding to primary treatment by complete regression of localized tumor. PMID- 2998093 TI - [Effect of levamisole on the course of chronic cytomegalovirus infection in women]. AB - The therapeutic effect of orally given levamisole in the treatment of women with chronic cytomegalovirus infection was studied. The patients were examined serologically, cytologically, virologically; the factors of cell-mediated immunity were also studied. Oral therapy with levamisole resulted in a definite increase of the cell-mediated immunity with a trend for decrease in antibody titres and reduced virus excretion. Evaluation of the clinical effect of levamisole therapy, however, revealed no distinct difference in the outcomes of pregnancy in the groups of women treated or not treated with levamisole. PMID- 2998094 TI - [Blocking of Sindbis virus replication in the body of infected mice using an inhibitor of chymotrypsin-like proteases]. AB - Parenteral administration of an inhibitor of chymotrypsin-like proteases (TPCK) to mice infected with alphavirus (Sindbis AR/339 strain) blocked virus replication in the brain and inhibited the development of viremia in the infected animals. The most likely mechanism of TPCK antiviral effect seems to consist in disturbance of proteolytic processing of viral proteins. PMID- 2998096 TI - [Spontaneous hepatitis in the crab-eating macaque exposed to immunodepressants]. AB - A case of spontaneous hepatitis was detected in experiments aimed at working out the conditions for reproduction of the immunosuppressed state in Macaca fascicularis with the purpose of subsequent infection of these monkeys with non A non B hepatitis virus transmitted by the fecal-oral route. One of 6 monkeys at the 8th day of the experiment was found to have developed an increase in the level of serum aminotransferases which grew progressively reaching high values by day 14. Fecal specimens from this monkey collected on the 5th day and later contained spherical virus-like structures 27 nm in diameter, antigenically identical with hepatitis A (HAV) virus. In the other 5 monkeys, no similar structures were found in fecal specimens throughout the experiment. The monkey with the signs of hepatitis was sacrificed on the 16th day of experiment, i. e. on the 8th day from the onset of hyperenzymemia. Immune electron microscopy of extracts of hepatic tissue and fecal specimens collected from this monkey has revealed 27 nm particles antigenically identical with HAV. The bulk of viral particles from the liver sedimented in cesium chloride buoyant density zone of 1.32 g/cm3, and from fecal specimens in the zone of 1.36 g/cm3. In the liver of this monkey, histological changes were found which are observed in acute hepatitis, and HAV antigen in hepatocyte cytoplasm was detected by the fluorescent antibody technique. It is suggested that the spontaneous disease of this monkey was due to natural infection with HAV which could be provoked by experimental immunosuppression. PMID- 2998095 TI - [Characteristics of the subvirion components formed after the treatment of Machupo virus with a nonionic detergent]. AB - Machupo virus labeled with radioactive precursors of RNA or protein synthesis was purified by isodensity ultracentrifugation in sucrose concentration gradient. The purified virus was disrupted with NP-40 nonionic detergent in the presence of 1 M KCl, and subvirion fractions were separated by ultracentrifugation in urografin density gradient. This treatment resulted in formation of two subvirion components with a buoyant density 1.25-1.26 and 1.10-1.12 g/cm3 in urografin concentration gradient. The subvirion fraction with the density of 1.25-1.26 g/cm3 contained high-molecular virion RNAs, a major protein of molecular weight of 64 kD, and seemed to be the virion nucleocapsid. The solubilised fraction (1.10-1.12 g/cm3) contained glycosylated protein 37 kD which seems to be a surface glycoprotein. PMID- 2998097 TI - [Procedures for enhancing the sensitivity of the counterimmunoelectro osmophoresis method in diagnosing viral infections]. PMID- 2998099 TI - AIDS: where do we go from here? PMID- 2998098 TI - AIDS: the significance of anti-HTLV-III antibodies. PMID- 2998100 TI - [Angiotensin-converting enzyme in patients with temporal arteritis]. AB - It is well documented in the literature that patients with active sarcoidosis exhibit elevated serum ACE activity. Since both temporal arteritis and sarcoidosis represent granulomatous inflammatory conditions, this study was undertaken to determine serum ACE activity in patients with temporal arteritis as well as in a control group. The serum ACE levels in patients with temporal arteritis were significantly lower than in the controls. Hence, granulomatous inflammation in temporal arteritis seems to differ radically from the granulomatous inflammation present in sarcoidosis. Serum ACE activity was increasing during cortisone treatment of patients with temporal arteritis, but dropped again as soon as cortisone therapy was discontinued. PMID- 2998101 TI - [Corticotropin releasing factor as an aid in the diagnosis of Cushing syndrome]. AB - 6 patients with Cushing's syndrome were investigated with regard to the effect of synthetic ovine corticotropin-releasing factor (o-CRF), administered as an intravenous bolus of 100 micrograms, on peripheral plasma concentrations of ACTH and cortisol. The purpose of this study was to evaluate the usefulness of this "CRF test" in the differential diagnosis of Cushing's syndrome as compared with conventional diagnostic procedures. 100 micrograms CRF caused a rise in plasma ACTH and cortisol in patients with bilateral adrenal hyperplasia (n = 3). However, in patients with cortisol-producing adrenal adenoma (n = 2) and ectopic ACTH overproduction (n = 1), no increase in plasma cortisol and ACTH was induced by exogenous CRF. We conclude from these findings that the CRF test will prove a valuable diagnostic tool to differentiate pituitary from extrapituitary forms of endogenous hypercortisolism in patients with Cushing's syndrome. PMID- 2998102 TI - Studies on the activity of Bendalina (bendazac L-lysine salt) as a scavenger of hydroxyl and superoxide radicals. AB - Bendalina (bendazac L-lysine salt) strongly inhibits the depolymerization of hyaluronic acid solutions by OH. or O2.- radicals. By viscometric determinations of these reactions the activity of Bendalina as a free radical scavenger was demonstrated. PMID- 2998104 TI - [Magnetic resonance tomography. 1. Introduction and physico-technical principles]. PMID- 2998105 TI - [Dibenzyran--alpha-receptor blocker for the therapy of functional disorders of bladder emptying]. PMID- 2998103 TI - The management of nonmetastatic locally advanced breast cancer using primary induction chemotherapy with hormonal synchronization followed by radiation therapy with or without debulking surgery. PMID- 2998107 TI - [Nuclear magnetic resonance tomography in the study of the liver and its diseases]. AB - Nuclear Magnetic Resonance (NMR) tomography has already attained a position in clinical use beside the established techniques ultrasound (US) und computerized tomography (CT) for the examination of the liver and its related diseases. In assessing its methodical significance one has to differentiate between focal liver lesions and damages of the liver parenchyma. Focal lesions may be identified with NMR just as safely as with US and CT. However, NMR produces additional information, especially in the echo-dense lesions in US, where both NMR and angio-CT can differentiate between echo-dense metastases, haemangiomas and fatty tumours. In damages of the liver parenchyma NMR seems to become the method of choice. Quantitative tissue differentiation is possible by determining the relaxation times. In fatty degeneration of the liver focal fatty infiltration and circumscribed cirrhotic changes can be discerned. It is possible to identify cirrhoses directly and not merely by indirect criteria as is the case with US and CT. NMR can also differentiate between aethylic (prolonged T1) and primary biliary cirrhosis (shortened T1). Haemochromatosis may be identified with CT, the accompanying secondary cirrhotic change, however, can only be detected by NMR. In liver cell carcinoma, e.g. secondary to cirrhosis, cancer nodes and cirrhotic regeneration nodes may be differentiated. Whole body NMR spectroscopy is still under research. Once it is perfected for clinical use it will change the entire laboratory medicine. PMID- 2998106 TI - [Results, complications and controlled after-care in thoracoscopic pleural and lung biopsy]. AB - Report on 546 transthoracic forceps biopsies from pleura and lung, selected under thoracoscopic observation. After inducing a diagnostic pneumothorax the biopsy is performed in general anaesthesia with a forceps introduced beside the thoracoscope. In 60%, the endoscopic-bioptic examination resulted in a definitive diagnosis. In the remaining cases too, the results of biopsy were usefull for further diagnostic and clinical conclusions. The number of complications is small. Main risks are bleeding, tension pneumothorax, and cutaneous emphysema. In diseases of pleura and in cases of disseminated small-size lung processes, the method permits in a high proportion a corroborated diagnosis. PMID- 2998109 TI - [Acquired immune deficiency syndrome (AIDS)]. AB - Acquired immune deficiency syndrome (AIDS) is a new disease caused almost certainly by a transmissible agent, which is most likely a retrovirus termed HTLV III. The disease is mainly spread by sexual, especially homosexual contact. Blood borne transmission is another recognized form of spreading of the disease; it seems, however, that the disease is not readily spread via casual, non-sexual, or other than blood-borne routes. Although the disease is still concentrated in the major metropolitan areas of the United States, it is now increasingly observed in several countries throughout the world. The basic characteristic of the disease is a profound dysregulation of the immune system as proved by a qualitative and quantitative defect of helper T-lymphocytes as well as by B-cell hyperactivity. Clinical manifestations are those of severe and life-threatening opportunistic infections and unusual neoplasms, particularly Kaposi's sarcoma. The mortality is extremely high and may well approach 100%. Therapeutic efforts include the treatment of opportunistic infections and the search for agents which may reconstitute immunologic competence. PMID- 2998108 TI - [Intraoperative sonography in surgical diseases of the liver]. AB - Intraoperative ultrasonography was performed in 24 patients with single or multiple liver metastases of colorectal cancer, in 4 patients with a hepatocellular carcinoma, in 2 patients with an hepatic abscess and in one patient with a focal nodular hyperplasia and one with a liver hemangioma. In 9 of 32 patients with inflammatory or malignant liver disease the tumors were not palpable or visible. These hepatic lesions were localized by intraoperative ultrasound. In 5 cases preoperative unknown hepatic tumors were diagnosed by intraoperative sonography. PMID- 2998110 TI - [Localization of pre-excitation in the WPW syndrome by analysis of the ventricular contraction course]. AB - Fourier phase analysis of gated radionuclide ventriculography (RNV) was applied in 23 patient for the identification of cardiac contraction patterns in WPW syndrome (controls with normal ventricular function and sinus rhythm, n = 30). The sequence and velocity of regional ventricular wall motion was determined and correlated to the results of electrophysiological studies. In 3/23 patients the contraction pattern was not different from controls. Indicating the preexcitation in 20/23 patients the earliest ventricular contraction was localized as follows: right atrial n = 5, paraseptal right n = 2, paraseptal left n = 6 and atrial left n = 7. In 15/16 patients nuclear data corresponded with electrophysiological endocardial mapping. Phase analysis of RNV provides a reliable non-invasive method for localization of preexcitation in WPW syndrome. PMID- 2998112 TI - Antigenic relationships between an isolate of the Western Subtype of tick-borne encephalitis virus and an inactivated vaccine derived from it. AB - High mutation rates resulting from the error prone replicases of RNA viruses could lead to antigenic alterations in viral products and pose significant problems during the manufacture of vaccines against RNA viruses. The production of a vaccine against tick-borne encephalitis virus has been monitored using both polyclonal sera and a library of monoclonal antibodies. Only a few antigenic changes were detected during the alteration of host cell from mouse brain to avian fibroblasts and upon subsequent expansion of the virus population during several rounds of replication. In addition, when the formalin inactivation process was monitored for antigenic change, virtually none was detected. PMID- 2998111 TI - Vaccination against enteric rota and coronaviruses in cattle and pigs: enhancement of lactogenic immunity. AB - Passive immunity against enteric viral infections is dependent upon the continual presence in the gut lumen of a protective level of specific antibodies. This article examines methods currently used to enhance the titre and duration of specific antibody in the mammary secretions of cows and pigs, with particular reference to rotavirus and coronavirus infections. In addition, some of the potential problems to be found in attempting to produce vaccines against these viral infections are outlined. PMID- 2998113 TI - Preparation of highly purified human cytomegalovirus envelope antigen. AB - A human cytomegalovirus (HCMV) envelope preparation was highly purified by immunoaffinity column chromatography using an anti-cellular-IgG column. The purified envelope induced high titre antibodies to HCMV in guinea-pigs. Analysis of the guinea-pig immune sera by RIA and immunofluorescence (IF) showed that this envelope preparation, unlike its unpurified counterpart, did not induce antibody to cellular contaminants. Dot-blot assay revealed viral proteins in the flow through fraction and cellular proteins in the bound fractions. Results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of flow through and bound fractions suggest that a number of proteins previously identified as virus-specific may, in fact, reflect cellular contamination of the envelope preparation, or crossreactivity of some virus-specific proteins with cellular proteins. PMID- 2998114 TI - Increased virulence of an infectious bursal disease live virus vaccine after passage in chicks. AB - A live infectious bursal disease virus vaccine increased in virulence when passaged six times in susceptible birds, as judged by damage to the bursa of Fabricius. It is suggested that the increase in virulence of this vaccine was due to the selection of a virulent subpopulation which had been present in the vaccine since its original isolation from the field and which had not been eliminated during its attenuation by passage in cell culture. The vaccine was then plaque purified and a virus strain was selected which protected against challenge, did not damage the bursa and appeared not to passage in birds. PMID- 2998115 TI - [Comparison of the receptor composition of breast tumors with their histological structure; effect of preoperative therapy]. PMID- 2998116 TI - Influence of base-pairing in the leader region on in vitro translation of Rous sarcoma virus RNA. AB - The capacity of the leader region of Rous sarcoma virus (RSV) RNA to act as a regulator of viral protein synthesis was tested in vitro. When DNA/RNA hybrids of sufficient length (greater than 90-100 nucleotides) are created within the leader, synthesis of Pr76gag is inhibited. The inhibition is dependent upon the length of the hybrid rather than its position with the exception that encumberance of the 5'-terminal 33 nucleotides does block translation. These findings demonstrate that physical alteration of the non-coding leader structure directly affects downstream initiation of protein synthesis. It is thus likely that biochemical or physiologic changes in vivo which alter the structure of the leader may affect regulation of viral protein synthesis. PMID- 2998117 TI - The complete sequence of bluetongue virus serotype 10 segment 3 and its predicted VP3 polypeptide compared with those of BTV serotype 17. AB - The complete sequence of the large RNA segment 3 (L3) of bluetongue virus serotype 10 (BTV-10) has been determined from DNA copies of the viral RNA cloned in the E. coli plasmid pBR322. The L3 viral RNA is 2772 nucleotides long with a single open reading frame of 2706 nucleotides. The L3 predicted primary gene product (VP3) is 103 342 daltons and has a net charge at neutral pH of -5. The sequence of the L3 RNA species differs by 126 point mutations from that of BTV-17 (i.e., 95.5% homology; see M. Purdy, J. Petre and P. Roy, J. Virol. 51, 754-759, 1984). The predicted L3 primary gene products of the two viruses differ by 9 amino acids. These differences correspond to 9 point mutations and represent 0.15% of the sites where nucleotide substitution could cause an amino acid change. By contrast, another 114 point mutations in the genome correspond to 6.5% of the available sites where nucleotide substitutions could be silent (i.e., where a nucleotide substitution may not cause an amino acid change). Three point mutations are in the 3' non-coding region of the RNA species. The quantitative differences between the coding and silent mutations are interpreted as representing the result of gene product conservation. PMID- 2998120 TI - [Cell patterns induced by human papilloma virus in samples sent to a cytodiagnostic central laboratory]. AB - Virus induced changes occur not so rarely in combination with precancerous and cancerous lesions. We compared the cytological and histological findings. The increasing frequency of this event is demonstrated by an analysis of the material seen in our laboratory, smears from more than 120,000 patients per year. It was possible to verify the viral etiology by the electron microscopical demonstration of virus particles. PMID- 2998121 TI - Tn3 as the molecular basis of ampicillin resistance in E. coli--an epidemiological survey. AB - Plasmids of 31 E. coli strains coding for the TEM-1 beta-lactamase were analysed for the molecular basis of this enzyme. In transposition experiments we could demonstrate that only 50% of the plasmids were able to transpose their ampicillin resistance gene. Two of the non-transposing structures were further examined. The 8.1 kb plasmid pBP738 contained Tn3 having suffered a point mutation within the transposase gene that could be complemented by an intact transposase. The 79 kb plasmid pBP749 carried a TEM-1 coding sequence, but the homology with Tn3 was limited to 1.18 kb. PMID- 2998118 TI - [Uni- or bilateral hepatoenterostomy in central bile duct occlusion?]. AB - With the help of different microscopic and macroscopic techniques 100 livers have been investigated to find out whether there are primary and secondary intrahepatic communications between the bile ducts of the right and the left lobe. The results of these investigations are compared with eight case reports. To find the best surgical approach to extensive tumorous blockage of the hilus it is basically necessary to know the anatomical situation in detail. PMID- 2998122 TI - [Metabolism of phosphate-limited Streptomyces cultures. III. The ambivalent effect of phosphates in nourseothricin-producing cultures of Streptomyces noursei JA 3890b]. AB - A common condition in the evolution of organisms and their metabolism seems to be a latent lack of available phosphate in the natural environment. Accordingly, the phosphate dependent metabolisms of the soil-living streptomycetes should be stamped by lack of phosphate, too. The biosynthesis of the streptothricin antibiotic nourseothricin by Streptomyces noursei 3890b is initiated by limitation of soluble phosphate in the fermentation medium. At the other side is shown that a certain rate of feeding of phosphate during the fermentation increases the nourseothricin biosynthesis. An ambivalente role of phosphate on the secondary metabolite biosynthesis is stated. The limitation of phosphate leads to a special physiological state of the producer, characterized by secondary product formation and dephosphorylating activities in cells. This state is temporally stabilized by the presence of a sufficient phosphate supply, realized by enzymatic hydrolysis of complex phosphate-containing substrates or by a direct feeding of inorganic phosphate to the fermentations. The occurrence of different physiological states in respect to the phosphate-dependent metabolism is described by S-shaped functions of the relationship between specific growth rate and the phosphate concentration in the medium. The special behaviour of Streptomyces noursei cells at phosphate limitation is discussed to be the result of the dephosphorylating activities in cells, hydrolyzing phosphoester-bonds of regulatory metabolites as well as energy-rich compounds. PMID- 2998123 TI - [A storage disease in a parakeet (Nymphicus hollandicus) morphologically similar to the systemic myoclonic disease (Lafora disease) in man and dog]. PMID- 2998119 TI - [Linitis plastica with infiltration of the entire gastrointestinal tract]. PMID- 2998124 TI - [Drinking water vaccination against mousepox (ectromelia)]. PMID- 2998125 TI - Application of an enzyme-linked immunosorbent assay (ELISA) involving monoclonal antibody for detection of BLV antibodies in individual or pooled bovine milk samples. PMID- 2998126 TI - [Contagiousness of monkey pox for humans: results of an investigation of 2 outbreaks of the infection in Zaire]. AB - The results of the investigation of two outbreaks of group monkeypox infection among humans (altogether 8 cases) in the zone of Bumba, Equatorial Province, Zaire, are presented. The primary source of infection in both outbreaks was not established, the outbreaks were supposedly caused by sick wild animals. Almost all persons affected by this infection were children aged 7 months to 7 years, never vaccinated against smallpox; the only exception was a 29-year old female patient, formerly vaccinated and revaccinated against smallpox. During one of the outbreaks the laboratory-confirmed transmission of infection from man to man was established in two generations. During the other outbreak there were grounds to suspect the transmission of infection in three generations, though the possibility of contacting infection from animals could not be completely ruled out. The existence of the inapparent form of monkeypox in humans was revealed. PMID- 2998127 TI - [Role of virus-induced autoreactive T-lymphocytes, T-suppressors and the serum factor regulating their activity in the pathogenesis of experimental infection caused by the Langat virus in mice]. AB - The precursors of autoreactive T-lymphocytes (PARTL) have been detected in the spleen of mice infected with Langat virus. When introduced into syngeneic recipients, PARTL differentiate in their lymph nodes into autoreactive T lymphocytes (ARTL) causing a fatal autoimmune disease in the syngeneic recipients in vivo and capable of destroying syngeneic cell cultures in vitro. In the thymus of mice infected with Langat virus T-suppressors (TS) inhibiting the differentiation of PARTL into ARTL have been detected. The serum of intact mice has been shown to contain the serum blocking factor (SBF) which suppresses the differentiation of PARTL and the activity of TS from donors having common H-2 haplotypes of the gene complex with serum donors. In the course of viral infection the decrease of SBF activity and, simultaneously, the activation of PARTL and TS occur. The activation of PARTL and TS in infected mice may be suppressed by the injection of the serum of intact donors identical in H-2 haplotypes. The injection of ARTL induced by Langat virus into syngeneic recipients infected with this virus provokes the transformation of asymptomatic infection into acute infection, while TS and SBF blocking the differentiation of PARTL protect the animals from death. PMID- 2998128 TI - [Use of thin-layer immune analysis for detecting antigens and antibodies]. PMID- 2998130 TI - The effect of naloxone on ATCH and beta-endorphin in patients with Cushing's disease. AB - Endogenous opiates may be important in the control of ACTH secretion in men. The effect of opiate receptor blockade by naloxone on ACTH, beta-endorphin-like substance and cortisol release was studied in healthy women and in 9 patients with Cushing's disease. In the healthy subjects, ACTH, beta-endorphin and cortisol levels were increased in response to naloxone. However, in 3 our of the 9 patients with Cushing's disease, a paradoxical decrease in serum ACTH, cortisol and beta-endorphin concentrations was observed after naloxone administration. In the patients with a paradoxical response to naloxone, transsphenoidal microadenomectomy was ineffective. PMID- 2998129 TI - Time dependency and reversibility of the effects of exclusive cyproterone acetate therapy on pituitary adrenal function in hirsute women. AB - The effects of cyproterone acetate (CA) administration on the pituitary-adrenal axis were studied in 30 hirsute females. The patients were treated continuously with a daily dose of 100 mg of CA for a maximal period of 12 months. Insulin induced hypoglycaemia and ACTH infusion were performed on pre-treatment conditions and after 1, 4, 6 and 12 months of CA treatment. From a clinical point of view, a dramatic improvement of hirsutism was evident after 6 months of therapy. The most commonly reported side effects were amenorrhoea and transient uterine haemorrhage. Apart from asthenia, no symptoms of adrenal insufficiency were noticed. No changes in pituitary-adrenal secretion were observed during the first 4 months of therapy. From the 6th month, a reduction in basal as well as stimulated cortisol levels was seen. Simultaneously, an enhanced ACTH response to hypoglycaemia was observed. Both effects became more pronounced after 1 year of treatment. There were no significant changes in ACTH basal values. Six months after discontinuation of the drug, adrenocortical reserve improved but was still slightly reduced when compared to pre-treatment range at that time. These findings suggest a time-dependent negative effect of CA on adrenal steroidogenesis which shows a reversible character 6 months after antiandrogen withdrawal. Therefore, steroid cover should be considered for intercurrent illness in patients treated for longer than 6 months with this therapeutic regime. PMID- 2998132 TI - Decreased prolactin responsiveness to thyrotrophin-releasing hormone and metoclopramide in hyperthyroidism. AB - To investigate whether prolactin (Prl) responsiveness to thyrotrophin-releasing hormone (TRH) differs in thyrotoxic and normal individuals, serum Prl was determined before and after iv injection of 200 micrograms TRH in 10 patients with untreated thyrotoxicosis and also in 9 normal subjects. Both the maximal Prl increment after TRH and the total Prl response, represented by the Prl incremental area, were significantly larger in the normal subjects compared with the thyrotoxic (max Prl increment 56 +/- 11 vs 15 +/- 3 ng/ml, P less than 0.001; Prl incremental area 3071 +/- 522 vs 579 +/- 171, P less than 0.001; mean +/- SEM). The maximal Prl increase after 15 mg oral metoclopramide (MET) was also significantly larger in the normal (125 +/- 13 ng/ml) than in the thyrotoxic subjects (60 +/- 13 ng/ml, P less than 0.01). When 200 micrograms TRH was injected iv 90 min after oral administration of 15 mg MET, an additional Prl increase was observed in normal individuals (21 +/- 6 ng/ml, P less than 0.01). In thyrotoxic patients, however, iv TRH failed to induce a significant increase in Prl after oral priming with MET (0 +/- 3 ng/ml). When 7 thyrotoxic patients, made euthyroid by 125I-treatment, were investigated according to the same protocol as the one mentioned above, they displayed normal Prl responses to iv TRH and to oral MET. Furthermore, they showed a significant Prl response to iv TRH after oral priming with MET (20 +/- 8 ng/ml, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2998131 TI - Effects of a 72-hour prostacyclin infusion on the hormone levels in patients with obliterative arterial disease. AB - Effects of a 72-h prostacyclin (PGI2) infusion (5 ng/kg/min) on hormone levels were studied in 11 patients (5 males, 6 females) suffering from obliterative arterial disease of the lower extremities. ACTH, cortisol, TSH, prolactin (Prl), GH, LH, FSH, T3, T4, calcitonin, parathyroid hormone (PTH), insulin, plasma renin activity (PRA), aldosterone and testosterone levels were measured at -15, 0, 30, 120, 240 min and 24, 48, 72 and 96 h after the infusion. During the first 240 min Prl and GH levels showed an increase that was thought to be either an effect of release of hormones or a consequence of stress. At the same time the thyroid hormones, T3, T4 and calcitonin decreased, presumably owing to an alteration in the blood flow to the thyroid gland. All these hormone levels returned to normal at 24 h in spite of the infusion continuing. PRA increased only during the second half of the infusion. No changes were found in the levels of ACTH, cortisol, TSH, LH, FSH, PTH, insulin, aldosterone and testosterone during the infusion. Five diabetics showed the same hormonal changes as the non-diabetics and their blood sugar levels remained unaffected during and after the procedure. PMID- 2998133 TI - Activation of adrenal adenyl cyclase by anti-thyroid plasma membrane antibodies. AB - We have previously shown that IgG isolated from rabbit anti-bovine thyroid plasma membrane (anti-BTPM) antiserum exhibits properties similar to thyroid stimulating antibodies (TSAb) in that it activates thyroid adenyl cyclase. In this study, the organ non-specificity of this reaction was investigated. It was observed that anti-BTPM IgG stimulated not only adenyl cyclase of bovine thyroid but also that of the adrenal. The stimulatory activities on the thyroid and adrenal adenyl cyclase were abolished by absorption of the IgG with bovine adrenal plasma membrane (BAPM). These results indicate that anti-BTPM antibodies, similar to TSAb, exert both thyroidal and extra-thyroidal effects. Thus anti-BTPM antibodies may be directed against antigenic determinants that are common to both thyroid and adrenal plasma membranes. Like the anti-BTPM IgG, anti-BAPM IgG also activated both thyroid and adrenal adenyl cyclase. However, when IgG of the anti BAPM antiserum was absorbed with thyroid plasma membranes, only the thyroid, but not the adrenal stimulating activity was abolished. It was concluded that the anti-BAPM antiserum contained antibodies directed against membrane antigens specific for the adrenal as well as common antigens shared by the thyroid. PMID- 2998134 TI - Thymus peptides interacting with opiate receptors. AB - The substances displacing labelled ligands from opiate receptors of the rat brain membrane fraction were found in the thymosin fraction 3 and acetoacid extract of the thymus by the radioreceptor assay. Comparison of the displacing activity of acetoacid extracts of perfused and non-perfused thymus and peripheral blood as well as an estimation of the blood content in the thymus allowed to conclude that blood does not participate in the ability of thymus preparations to bind to opiate receptors. On the basis of the enzymatic treatment data one can conclude that opiate receptor ligands present in thymus preparations are of peptide nature. The value of their sodium shift suggests that those peptides are partial agonists of morphine. The possible role of opioid peptides in the thymic endocrine function is discussed. PMID- 2998135 TI - Poly(L-lysine) enhances the release of reactive oxygen metabolites from human neutrophils. PMID- 2998136 TI - Bone marrow cyclic nucleotides (cAMP, cGMP) in phenylhydrazine-induced anemia. AB - The studies were carried out on male Wistar rats. The animals were divided into two groups: control and experimental. The hematocrit value and reticulocyte count were determined in blood samples collected from control and experimental rats. The experimental rats received subcutaneous injections of phenylhydrazine manufactured by Sigma for the duration of 3 days. On the 5th day blood samples were collected from all the control and experimental animals and determinations of hematocrit and reticulocyte count were repeated. cAMP levels were determined in bone marrow extracts by means of a radiocompetitive method. The cGMP level was determined by a radioimmunological assay. A significant elevation of cAMP level was detected in experimental rats, whereas the cGMP level changed only slightly. PMID- 2998138 TI - Demonstration of thiamine pyrophosphatase in human germ cells and Sertoli cells, a histochemical study. AB - The distribution of thiamine pyrophosphatase (TPPase) was studied in the human testicular biopsy tissue with the help of a modified histochemical gel method. The spermatogonia showed a large round strongly positive reaction zone in the supranuclear region. The pachytene spermatocytes exhibited a large semicircular TPPase reaction zone around the nucleus directing towards the lumen. The early round spermatids were characterized by a small, round, and weak supranuclear reaction zone. The elongated late spermatids were devoid of TPPase activity. The Sertoli cells possessed a "streamer" like formation of strong TPPase activity spreading from basal to apical portion. PMID- 2998137 TI - A histochemical investigation on the percutaneous absorption of vitamin D synthesized into the mammal epidermis. AB - The vitamin D transepidermis absorption was studied by means of a histochemical technique suitable to detect this vitamin and to discriminate it from cholesterol and its esters. Such technique shows vitamin D inside the mast cell granules. As the mast cell granules contain metachromatic substances its own histochemical reactivity must be previously blocked by methylation. After this treatment the mast cell granules do not stain by toluidine blue and do not react to the peracetic acid-toluidine blue reaction. However, the granules remains reactive to alkaline permanganate-toluidine blue and to alkaline permanganate-Schiff reactions. These results show that the mast cell granules do not contain cholesterol but they contain vitamin D. The lack of cholesterol suggests that vitamin D is not synthesized inside the granules. As the mast cells may appears within the epidermis or in close relationship with the epidermis, although it is placed into the superficial dermis, it was admitted that the mast cells uptake vitamin D contained inside the epidermis intercellular compartment. In such instances, the vitamin D synthesized by the keratinocytes enter the intercellular compartment, where its synthesis accomplishes, and migrate towards the basement membrane. At the basal epidermis layer or after passing through the basement membrane the vitamin D is taken up by mast cells, where it is stored inside its granules. PMID- 2998140 TI - Viral DNA sequences in human cytomegalovirus transformed hamster cell line at low passage levels. AB - The characteristics of a human cytomegalovirus (HCMV) transformed syrian hamster cell line (87-TRH-5) were examined. The cytomegalovirus DNA (HCMV-DNA) labelled in vitro by nick translation was used as a probe to detect viral DNA sequences in the 87-TRH-5 line. Cytomegalovirus specified DNA sequences were detected in cells examined up to passage 58, but were undetectable at higher passage levels. Six clones were derived from the 28th passage of 87-TRH-5 cells and examined for HCMV DNA sequences. Various amounts of HCMV-DNA were found. A tumour induced by passage 28 cells contained no detectable HCMV-DNA sequences. PMID- 2998142 TI - Cholinergic, opioid and glycine receptor binding sites localized in human spinal cord by in vitro autoradiography. Changes in amyotrophic lateral sclerosis. AB - Binding sites for the receptor ligands 3H-quinuclidinylbenzilate, 3H-alpha bungarotoxin (3H-alpha-Btx), 3H-etorphine and 3H-strychnine were localized autoradiographically at cervical, thoracic and lumbar levels of spinal cords from post-mortem human control subjects and subjects with amyotrophic lateral sclerosis (ALS). The highest densities of muscarinic binding sites were found in the motor neuron areas and in the substantia gelatinosa, while the grey matter binding was very low within Clarke's column. Both 3H-alpha-Btx and opioid receptor binding sites were numerous within the substantia gelatinosa, while glycine receptor binding sites were more uniformly distributed within the spinal grey matter. In ALS cases, muscarinic receptor binding sites were markedly reduced in motor neuron areas and slightly reduced in the dorsal horn, while the other binding sites studied were relatively unchanged. PMID- 2998141 TI - HCMV specific expression in HEL cells transformed by Xba I endonuclease fragmented HCMV-DNA. AB - The transfection of subconfluent monolayers of HEL cells with Xba I endonuclease fragmented HCMV-DNA resulted in a large number of morphologically transformed foci. The frequency of focus formation of Xba I fragmented DNA increased after TPA treatment of the cells. The morphologically altered cells showed specific reactivity with anti-HCMV serum pool in the nuclear and perinuclear region and in the cytoplasm. The HCMV-32P-labelled probe DNA has been specifically hybridized to the cells of morphologically altered foci. The autoradiographic grains were localized in both nucleic and cytoplasm of the cells showing a specific hybridization between the probe DNA and nuclear as well as polyribosomal RNA. PMID- 2998139 TI - Differentiation of blood vessels in the adipose tissue of lean and obese fetal pigs, studied by differential enzyme histochemistry. AB - Subcutaneous adipose tissue was obtained from fetuses removed from pregnant obese (Ossabaw) and lean (crossbred) sows at three stages of gestation (70, 90, and 110 days). Histochemical analysis for nucleo-side phosphatase (NPase), alkaline phosphatase (APase), and NADH tetrazoleum reductase (NADH-TR) was conducted on fresh-frozen cryostat sections. Age- associated changes in NPase and NADH-TR reactions in the arteriolar system were correlated with the morphological development of the medial layer of arterioles and arteries. For instance, a strong NPase reaction in small arterioles was associated temporally with the assumption of a normal smooth muscle cell morphology and arrangement in the medial layer. In the youngest fetuses, strong NADH-TR reactions were only evident in small and presumptive arterioles and venules (associated with fat cells). Little NADH-TR reactivity was evident in larger arterioles and venules in 70-day tissue. Arteries and large arterioles were distinguished from veins and venules (strong reactions vs. weak reactions) with NADH-TR and NPase reactions in the oldest fetuses. In the younger fetuses, the NPase distinction (arterioles vs. veinules) was obvious before NADH-TR distinction. Small adipocyte-associated vessels were APase positive in the youngest fetuses, but APase reactivity was limited to short segments of vessel between arterioles and capillaries in the oldest fetuses. With the following exceptions, all the above observations were independent of fetal strain. In obese fetuses (110 day) small venules and small arterioles were equally reactive for NPase activity. Capillaries in obese fetuses (110 day) were NADH-TR reactive, whereas no activity was evident in capillaries from lean fetuses (110 day).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2998143 TI - Quantitative vibration perception thresholds in patients under prolonged antiepileptic treatment. AB - Quantitative vibration perception threshold (VPT) measurements were performed on 134 healthy subjects and 147 epileptics undergoing prolonged (more than one year) antiepileptic treatment, 28 patients received diphenylhydantoin, 71 carbamazepine and 11 some other drug. 37 subjects received a combination of drugs, which included diphenylhydantoin and/or carbamazepine. VPT was measured quantitatively at the dorsum of the right foot with a device vibrating at 100 Hz. The mean of the four successive threshold values was calculated. VPTs of the epileptic groups treated with diphenylhydantoin, carbamazepine or combined medication were all significantly (P less than 0.001) higher than those of the control group. The group receiving other medication showed no difference in this respect. The percentage of patients with abnormal thresholds was about the same (10-16) in the epileptic groups. An exception was the group taking other medication, in which no abnormal VPTs were found. It is suggested that such quantitative measurement may be a useful screening method for detecting dysfunction in the peripheral nervous system due to prolonged antiepileptic treatment. It will, however, be necessary to test the clinical validity of the method for diagnosing manifest or subclinical polyneuropathies. PMID- 2998144 TI - Ethanol and polyneuropathy. AB - Two groups of alcoholics (30 patients each)--identified by the MALT score--were examined. Clinical and laboratory investigations showed no connection between thiamine, riboflavin, or Vitamin B6 deficiency and development of the polyneuropathy. Neither the polyneuropathy nor the diminished sensory conduction velocity were related to malnutrition. The relation between the duration of alcoholism and symptoms of polyneuropathy was highly significant in one group. The neurotoxicity of ethanol was confirmed in an experiment with rats. PMID- 2998145 TI - Na+/K+-ATPase activity and GABA uptake in astroglial cell-enriched fractions and synaptosomes derived from rats in the early stage of experimental hepatogenic encephalopathy. AB - Na+/K+-ATPase activity and GABA uptake were measured in the bulk isolated astrocytes and synaptosomes from rats in which an early, metabolic phase of hepatogenic encephalopathy (HE) was induced by the treatment with thioacetamide (TAA). Both the enzyme activity and the amino acid neurotransmitter uptake were increased above control in the astroglial fraction but remained unaffected in synaptosomes. The results lend support to the earlier observations that the astrocytes are the primary target cells in HE. Furthermore, they may be interpreted as indicating that the early astroglial reaction to HE comprises stimulation of the astrocytes' function, especially concerning clearance of K+ ions and neurotransmitters from the extracellular space of CNS. PMID- 2998146 TI - Ovarian steroid production in a woman with polycystic ovary syndrome associated with endometrial cancer. AB - A young woman with typical polycystic ovary syndrome (PCO) underwent laparotomy for moderately differentiated endometrial cancer. Specimens from the hyperplastic thecal and stromal tissue of the ovaries were incubated for 2 hours in the presence or absence of hCG, 100 IU/ml. Following incubation the tissue content of cyclic AMP and the amounts of progesterone (P), androstenedione (A), testosterone (T) and estradiol-17 beta (E2) in the incubation medium were analysed. For comparison, thecal cells from normal ovaries of regularly menstruating women were incubated under identical conditions. In vivo, the PCO ovaries secreted several fold greater amounts of T than normal ovaries. In vitro, the thecal cells were much more active, steroidogenically, than the stromal cells of the PCO ovary. Furthermore, the hyperplastic thecal cells of the PCO ovary produced several-fold greater amounts of androgens, and appeared more sensitive to stimulation with hCG, as compared with thecal cells from normal ovaries. The results indicate that in women with PCO associated with endometrial cancer the hyperplastic thecal cells are a significant site of abnormal androgen production and abnormal sensitivity to gonadotropin. PMID- 2998147 TI - Chlamydial cervicitis in women followed-up for human papillomavirus (HPV) lesions of the uterine cervix. AB - To assess the concomitant appearance of Chlamydia trachomatis and Human papilloma virus (HPV) (currently linked with the development of cervical cancer) in uterine cervix, a series of 250 women under continuous observation for cervical HPV lesions (with or without concomitant cervical intra-epithelial neoplasia (CIN)) were the subject of cervical culturing for C. trachomatis, as well as for IP-PAP (indirect immunoperoxidase) staining of the cervical biopsies with monoclonal antibody to C. trachomatis. Chlamydia-positive staining was found in only 2/204 biopsies (0.98%), whereas Chlamydia could be isolated from the cervix of as many as 26/250 (10.4%) women. In repeat cultures, Chlamydia was isolated in 39/936 specimens (4.2%), reflecting the effect of the treatment instituted. The results are discussed in terms of the suggested association of Chlamydia with CIN, as well as of the possible synergism between Chlamydia and HPV in cervical oncogenesis. The conclusion is drawn that Chlamydia and HPV are covariables of sexual behavior, their concomitant appearance in the uterine cervix most probably being ascribable to sexual promiscuity. PMID- 2998149 TI - Effects of dipyridamole on the glyceryl-trinitrate-induced inhibition of coronary artery muscle tone and platelet aggregation in relation to cyclic nucleotide metabolism. AB - The effects of glyceryl-trinitrate (GTN) and dipyridamole (DIP) on relaxation of bovine coronary arteries and on inhibition of aggregation of human platelets have been studied in vitro with special reference to the cyclic GMP (cGMP) system. GTN had a dose-dependent relaxant effect on bovine coronary arteries, and at a high concentration (10(-5) M) it had an inhibiting effect on platelet aggregation. The effects were associated with an increase in the cGMP levels of the tissues. DIP (5 X 10(-7) M respectively 5 X 10(-6) M) potentiated the coronary artery relaxation induced by GTN (10(-8) M) and the inhibition of platelet aggregation caused by GTN in the concentrations 10(-7)-10(-4) M. The potentiation was associated with higher levels of cGMP than those produced by GTN alone, at least in bovine coronary arteries. However, at a concentration of 10(-4) M, GTN, in combination with DIP, caused a significant fall in the cGMP level compared to GTN alone. GTN and DIP were not found to significantly increase the cAMP levels in the concentrations tested. DIP was shown to inhibit phosphodiesterase (PDE) from both platelets and bovine coronary arteries. This might be one of the possible mechanisms that can explain the above mentioned potentiation. It is suggested that the combination of DIP + GTN may be of some clinical importance since the potentiating effects were seen at concentrations comparable to the therapeutic plasma concentration for the respective drugs. PMID- 2998148 TI - Virus-induced central positional nystagmus in mice. AB - Geotropic direction-changing nystagmus in lateral body positions was observed in 4-week-old BALB/c mice after intracerebral injection with a temperature-sensitive mutant of mouse hepatitis virus. The positional nystagmus was detected already 2 days after infection and it lasted half a year at least. The nystagmic responses of the semicircular canals were also evaluated before and after infection. They were unaltered during the disease, which was clinically manifested by general weakness, ataxia and tremor. Histopathological examination 2 weeks after infection revealed demyelination in various parts of the CNS. PMID- 2998150 TI - Concomitant glucocorticoid treatment prevents the development of beta adrenoceptor desensitization in the guinea pig lung. AB - The aim of the present study was to investigate, whether concomitant administration of the synthetic glucocorticoid betamethasone (BM), theophylline (THEO), or the muscarinic antagonist ipratropium bromide (IPRA) could influence the desensitization-associated decrease of beta-adrenoceptors in the guinea pig lung during prolonged in vivo treatment with the beta 2-agonist terbutaline (TER). The animals were sacrificed 20 hrs after the last drug dosage and the lung membrane homogenates were prepared for 3H-dihydroalprenolol (3H-DHA) binding in vitro. Treatment with TER 200 micrograms/kg subcutaneously twice a day for five days decreased by 22% the maximum number of binding sites (Bmax) at saturation in comparison with the saline-treated controls. Concomitant administration of BM 2 mg/kg intraperitoneally abolished this effect of TER, whereas THEO 20 mg/kg or IPRA 5 micrograms/kg failed to modify it. None of the in vivo treatments affected the binding affinity of 3H-DHA. In vitro, TER inhibited in a concentration dependent manner 3H-DHA binding to the lung membranes of untreated guinea pigs. At high concentrations IPRA, but not THEO or BM, showed some binding to the beta receptors as well. Thus, it is concluded that glucocorticoids may prevent beta adrenoceptor desensitization in the lungs via an indirect mechanism, e.g. inhibition of phospholipase A2 enzyme. PMID- 2998151 TI - GABA inhibits inhalation anaesthetic-induced membrane fluidization: a spin label study in synaptic and phospholipid membranes. AB - The hydrophobic interaction of GABA (10(-3)-10(-9) M) with the inhalation anaesthetic enflurane was studied in synaptic plasma membranes of whole rat brain or of the striatum, synaptic mitochondrial membranes and dipalmitoyl lecithin (DPL) vesicles. Stearic acid spin labels, probing either the C-5 level (hydrophilic end) or the C-12 level (hydrophobic end) of the lipid bilayer, were used as indicators of fluidity. Inhalation anaesthetic at 2 mM fluidized the C-5 and the C-12 level of the membranes. A concentration of 0.4 mM produced consistently an increased order (stabilization) at the C-5 level in synaptic plasma membranes. GABA, added prior to the anaesthetic, caused a dose-related inhibition of the anaesthetic-induced fluidization. This restorative effect, which also occurred in DPL vesicles, was not prevented by the GABAA antagonists bicuculline or picrotoxin and was therefore probably not related to receptor mechanisms. When GABA was added after the anaesthetic (2 mM) the inhibition was seen only with 10(-3) M GABA. GABA did not influence (potentiate) the fluidity changes caused by 0.4 mM inhalation anaesthetic in synaptic membranes. Glutamate had a fluidizing effect, but no interaction with enflurane, at the C-5 level of DPL only at a high concentration (10(-3) M). The observed fluidization by enflurane and the restorative effect of GABA, probably through non-receptor hydrophobic interaction, may be involved in the mechanisms of toxic CNS effects. PMID- 2998152 TI - Effect of serum on isoproterenol-induced cyclic AMP accumulation in human lymphocytes. AB - The effects of autologous serum on basal and isoproterenol (IPR) or prostaglandin E1 (PGE1) stimulated adenosine 3',5' cyclic monophosphate (cAMP) levels were investigated in human lymphocytes. For all blood donors, serum (25% (v/v)) lowered the basal cAMP content. In contrast, the responsiveness of the lymphocyte cAMP accumulation to (-)-IPR was increased. This effect was most clearly demonstrable in bicarbonate buffered incubation medium (40-50% increase of maximal response), but was also seen in phosphate buffered medium (10-20% increase). Serum did not alter the sensitivity of the lymphocytes to IPR. The response to PGE1, which was a considerable more effective stimulator of cAMP accumulation than IPR, was not affected in any consistent way by serum. The results indicate that serum influences the regulation of lymphocyte cAMP and that this effect may partly be exerted at the level of the beta-adrenoceptors. PMID- 2998154 TI - [Leukotrienes and other products of lipoxygenases. Nature and biological profile]. PMID- 2998153 TI - Dual inhibitory action of ATP on adrenergic neuroeffector transmission in rabbit pulmonary artery. AB - The aim of this study was to determine whether the inhibitory action of ATP on sympathetic neuroeffector transmission in the isolated pulmonary artery is due to ATP itself or one of its dephosphorylated breakdown products, ADP, AMP or adenosine. Furthermore, the mechanism of the inhibitory action was investigated. ATP (10(-6)-3 X 10(-4) M), the degradation-resistant ATP-analogue, beta, gamma methylene-5'-triphosphate (10(-5)-3 X 10(-4) M), ADP (10(-6)-3 X 10(-4) M), AMP (10(-5)-3 X 10(-4) M), adenosine (10(-5)-3 X 10(-4) M) and 2-chloroadenosine (10( 7)-3 X 10(-4) M) reduced the contractions evoked by field-stimulation. This was also the case for prostaglandin E2 (3 X 10(-9)-3 X 10(-7) M), while prostaglandin F2 alpha (1.4 X 10(-8) M) slightly augmented the neurogenic response. The time course of the inhibitory effect of purinergic compounds on the stimulation evoked contractions was studied. In the case of ATP and ADP the inhibition was biphasic: an initial marked block (1 min. after drug addition) which in the continued presence of either compound recovered partially 10 min. later and then remained almost constant for another 90 min. The other purinergic agents caused a monophasic reduction. In the presence of indomethacin (5 X 10(-5) M), ATP and ADP also reduced the neurogenic contractions in a monophasic manner. Indomethacin did not alter the beta, gamma-methylene-5'-triphosphate-induced inhibition. Dilazep (3 X 10(-6) M) plus deoxycoformycin (3.6 X 10(-6) M), augmented the inhibitory effect of ATP. In contrast, theophylline (5 X 10(-5) M) did not alter the effect of ATP. The inhibitory effect of ATP (10(-4) M) on stimulation-evoked contractions was inversely proportional to the extracellular Ca2+ concentration (0.3-5.2 mM) and to frequency of stimulation (3-15 Hz). These results suggest that ATP initially causes a presynaptic inhibition of noradrenaline release evoked by field-stimulation. This phase I block is probably mainly due to an ADP mediated short-lasting release of prostaglandins of the E type. The continuous inhibition (phase II) is probably due to ATP and its metabolites, possibly mainly adenosine. The phase II inhibition may possibly involve a decreased entry of Ca2+ into adrenergic nerve terminals during depolarization. PMID- 2998155 TI - Skeletal muscle glycolysis during submaximal exercise following acute beta adrenergic blockade in man. AB - The present study describes the influence of beta-adrenergic blockade on glycogen utilization and lactate accumulation in skeletal muscle of exercising man. Twelve physically active men were examined during 25 min of continuous cycle exercise equivalent to 65% of their maximal oxygen uptake both with and without oral administration of 80 mg of propranolol (Inderal). Heart rate, oxygen uptake, rate of perceived exertion (RPE) and blood lactate concentration were measured during exercise. Muscle biopsies were obtained from m. vastus lateralis after 5 and 25 min of exercise. Beta-adrenergic blockade decreased steady state exercise heart rate by (mean +/- SD) 35 +/- 10 beats . min-1 (P less than 0.001) and oxygen uptake from 2.47 to 2.39 l . min-1 (P less than 0.01). Muscle glycogen decreased from the 5th to the 25th min of exercise, and beta-blockade had no significant effect on this decrease. In contrast to without drug, beta-blockade resulted in a decrease (P less than 0.05) in muscle lactate concentration from the 5th (6.9 mmol . kg-1 w./w.) to the 25th min (4.8 mmol . kg-1 w./w.). Similarly blood lactate levels were lower (P less than 0.05) with than without beta-blockade in the last but not the first 10 min of exercise. The alteration in muscle lactate concentration pattern following beta-blockade, may imply that adrenergic effects per se contribute to the stimulation of glycolysis during submaximal exercise, except in its earliest phase. Nevertheless, the effect is not great enough to produce substantial differences in glycogen utilization. PMID- 2998156 TI - The influence of the sympathetic impulse pattern on contractile responses of rat mesenteric arteries and veins. AB - Contractile responses to electrical field stimulation of excised small mesenteric arteries and veins of the rat were compared when stimuli were delivered in irregular bursts or at regular intervals. Spontaneously occurring skin vasoconstrictor impulses in a few-unit median nerve recording in man were stored on tape and used to trigger a stimulator. Two irregular stimulation sequences at average frequencies of 1.6 and 1.8 Hz, respectively, were used. In the arteries, average contractile responses were significantly greater at an irregular than at an even stimulation frequency, but in the veins, similar degrees of contraction were obtained with the two modes of stimulation. The frequency-response relationships to continuous regular stimulation showed the artery to respond less than the vein at low frequencies. This apparently explains the differences in behaviour between the vessels to irregular stimulation. The results show that not only the number of impulses, but also their pattern of occurrence, may influence the degree of vasoconstriction. Thus, the normal irregular sympathetic discharge pattern in itself has a bearing on the physiology of neuro-effector control mechanisms. PMID- 2998157 TI - Reduced uptake of noradrenaline into storage vesicles from the pregnant uterus. AB - A noradrenaline (NA) storage vesicle fraction was isolated from the uteri of virgin as well as pregnant guinea-pigs. The uptake of 3H-NA into respective vesicle fractions was compared. Total uptake of 3H-NA in the presence of Mg2+ (2.5mM) and ATP (2.5 mM) at 30 degrees C after 20 min was 6.0 +/- 1.4 pmol . mg-1 in uterus fractions from virgin guinea-pigs as compared to 1.87 +/- 0.41 pmol . mg-1 in uterus fractions from pregnant animals. When desipramine (5mM) was added in order to block neuronal uptake the corresponding figures were 4.75 +/- 0.92 and 0.51 +/- 0.18 pmol . mg-1, respectively. Thus in the presence of desipramine (5mM) uptake was reduced by 21.4% in fractions from virgin uteri but by 72.7% in fractions from pregnant uteri. Reserpine (0.1 microM) inhibited uptake of 3H-NA by 34% in fractions from virgin uteri and by 46% in fractions from pregnant uteri. Our results indicate a decreased amount of functional storage vesicles in uteri of guinea-pigs at term pregnancy and are consistent with the proposed degeneration of adrenergic nerves during pregnancy. PMID- 2998158 TI - Cerebrovascular responses to haemorrhagic hypotension in anaesthetized cats. Effects of alpha-adrenoceptor antagonists. AB - Haemorrhagic hypotension induces the phenomenon of cerebrovascular autoregulation and, concomitantly, involves an activation of the sympathetic nervous system. As brain vessels in cats have an atypical adrenoceptor distribution we studied the effects of an alpha-adrenoceptor antagonist on the autoregulatory response to haemorrhage. Cortical blood flow was studied by the H2 technique in chloralose anaesthetized cats subjected to a period of graded haemorrhage over 3 h. Three groups of cats were studied: control, i.e. those receiving saline (n = 10); yohimbine-treated (200 micrograms . kg-1 . h-1, n = 7); and prazosin-treated (50 micrograms . kg-1 . h-1, n = 6). In the control group, cortical blood flow remained relatively constant when mean arterial pressure was decreased from 102 +/- 1 mmHg (mean +/- SE) to approximately 50 +/- 1 mmHg; thereafter, blood flow decreased with decreasing perfusion pressure. In the arterial pressure range 64 55 mmHg, cortical blood flow was significantly higher in the yohimbine group (109 +/- 12 ml . 100 g-1 . min-1) compared to the control group (69 +/- 6 ml . min-1) and remained higher in the yohimbine-treated cats at more extreme levels of hypotension. Blood flow did not fall significantly in the yohimbine-treated cats until mean arterial pressures of 31 +/- 1 mmHg were attained. In the prazosin treated cats, flow began to decrease at arterial pressures even greater than those observed in the control group. Thus, there is a sympathetic vasoconstriction of brain arteries that is primarily mediated by alpha 2 adrenoceptors in the feline cerebrovascular bed. PMID- 2998160 TI - Immunological studies in a patient with the glucagonoma syndrome. AB - The glucagonoma syndrome is a rare clinical entity characterized by a glucagon producing tumor of the pancreas, necrolytic migratory erythema, weight loss and usually decreased glucose tolerance. Lately there has been increasing interest in the interaction of peptide hormones and the immune system, implicating a regulatory role of the peptide hormones on immune activation and function. We present a patient with metastasizing glucagonoma and highly elevated plasma levels of glucagon and pancreatic polypeptide. Normal numbers of peripheral blood lymphocytes with normal proportions of the T and B populations were seen. Within the T lymphocyte population the percentage of T4+ cells (helper phenotype) was increased with a concomitant decrease in T8+ cells (suppressor/cytotoxic phenotype), resulting in an abnormally high T4/T8 ratio of 6.7 (mean reference value 1.8). Functional tests demonstrated a normal tuberculin reaction and adequate lymphocyte responses in vitro to polyclonal activators. Furthermore we noticed a urine electrophoretic pattern consistent with a proximal tubular kidney defect. It is concluded that studying the immune system in patients with endocrine active pancreatic tumors may give clues on the influence of pancreatic peptide hormones on immune function and regulation. PMID- 2998159 TI - A method for in vitro studies on acid formation in human parietal cells. Stimulation by histamine, pentagastrin and carbachol. AB - Cells were isolated from human gastric mucosa on a large scale from gastric resection specimens and on a microscale from endoscopic biopsies by sequential incubations with pronase and collagenase. The accumulation of aminopyrine (AP) was used as an index of acid production in the parietal cells. Basal accumulation was about 0.2 pmol AP/10(4) parietal cells. Addition of histamine, db-cAMP, pentagastrin and carbachol increased the aminopyrine accumulation. Maximal accumulation was of the order of 1000-2800% of the control and was obtained after stimulation by 10(-4) M histamine and by 10(-3) M db-cAMP. Stimulation by pentagastrin and by carbachol reached 200 to 350% of the control. EC50 was 2 X 10(-6) M for histamine, 10(-8) M for pentagastrin, and 4 X 10(-6) M for carbachol. Human parietal cells were enriched from a mixture of gastric mucosal cells by isopycnic centrifugation on density gradients of Percoll. A parietal cell fraction with a purity of 83% was obtained. The density of human parietal cells was estimated to 1.06 g . ml-1. PMID- 2998162 TI - A theoretical study of the hydroxyl ion exchange for the fluoride ion in hydroxyapatite from enamel. PMID- 2998163 TI - Hormonal effects on cyclic AMP level in vivo in the myocardium, pancreas and tests of rats with chronic renal failure. PMID- 2998161 TI - Resonance Raman spectroscopy as a probe of heme protein structure and dynamics. AB - Our understanding of metalloporphyrin resonance Raman spectra has advanced to the point where it is possible to obtain detailed information about the structure of the heme group in situ in heme proteins. The porphyrin skeletal mode frequencies can be analyzed in terms of the ligation and spin state of the heme and may provide information about protein-induced stresses. The high-frequency region of the spectrum also contains bands due to vibrations of the porphyrin peripheral substituents, which are potentially monitors of the protein contacts. In the low frequency region, it is possible to locate bands, at least in some states of the heme protein, which are associated with vibrations of the axial ligands. They give direct information about the nature of the bonding to exogenous ligands or to the proximal protein residue. Thus, a variety of evidence is potentially available in the resonance Raman spectra from which a fairly complete picture of the heme site can be assembled for a particular protein in its various functional states. Detailed studies have been pursued for paradigmatic heme proteins, including myoglobin, hemoglobin, cytochrome c, horseradish peroxidase, and cytochrome oxidase. These studies provide a substantial data base from which the exploration of lesser known systems can be launched. Another extension of current knowledge to new frontiers is in the time domain, since pulsed lasers now make it feasible to carry out time-resolved resonance Raman studies on heme protein reactions. Time-resolved resonance Raman spectroscopy is capable of elucidating the temporal evolution of heme structure and provides a link between heme chemistry and protein dynamics. This link is being elucidated for hemoglobin and cytochrome c, where specific heme intermediates have been identified following ligand photodissociation or electron transfer. PMID- 2998165 TI - Ultrasonic evaluation of scrotal swellings. AB - The diagnostic value of ultrasonography for 101 scrotal swellings of 94 patients was assessed. The 101 swellings showed sonographically 87 extratesticular, 11 testicular and 3 combined lesions. This sonography was concluded to be a satisfactory diagnostic aid for scrotal cystic lesions, testicular tumors and the localization of scrotal lesions. PMID- 2998164 TI - [The use of 99mTc-dimercaptosuccinic acid renoscintigraphy in the evaluation of differential renal function]. AB - We studied the total and differential renal function by 99mTc-dimercaptosuccinic acid (DMSA) renoscintigraphy and present a formula to estimate the renal depth for the Japanese and the attenuation coefficient which influenced renal uptake. Total renal uptake of 99mTc-DMSA correlated well with creatinine clearance and with the PSP test, and there was a close correlation between its relative uptake and relative function as determined by 99mTc-diethylenetriaminepentaacetic acid (DTPA) renography. Therefore differential renal function test with 99mTc-DMSA renoscintigraphy was found to have clinical utility. We also demonstrated 99mTc DMSA renoscintigraphy provided useful morphological information. PMID- 2998166 TI - [Percutaneous renal cyst puncture]. AB - Ultrasound-guided renal cyst puncture was performed on 31 cysts which were then 95% ethanol instilled to prevent recurrence of cystic fluid. Ethanol was allowed to remain in place for 20 minutes and removed through the catheter. Morphological improvement was observed on IVP and Tc-99m-DMSA renal scintigram, and DMSA renal uptake rate increased slightly. The cystic wall became thicker, and cystic fluid did not remain any more. The renal tissue near the cyst was intact. One third of the patients had hotflush and/or somewhat drunken sense but these symptoms were only temporary. This method of therapy is a safe and effective way of treating renal cysts. PMID- 2998167 TI - The effect of intramuscularly administered vitamin D3 on serum vitamin D metabolites and electrolytes in vitamin D3 deficient dairy cows. PMID- 2998168 TI - Evaluation of the prophylactic potential of an immunomodulator against respiratory disease in calves. PMID- 2998169 TI - Unusual thoracoabdominal sites of metastases in testicular tumors. AB - Testicular tumors spread in a predictable manner to lymph nodes in the paraaortic chain and then to supradiaphragmatic nodes in the mediastinum and supraclavicular fossae. The most common sites of extranodal disease are the lungs and liver. In a series of 650 patients with testicular tumors who underwent CT examinations unusual sites of thoracoabdominal metastases were demonstrated in 20 patients (23 sites). The sites involved were kidney (six patients), adrenal glands (four patients), inferior vena cava (four patients), muscle (three patients), spleen (two patients), and stomach, pelvic cyst, seminal vesicles and prostate, and pericardium (one patient, each site). Renal metastases were either of soft-tissue density (four patients) or cystic (two patients). Cystic metastases were also identified in the spleen in two patients and in the adrenal gland in one patient. In three of the patients with inferior vena cava involvement, thrombus was seen to extend above the nodal mass over several centimeters. Metastases were demonstrated in the psoas, iliac, and middle gluteal muscles. These were separate from retroperitoneal nodal masses. The ability of CT to identify sites of metastases, which may remain undiagnosed by conventional staging procedures, is emphasized. PMID- 2998170 TI - Diffuse testicular disease: sonographic features and significance. AB - Sonographic findings in 26 patients with diffuse testicular disease were retrospectively analyzed for features to differentiate diffuse neoplasms from diffuse orchitis or infarct. Diffuse neoplasms were associated with moderate to marked enlargement, globular shape, lobulated contour, and heterogeneous texture of the testis; the epididymis and scrotal skin were normal. In diffuse inflammatory disease, the testis was mild to moderately enlarged, the oval shape and smooth contour were preserved, and the texture was generally homogeneous; the epididymis was often enlarged, and scrotal skin was almost always thickened. PMID- 2998172 TI - Left ventricular thrombi in association with normal left ventricular wall motion in patients with malignancy. PMID- 2998171 TI - CT-surgical correlation in pituitary adenomas: evaluation in 113 patients. AB - A retrospective study was undertaken in 113 patients with surgically proven pituitary adenomas to correlate the frequency, type, and location of computed tomographic (CT) abnormalities with surgical findings. There were 63 prolactin secreting, 19 growth-hormone-secreting, 12 adrenocorticotropic-hormone-secreting, two thyroid-stimulating-hormone-secreting, and 17 nonfunctioning adenomas. The 51 functioning and nonfunctioning macroadenomas had similar CT appearances. Only 34 secretory adenomas presented as discrete, focal, hypodense lesions; the rest were isodense with the adjacent pituitary gland. Secretory adenomas were clinically apparent earlier, and accordingly the abnormalities seen on CT were less developed. The location of the normal pituitary gland could not be determined by attenuation characteristics; only by infundibulum displacement or opposite to a discrete, focal, hypodense lesion could the gland location be predicted reliably. Adenomas with hemorrhage, infarction, and cyst formation were indistinguishable from those without these findings. CT was helpful in identifying the mass effect of macroadenomas; however, in microadenomas of all types CT abnormalities were uncommon. Thus, the diagnostic evaluation of the patient suspected of harboring a pituitary adenoma, particularly a microadenoma, must remain a joint effort based on clinical, radiographic, and endocrinologic data. PMID- 2998174 TI - Digestion of the polysaccharides of some cereal foods in the human small intestine. AB - The digestion and absorption of dietary starch and nonstarch polysaccharides (NSP) in the small intestine of man from oats, cornflakes, and white bread has been determined by feeding seven ileostomists test meals containing these foods and estimating carbohydrate recovery in the effluent. NSP, the main constituent of dietary fiber, was almost completely recovered from all three test meals, including the water soluble beta-glucan which is the main NSP in oats. Less than 0.6% of the starch in oats was recovered as starch with a further 1.2% as dextrins and maltose. 4% of cornflake starch however was recovered, of which the main part was resistant to alpha-amylase digestion in vitro unless specially dispersed. Similarly with white bread 2.5% of ingested starch reached the terminal ileum of which the greater part was starch resistant to alpha-amylase in vitro. Overall 5.8% of the carbohydrate in white bread, 5.3% in cornflakes, and 11.7% in oats was recovered. This study supports the view that human digestive enzymes do not break down dietary NSP. It also identifies a fraction of starch, RS, present in processed food which resists breakdown by alpha-amylase both in vitro and in the small intestine of man. PMID- 2998173 TI - Glycoconjugate localization with lectin and PA-TCH-SP cytochemistry in rat hypophysis. AB - Glycoconjugates were localized by light microscopy with lectin-peroxidase conjugates and by electron microscopy with the periodic acid-thiocarbohydrazide silver proteinate (PA-TCH-SP) sequence in immunocytochemically or morphologically identified cell types in rat pituitary. Lectin histochemistry demonstrated sialic acid and glycoconjugates with N-glycosidically linked oligosaccharides in gonadotrophs, thyrotrophs, and corticotrophs. Galactose penultimate to sialic acid was observed mostly in gonadotrophs. The terminal galactose-N acetylgalactosamine disaccharide was detected in a few gonadotrophs and in a moderate number of mammotrophs. Fucose was localized in only corticotrophs with two fucose-binding lectins and in thyrotrophs with another. Several different monosaccharides were seen in glycoconjugates in melanotrophs and in Herring bodies. Melanotrophs displayed heterogeneous staining with fucose-binding lectins. A small number of nonsecretory cells were also visualized in the pars distalis by virtue of their glycogen content. PA-TCH-SP staining revealed complex carbohydrates in secretory granules and some Golgi cisternae in all types of hormone-producing cells in the pars distalis except for the somatotrophs. Melanotrophs of pars intermedia exhibited stained secretory granules and irregular dense bodies containing a stained meshwork. Corticotrophs of the pars distalis lacked the latter bodies, although they form the same glycoprotein precursor hormone as melanotrophs. Lectin conjugates and the PA-TCH-SP sequence stained some groups of secretion granules in Herring bodies, possibly representing vasopressin-containing granules as well as other cell types in the pars nervosa. PMID- 2998175 TI - Cereal dietary fiber consumption and diverticular disease: a lifespan study in rats. AB - The relationship between consumption of dietary fiber (DF) from white bread, wholemeal bread, or bran and the development of diverticular disease of the colon has been investigated in a lifespan study using 1800 Wistar rats in nine diet groups. Use of the rat as a model for the human condition was validated by demonstration of significant relationships between fiber intake and fecal output and transit time, and the observation of true acquired diverticula, both single and multiple. Significant inverse relationships (mostly with p less than 0.001) were observed between the incidence of diverticula (and prediverticula) and the concentrations of fiber in the diets, measured by the neutral detergent fiber and Southgate methods. The study offers strong support to the Painter-Burkitt view of human diverticular disease as being due to fiber deficiency, if the extrapolation from rat to man is valid. On the same assumption, the amount of additional fiber required to be consumed in order to achieve a substantial reduction in incidence of the disease is very large. Effects of fiber on body weight, food intake, mineral levels, blood composition and properties, mortality, organ weights, and incidence of tumors and lesions are reported. Significantly fewer mammary tumors were found in rats fed the very high fiber stock diet than in those fed the purified diets. PMID- 2998176 TI - Effects of nutrients and nonnutrients on food intake. AB - Protein, trypsin inhibitor, and fiber can alter food intake by several unique mechanisms, but there may also be some common mechanisms. For example, both poorly digested proteins and fibers like guar gum and pectin lead to an accumulation of material in the small intestine and appear to delay nutrient absorption both in terms of the time course of absorption and, within the intestine, delaying its rate of progress to more distal regions of the gut. These factors could contribute to the reduction in food intake associated with ingestion of these substances. PMID- 2998177 TI - Short course prophylactic cranial irradiation for small cell lung cancer. AB - Ninety-one patients with small cell carcinoma of the lung were given a shortened, intensive course of prophylactic cranial irradiation consisting of 2,000 rad in five fractions. The CNS relapse rate was 21%, but in only one of 91 patients was the brain the first and only site of relapse. Acute toxicities consisting of headache (16%) and nausea and vomiting (15%) were observed. Results are compared with previous results from other studies of cranial irradiation. PMID- 2998178 TI - Computed tomography assisted volumetric analysis of primary liver tumor as a measure of response to therapy. AB - Serial computed tomography assisted volumetric analyses were made in 33 patients with hepatoma. Thirteen of 27 patients with tumor volumes less than 2,290 cc had a partial response to experimental therapy. However, only three of these patients also demonstrated a significant change in liver volume. Tumor volume determinations made by the method described are an accurate (+/- 10%) and reproducible way to measure response to therapy. In spite of small changes in total liver volume, there may be concomitantly substantial changes in tumor volume. PMID- 2998179 TI - Reye's syndrome associated with adenovirus infections. PMID- 2998180 TI - Effect of race and diet on human-milk vitamin D and 25-hydroxyvitamin D. AB - Vitamin D-deficiency rickets continues to be reported in infants fed human milk, and the importance of human milk as a source of vitamin D for infants is controversial. Furthermore, effects of race and of normally consumed maternal vitamin D intake on human-milk vitamin D have not been reported. Milk, serum, and three-day-diet diaries were obtained from 25 mother-infant pairs. Human-milk vitamins D3 and D2 and 25-hydroxyvitamin D3 were lower in blacks vs whites, whereas 25-hydroxyvitamin D2 did not differ. Total-milk vitamin D, but not 25 hydroxyvitamin D, correlated with vitamin D intake. Milk vitamin D2 specifically was correlated with vitamin D intake even after controlling for race. Infant serum 25-hydroxyvitamin D did not correlate with milk vitamin D or 25 hydroxyvitamin D; we speculate that the contribution of vitamin D from human milk in these infants is insignificant relative to the contribution from sunshine exposure. PMID- 2998181 TI - The use of dietary fiber in the management of simple, childhood, idiopathic, recurrent, abdominal pain. Results in a prospective, double-blind, randomized, controlled trial. AB - Recurrent abdominal pain (RAP) affects 10% to 18% of school-age children and is caused by obvious organic pathology in fewer than 10% of cases. Two recent studies do not support previous beliefs that most RAP is psychogenic. Studies have shown disorders of bowel motility in children with RAP similar to those of adult irritable bowel syndrome (IBS); controlled trials of additional dietary fiber in adult IBS have shown beneficial results. We did a randomized, double blind, placebo-controlled study in 52 children with RAP and demonstrated a clinically and statistically significant decrease in pain attacks (at least 50% fewer) in almost twice as many children who were given additional fiber as placebo. Compliance was excellent in both groups and side effects were few. Although the cause of RAP is poorly understood, it is hypothesized that the beneficial effect of added fiber is due to its effect on shortening transit time, as in IBS. PMID- 2998183 TI - Concomitant inheritance of alpha-thalassemia in beta 0- thalassemia/Hb E disease. AB - Concomitant inheritance of alpha-thalassemia in patients with beta 0 thalassemia/hemoglobin (Hb) E disease was detected by restriction endonuclease DNA mapping. Among 42 patients with beta 0-thalassemia/Hb E disease, seven were found to have an alpha-thalassemia-2 haplotype. Of these, five belonged to the rightward or 3.7-kb type of alpha-thalassemia-2 and the remaining two the leftward or 4.2-kb type. All the seven patients with alpha-thalassemia-2 haplotype had hemoglobin levels of 7.4 g/dl or above; those without detectable alpha-thalassemia had hemoglobin levels both higher and lower than 7.4 g/dl. The latter attended the clinic regularly, the former did occasionally. These findings suggest that concomitant inheritance of alpha-thalassemia can alleviate the severity of beta 0-thalassemia/Hb E disease. Failure to find alpha-thalassemia-1 haplotype in these patients suggests that concomitant inheritance of alpha thalassemia-1 with beta 0-thalassemia/Hb E might lead to so mild a condition that the individuals do not present clinically. The fact that many patients without a detectable alpha-thalassemia haplotype also had hemoglobin levels of 7.4 g/dl or higher suggests that there are additional factors responsible for the mildness of beta 0-thalassemia/Hb E disease. PMID- 2998182 TI - Dietary factors and the incidence of cancer of the stomach. AB - A case-control study of diet and stomach cancer was conducted during 1979-1982 in Toronto, Winnipeg, and St. John's Canada. Two hundred forty-six histologically verified cancer cases were individually matched by age, sex, and area of residence to 246 randomly selected population controls. Daily nutrient consumption values were calculated from quantitative diet history questionnaire data through use of the US Department of Agriculture Food Composition Data Bank, which was extended and modified for Canadian items. For the analysis, continuous conditional logistic regression methods were used. It was found that consumption of dietary fiber was associated with decreased risk of gastric cancer; the odds ratio estimate of trend was 0.40/10 g average daily intake of fiber (i.e., 0.40(1.5)/15 g, etc.) (p less than 10(-8)). Also, average daily consumption of nitrite, chocolate, and carbohydrate was associated with increasing trends in risk, with odds ratio estimates, respectively, 2.6/mg (p less than 10(-4)), 1.8/10 g (p less than 10(-4)), and 1.5/100 g (p = 0.015). While citrus fruit intake appeared to be somewhat protective (odds ratio = 0.75/100 g daily average, p = 0.0056), vitamin C intake was less so, and vitamin E not at all. Thus, a number of dietary components seem to be implicated in the pathogenesis of stomach cancer. PMID- 2998184 TI - Human polymorphonuclear leukocytes of the bone marrow, circulation, and marginated pool: function and granule protein content. AB - Polymorphonuclear leukocytes (PMN) demonstrate altered function during acute infections and after administration of corticosteroids. We questioned whether or not such changes are due to population shifts from functionally different compartments of the granulocyte pool. Volunteers were given epinephrine to induce demargination or hydrocortisone (HC) to promote egress of PMN from the bone marrow. PMN obtained before and after drug administration were compared for adherence, chemotaxis, luminol-enhanced chemiluminescence, and total content and release of lactoferrin (LF), myeloperoxidase (MPO), and beta-glucuronidase (beta glu). Epinephrine induced a significant neutrophilia of mature PMN (segmented neutrophils), but there were no changes in function or granule protein content. HC induced a significant neutrophilia with segmented neutrophils and immature PMN (bands). Circulating PMN obtained 4 hr after HC administration demonstrated less adherence, increased chemiluminescence, increased MPO release, and decreased MPO content. Band neutrophils, however, were more adherent than segmented PMN and showed a similar decrease in adherence following HC in vivo. Thus alteration of PMN adherence following intravenous corticosteroids is not due to an influx of immature neutrophils. On the other hand, it is possible that MPO content and release and capacity for oxidative metabolism change as PMN mature. PMID- 2998185 TI - Transient Passovoy defect during a febrile illness. AB - The Passovoy defect is a recently characterized hemorrhagic diathesis. We describe a patient with a febrile illness, possibly from Epstein-Barr (EB) virus, who acquired this defect transiently. Prothrombin time; assays for factors VIII, IX, XI, XII; and Fletcher (prekallikrein) and Fitzgerald (high molecular weight kininogen) factors were normal. No definite circulating inhibitor could be demonstrated. The transient Passovoy defect could possibly be ascribed to the infectious process or sulfisoxazole, which the patient had received. PMID- 2998186 TI - The natural history of familial hypopituitarism. AB - Familial hypopituitarism in the Hutterite Brethren is an autosomal recessive disorder involving sequential loss of anterior pituitary tropic hormones. Five individuals from two closely related families have been followed for 19 years. Both families are well integrated into the Hutterite community. Three sibs elected not to be treated with growth hormone and sex steroids. These sibs developed growth hormone and gonadotropin deficiency in the first decade of life, with subsequent loss of TSH function and finally ACTH deficiency in the third decade. The pattern of hormone loss differed in the second family, in that deficiency of growth hormone, gonadotropins, and TSH was evident in the first decade. A third family has been reported to have the same disorder and is from a different endogamous subdivision from that of the two families described here. Genealogical analysis of the three families shows that there are four ancestral couples common to the six parents. Thus all affected individuals are likely to be identical by descent for the same ancestral allele. The gene for hypopituitarism is not closely linked to the gene for growth hormone nor to the HLA region. PMID- 2998187 TI - Microcephaly, hypergonadotropic hypogonadism, short stature, and minor anomalies: a new syndrome. AB - Four sibs, three males and one female, had microcephaly, hypergonadotropic hypogonadism, short stature, and multiple congenital anomalies. They had five normal sibs and consanguineous parents. Findings in the affected sibs also included a narrow forehead, synophrys, micrognathia, abnormally folded pinnae, early loss of teeth in three, cubitus valgus in two, genu valgum, gynecomastia, and undescended testes in one. All sibs had normal chromosomes. Results of tests for growth hormone release and adrenocortical function were normal. Luteinizing hormone releasing hormone (LHRH) and human chorionic gonadotropin (hCG) stimulation tests were consistent with primary gonadal failure. Testicular biopsy, performed on two affected males, was normal in one and showed focal atrophy with decreased spermatogenesis in the other. The patients manifest a phenotype different from all other known types of hypergonadotropic hypogonadism and appear to represent a new MCA/MR syndrome. PMID- 2998188 TI - Bartter's syndrome: a unifying hypothesis. AB - The most proximate defect responsible for the pathogenesis of Bartter's syndrome remains uncertain. Although an abnormality in chloride reabsorption in the thick ascending limb of Henle has been postulated, renal clearance studies performed during oral water loading failed to disclose a reduction in fractional chloride reabsorption. We alternatively postulate that the underlying abnormality may reside in a generalized increase in cell sodium permeability. Elevated levels of cell sodium may secondarily stimulate Na-K-ATPase activity. In the cells of the distal nephron, stimulated Na-K-ATPase would lead to enhanced potassium secretion into the tubular fluid producing the characteristic potassium depletion. In addition, increased cell sodium influx may stimulate a sodium-calcium exchanger. If this process exists in vascular smooth muscle, it may result in reduction of cytosolic calcium activity. This effect and/or chronic potassium depletion may mediate the reduced vascular reactivity characteristic of this syndrome. PMID- 2998189 TI - Glomerular basement membrane splitting and microaneurysm formation associated with nitrosourea therapy. AB - A patient who developed renal insufficiency following nitrosourea therapy is reported. Light, immunohistochemical, and electron microscopic studies of the renal biopsy disclosed an unusual glomerular basement membrane injury. Light microscopy showed extensive basement membrane splitting and capillary aneurysm formation. Electron microscopic examination revealed an extensive subendothelial accumulation of electron-lucent granular material. The glomerular basement membrane was separated from the mesangium and showed splitting of the lamina densa. Immunofluorescent and immunoperoxidase staining of the glomeruli was negative for immunoglobulin, complement, and fibrinogen. This form of nitrosourea associated glomerular injury has not been described previously. PMID- 2998190 TI - Genital warts and cervical cancer. VII. An improved colposcopic index for differentiating benign papillomaviral infections from high-grade cervical intraepithelial neoplasia. AB - A new colposcopic sign (sharpness of peripheral margins) was graded into three objective categories representing subclinical papillomaviral infection, lower grade dysplasia, and grade 3 cervical intraepithelial neoplasia. Colposcopic features were prospectively recorded in 72 women and then correlated with histologic findings. Histologic diagnoses were evenly spread within the disease spectrum: 18 patients had subclinical papillomaviral infection without associated dysplasia; 15 had grade 1, 16 had grade 2, and 23 had grade 3 cervical intraepithelial neoplasia with or without koilocytotic atypia. Differences in the pattern of the peripheral margin were discriminatory throughout the entire diagnostic range. Predictive accuracy of this new colposcopic sign (79%) compared favorably with that of color (72%), vascular atypia (81%), and iodine staining (72%). Each criterion was independent of the other three. Hence, combining these four individual signs into a colposcopic index was 97% correct in forecasting approximate histologic findings. Because formulation of the colposcopic index is based on critical analysis rather than pattern recall, the use of this method will greatly simplify the otherwise arduous task of learning colposcopy. PMID- 2998191 TI - The effect of terodiline treatment in women with motor urge incontinence. Results from a double-blind study and long-term treatment. AB - The effects of terodiline were evaluated in 24 women with genuine motor urge incontinence: Twelve patients participated in a double-blind crossover study and 12 in a subsequent long-term study. All patients were investigated by simultaneous urethrocystometry before and after treatment. During the controlled study the subjects were treated with placebo or 37.5 mg of terodiline daily for two 3-week periods. The long-term study covered a period from 6 months to 3 years. A significantly higher increase in both bladder volume at urinary leakage (from 170 to 270 ml, p less than 0.001) and bladder capacity (from 320 to 390 ml, p less than 0.01) was registered after terodiline treatment compared to placebo. In the long-term study the effects on these parameters were still more pronounced with an increase in bladder volume at leakage (from 180 to 300 ml, p less than 0.001) and in bladder capacity (from 290 to 430 ml, p less than 0.0001). Subjective improvement with terodiline treatment was reported by all but two patients in the double-blind study and by all in the long-term study. Side effects such as dryness of the mouth were reported by four patients receiving terodiline in the double-blind study and by six in the long-term study. No patient discontinued the treatment. Terodiline seems to be a promising alternative for treatment of motor urge incontinence in women. PMID- 2998192 TI - The in vitro growth and characterization of the skeletal muscle component of Wilms' tumor. AB - Skeletal muscle differentiation within a Wilms' tumor is a well-documented histopathologic entity thought to occur at a relatively low incidence and influence prognosis. A serum-free hormonally defined growth medium has been developed, allowing the long-term growth of the skeletal muscle component of Wilms' tumors. Eight Wilms' tumors have been grown under these conditions. Three cases grew a homogeneous population of cells which ultrastructurally displayed all stages of myogenesis through myotubule formation. They also possessed immunoreactivity for skeletal muscle myosin and myoglobin and synthesized the M and B subunits of creatine kinase. Of interest was the finding that the ability to yield skeletal muscle cultures was limited to those cases which exhibited skeletal muscle fibers in vivo. This technique is also a very sensitive marker for identifying Wilms' tumors possessing a myoid component. A second serum-free hormonally defined medium has also been developed that supports the long-term culture of a unique cell type from Wilms' tumors which contain a myoid component. These cells are spindle-shaped and exhibit all of the characteristics of early myoblasts. PMID- 2998194 TI - Inflammation induced by bacterial cell wall fragments in the rat air pouch. Comparison of rat strains and measurement of arachidonic acid metabolites. AB - Streptococcal cell wall fragments, suspended in phosphate-buffered saline, were injected into a preformed subcutaneous air pouch in rats. The advantage of the air pouch model is the capacity for quantitation of exudative, cellular, and proliferative responses and soluble mediators. Accumulation of pouch fluid containing many leukocytes occurred during the first 3 days. Granulation tissue separable from the surrounding subcutaneous tissue developed by 6 days. Immunofluorescence and immunoperoxidase staining showed the presence of cell walls in inflammatory cells both in pouch fluid and in pouch tissue. Histologic features of this inflammation included an acute exudative phase with a predominantly neutrophil infiltration followed by a chronic phase characterized by fibroblast proliferation, formation of blood vessels, and infiltration with mononuclear cells. The lining of the pouch before injection of cell wall developed morphologic features of synovial membrane, which became more evident during the chronic phase of induced inflammation. Outbred Sprague-Dawley and inbred Lewis rats developed more pouch fluid, cell numbers in the pouch fluid, and granulation tissue than inbred Buffalo rats. The arachidonic acid metabolites, prostaglandin E2 and leukotriene B4, were measured in the pouch fluid, and more of each was produced in the Lewis than in the Buffalo strain. These measurements of inflammation are consistent with the relative susceptibility of these strains to cell-wall-induced arthritis. This model of inflammation can be used in the examination of the regulatory mechanisms of evolving chronic inflammation. PMID- 2998193 TI - Role of alpha 1-adrenoceptors in norepinephrine-induced cardiomyopathy. AB - This study practically delineated the contribution of alpha-adrenoceptor activation to the pathogenesis of norepinephrine (NE) cardiomyopathy. A total of 64 adult New Zealand white rabbits were used. NE cardiomyopathy was produced in rabbits by a 90-minute intravenous infusion of norepinephrine (2 micrograms/kg/min at infusion rate 0.382 ml/min). Arterial blood pressure and heart rate were constantly monitored. Arterial blood samples were obtained at 30 minute intervals for measurements of pH, blood gases, and glucose. Alpha adrenoceptor blocking agents, when employed, were given 15 minutes prior to the initiation of NE infusion. Two days after treatment the rabbits were killed. The hearts were examined microscopically and assigned a histologic score. Pretreatment with the alpha 1-adrenoceptor blocker prazosin at 50, 100, or 200 micrograms/kg significantly reduced NE-induced myocardial injury in a dose related manner. In contrast, the presence of alpha 2-adrenoceptor blocker yohimbine at 2.5 or 5.0 mg/kg was ineffective in preventing the formation of myocardial lesions. These findings suggest that NE cardiomyopathy may result largely from activation of the alpha 1-adrenoceptor system in the rabbit model. PMID- 2998195 TI - Multiparameter analyses of spontaneous nonthymic lymphomas occurring in NFS/N mice congenic for ecotropic murine leukemia viruses. AB - Mouse strains congenic for ecotropic retrovirus genes have a much higher frequency of spontaneous lymphomas than the background NFS/N strain. In this study, most of these lymphomas have been identified as B-cell in origin by morphologic features, identification of immunoglobulin class, and cell-surface antigens. The classification suggested by Pattengale and Taylor proved to be applicable to the lymphomas studied. Most were of large follicular center cells and are considered typical of the type formerly designated as "reticulum cell sarcoma, type B." Many lymphomas contained a large proportion of nonneoplastic cells which partially obscured their neoplastic component. The role of ecotropic murine leukemia viruses as etiologic agents for B-cell lymphomas remains equivocal. However, because the only difference between the NFS/N and congenic mice is the expression of viruses in the latter, it appears that these viruses are somehow involved in induction of B-cell lymphomas. PMID- 2998196 TI - Dramatic founder effects in Amerindian mitochondrial DNAs. AB - Southwestern American Indian (Amerindian) mitochondrial DNAs (mtDNAs) were analyzed with restriction endonucleases and found to contain Asian restriction fragment length polymorphisms (RFLPs) but at frequencies very different from those found in Asia. One rare Asian HincII RFLP was found in 40% of the Amerindians. Several mtDNAs were discovered which have not yet been observed on other continents and different tribes were found to have distinctive mtDNAs. Since the mtDNA is inherited exclusively through the maternal lineage, these results suggest that Amerindian tribes were founded by small numbers of female lineages and that new mutations have been fixed in these lineages since their separation from Asia. PMID- 2998198 TI - Atrial natriuretic factor inhibits Na-K-Cl cotransport in teleost intestine. AB - Addition of atrial natriuretic factor (ANF) to the contraluminal side of the intestinal mucosa of a marine teleost, the winter flounder Pseudopleuronectes americanus, inhibits short-circuit current, net transepithelial fluxes of Na and Cl, and the unidirectional influx of Rb across the brush border membrane. This action of ANF is closely mimicked by addition of 8-bromo-guanosine 3',5'-cyclic monophosphate (8-BrcGMP). In contrast to the intestine, the opercular epithelium of the flounder did not respond to the in vitro addition of either ANF or 8 BrcGMP. Because intestinal salt and water absorption diminishes when marine fish enter water of lower salinity, ANF may be an important hormonal regulator through which euryhaline fish adapt to varying salinities. PMID- 2998197 TI - ATP concentration gradients in cytosol of liver cells during hypoxia. AB - The activities of two ATP-requiring systems with different subcellular localizations were studied in cells in which average cellular ATP concentration was varied. The cytosolic ATP-sulfurylase activity varied linearly with the cellular ATP concentration; however, the plasma membrane Na+-K+-ATPase was substantially more sensitive to decreased ATP concentration. Under conditions where the cellular ATP concentration was lowered to 40% of control, Rb+ uptake was nearly zero. The results indicate that ATP-utilizing enzymes located in the plasma membrane in liver cells are exposed to a lower ATP concentration than are enzymes present in the cytosolic fluid surrounding the mitochondria. Analysis of radial diffusion of ATP from mitochondria, assuming that mitochondria are spherical and ATP consumption is zero order in ATP concentration, shows that the average ATP supply radius decreases as mitochondrial ATP production decreases. Hence, during limited ATP supply, enzymes with a greater average distance from mitochondria (e.g., plasma membrane ATPases) experience a more dramatic decrease in ATP concentration than enzymes in close proximity to mitochondria (e.g., cytoplasmic enzymes). Thus microheterogeneity of ATP supply can occur in cells without membranal compartmentation due to nonuniform distribution of ATP generating and ATP-consuming systems. PMID- 2998199 TI - Effect of pentobarbital on fructose 2,6-bisphosphate metabolism in isolated rat hepatocytes. AB - Addition of the commonly used anesthetic pentobarbital to hepatocytes from fed rats resulted in a dose-dependent decrease in the level of fructose 2,6 bisphosphate. At a concentration of pentobarbital (0.4 mM) that lowered fructose 2,6-bisphosphate by 60%, there was no significant change in the level of fructose 6-phosphate, ATP, or L-glycerol 3-phosphate. Higher concentrations of pentobarbital (2 mM) enhanced both glycolysis and glycogenolysis and fructose 2,6 bisphosphate levels were reduced to less than 10% of the control. Concomitant with these changes there was a decrease in ATP, glucose 6-phosphate, and fructose 6-phosphate and a two- and fivefold increase in ADP and AMP, respectively. In hepatocytes from starved rats pentobarbital also lowered ATP levels and inhibited gluconeogenesis but had no effect on either lactate production or the already low level of sugar diphosphate. However, in the fasted case pentobarbital completely prevented the 10-fold elevation of fructose 2,6-bisphosphate brought about by 30 mM glucose. The anesthetic had no effect on cAMP-dependent protein kinase activity or on pyruvate kinase activity in hepatocytes from fed or starved rats but caused reciprocal changes in the activities of the bifunctional enzyme 6 phosphofructo-2-kinase/fructose 2,6-bisphosphatase. Kinase activity was decreased and bisphosphatase activity was increased. These results suggest that the effects of pentobarbital on gluconeogenesis and glycolysis are due to inhibition of energy metabolism with elevated AMP levels causing activation of 6-phosphofructo 1-kinase and inhibition of fructose 1,6-bisphosphatase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2998200 TI - Morphine initiates migrating myoelectric complexes by acting on peripheral opioid receptors. AB - The role of peripheral and central opioid receptors in morphine-induced migrating myoelectric complexes (MMECs) was studied in conscious dogs implanted with silver silver chloride electrodes. In normal fasted dogs morphine (100-200 micrograms/kg iv) initiated phase III of the MMEC in the duodenum. Once initiated the MMEC propagated distally. This effect of morphine was blocked by the opioid receptor antagonists naloxone (2 mg/kg iv) and N,N-diallylnormorphinium bromide (4 mg/kg iv). Higher doses of morphine (300-600 micrograms/kg iv) initiated phase III activity in fed dogs as early as 20 min after feeding, while lower doses (150 micrograms/kg iv) initiated phase III activity routinely when administered 100 min after feeding. In dogs with bilateral vagotomies and bilateral thoracolumbar sympathetic chain ganglionectomies, morphine (150 micrograms/kg iv) initiated phase III activity in the duodenum, which then migrated distally. This study demonstrates that morphine initiates phase III of the MMEC by acting through peripheral opioid receptors. PMID- 2998201 TI - Prostaglandin-induced pepsinogen secretion from dispersed gastric glands from guinea pig stomach. AB - We examined the actions of several prostaglandins (PG) on pepsinogen secretion from dispersed gastric glands prepared from guinea pig stomach. Pepsinogen secretion was stimulated by PGA1, PGA2, PGB2, PGE1, and PGE2. PGE1 and PGE2 were 10 times more potent than PGA1 and PGA2. Reducing the incubation temperature from 37 degrees to 4 degrees C or adding carbonyl cyanide m-chlorophenylhydrazone reduced PG-stimulated pepsinogen secretion. PGE2-induced pepsinogen secretion was not altered by atropine or dibutyryl cGMP. Potentiation of pepsinogen secretion occurred with PGE2 plus carbachol or the calcium ionophore A23187 but not with PGE2 plus secretin or 8-bromo-cAMP. Isobutylmethylxanthine increased the potency and the efficacy of the action of PGE2 on pepsinogen secretion. These results indicate that PGE1, PGA2, PGB2, PGE1, and PGE2 can modulate pepsinogen secretion from dispersed gastric glands from guinea pig stomach. Moreover, potentiation of pepsinogen secretion occurs when PGE2 is combined with secretagogues whose actions appear to be mediated by changes in cellular calcium (carbachol and A23187) but not with secretagogues whose actions appear to be mediated by changes in cellular cAMP (secretin and 8-bromo-cAMP). These data suggest that PG-induced pepsinogen secretion may be mediated by changes in cellular cAMP. PMID- 2998202 TI - Metabolic effects of sodium bicarbonate in hypoxic lactic acidosis in dogs. AB - The metabolic effects of NaHCO3 therapy in hypoxic lactic acidosis were evaluated in the anesthetized dog. Hypoxic lactic acidosis was induced by ventilating the dogs with a hypoxic gas mixture of 8% O2/92%N2, resulting in arterial PO2 of less than 30 mmHg, pH below 7.20, bicarbonate less than 12 mM, and lactate more than 7 mM. In this situation lactate accumulates because of overproduction of lactate by gut and carcass in the presence of a diminished capacity of the liver to extract lactate. After the development of hypoxic lactic acidosis the dogs were treated for 60 min with either NaHCO3 or NaCl or had no therapy. Sixty minutes of either treatment resulted in further declines of blood pH and bicarbonate that were similar in all three groups. NaHCO3-treated animals, however, showed an increase in blood lactate that were significantly higher than those treated with NaCl or those that had no therapy. This could be explained by a significantly higher gut lactate production with NaHCO3 therapy than in the NaCl-treated group. Concomitantly NaHCO3-treated animals showed a decrement in liver and gut blood flow that did not occur with NaCl treatment. Only NaHCO3 therapy was associated with a further decrease of liver intracellular pH, which could be attributed to both an increase in the CO2 load to the liver and increased tissue lactate levels, which were not observed with NaCl or no therapy. Additionally, liver lactate extraction was not improved by administration of NaHCO3 or NaCl.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2998203 TI - Mechanism of bradykinin, ADH, and cAMP interaction in rabbit cortical collecting duct. AB - Vasopressin (ADH) and bradykinin (BK) have been shown to stimulate prostaglandin synthesis in rabbit cortical collecting tubules. We studied ADH and BK effects on osmotic water flow (Lp), Na transport (JNa), and transepithelial voltage (VT). Bath BK but not lumen BK blunted subsequent ADH hydroosmotic responses. This BK effect was prevented by ibuprofen or pertussigen pretreatment and was overcome with exogenous cAMP, suggesting that BK, via prostaglandins, interferes with ADH action on Lp at the cAMP generation step. In contrast, bath BK had no effect on bath-to-lumen (Jb-1Na) or lumen-to-bath (Jl-bNa) Na flux or on VT. As reported by others, ADH lowered Jl-bNa and depolarized VT; however, prostaglandin synthesis inhibitors neither prevented nor reversed these ADH effects. Together, these BK and ADH data do not support regulation of JNa by peptide-stimulated prostaglandins. Moreover, cAMP alone depolarized VT but had no effect on Jl-bNa. Therefore, ADH-induced depolarization of VT may at least partly owe to cAMP effects on VT independent of accompanying changes in JNa. As with Lp, bath BK blunted subsequent ADH effects on VT and, to a lesser extent, Jl-bNa; these BK effects on ADH action were also prevented by ibuprofen or pertussigen pretreatment. The data are consistent with the following model: 1) ADH depolarizes VT and increases Lp via cAMP; 2) ADH decreases JNa via neither cAMP nor prostaglandins; and 3) BK, via prostaglandins, inhibits the actions of ADH on Lp and VT at the inhibitory guanyl-nucleotide regulatory subunit of adenylate cyclase. PMID- 2998204 TI - Ontogeny of Na-K-ATPase activity in thick ascending limb and of concentrating capacity. AB - Na-K-ATPase activity in the thick ascending limb of Henle (TAL) and the capacity to concentrate urine was determined in 12-, 16-, 20-, 30-, and 40-day-old rats. The most pronounced increase in enzyme activity occurred between 16 and 20 days of age. The relation between enzyme activity in the cortical and medullary TAL was found to be the same in 16-, 20-, and 40-day-old rats, and most enzyme determinations were made in the medullary TAL. The development of Na-K-ATPase activity in TAL and urinary concentrating capacity followed the same pattern. The developmental increase in Na-K-ATPase activity and concentrating capacity between 16 and 20 days of age was accompanied by an increase in serum corticosterone level and was abolished by adrenalectomy. Treatment with glucocorticoid hormones precociously induced Na-K-ATPase activity and concentrating capacity in 13- to 16 day-old rats but had no effect on Na-K-ATPase activity in 17- to 20-day-old rats. The increase in enzyme activity from 20 to 40 days of age was accompanied by an increase in the single nephron glomerular filtration rate. The results suggest that Na-K-ATPase activity in the TAL is an important determinant of the concentrating capacity during development. The developmental surge in Na-K-ATPase activity and concentrating capacity between 16 and 20 days of age is probably set off by the rise in the serum corticosterone level. PMID- 2998205 TI - Characteristics of water diffusion in the rabbit proximal convoluted tubule. AB - Water diffusion in the in vitro microperfused rabbit proximal convoluted tubule was measured to assess the transepithelial pathway for water movement and the physical characteristics of the water permeation pathway. The measured diffusive water permeability (PDW(MEASURED] was high, 5 X 10(-3) cm/s, suggesting a transcellular transport route. Simultaneously measured n-butanol permeability (PDNB(MEASURED], an index of the cytoplasmic resistance to water diffusion, was 3.6 X 10(-3) cm/s. PDW(MEASURED], PDNB(MEASURED) and the free solution diffusion coefficients for water and n-butanol were used to derive a minimum membrane PDW (PDW(MEMBRANE] of 12.5 X 10(-3) cm/s. These data suggest that at least 60% of the transepithelial resistance to water diffusion resides in the cell cytoplasm. Measurement of the temperature dependence of PDW(MEASURED) and PDNB(MEASURED) gave an apparent activation energy of PDW(MEMBRANE) that was constant between 20 and 40 degrees C at about 4.3 kcal/mol. The organic mercurial sulfhydryl reagent p-chloromercuribenzene sulfonate, which has been shown to reduce diffusive water permeability in the red cell membrane by 50%, reduced PDW(MEMBRANE) by 54% without affecting PDNB(MEASURED). These last two independent lines of evidence are consistent with water diffusion through small, aqueous, protein-bounded channels. PMID- 2998206 TI - Ammonium replaces potassium in supporting sodium transport by the Na-K-ATPase of renal proximal straight tubules. AB - Ammonium is capable of replacing potassium to support the hydrolysis of ATP by the Na-K-ATPase in many tissues. Whether ammonium supports the transport function of the Na-K-ATPase in the kidney (where such a substitution could be physiologically important) has not been studied, however. To address this issue, we determined the rates of fluid absorption and bicarbonate absorption (measured as total carbon dioxide) in the proximal straight tubule of the rabbit where both processes are dependent on sodium transport by the Na-K-ATPase. Both fluid absorption and total carbon dioxide absorption were significantly inhibited by potassium removal from the bath and perfusate. When ammonium was included in the perfusate and bath (replacing potassium completely), both fluid absorption and bicarbonate absorption occurred at rates indistinguishable from the rates observed with potassium present (and ammonium absent). We conclude that ammonium can replace potassium in supporting sodium transport by the Na-K-ATPase of the proximal straight tubule. Interaction between ammonium and potassium on the Na-K ATPase could be important in this and other nephron segments. PMID- 2998207 TI - A uniform enzymatic method for dissociation of myocytes from hearts and stomachs of vertebrates. AB - A method is presented that consistently yields a large number of calcium-tolerant myocytes from mammalian, amphibian, and elasmobranch hearts and from mammalian stomach. The use of incubating solutions or cell harvesting techniques was not required. The time needed to isolate cells was shorter than previously reported values. Action potentials recorded from each cell type appear similar in configuration to that of the intact multicellular tissue. The isolated myocytes appear to tolerate long periods of electrophysiological experimentation using the "giga-seal" suction electrode technique of Hamill et al. (Pfluegers Arch. 391: 85 100, 1981). This method is ideally suited for comparative electrophysiological studies, since the procedure for cell isolation was not seriously modified according to the preparation or species used. PMID- 2998208 TI - Contribution of myocardial diffuse double-layer calcium to contractile function. AB - The role of diffuse double-layer calcium in cardiac excitation-contraction coupling was examined using rabbit interventricular septa, cultured neonatal rat myocardial cells, and gas-dissected sarcolemmal membranes. The diffuse double layer refers to the space directly adjacent to the sarcolemma where the ionic composition of the media is a direct function of the membrane surface potential. The divalent cation dimethonium was used as a specific probe for the diffuse double layer. According to Gouy-Chapman theory, replacement of sodium with sucrose should increase the amount of calcium located in this compartment. Dimethonium (10 mM) was found to decrease calcium uptake and contractility during low-sodium (33 mM) perfusion when the perfusate contained sucrose but not LiCl. Dimethonium did not decrease calcium uptake or contractility during control perfusion. The results suggest that calcium present in the myocardial diffuse double layer can be augmented or reduced in accordance with Gouy-Chapman theory. Changes in diffuse double-layer calcium are accompanied by small (7.8%) but significant changes in contractility. PMID- 2998209 TI - Hypothalamic obesity after hypophysectomy or adrenalectomy: dependence on corticosterone. AB - Recent studies have found that the hyperphagia and obesity resulting from lesions of the ventromedial hypothalamus (VMH) are both reversed and prevented by complete adrenalectomy. Several previous experiments, however, reported little or no suppression of VMH weight gain in hypophysectomized (HYPOX) rats. This study directly compared the effects of hypophysectomy and adrenalectomy on hypothalamic obesity in adult female rats. Complete adrenalectomy (i.e, stress-induced plasma corticosterone less than 1.0 micrograms/dl) totally suppressed abnormal weight gain in the first 20 days after VMH lesions but did not affect intracranial self stimulation. Hypophysectomy also resulted in suppression of weight gain, but the HYPOX-VMH rats nevertheless gained significantly more weight than HYPOX rats with sham lesions. However, the HYPOX-VMH animals had very low levels of plasma corticosterone and adrenocorticotropin (ACTH) (from residual pituitary tissue or of diencephalic origin), and incompletely adrenalectomized rats with similar low levels of plasma corticosterone gained an equal amount of weight after VMH lesions. It was concluded that adrenal glucocorticoid hormones play a largely permissive role in the VMH syndrome, with only very small levels required for the manifestation of obesity. PMID- 2998210 TI - Corticosterone: narrow range required for normal body and thymus weight and ACTH. AB - ACTH secretion appears to be under fairly tight negative feedback control by corticosteroids secreted from the adrenal cortex. In these studies we determined the circulating levels of a constant corticosterone signal that best restored body weight gain, thymus weight and ACTH levels to normal in bilaterally adrenalectomized rats given saline to drink. Young male rats were treated at the time of adrenalectomy with subcutaneously implanted pellets of wax or various ratios of corticosterone-cholesterol. Sham-adrenalectomized rats and adrenalectomized rats given corticosterone in the drinking fluid served as comparison groups. Rats were killed 3, 7, or 14 days after adrenalectomy. There was no difference in levels of plasma corticosterone in the morning and in the evening in pellet-implanted rats in contrast to the diurnal variation in the reference groups. Circulating corticosterone levels that best restored body weight, thymus weight, and resting and stress-induced ACTH levels to normal ranged between 4.5 and 7.4 micrograms/dl. Plasma corticosterone levels of 8-11 micrograms/dl were excessive and levels of 2-4 micrograms/dl were not adequate. We conclude that there is a very narrow range of plasma corticosterone compatible with normal growth rate, thymus mass and ACTH secretion. These results reveal the necessity for strict negative feedback regulation of ACTH secretion by corticosteroids. PMID- 2998211 TI - Transplacental diffusion and blood flow of gravid bovine uterus. AB - Electromagnetic blood flow transducers and uterine arterial, uterine venous, umbilical venous, fetal femoral arterial, and fetal femoral venous catheters were implanted in 11 cows on day 161 +/- 4 of gestation. Antipyrine (0.66 M) plus NaCl (0.16 M) dissolved in deuterium oxide (D2O), or H2O, was infused at a constant rate into the fetal femoral vein catheter. Concentrations of antipyrine and D2O in uterine arterial and venous blood and antipyrine in fetal arterial and umbilical venous blood, as well as middle uterine arterial blood flow (electromagnetic transducer), were determined. Antipyrine and D2O gave similar estimates (steady-state diffusion method) of gravid uterine blood flow. In addition, the slope of the regression of D2O on antipyrine estimates was not different (P greater than 0.10) from one. Electromagnetic transducers gave estimates of uterine blood flow that were 32-42% of those obtained with steady state diffusion but were correlated (P less than 0.05) with estimates obtained by use of both antipyrine and D2O. The transplacental clearance rate of antipyrine was similar (per kg placenta) to that observed in ewes. It was suggested that the maternal and fetal microvasculatures of the bovine placenta could have a concurrent arrangement with vascular shunts or maldistribution of flows, as has been suggested for the ewe. PMID- 2998212 TI - Acute volume expansion decreases adrenocortical sensitivity to ACTH and angiotensin II. AB - This study examined the plasma aldosterone and corticosteroid responses to a 60 min infusion of adrenocorticotropin (ACTH) or angiotensin (ANG) II started immediately after an acute isotonic saline volume expansion (0.5 ml . kg-1 . min 1 for 30 min). Five conscious dogs of either sex with exteriorized carotid loops were used in this repeated-design study. Volume expansion per se caused a 10% decrease in hematocrit, a 12.5% decrease in plasma protein, and a 2.7-mmHg increase in central venous pressure with no change in mean arterial pressure, heart rate, or plasma sodium. Volume expansion per se also resulted in significant reductions in vasopressin, plasma renin activity, ACTH, aldosterone, and corticosteroid levels. The aldosterone responses to ACTH and ANG II were significantly inhibited (46-71%) by acute volume expansion. The corticosteroid response to ACTH was 19-29% inhibited by volume expansion. We conclude that acute volume expansion significantly inhibits the adrenocortical sensitivity to its tropic hormones probably via alterations of synergistic factors. PMID- 2998214 TI - Juvenile (cellular) adenofibromas. A clinicopathologic study. AB - "Juvenile" adenofibromas that presented in 25 patients were reviewed. All of the patients were in the second decade of life. The tumors were solitary in 19 patients and multiple and bilateral in six patients. All were distinguished microscopically by prominent cellularity of both epithelium and stroma. Patients who presented with solitary tumors, regardless of size, microscopic pattern, or manner of excision, had no recurrence. In contrast, all patients who presented with multiple tumors developed additional benign masses, often requiring re excision. We believe that solitary "juvenile adenofibromas," regardless of size, should be excised so as to preserve as much breast tissue as possible. Those patients with multiple, bilateral tumors may anticipate recurrences, but malignant change is not seen. Tumors with this microscopic pattern also may occur, albeit uncommonly, in adults. PMID- 2998213 TI - Carry-over effects of marijuana intoxication on aircraft pilot performance: a preliminary report. AB - Ten experienced licensed private pilots were trained for 8 hours on a flight simulator landing task. They each smoked a cigarette containing 19 mg of delta 9 tetrahydrocannabinol (THC), and 24 hours later their mean performance on the flight task showed trends toward impairment on all variables, with significant impairment in number and size of aileron changes, size of elevator changes, distance off center on landing, and vertical and lateral deviation on approach to landing. Despite these deficits, the pilots reported no awareness of impaired performance. These results may have implications for performance of complex tasks the day after smoking marijuana. PMID- 2998215 TI - Beneficial effects of cyclosporine compared with azathioprine in cadaveric renal transplantation. AB - Recent reports have intimated that the use of antilymphocyte globulin in combination with azathioprine and steroids has ameliorated the beneficial affects of cyclosporine. We believe that even in the absence of significant statistical differences between patient survival rates and graft survival rates of cyclosporine-treated renal transplant patients compared with conventionally treated renal transplant patients, there are distinct advantages to cyclosporine use in renal transplantation. Twenty-three consecutive cadaveric renal transplant patients who received azathioprine, prednisone, and antilymphoblast globulin were compared with 23 cadaveric renal transplant patients who received cyclosporine and prednisone. Fewer statistically significant rejection episodes, multiple rejection episodes, and cytomegalovirus infections were demonstrated in those who received cyclosporine. Most notably, cyclosporine decreased the initial hospital stay, was associated with fewer readmissions, and therefore markedly reduced the initial cost of transplantation. PMID- 2998216 TI - A cluster of true appendicitis cases. AB - A cluster of cases of appendicitis occurred primarily in school-age boys in a small Texas town. The expected rate of appendicitis is 1.5 cases per 1,000 persons, or about 1 case per month in that town. However, in the spring of 1984, 13 cases, 10 in school-age boys, occurred. In eight of these patients, the initial onset of abdominal pain occurred over a 15 day period. A case controlled study of school-age patients indicated that sweets in the diet and consumption of local farm eggs may have been associated with the appendicitis. We hypothesize that a group of young male patients who were susceptible to appendicitis because of the high sugar content of their diets were exposed to a bacterium or virus that precipitated this outbreak of appendicitis. PMID- 2998217 TI - [The adrenergic system in pregnancy and labor]. PMID- 2998218 TI - Inhibition of benzodiazepine receptor binding by urinary extracts: effect of ethanol. AB - Two fractions which inhibit benzodiazepine receptor binding were isolated from both rat and human urine. The method used involved alkaline methanolysis followed by chloroform extraction and silicic acid chromatography. This method precludes artifactual formation of esters of beta-carboline carboxylic acid (BCC) during the extraction procedure. One of the fractions (fraction E) behaved similarly to methyl ester of BCC on thin-layer chromatography and also had a similar fluorescent spectra. Administration of ethanol to male Sprague-Dawley rats decreased the concentration and the total excretion of both inhibitory fractions in a dose-dependent manner. In a clinical study on males with different family histories of alcoholism, ethanol decreased excretion of fraction E. The excretion was not affected by placebo, and it was similar in family history-positive and family history-negative subjects. These results suggest that ethanol affects the gamma-aminobutyric acid-benzodiazepine receptor complex by changing release, metabolism, and/or excretion of an endogenous benzodiazepine ligand. PMID- 2998220 TI - Determination of picomole amounts of glycerate 3-phosphate, glycerate 2 phosphate, and phosphoenol pyruvate by an enzymatic assay coupled to firefly luciferase/luciferin luminescence. AB - A procedure for the determination of picomole amounts of glycerate 3-phosphate, glycerate 2-phosphate, and phosphoenol pyruvate is described. These metabolites were utilized by the glycolytic enzymes phosphoglycerate mutase, enolase, and pyruvate kinase to generate ATP which was determined by firefly luciferase/luciferin luminescence. The phosphoglycerate mutase used was of the glycerate 2,3-bisphosphate-independent type and was prepared from wheat germ. Stoichiometric conversion of glycerate 3-P, ranging in amount from 9 to 275 pmol, occurred after 25 min preincubation and required a narrow range of added mutase. The application of the procedure for determining these metabolites in suspensions of plant protoplasts is described. PMID- 2998219 TI - [Significance of endocrine parameters of stress]. AB - Thirty two male patients undergoing coronary bypass surgery were given low (group A, 0.01 mg/kg bw) and high dose (group B, 0.035 mg/kg bw) fentanyl anaesthesia. Haemodynamic and hormone responses were investigated from the beginning of anaesthesia until extracorporeal circulation (ECC) (group A: n = 16; group B: n = 16). Significant changes in haemodynamics occurred only in group A including an increase in heart rate (36%) and systolic arterial pressure (21%). Plasma vasopressin (ADH) levels rose significantly in both groups after the beginning or surgical procedure which was markedly less pronounced in patients with high fentanyl (group B). In group A (low dose) a second dose of fentanyl was given after sternotomy, which was followed by a significant decrease in ADH (80% from previous value). No significant variations could be demonstrated in plasma levels of cortisol, ACTH, and human growth hormone (HGH). The data stress the importance of plasma-vasopressin-levels in determining the endocrine stress response following trauma and operation. On the other hand there was a lack of correlation between trauma and pain and frequently reported patterns of the endocrine metabolic stress response. PMID- 2998221 TI - Cell membrane coating with glutaraldehyde: application to a versatile solid-phase assay for thyroid membrane proteins and molecules interacting with thyroid membranes. AB - In defined conditions, glutaraldehyde was shown to tightly bind cell membranes to flexible microtiter plates without significant alteration of the antigenic and functional properties of membrane proteins. In the presence of 0.06% glutaraldehyde, human thyroid membranes were bound to plastic firmly enough to resist numerous washing and flicking steps; the coated membranes remained almost unaltered with regard to monoclonal antibody and thyrotropin binding as well as adenylate cyclase and peroxidase activities. Based on the use of thyroid membrane coated microtiter plates, a versatile solid-phase assay was developed which allowed screening of anti-membrane monoclonal antibodies, detection of thyrotropin-displacing activity in hormone and antibody preparations, and monitoring of fractionation experiments of solubilized membrane antigens and thyrotropin receptor. It was concluded that the use of glutaraldehyde for coating cell membranes to flexible microtiter plates enabled the establishment of simple, rapid, and reliable assays for detection and quantitation of membrane proteins and molecules interacting with membranes. PMID- 2998222 TI - Radioenzymatic assay of angiotensin-converting enzyme inhibitors in plasma and urine. AB - A rapid, sensitive assay for angiotensin-converting enzyme (ACE) inhibitors is described. Biological samples were diluted with methanol to precipitate endogenous ACE and centrifuged. Supernatants were further diluted with 4-(2 hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 8. Diluted samples were incubated at 37 degrees C with the substrate [3H]hippurylglycylglycine and rabbit lung ACE for 45 min. Acid (1.0 N HCl) was then added, and the product, [3H]hippuric acid, was extracted into a water-immiscible scintillation cocktail. Drug standards were prepared in the biological matrix to correct for drug recovery. A computer program was used to convert radioactivity (dpm) to units of enzyme activity and then correlate enzyme activity with drug concentration. The ester prodrugs fosenopril and enalapril could be assayed down to 4 ng/ml in plasma after ester hydrolysis with NaOH. Drug disposition studies in rats, dogs, and monkeys have demonstrated that the method can be readily adapted to any ACE inhibitor and is suitable for determining drug bioavailability and pharmacokinetics. PMID- 2998223 TI - Quantitation of tissue calpain activity after isolation by hydrophobic chromatography. AB - A rapid and reliable method for quantitating tissue calpains (Ca2+-activated, neutral, thiol proteases) was developed using hydrophobic chromatography with phenyl-Sepharose. Calpains I and II isolated by this method are free of endogenous inhibitor(s) (calpastatin), activator(s), and nonspecific proteases. These calpains expose hydrophobic regions in the presence of Ca2+ and bind tightly to phenyl-Sepharose. Inactivation of bound calpain is prevented by the addition of leupeptin (20 microM). Calpains I and II bound initially by phenyl Sepharose in a Ca2+-dependent manner are then eluted successively on the basis of their Ca2+-independent binding to phenyl-Sepharose. Because calpastatin may prevent binding of calpain to phenyl-Sepharose by forming a protease-inhibitor complex in the presence of Ca2+, preadsorbing the protease to a suspension of phenyl-Sepharose beads initially in the absence of Ca2+ separates most of the calpain present in tissue extracts from calpastatin. The isolated calpains obtained are assayed by casein digestion. This quantitation procedure is suitable for measuring calpain activity in various tissues and cells including erythrocytes. PMID- 2998224 TI - Methods for the determination of atmospheric organic isocyanates. A review. PMID- 2998226 TI - 1,25-Dihydroxyvitamin D3 in teeth of rats and humans: receptors and nuclear localization. AB - Autoradiographic and biochemical studies were used to demonstrate 1,25 (OH)2 vitamin D3 target cells in teeth. Incisor pulp of rats and molar pulp of humans were incubated in vitro with 3H-1,25 (OH)2 vitamin D3. Subsequent frozen-section autoradiography revealed a large population of cells in the pulp of both incisors and molars which selectively concentrated radioactivity in their nuclei. Extracts of incisor pulp from mature rats were found to bind 3H-1,25 (OH)2 vitamin D3 and this binding was displaceable with excess 1,25 (OH)2 vitamin D3. Sucrose density analysis revealed that the protein in tooth pulp which binds 1,25 (OH)2 vitamin D3 sediments at 3.2-3.5S. The 1,25 (OH)2 vitamin D3 receptor of intestine and kidney also sediments in this region, indicating that the 1,25 (OH)2 vitamin D3 binding protein of tooth pulp is similar to that found in other target organs. These autoradiographic and biochemical data indicate that pulpal cells of mature rat and human teeth contain receptors for 1,25 (OH)2 vitamin D3. PMID- 2998227 TI - Angiotensin converting enzyme in semen and its possible role in capacitation. AB - Angiotensin Converting Enzyme (ACE) is present in the testis, epididymis and semen. But no physiological role has been assigned to ACE. The present study is to indicate possible role of ACE in capacitation. Semen was incubated in minimum capacitation in vitro. Epinephrine and serotonin were added to note the effect of biogenic amines. The leakage of ACE was very high in capacitated spermatozoa compared to spermatozoa in saline. Presence of amines in the saline induced ACE leakage. These results indicate that ACE may be involved in the capacitation. PMID- 2998225 TI - An immunohistochemical study on the mouse adenohypophysis with reference to the spatial relationship between GH cells and other types of hormone-producing cells. AB - The objective of the present immunohistochemical study was to determine whether the close spatial relationship between hormone-producing cells as described in rats also exists in the mouse adenohypophysis. In both immature and mature mice, GH cells were the only cell type that had a round shape throughout the pars distalis. All other types of secretory cells had angular or irregular shapes and were closely apposed to round GH cells. Thus, between GH and ACTH cells the same intimate relationship pertains as in rats. Unlike in rats, however, the juxtaposition of LH and Prl cells was observed only occasionally in mature female mice. The salient features of the mouse adenohypophysis were that most LH cells closely surrounded GH cells. These findings show that the cytoarchitectural interrelationship between adenohypophysial cells of mice differs from that of rats. PMID- 2998228 TI - Fatal intraoperative tumor embolism in a child with hepatoblastoma. PMID- 2998230 TI - Cost-effective applications of the Centers for Disease Control guidelines for prevention of nosocomial infections. PMID- 2998229 TI - An algorithm for the control of nosocomial varicella-zoster virus infection. AB - Inadvertent or uncontrolled introduction of varicella-zoster virus into the hospital environment occurs commonly and must be investigated in a systematic and efficient manner to minimize secondary spread to patients (particularly the immunocompromised) or hospital personnel. On the basis of a review of the literature and our practical experience with 11 such exposures to varicella zoster virus during a 2-year period, we have developed a working algorithm for such investigations. Index cases most often are children, resident physicians, students, young nurses, and ancillary personnel, or adult patients with herpes zoster. A negative or uncertain past history of this infection is an unreliable predictor of susceptibility among the exposed and should be confirmed by serology tests or delayed hypersensitivity skin testing. An incubation-contagion timetable, coupled with a stratification of risk among the exposed, permits a prioritized response in dealing with an introduction of varicella-zoster virus. The preemployment screening of all hospital workers for susceptibility to varicella-zoster virus should be considered as a practical and cost effective policy. PMID- 2998231 TI - Cost-effective application of the Centers for Disease Control Guideline for Handwashing and Hospital Environmental Control. PMID- 2998233 TI - Cost-effective application of the Centers for Disease Control Guideline for Prevention of Nosocomial Pneumonia. AB - Some parts of the Guideline are clearly cost-effective. Abandoning routine cultures of respiratory therapy equipment is cost-effective and should be adopted by any hospitals that have not done so already. Other practices such as the use of preoperative and postoperative instructions regarding deep breathing and incentive spirometry, and the policy of never reusing respiratory therapy equipment items that are intended for single use probably warrant further cost benefit analysis. Finally, there is increasing evidence that changing ventilator tubing every 24 hours is not cost-effective. Changing tubing every 48 hours appears to be safe and can save hospitals substantial sums of money. PMID- 2998232 TI - Cost-effective application of the Centers for Disease Control Guideline for Infection Control in Hospital Personnel. AB - I have tried to summarize the spirit of the Centers for Disease Control Guideline for Infection Control in Hospital Personnel. By eliminating certain unnecessary practices and preventing one infectious disease, we could have saved the hospital in these examples more than $18,000 in just 1 year. We must continue to evaluate and eliminate unnecessary practices while maintaining a safe environment for our employees. PMID- 2998234 TI - Cost-effective application of the Centers for Disease Control Guideline for Prevention of Surgical Wound Infections. AB - Surgical wound infections present a serious hazard to patients and an important legal and economic liability to health care providers. This Guideline furnishes infection control practitioners with several excellent cost-saving concepts and, if implemented, would result in a reduction in the rates of surgical wound infections. PMID- 2998235 TI - Atherosclerosis. I. A leiomyoproliferative disease of the arteries resulting from breakdown of the endothelial barrier to potent blood growth factors. II. Perspectives in atheroprophylaxis. AB - For a historical survey of the pathogenesis of atherosclerosis the reader is referred to references 1 and 2. Comprehension of the vessel-blood interface homeostasis hinges upon an understanding of the pathophysiology of angio-lymphoid relationships. Even in the smooth contact of the intact hydrophobic intimal lining with the marginal flow of the circulatory stream, small amounts of thrombin and small aggregates of aging platelets float by under physiologic conditions. Since endothelial cells of the vascular intima contain receptors for thrombin, filling these receptors with thrombin becomes a stimulus for the production of prostacyclin (PGI2) by the endothelial cells; PGI2 in turn inhibits adherence of the small platelet aggregates by ADP; homeostasis is maintained. The size of physiologic thrombin-producing platelet microaggregates is controlled by physiologic levels of antithrombin III. PMID- 2998236 TI - Comparison of intraosseous, central, and peripheral routes of sodium bicarbonate administration during CPR in pigs. AB - Obtaining venous access continues to be one of the most difficult problems faced by a physician caring for the pediatric patient in cardiac arrest. Our study examined the use of the intraosseous route (through the bone) to obtain venous access for sodium bicarbonate administration in a cardiac arrest model. Ventricular fibrillation was induced in 23 domestic swine. Cardiopulmonary resuscitation was performed for five minutes and sodium bicarbonate (1 mEq/kg) was administered through a peripheral IV line (n = 6), a central IV line (n = 5), or intraosseously (n = 6). Controls (n = 6) did not receive bicarbonate. Blood pH was sampled every two minutes for 30 minutes from the right ventricle, left ventricle, and femoral artery. An analysis of variance revealed that the central and intraosseous routes were significantly different (P less than .05) from the peripheral group, and that all three groups were significantly different (P less than .05) from the control. Pathology studies revealed only minor damage to bone when sodium bicarbonate was administered intraosseously. These data demonstrate that the intraosseous route is a rapid and effective alternative for venous access in a cardiac arrest model. PMID- 2998237 TI - Japanese encephalitis virus immunoglobulin M antibodies in porcine sera. AB - A solid-phase enzyme-linked immunosorbent assay (ELISA) was developed for detection of porcine immunoglobulin (Ig)M antibodies to Japanese encephalitis virus (JEV). Antibodies in sera were captured onto the solid phase of Microtiter plates sensitized with mouse monoclonal antibodies to porcine mu heavy chain. Virus antigen binding to the lawn of IgM was quantitated by subsequent binding of peroxidase-labeled human hyperimmune anti-JEV IgG, which in the final step, catalyzed a substrate color change. In sucrose density-gradient fractionated sera from recently infected pigs, the peak of ELISA JEV IgM activity corresponded to the peak of 18-S, 2-mercaptoethanol-sensitive hemagglutination-inhibiting (HAI) antibody activity. Within 2 to 3 days, JEV-infected sentinel pigs developed high JEV IgM activity; this activity decreased within 2 weeks. Among specimens collected from 99 random swine at abattoirs in Thailand during a period of low JEV transmission, none of 25 JEV HAI-negative sera had JEV IgM activity, 7 of 74 JEV HAI-positive sera did have JEV IgM activity, and the remaining 67 sera had readily detectable JEV HAI antibodies, but lacked JEV IgM. The JEV IgM solid phase ELISA was useful for rapidly diagnosing active or recent JEV infections in swine. PMID- 2998238 TI - Comparison of the herpesviruses of cattle by DNA restriction endonuclease analysis and serologic analysis. AB - Reference strains and field isolates of herpesviruses recovered from cattle in the United States were compared by restriction endonuclease (RE) analysis and the indirect fluorescent antibody test. As a result of these comparisons, 5 major biotypes of bovine herpesvirus (BHV) were defined. These types were (i) infectious bovine rhinotracheitis virus (BHV-1), (ii) bovine herpes mammillitis virus (BHV-2), (iii) malignant catarrhal fever (MCF) virus (herpesvirus alcelaphinae), (iv) the group of slow-growth isolates represented by the prototype strain Movar 33/63 (bovine cytomegalovirus candidate), and (v) the syncytia-forming Pennsylvania 47 strain. Bovine herpesvirus-1 and BHV-2 did not cross-react serologically with any other type of BHV tested. A low, but consistent level of serologic cross-reactivity was detected among MCF virus, the Movar group, and Pennsylvania 47. Several nonsyncytial, slow-growth strains, which were recovered from dissimilar clinical syndromes and were serologically related to Movar 33/63, exhibited similar DNA RE cleavage patterns, confirming their identity as members of a single type. There was no isolate from American domestic cattle similar to the African MCF virus, which has been sporadically isolated from exotic ruminants in the United States. The African MCF virus isolated during a MCF epizootic in a United States zoo exhibited some DNA RE cleavage differences in comparison with the MCF virus world prototype strain WC 11, indicating that strain diversity exists within this biotype. PMID- 2998239 TI - Appearance of ovine progressive pneumonia virus outside the United States. AB - A recently isolated Israeli retrovirus from a sheep with maedi-visna was compared with other retroviruses, using cDNA-RNA hybridization in solution. The Israeli isolate was shown to have close, if not identical, genetic homologic features with the ovine progressive pneumonia virus reported in the United States, rather than with those of the maedivisna viruses of European origin. PMID- 2998240 TI - Bluetongue and epizootic hemorrhagic disease in ruminants in Georgia: survey by serotest and virologic isolation. AB - The frequencies of precipitating antibodies to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in domestic ruminants and white-tailed deer (WTD) in Georgia were 36% and 32%, respectively (n = 2,200). The frequencies of seropositivity to BTV and EHDV were high among cattle (47% and 42%, respectively [n = 1,068]) and less so in WTD (36% and 34% [n = 414]). The frequencies among sheep were 34% for BTV and 29% for EHDV (n = 286), whereas among goats, seropositivity was 8% for BTV and 7% for EHDV (n = 433). Serum samples from northeastern Georgia (1 of the 4 regions in the survey) had the highest frequency of precipitating antibodies for BTV (45%) and EHDV (38%). The lowest frequency was in southeastern Georgia, with 29% seropositivity for BTV and 24% seropositivity for EHDV. Of the 175 farms or herds in the serosurvey, 70% included animals that had BTV-precipitating antibodies, and 67% included animals which had EHDV-precipitating antibodies. Seventeen viral isolates were obtained from individual animals on 9 different farms. Fifteen of the isolates were BTV--8 from cattle, 4 from sheep, and 3 from WTD; 8 of them were serotype 11, and 7 were serotype 17. Viral isolates from each of 2 WTD were identified as EHDV serotype 1 and serotype 2. Of the total 17 isolates, 11 were from clinically healthy ruminants, and 6 were from animals with clinical signs of BT or EHD. Five of the viral isolates originated from northeastern Georgia, 7 from the northwestern region, and 5 from the southwestern region; none was obtained from specimens from the southeastern region. PMID- 2998241 TI - Calicivirus isolation from three species of primates: an incidental finding. AB - Calicivirus isolations were made from 3 species of subhuman primates. Viruses were recovered from gingival lesions associated with periodontal disease in a spider monkey, from the oropharynx of a healthy silver leaf langur, and from the spleen of a lowland gorilla that had died of systemic coccidioidomycosis. Based on the results of cross-neutralization tests, all 3 isolates were serologically indistinguishable from a primate calicivirus Pan paniscus type 1. These isolations appeared to be incidental in nature and could not be associated causally with any specific disease entity. PMID- 2998242 TI - Prednisone inhibits late asthmatic reactions and the associated increase in airway responsiveness induced by toluene-diisocyanate in sensitized subjects. AB - To determine whether late asthmatic reactions and the associated increase in airway responsiveness induced by toluene diisocyanate (TDI) are linked to airway inflammation, we investigated whether they are inhibited by prednisone. Ten "sensitized" subjects were studied in 2 sets of experiments. In the first set, each subject was given no treatment and was studied before and for 8 h after exposure to TDI. In the second set, 2 to 4 wk later, each subject was studied before treatment and then during treatment with prednisone (50 mg once a day for 3 days, orally), both before and after exposure to TDI. To assess late asthmatic reactions to TDI, we measured FEV1 immediately before and after exposure, then hourly for 8 h. To assess changes in airway responsiveness, we measured the provocation dose (mg) of methacholine causing a 20% decrease in FEV1 (PD20FEV1) before and 8 h after exposure to TDI. When the subjects received no prednisone treatment, TDI caused late asthmatic reactions and increased airway responsiveness. By contrast, when the subjects received prednisone, TDI caused no late asthmatic reaction or increased airway responsiveness. Prednisone did not change baseline airway caliber or airway responsiveness. These results suggest that late asthmatic reactions and the associated increase in airway responsiveness induced by TDI in "sensitized" subjects may depend on the development of a steroid-responsive acute inflammatory reaction within the airways. PMID- 2998243 TI - Investigation of the protective effects of the antioxidants ascorbate, cysteine, and dapsone on the phagocyte-mediated oxidative inactivation of human alpha-1 protease inhibitor in vitro. AB - Oxidants derived from the atmosphere or from activated pulmonary phagocytes mediate functional inactivation of alpha-1-protease inhibitor (alpha-1-PI). Chronic exposure to these oxidants may cause emphysema. In this study we have investigated the effects of the antioxidants ascorbate, cysteine (10(-4) M to 10( 1) M), and dapsone (10(-6) M to 10(-3) M) on the oxidative inactivation of human alpha-1-PI by leukoattractant-activated polymorphonuclear leukocytes (PMNL) in vitro. During exposure of alpha-1-PI to stimulated PMNL in the presence of ascorbate and cysteine at concentrations of greater than 10(-4) M and dapsone at greater than 10(-6) M, the elastase inhibitory activity of alpha-1-PI was preserved. However, exposure of the alpha-1-PI to the antioxidants subsequent to PMNL-mediated oxidative inactivation was not associated with reactivation of elastase inhibitory capacity. Ascorbate, cysteine, and dapsone at concentrations that caused 50% protection of alpha-1-PI did not affect degranulation or the binding of radiolabeled leukoattractant to PMNL. It is suggested that the protective effects of the antioxidants are related to their ability to scavenge superoxide and oxidants generated by the PMNL-myeloperoxidase/H2O2/halide system. Because the effects of ascorbate and especially those of dapsone were observed at concentrations of these agents that are attainable in vivo, our results may have clinical significance. PMID- 2998244 TI - Airway hyperresponsiveness and inflammation induced by toluene diisocyanate in guinea pigs. AB - We examined the changes in airway responsiveness to increasing doses of an acetylcholine aerosol in anesthetized and ventilated guinea pigs 2, 6, or 24 h after exposure to 2 ppm toluene diisocyanate (TDI) or 2 h after exposure to air or 1 ppm TDI. Pulmonary resistance (RL) after the animals inhaled a buffered saline aerosol was used as baseline and was similar for air and TDI groups. The concentration of acetylcholine calculated to cause a 200% increase in RL was significantly lower for animals studied at 2 h (0.68%) or at 6 h (0.77%), but not at 24 h (2.39%), after TDI than for air animals (3.07%). The increase in airway responsiveness in the TDI-exposed animals was associated with histologic changes in the trachea and intrapulmonary airways. Exposure to 2 ppm TDI caused a patchy loss of cilia, shedding of epithelial cells into the airway lumen, and an influx of inflammatory cells into the trachea and other airways. In the lamina propria of the trachea, the concentration of extravascular polymorphonuclear leukocytes (PMN) was 13- to 26-fold greater in animals studied 2 or 6 h after exposure to 2 ppm TDI or at 2 h after 1 ppm TDI than in animals exposed to air. The concentration of PMN in the epithelium was significantly increased only in animals examined 2 h after 2 ppm TDI. Exposure to TDI also caused an influx of eosinophils into the tracheal mucosa. This influx occurred later and was more persistent than the influx of PMN. These results indicate that a single exposure to TDI can cause an increase in airway responsiveness that is associated with epithelial injury and acute airway inflammation. PMID- 2998245 TI - Diagnostic yield of bronchoalveolar lavage in pneumonitis occurring after allogeneic bone marrow transplantation. AB - Fifty-two bronchoalveolar lavages (BAL) were performed in order to investigate 46 episodes of pneumonitis that occurred after allogeneic bone-marrow transplantation. No complications have been attributed to this procedure. A specific etiologic diagnosis was obtained in 24 of 46 episodes (52%) by 26 of 52 BAL (50%). Cytomegalovirus (CMV), diagnosed by the presence of typical inclusions, was the pathogen most frequently identified by BAL (13 of 46 episodes) and was associated with other causes of pneumonia in 4 patients. The other causes of pneumonitis diagnosed by BAL were: giant-cell pneumonia: 1, aspergillosis alone: 3, Pneumocystis carinii: 1, Hemophilus influenzae: 3, isolated pulmonary hemorrhage: 3. One false negative (aspergillosis, n = 1) was diagnosed at autopsy. The overall mortality rate of these episodes was 24%. Thus, BAL appears to be a rapid and reproducible diagnostic method for monitoring pneumonitis in grafted patients, particularly CMV pneumonitis, and may avoid the need for surgical biopsy. PMID- 2998246 TI - Exacerbations of asthma in adults during experimental rhinovirus infection. AB - To determine the incidence of wheezing in adult asthmatics with rhinovirus infection, we exposed 21 asthmatic volunteers to 1 of 2 rhinovirus serotypes. Symptoms, spirometry, and histamine inhalation challenge were assessed prior to, daily during, and 3 wk after the rhinovirus infection. Four volunteers had fiberoptic bronchoscopy performed on the fourth study day. Nineteen volunteers became infected with rhinovirus; 17 of 19 had typical coryzal symptoms. Volunteers did not have significant changes in spirometry or histamine sensitivity during rhinovirus infection when taken as a group or when categorized by severity of asthma or severity of the clinical illness. A subgroup of 4 volunteers was identified that had a 10% or greater decrease in FEV1 and a parallel increase in histamine sensitivity during rhinovirus infection; these 4 volunteers were not otherwise distinguishable from the group as a whole. Rhinovirus was recovered from bronchoscopy specimens of 1 of the 4 infected volunteers bronchoscoped. Thus, exacerbations of wheezing occurred in the minority of experimental rhinovirus infections in adult asthmatics, suggesting that other viral pathogens may play a more important role in precipitating asthma attacks. PMID- 2998247 TI - Detection of the bleeding source from small intestine: intraoperative endoscopy and preoperative abdominal scintigraphy by technetium 99m pertechnetate. AB - Bleeding ulcerative lesions of the small intestine often present difficult diagnostic problems. Useful techniques to establish a diagnosis include abdominal scintigraphy using technetium 99-m pertechnetate and intraoperative endoscopic examination of the intestine. Our experience with these techniques is based on 25 patients who were treated in 1974-1983. In 18 adults, the diagnoses included Crohn's disease, non-specific ulcers and intestinal tuberculosis. In 6 of the 18 adults, the source of bleeding was difficult to determine during laparotomy and diagnosis was established by intraoperative endoscopic examination. In the seven children, the diagnoses included ectopic gastric mucosa and lymphoid hyperplasia. In the children, abdominal scintigraphy was used as a preoperative measure to detect the bleeding source. PMID- 2998248 TI - High and low spin state mixture in methemoglobin and metmyoglobin. AB - The mixture of low and high iron spin states is studied by electron spin resonance in methemoglobin and in metmyoglobin between 6K and 100K. The crystals contain iron (Fe3) exclusively in the high spin state, while powdered samples show a mixture of high and low spin iron. We detected, for the first time, the low spin state in metmyoglobin at low temperatures. The ratio of high to low spin concentrations (k-1) varies exponentially with inverse of temperature in both proteins, only the absolute value is greater in myoglobin. The slope of K-1 depends on the cooling rate and on the temperature range. The results are qualitatively explained assuming a temperature dependent distribution of crystical field around the cristal value, delta c. PMID- 2998249 TI - [Genetics and cancer]. PMID- 2998250 TI - [Prognostic factors in Wilms' tumor]. PMID- 2998251 TI - Primary human T-lymphotropic virus type III infection. AB - Primary infection with the human T-lymphotropic virus type III (HTLV-III) was documented in three patients by virus isolation during acute illness and concurrent or subsequent HTLV-III seroconversion. All patients had fevers, rigors, arthralgias, and myalgias. Additional symptoms included truncal maculopapular rash, urticaria, abdominal cramps, and diarrhea. Lymphocytic meningitis accompanied the febrile illness in two patients. The estimated incubation period was 4 to 6 weeks, and the symptoms lasted 2 to 3 weeks. Seroconversion occurred 8 to 12 weeks after presumed exposure and was manifested by a characteristic antibody response pattern. Physicians should consider the possibility of primary HTLV-III infection when evaluating patients who belong to one of the risk groups for the acquired immunodeficiency syndrome and who have prolonged febrile illnesses. PMID- 2998253 TI - Chronic Epstein-Barr virus disease: a workshop held by the National Institute of Allergy and Infectious Diseases. PMID- 2998252 TI - Diphtheria, tetanus, and pertussis: guidelines for vaccine prophylaxis and other preventive measures. Recommendation of the Immunization Practices Advisory Committee. Centers for Disease Control, Department of Health and Human Services. AB - This revision of the Immunization Practices Advisory Committee (ACIP) statement on diphtheria, tetanus, and pertussis updates the statement issued in 1981 and incorporates the 1984 supplementary statement on the risks of pertussis disease and pertussis vaccine for infants and children with personal histories of convulsions. It includes a review of the epidemiology of the three diseases, a description of the available immunobiologic preparations, and the appropriate immunization schedules. Also included are revisions in the schedule for combined diphtheria and tetanus toxoids, when pertussis vaccine is contraindicated, and revisions in the recommendations on precautions and contraindications to vaccine use, on immunization for infants and children who have underlying neurologic disorders, and on tetanus prophylaxis in wound management. PMID- 2998254 TI - S-2-(3-Aminopropylamino)ethyl phosphorothioic acid (WR-2721) in primary hyperparathyroidism. PMID- 2998255 TI - Foscarnet for cytomegalovirus retinitis. PMID- 2998256 TI - [Right endocarditis in disseminated aspergillosis]. AB - A 53 year old man with an anaplastic bronchial carcinoma was hospitalised for septic shock and acute respiratory distress after a cutaneous, probably staphylococcal infection, and died in spite of anti-staphylococcal antibiotherapy. The autopsy showed pulmonary, cardiac, cerebral and renal aspergillosis. A right heart aspergillous endocarditis, very rare in this pathology, was also discovered but there were no cardiac valves lesions. The patient was in an "immunodepressed" state as usually observed in pulmonary aspergillosis. The endocardial localisation of aspergillosis and the "pseudo miliary" appearances of the pulmonary lesion indicated an extra-pulmonary portal of entry, cutaneous or intravenous which is unusual in this pathology. This hypothesis is supported by previous reports of pulmonary aspergillosis where right heart endocarditis is exceptionally rare and by aspergillous left heart endocarditis after open heart surgery where pulmonary aspergillosis is absent. PMID- 2998257 TI - A comparison of X-ray diffraction and infrared spectrophotometric methods for the analysis of alpha-quartz in airborne dusts. PMID- 2998258 TI - [Large necrotic ulcerations caused by varicella-zona virus in an immunosuppressed patient]. PMID- 2998259 TI - [Ecology of tsetse flies in the preforested area of the Ivory Coast. Relation to human trypanosomiasis and possibilities for control]. AB - This paper gives the results of a tsetse fly research program in the preforested area of Ivory Coast. The main investigation tool was the Challier-Laveissiere's biconical trap. The ecodistribution, resting and pupal sites of the principal tsetse species, G. palpalis, G. pallicera and G. nigrofusca were described in the T. b. gambiense sleeping sickness focus of Vavoua. Population dynamics, host preferences, activity cycles were compared between tsetse populations in plantations and village surroundings. Man-fly contact was of high intensity where human habitat is scattered in plantations but in village tsetse flies used to feed mainly on pigs. Resettlement of scattered populations can be considered as a preventive measure against sleeping sickness transmission. Experiments on tsetse flies control by insecticides along edges, associated with insecticidal impregnated screens, were considered as promising. PMID- 2998260 TI - Prevalence of antibodies to bovid herpesvirus 1 (IBR-IPV), bovine virus diarrhoea, bovine respiratory syncytial parainfluenza 3, adeno A and adeno B viruses in indigenous and imported Moroccan cattle. AB - A serological survey on prevalence of antibodies to Bovid herpesvirus 1 (IBR-IPV) Bovine Virus Diarrhoea, Bovine Respiratory Syncytial, Parainfluenza 3, Adeno A and B viruses was performed in 524 cattle from different areas and management conditions in Morocco. General antibody prevalence was 62.8, 48.5, 70.4, 68.1, 9.0 and 12.4, for the six viruses, respectively. Higher prevalence and high antibody titers to most of the investigated viruses were found among cattle from extensive management systems, with few contacts with imported breeds, and without history of respiratory disease in the area. The significance of these findings is discussed with regards to the risk of the spreading of new diseases with the importation of foreign cattle into the country. PMID- 2998261 TI - Cortisol assays: guidelines for the provision of a clinical biochemistry service. PMID- 2998262 TI - Hormone studies in a case of virilisation due to ovarian tumour. AB - The investigation is described of a 62-year-old female patient who presented with severe virilisation; her plasma testosterone concentration was grossly elevated at 37.9 nmol/L. Measurement of plasma androstenedione, dehydroepiandrosterone (DHEA) and DHEA-sulphate, urinary 17-oxosteroids and urinary 'free' cortisol suggested an ovarian source of androgens. An ultrasound scan indicated the presence of an ovarian mass which was removed and classified as a Sertoli-Leydig cell tumour (arrhenoblastoma) combined with a mucinous cystadenoma. Following operation plasma testosterone levels returned to normal. The excessive pre operative testosterone production appeared to have had little effect on the plasma level of SHBG, since levels remained similar before and after removal of the tumour. Serum LH and FSH levels were higher post-operatively. PMID- 2998264 TI - Evaluation of two rapid methods for the detection of herpes simplex virus antigen in patient specimens. AB - An indirect immunofluorescent antibody procedure (IFA) for the detection and typing of herpes simplex virus (HSV) and an enzyme-linked immunosorbent assay (ELISA) procedure were compared with conventional viral culture. Specimens for culture and ELISA were inoculated into serum free viral transport medium (VTM) and, for IFA, onto slides provided in the kit. Tissue cultures (MRC-5 and primary rabbit kidney) were inoculated and examined daily for cytopathogenic effect (CPE). The remaining VTM was frozen at -70 degrees C until tested by the ELISA system. Slides for IFA were stained with HSV common and HSV-2 specific monoclonal antibodies. Of 155 specimens, 47 (30 percent) were unsatisfactory for the IFA test owing to an inadequate number of epithelial cells on the slides. Of 108 adequate specimens, 45 were culture positive; 39 were positive by the IFA test with a sensitivity of 87 percent and a specificity of 90 percent. Of the 39 positives, 29 (75 percent) were correctly classified as type 1 or type 2, six (15 percent) were typed incorrectly, and four (10 percent) were inadequate for typing by the IFA test. All 155 specimens were suitable for testing by the ELISA procedure. Of 55 specimens positive by culture, only 25 (sensitivity 45 percent) were positive by ELISA. However, the specificity was 100 percent. After incubation of two, three, and six days, the tissue cultures detected 71 percent, 89 percent, and 100 percent of the positives, respectively. PMID- 2998263 TI - Nickel-induced malignant tumors. AB - A series of malignant tumors experimentally induced by implanting Ni3S2 in gelatin capsules into the right hind limb of Fischer 344 rats is described. In this series, histology and ultrastructure revealed two major cell types. These are described as fibroblast-like and histiocyte-like. In addition, numerous giant cells, including large bizarre multinucleate giant cells with granular cytoplasm, were also seen. In this preliminary report, no more precise diagnosis will be given for this tumor than "malignant tumor of pluripotential origin". Further work is proposed to define more specifically its histogenesis. The appearances of the experimentally produced tumor are compared with certain tumors found in humans and also in experimentally induced tumors by Maruyama et al designated as malignant fibrous histiocytoma (MFH). PMID- 2998265 TI - Effects of wheat bran and porridge oats on hepatic portal venous volatile fatty acids in the pig. AB - Adult male pigs (40-60 kg of body weight) of the Kangaroo Island strain were surgically implanted with chronic indwelling hepatic portal venous cannulae. After a 24-hour fast the animals were given meals containing 500 g of either wheat bran or porridge oats and 200 g of sucrose and 2 litres of milk. With both cereal preparations plasma volatile fatty acids rose in the hepatic portal vein but the increase was significantly greater with wheat bran. Omission of sucrose and milk did not alter the response to porridge oats but diminished the response to wheat bran. These changes in plasma volatile fatty acids were unaffected by prior cooking of the cereals with hot water. With all test meals acetate and propionate were the major acids found, with butyrate contributing less than 8% of the total. This compositional profile was also found when the pigs were fed a commercial ration. The absence of butyrate differed from observations in the rat and reflected low concentrations of this acid in large bowel digesta. The difference in the response of the concentration of volatile fatty acids to feeding porridge oats and wheat bran in the pig was also the reverse of that found in the rat. These species differences may be of significance in relation to the choice of animal models for human fibre metabolism. PMID- 2998266 TI - The proton motive force in bacteria: a critical assessment of methods. PMID- 2998267 TI - Compartmentation in Dictyostelium. PMID- 2998268 TI - [Antibiotics and the environment]. PMID- 2998269 TI - [Metabolic characteristics of polyphosphates and other macroergic phosphorus compounds in relation to the degree of penicillin production and growth conditions of Penicillium chrysogenum]. AB - Metabolism of macroergic phosphorus compounds was studied in high- and low productive isogenic strains of Penicillium chrysogenum. It was shown that the levels of the high-polymer polyphosphates (fractions PP1, PP2 and PP3) in the strain intensively producing penicillin were 2-3 times higher than those in the low-productive strain by the 2nd day of the fermentation process (the period of penicillin production). The levels of pyrophosphate and ATP in the mycelium during the fermentation process did not significantly differ in the strains. The study on the relation between metabolism of the high-molecular polyphosphates and conditions of the culture growth and antibiotic production revealed that their accumulation was connected with biosynthetic processes giving rise to the growth of P. chrysogenum, while their consumption with penicillin production. The dynamics of the pyrophosphatase and polyphosphatase activity in the mycelium of the strains was studied. PMID- 2998270 TI - [Change in the natural immunity of mice after administration of blastolysin, a glycopeptide from the Lactobacillus bulgaricus cell wall]. AB - The effect of blastolysin, a glycopeptide of the cell walls of L. bulgaricus, on the resistance of mice to infections caused by S. typhimurium and B. pertussis, lysozyme levels in the blood serum and 5'-nucleotidase activity of peritoneal macrophages was studied. The changes in the nonspecific resistance of mice due to the effect of blastolysin depended on the administration route and the type of the developing infection. It activated the enzymatic activity of the peritoneal macrophages and changed the lysozyme levels in the blood serum of mice. The optimal protective effect of blastolysin was achieved on intramuscular injection in a dose of 100 micrograms. PMID- 2998272 TI - [Adenosine transformation into adenosine-5'-monophosphate by intact Erwinia herbicola cells]. AB - Strain 47/3 was isolated from the natural sources of Erwinia herbicola. The cells of this strain contain nucleoside phosphate transferase responsible for specific phosphorylation of the adenosine 5'-hydroxyl group. With the use of the strain intact cells, conditions for preparation of AMP were determined. The cells were grown in the meat-peptone broth supplemented with yeast extract. p Nitrophenylphosphate was used as a donor of phosphate groups. It was shown that the concentration of the reaction substrates, their ratio and PH of the reaction mixture were factors defining the cell activity. Under the optimal conditions (0.2 M acetate buffer, pH 4.5; concentrations of p-nitrophenylphosphate and adenosine 96 and 12 mg/ml respectively; concentration of the cells 1 per cent) the cells phosphorylated adenosine with a yield of 74 mol %. This indicates that the method may be used for production of AMP in preparative amounts. PMID- 2998271 TI - [Effect of antibiotics on the energy metabolism of circulating blood lymphocytes in mice]. AB - In high doses kanamycin, ristomycin and chloramphenicol had an inhibitory effect on the energy enzymes of the blood lymphocytes in intact and immunodeficiency mice. The inhibitory effect of chloramphenicol and penicillin was more pronounced in immunodeficiency animals. Cephaloridin had no effect on the energy metabolism of the lymphocytes in intact mice. It had a stimulating effect on the blood cells in immunodeficiency mice. PMID- 2998273 TI - [Importance of dactinomycin in the current therapy of tumors]. PMID- 2998274 TI - [Inhibitor of aminoglycoside phosphotransferases from Micromonospora sp.--a producer of sisomicin]. AB - A substance inhibiting the activity of aminoglycoside-3-'-phosphotransferases of both the actinomycetous and the bacterial origin was detected in the filtrates of the culture fluid and mycelium of Micromonospora sp. producing sisomicin. The activity of the substance against the aminoglycoside inactivating enzymes of different types was studied. It was shown that the quantity of the inhibitor in the culture fluid of Micromonospora sp. correlated with intensity of sisomocin production. Under conditions not providing production of the antibiotic the inhibitory activity was lacking. The inhibitor was purified by ion exchange chromatography on Amberlite CG-50 and KM-cellulose (NH+4 form), gel filtration through Sefadex G-50 and preparative paper chromatography. Stability of the inhibitor at different pH values and different temperatures, its sensitivity to certain enzymes and behaviour in high voltage electrophoresis were studied. The results of ultrafiltration showed that the molecular weight of the inhibitor was relatively low: less than 1000 D. PMID- 2998275 TI - Comparative anti-herpesvirus activities of 9-(1,3-dihydroxy-2 propoxymethyl)guanine, acyclovir, and two 2'-fluoropyrimidine nucleosides. AB - 9-(1,3-Dihydroxy-2-propoxymethyl)guanine (DHPG), was evaluated in cell culture and in animals for its inhibitory effect on herpes simplex viruses. Compounds run for comparison included acyclovir, 2'-fluoro-2'-deoxy-5-iodo arabinofuranosylcytosine (FIAC), and 2'-fluoro-2'-deoxy-5-methyl arabinofuranosyluracil (FMAU). In plaque reduction assays DHPG, acyclovir, FIAC, and FMAU were inhibitory to six herpes types 1 and 2 virus strains at concentrations of 0.2-2.4 microM. These concentrations were much lower than those required to inhibit Vero cell proliferation. In guinea pig vaginal infections, DHPG provided significantly greater inhibition of herpetic lesions than did acyclovir. In a herpes type 2 infection model in mice, DHPG, and FMAU were active at 5 mg/kg, whereas acyclovir and FIAC showed no statistically significant effect at 80 mg/kg. In a herpes type 1 encephalitis model, DHPG and FMAU were active at doses less than 10 mg/kg, with FMAU being about 4 times more potent than DHPG in that model. PMID- 2998277 TI - Surface potential changes on energization of mycoplasma cell membranes. AB - The membrane surface potential of mycoplasma cells was measured by changes in the partition between the membrane and the aqueous environment of the impermeable cationic amphipatic spin probe 4-(N,N-dimethyl-N-nonyl)ammonium-2,2,6,6 tetramethylpiperidine-1-oxyl (CAT9). Upon energization of glycolyzing mycoplasma cells, the outer surface of these membranes becomes more negatively charged. The effects of uncouplers further indicate that this change in surface potential appear to be dependent on the existence of a delta pH across the membranes. PMID- 2998276 TI - Topical butylated hydroxytoluene treatment of genital herpes simplex virus infections of guinea pigs. AB - The effect of topical treatment with butylated hydroxytoluene (BHT) was evaluated in primary and recurrent genital herpes simplex virus type 2 (HSV-2) infection of guinea pigs. In the first experiment, treatment with placebo, 5%, 10%, or 15% BHT was initiated 48 h after viral inoculation and continued 4 times daily for 15 days. During primary infection no differences in maximum lesion severity or titers of virus in lesions were observed, however, lesion duration was reduced in BHT-treated animals resulting in a significantly smaller lesion score-day area under the curve. In a second experiment using U.S.P. mineral oil as an additional placebo, BHT placebo and 15% BHT in a double blind trial, similar results were obtained. Treatment of the recurrent infection in either experiment failed to alter the number of recurrent episodes or days with lesions. PMID- 2998278 TI - Decreased amounts of core proteins I and II and the iron-sulfur protein in mitochondria from yeast lacking cytochrome b but containing cytochrome c1. AB - The effect of cytochrome b on the assembly of the subunits of complex III into the inner mitochondrial membrane has been studied in four mutants of yeast that lack a spectrally detectable cytochrome b and do not synthesize apocytochrome b. Quantitative analysis of intact mitochondria by immunoprecipitation or immunoblotting techniques with specific antisera revealed that the core proteins and the iron-sulfur protein were decreased 50% or more in the mitochondria from the mutants as compared to the wild type. Sonication of wild-type mitochondria did not result in any decrease in any of these proteins from the membrane; however, sonication of mitochondria from the four mutants resulted in a further decrease in the amount of these proteins suggesting that they are not as tightly bound to the mitochondrial membrane in the absence of cytochrome b. By contrast, the amounts of cytochrome c1 in the mitochondria, as determined both spectroscopically and immunologically, were not significantly affected by the absence of cytochrome b. In addition, no loss of cytochrome c1 was observed after sonication of the mitochondria suggesting that this protein is tightly bound to the membrane. These results suggest that the processing and/or assembly of these subunits of complex III into the mitochondrial membrane is affected by the absence of cytochrome b. PMID- 2998279 TI - Changes in pyrophosphatase activity during the de novo mineralization associated with cartilage and bone formation. AB - Pyrophosphatase was extracted from implants undergoing de novo mineralization in an in vivo model of matrix-induced endochondral bone formation. Before the onset of the mineralization of the plaques and after the mineralization process had been completed only one form of pyrophosphatase activity was observed. During the active deposition of calcium phosphate, however, a new, higher molecular weight form of pyrophosphatase activity was produced suggesting that this enzyme activity is associated with biological mineralization. This observation gives support to the earlier suggestion that inhibitors of calcium phosphate precipitation, such as pyrophosphate, must be removed from the site of mineralization before calcification can occur. This high-molecular-weight activity also appears to be associated with alkaline and/or acid phosphatase activity as determined by molecular exclusion chromatography. PMID- 2998280 TI - The orientation of phosphate-dependent glutaminase on the inner membrane of rat renal mitochondria. AB - Phosphate-dependent glutaminase is associated with the inner membrane of rat renal mitochondria. The orientation of this enzyme was characterized by comparing its sensitivity in isolated mitochondria and in mitoplasts to two membrane impermeable inhibitors. Mitoplasts were prepared by repeated swelling of mitochondria in a hypotonic phosphate solution. This procedure released greater than 70% of the adenylate kinase from the intermembrane space, but less than 10 and 25% of the marker activities characteristic of the inner membrane and matrix compartments, respectively. The addition of 20 microM p chloromercuriphenylsulfonate (pCMPS) caused a rapid inactivation of the purified glutaminase. In contrast, the glutaminase contained in isolated mitochondria and mitoplasts was only slightly affected by the addition of up to 2 mM pCMPS. Similarly, the activity in mitochondria and mitoplasts was not inhibited by the addition of an excess of inactivating Fab antibodies. However, a similar extent of inactivation occurred when either membrane fraction was incubated with concentrations of octylglucoside greater than 0.35%. Mitochondria were also treated with increasing concentrations of digitonin. At 0.4 mg digitonin/mg protein, all of the adenylate kinase was released but the glutaminase activity was either slightly inhibited or unaffected by the addition of pCMPS or the Fab antibodies, respectively. These studies establish that the glutaminase is localized on the inner surface of the inner membrane. Therefore, mitochondrial catabolism of glutamine must occur only within the matrix compartment. PMID- 2998281 TI - Properties of an acid phosphatase from Legionella micdadei which blocks superoxide anion production by human neutrophils. AB - The high-speed supernatant (100,000 g, 1 h) obtained after centrifuging a suspension of Legionella micdadei that had been freeze-thawed and sonicated contained (i) considerable acid phosphatase activity when assayed using 4 methylumbelliferyl phosphate (MUP) as the substrate, and a factor that blocked superoxide anion production by human neutrophils stimulated with f-Met-Leu-Phe. Chromatography of the extract on a hydroxylapatite column resolved two acids phosphatases (designated ACP1 and ACP2). Subsequent chromatography of ACP2 on a Sephadex G-150 column revealed coincident elution of phosphatase activity and neutrophil blocking activity. When heated at 45 degrees C for various periods of time, the phosphatase activity of the acid phosphatase preparation was lost at the same rate as the ability of the preparation to block superoxide anion production by neutrophils. Furthermore, preincubation of neutrophils and acid phosphatase together in the presence of a heteropolymolybdate complex that inhibits the phosphatase eliminated the effect of the L. micdadei phosphatase on neutrophil superoxide anion production. ACP2 had the following properties: pH optimum, 6.0; Km for MUP, 3.8 mM; isoelectric point, 4.5; substrate specificity, MUP greater than ADP greater than phosphoenolpyruvate greater than phosphothreonine greater than phosphoserine greater than phosphotyrosine; molecular weight (estimated by sucrose density gradient centrifugation and gel filtration chromatography), 71,000-86,000. These results indicate that a cell associated phosphatase may play a role in the virulence of L. micdadei. PMID- 2998282 TI - Isolation and properties of the soluble c-type cytochromes of the dinoflagellate Peridinium cinctum. AB - Four soluble cytochromes of the c type were isolated from the freshwater dinoflagellate Peridinium cinctum collected from Lake Kinneret, Israel. Cytochrome c with alpha-band maximum at 550 nm in the reduced state had a molecular mass of 10,200 Da, pI 7.4, and Em of 278 m V. This cytochrome was active in the respiratory chain of beef heart Keilin-Hartree particles. Cytochrome c-553 had a molecular mass of 13,200 Da, pI 4.9, and Em of 384 m V, and was active in light induced electron transport of Euglena gracilis chloroplast fragments. Cytochrome c-554 had a molecular mass of 13,500 Da, pI 4.4, and Em of 326 m V. This cytochrome was inactive in light induced electron transport but competed with cytochrome c-552 of Euglena in the assay. The acidic cytochrome c-557 was present in very small quantities. The properties of the soluble c-type cytochromes of P. cinctum are compatible with the classification of dinoflagellates as primitive eucaryotes. PMID- 2998283 TI - Regulation of respiration by Na+ and K+ in the halotolerant bacterium, Ba1. AB - In the obligate aerobe, moderate halophile bacterium, Ba1, the ion composition of the medium was found to have a profound influence on the response of the respiratory system to changes in the external pH. In the pH range 6.5 to 8.5 the respiratory activity either increased or decreased progressively, depending whether K+ or Na+ ions were omitted from the medium. A nearly constant rate of respiration was observed in the entire pH range when both K+ and Na+ were present simultaneously. The stimulatory effect of Na+ was expressed especially in the alkaline pH range, where it induced acidification of the intracellular milieu. It was manifest in whole cells as well as in inverted membrane vesicles, and was not affected by either uncoupler or inhibitor of H+-ATPase. In contrast, the respiratory stimulation induced by K+ was most prominent in the acidic pH range and was accompanied by alkalinization of the internal pH. The effect of K+ was observed only in intact cells. Agents which interfered with energy transfer suppressed the effect of K+. With ethanol as the electron donor, Na+ was found to decrease the extent of reduction of the cellular NAD+ in the aerobic steady state, and to cause increased reduction of the cytochromes. K+ had no appreciable effect on the extent of reduction of any component in the respiratory chain. The implications of the above findings are discussed in relation to the mechanism(s) involved in the cation-mediated regulation of respiration and intracellular pH. PMID- 2998284 TI - Fluorescence studies on the interaction between two cytochromes extracted from the yeast, Hansenula anomala. AB - The binding of cytochrome c to the cytochrome b2 core, both extracted from the yeast, Hansenula anomala, has been studied. Cytochrome b2 core heme is extracted and replaced by the fluorescent probe, 2-p-toluidinylnaphthalene-6-sulfonate (TNS). A dissociation constant in the range of 85 microM is found for the TNS apoprotein complex with a stoichiometry of 1:1. The interaction between the two proteins is followed by monitoring changes in the TNS fluorescence. We find the interaction between the cytochrome c and the apocytochrome b2 core to be dependent upon the ionic strength. The dissociation constant of this complex at 20 mM ionic strength is 6 +/- 2 microM with a 1:1 stoichiometry. This dissociation constant is similar to that estimated, by other researchers, for the dimer Zn cytochrome c-cytochrome b2 core complex. PMID- 2998285 TI - Escherichia coli integration host factor inhibits the NusA stimulation of RNA polymerase sigma subunit synthesis in vitro. AB - As reported previously, Integration Host Factor (IHF) stimulates cII expression but the stimulatory effect is prevented by the NusA protein (Peacock and Weissbach, 1985, Biochem. Biophys. Res. Commun. 127, 1026-1031). The interaction between IHF and the NusA protein has been investigated further in studies on the in vitro expression of the genes for the beta (rpoB) and sigma (rpoD) subunits of RNA polymerase, both known to be stimulated by NusA. The NusA stimulation of rpoD expression can be prevented by IHF, but IHF has no effect by itself on rpoD expression. IHF does not influence rpoB expression either in the presence or absence of NusA. PMID- 2998286 TI - Primary histiocytic dermatoses. AB - The physiology of the histiocyte (macrophage) in health and disease is reviewed briefly. An overview of the so-called primary malignant, pseudomalignant, and benign histiocytic disorders, excluding histiocytosis X, is presented. The malignant histiocytosis with erythrophagocytosis, the pseudomalignant histiocytic diseases (such as sinus histiocytosis with massive lymphadenopathy and regressing atypical histiocytosis), and the solitary lesions with histologic malignant and atypical storiform histiocytosis are described. Two groups of adult histiocytic diseases are reviewed; one is characterized by nonfamilial and familial histiocytic dermatoarthritis and the other by multiple widespread benign lesions, such as xanthoma disseminatum, generalized eruptive histiocytoma, nodular non-X histiocytosis, and various xanthomatous eruptions associated with paraproteinemia. Finally, multiple benign cutaneous histiocytic lesions of childhood, such as juvenile xanthogranuloma and congenital self-healing histiocytosis, are included. PMID- 2998287 TI - An electron spin resonance study of free radicals in black dust deposited in human lungs. AB - An Electron Spin Resonance measurement was conducted on black dust deposited in autopsied human lungs. Free radicals were detected in all specimens examined, and the spectra indicated the presence of two types of radicals, both of which are apparently singlet lines. The amount of the respective component differed from sample to sample. The component with the narrower width, 2.7 G, and the smaller g factor, 2.0025, is attributable to carbon radicals associated with the combustion of hydrocarbons, and especially its relevance to the experience of smoking was inferred. The other component, with a width of about 10 G and g factor fo 2.0037, was found to be more common among the specimens. The assignment of the latter component is not very clear, but an oxygen-related radical is a possible candidate. PMID- 2998288 TI - [Fetal rhabdomyomatous nephroblastoma]. PMID- 2998289 TI - [Trophoblastic tumor (of the placental site). Nosologic and prognostic considerations apropos of 4 cases]. PMID- 2998290 TI - A seroepidemiological study of cytomegalovirus and Epstein-Barr virus in rheumatoid arthritis and sicca syndrome. AB - Antibodies to cytomegalovirus (CMV) and Epstein-Barr virus capsid antigen (EBVCA) were examined in 41 patients with rheumatoid arthritis (RA), 26 patients with primary sicca syndrome, and 26 healthy subjects of similar age and sex. IgG antibody titres to EBVCA and CMV were similar in all three groups, apart from a trivial increase of antibodies to EBVCA in RA. False positive IgM anti-CMV antibodies detected in serum from one patient with sicca syndrome and 20 patients with RA were shown to be due to rheumatoid factors. These data did not support recent suggestions that patients with these diseases showed exaggerated immunological responses to either virus and emphasised the need to incorporate adequate laboratory and disease controls when seroepidemiological studies are performed on sera containing rheumatoid factors and autoantibodies. PMID- 2998291 TI - Acute thoracic inlet obstruction in achalasia with adenoid cystic and squamous cell carcinoma. AB - Respiratory symptoms due to compression of the trachea by the dilated esophagus in achalasia are extremely rare. A patient is presented whose respiratory manifestations included engorged neck veins and a neck swelling that fluctuated with respiration. He also had two malignant tumors in his dilated esophagus, a squamous cell carcinoma and an adenoid cystic carcinoma. PMID- 2998292 TI - Properties and regulation of GnRH receptors in the anterior pituitary and the testis of the rat: different response of Leydig cell LH and GnRH receptors to hormonal treatments. AB - The properties (association and dissociation rates, affinity and specificity) of GnRH receptors in the rat pituitary and the Leydig cells are very similar. In addition, in vivo administration for 5 or 11 days of 10 micrograms of GnRH to adult rats resulted in an increase (p less than 0.01) in both Leydig cell and pituitary GnRH receptors. In contrast, the same treatment caused a significant decrease (p less than 0.01) of Leydig cell LH receptor number. Furthermore, testosterone propionate (500 micrograms/day), which had no effect on Leydig cell LH receptors, caused a significant increase (p less than 0.01) of testicular GnRH receptors but a decrease (p less than 0.01) of pituitary GnRH receptors. Estradiol 17 beta valerate (5 micrograms/day) reduced (p less than 0.01) both testicular (5 days) and pituitary (5 and 11 days) GnRH receptors. Such treatment for 5 days also decreased (p less than 0.05) the Leydig cell LH receptors. No effect of dexamethasone and 17-hydroxyprogesterone was seen after 5 or 11 days of treatment. Thus, although pituitary and Leydig cell receptors appear similar, their regulation by exogenous hormone treatments is different. Furthermore, the regulation of Leydig cell GnRH and LH receptors also appears very different, suggesting different roles of the receptors in the control of the Leydig cell function. PMID- 2998293 TI - Effects of a met-enkephalin analogue on motility, O2 consumption, and ATP content of human spermatozoa. AB - Opioid narcotics are present in seminal plasma, although their physiological effect on spermatozoa is still unknown. This study reports data on metabolic parameters of human spermatozoa in the presence of a met-enkephalin analogue: D Ala2-Mephe4-Met-(o)-ol-Enkephalin, FK 33824, Sandoz, Basel, Switzerland (DAMME), and its receptor antagonist naloxone hydrochloride, Endo Laboratories, Garden City, New York. Our findings indicate that the metenkephalin analogue reduces sperm motility and cellular O2 consumption without affecting cellular ATP content and viability. The hypothesis that DAMME acts on adenylate-cyclase is briefly discussed. PMID- 2998294 TI - Some pharmacological actions of nisoxetine, a bicyclic inhibitor of noradrenaline uptake. AB - Some peripheral actions of the phenoxyphenylpropylamine, nisoxetine, have been compared with those of the tricyclic antidepressant, desipramine. The two drugs were equipotent in inhibiting the accumulation of 3H-noradrenaline by the sympathetic nerve terminals of the rat vas deferens; and, in low doses, equipotent in potentiating the actions of noradrenaline at both alpha 1- and alpha 2-adrenoceptors in this tissue. In the vas deferens, the alpha 1 adrenoceptor blocking action of desipramine was evident at concentrations of 0.1 mumol l-1 and above; nisoxetine was less potent. Neither drug exhibited antagonist actions at alpha 2-adrenoceptors. Nisoxetine was also less potent than desipramine in inhibiting responses to histamine, carbachol and bradykinin on the guinea-pig isolated ileum. Thus nisoxetine is the more specific of the two neuronal uptake inhibitors and may be a useful tool to block neuronal uptake in experiments designed to classify adrenoceptors in other tissues. PMID- 2998295 TI - Psychologic modulation of the human immune response to varicella zoster. AB - Psychoimmunology, the interrelationship between the brain/mind/psyche and the immune system, is now an established area of scientific research. Based on prior investigations we hypothesized that an experienced meditator could affect her delayed hypersensitivity reaction by a psychological process. A single-case study design was employed in which the subject was skin tested weekly with varicella zoster skin test reagent. After baseline immunologic studies, she was able, as hypothesized, to significantly reduce both the induration of her delayed hypersensitivity skin test reaction and in vitro lymphocyte stimulation to varicella zoster. Then, as predicted, she was able to allow her reaction to return to baseline. As a confirmation of what is to our knowledge this previously undescribed phenomenon, she was able to reproduce the entire sequence nine months later. It appears that this subject can intentionally modulate her immune response by a psychologic mechanism. PMID- 2998296 TI - Arrangement of genes TRP1 and TRP3 of Saccharomyces cerevisiae strains. AB - The tryptophan biosynthetic genes TRP1 and TRP3 and partly also TRP2 and TRP4 have been compared by the technique of Southern hybridization and enzyme measurements in twelve wild isolates of Saccharomyces cerevisiae from natural sources of different continents, in the commonly used laboratory strain S. cerevisiae X2180-1A and in a Kluyveromyces marxianus strain. We could classify these strains into four groups, which did not correlate with their geographical distribution. In no case are the TRP3 and TRP1 genes fused as has been found in other ascomycetes. Two strains were found which, in contrast to strain X2180-1A, show derepression of gene TRP1. Two examples are discussed to demonstrate the usefulness of Southern hybridizations for the identification of closely related strains. PMID- 2998297 TI - Abdominal midline incision closure. A multicentric randomized prospective trial of 3,135 patients, comparing continuous vs interrupted polyglycolic acid sutures. AB - A randomized prospective multicentric study was organized to compare results between techniques using continuous sutures and interrupted sutures in closing abdominal midline incisions. The suture material employed was polyglycolic acid. This study included 3,135 patients who were randomized between the two methods of closure and who were stratified according to the type of wound: clean, clean contaminated, and contaminated. The overall dehiscence rate was 1.6% in the continuous sutures group vs 2% in the interrupted sutures group. The dehiscence rate in the interrupted sutures group was significantly higher than in the continuous sutures group only in the stratum of contaminated wounds. The death rate was significantly higher in the interrupted sutures group. The number of needle sets was significantly less important when the continuous sutures technique was used. Continuous closure is preferable because it is more economic and expedient and also because it has the same incidence of wound dehiscence as interrupted sutures closure. PMID- 2998299 TI - Restriction endonuclease analysis of Aujeszky's disease (pseudorabies) virus DNA: comparison of Northern Ireland isolates and isolates from other countries. AB - Aujeszky's disease (AD; pseudorabies) viruses isolated in Northern Ireland over a 20 year period were compared with isolates from other parts of the world using restriction endonuclease analysis of virus DNA. When the numbers of Bam H1, Kpn 1 and Sal 1 restriction sites were considered, pathogenic Northern Ireland isolates resembled viruses isolated in England, Hungary and the U.S.A. and could be differentiated from viruses isolated in Denmark, Belgium and the Netherlands. The avirulent Northern Ireland isolate NIA4 and the Bartha vaccine strain were very similar to each other and could be distinguished from pathogenic isolates. While almost all the pathogenic viruses isolated in Northern Ireland from 1963 to 1983 appeared to possess the same number of restriction sites none of the viruses, even those made at the same farm during one outbreak of infection, were identical. The differences were confined to variation in the sizes of certain fragments which map in "variable" regions of the genome. PMID- 2998300 TI - Diabetogenic potential of coxsackie B viruses in nature. AB - Thirty-seven clinical isolates of coxsackievirus (CV) serotypes B-1, B-3, B-4, and B-5 were inoculated into male SJL mice. Twelve strains resulted in minor abnormalities of glucose metabolism in one or more of six infected mice (Tables 1 and 2). Sequential infection of male SJL mice with CVB-3, CVB-4, and CVB-5 resulted in abnormal glucose metabolism in 25 percent of the mice (Fig. 1). The glucose index of the abnormal animals was similar to that produced by sequential infection with reovirus and cytomegalovirus but less than that seen with more severe beta cell tropic agents such as streptozotocin or encephalomyocarditis virus. Infection of autoimmune New Zealand (NZB X NZW) F1 male mice with CBV-3, CVB-4, and CVB-5 resulted in transient elevation of the blood glucose concentration associated with acute acinar pancreatitis (Fig. 2). In spite of recent evidence that infection with the coxsackie B viruses can result in human diabetes mellitus, the diabetogenic potential of CVB field strains appears to be limited. Diabetes mellitus may occur as a rare event, limited to genetically susceptible hosts. Autoimmune mechanisms or repeated infection with other CVB serotypes may convert minimal beta-cell destruction into clinically overt disease. PMID- 2998298 TI - Stimulation of calmodulin by cadmium ion. AB - Cd2+, a serious environmental pollutant in certain industrial regions, accumulates in mammalian tissues with a very slow turnover. Using various criteria, we studied the ability of Cd2+ to substitute for Ca2+ in calmodulin (CaM), a ubiquitous Ca2+-binding protein that mediates many of the Ca2+ effects. CaM bound Cd2+ with a Kd of 4.5 microM, presumably to the Ca2+-binding sites. Binding of Cd2+ allowed CaM to bind 2 moles chlorpromazine, or to form a complex with skeletal muscle troponin-I, troponin-T, or phosphodiesterase. Complex formation with phosphodiesterase led to its activation, which was observed even in the presence of glutathione or cysteine, agents known to chelate Cd2+. This raises the possibility that one manifestation of Cd2+ toxicity may be through its activation of CaM, thus upsetting its normal regulation by a cellular flux of Ca2+. PMID- 2998302 TI - Characteristics of cell fusion induced by a caprine retrovirus. AB - The mechanism of cell-fusion induced by caprine arthritis-encephalitis virus (CAEV) was investigated. Following infection with concentrated CAEV, cell fusion occurred prior to the production of infectious progeny virus. The time of initial detection of cell fusion was dependent on the multiplicity of infection and was not detected until 5 hours after infection. The virus-induced cell fusion was only partially inhibited by ultraviolet irradiation of the virus and was not inhibited by cytosine arabinoside. The results indicated that fusion by CAEV was a slow response to direct interaction of input virus with cells (fusion from without) and that virus replication was not required. PMID- 2998301 TI - Loss of surface fibronectin after infection of cultured cells by HSV-1 and 2. AB - Fibronectin is lost from the surface of HSV infected cells during cell rounding. In order to investigate also the fate of fibronectin during the process of HSV induced cell-fusion, BHK, Vero as well as primary or secondary rabbit kidney cells were infected with HSV-1 strains producing cell-fusion. By immunofluorescence and immunoelectron microscopy a considerable loss of fibronectin after HSV infection could be demonstrated leaving only irregular clumps of fibronectin containing virus particles on the cell surface. Decrease and disarrangement of fibronectin was similar during cell rounding and cell fusion. Loss of Fibronectin was closely connected with the two types of the cytopathic effect (CPE) and could not be prevented by protease inhibitors. The immediate-early protein 175K is essential for induction of CPE and loss of fibronectin. The damage to the cell membrane during HSV infection shows certain analogous mechanisms with events induced by Cytochalasin B and might be explained by the loss of hypothetical fibronectin receptors. PMID- 2998303 TI - Influence of cellular functions on the evolution of persistent infections with Junin virus. AB - Vero cell cultures persistently infected with Junin virus and subjected to different cultural conditions were established. The production of infectious plaque-forming virus, ts mutants and interfering viral particles was determined at different times during 110 days after infection. Carrier cultures maintained in stationary conditions continuously released PFU while proliferating persistent cultures exhibited a cyclical pattern which tends to a rapid PFU disappearance. Concomitantly, in stationary cultures the production of interfering particles was delayed and was lower than in actively growing persistent cells. The metabolic state of the infected cells did not affect the release of ts mutants. The results suggest that a cellular function is involved on the regulation of Junin virus persistent infections. PMID- 2998304 TI - Ultrastructural studies on cell fusion induced by Epstein-Barr virus or N butyrate and 12-O-tetradecanoylphorbol-13-acetate. Brief report. AB - The morphological changes which occur in Raji cells during EBV induced fusion are described. Of particular interest is the formation of local contacts between cells, at these points the plasmalemmae of the two cells become disorganized and cytoplasmic bridges are formed. PMID- 2998306 TI - [Alkaline phosphatase and 5'-nucleotidase in early osteogenesis in man]. AB - By means of histochemical techniques at light and electron microscopic levels, as well as immunomorphological, biochemical and immunochemical methods localization and dynamics of alkaline phosphatase and 5'-nucleotidase contents have been determined in anlages of long tubular bones in 85 human embryos and prefetuses from the 6th up to 12th week of the intrauterine development, obtained as a result of artificial abortions in healthy women. The greatest activity of the enzymes studied is revealed in areas of an intensive osteogenesis and mineralization. Also, by means of the immunofluorescent method alkaline phosphatase of a placental type is revealed, that is not revealed, however, immunochemically. With increasing time of the intrauterine development, thermostability of alkaline phosphatase increases. PMID- 2998305 TI - Hypochlorite-serum reaction products inhibit porcine vascular endothelial cell growth in culture. AB - In vitro toxicity studies wer initiated in order to determine if chlorination affects vascular endothelial cells. Twelfth to twentieth passage porcine aortic vascular endothelial cells (PAE) were grown to confluency and replated in the presence of complete media (Eagle's minimum essential media supplemented with 20% fetal bovine serum) which had been preincubated for 30 minutes with 15.0 mg/liter chlorine. During a 72-hour exposure period, control PAE cells grew to confluency, an increase of approximately 9 fold in the number of cells/plate. Those cells exposed to media preincubated with 15.0 mg/liter chlorine derived from sodium hypochlorite increased only 6 fold. There was no sign of cell killing, but an apparent inhibition of cell division. No effect was seen when either the amino acid or vitamin component of the complete media was reacted at the 15.0 mg dose level. However, when the serum component was preincubated with 15.0 mg chlorine/liter as sodium hypochlorite, an inhibition in growth rate similar to the complete media occurred. PMID- 2998308 TI - Should corneal transplant donors be screened for human T-cell lymphotropic virus type III antibody? PMID- 2998307 TI - [Polymioclonia-opsoclonus: Kinsbourne's syndrome. Report of a case]. AB - Case report of a 9 years old boy with Kinsbourne's syndrome. This condition was characterized by the subacute onset of polymyoclonia, cerebellar ataxia and opsoclonus that set later, following an herpes zoster infection. Steroid therapy resulted in rapid dramatic improvement of neurological symptoms. PMID- 2998310 TI - Diagnostic considerations of tongue-base malignancies. AB - Forty-two patients with malignancies localized to the base of the tongue were treated at Sahlgrenska Hospital between 1971 and 1980. These patients were re analyzed with respect to symptomatology and clinical outcome. Pain in the mouth, throat, and ears as well as swallowing difficulties were the most frequent overt symptoms of disease. In general, patients experienced symptoms for at least 3 months before a positive tumor diagnosis was made. In all, 75% of the patients were found to have large tumors which extended beyond the base of the tongue. Most of the patients were treated with irradiation. The overall 3-year survival rate was 28%, while individual patient survival was related to the size of the primary tumor and to the occurrence of lymph node metastases. Careful attention to symptomatology may reduce delays in establishing an accurate diagnosis and consequently improve the prognosis for patients with these cancers. PMID- 2998309 TI - Type II collagen induced bone resorption in the temporal bone of rats: histological and immunohistochemical studies. AB - Bone resorption in the temporal bone of rats was induced by peripheral immunizations of heterogeneous type II collagen. Bone resorption was found at both the tympanic bone and the petrous bone of the otic capsule. Macrophages, fibroblasts and osteoclasts were found in the enlarged spaces of bone. Immunohistochemical studies demonstrated that macrophages, fibroblasts and osteoclasts produced collagenase and prostaglandins in the bone resorption processes, suggesting that these cells are responsible for the resorption observed. PMID- 2998312 TI - 'And could I have the AIDS test'? PMID- 2998311 TI - The expression of Ia-antigen on nasopharyngeal carcinomas xenografted into nude mice. AB - The expression of Ia-antigen on four different Epstein-Barr virus associated nasopharyngeal carcinomas xenografted into athymic mice could be detected by the monoclonal antibody OKIa. Xenografts of four additional head and neck tumors other than nasopharyngeal carcinoma and one xenograft of a metastatic melanoma cell line were negative for the Ia-antigen. Control antibodies OKT3, OKT4, OKT9, OKM1 and Leu7 were negative with all nasopharyngeal carcinomas and the non nasopharyngeal carcinoma xenografts. Complement receptors as the presumed receptors for the Epstein-Barr virus could not be detected on xenografted nasopharyngeal carcinoma cells but were found on freshly prepared peripheral blood lymphocytes as well as on the Epstein-Barr virus transformed lymphoblastoid cell line QIMR-WIL. The possible role of the Ia-antigen on nasopharyngeal carcinoma cells in respect to the Epstein-Barr virus association of this malignancy is discussed. PMID- 2998313 TI - Experimental infection of Culicoides brevitarsis from south-east Queensland with three serotypes of bluetongue virus. AB - Laboratory-reared C. brevitarsis (biting midges) were fed on sheep which had been experimentally infected with bluetongue serotype 1 (CSIRO 156), bluetongue serotype 20 (CSIRO 19) or bluetongue serotype 21 (CSIRO 154), or on cattle experimentally infected with bluetongue serotype 20 (CSIRO 19). Approximately 77 000 C. brevitarsis were exposed to sheep and 9000 to cattle. The average percentage feeding on sheep was 54% and on cattle 47%. In attempts to transmit virus by bite 3360 C. brevitarsis which had fed on viraemic sheep were held for 11-15 days before exposure to susceptible sheep. Although 11% of these insects fed, transmission of virus from sheep to sheep was not demonstrated. Estimated infection rates of C. brevitarsis for each serotype from sheep and serotype 20 from cattle were similar at 0.4% or lower. These low infection rates are one of the factors which make it unlikely that C. brevitarsis could be an efficient vector of bluetongue viruses in sheep in the field. PMID- 2998314 TI - Melphalan-resistant lymphoblastoid cell lines established from patients with ovarian cancer treated with cross-linking agents. AB - Lymphoblastoid cell lines (LCLs) were established from 83 patients with ovarian cancer by transformation of peripheral B lymphocytes with Epstein-Barr virus. Comparing the melphalan resistance of different groups of LCLs using the mean Do obtained from clonogenic survival assays, LCLs from melphalan-treated patients were significantly more resistant than LCLs from patients not treated with this drug. However, prior treatment of the patient with ionizing radiation was not associated with increased in vitro resistance of the LCL to this agent. In melphalan-treated patients where LCLs were established serially, the melphalan Do increased after further melphalan treatment in vivo and decreased when no further treatment was given. No correlation was found between age of donor and LCL resistance to any of the above agents. A group of 15 LCLs previously established from non-tumour donors was less resistant to melphalan than the LCLs from patients with ovarian cancer. In a group of 29 patients with advanced disease in whom the clinical response was known, LCL resistance to melphalan appeared to be associated with poor clinical response to cross-linking agents. These results suggest that B cell populations undergo long term, but not necessarily permanent, increases in resistance to melphalan. PMID- 2998315 TI - Glue sniffing neuropathy. AB - Three young men are described in whom a severe, subacute, predominantly motor peripheral neuropathy resulted from the deliberate inhalation of glue vapour. Weakness began after several years of daily glue sniffing and was marked in proximal as well as distal muscles. Muscle wasting was prominent at the time of presentation. Deterioration continued for several weeks after glue sniffing ceased. Peripheral nerve conduction was markedly slow and there was extensive denervation in the muscles. Characteristic changes were seen on sural nerve biopsy. The habit of glue sniffing is now widespread amongst Australian adolescents and this factor should be considered when any young person presents with a peripheral neuropathy. PMID- 2998316 TI - The inflammatory response in the synovium of a patient with Ross River arbovirus infection. AB - Ross River virus has been incriminated in the etiology of many sporadic and epidemic cases of polyarthritis in Australia and the Pacific. Both synovium and synovial exudate fluid recovered from the knee of an epidemic polyarthritis patient showed a predominantly mononuclear leucocyte infiltrate. Infectious virus could not be recovered from the synovial exudate. Functional natural killer cells were detected in the synovial fluid. Their level of cytotoxic activity was similar to that detected in the peripheral circulation. PMID- 2998318 TI - Ultrastructural examination as an aid to the diagnosis of canine pancreatic neoplasms. PMID- 2998317 TI - Human retroviruses. PMID- 2998320 TI - Persistence of passive immunity to porcine parvovirus. PMID- 2998319 TI - The isolation from cattle of 2 bluetongue viruses new to Australia. PMID- 2998321 TI - Isolation of parainfluenza virus from dogs. PMID- 2998323 TI - Angiographic appearances of islet cell tumours of the pancreas. PMID- 2998322 TI - Cancer and wart virus: a review. PMID- 2998324 TI - S1 nuclease removes DNA from eukaryotic metaphase chromosomes: cytological evidence. AB - Fixed and unfixed human chromosomes, as well as fixed rye chromosomes were treated with S1 nuclease, which specifically cleaves single stranded DNA. Subsequent staining with either acridine orange, ethidium bromide or Giemsa revealed that, contrary to what has previously been reported, S1 digestion extensively altered chromosomal morphology and staining intensity, although the alteration was more pronounced in fixed as compared to unfixed metaphases. A number of mechanisms, which may account for our findings, have been invoked: a) the presence in metaphase chromatin of B-DNA/Z-DNA transitional junctions, b) the induction, by alcohol: acid fixation procedure, of nicks within regular B-DNA conformation and c) the induction of sites available to S1 by torsional stress due to metaphase high condensation degree. PMID- 2998325 TI - Polysubstrate monooxygenases in Drosophila, mammals and man. AB - There is overwhelming evidence that polysubstrate monooxygenases play a central role in the metabolism of endogenous compounds as well as in the biotransformation of xenobiotics. These enzyme systems are of great importance in such diverse fields as insecticide resistance, mutagenicity, carcinogenicity, drug metabolism, etc. The constitutive and, in particular, the induced forms represent various products from a multigene family. This has first been shown for the mouse, but evidence is accumulating that this is also true for other mammals and for man. Also in insects a similar picture is emerging. If the regulation of cytochrome P-450 induction resembles in any way the other methods by which prokaryotes and eukaryotes cope genetically with the many forms of environmental selective pressures, it is very likely that most organisms have the genetic capacity to produce not only hundreds but probably thousands of inducible forms of cytochrome P-450 (Nebert et al., 1981). Doubtless, many fields from pest control to cancer prevention to drug safety will profit from the elucidation of the genetic mechanisms involved. PMID- 2998326 TI - Implications of 5'-nucleotidase and its inhibitor for cellular aging and cancer. PMID- 2998327 TI - Cellular senescence: factors modulating cell proliferation in vitro. AB - In our view, two major areas of the investigation of the aging process have been most fruitful over the past few years: namely, the genetic and hormonal strategies aimed at the understanding of in vitro cellular senescence. The genetic studies have primarily utilized cell fusion techniques and viral probes. Along with cell cycle studies involving the induction of thymidine kinase activity and TTP synthesis, the cell fusion studies are most consistent with a late G1 block in senescent cells. This effect would appear to be distinct from the G0 arrest of density-inhibited or mitogen-restricted cell populations. The hormonal studies which have centered on the regulation of cell proliferation have recently focused on peptide hormones. EGF has been of particular interest since it is so well characterized. This receptor system remains largely unchanged throughout the lifespan with the notable exception of the purified receptor associated, autocatalytic, tyrosine-specific kinase activity, which decreases with age. The functional significance of this decrease in enzyme activity is unknown, although its growth regulatory importance is implicated in several systems, and may well represent a critical early G0/G1 event which is absent in senescent cells. PMID- 2998328 TI - Metabolic changes during cardiac maturation. AB - Oxidation rates of palmitate, pyruvate and 2-oxoisocaproate and activities of cytochrome c oxidase and citrate synthase were assayed in heart homogenates of newborn and adult rats and of adult man. All activities doubled or increased more in rat heart at maturation. The rise was due to an increase of both mitochondrial activity and content. In human heart all activities and mitochondrial content were lower than in newborn rat heart. PMID- 2998329 TI - Developmental aspects of cardiac contractile proteins. AB - The change in the isomyosin complement of avian and mammalian hearts was examined during the embryonic period in primary cultures of embryonic myocytes and in the cross-section of the adult ventricular wall. The type of myosin was determined by immunofluorescence using Abs specific for heavy chains of V1 and V3 isomyosins and by cytochemical staining for Ca2+ activated myosin ATPase. Our analysis indicates that the first isomyosin to appear in both chambers of avian heart is of the V3 type (HC beta). With advancing development, however, the atria initiate the expression of HC alpha and repress that of HC beta while the ventricle retains HC beta. In cultured myocytes derived from rat embryos cellular heterogeneity was detected in response to thyroid hormone. The cells are not synchronized in their response. Two populations are discernible with the minor one being thyroid hormone insensitive. Heterogeneity of the cellular populations was also seen in the left ventricle of adult rabbits. Myocytes with a similar isomyosin complement appear clustered with a predominance of V1 in the epicardium. Heterogeneous myocytes are, however, also frequently seen connected by an intercalated disc. PMID- 2998330 TI - Long-term culture and characterization of the adult ventricular and atrial cardiac muscle cell. AB - Ventricular and atrial cardiac muscle cells isolated from the adult rat have been cultured and characterized. Once established in culture these myocytes resemble morphologically their in vivo counterparts except that they are flattened on the surface of the culture flask. These cultured cells possess differentiated ultrastructural characteristics including sarcomerically arranged myofilaments, appropriately organized sarcoplasmic reticulum, intercalated discs and a highly organized transverse tubular system. These cells regain the capacity to carry out semiconservative DNA replication; this capacity had previously been thought to have permanently been lost during early neonatal development. Other studies have shown that cultured ventricular myocytes resemble metabolically the in vivo and isolated perfused rat heart and that they possess an adenylate cyclase system which responds appropriately to adrenergic stimulation. These cells also appear electrophysiologically to resemble the intact adult heart. Long-term culture of adult cardiac muscle cells provides a new and unique system which can be used to study the structure and function of the adult mammalian ventricular and atrial cardiac myocyte. PMID- 2998331 TI - Characterization of sarcolemma from calcium-tolerant canine cardiocytes. AB - Large numbers of calcium-tolerant canine cardiocytes can be isolated from the collagenase-perfused canine myocardium. An average yield of 500 million cells provides abundant tissue for the preparation of subcellular fractions. Using nitrogen cavitation, along with extraction by 0.6 M potassium chloride/sucrose buffer, we have been able to prepare, after differential and sucrose gradient centrifugation, membrane fractions that are more than 100-fold enriched in sarcolemmal marker enzymes. This preparation of sarcolemma has the advantage of being essentially free of plasma membranes from endothelial, smooth muscle, and other cell types residing in the myocardium. PMID- 2998332 TI - Properties of caldesmon isolated from chicken gizzard. AB - Chicken gizzard smooth muscle contains two major calmodulin-binding proteins: caldesmon (11.1 microM; Mr 141 000) and myosin light-chain kinase (4.6 microM; Mr 136 000), both of which are associated with the contractile apparatus. The amino acid composition of caldesmon is distinct from that of myosin light-chain kinase and is characterized by a very high glutamic acid content (25.5%), high contents of lysine (13.6%) and arginine (10.3%), and a low aromatic amino acid content (2.4%). Caldesmon lacked myosin light-chain kinase and phosphatase activities and did not compete with either myosin light-chain kinase or cyclic nucleotide phosphodiesterase (both calmodulin-dependent enzymes) for available calmodulin, suggesting that calmodulin may have distinct binding sites for caldesmon on the one hand and myosin light-chain kinase and cyclic nucleotide phosphodiesterase on the other. Consistent with the lack of effect of caldesmon on myosin phosphorylation, caldesmon did not affect the assembly or disassembly of myosin filaments in vitro. As previously shown [Ngai & Walsh (1984) J. Biol. Chem. 259, 13656-13659], caldesmon can be reversibly phosphorylated. The phosphorylation and dephosphorylation of caldesmon were further characterized and the Ca2+/calmodulin dependent caldesmon kinase was purified; kinase activity correlated with a protein of subunit Mr 93 000. Caldesmon was not a substrate of myosin light-chain kinase or phosphorylase kinase, both calmodulin-activated protein kinases. PMID- 2998333 TI - Cytochrome c-551 and azurin oxidation catalysed by Pseudomonas aeruginosa cytochrome oxidase. A steady-state kinetic study. AB - The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a 'half-hearted' behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c 551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism. PMID- 2998334 TI - Control and production of leukotriene B4 in rat tumour and testicular Leydig cells. AB - As part of an investigation into the role of leukotrienes in steroidogenesis, the formation of leukotriene B4 was investigated in purified Leydig cells from rat testes and from a tumour by using a sensitive radioimmunoassay. Detectable levels were found in both Leydig cell types (70 pg/10(6) cells) and these remain unchanged during incubation for 60 min at 32 degrees C. Addition of the Ca2+ ionophore A23187 increased LTB4 production more than 6-fold within 10 min whereas steroidogenesis was not increased until after 20 min. In the presence of luteinizing hormone or luteinizing hormone releasing hormone agonist no increase in LTB4 was detected in the testis Leydig cells whereas luteinizing hormone stimulated testosterone production from 3.2 +/- 0.1 to 148.9 +/- 7.5 ng/10(6) cells during the same time period. Similar results were obtained with the tumour Leydig cells. The LTB4 was found to be rapidly secreted by the cells in all experiments. The basal and A23187-stimulated levels were inhibited by nordihydroguaiaretic acid, a lipoxygenase inhibitor. It is concluded that LTB4 is produced in Leydig cells and can be stimulated by high calcium levels, but that it is probably not required for the control of steroidogenesis. PMID- 2998335 TI - The study of conformational states of proteins by nuclear magnetic resonance. AB - By the use of examples, mainly of rather rigid proteins, we hope to have shown that conformational analysis of proteins is a problem that is not simply related to the conformational analysis of small molecules. The primary difficulties with proteins are (1) the multitude of possible conformers, (2) the complex dynamical behaviour and (3) the degree of co-operativity within the molecules. Any experimentally derived structural description of a protein is an attempt to represent some average of a complex time dependence. N.m.r. techniques have now reached the point where it is possible to use them to describe many detailed structural features of small globular proteins in solution and to detect and to describe conformational changes in such proteins. In addition, analysis is becoming possible of much less ordered regions of polypeptides, such as are found in less compact proteins, of for example myosin, histones and virus coat proteins, or in denatured states. The limits to the detailed conformational analysis of such proteins are likely to be ones of reality rather than method but the description of the properties shown in Table 1 is by its very nature an extremely important problem in conformational analysis of dynamic macromolecules. PMID- 2998336 TI - Effect of 1,25-dihydroxyvitamin D3 on cyclic AMP responses to hormones in clonal osteogenic sarcoma cells. AB - The effect of 1,25-dihydroxyvitamin D3 on adenylate cyclase responsiveness was studied in the clonal osteogenic sarcoma cell line, UMR 106-06, which responds to several bone active hormones. 1,25-dihydroxyvitamin D3 treatment had no consistent effect on basal formation of cyclic AMP in intact cells, but the responses to parathyroid hormone, isoproterenol, prostaglandin E2, salmon calcitonin and the plant diterpene, forskolin, were all attenuated, by up to 90%. The effect of 1,25-dihydroxyvitamin D3 was dose-dependent, with half-maximal effectiveness at 0.1 nM, and required 48 h treatment of cells before it became apparent. The relative potencies of other vitamin D3 compounds correlated closely with their relative affinities for the 1,25-dihydroxyvitamin D3 receptor and their biological activities in other systems. 1,25-dihydroxyvitamin D3 treatment had no effect on the kinetics of labelled calcitonin binding to UMR 106-06 cells. Furthermore, the fact that such a range of hormones was affected made a receptor mediated mechanism unlikely. Nucleotide stimulatory (Ns) unit activity was assayed after 1,25-dihydroxyvitamin D3 treatment and found to be unchanged. Islet activating protein, an inhibitor of nucleotide inhibitory unit (Ni) activity, failed to modify the 1,25-dihydroxyvitamin D3 effect. Thus the effect of 1,25 dihydroxyvitamin D3 appeared to be exerted beyond hormone receptor and nucleotide regulatory components of the adenylate cyclase complex. It is concluded that 1,25 dihydroxyvitamin D3 attenuates adenylate cyclase response to hormones by a direct or indirect action on the catalytic component of adenylate cyclase. PMID- 2998337 TI - Molecular identification and structural requirement of vasoactive intestinal peptide (VIP) receptors in the human colon adenocarcinoma cell line, HT-29. AB - The human colon adenocarcinoma cell line HT-29 in culture exhibits a cyclic AMP production system highly sensitive to vasoactive intestinal peptide (VIP), making HT-29 cells a unique cultured cell system for studying the mechanism of VIP action [Laburthe, Rousset, Boissard, Chevalier, Zweibaum & Rosselin (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2772-2775]. The quantitative characteristics of VIP receptors in HT-29 cells and their structural requirement and molecular size were studied. 125I-labeled VIP bound in a time-dependent manner to HT-29 cell homogenates. At equilibrium (60 min incubation at 30 degrees C), unlabelled VIP in the 0.01-10 nM concentration range competed with 125I-VIP for binding to cell homogenates. Scatchard analysis of binding data gave a straight line, indicating that VIP bound to a single population of sites with a KD of 0.12 +/- 0.02 nM and a capacity of 120 +/- 9 fmol/mg of protein. The structural requirement of these receptors was studied with peptides structurally related to VIP, either natural or synthetic. Several peptides inhibited 125I-VIP binding to HT-29 cell homogenates with the following order of potency, which is typical of the human VIP receptor: VIP (IC50 = 0.1 nM) greater than VIP-(2-28)-peptide (IC50 = 13 nM) greater than human growth hormone releasing factor (IC50 = 56 nM) greater than peptide histidine isoleucine amide (IC50 = 80 nM) greater than secretin (IC50 greater than 10 000 nM). To characterize the molecular component(s) of the VIP receptor in HT-29 cells, 125I-VIP was covalently bound to cell homogenates by using the cross-linker dithiobis(succinimidyl propionate). Sodium dodecyl sulphate/polyacrylamide-gel autoradiographic studies of affinity-labelled cell homogenates revealed two major bands, corresponding to 125I-VIP-protein complexes of Mr 66 000 and 16 000. The labelling of the Mr-66 000 component was specific, since it was abolished by native VIP, whereas that of the Mr-16 000 component was not. Densitometric scanning of autoradiographs indicated that the labelling of the Mr-66 000 complex was inhibited by low VIP concentrations in the 0.1-10 nM range (IC50 = 0.6 nM), but was unaffected by 1 microM-glucagon or octapeptide of cholecystokinin. It was also decreased by VIP-(2-28)-peptide with a potency 1% that of VIP. Assuming that one molecule of 125I-VIP bound per molecule of protein, one protein of Mr 63 000 was identified as a component of the VIP receptor in HT-29 cells. PMID- 2998338 TI - Gluconeogenesis from acetone in starved rats. AB - To non-anaesthetized rats starved for 3 days, [U-14C]acetone, NaH14CO3, L-[U 14C]lactate, [2-14C]acetate or D-[U-14C]- plus D-[3-3H]-glucose was injected intravenously. From the change in the plasma concentration of labelled acetone versus time after the injection, the metabolic clearance rate of acetone was calculated as 2.25 ml/min per kg body wt., and its rate of turnover as 0.74 mumol/min per kg. The extent and time course of the labelling of plasma glucose, lactate, urea and acetoacetate were followed and compared with those observed after the injection of labelled lactate, acetate and NaHCO3. The labelling of plasma lactate was rapid and extensive. Some 1.37% of the 14C atoms of circulating glucose originated from plasma acetone, compared with 44% originating from lactate. By deconvolution of the Unit Impulse Response Function of glucose, it was shown that the flux of C atoms from acetone to glucose reached a peak at about 100 min after injection of labelled acetone. In comparable experiments the transfer from lactate reached a peak at 14 min after the injection of labelled lactate. It was concluded that acetone is converted into lactate to a degree sufficient to account for the labelling of plasma glucose and is thus a true, albeit minor, substrate of glucose synthesis in starved rats. PMID- 2998339 TI - Ca2+-induced polyphosphoinositide breakdown due to phosphomonoesterase activity in chicken erythrocytes. AB - Treatment of chicken erythrocytes with ionophore A23187 and Ca2+ caused the breakdown of a large proportion of the cellular polyphosphoinositides. Since no diacylglycerol or phosphatidate was generated, but there was a small increase in the level of phosphatidylinositol, it was concluded that breakdown occurred as a result of phosphomonoesterase activation. Experiments with subcellular fractions showed that the phosphomonoesterase activity was present in the cytosolic fraction of the cells. PMID- 2998340 TI - Formation of positive supercoiled DNA by a nuclear factor from myeloma cells. AB - The formation of positive supercoiled DNA by an activity from a hypermutating myeloma line is reported. This activity forms positive supercoils from negative supercoiled DNA, it does not use positive supercoils to form negative ones and does not require an exogenous source of energy. The linking number changes by steps of 1, suggesting a type-I mechanism of action, and there seems to be an upper limit to the degree of positive supercoiling that can be achieved. Positive supercoiled DNA has to be taken into account as a possible structure of DNA in vivo for those functions where torsional stress is involved. PMID- 2998341 TI - Comparison of purified bovine heart and rat liver 6-phosphofructo-2-kinase. Evidence for distinct isoenzymes. AB - Rat liver and bovine heart 6-phosphofructo-2-kinase were purified by the same procedure. Compared with the liver enzyme, the heart enzyme had a smaller apparent Mr, different kinetic properties, was not inactivated by cyclic AMP dependent protein kinase, and contained less fructose-2,6-bisphosphatase activity. These differences suggest that heart and liver 6-phosphofructo-2-kinase are distinct isoenzymes. Likewise, 6-phosphofructo-2-kinase from rat heart and skeletal muscle was not inactivated on treatment with cyclic AMP-dependent protein kinase. PMID- 2998342 TI - A simple one-step procedure for the separation of calpain I, calpain II and calpastatin. AB - The soluble fraction from rabbit brain was adsorbed on a column of phenyl Sepharose. By applying a linear gradient with decreasing salt concentration and increasing pH, it was possible to separate calpain I and calpain II from each other and from the endogenous inhibitor calpastatin. Both enzymes were capable of degrading endogenously labelled neuronal proteins, including slowly axonally transported soluble proteins and rapidly transported membrane-bound proteins, as well as casein. PMID- 2998343 TI - The rate of substrate cycling between fructose 6-phosphate and fructose 1,6 bisphosphate in skeletal muscle from cold-exposed, hyperthyroid or acutely exercised rats. AB - The effects of cold-exposure, the hyperthyroid state and a single exercise bout in vivo on the maximal enzyme activities of 6-phosphofructokinase and fructose 1,6-bisphosphatase in vastus lateralis muscle and the rates of fructose 6 phosphate/fructose 1,6-bisphosphate cycling measured in epitrochlearis muscle in vitro were investigated. In all cases significant changes in substrate cycling rates were observed, whether in the absence of added hormones in vitro (acute exercise), or when stimulated by insulin plus adrenaline (cold-exposure), or with respect to the catecholamine-sensitivity of the cycling rate (the hyperthyroid state). PMID- 2998344 TI - A new site-specific endonuclease, ScaI, from Streptomyces caespitosus. AB - A new site-specific endonuclease has been isolated from Streptomyces caespitosus and named ScaI. Based on analysis of sequences around the restriction sites in pBR322 and pBR325, the recognition sequence of ScaI endonuclease was deduced to be a new hexanucleotide 5'-AGTACT-3'. The cleavage site was determined by comparing the ScaI-cleaved product of a primer-extended M13mp18-SCA DNA, which contains an AGTACT sequence, with dideoxy chain terminator ladders of the same DNA. ScaI was found to cleave the recognition sequence between the internal T and A, leaving flush ends to the cleaved fragments. PMID- 2998345 TI - The putative electrogenic nitrate-proton symport of the yeast Candida utilis. Comparison with the systems absorbing glucose or lactate. AB - Strain N.C.Y.C. 193 of Candida utilis was grown aerobically at 30 degrees C with nitrate as limiting nutrient in a chemostat. The washed yeast cells depleted of ATP absorbed up to 5 nmol of nitrate/mg dry wt. of yeast. At pH 4-6, extra protons and nitrate entered the yeast cells together, in a ratio of about 2:1. Charge balance was maintained by an outflow of about 1 equiv. of K+. Nitrate stimulated the uptake of about 1 proton equivalent during glycolysis or aerobic energy metabolism. Studies with 3,3'-dipropylthiadicarbocyanine indicated that the proton-linked absorption of nitrate, amino acids or glucose depolarized the yeast cells. Proton uptake along with lactate led neither to net expulsion of K+ nor to membrane depolarization. PMID- 2998346 TI - Simultaneous response of myocardial contractility and a major proteolytic process to beta-adrenergic-receptor occupancy in the Langendorff isolated perfused rat heart. AB - The Langendorff isolated rat heart was adapted to the study of minute-to-minute percentage changes in bulk protein degradation by using non-recirculating perfusion. Hearts were perfused at 8 ml/min at 35 degrees C with Krebs-Henseleit buffer containing 11 mM-glucose, and only hearts with regular ventricular rhythm were employed. Proteins were labelled by infusion of [3H]leucine for 0.5 h in vitro. A complete amino acid mixture was then added at 3 times normal rat extracellular concentrations. After labelling, the re-incorporation of [3H]leucine was competitively inhibited by addition of either 4 mM-leucine or 20 microM-cycloheximide. The residual unincorporated radioactivity and the preferentially labelled rapid-turnover proteins were eliminated during a 3 h preliminary perfusion period. The basal rate of release of [3H]leucine and percentage changes were then determined at 1 min intervals, by using each heart as its own control. Leucine metabolism was inconsequential to results. Exchange of intracellular leucine pools with extracellular leucine and subsequent release in effluent perfusate was 95% complete within approx. 2 min. The basal rate of protein degradation was unchanged by electrical stimulation of the heart rate to 360 beats/min or cessation of contractile activity by membrane depolarization under 25 mM-KCl. Infusion of the beta-agonist isoprenaline at 5-500 nM caused a graded inhibition of myocardial protein degradation within 5-6 min, with a maximum inhibition of 30%. This inhibition was sustained for at least 1 h of drug administration and was reversed within 4-6 min of cessation of isoprenaline or simultaneous infusion of 1 microM of the beta-receptor antagonist propranolol. Minute-to-minute adrenergic proteolytic control was a simultaneous co-variable with beta-receptor-mediated inotropic changes in right-intraventricular systolic pressure. Stoppage of the heart in asystole by the Ca2+-channel blocker nifedipine (0.7 microM) delayed the onset, but did not cause sustained reversal, of adrenergic-inhibited degradation, indicating the absence of a direct obligatory mechanistic linkage between the events of the contraction-relaxation cycle and protein degradation in this preparation. PMID- 2998347 TI - Characterization of Ca2+-activated protein phosphatase activity in exocrine pancreas. AB - Ca2+-activated protein phosphatase activity was demonstrated in mouse pancreatic acinar cytosol with alpha-casein and skeletal-muscle phosphorylase kinase as substrates. This phosphatase activity preferentially dephosphorylated the alpha subunit of phosphorylase kinase. After DEAE-cellulose chromatography, the Ca2+ activated phosphatase activity became dependent on exogenous calmodulin for maximal activity. Half-maximal activation was achieved at 0.5 +/- 0.1 microM Ca2+. Trifluoperazine completely inhibited Ca2+-activated phosphatase activity, with half-maximal inhibition occurring at 8.5 +/- 0.6 microM. Mn2+, but not Mg2+, at 1 mM concentration could substitute for Ca2+ in eliciting full enzyme activation. The apparent Mr of the phosphatase as determined by Sephadex G-150 chromatography was 93000 +/- 1000. Submitting active fractions obtained after Sephadex chromatography to calmodulin affinity chromatography resulted in the resolution of a major protein of Mr 55500 +/- 300. In conclusion, Ca2+-activated protein phosphatase activity has been identified in exocrine pancreas and has several features in common with Ca2+-activated calmodulin-dependent protein phosphatases previously isolated from brain and skeletal muscle. It is possible that this Ca2+-activated phosphatase may utilize as substrates certain acinar cell phosphoproteins previously shown to undergo dephosphorylation in response to Ca2+-mediated secretagogues. PMID- 2998349 TI - Comparative kinetic studies on the two interconvertible forms of Streptococcus faecalis inorganic pyrophosphatase. AB - In this work the two interconvertible forms of inorganic pyrophosphatase (EC 3.6.1.1) of Streptococcus faecalis were shown to differ in kinetics. The highly active form of the enzyme was more sensitive to the changes in the Mg2+ concentration, and thus also more sensitive to the inhibition caused by ATP, which competes with PPi for the chelation of Mg2+ ions. We have previously described a kinetic model for the less-active form of S. faecalis inorganic pyrophosphatase [Lahti & Jokinen (1985) Biochemistry 24, 3526-3530]. The kinetic model of the highly active enzyme form is proposed to be a modification of the model of the less-active form in which enzyme activation by free Mg2+ is necessary for the reaction to occur. In this model the enzyme exists in two states, referred to as R- and T-states. In the absence of ligands the enzyme is in the T-state. R-state, i.e. the catalytically active state, exists only in the presence of free Mg2+. Mg1PPi2- is the primary substrate, and free pyrophosphate is a weak inhibitor that cannot serve as a substrate for the highly active form of S. faecalis inorganic pyrophosphatase. This model closely resembles that previously presented for yeast inorganic pyrophosphatase. PMID- 2998350 TI - Effects of beta-casomorphin on dentate hippocampal field potentials in freely moving rats. AB - Intracerebroventricular administration of 166 nmoles of the exogenic opioid beta casomorphin (5) produced a potent and reversible depression of the compound action potential evoked in dentate granule cells by stimulation of the medial perforant path, whereas the extracellularly recorded excitatory postsynaptic potential is left unchanged. This in vivo effect of beta-casomorphin was obviously different from those observed previously in the CA 1 region in hippocampal slice experiments. The results suggest that more than one opioid mechanism determines the granule cell excitability. Some of the possible mechanisms involved in the effects of beta-casomorphins in the hippocampus are briefly discussed. PMID- 2998351 TI - Detection of a countertranscript in promyelocytic leukemia cells HL60 during early differentiation by TPA. AB - We have isolated several cDNA clones corresponding to the mRNAs expressed during early phase of differentiation of promyelocytic leukemia cells HL60 by TPA. Two interrelated clones were examined, one pHH81 and the other pHH58 possessing inserts of 180 and 730 base pairs, respectively. Northern blot analyses of poly(A) RNAs from induced cells revealed that the clone pHH81 hybridized with 4.3kb RNA, while the clone pHH58 hybridized with 4.3kb and, in addition 0.7kb RNA. Sequence determination of those cDNA clones and extensive Northern blot analyses revealed that the inserts of these clones were derived from 4.3kb mRNAs. 0.7kb RNA was hybridized with only 5' upstream region of the clone pHH58, especially with the strand designed to detect anti-sense RNA. Thus we concluded that 0.7kb RNA is a countertranscript of 4.3kb RNA expressed during early differentiation of HL60 cells. PMID- 2998348 TI - The metabolism of neuropeptides. Neurokinin A (substance K) is a substrate for endopeptidase-24.11 but not for peptidyl dipeptidase A (angiotensin-converting enzyme). AB - Both endopeptidase-24.11 and peptidyl dipeptidase A have previously been shown to hydrolyse the neuropeptide substance P. The structurally related peptide neurokinin A is also shown to be hydrolysed by pig kidney endopeptidase-24.11. The identified products indicated hydrolysis at two sites, Ser5-Phe6 and Gly8 Leu9, consistent with the known specificity of the enzyme. The pattern of hydrolysis of neurokinin A by synaptic membranes prepared from pig striatum was similar to that observed with purified endopeptidase-24.11, and hydrolysis was substantially abolished by the selective inhibitor phosphoramidon. Peptidyl dipeptidase A purified from pig kidney was shown to hydrolyse substance P but not neurokinin A. It is concluded that endopeptidase-24.11 has the general capacity to hydrolyse and inactivate the family of tachykinin peptides, including substance P and neurokinin A. PMID- 2998353 TI - cys154 Is important for lac permease activity in Escherichia coli. AB - The lac Y gene of Escherichia coli which encodes the lac permease has been modified by oligonucleotide-directed, site-specific mutagenesis such that cys154 is replaced with either gly or ser. Permease with gly in place of cys154 exhibits essentially no transport activity, while substitution of cys154 with ser also causes marked, though less complete loss of activity. The findings suggest that cys154 plays an important role in lactose:H+ symport. PMID- 2998352 TI - Association of increased polyamine levels with isoproterenol-stimulated mucin secretion in the rat submandibular gland. AB - Incubation of rat submandibular gland slices with 50 microM isoproterenol for 10 40 min stimulated mucin secretion and induced a 3- to 4-fold increase in tissue concentrations of the polyamines putrescine, spermidine and spermine. alpha Difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, suppressed the isoproterenol-induced increase in submandibular polyamines and inhibited mucin secretion. Exogenous putrescine restored tissue polyamine levels and partially reversed the inhibitory effect of alpha-difluoromethylornithine on mucin secretion. Rapid increases in polyamine levels appear to mediate isoproterenol-stimulated mucin secretion in the rat submandibular gland. PMID- 2998354 TI - Activated c-raf gene in a rat hepatocellular carcinoma induced by 2-amino-3 methylimidazo[4,5-f]quinoline. AB - A rat hepatocellular carcinoma, IQ7, induced by 2-amino-3-methylimidazo[4,5 f]quinoline (IQ) gave two transformants of NIH 3T3 cells on DNA mediated gene transfer. One of these transformants was examined further and secondary and tertiary transformants were obtained. The secondary transformant was tumorigenic in nude mice. The activated oncogene in this primary transformant was identified as rat c-raf by Southern blot analysis. PMID- 2998355 TI - Evaluation of a photolabile derivative of 1,25-dihydroxyvitamin D3 as a photoaffinity probe for 1,25-dihydroxyvitamin-D3 receptor in chick intestinal cytosol. AB - We evaluated the viability of 1 alpha, 25-dihydroxyvitamin D3-3 beta-[N-(4-azido 2-nitrophenyl)glycinate] (1,25-(OH)2-D3-ANG), an analog of 1 alpha, 25 dihydroxyvitamin D3 (1,25-(OH)2-D3) as a photoaffinity probe for 1,25-(OH)2-D3 receptor in chick intestinal cytosol. A competitive-binding assay revealed that chick intestinal cytosolic 1,25-(OH)2- D3 receptor bound to 1,25-(OH)2-D3-ANG approximately 20-times less effectively than it did to 1,25-(OH)2-D3. Irradiation of 1,25-(OH)2-D3- ANG in the presence of chick intestinal cytosolic preparation significantly diminished subsequent binding to 3H-1,25-(OH)2-D3, suggesting that the photoaffinity analog was covalently attached to the receptor. Therefore the nitroarylazide derivative of 1,25-(OH)2-D3 may be a valuable photoaffinity probe for the characterization of the 1,25-(OH)2-D3 receptor. PMID- 2998356 TI - Epidermal growth factor receptors in the human glioblastoma cell line SF268 differ from those in epidermoid carcinoma cell line A431. AB - Two established human tumor cell lines, epidermoid carcinoma line A431 and glioblastoma line SF268, were studied to compare the interaction of each with epidermal growth factor (EGF). SF268 cells bound [125I] EGF with 35-40 fold higher affinity than did the A431 cells. The EGF binding sites of both lines were photoaffinity labeled using 2,4-NAPS-[125I] EGF, a photoreactive derivative of EGF. Extracts of photolysed cells analyzed by SDS-PAGE showed a difference between the two cell lines in the high molecular weight component corresponding to the EGF receptor. EGF in a dose range from 0.3-200 nM had no effect on thymidine incorporation by SF268 cells, whereas thymidine incorporation by A431 cells was markedly inhibited by EGF. PMID- 2998358 TI - Structure-activity relationships of atrial natriuretic factor (ANF). III. Correlation of receptor affinity with relative potency on aldosterone production in zona glomerulosa cells. AB - The activity of various fragments of ANF as inhibitors of aldosterone secretion and as competitors of [125I] ANF (Arg101-Tyr126) binding to specific receptors was studied in bovine zona glomerulosa. Shortening or lengthening the N-terminal segment of ANF does not alter its biological activity while minimally altering affinity for its receptor. Removal of the C-terminal to Cys121 or expansion up to Arg128 leads to 1000-fold decrease in receptor affinity and activity. The results indicate the importance of the C-terminal segment of ANF in determining its active conformation. PMID- 2998357 TI - Multiple thyroid hormone binding sites on rat liver nuclear envelopes. AB - Nuclear envelopes relatively free of plasma membrane contamination were isolated from the male rat liver. Equilibrium binding of T3 to nuclear envelopes occurred after incubation for 3 h at 20 degrees C. Scatchard analysis revealed two classes of binding sites; a high affinity site having a KD of 1.8 nM with a maximum binding capacity of 14.5 pmol/mg protein and a low affinity site having a KD of 152.1 nM with a maximum binding capacity of 346.8 pmol/mg protein. No degradation of the radioligand occurred during incubation with the nuclear envelope. T4, rT3 and Triac competed effectively for the binding of T3 to the high affinity site whereas only T4 competed well for binding to the lower affinity site. The binding site was protease sensitive but not salt extractable. Multiple T3 binding sites having similar affinities have been reported on plasma membranes. An intriguing possibility is that membrane binding sites may be involved in translocation of thyroid hormone across membrane barriers. PMID- 2998359 TI - Biosynthesis of functionally active heparin cofactor II by a human hepatoma derived cell line. AB - Human plasma heparin cofactor II (HCII) inhibits thrombin by rapidly forming a stable, equimolar complex in the presence of heparin or dermatan sulfate. Cultured human hepatoma-derived cells (PLC/PRF-5) secreted (approximately equal to 200 ng/ml in 3 days) a protein of MW - 72 kD that was immunoisolated and immunoblotted with anti-HCII, co-migrated on SDS-PAGE with human plasma HCII, and formed covalent complexes with thrombin (MW - 101 kD) in the presence but not absence of heparin or dermatan sulfate; these complexes co-migrated with those obtained by incubating thrombin with human plasma under the same conditions. HCII was not detectable (less than 0.13 ng/ml) in post-culture medium from cultured human umbilical vein endothelial cells or human foreskin fibroblasts. PMID- 2998360 TI - The metal-mediated formation of hydroxyl radical by aqueous extracts of cigarette tar. AB - Aqueous extracts of cigarette tar produce hydroxyl radicals that are spin trapped by 5,5-dimethyl-1-pyrroline-N-oxide. The addition of catalase almost completely inhibits and superoxide dismutase partially inhibits spin adduct formation. The addition of ethylenediamine tetraacetic acid greatly increases the amount of hydroxyl radical adduct observed; in contrast, diethylenetriamine pentaacetic acid causes complete inhibition of spin adduct formation. We suggest that the hydroxyl radical arises from the metal-mediated decomposition of hydrogen peroxide, and that hydrogen peroxide is formed from the reduction of dioxygen by the semiquinones present in the cigarette tar. PMID- 2998362 TI - Increase in the type 2 insulin-like growth factor receptors in the rat kidney during compensatory growth. AB - We have observed an increase in binding of IGF-I and IGF-II to microsomes obtained from rat kidneys undergoing compensatory growth following contralateral nephrectomy. This increase was evident by the 4th day and it preceded observable growth of the kidney. Binding returned to control levels just prior to the flattening of the growth curve of the kidney. The increase was due to an increase in the type 2 binding sites, the only type unequivocally present, from 95.3 +/- 2.7 to 117.2 +/- 4.1 pM/150 micrograms of microsome protein at its maximum at 4 days. PMID- 2998361 TI - Selective inhibition of leukotriene B4 biosynthesis in rat pulmonary alveolar macrophages by dietary selenium deficiency. AB - Weanling male Fisher 344 rats were maintained on low selenium basal and Se supplemented diets for 38 weeks. A several fold reduction in the glutathione peroxidase activity of the lung and liver tissues in rats maintained on low Se basal diet established their Se-deficient status. Analysis of the supernatants from resting pulmonary alveolar macrophage suspensions showed negligible extracellular release of PGE2, TXB2 and LTB4 in both diet groups. A challenge with opsonized zymosan particles increased the release of the same three arachidonic acid metabolites by several fold in both diet groups. The differences between the two diet groups with respect to the secretion of the products of the cyclooxygenase pathway, PGE2 and TXB2 were negligible. By contrast, a significant reduction in the extracellular release of LTB4 was observed in cells from animals on low selenium basal diet. These results suggest a selective inhibition of LTB4 biosynthesis in pulmonary alveolar macrophages by dietary deficiency of selenium. PMID- 2998363 TI - Phorbol ester stimulates calcitonin secretion synergistically with A23187, and additively with dibutyryl cyclic AMP in a rat C-cell line. AB - The mechanism of action of 12-O-tetradecanoyl phorbol-13-acetate (TPA) on calcitonin secretion was studied in a rat C-cell line, rMTC 6-23. TPA stimulated calcitonin secretion at the concentration of 16nM. This effect was synergistically enhanced with calcium ionophore, A23187. Synthetic diacylglycerol, 1-oleoyl-2-acetyl-glycerol (OAG), also showed a synergism with A23187 on calcitonin secretion. When dibutyryl cyclic AMP was added with TPA, an additive effect was obtained. These data suggest that C-kinase might be a possible regulator of calcitonin secretion in addition to the cyclic AMP-mediated pathway. PMID- 2998364 TI - CSF-1 stimulates Na+K+-ATPase mediated 86Rb+ uptake in mouse bone marrow-derived macrophages. AB - 86Rb+ was used as an isotopic tracer for the measurement of K+-uptake into quiescent murine bone marrow-derived macrophages. 86Rb+ uptake was inhibited by ouabain indicating a Na+K+-ATPase is being measured. In support of this finding, increased sensitivity to ouabain inhibition was seen when the K+ content of the medium was reduced. A purified colony stimulating factor (CSF-1) was shown to stimulate the ouabain-sensitive 86Rb+ uptake in a dose-dependent manner. Such colony stimulating factor stimulation of 86Rb+ (K+) influx was rapid, with a maximal effect seen 10 minutes after growth factor addition followed by a gradual decrease. Thus increased Na+K+-ATPase activity was an early response of macrophages to the colony stimulating factor. PMID- 2998365 TI - Diaziquone as a potential agent for photoirradiation therapy: formation of the semiquinone and hydroxyl radicals by visible light. AB - When diaziquone was irradiated with 500 nm visible light, hydroxyl free radicals as well as the diaziquone semiquinone were produced. The diaziquone semiquinone is a stable free radical that exhibits a characteristic 5-line electron spin resonance (ESR) spectrum. Since hydroxyl free radicals are short lived, and not observable by conventional ESR, the nitrone spin trap 5,5-dimethyl-1-pyrroline-1 oxide (DMPO) was used to convert hydroxyl radicals into longer lived ESR detectable spin adducts. The formation of hydroxyl radicals was further confirmed by investigating reactions in which hydroxyl radical scavangers, sodium formate and dimethylsulfoxide, compete with the spin traps DMPO or POBN (alpha-(4-Pyridyl 1-oxide)-N- tert-butylnitrone) for hydroxyl free radicals. The products of these scavenging reactions were also trapped with DMPO or POBN. If drug free radicals and hydroxyl free radicals are important in the activity of quinone-containing antitumor agents, AZQ may have a potential in photoirradiation therapy or photodynamic therapy. PMID- 2998366 TI - Characterization of the Ca2+ coordination site regulating binding of Ca2+ channel inhibitors d-cis-diltiazem, (+/-)bepridil and (-)desmethoxyverapamil to their receptor site in skeletal muscle transverse tubule membranes. AB - Ca2+ inhibits (-)[3H]desmethoxyverapamil, d-cis-[3H]diltiazem and (+/ )[3H]bepridil binding to skeletal muscle transverse-tubule membranes with a half maximum inhibition constant, K0.5 = 5 +/- 1 microM. This value is close to that of the high affinity Ca2+ binding site which controls the ionic selectivity of the Ca2+ channel found in electrophysiological experiments suggesting that the Ca2+ coordination site which regulates the ionic selectivity is also the one which alters binding of the Ca2+ channel inhibitors investigated here. Ca2+ and ( )D888 bind to distinct sites. Occupation of the Ca2+ coordination site decreases the affinity of (-)D888 for its receptor by a factor of 5. Other divalent cations have the same type of inhibition behavior with the rank order of potency Ca2+ (K0.5 = 5 microM) greater than Sr2+ (K0.5 = 25 microM) greater than Ba2+ (K0.5 = 50 microM) greater than Mg2+ (K0.5 = 170 microM). PMID- 2998367 TI - Retinoic acid modulation of 1,25(OH)2 vitamin D3 receptors and bioresponse in bone cells: species differences between rat and mouse. AB - Retinoic acid (RA) caused a reduction in the level of 1,25(OH)2D3 receptors to 1/3 of control in rat osteoblast-like cells (ROB) while increasing the receptor level to 3-fold the control in mouse osteoblast-like cells (MOB). Scatchard analysis of receptor binding indicated that there was no change in affinity for 1,25(OH)2D3. The changes in receptor levels required time to develop and were dose-dependent. RA also modulated the ability of cells to respond to 1,25(OH)2D3 as measured by the induction of the enzyme 25(OH)D3-24 hydroxylase. Induction of enzyme activity by 1,25(OH)2D3 closely paralleled receptor level established by RA pretreatment. In MOB, the up-regulation of the receptor occurred despite the action of RA to inhibit DNA, RNA and protein synthesis. However, RA stimulation of 1,25(OH)2D3 receptor levels was blocked by the addition of cycloheximide or actinomycin D, indicating that the up-regulation required protein and RNA synthesis. The opposite effect of RA on mouse and rat cells suggests that important species-dependent factors modulate the action of retinoids on mammalian cells. PMID- 2998368 TI - Calcitonin gene-related peptide stimulates cyclic AMP formation in rat aortic smooth muscle cells. AB - In rat aortic smooth muscle cells in culture, calcitonin gene-related peptide stimulated cAMP formation in a dose-dependent manner, half-maximally effective at 0.5 to 1 nM. There was no effect on formation of cGMP, which was increased 300 fold in the same experiments by atriopeptin or sodium nitroprusside. The vasodilator effect of CGRP in rat aorta requires an intact endothelium, indicating that increase in vascular smooth muscle cAMP is not in itself sufficient to bring about relaxation. cAMP is probably a mediator of CGRP action in vascular smooth muscle. PMID- 2998369 TI - Human lung mast cell tryptase fails to activate procollagenase or degrade proteoglycan. AB - Pig synovial and human skin fibroblast procollagenases were treated with highly purified tryptase, the major proteinase of human mast cells, to determine whether this trypsin-like proteinase could activate the latent form of collagenase and so be involved in connective tissue breakdown. No significant activation of either human or pig procollagenase was found, but the highest concentration of tryptase partially destroyed procollagenase. Tryptase did not degrade type I collagen or proteoglycan. These data indicate that human mast cell tryptase does not contribute to connective tissue breakdown via procollagenase activation or via proteoglycan degradation. PMID- 2998370 TI - Inhibition of transmembrane movement and metabolism of platelet activating factor (PAF-acether) by a specific antagonist, BN 52021. AB - Incorporation of 1-[3H]-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine ([3H] PAF acether) into rabbit platelet phosphatidylcholine (PC) was inhibited by a specific antagonist, BN 52021 (IC50 5.6 X 10(-6) M, maximal effect, i.e 70% inhibition, at 10(-4) M). Under the same conditions, [3H] lyso-PAF-acether incorporation remained 9 fold lower, compared to PAF-acether, without any effect of BN 52021. Upon cell lysis, both phospholipids attained the same rate of metabolic conversion, corresponding to a 1.15-fold and a 12-fold increase for PAF acether and lyso-PAF-acether, respectively. In none of these cases was BN 52021 effective. It is concluded that transmembrane movement of the two phospholipids represents the limiting step of their metabolism. The higher rate of PAF-acether conversion by intact platelets could involve its binding to a membrane receptor, as suggested by the inhibitory effect of BN 52021, the significance of which is discussed. PMID- 2998371 TI - Phosphoinositides are not phosphorylated by the very active tyrosine protein kinase from the murine lymphoma LSTRA. AB - We studied the ability to phosphorylate phosphoinositides by 3 different subcellular preparations, and immunopurified tyrosine protein kinase (TPK) from two murine lymphoma cell lines induced by the Moloney murine leukemia virus: LSTRA with a very active TPK and MBL2 without significant TPK activity. We could not find any difference in the phosphorylation of phosphoinositides by these preparations. The TPK purified with two antibodies which phosphorylate actively tyrosine on exogenous substrate were unable to phosphorylate phosphoinositides. PMID- 2998372 TI - A genetic study of inositol trisphosphate involvement in phototransduction using Drosophila mutants. AB - Phosphatidylinositol 4,5-bisphosphate phosphodiesterase activity was found to be almost absent in the compound eyes of Drosophila visual mutant, norpA (no receptor potentials A). We compared the enzyme activities among independently isolated norpA alleles, each having a different degree of the vision defect. A close correlation was found between the size of receptor potentials (electroretinogram), phototactic behavior and the enzyme activity. The correlation exists not only among alleles, but also in a single, temperature dependent allele under different temperature; the enzyme activity of flies kept at 18 degrees C (phototactic) was about five times higher than that of the ones kept at 28 degrees C (blind). These results suggest that hydrolysis of phosphatidylinositol 4,5-bisphosphate is involved in phototransduction process in Drosophila eyes. PMID- 2998373 TI - Manganese stimulates incorporation of [3H]inositol into an agonist-insensitive pool of phosphatidylinositol in brain membranes. AB - In a particulate preparation from rat brain, manganese ions stimulate the incorporation of [3H]inositol into inositol phospholipids in a concentration dependent manner. Incubation with CDP-diacylglycerol (0.5 mM) alone had no effect on the incorporation of [3H]inositol but potentiated the stimulatory effect of manganese. Despite the increase in [3H]inositol incorporation into phosphatidylinositol, the carbachol-induced accumulation of [3H]inositol-1 phosphate was unaltered in membranes preincubated with manganese but when coincubated with CDP-diacylglycerol the carbachol-induced accumulation of [3H]inositol-1-phosphate was increased. These data suggest that manganese stimulates the incorporation of [3H]inositol into an agonist-insensitive pool of phosphatidylinositol. PMID- 2998374 TI - Activators of protein kinase C stimulate meiotic maturation of rat oocytes. AB - Agonistic analogs of gonadotropin releasing hormone can induce oocyte maturation in rat follicle-enclosed oocytes (1-5). Cyclic AMP does not rise following exposure of the ovarian follicle to GnRH (3) suggesting that cAMP-dependent protein kinase is not involved in the mechanism of GnRH action in this system. Protein kinase C, which is independent of cAMP, has recently been reported to mediate GnRH action in the pituitary (6-8). The possible involvement of this enzyme in the regulation of oocyte maturation has been tested in the present study. We report here that phospholipase C and direct activators of protein kinase C can mimic the response of rat oocytes to GnRH. These results suggest that GnRH-induced meiotic maturation of rat oocytes is mediated by the phospholipid-dependent protein kinase, protein kinase C. PMID- 2998375 TI - Rat brain and liver soluble phospholipase C: resolution of two forms with different requirements for calcium. AB - Two forms of phospholipase C, hydrolyzing specifically inositol phospholipids, are resoluted and partially purified from rat brain as well as liver cytosol by DEAE-cellulose followed by heparin-Sepharose, Sephacryl S-400, and aminohexyl Sepharose column chromatographies. With phosphatidylinositol as substrate, at pH 7.4 one is most active at 10(-6) M Ca2+ (Type I) whereas the other requires 10( 3) M Ca2+ (Type II). At pH 5.5 both Type I and II are active at 10(-3) M Ca2+ but essentially inactive at lower concentrations of this divalent cation. Both Type I and II hydrolyze preferentially polyphosphoinositides particularly at lower concentrations of Ca2+. PMID- 2998376 TI - Polyclonal anti-idiotypic opioid receptor antibodies generated by the monoclonal beta-endorphin antibody 3-E7. AB - Anti-idiotypic antibodies were raised in rabbits against the monoclonal beta endorphin antibody 3-E7. These antibodies inhibit beta-endorphin binding to the 3 E7 antibody, binding of 3H-diprenorphine to solubilized opioid receptors and the binding of 125I-beta-endorphin to rat brain membranes. Exposure of NG-108CC15 hybrid cells to anti-idiotypic antibodies produces an opioid-like inhibition of PGE1-stimulated cAMP accumulation. These data suggest that the antibodies raised by the anti-idiotypic route both bind to and activate opioid receptors. PMID- 2998377 TI - Partial characterization and solubilization of receptors for atrial natriuretic factor in rat glomeruli. AB - Specific receptors for atrial natriuretic factor (ANF) have been identified and solubilized in glomeruli from rat kidney. Radioiodinated synthetic ANF (Arg 101 Tyr 126) bound to a single class of high affinity (Kd 27 +/- 24 pM) sites with a density of 390 +/- 230 fmole/mg protein. The binding was time- and temperature dependent, saturable and reversible. The ANF-receptor complex was not affected by angiotensin II, ACTH or vasopressin. Solubilization with 10 mM 3-[(3 cholamidopropyl)-dimethylammonio]- 1-propane sulfonate (CHAPS) slightly increased the affinity for ANF (Kd 5.0 +/- 3.3 pM) without affecting the density (250 +/- 110 fmole/mg protein). Similar results were found with 1% Triton X-100. ANF related peptides interact generally in the same way with non-solubilized and solubilized receptors, indicating a fully preserved specificity of the receptors. PMID- 2998378 TI - Structural organization of the mouse proto-myb gene. AB - Proto-myb is a highly conserved cellular gene that is closely related to v-myb, the transforming gene of the avian myeloblastosis virus. We have isolated lambda clones encompassing 21 kbp of mouse DNA that contains portions of the proto-myb gene. Also, we have isolated a cDNA clone containing a 2.5 kbp insert corresponding to mouse proto-myb mRNA. By analyzing both the cloned DNAs and mouse genome DNA, we have constructed a restriction map for mouse proto-myb that extends over 50 kbp. PMID- 2998379 TI - The activation of the human neutrophil respiratory burst occurs only at temperatures above 17 degrees C: evidence that activation requires membrane fusion. AB - By considering the effects of temperature on the respiratory burst activity of human neutrophils stimulated by phorbol myristate acetate, we have found that activation only occurs at 17 degrees C and above. Between 20 and 30 degrees C, the rates of oxygen consumption rose dramatically in correspondence with cellular release of gelatinase and lactoferrin. Inasmuch as these mark tertiary and specific granules, respectively, a necessity for membrane fusion of one or both granules with the plasma membrane during triggering of the respiratory burst is very probable. PMID- 2998380 TI - Direct stimulation by thyrotropin-releasing hormone (TRH) of polyphosphoinositide hydrolysis in GH3 cell membranes by a guanine nucleotide-modulated mechanism. AB - Polyphosphoinositide hydrolysis was examined in membranes from thyrotropin releasing hormone (TRH)-responsive GH3 pituitary cells. [3H]Inositol phosphates (IP2 and IP3) were generated upon incubation of membranes from [3H]inositol labeled cells indicating the presence of a membrane-associated polyphosphoinositide phosphodiesterase (PPI PDE). Membrane PPI PDE activity was found to be stimulated by TRH and by GTP-gamma-S in Ca2+-modulated manner. In addition, TRH-stimulated PPI hydrolysis was potentiated by GTP. These results demonstrate direct in vitro effects of a hormone on PPI turnover and suggest the involvement of a GTP-binding component in transmembrane signalling by TRH. PMID- 2998381 TI - Differential up-regulation of microsomal and synaptic membrane mu opioid receptors. AB - Naltrexone was administered to rats for 7 days by osmotic minipump (5 mg/kg/day) and thereupon, forebrain mu opioid receptor levels in subcellular fractions were monitored by homologous displacement of [3H]D-ala2-mePhe4-gly-ol5 enkephalin binding. Microsomes displayed increases in mu receptor concentrations that were twofold greater than those associated with synaptic plasma membrane fractions (92 vs. 51%). Levels in crude membranes rose 77%. Binding affinities were unchanged. PMID- 2998382 TI - Autoxidation reactions of hemoglobin A free from other red cell components: a minimal mechanism. AB - Rates of autoxidation reactions are determined for normal human hemoglobin A preparations which are extensively purified to remove all other redox active red cell components. The effects of superoxide dismutase, catalase, and hydroxyl radical scavengers on the reaction provide evidence for superoxide formation as the rate determing step followed by fast reactions that involve peroxide and hydroxyl radical. These results support a minimum overall mechanism for heme iron(II) oxidation and dioxygen reduction to water. Side reactions also occur that result in the modification and precipitation of the protein moiety; catalase and hydroxyl radical scavengers reduce the extent of the side reactions. These studies provide insight into the basis of oxidant stress in the red cell. PMID- 2998383 TI - Membrane fatty acids, lipid peroxidation and adenylate cyclase activity in cultured neural cells. AB - Lipid peroxidation and basal adenylate cyclase activity have been examined in neuroblastoma cultured with a variety of exogenous fatty acids. Formation of cyclic AMP depended upon fatty acid type, with supplementation affecting activities in the order: linoleate greater than cis-vaccenate = linolenate greater than control (132.7, 72.6, 71.9 and 36.0 pmol cAMP formed/mg protein, respectively). Lipid peroxidation, measured by formation of malondialdehyde (MDA), also varied with fatty acid; however, there was little correlation between MDA production and basal cyclase activity. Inclusion of alpha-tocopherol in culture-medium blocked MDA formation without affecting cAMP accumulation. Fe2+ dependent induction of peroxidation was accompanied by a time-dependent inhibition of cyclase activity. PMID- 2998384 TI - The catalytic subunits of the (Na+,K+)-ATPase alpha and alpha(+) isozymes are the products of different genes. AB - The sequences of the first 14 amino acids of the (Na+,K+)-ATPase catalytic subunits from rat kidney (alpha) and rat brain axolemma (alpha(+)) have been determined. They are: (alpha), NH2-Gly-Arg-Asp-Lys-Tyr-Glu-Pro-Ala-Ala-Val-Ser Glu-His-Gly; (alpha(+)), NH2-Gly-Arg-Glu-Tyr-Ser-Pro-Ala-Ala-Glu-Val-Ala-Glu-Val Gly. Although they are highly homologous, it is clear these sequences are also sufficiently different to conclude they are the products of different genes, or at least different exons of the same, differentially spliced, gene. Among mammals, the amino terminal sequence of the kidney alpha chain is essentially invariant. Thus this section of the (Na+,K+)-ATPase molecule is more highly conserved in one tissue between several species than between different tissues in the same species. This may reflect upon the difference in function of the alpha and alpha(+) isozymes of (Na+,K+)-ATPase. PMID- 2998385 TI - Altered rhodopsin accessibility in the retinal dystrophic mouse. AB - Retinas obtained from 7-day-old rd mice show less reaction with antirhodopsin antisera than retinas from normal mice of the same age. Likewise, antisera prepared against synthetic peptides, which corresponds to the carboxyl terminus of rhodopsin, also react less with rd retinas from 7-day-old mice. In contrast, Western blots of denatured rhodopsin from rd vs. normal retinas of the same age indicate no change in the total quantity of this protein. These results demonstrate that in the 7-day-old rd mouse retina, rhodopsin is not altered in quantity; rather, it is less accessible to reaction with anti-rhodopsin antisera. Furthermore, these results suggest that the site of altered accessibility is on the carboxyl terminus of rhodopsin. PMID- 2998386 TI - Anti-rabbit thymus actin antibody inhibits proliferation of Epstein-Barr virus transformed human B cell line LA350: augmentation by cyclic AMP. AB - Antibody to actin isolated from the rabbit thymus gland exerted a dose-dependent inhibitory effect upon the proliferation of an Epstein-Barr virus-transformed human B cell line, LA350. Purified rabbit thymus actin specifically reversed the inhibitory effect of antibody by competing with surface actin on LA350. For example, LA350 proliferation at 48 hr was inhibited 90% at a 1:10 antibody dilution (p less than 0.001) but only 48% and 20% in the presence of 190 and 380 micrograms/ml of rabbit thymus actin, respectively (p less than 0.001 for reversal). Cyclic AMP augmented in a dose-related fashion the inhibitory effects of antibody, e.g., a 1:20 dilution of antibody inhibited 10, 19, 41, and 67% at 0.0, 0.5, 1.0, and 2.0 mM cAMP, respectively (p less than 0.001). We conclude that anti-actin antibody recognizes surface actin on a human B cell line and produces a functional inhibition of proliferation by a process that is augmented by cAMP. PMID- 2998387 TI - Monensin inhibits collagenase production in osteoblastic cell cultures and also inhibits both collagenase release and bone resorption in mouse calvaria cultures. AB - Monensin, a monovalent cation ionophore, inhibited collagenase production in mouse osteoblast-rich bone cell and clonal osteogenic cell cultures. Inhibition of parathyroid hormone-stimulated bone resorption by monensin was also studied in calvaria cultures. Collagenase activity levels in the medium decreased concomitantly with the inhibition of bone resorption by monensin, indicating that monensin inhibited bone resorption by blocking collagenase secretion from osteoblasts in bone explants. PMID- 2998388 TI - Poly(dG-dC) in the Z-form inhibits E. coli DNA polymerase I and AMV DNA polymerase activity. AB - The effect of Z-conformation of DNA on its template activity in DNA synthesis reactions in vitro has been studied. Normal poly(dG-dC) in the B-form, brominated and unbrominated in the Z-form have been compared for their template activity in DNA synthesis reactions mediated by AMV DNA polymerase and E. coli DNA polymerase I. The results indicate that poly(dG-dC) in the Z-form is totally inactive as a template for DNA synthesis and further that it is a strong competitive inhibitor of copying of the B-form DNA. PMID- 2998389 TI - Existence in animal tissues of adenosine triphosphate thiamin diphosphate phosphotransferase [EC 2.7.4.15]. AB - An enzyme which catalyzes the synthesis of thiamin triphosphate from thiamin diphosphate (TDP), thiamindiphosphate kinase (ATP:thiamin diphosphate phosphotransferase) [EC 2.7.4.15], was detected in animal tissues. The enzyme was partially purified (150-fold) from the cytosol fraction of guinea pig brain. The enzyme reaction required free (not protein-bound) TDP, ATP, Mg2+, and a cofactor, which is a low molecular weight and heat-stable compound. The enzyme activity was optimal at pH 11 and at 25 degrees C. A stoichiometric transfer of 32P from [gamma-32P]ATP to TDP was demonstrated. Km values for TDP and ATP were calculated to be 1.1 mM and 10 microM, respectively, and Vmax was 868 nmol/mg of protein/hr. The enzyme was found solely in the cytosol fraction of guinea pig brain and was also detectable in the skeletal muscle and heart. These results provide strong evidence for the existence of TDP kinase in animal tissues. PMID- 2998390 TI - Tetradecanoyl phorbol-13-acetate counteracts the responsiveness of cultured thyroid cells to thyrotropin. AB - We have studied the effects of TPA on the metabolism of porcine thyroid cells cultured for 1-4 days in the absence (control cells) and in the presence of 0.1 mU/ml TSH (TSH cells). The phospholipid turnover, evaluated after a 2 hr incorporation of 32P-phosphate into phospholipids, is markedly modified by the presence of TPA (1.5 microM, 2 hr) in the incubation medium of control and TSH treated cells. The total incorporation is 3-4 times higher than untreated cells, the labelling of phosphatidylinositol (PI) is slightly decreased or unchanged whereas that of phosphatidylcholine (PC) is strongly increased. The increased labelling of PI, promoted by an acute TSH treatment is counteracted by TPA. This TPA effect is not observed when prelabelled cells are challenged for 5 min with the drug. A similar effect is observed when 10 nM TPA is added in the culture medium for 20 hr. The addition of TPA does not affect significantly the protein iodine content in 3 or 4 days control cells incubated for 45 min or 2 hr with 125I-iodine, but dramatically decreases the very high iodination rate of TSH cells. We have tested the TPA effect on the cyclic AMP accumulation for the last 5 min of a 2 hr incubation. TPA inhibits by about 50-80% the stimulation evoked by TSH and only by 10% that evoked by forskolin (0.1 mM). These results suggest a possible link between the PC turnover and the adenylate cyclase responsiveness to TSH and the iodination rate. PMID- 2998391 TI - Effect of phosphatase inhibition of in vitro dopamine sulfation and 3' phosphoadenosine-5'-phosphosulfate catabolism in human brain. AB - The effects of inhibition of phosphatase activity in 100,000 g supernatant solution from human frontal cortex on dopamine (DA) conjugation were examined using the phosphatase substrate p-nitrophenyl phosphate (pNPO4). The increases in DA sulfation seen in the presence of pNPO4 suggested that inhibition of phosphatase activity in high speed supernatant solutions of brain may substantially alter the pattern of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) metabolism and subsequently the rate of DA sulfation. Accordingly, the effects of the pyrophosphate analog phosphonoacetic acid (PAA) on the extent of DA sulfation and PAPS metabolism were examined in 100,000 g supernatant solution from human frontal cortex. At concentrations up to 10 mM, PAA markedly reduced PAPS hydrolysis to inorganic sulfate and 3'-phosphoadenosine-5'-phosphate (PAP) and significantly extended the linear time period for the sulfation of DA. These findings suggest that the phosphatase enzymes that degrade PAPS to produce the end product inhibitor, PAP, and possibly other break-down products of PAP, play an important role in determining the observed levels of phenol sulfotransferase activity in tissue from human brain in vitro. PMID- 2998392 TI - Identification and regulation of alpha 2-adrenergic receptors in rabbit ileal mucosa. AB - The alpha 2-adrenergic receptors in rabbit ileal mucosal membranes can be identified by using [3H]clonidine. [3H]Clonidine bound to a homogeneous population of sites (30-120 fmoles/mg protein) with a KD of 2.2 nM at 25 degrees. Alpha-adrenergic agonists and antagonists competed with [3H] clonidine for the binding sites with an order of potency typical for alpha 2-receptors. Mg2+, Ca2+, or Mn2+ (2.4 mM) markedly increased the binding of [3H]clonidine. At the maximally effective concentration, Mg2+ increased both the binding affinity of [3H]clonidine and the number of receptor sites. Both NaCl and GppNHp, the guanyl nucleotide, inhibited [3H]clonidine binding. NaCl decreased the binding affinity of [3H]clonidine, with no appreciable effect on the number of receptor sites. These findings indicate that ileal mucosal alpha 2-receptors can exist in multiple affinity states, which can be regulated by divalent cations, NaCl, and guanyl nucleotides. It appears that NaCl and GppNHp regulate alpha 2-receptors in ileal mucosa by different mechanisms. PMID- 2998393 TI - Alteration of chemically induced hepatotoxicity by copper (II) (3,5 diisopropylsalicylate)2. AB - The effects of copper (II) (3,5-diisopropylsalicylate)2 (CuDIPS), which is a synthetic superoxide dismutase, on the hepatotoxicity of carbon tetrachloride and acetaminophen in fed and fasted animals were investigated. CuDIPS did not alter the covalent binding of metabolites of either of these chemicals to the hepatic endoplasmic reticulum. However, CuDIPS did inhibit the hepatotoxicity of carbon tetrachloride by inhibiting the induction of lipid peroxidation by carbon tetrachloride. CuDIPS had only a slight, and histologically insignificant, ability to decrease acetaminophen hepatotoxicity which is related to the inability of CuDIPS to prevent depletion of reduced glutathione by acetaminophen. The observation that fasting potentiates the hepatotoxicity of acetaminophen is emphasized, and the mechanism of this potentiation is suggested to be related to the depletion of reduced glutathione. PMID- 2998395 TI - Dose-dependent reduction of lipopolysaccharide pyrogenicity by polymyxin B. AB - Lipopolysaccharides (LPS) from Gram-negative bacteria are potent pyrogens in mammals. Polymyxin B (PB), a cationic polypeptide antibiotic, binds lipid A, the active moiety of LPS, with high affinity and abrogates several biological responses to LPS. We studied the effect of PB on pyrogenicity of purified LPS from E. coli 0111:B4 in rabbits. PB reduced the pyrogenic response to LPS in a dose-dependent manner at mass ratios (PB:LPS) from 5:1 to 100:1. Previous reports have suggested that PB is effective only at much higher doses. In our hands, PB itself is pyrogenic, unless previously gamma-irradiated. Our results confirm in vivo the anti-endotoxic action of PB. PMID- 2998394 TI - N-Ethyl-17(R,S)-methyl-(6aR,10aR)-delta 8-tetrahydrocannabinol-18-oic amide. Brain pharmacokinetics in mice, triglyceride/phospholipid partitioning and generalization to the discriminative stimulus properties of delta 9-THC in rats. AB - The title compound, designed as a model for the affinity moiety of a cannabinoid affinity gel was synthesized in tritiated form (sp. act. 7.27 mCi/mmole). To validate the affinity approach to isolate the putative THC receptor, the properties of the amide were studied. Upon i.p. injection in mice the amide reaches peak brain levels of 0.13% of the total dose after 15 min. Following i.v. injection, maximal brain concentrations of 1.9% are observed at 5 min. Compared to delta 9-THC, which distributes almost equally between triglyceride and phospholipid phases (51:49) the amide exhibits a strong preference for phospholipids (5:95) that can be interpreted as high relative membrane affinity. In rats trained in a water maze to discriminate between i.p. injections of 3 mg/kg delta 9-THC (ED50 = 1.8 mg/kg) and its vehicle, the amide was generalized to the training drug, being five times less potent (ED50 = 8.7 mg/kg) than delta 9-THC. This demonstration of cannabis-like activity indicates that the amide retains affinity to the postulated receptor and justifies the choice for the affinity ligand. PMID- 2998396 TI - The influence of tetracosactide and adrenal steroids on renal kallikrein activity and urinary kallikrein excretion in rats. AB - The effect of adrenal steroids (mineralo- and glucocorticoids) as well as that of the adrenocorticotrophic peptide tetracosactide (beta 1-24 corticotropin) on the renal kallikrein activity and on the urinary kallikrein excretion of rats was investigated. After the animals had been adapted to metabolic cages, they were injected with deoxycorticosterone acetate (15 mg/kg day), corticosterone (40 mg/kg day), both steroids combined or the vehicle (sesame oil). Additional groups of rats received tetracosactide (0.05, 0.1 or 0.2 mg/day) or the vehicle (100 microliter of 38 X 10(-3) M ZnCl2). After four days of treatment the urinary kallikrein excretion was higher in deoxycorticosterone-treated rats than in their controls. This increase was prevented when corticosterone was administered simultaneously. The renal kallikrein activity of corticosterone as well as that of deoxycorticosterone plus corticosterone-treated rats was subnormal. A dose related reduction of both the renal kallikrein activity and the urinary kallikrein excretion was observed 2 days after starting the tetracosactide administration. It may be concluded that a stimulation of the endogenous release of glucocorticoids in the rat reduces the renal kallikrein activity and that glucocorticoids can prevent the stimulating effect of mineralocorticoids. PMID- 2998397 TI - Adriamycin cardiomyopathy: implications of cellular changes in a canine model with mild impairment of left ventricular function. AB - The present study has examined early cellular effects of chronic adriamycin administration to dogs using a protocol (1 mg/kg/week to a total cumulative dose of 240 mg/m2) producing significant but small reductions in ejection fraction and stroke volume as determined echocardiographically prior to the development of clinical or radiological manifestations of heart failure. At this early phase of cardiomyopathy, significant reduction (P less than 0.05) in sarcoplasmic reticulum Ca2+, K+-ATPase was observed without any change in mitochondrial, lysosomal or sarcolemmal marker enzymes. Myocardial calcium (P less than 0.01) and glutathione (P less than 0.001) levels were increased significantly. Detailed analysis of myocardial phospholipid profiles failed to show any significant differences between control and treated dogs. In contrast, red cell membranes showed increased phosphatidylcholine (PC) and decreased phosphatidylserine (PS) contents, resulting in a significant increase in PC/PS ratio (P less than 0.05). No significant changes were detected in activities of catalase, superoxide dismutase or glutathione peroxidase in erythrocytes or myocardial tissue from control and adriamycin-treated animals. A significant (P less than 0.05) elevation in plasma sialic acid was observed following adriamycin treatment. Our results suggest that early adriamycin-induced damage is unlikely to result from alterations in cellular processes protecting tissues against oxidant injury. Regression analysis indicated that, of the various abnormalities observed, only the elevated myocardial calcium levels and the increases in plasma sialic acid correlated with the degree of myocardial functional impairment. Our findings suggest the presence of sarcolemmal alterations in Ca2+ handling in early adriamycin-induced myocardial injury and indicate that measurement of plasma sialic acid should be further investigated as a possible noninvasive indicator of impending adriamycin cardiotoxicity. PMID- 2998398 TI - Possible role of hydroxyl radicals in the metabolism of succinonitrile. PMID- 2998399 TI - Free radical damage to deoxyribose by anthracycline, aureolic acid and aminoquinone antitumour antibiotics. An essential requirement for iron, semiquinones and hydrogen peroxide. AB - Anthracycline, aureolic acid and aminoquinone antitumour antibiotics damage deoxyribose in cell-free systems when reduced in air by the enzyme ferredoxin reductase. Damage to deoxyribose is inhibited by the iron chelator desferrioxamine, the copper-containing protein caeruloplasmin and catalase but not by superoxide dismutase. Scavengers of the hydroxyl radical such as formate, butan-1-ol, ethanol and benzoate do not offer much protection, whereas mannitol and thiourea do. These findings point to a site-specific Fenton reaction in which the drug semiquinones reduce complexed iron and dioxygen leading to the formation of hydrogen peroxide and a ferrous complex. PMID- 2998400 TI - Effect of 3-aminobenzamide on antigenic variation of Trypanosoma brucei. AB - African trypanosomes, like Trypanosoma brucei, depend on antigenic variation to evade the immune response of the vertebrate host. An antigenic switch corresponds to the activation of a variable surface glycoprotein (VSG) gene from a large silent repertoire. Most switches require the duplicative transposition of a VSG gene, which involves strand breaks in DNA and subsequent repair. The nuclear enzyme adenosine-diphosphoribosyl transferase (ADPRT), which is dependent on the presence of DNA strand breaks for its activity, might be involved in this process because it has a regulatory role in DNA repair in all eukaryotic cells studied so far. In previous work, the presence of ADPRT activity was demonstrated in T. brucei. Moreover, it was also shown in isolated trypanosomes the ADPRT activity, which is stimulated by the induction of DNA strand breaks, could be blocked by the competitive inhibitor 3-aminobenzamide. Here we report experiments using rats which were infected with small numbers of T. brucei expressing VSG gene 118. After two days, the rats were coupled to a continuous intraperitoneal infusion system administrating 3-aminobenzamide in 0.9% NaCl (81.4 mM) at a rate of 0.65 ml/hr/rat for a period of up to five days. Control rats received only a 0.9% NaCl infusion. At days 1, 3 and 5, 250 microliters blood was obtained from a tail artery. Plasma 3-aminobenzamide was determined using a new high performance liquid chromatography method, developed for these experiments. In most rats the plasma concentrations were maintained between 0.8 and 1.2 mM. The rate of antigenic switching was determined by quantitating the fraction of trypanosomes that had lost their VSG 118 coat, using antibody against VSG 118 and a limiting dilution in mice. The average switching rate found was 2.0 X 10(-6) in controls and 1.3 X 10(-7) in drug-treated rats (15-fold reduction). This suggests that ADPRT is required for completing most antigenic switching events. We discuss the possibility that drug-resistant switching only involves non-duplicative VSG gene activation. PMID- 2998401 TI - Convulsant and anti-convulsant gammabutyrolactones bind at the picrotoxinin/T butylbicyclophosphorothionate (TBPS) receptor. PMID- 2998402 TI - [Synthesis and properties of an acyclic analog of 9-deazainosine and related compounds]. AB - An acyclic analogue of 9-deazainosine, 9-(2-hydroxyethoxymethyl)-9 deazahypoxanthine, and related compounds have been synthesized starting from 9 (hydroxyethyl)-9-deazahypoxanthine. The acyclo-9-deazainosine exhibited some cytotoxic activity. PMID- 2998403 TI - [Use of an RNAse inhibitor from the human placenta in specific fragmentation of high molecular weight RNA by ribonuclease H]. AB - Human placenta RNase inhibitor has been shown to suppress the interfering ribonucleases, virtually not affecting the RNase H activity. This allows the usage of the inhibitor in the course of site-specific cleavage of high molecular weight RNAs by ribonuclease H. PMID- 2998405 TI - [Synthesis of anomeric 5-trimethylgermyl-2'-deoxyuridines and study of their antiviral and cytotoxic properties]. AB - Glycosylation of silylated 5-trimethylgermyluracil with 2-deoxy-3,5-di-O-p-toluyl alpha-D-ribofuranosylchloride in dichloroethane in the presence of SnCl4 and subsequent deacylation led to anomeric 5-trimethylgermyl-2'-deoxyuridines. The alpha-nucleoside inhibits HSV-1 replication in vitro, blocks 2'-deoxyuridine incorporation into DNA of hepatoma 22A cells and incorporation of thymidine into DNA of cancer ovarian cells as well. Treatment with 125 mg/kg X 5 days of alpha nucleoside fails to increase the life-span of mice with leukemia P388. PMID- 2998406 TI - [Cloning of single-stranded synthetic DNA]. AB - The method for cloning a single-stranded synthetic DNA with the short complementary oligonucleotides, that form corresponding restriction sites, is proposed. The potency of the method is demonstrated by cloning a single-stranded polynucleotide A (93 nucleotide residues (n. r.] in plasmid vector pBR327. The polynucleotide A includes a leader structure of the human fibroblast interferon gene. Oligonucleotides (IV) (20 n. r.) and (VI) (16 n. r.) were taken as strengthening complements and to create the sticky ends for the restrictases HindIII and EcoRI. 72% of the obtained clones appeared to be hybrid. Four hybrid clones were analyzed, and three of them carried the desirable insertion. The primary structures of these insertions are confirmed by sequencing. PMID- 2998404 TI - [Synthesis of [cyclo(Glu gamma-----epsilon Lys(Gly)]ACTH-(5-14) undecapeptide. Biological and physico-chemical properties of analogs of ACTH-(5-10)- and ACTH-(5 14)-peptides]. AB - Modified corticotropin fragment - [Lys11 (Gly)]ACTH-(5-14)- and its cyclic analogue - [cyclo (Glu gamma----epsilon Lys (Gly)] ACTH-(5-14)-undecapeptides have been synthesized by classical approach. The cyclic structure has been fixed by amide bond between gamma-COOH group of glutamic acid and alpha-NH2 group of glycine coupled to the epsilon-NH2 group of lysine. Fragment condensation has been achieved by azide or dicyclohexylcarbodiimide methods. Cyclization has been performed using diphenylphosphorylazide. The melanotropic activity of the cyclicanalogue on isolated frog skin exceeds by two orders of magnitude that of the linear undecapeptide, however the steroidogenic activity in isolated cells of rat adrenal cortex is diminished by an order of magnitude as compared with that of the linear precursor. A similarity of the CD spectra for the cyclic ACTH peptides and their linear counterparts in water and trifluoroethanol points to the similarity and relative rigidity of their structures. PMID- 2998408 TI - Adenosine triphosphate pyrophosphohydrolase and neutral inorganic pyrophosphatase in pathologic joint fluids. Elevated pyrophosphohydrolase in calcium pyrophosphate dihydrate crystal deposition disease. AB - Adenosine triphosphate pyrophosphohydrolase (ATPPPH) and neutral inorganic pyrophosphatase activities were assayed in synovial fluids (SF) from 37 patients with a variety of arthropathies. ATPPPH activity was detected in all fluids, but was highest in patients with chronic chondrocalcinosis; its activity in patients with osteoarthritis was higher than that in patients with rheumatoid arthritis, gout, or pseudogout. ATPPPH activity correlated positively with SF pyrophosphate concentration and negatively with SF white blood cell count. Pyrophosphatase activity did not correlate with diagnosis, pyrophosphate level, or white blood cell count. PMID- 2998407 TI - Antiarthritic drugs containing thiol groups scavenge hypochlorite and inhibit its formation by myeloperoxidase from human leukocytes. A therapeutic mechanism of these drugs in rheumatoid arthritis? AB - We investigated the effect of antiarthritic drugs containing thiol groups, such as D-penicillamine, tiopronin (N-[2-mercaptopropionyl]glycine), sodium aurothiomalate, and aurothioglucose, on the chlorinating activity of myeloperoxidase purified from human leukocytes. Hypochlorite, the reactive product of the reaction catalyzed by myeloperoxidase, was effectively scavenged by these antiarthritic drugs, and in addition, D-penicillamine and tiopronin inhibited myeloperoxidase itself. The above-mentioned effects of these drugs were observed at concentrations that occur in the serum of rheumatoid arthritis patients treated with these agents. We suggest that the therapeutic effect of these antiarthritic drugs may be due to the protection of tissues against the reactive HOCI released by activated granulocytes at inflamed sites. PMID- 2998409 TI - Prevalence of monosodium urate and calcium pyrophosphate dihydrate crystals in postmortem knee synovial fluid. PMID- 2998410 TI - [Signet ring cell adenocarcinoma of the stomach in a 14-year-old patient]. PMID- 2998412 TI - Transposable elements in plants. Lecture held on the occasion of the recept of the Otto-Warburg-Medaille 1985. PMID- 2998411 TI - Maternal ethanol consumption: effect on (Na+-K+)-ATPase in rat offspring. AB - The activity of Mg2+-activated, ouabain-sensitive adenosine triphosphatase, (Na+ K+)-ATPase, was determined in homogenates of hypothalamus, cortex, cerebellum, and brain stem from the 19-day-old offspring of rats that were pair-fed control or (6.6%, v/v) ethanol liquid diets on a chronic basis prior to parturition. In the offspring of both control and ethanol-fed rats the specific activity of (Na+ K+)-ATPase was significantly (p less than 0.01) greater in the cortex than it was in the hypothalamus, brain stem or cerebellum (hypothalamus approximately brain stem approximately cerebellum). When the offspring of ethanol-fed and control rats were compared we observed no significant (p greater than 0.05) differences in the activity of (Na+-K+)-ATPase in any of the four brain regions examined. In addition, the results of kinetic analyses of cortical (Na+-K+)-ATPase were similar in the 19-day-old offspring of ethanol-fed rats and those whose mothers consumed either the control liquid diet or standard laboratory chow. The results of these studies suggest that the activity of the plasma membrane enzyme, (Na+ K+)-ATPase, was not affected in the 19-day-old offspring of ethanol-fed rats. PMID- 2998413 TI - Proteinase inhibitors of horse seminal plasma. A high molecular mass, acid soluble proteinase inhibitor. AB - Horse seminal plasma does not possess a proteinase inhibitor corresponding to human HUSI-I (human seminal plasma inhibitor). Instead a protein complex of high relative molecular mass (Mr) containing proteinase inhibitory activity was detected, which was called horse seminal plasma protein complex or HSPC. The compound had a broad enzyme-inhibiting spectrum. Its Mr was estimated to be 800 000 and it was composed of 7 different polypeptides with Mr values ranging from 11 000 to 30 000. Its carbohydrate content was between 3.5% and 5%. Despite the high molecular mass, the complex was soluble in diluted perchloric acid and did not lose its biological activity. The high recovery of seminal plasma protein (69%) after perchloric acid treatment, the unaltered immunoelectrophoretic precipitation pattern of the perchloric acid soluble part of seminal plasma, and the similarity of the polypeptide patterns of unfractionated seminal plasma and HSPC suggest that HSPC is one of the major components of horse seminal plasma. In addition to HSPC, horse seminal plasma contained a group of three electrophoretically distinguishable proteinase inhibitors, corresponding roughly to a Mr of 6500. They inhibited only trypsin. The similar Mr values and the identical narrow enzyme specificity suggest that they are isoinhibitors and may be analogues of human HUSI-II (human seminal plasma inhibitor). The lack of a HUSI-I analog in the horse is discussed in relation to a previously made observation that horse tracheobronchial fluid contains no detectable perchloric acid-soluble proteinase inhibitors. PMID- 2998414 TI - Glucostat capacity and metabolic zonation in rat liver after portocaval anastomosis. AB - The activities and zonal distribution of key enzymes of carbohydrate metabolism were studied in livers of rats after end-to-side portocaval anastomosis. Sham operated control animals with the same periods of interruption of hepatic blood supply as the shunted animals were pair-fed. The following alterations were observed: Food uptake was reduced to about 20% at the first postoperational day; it was then increased continuously to about 70% at day 8. Body weight, after a small 10% postoperational decrease, remained unaltered, but liver weight was lowered to 55% at day 8 and then stayed constant. The total glycogen reserves of the liver (g X 100 g body weight-1) were reduced, after a transient fall to about 10% at day 1-4, to about 25%. The total activity of the glucogenic phosphoenolpyruvate carboxykinase (mumol . min-1 X 100 g body weight-1) was diminished, after a transient increase to 190% and 150% at day 1 and 2 respectively, to about 55% from day 8 onwards. The total activity of the glucogenic glucose-6-phosphatase was lowered without a transient rise to about 30%. The total activities of the glycolytic pyruvate kinase isoenzyme L and glucokinase were decreased continuously to about 40% at day 8; that of the citrate cycle enzyme succinate dehydrogenase was lowered parallel with liver weight to 55%. The transient decrease of the glycogen reserves and the intermediate increase of the phosphoenolpyruvate carboxykinase capacity were due to the operational stress, since they were observed also in the sham-operated control animals. All other alterations, the decrease of liver weight and of the capacities of both gluconeogenic and glycolytic key enzymes, were specific for the portocaval anastomosis. The normal periportal to perivenous gradient of phosphoenolpyruvate carboxykinase of about 3.5:1, as measured in microdissected tissue samples, remained the same with specific activities reduced to about 80% each in the two zones. The normal periportal to perivenous gradient of pyruvate kinase L of about 1:1.7 was equalized with levels lowered to 35% and 23%, respectively, in the two zones. The normal periportal to perivenous gradients of glucose-6-phosphatase and succinate dehydrogenase, demonstrated histochemically, were essentially maintained with perivenous bridging occurring transiently at day 4 and 8.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2998415 TI - [Atherosclerosis: its cellular manifestations and the mechanisms of the development of the disease in human arteries]. PMID- 2998417 TI - Immunobiology of myasthenia gravis, experimental autoimmune myasthenia gravis, and Lambert-Eaton syndrome. PMID- 2998416 TI - [Dalargin--a peptide preparation with cytoprotective action]. AB - The role and possible clinical use of regulatory peptides, a new type of regulatory substances, is discussed. Special attention is paid to the opioid peptides and their analogues. The new drug dalargin has been developed on the basis of the endogenous opioid peptide leucine-encephalin. Its action has been studied using experimental models. It has been established that the optimal dose (10 micrograms/kg) of dalargin is effective in preventing ulceration in the cystamine duodenal ulcer rat model and the development of stomach erosive lesions in immobilization stress, reducing the degree of liver degeneration in CCl4 poisoning, etc. With an increase of the dose of dalargin the effect "escapes". Dalargin is primarily bound by delta-receptors, it produces no analgetic effect and does not enter the brain. Dalargin exhibits a pronounced cytoprotective and regeneratory action and may take an important place in treating the internal organ diseases. PMID- 2998418 TI - Human T-cell leukemia viruses (HTLV): a unique family of pathogenic retroviruses. PMID- 2998419 TI - [Opiate regulation of cyclic AMP levels in sympathetic ganglia in mammals]. PMID- 2998420 TI - [Preparation from an adult rat brain of a suspension of viable neurons, astrocytes and oligodendrocytes]. PMID- 2998421 TI - [In vitro study on the possible inflammatory mechanism of ferrous ions: effect on the production of oxygen free radicals]. PMID- 2998422 TI - Establishment and characterisation of cell lines from patients with lung cancer (predominantly small cell carcinoma). AB - Tissue samples from 59 patients with lung cancer have been used to establish cell lines in culture. The primary diagnosis was small cell carcinoma in all except four. Most of the samples were of bone marrow but pleural effusions, lymph node biopsies and skin metastases were also included. The samples were usually split between HITES serum-free medium and HITES plus 2.5% foetal calf serum. A total of 19 cell lines were established and characterised. One line is large cell anaplastic lung carcinoma, four are B-lymphoblastoid and fourteen are small cell lung cancer. Considerable heterogeneity in gross morphology, neuroendocrine differentiation (by electron microscopy) and content of the enzyme L-dopa decarboxylase was seen. The use of HITES plus 2.5% foetal calf serum resulted in better establishment of cultures than did serum-free HITES. PMID- 2998423 TI - Cyclic AMP binding proteins in human breast cancer. AB - The characteristics of a method for measuring cyclic AMP binding proteins in cytosols of human breast cancer are described. Using the assay, binding proteins were demonstrable in all of 100 tumour cytosols. Levels of binding in individual tumours varied from 0.8 to 15 pmol mg-1 cytosol protein (mean value 5 pmol mg-1 cytosol protein) and the dissociation constant ranged from 0.5 to 5.2 X 10(-8)M (mean 1.73 X 10(-8)M). Whilst replicate measurements within a single portion of tumour were reproducible (intra-assay coefficient of variation was between 4.5 and 7.8% and that for inter-assay variation was between 2.1 and 4.0%) there were often considerable differences in levels of binding proteins between different portions of the same tumour. Similar intra-tumour variations have been reported for other binding proteins and steroid receptors. The inter-relationships with such parameters may elucidate whether the differences are associated with variations in cellularity, cell type, or other specific factors. PMID- 2998424 TI - Thyrotrophin receptors, tumour radioiodine concentration and thyroglobulin secretion in differentiated thyroid cancers. AB - Tumour radioiodine concentration has been compared with serum thyroglobulin (Tg) and, in a few cases, with tumour complement of thyrotrophin receptors in patients with differentiated thyroid carcinoma. All tumours examined possessed TSH receptors. In most the complement was similar to that of normal thyroid tissue although all but one of the tumours had no detectable 131I concentration in vivo even with excess TSH stimulation. Elevated serum Tg (patient taking T4 in suppressive dose) was generally associated with tumours which had 131I concentrating function when stimulated by excess TSH. Some patients, however, had high serum Tg concentration but only low or indetectable tumour 131I uptake. We conclude that (a) measurement of tumour TSH receptor complement is unlikely to be useful in clinical management as tumours which do not significantly concentrate 131I in vivo may have a normal TSH receptor complement and (b) the capacity to secrete Tg is usually associated with 131I concentration but quantitatively the relationship varies considerably between tumours. PMID- 2998425 TI - Long-term effects of chemotherapy on lymphocyte chromosomes from patients treated for gestational trophoblastic tumours. AB - A cytogenetic follow-up study of patients treated with chemotherapy for gestational trophoblastic tumours was undertaken. In some cases, high levels of chromosome damage were found to persist in lymphocytes for several years after completion of therapy. These results are compared with those found in similar studies of non-malignant and other malignant diseases. The relevance of these findings to the risk of subsequent chemotherapy-induced malignancy is discussed. PMID- 2998426 TI - Duration of ENNG administration and its effect on histological differentiation of experimental gastric cancer. AB - An experimental trial in the induction of canine gastric cancers was conducted to study the relationship between the histological differentiation of adenocarcinoma and the duration of administration of the carcinogen, N-ethyl-N'-nitro-N nitrosoguanidine (ENNG). Twenty-three adult Beagle dogs were divided into three groups according to the duration of administration. Over 3 months administration, the total dose of ENNG per animal was 5.85 g, and only signet ring cell carcinomas and poorly differentiated adenocarcinomas were induced in the antral mucosa of the stomach in 5 of 10 recipients. During 6 and 9 months administration, the total doses per animal were 11.70 g and 17.55 g, well differentiated adenocarcinomas were observed in 12 of 13 animals and they coexisted with poorly differentiated adenocarcinomas and/or signet ring cell carcinomas. Atrophic hyperplastic gastritis and hyperplastic polyps were seen in the same stomach. The results of this study suggest that a greater amount of carcinogen, i.e., a higher total dose, is required for the development of well differentiated adenocarcinoma than for inducing poorly differentiated adenocarcinoma and signet ring cell carcinoma. PMID- 2998428 TI - Fine structure of the human gallbladder with cholesterosis with special reference to the mechanism of lipid accumulation. AB - Eight excised gallbladders with cholesterosis were studied by light and electron microscopy. Lipid droplets were found not only in the submucosa, but also in the infranuclear cytoplasm of epithelial cells. These contained well developed mitochondria and agranular reticulum. Lipid droplets were seen to form in the agranular reticulum. Macrophages were often present between the epithelial cells and the submucosa and contained numerous processes and well developed cell organelles, abundant lysosomes and lipid droplets. With the advance of lipid deposition, macrophages were filled with lipid droplets and became foam cells. It is suggested that these may become too large and rigid to pass through the endothelium of lymph vessels or that the lumen of the lymph vessels may be obstructed by large foam cells resulting in the destruction of these vessels and the accumulation of foam cells in the submucosa. PMID- 2998427 TI - Prolonged localisation of a monoclonal antibody against CEA in a human colon tumour xenograft. PMID- 2998429 TI - Methotrexate inhibits the leukotriene B4 induced intraepidermal accumulation of polymorphonuclear leukocytes. AB - The penetration of polymorphonuclear leukocytes (PMNs) into the epidermis following topical application of leukotriene B4 was assessed in the clinically uninvolved skin of psoriatic patients treated with methotrexate and of psoriatic patients without treatment, and in normal controls. Inhibition of PMN infiltration was observed in those patients treated with methotrexate while there was no significant difference between untreated psoriatic patients and normal controls. PMID- 2998430 TI - Demonstration of cellular retinoic acid binding protein in cultured human skin fibroblasts. AB - Previous studies have indicated that retinoids, such as all-trans-retinoic acid and 13-cis-retinoic acid, can modulate connective tissue metabolism in human skin fibroblast cultures. Such effects could be mediated through binding of these retinoids to specific cellular binding proteins. In the present study we have demonstrated cellular retinoic acid binding protein using both whole cell and cytosol binding assays with [3H]all-trans-retinoic acid or [3H]13-cis-retinoic acid as the ligand. Specific binding of [3H]all-trans-retinoic acid could be demonstrated by both techniques and the binding could be displaced by unlabelled all-trans-retinoic acid and 13-cis-retinoic acid, but not by retinol or RO-10 9359 (etretinate) in a 100-fold excess. Gel filtration chromatography of the cytosol proteins after incubation with [3H]all-trans-retinoic acid demonstrated that the specific binding protein had an apparent molecular weight of approximately 15 000 daltons. Thus, the cellular retinoic acid binding protein demonstrated in human skin fibroblasts may mediate the effects of the retinoids on connective tissue metabolism in these cells. PMID- 2998431 TI - Collagen prolyl hydroxylase inhibitor and reduced collagen formation in cotton pellet-induced granuloma and skin wound healing in rats. AB - P-1894B an inhibitor of collagen prolyl hydroxylase was applied topically to cotton pellet-induced granuloma and to skin lesions in rats. The total hydroxyproline content of the granulomata was significantly reduced by this agent. It also reduced the hydroxyproline content in the tissues surrounding skin lesions during the healing process, with delayed diminution in lesion size. These results suggest that collagen prolyl hydroxylase inhibitor P-1894B may be a suitable agent for topical treatment of hypertrophic scar tissue or keloid. PMID- 2998432 TI - Presence and distribution of carcinoembryonic antigen and lectin-binding sites in benign apocrine sweat gland tumours. AB - The presence and distribution of carcinoembryonic antigen (CEA) and lectin binding sites in benign apocrine sweat gland tumours was investigated using immunoperoxidase and immunofluorescent techniques respectively. CEA was present in apocrine hidrocystoma, hidradenoma papilliferum, and syringocystadenoma papilliferum. The distribution pattern of CEA was indistinguishable in each of the three types of tumour. The distribution of lectin-binding sites was similar in all cases of a given type of neoplasm, and the staining patterns were similar but not identical in hidradenoma papilliferum and syringocystadenoma papilliferum. However, the distribution of lectin-binding sites in apocrine hidrocystoma was different. These results could be useful in elucidating the nature of the cells of origin of benign apocrine sweat gland tumours and providing a basis for their classification. PMID- 2998433 TI - Peritoneal mesothelioma and malignant lymphoma in mice caused by fibrous zeolite. AB - Dust from the village of Karain containing the fibrous zeolite erionite, talc, and physiological saline were tested by intraperitoneal injection in 486 Swiss albino mice. Malignant tumours were found in 84 (41 mesotheliomas, 31 lymphomas, 1 peripheral epidermoid carcinoma, and 11 lymphomas and mesotheliomas together) of the 321 animals which died spontaneously within nine to 32 months after injection of Karain dust (26.1%). Three mesotheliomas and no lymphomas were found among 24 animals injected in the same way with talc during the same time (12.5%). In 46 control animals injected with physiological saline three mesotheliomas and one lymphoma were seen (8.7%). Thus Karain dust appears to be a potent carcinogen, causing both mesotheliomas and malignant lymphomas. PMID- 2998435 TI - The wart virus and genital neoplasia; a casual or causal association. PMID- 2998434 TI - Silicosis in jade workers. AB - The recent finding of cases of silicosis among jade workers in Hong Kong points to this disease being an occupational hazard. The source was found to be the silica flour that was added in a polishing process. Five cases are described together with the results of environmental investigation in a workplace. In three cases the disease was of early onset, rapidly progressive, and presented the features of galloping silicosis noted in other occupational exposures to silica flour. One patient had massive fibrosis and severe glomerulonephropathy, an association that has also been previously noted. One case showed evidence of active tubercular infection in addition to silicosis and two had healed lesions. Silica concentrations in the workplace during the suepect process were well above accepted threshold limit values. PMID- 2998436 TI - Natural history of cervical human papillomavirus (HPV) infections based on prospective follow-up. AB - To assess the natural history of human papillomavirus (HPV) lesions in the uterine cervix, a prospective follow-up of untreated lesions has been conducted since late 1981. The present report summarizes the data on 343 women with cervical HPV lesions currently followed-up for a mean of 18.7 (SD 15.2) months by colposcopy and PAP smears (group B) or by additional punch biopsy (group A). Initially these two groups were classified on the first PAP smears, presenting with HPV-induced cytopathic changes, and either with (group A) or without (group B) concomitant changes suggestive of cervical intraepithelial neoplasia (CIN). The clinical course of the HPV lesions could not be predicted adequately from the findings of the first PAP smears, as evidenced by the higher progression rate (15.4%) in the 214 women initially classified in group B, compared with 11.6% in the 129 women classified in group A. Furthermore, the number progressing to carcinoma in situ requiring conization was equal (seven patients) in the two groups. This necessitated a more flexible approach to follow-up, permitting transfer of patients between groups, which resulted in a final allocation of 261 women to group A, and 82 to group B. To date, 25% of the total of 343 HPV lesions have regressed, 61% have persisted, and 14% have progressed. Of the latter, a total of 14 (4.1%) have been coned due to progression to carcinoma in situ. The rate of regression seems to be inversely related, and progression directly related, to the degree of HPV-associated CIN.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2998437 TI - Human papillomavirus types 6 and 16 in multifocal intraepithelial neoplasias of the female lower genital tract. AB - Five women with multifocal intraepithelial neoplasia of the lower genital tract were investigated for the presence of human papillomavirus (HPV) infection by the method of DNA-DNA hybridization which detects the viral DNA. The DNA sequences of HPV types 6 and 16 were detected in each of the five patients and in each of the areas biopsied: cervix, vagina and vulva. DNA sequences of both viral types were also found in vulval intraepithelial neoplasia grades I-III and in cervical intraepithelial neoplasia grades I and III. The detection of HPV DNAs in multifocal lesions suggests a possible common aetiology for the lower genital tract intraepithelial neoplasias. PMID- 2998438 TI - Prevalence of human papillomavirus type 16 DNA sequences in cervical intraepithelial neoplasia and invasive carcinoma of the cervix. AB - The frequency of human papillomavirus (HPV) type 16 in premalignant and malignant lesions of the cervix was investigated and compared with the detection of HPV type 6. In cervical intraepithelial neoplasia (CIN) grades I-III HPV 6 was detected in 28% and HPV 16 in 62% of patients whereas 90% of malignant lesions contained HPV 16 only. In the CIN lesions there was an increase in HPV 16 detection as the severity of disease increased while the level of detection of HPV 6 decreased. Only three (18%) of the cervices that were colposcopically and histologically normal contained HPV genomes; although two of these three women had either a history of genital warts or a sexual partner with penile warts. PMID- 2998439 TI - Effect of in vitro fermentation using human faecal inoculum on the water-holding capacity of dietary fibre. AB - The water-holding capacities (WHC) of four sources of fibre were measured using dialysis membranes and osmotic-suction pressures of 45, 89 and 178 mosmol/1 (1, 2 and 4 atm). At all pressures, pectin had the highest WHC, followed by cabbage (Brassica oleracea) and lucerne (Medicago sativa) and then cellulose. A suction pressure of 89 mosmol/1 (2 atm) was used in the subsequent fermentation study since it had the lowest standard error of the mean and most closely approximated physiological conditions. The four fibres were anaerobically fermented in vitro with human faecal inoculum for 24 h. The WHC of the fermentation residues were measured. The potential water-holding capacity (PWHC), a function of the extent of fermentability and the WHC of the fermentation residues, was highest for lucerne, followed by cellulose, then cabbage and, finally, pectin. Only the PWHC values ranked the four fibres in the same order as in vivo values. It was concluded that the ethanol-insoluble residues containing unfermented fibre organic matter and microbial organic matter, both of which hold water, should be used to calculate PWHC and to predict the effect of fibre on rate of passage and faecal mass in humans. PMID- 2998441 TI - Iron availability from peas (Pisum sativum) and bread containing added pea testa in rats. AB - Iron retention in adult male rats given 3 g dried ground peas, immature and mature (Pisum sativum cv Dark-skin perfection) and leafless (Pisum sativum cv Filby), extrinsically labelled with 0.25 muCi 59Fe, was measured by whole-body counting. The Fe was less well absorbed (P less than 0.01) from the mature peas (0.251 (SE 0.021)) than from the immature (0.384 (SE 0.032] or leafless peas (0.344 (SE 0.026)). The availability of Fe from the leafless peas was compared with that of defatted soya-bean flour by the same technique. Significantly more Fe (P less than 0.005) was retained from the pea flour (0.471 (SE 0.013)) than from the soya-bean flour (0.377 (SE 0.022)). The effect of adding pea testa to bread (97.6 g/kg dry weight), as in the production of high-fibre white bread, on Fe availability was measured and compared with the availability of ferrous sulphate in young and adult male rats. There were no significant differences between the high-fibre and low-fibre breads in either age-group, although the older rats absorbed less Fe from all three sources. Retention from high-fibre bread, low-fibre bread and FeSO4 was as follows (mean with SE): young rats 0.452 (0.037), 0.475 (0.040) 0.541 (0.032); mature rats 0.363 (0.034), 0.366 (0.030), 0.471 (0.028). It was concluded that the addition of pea testa to white bread does not have detrimental effect on Fe availability. Immature and leafless peas appear to be a better source of available Fe than soya-bean flour, despite similar fibre levels, but with maturity the Fe in peas is rendered less available. PMID- 2998440 TI - Reduction of the phytate content of bran by leavening in bread and its effect on zinc absorption in man. AB - The effect of leavening of bread containing bran on the phytic acid content and on zinc absorption in man was studied. Twenty breads with leavening times varying from 0 to 120 h were prepared. The breads contained 250 g wheat bran/kg flour. The phytic acid content was determined after baking. The phytic acid content of bread containing bran was reduced to about 40% after 2 h of leavening and to 15% after 2 d. No further decrease was observed. Zn absorption from single meals was determined using a radioisotope technique. Forty-two students volunteered for these studies. They were served a breakfast of milk, butter, bread and 10, 16 or 30 g bran served either raw or baked into the bread with fermentation times of 15 min, 45 min, 3 h or 16 h. One meal contained no bran, but phytate and Zn were added in amounts equivalent to the content of 10 g bran. The amount and percentage of Zn absorbed increased at each bran level as fermentation was prolonged. The percentage of Zn absorbed was reduced by increased bran content in the meal. It is concluded that the fermentation of bread containing bran reduces the phytic acid content and increases Zn absorption from such bread. This may be of importance to people subjected to diets with a high cereal content, especially in combination with a low animal-protein intake. PMID- 2998442 TI - A new bioassay for assessment of copper availability and its application in a study of the effect of molybdenum on the distribution of available Cu in ruminant digesta. AB - Investigations were carried out on the feasibility of using an oral repletion technique in the rat to assess the bioavailability of copper in experimental sources providing no more than 250 micrograms Cu from any one source. Preliminary studies on the response in plasma Cu of partially Cu-depleted rats given repletion doses of 20-50 micrograms Cu as CuSO4/d on four consecutive days indicated that this index of Cu status was insufficiently sensitive to Cu dose. In contrast, the activity of cytochrome c oxidase (EC I.9.3.I) in the duodenal mucosa of partially Cu-depleted rats showed a measurable and uniform response to 10 micrograms Cu as CuSO4/d given on three consecutive days. Furthermore, when the rats were given 0, 2.5, 5.0 or 10.0 micrograms Cu/d, the increase in cytochrome c oxidase activity above that of the unsupplemented control group was linearly related to Cu dose. The mean response in cytochrome c oxidase activity in groups of eight rats was therefore used to assess the availability of Cu from experimental sources relative to that of Cu as CuSO4, only 240 micrograms Cu being required from each experimental material. The assay was used to study the effect of the Cu-antagonist molybdenum on the distribution of available Cu in digesta from sheep given dried grass either untreated (1.6 mg Mo/kg dry matter (DM)) or treated with ammonium molybdate (11.6 mg Mo/kg DM). The relative availability of Cu in untreated dried grass (75%) was substantially higher than in rumen (12%), duodenal (43%) or ileal (28%) digesta. In all cases, addition of Mo to the diet resulted in a substantial reduction in Cu availability. The effects of Mo on availability of Cu are discussed with special reference to the possible involvement of thiomolybdates in the Cu-Mo antagonism. PMID- 2998443 TI - Essential fatty acids in the liver and adipose tissue of genetically obese mice: effect of supplemental linoleic and gamma-linolenic acids. AB - Genetically obese mice (ob/ob) and their lean litter-mates were given diets iso energetically supplemented with sucrose, hydrogenated coconut oil, safflower oil or evening primrose (Oenothera biennis) oil. Weight gain over 15 weeks was significantly greater in the evening primrose oil-supplemented obese mice than in the other groups. In all the groups of obese mice, liver total phospholipids contained proportionally less linoleic acid and more dihomo-gamma-linolenic acid and arachidonic acid than did the lean controls. As a percentage of total fatty acids, n-3 essential fatty acids (EFA) in liver and adipose tissue lipids were significantly lower in the obese mice than in the lean controls. Supplementation with EFA-rich oils (safflower and evening primrose oil) increased the proportional composition of n-6 EFA and decreased the n-3 EFA more in the liver total phospholipids of the lean than the obese mice. PMID- 2998446 TI - The digestion of fibre by pigs. 2. Volatile fatty acid concentrations in large intestine digesta. AB - The effect of including lupin (Lupinus sp.) hulls, maize cobs, wheat bran and lucerne (Medicago sativa) stems in a basal fibre-free diet on the concentrations and the relative proportions of volatile fatty acids (VFA) in the proximal colon of pigs, 17-18 h after feeding, was studied. Concentrations of total VFA in the proximal colon increased with increasing levels of neutral-detergent fibre (NDF) intake, and this increase was highly dependent on the source of NDF in the diet. Molar proportions of the VFA were significantly affected by the level of NDF intake only in the cases of acetic and butyric acids, whereas the source of dietary NDF had a marked influence on the molar proportions of all acids. The results indicate that the extent of fermentative breakdown of fibre in the pig intestine can be influenced substantially by the type and the level of fibre in the diet. PMID- 2998444 TI - Abnormal essential fatty acid composition of tissue lipids in genetically diabetic mice is partially corrected by dietary linoleic and gamma-linolenic acids. AB - Genetically diabetic mice (db/db) and their non-diabetic litter-mates were maintained for 15 weeks on diets supplemented with safflower oil or evening primrose (Oenothera bienis) oil, both essential fatty acid (EFA)-rich sources, or hydrogenated coconut oil (devoid of EFA). Plasma glucose was higher in the diabetic mice supplemented with the oils than in the unsupplemented diabetic mice. In the oil-supplemented non-diabetic mice, plasma glucose did not differ compared with the unsupplemented non-diabetic mice. The proportional content of arachidonic acid in the phospholipids of the pancreas was significantly decreased in diabetic mice, an effect which was completely prevented by supplementation with safflower or evening primrose oil but not hydrogenated coconut oil. In the liver phospholipids of the diabetic mice, dihomo-gamma-linolenic acid was proportionally increased, an effect reduced by supplementation with safflower oil but not evening primrose or hydrogenated coconut oils. In the liver triglycerides of the diabetic mice, gamma-linolenic acid, dihomo-gamma-linolenic acid and arachidonic acid were all proportionally decreased, effects which were also prevented by safflower or evening primrose oil but not hydrogenated coconut oil. Alopecia and dry scaly skin were prominent in the diabetic mice but less extensive in the diabetic mice supplemented with EFA. PMID- 2998445 TI - The digestion of fibre by pigs. 1. The effects of amount and type of fibre on apparent digestibility, nitrogen balance and rate of passage. AB - The effects of the amount and the type of dietary fibre on the apparent digestibility (AD) by growing pigs of neutral-detergent fibre (NDF) and NDF components, on nitrogen balance and on the rate of passage of digesta were studied using a semi-purified basal diet and fibre in the forms of soya-bean hulls, lupin (Lupinus sp.) hulls, pea (Pisum sativum) hulls, wheat bran, maize hulls, maize cobs, oat hulls and lucerne (Medicago sativa) stems. Both the amount and the type of dietary fibre significantly influenced the AD of dietary dry matter, N and energy. The AD of NDF and of NDF components was markedly affected by the type and the amount of fibre in the diet. The proportion of NDF digested ranged from 0.016 to 0.905, of cellulose from 0.026 to 0.931 and of hemicellulose from 0.010 to 0.999. N retention by the pigs ranged from 12.9 to 25.8 g/d and with some fibres there was a tendency towards increased N retention with increasing intakes of NDF. Rate of passage of digesta, expressed as the 50 and 95% excretion times of stained feed particles, ranged from 22.2 to 85.1 h and 40.0 to 117.1 h respectively. Large individual variations in rate of passage occurred but, in general, the rate of passage tended to increase with increasing intakes of NDF. No strong associations between the rate of passage of digesta and apparent digestibility of NDF components were observed. The results suggest that the extent of fibre digestibility depends predominantly on the origin of the fibre and to a lesser extent on the amount of fibre in the diet. PMID- 2998447 TI - The digestion of fibre by pigs. 3. Effects of the amount and type of fibre on physical characteristics of segments of the gastrointestinal tract. AB - The aim of the study was to determine the relative effects of feeding growing pigs with graded amounts of neutral-detergent fibre (NDF) from various sources on the empty wet weight of segments of the pig gastrointestinal tract, on the weight and moisture content of their digesta, and on the pattern of digesta movement in them. Increased NDF intakes were associated with significantly higher wet weights of all gastrointestinal segments and increased lengths of the caecum. The lengths of both the small and the large intestines were unaffected by the ingestion by the pigs of different amounts of NDF from various sources. However, the caecum responded to these increased intakes of NDF by significant increases in length. The source of NDF in the diet was a factor that markedly influenced both the length and the weight of the distal colon. The nature of these increases in weight and length morphologically and their biological significance have not been determined. Despite some significant differences, neither the type nor the level of dietary NDF had any appreciable effect on the dry weight and on the proportion of dry matter (DM) of the contents in the contents in the stomach. As the level of NDF intake was increased, more undigested dietary material was found in all segments of the digestive tract of the pigs. The proportion of DM in the residues decreased progressively from the caecal contents to the contents of the distal colon. In most cases the degree to which the level of NDF intake affected the weight of the contents and the proportion of DM in them was highly dependent on the source of NDF in the diet. The distribution of the feed consumed in the morning and in the evening, as measured with stained feed particles and polyethylene beads, was extremely variable. It is concluded that prolonged intakes by pigs of diets containing high levels of fibre may lead to a hypertrophy and hence increased weight of segments of the gastrointestinal tract. PMID- 2998448 TI - Influence of feed intake and starvation on the magnitude of Na+,K+-ATPase(EC 3.6.1.3)-dependent respiration in duodenal mucosa of sheep. AB - Oxygen consumption and Na+,K+-ATPase(EC 3.6.1.3)-dependent (ouabain-sensitive) and -independent respiration were measured for duodenal mucosa biopsies from 10 month-old sheep given two levels of digestible energy (DE) intake (7.6-7.7 and 14.8 MJ lucerne (Medicago sativa) pellets/d) and following 48 h of starvation. The mucosal biopsies were determined to be structurally intact and free of adherent bacteria on histological and scanning-electron-microscope examinations. The use of D-glucose as a substrate during incubations did not elevate (P greater than 0.05) the respiration indices of the biopsies over those measured during acetate incubations. Glucose uptake did not (P greater than 0.05) influence the Na+,K+-ATPase-dependent respiration of the mucosal biopsies. Na+,K+-ATPase dependent respiration accounted for 50% of the total O2 consumption of the mucosal biopsies of sheep given the lower level of DE. Total O2 consumption of the duodenal mucosa was not (P greater than 0.05) increased when sheep were given the higher level of DE but Na+,K+-ATPase-dependent respiration of the mucosa was elevated (P less than 0.01) by 37% during this period. When sheep were starved for 48 h, total O2 consumption of the mucosal biopsies was not (P greater than 0.05) affected, however, Na+,K+-ATPase-dependent respiration of the biopsies dropped (P less than 0.01) by 45%. Na+,K+-ATPase-dependent respiration accounted for 61.3% of the O2 uptakes of mucosa from the sheep given the higher level of DE and 28.3% of the O2 uptake of mucosa from fasted sheep. PMID- 2998449 TI - Utilization of low-quality roughage by Bos taurus and Bos indicus cattle. 1. Rumen digestion. AB - Six Hereford and six Brahman steers were fed ad lib. Pangola grass (Digitaria decumbens) and Spear grass (Heteropogon contortus) hay alone and supplemented with rumen-degradable nitrogen and sulphur and minerals. The rumen digestion of the two feeds was determined by reference to the disappearance of substrate from nylon bags suspended in the rumen and withdrawn after intervals ranging from 8 to 120 h. The digestion of the unsupplemented Pangola grass diet occurred more rapidly in Brahmans than in Herefords and was associated with higher rumen ammonia concentrations in Brahmans (40 v. 16 mg/l). The rumen NH3 concentrations were increased to over 100 mg/l by supplementation. The digestion rate increased in both breeds after supplementation and the breed difference disappeared. Increases in digestion rate were not achieved above NH3 concentrations of 60-80 mg/l. Spear grass, especially the cell-wall-constituent fraction, was more resistant to digestion than Pangola grass. Digestion of the unsupplemented Spear grass diet proceeded more rapidly in Brahmans than in Herefords. The digestion rate in Brahmans were similar irrespective of whether the diet was supplemented or not. Supplementation increased digestion rate in Herefords. PMID- 2998450 TI - Utilization of low-quality roughage by Bos taurus and Bos indicus cattle. 2. The effect of rumen-degradable nitrogen and sulphur on voluntary food intake and rumen characteristics. AB - In a number of experiments voluntary food intake of three low-quality roughages, either alone or supplemented with rumen-degradable nitrogen and sulphur and minerals, was measured in Brahman (Bos indicus) and Hereford (Bos taurus) steers. The chaffed hays were Spear grass (Heteropogon contortus) (6.2 g N/kg organic matter (OM)), Pangola grass (Digitaria decumbens) (7.9 g N/kg OM), and Pangola grass (12.0 g N/kg OM). Rumen characteristics relating to rate of fluid outflow from the rumen were also determined. There was no significant difference between breeds in the dry-matter intakes of the unsupplemented diets which ranged from 11.3 to 17.8 g/kg body-weight (BW) by Herefords and from 11.8 to 16.1 g/kg BW by Brahmans. Supplementation of Spear grass with N and S significantly (P less than 0.05) increased intake by Herefords (24%) but not by Brahmans. When the lower-N Pangola grass was supplemented there was a significant increase in intake by both breeds with the magnitude of the response in Herefords (42%) (P less than 0.001) being greater than that in Brahmans (15%) (P less than 0.05). The intakes of both the supplemented Spear grass and the lower-N Pangola diets were significantly (P less than 0.05) greater by Herefords than Brahmans. There was no breed difference in intake when the higher-N Pangola grass was supplemented. Both breeds recorded an 8% intake response to supplementation, although the increase was only significant (P less than 0.05) in Herefords. The mean retention time of fluid in the rumen on the unsupplemented Pangola grass diet of lower N content was 12.7 h in Brahmans compared with 17.5 h in Herefords (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2998452 TI - Several methods for the measurement of dietary fibre. PMID- 2998451 TI - The digestion by cattle of silage-containing diets fed at two dry matter intakes. 1. Digestion of organic matter and nitrogen. AB - In a 4 X 4 Latin square experiment four cattle were given in two meals per d diets consisting of (g/kg dry matter (DM)) 500 barley, 400 grass silage and 100 soya-bean meal. The diets were given at either 1.15 (L) or 2.3 times (H) maintenance energy requirements and the soya-bean meal was either untreated (U) or formaldehyde-treated (T). A 24 h collection of duodenal digesta and a 7 d collection of faeces were made using chromium sesquioxide for flow estimation and 35S as a marker of microbial nitrogen entering the small intestine. Samples of rumen fluid were also taken for estimation of rumen pH, ammonia and volatile fatty acid concentrations. Spot samples of duodenal digesta were obtained after administration of Cr2O3-mordanted silage-fibre and soya-bean meal, to determine the rates of outflow of these markers from the rumen. Similar samples were also obtained after cessation of a continuous intraruminal infusion of ruthenium phenanthroline, 35S and CoEDTA. Incubations of each feedingstuff in porous synthetic fibre (psf) bags were carried out in the rumen and the rates of N disappearance from the bags determined. Increasing DM intake significantly (P less than 0.001) increased the quantities of organic matter (OM), total N and amino acid-N entering the small intestine and amounts subsequently voided in the faeces. Apparent digestibilities of OM and N were unaffected by DM intake; the proportions of total digestible OM digested in the rumen were significantly lower (P less than 0.01) at the higher level of DM intake. Formaldehyde treatment of the soya-bean meal increased the quantities of N entering the small intestine; these increases were not significant. Increased DM intake increased the quantities of both microbial N (P less than 0.001) and undegraded feed N (P less than 0.01) entering the small intestine; HCHO-treatment also significantly (P less than 0.05) increased the quantities of undegraded feed N entering the small intestine. The efficiency of microbial N synthesis within the rumen was not significantly affected by dietary treatments whereas apparent feed N degradability was reduced significantly (P less than 0.05) both by increasing DM intake and by HCHO-treatment of the soya-bean meal. Rates of disappearance of N from psf bags in the rumen were different for different feedingstuffs. However, for a given feedingstuff, the rate of N disappearance was not affected by the diets fed.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2998453 TI - The effect of dietary bagasse on the activities of some key enzymes of carbohydrate and lipid metabolism in mouse liver. AB - The effects of a 100 g/kg diet substitution of bagasse on the body-weight gain, food consumption and faecal dry weight of mice given a high-sucrose diet and on the activities of hepatic glucose-6-phosphate dehydrogenase (EC I.I.I.49), 6 phosphogluconate dehydrogenase (EC I.I.I.44), malate dehydrogenase (oxaloacetate decarboxylating) (NADP+) (EC I.I.I.40), ATP-citrate (pro-3S) lyase (EC 4.I.3.8), 6-phosphofructokinase EC 2.7.I.II), pyruvate kinase (EC 2.7.I.40) and fructose 1,6-bisphosphatase (EC 3.I.3.II) were studied. Bagasse had no effect on body weight gain, food consumption or faecal dry weight. Bagasse decreased the activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and phosphofructokinase expressed on a wet weight basis and on a protein basis. Bagasse decreased the activities of glucose-6-phosphate dehydrogenase and 6 phosphogluconate dehydrogenase expressed on a body-weight basis. These results suggest that bagasse decreases the flux through some pathways of hepatic lipogenesis when mice are given high-sucrose diets. PMID- 2998454 TI - Ytterbium acetate as a particulate-phase digesta-flow marker. AB - The ability of ytterbium acetabe (Yb acetate) to fulfil the requirements of a particulate-phase digesta-flow marker in a dual-phase marker system, and of the indigestible acid-detergent-fibre fraction of the feed (IADF) to act as a digesta flow marker, were examined using six mature wether sheep given a diet of dried grass (1 kg dry matter (DM)/d). CrEDTA was continuously infused (240 mg chromium/d) into the rumen of all sheep and Yb acetate was also continuously infused (100 mg Yb/d) into the rumen of three of the sheep. At this level of infusion the equilibrium concentration of Yb in rumen, duodenal and ileal digesta and in faeces could be reliably measured by atomic absorption spectrometry. Estimates of faecal DM excretion based on either Yb or IADF did not differ (P greater than 0.05) from that determined by total collection, whereas estimates based on Cr were significantly (P less than 0.05) lower. Urinary excretion accounted for 3.1% of the infused Cr but no Yb was detected in urine. Estimates of ileal DM flow, assuming total marker recovery, were similar (P greater than 0.05) with all three markers, whereas the estimate of duodenal DM flow based on IADF was lower (P less than 0.05) than the estimates based on either Cr or Yb. Compared with the infusion of Cr alone, the infusion of Cr and Yb had no effect (P greater than 0.05) on nutrient flows at the duodenum, ileum and in faeces nor on microbial degradative activity volatile fatty acid production and N metabolism in the rumen. Polyester bag and in vitro studies showed that pre-labelling the dried grass with up to 285 mg Yb/g DM did not affect its susceptibility to microbial degradation. The Yb in rumen, duodenal and ileal digesta was predominantly (greater than 90%) associated with the particulate matter but was not uniformly distributed and its concentration increased as particle size decreased. The use of CrEDTA and Yb acetate as a dual-phase marker system proved more reliable in estimating 'true' duodenal flow than the use of the individual markers when the digesta sample was unrepresentative. PMID- 2998455 TI - Kallikrein-related mRNAs of the rat submaxillary gland: nucleotide sequences of four distinct types including tonin. AB - We have determined the nucleotide sequence of four submaxillary gland mRNAs, designated PS, S1, S2, and S3, that encode kallikrein and kallikrein-like serine proteases. The four enzymes share between 74% and 86% amino acid sequence identity and are identical in length with the exception of single two amino acid deletions in the S2 and S3 enzymes. The PS enzyme appears to be a true tissue kallikrein. The S1 enzyme shares 86% amino acid sequence homology with the PS enzyme and retains key amino acid residues thought to be primary determinants of kallikrein cleavage specificity. The S2 enzyme is rat submaxillary tonin. The amino acid sequence of the S3 enzyme is identical with tonin at 84% of its amino acid positions and retains the same amino acid substitutions at positions likely to determine substrate cleavage preferences. PMID- 2998456 TI - Kinetic analysis of fusion of hemagglutinating virus of Japan with erythrocyte membrane using spin-labeled phosphatidylcholine. AB - HVJ* (hemagglutinating virus of Japan containing spin-labeled phosphatidylcholine in its envelope around 10 mol %) was adsorbed onto erythrocytes or erythrocyte ghosts at various doses, and the ESR spectrum of the virus-cell system was measured at 37 degrees C. The peak-height increase for the HVJ*-ghost system was satisfactorily analyzed on the basis of envelope fusion by a first-order kinetic equation with two different rate constants. The rate constant was obtained as k1 = 0.84 min-1 and k2 = 0.011 min-1, independent of the virus dose. The fraction of virus fused at the rate constant k1 decreased with the dose. However, the average number of fast-fusing viruses per cell was nearly independent of the dose, and the value was one to two. The peak-height increase in the HVJ*-erythrocyte system was caused by both envelope fusion and phospholipid exchange catalyzed by the virus-induced hemolyzate. At lower doses, where the virus-induced hemolysis was small and, therefore, the rate of phospholipid exchange was small, the peak height increase could be analyzed by the same kinetic equation with nearly the same rate constant value for k1 as that for HVJ*-ghosts. However, the k2 was larger than that for HVJ*-ghost, owing to the additional transfer by phospholipid exchange. PMID- 2998457 TI - NMR studies of the MgATP binding site of adenylate kinase and of a 45-residue peptide fragment of the enzyme. AB - Proton NMR was used to study the interaction of beta,gamma-bidentate Cr3+ATP and MgATP with rabbit muscle adenylate kinase, which has 194 amino acids, and with a synthetic peptide consisting of residues 1-45 of the enzyme, which has previously been shown to bind MgepsilonATP [Hamada, M., Palmieri, R. H., Russell, G. A., & Kuby, S. A. (1979) Arch. Biochem. Biophys. 195, 155-177]. The peptide is globular and binds Cr3+ATP competitively with MgATP with a dissociation constant, KD(Cr3+ATP) = 35 microM, comparable to that of the complete enzyme [KI(Cr3+ATP) = 12 microM]. Time-dependent nuclear Overhauser effects (NOE's) were used to measure interproton distances on enzyme- and peptide-bound MgATP. The correlation time was measured directly for peptide-bound MgATP by studying the frequency dependence of the NOE's at 250 and 500 MHz. The H2' to H1' distance so obtained (3.07 A) was within the range established by X-ray and model-building studies of nucleotides (2.9 +/- 0.2 A). Interproton distances yielded conformations of enzyme- and peptide-bound MgATP with indistinguishable anti-glycosyl torsional angles (chi = 63 +/- 12 degrees) and 3'-endo/O1'-endo ribose puckers (sigma = 96 +/- 12 degrees). Enzyme- and peptide-bound MgATP molecules exhibited different C4'-C5' torsional angles (gamma) of 170 degrees and 50 degrees, respectively. Ten intermolecular NOE's from protons of the enzyme and four such NOE's from protons of the peptide to protons of bound MgATP were detected, which indicated proximity of the adenine ribose moiety to the same residues on both the enzyme and the peptide. Paramagnetic effects of beta,gamma-bidentate Cr3+ATP on the longitudinal relaxation rates of protons of the peptide provided a set of distances to the side chains of five residues, which allowed the location of the bound Cr3+ atom to be uniquely defined. Distances from enzyme-bound Cr3+ATP to the side chains of three residues of the protein agreed with those measured for the peptide. The mutual consistency of interproton and Cr3+ to proton distances obtained in metal ATP complexes of both the enzyme and the peptide suggests that the conformation of the peptide is very similar to that of residues 1-45 of the enzyme. When this was assumed to be the case and when molecular models and a computer graphics system were used, MgATP could be fit into the X-ray structure of adenylate kinase in a unique manner such that all of the distances determined by NMR were accommodated.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2998459 TI - Reduction of oxidized cytochrome c by ascorbate ion. AB - The kinetics and mechanism of the reduction of oxidized cytochrome c by ascorbate has been investigated in potassium nitrate, potassium 4-morpholineethanesulfonate (KMes), potassium sulfate and potassium ascorbate media. The results are consistent with simple second order electron transfer from ascorbate dianion to cytochrome c and do not support electron transfer from an ascorbate dianion bound to the protein of the cytochrome as recently proposed by Myer and Kumar. A rate constant of 8 X 10(5) M-1 X s-1 (25 degrees C, ionic strength, 0.1) was found for the electron-transfer step. This rate constant is essentially independent of the specific ions used in controlling ionic strength. PMID- 2998458 TI - Extraction of mitochondrial membrane proteins into organic solvents in a functional state. AB - Protein-lipid complexes in organic solvents can be used as the starting material in the reassembly of functional planar and spherical bilayers (Montal, M., Darszon, A. and Schindler, H. (1981) Q. Rev. Biophys. 14, 1-79). The transfer of three enzymes of the inner mitochondrial membrane into organic solvents as protein-lipid complexes has been studied to understand better the extraction process. The enzymes studied were cytochrome c oxidase, ATPase and succinate dehydrogenase. These enzymes were transferred into hexane and diethyl ether in an active state, however, the activities extracted varied quantitatively, depending on the amount of protein of the starting preparation, the concentration of phospholipids and the cation employed. In all conditions cytochrome c oxidase was extracted with the highest yield and specific activity, and it was actually enriched in the organic extract. The values for succinate dehydrogenase and ATPase were lower, but their specific activities were similar to those of the starting material. This indicates that some membrane proteins are preferentially extracted into organic solvents in a functional state. The enzymes, as protein lipid complexes, are fairly stable in organic solvents; in a month of storage at 4 degrees C in hexane some enzymes loose less than 50% of their activity. PMID- 2998460 TI - Adenine nucleotide translocase-dependent anion transport in pea chloroplasts. AB - Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [14C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12-14-day-old plants was calculated to be 330 mumol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22 degrees C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phosphoenolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phosphoenolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis. PMID- 2998462 TI - Protective effect of Na+ and K+ against inactivation of (Na+ + K+)-ATPase by high concentrations of 2-mercaptoethanol at high temperatures. AB - Purified dog kidney (Na+ + K+)-ATPase (EC 3.6.1.3) was inactivated with high concentrations of 2-mercaptoethanol at 50-55 degrees C. The inactivation was prevented by NaCl or KCl, with KCl being more effective than NaCl (the former ion being about one order more efficient under a typical set of experimental conditions). A disulfide bond in the beta-subunit of the enzyme protein was prevented from reductive cleavage by NaCl or KCl in accordance with protection of the enzyme activity. Choline chloride did not exert a significant protective effect over a similar concentration range. (Na+ + K+)-ATPase was also inactivated with high concentrations of 2-mercaptoethanol in the presence of low concentrations of dodecyl sulfate. This inactivation was also prevented by NaCl or KCl, with the latter being again more efficient than the former. These results indicate that Na+ and K+ bound to their respective ion-binding sites on the alpha subunit exert a protective effect on a disulfide bond on the beta-subunit. This suggests some sort of interaction between the alpha- and the beta-subunits. PMID- 2998461 TI - Determination of osmotic volumes and pH gradients of plant membrane and lipid vesicles using ESR spectroscopy. AB - Volumes and pH gradients were determined with spin probes in liposomes and zucchini membrane vesicles by quantitating the internal concentrations of probes in the presence of an impermeable line-broadening agent, manganese + EDTA. Volume shrinkage in response to increasing external concentrations of MnEDTA was consistent with perfect osmotic behavior of both vesicle populations. Buffer additions were used to impose pH gradients on the vesicles; liposome gradients measured with a spin-labeled weak acid were slightly smaller than the maximum theoretical imposed gradients, whereas above a threshold magnitude, measured gradients for the plant membranes were significantly smaller than imposed gradients. However, the residual pH gradient in the zucchini vesicles decreased at about the same rate as the liposome gradient. Moreover, this residual gradient was not completely collapsed in the presence of the proton ionophore, FCCP, indicating that the vesicles were impermeable to ions; indeed, ion permeabilities of both vesicle preparations appeared to be similar during the slow phase of the pH gradient collapse. Thus, zucchini membrane vesicles are tightly sealed and appear to have a mechanism for dissipating pH gradients rapidly when these gradients exceed some threshold value. PMID- 2998463 TI - Phosphatase activity of (Na+ + K+)-ATPase. Ligand interactions and related enzyme forms. AB - The prevailing conformations of partially purified pig kidney (Na+ + K+)-ATPase interacting with ligands related to its phosphatase activity were determined following time-dependent trypsin digestion and inactivation as well as the amounts of Rb+ or Ca2+ bound to the enzyme after passage through cation-exchange resin columns. In the presence of 150 mM choline chloride, alone or with 3 mM MgCl2, 3 mM MnCl2 or 1 mM CaCl2, the major enzyme conformation was E1. Similar forms were seen with 5 mM p-nitrophenyl phosphate with and without 3 mM MgCl2. KCl, at 0.5 mM or 150 mM, produced an E2 enzyme state; the effects of 0.5 mM KCl were completely counteracted by 5 mM p-nitrophenyl phosphate. Under optimal conditions for phosphatase activity (3 mM MgCL2/5 mM p-nitrophenyl phosphate/10 mM KCl) the (Na+ + K+)-ATPase was in the E2 state. At low ionic strength and 20 degrees C and under 85% of maximal RbCl-stimulated phosphatase turnover (1 mM RbCl/3 mM MgCl2/5 mM p-nitrophenyl phosphate) no Rb+ occlusion could be detected. Ca2+, at low ionic strength and in the presence of 3 mM MgCl2, stimulated an ouabain-sensitive phosphatase activity. The rates of hydrolysis obtained wit 1 mM CaCl2 were similar to those seen with 0.5 mM KCl; under both conditions, similar patterns of trypsin digestion and inactivation of the enzyme were obtained. On the other hand, Ca2+ could not mimic Rb+ in its ability to induce an E2-occluding state. These results suggest that during phosphatase activity of (Na+ + K+) ATPase, the most abundant form is a non-occluding E2 and that at least one of the mechanisms of potassium stimulation of that activity it to take the enzyme into the E2 state. PMID- 2998465 TI - Temperature sensitivity of potassium flux into red blood cells in the familial pseudohyperkalaemia syndrome. AB - The temperature dependence of potassium flux into the red cells of normal and pseudohyperkalaemic individuals over the range 4-40 degrees C was measured using 86RbCl as tracer. Flux through the pump was measured as the ouabain-sensitive component (0.2 mM ouabain) and flux via Na+,K+-cotransport was measured as the decrease in the rate of K+ influx in the presence of 1 mM furosemide. The residual passive permeability of the red cell plasma membranes to K+ was that influx which was unaffected by either inhibitor. When Na+ influxes were measured, the ratio of Na+ to K+ transported via the furosemide-sensitive component was 1 over the full temperature range studied. The temperature sensitivity of K+ influx via the pump was normal as was the enzymic activity of the Na+,K+-ATPase. In contrast, the activity of the Na+,K+-cotransport system in pseudohyperkalaemics was more temperature sensitive than that of controls and affected individuals also showed a greater passive permeability to K+ at low temperatures. Red cell membranes from affected individuals have significantly increased amounts of phosphatidylcholine which are balanced, to a degree, by a decreased content of phosphatidylethanolamiane. It is proposed that in this example of familial pseudohyperkalaemia there is an alteration in the structure of the red cell plasma membrane which influences the temperature sensitivity of both its cotransport and passive permeability properties. PMID- 2998464 TI - Analysis of hexose transport in untransformed and sarcoma virus-transformed mouse 3T3 cells by photoaffinity binding of cytochalasin B. AB - The effect of simian virus 40 transformation on the hexose transport system in mouse embryo fibroblast Swiss 3T3 cells was examined. The concentration of hexose transporters was estimated by measuring D-glucose-inhibitable cytochalasin B binding. The binding of cytochalasin B to the plasma membranes of simian virus 40 transformed mouse 3T3 cells (SV3T3 cells) was significantly greater than that of 3T3 cells. On the other hand, cytochalasin B binding to the microsomal membranes of SV3T3 cells was decreased, and the total amount of binding to plasma and microsomal membranes was not significantly changed in both cell lines. The electrophoretic analysis demonstrated that both hexose-transporter components of Mr 46 000 and Mr 58 000 affinity labeled were responsible for an increase in the hexose transport by viral transformation. These results suggested that the higher hexose-transport activity of transformed cells is caused by a redistribution of transporter from intracellular membranes to plasma membranes. PMID- 2998467 TI - Effect of proteolysis on the electron spin resonance spectra of maleimide spin labeled erythrocyte membrane. AB - The ratio of low-field amplitudes of weakly and strongly immobilized signals of ESR spectra of a maleimide spin label bound to erythrocyte membranes (hw/hs) increases progressively during incubation at 37 degrees C. This increase is due to the 'self-digestion' of membrane proteins by endogenous proteinases and is attenuated by proteinase inhibitors. Digestion of membranes with chymotrypsin also increases the hw/hs ratio. These results suggest a need for a careful interpretation of data from spin-labeled membrane proteins, especially in experiments involving prolonged incubations of membrane preparations when the proteolytic effects may be significant. PMID- 2998466 TI - Solubilization and reconstitution of cholinephosphotransferase from sarcoplasmic reticulum: stabilization of solubilized enzyme by diacylglycerol and glycerol. AB - Cholinephosphotransferase (CDPcholine: 1,2-diacylglycerol cholinephosphotransferase, EC 2.7.8.2), which catalyzes the terminal step in phosphatidylcholine synthesis via the CDPcholine pathway, is present in sarcoplasmic reticulum from rabbit skeletal muscle (Cornell, R. and MacLennan, D.H. (1985) Biochim. Biophys. Acta 835, 567-576). The conditions for solubilization and reconstitution of this enzyme were investigated as a preliminary step towards its eventual purification. The activity was not released by treatment of membranes with 1 M KCl, but was solubilized after dissolution of membranes with detergents. Cholinephosphotransferase was inactivated by cholate, deoxycholate, Triton X-100, octylglucoside, Tween-20 or SDS at concentrations which solubilize the membrane. However, the activity could be fully recovered after reconstituting the membrane by adding excess lipid (soybean) and removing detergent by gel filtration, dialysis or by absorption to Bio-Beads. When the membrane was solubilized with octylglucoside or cholate at weight ratios of detergent: membrane protein of at least 10, the activity was irreversibly lost unless stabilizers were added with detergent. The substrate diacylglycerol and glycerol were effective stabilizers. PMID- 2998468 TI - On the water and proton permeabilities across membranes from erythrocyte ghosts. AB - The diffusional permeability of water across membranes from bovine and human erythrocyte ghosts was measured by a recently developed method which is based on the different indices of refraction of H2O and 2H2O. Resealed erythrocyte ghosts were prepared by a gel-filtration technique. Pd (2H2O/H2O) values of 1.2 X 10(-3) cm/s (human) and 1.7 X 10(-3) cm/s (bovine) were calculated at 20 degrees C. The activation energies of the water exchange were 23.5 kJ/mol (human) and 25.4 kJ/mol (bovine). Treatment of the ghosts with p-chloromercuribenzenesulfonic acid (PCMBS) led to a 60-70% inhibition of the diffusional water exchange. The pH equilibration across membranes of erythrocyte ghosts was measured by intracellular carboxyfluorescein. The rates of proton flux after pH-jumps (pH 7.3 to pH 6.1) were about 100-fold lower than those of the water exchange and dependent on the kind of anions present (Cl-, NO-3, SO2-4). The activation energies of proton flux were 60-70 kJ/mol. 4,4'-Diisothiocyanatostilbene-2,2' disulfonic acid (DIDS) inhibited the exchange by 97-98% and lowered the activation energy. The inhibitor of water exchange, PCMBS, increased the proton permeation rate by a factor of 4-5. It is assumed that the rate-limiting step for the proton permeation is determined by the anion exchange. Under this condition our results are not in accord with one channel as a common pathway for both the passive water and anion transport. PMID- 2998469 TI - Target analysis studies of red cell water and urea transport. AB - Radiation inactivation was used to determine the nature and molecular weight of water and urea transporters in the human red cell. Red cells were frozen to -50 degrees C in a cryoprotectant solution, irradiated with 1.5 MeV electrons, thawed, washed and assayed for osmotic water and urea permeability by stopped flow light scattering. The freezing and thawing process did not affect the rates of water or urea transport or the inhibitory potency of p chloromercuribenzenesulfonate (pCMBS) on water transport and of phloretin on urea transport. Red cell urea transport inactivated with radiation (0-4 Mrad) with a single target size of 469 +/- 36 kDa. 40 microM phloretin inhibited urea flux by approx. 50% at each radiation dose, indicating that urea transporters surviving radiation were inhibitable. Water transport did not inactivate with radiation; however, the inhibitory potency of 2.5 mM pCMBS decreased from 86 +/- 1% to 4 +/- 9% over a 0-2 Mrad dose range. These studies suggest that red cell water transport either required one or more low-molecular-weight proteins, or is lipid mediated, and that the pCMBS-binding site which regulates water flow inactivates with radiation. These results also suggest that red cell urea transport is mediated by a specific, high-molecular-weight protein. These results do not support the hypothesis that a band 3 dimer (190 kDa) mediates red cell osmotic water and urea transport. PMID- 2998470 TI - Detection of oxygen consumption during very early stages of lipid peroxidation by ESR nitroxide spin probe method. AB - Oxygen consumption during the very early stages of the spontaneous peroxidation of egg yolk phosphatidylcholine membranes was studied by monitoring the oxygen concentration in the aqueous phase of the sample using a spin-probe closed chamber method. The method depends on the broadening by oxygen of the proton superhyperfine lines of the electron spin resonance spectra of the nitroxide radical spin probe 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-yloxyl. It is concluded that this method is useful in monitoring lipid peroxidation and that it monitors the onset of the peroxidation process before the commonly used thiobarbituric acid assay detects the peroxidation products. PMID- 2998471 TI - [3H]Inositol incorporation into phosphoinositides of pig reticulocytes. AB - Phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) of pig reticulocytes were extensively labelled when these cells were incubated with [3H]inositol. In marked contrast, a total lack of [3H]inositol labelling of phosphoinositides was observed in mature erythrocytes. Phosphoinositides of both reticulocytes and mature erythrocytes were labelled with 32P but the labelling in reticulocytes was several-fold higher than in mature erythrocytes. Inclusion of Ca2+ (2 mM)+ ionophore A23187 (2 micrograms/ml) during the labelling experiments substantially reduced the radioactivity incorporation into phosphoinositides of reticulocytes. When [3H]inositol-prelabelled reticulocytes were treated with Ca2+ + A23187 the levels of radioactive PI and PIP2 did not change significantly. However, the PIP pool exhibited a remarkable sensitivity to Ca2+ as shown by a 75% increase in its radioactivity over the control. The ability to incorporate [3H]inositol into phosphoinositides remains transitorily intact in the reticulocyte stage. Thus, pig reticulocytes offer a suitable model in which to explore the physiological role of phosphoinositides in relation to cellular maturation process. PMID- 2998472 TI - Chymotryptic cleavage of alpha-subunit in E1-forms of renal (Na+ + K+)-ATPase: effects on enzymatic properties, ligand binding and cation exchange. AB - Chymotrypsin in NaCl medium at low ionic strength rapidly cleaves a bond in the N terminal half of the alpha-subunit of pure membrane-bound (Na+ + K+)-ATPase from outer renal medulla. Secondary cleavage is very slow and the alpha-subunit can be converted almost quantitatively to a 78 kDa fragment. The sensitive bond is exposed to cleavage when the protein is stabilized in the E1 form by binding of Na+ or nucleotides. The bond is protected in medium containing KCl (E2K form), but it is exposed when ADP or ATP are added (E1KATP form). Fluorescence analysis and examination of ligand binding and enzymatic properties of the cleaved protein demonstrate that cleavage of the bond stabilizes the protein in the E1 form with sites for tight binding of nucleotides and cations exposed to the medium. About two 86Rb ions are bound per cleaved alpha-subunit with normal affinity (Kd = 9 microM). The bound Rb+ is not displaced by ATP or ADP. The nucleotide-potassium antagonism is abolished and ATP is bound with high affinity both in NaCl and in KCl media. Na+-dependent phosphorylation is quantitatively recovered in the 78 kDa fragment, but the affinity for binding of [48V]vanadate is very low after cleavage. ADP-ATP exchange is stimulated 4-5-fold by cleavage; while nucleotide dependent Na+-Na+, K+-K+, or Na+-K+ exchange are abolished. Cleavage with chymotrypsin in NaCl at the N-terminal side of the phosphorylated residue thus stabilizes the E1 form of the protein and abolishes cation exchange and conformational transitions in the protein although binding of cations, nucleotides and phosphate is preserved. In contrast, cleavage with trypsin in KCl at the C-terminal side of the phosphorylated residue does not interfere with E1 E2 transitions and Na+-Na+ or K+-K+ exchange. This data support the notion that cation exchange and E1-E2 transitions are thightly coupled. PMID- 2998473 TI - Interaction of (Na+ + K+)-ATPase with artificial membranes. I. Formation and structure of (Na+ + K+)-ATPase-liposomes. PMID- 2998474 TI - Resonance Raman spectra for catalytic intermediates of cytochrome c oxidase detected with a mixed flow transient apparatus. AB - A novel technique was employed to collect resonance Raman spectra of an oxygenated intermediate of cytochrome c oxidase. Instead of laser pulses of high peak power, which may cause photodissociation, a continuous wave laser and a mixed flow apparatus were used. An intermediate formed within 450 microseconds after the reaction of cytochrome c oxidase with molecular oxygen could be detected. From the spectra it could be deduced that the most likely candidate for the intermediate would be a transient oxygenated species having the Fe2+ - O2 or Fe4+ = O heme in cytochrome a3 and the Fe2+ heme in cytochrome a. PMID- 2998475 TI - Binding of epidermal growth factor from man, rat and mouse to the human epidermal growth factor receptor. AB - Human, rat and mouse epidermal growth factors (EGF) bind to the same receptor on human placenta, but the binding characteristics differ. The apparent affinity constant (KA) for human EGF is higher (15 X 10(9) l/mol) than KA for rat EGF (10 X 10(9) l/mol). Mouse EGF binds with the lowest KA (5 X 10(9) l/mol). The pH optimum differs so that human and rat EGF bind with a pH optimum of 8.0, whereas mouse EGF binds with an optimum of pH 7.4. Half maximal dissociation is 130, 50 and 25 min for human, rat and mouse EGF, respectively. The structures of human, rat and mouse EGF differ somewhat. At least 11 of the first 24 residues differ. The N-terminal sequence of rat EGF is: Ala/Ser-Gly-X-Pro-Pro-Ser-Tyr-Asp-Gly-Tyr X-Lys-Asp-Gly-Gly-Val-X-Met-Ty r-Val -Glu. PMID- 2998477 TI - Cobalt(II) ion as a promoter of hydroxyl radical and possible 'crypto-hydroxyl' radical formation under physiological conditions. Differential effects of hydroxyl radical scavengers. AB - Co(II) ions react with hydrogen peroxide under physiological conditions to form a 'reactive species' that can hydroxylate aromatic compounds (phenol and salicylate) and degrade deoxyribose to thiobarbituric-acid-reactive material. Catalase decreases the formation of this species but superoxide dismutase or low concentrations of ascorbic acid have little effect. EDTA, present in excess over the Co(II), can accelerate deoxyribose degradation and aromatic hydroxylation. In the presence of EDTA, deoxyribose degradation by the reactive species is inhibited competitively by scavengers of the hydroxyl radical (.OH), their effectiveness being related to their second-order rate constants for reaction with .OH. In the absence of EDTA the scavengers inhibit only at much higher concentrations and their order of effectiveness is changed. It is suggested that, in the presence of EDTA, hydroxyl radical is formed 'in free solution' and attacks deoxyribose or an aromatic molecule. In the absence of EDTA, .OH radical is formed in a 'site-specific' manner and is difficult to intercept by .OH scavengers. The relationship of these results to the proposed 'crypto .OH' radical is discussed. PMID- 2998476 TI - Characterization of the binding of ferritin to the rat liver ferritin receptor. AB - The binding characteristics and specificity of the rat hepatic ferritin receptor were investigated using ferritins prepared from rat liver, heart, spleen, kidney and serum, human liver and serum, guinea pig liver and horse spleen as well as ferritins enriched with respect to either H- or L-type subunit composition, prepared by chromatofocusing of rat liver ferritin on Mono-P or by reverse-phase chromatography of ferritin subunits on ProRPC 5/10. No significant difference was apparent in the binding of any of the tissue ferritins, or of ferritins of predominantly acidic or basic subunit composition. However, serum ferritin bound with a lower affinity. The effect of carbohydrate on the ferritin-receptor binding was examined by glycosidase treatment of tissue and serum ferritins. Tissue ferritin binding was unaffected, while serum ferritin binding affinity was increased to that of the tissue ferritins. Inhibition of ferritin binding by lactoferrin was not due to common carbohydrate moieties as previously suggested but was due to direct binding of lactoferrin to ferritin. Therefore, carbohydrate residues do not appear to facilitate receptor-ferritin binding, and sialic acid residues present on serum ferritin may in fact interfere with binding. The results indicate that the hepatic ferritin receptor acts preferentially to remove tissue ferritins from the circulation. The lower binding affinity of serum ferritin for the ferritin receptor explains its slower in vivo clearance relative to tissue ferritins. PMID- 2998478 TI - Electron spin resonance studies of intact mammalian skeletal muscle. AB - Samples of skeletal muscle from mice, rats and man have been examined by conventional electron spin resonance techniques. One major free-radical signal with g value 2.0036-2.004 was detected in all intact muscle samples and homogenates at 77 K whereas this signal was not seen at room temperature. Other less prominant signals were also detected. Thirty minutes of excessive contractile activity of rat hind limb muscles was found to result in a leakage of intracellular creatine kinase enzyme into the blood plasma and also produced an average 70% increase in the amplitude of the major electron spin resonance signal. These data support the hypothesis that increased free-radical activity may play some role in muscle damage caused by extensive muscular activity. PMID- 2998480 TI - Effects of canavanine on the secretion of plasma proteins by Hep G2 cells. AB - Many secretory proteins contain an amino-terminal propeptide extension which is removed prior to secretion. The point of cleavage is usually marked by a basic pair of amino acids containing arginine. Canavanine, an analogue of arginine, is incorporated into protein and has been shown to inhibit the proteolytic processing of several of these prosecretory proteins. The addition of 3 mM canavanine to Hep G2 cells incubated with L-[35S]methionine inhibited the secretion of 11 plasma proteins studied. Of the secretory proteins studied only albumin is thought to contain a propeptide, which is marked by a pair of arginine residues at its point of proteolytic processing. Canavanine had varying effects on the secretion of plasma proteins; ranging from a 43-53% inhibition of secretion of alpha 1 antitrypsin and alpha 1 anti-chrymotrypsin to nearly abolishing (93% inhibition) secretion of transferrin. Canavanine also caused most of the proteins studied to migrate slower on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Two of the canavanine-treated proteins (albumin and transferrin) which underwent marked changes in electrophoretic mobility were more sensitive than untreated proteins to proteolysis by Staphylococcus Aureus V8 proteinase. The slower electrophoretic migration and the greater sensitivity to proteolysis of these proteins may be attributed to marked structural changes caused by the incorporation of canavanine. This suggests that the inhibition of plasma protein secretion by canavanine is not only due to an inhibition of the processing of proteins but may be caused by structural distortions of the secretory proteins. PMID- 2998479 TI - Studies on regulatory mechanisms of heme biosynthesis in hepatocytes from experimental-diabetic rats. AB - Isolated hepatocytes from rats with experimental diabetes exhibit increased content of cytochrome P-450 and cyclic AMP and normal activities of the regulatory enzymes delta-aminolevulinic acid synthase and ferrochelatase. The inducing effect exerted by phenobarbital on cytochrome P-450, delta aminolevulinic acid synthase and ferrochelatase biosynthesis and cyclic AMP content in diabetic hepatic cells is markedly greater than that observed in normal hepatocytes. This stimulatory response is neither enhanced by added dibutyryl cyclic AMP nor repressed by glucose. The present results suggest that the heme pathway of diabetic hepatocytes is more susceptible to porphyrinogenic factors. PMID- 2998481 TI - Accumulation of inositol phosphates and cyclic AMP in guinea-pig cerebral cortical preparations. Effects of norepinephrine, histamine, carbamylcholine and 2-chloroadenosine. AB - Norepinephrine and serotonin augment by about 2-fold the accumulation of cyclic [3H]AMP elicited by 2-chloroadenosine in [3H]adenine-labeled guinea-pig cerebral cortical slices. Histamine causes a 3-fold augmentation. The first two agents have no effect on cyclic AMP alone, while histamine has only a small effect alone. The augmentation of the 2-chloroadenosine response appears to be mediated by alpha 1-adrenergic, 5HT2-serotonergic and H2-histaminergic receptors. VIP elicited accumulations of cyclic AMP are also augmented through stimulation of alpha 1-adrenergic, 5HT2-serotonergic and H1-histaminergic receptors. Activation of these amine receptors also increases the turnover of phosphatidylinositols in [3H]inositol-labeled guinea pig cerebral cortical slices. Norepinephrine causes a 5-fold, serotonin a 1.2-fold, and histamine a 2.5-fold increase in accumulations of [3H]inositol phosphates. 2-Chloroadenosine, vasoactive intestinal peptide, baclofen, and somatostatin have no effect on phosphatidylinositol turnover, nor do the last two agents augment accumulations of cyclic AMP elicited by 2 chloroadenosine. The data suggest a possible relationship between turnover of phosphatidylinositol and the augmentations of the cyclic AMP accumulations elicited by biogenic amines in brain slices. PMID- 2998482 TI - Modulation by the ratio S-adenosylmethionine/S-adenosylhomocysteine of cyclic AMP dependent phosphorylation of the 50 kDa protein of rat liver phospholipid methyltransferase. AB - The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. S-Adenosylmethionine/S-adenosylhomocysteine ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio S adenosylmethionine/S-adenosylhomocysteine can modulate phosphorylation of a specific protein. PMID- 2998483 TI - Lysine binding to activated human platelets and its similarity to fibrinogen binding. AB - Platelet surface glycoproteins IIb-IIIa are considered to function as the binding site for fibrinogen. Fibrinogen binding is essential for platelet aggregation and several amines have been shown to inhibit this binding. The present study compares the binding properties of 125I-fibrinogen and [3H]lysine with platelets activated by the Ca2+ ionophore A23187. Many lines of similarities in the binding properties are apparent; however, several differences were also found. The similarities are listed below and the differences are pointed out in parentheses. Marked enhancement by platelet activation; deficiency of binding by thrombasthenic platelets lacking the glycoproteins IIb-IIIa; saturability (fibrinogen binding approaches saturation at more than 12 microM, within 10 min; lysine binding at more than 100 mM within 1 min); Ca2+-dependence (at 1 mM Ca2+ lysine binding is minute and fibrinogen binding is half-saturated); reversibility; the binding achieved within 10 min is exchangeable; dissociation depends upon time and external ligand concentration; inhibition by the oligoamines His-Lys and Lys4; inhibition by serum from a thrombasthenic patient who developed anti-glycoproteins IIb-IIIa antibodies; specificity; alanine neither binds to activated platelets nor inhibits fibrinogen binding; it thus appears that the lysine which associates with activated platelets is mostly bound onto the surface of the cells rather than being incorporated. Moreover, the major site of lysine binding seems to be the complexed glycoproteins IIb-IIIa. PMID- 2998484 TI - Effects of hemin on rat liver cyclic AMP-dependent protein kinases in cell extracts and intact hepatocytes. AB - Cyclic AMP-dependent protein kinases I and II, partially purified from rat liver cytosol, were inhibited 50% by 40 microM hemin and 100 microM hemin, respectively. With the purified catalytic subunit of cyclic AMP-dependent protein kinase, hemin caused non-competitive inhibition with respect to the peptide substrate and mixed inhibition with respect to ATP. Hemin also inhibited purified phosphorylase b kinase, indicating that hemin concentrations above 10 microM markedly inhibit multiple protein kinases. In isolated intact hepatocytes, hemin inhibited the glucagon-dependent activation of cyclic AMP-dependent protein kinases and the activation of glycogen phosphorylase. For both effects, high heme concentrations (40-60 microM) were required for 50% inhibition. Similar high levels of exogenous hemin inhibited total hepatocyte protein synthesis. By contrast, 5 microM hemin or less was sufficient to raise intracellular heme levels, as indicated by the relative heme-saturation of tryptophan oxygenase in hepatocytes. Hemin, 5 microM, completely repressed induction of 5-aminolevulinate synthase by dexamethasone in hepatocyte primary cultures. Such repression is unlikely to be mediated by inhibition of protein kinases. PMID- 2998485 TI - Affinity labeling of high-affinity alpha-thrombin binding sites on the surface of hamster fibroblasts. AB - The serine proteinase alpha-thrombin potently stimulates reinitiation of DNA synthesis in quiescent Chinese hamster fibroblasts (CCL39 line). 125I-labeled alpha-thrombin binds rapidly and specifically to CCL39 cells with high affinity (Kd approximately 4 nM). Binding at 37 degrees C was found to remain stable for 6 h or more during which time no receptor down-regulation, ligand internalization and/or degradation could be detected. The structure of alpha-thrombin receptors on CCL39 cells was identified by covalently coupling 125I-alpha-thrombin to intact cells using a homobifunctional cross-linking agent, ethylene glycol bis(succinimidyl succinate). By resolution in sodium dodecyl sulfate polyacrylamide gel electrophoresis we observed the specific labeling of a major alpha-thrombin-binding site of Mr approximately 150 000 revealed as a 125I-alpha thrombin cross-linked complex of Mr approximately 180 000. Independent of chemical cross-linking, 125I-alpha-thrombin also formed a covalent complex with a minor, 35 000 Mr, membrane component identified as protease nexin. Two derivatives of alpha-thrombin modified at the active site are 1000-fold less than alpha-thrombin for mitogenicity. These two non-mitogenic derivatives bound to cells with similar affinity and maximal binding capacity as native alpha thrombin, and affinity-labeled the receptor subunit of Mr 150 000. When present in large excess, during incubation of cells with alpha-thrombin, these binding antagonists were ineffective in blocking alpha-thrombin-induced DNA synthesis. These data suggest that the specific 150 000 Mr binding sites that display high affinity for alpha-thrombin do not mediate induction of the cellular mitogenic response. PMID- 2998486 TI - [Effect of bacterial toxins on the GTPase activity of transducin from bovine rod outer segments]. AB - The effects of choleragen- and pertussis toxin (PT)-induced ADP-ribosylation on the GTP-binding protein transducin (TD) from retinal rod outer segments (ROS) have been studied. It has been shown that both toxins cause inhibition of the TD GTPase activity. PT inhibited the GTPase by 30-40% in "native" ROS and by 70-80% in homogeneous TD. Choleragen, in contrast with PT, had no effect on the GTPase activity of homogeneous TD, but was as effective as PT in membrane preparations. The effects of both toxins on the GTPase activity of TD were found to be dependent on the chemical structure of the guanyl nucleotide present in the vehicle. The data obtained suggest that PT and choleragen differ in their specificity for the TD-guanyl nucleotide complex. The former can interact with free TD as well as with the TD-GDP complex, while the latter affects only the TD GTP complex. PMID- 2998487 TI - [Complex type of kinetics of fructose-1,6-diphosphate from the rat and rabbit liver]. AB - Studies on rat and rabbit liver fructose 1.6-bisphosphatase inhibition by AMP showed that with an increase in EDTA concentration the hyperbolic AMP inhibition curve is transformed into a sigmoidal one. At intermediate EDTA concentrations, the kinetic curves have a plateau. The appearance of the intermediate plateau may be due to the superposition of kinetic curves corresponding to two enzyme forms simultaneously present in the assay mixture. One of these forms deprived of endogenous Me2+ (presumably Zn2+) is inhibited by AMP in a cooperative manner, while the other one retains Me2+ which prevents the cooperative response of the enzyme to AMP. PMID- 2998488 TI - [Thiol-dependent generation of superoxide radical induced by menadione and vicasol]. AB - It was demonstrated that in the presence of blood serum menadione and vicasol reduce cytochrome c in two ways, i. e., via O2-. -dependent and O2-. -independent routes. T ability of the serum to generate O2-. in the presence of menadione is retained after dialysis. Albumin can also act as an electron donor during O2-. generation mediated by menadione; however, this ability of the serum does not correlate with the presence of albumin in it. N-Ethylmaleimide and Cu2+ inhibit the ability of albumin and blood serum to induce menadione-mediated generation of O2-. It is concluded that in the presence of thiol-containing proteins and O2-. vitamin K3 forms an oxidative system generating O2-. PMID- 2998489 TI - [Interaction of sarcolysine with beta-adrenoreceptors in tumor cells]. AB - The sites of specific binding of 3H-L-dihydroalprenolol (3H-DHA) were identified on the surface of ascites sarcoma 37 cells, using competitive displacement and binding of the beta-adrenergic antagonists, 3H-DHA and L-propranolol. These binding sites possessed the properties of beta-adrenergic receptors coupled with adenylate cyclase. Analysis of 3H-DHA binding by the Scatchard method revealed the presence of beta-adrenergic receptors of two types, i. e., with a high (Kd = 0.9-1.0 nM) and low (Kd = 15-20 nM) affinity for 3H-DHA. The number of high affinity receptors was (5.0-7.5) X 10(3); that of low affinity receptors was (20 30) X 10(3) on a per cell basis. Sarcolysine at concentrations of 1-10 microM displaced receptor-bound 3H-DHA, competed with the ligand for the common binding sites and caused, similar to isoproterenol, a short-term elevation of the intracellular cAMP content. Sarcolysine within the same concentration range (2.5 25 microM) caused non-competitive inhibition of the cAMP phosphodiesterase (PDE2) activity of plasma membranes isolated from ascites sarcoma 37 cells. The data obtained point to the functional coupling between beta-adrenergic receptors, adenylate cyclase and membraneous PDE2 of tumour cells as well as to its possible role in the antitumour effect of sarcolysine. PMID- 2998490 TI - [Modification of radiosensitivity and the cyclic AMP system]. AB - The results of the experimental papers of the Soviet and foreign investigators concerning the role of cyclic AMP in modification of radiosensitivity of biological objects and the participation of cAMP system in the realization of radioprotective effect of various radioprotectants have been summarized. The action of radioprotectors on the cAMP level in cells and tissues of organism and their effect on the proteins activity of cAMP system has been considered. Particular attention has been given to the mechanisms of the radio-protective action of catecholamines on mammalian cells in vitro. On the grounds of the authors' own and literary data has been supposed that beta-adrenergic receptors and cAMP system, coupled with them, are obligatory for the performance of the radioprotective potency of catecholamines. The methodological problems of radiobiological experiments and adequate interpretation of results in compliance with terms of biochemistry and molecular endocrinology is discussed. PMID- 2998491 TI - Hormonal and metabolic changes in the perinatal period. AB - A review of some hormonal and metabolic changes occurring during the four stages of the perinatal period is presented. Glucocorticoids and insulin are the hormones that mediate liver glycogen accumulation during late fetal stage. In the presuckling period, muscle glycogenolysis supplies the lactate moieties that are oxidized by the neonatal tissues, representing the alternative substrate until glucose and ketone bodies become available. The postnatal increase in plasma catecholamine concentrations and the decrease in the insulin/glucagon ratio triggers liver glycogenolysis and gluconeogenesis, and hence postnatal hypoglycemia is reversed. In the suckling period, the oxidation of fatty acids, ketone bodies utilization and active gluconeogenesis supply the bulk of the energy and carbon components required to support the rapid growth rate of this period. The increase in the insulin/glucagon ratio that occurs with the change to a carbohydrate-rich diet starts the induction of lipogenesis at weaning. PMID- 2998492 TI - Insulin and glucagon during the perinatal period: secretion and metabolic effects on the liver. AB - Insulin and glucagon are detected in the plasma of most species early in gestation. In the fetus at term, insulin and glucagon secretion can be modified by long-term changes in glucose concentration but the responsiveness of A and B cells to glucose is lower than in the adult. The plasma insulin/glucagon molar ratio is high in the fetus at term, then decreases dramatically immediately after birth and remains low during the first hours of extrauterine life. This situation results in favored hepatic glycogen storage and prevented gluconeogenesis in utero, and sharp glycogen breakdown and active gluconeogenesis during the early postnatal period. PMID- 2998493 TI - Contribution of brown fat to the neonatal thermogenesis. AB - Homeothermic animals must maintain their body temperatures within a very narrow range. Large homeotherms face problems with dissipation of metabolic heat: for small homeothermic animals heat losses at most environmental temperatures are far in excess of normal metabolic heat production. Accordingly, brown adipocytes exhibit in mammalians a very rapid process of morphological functional maturation perinatally. This thermogenesis occurred without electrical activity in the skeletal muscle and it is therefore termed 'nonshivering thermogenesis'. The anatomical site of nonshivering thermogenesis is mainly the brown adipose tissue in the small newborn mammals. In animals reared under thermoneutral conditions the tissue subsequently involutes and nonshivering thermogenic capacity is largely lost. Cold- and diet-induced adaptation may slow or prevent this involution. The decline in the extent of nonshivering thermogenesis during development at thermoneutral temperatures is reflected by an alteration in the bioenergetic properties of the isolated brown fat mitochondria. PMID- 2998494 TI - Vitamin D3 3 beta sulfate has less biological activity than free vitamin D3 during pregnancy in rats. AB - The biological activities of free (D3) and sulfoconjugated (SD3) vitamin D3 were compared after 6 weeks of oral administration to D-deficient (-D) female rats which were mated in the meantime. Mothers and pups were sacrificed 1-2 days following parturition and mineral and hormonal plasma status was determined in mothers and bone mineral determinations and bone histomorphometric studies performed. In newborns, plasma levels of Ca, P and 25-hydroxyvitamin D (25(OH)D) were measured. After parturition, -D mothers had decreased body weight (BW) as well as decreased plasma levels of Ca, P and 1,25-dihydroxyvitamin D (1,25(OH)2D) associated with undetectable levels of 25(OH)D. Plasma levels of immunoreactive calcitonin and parathormone, by contrast, were higher than in vitamin D-replete (+D) control mothers. Bone histomorphometric analysis showed osteomalacia and secondary hyperparathyroidism in -D mothers. After parturition, -D +SD mothers had reduced BW compared to D-treated mothers and the plasma parameters measured were abnormal. Almost all bone histomorphometric parameters were found to be intermediate between +D and -D groups without reaching values of +D mothers. By contrast, -D +D mothers had most of the bone formation parameters identical to those of +D mothers. However, bone resorption was still higher while plasma levels of P and 25(OH)D remained slightly, but significantly lower than in +D mothers. In pups, plasma Ca in both D3- and SD3-treated groups was similar to values in +D-treated rats. However, pups from SD3-treated mothers still showed plasma levels of P and 25(OH)D lower than in +D pups. In conclusion, treatment with SD3 in -D mother rats significantly improves the biochemical plasma parameters of pups, but complete normalization can be achieved only in the D3 treated group. Our results show that when administered at equal amounts, SD3 has a much lower biological activity than D3 in -D female rats and cannot therefore replace vitamin D3 particularly during pregnancy. PMID- 2998495 TI - Effects of corticosteroids and pancreatic hormones on carbamyl phosphate synthetase-I and ornithine transcarbamylase activities in fetal rat liver. AB - The effects of corticosteroids and pancreatic hormones on two mitochondrial enzymes of ureagenesis, carbamyl phosphate synthetase-I (CPS-I) and ornithine transcarbamylase (OTC), were investigated and compared in fetal rat liver. Supplementing hydrocortisone acetate (50 micrograms) to 18.5-day-old fetuses significantly increased CPS-I activity (by 36%) and decreased OTC activity (by 23%). An actinomycin D supply (2 micrograms) to 18.5-day-old fetuses prematurely increased OTC activity and decreased fetal insulin level (by 42%). This treatment had no effect on CPS-I activity. Glucagon supply (25 micrograms) during the late fetal period increased both activities within 2 h, while dibutyryl-cAMP enhanced OTC activity 17 h later. These results suggested that the fetal development of CPS-I activity was under the control of corticosteroids and glucagon. In contrast, corticosteroid hormones produced an inhibitory effect on OTC activity. This might be explained by the permissive effect of corticosteroids on insulin action, since insulin might act as a repressor in utero of enzyme development. Thus, the paradoxical effect of actinomycin D on OTC activity was probably due to the decrease in fetal insulinemia. PMID- 2998497 TI - Vasoactive intestinal peptide: a novel stimulator of steroidogenesis by cultured rat granulosa cells. AB - Vasoactive intestinal peptide (VIP) and VIPergic nerve fibers are present in the ovaries of several mammalian species, suggesting a possible ovarian action of VIP. We have investigated the direct effects of synthetic porcine VIP on rat granulosa cell steroidogenesis in vitro. The cells were obtained from immature, hypophysectomized, estrogen-primed rats, and cultured in a serum-free medium for 24 h in the absence or presence of varying amounts of VIP. Medium steroids were then determined by specific radioimmunoassay. Vasoactive intestinal peptide dose dependently stimulated progesterone, 20 alpha-hydroxypregn-4-ene-3-one (20 alpha OH-progesterone), and estrogen production with an approximate ED50 value of 3 X 10(-8) M. Maximum steroid production induced by VIP ranged from 15% to 28% of that seen with maximal follicle-stimulating hormone (FSH) stimulation. In contrast to the ability of FSH to induce luteinizing hormone (LH) receptor formation, treatment with VIP did not increase [125I]iodo-human chorionic gonadotropin (hCG) binding to granulosa cells. The ability of several gastrointestinal peptides, having 17-44% sequence identity to VIP, to stimulate granulosa cell steroidogenesis was also tested. The most closely related peptide, PHM-27 was less effective than VIP, and the least closely related, secretin and glucagon, were ineffective at 10(-6) M. Vasoactive intestinal peptide seems to act at least partly through cyclic 3',5'-adenosine monophosphate (cAMP)-dependent processes: addition of a phosphodiesterase inhibitor significantly potentiated the VIP stimulation of granulosa cell steroidogenesis, and VIP was capable of producing a dose- and time-dependent increase in both intracellular and medium cAMP levels. Vasoactive intestinal peptide stimulation of estrogen production seemed to be a result of increased aromatase activity. The increased progesterone production was associated with increased pregnenolone production, increased rate of conversion of pregnenolone to progesterone via 3 beta-hydroxysteroid dehydrogenase, and decreased metabolism of progesterone via 20 alpha hydroxysteroid dehydrogenase. These results indicate that VIP exerts a specific action on granulosa cells to increase estrogen and progestin production. The observed direct effects of VIP, coupled with its identification in the ovary, suggest that VIP may be a physiologically important regulator of ovarian activity. PMID- 2998496 TI - The outcome of pregnancy after chemotherapy for gestational trophoblastic disease. AB - Thirteen pregnancies in 11 women previously treated with cytotoxic chemotherapy for GTD were studied. There were 11 live full-term babies (84.6%), 2 blighted ova (15.4%), 1 placenta accreta (7.7%) and 1 fetal anomaly (7.7%). Unlike other series, 69% of the pregnancies occurred within 1 year after the termination of chemotherapy. The rate of successful pregnancy was not different from that reported by others. Prenatal elimination of abnormal embryos after conception rather than wastage of damaged oocyte before conception might explain this observed low incidence of abnormal birth in GTD patients treated with cytotoxic chemotherapy. PMID- 2998498 TI - Evidence that dissociation, not intracellular degradation, is the major pathway for removal of receptor-bound 125I-human chorionic gonadotropin in cultured rat luteal cells. AB - The nature of the labeled products released by cultured rat luteal cells pulse labeled with 125I-human chorionic gonadotropin (hCG) was examined. After pulse labeling in a 3-h incubation, the cells containing receptor-bound 125I-hCG were incubated in fresh medium in the absence of 125I-hCG up to 48 h. The medium was collected at different time intervals and analyzed to determine the extent of degradation of 125I-hCG. The amounts of radioactivity remaining associated with the cells at these time intervals were also determined. Most of the released radioactivity could be precipitated with 10% trichloracetic acid and was identical in molecular weight to intact 125I-hCG as determined by gel filtration chromatography. After 20 h of reincubation, only less than 50% of the initially bound hormone remained on the cells. At this time point the cells were capable of rebinding 125I-hCG at levels comparable to the original when incubated with a fresh dose of the labeled hormone. The rebinding ability was not a result of de novo receptor synthesis since cycloheximide had no effect on this process. The results indicate that dissociation is the major pathway for release of hCG bound to cultured rat luteal cells and that receptors become functional again after dissociation of the hormone by a cycloheximide-independent process. PMID- 2998500 TI - Antibodies to human T-cell leukemia virus (HTLV-1) in non human primates from Senegal. PMID- 2998499 TI - Neuron specific enolase: a marker of (small cell) cancers of neuronal and neuroendocrine origin. AB - Neuron specific enolase (NSE) has been demonstrated to be a marker for tumours of neuroendocrine origin. Serum NSE levels have been shown by several groups to be of help in the identification of advanced small cell lung cancers and neuroblastoma. The lack of sensitivity to small tumour burdens and lack of specificity of small increments above normal restricts the test as a diagnostic aid in early stage disease. The main use would seem to be in the monitoring of chemotherapy and follow up after completing treatment as sequential measurements enable relatively small changes in level to be interpreted. PMID- 2998501 TI - Double-strand cleavage at a two-base deletion mismatch in a DNA heteroduplex by nuclease S1. AB - A two-base deletion mismatch was generated in a DNA heteroduplex by hybridization of two linear plasmid DNA molecules differing only by the presence of a two-base deletion in one of them. The heteroduplex was shown to be sensitive to double strand cleavage by nuclease S1, thus demonstrating the potential value of single stranded probes for the detection of polymorphisms in genomic DNA due to very small deletions. PMID- 2998502 TI - The effect of osmotic pressure of aqueous PEG solutions on red blood cells. AB - A drastic increase of the intracellular microviscosity of red blood cells in the presence of polyethylene glycol (PEG) was established by electron spin resonance using the small spin label molecule 2,2,6,6-tetramethyl-piperidine-N-oxyl-4-one (TEMPONE). The effective osmotic pressure of PEG solutions stressing the cells was estimated by comparison with those cytoplasmic rotational correlation times of TEMPONE measured in NaCl or sucrose containing media of known osmotic pressure. PMID- 2998503 TI - Study of the effect of varying hematocrit on free deformation and orientation of erythrocytes in flow. AB - The electron paramagnetic resonance (EPR) spin label method was used to investigate the effect of varying hematocrit on the deformation and orientation behavior of erythrocytes in shear flow. The relative EPR spectral change due to flow, which we use as a measure of the average deformation and orientation of erythrocytes, was observed as a function of the hematocrit. The profile generally shows a rising and a declining phase with the maximum in-between. The position of the maximum with respect to the hematocrit and the level of the spectral change are influenced by the suspending medium viscosity, osmolarity, and depend upon modifications of the red cell properties such as the internal viscosity, area-to volume ratio and membrane rigidity. Results show that there is an upper limit of free deformation and orientation of the cell for a given hematocrit value. A possible role of the cell-cell interaction is discussed in restricting the space around a cell which is required for free deformation and orientation. A significance of the findings is that the actual deformation and orientation of an ensemble of cells which give rise to the EPR spectral change in flow is determined not only by the single cell deformability but also by the way the cells interact with each other under a given fluid dynamic condition. PMID- 2998505 TI - [Neurochemical mechanisms of the analgesic effect of L-DOPA]. AB - Experiments on alert rats have shown that, with pre-inhibition of peripheral DOPA decarboxylase, L-DOPA suppresses behavioral nociceptive reactions without changing hemodynamic ones. Analgetic effect of L-DOPA appears to be related to the stimulation of brain postsynaptic alpha1-adrenoceptors and the drug action on opioidergic systems. PMID- 2998504 TI - [Structural changes in membrane lipids of the sarcoplasmic reticulum of skeletal muscles during hypercholesterolemia]. AB - Membrane lipid phase has been studied in the sarcoplasmic reticulum (SR) of skeletal muscles, using spin probes. Hypercholesterolemia was found to increase rotation and decrease hydrophobicity of water-soluble probe medium located inside SR vesicles. This is probably indicative of the reduction in SR vesicle membranes. At the same time reduced rotation and increased hydrophobicity and regularity of fat-acid probe micro-environment are observed in hypercholesterolemia, with the differences disappearing nearer to the centre of the membrane. It is suggested that POL activation and cholesterol accumulation in SR membranes in hypercholesterolemia lead to greater density of phospholipid molecules in the membrane. PMID- 2998506 TI - [Effect of gamma-aminobutyric acid, its agonists and antagonists on uterine smooth muscle]. AB - Experiments on isolated strips of the rabbit uterus showed the ability of GABA, GABAA-receptor agonist (diazepam) and GABAB-receptor antagonist (phenibut) to inhibit uterine contractility. GABAA-receptor antagonist (bicuculline) had a stimulating effect on contractility. It is assumed that GABA-ergic system plays an important role in the regulation of functional inhibition of contractile activity in the rabbit uterus, with GABA agonists regarded as potential gravidoprotectors in uterine hyperactivity or threatening miscarriage. PMID- 2998507 TI - [Interaction of ethanol with opiate receptors]. AB - Addition of ethanol to rat brain homogenate containing opiate receptors inhibits at a concentration of 50 mM the stereospecific binding of 3H-naloxone at 37 degrees C but not at 0 degree C, with the ID50 being 462 mM under these conditions. The temperature-dependent inhibition of the ligand binding suggests that ethanol does not compete with naloxone for specific binding sites of opiate receptors and changes the structure of lipids in biological membranes. Scatchard's analysis has demonstrated that apart from a decrease in the number of highly affinity binding sites of 3H-naloxone, the total amount of the binding sites remains unchanged both in the presence and absence of ethanol and constitutes 453 and 549 fmol/mg protein. It is assumed that ethanol might interconvert highly and low-affinity binding sites. Analysis of the effect of ethanol on 3H-naloxone binding with opiate receptors contained by synaptic membranes obtained from animals with varying predisposition to voluntary alcoholization has shown that ethanol inhibits to a greater degree ligand binding with membranes obtained from rats predisposed to alcoholization. The possibility of the involvement of receptors in the biochemical mechanisms by which the initial alcoholic motivation is effected is under discussion. PMID- 2998508 TI - [Effect of polysaccharides on the cyclic nucleotide content and phosphodiesterase activity in the organs of mice with Lewis lung carcinoma]. AB - Prodigiozan and zymosan were shown to have different effects both on the intracellular content of cyclic nucleotides and pulmonary metastases formation in mice with Lewis' carcinoma. The authors believe that determination of cyclic nucleotide ratio in immunocompetent organs (thymus and spleen) may be used as an additional criterion for identification of new antitumour immunomodulators. PMID- 2998509 TI - Transformed T lymphocytes infected by a novel isolate of human T cell leukemia virus type II. AB - Human T cell leukemia virus type II (HTLV-II) has been isolated from a patient (Mo) with features of leukemic reticuloendotheliosis (LRE) and from a patient with acquired immunodeficiency syndrome (AIDS). We have obtained another isolate of HTLV-II from a patient (CM) with severe hemophilia A, pancytopenia, and a 14 year history of staphylococcal and candidal infections but no evidence of T cell leukemia/lymphoma, AIDS, or LRE. Fresh mononuclear cells and cultured lymphocytes from CM express retroviral antigens indistinguishable by molecular criteria from HTLV-IIMo. Leukocyte cultures from CM yield hyperdiploid (48,XY, +2, +19) continuous lymphoid lines; human fetal cord blood lymphocytes (CBL) are transformed by cocultivation with these CM cell cultures but retain normal cytogenetic constitution. Electron microscopic examination of the CM cultures and transformed CBL reveals budding of extracellular viral particles, intracellular tubuloreticular structures, and viral particles contained within intracellular vesicles. CM cell cultures and the transformed CBL do not require exogenous interleukin 2, have T cell cytochemical features and mature T helper phenotypes, and exhibit minimal T helper and profound T suppressor activity on pokeweed mitogen-stimulated differentiation of normal B cells. These characteristics, which are similar to those observed with the first HTLV-II isolate, may represent properties of all HTLV-II-infected T cells. PMID- 2998510 TI - Transforming genes in human leukemia cells. AB - High-molecular weight DNAs of fresh bone marrow cells from 32 patients with fresh leukemia were assayed for the presence of transmissible activated transforming genes by a DNA-mediated gene transfer technique using NIH/3T3 cells. DNAs of bone marrow cells from four of the 32 patients induced transformation of NIH/3T3 cells. Two of the four cases, a chronic myelogenous leukemia and an acute lymphocytic leukemia, contained activated N-ras oncogenes. Molecular cloning and nucleotide sequence analysis revealed that the lesion responsible for the transforming activity was localized to a single nucleotide transition from guanine to thymine in codon 12 of the predicted protein in each of the two cases. These observations indicate that activation of N-ras oncogenes is independent of the specific stage of cell differentiation or the leukemia phenotype. The other two transforming genes associated with an acute myelogenous leukemia and an acute lymphocytic leukemia showed homology neither with members of the ras gene family nor with the human Blym-1 gene. Thus, the NIH/3T3 transfection assay frequently detects activated N-ras oncogenes in human leukemias, while other transforming genes, distinct from the ras gene family, can be detected in some leukemias by the transfection assay. PMID- 2998512 TI - Acute nonlymphocytic leukemia, preleukemia, and solid tumors following intensive chemotherapy of small cell carcinoma of the lung. AB - Six of 796 patients treated with intensive combination chemotherapy for small cell carcinoma of the lung developed overt acute nonlymphocytic leukemia (ANLL) (three patients) or preleukemia with severe refractory cytopenia and clonal cytogenetic abnormalities in bone marrow cells (three patients). The latent period to development of preleukemia or leukemia was less than two years in four of the six patients. The cumulative risk of preleukemia and leukemia according to a Kaplan-Meier estimate was 14.0% +/- 6.9% (mean +/- SE) four years after the start of treatment. The relative risk of overt ANLL was 77, since three cases were observed v 0.039 cases expected, based on the age- and sex-specific incidence of acute nonlymphocytic leukemia in the general Danish population. The risk of secondary solid tumors was not increased. The possible causes of the exceptionally early appearance and very high cumulative risk of leukemic complications found in the present study, as compared to previous experience in other malignant diseases, is discussed, including the implications for future therapy of patients with small cell lung cancer. PMID- 2998511 TI - Establishment of a novel human megakaryoblastic leukemia cell line, MEG-01, with positive Philadelphia chromosome. AB - A megakaryoblastic cell line, designated MEG-01, was established from the bone marrow of a patient with blast crisis of Philadelphia (Ph1) chromosome-positive chronic myelogenous leukemia. MEG-01 cells grew in single-cell suspension with a doubling time of 36 to 48 hours. Under the usual culture conditions, approximately half of the cells adhered to the culture flask with extention of pseudopods. MEG-01 cells were positive for the periodic acid-Schiff reaction, alpha-naphthyl acetate esterase, and acid phosphatase, and negative for myeloperoxidase, alpha-naphthyl butyrate esterase, naphthol AS-D chloroacetate esterase, and alkaline phosphatase. Ultrastructural platelet peroxidase was positive in MEG-01 cells. Cytoplasmic factor VIII (FVIII)-related antigen was weakly positive in larger MEG-01 cells by both an indirect immunofluorescent technique with monoclonal antibodies and a direct immunoperoxidase technique using horseradish peroxidase-conjugated conventional rabbit anti-human FVIII antibody. Platelet glycoprotein (GP) IIb/IIIa antigen was uniformly demonstrated on the surface of MEG-01 cells by both indirect immunofluorescent and immunoperoxidase techniques using antiplatelet GP IIb/IIIa monoclonal antibodies; platelet GP lb antigen was demonstrated only in the cytoplasm of larger MEG-01 cells. MEG-01 cells possessed no markers for B or T lymphocytes or for myeloid cells. Chromosome analysis of this cell line revealed a human male hyperdiploid karyotype with a modal chromosome number of 56 to 58. The Ph1 chromosome was observed in all karyotypes analyzed. This novel human megakaryoblastic cell line may provide a useful model for the study of human megakaryopoiesis and of the biosynthetic mechanisms of proteins unique to megakaryocytic lineage. PMID- 2998513 TI - Restriction fragment length polymorphisms as markers of engraftment in allogeneic marrow transplantation. AB - We have used DNA hybridization techniques employing restriction fragment length polymorphisms (RFLPs) to quantitate the level of donor cell engraftment in bone marrow transplantation recipients. The genetic origin of the bone marrow cells and various peripheral blood populations was analyzed in 14 patients. We found at least one informative polymorphism for each donor-recipient pair. Additional markers of engraftment included cytogenetic analysis, HLA typing, and red cell typing. By DNA analysis, four patients had complete engraftment, five had partial engraftment, and five had no evidence of donor cell engraftment. In three cases, DNA analysis permitted detection of minor populations (5% to 10%) of donor or host cells. Eight of fourteen patients were evaluable for chimerism posttransplant by cytogenetic analysis. In five cases, cytogenetic results were completely concordant with DNA analyses. In two cases of apparent autologous recovery, as assessed using RFLPs, a small number of cells of donor karyotype was seen. In one other case, a small number of cells of host karyotype was not detected by RFLP studies. HLA typing in three partially engrafted patients was purely either of donor or host type. Red cell typing was discordant with DNA and/or cytogenetic results in four of eight cases. We conclude that DNA analysis at a limited number of informative genetic loci is useful for quantitating the degree of engraftment in multiple populations of nondividing cells following allogeneic bone marrow transplantation. PMID- 2998514 TI - Inhibition of dog platelet function by in vivo infusion of F(ab')2 fragments of a monoclonal antibody to the platelet glycoprotein IIb/IIIa receptor. AB - To assess the potential of monoclonal antibodies that inhibit platelet function in vitro as in vivo therapeutic agents, F(ab')2 fragments (0.17 to 0.81 mg/kg) of a murine monoclonal antibody (7E3) that binds to platelet glycoproteins IIb and/or IIIa and blocks platelet aggregation induced by ADP were infused into three dogs. Soon after infusion, platelets recovered from the dogs showed a decreased aggregation response to adenosine diphosphate, with the highest dose producing nearly total inhibition. These platelets also showed decreased ability to bind 125I-7E3, which was assumed to reflect occupancy of the sites by the unlabeled F(ab')2 fragments. At the highest dose, the binding decreased by 85%, reflecting the binding of approximately 44,000 molecules of 7E3 F(ab')2 per platelet. Platelet counts decreased after antibody infusion by less than 20%, and none of the dogs showed spontaneous bleeding. Both the aggregation and binding results reverted toward normal within one day. We conclude that it is possible to profoundly inhibit platelet function by in vivo infusion of F(ab')2 fragments of a monoclonal antiplatelet antibody without producing spontaneous hemorrhage or significant thrombocytopenia. PMID- 2998516 TI - Pharmacological action of 5'-methylthioadenosine on isolated rabbit aorta strips. AB - The effects of 5'-methylthioadenosine (MTA) and adenosine analogues on isolated rabbit thoracic aorta strips were studied in vitro. High concentrations (500 1,000 microM) of adenosine analogues produced dose-dependent relaxation in isolated rabbit thoracic aorta strips. The relative potencies of relaxant effect were MTA greater than N6-phenylisopropyladenosine greater than 2-chloroadenosine on a molar basis. MTA (50-1,000 microM) suppressed the contraction induced by norepinephrine in isolated rabbit thoracic aorta strips in a concentration dependent manner. Nucleoside uptake inhibitor dipyridamole did not impair the MTA actions. Pretreatment of the aorta strips with theophylline, an adenosine receptor antagonist, blocked the actions of MTA. MTA showed a relaxant effect in KCl-contracted aorta suggesting that MTA did not affect the metabolism or reuptake of norepinephrine. The present experiments suggest that MTA has a pharmacological action on the arterial smooth muscle cells mediated through adenosine receptors. PMID- 2998515 TI - Antibodies to human T-cell leukemia virus type III in hemophiliacs from Spain. AB - We tested serum samples from 50 hemophiliacs from Sevilla, Spain, for antibody to HTLV-III by indirect membrane immunofluorescence (IMI) and radioimmunoprecipitation with SDS polyacrylamide gel electrophoresis (RIP SDS/PAGE). All had received commercial clotting factors from the United States with the exception of one hemophiliac who had never been transfused. Thirty-four (68%) reacted with HTLV-III-infected cells (H9/HTLV-III) by both methods, but not with the uninfected line (H9). Of 41 hemophilia-A patients tested, 28 (68%) were positive, and of nine hemophilia-B patients, six (66%) were positive. The nontransfused hemophilia-B patient was negative for antibody to HTLV-III by both methods. One patient with clinical AIDS tested positive as did six of seven with chronic unexplained lymphadenopathy. The eight individuals with AIDS or lymphadenopathy all had hemophilia A. We conclude that exposure to HTLV-III is widespread among asymptomatic hemophiliacs in Spain. PMID- 2998517 TI - [Papillomavirus infection and cervical intraepithelial neoplasia (CIN). Study of CINIII in 2 series of conization and hysterectomies (1957-1968 and 1981-1983)]. AB - At the Institute of Pathology and Applied Cytology, 619 cases of conization or hysterectomies for CIS had been reviewed, through the study of 6,439 slides: 263 cases from the years 1957-1968, 287 cases from the years 1981-1983, have been studied. In the 1957-1968 group, a condylomatous lesion was associated with CINIII/CIS in 60% and in 79% in the 1981-1983 group. The relationship between the condylomatous lesions and the CIS, the grade of these lesions and the mean age of the patients had been examined. In the both groups, the HPV signs had been more frequently discovered in the younger women. But a difference of 4 years was found between the patients with CINIII without any sign of HPV infection and those with a CINIII/CIS associated with a condyloma. These data support the hypothesis of a lessening and a later disappearance of the HPV signs when the neoplasias become more severe. In both groups, the relationship between the signs of HPV infection and the grade of the cervical epithelial atypias are exactly the same. These lesions, more often extended, with a transitional passage between CIN with HPV cytopathological effects and CIS, comfort the hypothesis of a straight relationship between HPV infection and carcinoma of the cervix. PMID- 2998518 TI - Beta-adrenergic receptor structure, synthesis, antibodies and human disease. AB - Lung beta 2-adrenergic receptors have been isolated using a covalent affinity label and monoclonal and autoantibodies with specificity toward the receptor. The beta 2-receptor monomer has a molecular mass of 58-64,000 daltons. Target size analysis indicates that the beta 2-receptor exists as a dimer in lung membranes. The half life of the beta 2-receptor on cultured lung cells is on the order of 20 30 h. Glucocorticoids induce the synthesis of beta 2-receptors, resulting in a doubling of the membrane concentration of receptors in 24 h. Autoantibodies to beta 2-adrenergic receptors may play a role in beta 2-receptor associated human diseases including asthma. Autoantibodies to beta 2-receptors in humans are associated with decreased sensitivity of beta 2-receptor functions and increased responsiveness of alpha-adrenergic and muscarinic cholinergic receptors. PMID- 2998519 TI - Prednisone, indomethacin and airway responsiveness in toluene diisocyanate sensitized subjects. AB - We investigated whether late asthmatic reactions and the associated increase in airway responsiveness induced by toluene diisocyanate (TDI) in sensitized subjects are inhibited by indomethacin and/or prednisone. Four sets of experiments were conducted in five subjects sensitized to TDI. To assess late asthmatic reactions to TDI, FEV1 was measured immediately before and after exposure to TDI and then hourly for 8 h. To assess change in airway responsiveness, the provocative dose (mg) of methacholine that caused a decrease in FEV1 of 20% (PD20FEV1) before treatment, and then before and after exposure to TDI was measured. In the first set of experiments, each subject was given no treatment and was studied before and 8 h after exposure to TDI; in the other two sets, each subject was studied before treatment, then during treatment with indomethacin (50 mg q.i.d. for 3 days, orally) or prednisone (50 mg once a day, for 3 days, orally), both before and 8 h after TDI exposure. In a fourth series of experiments, each subject was again given no treatment and studied before and 8 h after TDI. When the subjects were given no treatment or indomethacin, TDI caused late asthmatic reactions and increased airway responsiveness to inhaled methacholine. In contrast, when the subjects were given prednisone, TDI caused neither late asthmatic reactions nor increased airway responsiveness. Treatment with indomethacin and prednisone did not change baseline FEV1 and airway responsiveness. These results suggest that release of prostaglandins does not contribute to late asthmatic reactions and the associated increase in airway responsiveness induced by TDI. Inflammatory mediators inhibited by prednisone but not by indomethacin may be involved. PMID- 2998520 TI - Interaction between cholinergic and adrenergic synapses of the rat subfornical organ and the thirst-inducing effect of angiotensin II. AB - Previous studies by our group and other authors have demonstrated that application of carbachol or angiotensin II to the subfornical organ (SFO) of satiated rats causes an intense thirst-inducing response. It has also been demonstrated that muscarinic cholinergic synapses are mainly involved in the thirst-inducing effect of carbachol, with a secondary role played by nicotinic receptors. The beta-adrenergic pathways of the SFO have also been shown to participate in the regulation of water intake. The present study was designed to investigate the possible interaction between cholinergic and adrenergic neurons of the subfornical organ and the effect of angiotensin II and carbachol in the regulation of water intake by this structure. The intense water intake induced by injection of 2 nmol carbachol into the SFO was markedly reduced when different doses of propranolol (20, 40, 80, and 160 nmol) were previously injected. The response to carbachol, however, was not changed by previous treatment with regitine (20, 40, and 80 nmol). Injection of 0.1 to 4.0 ng angiotensin II into the SFO caused a dose-dependent increase in water intake. When the 4 ng dose of angiotensin was injected into the SFO after an injection of atropine (20, 40, and 80 nmol), complete absence of water intake was observed, the same occurring when propranolol was previously injected at doses of 40 and 80 nmol. The thirst inducing effect of angiotensin II (4 ng) was not changed by previous injection of hexamethonium (20, 40, 80, and 160 nmol) or phentolamine (20, 40, and 80 nmol). These results permit us to suggest that angiotensin II and carbachol induce thirst when applied to the SFO by acting through independent systems. The participation of beta-adrenergic receptors in the thirst-inducing effect of angiotensin II and carbachol was also demonstrated, as well as the participation of muscarinic cholinergic receptors in the thirst-inducing effect of angiotensin II injected into the SFO. PMID- 2998521 TI - Opioid receptor types on adrenergic nerve terminals of rabbit ear artery. AB - Methionine enkephalin, leucine enkephalin, [D-Ala2, D-Leu5] enkephalin, alpha neoendorphin, beta-endorphin, dynorphin (1-13) and ethylketocyclazocine inhibited the contractions of rabbit ear artery ring segments elicited by transmural nerve stimulation at 8 Hz. Ethylketocyclazocine, dynorphin (1-13) and leucine enkephalin produced partial inhibition, their apparent intrinsic activities (alpha) being 0.57, 0.75 and 0.66, respectively. Morphine and normorphine, which are agonists at mu-receptors, did not inhibit the response of the artery. Naloxone antagonized the actions of opioids and ethylketocyclazocine, and was more effective against methionine enkephalin, leucine enkephalin and [D-Ala2, D Leu5] enkephalin than against alpha-neoendorphin, ethylketocyclazocine and dynorphin (1-13). The pA2 values of naloxone against so-called delta-agonists were approx. 8.5, and against so-called kappa-agonists were approx. 7.7. The supposed kappa-antagonist, Mr2266, was more effective than naloxone in antagonizing the actions of alpha-neoendorphin, and the kappa-agonists dynorphin (1-13) and ethylketocyclazocine. The pA2 values of Mr2266 against kappa-agonists were 8.5-9.0, and against delta-agonists were 7.8 or less. The opioid peptides and opioids tested did not cause dilatation of the artery previously contracted with histamine. These results suggest that the opioid peptides and ethylketocyclazocine acted on opioid receptors at adrenergic nerve terminals in the ear artery. The opioid receptors appear to be of the delta- and kappa-types, not the mu-type. PMID- 2998522 TI - Evidence for an A2-subtype adenosine receptor on pancreatic glucagon secreting cells. AB - The effects of a 5'-substituted analogue of adenosine, 5'-N ethylcarboxamidoadenosine (NECA) have been studied on glucagon secretion in vitro, using the isolated pancreas of the rat perfused in the presence of glucose (2.8 mM). NECA provoked a peak of glucagon secretion, the kinetics of which were comparable to those previously obtained with adenosine. The effect was concentration-dependent and appeared at nanomolar concentrations. The EC50 was approximately 4 X 10(-8) M. A comparison of relative potency between adenosine and NECA showed that NECA was about 800 fold more potent than adenosine in inducing glucagon secretion. Theophylline (50 microM) considerably decreased the peak of glucagon secretion induced by 1.65 microM NECA and totally suppressed the effect of 16.5 nM NECA. These results indicate the involvement of an adenosine receptor. These and other previous results (low stereoselectivity of N6 phenylisopropyladenosine) provide evidence for an adenosine receptor of the A2 subtype being involved in glucagon secretion. PMID- 2998523 TI - Potentiation by cholinoceptor agonists of contractions to field stimulation of rat vas deferens. AB - Cholinoceptor agonists (arecoline congruent to carbachol greater than acetylcholine greater than pilocarpine) potentiated contractions to field stimulation of rat vas deferens via the activation of an atropine-sensitive muscarinic receptor. The potentiating effect of carbachol was dependent on the level of calcium in the medium, being more potent at higher calcium concentrations. The potentiating effect of carbachol was more pronounced in the epididymal than in the prostatic segment but was not attenuated by prazosin, an alpha 1-adrenoceptor antagonist. Carbachol did not significantly modify the direct contractile effects of noradrenaline nor alter the field-stimulation evoked release of noradrenaline from the epididymal vas deferens. It is concluded that the potentiating effect of cholinoceptor agonists on the contractions to field stimulation in the rat vas deferens was not a result of an enhancement of adrenergic neurotransmission. PMID- 2998524 TI - Effects of indomethacin, piroxicam and selected prostanoids on gastric acid secretion by the rat isolated gastric mucosa. AB - The effects of the cyclo-oxygenase inhibitors indomethacin and piroxicam have been investigated on histamine- and dibutyryl cyclic AMP-induced acid secretion in the rat isolated gastric mucosa. The relative potencies of a number of prostanoids as inhibitors of histamine-induced acid secretion were determined in an attempt to classify the prostaglandin receptor mediating this response. Indomethacin (8 X 10(-9) - 2.7 X 10(-6) M) and piroxicam (3 X 10(-6) M) potentiated the secretory responses elicited by histamine. This effect might be due to inhibition of the biosynthesis of antisecretory prostanoids. Indomethacin (2.7 X 10(-6) M) and piroxicam (3 X 10(-6) M) also potentiated the secretory response to dibutyryl cyclic AMP, but since prostaglandin E2 (PGE2, 10(-5) M) did not inhibit this secretory response, the mechanism of the potentiation may differ from that of histamine. The potency of the thromboxane mimetic U-46619 as an inhibitor of histamine-induced acid secretion was markedly reduced in the presence of indomethacin, suggesting that U-46619 may release endogenous antisecretory prostanoids. In the presence of indomethacin (2.7 X 10(-6) M) all the prostanoids tested produced concentration-related inhibitions of histamine induced gastric acid secretion. PGE-analogues were the most potent compounds, the rank order of potency being 16, 16 dimethyl PGE2 greater than PGE2 greater than PGF2 alpha greater than U-46619 greater than PGD2 greater than PGI2. This order of potency is very similar to that obtained in smooth muscle preparations containing 'EP' receptors, suggesting that this receptor type also mediates inhibition of histamine-induced acid secretion in the rat. PMID- 2998525 TI - Evidence for two mechanisms of depolarization associated with alpha 1 adrenoceptor activation in the rat anococcygeus muscle. AB - Membrane potential responses in the rat isolated anococcygeus to bath-applied noradrenaline and field stimulation have been investigated by use of intracellular microelectrode and combined extracellular electrical and mechanical (sucrose gap) recording techniques. Intracellular recordings were made usually from tissues immobilized with hypertonic Krebs solution. Bath-application of noradrenaline produced depolarizations which consisted of two components; an initial 'fast' phase which peaked within 1-2 s and which was followed by a 'slow' sustained depolarization. Both components were concentration-dependent. Noradrenaline could also evoke oscillations in membrane potential which, unlike the 'fast' component of depolarization, were prevented by conditioning hyperpolarization of the membrane and were evoked by direct membrane depolarization with externally applied current pulses. Thus, the oscillations are voltage-dependent phenomena. Replacement of the external NaCl of the Krebs solution with an equimolar amount of Na benzenesulphonate abolished the noradrenaline-evoked 'fast' depolarization while the 'slow' phase was unaffected. This suggests that two mechanisms of depolarization are activated in this muscle by the bath-application of noradrenaline. The adrenergic excitatory junction potential was also abolished in Na benzenesulphonate. Prazosin reduced both the 'fast' and 'slow' components of depolarization produced by noradrenaline indicating their mediation by alpha 1-adrenoceptors. The membrane potential (-29 mV) at the maximum amplitude of the 'fast' depolarization was similar to the equilibrium potential (-27 mV) for the depolarization evoked by ionophoretically applied noradrenaline and which was obtained by extrapolation from the relationship between amplitude of the ionophoretic response and membrane potential displacement in the partition chamber. These results suggest that the 'fast' depolarization and the ionophoretic response are due to an increased membrane conductance, possibly to chloride. PMID- 2998526 TI - Neuronal muscarinic receptors attenuate vagally-induced contraction of feline bronchial smooth muscle. AB - In anaesthetized cats, stimulation of the vagus nerves produced bradycardia and a bronchoconstriction which was measured as an increase in lung resistance (RL) and a fall in dynamic lung compliance (Cdyn); these effects were abolished by atropine. Gallamine potentiated vagally-mediated changes in RL and Cdyn at doses that blocked muscarinic receptors in the heart and inhibited neuromuscular transmission. (+)-Tubocurarine and suxamethonium did not affect the response of the lung or the heart to vagal stimulation. Bronchoconstriction induced by intravenous acetylcholine was not potentiated by gallamine, indicating that postsynaptic muscarinic receptors in the lung and changes in muscle tone were not involved. Potentiation of vagally-induced bronchoconstriction appears to be due to blockade of inhibitory muscarinic receptors located in the pulmonary parasympathetic nerves innervating both central and peripheral airways. Pilocarpine was an agonist for these neuronal receptors as it inhibited vagally induced bronchoconstriction at low doses (10 ng to 1 microgram kg-1). The results demonstrate that gallamine is an antagonist and pilocarpine an agonist at neuronal muscarinic receptors which attenuate parasympathetic nerve activity in feline lung. PMID- 2998528 TI - Adrenergic receptors in depression. Effects of electroconvulsive therapy. AB - Platelet alpha 2-and lymphocyte beta 2-adrenoceptor densities, plasma noradrenaline and serum cortisol were measured before, during and one week after a course of EEG-monitored electroconvulsive therapy, in nine depressed patients. A 50% fall in Hamilton Depression Rating scores occurred after a fairly consistent total seizure time, regardless of the amount of ECT given. Platelet alpha 2-adrenoceptor densities showed a statistically significant fall after three ECTs, but were unchanged after the full course of ECT and were independent of clinical change. Lymphocyte beta 2-adrenoceptor densities were unaltered. Plasma noradrenaline concentrations were initially high, and fell with ECT in a manner paralleling clinical recovery. Plasma noradrenaline may be a more useful index of central changes during antidepressant treatment than peripheral blood cell receptor densities. PMID- 2998529 TI - Computed tomography and ultrasound of a urachal cancer. PMID- 2998527 TI - Cl-/Ca2+-dependent L-glutamate binding sites do not correspond to 2-amino-4 phosphonobutanoate-sensitive excitatory amino acid receptors. AB - A series of phosphono and phosphino analogues of glutamate were used to compare the pharmacological properties of (a) Cl-/Ca2+-dependent, 2-amino-4 phosphonobutanoate (AP4)-sensitive L-[3H]-glutamate binding sites in rat brain synaptic plasma membranes (SPMs) and (b) AP4-sensitive excitatory synaptic responses by use of electrophysiological techniques. In the presence of Cl- and Ca2+, L-[3H]-glutamate bound to SPMs with Kd 804 nM and Bmax 53 pmol mg-1 protein. The AP4-sensitive (Ki 7.3 microM) population of binding sites represented 61% of L-glutamate specifically bound. omega-Substituted analogues of AP4 were potent inhibitors of L-[3H]-glutamate binding (Ki values 2.4-38 microM), whereas N-substituted compounds or propionic acid derivatives were inactive. Experiments with AP4 alone and in combination with other analogues demonstrated that the primary target of all substances was the AP4-sensitive population of L glutamate binding sites. In the hippocampal slice in vitro, AP4 antagonized lateral perforant path-evoked field potentials with an IC50 of 2.7 microM. In contrast to their actions at AP4-sensitive L-glutamate binding sites, all other compounds (except for the omega-carboxymethylphosphino analogue, IC50 19 microM) were weak or inactive as antagonists of this synaptic response (IC50 values greater than 100 microM). Inactive compounds which exhibited activity in the binding assay did not reverse the synaptic depressant effects of AP4, indicating that they were neither agonists nor antagonists at AP4-sensitive synapses. 4 The lack of correspondence between (a) the Cl- /Ca2 -dependent, AP4-sensitive population of L- [3H]-glutamate binding sites and (b) AP4-sensitive synaptic responses indicates that these binding sites are not the receptors through which AP4 exerts its neuropharmacological effects. The possibility that Cl- /Ca2+ dependent 'binding sites' represent transport into resealed SPM vesicles is discussed. 5 Electrophysiological data demonstrate that AP4-sensitive synaptic receptors display a high degree of ligand selectivity. High antagonist potency is shown only by glutamate analogues with unmodified alpha-amino and alpha-carboxyl groups, and with a bifunctional (dianionic) omega-terminal. PMID- 2998530 TI - Stromal radiosensitivity: influence of tumour type on the Tumour Bed Effect assay. PMID- 2998531 TI - Assessment of thallium-pertechnetate subtraction scintigraphy in hyperparathyroidism. AB - Reliable techniques for detecting and localising abnormal parathyroid tissue have been a persistent problem. We have evaluated thallium-pertechnetate subtraction scintigraphy in a prospective study of 40 patients with clinical and biochemical evidence of hyperparathyroidism prior to parathyroid surgery. Four patients were excluded as they were shown to have goitre, making subtraction scanning non diagnostic. 89% of parathyroid adenomas (totalling 27 glands in 26 patients) and 41% of hyperplastic glands (17 glands in 6 patients) were accurately localised prior to surgery. These included three retrosternal glands, four patients with renal failure and tertiary hyperparathyroidism and five patients who had previously undergone neck exploration. The apparent discrepancy between detecting hyperplastic and adenomatous glands was associated with the smaller size of the former. For both types of gland, scintigraphy successfully located parathyroids 0.6 g or more in weight. These results suggest that this simple and non-invasive method is a useful technique for locating parathyroid tissue before parathyroid surgery. PMID- 2998533 TI - Urethral syndrome (abacterial cystitis)--search for a pathogen. AB - Thirty-one women with the recurrent urethral syndrome (abacterial cystitis) had extensive microbiological, cytological and histological investigations. A Streptococcus species was isolated from the bladder aspirate of one patient, a wall-deficient Streptococcus species from two others and both a Lactobacillus species and Ureaplasma urealyticum from another. A possible microbiological cause was therefore identified in the bladder in only 4 of the 31 patients. A Lactobacillus species was isolated from the bladder aspirate of one control subject. Lactobacilli were grown in the voided urine of the majority of patients and in seven of the eight control subjects. Chlamydia trachomatis and Herpes simplex were isolated from the cervix and the urethra of one patient. Urethral cytology was normal in all patients. Trigonitis was noted at cystoscopy in 26 of the 31 patients. Bladder biopsies showed squamous metaplasia in 15 and lymphocytic infiltration of the lamina propria in 29, giving support to an inflammatory aetiology of this enigmatic syndrome. PMID- 2998532 TI - Use of the discriminant index in dynamic treatment to reduce recurrence of calcium oxalate kidney stones. AB - Treatment with phosphates, thiazides and allopurinol was undertaken in 54 idiopathic calcium oxalate stone formers, 38 of whom were recurrent stone formers. The patients were followed up for 1 1/2 to 4 years (mean 2.6). During the same period at the pre-treatment stage the patients formed 80 stones, but during therapy only one stone was formed. A dynamic scheme of therapy was used. Each patient was tested before the start of drug treatment by the discriminant index (DI) method, which measures the overall inhibitory potential to calcium oxalate crystallisation. About 10 days after the start of treatment the DI was tested again. If the response was positive, therapy was continued; if not, the patient was given another drug. Adjustments were made as required. The stopping of stone formation correlated well with the DI prediction but less well with the hypocalciuric effect of the drugs. PMID- 2998534 TI - Carcinoid tumour of the prostate associated with inappropriate ACTH secretion. PMID- 2998535 TI - Chemodectomas of the neck: the response to radiotherapy. AB - In the 16 year period 1965-81, 12 patients with head and neck chemodectomas received radiotherapy. In only 1 patient did the tumour fail to respond, due to metastases, and 9 showed complete resolution of the tumour. As this form of treatment is associated with few complications, we conclude that it is the treatment of choice in those patients who are unfit for surgery and those whose tumours are technically unresectable. PMID- 2998537 TI - Large hepatocellular cancers: hepatic resection or liver transplantation. PMID- 2998536 TI - Severe hypotension after first dose of enalapril in heart failure. AB - The new, long acting converting enzyme inhibitor enalapril was given to 26 patients with moderate to severe heart failure. In 23 cases the mean systolic blood pressure fell from 120 (SD 22) to 108 (25) mm Hg without adverse effects. Profound hypotension with severe bradycardia and sweating, however, occurred in three patients, most pronounced two to four hours after the first dose. The haemodynamic and biochemical changes in these patients were similar to those seen in patients with severe symptomatic hypotension after the first dose of the converting enzyme inhibitor captopril, except that with enalapril the changes occurred later and were longer lasting. Evidence of myocardial damage and reversible renal failure was seen in one patient, and acute reversible deterioration in renal function occurred in one other. In patients with heart failure converting enzyme inhibitors should be administered initially under strict medical supervision with appropriate facilities available for dealing with occasional profound hypotension. PMID- 2998538 TI - Infiltrating lobular carcinoma of the breast. PMID- 2998539 TI - Early neurological complications of coronary artery bypass surgery. AB - A prospective study of 312 patients undergoing elective coronary artery bypass surgery was undertaken to determine the incidence, severity, and functional impact of postoperative neurological complications. Detailed evaluation of the patients showed that neurological complications after surgery were common, occurring in 191 of the 312 patients (61%). Although such a high proportion of the total developed detectable changes, serious neurological morbidity was rare. Neurological disorders resulted in death in only one patient (0.3%) and severe disability in only four (1.3%). Forty eight patients were mildly disabled during the early postoperative period, and the remaining 138 with neurological signs had no serious functional disability. The postoperative neurological disorders detected included one death from cerebral hypoxic damage. Prolonged depression of conscious level was observed in 10 patients (3%) and definite stroke in 15 (5%); 78 (25%) developed ophthalmological abnormalities and 123 (39%) primitive reflexes; postoperative psychosis was observed in four (1%); and 37 (12%) developed disorders of the peripheral nervous system. The incidence of serious neurological problems such as fatal cerebral damage, stroke, and brachial plexopathy is in accordance with experience elsewhere. Lesser abnormalities, whose detection required detailed neurological examination, were much commoner than expected from previous reports. PMID- 2998541 TI - Sugar, fat, and the risk of colorectal cancer. AB - The habitual diet of 50 patients with large bowel cancer, as assessed by a dietary history method, was compared with that of 50 closely matched controls. Patients were included only if their symptoms were unlikely to have changed previous eating habits. The mean daily intakes of all major nutrient classes and of dietary fibre were estimated. Patients with large bowel cancer consumed 16% more energy than controls (mean (SEM) daily intake 9.92 (0.41) v 8.56 (0.32) MJ (2370 (98) v 2046 (76) kcal), respectively; p less than 0.0001), mainly in the form of carbohydrate (21% more; 282.6 (13.7) v 233.4 (10.5) g; p less than 0.0001) and fat (14% more; 100.8 (4.3) v 88.4 (3.2) g; p less than 0.001). The extra carbohydrate was largely in the form of sugars depleted in fibre and the extra fat as combinations of fat and such sugars. As the selection criteria used make it unlikely that this eating pattern was caused by the disease the data suggest that a high intake of sugars depleted in fibre and fat predisposes to the development of large bowel cancer. PMID- 2998543 TI - Risk factors for cytomegalovirus infection and disease after renal transplantation. PMID- 2998542 TI - ABC of nutrition. Some principles. PMID- 2998540 TI - Five cases of cyclical Cushing's syndrome. AB - Reported cases of cyclical Cushing's syndrome are rare. Of 14 successive patients with Cushing's syndrome nine collected sequential urine samples for the estimation of cortisol:creatinine ratio. Five had cyclical Cushing's syndrome while two had considerable variation in urinary cortisol excretion without a cyclical pattern being established. Two of the five patients with a cyclical syndrome had paradoxical responses to dexamethasone. In only one patient with a cyclical pattern did the cortisol:creatinine ratio fall after treatment with bromocriptine or cyproheptadine, or both. The high incidence of the cyclical form of Cushing's syndrome has important clinical implications. A high index of suspicion of the syndrome is required in patients with symptoms or signs of Cushing's syndrome but with normal cortisol values, in patients with fluctuating cortisol values, and in patients with anomalous responses to dexamethasone. Because of possible variations in steroidogenesis the results of drug studies in Cushing's syndrome must be interpreted cautiously. PMID- 2998544 TI - Ear tattooing as a method of spread of bovine leukosis virus infection. PMID- 2998546 TI - Muscular rigidity and delineation of a dopamine-specific neostriatal subregion: tonic EMG activity in rats. AB - In order to investigate the role of neostriatal dopamine receptors in muscular rigidity we have studied catalepsy and spontaneous muscle tone in rats receiving haloperidol injections into various parts of the neostriatum. It was found that low doses of haloperidol (250-750 ng/0.5 microliter) induced a tonic activity in the EMG of the gastrocnemius-soleus muscle when injected into the most rostral part of the neostriatum (A 8620-9650). This tonic EMG activity was found to be both dose-dependent and dopamine-specific: the haloperidol effect of 500 ng could be inhibited by 500 ng apomorphine. No muscular rigidity could be observed when haloperidol (500 ng/0.5 microliter) was injected further caudally into the neostriatum or into the medial part of the nucleus accumbens. With regard to the haloperidol-induced catalepsy as measured by means of the bar test, a similar distribution of effective and ineffective injection sites was observed; however, higher doses of haloperidol (1.5-2.5 micrograms/0.5 microliter) were required. Thus catalepsy and muscular rigidity were found to be closely related phenomena within the neostriatum. Finally, it is discussed how information relevant to tonic EMG activity is transmitted from the substantia nigra pars compacta to the superior colliculus and the ventromedial thalamus via the neostriatum and the substrate nigra pars reticulata. PMID- 2998545 TI - Frequency-dependent effects of phenytoin on frog junctional transmission: presynaptic mechanisms. AB - The action of the antiepileptic drug, phenytoin, on junctional transmission at various frequencies of synaptic activation was studied in frog nerve-muscle preparation. Intracellular recordings were made from muscle end-plates, and extracellular focal and subsendothelial recordings were obtained from motor nerve terminals and their parent axons, respectively. When the motor nerve was stimulated at 100-200 Hz, exposure to the drug (0.1-0.3 mM) induced intermittent failures of junctional transmission which appeared faster as the rate of stimulation was increased. At these and at lower stimulation frequencies (30-50 Hz), in which failures of transmission occurred only rarely, phenytoin markedly limited the buildup of end-plate potential amplitude during the period of repetitive nerve stimulation (tetanic potentiation). Several lines of evidence suggest that both drug effects are consequent to a frequency-dependent depression of the action potential at motor axons and terminals, which could lead to an intermittent conduction block at the higher rates of stimulation. The selective action of phenytoin on high frequency synaptic transmission may contribute to the specificity shown by this drug in suppressing epileptic seizures while sparing neuronal activity. PMID- 2998547 TI - The role of monosialoganglioside GM1 in the synaptic plasticity: in vitro study on rat hippocampal slices. AB - Rat hippocampal slices were incubated with neuraminidase from Vibrio Cholerae. This enzyme liberates sialic acid from polysialogangliosides converting them into monosialoganglioside GM1. Thus, the tissue is enriched in GM1 content. Another set of slices was incubated with GM1 itself. Both treatments increased the magnitude of potentiation of synaptic response recorded from pyramidal cell layer following high frequency stimulation of Schaffer collateral-commissural fibers. It is concluded that enrichment of synaptic membranes in GM1 enhances the ability of these nerve endings to be potentiated. PMID- 2998548 TI - Regulation of rat brain angiotensin II (AII) receptors by intravenous AII and low dietary Na+. AB - Previous studies have shown the presence of specific AII receptors at several areas of the brain. The purpose of this study was to examine by radioreceptor assay the effect of intravenous AII infusion (5 or 25 ng/kg/min) and low dietary Na+ (less than 8 mmol/100 g) on AII receptors in five brain regions: the olfactory lobes (OLF), hypothalamus/thalamus/septum (HTS), midbrain (MID), cerebellum (CER) and medulla (MED). Scatchard analysis of binding data from control rats showed significant (P less than 0.01 ANOVA) differences between brain areas in both Ka (1.54 OLF, 1.87 HTS, 1.25 MID, 1.33 MED, 0.77 CER x 10(9) M-1) and Ro (321 OLF, 224 HTS, 203 MID, 145 MED, 41 CER fmol/g tissue). Following the i.v. infusion of AII for 4-7 days, marked changes were observed in the areas with a porous BBB, the HTS and MED. Both the Ka [3.20 (HTS) and 0.67 (MED) x 10(9) M-1] and Ro [116 (HTS) and 249 (MED) fmol/g tissue] changed. In addition, decreases in Ro were also observed in the OLF (241 fmol/g tissue) and CER (21 fmol/g tissue), areas which have not been considered as being accessible to blood borne AII. A low Na+ diet for 21-30 days changed the Ka and Ro in all five regions but not in similar directions. Furthermore, with the exception of the OLF the direction of change was not similar to that caused by i.v. infusion of AII. It was concluded that AII receptor sites in the rat brain differ from each other in both receptor properties in their response to such regulatory factors as AII Na+ depletion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2998550 TI - Modification of the projection from the sensory cortex to the motor cortex following the elimination of thalamic projections to the motor cortex in cats. AB - Examination of the projection from area 2 of the sensory cortex to the motor cortex revealed substantial changes following lesion of the ventrolateral nucleus of the thalamus. These observed changes were as follows. (1) The polarity of the evoked potentials elicited by area 2 stimulation reversed in the depth of the motor cortex whereas in normal animals, there was no reversal. (2) The amplitude of area 2-elicited EPSPs in the motor cortical neurons became greater following the lesion of VL. (3) The shape of the observed EPSPs was characterized by multiple peaks whereas in normal animals, the EPSPs were generally smooth and monophasic. (4) Neurons receiving a short-latency input from area 2 were distributed throughout the depths of the motor cortex whereas in normal animals, they were located only in the upper layers (layers II and III). (5) Intracellular injection of HRP revealed that the neurons receiving short-latency input were not restricted to typical stellate type cells, but also included bipolar or bitufted neurons with elongated cell bodies and polarized arborizations. These neurons were located in the superficial (II and III) as well as in the deep (V) layer. It is concluded that the elimination of thalamic input resulted in the reinforcement of the corticocortical input to the motor cortex. The subsequently observed corticocortical projection extended to neurons did not originally innervated by the association fibers. The results suggested that functional recovery following thalamic lesion is partly due to reorganization of projections from the sensory cortex to the motor cortex. PMID- 2998549 TI - Anatomical and physiological properties of the projection from the sensory cortex to the motor cortex in normal cats: the difference between corticocortical and thalamocortical projections. AB - Details of the distribution of terminal sites of the projection fibers from area 2 of the sensory cortex to the motor cortex were studied and compared with the distribution of terminals from the ventrolateral (VL) nucleus of the thalamus to the motor cortex. The results obtained were as follows: Intracortical microstimulation (ICMS) in area 2 produced measurable short-latency EPSPs only in neurons located in layers II and III of the motor cortex, whereas VL stimulation produced short-latency EPSPs in neurons throughout the depths of the motor cortex. The time from the beginning to the peak of the EPSPs was not significantly different for area 2- and VL-elicited EPSPs suggesting that there was no systematic difference between effective terminal sites for both inputs. However, there was a difference when a given neuron received both inputs suggesting that there was a segregation between the two inputs within a given cell. The majority of area 2-elicited EPSPs were smooth and monophasic, but some (40%) of them showed double peaks indicating that some neurons received mono- and disynaptic inputs from area 2. Intracellular injections of HRP suggested that neurons receiving input from area 2 were predominantly multipolar non-pyramidal neurons in layers II and III whereas neurons receiving thalamic input were pyramidal as well as non-pyramidal cells. Field potentials in the motor cortex evoked by area 2 stimulation did not change polarity in the depths of the cortex and therefore, differed from the VL-evoked potentials suggesting differences in the mechanisms of generating the electrical fields. It is concluded that association fibers effective for producing EPSPs terminate primarily on non pyramidal cells in layer II and III whereas VL fibers terminate not only on pyramidal but also on non-pyramidal cells in layers III and V. This study provided a basis for examining the modifiability of association fibers after elimination of VL input to the motor cortex which is reported in the following paper. PMID- 2998551 TI - Distribution of corticocortical and thalamocortical synapses on identified motor cortical neurons in the cat: Golgi, electron microscopic and degeneration study. AB - Details of the terminal connection of corticocortical and thalamocortical fibers on pyramidal and stellate neurons in the cat motor cortex were studied using the electron microscope in combination with the Golgi and axonal degeneration techniques. Corticocortical terminals were examined in 23 identified neurons of which 11 were pyramidal and 12 were stellate. Stellate neurons located in layer III received many degenerating terminals (average 8.4 +/- 2.2 per unit length of dendrite (ULD)) and the majority of these (95%) were found on the proximal dendrites or on the cell bodies. The pyramidal neurons received fewer degenerating terminals (average 2.1 +/- 0.27/ULD) and these were located on more distal dendritic shafts or on dendritic spines. The majority of these synapses were of the asymmetric type. Thalamocortical terminals were examined in 9 pyramidal and 9 stellate neurons. Pyramidal neurons received many terminals (average 6.0 +/- 1.23/ULD) and these were found on the basal as well as the apical dendrites and on dendrite spines. Stellate neurons received fewer terminals (average 4.2 +/- 0.64/ULD) and were located primarily on proximal dendritic shafts. The majority of these synapses were of the asymmetric type. The functional role of these synapses is discussed in relation to the physiological results reported in the preceding paper. PMID- 2998552 TI - Morphological and electrophysiological study of sprouting of corticorubral fibers after lesions of the contralateral cerebrum in kitten. AB - The appearance of crossed corticorubral projections following ablations of the ipsilateral cortex is shown to result from the formation of new connections and is not due to the preservation of pre-existing bilateral connections. At least some of these crossed projections are collaterals of the pyramidal tract. Post tetanic potentiation can be demonstrated both intra- and extracellularly following ipsilateral cerebral peduncle stimulation whereas no changes in excitatory postsynaptic potential amplitude are observed following contralateral cerebral peduncle stimulation. PMID- 2998553 TI - Disturbed GABAergic transmission in mutant Han-Wistar rats: further evidence for basal ganglia dysfunction. AB - A mutant strain of Wistar rats which carries an autosomal gene defect is characterized by a progressively developing hyperexcitability, tremor, olfactory and gustatory movements, bradykinesia, ataxia and a pathologically increased muscle tone of hindlimbs which can be measured by recording tonic activity in the electromyogram (EMG) of the gastrocnemius-soleus muscle. The activity of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) and the receptor binding of GABA as estimated by [3H]GABA binding to synaptic membranes were examined in olfactory bulbs, frontal cerebral cortex, corpus striatum, hippocampus, thalamus, hypothalamus, tectum, substantia nigra, medulla oblongata, cerebellum, and pons of mutant rats. Mutant rats exhibit a lower activity of GAD in synaptosomal fractions of olfactory bulbs and substantia nigra whereas GAD activity within the pons was increased. The changes in the activity of GAD were accompanied by alterations in [3H]GABA binding to synaptic membranes: GABA binding was significantly elevated in the olfactory bulbs and the substantia nigra, but it was markedly reduced in the pons. The functional importance of impaired nigral GABAergic transmission in mutant rats was demonstrated by the fact that intranigral injection of the GABA agonist muscimol reduced the tonic extension of the hindlimbs as indicated by reduced tonic EMG activity of the gastrocnemius soleus muscle, while intranigral injection of the GABA antagonist bicuculline increased the disturbance. PMID- 2998554 TI - Pharmacological protection of reoxygenation damage to in vitro brain slice tissue. AB - Pharmacological protection of central neural function against damage by hypoxia and reoxygenation was studied electrophysiologically and biochemically in hippocampal brain slices. Hypoxia causes a loss of both orthodromically and antidromically evoked potentials in CA1 pyramidal cell neurons. Damage due to hypoxia lasting more than 10 min cannot be restored by reoxygenation. Following pretreatment with methylprednisolone (10(-5) M), indomethacin (10(-5) M) or allopurinol (10(-5) M), reoxygenation after 10 min of hypoxia resulted in complete recovery of the evoked activity. Na+,K+-ATPase activity was not reduced by 10 min of hypoxia, but was reduced by 50% during the first 10 min of subsequent reoxygenation. Allopurinol (10(-5) M) protected against loss of this enzyme activity. The protective action by these drugs of both electrophysiological and biochemical aspects of neural function is consistent with the hypothesis that secondary ischemic damage is caused by the formation of oxygen-derived free radicals during reperfusion. PMID- 2998555 TI - Synaptogenesis in the neonatal preoptic area grafted into the aged brain. AB - The medial preoptic area (POA) of newborn female rats was transplanted into the third ventricle of young adult (2 months of age) and aged (27 months of age) female rats. The grafted POA tissues were examined ultrastructurally 2 months after transplantation. The mean numbers of axodendritic shaft and spine synapses in the POA transplants in both young adult and aged female rats were much greater than those in the medial POA of newborn female rats. These results suggest that the neuronal substrates in the medial POA grafts develop well in the brain of the aged female rats. PMID- 2998557 TI - Stress-induced activation of the hippocampal cholinergic system and the pituitary adrenocortical axis. AB - The septo-hippocampal cholinergic system in rats undergoes rapid activation after acute stress. This is expressed by rapid increases both in high affinity choline uptake and newly synthesized acetylcholine release. Administration of ACTH or corticosterone at a high dose led 10 min later to changes comparable to those observed after acute stress. Choline uptake and acetylcholine release were also elevated 2 days after adrenalectomy. The effects of adrenalectomy could be attenuated by corticosterone, but not by ACTH treatment. The results demonstrate that (a) after short term stress the septo-hippocampal cholinergic system is activated secondary to activation of the pituitary-adrenocortical axis and (b) major changes in circulating corticosterone can modulate the activity of the hippocampal cholinergic synapse. PMID- 2998556 TI - Distribution of calpain I, an enzyme associated with degenerative activity, in rat brain. AB - The calcium-activated protease calpain I was localized in rat brain by immunocytochemistry. Calpain I-like immunoreactivity (CLI) was prominent in several structures in which degeneration is an ongoing feature, e.g. spinal motoneurons, olfactory nerve. Also noteworthy was the presence of CLI in regions susceptible to age-related pathologies, e.g. cerebellar Purkinje cells, substantia nigra and subiculum. This distribution suggests that calpain I may be involved with both normal and pathological neuronal degeneration. PMID- 2998558 TI - Individual differences in non-regulatory ingestive behavior and catecholamine systems. AB - Animals that eat and/or drink in response to electrical stimulation of the lateral hypothalamus (ESLH-pos) are more responsive to both schedule-induced polydipsia (SIP) tests and a series of amphetamine (AMPH) injections than animals that do not exhibit these behaviors (ESLH-neg). Moreover, prior exposure to the behaviorally activating SIP experience, or to AMPH, permanently transformed the ESLH-neg animals into animals that reliably ate or drank during ESLH. Prior treatment with AMPH also increases the water consumed during subsequent SIP tests. Thus, initial of induced differences in sensitivity to activating experiences can determine behavioral propensities. PMID- 2998559 TI - Sites of action of brain-gut peptides in cultured neurons of rat brainstem. AB - Using cultures of dissociated neurons from the lower brainstem of 14- to 15-day old rat embryos, we studied a site of action of a brain-gut peptide by determining whether neuronal responses to a test peptide are abolished or not after replacement of normal medium with low Ca2+- high Mg2+ medium. VIP, secretin and CCK-4 may act on the postsynaptic membrane, while motilin and neurotensin may act on the presynaptic terminal. Somatostatin and bombesin may work either presynaptically or postsynaptically. PMID- 2998560 TI - Dual effect of calmidazolium (R 24571) on transmitter release at the frog neuromuscular junction. AB - The effect of the non-phenothiazine calmodulin-inhibitor R 24571 on transmitter release at the frog neuromuscular junction has been investigated. MEPP frequency was reduced by 25-40% at low concentrations of the drug (2-5 X 10(-7) M) but increased at higher concentrations. These effects were independent of [Ca2+]o. EPP quantal content was increased at low levels of release (quantal content 0.5 1.5) and was unaffected at higher levels (quantal content 30-40). It is concluded that R 24571 has both inhibitory and stimulatory effects, but that evoked release of transmitter is insensitive to the inhibitory action of the drug. PMID- 2998561 TI - Low doses of ethanol activate dopaminergic neurons in the ventral tegmental area. AB - In unanesthetized rats the intravenous administration of low doses of ethanol (0.125-0.5 g/kg) produced a dose-dependent increase (30-80%) in the firing rate of dopaminergic (DA) neurons in the Ventral Tegmental Area (VTA). In agreement with previous observations, a dose range between 0.5 and 2 g/mg of ethanol was needed to produce comparable stimulant responses in DA neurons of the Substantia Nigra Pars Compacta. However, in anesthetized rats, doses of ethanol up to 1 g/kg failed to activate VTA-DA neurons. The high sensitivity of VTA-DA neurons to ethanol activation suggests that they might be involved in the reinforcing properties of the drug. PMID- 2998562 TI - Behavioural structure and mechanisms of anorexia: calibration of natural and abnormal inhibition of eating. AB - The study of experimentally induced anorexia poses a problem for investigations of the processes controlling food intake. Inhibition of food consumption may arise from a specific intervention in a physiological system controlling nutritional requirements or from non-specific changes leading to the suppression or contamination of behaviour. The present experiment used the analysis of the structure of behaviour to distinguish between normal anorexia (natural development of satiation) and pathological anorexia brought about by intestinal discomfort (injection of lithium chloride) or adulteration of food (quinine added to diet). The treatments produced marked changes in parameters of feeding and in the frequencies of behaviours associated with eating. Both lithium chloride and quinine treatments gave rise to a slow rate of eating accompanied by a disordered temporal sequence of eating, grooming and resting. This behavioural calibration of anorexia can contribute to the behavioural pharmacology of feeding by helping to diagnose drugs which facilitate normal processes of satiation and those which act via a non-specific disruption of behaviour. PMID- 2998563 TI - Effects of kappa opiate agonists on palatable food consumption in non-deprived rats, with and without food preloads. AB - There is increasing evidence to suggest that kappa opiate receptors may be importantly involved in the mediation of feeding responses in the rat. A series of experiments is reported in which the effects of four kappa receptor agonists (ethylketocyclazocine, U-50,488H, tifluadom, bremazocine) on the consumption of a highly palatable diet were investigated. Under one condition, non-deprived male rats were administered drug treatments before a 30 min feeding test. Bremazocine (0.1 mg/kg) and ethylketocyclazocine (3.0 mg/kg) both significantly decreased the level of food consumption. In contrast, U-50,488H and tifluadom each produced significant increases in food intake. In a second condition, non-deprived male rats were first allowed to consume some of the palatable diet to achieve partial satiation, prior to the administration of the drug treatments. In this case, evidence for hyperphagic effects of all four kappa agonists was obtained, within the first 30 min access to the palatable diet. Thus, hyperphagia occurred with 0.01 mg/kg bremazocine and 0.1 mg/kg ethylketocyclazocine. We conclude that some kappa agonists have mixed stimulant/inhibitory effects on food intake, whereas others are more consistent in producing hyperphagia. In neither condition did morphine (0.3-10.0 mg/kg) show any hyperphagic effect. Our data support an involvement of kappa opiate receptors in mechanisms which control palatable food consumption in non-deprived rats. PMID- 2998564 TI - The involvement of brain structures in the adjuvant effect of muramyl dipeptide. AB - With the aid of small electrolytic lesions we have studied the possible participation of brainstem structures in the adjuvant activity of muramyl dipeptide (MDP). The results suggest the involvement of the serotonergic groups B6.7.8 and serotonergic pathways in the upper region of the reticular formation. Since lesions in the caudal parts of reticular formation in the area of aminergic groups A1.3.5.7 with the participation of corresponding pathways also influenced the adjuvant effect of MDP the possible role of noradrenaline is also implicated. PMID- 2998565 TI - Glycine receptor distribution in mouse CNS: autoradiographic localization of [3H]strychnine binding sites. AB - Biochemical and electrophysiological studies of mammalian CNS indicate that the amino-acid, glycine, is a major inhibitory neurotransmitter whose location is, for the large part, confined to the spinal cord and brain stem. In this study, autoradiographs of C57BL/6J mouse brain sections labeled with [3H] strychnine, a potent antagonist of glycine, were used to map the distribution of glycine receptors in the CNS. Autoradiographs showed highly localized areas of grain density, which confirmed the gross distributions described in homogenate binding studies and gave a more precise regional localization of glycine receptors in this animal. The highest overall labeling was observed in the spinal cord and medulla; areas of highest grain density included the dorsal horn of the spinal cord, the cranial nerve nuclei, the dorsal column nuclei and nuclei of the medullary reticular formation. A decrease in overall grain density was observed rostrally throughout the midbrain and pons; in caudal regions, however, dense labeling was observed over the trigeminal, vestibular and facial nuclei and over the major nuclei of the auditory system. In more rostral areas, the interpeduncular nucleus and the substantia nigra were also clearly delineated, as were certain thalamic nuclei. The cerebellum, cortex, hippocampus and olfactory bulbs showed only very low levels of grain density. In summary, it appears that high concentrations of glycine receptors in the brain and spinal cord may be preferentially localized to neurons involved in the processing of information originating from exteroceptive sensory mechanoreceptors. PMID- 2998566 TI - [Alpha-adrenolytic activity of substance DH 1011]. PMID- 2998567 TI - [Arterial hypertension, a public health problem in black Africa]. PMID- 2998568 TI - [Geographic differentiation of the mitochondrial genome in Mus spretus Lataste]. AB - Analysis of mitochondrial DNA variability of the Mouse M. spretus over its whole living area reveals genetic isolation and phylogenetic independence of two geographical groups. The recent history of the species is discussed in the light of these data. PMID- 2998569 TI - [Preparation of iodinated standards for quantitative radioautography in vitro using a tritium-sensitive film]. AB - In the present study we developed iodinated 125I-standards and tested the response of tritium-sensitive sheet film (3H-Ultrofilm) to varying concentrations of 125I prepared from brain "paste" mixed with mono-iodinated TiTx gamma toxin. Results have practical implications since they allow quantitative measurements of autoradiograms obtained with 125I-coordinats. An example is shown for 125I neurotensin binding sites on rat brain sections. PMID- 2998570 TI - [Adaptation to the cell line PLC/PRF/5 of hepatitis A virus released in the cell culture medium]. AB - The propagation of hepatitis A virus (HAV), CF53 strain, released without any cytopathic effect into the PLC/PRF/5 cells supernatant, was studied in the course of six serial passages (6th to 11th). The decrease (from 5 to 1 week) of incubation time required to detect HAV, by RIA, in culture supernatant, the increase in Hepatitis A antigen (from 777 to 10,038 c.p.m./50 microliter) and infectivity titre (from 10(3.0) TCID 50/ml to 10(4.5) TCID 50/ml) were consistent with the adaptation of this virus to the cell line PLC/PRF/5. PMID- 2998571 TI - [Effect of cycloheximide on nuclear triiodothyronine receptors in the pituitary of the normal and hypothyroid rat]. AB - Cycloheximide (Cy), an inhibitor of protein synthesis was found to provoke a dose dependent decrease of the hypophysis T3 nuclear receptors (T3nR) concentration in normal rats. In thyroidectomized rats, the reduced T3nR density was found to be normalized within 3 hrs. after a single injection of T3. Pretreatment with Cy inhibited the T3 effect on its own receptors, whereas Cy given after T3 was partially or not effective. These data suggest that the half-life of T3nR in the hypophysis is short (about 3 hrs.), and that it depends on protein neosynthesis. PMID- 2998573 TI - Classification of idiopathic hypercalciuric patients by isotopic calcium absorption: a comparison with oral calcium tolerance test. AB - To test the accuracy of calcium tolerance test in estimating calcium absorption, we have measured the radioactive calcium absorption (expressed as Fx) in 27 patients with IH and renal calcium stones. The results of this test were compared with those of a standard oral calcium tolerance test. Although only seven of nine AH patients displayed normal fasting calcium excretion, they all displayed Fx values above normal and a normal parathyroid activity. Conversely, only 5 of our 18 RH patients demonstrated a hyperabsorption of radioactive calcium and an elevation in iPTH and cAMP above normal limits, yet all of them showed an increased calciuric response to an oral calcium challenge. Calcium absorption was inversely related to iPTH (r = -082; P less than 0.001) and cAMP (r = -064 P less than 0.05) in AH, but directly proportional to these parameters (r = 0.62 P less than 0.001 and r = 0.46 P less than 0.05, respectively) in RH patients. In view of these results, two ratios, iPTH/Fx and cAMP/Fx were used to discriminate between the two groups of patients. Both ratios were over normal limits in all RH patients and within normal range in all but one AH patient. Furthermore, no overlap was found between the two groups. Conversely, we were unable to completely separate AH from RH subjects on the basis of the oral calcium tolerance test, since in both groups the fasting and the absolute (or percentage) changes in urinary calcium, cAMP and blood iPTH levels following oral calcium loading, overlapped in each instance.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2998572 TI - Human bone cells in vitro. AB - Human bone cell cultures were established by maintaining collagenase-treated bone fragments in low Ca++ medium. The resulting cell cultures exhibited a high level of alkaline phosphatase activity and produced a significant increase in intracellular cAMP when exposed to the 1-34 fragment of human parathyroid hormone. With continued culture, the cells formed a thick, extracellular matrix that mineralized when cultures were provided daily with normal levels of calcium, fresh ascorbic acid (50 micrograms/ml) and 10 mM beta-glycerol phosphate. Biosynthetically, these cells produced type I collagen (without any type III collagen), and the bone-specific protein, osteonectin. In addition, the cells produced sulfated macromolecules electrophoretically identical to those positively identified as the bone proteoglycan in parallel cultures of fetal bovine bone cells. This technique provides a useful system for the study of osteoblast metabolism in vitro. PMID- 2998574 TI - In vitro use of vitamin D3 metabolites: culture conditions determine cell uptake. AB - This report demonstrates that routine variations in cell culture conditions dramatically affect the amount of vitamin D3 metabolites to which cultured cells have access. Increasing the concentration of a metabolite in the medium increases the amount of the metabolite in the cell compartment. Increasing the volume of medium in the culture dishes (while maintaining a constant metabolite concentration) also increases the amount of metabolite in the cell compartment. Moreover, daily changes of the medium containing fresh metabolite increase the amount of the metabolite in the cell compartment as well. These variables may explain the inability of different laboratories to duplicate dose-response curves. PMID- 2998575 TI - Changes in vitamin D metabolites and bone histology in rats during recovery from rickets. AB - The relative roles of 25-hydroxyvitamin D (25-OHD), 1,25-dihydroxyvitamin D (1,25 (OH)2D) and 24,25-dihydroxyvitamin D (24,25-(OH)2D) in bone mineralization are largely unknown. Young vitamin D depleted rats were fed increasing amounts of vitamin D and grouped radiologically in accordance with the rat line test. They ranged from severely rachitic to normal. Radiology was correlated with serum levels of 25-OHD, 1,25-(OH)2D, 24,25-(OH)2D, ionized calcium, magnesium, and phosphate, with bone histology, and with the total mineral content of the animals. Serum 1,25-(OH)2D rose in a linear fashion to supranormal values during bone healing and correlated with the radiological degree of rickets. Serum 25-OHD was below detection limit in the most rachitic and low in the radiologically normal rats, whereas 24,25-(OH)2D was low in all groups. These two metabolites showed no correlation with the radiologic, histologic or biochemical parameters. In rachitic rats, 1,25-(OH)2D appears to play a major role in bone healing and possibly exerts a direct effect on bone cells. It cannot be ruled out, however, that the effect is mediated through a rise in serum levels of calcium and phosphorus, although signs of bone healing were seen in the presence of a subnormal calcium X phosphorus product. Initiation of mineralization can take place with unmeasurable 25-OHD, and 24,25-(OH)2D seems to be without importance. PMID- 2998576 TI - Interactions between aluminum and the actions and metabolism of vitamin D3 in the chick. AB - The effects of intraperitoneal injections of aluminum chloride were tested on the intestinal calcium absorption and bone calcium mobilization responses to vitamin D3 and 1,25(OH)2D3, as measured by bioassay in chicks. Aluminum at 5 mg/kg given 5 days before the bioassay in vitamin D-deficient chicks, partially blocked the intestinal calcium absorption response to low (0.65 and 3.2 nmol), but not to higher (32 nmol) doses of vitamin D3. The responses to all doses (0.32-2.1 nmol) of 1,25(OH)2D3 were partially blocked by aluminum treatment. Serum calcium values were elevated in vitamin D-deficient chicks by aluminum administration, but no consistent effects of the treatment on bone calcium mobilization in response to vitamin D3 or 1,25(OH)2D3 were noted. Aluminum treatment in vivo led to decreased 25-OH-D3-1-hydroxylase activity subsequently measured in renal homogenates; under a variety of conditions, no direct effect of aluminum on 25-OH-D3 metabolism by primary cultures of chick kidney cells was observed. The results suggest that the ability of the intestine to respond normally to 1,25(OH)2D3 may be compromised by exposure to high levels of aluminum and that the effect of this element on 25-OH D3 metabolism observed in vivo may not be exerted by direct action on the renal cell. PMID- 2998577 TI - Effects of thionaphthene 2-carboxylic acid and related compounds on bone resorption in organ culture. AB - We have compared the effects of thiophene 2-carboxylic acid (TCA) and a number of sulfur- and nitrogen-containing analogs for their ability to inhibit bone resorption in organ cultures of fetal rat long bones. Four compounds,- thionaphthene-2-carboxylic acid (TNCA), dibenzo-thiophene-4-carboxylic acid, indole-2-carboxylic acid and carbazole-1-carboxylic acid--caused a dose-related inhibition of PTH-stimulated bone resorption, although TCA was ineffective in this system. TNCA at 3 X 10(-4) M or 10(-4) M was the most potent inhibitor of PTH-stimulated bone resorption and was selected for further study. TNCA also inhibited stimulation of resorption by prostaglandin E2 and 1,25-dihydroxyvitamin D. Unlike calcitonin, the effect of TNCA was persistent and did not show escape. Moreover, TNCA could inhibit resorption in bones that had previously escaped from calcitonin. TNCA did not appear to be a nonspecific toxin, since it did not decrease incorporation of [3H]thymidine or [3H]proline into fetal rat long bones. The fact that resorption in unstimulated cultures was only decreased when the control rates were high also argues against nonspecific toxicity. Moreover, this suggests that TNCA will be most effective under conditions of accelerated bone resorption when an inhibiting effect is most desirable. PMID- 2998578 TI - The in vitro formation of sulfates and glucuronides of estrogens by adult and fetal ovine tissues. AB - Incubation of nanomolar concentrations of [3H]estrone with ovine liver slices from adult and fetal animals demonstrated, in particular, the production of estrogen sulfates together with smaller amounts of glucuronides, even although microsomal estrogen glucuronyltransferase (GT) and sulfatase activities were high, especially in adult tissue. [3H]Estriol was conjugated almost exclusively as sulfate under the same experimental conditions. Slices of maternal and fetal kidney medulla were also strikingly active in promoting estrogen sulfate production as were slices of fetal kidney cortex. Adult kidney cortex conjugated estrogen only in the glucuronide form. These data indicate the possibility that maternal and fetal liver and kidney might contribute to the high circulating level of estrone sulfate in the pregnant sheep. Through the use of [3H]estrone and [3H]estrone sulfate as substrates, it was possible to demonstrate that adult slices of kidney medulla possessed relatively low sulfatase, considerable sulfotransferase (ST), and virtually no GT activity, whereas cortex had high sulfatase, little or no ST, and low, though demonstrable, GT activity. The ST activity of kidney high-speed supernatants was stimulated by the presence of sulfhydryl groups, whereas that in liver was not. Enzymic reduction of estrone and (or) estrone sulfate by liver and kidney slices indicated that, in the former, 17 alpha-reduction prevailed and, in the latter with the exception of the maternal medulla, 17 beta-reduction was the main pathway, particularly in the fetus. PMID- 2998579 TI - Receptor interactions controlling lipoprotein metabolism. AB - Lipoprotein receptors play a central role in lipoprotein metabolism and a major role in cholesterol homeostasis. The most completely characterized lipoprotein receptor is the LDL (low density lipoprotein) or apo-B,E(LDL) receptor. The apo B,E(LDL) receptor is present on both hepatic and extrahepatic cells and is responsible for the metabolism of a major portion of plasma LDL. Binding and internalization of LDL particles by this receptor initiates a series of intracellular events, resulting in the regulation of cellular cholesterol metabolism. In addition to the apo-B on LDL interacting with the apo-B,E(LDL) receptor, the apo-E on apo-E-containing lipoproteins is also capable of interacting and regulating intracellular cholesterol metabolism. The liver has also been shown to contain a second distinct lipoprotein receptor that is specific for apo-E. This receptor has been demonstrated on hepatic membranes from humans, dogs, and swine and is referred to as the apo-E receptor. This receptor may be responsible for the clearance of chylomicron remnants from plasma by the liver and may participate in reverse cholesterol transport. Thus, apo-E is a major determinant in lipoprotein metabolism and cholesterol homeostasis. The receptor binding properties of apo-E are well characterized, and a series of structural variants, several with lipoprotein binding defects, have been identified. Studies of the binding activity of these receptor-defective apo-E variants have helped to define the receptor binding domain of apo-E.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2998581 TI - Carcinoma of the tongue: the Montreal General Hospital experience from 1979 to 1984. AB - In a retrospective study of 54 patients with primary carcinoma of the tongue seen at the Montreal General Hospital from 1979 to 1984, the overall 2-year survival for those with stage I disease was 92%. Surgery alone was the initial treatment in 84% of these patients. Disease recurred in 36%. In stage II patients, 8 of 12 had surgery followed by radiotherapy; 37% had recurrent disease. Surgery alone was used in 4 of 12 and with recurrence in 3. Overall survival in stage II was 83% at 2 years. In cases of localized disease, the survival rate for surgery alone was not significantly different from that after surgery plus radiotherapy. Combined surgery plus radiotherapy might offer better regional control in stage II disease. Of 18 stage III patients, 7 were treated by surgery followed by radiotherapy; 3 died and 4 are disease free. All had commando procedures. Radiotherapy alone was used in seven and six of them had recurrences. Overall, the 2-year survival for stage III was 61%. Control of regional disease was achieved in 57% of those who had surgery plus radiotherapy as opposed to 39% overall. Stage IV patients had a 2-year survival of 10%. Nine of the 11 received chemotherapy followed by radiotherapy. This combination did not improve survival and most of these patients died within 1 year. PMID- 2998580 TI - Restriction-modification systems in Streptomyces antibioticus. AB - Several restriction systems were detected in different strains of Streptomyces antibioticus by using actinophages as biological indicators. Adsorption of phages to the bacteria, together with the study of the efficiency of plating gave an initial indication of restriction in three strains. The alternation of efficiency of plating values obtained from restricting and nonrestricting hosts, gave evidence for the presence of a restriction-modification system in another strain. No common modification systems were detected among the different strains tested. Two specific endonucleases with a possible role in restriction were detected in strains ATCC 11891 and ETH 7451, respectively. PMID- 2998582 TI - AIDS hysteria: a contagious side effect. PMID- 2998583 TI - Marker chromosomes of the long arm of chromosome 1 in endometrial carcinoma. AB - Cytogenetic studies were performed on endometrial specimens of four patients with hyperplasia, six with adenocarcinoma, and one with a mixed mesodermal tumor. Except for one cell, all 65 cells from the hyperplastic specimens had a normal female karyotype. However, a total of 92 cells from the five adenocarcinoma specimens had chromosome abnormalities, though all 20 cells from a specimen of a well differentiated adenocarcinoma showed a normal karyotype. The chromosome number and morphology of the aneuploid cells had minimal changes. The modal number of chromosomes was pseudodiploid in one case and hyperdiploid in four cases. Three kinds of structural abnormalities involving chromosomes #1 were identified to be of clonal origin: del(1p21) in two cases, tdic(1;16)(p21;q24) in one case, and i(1q) markers in two cases. Because the carcinoma cells had two chromosomes #1 of normal morphology, the presence of the marker chromosome led to partial trisomy or tetrasomy of the long arm of chromosome #1. This involvement may be assumed to represent a karyotypic change characteristic of some adenocarcinomas of the endometrium. Complex karyotypes with many rearranged chromosomes were observed in cells from the mixed mesodermal tumor. The karyotypic differences between endometrial carcinoma and the mixed medodermal tumor suggest that the genesis (and its mechanism) of the former may differ from that of the latter. PMID- 2998584 TI - The cytogenetics of Wilms' tumor. AB - A close association has been demonstrated between the congenital deletion 11p13 and predisposition to Wilms' tumor. Recent cytogenetic studies on Wilms' tumor cells from normal children strongly suggests that somatic changes in the short arm of chromosome #11 play an important role in the development of this tumor. The application of improved cytogenetic techniques coupled with molecular biologic analysis may help resolve questions regarding the requirement of additional changes (to alterations of 11p13) in order to evoke complete transformation leading to malignancy. PMID- 2998585 TI - In situ hybridization--application to gene localization and RNA detection. AB - In situ hybridization offers a direct approach for localization and quantitation of nucleic acid sequences in cellular preparations. Recent improvements in technology and methodology make possible the detection of DNA and RNA of relatively low copy number. For example, development of in situ hybridization methods for detection of single copy DNA sequences on mitotic chromosomes has led to general use of this technique for gene mapping of the human genome. More recently, improvements in methodology for detection of low abundancy RNA make possible a facilitated analysis of gene expression, both from cellular genes and exogenous sequences, such as viral genomes. In situ hybridization is now a powerful method for studying nucleic acid organization and function in normal cells, as well as in malignant cells, which should contribute to better understanding of the cell transformation process. PMID- 2998586 TI - Gene amplification in cancer: a molecular cytogenetic approach. AB - Many human tumors have been shown to exhibit cytologic evidence of gene amplification. In several instances the amplified genes have proven to be cellular oncogenes. We present an approach for combining cytogenetic and molecular biological techniques to systematically investigate this phenomenon. PMID- 2998587 TI - Effects of zinc during culture of an insulin-producing rat cell line (RINm5F). AB - The effects of various Zn2+ concentrations on cell proliferation, insulin secretion and contents of insulin and zinc were studied in a clonal cell line (RINm5F) established from a transplantable rat islet tumor. The RINm5F cells were equally effective in proliferating and releasing insulin at zinc concentrations ranging from 0.013 to 0.073 mM. The percentage of cells able to exclude trypan blue was significantly less in cultures with 0.073 mM Zn in the medium. Increasing the extracellular concentrations of Zn2+ to 0.044 and 0.073 mM, respectively, resulted in a 40-60% reduction in the cellular content of insulin. There was a significant increase (73%) in the cellular content of zinc only when increasing the extracellular concentration of the element to 0.073 mM. The addition of 0.2 mM EGTA to a zinc-deficient medium had no effect on proliferation, insulin release or content of insulin and zinc, indicating the presence of a stable endogenous pool of zinc maintaining the function of the RINm5F cells for at least 5 days in culture. PMID- 2998588 TI - Glutathione peroxidase, glutathione S-transferase and glutathione reductase activities in normal and neoplastic human breast tissue. AB - Glutathione peroxidase (GSH-Px), glutathione S-transferase (GSH-Tr) and glutathione reductase (GSSG-Rx) activities have been determined in normal and neoplastic human breast tissues. Large interindividual variations in the activities of all enzymes tested were found in both tumor and non-tumor specimens. In general a significant increase in the activities of the 3 enzymes was found in tumors, whereas in fibroadenoma they were as high as in healthy tissues. When a comparison was made between normal and neoplastic tissues of the same individual, GSH-Tr and GSSG-Rx activities were found to be higher in 15 and 11 cases, respectively, out of 17. GSG-Px activity was higher in all cases. From measurement of GSG-Px activity with both H202 and cumene hydroperoxide, it was deduced that human breast contains only the selenium-dependent form. PMID- 2998590 TI - Non-cytotoxic activity of pyran copolymer-induced macrophages associated with potentiation of tumour vaccine in recipient mice. AB - Mice inoculated with both L1210 murine tumour vaccine and pyran copolymer were more resistant to L1210 than those inoculated with either of these agents alone. Rabbit anti-mouse thymocyte globulin and silica reduced the augmented resistance of these mice, suggesting the involvement of activated anti-tumour T cells and macrophages in the augmented resistance. We studied the activation of these two cells separately and examined the possible contribution of pyran copolymer induced peritoneal cells to the augmented resistance to an inoculation of live tumour. Pyran copolymer-induced peritoneal cells endowed the tumour vaccine primed mice, but not unprimed mice, with resistance to implanted L1210 and, among those peritoneal cell populations, macrophages but not T cells were responsible for this effect since the activity was associated with a cell population which was adherent to nylon wool columns, sensitive to silica and insensitive to anti Thy 1.2 antibody plus complement. The pyran copolymer-induced peritoneal cells had very little antiproliferative activity when tested against L1210 in vitro and mice inoculated with these peritoneal cells did not survive a challenge of live L1210 cells much longer (less than 1 day) than L1210 inoculated control mice. Furthermore, the survival of L1210 vaccine-primed mice inoculated with one-tenth the amount of live L1210 (10(2)) was still much shorter than that of mice primed with L1210 vaccine plus pyran copolymer and challenged with ten times as many (10(3)) live L1210 cells. Therefore, direct tumoricidal activity was probably not a major factor in the in vivo immunological augmenting activity of the pyran copolymer-induced macrophages. PMID- 2998589 TI - A p50 surface antigen restricted to human urinary bladder carcinomas and B lymphocytes. AB - We have previously described the derivation of a monoclonal antibody, S2C6, to a novel 50 Kdalton antigen associated with human urinary bladder carcinoma. No reactions were obtained with carcinomas of unrelated origin or with normal urothelial cells. However, the antibody also reacted with a similar antigen on some cell lines of B lymphocyte origin. Using large panels of target cells we have now shown that this reactivity was entirely restricted to cells of the B lineage within the haematopoietic system. As opposed to its apparent restriction to malignant cells of the urothelium, the S2C6 antigen was expressed by normal B lymphocytes as well as by many malignant B cells (chronic lymphocytic leukaemia, hairy cell leukaemia and immunocytoma). Pre-B cells derived from acute lymphocytic leukaemia and plasma cells from multiple myeloma lacked the antigen. Expression was significantly enhanced on cultured B cells from Burkitt lymphomas and on Epstein-Barr virus-transformed lymphoblastoid cell lines including those of the pre-B phenotype derived from fetal bone marrow. As judged from the molecular size and the distribution pattern displayed by the S2C6 antigen it appears to be distinct from other B cell antigens previously described. A possible relation of the S2C6 antigen to a receptor for B cell growth factors is discussed. PMID- 2998591 TI - Characterization of three new variant type cell lines derived from small cell carcinoma of the lung. AB - Three new, well growing cell lines (GLC-1, GLC-2, and GLC-3) have been established from small cell lung carcinoma (SCLC) and characterized. A subclone (GLC-1-M13) markedly different from its parent line GLC-1 was also isolated and characterized. Cytogenetic analysis of the cell lines revealed deletions in the short arm of chromosome 3 as a most consistent chromosomal aberration. The deleted region was not identical in all metaphases, 3p(21-23) being the shortest region of overlap. Despite their SCLC origin GLC-1, GLC-2, and GLC-3 do not show pronounced SCLC differentiation features. Neurosecretory granula were very rare (GLC-1) or completely absent (GLC-2 and GLC-3), whereas the SCLC-related enzyme and hormone markers L-3,4-dihydroxyphenylalanine decarboxylase, neuron-specific enolase, creatine kinase BB, and bombesin-like immunoreactivity were variably expressed. Although the subclone GLC-1-M13 was derived from the poorly differentiated GLC-1, it behaved according to the above criteria as a differentiated "classic" SCLC cell line. When assessed with specific monoclonal antibodies the different cell lines appeared to express different subsets of intermediate filament proteins, indicative for different stages and directions of differentiation: "undifferentiated" (GLC-1 and GLC-2); "neural tissue related" (GLC-2); "simple epithelium" related (GLC-1-M13); and a combination of simple and squamous epithelium related (GLC-3). We conclude that GLC-1, GLC-2, and GLC-3 represent dedifferentiated forms of SCLC, related to the recently described "variant" type of SCLC, whereas the clonal derivate GLC-1-M13 behaves like a differentiated "classic" SCLC cell line. PMID- 2998592 TI - Changes in nuclear proteins on transformation of rat epithelial thyroid cells by a murine sarcoma retrovirus. AB - Two-dimensional electrophoresis has been used to document changes in nuclear proteins following viral transformation of an epithelial cell line exhibiting differentiation markers. After transformation, these markers are lost, and the cells become tumorigenic and capable of growth in soft agar. A sharp rise in the phosphorylation of histones H1, H2A, and ubiquitinated H2A is seen on transformation, together with the appearance of three phosphorylated proteins that are extractable by perchloric acid and appear related to high mobility group Protein 14, a constituent of active chromatin. Since comparison is made between normal and transformed cells that are each grown to confluence and since there is little difference between their observed growth rates, the changes seen represent intrinsic differences between the cell lines and are thus a direct reflection of the process of transformation. PMID- 2998593 TI - Stage-dependent induction of prenatal tumors in mice by the Kirsten and Moloney strains of murine sarcoma viruses. AB - The Moloney (MoMSV) and Kirsten (KiMSV) strains of murine sarcoma viruses are known to induce mesenchymal sarcomas upon infection of newborn rodents. To determine their activity in mouse embryos, 11- to 15-day-pregnant CD-1 mice were laparotomized, and the single implants were inoculated into the abdominal portion of the embryonal body with an average of 15 and 1500 focus-forming particles/g of body weight of the MoMSV and KiMSV viruses, respectively. Another group of less than 1-day-old pups was given a comparable amount of either virus. Tumors appeared in the young within the first few weeks of life with incidences and histological types dependent on the gestational day and the viral strain inoculated. Mixed mesenchymal sarcomas at or near the site of inoculation and vascular tumors of the brain were by far the most frequent neoplasms observed in the newborn. With MoMSV there was an increased incidence of sarcomas with advancing age at treatment, being 0% at 11 days of pregnancy and 96% in newborn (P for trend, less than 0.025). By contrast, KiMSV caused an incidence of sarcomas below 20% throughout (P for trend, greater than 0.05). Brain tumors were identified in the several MoMSV and KiMSV groups, with a peak value of 43% following the inoculation of both viruses into 13- and 15-day-old embryos, respectively. While the total incidence of these tumors was significantly different from controls, no positive trend by day of treatment was found among the MoMSV and KiMSV viruses (P less than 0.05). The tumors were mainly capillary angiomas, but a few cavernous angiomas were also detected. In addition, eight pups which were given injections of both viruses at developmental Days 11 to 13 had tumors of the choroid plexus. In many instances, newborn pups were affected by multiple vascular abnormalities of the brain, including capillary telangiectases and multiple hemorrhagic areas. No such lesions nor tumors at any site were found among the control animals. The present results are important not only because of the evidence that Swiss embryos respond selectively to the carcinogenic effects by murine sarcoma viruses, but also because they offer the opportunity to dissect directly in vivo the mechanisms underlying the stage related sensitivity of prenatal mice to oncogenic retroviruses. PMID- 2998594 TI - Expression of epidermal growth factor and transforming growth factor-beta in human salivary gland adenocarcinoma cell line. AB - Neoplastic intercalated duct cells that originated from a human submandibular salivary gland release transforming growth factor and human epidermal growth factor into serum-free medium. The transforming growth factor with the soft agar colony-forming activity when assayed in the presence of mouse epidermal growth factor on normal rat kidney clone 49F indicator cells, but without mitogenic action to quiescent 3T3 cells, does not compete with epidermal growth factor for receptor binding, is heat and acid resistant but trypsin and dithiothreitol sensitive, and therefore is of the transforming growth factor-beta class. The immunoreactive human epidermal growth factor is detected by radioimmunoassay on certain fractions obtained by the Bio-Gel P-60 chromatography of neoplastic epithelial duct cells originated from a human submandibular salivary gland conditioned medium and is biologically very similar to mouse epidermal growth factor. Moreover, the presence of human epidermal growth factor in cultured neoplastic epithelial duct cells originated from a human submandibular salivary gland is demonstrated by the peroxidase:antiperoxidase method. PMID- 2998595 TI - Diversity of human pancreatic cancer cell proteinases: role of cell membrane metalloproteinases in collagenolysis and cytolysis. AB - In this study we have examined the tissue-destructive proteinases of human pancreatic ductal cancer cell lines derived initially from xenogenic transplants. Cancer cell organelles were isolated following nitrogen cavitation using sucrose density gradient centrifugation. Serine, cysteine, and metalloproteinases were assayed using radiolabeled protein and synthetic substrates. Tumor-induced RBC lysis was quantitated by measuring the release of isotope from 59Fe-labeled RBCs co-cultivated with tumor cells or subcellular fractions. Enzyme inhibitors with specificity toward different classes of proteinases were used in the above assays to categorize the enzymes responsible for substrate degradation. Results indicated that intact pancreatic cancer cells (RWP-1 and RWP-2 cell lines), cell homogenate, and cytosol contain proteinases which were able to degrade [3H]collagen (type I) and [3H]gelatin and lyse normal RBCs. Cancer cell membrane fractions were enriched in collagenolytic, gelatinolytic, and cytolytic activities which could be abrogated by EDTA but not by inhibitors of serine or cysteine proteinases, which indicates that metalloproteinases are the active enzymes in these assays. Although plasminogen activator and cysteine proteinases were also enriched in the tumor cell membranes, these activities were not required for collagen degradation or cytolysis. We conclude that human cancer cell membrane proteinases are advantageously situated to facilitate damage to surrounding normal tissues. PMID- 2998596 TI - 1-beta-D-arabinofuranosylcytosine metabolism and incorporation into DNA as determinants of in vivo murine tumor cell response. AB - In this study, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) formation, retention, and incorporation into DNA were simultaneously evaluated in vivo in mice bearing leukemia cells sensitive to 1-beta-D arabinofuranosylcytosine (ara-C) (L1210/0), leukemia cells resistant to ara-C (L1210/R), P288, and lymphosarcoma P1798, namely cells characterized by differential sensitivity to ara-C. In L1210/R cells, resistance to ara-C was correlated with low deoxycytidine-cytidine kinase activity (0.04 nmol/mg protein/min), with a low level of intracellular accumulation of ara-CTP, with a low level of incorporation of ara-C into DNA, and with no significant inhibition of thymidine incorporation into DNA. Thus a simple measurement of the intracellular pool of total ara-C nucleotides is sufficient to identify cells with this type of resistance. In contrast, in cells with sufficient deoxycytidine cytidine kinase activity (greater than 0.1 nmol/mg protein/min), the factors determining the quality of response to ara-C could be distinguished as follows: (a) those which are responsible for in vitro cytotoxicity (producing in vivo cytoreduction); and (b) those which are responsible for in vivo selectivity (producing long term survivors). In P288 cells which are sensitive in vitro to ara-C, the determining factor for this sensitivity is the amount of ara-CTP formed which produced greater than 80% inhibition of thymidine incorporation into DNA. The lack of antitumor activity in vivo, however, was due to similarities in ara-CTP retention in target tumor cells (P288) and normal bone marrow cells. In both cases, ara-CTP retention at 4 h was less than 10% of the value obtained at 30 min. In contrast, in cells such as L1210 and P1798 long term survivors (cures) were directly correlated with higher ara-CTP retention. For example, 4 h after drug administration, ara-CTP retentions were 20, 82, and 6% for L1210, P1798, and bone marrow cells, respectively. At 24 h, 20% ara-CTP was retained intracellularly by P1798 tumor cells. In summary, results presented herein demonstrate the importance of differential ara-CTP retention as the most critical determinant of response for the induction of long term survivors, and ara-C incorporation into DNA by tumor cells after in vivo treatment appears to be less significant. These data also demonstrate close correlation between ara-CTP pools, retention, and the extent of inhibition of recovery of thymidine incorporation into DNA. PMID- 2998597 TI - Photoenhancement of lipid peroxidation associated with the generation of reactive oxygen species in hepatic microsomes of hematoporphyrin derivative-treated rats. AB - Hepatic microsomes prepared from rats pretreated with hematoporphyrin derivative (HPD) undergo rapid enhancement of lipid peroxidation in the presence of solar radiation (approximately 400 nm). Quenchers of singlet oxygen, including 2,5 dimethylfuran, histidine, and beta-carotene, and inhibitors of the hydroxyl radical, including benzoate, mannitol, and ethanol, largely protected against the enhancement of lipid peroxidation caused by HPD photosensitization. Catalase, a scavenger of hydrogen peroxide and superoxide dismutase, a scavenger of superoxide anion, had little or no protective effect against HPD-photosensitized enhancement of lipid peroxidation. Our data indicate that in vitro irradiation of hepatic microsomes prepared from HPD-treated rats results in the generation of both singlet oxygen and hydroxyl radical. These reactive moities are associated with a rapid increase in microsomal lipid peroxidation which may explain the unique susceptibility of membranous components of cells to this type of phototoxic injury. PMID- 2998598 TI - Partial purification and characterization of a hepatocyte growth factor produced by rat hepatocellular carcinoma cells. AB - Serum-free medium conditioned by confluent cultures of JM1 of JM2 rat hepatocellular carcinoma cells stimulated DNA synthesis in primary cultures of adult rat hepatocytes in a dose-dependent, saturable manner and in the absence of epidermal growth factor. The hepatotrophic activity was nondialyzable in Mr 50,000 cutoff membranes, heat (60 degrees C) and acid stable, and sensitive to trypsin and dithiothreitol treatment. Gel filtration of concentrated JM1 or JM2 conditioned medium on Sephadex G-100 separated the activity into two regions, the major broad peak migrating with apparent Mr 25,000. Chromatography fractions active in the hepatocyte proliferation bioassay also inhibited specific binding of iodinated epidermal growth factor to cultures of A431 carcinoma cells and rat hepatocytes. These results suggest that neoplastic liver cells synthesize and secrete polypeptide growth factors which can bind to epidermal growth factor receptors and stimulate proliferation of normal adult rat hepatocytes in primary culture. PMID- 2998599 TI - Changes in receptor occupancy and growth factor responsiveness induced by treatment of a transformed mouse embryo cell line with N,N-dimethylformamide. AB - Treatment of the transformed mouse embryo fibroblast cell line (AKR-MCA) with N,N dimethylformamide (DMF) results in a reversion to the nontransformed AKR-2B cell line phenotype. AKR-MCA cells grown in the presence of 1% DMF showed a 2-fold increase in the sites for epidermal growth factor (EGF) binding. However, most of these sites were occupied by an endogenous ligand. The EGF receptor was unoccupied in untreated AKR-MCA cells. The increased receptor occupation was paralleled by an increase in the mitogenic response to EGF. Treatment of these cells with 1% DMF resulted in a 6-fold stimulation of mitogenesis by EGF. The ability to respond to nutrient replenishment (a property of growth-arrested AKR MCA cells) was lost within 24 h of DMF treatment. Upon removal of DMF from the cells, both the mitogenic response to EGF and the occupation of the EGF receptor by endogenous ligands were lost. Treatment of the AKR-2B cell line with DMF had little effect on its growth properties. Therefore, DMF altered the growth control response and growth factor binding of AKR-MCA cells in a reversible, noncytotoxic manner. PMID- 2998600 TI - Expression of antigens coded in murine leukemia viruses on thymocytes of allogeneic donor origin in AKR mice following syngeneic or allogeneic bone marrow transplantation. AB - Removal of T-lymphocytes from marrow inoculum with monoclonal antibody plus complement permitted establishment of long-lived allogeneic chimeras between C57BL/6 and AKR/J mice. Development of leukemia was prevented for 15 mo. Protection from leukemia occurred with both young (4 wk) and older (4 mo) recipients. AKR mice reconstituted with syngeneic marrow or control AKR mice all developed leukemia-lymphoma before 1 yr of age. During spontaneous lymphomagenesis in AKR mice, amplified expression of gag or env gene-coded virus antigens on the surface of thymocytes preceded leukemia development and evidence for amplification of other virus genes. These changes generally appeared before 6 mo. Similar viral gene expression and viral gene amplification occurred in the thymus and spleen cells of leukemia-resistant chimeric mice. Using monoclonal antibodies to Mr 70,000 glycoprotein epitopes characteristic of ecotropic, xenotropic, or dualtropic viruses, antigens marking each virus form were found on thymocytes of allogeneic 4-wk and 4-mo chimeras as well as on the cells of AKR mice and of AKR mice reconstituted with syngeneic marrow. Flow cytometric analysis showed amplification of the virus genes in mice protected from leukemia lymphoma by allogeneic bone marrow transplantation from leukemia-resistant mice. Allogeneic chimeras and syngeneically transplanted mice both showed evidence of accelerated viremia and of recombinant virus formation. The findings suggest that an event essential to leukemogenesis which occurs within the AKR lymphoid cells or their environment is lacking in the allogeneic chimeras. The nature of this influence of a resistance gene or genes introduced into AKR mice by allogeneic bone marrow transplantation deserves further study. PMID- 2998601 TI - Production of hydroxyl-free radical by reaction of hydrogen peroxide with N methyl-N'-nitro-N-nitrosoguanidine. AB - Production of a hydroxyl free radical (.OH) by reaction of hydrogen peroxide (H2O2) with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was examined by electron spin resonance using the X OH spin trapping agent 5,5-dimethyl-1-pyrroline-1 oxide (DMPO). The electron spin resonance spectra of the H2O2-MNNG-DMPO system after exposure to light at an intensity of 0.03 mW/cm2 for 5 min, and the DMPO- (.OH) spin adduct (2-hydroxy-5,5-dimethyl-1-pyrroline-1-oxide) generated by use of Fenton's reagent showed the same hyperfine structure and g-value. The signal of the DMPO adduct obtained in the H2O2-MNNG-DMPO system disappeared on addition of the .OH scavenger sodium benzoate. The addition of another .OH scavenger, ethanol, resulted in the appearance of a new signal due to trapping of the alpha hydroxyethyl radical. These results show that .OH was formed in the H2O2-MNNG DMPO system. The typical signal of the DMPO-(.OH) spin adduct was not observed in the system in the absence of light. The amount of DMPO-(.OH) spin adduct increased with increase in the concentration of H2O2 when the MNNG level was kept constant, and it changed with the concentration of MNNG at a constant H2O2 level, indicating that .OH was produced by the interaction of MNNG with H2O2. In the absence of H2O2, complicated trapped signals appeared in the spectrum of the MNNG DMPO system in the light, but these signals were not observed when the system was kept in the dark. In the absence of MNNG, the H2O2-DMPO system did not show any signal, even in the light. These results indicate that interaction of free radicals derived from MNNG with H2O2 on exposure to light resulted in .OH production. PMID- 2998602 TI - Detection of tumor-associated antigens in the sera of lung cancer patients by three monoclonal antibodies. AB - One hundred sixty-one sera from lung cancer patients, including 46 samples from patients who had not yet received treatment were screened for tumor-associated antigens with 3 monoclonal antibodies, CSLEX1, CSLEA1, and CLEX5, by a new cell binding inhibition assay. We had previously determined that the antigens recognized by CSLEX1 and CSLEA1 are sialosylated Lewisx and sialosylated Lewisa, respectively. Either of these two antibodies alone reacted with about 65% of the 46 untreated patients' sera. Eighty-seven % of the 46 showed positive results with at least one of the two antibodies. The CLEX5 monoclonal antibody is presented here as recognizing a potential tumor-associated antigen. CLEX5 reacted with 54% of the 46 sera from nontreated lung cancer patients. When the results for all three antibodies were combined, the percentage of positive sera was 89% (of 46). Some interesting patterns in the serum levels of the antigens detected by these antibodies were observed. Levels of sialosylated Lewisx were significantly higher in sera from nontreated advanced stage (III and IV) patients (P less than 0.0003). In addition, levels of the antigens detected by CSLEX1 and CSLEA1 were dependent on whether or not the patient had been receiving treatment. These observations suggest potential applications of monoclonal antibodies to diagnosis and monitoring of therapies. PMID- 2998603 TI - Curative cancer chemotherapy. AB - Cancer chemotherapy provides variably effective treatment for the majority of forms of human cancer and curative treatment for some 12 categories of cancer. Curative treatment is defined as the proportion of patients who survive beyond the time after which the risk of treatment failure approaches zero, i.e., the disease-free survival plateau. This progress has resulted from a closely integrated scientific effort, including drug development, pharmacology, preclinical modeling, experimental design with respect to clinical trials, quantitative criteria for response, and a series of clinical trials (initially in children with acute lymphocytic leukemia) in which the importance of complete remission, of dose and schedule, of sequencing chemotherapeutic agents, of pharmacological sanctuaries, and particularly of combination chemotherapy was studied. The principles derived from these studies, particularly those relating to combination chemotherapy, resulted in curative treatment for disseminated Hodgkin's disease, non-Hodgkin's lymphoma, pediatric solid tumors, testicular cancer, and limited small cell lung cancer. Many patients with certain stages of solid tumors, such as breast cancer and osteogenic sarcoma, are at high risk of having disseminated microscopic disease. Experimental studies indicate that treatment which is only partially effective against macroscopic disease is much more effective against microscopic tumors. Therefore chemotherapy is administered immediately following control of the primary tumor in patients at high risk of having disseminated microscopic disease, a treatment known as adjuvant chemotherapy. This program has been highly successful in increasing the cure rate in patients with pediatric solid tumors and in prolonging disease-free survival in patients with premenopausal breast cancer. Given dissemination of the technology, it is estimated that 15,000-30,000 patients per year are potentially curable in the United States. Curability of cancer by chemotherapy generally is inversely related to age, i.e., the above tumors are most common in children and young adults. There are new and promising treatment strategies, such as neoadjuvant chemotherapy and autologous bone marrow transplantation. The revolution in molecular and cellular biology is providing an increase in targets, rationale, and opportunity for more effective and novel chemotherapeutic approaches. PMID- 2998604 TI - Tumor invasion and metastases--role of the extracellular matrix: Rhoads Memorial Award lecture. PMID- 2998605 TI - Enhanced replication of murine cytomegalovirus in murine leukemic lymphocytes. AB - Replication in vitro of murine cytomegalovirus was found to be enhanced in lymphoid cells from leukemic (AKR/J) mice as compared with similar cells from nonleukemic animals. Prolonged productive murine cytomegalovirus infection in lymphoid organs in vivo was demonstrable only in leukemic AKR/J mice. Latent nonproductive murine cytomegalovirus infection established in young nonleukemic AKR/J mice was invariably reactivated and expressed in salivary glands and lymphoid organs after these animals became leukemic. PMID- 2998606 TI - Transferrin binding to two human colon carcinoma cell lines: characterization and effect of 60-Hz electromagnetic fields. AB - 125I-Labeled human transferrin was used to study the binding of transferrin to Colo 320 DM and Colo 205 human cell lines derived from adenocarcinomas of the colon. Although transferrin uptake was greater in both cases at 37 degrees than at 4 degrees it was found that slightly greater than two-thirds of the transferrin associated with the cells at 37 degrees was not bound to surface receptors but rather had been internalized by the cells. Subsequent analysis of true surface binding at 4 degrees by Scatchard analysis allowed determination of the number of transferrin receptors as well as association constants for the interaction. The number of transferrin receptors per cell was found to be inversely related to the cell density of the cultures from which cells were removed for study. Association constants were unaffected by cell density, with average values of 1.2 and 5.4 X 10(8) M-1 obtained for Colo 320 DM and Colo 205, respectively. Additionally, maximum theoretical numbers of receptors of 1.05 X 10(5)/cell for Colo 320 DM and 1.39 X 10(5)/cell for Colo 205 were determined. Furthermore, exposure of Colo 205 cells to three different experimental situations, i.e., 60 Hz-generated electric field only (E+, 300 mA/m2rms), magnetic field only (M+, 1.0 gauss rms), and combined electric + magnetic fields at these intensities (E+M+), altered the expression of transferrin receptors as compared to a concurrently run unexposed control population of cells (E-M-). In three separate experiments the number of transferrin receptors quantitated on both M+ and E+M+ cells was independent of cell culture density and was close to or exceeded the maximum theoretical number of receptors determined for this cell line. In contrast, E+ cells expressed fewer transferrin receptors than was predicted on the basis of cell culture density. PMID- 2998607 TI - Expression of epidermal growth factor receptors on human cervical, ovarian, and vulval carcinomas. AB - We describe the properties of two monoclonal antibodies produced to a synthetic peptide consisting of residues 985 to 996 from the cytoplasmic domain of the epidermal growth factor (EGF) receptor. We have examined a group of ten human tumors including cervical, ovarian, and vulval carcinomas for expression of EGF receptors by immunohistological staining using one of these antibodies and another monoclonal antibody to the extracellular domain of the molecule. The tumors were examined using a sensitive amplified enzyme system and a less sensitive indirect staining method. There was generally a good correlation in staining intensity with the two monoclonal antibody reagents. Both antibodies showed strong staining of squamous cell carcinomas and usually weak or heterogeneous patterns with the adenocarcinomas. Samples of each tumor were solubilized in detergent and analyzed for the presence of functional EGF receptors by immunoprecipitation and autophosphorylation. Three of the squamous cell tumors gave labeled bands, Mr 170,000, on sodium dodecyl sulfate:polyacrylamide gels. DNA was extracted from seven of the tumors and digested with two restriction endonucleases, and the fragments were analyzed on Southern blots using probes representing the extracellular and cytoplasmic domains of the molecule. The tumor DNA showed no apparent rearrangements or amplifications when compared to the EGF receptor gene in human placental DNA. These results suggest that there is a high level of EGF receptors on some squamous cell tumors. PMID- 2998608 TI - Conventional and immunocolloidal gold electron microscopy of eight simian retroviruses closely related to human T-cell leukemia virus type I. AB - Simian retroviruses closely related to human T-cell leukemia virus type I (HTLV I) were isolated from 8 species, examined by both conventional and thin section immunocolloidal gold electron microscopy, and compared with HTLV-I. Mature forms of simian viruses were found in extracellular aggregates and within cytoplasmic vacuoles. They were morphologically similar to each other and to HTLV-I. They consisted of a seemingly smooth envelope and a centrally located nucleoid. Their size varied considerably among species and also within the same species; this is characteristic of this group of retroviruses. No budding particles of simian viruses were observed. Thin section immunocolloidal gold electron microscopy using various human and simian sera showed that simian viruses were antigenically related to each other and to HTLV-I. One drawback of this otherwise very useful technique was the difficulty in identifying virions because of the poor preservation of their fine structure by fixation with glutaraldehyde alone. This was overcome by using materials prepared for conventional electron microscopy, in which virions showed weak but specific reactions with gold particles after deosmification and antigen restoration with sodium metaperiodate. PMID- 2998609 TI - Clonal variation in the production of a platelet-derived growth factor-like protein and expression of corresponding receptors in a human malignant glioma. AB - Two cell lines (U-343 MG and U-343 MGa) with different phenotypic characteristics were established from the same human glioblastoma multiforme biopsy. Previous studies have shown that a clonal derivative (Cl 2) of the U-343 MGa line produces a PDGF-like growth factor. In the present investigation glioma PDGF production and 125I-PDGF binding were found to be differently expressed in U-343 MG, U-343 MGa, and U-343 MGa Cl 2 cultures, providing evidence for a clonal variation in these properties. In order to investigate this point further, several clones were derived from low (23 clones) and high (30 clones) passage U-343 MGa cultures, as well as from U-343 MGa Cl 2 cells (30 clones). The clones could be divided into 4 groups according to morphology and growth pattern. A determination of the amount of PDGF receptor competing activity in serum-free conditioned media gave evidence for a clonal variation in the production of glioma PDGF, corresponding to 0-87 ng of authentic PDGF per ml. There was also a considerable range in 125I-PDGF binding (0-44 fmol of tracer bound per 10(6) cells). Scatchard plots performed on two clones confirmed the presence of saturable, high affinity PDGF receptors. High passage cultures were found to give rise to a higher number of high producing clones than did low passage cultures. There appeared to be a negative correlation between production of glioma PDGF and binding of 125I-PDGF, probably due to the receptor blocking activity of the endogenous growth factor. However, the presence of clones, apparently devoid of both glioma PDGF production and 125I PDGF binding, suggests a true clonal variation in these two parameters. The growth rate in serum-free medium was found to correlate fairly well to the extent of glioma PDGF production. Production of glioma PDGF was found to have a morphological correlate and be most prominent among clones of "immature" looking, tightly growing cells. Clones that had large star-shaped cells with some resemblance to normal glia-like cells in culture were found to have a low production and a high 125I-PDGF binding capacity. PMID- 2998610 TI - High incidence of amplification of the epidermal growth factor receptor gene in human squamous carcinoma cell lines. AB - Southern blot-hybridization analysis of DNAs from human tumors demonstrated amplification of the epidermal growth factor (EGF) receptor gene in 10 of 12 squamous cell carcinoma cell lines tested and in none of 18 tumor cell lines of nonsquamous cell carcinomas. The degree of amplification in the squamous cells varied from 2- to 50-fold relative to the epidermal keratinocyte. Hybridization analysis of the RNA showed that the amplification of the EGF receptor gene is accompanied with an increase of the 5.6 kilobases of EGF receptor mRNA. Scatchard plot analysis and sodium dodecyl sulfate-polyacrylamide gel analysis of the EGF receptor revealed that the synthesis of the EGF receptor is also greater in the cells with amplified EGF receptor gene. In contrast, Southern blot analysis of DNAs of primary tumors showed that incidence of amplification of the EGF receptor gene in squamous cells (1 of 6) was almost as frequent as in nonsquamous cells (1 of 4). These results show that amplification of the EGF receptor gene is commonly found in various tumors. In addition, our data suggest that primary squamous cell carcinomas with amplified EGF receptor gene may readily adapt to growth in tissue culture. PMID- 2998611 TI - Effect of hyperthermia on the antiproliferative activities of murine alpha-, beta , and gamma-interferon: differential enhancement of murine gamma-interferon. AB - Fever is frequently an important side effect of interferon (IFN) therapy. Studies have shown that culturing interferon-treated cells at elevated temperature heightens the antiproliferative activity of IFN-alpha and IFN-beta. Since IFN gamma has also been shown to be a potent antiproliferative agent, the effect of elevated temperature on IFN-gamma activity was compared to its effect on IFN alpha and IFN-beta. Mouse B-16 melanoma cells were simultaneously cultured under cloning conditions at a range of temperatures (37.3, 38.1, 38.6, and 39.4 degrees C) in the presence of MuIFN-alpha, MuIFN-beta, and MuIFN-gamma. The antiproliferative activities of all three interferons were enhanced by incubation at the elevated temperatures. However, the elevated temperatures had a more dramatic enhancing effect on the antiproliferative activity of MuIFN-gamma (10 fold enhancement) than of either MuIFN-alpha or MuIFN-beta (2.9- and 3.4-fold enhancement, respectively). Next, the enhancing effect of elevated temperature (39.4 degrees C) was examined for a range of interferon concentrations. The degree of the enhancing effect increased with increasing concentrations of MuIFN gamma but not with increasing concentrations of MuIFN-alpha or MuIFN-beta. Enhancing effects of temperature as high as 14-fold were observed for 100 units of MuIFN-gamma/ml. This dramatic enhancement was observed for both natural and recombinant MuIFN-gamma and was neither a function of greater relative perception of MuIFN-gamma titer at elevated temperature nor a function of greater relative stability of MuIFN-gamma at the elevated temperature. The differential enhancement of MuIFN-gamma activity by elevated temperature appeared to be specific for the antiproliferative activity, since the antiviral activity of MuIFN-gamma was not relatively more enhanced at 39.4 degrees C than were the antiviral activities of MuIFN-alpha and MuIFN-beta. These results suggest that fever may be an important factor in maximizing the antitumor effects of MuIFN gamma and perhaps of human IFN-gamma. They also raise the possibility that a combination treatment regimen of hyperthermia and interferon therapy, particularly IFN-gamma therapy, may provide a significant antitumor effect. PMID- 2998612 TI - Cellular and molecular changes during chemical carcinogenesis in mouse skin cells. PMID- 2998614 TI - Transforming genes of human malignancies. PMID- 2998613 TI - Regulation of cell differentiation and tumor promotion by 1 alpha,25 dihydroxyvitamin D3. PMID- 2998615 TI - Stabilization of Z-DNA conformation by chemical carcinogens. PMID- 2998616 TI - Heart injury in head-injured adolescents. AB - Of 19 adolescents (ages 10-18) admitted consecutively because of major blunt impact trauma, 15 had head injuries (Glasgow coma scales 4-15). Eight had cardiac injury (42%), as demonstrated by cardiac wall-motion studies utilizing ECG-gated radionuclide angiography. Of the head-injured patients, 7 had cardiac injury (47%), and of these, one died in cardiac shock. Significant cardiac injury is known both experimentally and clinically to escape detection by conventional methods and a compromised cardiac output may bode ill for a damaged brain if cerebral perfusion pressure is in jeopardy. PMID- 2998617 TI - Sialyltransferase and nucleoside diphosphatase as markers for tumor monitoring. AB - In this study we report serum sialyltransferase and nucleoside diphosphatase activities of patients with malignant tumors of various primary sites and extent, prior to and during chemotherapy. Enzyme levels were compared to clinical and laboratory parameters. The sialyltransferase and uridine diphosphatase (UDPase) activities in samples of 43 patients with advanced ovarian cancer was four to ten fold above the normal mean value (sialyltransferase 85.1 +/- 58 pmol/hr/ml and UDPase 26.6 +/- 7.2 nmol/hr/ml). After effective chemotherapy with adriamycin and cisplatin, the enzyme activity decreased markedly. In cases of complete remission, enzyme activity decreased to the normal range. In three cases after initial response for several months a rise of both enzymes was observed before any other biochemical finding of the forthcoming relapse. Similar patterns were observed in testicular cancer (6 cases). Clinical correlation is also obvious in other tumors except malignant lymphomas. Our findings show that the activities of these enzymes correlated with the clinical course, and therefore they can be the basis for clinical application for tumor monitoring, especially during chemotherapy. PMID- 2998618 TI - Plasma cyclic nucleotide levels in monitoring acute leukemia patients. AB - To verify the clinical usefulness of plasma cyclic nucleotide determination as a tumor marker, levels were measured in 52 normal subjects and in 106 acute leukemia patients. In untreated patients plasma cyclic GMP (cGMP) levels were markedly elevated, whereas cyclic AMP levels did not significantly differ from those of normal subjects. Plasma cGMP levels normalized in all patients who attained complete remission and remained in the normal range during all the remission period. In the patients who relapsed, an early increase in cGMP levels to the pretreatment values was observed, thus suggesting that their determination may be of clinical relevance in monitoring the patients' response to treatment. PMID- 2998619 TI - Enzyme activities in human breast tumor cells and sera. AB - Galactosyltransferase (GalTF), sialyltransferase (SiaTF), fucosyltransferase (FucTF), 5'-nucleotidase (5'Nucl), and ADP-ribosyltransferase (RibTF) were determined in three subcellular fractions of tumor cells and adjacent control tissue from 20 patients with small primary infiltrating ductal adenocarcinomas of the breast. Viable, as pure tumor cell populations as possible were isolated, subfractionated, and their enzyme levels compared to those in the patients' sera. The activities in tumor cells of the three glycosyltransferases were two- to seven-fold higher, whereas 5'-Nucl and RibTF showed reduced activities when compared to adjacent noninvolved tissue. Serum GalTF and SiaTF were slightly elevated in early mammary carcinoma, whereas FucTF, 5'Nucl, and RibTF were decreased in comparison with a control group. The proposed tumor origin of circulating enzymes could not be confirmed. Surprisingly, only for RibTF could a correlation between tumor and serum activity be established; a weak correlation was found for SiaTF. However, no such relationship could be determined for GalTF, FucTF, or 5'Nucl. In conclusion, the enzyme profile of the tumor cell does not, except for RibTF, appear in the serum. Serum enzyme profiles, therefore, do not permit detection of the early stages of breast cancer. A high correlation between RibTF activity and cytosol estrogen and progesterone receptor levels has been determined in tumor cells, possibly indicating slower growing, more differentiated types of breast tumors. PMID- 2998620 TI - Potential for personal modification of risk for developing colon cancer. AB - Research in varied populations, in appropriate animal models, and through other laboratory techniques, in great part fostered through the National Large Bowel Cancer Program of the National Cancer Institute, has provided a reasonable basis for assessing environmental elements as to risk for large bowel cancer. It was noted that the term large bowel cancer needs to be specifically related to subsections of the large bowel that appear to have different risk factors. For the major type of neoplastic disease in the large bowel, that in the descending and sigmoid colon, there is a good association with nutrition and specific nutritional elements. The risk of this type of colon cancer is proportional to the customary dietary fat intake--high in the western world and low in the Orient. It is inversely proportional to stool bulk, itself related to cereal fiber intake. These two major elements are sufficiently secure as to underlying scientific data and understanding of mechanisms to permit utilizing them for personal modification of risk. Thus, a dietary regimen low in total fat, 20% of calories, and higher in cereal fiber, of the order of 30 g per day, are indicated and would serve to reduce risk not only in the general population, but most likely also in patients who have been successfully treated through conventional modalities. There are also suggestions that regular intake of yellow and green vegetables, of foods with calcium salts, selenium, and other micronutrients, lower risk even more. Further research is needed to provide the data base necessary for deliberate interventions with these agents. PMID- 2998621 TI - Large bowel cancer: prospects for control. AB - The incidence of large bowel cancer, a major cancer in the western world, varies significantly for different segments of the colon as a function of geographic pathology, but the rectal cancer incidence shows smaller distinctions in different countries. Cancer in the descending and sigmoid colon relate to western lifestyle, particularly the high level of dietary fat (40-45% of calories). The lower risk seen in Finland is associated with a high intake of cereal fibers. The current information base with regard to the nutritional factors that show an enhancing effect by high-fat intake and protective effects of agents increasing stool bulk, such as cereal and vegetable fibers, is sufficiently convincing to allow recommendations for intervention trials using fat and fiber. Optimal levels for disease prevention are different from normally accepted and traditional intakes of fat and fiber, and measurable parameters such as serum cholesterol levels, stool weight, fecal bile acid concentration, and perhaps fecal mutagens reflect averages derived from the intake of a high-fat, low-fiber diet that are not optimal. Optimal recommendations are for a dietary level of 20-25% of fat calories and about 30 g of total fiber from whole grain cereals and cruciferous vegetables. Such measures (low-fat, high-fiber) have the advantage of having no obvious adverse effects and can be implemented now on a public and personal basis while additional options in preventive medicine are being explored. PMID- 2998622 TI - Scanning X-ray microradiographic study of the formation of caries-like lesions in synthetic apatite aggregates. PMID- 2998623 TI - Quantitative autoradiographic characterization of receptors for angiotensin II and other neuropeptides in individual brain nuclei and peripheral tissues from single rats. AB - Autoradiographic techniques coupled with computerized microdensitometry and comparison with 125I standards were used to characterize and quantitate receptors for neuropeptides in rat brain and adrenal and pituitary glands. These techniques are rapidly performed, anatomically precise, and more sensitive than membrane binding techniques. They permit the determination of complete saturation curves and Scatchard analysis in discrete nuclei of the rat brain and in single rat pituitary and adrenal glands. Angiotensin II (AII) receptors were quantitated after incubation of 16-micron tissue sections with the AII agonist 125I-[Sar1] AII. High-affinity, high-density AII receptors were present in the organon subfornicalis, organon vasculosum laminae terminalis and nuclei triangularis septalis, suprachiasmatis, and paraventricularis of the rat and in rat adrenal capsule-zona glomerulosa area, adrenal medulla, and anterior pituitary. These techniques could be used for precise localization and quantitation of other neuropeptide receptors in single rat brain nuclei, after optimizing the assay conditions and provided that suitable 125I ligands are available. PMID- 2998624 TI - Quantitative autoradiography of the development of mu opiate binding sites in rat brain. AB - The regional developmental appearance of mu binding sites in rat brain was examined by quantitative autoradiography of 3H-dihydromorphine binding in rats 2, 14, 21, and 28 days old. Labeling with 3H-dihydromorphine was heterogeneous in adult rat brains, as previously reported by other laboratories. Levels of 3H dihydromorphine binding ranged from approximately 250 nCi/g tissue in the interpeduncular nucleus and 100 nCi/g tissue in the habenula to 40 nCi/g tissue in the hypothalamus and periaqueductal gray. Some areas, particularly white matter regions, had no detectable specific binding. The density of 3H dihydromorphine binding increased in all regions between 2 and 28 days of age. The increases in 3H-dihydromorphine binding in various regions of rat brain developed at different rates. Maximal densities were seen by 14 days of age in most regions examined, including the caudate, hippocampus, amygdala, and hypothalamus. Binding in the medial thalamus and quadrigeminal plate, however, did not reach maximal levels until 21 days. Although quantitative autoradiography offers major advantages in the examination of the regional distribution of opiate binding sites, variability both between sections from the same brain and between sections from different brains demonstrate some of the difficulties associated with this type of experimental approach. PMID- 2998625 TI - Long-term potentiation and 4-aminopyridine. AB - Long-term potentiation (LTP) of excitatory postsynaptic potentials (epsp's) was investigated with extracellular field potential recording in hippocampal slices from rats. In the presence of 100 microM 4-aminopyridine (4-AP) the probability of eliciting LTP was unchanged or increased; the extent of potentiation was not significantly different from normal. During LTP saturation, 4-AP further enhanced the epsp. These data are inconsistent with an involvement of A-current reduction in LTP. PMID- 2998626 TI - Amiloride inhibits protein synthesis and lowers the intracellular pH in exponential growing Yoshida rat ascites hepatoma (AH 130) cells: evidence for a role of the Na+/H+ exchanger. AB - We have previously demonstrated in a rat ascites hepatoma cell line (Yoshida AH 130) the presence of a glucose-activatable and amiloride sensitive Na+/H+ exchange (Cell Biol. Int. Rep., 1984, 8, 297-307). Amiloride is known to inhibit this exchange and to cause a cytoplasmic acidification, with inhibition of protein and DNA synthesis, in cells induced to grow. Amiloride appears also to penetrate the cells and to inhibit directly protein synthesis. In the present report we describe experiments in which the activity of amiloride (0.1, 0.4 and 3.0 mM) on protein synthesis and the internal pH of cells was compared in exponential growing and stationary phase Yoshida ascites cells. In phosphate buffered medium and Na+ out = 147 mM no inhibition of protein synthesis (3H-leu incorporation into total cell protein) and no internal acidification (14C-DMO distribution between intra- and extracellular volume) were produced by 0.1 and 0.4 mM amiloride in exponential growing cells. In stationary phase cells, on the contrary, 0.4 mM amiloride inhibited protein synthesis by 60% without decreasing the internal pH. When the Na+ out was lowered to 25 mM, to reduce competition with amiloride, and/or all Na+ out was substituted with choline, 0.1 and 0.4 mM amiloride markedly inhibited protein synthesis and decreased the internal pH in exponential growing cells. No apparent inhibition occurred in stationary phase cells under the same conditions, possibly due to a preexistent internal acidification, with severe decrease of protein synthesis. Fluorimetric studies of amiloride "binding" to ascites cells showed that a reduced number of amiloride receptor sites could exist in Yoshida hepatoma cells at the stationary phase of growth.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2998628 TI - Apoprotein-B binding sites in estrogen-treated rabbit liver: quantitative immunoelectron microscopy. AB - Normal and estrogen-treated rabbit livers were perfused with iodinated very low density lipoproteins (125I-VLDL) and binding and cellular distribution of apolipoprotein (Apo)-B rich lipoproteins in hepatocytes was analyzed using quantitative immunoelectron microscopic and autoradiographic techniques. Apo-B containing particles were bound to and internalized through the formation of endocytotic pits and vesicles with considerably more binding to estrogen-treated specimens (2-fold). Thus, estrogen treatment stimulates Apo-B binding and subsequent internalization of the Apo-B containing particles by increasing the number of endocytotic pits. In contrast to the estrogen induced liver distribution change of Apo-E reported previously, the Apo-B distribution was not changed due to estrogen treatment. PMID- 2998627 TI - Effects of phosphatase inhibitors on nuclease activity. AB - Nucleases such as DNase I, which selectively digest chromatin, are inhibited by several commonly used phosphatase inhibitors including sodium bisulfite. Two inhibitors, sodium arsenate and fructose-1,6-diphosphate, did not significantly inhibit nuclease action. Two other effective phosphatase inhibitors, p chloromercuriphenyl sulfonate and 5,5'-dithiobis(2-nitrobenzoate), can be used during nuclei isolation and then washed out of nuclei before nuclease digestion. Using this procedure, 1mM p-chloromercuriphenyl sulfonate is as effective as 50mM bisulfite in retaining the phosphatase-sensitive mitotic phosphorylations of histones H1 and H3. PMID- 2998629 TI - [Delayed hypersensitivity to polioviruses in patients with amyotrophic lateral sclerosis]. PMID- 2998630 TI - Carcinogenicity and metabolic activation of hexestrol. AB - The carcinogenic activity of the synthetic estrogen hexestrol was measured in male Syrian hamsters. Between 90% and 100% of the animals treated with hexestrol or with 3',3",5',5"-tetradeuteriohexestrol, implanted subcutaneously as 25-mg pellets, were found with renal carcinoma after 6-7 months. In vitro hexestrol metabolism, mediated by phenobarbital-induced rat liver microsomes, led to the formation of 3'-hydroxyhexestrol. This metabolite was identified by comparison with authentic reference material synthesized by oxidation of hexestrol with Fremy's salt. Diethylstilbestrol could not be detected as a metabolite. In urine of male Syrian hamsters, 3'-hydroxyhexestrol, 3'-methoxyhexestrol, 1 hydroxyhexestrol, and other hydroxylated and/or methoxylated hexestrol metabolites were identified. Again, diethylstilbestrol was not detectable as a hexestrol metabolite in vivo. The reactivity of 3'-hydroxyhexestrol was then studied to determine if this catechol estrogen played a role in hexestrol carcinogenicity. Horseradish peroxidase catalyzed the oxidation of 3' hydroxyhexestrol to 3',4'-hexestrol quinone. This oxidation reaction could also be carried out non-enzymatically using silver oxide or silver carbonate on celite as oxidants. The quinone was unstable (t1/2 in methylene chloride: 53 min). It reacted with sulfur-containing compounds such as mercaptoethanol by Michael addition to form 3'-(2-hydroxyethylthio)-5'-hydroxyhexestrol. 3',4'-Hexestrol quinone reacted with simple amines such as ethylamine to form N-ethyl aminohexestrol. The chemical reactions described above were carried out to test the reactivity of identified or suspected metabolic intermediates of hexestrol. It was concluded that carcinogenicity of hexestrol was not based on its conversion to diethylstilbestrol. Rather, catechol estrogen formation may be necessary for the carcinogenic action of hexestrol in analogy to events observed earlier with estradiol. PMID- 2998631 TI - Interaction of menadione (2-methyl-1,4-naphthoquinone) with glutathione. AB - The interaction of menadione with reduced glutathione (GSH) led to a removal of menadione and formation of menadione-GSH conjugate and glutathione disulfide (GSSG). The changes in thiol level were essentially biphasic with an initial rapid decrease in GSH and appearance of GSSG (less than 1 min) followed by secondary less pronounced changes. The interaction of menadione and GSH caused an oxygen uptake and both superoxide anion radical and hydrogen peroxide were produced during the reaction, the amount dependent on the GSH/menadione ratio. Catalase did not protect against the initial decrease in GSH level but markedly inhibited the secondary changes while superoxide dismutase had little effect. These results suggest that the initial changes in thiol level are the result in part of a redox reaction between menadione and GSH as well as conjugate formation, whilst the secondary changes reflect conjugate formation and the activity of other oxidants such as hydrogen peroxide. The potential biological significance of this reaction was investigated using hepatocytes depleted of reduced pyridine nucleotides and thus not able to perform enzyme-catalyzed reduction of menadione. In these cells menadione induced GSSG formation at a rate similar to that observed in control cells. This suggests that quinone-induced oxidative challenge caused by the chemical interactions of a quinone and glutathione may have biological relevance. PMID- 2998632 TI - Degradation kinetics of sodium sulbactam in aqueous solutions. PMID- 2998633 TI - Change in digitonin-stimulated superoxide anion production by guinea pig polymorphonuclear leukocytes in response to the presence of calcium ion. PMID- 2998634 TI - Effect of eicosapentaenoic acid on mouse peritoneal exudate cells. PMID- 2998635 TI - Cellular retinoid binding proteins. AB - The cellular retinol-binding protein (CRBP) and the cellular retinoic acid binding protein (CRABP) have similar physicochemical characteristics. The amino acid sequences of rat CRBP and bovine CRABP have been elucidated and they display 40% sequence identity. Both protein sequences appear to be evolutionarily highly conserved. The amino acid sequence of human CRBP, deduced from a cDNA-clone, is 96% identical to the rat CRBP sequence. CRBP and CRABP are members of a protein family, all members of which may bind hydrophobic ligands and interact with membrane components. All members of the protein family are probably related in tertiary structure and might interact with membrane components through two regions with a high probability for alpha-helix. The tissue distribution of CRBP and CRABP, together with their relation to lipid transporting proteins suggests that CRBP and CRABP are cellular transporting proteins for retinol and retinoic acid, respectively. PMID- 2998636 TI - Phosphatidylinositol transfer proteins: structure, catalytic activity, and physiological function. AB - Among the diverse lipid transfer proteins which are found in tissues and biological fluids are those which exhibit a specificity toward phosphatidylinositol and phosphatidylcholine, with a preference for the former. Phosphatidylinositol transfer proteins (PI-TPs) have been purified from several eukaryotic sources; those present in bovine brain and heart have been extensively studied. This review examines the tissue distribution of PI-TPs and the means by which transfer activity is measured using natural and artificial membranes. The interaction of these proteins with lipid monolayers and bilayers is discussed in terms of phospholipid fatty acyl and polar head group compositions. The inhibition of transfer activity by sulfhydryl agents and amphiphilic amines is summarized. The metabolism of the phosphoinositides is considered and a role for PI-TPs is proposed. PMID- 2998637 TI - Prenatal diagnosis of hemoglobinopathies by DNA analysis. AB - Restriction site polymorphisms are normal inherited variations in DNA that can be readily detected by restriction endonuclease analysis. Currently, 17 such polymorphisms are recognized within a 60 kb (kilobase) stretch of DNA which includes the beta-globin gene complex. Because of their proximity to the beta globin gene, often these restriction site polymorphisms can be used to predict inheritance of beta-globin variants that produce disease. For example, restriction site polymorphisms can be used for prenatal diagnosis for the large majority of couples at risk of having a child with beta-thalassemia. When each member of such a couple is heterozygous at one or more of these 17 sites, family studies are usually successful in determining which forms of the polymorphism are co-inherited with the beta-thalassemia genes in that particular family. Subsequently, study of fetal DNA isolated from amniocytes obtained by midtrimester amniocentesis or from chorionic villi obtained by first trimester chorion biopsy will reveal which DNA polymorphisms that fetus has inherited. By deductive reasoning one can then predict which beta-globin genes it has co inherited. Because of the general nature of these polymorphisms, which are related to the beta-globin gene and its variants only because of their proximity on chromosome 11, they are potentially useful in the prenatal diagnosis of any beta-chain hemoglobinopathy. Some hemoglobinopathies (including alpha thalassemia, sickle cell anemia, and some cases of beta-thalassemia) can be detected directly by DNA analysis. In these cases in utero diagnosis does not need to rely on restriction site polymorphisms, which require preliminary family studies and are not applicable in all at risk pregnancies. Recently, genetic probes, which are necessary for detecting restriction site polymorphisms, have been isolated for sequences of several genes whose protein products are important in blood coagulation. These include probes for all three genes whose polypeptide products combine to form the fibrinogen molecule as well as probes for the prothrombin, Factor IX, Factor VIII, and antithrombin III genes. Defects in these genes are expected to be the causes of afibrinogenemia, prothrombin deficiency, hemophilia B, hemophilia A, and antithrombin III deficiency, respectively. From experience with other genes, it is expected that restriction site polymorphisms within and/or flanking these genes will be found.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2998638 TI - Hydroxyl radicals do not crosslink a DNA-lysozyme complex. AB - The ionic complex between lysozyme and either Escherichia coli DNA or pBR322 DNA was not crosslinked by two systems capable of producing nanomolar amounts of hydroxyl radicals, the oxidation of xanthine by xanthine oxidase and the iron catalyzed oxidation of ascorbic acid. Nor did effective crosslinking occur with micromolar quantities of hydroxyl radicals raised by the addition of adenosine nucleotides to ferrous iron and hydrogen peroxide. In this case, radical content was estimated by colorimetric analysis of formaldehyde following hydroxyl radical oxidation of dimethyl sulfoxide. Similar amounts of radicals generated by pulse radiolysis in a nitrous oxide atmosphere failed also to induce crosslinking. These findings do not support a role for hydroxy radicals in the N-acetoxy-2 acetylaminofluorene induced crosslinking of DNA to lysozyme proposed earlier. PMID- 2998639 TI - Genotoxicity of dimethylnitrosamine in the presence of chrysotile asbestos UICC B and xonotlite. AB - Interactions of particulates with chemical genotoxic agents may play an important role in the induction of carcinogenesis. With respect to bronchogenic cancer, the synergism associated with combined exposure to asbestos and tobacco smoke is a well-documented phenomenon. The present work focused on chrysotile asbestos and xonotlite. The latter is a fibrous calcium silicate which is increasingly being used to replace asbestos in various industrial applications. The study was aimed at testing the possible interaction of these materials with dimethylnitrosamine (DMN), a genotoxic component of tobacco smoke. The capacity of fibers to interfere with the genotoxic response elicited by DMN in the UDS/hepatocyte assay system specifically designed for sensitive detection of short-patch DNA repair, was looked for. The properties of the selected fibers with respect to binding affinity towards DMN were also examined. PMID- 2998640 TI - Pharmacology of platelet inhibition in humans: implications of the salicylate aspirin interaction. AB - The current dispute over the effects of "low" vs "high" doses of aspirin should take into consideration the pharmacokinetics of this drug. In fact, different pharmaceutical formulations of aspirin may deliver little or no aspirin to the systemic blood. This was the case, for instance, in healthy volunteers taking 320 mg of compressed aspirin or 800 mg of enteric-coated aspirin. In all instances thromboxane B2 generation in serum was fully inhibited. Platelet cyclooxygenase might therefore be effectively acetylated by exposure to aspirin in the portal circulation, whereas vascular cyclooxygenase could be spared. Thus aspirin formulations ensuring complete first-pass deacetylation should be sought rather than "low" or "high" doses of unspecified aspirin formulations. Regardless of the type and dose of aspirin administered, salicylate is formed and accumulates in the circulation. It may antagonize the effects of aspirin on cyclooxygenase, at least in acute conditions. As an example, after administration of 1 g of salicylate to healthy volunteers, when plasma levels of the drug were about 75 micrograms/ml, the effect of 40 mg iv aspirin (given 40 min later) on platelet cyclooxygenase and aggregation was significantly diminished. In contrast, in patients undergoing saphenectomy, the same dose of salicylate (1 g) gave plasma drug levels of about 25 micrograms/ml; salicylate was unable to prevent the inhibitory effect on platelets of 40 mg iv aspirin (given 1 hr later) but did act on vascular prostacyclin. Thus the combination of salicylate with aspirin at an appropriate dose and blood level ratio may result in almost complete dissociation of the drug's effect on platelets and vessels in man.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2998641 TI - Thromboxane A2 and prostaglandin endoperoxide receptors in platelets and vascular smooth muscle. AB - 9,11-Dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyphenyl)-13, 14-dihydro-13 aza-15 alpha beta-omega-tetranor-TXA2 (I-PTA-OH), a recently synthesized thromboxane (TX) A2/prostaglandin (PG) H2 receptor antagonist, was shown to be a competitive antagonist of human platelet aggregation induced by the stable endoperoxide analog U46619. This antagonism was due to competitive blockade of the platelet TXA2/PGH2 receptor since I-PTA-OH did not antagonize the first phase of ADP-induced aggregation which is TXA2/PGH2 independent, nor did it inhibit TXA2 synthesis. In addition, analysis of dose-response curves to U46619 (0.1 to 40 microM) in the presence of increasing concentrations of I-PTA-OH (0.5 to 10 microM) showed that I-PTA-OH produced a parallel rightward shift of the dose response curve. Further analysis of the data in the form of a Schild plot yielded a straight line with a slope (m = 1.03) not significantly different from -1. These results are consistent with the notion that I-PTA-OH acts as a competitive antagonist of the TXA2/PGH2 receptor. PMID- 2998643 TI - Elimination of the "chromogen oxidase" activity of bilirubin oxidase added to obviate bilirubin interference in hydrogen peroxide/peroxidase detecting systems. AB - The use of bilirubin oxidase to remove interference by bilirubin in hydrogen peroxide/peroxidase detecting systems is hampered by its inherent "chromogen oxidase" activity (its ability to oxidize the chromogens used in the systems). This unwanted activity is greater than 99% inhibited by 0.5 mmol/L cyanide, 97% inhibited by 20 mmol/L azide. At these same concentrations, they inhibit bilirubin oxidase activity by 95% and 73%, respectively. Sequential addition of reagents allows the use of bilirubin oxidase without interference by the chromogen oxidase activity. PMID- 2998642 TI - AH23848: a thromboxane receptor-blocking drug that can clarify the pathophysiologic role of thromboxane A2. AB - Despite numerous suggestions in the literature that thromboxane A2 is involved in a variety of occlusive vascular diseases, no definitive evidence is available. Arguments have been presented to support the view that such evidence can only come from clinical studies with a highly specific thromboxane receptor-blocking drug. We have now identified such a drug, AH23848, in our laboratories. Preliminary experiments with AH23848, ([1 alpha (Z), 2 beta,5 alpha]-(+/-)-7-[5 [[(1,1'-biphenyl)-4-yl]methoxy]-2-(4-morpholin yl)-3-oxocyclopentyl]-4-heptenoic acid), show that it is a potent, specific thromboxane receptor-blocking drug that is orally active and has a long duration of action. It should be a valuable tool in elucidating any physiologic or pathologic role of thromboxane A2. PMID- 2998644 TI - I-cell disease and pseudo-Hurler polydystrophy: heterozygote detection and characteristics of the altered N-acetyl-glucosamine-phosphotransferase in genetic variants. AB - The human disorders I-cell disease and pseudo-Hurler polydystrophy (also known as mucolipidosis II and III, respectively) are caused by an inherited deficiency of UDP-GlcNAc: lysosomal enzyme precursor GlcNAc-P transferase activity. The most common genetic variants of these diseases (complementation group A) can be identified in homozygotes and heterozygotes using a GlcNAc-P transferase assay with artificial acceptors and commercially available radiochemicals. The kinetic characteristics of the residual GlcNAc-P transferase activity in complementation group A fibroblasts indicates that the low activity is due to a low Vmax. The measured Michaelis-Menten constants for the substrates UDP-GlcNAc and alpha methyl mannoside are in the normal range. Homozygotes and heterozygotes of another less common variant of pseudo-Hurler polydystrophy (complementation group C) have normal activity and normal kinetic characteristics with this assay using alpha-methyl mannoside as the acceptor substrate. Several PHP variants with unusual characteristics are discussed. PMID- 2998645 TI - Kinetic studies on neurotoxin-binding inhibitory antibodies in myasthenia gravis. AB - A method for measuring antibodies which inhibit the binding of 125I-alpha bungarotoxin to the acetylcholine receptor was developed. The procedure allows the detection of inhibitory activity in 70% of sera from the patients with myasthenia gravis. Kinetic analysis showed that (1) a linear dose-response exists between logarithmic concentrations of any test serum and inhibitory activity, (2) the slope of the inhibition line is identical among various myasthenic sera and, (3) the slope of line induced by various cholinergic ligands was comparable to that obtained by myasthenic sera. The inhibitory activity correlated with the titer of anti-acetylcholine receptor antibody measured by an immunoprecipitation method (r = 0.76, p less than 0.01). PMID- 2998646 TI - Serum angiotensin converting enzyme activity in patients with psoriasis. AB - Serum angiotensin converting enzyme activity is frequently increased in patients with active sarcoidosis. In spite of a reported association between sarcoidosis and psoriasis, serum angiotensin converting enzyme activities have not been reported for patients with psoriasis. We found the mean (SD) angiotensin converting enzyme activity for 51 healthy subjects was 18.6 (5.8) kU/l, but for 52 patients with psoriasis without coexisting sarcoidosis, it was 28.3 (6.7) kU/l. There is a significant difference between these means (p less than 0.01). Forty-two percent (22/52) of the psoriasis patients had an increased serum angiotensin converting activity. Other diseases sometimes associated with an increased serum angiotensin converting enzyme activity were excluded as possible causes of a elevated activity in our patients with psoriasis. We conclude that almost half of the patients with psoriasis will have an elevated serum angiotensin converting enzyme activity, even when coexisting sarcoidosis is absent. PMID- 2998647 TI - High molecular weight vasopressin: detection of a large amount in the plasma of a patient. AB - Using a sensitive and specific radioimmunoassay for arginine-vasopressin, we have searched for the presence of high molecular weight (HMW) vasopressin in the plasma of a patient with the syndrome of inappropriate antidiuretic hormone secretion (SIADH) and an oat cell carcinoma of the lung. After incubation in 8 mol/l urea, one millilitre of the plasma from this patient was fractionated on a Sephadex G-50 column and the immunoreactive vasopressin content was evaluated before and after trypsin treatment of the eluted fractions. A wide peak of apparent mol. wt. 2500-6000 daltons was revealed only after tryptic digestion of each fraction. This peak contained the equivalent of 1900 pg of vasopressin. A tryptic digest of this peak, rechromatographed on Sephadex G-25, gave two small peptides the major one eluting at a position identical to vasopressin. These results demonstrate the presence of a large amount of HMW vasopressin in the plasma of a patient with SIADH and oat cell carcinoma of the lung. PMID- 2998648 TI - An analysis of thyrotrophin receptor binding and thyroid stimulating activities in a series of Graves' sera. AB - Improved receptor and bioassays have been used to compare TSH receptor binding and thyroid stimulating activities in unextracted sera from 110 patients with Graves' disease. The two parameters showed a significant correlation (r = 0.65; P less than 0.001) although there were some clear discrepancies. Dose-response studies in 17 sera showed that both receptor binding and thyroid stimulating responses always increased with increasing doses of serum. In patients who were in relapse or remission following antithyroid drug treatment, the results of both bio- and receptor assays correlated well with disease activity with only one clear discrepancy which could have been attributable to the coexistence of autoimmune stimulation and destruction of the thyroid. PMID- 2998650 TI - Extra-endocrine functions of vitamin D. PMID- 2998649 TI - Familial male pseudohermaphroditism with gynaecomastia due to 17 beta hydroxysteroid dehydrogenase deficiency. A report of 3 cases. AB - Three sisters with male pseudohermaphroditism due to 17 beta-hydroxysteroid dehydrogenase deficiency are described. On the basis of a 46 XY karyotype and female phenotype all subjects were thought to have the testicular feminization syndrome. At puberty the two older patients developed signs of virilization and gynaecomastia. In these patients the plasma androstenedione level was 4-5 times higher than normal, whilst the plasma testosterone level was low compared to the normal range and, under basal conditions, their plasma androstenedione to testosterone ratio was 20-25 times higher than normal. Interestingly, in the third, prepubertal case, the basal androstenedione to testosterone ratio was normal but became six times higher than normal after hCG stimulation. These data support the diagnosis of male pseudohermaphroditism due to 17 beta-hydroxysteroid dehydrogenase deficiency and underline the diagnostic value of the hCG stimulation test prepubertally. PMID- 2998651 TI - Failure to suppress C-peptide secretion by euglycaemic hyperinsulinaemia: a new diagnostic test for insulinoma? AB - In order to study the suppression of C-peptide secretion in 5 patients with insulin-producing tumours or beta cell hyperplasia, we raised and maintained plasma insulin at a high physiological level and kept plasma glucose unchanged for 2 h with combined infusions of glucose and insulin (insulin clamp technique). No suppression of C-peptide secretion was seen in any of the patients, in contrast with a 35-65% decline seen in each of 17 healthy control subjects. In 3 patients surgical removal of beta cell adenoma normalized the response, whereas it remained unchanged in a patient with beta cell hyperplasia after partial pancreatectomy and in another with inoperable carcinoma. These results indicate that insulin secretion by insulinomas is characterized by lack of suppression by insulin. Measurement of the insulin-insulin feedback loop by the clamp technique may provide a rapid test to reveal autonomous insulin secretion without the risk of hypoglycaemia. PMID- 2998652 TI - I-cell disease: clinical studies of 21 Japanese cases. AB - Clinical pictures of 21 cases with I-cell disease patients, 12 males and 9 females, were analyzed. Characteristic coarse facial features and shortness of stature were observed in all cases. In general, the motor development was found to be more severely retarded than the mental development of the patients. Rather little involvement of the nervous system seemed to cause somewhat acceptable mental development in some cases, and also cause the absence of epileptic seizures in all cases. Involvement of the cardiovascular system, especially progressive hypertrophic cardiomyopathy, could be highly responsible for frequent sudden death of I-cell disease patients. PMID- 2998653 TI - Linkage relationships of paraoxonase (PON) with other markers: indication of PON cystic fibrosis synteny. AB - The linkage relationships of the serum arylesterase paraoxonase (PON) was examined in our Danish material of normal families and in Danish and English cystic fibrosis families. Highest lod scores were found between PON and cystic fibrosis. The combined lod score for this relationship was z = 2.69 at theta = 0.07 in males and theta = 0.00 in females. When scored in accordance with a tentative three allele model for PON, the score was z = 3.70 at the same theta values. Linkage studies for PON against 64 other polymorphic marker systems did not give any lod score above +1.3 and PON still remains chromosomally unassigned. By the present screening about 2/3 of the genome could tentatively be excluded as the region of PON and cystic fibrosis. PMID- 2998654 TI - A study of DNA polymorphisms around the human apolipoprotein AI gene in hyperlipidaemic and normal individuals. AB - We have used a 2.2 kb fragment of the human apolipoprotein AI (apo AI) gene to screen a number of unrelated individuals for common restriction fragment length polymorphisms (RFLPs) of the gene. As well as the previously reported SstI RFLP (allele frequencies in normolipidaemic individuals 0.94 and 0.06) we have detected RFLPs with the enzymes PstI and XmnI (allele frequencies in normolipidaemic individuals 0.88 and 0.12 for both polymorphisms). In the population studied, the RFLPs appear to be in linkage equilibrium and can be used in conjunction as a haplotype, with a PIC value (polymorphism information content) of 0.5. Significant differences in allele frequency were observed between subgroups of hyperlipidaemic patients and normolipidaemic controls. There is no strong population association in our patient group between any allele of the RFLPs studied and hypertriglyceridaemia. PMID- 2998655 TI - Norrie disease caused by a gene deletion allowing carrier detection and prenatal diagnosis. AB - Carrier determination and prenatal diagnosis in Norrie disease (ND) has so far not been reported. We describe a kindred with 4 members affected by ND in which a deletion comprising gene locus DXS7 on the short arm of the X chromosome defined by probe L1.28 causes the disorder. This allowed us to predict via chorion villus biopsy that a male foetus of a carrier woman is unaffected. PMID- 2998656 TI - Monocyte function in intravenous drug abusers with lymphadenopathy syndrome and in patients with acquired immunodeficiency syndrome: selective impairment of chemotaxis. AB - We have investigated monocyte function in 17 intravenous drug abusers with the clinical and laboratory features of lymphadenopathy syndrome (LAS). LAS patients had normal numbers of circulating monocytes. Monocytes from LAS patients were comparable to cells from normal donors in terms of phagocytosis of latex beads, interleukin-1 secretion, O2- release and killing of antibody-sensitized lymphoma cells or actinomycin D pretreated WEHI 164 cells. In contrast 13 out of 17 LAS subjects tested in this respect as well as six out of nine AIDS patients showed a marked defect of monocyte chemotaxis. Thus monocytes from patients with LAS or AIDS have a selective defect of monocyte chemotaxis. PMID- 2998657 TI - Characterization of monoclonal antibody specific for human type II collagen: possible implication in collagen-induced arthritis. AB - We obtained monoclonal antibodies specific for human type II collagen and characterized them using human collagen type I, II, III and V and tropocollagen A (3/4) (TCA) and tropocollagen B (1/4) (TCB) fragments of type II collagen which were obtained by digestion with tadpole collagenase. These antibodies were of the IgG2a class and specific for the conformational determinant of TCA fragment of type II collagen. When injected intravenously into DBA/1J mice, one of the monoclonal antibodies induced arthritis, which was characterized by early onset, mildness in severity and preferential localization mainly in the peripheral joints of the lower extremities. These results suggest that, at least, one of the arthritogenic determinants of type II collagen for collagen-induced arthritis of mice exists in the three quarter region from the N-terminus of type II collagen. PMID- 2998658 TI - Detection and preliminary characterization of a transmissible agent inducing autoantibody against Golgi-antigen. AB - A transmissible agent inducing autoantibody has been found in association with tumours induced in STU mice with the progressor strain of Moloney sarcoma virus (Mo-MSV). The activity of the agent detectable in serum from tumour-bearing hosts was expressed by development of autoantibody against Golgi-associated evolutionary conserved antigen (Weiland et al. 1984). This agent was tentatively designated 'AGIA', anti-Golgi inducing agent, on account of its most remarkable biological activity. Concomittant with autoantibodies were cytotoxic antibodies reactive with a Mo-MSV non-producer transformant (Sac). Both antibody activities were regularly detectable 2 weeks after inoculation of the agent. At that time the antibody containing serum possessed an infectivity titre of approximately 10(7.5) ID50/ml. Signs of illness were not observed during this period. The antibody-inducing agent was lost from progressor Mo-MSV transformants during their first passage in culture. Neither murine embryo fibroblasts nor murine tumour cells were permissive for propagation of the agent in vitro. PMID- 2998659 TI - Production of a monoclonal antibody to a membrane antigen of human T-cell leukaemia virus (HTLV1/ATLV)-infected cell lines from a systemic lupus erythematosus (SLE) patient: serological analyses for HTLV1 infections in SLE patients. AB - Human T-cell leukaemia virus (HTLV1/ATLV), which causes adult T cell leukaemia (ATL), is an infectious, lymphotrophic retrovirus unique for humans. The present study was undertaken to determine whether HTLV1 had any pathogenetic role for systemic lupus erythematosus (SLE). The incidence of antibodies to ATL cell associated antigens (ATLA) in sera from patients with SLE and other collagen diseases was investigated by an indirect immunofluorescent cytoplasmic staining of an HTLV1-infected cell line (MT-1). A radioimmunoassay was also performed to detect antibodies to HTLV1 protein and crude membrane fraction derived from an HTLV1-producing cell line MT-2. Furthermore, an Epstein-Barr virus (EBV) transformed B cell line (ES-1) was constructed from an SLE patient, which produced a monoclonal antibody (IgG, lambda) reactive to an HTLV1-related cell membrane antigen expressed on MT-1 and MT-2 cells. The specific reactivity of the monoclonal antibody was analysed by an indirect immunofluorescent cell-membrane staining and a microcytotoxicity test. The incidence of anti-ATLA antibodies was not different among SLE and other collagen diseases. The monoclonal antibody produced by ES-1 stained and killed HTLV1-infected cell lines specifically, but did not react with other human lymphoid cell lines. This monoclonal antibody failed to react with peripheral blood mononuclear cells (PBMC), mitogen-induced T cell blasts, and iododeoxyuridine-treated T cells from SLE patients. Thus, a possible role of HTLV1 in the aetiology of SLE was not established. PMID- 2998660 TI - Self-reactive B lymphocytes detected in young adults, children and newborns after in vitro infection with Epstein-Barr virus. AB - B-lymphocytes from healthy children and young adults who were seronegative for autoantibodies and B lymphocytes from umbilical cord blood of newborns were induced to secrete a variety of autoantibodies upon infection with Epstein-Barr virus. Such autoantibody-secreting clones were obtained from different lymphoid tissues and occurred at frequencies of 1 in 10(6)-10(7) mononuclear cells. The autoantibodies were exclusively of the IgM class. They recognized normal cellular components, such as cytoplasmic, nuclear and cytoskeletal antigens. The fact that self-reactive clones were not efficiently eliminated during ontogeny suggests that suppressor mechanisms might be responsible for normal self tolerance. PMID- 2998662 TI - Need for hepatocellular carcinoma screening before renal transplantation in HBs +, HBe +, western African. AB - We report a case of fulminant hepatocellular carcinoma discovered 50 days after renal transplantation. The recipient was a young Senegalese, hepatitis B virus chronic carrier. The pre-transplant check-up was normal, and the tumor was latent until its dramatic expression. Progression of hepatitis B liver disease occurs in immuno-suppressed renal transplant recipients, which often leads to chronic active hepatitis, cirrhosis and hepatocellular carcinoma, with a high risk of death due to liver disease. The early discovery of the tumor in this patient emphasizes the necessity for complete hepatic screening before transplantation in african, hepatitis B virus chronic carrier recipients. Moreover, the accumulation of risk factors for hepatocellular carcinoma: hepatitis B virus, food mycotoxins (aflatoxin), parasitic infestation and immunosuppression with transplantation is stressed. PMID- 2998663 TI - Hemodynamic and metabolic effects of enalapril in patients with heart failure. AB - Placebo and enalapril were added on a double-blind basis to conventional treatment in 14 patients with congestive heart failure (CHF), New York Heart Association class II-III. The patients were followed for 14 weeks and their performance was evaluated by a treadmill test, ejection fraction by nuclear scan, cardiothoracic ratio, and Yale Scale score. Metabolic studies were done to test any adverse effects of the drugs. Enalapril decreased arterial pressure and cardiothoracic ratio, and increased ejection fraction. Placebo exerted no significant effects. However, both drugs improved treadmill time and Yale Scale score. No adverse metabolic or clinical effects were observed with either drug. Based on these limited observations we conclude that: Enalapril is a useful ancillary agent to conventional treatment of CHF; it exerts its effects through afterload and preload reduction; and it is safe and well tolerated and has a prolonged duration of action. PMID- 2998661 TI - Enhanced viral inhibition of lymphocyte mitogenesis in patients with advanced breast cancer. AB - Virus particles are frequently able to non-specifically inhibit the capacity of human peripheral blood lymphocytes to respond to mitogenic or antigenic stimuli. In the case of breast cancer patients with advanced disease, the quantity of virus required to abrogate responsiveness to phytohaemagglutinin (PHA) was approximately four-fold less than that found when cells from healthy donors were employed. The results show that such virus co-incubated cultures are deficient with regard to their ability to synthesize detectable quantities of T cell growth factor (TCGF) activity, and that the extent of responsiveness to PHA in each case corresponds roughly to the amount of TCGF activity that is present in the cultures. While the addition of exogenous purified TCGF to cultures containing virus, normal cells and stimulus caused a reversal of the usual inhibitory effect, this finding was generally not obtained in the case of lymphocytes obtained from patients with advanced breast cancer. These data suggest that one mechanism of explaining diminished cellular immune responsiveness in breast cancer patients may be a relative inability of appropriate cells or subsets of cells to respond effectively to TCGF. PMID- 2998665 TI - Fingerprint inclusions in non-vacuolated lymphocytes in juvenile neuronal ceroid lipofuscinosis. AB - Three patients afflicted with juvenile neuronal ceroid-lipofuscinosis (NCL) have been found to harbor fingerprint lipopigments in the absence of vacuoles in their lymphocytes, in contrast to the majority of juvenile NCL patients in whose lymphocytes lipopigments with fingerprint profiles are usually located within vacuoles. These three patients may belong to clinical subtypes of juvenile NCL: the first patient to an early juvenile or a transitional form, the two latter patients to a protracted form. PMID- 2998666 TI - Advances in the management of bone tumors. PMID- 2998664 TI - Adreno-leukodystrophy (adreno-testiculo-leukomyelo-neuropathic-complex). AB - We have learned much about adreno-leukodystrophy (ALD) since the first case report by Siemerling and Creutzfeld [1923]. Many aspects of this disease, however, are still enigmatic and worthy of investigation (see Pathogenesis). We now realize that ALD is a constellation of clinical and pathologic presentations, all of which presumably are caused by an X-linked genetic defect in the handling of fatty acids [Migeon et al. 1981, Ogino and Suzuki 1981, Singh et al. 1984]. At the present time, and for the sake of this discussion, adreno-leukodystrophy is subdivided into 5 major clinical types: classical, X-linked juvenile ALD; X linked adult ALD; adrenomyeloneuropathic variant (AMN); female ALD; and neonatal ALD. A sixth type, which has only pathologic and pathogenetic relevance, is the fetal form [Powers et al. 1982]. Patients may have clinical or subclinical involvement of only one organ system (e.g., adrenal) or they may have any combination of adrenal, testis, brain, spinal cord, and peripheral nerve disease [O'Neill et al. 1981]. The most common type is the classical juvenile form, followed by the AMN variant. X-linked adult ALD is uncommen and female ALD is rare. The precise nosologic placement of the neonatal form is still debated and will be discussed more fully below. Although satisfactory treatment of central nervous system demyelination in ALD and system degeneration in AMN awaits a better understanding of their pathogenesis, we are now able to control this disease complex by carrier identification and genetic counseling or by in utero detection and therapeutic abortion [Moser et al. 1984]. PMID- 2998667 TI - Virus-like intranuclear inclusions in giant cell tumor of bone. AB - An ultrastructural study of 13 cases of typical giant cell tumor of bone (GCT) revealed the presence of virus-like intranuclear inclusions (INI), morphologically identical to those reported in Paget's disease of bone. In two giant cells of two different patients, INI were found only after careful survey of a great number of cells. This finding, coupled with the scarcity of GCT with similar inclusions reported in the literature, reveals the extreme rarity of this finding and leaves open to discussion the specificity of these INI even in Paget's disease, as well as the possible etiological significance in giant cell tumor of bone. PMID- 2998668 TI - [Experimental pyridoxine neuropathy--an electrophysiological and histological study in rabbits]. PMID- 2998669 TI - [A case of hypothyroid myeloneuropathy with giant axon--on the findings of sural nerve biopsy]. PMID- 2998670 TI - Technetium-99m pyrophosphate scintigraphy for the detection of acute myocardial infarction. How useful is it? AB - To evaluate the contribution of Tc-99m pyrophosphate scintigraphy (TPS) on the overall management of patients suspected of having acute myocardial infarction (AMI), hospital records of 58 consecutive patients who underwent TPS, were evaluated in depth. The results indicate that TPS was essential for the diagnosis of AMI in 16% of the patients. TPS was most rewarding in perioperative patients and in patients with borderline or uninterpretable electrocardiographic and enzyme changes. Also, in some cases, TPS was able to confirm or exclude the diagnosis of AMI prior to the confirmation by serial electrocardiograms (ECG) and serial enzyme changes. TPS was less rewarding in patients with clinically low index of suspicion for AMI. It may also be confusing in patients with high clinical likelihood of AMI and a history of prior myocardial infarction because of the possibility of persistently positive TPS in some of these patients. Considering the limitations of ECGs, the cardiac enzymes, and atypical clinical presentations in the patient population we evaluated, TPS appears to be fairly accurate when the scintigraphic findings are compared with the final diagnosis at the time of discharge from the hospital. PMID- 2998671 TI - Thyroid scintigraphy for the detection of radiation-induced thyroid cancer. AB - Thyroid scintigraphy with Tc-99m pertechnetate was performed in 249 patients who received radiation therapy for abnormalities in the head or neck in order to determine the role of this examination in the detection of abnormal nodules arising from cancer. These patients received a mean total dose of about 10.1 Gy. The mean follow-up period was 39 years. All patients underwent physical examination without prior knowledge of the scintigram. Scintigrams were evaluated without prior knowledge of the physical examination. In 158 cases, both the physical examination and scintigraphy were negative. In 64 cases, both examinations were positive. In ten patients, the physical examination was positive and scintigraphy was negative and vice versa in 17 patients. Of 249 patients, 28 ultimately underwent thyroid surgery; a total of four had carcinoma. A cost-benefit relationship as to routine scintigraphy as a screening procedure is presented. If patients are first screened by palpation, a number of abnormal nodules will be missed. In addition, a considerable number with positive palpation would probably undergo surgery unnecessarily. From a clinical and financial point of view, it is believed that scintigraphy is the examination of choice for screening for radiation-induced thyroid malignancies. PMID- 2998672 TI - A thyroid nodule representing metastatic renal carcinoma. AB - Metastatic neoplasms to the thyroid that become clinically apparent are rare, but a patient that presents with a thyroid nodule and a history of a prior malignancy elsewhere (especially a renal neoplasm) should be thought to have a metastatic nodule first and a nodule of thyroid origin second. This report describes a 66 year-old woman who presented with a large symptomatic thyroid nodule and a history of a right nephrectomy for renal carcinoma two years previously. PMID- 2998673 TI - Reverse discordant behavior in microfollicular adenoma of thyroid. Case report. AB - A patient with reverse discordant behavior between Tc-99m pertechnetate and I-123 in a solitary microfollicular adenoma is presented and possible mechanism is discussed. PMID- 2998674 TI - The clinical value of scintigraphic brain scanning. Experience at the Hillbrow Hospital, Johannesburg, South Africa. AB - Patients were referred to the Department of Nuclear Medicine for brain scintigraphy to be screened for possible intracranial pathology. These referrals were made in order to reduce the heavy load on the transmission computerized tomography (TCT) facilities. Great clinical importance, therefore, has been attached to scintigraphic findings; this emphasizes the need for an accurate assessment of the predictive value of this procedure. PMID- 2998675 TI - Inadequate aldosterone response to hyperkalemia during angiotensin converting enzyme inhibition in chronic renal failure. AB - To assess the mechanism involved in hyperkalemia during angiotensin converting enzyme inhibition with captopril in chronic renal failure, captopril, 150 mg/day, was administered to 16 patients with hypertension with plasma creatinine levels between 1.6 and 12.4 mg/dl. After 4 weeks of therapy, plasma potassium levels increased from 3.9 +/- 0.1 to 5.5 +/- 0.2 mEq/L (P less than 0.001) and the final plasma potassium levels correlated with plasma creatinine levels (r = 0.67; P less than 0.01). In six patients with plasma creatinine levels greater than or equal to 3 mg/dl, aldosterone excretion decreased after 4 weeks of captopril, from 7.5 +/- 3.1 to 1.8 +/- 0.5 micrograms/24 hr, whereas plasma renin activity increased from 0.6 +/- 0.2 to 4.4 +/- 1.1 ng/ml/hr (P less than 0.05). This was associated with increases in plasma potassium levels from 3.9 +/- 0.2 to 5.4 +/- 0.4 mEq/L (P less than 0.005) and a significant reduction in fractional excretion of potassium from an average of 34% to 25%. No significant changes in plasma creatinine levels were observed during therapy. There was a significant positive correlation between aldosterone excretion and the potassium excretion fraction (r = 0.53; P less than 0.01). Increases in plasma potassium levels were not able to increase aldosterone excretion, although the greater the plasma potassium level attained, the smaller the reduction in aldosterone excretion (r = 0.47; P less than 0.05). Our results indicate that adequate aldosterone production is essential to preserve potassium homeostasis in chronic renal failure. Moreover, angiotensin II appears necessary for an adequate aldosterone response to potassium stimulation. PMID- 2998676 TI - The effect of renal function on enalapril kinetics. AB - Enalapril maleate (MK-421), a nonmercapto-containing angiotensin converting enzyme (ACE) inhibitor, is converted in vivo to enalaprilat (MK-422), the active diacid. We evaluated serum profiles and urinary excretion of oral enalapril maleate in patients with renal disease (group I, creatinine clearance less than 3 ml/min, patients undergoing dialysis, n = 10; group II, creatinine clearance 10 to 79 ml/min, n = 9) compared with healthy subjects (group III, creatinine clearance greater than 80 ml/min, n = 10). Group I received a 10 mg dose during a day while not receiving dialysis and a 10 mg dose 1 hour before dialysis 2 weeks later. Groups II and III received a single 10 mg dose. Blood samples and urine were collected for 48 hours. Impaired renal function resulted in elevated serum and plasma concentrations of enalapril maleate and decreased excretion rates and urinary recovery of enalapril maleate and enalaprilat. The data suggest an apparent increase in the extent of metabolism of enalapril maleate to enalaprilat or an increase in nonrenal elimination of unchanged enalapril maleate in renal disease compared with normal health. Enalaprilat was dialyzable. PMID- 2998677 TI - Sulbactam kinetics and excretion into breast milk in postpartum women. AB - We gave intravenous infusions of sulbactam, a beta-lactamase inhibitor, in combination with ampicillin or cephalothin to women 2 days after cesarean section delivery. The elimination t1/2 was 1.0 hours, the volume of distribution at steady state was 268 ml/kg, and renal clearance was 295 ml/min. These values are similar to those in normal young men and in surgical patients and suggest that dose regimens of sulbactam will not need adjustment in the postpartum period. Sulbactam concentrations in breast milk averaged 0.5 micrograms/ml, a value similar to that of several beta-lactam antibiotics. PMID- 2998678 TI - Comparative study of hormonal counter-regulation during GCIIS-guided insulin hypoglycemia tests using human insulin (recombinant DNA) and pork insulin. AB - Human insulin (BHI, recombinant DNA) and pork insulin (PI) were compared in 10 healthy volunteers. Using a glucose controlled insulin infusion system for the performance of the insulin hypoglycemia test (IHT), a comparable dosage of both insulins had to be infused (BHI 0.129 +/- 0.007 vs PI 0.115 +/- 0.01 U/kg; mean +/- SEM). Blood glucose slopes and nadirs did not differ significantly (BHI 30 +/ 2 vs PI 29 +/- 2 mg/dl). There was no difference in C-peptide inhibition (minimum for BHI 0.50 +/- 0.08 vs PI 0.42 +/- 0.08 micrograms/l). Maximum hormone responses were identical for ACTH (BHI 78.4 +/- 11.3 vs PI 76.0 +/- 8.7 pg/ml), cortisol (BHI 246 +/- 20 vs PI 252 +/- 15 ng/ml) and GH (BHI 43.8 +/- 7.3 vs PI 49.4 +/- 6.7 ng/ml). Peak levels of prolactin did not differ significantly (BHI 1,335 +/- 315 vs PI 1,766 +/- 614 microU/ml). The urinary excretion pattern of epinephrine in three 120 min periods before, during and after IHT was identical (before IHT: BHI 0.9 +/- 0.2 vs PI 0.6 +/- 0.1 micrograms/120 min; during IHT: BHI 12.6 +/- 2.2 vs PI 13.4 +/- 2.5 micrograms/120 min; after IHT: BHI 2.5 +/- 0.7 vs PI 3.7 +/- 1.3 micrograms/120 min). No differences in the minima of serum potassium levels were observed (BHI 3.38 +/- 0.04 vs PI 3.33 +/- 0.05 mmol/l). We conclude that the biological effects of human insulin and pork insulin are comparable. Our data do not support the assumption of a different hypothalamic handling of human insulin (recombinant DNA) and porcine insulin. PMID- 2998679 TI - Renal handling of 125I-labelled insulin in the hen. AB - Renal handling of 125I-insulin was studied using a modification of the Sperber technique. Results showed 125I-insulin to be extracted at the peritubular side of the nephron in a process that was competitively inhibited by increasing amounts of unlabelled insulin, but not ACTH, in the injection mixture. When unlabelled insulin instead was injected 30 sec after the labelled insulin it showed significantly less interference with peritubular extraction of 125I-insulin, indicating strong attachment to the cell membrane or possible internalization of 125I-insulin into proximal tubular cells. Light microscope autoradiography 1 min after injection of 125I-insulin showed grains over proximal tubules only. On the ligated side localization was preferably peritubular while on the control side it was luminal. Electron microscope autoradiography showed sparsely distributed grains, however, frequently located over basal parts of proximal tubular cells. Pretreatment with lysine hydrochloride lowered renal extraction of 125I-insulin and increased urinary recovery of iodine label bilaterally. 125I-glucagon and 125I-C-peptide were not extracted from the peritubular circulation. In conclusion, the model has provided evidence of a rapid and significant peritubular extraction of 125I-insulin by proximal tubular cells in a process probably involving specific insulin receptors. Following receptor binding probably only minor amounts of 125I-insulin enters the proximal tubular cells, while the greater part is degraded at the cell surface or released into the circulation. PMID- 2998680 TI - Biliary drainage by ultrasound-guided puncture of the left hepatic duct. AB - Percutaneous transhepatic biliary drainage under ultrasonic guidance was performed in 38 patients with obstructive jaundice due to malignancy (49 intubations). The method was used for palliation in 33 patients and for pre operative drainage because of cholangitis in five patients. Puncture of the left lobar ducts was the method of choice (35 patients). Only in cases of poor visualisation of the left biliary ducts was right-sided drainage performed (three patients). Combined left- and right-sided drainage was necessary in nine patients. All attempts with ultrasound-guided punctures were successful. There were no complications related to the punctures. Delayed complications were cholangitis (10 patients) and bleeding (one patient). The advantages of the method compared with conventional percutaneous transhepatic biliary drainage and the advantages of the left liver lobe drainage are outlined. PMID- 2998681 TI - The value of radionuclide venography in superior vena caval obstruction. AB - A prospective study on 27 patients with suspected obstruction of the superior vena cava (SVC) and 10 control patients with no known chest disease was undertaken to determine the value of radionuclide venography in aiding diagnosis and treatment of the condition. The technique proved simple, safe and non invasive and could rapidly confirm or exclude the diagnosis when in doubt. In addition, the technique had a role in radiotherapy planning, highlighting unsuspected superior mediastinal disease not visible on the chest radiograph. However, follow-up post-treatment scans were of little help due to the good clinical markers of obstruction of the SVC. The normal and abnormal scan appearances are described. PMID- 2998682 TI - Contrast agents in magnetic resonance imaging. AB - With the advent of magnetic resonance imaging (MRI) it has become apparent that paramagnetic contrast agents may have a place in clinical practice. The mechanism of action, development, techniques of use and initial animal and clinical results are reviewed. Gadolinium diethylene triamine penta acetic acid (Gd3+-DTPA) has proved an effective paramagnetic contrast agent in experimental animals and clinical trials with this agent commenced in November-December 1983. Gd3+-DTPA will cross a damaged blood-brain barrier, is excreted mainly by glomerular filtration and is distributed mainly in the extracellular space. No short-term toxicity has been detected. Long-term toxicity is, as yet, unknown. Optimum dose, pulse sequences and timing of imaging remain to be determined by further studies but Gd3+-DTPA shows promise as a useful addition to MRI. PMID- 2998683 TI - Internal biliary drainage and local radiotherapy with iridium-192 wire in treatment of hilar cholangiocarcinoma. AB - Curative surgery is not possible in the vast majority of patients who present with hilar cholangiocarcinoma. Palliative therapy to relieve jaundice, either at laparotomy or percutaneously, is therefore necessary. The mean survival of these patients is of the order of 8.5 months (Wheeler et al., 1981). We report a significant increase in mean survival to 16.8 months in patients treated with internal biliary drainage when combined with local irradiation to the tumour with iridium-192. PMID- 2998684 TI - Urinary adenosine cyclic 3',5'-monophosphate in idiopathic calcium stone-formers. PMID- 2998685 TI - Biochemical and morphometric properties of mitochondrial populations in human muscle fibres. AB - Two mitochondrial subpopulations were evaluated with biochemical and morphological techniques in human gastrocnemius muscle of 10 patients with peripheral arterial insufficiency and 12 control individuals. The subsarcolemmal mitochondria were released by gentle homogenization, with a recovery of 32-37%, and the intermyofibrillar by enzymic digestion and further mechanical disintegration, recovery 18-21%. The subsarcolemmal mitochondria were morphologically defined as those located within 2 micron from the sarcolemma membrane and the intermyofibrillar mitochondria as those located in the rest of the fibre. In the controls the intermyofibrillar mitochondria had a lower respiratory ratio than the subsarcolemmal, owing to a higher state II respiration. The subsarcolemmal space, which contained 25% of the mitochondria, had a mitochondrial volume density two- to three-fold that of the intermyofibrillar space in the controls. The patients, who had a 48-64% higher oxidative enzyme capacity in their muscle tissue, had higher respiratory rate and respiratory control index with similar ADP/O ratio in the subsarcolemmal fraction in comparison with the controls. The citrate synthase activity was higher in both mitochondrial fractions of the patients. The volume densities of mitochondria, total as well as for both subpopulations, were also higher in the patients, which was further reflected in higher yields of mitochondrial protein. The results demonstrate that both subpopulations of muscle mitochondria are able to adapt quantitatively and/or qualitatively. Furthermore, they show that the increased oxidative enzyme capacity of the patients is associated with an increased quantity of both mitochondrial populations and a qualitative improvement of the respiratory activity of the subsarcolemmal mitochondria. PMID- 2998686 TI - Alpha-adrenoceptor changes after oestrogen treatment in platelets and other tissues in female rabbits. AB - alpha 2-Adrenoceptors on blood platelets have been widely used as a model for alpha-adrenoceptors in less accessible tissues. The effect of oestrogen (200 micrograms/day intramuscularly) on alpha 2-adrenoceptor number and function was studied in immature female rabbits. alpha 2-adrenoceptor number was measured in whole platelets, and membrane preparations of forebrain, hindbrain, spleen and kidney by radioligand binding. alpha 2-Adrenoceptor function was examined by measuring platelet aggregation in vitro and circulatory responses to selective alpha 2-adrenoceptor agonists in vivo. Oestrogen treatment resulted in a significant decrease in platelet alpha 2-adrenoceptor number and function. However, no changes were observed either in receptor number in other tissues or in responses to alpha 2-agonists in vivo. The results suggest that oestrogen modulation of rabbit platelet alpha 2-adrenoreceptor number and function may be different from that of brain, kidney and spleen. Caution should be exercised in extrapolating results from platelets to alpha-adrenoceptors at other sites. PMID- 2998687 TI - The characterization and energetic potential of brown adipose tissue in man. AB - In adult man, brown fat can be detected in perinephric fat depots by visual inspection, electron microscopy and nucleotide binding to the tissue-specific uncoupling protein. The 32 kDa uncoupling protein is functionally active, showing a nucleotide-sensitive conductance to protons and an uncoupling response to fatty acids. The amount of uncoupling protein in human mitochondria is equivalent to that in a partially cold-adapted guinea pig, indicating some potential for thermogenesis. Respiratory capacity measurements indicate that the total perinephric fat in adult man can only account for one-fivehundredth of the whole body response to infused noradrenaline. Thus, although brown fat has been found to be quantitatively important in animal studies, considerable caution must be exercised in extrapolating its significance to adult man. PMID- 2998688 TI - Sleep apnoea in alcoholic patients after withdrawal. AB - Respiration during sleep was studied in 16 withdrawn alcoholic patients and in 12 control subjects. The alcoholic patients had increased numbers of central (P less than 0.01) and of obstructive (P less than 0.05) apnoea and of hypopnoea episodes (P less than 0.01) as compared with controls. Significant positive associations were found between the frequencies of central apnoea (P less than 0.05) or hypopnoea (P less than 0.01) and clinical evidence of central nervous system damage in the alcoholic patients. Hypopnoea also showed a significant association with vagal neuropathy (P less than 0.05), assessed by tests of cardioreflexes. We conclude that abnormal respiratory events are common in abstinent alcoholic patients and that they are likely to be at least partly related to nervous damage. PMID- 2998689 TI - Evidence against a role for superoxide ions in the injury of nephrotoxic nephritis in rats. AB - The accelerated model of nephrotoxic serum nephritis (NTSN) was produced in 12 Sprague-Dawley rats. Six of these rats were administered superoxide dismutase (EC 1.15.1.1; SOD) subcutaneously (8 mg/kg) 8-hourly for 4 days. The first dose was given 6 h before the nephrotoxic serum (NTS). The progression of renal disease was monitored by following (i) albumin excretion, (ii) serum creatinine and creatinine clearance and (iii) renal histopathology and immunofluorescence. There was no evidence that SOD influences the course of NTSN. SOD was scarcely excreted by control rats or rats with NTSN. PMID- 2998690 TI - Effect of natural and synthetic atrial natriuretic factor on arterial blood pressure, natriuresis and cyclic GMP excretion in spontaneously hypertensive rats. AB - The differential effects of extracted and synthetic atrial natriuretic factor (ANF) on arterial blood pressure, natriuresis, and cyclic GMP excretion were studied in normotensive (WKY) and spontaneously hypertensive (SHR and SHRSP) rats. Atrial extracts or synthetic (101-126)-ANF decreased arterial blood pressure in all tested animals, but the blood pressure-lowering effect was more pronounced in hypertensive than in normotensive rats. ANF-induced diuresis and natriuresis were two- to three-fold higher in the hypertensive groups. However, a several-fold increase in total urinary cyclic GMP level after the infusion of ANF was essentially equal in the three groups. Our data suggest that acute infusion of ANF reveals a defect of sodium and water handling in SHR. It is possible that this defect is located at the distal nephron, and is made apparent by the action of ANF on glomeruli via a cyclic GMP-induced vascular effect. PMID- 2998692 TI - Diseases of the peripheral motor-sensory unit. PMID- 2998691 TI - The mechanisms of action of noradrenaline on ouabain-sensitive rubidium uptake in guinea-pig myocardium. AB - The mechanism by which noradrenaline stimulates ouabain-sensitive rubidium uptake in guinea-pig myocardial tissue has been studied. The stimulatory action of low concentrations of noradrenaline was reversed by high doses of propranolol (10(-5) mol/l) in atrial tissue, but was not reversed by alpha- or beta-, or combined alpha- and beta- adrenoceptor antagonists in ventricular tissue. Rubidium uptake was also found to increase with increasing extracellular potassium concentration [( K]o). The percentage values of stimulation by noradrenaline decreased with increasing [K]o. Noradrenaline had no effect on the rate of ATP splitting by an isolated membrane preparation of Na,K-ATPase. It is proposed that noradrenaline stimulates active cation transport by either (a) an effect secondary to increased passive efflux of K ions, or (b) an action at a novel adrenergic receptor, distinct from the ATPase enzyme itself. PMID- 2998693 TI - [Collateral effects of an anti-arrhythmic agent: amiodarone]. PMID- 2998694 TI - In vitro antimicrobial activity of cefoperazone-sulbactam combinations against 554 clinical isolates including a review and beta-lactamase studies. AB - Cefoperazone was tested against 554 clinical isolates alone and with sulbactam in three combinations. The addition of sulbactam in low concentrations (less than or equal to 4 micrograms/ml) improved the spectrum of cefoperazone principally against gram-negative bacilli such as Acinetobacter species, some Pseudomonas species, and beta-lactamase-positive Enterobacteriaceae. Nearly all of the spectrum increase was achieved at a sulbactam level of less than or equal to 2 micrograms/ml. Sulbactam was found to be an effective antimicrobial agent against Acinetobacter species (MIC50, 1.0 microgram/ml), Pseudomonas acidovorans (MIC50, 2.0 micrograms/ml), Neisseria gonorrhoeae (MIC50, less than or equal to 0.5 microgram/ml), and N. meningitidis (MIC50, less than or equal to 0.5 microgram/ml). Sulbactam had a higher affinity and binding constant for the plasmid-mediated beta-lactamases such as TEM-1 and TEM-2 compared to cefoperazone (greater than or equal to 10-fold difference). This finding was important as cefoperazone can be hydrolyzed at a moderate rate by the highly efficient TEM enzymes (less than 2% of clinical Escherichia coli isolates). Sulbactam increased the susceptibility (less than or equal to 16 micrograms/ml) of 220 isolates of Enterobacteriaceae to cefoperazone from 88.6 to 96.3% when 4.0 micrograms/ml of sulbactam was added. The cefoperazone antimicrobial activity was also increased against the nonenteric bacilli from a 69.5 to a 87.4% total inhibition. MICs among cefoperazone-susceptible gram-negative and gram-positive strains were routinely decreased 2- to 32-fold, as calculated from MIC90 results. Therefore, sulbactam should predictably increase the antimicrobial spectrum and clinical effectiveness of cefoperazone against nosocomial and other pathogens such as the plasmid-containing enteric bacilli, Bacteroides species and Acinetobacter species, and possibly provide the opportunity to reduce dosage schedules for infecting species already susceptible to cefoperazone alone. PMID- 2998695 TI - Serum IgA anti-hepatitis A virus as detected by enzyme-linked immunosorbent assay. Diagnostic significance in patients with acute and protracted hepatitis A. AB - A capture enzyme-linked immunosorbent assay for the detection of serum IgA against hepatitis A virus has been developed. The test was highly specific, and the time course of detectability of IgA anti-HAV was longer than six months in 42/42 patients with acute or protracted hepatitis A followed prospectively, but shorter than two years in 14/14 patients tested 21-24 months after diagnosis of acute hepatitis A. IgM anti-HAV were at detectable levels in only 1/42 cases tested six months after the clinical onset. The detection of serum IgA anti-HAV is a simple and specific method to differentiate protracted cases of hepatitis A from non-A, non-B hepatitis. PMID- 2998696 TI - [Diagnostic problems posed by respiratory infections of dogs]. AB - The following viruses as well as bacteria and mycoplasma have been isolated from dogs with contagious respiratory disease: canine distemper virus; Canine adenoviruses (type 1 and 2); Parainfluenza type 2 (SV5); Reovirus type 1; Canine Herpesvirus; Bordetella bronchiseptica, Streptococcus, Pasteurella, Staphylococcus and Mycoplasma. The occurrence of these agents can be in direct relationship with: the evolution of a systemic disease; respiratory disorders being a regular or inconsistant symptom of this disease; the evolution of a disease restricted to the respiratory tract; the tropism of the bacterial or viral agent is exclusively respiratory; secondary bacterial complications to a primary viral infection; saprophyte state or latency without pathologic significance. These various infectious agents are implicated alone or in mixed infections and the wide variety of clinical symptoms don't allow to precise a clinical diagnosis. We will try to bring some bases allowing, by the help of laboratory an etiologic diagnosis. This diagnosis is essential for providing an efficient prevention. We will approach some parameters which we have been confronted with as regards Canine Distemper and Canine Adenovirosis. Our purpose is, through these examples of the canine pathology, to confirm and complete some other similar situations which can appear in other animal species. PMID- 2998697 TI - Diagnosis and prophylaxis of infectious bovine rhinotracheitis: the role of virus latency. AB - Efficient methods of diagnosis and prophylaxis of infectious bovine rhinotracheitis must consider the concept of latency of the etiological agent, infectious bovine rhinotracheitis virus (Bovine herpesvirus 1; BHV 1). The identification of BHV 1 in nasal mucus samples or a rise in specific antibodies have to be cautiously interpreted, because they can signify either a primary infection or a reexcretion of the virus after reactivation. The isolated virus can also either be a vaccine or a virulent strain. Another aspect of BHV 1 infection diagnosis is the detection of latent carriers, which are able to transmit the virus to uninfected animals; delayed hypersensitivity test seems to be a good candidate. The classical methods of prophylaxis protect the animal against the disease, but they should also impede the reexcretion of virulent strains by latent carriers. Since, in several countries, attenuated viruses are used as vaccines, a special emphasis has to be laid on the persistence of these vaccine viruses in a latent form in the bovine population. PMID- 2998698 TI - Single photon emission computed tomography. AB - Single photon emission computed tomography (SPECT) is becoming an increasingly important part of routine clinical nuclear medicine. By providing tomographic reconstructions in multiple planes through the patient, SPECT expands the clinical applications in nuclear medicine as well as providing better contrast, edge definition and separation of target from background activities. Imaging techniques have been developed for the evaluation of regional cerebral blood flow using radiolabeled amines. Thus cerebral functional imaging can be used in the diagnosis of acute cerebral infarction, cerebral vascular disease, dementia and epilepsy. SPECT plays a complementary role in the evaluation of coronary artery disease, particularly when it is coupled with thallium-201 and exercise testing. SPECT extends our diagnostic capabilities in additional areas, such as liver and bone scintigraphy as well as tumor imaging with gallium-67. PMID- 2998700 TI - Calcified mucinous adenocarcinoma of the stomach--the CT appearances. AB - The CT appearances of calcified mucinous adenocarcinoma of the stomach have not been previously reported. This is a rare tumour with a high propensity to calcify, the calcifications typically are small and spiculated. A case is described where the condition was suspected in the plain films of the abdomen and confirmed by CT examination of the stomach. PMID- 2998699 TI - Recognition of lumbar disc disease with magnetic resonance imaging. AB - Ten normal adult volunteers, 75 patients with low back pain and/or lumbar radiculopathy, 16 patients following chymopapain treatment, 14 patients with recurrent symptoms following disc surgery, and two patients with distal cord compression were scanned on Fonar 3000 permanent magnet scanner. Of all the patients 98 had additional computed tomography scans (CT) of the lumbar spine and 82 had myelography. Lumbar magnetic resonance imaging (MRI) and CT scans were both diagnostic in cases of herniated and extruded discs. MRI scan showed more information concerning the degenerative state of the intervertebral discs. It was relatively more accurate in detecting, small bulging and herniated discs without ruptured anulus and the relation of the migrated fragments of extruded discs to both the back of the vertebrae and the thecal sac. Moreover, lumbar MRI matched the clinical response of disc disease to chymopapain treatment more than lumbar CT scan. In addition, the MRI studies differentiated more accurately postoperative epidural fibrotic changes from recurrent herniated and/or extruded disc and detected distal spinal cord abnormalities. CT scan easily detected laterally herniated lumbar discs. Myelography was the diagnostic study in cases of arachnoiditis. PMID- 2998701 TI - Update: evaluation of human T-lymphotropic virus. Type III/lymphadenopathy associated virus infection in health-care personnel--United States. Centers for Disease Control. PMID- 2998702 TI - Age-related changes of calpain II and alpha-crystallin in the lens of hereditary cataract (Nakano) mouse. AB - The age-related changes of calpain II (high-Ca2+-requiring form of Ca2+-dependent cysteine proteinase; EC 3.4.22.17) and alpha-crystallin in the lens of hereditary cataract (Nakano; cac/cac) mouse were studied. Before the onset of the cataract formation, i.e., at the end of the 2nd week after birth, the calpain activity in Nakano mice was as high as that in the control ICR mice, but it decreased rapidly as the cataract progressed to completion during the 4th and the 12th week. Marked degradation of lens proteins ensued between the 2nd and the 4th weeks, and one of these proteins was identified, using monospecific antibodies, as B chain of alpha crystallin. A chain of alpha-crystallin was not degraded in vivo, in contrast to its known susceptibility to calpain in vitro. The present data suggest that in Nakano mice, calpain may be involved in the onset or early stage of the cataract formation. PMID- 2998703 TI - Hyperpigmentation, vitiligo, and Addison's disease. AB - Addison's disease is an uncommon disorder whose dermatologic manifestations range from vitiligo to hyperpigmentation. The association of adrenal autoantibodies and vitiligo has made the latter a possible cutaneous marker for an autoimmune cause. The other cutaneous marker, hyperpigmentation, is now more clearly understood on the basis of a prohormone common to both adrenocorticotrophic hormone (ACTH) and melanocyte-stimulating hormone (MSH). PMID- 2998704 TI - Cushing's syndrome due to ectopic ACTH production: cutaneous manifestations. AB - Ectopic production of adrenocorticotrophic hormone (ACTH) usually results in hypokalemic myopathy but is occasionally associated with florid Cushing's syndrome. We present a patient with Cushing's syndrome associated with a small cell undifferentiated lung carcinoma. We describe the skin changes observed in this patient and review the cutaneous manifestation of Cushing's syndrome, and present current knowledge of the effects of glucocorticoids on the skin. PMID- 2998706 TI - Symptomatic solitary granular cell tumor of the trachea. AB - The granular cell tumor is a neoplasm that has generated considerable controversy. A rare case of solitary granular cell tumor located in the cervical trachea is described with a new mode of therapy using the carbon dioxide laser. PMID- 2998707 TI - [Value and limitations of computerized axial tomography in the diagnostic study of synovial sarcoma (study of l3 case reports)]. PMID- 2998708 TI - [A statistical study of 34 patients operated on for esophageal achalasia in the Ivory Coast]. PMID- 2998705 TI - Quantitative cytochemical differences between young and old patients with lung cancer. AB - In order to study differences in the tumor growth and frequency of metastases between younger and older patients with lung cancer, we investigated the nuclear DNA and nuclear protein according to age by means of cytophotometry after combined staining with Feulgen and Naphthol Yellow S. On the basis of Feulgen Naphthol Yellow S staining method, 13 patients less than 40 years old and 16 patients older than 70 years were investigated. Our results showed no significant difference in nuclear DNA contents between young and old patients, but there were significantly higher nuclear protein contents (p less than 0.05) and nuclear protein to nuclear DNA (NP/DNA) ratios (p less than 0.001) in young patients than in old patients. This suggests that young patients may have higher tumor proliferation (high nuclear protein contents and NP/DNA ratios). The lower nuclear protein content and NP/DNA ratio of older cases is in keeping with the general phenomenon of slower tumor growth and less frequent metastases in such cases. PMID- 2998710 TI - Progress in chronophysiology of peptide hormones with emphasis on clinical application of analogues. AB - It is now established that peptide hormones are among the most versatile substances involved in intercellular communications. By substitution of one or more natural aminoacids, numerous peptide analogues have been synthesized with increased duration of action and potency with respect to endogenous hormones. Most of these effects are programmable as a function of dose and timing of administration. Not surprisingly, the chronobiological use of hormone-related peptides may represent a valuable tool providing new approaches to diagnosis and therapy of different disease states. One pertinent example of the applicability of peptide analogues refers to a new short chain ACTH analogue (ACTH 1-17; Synchrodyn) that has been proved useful to reset or preset the rhythmic ordering of a number of functions and to provide, furthermore, information about specific pathogenetic mechanisms and/or subtle alterations of the adrenal function. A large body of evidence indicates that the same concepts apply to most actions of the gonadotropin-releasing hormone (LH-RH) related analogues. The efficacy of the so-called low-dose pulsatile LH-RH therapy in the management of hypogonadotropic hypogonadism and of some forms of infertility seems now established. The use of potent analogues is providing new approaches to the heretofore unsuccessful therapies in a number of disease conditions. In this light, chronoendocrinology and biotechnology have to be mutually supporting. Novel clinical applications are expected from the emerging use of portable instrumentation designed to record endogenous data and/or to deliver drugs according to a temporal program. PMID- 2998712 TI - Synthesis of collagen: chemical regulation of post-translational events. AB - Collagen biosynthesis involves many unique post-translational events. Inhibition of some of these will lead either to decreased formation of the extracellular collagen fibres or to an accumulation of fibres with altered functional properties. The events that would seem most suitable targets for chemical regulation are triple helix formation, the cleavage of propeptides from the procollagen molecules and cross-link formation. Attempts have recently been made to develop inhibitors of prolyl 4-hydroxylase in particular, as inhibition of this enzyme will prevent triple helix formation and thus lead to a non-functional protein. Prolyl 4-hydroxylase is inhibited competitively with respect to ferrous ion by several bivalent cations, especially zinc, with respect to 2-oxoglutarate by pyridine 2,5-dicarboxylate, pyridine 2,4-dicarboxylate, 3,4-dihydroxybenzoate and many related compounds, with respect to oxygen by superoxide dismutase-active copper chelates and with respect to the peptide substrate by a number of peptides. Triple helix formation can also be inhibited by administering certain proline analogues such as cis-4-hydroxyproline and L-azetidine-2-carboxylic acid, which are incorporated into proteins in place of proline. Only preliminary data are available on the possibilities for using any of these substances to inhibit collagen accumulation in fibrotic processes. PMID- 2998709 TI - Organization of a repetitive human 1.8 kb KpnI sequence localized in the heterochromatin of chromosome 15. AB - We have isolated a repetitive 1.8 kb KpnI DNA sequence which is amplified in the homogeneously staining regions of a human melanoma cell line. Under low stringency conditions this sequence (D15Z1) hybridized in situ to the centromeric heterochromatin of chromosomes 1, 9, 15p, 16, and distal Yq as well as to the short arms of the other acrocentric chromosomes. Under conditions of high stringency, labelling was predominantly on the short arm of chromosome 15. D15Z1 was shown to be present at approximately 3,000 copies per haploid genome and organized in long tandem arrays showing restriction site heterogeneity. Sequences homologous to D15Z1 were highly enriched in the less dense shoulder region of a Ag+-Cs2SO4 gradient. Analysis of D15Z1 indicated that this sequence is composed of tandemly arranged imperfect repeats of the consensus 5' AATGG 3' similar to previously identified satellite III sequences. Digestion of D15Z1 with HinfI resulted in a series of restriction fragments making up a subset of the HinfI ladder components of satellites III and IV. These data suggest that D15Z1 represents a chromosome 15 specific domain of human satellites III or IV and that it makes up the major fraction of the heterochromatin of this chromosome. Possible relationships between this sequence and the cytochemical staining properties of human chromosomes with distamycin A/DAPI, D280/170, and antiserum to 5-methylcytosine are discussed. PMID- 2998711 TI - Structural and functional studies on the interstitial collagen genes. AB - An understanding of the molecular mechanisms which control expression of the type I and III collagen genes may provide a rational basis for the design of more effective therapeutic approaches to fibrotic diseases. The structure of the interstitial collagen genes is reviewed and potential sites which could control their expression are examined. One approach to the study of the regulation of these genes consists in DNA-mediated gene transfection experiments and is discussed in this paper. PMID- 2998713 TI - The turnover and degradation of collagen. AB - The interstitial collagens are degraded predominantly extracellularly, by specific collagenases (metalloproteinases) capable of cleaving the helical region across the three chains at a similar locus, solubilizing the cleaved products from the fibril. Other neutral proteinases may also function in this role by cleaving near cross-links in the fibril. Collagen type, molecular aggregation and small changes in temperature all markedly affect rates of collagenolysis in the fibril. Regulation of collagenolysis is also modulated at the levels of (1) cellular production of latent collagenase (procollagenase), (2) activation of latent collagenase, and (3) production of collagenase inhibitors. Fibroblastic cells and certain macrophages are probably the predominant sources of collagenases in inflammation; an enzyme in polymorphonuclear leucocytes (neutrophils) is distinct from the tissue enzyme. Molecules such as mononuclear cell factor (MCF), homologous with interleukin 1, which augment cellular collagenase production in inflammation, are derived from monocytes. The mechanisms of augmented collagenase production involve new protein synthesis and, if this augmentation is analogous to that produced by urate crystals, it is probably associated with increased levels of procollagenase mRNA. MCF production is itself controlled by products of lymphocytes as well as by interactions of monocytes with the Fc portion of immunoglobulins and components of the extracellular matrix. Activation of latent (pro)collagenase probably occurs in vivo through the action of neutral proteinases such as plasmin (through plasminogen activator). These effects may be indirect and exerted through proteolytic activation of a procollagenase activator. Tissue inhibitors act to regulate the active collagenase. PMID- 2998714 TI - Cell kinetic studies of in situ human brain tumors with bromodeoxyuridine. AB - At the time of surgery, 18 patients with various brain tumors were given a 1-h i.v. infusion of bromodeoxyuridine (BrdUrd), 150-200 mg/m2. At an infusion rate of 200 mg/m2/h, serum BrdUrd levels of 8 microM were achieved. After the infusion, tumor tissue was obtained and divided into two portions. One portion was fixed in 70% ethanol, embedded in paraffin, and sectioned; the sections were deparaffinized, denatured with 2 N HCl, and reacted with monoclonal antibodies against BrdUrd (anti-BrdUrd MAb). BrdUrd-labeled nuclei were demonstrated satisfactorily by an indirect peroxidase method. The other portion was dissociated into single cells with a DNase enzyme cocktail and reacted with FITC conjugated anti-BrdUrd MAb to determine the percentage of BrdUrd-labeled cells or with chromomycin A3 for DNA analysis. The single-cell suspensions were analyzed by flow cytometry. The fraction of S-phase cells in the tissue sections was similar to both the percentage of BrdUrd-labeled nuclei and the S-phase fraction determined by flow cytometric analysis. The results obtained with BrdUrd-labeled nuclei were similar to those obtained from previous autoradiographic studies of various brain tumors exposed to a pulse of 3H-thymidine. Since BrdUrd is not radioactive and is nontoxic at the dosage used, these techniques, together with the histopathological diagnosis, may help to predict the biological malignancy of individual tumors. PMID- 2998715 TI - Liberation of hydrogen from gastric acid following administration of oral magnesium. AB - We are in the process of developing a noninvasive test for gastric acid secretion based on the reaction of orally administered magnesium metal with gastric acid: Mg + 2HCl in equilibrium with MgCl2 + H2. We hypothesized that the hydrogen gas thus evolved could be detected in exhaled air and belches and that the amount of hydrogen released could be related to the amount of acid in the stomach. To validate this hypothesis, we gave magnesium to two groups of young adult volunteers following either betazole stimulation or cimetidine inhibition of acid secretion. In group I we gave subcutaneous betazole and gave magnesium in doses from 10 to 200 mg. In group II we gave oral betazole and used a constant dose of 150 mg of magnesium. In both groups we consistently detected significant increases in breath and belch hydrogen following magnesium in the betazole stimulated volunteers. This response was blocked by cimetidine. The magnitude of the response was related to the magnesium dose, with 150 mg appearing to induce a maximum response. Administration of oral magnesium up to 200 mg was not associated with any untoward effects. We conclude that magnesium led to the release of hydrogen gas in vivo and that the quantity of hydrogen gas recovered was related to the amount of gastric acid. With further development, this principle might be used to develop a simple noninvasive test for gastric acid secretion. PMID- 2998717 TI - [3'-hydroxy-T-2 toxin--a new Fusarium sporotrichiella mycotoxin]. PMID- 2998718 TI - Variation in prophylactic antibiotic use in pediatric orthopedic surgery. AB - Fifty-two pediatric patients undergoing orthopedic surgery were evaluated to determine the pattern of prophylactic antibiotic use. Of the 50 patients receiving antibiotics, 43 patients were given the antibiotic prior to surgery; 38 patients received the drug within two hours prior to surgery. All patients received the postoperative antibiotic for a duration ranging from 2 to 14 days. Thirty-one patients received the postoperative antibiotic for longer than 48 hours without documentation of infection. Cephapirin and/or cefazolin were the most common antibiotics used. Due to fixed postoperative doses (0.5-2 g), the dose/kg/d exceeded the maximum recommended therapeutic doses in one-third of the patients. The results of this study illustrate that the use of prophylactic antibiotics in pediatric orthopedic surgery can be improved to maximize benefits and minimize costs and the potential for adverse effects. Until data from controlled studies become available, it seems appropriate to follow the Centers for Disease Control guidelines in establishing hospital policy for surgical prophylaxis. PMID- 2998719 TI - ACE inhibitors: enalapril and captopril compared. PMID- 2998720 TI - Anti-D immunoglobulin prophylaxis--are we doing enough? PMID- 2998721 TI - [Diagnosis of Cushing's syndrome]. PMID- 2998716 TI - Rodent models for carcinoma of the colon. PMID- 2998722 TI - [Therapy of Cushing's syndrome]. PMID- 2998724 TI - [Method and program for evaluating and correcting the parameters characterizing hormone-receptor interaction]. PMID- 2998725 TI - [Cyclic adenosine monophosphate content in the lymphocytes of cancer patients]. AB - Examinations have been carried out on 83 cancer patients (aged 34-72), 24 patients with atherosclerosis (aged 38-68) and 34 healthy persons (aged 20-69). The cAMP concentration decrease with age in lymphocytes of healthy persons is confirmed. This index in patients with lung carcinoma, breast carcinoma in remission and in patients with atherosclerosis was identical with that of healthy persons, but in patients with breast and corpus uteri carcinomas without metastases it was higher than in healthy persons of the similar age. The amount of cAMP in lymphocytes is compared with the magnitude of the blast transformation and with the amount of cholesterol in blood and lymphocytes in examined persons. The cAMP level in lymphocytes is observed to increase in the process of hyperlipidemia disappearance in cancer patients and in those with atherosclerosis. PMID- 2998726 TI - [Generators of somatosensory evoked potentials following leg nerve stimulation]. AB - Stimulation of the tibial nerve results in complex spinal potentials evoked from the lumbosacral segments of the spinal cord. These potentials suggest the interaction of different generators. Characteristically a triphasic potential can be recorded from L1. It is composed out of an afferent volley in the dorsal roots (R-response) a synaptically strengthened S-response (N21) and a descending reflexly evoked ventral root discharge (A-wave). Above cervical segments of the spinal cord the evoked potentials are probably composed of the afferent volley in the posterior columns (N24), the activity of the nucleus gracilis (N27) and the volley in the medial lemniscus (P30). The primary cortical response P40 is regarded as the specific activity of the somatosensory foot area. PMID- 2998723 TI - Meptazinol. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic efficacy. AB - Meptazinol is a new opioid-type analgesic with mixed agonist/antagonist properties. It may be given orally, intravenously or intramuscularly. In studies in patients with moderate to severe pain of various aetiologies, usually following surgery or in obstetrics, the characteristics of analgesia with meptazinol were comparable to those seen with equianalgesic doses of pentazocine, pethidine or a combination of dextropropoxyphene and paracetamol. Preoperative use and use as a component of anaesthesia require further investigation before conclusions may be drawn on its effectiveness in these areas. Onset of action, recorded in a few studies, was faster than that with the other analgesics but duration was shorter than that of morphine, buprenorphine and pentazocine. Only a small number of patients with chronic pain have received long term therapy with meptazinol; in such patients there was no need for increased doses as treatment progressed. Respiratory depression has only been observed in patients receiving meptazinol as a premedication or while undergoing anaesthesia. Similarly any haemodynamic changes have been limited to preoperative patients or patients undergoing anaesthesia. Like other agonist/antagonist analgesic drugs, the abuse potential of meptazinol seems relatively low, but only wider clinical use for longer periods can establish this with certainty. The most commonly reported side effects have been gastrointestinal in nature, and although the incidence of central nervous system side effects has been relatively low, drowsiness and dizziness have caused occasional problems. Thus, meptazinol is a relatively potent but safe addition to the analgesics available for treatment of the patient with moderate to severe pain. PMID- 2998727 TI - [Generators of somatosensory evoked potentials following stimulation of nerves of the arm]. AB - The cervical evoked potentials recorded above the 7th spinous process represent activity from the dorsal roots (N 11a) and the dorsal horn (N 13a), respectively. The upper neck responses are attributed to ascending activity in the dorsal column (N 11b) and the activation of the cuneate nucleus (N 13b). Scalp-leads from the contralateral sensory hand area using an Fz-reference consistently exhibit a sequence of components with successively more rostrally located generator sites: P 15 in or near the thalamus, N 20/P 25 in the primary sensory cortex and the following components in the parietal association fields (area 5 and 7). PMID- 2998728 TI - [Methodologic effects in somatosensory evoked potential diagnosis (stimulus frequency, filter, stimulation and recording site)]. AB - The importance of the parameters stimulation rate, filter, stimulation- and recording place is presented; thus, a reduction in the low-pass filter frequency produces an increase in latency and an increase in the high-filter a decrease in latency of all SEP-components. A proper choice of the filters leads to a selection of the relevant signals; in a topic classification of the SEP generator the referential electrode must in any case be taken into consideration. PMID- 2998729 TI - [Effect of body size, arm length, sex and temperature on somatosensory evoked potential latency]. AB - Beside the technical factors different biological factors contribute to the variability of the somatosensoric evoked potentials. In 30 volunteers the dependence of the latency of the spinal components N9, N13 (recorded at C7), of the cortical component N20 and of the interpeak interval N13-N20 from the body size and length of the arm was investigated. A significant dependence at the 1% level (p less than 0.001) was found for the latencies of N9, N13 and N20, whereas the interpeak interval N13-N20 was significant at the 5% level (p less than 0.05). Similarly in 20 further volunteers a significant dependence of the components N1, P1, N2, P2 and N3 from the body size at the 1% (p less than 0.001) after distal stimulation of the tibial nerve was observed. Using a covariance analysis it could be shown, that even after a body size correction, in females shorter latencies occur than in males. Furtheron we studied the effect of the temperature on the SSEP-latencies, by heating the upper and lower extremity stepwise from 31 degrees C to 37 degrees C using an infrared (DISA-Regler-System) lamp. As no significant dependence could be observed, we believe that the effect of temperature on latencies is so small, that in usual conditions no correction is needed. On the contrary a correction with body size or arm length is necessary, to avoid wrong positive or negative results. PMID- 2998730 TI - Midmotoraxonal reexcitation in human peripheral nerve. PMID- 2998731 TI - The effects of chronic prednisone administration on intestinal receptors for 1,25 dihydroxyvitamin D3 in the dog. AB - Glucocorticoid inhibits intestinal calcium absorption. To further explore the mechanism of this inhibition, we studied dogs during the administration of oral prednisone (1.2-1.5 mg/kg X day) for 20 to 28 weeks in comparison to untreated dogs. Prednisone administration had no effect on serum 25-hydroxyvitamin D concentrations, but was accompanied by a fall in serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations from 87 +/- 20 pM (control) to 62 +/- 28 pM (prednisone-treated; P less than 0.01). Cytosol prepared from the duodenal, jejunal, and ileal mucosa of control dogs was found to contain a specific 3.2S [3H]1,25-(OH)2D3 binder analogous to the binder that has been observed in the intestine of other species and in other tissues. The apparent concentration of this binder decreased progressively from duodenum to ileum. Prednisone administration increased the apparent duodenal concentration of the binder from 170 +/- 91 (control) to 363 +/- 124 fmol/mg protein (prednisone-treated; P less than 0.025). The intestinal content of calcium-binding protein also declined progressively from the duodenum to the ileum, but was not affected by prednisone administration. These data suggest that events other than alterations in intestinal 1,25-(OH)2D3 receptors must mediate the inhibition of intestinal calcium absorption during chronic glucocorticoid administration. PMID- 2998732 TI - Normal and recombinant human growth hormone administered by constant infusion feminize catechol estrogen formation by rat liver microsomes. AB - Human GH or recombinant DNA-derived human GH administered to normal mature male rats by constant infusion decreased hepatic 2-hydroxylation of estradiol to female levels, as measured by 3H2O release and isolation of the catechol estrogen product. PRL had no effect under the same conditions. The maximum response to GH was attained at an infusion rate of 0.02 IU/h . kg after 5-7 days, and a significant change was observed within 1-2 days. The effect of GH was primarily on hydroxylation of C-2 of the estrogen, as demonstrated by comparative studies with estradiol labeled with 3H at C-4 or C-6,7, and appeared to be mediated by the cytochrome P-450 system of the liver microsomes. Ascorbic acid at 1 mM did not affect 2-hydroxylation significantly while protecting the catechol estrogen produced from further oxidation. The results indicate that GH has the potential to regulate estrogen metabolism in the liver and provide evidence for another component in the hypothalamic-pituitary-liver axis. PMID- 2998733 TI - Ontogeny of receptors for insulin-like peptides in chick embryo tissues: early dominance of insulin-like growth factor over insulin receptors in brain. AB - Recently, we confirmed early data showing deleterious effects of exogenous insulin on chick embryos at 2 days of development, although insulin receptors were not clearly demonstrable until days 3-4. Now we report that insulin-like growth factor (IGF) receptors are present in whole embryos on day 2. The developmental patterns of [125I]IGF-I and [125I]IGF-II binding to brain were similar, and IGF-I showed approximately a 2-fold higher binding than IGF-II; there was a sharp increase from days 3 to 6, and a subsequent gradual fall during the second and third weeks of ontogeny. Competitive binding experiments with unlabeled analogs suggested that both labeled IGFs were binding to type I IGF receptors, and insulin interacted with them. The temperature and pH dependence were relatively higher than those for some other known IGF receptors. We have previously reported that [125I]insulin binding to brain is barely detectable on day 3 and shows a progressive rise throughout the rest of embryonic life. The pattern of IGF and insulin receptors appears to be organ specific, since nonneural tissues such as heart, liver, and limb buds showed different binding profiles in ontogeny. We conclude from these data that IGF receptors develop in chick embryo brain before insulin receptors and probably can mediate effects of IGF and insulin at early stages of embryogenesis. PMID- 2998734 TI - Adenosine inhibits prolactin and growth hormone secretion in a clonal pituitary cell line. AB - Although purine nucleosides have been shown to regulate the secretion of several peptide and steroid hormones, effects on pituitary hormone release have not been reported. We show here that in the clonal GH4C1 pituitary cell line maximal concentrations of adenosine (greater than or equal to 50 microM) inhibited PRL and GH secretion by 40%. Adenosine deaminase abolished the inhibitory effect of adenosine but not that of SRIF or (-)N6(R-2-phenylisopropyl)adenosine (PIA), a nonhydrolyzable adenosine analog. Furthermore, this enzyme increased basal secretion by 50%, and analysis of the incubation medium by HPLC demonstrated that the cells secreted biologically effective concentrations of adenosine. These results indicate that adenosine produced in culture tonically inhibits hormone release. In other target cells, adenosine inhibition is mediated by two types of binding sites: an extracellular Ri-site requiring an intact ribose moiety or an intracellular P-site requiring an intact purine ring. Four lines of evidence indicate that in GH4C1 cells, adenosine acts at an Ri-site. PIA, an Ri-site specific agonist, was a potent inhibitor of hormone release (ED50 = 30 nM). Theophylline, an Ri-site antagonist, competitively inhibited the action of PIA (Ki = 2.4 microM). 3) 2'5'-Dideoxyadenosine, a P-site-specific agonist, did not inhibit PRL release even at a concentration of 1 mM. 4) Dipyridamole, an adenosine uptake inhibitor, did not reduce adenosine inhibition. In addition to its effect on basal secretion, PIA inhibited stimulation of hormone release by vasoactive intestinal peptide and TRH. PIA also reduced vasoactive intestinal peptide-stimulated cAMP accumulation by 75%, consistent with its action to inhibit adenylate cyclase via Ri receptors in other targets. Since PIA inhibition of PRL release and cAMP accumulation was not additive with the effects of SRIF and carbamyl choline, these inhibitors may act via a common rate-limiting step. Our results demonstrate that adenosine activates an Ri-type of adenosine receptor in GH4C1 cells and that the production of adenosine under normal culture conditions causes autocrine inhibition of secretion. PMID- 2998735 TI - Catechol estrogen formation by pig blastocysts during the preimplantation period: biochemical characterization of estrogen-2/4-hydroxylase and correlation with aromatase activity. AB - Formation of the catechol estrogens 2- and 4-hydroxyestradiol (2-OHE2 and 4-OHE2) from estradiol by pig blastocysts was studied using a direct product isolation assay for estrogen-2/4-hydroxylase (E-2/4-H). Blastocyst E-2/4-H activity was characterized biochemically using homogenates of blastocysts obtained on day 12 of pregnancy. This information was used to establish appropriate incubation conditions for the assay of E-2/4-H activity in blastocysts during the preimplantation period. Catechol estrogen formation was linear with time for up to 30 min and with blastocyst protein concentrations of up to 100 micrograms in a reaction volume of 150 microliters. The E-2/4-H activity of pig blastocysts was maximal at pH 7.9 and was not affected by the nonionic detergent Tween-80. The E 2/4-H activity was dependent on nicotinamide cofactor, with NADPH preferred over NADH for 2-OHE2 formation. The predominant catechol estrogen formed was 2-OHE2: maximum velocities (Vmax) for the formation of 2- and 4-OHE2 were 1570 and 174 pmol/mg protein . 30 min, respectively. The apparent Km values with respect to estradiol for 2- and 4-OHE2 were similar, 4.39 and 4.27 microM, respectively. Blastocyst E-2/4-H activity was detectable in one of two samples of blastocysts from day 10 of pregnancy (4.4 pmol 2-OHE2/mg protein . 30 min), increased to a maximum on days 12 and 13 (628 +/- 153 and 516 +/- 227 pmol 2-OHE2/mg protein . 30 min, respectively), and declined by day 14 (63.2 +/- 32.9 pmol 2-OHE2/mg protein . 30 min). The activity of E-2/4-H was positively correlated with aromatase activity assayed in the same tissue samples from days 10-14 of pregnancy. The surge in E-2/4-H activity coincides with several of the critical events that occur near the time of implantation. Our findings are consistent with the hypothesis that catechol estrogens mediate some of the actions of estrogens in early pregnancy in the pig. PMID- 2998736 TI - Light and agonist alter vasoactive intestinal peptide binding and intracellular accumulation of adenosine 3',5'-monophosphate in the rat pineal gland. AB - We examined the effects of environmental light and prior treatment with an agonist on vasoactive intestinal polypeptide (VIP) binding and VIP stimulation of cAMP accumulation in the rat pineal gland. VIP binding to pinealocytes and cAMP accumulation in response to VIP were significantly increased in animals kept exposed to constant light compared to those in animals experiencing a dark night before the experiments. Scatchard analysis of [125I]VIP binding indicated the presence of two classes of binding sites: high affinity, low capacity sites and low affinity, high capacity sites. The increased VIP binding to pinealocytes in rats maintained in constant light was attributed to an increase in the number of available VIP receptors at both high and low affinity sites. The affinity of VIP binding to cells was not affected by exposure of the animals to light. VIP stimulation of cAMP accumulation was not inhibited by d,l-propranolol. Prior treatment of pinealocytes with VIP decreased [125I]VIP binding by reducing the number of receptors and significantly inhibited subsequent VIP stimulation of cAMP accumulation. Prior treatment with norepinephrine did not alter the number of VIP receptors. Our results strongly suggest that VIP is a neuromodulator of pineal function. PMID- 2998737 TI - The early ontogenesis of thyroid hormone receptor in the rat fetus. AB - We have determined the concentration of thyroid hormone receptor binding sites in nuclear extracts derived from rat fetal organs throughout gestation and the postnatal period. Before day 14 of gestation nuclear extracts were obtained from whole fetuses. No receptor binding activity could be detected at day 12 of gestational age, and small amounts were detected at day 13 (maximum binding capacity less than 50 fmol/mg DNA). The receptor could be measured in pools of individual organs from day 14 (brain) or from day 16 (heart, liver, and lung) onwards. The order of analog binding affinity at 14 days was triiodothyroacetic acid = T3 greater than T4 greater than rT3, suggesting that at 14 days of fetal age the receptor has the same binding specificity as the receptor from mature tissues. In brain, the concentration of binding sites increased from 77 fmol/mg DNA at 14 days to 210 fmol/mg DNA at 17 days, remaining at this level until birth. Receptor concentration was identical whether the binding assays were performed on purified nuclei or nuclear extracts. There was no effect of maternofetal hypothyroidism on receptor concentration in the brain at 21 days of gestational age. Lung concentrations of receptor also remained constant during the fetal period. During the postnatal period, there was an increase in receptor concentration in brain and lung, with maximum levels at day 6. The pattern of receptor development in heart and liver was different, since its concentration increased progressively throughout the fetal and postnatal periods towards the levels found in adult rat tissues. The results suggest that the appearance of the thyroid hormone receptor coincides with that of the first fetal thyroid gland structures, but that it occurs much before thyroid function is fully established. As far as the receptor is concerned, fetal tissues have the potential to respond to thyroid hormone as early as the 13th day of gestational age. PMID- 2998739 TI - Developmental changes in the binding of follicle-stimulating hormone (FSH) to testicular preparations of mice and the effects of hypophysectomy and administration of FSH on the binding. AB - Changes with age in testicular FSH binding and the effects of hypophysectomy and administration of FSH on FSH binding were studied in mice. The binding per unit testicular weight reached a peak at 10-20 days of age and it rapidly decreased during 20-37 days. The total binding per two testes increased from 4 days of age, reaching a peak at 31 days, and decreased thereafter when the testis still continued growing. Scatchard plot analyses of the binding showed that the equilibrium constant of dissociation (Kd) was about 3 X 10(-10) M regardless of age, and the changes in FSH binding were due to changes in the number of binding sites. Hypophysectomy at 90 days of age induced a significant decrease in the testicular weight, but the concentration of FSH binding sites markedly increased 16 days after the operation. In hypophysectomized mice the total number of FSH binding sites was increased 1.8-fold as compared with intact mice. In contrast, hypophysectomy induced a decrease in the total number of LH-binding sites. Injections of 100 micrograms NIH-FSH-P-2 for 10 days to hypophysectomized mice significantly decreased the concentration and the total number of FSH binding sites as compared with those in saline-injected hypophysectomized mice. These results suggest that FSH reduces its own receptors in the testis of mice. PMID- 2998738 TI - Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. AB - Two receptor systems for corticosterone (CORT) can be distinguished in rat brain: mineralocorticoid-like or CORT receptors (CR) and glucocorticoid receptors (GR). The microdistribution and extent of occupation of each receptor population by CORT were studied. The CR system is restricted predominantly to the lateral septum and hippocampus. Within the hippocampus, the highest density occurs in the subiculum +/- CA1 cell field (144 fmol/mg protein) and the dentate gyrus (104 fmol/mg protein). Affinity of CR for CORT was very high (Kd, approximately 0.5 nM). The GR system has a more widespread distribution in the brain. The highest density for GR is in the lateral septum (195 fmol/mg protein), the dentate gyrus (133 fmol/mg protein), the nucleus tractus solitarii and central amygdala. Substantial amounts of GR are present in the paraventricular nucleus and locus coeruleus and low amounts in the raphe area and the subiculum + CA1 cell field. The affinity of GR for CORT (Kd, approximately 2.5-5 nM) was 6- to 10-fold lower than that of CR. Occupation of CR by endogenous ligand was 89.5% during morning trough levels of pituitary-adrenal activity (plasma CORT, 1.4 micrograms/100 ml). Similar levels of occupation (88.7% and 97.6%) were observed at the diurnal peak (plasma CORT, 27 micrograms/100 ml) and after 1 h of restraint stress (plasma CORT, 25 micrograms/100 ml), respectively. Furthermore, a dose of 1 microgram CORT/100 g BW, sc, resulted in 80% CORT receptor occupation, whereas GR were not occupied. For 50% occupation of GR, doses needed to be increased to 50-100 micrograms/100 g BW, and for 95% occupation, a dose of 1 mg CORT was required. The plasma CORT level at the time of half-maximal GR occupation was about 25 micrograms/100 ml, which is in the range of levels attained after stress or during the diurnal peak of pituitary-adrenal activity. Thus, CR are extensively filled (greater than 90%) with endogenous CORT under most circumstances, while GR become occupied concurrent with increasing plasma CORT concentrations due to stress or diurnal rhythm. We conclude that CORT action via CR may be involved in a tonic (permissive) influence on brain function with the septohippocampal complex as a primary target. In view of the almost complete occupation of CR by endogenous hormones, the regulation of the CORT signal via CR will, most likely, be by alterations in the number of such receptors. In contrast, CORT action via GR is involved in its feedback action on stress-activated brain mechanisms, and GR occur widely in the brain.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2998740 TI - Opiate receptors are present in the rat testis. Identification and localization in Sertoli cells. AB - We have characterized opioid binding sites in the Sertoli cells of adult and 18 day-old rat testes. Maximal specific etorphine binding was attained after 30 min at 4 C. The binding was reversible, with association and dissociation rate constants of 0.98 X 10(5) M-1 min-1 and 0.33 min-1, respectively. Scatchard analyses and saturation curves revealed a single class of high-affinity, low capacity binding sites. No opioid binding was observed in Leydig cell cultures. Exposure to opioids for 3 days caused a significant increase in [3H]etorphine specifically bound to the Sertoli cells that was completely prevented by naloxone, demonstrating opioid up-regulation of its own receptor. Chronic opioid treatment of the cultures significantly inhibited androgen-binding protein production, and this effect was prevented by naloxone. Since the circulating concentrations of endorphins (10(-12) M) are lower than the Kd of testis opiate receptors, it is conceivable that opioids of Leydig cell origin act on the specific high-affinity receptors of the Sertoli cells, and may play a role in modulating their function. PMID- 2998741 TI - Gastroduodenal cytomegalovirus infections after renal transplantation- fiberscopic observations. PMID- 2998742 TI - Activation of cAMP dependent protein kinase during surfactant release from type II pneumocytes. AB - Release of surfactant from pulmonary type II epithelial cells was stimulated by the beta-adrenergic agonist terbutaline and the diterpene forskolin. Cytosolic cyclic adenosine monophosphate (cAMP) concentrations increased significantly following exposure to terbutaline or forskolin and reached maximal levels within 5 min after treatment. Terbutaline and forskolin had a synergistic effect on cytosolic cAMP levels when added simultaneously. cAMP-dependent protein kinase activity was identified in cytosolic preparations of type II pneumocytes by phosphorylation of the peptide substrate Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) and binding of 3H-cAMP to the regulatory components of cAMP-dependent protein kinase. Type I and type II regulatory subunits of the cANP-dependent kinase were present in approximately equal concentrations in type II cell cytosol. Activation ratio of cAMP-dependent protein kinase in cultured type II cells increased significantly in the presence of terbutaline, forskolin, or terbutaline plus forskolin. Activation ratios increased from 0.45 +/- 0.03 for control cells to 0.96 +/- 0.06 for cells exposed to terbutaline (10 microM) plus forskolin (5 microM) for 20 min. Release of 3H-phosphatidylcholine was also stimulated by terbutaline and forskolin. Effects of terbutaline and forskolin on surfactant release were approximately additive. Our results demonstrated increased cytosolic cAMP levels, increased cAMP-dependent protein kinase activation ratios, and subsequent augmented surfactant release from isolated type II pneumocytes in response to terbutaline and forskolin. These data support a role for activation of cAMP-dependent protein kinase as a mediator of surfactant release and document the utility of forskolin for study of cAMP-mediated effects in isolated type II cells. PMID- 2998744 TI - Effects of ozone on phospholipid synthesis by alveolar type II cells isolated from adult rat lung. AB - Isolated alveolar type II cells were exposed to ozone by gas diffusion through the thin Teflon bottom of culture dishes. After exposure, type II cells were further incubated in the presence of labeled substrates to assess the capacity to synthesize surfactant lipids. The incorporation of [Me-14C]choline into both total and disaturated phosphatidylcholines in inhibited to 50% of the control values under conditions that result in a diffusion of 0.4 microgram O3/18 cm2 dish per 2.5 h. The incorporation rates of [1-14C]palmitate, [1-14C]acetate, D[U 14C]glucose, and [1,3-3H]glycerol into phosphatidylcholines are also lower after ozone exposure. Moreover, the synthesis of phosphatidylglycerols and phosphatidylethanolamines from these substrates is also inhibited by exposure of type II cells to ozone. These incorporation studies indicate that the effect of ozone is early in the biosynthetic pathway, probably at the step catalyzed by the enzyme glycerolphosphate acyltransferase. Determination of the activity of this enzyme after the ozone exposure shows that it is decreased, whereas the activity of lysophosphatidylcholine acyltransferase is increased. The activity of choline phosphotransferase also appears to be decreased after exposure of type II cells to ozone, although this enzyme was less susceptible than glycerolphosphate acyltransferase. Studies with the sulfhydryl reagent 5,5'-dithiobis (2 nitrobenzoic acid) indicate a positive correlation between the effect of this compound on enzyme activities in sonicated type II cells and the sensitivity of these enzymes in intact cells to ozone. This suggests that the effect of ozone on the synthesis of surfactant lipids is at least partially exerted via oxidation of the sulfhydryl groups of glycerolphosphate acyltransferase. PMID- 2998743 TI - Metabolism of leukotrienes by adult and fetal human lungs. AB - The metabolism of leukotriene (LT) A4, B4, C4, D4, and E4 was studied using both bioassay and reversed phase high performance liquid chromatography (RP-HPLC) methods. The incubation of 20,000 g supernatants of homogenates of human adult lung with LTA4 and LTC4 for various periods of time produced substances of higher biologic activity than the controls (without incubation) when measured on strips of guinea pig lung parenchyma and ileum. RP-HPLC analyses of the incubation media revealed the formation of LTB4, LTC4, LTD4, and LTE4 from LTA4 and the formation of LTD4 and LTE4 from LTC4. LTD4 was converted to LTE4 whereas LTB4 and LTE4 were not catabolized to an appreciable extent during a 2-h incubation period. Supernatants (20,000 g) of human fetal lung homogenates also contain the enzymatic activities to transform LTC4 into LTD4 and LTE4; however, LTA4 was mainly converted to LTB4 and to products of the nonenzymatic hydrolysis of LTA4 such as the delta 6-trans-LTB4, delta 6 -trans-12-epi-LTB4 and the 5,6 dihyroxyeicosatetraenoic acids; much smaller quantities of the peptidoleukotrienes were formed than in adult lung homogenates. PMID- 2998746 TI - Structure-activity relationships in the free-radical metabolism of xenobiotics. AB - Many xenobiotics, including naturally occurring compounds, drugs, and environmental agents, are metabolized both in vivo and in vitro to free-radical intermediates. The one-electron reduction of nitroaromatic compounds, quinones, and a wide variety of other chemicals is catalyzed enzymatically by a number of reductases and dehydrogenases. Structure-activity studies have shown that the cytotoxicities of nitroaromatic compounds and quinones are related to their one electron reduction potentials (E1(7)). Other factors such as oil:water partition coefficients may also be important. Xenobiotics may also be oxidized to free radicals by peroxidases. Hammett's rules apply to the one-electron oxidation of simple meta- or para-substituted phenols and amines by horseradish peroxidase, compound I. PMID- 2998745 TI - The role of structure in the disposition of halogenated aromatic xenobiotics. AB - Halogenated aromatic xenobiotics such as the chlorinated and brominated biphenyls, naphthalenes, dibenzodioxins, and dibenzofurans are widespread environmental contaminants. The number, position, and nature of the halogen atoms as well as the structure of the aromatic rings influence the disposition of these chemicals in living systems. Absorption is governed primarily by the physical properties of lipophilicity and solubility. Distribution through the blood occurs by nonspecific binding to plasma proteins and cellular components. Liver and adipose tissue are the major depots. Metabolism is a prerequisite for excretion. The highly substituted isomers tend to be resistant to metabolism. The route of excretion shifts from urine to feces with increasing size and number of halogen atoms. Although pharmacokinetic modeling has allowed some predictions to be made from one compound to another or across species, more information on metabolism is required in order to improve the ability to predict the disposition in humans of this class of toxic environmental pollutants. PMID- 2998748 TI - Receptor-dependent mechanisms of glucocorticoid and dioxin-induced cleft palate. AB - Glucocorticoids (triamcinolone) and dioxins (TCDD) are highly specific teratogens in the mouse, in that cleft palate is the major malformation observed. Glucocorticoids and TCDD both readily cross the yolk sac and placenta and appear in the developing secondary palate. Structure-activity relationships for glucocorticoid- and TCDD-induced cleft palate suggest a receptor involvement. Receptors for glucocorticoids and TCDD are present in the palate and their levels in various mouse strains are highly correlated with their sensitivity to cleft palate induction. Receptors for glucocorticoids appear to be more prevalent in the palatal mesenchymal cells whereas those for TCDD are probably located in the palatal epithelial cells. Glucocorticoids exert their teratogenic effect on the palate by inhibiting the growth of the palatal mesenchymal cells whereas TCDD alters the terminal cell differentiation of the medial palatal epithelial cells. PMID- 2998750 TI - The molecular basis of chemical toxicity. AB - Studies of structure-activity relationships and molecular mechanisms of action are fundamental to our understanding of the harmful effects of chemicals on the environment and their more direct effects on human health. It is important to identify factors that determine toxicological effects of foreign chemicals in biological systems and to assess our knowledge about chemical mechanisms of toxicity. Several fundamental mechanisms underlying toxic action are described, and the importance of studying receptor-substrate interactions is stressed. The Ah receptor is cited as an example of a protein-small molecule interaction associated with extreme acute toxicity in laboratory animals. It is recognized that reliable attempts at predictive toxicology across compound classes through structural and theoretical chemistry approaches must be based on sound knowledge about mechanisms of action at the molecular level. Such studies also contribute to the knowledge base in biomedical and physical sciences. PMID- 2998747 TI - Effects of structure on binding to the 2,3,7,8-TCDD receptor protein and AHH induction--halogenated biphenyls. AB - The quantitative structure-activity relationships (QSARs) for polychlorinated biphenyl (PCB) congeners have been determined by comparing the EC50 values for three in vitro test systems, namely, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction in rat hepatoma H-4-II-E cells and competitive binding avidities to the rat cytosolic receptor protein (using 2,3,7,8-tetrachlorodibenzo-p-dioxin as a radioligand). For several PCB congeners that are in vivo inducers of rat hepatic microsomal AHH, there was a linear correlation between the -log EC50 values for receptor and the -log EC50 values for AHH (or EROD) induction; moreover, a comparable linear relationship was observed between the -log EC50 values for AHH and EROD induction. Previous in vivo studies have shown that the most active PCB congeners 3,3',4,4'-tetra-, 3,4,4',5-tetra-, 3,3',4,4',5-penta-, and 3,3',4,4',5,5'-hexachlorobiphenyl, cause many of the biologic and toxic effects reported for the highly toxic halogenated aryl hydrocarbon, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Moreover, the monoortho-substituted homologs of the four coplanar PCBs also elicit comparable in vivo biologic and toxic responses. It was evident from the QSARs for PCBs that there was an excellent correspondence between the in vivo and in vitro potencies of the individual PCB congeners. The effects of substituents on both receptor binding and AHH/EROD induction was determined for a series of 4'-substituted (X) 2,3,4,5-tetrachlorobiphenyls (where X = H, Cl, Br, I, OH, OCH3, NO2, COCH3, F, CF3, CH3, C2H5, i-C3H7, n-C4H9 and t-C4H9). Not unexpectedly, there was a linear relationship between the -log EC50 values for AHH and EROD induction, and these results confirm that both enzymatic oxidations are catalyzed by the same cytochrome P-450 isozyme(s). The effects of substituent structure on receptor binding for 12 substituents was subjected to multiple regression analysis which correlates the relative binding affinities of the compounds with the physical chemical characteristics of the substituents. The analysis gave the following equation: log (1/EC50) = 1.53 sigma + 1.47 pi + 1.09 HB + 4.08 for n = 12, s = 0.18, r = 0.978; where n is the number of substituents, s is the standard deviation, r is the correlation coefficient, and sigma = electronegativity, pi = hydrophobicity (log P) and HB = hydrogen bonding capacity for the substituent groups.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2998751 TI - Metallic elements in crude and milled chrysotile asbestos from Quebec. AB - Crude and milled fibers from different asbestos mines in Quebec have been analyzed for their metallic content. Crude fibers present a few metal concentrations that are characteristic for each mine. Manganese and iron can be used as discriminators, zinc and cobalt are constantly present in minute quantities, and calcium is virtually absent from crude fibers. Milled fibers are enriched in metals by factors ranging from 1.3 to greater than 15. Chromium and nickel concentrations are many times higher than in crude fibers and iron content is increased by more than 100%. Total metal content in milled fibers is mainly due to nonchrysotile material. PMID- 2998752 TI - Effects of prolonged inhalation of silica and olivine dusts on immune functions in the mouse. AB - Immunologic responses were determined in Balb/c mice following intermittent silica or olivine inhalations for 150, 300, or 570 days. Animals dust-exposed for 570 days were tested immediately postexposure, while those exposed for 150 or 300 days were tested immediately or were rested for 30 or 150 days as a measure of possible recovery from effects of the dust inhalations. Silica inhalation suppressed the number of specific plaque-forming cells (PFC) in the spleen produced in response to aerosolized Escherichia coli bacteria. When tested after 570 days, silica inhalation also reduced the ability of alveolar macrophages to phagocytize Staphylococcus aureus in vitro. Olivine inhalation also suppressed splenic PFCs and alveolar macrophage phagocytosis, but to a lesser degree than silica. In animals tested after 570 days of dust exposure, it was determined that the ability to lyse allogeneic tumor cells in vitro was impaired by olivine slightly more than by silica, while antibody-dependent cell-mediated cytotoxic and mitogenic responses by splenic lymphocytes were unchanged by inhalation of either dust. The effects of increased exposure periods, and of recovery periods after exposure, were confounded by age-related immunologic changes which were present after the longer exposures. PMID- 2998749 TI - 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) as a potent and persistent thyroxine agonist: a mechanistic model for toxicity based on molecular reactivity. AB - TCDD and thyroxine have common molecular reactivity properties which enable them to present a planar face and lateral halogens in interactions with proteins. These molecular properties are consistent with the structure-toxicity relationship for TCDD and related compounds. Biological evidence is discussed including preliminary studies on the effects of TCDD exposure on tadpole growth and development which is consistent with the possible thyroxine-like activity of TCDD. The work suggests the possibility that toxicity is at least in part the expression of potent and persistent thyroid hormone activity (responses induced by TCDD which qualitatively correspond to those mediated by thyroid hormones). A mechanism for toxicity is proposed which involves receptor proteins; the planar aromatic system controls binding to cytosolic proteins and halogen substituents regulate binding to nuclear proteins. This simple model based on molecular reactivity sheds light on the diversified effects of TCDD and related compound toxicity and on certain thyroid hormone action. The model also permits predictions to be made with regard to the toxicity and thyroid hormone activity of untested compounds. In addition, the model suggests a general mechanism for hormone action based on metabolically regulated differential and cooperative protein receptor binding events in cellular compartments which can explain agonism, antagonism and potentiation within the framework of receptor occupancy theory. PMID- 2998753 TI - Studies on inositolphosphatase in rat small intestine. AB - The possibility that inositolphosphatase differs from other intestinal phosphatases was tested by comparing several enzymatic characteristics of phosphatase activities of rat intestinal homogenate acting on various specific substrates. Optimum pH and temperature, Km, Vmax, heat stability, inhibition and metal ion requirement studies suggest that inositolphosphatase differs from phytase and p-nitrophenylphosphatase. Furthermore, we found that inositolphosphatase activity was about 2 times higher in duodenum and jejunum than ileum. It sedimented (90-100%) with a high-speed particulate fraction of mucosal homogenate; 42% of the activity was separated with the brush border membrane isolated from mucosal homogenate. Partial separation by gel filtration on Sephadex G200 and chromatography on phenyl Sepharose CL 4B provided additional evidence to suggest that inositolphosphatase and phytase are different enzymes. PMID- 2998754 TI - Regulation of high density lipoprotein receptors in cultured macrophages: role of acyl-CoA:cholesterol acyltransferase. AB - The interaction of human serum high density lipoproteins (HDL) with mouse peritoneal macrophages and human blood monocytes was studied. Saturation curves for binding of apolipoprotein E-free [125I]HDL3 showed at least two components: non-specific binding and specific binding that saturated at approximately 40 micrograms HDL protein/ml. Scatchard analysis of specific binding of apo E-free [125I]-HDL3 to cultured macrophages yielded linear plots indicative of a single class of specific binding sites. Pretreatment of [125I]HDL3 with various apolipoprotein antibodies (anti apo A-I, anti apo A-II, anti apo C-II, anti apo C III and anti apo E) and preincubation of the cells with anti-idiotype antibodies against apo A-I and apo A-II prior to the HDL binding studies revealed apolipoprotein A-I as the ligand involved in specific binding of HDL. Cellular cholesterol accumulation via incubation with acetylated LDL led to an increase in HDL binding sites as well as an increase in the activity of the cytoplasmic cholesterol esterifying enzyme acyl-CoA:cholesterol acyltransferase (ACAT). Incubation of the cholesterol-loaded cells in the presence of various ACAT inhibitors (Sandoz 58.035, Octimibate-Nattermann, progesterone) revealed a time- and dose-dependent amplification in HDL binding and HDL-mediated cholesterol efflux. It is concluded that the homeostasis of cellular cholesterol in macrophages is regulated in part by the number of HDL binding sites and that ACAT inhibitors enhance HDL-mediated cholesterol efflux from peripheral cells. PMID- 2998755 TI - Rapid and reversible translocation of the catalytic subunit of cAMP-dependent protein kinase type II from the Golgi complex to the nucleus. AB - In unstimulated interphase bovine epithelial (MDBK) cells, both regulatory (R II) and catalytic (C) subunits of the type II enzyme of cAMP-dependent protein kinase (cAMP-dPK II) are associated with the Golgi complex. However, as demonstrated by indirect immunofluorescence microscopy, within 5 min after stimulation of adenylate cyclase by forskolin, the C subunit dissociates from the Golgi associated R II and becomes diffusely distributed. With increasing time of forskolin treatment, C subunits accumulate in the nucleus, while R II subunits remain associated with the Golgi complex. The effect of forskolin is rapidly reversible in that C subunits begin to reassociate with the Golgi complex within a few minutes after drug removal. C subunit translocations similar to those produced by forskolin also occur after treatment of MDBK cells with dibutyryl cAMP, confirming that the observed effects are most likely mediated by elevation of intracellular cAMP levels. These results suggest that nuclear translocation of activated protein kinase subunits may represent an important link between hormonal stimuli and physiological responses. PMID- 2998756 TI - Transport of proteins into mitochondria: a potassium diffusion potential is able to drive the import of ADP/ATP carrier. AB - The transfer of cytoplasmically synthesized precursor proteins into or across the inner mitochondrial membrane is dependent on energization of the membrane. To investigate the role of this energy requirement, a buffer system was developed in which efficient import of ADP/ATP carrier into mitochondria from the receptor bound state occurred. This import was rapid and was dependent on divalent cations, whereas the binding of precursor proteins to the mitochondrial surface was slow and was independent of added divalent cations. Using this buffer system, the import of ADP/ATP carrier could be driven by a valinomycin-induced potassium diffusion potential. The protonophore carbonylcyanide m-chlorophenyl-hydrazone was not able to abolish this import. Imposition of a delta pH did not stimulate the import. We conclude that the membrane potential delta psi itself and not the total protonmotive force delta p is the required energy source. PMID- 2998757 TI - Cell-type preference of immunoglobulin kappa and lambda gene promoters. AB - Immunoglobulin gene constant regions are known to be associated with strictly tissue-specific enhancer elements. Until recently the promoter of the variable region, which becomes linked to the constant region by somatic rearrangement, could have been viewed as a passive recipient of the enhancer stimulus. Here we show that the promoters of the immunoglobulin kappa and lambda light chain genes are approximately 20-30 times more active in lymphoid cells than in non-lymphoid cells. To avoid the problem of differential mRNA stability upon transfection of immunoglobulin genes into non-lymphoid cells we have constructed chimeric genes. All kappa mRNA sequences were progressively deleted to fuse the kappa gene promoter to a globin gene coding body. A similar chimeric gene was constructed with the promoter of the lambda gene. The cell-type preference of the promoter may be exploited during B-lymphocyte differentiation to regulate the immunoglobulin gene promoter independently from the enhancer. PMID- 2998758 TI - DO beta: a new beta chain gene in HLA-D with a distinct regulation of expression. AB - The HLA-D region of the human major histocompatibility complex encodes the genes for the alpha and beta chains of the DP, DQ and DR class II antigens. A cDNA clone encoding a new class II beta chain (designated DO) was isolated from a library constructed from mRNA of a mutant B-cell line having a single HLA haplotype. Complete cDNA clones encoding the four isotypic beta chains of the DR1, DQw1, DPw2 and putative DO antigens were sequenced. The DO beta gene was mapped in the D region by hybridization with DNA of HLA-deletion mutants. DO beta mRNA expression is low in B-cell lines but remains in mutant lines which have lost expression of other class II genes. Unlike other class II genes DO beta is not induced by gamma-interferon in fibroblast lines. The DO beta gene is distinct from the DP beta, DQ beta and DR beta genes in its pattern of nucleotide divergence. The independent evolution and expression of DO beta suggest that it may be part of a functionally distinct class II molecule. PMID- 2998760 TI - Chromatin structure of transcriptionally active and inactive human c-myc alleles. AB - To help elucidate the mechanism of regulation of the c-myc gene we have characterised the DNase I hypersensitive sites around this gene in the human promyelocytic leukaemia cell line HL60, which carries amplified c-myc, and in Ramos, a Burkitt's lymphoma cell line with a translocation close to exon 1 of c myc. Although dividing HL60 cells display a pattern of DNase I hypersensitive sites which is similar to that of previously described c-myc genes in B cells (lymphoblastoid and Burkitt's lymphoma), changes were found in DNase I hypersensitive sites upon differentiation of the HL60 cell line (accompanied by decreased c-myc transcription). Lack of c-myc transcription coincides with the loss of several DNase I hypersensitive sites and the reduction in intensity of a further site. A similar pattern was also seen in the inactive allele of the Burkitt's lymphoma cell line Ramos. A striking feature of both differentiated HL60 cells and of the inactive allele in Ramos is the quantitative maintenance of a DNase I hypersensitive site which occurs approximately 2.5 kb upstream of the c myc gene promoters. PMID- 2998759 TI - Members of novel VH gene families are found in VDJ regions of polyclonally activated B-lymphocytes. AB - Four potentially productive and two non-productive VDJ gene segments were isolated from the DNA of mouse B-lymphocytes which had been polyclonally activated by bacterial lipopolysaccharide (LPS). Three VDJ regions exhibit VH genes which stem from two novel VH gene families. The complexity of these families is 5-9 genes. One of the non-productive VDJ regions exhibits a D segment which may have been generated by joining of two DSP2 segments. Both non productive VDJ regions appear to contain rearranged pseudo VH genes. Three potential somatic mutations distributed over two productive VDJ regions are observed. PMID- 2998761 TI - Transforming p21 ras protein: flexibility in the major variable region linking the catalytic and membrane-anchoring domains. AB - The mammalian p21 ras proteins contain a 20-amino acid region that is highly divergent, in contrast to the strong sequence conservation that is common to other regions of these proteins. This major variable region is located near the C terminus just upstream from a conserved cysteine residue that is required for post-translational processing, membrane localization and transforming activity of the proteins. We have now used the viral oncogene (v-rasH) of Harvey sarcoma virus to study the major variable region by deleting or duplicating parts of the gene. Reducing this region to five amino acids or increasing it to 50 amino acids has relatively little effect on the capacity of the gene to induce morphological transformation of NIH 3T3 cells. Assays of GTP binding, GTPase and autophosphorylating activities of such mutant v-rasH-encoded proteins synthesized in bacteria indicated that the sequences that encode these biochemical activities are located upstream from the major variable region. In the context of transformation, we propose that the region of sequence heterogeneity serves principally to connect the N-terminal catalytic domain with amino acids at the C terminus that are required to anchor the protein in the membrane. PMID- 2998762 TI - The nucleotide sequence of the human int-1 mammary oncogene; evolutionary conservation of coding and non-coding sequences. AB - The mouse mammary tumor virus can induce mammary tumors in mice by proviral activation of an evolutionarily conserved cellular oncogene called int-1. Here we present the nucleotide sequence of the human homologue of int-1, and compare it with the mouse gene. Like the mouse gene, the human homologue contains a reading frame of 370 amino acids, with only four substitutions. The amino acid changes are all in the hydrophobic leader domain of the int-1 encoded protein, and do not significantly alter its hydropathic index. The conservation between the mouse and the human int-1 genes is not restricted to exons; extensive parts of the introns are also homologous. Thus, int-1 ranks among the most conserved genes known, a property shared with other oncogenes. PMID- 2998763 TI - Retroviral characteristics of the long terminal repeat of murine E.Tn sequences. AB - E.Tn sequences form a family of long moderately repeated sequences which are abundantly transcribed in the pluripotent cell lineage between day 3.5 and 7.5 of early mouse embryogenesis. The structure of the long terminal repeat (LTR) bordering the E.Tn sequences has been investigated by nucleotide sequencing, primer extension and S1 mapping experiments. This has allowed the identification of U3, R and U5 domains, and of several other structural features all of which are characteristics of retroviral LTRs. PMID- 2998764 TI - The regulatory chain in the p36-kd substrate complex of viral tyrosine-specific protein kinases is related in sequence to the S-100 protein of glial cells. AB - The major cytoplasmic target of various tyrosine-specific protein kinases is a 36 kd protein (p36). This protein can exist as a monomer or as a complex with a small subunit which seems to have a regulatory function. Amino acid sequence analysis of the small subunit from porcine intestine documents a unique polypeptide of 95 residues with a calculated mol. wt. close to 11 kd (p11). Since an immunologically related subunit of the same electrophoretic mobility is also found in the corresponding complex of chicken intestine p11 is well conserved across species. Unexpectedly, the sequence of p11 shows a high homology with the glia-specific protein S-100 whose biological function is not known. Although both proteins are dimers of rather small polypeptides we have not been able to detect in our preparations of p11 the moderate Ca2+ binding known for S-100. Certain implications of this sequence relation are discussed. PMID- 2998765 TI - Topoisomerase I phosphorylation in vitro and in rapidly growing Novikoff hepatoma cells. AB - Changes in phosphorylation modulate the activity of topoisomerase I in vitro. Specifically, enzymatic activity is stimulated by phosphorylation with a purified protein kinase (casein kinase type II). The purpose of this study was to compare the sites that are phosphorylated in vitro by casein kinase type II with the site(s) phosphorylated in vivo in rapidly growing Novikoff hepatoma cells. Topoisomerase I labeled in vitro was characterized by three major tryptic phosphopeptides (I-III). Separation of these peptides by a C18-reverse phase h.p.l.c. column resulted in their elution at fractions 18 (I), 27 (II) and 44 (III) with 17%, 22.5% and 33% acetonitrile, respectively. In contrast, only one major phosphopeptide was identified by h.p.l.c. in topoisomerase I labeled in vivo. This phosphopeptide eluted at fraction 18 corresponding to the elution properties of phosphopeptide I labeled in vitro. It also co-migrated with tryptic phosphopeptide I when subjected to high-voltage electrophoresis on thin-layer cellulose plates. Preliminary experiments suggest that phosphorylation occurs at a serine residue six amino acids from the N-terminus of the peptide. These data indicate that topoisomerase I is phosphorylated in vivo and in vitro within the same tryptic peptide and suggest that topoisomerase I is phosphorylated in vivo by casein kinase II. PMID- 2998766 TI - alpha-Thrombin-induced early mitogenic signalling events and G0 to S-phase transition of fibroblasts require continual external stimulation. AB - In resting Chinese hamster fibroblasts (CCL39) alpha-thrombin rapidly stimulates several biochemical events implicated in the mitogenic response, including the breakdown of inositol phospholipids, activation of a plasma membrane Na+/H+ antiporter, phosphorylation of ribosomal protein S6 and increased expression of the proto-oncogene c-myc. Complete removal of the growth factor during cellular G0/G1 transit precludes the re-initiation of DNA synthesis. The present study was designed to examine the fate of alpha-thrombin-activated early events following growth factor inactivation. In cells stimulated for 30 min with alpha-thrombin, neutralization of the growth factor results in: (i) immediate arrest of inositol phosphate formation, (ii) rapid inactivation of Na+/H+ exchange, (iii) deactivation of the S6 phosphorylating system and (iv) strong reduction of c-myc mRNA level. Our findings that commitment for DNA synthesis as well as persistent activation of 'early' cellular events requires continual growth factor stimulation suggest that: (i) growth factor-induced transmembrane signals have a short life and (ii) the generation of these signals during the 8 h of the pre replicative phase is required for G0-arrested cells to enter the S phase. PMID- 2998769 TI - Efficient expression of an Epstein-Barr nuclear antigen in Drosophila cells transfected with Epstein-Barr virus DNA. AB - In a search for exogenous promoters which function in cultured Drosophila cells, we have co-transfected a D. melanogaster cell line with an Epstein-Barr virus (EBV) cosmid clone which encodes the Epstein-Barr nuclear antigen (EBNA-1). Here we report that Drosophila cells containing stably integrated copies of EBNA-1 encoding DNA synthesise a polypeptide of mol. wt. identical to that of authentic EBNA-1, which is detectable with EBNA-positive but not EBNA-negative human serum. As in EBV-transformed lymphoblastoid cells, this neo-antigen is associated with the nucleus of transfected cells suggesting that cellular localisation signals which operate in mammalian cells are also recognised in insect cells. PMID- 2998768 TI - Structural prerequisites of simian virus 40 large T antigen for the maintenance of cell transformation. AB - We have investigated the functional roles of two structural subsets of simian virus 40 (SV40) large T antigen, namely homo-oligomers and complexes with the host cellular p53 protein, for the transformed phenotype. We examined T antigen produced in cells transformed by temperature-sensitive SV40 large T mutants: heat sensitive or unrestricted SV40 tsA58-transformed rat cells and unrestricted tsA1499 transformants. In both unrestricted cell lines, T antigen was temperature sensitive only for the formation of fast sedimenting homo-oligomers. Corresponding to our recent observations obtained with tsA1499-infected monkey cells, in tsA1499 transformants large T was competent to form stable T-p53 complexes independently of the temperature. However, T antigen coded for by tsA58, which is heat-sensitive for binding to p53, occurred in stable complexes with this protein in unrestricted tsA58 transformants under all conditions. Furthermore, in both unrestricted transformants T-p53 complexes arise in the absence of homo-oligomers of T antigen. In conclusion, T antigen homo-oligomers are not involved in cell transformation, whereas T-p53 complexes may be involved in the maintenance of this phenotype. PMID- 2998767 TI - T antigen and template requirements for SV40 DNA replication in vitro. AB - A cell-free system for replication of SV40 DNA was used to assess the effect of mutations altering either the SV40 origin of DNA replication or the virus-encoded large tumor (T) antigen. Plasmid DNAs containing various portions of the SV40 genome that surround the origin of DNA replication support efficient DNA synthesis in vitro and in vivo. Deletion of DNA sequences adjacent to the binding sites for T antigen either reduce or prevent DNA synthesis. This analysis shows that sequences that had been previously defined by studies in vivo to constitute the minimal core origin sequences are also necessary for DNA synthesis in vitro. Five mutant T antigens containing amino acid substitutions that affect SV40 replication have been purified and their in vitro properties compared with the purified wild-type protein. One protein is completely defective in the ATPase activity of T antigen, but still binds to the origin sequences. Three altered proteins are defective in their ability to bind to origin DNA, but retain ATPase activity. Finally, one of the altered T antigens binds to origin sequences and contains ATPase activity and thus appears like wild-type for these functions. All five proteins fail to support SV40 DNA replication in vitro. Interestingly, in mixing experiments, all five proteins efficiently compete with the wild-type protein and reduce the amount of DNA replication. These data suggest that an additional function of T antigen other than origin binding or ATPase activity, is required for initiation of DNA replication. PMID- 2998770 TI - Identification of a sequence element in the promoter of the Drosophila melanogaster hsp23 gene that is required for its heat activation. AB - The expression of Drosophila melanogaster hsp23-Escherichia coli beta galactosidase hybrid genes containing different segments of the 5' non transcribed sequence of the hsp23 gene has been examined at the RNA and protein levels in Xenopus oocytes. Transcription of the hybrid genes is initiated correctly. Mutant genes with hsp23 gene promoter segments of at least 140 bp in length are strongly heat-activated while genes with shorter promoter segments are expressed constitutively and at low levels. This maps an element required for the heat-controlled expression of the D. melanogaster hsp23 gene to a region, approximately 140 bp upstream from the start of the transcription site, which contains a sequence (CGAGAAGTT-TCGTGT) that is closely related to the one responsible for the heat regulation of the hsp70 gene. These findings demonstrate the importance of this regulatory sequence for a second hsp gene and support the notion that hsp genes are heat-regulated by a common mechanism. The functional element in the hsp23 gene promoter is located greater than 80 bp further upstream from the TATA box than the relevant element in the hsp70 gene promoter. Even though other related sequences are present further upstream and downstream from the functional element, they play at most an auxiliary role in the regulation of hsp23 gene expression. PMID- 2998771 TI - Identification of a telomeric DNA sequence in Plasmodium berghei. AB - A fragment of Plasmodium berghei DNA was cloned using a technique designed to select for telomeric sequences. The cloned fragment recognizes Bal31-sensitive bands in P. berghei genomic digests. It contains at its distal end at least 70 tandem repeats of the heptanucleotide sequence CCCTGAAA. The presence of natural single strand discontinuities in the telomeric regions of P. berghei DNA is demonstrated by the selective incorporation of deoxyribonucleoside triphosphates in the absence of DNase. The number of copies of the cloned sequence present in each genome agrees with an estimate of 6-12 chromosomes per nucleus. PMID- 2998773 TI - Cell type-specific transcriptional enhancement in vitro requires the presence of trans-acting factors. AB - Cell-specific transcriptional enhancement was observed, depending on the enhancer sequences, using nuclear extracts prepared from B-cells, T-cells and HeLa cells. SV40 enhancer stimulated in vitro transcription up to 15-fold in all three cell extracts, whereas transcriptional potentiation in vitro by IgC mu and LPV enhancers was only seen in B- and T-cell extracts. Thus, the cell type specificity seen in vivo can be reproduced in vitro. The transcriptional enhancement requires the presence of enhancer sequences in cis and also of a common factor interacting in trans with all three enhancer sequences. Interestingly, first experiments indicate the additional presence of cellular factors in T-cell and most prominently in HeLa cell extracts which can reduce the enhancer activity of C mu and LPV. PMID- 2998772 TI - Structure and regulated expression of genes encoding fructose biphosphate aldolase in Trypanosoma brucei. AB - Low stringency hybridisation with a rabbit aldolase cDNA was used to select cDNA clones encoding fructose biphosphate aldolase in Trypanosoma brucei. A clone which is almost full length encodes a protein of 41 027 daltons which has 50% identity with rabbit aldolase A and slightly lower homology with B-type aldolases. The homologous mRNA is at least 6-fold more abundant in bloodstream trypomastigotes than in procyclic forms, as expected from measurements of enzyme activity. Genomic mapping results indicate that trypanosomes have four copies of the aldolase gene arranged as two copies of a tandem repeat. The protein has a short N-terminal extension (relative to other known aldolases) which could be involved in the glycosomal localisation of the enzyme. PMID- 2998774 TI - The cloning and characterization of the bacteriophage D108 regulatory DNA-binding protein ner. AB - From the transposable Mu-like bacteriophage D108 we have cloned the ner gene under the control of the lac UV5 promoter in the expression vector pOP95-15. The recombinant plasmid, pPT011, overproduced the 8-kd D108 ner protein (visualized by in vitro-coupled transcription-translation) and served as a substrate for DNA sequencing of the D108 ner gene. The ner protein of D108 was found to be 48% homologous to the Mu ner protein, though the DNA sequences that encode these proteins are quite divergent. We used the retardation of migration of 32P labelled DNA restriction fragments by ner-containing crude protein extracts in polyacrylamide gels (band competition assay) to determine which DNA restriction fragment(s) contained the ner-binding sites. DNA footprinting using crude extracts physically identified the 47-bp DNA sequence that the ner protein was interacting with in the D108 early gene regulatory region. This sequence is located 10 bp downstream from the presumed D108 early gene transcription initiation site. Therefore, by binding strongly to this 47-bp DNA sequence, the D108 ner protein can regulate D108 early gene transcription. PMID- 2998775 TI - Major light-harvesting polypeptides encoded in polycistronic transcripts in a eukaryotic alga. AB - By sequence analysis of previously identified fragments and low stringency hybridization of an identified gene for a phycobiliprotein subunit to total plastid DNA, we have identified four phycobiliprotein subunit genes in a eukaryotic alga, Cyanophora paradoxa. The four phycobiliprotein subunits, alpha and beta of phycocyanin (PC) and allophycocyanin (APC), comprise the bulk of the light-harvesting complex in this alga. The alpha and beta subunit genes encoding each phycobiliprotein (the products of which are required in a 1:1 ratio in the light-harvesting complex) are contiguous; however, the genes for different phycobiliproteins, PC and APC, are located in different regions of the genome. The two PC subunit genes are in the small single copy region of the plastid genome whereas the APC subunit genes are in the large single copy region and the two sets of phycobiliprotein genes are transcribed from opposite strands. The alpha and beta subunits of both PC and APC are encoded in dicistronic transcripts and this arrangement may provide a mechanism by which the two subunits can be synthesized in equimolar amounts. Levels of the PC transcript are approximately five times that of the APC transcript which may reflect the relative abundance of their gene products in the phycobilisome. The 5' ends of the transcripts for PC and APC were mapped and the regulatory regions identified. Several features of the promoter regions for these highly transcribed genes are described. PMID- 2998776 TI - Highly purified fibroblast-derived growth factor, an SV40-transformed fibroblast secreted mitogen, is closely related to platelet-derived growth factor. AB - Fibroblast-derived growth factor (FDGF), a polypeptide secreted by an SV40 transformed baby hamster kidney cell line (SV28), was purified approximately 1000 fold from SV28-conditioned medium. FDGF, which gave a single band on SDS polyacrylamide gel electrophoresis, is a hydrophobic and cationic protein of apparent mol. wt. 31 000 containing disulphide-linked polypeptides. This factor is positive in Western blots using human platelet-derived growth factor (PDGF) anti-serum. FDGF is a potent mitogen for Swiss 3T3 cells; half-maximal stimulation of DNA synthesis was achieved at a concentration of approximately 1 nM, comparable with those for human and porcine PDGF. FDGF inhibits EGF binding to Swiss 3T3 cells, as does PDGF. The coincidence of the physical, biological and immunological characteristics of FDGF and PDGF strongly suggests that they are closely related in structure. PMID- 2998777 TI - EBV-inducing factor from platelets exhibits growth-promoting activity for NIH 3T3 cells. AB - An Epstein-Barr virus-indicating factor (EIF) has been purified from serum and platelets. We show here that highly purified preparations of platelet EIF exhibit growth-promoting activity for NIH 3T3 cells maintained in platelet-poor plasma. The Epstein-Barr virus (EBV)-inducing activity and growth-promoting activity co elute upon gel chromatography under non-dissociating as well as dissociating conditions and co-migrate in SDS-gel electrophoresis, supporting the notion that both activities reside on the same molecule. Furthermore, both activities require a pH shock for full activity and act in the same concentration range. The growth promoting activity of EIF can be differentiated from that of platelet-derived growth factor (PDGF), biologically (on the basis of differential response of cell lines to both factors), biochemically (on the basis of differences in isoelectric points and mol. wts. and the requirement of EIF to become activated by a pH shock) and by the lack of inhibition of EIF by antibody to PDGF. PMID- 2998778 TI - Activation of cellular promoters during herpes virus infection of biochemically transformed cells. AB - At least two of the immediate-early (IE) products of herpes simplex virus-1 (HSV 1) are responsible for the activation of transcription from viral early promoters. This process appears not to be promoter specific since several unrelated viral and cellular plasmid-borne promoters can also be activated in short-term transfection assays. This paper describes experiments that show that cellular promoters integrated into the host genome can also be activated during viral infection, and that this process is brought about by IE gene products. Biochemically transformed cell lines were isolated following transfection of plasmids containing the human epsilon-globin promoter linked to the herpes thymidine kinase coding region (as selectable marker), and an unselected rabbit beta-globin gene. Infection of some, but not all, such cell lines with HSV-1 resulted in a rapid and considerable stimulation of the integrated epsilon- and beta-globin promoters. Both promoters could also be activated (albeit less efficiently) during pseudorabies virus infection, and after introduction by transfection of plasmids containing HSV IE genes. The implications of these results for viral-host interactions and the mechanism of viral-induced promoter activation are discussed. PMID- 2998779 TI - Structure and cell-specific expression of a cloned human retinol binding protein gene: the 5'-flanking region contains hepatoma specific transcriptional signals. AB - Human plasma retinol binding protein (RBP) is coded by a single gene and is specifically synthesized in the liver. We have characterized a lambda clone, from a human DNA library, carrying the gene coding for plasma RBP. Southern blot analysis and DNA sequencing show that the gene is composed of six exons and five introns. Primer elongation and S1 mapping experiments allowed the definition of the initiation of transcription and the identification of the putative promoter. The 5'-flanking region of the RBP gene was fused upstream to the coding sequence of the bacterial enzyme chloramphenicol acetyl transferase (CAT): the chimeric gene was introduced, by calcium phosphate precipitation, into the human hepatoma cell line Hep G2 and into HeLa cells. Efficient expression of CAT was obtained only in Hep G2. Primer elongation analysis of the RNA extracted from transfected Hep G2 showed that initiation of transcription of the transfected chimeric gene occurs at a position identical to that of the natural gene. Transcriptional analysis of Bal31 deletions from the 3' end of the RBP 5'-flanking DNA allowed the identification of the RBP gene promoter. PMID- 2998780 TI - Nucleotide sequence of cDNA clones of the murine myb proto-oncogene. AB - We have isolated cDNA clones of murine c-myb mRNA which contain approximately 2.8 kb of the 3.9-kb mRNA sequence. Nucleotide sequencing has shown that these clones extend both 5' and 3' to sequences homologous to the v-myb oncogenes of avian myeloblastosis virus and avian leukemia virus E26. The sequence contains an open reading frame of 1944 nucleotides, and could encode a protein which is both highly homologous, and of similar size (71 kd), to the chicken c-myb protein. Examination of the deduced amino acid sequence of the murine c-myb protein revealed the presence of a 3-fold tandem repeat of 52 residues near the N terminus of the protein, and has enabled prediction of some of the likely structural features of the protein. These include a high alpha-helix content, a basic region toward the N terminus of the protein and an overall globular configuration. The arrangement of genomic c-myb sequences, detected using the cDNA clones as probes, was compared with the reported structure of rearranged c myb in certain tumour cells. This comparison suggested that the rearranged c-myb gene may encode a protein which, like the v-myb protein, lacks the N-terminal region of c-myb. PMID- 2998781 TI - The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix. AB - Yeast cytochrome c oxidase subunit IV (an imported mitochondrial protein) is made as a larger precursor with a transient pre-sequence of 25 amino acids. If this pre-sequence is fused to the amino terminus of mouse dihydrofolate reductase (a cytosolic protein) the resulting fusion protein is imported into the matrix space, and cleaved to a smaller size, by isolated yeast mitochondria. We have now fused progressively shorter amino-terminal segments of the subunit IV pre sequence to dihydrofolate reductase and tested each fusion protein for import into the matrix space and cleavage by the matrix-located processing protease. The first 12 amino acids of the subunit IV pre-sequence were sufficient to direct dihydrofolate reductase into the mitochondrial matrix, both in vitro and in vivo. However, import of the corresponding fusion protein into the matrix was no longer accompanied by proteolytic processing. Fusion proteins containing fewer than nine amino-terminal residues from the subunit IV pre-piece were not imported into isolated mitochondria. The information for transporting attached mouse dihydrofolate reductase into mitochondria is thus contained within the first 12 amino acids of the subunit IV pre-sequence. PMID- 2998783 TI - Inhibition of calf thymus type II DNA topoisomerase by poly(ADP-ribosylation). AB - The effect of poly(ADP-ribosylation) on calf thymus topoisomerase type II reactions has been investigated. Unknotting of phage P4 head DNA, and relaxation and catenation of supercoiled PM2 DNA are inhibited. We conclude that the inhibition results from poly(ADP-ribosylation) on the following grounds. Firstly, the enzyme poly(ADP-ribose) (PADPR) synthetase and NAD are required, secondly, the competitive synthetase inhibitor nicotinamide abolishes topoisomerase inhibition, and thirdly, the polymer alone is not inhibitory. The mechanism of inhibition appears to be disruption of the strand cleavage reaction. A topoisomerase-DNA complex can be formed that upon treatment with protein denaturant at low ionic strength results in strand cleavage. The amount of DNA present in such a cleavable-complex progressively decreased following pretreatment of topoisomerase type II with PADPR synthetase and increasing concentrations of NAD. Treatment of the pre-formed complex with NAD and PADPR synthetase had no effect on its salt-induced dissociation. This suggests that either poly(ADP-ribosylation) has no influence on dissociation of topoisomerase, in contrast to association, or topoisomerase is not accessible to the synthetase when bound to DNA. Similar data were obtained with calf thymus type I topoisomerase. PMID- 2998782 TI - Identification and molecular analysis of a third Aspergillus nidulans alcohol dehydrogenase gene. AB - An Aspergillus nidulans functional cDNA encoding an alcohol dehydrogenase (ADH) was isolated by its ability to complement an adh1 mutation in Saccharomyces cerevisiae. Alignment of the cDNA and cloned genomic DNA sequences indicated that the ADH gene contains two small introns. The presence of ethanol in the growth medium was shown to result in ADH mRNA accumulation presumably due to transcriptional induction of the gene. However, ADH mRNA accumulation was at most only partially repressed by the presence of glucose. The ADH gene characterized here is designated ADH3 since it is distinct from the alcA gene which encodes ADH I and appears distinct from the gene which encodes ADH II. We demonstrated that the first intron in the A. nidulans ADH3 gene was not efficiently spliced in S. cerevisiae whereas the promoter region was utilized weakly. We also present a comparison of the primary structure of A. nidulans ADH III with the alcohol dehydrogenases of S. cerevisiae and Schizosaccharomyces pombe. PMID- 2998785 TI - The effect of anaesthetic induction with etomidate on the endocrine response to surgical trauma. AB - In order to investigate the effects of anaesthetic induction with a single bolus dose of etomidate on the endocrine response to a standard surgical stress, five patients were randomly allocated to receive etomidate and five to receive thiopentone as an induction agent prior to elective inguinal hernia repair. The group receiving etomidate showed a significant suppression in the circulating level of cortisol at 90 and 120 min post-induction (P less than 0.001) and an elevation in their plasma adrenocorticotrophic hormone at 240 and 360 min post induction (P less than 0.001). The effect of a bolus dose of etomidate upon circulating cortisol levels was transient and no cardiovascular instability was noted during the study. The etomidate group also showed suppression of their circulating testosterone levels at 90 min post-induction (P less than 0.001), suggesting that etomidate inhibits steroidogenesis at a site other than 11 beta hydroxylase. PMID- 2998784 TI - Definition of three resolvase binding sites at the res loci of Tn21 and Tn1721. AB - The dual functions of resolvase, site-specific recombination and the regulation of its own expression from tnpR, both require the interaction of this protein with the DNA sequence at res, but the specificity of this interaction differs between groups of Tn3-like elements. In this study, DNA fragments that contained res from Tn21 or Tn1721 were subjected to either cleavage by DNase I or methylation by dimethyl sulphate in the presence of the purified resolvase from Tn21 or Tn1721. These experiments showed that each resolvase bound to the same three sites (I, II and III) within res from Tn1721 and to an equivalent series of three sites on Tn21: the differences in the amino acid sequences of the two proteins did not affect their interaction with either DNA. The DNA sequences at each site had some similarities and, in conjunction with data from the related transposon Tn501, a consensus was established. However, the three sites are functionally distinct: site I (tnpR-distal) spans the recombination cross-over point and sites II and III (tnpR-proximal) overlap the promoter of tnpR. The binding sites on these transposons were compared with those in the gamma delta/Tn3 system: the similarities between the two groups of transposons revealed some general features of resolvase-DNA interactions while the differences in fine structure elucidated the specificity of each resolvase. PMID- 2998788 TI - Orientation of rat liver cytochrome c oxidase subunits investigated with subunit specific antisera. AB - The orientation of rat liver cytochrome c oxidase subunits in the inner mitochondrial membrane was investigated with monospecific antisera against subunit II and nine nuclear-coded subunits. Mitoplasts were incubated with the antisera and the amount of bound antibodies was determined either directly with fluorescein-conjugated protein A or indirectly by back-titration of unbound antibodies with a nitrocellulose immunoassay. All subunits were found oriented to the cytosolic side, except subunits VIb and VIIc which did not react with their corresponding antisera. Antisera against subunits I, III and Vb were not available. PMID- 2998786 TI - Physical performance and serum potassium under chronic beta-blockade. AB - Various publications have described a beta 2-receptor regulated potassium transport system in the cellular membrane of human skeletal muscle. To examine the suggestion that serum potassium alterations are among the causes of premature muscular fatigue during physical exercise under pharmacological blockade of beta receptors, we have compared the influence of sustained blockade with a beta 1 selective blocker and a nonselective beta-blocker on the levels of serum potassium before, during and after a physical exercise test. 63 healthy physical education students received in random order and under double blind conditions either 100 mg Metoprolol (beta 1-selective) or 80 mg Propranolol (non-selective), or placebo daily for 3 months. Serum potassium was measured before, during (at 150 Watt and at the end of exercise) and after a bicycle exercise with a stepwise increase in work loads. After three months of beta-blocker treatment serum potassium levels during exercise were significantly higher than in control subjects receiving the placebo, and it took longer for the serum potassium levels to return to the resting level in the beta-blocker treated subjects. At rest, however, the levels were not found to be statistically different. In the subjects receiving Propranolol the post-exercise serum potassium levels were higher than in the subjects receiving Metoprolol. Three days after cessation of the medication these differences were no longer perceptible. Our findings confirm the existence of a beta-receptor regulated potassium transport system in human skeletal muscle and indicate that the transmembranous potassium transport in human skeletal muscle is predominantly regulated via beta 2-receptors, although beta 1-receptors seem also to be involved.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2998787 TI - Chemotherapy of herpesvirus infections: present successes and future hopes. PMID- 2998789 TI - Characterization of extracellular matrix-associated glycosaminoglycans produced by untransformed and transformed bovine corneal endothelial cells in culture. AB - A cloned bovine corneal endothelial cell line was transformed in vitro by simian virus 40, and the subendothelial extracellular matrix-associated sulfated glycosaminoglycans synthesized by the cells were isolated and compared with their untransformed counterpart. The transformed endothelial cells grew at faster rates to higher stationary cell densities in the absence of fibroblast growth factor than did the untransformed cells. On a per-cell basis, the transformed cells produced slightly lower amounts of sulfated glycosaminoglycans. The rate of production of sulfated glycosaminoglycans in extracellular matrix increased during seven days of culture. At confluency the extracellular matrix-associated sulfated glycosaminoglycans synthesized by the untransformed endothelial cells consisted of about 80% heparan sulfate and about 20% chondroitin sulfate. Extracellular matrix-associated sulfated glycosaminoglycans of transformed endothelial cells were composed of about 70% heparan sulfate and about 30% chondroitin sulfate plus dermatan sulfate. High-speed gel permeation chromatography profiles on Fractogel TSK HW-55(S) of matrix-associated heparan sulfate from untransformed and transformed endothelial cells were very similar, and gave single peaks (Kav = 0.19). Apparent Mr estimated from the eluting position of the peaks were approximately 47000. Heparan sulfate from both untransformed and transformed endothelial cells was degraded by incubation with a metastatic B16 melanoma cell lysate containing heparanase (heparan-sulfate specific endo-beta-glucuronidase). The eluting position of the heparan sulfate degradation products on gel permeation column were similar (Kav = 0.43). Size analysis and anion-exchange chromatography of the degradation products after nitrous acid deamination at low pH indicated that the degree of N-sulfation of heparan sulfate was similar in untransformed and transformed endothelial cells. The results indicated that transformation of endothelial cells only slightly changes the molecular nature of subendothelial matrix-associated sulfated glycosaminoglycans. PMID- 2998790 TI - Substrate specificity and stereospecificity of calf spleen phosphodiesterase towards deoxyribonucleosidyl 3'-(4-nitrophenyl phosphates) and phosphorothioates. AB - Phosphodiesterase from calf spleen exhibits nucleotidyltransferase activity when incubated with either the (PR) or the (PS) diastereomer of thymidyl 3'-(4 nitrophenyl phosphorothioate). Thymidylyl(3'-5')thymidyl phosphorothioate 3'-(4 nitrophenyl phosphorothioate) was identified as the main product of the enzyme catalyzed reaction and the absolute configuration at the internucleotide phosphorus atom of the product was determined. The nucleotidyltransferase reaction is shown to proceed with retention of configuration at phosphorus, implying involvement of a double displacement mechanism with the formation of a nucleotidylated enzyme intermediate. To study the substrate specificity of spleen phosphodiesterase a series of deoxyribonucleosidyl 3'-(4-nitrophenyl phosphates) and phosphorothioates were synthesized, and Km and V parameters for each substrate were measured. The results obtained show virtually no specificity for substrates with different nucleosidyl moieties, while about a 20 - 30-fold drop in V and a slight increase in Km values is observed for phosphorothioate analogues as compared with corresponding phosphates. The enzyme showed no significant stereoselectivity towards phosphorothioates of opposite configurations at phosphorus. PMID- 2998791 TI - Identification of a 150-kDa membrane component which is modulated by parathyroid hormone. AB - Monoclonal antibodies have been produced against primary bone cells obtained from the collagenase digestion of mouse cranial bone. Antibodies were selected on the basis of their immunoglobulin class and those which were identified as IgG were further screened for their ability to inhibit cAMP accumulation in response to sub-maximal doses of the 1-34 amino-terminal peptide of bovine parathyroid hormone, bPTH(1-34). Nine hybridoma clones were subsequently characterized as inhibitory with respect to parathyroid hormone (PTH) responses in intact mouse cranial bone and which also identified a variety of membrane components from detergent extracts of surface-labeled primary bone cells. Five of these antibodies immunoprecipitated a membrane component with Mr of 80 000 that appeared to be a major component of the extract susceptible to surface-labeling with 125I. All nine monoclonal antibodies were shown to bind to a suspended-cell preparation of primary bone cells with 2-3 orders of magnitude greater binding than that of control antibodies. Using this assay, one clone, designated 3G12 IgG, was observed to exhibit desensitization effects at the binding level with a time course and dose dependency for PTH pre-incubation that was similar to the establishment of the refractory state in other systems. In addition, the desensitization effect occurred at 37 degrees C but not at 4 degrees C. This antibody was shown to bind saturably to both intact mouse cranial bone and primary bone cells with an apparent affinity constant (Ka) in the range of 10(9) M. Inhibition of bone cAMP accumulation in response to 2.5 nM bPTH(1-34) was directly correlated to the binding of 3G12 IgG to intact mouse calvariae. A maximum inhibition of approximately 85% was observed. 3G12 IgG immunoprecipitated a single membrane component, Mr 150 000, from NP-40 detergent extracts of 125I labeled primary mouse bone cells. The molecular mass of this component was also 150 000 daltons when run on polyacrylamide gel slabs under non-reducing conditions. Control and PTH-pre-treated bone cells were surface-labeled, detergent-solubilized and immunoprecipitated with 3G12 IgG in order to investigate further the desensitization effect at the molecular level. Incubation of bone cells with 1 microgram/ml bPTH(1-34) for 45 min at 37 degrees C caused an increased susceptibility to surface-labeling with 125I that was approximately three-fold higher in specific activity than that of control cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2998792 TI - The influence of ganglioside insertion into brain membranes on the rate of ganglioside degradation by membrane-bound sialidase. AB - Microsomal membranes isolated from calf brain contain a sialidase which cleaves ganglioside substrates naturally occurring within these membranes as well as exogenously added [3H]ganglioside GD1a. Micelles of [3H]ganglioside GD1a bind to the microsomal membranes in two steps. The first step, called adsorption, is fast and reversible by treatment with trypsin; the second step, called uptake, is slower and not reversible. The product of the enzymic degradation, [3H]ganglioside GM1, is exclusively located in the ganglioside pool taken up by the sialidase-bearing membranes, and not in the trypsin-releasable pool. Electron spin resonance (ESR) studies using a spin-labelled analogue of ganglioside GD1a indicate that the ganglioside uptake by microsomal membranes is accompanied by the disappearance of the micellar structure and by the 'dilution' of the probe molecules with membrane lipids. These findings suggest that exogenously added ganglioside substrate inserts into the microsomal membrane before it is recognized as substrate by the membrane-bound sialidase. Therefore, the influence of pH, ionic strength and membrane-fluidizing agents on the degradation rate measured with exogenous ganglioside GD1a does not only reflect kinetic parameters of the enzymic reaction itself but also the velocity of ganglioside insertion. Increasing ionic strength reduces the degradation rate. The acceleration of insertion with falling pH values shifts the measured pH optimum of the ganglioside degradation to lower values (pH 3.6) and masks the substantial residual sialidase activity at pH 5-7. The membrane-fluidizing alcohol n-hexanol greatly accelerates ganglioside insertion as well as ganglioside degradation. The latter was clearly demonstrated by studying the hydrolysis of endogenous ganglioside substrates, and is due to a decrease of the apparent Km value and an increase in the Vmax value. The Vmax value was also enhanced by freezing and thawing of the microsomal membranes. PMID- 2998793 TI - Binding stoichiometry of a fluorescent cGMP analogue to membranes of retinal rod outer segments. AB - The high-affinity binding of the cGMP analogue 8-(5-thioacetamidofluorescein) cGMP to rod outer segment membranes depleted of peripherally bound proteins has been defined by equilibrium dialysis (mean +/- SD): membranes contain about one cGMP binding site per 130 rhodopsin molecules; the concentration of free ligand for half saturation is 2.0 +/- 0.6 microM; the apparent Hill coefficient of the bound versus free ligand relationship is 1.7 +/- 0.5; half saturation of the binding sites is sufficient for 85% activation of calcium permeability. A gating mechanism is proposed. PMID- 2998794 TI - Isolation and characterization of a blue copper protein from Thiobacillus versutus. AB - The blue copper protein induced during growth of Thiobacillus versutus on methylamine was purified and characterized. It is an acidic protein (isoelectric point 4.7), contains one Cu2+ ion/enzyme molecule, is a monomeric protein (molecular mass about 14 kDa), has a maximum in its absorption spectrum at 596 nm (molar absorption coefficient 3.9 X 10(3) M-1 cm-1), shows an axial type-I electron paramagnetic resonance spectrum (g parallel = 2.239, g perpendicular = 2.046 and A parallel = 5.6 mT) and has a redox potential (Eo) of + 260 mV. In view of these properties and in view of the fact that the protein is active as an electron carrier between methylamine dehydrogenase and cytochrome c, it is concluded that it is similar to the amicyanins isolated from Methylomonas sp. strain J and Pseudomonas sp. strain AM 1. PMID- 2998795 TI - Computer-assisted radionuclide perfusion study in solitary cold thyroid nodules for diagnosis of malignancy. AB - A computer-assisted radionuclide perfusion study was performed in 25 cases of solitary 'cold' thyroid nodules to assess their vascularity relative to that of normal adjoining thyroid tissue. Hypervascularity was seen in 3 malignant nodules; 1 follicular adenoma also showed increased vascularity. The 21 nodules demonstrating equal vascularity or avascularity with respect to normal thyroid tissue were benign in nature. Computer-generated time-activity curves were helpful in distinguishing hypervascular from equally vascular nodules. This technique, in conjunction with clinical evaluation, can be of value in deciding whether patients should undergo surgery or receive conservative management. PMID- 2998796 TI - Cardiac function in acute hypothyroidism. AB - It has been established that chronic hypothyroidism may affect cardiac function by several mechanisms. It is not known how long the patient has to be hypothyroid for cardiac involvement to develop. This study was undertaken to assess the effect of a short period of hypothyroidism (10 days) on cardiac function. Nine patients who had had total thyroidectomy, had received ablative radioiodine for thyroid cancer and were euthyroid on replacement therapy were studied while both euthyroid and hypothyroid. Cardiac assessment was performed by X-ray, ECG, echocardiography and gated blood-pool scans. After 10 days of hypothyroidism, the left-ventricular ejection fraction failed to rise after exercise in 4 of the 9 patients studied, which was significant (P less than 0.002). No significant changes in cardiac size or function at rest were detected. This functional abnormality in the absence of any demonstrable change in cardiac size and the absence of pericardial effusion with normal basal function suggest that short periods of hypothyroidism may reduce cardiac reserve, mostly because of alterations in metabolic function. PMID- 2998797 TI - Radiotoxicity of 11C-methionine measured by the accumulation of DNA strand breaks in mammalian cells. AB - The radiotoxic effects of L-[methyl-11C] methionine were estimated by measuring the accumulation of DNA strand breaks. CHO, Chinese-hamster cells, were incubated in 11C-methionine-containing medium for 60 min at 37 degrees C. The number of unrepaired DNA strand breaks was then examined by the DNA-unwinding method. For comparison, cells were also externally irradiated under similar conditions with gamma radiation (137Cs) or positrons (11C). The relative biological effectiveness of 11C-methionine was estimated to be about 1. PMID- 2998798 TI - High-yield preparation of porcine hepatocytes for long survival after transplantation in the spleen. AB - The creation of an auxiliary liver by autotransplantation of liver parenchymal cells into the spleen has mainly been studied in rats for the treatment of acute liver failure. In order to apply this procedure to humans with chronic liver insufficiency the aim of this work was: To demonstrate that hepatocytes can survive for long periods after autotransplantation into the spleen; to increase the yield of the isolation of hepatocytes obtained from pig livers since this animal has a more fibrous liver than rats or normal humans and consequently one which is more difficult to dissociate. In 21 pigs isolated hepatocytes were obtained with in collagenase dissociation technique, the yield being 1-3 X 10(7) cells per gram of liver and the viability 70-95%. The hepatocytes survived and maintained normal morphological and histochemical characteristics up to 7 months after transplantation, the date of sacrifice of the last animal. PMID- 2998799 TI - The heterogeneity of human cancers and its influence on metastases and therapy. PMID- 2998800 TI - The sebaceous gland antigen defined by the OM-1 monoclonal antibody is expressed at high density on the surface of ovarian carcinoma cells. AB - A monoclonal antibody, designated OM-1, was raised against ovarian serous papillary cystadenocarcinoma (stage IV) cells. This antibody was found to react strongly with primary and metastatic ovarian serous cystadenocarcinomas and endometrioid carcinomas but the antigen detected was either absent or at very low levels in ovarian mucinous adenocarcinomas, clear cell carcinomas, benign serous and mucinous cystadenomas and Brenner tumours. The OM-1 antibody gave no detectable reaction with 93 other human tumours, including examples of breast and colon adenocarcinomas. In normal tissues the OM-1 antibody reacted with normal sebaceous gland cells, lung type II pneumocytes and placental syncytial trophoblasts. In the normal ovary OM-1 reactivity was confined to extremely weak staining of the surface epithelium. No reaction with any other ovarian cell type could be detected. No evidence of reaction with other normal cell populations present in 24 adult and seven foetal tissues was found. The antigen detected is compared with other ovarian tumour-associated antigens. The OM-1 antibody is likely to prove of value in the detection and diagnosis of ovarian carcinoma. PMID- 2998801 TI - Importance of the concomitant presence of palpable MOPC-315 tumor in stimulation of splenocytes by C-type MOPC-315 virus in vitro. AB - BALB/c mice inoculated with MOPC-315 tumor cells developed an antiviral response against C-type particles extracted from subcutaneous tumors of plasmacytoma bearing mice as shown by in vitro stimulation of spleen cells from tumor-bearing mice by virus-containing preparations. Induction of blastogenic response by virus containing preparations was found to occur in unfractionated spleen cell populations, the glass-wool non-adherent fraction (depleted of macrophages and tumor cells) and the nylon-wool non-adherent (T-enriched) fraction of spleen cells. The antiviral response was no more detectable in spleens of tumor-bearing mice cured by melphalan. Cured mice developed a strong antitumor immune response as shown by their resistance to challenge with a tumorigenic dose of MOPC-315 tumor cells. However, challenge with tumor cells of cured, resistant mice did not induce reappearance of antiviral response. PMID- 2998802 TI - Double blind study of the influence of co-dergocrine on platelet parameters in healthy volunteers. AB - In 20 healthy volunteers double-blind randomized study was done to examine the effect of co-dergocrine (Hydergine) on platelet function. All the volunteers were pretreated with 3 X 1 placebo for 1 week, followed by the randomized study in which 10 volunteers each received drug or placebo for 6 weeks. Various platelet function parameters, such as the peripheral platelet count, plasma thromboxane B2, platelet sensitivity to the antiaggregatory prostaglandins PGI2, PGE1 and PGD2, ADP-induced aggregation, collagen-induced aggregation, the WU-test, the platelet proteins platelet factor 4 and beta-thromboglobulin, malondialdehyde, circulating endothelial cells and the intracellular c-AMP level in platelets were examined. Treatment with co-dergocrine decreased platelet activity to a significant extent, as shown by a number of platelet function parameters, such as thromboxane B2, WU-test, beta-thromboglobulin, platelet factor 4, malondialdehyde and ADP-induced aggregation. The findings suggest that co-dergocrine might be able to decrease platelet activity and improve interaction with the walls of to a blood-vessel significant degree. PMID- 2998804 TI - Partial purification and biological properties of an eosinophil-activating factor. AB - A protein (eosinophil-activating factor, EAF), which enhances the capacity of human peripheral blood eosinophils to kill antibody-coated schistosomula of Schistosoma mansoni, has been partially purified from supernatants of cultured peripheral blood mononuclear cells by sequential chromatography on Sephacryl S200 and DEAE-cellulose. This protein is acidic with a molecular mass on gel filtration of 40 +/- 7 kDa. It not only enhances the activity of eosinophils against schistosomula but also increases their ability to lyse antibody-coated, herpes simplex virus-infected Chang liver cells. It enhances the production of superoxide and hydrogen peroxide by eosinophils that occurs both spontaneously and in response to opsonized zymosan. However, increased respiratory burst activity does not appear to be responsible for the enhancement of eosinophil mediated killing of schistosomula, since a comparable or greater increase in hydrogen peroxide production is induced by column fractions that have little or no effect on schistosomulum killing. EAF enhances eosinophil degranulation, both spontaneously and after incubation with opsonized zymosan. Enhanced degranulation is associated with release of eosinophil peroxidase and eosinophil cationic protein. These findings suggest that EAF enhances the capacity of eosinophils to kill parasites by increasing the extent of eosinophil degranulation and the amount of toxic granule proteins that are secreted. PMID- 2998806 TI - Biosynthesis of complement protein D by HepG2 cells: a comparison of D produced by HepG2 cells, U937 cells and blood monocytes. AB - The biosynthesis of complement protein D of the alternative pathway by HepG2 cells, a human hepatocyte cell line, was studied and compared to the biosynthesis of D by U937 cells and blood monocytes. Increasing amounts of antigenic D were detected in HepG2 cell culture supernatants by radioimmunoassay. The kinetics of D synthesis and secretion by HepG2 cells was followed in a pulse-chase study using [35S]cysteine. As analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography, only a single D band was seen intra- and extracellularly and both forms had the same apparent molecular weight as D synthesized by U937 cells or purified from serum. Treatment of HepG2 and U937 cells with canavanine, an arginine amino acid analog, to inhibit intracellular processing resulted in slight depression of the apparent molecular weight of D synthesized by these cells. D synthesized by blood monocytes had an apparent molecular weight similar to that synthesized by HepG2 and U937 cells, suggesting that these cell lines do not synthesize and process D differently than normal monocytes. The data demonstrate that the hepatocyte is a site of D synthesis and suggest that D is not synthesized as a precursor molecule. PMID- 2998803 TI - Effect of guar gum on glipizide absorption in man. AB - The effect of 4.75 g guar gum, a fibre preparation, on the absorption of 2.5 mg glipizide has been studied in 10 healthy volunteers given a standard breakfast. Three different experimental protocols were used: glipizide without guar gum (Treatment 1), glipizide with guar gum (Treatment 2) and guar gum 30 min after the drug together with breakfast (Treatment 3). The serum glipizide at 30 minutes was higher during Treatment 2 than Treatment 3 (p less than 0.01), but neither differed from the control treatment. The AUCs for glipizide were calculated up to 8 hours. They did not differ significantly between the treatment, although there was a non-significant trend to lower values during Treatment 3. Serum insulin and blood glucose levels were determined up to 3 h. Corresponding to differences in the glipizide concentration, serum insulin was highest and blood glucose lowest at 30 minutes during Treatment 2. According to this single dose study, guar gum does not have any substantial deleterious effect on the absorption of glipizide. The lack of effect may be due to the complete gastrointestinal absorption of glipizide. PMID- 2998805 TI - Isolation and characterization of Abelson murine leukemia virus-transformed mast cell lines from midgestation embryonic placenta. AB - Abelson murine leukemia virus was used to transform cells of the midgestation embryonic placenta. The frequency of transformed foci in semisolid agarose was highest when cells were isolated at 10 days of gestation and cell lines could be established in liquid culture. The continuous cell lines express characteristics of cultured mast cells, including surface antigens which are shared with lymphocytes and mononuclear phagocytes. These results imply a relationship between the transforming gene product and the mast cell growth factor interleukin 3. PMID- 2998808 TI - Evaluation of different beta-carbolines in Mongolian gerbils with reflex epilepsy. AB - Three benzodiazepine (BZ) receptor ligands of the beta-carboline group, namely the BZ receptor agonist ZK 93 423, the partial agonist ZK 91 296, and the antagonist ZK 93 426, were studied in epilepsy-prone Mongolian gerbils with different seizure types. Diazepam and clonazepam were included in these studies for comparison. In vivo binding studies in gerbils showed that all compounds were potent in displacing [3H]lormetazepam from binding sites in cerebellum and forebrain. Except for ZK 93 426, all drugs proved capable of dose dependently protecting gerbils from minor (myoclonic) and major (tonic-clonic) seizures induced by air blast stimulation. For ZK 93 423, ZK 91 296, diazepam and clonazepam, a highly significant correlation was found between anticonvulsant ED50S and ED50S for displacement of [3H]lormetazepam binding. Calculation of receptor occupancy revealed that beta-carbolines and benzodiazepines displayed anticonvulsant effects in gerbils at low occupancy (8-15% in forebrain). Even at almost total receptor occupancy, the BZ receptor antagonist ZK 93 426 was without any effect on seizure behaviour but antagonized the anticonvulsant effect of ZK 91 296. In contrast to diazepam, ZK 91 296 was devoid of any sedative side effects even at 90% receptor occupancy. The data suggest that anticonvulsant beta carbolines deserve interest as a new type of anticonvulsant drug. PMID- 2998807 TI - Ceruletide inhibits water intake in deprived mice: comparison with morphine and the enkephalin analogue, FK 33-824. AB - Subcutaneous injections of ceruletide (caerulein diethylammonium hydrate, CER) reduced dose-dependently the water intake in male NMRI mice deprived of water for 18 h. The ED50 for this effect was 5.5 (3.70-7.94) nmol/kg, which is 3.7 times more than the corresponding food intake inhibiting dose. Also inhibitory but much less potent than CER were (in decreasing order) FK 33-824, morphine and naloxone. Naloxone was an antagonist to both FK 33-824 and morphine but not to CER, thereby separating CER from the opioids. When water intake reducing doses of CER (15 nmol/kg) and FK 33-824 (850 nmol/kg) were combined, the two peptides were not additive but antagonized each other. Together, the present and previous results suggest that pharmacological inhibition of food and water intake have different characteristics. PMID- 2998809 TI - Cardiovascular effects of centrally perfused clonidine. AB - Earlier work has indicated that the systemic cardiovascular actions of clonidine might be mediated by caudal brainstem centers, especially the nucleus of the solitary tract (NTS). This study sought to define the mode of clonidine action on the NTS more explicitly using the technique of push-pull perfusion on urethane anesthetized rats. The NTS of stereotaxically mounted subjects was unilaterally perfused with an artificial cerebrospinal fluid at 25 microliter/min. Clonidine was added to the medium at concentrations of 5 to 500 microM, without interruption of flow, for test periods of 10 min. Systemic drug actions were expressed in terms of mean arterial pressure (MAP) and heart rate (HR), both of which were recorded continuously throughout the experiment. Decreases occurred in both MAP and HR following clonidine perfusion at all concentrations. However, the dose-effect relationship for the blood pressure response was dependent to some extent on control pressure. When this was considered as a variable, the drug induced pressure effects were significantly dose-dependent. Control HR values were more stable than pressure and dose-related decreases following clonidine administration were highly significant. The clonidine concentrations investigated here were considerably lower than those previously studied by microinjection. The observed dose-related depression of MAP and HR under basal conditions may be related to specific alpha 2-adrenergic receptor activation of the NTS. PMID- 2998810 TI - A comparative study of electrical field stimulation of the guinea-pig, ferret and marmoset urinary bladder. AB - The spontaneous and electrically evoked activity was examined in guinea-pig, ferret and marmoset urinary bladder. Electrical field stimulation of detrusor strips in vitro induced a rapid, frequency-dependent contraction with a maximum response at 40 Hz. This contraction was partly decreased by either atropine (0.29 microM) or desensitisation to alpha, beta-methylene ATP (alpha, beta-MeATP) (5 microM), and was totally blocked by a combination of the two. Atropine blocked responses to carbachol (30 microM) but not to ATP (80 microM), whereas desensitisation to alpha, beta-MeATP blocked those to ATP but not to carbachol. The nature of the excitatory neurotransmission mechanism in the bladder of those species examined in the present experiments was shown to be similar to that of other species described previously. PMID- 2998811 TI - Differential effects of cations and guanyl nucleotides on agonist and antagonist binding to rat adrenal and uterine angiotensin II receptors. AB - The effects of various modulators (cations, Gpp(NH)p) of hormone-receptor interaction were tested on agonist [( 125I]angiotensin II) and antagonist (125I [Sar1,Ala8]angiotensin II) binding to membrane particles from the rat adrenal zona glomerulosa and uterine smooth muscle. The two radioiodinated peptides labeled the same population of binding sites. Sodium ion (140 mM) induced a 2 fold increase in the affinity of adrenal angiotensin II receptors for the agonist (Ka = 2.15 nM-1, vs. 1.01 nM-1 for controls), but decreased antagonist binding by reducing the number of available receptors by up to 50% in both adrenal and uterine membrane particles. Potassium ion only inhibited antagonist binding. Calcium and magnesium ions (0-10 mM) increased agonist binding and decreased antagonist binding to adrenal and uterine angiotensin II receptors, an effect mediated by changes in both affinity and number of receptors for the two peptides. The non-hydrolyzable GTP analog, Gpp(NH)p (10(-9) - 10(-4) M) decreased the affinity of angiotensin II receptors for the agonist by up to 50%, but did not affect antagonist binding to the receptor. Thus, there were marked differences in the sensitivity of agonist and antagonist peptides of angiotensin II to the modulatory effect of cations and guanyl nucleotides on ligand-receptor interaction. It is suggested that these differences may be important in determining the activatory/inhibitory properties of angiotensin peptides. PMID- 2998812 TI - In vivo opiate receptor binding of oripavines to mu, delta and kappa sites in rat brain as determined by an ex vivo labeling method. AB - The relative in vivo receptor affinities of three oripavine drugs given subcutaneously were determined at the mu, delta and kappa type of opiate binding sites in rat brain. The oripavines include the agonist etorphine, the antagonist diprenorphine and the mixed agonist-antagonist buprenorphine. With the use of mu, delta and kappa specific labeling conditions in brain homogenates immediately after sacrifice (ex vivo labeling), the method relies on the assay of those receptor sites that remain unbound in vivo. Because of the slow receptor binding kinetics of the oripavines, little or no dissociation of the in vivo ligand occurs during the ex vivo labeling period. All three drugs displayed lower affinity in vivo at the delta sites relative to mu sites, whereas the kappa affinities were highly variable. Etorphine displayed considerable mu selectivity, while burpenorphine's affinity at the mu and kappa sites was similar. The apparent in vivo binding affinities obtained from the ex vivo labeling approach are compatible with previous results where tracers were applied in vivo. The dramatic differences of the in vivo and in vitro opiate receptor binding properties of the oripavines demonstrate the need for in vivo receptor binding parameters in the analysis of the function of individual receptor types. PMID- 2998813 TI - An investigation of age-related changes in pre- and postjunctional alpha adrenoceptors in human saphenous vein. AB - The responsiveness of prejunctional alpha 2-, postjunctional alpha 1- and postjunctional alpha 2-adrenoceptors was examined in human isolated saphenous veins from male patients in the age range 37-70. There was no age-related alteration in the prejunctional potency of the alpha 2-adrenoceptor agonist xylazine for inhibiting the stimulation-evoked overflow of tritium in tissues preincubated with [3H]noradrenaline. The alpha 2-adrenoceptor antagonist yohimbine (0.01-1 microM) and the alpha 1-adrenoceptor antagonist prazosin (0.1-1 microM) significantly reduced stimulation-evoked contractions in a concentration dependent manner. There was no significant age-related correlation for the potency of prazosin but there was a significant negative correlation between the potency of yohimbine and age (r = 0.70, n = 11, P less than 0.05), i.e. the potency of yohimbine decreased with increasing age. The decreased postjunctional potency of yohimbine may reflect a loss of alpha 2-adrenoceptors with increasing age. PMID- 2998814 TI - Platelet alpha 2-adrenoceptors in heroin addicts during withdrawal and after treatment with clonidine. AB - The density of platelet alpha 2-adrenoceptors, quantitated by means of the binding of [3H]clonidine and [3H]yohimbine, and the aggregation response induced by adrenaline were investigated in thirty-two heroin addicts during spontaneous withdrawal. The number of binding sites for [3H]clonidine, but not for [3H]yohimbine, was significantly increased during withdrawal and the increase followed a time course related to the severity of the abstinence syndrome. There was a positive and significant correlation between the severity of withdrawal and the density of platelet alpha 2-adrenoceptors. Concomitantly, the adrenaline induced platelet aggregation was potentiated. Treatment with clonidine led to significant decreases in receptor densities as well as in functional responses. These results suggest that only alpha 2-adrenoceptors in the agonist state (i.e. number of binding sites for [3H]clonidine) are modulated during the development of the heroin withdrawal syndrome. PMID- 2998816 TI - Atropine- and tetrodotoxin-resistant motor activity, responsive to 4 aminopyridine, present in isolated rabbit jejunum. AB - The effects of 4-aminopyridine on the spontaneous contractile activity of rabbit jejunum segments were studied. Apart from its effects on the cholinergic component of the spontaneous activity, the drug was found to be effective to increase an atropine-, tetrodotoxin-resistant activity of the preparation, suggesting either a direct effect on intestinal smooth muscle cells or an indirect effect through an unidentified excitatory innervation. PMID- 2998815 TI - Mode of analgesic action of dipyrone: direct antagonism of inflammatory hyperalgesia. AB - Dipyrone blocked carrageenin-induced oedema and hyperalgesia in a dose-dependent manner. In contrast with indomethacin, paracetamol and acetyl salicylic acid, much lower doses of dipyrone were necessary for blocking hyperalgesia (ED50 = 19 mg/kg, i.p.) than oedema (180 mg/kg, i.p.) Dipyrone administered intraperitonially or intraplantarly was able to antagonise PGE2-, isoprenaline- and calcium chloride-induced hyperalgesia, effects which are not observed with non-steroid anti-inflammatory drugs. Systemic or local administration of dipyrone had no effect upon Db-cAMP-induced hyperalgesia while a centrally acting analgesic, morphine, given systemically, was highly effective. These results support our suggestion that the mechanism of action of dipyrone is different from that of classical non-steroidal anti-inflammatory drugs. Although the site of action is peripheral its analgesic effect does not derive from inhibition of the synthesis of prostaglandins but is exerted via direct blockade of the inflammatory hyperalgesia. PMID- 2998817 TI - Stimulation of protein kinase C reduces ACTH-induced excessive grooming. PMID- 2998818 TI - DMI, Wy-45,030, Wy-45,881 and ciramadol inhibit locus coeruleus neuronal activity. AB - Wy-45,030 and Wy-45,881 block the uptake of norepinephrine and serotonin in rat brain synaptosomal preparations and share several in vivo and in vitro effects with known tricyclic antidepressants. To further characterize their activity, these compounds were compared to desipramine and ciramadol in electrophysiological studies of their acute effects on noradrenergic neuronal activity. All four compounds inhibited locus coeruleus neuronal activity with a rank order of potency of desipramine greater than Wy-45,881 greater than Wy 45,030 greater than ciramadol. Administration of the alpha-adrenergic blocking drug, piperoxane, increased locus coeruleus firing rate after desipramine, Wy 45,030 and Wy-45-881. Pretreatment with naloxone prevented the reduction in locus coeruleus impulse flow observed after ciramadol administration but had no effect on the inhibition produced by Wy-45,030. Wy-45,030 and Wy-45,881, like classical antidepressants, appear to inhibit locus coeruleus neuronal firing by potentiating neuroinhibitory transmission of locus coeruleus neurons by blocking the uptake of norepinephrine into presynaptic terminals. PMID- 2998819 TI - Adenosine receptor activation in brain reduces stress-induced ulcer formation. AB - Rats restrained in a cold environment for 3 h developed a high incidence of gastric ulcers. Administration of adenosine receptor agonists prior to a restraint period significantly reduced ulcer formation and severity, and lowered plasma corticosterone levels. This protective effect was blocked by 8 phenyltheophylline, a methylxanthine type adenosine receptor antagonist able to permeate the blood-brain barrier. This finding together with the absolute and relative order of potencies with which adenosine receptor agonists produced their effects suggests that CNS adenosine A1 receptors are involved in blocking and methylxanthines in exacerbating stress-induced gastric pathology. PMID- 2998820 TI - Regulation of [35S]t-butylbicyclophosphorothionate binding sites in rat brain by GABA, pyrethroid and barbiturate. AB - GABA regulates the binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) within the GABA receptor-ionophore complex by decreasing the rate of radioligand association and increasing the rate of dissociation but in different ways for the EDTA/water-dialyzed rat brain membranes and a solubilized preparation obtained on treatment with the zwitterionic detergent CHAPS. In the membranes, GABA at 0.3-1 microM is a non-competitive inhibitor of [35S]TBPS binding, affecting the density of binding sites but not the affinity of the receptor, while at 5 microM both the apparent density and affinity are significantly decreased. On treatment with CHAPS the solubilized preparation and the corresponding pellet fraction become less sensitive to GABA which even at 5 microM acts only as a non-competitive inhibitor. CHAPS solubilization decreases the sensitivity of the receptor [35S]TBPS complex to GABA-induced dissociation. GABA at micromolar levels also greatly influences the action of compounds within the TBPS domain, facilitating and modulating displacement of [35S]TBPS from EDTA/water-dialyzed membranes by the alpha-cyanopyrethroid cypermethrin and the barbiturate 5-(1',3' dimethylbutyl)-5-ethylbarbiturate. Large differences in the Hill numbers indicate that different mechanisms may be involved in GABA modulation of the pyrethroid and barbiturate sites. In contrast, GABA does not effect [35S]TBPS displacement by photoheptachlor epoxide which acts directly at the TBPS binding site. PMID- 2998821 TI - Suppression of positive inotropic and toxic effects of cardiac glycosides by amiloride. AB - Effects of amiloride on the inotropic and toxic actions of cardiac glycosides were examined using left atrial muscle isolated from guinea pig heart. Preincubation of atrial muscle with amiloride significantly decreased the maximum positive inotropic effect of dihydrodigoxin but failed to reduce that of isoproterenol. Amiloride prevented the contracture and significantly reduced the incidence of arrhythmias induced by 2 microM digoxin. Similar experiments examining 5 microM digoxin-induced arrhythmias showed that amiloride increased both the time required to produce arrhythmias and the fractional occupancy of sarcolemmal Na,K-ATPase by digoxin at the onset of arrhythmias. The antagonism of cardiac glycoside actions was best observed during the decline in developed tension elicited by amiloride subsequent to its initial positive inotropic effect. Amiloride had no effect on binding site concentration for ATP-dependent [3H]ouabain binding but decreased affinity of the binding sites for ouabain in membrane preparations obtained from guinea pig heart. Furthermore, amiloride inhibited Na,K-ATPase activity and increased the IC50 value for ouabain inhibition of the enzyme. These results indicate that amiloride antagonizes the positive inotropic and toxic effects of cardiac glycosides. Possible mechanisms for the antagonism include inhibition of sarcolemmal Na+/Ca2+ or Na+/H+ exchange. PMID- 2998822 TI - Effects of atrial natriuretic factor, sodium nitroprusside, and acetylcholine on cyclic GMP levels and relaxation in rat aorta. AB - The purpose of this study was to investigate the mechanisms whereby an endothelium-dependent vasodilator, acetylcholine, a nitrovasodilator, sodium nitroprusside and atrial natriuretic factor (atriopeptin II), elevate cyclic GMP levels and induce relaxation in rat thoracic aorta. Methylene blue inhibited the elevated cyclic GMP levels and relaxation due to sodium nitroprusside and acetylcholine, but not those to atriopeptin II. Cyanide inhibited relaxations to all three vasodilators, but inhibited the elevated cyclic GMP levels in response to only nitroprusside and acetylcholine. The reducing agents sodium borohydride, dithiothreitol, sucrose and isoproterenol all inhibited the elevated cyclic GMP levels due to nitroprusside and acetylcholine, while the increased cyclic GMP levels with atriopeptin II were unaffected by sodium borohydride, sucrose and isoproterenol. The effects of the reducing agents on relaxation induced by the vasodilators were difficult to interpret due to their nonspecific contractile and relaxant properties. Agents and procedures known to inhibit the Na+, K+-pump and relaxation to endothelium-dependent vasodilators and nitroprusside, including ouabain, K+-free, Mg2+-free and low Na+ Krebs-Ringer bicarbonate solution, all partially inhibited relaxations to atriopeptin II. Relaxations to atriopeptin II were also inhibited in tissues contracted with KCl. The present results suggest that the mechanism of atrial natriuretic factor-induced increased cyclic GMP levels, in contrast to that of nitroprusside and acetylcholine, does not involve the formation of free radicals, a reducible species or interaction with heme. Furthermore, the cyclic GMP formed in response to nitroprusside, acetylcholine and atrial natriuretic factor mediates relaxation through a common mechanism that may be functionally antagonized by agents and procedures which result in membrane depolarization. PMID- 2998823 TI - Synergistic interaction of 4-aminopyridine with neostigmine at the neuromuscular junction. AB - The pre- and postsynaptic events at the neuromuscular junction were studied in vitro in rat skeletal muscle exposed to clinically significant concentrations of 4-aminopyridine (4-AP), neostigmine or combinations of the two drugs. Simultaneous application of 4-AP and neostigmine produced increases in the amplitudes of nerve-evoked end-plate potentials which were significantly greater than the summed effects of the drugs applied individually. Such synergism was present at the junctions where transmission was blocked either postsynaptically by d-tubocurarine or presynaptically by low [Ca2+]0 and high [Mg2+]0. Quantal content analysis in the latter preparation indicated that the evoked release of acetylcholine was potentiated significantly more than the amplitude of spontaneous miniature end-plate potentials, suggesting that the site of synergism is predominantly presynaptic. For symptomatic relief and long-term management of the neuromuscular junction disorders, we propose a combined medication of aminopyridine and anticholinesterase at reduced dosages. Such therapy would minimize adverse effects and be particularly effective in the treatment of such presynaptic disorders as the Lambert-Eaton myasthenic syndrome and botulism. PMID- 2998824 TI - Biochemical mechanisms of regulation of mucus secretion by prostaglandin E2 in rat gastric mucosa. AB - The effects of prostaglandin E2 (PGE2) and inhibitors of RNA and protein synthesis on rat gastric mucosa were investigated in order to study the cellular and biochemical mechanisms involved in the PGE2-stimulated formation and secretion of gastric mucus. It was shown that PGE2 caused significant stimulation of gastric mucus secretion and this effect of PGE2 was inhibited by cycloheximide but not actinomycin D. The influence of PGE2 on the in vitro incorporation of N acetyl-[3H]glucosamine and 14C-labelled amino acids in to the glycoproteins representing a major mucus component of the isolated gastric mucosa cells was also studied. The stimulatory effect of PGE2 on incorporation of labelled precursors into glycoproteins of gastric cells was also inhibited by cycloheximide. These results suggest that the effect of PGE2 on mucus production requires ongoing protein synthesis. cAMP can fully reproduce the effect of PGE2 on the formation and the secretion of gastric mucus. The binding of [3H]PGE2 to rat gastric non-parietal cell fractions consisting predominantly of mucoid cells correlated with the ability of PGE2 to increase adenylate cyclase activity in these cells. PGE2 had no effect on adenylate cyclase activity in cell suspensions enriched in parietal cells. These data suggest further that the stimulatory effect of PGE2 on mucus secretion may be mediated by cAMP as a messenger. PMID- 2998825 TI - Comparison of the effects of detomidine and xylazine on some alpha 2-adrenoceptor mediated responses in the central and peripheral nervous systems. AB - The effects of detomidine, a novel veterinary sedative analgesic, on some alpha 2 adrenoceptor-mediated responses in the central and peripheral nervous systems were studied. In pithed rats, detomidine was a very potent agonist at both pre- and postsynaptic alpha 2-adrenoceptors. Doses of 1.9 micrograms/kg and 6.5 micrograms/kg inhibited electrically induced tachycardia by 50% and increased mean blood pressure by 50 mmHg, respectively. In comparison, xylazine, though similar in specificity, was 40 times less potent than detomidine in this preparation. In unanaesthetized rats, detomidine both caused sedation and induced complex changes in body temperature. Low doses caused decreases in rectal temperature but these were reversed as the dose was increased. The decrease in rectal temperature could be blocked by yohimbine. Prazosin somewhat inhibited but did not eliminate the hyperthermia seen with the very high doses of detomidine. Xylazine caused much more severe falls in rectal temperature which could not be completely antagonized by alpha 2-adrenoceptor blockade. Both detomidine and xylazine caused dose-dependent mydriasis in anaesthetized rats, detomidine being about 10 times more potent than xylazine. The mydriatic effects of detomidine could be prevented by alpha 2- but not by alpha 1-adrenoceptor blockade. It is concluded that detomidine is a potent and rather specific alpha 2-adrenoceptor agonist in the central and peripheral nervous systems. In comparison with xylazine, detomidine has higher potency and greater specificity, especially at central alpha 2-adrenoceptors. PMID- 2998826 TI - Pharmacological characterization and regional distribution of alpha-noradrenergic binding sites of rat spinal cord. AB - In order better to interpret their physiological role in rat spinal cord, we characterized binding sites of [3H]WB-4101 and [3H]p-aminoclonidine ( [3H] PAC), and determined their regional distribution. These binding sites have characteristics required for, respectively, alpha 1 and alpha 2 receptors of norepinephrine. Binding to these sites is saturable, with Kd values of 0.38 nM and 35 nM for high and low affinity binding sites respectively of [3H]WB-4101; and 1.7 nM, for a single binding site of [3H]PAC. For whole cord, Bmax values are 52 and 320 (high and low affinity sites respectively); and 21 fmol/mg protein. Catecholamines compete stereoselectively for these sites, while selected noradrenergic agents compete with an order of potency corresponding to their relative activity at the alpha 1 and alpha 2 receptors. We conclude that spinal alpha 1 and alpha 2 binding sites have the same pharmacologic properties as corresponding peripheral sites. The alpha 2 and, to a lesser degree, the alpha 1 binding sites vary in concentration with region. Our results support the contention that alpha 2 binding sites subserve neuronal function in the spinal cord. PMID- 2998827 TI - Estrogen effects on nigral glutamic acid decarboxylase activity: a possible role for catecholestrogen. AB - Repeated but not single injections of estradiol benzoate significantly reduced nigral glutamic acid decarboxylase (GAD, EC 4.1.1.15). A single injection of the catecholestrogen 2-hydroxyestradiol produced similar results. Tolerance developed to the latter effect, as reflected by the lack of nigral GAD activity changes in rats repeatedly injected with 2-hydroxyestradiol. Repeated injection of the antiestrogen tamoxifen not only failed to antagonize the action of estradiol benzoate but itself reduced nigral GAD activity. Hypophysectomy, which itself decreased nigral GAD activity prevented the lowering effects of either repeated estradiol benzoate administration or single 2-hydroxyestradiol injection on the enzymatic activity. PMID- 2998829 TI - Serologic and virologic surveys on feline herpesvirus and feline calicivirus infections in cats for experimental use. AB - Serologic survey and virus isolation of feline herpesvirus (FHV) and feline calicivirus (FCV) were performed on cats used for research at the Laboratory Animal Research Centre, The University of Tsukuba, over the period from 1978 to 1981. Of the 507 mature and immature cats, 4 months old or older, 102 (20.1%) had HI antibody against FHV and 412 (81.3%) SN antibody against FCV. Some 23 (16.2%) and 76 (53.5%) kittens among 142 younger than 4 months had antibodies against FHV and FCV, respectively. Both the antibodies in kittens were considered to be maternally derived. The FCV antibody rate was especially high in cats weighing 2.5 kg (males) and 2.0 kg (females) or more, which were regarded as 8 to 10 months of age. An attempt was made to isolate the viruses from the oropharynx and conjunctiva of clinically healthy mature or immature cats and kittens. As the result, either one or both of the viruses were isolated from 31 of 75 mature and immature cats, and isolation rates of FHV and FCV were 6.7% and 36.0%, respectively. On the other hand, no virus was detectable in 16 kittens. PMID- 2998828 TI - Diphenylalkylamine calcium antagonists interact with alpha-adrenoceptor binding sites in aortic membranes. AB - Some interactions of calcium antagonists with [3H]prazosin and [3H]yohimbine binding sites were investigated in bovine aorta membranes. Diphenylalkylamines (flunarizine, cinnarizine and bepridil) acted as competitors of the two ligands with Ki values in the microM range. With the exception of verapamil, reference compounds (nifedipine, Bay-K 8644, diltiazem) and the peripheral benzodiazepine receptor antagonist PK 11195 did not displace the ligands. The apparent affinity of the diphenylalkylamines for alpha-adrenoceptor was consistent with the concentrations producing vasodilatation. PMID- 2998830 TI - Evaluation of tri-combinant vaccine for feline herpesvirus, calicivirus and panleukopenia virus infections in Japanese native cats. AB - Tri-combinant vaccine consisting of attenuated feline herpesvirus (FHV) and feline calicivirus (FCV) and inactivated feline panleukopenia virus (FPLV), were evaluated for safety and efficacy, using Japanese native cats and the viral strains isolated in Japan. Thirty-eight 9- to 12-week-old kittens were inoculated intramuscularly and subcutaneously with the vaccine. Consequently, no adverse reaction was found, and protective efficacy was confirmed by challenge tests with the virulent strains of each virus. Serum-neutralizing antibodies against FCV and FPLV were maintained for at least one year after vaccination, whereas antibody against FHV disappeared in two cases at 24 weeks after vaccination. Application of this vaccine seemed effective for control of feline viral disease in cats for experimental use. PMID- 2998831 TI - Immunohistochemical detection of mammary tumor virus antigens in sweat and sebaceous glands of mice. AB - Mammary tumor virus (MTV) antigens in both sexes of GRS/A, SHN and C3H mice were examined in the sweat and sebaceous glands by immunoperoxidase technique using antiserum against gp52, envelope protein, or p27, core protein. Balb/c mice were used for reciprocal foster nursing with these inbreds to discriminate the expression of endogenous MTV from that of exogenous MTV. Both antigens were first detected around the age of 4 months in the sweat glands of mice with endogenous GR- or SHN- MTV. A linear staining of gp52 was seen along the luminal borders of glandular cells, and the reaction products for gp52 were demonstrated on the apical cell membranes, where no virion could be seen ultrastructurally. A diffuse staining of p27 was found in the cytoplasm of some glandular cells, where MTV particles could not be detected. In the sebaceous gland of the same mice, however, only p27 was first detected at the age of 60 days. A dot-like staining of p27 was found in the perinuclear region of some glandular cells, where an aggregation of intracytoplasmic A particles could be seen under an electron microscope. These positive stainings were unrelated to sex. In such skin appendages of all examined C3H mice and Balb/c mice with GR- or SHN- MTV, no antigen expression could be seen up to the age of 500 days. Therefore, some genes might be able to regulate the expression of endogenous MTV antigens in the skin appendages, while their glandular cells would have no receptor for exogenous MTV, namely the so-called "milk factor". PMID- 2998832 TI - [Effects of repeated injections of epinephrine on lipolysis in rats]. AB - The effects of repeated injections of epinephrine on lipolysis in rats were investigated in vivo and in vitro. By daily subcutaneous injections of epinephrine (100 micrograms/kg) for 20 days, the weight of epididymal adipose pads were significantly decreased. By repeated injections of epinephrine for 20 days, the extent of increase in plasma nonesterified fatty acid (NEFA) levels after epinephrine injection became smaller, and the amount of epinephrine-induced NEFA and cyclic AMP release from epididymal adipose tissue were decreased. These results suggested that the lipolytic response to epinephrine in rats was decreased by repeated injections of epinephrine. PMID- 2998834 TI - Virosome-mediated implantation of red cell band 3 into the plasma membrane of cultured hepatoma cells. AB - A method for implanting exogenous membrane proteins into recipient hepatoma cells is described. Red cell band 3 and Sendai virus envelope proteins HN and F were extracted from their respective sources and purified by centrifugation to equilibrium through sucrose step gradients in the presence of octyl-beta-D glucopyranoside. 0.05-0.15 micron vesicles were formed by adding lipid to combined detergent solubilized, isolated membrane proteins and removing detergent by dialysis. The vesicles were hybrid band 3-Sendai envelope vesicles and not a mixture of two distinct vesicle types as judged by (1) the ability of Sendai specific antibody to immunoprecipitate greater than 99% of band 3 from vesicle suspensions and (2) comigration of band 3 and Sendai envelope proteins on isopyknic sucrose density gradients. The hybrid vesicles (virosomes) were not fusogenic but did bind to cultured hepatoma cells in the cold. Subsequent treatment of virosomes absorbed onto cultured cells with polyethylene glycol resulted in a stable association of 2-10% of added band 3 and Sendai envelope proteins with the cells. Efficient transfer of virosome-associated band 3 to the cells was dependent on both lipid and Sendai envelope proteins. Fluid phase marker transfer, immunofluorescence, and protease digestion experiments demonstrate that the majority of the virosomes were implanted into recipient hepatoma membranes and not simply adsorbed onto their surface or immediately endocytosed. The hybrid membrane protein-viral envelope vesicles thus offer an efficient means for insertion of foreign proteins into the membranes of recipient cultured cells. PMID- 2998833 TI - Cell cycle effects of iron depletion on T-47D human breast cancer cells. AB - T-47D human breast cancer cells grown in culture medium containing low concentrations of fetal calf serum (FCS) proliferated very slowly, with an accumulation of cells in the G2 phase of the cell cycle, increased polyploid cells, and increased expression of transferrin receptors. Cell proliferation was stimulated by the addition of human transferrin or ammonium ferric citrate to the medium. Growth inhibition and accumulation of G2-phase cells could also be produced in T-47D cells grown in medium containing 10% FCS by the addition of the iron chelator, desferrioxamine. It is concluded that cellular deprivation of iron and/or transferrin is the major cause of reduced proliferation rates and G2-phase arrest which accompany the culture of these cells in medium supplemented with low concentrations of FCS. PMID- 2998835 TI - Degradation of exogenous membrane proteins implanted into the plasma membrane of cultured hepatoma cells. AB - The degradation of radiolabeled red cell band 3 and Sendai envelope proteins was studied after band 3 virosomes were fused with hepatoma cells as previously described (Hare, J E & Huston, M, Exp cell res 161 (1986) 317) [26]. 125I-band 3 (T1/2 = 13-14 h), Sendai HN (T1/2 = 37-40 h), and F (T1/2 = 21-23 h) envelope proteins were degraded by an apparent first-order process that was greater than 90% sensitive to 20 mM NH4Cl. 125I-Sendai envelope proteins were degraded at approximately similar rates when hepatoma cells were fused with intact virus, isolated viral membrane, or band 3 virosomes. There thus appears to be distinct heterogeneity among the degradation rates of implanted polypeptides dependent on structural aspects of each. To identify the subcellular site of membrane protein degradation, band 3 was labeled with membrane impermeant [14C]sucrose and implanted into hepatoma plasma membranes. After replating, trichloroacetic acid (TCA)-soluble label was found to accumulate in the lysosomal compartment of fractionated cells. The results identify the lysosome as the ultimate site of plasma membrane protein degradation, but suggest that plasma membrane proteins are selectively rather than non-selectively delivered to this compartment. PMID- 2998836 TI - Epidermal growth factor receptor expression related to differentiation capacity in normal and transformed keratinocytes. AB - Epidermal growth factor (EGF) and Ca2+ have been indicated to play a major role in skin development. We have used normal keratinocytes, SV40-transformed keratinocytes (SVK14) and various squamous carcinoma cell (SCC) lines as in vitro model system to study the effect of the extracellular Ca2+ concentration of EGF receptor expression in relation to the capability of cells to differentiate. The cell lines used exhibit a decreasing capacity to differentiate in the order of keratinocytes approximately SVK14 greater than SCC-12F2 greater than SCC-15 greater than SCC-12B2 greater than SCC-4, as judged from Ca2+-ionophore-induced cornified envelope formation. Under normal Ca2+ conditions, all cell lines (except for SCC-15) exhibited two classes of EGF-binding sites. The number of low affinity binding sites increased considerably as cells were less able to differentiate, while the apparent dissociation constant (kd) was similar in all cell lines. In contrast, the properties of high-affinity EGF binding varied in the various cell lines without a clear relationship to the degree of differentiation capacity. Lowering the extracellular Ca2+ concentration to 0.06 mM resulted in a decrease of Ca2+ ionophore-induced cornified envelope formation, demonstrating the decreased ability to differentiate under these conditions. The decreased ability to differentiate was accompanied by a marked increase in the number of EGF-binding sites, but without a change of the kd. Furthermore, no high affinity EGF-binding sites were detectable under these conditions. Finally, addition of Ca2+ to low Ca2+-cultured cells caused a rapid decrease of EGF binding in all cell lines, most prominently in normal keratinocytes and SCC-12F2 cells. The data presented demonstrate: The combination of normal keratinocytes, SVK14 and the various SCC lines provides an attractive model system to study differentiation in vitro; EGF-receptor expression is related to the state of differentiation, both phenomena being sensitive to the external Ca2+ concentration; and EGF-receptor expression is related to the capability of cells to differentiate. PMID- 2998837 TI - Large T antigen-rich viral DNA replication loci in SV40-infected monkey kidney cells. AB - The nuclear distribution of the large T antigen (T-Ag) during lytic infection of CV1 monkey kidney cells with SV40 virus was studied by immunoelectron microscopy. The viral protein was associated with the cellular chromatin and also accumulated within a small number of clearly delimited areas of the nucleoplasm. These T-Ag rich areas were devoid of viral particles but contain 3-10 nm DNA filaments in an amorphous matrix. We have named these areas 'viral DNA/T-Ag loci.' The combination of the immunostaining for T-Ag with ultrastructural autoradiography revealed that these viral DNA/T-Ag loci were the sites of active SV40 DNA synthesis. We suggest that the viral DNA/T-Ag loci may represent definite structural domains specifically involved in viral DNA replication regulated by SV40-T antigen. PMID- 2998838 TI - Age-related changes in collagenase expression in cultured embryonic and fetal human skin fibroblasts. AB - Since skin collagenase is required for initiation of the degradation of types I and III collagens, the major collagens of the human dermis, we examined its expression during embryonic and fetal development. When using skin fibroblasts cultured from human embryos and fetuses, immunoreactive collagenase concentrations were strongly correlated with estimated gestational age (p less than 0.001), with levels at 7-8 weeks of gestation that were about one-twentieth of those in the 29-week cell cultures. In crude culture medium, the apparent catalytic efficiency (activity per unit immunoreactive protein) was variable, an observation attributable in part to variable expression of a collagenase inhibitory protein. Following chromatographic purification, four of ten fetal collagenases were found to have greater than or equal to 4-fold decrease in specific activity, suggesting that these particular fetal collagenases may be structurally and/or catalytically altered. Since the decreased levels of immunoreactive protein suggested that decreased enzyme synthesis was the major mechanism, we examined collagenase synthesis in a cell-free translation system. Here, we quantitated collagenase expression in the culture medium of intact cells prior to harvesting mRNA. Compared with the intact adult cells, the fetal cells had 3-17 times less collagenase activity in the medium, while in cell-free translation there was a 2- to 3-fold decrease in collagenase synthesis. These data suggest that decreased in vitro expression is correlated with decreased levels of translatable collagenase mRNA but that other factors, such as the collagenase inhibitor and altered specific activity of the enzyme, may be important in modulating collagenase activity. PMID- 2998839 TI - Low temperature-induced cell surface membrane vesicle shedding is associated with DNA fragmentation. AB - Temperature shift conditions of 0 degree to 22 degrees C or 0 degree to 37 degrees C induce the formation and shedding of membrane vesicles (MV) from P815 tumor cell surfaces. When the MV shedding process takes place at 22 degrees C it occurs without changes in cell surface membrane permeability, whereas at 37 degrees C, changes in permeability to 51Cr and trypan blue do occur, thus mimicking the lymphocyte-mediated lytic process of tumor cells [1]. The present studies demonstrate that nuclear DNA fragmentation also occurs in both 0 degree to 22 degrees C and 0 degree to 37 degrees C temperature shifts but cell surface membrane permeability to DNA fragments occurs only in the latter condition, i.e., 0 degree to 37 degrees C. The microtubule-stabilizing agent deuterium oxide (D2O) inhibited the MV shedding process, the changes in membrane permeability, and DNA fragmentation. When P815 cells which had been induced to shed MV by the 0 degree to 22 degrees C temperature shift were labeled with 51Cr and used as targets for alloimmune lymphocytes, they were found to be as susceptible to T-cell lysis as control P815 cells. This result indicates that the lytic effect of alloimmune T lymphocytes can be exerted at the target cell surface membrane level independently of nuclear DNA fragmentation. PMID- 2998840 TI - Reversion of bovine papillomavirus-induced transformation and immortalization by a xanthate compound. AB - Bovine papilloma virus-transformed hamster embryo fibroblasts (HEF-BPV) reacted to exposure to tricyclodecan-9-yl-xanthogenate (D609) with immediate reversion to the growth kinetics and the flat morphology of the untransformed parental cells. After six population doublings in the presence of D609, clones which displayed an untransformed morphology in the absence of D609 arose with a high frequency (90%). Such clones had reacquired a limited in vitro lifetime and had lost the ability to induce tumors in athymic nude mice. At the molecular level the revertant clones had lost all extrachromosomal monomeric BPV-1 DNA molecules. Only high molecular weight (HMW) oligomeric BPV-1 DNA that was probably integrated into the cellular genome was still detectable in a methylated transcriptionally inactive state. In contrast to transformed cells, the revertant clones no longer transcribed BPV-1-specific mRNA molecules, but were stimulated by a tumor promoter to transient viral gene expression. This article provides direct evidence for the complete reversibility of the property of "immortality". PMID- 2998841 TI - Cell configuration and adhesive properties of metastasizing and non-metastasizing BSp73 rat adenocarcinoma cells. AB - The pattern of cell substrate interaction, the cell surface composition and the organization of cytoskeletal elements was studied in tumour cell variants of the BSp73 rat adenocarcinoma displaying different metastatic capabilities and cell configuration. The non-metastasizing AS variant cells adhered to the substrate and spread via vinculin-containing focal contacts. These cells also synthesized, secreted and assembled fibronectin at the pericellular area. The metastasizing ASML variant cells adhered to the substrate at a slower rate via thick cytoplasmic protrusions, but were removed from the substrate by trypsin-EDTA slower than the non-metastasizing AS variant cells. The ASML cells also synthesized very low levels of both vinculin and fibronectin, displayed a diffuse pattern of actin and tubulin organization, and were unable to spread on the substrate. Spreading could not be induced in the ASML cells by seeding the cells on an extracellular matrix derived from bovine corneal endothelial cells or on concanavalin A (conA)-coated substrates, or by the addition of db-cAMP to the medium. The metastasizing cells expressed a unique and abundant cell surface glycoprotein of Mr 170 000 which was also shed into the growth medium. The relationships among the adhesive properties, the organization of cell surface components and of the cytoskeleton in the tumour cell variants, and the expression of their metastatic phenotype is discussed. PMID- 2998842 TI - Modulation in protein phosphorylation during G2 phase of the cell cycle in two cell mouse embryos. AB - The protein phosphorylation activities in extracts were assayed for 2-cell mouse embryos at three stages of the G2 phase of the cell cycle. The 2-cell embryos were unique in having a prolonged G2 phase and so easily staged at early G2 (EG2), middle G2 (MG2) and late G2 (LG2) by timing the embryo isolation from pregnant mice. The embryo extracts were used both as sources of protein kinases and their substrates. The phosphoproteins of the extracts were labelled with [gamma-32P]ATP and separated by electrophoresis on SDS-polyacrylamide gels. The present study revealed that protein phosphorylation increased 3-6-fold during the progression of 2-cell embryos from EG2 to LG2 and the level of protein phosphorylation at any stages was greatly decreased by the presence of cAMP. Thus, the protein phosphorylation system of 2-cell mouse embryos seems to differ from those reported systems in mammals in its negative dependence on cAMP. PMID- 2998843 TI - Neurofilament phosphorylation in development. A sign of axonal maturation? AB - Monoclonal antibodies to the 200K neurofilament (NF) protein selectively decorated axons in tissue sections. Dilution of the antibodies in phosphate buffer and digestion with phosphatase abolished the stain. With conventional monoclonal and polyclonal NF antibodies, i.e. antibodies decorating NF regardless of their location (axons, perikarya and dendrites), the staining was not affected by this treatment. With all antibodies, axon-specific and conventional, the staining was abolished by trypsin digestion. Subsequent digestion with phosphatase did not restore the staining. Compared with conventional NF antibodies, staining with axon-specific anti-NF 200K was a late phenomenon in chick embryo development. NF 200K immunoreactivity was first observed in peripheral nerves and in the anterior columns of the spinal cord on day 10. Sensory ganglia and optic nerve fibers were negative. With conventional NF antibodies these structures were stained on days 4 and 5, respectively. In the following days of development the study was confined to the retina, optic nerves, cranial peripheral nerves and sensory ganglia. Up to day 16, bundles of thin peripheral nerve fibers, strongly decorated by conventional NF antibodies, did not stain with anti-NF 200K in double labelling experiments. Nerve bundles emerging from the ganglia were also negative, although some thick nerve fibers within the ganglia were stained. NF 200K immunoreactivity was first observed on day 17 in the optic nerve and in the layer of optic nerve fibers. At this time, staining was confined to the bundle emerging from the temporal side of the retina. In newborn chicken, only few fibers stained with anti-NF 200K in the nasal bundle, while the temporal bundle was well stained. It is suggested that the NF 200K antibodies reacted with a phosphorylated epitope in the axon, and that NF phosphorylation is a late event in ontogenesis probably related to axonal maturation. PMID- 2998844 TI - The lateral mobility of cell membrane components is not altered following cell fusion induced by Sendai virus. AB - The interaction of Sendai virus glycoproteins with cell membranes was proposed to increase the lateral mobility of membrane proteins, enabling membrane fusion and the aggregation of intramembrane particles by thermotropic separation (Volsky, DJ & Loyter, A, Biochim biophys acta 514 (1978) 213 [13]; Maeda, T et al. Exp cell res 123 (1979) 333 [15]; and Kim, J & Okada, Y, Exp cell res 132 (1981) 125 [44]). In order to test this hypothesis, we employed fluorescence photobleaching recovery to investigate the effects of Sendai virus-induced fusion on the lateral mobility of membrane proteins and lipids in a variety of cell types (human erythrocytes, BHK21, HeLa, 3T3 NIH, and mouse spleen lymphocytes). The results of the lateral diffusion measurements demonstrate that no significant alterations occur in the lateral motion of membrane proteins or a fluorescent phospholipid on all the cell types examined, including cells which revealed high susceptibility to the virally mediated fusion (human erythrocytes and BHK21 cells). These findings suggest that a permanent increase in the lateral mobility of cell surface components does not generally occur during Sendai virus-induced cell fusion, and thus cannot play a role in the fusion mechanism. The possible involvement of transient alterations in the lateral mobility of membrane components in the fusion mechanism is discussed. PMID- 2998845 TI - Quiescent human diploid fibroblasts. Common mechanism for inhibition of DNA replication in density-inhibited and serum-deprived cells. AB - The mechanism for cessation of proliferation in density-inhibited quiescent human diploid fibroblasts (HDF) and serum-deprived quiescent HDF was compared in two ways. Density-inhibited HDF were fused to either replicating HDF or SV40 transformed HDF and DNA synthesis was measured in the resulting heterokaryons. DNA synthesis was inhibited in the replicating HDF nuclei in heterokaryons in a way that suggested that entry into S phase was blocked, but ongoing DNA synthesis was not inhibited. In contrast, DNA synthesis was induced in the quiescent nuclei in heterokaryons formed with SV40-transformed HDF. Previous experiments had shown that serum-deprived HDF also behave in this way in heterokaryons. To test this similarity further, we examined the inhibitory activity of cell membranes prepared from both types of quiescent HDF. We found that both types of quiescent HDF contain DNA synthesis-inhibitory activity that is (1) effective on replicating HDF; (2) ineffective on SV40-transformed HDF; (3) sensitive to heat and trypsin. Thus, these results support the hypothesis that both density inhibited HDF and serum-deprived HDF share a common mechanism for arrest in G1 phase. They also suggest that a membrane-bound protein plays a role in the inhibition of DNA synthesis in quiescent HDF. PMID- 2998846 TI - Systematic shut-off of the hormone receptors in intraspecific adrenal x Leydig cell hybrids. AB - The mouse Y1 adrenal cell line was fused with mouse Leydig cells in primary culture. The selected hybrids were examined for their response to gonadotropin (hCG) and ACTH. None of them bound specifically [125I]hCG, nor did they augment their cAMP production in response to gonadotropin or ACTH stimulation, whereas their adenylate cyclase remained responsive to forskolin and cholera toxin, thus indicating a repression of hCG receptor synthesis and probably a loss of ACTH receptors, rather than a lesion of the coupling between the hormone receptor complex and the adenylate cyclase. Basal pregnenolone production in 17 hybrids was close to that of Leydig and Y1 cells and was enhanced after 8-bromo adenosine 3',5'-monophosphate (8-Br-cAMP) stimulation in 11 of them. Therefore, the negative control leading to the extinction of both parental functions acts preferentially at the first step of steroidogenesis, i.e., the gene(s) coding for the hormone receptors. PMID- 2998847 TI - Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli spermatogenic cell co-cultures. AB - Sertoli-spermatogenic cell co-cultures prepared from sexually immature rats (20 22 days old) and maintained in serum-free, hormone/growth factor-supplemented medium were used to determine the cell-specific localization of the growth factor somatomedin-C (SM-C). SM-C localization studies were carried out by indirect immunofluorescence using a monoclonal antibody (sm-1.2) to SM-C. In cultured rat hepatocytes, Sertoli and testicular peritubular cells, SM-C immunoreactivity was observed as a diffuse distribution of discrete immunofluorescent granules. Radio immunoassay experiments using a rabbit antibody against human SM-C showed that testicular peritubular cells and Sertoli cells in primary culture accumulated SM C in the medium. In spermatogenic cells co-cultured with subjacent Sertoli cells, immunoreactive SM-C was associated with pachytene spermatocytes but not with spermatogonia or early meiotic prophase spermatocytes (leptotene or zygotene). Both Sertoli cells and pachytene spermatocytes displayed binding sites for exogenously added SM-C. SM-C6 binding to spermatocytes reaching an advanced stage of meiotic prophase suggests a possible role of this growth factor in the meiotic process. PMID- 2998848 TI - Ocular responses to superoxide generated by intraocular injection of xanthine oxidase. AB - Xanthine oxidase (XaO) was injected into the anterior chamber of rabbit eyes by a closed circuit perfusion system. Doses of 1.5 milliunits (mU) or greater produced a maximal leucocyte accumulation after 4 hr, with an initial elevation of ocular pressure in the first 15 min. Similar experiments on rats with intravitreal injections of 0.1-1.5 mU of XaO resulted in a significant accumulation of leucocytes after 5 hr which, at the highest dose of XaO, was partly due to traces of bacterial endotoxin in the XaO. However, in endotoxin-desensitized rats the response to 1.5 milliunits XaO was seven-fold greater than the response to endotoxin alone. Simultaneous administration of xanthine (Xa) substrate with XaO was not required to elicit cell infiltration into the anterior chamber. Dialyzed enzyme was also effective but boiling abolished the response. Addition of XaO to rabbit aqueous humor in vitro decreased the ascorbate content, consistent with the generation of superoxide from an endogenous substrate. The results suggest that enzymatically active XaO, which can cause intraocular generation of superoxide from an XaO substrate present in aqueous humor, initiates the chemotactic response. A chemotactic agent may be generated from superoxide reacting with endogenous precursors in aqueous humor or by selective activation of the lipoxygenase pathway of arachidonic acid metabolism in adjacent tissues. PMID- 2998849 TI - High concentration of retinoic acid binding protein in a retinoblastoma. PMID- 2998850 TI - Studies on the eye lens in poikilothermal animals. I. Comparative studies on cation maintenance systems in rainbow trout and rat lenses. AB - The response of the poikilothermal lens to various incubation temperatures in vitro was compared with that of the homothermal lens. The rainbow trout lens was used as the poikilothermal lens and the rat lens as the homothermal lens. In contrast to rat lenses, cataract developed at 37 degrees C in rainbow trout lenses, which was called 'warm cataract'. Warm cataract developed not only when lenses were incubated in vitro but also when rainbow trout were kept in water at 37 degrees C. Water, Na+, Ca2+ and insoluble protein increased and K+ and Mg2+ decreased in warm cataract lenses, but GSH and soluble protein sulfhydryl levels did not change. This cataract was irreversible after only 5 min incubation at 37 degrees C. On the other hand, rainbow trout lenses remained transparent without the change of cation balance at 0-25 degrees C while cold cataract developed in rat lenses. Na,K-ATPase activity was detected at 0 degrees C in rainbow trout lens homogenates, but not in rat lens homogenates. Na+-K+ ratio (Na+/K+) increased when the rainbow trout lens was treated with ouabain at 0 degrees C. In the rainbow trout lens, lactic acid was produced continuously for 30 days at 0 degrees C while it was not in the rat lens between 1 hr and 10 days after. These results strongly suggest that Na,K-ATPase acts as a cation pump at 0 degrees C and that ATP is supplied by glycolysis in the rainbow trout lens in order to maintain the transparency. The above results also suggest that enzymes and membrane structures in rainbow trout lens are adapted to a cold-temperature habitat and that Na,K-ATPase and anaerobic glycolysis are important for the maintenance of lens transparency at low temperatures. PMID- 2998851 TI - Studies on the eye lens in poikilothermal animals. II. Stimulation of anaerobic glycolysis in rainbow trout lenses incubated with Ca2+-free medium. AB - The effects of Ca2+-free medium on cation levels and lactic acid production of rainbow trout lens incubated at various temperatures were examined in order to study passive cation transport system. Na+-K+ ratio (Na+/K+) increased when the lens was treated with Ca2+-free medium, but the effect of Ca2+-free medium was lowest between 15 and 20 degrees C which was the optimum water temperature for rainbow trout. Lactic acid production was stimulated between 15 and 20 degrees C in Ca2+-free medium. It was also stimulated at 37 degrees C in rat lenses incubated with Ca2+-free medium. However, the production was suppressed in ouabain-treated rainbow trout lenses. The suppression of Na+/K+ increase between 15 and 20 degrees C disappeared when the lens was treated with both Ca2+-free medium and ouabain. These results suggest that Na,K-ATPase activity and glycolysis were stimulated to prevent the increase of Na+/K+ in Ca2+-free medium. In addition, these results indicate that the optimum temperature was between 10 and 20 degrees C for the rainbow trout lens incubation in vitro, which is also the optimum water temperature in vivo. PMID- 2998852 TI - Calcium-dependent regulation of adenylate cyclase and phosphodiesterase activities in bovine lens: involvement of lens calmodulin. AB - When calmodulin levels were determined in bovine lens layers at different ages, the values in the epithelial cell layer were strikingly higher than in the cortical layer and higher than in the nucleus. In the epithelial cell layer, except in very old animals, the calmodulin levels were maintained in adult animals. In the lens nucleus the extremely low level of calmodulin decreased during aging. In the cortical layer there were no systematic changes of calmodulin level during aging. In vitro, low calcium concentrations (micromolar) activated and higher calcium concentrations (over 100 microM) inhibited the lens adenylate cyclase activity. cAMP and cGMP degradation by phosphodiesterase was activated by calcium. It is suggested that calmodulin might be involved in the regulation of both adenylate cyclase and phosphodiesterase activities of lens epithelial cells and that free calcium plays a well-defined role in cAMP synthesis. PMID- 2998854 TI - Cathepsin B and D, and Ca2+-dependent neutral protease activities in the retina of taurine-depleted rats. AB - Rats were treated with guanidinoethanesulfonic acid (GES), a taurine transport inhibitor, which reduces the tissue content of taurine. The quantity of taurine in the rat retinas after GES treatment was reduced by 46% after the first week, 60% after the second week, and 67% after the third week. Activities of cathepsin B and D were not significantly altered when calculated on the basis of either protein or DNA content in the taurine-depleted retinas. However, cytosolic Ca2+ dependent neutral protease activity in retinas from GES-treated rats increased by 55% after the third week. PMID- 2998853 TI - Tyrosinase activity in the uveal tissue of the adult bovine eye. AB - Tyrosinase activity in crude extracts from various tissues of the adult bovine eye was examined biochemically. Enzyme activity was measured by using L-3,4 dihydroxyphenylalanine (L-DOPA) as substrate and determining colorimetrically by an increase in absorbancy at 400 or 475 nm. Tyrosinase activity was found in the ciliary body, iris, and choroid with the ciliary body having the highest enzyme activity. The enzyme was 1324-fold purified from the crude extract of the ciliary body by ammonium sulfate fractionation, trypsin digestion, followed by chromatography on Sephacryl S-200, hydroxylapatite, and DEAE-cellulose columns. The apparent Km value for L-DOPA was 0.2 mM and the molecular weight of the enzyme was estimated to be 70000 by gel method. The enzyme activity was markedly reduced by phenylthiourea and diethyldithiocarbamate, specific inhibitors of tyrosinase. PMID- 2998856 TI - Two components of long-term potentiation in mossy fiber-induced excitation in hippocampus. AB - Experiments were made in thin transverse sections of the guinea pig hippocampus to clarify whether tetani to a heterosynaptic input can induce long-term potentiation (LTP) in the absence of seizure discharges and whether the heterosynaptic LTP occurs in a monosynaptic or polysynaptic pathway. Early and late responses were differentiated in field potentials elicited in region CA3 by mossy fiber stimulation. The LTP of the early response was specific to a tetanized pathway, whereas heterosynaptic LTP was observed in the late response in the absence of seizure discharges. The magnitude of LTP of the early response was significantly smaller than that of the late response. LTPs of the early and late responses were not induced at a low Ca2+ concentration. A brief exposure to a high Ca2+ solution resulted in a long-lasting potentiation of both the early and late responses. Counterparts to the early and late responses were recorded from the distal dendritic layer in reversed polarities. The intracellular counterpart to the late response was a slow depolarization superimposed on the peak and falling phase of excitatory postsynaptic potentials. These results suggest that LTP of the early response reflects enhanced transmission through synapses between mossy fibers and CA3 neurons and LTP of the late response reflects modified interaction among postsynaptic neurons. PMID- 2998855 TI - Association of lipid peroxidation during luteal regression in the rat and natural aging in the rotifer. AB - Lipid peroxides (LP) were measured in whole homogenates and membrane fractions from luteinized rat ovaries and in homogenates of aging rotifers. There was a significant increase in the accumulation of LP as plasma progesterone levels declined during luteal regression. Lipid peroxide levels also increased with age in rotifers. This study indicates that lipid peroxide accumulation is associated with cellular breakdown and suggests that the rat corpus luteum may be used as a mammalian aging model. PMID- 2998857 TI - Mechanism of intestinal adaptation in rats with acute renal failure. AB - Acute renal failure (ARF) was experimentally induced in rats and the specific activity of mucosal Na-K-ATPase activity in segments of the small intestine and colon was measured. Bilateral nephrectomy (BN) resulted in a significant evaluation of the enzyme activity in all segments examined. With an additional procedure of adrenalectomy (BN + Ax), the enzyme activity failed to show any increase in ARF rats produced by BN. However, a supplementation of a maintenance dose of dexamethasone to adrenalectomized ARF rats (BN + Ax + DM 10) resulted in a significant resumption of the activity in all intestinal segments, although its increase was insignificant in the duodenum. Addition of a high dose of DOCA (BN + Ax + DOCA 500) was effective in increasing the enzyme activity only in the colon but not in the small intestine. With a high dose of DM or a maintenance dose of DM plus a high dose of DOCA (BN + Ax + DM 30 or BN + Ax + DM 10 + DOCA 500), there was an increase in the enzyme activity of all intestinal segments. In ARF rats induced by bilateral lower ureteral ligation (BLUL), the enzyme activity did not show any increase at all. Addition of a high dose of DOCA to this animal model (BLUL + DOCA 500) brought about the increase of the enzyme activity in the intestinal segments but for the jejunum.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2998858 TI - Chemical evidence for interactions between vitamins E and C. AB - Experimental proof is provided for interactions between radicals of vitamin E/vitamin C as generated by air-oxidized lipids (liquid fraction of subcutaneous chicken fat). Using ESR spectroscopy, hydrogen atom exchange is shown to take place between vitamin C and the radical of vitamin E. Sequential consumption of these two vitamins in oxidized lipid, first vitamin C then vitamin E, is demonstrated by means of differential pulse polarography. These results elucidate the in vitro radical scavenging functions attributed to vitamin E and vitamin C as well as their synergism in lipid antioxidation. PMID- 2998859 TI - Lithium suppresses hibernation in the Turkish hamster. AB - Daily uptake of lithium salt (LiCl) in the drinking water at a rate of over 100 micrograms/g b.wt (or 2.4 mEq/kg) reduced or suppressed natural torpidity (hibernation) in the Turkish hamster (Mesocricetus brandti). The data indicate a direct influence of lithium on clock-related functions controlling the hibernation process rather than indirect effects by preventing gonadal regression and thereby also hibernation. PMID- 2998861 TI - The effect of sulfhydryl compounds on the catalytic activity of Cu, Zn-superoxide dismutase purified from rat liver. AB - Sulfhydryl compounds such as reduced glutathione, cysteine and 2 mercaptopropionylglycine, a hepato-protective agent, activated Cu, Zn-superoxide dismutase purified from rat liver at low concentrations (below 10 microM). Furthermore we found evidence indicating that this activation is achieved by reducing Cu2+ present in the catalytic site of the dismutase, and thereby promoting the dismutation of superoxide anions. PMID- 2998860 TI - Benzamide potentiation of behavioral apomorphine-induced effects; mechanism involved. AB - A new N-pyridinyl benzamide was found to potentiate strongly the effects of apomorphine on the motility of reserpinized mice and on circling behavior. Since dopaminergic agonist activity could not account for this potentiation, involvement of alpha 2-adrenergic agonist activity provided the only consistent explanation. PMID- 2998862 TI - Selective protection of cells against X-irradiation. Isoproterenol protects only those cells that possess beta-adrenoreceptors. AB - In mixed culture of Chinese hamster fibroblasts, clone 431, and transformed murine L fibroblasts, clone B-82, isoproterenol was found to protect only 431 cells against ionizing radiation. It was shown that 431 cells, in contrast to B 82 cells, possess beta-adrenoreceptors, and the radioprotective effect of isoproterenol can be realized only if this agent interacts with beta adrenoreceptors coupled with the cAMP system. Since malignization often causes the disappearance of beta-adrenergic and other hormone receptors, the combined culturing and irradiation of the cells studied can be regarded as a model of the growth of malignant cells (B-82) among normal tissue cells (431 cells) under conditions of radiation therapy. A possibility of selective protection against radiation damage of normal tissue cells, with retention of the former radiosensitivity of tumor cells, is discussed. PMID- 2998864 TI - Effects of lead inclusion bodies on subcellular distribution of lead in rat kidney: the relationship to mitochondrial function. AB - The effect of Pb pretreatment on the subcellular binding of a tracer dose of 203Pb was studied in kidneys of rats in which intranuclear and cytoplasmic inclusion bodies had been induced by a single ip injection of Pb acetate (50 mg Pb/kg) 6 days earlier. Results of subcellular fractionation studies in rats injected iv with 203Pb 24 hr prior to sacrifice demonstrated that 203Pb activity was about 1.5 times higher in kidney homogenates and mitochondrial fractions of control compared to Pb-pretreated rats. Cytosolic 203Pb activity in control rats was 5 times higher than that in Pb-pretreated rats. In contrast, Pb pretreatment increased the 203Pb binding capacity to the nuclear and inclusion body fractions by 7 and 20 times, respectively, compared with controls. Pb pretreatment decreased total mitochondrial 203Pb binding but resulted in a higher proportion of 203Pb bound to the inner membrane and matrix fractions relative to the controls. After in vitro incubation of control renal mitochondria with 203Pb the binding to inner and outer membranes and matrix fractions increased with increasing concentration of unlabeled lead added to the incubation. Deposits on the inner membrane of isolated mitochondria from Pb-pretreated rats were observed by isotonic ammonium molybdate negative staining and these mitochondria also showed decreased respiratory control ratios (RCRs). Succinate-mediated respiration rates and membrane binding of the fluorescent probe ethidium bromide were not affected by Pb pretreatment. These data indicate that lead influences its own subcellular distribution in the kidney following Pb pretreatment as shown by the increase of nuclear and inclusion body binding and increase of mitochondrial inner membrane and matrix binding of lead. The mitochondrial inner membrane shows a preferential affinity for lead following in vivo treatment which can be correlated with impairment of a specific inner membrane function (depressed RCRs). Lead exposure did not alter the activity of the mitochondrial membranes to undergo energy linked conformational changes. PMID- 2998863 TI - Action of TSH on nuclear ADP-ribosylation in dog thyroid slices. AB - Treatment of dog thyroid slices with thyrotropin (TSH) results in an increase in ADP-ribosylation in nuclei isolated thereafter. This increase is time-dependent and is observed with concentrations of TSH eliciting physiological responses. The technique described here does not involve permeabilization of cell membranes, thereby avoiding artefacts which could arise from hypotonic shock. Cyclic AMP mimicked the stimulatory action of TSH. PMID- 2998865 TI - Biological properties of griseolic acid, a cyclic AMP phosphodiesterase inhibitor with an adenine group. AB - Griseolic acid inhibited cAMP phosphodiesterase (PDE) at low concentrations, the I50 being of the order of 0.01-0.1 microM. Administration of griseolic acid to rats increased the cAMP level in liver and plasma several-fold. It increased glycogen degradation in mouse liver and stimulated lipolysis in isolated rat fat cells. Griseolic acid did not block the adenosine-elicited accumulation of cAMP in guinea pig brain slices. It had no effect on cAMP-dependent protein kinase from rat liver nor on the adenyl cyclase from rat brain. PMID- 2998866 TI - Intact human lymphocyte membranes respond to muscarinic receptor stimulation by oxotremorine with marked changes in microviscosity and an increase in cyclic GMP. AB - The muscarinic agonist oxotremorine produced a linear dose-dependent increase in membrane fluidity of intact and viable human lymphocytes in vitro. This effect proved to be receptor-mediated because preincubation with 10(-5)M atropine shifted the dose-response curve one order of magnitude rightward. Pirenzepine preincubation did not affect membrane fluidity variation. A cGMP increase was also found after oxotremorine treatment. The results are discussed in terms of possible modulation of guanyl cyclase and adenyl cyclase through membrane fluidity variations. PMID- 2998867 TI - Stimulation of specific GTPase activity by vasopressin in isolated membranes from cultured rat hepatocytes. AB - Membranes were isolated by isotonic homogenization and differential centrifugation from rat hepatocytes cultured overnight. The specific GTPase activity of the membranes was 1-1.3 pmol gamma-labelled GTP hydrolysed/mg protein per min in the presence of 1.2 mM Na+, 2 mM EGTA, 1 mM ATP and 0.2 mM 5-adenylyl imidodiphosphate. Under these conditions there was a stimulation of specific GTPase activity of no more than 20% by 11-115 nM vasopressin. No effect of vasopressin was seen in the presence of 1.7 microM free Ca2+ or 100 mM Na+. The findings indicate that vasopressin is able to influence GTPase activity as well as accelerate phosphoinositide breakdown in rat hepatocytes. PMID- 2998868 TI - Oxidation of nitroxide radicals by an iron-hydrogen peroxide-amino acid system. AB - The oxidation of nitroxide radicals by the reaction of hemoglobin with hydrogen peroxide occurred using both FeSO4 and amino acids instead of hemoglobin. Tyrosine, phenylalanine, tryptophan, methionine and cysteine in globin were connected with the formation of the oxoammonium cation. Of these amino acids, tyrosine was especially effective in its oxidation. It was found that both the binding of nitroxide radicals to ferric iron and the activation of amino acids by hydroxyl radicals were important in the radical oxidation. PMID- 2998869 TI - Enhancement of O2- generation and tumoricidal activity of murine macrophages by a monosaccharide precursor of Escherichia coli lipid A. AB - The effects of a monosaccharide precursor of Escherichia coli lipid A (lipid X) on murine macrophages were studied. Lipid X is a diacylglucosamine 1-phosphate bearing beta-hydroxymyristoyl groups at positions 2 and 3. Lipid X, as well as lipopolysaccharide and lipid A, enhanced O2- generation in mouse peritoneal macrophages and a macrophage-like cell line, J774.1, and further induced the tumor-cytotoxic activity of peritoneal macrophages. Elimination of a 1-phosphate or 3-O-beta-hydroxymyristoyl groups are essential for the elevated O2- generation and induction of tumoricidal activity due to lipid X. PMID- 2998870 TI - Rapid isolation of animal mitochondrial DNA by alkaline extraction. AB - A simple technique for rapid isolation of mitochondrial DNA (mtDNA) from animal cells is described. The method is based on the selective alkaline denaturation procedure of Birnboim and Doly [(1979) Nucleic Acids Res. 7, 1513-1523] and avoids the use of CsCl gradient centrifugation. The yield of mtDNA is comparable to that obtained by standard techniques. This DNA is sufficiently pure for restriction analysis and cloning of mtDNA fragments. PMID- 2998871 TI - Expression of genes for subunits of plant-type RuBisCO from Chromatium and production of the enzymically active molecule in Escherichia coli. AB - A DNA fragment containing genes for both large (A) and small (B) subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from a photosynthetic bacterium Chromatium vinosum was ligated with vectors for expressing unfused proteins and introduced into cells of Escherichia coli. The expressers of RuBisCO were screened on agar plates using the specific antibody raised against the native enzyme from Chromatium. The production of both subunits A and B in the expressers was demonstrated by an immunoblotting experiment. The amount of RuBisCO produced in the E. coli cells was as high as 15% of the total soluble protein after induction with isopropyl-beta-D-thiogalactoside. The specific activity of enzyme molecules produced in E. coli was nearly the same as that of the original Chromatium enzyme. On gel filtration high-performance liquid chromatography the two enzymes showed identical elution behavior, strongly indicating their similar quaternary structures. PMID- 2998872 TI - Spin label studies of the essential sulfhydryl group environment in chicken liver fructose-1,6-bisphosphatase. AB - The local environment of the essential sulfhydryl groups in chicken liver fructose-1,6-bisphosphatase has been investigated by ESR techniques using a series of iodoacetamide spin labels, varying in chain length between the iodoacetate and nitroxide free radical group. The ESR spectrum of spin-labeled chicken liver fructose-1,6-bisphosphatase showed that the sites of labeling were highly immunobilized when the enzyme was chemically modified by spin label iodoacetate, suggesting that the sulfhydryl groups of the protein are in a small, confined environment. From the change in the ESR spectra of these nitroxides as a function of chain length, we conclude that the sulfhydryl group is located in a cleft approx. 10.5A in depth. PMID- 2998873 TI - Action of cobra venom cardiotoxin on chick embryonal fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus. AB - The cytolytic action of cardiotoxin analogue III from the venom of the Formosan cobra on chick embryonal fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus was investigated. The 50% effective dose of the toxin for the cells cultured at a non-permissive temperature (41 degrees C) or for noninfected normal cells was about 8 micrograms/ml whereas the value was 2 micrograms/ml for the cells cultured at a permissive temperature (36 degrees C). This indicates that the transformed cells became more susceptible to the cytolytic action of the toxin than the non-transformed cells. PMID- 2998874 TI - Activation of the PCSM-protein phosphatase by a Ca2+-dependent protease. AB - The inhibitor-1 phosphatase but not the phosphorylase phosphatase activity of a newly discovered 250 kDa polycation-stimulated (PCSM) protein phosphatase in rabbit skeletal muscle is increased up to 10-fold by a Ca2+-dependent protease. The enzyme-directed protease effect to which the PCSH and PCSL phosphatases are insensitive was progressively lost during purification of the enzyme. This could be explained by either a slow conversion of the enzyme to an active form of the enzyme with a change in specificity, or the loss of a protease-sensitive inhibitor of the inhibitor-1 phosphatase activity, resulting in a PCS phosphatase characterized by its high inhibitor-1/phosphorylase alpha activity ratio. The Ca2+-dependent protease is completely inhibited by EGTA or leupeptin. PMID- 2998876 TI - High enkephalyl peptide degradation, due to angiotensin-converting enzyme-like activity in human CSF. AB - The metabolism of enkephalin peptides was studied in human cerebrospinal fluid. The degradation rates of (Leu)-enkephalin and (Leu)-enkephalin-Arg6 were compared and the latter was degraded at a 10-fold higher rate. The major enzyme activity was investigated by Mr determination and inhibition experiments, showing marked similarity with angiotensin-converting enzyme. PMID- 2998875 TI - Parallel inactivation of alpha 2-adrenergic agonist binding and Ni by alkaline treatment. AB - Alpha 2-Adrenergic receptor-mediated inhibition of adenylate cyclase requires the guanine nucleotide-binding protein, Ni. This protein may also be required for stabilization of high-affinity alpha 2-adrenergic agonist binding. Human platelet membranes treated under alkaline conditions (pH 11.5) exhibited a selective loss of high-affinity agonist binding as measured by p-[3H]aminoclonidine and [3H]UK 14,304. Binding of the antagonist [3H]yohimbine was largely unaffected with retention of greater than 60% of control binding sites. Ni, determined by pertussis toxin-catalyzed [32P]ADP-ribosylation of cholate extracts from alkaline treated membranes, was also markedly reduced. The parallel loss of alpha 2 agonist binding and Ni provides additional evidence that Ni is required for alpha 2-adrenergic agonist binding. PMID- 2998877 TI - Delta and mu opiate receptor probes: fluorescent enkephalins with high receptor affinity and specificity. AB - The fluorescent amino acid, L-1-pyrenylalanine (Pya) was incorporated into [D Ala2,Leu5]enkephalin and its methyl ester at position 4 or 5. Pya-enkephalins showed strong fluorescent intensity and displayed high binding affinity for opiate receptors. Pya4-enkephalins showed high specificity for the mu receptors, while Pya5-enkephalins showed high specificity and selectivity for the delta receptors. Particularly, [D-Ala2,Pya5]enkephalin was as potent as the most utilized delta-specific ligand of [D-Ala2,D-Leu5]enkephalin (DADLE), and yet its delta-selectivity was about 5-times greater than that of DADLE. Thus, Pya enkephalins per se can be utilized as a fluorescent probe or tracer for the opiate receptor-binding assays. PMID- 2998878 TI - Structure of the rat pro-opiomelanocortin (POMC) gene. AB - The gene encoding pro-opiomelanocortin (POMC) presents unique regulatory features. In particular, glucocorticoids inhibit transcription of the POMC gene in the anterior pituitary, but not in the intermediate pituitary. In order to study the mechanism leading to transcriptional inhibition of POMC by glucocorticoid and the interaction of the glucocorticoid receptor complex with specific DNA sequences along the POMC gene, we have cloned the rat POMC gene and determined its structure. The gene is composed of three exons and appears to be present at a single copy per haploid genome. Besides the usual regulatory signals like 'TATA' and 'CCAAT' boxes, the upstream region contains sequences homologous to known enhancer sequences and to the glucocorticoid receptor binding site observed in glucocorticoid-responsive genes. PMID- 2998879 TI - Fusion of Sendai virions with phosphatidylcholine-cholesterol liposomes reflects the viral activity required for fusion with biological membranes. AB - Sendai virus envelopes were reconstituted after solubilization of intact virions with either Triton X-100 or octylglucoside. Envelopes obtained from Triton X-100, but not from octylglucoside solubilized virions, were hemolytic and promoted cell cell fusion. Fluorescence dequenching studies [using N-4-nitrobenzo-2-oxa-1,3 diazole phosphatidylethanolamine-bearing viral envelopes] revealed that both preparations fused with negatively charged phospholipids. Fusion with phosphatidylcholine (PC)/cholesterol (chol) liposomes was promoted only by the hemolytic viral envelopes. Fluorescence dequenching studies, using intact virions bearing octadecylrhodamine B chloride, revealed that intact virions fused with PC/chol as well as with negatively charged phospholipids. Only fusion with PC/chol liposomes was inhibited by phenylmethylsulfonyl fluoride and dithiothreitol, reagents which are known to block the viral ability to fuse with biological membranes. PMID- 2998880 TI - Location of two of the introns in the antithrombin-III gene. AB - At least two of the introns in the antithrombin-III (AT-III) gene are located in positions different from those of the other three proteins in this superfamily for which the gene structures are known, namely, ovalbumin, alpha 1-antitrypsin and angiotensinogen. In another part of the 3'-portion of the AT-III gene there is no intron where each of the other three gene structures has one. PMID- 2998881 TI - Membrane-bound N-acetyl-beta-glucosaminidase. Different binding specificity in control and I-cell disease livers. AB - Several lysosomal enzymes solubilized at pH 4 from saponin-treated membranes showed markedly variable affinities to the bovine liver phosphomannosyl receptor in both bovine and human livers. The enzymes from I-cell disease liver did not bind the receptor in spite of normal intracellular activities. N-Acetyl-beta glucosaminidase was effectively released from the membrane preparation of a control liver by mannose 6-phosphate, and other sugars showed little effect in this experiment. However, in the I-cell disease liver, dissociation occurred not by mannose 6-phosphate but by other sugars, such as fucose, mannose and N-acetyl D-glucosamine. These results indicate the presence of an alternate transport system other than the pathway mediated by the mannose 6-phosphate receptor, and the role of other sugar-binding proteins is discussed in intracellular processing and transport of newly synthesized enzymes. PMID- 2998883 TI - Presence of membrane-associated phosphatidate phosphohydrolase activity in cultured islets and its stimulation by glucose. AB - The cellular location at which exogenous phosphatidic acid is hydrolysed in cultured neonatal rat islets was examined. Phosphatidate phosphohydrolase activity could be demonstrated in both whole cell sonicates and isolated plasma membranes. In the whole cell fraction phosphatidic acid hydrolysis to diacylglycerol was stimulated 43% by the presence of Mg2+. The activity present in isolated membranes was totally dependent on the presence of Mg2+ and was increased in plasma membranes from glucose-stimulated islets. Following exposure of islets to low glucose concentrations, raising the Ca2+ concentration from 150 nM to 40 microM in the presence of Mg2+ did not affect the formation of diacylglycerol in whole cell fractions or plasma membranes. These results indicate the presence within the islet of membrane-bound phosphatidate phosphohydrolase activity and demonstrate its activation by glucose. PMID- 2998882 TI - Polymyxin B inhibits phorbol 12-myristate 13-acetate, but not chemotactic factor, induced effects in rabbit neutrophils. AB - The addition of the amphipathic polycationic antibiotic polymyxin B to a suspension of rabbit neutrophils results in inhibiton of the agonist (secretion of secondary granules) and antagonist (inhibition of chemotactic factor induced degranulation) properties of phorbol 12-myristate 13-acetate. On the other hand, polymyxin B does not inhibit the degranulation of the neutrophils that is induced by chemotactic factors. These results imply that the role of protein kinase C in the initiation of neutrophil functions in response to the addition of chemotactic factors is less critical than previously thought. In addition, the reversal of the inhibitory properties of phorbol esters by polymyxin B indicates that the former are mediated by the ability of the tumor promoters to activate protein kinase C. These results thus strengthen the hypothesis that protein kinase C plays important roles in the regulation (as contrasted to initiation) of neutrophil functions. PMID- 2998884 TI - Distribution of atrial natriuretic factor receptors in dog kidney fractions. AB - Specific receptors for atrial natriuretic factor were studied in purified glomeruli, proximal tubules, thick ascending limbs of Henle's loops and collecting ducts from dog kidney. Glomeruli contain the highest concentration of receptor sites (pK = 9.9, Bmax = 200 fmol/mg protein), followed by collecting ducts (pK = 9.4, Bmax = 150 fmol/mg). Low levels of receptor sites were also detectable in thick ascending limbs of Henle's loops (pK = 9.4, Bmax = 36 fmol/mg) while proximal tubules were completely devoid of specific binding sites. These results indicate that the glomeruli appear to be the primary site of interaction of atrial natriuretic factor in kidney cortex but that it might also act in the medulla on lower nephron tubular function. PMID- 2998885 TI - Total thyroidectomy: the treatment of choice in differentiated thyroid carcinoma? AB - A review of 46 patients with differentiated thyroid cancer, diagnosed and treated in the St. Radboud Hospital from 1977 till 1984, is presented. The age of the patients ranged from 16 to 80 years. There were 39 women and 7 men. Thirty of 31 patients with papillary carcinoma and 13 of 15 patients with follicular carcinoma underwent total thyroidectomy. If less than total thyroidectomy had been performed, 13 (43%) patients with papillary cancer and 2 (15%) with follicular cancer would have had cancer left in the residual lobe. The complication rate was acceptable, two cases of permanent hypoparathyroidism, one recurrent nerve palsy. During a short follow-up period of 7 years maximum already 6 patients older than 60 years with papillary carcinoma had died, 5 of widespread cancer (16.6%) and one of an unrelated disease. Three patients developed local recurrences, on in the trachea and 2 outside the thyroid bed. One patient with follicular carcinoma, who had undergone a lobectomy, developed recurrent disease. These figures plus the increased risk of complications in a second neck exploration suggest that total thyroidectomy is the treatment of choice for patients with differentiated thyroid cancer. Total thyroidectomy can be done without mortality and without significant morbidity. PMID- 2998886 TI - Reduction in the stearic to oleic acid ratio in human malignant liver neoplasms. AB - Total lipid extracts of liver tissue from 14 patients with primary and secondary liver tumours were analysed for relative values of saturation of 18 carbon chain length fatty acids (C18FA). The saturation indices (ratio C18S:C18u) of the tumour areas were significantly and consistently lower than the corresponding values in the non-tumour areas (P less than 0.001). It is proposed that the relative increase in unsaturated C18FA (oleic acid) could prove to be a chemical marker reflecting deficient cellular control of the desaturation of stearic acid. PMID- 2998887 TI - Ryanodine block of calcium oscillations in heart muscle and the sodium-tension relationship. AB - The control of tension is examined in cardiac Purkinje fibers. We show, in accordance with earlier results (14, 15), that tension produced by this preparation is a steep power function of intracellular sodium. With the aid of ryanodine, a pharmacological agent that blocks spontaneous and spatially asynchronous calcium release from the sarcoplasmic reticulum (SR), we investigate the influence that such calcium fluctuations have on tension. We find that even when we control for alterations of intracellular pH, the presence of such fluctuations reduces the dependence of tension on intracellular sodium. We present a simple model that can explain how the presence of oscillations of intracellular calcium leads to a reduction of the slope of the tension-calcium relationship. We show, furthermore, that when the oscillations are spatially asynchronous, this reduction of slope is even greater. The modeling takes account of the known relationships between tension and calcium and tension and sarcomere length. We conclude that the effect of ryanodine to steepen the tension-sodium relationship can be explained by ryanodine's blocking calcium release from the SR, thereby abolishing oscillations of intracellular calcium. PMID- 2998889 TI - [Change in the status of sensory inputs into the secondary neurons of the olfactory bulb in response to adequate stimulation]. PMID- 2998888 TI - [Features of the properties of neurons of different structuro-functional groups of the parietal association cortex]. AB - Neuronal responses in parietal associative cortex to different kinds of stimulation were studied in anaesthetized and immobilized cats. Analysis of responses to white matter stimulations (area 5) divided the neurons into two groups: those with cortical output (29%) and those with cortical input as well as intermediate neurons related to processing of information in cortical circuits (71%); the inhibition on white matter stimulation was shorter in the former neurons, their responsiveness to peripheral stimulation being better than in the latter group. PMID- 2998890 TI - [Effect of enkephalins on the peripheral effects of adrenalin]. AB - In 140 white male rats, effects of lei-enkephalin synthetic analogue and adrenaline on heart rate, cAMP concentration in the heart and liver, glycogen concentration in the liver, parathormone content in the blood plasma, were studied. The lei-enkephalin analogue was shown to hinder the changes of these parameters induced with adrenaline. Enkephalins seem to suppress the effect of catecholamines upon adenylatcyclase and to reduce the cAMP level in tissues. PMID- 2998891 TI - [Intensification of vagal inhibition of the work of the heart by a sympathetic nerve]. AB - Stimulation of the stellate ganglion sympathetic nerve added to stimulation of the vagus under conditions of obsidan suppression of beta-adrenoreceptors, intensifies the vagal inhibitory effect on the heart work. Possible participation of adrenergic and serotoninergic structures in the mechanism of the inhibitory phenomenon, was investigated. PMID- 2998892 TI - [Effect of temperature on the adrenoreactivity of the small intestine in the rat]. AB - In isolated sections of the rat small intestine, adrenergic responses to phenylephrine (P), noradrenaline (NA) and isopropylnoradrenaline (IN) were studied at various temperatures of the incubation medium. Raise of the temperature from 33 to 37 degrees C increased the sensitivity of adrenoreceptors to P and NA 3.2-and 2.1-fold but did not change the sensitivity to IN. This change of adrenoreactivity was eliminated by phentolamine blockade of alpha adrenoreceptors, and the raise of temperature always produced a decrease in maximal reaction of the small intestine. The change of adrenoreceptors of the small intestine smooth muscles seems to be related primarily to the rearrangement of alpha-adrenoreceptors. PMID- 2998893 TI - [Changes of peripheral mononuclear cell subpopulations in autoimmune thyroid diseases]. AB - To estimate abnormalities in humoral or cellular immunity that relate to the etiologies of Graves' disease (GD) and Hashimoto's thyroiditis (HT), peripheral mononuclear cell subpopulations were enumerated by an immunofluorescence technique using monoclonal antibodies. Also antithyrotropin receptor antibodies (thyrotropin-binding inhibitor immunoglobulin, TBII) were measured by the radioreceptor assay according to Smith's method; and antithyroglobulin antibodies (TGHA) and antithyroid microsomal antibodies (MCHA), by the tanned red cell hemagglutination technique. The data obtained were analyzed for the count of peripheral total white cells, lymphocytes and granulocytes, and to the level of serum free thyroxine (T4), free T4 index (FT4I) and free triiodothyronine index (FT3I). Peripheral white cells tended to be decreased in some cases of GD and HT. The absolute count of lymphocytes was slightly increased in GD but did not change in HT. On granulocytes (neutrophils mainly), the absolute count was considerably decreased in hyperthyroid GD. In this disease, FT3I showed significantly positive correlations to the percentage and absolute count of peripheral lymphocytes, whereas FT4I revealed significantly negative correlations to the percentage and count of granulocytes. These facts indicate that lymphocytosis and granulocytopenia in GD might be ascribed partially to the direct effects of thyroid hormones. The percentage of peripheral OKT3-positive cells (common T lymphocytes) was significantly decreased in untreated cases of GD, showing negative correlations to FT4, FT4I and FT3I. The absolute count of OKT4-positive cells (inducer/helper T lymphocytes) was increased in untreated cases of GD and hyperthyroid and euthyroid cases receiving antithyroid drugs (ATD) for GD. But peripheral OKT8-positive cells (suppressor/killer T lymphocytes) was significantly decreased in HT. The percentage of peripheral OKT8-positive cells in GD was also decreased in untreated and remitted cases, but slightly more increased in ATD-medicated cases than in nonmedicated cases. The ratios of OKT4 positive cells to OKT8-positive cells (OKT4+/OKT8+ ratios) were significantly increased in untreated, ATD-medicated euthyroid and remitted cases of GD, and in HT. It seems that ATD may exert direct effects on OKT8-positive cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2998894 TI - [A simple and highly sensitive radioimmunoassay for 8-arginine vasopressin in human plasma using a reversed-phase C18 silica column]. AB - A highly sensitive and simple radioimmunoassay for the measurement of 8-arginine vasopressin (AVP) in human plasma has been developed. The dose response curve ranges from 0.025 to 8 pg/tube. This simple extraction technique employing an ODS C18 column recovered 87.1 +/- 10.4 (mean +/- SD)% of AVP in the range of 1-10 pg/ml added to 0.5 ml plasma. Determination of AVP in each fraction of plasma, which was gel-filtrated through a Sephadex G25 (1 X 25 cm), revealed that the fraction of plasma AVP was superimposed on that of authentic AVP, and interference of non-specific substances was completely eliminated by an ODS C18 column. Using the assay, 13 of 16 patients with diabetes insipidus (DI) showed plasma AVP concentrations ranging from 0.03 to 0.21 pg/ml, and the other 3 patients had less than 0.03 pg/ml. The AVP concentrations of DI were clearly distinguished from those obtained in normal subjects (0.30-4.20 pg/ml, n = 65). The within and between assay variability was about 10% each. Plasma AVP concentrations (mean +/- SD) of normal subjects (n = 6) standing, sitting, and supine after an overnight of fluid deprivation were 2.41 +/- 1.15, 1.95 +/- 0.85 and 0.97 +/- 0.48 pg/ml (30 min after) respectively. Plasma AVP concentrations of normal subjects (n = 6) after water load (20 ml/kg wt) were clearly reduced from 1.89 +/- 1.00 (before) to 0.42 +/- 0.21 (standing for 60 min) and also from 0.89 +/- 0.41 to 0.40 +/- 0.22 pg/ml (supine for 60 min). PMID- 2998895 TI - [Studies on the pathogenesis of subacute thyroiditis]. AB - Despite the fact that subacute thyroiditis (SAT) is a common disease, its pathogenesis is still uncertain. At present, viral infection is simply thought to be a possible pathogenic factor. In Caucasians and Chinese, however, a strong association has recently been found between HLA-Bw35 and SAT. Therefore, genetic factors are also thought to participate in the onset of SAT. In this study we have further attempted to clarify the pathogenesis of this disease in this regard. PROTOCOL: HLA typing for A, B and C loci (class I antigens) was performed on 89 patients with SAT (males: 7, females: 82) aged from 23 to 66 years, and DR and MT loci (class II antigens) were investigated in 55 patients. In 12 patients, viral antibody titers were measured as soon as possible after onset and two weeks later. The viral antibodies evaluated were those of Influenza A and B, Coxsackie A9, B1, B2, B3, B4, B5 and B6, Echo 3, 7, 11 and 12, Parainfluenza 1, 2, 3 and 4, and Adeno 8 virus. The following results were obtained: In class I HLA typing, the frequency of HLA-Bw35 in SAT was 67.4%, which was significantly (p less than 0.0001) higher than that in the control (14.1%). On the other hand, the frequency of Cw1 in SAT (14.6%) was significantly (p less than 0.01) lower than that of the control (32.1%), and that of Cw3 (65.2%) was significantly (p less than 0.01) higher than that of the control (46.5%). In class II HLA typing, the frequency of DRw8 in SAT was 38.2%, which was significantly (p less than 0.05) higher than that of the control (21.9%). Other antigens showed no significant changes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2998896 TI - [The effects of dopamine on the release of immunoreactive beta-endorphin-like peptide from the dispersed cells of the rat neurointermediate lobe]. AB - The intermediate lobe of the rat pituitary gland is a homogeneous population of cells which synthesize and secrete various peptides related to ACTH and lipotropin derived from a common precursor, proopiomelanocortin. Catecholamine beta-receptor (beta-adrenoceptor) and dopamine receptor which are present in the intact cells of the intermediate lobe, remain functional in the enzymatically dispersed cells. In this study we investigated the mechanism by which beta endorphin is released from the dispersed cells of the neurointermediate lobe of the rat pituitary gland. 1-Isoproterenol stimulated the release of immunoreactive beta-endorphin-like peptide (IR-beta-Ep) and the accumulation of adenosine 3', 5' monophosphate (cAMP). On the other hand, dopaminergic drugs, apomorphine, bromocriptine, dopamine, lergotrile and lisuride, decreased the rate of release of IR-beta-Ep. Dopamine also inhibited the stimulatory effects of 1-isoproterenol on the release of IR-beta-Ep and cAMP accumulation. Dopamine antagonists, fluphenazine and sulpiride, diminished the inhibitory effects of dopamine on the release of IR-beta-Ep and cAMP accumulation which were stimulated by 1 isoproterenol. it has been reported that cholera toxin enhanced the release of IR beta-Ep and the accumulation of cAMP in the rat neurointermediate lobe. After preincubation in the incubation medium containing 30 nM cholera toxin for 2 hours, the cells (CT cells) showed spontaneous release of IR-beta-Ep and cAMP accumulation. 1-Isoproterenol had no effect on the release of IR-beta-Ep and cAMP accumulation in CT cells. Dopamine, however, inhibited both the release of IR beta-Ep from CT cells and cAMP accumulation in CT cells. These results suggest that dopamine may be involved in the regulatory mechanism of IR-beta-Ep release from the dispersed cells of the rat neurointermediate lobe. PMID- 2998897 TI - [Studies on the feedback regulation of pulsatile luteinizing hormone secretion. II. Role of the catecholaminergic system in estrogen-induced suppression of the frequency of pulsatile LH secretion in ovariectomized rats]. AB - The pattern of pulsatile secretion of luteinizing hormone (LH) is affected by the circulating gonadal hormone levels. In the rat the frequency and amplitude of the LH pulse increase after ovariectomy and decrease after estrogen replacement. Since estrogen is known to affect the catecholaminergic activities in the rat hypothalamus, the present study examined whether the catecholaminergic mechanisms are involved in the estrogen-induced suppression of pulsatile LH secretion. Recently, we have elucidated that the preoptic suprachiasmatic area (POSC) is a specific site of action of estradiol in reducing the LH pulse frequency. Subsequently, in this paper, we report the effects of various catecholamine synthesis inhibitors and synaptic blockers on the basal pulsatile LH secretion and on the frequency suppression induced by local implantation of EB in the POSC. Female rats of the Wistar strain were ovariectomized about 4 weeks before the experiment. Blood samples were obtained at 6-min intervals for 4 h without anesthesia through the indwelling atrial catheter. The rats were given i.p. injection with the drug or the vehicle 1 or 3h before bleeding. The steroid was implanted into the POSC via the chronically-implanted cannula 1 h after the initiation of the bleeding. Serum LH concentrations were determined by radioimmunoassay. The following results were obtained. EB implantation into the POSC suppressed the frequency of existing pulsatile LH secretion in vehicle treated rats. Pretreatment of the rat with alpha-methyl-p-tyrosine (AMPT), AMPT + threo-dihydroxyphenylserine (DOPS) or pimozide did not affect the basal pulsatile LH secretion but prevented suppressive effect of EB implantation on the LH pulse frequency. In rats pretreated with diethyldithiocarbamate (DDC) or phenoxybenzamine the basal pulsatile LH secretion was abolished. Further suppression by EB implantation was not clear. Pretreatment of the rats with propranolol did not affect the basal pulsatile LH secretion nor the EB-induced suppression of the LH pulse frequency. These results support the hypothesis that the alpha-adrenergic mechanism is required for maintaining the basal pulsatile LH secretion in ovariectomized rats. Furthermore, the dopaminergic system is obligatorily necessary for the manifestation of the inhibitory action of EB implanted in the POSC on the LH pulse frequency. PMID- 2998898 TI - A highly specific detection method for diethylstilbestrol in bovine urine by radioimmunoassay following high performance liquid chromatography. AB - A specific radioimmunoassay (RIA) for diethylstilbestrol (DES) is presented following purification of sample extracts by isocratic reversed phase high performance liquid chromatography. Applications are given of a forensic investigation for the control of DES in bovine urine. Specificity of HPLC-RIA is compared with that of RIA's with other (chromatographic) purification procedures and with gas chromatography-mass spectrometry. Implications of the use of these techniques in practice and the use of various DES-antisera are discussed. PMID- 2998899 TI - [Solid epidermoid hidradenoma of the large toe]. PMID- 2998900 TI - Significance of glycosidases and phosphatases in Cercopithecus and Macaca cells. AB - When selected ratios of different glycosidases and phosphatases from primary monkey kidney cells or from monkey kidney cell lines are presented graphically, characteristic patterns do evolve. Three different subtypes of Vero cells show similar glycosidase patterns. The Vero subtypes tested show glycosidase patterns that are closely similar to those of primary cells of Cercopithecus aethiops. Glycosidase patterns of BS-C-1 and CV-1 cells are less similar to those of primary Cercopithecus cells than are those of Vero cells. Primary kidney cells from Macaca cynomolgus show significantly different glycosidase patterns compared with those of different Cercopithecus cells. The distinct glycosidase patterns can be used to classify the tested cell lines in relation to each other. PMID- 2998901 TI - Mitogenic effects of certain cathepsins and calciferin on the intact liver in vivo. AB - Calciferin, a new parathyroid hormone stimulating the release of cathepsins D and L (but not B) from isolated lysosomes, or the release of cathepsin D from erythrocytes or ghosts in vitro, elevated free cathepsin D in the blood, and at the same time stimulated DNA synthesis in the intact liver when it was injected into mice. Both calciferin and free cathepsin D in the blood (rats) were elevated concomitantly soon after 70% hepatectomy, reaching a peak around 5 hr. The cathepsin D-elevation was almost proportional to fractional hepatectomies. Cathepsin L (but not B), when injected intraperitoneally into mice, stimulated DNA synthesis and mitosis in the intact liver much like cathepsin D, the effect of which was reported earlier. In contrast to the mitogenic effects of calciferin or cathepsins (D and L) in vivo, only cathepsin L (but not cathepsin D or calciferin) in low concentrations appeared to stimulate DNA synthesis in the cultured liver cells, and also stimulated adenylate cyclase of isolated liver plasma membranes in vitro. Dibutyryl-cyclic AMP in concentrations lower than 10( 5) M also stimulated DNA synthesis in cultured liver cells. PMID- 2998903 TI - Ethane dimethane sulphonate (EDS) specifically inhibits LH stimulated steroidogenesis in Leydig cells isolated from mature rats but not in cells from immature rats. AB - Effects of ethane dimethane sulphonate (EDS) on the pattern of protein synthesis, steroid production and ATP levels in isolated Leydig cells have been investigated. After incubation of Leydig cells isolated from mature rats with EDS (75 micrograms/ml) for 3-5 h, the synthesis of a 33 kDA and 50 dKa protein and LH stimulated steroid production was inhibited, but the LH stimulated cAMP production and conversion of 22R-hydroxycholesterol to testosterone were not affected. Busulphan or ethyl methyl sulphonate (EMS) at similar molar concentrations had no effect on steroid production. After 24 h incubation with EDS Leydig cells were detached from the plastic surface and had rounded up, but the cellular ATP levels were the same as in control cells. Leydig cells were destroyed after incubation with EMS 2000 micrograms/ml for 24 h. EDS had no detectable effects on steroid production by isolated Leydig cells from mice, from Leydig cell tumour tissue or from immature rats, nor on rat adrenal cells or on LH and FSH secreting pituitary cells. The data indicate that EDS specifically inhibits LH regulated functional properties of mature Leydig cells possibly via alkylation of proteins. EDS could be a valuable tool to study possible regulator proteins for control of steroidogenesis in Leydig cells from adult rats. PMID- 2998902 TI - The phosphorylation of proteins: a major mechanism for biological regulation. Fourteenth Sir Frederick Gowland Hopkins memorial lecture. PMID- 2998904 TI - alpha-Melanotropin-induced changes in protein phosphorylation in melanophores. AB - To investigate a possible role of protein phosphorylation in the mechanism of action of alpha-MSH, excised tail-fins of Xenopus tadpoles were incubated with or without alpha-MSH. After homogenization, in vitro endogenous protein phosphorylation was assayed using [gamma-32P]ATP. alpha-MSH treatment of intact tail-fins, producing full pigment dispersion, resulted in a 5-fold increase in 32P-incorporation into a 53 kDa protein band. This increase in 53 kDa phosphorylation was completely reversible. The increase was not found in homogenates from the melanophore-free part of the alpha-MSH-treated tail-fins. Phosphorylation of the 53 kDa protein could be detected in homogenates of alpha MSH-treated primary cultured melanophores. Incubation of tail-fins with ACTH1-24, an alpha-MSH-like peptide producing full pigment dispersion, also induced an increase in 53 kDa phosphorylation. A structurally related peptide (ACTH15-24) and an unrelated peptide (LH-RH), neither of which induced pigment dispersion, were ineffective in stimulating 53 kDa phosphorylation. Injection of white adapted tadpoles with 1 micrograms of alpha-MSH or adaptation of tadpoles to a black background also resulted in a significant increase in 53 kDa phosphorylation. alpha-MSH added to the homogenates did not affect 53 kDa phosphorylation, indicating that alpha-MSH acts through a receptor-mediated mechanism. The increase in 53 kDa phosphorylation measured in vitro (post hoc), most likely reflects an alpha-MSH-induced decrease in 53 kDa phosphorylation in vivo. Our results strongly suggest that a decrease in 53 kDa phosphorylation is involved in the mechanism of action of alpha-MSH on melanophores. PMID- 2998906 TI - Hormone binding modified endogenous proteolysis of LH/hCG receptors in rat ovarian plasma membranes. AB - Purified rat ovarian plasma membranes were subjected to incubation under conditions where luteinizing hormone receptors were either free or bound to hCG. When receptor proteolysis was followed by labeling the receptor with tritiated borohydride or [125I]hCG, occupied receptors were found to be more accessible to endogenous proteinases than unoccupied receptors. They exhibited greater rates of degradation and also produced an additional degradation product upon proteolysis. Degradation of other plasma membrane (glyco)polypeptides, however, was not affected by hormone binding. These results indicate that hCG binding induces a conformational or a structural change in its receptor, thereby increasing its susceptibility to endogenous plasma membrane proteinases. PMID- 2998905 TI - Involvement of cyclic AMP, iodide and metabolites of arachidonic acid in the regulation of cell proliferation of isolated porcine thyroid follicles. AB - Experiments with primary cultures of isolated porcine thyroid follicles were performed in serum-free well-defined medium to investigate different pathways that may be involved in the regulation of thyroid cell growth. The incorporation of [3H]thymidine into DNA within 72 h was about 25-fold with fetal calf serum (FCS, 1%), 20-fold with epidermal growth factor (EGF, 1 ng/ml) and 3.5-fold with insulin (10 micrograms/ml) as compared to controls. Bovine TSH significantly reduced the basal and insulin-induced growth rate at concentrations of 10(-6) to 10(-4) U/ml and 10(-4) U/ml, respectively. Forskolin stimulated cyclic AMP accumulation in thyroid cells and significantly reduced FCS-, EGF- or insulin induced growth. In contrast, a 2- to 7-fold increase in FCS-, insulin- or EGF induced growth rate was found, when cyclic AMP formation was inhibited by 2',5' dideoxyadenosine (DDA). Iodide was stimulatory at low concentrations (1 microM) and inhibitory at higher concentrations (40-80 microM) on FCS-induced growth rate. The inhibitory effect of iodide was blocked by propylthiouracil (PTU), indicating that an iodinated compound is responsible for this effect. Indomethacin, a cyclooxygenase inhibitor, did not inhibit EGF- and insulin induced growth up to a concentration of 100 microM. However, nordihydroguaiaretic acid (NDGA) and BW-755C, which are lipoxygenase inhibitors, strongly inhibited the growth of thyroid cells at micromolar concentrations. These data clearly show that (1) bovine TSH is not a growth factor for isolated thyroid cells in vitro, (2) thyroid cell proliferation, induced by FCS, EGF and insulin is under negative control of cyclic AMP. (3) Iodide controls dose-dependently thyroid cell growth by iodinated metabolites, probably modulating 2 different pathways: (a) at low iodide concentrations, an iodinated compound enhances the growth rate by inhibition of cyclic AMP formation, and (b) at high concentrations, iodide diminishes the growth rate by inhibiting the response to growth factors. (4) Metabolite(s) of lipoxygenase appear to be involved in intracellular signal transduction evoked by growth factors in thyroid cells. PMID- 2998907 TI - Interaction of thyroid hormone and nutritional signals on thyroid hormone action. AB - The interaction between thyroid hormone (T3) and nutritional signals has been of interest for nearly a century. Thus, enhanced glucose production, absorption and utilization are associated with hyperthyroidism, whereas diminished glucose utilization and lipogenesis characterize hypothyroidism. Recent studies have uncovered what appears to be yet another area of interaction at the molecular level. On the one hand, a marked overlap exists between the changes in rat hepatic mRNA activity profile induced by hyperthyroidism and high carbohydrate administration. On the other hand, the patterns produced by hypothyroidism, starvation and diabetes are characterized by oppositely directed shifts. These findings may be due, in part, to a synergistic relationship between carbohydrate feeding and T3 administration in the induction of many hepatic lipogenic enzymes and their respective mRNAs. Studies both in the intact rat as well as in isolated hepatocyte cultures indicate that this synergism arises from the ability of T3 to multiply an intracellular signal derived from the metabolism of glucose. The development of recombinant DNA techniques can now be applied to the study of the interaction of T3 with nutritional signals. Initial efforts have demonstrated a hepatic mRNA (mRNAS14) rapidly responsive to both T3 and carbohydrates. With this probe, studies are under way to define the precise molecular mechanisms by which T3 and carbohydrates interact to influence gene expression. PMID- 2998908 TI - Chronic and acute effects of forskolin on isolated thyroid cell metabolism. AB - The chronic treatment (2 days or more) of cultured thyroid cells with 1-10 microM forskolin (forskolin-treated cells) sensitizes the response of adenylate cyclase to further acute stimulation by 100 microM forskolin or 10 mU/ml thyrotropin (TSH). This positive regulation, similar to that produced by 0.1 mU/ml TSH (TSH treated cells), is obtained between 2 and 3 days of culture. The acute response to TSH or forskolin of cells treated for 4 days with forskolin increases with the concentration of forskolin present during the chronic treatment. This result is different from that obtained after a chronic treatment with TSH which induces refractoriness beyond 0.1 mU/ml. These cells are then desensitized to TSH but not to forskolin. When both agonists are mixed together, their acute effect is additive on control, TSH- and forskolin-treated cells. The chronic treatment of cultured thyroid cells with 1-10 microM forskolin produces, just like 0.1 mU/ml TSH, a chronic phospholipid effect characterized by enhanced incorporation of 32Pi into phosphatidylinositol (PI) and phosphatidic acid. The acute challenge of these cells with 100 microM forskolin evokes a reverse phospholipid effect, i.e. a decreased incorporation of 32Pi into PI. The acute stimulation of TSH-treated cells with TSH produces a reverse phospholipid effect whereas the acute stimulation of forskolin-treated cells with TSH gives a normal phospholipid effect as it does on control cells. These results show that the observed effects of TSH on cAMP accumulation and phospholipid turnover are not independent and are regulated in an inverse reciprocal pattern. PMID- 2998909 TI - Separation and analysis of two plasma membrane fractions from bovine thyroid which differ in TSH binding and TSH activation of adenylate cyclase. AB - A tissue disruption technique leading to the separation of thyroid epithelial cell components from interfollicular material has been used to study the distribution and the properties of membrane adenylate cyclase originating from intraglandular thyroid and non-thyroid cells. Bovine thyroid fragments were forced through a metallic sieve. The material which filtrates was composed of open cells and cell debris (fraction A); the material remaining on the sieve contained the basal lamina and the interfollicular material as shown by photon and electron microscopic observations (fraction B). Homogenates (HA and HB) were prepared from fractions A and B and centrifuged on a 41% sucrose layer to prepare membrane fractions: MA and MB, which were tested for the presence of adenylate cyclase, TSH-responsive adenylate cyclase and 125I-labelled TSH binding activity. HA and HB contained respectively 70% and 30% of the total thyroid adenylate cyclase activity. MA and MB were similarly enriched in 5'-nucleotidase and adenylate cyclase: 8- to 10-fold as compared to the corresponding homogenates. MA and MB exhibited a marked difference in the response to TSH: TSH either alone or in the presence of Gpp(NH)p stimulated the adenylate cyclase of MA and did not have any effect on MB. Fractionation of MA by isopycnic centrifugation on Percoll gradients yielded a membrane peak exhibiting a TSH-responsive adenylate cyclase activity and a 125I-labelled TSH binding activity displaceable by an excess of unlabelled TSH. A membrane peak at the same density was obtained from MB but its adenylate cyclase did not respond to TSH and there was no specific binding of labelled TSH. Our data indicate that an important fraction of membrane adenylate cyclase of the thyroid does not seem to be coupled with TSH receptor; the major part of this fraction (MB) likely originates from intraglandular non-thyroid epithelial cells. The separation of this membrane fraction from the thyroid cell plasma membrane fraction (MA) allows to increase the response of this latter fraction to TSH. PMID- 2998910 TI - Virus-induced murine diabetes. Enhancement by immunosuppression. AB - Adult, male ICR Swiss mice are susceptible to the diabetogenic effects of the D variant of encephalomyocarditis virus (EMC-D) in contrast to adult C3H/HeJ male mice, which are relatively resistant. To date, experimental evidence suggests that the immune system plays a role in the pathogenesis of this infection. We have investigated the potential involvement of the immune system in the pathogenesis of EMC-D-induced diabetes using cyclosporin-A (CyA), a potent immunosuppressive drug. The data show that treatment with CyA results in increased severity and incidence of diabetes in susceptible ICR Swiss mice and induction of diabetes in resistant C3H/HeJ mice. It is concluded that immune mediation probably is not involved in the early pathogenesis of EMC-D-induced diabetes in mice. PMID- 2998911 TI - HLA heterogeneity of insulin-dependent diabetes mellitus at diagnosis. The Pittsburgh IDDM study. AB - Although some previous studies have suggested that insulin-dependent diabetes mellitus (IDDM) is a heterogeneous condition with variant forms being associated with HLA-DR types, the evidence, thus far, is conflicting. To address this issue, we have examined the presenting characteristics of a consecutive admission series of 200 newly diagnosed cases of IDDM from the Children's Hospital of Pittsburgh. Because HLA-DR frequencies vary by race, data are presented only for the 172 white cases with complete HLA-DR typing. HLA-DR3 was found more frequently among male cases and DR4 among female cases (P less than 0.005). Generally, patients with DR4 presented with a severer clinical picture, being more likely to have impaired consciousness and significant dehydration. In addition, patients with DR4 were more likely to be acidotic, ketotic, and to more frequently report a recent viral infection. This latter finding was supported by a greater frequency of antibodies to Coxsackie-B viruses in the DR4 cases at presentation. These results therefore suggest that there is considerable heterogeneity in IDDM, at least in presenting characteristics, according to HLA-DR type. PMID- 2998912 TI - A murine model of insulin-dependent diabetes mellitus resulting from the cumulative effects of the nondiabetogenic strain of encephalomyocarditis virus and a single low dose of streptozocin. AB - The induction of insulin-dependent diabetes in outbred male and female mice was examined using a combination of the usually nondiabetogenic B-variant of encephalomyocarditis (EMC-B) virus and single low doses of streptozocin (STZ). Neither EMC-B virus nor low doses of STZ were overtly diabetogenic when administered alone; however, when these two insults occurred 1 day apart, diabetes resulted in male but not in female mice. The induction of diabetes was dependent on the time interval between these two insults, since EMC-B virus and STZ given 4 days apart did not induce diabetes. Unexpectedly, when the order of these two insults was reversed, diabetes occurred. The absence of diabetes when EMC-B virus was given before STZ suggested the possibility that virus-induced interferon blocked the cytotoxic effects of STZ. This suggestion was supported by the observation that an antiserum against beta interferon abrogated the virus mediated protection against STZ-mediated cytotoxicity. Also, Poly I:C administered before a single diabetogenic dose of STZ delayed the onset of severe hyperglycemia. PMID- 2998913 TI - Beta-endorphin modulation of the glucoregulatory effects of repeated epinephrine infusion in normal dogs. AB - Successive epinephrine infusions were used as a partial model to examine hormonal and metabolic responses to repeated stress stimuli. As both the endogenous opiates and epinephrine are released in response to stress, we have also studied interactions between epinephrine and B-endorphin. Epinephrine (0.1 microgram/kg . min) was infused for 60 min, followed by a 60-min recovery, in nine normal, conscious dogs. In a similar study, B-endorphin (0.06 microgram/kg . min) was given 30 min before epinephrine, then continuously infused throughout the study (N = 4 dogs). When epinephrine was infused, levels rose to 600-800 pg/ml. The changes in glucagon, B-endorphin, FFA, and hepatic glucose production were similar during both epinephrine infusions, but there was a diminished insulin response, a greater decrease in glucose metabolic clearance, and a greater increase in plasma glucose with the second epinephrine infusion. When B-endorphin was given, plasma levels increased to 5.3 ng/ml. Compared with the infusion of epinephrine alone, there was a much greater rise in plasma glucose due to greater suppression of glucose metabolic clearance. With the second epinephrine infusion, however, the changes in glucose concentration were not substantially different from those seen during the second infusion of epinephrine alone, as both hepatic glucose production and glucose metabolic clearance were suppressed. B-endorphin diminished the insulin and glucagon responses during the first epinephrine infusion and abolished them during the second, but did not alter the FFA, ACTH, or cortisol responses to epinephrine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2998915 TI - "Conventional" and high carbohydrate/high fibre/low fat diets in adults with established type 1 (insulin-dependent) diabetes. PMID- 2998914 TI - Photoaffinity labelling of hepatic plasma membranes suggests two classes of hepatic insulin receptor. AB - Photoaffinity labelling of hepatic insulin receptors revealed specifically labelled bands of 130, 90 and 40 kDa. Endogenous protease activity in hepatic plasma membranes, as well as contaminating proteases present in preparations of clostridial collagenase, degraded some of the 130-kDa insulin-binding subunit to a 115-kDa form. However, a large proportion of the 130-kDa subunits were resistant to degradation, suggesting the presence of two classes of insulin receptor in hepatic plasma membranes. In one class the 130-kDa subunit was sensitive to proteolysis, while in the other it was not. In contrast, the 130-kDa receptor subunits of adipose tissue were all resistant to such degradation. Scatchard analysis of control and collagenase-treated plasma membranes demonstrated that conversion of the 130-kDa subunit to a 115-kDa form did not affect the insulin-binding characteristics of the receptor. It was also apparent that insulin binds to a single class of high-affinity sites in hepatic plasma membranes. PMID- 2998916 TI - [Assay of trace elements in the serum by the PIXE method in patients with Crohn's disease]. AB - Concentrations of trace elements were determined by the PIXE method (particle induced X-ray emission) in 43 serum samples from 29 Crohn's disease patients and compared with the results obtained from a control group of 100 healthy subjects. Most of the patients were outpatients whose disease was quiescent or moderately active. Half of them had a good nutritional state. Mean serum selenium level was significantly higher in the Crohn's disease group than in the control group. A negative correlation was found between serum selenium and C-reactive protein levels. Mean serum bromine was normal in the Crohn's disease group, but there was a positive correlation between serum bromine and fibrinogen and C-reactive protein levels and leucocytosis. Mean serum copper concentration was higher in women than in men in both groups. In Crohn's disease patients, copper serum concentration was increased and correlated positively to fibrinogen and C reactive protein concentrations, erythrocyte sedimentation rate and thrombocytosis, and negatively to hematocrit. Copper serum level could be used as a marker of Crohn's disease activity. Mean serum zinc level was decreased in the Crohn's disease group. There was a positive correlation between serum zinc level and erythrocyte sedimentation rate and fibrinogen concentration. PMID- 2998917 TI - Colonic metabolism of wheat starch in healthy humans. Effects on fecal outputs and clinical symptoms. AB - To study the intracolonic digestion of starch, 5 healthy volunteers were maintained on a constant diet for 7 days. On the fourth day, the cecum was intubated and a suspension of raw wheat starch (50 g, in 500 ml of 154 mM NaCl and containing 10 g of polyethylene glycol 4000) was infused into the distal ileum at 2 ml/min. Hydrogen excretion in breath was measured, cecal contents were sampled, and symptoms were recorded. For the 2-3 days before and after starch infusions, fecal weight, pH, and percentage of dry matter were monitored; fecal outputs of starch, volatile fatty acids, lactic acid, ethanol, polyethylene glycol, alpha-amylase, nitrogen, and ammonia were also measured. A lactulose (10 g) hydrogen breath test was performed 5-7 days after the starch infusions. After the infusion of starch, concentrations of lactic and volatile fatty acids increased and pH decreased markedly in cecal contents. None of the fecal values changed significantly after starch, however, indicating that carbohydrate catabolism was nearly complete and that the colon absorbed the catabolic products efficiently. Abdominal symptoms, especially bloating, were noted by all subjects, and 2 subjects complained of cramping pain. No subject experienced diarrhea. The amounts of starch metabolized in the colon (47.3 +/- 2.9 g), as calculated from the excretion of H2 in breath compared to the hydrogen breath test after lactulose, were close to the actual load (50 g). PMID- 2998918 TI - Pseudomyxoma peritonei associated with colloid carcinoma of the pancreas. AB - A case of pseudomyxoma peritonei associated with a mucin-rich, "colloid" adenocarcinoma of the pancreas is described. This previously unreported association was characterized by an extremely rapid clinical course, transerosal spread of the neoplastic process, and the consistent presence of intracytoplasmic lumina in all cells, an ultrastructural feature thought to characterize malignant lesions. PMID- 2998919 TI - Structure-activity relationship of subtypes of cholecystokinin receptors in the cat lower esophageal sphincter. AB - Cholecystokinin (CCK) causes relaxation of the cat lower esophageal sphincter (LES) by stimulating CCK receptors on the noncholinergic, nonadrenergic inhibitory neurons and causes contraction by stimulating CCK receptors on the sphincter muscle. Studies were performed in anesthetized cats to identify differences between the two CCK receptors by investigating the structure-activity relationships of various fragments of CCK or gastrin molecule. Lower esophageal sphincter pressures were monitored continuously, using continuously perfused catheters, and agents were administered intravenously or close intraarterially. Based on their doses and the presence or absence of tetrodotoxin pretreatment, CCK analogues produced either relaxation or contraction of the sphincter. The relative potencies of CCK analogues on the inhibitory (neural) response were CCK 8 greater than G-17-I greater than or equal to dCCK greater than CCK 4(1:1/14,000: 1/15,000: 1/335,000). Sulfated gastrin was nearly as potent as CCK 8. The relative potency of these agents on the contractile (muscle) response was CCK-8 = G-17-I greater than or equal to dCCK-8 greater than CCK-4 (1:1:1/4.5: 1/2000). Deamidated CCK-8 was inactive. Proglumide shifted the dose-response curves of the inhibitory as well as excitatory effects of CCK analogues to the right. These studies show that there are two distinct species of CCK receptors: (a) The CCK alpha receptors, present on the inhibitory neurons, are very discriminative and are critically dependent on SO4; and (b) the CCK beta receptors, present on the sphincter muscle, are not discriminative and are not critically dependent on SO4. Nonsulfated gastrin may share the CCK beta receptors with CCK. PMID- 2998920 TI - Independence of salt gland function and adrenocortical activity in ducks (Anas platyrhynchos). AB - In immature ducklings injected with hypertonic saline the volume of extrarenal salt gland secretion was unaffected by prior treatment (immediately or 4 hr before salt loading) with long-lasting adrenocorticotrophin (ACTH 100 iu/kg, im) or exogenous corticosterone (1.0 mg/kg, im). Pretreatment with metyrapone, an 11 beta-hydroxylase blocker, (80 mg/kg, im) 4 hr before salt loading did not effect the volume of salt gland secretion but delayed the onset of extrarenal excretion. Salt gland function was suppressed in birds pretreated with metyrapone immediately prior to salt loading. The intravenous administration of metyrapone after salt loading immediately reduced salt gland activity, which remained suppressed for at least 40-50 min thereafter. The inhibitory effect of metyrapone on salt gland activity was not counteracted by the subsequent or simultaneous administration of exogenous corticosterone to salt-loaded ducks. These results suggest that acute alterations in adrenocortical activity are not causally responsible for changes in salt gland function. PMID- 2998921 TI - Changes in 3H-leucine enkephalin binding in spinal cord of frog after dorsal rhizotomy, cordotomy, transcutaneous stimulation and temperature variations: correlation with nociceptive sensitivity. AB - In the frog spinal cord about 50% of the 3H-leucine enkephalin (3H-LE) binding sites (b.s.) were blocked by an endogenous ligand. Three days after deafferentation and cordotomy the number of free b.s. increased by 44 and 56%, respectively. In spinal frogs the threshold of the flexor reflex responses evoked by nociceptive stimuli decreased. More than 7 days after deafferentation and cordotomy the number of both total and free 3H-LE b.s. decreased, while the threshold of the flexor reflex responses returned to that before spinalization. Transcutaneous electrical stimulation (TES) of the hind limbs (30 Hz, 5 minutes) in frogs spinalized 3 hours earlier increased 3H-LE binding at low intensities of stimulation (0.2 mA) and decreased the threshold of the flexor reflex responses. TES at higher intensities (1.0 mA) decreased 3H-LE binding and increased the threshold. Three days after spinalization TES even at low intensity diminished 3H LE binding and raised flexor reflex threshold. A decrease in the number of free 3H-LE b.s. was found when the frog body temperature was elevated (from 15 to 24 degrees C) or lowered (from 15 to 1 degrees C) for 14 days and was accompanied by an increase in flexor reflex threshold. The data suggest the existence of an endogenous opioidergic system in the frog spinal cord which has a high degree of tonic activity. PMID- 2998922 TI - Transposition of plasmid-borne Tn10 elements does not exhibit simple length dependence. AB - The transposition frequencies of Tn10 elements from the bacterial chromosome to an F epitome decrease 40% for every kilobase increase in transposon length. The basis for this relationship is not known. We have now examined complemented transposition of defective Tn10 elements off small multicopy plasmids. We find that length dependence in this situation is either reduced or absent, depending on the specific class of transposition events involved. These observations can be interpreted as evidence against the model that chromosomal length dependence occurs because of decay of a transposition-associated replicative complex. This interpretation is consistent with unrelated experiments suggesting that Tn10 transposition is normally nonreplicative. Alternative explanations of length dependence phenomena are discussed. PMID- 2998924 TI - Acquired immune deficiency syndrome (AIDS) and consultation-liaison psychiatry. AB - Acquired Immune Deficiency Syndrome (AIDS) is a new highly lethal transmissible syndrome that occurs primarily in identified high-risk groups. The number of AIDS cases has been doubling approximately every 6 months in the United States since 1981. A large number of healthy HTLV-III seropositive individuals, and a significant number of individuals with AIDS-related complex (ARC), who are at increased risk for eventual development of AIDS, have been identified. At least one third of AIDS patients develop neurologic disease prior to death. Organic mental disorders are frequent in AIDS and can have devastating consequences. Severe psychologic distress and functional psychiatric syndromes are also common. The psychosocial effects of AIDS for patients, family and friends, and health care professionals are discussed in relationship to the psychosocial consequences of other serious medical illnesses including cancer. An ideal comprehensive program to meet the needs of "AIDS affected" individuals is presented, as are the authors' views on the tasks of C-L psychiatrists in participating in the comprehensive care of these individuals. PMID- 2998923 TI - Isolation and characterization of mutations in the beta-tubulin gene of Saccharomyces cerevisiae. AB - Of 173 mutants of Saccharomyces cerevisiae resistant to the antimitotic drug benomyl (BenR), six also conferred cold-sensitivity for growth and three others conferred temperature-sensitivity for growth in the absence of benomyl. All of the benR mutations tested, including the nine conditional-lethal mutations, were shown to be in the same gene. This gene, TUB2, has previously been molecularly cloned and identified as the yeast structural gene encoding beta-tubulin. Four of the conditional-lethal alleles of TUB2 were mapped to particular restriction fragments within the gene. One of these mutations was cloned and sequenced, revealing a single amino acid change, from arginine to histidine at amino acid position 241, which is responsible for both the BenR and the cold-sensitive lethal phenotypes. The terminal arrest morphology of conditional-lethal alleles of TUB2 at their restrictive temperature showed a characteristic cell-division cycle defect, suggesting a requirement for tubulin function primarily in mitosis during the vegetative growth cycle. The TUB2 gene was genetically mapped to the distal left arm of chromosome VI, very near the actin gene, ACT1; no CDC (cell division-cycle) loci have been mapped previously to this location. TUB2 is thus the first cell-division-cycle gene known to encode a cytoskeletal protein that has been identified in S. cerevisiae. PMID- 2998925 TI - [Integration of various plasmids into the Bacillus subtilis chromosome]. AB - Some features of integration of temperature-sensitive pE194, pGG10 and pGG20 plasmids into the Bacillus subtilis chromosome were studied. Several auxotrophic mutations were obtained using insertion of these plasmids into the chromosome. The sites of plasmids for illegitimate recombination were determined. It was shown that the integration into the Bac. subtilis chromosome is characteristic not only for the plasmid pE194 but is the property of Staphylococcus aureus plasmid pC194 and Escherichia coli pBR322 plasmid. The influence of different Bac. subtilis rec mutations on the frequency of integration was studied. PMID- 2998926 TI - [Phage-transposon interaction: the cip locus of prophage D3112 responsible for the inhibition of integration and transposition of related phage B39 of Pseudomonas aeruginosa]. AB - Bacterial cells lysogenic for D3112, a transposable Pseudomonas aeruginosa phage restrict the growth of a related heteroimmune B39 phage. The lysogens are divided into two different types PAO(D3112). In the lysogens of the type I the efficiency of B39 growth only decreases slightly, the lysogens of the type II restricting completely the growth of this phage (e.o.p. is less than 10(-7). As shown by the results of Southern hybridization experiments, lysogens of the type I are monolysogens, while those of the type II are double or polylysogens. Restriction of B39 in PAO(D3112) is caused by expression of a locus in the D3112 genome. The locus has been termed as cip (control of interaction of phages). The cip locus was mapped at the interval 1.3-2.45 kb of the D3112 physical map using different deletion derivatives of D3112. Expression of cip only takes place in the prophage state and not during the phage lytic development. When expressed, cip affects the early steps in the growth of B39 lowering the level of integration and transposition processes; the effect is not dependent on the way of initiation of the lytic cycle (through prophage induction or infection). PMID- 2998927 TI - [Expression of Pseudomonas aeruginosa transposable phages in Pseudomonas putida cells. I. The establishment of a lysogenic state and the effectiveness of lytic development]. AB - Expression of transposable phages (TP) of Pseudomonas aeruginosa in the cells of P. putida was studied. The high efficiency of phage lytic development was shown both as a consequence of zygotic induction after transfer of the RP4::TPc+ plasmid into nonlysogenic recipients, and as a result of heat induction of lysogens PpG1 (D3112cts15). The high phage yield (20-25 particles of D3112cts phage per one cell of P. putida) is an evidence for a high level of transposition in the cells of this bacterial species. Plasmids RP4::TP are transferred into cells of PpG1 and PAO1 with similar frequency. However, the efficiency of establishment of the lysogenic state is lower in PpG1. Transposable phages of P. aeruginosa can integrate into the chromosome of PpG1 producing stable inducible lysogens. The presence of RP4 in the P. putida cells is not necessary for expression of transposable phages. The transposable phage D3112cts15 can be used in experiments of interspecies transduction of plasmids and chromosomal genes. PMID- 2998928 TI - Analysis of genes for 5S rRNA from the cricket, Acheta domesticus: two classes of repeating units. AB - To examine the modulation of 5S rRNA gene activity during development in the cricket, Acheta domesticus, 5S X DNA was isolated from a lambda Charon 4 genomic library and characterized. Southern blot analysis of cloned A. domesticus genomic DNA revealed that restriction fragments of 3.0 and 2.1 kb represent two size classes of 5S X DNA repeating units; over 90% of the repeats measure 3.0 kb. Restriction analysis of two 5S X DNA clones suggests that the 2.1-kb repeats are not randomly interspersed within clusters of the larger 3.0-kb repeating units. Heteroduplex and restriction mapping of several clones indicate that the spacers of both repeating units account for their unusual length. The major difference between the two classes of repeats may lie in 0.9-kb spacer sequences to the 3.0 kb repeats. PMID- 2998930 TI - Omega mutagenesis in gram-negative bacteria: a selectable interposon which is strongly polar in a wide range of bacterial species. AB - We have used the 2.0-kb DNA fragment omega [Prentki and Krisch, Gene 29 (1984) 303-313] to mutagenize in vitro a broad-host-range plasmid carrying the entire meta-cleavage pathway of the Pseudomonas putida TOL plasmid pWW0. The mutant plasmids were subsequently introduced by conjugal mobilization into a variety of Gram-negative bacteria. The omega fragment carries a selectable marker (aadA+; SpcR/SmR), which is expressed in all species tested, as well as flanking transcription and translation termination signals and synthetic polylinkers. Expression of the plasmid-borne catechol 2,3-dioxygenase (C23O) gene, situated downstream from the site of omega insertion, was substantially reduced in all strains tested. The transcription terminators originally cloned from bacteriophage T4 gene 32, are apparently functional in a wide range of hosts. Insertional mutagenesis with the omega 'interposon' can thus be used in a wide variety of species, with the advantages of a positive selection for the presence of the fragment, the termination of RNA and protein synthesis beyond the site of insertion, and genetic stability of the resulting mutation. PMID- 2998929 TI - Plasmid vectors designed for the analysis of transcription termination signals. AB - We have constructed synthetic operons in which two genes (cat and lacZ or cat and galK) were placed in tandem under the control of the bacteriophage lambda oLpL operator and promoter. Restriction sites were introduced between the promoter and the proximal cat gene or between the cat and lacZ or galK genes. In the latter case, introduction of a transcriptional terminator between the two structural genes should affect only the distal gene. Thus, following induction, the expression of the cat gene serves as an internal control, compensating for changes due to plasmid copy number or possible decrease in transcription initiation. We used these plasmids to select a lambda DNA fragment which includes the N-unresponsive tJ transcriptional terminator. This DNA fragment was inserted between the cat and galK genes. Enzymatic assays of these two gene activities following induction indicate that transcripts initiated at the pL promoter under N+ conditions terminate at tJ between the two genes. S1-nuclease analysis showed that these transcripts terminate at several sites in the tJ region. Similar results were obtained whether the host cells were RNaseIII+ or RNaseIII-. As a control, we showed a complete antitermination of the lambda t'I terminator under similar conditions, indicating that a sufficient amount of the N gene product is made from one N gene copy to suppress terminators carried on multicopy plasmids. PMID- 2998931 TI - Restriction endonuclease mapping of three plasmids from Bacillus thuringiensis var. israelensis. AB - Restriction endonuclease cleavage site maps have been constructed of plasmids pTX14-1, pTX14-2, and pTX14-3 from Bacillus thuringiensis var. israelensis (Bti). PMID- 2998932 TI - A promoter for Bacillus subtilis expression vector. AB - A 117-bp EcoRI-PstI fragment with strong promoter activity (P1 promoter) was cloned from Bacillus subtilis chromosomal DNA and sequenced. The P1 promoter was shown to contain a putative -35 region (TTTACT) and -10 region (TAGATT), and promotes expression of cloned human interleukin-2 (IL-2) and human interferon gamma (IFN-gamma) genes in B. subtilis. PMID- 2998933 TI - Evidence for autoregulation of the nusA-infB operon of Escherichia coli. AB - Analysis of three different nusA mutant strains suggests that the expression of the nusA-infB operon of Escherichia coli is regulated autogenously by the nusA gene product, a protein known to mediate transcription termination and antitermination. The cellular amounts of NusA and IF2 (infB) proteins are enhanced by a nusAts mutation which causes reduced transcription-termination activity. A nusAam mutant carrying the am ts suppressor, supFts6, overproduces the IF2 protein when the amount of NusA protein is reduced by the thermal inactivation of the supFts6. A modified form of NusA with the cat protein of Mr of 24 000 attached to the C terminus of NusA is overproduced compared to the wild type NusA and causes the overproduction of IF2. PMID- 2998934 TI - Novel structure of a human U6 snRNA pseudogene. AB - A genomic DNA library containing human placental DNA cloned into phage lambda Charon 4A was screened for snRNA U6 genes. In vitro 32P-labeled U6 snRNA isolated from HeLa cells was used as a hybridization probe. A positive clone containing a 4.6-kb EcoRI fragment of human chromosomal DNA was recloned into the EcoRI site of pBR325 and mapped by restriction endonuclease digestion. Restriction fragments containing U6 RNA sequences were identified by hybridization with isolated U6[32P]RNA. The sequence analysis revealed a novel structure of a U6 RNA pseudogene, bearing two 17-nucleotide(nt)-long direct repeats of genuine U6 RNA sequences arranged in a head-to-tail fashion within the 5' part of the molecule. Hypothetical models as to how this type of snRNA U6 pseudogene might have been generated during evolution of the human genome are presented. When compared to mammalian U6 RNA sequences the pseudogene accounts for a 77% overall sequence homology and contains the authentic 5'- and 3'-ends of the U6 RNA. PMID- 2998935 TI - Cloning vectors derived from the Pseudomonas plasmid pVS1. AB - The Pseudomonas plasmid pVS1, which has about seven copies, was reduced to a minimal replicon and used to construct stable gene-cloning vehicles. The host for all cloning experiments was P. aeruginosa strain PAO. Two nonmobilizable plasmids, pME260 and pME290, and one RP1-mobilizable plasmid, pME285, were constructed. The vectors pME260 (6.3 kb) and pME290 (6.8 kb) carry the Tn801 bla gene specifying carbenicillin (Cb) resistance, a good selective marker in Pseudomonas, and the Tn903 aph gene encoding kanamycin (Km) resistance, with useful restriction sites for insertional inactivation. The Mob+ vector pME285 (10.6 kb) carries the aph gene and the Tn501-derived merRTCA genes coding for mercuric ion resistance, another good selective marker in Pseudomonas. The hypothetical merD gene, which may follow the merA gene in Tn501 but is absent from pME285, appeared to be dispensable for mercuric ion resistance in P. aeruginosa. The Mob- vector pME290 could be introduced by transformation and maintained in strains of P. aeruginosa, P. fluorescens, P. putida, P. acidovorans, P. stutzeri, P. mendocina, P. cepacia, and P. syringae. The plasmid was compatible with IncP-1 and IncP-4 replicons. PMID- 2998936 TI - Studies on deo operon regulation in Escherichia coli: cloning and expression of the cytR structural gene. AB - The structural gene that encodes one repressor (the cytR-encoded repressor) of the Escherichia coli deo operon has been cloned from a lambda dmet transducing phage into the multicopy plasmid pBR322 by selecting for ApR, Lac- transformants of E. coli SS110(delta lac, cytR, tsx::lac). Restriction maps for the cytR+ plasmids have been generated and the position of the cytR gene on the cloned insert of these plasmids has been determined through deletion analysis. Results from maxicell experiments employing pCB001 and its cytR- derivatives suggest that the cytR gene encodes a protein with a subunit Mr of 37 000. In contrast to the complete repression of the deo operon obtained when deoR+ plasmids were introduced into E. coli SS201 (deoR, cytR), transformation of this DeoR-, CytR- strain with any of the cytR+ plasmids yields only clones which have phenotypes and Deo enzyme levels characteristic of a DeoR- single mutant. The data presented in this study are consistent with the interpretation that, in E. coli, the deoR encoded repressor controls deo operon transcription initiating from both deo promoter-operator sites, PO1 and PO2. In contrast, the cytR-encoded repressor regulates deo operon expression only through deo promoter-operator site PO2. PMID- 2998937 TI - Nucleotide sequence and organisation of the gua promoter region of Escherichia coli. AB - Overlapping restriction fragments of DNA carrying the gua promoter region of Escherichia coli have been cloned using promoter-probe plasmids. Antibiotic resistance conferred by the constructed plasmids is repressed by guanine and enhanced by adenine, two features characteristic of expression of the gua operon. The nucleotide sequence of these fragments reveals the gua promoter 43 bp upstream of the translational start codon for inosine 5'-monophosphate (IMP) dehydrogenase. The promoter is preceded by an A + T-rich region and several potential polymerase secondary binding sites, and is immediately followed by a G + C-rich discriminator, suggesting that the gua operon may be under stringent control. A sequence with twofold symmetry overlaps both promoter and discriminator and is therefore located where repressor binding could interfere with transcription initiation. A stem and loop can be formed from the leader mRNA, thus sequestering the ribosome-binding site. PMID- 2998938 TI - Nucleotide sequence of the chicken cardiac alpha actin gene: absence of strong homologies in the promoter and 3'-untranslated regions with the skeletal alpha actin sequence. AB - The entire nucleotide sequence of the chicken cardiac alpha-actin (CC alpha A) gene has been determined. This is the first complete sequence of a cardiac actin gene that includes the promoter region, cap site, all the introns, and the polyadenylation site. The gene contains six introns, five of which interrupt the coding region at amino acids (aa) 41, 150, 204, 267, and 327. The first intron is in the 5'-noncoding region and is 438 bp in length. The CC alpha A gene encodes an mRNA of approx. 1400 bp with 5'- and 3'-untranslated region of 59 and 184 nucleotides (nt), respectively. Like the chicken skeletal alpha-actin gene, the CC alpha A gene has the codon for the aa cysteine between the initiator ATG and the codon for the N-terminal aspartic acid residue of the mature protein. There are no strong homologies (less than 13 consecutive nt) in the promoter or 3' untranslated regions between the CC alpha A and chicken skeletal alpha-actin genes even though both are expressed in skeletal muscle during development. However, the 3'-untranslated region of the CC alpha A gene demonstrates significant sequence homology (76% over a 200-nt region) with the same region in the partial sequence of the human cardiac gene. The conservation of these sequence homologies between identical isoforms rather than the different alpha actin genes suggests these conserved regions may have a role in regulation rather than tissue-specific expression, as previously proposed. PMID- 2998939 TI - The traM gene of the resistance plasmid R1: comparison with the corresponding sequence of the Escherichia coli F factor. AB - The 7.7-kb EcoRI fragment of the resistance plasmid R1 contains the gene for the TraM protein. The sequence was identified by the presence of an open reading frame (ORF) which is preceded upstream by two promoter sequences. Both these promoters were found to be active, although the more distant one predominates, as was judged by the relative abundance of mRNA of the expected length. The TraM protein could be synthesized in an in vitro DNA-dependent protein synthesis system if the DNA of the corresponding region was supplied as template. Comparison of the traM genes of R1 and the F factor showed a high degree of similarity, although a number of mutations, especially near the 5' terminus, introduce specific amino acid (aa) changes. Two-thirds of the 3' sequences differ mainly in silent mutations; hence the aa sequence of the corresponding carboxy terminal portion of the protein is highly conserved. The 5' and 3' untranslated regions of the mRNAs show little homology. One of the promoter regions, the ribosome-binding sequences, and the transcription termination sites are located at comparable positions but differ in details. PMID- 2998940 TI - One-step gene replacement in yeast by cotransformation. AB - A general method to replace chromosomal DNA sequences of Saccharomyces cerevisiae by any in vitro modified DNA sequence has been developed and was applied to the PHO5 locus on chromosome II. A recipient strain was constructed in which part of the chromosomal PHO5 sequence was substituted by the URA3 gene. Replacement of this pho5-URA3 substitution by pho5 mutant alleles was achieved in one step by cotransformation with a pho5 DNA fragment and the self-replicating plasmid YEp13, which contains the LEU2 gene as a selectable marker. Leu+ transformants were selected, and the replacement events at the PHO5 locus were detected by their Ura phenotype (1-4% of the Leu+ were Ura-). In a similar way the PHO5 coding sequence was replaced by the sequence coding for human tissue-type plasminogen activator (t-PA). PMID- 2998941 TI - The aminoglycoside-resistance operon of the plasmid pSa: nucleotide sequence of the streptomycin-spectinomycin resistance gene. AB - The nucleotide sequence of the probable C terminus of the kanamycin-resistance gene (KmR) and the probable complete sequence of the streptomycin-spectinomycin resistance gene (SpR) of the IncW plasmid pSa have been determined. The two genes appear to be oriented in the same direction and separated by a spacer region of 53 bp, with transcription proceeding from the KmR gene into the SpR gene. An RNA transcript encompassing the C terminus of the KmR gene, the 53-base spacer, and the N terminus of the SpR gene has the potential to form a stem-loop structure with a free energy value of -68 kcal/mol. The SpR gene of pSa has extensive sequence homology with the aadA gene of the plasmid R538-1. Comparison of the proposed amino acid sequence of the KmR protein of pSa with those of two aminoglycoside phosphotransferases revealed a region of potential homology with those proteins. PMID- 2998942 TI - High-level expression of the bovine growth hormone gene in heterologous mammalian cells. AB - The gene coding for bovine growth hormone (bGH) was isolated from a lambda-phage library constructed using bovine pituitary DNA partially digested with MboI. Expression of this gene transfected into mouse and monkey cells was studied. CV-1 monkey cells transfected with simian virus 40 (SV40) vectors containing the intact bGH gene, including the putative promoter region, did not express bGH. However, replacement of the bGH promoter with the mouse metallothionein-I (MT) promoter resulted in high-level synthesis and secretion of bGH. These results show that the bGH promoter functions poorly in CV-1 cells but CV-1 cells process and translate the bGH mRNA accurately. The MT-bGH chimeric gene was used to establish permanent bGH-secreting mouse C127 cell lines using the 69% transforming fragment of bovine papilloma virus (BPV) as the vector. One such cell line produced high levels of bGH and secreted it into the medium efficiently. Secreted bGH is processed accurately and is bioactive as judged by its ability to bind to rabbit liver membrane preparations. PMID- 2998943 TI - Cloning and analysis of the promoter region of the erythromycin resistance gene (ermE) of Streptomyces erythraeus. AB - A DNA fragment containing the coding and regulatory sequences of the erythromycin (Er) resistance (ermE) gene of the Er produces Streptomyces erythraeus was cloned in Streptomyces lividans using the plasmid vector pIJ61. The approximate location and orientation of ermE were deduced from studies of its expression after subcloning in Escherichia coli. Sequences responsible for transcription of ermE in Streptomyces were studied by nucleotide (nt) sequencing, high resolution S1 and exonuclease VII mapping, in vitro transcription and in vivo promoter-probing. Tandemly arranged promoters of typical prokaryotic appearance initiate transcription of the coding region of ermE; a promoter of similar sequence was identified that initiates transcription of a likely coding region running in the opposite direction to ermE. It is suggested that these sites represent a class of vegetatively expressed Streptomyces promoter that is utilised by a form of RNA polymerase holoenzyme that also recognizes typical promoters of other bacterial genera. PMID- 2998944 TI - Growth hormone gene expression in eukaryotic cells directed by the Rous sarcoma virus long terminal repeat or cytomegalovirus immediate-early promoter. AB - The cytomegalovirus (CMV) immediate-early (IE) gene-regulatory region was found to be three- to fourfold more efficient than the Rous sarcoma retroviral long terminal repeat (LTR) in promoting expression of the bovine growth hormone (bGH) gene by rat GH3 cells. PMID- 2998945 TI - A shuttle vector plasmid for studying carcinogen-induced point mutations in mammalian cells. AB - We have constructed a shuttle vector plasmid for studying mutagenesis in mammalian cells. The plasmid replicates in cell lines permissive for SV40 virus as well as in the bacterium Escherichia coli and carries a bacterial suppressor tRNA gene (supF) that can serve as a mutagenesis marker. The plasmid replicates as efficiently as SV40 virus in African Green Monkey kidney CV1 cells, indicating that all traces of the inhibitory sequences normally found in pBR322 and its derivatives have been removed. The design of the plasmid and the small size of the mutagenesis target gene decrease the probability of recovering spontaneous deletion mutations that have been shown to occur at high frequency during passage in mammalian cells. The frequency of spontaneous-mutant plasmids recovered after passage in CV1 cells is substantially lower than with other vectors described previously. When the plasmid DNA is treated with UV radiation before passage in CV1 cells, mutants are observed at a frequency about 20-fold above the spontaneous background. PMID- 2998946 TI - Nucleotide sequence of the phosphoenolpyruvate carboxylase gene of the cyanobacterium Anacystis nidulans. AB - Nucleotide sequence of the open reading frame (ORF) for the phosphoenolpyruvate carboxylase gene (ppc) of the cyanobacterium Anacystis nidulans was determined. The ORF consists of 3159 bp and codes for 1053 amino acid (aa) residues. The codon usage of the ppc of A. nidulans is not so markedly different from that of the Escherichia coli ppc, yet, in A. nidulans the preferred codons are AAG for lysine and CCC for proline, whereas those are seldom used in the E. coli ppc. PMID- 2998947 TI - Use of the deoxyinosine-containing probe to isolate and sequence cDNA encoding the fusion (F) glycoprotein of Sendai virus (HVJ). AB - A synthetic 20-mer based on the known amino acid (aa) sequence of the N-terminus of Sendai virus F1 polypeptide was synthesized. Using this dI-probe, which contained deoxyinosines at all six ambiguous codon positions, we isolated clones carrying cDNAs for the F mRNA of Sendai virus. Nucleotide (nt) sequence analysis revealed a long open reading frame (ORF) that encodes a protein of 565 aa. Thus, this type of dI-probes should prove useful for selecting cDNA clones, when the aa sequence is known and is characterized by high codon redundancy. PMID- 2998949 TI - Two mammalian cell systems for propagation of the hepatitis B virus genome in extrachromosomal and chromosomally integrated states: production of the surface and e antigens. AB - A recombinant plasmid consisting of (i) the entire genome of hepatitis B virus (HBV) DNA, (ii) the replication origin of SV40 virus, and (iii) a deletion derivative of pBR322 was introduced either into COS cells of monkey origin which constitutively express SV40 large T antigen, or into thymidine kinase(TK) deficient mouse L cells together with the TK DNA of Herpes simplex virus. In the COS cell system, the transfecting recombinant DNA replicates via SV40 origin and is maintained in an autonomously replicating state. The cells carrying these extrachromosomal elements express the hepatitis B surface antigen gene at moderate rate, and release the products into the culture medium. However, neither core antigen nor e antigen expression was detected in this system. In the L cell system, the transformed L cells carry the recombinant DNA in a chromosomally integrated state. Such cells express the surface antigen gene at high rate, and release the products into the culture medium. This system also excretes the e antigen into the culture medium. The core antigen was not detected. PMID- 2998948 TI - Versatile expression vectors for high-level synthesis of cloned gene products in Escherichia coli. AB - We have constructed a set of expression vectors which contain synthetic DNA sequences comprising a computer-generated model ribosomal binding site located downstream from the tightly regulated phage lambda pL. promoter. These vectors have been used in several laboratories to produce significant amounts of eukaryotic and prokaryotic gene products in Escherichia coli, either as fusion proteins (with two to nine extra N-terminal amino acids) or as proteins containing the naturally occurring amino terminus. For inserting DNA sequences downstream of an initiation codon, we used synthetic oligonucleotides to introduce multiple-use restriction sites recognized by EcoRI, BamHI and ClaI which generate termini complementary to those of a variety of enzymes (e.g., EcoRI, MboI, TaqI, and HpaII), in addition to their own. A set of three of these vectors was made to accommodate all three translational reading frames. In combination, the features of these vectors afford useful advantages over expression vectors previously described, especially for the application of shot gun cloning of genomic DNA to generate expression libraries. PMID- 2998950 TI - Expression of the gag gene of human T-cell leukemia virus type I in Escherichia coli and its diagnostic use. AB - An expression plasmid, pHY202, was constructed which directs the synthesis of a fusion protein encoded by the gag sequence of human T-cell leukemia virus type I (HTLV-I) inserted into the lacZ' gene. Escherichia coli cells harboring pHY202 produced the 43-kDal LacZ'-Gag fusion protein with a yield of approx. 0.3% of total soluble proteins. The fusion protein is specifically recognized by monoclonal antibodies against the Gag proteins p19 and p24, and could be applicable for the diagnosis of HTLV-I infection, because almost all sera from HTLV-I carriers gave a positive response in the enzyme-linked immunosorbent assay (ELISA) employing the LacZ'-Gag hybrid protein purified by immunoaffinity column chromatography. PMID- 2998952 TI - The nucleotide sequence of a spore germination gene (gerA) of Bacillus subtilis 168. AB - The nucleotide sequence of a 2.1-kb fragment of Bacillus subtilis DNA that contains part of the spore germination locus, gerA, has been determined. An open reading frame (ORF) of 1440 bp (480 codons) has been identified which corresponds to the previously located complementation unit I of the gerA locus. The orientations of transcription of the gerA and of the adjacent fumarase (citG) gene are divergent. The deduced polypeptide product of the gerA gene, of Mr 53 506, contains both hydrophobic and hydrophilic domains and is likely to be membrane-associated. PMID- 2998951 TI - Cloning and localization of the Bacillus subtilis chromosome replication terminus, terC. AB - A 10.9-kb segment of the Bacillus subtilis 168 chromosome has been cloned in an Escherichia coli plasmid and shown to contain terC (the replication terminus of the chromosome). The terC-containing portion of this plasmid has been subcloned within each of two overlapping fragments of DNA, 1.75 and 1.95 kb, again in E. coli plasmids. These have afforded a more precise definition of the location of terC in the B. subtilis chromosome and provided material for a detailed analysis of the structure and functioning of this site. PMID- 2998953 TI - Dysdifferentiative nature of aging: age-dependent expression of MuLV and globin genes in thymus, liver and brain in the AKR mouse strain. AB - The amount and sequence complexity of RNA transcribed by the murine leukemia virus (MuLV) genome and to the alpha- and beta-globin genes in thymus, brain and liver were measured throughout the life span of AKR mice using a cDNA X RNA hybridization technique. RNA complementary to the complete sequence of specific cDNA probes for both MuLV and globin was found in nuclei and cytoplasm of thymus, brain and liver at all ages studied. No significant age-dependent change in the amount or sequence complexity of globin RNA was detected. The amount of MuLV RNA in both nuclei and cytoplasm of thymus increased about five times from 2 to 5 months of age, but no significant change was observed over this age range in MuLV RNA from liver and brain. By the age of 10 months, most of the mice had developed leukemia and the thymus showed a further increase in the amount of MuLV RNA. Nuclear RNA from liver and brain then showed a significant increase in the amount of MuLV, but no change in the amount of MuLV was found in the cytoplasm. No qualitative age-dependent changes in the MuLV RNA sequence complexity in thymus, liver and brain were detected. These results suggest that in the AKR mouse strain for the three tissues studied, an age-dependent relaxation occurs in the repression of the MuLV genome but not for alpha- and beta-globin genes. The rate of the depression of MuLV genes in the short-lived AKR mouse strain appears similar to that of the long-lived C57BL/6J mouse strain. This age-dependent relaxation of MuLV genes appears to be limited to the nuclei. Thus, the presence of a specific regulatory system for the transport of MuLV RNA through the nuclear membrane which does not deteriorate with age is indicated. PMID- 2998954 TI - Age-related reduction of beta-adrenoceptor sensitivity in rat heart occurs by multiple mechanisms. AB - Myocardial responsiveness to catecholamines was evaluated by measuring isoproterenol-stimulated adenylate cyclase activities in myocardial particulate fractions from Fischer 344 rats of 3, 12, and 24 months of age. Dose-response curves of isoproterenol revealed a progressive increase in the activation constant (Kact) with advancing age. In addition, the maximal velocity (Vmax) for 12- and 24-month-old groups was about 20-25% lower than for the 3-month-old group. Analysis of receptor-binding data and nonreceptor-mediated enzyme activities suggests that the age-related decrease in Vmax for isoproterenol may result from a loss of myocardial beta-receptors, whereas the increase in Kact is probably due to a deficit in the postreceptor components of the receptor-cyclase complex. PMID- 2998955 TI - [Industrial hygiene in the manufacture of chlorine-free potassium fertilizers]. PMID- 2998956 TI - [Quantitative study of calmodulin and sperm motility in fertile euspermic and infertile asthenozoospermic individuals]. PMID- 2998958 TI - Invasion potential of human choriocarcinoma cell lines and the role of lytic enzymes. AB - The chick chorioallantoic membrane (CAM) was used as an assay system to examine the invasive potential of human choriocarcinoma cell lines. When 5 X 10(6) cells were inoculated into the CAM at the 10th day of postfertilization, three of eight cell lines formed extensively invasive tumors within the CAM. A tendency to correlation between the tumorigenic potential of cell lines in hamster cheek pouches and their invasive potential in the CAM was noted. In addition, the invasive capacity of cell lines correlated well with the amount of collagenase but did not correlate with the amount of plasminogen activator or cathepsin B secreted by them. It is concluded that a heterogeneity of invasive potential in the CAM exists among human choriocarcinoma cell lines and the role of the collagenase secreted by them is suggested. PMID- 2998957 TI - Phase II trial of medroxyprogesterone acetate in advanced ovarian cancer: an EORTC Gynecological Cancer Cooperative Group Study. AB - Progestin therapy of ovarian carcinoma in the past has been reported to lead to varying response rates. A multicenter phase II study of high-dose MPA was conducted in 53 patients with epithelial ovarian cancer who had received adequate trials of conventional therapy with cytotoxic agents. Forty-one patients were included for response and toxicity evaluations. Only one partial response has been recorded with a duration of 20 weeks. Stabilization of disease was observed in 7 patients. The present investigation shows that MPA given at the present high dose is not effective in patients extensively pretreated with chemotherapy. PMID- 2998959 TI - Granular cell myoblastoma of the vulva. PMID- 2998960 TI - Neuroendocrine (Merkel cell) carcinoma of the vulva: a case report and review of the literature. AB - The clinical and pathologic features of a vulvar neuroendocrine (Merkel cell) neoplasm are presented. Cytologic studies of material obtained from needle aspiration suggested that the tumor was a small cell neoplasm possibly of neuroendocrine derivation. The light-microscopic findings of sheets of small, uniform cells were consistent with a diagnosis of neuroendocrine tumor. The electron-microscopic characteristics, including the presence of neurosecretory granules, confirmed the diagnosis of a neuroendocrine (Merkel cell) carcinoma. Regional lymph node metastases were present at the time of initial surgery, and both local and distant metastases developed 8 months later. A comprehensive pretreatment metastatic evaluation is recommended. The role of chemotherapy for primary therapy is considered. PMID- 2998961 TI - Effects of variations in thyroid hormone serum concentrations on serum ACE activity. AB - Serum angiotensin converting enzyme activities were significantly increased in 26 untreated hyperthyroid patients (20.3 +/- 5.4 U/ml; P less than 0.001) compared with healthy control subjects (13.1 +/- 2.3 U/ml). In 12 patients a significant fall in enzyme activities was observed after treatment compared with pretreatment serum ACE levels (P less than 0.001). Eight patients with hypothyroidism (15.7 +/ 5.1 U/ml) and 11 athyreotic patients, totally thyroidectomized for well differentiated thyroid cancer, showed no significant differences in serum ACE activities (14.3 +/- 2.2 U/ml) compared with control subjects. After thyroid hormone supplementation a significant increase in serum ACE activity (P less than 0.05) was found in the athyreotic patients. Addition of increasing amounts of L thyroxine to a serum sample of an athyreotic patient showed no significant effect on ACE activity in vitro. We suggest that the elevated serum ACE activity in hyperthyroidism is not from the thyroid gland, but represents a direct effect of thyroid hormone on ACE synthesis and/or release from endothelial cells. PMID- 2998962 TI - Dexamethasone suppresses cortisol but not ACTH and beta-endorphin plasma concentration in healthy man. PMID- 2998963 TI - Screening methods for early detection of hepatocellular carcinoma. AB - The value of various screening methods in the detection of early hepatocellular carcinoma was investigated in 95 patients with cirrhosis. Infusion hepatic angiography and computed tomography with angiography were performed yearly, ultrasound every 3 months, and determination of serum alpha-fetoprotein levels every 2 months. "Space-occupying lesions" suspicious for hepatocellular carcinoma were found in 13 of the 95 cases (13.7%). Detection rates of "space-occupying lesions" were 77% for infusion hepatic angiography, 77% for computed tomography with angiography and 54% for ultrasonography, respectively. In 8 of the 13 cases, "space-occupying lesions" were subsequently confirmed as hepatocellular carcinoma by operative findings or clinical course. Serum alpha-fetoprotein levels were negative in 3 of the 8 hepatocellular carcinoma-confirmed cases, and 3 of the remaining 5 cases demonstrated levels above 400 ng per ml at the time of diagnosis. A radical resection of hepatocellular carcinoma was successfully performed in two cases. Although it was difficult to differentiate hepatocellular carcinoma from other lesions in the case of "space-occupying lesions" smaller than 2 cm in diameter, the results suggest that regularly scheduled screening may be useful to detect early hepatocellular carcinoma. PMID- 2998965 TI - Modeling of substrate elimination by the liver: has the albumin receptor model superseded the well-stirred model? PMID- 2998964 TI - Effect of glycoursodeoxycholate on precipitation of calcium carbonate. AB - The potential role of bile salts in preventing calcium carbonate precipitation was investigated by studying their interaction of Ca2+ and their inhibitory effects on calcium carbonate formation. Glycochenodeoxycholate micelles bound more calcium than did glycocholate. At bile salt concentrations exceeding 12.5 mM, glycoursodeoxycholate bound calcium as well as glycochenodexycholate did. Similar results for calcium binding were observed in mixed micelles of bile salts and lecithin. In bicarbonate (25 or 50 mM) and CaCl2 (10 mM) solutions, calcium carbonate formation was inhibited by the bile salts. Glycoursodeoxycholate and glycochenodeoxycholate (25 mM) prevented calcium carbonate formation which was delayed by glycocholate. This effect is not due to differences between both series of bile salts for calcium binding since glycoursodeoxycholate or glycochenodeoxycholate (25 mM) more efficiently prevented calcium carbonate precipitation than did 35 mM glycocholate in spite of the same Ca2+ binding. These results suggest that some bile salts may have a specific role in preventing calcium precipitation in bile. The mechanism is unknown. The physical properties of glycoursodeoxycholate and glycochenodeoxycholate do not support a role for CaCO3 precipitation in gallstone calcification during litholytic therapy. PMID- 2998966 TI - Massive intravascular hemolysis without serum haptoglobin depletion. AB - Hyperplasia of marrow histiocytes with extensive hemophagocytosis was found in a patient with cytomegalovirus infection. He experienced massive intravascular hemolysis, but, unexpectedly, no depression of the serum haptoglobin level was found by either single radial immunodiffusion or rate nephelometry. The unexpectedly high haptoglobin value may have been the result of "blockade" of the monocyte-macrophage system, with resultant failure to clear haptoglobin hemoglobin complex rapidly from the circulation. Use of the techniques described for the indirect estimation of unbound serum haptoglobin alone should avoid confusing results in similar clinical circumstances. PMID- 2998967 TI - Malignant fibrous histiocytoma of the heart presenting as unilateral pulmonary thromboembolism and infarct. AB - A malignant fibrous histiocytoma of the heart located at the pulmonic valve and thrombotic occlusion of branches of the left pulmonary artery with pulmonary infarct were found at autopsy in a 77-year-old man. The thrombi contained malignant cells. The patient had undergone left upper lobectomy four years earlier for thromboembolism with infarct, and review of the slides from that procedure revealed similar malignant cells within thrombi. This case is remarkable for the slow growth of the neoplasm and the prolonged survival of the patient, without specific therapy. PMID- 2998968 TI - Concerning collagen types in fibrosarcoma. PMID- 2998969 TI - Close linkage between Norrie disease, a cloned DNA sequence from the proximal short arm, and the centromere of the X chromosome. AB - Norrie disease (ND) is an X-linked recessive disorder with congenital blindness (atrophia bulborum hereditaria, pseudoglioma). Six kindreds segregating for ND were studied for linkage with polymorphic markers of the human X chromosome. No recombination was observed between the ND-locus (NDP) and the DXS7 locus, the latter followed as a DNA-restriction fragment length polymorphism, detected by the recombinant DNA probe L1.28, and assigned to the region Xp11.2-Xp11.3. The maximum lod scores are zeta = 3.81 at theta = 0.00. Linkage data between NDP and the other genetic markers used in the present study are in keeping with this assignment of the mutation to the proximal Xp. PMID- 2998970 TI - DNA-polymorphic patterns linked to the beta-globin genes in German families affected with hemoglobinopathies and thalassemias: a comparison to other ethnic groups. AB - DNA haplotype constellations of the beta-globin gene cluster have been analyzed in German families with hemoglobinopathies (Hb Freiburg, Hb Koln, Hb Presbyterian) and beta-thalassemias. The polymorphic patterns obtained were compared to those found in families from Greece, Italy, and Turkey affected by beta-thalassemia syndromes. With the combined analysis of seven restriction site polymorphisms a DNA-diagnostic prediction for additional offspring could be made with an overall frequency of 75% in the four ethnic groups. PMID- 2998971 TI - Allelic variation adjacent to the human insulin and apolipoprotein C-II genes in different ethnic groups. AB - We have used DNA probes for the human insulin gene and apolipoprotein C-II (apo C II) gene to determine the extent of allelic variation in different ethnic groups. The distribution of an apo C-II DNA polymorphism revealed by the restriction endonuclease Taq I showed no significant variation amongst racial groups; in contrast, an insulin gene-related DNA polymorphism showed marked variability. In Japanese, Chinese, and Asian Indian groups there was an increased frequency of homozygosity for the class 1 allele compared to Caucasian groups (P less than 0.001, P less than 0.01, and P less than 0.05, respectively). In Caucasian, Japanese, Chinese, and Asian Indian groups no class 2 allele was observed; but in the Negroid populations (African and West Indian) the class 2 allele frequencies were 0.23 and 0.25 respectively. Possible reasons for this variation in allele distribution are considered in relation to disease associations. PMID- 2998972 TI - The isolation, characterisation, and chromosomal assignment of the gene for human 3-hydroxy-3-methylglutaryl coenzyme A reductase, (HMG-CoA reductase). AB - We have used a cDNA clone for Chinese hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase to isolate a genomic recombinant for human HMG-CoA reductase. The identity of the gene was confirmed by partial sequence analysis. Several unique fragments that will be useful for restriction fragment length polymorphism (RFLP) studies were identified. In situ hybridisation of a 2.6 kb unique fragment of the gene to human metaphase chromosomes localised the human HMGCoA reductase gene to human chromosome 5q12. PMID- 2998973 TI - Restriction fragment length polymorphism detected by human salivary amylase cDNA. AB - The plasmid clone which contains human salivary amylase cDNA was used to detect restriction fragment length polymorphisms (RFLPs). After double digestion with Pst 1 and Bam H1, a polymorphism with two alleles was observed. In Japanese, frequencies of these alleles, tentatively called 5.7 kb and 6.5 kb fragment alleles, are 0.55 and 0.45, respectively. PMID- 2998974 TI - [Pathogenesis of atopic dermatitis]. AB - Atopic dermatitis (AD) is a familial inflammatory skin disorder which is characterized by extreme pruritus, the typical morphology and distribution, the chronic or chronically relapsing time course and the personal or family history of atopy (asthma, allergic rhinitis, atopic dermatitis). However, there exists a variety of additional features which are either less specific or relatively rare. Although this disease has been well-known since the beginning of the century, the pathogenesis is not clearly understood at present. This review summarizes the reported deviations of the immune system as well as the alterations of the mediators of inflammation and the abnormalities of cyclic nucleotide regulation. These findings will be correlated with clinical symptoms. In particular the following topics were taken into consideration: association with HLA-antigens, elevation of serum IgE and generation of IgE immune complexes, numerical and functional deficiencies of T-suppressor cells, involvement of granulocytes, alterations of mediators of inflammation and especially the observations on the cyclic adenosine monophosphate (cAMP)-phosphodiesterase. These extremely complex findings based on the interaction between disregulation of the autonomous nervous system and alterations of the immune system may provide a better understanding of the pathogenesis of atopic dermatitis. PMID- 2998975 TI - [Cytomegalovirus infections after kidney transplantation and passive immunization]. AB - Cytomegalovirus infection after renal transplantation impairs the survival rate of patient and graft. An incidence of 24 to 92% of CMV infections after renal transplantation is reported, but only 15 to 77% of these patients show clinical symptoms. Contaminated donor organs, blood transfusions and reactivation by immunosuppression are the main causes of this infection. Active immunisation cannot prevent the reactivation of the infection, but also antiviral agents can hardly influence the course of the disease. Passive immunisation showed promising effects in bone-marrow transplantation and is now also tried in renal transplantation. The first results of a randomized study do not allow a final conclusion, but show less clinical symptomatic CMV infections, and statistically significant less herpes simplex infections in the group receiving anti CMV IgG prophylaxis. PMID- 2998976 TI - [Cytomegalovirus infection after kidney transplantation--importance of the primary infection]. AB - Even though for many reasons (shortage of organs, histocompatibility, lack of time to await the results) an organ transplantation from a sero-positive donor to a sero-negative recipient will not be unavailable, CMV-serology should be checked as early as possible, if possible even before transfusions or their donors since the virus remains infectious over several weeks in the leucocytes. The prophylactic use of hyperimmunoglobulin is justified in cases where organs or blood from sero-positive donors are given to sero-negative immunosuppressed patients because complications arising from the infection can be life-threatening in these patients. PMID- 2998977 TI - [Is CMV-hyperimmune serum prophylaxis after kidney transplantation always meaningful?]. AB - CMV-infections are a common and dreadful complication in immunosuppressed patients after kidney transplantation. This randomized, controlled clinical study investigates the influence of CMV-hyperimmune serum (HIS)-prophylaxis on infection incidence and severity of illness with regard to the type of immunosuppression employed. Though the clinical manifestation of the disease is lowered by HIS in cyclosporin-treated patients, there is a higher infection incidence with increased severity in HIS-treated ATG/Imurek patients compared to the respective control. PMID- 2998979 TI - [Experiences with cytomegalovirus hyperimmunoglobulin after bone marrow transplantation]. AB - The effects of an intravenous hyperimmune cytomegalovirus globulin after bone marrow transplantation are reported. From day -1 22 patients received 2 ml/kg body weight every two weeks during the first four months after BMT. Infusions were tolerated without any side effects. In three patients a CMV-infection could be documented which was symptomatic in two of them. A further patient experienced a CMV infection during the pretransplant phase before hyperimmune globulin had been administered. The most severe case presented as a nonlethal interstitial pneumonia. PMID- 2998978 TI - [Incidence and clinical relevance of cytomegalovirus diseases using cyclosporine compared to conventional immunosuppression]. AB - Since transplanted patients have been treated with cyclosporin A after kidney transplantation, the whole immunosuppression has changed. Combined with the specific T-cell suppression, steroids were administered in much lower concentrations. ALG and ATG combined with cyclosporin were avoided because of the immunoproliferative syndrome. Changing in immunosuppression reduced the incidence of CMV-infections from 4.3% to 3.1% in our center. Beside the apparent infection, severe illness was significantly reduced. Under conventional immunosuppression, in 1.7% a loss of transplantate could be observed. This was not the case under cyclosporin A. Since half a year the immunosuppression has changed and triple drug usage was common in high responder patients. It seems that CMV-infections will become more relevant than in former days. Important for us is the recognition of risk groups at the point of transplantation. In these patients a passive vaccination with CMV-hyperimmunoglobulin should be performed. PMID- 2998980 TI - [Cytomegalovirus hyperimmune globulin and cytomegalovirus-tested blood products- a pilot study in bone marrow graft recipients]. AB - Nineteen bone marrow transplant recipients had cytomegalovirus prophylaxis using a commercial hyperimmune globulin. Seronegative donors were selected for blood component substitution. The incidence of seroconversion was 25%. One patient acquired fatal cytomegalovirus associated interstitial pneumonitis. The value of this combined cytomegalovirus prophylaxis in bone marrow transplant recipients in comparison to sole passive immunization is recommended to be the subject of a controlled trial. PMID- 2998982 TI - Asynchronous regulation of mouse H-2D and beta-2 microglobulin RNA transcripts. AB - The major transplantation (or H-2) antigens in the mouse are cell-surface glycoproteins composed of a heavy chain and a light chain, the beta-2 microglobulin (beta 2m). The expression of these proteins is regulated during development. Embryonic cells at early stages of development do not express these proteins. On the other hand, these molecules are present on the surface of all adult somatic cells. We investigated whether the expression of both chains was coordinately regulated. Using specific single-stranded DNA probes in an S1 nuclease analysis, we compared the relative amounts of H-2D and beta 2m transcripts in normal tissues, in transformed cells, and during embryonic development. Our results show that (1) the steady state level of beta 2m transcripts varies from one adult organ to another, while that of H-2D transcripts stays approximately the same; (2) upon transformation, the amount of H-2D-specific mRNA increases drastically, while the beta 2m mRNA level remains constant; (3) whereas the quantity of beta 2m mRNA increases during early development, the amount of H-2D mRNA remains at a very low level. These data suggest that the regulation of H-2D and beta 2m genes are not identical and that their activation during development is not synchronous. PMID- 2998981 TI - Different roles for cytosine methylation in HLA class II gene expression. AB - We studied the role of cytosine methylation in the control of HLA class II gene expression in isogenic sets of cells whose members differ in their expression of HLA class II genes. These included: T5-1, 6.1.6, and P30, which are a class II expressing B-cell line, a class II nonexpressing mutant derived from T5-1, and an HLA-DR expressing partial revertant derived from 6.1.6, respectively; the class II expressing B-cell line, SB, and the class II non-expressing T-cell line, HSB, from the same individual. The use of sets of cells that differ in the way their class II genes are regulated allows us to study how that difference is reflected in the methylation state of their class II genes. At least five out of six class II genes in nonexpressing cells have a CpG site that is demethylated, when compared with the same class II gene in the respective expressing cells. The results presented in this paper indicate that most methylation changes in and around class II genes have a correlation with their state of expression. Some of these changes reflect rather than determine the state of expression. Other methylation changes appear to directly affect expression, whereas some methylation differences neither correlate with nor influence gene expression. Although 5-azacytidine does not affect class II expression in T5-1 or 6.1.6, it does induce expression in HSB. This indicates that the basis for nonexpression of class II genes is different in 6.1.6 and HSB. PMID- 2998985 TI - The beginnings of the Burkitt's lymphoma story. PMID- 2998984 TI - Burkitt's lymphoma in Europe. AB - Burkitt's lymphoma (BL) is the most frequent childhood non-Hodgkin's malignant lymphoma (NHML) in Europe and accounts for 5 to 10% of adult NHML. Age distribution is similar to that of endemic BL, with a male:female ratio of 3.7:1. Epstein-Barr virus (EBV) association is found in 15% of cases. A better definition of this monoclonal B-cell malignant proliferation is cytogenetic (i.e., 8;14 or variant translocation). Abdominal masses are initially present in 70% of cases, whereas the jaw is involved in only 4%. The disease is characterized by its overwhelming evolution in the absence of therapy. However, complete remission is usually obtained after the first chemotherapy regimen. In the past, death has been related to cerebrospinal fluid involvement, local recurrence or secondary marrow involvement. Today, it is expected that more than 80% of BL cases will be cured, i.e., 100% of stages I and II abdominal, 70% of stage II non-abdominal and stage III, and 50% of stage IV. Ninety percent of patients alive with no evidence of disease eight months after complete remission can be considered as definitively cured. PMID- 2998986 TI - Clinical features of Burkitt's lymphoma in the USA. PMID- 2998983 TI - Linkage of a 7S RNA sequence and kappa light chain genes in the mouse. AB - A mouse 7S RNA cDNA plasmid clone was employed to identify and map DNA restriction fragment variants using recombinant inbred (RI) and congenic mouse strains. More than a dozen such restriction variants were identified and mapped to different regions of the mouse genome. One such variant, designated Rn7s-6, showed close linkage to the Ly-2,3-Igk-V (T lymphocyte antigens 2 and 3, kappa immunoglobulin variable region) cluster of markers on chromosome 6. No recombinants were detected among three of these markers in 59 RI strains. On the basis of these data, the Rn7s-6 sequence may be placed within 1.3 centimorgans of Ly-3 and one of the Igk-V-region markers, Igk-Ef1. Two mouse stocks with previously identified crossovers within the Ly-2,3-Igk-V region were used to sublocalize Rn7s-6. The results are consistent with the gene order (Ly-2, Ly-3) (Rn7s-6, Igk-Ef1)-Igk-Ef2. Several mouse plasmacytomas, known to have various parts of the kappa chain complex deleted, retain the Rn7s-6 sequence. The Rn7s-6 variant is a plus/minus variant; no sequence allelic to Rn7s-6 is found in inbred strains that share the Ly-3a-Igk-Ef1a haplotype. PMID- 2998987 TI - Epstein-Barr virus and Burkitt's lymphoma worldwide: the causal relationship revisited. AB - Burkitt's lymphoma (BL) in tropical Africa represents by far the most common tumour in children between 0 and 14 years of age, 97% of the tumours being associated with Epstein-Barr virus (EBV). In North Africa, the tumour is about ten times less frequent than in equatorial Africa, but, according to reports from Algeria, 85% of the cases appear to be associated with EBV. In Western countries, BL represents about 3% of childhood tumours, 10 to 15% of them EBV-associated. Thus, from the northern industrialized countries to the equatorial developing countries, increasing incidences of lymphomas of the BL type are paralleled by an increasing proportion of EBV-associated cases. The Ugandan BL prospective study showed that high antibody titres to viral capsid antigen (VCA) preceded BL development by many years, with a quantifiable relationship between the level of VCA antibodies and tumour risk. If an early and/or massive EBV primary infection seems to represent the critical event for BL development in equatorial Africa, the favourable conditions for EBV-associated tumours in North Africa and in Europe remain to be investigated. Malaria appears to favour BL development through an EBV-specific T-cell immune deficiency. Chromosomal translocations and oncogene activation, considered as the final step in lymphoma development, do not appear to be related to EBV. Intervention against the virus may represent the ultimate proof of a causal relationship between EBV and the majority of BL cases around the world. PMID- 2998988 TI - Historical background; Burkitt's lymphoma and Epstein-Barr virus. PMID- 2998989 TI - Epidemiology of Burkitt's lymphoma: other risk factors. AB - Events surrounding infection and disease in infectious mononucleosis are a model for epidemiological factors that may affect the phase of initiation, the phase of promotion and the phase of tumour development in African Burkitt's lymphoma (BL). Consideration of the circumstances of early Epstein-Barr virus (EBV) infection as the initiator, and of malaria as the promoter, of African BL reveal some shortcomings in the evidence incriminating malaria. Possible factors leading to the emergence of the clinical tumour suggest that other factors may be involved. The absence of EBV and malarial infection in some cases of American BL indicate that they are neither necessary nor sufficient causes of the tumour in all settings. PMID- 2998991 TI - Role of Epstein-Barr virus in the etiology of Burkitt's lymphoma. AB - Although Epstein-Barr virus (EBV) was discovered in cultured Burkitt's lymphoma (BL) cells, its exact role remains unclear. Viral genome is found in 95-98% of endemic BL and 15-20% of non-endemic BL. Children destined to develop BL in Africa show elevated titres of viral capsid antibodies one to two years preceding emergence of BL. A multistep process follows early EBV infection during early childhood. Immune deficiency probably permits continuation of the infections, with smouldering polyclonal B-cell proliferation proceeding. Final steps in the pathogenesis consist of cytogenetic and molecular conversion to monoclonal BL. Reciprocal chromosomal translocations involve breakpoints containing c-myc, heavy and light-chain Ig loci. Activation of oncogenes, c-myc and B-lym, may be essential in the molecular pathogenesis of BL. A spectrum of EBV-induced pathological entities is found in individuals with X-linked lymphoproliferative and acquired immune deficiency syndromes. Lymphoma identical to endemic BL occurs in these immune-deficient patients. Non-endemic BL is possibly due to immune defects, initiators and promoters of B-cell proliferation, which may not be identical to factors in endemic BL; however, cytogenetic events and activation of oncogenes may be pathways of both endemic and non-endemic BL. PMID- 2998992 TI - Cell-mediated immunosurveillance mechanisms and the pathogenesis of Burkitt's lymphoma. AB - Paired Epstein-Barr virus (EBV)-carrying cell lines have been established from Burkitt's lymphoma (BL) patients, one of each pair being the BL cell line derived from the malignant cells of the tumour, the other, the lymphoblastoid cell line (LCL) derived from the patient's normal B cells by experimental infection with the virus. Comparative studies have shown the following: (1) All the lines were to some extent sensitive to in-vitro activated natural-killer cells, individual pairs differing as to whether BL or LCL cells were more susceptible. (2) For six of the seven pairs tested, the BL cell line was clearly sensitive to allo specific (anti-class 1 HLA) effector T cells, although levels of lysis were slightly below those observed for the corresponding LCL; only one BL cell line showed evidence of a dramatic reduction of HLA antigen expression, and this line was insensitive to allo-specific cytolysis. (3) For two of the three pairs tested to date, EBV-specific cytotoxic T-cell preparations from HLA antigen-matched donors lysed the LCL but not the BL cell line, despite the latter's apparent expression of the relevant restricting antigens. In both of these cases, it was known that the tumour arose in vivo in the face of prevailing EBV-specific T-cell surveillance. An escape of the malignant cells from such surveillance may therefore be important in the overall pathogenesis of EBV genome-positive BL. PMID- 2998990 TI - A preliminary report of epidemiological studies of Burkitt's lymphoma, Epstein Barr virus infection and malaria in North Mara, Tanzania. PMID- 2998993 TI - The cytogenetics of human B lymphoid malignancy: studies in Burkitt's lymphoma and Epstein-Barr virus-transformed lymphoblastoid cell lines. AB - Cells from Burkitt's lymphoma (BL) and from the majority of human B-cell neoplasms show karyotypic changes that characteristically involve chromosomal breakage and recombination in addition to some chromosome gains. These aberrations increase as the tumours progress in vivo, and a similar tendency is seen in BL-derived lymphoid lines in vitro. Epstein-Barr virus (EBV)-transformed lymphoblastoid lines of non-malignant origin also develop karyotypic abnormalities on prolonged culture, but these are predominantly nonrandom gains of whole chromosomes (i.e., non-disjunction events). They have never been observed to acquire the 8;14 translocation, which is an almost constant feature of BL. Nevertheless, there is some concordance between the pattern of chromosome gains found in long-term cultured lymphoblastoid lines and that seen in direct preparations from B-cell neoplasms. Many of the lymphoblastoid lines that have become aneuploid are tumorigenic in immunosuppressed mice, indicating that EBV transformed human B cells can acquire a malignant phenotype in the absence of specific chromosomal translocations. It is suggested that the predominance of chromosomal breakage and recombination events in the karyotypic evolution of BL and other lymphoid neoplasms comes about because chromosomal instability (which varies within a population) is a major risk factor for lymphoid malignancy, interacting with other risk factors, including impaired T-cell function and EBV to determine the clinical and epidemiological patterns of BI and related neoplasms. PMID- 2998994 TI - Prevention of endemic Burkitt's lymphoma. PMID- 2998996 TI - Cytogenetics of Burkitt's lymphoma-leukaemia: a review. AB - Two types of chromosomal abnormality have been found in Burkitt's lymphoma leukaemia. Three specific translocations, t(8;14), t(8;22) and t(2;8), having in common 8q24 band involvement, are thought to be present in the overwhelming majority of cases. These stereotyped translocations have been shown in many cases to be related to DNA molecular rearrangements of the immunoglobulin genes and the c-myc oncogene. Secondary chromosomal abnormalities, some of them nonrandom, have also been described. Their possible relation to other oncogene involvement and to the Epstein-Barr virus genome is discussed. PMID- 2998995 TI - Biochemistry of latent Epstein-Barr virus infection and associated cell growth transformation. AB - There is sufficient knowledge of the biochemistry of Epstein-Barr virus (EBV) persistence and gene expression in latent growth-transforming infection and of the persistence and expression of other oncogenic viruses to permit interesting and possibly useful comparisons. Most smaller oncogenic viral genomes usually persist solely as integrated DNAs despite their ability to circularize. Papilloma and hepatitis viruses may persist as episomes, and parts of their genomes may integrate. Usually, only the oncogenic fragment of adenovirus DNA is integrated into cell DNA. In contrast, the entire EBV genome persists in cells as an episome or as integrated DNA. Thus, EBV may have novel mechanisms to maintain its complete genome as an episome or as a complete integrated virus DNA. Three viral genes are expressed in latently EBV-infected growth-transformed cells, each of which encodes one RNA and one protein. Two of the proteins are probably nuclear DNA-binding proteins; the third is probably a membrane protein. Thus, the repertoire of genes expressed is similar in complexity and intracellular distribution to that expressed by papova and adenoviruses in cellular transformation. The papova and adenovirus-transforming genes are partially analogous to retrovirus oncogenes. This similarity cannot as yet be extended to EBV. There is no homology at the DNA-sequence or protein-sequence level between EBV and other viral or cell oncogenes. Thus, it remains important to pursue analysis of the EBV-transforming genes. Identification of these genes is a first step in discerning their function in latent growth-transforming cell infection. Parts of each of these genes are being made in bacteria. The bacterial products enable us to make antisera that are specific for each of the viral proteins. These antisera can also be used to identify the viral proteins within latently infected growth-transformed cells or within cells stably expressing transfected virus genes. The antisera can also be used to study the association of Epstein Barr nuclear antigen (EBNA) 1 and 2 with DNA and of the lymphocyte-determined membrane antigen (LYDMA) with the cell membrane. The three genes must be introduced into nontransformed cells to determine whether, alone or in combination, they are sufficient to accomplish cell growth transformation. PMID- 2998997 TI - Burkitt's lymphoma in Algeria. PMID- 2998998 TI - An empirical study of a hospital-based home care program. AB - Although it is widely perceived that home nursing care reduces the utilization of hospital services, and thus the cost of care, the magnitude of the savings is not clear. In this study of a hospital-based home nursing care program, we compared the medical process at two hospitals, one with and one without a home nursing department. Regression analysis showed that home nursing care significantly reduced both the length of hospital stays and the number of follow-up visits to outpatient clinics. After accounting for the cost of the home nursing program, however, we found that the program did not significantly reduce overall hospital expenditures. PMID- 2999000 TI - Effect of streptococcal lipoteichoic acid on prolyl hydroxylase activity as related to collagen formation in mouse fibroblast monolayers. AB - Dried and wet mouse fibroblast monolayers with labeled collagenous substrate were used to study the effects of lipoteichoic acid (LTA) on cellular prolyl hydroxylase activity. LTA is a scavenger of cations, and Fe2+ is essential for prolyl hydroxylase activity. Surprisingly, addition of LTA to dried monolayers resulted in increased prolyl hydroxylase activity, whereas preincubation of Fe2+ with LTA only negated this increase. However, significant inhibition of enzyme activity by wet monolayers occurred whether LTA was added directly to the test system or whether it was used after preincubation with Fe2+. These data suggest that LTA causes membrane perturbations. Also, that the binding of LTA to the membrane of dried and wet monolayers appears to be decidedly different when based on the subsequent availability of Fe2+ for cellular prolyl hydroxylase activity. The ability of LTA to act as a cationic exchanger and the presence of intracellular Fe2+ inaccessible to LTA probably accounted for the lack of complete inhibition of prolyl hydroxylase activity by this amphiphile in the wet cell system. Considerably less iron was needed to negate the partial inhibition of prolyl hydroxylase activity by LTA in viable cells than was needed to restore the increased enzyme activity by this amphiphile in equivalent dried preparations. These and other results showed that, although LTA does not affect collagen polypeptide chain formation in wet monolayers, its involvement at the molecular level does result in a marked decrease in the hydroxylation of collagenous peptidyl prolyl residues through LTA interaction with Fe2+. This reduction in prolyl hydroxylase activity equaled the reduction in hydroxylation of collagenous protein in fibroblast monolayers caused by LTA reported earlier (O. Leon and C. Panos, Infect. Immun. 40:785-794, 1983). Therefore, these data suggest that partial inhibition of prolyl hydroxylase activity is directly related to the synthesis of defective collagen by wet fibroblast monolayers exposed to minute amounts of group A, type 12 streptococcal LTA. Use of LTA also showed that complete inhibition of hydroxyproline formation is not required for the continued formation and accumulation of defective collagenous protein by these monolayers. PMID- 2998999 TI - Dissection of macrophage tumoricidal and protozoacidal activities using T-cell hybridomas and recombinant lymphokines. AB - Macrophage (M phi) phenotype and function can be modulated by various T-cell lymphokines (LK). The alteration of M phi phenotype is a result of LK concentration, duration of exposure, and the level of M phi activation when obtained from in vivo sources through elicitation by either sterile irritants or cellular immune mechanisms. To dissect M phi activation into discrete signals, we constructed T-cell hybridomas by fusing hypoxanthine-aminopterin-thymidine sensitive BW5147 cells with nylon wool-purified, concanavalin A-stimulated T cells. The resulting T-cell hybrids were screened for their ability to (i) protect M phi from the cytopathic effect of Naegleria lysates, (ii) induce class II major histocompatibility complex gene product (Ia antigen) expression, (iii) increase tumoricidal and cytostatic activity, and (iv) alter ectoenzyme profiles on either resident or thioglycolate-elicited M phi. Two hybridomas (T-3 and T-9) were selected for further evaluation because of their activity patterns. Supernatants from T-3 and T-9 were compared with cloned gamma-interferon (IFN gamma) for alterations of biological activities. Both T-3 and T-9 were able to protect resident-M phi cells from Naegleria lysate but had no protective effect on thioglycolate-induced M phi. T-9 supernatant had patterns of activity similar to IFN-gamma, whereas T-3 patterns were different. The addition of anti- IFN gamma removed T-9 cytostatic activity while not affecting T-3-induced activity. The LK inducing protection from the cytopathic effect of Naegleria lysate is not IFN-gamma but another molecular moiety. We conclude that the activation of M phi for the destruction of tumor cells and amoebae may occur via different mechanisms. PMID- 2999001 TI - Oxidative metabolism in cord blood monocytes and monocyte-derived macrophages. AB - Little is known about phagocytosis-associated oxidative metabolism in mononuclear phagocytes from the human neonate. We investigated this phenomenon in monocytes from the cord blood of term newborn infants by measuring generation of superoxide anion (O2-) and hydroxyl radical (X OH) after stimulation with opsonized zymosan or phorbol myristate acetate. Production of these microbicidal oxygen metabolites by monocytes from neonates and healthy adult volunteers was equivalent. When cultured in the presence of the macrophage activator lipopolysaccharide or muramyl dipeptide, monocytes from neonates and adults differentiated into cells with the appearance of macrophages and with an enhanced capacity to release O2- compared with cells cultured in the absence of an activator. Monocyte-derived macrophages from neonates produced only slightly less O2- than did adult cells. Thus, unlike the cord blood neutrophil, which exhibits abnormalities in oxidative metabolism, the cord blood mononuclear phagocyte has a respiratory burst that is quantitatively comparable to that of the adult cell. PMID- 2999002 TI - Epidemic of LAV/HTLV III infection in drug addicts in Milan: serological survey and clinical follow-up. AB - A clinico-epidemiological study is reported concerning a group of 306 parenteral drug addicts (PDAs), 71 of whom were affected with the lymphadenopathy syndrome (LAS); all were followed-up between 1981 and 1984. Although full-blown acquired immune deficiency syndrome (AIDS) was observed only in one case, none of the other patients examined have undergone complete recovery so far. The results of our study point to a wide circulation of LAV/HTLV III among our group of PDAs, starting at least as early as 1981 and preceding by a few months the development of clinical signs and symptoms of LAS. A peak incidence of the latter was observed during the winter of 1983/1984, running parallel with a marked increase in seropositives for LAV/HTLV III antibody. Drug addiction, sexual promiscuity and a low standard of living all seem to play a decisive role in the spread of the infection and, consequently, of the diseases related to it (LAS, AIDS-related complex and AIDS). In fact, PDAs appear to represent the major source of the disease in Italy. PMID- 2999003 TI - Adhesion of guinea pig polymorphonuclear leukocytes to autologous aortic strips: influence of chemotactic factors and of pharmacological agents which affect arachidonic acid metabolism. AB - In superfusion experiments, the complement peptide C5a-desArg and the leukotriene B4 (LTB4) enhanced adhesion of guinea pig polymorphonuclear leukocytes to autologous aortic strips (threshold at about 10(-8) M, maximal effects at 10(-7) M). C5a-desArg acted primarily by stimulation of the leukocytes: pretreatment of them with the peptide abolished their response by deactivation, whereas pretreatment of the endothelium did not affect adhesion. However, the endothelium obviously cooperated in the response: enhanced adhesion was obtained only when the leukocytes were exposed to C5a-desArg while in contact with the endothelium. The cooperation is most probably due to release of arachidonic acid from endothelium and formation of lipoxygenase products (LTB4?) therefrom by the stimulated leukocytes. Incubation of leukocytes with nordihydroguaiaretic acid or with relatively high concentrations of indomethacin--both known to inhibit lipoxygenases-- lowered the effect of C5a-desArg, but not that of LTB4 nor the spontaneous adhesion. On the other hand, the stable prostacyclin analogue ZK 36 374 decreased C5a-desArg-induced adhesion, while pretreatment of the aortic strips with indomethacin increased it. These results suggest that endogenous prostacyclin may also play a role in this system by reducing adhesion. PMID- 2999004 TI - Ionizing radiation dosimetry in the absorbed dose range 0.01-50 MGy based on resistance and ESR linewidth measurements of organic conducting crystals. AB - The materials studied in the present work as high-dose dosimeters are members of a large class of molecular crystals which are organic conductors of electricity. Very different from each other in the details of their molecular and crystal structures, they all behave in the same way when subjected to increasing high doses of radiation, at least from the point of view of their electronic transport properties, because of the quasi-one-dimensional character of the conduction process. Their resistivities increase exponentially with the absorbed dose while their electron spin resonance (ESR) linewidths decrease exponentially. Very small single crystals less than 10 micron thick can be used as dosimeters in the dose range 0.01-50 MGy for gamma rays as well as for electron irradiations, by applying four probe resistance measurements. Only a few compounds over a large number of candidates have been irradiated in the present work with gamma-rays, low energy x-rays and electrons. In some favourable cases the energy and temperature dependences of the dosimeters have been checked experimentally. Their mass energy absorption coefficients and electron stopping powers have been also calculated. It is hoped to extend this kind of dosimetry to lower and higher doses by trying new compounds from the large family of organic conductors or by improving the resistivity and ESR measurement techniques. PMID- 2999006 TI - Evidence for increased epidermal growth factor receptors in human sarcomas. AB - The results of an immunocytochemical study of the epidermal growth factor receptor (EGFR) in 35 human soft-tissue sarcomas, using a murine monoclonal antibody (MAb) EGF-R1, are reported. In many of the tumours staining was stronger than in the adjacent stroma, suggesting increased levels of receptor. Particularly strong staining was seen in one epithelioid sarcoma and in the spindle-cell component of a synovial sarcoma. Binding studies carried out on an epithelioid sarcoma cell line established from one of the specimens, using radiolabelled EGF, showed that approximately 8% of the receptors were of high affinity with a dissociation constant (KD) of approximately 10(-10)M, while the remainder were of lower affinity with a KD of 10(-9)M. The cells expressed a total of 1.7 X 10(6) receptors/cell which is equivalent to that found in some epidermoid tumours where gene amplification has been demonstrated. These data suggest that, as with other tumours recently reported, increased levels of epidermal growth factor receptor may be related to transformation. PMID- 2999005 TI - Prevalence of HTLV-I in Arctic regions. AB - Sera of native inhabitants of Arctic regions were assayed for antibodies to HTLV I by the ELISA technique followed by competition experiments to confirm antibody specificity. Residents of 7 widely separated Alaskan villages exhibited prevalence rates of 0 to 12% for HTLV-I antibodies. Less than 1% of Greenland Eskimos were HTLV-I antibody-positive. Residents of 3 northern Swedish regions ranged in HTLV-I antibody prevalence from 0 to 5%. Sera of healthy native inhabitants of Alaska and northern Sweden were similarly assayed for antibodies to HTLV-II. No additional sera were shown to be positive for HTLV-II antibodies. While some of the HTLV-I antibody-positive sera exhibited cross-reactivity with HTLV-II antigens, competition experiments using disrupted HTLV-II or purified HTLV-I p24 as test antigens indicated that the primary antibody response in all cases tested was elicited by HTLV-I. Our results show that HTLV-I distribution is not restricted to endemic areas in warm, humid climates, but extends to Arctic regions. Within these regions, HTLV-I exhibits the same restricted distribution seen in other areas where virus infection is prevalent. The Arctic does not seem to be a reservoir for HTLV-II infection. The origin of HTLV-I in Arctic areas is not known. One may speculate that foreign visitors introduced the virus into Aleut and Lapp populations, and that it has been maintained there and restricted in its distribution as a result of close familial relationships. PMID- 2999007 TI - Autoimmune basis for visual paraneoplastic syndrome. PMID- 2999008 TI - Segmental syringocystadenoma papilliferum in an unusual location. PMID- 2999010 TI - Acquired C1-inhibitor deficiency associated with antiidiotypic antibody to monoclonal immunoglobulins. PMID- 2999009 TI - Paget's 1874 article on the breast. Modern misconceptions. PMID- 2999011 TI - Fat cell adrenoceptors: inter- and intraspecific differences and hormone regulation. AB - The present review summarizes recent data on fat cell adrenoceptors with the aim of clarifying the role played by catecholamines in the regulation of adipocyte metabolism. Part of the review is focused on the possible interest of animal models for the study of catecholamine-mediated effects in human fat cells. It is now clearly demonstrated that human, hamster, dog and rabbit fat cells possess three basic types of adrenoceptor: the beta 1-, alpha 2- and alpha 1 adrenoceptors identified in biological assays or binding studies with selected radioligands. The rat is an exception in the species commonly studied as catecholamines exert an exclusive lipolytic effect through beta-adrenoceptor stimulation, there are no alpha 2-adrenoceptors in rat white fat cells although an alpha 1-adrenoceptor does exist. In human fat cells, physiological amines are lipolytic or antilipolytic. Binding studies have revealed that alpha 2 adrenoceptors are three to four times more numerous than beta 1-adrenoceptors. Moreover physiological amines, in particular epinephrine, have a higher affinity for alpha 2-sites than for beta 1-sites. Dose-response studies of the effect of epinephrine on adenosine-deaminase or isoproterenol-stimulated fat-cells demonstrate an inhibitory effect of epinephrine on lipolysis promoted by stimulation of alpha 2-adrenoceptors which occurs before the commonly described beta 1-adrenergic effect which promotes stimulation of lipolysis. This aspect and its putative physiological interest is described and discussed. Intraspecific variations in adrenergic responses of adipocytes have been briefly analysed. The appearance and disappearance of alpha 2-adrenoceptors according to the extent of adipose tissue and increment of fat cell size are discussed. Variations of adrenergic responsiveness during fasting, calorie restriction or chronic stimulation of the adipocytes by physiological amines are also discussed. PMID- 2999012 TI - Molecular mechanisms for hormonal control of adipose tissue lipolysis. AB - The fast-acting lipolytic hormones and insulin regulate adipose tissue lipolysis through control of the activity of hormone-sensitive lipase. This enzyme catalyzes the rate limiting step of adipose tissue lipolysis--the hydrolysis of stored triacylglycerols. The isolated enzyme is rapidly phosphorylated and activated by cyclic AMP-dependent protein kinase, with 1 mol of phosphate incorporated per mol of lipase Mr = 84000 subunit into a single serine residue. The enzyme is dephosphorylated and deactivated by protein phosphatases type 1, 2A and 2C. In the intact, isolated adipocytes the enzyme incorporates phosphate in the absence of hormonal stimulation into a specific 'basal' phosphorylation site. The phosphorylation of this 'basal' site (into a serine residue) is not accompanied with any change of the activity of the enzyme and is not influenced by hormones. The fast-acting lipolytic hormones induce a phosphorylation of another serine residue in a 'regulatory' phosphorylation site, which is identical to that phosphorylated in the isolated enzyme by cyclic AMP-dependent protein kinase. Following the phosphorylation of the 'regulatory' site the activity of the lipase, and consequently the rate of lipolysis, is increased almost 50-fold. Insulin causes a rapid net dephosphorylation of the lipase and exerts its well known anti-lipolytic action. Half-maximal inhibition of both phosphorylation and activity occurs at an insulin concentration of about 25 pM. The mechanism(s) whereby insulin causes its effects is unknown but apparently to a large extent involve reduction of the cellular cyclic AMP level. PMID- 2999013 TI - Participation of alpha-adrenoreceptors in brown adipose tissue thermogenesis in vivo. AB - Norepinephrine-induced BAT thermogenesis in anaesthetized cold-acclimated rats was markedly inhibited by alpha1-adrenoreceptor antagonists (eg prazosin). The inhibition was relieved by infusion of alpha1-agonists (eg phenylephrine). The beta-agonist, isoproterenol, was a relatively poor activator of BAT thermogenesis in vivo, but became a potent activator when combined with doses of phenylephrine that when given alone did not stimulate BAT thermogenesis. Phenylephrine also potentiated the thermogenic response of BAT to suboptimal doses of norepinephrine. The effects of prazosin and phenylephrine on BAT thermogenesis were shown to occur at the level of the BAT itself. PMID- 2999015 TI - Brown adipose tissue thermogenesis and the energetics of lactation in rodents. AB - Brown adipose tissue thermogenesis has been shown to be suppressed during lactation in rats and mice. In parallel with this suppression there is a reduction in the capacity for non-shivering thermogenesis in the whole animal. The extent to which thermogenesis is reduced in lactation is related to litter size, the larger the litter the greater being the reduction. It is argued that the suppression of brown adipose tissue thermogenesis during lactation results in a substantial economy in the non-lactational component of maternal energy expenditure. PMID- 2999014 TI - Regulation of energy expenditure in brown adipose tissue. AB - The endocrinological and biochemical mechanisms controlling energy expenditure in brown adipose tissue at the cellular as well as at the total tissue levels are briefly reviewed. Thermogenesis in brown adipose tissue is principally controlled by the activity of hormone-sensitive lipases that represent the 'flux-generating' step in the stimulus-calorigenesis sequence. Long chain fatty acids are the physiological messengers regulating mitochondrial respiration. Agents stimulating brown adipocyte lipolysis (catecholamines, glucagon, methylxanthines) also stimulate respiration, and conversely, agents inhibiting lipolysis (adrenergic antagonists, insulin, prostaglandins) also inhibit respiration. This indicates that lipolysis and respiration are functionally coupled in brown adipose tissue. On the other hand, brown adipose tissue thermogenic capacity increases during cold acclimation or adaptation to hyperphagia. Brown adipocyte proliferation and differentiation from precursor cells (interstitial cells and brown preadipocytes) represent the fundamental phenomena explaining the increase capacity of cold acclimated and/or hyperphagic animals for responding calorigenically to catecholamines. Physiological situations associated with a stimulation of energy expenditure and a negative energy balance (cold acclimation, exercise training, caffeine consumption) generally induce a stimulation of adipocyte proliferation in brown adipose tissue that is accompanied by a simultaneous inhibition of cell proliferation in white adipose tissue. The physiological significance of these metabolic adaptations is to modulate the capacity of homeothermic animals for energy expenditure in accordance with energy requirements. PMID- 2999016 TI - Distinct behavior of beta-endorphin and corticotropin toward leucine aminopeptidase action. AB - Reactions of human beta-endorphin, corticotropin and their synthetic analogs with leucine aminopeptidase have been investigated. The results confirmed previous findings that beta-endorphin is resistant to the aminopeptidase action whereas corticotropin is not. Beta-endorphin-(1-5) is completely digested by the enzyme while beta-endorphin-(1-17) is resistant. In contrast, the NH2-terminal 7 residues in corticotropin are removed readily by leucine aminopeptidase. This is confirmed by the observation that human corticotropin-(7-38) is not hydrolyzed by the enzyme. This contrasting behavior of the two hormones toward leucine aminopeptidase may be related to differences in their conformational structures. PMID- 2999017 TI - Studies on opioid receptor selectivity of beta-endorphin antagonists. AB - Opioid receptor selectivity of several beta-endorphin (beta-EP) analogs which antagonize beta-EP-induced analgesia has been assessed using partially selective binding assays. Although the apparent affinity dissociation constant of beta-EP in these assays varies from 0.2 to 360 nm, the potency of beta-EP antagonists relative to beta-EP remains largely unchanged. It is unlikely that differences in receptor affinities can account for the antagonist properties of these analogs in vivo. PMID- 2999019 TI - Effects of ACTH and prednisone on mood: incidence and time of onset. AB - Prior studies of mood and cognitive changes produced by ACTH and glucocorticoids have not characterized accurately the incidence or time of onset of these changes. In this study mood and cognitive reactions of fifteen medical patients treated with ACTH or prednisone were studied prospectively. ACTH produced a lessening of dysphoria by the third treatment day, and mild euphoric reactions occurred in three of seven of the patients treated. Prednisone produced a reduction of dysphoria by the seventh day, but no euphoric reactions in the eight patients treated with it. Neither medical symptom improvement nor elevation of plasma cortisol levels in the patients given ACTH appeared to account for the results. The mechanism of the observed mood change remains to be elucidated. PMID- 2999020 TI - Matrix isolation of free radicals from carbohydrates. II. Reactions of H. with glucose and derivatives in acidic glasses. AB - Photolytically produced H.-atoms in 6 mol dm-3 H2SO4/H2O glasses trapped at 77 K react upon annealing to 130 K with dissolved carbohydrates to form carbon-located free radicals by abstraction of carbon-bound protons. Analysis of electron spin resonance (e.s.r.) spectra at various annealing stages from alpha- and beta-D glucose together with 6,6-d2-D-glucose, 6-deoxy-D-glucose, 2-deoxy-D-glucose, glucose-1-phosphate, D-xylose, D-allose and D-mannose indicates radical formation at all possible carbon sites with a strong preference for C1 and a somewhat enhanced contribution of C4 over the statistical expectation. The corresponding component spectra are analysed either by spectra isolation or simulation and their parameters are given. Intramolecular radical transformation at temperatures of 140-160 K is explained by acid-catalysed H2O-elimination. The findings are discussed in relation to the radiation-chemistry of aqueous glucose solutions. We thus show that the system of photolyzed Fe2+ in acidic glasses at low temperatures containing 10 mmol dm-3 carbohydrate is suitable for studying H(D.) reactions by means of e.s.r. spectroscopy. Unlike previously used glasses containing carbohydrates, contributions of oxidation and reduction by direct effects or mixtures of direct and indirect effects and phase-effects due to incomplete glass formation are avoided. PMID- 2999018 TI - Alteration of the blood-retina barriers in cases of viral retinitis. AB - This paper presents three cases of serologically documented viral retinitis, and the great value of fluorescein angiography in outlining the structural abnormalities and the site of the lesions. Of our series in the case of influenza retinitis, the fluoroangiographic findings showed dye leakage from retinal vessels in the posterior pole. The dye leakage did not appear completely in the sites of macular edema. This edema was similar to the clinical appearance of cotton-wool spots and was arranged in a star-like pattern. One of the cases of cytomegalovirus retinitis (Case 3), a previously healthy adult with dysfunction of the cellular immune system, seems to be a further example of an inflammation in the inner retina presenting cotton-wool spots at the early stage. Case 2, a previously healthy adult, fulfilled the criteria for Vogt-Koyanagi-Harada syndrome. The patient had serologically documented cytomegalovirus infection with dysfunction of the cellular immune system. The fluorescein angiographic examination showed alteration both in the inner and outer blood-retina barriers as it is characteristic in cases of Harada syndrome. The cytomegalovirus infection might be assumed to play a role in the clinical picture as well as in the etiology of this disease. PMID- 2999023 TI - AIDS update Part 2. PMID- 2999022 TI - Dysenzymia induced by hexavalent chromium in rat liver. AB - The alterations in the distribution and activity of certain key enzymes, viz. alkaline phosphatase, acid phosphatase, glucose-6-phosphatase, cholinesterase and lipase, have been determined in the liver of rats (Rattus rattus albino) after experimental poisoning with hexavalent chromium. The histochemical and biochemical observations presented herewith provide visual evidence of chromium induced inhibition of all these enzymes except lipase, which was found to be stimulated insignificantly. The results have been interpreted in terms of changes in the micro-environment of the cell, formation of apo-enzymes, metal-protein complexes, oxidative phosphorylation and finally with liver function. PMID- 2999021 TI - Ion transport regulation by prostaglandins in mouse macrophages. AB - Although the prostaglandins PGE1, PGE2 and PGF2 alpha had no effect on ion transport in isolated human erythrocytes, they modulated ion transport in isolated mouse macrophages, apparently through the mediation of cAMP, by inhibiting the NA+, K+ cotransport system, stimulating the Na+, K+ pump, and stimulating the Na+: Ca++ exchange mechanism. PMID- 2999025 TI - Viral hepatitis. PMID- 2999024 TI - Inactivation of herpesvirus on CPR manikins utilizing a currently recommended disinfecting procedure. AB - The adequacy of a currently recommended protocol for disinfecting CPR manikins was investigated. Known quantities of Herpes simplex type 1 virus were applied to various sites on both adult and infant manikin heads, exposed to disinfectant under simulated classroom conditions, and then assayed for infectious virus. Results indicate that the disinfecting protocol and disinfectant are adequate for inactivating herpesvirus on CPR manikins, but that care must be exercised to ensure thorough cleaning. PMID- 2999027 TI - Togaviridae. AB - The family Togaviridae comprises four genera: Alphavirus (with 26 species), Rubivirus (one species), Pestivirus (three species), and Arterivirus (one species). The main characteristics of the member viruses are: (i) the virus particles are spherical, 50-70 nm in diameter, including an envelope with surface projections that incorporate two or three polypeptides, usually glycosylated; (ii) the nucleocapsid comprises a core protein and a single strand of positive sense RNA, molecular weight about 4 X 10(6); where characterized, the RNA has an m7G 'cap' at the 5' end and is polyadenylated at the 3' end; (iii) maturation occurs by budding of spherical nucleocapsids 30-35 nm in diameter, with proven or presumed icosahedral symmetry, through cytoplasmic membranes. Where characterized, translation of structural proteins occurs on subgenomic messenger RNA(s); these appear to represent the 3' end of the genome. Nearly all alphavirus species are transmitted by mosquitoes. Transmission also occurs transovarially (Alphavirus) or transplacentally (Rubivirus and Pestivirus). Members of a genus are serologically related, but are not related to members of other genera. PMID- 2999026 TI - Contribution of paramagnetic trace elements to the spin-lattice relaxation time in the liver. AB - The relaxation times of water protons in rat liver tissue were measured with a NMR spectrometer at 20 MHz. The paramagnetic trace elements Cu, Fe, and Mn were determined by neutron activation analysis. No shortening of T1 could be observed when liver Cu or Fe concentration was increased in the microgram range. T1 was strongly correlated with the liver Mn concentration of untreated animals and animals whose liver Mn concentration was artificially increased or decreased by intravenous injection of manganous acetate or a metal chelating agent with high affinity for hepatobiliary excretion. Deviations from this Mn-T1 correlation were found in the initial phase of liver cirrhosis induced by thioacetamide (elongated T1, normal Mn concentration) and after stimulation of liver growth by phenobarbital (normal T1, decreased Mn concentration). An increased or decreased enhancement factor for Mn may have contributed to the observed deviations during phenobarbital and thioacetamide treatment. PMID- 2999028 TI - Etiologic studies of the 1983 and 1984 outbreaks of epidemic diarrhea in Guangxi. AB - Studies are reported on two outbreaks of epidemic diarrhea in China involving 19,007 patients. The first outbreak occurred in the northern part of the Guangxi Autonomous Region and extended south from April through September 1983. In June 1984 a second outbreak of 6,570 cases occurred in Guanyang, a county in the northern part of Guangxi. 125 fecal samples from the two outbreaks were cultured for bacteria; all of the samples except one were negative. Rotavirus particles were detected in 44.1% (15/34) of stool specimens examined by immune electron microscopy. The two outbreaks appeared to be caused by non-group-A rotaviruses, based on the pattern of electrophoretic migration of the RNA genome segments in polyacrylamide gels and on the lack of a detectable common group antigen(s) when tested by an enzyme-linked immunosorbent assay for group A rotaviruses. Complement-fixation tests also indicated that 37.5% of convalescent-phase sera from patients produced antibody to the isolated rotavirus. The total molecular weight of the RNA segments in the new Guangxi strain was calculated to be 10.69 X 10(6), with the 11 genome segments having molecular weights (X 10(6)) of 2.19, 1.83, 1.56, 1.53, 0.82, 0.81, 0.60, 0.46, 0.35, 0.30, and 0.24. This study also reports comparative detection rates for direct and immune electron microscopy and for PAGE. PAGE proved to be the most sensitive detection method for these new rotaviruses. PMID- 2999029 TI - Beneficial role of a nonpathogenic orbi-like virus: studies on the interfering effect of M14 virus in mice and mosquitoes infected with Japanese encephalitis virus. AB - M14 virus, isolated from Culex tritaeniorhynchus mosquitoes collected in a Beijing suburb, was identified as a noncytopathogenic orbi-like virus. It was found to interfere with the growth of Japanese encephalitis (JE) virus, a mosquito-borne virus which infects humans, pigs, and horses in much of Asia, including China. JE virus is transmitted by C. tritaeniorhynchus mosquitoes and causes encephalitis in humans and horses and abortion in pigs. Because it had potential as an interfering agent for the biological control of JE, the M14 virus was characterized and its interfering effect was studied in mice and in C. tritaeniorhynchus mosquitoes. PMID- 2999031 TI - Mouse polyoma virus and adenovirus replication in mouse cells temperature sensitive in DNA synthesis. AB - Mouse adenovirus multiplies, apparently without impediment, in temperature inactivated ts A1S9, tsC1 and ts2 mouse fibroblasts. Thus, the DNA of mouse adenovirus can replicate in the absence of functional DNA topoisomerase II, a DNA chain-elongation factor, and a protein required for traverse of the G1/S interface, respectively, encoded in the ts A1S9, tsC1 and ts2 genetic loci. These results are compared with those obtained with polyoma virus. PMID- 2999030 TI - Effects of cyclosporine on the pathogenesis of primary cytomegalovirus infection in the guinea pig. AB - The effects of cyclosporin A (CsA) upon the pathogenesis of primary cytomegalovirus (CMV) infection were investigated. Hartley and strain 2 guinea pigs were inoculated subcutaneously on day 0 with 10(4) TCD50 of virulent, salivary-gland-passaged guinea pig CMV and received oral CsA (20 mg/kg/day) for 14 days. CMV-infected, CsA-treated animals lost approximately 20% of their total body weight in 14 days and had more than twice the rate of CMV isolation from internal organs when compared with untreated controls, despite similar rates of viremia. Internal organs of CsA-treated animals demonstrated widespread viral inclusions and minimal inflammatory response to the presence of CMV-infected cells. The mean change in peripheral blood lymphocyte values for CMV-infected, CsA-treated Hartley guinea pigs was -2,867 +/- 2,955 (mean +/- SD) lymphocytes/microliters as compared to +10,933 +/- 7,583 in CMV-infected, untreated controls (p less than 0.01). In the guinea pig model, CsA administration had adverse effects upon the pathogenesis of primary CMV infection. PMID- 2999032 TI - Histopathological aspects of tissue reactivity to suture materials in microsurgical arterial, anastomosis. AB - In order to assess tissue reactivity to synthetic suture material in microsurgery, a histological study was carried out in 20 rabbits divided into two groups: "A" and "B". The aorta of the animals was sectioned and subsequently anastomosed using polyglycolic acid in the first group and monofilament nylon in the second group. The animals were sacrificed at 10, 20, 40, 60 and 90 days after surgery, and the specimens were studied both macroscopically and microscopically. Tissue reactivity during the initial 40 days had similar histological characteristics in both groups evidentiating a perisuture granuloma with macrophages and giant cells. The second period, 40 to 90 days after surgery, was characterized by a tissue reactivity specific for each type of suture. In group "A", the inflammatory process diminished in parallel to the absorbtion of polyglycolic acid with "restitutio ad integrum" of the vessel wall. In group "B", the inflammatory process persisted due to the presence of non absorbable monofilament nylon, thus causing a fibrosclerotic transformation of the vessel wall. The results of this study suggest that the most suitable suture material for anastomosing living tissues are absorbable sutures. PMID- 2999033 TI - [Danger to the human caused by animal poxvirus following discontinuation of mandatory vaccination against smallpox]. AB - The discontinuation of smallpox vaccination will lead to a gradual decrease or disappearance of immunity to poxviruses of the genus Orthopoxvirus. It is discussed whether orthopoxviruses of animals may then constitute a potential danger to man, with respect to their mutagenic and adaptation capabilities as well as their possible genetic interactions. Infection of man with these viruses is generally possible, but at present, not of acute importance. Monkeypox and cowpox viruses, as well as their variants occurring in carnivores and rodents, are of primary interest for individuals who have not been vaccinated against smallpox. Suggestions are put forward on how the human population, as well as domestic and laboratory animals, may be protected against infection with orthopoxviruses originating from animals. In particular, it is recommended that endangered groups of people should receive voluntary vaccination with genetically stable, attenuated vaccinia virus strains. PMID- 2999034 TI - Estimates of lifetime lung cancer risks resulting from Rn progeny exposure. AB - Data on five mining populations exposed to Rn progeny have been used to estimate the lifetime risk of lung cancer resulting from occupational and environmental exposure under current standards. Slopes of dose-response relations for lung cancer show a tendency to decrease with increasing dose. Our best estimate of curvilinearity is given by raising dose to the power 0.92 +/- 0.07, but the improvement in fit beyond simple linearity is not significant. On the other hand, the addition of a cell-killing term significantly improves the fit of the linear model. In any event, linear extrapolation is unlikely to underestimate the excess risk at low doses by more than a factor of 1.5. However, these inferences about curvilinearity are highly subject to error from the choice of reference populations, dosimetry, and latency. Under the linear-cell-killing model, our best estimate of excess relative risk is 2.28 +/- 0.35 per 100 working level month (WLM) (a doubling dose of 44 WLM). Attributable risks in these five studies range from 3.4-17.8 per 10(6) person-yr WLM-1. Risks from Rn progeny appear to interact with age and smoking in a form intermediate between additive and multiplicative. The "relative risk" model is therefore preferable for projecting lifetime risks, but life-table projections are described for a wide variety of assumptions. Our best estimate of the effect of a 50-yr occupational exposure to 4 WLM yr-1 is 130 excess lung cancer deaths per 1000 persons (0.65 per 1000 person-WLM), with a range from 60-250 per 1000. Similar calculations for lifetime exposure to an additional 0.02 working level (WL) beyond normal background produces an estimate of 20 excess lung cancers per 1000 persons. PMID- 2999036 TI - Radiation measurements in a labyrinth penetration at a high-energy proton accelerator. AB - The efficient design of access penetrations at high-energy proton accelerators is desirable for both economic and personnel protection reasons. This paper reports on a series of measurements made in a personnel access labyrinth which viewed an A1 target bombarded by 400-GeV protons from the Fermilab Tevatron. Measurements of absorbed dose in the labyrinth using tissue-equivalent ion chambers were consistent with theoretical predictions of both the relative attenuation through the penetration and the absolute magnitude near the target. The multisphere technique was used to determine the neutron energy spectrum in one section of the labyrinth. A recombination chamber was used to measure the quality factor of the radiation field in two sections of the labyrinth. Good agreement with the quality factor deduced from the multisphere result was obtained at the same measurement location. PMID- 2999035 TI - The application of electrets to passive Rn progeny dosimeters. AB - The theoretical and experimental bases are presented for development of a passive electret dosimeter for Rn progeny. The mechanism of aerosol collection is described, and experiments to develop a suitable aerosol collecting element (electret) for a passive Rn progeny dosimeter are reported. PMID- 2999037 TI - Neutron spectrum measurements at a 40-MeV proton cyclotron. AB - A set of seven activation reactions has been selected for neutron spectral analysis in the environment of a proton-cyclotron target. This choice of reactions: 59Co(n, p) 59Fe, 59Co(n, 2n) 58Co, 59Co(n, 3n) 57Co, 197Au(n, gamma) 198Au, 197Au(n, 2n) 196Au, 197Au(n, 4n) 194Au, 27Al(n, alpha) 24Na, analyzed by means of a Ge(Li) detector, reduces to a minimum of three the number of activation detectors employed, and makes possible convenient and accurate spectral measurements to at least 40 MeV. Criteria for selection of the activation materials from a list of candidates are discussed. A detailed comparison of the unfolding programs LYRA and SAND is made, and reasons are given for our choice of SAND in our application. Spectra of neutrons emitted at 0 degrees, 45 degrees and 90 degrees from thick targets of A1, Fe, Cu, Ta and stainless steel, irradiated by 40-MeV protons at the Milan AVF cyclotron, using the analysis technique described, are given and discussed. PMID- 2999038 TI - Performance of a passive electret dosimeter for Rn progeny. PMID- 2999039 TI - The relative effect of ventilation on the potential alpha energy from 222Rn and 220Rn progeny. PMID- 2999040 TI - [Abnormal germinal cells. The viewpoint of the internist]. PMID- 2999041 TI - [Measuring the DNA distribution pattern in human testicular tumors]. PMID- 2999042 TI - [Aggregated familial occurrence of testicular tumors]. PMID- 2999043 TI - Vitamin D and cyclic nucleotide changes in response to calcitonin in man. PMID- 2999044 TI - A new X-ray diffraction method for the quantitative analysis of free silica in the airborne dust in working environment. PMID- 2999045 TI - Psittacine inclusion body hepatitis in an aviary. AB - Psittacine inclusion body hepatitis (also known as Pacheco's parrot disease) was believed to be responsible for fatal necrotizing hepatitis and splenitis in a variety of psittacine birds from a private aviary. Splenic cells and degenerative hepatocytes around the outer zone of necrotic areas had margination of nuclear material and large intranuclear inclusion bodies. Clinical signs consisted of weakness, anorexia, vomiting, loose feces, and slight ruffling of feathers. The source of the infection was undetermined, but could have been associated with 3 Patagonian conures within the aviary. Patagonian conures are well-recognized as clinically normal carriers. The outbreak was limited by strict quarantine and disinfection of the aviary for 14 days. PMID- 2999047 TI - Morpho-quantitative analysis of nuclear inclusions in periaqueductal grey matter neurons in the cat. AB - The morpho-quantitative analysis carried out in the neuronal population of the periaqueductal grey matter of the cat has shown that nuclear inclusions are mainly of the filamentous type and that they are distributed predominantly in the external region, i.e. in the part of the periaqueductal grey matter situated furthest from the cerebral aqueduct, where 30% of the cells contain nuclear inclusions. In the internal region, i.e. in the part nearest the subependymal zone, only 2% of the neurons have nuclear inclusions. The glia in the internal region is more abundant and surrounds each nerve cell body while in the external zone of the periaqueductal grey matter it is scanty and does not delimit the neuronal soma. This difference suggests that there may be a relationship between the incidence of nuclear inclusions and the neuron/glia ratio. PMID- 2999046 TI - Ultrastructure of the hepatocytes in a vertebrate liver without bile ducts. AB - Thin sections and freeze fracture replicas were used to study the structure of the hepatocytes of the parasitic adult lamprey (Petromyzon marinus L.). Despite the absence of bile ducts and bile canaliculi, the hepatocytes have some features which resemble those of cells in the livers of other vertebrates. Hepatocytes are characterised by large gap junctions, many cytoplasmic inclusions, and large deposits of iron. The latter is present throughout the cytoplasmic matrix and within large inclusion bodies which may arise through sequestration of parts of the cytoplasm by membrane isolation. There is no evidence for the involvement of hepatocytes in glucose metabolism but their fine structure reflects the production of bile products and the processing of lipoproteins. The accumulation of bile products within cytoplasmic inclusions resembles the situation resulting from biliary atresia or other cholestatic conditions in higher organisms. There is little folding of the plasma membrane facing the perivascular space (of Disse), perhaps indicating limited involvement of this surface in the transport of bile products. Nerve endings in close apposition to hepatocytes suggest possible nervous control or metabolic function or the presence of sensory receptors in lamprey liver. PMID- 2999048 TI - Nuclear bodies in the caecum of the rabbit. AB - The structure and cytochemistry of nuclear bodies in the caecum of the rabbit are described and a developmental pathway from morphologically simple to complex forms is proposed. Simple nuclear bodies are proteinaceous in nature while complex bodies possess a proteinaceous capsule with some associated ribonucleoprotein. The interna of complex bodies possess various individual components consisting either of deoxyribonucleoprotein, ribonucleoprotein, or unassociated proteins. The significance of these nuclear bodies in caecal biology is discussed. PMID- 2999049 TI - The stimulation of bioluminescence in Photobacterium leiognathi as a potential prescreen for antitumor agents. AB - The stimulation of bioluminescence in Photobacterium leiognathi has previously been described as a test for genotoxic compounds. An adaptation of this procedure has been developed which uses a dim variant of P. leiognathi and permits the prescreening of microbial fermentation broths for potential antitumor agents. Bioluminescence in this organism was stimulated by compounds which bind to DNA or affect DNA synthesis. Antibiotics with target sites such as protein, cell wall or RNA synthesis, did not alter bioluminescence. Fermentation broths from over 5,000 soil isolates were prescreened in this assay and 95 (1.6%) were defined as active. Further analysis of selected cultures suggested that about half produced compound(s) with DNA-binding activity. These results suggest that the photobacterium induction assay (PIA) may be useful as a prescreen for potential antitumor agents. The assay is rapid, simple and requires only microgram quantities of material for testing. PMID- 2999050 TI - Cloning and expression of an APH(3')-III phosphotransferase from Staphylococcus aureus in Streptomyces lividans. AB - An aminoglycoside 3' type III phosphotransferase derived from Staphylococcus aureus plasmid pRN1956 was cloned on the high copy number Streptomycetes vectors pIJ702 and pIJ704. Streptomyces lividans transformants carrying the hybrid plasmids show a resistance pattern towards aminoglycoside antibiotics comparable to the resistance pattern of S. aureus. The APH(3')-III with expanded spectrum of resistance, is a useful additional marker for gene cloning in Streptomycetes. PMID- 2999052 TI - Digestibility of fiber components and reproductive performance of sows fed high levels of alfalfa meal. AB - Two experiments were conducted with second-parity sows fed either 5 or 50% alfalfa meal diets (Exp. 1) or 5, 50 or 95% alfalfa meal diets (Exp. 2) beginning 30 d after breeding and continuing through lactation, 21 d postpartum. Diets in both experiments were calculated to be equal in crude protein, but different in metabolizable energy content. Feed intake was restricted to 2 kg/d during gestation and ad libitum during lactation. Sows were tethered in metabolism crates 45 d after breeding. Total feces were collected during two 5-d collection periods, 60 and 100 d after breeding. Digestibilities of dry matter, fiber components (neutral detergent fiber, acid detergent fiber, hemicellulose and cellulose), protein and energy were determined in both experiments for all diets and periods of gestation. In both experiments, there was a reduction (P less than .05) in digestibility of dry matter, fiber components, protein and energy with increasing fiber levels for both periods of gestation. In Exp. 1, the decrease in digestibility of dry matter, neutral detergent fiber, acid detergent fiber, cellulose and energy was greater at 100 d gestation than at 60 d in sows fed 50% alfalfa. In Exp. 2, 50 and 95% dietary alfalfa reduced 60-d digestibilities of dry matter, all fiber constituents, energy and protein; these digestibilities were further reduced (P less than .05) at 100 d of gestation. In Exp. 1, weight gains of sows fed 5 and 50% alfalfa diets from breeding to 109 d gestation, were 42 and 18 kg/sow (5 greater than 50% level, P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2999051 TI - Evidence of collateral axonal projections to the superior olivary complex. AB - In this study we investigated the collateral axonal projections to the superior olivary complex using the combined anterograde and retrograde transport of wheat germ agglutinin conjugated with horseradish peroxidase. Small injections of this tracer were placed in the lateral or medial superior olivary nuclei in cats, and the location of anterograde label in the alternate nuclei of the superior olivary complex was determined. Injections of [3H]leucine were also placed in these nuclei for control purposes. After wheat-germ agglutinin-horseradish peroxidase injections in the lateral superior olivary nucleus anterograde label was observed bilaterally in the medial superior olivary nuclei. Likewise, after injections in the medial superior olivary nucleus anterograde label was observed in the contralateral medial and lateral superior olivary nuclei. The topography of the anterograde label was always precise and varied predictably as a function of the injection site. Most retrogradely labeled cells were located in the ipsilateral anteroventral cochlear nucleus and the medial nucleus of the trapezoid body. Various interpretations of the data are considered. Our primary conclusion is that cells in the anteroventral cochlear nucleus are a major source of collaterals to both the ipsilateral and contralateral nuclei of the superior olivary complex. PMID- 2999054 TI - Influence of realimentation diet on recovery of rumen activity and feed intake in beef steers. AB - Two trials were conducted to determine the influence of realimentation diet energy, protein, B-vitamin (BV) and Lactobacillus acidophilus (LAC) content on recovery of rumen activity and feed consumption in beef steers. In trial 1, ruminal-fistulated steers were fasted and refed 1) prairie hay, 2) 10% protein (LCP), 3) 12.5% protein (MCP), 4) LCP + BV or 5) LCP + LAC. In trial 2, calves were fasted and refed 1) 60% cottonseed hulls-40% alfalfa dehy (high roughage), 2) LCP, 3) 15% protein (HCP), 4) LCP + BV or 5) LCP + LAC. Rumen fermentative capacity declined 74% (P less than .05) during feed and water deprivation, but returned to control levels by d 7 of realimentation. On d 3 of realimentation, steers fed the LCP and MCP diets had molar proportions of ruminal butyrate in excess of 35%. Steers fed the hay, LAC and BV diets did not have a high butyrate fermentation. In trial 2, calves lost about 15% of their body weight during feed and water deprivation. Calves fed the high roughage diet appeared to return to prefast feed and energy intakes more slowly than steers fed the medium roughage diets. Results of this study indicate that rumen fermentative capacity is a factor limiting feed intake in fasted calves for 7 to 14 after the reintroduction of feed and water. PMID- 2999053 TI - Monensin effects on digestibility, ruminal protein escape and microbial protein synthesis on high-fiber diets. AB - The influence of monensin level (0, 6.1, 12.2, 18.3 and 36.6 ppm) on diet fiber digestibility, microbial protein synthesis and ruminal escape of dietary protein was evaluated in two steer metabolism trials. A growth trial was conducted to study possible interactions of forage quality and monensin level. In metabolism trial 1, four ruminal-cannulated steers were assigned to four monensin levels in a 4 X 4 Latin square design to measure fiber digestibility, rate of passage and protein metabolism. In metabolism trial 2, five duodenal-cannulated steers were assigned to five monensin levels in a 5 X 5 Latin square design to measure fiber digestibility, bacterial N flow and plant N flow. In the two metabolism trials, the level of monensin influenced organic matter (OM) digestibility, neutral detergent fiber (NDF) digestibility and ruminal NDF digestibility quadratically, with the intermediate levels of monensin being superior either to the high level of monensin or no monensin. A quadratic increase in particulate disappearance rate (P = .09) and no effect (P = .95) on liquid disappearance were also observed in trial 1. In trial 1, monensin level quadratically decreased (P = .10) the bacterial protein concentration and increased (P = .02) the ratio of total N:diaminopimilic acid in whole rumen contents. In trial 2, no overall difference in duodenal N flow (P = .64) or flow of individual amino acids (P = .46) was observed. In the growth trial, no interaction of cornstalk quality and monensin was observed (P less than .38).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2999055 TI - Effects of sodium bicarbonate on nitrogen balance, bacterial protein synthesis and sites of nutrient digestion in sheep. AB - Two experiments were conducted to determine effects of sodium bicarbonate (NaHCO3) on nitrogen (N) balance, ruminal N metabolism and site and extent of nutrient digestion in sheep fed 75% concentrate diets. A 2 X 2 factorial arrangement of treatments was employed in both trials with experimental diets balanced for 10.5 or 12.0% crude protein and containing 0 or 3.5% NaHCO3. In experiment 1, 12 lambs were allotted to four diets for two periods in a randomized complete-block design. Dry matter (DM) digestibility was increased (P less than .10) by NaHCO3 supplementation, but organic matter (OM) digestibility was unaffected by treatment. Apparent N digestibility was not affected by NaHCO3 addition but was increased (P less than .0001) at the higher level of protein. Ruminal pH (4 h postfeeding) was increased (P less than .01) by NaHCO3 supplementation. Sodium bicarbonate had no effect on molar proportions of acetate or propionate, but increased molar proportion of butyrate (P less than .10) in ruminal fluid. Mean N retention (g/d) was increased (P less than .05) at the higher protein level, but was not affected by NaHCO3. In experiment 2, four ruminal- and duodenal-cannulated wethers were utilized in a 4 X 4 Latin square design. Sodium bicarbonate addition increased ruminal pH (P less than .05) 2 h postfeeding but did not affect ruminal ammonia (NH3) levels, total VFA concentration or ruminal fluid dilution rates. Molar proportion of acetate was increased (P less than .01) by NaHCO3 at the lower protein level. Ruminal particulate dilution rates were increased (P less than .05) by NaHCO3 addition. Ruminal, postruminal and apparent total tract digestibilities of OM and neutral detergent fiber (NDF) were unaffected by NaHCO3 supplementation. Sodium bicarbonate decreased (P less than .05) ruminal starch digestion at the lower protein level but increased (P less than .05) it at the higher protein level. Bacterial N flow (g/d) at the duodenum and efficiency of bacterial protein synthesis were increased (P less than .10) by NaHCO3 additions. PMID- 2999056 TI - A note on the use of metal species in microbiological tests involving growth media. AB - The feasibility of using traditional growth media for biological testing of metal species, for example as potential microbiocides, was investigated. Significant interactions between both of the representative metal species studied, Cu2+ and FeEDTA, and the test media were found. It is recommended that the use of growth media for tests on metal species should be avoided. PMID- 2999057 TI - Transposable multiresistance. PMID- 2999058 TI - Trimethoprim resistance of Escherichia coli in outpatients in Finland after ten years' use of plain trimethoprim. AB - Development of trimethoprim resistance among Escherichia coli collected from urine samples in three areas in Finland was studied in 1978-1984. Three different trends of development of resistance were found: in the Turku area resistance has increased evenly during 1978-1982 from 5.4% to 10.1%, but thereafter a plateau seems to have been reached; in the Helsinki area resistance increased rapidly from 2.9% in 1980 to 11.1% in 1984, possibly due to the spread of a trimethoprim resistance transposon Tn7, which occurred significantly more often among E. coli strains in this area than in the Turku area; in the Rovaniemi area resistance has been at a plateau level, between 3.1 and 5.7%, during the whole study. No clear correlation between the consumption of trimethoprim and the level of resistance was found. The frequency of trimethoprim resistance in E. coli isolated from patients less than 65 years was 5.2% and in those isolated from patients greater than or equal to 65 years 14.7%. PMID- 2999060 TI - Chronic electrical stimulation of nongrafted and grafted skeletal muscles in rats. AB - Our purpose was to determine the effects of chronic electrical stimulation on the structure and function of neve-intact grafts in rats. Fourteen days after grafting, extensor digitorum longus (EDL) grafts (n = 6) and nongrafted EDL muscles (n = 4) were stimulated 8 h/day at 10 Hz for 26 days. Measurements were made subsequently of cytochrome c concentration, capillary density, contraction and relaxation times, developed tension, and the resistance to fatigue. Compared with contralateral nonstimulated grafts, chronically stimulated grafts demonstrated a 65% greater cytochrome c concentration, 45% greater number of capillaries per millimeter squared, 30% greater resistance to fatigue, 35% longer contraction time, 30% longer relaxation time, and 30% lower maximum tetanic tension. The differences that resulted from the stimulation of nongrafted EDL muscles were significant but of less magnitude. Chronic stimulation of 8 h/day provided a mixed stimulus for adaptation that enhanced the metabolic and endurance characteristics of fibers in muscles and grafts, but decreased the total fiber cross-sectional area and development of force. PMID- 2999059 TI - Exercise training and its effects with renal hypertensive rats. AB - The influence of endurance training on functional capacity [maximal O2 consumption (VO2 max)], caudal arterial blood pressure, and myocardial capillary density were investigated in normotensive rats and rats made hypertensive using the two-kidney one-clip approach (Goldblatt's hypertension). Male Sprague-Dawley rats were assigned to sham (N: 120-140 mmHg), moderately hypertensive (MH = 0.30 mm clips, 150-170 mmHg), or severely hypertensive (SH = 0.25-mm clips, 190-230 mmHg) groups. Rats designated to be runners (T) were exercised on a motor-driven treadmill equal to 50-70% of their VO2 max values for 8-12 wk. Compared with their nontrained (NT) controls, training was associated with significantly higher VO2 max values (12-15%) and muscle cytochrome-c oxidase activities (33-78%). Resting systolic blood pressure was not significantly changed in the N-and MH-T subgroups; however, it was 20-30 mmHg higher in the SH-T subgroup. Mean absolute heart weight for only the N-T group was significantly heavier than their NT controls. However, the mean predicted heart weights (heart wt = 0.639 X body wt of N-NT + 0.001 g) of the two SH groups were significantly higher than expected. The SH-T group had a lower (11%) subepicardial capillary density mean than its NT control and significantly fewer capillaries in the subendocardial region than the other five subgroups. It was concluded that moderate exercise training appeared to be detrimental to rats with severe hypertension because it increased resting blood pressure and decreased myocardial capillary density, even though it improved their functioning capacity. PMID- 2999061 TI - Decompression outcome following saturation dives with multiple inert gases in rats. AB - This investigation examined the question of whether gas mixtures containing multiple inert gases provide a decompression advantage over mixtures containing a single inert gas. Unanesthetized male albino rats, Rattus norvegicus, were subjected to 2-h simulated dives at depths ranging from 145 to 220 fsw. At pressure, the rats breathed various He-N2-Ar-O2 mixtures (79.1% inert gas-20.9% O2); they were then decompressed rapidly (within 10 s) to surface pressures. The probability of decompression sickness (DCS), measured either as severe bends symptoms or death, was related to the experimental variables in a Hill equation model incorporating parameters that account for differences in the potencies of the three gases and the weight of the animal. The relative potencies of the three gases, which affect the total dose of decompression stress, were determined as significantly different in the following ascending order of potency: He less than N2 less than Ar; some of these differences were small in magnitude. With mixtures, the degree of decompression stress diminished as either N2 or Ar was replaced by He. No obvious advantage or disadvantage of mixtures over the least potent pure inert gas (He) was evident, although limits to the expectation of possible advantage or disadvantage of mixtures were defined. Also, model analysis did not support the hypothesis that the outcome of decompression with multiple inert gases in rats under these experimental conditions can be explained totally by the volume of gas accumulated in the body during a dive. PMID- 2999063 TI - Staging and treatment of small cell lung cancer. PMID- 2999064 TI - Is there a place for surgery in small cell lung cancer? PMID- 2999062 TI - p-Aminohippurate transport in canine tracheal epithelium. AB - The unidirectional fluxes of 20, 100, 500, and 2,000 microM rho-aminohippurate (PAH) were measured under open- and short-circuit conditions in canine tracheal epithelium mounted as flat sheets in Ussing chambers. In tissues pretreated with mucosal indomethacin (10(-6) M) and amiloride (10(-4) M), unidirectional PAH fluxes under short-circuit conditions increased with increasing bath concentrations but there was no significant net PAH transport. After stimulation of chloride secretion by mucosal cyclic adenosine 3',5' -cyclic monophosphate (cAMP 10(-3) M), there was a significant increase in the secretory flux of PAH and a significant decrease in the absorptive flux of PAH. This resulted in net PAH secretion that demonstrated saturation kinetics with an apparent Michaelis Menten constant of 754 microM by Lineweaver-Burk analysis. Intracellular concentrations of PAH were 0.4-1.2 times bath concentrations after pretreatment with indomethacin and amiloride and increased to 2.6-3.3 times bath concentrations after cAMP. Under open-circuit conditions, secretory PAH flux decreased and absorptive flux increased resulting in net PAH absorption. We conclude from these early studies that the canine tracheal epithelium possesses a specialized system for the transport of organic anions in the airways and that this transport system may share many similarities with organic anion transport in the kidney. PMID- 2999065 TI - Surgery in non-small cell lung cancer. PMID- 2999066 TI - Poland's syndrome. Including ultrasonography of the pectoralis muscle as a new diagnostic modality. PMID- 2999067 TI - Escherichia coli rep gene: identification of the promoter and N terminus of the rep protein. AB - The functional Escherichia coli rep gene, which encodes the Mr 67,000 Rep helicase, has been localized within a 2.55-kilobase sequence. Its regulatory region has been characterized by the use of rep-lacZ fusions. The direction of transcription of the rep gene is clockwise on the E. coli chromosome, as are the nearby ilvC and rho genes. The sequence of the rep control region was determined, and putative regulatory sequences were identified; no evidence for autoregulation of expression was obtained. Transcription of the gene was not enhanced during the SOS response. The location of the promoter and the beginning of the protein were confirmed by S1 nuclease mapping of the 5' end of rep mRNA and determination of the NH2-terminal sequence of the rep protein. PMID- 2999069 TI - Gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri: cloning and expression in Escherichia coli. AB - A library of cloned Spiroplasma citri genomic sequences was constructed by incorporating HindIII digestion fragments into the plasmid vector pBR328. Immunological screening allowed the identification of a recombinant plasmid containing the gene for spiralin, the major membrane protein of S. citri. The spiralin produced by the Escherichia coli transformant was characterized by immunological detection with monoclonal antibody after Western blotting of two dimensional (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide) electrophoresis gels and by partial proteolytic mapping. The gene for spiralin occurred within a 6.5-kilobase-pair cloned DNA fragment. Spiralin in E. coli was produced regardless of the orientation of the insert within the pBR328 vector. A spiroplasmal DNA sequence which acted as a promoter in E. coli was cloned along with the structural spiralin gene which is expressed in E. coli from that sequence. PMID- 2999068 TI - Characterization of a gamma-glutamyl kinase from Escherichia coli that confers proline overproduction and osmotic tolerance. AB - Mutation(s) in the proBA operon of Escherichia coli confers proline overproduction and enhanced osmotic tolerance in enteric bacteria (L. N. Csonka, Mol. Gen. Genet. 182:82-86, 1981; M. J. Mahan and L. N. Csonka, J. Bacteriol. 156:1249-1262, 1983). A glutamate-dependent ATPase assay was developed and used to determine proB-encoded gamma-glutamyl kinase activity in the absence of glutamate-gamma-semialdehyde dehydrogenase. This assay indicated that the feedback insensitivity of mutant gamma-glutamyl kinase was independent of glutamate-gamma-semialdehyde dehydrogenase. However, the capacity of glutamate gamma-semialdehyde dehydrogenase from the osmotolerant mutant to interact with the kinase was altered in thermal stability, suggesting that mutations in both proB and proA may be required for osmotolerance. PMID- 2999071 TI - Genetic organization and transcription from the gene (trmA) responsible for synthesis of tRNA (uracil-5)-methyltransferase by Escherichia coli. AB - The enzyme catalyzing the formation of 5-methyluridine (ribothymidine) in tRNA of Escherichia coli is tRNA (uracil-5)-methyltransferase (EC 2.1.1.35). A 2.8 kilobase EcoRI chromosomal DNA fragment contains trmA, the structural gene for this enzyme. Subcloning, transcription in vitro, Tn5 insertion mutagenesis, and transcriptional fusion experiments were performed to establish the gene organization of the trmA region on the E. coli chromosome. trmA is a monocistronic operon. The trmA promoter was localized by in vitro experiments, and the direction of transcription was shown to be counterclockwise on the standard E. coli K-12 chromosomal map. The level of transcription of trmA in vitro and the expression of protein in minicells equal those of the bla gene of plasmid pBR322. PMID- 2999070 TI - Transcription of the Escherichia coli fumarate reductase genes (frdABCD) and their coordinate regulation by oxygen, nitrate, and fumarate. AB - The fumarate reductase enzyme complex allows Escherichia coli to grow anaerobically with fumarate as a terminal electron acceptor for oxidative phosphorylation when the preferred compounds oxygen and nitrate are not available. We used the pKO promoter test vectors to identify a single promoter for the frdABCD genes which encode fumarate reductase. Expression of galactokinase from the frd promoter-galK operon fusion plasmid was repressed by oxygen and by nitrate and was induced by fumarate, indicating that frd gene expression is regulated at the transcriptional level by these terminal electron acceptors. S1 nuclease analysis, using a single-stranded DNA probe from the frd promoter region and mRNA isolated from a fumarate reductase-induced culture, revealed that the frd mRNA transcript initiates with an adenine residue 93 bases prior to the start of frdA translation. No promoters internal to the frd genes were revealed with the plasmid promoter screening system. S1 nuclease analysis revealed that the frd mRNA terminates in a uridine-rich region centered at 46 bases after the last codon of frdD. A stem and loop structure previously described as the growth rate-dependent attenuator for the linked ampC gene precedes the frd mRNA terminus. This result confirms the proposal that the stem and loop structure serves the dual role of a frd terminator anaerobically and an ampC attenuator aerobically. The four frd genes encoding the subunits of the fumarate reductase complex thus comprise an operon which is regulated at the transcriptional level in response to the cellular availability of the alternate electron acceptors oxygen, nitrate, and fumarate. PMID- 2999072 TI - Insertional mutagenesis of the lon gene in Escherichia coli: lon is dispensable. AB - The lon gene of Escherichia coli codes for an ATP-dependent protease. Mutations in lon cause a defect in the intracellular degradation of abnormal and mutant proteins and lead to a number of phenotypic changes, such as UV sensitivity and overproduction of capsular polysaccharide. We have isolated lambda transducing phage carrying the lon gene and used the lon phage as a target for insertional mutagenesis by a defective transposon Tn10 to produce lon::delta 16 delta 17Tn10 derivatives. The delta 16 delta 17Tn10 (hereafter called delta Tn10) elements were inserted at sites throughout the lon gene and disrupted the coding region between 15 and 75% of the distance from the amino-terminal end. Radioactive labeling of proteins in vivo in cells infected with different lambda lon::delta Tn10 phage demonstrated that the insertions resulted in the synthesis of truncated Lon proteins. The lon::delta Tn10 mutations, when crossed from the phage into the bacterial chromosome, abolished the synthesis of intact Lon protein, as assayed by antibody on Western blots. An analysis of the protein degradative ability of lon::delta Tn10 cells suggests that although the insertions in lon caused a reduction in ATP-dependent protein degradation, they did not completely eliminate such degradation either in vivo or in vitro. The lon::delta Tn10 mutations and a lon deletion retaining only the amino-terminal 25% of the gene did not affect the energy-dependent degradation of proteins during starvation and led to only a 40 to 60% reduction in the ATP-dependent degradation of canavanine-containing proteins and puromycyl peptides. Our data provide clear evidence that energy-dependent proteolytic enzymes other than Lon exist in E. coli. PMID- 2999073 TI - Efficient synthesis and secretion of a thermophilic alpha-amylase by protein producing Bacillus brevis 47 carrying the Bacillus stearothermophilus amylase gene. AB - Bacillus subtilis and Bacillus brevis 47-5, carrying the Bacillus stearothermophilus alpha-amylase gene on pUB110 (pBAM101), synthesized the same alpha-amylase as the donor strain as determined by the enzyme's thermal stability and NH2-terminal amino acid sequence. Regardless of the host, the 34-amino acid signal peptide of the enzyme was processed at exactly the same site between two alanine residues. B. brevis 47-5(pBAM101) secreted the enzyme most efficiently of the hosts examined, 100, 15, and 5 times more than B. stearothermophilus, Escherichia coli HB101(pH1301), and B. subtilis 1A289(pBAM101), respectively. The efficient secretion of the enzyme in B. brevis 47-5(pBAM101) was suggested to be due to the unique properties of the cell wall of this organism. PMID- 2999075 TI - Tn4400, a compound transposon isolated from Bacteroides fragilis, functions in Escherichia coli. AB - Transfer factor pBFTM10, isolated from the obligate anaerobic bacterium Bacteroides fragilis, carries a clindamycin resistance determinant which we have suggested is part of a transposable element. DNA homologous to this determinant is found in many Clnr Bacteroides isolates, either in the chromosome or on plasmids. We have now established that Ccr resides on a transposon, Tn4400. In addition to the Ccr determinant that functions under anaerobic conditions in B. fragilis, Tn4400 also carries a determinant for tetracycline resistance (Tcr) which only functions in Escherichia coli under aerobic conditions. The presence of Tn4400 on pBFTM10 does not confer tetracycline resistance on B. fragilis cells containing it. DNA from pBFTM10 was cloned in E. coli, with pDG5 as the cloning vector, to form pGAT500. Using a mobilization assay involving pGAT500 and an F factor derivative, pOX38, we determined that a 5.6-kilobase region of pBFTM10 DNA was capable of mediating replicon fusion and transposition. Most of the mobilization products resulted from inverse transposition reactions, while some were the result of true cointegrate formation. Analysis of the cointegrate molecules showed that three were formed by the action of one of the ends of Tn4400 (IS4400), and one was formed by the action of the whole element (Tn4400). The cointegrate molecule carrying intact copies of Tn4400 at the junction of the two plasmids could resolve to yield an unaltered donor plasmid (pGAT500) and a conjugal plasmid containing a copy of Tn4400 or a copy of one insertion sequence element (pOX38::Tn4400 or pOX38::IS4400). Thus, Tn4400 is a compound transposon containing active insertion sequence elements as directly repeated sequences at its ends. PMID- 2999074 TI - Characterization and sequence analysis of pilin from F-like plasmids. AB - Conjugative pili are expressed by derepressed plasmids and initiate cell-to-cell contact during bacterial conjugation. They are also the site of attachment for pilus-specific phages (f1, f2, and QB). In this study, the number of pili per cell and their ability to retract in the presence of cyanide was estimated for 13 derepressed plasmids. Selected pilus types were further characterized for reactivity with anti-F and anti-ColB2 pilus antisera as well as two F pilus specific monoclonal antibodies, one of which is specific for a sequence common to most F-like pilin types (JEL92) and one which is specific for the amino terminus of F pilin (JEL93). The pilin genes from eight of these plasmids were cloned and sequenced, and the results were compared with information on F, ColB2, and pED208 pilin. Six pilus groups were defined: I, was F-like [F, pED202(R386), ColV2-K94, and ColVBtrp]; IIA was ColB2-like in sequence but had a lowered sensitivity to f1 phage due to its decreased ability for pilus retraction [pED236(ColB2) and pED203(ColB4)]; IIB was ColB2-like but retained f1 sensitivity [pED200(R124) and pED207(R538-1)]; III contained R1-19, which had a ColB2-like amino terminus but had an additional lysine residue at its carboxy terminus which may affect its phage sensitivity pattern and its antigenicity; IV was R100-1-like [R100-1 and presumably pED241(R136) and pED204(R6)] which had a unique amino-terminal sequence combined with a carboxy terminus similar to that of F. pED208(Folac) formed group V, which was multipiliated and exhibited poor pilus retraction although it retained full sensitivity to f1 phage. The pED208 pilin gene could not be cloned at this time since it shared no homology with the pilin gene of the F plasmid. PMID- 2999076 TI - Binding of polycationic antibiotics and polyamines to lipopolysaccharides of Pseudomonas aeruginosa. AB - Polycations, such as aminoglycoside and peptide antibiotics, and naturally occurring polyamines were found to bind to the lipopolysaccharide of Pseudomonas aeruginosa and alter its packing arrangement. Binding of cations was measured by the displacement of a cationic spin probe from lipopolysaccharide into the aqueous environment upon addition of competitive cations. The level of probe displacement was dependent on the concentration and charge of the competing cation, with the more highly charged cations being more effective at displacing probe. The relative affinity of several antibiotics for lipopolysaccharide correlated with their ability to increase outer membrane permeability, while the relative affinity of several polyamines correlated with their ability to stabilize the outer membrane. Probe mobility within the lipopolysaccharide head group was shown to be decreased by cationic antibiotics and unaltered or increased by polyamines. We propose that antibiotic permeability and disruption of outer membrane integrity by polycationic antibiotics results from binding of the antibiotic to anionic groups on lipopolysaccharide with a consequent change in the conformation of lipopolysaccharide aggregate structure. PMID- 2999077 TI - Two functions of the E protein are key elements in the plasmid F replication control system. AB - By using a plasmid carrying a translational fusion between the E gene of the IncFI plasmid F and the lacZ gene, we located the operator of the autogenously regulated E gene to an inverted repeat overlapping the E-gene promoter and showing perfect homology to part of the sequence found in all the direct repeats of two regions exerting an inhibitory effect on F replication, incB and incC. Excess E protein provided in trans to an F plasmid increased the replication frequency of the F plasmid. This stimulatory effect was counteracted by increased dosages of incB or incC. A model is proposed for the replication control system of F in which the key elements are autoregulation of E-gene expression and titration of E protein by incB and incC. PMID- 2999078 TI - Bacteriophage SPO2-mediated plasmid transduction in transpositional mutagenesis within the genus Bacillus. AB - A single copy of the Streptococcus faecalis transposon Tn917, located in the Bacillus subtilis chromosome, was able to transpose onto the SPO2 cos plasmid pPL1017, which codes for chloramphenicol resistance and contains the bacteriophage phi 105 immunity region. Selection for pPL1017::Tn917 chimeras was performed by SPO2-mediated plasmid transduction of transposon-borne resistance to macrolide-lincosamide-streptogramin B antibiotics (MLSr). The transposition of Tn917 onto plasmid pPL1017 occurred with a frequency of 10(-5) and was dependent on the presence of a subinhibitory dose of erythromycin. Twelve chimeras were subjected to genetic and physical analyses. Two Cams transductants harbored plasmids whose chloramphenicol acetyltransferase genes had been insertionally inactivated by Tn917. Several transpositions in the vicinity of the phi 105 immunity region were detected. However, all of the 300 MLSr, Camr transductants screened were immune to phi 105 infectious activity. One pPL1017::Tn917 chimera, pLK200, was transferred by SPO2 plasmid transduction into the Bacillus amyloliquefaciens prototrophic strain DSM7. Plasmid pLK200 was effective in the mutagenesis of the DSM7 chromosome and yielded auxotrophs at a frequency of 0.5 to 5.3%. Generation of auxotrophs was also dependent on the presence of a subinhibitory dose of erythromycin. Forty-four auxotrophs representing at least nine amino acid requirements were recovered. PMID- 2999079 TI - Regulation of guaC expression in Escherichia coli. AB - The guaC gene encodes GMP reductase, which converts GMP to inosine monophosphate. Regulation of guaC expression was examined by use of guaC-lac fusions created by Mu d1(lac). In these strains, beta-galactosidase is induced by guanine derivatives, and this induction is prevented by adenine. Our previous implication that glutamine acts as a negative effector of transcription was confirmed by showing that glutamine analogs (diazo-oxo-norleucine and methionine sulfoximine) can also induce beta-galactosidase. GMP was implicated as a likely candidate for the in vivo inducer by introducing a gpt block to prevent the conversion of guanine to GMP and a deoD block to prevent the interconversion of guanine and guanosine. Regulatory mutants were isolated by growth on lactose plus adenine. Though these showed high constitutive levels of beta-galactosidase, they were normal for the regulation of GMP reductase when the fusion was corrected by transduction to guaC+ or when guaC+ was introduced by plasmid complementation. The regulatory mutants were linked to guaC. PMID- 2999080 TI - Isolation and characterization of the DNA region encoding nodulation functions in Bradyrhizobium japonicum. AB - The DNA region encoding early nodulation functions of Bradyrhizobium japonicum 3I1b110 (I110) was isolated by its homology to the functionally similar region from Rhizobium meliloti. Isolation of a number of overlapping recombinant clones from this region allowed the construction of a restriction map of the region. The identified nodulation region of B. japonicum shows homology exclusively to those regions of R. meliloti and Rhizobium leguminosarum DNA known to encode early nodulation functions. The region of homology with these two fast-growing Rhizobium species was narrowed to an 11.7-kilobase segment. A nodulation defective mutant of Rhizobium fredii USDA 201, strain A05B-2, was isolated and found to be defective in the ability to curl soybean root hairs. Some of the isolated recombinant DNA clones of B. japonicum were found to restore wild-type nodulation function to this mutant. Analysis of the complementation results allows the identification of a 1.8-kilobase region as essential for restoration of Hac function. PMID- 2999081 TI - Cloning, structure, and expression of the Escherichia coli K-12 hisC gene. AB - We used an expression vector plasmid containing the Escherichia coli K-12 histidine operon regulatory region to subclone the E. coli hisC gene. Analysis of plasmid-coded proteins showed that hisC was expressed in minicells. A protein with an apparent molecular weight of 38,500 was identified as the primary product of the hisC gene. Expression was under control of the hisGp promoter and resulted in very efficient synthesis (over 100-fold above the wild-type levels) of imidazolylacetolphosphate:L-glutamate aminotransferase, the hisC gene product. The complete nucleotide sequence of the hisC gene has been determined. The gene is 1,071 nucleotides long and codes for a protein of 356 amino acids with only one histidine residue. PMID- 2999083 TI - Recombination sites in plasmid drug resistance gene amplification. AB - The resistance plasmid NR1 derivative pRR330 consists of a neomycin-kanamycin resistance gene (neo-kan) flanked by directly repeated sequences of both insertion element IS1 DNA (768 base pairs) and 840 base pairs of DNA which are a part of the chloramphenicol acetyltransferase (cam) gene. Most Escherichia coli cell populations that were cultured in high neomycin concentrations carried plasmids whose neo-kan gene amplification was mediated either by IS1 DNA or by cam DNA as homologous recombination sites. This suggests that the final amplified cell populations were the descendants of a single cell. PMID- 2999082 TI - lac Up-promoter mutants with increased homology to the consensus promoter sequence. AB - Four lac promoter mutants were constructed. The mutations increased the homology between the lac promoter and the consensus promoter sequences by introducing the consensus -10 and -35 regions and the consensus spacing of 17 residues between these two regions. The promoter mutants were cloned into a pBR322-derivatized vector upstream from the lacZ gene, and levels of beta-galactosidase were an indication of promoter activity. All mutants exhibited higher activity than did the wild-type promoter. PMID- 2999084 TI - F'-coded, temperature-sensitive lambda cI857 repressor gene for easy construction and regulation of lambda promoter-dependent expression systems. AB - We describe the construction and properties of an F' factor which carries the temperature-sensitive cI857 allele of the repressor gene of coliphage lambda and which lacks the lambda cro function. This episome can easily be transferred to any F- and F' Escherichia coli strain, thus facilitating the construction and regulation of lambda promoter-dependent expression systems without the use of defective prophages. PMID- 2999085 TI - Excretion of alkaline phosphatase by Escherichia coli K-12 pho constitutive mutants transformed with plasmids carrying the alkaline phosphatase structural gene. AB - Escherichia coli alkaline phosphatase constitutive mutants carrying a pst or a phoS mutation and a plasmid-bearing gene phoA+ excreted into the growth medium up to 50% of the total alkaline phosphatase production. This excretion was pH dependent and did not involve drastic modifications of the cell envelope. Alkaline phosphatase accounted for 80% of total released proteins. Amplification of gene phoA+ was a necessary condition for excretion to occur. When the beta lactamase structural gene bla+ was coamplified with gene phoA+, both enzymes were excreted. pst-transformed excretory strains did not show the pleiotrophic phenotype previously described for lky mutants. PMID- 2999086 TI - Proton motive force in washed cells of Rhizobium japonicum and bacteroids from Glycine max. AB - The components of the proton motive force (delta p), namely the membrane potential and the transmembrane pH gradient, were measured in washed cells of Rhizobium japonicum CC705 grown in cultures (5% O2-95% N2) in the presence of 10 mM KNO3 and in bacteroids from Glycine max. The delta p and its components remained reasonably constant in cells as well as in bacteroids at various stages of growth. The effects of uncouplers and ATPase inhibitors on the delta p and its components were determined in both cultured cells and bacteroids. The data indicated that a respiration-driven H+ translocation is the source of the delta p in both cultured cells and bacteroids. PMID- 2999087 TI - Nucleotide sequence of the gene determining plasmid-mediated citrate utilization. AB - The citrate utilization determinant from transposon Tn3411 has been cloned and sequenced, and its polypeptide products have been characterized in minicell experiments. The nucleotide sequence was determined for a 2,047-base-pair BglII restriction endonuclease fragment that includes the citrate determinant. This region contains an open reading frame that would encode a 431-amino-acid very hydrophobic polypeptide and which is preceded by a reasonable ribosomal binding site. However, the single polypeptide found in minicell experiments had an apparent molecular weight of 35,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PMID- 2999088 TI - Cloning and DNA sequence of a plasmid-determined citrate utilization system in Escherichia coli. AB - The citrate utilization determinant from a large 200-kilobase (kb) naturally occurring plasmid was previously cloned into the PstI site of plasmid vector pBR325 creating the Cit+ tetracycline resistance plasmid pWR61 (15 kb). Tn5 insertion mutagenesis analysis of plasmid pWR61 limited the segment responsible for citrate utilization to a 4.8-kb region bordered by EcoRI and PstI restriction nuclease sites. The 4.8-kb fragment was cloned into phage M13, and the DNA sequence was determined by the dideoxyribonucleotide method. Within this sequence was a 1,296-base-pair open reading frame with a preceding ribosomal binding site. The 431-amino-acid polypeptide that could be translated from this open reading frame would be highly hydrophobic. A second long open reading frame with the potential of encoding a 379-amino-acid polypeptide preceded the larger open reading frame. Portions of the 4.8-kb fragment were further subcloned with restriction endonucleases BglII and BamHI, reducing the minimum size needed for a citrate-positive phenotype to a 1.9-kb BamHI-BglII fragment (which includes the coding region for the 431-amino-acid polypeptide, but only the distal 2/3 of the reading frame for the 379-amino-acid polypeptide). Citrate utilization results from a citrate transport activity encoded by the plasmid. With the 4.8-kb fragment (as with larger fragments) the citrate transport activity was inducible by growth on citrate. On transfer from glucose, succinate, malate, or glycerol medium to citrate medium, the Cit+ Escherichia coli strains showed a delay of 36 to 48 h before growth. PMID- 2999090 TI - The acquired immunodeficiency syndrome. PMID- 2999089 TI - dfp Gene of Escherichia coli K-12, a locus affecting DNA synthesis, codes for a flavoprotein. AB - The cloned dfp gene complements dna-707 (now designated dfp-707), a temperature sensitive conditionally lethal mutation that results in a slow cessation of DNA synthesis while protein synthesis is maintained. In vitro and in vivo experiments failed to demonstrate a specific defect in the initiation of DNA replication, and turn-off of DNA synthesis at high temperature was slower than that of a typical initiation (dnaA) mutant. The gene was localized, and its product was identified through the construction and analysis of deletion and insertion mutants of dfp containing plasmids. dfp is located between the rpmB and dut genes at 81 min on the linkage map of Escherichia coli K-12. It is transcribed clockwise, independently of dut. The ability of a plasmid to complement a chromosomal dfp 707 mutation was correlated with its ability to produce a 45-kilodalton polypeptide. The purified protein contained 1 mol of flavin mononucleotide per mol of polypeptide. PMID- 2999091 TI - Transfusion associated AIDS. PMID- 2999092 TI - The specific modification of histidyl residues of inorganic pyrophosphatase from Bacillus stearothermophilus by photooxidation. AB - Photooxidation of inorganic pyrophosphatase [pyrophosphate phosphohydrolase EC 3.6.1.1] from Bacillus stearothermophilus in the presence of rose bengal resulted in rapid loss of enzymatic activity. The pH profile of the inactivation rate by the photooxidation showed an inflection point around pH 6.8, suggesting the involvement of histidyl residues in the inactivation. Amino acid analysis revealed that the loss of enzymatic activity was accompanied by the destruction of 3 histidyl residues per molecule. The presence of Mg2+ alone afforded partial protection against the inactivation, whereas inorganic pyrophosphate, the substrate, showed almost no protective effect against inactivation. The photooxidation of inorganic pyrophosphatase altered the circular dichroism spectrum and the difference UV spectrum induced by Mg2+ in the near ultraviolet region. These results suggested that histidyl residues appear to be located at the binding site of Mg2+ and may contribute to the conformational change induced by Mg2+. PMID- 2999093 TI - Effect of cetiedil on the superoxide-generating system of porcine neutrophils. AB - Cetiedil, alpha-cyclohexyl-3-thiopheneacetic acid 2-(hexahydro-1H-azepin-1-yl) ethyl ester, was found to inhibit the generation of superoxide (O2-) by porcine neutrophils exposed to various stimulators. The concentration of cetiedil required for 50% inhibition was about 45 microM when neutrophils were stimulated by phorbol myristate acetate. Cetiedil not only decreased the rate of generation of O2-, but prolonged the lag time prior to the production of O2-. The inhibitory effect of cetiedil on the O2(-)-generating activity of the NADPH oxidase in the membrane vesicles was less than that on whole cells; the concentration of cetiedil necessary for 50% inhibition was about 250 microM. To study the mechanism of cetiedil's effect on the membrane, the transmembrane potential of neutrophils and the intracellular free Ca2+ concentration were monitored by using fluorescence probes, diS-C3-(5), and quin-2, respectively. Cetiedil caused depolarization of the membrane potential and increased the intracellular free Ca2+. These results indicate that integrity of ionic distribution is necessary to activate the O2(-)-generating system of neutrophils. PMID- 2999094 TI - Purification and properties of myo-inositol-1-phosphatase from rat brain. AB - myo-Inositol-1-phosphatase [EC 3.1.3.25] was purified from a cytosolic fraction of rat brain. The purified enzyme appeared homogeneous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 29,000. The molecular weight of the native enzyme was 55,000 as determined by molecular sieve chromatography. These values indicated that the native enzyme was composed of two identical subunits. The isoelectric point of the enzyme was 4.6. The enzyme hydrolyzed inositol-1-phosphate, 2'-AMP, 2'-GMP, beta-glycerophosphate, and alpha glycerophosphate; the ratio of the reaction rates was 100 : 84 : 73 : 64 : 32. The Km values for inositol-1-phosphate, 2'-AMP, and beta-glycerophosphate were 1.2 X 10(-4) M, 1.9 X 10(-4) M, and 7.7 X 10(-4) M, respectively. Mn2+ and Ca2+ were strong competitive inhibitors against Mg2+, with Ki values of 3 microM and 20 microM, respectively. This result suggests that myo-inositol-1-phosphatase might be regulated by intracellular Ca2+ and/or Mn2+. Li+, which is known to show a therapeutic effect on manic-depressive disease and also to prolong the intrinsic periods of circadian rhythms in various organisms, was a potent uncompetitive inhibitor and inhibited 50% of the activity at 1 mM. The possibility that myo-inositol-1-phosphatase and inositol phospholipid metabolism are involved in circadian rhythm oscillation is discussed in terms of Li actions. PMID- 2999095 TI - Hydrolytic and autolytic behavior of two forms of calcium-activated neutral protease (CANP). AB - Some endogenous substrates were incubated with two forms of calcium-activated neutral protease (CANP) with high (muCANP) and low (mCANP) sensitivities to calcium ions. In addition to analyses of the processes of their degradation, changes in the molecular properties of these CANPs were also examined. Among the tested substrate proteins, the myosin heavy chain of rabbit skeletal muscle myofibrils and spectrin or band 3 protein of human erythrocyte membranes were degraded relatively rapidly. So far as these proteins were concerned, a higher degradation velocity was observed for muCANP than for mCANP. Vimentin from ascites tumor cells was degraded most rapidly and no difference was observed in degradation velocity between muCANP and mCANP. In all cases, muCANP and mCANP produced different proteolytic peptide fragments, suggesting the different substrate-specificities of these CANPs. The degradation of substrates always accompanied the autodigestion of CANPs, and the small subunits of both CANPs were degraded in the early stage of the autodigestion. The large subunit of muCANP (79K) was converted to a 76K polypeptide via a 77K polypeptide as an intermediate. The autodigested muCANP with 76K polypeptide retained sufficient protease activity and, moreover, its calcium-sensitivity was higher than that of intact muCANP. The possibility is thus proposed that restricted autodigestion is a necessary activation step for the appearance of activity of muCANP. No such transition was observed for mCANP. PMID- 2999096 TI - Cytochrome c oxidase of Pseudomonas AM 1: purification, and molecular and enzymatic properties. AB - Cytochrome c oxidase (cytochrome aa3-type) [EC 1.9.3.1] was purified from Pseudomonas AM 1 to an electrophoretically homogeneous state and some of its properties were studied. The oxidase showed absorption peaks at 428 and 598 nm in the oxidized form, and at 442 and 604 nm in the reduced form. The CO compound of the reduced enzyme showed peaks at 432 and 602 nm. The enzyme molecule was composed of two kinds of subunits with molecular weights of 50,000 and 30,000 and it contained equimolar amounts of heme a and copper atom. The enzyme rapidly oxidized Candida krusei and horse ferrocytochromes c as well as Pseudomonas AM 1 ferrocytochrome c. The reactions catalyzed by the enzyme were strongly inhibited by KCN. PMID- 2999097 TI - Target size of 5'-nucleotidase in smooth muscle. AB - Target size of the 5'-nucleotidase in six different smooth muscles was determined by radiation inactivation. The enzyme in the soluble fraction of rat myometrium and vas deferens gave a target size of approximately 80,000 daltons. The plasma membrane bound 5'-nucleotidase however, gave target size of 80,000 to 110,000 daltons in rat gastric fundus and vas deferens and dog stomach and ileum, 135,000 daltons in rat mesenteric artery and 210,000 daltons in rat myometrium. PMID- 2999099 TI - Angiotensin-converting enzyme from human tissues. Physicochemical, catalytic, and immunological properties. AB - Angiotensin-converting enzyme was purified from human lung, kidney, testis, blood plasma, and seminal plasma using a facile two-step protocol which included affinity chromatography on Sepharose-bound lisinopril followed by either gel filtration or hydroxylapatite chromatography. Molecular mass for converting enzyme from all sources except testis was 140 kDa. That from testis consisted of both a 90- and a 140-kDa form in a 4:1 ratio. Detergent-extracted membrane-bound converting enzyme aggregated on gel filtration chromatography, while trypsin extracted and soluble converting enzyme did not. Comparison of detergent extracted and trypsin-extracted membrane-bound converting enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing indicated that the membrane binding sequence contributed minimally to the size and charge of the enzyme. Catalytic and kinetic properties assessed by interaction with substrates, inhibitors, and anti-converting enzyme immunoglobulin were similar for all forms and sources of converting enzyme. Enzyme-linked immunosorbent assay revealed only partial homology between the 90- and 140-kDa forms of the enzyme. PMID- 2999098 TI - Identification of the receptor for atrial natriuretic factor on cultured vascular cells. AB - Binding experiments with 125I-atrial natriuretic factor (ANF) followed by covalent attachment with disuccimidyl suberate show that the peptide binds predominantly to a protein of apparent molecular mass of 66,000 daltons on the cell surface of cultured bovine aortic smooth muscle cells. A minor protein species of 180,000 Mr is also visualized after cross-linking. Endothelial cells, however, whose ANF binding parameters differ substantially from smooth muscle cells, also appear to have qualitatively identical 125I-ANF binding proteins. The identity of these putative proteins, as the ANF receptor, is confirmed by findings that covalent attachment of 125I-ANF is saturable, concentration dependent, and competed by nanomolar concentrations of unlabeled ANF. Furthermore, other peptide hormones such as angiotensin II, glucagon, or insulin are ineffective in competing for 125I-ANF binding and cross-linking to the receptor. PMID- 2999100 TI - Isolation and characterization of yeast strains carrying mutations in the glyceraldehyde-3-phosphate dehydrogenase genes. AB - Mutant yeast strains were constructed which carry insertion mutations in each of the glyceraldehyde-3-phosphate dehydrogenase structural genes which have been designated TDH1, TDH2, and TDH3. Haploid strains carrying mutations in TDH1 and TDH2 as well as TDH1 and TDH3 were isolated from crosses between strains carrying the appropriate single mutations. The three single mutants as well as the two double mutants grow at wild type rates when ethanol is used as carbon source. Mutant strains lacking only a functional TDH2 allele or a TDH3 allele grow at 50 and 75% of the rate observed for wild type cells, respectively, when glucose is used as carbon source. No growth phenotype was observed for strains lacking only a functional TDH1 allele when either fermentable or nonfermentable carbon sources were used. Evidence is presented that strains lacking functional TDH2 and TDH3 alleles are not viable. These data demonstrate that the presence of a functional TDH2 or TDH3 allele is required for cell growth. PMID- 2999101 TI - The generation and subsequent fate of glutathionyl radicals in biological systems. AB - Horseradish peroxidase-catalyzed oxidation of p-phenetidine in the presence of either glutathione (GSH), cysteine, or N-acetylcysteine led to the production of the appropriate thioyl radical which could be observed using EPR spectroscopy in conjunction with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide. This confirms earlier work using acetaminophen (Ross, D., Albano, E., Nilsson, U., and Moldeus, P. (1984) Biochem. Biophys. Res. Commun. 125, 109-115). The further reactions of glutathionyl radicals (GS.), generated during horseradish peroxidase-catalyzed oxidation of p-phenetidine and acetaminophen in the presence of GSH, were investigated by following kinetics of oxygen uptake and oxidized glutathione (GSSG) formation. Oxygen uptake and GSSG generation were dependent on the concentration of GSH but above that which was required for maximal interaction with the primary amine or phenoxy radical generated during peroxidatic oxidation of p-phenetidine or acetaminophen, suggesting that a secondary GSH-dependent process was responsible for oxygen uptake and GSSG production. GSSG was the only product of thiol oxidation detected during peroxidatic oxidation of p-phenetidine or acetaminophen in the presence of GSH, but under nitrogen saturation conditions its production was reduced to 8 and 33% of the corresponding amounts obtained under aerobic conditions in the cases of p-phenetidine and acetaminophen, respectively. Nitrogen saturation conditions did not affect horseradish peroxidase-catalyzed metabolism. This shows that the main route of GSSG generation in such reactions is not by dimerization of GS. but via mechanism(s) involving oxygen consumption such as via GSSG-. or via GSOOH. PMID- 2999102 TI - Interaction of plasma gelsolin with G-actin and F-actin in the presence and absence of calcium ions. AB - Plasma gelsolin formed a very tight 1:2 complex with G-actin in the presence of Ca2+, but no interaction between gelsolin and G-actin was detected in the presence of excess EGTA. However, the 1:2 complex dissociated into a 1:1 gelsolin:actin complex and monomeric actin when excess EGTA was added. Plasma gelsolin bound tightly to the barbed ends of actin filaments and also severed filaments in the presence of Ca2+ and bound weakly to the filament barbed end in the presence of EGTA. The 1:2 gelsolin-actin complex bound to the barbed ends of filaments but did not sever them. By blocking the barbed end of filaments with plasma gelsolin, we determined the critical concentration at the pointed end in 1 mM MgCl2 and 0.2 mM ATP to be 4 microM. The dissociation rate constant for ADP-G actin from the pointed end was estimated to be about 0.4 s-1 and the association rate constant to be about 5 X 10(4) M-1 s-1. Finally, we obtained evidence that plasma gelsolin accelerates but does not bypass the nucleation step and, therefore, that the concentration of gelsolin does not directly determine the concentration of filaments polymerized in its presence. Thus, gelsolin-capped filaments may not provide an absolutely reliable method for determining the rate constant for the association of ATP-G-actin at the pointed ends of filaments, but a reasonable estimate would be 1 X 10(5) M-1 s-1 in 1 mM MgCl2 and 0.2 mM ATP. PMID- 2999104 TI - Isolation and characterization of thromboxane synthase from human platelets as a cytochrome P-450 enzyme. AB - Thromboxane synthase from human platelets was purified to apparent homogeneity by conventional chromatographic techniques. A 423-fold enrichment over the specific content in the 100,000 X g sediment from platelet homogenates was obtained. The enzyme gave a single band on sodium dodecyl sulfate-gel electrophoresis corresponding to a monomeric molecular weight of 58,800. One heme per polypeptide chain was present, and by optical and EPR spectroscopy a close analogy to the group of cytochrome P-450 proteins was established. From its substrate prostaglandin H2, the stable end product thromboxane B2 is formed with a specific activity of 24.1 mumol min-1 mg of protein-1 which corresponds to a molecular activity of 1628 min-1. The enzyme formed 12L-hydroxy-5,8,10-heptadecatrienoic acid together with thromboxane B2 in a 1:1 ratio. Both products were identified by gas chromatography-mass spectrometry analysis. As reported previously for platelet microsomes (Ullrich, V., and Haurand, M. (1983) Adv. Prostaglandin Thromboxane Leukotriene Res. 11, 105-110), the pure hemoprotein spectrally interacts with pyridine- or imidazole-based inhibitors and for the potent inhibitor imidazo-(1,5-a)pyridine-5-hexanoic acid a stoichiometric binding to the heme was shown. Substrate analogs with a methylene group replacing the oxygen in either the 9- or 11-position caused difference spectra showing spectral shifts towards 387 and 407 nm, respectively. The identification of thromboxane synthase as a P-450 protein suggests that the heme-thiolate group of the enzyme is required to split and activate the endoperoxide bond of prostaglandin H2. PMID- 2999105 TI - Novel purification of cytochrome c1 from mitochondrial Complex III. Reconstitution of antimycin-insensitive electron transfer with the iron-sulfur protein and cytochrome c1. AB - Complex III of beef heart mitochondria was separated into the iron-sulfur protein and the complex devoid of it as described previously (Shimomura, Y., Nishikimi, M., and Ozawa, T. (1984) J. Biol. Chem. 259, 14059-14063). From the latter preparation, cytochrome c1 was subsequently purified by detergent-exchange chromatography on a phenyl-Sepharose column and DEAE-Sepharose column chromatography. In the former chromatography, the resolution of the iron-sulfur protein-depleted complex was achieved by changes of detergents on the surface of the complex; nearly homogeneous cytochrome c1 was eluted from the column with dodecyl octaethylene glycol mono-ether after dissociation of core proteins and subunit VI with guanidine and cholate. The purified cytochrome c1 consists of a single polypeptide as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 39 nmol of heme/mg of protein. The isolated iron sulfur protein catalyzes reduction of cytochrome c by ubiquinol, which is insensitive to antimycin, at a rate of 0.03 mumol of cytochrome c reduced/min/nmol of protein, while the purified cytochrome c1 has no such catalytic activity. When cytochrome c1 and the iron-sulfur protein form a complex, the rate of cytochrome c reduction increases to 0.12 mumol/min/nmol of the iron-sulfur protein. In this reaction, cytochrome c1 mediates antimycin insensitive electron transfer from the iron-sulfur protein to cytochrome c, thereby constituting a pathway of electrons: ubiquinol----iron-sulfur protein--- cytochrome c1----cytochrome c. The complex formation between the iron-sulfur protein and cytochrome c1 was verified by binding of cytochrome c1 to a column of protein A-Sepharose to which the iron-sulfur protein was linked with immobilized anti-iron-sulfur protein antibody. The electron-transfer activity of the mixture is at a comparable level to that of antimycin-inhibited Complex III, and both activities are partially sensitive to superoxide dismutase. Thus, the above described coupling of the iron-sulfur protein and cytochrome c1 is considered as reconstitution of the antimycin-insensitive pathway of electrons in Complex III. PMID- 2999106 TI - Purification of an active opioid-binding protein from bovine striatum. AB - We report the purification to apparent homogeneity of an active opioid-binding protein solubilized from bovine striatal membranes. The purification was accomplished in two steps: affinity chromatography on beta naltrexylethylenediamine (NED)-CH-Sepharose 4B followed by lectin affinity chromatography on wheat germ agglutinin-agarose. The ligand affinity-purified fraction exhibits stereospecific and saturable binding of opiates and is heat sensitive. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the NED-purified material gave 6-8 bands by silver staining or autoradiography of radioiodinated material. Under nondenaturing conditions, the NED-purified material elutes in a molecular mass range between 300 and 350 kDa from gel exclusion chromatography on Ultrogel AcA-34. The specific activity of the affinity-purified fraction (800-1500 pmol/mg protein) is enriched 4000 to 7000-fold over that of the membrane-bound or unpurified soluble receptor. Further purification (10-20-fold) is achieved by chromatography of the NED eluate on wheat germ agglutinin-agarose. The eluted fraction shows a single protein (65 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified material is an acidic glycoprotein with a pI of 6.0-6.3 and binds opiates with a specific activity (12,000-15,000 pmol/mg) that is 65,000 to 75,000-fold greater (theoretical, 77,000-fold) than that of the membrane-bound or crude soluble receptors. PMID- 2999103 TI - Photoaffinity labeling of the V1 vasopressin receptor in plasma membranes from rat liver. AB - Photoaffinity labeling experiments were performed with membranes from rat liver containing V1 vasopressin receptors. Photoreactive analogues of [1-beta mercaptopropionic acid]vasopressin [( Mpa1], vasopressin, or deamino-vasopressin) retaining a high binding affinity (apparent dissociation constants: 5 X 10(-9) M 3 X 10(-8) M) and agonistic properties were used. The tritium-labeled analogue [Mpa1,Lys(N epsilon-4-azidobenzoyl)8]vasopressin preferentially and specifically labels a 30-kDa polypeptide and with lower efficiency a 38-kDa polypeptide. The analogue [Mpa1,Dab4(N gamma-(N-4-azido-2-nitrophenyl-beta-Ala4]arginine vasopressin specifically labels the 38-kDa polypeptide. The labeling of these two membrane proteins is completely suppressed by an excess of arginine-vasopressin; bradykinin or angiotensin II do not inhibit the incorporation of the reactive vasopressin analogues into these proteins. The results suggest that the rat hepatic V1 receptor exists in the plasma membrane in an oligomeric form composed of two subunits with a molecular mass of 30 and 38 kDa. PMID- 2999107 TI - The gene encoding the large subunit of human RNA polymerase II. AB - As a first step to approach the structural and functional analysis of DNA dependent RNA polymerase II (EC 2.7.7.8), we have isolated genomic sequences for the large subunit of the human enzyme. The sequences homologous to Drosophila RNA polymerase II large subunit sequences are present in the genome as single copy genes, when assayed at high stringency. The polypeptide information is encoded in a mRNA of 7.35 kilobases, as determined by Northern blot analysis. In vitro translation reveals a polypeptide of 220 kDa, similar in electrophoretic mobility to the largest subunit of the enzyme. A fusion-polypeptide synthesized in bacteria contains a region that cross-reacts with anti-RNA polymerase II antiserum. Antiserum directed against the purified fusion protein reacts with the large subunit of RNA polymerase II, whether in the intact IIA (220 kDa) or in the degraded IIB (180 kDa) forms. Moreover, the antifusion protein antibody inhibits not only the purified calf thymus RNA polymerase II activity but also specific RNA polymerase II transcription in a HeLa cell extract. Thus, the DNA fragment isolated contains structural and functional domains of the human RNA polymerase II large subunit. PMID- 2999108 TI - Isolation and properties of two actin-binding domains in gelsolin. AB - Gelsolin is a Ca2+-sensitive 90-kDa protein which regulates actin filament length. A molecular variant of gelsolin is present in plasma as a 93-kDa protein. Functional studies have shown that gelsolin contains two actin-binding sites which are distinct in that after Ca2+-mediated binding, removal of free Ca2+ releases actin from one site but not from the other. We have partially cleaved human plasma gelsolin with alpha-chymotrypsin and identified two distinct actin binding domains. Peptides CT17 and CT15, which contain one of the actin-binding domains, bind to actin independently of Ca2+; peptides CT54 and CT47, which contain the other domain, bind to actin reversibly in response to changes in Ca2+ concentration. These peptides sequester actin monomers inhibiting polymerization. Unlike intact gelsolin, neither group of peptides nucleates actin assembly or forms stable filament end caps. CT17 and CT15 can however sever actin filaments. Amino acid sequence analyses place CT17 at the NH2 terminus of gelsolin and CT47 at the carboxyl-terminal two-thirds of gelsolin. Circular dichroism measurements show that Ca2+ induces an increase in the alpha-helical content of CT47. These studies provide a structural basis for understanding the interaction of gelsolin with actin and allow comparison with other Ca2+-dependent actin filament severing proteins. PMID- 2999109 TI - A single gene codes for the kinase and phosphatase which regulate isocitrate dehydrogenase. AB - The gene which codes for isocitrate dehydrogenase kinase/phosphatase of Escherichia coli, aceK, has been cloned. Physical and functional mapping of this clone indicated that both the isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase activities are encoded by an 1800-base pair sequence. This sequence produced a polypeptide with an apparent molecular weight of 66,000, which is identical to that of the purified protein. Since a protein of this size would require an 1800-base pair coding sequence, we conclude that isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase are expressed from a single gene. This strongly suggests that both activities reside on the same polypeptide chain. The cloning of aceK was made possible by the fortuitous addition of a second origin of replication to the expression vectors which were employed. These expression vectors were found to inhibit the growth of E. coli on the minimal acetate selective medium. The inclusion of a second origin of replication reduced the copy number and so reduced the inhibitory effects of these vectors. Control of the copy number through the addition of replication origins may have a general facility when manipulating plasmids which are potentially toxic to E. coli. PMID- 2999110 TI - Screening for thermostable mutant of kanamycin nucleotidyltransferase by the use of a transformation system for a thermophile, Bacillus stearothermophilus. AB - A structural gene of kanamycin nucleotidyltransferase cloned into a single stranded bacteriophage M13 was subjected to mutagenesis with hydroxylamine. Having recloned the mutagenized gene of the enzyme in a vector plasmid pTB922, the recombinant plasmid was used to transform Bacillus stearothermophilus with a purpose of screening for the more thermostable enzyme than the wild type. Out of greater than 8 X 10(3) transformants, 12 clones that were suspected to harbor the mutant gene encoding the more thermostable enzyme were isolated by shifting from a permissive (55 degrees C) to a nonpermissive (61 degrees C) temperature that inactivates the wild-type enzyme. DNA sequence analysis of the mutant genes revealed two types of mutation of single base substitution and hence a single amino acid replacement. The first type was the replacement of an aspartate by a tyrosine at position 80 of the wild-type enzyme, while the second was that of a threonine by a lysine at position 130. Purified enzymes from the two mutant genes were confirmed to be substantially more thermostable than the wild type in vitro. The method of screening for a thermostable kanamycin nucleotidyltransferase presented here could be applied to any other enzyme, if a transformation system of a thermophile were available. Indeed, thermostable mutants with a subtle amino acid change would be of value for better understanding of forces and interactions that contribute to the stability of a protein. PMID- 2999111 TI - Purification of the HhaII restriction endonuclease from an overproducer Escherichia coli clone. AB - An Escherichia coli K12 strain carrying the HhaII methylase and restriction genes on two separate compatible plasmids, pSK5 and pSK7, is used to overproduce the restriction endonuclease. Plasmid pSK5 expresses the methylase gene constitutively from its chloramphenicol resistance gene promoter, and plasmid pSK7 expresses the restriction endonuclease under control of the lacUV5 promoter. Induction of the two-plasmid clone with 1 mM isopropyl-1-thio-beta-D galactopyranoside results in a 15-fold increase in HhaII endonuclease activity. The enzyme has been purified to apparent homogeneity. It migrates as a 23 kilodalton polypeptide on denaturing sodium dodecyl sulfate-polyacrylamide electrophoretic gels and as a 52-kilo-dalton native protein dimer on a high pressure liquid chromatography sizing column. PMID- 2999112 TI - Catalytic properties of the HhaII restriction endonuclease. AB - The catalytic properties of the HhaII restriction endonuclease were studied using plasmid pSK11 DNA containing a single 5'-G-A-N-T-C HhaII cleavage site as substrate. Reactions were followed by two methods: 1) gel electrophoretic analysis of nicked circular and linear DNA products, or 2) release of 32P-labeled inorganic phosphate from specifically labeled HhaII sites in a reaction coupled with bacterial alkaline phosphatase. The enzyme is optimally active at 37 degrees C in 10 mM Tris-HCl (pH 9.1) and 4-10 mM MgCl2 without added NaCl. Activity is stabilized by the presence of 2-mercaptoethanol and 0.2% Triton X-100 or 50 microgram/ml bovine serum albumin. At enzyme concentrations below 10 nM and using pSK11 as substrate, initial kinetic rates were dependent on the order of mixing of reactants. A lag of 3-4 min was observed if enzyme or substrate was added last. Preincubation of substrate and enzyme followed by initiation of the reaction with MgCl2 or preincubation of the enzyme with nonspecific DNA followed by initiation with substrate eliminated or reduced the lag, respectively, and speeded up the reactions. Under a wide range of reaction conditions, nicked pSK11 DNA accumulated early, while linear molecules appeared later, suggesting that HhaII cleaves one strand at a time in separate binding events. The apparent Km for covalently closed pSK11 DNA molecules was approximately 17 nM, and the turnover number for the conversion of covalent to nicked sites was 1.1 single strand scissions/min. Pre-steady state kinetic analysis indicated that cleavage of the first phosphodiester bond in a site is first order with a rate constant of about 0.8 min-1, while cleavage of the second phosphodiester bond is first order with a rate constant of about 0.2 min-1. PMID- 2999113 TI - Characterization of a yeast nuclear gene (MST1) coding for the mitochondrial threonyl-tRNA1 synthetase. AB - The wild-type yeast nuclear gene MST1 complements mutants defective in mitochondrial protein synthesis. The gene has been sequenced and shown to code for a protein of 54,030 kDa. The predicted product of MST1 is 36% identical over its 462 residues to the Escherichia coli threonyl-tRNA synthetase. Amino acylation of wild-type mitochondrial tRNAs with a mitochondrial extract from mst1 mutants fail to acylate tRNAThr1 (anticodon: 3'-GAU-5') but show normal acylation of tRNAThr2 (anticodon: 3'-UGU-5'). These data suggest the presence of two separate threonyl-tRNA synthetases in yeast mitochondria. Antibodies were prepared against a trpE/MST1 fusion protein containing the 321 residues from the amino-terminal region of the E. coli anthranilate synthetase and 118 residues of the mitochondrial threonyl-tRNA synthetase. Antibodies to the fusion protein detect a 50-55-kDa protein in wild type yeast mitochondria but not in mitochondria of a strain in which the chromosomal MST1 gene was replaced by a copy of the same gene disrupted by insertion of the yeast LEU2 gene. The ability of the mutant with the inactive MST1 gene to charge tRNAThr2 argues strongly for the existence of a second threonyl-tRNA synthetase gene. PMID- 2999114 TI - MSW, a yeast gene coding for mitochondrial tryptophanyl-tRNA synthetase. AB - E569 and E606 are noncomplementing pet mutants of Saccharomyces cerevisiae. Both strains are defective in mitochondrial protein synthesis and as a result exhibit a pleiotropic deficiency in respiratory components that are translated on mitochondrial ribosomes. The wild type gene MSW capable of complementing the protein synthesis defect has been cloned by transformation of one of the mutants with a genomic library of wild type yeast nuclear DNA. The cloned gene has been sequenced and shown to code for a protein with a molecular weight of 42,414 which is 37 and 39% identical to the tryptophanyl-tRNA synthetases of Escherichia coli and Bacillus stearothermophilus, respectively. A strain containing an insertion in the chromosomal copy of MSW was constructed by in situ gene replacement. This mutant fails to charge mitochondrial tryptophanyl-tRNA providing further evidence that MSW is the structural gene for mitochondrial tryptophanyl tRNA synthetase. The existence of another gene coding for the cytoplasmic tryptophanyl-tRNA synthetase is inferred from the observation that mutations in MSW are not lethal but only result in a respiratory deficiency. PMID- 2999115 TI - Zn2+, not Ca2+, is the most effective cation for activation of dolichol kinase of mammalian brain. AB - The cation specificity of dolichol kinase of mammalian brain and the potential involvement of a Ca2+-calmodulin system in regulation of this enzyme have been studied. Among 10 divalent cations examined, Zn2+ was found to be most effective for the activation of dolichol kinase of rat and calf brain and cultured C-6 glial cells. The activations with Ca2+, Co2+, and Mg2+ were 53%, 32%, and 18% of the full activation with Zn2+, respectively. No combinations of the cations could activate the enzyme as much as Zn2+ alone. A role for a Ca2+-calmodulin system in the regulation of brain dolichol kinase was not supported by our data. First, the concentration of free Ca2+ required for the maximum activation of dolichol kinase was two to three orders of magnitude greater than the concentration required by typical calmodulin-dependent enzymes. Second, neither the depletion of calmodulin from the microsomal fraction nor the addition of exogenous calmodulin caused an alteration in the activation of dolichol kinase by Ca2+ (or Zn2+). Third, antagonists of calmodulin failed to suppress the activation of the enzyme by Ca2+ (or Zn2+). The data raise the possibility that Zn2+ is involved in the regulation of dolichol kinase in brain. PMID- 2999116 TI - The turnover of thrombin-thrombomodulin complex in cultured human umbilical vein endothelial cells and A549 lung cancer cells. Endocytosis and degradation of thrombin. AB - We have prepared a monoclonal antibody directed against human thrombomodulin. We used the antibody to measure thrombomodulin molecules in cultured human endothelial cells from umbilical vein and in a human lung cancer cell line (A549). Endothelial cells contain approximately 30,000-55,000 molecules of thrombomodulin/cell while the A549 cell has about 1/4 of this number. About 50 60% of thrombin binding sites on endothelial cells are thrombomodulin, while about 90% of thrombin binding sites on A549 cells are thrombomodulin. Exposure of these cells to thrombin decreased thrombomodulin on the cell surface suggesting that internalization of thrombin-thrombomodulin occurred. The internalized 125I thrombin was degraded in the cells and thrombomodulin reappeared on the cell surface after 30 min, suggesting the recycling of thrombomodulin. The rate of protein C activation correlated with the presence of the thrombin-thrombomodulin complex on the cell surface. The binding of thrombin to cell-surface thrombomodulin accelerates protein C activation; the subsequent internalization of the thrombin-thrombomodulin complex is associated with cessation of protein C activation. Therefore, endocytosis of thrombin-thrombomodulin may serve to control protein C activation. The uptake and degradation of thrombin bound to thrombomodulin may provide a mechanism for clearance of thrombin from the circulation. PMID- 2999117 TI - Hydroxyl radical attack on dopamine. AB - Hydroxyl radicals were generated in the presence of 1 mM dopamine (3,4 dihydroxyphenylethylamine) at pH 7.2 (50 mM phosphate buffer) by the following two mechanisms: 1) a classic Fenton-type reaction between hydrogen peroxide and a ferrous chelate (ferrous diethylenetriaminepentaacetate) and 2) the cyclical redox reactions of iron-EDTA/ascorbate. Three ring-monohydroxylated products of dopamine were detected by high performance liquid chromatography with electrochemical detection: 2-hydroxydopamine, 5-hydroxydopamine, and 6 hydroxydopamine in an approximate ratio of 3:2:1. Scavengers of hydroxyl radicals (dimethyl sulfoxide, mannitol, ethanol) suppressed the yields of products in a concentration-dependent manner. The formation of nonphysiologic hydroxylated forms of dopamine can provide a probe for the formation of hydroxyl radicals in dopamine neurons. PMID- 2999118 TI - Corticotropin releasing factor increases proopiomelanocortin messenger RNA in mouse anterior pituitary tumor cells. AB - The ability of corticotropin releasing factor (CRF) to stimulate adrenocorticotropin (ACTH) synthesis in corticotrophs was assessed by measuring total cell content of ACTH and the levels of proopiomelanocortin (POMC) mRNA in a cloned tumor cell line of the mouse anterior pituitary (AtT-20/D16-16). CRF treatment caused a time-dependent increase in POMC mRNA levels (measured using a hybridization technique) as well as elevating total ACTH content in AtT-20 cells. The increase in POMC mRNA levels preceded changes in ACTH content and slowly returned toward control levels after CRF withdrawal. The rise in POMC mRNA levels following CRF stimulation appeared to be specific since beta-actin mRNA levels were not affected by CRF treatment. Both 8-bromo-cAMP and phorbol ester increased POMC mRNA levels in AtT-20 cells, suggesting that CRF may act through different protein kinases to regulate the POMC gene. CRF appears to activate the POMC gene since treatment of the AtT-20 cells with the peptide increased the levels of an RNA species in the nuclei having the expected molecular weight of the transcript of the POMC gene. The results indicate that continued exposure of corticotrophs to CRF induces long term increases in the ACTH synthetic capacity of those cells. PMID- 2999119 TI - The mannose-permease of the bacterial phosphotransferase system. Gene cloning and purification of the enzyme IIMan/IIIMan complex of Escherichia coli. AB - The mannose-permease complex of the phosphoenolpyruvate-dependent phosphotranferase system exhibits two apparently unrelated activities. It mediates active transport concomitant with phosphorylation of mannose, 2 deoxyglucose, and a number of other hexoses, and it is required for penetration of bacteriophage lambda DNA across the cytoplasmic membrane of Escherichia coli. A cloned fragment of E. coli chromosome restores mannose-fermentation in, and confers lambda sensitivity to, an E. coli strain with a mutation in the gene for the phosphoenolpyruvate-dependent mannose uptake. Using complementation analysis of phosphotransferase activity and of lambda sensitivity, a 3.8-kilobase pair fragment was shown to carry two adjacent genes, ptsM and ptsL. Although each gene has a promoter of its own, transcription of ptsL has a positive polar effect on the transcription of ptsM. A complex of two proteins, IIMan and IIIMan, was purified to homogeneity from an overproducing strain. IIMan is encoded by gene ptsM, IIIMan by gene ptsL. IIMan, a 27-kDa protein, is the transmembrane component of the complex. IIIMan, a 35-kDa protein, exists as a dimer and is found both membrane-associated and free in the cytoplasm. IIIMan can be phosphorylated in a phosphoenolpyruvate-dependent reaction, while phosphorylation of IIMan could not be detected. IIMan and IIIMan are both required for phosphorylation of 2-deoxyglucose in vitro, while IIMan alone is sufficient to confer lambda sensitivity. PMID- 2999120 TI - Preparation and properties of ferrous chloroperoxidase complexes with dioxygen, nitric oxide, and an alkyl isocyanide. Spectroscopic dissimilarities between the oxygenated forms of chloroperoxidase and cytochrome P-450. AB - Extensive spectroscopic investigations of chloroperoxidase and cytochrome P-450 have consistently revealed close similarities between these two functionally distinct enzymes. Although the CO-bound ferrous states were the first to display such resemblance, additional comparisons have focused on the native ferric and ferrous and the ligand-bound ferric derivatives of the enzymes. In order to test the extent to which the spectral properties of the two enzymes match each other, we have prepared the NO, alkyl isocyanide, and O2 adducts of ferrous chloroperoxidase, the latter two for the first time. As expected, the NO adducts of the two proteins have similar UV-visible absorption and magnetic circular dichroism spectra; the same behavior is observed for the alkyl isocyanide complexes. Unexpectedly, the dioxygen adduct of ferrous chloroperoxidase (i.e. Compound III), generated in cryogenic solvents at -30 degrees C by bubbling with O2, is spectrally distinct from oxy-P-450-CAM. Identification of this derivative as oxygenated chloroperoxidase is based on the following criteria: It is EPR silent at 77 K. The bound O2 is dissociable as judged by the uniform conversion to the CO-bound form. Oxy-chloroperoxidase autoxidizes to form the native ferric enzyme without detectable intermediates at a rate comparable to that determined for oxy-P-450-CAM. Oxy-chloroperoxidase exhibits optical absorption (lambda nm (epsilon mM) = 354 (41), 430 (94), 554 (16.5), 587 (12.5)) and magnetic circular dichroism spectra that are clearly distinct from those of histidine-ligated heme proteins such as oxy-myoglobin or oxy-horseradish peroxidase. Surprisingly, several of its spectral properties, namely the red-shifted Soret peak and discrete alpha peak, are also unlike those of oxy-P-450-CAM. Since considerable evidence has accumulated supporting the ligation of an endogenous thiolate to the heme iron of chloroperoxidase, as has been established for the P-450 enzyme, the observed dissimilarities suggest that the electronic properties of the two dioxygen adducts are quite sensitive to differences in their active site heme environment. This, in turn may be related to the functional differences between the two enzymes. PMID- 2999121 TI - Photooxidation of porphyrin in Mg-substituted horseradish peroxidase. AB - Upon photoirradiation under aerobic conditions, the porphyrin prosthetic group in Mg-substituted horseradish peroxidase was oxidized to a mixture of its pi-cation radical and an oxidized product with an absorption band at 448 nm. The 448 nm compound was then converted to a 489 nm compound in the dark and the activation energy for the conversion was 19.3 kcal/mol. About 1 mol of O2 was consumed per mol of the 448 nm compound formed and no O2 consumption was seen in the dark reaction. The substitution of ethyl groups (meso) and hydroxyethyl groups (hemato) for the vinyl groups in protoporphyrin IX did not have an effect on the result. Under anaerobic conditions and in the presence of a suitable electron acceptor, the only photooxidation product of porphyrin was its pi-cation radical. The formation of hydroxyl radicals during irradiation under aerobic conditions was confirmed by the spin-trapping method. The formation of the above two radicals could be followed by ESR spectroscopy separately at a fixed magnetic field which was set to maximize each ESR signal. The rate of hydroxyl radical formation depended linearly on the concentration of Mg peroxidase. The photooxidation of porphyrin was slow and gave nonspecific product(s) when Mg protoporphyrin IX was present in the heme crevice of apomyoglobin or free in solution. PMID- 2999122 TI - Kidney epithelial cells of monkey origin (BSC-1) express a sodium bicarbonate cotransport. Characterization by 22Na+ flux measurements. AB - Na movement across the plasma membranes of confluent monolayers of monkey kidney epithelial cells (BSC-1) was studied using 22Na+ uptake and efflux techniques in the presence of 10(-4) M ouabain. In the presence of 28 mM bicarbonate, uptake was inhibited by both 10(-3) M amiloride and 10(-3) M 4,4'diisothiocyanostilbene 2,2'-disulfonic acid (DIDS). In DIDS-pretreated cells, 10(-3) M amiloride led to a further reduction of 22Na+ uptake, while 10(-5) furosemide was ineffective. DIDS also inhibited sodium efflux, indicating that the DIDS-sensitive pathway mediates both influx and efflux of 22Na+. DIDS-sensitive 22Na+ uptake, as studied in the presence of both 10(-4) M ouabain and 10(-3) M amiloride, was abolished by the absence of bicarbonate, which could not be substituted by other plasma membrane-permeable buffers. In 28 mM HCO3-, DIDS-sensitive uptake of 28 mM Na+ was cis-inhibited by 124 mM Na+, but no significant inhibition by K+ or Li+ was found. DIDS-sensitive 22Na+ uptake was a saturable function of both Na+ concentration (apparent Km between 20 and 40 mM at 28 mM HCO3-) and HCO3- concentration (apparent Km between 7 and 14 mM at 151 mM Na+). Intracellular microelectrode measurements showed that net Na+ transport in the presence of HCO3 is electrogenic, i.e. that there is anion cotransport with Na+. This effect is abolished by 1 mM DIDS. It is concluded that monkey kidney epithelial cells possess a stilbene-sensitive, electrogenic sodium bicarbonate symport, which may play an important role in bicarbonate reabsorption in the mammalian kidney. PMID- 2999123 TI - Nucleoside diphosphate regulation of overall rates of protein biosynthesis acting at the level of initiation. AB - A sensitive assay method developed to examine the effects of subtle, physiologically relevant, changes in the levels of adenine and guanine mono-, di , and triphosphorylated nucleotides specifically on the initiation of protein synthesis is described. Initiation rates are quantified by measuring the amount of protein synthesis resulting from the run-off of ribosomes which have initiated during defined intervals in a modified in vitro protein-synthesizing system developed from Ehrlich ascites tumor cell lysates (Henshaw, E.C., and Panniers, R. (1983) Methods Enzymol. 101, 616-629). The modifications include the attenuation of the ATP-regenerating system so that the relative nucleotide levels more nearly reflect actual intracellular conditions. With this system the rate of initiation is highly sensitive to changes in the ADP:ATP and GDP:GTP ratios, but indifferent to the absolute levels of either diphosphate. While the tight coupling of these two ratios by endogenous nucleoside diphosphate kinase activity prevents the independent manipulation of either ratio, the data do eliminate both AMP and GMP per se as inhibitory species. The close agreement of our data calculated in terms of energy charge to previously published results on overall rates of protein synthesis in rat thymocytes (Mendelsohn, S.K., Nordeen, S.K., and Young, D.A. (1977) Biochem. Biophys. Res. Commun. 79, 53-60) continues to suggest a physiologically relevant regulatory influence of subtle changes in nucleotides acting at the level of the initiation reaction. PMID- 2999124 TI - Cholesterol-rich intracellular membranes: a precursor to the plasma membrane. AB - The disposition of newly synthesized sterols in cultured human fibroblasts has been examined in this study. We began by demonstrating that cholesterol mass and exogenously added [3H]cholesterol both are markers for the plasma membrane, perhaps better than 5'-nucleotidase. Cells were incubated with radioactive acetate to label their endogenous sterols biosynthetically, treated with cholesterol oxidase to convert plasma membrane cholesterol to cholestenone, and then homogenized and spun to equilibrium on sucrose gradients. The density gradient profiles of the various organelles were monitored using these markers: plasma membrane, radioactive cholestenone; smooth endoplasmic reticulum, 3 hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase); and Golgi apparatus, galactosyltransferase. The buoyant density profiles of radioactive intracellular cholesterol and lanosterol both had a peak at 1.12 g/cm3, similar to 5' nucleotidase and galactosyltransferase but not to HMG-CoA reductase. This result suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. Digitonin treatment shifted the profiles of both plasma membrane and intracellular cholesterol to higher densities. Pretreatment of intact cells with cholesterol oxidase abolished the digitonin shift of plasma membranes but not the intracellular cholesterol, indicating that these two membrane pools are not entirely physically associated. Because intracellular cholesterol was shifted more than any of the organelle markers, it must reside in a separate membrane. Since digitonin selectively shifts the density of membranes rich in cholesterol, we infer that newly synthesized cholesterol accumulates in such membranes prior to its delivery to the plasma membrane. Taken together, these results suggest that cholesterol may be concentrated for delivery to the plasma membrane by being synthesized from a sterol precursor such as lanosterol in a discrete but undefined intracellular membrane. PMID- 2999125 TI - Structural elucidation of the disulfated oligosaccharide from bovine lutropin. AB - The Asn-linked oligosaccharides of the pituitary hormone lutropin (LH) contain both sulfate and GalNAc. Bovine pituitary explants incorporate [3H]glucosamine, [3H]mannose, [3H]fucose, and [35S]sulfate into the Asn-linked oligosaccharides of LH. Endoglycosidase F or N-glycanase releases the [3H]glucosamine- and [3H]mannose-labeled oligosaccharides from the protein, which resolve on anion exchange high pressure liquid chromatography as neutral (S-0), mono- (S-1), and disulfated (S-2) species. Based on sequential enzyme digestion, methylation, periodate oxidation, and nuclear magnetic resonance studies, the proposed structure for S-2 is as follows: formula see text. Sulfate is confined to position 3 or 4 of GalNAc based on periodate and methylation data and can be removed by methanolysis. The presence of beta-linked GalNAc at a position typically occupied by Gal has not previously been observed. PMID- 2999126 TI - trans-2,5-Bis-(3,4,5-trimethoxyphenyl)tetrahydrofuran. An orally active specific and competitive receptor antagonist of platelet activating factor. AB - trans-2,5-Bis(3,4,5-trimethoxyphenyl)tetrahydrofuran (L-652,731) is found to be a potent and orally active platelet activating factor (PAF)-specific and competitive receptor antagonist. It potently inhibits [3H]PAF (1 nM) binding to receptor sites on rabbit platelet membranes with an ED50 of 2 X 10(-8) M under the assay condition without the addition of mono- or divalent cations. In a comparative study, it is more potent than CV-3988, kadsurenone, and ginkgolide B as a receptor antagonist. The equilibrium dissociation constants (KB) of L 652,731 obtained either from the inhibition of receptor binding or from the inhibition of PAF-induced aggregation of gel-filtered rabbit platelet are 2.7 X 10(-8) and 2.1 X 10(-8) M, respectively. The agreement of these KB determinations based on receptor and cellular function suggests that L-652,731 does not inhibit other steps following PAF-receptor binding. L-652,731 does not antagonize the binding of several radioligands to their respective receptor. It shows no inhibitory effect on platelet aggregation induced by other aggregating agents including thrombin, collagen, A-23187, arachidonic acid, epinephrine, and ADP. L 652,731 is orally active; it inhibits PAF-induced rat cutaneous vascular permeability with an ED50 of 30 mg/kg orally. Significant inhibitory results of L 652,731 suggest that PAF may be partially involved in cutaneous vascular permeability induced by histamine and bradykinin. PMID- 2999127 TI - The role of compartmentation and glycerol kinase in the synthesis of ATP within the glycosome of Trypanosoma brucei. AB - Glycosomes, purified from trypomastigote forms of Trypanosoma brucei, contained all the enzymes necessary to convert glucose to alpha-glycerophosphate and 3 phosphoglycerate. The multienzyme reaction which produces 2 alpha glycerophosphate, 2 ADP, and 2 NAD+ from 1 glucose, 2 ATP, and 2 NADH was studied spectrophotometrically. Intact glycosomes, suspended with 5.6 mM alpha glycerophosphate and 2 mM ADP, produced ATP inside the glycosomes for glucose phosphorylation at a rate of 0.7 mumol/min/mg protein, so confirming the feasibility of producing ATP from alpha-glycerophosphate and ADP catalyzed by glycosomal glycerol kinase, and coupling this ATP production to the ATP-requiring stages of glycolysis. No evidence was found for direct channeling of the ATP generated by glycerol kinase and either hexokinase or phosphofructose kinase in glycosomal enzyme complexes cross-linked by dimethyl suberimidate treatment of intact glycosomes prior to solubilization of their membrane. Compartmentation of glycolytic intermediates, enzymes, and ATP inside isolated glycosomes was demonstrated by their inaccessibility to exogenous enzymes. We conclude that the compartmentation of the glycosome and the efficient production of ATP in the glycosome from whole cell concentrations of sn-glycerol 3-phosphate and ADP account for the observed whole cell production of equimolar glycerol from glucose with net ATP synthesis by T. brucei under anaerobic conditions. PMID- 2999128 TI - A monoclonal antibody capable of modulating opioid binding to rat neural membranes. AB - A monoclonal antibody capable of inhibiting opioid binding to rat neural membranes has been produced. Spleen cells from a BALB/c mouse, immunized with a partially purified opioid receptor complex, were fused with P3-X63.Ag8.653.3 myeloma cells. The cell line OR-689.2.4 secreted an IgM that was capable of partially inhibiting opioid binding to rat neural membranes under equilibrium binding conditions, while not affecting the binding of nonopioid ligands. Control mouse immunoglobulins and heat-denatured OR-689.2.4 did not inhibit opioid binding to membranes. The purified immunoglobulin inhibited the binding of [3H]dihydromorphine in a titrable, saturable, and reversible manner, as well as the binding of the delta-ligand [3H][D-Ala2,D-Leu5]enkephalin, the kappa-ligand [3H] ethylketocyclazocine, and 3H-labeled antagonists. In addition to blocking the binding of opioids to membranes, the immunoglobulin could also displace bound [3H]dihydromorphine from neural membranes. The 125I-labeled immunoglobulin specifically bound to neural membranes with a Kd of 1.3 nM and a maximal number of binding sites of 41.8 fmol/0.25 mg of membrane protein. In a titrable manner, the immunoglobulin precipitated opioid binding sites from a solubilized preparation of neural membranes. When OR-689.2.4 conjugated to Sepharose was incubated with the partially purified opioid receptor complex, labeled with 125I, a 35,000-dalton protein was specifically bound by the immunoglobulin. This antibody provides a tool for probing the multiple opioid binding sites. PMID- 2999129 TI - Regulation of deoxyadenosine and nucleoside analog phosphorylation by human placental adenosine kinase. AB - The enzymes responsible for the phosphorylation of deoxyadenosine and nucleoside analogs are important in the pathogenesis of adenosine deaminase deficiency and in the activation of specific anticancer and antiviral drugs. We examined the role of adenosine kinase in catalyzing these reactions using an enzyme purified 4000-fold (2.1 mumol/min/mg) from human placenta. The Km values of deoxyadenosine and ATP are 135 and 4 microM, respectively. Potassium and magnesium are absolute requirements for deoxyadenosine phosphorylation, and 150 mM potassium and 5 mM MgCl2 are critical for linear kinetics. With only 0.4 mM MgCl2 in excess of ATP levels, the Km for deoxyadenosine is increased 10-fold. ADP is a competitive inhibitor with a Ki of 13 microM with variable MgATP2-, while it is a mixed inhibitor with a Ki and Ki' of 600 and 92 microM, respectively, when deoxyadenosine is variable. AMP is a mixed inhibitor with Ki and Ki' of 177 and 15 microM, respectively, with variable deoxyadenosine; it is a non-competitive inhibitor with a Ki of 17 microM and Ki' of 27 microM with variable ATP. Adenosine kinase phosphorylates adenine arabinoside with an apparent Km of 1 mM using deoxyadenosine kinase assay conditions. The Km values for 6 methylmercaptopurine riboside and 5-iodotubercidin, substrates for adenosine kinase, are estimated to be 4.5 microM and 2.6 nM, respectively. Other nucleoside analogs are potent inhibitors of deoxyadenosine phosphorylation, but their status as substrates remains unknown. These data indicate that deoxyadenosine phosphorylation by adenosine kinase is primarily regulated by its Km and the concentrations of Mg2+, ADP, and AMP. The high Km values for phosphorylation of deoxyadenosine and adenine arabinoside suggest that adenosine kinase may be less likely to phosphorylate these nucleosides in vivo than other enzymes with lower Km values. Adenosine kinase appears to be important for adenosine analog phosphorylation where the Michaelis constant is in the low micromolar range. PMID- 2999130 TI - The primary sequence of Ricinus communis agglutinin. Comparison with ricin. AB - A mixture of synthetic oligonucleotides representing all possible sequences of a peptide present in the ricin B chain has been used to screen a cDNA library constructed using ripening castor bean seed poly(A+) RNA. The eight largest recombinant plasmids selected, by hybridization, a single mRNA species whose translational product was identified as preprolectin by immunoprecipitation. Restriction enzyme analysis of these clones demonstrated that two classes were present representing sequences complementary to two distinct but closely related preprolectin mRNA species. The nucleotide sequence of the cloned cDNA from one of these classes encodes preproricin and has been presented elsewhere (Lamb, F. I., Roberts, L. M., and Lord, J. M., (1985) Eur. J. Biochem. 148, 265-270). The nucleotide sequence of the second class is presented here and shown to represent prepro-Ricinus communis agglutinin. The entire coding sequence was deduced from two overlapping cDNA clones having inserts of 1668 and 1151 base pairs. The coding region defines a preproprotein with a 24-amino acid N-terminal signal sequence preceding the A chain (266 amino acids) which is joined to the B chain (262 amino acids) by a 12-amino acid linking peptide. The protein was confirmed as R. communis agglutinin since the deduced B chain N-terminal sequence corresponds exactly with that determined for purified R. communis agglutinin B chain over a region where several residue differences occur in the ricin B chain. The nucleotide and deduced amino acid sequences of the R. communis agglutinin precursor are compared with those of the ricin precursor. PMID- 2999131 TI - Reductive alkylation with oxidized nucleotides. Use in affinity labeling or affinity chromatography. AB - A study has been made of the products of a reaction of oxidized ribonucleotides with a primary amine. As a model reaction, periodate-oxidized adenosine was combined with glycine in the presence of NaCNBH3. The purified major product of this reaction, adenine 9,2'-(4'-carboxymethyl-6'-hydroxymethylmorpholine), was characterized by 13C and 1H NMR spectroscopy, ultraviolet spectroscopy, and thin layer chromatography. When used to generate affinity columns, oxidized adenosine or oxidized ATP formed stable products with immobilized diaminohexane when treated with NaCNBH3. Failure to treat with NaCNBH3 yielded an unstable affinity matrix. These results are used in the interpretation of differing results when oxidized nucleotides have been used as affinity labels for different proteins. PMID- 2999132 TI - The properties and regulation of pantothenate kinase from rat heart. AB - Pantothenate kinase (ATP:D-pantothenate 4'-phosphotransferase, EC 2.7.1.33), the first enzyme in the pathway of CoA synthesis, was partially purified from rat heart. A study of the properties of the kinase showed that it possesses a broad pH optimum between 6 and 9, is activated or inhibited nonspecifically by various anions, and has MgATP as the nucleotide substrate. The Km for MgATP is 0.6 mM and that for pantothenate is 18 microM. CoA and acyl esters of CoA are inhibitors of the kinase with the inhibition by acetyl-CoA being only slightly greater than that by free CoA. The inhibition by free CoA is uncompetitive with respect to pantothenate concentration, with a Ki for inhibition of 0.2 microM. L-Carnitine was found to be a nonessential activator of the kinase. This compound had no effect by itself but specifically reversed the inhibition of the kinase by CoA. The Ka for deinhibition by L-carnitine is 0.27 mM. Free carnitine content was measured in perfused hearts and is found to vary in correlation with perfusion conditions that are known to alter rates of intracellular phosphorylation of pantothenate. These properties of pantothenate kinase provide a potential mechanism for the control of CoA synthesis. The enzyme is regulated by feedback inhibition by CoA and its acyl esters and this inhibition is modified by changes in the concentration of free carnitine. PMID- 2999133 TI - Control of K+ influx in 3T3 cells transformed by a conditional mutant of Rous sarcoma virus. AB - Mouse 3T3 cells transformed by a conditional mutant of Rous sarcoma virus (LA90) can assume either a normal or a transformed phenotype, depending on the temperature of cultivation. These cells (LA90) were arrested at the G0/G1 phase of the cell cycle by starvation for serum growth factors at the nonpermissive temperature (39 degrees C). Release from the G0/G1 phase by serum growth factors resulted in a rapid stimulation of Rb+ influx. To investigate whether the stimulation of Rb+ influx is obligatory for cell proliferation, the cultures were released from the G0/G1 phase by a temperature decrease in the absence of serum. A temperature decrease from 39 to 32 degrees C activated the viral pp60src gene mitogenic activity. Under these conditions, no rapid stimulation of Rb+ influx was observed. These results suggest that the rapid stimulation of Rb+ influx induced by serum growth factors is not an essential signal for cell release from the G0/G1 phase. However, a delayed increase in Rb+ influx concomitant with an increase in the cell content of K+ was observed in the cultures released from the G0/G1 phase by temperature decrease in the absence of serum growth factors. We found that the LA90 cells incubated at the permissive temperature (32 degrees C) secreted a mitogenic activity into the medium. Moreover, the conditioned medium from cultures incubated at 32 degrees C, but not at 39 degrees C, stimulate Rb+ influx in G0/G1 cells. These results indicate that Rous sarcoma virus pp60src induces a slow autocrine secretion of a mitogenic activity. This mitogenic activity slowly modulates the K+ content. Therefore, the slow elevation in cellular content of K+ is proposed to be an obligatory event for proliferation in normal and transformed cells. PMID- 2999134 TI - The covalent structure of the phase-1 flagellar filament protein of Salmonella typhimurium and its comparison with other flagellins. AB - In order to circumvent problems associated with direct chemical analysis of the phase-1 flagellar filament protein (flagellin) of Salmonella typhimurium, the covalent structure was determined by recombinant DNA procedures. The corresponding structural gene (H-1i) was cloned into plasmid pBR322 in a 4.3 kilobase fragment produced by EcoRI digestion of chromosomal DNA, and the nucleotide sequence of the region specifying the flagellar protein was determined. Comparison of the data obtained with the limited information available for other salmonellar flagellins supported the concept that both ends of the molecule are conserved in this genus. Additionally, a conservation of base sequence in the region of H-1 genes coding for the N-terminal end of flagellins was apparent, suggesting that this area may have an additional regulatory role. The i flagellin was found to be unrelated to proteins in the NBRF data base with the exception of other flagellins. The three flagellins which have been sequenced to date (those produced by Bacillus subtilis, Caulobacter crescentis, and phase-1 S. typhimurium) show homologies in amino acid sequence at both the N-terminal and C-terminal ends despite large differences in their total molecular weight, and comparison suggests that B. subtilis and Salmonella are more closely related to each other than either is to Caulobacter. PMID- 2999135 TI - R 59 022, a diacylglycerol kinase inhibitor. Its effect on diacylglycerol and thrombin-induced C kinase activation in the intact platelet. AB - R 59 022 (6-[2-[4-[(4-fluorophenyl) phenylmethylene)-1-piperidinyl]ethyl]-7 methyl-5H-thiazolo[3,2-alpha] pyrimidin-5-one) was found to inhibit diacylglycerol kinase in human red blood cell membranes at concentrations where polyphosphoinositide phosphodiesterase, phosphatidylinositol kinase, and phosphatidylinositol 4-phosphate kinase activity remained unaffected. The concentration needed for half-maximal inhibition (IC50) was 2.8 +/- 1.5 X 10(-6) M for the kinase acting on endogenous diacylglycerol and 3.3 +/- 0.4 X 10(-6) M when 1-oleoyl-2-acetylglycerol (OAG) was added exogenously as substrate. In intact platelets, R 59 022 inhibits the phosphorylation of OAG to 1-oleoyl-2 acetylglyceryl-3-phosphoric acid (OAPA) (IC50: 3.8 +/- 1.2 X 10(-6) M); concomitantly the stimulation of protein kinase C activity by OAG was amplified. When in platelets inositol lipid turnover is accelerated by thrombin, further addition of R 59 022 results in a marked elevation of diacylglycerol levels, a decreased formation of phosphatidic acid and an increased protein kinase C activity as compared with the controls. It is concluded that in studies on the signal-transducing system coupled to inositol lipid metabolism R 59 022 might occupy a role comparable to cyclic AMP phosphodiesterase inhibitors, since it potentiates the effect of the putative second messenger diacylglycerol by preventing its rapid metabolism. PMID- 2999136 TI - Inhibition by islet-activating protein of a chemotactic peptide-induced early breakdown of inositol phospholipids and Ca2+ mobilization in guinea pig neutrophils. AB - Receptors for a chemotactic peptide (fMet-Leu-Phe) in guinea pig neutrophils were primarily coupled to phospholipase C catalyzing breakdown of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate, which was in turn responsible for intracellular Ca2+ mobilization. These early responses of neutrophils to fMet Leu-Phe, eventually leading to O2- generation, were abolished by prior exposure of cells to islet-activating protein (IAP), pertussis toxin, which had been reported to bring about ADP-ribosylation of a membrane Mr = 41,000 protein (Okajima, F., and Ui, M. (1984) J. Biol. Chem. 259, 13863-13871). The IAP substrate, probably the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase (Ni) or an analogous protein, is hence proposed to mediate fMet-Leu-Phe receptor-linked activation of the phospholipase C. In support of this proposal, A23187 and phorbol myristate acetate which stimulate arachidonate release or O2- generation by-passing these early processes of signaling were effective in IAP-treated cells as well. Release of arachidonic acid and accumulation of inositol 1-monophosphate in delayed response to fMet-Leu Phe were also abolished by the IAP treatment of cells, despite the fact that slowly-onset inflow of Ca2+ which must be responsible for these delayed responses was observed in these IAP-treated cells. Thus, the IAP substrate may play an additional role in Ca2+-dependent activation of somehow compartmentalized phospholipases. PMID- 2999137 TI - Discriminative insulin antagonism of stimulatory effects of various cAMP analogs on adipocyte lipolysis and hepatocyte glycogenolysis. AB - Although insulin effectively blocked hormone-stimulated glycerol output in adipocytes or phosphorylase activation in hepatocytes, the inhibitory effect of insulin on cAMP analog-stimulated cells depended on the cAMP analog used. Of the 20 analogs tested in adipocytes and 13 tested in hepatocytes, the effects of about half of them were effectively blocked by insulin, whereas the effects of many of them were not inhibited at all. In order to approach the explanation for this discriminative insulin action, the inhibitory effects of insulin on the responses to the analogs in the intact cells were correlated with the in vitro cAMP analog specificity for the hepatocyte cAMP-dependent protein kinase isozymes and the low Km, hormone-sensitive phosphodiesterases from both cell types. No correlation was found between insulin resistance of analog-stimulated hepatocyte phosphorylase and the concentration of analog required in vitro for half-maximal activation of either type I or type II cAMP-dependent protein kinase from hepatocytes. However, a good correlation was found between insulin resistance of cAMP analog-stimulated responses and the analog I50 values for the phosphodiesterase from both cell types. Using a new method capable of measuring hydrolysis at low analog concentrations, several of those analogs which had relatively low, but not high, phosphodiesterase I50 values were shown to be directly hydrolyzed by the low Km adipocyte phosphodiesterase. The insulin inhibition of cell responses when stimulated by hydrolyzable analogs, but not by poorly hydrolyzable analogs, is best explained by insulin stimulation of the low Km phosphodiesterases from both cell types. PMID- 2999138 TI - Irreversible inactivation of the beta-adrenoreceptor by a partial agonist. Evidence for selective loss of the agonist high affinity binding sites. AB - The catecholamine derivatives aminomenthylnorepinephrine (compound 1) and bromoacetylaminomenthylnorepinephrine (compound 2) were synthesized and their interaction with the rat lung beta-adrenoreceptor was characterized. Compared to (-)-isoproterenol, compounds 1 and 2 were 10 and 280 times less potent, respectively, at inhibiting (-)-[3H]dihydroalprenolol binding. At pH 7.4, all 3 compounds induced a loss of receptors (40-60%) which could be recovered by treatment with guanyl-5'-yl imidodiphosphate (Gpp(NH)p). However, at pH 8.1 Gpp(NH)p treatment did not recover those receptors lost by compound 2 only. The compound 2-induced receptor loss at pH 8.1 was time-dependent, prevented by propranolol but unaffected by Gpp(NH)p or after membrane heating at 50 degrees C which prevented the formation of the agonist high affinity binding state. Although, the maximal receptor loss as measured by [3H]dihydroalprenolol was 40 60%, more than 80% of the receptors were lost when measured by direct agonist binding, and the receptors left showed little agonist high affinity binding state formation. In rat reticulocyte membranes, compounds 1 and 2 stimulated adenylate cyclase activity with intrinsic activities of 0.55 and 0.31, respectively. However, at pH 8.1, compound 2 initially stimulated the enzyme followed by a blockade. These data indicated that both compounds 1 and 2 were partial beta adrenoreceptor agonists and, at pH 8.1, compound 2 appeared to bind irreversibly only to those lung receptors able to form the agonist high affinity binding state. Furthermore, after irreversible binding, compound 2 appeared to act as an antagonist. PMID- 2999140 TI - Characterization of cDNA and genomic sequences corresponding to an embryonic myosin heavy chain. AB - We report here the isolation and characterization of cDNA and genomic sequences corresponding to a rat embryonic myosin heavy chain (MHC) protein. This gene, which is present as a single copy in the rat genome, comprises about 25 kilobase pairs of DNA and contains approximately 80% intronic sequences. The embryonic MHC gene belongs to a highly conserved multigene family, and exhibits a high degree of nucleotide and amino acid sequence conservation with other sarcomeric MHC genes from nematode to man. S1 nuclease mapping experiments using cDNA and genomic probes show that this MHC gene is transiently expressed during skeletal muscle development. Its mRNA is detected in fetal skeletal muscle during early development and persists up to 2 weeks after birth with the overlapping expression of neonatal and adult skeletal MHC mRNAs. However, this MHC is not expressed in the adult skeletal muscle with the exception of extraocular muscle fibers. The transient expression during muscle development of the isoform produced by this gene and its sequential replacement by other MHCs raises interesting questions about the mechanism controlling MHC isozyme transitions and the physiological significance of the individual MHCs in muscle fibers. PMID- 2999139 TI - Reconstitution of catecholamine-stimulated adenylate cyclase activity using three purified proteins. AB - beta-Adrenergic receptors, the GTP-binding regulatory protein that stimulates adenylate cyclase (Gs), and adenylate cyclase were each purified and reconstituted into unilamellar vesicles composed of phosphatidylethanolamine and phosphatidylserine (3:2, w/w). The molar ratio of receptor:Gs:adenylate cyclase was estimated to be about 1:10:1. Adenylate cyclase activity in the vesicles was stimulated up to 2.6-fold by beta-adrenergic agonists. Stimulation was dependent on the presence of guanine nucleotide, displayed appropriate beta-adrenergic selectivity and stereoselectivity for agonists, and was blocked appropriately by beta-adrenergic antagonists. Therefore, while additional proteins may modulate adenylate cyclase activity in native membranes, these results show that these three proteins are sufficient for the expression of hormone-stimulated adenylate cyclase. PMID- 2999141 TI - The structure and organization of a proline-rich protein gene of a mouse multigene family. AB - One gene of the mouse proline-rich protein multigene family was cloned on a 3.6 kilobase pair EcoRI/BglII DNA fragment from a (partial) Sau3A bacteriophage library of CD-1 mouse chromosomal DNA. Phage harboring the gene were identified by plaque hybridization using 32P-labeled proline-rich protein cDNA inserts from clones pRP33 and pMP1 obtained from rat and mouse, respectively. The transcriptional unit includes three exonic sequences separated by 1434 base pairs (intron I) and 450 base pairs (intron II). The complete primary structure of the gene and the 5' and 3' flanking regions (3595 base pairs) were determined by the Maxam and Gilbert (Maxam, A.M., and Gilbert, W. (1980) Methods Enzymol. 65, 499 560) sequencing method. The DNA on the 5' side of exon I contains several sequences that may be involved in the induction and expression of this mouse gene. These sequences include putative regulatory sites such as those considered to be inducible by cAMP and steroids, Z-DNA and enhancer sequences and the expected TATAA and CAAT boxes. The mature protein coding region, exon II, is not interrupted with intron sequences. Exon III is located in the nontranslated region and contains the poly(A) addition site. The deduced amino acid sequence showed that the protein encoded by this gene contains 13 tandemly repeat regions, each 14 amino acids in length, with the prototype sequence PPPPGGPQPRPPQG. Each amino acid within the repeat has a favored codon. The consensus DNA sequence for each repeat is CCA CCA CCA CCA GGA GGC CCA CAG CCG AGA CCC CCT CAA GGC. The high degree of conservation of both nucleotide and amino acid sequences within the repeat region suggests that proline-rich protein genes likely evolved by gene duplication of a 42-base pair internal repeat. PMID- 2999142 TI - The effects of processing inhibitors of N-linked oligosaccharides on the intracellular migration of glycoprotein E2 of mouse hepatitis virus and the maturation of coronavirus particles. AB - We have studied the effects of tunicamycin and inhibitors of the processing of N linked glycans including N-methyl-1-deoxynojirimycin, castanospermine, mannodeoxynojirimycin, and swainsonine on the transport of glycoprotein E2 and the intracellular maturation of the coronavirus mouse hepatitis virus A59. Indirect immunofluorescence staining with monoclonal antibodies revealed that glycoprotein E2 exhibits different antigenic properties depending on the presence and on the structure of the N-linked oligosaccharides and that efficient transport of glycoprotein E2 to the plasma membrane requires the removal of glucose residues. In the presence of tunicamycin in the nonglycosylated E2 apoprotein was synthesized in normal amounts and readily acylated throughout the infectious cycle. This E2-species could not be detected on the surface of mouse hepatitis virus A59-infected cells with indirect immunofluorescence staining or lactoperoxidase labeling. N-Methyl-1-deoxynojirimycin and castanospermine, both of which selectively inhibited the processing glucosidases, caused a drop in virion formation by two log steps and a drastic delay in the surface expression of glycoprotein E2. The E2 species synthesized under such conditions was acylated but accumulated intracellularly in a compartment distinct from the Golgi. Concomitantly, synthesis of the matrix glycoprotein E1 of mouse hepatitis virus A59 was drastically impaired. Mannodeoxynojirimycin and swainsonine, which block later stages of the processing pathway, had less or no effect on the transport of glycoprotein E2 and the formation of virus particles. PMID- 2999143 TI - Distinct biologically active receptors for insulin, insulin-like growth factor I, and insulin-like growth factor II in cultured skeletal muscle cells. AB - The expression of insulin-like growth factor (IGF) receptors at the cell surface and the changes in IGF responsiveness during differentiation were studied in the L6 skeletal muscle cell line. Throughout the entire developmental sequence, distinct receptors for IGF I and IGF II that differed in structure and peptide specificity could be demonstrated. During differentiation, both 125I-IGF I and 125I-IGF II binding to the L6 cells decreased as a result of a 3-4-fold reduction in receptor number, whereas 125I-insulin binding increased. Under nonreducing conditions, disuccinimidyl suberate cross-linked 125I-IGF I and 125I-IGF II to two receptor complexes with apparent Mr greater than 300,000 (type I) and 220,000 (type II). Under reducing conditions, the apparent molecular weight of the type I receptor changed to Mr 130,000 (distinct from the 120,000 insulin receptor) and the type II receptor changed to 250,000. IGF I and IGF II both stimulated 2-deoxy D-glucose and alpha-aminoisobutyric acid uptake in the L6 cells with a potency close to that of insulin, apparently through interaction with their own receptors. The stimulatory effects of IGF II correlated with its affinity for the type II but not the type I IGF receptor, as measured by inhibition of affinity labeling, whereas the effects of IGF I correlated with its ability to inhibit labeling of the type I receptor. In spite of the decrease in type I and type II receptor number, stimulation of 2-deoxy-glucose and alpha-aminoisobutyric acid uptake by the two IGFs increased during differentiation. PMID- 2999144 TI - Leader peptidase catalyzes the release of exported proteins from the outer surface of the Escherichia coli plasma membrane. AB - Leader peptidase cleaves the amino-terminal leader sequences of many secreted and membrane proteins. We have examined the function of leader peptidase by constructing an Escherichia coli strain where its synthesis is controlled by the arabinose B promoter. This strain requires arabinose for growth. When the synthesis of leader peptidase is repressed, protein precursors accumulate, including the precursors of M13 coat protein (an inner membrane protein), maltose binding protein (a periplasmic protein), and OmpA protein (an outer membrane protein). These precursors are translocated across the plasma membrane, as judged by their sensitivity to added proteinase K. However, pro-OmpA and pre-maltose binding protein are retained at the outer surface of the inner membrane. Thus, leader peptides anchor translocated pre-proteins to the outer surface of the plasma membrane and must be removed to allow their subsequent release into the periplasm or transit to the outer membrane. PMID- 2999145 TI - Calmodulin plus cyclic AMP-dependent phosphorylation of a Mr 22,000 pituitary protein. AB - Protein phosphorylation was examined in cytosolic extracts of adult rat anterior pituitary. In the presence of both cyclic AMP and calmodulin, the phosphorylation of a Mr 22,000 protein was markedly stimulated. Cyclic AMP and calmodulin must both be present in order for this effect to be observed; cyclic GMP does not substitute for cyclic AMP, and the effect is abolished by either trifluoperazine or the heat-stable inhibitor of cyclic AMP-dependent protein kinase. Two dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that there are three molecular species of the Mr 22,000 phosphoprotein, with pI values ranging from 6.8 to 8.1. Phosphorylation of this protein is maximally stimulated by 5 microM cyclic AMP and 5.7 microM calmodulin. The effect of cyclic AMP plus calmodulin is enhanced by preincubation and requires a divalent cation; maximal phosphorylation takes place at 100 microM Mn2+, although higher concentrations of Mg2+ and Co2+ support an equivalent degree of phosphorylation. Cyclic AMP plus calmodulin-dependent protein phosphorylation was not detected in other rat tissues surveyed, including brain, testes, adrenal, kidney, liver, spleen, skeletal muscle, pineal, or posterior pituitary. These results help to explain the previous findings of Brattin and Portanova (Brattin, W.J., Jr., and Portanova, R. (1981) Mol. Cell. Endocr. 23, 77 90) of in vivo but not in vitro phosphorylation of three Mr 20,000 anterior pituitary proteins and indicate a possible point of convergence for calcium and cyclic AMP actions in the anterior pituitary. PMID- 2999146 TI - 5-Methyltryptamine stimulates phospholipase C-mediated breakdown of exogenous phosphoinositides by blowfly salivary gland membranes. AB - 5-Methyltryptamine, through a GTP-dependent mechanism, stimulated breakdown of endogenous [3H]inositol-labeled phosphoinositides in membranes prepared from blowfly salivary gland homogenates through a phospholipase C exhibiting a pH optimum of approximately 7.0. Unlabeled membranes, prepared from salivary gland homogenates, hydrolyzed exogenous [3H]phosphatidylinositol 4,5-bisphosphate substrate with generation of labeled inositol phosphates. Inositol trisphosphate formation was increased approximately 200% by 10 microM guanosine 5'-(O-thio) trisphosphate (GTP gamma S) within 30 s. 5-Methyltryptamine, in the presence of 10 microM GTP gamma S, increased the rate of inositol trisphosphate formation by approximately 500% within 30 s. Half-maximal activation of hormone-stimulated breakdown of exogenous substrate required approximately 0.05 microM GTP gamma S. [3H]Phosphatidylinositol was also hydrolyzed during incubation with membranes, resulting in the generation of inositol, glycerol phosphoinositol, and inositol monophosphate. Formation of inositol monophosphate was stimulated approximately 30% by 10 microM GTP gamma S and 10 microM 5-methyltryptamine. Neither inositol nor glycerol phosphoinositol formation was affected by hormone. These results indicate that in a cell-free system from blowfly salivary glands, 5 methyltryptamine, through a GTP-dependent mechanism, directly activates a phospholipase C which mediates phosphoinositide hydrolysis. PMID- 2999147 TI - The formation of inositol 1,2-cyclic phosphate on agonist stimulation of phosphoinositide breakdown in mouse pancreatic minilobules. Evidence for direct phosphodiesteratic cleavage of phosphatidylinositol. AB - It is generally thought that formation of inositol 1,2-cyclic phosphate (IcP) on agonist-stimulated "breakdown" of endogenous phosphatidylinositol in intact cells would provide strong evidence for the direct phosphodiesteratic cleavage of phosphatidylinositol. We report here that on ionophoresis of extracts of pancreatic minilobules incubated with the cholecystokinin/pancreozymin congener, caerulein, the usual inositol phosphates, i.e. inositol 1-phosphate (IP), inositol 4,5-bisphosphate (IP2), and inositol 1,4,5-trisphosphate (IP3) were seen. In addition, an [3H]inositol-labeled unknown was present with the correct electrophoretic mobility of IcP. There was only a trace of "IcP" in the unstimulated pancreatic minilobules. Several lines of evidence indicate that the unknown peak was IcP. 1) It ran on ionophoresis with standard [14C]IcP, and the ratio of 3H to 14C for each point on the peak was a constant within experimental error. 2) The putative IcP peak which had been eluted from the electropherogram also coincided with standard [14C]IcP on paper chromatography. 3) On mild acid hydrolysis in the presence of standard 14C-labeled IP, the putative [3H] IcP peak disappeared and appeared in the exact position of the standard [14C]IP peak, as to be predicted of IcP. The formation of IcP on agonist stimulation supports direct phosphodiesteratic cleavage of phosphatidylinositol on stimulation of phosphoinositide breakdown in pancreatic minilobules. PMID- 2999148 TI - The Euglena gracilis chloroplast ribulose-1,5-bisphosphate carboxylase gene. I. Complete DNA sequence and analysis of the nine intervening sequences. AB - The nucleotide sequence of 6225 base pairs (bp) of Euglena gracilis chloroplast DNA including the complete DNA sequence of the chloroplast-encoded ribulose-1,5 bisphosphate carboxylase large subunit gene along with the flanking DNA sequences is presented. The gene is greater than 5.5 kilobase pairs in length and is organized as 10 exons coding for 475 amino acids, separated by 9 introns. The exons range in size from 45 to 438 bp, while the introns range in size from 382 to 568 bp. The introns have highly conserved boundary sequences with the consensus, 5'-N GTGTGGATTT...(intron)...TTAATTTTAT N-3'. The introns are 82-85 mol% AT, with a pronounced T greater than A greater than G greater than C base bias in the RNA-like strand. They do not appear to encode any polypeptides. In addition, the introns have a conserved sequence 30-50 bp from their 3'-ends with the consensus, 5'-TACAGTTTGAAAATGA-3'. The 5'-TACA sequence bears some homology to the 5'-end of the TACTAACA sequence found in a similar location in yeast nuclear mRNA introns. The conserved sequences of the Euglena rbcL introns may be indicative of a splicing mechanism similar to that of eucaryotic nuclear mRNA introns and group II mitochondrial introns. PMID- 2999149 TI - Role of Ni in coupling angiotensin receptors to inhibition of adenylate cyclase in hepatocytes. AB - Angiotensin II can inhibit glucagon-stimulated cyclic AMP production in hepatocytes and adenylate cyclase activity in hepatic membranes. Pertussis toxin, an exotoxin produced by Bordetella pertussis, was used to investigate the role of the inhibitory guanine nucleotide-binding regulatory protein of adenylate cyclase (Ni) in coupling angiotensin receptors to the adenylate cyclase system. An assay was developed using [32P] NAD+ to quantitate the amount of Ni protein in the membrane and the extent of its ADP-ribosylation catalyzed by toxin. The ability of angiotensin to inhibit adenylate cyclase and interact with its receptor was compared with the degree of modification of Ni in membranes prepared from isolated hepatocytes. In control membranes angiotensin II inhibited basal adenylate cyclase by 35%. When all of the Ni molecules in the membrane were ADP ribosylated, angiotensin did not inhibit adenylate cyclase. However, the attenuation of angiotensin's effect on cyclase was not linearly correlated with the degree of modification of Ni; ADP-ribosylation of greater than 80% of the Ni was required before a reduction of the angiotensin effect was observed. A possible explanation for this finding is an excess of Ni molecules in the membrane (approximately 3.4 pmol/mg of membrane protein) over angiotensin II receptors (approximately 1.2 pmol/mg of membrane protein). 125I-angiotensin bound to sites in the membrane with two affinities. Computer fitting of the binding isotherms yielded parameters of N1 = 279 fmol/mg protein, Kd1 = 0.2 nM; N2 = 904 fmol/mg protein, Kd2 = 1.4 nM. When all of the Ni molecules in the membrane were ADP-ribosylated, angiotensin bound to only one site with binding parameters of N = 349 fmol/mg protein, Kd = 0.4 nM. GTP-gamma-S caused a 7-fold increase in the Kd of this site to 2.7 nM. Overall, the data indicate that the Ni protein mediates the effect of angiotensin on adenylate cyclase. The observation that GTP gamma-S can markedly decrease the affinity of angiotensin receptors when all Ni molecules are ADP-ribosylated suggests that angiotensin receptors may couple to other GTP-binding proteins which may mediate the effects of angiotensin in other signal transduction systems. PMID- 2999150 TI - Metabolism of diethylstilbestrol by horseradish peroxidase and prostaglandin-H synthase. Generation of a free radical intermediate and its interaction with glutathione. AB - Diethylstilbestrol is carcinogenic in rodents and in humans and its peroxidatic oxidation in utero has been associated with its carcinogenic activity. Horseradish peroxidase-catalyzed oxidation of [14C]diethylstilbestrol and [14C]diethylstilbestrol analogs induced binding of radiolabel to DNA only when the compound contained a free hydroxy group (Metzler, M., and Epe, B. (1984) Chem. Biol. Interact. 50, 351-360). We have found that horseradish peroxidase or prostaglandin-H synthase-catalyzed oxidation of diethylstilbestrol in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide caused the generation of an ESR signal indicative of a free radical intermediate (aN = 14.9 G, aH = 18.3 G). The identity of the trapped radical could not be identified on the basis of published hyperfine coupling constants, but the observation that horseradish peroxidase-catalyzed oxidation of 1-naphthol produced an identical ESR signal suggests that the radical was either a phenoxy or phenoxy-derived radical. During horseradish peroxidase-catalyzed oxidation of diethylstilbestrol in the presence of glutathione the thiol reduced the diethylstilbestrol radical to generate a thiyl radical. This was shown by a thiol-dependent oxygen uptake during horseradish peroxidase-catalyzed oxidation of diethylstilbestrol and the observation of an ESR signal consistent with 5,5-dimethylpyrroline-N-oxide glutathionyl radical adduct formation. A diethylstilbestrol analog devoid of free hydroxy groups, namely diethylstilbestrol dipropionate, did not produce an ESR signal above control levels during horseradish peroxidase-catalyzed metabolism in the presence of 5,5-dimethylpyrroline-N-oxide. Thus, free radicals are formed during peroxidatic oxidation of diethylstilbestrol and must be considered as possible determinants of the genotoxic activity of this compound. PMID- 2999151 TI - Regulation of peptide amidation in cultured pituitary cells. AB - The intermediate lobe of the pituitary contains the alpha-amidated peptide alpha melanotropin and high levels of a copper and ascorbate-dependent peptidylglycine alpha-amidating monooxygenase (PAM) capable of converting peptides terminating in -X-Gly into amidated products (-X-NH2). As reported previously, the ability of cultured intermediate pituitary cells to produce alpha-amidated alpha melanotropin declined rapidly. A decline in PAM activity assayed in vitro under optimized conditions failed to account quantitatively for the lack of production of alpha-amidated product, while a 100-fold decline in cellular levels of ascorbate could account for the lack of production of alpha-amidated product. Incubation of intermediate pituitary cultures with ascorbate partially restored the ability of the cells to produce alpha-amidated product without significantly increasing the level of PAM activity. In intermediate pituitary cultures made competent to produce alpha-melanotropin by addition of ascorbate, the actual extent of amidation occurring was modulated by the presence of specific secretagogues (bromocriptine or corticotropin-releasing factor). Cultured anterior pituitary cells showed a similar rapid 3-fold decline in PAM activity assayed in vitro under optimized conditions. Cellular levels of ascorbate also declined rapidly to levels 100-fold below those in the intact anterior pituitary. The addition of ascorbate to the anterior pituitary cultures rapidly restored the enzyme activity assayed in vitro to the levels in the initial cell suspension. Thus, production of amidated product peptide may be regulated by cellular levels of ascorbate, by cellular levels of PAM activity, and by the concentration of specific secretagogues to which the cells are exposed. PMID- 2999152 TI - Relationships within the family of GTP-binding proteins isolated from bovine central nervous system. AB - Four members of a family of GTP-binding proteins (G-proteins) which translate stimulation of extracellular receptors into regulation of intracellular enzymes were isolated from the bovine central nervous system. These proteins were examined for functional similarities and cross-reactivity with antibodies to the G-protein (transducin, Gt) from the photoreceptor system. Two proteins, Gs and Gi, can be distinguished by their respective abilities to stimulate or inhibit adenylate cyclase. The activated alpha subunits of Gt and a fourth member of the family, Go, did not affect this enzyme. Gt was shown to be unique in its ability to stimulate cGMP-dependent phosphodiesterase. While functionally diverse, the G proteins were shown to have some common antigenic properties. Antibodies directed against the beta subunit of Gt recognize the beta 36 subunits of all preparations but not a putative second beta 35 subunit. Antibodies specific for the alpha subunit of Gt did not recognize other alpha subunits when immune blots from sodium dodecyl sulfate gels were examined. However, Go alpha, but not Gs alpha or Gi alpha, reacted strongly with the antibodies when the native subunit was spotted directly. This suggests that Go alpha and Gt alpha have homologous structural determinants. An antiserum that recognized Gt gamma did not recognize gamma subunits from other sources. These data support the proposed diversity of function and similarity of structure among the four G-proteins. The alpha and potentially gamma subunits appear to be responsible for the specificity of function. PMID- 2999154 TI - Purification and properties of UDP-GlcNAc:dolichyl-phosphate GlcNAc-1-phosphate transferase. Activation and inhibition of the enzyme. AB - The GlcNAc-1-P transferase was solubilized from pig aorta microsomal fractions using 0.5% Nonidet P-40. The activity of the solubilized enzyme was stimulated by exogeneously added phospholipids in the order phosphatidylglycerol greater than phosphatidylinositol greater than phosphatidylserine. When the enzyme was stored in 20% glycerol containing 20 micrograms of phosphatidylglycerol/mg of protein, more than 80% of the activity remained after storage for 6 days at 0-4 degrees C. On the other hand, in the absence of the stabilizers, the enzyme lost most of its activity within 24 h. The transferase was purified about 68-fold using ammonium sulfate and DEAE-cellulose fractionation. The DEAE-cellulose chromatography separated a heat-stable factor from the enzyme, which when added back to the partially purified enzyme stimulated about 5-fold. With this partially purified enzyme, the Km for UDP-GlcNAc was found to be 1 X 10(-7) M, and that for dolichyl P about 1 X 10(-6) M. The stimulatory factor increased the Vmax for both UDP GlcNAc and dolichyl-P 5-10-fold, but the Km values remained the same. The pH optimum for the enzyme was between 7.4 and 7.6, and either Mn2+ (1 mM) or Mg2+ (10 mM) was required for optimum activity. The GlcNAc-1-P transferase was also stimulated by the addition of GDP-mannose (or other purine sugar nucleotides) or dolichyl-phosphoryl-mannose to the incubation mixtures. These two compounds acted in different ways on the enzyme since their stimulatory effects were additive. The effect of GDP-mannose was found to be due to protection of the substrate, UDP GlcNAc, from degradation, but the effect of dolichyl-P-mannose remains to be established. In addition, the stimulations shown by phosphatidylglycerol, GDP mannose, and factor, or phosphatidylglycerol, dolichyl-P-mannose, and factor, were all additive, indicating that they were acting at different sites on the enzyme. The transferase was quite sensitive to the action of sulfhydryl reagents such as N-ethylmaleimide or p-chloromercuribenzene sulfonate, and was rapidly inactivated in their presence. The enzyme could be protected to the extent of about 50% when all of the substrates (UDP-GlcNAc, dolichyl-P, Mn2+) were added before the addition of the sulfhydryl reagents. PMID- 2999153 TI - Mitochondrial uncoupling protein from mouse brown fat. Molecular cloning, genetic mapping, and mRNA expression. AB - We have identified cDNAs clones for several cold-inducible mRNAs from the brown adipose tissue of mice. pCIN-1, a plasmid with a 900-base pair insert, encoded the mitochondrial uncoupling protein (UCP) as determined by the ability of the cDNA insert to select, by hybridization, an mRNA that could be translated into a 32,000-Da protein immunoprecipitable with anti-UCP antibodies. Nine tissues were analyzed; however, UCP cDNA hybridized to an mRNA species of 1.6 and 2.0 kilobase pairs only in brown adipose tissue. A maximum induction of 10-fold occurred within 6 h of exposure to cold (5 degrees C). A BamHI restriction fragment polymorphism detected by Southern blot analysis of genomic DNA in recombinant inbred mouse strains allowed us to map the UCP gene to Chromosome 8. The analysis of the UCP gene expression in diabetic (db) and obese (ob) mice maintained at 27 degrees C for 3 days followed by cold exposure for 4 h at 5 degrees C indicated that UCP mRNA levels in mutant mice were unaffected at 27 degrees C and only slightly reduced at 5 degrees C. Accordingly, the inability of diabetic and obese mice to thermoregulate is not associated with a lack of UCP mRNA induction. PMID- 2999156 TI - Quantitative analysis of the accumulation of Zein mRNA during maize endosperm development. AB - In order to characterize the heterogeneity and expression of maize zein genes, we constructed and characterized a cDNA library of endosperm mRNAs. Clones from the library that were of sufficient size to be full-length or near full-length copies of zein mRNA were characterized by restriction enzyme mapping and cross hybridization analysis. Based on these comparisons we found three classes of zein sequences corresponding to proteins of Mr 22,000, five corresponding to proteins of Mr 19,000, and a single one corresponding to a protein of Mr 15,000. Representative clones from these nine groups were used as probes to measure levels of the corresponding mRNAs in developing endosperms. It was found that these groups represent varying amounts of transcripts that range from 2 to 20% of the total endosperm mRNA population. For the Mr 19,000 and Mr 22,000 zein clones there is a correlation between the amount of mRNAs and the apparent number of genes in the genome. The relative level of mRNA for the Mr 15,000 zein was found to be 3 times that of the Mr 22,000 and Mr 19,000 zeins, suggesting that these genes are transcribed at a higher rate during endosperm development or that their mRNAs are more stable. PMID- 2999155 TI - Molecular cloning of cDNAs cognate to genes sensitive to hormonal control in rat liver. AB - Poly(A)-RNAs were prepared from livers of rats treated with hydrocortisone and cycloheximide, then enriched for large mRNAs by successive sucrose gradients and gel electrophoresis. The size-selected RNAs were used as templates for synthesis of double-stranded cDNAs that were cloned in Escherichia coli using the pBR322 plasmid vector. Recombinant plasmids characterized as carrying inserts of potential interest were further analyzed by differential hybridization to mRNAs from untreated and hydrocortisone-treated rats. Seven of the cloned cDNAs were identified as complementary to mRNAs whose content in liver is sensitive to modulation by the steroid. Further screening for hormonal responsiveness revealed that two of the cloned cDNAs hybridize to a 3.4-kilobase mRNA that is rapidly induced in liver by treatment with insulin or cAMP as well as by hydrocortisone; restriction enzyme analysis demonstrated that the cDNAs from these two clones are derived from the same mRNA. In isolated nuclei, the rate of transcription of this mRNA is increased by each of the inducing hormones. This unusually regulated mRNA codes for a protein of 53 kDa on denaturing gels, undergoes rapid intracellular degradation that is prevented by cycloheximide, and appears to be the product of a single copy gene. PMID- 2999157 TI - Nucleotide sequence analysis of zein mRNAs from maize endosperm. AB - A comparison of the DNA and protein sequences of a group of zein cDNA clones reveals that they share extensive sequence homology and probably originated from a common ancestral gene. A comparison of clones corresponding to Mr 22,000 polypeptides shows they are 92% homologous, while five clones corresponding to the Mr 19,000 zeins vary in homology from 75 to 95%. The clones corresponding to the Mr 22,000 proteins are 60-65% homologous to clones encoding the Mr 19,000 zein proteins. A clone corresponding to the Mr 15,000 zein has little homology to either the Mr 22,000 or 19,000 zeins. Clones corresponding to both the Mr 22,000 and 19,000 zeins have two putative polyadenylation signals. S1 nuclease mapping indicates that the first polyadenylation signal following the stop codon is utilized by the Mr 22,000 sequences, while primarily the second polyadenylation signal is utilized by the Mr 19,000 sequences. PMID- 2999158 TI - Newly synthesized G protein of vesicular stomatitis virus is not transported to the Golgi complex in mitotic cells. AB - Newly synthesized G protein of vesicular stomatitis virus is not transported to the surface of cultured mammalian cells during mitosis (Warren et al., 1983, J. Cell Biol. 97:1623-1628). To determine where intracellular transport is inhibited, we have examined the post-translational modifications of G protein, which are indicators of specific compartments on the transport pathway. G protein in mitotic cells had only endo H-sensitive oligosaccharides containing seven or eight mannose residues, but no terminal glucose, and was not fatty acylated. These modifications were indicative of processing only by enzymes of the endoplasmic reticulum (ER). Quantitative immunocytochemistry was used as an independent method to confirm that transport of G protein out of the ER was inhibited. The density of G protein in the ER cisternae was 2.5 times greater than in infected G1 cells treated similarly. Incubation of infected mitotic cells with cycloheximide, which inhibits protein synthesis without affecting transport, did not result in a decrease in the density of G protein in the ER cisternae, demonstrating that G protein cannot be chased out of the ER. These results suggest that intracellular transport stops at or before the first vesicle mediated step on the pathway. PMID- 2999159 TI - Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein. AB - Rotavirus, a non-enveloped reovirus, buds into the rough endoplasmic reticulum and transiently acquires a membrane. The structural glycoprotein, VP7, a 38-kD integral membrane protein of the endoplasmic reticulum (ER), presumably transfers to virus in this process. The gene for VP7 potentially encodes a protein of 326 amino acids which has two tandem hydrophobic domains at the NH2-terminal, each preceded by an in-frame ATG codon. A series of deletion mutants constructed from a full-length cDNA clone of the Simian 11 rotavirus VP7 gene were expressed in COS 7 cells. Products from wild-type, and mutants which did not affect the second hydrophobic domain of VP7, were localized by immunofluorescence to elements of the ER only. However, deletions affecting the second hydrophobic domain (mutants 42-61, 43-61, 47-61) showed immunofluorescent localization of VP7 which coincided with that of wheat germ agglutinin, indicating transport to the Golgi apparatus. Immunoprecipitable wild-type protein, or an altered protein lacking the first hydrophobic sequence, remained intracellular and endo-beta-N acetylglucosaminidase H sensitive. In contrast, products of mutants 42-61, 43-61, and 47-61 were transported from the ER, and secreted. Glycosylation of the secreted molecules was inhibited by tunicamycin, resistant to endo-beta-N acetylglucosaminidase H digestion and therefore of the N-linked complex type. An unglycosylated version of VP7 was also secreted. We suggest that the second hydrophobic domain contributes to a positive signal for ER location and a membrane anchor function. Secretion of the mutant glycoprotein implies that transport can be constitutive with the destination being dictated by an overriding compartmentalization signal. PMID- 2999161 TI - Cell anchorage determines whether mammary tumor virus glycoproteins are processed for plasma membranes or secretion. AB - The subcellular localization of mouse mammary tumor virus (MMTV) glycoproteins was analyzed in infected and cloned rat hepatocarcinoma cells cultured with the MMTV transcriptional inducer dexamethasone. When reacted with protein A-coated erythrocytes in the presence of antisera specific for viral glycoproteins or with fluorescent antisera, only some of the cells acquired surface label. This diversity was dependent on cell anchorage to the substratum. In general, the more rounded, less adherent cells contained the MMTV glycoproteins on their surfaces, whereas the flatter, more adherent cells did not. After a change in adherence, a delay preceded complete remodeling of the plasma membranes. Fluorescent antibody studies of fixed cells and analyses of viral glycoprotein synthesis and shedding using L-[35S]methionine indicated that the different expression of MMTV glycoproteins in round versus flat cells is caused by a switch in posttranslational processing. In round cells, the MMTV-encoded precursor glycoprotein is proteolytically cleaved and then transported to plasma membranes as a complex of two subunits, the smaller being the membrane anchor. In flat adherent cells, the smaller subunit is rapidly degraded in an intracellular organelle and the larger is then secreted into the medium. As indicated by labeling of cells with 125I, the concentrations of several host-encoded plasma membrane components are also influenced by cell anchorage. We propose that this switch in cell surfaces and in secretions dependent upon cell-substratum attachments may be a common control mechanism important for embryogenesis, wound healing, and cancer. PMID- 2999160 TI - In vitro reconstitution of exocytosis from plasma membrane and isolated secretory vesicles. AB - We describe the reconstitution of exocytotic function through recombination of purified cortical secretory vesicles (CVs) and plasma membrane from sea urchin eggs. CVs were dislodged from a cell surface complex preparation by gentle homogenization in an isotonic dissociation buffer, and purified by differential centrifugation. CV-free plasma membrane fragments were obtained by mechanically dislodging CVs from cortical lawn (CL) preparations with a jet of CL isolation buffer. This procedure produced a "plasma membrane lawn" preparation, consisting of plasma membrane fragments attached via their vitelline layer (an extracellular glycocalyx) to a polylysine-coated microscope slide. When freshly prepared CVs were incubated with plasma membrane lawns, CVs reassociated with the cytoplasmic face of the plasma membrane, forming an exocytotically competent, reconstituted cortical lawn (RL). Exocytosis in RLs was monitored by phase-contrast microscopy, and quantitated with a sensitive microphotometric assay. Half-maximal exocytosis in RLs occurred at 18.5 microM free Ca2+; half-maximal exocytosis in control lawns occurred at 5.7 microM free Ca2+. Greater than 90% of the purified CVs that were not attached to a plasma membrane lawn remained intact when bathed in a buffer containing millimolar Ca2+. This result excluded the possibility that Ca2+ triggered CV lysis was responsible for our observations, and confirmed that the association of CVs with the plasma membrane was required for exocytosis in RLs. Evidence that the Ca2+-stimulated release of CV contents in CLs and RLs is the in vitro equivalent of exocytosis was obtained with an immunofluorescence-based vectorial transport assay, using an antiserum directed against a CV content protein: stimulation of RLs or partially CV-depleted CLs with Ca2+ resulted in fusion of the CV and plasma membranes, and the vectorial transport of CV contents from the cytoplasmic to the extracytoplasmic face of the egg plasma membrane. PMID- 2999162 TI - Differential effects of the tumor promoter phorbol-12-myristate-13-acetate on the morphological and biochemical differentiation of N-18 mouse neuroblastoma cells. AB - The phorbol ester tumor promoter phorbol-12-myristate-13-acetate (PMA) was found to have differential inhibitory effects on the expression of morphological and biochemical differentiation of N-18 mouse neuroblastoma cells. PMA completely inhibited neurite extension and associated growth characteristics and partially inhibited the increased expression of R1 cAMP-binding protein; PMA had no effect on the induction of acetylcholinesterase activity in cells prompted to differentiate either by treatment with 1 mM dibutyryl cAMP or by serum deprivation. 4-alpha-Phorbol-12, 13-didecanoate, an inactive analogue of phorbol ester tumor promoter, was without effect. The implications of these findings concerning the mechanism of action of phorbol ester tumor promoters in the control of cell differentiation are discussed. PMID- 2999163 TI - Insulin stimulation of glucose transport and metabolism in a human Wilms' tumor derived myoblast-like cell line: modulation of hormone effects by glucose deprivation. AB - The effects of insulin and glucose on parameters of metabolism were investigated in myoblast-like (MBL) cells, a human myoblast-like cell line derived from a Wilms' tumor. Insulin responses were studied after 4 hr pre-incubation in serum free media, with or without 5 mM glucose. Insulin was added during the last 2 hr. Glucose starvation markedly increased basal glucose transport (measured as 2 deoxyglucose uptake) as well as the net uptake of [14C]glucose and [14C]glucose incorporation into glycogen. Insulin stimulated net glucose uptake and incorporation into glycogen in a dose-dependent manner in glucose-fed and starved cells. These insulin responses were markedly enhanced in glucose-starved cells. Insulin accelerated 2-deoxyglucose transport in glucose-fed cells but did not further stimulate basal glucose transport in glucose-deprived cells. Insulin increased the incorporation of [3H]leucine into protein in glucose-fed or starved MBL cells equally. The dose of insulin required for half-maximal insulin responses was similar for all parameters studied. Cycloheximide did not prevent the increased basal glucose incorporation in glucose-starved cells, but markedly inhibited the insulin response, while in glucose-fed cells, cycloheximide stimulated basal glucose incorporation. We conclude that MBL cells resemble fibroblasts in their insulin-independent stimulation of glucose transport in response to glucose-deprivation; when provided with glucose, they respond to insulin like fibroblasts. However, after brief glucose-starvation, the stimulated glucose transport system is no longer insulin-responsive in MBL cells, while pathways leading to the synthesis of macromolecules demonstrate preserved or enhanced stimulation by insulin, suggesting that these cells may serve as models to study the regulation of receptor-response coupling by the metabolic milieu. PMID- 2999165 TI - Separation of murine megakaryocytes and their progenitors on continuous gradients of Percoll. AB - Murine bone marrow was separated on continuous Percoll density gradients to analyze the distribution of cells of the megakaryocyte lineage. Eighty-seven percent of the recovered megakaryocytes were found in fractions of density less than 1.058 g/cm3, with 63% of these cells found between 1.020 and 1.036 g/cm3. When megakaryocytes were classified according to size, 92% of the large (greater than or equal to 18 micron) acetylcholinesterase (AchE) positive cells were found in the least dense fractions (1.016-1.039 g/cm3), whereas 86% of the small (less than or equal to 10.6 micron) AchE positive cells were found in fractions of higher density (1.039-1.078 g/cm3). The distribution of enzymatic AchE activity of the separated fractions corresponded to the location of the histochemically positive cells. When ploidy measurements were made of various fractions, most of the high ploidy (32N and 64N) cells were found at low density (1.028-1.036 g/cm3), whereas no cells greater than 4N were found at density greater than 1.071 g/cm3. Thus, large AchE positive cells and the cells of highest ploidy were found at lower densities of Percoll, while small AchE positive cells and cells of low ploidy were found at higher densities. An exception to this inverse relationship was found in fractions of lowest density (less than 1.030 g/cm3) where an anomalous distribution of size and ploidy was found. The majority of megakaryocytic colony-forming cells (CFU-MK) were found at high density, as were the granulocyte-macrophage colony-forming cells (CFU-GM; approximately 1.074 g/cm3). The density distribution of the incorporation of tritiated thymidine into liquid marrow cultures was concordant with the high density distribution of colony-forming cells. The data show that megakaryocytic maturity and Percoll density varies inversely and that fractionation of marrow on continuous Percoll gradients may be a useful method for the separation and/or enrichment of megakaryocytes at different stages of differentiation. PMID- 2999166 TI - Effects of EGF and thrombin on inositol-containing phospholipids of cultured fibroblasts: stimulation of phosphatidylinositol synthesis by thrombin but not EGF. AB - The effects of growth factors on inositol-containing phospholipids were investigated to test the hypothesis that alterations in their metabolism are involved in mitogenic stimulation. Thrombin and EGF stimulated comparable increases in the synthesis (30-50%) and degradation (20-40%) of phosphatidylinositol 4-monophosphate (DPI) and phosphatidylinositol 4,5 bisphosphate (TPI) in a cell line which is mitogenically responsive to both growth factors. The increases in synthesis were time and dose dependent in a manner which was consistent with their involvement in mitogenesis; the increases were observed only under conditions where a mitogenic response occurred. While it has been suggested that an increased synthesis of phosphatidylinositol (PI) is coupled to the stimulation of DPI and TPI synthesis, we found that thrombin stimulated an early synthesis PI but EGF did not. To further evaluate the involvement of PI in thrombin-stimulated cell division we determined the time and dose dependence of the stimulated PI synthesis and found that it also occurred in a manner which was consistent with its involvement in thrombin-stimulated cell division. Furthermore, the stimulated PI synthesis was not observed with nonmitogenic proteases or in cell lines which were not responsive to thrombin. These results demonstrate that the metabolism of DPI and TPI appears closely related to the mitogenic response generated by EGF and thrombin. However, an early stimulation of PI synthesis is not coupled to this metabolism and is not necessary for mitogenic stimulation by EGF. Thus, a stimulation of PI synthesis is not a valid measure of alterations in inositol-containing phospholipids and what has been termed the "PI response." PMID- 2999164 TI - Characterization of permeation pathways in the plasma membrane of human erythrocytes infected with early stages of Plasmodium falciparum: association with parasite development. AB - Human intraerythrocytic malarial parasites (Plasmodium falciparum) induce permeability changes in the membrane of their host cells. The differential permeability of infected erythrocytes at various stages of parasite growth, in combination with density gradient centrifugation, was used to fractionate parasitized cells according to their developmental stage. By this method it was possible to obtain cell fractions consisting essentially of erythrocytes infected with the youngest parasite stage (i.e., rings). These preparations were used for the measurement of transport of various solutes. It is shown that permeabilization of host erythrocyte membrane appears as early as 6 h after parasite invasion of the erythrocyte and increases gradually with parasite maturation. Since the selectivity for several different solutes and the enthalpy of activation of transport remain unaltered with maturation-related increase of permeability, it is concluded that the number of transport agencies in the host cell membrane increases with parasite maturation. Evidence is presented to indicate the need for parasite protein synthesis as an essential factor for the generation of the new permeability pathways. PMID- 2999168 TI - Cellular redistribution of beta-adrenergic receptors in a human astrocytoma cell line: a comparison with the epidermal growth factor receptor in murine fibroblasts. AB - The redistribution of beta-adrenergic receptors (beta-AR) during agonist-induced desensitization has been compared to the process of receptor-mediated endocytosis of epidermal growth factor (EGF) in human astrocytoma cells (1321N1). [125I]EGF exhibited saturable binding to high affinity (KD = 1-2 nM) receptor sites on intact 1321N1 cells. [125I]EGF was found to internalize rapidly using an acid wash technique to remove surface bound hormone. Sucrose density gradient fractionation following exposure to EGF revealed a redistribution of EGF binding sites from high density (heavy peak) to low density (light peak) regions of the gradient. The light peak binding probably represents EGF in internalized vesicles formed during endocytosis. Low temperature (4 degrees C) or the presence of the lectin concanavalin A (con A) inhibited this ligand-induced movement of EGF receptors. When cells were incubated simultaneously with EGF and the beta-AR agonist isoproterenol, both receptors were found to co-migrate in the low density regions of sucrose gradients. No evidence of heterologous ligand-induced receptor endocytosis was found. These results suggest that the EGF receptors and beta-AR are processed in parallel by 1321N1 cells. PMID- 2999167 TI - Transferrin receptor regulation is coupled to intracellular ferritin in proliferating and differentiating HL60 leukemia cells. AB - Cultured myeloid leukemia cells display transferrin receptors but decrease receptor display after differentiation induction or accumulation of intracellular iron. To determine whether regulation of transferrin receptors and ferritin were linked under these disparate conditions, serum-free and fetal bovine serum (FBS) cultures of HL60 promyelocytic leukemia cells were used to investigate relationships between transferrin receptor display and intracellular ferritin. Using 125I-transferrin binding and immunofluorescence staining for transferrin receptors, HL60 cells cultured in serum-free, transferrin-free medium expressed fewer transferrin receptors and contained increased ferritin when compared to cells cultured with FBS or transferrin supplemented, serum-free medium. When placed in medium containing transferrin, cells previously grown in transferrin free medium rapidly re-expressed transferrin receptors and decreased their ferritin content. HL60 cells induced to differentiate into granulocytes or macrophages also decreased transferrin receptor display and increased their ferritin content. Transferrin receptor display and ferritin content in both proliferating and differentiating myeloid leukemia cells are inversely related and their regulation is closely linked. Regulation of transferrin receptor display and ferritin synthesis may be important events regulating myeloid cell growth and differentiation. PMID- 2999170 TI - Chromatography of radiolabelled anions using reversed-phase liquid chromatographic columns. PMID- 2999169 TI - High-performance liquid chromatography of in vitro synthesized poly(ADP-ribose) on ion-exchange columns, separation of oligomers of varying chain length and estimation of apparent branching. AB - Separated macromolecular fractions of in vitro synthesized poly(ADP-ribose) by liver nuclei were subjected to ion-exchange chromatography in a programmed high performance liquid chromatographic elution system. The effects of ionic strength, pH and temperature on the separation of poly(ADP-ribose) chains were determined. Short chain oligomers (up to n = 11) were fractionated into individual components by baseline separation. Each fraction was analyzed for chain length. Trace amounts of Ado(P)Rib(P)Rib(P) found in phosphodiesterase digests were taken as indication of apparent branching. In phosphodiesterase digests of the shorter oligomers, besides traces of the above component, two other digestion products were also observed, presumably representing oligomer termini, one terminal fragment being dominant in short oligomers. Medium and long chain oligomers were partly resolved to individual components, and especially the long oligomers exhibited marked temperature dependent elution patterns. Apparent branching increased with increasing chain length up to about 3% for n = 44 and components presumably indicating termini diminished to mere traces. The adenine spectra of all fractions identified individual components. PMID- 2999172 TI - Solid phase extraction system for vitamin D and its major metabolites in human plasma. AB - A new procedure using C18 and silica cartridges for the extraction and subsequent separation of vitamin D and its major metabolites from plasma has been developed and compared to a conventional extraction procedure with respect to lipophilic material extracted as evaluated by high-performance liquid chromatographic profiles. The C18 cartridges were efficient in extracting all compounds tested while subsequent chromatography of the extract on silica cartridges was effective in resolving vitamin D and its metabolites based on increasing polarity. High performance liquid chromatographic profiles of each silica cartridge fraction clearly demonstrated that the newly conceived solid phase extraction was superior to conventional extraction methods with respect to cleanliness of sample fractions. This difference in lipophilic load between the new and conventional extraction systems was most apparent in the vitamin D and 25-hydroxyvitamin D containing fractions. The new extraction system can be used when total extraction and subsequent analysis of vitamin D and its major metabolites is desired. PMID- 2999171 TI - Quantitation of sulfidopeptide leukotrienes by reversed-phase high-performance liquid chromatography. PMID- 2999173 TI - Detection and measurement of fat cell-binding immunoglobulins: a new method applicable to the diagnosis and study of Graves' disease. AB - This report describes a new method for detecting and quantitating those immunoglobulins G (IgG) in serum that are related to Graves' disease. The method is based on previous observations which indicate that the guinea pig fat cell membrane (FCM) is capable of binding Graves'-specific IgG, but does not bind the IgG common to Graves' disease and Hashimoto's disease, such as antimicrosomal antibodies. Crude FCM preparations were iodinated by a lactoperoxidase technique and were then treated with Triton X-100 to yield a solubilized radioiodinated FCM (SFCM) preparation. SFCM, which retained bovine (b) TSH binding and Graves'-IgG binding properties, provided a radioactively labeled receptor with which to test for the presence of fat cell-binding IgG (FBI) in immunoprecipitates prepared by reacting these IgG with antibody against the Fc fragment of human IgG. FBI values (percentage of added SFCM bound to immunoprecipitate; mean + SD) in IgG from 16 patients with thyrotoxicosis caused by Graves' disease (6.0 +/- 1.7) were completely separated from those in IgG from 16 normal subjects (0.4 +/- 0.3). IgG from 2 hypothyroid patients with Hashimoto's disease, which were strongly positive in the TSH binding inhibition (TBI) assay, yielded FBI values within the range in Graves' disease, but values in TBI-negative IgG from 15 other patients with Hashimoto's disease were normal (0.0 +/- 0.9). Moderately false positive FBI values were found in the IgG of 15 patients with rheumatoid arthritis or systemic lupus erythematosis, all rheumatoid factor positive, 3 of which were also TBI positive. In IgG from Graves' disease and those from patients with TBI-positive collagen-vascular disease, binding of SFCM was inhibited by bTSH in a dose dependent manner. As with binding of TSH to thyroid plasma membranes, similar but less potent inhibition of binding of IgG to SFCM was produced by LH, FSH, and hCG, but not by insulin, glucagon, PRL, or ACTH. FBI values in TBI-negative IgG from patients with collagen-vascular disease were also decreased by TSH, but higher concentrations of bTSH were required. In 40 IgG from among the various clinical groups tested, a significant correlation was found between FBI values and TBI activity (r = 0.48; P less than 0.01). In addition, among 10 IgG from Graves' disease and 6 from collagen-vascular disease patients, a very close correlation (r = 0.89; P less than 0.001) was noted between their TBI activity and the extent to which their FBI values were decreased by a standard concentration of bTSH.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2999175 TI - Receptor-positive hereditary resistance to 1,25-dihydroxyvitamin D: chromatography of hormone-receptor complexes on deoxyribonucleic acid-cellulose shows two classes of mutation. AB - We used cultured skin fibroblasts from patients with hereditary resistance to 1,25-dihydroxyvitamin D [1,25-(OH)2D] and normal hormone binding to soluble extract from cells [i.e. receptor-positive resistance to 1,25-(OH)2D] to characterize DNA binding of the receptor for 1,25-(OH)2D. Occupied receptor was generated by incubating soluble extracts from cells with [3H]1,25-(OH)2D3; occupied receptor was applied to columns of DNA-cellulose and then eluted with linear gradients of KCl. Occupied receptors of cells from other sources eluted as a single peak at 0.20-0.26 M KCl; this elution pattern was independent of tissue (skin, breast cancer, or osteosarcoma) or species (human or rat) of origin of the receptors. With cells from two kindreds in whom there was mildly decreased localization of the hormone-receptor complex to the nucleus in vitro, occupied receptor interacted abnormally with DNA-cellulose (elution at 0.09-0.13 M KCl vs. normal at 0.20-0.26 M KCl); this suggested mutation(s) that affected a DNA binding domain of the receptor in these two kindreds. With receptor-positive cells from two other kindreds in whom there was unmeasurable hormone localization to the nucleus, the elution pattern of occupied receptors from DNA-cellulose was normal; this suggested mutation(s) which did not affect the same DNA-binding site. We conclude that our demonstration of two distinct elution profiles from DNA-cellulose reflects two independent classes of mutation, either of which can cause receptor-positive resistance to 1,25-(OH)2D. PMID- 2999176 TI - Characterization of a gonadotropin-releasing hormone receptor site in term placenta and chorionic villi. AB - The properties of GnRH receptor sites in the human placenta were analyzed by binding studies performed in particulate and solubilized receptor preparations. The binding affinities (Ka) of the membrane receptor of term placenta for the GnRH superagonists [D-Lys6]- and [D-Ala6]des-Gly10- GnRH-N-ethylamide were 1.6 X 10(6) and 5.4 X 10(5) M-1, respectively. The binding affinities of native GnRH and a potent GnRH antagonist were similar to those of the superagonists (1.1 and 2.0 X 10(6) M-1, respectively). The placental sites could be solubilized by extraction with the detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane sulfonate, with retention of 40-45% of the specific binding activity, though with a significant decrease in binding affinity. The sites were also solubilized with sodium dodecyl sulfate after covalent labeling with a photoreactive 125I-labeled [D-Lys6] GnRH agonist derivative. Analysis of the solubilized hormone-receptor complex on polyacrylamide gradient gels showed a single band of 53,700 mol wt, similar to the mol wt of the pituitary GnRH receptor in other species. It is clear that the placental receptor differs markedly from the GnRH receptor of the pituitary gland in its low binding affinity and lack of selectivity for GnRH analogs. However, it is possible that the human placental receptor for GnRH could serve as a low affinity regulatory site for locally formed GnRH or related low affinity regulatory site for locally formed GnRH or related peptides within the placenta, and that the placental GnRH system has a significant role in the maintenance of pregnancy. PMID- 2999174 TI - A new serum-based assay for fat cell-binding immunoglobulins: application to the detection of the thyrotropin receptor antibodies of Graves' disease. AB - In an accompanying report, we describe a new test for detecting and quantitating those immunoglobulins G (IgG) related to the presence of hyperthyroidism in Graves' disease. In this procedure, an immunoprecipitate formed between the test IgG and antiserum against the Fc portion of the human IgG is incubated with 125I labeled solubilized guinea pig fat cell membranes (SFCM). The proportion of added 125I bound to the immunoprecipitate is a measure of fat cell-binding IgG (FBI) in the test preparation. In this report we describe an improvement of the basic technique that permitted its use with serum. Here, the test specimen of serum was allowed to interact with anti-Fc IgG coupled to beads of Sepharose-4B. SFCM were then added, and the test proceeded as in the IgG-based procedure. Serum FBI values were decreased in a dose-dependent manner by bovine (b) TSH and, with lesser potency, by other glycoprotein hormones (bLH, bFSH, and hCG). Further, in experiments with sera and their corresponding IgG fractions from patients in the various groups studied, both individual serum FBI values and the extent to which they were decreased by the addition of bTSH were closely correlated with the TSH binding inhibitory (TBI) activity of the corresponding IgG fractions. These findings indicate that FBI values in the serum-based test, as in the IgG-based test, reflect mainly the concentration of IgG that bind to the TSH receptor in SFCM. Two entirely separate evaluations of the serum-based FBI test were carried out. In the first, in sera from 21 patients with Graves' hyperthyroidism, FBI values (mean +/- SD, 1.6 +/- 0.6%) were completely separated from those in normal sera (-0.6 +/- 0.3%; n = 20), TBI-negative sera from patients with Hashimoto's disease (-0.3 +/- 0.3%; n = 21), and sera from patients with collagen-vascular disease (0 +/- 0.3%; n = 16). Positive results were also obtained in sera from 2 patients with TBI-positive Hashimoto's disease and 2 with diabetes mellitus associated with anti-insulin receptor antibodies (type B diabetes mellitus). In the latter, however, abnormal FBI values were not decreased by bTSH, but were decreased by insulin, which, conversely, had no effect on the elevated FBI values found in Graves' hyperthyroidism. In the second evaluation, FBI values were measured in 34 sera from normal subjects and 38 sera from patients with hyperthyroidism due to Graves' disease; 14 samples in the former group and 16 in the latter group were studied without knowledge of the diagnosis.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2999177 TI - The effect of serum prolactin on plasma adrenal androgens and the production and metabolic clearance rate of dehydroepiandrosterone sulfate in normal and hyperprolactinemic subjects. AB - Hyperprolactinemic patients may have increases in plasma dehydroepiandrosterone (DHA) and dehydroepiandrosterone sulfate (DHAS). We examined the effect of lowering serum PRL with bromocriptine or pituitary surgery on the serum concentrations of adrenal androgens and on the production rate (PR) and MCR of DHAS in eight hyperprolactinemic women (HP). We also examined the effect of bromocriptine therapy on adrenal androgens in five normal men. Serum DHAS was elevated in HP compared to normal women (mean +/- SEM, 254 +/- 28 vs. 182 +/- 13 microgram/dl; P less than 0.04). Serum DHA and androstenedione (delta 4) in HP were not significantly different from normal. Serum PRL fell from 160 +/- 16 to 37 +/- 9 ng/ml during or after treatment. Mean 24-h serum DHAS fell from 198 +/- 30 to 106 +/- 17 micrograms/dl (P less than 0.001) with treatment, without a change in the mean 24-h serum cortisol concentration (6.2 +/- 0.4 vs. 6.6 +/- 0.4 micrograms/dl). Thus, the DHAS to cortisol (DHAS/F) ratio fell significantly (32 +/- 5 to 17 +/- 4; P less than 0.001). This was also true of the DHAS/F ratio during ACTH stimulation (8 +/- 1 to 6 +/- 1; P less than 0.02). Similar changes were found in basal and ACTH-stimulated DHA/F ratios, whereas the basal and ACTH stimulated delta 4/F ratios did not change significantly with treatment. Treatment lowered the PR of DHAS from 27 +/- 5 to 17 +/- 3 mg/24 h (P less than 0.03) and increased the DHAS MCR from 16 +/- 2 to 21 +/- 3 liters/24 h (P less than 0.01). Bromocriptine treatment of normal men lowered serum PRL from 15 +/- 2 to less than 2.5 ng/ml. There were no significant changes in the basal and ACTH stimulated serum DHAS/F, DHA/F, or delta 4/F ratios or DHAS PR and MCR during bromocriptine therapy. The failure of bromocriptine to significantly alter these steroids in normal men suggests that bromocriptine was not directly responsible for the changes in HP treated with this drug. A mechanism for the increased PR of DHAS in HP was sought by examining the serum concentrations of the steroid biosynthetic intermediates relevant to DHAS production. Lowering serum PRL was associated with a decrease in basal and ACTH-stimulated 17-hydroxypregnenolone/17 hydroxyprogesterone and DHA/delta 4 ratios, suggesting an increase in 3 beta hydroxysteroid dehydrogenase/delta 4,5-isomerase activity. However, increased gonadal secretion of the delta 4-steroids may have occurred with the fall in serum PRL.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2999178 TI - Stimulation of insulin secretion by a rapid intravenous calcium infusion in patients with beta-cell neoplasms of the pancreas. AB - The effects of calcium on fasting plasma insulin and glucose levels were compared in 16 normal subjects and 11 patients with beta-cell neoplasms of the pancreas. Calcium was administered iv either as a rapid calcium infusion (RCI; 2 mg/kg in 1 min) or as a long calcium infusion (LCI; 12 mg/kg in 3 h). In normal subjects, the RCI produced a rise in mean plasma insulin from 11 +/- 1 (+/- SEM) microU/ml basally to a peak of 18 +/- 2 microU/ml (P less than 0.001). No consistent pattern of change in insulin levels occurred during the LCI, and plasma glucose levels did not change significantly with either test. In the patients with beta cell neoplasms, the RCI resulted in a rapid increase in mean plasma insulin from 36 +/- 6 microU/ml to a peak level of 312 +/- 67 microU/ml (P less than 0.002). With the LCI, a more gradual rise in insulin from 35 +/- 11 to 92 +/- 36 microU/ml occurred (P less than 0.002). The mean increase in insulin in the patients with beta-cell neoplasms was significantly greater for the RCI than for the LCI (P less than 0.01). Pronounced increments in plasma insulin occurred in all 11 patients after the RCI, but in only 3 of 8 patients during the LCI. Plasma glucose levels declined significantly from 69 +/- 7 to 56 +/- 8 mg/dl during the RCI (P less than 0.05) and from 69 +/- 8 to 49 +/- 7 mg/dl during the LCI (P less than 0.005). Symptomatic hypoglycemia developed in 3 patients during the LCI but did not occur after the RCI. These data indicate that calcium is a more effective insulin secretagogue in patients with beta-cell neoplasms when administered as an RCI than as an LCI, and suggest that the RCI may be a useful test for the diagnosis of insulin-secreting tumors. PMID- 2999179 TI - Evidence for normal antidiuretic responses to endogenous and exogenous arginine vasopressin in patients with guanine nucleotide-binding stimulatory protein deficient pseudohypoparathyroidism. AB - In six patients with pseudohypoparathyroidism (PHP) who were deficient in guanine nucleotide-binding stimulatory protein (Ns) activity, the response to endogenous arginine vasopressin (AVP) was tested during water deprivation. Hourly plasma osmolality (Posm), urinary osmolality (Uosm), and urinary AVP (UAVP) values were compared to those in normal subjects. The Uosm vs. Posm and the UAVP vs. Uosm relationships of the patients were all within the normal range. Four patients with Ns-deficient PHP were subjected to maintained water loads and infused with AVP at three different rates for 1 h each to assess their responses to exogenous AVP. Urinary volume and osmolality values from the final 30 min of each infusion rate were measured. All volume values except 1 were within 1.6 SD of normal, and all osmolality values except 1 were within 1.1 SD of normal. In conclusion, these studies indicate that these six patients with Ns-deficient PHP are not resistant to the antidiuretic (cAMP-mediated) action of endogenous or exogenous AVP, in contrast to the previously documented resistance of patients with Ns-deficient PHP to the actions of PTH, TSH, glucagon, and gonadotropins. PMID- 2999181 TI - Characterization of insulin, insulin-like growth factors I and II, and growth hormone receptors on human leukemic lymphoblasts. AB - Receptors for insulin, insulin-like growth factors I and II (IGF-I and IGF-II), and human GH were studied in 6 T- and 12 B-lymphoblast cell lines isolated from patients with lymphoid malignancies. These cell lines have been maintained in continuous culture with stable chromosome, immunophenotype, and enzyme characteristics for an 8- to 21-month period. Four of 6 T-cell lines expressed IGF-I, but not insulin, receptors. One T-cell line bound only insulin, and 1 T cell line bound both hormones. Conversely, 9 of 12 B-cell lines had insulin, but not IGF-I, receptors. Two of these lines bound both hormones, and 1 line did not bind either hormone. IGF-II binding was less than 1.5%/10 million cells for all lines and was less than 1% for 13 of the 18 lines. Specific binding of GH was undetectable in all cell lines. Time, temperature, and pH dependence of peptide binding were characterized for the T-cell IGF-I receptor and the B-cell insulin receptor and were consistent with previously described models for these receptors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cross linked receptors revealed an apparent mol wt greater than 300K unreduced and a 130K binding subunit after dithiothreitol reduction for both the T-cell IGF-I and the B-cell insulin receptor. These cell lines thus provided a unique opportunity for the study of growth factor receptors in fully characterized clonal populations of human lymphoblasts. We conclude that specific receptors for IGF-I and insulin are present on T- and B-lymphoblasts; divergence of these receptors appears to be a characteristic of lymphoblasts, since T-cells preferentially expressed IGF-I receptors and B-cells expressed insulin receptors; and IGF-II binding was relatively low, while specific GH binding was undetectable on both T- and B-lymphoblasts. These results suggest that insulin and IGF-I may play a role in lymphocyte differentiation and metabolism. PMID- 2999180 TI - Impairment of prednisolone disposition in women taking oral contraceptives or conjugated estrogens. AB - Companion studies were designed to determine the effects of oral contraceptives and conjugated estrogens on the pharmacokinetics of prednisolone. Twenty-four normal women entered the studies, including six young women taking oral contraceptives and six age-matched control women, and six postmenopausal women receiving conjugated estrogens and six age-matched postmenopausal women. All received 0.53 mg/kg prednisolone phosphate, iv. Significant decreases (P less than 0.05) in the clearance and volume of distribution and significant increases in the half-life were found for both total and unbound prednisolone in the women taking oral contraceptives compared to values in the young control women. A significant decrease in the unbound clearance and increases in the total and unbound half-lives of prednisolone were found in the women receiving conjugated estrogens compared to values in the postmenopausal control women. Total clearance and volume of distribution were unchanged by conjugated estrogen therapy. Administration of prednisolone to women receiving estrogen-containing oral contraceptives or conjugated estrogens results in exposure of these women to increased concentrations of unbound prednisolone for increased periods of time. Increases in the pharmacological and toxic effects of prednisolone might be expected in these women. PMID- 2999182 TI - Relationships among vitamin D, 25-hydroxyvitamin D, and vitamin D-binding protein concentrations in the plasma and milk of human subjects. AB - We measured plasma and milk concentrations of vitamin D2, vitamin D3, 25 hydroxyvitamin D2 (25OHD2), 25-hydroxyvitamin D3 (25OHD3), and vitamin D-binding protein (DBP) in a group of lactating women. All vitamin D compounds were quantitated using competitive protein binding assay, while DBP concentrations were determined by rocket electrophoresis. Vitamin D3 was the most abundant vitamin D compound in human milk, followed by vitamin D2, 25OHD3, and, finally, 25OHD2. The average vitamin D activity in milk was between 33-68 IU/liter, depending on the criterion of biological activity used. DBP concentrations in milk were approximately 3% of those in plasma. Significant relationships were found between plasma and milk levels for all vitamin D compounds. The milk to blood concentration ratio was greatest for vitamin D2, followed by vitamin D3, 25OHD2, and 25OHD3. (Thus, the parent compounds gained access into milk in a much more efficient fashion than their 25-hydroxy metabolites. It is postulated that this differential translocation is controlled by the DBP in the circulation.) There was no significant correlation between plasma and milk DBP concentrations, nor were milk DBP concentrations related to the vitamin D content of milk. This investigation supports the concept that the nutritional status of lactating mothers affects the vitamin D sterol potential of her milk which, in turn, would likely have an effect on the vitamin D status of her nursing infant. PMID- 2999183 TI - Human pituitary tissue secretes a potent growth factor for chondrocyte proliferation. AB - We report the secretion from human pituitary tumor fragments in organ culture of a potent mitogen for chondrocyte proliferation. Primary human pituitary cell and organ cultures were established from pituitary fragments obtained from patients with acromegaly, prolactinomas, and nonfunctional adenomas. The conditioned culture medium contained a mitogenic factor(s) that stimulated rabbit fetal chondrocyte proliferation, causing up to an 8-fold increase in cell number when added to Ham's F-10 medium in the presence of 10% fetal bovine serum. Blood leaking into the surgical field after the adenomectomy is known to contain very high concentrations of pituitary hormones. Serum samples, obtained from this venous "ooze" collected at the site of pituitary surgery, also were found to contain chondrocyte growth-promoting activity. Some venous serum samples stimulated chondrocyte proliferation in a dose-dependent manner down to a 1:10 dilution of 1 microliter serum, indicating that the material being secreted was very potent indeed. Gel filtration on Sephadex G-100 and analytical gel isoelectric focusing of culture media or serum samples from the pituitary fossa demonstrated that the growth factor secreted from the pituitary tumor fragments as well as from the venous serum is similar, if not identical, to chondrocyte growth factor (mol wt, 43,000; pI 7.6-7.9) purified from human pituitaries collected at autopsy. These results suggest that the chondrocyte growth-promoting factor(s) may not only be secreted by pituitary tumor fragments but by normal human pituitary tissue as well. PMID- 2999184 TI - Sulfohydrolase activity for estrone sulfate and dehydroepiandrosterone sulfate in human fetal membranes and decidua around the time of parturition. AB - We examined the distribution and kinetic parameters of sulfohydrolase activity in human amnion, chorion, and decidua using estrone sulfate (E1S) and dehydroepiandrosterone sulfate as substrates. Amnion contained low levels of sulfatase activity. Chorion had active sulfohydrolase activity for both substrates, but a significantly greater maximum velocity (Vmax) for E1S. The Km was not different between the two substrates. However, there was a slight but statistically significant decrease in Km and increase in Vmax for sulfohydrolase activity using E1S in chorion from patients delivering vaginally after the spontaneous onset of labor compared to those delivering by elective cesarean section before the onset of labor but at a similar gestational age. Decidua possessed sulfohydrolase for E1S with similar Km and Vmax as chorion. There were no changes occurring around the onset of labor. Using dehydroepiandrosterone sulfate as substrate, the decidua had a similar Km as the chorion, but its Vmax was significantly less. In both tissues for both substrates, the enzyme had highest specific activity in the 105,000 X g pellet, with almost no activity in the soluble fraction. The greatest total sulfohydrolase activity was contained in the 800 X g pellet despite several methods of homogenization and washing of the 800 X g pellet. We conclude that the sulfohydrolase activity of human chorion and decidua may be an important factor in regulating free steroid levels within the pregnant uterus. The significant change in the kinetic parameters of E1S sulfatase may partially explain the increased ability of chorion to hydrolyze E1S which occurs in association with the spontaneous onset of labor. PMID- 2999185 TI - [Biochemical study of estrogen receptor and immunohistochemical localization of endogenous estradiol in human gastric cancer]. PMID- 2999186 TI - Comparison of six methods for the detection of antibody to cytomegalovirus. AB - Five commercial assays were compared to a standardized complement fixation (CF) test for the detection of antibody to cytomegalovirus. Two hundred and thirty serum specimens were analyzed. In addition, nine pairs of acute- and convalescent phase sera were tested by two of the commercial assays. The assays were compared as to sensitivity, specificity, and positive and negative predictive value, as well as incidence of false-positive and -negative results. Samples which did not agree in all the assays were retested and tested with an indirect fluorescent antibody assay. Of 228 specimens, 103 (45.2%) were positive by CF. Of the 230 samples, 2 (0.9%) were inconclusive by CF and readable in the other assays. Of the 230 specimens, 97 (42.2%) were positive by an enzyme immunoassay (EIA; Litton Bionetics), 100 (43.5%) were positive by a second EIA (Abbott Laboratories), 104 (45.2%) were positive by a third EIA (M. A. Bioproducts). One hundred and eight (47.0%) were positive by indirect hemagglutination (IHA; Cetus Corporation), and 110 (47.8%) were positive by latex agglutination (LA; Hynson, Westcott and Dunning). Sensitivity and specificity were similar with all the assays (93 to 100%). The greater numbers of positive results by IHA and LA were confirmed by repeat CF testing at less than 1:8 dilution, and by indirect fluorescent-antibody assay. Acute- and convalescent-phase serum pairs showed a significant rise in antibody titer when tested by anticomplement immunofluorescence, IHA, and LA. There was good agreement among the assays, with LA having the highest sensitivity. PMID- 2999187 TI - Effect of heat and fresh human serum on the infectivity of human T-cell lymphotropic virus type III evaluated with new bioassay systems. AB - MT-4 cells, which are a human T-cell lymphotropic virus type I (HTLV-I)-positive cell line highly permissive to HTLV-III infection, were used to detect the biologically active virus. For quantitation of the virus, induction of HTLV-III specific antigen(s) and inhibition of DNA synthesis in infected MT-4 cells were assessed by indirect immunofluorescence and by a proliferation assay measuring [3H]thymidine uptake, respectively. HTLV-III was fully inactivated by treatment at 56 degrees C for 30 min. It was not inactivated by treatment with fresh anti HTLV-III-negative serum. Thus, these assay systems with MT-4 cells would be useful in further studies on acquired immune deficiency syndrome. PMID- 2999188 TI - In vitro protective effect of bacteria-derived bovine alpha interferon I1 against selected bovine viruses. AB - We used bacteria-derived bovine alpha-interferon I1 (Bo IFN-alpha I1) to study its antiviral effect in a bovine turbinate cell line on bovine diarrhea virus, infectious bovine rhinotracheitis virus, parainfluenza 3 virus, and pseudorabies virus. We based our study upon replicate tests for each strain by using a block titration system with various concentrations of Bo IFN-alpha I1 against various concentrations of virus. The data were compiled in two-axis tables (replicate X concentration) and were statistically analyzed by the Spearman-Karber method. An increase in the concentration of Bo IFN-alpha I1 enhanced its protective effect against every test virus strain. Bo IFN-alpha I1 had a marked in vitro effect on the bovine diarrhea viral strains. It demonstrated less protection against the pseudorabies and parainfluenza 3 viruses. Its effectiveness against the two infectious bovine rhinotracheitis viral strains was lesser and of a low order. PMID- 2999189 TI - Specific detection and typing of adenovirus types 40 and 41 in stool specimens by dot-blot hybridization. AB - A dot-blot hybridization test was developed which allowed the direct detection of fastidious enteric adenovirus DNA in stool specimens from children with diarrhea and simultaneous typing of the viruses as adenovirus type 40 (Ad40) or Ad41. Cloned PstI fragments of Ad40 and Ad41 were used as 32P-labeled probes in the test, which allowed detection of picogram quantities of viral DNA in 20 to 40 microliter of stool suspension. Results were obtained within 48 h. The type specificity of the test was evaluated with 76 specimens known to contain either Ad40 or Ad41 by restriction enzyme analysis. Sixty-one specimens had sufficient DNA to be detected without any removal of protein. Thirty-one adenoviruses were typed as Ad40, and 30 were typed as Ad41, giving 100% correlation with the results of restriction enzyme analysis. The other 15 specimens were detected and typed as Ad40 or Ad41 only after removal of protein by a phenol extraction method. The dot-blot hybridization method is particularly useful for identifying those Ad40 and Ad41 strains which defy all attempts at culture and will be a useful tool in the epidemiology of fastidious enteric adenovirus infections. PMID- 2999190 TI - Combined immunoaffinity cDNA-RNA hybridization assay for detection of hepatitis A virus in clinical specimens. AB - To apply cDNA-RNA hybridization methods to the detection of hepatitis A virus (HAV) in clinical materials, we developed a two-step method in which a microtiter based, solid-phase immunoadsorption procedure incorporating a monoclonal anti-HAV capture antibody was followed by direct blotting of virus eluates to nitrocellulose and hybridization with 32P-labeled recombinant HAV cDNA. This immunoaffinity hybridization method is simple and involves few sample manipulations, yet it retains high sensitivity (10- to 30-fold more than radioimmunoassay) and is capable of detecting approximately 1 X 10(5) to 2 X 10(5) genome copies of virus. The inclusion of the immunoaffinity step removes most contaminating proteins and thus facilitates subsequent immobilization of the virus for hybridization. It also permits positive hybridization signals to be related to specific antigens and adds a level of specificity to the hybridization procedure. When the method was applied to 23 fecal specimens collected from individuals during week 1 of symptoms due to hepatitis A, 13 specimens were found to be reproducibly positive for HAV RNA by immunoaffinity hybridization, whereas only 11 contained viral antigen detectable by radioimmunoassay. PMID- 2999191 TI - Detection of bovine herpesvirus 1 DNA immobilized on nitrocellulose by hybridization with biotinylated DNA probes. AB - A molecular hybridization technique using biotinylated DNA probes was used to detect bovine herpesvirus 1 (BHV-1) nucleic acid species immobilized on nitrocellulose. Seventeen recombinant plasmids containing HindIII restriction fragments of the BHV-1 genome were compared for their ability to detect immobilized BHV-1 DNA from purified virus and infected cells. One probe, pCB2, labeled by nick translation with either 3H or biotin, detected as little as 10 pg of viral DNA. In time course experiments, BHV-1 DNA could be detected by 2 h postinfection in 10(6) infected cells. BHV-1 DNA was detected in nasal swabs and exudate from experimentally infected cattle, even when specimens had been stored for over a year. In a retrospective study of a respiratory disease outbreak in a feedlot, hybridization was compared with virus isolation for diagnosis of BHV-1 infections. The sensitivity rate was 0.68 with virus isolation as the referent standard. Blot hybridization provides a novel approach with unique applications for the diagnosis of bovine herpesvirus infections. PMID- 2999193 TI - Cytomegalic virus periventriculitis: a sonographic picture mimicking ventricular hemorrhage. PMID- 2999192 TI - The effects of a calcium dependent protease on the ultrastructure and contractile mechanics of skinned uterine smooth muscle. AB - In situ substrates for a vascular smooth muscle calcium-dependent protease (CDP) were investigated using a chemically skinned uterine smooth muscle preparation. Treatment of skinned smooth muscles with CDP had no effect on the total content of actin and myosin. Electron microscopical observations demonstrated that membrane plaques, cytoplasmic dense bodies, and intermediate filaments were all degraded by CDP. In addition, CDP reduced both isometric force and isotonic shortening velocity of contracted muscles in a concentration and time-dependent manner. Treatment of contracting muscles with CDP resulted in a condensation of myofilaments away from the plasma membrane concurrent with the loss of contractility. The condensation of myofilaments was ATP-dependent and could be inhibited by removal of ATP prior to proteolysis. The effects of proteolysis on smooth muscle ultrastructure and contractility support previously proposed models which assign a role to cytoskeletal elements in coordinating the molecular interaction of actomyosin to produce muscle contraction. The loss of cytoskeletal structures following protease treatment suggests that one of the functions of CDP in smooth muscle may be the disassembly of the cell cytoskeleton. PMID- 2999195 TI - Presence of epidermal growth factor receptor as an indicator of poor prognosis in patients with breast cancer. AB - Epidermal growth factor receptors are present in some breast cancers in man, and there is an inverse relation to oestrogen receptor state. We assessed the presence of epidermal growth factor receptors as a single prognostic indicator in a series of breast tumours by comparing this with the Bloom and Richardson scores for these tumours. One hundred and eight ductal tumours were examined for epidermal growth factor receptors by radioligand binding. There was a significant (p less than 0.01) correlation between the presence of the growth factor receptor and poor prognosis as assessed by the Bloom and Richardson score, suggesting that epidermal growth factor receptor state could be a useful prognostic marker. Epidermal growth factor receptor state was not significantly correlated with the lymph node state but showed a tendency to be associated with large tumours. PMID- 2999196 TI - Cellular mechanisms of digitalis action. AB - Although the therapeutic actions of digitalis glycosides have been known for over 200 years, their direct inotropic actions on the heart were not established until the last 50 years. Digitalis has undergone intense research, particularly with respect to its mechanisms of action. Many authors have claimed to have found the true mechanism of action, compounding the complexity of literature. Recent subcellular studies have pointed to specific areas of action of the digitalis glycosides. Each discovery has been dependent on the greater understanding of the electrophysiologic characteristics of cardiac muscle and excitation-contraction coupling. The current hypothesis suggests that digitalis specifically inhibits Na K ATPase. This produces an elevation in intracellular sodium level that in turn produces an increase in the intracellular calcium level. The increased quantities of calcium available to the contractile elements of cardiac muscle provide the observed increased inotropy. PMID- 2999197 TI - New advances in the assessment and treatment of digitalis toxicity. AB - The narrow margin between the therapeutic and toxic doses and serum levels of cardiac glycosides results in a high incidence of digitalis toxicity. This common problem has led to the development of methods for determining serum glycosides concentrations. It is clear that overlap of serum digoxin levels occurs between groups of patients with and without evidence of toxicity. In spite of these difficulties, use of serum digoxin measurement has been reported to be associated with a lower incidence of digitalis intoxication in clinical practice. When digitalis toxicity does develop, it is generally of two types: disturbances of impulse formation and disturbances of conduction. Therapeutic interventions may include antiarrhythmic drugs, pacemaker placement, and, in the most severe cases, administration of cardiac glycosides-specific antibodies. Recent studies have shown that monoclonal digoxin-specific antibodies and Fab fragments obtained by somatic cell fusion are effective in reversing advanced and otherwise lethal digoxin intoxication. The homogeneity of this antibody offers attractive possibilities for improving our ability to treat advanced digitalis intoxication safely and effectively. PMID- 2999198 TI - The organization of the neonatal rat's brainstem trigeminal complex and its role in the formation of central trigeminal patterns. AB - The present study delimits the relationship of primary trigeminal afferents to their targets, the brainstem trigeminal nuclei of the neonatal rat. Previously, the brainstem trigeminal complex of the rat has been subdivided on the basis of either cytoarchitectonics or patterns of succinic dehydrogenase activity into the principal sensory nucleus and the three subnuclei of the spinal trigeminal nucleus, oralis, interpolaris, and caudalis. In this paper, we demonstrate that each of these subdivisions can also be identified by its pattern of primary trigeminal afferents. In addition, we demonstrate that the terminations of these afferents are distributed in a punctate fashion which correlates with vibrissae related patterns of histochemical staining. Further, vibrissae removal in the neonatal rat at any age studied results in a corresponding deafferentation of both the principal sensory nucleus and all subnuclei of the spinal trigeminal nucleus. This same procedure has a graded, age-dependent effect on the vibrissae related pattern of cytochrome oxidase staining in somatosensory cortex. On this basis, we conclude that vibrissae-related pattern formation in the central trigeminal system can be best understood in terms of a single "sensitive" period for the entire system. We hypothesize that this is the period during which an interaction normally occurs between primary trigeminal afferents and target neurons of the principal sensory nucleus. PMID- 2999199 TI - The alterations in pathogenicity and immunogenicity of a Kenya sheep and goat pox virus on serial passage in bovine foetal muscle cell cultures. AB - A Kenyan sheep and goat pox virus was attenuated by serial passage in bovine foetal muscle cell cultures. The pathogenicity of the strain was lost between the 15th and 20th passages. Serum-neutralizing antibody developed after inoculation with passages tested up to the 50th. These passages appeared to protect animals against laboratory challenge by intra-dermal titration. The 18th passage was successfully and extensively used to control the disease under field conditions. PMID- 2999194 TI - Myelodysplastic syndromes: pathogenesis, functional abnormalities, and clinical implications. AB - The myelodysplastic syndromes represent a preleukaemic state in which a clonal abnormality of haemopoietic stem cell is characterised by a variety of phenotypic manifestations with varying degrees of ineffective haemopoiesis. This state probably develops as a sequence of events in which the earliest stages may be difficult to detect by conventional pathological techniques. The process is characterised by genetic changes leading to abnormal control of cell proliferation and differentiation. Expansion of an abnormal clone may be related to independence from normal growth factors, insensitivity to normal inhibitory factors, suppression of normal clonal growth, or changes in the immunological or nutritional condition of the host. The haematological picture is of peripheral blood cytopenias: a cellular bone marrow, and functional abnormalities of erythroid, myeloid, and megakaryocytic cells. In most cases marrow cells have an abnormal DNA content, often with disturbances of the cell cycle: an abnormal karyotype is common in premalignant clones. Growth abnormalities of erythroid or granulocyte-macrophage progenitors are common in marrow cultures, and lineage specific surface membrane markers indicate aberrations of differentiation. Progression of the disorder may occur through clonal expansion or through clonal evolution with a greater degree of malignancy. Current attempts to influence abnormal growth and differentiation have had only limited success. Clinical recognition of the syndrome depends on an acute awareness of the signs combined with the identification of clonal and functional abnormalities. PMID- 2999200 TI - The prevalence of antibody to camel pox virus in six different herds in Kenya. AB - Serum neutralizing antibody to camel pox virus was found in 5 out of 6 camel herds in Kenya. This was not related to recently observed clinical disease in the herds. PMID- 2999201 TI - The discovery of calmodulin--an historical perspective. Dedicated to the memory of Shiro Kakiuchi (1929-1984), a quiet leader in calcium research. PMID- 2999203 TI - The binding of cyclic nucleotide analogs to a purified cyclic GMP-stimulated phosphodiesterase from bovine adrenal tissue. AB - The specificity of binding of [3H]cGMP to purified bovine adrenal cGMP-stimulated phosphodiesterase was investigated by adding increasing concentrations of unlabelled analogs of cAMP and cGMP. The data show a perfect correlation between the potencies of stimulation of cAMP phosphodiesterase activity and displacement curves of [3H]cGMP binding. Since the Sp and Rp diastereomers of adenosine 3',5' monophosphate behaved as a cAMP-dependent protein kinase agonist and antagonist, respectively, the possible biological activity of these compounds and the corresponding cGMP analogs (cGMPS Sp and Rp) on the cGMP-stimulated phosphodiesterase was investigated. The data show no regioselectivity in binding nor on activation of one of the two (Sp) or (Rp) isomers. PMID- 2999202 TI - A simple assay for cyclic adenosine 3':5'-monophosphate in human saliva. AB - Methods specially designed for the assay of cyclic adenosine 3':5'-monophosphate (cAMP) in human saliva have not previously been published. Methods for measurements in plasma or tissue preparations are inapplicable to saliva due to interference. This interference can be reduced by calibrating standard solutions with reagent blanks prepared from saliva pre-treated to remove cAMP (by charcoal adsorption). Further, human saliva contains only small amounts of cAMP (approximately 0.2 pmol/50 microliters saliva). The method presented provides the necessary sensitivity (lower detection limit: 0.02 pmol/50 microliters) obtained by adjustments of tracer and binding protein concentrations. The method is simple and has a comparatively great capacity for the number of test tubes to be assayed. PMID- 2999204 TI - Differences in the mode of action of 1-oleoyl-2-acetyl-glycerol and phorbol ester in platelet activation. AB - The ADP-hydrolyzing enzyme apyrase inhibited platelet aggregation and phosphorylation of a 40,000 dalton platelet protein (P40) induced by 1-oleoyl-2 acetyl glycerol (OAG), indicating a dependence on secreted ADP. Apyrase also enhanced OAG-induced potentiation of forskolin or prostaglandin I2 activation of cyclic AMP formation in platelets. Cyclic AMP formation induced by OAG alone could be demonstrated in the presence of a phosphodiesterase inhibitor. Elevation of cyclic AMP level inhibits platelet aggregation so that secreted ADP may be required to inhibit OAG-activated adenylate cyclase for aggregation to proceed. In contrast, apyrase only partially affected phosphorylation of P40 and aggregation induced by the tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA). TPA caused marked inhibition of forskolin-stimulated cyclic AMP formation. TPA inhibition of cyclic AMP formation was largely reversed by apyrase, indicating that it was mainly due to release of ADP. PMID- 2999205 TI - Correlation between mammary prolactin receptors of lactating mice and litter weight. AB - Correlations between numbers and dissociation constants of mammary prolactin receptors of lactating mice and litter weight were examined. The apparent numbers and dissociation constants of mammary prolactin receptors were obtained from Scatchard plots with inhibition of iodine-125 prolactin binding by various concentrations of unlabeled prolactin. Litter weight measured 5 h after separation from the mother showed a nearly quadruple, almost linear increase during the first 15 d after birth. The dissociation constant for prolactin binding was fairly constant through lactation and did not correlate with either number of prolactin receptors or litter weight. The number of prolactin receptors in mammary cells increased rapidly in early lactation, reached a maximum at mid lactation, began to decrease thereafter, and was correlated closely with litter weights on d 5 (.70) and 10 (.64) postpartum, suggesting that number of prolactin receptors represents the lactational potential of the lactating mouse. PMID- 2999206 TI - The effect of a fluoride dentifrice containing an anticalculus agent on dental caries in children. AB - In this double-blind caries study, 1160 Taiwanese children (ages 8-15) completed a program using a test dentifrice containing 1.243 percent sodium fluoride and soluble pyrophosphates, or a control dentifrice without these agents. The average reduction of new carious tooth surfaces was 39 percent with the sodium fluoride dentifrice. PMID- 2999207 TI - Herpes vaccine effective in animal studies. PMID- 2999208 TI - Issues in exercise-induced asthma. AB - It is concluded that challenge by exercise and ISH induces asthma by the same mechanism, the protective effect of water vapor is evidence that the events that lead to bronchial smooth muscle contraction begin in the airway lumen, it is the loss of water rather than the loss of heat from the airways that is the primary stimulus to EIA and HIA, the mechanism by which water loss induces asthma is by increasing the osmolarity of the epithelial fluid, in some subjects with asthma, cooling of the airways enhances the response to water loss, the increase in osmolarity stimulates the production and release of bronchoactive substances from mast cells and epithelial cells, vagal afferent pathways are activated by changes in osmolarity and by the released mediators, and vagal efferent activity may be modified by alpha-adrenoceptor antagonists and SCG. PMID- 2999209 TI - Short-term effects of ovariectomy: the opioid control of LH secretion in fertile climacteric and postmenopausal women. AB - The aim of this study was to evaluate the activity of opiate receptors involved in the regulation of LH secretion in relationship to ovariectomy. Menstruating fertile (n = 5) and climacteric (n = 7) patients and postmenopausal (n = 5) women who underwent therapeutical bilateral ovariectomy were studied in the first week postsurgery and LH plasma levels were evaluated after naloxone (4 mg in bolus plus 4 mg infusion/90 min), LHRH (10 micrograms + 10 micrograms iv) and saline administration. Two groups of fertile (n = 6) and postmenopausal (n = 6) subjects were studied as controls. Since the LH responsiveness to naloxone was impaired in climacteric patients after ovariectomy, the test was repeated in 5 of them after 1 and 6 months of estrogen-gestagen treatment (conjugated estradiol + noretisterone acetate), showing a significant increase in all patients in both cases. In four subjects treated with only gestagen, naloxone was still unable to significantly modify LH plasma levels. These results indicate that ovariectomy affects the activity of opiate receptors, resulting in the first week postsurgery LH rise inversely related to basal LH levels. Furthermore, these results indicate that one or six cycles of estrogen-gestagen treatment in ovariectomized patients similarly induces a restoration of the opiate receptors neuroendocrine activity. PMID- 2999210 TI - Regional distribution of nuclear T3 receptors in rat brain and evidence for preferential localization in neurons. AB - We examined the distribution of nuclear T3 in mature rat brain with the aim of determining specific targets of thyroid hormones within this tissue. Saturation experiments, performed in 9 different structures of the brain and in 4 parts of the cortex, revealed the presence of a single class of binding sites with a mean Ka of 0.53 X 10(10) M-1. The highest concentrations of receptors were found in the amygdala (0.523 +/- 0.025 ng T3/mg DNA, Mean +/- SE) and the hippocampus (0.438 +/- 0.071 ng T3/mg DNA) while the lowest were in the brain stem (0.058 +/- 0.003 ng T3/mg DNA) and the cerebellum (0.079 +/- 0.026 ng T3/ml DNA). The receptor was not uniformally distributed within the cerebral cortex, its concentration being relatively high in the central sections and intermediate in the remaining portions. The cell type distribution of the T3 receptor was studied by separating glial and neuronal nuclei on a discontinuous sucrose gradient. There was no detectable specific T3 binding in the fraction of oligodendrocyte nuclei (approximately 95% pure). Conversely, the neuron-enriched fraction (approximately 60%) showed a significant increase in receptor concentration compared to total nuclei (35-40% neurons): 0.857 +/- 0.196 vs 0.511 +/- 0.095 ng T3/mg DNA (p less than 0.01) in the cortex and 0.425 +/- 0.018 vs 0.234 +/- 0.24 ng T3/mg DNA (p less than 0.01) in the forebrain. The absence of nuclear T3 receptors in oligodendrocytes may have important implications on the mechanism of action of thyroid hormone in myelination. PMID- 2999211 TI - Persistence of a circadian rhythmicity of glucocorticoid secretion in a patient with Cushing's syndrome: study before and after unilateral adrenalectomy. AB - A 47-year-old woman affected by Cushing's syndrome due to an adrenal adenoma is described. An altered but rhythmometrically apparent cortisol secretory rhythm was detected using the single-cosinor computation. In fact serum cortisol levels and urinary excretion of 17-OHCS were elevated in the PM hours, particularly between 14:00-18.00 h and 18:00-22:00 h, and normal between 02:00-10:00 h. The patient was cured by unilateral adrenalectomy and one year later the circadian rhythm of corticosteroids secretion was investigated again. A normal rhythm of cortisol secretion and of 17-OHCS urinary excretion was found. Though it may be hypothesized that factors intrinsic to the tumoral adrenal cells were responsible for the rhythmic, but phase-shifted, hormonal release, the cause of the persistent and abnormal cortisol secretory rhythm is unknown. PMID- 2999213 TI - [Mixed malignant Mullerian tumors of the uterus]. AB - Mixed malignant Mullerian tumours have an epithelial component and a connective tissue component both of which are malignant. The exact nature of each gives rise to a large number of clinical varieties and that has given rise to a confusing nomenclature. All have in common that they occur after the menopause and that they appear to be banal but that their prognosis is awful. The frequency of these tumours seems to be increasing. It is essential to recognise them because their treatment has to be more profound than that carried out for endometrial adenocarcinoma. The initial treatment of the lesions is by surgery and radiotherapy. Chemotherapy is to be reserved to improve the prognosis which itself is linked to the amount of distant metastases. PMID- 2999212 TI - Multifactorial control of the 24-hour secretory profiles of pituitary hormones. PMID- 2999214 TI - [In situ breast carcinoma developing in a fibroadenoma. Apropos of 4 case reports]. AB - There were 4 cases of fibro-adenoma which, when examined histologically, showed a lobular or canalicular carcinoma in situ. These are reported. The results as compared with those in the literature suggest to the authors that fibro-adenoma should be removed. PMID- 2999215 TI - [Luteinized cystic hyperplasia of the ovaries in normal pregnancy. Diagnostic and nosological problems]. AB - The authors report a new case of luteinized hyperplastic cystic ovary in a pregnancy that was in every other respect normal. This syndrome of ovarian hyperstimulation is most often found in patients suffering from trophoblastic diseases or following iatrogenic accidents. It is exceptionally rare in a non molar pregnancy. The comparison with the other form of ovarian hyperactivity that gives rise to a luteoma of pregnancy emphasizes the questions that have to be answered of the hormonal mechanism leading to maternal and fetal virilisation that complicate these two pseudo-tumours and how they are handed on from one generation to the other, and what the mechanisms is of placental HCG in this abnormal condition. PMID- 2999216 TI - RNA genome electrophoretic analysis of rotavirus from feces of epidemic adult diarrhoea occurring in Shandong Province, China. PMID- 2999217 TI - Association of a type-2 canine adenovirus with an outbreak of diarrhoeal disease among a large dog congregation. PMID- 2999218 TI - Clinical and biochemical aspects of bovine rotavirus infection in Iraq. PMID- 2999219 TI - Anorectal motility and rectal sensitivity in chronic idiopathic constipation: effect of high-fiber diet. AB - The rectoanal inhibitory reflex and rectal sensitivity were evaluated in 13 patients with idiopathic chronic constipation and in 13 control subjects. A double balloon probe was used to stimulate and record the rectoanal inhibitory reflex and rectal sensitivity. The results showed a linear relationship between relaxation amplitude and the logarithm of rectal distending volume, and between relaxation duration and the logarithm of rectal distending volume, with a significant reduction of relaxation amplitude in the patient group. The rectal sensitivity thresholds were significantly increased in patients compared with controls. In 10 additional patients with chronic constipation, motility and sensitivity parameters were evaluated before and after 28 days of high-fiber diet. After diet, the values of relaxation amplitude were significantly increased, returning within normal range, but sensitivity parameters did not change. In conclusion, it appears that in adult chronic idiopathic constipation, anorectal motility and rectal sensitivity are altered, and that only motor abnormalities are corrected by a high-fiber diet. PMID- 2999220 TI - Hepatocellular carcinoma related to hepatitis B virus in a patient with Waldenstrom's macroglobulinemia. AB - A patient with long-standing Waldenstrom's disease and cryoglobulinemia, treated with chlorambucil, developed hepatocellular carcinoma. Although HBs antigen, anti HBs, and anti-HBc antibodies were not detected in his serum by conventional polyclonal radioimmunoassays, immunofluorescence techniques showed HBs antigen to be present in hepatocytes. PMID- 2999221 TI - Hepatocellular carcinoma in a patient with precirrhotic primary biliary cirrhosis. AB - Hepatocellular carcinoma is generally associated with long-standing chronic liver disease of diverse etiology, most commonly HBsAg-positive chronic active hepatitis, hemochromatosis, or alcoholic liver disease. Patients with primary biliary cirrhosis have only rarely developed a subsequent hepatocellular carcinoma. We report such a patient, a 77-year-old woman with an early, precirrhotic stage of primary biliary cirrhosis who developed a hepatoma. PMID- 2999222 TI - 5'-Nucleotidase activity and substrate affinity in digenetic trematodes. AB - 5'-nucleotidases of eight species of digenetic trematodes were studied using five different substrates. All species showed the following preferential order of substrate affinity; AMP greater than CMP greater than GMP greater than TMP greater than UMP. It was observed that different species occupying similar habitats possessed closely related levels of enzyme activities. The function of 5'-nucleotidases in trematodes is also suggested. PMID- 2999224 TI - Hepatic encephalopathy. Experimental studies in a rat model of fulminant hepatic failure. AB - A new approach to pathogenetic study of hepatic encephalopathy was recently undertaken in order to identify the neurological alterations of the brain which characterize the coma. In this study attention was firstly addressed to a correct and objective evaluation of the comatose state in rats with fulminant hepatic failure induced by galactosamine. For this purpose visual evoked potentials were utilized since this electrophysiological test proved reliable and sensitive on the basis of an extensive pharmacological study. Two different stages of coma were identified in the rat and they were named mild and severe. Receptor binding studies performed on brain membranes of these rats show in the mild stage an increased number of low and high affinity GABA receptors and a decreased affinity of dopamine receptors. The severe stage is characterized by the persistence of only high affinity GABA receptors and a reduced number of dopamine receptors. This imbalance between inhibitory and excitatory receptor systems may explain the generalized central nervous system depression which characterizes the hepatic encephalopathy while the increased number of benzodiazepine receptors found in both stages of coma may account for the brain supersensitivity to sedative administration of patients with liver disease and for the sedative-induced episodes of coma. These receptor alterations may be attributed to a disuse and/or a partial degeneration of nerve terminals due to peripheral neurotoxins (i.e., ammonia, mercaptans, short chain fatty acids) and the decrease of glutamate decarboxylase activity and of zinc levels in brain tissues seems to be respectively a direct and an indirect demonstration of this phenomenon. Bearing in mind the supersensitivity of the GABA-benzodiazepine receptor system and their reciprocal interaction, a benzodiazepine antagonist was administered to rats in mild stage of encephalopathy. Electrophysiological and benzodiazepine binding studies demonstrated that this treatment can temporarily counteract some of the neurological disturbances of the earlier stage of coma and act as antidote of the sedative-induced episodes of coma. PMID- 2999223 TI - Carbohydrate intolerance associated with reduced hepatic glucose phosphorylating and releasing enzyme activities and peripheral insulin resistance in alcoholics with liver cirrhosis. AB - Carbohydrate intolerance was investigated in 8 alcoholics with liver cirrhosis and in controls. Indices of carbohydrate metabolism, glucose and insulin levels after glucose loading, were compared with glucose phosphorylating (glucokinase, hexokinase) and releasing (glucose-6-phosphatase) enzymes. Comparison was also made with pericellular collagen in liver biopsies and with insulin sensitivity assessed by the euglycemic clamp technique and with conventional liver function tests including oral antipyrine test. Glucokinase activity was low or absent, hexokinase activity increased and the GK/HK ratio reduced. Glucose-6-phosphatase activity was lowered and insulin sensitivity decreased. Pericellular collagen was increased (P less than 0.001) and related to the fasting glucose (r0.593) and insulin levels (r0.526). Blood glucose was related to antipyrine metabolism (r 0.727) but not to the other liver tests. Glucose intolerance in cirrhosis seems to be associated with reduced glucose phosphorylating and liberating enzyme activities. Hyperinsulinaemia, developing secondarily, may then lead to insulin resistance. PMID- 2999225 TI - 32-year-old AIDS patient with progressive pulmonary infiltrates. PMID- 2999226 TI - Skin diseases: current concepts, therapy. 5. Virus infections. PMID- 2999227 TI - Epidemic Coxsackie B virus infection in Johannesburg, South Africa. AB - A particularly extensive epidemic of Coxsackie B3 virus infection occurred in Johannesburg in the spring and summer of 1984. A total of 142 positive cases were diagnosed by isolation of the virus from stools and other specimens (60) or by serology (82). Coxsackie B3 accounted for 87% of the isolations and was also the dominant serotype on serology. The outbreak involved predominantly children and young adults, with no apparent sex differences being noted. The majority of specimens came from the white population and no significant difference in age or sex distribution could be observed between the two race groups. The major clinical presentation in the white group was Bornholm disease followed by cardiac involvement and then meningoencephalitis. In the black group, however, myocarditis was the major clinical presentation, which is of particular interest taking into account the extremely high incidence of acute rheumatic carditis in this population and the prevalence of chronic cardiomyopathy. PMID- 2999228 TI - Cross-reactions of immunoglobulin M and G antibodies with enterovirus-specific viral structural proteins. AB - We analysed the reactivity of enterovirus-specific human IgM and IgG antibodies with the structural proteins of different enteroviruses by the immunoblot technique. In general, all immunoglobulin G antibodies of the tested sera reacted with capsid polypeptide VP 1 of the viruses tested (echoviruses 9 and 11, coxsackievirus B3 and poliovirus 2). In contrast, enterovirus specific immunoglobulin M antibodies of adults reacted with capsid polypeptides VP 1, VP 2, and/or VP 3 of the viruses mentioned above. The reactions with VP 2 and/or VP 3 were often stronger than with VP 1. IgM antibodies from sera of newborns infected by echovirus 11 reacted with VP 1 and VP 2/3 of echovirus 11 and also with VP 2 and VP 3 of poliovirus 2. Preabsorption experiments indicate that cross reactive IgG antibodies react with epitopes of VP 1 not present on the surface of intact virus particles. The results from the immunoblot technique were compared to data from microneutralization tests and M-antibody capture radioimmunoassays. PMID- 2999229 TI - Vascular neuroeffector function in two-kidney, one clip hypertensive dogs. AB - In control dogs and those made hypertensive for 1 and 8 months by partially occluding a renal artery, contractile responses of mesenteric artery strips to adrenergic nerve stimulation and to norepinephrine, plasma renin activity and vascular angiotensin converting enzyme (ACE) activity were compared. Contractile responses to norepinephrine were potentiated in the artery strips from 8-month hypertensive dogs; however, the response to electrical stimulation of adrenergic nerves was not influenced. Contractions induced by the nerve stimulation were potentiated by a low concentration (2 X 10(-10) mol/l) of angiotensin (ANG) II; the potentiating effect was enhanced in 8-month-hypertensive dog arteries. 3H overflow evoked by adrenergic nerve stimulation was increased by ANG II to a greater extent in superfused mesenteric artery strips obtained from hypertensive (8-month) dogs, previously soaked in 3H-norepinephrine. Angiotensin converting enzyme activity was markedly greater in 8-month-hypertensive dog mesenteric arteries than in normotensive dog arteries. It may be concluded that the hypertension is maintained by increased sensitivity of post-synaptic alpha 1 adrenoceptors and pre-synaptic ANG receptors and increased vascular ACE activity, possibly promoting the production of ANG II in the vascular wall. PMID- 2999230 TI - Physiological responses to angiotensin II infusion during chronic angiotensin converting enzyme inhibition in dogs on normal, low and high sodium intake. AB - The function of the renin-angiotensin system (RAS) is influenced by changes in sodium balance and angiotensin II (ANG II) levels. In previous studies it has been difficult to assess the physiological effects on blood pressure homeostasis of different rates of ANG II infusion and of different levels of sodium in the diet. The present study examined the quantitative effects of ANG II in conscious dogs on low, normal and high sodium intake with the endogenous RAS blocked by continuous intravenous infusion of enalapril [MK-421,N-(1(S)-carbethoxy-3-phenyl propyl)-L-alanyl-L-proline maleate]. Conscious dogs on three different sodium diets, low, normal and high (5, 30 and 250 mmol/day), were infused continuously with enalapril, 4 mg/kg/day, and studied with superinfused ANG II at rates of 0, 1, 3, 6 and 12 ng/kg/min, each period lasting 1 week. Converting enzyme inhibitor (CEI) decreased mean arterial blood pressure (MAP) equally in dogs on low and normal sodium intake by 20 mmHg, but did not have a significant effect in dogs on high sodium intake. The initial infusion of ANG II at the lowest rate had a pronounced effect on MAP in dogs on normal and high sodium, but had no effect in the sodium depleted dogs. However, at the higher rates of infusion, the angiotensin increased the pressure equally at all levels of sodium intake.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2999231 TI - Tumor promoters in conjunction with calcium ionophores mimic antigenic stimulation by reactivation of alloantigen-primed murine T lymphocytes. AB - Antigen binding to its specific receptor on T cells initiates a series of intracellular events that result in cell differentiation, activation, and clonal expansion. However, the mechanism by which these antigen-occupied receptors induce the transmembrane signal transduction needs clarification. Because this mechanism appears to involve an increase in intracellular free Ca2+ concentration and activation of protein kinase C (PKC), we tested the effect of Ca2+ ionophores and PKC activators on alloantigen-specific primary mixed leukocyte culture cells. Both calcium ionophores, A23187 and ionomycin, in conjunction with 12-O tetradecanoylphorbol 13-acetate (TPA) mimicked the effect of antigen or interleukin 2 (IL 2) by inducing strong proliferative and alloantigen-specific cytotoxic responses. In addition, Ca2+ ionophore and TPA induced IL 2 receptor expression and IL 2 secretion. The capacity of other phorbol esters or a non phorbol ester tumor promoter (teleocidin) to replace TPA in induction of cell activation correlated with their ability to bind to and to activate PKC. In addition, the synergistic effect of Ca2+ ionophore and TPA was blocked by either a Ca2+ chelator (EGTA) or cAMP, which is thought to inhibit phosphatidylinositol metabolism. To determine whether the induction of this cytotoxic activity was mediated by a direct effect of Ca2+ ionophore and TPA on cytotoxic T (Tc) cells or was secondary to IL 2 secretion by activated helper T (Th) cells, we tested the effect of Ca2+ ionophore and TPA on isolated populations of cloned, alloantigen-specific Th and Tc cells. Both agents induced cell proliferation and IL 2 production by Th cells, but not by Tc cells. Activation of mixed clones of Th and Tc cells, but not of Tc cells alone, resulted in cytotoxic activity, an effect that could be blocked by anti-IL 2 receptor antibodies. The results thus demonstrate that an increased concentration of intracellular Ca2+ in conjunction with PKC activation can bypass the signal provided by antigen-receptor interaction on Th cells, but does not substitute for IL 2 in activating cytotoxicity by isolated Tc cells. PMID- 2999232 TI - A potential role for adrenocorticotropin in regulating human B lymphocyte functions. AB - Adrenocorticotropin (ACTH) was found to enhance the growth and differentiation of human B lymphocytes. By using highly purified preparations of human tonsillar B cells, the effects of ACTH on the growth and differentiation of in vitro activated B cells were examined. Optimal concentrations of ACTH were found to increase the proliferation of activated B cells by twofold to threefold when ACTH was present in culture with either a B cell growth factor or recombinant interleukin 2 (IL 2). ACTH had essentially no effects when added to cultures of activated B cells in the absence of the growth factor. Additionally, when ACTH was added in conjunction with an optimal concentration of either a B cell differentiation factor or IL 2 to cultures of activated B cells, the combination of ACTH and factor enhanced Ig secretion by twofold compared with the factor alone. In the absence of the differentiative signal, ACTH had minimal effects on Ig production. Only the first 24 amino acid fragments of ACTH were required to enhance B cell growth and differentiation when combined with the appropriate, more classical signals. Thus, ACTH may have a physiologic role in regulating human B cell function. PMID- 2999233 TI - Requirement of Ia-positive accessory cells in the MLR response against class II antigen on human B cell tumor line. AB - In this report we have made a comparative study of the capacity of normal human stimulator cells and Epstein-Barr virus-transformed human B cell line Wa (EBV-Wa) cells to stimulate alloreactive T cells. Class II antigen (presumably HLA-DR4 determinant) on EBV-Wa cells was shown to act as a stimulating molecule in the mixed lymphocyte reaction (MLR) through a blocking study by using anti-Ia antibodies. Furthermore, it was found that HLA-DR-positive accessory cells in the responder population were required to elicit MLR responses against HLA-DR antigen on EBV-Wa cells. In contrast, HLA-DR-positive accessory cells in the responding cell population were not essential for elicitation of MLR responses against HLA DR antigen on normal allogeneic peripheral blood mononuclear cells, as reported. The cell-cell interaction between responder HLA-DR-positive accessory cells and responding T cells in a major histocompatibility complex (MHC)-restricted manner was required for eliciting MLR responses against class II antigen on EBV-Wa cells such as antigen-presenting cell-T cell interaction in soluble antigen-specific T cell proliferative responses. The function of HLA-DR-positive accessory cells in the responder population could not be substituted for by the presence of interleukin 1. Furthermore, there was no obvious correlation between the degree of surface HLA-DR antigen expression on EBV-Wa cells and its stimulating ability. Thus, two distinct types of allo-class II, antigen-specific T cell activation between normal human stimulator cells and EBV-Wa cells were shown to exist. PMID- 2999234 TI - Leukotrienes augment interleukin 1 production by human monocytes. AB - The effects of leukotrienes (LT) on production of interleukin 1 (IL 1) by human peripheral blood monocytes were examined. LTB4 enhanced IL 1 production by lipopolysaccharide (LPS)-stimulated monocytes twofold to threefold, and the most efficient concentrations of LTB4 were 10(-8) to 10(-7) M. LTD4 also enhanced IL 1 production, but to a lesser extent than LTB4. Adherence-purified, but otherwise unstimulated, human monocytes could also be induced to produce IL 1 in response to LTB4. Similarly, IL 1 production by monocytes stimulated with the known IL 1 inducers muramyl dipeptide, silica, or zymosan was also enhanced by LTB4. Inhibition of cyclooxygenase with use of indomethacin during IL 1 production by LPS-treated monocytes enhanced thymocyte response to IL 1, but LTB4 further enhanced IL 1 production when added to indomethacin-treated monocyte cultures. Neither LTB4 nor indomethacin had any direct effect on thymocyte proliferation. Optimal enhancement of IL 1 production occurred when LPS and LTB4 were present together at the initiation of the 24-hr monocyte culture. Significant enhancement was also observed, however, when monocyte cultures were either preincubated with LTB4 before addition of LPS or cultured with LPS alone for 3 hr before addition of LTB4. These results indicate that leukotrienes can modulate IL 1 production by human monocytes and suggest that they may play a role in IL 1-mediated functions of monocytes in inflammatory and immune reactions. PMID- 2999235 TI - Recombinant interleukin 1 suppresses lipoprotein lipase activity in 3T3-L1 cells. AB - Recombinant murine interleukin 1 (rIL 1) inhibits 3T3-L1 cell expression of lipoprotein lipase (LPL) activity when present in exceedingly dilute concentration (less than 10(-15) M). The extreme sensitivity of the adipocyte system to rIL 1 far exceeds that of the standard lymphocyte-activating factor assay. However, enzyme suppression is incomplete; even at micromolar concentrations, rIL 1 causes only about a 50% reduction in LPL activity. By contrast, cachectin (tumor necrosis factor) achieves nearly complete LPL suppression at subnanomolar concentrations. Concentrated solutions of rIL 1 are incapable of competing with radiolabeled cachectin for binding sites on 3T3-L1 cells. rIL 1-induced LPL suppression is abolished by the addition of a specific IL 1 neutralizing antiserum to the assay system. rIL 1 appears capable of influencing adipocyte expression of LPL, but apparently acts through a different mechanism than cachectin/TNF. PMID- 2999236 TI - Cachectin/tumor necrosis factor: production, distribution, and metabolic fate in vivo. AB - A highly specific radioreceptor assay for cachectin/tumor necrosis factor (TNF) was utilized to measure the time course of lipopolysaccharide (LPS)-induced hormone production in rabbits. Cachectin/TNF bioactivity was monitored in the same serum samples by measuring lipoprotein lipase (LPL) suppression in 3T3-L1 cells. Cachectin/TNF is produced in large quantities by LPS-treated rabbits without priming by bacillus Calmette Guerin, C. parvum, or other agents. Nanomolar concentrations of the hormone are achieved, with peak levels occurring at 2 hr postinjection; the hormone is rapidly cleared thereafter. In separate studies, mice were used to assess the distribution and metabolic fate of cachectin/TNF. Radioiodinated hormone is cleared from the plasma with a half-life of 6 to 7 min. Studies of the tissue distribution of label after injection demonstrate that liver, kidneys, skin, and gastrointestinal tract take up most of the hormone. Electrophoretic analysis of tissues recovered from injected animals suggests that the hormone is very rapidly degraded after binding. PMID- 2999237 TI - TCGF(IL 2)-receptor inducing factor(s). II. Possible role of ATL-derived factor (ADF) on constitutive IL 2 receptor expression of HTLV-I(+) T cell lines. AB - Interleukin 2 (IL 2) receptor (IL 2-R) is constitutively expressed on T cell lines established from the patients with adult T cell leukemia (ATL), which is a human T cell leukemia lymphoma virus (HTLV-1)(+) T4(+)-leukemia endemic in Japan, the United States, and other countries. Many of these cell lines continuously produce an acidic lymphokine, ATL-derived factor (ADF), which preferentially induces the synthesis and expression of IL 2-R on a sensitive HTLV-1(-) non-T cell line (YT). The induced IL 2-R was characterized by the binding of 125I-IL 2 and flow cytometry by using fluoresceinated anti-human IL 2-R monoclonal antibodies (anti-Tac). Scatchard analysis with 125I-IL 2 showed ADF induced high affinity receptor sites on YT cells. To test the possibility that ADF produced by HTLV-1(+) T cells is involved in the abnormal expression of IL 2-R, we studied the effect of ADF on an HTLV-1(+) IL 2-dependent T cell line (ED) in which the beta-chain gene of the T cell antigen receptor (T beta) was rearranged. Unlike IL 2-independent HTLV-1(+) cell lines that constitutively expressed Il 2-R, the IL 2 R expression on ED cells declined in the absence of crude IL 2 or recombinant IL 2. When either ADF or recombinant IL 2 was added to the culture of ED cells, there was a dose-dependent enhancement of IL 2-R expression in 24 hr. ADF and IL 2 showed a synergism in the IL 2-R induction, and both factors were needed to induce the maximal receptor expression in these T cells. The lack of IL 2 production by ADF-treated YT, as well as ED cell line suggested IL 2 may not be involved in the IL 2-R induction by ADF. Northern blot hybridization with human IL 2-R cDNA probe showed the increase of IL 2-R mRNA in YT cells after ADF treatment. ADF also enhanced IL 2-R expression of a rat T cell line transformed by HTLV-1(TARS-1), as demonstrated with anti-rat IL 2 receptor monoclonal antibodies (ART-18). An ADF-like IL 2-R-inducing factor was also detected in the conditioned medium of two HTLV-1(+) rat T cell lines (TARL-2 and TART-1), which constitutively expressed a higher number of Il 2-R than TARS-1 cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2999238 TI - Inhibition of lymphocyte and neutrophil chemotaxis by pertussis toxin. AB - The cells of the mammalian immune system possess special migratory properties within their in vivo environment, a surveillance characteristic that is thought to be important in the protection of the organism from transformants and exogenous pathogens. Pertussis toxin (PT) has been shown to disrupt the intensity of this process by seriously affecting lymphocyte recirculation in vivo. The mechanisms responsible for this inhibition were investigated by using the in vitro model systems of polymorphonuclear leukocyte and lymphocyte chemotaxis. The type of inhibition that was observed in these in vitro assay systems was quite similar to that observed in vivo, because PT could depress chemotaxis in vitro as well as the accumulation of radiolabeled lymphocytes and neutrophils within a peripheral site of inflammation in vivo. The alterations in neutrophil motility were found to be associated with a stimulus-specific inhibition of the triggering of superoxide anion generation and lysosomal secretion. Some inhibition of neutrophil adherence to plastic surfaces was also observed, most notably after augmentation of adherence with the chemoattractant fMLP. The observed alterations in cellular function after PT treatment occurred in the absence of defects in chemoattractant binding to the neutrophil cell surface, or of membrane potential changes stimulated by ligand binding. The effect of PT in this system was found to be associated with an abnormality in the regulation of intracellular free calcium, suggesting that the substrate for PT in neutrophils is involved in the regulation of calcium ion channels. PMID- 2999240 TI - Organization of rabbit immunoglobulin genes. I. Structure and multiplicity of germ-line VH genes. AB - Two rabbit germ-line VH gene segments have been isolated from a recombinant phage DNA library. Nucleotide sequence analysis indicates that both of the genes share structural and regulatory features common to mouse and human VH genes, although one appears to be a pseudogene. Comparison of the protein sequences encoded by these genes to the protein sequences of rabbit immunoglobulin V regions indicates that both genes encode VH a-negative-like molecules. Quantitative genomic blot analysis with a VH probe capable of recognizing most, if not all, germ-line VH genes indicates that there are approximately 100 VH genes in the haploid genome of rabbits. The average spacing between the germ-line VH genes was determined to be approximately 6 kb. The molecular basis for the allelic inheritance of rabbit VH allotypes is discussed in view of the structural organization of germ-line VH genes. PMID- 2999239 TI - The immune response to mouse hepatitis virus: expression of monocyte procoagulant activity and plasminogen activator during infection in vivo. AB - After infection with 10(3) plaque-forming units of mouse hepatitis virus strain 3 (MHV-3) in vivo, peripheral blood mononuclear cells and splenic cells expressed procoagulant activity (PCA) in a pattern directly correlating with susceptibility to disease. Mononuclear cells from BALB/cJ mice, a strain which is fully susceptible to MHV-3, expressed a greater than 500-fold increase in PCA. PCA was first detected within 12 hr of infection; prior to histologic evidence of disease and viral replication, it reached maximal levels 48 hr post-infection (p.i.) and persisted until the death of the animals 5 to 7 days p.i. Mononuclear cells from C3HeB/FeJ mice expressed a significant but lesser titer of PCA, with elevated PCA persisting throughout the chronically infected state until death of the animals 4 to 6 mo p.i. Basal levels of PCA were detected in mononuclear cells from fully resistant A/J mice despite the presence of large amounts of virus in livers, spleens, and sera from these animals. When mononuclear cells expressing high PCA were subfractionated, monocytes were found to be the cellular source of greater than 96% of the PCA activity. Increased plasminogen activator activity was found in monocytes from resistant A/J mice at the time when PCA was markedly elevated in BALB/cJ and C3HeB/FeJ mice. This activity persisted for 5 to 7 days p.i., but was undetectable 10 days p.i. at a time when the mice had cleared the virus from their blood streams. These observations suggest that monocyte PCA may be important in the pathogenesis of MHV-3 disease, whereas the production of monocyte plasminogen activators may contribute to resistance of A/J mice to MHV-3 induced liver disease. PMID- 2999241 TI - Heterogeneity of EBV-transformable human B lymphocyte populations. AB - Although most human B cells express receptors for Epstein Barr virus (EBV), few (usually less than 1%) are readily transformed into B lymphoblastoid cell lines after exposure to EBV. Transformable cells previously have been found to be mostly resting B lymphocytes. We recently developed a limiting dilution culture system which permits the growth of EBV-transformed B lymphocytes with high efficiency. Because in this system up to over 30% of peripheral blood- or tonsil derived B cells respond to EBV, we re-examined the properties of EBV transformable cells. Frequencies of transformable lymphocytes were determined by Poisson analysis. EBV-susceptible B cells committed to IgM, IgG, or IgA secretion were found to occur in the range of 3 to 27, 0.1 to 6, and 0.1 to 5 per 100 B cells, respectively. Under our culture conditions, a major proportion of the IgM committed cells derived from large lymphocytes which appeared to have entered the cell cycle. This population contains most of the EBV-responsive cells detected and, therefore, most of the additional cells responding in our culture system. In contrast, precursors of IgG- or IgA-producing lymphoblast lines were small cells with DNA contents typical for the G0/G1 phases of the cell cycle. Anti immunoglobulin antibodies were used in panning experiments to separate B cell subpopulations which expressed different immunoglobulin isotypes on their surface. In limiting dilution cultures of these purified B lymphocytes subsets, it was found that virtually all precursors of IgM-producing cell lines expressed surface IgM (sIgM) before their infection and transformation by EBV. The "cloning efficiency" of positively selected, large sIgM+ cells approached 100%. In contrast, sIgG or sIgA were found only on cells committed to the production of IgG or IgA, respectively. The expression of sIgD was examined by using sequential panning procedures. Virtually all IgM-committed lymphocytes and a subset of cells committed to IgA secretion were found among the sIgD+ cells. The majority of cells committed to IgA production and all IgG-committed cells were found in the sIgD- B cell population. Our findings indicate that the EBV-susceptible B cell subset of normal lymphocytes is heterogeneous with respect to cell size, cell cycle, sIg determinants, and efficiency of transformation. On the basis of our findings and previous reports, we propose a model in which transformability is a B cell-inherent property. Factors unrelated to the virus but present in our culture system appear responsible for the enhanced vulnerability to viral transformation in some cells which entered into the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2999243 TI - Interferon-induced inhibition of receptor-mediated endocytosis of colony stimulating factor (CSF-1) by murine peritoneal exudate macrophages. AB - Apart from its characteristic antiviral activity, interferon (IFN) also exerts a variety of biologic effects on macrophages. We have studied the effect of IFN on the expression of the colony-stimulating factor receptors (CSF-1 receptors) by murine peritoneal exudate macrophages (PEM). At 37 degrees C, murine IFN decreased the expression of the CSF-1 receptor activity in a time- and dose dependent fashion by PEM from both endotoxin-sensitive (C3H/Sn) and endotoxin resistant strains (C3H/HeJ) of mice. Scatchard analysis from the binding data suggests that the decreased expression of CSF-1 receptors is a result of decreased number of receptors rather than a decreased binding affinity. When IFN was incubated with anti-IFN before the addition to cultures, the effect was completely abolished indicating that this activity resides in the same molecules as IFN. The suppressed CSF-1 receptor activity on PEM by IFN appeared to be stable. Removal of added IFN never resulted in a full recovery of CSF-1 binding activity by PEM even after prolonged incubation (7 days). IFN also inhibited the receptor-mediated uptake and utilization of CSF-1 molecules by treated cells, which appeared to be a direct effect of the decreased number of CSF-1 receptors. Treatment of PEM with dexamethasone, prostaglandin, transferrin, insulin, or dibutyryl cAMP failed to suppress both the expression of CSF-1 receptors and CSF 1 utilization by PEM. These studies suggest that IFN may play a role in the regulation of both macrophage production and differentiation via the modulation of specific membrane receptors and inhibition of receptor-mediated CSF-1 endocytosis. PMID- 2999242 TI - Sequential synergistic effect of interleukin 2 and interferon-gamma on the differentiation of a Tac-antigen-positive B cell line. AB - The presence of Tac-antigen (Tac-Ag) on human B lymphocytes and its functional significance with regard to the ability of interleukin 2 (IL 2) to modulate B cell differentiation is currently an area of high interest. An Epstein-Barr virus transformed B cell line (CB) that secretes IgG was 30 to 40% Tac-Ag+ and was used as a model for examining the role of Tac-Ag and IL 2 in B cell differentiation. Recombinant IL 2 alone was found to have a modest but significant effect on CB in enhancing IgG secretion, increasing the plaque-forming cell response from 637 to 1734 at high concentrations (1000 U/ml IL 2) and to 888 at lower concentrations (100 U/ml). In contrast, recombinant interferon-gamma (IFN-gamma) alone had no effect on the differentiation of CB. However, both factors together showed marked synergy in increasing the number of plaque-forming cells to over 3000 by using only 10 U/ml of IFN-gamma and 100 U/ml of IL 2. These two factors were shown to act sequentially in that IL 2 was needed initially, while IFN-gamma was required for the next differentiation step into IgG-secreting cells. The effect of IL 2 on stimulating differentiation was blocked by anti-Tac, indicating that the action of IL 2 is mediated through its Tac-Ag receptor. CB cells were also sorted into Tac+ and Tac- populations and were cultured separately. In 2 wk, both populations reverted to the pattern of the original cell line. Moreover, cell cycle analysis when using double staining procedures indicated that Tac-Ag on the cell surface of CB appears and disappears according to the stage of the cell cycle, and that Tac is most strongly expressed in the S and G2 + M phases. Thus, the present study suggests that certain B cells are capable of responding to sequential stimulation by IL 2 and IFN-gamma with terminal differentiation into Ig-secreting cells, and that the amount of Tac-Ag expression is cell cycle dependent. PMID- 2999245 TI - Chemotaxis of large granular lymphocytes. AB - The hypothesis that large granular lymphocytes (LGL) are capable of directed locomotion (chemotaxis) was tested. A population of LGL isolated from discontinuous Percoll gradients migrated along concentration gradients of N formyl-methionyl-leucyl-phenylalanine (f-MLP), casein, and C5a, well known chemoattractants for polymorphonuclear leukocytes and monocytes, as well as interferon-beta and colony-stimulating factor. Interleukin 2, tuftsin, platelet derived growth factor, and fibronectin were inactive. Migratory responses were greater in Percoll fractions with the highest lytic activity and HNK-1+ cells. The chemotactic response to f-MLP, casein, and C5a was always greater when the chemoattractant was present in greater concentration in the lower compartment of the Boyden chamber. Optimum chemotaxis was observed after a 1 hr incubation that made use of 12 micron nitrocellulose filters. LGL exhibited a high degree of nondirected locomotion when allowed to migrate for longer periods (greater than 2 hr), and when cultured in vitro for 24 to 72 hr in the presence or absence of IL 2 containing phytohemagluttinin-conditioned medium. The chemotactic LGL was HNK 1+, OKT11+ or HNK-1+, OKT11- on the basis of monoclonal antibody and complement depletion. They did not bear either T cell or monocyte cell surface markers, exhibiting an OKT3-, OKT4-, OKT8-, OKM1-, and MO2- phenotype, and did not form E rosettes at 29 degrees C, which is characteristic of lytic NK cells in contrast to T cells. Furthermore, a rat LGL leukemia (RNK) exhibited a chemotactic response to both f-MLP and casein. LGL chemotaxis to f-MLP could be inhibited in a dose-dependent manner by the inactive structural analog CBZ-phe-met, and the RNK tumor line specifically bound f-ML[3H]P, suggesting that LGL bear receptors for the chemotactic peptide. PMID- 2999244 TI - Modulation of cyclic AMP-dependent protein kinase isozyme expression associated with activation of a macrophage cell line. AB - We have used the RAW 264.7 macrophage (MO) cell line to study cAMPdPK isozymes during activation by lymphokine (LK) and lipopolysaccharide (LPS). Untreated cells were found to have two isozymes of cAMPdPK in their cytosol. PKI and PKII were differentiated based on the Mr of their regulatory subunits (RI, 45,500; and RII, 52,000, respectively) as determined by photoactivated incorporation of the cAMP analog 8-N3-[32P]cAMP. Loss of the RI subunit of PKI occurred in association with activation of the cell line by suboptimal concentrations of LK and LPS (1/40 dilution, 1 ng/ml) or high concentrations of LPS alone (10 ng/ml to 100 micrograms/ml). No modulation of the RII subunit of PKII was observed under these conditions. The loss of RI was dependent on the addition of a triggering signal to the MO. Treatment of RAW 264.7 cells with LK alone at dilutions from 1/10 to 1/1280 was not sufficient to cause a disappearance of the RI subunit from the cytosol or to induce antitumor activity. The addition of a suboptimal concentration of LPS after LK or a high dose of LPS alone was required for acquisition of cytolytic activity and loss of RI. The kinetics for the disappearance of RI from treated cells were found to be identical after activation with either LK and LPS or high concentrations of LPS alone. RI could no longer be detected in the cytosol 8 hr after the addition of activating agents. The antitumor activity of the RAW 264.7 cell line was transiently expressed after activation. Cells no longer exhibited tumoricidal activity 48 hr after the removal of activating agents. It was observed that the loss of cytolytic function was accompanied by the reexpression of RI in the cytosol. This study provides evidence that modulation of cAMPdPK isozymes occurs during activation, suggesting a potential mechanism for controlling the effects of cAMP on the MO. PMID- 2999246 TI - Expression of CTL-defined, AKR/Gross retrovirus-associated tumor antigens by normal spleen cells: control by Fv-1, H-2, and proviral genes and effect on antiviral CTL generation. AB - In previous studies we have characterized H-2-restricted cytolytic T lymphocytes (CTL) type specific for Gross cell surface antigen-positive tumor cells induced by AKR/Gross leukemia viruses. The generation of such CTL was shown to be controlled by at least three genetic loci including H-2 and Fv-1. The Fv-1n phenotype was able to negate positive immune response gene effects of the H-2b haplotype. Fv-1n-mediated inhibition appeared to operated by allowing the early expression by normal cells of N-ecotropic leukemia virus-related antigens recognized by the antiviral CTL, perhaps via tolerance induction. In the present study, the expression of CTL-defined viral antigens by normal cells is further considered. Possible gene dosage effects by H-2 as well as Fv-1 and the other virus-related (V) genes, including proviral structural loci, were examined by comparison of a panel of congenic and F1 mice. These experiments indicated that the quantitative level of expression of CTL-defined viral antigens was primarily controlled by the Fv-1 genotype. Gene dosage effects were also observed for the V genes and, in some situations, for H-2. The importance of the early display of viral antigens by normal cells was underscored by the inability of those mice to generate specific antiviral CTL responses. Even strains expressing low levels of viral antigens, such as responder X nonresponder (AKR.H-2b:Fv-1b X AKR.H-2b)F1 mice, failed to respond. These results are discussed with respect to the inability of mice of the AKR background to respond with specific antiviral CTL generation and in light of their high incidence of spontaneous leukemia. PMID- 2999247 TI - Immunologic induction of malignant lymphoma: graft-vs-host reaction-induced B cell lymphomas contain integrations of predominantly ecotropic murine leukemia proviruses. AB - The induction of a graft-vs-host reaction in (BALB/c X A)F1 mice by i.v. injection with BALB/c lymphoid cells leads to a lymphoid hyperplasia that may progress to malignant lymphoma. In the present paper, the following aspects of graft-vs-host-reaction lymphomagenesis were studied: 1) the cellular requirements for the induction of lymphomas, 2) their cellular origin, and 3) the role of murine leukemia viruses. The development of graft-vs-host-reaction lymphomas was found to be mediated by donor T cells and to require the presence of histoincompatibility between donor and host. Histologically, the vast majority of these lymphomas were either of follicular center cell or of immunoblastic type, whereas immunoperoxidase studies showed that they were virtually all B cell derived. Most of the lymphomas were of host origin. In the DNA of approximately 80% of the lymphomas, integrated murine leukemia virus proviruses were detected. In the B cell lymphoma DNA, integrated ecotropic proviruses prevailed, but recombinant murine leukemia virus and/or deleted murine leukemia virus genomes were also detected in some tumor DNA. PMID- 2999248 TI - Macrophage cytotoxicity: interleukin 1 as a mediator of tumor cytostasis. AB - Purified macrophage interleukin 1 (IL 1) induced a concentration-dependent inhibition of the proliferation of two commonly used tumor cell target lines, the human myeloid K562 and the murine T lymphoma Eb. In contrast, mastocytoma-derived P815 cells were not inhibited. The cytostatic action of IL 1 was not associated with direct cytotoxicity and was only partially reversible. PGE or interferon did not appear to mediate these effects. IL 1 treatment of the multipotential K562 cells revealed no morphologic evidence for the induction of specific differentiation. FACS analysis of IL 1-treated K562 cells showed a rapid decrease in transferrin receptor density, and a more delayed, but highly significant, increase in HLA-A,B,C antigen density. These findings provide one explanation for the frequently reported macrophage cytostatic actions against tumor cells, and indicate as well that IL 1, like interferon, may enhance the expression of Class I MHC antigens. These observations further extend the range of IL 1 actions and underscore the fundamental and direct role of this monokine in macrophage antitumor activity. PMID- 2999249 TI - In situ enzyme immunodetection of surface or intracellular bacterial antigens using nitrocellulose sheets. AB - We describe an immunological method which allows the in situ colorimetric detection of translated DNA fragments in bacteria. In the absence of lysis only cell surface proteins are detected. For cytoplasmic proteins, lysis is required. The procedure comprises the following steps: bacteria are lysed, the proteins are transferred onto a disc of nitrocellulose sheet, the remaining protein sites are blocked, the disc is successively soaked in a solution of antibodies specific for the protein to be detected and in a solution of peroxidase-labelled anti-IgG antibody solution. Finally, the immune complexes are made visible by enzyme substrate incubation. We describe the application of this method to the detection of the LamB protein, the LacZ protein, and a LamB-polio VP1 chimera translated from cloned DNA fragment in E. coli. PMID- 2999250 TI - Production of LTB4-like chemotactic arachidonate metabolites from human keratinocytes. AB - Keratinocytes have recently been recognized as a source of mediators of cellular immune function. We present here further data on the production of 5-lipoxygenase dependent arachidonate metabolites from freshly isolated human epidermal cells. Stimulation of cells with arachidonic acid or the calcium ionophore A 23187 alone or together caused a dose- and time-dependent release of chemotactic activity which was maximal during the first 10 min and which continued for up to 18 h. Indomethacin (10(-6) M) enhanced and compound BW 755C (20 micrograms/ml), a lipoxygenase inhibitor, decreased release. The chemotactic activity was heat stable for 30 min at 56 degrees C and was extractable into ether at pH 3.0. Analysis of 15- and 30-min supernatants showed coelution of biologic activity and of leukotriene B4 (LTB4), as measured by radioimmunoassay, at marker positions of LTB4 and of 20-OH-LTB4. Elimination of Langerhans cells did not alter the secretion of chemotactic lipids, suggesting that keratinocytes are the main source of potent, biologically active, lipoxygenase-dependent arachidonate metabolites. PMID- 2999252 TI - Low endemicity and low pathogenicity of rotaviruses among rural children in Costa Rica. AB - Rotaviruses were prospectively studied in 51 rural Costa Rican children from birth to two years. Samples of feces were collected weekly over a 33-month period. Rotavirus was detected in 45 (1.04%) of 4,317 fecal specimens; 39 infections were documented (an incidence of 0.5 infection per child-year), only five of which were associated with diarrhea (a pathogenicity of 12.8%). Secretory antibody in fecal extracts, detected in six of 39 infections, was short lived and did not protect against reinfection. Serum antibody was present in 69.6% of two year-old children, but was not detected in 18.8% with documented infections. On the other hand, serum antibody was present in six of 14 children in whom rotavirus was not detected, thus increasing the overall incidence to 0.6 infection per child-year. The combination of prolonged breast-feeding, exposure to a lower infecting dose (compared with urban children), and a higher standard of hygiene than expected may explain the low incidence and low pathogenicity of rotavirus among these rural children. PMID- 2999251 TI - Preferential lectin binding to specific layers of rat oral epithelium and modification by enzyme pretreatment. AB - Three fluorescein-labeled lectins were shown to bind differentially to cell surfaces in different epithelial layers of rat oral mucosa regardless of the age or the site of origin of the tissue. Griffonia simplicifolia (GS-1-B4), specific for alpha-D-galactosyl end groups, labeled basal cells only; Ulex europeus (Ulex 1) specific for alpha-L-fucosyl groups labeled spinous cells; and Bandeiraea simplicifolia (BSII), specific for N-acetyl-D-glucosamine, labeled cornified cells. Pretreatment of sections with alpha-galactosidase completely abolished the staining of basal cells by GS-1-B4, but had no effect on the staining of spinous cells by Ulex 1. In contrast, alpha-fucosidase abolished the staining of spinous cells by Ulex 1 and caused staining of both basal and spinous cells by GS-1-B4. Neuraminidase and chondroitinase ABC produced results similar to one another, with staining of basal cells by GS-1-B4 and labeling of both basal and spinous cells with Ulex 1. beta-galactosidase, beta-glucuronidase, and testicular hyaluronidase did not affect the staining pattern of GS-1-B4 or Ulex 1, whereas chymotrypsin completely abolished any staining with either lectin. The results demonstrate a complex arrangement of cell surface carbohydrates in the epithelium of rat oral mucosa. The findings indicate a possible simplification in the spatial arrangements of cell surface carbohydrates during the differentiation of basal to spinous cells. PMID- 2999253 TI - Characterization of serotypes of human rotavirus strains by solid-phase immune electron microscopy. AB - Serotyping of human rotaviruses (HRVs) by neutralization requires the previous adaptation of strains to growth in cell cultures, which is often unsuccessful. By using the solid-phase immune electron microscopy (SPIEM) technique with protein A and type-specific, cross-adsorbed, polyclonal immune sera, we divided 40 previously typed culture-adapted strains into the same four serotypes distinguished by neutralization, but HRV strains could also be typed directly on stool extracts. Of 171 HRV strains tested by SPIEM, 163 were typed as a single serotype, two were shown to be mixed serotypes, three could not be typed since they were partially antibody-coated, and three were lacking the common group antigen (atypical rotaviruses or pararotaviruses). In addition, strains of serotype 4 could be classified by SPIEM into two subtypes, 4A and 4B. Overall, strains of serotype 1 were detected in nearly 50% of the 171 HRV-positive stools examined by SPIEM, strains of either serotype 2 or 4 in about 20%, and strains of serotype 3 in nearly 10%. Although the data were too scarce to allow for definite conclusions, the prevalence of the four serotypes appeared to change with the geographic area and the year. PMID- 2999254 TI - Maternal antibody-mediated protection against gastroenteritis due to rotavirus in newborn mice is dependent on both serotype and titer of antibody. AB - In order to evaluate the role of passively acquired, rotavirus-specific antibodies in protection against diarrhea, we inoculated mouse dams with rotaviruses of various serotypes, and their newborns were orally challenged with a primate rotavirus (simian SA-11). Dams were immunized by using a regimen that included repeated inoculations administered either orally or intraperitoneally with adjuvant. The serum antibody response detected in dams by radioimmunoassay and plaque-reduction neutralization after parenteral immunization was approximately 15-fold and 80-fold greater, respectively, than that found after oral "hyperimmunization." Parenteral immunization with rotavirus serotypes either homotypic or heterotypic to the challenge virus protected suckling mice against diarrhea; protection was closely correlated with the in vitro neutralizing activity of maternal serum against the challenge virus. Oral immunization with only rotavirus strains homotypic to the challenge virus afforded protection; the lower immune response after oral immunization with rotaviruses heterotypic to the challenge virus resulted in a titer of neutralizing antibody to the challenge virus below the protective threshold. From our current studies it appears that antibody-mediated passive protection against rotavirus challenge is dependent on both serotype and titer of antibody. PMID- 2999256 TI - The source of murine cytomegalovirus in mice receiving kidney allografts. AB - The sources of cytomegalovirus (CMV) infection in kidney transplant recipients include reactivation of latent endogenous virus in the recipient or reactivation of latent virus in donated blood or kidney. In the present study, kidneys from mice latently infected with one strain of murine CMV were transplanted into either uninfected recipients or recipients latently infected with a different strain of murine CMV; the recipients were immunosuppressed, subsequently were cultured for murine CMV, and the infecting strain was characterized. The results show that reactivation of latent murine CMV from the donated kidney can be the source of active infection in previously uninfected recipients. When the recipient had been previously infected, however, reactivation of the endogenous recipient strain of murine CMV was the source of active infection in 10 of 12 instances. At no time were both exogenous and endogenous strains of virus reactivated simultaneously. These studies indicate that donor kidney may be the source of latent virus in the uninfected recipient but that endogenous virus predominates in previously infected recipients. PMID- 2999255 TI - Morbidity of cytomegalovirus infection in recipients of heart or heart-lung transplants who received cyclosporine. AB - Forty-four heart and five heart-lung transplant recipients with cytomegalovirus (CMV) infection were investigated for risk factors associated with symptomatic CMV infection (17 patients) and CMV pneumonia (eight patients). Symptomatic infection was associated with primary rather than reactivated infection (P less than .005), younger age (P less than .005), heart-lung transplantation (P less than .001), and significant rises in titer of antibody to the early antigen of Epstein-Barr virus (P less than .001). Among recipients of heart transplants, patients with cardiomyopathy more often had symptomatic disease due to CMV (P less than .05). CMV pneumonia was associated with heart-lung transplantation and, in patients with primary CMV infection, earlier positive cultures for CMV after transplantation (P less than .02). CMV viremia was found in all patients with symptomatic infection, including the eight patients with CMV pneumonia, and the frequency of positive buffy coat cultures for CMV was significantly higher in patients with symptoms than in patients without symptoms (P less than .001). Neither symptomatic CMV infection nor CMV pneumonia was significantly associated with the use of antithymocyte globulin, restricted to therapy for rejection, and the use of high doses of acyclovir in 11 patients had no demonstrable impact on CMV culture positivity. PMID- 2999257 TI - New observations regarding killing of fibroblasts infected with herpes simplex virus: cooperation between elutable factor and peripheral mononuclear cells. AB - With a standard chromium release assay, natural killing (NK) activity of peripheral mononuclear cells (PMCs) from 28 individuals was compared based on the ability of sera to support antibody-dependent cell-mediated cytotoxicity (ADCC) for cells infected with herpes simplex virus (HSV). PMCs from all 20 individuals whose sera produced ADCC were capable of killing HSV-infected cells compared with none of the PMCs from the eight individuals whose sera did not produce ADCC (mean specific release, 33.2% vs. 6.8%). Results could not be explained by contaminating serum in the assay or by ineffective NK by the PMCs from the eight negative subjects because many of them killed the k562 myeloid cell line as effectively as PMCs from other individuals. In addition, NK could be eliminated by preincubation of effector cells at 37 C, and the capacity to kill by PMCs could be reconstituted by incubation in serum. Killing was more a function of source of serum rather than source of cells. PMID- 2999258 TI - Uptake of [125I]iododeoxycytidine by cells infected with herpes simplex virus: a rapid screening test for resistance to acyclovir. AB - A rapid screening test for resistance to acyclovir, mediated by a lack of thymidine kinase (TK) activity in herpes simplex virus (HSV), was developed by utilizing the uptake of [125I]iododeoxycytidine (IdC) by infected Vero cells. Cells infected with TK+ virus demonstrate uptake of IdC within 3 hr of infection. The assay can detect as few as 690 pfu of virus. Cells infected with TK virus have an uptake of IdC similar to that of uninfected cells, whereas cells infected with TK+ generally have an uptake greater than 10 times that of uninfected cells if tested when obvious viral cytopathic effect is present. All 19 clinical HSV isolates tested were correctly identified as TK+. Of 17 blinded HSV isolates tested, all six TK- isolates were correctly identified. A single strain with an ID50 of 3.4 micrograms/ml and an altered TK substrate specificity was incorrectly classified as TK+. The assay is a useful, rapid screening test for viral TK activity. PMID- 2999259 TI - Glycoproteins of human parainfluenza virus type 3: characterization and evaluation as a subunit vaccine. AB - The envelope glycoproteins of human parainfluenza type 3 virus were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reactivity with specific monoclonal antibodies. The molecular weight of the hemagglutinin neuraminidase (HN) glycoprotein was found to be 72,000, and the fusion (F) glycoprotein appeared to consist of 74,000 (F0) or 56,000 (F1) species. Envelope glycoproteins were solubilized with octyl-glucoside and, after removal of the detergent by dialysis, were used for immunization of hamsters. Other animals were immunized with a formalin-inactivated preparation of whole virus. A single subcutaneous immunization with these antigen preparations induced a serum antibody response to the HN and F glycoproteins, as determined by plaque neutralization, hemagglutination inhibition, inhibition of virus-induced cell fusion, and immune precipitation tests. An IgG antibody response to both glycoproteins was also observed in bronchial washings. Animals immunized with the highest dose of envelope glycoproteins showed complete protection from challenge infection, whereas immunization with inactivated virus did not completely protect animals. PMID- 2999261 TI - Determination of infection and immunity to varicella-zoster virus with an enzyme linked immunosorbent assay. PMID- 2999262 TI - Texas, teenagers, and CMV. PMID- 2999260 TI - Secretory and serum immunoglobulin class-specific antibodies to poliovirus after vaccination. AB - Immune responses in serum and saliva were studied in Pakistani children by enzyme linked immunosorbent assay after natural exposure to poliovirus and vaccination with live or inactivated poliovirus vaccines. Swedish children unexposed to wild poliovirus who had almost 100% vaccination coverage with inactivated vaccine at 8, 9, and 18 months and at 5 years of age were analyzed for comparison. Natural exposure induced secretory IgA (SIgA) antibodies to poliovirus in the saliva of Pakistani infants at one month of age that reached adult levels at six months. No difference in levels of salivary antibody at eight months was observed between groups vaccinated with either live or inactivated vaccines. Vaccination with live or inactivated vaccine starting at 2 or 3 months of age resulted in high titers of IgG antibody to poliovirus in serum, the highest of which occurred after four doses of live vaccine. In Sweden, an increase of antibody in serum was observed after the third vaccination. IgA antibodies continued to increase subsequently, whereas IgG antibodies reached a plateau. The SIgA response in saliva initially appeared on the third vaccination, with a significant increase after the fourth. Repeated vaccination with inactivated poliovirus vaccine induces specific SIgA antibodies. Adults all had SIgA antibodies to poliovirus in saliva. PMID- 2999263 TI - Cytomegalovirus antibodies of patients in the Gizan area of Saudi Arabia. PMID- 2999264 TI - Development of large granular lymphocytes in owl monkeys following EBV inoculation. PMID- 2999266 TI - Rotaviruses and immunobiologic failures. PMID- 2999265 TI - IgM neutralizing antibody to hepatitis A virus. PMID- 2999267 TI - [Cardiac involvement in systemic amyloidosis: myocardial scintigraphic evaluation]. AB - To assess the clinical significance of technetium-99m-pyrophosphate (Tc-99m-PYP), -methylene diphosphonate (Tc-99m-MDP) and thallium-201 (Tl-201) myocardial scintigraphy in the diagnosis of cardiac amyloidosis and in the differential diagnosis of cardiac diseases, 12 patients with biopsy-proved systemic amyloidosis (seven with familial amyloid polyneuropathy (FAP) and five with primary amyloidosis) were investigated. The results obtained were as follows: In 10 patients (six with FAP and four with primary amyloidosis) studied by Tc-99m PYP scintigraphy, two (FAP one, primary amyloidosis one) had diffusely positive myocardial uptake, which was of greater intensity than that of the sternum. Six (four FAP; two primary amyloidosis) also had diffusely positive myocardial uptakes, but the intensity was less than that of the sternum. The remaining two (one FAP; one primary amyloidosis) had only equivocal myocardial uptakes. Two of these patients also had hepatic uptakes and another had both hepatic and thyroid uptakes. The intensity of myocardial uptake of Tc-99m-PYP in patients with echocardiographic left ventricular hypertrophy and/or highly refractile myocardial echoes, so-called granular sparkling appearance (GS) was slightly greater than that in patients with neither myocardial hypertrophy nor GS. FAP had slightly less intensity than primary amyloidosis. In 29 persons with other cardiac diseases and normal subjects examined by Tc-99m-PYP scintigraphy, seven (two dilated cardiomyopathy; two sarcoidosis; three hypertensive heart disease) also had diffusely positive myocardial scans of mild or moderate degree. However, none of them had marked myocardial or other tissue uptakes. Both Tc-99m-PYP and MDP scintigraphic studies were performed in four patients (three FAP; one primary amyloidosis). In Tc-99m-MDP scintigraphy, diffusely positive myocardial uptakes were observed in two patients with FAP and the remaining two had negative scans. The intensity of Tc-99m-MDP myocardial uptake in each patient was significantly lower than that of Tc-99m-PYP uptake. Tl-201 scintigraphy was carried out in 10 patients (six FAP; four primary amyloidosis). Left ventricular hypertrophy was found in six patients and right ventricular visualization in five. Although electrocardiograms in seven of 10 patients showed QS patterns in the right to mid precordial leads, similar to that seen in antero-septal and extensive anterior myocardial infarctions, neither myocardial perfusion defect nor low uptake on Tl 201 images was detected in nine of them. The scintigrams in another one, which showed low uptake at the apical portion of the left ventricle, were considered normal.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2999268 TI - [Use of calcium phosphate ceramics in periodontal therapy. 4. Radiographic quantitative analysis to determine the prognosis in hydroxyapatite use]. PMID- 2999269 TI - [A clinical evaluation of porous hydroxyapatite ceramics implants in human periodontal bone defects--results after 1 year]. PMID- 2999270 TI - [Effect of saliva on fluorapatite formation on tooth surfaces using a silica gel method]. PMID- 2999271 TI - [Serum neuron-specific enolase in patients with lung cancer]. PMID- 2999272 TI - Comparison of superoxide generation and luminol-dependent chemiluminescence with eosinophils and neutrophils from normal individuals. AB - Although eosinophilia is found in many allergic and hypersensitivity diseases, the function of the eosinophil is not clearly established. To evaluate and characterize this function, anticoagulated blood from normal subjects was separated into purified populations of both eosinophils and neutrophils by a modified method for Percoll gradients. With this separation procedure, highly purified populations of eosinophils (95.0% +/- 2.1%) and neutrophils (97.2% +/- 0.4%) were obtained. Functional response of these two isolated granulocyte cell types was measured by luminol-dependent chemiluminescence (CL) and superoxide generation to opsonized zymosan and phorbol 12-myristate 13-acetate (PMA). Both the eosinophil and neutrophil peak CL response and superoxide generation to zymosan (1 mg), in the presence of autologous serum (10%), were identical. In contrast, when PMA (10(-4) to 10(0) micrograms/ml) was the stimulant, eosinophil CL was at least twofold greater than the neutrophil light emission (1,595,741 +/- 122,435 cpm/5 X 10(5) cells vs. 765,448 +/- 24,171 cpm/5 X 10(5) cells; n = 6). This same differential in responsiveness was seen in superoxide generation. Thus, under certain conditions the eosinophil's respiratory burst may be greater than that of the neutrophil, and this differential in metabolic activity may contribute directly to the eosinophil's inflammatory potential. PMID- 2999273 TI - Isozymes of human erythrocyte pyrimidine 5'-nucleotidase. AB - A scheme for the partial purification of human erythrocyte pyrimidine nucleotidase isozymes is presented. The characteristics of three of these were distinctively established. One isozyme, designated pyrimidine nucleotidase (PyrNase), is active for uridine monophosphate (UMP) and cytidine monophosphate (CMP), but not for thymidine monophosphate (dTMP). Two other isozymes, designated thymidine nucleotidase I (ThyNase I) and thymidine nucleotidase II (ThyNase II), were active for dTMP, but less so for UMP and CMP. Molecular weights of PyrNase, ThyNase I, and ThyNase II were estimated as 52,000, 52,000, and 48,000, respectively. Isoelectric points of these isozymes were 5.22, 4.90, and 4.68, respectively. Kinetic study revealed that the Km value for dTMP of ThyNase I was 3.83 mmol/L and of ThyNase II was 6.26 mmol/L. PMID- 2999275 TI - Synchronous parotid tumors of different histological types in association with metastasizing hypopharyngeal carcinoma. AB - At operation for metastasizing carcinoma of the piriform sinus a patient was found to have two separate parotid tumors, one a pleomorphic adenoma and the other a papillary cystadenoma lymphomatosum (Warthin's tumor). These tumors presented as palpable masses on the same side as a lymph node involved with carcinoma. Synchronous parotid tumors of different types are rare; previous reports in the literature show that this combination of pleomorphic adenoma and Warthin's tumor is the most common. We have found no previous report of multiple parotid neoplasms in a patient with coexistent squamous carcinoma of the head and neck. PMID- 2999276 TI - Congenital pleomorphic adenoma of the nasopharynx (report of a case). AB - A case of congenital salivary gland tumour occurring in the nasopharynx is reported. Congenital neoplasms of the head and neck (of any histological type) and congenital tumours of the nasopharynx are discussed and the literature is reviewed. PMID- 2999277 TI - Granular cell tumours of the oesophagus and larynx. AB - Two cases of granular cell tumour of the oesophagus and one case of granular cell tumour of the larynx are reported. The light and electron microscope findings are discussed, as well as the much debated histogenesis of such tumours. PMID- 2999274 TI - Aggregation of chymotrypsin-treated thrombasthenic platelets is mediated by fibrinogen binding to glycoproteins IIb and IIIa. AB - Previous experiments demonstrated that chymotrypsin, but not adenosine diphosphate (ADP), exposed fibrinogen binding sites on platelets from patients with Glanzmann's thrombasthenia. Three of these patients have been reexamined, and previous observations were confirmed. The quantity of iodine 125-labeled glycoprotein IIb (GPIIb) and glycoprotein IIIa (GPIIIa) on the platelets of these patients was considerably less than normal but was detectable by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. The amount of residual GPIIb and GPIIIa as measured by binding studies with radiolabeled monoclonal antibodies was between 3% and 12% of the normal value. Platelet suspensions from these patients did not aggregate with fibrinogen and did not bind 125I-fibrinogen on stimulation with ADP. However, incubation of these platelets with chymotrypsin or pronase resulted in fibrinogen binding and platelet aggregation. Monoclonal antibodies specific for the GPIIb GPIIIa complex blocked both the fibrinogen binding and the aggregation of enzyme treated platelets. The treatment of washed platelets of a fourth thrombasthenic patient with ADP or with chymotrypsin failed to result in fibrinogen binding and aggregation. However, the level of GPIIb and GPIIIa on these platelets as measured by a Western blot technique and by monoclonal antibody binding amounted to less than 0.35% to 0.5% of normal values. In conclusion, fibrinogen binding sites exposed on thrombasthenic platelets by chymotrypsin are derived from GPIIb GPIIIa molecules. Aggregation of chymotrypsin-treated thrombasthenic platelets by fibrinogen appears to represent a sensitive test for detection of functionally active GPIIb-GPIIIa complex on the platelet surface. PMID- 2999278 TI - The interaction of murine cytomegalovirus with murine neutrophils: effect on migratory and phagocytic activities. AB - To investigate the interaction of cytomegalovirus (CMV) with neutrophils, we studied the effects of in vitro incubation of murine neutrophils with murine CMV (MCMV). Neutrophils incubated with MCMV for 4 h had depressed chemotactic activity, mean chemotactic index of 1.34 +/- 1.43 for MCMV-treated neutrophils vs 3.22 +/- 2.11 for controls. Engulfment of latex spheres by MCMV-treated neutrophils was also reduced. These differences were not attributable to loss of cell viability. UV-inactivation of MCMV pools abolished the inhibitory effects of MCMV, indicating that these effects were related to infectivity of the virus. Electron microscopic studies at 4 h demonstrated virus particles within the phagosomes of occasional neutrophils. These studies demonstrate that neutrophil functions can be altered by an in vitro interaction with infectious CMV and suggest that a direct effect of CMV on neutrophils could account for the abnormal neutrophil functions observed during animal CMV infections. PMID- 2999279 TI - Serum activity and hepatic secretion of lecithin:cholesterol acyltransferase in experimental hypothyroidism and hypercholesterolemia. AB - Lecithin:cholesterol acyltransferase (LCAT), the major cholesterol esterifying enzyme in plasma, plays an important role in the removal of cholesterol from peripheral tissues. This study in rat focuses upon the effects of hypothyroidism and cholesterol feeding on serum activity and hepatic LCAT secretion. To obviate the effect that inclusion of high concentrations of cholesterol in the rat serum may have on the proteoliposome used in the assay of LCAT, very low and low density lipoproteins (VLDL and LDL) were removed by ultracentrifugation at d 1.063 g/ml. The molar esterification rate in the euthyroid VLDL + LDL-free serum was found to be 0.94 +/- 0.06 compared to 0.67 +/- 0.05 in hypothyroid rats and 1.56 +/- 0.14 in hypercholesterolemic rats. LCAT secretion by suspension cultures of hepatocytes from hypercholesterolemic rats was found to be significantly depressed when compared to that for euthyroid and hypothyroid animals. Secretion by hepatocytes from hypothyroid rats was depressed for the first 0-4 hr, but rapidly recovered. The depressed secretion of LCAT by hepatocytes from hypercholesterolemic rats correlates with the appearance in the media of apoE rich, discoidal HDL. Discoidal HDL was six times more effective as a substrate for purified human LCAT than HDL from hypercholesterolemic serum, and twice as effective as serum and nascent HDL from euthyroid animals. It is concluded that the depressed LCAT activity in serum from hypothyroid rats is due to a depressed hepatic secretion of the enzyme and that the elevated serum activity of hypercholesterolemic rats may be related to a defect in LCAT clearance. Finally, the appearance of discoidal HDL in the medium upon culture of hepatocytes from hypercholesterolemic rats appears to be due to an inhibition of LCAT secretion by these cells. PMID- 2999281 TI - Improved glucose tolerance induced by long term dietary supplementation with hairy basal seeds (Ocimum canum sim) in diabetics. PMID- 2999280 TI - Isolation, characterization, and uptake in human fibroblasts of an apo(a)-free lipoprotein obtained on reduction of lipoprotein(a). AB - Treatment of native human Lp(a) under nondenaturing conditions with dithiothreitol yielded both a lipoprotein particle and a lipid-free protein component that could be separated by either ultracentrifugation at d 1.063 g/ml or heparin-Sepharose chromatography. The protein component only showed antigenicity against anti-Lp(a) but not against anti-B. It was heterogeneous according to SDS polyacrylamide gel electrophoresis (PAGE) consisting of two bands, a major band with molecular weight similar to apoB and a minor band with slightly lower molecular weight. The lipoprotein particle was similar to LDL with regard to its electrophoretic mobility, lipid-protein composition, its apparent molecular weight according to gel-exclusion chromatography, and its apoprotein content; only apoB was found to be present by SDS-PAGE and immunochemical analysis. This lipoprotein also proved to be identical to LDL in its uptake by the receptor-mediated LDL-pathway in cultured human fibroblasts as shown by the similarity of the concentration-dependent binding, internalization, and degradation curves at 37 degrees C of the 125I-labeled lipoproteins. Normal Lp(a) was not taken up as readily as either its reduced lipoprotein component or LDL in the various steps of the receptor-mediated pathway. The maximal capacity for Lp(a) in the degradation assay was only 25% of that of LDL and it had a fourfold higher Km. It is therefore probable that the LDL-receptor-mediated pathway is not a major route for the clearance of Lp(a) in vivo. These studies suggest that Lp(a) is, in essence, an LDL-particle to which the protein (a) is attached through disulfide bonds to apoB. PMID- 2999282 TI - Characterization of a specific insulin-like growth factor-I/somatomedin-C receptor on high density, primary monolayer cultures of bovine articular chondrocytes: regulation of receptor concentration by somatomedin, insulin and growth hormone. AB - In order to obtain a phenotypically stable cell population of chondrocytes, high density primary monolayer cultures of bovine articular chondrocytes were established. Using these cultures, a specific insulin-like growth factor I/somatomedin-C (IGF-I/SM-C) receptor was demonstrated and characterized. At 15 degrees C steady-state binding was attained by 5 h, and averaged 25% per 2.2 X 10(6) cells. Fifty per cent displacement of 125I-labelled IGF-I/SM-C by unlabelled IGF-I/SM-C occurred at concentrations of only 2.3 ng/ml, whereas IGF II and porcine insulin were approximately 15- and 1000-fold less potent respectively. Scatchard analysis gave a linear plot, with a calculated association constant of 2.26 X 10(9) l/mol and a receptor number of 15 400 sites per cell. Preincubation of chondrocyte monolayers with either IGF-I SM-C or porcine insulin at 37 degrees C for 20 h resulted in reduction of 125I-labelled IGF-I/SM-C binding in a dose-dependent manner, although higher concentrations were required with insulin. More than 40% down-regulation of the receptor occurred with IGF-I/SM-C at concentrations of 10 nmol/l and nearly 70% reduction at 50 nmol/l. Interestingly, after preincubation with either human (h) or bovine (b) GH, 40% down-regulation of 125I-labelled IGF-I/SM-C binding was observed at concentrations of 10 mumol/l. Local production of IGF-I/SM-C by chondrocytes in response to GH stimulation may have occurred, but, because only 120 pmol IGF-I/SM C and less than 30 pmol IGF-I/SM-C per litre were recovered from serum-free conditioned media preincubated with bGH and hGH respectively, this was not established.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2999283 TI - The functional significance of glycosylation of pro-opiomelanocortin in melanotrophs of the mouse pituitary gland. AB - Pro-opiomelanocortin (POMC) is a glycoprotein precursor for a number of neuropeptides and peptide hormones. The functional significance of the glycosylation of POMC has never been established. Using the antibiotic tunicamycin to block glycosylation of the prohormone in the mouse pars intermedia, we have compared processing of non-glycosylated prohormone with that of glycosylated prohormone in pulse-chase experiments. The peptides produced from non-glycosylated prohormone were shown to be correct cleavage products. Therefore it was concluded that, with the possible exception of peptides from the N terminal region of the prohormone, the carbohydrate on POMC plays no role in directing cleavage or in protecting the prohormone from random proteolysis. Tunicamycin treatment retarded N-terminal acetylation of melanotrophin but had no apparent effect on acetylation of beta-endorphin. The mouse pars intermedia synthesizes two forms of POMC which differ in their degree of glycosylation. Our results indicated that, during secretion, the melanotrophs make no distinction between peptides derived from the two prohormones. PMID- 2999284 TI - Regulation of testosterone production in Leydig cells from fetal mice under dynamic conditions: effect of human chorionic gonadotrophin and 8-bromo-cyclic AMP. AB - The temporal release of testosterone by Leydig cells from 18-day-old mouse fetuses in response to human chorionic gonadotrophin (hCG) and to 8-bromocyclic AMP (8-bromo-cAMP) was investigated under short-term incubation (180 min) conditions. A rapid and large increase in testosterone release was induced by a 5 min exposure to hCG (20 i.u./l) or 8-bromo-cAMP (10 mmol/l). The testosterone response of fetal Leydig cells to the two gonadotrophic stimuli was Gaussian in distribution with a peak value of testosterone by 15-20 min. Repeated exposure to hCG resulted in a reduced testosterone response but an increased accumulation of cAMP. The apparent resistance of fetal Leydig cells to hCG could not be overcome either by increasing the hCG concentration (to 2000 i.u./l) or by exposing the cells to 8-bromo-cAMP (10 mmol/l). Continuous exposure to hCG (200 i.u./l) divided into multiple small doses (each 8 i.u./l) induced testosterone secretion with different kinetic characteristics: a three-fold longer time-lag between hormone exposure and the peak value; a twofold greater testosterone response (P less than 0.001) and a gradual decrease of testosterone secretion. Oestradiol significantly reduced basal and hCG-stimulated testosterone production only at a high concentration (10 mumol/l). These results indicate that continuous or pulsatile exposure to hCG can induce refractoriness of fetal Leydig cells. The similarity between the actions of hCG and 8-bromo-cAMP on fetal steroidogenesis suggests that this rapid defect is not primarily due to a depletion of gonadotrophin receptors but results from disruption of regulatory mechanisms at the post-receptor level. PMID- 2999285 TI - Endogenous opioid peptides and hypothalamic neuroendocrine neurones. AB - This consideration of the influence of endogenous opioid peptide systems on GnRH and oxytocin neurones serves to illustrate some of their possible regulatory interactions with other neuroendocrine systems. Opioids are known to influence the secretion of all the anterior pituitary hormones (see Grossman & Rees, 1983) and these effects are likely to be mediated, at least in part, in the hypothalamus. For example, inhibitory effects of opioids have also been described on secretion from the median eminence of somatostatin (Drouva et al. 1981b) and dopamine (Wilkes & Yen, 1980), and this site of action probably accounts for at least some of the stimulatory effects of exogenous opioids on plasma growth hormone and prolactin levels respectively. For the GnRH neurones the influence of endogenous opioid neurones, possibly the arcuate beta-endorphin system, appears to be mediated indirectly by inhibiting release of excitatory or facilitatory monoamines. This opioid-adrenergic interaction itself appears to be central in the regulation of gonadotrophin secretion and mediation of the feedback effects of gonadal steroids in the brain. The steroids may act directly on both adrenergic and opioid neurones, altering monoamine metabolism and release which may, in turn, regulate numbers of adrenergic receptors perhaps located on the GnRH neurones. Opioid peptide levels are also modulated by steroids probably reflecting altered synthesis and/or processing of precursors. Regulation of the opioid-adrenergic input may not only acutely affect the secretory output of the GnRH neurones but also influence synthesis or processing of GnRH itself (see Kalra & Kalra, 1984) and its degradation by hypothalamic peptidases (Advis, Krause & McKelvy, 1983). Oxytocin neurones demonstrate three further levels of interaction with endogenous opioid peptides. First the anatomical organization of the oxytocin neurones has enabled a clear demonstration of the action of opioids close to the secretory terminals to uncouple the generation of electrical activity from release of peptide. Secondly, both the oxytocin and the neighbouring vasopressin neurones themselves synthesize, process and package opioid peptides. These neurones thus provide a clear example of co-existence of several biologically active products in individual neurones. The relative expression of the different gene products may prove to be a further level of control of opioid influences on the oxytocin and vasopressin neurones.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2999286 TI - Effects of nutritional and mechanical properties of food on ruminative behavior. AB - Previous studies have identified a reliable relation between the quantity of food ingested and ruminating in profoundly retarded individuals and have established some parametric characteristics of this relation. The present study investigated three different properties of food that may influence this relation. Experiment 1 examined the role of stomach distention produced by including in the subject's diet wheat bran in amounts equivalent to and exceeding the calculated amount of crude fiber in the starch-satiation diet reported by Rast, Johnston, Drum, and Conrin (1981) and Rast, Johnston, and Drum (1984). There was a decrease in ruminating, although this decrease was smaller and more gradual than in the starch-satiation condition. Experiment 2 showed that increasing calories without increasing food volume resulted in a gradual and moderate decrease in ruminating. Experiment 3 replicated and extended the first two experiments by varying both caloric intake and stomach distention as well as oropharyngeal and esophageal stimulation in a different sequence of conditions. All variables exerted some control over responding. However, the large and immediate effects of the starch satiation procedure occurred only when subjects were permitted to consume unlimited quantities. PMID- 2999287 TI - Biological activities of a human pluripotent hemopoietic colony stimulating factor on normal and leukemic cells. AB - We studied the biological effects of pluripoietin, a human pluripotent hemopoietic colony-stimulating factor (CSF) purified from the 5637 bladder carcinoma cell line. We found that this human CSF appears to be a unique hemopoietic growth factor, differing from interleukin 3 (IL-3) by virtue of its leukemia differentiating activity in mouse and man, and from mouse granulocyte CSF, which does have differentiation-inducing activity, but lacks pluripoietic activity. In addition, differences from IL-3 were observed in cross-species activity on normal and leukemic cells. PMID- 2999288 TI - Diversity in the germline antibody repertoire. Molecular evolution of the T15 VN gene family. AB - The T15 heavy chain variable region (VH) gene family in BALB/c mice includes four elements each greater than 88% homologous with the other. One of these elements, V1, encodes virtually all of the VH regions in BALB/c antiphosphorylcholine antibodies, while another element, V3, is a pseudogene and cannot be transcribed or translated. We have examined the structural features of this VH gene family in other mouse strains and, in particular, have cloned and sequenced the alleles of these gene segments present in B10.P mice. Each of the four B10.P sequences can be matched with its allelic counterpart in BALB/c mice. This represents the first successful analysis of allelism in antibody variable region gene segments. The V1B10.P allele, like its BALB/c counterpart, encodes most of the known phosphorylcholine binding heavy chains from C37BL/6 mice. Similarly, the V3B10.P gene segment is a pseudogene like V3BALB, although only two of four abnormalities present in the BALB/c allele are also present in the B10.P allele. Careful analysis of the specific substitutions observed in the T15 VH gene family suggests that environmental selection for functional combining regions contributes significantly to the pattern of variation in the germline antibody repertoire. In addition, evidence is presented supporting frequent gene conversion events in the divergence of antibody genes. PMID- 2999289 TI - Cachectin/tumor necrosis factor stimulates collagenase and prostaglandin E2 production by human synovial cells and dermal fibroblasts. AB - Cachectin/TNF (tumor necrosis factor), an endotoxin-induced murine macrophage hormone implicated in the pathogenesis of cachexia and shock, has been found capable of stimulating collagenase and prostaglandin E2 (PGE2) production by isolated human synovial cells and dermal fibroblasts. This bioactivity associated with cachectin is comparable to that observed with the monokine interleukin 1 (IL 1), previously suggested as the major mediator of proteolysis. The ability of cachectin/TNF to stimulate collagenase and PGE2 production suggests that it may play a role in tissue destruction and remodelling, as these processes occur in inflammatory diseases. PMID- 2999290 TI - Comparison of substrates for measuring serum choline esterase activity in hepato biliary disease. AB - The efficacy of various substrates for measuring serum choline esterase for the evaluation of hepatic function was studied using o-toluoyl- and succinyl-choline, and acetyl, butyryl- and propionyl-thiocholine. In hepatic disease, the serum choline esterase activity with these substrates was decreased at a similar rate, showing no significant difference. In 78 - 84% of cases with hepatic cirrhosis the enzyme activity with these substrates was less than 50% of the average level of normal individuals, but in acute and chronic hepatitis only 4-9 and 12-14% of patients showed these lower values, respectively. The present study indicates the usefulness of sequential monitoring of serum choline esterase activity with any of these substrates for assessing hepatic disease, particularly cirrhosis, and for monitoring the course of hepatic disease. PMID- 2999292 TI - Cannabinoids in blood and urine after passive inhalation of Cannabis smoke. AB - To test the possibility that cannabinoids are detectable following passive inhalation of Cannabis smoke the following study was performed. Five healthy volunteers who had previously never used Cannabis, passively inhaled Cannabis smoke for 30 min. Cannabis smoke was provided by other subjects smoking either marijuana or hashish cigarettes in a small closed car, containing approximately 1650 L of air. delta 9-Tetrahydrocannabinol (THC) could be detected in the blood of all passive smokers immediately after exposure in concentrations ranging from 1.3 to 6.3 ng/mL. At the same time total blood cannabinoid levels (assayed by radioimmunoassay [RIA] ) were higher than 13 ng/mL in four of the volunteers. Both THC and cannabinoid blood concentrations fell close to the cutoff limits of the respective assays during the following 2 h. Passive inhalation also resulted in the detection of cannabinoids in the urine by RIA and enzyme multiple immunoassay technique (EMIT) assays (above 13 and 20 ng/mL, respectively). It is concluded that the demonstration of cannabinoids in blood or urine is no unequivocal proof of active Cannabis smoking. PMID- 2999291 TI - Dichotomous effects of forskolin on somatic and germ cell components of the ovarian follicle: evidence of cAMP involvement in steroid production and action. AB - The role of cyclic AMP (cAMP) in ovarian follicular functions in Rana pipiens was investigated with the use of the adenylate cyclase stimulator, forskolin, which is thought to elevate intracellular level of cAMP. Effects of forskolin on oocyte germinal vesicle breakdown (GVBD) and on progesterone production by the follicles were assessed during the course of in vitro culture. Addition of forskolin to culture medium suppressed both progesterone-and frog pituitary homogenate (FPH) induced meiotic maturation of the oocytes. Inhibitory effects of forskolin were essentially reversible and forskolin completely inhibited GVBD when added during the first four hours of incubation following exposure to progesterone. Forskolin alone stimulated a low level progesterone production by isolated follicles, but markedly stimulated progesterone production when it was supplemented with a low dose of FPH (0.005 pituitary equivalent/ml). Thus, forskolin acts synergistically with FPH on follicle cells to stimulate progesterone production. A higher dose of FPH (0.05 pitui. eq./ml) produced no additional synergistic effect of forskolin. Therefore, forskolin appears to have two contradictory functions in ovarian follicles: it augments FPH induced follicle secretion of meiosis initiator, progesterone, and simultaneously suppresses the maturation of the oocytes triggered by exogenous progesterone or FPH. The data presented indicate that there are two independent adenylate cyclase systems in the ovarian follicles which have separate functions: one in the follicle cells and the other in the oocyte. The two enzyme systems are thus compartmentalized and regulate different biological functions using the same messenger, cAMP. The data provide evidence that in amphibians, as in mammals, pituitary hormones regulate steroid hormone production by follicle cells via a cyclic AMP system. Thus, control of oocyte maturation induction appears to be determined by the relative levels of cAMP present in the follicle cells and oocytes. PMID- 2999293 TI - Mechanism of basolateral membrane H+/OH-/HCO-3 transport in the rat proximal convoluted tubule. A sodium-coupled electrogenic process. AB - In order to examine the mechanism of basolateral membrane H+/OH-/HCO-3 transport, a method was developed for the measurement of cell pH in the vivo doubly microperfused rat proximal convoluted tubule. A pH-sensitive fluorescein derivative, (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein, was loaded into cells and relative changes in fluorescence at two excitation wavelengths were followed. Calibration was accomplished using nigericin with high extracellular potassium concentrations. When luminal and peritubular fluids were pH 7.32, cell pH was 7.14 +/- 0.01. Decreasing peritubular pH from 7.32 to 6.63 caused cell pH to decrease from 7.16 +/- 0.02 to 6.90 +/- 0.03. This effect occurred at an initial rate of 2.4 +/- 0.3 pH units/min, and was inhibited by 0.5 mM SITS. Lowering the peritubular sodium concentration from 147 to 25 meq/liter caused cell pH to decrease from 7.20 +/- 0.03 to 6.99 +/- 0.01. The effect of peritubular sodium concentration on cell pH was inhibited by 0.5 mM SITS, but was unaffected by 1 mM amiloride. In addition, when peritubular pH was decreased in the total absence of luminal and peritubular sodium, the rate of cell acidification was 0.2 +/- 0.1 pH units/min, a greater than 90% decrease from that in the presence of sodium. Cell depolarization achieved by increasing the peritubular potassium concentration caused cell pH to increase, an effect that was blocked by peritubular barium or luminal and peritubular sodium removal. Lowering the peritubular chloride concentration from 128 to 0 meq/liter did not affect cell pH. These results suggest the existence of an electrogenic, sodium coupled H+/OH-/HCO-3 transport mechanism on the basolateral membrane of the rat proximal convoluted tubule. PMID- 2999294 TI - Circadian regulation of retinomotor movements. I. Interaction of melatonin and dopamine in the control of cone length. AB - In lower vertebrates, cone retinomotor movements occur in response to changes in lighting conditions and to an endogenous circadian clock. In the light, cone myoids contract, while in the dark, they elongate. In order to test the hypothesis that melatonin and dopamine may be involved in the regulation of cone movement, we have used an in vitro eyecup preparation from Xenopus laevis that sustains light- and dark-adaptive cone retinomotor movement. Melatonin mimics darkness by causing cone elongation. Dark- and melatonin-induced cone elongation are blocked by dopamine. Dopamine also stimulates cone contraction in dark adapted eyecups. The effect of dopamine appears to be mediated specifically by a dopamine receptor, possibly of the D2 type. The dopamine agonist apomorphine and the putative D2 agonist LY171555 induced cone contraction. In contrast, the putative D1 agonist SKF38393-A and specific alpha 1-, alpha 2-, and beta adrenergic receptor agonists were without effect. Furthermore, the dopamine antagonist spiroperidol not only blocked light-induced cone contraction, but also stimulated cone elongation in the light. These results suggest that dopamine is part of the light signal for cone contraction, and that its suppression is part of the dark signal for cone elongation. Melatonin may affect cone movement indirectly through its influence on the dopaminergic system. PMID- 2999295 TI - Environmental regulation of carbohydrate metabolism by Streptococcus sanguis NCTC 7865 grown in a chemostat. AB - Carbohydrate metabolism by the oral bacterium Streptococcus sanguis NCTC 7865 was studied using cells grown in a chemostat at pH 7.0 under glucose or amino acid limitation (glucose excess) over a range of growth rates (D = 0.05 h-1-0.4 h-1). A mixed pattern of fermentation products was always produced although higher concentrations of lactate were formed under amino acid limitation. Analysis of culture filtrates showed that arginine was depleted from the medium under all conditions of growth; a further supplement of 10 mM-arginine was also consumed but did not affect cell yields, suggesting that it was not limiting growth. Except at the slowest growth rate (D = 0.05 h-1) under glucose limitation, the activity of the glucose phosphotransferase (PTS) system was insufficient to account for the glucose consumed during growth, emphasizing the importance of an alternative method of hexose transport in the metabolism of oral streptococci. The PTS for a number of sugars was constitutive in S. sanguis NCTC 7865 and, even though the cells were grown in the presence of glucose, the activity of the sucrose-PTS was highest. The glycolytic activity of cells harvested from the chemostat was affected by the substrate, the pH of the environment, and their original conditions of growth. Glucose-limited cells produced more acid than those grown under conditions of glucose excess; at slow growth rates, in particular, greater activities were obtained with sucrose compared with glucose or fructose. Maximum rates of glycolytic activity were obtained at pH 8.0 (except for cells grown at D = 0.4 h-1 where values were highest at pH 7.0), while slow growing, amino acid-limited cells could not metabolize at pH 5.0. These results are discussed in terms of their possible significance in the ecology of dental plaque and the possible involvement of these bacteria in the initiation but not the clinical progression of a carious lesion. PMID- 2999296 TI - Effects of imidazole- and triazole-derivative antifungal compounds on the growth and morphological development of Candida albicans hyphae. AB - Six azole-derivative antifungal compounds affected several aspects of Candida albicans hyphal development with only a relatively small degree of inhibition of growth rate, measured in terms of ATP concentration, whereas amphotericin B and 5 fluorocytosine affected morphology only when they also substantially inhibited fungal growth rate. At 10(-8) M, all the azoles tested inhibited branch formation by C. albicans hyphae. At 10(-7) M and higher concentrations, clotrimazole and miconazole strongly suppressed emergence of new hyphal outgrowths from parent yeast cells, whereas ICI 153066 and itraconazole had little effect on this phenomenon and ketoconazole and tioconazole had intermediate effects. At the highest concentrations tested (10(-5) M) hyphal development was ultimately arrested by the azole compounds and the fungus grew predominantly in the form of budding yeast cells; however, none of the azole antifungals prevented initial emergence of an apparently normal germ tube. The antifungals only exerted their morphological effects when they were present in the culture medium: removal of the compounds after exposure of C. albicans to them led to reversion to normal growth. PMID- 2999297 TI - Derepression of sporulation and synthesis of mycobacillin and dipicolinic acid by guanosine 3':5'-cyclic monophosphate under conditions of glucose repression in Bacillus subtilis. AB - Dibutyryl cyclic GMP, but not dibutyryl cyclic AMP, derepresses sporulation and synthesis of mycobacillin and dipicolinic acid under conditions of glucose repression in Bacillus subtilis strain B34. Neither of these compounds appears to affect sporulation and synthesis of mycobacillin and dipicolinic acid in this strain under normal physiological conditions. Mutants insensitive to glucose repression were indifferent to the addition of either of the nucleotides both in the presence and in the absence of glucose. A role for dibutyryl cyclic GMP in annulling the repressing effect of glucose on sporulation and on synthesis of mycobacillin and dipicolinic acid is thus indicated. PMID- 2999298 TI - Molecular cloning and expression of a xylanase gene of alkalophilic Aeromonas sp. no. 212 in Escherichia coli. AB - A gene coding for a xylanase activity of alkalophilic Aeromonas sp. no. 212 (ATCC 31085) was cloned in Escherichia coli HB101 with pBR322. Plasmid pAX1 was isolated from transformants producing xylanase, and the xylanase gene was located in a 6.0 kb Hind III fragment. The pAX1-encoded xylanase activity in E. coli HB101 was about 80 times higher than that of xylanase L in alkalophilic Aeromonas sp. no. 212. About 40% of the enzyme activity was observed in the periplasmic space of E. coli HB101. The pAX1-encoded xylanase had the same enzymic properties as those of xylanase L produced by alkalophilic Aeromonas sp. no. 212, but its molecular weight was lower (135 000 vs 145 000, as estimated by SDS polyacrylamide gel electrophoresis). PMID- 2999299 TI - The generation of metronidazole radicals in hydrogenosomes isolated from Trichomonas vaginalis. AB - The nitro radical-anion of the anti-trichomonal drug metronidazole has been detected by electron spin resonance spectrometry under anaerobic conditions in suspensions of intact hydrogenosomes isolated from the parasitic protozoon Trichomonas vaginalis. Metronidazole reduction was driven by pyruvate, but progressive damage to the radical generating system was observed. Quenching of signals due to metronidazole radicals by chromium oxalate suggests that the radicals generated within the organelle can cross the hydrogenosomal membrane into the external medium. Even if a similar process of radical migration occurs in vivo, it seems likely that intrahydrogenosomal damage may explain drug action. PMID- 2999300 TI - Immunochemical characterization of the outer membrane complex of Serratia marcescens and identification of the antigens accessible to antibodies on the cell surface. AB - Serratia marcescens New CDC O14:H12 contains major outer membrane proteins of 43.5 kDal, 42 kDal (the porins) and 38 kDal (the OmpA protein) which can be separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Immunoblotting of whole cell or outer membrane preparations using antiserum raised against the whole cells revealed similar complex patterns of antigens. The OmpA protein was the major immunogen, although six other outer membrane proteins were also detected; the porins reacted only weakly with antibodies in this system. Immunoabsorption of antisera with whole cells showed that only the O antigenic chains of lipopolysaccharide and the H (flagella) antigens were accessible to antibody on the cell surface. Failure to detect the OmpA protein and other envelope antigens in this way suggests that their antigenic sites are not able to react with antibodies and are possibly masked by the O antigen. PMID- 2999301 TI - Protease production during sporulation of germination mutants of Bacillus subtilis and the cloning of a functional gerE gene. AB - Early in sporulation, cells of wild-type Bacillus subtilis produce three proteases (b, c and d) with monomeric Mr values of about 65 000, 53 000 and 43 500, and a further protease, e (Mr about 30 000) at the time of coat assembly. An additional protease, f (Mr about 15 000) appears transiently in sporangia at about the time of spore release. Three strains with defective spore coats were examined for alterations in sporulation proteases. A strain carrying the gerE36 mutation produces b, c and d normally, fails to produce e and accumulates f on or in its spores. A strain carrying the spoVIC610 mutation produces normal quantities of proteases b, c and d, but has a reduced amount of proteases e and f. A strain carrying both the gerE36 and the spoVIC610 mutations accumulates neither protease e nor f. The wild-type allele of the gerE gene was cloned in the vector, phage phi 105J9. Complementation tests with the cloned gene showed that the gerE36 mutation is recessive to the wild-type allele. PMID- 2999303 TI - Tn2440, a composite tetracycline resistance transposon with direct repeated copies of IS160 at its flanks. AB - The tetracycline resistance region of the multi-resistance plasmid pBP16 is flanked by direct repeats of the insertion sequence IS160. The tetracycline resistance region plus the flanking IS elements can transpose as a discrete unit. The composite transposon, designated Tn2440, has a size of 4.0 kb. PMID- 2999302 TI - Genetic and biochemical characterization of the red gene cluster of Streptomyces coelicolor A3(2). AB - Production of the red antibiotic, undecylprodigiosin, by Streptomyces coelicolor A3(2) was studied by DNA cloning and biochemical analysis. Over 21 kb of genomic DNA were cloned, in several segments, into plasmid vectors. The cloned DNA 'complemented' several specific mutations in the red gene cluster. Four red genes (redA, B, E, and F) were mapped to different regions within the cloned DNA. Screening with redE probes for DNA homologies among various streptomycetes revealed hybridizing DNA in three strains, one of them not known to synthesize prodigiosin pigments. Biochemical studies using protoplasted cells revised our interpretation of the nature of redE and redF mutations. Two forms of undecylnorprodigiosin: S-adenosylmethionine O-methyltransferase activity on gel filtration columns were detected: a very high molecular mass peak (greater than 5 MDal) and a 49 kDal) and a 49 kDal peak. Analyses of extracts from red mutants suggested that these two forms are related, and that at least the redE and redF gene products are necessary for O-methyltransferase activity in vivo. Lack of activity of the redE gene in a heterologous host, S. glaucescens, is consistent with the necessity for a biosynthetic complex involving several red gene products for efficient expression. Experiments in liquid antibiotic production medium indicated that prodigiosin compounds in S. coelicolor are examples of 'secondary metabolites' whose synthesis lags behind that of cell mass. The peak of specific activity of O-methyltransferase coincided with the 'late exponential' phase of growth. Thus, understanding the genetic regulation of undecylprodigiosin biosynthesis in S. coelicolor may be relevant to other antibiotic production pathways, and perhaps to 'secondary' metabolism in general. PMID- 2999304 TI - Isolation and preliminary characterization of lytic and lysogenic phages with wide host range within the streptomycetes. AB - Examination of approximately 700 soil samples yielded about 100 actinophages. Restriction analysis of phage DNA indicated that 57 are unique, and of these, 20 produce turbid plaques on one or more of the streptomycetes tested. Five phages are shown to insert into the genome of Streptomyces avermitilis. None of the phages was able to perform generalized transduction of S. avermitilis. PMID- 2999305 TI - Neutralization of yellow fever virus studied using monoclonal and polyclonal antibodies. AB - Monoclonal and polyclonal antibodies with known specificity for either the 54K envelope glycoprotein or the 48K non-structural glycoprotein of yellow fever (YF) virus-infected cells were studied in plaque reduction neutralization tests. Viruses employed in the tests comprised wild-type and vaccine strains of YF and a selection of other flaviviruses. Of 17 monoclonal antibodies examined, six of the 54K-specific antibodies neutralized at least one YF preparation. Both vaccine and wild-type YF viruses varied in their susceptibility to neutralization and there were also differences between individual 17D vaccine strains. The monoclonal antibodies produced a range of titres with the different viruses, the most potent, 864, leaving no non-neutralizable fraction. Addition of anti-globulin, complement or other antibodies did not affect the results. YF-neutralize antibodies which bound to other flaviviruses did not necessarily neutralize them; hence, neutralization could be defined as either homotypic, heterotypic or both homotypic and heterotypic. A polyclonal antiserum and a broadly reacting monoclonal antibody produced almost identical neutralization results in tests with wild-type YF viruses. In contrast, the polyclonal antiserum produced higher titres with vaccine strains of YF. In mouse passive protection experiments on the other hand, the monoclonal antibody did not differentiate between these viruses. PMID- 2999307 TI - Production of a monoclonal antibody against an epitope on HeLa cells that is the functional poliovirus binding site. AB - Cell lines of primate origin carry receptors on their plasma membrane which are responsible for the specific binding of poliovirus. This paper describes the isolation and characterization of a monoclonal antibody reacting with the plasma membrane of HeLa cells. The antibody (D171) was selected for its protection of HeLa cells against the cytopathic effect of poliovirus type 1. This protection was found to extend to all three viral serotypes, while the replication of five other viruses in HeLa cells was not affected. The 125I-labelled purified antibody did not react with cell lines derived from pig, dog or rodents but bound specifically to all lines of human or primate origin. Immunoglobulin or Fab fragments of D171 prevented the binding of 35S-labelled poliovirus to HeLa cells. Conversely, nearly all binding sites of 125I-labelled D171 immunoglobulins or Fab fragments could be blocked after preincubation of HeLa cells with poliovirus. These results indicate that D171 recognizes the poliovirus receptor site on different susceptible cells and that practically all D171 binding sites are involved in the specific attachment of poliovirus to the plasma membrane. To determine whether the epitope recognized by D171 could be separated from the receptor for poliovirus, human-mouse cell hybrids were prepared and analysed. In all 40 clones tested, the susceptibility to poliovirus correlated with the binding of D171. PMID- 2999306 TI - delta-9-Tetrahydrocannabinol decreases host resistance to herpes simplex virus type 2 vaginal infection in the B6C3F1 mouse. AB - The effect of delta-9-tetrahydrocannabinol (Delta-9-THC) on host resistance to herpes simplex virus type 2 (HSV-2) vaginal infection in the B6C3F1 mouse was determined. Animals were given Delta-9-THC or vehicle on days -1 to 2, or cyclophosphamide on day -1 or on days -1 to 2. HSV-2 was introduced intravaginally on day 0. Host resistance to virus infection was assessed by comparing frequency and severity of lesions, virus shedding and mortalities. Replicate groups of animals were bled on days 5, 8, 10, 14 and 21 post-viral inoculation to allow for screening for viraemia and for definition of the effect of Delta-9-THC on the humoral response. Animals were also employed for determination of delayed hypersensitivity responses (DHR). Virus-infected animals treated with 100 mg/kg Delta-9-THC exhibited greater severity of herpes genitalis, higher mortalities and higher mean titres of virus shed from the vagina. Suppression of the humoral response to HSV-2 occurred in animals treated with this dose of drug when compared to virus-infected vehicle controls. A delay in the onset of the DHR to HSV-2 was observed in animals receiving 100 mg/kg Delta-9-THC when compared with those receiving vehicle. These results indicate that Delta-9-THC decreases host resistance to HSV-2 vaginal infection in the B6C3F1 mouse. This decreased resistance is associated with suppression of the immune response to primary infection with HSV-2. PMID- 2999308 TI - Two initiation sites for foot-and-mouth disease virus polyprotein in vivo. AB - Typically, the translation of eukaryotic mRNAs into protein is initiated at a single site. However, we have recently shown that not one but two primary products, P20a and P16, are translated from the 5' end of the coding region of the genome of foot-and-mouth disease virus (FMDV). In this paper we show by partial protease digestion of these proteins that they differ only at their N termini, thus confirming the presence of two initiation sites for translation of FMDV RNA. Sequence analysis of two subtypes of the virus (A10 and A12) confirms the presence of two initiator AUG codons in the expected position on the genome. By correlation with protein synthesis data from these subtypes it appears that the relative use of each initiation site is dependent on its surrounding nucleotide sequence. In addition, the ratio of the two proteins when synthesized in vitro differs markedly from that when they are synthesized in vivo, suggesting the presence of a control mechanism for synthesis of P20a in vivo which may be absent in vitro. We also show that the cleavage site between these two proteins and the structural protein precursor, P88, is located closer to the N terminus of the polyprotein than has previously been reported. PMID- 2999309 TI - Oligonucleotide fingerprint analysis of coxsackievirus A10 isolated in Japan. AB - Eight coxsackievirus A10 strains isolated in 1978 and in 1981 and 1982 from patients with hand, foot-and-mouth disease and with herpangina at a dispensary in Matsue city were compared by RNA fingerprinting techniques. The oligonucleotide maps of the four 1978 isolates were related to each other by 85 to 93% with respect to their large T1 oligonucleotides. In contrast, the oligonucleotide maps of the four 1981 and 1982 isolates were very different from each other. Co electrophoresis experiments revealed that the 1981 and 1982 strains shared only 17 to 34% of their large oligonucleotides. In addition, some large oligonucleotides were found in most of the fingerprint maps of isolates from 1978 to 1982, suggesting that there are regions in the genome of coxsackievirus A10 which are not subject to mutational changes. PMID- 2999310 TI - Intermolecular recombination of the herpes simplex virus type 1 genome analysed using two strains differing in restriction enzyme cleavage sites. AB - Intermolecular recombination of herpes simplex virus type 1 (HSV-1) was studied by analysing the segregation of strain-specific restriction enzyme cleavage sites among progeny viruses produced after co-infection by two HSV-1 strains differing in eight restriction enzyme cleavage sites. Out of 93 progeny viruses examined, 51 clones were recombinant, and crossover sites of the recombinants were mapped on the HSV-1 genome. These sites were distributed evenly in the unique sequence of the L component (UL) and the recombination frequency in UL was estimated to be 1.12 per genome length, or 0.007 per kilobase pair. No evidence was obtained to support the existence of enhanced intermolecular recombination events in the regions containing inverted repeats and the L-S junction in comparison with the recombination frequency in UL. The finding of recombinants in an arrangement that minimized the number of crossover events suggested the participation of both of two arrangements of the L component of parental DNA (P or IS, and IL or ISL) in the generation of the recombinants. The possibility of a preference for P or IS over IL or ISL arrangements remains to be determined. PMID- 2999311 TI - A proposal for naming adenovirus genome types, exemplified by adenovirus type 6. AB - A numerical code to denominate adenovirus (AV) genome types is proposed. Seven restriction endonuclease patterns are listed in alphabetical order (BamHI, BGlII, BstEII, EcoRI, HindIII, KpnI, SmaI); patterns deviating from those of the prototype (1) are named 2, 3 etc. depending on the chronological order of the respective isolates. Thus AV6/3:1231121 is a type 6 strain deviating from the prototype in three patterns, i.e. those of BglII, BstEII and KpnI. From 24 AV6 isolates, six were identical with the prototype, whereas the other strains represented 13 different genome types. PMID- 2999312 TI - Antibody to the 32K structural protein of infectious bursal disease virus neutralizes viral infectivity in vitro and confers protection on young chickens. AB - Chicken sera containing IgG antibodies specific for the 32 000 (32K) mol. wt. structural polypeptide of infectious bursal disease (IBD) virus, as assessed by Western blotting, neutralized the in vitro infectivity of tissue culture-adapted IBD virus. When injected into young chickens, the serum passively protected them from challenge with pathogenic IBD virus. Chickens immunized with the 32K structural polypeptide of IBD virus, prepared by electroelution from SDS-PAGE gels, produced antibody detectable by ELISA and the virus neutralization assay, while chickens immunized with the 37K or 41.5K viral polypeptides synthesized antibody detectable by ELISA, but only very low levels of virus-neutralizing antibody. The immunoglobulin fraction of sera obtained from chickens immunized with the 32K polypeptide, but not the 41.5K polypeptide, passively protected chickens from infection with IBD virus. It is concluded that the 32K polypeptide is a major protective immunogen of IBD virus. PMID- 2999313 TI - Reassortment of human rotaviruses carrying rearranged genomes with bovine rotavirus. AB - Rotaviruses isolated from chronically infected immunodeficient children were previously shown to contain RNA yielding abnormal migration profiles on gels: normal RNA segments were lost or decreased in concentration, and additional bands of dsRNA were found which were derived (rearranged) from genome segments of lower molecular weight by concatemer formation. These viruses grew very slowly during passage in secondary rhesus monkey kidney cells. Upon superinfection with the tissue culture-adapted UK Compton strain of bovine rotavirus (BRV) extensive genome reassortment occurred. Clones with the following reassorted genome patterns were isolated: (i) RNA segments 5 or 6 of BRV were replaced by the corresponding RNA segments of human rotavirus; (ii) RNA segments 9 or 11 of BRV were replaced by different rearranged bands of RNA of human rotavirus; (iii) reassortants were observed containing more than one segment/rearranged band of human rotavirus RNA in different combinations. The reassortant viruses possessed functional proteins coded for by the genome segments and/or by rearranged bands of RNA of the human rotaviruses. Rearrangement of parts of the rotavirus genome may be a mechanism of evolution of these viruses. PMID- 2999314 TI - Release of progeny virus from cells infected with simian rotavirus SA11. AB - Analysis of cells infected with simian rotavirus SA11 at late times of infection indicated that the particles were associated with membranes and the cytoskeleton. Although a large amount of cellular and non-structural viral proteins were released at these times, probably by cellular lysis, only virus with an outer layer was found outside the cells, while virus without an outer layer remained associated with the cells, probably with membranes and the cytoskeleton. Inhibition of glycosylation by tunicamycin did not abolish cell lysis but inhibited the liberation of particles and the non-glycosylated precursors of the structural and non-structural viral glycoproteins. These results indicate that immature virus was tightly associated with the structural matrix of the cell. PMID- 2999315 TI - Rapid inactivation of rotaviruses by exposure to acid buffer or acidic gastric juice. AB - Inactivation rates of three bovine and several primate-origin rotaviruses were determined during exposure to acid buffers at pH 2.0, pH 3.0 or pH 4.0. Each rotavirus was inactivated at pH 2.0 (the acidity most resembling the normal fasting stomach) very rapidly, with half-lives for infectivity determined to be 1 min or less. Each rotavirus was inactivated at a much slower rate at pH 3.0; inactivation at pH 4.0 was minimal. No remarkable differences in acid resistance between different rotavirus strains were detected. Although these determinations were performed at room temperature (23 degrees C), experiments at diverse temperatures indicated an even more rapid rate of viral inactivation by acid at normal body temperature (37 degrees C). Studies of rotavirus exposed to natural human gastric juice at pH 1.8 or pH 2.1 revealed a rate of virus inactivation similar to that observed with glycine buffer of identical pH. PMID- 2999316 TI - Isolation of feline rotaviruses and their relationship to human and simian isolates by electropherotype and serotype. AB - Rotaviruses were detected by electron microscopy in the faecal specimens of six clinically well cats and virus was subsequently isolated from four of them. Analysis of the RNA of the isolates showed the existence of three electrophoretic types characteristic of the 'long' RNA electrophoretic pattern exhibited by rotaviruses. All feline isolates were neutralized only by antiserum to SA11 rotavirus, indicating that these isolates were serotype 3 rotaviruses. Antiserum prepared against a feline strain neutralized all the feline isolates as well as SA11 but showed no neutralizing activity against human isolates of serotype 1, 2 or 4. PMID- 2999318 TI - Survival characteristics of airborne human coronavirus 229E. AB - The survival of airborne human coronavirus 229E (HCV/229E) was studied under different conditions of temperature (20 +/- 1 degree C and 6 +/- 1 degree C) and low (30 +/- 5%), medium (50 +/- 5%) or high (80 +/- 5%) relative humidities (RH). At 20 +/- 1 degree C, aerosolized HCV/229E was found to survive best at 50% RH with a half-life of 67.33 +/- 8.24 h while at 30% RH the virus half-life was 26.76 +/- 6.21 h. At 50% RH nearly 20% infectious virus was still detectable at 6 days. High RH at 20 +/- 1 degree C, on the other hand, was found to be the least favourable to the survival of aerosolized virus and under these conditions the virus half-life was only about 3 h; no virus could be detected after 24 h in aerosol. At 6 +/- 1 degree C, in either 50% or 30% RH conditions, the survival of HCV/229E was significantly enhanced, with the decay pattern essentially similar to that seen at 20 +/- 1 degree C. At low temperature and high RH (80%), however, the survival pattern was completely reversed, with the HCV/229E half-life increasing to 86.01 +/- 5.28 h, nearly 30 times that found at 20 +/- 1 degree C and high RH. Although optimal survival at 6 degree C still occurred at 50% RH, the pronounced stabilizing effect of low temperature on the survival of HCV/229E at high RH indicates that the role of the environment on the survival of viruses in air may be more complex and significant than previously thought. PMID- 2999317 TI - Characterization of streptococcal bacteriophage c6A. AB - Bacteriophage c6A is a lytic phage that infects strains of Streptococcus lactis. Infection of S. lactis C6 under standard conditions yielded 124 +/- 8 p.f.u. per infected cell after a latent period of 25 min at 30 degrees C. The virion of c6A was shown to contain at least 12 polypeptides and a 21.9 kilobase double stranded, linear DNA genome with complementary 5'-protruding single-stranded termini. The (G + C) content of this DNA was estimated to be 36.7%. A restriction map was constructed which indicates that a number of restriction endonucleases did not digest the DNA and that others cleaved with a much lower frequency than expected. PMID- 2999320 TI - Inhibition of herpes simplex virus type 1-induced interferon synthesis by monoclonal antibodies against viral glycoprotein D and by lysosomotropic drugs. AB - Components of herpes simplex virus remained bound to the diploid cell membrane after nucleocapsid penetration into the cytosol. These components enabled the infected cells to induce interferon-alpha (IFN-alpha) in peripheral blood mononuclear cells even when the infected cells were fixed by glutaraldehyde. Monoclonal antibodies directed against the major viral glycoprotein D could neutralize their IFN-alpha-inducing capacity. Thus, the process of IFN induction does not require uptake and penetration of the inducer into the effector cells. The process was, however, sensitive to lysosomotropic drugs. These data suggest that a membrane receptor is involved in the IFN-alpha induction mechanism. PMID- 2999319 TI - Detection and localization of the v-myb(AMV) gene products of avian myeloblastosis virus by a synthetic peptide antiserum. AB - An antiserum made against a synthetic peptide from an internal region of the predicted amino acid sequence of the avian myeloblastosis virus (AMV) transforming v-myb(AMV) gene identified two products, p46v-myb(AMV) and p32v myb(AMV), which were localized in the nucleus of AMV-transformed myeloblasts. We propose that these proteins are the in vivo products of the v-myb(AMV) gene and thus the transforming protein(s) of AMV. PMID- 2999321 TI - Restriction endonuclease analysis of bovine herpesvirus 1 DNA and nucleic acid homology between isolates. AB - Isolates of bovine herpesvirus 1 (BHV-1) are associated with a variety of clinical manifestations. To determine if a single form of BHV-1 was responsible for the different virus-associated diseases or whether subpopulations of various isolates produced different clinical symptoms, studies were initiated to examine the DNA restriction enzyme patterns and nucleic acid homology between virus isolates from respiratory infections and other clinical syndromes. Differences between the genomes of several virus isolates were detected using DNA restriction enzyme analyses. However, nucleic acid hybridization studies of the virus DNAs using filter and liquid hybridization indicated at least a 95% genetic homology between the virus isolates from different types of infections. Additionally, these studies demonstrated that the DNA of BHV-1 had an average molecular weight of 84 X 10(6). PMID- 2999322 TI - High frequency of Coxsackie-B-virus-specific IgM in children developing type I diabetes during a period of high diabetes morbidity. AB - Twenty-four consecutive children with newly diagnosed insulin-dependent (type I) diabetes mellitus (IDDM) were investigated for a history of infectious disease. Thirteen of the 24 (54%) patients reported symptoms of acute infection within two months before diabetes was diagnosed. The mean age was 8.5 years and 15 (63%) of the patients were girls. No clear seasonal variation in onset was seen. Coxsackie B (CB)-virus-specific IgM responses were detected by reverse radioimmunoassay (RIA) in 16 of the 24 (67%) patients on the day of diagnosis of IDDM. The highest titre was usually recorded at that time, but with some the highest titre was found with a second serum obtained three to seven weeks after diagnosis. Thereafter the titres declined, and after six months IgM was detected only in a few patients. Thirteen patients displayed monotypic IgM responses, whereas three patients showed ditypic responses. Among the former, IgM was recorded against Coxsackie B4 (CB4) in four, B5 (CB5) in three, B1 (CB1) in two, B2 (CB2) in two, and B3 (CB3) in two patients. The ditypic responses were against CB2 and CB3, CB3 and CB4, and CB5. No CB-virus-specific IgM was detected in sera, found during the same period, from age-matched nondiabetic children without evidence of infection. In neutralisation (NT) tests, antibodies to the homotypic virus were found in 12 of the 16 diabetic patients showing CB-virus-specific at the time of diagnosis. A significant rise in NT titre was demonstrated in three of these patients. No significant clinical difference was noted between IgM positive and IgM negative patients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2999323 TI - Cell-mediated immune responses to BK virus in normal individuals. AB - A lymphoproliferative assay was developed to study cell-mediated immunity (CMI) to BK virus (BKV), a human papovavirus, in healthy volunteer subjects. Responses to ultraviolet-inactivated antigen prepared from BKV-infected fibroblasts were compared to those elicited against a mock antigen preparation and an unrelated control antigen (tetanus toxoid, TET). CMI to BKV and TET were contrasted with humoral immunity as measured by enzyme-linked immunosorbent assay (ELISA). Specificity of the assay was confirmed by absence of response to mock antigen in all subjects studied. Positive response to BKV antigen was observed in all of 15 seropositive individuals but not in 5 neonates or 1 seronegative child. Similarly, all TET seropositive (n = 13) but no seronegative subjects (n = 2) responded to TET. The magnitude of lymphoproliferation to either antigen did not correlate with antibody titer. Additionally, the frequency of peripheral blood BKV-specific proliferating lymphocytes was determined by limiting dilution analysis (LDA). The frequency was approximately tenfold less than that observed for TET in the same group of subjects (1/30,300 vs 1/2,700). This may be due to differences in route and frequency of antigen exposure, both of which are unknown, at present, for BKV. PMID- 2999324 TI - A murine monoclonal antibody recognising a single glycoprotein within a human cytomegalovirus virion envelope glycoprotein complex. AB - Nonionic detergent solubilised polypeptides from highly purified human cytomegalovirus virions were used as immunogens to produce murine monoclonal antibody secreting hybridomas. One monoclonal antibody was shown, by immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis (SDS PAGE), to precipitate three glycoproteins with molecular weights 52, 95, and 130 (all X 10(3)) and one minor component with a molecular weight of 50 X 10(3). When virion envelope components were first separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes, this monoclonal antibody recognised two related components with molecular weights 50 and 52 (both X 10(3)). Immunofluorescence studies suggested that these viral antigens were associated with membrane systems of virus-infected cells and were particularly abundant late in infection. PMID- 2999325 TI - Factors influencing the sensitivity of herpes simplex virus detection in clinical specimens in a simultaneous enzyme-linked immunosorbent assay using monoclonal antibodies. AB - A rapid simultaneous enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was investigated for herpes simplex virus (HSV) detection. All HSV isolated (n = 127) were detected, whereas no response was obtained with HSV negative preparations. Equivalent results were obtained from 275 of 277 clinical specimens in the monoclonal ELISA and in an ELISA using polyclonal antibodies, confirming that appropriately selected monoclonal antibodies may be as efficacious as polyclonal antibodies in antibody-based assays. In clinical specimens, the rate of HSV detection (sensitivity) relative to tissue culture isolation was low for both assays, and the major factor responsible for this was the low concentration of virus present in some specimens. The sensitivity of ELISA obtained in routine use varied with different panels of unselected specimens and was related to the speed of development of the cytopathic effect. These results emphasise the need for caution in assigning a definitive sensitivity level to ELISA tests evaluated on different panels of specimens. PMID- 2999326 TI - Virus-specific antibody-producing cells in blood and cerebrospinal fluid in acute Japanese encephalitis. AB - During an epidemic of Japanese encephalitis (JE) in northern Thailand, cerebrospinal fluid (CSF) leukocytes and blood leukocytes from 28 patients with suspected JE were tested for spontaneous in vitro synthesis of antibodies to JE virus (JEV). Sixteen patients were subsequently proven to be infected with JEV. Supernatant fluids of three-day cultures of unstimulated peripheral blood mononuclear leukocytes or unstimulated unfractionated CSF leukocytes were tested for JEV IgM and IgG antibodies with isotype-specific "antibody capture" radioimmunoassays. Blood-derived leukocytes from all sixteen JEV-infected patients and CSF-derived leukocytes from four JEV-infected patients synthesized JEV antibodies. Blood-derived and CSF-derived leukocytes from all 12 patients with central nervous system infections caused by agents other than JEV uniformly failed to synthesize JEV antibodies. Virus-specific antibody-producing cells can be detected in the blood and CSF early in the clinical course of acute JE. PMID- 2999327 TI - Benzodiazepine receptors: multiple receptors or multiple conformations? AB - Several lines of evidence from reversible binding studies seem to indicate there are at least two "central" benzodiazepine receptor subtypes, the BZ1 and BZ2 receptors. Irreversible binding studies, using 3H-flunitrazepam as a photoaffinity label for benzodiazepine receptors, not only are in perfect agreement with the data from reversible binding studies but extend these studies by identifying P51, a protein with apparent molecular weight 51,000, as a protein associated with the BZ1 receptor and by suggesting that the BZ2 receptor might actually consist of several different benzodiazepine receptors associated with different and distinct proteins irreversibly labeled by 3H-flunitrazepam. Other reversible binding studies have accumulated indicating the existence of several different conformations of benzodiazepine receptors. Irreversible binding studies support this conclusion and in addition suggest the existence of four different benzodiazepine binding sites within the GABA-benzodiazepine receptor complex. It is therefore hypothesized that there are several different GABA-benzodiazepine receptor subtypes all of which have four distinct benzodiazepine binding sites which can exist in at least three different but freely interconvertible conformations. This hypothesis can account for all experimental observations obtained so far and might partially explain the distinct clinical effects of structurally similar benzodiazepines. PMID- 2999328 TI - Clonidine and a beta-agonists induce hyperthermia in rats at high ambient temperature. AB - The effects of the alpha-agonist clonidine and the beta-agonist clenbuterol on body temperature of rats kept at high ambient temperature (28 degrees C) were studied. Both drugs induced a dose-dependent significant increase in temperature. The clonidine-induced hyperthermia was blocked by various alpha 2-antagonists, yohimbine, rauwolscine and RX 781094 and the alpha 1-antagonists, prazosin and corynanthine but not by 1-propranolol, spiperone, metergoline. The hyperthermic effect of clonidine was potentiated in rats after a lesion of the central noradrenergic terminals by DSP-4. The clenbuterol-induced hyperthermia was counteracted by 1-propranolol, yohimbine and rauwolscine but not by atenolol, prazosin, spiperone, metergoline. These observations indicate that clonidine- and clenbuterol-induced hyperthermia is mediated by alpha 2-(postsynaptic) and beta adrenoceptors, respectively. Moreover, in the latter effect alpha 2-adrenoceptors are involved. The simple temperature measurement can thus be used as a preliminary indicator of central alpha 2- or beta-agonistic properties of the screened drug. PMID- 2999329 TI - Adaptive and differential changes on beta- and alpha 2-adrenoreceptors mediated hyperthermia after chronic treatment with antidepressant drugs in the rat kept at high ambient temperature. AB - We found previously that the beta-agonist clenbuterol and the alpha-agonist clonidine produced hyperthermia in rats kept at high ambient temperature, which effects were mediated by beta- and alpha 2-adrenoceptors, respectively. In the present paper this observation was used for testing the responsiveness of beta- or alpha 2-adrenoceptors, changed by a pharmacological manipulation, i.e., by chronic treatment with antidepressants. The animals were pretreated with desipramine, imipramine or amitriptyline twice a day for 1 or 2 weeks. All antidepressants significantly attenuated the clenbuterol-induced hyperthermia after 2 weeks of treatment. The effect of desipramine was stronger than that of the other antidepressants and appeared as little as 1 week after the treatment. The hyperthermic effect of clonidine was significantly reduced by repeated treatment with desipramine, increased after 2 weeks administration of imipramine, whereas amitriptyline produced no significant changes. In conclusion, these data suggest that, after repeated treatment, the antidepressants tested produce an adaptive decrease in function of beta-adrenoceptors while the same drugs exert differential effects on alpha 2-receptors. Moreover, clenbuterol induced hyperthermia may be a useful test for examining possible functional changes in beta-adrenoceptor sensitivity. PMID- 2999330 TI - Derivatives of Co(II), Co(III), Ni(II), Cu(II), and Zn(II) with 5' AMP. Use as enzymatic labels with glycogen phosphorylase B. AB - Some new derivatives of Co(II), Co(III), Ni(II), Cu(II), and Zn(II) with 5' AMP have been obtained, characterized by elemental analysis, infrared, electronic, and fluorescence spectroscopy. The activities of these complexes as substitutes of 5' AMP as allosteric activators of glycogen phosphorylase b have been tested. The derivatives that have no interaction with the phosphate group are good analogs of the natural allosteric activator; the complexes that have direct bonding between metallic ion and phosphate groups do not activate the enzyme. PMID- 2999331 TI - Direct evidence of nitrogen coupling in the copper(II) complex of bovine serum albumin by S-band electron spin resonance technique. AB - ESR spectra of the tight binding Cu(II) complex of bovine serum albumin (BSA) has been studied using S-band. At physiological pH, only one form of copper binding to BSA was detected from the ESR spectra. From previous X-band ESR spectra, nitrogen superhyperfine splittings were observable in the g perpendicular region; however, the resolution of the g parallel region was not sufficient to confirm the exact donor atoms of the complex. Using low-frequency ESR (2-4 GHz) at 77 K, we have resolved the nitrogen superhyperfine structure in the g parallel region. A computer simulation method has been developed for distinguishing between three and four nitrogen donor atoms. The Hyde-Froncisz theory of g and A strain broadening has been modified to use a field-swept calculation for the line shape. The observed intensity pattern and the computer simulation of such spectra positively confirm the structure of Cu(II) ion coordinated to four in-plane nitrogen atoms in frozen aqueous solutions of Cu(II)-BSA complexes at physiological pH. This is the first time that this binding site has been confirmed on the protein instead of a protein fragment or model compound. This work is another example of the usefulness of the S-band ESR technique for characterizing the metal-protein interactions when random variation in g factors cause line broadening in conventional X-band ESR spectra. PMID- 2999332 TI - Polyamine regulation of the microtubule-associated protein kinase. AB - Microtubule protein prepared by cycles of assembly-disassembly contains a cyclic AMP-dependent protein kinase that phosphorylates the high-molecular-weight microtubule-associated protein MAP-2. The polyamine spermine at 2mM affected the phosphorylation of MAP-2 in a manner that depended on the cyclic AMP concentration. At cyclic AMP concentrations below 10(-6) M, spermine increased the rate of phosphorylation, while at cyclic AMP concentrations above 10(-6) M, spermine decreased the rate of phosphorylation. Spermine also decreased the final extent of cyclic AMP-dependent phosphorylation but did not affect the protein substrate specificity of the microtubule-associated protein kinase. MAP-2 was the principal substrate both in the presence and in the absence of spermine. Because of these results, we propose that microtubule protein phosphorylation may be regulated in vivo by spermine as well as by cyclic AMP levels. PMID- 2999333 TI - Postnatal development of proteins associated with different benzodiazepine receptors. AB - The postnatal development of several proteins irreversibly labeled by [3H]flunitrazepam in membranes from rat cerebral cortex was investigated. It was demonstrated that in the early postnatal days proteins with apparent molecular weights 55,000 and 59,000 were predominantly labeled whereas irreversible labeling of a protein with apparent molecular weight 51,000 started to predominate only in the second postnatal week. Irreversible labeling of another protein with apparent molecular weight 62,000 was weak throughout development. All these proteins seem to be associated with central benzodiazepine receptors. Irreversible labeling at various time points after birth seems to parallel the postnatal development of these proteins, and the different time course of development and different binding properties of the individual proteins support the hypothesis that these proteins are associated with separate and distinct benzodiazepine receptor subtypes. The pharmacological properties of the individual receptor subtypes seem to be fully developed in the early postnatal days, and therefore newborn animals seem to be a good model system for the investigation of properties and function of these various benzodiazepine receptor subtypes. PMID- 2999334 TI - Enhancement of ATPase activity by a lipid peroxide of arachidonic acid in rat brain microvessels. AB - The effects of 15-hydroperoxyarachidonic acid (15-HPAA) on Na+, K+- and Mg+ ATPase activities in the blood-brain barrier (BBB) were examined using rat brain microvessels (MV). 15-HPAA markedly stimulated these ATPase activities in MV at low concentrations whereas the synaptosomal Na+, K+-ATPase activity was inhibited in a dose-dependent manner. Further neurochemical analysis revealed that this stimulatory effect of 15-HPAA in MV was not due to a simple detergent-like action of the compound on the membranes but rather to stimulation of the phospholipase A2 and lipoxygenase activity within MV. In addition, it was shown that free radical reactions were involved in the mechanism. Since such anti-edema drugs as 1,2-bis(nicotinamido)propane were proved to be potent suppressors of the enhanced ATPase activity, further speculations on the role of this effect for ischemic brain edema are offered. PMID- 2999335 TI - Abnormality of alpha 1-adrenergic receptors in the frontal cortex of epileptic rats. AB - We found that the binding of [3H]prazosin, a selective ligand for alpha 1 adrenergic recognition sites, is significantly lower in the frontal cortex of the genetically epilepsy-prone rats (GEPRs), as compared with normal Sprague-Dawley rats. Scatchard analysis reveals a decrease in the Bmax of [3H]prazosin binding with no change in the apparent KD, suggesting that there are fewer alpha 1 adrenergic recognition sites in the frontal cortex of the GEPR. This abnormality is associated with a reduced capacity of norepinephrine (NE) to stimulate [3H]inositol monophosphate ([3H]IP1) formation in frontal cortex slices prelabeled with [3H]inositol. No significant differences in [3H]prazosin binding as well as NE-stimulated [3H]IP1 formation have been observed in other brain regions including hippocampus, corpus striatum, and inferior colliculus. These results indicate that a deficit in the alpha 1-adrenergic receptor system in the frontal cortex may play a role in the seizure process in the GEPR. PMID- 2999336 TI - Beta 2-adrenergic receptors on peripheral nerves. AB - We report that peripheral nerves have a functional adenylate cyclase-coupled beta adrenergic receptor. The pharmacological specificity of this receptor is shown to be of the beta 2 subtype. Two peripheral nerves, the sciatic from the frog and rat and the vagus from the rat, responded to beta 2-agonists with 10-50-fold increases in intracellular cyclic AMP level. This increase was inhibited by the beta-adrenergic antagonist propranolol. In contrast, a central nerve tract, the corpus callosum, responded to isoproterenol with only a minimal one- to twofold increase in cyclic AMP level. These studies demonstrate that peripheral nerves have beta 2-adrenergic receptors that are responsive to exogenously applied catecholamines and suggest a role for these ligands in the previously described modulation of axonal conduction. PMID- 2999337 TI - Incorporation of tritiated galactose into galactocerebroside by cultured rat oligodendrocytes: effects of cyclic adenosine 3',5'-monophosphate analogues. AB - Cells dissociated from the forebrains of 21-day-old rats were enriched in oligodendroglia by Percoll gradient centrifugation, seeded on polylysine-coated surfaces, and cultured in a serum-containing medium. Incorporation by the cultures of tritium from D-[3H]galactose into the galactosyl residue of galactocerebroside (galC) increased in an almost linear fashion for 48 h with 1-8 muCi of D-[3H]galactose (30 mCi/mumol) per milliliter medium. Treatment for 2 days (day 1-3 after seeding) with 10(-4) M or 10(-3) M dibutyryl cyclic adenosine 3',5'-monophosphate (db cyclic AMP) or 10(-4) M 8-bromo cyclic AMP stimulated galC radiolabelling. Incorporation of D-[3H]galactose into galC during a terminal 48-h radiolabelling period was not stimulated when the cells were continuously treated with these cyclic AMP analogues for 8 rather than 2 days. PMID- 2999338 TI - Subcellular localization of "peripheral-type" binding sites for benzodiazepines in rat brain. AB - The binding of [3H]Ro 5-4864, a specific ligand for "peripheral-type" benzodiazepine binding sites and [3H]Ro 15-1788, a specific ligand for the central benzodiazepine receptors, was determined in subcellular fractions of rat brain. As previously reported, the highest levels of "peripheral-type" benzodiazepine binding sites and benzodiazepine receptors were found in the crude P1 and P2 fractions, respectively. Purification of these crude fractions revealed that high levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding were present in the mitochondrial and synaptosomal fractions. In contrast, the purified nuclei and myelin contained low levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding. PMID- 2999339 TI - A monoclonal antibody raised to corpus callosum extract reacts with 2',3'-cyclic nucleotide 3'-phosphohydrolase. AB - Monoclonal antibody against 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with delipidated white matter from rat corpus callosum. The antibody was characterized by solid-phase radioimmunoassay, immunoblot of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoprecipitation from C6 glioma cells, and indirect immunofluorescence staining of monolayer cultures containing oligodendrocytes. The monoclonal antibody bound specifically to an intracellular antigen of oligodendrocytes, but not to Schwann cells, astrocytes, neurons, or fibroblast cytoplasm. The immunoblot of SDS-PAGE of CNS myelin showed that the antibody identified two protein bands at 48,000 and 50,000 molecular weight. These proteins were not identified in peripheral nervous system myelin. The monoclonal antibody immunoprecipitated CNP enzyme activity from extracts of C6 glioma cells. This monoclonal antibody should prove useful in further study of this myelin-specific enzyme in CNS myelin and in cells responsible for myelin production. PMID- 2999340 TI - Surgical sympathetic denervation increases alpha 1-adrenoceptor-mediated accumulation of myo-inositol trisphosphate and muscle contraction in rabbit iris dilator smooth muscle. AB - Sympathetic denervation of the iris muscle produces increases in both the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) and in muscle contraction in response to norepinephrine (NE). To shed more light on the biochemical basis underlying this supersensitivity we investigated: the effects of NE on PIP2 breakdown, measured as myo-inositol trisphosphate (IP3) accumulation, and on muscle contraction in normal and denervated rabbit iris dilator; and the effects of denervation on selected biochemical properties of this muscle. The data obtained from these studies can be summarized as follows: The EC50 values (microM) for NE-induced IP3 accumulation in normal and denervated dilators were 14 and 3, respectively. This accumulation of IP3 was blocked by prazosin (1 microM). The EC50 values (microM) for NE-induced contraction for the normal and denervated muscles were 10 and 0.6, respectively. The NE-induced muscle contraction was blocked by prazosin (1 microM). The t1/2 values (s) for IP3 accumulation in normal and denervated muscles were 31 and 11, respectively, and for contraction the values were 19 and 9, respectively. Denervation increased significantly (15-18%) the basal labelling of phosphoinositides from myo [3H]inositol, but not from 32P or [14C]arachidonic acid. Denervation had little effect on the activities of the enzymes involved in phosphoinositide metabolism. However, the activities of protein kinase C and Ca2+-ATPase increased in the denervated muscle. It is concluded that sympathetic denervation of the iris dilator renders the coupling between alpha1 receptors and PIP2 breakdown into IP3 and 1,2-diacylglycerol (DG) more efficient.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2999341 TI - Cytochemical localization of 5'-nucleotidase in the enteric ganglia and in smooth muscle cells of the guinea-pig. AB - Cytochemical techniques were used to study the localization of 5'-nucleotidase in the enteric ganglia and in smooth muscle cells of the guinea-pig ileum, iris and vas deferens. Enzymatic activity was revealed in plasma membranes and caveolae of smooth muscle cells and in neurons and neuroglia of the enteric ganglia. The strongest activity was seen in the membrane of the smooth muscle cells, especially the caveolae intracellulares, and this was interpreted to indicate a high level of purine utilization and involvement in calcium translocation by these cells. The ganglia displayed enzymatic activity in the membranes of non specialized neuron-to-glia boundaries as well as at some synaptic specializations. This finding is consistent with a possible release of adenine nucleotides within the ganglia. PMID- 2999342 TI - An attempt to transfer radiation resistance to an ataxia-telangiectasia cell line. AB - Fibroblasts from ataxia-telangiectasia (AT) are hypersensitive to the lethal effects of ionizing radiation. Genomic DNA from normal human cells was transfected together with the selectable bacterial marker, gpt, on plasmid pSV2 into an SV40-transformed AT line, AT5BIVA. One radiation resistant clone (67) was recovered following repeated cycles of gamma-irradiation from a population of 90,000 clones. The normal level of radiation resistance has been maintained for at least 11 months in the absence of further selection by radiation. The resistant clone is not a contaminant as determined by isoenzyme analysis, carries one copy of the gpt gene, and its DNA synthesis is inhibited after radiation to an extent intermediate between that of AT and normal cells. It is not yet established whether clone 67 is a bona fide transformant or arose as a consequence of mutation of the parent AT line. PMID- 2999343 TI - Immune response to Epstein-Barr virus (EBV) in ataxia-telangiectasia: EBV specific antibody patterns and their relation to cell-mediated immunity. AB - Epstein-Barr virus (EBV)-specific antibody titers were investigated in 60 patients with ataxia-telangiectasia (AT) and 22 healthy members of their families. In addition, we studied 36 patients with primary immunodeficiencies, Behcet disease, and other conditions and 61 unrelated healthy controls. Twenty seven AT patients were examined sequentially at intervals varying from 2 months to 8 years. The AT patients showed an increased incidence (66.6%) of high antibody titers (greater than or equal to 1:320) to viral capsid antigen (VCA) and also a high incidence (35%) of antibody titers to early antigens (EA), but low titers (less than 1:10) of antibodies to the EBV-associated nuclear antigen (EBNA) in 35% of the patients. The geometric mean titers (GMT) of antibodies to VCA were five to six times higher; those of anti-EBNA were five times lower in AT patients as compared with control groups. In serial determinations, anti-VCA and anti-EBNA titers remained constant with the exceptions of two patients who developed ALL and Hodgkin lymphoma. The patients with other diseases did not differ significantly from the controls, with the exception of lower titers (less than 1:10) of anti-EBNA (52.8%). AT patients with low anti-EBNA titers tended to have more advanced T-cell deficiencies than those with moderate anti-EBNA titers, as detected by total lymphocyte and E-rosetting cell counts and skin test responses. The percentage of patients with low serum IgA levels was found to be higher in the low anti-EBNA group than in the moderate anti-EBNA group (44.5 vs 20%). PMID- 2999344 TI - Defective allosuppression in patients with ataxia-telangiectasia. AB - We describe a novel in vitro assay system that detects the generation of suppressor T cells after exposure of human lymphocytes to class I alloantigens. We have used this system to study immune functions in a group of seven patients with ataxia-telangiectasia (AT). Normal T lymphocytes exposed to cells differing at the A and B locus histocompatibility locus antigens (HLA) become activated for suppression of Epstein-Barr virus (EBV)-induced immunoglobulin (Ig) production. In contrast to the normal, T cells from patients with AT demonstrate no inhibitory effect after allostimulation. These data indicate that patients with AT have a profound defect involving responses to class I antigens of the major histocompatibility complex (MHC). PMID- 2999345 TI - Iodine 131 antiferritin, a new treatment modality in hepatoma: a Radiation Therapy Oncology Group study. PMID- 2999346 TI - Neurologic, neuropsychologic, and computed cranial tomography scan abnormalities in 2- to 10-year survivors of small-cell lung cancer. AB - In order to evaluate the relationship between neurologic function and cranial irradiation, 20 patients treated on National Cancer Institute (NCI) small-cell lung cancer (SCLC) trials who were alive and free of cancer 2.4 to 10.6 years (median, 6.2) from the start of therapy were studied. All were tested with a neurologic history and examination, mental status examination, neuropsychologic testing, and review of serial computed cranial tomography (CCT) scans. Fifteen patients had been treated with prophylactic cranial irradiation (PCI), two patients with therapeutic cranial irradiation, and three received no cranial irradiation. All patients but one were ambulatory and none were institutionalized. Fifteen patients (75%) had neurologic complaints, 13 (65%) had abnormal neurologic examinations, 12 (60%) had abnormal mental status examinations, 13 (65%) had abnormal neuropsychologic testing, and 15 (75%) had abnormal CCT scans. Compared with those given low-dose maintenance chemotherapy during PCI using 200 to 300 rad per fraction, patients who were given high-dose induction chemotherapy during the time of cranial irradiation or large radiotherapy fractions (400 rad) were more likely to have abnormal mental status examinations (6/6 v 4/9) and abnormal neuropsychologic tests (6/6 v 4/9), but no major difference in CCT findings was present. CCT scans in the majority of cases (11/18) showed progressive ventricular dilatation or cerebral atrophy up to 8 years after stopping therapy. We conclude neurologic abnormalities are common in long-term survivors of SCLC, and may be more prominent in patients given high dose chemotherapy during cranial irradiation or treated with large radiotherapy fractions. The CCT scan abnormalities are common and progressive years after prophylactic cranial irradiation and chemotherapy are stopped. PMID- 2999347 TI - Comparative electrophysiology of pyramidal and sparsely spiny stellate neurons of the neocortex. AB - Slices of sensorimotor and anterior cingulate cortex from guinea pigs were maintained in vitro and bathed in a normal physiological medium. Electrophysiological properties of neurons were assessed with intracellular recording techniques. Some neurons were identified morphologically by intracellular injection of the fluorescent dye Lucifer yellow CH. Three distinct neuronal classes of electrophysiological behavior were observed; these were termed regular spiking, bursting, and fast spiking. The physiological properties of neurons from sensorimotor and anterior cingulate areas did not differ significantly. Regular-spiking cells were characterized by action potentials with a mean duration of 0.80 ms at one-half amplitude, a ratio of maximum rate of spike rise to maximum rate of fall of 4.12, and a prominent afterhyperpolarization following a train of spikes. The primary slope of initial spike frequency versus injected current intensity was 241 Hz/nA. During prolonged suprathreshold current pulses the frequency of firing adapted strongly. When local synaptic pathways were activated, all cells were transiently excited and then strongly inhibited. Bursting cells were distinguished by their ability to generate endogenous, all-or-none bursts of three to five action potentials. Their properties were otherwise very similar to regular-spiking cells. The ability to generate a burst was eliminated when the membrane was depolarized to near the firing threshold with tonic current. By contrast, hyperpolarization of regular spiking (i.e., nonbursting) cells did not uncover latent bursting tendencies. The action potentials of fast-spiking cells were much briefer (mean of 0.32 ms) than those of the other cell types.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2999348 TI - Cross-reinnervated motor units in cat muscle. I. Flexor digitorum longus muscle units reinnervated by soleus motoneurons. AB - The properties of flexor digitorum longus (FDL) muscles and of individual motor units were studied in cats 30-50 wk after self-reinnervation by FDL motoneurons (FDL----FDL) or cross-reinnervation by soleus (SOL) motoneurons (SOL----FDL). Individual motor units were functionally isolated by intracellular recording and stimulation of identified SOL alpha-motoneurons. Glycogen-depletion methods permitted histochemical study of muscle fibers belonging to physiologically characterized muscle units. The observations were compared with data from normal cat FDL muscles and motor units (27). Intentionally self-reinnervated FDL muscles (FDL----FDL; n = 5) were normal in size and wet weight. FDL----FDL motor units could be classified into the same physiological categories found in normal FDL [types: fast contracting, fatigable (FF), fast contracting, fatigue resistant (FR), and slow (S); n = 24], with approximately the same proportions as normal. The histochemical muscle fiber types associated with these categories were also qualitatively normal although there was evidence of marked distortion of the normal histochemical mosaic. These data confirm other studies of self reinnervation and suggest that self-reinnervation can produce complete interconversion of muscle fiber types. Cross-reinnervation of FDL muscle by SOL motoneurons (SOL----FDL; n = 12) produced muscles that were smaller (about half the normal wet weight) and more red than normal. SOL----FDL muscle contracted more slowly than normal or FDL----FDL muscles and had much higher proportions of histochemical type I muscle fibers. In those SOL----FDL muscles, in which little or no unwanted self-reinnervation could be demonstrated, greater than 95% of the muscle fibers were type I. Forty-one individual motor units in SOL----FDL muscles were isolated by intracellular penetration in functionally identified SOL alpha motoneurons. Their muscle units were all type S by physiological criteria (absence of "sag" in unfused tetani and marked resistance to fatigue). SOL----FDL muscle units had contraction times and fatigue properties that were essentially identical to those of type S units in the normal FDL. All of the seven units, successfully studied by glycogen depletion, exhibited histochemical type I fibers. SOL motoneurons that innervated FDL muscle units had slightly shorter afterhyperpolarization durations than normal SOL cells, but axonal conduction velocities were normal.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2999349 TI - Cross-reinnervated motor units in cat muscle. II. Soleus muscle reinnervated by flexor digitorum longus motoneurons. AB - The properties of whole soleus (SOL) muscles and of individual motor units were studied in cats 30-50 wk after self-reinnervation by soleus (SOL) motoneurons (SOL----SOL) or cross-reinnervation by flexor digitorum longus (FDL) motoneurons (FDL----SOL). As in the preceding paper (22), intracellular and glycogen depletion methods were used to examine the physiological and histochemical properties of individual motor units. The results were compared with data from normal SOL motor units (8, 12). Intentionally self-reinnervated SOL muscles (SOL- --SOL; n = 6) were normal in size and wet weight, and all of the five SOL----SOL motor units studied had physiological and histochemical characteristics that matched those of normal SOL units. Cross-reinnervation of SOL by FDL alpha motoneurons (FDL----SOL; n = 7) produced muscles with wet weights and appearance essentially identical to normal SOL. However, whole-muscle twitch contraction times were much shorter (mean 60.4 ms) than those of normal (mean 136.9 ms, n = 18) or SOL----SOL muscles (mean 115.3 ms; n = 6). Despite this difference, none of the FDL----SOL muscles contained more than 7% histochemical type II muscle fibers, all of which were type IIA. Normal cat SOL muscles can contain up to 5% type IIA fibers, but none of our SOL----SOL muscles showed any type II fibers. Two FDL----SOL muscles had significant amounts of unintended self-reinnervation, permitting side-by-side comparison of FDL----SOL and SOL----SOL muscle fibers. The twitch contraction times of the two populations differed markedly, but they were histochemically indistinguishable except for the fact that SOL----SOL fibers had high neutral fat content (as do normal SOL fibers), whereas FDL----SOL showed much lower fat content. The 23 FDL----SOL muscle units studied were classified as physiological type S by criteria ("sag" test and fatigue resistance) used to identify motor-unit types in normal cat muscles. All five of the FDL----SOL units studied histochemically after glycogen depletion showed the type I histochemical profile, which is characteristic of the normal cat SOL. In marked contrast to the preceding study, cross-reinnervation of cat SOL by FDL motoneurons produced no conversion of muscle-unit properties into those associated with fast-twitch unit types, despite significant decreases in isometric twitch contraction time. The altered twitch speed was not associated with evident changes in conventional myofibrillar adenosine triphosphatase (ATPase) histochemistry.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2999350 TI - Kinesiological studies of self- and cross-reinnervated FDL and soleus muscles in freely moving cats. AB - The activity patterns in self- and cross-reinnervated flexor digitorum longus (FDL) and soleus (SOL) muscles were examined during natural movements in awake, unrestrained cats in which electromyographic (EMG) electrodes, tendon-force gauges, and muscle-length gauges had been chronically implanted under anesthesia and aseptic conditions. Kinesiological data were recorded between 13 and 22 mo after nerve surgery. Self-reinnervated FDL and SOL muscles (i.e., FDL----FDL and SOL----SOL, respectively) exhibited locomotor activity patterns that were the same as observed in normal, unoperated FDL and SOL muscles (26). FDL----FDL muscles exhibited primarily brief bursts of activity in early swing, just after the toes had left the ground, whereas SOL----SOL muscles showed bursts of activity just before and during stance. In contrast, the cross-reinnervated muscles (both SOL----FDL and FDL----SOL) that had little or no unwanted self reinnervation showed the patterns of activity that are associated with the innervating foreign motoneurons. That is, cross-reinnervated SOL----FDL muscles were intensely active in quadrupedal standing and, during the stance phase of stepping, producing large force transients while actively lengthening. Conversely, cross-reinnervated FDL----SOL muscles were active mainly in short bursts at the onset of the swing phase of stepping, just after the foot had left the ground. There was considerable modulation of EMG and peak force output in FDL ---SOL muscles with changing speed of locomotion, whereas little modulation was evident in SOL----FDL muscles. The activity patterns in self- and cross reinnervated FDL and SOL muscles were also recorded during scratch and paw shaking reflexes. As in locomotion, the observed patterns were in all cases consistent with those expected for the innervating motor pool rather than the innervated muscle. Muscles that had been dually reinnervated by both the original and foreign motor pools displayed activity patterns that were a mixture of the FDL and SOL activity patterns described above. The present results demonstrate that motoneuron activation patterns remain qualitatively unaltered when their motor axons reinnervate foreign muscles. In addition, the observations permit some quantitative estimates of the degree to which cross-reinnervated muscles are subjected to patterns of motoneuron activity and to conditions of mechanical loading that are markedly different from those in the self-reinnervated or normal conditions. PMID- 2999351 TI - Effects of groups of propriospinal interneurons on fictive swimming in the isolated spinal cord of the lamprey. AB - Fictive swimming activity was induced in isolated spinal cords of adult lampreys Ichthyomyzon unicuspis and Petromyzon marinus by addition of D-glutamate or N methyl-D,L-aspartate (NMA) to the bathing fluid. Propriospinal interneurons are defined as nerve cells within the spinal cord with projections longer than 1 segment. The hypothesis that propriospinal interneurons contribute to intersegmental coordination during fictive swimming was tested using electrical stimulation, extracellular recording, and separated compartments. Stimulation of the split caudal end of the spinal cord indirectly excited ascending propriospinal interneurons, which enhanced and entrained bursts in rostral contralateral ventral roots. Indirect electrical stimulation of descending propriospinal interneurons could delay and diminish bursts in caudal contralateral ventral roots. Extracellular recordings from the rostral and caudal split ends of the spinal cord sometimes showed spike activities in phase with contralateral or ipsilateral ventral roots. Inhibition of 1-3 segments by spot applications of glycine or gamma-aminobutyric acid (GABA) did not interrupt normal coordination or rostrocaudal phase lag. When a middle region of spinal cord was inhibited in a compartment with GABA or glycine, the caudal spinal cord could entrain the bursts in rostral ventral roots. In a few preparations the caudal region induced antiphasic bursts in previously silent rostral roots through the inhibited region. The maximum separation for caudal-upon-rostral antiphasic entrainment was approximately 20 segments in Ichthyomyzon and 36 segments in Petromyzon. Increased concentrations of an excitatory amino acid in a rostral compartment could produce descending entrainment of bursts in an adjacent caudal compartment at a higher frequency with rostrocaudal phase lag. The rostral upon-caudal entrainment could still occur through spot applications of GABA or glycine but not through long inhibited regions. Two hypothetical groups of propriospinal interneurons are proposed for the coordination of swimming activities in the isolated spinal cords of adult lampreys. 1) Crossed, ascending interneurons may be excited in phase with nearby motoneurons and may excite and entrain rostral pattern generators on the opposite side. 2) Short, commissural interneurons may be excited in phase with nearby motoneurons and may inhibit contralateral generators. PMID- 2999352 TI - Malignant cerebellar astrocytoma. Report of five cases. AB - Five cases of malignant cerebellar astrocytoma observed during a seven-year period are reported. The analysis of the cases allows us to conclude that malignant cerebellar astrocytoma represents a true tumoral entity quite distinct from cerebellar glioblastoma as well as from medulloblastoma. A perfect correspondence between the histological malignancy and the duration of survival has been noted in all the cases. PMID- 2999354 TI - Second opinions: when needed? PMID- 2999353 TI - Photon deficient bone metastasis of hepatocellular carcinoma with avid gallium-67 uptake. AB - While bone metastases producing photon deficient defects on bone scintigraphy have previously been reported, this finding has not been emphasized for hepatocellular carcinoma (HCC). Furthermore, "filling-in" of such photon deficient defects with 67Ga at skeletal sites of metastatic HCC has not been described. In this case report, the combination of a photon deficient defect on bone scintigraphy and avid accumulation of 67Ga in this same area was of value in confirming the diagnosis of metastatic HCC. PMID- 2999356 TI - Meeting the terminal functions of a baccalaureate curriculum: the students' perception. AB - Nursing faculty have established terminal functions which graduates are expected to be able to accomplish after successfully completing the nursing program. Students have asked themselves and faculty if they are ready to perform as professional nurses after graduation. This study utilized the terminal functions established by one medium-size state university to obtain data regarding the students' perceptions of their competencies based on the terminal functions. The assessment was conducted at the time of graduation and again one year post graduation. The data may provide other faculty with a method to further evaluate nursing curricula and illustrate the changes in graduates' perceptions of their abilities over time. PMID- 2999355 TI - Changing the locus of control of registered nurse students with a futuristic oriented course. AB - Locus of Control research has established that college students are becoming more external, and that internality is preferable. If an external locus of control is associated with poor self-esteem, alienation, depression, and "burn out" in nurses as it is with other individuals, then we need to reverse this trend. Lefcourt (1976) tells us that in the future, methods fostering internality will be crucial. Toffler (1980) says that to break out of the rigid mentality of the Second Wave, future-oriented nurses must be self-responsible. Self-responsible, internally controlled practitioners will enable themselves and the nursing profession to grow as they will anticipate and participate in the selection of the knowledge, skills, and characteristics they need to grow. The purpose of this study was to determine if a course which explores the transition into a futuristic-oriented professional nursing role would move registered nurse students in a baccalaureate program toward stronger internality. A basic pretest intervention-post test experimental design was used and results of an analysis of covariance significantly determined the efficacy of the course in encouraging greater internality, F (3,60) = 49.55, p less than .0001. PMID- 2999357 TI - Disciplinary boundary maintenance in nursing education. AB - This paper reports the results of two small-scale surveys examining the extent to which nursing education is reflecting patterns of insularity from other disciplines. One survey examined employment of non-nurse faculty and mechanisms for delivering non-nursing content in 16 baccalaureate and higher degree nursing programs; the other examined the disciplinary origins of required readings in 44 nursing courses. Non-nurse faculty were employed in half of the schools surveyed; however, non-nursing content was delivered primarily by nurses with advanced preparation in another discipline. There was heavy, but not total reliance on nursing authorship and journals in the courses surveyed. A pattern of relatively high insularity was inferred from reported and anticipated decreases in non-nurse faculty, the rarity of joint appointments, heavy reliance on nurses to deliver non-nursing content and use of high proportions of nursing literature. PMID- 2999358 TI - Determining the use of physical assessment skills in the clinical setting. AB - This study examined the clinical application of physical assessment skills by baccalaureate nurses. Fifty-nine pediatric staff nurses practicing in two different hospitals in Southern California completed a questionnaire which surveyed how frequently assessment skills were utilized and what deterrents, if any, inhibit their use. Approximately one-third of the 36 skills listed were utilized daily by at least 74% of the participants, and respondents selected a variety of deterrents to the utilization of other skills. There was no significant difference in the utilization of skills between nurses who had taken a separate course in physical assessment and those from an integrated curriculum. PMID- 2999359 TI - Applying the Rasch Model to test administration. AB - The Rasch Model is offered as a valuable means to analyze what occurs when students take tests. Unlike Classical Test Theory, the Rasch Model is not sample dependent; it generates data about the test items and the students who took the test. Test item values, item fit statistics, person measures and person fit statistics are some of the results obtained when one uses this model. Through the application of the Rasch Model described here, one may assess its value particularly to nurse educators who address many variables when administering tests. This model facilitates analysis of these variables. Additionally, it allows for comparison of test item values and person measures on the same linear scale. The result of the application of the Rasch Model is a more refined measurement of test item difficulty and student performance. PMID- 2999361 TI - Psychotherapeutic techniques and methods applied in teaching human sexuality. PMID- 2999360 TI - The impact of successful laboratory system on the teaching of nursing skills. AB - The laboratory guided practice approach has been found an effective strategy for teaching nursing skills to large numbers of students. It provides flexibility in meeting individualized needs and serves to decrease stress levels of both students and faculty. At the School of Nursing, University of N.C. at Chapel Hill, it has served to establish more uniform clinical expectations of students and greater correlation between skills level and clinical site placement, as well as integration of skill components into clinical courses. These outcomes and the positive evaluations discussed above help to validate this strategy as a practical and effective way to approach the teaching of clinical nursing skills. PMID- 2999362 TI - Computer assisted test bank. PMID- 2999363 TI - The demonstration of a joint faculty/practice position. AB - When the final report was presented at the end of the second semester, suggestions were made to continue the position and also to expand it to other clinical areas. The decision was made to continue the position for a third semester and to seek a reciprocal arrangement by a nurse in practice. In such a position, a nurse in practice would take a leadership role in bringing strengths from practice to nursing education. Ideally joint positions will occur in other clinical areas. Each nurse in a joint position would strive to provide high quality of care, achieve academic excellence and conduct research. Although the author is no longer in the joint position, the results of this experience have been rewarding. Both institutions seek to develop similar positions. Communication and collaboration between practice and education have improved as each group recognizes the strengths of the other. An increase of sharing of skills and knowledge is observed. The author continues to provide support for activities which were begun during this experience. Many benefits have been realized from this experience; a parent education program continues to expand through the efforts of the nursing staff, the support group is on going under the supervision of the Head Nurse. A warm welcome awaits the author when she arrives on the pediatric unit for a visit! The author's overall goal to demonstrate a mechanism to improve communication and collaboration between nursing education and practice has been realized. Nurses have grown from this experience, patients have received improved nursing care and nursing knowledge has been augmented. This has been a vital experience in nursing. PMID- 2999364 TI - [Adenoid cystic carcinoma--a clinicopathologic study of 12 cases]. PMID- 2999366 TI - Immunoelectron microscope demonstration of the basement membrane components laminin and type IV collagen in the dermal cylindroma. AB - Specific antisera to the human laminin P1 fragment and the 7S domain of type IV collagen were used to investigate the ultrastructural location of these main basement membrane (BM) components in a dermal cylindroma. Thick frozen sections were treated by the immunoperoxidase method, postfixed, embedded in epon and then sectioned for electron microscopic examination. Laminin and type IV collagen were detected in all layers of the cylindroma BM, together with non-specifically stained, diffusely distributed fibrillar structures, possible microfibrils and anchoring fibrils or residual stromal collagen fibres. The mixed presence of the BM components indicates that these substances codistribute rather than occurring as separate layers even when forming neoplastic basement membranes. PMID- 2999365 TI - Characterization of tumour cells in malignant fibrous histiocytomas and other soft tissue tumours in comparison with malignant histiocytes. I. Immunohistochemical study on paraffin sections. AB - We have studied the possible origin of histiocytic cells, present in fibrous histiocytomas (MFH) by using immunohistochemistry to demonstrate lysozyme, alpha 1-antitrypsin, alpha 1-antichymotrypsin and receptors for peanut and soy bean agglutinin in tumour cells of MFH compared with their presence in tumour cells of malignant histiocytosis (MH) ('true' histiocytic lymphoma, 'true' histiocytic sarcoma). We included in this study a number of other soft tissue tumours (STT). Lysozyme was detected in half of the cases of malignant histiocytosis (n = 16) but in only two out of 77 MFH. alpha 1-Antitrypsin and alpha 1-antichymotrypsin usually occurred together although the latter was seen in more cases. Both markers were present in majority of cases of MH whereas they were detected in a minority of cases of MFH. MFH cases of the storiform subtype were less frequently stained than the pleomorphic or giant cell subtypes. Receptors for peanut or soy bean agglutinin were detected in nearly all MH cases, whereas their presence was only detected in a small number of MFH. Lysozyme was not detectable in other STT. alpha 1-Antitrypsin and alpha 1-antichymotrypsin were uncommonly present in other STT, except in osteosarcoma and rhabdomyosarcoma. These markers therefore have a limited value as indicators of a possible histiocytic origin of MFH. Lectins showed weak affinity for other STT. In accordance with others, we therefore conclude that the progenitor cell of MFH has to be sought within the undifferentiated mesenchymal cells and that histiocytes themselves probably do not give rise to MFH. PMID- 2999367 TI - Technetium thyroid uptake ratios in pediatric Graves disease. AB - Patients with Graves disease were prospectively followed by means of three 99mtechnetium thyroid uptake ratios. These three ratios were greater than 90% sensitive and specific for the detection of hyperthyroidism in the patient with untreated Graves disease. Twelve of 15 patients experienced prolonged remission after normalization of the ratios. These ratios exhibit significant linear correlation with serum thyroxine and triiodothyronine concentrations (r = 0.4 0.6, P less than 0.01) and are a very sensitive index of medical oversuppression of thyroid function. PMID- 2999368 TI - Isolated ACTH deficiency in childhood: lack of response to corticotropin releasing hormone alone and in combination with arginine vasopressin. PMID- 2999369 TI - Transfusion transmission of cytomegalovirus confirmed by restriction endonuclease analysis. PMID- 2999370 TI - Pseudohermaphroditism, glomerulopathy, and Wilms tumor (Drash syndrome) PMID- 2999371 TI - An in vivo model to study migration of cells and orientation of connective tissue fibers in simulated periodontal spaces. PMID- 2999372 TI - Utility of 7,7,8,8-tetracyanoquinodimethane and p-chloranilic acid in the qualitative and quantitative analysis of pentazocine. PMID- 2999373 TI - [Isolation and analgesic mechanism of the opioid analgesic neuropeptide, kyotorphin (Tyr-Arg)]. PMID- 2999375 TI - Adsorption of hypochlorite by aluminum hydroxide. PMID- 2999374 TI - Histology and ultrastructure of kidney tissue from ringed turtle doves that ingested lead. AB - Ringed turtle doves (Streptopelia risoria) ingested 4 x 122 mg lead pellets or lead acetate (75 micrograms Pb/g body weight) and their kidneys were examined by histological and electron microscopic techniques. Doves that received lead treatments had readily discernable lead intranuclear inclusion bodies in cells of the proximal convoluted tubules. At 33,000 X, the inclusions had a characteristic dense central core, and outer fibrillary zone. Necrosis in the proximal tubular area was seen as deterioration of cell cytoplasm and reduction in mitochondria. In doves ingesting lead, histological evaluation of proximal convoluted tubular cells revealed acid-fast pinkish granules in cell nuclei. Inclusions from rapidly as 1 dove that died after receiving the first of two lead doses (75 micrograms Pb/g body weight/day) had relatively small intranuclear inclusions in cells of the proximal convoluted tubules. PMID- 2999376 TI - Disposition of pentopril, a new orally active angiotensin-converting enzyme inhibitor, and its active metabolite in rats. AB - The disposition characteristics of pentopril (the ethyl ester) and its active carboxylic acid metabolite (CGS 13934) were determined in conscious rats after separate intravenous administrations of both compounds. The relationship between plasma concentration and pharmacological effect was also evaluated. The extent of apparent bioavailability of the active metabolite was determined after oral administration of pentopril. Pharmacokinetic parameters were calculated from the plasma concentration-time data for both the parent drug and its active metabolite after their separate intravenous administrations using a one-compartment model for the drug and a two-compartment model for the metabolite. The elimination half life for the drug was approximately 1 min. The elimination half-life for the metabolite was 13 min (SD, +/- 3.5, n = 4) after its direct intravenous administration, but increased to an apparent half-life of 20 min (SD +/- 5, n = 5) when formed in vivo as a metabolite. Comparison of the formation rate of the metabolite and the elimination rate of the parent drug indicated that the parent drug was rapidly and completely hydrolyzed to the acid metabolite as soon as it reached the systemic circulation. No parent drug was detected in plasma after its oral administration. The apparent bioavailability of the acid metabolite was 66% after oral drug administration. A close relation between inhibition of pressor response to angiotensin I (AI) and plasma concentration of the active metabolite was observed when plotted against time after drug or metabolite administration. A Michaelis-Menten function correlated (multiple r2:0.995) well between effect and plasma metabolite concentration with mean concentration for 50% of maximum inhibition, IC50, of 3.6 X 10(-7) M (0.11 microgram/mL). PMID- 2999377 TI - Assay for mitolactol and its bifunctional alkylating metabolites in plasma. AB - A method involving precolumn derivatization and high-performance liquid chromatography is described for the measurement of mitolactol levels in plasma. The basis of the assay is the reaction at pH 7.4 and 50 degrees C of mitolactol with diethyldithiocarbamate to form 1,6-bis(diethyldithiocarbamoyl)-2,3,4,5 tetrahydroxyhexane. This derivative is then extracted into chloroform, resolved by normal-phase chromatography, and detected by UV (254 nm) absorbance. The method quantitates the sum of mitolactol and its active bifunctional metabolites, bromoepoxydulcitol and dianhydrogalactitol, in plasma down to concentrations of 0.5 microM. The pharmacokinetic parameters of the drug in mice have been determined following the intraperitoneal injection of either 20 or 100 mg/kg of body weight. Absorption from the peritoneal cavity was largely complete by 5 min. Parameters obtained include a first-order elimination constant, k = 0.92 X 10(-2) min-1 and an apparent volume of distribution, Vd = 0.78 L/kg. For a 100-mg/kg dose, the area under the concentration-time curve was 49 mM X min, and the mean peak drug concentration was reached at 40 min following intraperitoneal injection. Concentrations of mitolactol in total plasma and in plasma ultrafiltrates were identical, indicating that the drug is not (less than 4%) reversibly bound to plasma proteins. PMID- 2999378 TI - Incidence of bubbles on samples cast in a phosphate-bonded investment. PMID- 2999379 TI - Hydroxyapatite for alveolar ridge augmentation: indications and problems. AB - The increased use of hydroxyapatite for augmentation of residual alveolar ridges has created some problems in the subsequent fabrication of complete denture prostheses. Although hydroxyapatite appears to be an effective substitute for resorbed alveolar ridges, its use is not indicated for all patients. In addition, the premise that "if a little is good, a lot is better" does not apply. The following conclusions can be drawn. Augmentation should not be considered if vestibular extension will provide acceptable results. Placement of hydroxyapatite should improve the contour and amount of the residual alveolar ridge. Use as little as possible to accomplish the goal. Preprosthetic surgery should permit vertical as well as horizontal extension of the denture. Augmentation should not result in the need to use lining mucosa to support a complete denture. Vestibular extensions are indicated for most augmented alveolar ridges. The relative strength of the strong arch should not be increased in prognathic or retrognathic ridge relationships. Preprosthetic surgery should be a combined surgical prosthodontic treatment endeavor. If a foundation is created that cannot be used effectively for the support, retention, and stability of an intended prosthesis, little benefit is provided for the patient. Small variations in intended treatment can significantly increase success of the prosthesis (Fig. 13). Unfortunately, the relative ease of hydroxyapatite placement has led to its use in patients who do not require augmentation and augmentation in amounts and regions that are not conducive to improving denture success. A coordinated effort between the surgeon and the prosthodontist will usually result in treatment that provides the best potential foundation for a specific patient. PMID- 2999380 TI - Subcellular distribution of hydrolases in Naegleria fowleri. AB - The presence and particle association of various hydrolytic enzymes in Naegleria fowleri has been studied in whole cell extracts of trophozoites in an effort to establish authentic markers for surface membrane and lysosomal components. Evidence from the experiments reported here indicates that in N. fowleri a) acid proteinase, N-acetylglucosaminidase, and acid phosphatase are associated with cytoplasmic granules closely resembling lysosomes; b) 5'-nucleotidase is associated with the surface membrane, probably on the external surface; c) aspartate aminotransferase is associated with mitochondria; d) alpha-D glucosidase and an aminopeptidase have bimodal distributions, activity being associated with both the surface membrane and lysosomal particles. PMID- 2999381 TI - Involvement of local adrenergic receptors in the process of ovulation in gonadotrophin-primed immature rats. AB - Immature female rats were primed with 4 i.u. PMSG at 08:00 h of Day 26. This results in ovulation in the morning of Day 29. The number of ovulations was counted in terms of newly formed corpora lutea in the morning of Day 30. Various adrenergic drugs were delivered into the ovarian bursa bilaterally in the afternoon of Day 27 to study their effect on ovulation. A methyl cellulose gel solution was used as vehicle to minimize leakage from the bursa. Noradrenaline, terbutaline and 4-aminopyridine significantly enhanced the number of corpora lutea compared to control ovaries injected with gel vehicle alone. The effect of terbutaline was counteracted by propranolol. Phentolamine partly blocked the noradrenaline-induced enhancement and the antagonist alone significantly reduced the number of ovulations. The results indicate that stimulation of alpha adrenergic receptors (probably via actions in the follicle wall) as well as beta receptors (influencing steroid-producing cells) may interfere with the ovulation process. PMID- 2999382 TI - Characterization of the maturational changes induced by a GnRH analogue in the rat ovarian follicle. AB - The GnRH analogue [D-Ser(t-Bu)6]des-Gly10-GnRH-N-ethylamide (GnRHa, 2 micrograms/rat) or hCG (4 i.u./rat) was administered to hypophysectomized, PMSG primed immature female rats. Oocyte maturation was initially detected by 2 h after GnRHa administration but the response to hCG was observed only after 4 h. Initiation of GnRHa-induced ovulation also preceded the response to hCG by 2 h. Maximal response to both these hormones was obtained at 10 and 14 h after hormone administration for oocyte maturation and ovulation respectively. The number of oocytes ovulated after GnRHa was significantly lower than that with hCG (29 +/- 4 and 50 +/- 7 per rat respectively; P less than 0.05). Expansion of the cumulus mass and secretion of mucoid material, which are characteristic responses to LH, were also observed after GnRHa administration. However, while the action of 5 micrograms ovine LH/ml on the cumulus cells was mediated by cAMP, no accumulation of the nucleotide could be detected in follicles exposed to GnRHa (10(-7) M). We conclude that even though GnRHa and LH/hCG seem to elicit similar responses in the ovarian follicle they differ in their kinetics, their efficiency and the mediator of their action. PMID- 2999384 TI - Photoperiodic modulation of testicular LH receptors in the bank vole (Clethrionomys glareolus). AB - The temporal changes in testicular binding of 125I-labelled hCG in juvenile bank voles (18 days of age, born and reared in a 18L:6D photoperiod) exposed to a long (18L:6D, Group L) or short (6L:18D, Group S) photoperiod for 0, 3, 7, 14 and 42 56 days were investigated. During testicular maturation, in Group L, there was a slight initial decrease in LH receptor numbers per testis followed by a marked prepubertal rise during the initial phase of rapid testicular growth after which a decrease took place. In Group S, during testicular regression, the temporal changes in LH receptor numbers per testis resembled those of Group L except that the corresponding increase in hCG binding during the initial week was considerably less marked and the receptor numbers remained thereafter at a significantly lower level than in Group L. Leydig cell count indicated that the observed changes in LH receptors per testis were due to changes in the number of Leydig cells as well as in LH receptors per Leydig cell. The present results indicate, that (1) photoperiod is an important modulator of testicular LH receptor numbers in this species, (2) photoperiod or age has no significant effect on the binding affinity of LH receptors, (3) short photoperiods arrest the induction of LH receptors as well as the increase in Leydig cell numbers associated with normal testicular maturation, and (4) changes in LH receptor numbers per testis correlate well with the photoperiod-induced changes in androgen biosynthesis, spermatogenesis and Leydig cell morphology observed in our previous studies. PMID- 2999383 TI - Purification of rabbit endometrial plasma membranes from receptive and non receptive uteri. AB - We have developed a method for isolation of plasma membranes from rabbit endometrium, with high yield and purification. Endometrial homogenates are precipitated with calcium chloride and the resulting supernatant is fractionated by centrifugation in a self-forming gradient of 20% Percoll. Before fractionation, the intact luminal epithelial surface was labelled with 125I labelled soyabean agglutinin. Between buoyant densities of 1.015 and 1.017 g/ml, a discrete peak of surface label was obtained, which coincided with activities for 5'-nucleotidase and alkaline phosphatase, enzyme markers for the plasma membrane. This peak was well separated from the majority of cellular protein, and from marker enzyme activities for mitochondria and microsomes (NADH cytochrome C reductase) and lysosomes (acid phosphatase). Electron microscopy of the purified membranes showed membrane sheets and vesicles free from other cellular organelles. Analysis of detergent-soluble membrane proteins, fractionated by concanavalin A-affinity chromatography, revealed differences in the protein pattern of membranes from uteri of rabbits receptive (Day 6 of pregnancy) and non receptive (Day 3) for implantation. The method will be useful for generation of immunological and affinity probes for surface antigens involved in ovoimplantation. PMID- 2999386 TI - Continuous infusion of oxytocin prevents induction of uterine oxytocin receptor and blocks luteal regression in cyclic ewes. AB - Continuous intravenous infusion of oxytocin (3 micrograms/h) between Days 13 and 21 after oestrus delayed return to oestrus by 7 days (length of cycle 23.3 +/- 0.6 days compared to 16.6 +/- 0.2 days in control ewes). At a lower infusion rate (0.3 micrograms/h) oxytocin delayed luteolysis in only 2 of 5 ewes. Treatment from Day 14, when luteolysis had already begun, was ineffective. Delay of luteal regression by oxytocin had no effect on the length of subsequent cycles. Measurement of circulating progesterone concentrations and luteal weight showed that prolongation of the oestrous cycle was due to prevention of luteal regression. Luteal regression and behavioural oestrus were induced during continuous oxytocin administration begun on Day 13 when cloprostenol was given on Day 15 (mean cycle length, 17.3 +/- 0.21 days). Continuous oxytocin infusion from Day 13 blocked the rise in uterine oxytocin receptor concentrations which normally precedes oestrus. Mean receptor concentrations in caruncular and intercaruncular endometrium and in myometrium were 76, 36 and 9 fmol/mg protein on Day 17 in ewes receiving continuous oxytocin (3 micrograms/h); in control ewes these values were 675, 638 and 130 fmol/mg protein respectively at oestrus. Receptor concentrations on the day of oestrus in ewes receiving oxytocin and cloprostenol were not significantly different from those in control ewes (649, 852, and 109 fmol/mg protein respectively). Since cloprostenol, a PGF-2 alpha analogue, overcame the antiluteolytic action of oxytocin, it is suggested that continuous oxytocin treatment may inhibit uterine production of PGF-2 alpha, possibly by down regulating the uterine oxytocin receptor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2999385 TI - Effect of ACTH on contralateral testicular damage and cytotoxic antisperm antibodies after unilateral testicular ischaemia in the rat. AB - Unilateral testicular ischaemia in the rat results in morphological damage in the contralateral testis (sympathetic orchiopathia). An increase in serum cytotoxic antisperm antibodies and serum IgM levels seen in this condition when compared to controls was reduced by daily i.m. administration of a synthetic adrenocorticotrophic hormone (ACTH) for 7 days. ACTH would appear to be acting as an immunosuppressant, possibly reducing the effect of this autoimmune condition on subsequent fertility. PMID- 2999387 TI - Regulation of testicular LH/hCG receptors in golden hamsters (Mesocricetus auratus) during development. AB - During prepubertal development in the golden hamster, there are major age-related changes in the number of testicular LH/hCG receptors. Between 22 and 35 days of age, there was greater than 10-fold increase in testicular LH/hCG receptors, followed by a decrease at Day 37. Concomitant with, but preceding slightly, the changes in receptors, were increases in plasma LH and FSH and most noticeably prolactin concentrations, between Days 10 and 20 of age. Inhibition of the increases in plasma levels of prolactin by daily injections of bromocriptine, between 14 and 31 days of age, resulted in suppressed testicular and seminal vesicle weights, and decreased content and concentration of testicular LH/hCG receptors. Similarly, the premature increase in plasma prolactin concentrations in prepubertal hamsters between 6 and 20 days of age, by means of ectopic pituitary transplants, resulted in increased testicular and seminal vesicle weights, as well as an increase in the concentration of testicular LH/hCG receptors. These results strongly suggest that increases in plasma prolactin values during development are important in enhancement of the development of testicular LH/hCG receptors. PMID- 2999388 TI - Unifying concept of pelvic floor disorders and incontinence. AB - Denervation of pelvic floor sphincter muscles is a feature of pelvic floor disorders. When severe, it may be accompanied by stress incontinence of faeces, or of urine. The extent of chronic partial denervation of these pelvic floor muscles can be quantified by electromyography (EMG), and its cause identified by electrophysiological studies of the motor innervation of these striated muscles. Damage to this innervation is often initiated by childbirth, but appears to progress during a period of many years so that the functional disorder usually presents in middle life. Incontinence develops in some patients, but not in others. This can be predicted by the severity of the abnormalities found in EMG studies of the pelvic sphincter musculature and motor latency studies of its innervation. The results of such investigations in the six common types of pelvic floor disorder are presented. Recognition of the causative factors leading to damage to the innervation of the pelvic sphincter muscles implies new approaches to treatment and to prevention of pelvic floor disorders and incontinence. PMID- 2999389 TI - Hepatocellular carcinoma masquerading as malignant teratoma. PMID- 2999390 TI - Potential check on heterosexual transmission of AIDS. PMID- 2999391 TI - Small cell carcinoma of the bronchus: a rare cause of haematuria from a metastasis in the urinary bladder. PMID- 2999393 TI - Synthesis and pharmacology of the potent angiotensin-converting enzyme inhibitor N-[1(S)-(ethoxycarbonyl)-3-phenylpropyl]-(S)-alanyl-(S)-pyroglutamic acid. AB - Structure 3a, a potent angiotensin-converting enzyme inhibitor, was prepared in five steps from L-(+)-alpha-amino-4-phenylbutyric acid by construction of the activated side-chain ester 16, displacement with L-pyroglutamate ester anion, and deblocking. Diastereomer separation was accomplished by chromatography at the diester stage, 17. Pharmacological assays established that 3a parallels enalapril in its ability to inhibit converting enzyme and lower blood pressure. PMID- 2999392 TI - Corticotrophin injections to treat cigarette withdrawal symptoms. PMID- 2999394 TI - Angiotensin converting enzyme inhibitors: structure-activity profile of 1 benzazepin-2-one derivatives. AB - The preparation of a series of 3-amino-2-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine 1-acetic acid derivatives 5a-y by reductive amination of 2,3,4,5-tetrahydro-1H-1 benzazepine-2,3-dione (7) with L-amino acid derivatives is described. The compounds were tested for inhibition of angiotensin converting enzyme. The structure-activity profile of the series is discussed. Compound 5a was especially potent when tested in dogs for inhibition of angiotensin I pressor response, having an ID50 = 0.07 mg/kg po. PMID- 2999395 TI - Angiotensin converting enzyme inhibitors: N-substituted D-glutamic acid gamma dipeptides. AB - The preparation of two series of N-carbobenzoxy-gamma-D-glutamyl secondary 2S amino acids and (N-substituted gamma-D-glutamyl)indoline-2(S)-carboxylic acid dipeptides is described. In vitro inhibition of angiotensin converting enzyme (ACE) is reported for each compound, and the structure-activity relationship is discussed. Oral and iv inhibition of AI pressor response in vivo of selected compounds in Table II is also discussed. The most potent compounds in vitro, 3 and 6a, had an ACE IC50 of 7 and 2.7 X 10(-9) M, respectively. PMID- 2999396 TI - GABA agonists. Resolution, absolute stereochemistry, and enantioselectivity of (S)-(+)- and (R)-(-)-dihydromuscimol. AB - (RS)-5-(Aminomethyl)-2-isoxazolin-3-ol (dihydromuscimol, DHM) is a potent 4 aminobutyric acid (GABA) agonist, the inhibitory effects of which on neurons are sensitive to the antagonist bicuculline methochloride (BMC), and it also interacts with the GABA uptake system in vitro. (S)-(+)-DHM (4) and (R)-(-)-DHM (5) were obtained in optically pure forms via resolution of tert-butyloxycarbonyl protected DHM (1) using cinchonidine as the only resolving agent. The optical purity and absolute stereochemistry of 4 and 5 were established by chemical correlation to the (S)-(+) enantiomer of 3-hydroxy-4-aminobutyric acid (GABOB). While 4 was a specific and potent BMC-sensitive GABA agonist in vivo and in vitro, possibly the most potent GABA agonist so far described, the inhibition of GABA uptake by DHM proved to reside exclusively in the (R)-(-) enantiomer (5). The affinity of 5 for BMC-sensitive GABA receptor sites in vitro was some 50 times lower than that of 4. Compounds 4 and 5 can be considered semirigid isosteres of the conformationally flexible GABA analogues (S)-(+)- and (R)-(-) GABOB, respectively, which show a very low degree of enantioselectivity with respect to GABA synaptic mechanisms. This correlation between the degree of enantioselectivity and conformational mobility of chiral GABA analogues might be of importance for the design of new drugs with specific actions at synapses at which GABA is the transmitter. PMID- 2999397 TI - Dog coronary artery adenosine receptor: structure of the N6-alkyl subregion. AB - The moderately potent and stereoselective coronary vasoactivity of N6-[1-phenyl 2(R)-propyl]adenosine (1) is the basis for the present study that maps the N6 region of the coronary artery adenosine receptor by means of the structure coronary vasoactivity relationships of 81 analogues of 1 in the open-thorax dog. Stereoselectivity is a general property of N6-substituted adenosines that have a chiral center adjacent to N6. The activity ratio of 1 to its S diastereomer is 10, the result of the positive interaction with the receptor of the propyl C-3 group of the R diastereomer in combination with the steric hindrance exerted by this group of the S diastereomer. Replacing the benzyl moiety of 1 by an ethyl, phenyl, phenethyl, or naphthyl group lowers potency of the R diastereomer and, accordingly, the R/S ratio. Propyl C-1 of 1 interacts with a receptor region large enough to accommodate three methylene residues and the propyl C-3 residue with a separate region large enough to accommodate two. The receptor subregion that interacts with the propyl C-1 of 1 is more tolerant of bulk and of polar substituents than the subregion that interacts with propyl C-3. Evidence bearing on the possible contribution of N6 to activity, e.g. through hydrogen bonding, is ambiguous. These results support a provisional model of the N6-alkyl subregion. PMID- 2999399 TI - Novel nonnarcotic analgesics with an improved therapeutic ratio. Structure activity relationships of 8-(methylthio)- and 8-(acylthio)-1,2,3,4,5,6-hexahydro 2,6-methano-3-benzazocines. AB - Conversion of the 8-phenolic 1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocines to the corresponding 8-thiophenolic analogues was achieved by three different routes. Diazotization of 8-amino-2,6-methano-3-benzazocine (2) followed by the reaction with CH3SNa afforded 8-(methylthio)-1,2,3,4,5,6-hexahydro-2,6-methano-3 benzazocine (3). Another route using Grewe cyclization was also examined for the synthesis of 3. As the most effective route, Newman-Kwart rearrangement of benzazocines was selected and closely investigated. 8-(N,N Dimethylthiocarbamoyl)oxy derivatives (6a-e) rearranged to 8-(N,N dimethylcarbamoyl)thio derivatives (7a-e) in good yields. Reductive cleavage of 7a-e and subsequent methylation or acylations gave the title compounds (3, 8-24). Although analgesic activities of sulfur-containing benzazocines decreased compared to the corresponding hydroxy compounds, the N-methyl derivative (S metazocine, 8) showed potent analgesic activity. PMID- 2999398 TI - Structure-activity relationships among benextramine-related tetraamine disulfides at peripheral alpha-adrenoreceptors. AB - Several N,N''-(dithiodi-2,1-ethanediyl)bis[N'-(arylmethyl)-1,6-hex anediamines] were prepared and evaluated for their blocking activity on postsynaptic alpha 1 adrenoreceptors in the isolated rat vas deferens. The results were compared with those obtained for benextramine (1). N,N''-(Dithiodi-2,1-ethanediyl)bis[N' (pyrrol-2-ylmethyl)-1, 6 -hexanediamine] (pyrextramine, 29) was the most potent among the tetraamine disulfides investigated. Thus, it was selected for further pharmacological evaluation to assess its receptor specificity. At a concentration of 10 microM it did not affect the responses elicited by 5-hydroxytryptamine and histamine in guinea pig ileum and by isoproterenol in guinea pig atria and tracheal chain. Furthermore, it was more specific than benextramine (1) toward the muscarinic receptor, being significantly less potent in inhibiting the carbachol-induced response in rat jejunum. These results show that pyrextramine (29) is an irreversible alpha-blocking agent that is more potent and specific than benextramine (1). In conclusion, 29 may be a useful tool in the elucidation and characterization of the peripheral alpha 1-adrenoreceptor. PMID- 2999400 TI - N-(1,3,4,6,7,12b-hexahydro-2H-benzo[b]furo[2,3-a]quinolizin -2-yl)-N- methyl-2 hydroxyethane-sulfonamide: a potent and selective alpha 2-adrenoceptor antagonist. PMID- 2999401 TI - Synthesis and activity profiles of novel cyclic opioid peptide monomers and dimers. AB - A new family of cyclic opioid peptide analogues of the type H-Tyr-D-Xxx-Phe-Yyy NH2 was obtained through amide bond formation between side chain amino and carboxyl groups of Orn (or Lys) and Asp (or Glu) residues substituted in positions 2 and 4 of the peptide sequence. Peptides were synthesized entirely by solid-phase techniques, and aside from the cyclic monomers, cyclization on the benzhydrylamine resin also produced side chain linked antiparallel cyclic dimers due to intersite reaction. In binding studies based on displacement of mu- and delta-opioid receptor-selective radiolabels from rat brain membranes the highly rigid cyclic monomer H-Tyr-D-Orn-Phe-Asp-NH2 (1) (containing a 13-membered ring) was shown to be one of the most selective mu-receptor ligands reported to date, whereas the corresponding cyclic dimer, (H-Tyr-D-Orn-Phe-Asp-NH2)2 (1a), was nonselective. The difference in receptor selectivity observed between 1 and 1a is a consequence of the different conformational constraints present in the cyclic monomer and dimer. In contrast to 1, the conformationally less restricted cyclic analogue H-Tyr-D-Lys-Phe-Glu-NH2 (3) (15-membered ring) showed no receptor preference. Qualitatively similar potency relationships were observed in the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays. However, in the case of analogues 1 and 3 discrepancies observed between potencies determined in the mu-receptor-representative GPI bioassay and in the mu-receptor-selective binding assay seemed to indicate that the conformational constraint present in these compounds may produce an "efficacy" enhancement. Corresponding analogues containing an Asp (or Glu) residue in the 2-position and an Orn (or Lys) residue in the 4-position showed similar selectivity relationships, but better agreement between bio- and binding assay data. These results indicate that incorporation of various conformational constraints into opioid peptides permits manipulation of both receptor selectivity and efficacy. PMID- 2999402 TI - 3-Phenyl-1-indanamines. Potential antidepressant activity and potent inhibition of dopamine, norepinephrine, and serotonin uptake. AB - A series of 3-phenyl-1-indanamines was synthesized and tested for potential antidepressant activity and for inhibition of dopamine (DA), norepinephrine (NE), and serotonin (5-HT) uptake. Trans isomers were generally potent inhibitors of DA, NE, and 5-HT uptake, while cis isomers preferentially inhibited the uptake of 5-HT. The affinity for the DA-uptake site was very dependent on the aromatic substitution pattern where highest potency was found for 3',4'-dichloro substituted compounds (45). This substitution pattern also resulted in high affinity for the NE-and 5-HT-uptake sites, but potent 5-HT-uptake inhibiting activity could also be obtained with other substitution patterns. Only small amines could be accommodated at the 5-HT-uptake site while larger amines such as piperazine could be accommodated both at the DA-and NE-uptake sites. The observed structure-activity relationships were explained from the results of superimpositions of a trans (45) and cis (72) isomer with 5-HT and DA, respectively, in relation to a proposed three-point binding of the uptake inhibitors at the uptake sites. Finally, comparison of the structures of the 3 phenyl-1-indanamines with other newer bicyclic catecholamine- and/or serotonin uptake inhibitors revealed common structural elements important for potent DA-, NE-, and/or 5-HT-uptake inhibition. PMID- 2999403 TI - New antiallergic pyrano [3,2-g]quinoline-2,8-dicarboxylic acids with potential for the topical treatment of asthma. AB - A number of antiallergic pyranquinolinedicarboxylic acid derivatives with potential for the topical treatment of asthma have been synthesized. All the compounds have been evaluated against rat passive cutaneous anaphylaxis and in a dog hypotension screen. This is the first detailed description of the application of the latter screen for the identification of antiallergic agents. Two compounds, disodium 9-ethyl-6, 9-dihydro-4,6-dioxo-10-propyl-4H-pyrano [3,2 g]quinoline-2,8-dicarboxylate (86) and disodium 6-(methylamino)-4-oxo-10-propyl 4H-pyrano[3,2-g]-quinoline-2, 8-dicarboxylate (72), were selected and further evaluated for their ability to induce phosphorylation of a 78000 molecular weight protein associated with the rat peritoneal mast cell. Their ability to inhibit histamine release from these cells and from a mucosal mast cell preparation has also been evaluated. These compounds, nedocromil sodium (TILADE 86) and minocromil (the free acid of 72), are at present undergoing therapeutic evaluation. The rationale for the screening procedure and the relevance of the second carboxylic acid function of these dibasic acids to receptor binding are discussed. PMID- 2999405 TI - Pyrimidinones. 1. 2-Amino-5-halo-6-aryl-4(3H)-pyrimidinones. Interferon-inducing antiviral agents. AB - Interferon induction and antiviral activity was discovered with 2-amino-5-bromo-6 phenyl-4(3H)-pyrimidinone. An analogue study incorporating a series of 2-amino-5 substituted-6-arylpyrimidinones revealed that the most potent interferon inducers were mono- and difluorophenyl analogues. These same analogues were also potent antiviral agents against Semliki Forest virus and herpes simplex type 1. In addition the monomethoxyphenyl analogues were potent antiviral agents but weak interferon inducers. Relatively modest structural changes led to dramatic changes in bioactivity. There was a relatively poor correlation between levels of circulating interferons induced and systemic antiviral activity. PMID- 2999404 TI - Factors affecting binding of trans-N-[2-(methylamino)cyclohexyl]benzamides at the primary morphine receptor. AB - In this paper, we describe the synthesis of a series of trans-N-[2 (methylamino)cyclohexyl]benzamides possessing morphine-like pharmacological properties. The affinity of the compounds for the agonist and antagonist states of the mu opioid receptor has been established by means of an in vitro binding assay. We have investigated the geometry and electronic structure of the molecules using molecular mechanics and an ab initio SCF-MO procedure with FSGO basis sets. Comparison to naloxone reveals properties of possible importance in receptor association. We have considered both the S,S and R,R isomers in the binding model. Statistical analyses imply that three factors play a significant role in binding: (1) membrane-water partitioning, (2) the capacity of the aromatic ring and amine N-substituent to act as electron acceptors, (3) the conformational energy required to attain the binding configuration. PMID- 2999406 TI - Synthesis and biological activities of some pseudo-peptide analogues of tetragastrin: the importance of the peptide backbone. AB - Pseudo-peptide analogues of the C-terminal tetrapeptide of gastrin, in which a peptide bond has been replaced by a CH2-NH bond, i.e. (tert-butyloxycarbonyl)-L tryptophyl-psi (CH2-NH)-L-leucyl-L-aspartyl-L-phenylalanine amide (8), (tert butyloxycarbonyl)-L-tryptophyl-L-leucyl-psi (CH2-NH)-L-aspartyl-L-phenylalanine amide (13), (tert-butyloxycarbonyl)-L-tryptophyl-L-leucyl-L-aspartyl-psi (CH2NH) L-phenylalanine amide (20), were synthesized. The pseudo-peptides 8 and 13 were shown to have the same affinity as (tert-butyloxycarbonyl)-L-tryptophyl-L-leucyl L-aspartyl-L-phenylalanine amide (21) for the gastrin receptor on isolated mucosal cells. The pseudo-peptide 20 exhibited lower affinity (IC50 congruent to 10(-5) M). The biological activity of these pseudo-peptides was studied on acid secretion in the anesthetized rat. Compound 8 stimulated acid secretion, identically with that of 21. Compound 13 did not exhibit any agonist activity but was able to antagonize the action of gastrin (ED50 = 0.3 mg/kg). Compound 20 did not show any agonist activity but was able to inhibit gastrin-induced acid secretion, with lower potency (ED50 = 15 mg/kg). The importance of the peptide bonds in the mode of action of gastrin is discussed, and a hypothetical approach of the mechanism of action is presented. PMID- 2999407 TI - [[(4,5-Dihydro-2-oxazolyl)phenoxy]alkyl]isoxazoles. Inhibitors of picornavirus uncoating. AB - A series of [[(4,5-dihydro-2-oxazolyl)phenoxy]alkyl]isoxazoles has been synthesized and evaluated as antipicornavirus agents. The effect of alkyl groups in the 4- and 5-position of the oxazoline ring, as well as the alkyl chain length, on antiviral activity was examined. Compound 14 was evaluated in vivo and was found to significantly reduce mortality at an oral dose of 4 mg/kg in mice infected intracerebrally with poliovirus-2. Compound 14 was also effective in preventing paralysis when administered intraperitoneally to mice infected subcutaneously with a lethal dose of ECHO-9 virus. On the basis of the results of these studies, compound 14 is a strong candidate for clinical evaluation as a systemic agent for the treatment of picornavirus infections. PMID- 2999408 TI - Hybromet: a ligand for purifying opioid receptors. AB - Condensation of the Grignard reagent derive from 2-[4-(allyloxy)phenyl]ethyl bromide (4b) with 7 alpha-acetyl-6,14-endo-ethenotetrahydrothebaine (5) furnished the (R) tertiary carbinol, 7, which upon methoxymercuration followed by treatment with the KBr gave the bromomercurio compound 10 (Hybromet). The corresponding N cyclopropylmethyl analogue, 11, was prepared also. The bromomercurio compound, 1, and the mercaptobenzothiazole derivative, 3, gave allyl phenyl ether when treated with BAL at room temperature. Similar treatment of 10 with BAL gave 7 in high yield. Binding studies using rat brain homogenates indicated that 7, 13, and 14 have moderately high affinities for mu rather than delta binding sites. Although much weaker, 10 showed preferential mu binding also. These results along with the fact that 10 reacted smoothly with sulfhydryl groups suggest that Hybromet would be a suitable ligand for use in affinity chromatography. PMID- 2999409 TI - 3,4-O-diacetylisoproterenol. Preparation, structure proof, and beta-receptor effect. AB - Direct acetylation of isoproterenol by selective O-acetylation using CH3COCl/CF3COOH was shown to lead to the formation of 2-(3,4-diacetoxyphenyl)-2 chloro-N-isopropyl-1-ethanamine and not to 3,4-O-diacetylisoproterenol. The latter was prepared by reduction of 3,4-diacetoxy(2-isopropylamino)acetophenone and its structure confirmed by IR, 1H, 13C NMR, mass spectral, and elemental analysis. The two compounds were tested for activity on beta-receptors. Efficacy and affinity on beta 1-receptors were found identical with the effect of isoproterenol. So was efficacy on beta2-receptors, while affinity was lower for the chloro compounds than for isoproterenol and diacetylisoproterenol which exhibited identical affinity. PMID- 2999410 TI - Antibody-dependent cell-mediated cytotoxicity by human lymphocytes: a regulatory role for the sheep red blood cell receptor. AB - Antibody-dependent cell-mediated cytotoxicity (ADCC) is a cytolytic mechanism whereby unimmunized lymphoid cells lyse antibody-sensitized target cells. The cells mediating ADCC are referred to functionally as killer cells (K cells). K cells are a heterogeneous population of lymphocytes based on cell membrane marker criteria but may be morphologically homogeneous. It is well established that K cells must have an Fc receptor (FcR) appropriate for the class of target cell bound antibody, and that physical interaction between the K cell's FcR and the Fc portion of target cell-bound antibody is required for the initiation of lysis. Relatively little is known about the regulatory mechanisms presumed to exist and modulate ADCC reactions. We have made 2 observations that led us to investigate the possibility that the sheep erythrocyte (SRBC) receptor (ER) on human cells might be involved as a regulator of ADCC. First, SRBC are more efficiently lysed by human lymphocytes than are other erythrocyte target cells, and second, although nonsensitized bystander cells are usually not lysed during ADCC reactions, SRBC are consistently lysed. Removal of E-rosetting capacity of lymphocytes by trypsin treatment selectively decreases lysis of IgG sensitized SRBC. Furthermore, lysis of nonsensitized bystander SRBC is also inhibited by trypsin treatment of effector cells. ADCC is also decreased by blocking the ER with simple sugars or monoclonal antibody so that E-rosettes are inhibited. In contrast to trypsin treatment, which selectively inhibits lysis of SRBC, ER specific monoclonal antibody and simple sugars decrease cytolysis against all target cells tested. PMID- 2999411 TI - Evolution of two actin genes in the sea urchin Strongylocentrotus franciscanus. AB - The complete nucleotide sequences of two chromosomally linked actin genes from the sea urchin Strongylocentrotus franciscanus are presented. The genes are separated by 5.7 kilobases, occur in the same transcriptional orientation, and contain introns in identical positions. The structures and nucleotide sequences of the two genes are extremely similar, suggesting that they arose through a recent duplication. Comparison of the nucleotide sequences of the genes allows inferences to be made about mutational mechanisms active since the duplication event. Whereas point mutations predominate in the coding regions, the introns and flanking DNA are more heavily influenced by a variety of events that cause simultaneous changes in short regions of DNA. PMID- 2999413 TI - Caution: AIDS virus present--handle with care! PMID- 2999415 TI - Clinical use of tricalciumphosphate and hydroxyapatite in maxillofacial surgery. PMID- 2999414 TI - The use of hydroxylapatite to coat subperiosteal implants. PMID- 2999412 TI - Rat LINE1: the origin and evolution of a family of long interspersed middle repetitive DNA elements. AB - We present approximately 7.0 kb of composite DNA sequence of a long interspersed middle repetitive element (LINE1) present in high copy number in the rat genome. The family of these repeats, which includes transcribing members, is the rat homologue of the mouse MIF-Bam-R and human Kpn I LINEs. Sequence alignments between specimens from these three species define the length of a putative unidentified open reading frame, and document extensive recombination events that, in conjunction with retroposition, have generated this large family of pseudogenes and pseudogene fragments. Comparative mapping of truncated elements indicates that a specific endonucleolytic activity might be involved in illegitimate (nonhomologous) recombination events. Sequence divergence analyses provide insights into the origin and molecular evolution of these elements. PMID- 2999416 TI - Cytochemical study of macrophage lysosomal inorganic trimetaphosphatase and acid phosphatase. AB - Cytochemical investigations have associated acid inorganic trimetaphosphatase (TMPase) activity with the lysosomes of certain cell types. We have used the modified staining technique of Berg to show that this enzyme activity is present in normal mononuclear phagocytes and macrophage cell lines. We have found this enzyme activity to be present in murine RAW264 macrophages, in human U937 macrophages, in normal human blood monocytes, and in guinea pig peritoneal macrophages. All of the RAW264 and U937 macrophages showed intense TMPase activity. Many of the human monocytes and most of the guinea pig macrophages were labeled by this method. The reaction product was associated with the lysosomes of these cell types. The lysosomal staining-pattern was similar to that of acid phosphatase. Differences with regard to Golgi staining were noted. This indicates that TMPase is a lysosomal enzyme of mammalian macrophages. The distinction between TMPase and acid phosphatase activity has been demonstrated by measuring the pH optimum of each enzyme. Using substrates identical to those of the ultrastructural cytochemistry, we show that the pH optimum of TMPase is 4.0 and that of acid phosphatase is 5.0. The enzymatic activities are therefore ultrastructurally and biochemically distinct. Following phagocytosis of latex, yeast (Saccharomyces cerevisiae), or Corynebacterium parvum, TMPase has been found to be associated with phagosomes. This enzyme may take part in the degradation of phagocytosed materials, particularly microorganisms which contain inorganic polyphosphates and metaphosphates. PMID- 2999417 TI - The role of liver endothelium in the binding and uptake of ceruloplasmin: studies with colloidal gold probe. AB - To determine the mode of uptake of ceruloplasmin (CP) by liver, the protein was labeled with colloidal gold and infused into the portal vein. In cold almost all probes bound to the sinusoidal endothelium, and at 37 degrees C internalization via a system of coated pits and vesicles occurred. Only rarely did the probe appear to bypass the endothelium, moving to the abluminal side through the gaps between endothelial cells. In the endothelial cytoplasm, the probe was seen in coated vesicles, endosomes, tubules, and large vesicles which may have formed by fusion of endosomes and tubules. Moreover, externalization of the probe to the abluminal side was noted, and this also occurred via a system of coated vesicles. The findings suggest that the uptake of CP in the liver may be primarily a transendothelial phenomenon (transcytosis). PMID- 2999419 TI - Synthetic glycoprotein D-related peptides protect mice against herpes simplex virus challenge. AB - Glycoprotein D (gD) of herpes simplex virus (HSV) protects mice from a lethal challenge by either HSV type 1 (HSV-1; oral) or HSV-2 (genital). We evaluated whether synthetic peptides representing residues 1 through 23 of gD (mature protein) can be used as a potential synthetic herpesvirus vaccine. The immunogenicity of the peptides was demonstrated by the biological reactivity of antipeptide sera in immunoprecipitation and neutralization assays. All sera which immunoprecipitated gD had neutralizing against both HSV-1 and HSV-2. The highest titers were found in animals immunized with the longest peptides. The region of residues 1 through 23 was immunogenic regardless of whether the type 1 or type 2 sequence was presented to the animal. Immunization of mice with gD or synthetic peptides conferred solid protection against a footpad challenge with HSV-2. However, the peptides were not as effective as gD in protection against an intraperitoneal challenge. The results suggested that synthetic vaccines based on gD show promise and should be more rigorously tested in a variety of animal models. PMID- 2999418 TI - Sequences involved in determining the locations of the 5' ends of the late RNAs of simian virus 40. AB - The 5' ends of the simian virus 40 (SV40) late RNAs are heterogeneous in location, spanning a 300-nucleotide region from residues 28 to 325. To examine whether upstream or downstream measuring functions analogous to the TATA box play roles in positioning the 5' ends of these RNAs, we determined by S1 and primer extension mapping the locations of the 5' ends of the late viral RNAs made in monkey cells infected with: (i) three wild-type strains of SV40 that contain tandem duplications of the enhancer region that are 64, 85, and 91, rather than 72, base pairs in length; (ii) four viable mutants that contain alterations in the 21-base-pair tandem repeats; and (iii) four viable mutants that possess small deletions or insertions at or near the major cap site at residue 325. Most of the 5' ends of the RNAs were identical in location to those seen with wild-type strain 776. The only exceptions were the absence of RNAs whose 5' ends mapped to within three bases upstream or downstream of a sequence alteration. In addition, the sequences within residues 251 to 277 that function as transcriptional initiation sites in wild-type strain 776 also did so in their second locations in the wild-type strains in which these sequences are duplicated. Differences were noted in the relative abundances of the numerous 5' ends of the late RNAs, even among the wild-type strains. These findings indicate that many (and likely all) of the approximately two dozen locations of 5' ends of SV40 late RNAs are each determined largely by sequences within their immediate vicinity. However, sequences somewhat removed from these transcriptional initiation sites may modulate the efficiencies with which they are utilized. PMID- 2999420 TI - Polyomavirus middle T protein encoded by a retrovirus transforms nonestablished chicken embryo cells. AB - A murine retrovirus encoding the middle T protein of polyomavirus infected and transformed nonestablished chicken embryo cells. The infected cultures formed colonies in soft agar-containing medium and released infectious transforming virus. Middle T protein expressed in the transformed chicken cells associated with p60c-src and, in immunoprecipitates, enhanced the tyrosine protein kinase activity of p60c-src. PMID- 2999421 TI - Sequence alterations in temperature-sensitive M-protein mutants (complementation group III) of vesicular stomatitis virus. AB - Sequences were determined of the coding regions of the M-protein genes of the Glasgow and Orsay strains of vesicular stomatitis virus (Indiana serotype) and of two group III (M-protein) mutants derived from each wild type. Synthetic primers were annealed with viral genomic RNA and extended with reverse transcriptase. The resulting high-molecular-weight cDNA was sequenced directly. Both Glasgow and Orsay wild types differed in 13 bases from a clone of the San Juan strain sequenced by J. K. Rose and C. J. Gallione (J. Virol. 39:519-528, 1981). Six of these base changes caused amino acid changes in each wild type, whereas seven were degenerate. The Orsay and Glasgow sequences resembled each other more closely than either resembled that of Rose and Gallione, differing in eight nucleotides and four amino acids. Each of the four mutants, however, differed from its parent wild type in only one or two point mutations. Every mutation caused a change either from or to a charged amino acid; the change for tsG31 was Lys (position 215) to Glu, the change for tsO23 was Gly (position 21) to Glu, the change for tsO89 was Ala (position 133) to Asp, the changes for tsG33 were Lys (position 204) to Thr and Glu (position 214) to Lys. The charge differences predicted from these amino acid changes was confirmed by nonequilibrium pH gradient electrophoresis for tsG31, tsG33, tsO23, and the two wild types. These mutations affect residues spanning nearly 85% of the linear sequence, although the mutants possess nearly identical phenotypic properties. PMID- 2999422 TI - Roles of helper and defective retroviral genomes in murine erythroleukemia: studies of spleen focus-forming virus in the absence of helper. AB - Retroviruses that cause acute oncogenesis are generally complexes of a replication-competent helper virus and a replication-defective component. However, the pure defective components have not been previously available. We prepared the defective spleen focus-forming virus component of Rauscher erythroleukemia virus (R-SFFV) by transfecting a colinear R-SFFV DNA clone into a retroviral packaging cell line (psi 2 cells). The transfected cells released virus (psi 2/SFFV) that was free of helper virus and that induced erythropoietin dependent erythroid burst formation in bone marrow cultures. When injected into normal adult NIH/Swiss mice in moderate doses, psi 2/SFFV caused a rapid splenic erythroblastosis that regressed. Extensive erythroblastosis could be maintained by repeated injections of psi 2/SFFV into anemic mice or by the addition of a helper virus. We conclude that R-SFFV alone causes proliferation but not immortalization of a population of erythroblasts that is normally replenished from a precursor stem cell pool. Because these precursor cells are inefficiently infected, a single moderate inoculum of psi 2/SFFV causes a wave of erythroblastosis. The properties of the proliferating erythroblasts are substantially determined by the R-SFFV viral component. PMID- 2999423 TI - Multiple spliced and unspliced transcripts from human cytomegalovirus immediate early region 2 and evidence for a common initiation site within immediate-early region 1. AB - Human cytomegalovirus immediate-early (IE) region 2 (0.732 to 0.740 map unit) begins 35 nucleotides downstream of IE region 1 (Stenberg et al., J. Virol. 49:190-199, 1984). A series of mRNAs that have different splicing patterns are transcribed from region 2. There is an unspliced 1,589-nucleotide exon present in minor amounts and two spliced exons (836 and 289 nucleotides) present in larger amounts. The IE region 2 exons were found to be spliced onto the first three exons of region 1. Therefore, under IE conditions the region 1 promoter regulatory region can promote transcription of region 2. Promoter sequences (i.e., CAAT and TATA boxes) are found upstream of the 5' end of IE region 2 but presumably function poorly at IE times after infection. The transcriptional regulation of these IE genes and the possible functional roles of the proteins are discussed. We postulate that a series of unique but related proteins are made from the region 2 transcripts. Some of these proteins should contain the same 169 amino-terminal residues as the major IE 72-kilodalton protein encoded by IE region 1 (Stenberg et al., J. Virol. 49:190-199, 1984). Variations in the amino acid sequences of the region 2 proteins could occur at either the amino terminus, the carboxy terminus, or both termini. PMID- 2999424 TI - Autoregulation of the human cytomegalovirus major immediate-early gene. AB - The gene coding for the human cytomegalovirus major immediate-early 72-kilodalton protein was cloned into simian virus origin of DNA replication plasmid pSVOd. Transfection of this plasmid (pSVCC2) into cells constitutively expressing the simian virus 40 T-antigen resulted in readily detectable levels of immediate early region 1-specific RNA and protein. Partial restriction enzyme digestion of pSVCC2 was used to generate specific amino acid deletions within the 72 kilodalton protein. Mutant delta S12, which contained a deletion of 145 amino acids at the carboxy terminus of the protein, accumulated at least 10 times more immediate-early region 1 RNA than wild-type pSVCC2 did. In contrast, normal levels of delta S12-specific RNA were detected in cells cotransfected with wild type pSVCC2. Therefore, the wild-type gene was capable of suppressing transcription of the mutant gene. Our results suggest that the wild-type major immediate-early protein of cytomegalovirus autoregulates transcription of immediate-early region 1 and that one of the regulatory domains is within the carboxy-terminal 145 amino acids of the viral protein. PMID- 2999425 TI - Characterization of ribosome binding on Rous sarcoma virus RNA in vitro. AB - We determined the sites at which ribosomes form initiation complexes on Rous sarcoma virus RNA in order to determine how initiation of Pr76gag synthesis at the fourth AUG codon from the 5' end of Rous sarcoma virus strain SR-A RNA occurs. Ribosomes bind almost exclusively at the 5'-proximal AUG codon when chloride is present as the major anion added to the translational system. However, when chloride is replaced with acetate, ribosomes bind at the two 5' proximal AUG codons, as well as at the initiation site for Pr76gag. We confirmed that the 5'-proximal AUG codon is part of a functional initiation site by identifying the seven-amino acid peptide encoded there. Our results suggest that (i) translation in vitro of Rous sarcoma virus virion RNA results in the synthesis of at least two polypeptides; (ii) the pattern of ribosome binding observed for Rous sarcoma virus RNA can be accounted for by the modified scanning hypothesis; and (iii) the interaction between 40S ribosomal subunits or 80S ribosomal complexes is stronger at the 5'-proximal AUG codon than at sites farther downstream, including the initiation site for the major viral proteins. PMID- 2999426 TI - Simian virus 40 large T antigen oligomers: analysis of electrophoresis in the absence of detergent. AB - Large T antigen of simian virus 40 is found as monomeric and oligomeric species in transformed cells. These can be demonstrated in cell extracts by velocity centrifugation in sucrose gradients. We analyzed them further in a transformed human line cell (SV80) and a transformed mouse line cell (SVT2). Individual fractions from sucrose gradients were subjected to polyacrylamide gel electrophoresis in the absence of detergent. T-antigen species were then detected by protein blotting and antibody overlay with polyclonal anti-D2 T antibody or monoclonal Pab419, Pab101, or Pb1700 antibody. The rapidly sedimenting species (14S and larger) of large T antigen from both cell lines reproducibly showed two major bands with estimated molecular weights of 670,000 and 850,000. A third band of 1,200,000 was more prominent in SVT2 cells than in SV80 cells. In SV80 cells the slowly sedimenting species of large T antigen (5S to 11S) contained two reproducible bands. A band with a molecular weight of 95,000 was the predominant one in all fractions between 5S and 11S. A relatively minor band with a molecular weight of 230,000 was found in fractions between 9S and 11S. The low-molecular weight forms were seen in SVT2 cells only when a prominent peak at 5S to 7S was present, that is, when extracts were stored before analysis. In fresh extracts, the low-molecular-weight bands and slowly sedimenting forms were absent. PMID- 2999427 TI - Biological and biochemical differences between variants of spleen focus-forming virus can be localized to a region containing the 3' end of the envelope gene. AB - Two variants of the spleen focus-forming virus (SFFV), SFFVP and SFFVA, induce acute erythroleukemia in mice but differ in their effects on erythroid cells as well as in the posttranslational modification of the product of their envelope genes. To localize the region of the SFFV envelope gene responsible for these differences, we utilized a recombinant virus containing the 3' half of the SFFVP env gene, where the vast majority of the differences between SFFVP and SFFVA reside, and the SFFVP long terminal repeat (LTR) on an SFFVA background. Analysis of the recombinant virus indicates that it is capable of inducing all of the biological effects previously associated with SFFVP, including the ability to proliferate and differentiate without the need for erythropoietin. In addition, the env gene product of the recombinant virus can be detected on the cell surface, a property previously associated only with SFFVP. Although the recombinant virus also contains LTR sequences from SFFVP, we do not believe it is likely that the four LTR nucleotides that are unique to SFFVP are responsible for the biological or biochemical differences observed. These results strengthen the argument that the SFFVP env gene product acts at the cell surface to alter the hormonal requirements for erythroid cell growth and differentiation. PMID- 2999428 TI - Three trans-acting regulatory proteins of herpes simplex virus modulate immediate early gene expression in a pathway involving positive and negative feedback regulation. AB - trans-Acting regulatory components of herpes simplex virus were studied in a transient assay system by the analysis of expression of recombinant constructs which contain virus delayed-early (DE) or immediate-early (IE) upstream promoter regulatory regions linked to the bacterial gene for chloramphenicol acetyltransferase (CAT). These recombinant CAT constructs were cotransfected into Vero cell cultures together with intact genes for the IE175K protein, the IE110K protein, or the late component, Vmw65. We demonstrate specific functional interactions between the trans-acting factors and their appropriate cis-acting regulatory signals. Thus, the IE175K protein stimulated expression only from the DE-CAT constructs, and the late Vmw65 protein stimulated expression only from the IE-CAT construct. Unexpectedly, however, the IE110K protein stimulated expression from both DE- and IE-CAT constructs. Furthermore, the IE175K protein inhibited both basal levels and IE110K- or Vmw65-activated levels of expression from its own promoter-regulatory region in the IE-CAT construct. These results provide direct evidence for a negative autoregulatory role of IE175K protein on its own expression at the transcriptional level and demonstrate differences in functional properties of the IE175K and IE110K proteins, which we speculate may reflect different mechanisms of action of the two proteins. PMID- 2999429 TI - Patterns of methylation of polyomavirus DNA in polyoma-transformed rat cells. AB - The patterns of methylation of integrated polyomavirus (Py) DNA and flanking cellular sequences were determined in an inducible line of Py-transformed rat cells, designated LPT, by an analysis of cleavage patterns of LPT DNA generated by the restriction enzymes MspI, HpaII, and HhaI. The Py DNA in LPT cells is integrated into a single chromosomal site and includes whole viral genomes arranged in a head-to-tail configuration. Amplification of the viral DNA and synthesis of infectious virus can be induced in these cells by treatment with carcinogens. The experiments reported here show that in uninduced LPT cells only the late Py genes, which encode the Py capsid proteins, and sequences flanking one of the two viral DNA-cell DNA junctions are methylated. The early genes, encoding the Py T antigens, and sequences flanking the second junction are unmethylated. Since only the early genes are transcribed and translated in uninduced LPT cells, it is apparent that an inverse correlation exists between transcription and methylation of the integrated Py genes in LPT cells, as reported in other cellular and viral systems. The patterns of methylation of integrated Py genomes were also examined in cells of a subclonal derivative of the LPT line in which the virus cannot be activated. In these cells, not only the late Py genes were found to be methylated, but also the 3' portion of the early gene which encodes the Py large T antigen. These findings may have implications for understanding Py induction in LPT cells. PMID- 2999430 TI - Two independent mutations are required for temperature-sensitive cell transformation by a Rous sarcoma virus temperature-sensitive mutant. AB - We molecularly cloned the src coding region of tsNY68, a mutant of Rous sarcoma virus temperature sensitive (ts) for transformation, and constructed a series of ts wild-type recombinant src genes. DNA containing the hybrid genes was transfected into chicken cells together with viral vector DNA and helper viral DNA, and infectious transforming viruses were recovered. Characterization of these recombinant viruses indicated that at least two mutations are present in the 3' half of the mutant src gene, both of which are required for ts. Nucleotide sequence analysis revealed three differences in the deduced amino acid sequence compared with the parental virus. Two of these changes, a deletion of amino acids 352 to 354 and an amino acid substitution at position 461, are responsible for the ts phenotype. PMID- 2999431 TI - Construction and analysis of additional adenovirus substitution mutants confirm the complementation of VAI RNA function by two small RNAs encoded by Epstein-Barr virus. AB - Adenovirus VAI RNA is essential for the efficient initiation of translation of viral mRNAs at late times after infection. Recently, by constructing an adenovirus type 5 substitution mutant, we showed that the Epstein-Barr virus encoded two small RNAs complemented for the VAI RNA function in the adenovirus type 5 lytic growth (Bhat and Thimmappaya, Proc. Natl. Acad. Sci. USA 80:4789 4793, 1983). This observation was based on our inability to propagate an adenovirus type 5 mutant lacking functional VAI and VAII genes. Subsequently, it was found that this mutant was viable and able to grow to a low titer. Therefore, we examined the complementation of the VAI RNA function by the Epstein-Barr virus encoded RNAs by constructing additional adenovirus type 5 substitution mutants containing multiple copies of the Epstein-Barr virus-encoded RNA genes in nonessential early transcriptional region III. The new substitution mutants synthesized viral polypeptides at late times at levels comparable to those observed in wild type-infected cells. Our results convincingly demonstrated that the two Epstein-Barr virus-encoded RNAs can efficiently complement for the VAI RNA-mediated translational defect in adenovirus-infected cells. PMID- 2999432 TI - Effect of herpes simplex virus types 1 and 2 on surface expression of class I major histocompatibility complex antigens on infected cells. AB - Cytotoxic T lymphocytes (CTL) generated in C57BL/6 (H-2b) mice in response to infection with the serologically distinct herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) were cross-reactive against target cells infected with either serotype. However, HSV-2-infected cells were shown to be much less susceptible to CTL-mediated lysis, and analysis through the use of HSV-1 X HSV-2 intertypic recombinants mapped the reduced susceptibility to a region contained within 0.82 to 1.00 map units of the HSV-2 genome. The study reported here was undertaken to determine the possible reasons for the reduced susceptibility of HSV-2-infected cells to lysis by CTL. Competition for the specific lysis of labeled HSV-1 infected cells by either HSV-1- or HSV-2-infected, unlabeled inhibitor cells and frequency analysis of the CTL precursor able to recognize HSV-1- and HSV-2 infected cells suggested that the reduced susceptibility of HSV-2-infected cells to lysis could be explained, at least in part, by reduced levels of target cell recognition. A determination of the surface expression of the critical elements involved in target cell recognition by CTL following infection with HSV-1 or HSV 2 revealed that all the major HSV-specific glycoprotein species were expressed. Infection with both HSV-1 and HSV-2 caused a reduction in the expression of the class I H-2 antigens. However, this reduction was much greater following infection with HSV-2. This suggested that one important factor contributing to reduced lysis of HSV-2-infected cells may be the altered or reduced expression of the class I H-2 self-antigens. PMID- 2999433 TI - Membrane association of the transforming protein of avian sarcoma virus UR2 and mutants temperature sensitive for cellular transformation and protein kinase activity. AB - The localization of the transforming protein P68gag-ros of avian sarcoma virus UR2, which has a hydrophobic region at the N terminus of its ros-specific tyrosine kinase-encoding sequence, was examined by subcellular fractionation. P68 behaved as an integral membrane protein associated with the plasma membrane of transformed cells. P68 became membrane associated very rapidly in its biogenesis. Three temperature-sensitive mutants of UR2 were isolated and characterized. Cells infected with the mutants were temperature sensitive for morphological alteration and colony formation. The mutant P68 proteins were membrane associated in mutant infected cells regardless of the temperature but were active as protein kinases only at the permissive temperature. The results suggest that P68 is a membrane associated protein whose kinase activity plays a crucial role in UR2-mediated cell transformation. PMID- 2999435 TI - Epstein-Barr virus genome may encode a protein showing significant amino acid and predicted secondary structure homology with glycoprotein B of herpes simplex virus 1. AB - We report significant sequence and predicted secondary structure homology between the herpes simplex virus 1 glycoprotein B (gB) and a protein predicted to be encoded by the BALF4 reading frame of Epstein-Barr virus (EBV). Homology was detectable at the DNA level and was highly significant at the protein level and when evolutionary substitution frequencies of amino acids in related proteins were taken into account. Hydropathic analyses predicted that the two proteins possess conserved N-terminal and C-terminal hydrophobic domains. The N-terminal hydrophobic domains share features in common with known cleavable membrane insertion signal sequences. The amino acid sequences of the C-terminal hydrophobic domains predict three adjacent membrane-spanning segments as had been previously predicted for gB. In an alignment of the two amino acid sequences, 247 of 903 gB residues had a matched pair in the BALF4 sequence, and 247 of 854 BALF4 residues were found to have a matched pair in the gB sequence. In addition, all 10 cysteine residues located outside the predicted signal sequence of both proteins were conserved, as were four predicted N-linked glycosylation sites. In all, 43% of the residues in the aligned sequences are predicted to possess equivalent secondary structures. gB is a virion envelope glycoprotein required for virus entry into cells. The domain of gB determining the rate of entry into cells has been mapped; the predicted structure of this domain in gB and the predicted EBV protein are almost identical. Similarly, the cytoplasmic domain of gB postulated to interact with submembrane proteins was also nearly identical in predicted structure to that of the EBV protein. These results suggest that EBV encodes a protein similar in structure and function to the herpes simplex virus 1 gB. PMID- 2999434 TI - Molecular and biological characterization of the endogenous ecotropic provirus of BALB/c mice. AB - We have isolated two identical molecular clones of the single, endogenous ecotropic provirus of BALB/c mice. The BALB/c clones are approximately 1/10 as infectious as an exogenous proviral clone derived from AKR mice, p623. Transfection of mouse cells with each BALB/c proviral clone yielded XC-negative, N-tropic, ecotropic virus. Cotransfection of subgenomic fragments of p623 and the BALB/c provirus did not increase infectivity to the level observed for p623; however, a 292-base-pair fragment of the p623 env gene was found to rescue XC plaque formation. Sequence analysis showed that the XC-negative BALB/c provirus differed from the XC-positive AKR-derived provirus at a single nucleotide at the junction of the gp70 and p15E envelope proteins. Extensive sequence analysis of the BALB/c endogenous provirus showed that it differed from the sequence of the AKR-derived provirus at approximately 0.5% of 4,500 sequenced nucleotides. In addition, the BALB/c long terminal repeat contains a single copy of the enhancer containing sequences that are repeated twice in p623. The limited variation between the ecotropic proviruses of BALB/c mice and AKR mice suggests that few cycles of reverse transcription separate these viral genomes. PMID- 2999436 TI - Synthesis of the Ad2+ND5-specified 42K protein is regulated posttranscriptionally in abortively infected monkey cells. AB - In abortive infections of monkey cells by Ad2+ND5, the synthesis of the simian virus 40-specific 42,000-molecular-weight (42K) protein was reduced approximately 10-fold compared with a productive coinfection by Ad2+ND5 plus Ad2hr400 and about 20-fold compared with productive infections by Ad2+ND5 plus simian virus 40 or by Ad2+ND2 alone. However, the level of Ad2+ND5-specific mRNA was depressed twofold or less in abortive infections compared with productive infections. Moreover, the 42K mRNA isolated from abortive Ad2+ND5 infections translated in vitro with the same efficiency as the mRNA isolated from productive coinfections. This is analogous to the block to synthesis of the adenovirus fiber polypeptide in monkey cells (Anderson and Klessig, J. Mol. Appl. Genet. 2:31-43, 1983). Also like fiber protein, the increased level of the 42K protein found in productive infections was due to enhanced synthesis, not increased stability of the protein. Our results suggest that the synthesis of the Ad2+ND5-specified 42K protein and the adenovirus fiber protein are regulated in similar posttranscriptional manners. PMID- 2999437 TI - Similar regulation of the synthesis of adenovirus fiber and of simian virus 40 specific proteins encoded by the helper-defective Ad2+SV40 hybrid viruses Ad2+ND5 and Ad2+ND4del. AB - Human adenoviruses fail to multiply effectively in monkey cells. The block to the replication of these viruses can be overcome by coinfection with simian virus 40 (SV40) or when part of the SV40 genome is integrated into and expressed as part of the adenovirus type 2 (Ad2) genome, as occurs in several Ad2+SV40 hybrid viruses, such as Ad2+ND1, Ad2+ND2, and Ad2+ND4. The SV40 helper-defective Ad2+SV40 hybrid viruses Ad2+ND5 and Ad2+ND4del were analyzed to determine why they are unable to grow efficiently in monkey cells even though they contain the appropriate SV40 genetic information. Characterization of the Ad2+ND5-SV40 specific 42,000-molecular-weight (42K) protein revealed that this protein is closely related, but not identical, to the SV40-specific 42K protein of the SV40 helper-competent Ad2+ND2 hybrid virus. Although the minor differences between these proteins may be sufficient to account for the poor growth of Ad2+ND5 in monkey cells, the most striking difference between helper-competent Ad2+ND2 and helper-defective Ad2+ND5 is in the production of the SV40-specific protein after infection of monkey cells. Whereas synthesis of the SV40-specific proteins of Ad2+ND2 is very similar in human and in monkey cells, production of the 42K protein of Ad2+ND5 is dramatically reduced in monkey cells compared with human cells. Similarly, the synthesis of the SV40-specific proteins of Ad2+ND4del is markedly reduced in monkey cells. Thus, it is likely that both Ad2+ND5 and Ad2+ND4del are helper defective because of a block in the production of their SV40-specific proteins rather than because their SV40-specific proteins are nonfunctional. This block, like the block to adenovirus fiber synthesis, is overcome by coinfection with SV40, with helper-competent hybrid viruses, or with host range mutants of adenoviruses. This suggests that the synthesis of fiber and the synthesis of SV40-specific proteins are similarly regulated in Ad2+SV40 hybrid viruses. PMID- 2999438 TI - Transcriptional and translational mapping and nucleotide sequence analysis of a vaccinia virus gene encoding the precursor of the major core polypeptide 4b. AB - We prepared antiserum that reacted with a major core polypeptide of approximately 62,000 daltons (62K polypeptide), designated 4b, and its 74K precursor, designated P4b. A cell-free translation product of vaccinia virus late mRNA that comigrated with P4b was specifically immunoprecipitated. The late mRNA encoding P4b hybridized to restriction fragments derived from the left end of the HindIII A fragment and to a lesser extent from the right side of the HindIII D fragment. A polypeptide that comigrated with P4a, the precursor of another major core polypeptide, was synthesized by mRNA that hybridized to DNA segments upstream of the P4b gene. Complete nucleotide sequence analysis of the P4b gene revealed an open reading frame, entirely within the HindIII A fragment, that was sufficient to encode a 644-amino-acid polypeptide of 73K. The 5' end of the P4b mRNA was located at or just above the translational initiation site. PMID- 2999439 TI - Replication of cytomegalovirus in human arterial smooth muscle cells. AB - Cytomegalovirus (CMV) strain AD-169 replicated in smooth muscle cell (SMC) cultures derived from human umbilical arteries, producing enveloped infectious virions. However, unlike the effects of CMV on fully permissive human lung fibroblasts, the effects of strain AD-169 on SMC cultures were delayed and prolonged, resulting in extended survival of a fraction of the starting population. This period of survival did not exceed the life-span of the control SMC cultures. Infectious CMV continued to be isolated from the surviving SMC cultures after extinction of the original inoculum by dilution and after treatment of the cultures with CMV neutralizing antibody. The implications of these findings for the pathogenesis of atherosclerosis are discussed. PMID- 2999440 TI - Human cytomegalovirus-induced DNA polymerase and its interaction with the triphosphates of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil, 5-iodocytosine, and -5-methylcytosine. AB - Human cytomegalovirus-induced DNA polymerase and cellular DNA polymerase alpha were purified by successive chromatography on DEAE-cellulose, phosphocellulose, heparin agarose, and single-stranded DNA agarose columns. The purified virus induced DNA polymerase was resolved to consist of two polypeptides corresponding to molecular weights of 140,000 and 58,000, as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Virus-induced DNA polymerase and cellular alpha polymerase were examined for their sensitivities to the triphosphates of 1 (2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAUTP), -5 iodocytosine (FIACTP), and -5-methylcytosine (FMACTP). The inhibitive effects of these triphosphates on the DNA polymerases were competitive with regard to the natural substrates; thus FMAUTP competes with dTTP, and FIACTP and FMACTP compete with dCTP. The inhibition constants (Ki) for FMAUTP, FIACTP, and FMACTP of virus induced DNA polymerase are 0.06, 0.30, and 0.47 microM, respectively. Cellular DNA polymerase alpha is much less sensitive to these inhibitors, and its Ki values for FMAUTP, FIACTP, and FMACTP are 0.45, 3.10, and 2.90 microM, respectively. In addition, human cytomegalovirus-induced DNA polymerase, but not cellular DNA polymerase alpha, can utilize these analog triphosphates as alternate substrates for their corresponding natural deoxyribonucleoside triphosphates in in vitro DNA synthesis. PMID- 2999441 TI - Localization of the coding region for an Epstein-Barr virus early antigen and inducible expression of this 60-kilodalton nuclear protein in transfected fibroblast cell lines. AB - Expression of a component of the Epstein-Barr virus early antigen (EA) complex has been studied in fibroblast cells transfected with both wild-type and P3HR-1 defective DNA fragments covering the BamHI-M-S region of the Epstein-Barr virus genome. Baby hamster kidney (BHK) cells transfected with the BglII-J fragment and stained with human serum that was positive for the diffuse component of EA [EA(D)] in an indirect immunofluorescence assay exhibited positive nuclear staining in 5% of the cell population. Cleavage of BglII-J before transfection with the restriction enzyme BglII, StuI, HindIII, or PvuII did not affect EA expression, whereas prior cleavage with BamHI or EcoRI reduced or eliminated synthesis of EA. These observations were confirmed by using individual cloned subfragments. A Bal 31 deletion clone (pTS1) in which the HindIII and StuI sites were eliminated retained activity, whereas a clone (pTS5) in which the deletion extended closer to the EcoRI site had greatly reduced activity. Transfection of the individual BamHI-M or BamHI-S fragments, which span BglII-J, also resulted in little or no EA expression. The 2.1-kilobase biologically active region defined by these experiments corresponds precisely to the BMLF1 open reading frame. Immunoblot analyses of BHK cells transfected with either P3HR-1 defective DNA clones or the BglII-J wild-type fragment identified the product of this EA(D) coding region as a family of polypeptides consisting of a major 60-kilodalton product and minor 45- and 50-kilodalton species. In latently Epstein-Barr virus infected lymphocytes these early antigens are not expressed, but can be induced by treatment of the cultures with sodium butyrate or phorbol esters. Using the BglII-J and pTS6 clones that were positive in transient assays, we also established Neor coselected BHK and Vero cell lines which showed similar regulated expression of the 60-kilodalton EA(D) protein. In these cell lines constitutive expression of EA(D) was limited (0.1% positive by indirect immunofluorescence and undetectable by immunoblot analysis). However, expression of EA(D) could be induced by treatment with sodium butyrate. In the induced cultures, up to 30% of the cells were EA(D) positive by immunofluorescence, and there was a concomitant appearance of the 60-kilodalton EA(D) polypeptide. PMID- 2999442 TI - A second Epstein-Barr virus early antigen gene in BamHI fragment M encodes a 48- to 50-kilodalton nuclear protein. AB - We used antiserum raised against the bacterially synthesized product of one of the open reading frames in Epstein-Barr virus (EBV) BamHI fragment M to demonstrate that this reading frame (BMRF1) codes for a nuclear protein of the diffuse early antigen (EA) class. In indirect immunofluorescence assays, the rabbit anti-BMRF1 antiserum gave nuclear staining in approximately 5% of Raji cells which had been treated with sodium butyrate, and positive fluorescence was observed in both acetone- and methanol-fixed cells. Uninduced Raji cultures contained less than 0.1% positive cells regardless of whether indirect immunofluorescence or anti-complement immunofluorescence was used. In immunoblot analyses, the rabbit serum identified a family of polypeptides of 46 to 55 kilodaltons (kDa) in total protein extracts from B95-8 cells or from butyrate induced Raji cells. In both cell types, the dominant polypeptides were the 48- and 50-kDa species. This same family of polypeptides was identified when the immunoblots were reacted with the R3 monoclonal antibody, and we concluded that this antibody also recognized the product of the BMRF1 open reading frame. Fibroblast cell lines containing EBV BamHI fragment M were established by cotransfection of baby hamster kidney cells with BamHI-M and the gene for neomycin resistance. Aminoglycoside G418-resistant colonies which showed evidence for EBV antigen expression in immunofluorescence assays were selected, and clonal cell lines were established. After 3 to 4 months of passaging, constitutive synthesis of EA was no longer detectable in these cell lines either by immunofluorescence or by immunoblot analysis. However, in the one cell line examined, synthesis of the 48- to 50-kDa EA was induced by treatment of the culture with sodium butyrate. Thus, the regulation of expression of this EA in transfected fibroblasts is analogous to that seen in Raji lymphoblasts. We showed previously that BamHI fragment M also contains the coding sequences for a 60-kDa nuclear EA, and hence BamHI-M encodes two separate components of the diffuse EA complex. PMID- 2999443 TI - Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90K cleavage fragments. AB - In the murine coronavirus mouse hepatitis virus, a single glycoprotein, E2, is required both for attachment to cells and for cell fusion. Cell fusion induced by infection with mouse hepatitis virus strain A59 was inhibited by the addition of monospecific anti-E2 antibody after virus adsorption and penetration. Adsorption of concentrated coronavirions to uninfected cells did not cause cell fusion in the presence of cycloheximide. Thus, cell fusion was induced by E2 on the plasma membrane of infected 17 Cl 1 cells but not by E2 on virions grown in these cells. Trypsin treatment of virions purified from 17 Cl 1 cells quantitatively cleaved 180K E2 to 90K E2 and activated cell-fusing activity of the virions. This proteolytic cleavage yielded two different 90K species which were separable by sodium dodecyl sulfate-hydroxyapatite chromatography. One of the trypsin cleavage products, 90A, was acylated and may be associated with the lipid bilayer. The other, 90B, was not acylated and yielded different peptides than did 90A upon limited digestion with thermolysin or staphylococcal V8 protease. Thus, the cell fusing activity of a coronavirus required proteolytic cleavage of the E2 glycoprotein, either by the addition of a protease to virions or by cellular proteases acting on E2, which was transported to the plasma membrane during virus maturation. There is a striking functional similarity between the E2 glycoprotein of coronavirus, which is a positive-strand RNA virus, and the hemagglutinin glycoprotein of negative-strand orthomyxoviruses, in that a single glycoprotein has both attachment and protease-activated cell-fusing activities. PMID- 2999444 TI - Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: host-dependent differences in proteolytic cleavage and cell fusion. AB - Cell fusion induced by infection with mouse hepatitis virus strain A59 (MHV-A59) varied markedly in extent and time course in four different murine cell lines. When inoculated at a multiplicity of 3 to 5 PFU per cell, the Sac-, L2, and DBT cell lines began to fuse by 7 h, were fused into confluent syncytia by 9 to 12 h, and peeled from the substrate by 10 to 14 h. These virulent virus-cell interactions were in striking contrast to the moderate interaction of MHV-A59 with the 17 Cl 1 cell line, in which only small syncytia were observed 18 h postinoculation, and greater than 50% of the cells remained unfused by 24 h. The yield of infectious virus produced by 17 Cl 1 cells was 10-fold higher than the yields from the other three cell lines. The processing of the nucleocapsid protein, the membrane glycoprotein E1, and the peplomeric glycoprotein E2 were found to differ significantly in the four cell lines. Since the E2 glycoprotein is responsible for virus-induced cell fusion, we attempted to correlate differences in cellular processing of E2 with differences in fusion of infected cells. The predominant intracellular form of E2 in all cell lines was the 180K species. Pulse-chase experiments showed that a small portion of the 17 Cl 1 cell associated 180K E2 was cleaved by 1 h after synthesis to yield 90K E2, shown in the preceding paper to consist of two different glycoproteins called 90A and 90B (L. S. Sturman, C. S. Ricard, and K. V. Holmes, J. Virol. 56:904-911, 1985). This cleavage occurred shortly before the release of virions from cells, as shown by pulse-chase experiments. After budding at intracellular membranes, virions released into the medium by the four cell lines contained different ratios of 180K to 90K E2. Virions from Sac- cells, which contained 100% 90K E2, fused L2 cells rapidly without requiring virus replication, whereas virions from 17 Cl 1 cells, which had 50% 90K E2, required trypsin activation to induce rapid fusion (Sturman et al., J. Virol. 56:904-911, 1985). The addition of protease inhibitors to the medium markedly delayed L2 cell fusion induced by MHV infection. The extent of coronavirus-induced cell fusion does not depend solely upon the percent cleavage of the E2 glycoprotein by cellular proteases, since extensive fusion was induced by infection of L2 and DBT cells but not 17 Cl 1 cells, although all three cell lines cleaved E2 to the same extent.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2999445 TI - Recombination and oligonucleotide analysis of guanidine-resistant foot-and-mouth disease virus mutants. AB - Guanidine resistance (gr) mutations of foot-and-mouth disease virus were mapped by recombining pairs of temperature-sensitive mutants belonging to different subtypes. In each cross, one parent possessed a gr mutation. Recombinants were isolated by selection at the nonpermissive temperature and assayed for the ability to grow in the presence of guanidine. From the progeny of three crosses, four different types of recombinant were distinguished on the basis of protein composition and RNA fingerprint. The sequences of the RNase T1-resistant oligonucleotides were determined and located in the full-length sequence of foot and-mouth disease virus. The resulting maps show that (i) each recombinant was generated by a single genetic crossover, and (ii) both of the gr mutations studied were located within an internal 2.9-kilobase region which spans the P34 gene. This supports our hypothesis that guanidine inhibits the growth of foot-and mouth disease virus by acting on nonstructural polypeptide P34. Additional evidence was provided by RNA fingerprinting gr mutants. In two of four cases the gr mutation was associated with a change in an oligonucleotide located near the 3' end of the P34 gene; in one of these the nucleotide substitution was identified. PMID- 2999446 TI - Cellular DNA regions involved in the induction of rat thymic lymphomas (Mlvi-1, Mlvi-2, Mlvi-3, and c-myc) represent independent loci as determined by their chromosomal map location in the rat. AB - The induction of thymic lymphomas by Moloney murine leukemia virus in the rat is linked to provirus integration in at least four independent cellular DNA regions (Mlvi-1, Mlvi-2, Mlvi-3, and c-myc). Because sequences homologous to at least three of these regions (Mlvi-1, Mlvi-2, and c-myc) map to chromosome 15 in the mouse, the question was raised whether they are closely linked in the rat genome and whether provirus integration in any one of these regions affects the same functional domain in rat DNA. In this study, we identified the chromosomal map location of Mlvi-1, Mlvi-2, and Mlvi-3 in the rat by using mouse-rat somatic cell hybrids that lose the rat chromosomes. The results showed that Mlvi-1 maps similarly to c-myc to chromosome 7, and Mlvi-2 maps to chromosome 2. Mlvi-3 probably maps to chromosome 15. We conclude that Mlvi-1, Mlvi-2, and Mlvi-3 are separate and independent genetic loci. Although Mlvi-1 and c-myc map to the same chromosome, they are not related, as determined by hybridization and restriction endonuclease mapping. The chromosomal map location of Mlvi-1 to chromosome 7 and Mlvi-2 to chromosome 2 is interesting, since chromosomal aberrations involving these two chromosomes are reproducibly observed in rat neoplasias induced by a variety of agents. PMID- 2999447 TI - Analysis of a deleted MC29 provirus: gag sequences are not required for fibroblast transformation. AB - Recovered avian myelocytomatosis virus HBI is an MC29-related virus that induces lymphoid tumors in chickens rather than the predominant neoplastic disease induced by wild-type MC29 (namely, endotheliomas). An analysis of the structure of the HBI provirus(es) in the tumors demonstrated that the provirus(es) could be either full size or deleted. One tumor was found to be clonal in that it contained a single provirus which had been partially deleted; this raised a question concerning the role of this provirus in the maintenance of tumor growth. To characterize the detailed structure of this provirus and determine its biological activity, it was molecularly cloned from tumor DNA. Sequencing confirmed that the provirus contained a deletion which effectively removed the whole gag gene. However, the provirus was shown to encode a myc-specific protein, presumably initiating from within the myc gene, and to be biologically active when it was transfected onto quail embryo fibroblasts. Our results suggest that myc alone is sufficient to transform quail embryo fibroblasts and to maintain tumor growth in vivo. PMID- 2999450 TI - Nucleotide sequence of HBI, a novel recombinant MC29 derivative with altered pathogenic properties. AB - HBI is a recombinant avian retrovirus with novel pathogenic properties that was derived from the myc-containing virus MC29. In contrast to MC29, which causes endotheliomas in chickens, HBI induces lymphoid tumors. The results of molecular cloning and nucleotide sequencing of HBI reported here show that the virus contains sequences derived from both c-myc and ring-neck pheasant virus, in addition to MC29. The 3' half of the myc gene was largely replaced by c-myc sequences, and most of the long terminal repeat and gag regions were replaced by ring-neck pheasant virus sequences. The long terminal repeat contained a triplicate sequence which was homologous to the core enhancer sequence of the simian virus 40 72-base-pair repeat. The significance of these changes in relation to the unusual biological properties of the virus are discussed. PMID- 2999448 TI - Effects of sodium n-butyrate on entry into S phase in resting rat 3Y1 cells infected with simian virus 40. AB - In quiescent rat 3Y1 fibroblasts infected with simian virus 40 (SV40), sodium butyrate elongated the time lag before entry into S phase in a concentration dependent fashion. In spite of the elongated time lags, SV40-infected cells entered S phase in a very synchronous mode, irrespective of the butyrate concentrations. The elongated time lag seemed to be at least partially due to a delayed synthesis and a delayed accumulation of large T antigen caused by butyrate. The entry into S phase was also delayed even when butyrate was added to the cultures after expression of T antigen to an extent sufficient for untreated cells to enter S phase. This suggests that butyrate may also inhibit a cellular event(s) that is required for entry into S phase after expression of the T antigen. In contrast, serum-stimulated cells were more sensitive to butyrate with respect to entry into S phase than SV40-infected cells, and the distribution of the time lag among cell populations increased (i.e., asynchrony in entry into S phase increased) with an increase in the butyrate concentration. PMID- 2999449 TI - Sequences from polyomavirus and simian virus 40 large T genes capable of immortalizing primary rat embryo fibroblasts. AB - We developed a procedure to evaluate quantitatively the capacity of subgenomic fragments from polyomavirus and simian virus 40 (SV40) to promote the establishment of primary cells in culture. The large T antigen from both of these viruses can immortalize primary rat embryo fibroblasts. Both antigens have amino terminal domains that retain biological activity after deletion of other parts of the polypeptide chain. However, this activity varies considerably among various mutants, presumably because of alterations in the stability or conformation of the truncated polypeptides. The polyomavirus middle T gene alone immortalizes at a low efficiency, which indicates that this oncogene can have both immortalization and transformation potentials depending on the assay system chosen. We generated deletions in the polyomavirus and SV40 large T genes to localize more precisely the functional domains of the proteins involved in the immortalization process. Our results show that the region of the SV40 large T antigen involved in immortalization is localized within the first 137 amino acid residues. This region is encoded by the first large T exon and a small portion from the second exon which includes the SV40 large T nuclear location signal. The polyomavirus sequence involved in immortalization comprises a region from the second large T exon, mapping between nucleotides 1016 and 1213, which shares no homology with SV40 and is thought to be of cellular origin. We suggest that this region of the polyomavirus large T gene functions either as a nuclear location signal or as part of the large T protein sequence involved in DNA binding. PMID- 2999453 TI - Markers for disease genes open new era in diagnostic screening. PMID- 2999451 TI - Structure and evolution of two related transcription units of Epstein-Barr virus carrying small tandem repeats. AB - Two regions of the Epstein-Barr virus (EBV) genome carrying partially homologous clusters of short tandem repeats (NotI and PstI repeats) flanked by 1044 and 1045 base pairs with almost complete homology (DL and DR, left and right duplication, respectively) were most abundantly transcribed into poly(A)+ mRNA after induction with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. The nucleotide sequence of both repeat clusters and the conserved upstream regulatory sequences from the M-ABA EBV strain are presented. Nearly the whole part of the sequences coding for the RNAs is covered by the NotI and PstI repeats, respectively. The regulatory sequences for these genes are located in the homologous regions of 1044 and 1045 base pairs (DL and DR, respectively). A CAAT box, a TATA box, and other herpes simplex virus-like elements were identified for both transcription units. The initiation points and the 3' ends of both inducible RNAs were mapped by S1 nuclease analysis. Both genes have open reading frames and may potentially code for proteins with repetitive amino acid compositions. The structure of these two inducible EBV genes is discussed, and an evolutionary model is proposed for the generation of gene duplication in the M-ABA strain of EBV. PMID- 2999454 TI - Laboratory detection of marijuana use. PMID- 2999452 TI - Activity of the cytomegalovirus genome in the presence of PPi analogs. AB - PPi analogs and esters of these were studied for their effect on cytomegalovirus (CMV) multiplication. Five aromatic monoesters of phosphonoformate esterified either in the phosphono or the carboxylic group and two diesters were demonstrated to inhibit CMV DNA synthesis and late viral protein synthesis. In a direct assay, the monoesters but not the diesters inhibited CMV DNA polymerase activity. The production of early CMV antigens was not inhibited by any of the compounds. After incubation with either drug for periods up to 7 days, renewed viral production occurred on withdrawal of the compound. All inhibitory esters as well as PPi analogs showed a CMV multiplicity dependence. This was demonstrated both for CMV strain Ad.169 and for all tested CMV isolates. Evidence was found that the esters are hydrolyzed to phosphonoformate and, therefore, may be of importance as useful prodrugs in the specific therapy of CMV infections. The general phenomenon of reversibility to the productive state and the multiplicity dependence of CMV are important factors in any treatment schedule. PMID- 2999456 TI - Leads from the MMWR. Recommendations for assisting in the prevention of perinatal transmission of HTLV-III/LAV and acquired immunodeficiency syndrome. PMID- 2999455 TI - Prevention of herpesvirus infections in renal allograft recipients by low-dose oral acyclovir. AB - Forty patients with serum antibody against herpes simplex virus (HSV) were enrolled in a randomized, placebo-controlled, double-blind investigation of acyclovir given orally in a low dosage as prophylaxis against recurrent HSV infection after renal transplantation. During 30 postoperative days of medication, 14 of 21 placebo-treated and one of 19 acyclovir-treated patient(s) developed reactivation of HSV infection. Eleven of the former, but not the latter, had herpetic lesions. The protection against active infection with HSV during the period of prophylaxis with acyclovir is statistically highly significant. From 30 to 90 days after transplantation when no antiviral medicine was given, 60% (3/5) of the remaining placebo recipients and 44% (7/16) of the acyclovir patients developed active HSV infections. Herpetic lesions occurred in two of three and two of seven of infected people in the respective groups. No adverse effects of the drug were observed. The results show that HSV infections in immunosuppressed renal allograft recipients can be safely prevented, deferred, and ameliorated by an initial period of prophylaxis with a low dose of oral acyclovir. PMID- 2999457 TI - The incidence of cervical and vaginal dysplasia after exposure to DES. PMID- 2999458 TI - Immunoreactive renin in human brain: distribution and properties. AB - Readily detectable levels of renin activity were demonstrated in the human brain. This activity was inhibited by specific antibody raised against human renal renin, indicating that it was not due to the nonspecific action of proteases such as cathepsin D. The pineal gland was found to be the richest source of renin followed by the pituitary, hypothalamus and hippocampus. The substantia nigra, caudate nucleus, putamen and thalamus contained moderately high concentrations of renin. The brain renins from pineal and pituitary glands shared some biochemical features with well-known kidney renin, such as molecular weight (46,000 daltons for pineal renin; 37,000-45,000 daltons for pituitary renin), optimum pH (6.0 7.0), the presence of trypsin activatable inactive renin, and a glycoprotein nature. However, the electrofocusing pattern of renin from pituitary tissue (pI = 4.43, 5.77) differed from that of plasma and kidney enzymes heretofore reported, a discrepancy which could be interpreted as evidence for the endogeneous synthesis of renin in the brain tissue. Furthermore, a high activity of immunoreactive renin was found in human neuroblastoma tissue. The biochemical characteristics of the neuroblastomal renin were generally similar to the known properties of kidney renin in many respects, providing evidence of the presence of the renin-angiotensin system within human neuronal cells. PMID- 2999459 TI - Characterization of alpha- and beta-adrenergic and angiotensin receptors in cultured vascular smooth muscle cells of rat aorta. AB - To study the cellular mechanism of vascular responsiveness by vasoactive hormones, such as catecholamines and angiotensin (A), the vascular smooth muscle cells (VSMC) of two clonal cell lines (A7r5 and A10) from rat embryo, and of adult rat aorta were established in culture. Binding studies using 125I-labeled hydroxyphenylethylaminoethyltetralone as an alpha-adrenergic ligand and 125I labeled-iodocyanopindolol as a beta-adrenergic ligand, revealed that cultured VSMCs contain both alpha- and beta-adrenergic receptors; the binding was specific, rapid, reversible, and saturable. alpha-Adrenergic receptors appear to be a single class of high-affinity binding sites with an apparent dissociation constant (Kd) of approximately 2 X 10(-10) M and a maximal binding capacity (Bmax) of approximately 300,000-400,000 sites/cell, and exclusively of alpha 1 subtype that is responsible for smooth muscle contraction. On the other hand, beta-adrenergic receptors show almost comparable characteristics with the apparent Kd of approximately 0.7-1.1 X 10(-10) M and Bmax of approximately 50,000 130,000 sites/cell, and consist predominantly of beta 2-subtype that mediates smooth muscle relaxation. Furthermore, beta-adrenergic receptors are coupled to adenylate cyclase system, of which activation by beta-agonists induces intracellular cyclic AMP formation. In contrast, the binding of 125I-labeled-AII was demonstrated only in A7r5. The binding declined rapidly during incubation possibly due to faster degradation of AII by proteolytic enzyme(s). AII receptors appear to be a single class of high-affinity binding sites with the apparent Kd of approximately 0.9 X 10(-10) M and Bmax of approximately 11,000 sites/cell, of which affinity is higher than any of vascular AII receptors previously reported.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2999460 TI - Sensitive assay and kinetic property of urinary inhibitor of Na+, K+-ATPase in sodium-loaded patients with essential hypertension. AB - A sensitive assay method to evaluate the inhibitor of Na+, K+-ATPase in human urine was developed by measuring the inorganic phosphate liberated from ATP in vitro using Na+, K+-ATPase from porcine cerebral cortex. Ouabain inhibited the Na+, K+-ATPase by competing with the potassium ion (an apparent Ki = 2.6 +/- 0.89 X 10(-8) M, n = 8) under the condition of 100 mM NaCl, 4.5 mM MgSO4 and 0.56 mM ATP. The apparent Km value of KCl was 0.4 mM. Factors inhibiting Na+, K+-ATPase were detected in the post-salt fraction on Sephadex G-15 chromatography following the ethanol extraction of lyophilized fresh urine of sodium loaded human subjects (300 meq Na+/day, for 4 days) with essential hypertension. Two active fractions around the 400 daltons following salt were eluted on Sephadex G-15 chromatography. The slower eluted factor competed kinetically with potassium ion, but the inhibitory activity was lost within two days during storage at 4 degrees C. The faster-eluted inhibitor lost its activity within a day. These results indicate that the unstable inhibiting factors of Na+, K+-ATPase exist in human urine and one of these factors inhibits ouabain sensitive Na+, K+-ATPase by binding to the potassium binding site (or very close to it), which exists at the outer surface of the cell membrane of this enzyme. PMID- 2999461 TI - [Effects of cigarette smoking on endocrine function in man]. PMID- 2999462 TI - [Histological classification of breast cancer]. AB - The characteristics of histological classification of breast cancer by Stewart F.W., WHO and Japan Mammary Cancer Society (published in 1971 and revised in 1984) are discussed. The classification of JMCS is fundamentally the same as WHO classification with a subclassification of invasive ductal carcinoma. Invasive ductal carcinoma has three subgroups; papillotubular, solid-tubular and scirrhous carcinoma. In these three subgroups, papillotubular carcinoma shows mainly ductal spread, well histologic differentiation, the lowest lymph node metastasis and the best prognosis. Scirrhous carcinoma shows diffuse stromal infiltration, poor histologic differentiation, the highest metastasis and the worst prognosis. Solid tubular carcinoma with expansive stromal invasion shows intermediate biological behavior. Thus, the JMCS classification contains in itself not only histologic type but also the mode of spread, grade of histologic differentiation, ratio of lymph node metastasis and prognosis. PMID- 2999463 TI - [A clinicopathological study of 160 cases of mucinous carcinoma of the breast]. AB - A clinicopathological study was performed on characteristics of mammary mucinous carcinoma and its 3 pathological subgroups: "gelatinous type"--with predominant mucin component, "epithelial type"--with predominant epithelial component, and "combined type"--with a small part of infiltrating ductal carcinoma and mucinous pattern. Among 4,653 primary breast cancers mastectomized at the C.I.H. between 1946 and 1981, 160 or 3.4% was mucinous carcinoma. They were divided into 106 gelatinous type, 20 epithelial type and 34 combined type. 10-year survival rate of mucinous carcinoma (75.3%) was better than whole breast cancer (65.6%). And it was 90.7%, 82.4% and 67.6% in gelatinous, epithelial and combined type, respectively. PMID- 2999465 TI - [A case of abdominal wall fistula due to cholangioma]. AB - A 59-year-old woman was admitted to our hospital complaining of intractable abdominal wall fistula. Several examinations led to the suspicion of a fistula due to liver cell tumor. Histologically, the tumor was diagnosed as cholangioma. Chemotherapy was very effective after fistulectomy, and to date, she has been doing well for 34 months. PMID- 2999466 TI - [The outbreak of five lymphoid malignancies in one family during seven years]. PMID- 2999464 TI - [Clinical study on tissue polypeptide antigen (TPA) in gastric cancer]. AB - We evaluated whether assay of tissue polypeptide antigen (TPA) in the serum ia valuable for the determination of cancer stages compared to other tumor markers such as CEA, AFP, and ferritin. The study population consisted of 79 gastric cancer patients and 212 patients with benign gastroenteric disease. The percentage of positive cases for TPA (higher than 200u/l) was 41% in gastric cancer and 20% in active peptic ulcer. Serum TPA levels in well differentiated carcinoma and signet ring cell carcinoma were higher than that in other histological types. Serum TPA levels correlated well with the stage of the gastric cancer. PMID- 2999467 TI - [Cytodiagnosis from the surgical viewpoint. B. Use of cytodiagnosis in breast cysts]. PMID- 2999468 TI - [Diagnostic criteria standards in aspiration biopsy cytodiagnosis A. Diagnosis of benign cases]. PMID- 2999469 TI - [Study on the effects of various factors on platelet adhesion]. PMID- 2999470 TI - [Receptors on the platelet membrane]. PMID- 2999471 TI - [Pitfalls in the diagnosis of hepatocellular carcinoma]. PMID- 2999472 TI - [Detection and qualitative evaluation of small hepatocellular carcinoma by ultrasonography--matters to be heeded and limitations]. PMID- 2999473 TI - [Evaluation of CT in hepatocellular carcinoma]. PMID- 2999474 TI - [Differential diagnosis of hepatocellular carcinoma by angiography]. PMID- 2999475 TI - [Differential arteriographic and CT arteriographic diagnosis of hepatocellular carcinoma]. PMID- 2999476 TI - [Differential diagnosis of hepatocellular carcinoma and cavernous hemangioma; possibility of progress evaluated by magnetic resonance imaging]. PMID- 2999477 TI - [Plain abdominal X-ray images and plain CT images after intrahepatic-arterial infusion of lipiodol]. PMID- 2999478 TI - [Simplification of selective internal mammary arteriography by the "ITM catheter"]. PMID- 2999479 TI - [An immunofluorescence study of the papilloma virus in viral warts]. PMID- 2999480 TI - [Endoscopic ultrasonography in the diagnosis of the degree of vertical invasion of gastric cancer]. PMID- 2999481 TI - [Cell culture of human insulinoma]. PMID- 2999482 TI - [A new sampling tube using charcoal and silica gel mixture for measurement of organic solvents]. PMID- 2999483 TI - Pharmacokinetic study of a human recombinant interferon (Re-IFN-alpha A) in cynomolgus monkeys by 2'-5' oligoadenylate synthetase assay. AB - We evaluated 2'-5'Oligoadenylate (2-5 A) synthetase assay for pharmacokinetic study of human interferon (IFN) in cynomolgus monkeys. The enzyme was induced in primary cultures of cynomolgus monkey kidney (PMK) cells as well as in FL cells in response to human recombinant IFN-alpha A treatment. The enzyme activity increased with IFN dose and, in parallel with the enzyme elevation, developed the antiviral state of the cells. The enzyme activity induced in the peripheral blood lymphocytes peaked at 6 to 12 hr after iv or im administration. The peak level of the enzyme activity depended on the IFN concentration of the blood and the activity rapidly decreased as serum IFN was cleared from the blood. These results indicate that human recombinant IFN-alpha A induces 2-5 A synthetase in monkey cells both in vitro and in vivo, and that the enzyme assay can be used to quantitatively monitor the host response after IFN administration. PMID- 2999484 TI - [Clinical evaluation of 201Tl-chloride scintigraphy in breast tumors]. PMID- 2999485 TI - Clinical application of monoclonal antibodies to small cell lung cancer. AB - Monoclonal antibodies to small cell lung cancer (SCLC) were produced by fusion of P3X63/AgU1 mouse myeloma cells with spleen cells from BALB/c mice immunized against intact cells of SCLC tumors grown in BALB/c athymic nude mice. The antibody TFS-2 demonstrated "pancarcinoma" reactivity showing binding to non small cell lung cancer (NSCLC) and carcinomas derived from other organs such as stomach, pancreas, and colon. This antibody failed to react with a variety of normal tissues including lung, bone marrow, spleen, stomach, colon, and pancreas. TFS-2 showed complement-mediated cytolysis of target cells. TFS-2 had no significant effects on hematopoietic stem cells. This antibody will serve as a powerful tool for the in vitro removal of tumor cells from bone marrow, thus facilitating high-dose chemotherapy of SCLC with autologous bone marrow transplantation. In contrast, TFS-4 monoclonal antibody reacted specifically with SCLC but not with NSCLS tumors. This antibody can distinguish SCLC from NSCLC tumors in histologic diagnosis at transbronchial biopsy, and in cytological identification of tumor cells in malignant effusion, and in infiltrated bone marrow. Radiolabeled TSF-4 specifically accumulated into SCLC tumors that were implanted subcutaneously into athymic nude mice. Thus, the antibody will be useful not only for diagnosis of lung cancer but also for targeting anti-tumor agents. PMID- 2999486 TI - Changes in action potential and beta-adrenergic effects of the circular muscle of postpartum rat uterus. AB - Spike potential dominated in the circular muscle of postpartum rat uterus during the period between 0 and 15 hr after the delivery of the first newborn. During the postpartum period ranging between 20 and 48 hr, the plateau potential was dominant. Application of 10(-9) M isoprenaline strongly depressed the contraction during early postpartum period (0-15 hr), and the depressant effect was much smaller thereafter. In vivo treatment of postparturient rats with estradiol-17 beta (50 micrograms) or progesterone (50 micrograms) for 2 days did not alter the postpartum change in action potential or the effect of isoprenaline. The postpartum changes in muscle properties mentioned above were prevented in the distended portion of uterus, when several fetuses and placentas were artificially kept inside the uterus for 2 days, while other fetuses were delivered out. The hormonal influences on the circular muscle of postpartum rat uterus were discussed in view of the above experimental findings. PMID- 2999487 TI - Binding characteristics of 3H-dihydroalprenolol to beta-adrenergic receptors of rat brain: influence of exo- and endo-glycosidases and glycopeptidase. AB - The significance of carbohydrate moieties containing the beta-adrenoceptor molecule in the rat brain was examined using radioligand binding assay methods. Thus, this experiment was designed to assess the effects of exoglycosidase (alpha D-mannosidase and neuraminidase), endoglycosidase (endoglycosidase D and endoglycosidase H), and glycopeptidase A on the affinity of beta-adrenoceptor. The main reason why five kinds of enzymes were used in the present study is that they can hydrolyze different carbohydrate molecules from cell membranes. Rat brain was used and beta-adrenoceptor binding assay was carried out using 3H dihydroalprenolol (3H-DHA) as a ligand. 3H-DHA binding to beta-adrenoceptors was sensitive to very low concentration of endoglycosidase H and glycopeptidase A, thus indicating that the treatments with these enzymes of rat brain membrane appear to decrease the number of beta-receptor binding sites. On the other hand, the treatment with neuraminidase, endoglycosidase H, and glycopeptidase A of the membrane induced lower values of the dissociation constant (Kd) than those of the control. alpha-D-mannosidase and endoglycosidase D are without effect in spite of the removal of hexose contents and total carbohydrate contents with these treatments, respectively. These results imply that complex type N-linked acidic carbohydrate chains containing neuraminic acid and high mannose type N-linked carbohydrate chains, which are hydrolyzed with endoglycosidase H and glycopeptidase A, of the rat brain membrane containing beta-adrenoceptor molecules play a crucial role in the drug-receptor interaction. PMID- 2999488 TI - Chronic effects of enalapril on blood pressure, stroke, plasma renin, urinary electrolytes and PGE2 excretion in stroke-prone spontaneously hypertensive rats. AB - Antihypertensive effect of enalapril (MK-421), an orally active non-sulfhydryl containing converting enzyme inhibitor, was examined in stroke-prone spontaneously hypertensive (SHRSP) rats. The treatment was started at 14-15 weeks of age with tail blood pressure over 240 mmHg and was continued for 11 weeks. We used captopril as the reference drug. The dose of enalapril and captopril was 10 and 30 mg/kg per day, p.o., respectively. Enalapril showed a sustained antihypertensive effect from the 1st to the 11th week of the treatment. This antihypertensive effect was substantiated by the good increase in body weight; decrease in heart weight; decrease in incidences of vascular disease, nephrosclerosis, stroke and death. Enalapril treatment also prevented the increases in urine volume, and excretion of osmotically active solutes, Na, Cl and K with age. Captopril treatment showed about the same antihypertensive effect. No side effects were seen in the enalapril or captopril treated group. The antihypertensive potency of enalapril was about 3 times more than that of captopril. Enalapril and captopril slightly increased plasma renin concentration. Urinary excretion of PGE2 was not changed by enalapril or captopril treatment. These results clearly demonstrate the efficacy of long-term treatment with enalapril to prevent development of malignant hypertensive cardiovascular disease in SHRSP rats. PMID- 2999490 TI - [Malignant fibrous histiocytoma of the chest wall showing multiple shadows on the chest X-ray]. PMID- 2999489 TI - Spin label study of the effect of ticlopidine on platelets. AB - The effect of ticlopidine on platelet membrane fluidity was investigated using a spin label technique. Ticlopidine, when orally administered to rats, increased both the order parameter and the motion parameter, indicating a decrease in the membrane fluidity of platelets. On the other hand, the order parameter and the motion parameter decreased markedly when the platelets were aggregated by thrombin. Ticlopidine inhibited the thrombin-induced aggregation of platelets and caused a slight increase in order parameter and motion parameter in thrombin aggregated platelets. Judging from sodium dodecyl sulfate polyacrylamide gel electrophoresis, ticlopidine did not modify the electrophoretic pattern of platelet proteins appreciably. Ticlopidine decreased cholesterol/phospholipids molar ratio and increased slightly total amounts of proteins of the platelets. These results indicate that the inhibitory action by ticlopidine was accompanied by changes in membrane fluidity, and these changes were due to a perturbation of the membrane phospholipid core of the platelets by ticlopidine and/or its metabolites. PMID- 2999492 TI - A cytomegalovirus isolated from swine testicle cell culture. PMID- 2999491 TI - [Changes in the serum anti-virus antibody titer following open-heart surgery and the effect of human immune globulin]. PMID- 2999493 TI - In vitro studies on the breakdown of canine erythrocytes exposed to the onion extract. PMID- 2999494 TI - Histopathology and hematology of acute infection with mouse hepatitis virus, MHV 3 in mice with different susceptibility. PMID- 2999496 TI - Effects of cytostatic drugs and 40.5 degrees C hyperthermia on human bone marrow progenitors (CFU-C) and human clonogenic tumor cells implanted into mice. AB - Human spontaneous tumors after surgical resection were implanted into NMRI nude mice. Clonogenic cells from these tumors (4 malignant melanomas, 2 squamous cell carcinomas of the lung, and 1 small cell carcinoma of the lung) were cultured in a methylcellulose monolayer assay. Dose-response curves with 7 cytostatic drugs (doxorubicin, bleomycin, dactinomycin, cisplatin, vincristine, vinblastine, and melphalan) were assessed at 37 degrees C and after a 2-hour pulse at 40.5 degrees C. In addition, bone marrow cells (CFU-C) were plated in the same assay. Dose response curves were assessed with the same drugs under the same conditions. Hyperthermic treatment without drugs did not alter the colony formation of tumor and bone marrow cells. Bone marrow samples from different donors showed homogeneous response patterns; the tumor probes revealed sensitivity patterns typical for each tumor. In 13 of 48 tumor-drug combinations a thermal enhancement of the drug effects was observed, whereas there was no significant difference between normothermic and hyperthermic cultures in the bone marrow cultures. A comparison of colony and bone marrow dose-response curves suggested that a hyperthermic enhancement can occur in single cases. However, this phenomenon seems to be due to individual properties of the tumor. PMID- 2999495 TI - Infectivities of bovine leukemia virus in peripheral blood lymphocytes from naturally infected cattle and their relation to persistent lymphocytosis and antibody titers. PMID- 2999497 TI - T8 and T3 surface glycoproteins in human T-cell mediation of leukocyte adherence inhibition to extracts of autologous cancer. AB - Monoclonal antibodies (MAb's) [anti-Leu-1, anti-Leu-2a (T8), anti-Leu-3a (T4), and anti-Leu-4 (T3)] were used to elucidate the type of T-cell mediating leukocyte adherence inhibition (LAI) and the role of T-cell surface glycoproteins in LAI. T8+ (Leu-2a+) and T4+ (Leu-3a+) subtypes were isolated by panning. T8+ (Leu-2a+) cells showed LAI to extracts of autologous cancer, whereas the T4+ (Leu 3a+) subset had no LAI response. Moreover, MAb to the T8+ (Leu-2a+) glycoprotein negated T-cell LAI, but MAb to Leu-1+ or to T4+ (Leu-3a+) did not negate T-cell LAI to autologous cancer extracts. The T3+ (Leu-4+) differentiation also was essential for T-cell LAI to autologous cancer extracts since anti-Leu-4 (T3) negated the response. Since LAI to autologous cancer extracts depends ultimately on leukotriene and other oxidative metabolites of arachidonic acid generated by the T-cell binding tumor antigen, the effect of MAb on LAI induced by leukotriene B4 isomer III. 5(S), 12(R)-dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (LTB4) was examined. Authentic LTB4 induced nonadherence of 34% of adherent T cells, and this effect was not negated by anti-Leu-1, anti-T8 (Leu-2a), or anti T4 (Leu-3a). However, anti-T3 (Leu-4) abrogated LTB4-induced LAI of pure T-cells without any effect on the basic adherence properties of T-cells. The present findings indicated that LAI to autologous cancer extracts was mediated by T-cells of the T8+ phenotype when they recognize tumor antigen and polymorphic major histocompatibility complex determinants on autologous cancer membranes. Moreover, differentiation glycoproteins T8+ (Leu-2a+) and T3+ (Leu-4+) on the surface of the responding effector T-cells performed distinct biologic functions that enabled the tumor antigen to trigger T-cell LAI. PMID- 2999498 TI - Sodium-potassium ATPase activity mediates cyst formation in metanephric organ culture. AB - To study the possible role of altered transtubular transport in renal tubular cyst formation, the ontogeny of renal Na-K ATPase was studied during glucocorticoid-induced cystic metanephric tubular development in serum-free, murine organ culture (SFMOC). Utilizing an enzyme-linked kinetic microassay, a developmental profile of total ATPase and specific Na-K ATPase activity was established for control (CON) and glucocorticoid-induced cystic organ culture (CY) explants. During 120 hr of CON and CY organ culture nephrogenesis total Na-K ATPase activity, specific Na-K ATPase activity, and the Na-K ATPase: total ATPase ratio progressively increased, simulating normal in vivo murine enzyme development. However, from 48 to 120 hr of organ culture, CY showed significant increases in Na-K ATPase activity when compared to CON at similar stages of development. Na-K ATPase activity (expressed as nmoles . min-1 . mg protein -1, mean +/- SD) was, at: 48 hr, CY 13.1 +/- 0.7 vs. CON 11.0 +/- 0.9 (P less than 0.01); 72 hr, CY 16.4 +/- 1.1 vs. CON 12.2 +/- 0.7 (P less than 0.001); 96 hr, CY 35.4 +/- 4.9 vs. CON 13.7 +/- 0.4 (P less than 0.001); and 120 hr, CY 26.1 +/- 1.4 vs. CON 16.3 +/- 0.9 (P less than 0.001). The initial differences in CY enzyme activity preceded the earliest ultrastructural evidence of cyst formation by 18 to 24 hr, while subsequent increases in Na-K ATPase activity in CY paralleled progressive tubular cyst formation. Tubular cyst formation in CY could be largely prevented by daily incubation of explants with ouabain, 0.2 mM (final concentration) X 120 min, without deleterious effects on overall metanephric development.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2999499 TI - Interstitial nephritis associated with cytomegalovirus infection. AB - Cytomegalovirus (CMV) infection may cause impairment of renal graft function and glomerular and interstitial injury. Whether renal lesions are the consequence of infection or of decreased graft tolerance is uncertain. We studied autogenous renal tissues obtained from two infants with CMV infection. Light and electron microscopy revealed interstitial nephritis, but not glomerulopathy. Analysis of frozen tissues using monoclonal antibodies by indirect immunofluorescence demonstrated that most infiltrating cells were T cells (OKT3+), the majority of which reacted with OKT8. In contrast, tissues obtained from one individual prior to CMV infection, from individuals with end-stage kidney disease, and from normal renal donors revealed either balanced proportions of OKT4+ and OKT8+ cells or a preponderance of the former. Thus, CMV infection may be associated with interstitial nephritis involving a characteristic subpopulation of T cells. PMID- 2999500 TI - [Kaposi's sarcoma and AIDS]. AB - Kaposi's sarcoma occurs as a complication in numerous cases of AIDS, the much discussed acquired immune deficiency syndrome. Considering the latter's near epidemic incidence, the clinical and histological features of Kaposi's sarcoma are recalled to mind. Hitherto only sporadically recorded in non-African countries, the disease is now more frequently observed also in the USA and Central Europe. In textbooks of ophthalmology Kaposi's sarcoma has so far been grossly neglected. Therefore, on the basis of historical facts taken exclusively from original papers, Kaposi's sarcoma is reconsidered with special reference to ophthalmological aspects. PMID- 2999501 TI - [Chickenpox prevention in patients at risk with a special immunoglobulin]. AB - Chickenpox, which normally is an innocuous disease in children, may cause serious sequelae in immunocompromised hosts. An open prospective study is presented, that comprised 96 of such patients. They were treated with 0,2 ml/kg Varicella-Zoster Immunoglobulin (VZIG) prophylactically or after exposure. The immunoglobulin was given in between 72 hours after known contact, newborns received the injection at the day of delivery. After that the patients were followed for six weeks for documentation and assessment of protection. In 85 (88,5%) cases there was no manifestation of varicella. 10 patients showed mild forms of the disease; 3 of these had got the preparation within the recommended time gap of 72 hours after exposure. One child died from varicella pneumonia and haemorrhage. In this case the preparation had only been given 10 days after contact. PMID- 2999502 TI - [Myeloperoxidase deficiency as a cause of recurrent infections]. AB - Myeloperoxidase (MPO) deficiency is a common hereditary leukocyte function defect. A two year old girl with MPO-deficiency suffered from recurrent skin infections. No MPO-activity was detectable in leukocytes of her peripheral blood smears, while NBT reduction and chemotactic activity was normal. The quantitative enzyme determination in leukocyte sonicates confirmed the total MPO-deficiency in the girl's leukocytes and a partial MPO-deficiency in the cells of her mother. The patient leukocytes demonstrated also an impaired chemiluminescence. PMID- 2999503 TI - [Gyrase inhibitor]. PMID- 2999505 TI - [Acute suppurative cholangitis]. PMID- 2999504 TI - Changing biclonal gammopathy due to different lymphocyte clones in acquired immunodeficiency syndrome with Kaposi's sarcoma. AB - We report the first case of acquired immunodeficiency-syndrome (AIDS) with Kaposi's sarcoma which at the same time has also a changing biclonal gammopathy (initial: double IgG-paraproteinaemia, later on IgA-IgG double-paraproteinaemia) due to different lymphocyte clones. The relationship of a B-cell neoplasia to a concurrent disease of T-cells is discussed. PMID- 2999506 TI - [Cytochrome C in the combined therapy of patients with cerebral and coronary circulatory disorders]. PMID- 2999507 TI - [Prevention of decompression sickness in rats using drugs and surface-active agents]. PMID- 2999508 TI - Inactivation of bacteria and viruses in water: passage of germicidal ultraviolet light through Teflon. AB - Teflon pipe as used in a water purification system transmitted germicidal ultraviolet (UV) light to inactivate Pseudomonas aeruginosa and poliovirus. The information is useful for animal care workers and others concerned with the prevention of microbial growth in water systems such as deionizers and distilled water. Of special significance is that there is a plastic that transmits UV light. PMID- 2999509 TI - Pulmonary endothelial cell killing by human neutrophils. Possible involvement of hydroxyl radical. AB - Human blood neutrophils stimulated by a variety of agents were shown to have cytotoxic effects on bovine pulmonary artery endothelial cells. Effective agonists included immune complexes, opsonized zymosan and 12-O-tetradecanoyl phorbol acetate. Unstimulated human neutrophils and neutrophils stimulated with N formyl-methionyl-leucyl-phenylalanine or with platelet-activating factor failed to induce significant killing even though secretory release of lysosomal enzymes occurred. In comparing the effects of the different agonists, endothelial cell killing showed a better correlation with the production of H2O2 than with the generation of O2-. Endothelial cell killing by stimulated human neutrophils was inhibited by catalase but not by soybean trypsin inhibitor or superoxide dismutase. Killing was also inhibited by two scavengers (N, N-dimethylthiourea and D-mannitol) of hydroxyl radical and by deferoxamine mesylate, an iron chelator. Iron-saturated deferoxamine mesylate was significantly less effective in protecting the endothelial cells against killing. Agents that were protective against endothelial cell killing did not interfere with the generation of O2- in stimulated neutrophils. These results suggest that leukocyte-induced endothelial cell killing in vitro can be induced by some but not all agonists for neutrophils and that the killing is oxygen-dependent and may be directly due to hydroxyl radical production. PMID- 2999510 TI - Identification of a subpopulation of primary granules in human neutrophils based upon maturation and distribution. Study by transmission electron microscopy cytochemistry and high voltage electron microscopy of whole cell preparations. AB - Two main classes of primary granules have been identified in human neutrophils by peroxidase cytochemistry and electron microscopy, spherical granules and elongated granules, containing crystals with well-defined periodicity. In the present study, we report another subpopulation of primary granules, distinguishable from the other primary granules by their small size, strong peroxidase reactivity under selective incubation conditions with diaminobenzidine, stage of neutrophil maturation at which they appear, and distinctive morphological characteristics. Early promyelocytes, reacted with diaminobenzidine before fixation, demonstrate no reactivity in the large primary granules, but strong peroxidase activity in the endoplasmic reticulum and Golgi apparatus. At the end of the promyelocyte stage of maturation, when the endoplasmic reticulum decreases in peroxidase reactivity, small, round or elongated granules (100 to 200 nm) are observed, reacting strongly for peroxidase. These small granules persist during maturation. The data suggest that these small granules are packaged after the large primary granules and before the secondary granules. All three classes of primary granules exhibit peroxidase activity when fixed prior to incubation in diaminobenzidine at neutral or alkaline pH. Distinctive morphological characteristics of the small primary granules are observed in circulating neutrophils. The small granules are arranged in chains or clusters primarily at the cell margin or uropod in moving cells and are aligned along the axis of cell polarity. This granule association is more evident in adherent neutrophils, particularly after short incubation with phorbol myristate acetate. Chains consisting of as many as 17 small granules are observed. Stereo high voltage electron microscopy of whole-mount preparations of adherent neutrophils reveals chains of small granules apparently interconnected by microtrabeculae. Thus, the small granules observed in thin sections do not represent a transverse section of elongated granules, and the interconnection of granules by microtrabeculae may determine the arrangement of granules. These data support the existence of subpopulations of primary granules which contain distinct forms of myeloperoxidase. PMID- 2999511 TI - Binding of bovine thyrotropin to specific sites in thyroid tissue from control and hemithyroidectomized rats. AB - The binding of 125I-bovine thyrotropin to thyroid particulate fractions from sham operated (control) and hemithyroidectomized rats was compared to determine if a change in either the number of bovine thyroid-stimulating hormone (bTSH) binding sites or their affinity for bTSH occurs in physiological situations that evoke changes in the intensity of thyroid stimulation. Following hemithyroidectomy serum TSH levels increase and the remnant thyroid lobe enlarges. Because of compensatory thyroid hypertrophy the concentration of TSH binding sites in the thyroid glands from hemithyroidectomized and control rats was related to particulate protein concentration, to the degree of thyroid cellularity as indicated by DNA concentration, and to the concentration of the plasma membrane markers, 5'-nucleotidase and magnesium-dependent ATPase. In each of four experiments, saturation studies revealed that the maximum specific binding of TSH per unit particulate protein and per thyroid lobe was greater in particulates from remnant than from control thyroid lobes. When related to DNA concentration, the concentration of TSH binding sites in remnant lobes was approximately twice that in control lobes. Because of an increase in plasma membrane markers per lobe after hemithyroidectomy, however, there was no difference in the number of TSH binding sites when related to the concentrations of the membrane marker enzymes in the particulate fractions. As judged from Scatchard analysis, the affinity of TSH binding was lower in remnant than in control lobes. This was partially but not completely due to the increased concentration of particulate protein in the remnant thyroid. These experiments demonstrate that the increase in serum TSH levels after hemithyroidectomy in the rat is associated with alterations in TSH receptor capacity and affinity. PMID- 2999512 TI - Androgen regulates MMTV RNA in the short-term in S115 mouse mammary tumour cells. AB - This report demonstrates that androgens as well as glucocorticoids can regulate MMTV RNA production in the short term. In S115 mouse mammary tumour cells, MMTV RNA accumulation is regulated within hours by androgen, at a time before any increase in DNA synthesis can be detected, thus providing a marker of an early postreceptor molecular event in steroid action on these cells. Androgen acts via its own receptor and not by cross-binding to the glucocorticoid receptor. The effects are at transcription and not just on stabilisation of RNA because they are blocked by actinomycin D. However, the androgen action shows some partial dependence on simultaneous protein synthesis since cycloheximide is inhibitory. The androgen regulation of MMTV RNA is compared and contrasted with that by glucocorticoids in these cells. PMID- 2999513 TI - Strain differences in the induction of mono-oxygenase activity in mouse skin by topical clobetasol propionate: evidence of a role for the hr locus. AB - The effect of the topical application of clobetasol propionate on cutaneous ethoxycoumarin O'dealkylation (EOD) has been studied in various strains of mice. Clobetasol propionate markedly increased cutaneous EOD activity in adult hairless mice only. Similar treatment of adult haired C57BL/6J mice, or adult haired DBA/2J mice had no significant effect on cutaneous EOD activity. In contrast 3 methylcholanthrene induced cutaneous EOD activity in both hairless and C57 strains to a far greater extent than in the DBA strain. EOD activity in hairless mice non-responsive to polycyclic hydrocarbons, derived by selective breeding of hairless and DBA strains was induced by clobetasol propionate to a similar extent to that observed in responsive hairless strains. Hepatic EOD activity was not induced by clobetasol propionate in any of the strains tested. Strain differences in the induction of EOD by clobetasol propionate were not related to differences in either the concentration of cytosolic glucocorticoid receptor in the skin, the dissociation constant of the cytosolic receptor, or differences in percutaneous absorption. Polycyclic hydrocarbons did not compete with triaminolone acetonide for binding to the cytosolic glucocorticoid receptor. Strain differences in the induction of EOD activity by clobetasol propionate appear therefore not to be related to strain differences in either the Ah receptor of the glucocorticoid receptor, but to be regulated by the hr locus. PMID- 2999514 TI - An experimental test of telephone aftercare contacts with alcoholics. AB - Alcoholics from two hospital-based treatment centers participated in an experimental test of the effects of extended aftercare on inpatient recovery rates. At discharge from inpatient treatment, subjects were randomly assigned either to an experimental group scheduled to be called by a center counselor every 2 weeks for 1 year or to a control group that experienced only the usual treatment. Follow-up interviews conducted approximately 12 months after hospital discharge found that the experimental group had no higher recovery rates than the control group. There was weak evidence that the calls reduced the burden that alcoholics place on community control and service agencies. There was no evidence that either the phone calls were more effective for some patients than for others or that some kinds of phone calls were more effective than others. Although most subjects said they liked the calls, wanted them to continue and perceived them as "good treatment," only one subject gave the calls credit for helping him maintain sobriety. PMID- 2999515 TI - Demographic variables as predictors of alcoholism treatment outcome. AB - Interactions of 18 demographic factors with alcoholism treatment outcome and with aftercare participation were studied. For 2 years subsequent to discharge from a 30-day inpatient program, 1210 alcoholic men veterans were followed up and grouped with regard to (1) posttreatment status (abstainers, improved, unimproved, unclassified, deceased) and (2) in terms of responders (abstainers and improved) and nonresponders (unimproved). Classifications were based on comparing each subject's 2-year posthospital drinking status with his 2-year prehospital drinking pattern. Seven variables were found to discriminate between the groups in both analyses. Those responding to treatment tended to be older, married and employed at admission, had more days of prehospital abstinence, were less likely to have had prior hospitalizations, and were more likely to participate in aftercare and to visit more frequently. In multivariate analyses, however, only the last two variables showed promising predictive ability, whereas the variables of days sober, age and married contributed only slightly to the prediction of treatment outcome. An effort to find variables that might predict aftercare participation was unsuccessful; the combined contribution of four factors amounted to only 5% of the total variance. PMID- 2999516 TI - Supramolecular organization of glycolytic enzymes. AB - On the basis of the analysis of the data on adsorption of glycolytic enzymes to structural proteins of skeletal muscles and to the erythrocyte membranes, the data on enzyme-enzyme interactions and the data on the regulation of activity of glycolytic enzymes by cellular metabolites, the structure of the glycolytic enzymes complex adsorbed to a biological support has been proposed. The key role in the formation of multienzyme complex belongs to 6-phosphofructokinase. The enzyme molecule has two association sites, one of which provides the fixation of 6-phosphofructokinase on the support and another is saturated by fructose-1,6 bisphosphate aldolase. The multienzyme complex contains one tetrameric molecule of 6-phosphofructokinase and two molecules of each of other glycolytic enzymes. Hexokinase is not a part of the complex. The molecular mass of the multienzyme complex is about 2.6 X 10(6) daltons. The multienzyme complex has symmetry axis of second order. The formation of the multienzyme complex leads to the compartmentation of glycolytic process. The problem of integration of physico chemical mechanisms of enzyme activity regulation (allosteric, dissociative and adsorptive mechanisms) is discussed. PMID- 2999517 TI - Changes of H-2 antigen expression on thymocytes during leukemia development by radiation leukemia virus. AB - The expression of antigens encoded by the K and D region genes of the major histocompatibility complex on thymocytes of BL/6 mice infected with Radiation Leukemia Virus (RadLV) variants, A-RadLV to which they are sensitive or D-RadLV to which they are resistant, was investigated. Reduced thymus cellularity due to the thymolytic effect of both RadLV variants (30-40% cell reduction within 24 h after intrathymic virus injection) was accompanied with elevated H-2 expression on thymocytes. A high density of H-2D and to a lesser degree increased expression of H-2K were observed following infection with both virus variants. This elevated H-2 expression was maintained transiently for 6-7 weeks in the resistant situation and persisted in the sensitive situation until overt leukemia developed. The occurrence of A-RadLV transformed cells in 75% of the tested thymuses within 10 days after infection (vs 16% in D-RadLV treated mice) and their further expansion until overt leukemia developed could explain the continued expression of elevated H-2 expression on thymocytes in the sensitive situation. The majority (85%) of the primary A-RadLV induced leukemias tested expressed more H-2D/H-2K gene products than normal thymocytes. We conclude that leukemia development due to RadLV infection is not associated with the reduction or disappearance of H-2D/H-2K gene products. PMID- 2999518 TI - Prognostic value of 5'nucleotidase in acute lymphoblastic leukemia with the common-ALL phenotype. AB - 5'-Nucleotidase (5'NT) is an enzyme found on the surface membrane of leukemic cells with the c-ALL phenotype. We studied 79 children with acute lymphoblastic leukemia (ALL). Thirty six of them had the c-ALL phenotype. Within this c-ALL group, ten had 5'-NT positive leukemic cells. Clinical data were available in 33 c-ALL cases. Sex, age and initial white blood cell count were comparable between 5'NT positive and 5'NT negative c-ALL cases. In the 5'NT positive group the probability of complete continuous remission was significantly lower than in the 5'NT negative group (p less than 0.05). PMID- 2999519 TI - The reversal of feline retroviral-induced suppression of lymphocyte Con A receptor mobility by indomethacin and PGE2. AB - Ultraviolet light-inactivated feline leukemia virus (FeLV) and its 15,000 dalton envelope protein (p15E) inhibited concanavalin A receptor motility of feline peripheral lymphocytes (PBL). In contrast, the virus had no effect on immunoglobulin capping of feline PBL. The inhibitory action of FeLV and FeLV p15E was reversed by the addition of indomethacin. The indomethacin effect was titratable and gave significant reversal between 1 X 10(-5) and 1 X 10(-10) M. The indomethacin inhibition of the prostaglandin synthesis does not appear to be the mechanism of action in its ability to reverse FeLV suppression of Con A receptor mobility. Since the addition of prostaglandin E2 (PGE2) (1.0 microM) was also able to reverse the FeLV-induced inhibition and neither indomethacin nor PGE2 had an observable effect on Con A receptor mobility of normal PBL without FeLV present. PBL from FeLV-infected cats were sensitive to indomethacin (1.0 10.0 microM) and an indomethacin-related increase in cap formation was observed. Since both indomethacin and PGE2 were able to reverse the FeLV-induced suppression it was concluded that their mechanism of action may be through a common site. In addition, indomethacin may prove useful as an immune response modifier for therapy in FeLV-infected cats. PMID- 2999520 TI - Immunoglobulin G antibodies binding to a synthetic peptide deduced from the nucleotide sequence of the env gene of HTLV I in patients with leukemia and rheumatoid arthritis, HLA sensitized persons and blood donors. AB - A synthetic pentadecapeptide preparation, env 406-420, with an amino acid sequence deduced from the envelope glycoprotein gene of human T cell leukemia virus type I (HTLV I), was used as the antigen in an enzyme immunoassay for immunoglobulin G antibodies, exploring its usefulness for seroepidemiological purposes. The frequency of reactivity in the test groups, presented in decreasing order was: patients with rheumatoid arthritis; multitransfused nonleukemic patients; Japanese cases of adult T cell leukemia (ATL); HLA sensitized persons; Swedish cases of adult acute leukemia; and Swedish blood donors. Three American cases of ATL and 12 HTLV I seropositive monkeys did not react. In RF positive sera from patients with rheumatoid arthritis, no quantitative correlation between RF activity and anti-env 406-420 activity was seen. Anti-env 406-420 positive sera did not react or reacted only weakly with four control peptide preparations with different amino acid sequences. The experience with oligopeptide serology still is limited. Our results illustrate that unexpected cross-reactions which are hard to interpret can occur. Although absorption experiments indicated an HTLV I specific component of the reactivity, antibodies against epitopes of allo- and auto-immune specificity may also have participated. PMID- 2999521 TI - In vitro induction of CFU-S proliferation by a non viral splenic activity from myeloproliferative sarcoma virus infected mice. AB - The myeloproliferative sarcoma virus (MPSV) induces a myeloproliferative syndrome in DBA/2 mice. It is characterized by a considerable increase in the number (100 fold) and in the concentration (10-fold) of pluripotent hematopoietic stem cells detected in vivo (CFU-S) in the spleens of infected animals. Prior studies have shown the presence of a mixed-colony promoting activity (MPA) in neoplastic spleens. In the presence of a small quantity of erythropoietin, MPA induces the proliferation and differentiation of pluripotent hematopoietic stem cells, detected in vitro (Mix-CFU). We tested the effect of factors produced by neoplastic spleen cells on the proliferation of day 10 CFU-S and their entry into the cell cycle. This was done by comparing the number of day 10 CFU-S present in suspensions of normal bone marrow cells incubated for 2 days on agar underlays containing cells from either normal or neoplastic spleens. Our results show the existence of an activity secreted by cells from the spleens of MPSV-infected animals which starts CFU-S cycling and which is physically distinct from MPSV. The presence of this activity, whose identity with MPA remains to be proven, would enable us to explain the proliferation of CFU-S in the course of the disease. PMID- 2999523 TI - Partial purification and characterization of latent human leukocyte collagenase. AB - Latent and active collagenase were extracted from human polymorphonuclear leukocytes. Separation of the two forms of the enzyme was performed by gel filtration on Sepharose 6 B. The latent form of the enzyme was detected from chromatographic fractions after a brief treatment with trypsin or exposure of the fractions to the sulfhydryl reagent phenylmercuric chloride. Latent enzyme eluted before active enzyme from the column, indicating a higher apparent molecular weight. Partially purified latent enzyme exhibited an apparent molecular size of 70-75 kDa as estimated by gel filtration. A value of 50-55 kDa was obtained for active enzyme. Without activation the latent enzyme did not degrade soluble collagen substrate. This was demonstrated by a quantitative viscometric assay and also by sodium dodecyl sulfate polyacrylamide gel electrophoresis, when no typical cleavage products of collagen could be seen. Latent enzyme could not be obtained unless serine protease inhibitors were present during the extraction and purification procedures. The effects of the activators trypsin, phenylmercuric chloride, phenylmethyl sulfonyltrypsin, and N-ethylmaleimide on the latent human polymorphonuclear leukocyte collagenase were studied. Contrary to the suggestion that inactive proteases activate latent human polymorphonuclear leukocyte collagenase, the inactive phenylmethyl sulfonyl-trypsin could not activate latent collagenase. PMID- 2999524 TI - Dietary fibre consumption and its association with large bowel cancer in man. AB - The hypothesis that lack of fibre in the diet is responsible for a variety of large bowel problems, including cancer, has stimulated much discussion and research over the past 15 years. However, the epidemiological examination of this hypothesis has been hampered by the absence of data on the fibre content of most of the world's foods. In studies in Britain and Scandinavia where consumption of the chemical fraction of dietary fibre, the non-starch polysaccharides, has been determined using accurate methods, significant negative association between colon cancer occurrence and NSP consumption have been shown. Fibre may therefore be protective to populations otherwise assumed to be at risk from a westernised type of diet. At present, methodological problems preclude the use of case-control studies in confirming or refuting these associations. PMID- 2999526 TI - [Treatment and criteria for cure of acromegaly]. PMID- 2999525 TI - The effect of zinc on normal and neoplastic T-lymphocyte proliferation. AB - After 24-72 h of PHA-stimulation, T-cells expressed the transferrin receptor. This receptor facilitates zinc uptake. Zinc transferrin stimulated DNA synthesis in pre-activated or activated, but not in resting T-cells. The regulatory nuclear protein matrix fraction increased from 5 to 40% of the total nuclear protein material in lymphocytes simultaneously with the initiation of DNA synthesis. In contrast, optimal concentration (0.1-0.4 mM) of zinc salts induced a mitogenic response in transferrin-receptor negative resting, but not in PHA-activated or leukemic T-cells. Higher concentrations were toxic. These findings can explain earlier reports on the effect of zinc on immunocompetence in zinc deficient mice and enteropathic acrodermatitis as well as present findings of a normalization of the T-suppressor-cell number in immunosuppressed patients. PMID- 2999527 TI - [Osteohypoplastic angiomatosis. Servelle's syndrome]. AB - One case of osteohypoplastic angiomatosis on the right leg, emphasizing the clinical-radiological characteristics and the nosological difficulties of this syndrome. We point out surgery and continous compression as elective treatments, since all other methods proved ineffective. PMID- 2999528 TI - [Cancer of the breast in the male. Presentation of 2 cases]. AB - We present two cases of adenocarcinome of the male mammary gland, without past history of gynecomastia, cirrhosis, malnutrition, hyperoestrogenism, estron therapy for prostatic cancer, testicular tumor, Klinefelter Syndrome, parasitosis, trauma or irradiation. Male mammary adenocarcinoma is rare in all hospital. In our center we found two cases among 204 female mammary adenocarcinomas, that is one porcent, in a revision between 1971 and 1980. Most male mammary adenocarcinomas are of the ductal/infiltrative type, early metastasis to regional ganglia or by continuity to adjacent skin. PMID- 2999529 TI - [Multiple granular cell myoblastomas. Clinical findings]. AB - Three cases of multiple granular cell myoblastoma are reported. In the discussion the authors emphasize the frequency, pathogeny, evolution and new classification of this type of proliferative disorders. PMID- 2999522 TI - Lung surfactant and pulmonary toxicology. PMID- 2999530 TI - Receptor-receptor interactions in the central nervous system. A new integrative mechanism in synapses. PMID- 2999531 TI - Recent developments in the design of angiotensin-converting enzyme inhibitors. AB - Orally-active angiotensin-converting enzyme inhibitors are rapidly establishing themselves in the therapy of hypertension and congestive heart failure. Concerted efforts in a number of laboratories have now led to the discovery or synthesis of an unparalleled variety of potent inhibitors. The manner in which several of these inhibitors bind to ACE is beginning to be understood. It is hoped that some of the insights to be derived from the SAR and structural studies done with ACE inhibitors will be applicable to other enzyme targets as well. The success of ACE inhibitors as pharmacological tools and in the clinic will also quite certainly encourage future efforts to develop new enzyme inhibitor approaches to drug therapy. PMID- 2999532 TI - [Can hepatitis B virus and AIDS-associated retrovirus (LAV/HTLV-III) be transmitted by insects?]. PMID- 2999533 TI - [Practically relevant electrodiagnosis in facial and recurrent nerve pareses. A review]. AB - Following a general description of pathophysiological changes of an injured peripheral nerve, a survey is presented in which those electrodiagnostic tests that are of practical relevance today in cases of facial and recurrent nerve pareses, are defined and the methods used are described (electromyography, neuromyography, blink reflex, reflex myography of the larynx). Typical electrodiagnostic findings are explained and demonstrated, using examples from our own case material. The relative merits and diagnostic efficiency of separate and combined findings are discussed. PMID- 2999534 TI - [Radiotherapy of nasopharyngeal tumor. Evaluation of personal results, consequences for irradiation technic]. AB - In this paper we present our results of radiotherapy of thirty patients with nasopharyngeal tumour from 1960-1983. Before therapy, 19% of the cases were diagnosed as a T1 tumour, 57% of the patients showed a T3 or T4 tumour. In 24% of the patients, lymph node metastases could be recognised in pretherapeutic staging, whereas in 5% distant metastases were evident. In more than 50% of the cases tumour recurrence or metastases occurred within one year, in 47% of these patients there was local recurrence, in 35% lymph node metastases. The 5-year survival rate was 28%. Basing on an assessment of our results and considering the published data, we changed our radiotherapy technique to a large-field radiotherapy including the tumour region and the lymph vessels. The advantages of this technique and the possible side effects are discussed. PMID- 2999535 TI - [Retinitis in a female patient with a prolonged febrile condition caused by Epstein-Barr viruses]. PMID- 2999536 TI - Hepatic adrenoceptors involved in the glycogenolytic response to exogenous (-) norepinephrine in the dog liver in vivo. AB - Effects of various sympathomimetic amines on the hepatic glucose mobilization were studied in anesthetized dogs. Phenylephrine (30, 100, 300 micrograms), isoproterenol (0.1, 1, 10 micrograms) and (-)-norepinephrine (0.5, 5, 50 micrograms) were injected into the common hepatic artery in three separate groups of dogs. Dose-dependent increases in hepatic venous glucose concentration were observed following the injections of these drugs. Aortic glucose concentration also increased significantly, but to a lesser extent as compared with that in hepatic venous blood. Peak responses were obtained 3 to 5 min after the drug administrations. The increases in hepatic venous glucose concentration induced by the injections of (-)-norepinephrine were significantly diminished to a similar extent in dogs treated with either phentolamine (2 mg/kg, i.v.) or (-) propranolol (0.2 mg/kg, i.v.). The results indicate that in the dog liver in vivo, both hepatic alpha- and beta-adrenoceptors can be involved in the hepatic glycogenolysis. The glycogenolytic response to exogenously administered (-) norepinephrine is mediated via alpha- as well as beta-adrenoceptors in the liver of anesthetized dogs. PMID- 2999537 TI - Mechanisms mediating canine renal vasoconstriction induced by nicotine infusion. AB - We investigated the respective contributions of the renin-angiotensin and alpha adrenergic systems to nicotine-induced, canine, renal vasoconstriction by using saralasin (4 micrograms/kg/min) and phentolamine (25 micrograms/kg/min) blockade respectively. Nicotine infusion (0.024 mg/kg/min) increased mean arterial blood pressure (MABP) (114 +/- 3.0 to 219 +/- 8.0 mmHg) and decreased total renal blood flow (TRBF) (3.12 +/- 0.34 to 1.60 +/- 0.37 ml/min/g). Nicotine infusion produced a significantly lesser blood flow in outer cortex (OC), inner cortex (IC), and outer medulla (OM) compared to control dogs. The intrarenal-artery infusion of saralasin or phentolamine had no effect on the nicotine-induced MABP changes. Phentolamine infusion prior to nicotine resulted in a significantly greater TRBF (P less than 0.01), OC (p less than 0.001), IC (p less than 0.001) and OM (p less than 0.01) flow than in the group that received nicotine only. Saralasin pretreatment prior to nicotine resulted only in a significantly (p less than 0.01) greater OC flow than nicotine only. Our data suggest that while angiotensin II mediates a portion of the action of nicotine on the OC renal vasculature, the alpha adrenergic system predominates as the mediator of nicotine-induced renal vasoconstriction in the first 7 minutes of nicotine infusion. PMID- 2999538 TI - Vasopressin inhibition of cyclic AMP accumulation and effects on the learned response in inbred mouse strains. AB - The effects of a single injection of arginine vasopressin on the cyclic AMP responses of cerebral cortex slices to noradrenaline, forskolin and 2-chloro adenosine were tested in six inbred mouse strains. The noradrenaline response was reduced in one strain and that to 2-Cl-adenosine in 2 strains. There was a positive correlation between the differences in the cyclic AMP response to 2-Cl adenosine between control and AVP-treated mice and the differences in latencies during the extinction period of conditioned avoidance response learning. PMID- 2999539 TI - Blockade of ethanol induced conditioned taste aversion by 3-amino-1,2,4-triazole: evidence for catalase mediated synthesis of acetaldehyde in rat brain. AB - This investigation seeks to present evidence for the oxidation of ethanol in the brain via the peroxidatic activity of catalase and simultaneously provide evidence for the role of central acetaldehyde (ACH) in the mediation of an ethanol-induced conditioned taste aversion (CTA). Ethanol is capable of inducing a conditioned taste aversion. Pretreatment with the catalase inhibitor, 3-amino 1,2,4-triazole (AT), shows an attenuation of this ethanol-induced CTA. Animals receiving ethanol injections showed a CTA to a novel solution paired with a drug administration, while ethanol injected animals pretreated with AT did not show a CTA to ethanol administration. This effect of AT appears to be specific to the effects of ethanol as CTA's to morphine and lithium chloride were not affected by AT pretreatment. Peripheral levels of ethanol were the same in all animals regardless of pretreatment indicating that AT had no effect on peripheral levels of ethanol. These data increase support for the notion that acetaldehyde is produced directly in the brain and that it may be the agent mediating some of the psychopharmacological properties of ethanol. PMID- 2999540 TI - Neuropeptide Y (NPY) binding sites in rat brain labeled with 125I-Bolton-Hunter NPY: comparative potencies of various polypeptides on brain NPY binding and biological responses in the rat vas deferens. AB - The binding of biologically active 125I-Bolton-Hunter (BH)-NPY to rat brain membranes was saturable and reversible and regulated by inorganic cations and guanyl nucleotides consistent with other neurotransmitter receptor systems. The concentration of specific 125I-NPY binding differed in various brain regions, being highest in the hippocampus and lowest in the cerebellum. Scatchard analysis of 125I-NPY binding showed a single class of receptor sites with a Kd = 0.1 nM and Bmax of 3 pmole/g tissue in hippocampus. Peptide YY, porcine and human NPY inhibited the specific 125I-BH-NPY binding with IC50 values of 50-120 pM. In contrast, human NPY free acid and pancreatic polypeptides from human (HPP), rat (RPP) and avian (APP) sources were much weaker (IC50 greater than or equal to 300 nM). The rank order of potencies for NPY analogs and the inactivity of APP and HPP fragment (31-36) on brain binding appeared to correlate with their relative activities in inhibiting contractions of the field-stimulated rat vas deferens. However, PYY, HPP and RPP exhibited activity in the field-stimulated rat vas deferens indicative of a possible action upon sites distinct from the brain NPY binding site. PMID- 2999541 TI - GABA, depressants and chloride ions affect the rate of dissociation of 35S-t butylbicyclophosphorothionate binding. AB - The dissociation of 35S-TBPS was studied from binding sites of rat cerebral cortex. Monophasic dissociation plots became polyphasic and accelerated in the presence of micromolar concentrations of GABA suggesting the involvement of low (or super-low) affinity GABA receptors. The presence of the depressants etazolate, R(-)MPPB and ethanol resulted in similarly accelerated dissociation patterns. In contrast, the convulsants S(+)MPPB and pentamethylenetetrazol did not significantly affect the dissociation of TBPS. Dissociation initiated by dilution was not affected either by an excess of picrotoxin or by varying the equilibrium occupancy of the TBPS sites. These findings rule out the possibility of a kinetic cooperativity for the binding of convulsants. The removal of chloride ions also enhanced the rate of TBPS dissociation. Kinetic heterogeneity of the TBPS binding sites can be interpreted with allosteric interactions mediated by various sites at the GABA receptor complex coupled to different states of the chloride ionophore. PMID- 2999542 TI - delta 9-Tetrahydrocannabinol elicited ipsilateral circling behavior in rats with unilateral nigral lesion. AB - The present study was designed to examine the influence of delta 9 tetrahydrocannabinol (THC) on the central dopaminergic system using circling behavior. THC 5 mg/kg i.p. produced ipsilateral circling in rats with unilateral nigral lesion by 6-hydroxy-dopamine. THC-induced ipsilateral circling was completely antagonized by 0.2 mg/kg of haloperidol. These findings suggest that THC may cause a presynaptic stimulation of nigrostriatal dopaminergic neurons. PMID- 2999543 TI - Differential opiate influences on food hoarding and intake in the deer mouse, Peromyscus maniculatus. AB - The feeding behavior of the deer mouse, Peromyscus maniculatus, includes food hoarding as well as ingestion. In this animal the mu opiate agonist, morphine, and the kappa opiate agonist, U-50, 488H, selectively stimulate food hoarding and ingestion, respectively. This suggests that mu and kappa opiate systems may differentially mediate primary components of natural feeding behavior. PMID- 2999544 TI - gamma-Aminobutyric acid in peripheral tissues. AB - Significant amounts of gamma-aminobutyric acid (GABA), an endogenous amino acid, are present in mammalian peripheral tissues. This finding led to the suggestion that GABA may act as a neurotransmitter in the peripheral nervous system as it does in the central nervous system. This review deals with recent identification of GABA in the autonomic nervous system and the possible functional role of GABA in neuronal and non-neuronal tissues. The identification of GABA in the autonomic nervous system has paved the way for new approaches in pharmacological investigations. PMID- 2999546 TI - Collagen accumulation can continue with decreased prolyl hydroxylase activity in the liver. AB - A single dose of dimethylnitrosamine dose-relatedly increased total hepatic hydroxyproline content in rats 14 days after the dosing. In cases of 35 mg/kg of dimethylnitrosamine, it increased rapidly to 1.7 times the normal level within 14 days. This increase persisted thereafter until 84 days. Hepatic collagen prolyl hydroxylase activity was 1.8 times the normal level by the fourth day after the dosing but normalized within 14 days. It decreased further to levels significantly lower than those in normal rats at 28, 56 and 84 days. Serum glutamic pyruvic transaminase activity was about 13 times the normal level at 2 days and normalized after 7 days. On histology, fiber developed in necrotic areas around the central veins after 7 days and remained after 28 days when the necrosis had already disappeared. These results suggest that abnormal collagen can continue to increase in the state of decreased collagen prolyl hydroxylase activity in the liver. PMID- 2999545 TI - Multiple opiate receptors may be involved in suppressing gamma-aminobutyrate release in substantia nigra. AB - Slices of rat substantia nigra were preloaded with tritiated gamma-aminobutyrate (GABA) or dopamine (DA) and perfused with Krebs solution containing 5 microM aminooxyacetic acid or 10 microM nialamide to inhibit the catabolism of GABA and DA respectively. Repeated brief exposures to high potassium medium (+ 30 mM K+ for 1 min) evoked a consistent pattern of calcium-dependent 3H efflux against which the effects of opiates (10-400 microM) were assessed. Opiate agonists inhibited K+-induced 3H-GABA efflux in the following decreasing order of potency: bremazocine greater than D-Ala2-Met5-enkephalinamide (ENK) greater than SKF 10047 much greater than morphine, consistent with the participation of kappa, delta, sigma and to a lesser extent mu opiate receptors respectively. Naloxone (1 microM) partially antagonised the response to morphine and ENK, while ICI 154129 attenuated ENK only. Save for a GABA-releasing action of SKF 10047 at high doses, none of the compounds altered basal outflow of 3H-GABA. Naloxone, in the dose range 10-400 microM, also significantly inhibited depolarisation-induced release of 3H-GABA. In parallel experiments none of the compounds tested were found to influence 3H-DA release in concentrations up to 40 microM, but thereafter suppressed K+-induced 3H-DA outflow indiscriminately. The results are discussed with reference to the possible mechanism(s) via which injected and endogenous opiates may affect motor performance by attenuating GABA transmission in the nigra. PMID- 2999547 TI - Immunoassayable adrenocorticotropin in peripheral organs: concentrations during early development. AB - Although many have identified immunoassayable adrenocorticotropin (ACTH) in sites outside the pituitary (brain, gastrointestinal tract), there is relatively little information regarding immunoassayable ACTH in other major peripheral organs. Several major peripheral organs (pancreas, liver, kidney, heart) of rats were found to contain variable amounts of immunoreactive (IR-) ACTH which appeared to be authentic IR-ACTH on the basis of parallelism to ACTH1-39 upon serial dilution of extracts and gel filtration chromatography. Concentrations of IR-ACTH in peripheral organs were also studied to determine if changes occur during early development. Concentrations of IR-ACTH did not show significant changes in liver and heart at various ages between 10 and 80 days, but IR-ACTH in pancreas and kidney (day 10 vs. 80) did show significant decrements with aging. PMID- 2999548 TI - Bisphosphonates and bone resorption: effects on collagenase and lysosomal enzyme excretion. AB - When added to cultures of parathyroid hormone (PTH)-stimulated bones, dichloromethylenebisphosphonate (C12MBP) and 3-amino-1-hydroxypropydilene-1,1 bisphosphonate (AHPrBP) inhibit completely and in a parallel manner the development of resorption lacunae, the loss of calcium by the explants and their PTH-induced excretion of lysosomal hydrolases (beta-glucuronidase and N-acetyl beta-glucosaminidase). The loss of collagen (hydroxyproline) by the bones is usually less inhibited than their loss of calcium and their heparin-induced excretion of collagenase is unaffected. To interpret these data, it is proposed that these bisphosphonates act more on the activity of osteoclasts, suppressing simultaneously their excretion of lysosomal enzymes and their erosion of mineralized bone matrix, than on that of other cell types (osteoblasts ?) responsible for collagenase production and the removal of uncalcified collagen. PMID- 2999549 TI - Platelet 3H-imipramine binding distinguishes depression from Alzheimer dementia. AB - Platelet 3H-imipramine binding and serotonin uptake were studied simultaneously in normal subjects and in depressed, parkinsonian and Alzheimer's disease patients to investigate the usefulness of these variables in the diagnosis of depression in the elderly. Whereas Vmax of platelet serotonin uptake was significantly reduced in all patient groups compared to age matched normal subjects, the density of 3H-imipramine binding was reduced in depressed patients only. The lower Bmax values in depressed patients was independent of patient age. These data suggest that platelet 3H-imipramine binding may be a useful laboratory index which discriminates depression from dementia in the elderly. PMID- 2999550 TI - The relative incorporation of arachidonic and eicosapentaenoic acids into human platelet phospholipids. AB - The incorporation of arachidonic acid (AA) as compared to eicosapentaenoic acid (EPA) into human platelet phospholipids was tested by incubating washed platelets with a known mixture of [3H]AA and [14C]EPA. Following incubation, the platelet lipids were extracted, the individual phospholipids--phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE)- were separated by thin layer chromatography, and their corresponding [3H]/[14C] ratios were determined. Based on a [3H]/[14C] ratio of unity for the substrate mixture, the PC, PS, PI and PE exhibited ratios of 0.55, 0.93, 1.12 and 0.74, respectively, which were significantly different from 1.00 in all instances except in the case of PS. These results indicate that PC and PE selectively incorporated EPA, while PI showed preference toward AA. These selectivities may account partly for the differing AA/EPA mass ratios that have been observed among the individual phospholipids of human subjects consuming fish oils. PMID- 2999552 TI - [Radionuclide study of patients with bone tumors in organ-sparing operations]. PMID- 2999553 TI - [Radioimmunologic evaluation of the prognosis and effective therapy of patients with small cell lung cancer]. AB - Radioimmunoassay was used to determine CEA, ACTH, calcitonin (CT), parathyroid hormone (PTH) and cortisol levels in the blood plasma and serum of small-cell lung carcinoma patients before and after chemotherapy. The prognostic value of the determination of CEA, ACTH and CT levels was shown. In 74% of patients, progression of the disease was accompanied by a rise of the CEA level, successful treatment by a decrease in the CEA level. A similar correlation between tumor activity and ACTH, CT and PTH levels was shown in 50.44 and 47% of the patients respectively. PMID- 2999551 TI - Non-soluble dietary fiber effects on lipid absorption and blood serum lipid patterns. AB - Generalized effects of dietary fiber on lipid absorption and blood serum lipid patterns of humans have not been defined and may not even exist. The term dietary fiber covers a wide variety of materials with different chemical and physical characteristics. The ability of pectins and mucilages, often classed as soluble fibers, to lower blood and liver lipids has been demonstrated repeatedly and consistently. However, demonstrated hypolipidemic effects of feeding such non soluble fibers as cellulose, hemicellulose and bran are by no means consistent. On the basis of pooled data, it appears that hypolipidemic response or non response of humans to inclusion of non-soluble fibers in diets is in part related to the degree of fecal bulking as a result of in vitro water holding capacity and in part related to pre-study blood serum lipid levels of the individual subjects. PMID- 2999554 TI - [Scintigraphy of knee joints in inflammatory dystrophic diseases]. PMID- 2999555 TI - [Gamma-topography in the diagnosis of rhinosinogenic brain abscesses]. PMID- 2999556 TI - [Use of radionuclides for evaluating reparative osteogenesis in purulent infections]. PMID- 2999557 TI - [Poliomyelitis in New Caledonia: epidemiological data, post-vaccinal seroconversion, prophylactic strategy]. AB - After an outbreak in 1956, poliomyelitis appears to get a sporadic development. Consequently, it has not been a major medical concern. In 1982, 5 cases of paralytic manifestations due to a polio virus of type 1 underlined the relationship between this reappearance of the disease, some neglecting in mass vaccination and some deficiency in sanitary conditions of the surroundings. A serologic monitoring, complementary to that survey, has been conducted up to 1984. It has shown that, in a number of zones of the territory, a natural immunization has appeared quickly. This immunization is of the same type that in some underdeveloped tropical countries: at 6, 93,3% of the children present protective antibodies against 1 type of virus, 40% against 2 types and 6,7% against the 3 types. Vaccination with non-active vaccine, adopted because the risks of interference linked to the frequency of excretion of the enteroviruses, is efficient. After a primo-vaccination followed by a booster dose one year later, sero-conversion ranges from 95,3% to 100% for type 1, 83,3% to 96,5% for type 2, 75% to 84,9% for type 3. On the other hand, sero-conversion observed one month after the second vaccination appeared to be unsatisfactory to lighter the vaccinal time-table. PMID- 2999558 TI - [Immune response as a function of the nutritional status in young children 1 to 3 years of age in the south of Ivory Coast]. AB - 177 children between 1 and 3 years (74 well-nourished, 55 suspected of protein/calorie deficiency, 48 under nourished) were vaccinated (tuberculosis, diphtheria, tetanus, whooping-cough, polio); one month after the third dose of DTWC polio, we proceeded to apply the Merieux multitest and to check for intradermal reaction to the tuberculin: This study has shown that the Merieux multitest gives results comparable to the classical intradermal method with tuberculin. The multitest makes it possible to explore simultaneously seven different antigens under perfectly comparable conditions from the standpoint of precision and standardization (diphtheria, tetanus, tuberculin, Streptococcus, Proteus, Trichophyton and Candida). This exploration has shown that there is no significant difference in terms of the nutritional condition of the children. Well-nourished children, under-nourished children and children suspected of a deficiency react in the same manner to antigenic attractions whether vaccinal or spontaneous. This study would seem to suggest, therefore, that the nutritional conditions of a child need not to be taken into account when administering a vaccine. PMID- 2999559 TI - [Report on 5 years of expanded program of vaccination in an experimental zone in Ivory Coast]. AB - Started in june 1978 in the Abengourou District (Ivory Coast) the expanded program on immunization (E.P.I.) was assessed in june 1983 by the mean of a vaccinal survey, after five years of implementation. This survey was conducted in both urban circle where E.P.I. was fully in operation from 1978, and rural circle where it was progressively implemented to reach about 3/4 of the villages by 1983. The authors report and compare the findings in terms of global vaccinal coverage towards the six diseases covered by the E.P.I. in town (71,7%) and in rural area (42,6%). Their report includes the results of a survey about the sanitary protection of the children and about the respect of the ideal immunization schedule and recommended in Ivory Coast (39,9% in town and 6,2% in rural area). In considering measles, they attempt to understand the true benefit of such a plan in terms of reduced mortality and morbidity rate from 15,8% to 0,9%. Considering their findings, the authors recommend to concentrate efforts in two directions for the coming years. To better cover the rural zones. To better respect the ideal immunization schedule. PMID- 2999560 TI - [Polish asbestos and its fibrogenic effect. II. Identification and fibrogenic effect of fibrous minerals occurring in road stone deposits]. AB - Fibrogenic properties of a fibrous mineral occurring in road stone deposits mined at Naslawice at the Lower Silesia have been tested. Diffractometric and infrared absorption spectra tests identified this mineral as antigorite. The content of free crystalline silica was 1.3%. The dust (50 mg) obtained from the test mineral when intratracheally administered to experimental animals as a suspension in physiological NaC1 solution yielded statistically significant increases of the lung weight and hydroxyproline content, as compared to controls. The mean weight of experimental animals' lungs after 3 months was 1898.4 mg, after 6 months- 2116.8 mg, after 9 months--2878.4 mg. The control animals' lung weight was 1409.6 mg after 3 months, 1634.4 mg after 6 months and 1939.9 mg after 9 months. Hydroxyproline content in experimental animals' lungs was 5.1 after 3 months, 5.8 mg after 6 months and 8.6 mg after 9 months. In controls, hydroxyproline content in lungs was, respectively: 3.5; 3.9 and 4.0 mg after 3, 6, and 9 months of the experiment. However, the process of lung fibrosis when affected by antigorite from Naslawice was slower than under effects of antigorite from Szklary, which may result from almost 10 times fewer numbers of fibrous particles in the dust obtained from antigorite from Naslawice. PMID- 2999561 TI - Diabetic lipoprotein deficient serum: its effect in low density lipoprotein (LDL) uptake and degradation by fibroblasts. AB - Low density lipoproteins (LDL) isolated from poorly controlled diabetic patients are known to be taken up and degraded by fibroblasts at a lower rate than LDL isolated from normal subjects. This aberrant metabolic behavior has been attributed to a diabetic-related abnormality in LDL composition yet to be characterized. The studies reported in this article show that the decrease in uptake and intracellular degradation of LDL from diabetic patients is further enhanced when the cells are exposed to lipoprotein deficient serum (LPDS) isolated from the same poorly controlled diabetic patients. Comparative studies of the composition of LPDS obtained from normal donors and poorly controlled diabetic patients showed an increase in saturated and total unesterified fatty acids (UFA), lecithin, apolipoprotein A1, and immunoreactive insulin in the LPDS from diabetic patients. We postulate that exposure of cells to LPDS obtained from poorly controlled diabetic patients may induce changes in the composition of the fibroblast membrane and alter its fluidity, leading to further decrease in the uptake and degradation of LDL. During poor diabetic control, cell membrane changes, and modification of LDL composition are likely to act either additively or synergistically to induce an abnormal LDL-cell interaction. This abnormal interaction may be a relevant factor to explain the greater incidence of arteriosclerosis in diabetes mellitus. PMID- 2999563 TI - Cloning and expression of the Escherichia coli rho gene in a plasmid vector. AB - In order to further elucidate the role of Rho protein on transcription termination and cells growth control, we have subcloned by two steps the rho+ structural gene of Escherichia coli from Lambda rho+524 into a plasmid vector. The resulting plasmid pEG25 contains a 2.9 kbp insert which is able to complement several different rho mutations and to express a functional Rho protein in U.V. irradiated maxicells. PMID- 2999564 TI - Tryptophanase activity in different toxigenic and nontoxigenic strains of Vibrio cholerae: effect of glucose. AB - Tryptophanase activity was measured in eight different toxigenic and nontoxigenic strains of Vibrio cholerae (V. cholerae) in presence and absence of inducer tryptophan (2 mM). Stimulation of enzyme activity was observed in both toxigenic and nontoxigenic strains of V. cholerae in presence of inducer. Tryptophanase activity remained much higher in toxigenic strains than that in nontoxigenic strains. Low levels of enzyme activity in nontoxigenic strains could be increased by the addition of exogenous cyclic AMP. A lower concentration of glucose (0.25 gm%) in culture medium produced no inhibitory effect on enzyme activity. But a higher concentration of glucose (3 gm%) repressed the tryptophanase activity. The repressive effect of glucose could be reversed by the addition of exogenous cyclic AMP. PMID- 2999562 TI - The role of calcium in the induction of refractoriness to cyclic AMP stimulation by TSH. AB - An initial exposure of beef thyroid slices to 25 mU/mL thyroid-stimulating hormone (TSH) for two hours induces a diminished stimulation of cyclic adenosine monophosphate (AMP) production upon subsequent readdition of TSH but does not modify the effect of prostaglandin E1 (PGE1). Incubation of thyroid slices in calcium-free buffer with or without 2 mmol/L ethylene glycol bis (beta-aminoethyl ether)--N,N' = tetracetic acid (EGTA) prevented desensitization induced by TSH and PGE1, to the subsequent stimulation by TSH and PGE1, respectively, despite the presence of calcium in subsequent incubations. TSH-induced desensitization was not modified by increasing the calcium concentration up to 50 mmol/L in the initial incubation. However, the stimulatory effect of TSH upon cyclic AMP levels was decreased as the calcium concentration in the first incubation was increased. In the presence of at least 1 mmol/L calcium, an initial incubation of thyroid slices with 20 mumol/L ionophore A-23187 decreased the stimulation of cyclic AMP by 25 mU/mL TSH added to the slices for the first time during a subsequent incubation. Under these conditions, A-23187 had no effect on PGE1 stimulation of cyclic AMP. These results indicate that calcium may play a role in the TSH induced, but not PGE1, desensitization of cyclic AMP formation. PMID- 2999565 TI - Gangliosides in early interactions between vesicular stomatitis virus and CER cells. AB - In the present report an attempt was made to elucidate the role of gangliosides in early interactions between vesicular stomatitis virus (VSV) and CER cells. Research was carried out to test the ability of gangliosides from mammal brains and from CER cells to inhibit viral attachment to susceptible cells. The incubation of VSV in the presence of gangliosides decreased the subsequent infection of CER cells by the virus. When similar experiments were performed with gangliosides inserted in liposomes the inhibition of infection was enhanced. Since carbohydrate moieties could participate to rhabdovirus binding as a part of a glycolipid receptor, CER cells were subjected to the action of glycosidases and these produced a fall in the viral attachment. Deglycosilated CER cells reacquired their susceptibility to virus infection after coating with gangliosides immediately after enzyme treatment. Results obtained show the participation of gangliosides in the receptorial structure for vesiculovirus of susceptible CER cells. PMID- 2999566 TI - Studies on pathogenesis of buffalo pox virus in rabbits: quantitative assay of virus in different organs. AB - The buffalo pox virus was found to multiply in the skin, the primary site of inoculation with an eclipse phase of 12 hr. The virus was then detected in the skin after 15 hr followed by its appearance in regional lymph nodes 36 hr postinoculation. Primary viremia was detected 48 hr postinoculation, followed by detection of virus in the lungs, liver, and spleen. The virus multiplied in the lungs on day 4 and in the liver and spleen on day 5 postinoculation and its release led to secondary viremia. In a follow-up from day 7 to 14 postinoculation, the virus was detected in the kidneys, stomach, intestines, and gonads. PMID- 2999567 TI - Enhancement of influenza virus hemolysis by physical and serological treatments. AB - The mode of hemolysis by influenza A virus was compared with that of Sendai virus. The WSN strain of influenza virus grown in either eggs or MDCK cells expressed hardly any hemolytic activity by itself. Treatment of the MDCK cell grown WSN virus with sonication or freezing and thawing moderately enhanced the hemolytic activity, but the maximum level attainable was considerably lower than that of Sendai virus. A high level of hemolytic activity comparable to that of Sendai virus was obtained only after treatment of the virus with antibody and complement. An electron microscopic study revealed that non- or low-hemolytic WSN virions were not permeable to uranyl acetate stain in contrast with the hemolytic virions obtained after treatment with antibody and complement, indicating that the hemolytic virions had sustained some injury to their envelopes. These phenomena were comparable to those found with Sendai virus, showing that damage to the envelope is also responsible for the hemolysis of influenza virus. The influenza viruses, however, remained spherical after every treatment and the stain did not penetrate into the core of the virion. These observations suggest that the envelope of influenza virus is more rigid than that of Sendai virus but that the hemolytic process of influenza virus is nevertheless mediated through envelope-membrane fusion as in the case of Sendai virus. PMID- 2999568 TI - A field study of infection with human T-cell leukemia virus among asian primates. AB - Asian nonhuman primates were surveyed seroepidemiologically for natural infection with human T-cell leukemia virus (ATLV/HTLV) or a closely related agent. Materials from various primates (three genera [Macaca, Presbytis, and Hylobates], 17 species, totalling 1,079 animals) under natural conditions were obtained in the field study. Virus infection was determined by the indirect immunofluorescence test using HTLV-specific antigens. Animals seropositive for HTLV were found only among macaques originating from various localities, toque monkeys in Sri Lanka (17.5%), crab-eating macaques in Thailand (1.3%), stumptailed macaques in Thailand (1.5%), rhesus monkeys in Thailand (3.3%), and Celebes macaques in Indonesia (16.9%). Langurs and gibbons were seronegative. Thus the wide distribution of HTLV in nature among various macaques suggests that the introduction of this virus into primates occurred in ancient times. PMID- 2999569 TI - Fitness: a new look at an old term (measurements of human aerobic performance). AB - Despite the increased public interest in being fit, there is no universally accepted definition of "fitness." Various reports equate fitness to oxygen consumption with exercise. This genetically determined V02 maximum is the limiting factor in high-intensity, short duration maximal aerobic exercise. For the nonathelete, however, this capacity may not be an accurate assessment of fitness. Instead, the ability of this individual to do less vigorous work of greater duration may be the best estimate of his fitness. Energy expenditure, a requirement of work, is obtained from the metabolism of carbohydrates and lipids. A measure of the relative utilization of these substrates is the respiratory quotient (R.Q.). Initial phases of exercise depend primarily on carbohydrate metabolism which is reflected by an RQ of 1.0. As exercise continues, substrate utilization relies upon an increasing percentage of free fatty acid metabolism and the R.Q. will approach 0.7. As nonathletes are trained, there is more rapid shift to free fatty acid metabolism which is reflected by a lower R.Q. in the early phases of exercise. The serial measurement of R.Q. as an individual steadily exercises below his anaerobic threshold will reflect his ability to utilize lipids as an energy source. The greater the rate of change of R.Q. with time, the greater is his ability to metabolize lipids. Increased utilization of lipids during exercise indicates his augmented capacity to do submaximal long duration work, therefore, fitness. PMID- 2999570 TI - Endogenous tumor lectins: a new class of tumor markers and targets for therapy? AB - Endogenous lectins of normal tissues can play a functional role in recognition processes and cell adhesion. These functions are areas of particular relevance to tumor growth and metastasis. Our initial results on endogenous lectins of different tumors lead to the working hypothesis that the pattern of endogenous lectins is qualitatively and quantitatively different between different types of tumors and between tumors and normal, nonmalignant tissues. The endogenous lectins may thus prove to be potentially important in establishing a new concept for a rational lectin-based type of diagnosis and therapy of various tumors. PMID- 2999571 TI - Synthetic marijuana for nausea and vomiting due to cancer chemotherapy. PMID- 2999572 TI - Augmentation of the neutralisation test for type 1 HSV: evidence of high representation of neutralising antibody in the adult community. AB - Optimal neutralisation of type 1 herpes simplex virus was obtained by reacting undiluted human serum with virus for 4 h at 37 degrees C, followed by addition of antihuman globulin for 20 min; under these conditions it was possible to detect neutralising antibody activity in 40 of 45 human sera (88%) previously adjudged to be negative by conventional neutralisation tests. PMID- 2999573 TI - Serum interferon and clinical manifestations of infection with human T lymphotropic virus type III. AB - Sera from 32 homosexual men were studied for the presence of antibodies against human T-lymphotropic virus type III (HTLV III) and acid-labile interferon (IFN) alpha. Infection with HTLV III was found to be associated with the presence of serum IFN. IFN was detected in 74% of sera from male homosexuals with HTLV III antibody, but in only 6% of sera from antibody-negative individuals. More than 80% of sera from HTLV III-infected patients with the acquired immune deficiency syndrome (AIDS) or pre-AIDS conditions (generalized lymphadenopathy or the AIDS related complex) were positive for IFN, while IFN was not present in sera from healthy homosexual men with HTLV III antibody. In conclusion, the presence of serum IFN may be predictive of the development of AIDS or pre-AIDS conditions in male homosexuals exposed to HTLV III. PMID- 2999574 TI - Effect of hypertonic conditions on protein synthesis in MA104 cells infected with human rotavirus. AB - When a high NaCl concentration was used to decrease selectively the synthesis of cell proteins, the synthesis of most cellular polypeptides was greatly diminished relative to human rotavirus proteins. Thus, in the presence of 150 mM excess NaCl, 11 viral polypeptides were clearly identified. However, hypertonic conditions also reduced viral protein synthesis to a different extent with individual proteins. No significant changes in viral protein synthesis occurred during incubation under the hypertonic condition for up to 6 h, and infectious virus yields of MA104 cells incubated in the hypertonic medium did not differ from the yields of untreated MA104 cells. These results indicate that hypertonic conditions provide a useful tool for qualitative studies of viral protein synthesis in human rotavirus infected cells. PMID- 2999575 TI - Effects of maternal beta sympathomimetic therapy on the neonate. PMID- 2999576 TI - Recommendations for assisting in the prevention of perinatal transmission of human T-lymphotropic virus type III/lymphadenopathy-associated virus and acquired immunodeficiency syndrome. PMID- 2999577 TI - 1985 STD Treatment Guidelines. PMID- 2999578 TI - Temporal trends in the incidence of malformation in the United States, selected years, 1970-71, 1982-83. PMID- 2999579 TI - Evolution of the single copy alpha A-crystallin gene: differently sized mRNAs of mammals and birds show homology in their 3' non-coding regions. AB - alpha A-Crystallin, a major structural polypeptide of the vertebrate eye lens, is evolutionarily highly conserved. We have analyzed the corresponding nucleic acid sequences in both genomic DNA digests as well as in lens cytoplasmic RNA preparations from a wide variety of vertebrates by blot hybridization with cloned rat alpha A2-crystallin cDNA probes. The probes are not able to hybridize under any conditions to RNA and DNA derived from fishes and amphibia, but do show substantial homology with the sequences of mammals, birds and reptiles. The alpha A-crystallin gene, which has been isolated from a hamster gene library occurs only once in the haploid genome. Coding and 3'-untranslated regions of alpha A2 crystallin mRNA are conserved among all mammals and birds examined. However, the regions comprising the conserved sequences are differently represented in the ultimate mRNA. The alpha A2-mRNA 3'-non-coding regions of reptiles and birds are 300-550 bases longer than those of mammals. Some rodents produce next to the alpha A2-mRNA another messenger that encodes the alpha AIns-polypeptide possessing an insertion of 22/23 amino acid residues between positions 63 and 64 of the alpha A2-polypeptide chain. alpha A2 and alpha AIns-mRNA are generated from a single gene as major and minor species, respectively, in a proportion which is similar to the ratio of the polypeptides found in vivo and in vitro. The size heterogeneity of the alpha A2-mRNA from most mammals examined is due to the variable size of the poly(A) tail. PMID- 2999580 TI - Isolation of a protein labeled with diisopropyl fluorophosphate on stimulation of polymorphonuclear leukocytes with immune complexes. AB - As demonstrated by others, diisopropyl fluorophosphate (DFP) markedly inhibits the O2- generation from guinea-pig polymorphonuclear leukocytes (PMN) stimulated by an antibody complex with ovalbumin (Ag-Ab complex), and also the intracellular uptake of antibody-sensitized erythrocytes by the cells. However, when PMN were treated with DFP and washed to remove the inhibitor, they again became able to exhibit the O2- -generating and phagocytic activities. The [3H]DFP-labeling of intact PMN followed by solubilization with Triton N101, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the existence of several [3H]DFP-labeled proteins with different mol. wts, which disappeared on pretreatment of cells with cold DFP. However, stimulation of DFP-pretreated PMN with Ag-Ab complex in the presence of [3H]DFP resulted in the appearance of a [3H]DFP-labeled, membrane-bound protein with a mol. wt of 40,000. This protein was isolated by affinity chromatography of the solubilized PMN and phagosomes on anti-Ig antibody-Sepharose 4B. Although the enzymatic properties of the protein are not clear, the results so far obtained suggest that it is a putative, stimulus-activated serine protease participating in the triggering events leading to the activation of NADPH oxidase responsible for the respiratory burst and the formation of phagosomes. PMID- 2999581 TI - Studies on the mechanism of natural killer cell-mediated cytotoxicity. VI. Characterization of human, rat, and murine natural killer cytotoxic factors. AB - Recent evidence has implicated natural killer cytotoxic factors (NKCF) as the lytic mediators of NK cell-mediated cytotoxicity reactions. The objective of this study was to examine and compare some of the biochemical and functional characteristics of human, rat, and murine NKCF. Supernatants containing NKCF were generated by stimulating effector cells with Con A or U937 (for human PBL) or YAC 1 (for rodent spleen cells) and tested for cytotoxic activity in a 20-hour (rodent) or 24-hour (human) 51Cr release assay. NKCF activity was inactivated by heating to 63 degrees C, 8 M urea, pH 2, and reduction and alkylation. These factors were highly sensitive to trypsin, moderately sensitive to papain and resistant to neuraminidase. Adsorption of human NKCF to U937 cells is inhibited by mannose-6-phosphate and adsorption of rodent NKCF to YAC-1 cells is inhibited by alpha-methyl-D-mannoside and fructose-6-phosphate. Oxidation of NKCF with sodium periodate abolished lytic activity. Pretreatment of NKCF with Con A but not pretreatment of target cells inhibited lytic activity. NKCF activity eluted in a single broad band of apparent MW of 15,000-40,000 after fractionation by HPLC gel permeating chromatography. Pooled fractions containing NKCF activity were subjected to some of the same tests performed on whole supernatants. Test result with semipurified NKCF confirmed that these factors are inactivated by trypsin or sodium periodate and that mannose-6-phosphate inhibits their binding to target cells. There were no major differences observed in NKCF produced by the three different species whether stimulated by Con A or NK-sensitive tumor cells. The evidence indicates that NKCF are glycoproteins in which disulfide bonding is essential for lytic activity. Furthermore, it appears that carbohydrate residues expressed on NKCF molecules are involved in the binding of these factors to the target cell membrane. PMID- 2999582 TI - [Electron microscope studies in adenoid cystic carcinoma of the trachea]. PMID- 2999583 TI - [Hypertrophy of type II pneumocytes in rats following exposure to DQ-12]. PMID- 2999584 TI - Multiple synchronous scar-related lung carcinomas: report of a case and review of the literature. PMID- 2999585 TI - Malignant fibrous histiocytoma of the maxillary sinus. PMID- 2999586 TI - Effect of novobiocin on the frequencies of chromatid-type aberrations and sister chromatid exchanges following gamma-irradiation. AB - The effect of novobiocin on the frequencies of chromatid-type aberrations and SCEs was examined in Chinese hamster V79 cells which were exposed to gamma-rays and post-treated with novobiocin. While no chromatid aberrations were induced in the unirradiated cells by novobiocin, the frequency of SCEs was slightly increased by treatment with novobiocin alone. Irradiation of G2 cells produced multiple chromatid-type aberrations and post-treatment of the irradiated cells with novobiocin resulted in a significant increase of the aberrations, including chromatid gaps and breaks. In contrast, novobiocin failed to increase the frequency of SCEs induced by gamma-rays when the irradiated cells were post treated with novobiocin. PMID- 2999589 TI - Calmodulin and Ca2+-dependent cyclic AMP phosphodiesterase activity in Trypanosoma cruzi. AB - Calmodulin has been purified from Trypanosoma cruzi epimastigote forms by ion exchange chromatography, gel filtration and affinity chromatography on 2-chloro 10-(3-aminopropyl)phenotiazine-Sepharose. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, the factor showed a polypeptide band with an apparent molecular weight of 16 000. In addition, cyclic AMP phosphodiesterase activity from T. cruzi epimastigote forms was purified by ion-exchange chromatography and affinity chromatography on a brain calmodulin-Sepharose column. The enzyme was activated by homologous calmodulin as well as by bovine brain and Neurospora crassa calmodulins. The activation required micromolar concentrations of Ca2+ and it was blocked by EGTA and by some neuroleptic drugs such as chlorpromazine, fluphenazine and compound 48/80. Activations were observed at micromolar concentrations of cyclic AMP as substrate. In addition, T. cruzi calmodulin was also active in bringing about the stimulation of brain phosphodiesterase. PMID- 2999587 TI - The MR P-type transposable elements and the genetic activities of mutagens and carcinogens in Drosophila melanogaster. I. N,N-Dimethylnitrosamine (DMN). AB - A technique was developed for the assay of the genetic activities of carcinogens in both the soma and germ line in the course of early larval development and to assess the extent of their modification through the introduction into the genome of the MR IInd autosome P-type transposable elements. The influence of MR on genotoxicity for a given treatment was indicated by the relative frequencies of non-MR (Cy) to MR-carrying sibs emerging within the same cultures. The viable genetic changes in the soma were classified as recombinational or mutational events on the basis of the comparative yields of mosaic sectors in females heterozygous for the markers y w sn carried in standard order (XS) or multiply inverted (XIn) X-chromosomes. The results with this technique are here described for the carcinogen DMN. In the absence of MR, the topical application of DMN induced no larval lethality up to the highest tested doses (20 mM), but raised the yields of the somatic sectors for all the test markers in the emerging females in accordance with a linear dose fit. Comparison of the XS and XIn data indicated that somatic mutagenesis by DMN entailed the induction of recombinational and mutational events in roughly equal proportions. The introduction of MR into the genome, whether patro- or matroclinously, resulted in dramatic and proportionately equivalent enhancements in the activities of DMN, both with respect to the induction of larval lethality and somatic sectoring. These activities increased exponentially with dose, following a 4th-degree polynomial course up to 10 mM, when larval lethality approached 100%. At lower dose levels, the yields of all sector types in the viable females also followed comparable polynomial curves at different heights, except for y sn in the XIn series, where sector recovery remained at the control level throughout the examined dose range. Analysis of the sector size distribution for eye and bristle mosaicism in the XS control and DMN-treated series gave statistically comparable mean values, irrespective of the presence or absence of MR, indicating that DMN alone, or in conjunction with the P elements, did not alter the timing of aberrant clone initiation. In contrast, estimates of the genetic induction events in the somatic primordia with 5 mM DMN indicated greatly increased response in the presence of MR, which was in excess of 20-fold for the mutational changes involving the sn locus.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2999588 TI - Chromosomal aberrations induced by the restriction endonuclease Alu I in Chinese hamster ovary cells: influence of duration of treatment and potentiation by cytosine arabinoside. AB - Induction of chromosomal aberrations by the restriction endonuclease Alu I in Chinese hamster ovary cells (CHO) has been studied. Treatment of cell pellets with Alu I for a time as short as 1 min was found to induce significant increase in the frequency of chromosomal aberrations. Alu I was found to be effective both in trypsinized cells as well as in cells which were collected with a rubber policeman, indicating that trypsinization of cells is not a prerequisite for the entry of the enzyme into the cells. Treatment of cells with Alu I in the presence of 1-beta-D-arabinosylcytosine (ara C) led to an increase in the induced frequency of aberrations, most probably due to the inhibition of ligation of DNA strand breaks by ara C. PMID- 2999590 TI - Identification of the Echinococcus (hydatid disease) organisms using cloned DNA markers. AB - Cloned DNA fragments of the ribosomal RNA gene of Schistosoma mansoni hybridise strongly to Echinococcus DNA following restriction endonuclease and Southern transfer analysis. Individuals within a strain of E. granulosus exhibit identical patterns of hybridisation. However, the hybridisation patterns show significant differences between E. granulosus and E. multilocularis, and between the horse and sheep strains of E. granulosus. This technique represents a powerful, additional method for the identification and characterisation of new isolates of E. granulosus and E. multilocularis. PMID- 2999591 TI - Isolation of HTLV-III from cerebrospinal fluid and neural tissues of patients with neurologic syndromes related to the acquired immunodeficiency syndrome. AB - We conducted virus-isolation studies on 56 specimens from the nervous system of 45 patients in order to determine whether human T-cell lymphotropic virus Type III (HTLV-III) is directly involved in the pathogenesis of the neurologic disorders frequently encountered in the acquired immunodeficiency syndrome (AIDS) and the AIDS-related complex. We recovered HTLV-III from at least one specimen from 24 of 33 patients with AIDS-related neurologic syndromes. In one patient, HTLV-III was isolated from the cerebrospinal fluid during acute aseptic meningitis associated with HTLV-III seroconversion. HTLV-III was also isolated from cerebrospinal fluid from six of seven patients with AIDS or its related complex and unexplained chronic meningitis. In addition, of 16 patients with AIDS related dementia, 10 had positive cultures for HTLV-III in cerebrospinal fluid, brain tissue, or both. Furthermore, we cultured HTLV-III from the spinal cord of a patient with myelopathy and from the sural nerve of a patient with peripheral neuropathy. These findings suggest that HTLV-III is neurotropic, is capable of causing acute meningitis, is responsible for AIDS-related chronic meningitis and dementia, and may be the cause of the spinal-cord degeneration and peripheral neuropathy in AIDS and AIDS-related complex. PMID- 2999593 TI - Burkitt's and other non-Hodgkin's lymphomas in adults exposed to a visitor from Africa. PMID- 2999594 TI - HTLV-III, AIDS, and the brain. PMID- 2999592 TI - Intra-blood-brain-barrier synthesis of HTLV-III-specific IgG in patients with neurologic symptoms associated with AIDS or AIDS-related complex. AB - Intra-blood-brain-barrier production of virus-specific antibody is good evidence of infection within the blood-brain barrier. Patients with the acquired immuno deficiency syndrome (AIDS) have an increased incidence of neurologic abnormalities--i.e., unexplained, diffuse encephalopathy manifested clinically as chronic progressive dementia. To define the role of human T-cell lymphotropic virus Type III (HTLV-III), the etiologic agent of AIDS, in the pathogenesis of neurologic dysfunction, we compared cerebrospinal fluid and serum from patients with neurologic symptoms associated with AIDS and the AIDS-related complex for the presence of antibodies directed against HTLV-III. Antibodies directed against HTLV-III antigens were detected by four immunologic tests: a fixed-cell immunofluorescence assay, an enzyme-linked immunosorbent assay, immunoblots of viral lysates, and immunoprecipitation of cellular lysates. All patients were seropositive, and 22 of 23 (96 per cent) had HTLV-III-specific antibodies in their cerebrospinal fluid. Unique oligoclonal IgG bands were detected in the cerebrospinal fluid, and the rate of IgG synthesis within the blood-brain barrier was elevated. In eight of nine patients tested, the enzyme-linked immunosorbent assay showed that the percentage of HTLV-III-specific IgG in cerebrospinal fluid was higher than in serum, suggesting that HTLV-III infection of neurologic tissue occurs in the majority of patients with neurologic disease associated with AIDS or its related complex. PMID- 2999595 TI - Replication of Epstein-Barr virus within the epithelial cells of oral "hairy" leukoplakia, an AIDS-associated lesion. AB - We conducted a study to identify the viruses in tissue specimens of oral "hairy" leukoplakia, a lesion that is found in immunosuppressed male homosexuals and that is associated with the subsequent development of the acquired immunodeficiency syndrome. When stained for papillomavirus core antigen, 49 of 67 biopsy specimens (73 per cent) yielded positive results in epithelial-cell nuclei. Electron microscopy showed papillomavirus-like particles in all of 25 specimens, and the herpes-type virus described in a previous report was seen in 23 of the 25 specimens. Three specimens had both types of particle in the same individual epithelial cells. Immunofluorescence for herpes simplex virus, varicella-zoster virus, and cytomegalovirus gave negative results in all cases, but 19 of 20 specimens showed intense nuclear staining in epithelial cells for the viral capsid antigen of Epstein-Barr virus (EBV). DNA hybridization using EBV probes in Southern blots demonstrated EBV DNA in all of 13 specimens and found 200 or more viral DNA molecules per cellular genome in 11 of the 13. The whole EBV genome was also demonstrated in the specimens and found to be in linear virion form. We conclude that EBV replicates within the epithelial cells in hairy leukoplakia. PMID- 2999596 TI - The prospects for and pathways toward a vaccine for AIDS. PMID- 2999597 TI - Infrequency of isolation of HTLV-III virus from saliva in AIDS. PMID- 2999598 TI - Radon daughters and lung cancer. PMID- 2999599 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 52-1985. Transverse myelopathy and later development of multiple intracerebral lesions in a 64-year-old man. PMID- 2999600 TI - HTLV-III/LAV-antibody-positive soldiers in Berlin. PMID- 2999601 TI - Cough and wheeze caused by inhibitors of angiotensin-converting enzyme. PMID- 2999602 TI - Tobacco use, treatment strategies, and pharmacological adjuncts: an overview. PMID- 2999603 TI - Epstein-Barr virus. Dream or reality of a vaccine? PMID- 2999604 TI - Protection of cottontop tamarins against Epstein-Barr virus-induced malignant lymphoma by a prototype subunit vaccine. AB - Epstein-Barr (EB) virus is one of the five herpesviruses of man. Strong links between this agent and the chain of events causing two human cancers, endemic Burkitt's lymphoma and undifferentiated nasopharyngeal carcinoma, have long been evident (reviewed in ref. 1). Because of this, and because of the very high incidence of nasopharyngeal carcinoma in certain large populations, it was suggested in 1976 that a vaccine should be developed against EB virus to prevent infection and thereby reduce tumour incidence amongst those at risk. The virus determined membrane antigen (MA) was proposed as immunogen because it was known to elicit naturally occurring virus-neutralizing antibodies in man and because analogous antigens had been shown to act as effective experimental vaccines for preventing the herpesvirus-induced lymphomas of Marek's disease in chickens. Progress has been achieved in defining, quantifying and preparing MA molecules, and in enhancing their immunogenicity; a sensitive assay for antibodies to MA has been elaborated. Here we report that isolated cell membranes expressing MA, or purified MA glycoprotein of relative molecular mass (Mr) 340,000 (gp340), have been used to vaccinate cottontop tamarins (Saguinus oedipus oedipus), and that animals receiving either preparation were protected against the effects of a 100% tumour-inducing challenge dose of EB virus. PMID- 2999605 TI - A role for Ni in the hormonal stimulation of adenylate cyclase. AB - The best understood system for transduction of extracellular messages into intracellular signals is the hormone receptor-coupled adenylate cyclase. In such systems receptors are functionally coupled to the enzyme by two special proteins, termed the stimulatory and inhibitory guanine nucleotide regulatory proteins (Ns and Ni, respectively). These proteins, thought to mediate, respectively, stimulatory and inhibitory influences on the adenylate cyclase, are members of a larger class of heterotrimeric guanine nucleotide regulatory proteins involved in membrane signal transduction. We have studied the interactions of the various components of the adenylate cyclase system by co-reconstituting pure beta adrenergic receptors, pure Ns and Ni, and functionally resolved preparations of the catalyst in phospholipid vesicles. In the absence of Ni, beta-adrenergic receptor/Ns-mediated catecholamine stimulation of the enzyme is relatively modest (approximately 1.3-fold). Surprisingly, however, when Ni is also present, stimulation increases dramatically (up to 7-8-fold) because of a greater suppression of basal relative to agonist-stimulated enzyme activity. Thus, Ni may actually be required for maximal agonist stimulation as well as for inhibition of the adenylate cyclase. PMID- 2999606 TI - Regulation of the association of membrane skeletal protein 4.1 with glycophorin by a polyphosphoinositide. AB - Many of the physical properties of the erythrocyte membrane appear to depend on the membrane skeleton, which is attached to the membrane through associations with transmembrane proteins. A membrane skeletal protein, protein 4.1, is pivotal in the assembly of the membrane skeleton because of its ability to promote associations between spectrin and actin. Protein 4.1 also binds to the membrane through at least two sites: a high-affinity site on the glycophorins and a site of lower affinity associated with band 3 (ref. 11). The glycophorin-protein 4.1 association has been proposed to be involved in maintenance of cell shape. Here we show that the association between glycophorin and protein 4.1 is regulated by a polyphosphoinositide cofactor. This observation suggests a mechanism which may explain the recently reported dependence of red cell shape on the level of polyphosphoinositides in the membrane. PMID- 2999607 TI - Testing for cystic fibrosis. PMID- 2999608 TI - Topology of signal recognition particle receptor in endoplasmic reticulum membrane. AB - The signal recognition particle (SRP) receptor is an integral membrane protein of the endoplasmic reticulum which, in conjunction with SRP, ensures the correct targeting of nascent secretory proteins to this membrane system. From the complementary DNA sequence we have deduced the complete primary structure of the SRP receptor and established that its amino-terminal region is anchored in the membrane. The anchor fragment and the cytoplasmic fragment contribute jointly to a functionally important region which is highly charged and may function in nucleic acid binding. PMID- 2999609 TI - Bovine opsin has more than one signal sequence. AB - By deletion of selected segments from a bovine opsin complementary DNA clone and subsequent analysis of transcripts in a cell-free translation-translocation system, we have localized two out of four theoretically conceivable signal sequences required for the integration of opsin into microsomal membranes. PMID- 2999610 TI - Loss of genes on the short arm of chromosome 11 in bladder cancer. AB - Recent studies have shown that normal cellular sequences on chromosome 13 are lost during the development of retinoblastomas and that sequences on chromosome 11 are similarly lost during the development of Wilms' kidney tumours and embryonal tumours. Cells from these tumors have been found to contain either the paternal or maternal copies of loci on the affected chromosome, but not both. Thus, the somatic loss of heterozygosity for sequences on chromosome 13 or 11 is hypothesized to result in homozygosity for a recessive mutant allele on these chromosomes, and in this way the chromosomal loss may contribute to the development of these tumours. We sought to investigate whether similar losses of heterozygosity for chromosome 11 sequences occurred in a common adult tumour. We chose to analyse bladder cancers, since such cancers are common in the adult population and are derived from urogenital tissue, as are Wilms' tumours. We examined constitutional and tumour genotypes at loci on the short arm of chromosome 11 (11p) in 12 patients with transitional cell carcinomas. In five tumours, we observed the somatic loss of genes on 11p resulting in homozygosity or hemizygosity of the non-deleted alleles in the tumour cells. Our results show that the frequency of loss of 11p sequences in bladder cancer approaches that seen in Wilms' tumour (42% compared with 55%), and suggest that recessive genetic changes involving sequences on 11p may contribute to the development of bladder neoplasms. PMID- 2999611 TI - A polymorphic DNA marker linked to cystic fibrosis is located on chromosome 7. AB - Although cystic fibrosis (CF) is among the most common inherited diseases in Caucasian populations, the basic biochemical defect is not yet known. CF is inherited as an autosomal recessive trait apparently due to mutations in a single gene, whence the efforts made to identify the genetic locus responsible by linkage studies. Two markers have recently been identified that are genetically linked to CF: one is a genetic variation in serum level of activity of the enzyme paraoxonase, and the other is a restriction fragment length polymorphism (RFLP) identified with a randomly isolated DNA probe. We report here that the genetic locus DOCRI-917 defined by the cloned DNA probe is located on chromosome 7. PMID- 2999612 TI - Localization of cystic fibrosis locus to human chromosome 7cen-q22. AB - Cystic fibrosis (CF) is the most common genetic disease in Caucasian populations, with an incidence of 1 in 2,000 live births in the United Kingdom, and a carrier frequency of approximately 1 in 20. The biochemical basis of the disease is not known, although membrane transport phenomena associated with CF have been described recently. Consanguinity studies have shown that the inheritance of CF is consistent with it being a recessive defect caused by a mutation at a single autosomal locus. Eiberg et al. have reported a genetic linkage between the CF locus and a polymorphic locus controlling activity of the serum aryl esterase paraoxonase (PON). The chromosomal location of PON, however, is not known. Linkage to a DNA probe, DOCR1-917, was also recently found at a genetic distance of approximately 15 centimorgans (L.-C. Tsui and H. Donnis-Keller, personal communication), but no chromosomal localization was given. Here we report tight linkage between the CF locus and an anonymous DNA probe, pJ3.11, which has been assigned to chromosome 7cen-q22. PMID- 2999613 TI - Identification of the gene responsible for human T-cell leukaemia virus transcriptional regulation. AB - Human T-cell leukaemia viruses (HTLVs) have genomic organization distinct from that of other replication-competent retroviruses, possessing four genes, gag, pol, env and chi. The unique fourth gene, chi (also referred to as lor), is located between env and the 3' long terminal repeat (LTR), encoding a protein of relative molecular mass 40,000 for HTLV-I and 37,000 for HTLV-II, located in the nucleus of infected cells. HTLV-I is the causative agent of adult T-cell leukaemia (ATL), a T-lymphocyte malignancy, while HTLV-II has been found associated with a T-cell variant of hairy cell leukaemia. Both viruses immortalize T cells in vitro. However, the mechanism of cellular transformation induced by HTLV is not known as there seems to be no common site of provirus integration in primary ATL cells and the virus contains no classical oncogene sequences. These observations have provoked speculation that the unique and strongly conserved chi protein (85% amino-acid homology between HTLV-I and -II) is involved in HTLV leukaemogenesis. Recent mutagenesis experiments in our laboratory have shown that the chi gene is essential for HTLV replication. It has also has been shown that the LTRs of HTLV and the related bovine leukaemia virus (BLV) are activated in trans in virus-infected cells, and, although such experiments did not directly demonstrate a role for the chi protein in transcriptional activation, it has been suggested that the chi protein is responsible for the transcriptional activation of the LTR and may be involved in cellular transformation. We have now developed a transient co-transfection assay which demonstrates that transcriptional activation of the HTLV LTR is mediated solely by the chi protein and that no other virus genes are required. PMID- 2999614 TI - Dissociation of transforming and trans-activation functions for bovine papillomavirus type 1. AB - It has been shown that genetic information encoded by the 3' open reading frames (ORFs), E2, E3, E4 and E5, of bovine papillomavirus type 1 (BPV-1), is sufficient to induce cellular transformation of certain mouse cells. The product of the E2 ORF has further been shown to be responsible for the trans-activation of a transcriptional regulatory element located in the noncoding region (NCR) of the BPV-1 genome. To examine whether or not the E2 trans-activation function is encoded by the same gene that encodes the 3' ORF viral transformation function, we have now analysed the expression of the trans-activation function in series of mouse C127 cells transformed by BPV-1 deletion mutants. In addition, using mutated complementary DNA clones generated by the insertion of a premature translational termination linker into different sites of a BPV-1 cDNA clone containing the 3' ORFs intact, we demonstrate that transformation and transcriptional trans-activation functions can be dissociated and that they map respectively to the E5 and E2 ORFs. PMID- 2999615 TI - Effect of forskolin on platelet deaggregation and cyclic AMP generation. AB - The diterpene, forskolin, induced a partial deaggregation of ADP- or collagen aggregated human platelets in vitro. An increase in platelet cyclic AMP by forskolin was assumed to mediate the platelet deaggregation. PGE1 also deaggregated these platelets, and a combination of forskolin and PGE1 produced deaggregation greater than the maximum which could be obtained with each agent alone. A greater than additive effect was observed on the platelet cyclic AMP level in the presence of both forskolin and PGE1. No additive effect was observed in the phosphorylation of molecular weight (Mr) 21K polypeptide using forskolin (0.1 mmol/l) and PGE1 (5 mumol/l) suggesting that although cyclic AMP is responsible for the deaggregation process a mechanism other than phosphorylation through cyclic AMP-dependent protein kinase may be responsible for the effect of forskolin on platelet deaggregation. PMID- 2999616 TI - The affinity of (-)-propranolol for beta 1- and beta 2-adrenoceptors of human heart. Differential antagonism of the positive inotropic effects and adenylate cyclase stimulation by (-)-noradrenaline and (-)-adrenaline. AB - An appraisal of the affinity of (-)-propranolol was made for beta-adrenoceptors of isolated heart preparations and myocardial membrane particles from patients undergoing open heart surgery. In order to eliminate possible distorting influences of neuronal and extraneuronal uptakes of catecholamines on the affinity estimates for (-)-propranolol, isolated tissues were pretreated once with 5 or 10 mumol/l phenoxybenzamine for 2 h. Phenoxybenzamine caused potentiation of the positive inotropic effects of (-)-noradrenaline and (-) adrenaline but not of (-)-isoprenaline; potentiation was more pronounced in atrial than in ventricular preparations. Potentiation was greater for (-) noradrenaline than for (-)-adrenaline. It is concluded that the concentration of physiological catecholamines at the human heart beta-adrenoceptors is limited by neuronal capture but not by extraneuronal uptake. The antagonism of the positive inotropic effects of (-)-adrenaline and (-)-noradrenaline by (-)-propranolol was simple competitive in left ventricular myocardium of patients with mitral lesion. The effects of (-)-adrenaline and (-)-noradrenaline were antagonized to similar extent by (-)-propranolol. An equilibrium dissociation constant KB (-log mol/l) of 8.6 was estimated for (-)-propranolol. In atrial preparations the inotropic effects of (-)-adrenaline were antagonized significantly more by (-)-propranolol than those of (-)-noradrenaline. KB-Values (-log mol/l) of 8.9 [against (-) adrenaline] and 8.5 [against (-)-noradrenaline] were estimated for (-) propranolol. Concentration-effect curves for the stimulation of adenylate cyclase of both atrium and ventricle were biphasic for (-)-noradrenaline and monophasic for (-)-adrenaline. The high-sensitivity and low-sensitivity components of (-) noradrenaline comprised 1/3 and 2/3, respectively, of maximum cyclase stimulation. As expected from beta 1-adrenoceptors, the high-sensitivity component of the curve for (-)-noradrenaline was selectively antagonized by (-) bisoprolol; as expected from beta 2-adrenoceptors, the low-sensitivity component was selectively antagonized by ICI 118,551. (-)-Propranolol antagonized the effects of (-)-noradrenaline mediated by beta 2-adrenoceptors 2 to 3 times more potently than the effects mediated by beta 1-adrenoceptors. (-)-Propranolol competed with 3H-(-)-bupranolol for binding to left ventricular beta adrenoceptors. An equilibrium dissociation constant (-log mol/l) of 8.6 was estimated for (-)-propranolol.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2999617 TI - Uncoupling between beta-adrenoceptors and adenylate cyclase in dog ischemic myocardium. AB - We evaluated the effects of ischemic injury on the myocardial adenylate cyclase system, 5 h after ligation of the left anterior descending coronary in 5 anesthetized dogs. Crude cardiac membrane preparations were isolated from control and ischemic areas of ventricular myocardium and tested for: 1. L (125I)iodocyanopindolol binding, in the absence and presence of +/- -isoprenaline and GTP, and 2. adenylate cyclase activity. The density of beta-adrenoceptors increased by 35% in membranes from ischemic areas while the proportion of receptors in a high affinity state for +/- -isoprenaline decreased from 43% to 20%. Adenylate cyclase activities in the basal state and under stimulation with NaF, forskolin, Gpp(NH)p, +/- -isoprenaline and VIP were all markedly and similarly reduced, being only about 30% of comparable activities in membranes from control areas. The +/- -isoprenaline subsensitivity of cardiac adenylate cyclase can, thus, be attributed to a defective enzymatic system and not to a reduction in the number of beta-adrenoceptors implying that the internal components of the system were more sensitive to acute ischemia than the outward oriented hormone receptors. It is tempting to ascribe this uncoupling to a functional depletion in the guanine nucleotide-binding regulatory protein Ns that might reflect a loss of high energy phosphate stores including GTP. PMID- 2999619 TI - Isolated ACTH deficiency. PMID- 2999621 TI - [Consensus meeting on the diagnosis of solitary thyroid nodules]. PMID- 2999620 TI - [Solitary cold thyroid nodule; the value of various diagnostic methods checked against the histological findings after surgery: a retrospective study in 106 patients]. PMID- 2999622 TI - [LAV/HTLV-III infection after a one-time sexual contact with an AIDS patient]. PMID- 2999618 TI - Evidence for an A2 adenosine receptor in guinea pig lung. AB - Adenosine receptors in guinea pig lung were characterized by measurement of cyclic AMP formation and radioligand binding. 5'-N-Ethylcarboxamidoadenosine (NECA) increased cyclic AMP levels in lung slices about 4-fold over basal values with an EC50 of 0.32 mumol/l. N6-R-(-)-Phenylisopropyladenosine (R-PIA) was 5 fold less potent than NECA. 5'-N-Methylcarboxamidoadenosine (MECA) and 2 chloroadenosine had EC50-values of 0.29 and 2.6 mumol/l, whereas adenosine and inosine had no effect. The adenosine receptors in guinea pig lung can therefore be classified as A2 receptors. Several xanthine derivatives antagonized the NECA induced increase in cyclic AMP levels. 1,3-Diethyl-8-phenylxanthine (DPX; Ki 0.14 mumol/l) was the most potent analogue, followed by 8-phenyltheophylline (Ki 0.55 mumol/l), 3-isobutyl-1-methylxanthine (IBMX; Ki 2.9 mumol/l) and theophylline (Ki 8.1 mumol/l). In contrast, enprofylline (1 mmol/l) enhanced basal and NECA stimulated cyclic AMP formation. In addition, we attempted to characterize these receptors in binding studies with [3H] NECA. The KD for [3H]NECA was 0.25 mumol/l and the maximal number of binding sites was 12 pmol/mg protein. In competition experiments MECA (Ki 0.14 mumol/l) was the most potent inhibitor of [3H]NECA binding, followed by NECA (Ki 0.19 mumol/l) and 2-chloroadenosine (Ki 1.4 mumol/l). These results correlate well with the EC50-values for cyclic AMP formation in lung slices. However, the Ki-values of R-PIA and theophylline were 240 and 270 mumol/l, and DPX and 8-phenyltheophylline did not compete for [3H] NECA binding sites. Therefore, a complete characterization of A2 adenosine receptors by [3H]NECA binding was not achieved.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2999623 TI - [Miniature currents of the endplates of the muscle fibers of the diaphragm of the rat after inhibition of acetylcholinesterase with galanthamine]. AB - Miniature end-plate currents (MEPC) in rat diaphragm were studied with voltage clamp technique when synaptic acetylcholinesterase (AChE) was inhibited with different concentrations of galanthamine. The MEPC amplitude and time course were increased progressively with galanthamine concentrations in the range of 3.16 X 10(-8) - 10(-6) g/ml. The decay of MEPC was always exponential. The input resistance of muscle fibres increased. Galanthamine (10(-5) g/ml) produced a curare-like action: the amplitude and duration of MEPC were less as compared with those at galanthamine concentration 10(-6) g/ml, the decay of MEPC became biphasic. During washing out of the drug, the duration of MEPC began to increase and then to diminish, returning to the initial value 3 hours later. The decay of MEPC became exponential. A positive correlation was found between half-decay time and amplitude of MEPC both in the presence and in the absence of anticholinesterase. It is supposed that the functional role of synaptic AChE in limiting the postsynaptic effect of acetylcholine is not so significant as it is usually considered, therefore it is possible to use the parameters of MEPC for the estimation of functional AChE activity. PMID- 2999625 TI - [Multi-component synaptic potentials of rubrospinal neurons of the cat induced by corticofugal spike trains]. AB - Compound nature of EPSPs in rubrospinal neurons evoked by stimulation of the sensorimotor and associative parietal region of the cerebral cortex was shown in acute experiments on nembutalized cats by means of intracellular technique. Monosynaptic nature of the first two components of EPSPs evoked by corticofugal impulses propagating with an average velocity of 18.5 m/s and 7.5 m/s was revealed. These components are supposed to arise as a result of activation of slow conducting pyramidal and corticorubral neurons. In some rubrospinal neurons the first EPSP component evoked by corticofugal impulsation had a fast rising phase and reflected activation of axo-somatic synapses. The results are discussed in the light of the mechanisms of reorganization of cortical synaptic inputs to the red nucleus neurons. PMID- 2999624 TI - [Effect of vasopressin and oxytocin on evoked activity of dorsal horn cells of an isolated segment of spinal cord in rat pups]. AB - Effects of vasopressin and oxytocin on field potentials and action potentials of single cells in dorsal horn evoked by dorsal root stimulation were studied in isolated spinal cord segment of 2-3 weeks old rats. Both investigated neuropeptides depressed postsynaptic wave of the field potentials and evoked discharges of single cells. The participation of the hypothalamospinal neurohormonal system in modulation of sensory information in dorsal horn is discussed. PMID- 2999626 TI - [Analysis of the components of synaptic potentials induced by corticofugal spikes in the rubrospinal neurons of the cat]. AB - EPSPs of rubrospinal neurons evoked by stimulation of the sensorimotor cortex were studied in nembutalized cats by means of intracellular recordings. Participation of the corticospinal input in the genesis of EPSPs mentioned was revealed by selective activation of corticospinal fibres on the level of medullary pyramids as well as by studying peculiarities of their interaction with the effects of cortical stimulation. It was shown that both predominantly slow conducting corticospinal and corticorubral neurons participate in the genesis of the first two components of the complex EPSPs. Problems of cellular composition and mechanisms of corticofugal influences on the red nucleus neurons are discussed. PMID- 2999628 TI - The etiology of underlying liver lesions in 70 autopsied cases of hepatocellular carcinoma. AB - In 70 patients with hepatocellular carcinoma without history of antineoplastic chemotherapeutic drugs, anabolic and contraceptive steroids, representative sections of nonneoplastic liver tissue were examined for the presence of etiological markers. Hepatitis B surface antigen-positive hepatocytes were found in 16 (22.8%), alpha-1-antitrypsin globules in 3 (4.2%), Mallory bodies in 9 (12.8%), acicular inclusions in 1 (1.4%), diffuse giant mitochondria in 2 (2.8%), copper-binding protein in 25 (35.7%), greater amount of hemosiderin in 9 (12.8%) cases. Thorotrast was not detected. One or more markers were seen in 38 (54.3%) cases, most frequently in association with liver cell dysplasia and alcoholism. The presence of hepatitis B surface antigen in livers with dysplastic foci was highly significant as compared to organs showing no dysplasia. Only the mentioned antigen and the alpha-1-antitrypsin globules were found to indicate the etiology of the underlying liver lesion. The value of the other markers was found inconsistent in etiological diagnosis. PMID- 2999627 TI - No radiosensitivity-related change in plasma membrane properties in X- or gamma irradiated L5178Y-R and L5178Y-S cells. AB - Two strains of murine lymphoma cells, L5178Y-R (LY-R) and L5178Y-S (LY-S) differ in radiosensitivity (D0 ca 1 and 0.5, respectively), in Na+/K+ and Mg2+-ATPase activities and susceptibility to heat; their fatty acid composition is also slightly different. Nevertheless, neither Na+-dependent amino acid uptake, nor membrane fluidity change after X- or gamma-irradiation (10 Gy under aerobic conditions, 27 Gy under extreme hypoxia). Although in LY-S cells there is a decrease in partition coefficient in a two-phase system, which indicates a late (24 h after irradiation) change in surface charge, this change is not related to viability, membrane fluidity, amino acid transport and survival. PMID- 2999629 TI - [Serogenetic polyneuritis following enzyme nucleolysis]. PMID- 2999630 TI - The effects of oral furosemide on the response of urinary excretion of cyclic adenosine monophosphate and phosphate to parathyroid extract in normal subjects. AB - We studied the effects of oral furosemide, 80 mg/day for 7 days, on the response of urinary excretion of phosphate and cyclic AMP to exogenous parathyroid extract (PTE) in 6 normal subjects. All 6 subjects had marked increases in urinary calcium and a significant increase in urinary cyclic AMP from the control to the furosemide periods: this suggests that furosemide-induced hypercalciuria produced elevated parathyroid activity. After treatment with furosemide, the response of urinary cyclic AMP and phosphate to PTE was blunted. During the subsequent calcium infusion (4 mg/kg), urinary cyclic AMP was suppressed to subnormal values, and the response to PTE returned to normal. The evidence suggests that furosemide may blunt the response to PTE, perhaps as a result of the elevated parathyroid activity produced by furosemide-induced hypercalciuria and lowering of plasma-ionized calcium. This blunting effect of furosemide on the response of urinary phosphate and cyclic AMP to PTE should be considered in the evaluation of parathyroid function in patients taking furosemide. PMID- 2999632 TI - [ACNU delivery to malignant glioma tissue by osmotic blood brain barrier modification with intracarotid infusion of hyperosmoral mannitol]. AB - Drug delivery to the tumor has been one of the major subjects in the field of brain tumor chemotherapy because of blood brain barrier. Recent studies including quantitative autoradiographic studies revealed that blood brain barrier is present and intact in the brain adjacent to tumor where viable tumor cells are infiltrating, and also in the tumors which are early in the development. In 1972 Rapoport et al demonstrated that it is possible to transiently and reversibly open the blood brain barrier by an intracarotid infusion of a hyperosmoral solution. This technique is found to increase cerebrovascular permeability to chemotherapeutic agents. Six cases of glioma, including 4 astrocytoma grade 4, 1 astrocytoma grade 3, 1 astrocytoma grade 2, were treated during operation with intracarotid infusion of ACNU 100 mg/body/5 min. (1.3-2.2 mg/kg) following intracarotid infusion of 20% mannitol 200 ml (1.3-1.6 ml/sec) through the catheter in the internal carotid artery set preoperatively, and ACNU concentration in tumor tissues and blood were measured at 5, 10, 15, 20, 25, 30, 40, 60 minutes after that. On every case mannitol contrast enhancement CT was studied by the intracarotid infusion of 60% conray 100 ml/5 min. following the intracarotid infusion of 20% mannitol 200 ml comparing with contrast enhancement CT and plain CT. Maximum ACNU concentrations in blood were 2.12-4.12 micrograms/ml (mean 3.1 +/- 0.74) at 5 min. after the intraarterial administration of mannitol and ACNU on every case. At 20 min. following the administration ACNU levels were decreased to half level (mean 1.49 +/- 0.42 microgram/ml) and 0.58 +/- 0.18 microgram/ml at 60 min.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2999633 TI - Prenatal exposure of rats to antidepressant drugs down-regulates beta adrenoceptors and 5-HT2 receptors in cerebral cortex. Lack of correlation between 5-HT2 receptors and serotonin-mediated behaviour. AB - Changes in the binding of beta-adrenoceptors and 5-HT2 receptors and in behaviour mediated by serotonin were studied after either acute treatment or prenatal exposure of rats to antidepressant drugs. Chlorimipramine, iprindole and mianserin reduced the density of [3H]dihydroalprenolol-labelled beta adrenoceptors and of [3H]spiperone-labelled 5-HT2 receptors in 25-day-old rats after prenatal exposure to these drugs from gestational day 6 to delivery. Prenatal exposure to nomifensine reduced also the number of beta-adrenoceptors but, in contrast, the density of 5-HT2 receptors and the KD for binding of [3H]spiperone were markedly increased. Acute treatment of 25-day-old rats with the same antidepressants did not modify in any case the characteristics of binding to beta-adrenergic or 5-HT2 receptors. The behavioural syndrome induced by 5-hydroxytryptophan and clorgyline was only antagonized on acute treatment by mianserin. Chronic treatment in utero with the antidepressants produced varied effects on the serotonin syndrome and there was no correlation between the number of 5-HT2 receptors and the intensity of the behaviour mediated by serotonin. The observed changes in beta-adrenergic and 5-HT2 receptors after prenatal exposure to antidepressant drugs appear to be more marked and longer-lasting than those induced by chronic treatment of adult rats. PMID- 2999631 TI - Neurochemical studies on imprinting behavior in chick and duckling. AB - The effect of lesions in the parts of medial hyperstriatum ventrale (MHV) or lateral neostriatum (LN) on imprinting behavior was examined. Bilateral lesions in MHV severely impaired imprinting behavior in ducklings, but lesions in LN did not impair such behavior. On the other hand, the birds lesioned in MHV still had brightness discriminative learning ability with perfect performance of visuomotor coordination. The neurotransmitter candidates controlling imprinting behavior in MHV were investigated using blockers of neurotransmission. 6-Hydroxydopamine (6 OHDA) and haloperidole injected into MHV did not have specific effect on imprinting behavior although catecholamine contents in MHV was clearly decreased and locomotor activity was strongly suppressed. Atropine injection into MHV caused significant impairment of following behaviour, but locomotor activity was not affected. The injection of atropine into other region did not have any effect. alpha-Bungarotoxin showed no effect on either imprinting or locomotor activity. Kainic acid injection into MHV caused the decrease of glutamate content in the MHV region and it reached maximum at 24 hours after injection. However, imprinting behavior was already impaired at 3 hours after injection, but at 24 hours it had been recovered. A same effect was observed by glutamate injection. The effect of protein synthesis inhibitor on the acquisition process of imprinting was investigated. The acquisition of imprinting was significantly impaired by cycloheximide (CHX) injection into MHV within 2 hours after the first exposure to the imprinting stimulus. When CHX was given 24 hours after, imprinting behavior was not affected. PMID- 2999634 TI - The interaction between benzodiazepine antagonists and barbiturate-induced cerebrovascular and cerebral metabolic depression. AB - It has been reported that pentobarbital facilities binding to benzodiazepine receptors binding at anesthetic concentrations and that this action may play a role in the anesthetic potency of this barbiturate. The interaction between pentobarbital and benzodiazepine receptors was tested with Ro 15-1788 which is reported to be a pure benzodiazepine antagonist and 3-hydroxymethyl-beta carboline (3-HMC), an antagonist which has inverse activity alone. Cerebral blood flow (CBF) and cerebral oxygen consumption (CMRO2) were measured in rats after injections of pentobarbital with and without the antagonists. Pentobarbital produced dose-dependent decreases in cerebral blood flow and cerebral oxygen consumption at 15 and 30 mg/kg. The antagonist Ro 15-1788 (10 mg/kg) stimulated cerebral blood flow and cerebral oxygen consumption alone but did not alter the cerebral depression produced by pentobarbital. The cerebral metabolic stimulation produced by Ro 15-1788 was unexpected since the drug is reported to be a pure antagonist without agonistic activity, but the lack of effect on pentobarbital induced cerebral depression is consistent with other reports. 3-Hydroxymethyl beta-carboline at 10 mg/kg did not stimulate cerebral blood flow and cerebral oxygen consumption but significantly antagonized the decrease in cerebral oxygen consumption produced by 15 mg/kg pentobarbital. 3-Hydroxymethyl-beta-carboline had no significant effect on decreases in cerebral blood flow and cerebral oxygen consumption produced by phenobarbital, a barbiturate which is reported not to alter binding to benzodiazepine receptors. The ability of 3-HMC to antagonize the effects of pentobarbital would be consistent with an action of both drugs at the benzodiazepine receptor but not by altering binding to an endogenous receptor. PMID- 2999635 TI - Effects of gamma-vinyl-GABA on the human electroencephalogram. AB - Gamma-vinyl-GABA (GVG) is a new anticonvulsant drug that significantly raises the level of the gamma-aminobutyric acid in the brain (GABA). The effects of gamma vinyl-GABA on the human electroencephalogram were studied to assess the role of GABAergic mechanisms on electrocortical activity. Serial EEGs were recorded in 15 epileptic patients undergoing a controlled clinical trial of gamma-vinyl-GABA. The effects of gamma-vinyl-GABA on alpha, beta, or theta activity, sleep spindles and epileptiform activity were studied. No changes could be detected in any of the intrinsic brain rhythms. Three patients showed a mild amelioration of epileptiform activity; no increase in epileptiform activity was seen. PMID- 2999637 TI - Cerebrospinal fluid anion transport: studies of pertechnetate in unanesthetized sheep. AB - Perfusion of the ventriculocisternal system in unanesthetized sheep was used to quantify the pathways by which the pertechnetate anion (TcO4-) may leave the cerebrospinal fluid space. The largest fraction (about 89%) was removed from the fluid before it reached the cisterna magna; this extraction was significantly inhibited by iodide ion in the perfusion fluid. One-fifth of the extracted pertechnetate remained in the head of the animal for over 3 hours. Only about 3% of the infused pertechnetate was resorbed with the bulk flow of cerebrospinal fluid through the arachnoid villi. These results are consistent with the concept of a specific brain channel for removal of anions. As pertechnetate and iodide ion are thought to be transported by the same mechanisms, the physiological role of this channel may be maintenance of a very low iodide concentration in the extracellular fluid of the brain. PMID- 2999636 TI - Choroid plexus Na+/K+-activated adenosine triphosphatase and cerebrospinal fluid formation. AB - The quantitative relationship between intraventricular fluid formation and choroid plexus Na+/K+-activated (transport) adenosine triphosphatase (ATPase) was studied in rabbit and dog by perfusing the ventricular system with a solution containing ouabain (10(-8) to 10(-3) M). The effect of ouabain in the same range of concentrations on ATPase activity was also measured by the release of inorganic orthophosphate from in vitro choroid plexus tissue. Normally, about 20 to 25% of ATPase activity in lateral ventricle plexus is Na+/K+-activated, and this component is almost completely inhibited by ouabain in a concentration of 10(-4) M. At this concentration, the rate of intraventricular cerebrospinal fluid (CSF) formation is decreased some 70 to 80% in dog and rabbit. The results of this study suggest that a portion of the intraventricular fluid formation is insensitive to cardiac glycoside inhibition and that either there are two different mechanisms responsible for choroid plexus fluid formation or there is a significant non-ATPase dependent extrachoroidal source of CSF. PMID- 2999638 TI - Selective conventional arteriography and computed tomography with and without contrast infusion: minimal requirements for the assessment of transient ischemic attacks. AB - The authors report two unusual cases referred for extracranial bypass surgery. These patients presented with symptoms of transient ischemic attacks (TIAs) and demonstrated the necessity of conventional selective angiography and computed tomography (CT), with and without contrast infusion, as part of the preoperative assessment. The authors discuss how digital subtraction angiography alone is inadequate and how CT without contrast enhancement may fail to visualize lesions that may exhibit TIA-like symptoms. In the face of the multiplicity of preoperative tests used to evaluate patients with TIA symptoms, the authors stress the importance of selective conventional angiography and CT with and without contrast administration. PMID- 2999640 TI - Drug-induced field potential changes in dopaminergic target areas after electrical stimulation of the rat mesencephalon. AB - Changes of field potentials electrically evoked from the mesencephalon were studied in two dopaminergic target areas. The modulation of the responses in prefrontal cortex and striatum induced by intravenous injection of various compounds influencing central dopaminergic transmission is followed. In general, drugs with a down-regulating effect on dopaminergic impulse activity produced a significant diminution of the response, whereas compounds enhancing dopaminergic impulse flow produced an increase of the signal amplitude. The results are discussed in terms of the usefulness of the evoked potential as a screening procedure for monitoring dopaminergic impulse flow during the evaluation of new drugs against schizophrenia. PMID- 2999639 TI - Contrasting actions of naloxone in experimental spinal cord trauma and cerebral ischemia: a review. AB - Endorphins have been implicated in the pathophysiology of both spinal cord injury and cerebral ischemia. This review examines the nature of the experimental evidence to support this hypothesis. Present studies suggest that naloxone administration improves neurological function and outcome in the setting of the spinal cord trauma by centrally inhibiting an opiate receptor-mediated diminution of spinal cord flow. In the setting of spinal shock, naloxone administration is associated with improvement in vital sign and cardiovascular parameters as measured by mean arterial pressure, cardiac output, body temperature, and ventilation. Experiments using a variety of animal stroke models similarly support the notion that naloxone improves neurological function in the setting of cerebral ischemia by a stereospecific opiate receptor-mediated effect, but this improvement does not seem to be accompanied by augmentation of blood flow to affected areas of the brain or by any improvement in vital signs or cardiovascular parameters as seen in spinal cord trauma. A variety of mechanisms are discussed to explain these observations. The therapeutic implications of administering opiate agonists and antagonists in the setting of neurological deficits are outlined for the neurosurgeon. PMID- 2999641 TI - gamma-Aminobutyrate sensitivity does not change during long-term potentiation in rat hippocampal slices. AB - Long-term potentiation is a long-lasting enhancement of synaptic efficacy following brief, high-frequency, repetitive stimulation of a monosynaptic input. Intracellular recordings have shown that the inhibitory postsynaptic potential changes in amplitude during long-term potentiation. Yet how this may occur is unclear. To test for a possible alteration in postsynaptic sensitivity to the recurrent inhibitory transmitter gamma-aminobutyrate, we have examined the effect of gamma-aminobutyrate, focally applied to the hippocampal CA1 cell-body layer, on the extracellular recorded action potential (population spike). We found that the degree, duration, dose-dependence and time-course of inhibition produced by gamma-aminobutyrate are unchanged during long-term potentiation. This suggests that a change in sensitivity of CA1 pyramidal cells to the transmitter gamma aminobutyrate is not the reason for the alteration in the inhibitory postsynaptic potential during long-term potentiation. PMID- 2999642 TI - Immunocytochemical and electron microscopic study of serotonin neuronal organization in the dorsal raphe nucleus of the monkey. AB - Serotonin neurons in the dorsal raphe nucleus were identified using an antibody to a serotonin-bovine serum albumin conjugate and the peroxidase anti-peroxidase method. Nerve cell bodies showing serotonin-like immunoreactivity ranged in size from 15 to 22 micron in diameter; their dendrites were also immunoreactive. Immunostaining was present in the cytoplasmic matrix, outer membranes of mitochondria, rough endoplasmic reticulum, multivesicular bodies and dense-cored vesicles. Heavily immunoreactive axonal varicosities contained small round vesicles (18-35 nm) and larger dense-cored vesicles (50-90 nm). Both unmyelinated (0.2-0.5 micron) and myelinated (0.8-1.1 micron) serotonin-like immunoreactive axons were found, often interspersed within bundles of similar caliber unlabeled axons. Serotonin-like immunoreactive somata and dendrites were postsynaptic to numerous unlabeled terminals that contained either (a) clear round vesicles (18 25 nm) with many small dense-cored vesicles (30-50 nm), (b) clear round vesicles (18-25 nm) with large dense-cored vesicles (90-110 nm) or (c) clear round vesicles (18-25 nm) with or without flat vesicles. In addition pairs of unlabeled terminals formed crest synapses onto serotonin-like immunoreactive dendritic spines. This variety of unlabeled terminals making contact with serotonin-like immunoreactive elements suggests that several neuronal systems with possibly different transmitters may regulate serotonin raphe neurons. We occasionally observed serotonin-like immunoreactive dendrites and terminals in apposition to other serotonin-like immunoreactive dendrites with membrane specializations at the site of contact. This might represent a possible site for the self inhibition of serotoninergic neurons reported in physiological studies of the serotonin system in the dorsal raphe nucleus. PMID- 2999644 TI - [Chemotherapy of pulmonary carcinoma. Considerations and a proposal of a system of the evaluation of clinical course]. AB - With absolutely no attempt to reject chemotherapy of lung cancer as useless, particularly now that extremely effective antiblastic products are available, it is however suggested that we should think carefully before adopting treatments of doubtful efficacy that may prolong the patient's life but also increase his suffering in that they produce extremely unpleasant side effects without having much influence on the basic symptoms. In such cases it may be the physician's painful duty to withhold treatment. PMID- 2999643 TI - Effects of adenosine 5'-monophosphate and adenosine 5'-triphosphate on functionally identified units in the cat spinal dorsal horn. Evidence for a differential effect of adenosine 5'-triphosphate on nociceptive vs non nociceptive units. AB - A study was done of the effects of iontophoretic application of adenosine 5' monophosphate (AMP) and adenosine 5'-triphosphate (ATP) on functionally identified neurones in the spinal dorsal horn of the cat. AMP depressed nearly two-thirds of the 32 neurones tested regardless of functional type; the remainder were unaffected. ATP, on the other hand, had three types of effect: depression, excitation and a biphasic effect which consisted of excitation followed by depression. A significant difference was found when a comparison was made of the frequency of occurrence of each of these three types of effect in the samples of non-nociceptive (n = 18) and of wide dynamic range neurones (n = 42): of non nociceptive neurones 61% were excited, 11% were depressed, 6% had a biphasic response and 22% were unaffected; of wide dynamic range neurones 45% had a biphasic response, 19% were depressed, 14% were excited and 21% were unaffected (chi 2 = 16.2, P less than 0.005). The depressant effects of both AMP and ATP and the depressant phase of the biphasic effect of ATP seem to be mediated through activation of P1-purinergic receptors because these effects were blocked by theophylline, a P1-purinergic antagonist [Burnstock (1978) In Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach, pp. 107-118]. Thus the biphasic effect appears to consist of excitatory and depressant responses in the same neurone. The differential effects of ATP on non-nociceptive vs wide dynamic-range neurones are similar to the differential effects on these neurones observed during activation of low-threshold primary afferents. This similarity, together with evidence that ATP can be released from primary afferent neurones [Holton and Holton (1954) J. Physiol., Lond. 126, 124-140; Holton (1959) J. Physiol., Lond. 145, 494-504], prompts us to suggest that ATP may be a chemical mediator of effects of low-threshold primary afferent inputs in the spinal dorsal horn. PMID- 2999646 TI - [A rare case of Krukenberg's tumor. Echographic evaluation and surgical treatment]. PMID- 2999645 TI - [Severe arterial spasm and secondary thromboses of the limbs in chronic ergotism]. AB - A 40 year old woman who took a daily dose of 2-4 mg of Ergotamine Tartrate (Cafergot) regularly for 6 years to combat persistent migraine, was treated for a non-atherosclerotic arterial disease, severe arteriospasm of the great limb arteries, hyperplasia of the intima and segmental thrombosis. Binding of adrenergic alpha and beta receptors was investigated. Surprisingly it was found that the number of adrenergic beta receptors was significantly lower thant that of a healthy woman of the same age used as a control while the number of alpha receptors was not significantly different. This action of ergotamine on beta receptors could be explained by a dopamine-mimetic stimulation, due to the central nervous system, that could lead to the preferential regulation of beta receptors rather than alpha receptors, almost as a protective mechanism of alpha receptors. PMID- 2999647 TI - Immunosuppressive treatment of multiple sclerosis. PMID- 2999648 TI - Effects of noradrenaline on some potassium currents in CA1 neurones in rat hippocampal slices. AB - Pyramidal (CA1) cells in rat hippocampal slices were voltage clamped using a single electrode voltage clamp. In the presence of tetrodotoxin (TTX), depolarizing pulses from holding potentials of -60 to -70 mV elicited a slow inward calcium (Ca2+) current and two outward potassium (K+) currents: an A current and a slower, Ca2+-dependent K+ current. Noradrenaline (NA) (20 microM) depressed the amplitude of the K+ currents without affecting the Ca2+ current. The effect of NA could be blocked with propranolol and was mimicked by isoprenaline, suggesting that NA depresses the K+ currents by binding to beta receptors. PMID- 2999649 TI - Metabolic and structural correlates of the vibrissae representation in the thalamus of the adult rat. AB - Cytochrome oxidase (CO) histochemistry was used to examine patterns of metabolic activity in the ventral posteromedial nucleus of the adult rat thalamus. In sections cut in an oblique horizontal plane, CO staining reveals distinct patches of heightened activity arranged in a fashion remniscent of the pattern of vibrissae on the contralateral face and which corresponds to the known somatotopic organization of the nucleus. The CO-reactive zones coincide with oval cylinders of thalamic neurons that appear to be anatomically linked with corresponding barrels in the contralateral somatosensory cortex. PMID- 2999650 TI - Limbic, hypothalamic, cortical and spinal regions are enriched in receptors for thyrotropin-releasing hormone: evidence from [3H]ultrofilm autoradiography and correlation with central effects of the tripeptide in rat brain. AB - Light microscopic autoradiographic localization of specific recognition sites for thyrotropin-releasing hormone (TRH) was determined on thin sections of rat brain using the potent analogue [3H](3-Me-His2)-TRH ([3H]MeTRH). Microdensitometric analysis of the relative optical densities of TRH receptor labelling revealed the following brain regional enrichment: lateral and cortical amygdaloid nuclei greater than ventral dentate gyrus greater than n. accumbens greater than medial septum greater than piriform cortex greater than paraventricular thalamic and hypothalamic nuclei greater than preoptic area greater than diagonal band of Broca greater than lateral septum greater than I-IV layers of frontoparietal cortex greater than dorsal hippocampus greater than olfactory tubercle greater than caudate putamen; globus pallidus. In the spinal cord the apparent relative enrichment of TRH receptors was: substantia gelatinosa = central canal gray greater than ventral gray greater than dorsal gray (layers III-VII) greater than white matter. This heterogeneous distribution of TRH binding sites correlated well with our previous data obtained from membrane binding studies. Furthermore, the specific anatomical localization of receptors for TRH in many nuclei was consistent with those loci involved in the mediation of many physiological and behavioural actions of the peptide in rodent brain. PMID- 2999651 TI - Ascorbic acid does not modulate stimulated dopamine release: in vivo voltammetric data in the rat. AB - Electrical stimulation of the nigrostriatal pathway released dopamine (DA) in the striatum of the anaesthetized rat. The level of DA released by 10-s stimulus trains was measured by high-speed cyclic voltammetry. Metoclopramide (10 mg/kg) increased DA release by approximately 20%. Apomorphine (1.76 mg/kg) caused a approximately 40% decrease in release which was blocked by metoclopramide. Ascorbate (1.76 g/kg) had no effect on stimulated DA release. Furthermore, pretreatment of rats with ascorbate trebled the striatal extracellular ascorbate level, but failed to modify the effects of metoclopramide and apomorphine on DA release. We conclude that ascorbate has no effect on the presynaptic autoreceptors that modulate striatal DA release in vivo. PMID- 2999652 TI - Substantia nigra projection to medullary reticular formation: relevance to oculomotor and related motor functions in the cat. AB - Wheat germ lectin-horseradish peroxidase was injected into the medial medullary reticular formation in 4 cats. Retrogradely labeled cell bodies were observed in the ipsilateral substantia nigra pars reticulata and bilaterally in the superior colliculus, oculomotor nucleus and fastigial nucleus of the cerebellum. The nigral projection to this reticular formation area, which receives diffuse afferents from cortex, cerebellum, oculomotor system and vestibular nucleus and projects to spinal cord, may be of some relevance in understanding the nigral role in postural, head- and eye-movement control. PMID- 2999653 TI - Autoradiographic and quantitative study of benzodiazepine-binding sites in human hippocampus. AB - Benzodiazepine-binding sites were studied on frozen sections of 5 human hippocampi, using autoradiographic and biochemical techniques. The affinity, density, distribution and heterogeneity (two types) of sites were investigated using [3H]flunitrazepam as a ligand, clonazepam or C1 218872 as displacing agents. The autoradiographic images evidence a differential distribution of the binding sites in the histologic layers of the hippocampus. Subtypes I and II coexist in the same proportion in the three layers exhibiting the highest densities of binding sites (stratum granulosum and pyramidale, deep layer of stratum radiatum). The Kd, Bmax and Ki values found here are analogous to those described in animal studies, but the anatomical distribution of the sites in human hippocampus seems to differ slightly from that previously described in that of the rat. PMID- 2999654 TI - Neurohypophysial peptides depress cholinergic transmission in a mammalian sympathetic ganglion. AB - The actions of arginine-vasopressin (AVP) and oxytocin (OXT) were investigated in the rat superior cervical ganglion (SCG). At micromolar concentrations AVP decreased the amplitude of fast excitatory postsynaptic potentials (f-EPSPs) evoked by preganglionic stimulation and in many cells depolarized the postsynaptic membrane. Both effects were reversibly abolished by a potent vasopressor antagonist. The peptide decreased the frequency of spontaneous miniature EPSPs and the quantal content of the f-EPSPs without affecting the sensitivity of the ganglion cells to acetylcholine. OXT exerted the same effects as AVP but was less powerful. It was concluded that neurohypophysial peptides exert a dual pre- and post-synaptic action mediated by specific receptors. PMID- 2999655 TI - The role of hippocampus in memory: a hypothesis. AB - The role of the hippocampus in memory storage in the mammalian brain is examined. The intrinsic anatomical organization of the hippocampus is such that a multidimensional mapping of other brain regions is represented. Emerging knowledge of the cortico-limbic-subcortical anatomy suggests that the hippocampal representations preserve the topological features of the targets and possess reciprocal connectivity. Long-Term Potentiation (LTP) is a prominent physiological characteristic of hippocampal synapses and is a promising candidate mnemonic device. The hypothesis is advanced that the pattern, or index, of specific neocortical (and other) areas activated by an experiential event is represented, or indexed, in the hippocampus by means of LTP. This hypothesis, termed the Memory Indexing Theory, suggests that experiential events are initially stored in an index of neocortical locations maintained in hippocampus. Subsequently, other regions, notably neocortex itself, permanently encode these experiential events and the interrelationships between them. PMID- 2999656 TI - Strategies in neuropeptide receptor binding research. AB - Strategies and general approaches used in neuropeptide receptor binding assays are described. Special attention is given to the nature of the ligand, its physical and chemical stability and the demonstration of an appropriate ligand selectivity pattern. Examples are given to illustrate critical aspects of neuropeptide receptor binding assays. Strong correlation between binding and bioassay data is also stressed. PMID- 2999658 TI - Electron microscopic findings in faeces from children with gastroenteritis. AB - A total of 788 faecal samples from children in the Wellington region with symptoms of gastroenteritis were examined by immune electron microscopy. Virus was seen in 211 specimens; 87% of these were from infants less than three years of age. A viral diagnosis was made in 27% of cases consisting of rotaviruses (19%), adenoviruses (5%), and small round viruses (3%). Both rotaviruses and adenoviruses were most prevalent during the winter months, while the small round viruses peaked in summer and winter. The importance of fastidious adenoviruses is illustrated-of the 38 separate sightings only one was recovered in tissue culture. The value of the electron microscope as a rapid diagnostic tool in these cases is shown as it is often the only method of identification available. PMID- 2999659 TI - Adverse reactions to vaginal pessaries, enalpril and triazolam. PMID- 2999657 TI - New concepts in cocaine addiction: the dopamine depletion hypothesis. AB - Euphoric properties of cocaine lead to the development of chronic abuse, and appear to involve the acute activation of central DA neuronal systems. This is based upon known effects of cocaine on DA neurons, and the role played by DA in reward states and self-stimulation behavior. With chronic cocaine use, neurotransmitter and neuroendocrine alterations occur. DA depletion is hypothesized to result from overstimulation of these neurons and excessive synaptic metabolism of the neurotransmitter. DA depletion may underlie dysphoric aspects of cocaine abstinence, and cocaine urges. Neurochemical disruptions caused by cocaine are consistent with the concept of "physical" rather than "psychological" addiction. Possible pharmacological interventions in cocaine addiction are outlined and the psychological approach to these patients is discussed. PMID- 2999660 TI - Abdominal scintigraphy with 99Tcm-labelled red cells in the detection of rebleeding from peptic ulcers. AB - The technique of 99Tcm-labelled red cell scintigraphy as a means of detecting rebleeding was investigated in 33 patients with bleeding peptic ulcers. Scintigrams were performed twice during the 24 h period succeeding diagnostic endoscopy. There was scintigraphic evidence of rebleeding in 23 patients but this was clinically manifest in 14 patients only. Thirteen of the 14 patients with clinical rebleeding had positive scintigrams while only in one patient with clinical rebleeding was the scintigram negative (P less than 0.05). These results show that rebleeding is common and often clinically inapparent during the first 24 h following gastroscopy but that in the absence of scintigraphic rebleeding serious clinical rebleeding is unlikely to occur. PMID- 2999662 TI - Amenorrhea and endometrial atrophy. PMID- 2999661 TI - Persistence of endometrial activity after radiation therapy for cervical carcinoma. AB - Radiation therapy is a proved treatment for cervical carcinoma; however, it destroys ovarian function and has been thought to ablate the endometrium. Estrogen replacement therapy is often prescribed for patients with cervical carcinoma after radiation therapy. A review of records of six teaching hospitals revealed 16 patients who had endometrial sampling for uterine bleeding after standard radiation therapy for cervical carcinoma. Fifteen patients underwent dilatation and curettage, and one patient underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy when a dilatation and curettage was unsuccessful. Six patients had fibrosis and inflammation of the endometrial cavity, seven had proliferative endometrium, one had cystic hyperplasia, one had atypical adenomatous hyperplasia, and one had adenocarcinoma. Although the number of patients who have an active endometrium after radiation therapy for cervical carcinoma is not known, this report demonstrates that proliferative endometrium may persist, and these patients may develop endometrial hyperplasia or adenocarcinoma. Studies have indicated that patients with normal endometrial glands have an increased risk of developing endometrial adenocarcinoma if they are treated with unopposed estrogen. Patients who have had radiation therapy for cervical carcinoma should be treated with estrogen and a progestational agent to avoid endometrial stimulation from unopposed estrogen therapy. PMID- 2999663 TI - Hepatic metastases in gestational trophoblastic disease. AB - From 1975 to 1983, 195 patients were treated for persistent or metastatic gestational trophoblastic disease. Fifteen patients with liver metastases were analyzed. All were treated with chemotherapy alone, none received hepatic irradiation, and no patient bled from her hepatic metastases. Thus, the need for prophylactic hepatic irradiation to prevent hemorrhage is doubtful. The good results obtained in the studied patients emphasize the significance of using vigorous primary multiagent chemotherapy in high-risk gestational trophoblastic disease patients. PMID- 2999664 TI - Adenoid cystic carcinoma of Bartholin gland. AB - Five cases of adenoid cystic carcinoma of the Bartholin gland, a rare vulvar tumor, are reviewed with respect to clinical and pathological characteristics. Histologic transition from normal Bartholin gland to adenoid cystic carcinoma was evident in two cases. Two patients developed the tumor in association with pregnancy. Local recurrences are common and may precede distant metastases, pulmonary being the most common. Patients with repetitive local recurrence or pulmonary metastases may have slowly progressive disease and survive for many years. This is reflected in the disparity between the progression-free interval and survival curves. The recommended primary treatment is wide local excision, obtaining clear margins, and an ipsilateral inguinal lymphadenectomy. PMID- 2999665 TI - Human lymphoblastoid interferon (Wellferon) in primary therapy of two children with condylomata acuminata. AB - Human lymphoblastoid interferon (Wellferon) was administered parenterally to two prepubertal girls as primary therapy for genital condylomata acuminata. One child probably had contracted the disease as the result of sexual molestation. Both patients experienced minimal side effects during therapy and underwent complete remission within six weeks. These cases support use of interferon as primary therapy in young patients with extensive condylomata and in those who may have contracted the disease as the result of sexual abuse. PMID- 2999666 TI - Effect of vagus nerve on the action potential duration of ventricular cell of dog. I. Effect of stimulation of vagus nerve and atropine. PMID- 2999667 TI - [Follow-up patient care--consultation centers for cancer patients, why unpublicized?]. PMID- 2999668 TI - [Simultaneous determination of Ca 125 and D-dimer in plasma and ascites in ovarian cancer]. AB - In 37 patients with ovarian carcinoma stages Ic to IV the values of preoperatively determined tumour markers and the values of the follow-ups were examined in a trial to observe the course of the disease. The following sensitivity was recorded: Ca 125 92%, D-dimer 100%, TPA 76%, CEA 29%, CA 19-9 15%. Ca 125, D-dimer and TPA are unsuitable as follow-ups. They are also detectable in ascites. PMID- 2999669 TI - [Radiotherapy in primary radically operated ovarian cancer]. AB - The basis of primary treatment is consequent surgery. Radiation therapy is not necessary for borderline tumors. An effect has been seen in stages Ib to III with a small amount of post-operative residual tumor. A comparison with the value of alternative chemotherapy has not yet been made. Instillation of the radioisotopes is effective. The value of radiation therapy after successful chemotherapy of stages III/IV has not yet been investigated enough. The results of radiation therapy after surgical and chemotherapeutical failure are poor. PMID- 2999670 TI - Birth defects in three common pediatric malignancies; Wilms' tumor, neuroblastoma and Ewing's sarcoma. AB - During the period 1965-1980, 84 patients with Wilms' tumor, neuroblastoma and Ewing's sarcoma were treated at the University of Rochester Medical Center. All patients were evaluated for the presence of congenital abnormalities. Ten of 34 (29%) patients with Wilms' tumor, 3 of 32 (9%) patients with neuroblastoma, and 0 of 18 patients with Ewing's sarcoma were so effected. In the patients with Wilms' tumor, 5 children had more than one abnormality. In this group, types of defects included genitourinary in 10 patients, central nervous system in 3, and other structural defects in 7. In the patients with neuroblastoma, the abnormalities were dissimilar. As expected, most of the 13 patients with congenital malformations were detected as having defects prior to the diagnosis of malignancy. For the patients with Wilms' tumor, median age at diagnosis for the entire group and for the subgroup with defects was 3 years, with age ranges similar. Male to female ratios were 1.3:1 and 1:1, respectively. Seventy percent of each group survived more than 5 years. For the patients with neuroblastoma, median age (range) for the entire group was 16 months (0-12 years). The patients with defects had ages of 5 days, 1 month and 9 months at diagnosis. Male to female ratio for the entire group was 0.6:1 and survival was 63% at 3 years (range 3-23 years). Those with defects are alive 3-8 years from diagnosis. PMID- 2999671 TI - Treatment of vitreous hemorrhage in Rh-positive patients by intravitreal injection of anti-Rho-immunoglobulin. AB - 22 Rh-positive patients suffering from vitreous hemorrhage caused by injuries to the eye, hypertension, or associated with subarachnoid hemorrhage, were treated with anti-Rh-immunoglobulin by intravitreal injection. 8 patients (36.4%) needed only one immunoglobulin injection and rapidly regained their former visual acuity. In 2 cases with repeated hemorrhage the result of the treatment was not satisfactory. The incidence of adverse effects was low. PMID- 2999672 TI - Identification and partial purification of a nucleotide-stimulated protease in rat retina. AB - A nucleotide-stimulated protease of rat retina was partially purified by DEAE cellulose column chromatography. A 50-fold increase of an ATP-stimulated protease activity over a cytosol fraction was achieved by this chromatography. Maximum stimulation of protease activity by ATP was achieved at 2 mM. The pH optimum of the ATP-stimulated protease activity was between 7.5 and 8.5. 2 mM GTP stimulated the protease activity by 91%. 5 mM cyclic AMP stimulated the protease activity by 73% while 5 mM cyclic GMP stimulated the activity by 84%. The possible role of the nucleotide-stimulated protease in retina and potential involvement of the protease with inherited retinal dystrophy are discussed. PMID- 2999673 TI - Comparison of leukotrienes as conjunctival microvascular permeability factors. AB - The effect of leukotrienes (LT) B4, C4, D4 and E4 on the microvascular permeability of the palpebral conjunctiva was investigated in the hamster. Microvascular permeability was quantitatively measured as extravascular albumin content by a technique which employs radiolabeled serum albumin and red blood cells. The natural 5S, 6R-LTD4 stereoisomer approached 1,000 times the potency of 5R, 6S-LTD4, indicating a stereoselective response to LTD4. 5S, 6R-LTD4 was approximately 100 times more active than LTC4, and LTE4 appeared no more active than 5R, 6S-LTD4. LTB4 was essentially inactive over a dose-range of 5-500 ng. These results indicate that the conjunctival microvasculature may vary markedly in terms of vasopermeability response to different LTs. PMID- 2999674 TI - Corneal alpha-galactosidase deficiency in macular corneal dystrophy. AB - Glycosidases, which cleave sugar molecules from complex glycopolymers, have been previously quantified in normal human cornea in our laboratory. Data quantifying glycosidases in macular corneal dystrophy are lacking. Tissue obtained at keratoplasty from patients with macular dystrophy and normal corneas obtained from eye bank eyes were used to determine levels of glycosidase activity. A fluorometric technique was employed using 4-methyl-umbelliferyl-glycosides as substrates. The corneal tissues were homogenized, centrifuged, and the supernatants assayed for enzyme activity. Specific activities (mumol/mg protein/hour) were determined and Km and Vmax values were obtained for all but one enzyme. Activity of alpha-galactosidase was significantly lower in cornea tissue and keratocytes from macular corneal dystrophy compared to normal. PMID- 2999675 TI - Magnetic resonance scanning in orbital tumor diagnosis. AB - We have compared the accuracy of magnetic resonance scanning (MRI) versus computed tomography (CT) in the differentiation of lateral orbital masses. The MRI results did not improve our ability to accurately diagnose malignant epithelial and lymphoid tumors. PMID- 2999677 TI - Incentives for planned patient education. PMID- 2999676 TI - Microwave sterilization of nitrous oxide nasal hoods contaminated with virus. AB - Although there exists a desire to eliminate the possibility of cross-infection from microbial contaminated nitrous oxide nasal hoods, effective and practical methods of sterilization in a dental office are unsatisfactory. Microwaves have been used to sterilize certain contaminated dental instruments without damage. In this study nasal hoods contaminated with rhinovirus, parainfluenza virus, adenovirus, and herpes simplex virus were sterilized in a modified microwave oven. Ninety-five percent of the virus activity was destroyed after 1 minute of exposure of the contaminated nasal hoods to microwaves. By the end of 4 minutes, complete inactivation of all four viruses was found. Repeated exposure of the nasal hoods to microwaves resulted in no damage to their texture and flexibility. Microwave sterilization may potentially provide a simple and practical method of sterilizing nitrous oxide anesthesia equipment in a dental or medical practice. PMID- 2999678 TI - [Glomus tumors of the fingers and hand (Barre-Masson disease)]. PMID- 2999679 TI - [Synacthen depot-induced liver damage in the therapy of secondary generalized epilepsies in childhood]. PMID- 2999680 TI - The effect of splenectomy on resistance of mice to Entamoeba histolytica infection. AB - The role of the spleen in amoebic infection was examined in mice, using strains selected as being either genetically-susceptible (C57BL/6) or genetically resistant (A/J) to amoebiasis. Splenectomized and sham-operated animals were inoculated intracaecally with 2.5 X 10(5) polyxenic trophozoites of E. histolytica at 6, 12 and 15 days post-splenectomy. The animals were killed 6 or 12 days after infection and the parasite burden was evaluated. Removal of the spleen in both susceptible and resistant mouse strains rendered these hosts extremely resistant to amoebic infection by this criterion. Gross examination of the caeca of non-splenectomized, genetically-susceptible mice showed numerous ulcers over the mucosal surface when compared to the splenectomized group which had superficial lesions or none. These observations suggest that the spleen plays a suppressive role in early anti-amoebic resistance. PMID- 2999681 TI - Endocrine responses of protein-malnourished rats infected with Nippostrongylus brasiliensis (Nematoda). AB - During the course of a primary infection of Nippostrongylus brasiliensis in protein-malnourished rats, plasma concentrations of corticosterone were found to decrease from 0.89 +/- 0.03 at the start of the infection to 0.28 +/- 0.08 mumol/l, 9 days post-infection (p.i.). Similarly, adreno-corticotrophic hormone levels were also observed to fall from 62.09 +/- 12.06 to 20.54 +/- 1.81 by 5 days p.i. and to 35.12 +/- 16.61 nmol/l by 9 days p.i. However, no significant changes were seen in the dry weight of the adrenal glands other than those expected in relation to changes in body weight. Concentrations of both plasma tri iodothyronine and thyroxine were also severely depleted over the same period from 5.33 +/- 0.34 to 1.09 +/- 0.42 and 104.43 +/- 10.4 to 34.12 +/- 8.95 nmol/l respectively. This was considered not to be due to any change in the capacity of the plasma to bind these hormones. Results for insulin were highly variable, but overall were lower from protein-malnourished rats than results documented for well-nourished rats and a reduction from 18.07 +/- 3.17 to 6.08 +/- 2.18 mu units/ml, 9 days p.i. was observed. The results are discussed in relation to the role of these hormones in protein metabolism in a protein-malnourished animal. PMID- 2999682 TI - Detection of Ross River virus immunoglobulin M antibodies by enzyme-linked immunosorbent assay using antibody class capture and comparison with other methods. AB - An enzyme-linked immunosorbent assay based on antibody class capture was developed for the detection of Ross River virus-specific immunoglobulin M antibodies (RRV IgM). The assay was specific, reproducible and precise. When compared with conventional tests for the detection of RRV IgM, such as hemagglutination inhibition following sucrose density gradient centrifugation and indirect enzyme-linked immunosorbent assay, the class capture assay was more sensitive. In 186 sera which were collected from 39 patients with RRV infection over a period of 1-4 yr from onset of initial symptoms, RRV IgM persisted for at least 1-2 yr. Sera were tested both at a single dilution from which the results were expressed as a binding index and in a dilution series in which they were expressed as an antibody titre. Binding index values gave better discrimination between sera collected during acute and later phases of the disease and may be of greater value than antibody titres in the diagnosis of RRV infection. PMID- 2999683 TI - [Role of the cAMP/cGMP ratio in the post-stress activation of the primary immune response]. PMID- 2999684 TI - Diagnostic imaging of pediatric abdominal masses. AB - This overview delineates the clinical and pathological features of various abdominal mass lesions found in neonates and in older infants and children. The application and limitations of imaging procedures currently available are reviewed with emphasis on the principles and advantages of advanced imaging techniques. Selected pediatric abdominal masses are discussed and their radiologic features illustrated. Integrated imaging strategies for abdominal masses in the newborn and in the older infant and child are proposed. PMID- 2999685 TI - Estrogen stimulation of surfactant synthesis. AB - Administration of 17 beta-estradiol to pregnant rabbits accelerates fetal lung maturation and stimulates surfactant production: the hormone increases the amount of surfactant in fetal lung lavage, increases the rate of phosphatidylcholine synthesis, depletes fetal lung glycogen, and accelerates morphologic maturation of the fetal lung. Both estrogens and glucocorticoids stimulate fetal lung cholinephosphate cytidylyltransferase in a number of in vivo and in vitro systems and there is increasing evidence that this enzyme may be of particular importance in the regulation of phosphatidylcholine synthesis. Estrogen appears to increase the catalytic activity rather than the amount of cholinephosphate cytidylyltransferase. This action of estrogen is mediated by phospholipids. PMID- 2999686 TI - Role of cyclic adenosine monophosphate in rat lung development. AB - The objective of this study was to identify the biochemical mechanisms concerned with pulmonary growth and development. The data show that cyclic adenosine monophosphate (cAMP), adenylate cyclase, cAMP phosphodiesterase, and their regulation by intracellular modulators are important to the development of rat lungs. The presence in rat lung cytoplasm of factors modulating adenylate cyclase activity is described. These factors appear to be important physiologically as they are present in vivo, they appear in the cytoplasm at a specific age, and their activity is altered by diabetes and adrenalectomy and restored to original levels by administration of insulin and dexamethasone, respectively. The cytoplasmic activation of adenylate cyclase appears to be due to multiple proteins that can be resolved into less active components by DEAE-cellulose chromatography. Recombination of these proteins not only restored activity to the original level but actually resulted in more than additive activation, indicating some interdependence and positive cooperativity among the different components to maximally stimulate adenylate cyclase activity. The rat lung cytoplasmic activator protein regulates adenylate cyclase by a mechanism different from those reported for epinephrine, NaF, 5'-guanylimidophosphate, and calmodulin. PMID- 2999688 TI - Somatomedin in fetal growth. AB - The somatomedins comprise a family of insulin-like peptide growth factors, whose concentrations in postnatal life are growth hormone-dependent, that stimulate sulfate uptake by cartilage and that stimulate the proliferation of a variety of types of cultured cells. Several lines of evidence now support the concept that the somatomedins stimulate fetal growth: 1) they act as mitogens for a number of embryonic and fetal derived cells grown in culture; 2) specific plasma membrane receptors for somatomedins exist in many fetal tissues; 3) multiple fetal tissues appear to be capable of somatomedin synthesis; 4) in some studies blood somatomedin concentrations correlate with birth size. It is proposed that the somatomedins might act at or near their sites of synthesis, and that under the influence of placental lactogen, as well as other factors, they have a significant stimulatory effect on both organ-specific and generalized somatic fetal growth. A hypothesis that integrates the possible growth-promoting actions of both classic hormones and growth factors in the fetus is presented. PMID- 2999689 TI - Microsomal C-25 hydroxylation of [3H]-vitamin D3 by the fetal and neonatal rat liver. AB - The liver microsomal C-25 hydroxylation of [3H]-vitamin D3 was evaluated in 19- and 22-day-old rat fetuses, and in 1-, 2-, 3-, 14-, 30-, and 60-day-old pups. The hepatic production of [3H]-25-hydroxyvitamin D3 by the 19- and 22-day-old fetuses was evaluated at 2.3 +/- 0.6 and 5.9 +/- 0.6 fmol . min-1 . 15 mg-1 microsomal protein, respectively. Values stayed unchanged during the 1st day after birth but increased on days 2 and 3 of chronologic age. Thereafter, the activity remained at a plateau of 11.2 +/- 0.6 fmol . min-1 . 15 mg-1 microsomal protein with no further statistically significant increase in the activity of the vitamin D3-25 hydroxylase through 60 days of chronologic age. The microsomal cytochrome P-450 specific content increased during the perinatal period with values ranging from 0.15 nmol . mg protein-1 in 22-day-old fetuses to 0.44 in 60-day-old rats; the developmental pattern of the cytochrome P-450 was similar to that observed for the vitamin D3-25 hydroxylase activity. When the amount of [3H]-25-hydroxyvitamin D3 formed was expressed in relation to the amount of enzyme present in the reaction medium, a constant C-25 hydroxylation capacity in all age groups was observed suggesting that the cytochrome P-450 isoenzyme responsible for the C-25 hydroxylation of vitamin D3 may be constitutionally determined and that its appearance originates during fetal life. PMID- 2999687 TI - Fetal pulmonary beta-adrenergic receptors: characterization in the human and in vitro modulation by glucocorticoids in the rabbit. AB - At least two developmental responses necessary to prepare the fetal lung to serve as a gas-exchange organ, the release of surface-active material and the reabsorption of alveolar water, can be stimulated by beta-adrenergic agonists. The sensitivity of these responses increases dramatically in late gestation. beta Adrenergic receptors can be identified by radioligand binding and are present in human fetal lung as early as 16 weeks of gestation. The temporal relationship of the increases in both pulmonary beta-receptors and plasma-free cortisol in the fetal rabbit during gestation suggests that endogenous glucocorticoids may cause increased concentration of pulmonary beta-receptors. Treatment of pregnant rabbits at 24 or 25 days of gestation results in precocious increases in both fetal lung beta-receptors and agonist-specific, high-affinity binding. The increase in receptor concentration with glucocorticoid is not dependent on other endocrine response inasmuch as 0.1 microM dexamethasone increases beta-receptor concentrations at 24 and 48 hours of incubation in cultures of fetal rabbit lung organ. This effect of glucocorticoid to increase beta-receptor concentration and high-affinity binding may explain the increased fetal pulmonary beta-adrenergic response at term and may be responsible in part for the reduction in neonatal respiratory syndrome seen after antenatal glucocorticoid therapy. PMID- 2999690 TI - Spermatic cord torsion: diagnostic limitations. AB - To distinguish spermatic cord torsion from other intrascrotal pathology, scrotal ultrasound and radionuclide scanning have been highly recommended on the basis of both clinical and experimental studies. We review the data from six patients in whom ultrasound or nuclear medicine examination was misleading. We emphasize that history, physical examination, and urinalysis remain the cornerstones of the diagnosis of spermatic cord torsion. Scrotal ultrasound and nuclear medicine scans are useful adjuncts and are reassuring when in agreement with the clinical picture. However, they are not 100% sensitive or specific, and a negative study should not prevent emergency operative exploration of a clinically suspicious lesion. PMID- 2999691 TI - Progression of neurovisceral storage disease with supranuclear ophthalmoplegia following orthotopic liver transplantation. AB - A 7-year-old girl with progressive ataxia, spasticity, supranuclear ophthalmoplegia, and sea-blue histiocytes in her bone marrow underwent orthotopic liver transplantation for hepatocellular carcinoma. After an initial period of stabilization, she has shown progression of neurologic symptoms with recurrence of storage material in the transplanted liver. PMID- 2999692 TI - [Mechanism of action of euphylline in atopic bronchial asthma in children]. PMID- 2999693 TI - [Correlation of myeloperoxidase and alkaline phosphatase activity of the blood neutrophils in respiratory diseases in children]. PMID- 2999694 TI - Influence of chronic ADH treatment on adenylate cyclase and ATPase activity in distal nephron segments of diabetes insipidus Brattleboro rats. AB - The medullary thick ascending limb (MAL), but not the medullary collecting tubule (MCT), has been shown to have an impaired adenylate cyclase (AC) responsiveness to ADH and a selective hypoplasia in Brattleboro diabetes insipidus (DI) rats. Since chronic ADH administration has been found to increase epithelium volume and basolateral membrane surface area in MAL but not in MCT, we investigated whether chronic ADH infusion would affect the hormone-sensitive AC and the Na-K-ATPase activity--two markers of the basolateral membrane--in single micro-dissected portions of thick ascending limb and collecting tubule in DI rats. Results indicate that 1. in MAL of ADH-treated rats, AC responses to in vitro AVP and glucagon and Na-K-ATPase activity increased to the same extent as did epithelium volume (60-80%); 2. changes in the other segments were independent of any morphological alteration. In the cortical thick ascending limb, AVP and glucagon sensitive AC decreased by 30-40% whereas Na-K-ATPase activity did not change. In the collecting tubule, AC response to in vitro AVP was not altered by ADH treatment but glucagon-sensitive AC dropped by 50% and Na-K-ATPase activity doubled, independently of any variation in plasma aldosterone and glucagon levels. These results show that, in the MAL, the ADH-induced variations in enzyme activity are a reflection of the enlargement of the basolateral membrane surface area. Further studies are needed to clarify the origin of enzymatic alterations in the other segments. PMID- 2999695 TI - [Local effect of radiation therapy for adenocarcinoma of the cardia--histological analysis of the preoperatively irradiated cases]. PMID- 2999696 TI - T4-induced alpha- and beta-glucosyltransferase: cloning of the genes and a comparison of their products based on sequencing data. AB - Bacteriophage T4 alpha- and beta-glucosyltransferases link glucosyl units to the 5-HMdC residues of its DNA. The monoglucosyl group in alpha-linkage predominates over the one in beta linkage. Having recently reported on the nucleotide sequence of gene alpha gt (1) we now determined the nucleotide sequence of gene beta gt. The genes were each cloned on a high expression vector under the control of the lambda pL promoter. After thermo-induction the proteins were isolated and purified to homogeneity. To verify that the translational starting sites and the proposed reading frames are effective in vivo the sequence of the first 31 amino acid residues from gp alpha gt and the first 30 amino acid residues from gp beta gt were determined by Edman degradation. The primary structures of the two proteins seem to have only limited structural similarities. The results are discussed comparing secondary structure predictions and homologies with other proteins from the protein sequence database of the Protein Identification Resource. PMID- 2999697 TI - Direct cloning of specific genomic DNA sequences in plasmid libraries following fragment enrichment. AB - We describe a simple method to directly clone any DNA fragment for which a flanking restriction enzyme map is known. Genomic DNA is digested with multiple enzymes cutting outside the fragment to be cloned, selected by electroelution from an agarose gel, and cloned directly into a plasmid vector. It is only necessary to screen 10-1000 colonies and recombinant DNA is ready for immediate molecular analysis without further subcloning. The use of this technique is demonstrated for the cloning of a sequence from within the human alpha-globin complex that was previously shown to be "unclonable" in bacteriophage and cosmid vectors and which is a multiallelic general genetic marker, as well as both beta globin alleles from an individual with beta-thalassaemia. PMID- 2999698 TI - Molecular cloning and structure of the human interleukin 2 receptor gene. AB - We have cloned the IL-2 receptor gene from human genomic DNA libraries using IL-2 receptor cDNA as probe. The genomic DNA segments that hybridized with cDNA were subcloned in M13 phages and their sequences were determined. The nucleotide sequences showed that the IL-2 receptor gene was encoded by eight exons and that the coding region sequences agreed completely with that of the IL-2 receptor cDNA cloned from a cell line derived from adult T cell leukemia (ATL), in which IL-2 receptors are expressed abnormally. The nucleotide sequence of the 5'-flanking region had a putative promotor region, which had some homology with the human IL 2 gene. Transcription initiation sites were clustered about 25 bp 3' to the TATA box as assessed by primer extension analysis. These sites for normal and ATL T cells were the same. Exons 2 and 4 encoding the extracytoplasmic portion had significant homology, suggesting that the two exons are derived by duplication of an ancestral exon. Exon 2 contained six cysteine residues, four of which are conserved at the corresponding positions in exon 4. PMID- 2999699 TI - The nucleotide sequence of the nitrogen regulation gene ntrB and the glnA-ntrBC intergenic region of Klebsiella pneumoniae. AB - The nucleotide sequence of the Klebsiella pneumoniae ntrB gene and the glnA-ntrBC intergenic region has been determined. NtrB encodes a 38,409 Dalton polypeptide with a potential DNA-binding domain between residues 67 and 86. This N-terminal domain may play a role in the co-operative control of ntr-regulated promoters by the ntrB and ntrC products. Mapping of in vivo transcripts with S1 nuclease identified three transcripts in the glnA-ntrBC intergenic region. Two transcripts originate upstream of glnA; one reading through into ntrBC and one terminating at a sequence resembling a rho-independent terminator between glnA and ntrBC. A third transcript originates from the ntrBC promoter which has a consensus binding site for the ntrC product in the -10 region. Comparison of the glnA-ntrBC intergenic sequences from K. pneumoniae, Escherichia coli and Salmonella typhimurium has identified a number of conserved features and some significant differences. PMID- 2999701 TI - Promoter selectivity of E. coli RNA polymerase: analysis of the promoter system of convergently-transcribed dnaQ-rnh genes. AB - Promoter properties were analyzed for the convergently-overlapped E. coli genes coding for the DNA polymerase III epsilon subunit (dnaQ) and the ribonuclease H (rnh). The rates of open complex formation for a single promoter of the rnh gene and two tandem promoters of the dnaQ gene were constant whether they are located on a single DNA fragment or separated into individual fragments. The relative expression levels of these three promoters, as measured using an in vitro mixed transcription system, varied differentially depending on the concentration of RNA polymerase. At low enzyme concentrations, the downstream promoter (P2) of the dnaQ gene was utilized preferentially, but the upstream promoter (P1) was utilized as well when the enzyme concentration was increased. This indicates different physiological roles between the two dnaQ promoters. The level of rnh transcription was as low as that of dnaQ-1 RNA synthesis but the rnh promoter was utilized as well as the dnaQ P2 promoter when it was separated from the dnaQ promoters. This implies a promoter interference between the convergently transcribed genes. PMID- 2999700 TI - The nucleotide sequence of the nitrogen-regulation gene ntrA of Klebsiella pneumoniae and comparison with conserved features in bacterial RNA polymerase sigma factors. AB - The nucleotide sequence of the Klebsiella pneumoniae ntrA gene has been determined. NtrA encodes a 53,926 Dalton acidic polypeptide; a calculated molecular weight which is significantly lower than that determined by SDS polyacrylamide gel analysis. NtrA is followed by another open-reading frame (orf) of at least 75 amino acids. In the spacer region between ntrA and orf there are no apparent transcription termination or promoter sequences and therefore orf may be co-transcribed with ntrA. Previous authors have proposed that NtrA could act as an RNA polymerase sigma factor but the NtrA amino acid sequence does not show a high level of homology to any known sigma factor. However analysis of sequences of five sigma factors from E. coli and B. subtilis has identified two conserved sequences at the C-terminal end of all these polypeptides. These sequences resemble those found in known site-specific DNA-binding domains and may be involved in recognition of conserved -35 and -10 promoter sequences. A similar pair of sequences is present at the C-terminus of NtrA and could play a role in recognition of ntr-activatable promoters. PMID- 2999702 TI - Localisation and characterization of a new rho-dependent transcription terminator from bacteriophage T5. AB - Relatively few rho-dependent terminators have been described in the literature. This manuscript describes another such terminator, isolated from phage T5. Functional analysis, involving the generation of deletion subclones, has permitted the localization of the terminator on a 413 bp fragment. Attempts to further reduce the size of this fragment resulted in loss of terminator activity. DNA sequence analysis of the terminator region supports the model whereby a rho dependent terminator is composed of a long region of non-translated unstructured DNA, which permits rho binding, followed by RNA polymerase pausing sites where termination (in the presence of rho) may occur. The results agree with the currently held hypothesis that, despite the many similarities found between various rho dependent termination sequences, no consensus can be defined for either the rho binding or the rho termination sites (1,2). PMID- 2999703 TI - Correct removal by splicing of a Neurospora intron in yeast. AB - Processing of intron-containing nuclear messenger RNAs in yeast require an internal conserved sequence (ICS) element, UACUAAC. Similar elements (ugCUAGAC) have been identified in sequences interrupting nuclear genes of the related ascomycete Neurospora crassa. To examine the structural splicing requirements in yeast, we constructed hybrid genes containing the intron of the Neurospora histone H3 gene and cloned them into high copy number yeast vectors. Subsequently we analyzed the RNAs transcribed in yeast from the fusion genes by Northern analysis and primer extended sequencing. It turned out that the Neurospora intron, which contains the sequence element UGCUAAC, can be removed, though very inefficiently, provided that it is located near the 5'-end of the primary transcript. This proves that an A at the second position of the ICS is no absolute requirement for splicing in yeast. In addition, the results indicate that the yeast splicing machinery is intron-position dependent. PMID- 2999704 TI - DNA sequences regulating human beta globin gene expression. AB - Human delta globin is expressed at approximately 1-2% of the level of human beta globin in erythroid cells despite the marked homology between these two globins. To determine the DNA sequences responsible for this effect, delta and beta globin genes and fusion products of these genes constructed in vitro were transfected and expressed in HeLa cells. The results indicate that when the small intervening sequence of the beta gene (beta IVS 1) is replaced by delta IVS 1, expression of the chimeric gene is the same as that of the normal beta globin gene. By contrast, when the large intervening sequence of the beta gene (beta IVS 2) is replaced by delta IVS 2, expression of the chimeric gene is markedly reduced. These results suggest that there are signals within IVS 2 of the delta and beta genes which affect their relative expression. PMID- 2999706 TI - A modular system for the assay of transcription regulatory signals: the sequence TAATGARAT is required for herpes simplex virus immediate early gene activation. AB - A modular system for assaying the activity of transcriptional regulatory signals based on herpes simplex virus (HSV) promoter and terminator sequences linked to the bacterial chloramphenicol acetyltransferase (CAT) gene has been used to study activation of HSV immediate early (IE) gene expression. Insertion of the SV40 72 base pair (bp) repeat increased mRNA levels by 15-fold thus demonstrating the ability of the HSV IE promoter to respond to a heterologous enhancer. A fragment containing part of the intergenic region located between HSV-2 immediate early (IE) genes-3 and -4/-5 increased mRNA levels by 5-fold in response to transactivation by an HSV virion structural polypeptide. The HSV activator fragment increased mRNA levels by 2-fold in the absence of transactivation indicating that cellular proteins are involved in IE gene expression. From HSV 1/HSV-2 DNA sequence comparisons we previously proposed that a DNA sequence, consensus TAATGARAT, present upstream of all HSV-1 and HSV-2 IE genes was required for the co-ordinate induction of IE genes. We show here that a synthetic oligonucleotide containing TAATGARAT conferred the ability to stimulate CAT activity only on transactivation: two copies of TAATGARAT stimulated expression by 2-fold while six copies gave an 8-fold increase. This activation, which was not dependent on orientation of the TAATGARAT sequence, directly demonstrates that TAATGARAT is a component of the IE gene activation sequence. PMID- 2999705 TI - Sequence analysis of a KpnI family member near the 3' end of human beta-globin gene. AB - We determined the complete nucleotide sequence (6125 bp) of a full-length member of human KpnI family, designated T beta G41, which is located about 3 kb downstream from the beta-globin gene. Comparison of the sequence with the KpnI family sequence compiled by Singer revealed that a new 131 bp sequence is present in the T beta G41. Hybridization analyses showed that a few thousand of human KpnI family members are carrying this additional sequence. Computer search of DNA databases for T beta G41-homologous sequence showed that some T beta G41 homologous sequences were closely associated with pseudogenes. The T beta G41 sequence also showed significant sequence homology with ChBlym-1, a transferrin like transforming gene of chicken. Furthermore, an amino acid sequence deduced from the T beta G41 nucleotide sequence revealed a relatively-high homology to those of human transferrin and lactotransferrin. PMID- 2999709 TI - An anonymous single copy chromosome 21 probe, DS21D2, associated with a frequent RFLP. PMID- 2999708 TI - Comparative nucleotide sequence analysis of two types of larval beta-globin mRNAs of Xenopus laevis. AB - The complete nucleotide sequence of the cDNA insert of the clone pXGL25 derived from the larval beta II-globin mRNA of Xenopus laevis has been determined. The sequence of 593 nucleotides represents part of the 5'nontranslated region, the coding region for 146 amino acids and the entire 3'nontranslated region. It diverges from the related larval beta I-sequence by 24.9% in the coding region. Alignment of the 5' and 3'nontranslated regions of the two related larval beta sequences to maximum matching resulted in 31.2% and 46.7% divergence, respectively. Divergence between the corresponding adult and larval sequences considerably exceeds that of related larval sequences, suggesting that larval genes may have arisen by gene duplication prior to genome duplication. In contrast to mammalian beta-globin mRNAs, replacement and silent base substitutions are equally abundant, thus indicating less functional constraint on the larval Xenopus laevis beta-globin chains. The larval beta I- and beta II globins diverge by 30.8% and show most variation in the alpha 1/beta 2-chain interaction sites. PMID- 2999707 TI - DNA sequence of the herpes simplex virus type 1 gene whose product is responsible for transcriptional activation of immediate early promoters. AB - Previous work has shown that transcriptional activation of herpes simplex virus type 1 (HSV-1) immediate early genes is mediated by a protein species (Vmw65) present in the tegument of infecting virions. This paper describes DNA sequence analysis and mRNA mapping of the Vmw65 gene in HSV-1 strain 17. The Vmw65 coding region was identified as a 490 codon sequence encoding a polypeptide of molecular weight 54,342 and characterised by a high proportion of charged amino acid residues. A homologue to Vmw65 was detected in the genome of varicella-zoster virus, another human herpesvirus. Apart from its role in trans-activation, Vmw65 is a major constituent of the virion. Its possible significance in virus structure is discussed. PMID- 2999710 TI - Regulation of gene amplification and expression in cells that constitutively express a temperature sensitive SV40 T-antigen. AB - Simian cells have been transformed with SV40 origin-defective recombinant plasmids containing the tsA209 T-antigen gene. These plasmids contain deletions of either 5 or 52 nucleotides that include the BglI site at the SV40 ori, are defective for replication in COS-1 cells but retain a functional SV40 early promoter. Two cell lines transformed with these plasmids, U4 and S7, and their respective clonal derivatives E5 and F11, contain the tsA209 T-antigen gene integrated into the cell DNA and express T-antigen as detected by immunoprecipitation and immunofluorescence. These cells behave as ts-COS cells, since they complement in a temperature dependent manner the replication of an SV40 derived recombinant plasmid. When transfected with recombinant plasmids containing the chloramphenicol acetyl transferase (CAT) gene cloned into SV40 replicons, ts-COS cells were able to regulate the induction of the CAT activity by temperature. The ratios of CAT activity observed at permissive versus restrictive temperature were in the range of 20-400. Thus, these ts-COS cells are useful systems for the regulated expression of cloned genes in simian cells. PMID- 2999711 TI - Alpha-amanitin insensitive transcription of the human epsilon-globin gene. AB - In vitro transcription was used to show that RNA polymerase III is responsible for the initiation of transcription at a position 200 bp upstream from the epsilon-globin major cap site. High levels of -200 transcription interferes with the RNA polymerase II major cap site transcription. Using DNA mediated transient expression, the ratio of -200 to +1 transcription can be modulated by either the direction of replication or the presence of an enhancing element in the vector. We suggest that this heterogeneous usage of cap sites is not related to epsilon globin gene transcription in vivo, but is instead the result of a combination of factors inherent to transient expression experiments. PMID- 2999712 TI - Genomic cloning and characterization of a ricin gene from Ricinus communis. AB - A genomic clone that specifies a single polypeptide precursor for ricin, a toxic lectin of Ricinus communis (castor bean), was isolated, sequenced and Sl mapped. The gene encodes a 64 kDa precursor which contains, in the following order: a 24 or 35 amino acid signal peptide, the A chain, a 12 amino acid linker peptide, and the B chain. The 5'-end of the ricin mRNA maps approximately 35 bases upstream from the first methionine codon. Two putative TATA boxes and a possible CAAT box lie in the 5'-flanking region. Two possible polyadenylation signals were found in the 3' flanking region. No introns were found, which is typical of other lectin genes that have been sequenced. Southern blot analysis suggests that the castor bean genome contains approximately six ricin-like genes. PMID- 2999713 TI - Functional activity and chromatin configuration of SV40 enhancer injected in Xenopus laevis oocytes. AB - The SV40 enhancer microinjected into the nuclei of X. laevis oocytes is able to activate transcription about 100-fold when cloned upstream of the SV40 early promoter region or about 10-fold regardless of its orientation when located at a long distance from a test gene (bacterial chloramphenicol acetyl transferase). This effect is qualitatively and quantitatively similar to that observed when analogous constructions were transfected into mammalian cells. Making use of a direct labelling technique for the analysis of the chromatin structure we could show that the SV40 enhancer region in the microinjected plasmids is particularly accessible to cleavage with MNase or DNAse I. Single sites in the 72 bp repeats have been mapped at the nucleotide level. PMID- 2999714 TI - DNA sequence of the region in the genome of herpes simplex virus type 1 containing the genes for DNA polymerase and the major DNA binding protein. AB - In the long unique region of the genome of herpes simplex virus type 1 (HSV-1), the genes for DNA polymerase and the major DNA binding protein are arranged in a head to head manner, with an origin of DNA replication (termed OriL) located between them. This paper reports an 8400 base pair DNA sequence containing both genes and the origin, obtained mostly by M13/dideoxy analysis of plasmid cloned fragments. Amino acid sequences of the two proteins were deduced. Homologues of both genes were detected in the genome sequence of the distantly related Epstein Barr virus (EBV). Arrangement of these HSV-1 and EBV genes differs in genome location and in relative orientation. A part of HSV-1 DNA polymerase was found to be similar to a sequence in adenovirus 2 DNA polymerase, but the significance of this is unclear. Since a DNA sequence in the locality of OriL deletes on plasmid cloning, this region was analysed using virus DNA. A palindrome with 72-residue arms was found, which shows great similarity to the better characterized origin, OriS. PMID- 2999715 TI - Polymorphism of the 3' open reading frame of the virus associated with the acquired immune deficiency syndrome, human T-lymphotropic virus type III. AB - The genome of the virus associated with the acquired immune deficiency syndrome (AIDS), human T-lymphotropic virus type III (HTLV-III), includes two open reading frames, not found in other retroviruses. One of these, designated 3' open reading frame (3'orf) is 648 base pairs (bp) in length, and overlaps with the 3' long terminal repeat (LTR) sequences. Sequences of additional HTLV-III clones were determined in order to estimate the level and location of variation within 3'orf, to gain some insight into the function of its protein product. Newly determined sequences are reported for 3'orf of two unintegrated clones of HTLV-III and three cDNA clones made from virion RNA derived from the same cell line infected with pooled blood samples of different patients with AIDS or AIDS-related complex symptoms (ARC). In addition, sequences for 3'orf were derived from an unintegrated viral clone derived from a different cell line infected with a distinct isolate from a single patient. These sequences are compared to those previously reported for six other viral clones. Sequences of 3'orf differ among clones by 1.1-10.4% bp and 2.4-17.0% of predicted amino acids. This represents significantly greater sequence variation than is found in the entire genome on average. Moreover, a functional proviral clone has a termination codon at amino acid residue 124 of this open reading frame. This raises questions concerning the structure, and regulation of expression of the protein encoded by 3'orf. PMID- 2999716 TI - Discrete size classes of monkey extrachromosomal circular DNA containing the L1 family of long interspersed nucleotide sequences are produced by a general non sequence specific mechanism. AB - The L1 family of long interspersed nucleotide sequences (LINES) has recently been identified and characterized in the small polydisperse circular DNA (spc-DNA) populations of monkey (1), human (2) and mouse (3) cells. In monkey spc-DNA, the L1 (also known as Kpn I) family is present in discrete size classes (ranging from 300 to 6000 base pairs (bp)) which appear to be generated by non homologous recombination events within chromosomal elements. In this communication it is shown that different regions of the consensus L1 family are present at different frequencies in monkey spc-DNA (as they are in chromosomal DNA), that all regions of the family are present in extrachromosomal DNA, and that each region appears to be represented in an identical discrete spc-DNA size distribution. This size distribution reflects a non-sequence specific mechanism that generates spc-DNA size classes by chromosomal DNA recombination events that are in some way constrained to occur between sites separated by relatively defined lengths. PMID- 2999718 TI - p49f, A highly polymorphic probe, that detects Taq1 RFLPs on the human Y chromosome. PMID- 2999717 TI - Modulation of fibronectin gene activity in chick embryo fibroblasts transformed by a temperature-sensitive strain (ts68) of Rous sarcoma virus. AB - Transcriptional regulation of the fibronectin gene is a major mechanism for lowering steady-state levels of fibronectin mRNA in chick embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSV) (1). In the present study, we have measured the change of transcriptional activity of the fibronectin gene in CEF transformed by a temperature-sensitive strain of RSV (ts68). Ts68-CEF maintained at either 35 degrees C or 41 degrees C were shifted to 41 degrees C or 35 degrees C, respectively, at 5-hour intervals, and isolated nuclei were used in runoff transcription assays. Nuclear RNA labeled with [alpha-32P]UTP was hybridized to DNA fragments encoding the src gene, the beta-actin gene and the fibronectin gene. In shift-up (35 degrees C----41 degrees C) and shift-down (41 degrees C--- 35 degrees C) experiments, src gene and beta-actin gene activities in ts68-CEF nuclei remained relatively unchanged. In ts68-CEF shifted to the nonpermissive temperature (41 degrees C), a lag time of at least 5 hours was followed by a 4- to 5-fold increase in fibronectin specific RNA 15 hours after the shift. When cells were shifted to the permissive temperature (35 degrees C), a 4- to 5-fold decrease in fibronectin RNA was apparent within 5 hours of the temperature shift and a 17- to 18-fold decrease was observed 15 hours after the shift. The relatively slow rates of changes of fibronectin gene activity in shift-up experiments suggest that the effect of p60src on fibronectin gene activity is indirect. PMID- 2999719 TI - Facts about fibre. PMID- 2999721 TI - Anorexia and weight loss: indicators of cachexia in small cell lung cancer. AB - A study was conducted to determine the incidence and extent to which anorexia, a decrease in spontaneous food intake, contributes to the occurrence of cancer cachexia. Data for ten male subjects with small cell carcinoma of the lung are reported for a five-month period following diagnosis. Although body weights of the subjects at the time of diagnosis averaged less than 95% of the usual weight (weight 6 months prior to diagnosis), they were greater than 109% of the mean ideal weight. At five months, the mean weight (N = 8) was 88% of the preillness weight. From the time of diagnosis, there was a mean loss of 7.2 kg (15.8 lb). The urinary creatinine excretion was below the normal range, whereas the urinary urea nitrogen values were within the normal range. At the time of diagnosis, the mean triceps skin-fold measurements were approximately 80% of the standard reference for males. During the five-month period, the mean midarm muscle circumference determinations remained greater than 90% of the reference standard. The mean serum transferrin values were 10% or more below the reported lower range of normal, whereas the great majority of the serum albumin values were 3.0 g/dl or above during the five-month period. The mean caloric intake of 2,204 kcal at the time of diagnosis was only 86% of the estimated basal energy expenditure (BEE) times a factor of 1.5 used to account for moderate activity. Four months following diagnosis, the mean caloric intake had fallen to 1,702 kcal, only 67% of the BEE X 1.5 (calculated from the weight at diagnosis). The findings provide evidence of a decline in spontaneous food intake, a small decrease in body fat, and a greater than 13% weight loss. The oral intake was less than adequate for any activity beyond the basal state. Decreased intake could account for most of the weight loss observed in the subjects. PMID- 2999720 TI - Effect of N-(4-hydroxyphenyl)retinamide on murine mammary tumor cells in culture. AB - The effects of N-(4-hydroxyphenyl)retinamide (HPR), a synthetic analogue of vitamin A, on cell morphology, cell cycle kinetics, cytoplasmic matrix, and expression of murine mammary tumor virus (MuMTV) in MuMTV-infected murine mammary tumor cells (GR-3A) were determined. Cellular uptake of HPR was rapid and linear, with zero-order kinetics, during the first 30 minutes of incubation. Flow cytometric analysis of cells treated with nontoxic levels of HPR (10 microM) for 48 hours revealed a reduction in percent cells in the DNA synthetic (S) phase of the cell cycle with a concomitant increase in percent cells in the G1 phase of the cell cycle. Dexamethasone-stimulated MuMTV expression was not affected after 48 hours of HPR exposure, whereas the virus expression was significantly reduced in cells treated with HPR for seven days. The reduction in MuMTV expression was preceded by changes in cell morphology (decreased cell-cell contact and reduced cell flattening) and altered F-actin aggregation. Continuous exposure to HPR (10 microM) for 14 days resulted in reduced cell proliferation rates and decreased cell plating efficiency of GR-3A cells. Taken together, these results indicate that HPR is rapidly incorporated into GR-3A cells and that the effects of HPR on cell profileration, cytoskeletal organization, and cell morphology appear to precede the effects of this retinoid on the expression of the etiological agent of murine mammary tumorigenesis, MuMTV. PMID- 2999723 TI - Conversion of atriopeptin II to atriopeptin I by atrial dipeptidyl carboxy hydrolase. AB - We are examining the substrate specificity of atrial dipeptidyl carboxyhydrolase, a membrane-bound metallo enzyme that we isolated from bovine atrial tissue homogenates. This enzyme readily removes the dipeptide, Phe-Arg, from Bz-Gly-Ser Phe-Arg, a stand-in substrate for atriopeptin II, one of several atrial natriuretic factors. We now report that the atrial enzyme cleaves the C-terminal dipeptide, Phe-Arg, from atriopeptin II to form atriopeptin I. The km (pH 7.5) is 25 microM and the ratio of relative Vmax/km as a measure of substrate specificity indicates that atriopeptin II is a 240-fold better substrate than Bz-Gly-His-Leu. Only Phe-Arg was detected as a hydrolysis product, indicating that sequential cleavage of Asn-Ser from atriopeptin II does not occur, and that atriopeptin I is not a substrate. Bz-Gly-Asn-Ser was as good a substrate for the atrial enzyme as Bz-Gly-His-Leu, but Bz-Cys(bzl)-Asn-Ser was not hydrolyzed. This result suggests that the presence of an intact disulfide bond or an S-alkylated residue in the P1 position of a substrate (as in atriopeptin I) prevents hydrolysis by the atrial enzyme. Comparative studies were made with the angiotensin I converting enzyme. Atriopeptin II was not a substrate. The stand-in substrates for atriopeptin I, Bz Cys(bzl)-Asn-Ser and Bz-Gly-Asn-Ser were barely hydrolyzed, which by itself suggests that atriopeptin I is not a substrate of the angiotensin converting enzyme. Our results strongly suggest that atriopeptin II is converted to atriopeptin I and that hydrolysis is mediated by the atrial enzyme. The angiotensin I converting enzyme plays no role in processing these peptides. We suggest that the atrial enzyme be named atrial peptide convertase. PMID- 2999722 TI - Comparison between excessive grooming induced by bombesin or by ACTH: the differential elements of grooming and development of tolerance. AB - Bombesin and ACTH-(1-24) induce a dose dependent increase in grooming behavior. Lower doses of bombesin induce a more general type of compulsive grooming in which most elements are involved, whereas higher amounts of bombesin induce a shift towards the element scratching at the cost of bodily grooming and sexual grooming. In contrast ACTH-(1-24) induces a dose dependent increase of all elements of grooming. It is concluded that the grooming displayed by animals treated with ACTH-(1-24) or with bombesin is of a completely different nature. In addition it is observed that under the conditions used tolerance occurs for the grooming inducing effect of ACTH-(1-24), but not for that of bombesin. Moreover, it appears that no cross tolerance exists between bombesin and ACTH-(1-24). PMID- 2999724 TI - Central effects of growth hormone-releasing factor (GRF) on intestinal motility in dogs: involvement of dopaminergic receptors. AB - The effects of intracerebroventricular (ICV) and intravenous (IV) administration of human pancreatic growth hormone-releasing factor (hpGRF) on gastro-intestinal motility were examined in fasted and fed conscious dogs equipped with chronically implanted strain-gauges on the antrum and the jejunum. During the fasted state, hpGRF injected ICV at 0.1 micrograms . kg-1 or IV at 0.5 micrograms . kg-1 did not affect the cyclic occurrence of the migrating motor complex (MMC). This pattern was normally disrupted for 8-10 hours by a daily standard meal. Injected ventricularly (0.1 micrograms . kg-1) but not intravenously (0.5 micrograms . kg 1) 10-15 min after the daily meal, hpGRF significantly reduced (p less than 0.01) the duration of the jejunal fed pattern (2.0 +/- 1.4 vs. 8.4 +/- 1.1 hours for control) but not that of the stomach. This effect persisted when hpGRF (0.1 micrograms . kg-1 ICV) was administered after indomethacin (2 mg . kg-1 IM), naltrexone (0.1 mg . kg-1 IV) or domperidone (1 mg . kg-1 IV) but was abolished by a previous IV injection of metoclopramide (1 mg . kg-1). It was concluded that hpGRF is able to act centrally to control the pattern of jejunal motility in fed but not in fasted dog, its effect being probably mediated through dopaminergic pathways. PMID- 2999725 TI - Specific arginine vasopressin binding in particulate membrane from rat aorta. AB - Arginine vasopressin (AVP) has been shown to have direct pressor effects on vascular smooth muscle. We have characterized a specific binding site for AVP in rat aorta membranes. We identified a specific binding site for AVP with a Kd of 1.6 nM and Bmax of 48 pM/mg protein. The time course, pH dependence, and temperature dependence were consistent with those found for other peptide receptors. Analogues of AVP competed with tritium labelled AVP for binding to the aortic vascular receptor in direct proportion to their pressor activities. PMID- 2999726 TI - Degradation of alpha and beta neo-endorphin by rat brain membrane peptidases. AB - Fractionation of Triton-solubilized rat brain membranes on diethylaminoethyl cellulose resolves two peptidases which hydrolyze beta-neo-endorphin. One of these peptidases was identified as Angiotensin Converting Enzyme by (a) its sensitivity to inhibition by the specific inhibitors MK422 and captopril, (b) by the identification of reaction products, and (c) by comparison to authentic angiotensin converting enzyme. In contrast, alpha-neo-endorphin hydrolysis by angiotensin converting enzyme could not be detected. The second enzyme active on beta-neo-endorphin was identified as an aminopeptidase. This aminopeptidase is identical to the previously described enkephalin-degrading aminopeptidase. The possible involvement of these enzymes in the metabolism of opioid peptides is discussed. PMID- 2999728 TI - Acetylation of alpha MSH and beta-endorphin by rat neurointermediate pituitary secretory granule-associated acetyltransferase. AB - ACTH(1-8) and ACTH(9-13)NH2 were used as potential enzyme inhibitors to begin examining the relationship between the acetylation of ACTH- and beta-endorphin related peptides. ACTH(1-8) was a potent inhibitor of the acetylation of both ACTH- and beta-endorphin-related peptides, whereas ACTH(9-13)NH2 was an effective inhibitor only of the acetylation of ACTH-related substrates. This inhibition pattern indicated that there may be an unusual interaction between some ACTH- and beta-endorphin-related peptides as substrates for the acetyltransferase. Utilizing HPLC to separate ACTH- and beta-endorphin-related peptides present in the same reaction mixture, ACTH(1-14) and beta-endorphin(1-27) at Km and saturating concentrations were used as substrates to examine the ability of one peptide substrate to affect the acetylation of the other. It was observed that the acetylation of ACTH(1-14), even at Km concentration, was relatively unaffected by the presence of beta-endorphin(1-27). However, the acetylation of beta-endorphin(1-27) was significantly reduced by the presence of ACTH(1-14). This preferential acetylation of ACTH-related peptides over the acetylation of beta-endorphin-related peptides might have physiological importance under some conditions. PMID- 2999727 TI - ACTH 1-24-induced potentiation of norepinephrine contractile responses in aortic strips from spontaneously hypertensive (SH) and normotensive (WKY) rats. AB - Norepinephrine (NE)-induced contractile responses were less in aortic strips from SH compared to WKY rats. ACTH 1-24 potentiated NE responses in both SH and WKY aortic strips. This effect was more potent in SH aortic strips. NE-induced contractions in SH aortic strips were less sensitive to changes in external Ca2+ levels than were those of WKY aortic strips. ACTH 1-24 did not potentiate NE responses under low external Ca2+ conditions in SH aortic strips or under high external Ca2+ conditions in WKY aortic strips. The greater sensitivity of NE responses following ACTH 1-24 in SH aortic strips may imply that this peptide is modulating a mechanism related to an impaired contractility and that Ca2+ plays a key role in the observed effects. PMID- 2999729 TI - Cholecystokinin octapeptide analogues stable to brain proteolysis. AB - Based on recent findings identifying the initial degradative cleavage of CCK-8 at the Met3-Gly4 bond by a metalloendopeptidase, two analogues of CCK-8 with D-Ala and D-Trp substitutions at the Gly4 position were synthesized as stable analogues. Their stability to proteolysis by brain membranes and their binding potency at central CCK receptors were quantified. Both peptides are stable to degradation by peptidases in cortical synaptic membrane preparations. The analogues are nearly equipotent to CCK-8 in their affinities for inhibition of 125I-CCK-33 binding to guinea pig cortical membranes. L-Ala and L-Trp substituted peptides were synthesized for comparison. Both these peptides are degraded by synaptic membranes and the L-Trp substituted peptide possesses a greatly reduced affinity for central CCK receptors. Therefore, the structure of CCK due to the D conformation of Gly is more capable of interacting with brain CCK receptors. Further conformational analysis will establish whether the stabilized structure is a beta-bend or a beta-turn. Since these peptides are highly potent and stable to brain proteolysis they may be useful as stable CCK analogues for in vivo application. PMID- 2999730 TI - Functional relations of crab molt-inhibiting hormone and neurohypophysial peptides. AB - Biological and immunological relationships between molt-inhibiting hormone (MIH) activity in eyestalk ganglia extracts of the crab, Cancer antennarius Stimpson, and peptides of the vasopressin-oxytocin family were assessed. Lysine vasopressin (LVP), arginine vasopressin (AVP), vasotocin (VT), and oxytocin (OT) mimicked MIH action by inhibiting ecdysteroid production of Y-organ segments in vitro with the relative potencies LVP greater than AVP greater than VT much much greater than OT. The inhibitory effect was reversible and specific (6 other peptides did not alter Y-organ activity). MIH and LVP increased Y-organ cyclic adenosine 3',5' monophosphate (cAMP) levels dose-dependently and with identical time course in which the rise in cAMP preceded inhibition of ecdysteroid production. The synthetic vasopressin antidiuretic agonist 1-deamino-8-D-AVP (dDAVP) inhibited Y organ steroidogenesis dose-dependently; the vasopressin analog ([1(B-mercapto beta, beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine[AVP) (d(CH2)5Tyr(Me)AVP), a vasopressor antagonist, had no effect on basal or MIH suppressed steroidogenesis. AVP antiserum abolished the inhibitory action of MIH, LVP, and AVP. Competitive binding curves for MIH, LVP, AVP, VT, and OT with the AVP antiserum suggested that MIH is most closely related to LVP. MIH may be structurally related to the vasopressins and act on Y-organ cells via type V2 (cAMP-linked) receptors. PMID- 2999731 TI - Administered peptides inhibit the degradation of endogenous peptides. The dilemma of distinguishing direct from indirect effects. AB - Virtually all peptides are biologically active following central administration as a consequence of both direct and indirect cellular actions. Direct effects are mainly interactions with specific membrane receptors but may include unions with other components of the receptor/effector complex. Significant indirect biological effects of exogenous peptides, including apparent secretagogue effects on endogenous peptides largely overlooked in practice, result from extensive competition with endogenous peptides for degradative enzymes (peptidases). A consequence of this competition is enhancement of tonic or intermittent activity of endogenous peptides. The pharmacological profile of any peptide reflects or includes, therefore, the spectrum of endogenous peptides that is protected from peptidase action. It is likely that certain pharmacologically active peptides, including a large number of di-, tri- and oligo-peptides, elicit responses mainly or exclusively by competing for peptidases. Therefore, reliable estimates of the relative contributions of direct and indirect actions of exogenous peptides may be difficult, if not impossible, to obtain. PMID- 2999732 TI - CRF immunoreactive peptides in the human hypophysis: a cautionary note. AB - By monitoring with a non competitive enzyme linked immunosorbent assay (ELISA), corticotropin releasing factor (CRF)-like immunoreactive material was isolated from the human hypophysis. After acid extraction of peptides from frozen human hypophyses, the purification was achieved by affinity chromatography using purified anti-ovine-CRF IgG bound to a solid phase and then by two HPLC steps using an alkylsilane-bonded large pore size silica. Two CRF-like peptides were purified: discrete immunoreactive peaks coinciding with an optical density peak at 215 nm. Although these peptides were recognized by ELISA, they were not recognized in an RIA using the same anti-ovine-CRF serum and ovine CRF-41 as tracer. Neither of these CRF-immunoreactive peptides had any effect on either the spontaneous or stimulated ACTH release in the perfused isolated anterior pituitary cell bioassay. PMID- 2999733 TI - Influence of CCK and bombesin on ACTH and cortisol secretion in the conscious dog. AB - Bombesin and cholecystokinin (CCK) have a variety of similar actions. Previous investigations have demonstrated that IP injections of bombesin and CCK-33 increased corticosterone secretion in conscious, freely-moving, fed rats. In this study bombesin or CCk-8 was administered by continuous, intravenous infusion to conscious, awake, fasted, mongrel dogs. Following a 30-40 minute control infusion, a progressively-increasing, stepwise infusion of either bombesin (0.1, 1.0 and 2.0 micrograms/kg-hr) or CCK-8 (62.5, 125, and 250 ng/kg-hr) was administered. Each drug dose was infused for 40-45 minutes and blood samples were drawn at 20-22.5 minutes intervals. Bombesin caused significant, dose-dependent increases in plasma cortisol (286 +/- 39% of control) and plasma ACTH (176 +/- 33% of control). CCK-8 had no consistent effect on either cortisol or ACTH secretion. Whether the lack of effect of CCK-8 in dogs, as compared to rats, is due to species variations or to the differing experimental designs is unknown. PMID- 2999734 TI - [Evaluation of the immunologic system in patients with small cell and squamous cell lung cancer during the early stage of treatment]. PMID- 2999735 TI - [The immunologic and enkephalin-endorphin systems]. PMID- 2999736 TI - Human mucinous breast carcinomas and their lymph node metastases. A histological review of 247 cases. AB - In a histologic reevaluation of 247 primary human mucinous breast carcinomas, 207 tumors fullfilled the criteria for further histopathological evaluation. The criteria for entrance in this survey are that at least 25% of the tumor consists of areas of extracellular mucin with small islands of solid epithelial tumor tissue floating in the mucin, and that the extracellular mucin should comprise at least 33% of the total tumor volume. The 247 carcinomas that have been further evaluated have been subclassified into two groups: "pure" mucinous breast carcinomas that consist solely of tumor tissue with extracellular mucin production (95 tumors), and "mixed" mucinous carcinomas that also contain infiltrating carcinoma without extracellular mucin (112 tumor). A significantly greater number of mixed carcinomas than pure carcinomas have an aggressive growth pattern--as defined by tumor size, adherence to overlying skin/bottom fascie, and primary axillary lymph node metastases. The histogenesis of the mucinous carcinomas is briefly discussed in relation to the present observations and the literature. The importance of clearly distinguishing between the mixed and the pure mucinous carcinomas in the diagnosis of these tumors is emphasized. PMID- 2999737 TI - New central dopamine agonists. AB - Recently, the importance of the dopamine receptor agonists has increased in the treatment of parkinsonism, different endocrinological diseases and cardiovascular illness. In the therapy some well known drugs, derivatives of ergot groups e.g. bromocriptine, lisuride and pergolide, have been found useful. In the Institute for Drug Research numerous semi-synthetic elymoclavine derivatives were synthesized during the past years, and the influence of these new compounds on both the central and peripheral dopamine transmission was examined. Among the different ergot derivatives compound GYKI-32 887 seemed to be the most effective dopamine agonist and it was selected for preclinical investigation. The endocrinological effects and the pre- and postsynaptic dopamine receptor stimulant activity of this new compound are summarized. GYKI-32 887 was more potent than bromocriptine as regards its inhibitory effect on prolactin secretion and antiparkinsonian efficacy. Besides the strong dopamine receptor stimulant action this new ergoline compound, contrary to bromocriptine, inhibits the convulsive action of bicucullin. It may be assumed that the GABA receptor agonistic effect of GYKI-32 887 would be also valuable in the treatment of various form of dyskinesias. PMID- 2999738 TI - Regulation of the activity of protein kinases by endogenous heat stable protein inhibitors. AB - Protein kinase activities are regulated by endogenous thermostable protein inhibitors. Type I inhibitor is a protein of MW 22,000-24,000 which inhibits specifically cyclic AMP-(cAMP) dependent protein kinase (APK) as a competitive inhibitor of catalytic subunits of the enzyme. Type I inhibitor activity changes inversely according to the activation of adenylate cyclase and the changes in cAMP content in tissues. It seems that type I inhibitor serves as a factor preventing spontaneous cAMP-dependent phosphorylation in unstimulated cell. The other thermostable protein which inhibits APK activity has been found in Sertoli cell-enriched testis (testis inhibitor). Physiological role of the testis inhibitor is unknown. Type II inhibitor is a protein of MW 15,000 which blocks phosphorylation mediated by cAMP and cyclic GMP (cGMP) dependent (APK and GPK) and cyclic nucleotide independent protein kinases as a competitive inhibitor of substrate proteins. Activity of this inhibitor specifically changes in reciprocal manner to the changes in cGMP content. It seems that type II inhibitor serves as a factor preventing the phosphorylation catalyzed by GPK when cGMP content is low. Stimulation of guanylate cyclase and activation of GPK is followed by a decrease of type II inhibitor activity. This change in relationship between activities of GPK and type II inhibitor allows for effective phosphorylation catalyzed by this enzyme when cGMP content is increased. PMID- 2999739 TI - Catalepsy, hypermotility, increase of striatal acetylcholine release induced by morphine and Met-enkephalin as affected by prolonged hydrocortisone and ACTH treatment. AB - In the rats treated with ACTH or hydrocortisone for 14 days the catalepsy induced by morphine was almost completely inhibited, while the haloperidol induced catalepsy remained unchanged. The morphine induced hypermotility was altered neither by prolonged treatment with ACTH nor by an acute glucocorticoid administration. Inhibition of Na/K ATPase by ouabain led to an increase of striatal acetylcholine (Ach) release, which was enhanced by Met-enkephalin. This effect of the opioid peptide was not demonstrable in the striata of ACTH or hydrocortisone pretreated rats. It is concluded that glucocorticoids are regulatory factors of the striatal opiate neurotransmission possibly via altered receptorial mechanisms. PMID- 2999740 TI - Cutaneous histiocytosis syndromes. AB - Cutaneous histiocytosis may take two principal forms. It is either a benign proliferative process or a relentless, progressive process with a poor prognosis. In histiocytic medullary reticulosis, histiocytes demonstrate nuclear atypia and the outcome is uniformly fatal. Benign cephalic histiocytosis X causes lesions similar to those of histiocytosis X, but Langerhans' cells are absent. In congenital self-healing histiocytosis X, the Letterer-Siwe-like cutaneous infiltrate contains Langerhans' cells, but the lesions heal spontaneously without treatment. The nodular cutaneous lesions of juvenile xanthogranuloma appear in infancy and resolve without treatment; however, the higher percentage (10%) of associated ocular lesions may lead to glaucoma and blindness. In histiocytosis X, the cutaneous lesions show a marked proliferation of Langerhans' cells, with prognosis dependent on the patient's age and the extent of organ dysfunction. Patients who survive the acute form of the disease may develop diabetes insipidus, growth retardation, pulmonary fibrosis, and biliary cirrhosis. A subtle immunologic defect has been identified in patients with histiocytosis X, yet the pathogenesis of the disease is still speculative. Familial disease occurring in early infancy should be differentiated from complete or partial immunodeficiency syndromes. Guidelines for evaluating patients with cutaneous histiocytosis are reviewed. PMID- 2999741 TI - External eye diseases. AB - Diseases of the external eye can have several causes and many have similar symptoms, but a precise diagnosis of the specific disorder is crucial for proper treatment. Evaluation should begin with a complete medical history to ascertain any systemic disease that may affect the eyes. Physical examination should include determination of visual acuity, inspection of the tarsal and bulbar conjunctiva, and microscopic evaluation of the cornea. When external inflammatory disease is present, laboratory testing is required. Smears and cultures are the only reliable methods to determine the specific organism causing the infection. Treatment is then aimed at eradicating the underlying microbes. PMID- 2999742 TI - Response of B complex haplotypes B22, B24, and B26 to Rous sarcomas. AB - Five individual male matings of line UNH 105 New Hampshires, in which all males and most females were either B22/B24 or B22/B26, produced 462 progeny that fell into six B complex genotypes: B22/B22, B24/B24, B26/B26, B22/B24, B22/B26, and B24/B26. The genotypes of parents and offspring were determined by blood typing for B alloantigens using a panel of antisera. Six-week-old chickens were inoculated with Rous sarcoma virus (RSV). Resulting tumors were scored for size six times over a 10-week period; based upon these scores, a tumor profile index (TPI) was assigned to each chicken as a criterion of immunological response. The B22/B26 hosts showed the greatest mean response (TPI 3.3) and B24/B24 chickens the lowest response (TPI 4.4), the difference being statistically significant. Dominance in the response to sarcoma was observed when either the B22 or B26 haplotype combined with the B24 haplotype and compared with the appropriate corresponding homozygotes, and when the B22 or B26 heterozygote was compared with B22/B22 and B26/B26 homozygotes. PMID- 2999743 TI - Infection and immunity with a virus isolate from turkeys. AB - Avian pox virus was isolated from cutaneous pox lesions removed from turkey breeders that had been vaccinated three times with a commercial fowl pox vaccine. In three cross-immunization experiments with turkeys and two with chickens, the turkey pox isolate, designated NC5271, proved immunologically different from fowl, pigeon, and quail pox viruses. Significant protection against NC5271 virus infection and inducement of pox lesions was only attained when the homologous isolate was used as a vaccine. The potential need in the field for such a vaccine was discussed. PMID- 2999744 TI - Performance of broiler progeny of breeder flocks vaccinated with inactivated oil emulsion malabsorption syndrome virus vaccine. AB - Broiler breeder pullets were vaccinated at 20 to 24 weeks of age with an inactivated, oil emulsion vaccine containing the CO8 strain of avian reovirus. The vaccination induced a high and uniform antibody response that peaked 4 to 5 months postvaccination and persisted up to 11 months postvaccination. Broiler production parameters in progeny of vaccinated breeders were compared weekly with parameters of the total broiler production. There was a consistent improvement in body weight at processing time and a reduction in total production cost in progeny of vaccinated parents. There was also a reduction of percent of parts condemned during the first part of the trial. There were no consistent differences in feed conversion or condemnation of whole carcasses. Progeny of vaccinated parents had reduced livability. This effect was interpreted to be due to the smaller egg size of young breeder flocks. PMID- 2999747 TI - An epidemic of erythema infectiosum in a school. PMID- 2999745 TI - Phosphorus phase feeding and uterine and isthmus mucosal enzymes and minerals in relation to soft-shelled and shell-less egg production. AB - The relationships among phosphorus phase feeding, egg shell quality, and the activities and concentrations of several enzymes and minerals in the uterine and isthmus mucosae of hens at the time of oviposition were investigated. During the first 8 months of production (Phase 1), layer diets contained .3, .5, or .7% available phosphorus. Between 9 and 12 months of production (Phase 2), dietary available phosphorus was either increased or decreased by .2% phosphorus, or was left unchanged. No significant differences due to Phase 1 diets were demonstrated for hard-shelled (HS), soft-shelled (SS), or shell-less (SL) egg production, livability, egg weight, or specific gravity. Phase 2 diets had no significant effect on SS or SL egg production, livability, or egg specific gravity; however, decreasing dietary phosphorus reduced egg weight. Levels as high as .9% had no effect on specific gravity or HS egg production, while .1% dietary phosphorus was detrimental to HS egg production and feed consumption. No significant differences due to dietary available phosphorus or egg type (SS vs. HS) were demonstrated for uterine or isthmus mucosal enzyme activities or mineral contents, with one exception. Higher inorganic phosphorus concentrations were found in the uterus of HS egg layers when compared to levels in the uterus of SS egg layers and the isthmus of HS and SS egg layers. Acid phosphatase and carbonic anhydrase activities, and total calcium levels were significantly higher in the isthmus than the uterus, while alkaline phosphatase and pyrophosphatase activities, and inorganic phosphorus levels were significantly higher in the uterus than the isthmus.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 2999746 TI - Adrenal cortical response of tom poults. AB - In each of two trials, plasma corticosterone (B) was measured in Large White turkey tom poults after the following treatments were applied: 1) .9% saline injected; 2) cold water immersion, and 3) adrenocorticotropic hormone (ACTH) injected (10 IU/kg body weight). Poults were treated at 3- to 4-day intervals from the day of hatching to 21 days of age. Plasma samples were obtained at 1, 2, 3, 4, and 6 hr posttreatment. In both trials, there was a depression in B levels within the first 3 hr following ACTH or cold water immersion treatment. Significant increases in plasma B levels of the cold water treatment occurred at 4 hr posttreatment in Trial 1 in 7-day-old poults and in Trial 2 in 21-day-old poults. A significant adrenal cortical response to ACTH injection was observed in 3- and 7-day-old poults at 6 hrs posttreatment in Trial 2. Plasma B concentrations were also measured in three groups of nontreated Large White tom poults on the day of hatching at a commercial hatchery. Plasma samples were obtained from poults in incubators at 1000 hr, immediately following commercial processing procedures at 1030 hr, and at poult placement at 1330 hr. Plasma B levels of poults sampled in the incubator and after processing were similar. However, B levels of poults sampled at placement were increased significantly above the other two groups. PMID- 2999749 TI - [Multiple primary bronchial cancers: squamous epithelial cancer and metachronous small-cell cancer--a case report and literature review]. PMID- 2999748 TI - [Gyrase inhibitor in pneumology. Ofloxacin--a new broad spectrum antibiotic in acute and chronic respiratory tract infections]. PMID- 2999750 TI - [Studies using Ulex europaeus agglutinin I (UEA I) on normal tissue and synovial sarcomas, mesotheliomas and carcinomas]. PMID- 2999751 TI - [Primary malignant fibrous polymorphic histiocytoma of the aorta]. PMID- 2999753 TI - [Acute erythroblastopenia disclosing homozygous beta-thalassemia. Role of parvovirus infection]. AB - An acute, transient aplastic crisis in a 15-month old boy revealed the presence of homozygous beta-thalassaemia. The crisis was very likely due to a parvovirus infection, in view of the presence of specific IgM at the onset and of seroconversion to total antibodies. Later, requirements for transfusions were in favour of an intermediate type thalassaemia. The responsibility of the parvovirus is discussed in the light of recent data concerning the inhibitory action of this virus on bone marrow erythropoiesis. PMID- 2999752 TI - In vitro secretory patterns of human chorionic gonadotrophin, placental lactogen and pregnancy-specific beta 1-glycoprotein. AB - The control of secretion of the placental hormones human chorionic gonadotrophin (hCG) and human placental lactogen (hPL), and the trophoblastic protein pregnancy specific beta-glycoprotein (SP1), is not well understood. During pregnancy, the hCG concentrations peak in the first trimester then decrease, while hPL and SP1 increase steadily throughout gestation. In order to determine whether the discordance between hCG secretion and that of hPL and SP1 observed in vivo also occur in vitro, we cultured placental explants with and without dibutyryl cyclic AMP (dbcAMP) and theophylline. Between 5 and 12 explants were used for each treatment in each experiment. The concentration of the proteins secreted into the media each day was measured by specific radioimmunoassays. The quantities of hPL and SP1 secreted per day declined in a parallel fashion after 24 hours under both basal and dbcAMP-stimulated conditions. The hCG output progressively decreased in the unstimulated cultures until 48 hours, at which time an increase in hCG secretion was observed. The dbcAMP-stimulated placentae significantly increased their hCG output at both 48 and 72 hours. These data show that hCG secretion is regulated differently from that of hPL and SP1. The results do not negate the possibility that term placental tissue may contain an inhibitor of hCG release that is removed by experimental manipulation in vitro. PMID- 2999754 TI - [Elevation of serum angiotensin converting enzyme levels in Hodgkin's lymphoma]. PMID- 2999755 TI - [Insulinoma: diagnostic elements. 13 cases]. AB - The mean age of the 13 patients studied (9 women, 7 men) was 50.5 +/- 15.7 years. The disease was discovered on account of malaise (3 cases), behavioural disorders (4 cases), coma (3 cases), syncope (1 case) or right hemiparesis (1 case) or in the course of systematic examination (1 case). Eleven patients consulted for evaluation of hypoglycaemia and 2 for behavioural disorders. The history was characteristic, with malaise, loss of consciousness, severe neurological disorders (seizures, hemiparesis, hemiplegia or coma) and psychiatric disorders. These symptoms typically occurred in the morning before breakfast or between meals in 9 patients, and atypically at any point of time or after meals in 4 patients. Their hypoglycaemic nature was demonstrated by blood glucose determination in 11/13 cases and by response to ingestion of sugar in 12/13 cases. The mean period elapsed between the initial symptoms and the final diagnosis was 20.3 +/- 17.3 months. Inappropriate insulin secretion was elicited a.m. before breakfast, during Conn's diet or fasting test, or by calculating the blood insulin/glucose ratio or Turner's coefficient. Prior to surgery, the insulinoma was located by ultrasonography in 3/8 cases, by computerized tomography in 2/6 cases, by selective arteriography in 6/11 cases, and by phlebography with spleno-portal catheterization and staged sampling for insulin and C-peptide assays in 8/9 cases. Histological examination after surgery (11 cases) or necropsy (1 case) showed an adenoma without evidence of malignancy. PMID- 2999757 TI - [Hyperinsulinic euglycemic clamp. Another approach to the diagnosis of insulinoma]. PMID- 2999756 TI - [Meningoencephalitis in a primary cytomegalovirus infection. Localization to the brain stem with bilateral subclinical optic neuritis]. AB - A female adult without previous medical or surgical pathology nor specific treatment developed meningoencephalitis localised to the brainstem associated with bilateral subclinical optic neuritis. Serology showed that the disease was related to primary infection with cytomegalovirus. The patient spontaneously recovered. PMID- 2999758 TI - [Won't the metyrapone test have a place any more in the evaluation of anterior pituitary corticotropic deficiencies?]. PMID- 2999759 TI - [Use of Cibacron blue F 3GA-CL-sepharose 6B for the purification of T4 DNA- and RNA-ligases]. AB - The sorption capacity of the dye cibacron blue F3GA, immobilized on CL-Sepharose 6B and other support matrices, in respect to DNA- and RNA-ligases T4 was being studies. Cibacron blue F3GA immobilized on CL-Sepharose 6B binds a three-fold amount of DNA-ligase in comparison to RNA-ligase. The enzyme chromatography on cibbacron blue F3GA-CL-Sepharose 6B revealed a stronger linkage between DNA ligase T4 and the sorbent than between RNA-ligase T4 and the sorbent. Elution was performed with potassium chloride. DNA-ligase T4 was eluted with 0.25-0.5 M KCl and RNA-ligase T4 with 0.08-0.18 M KCl. Since deoxyexonuclease contaminants possess stronger bonds with the sorbent than ligases, elution of deoxyexonucleases occurs at higher concentrations of KCl. Chromatography of enzymes on cibacron blue F3GA-CL-Sepharose 6B allows one to obtain DNA- and RNA ligases essentially free of DNase and RNase contaminants. PMID- 2999760 TI - [Cytoreceptors in the molecular mechanisms of hormone action]. AB - The author considered and analysed the phenomenological model of the functional structural organization of a hormonal receptor molecule. He also defined the main types of hormone reception by target cells: intracellular and membranous. The paper presents some data on the structure and physicochemical characteristics of different hormone receptors, regularities and mechanisms of reception of steroid, thyroid and protein-peptide hormones (as well as catecholamines). Routes of the initiation of hormonal effects at the molecular level were shown. A special analysis was applied to the adenylate cyclase, calcium and protease routes of the initiation of hormonal effects from the cell surface. In conclusion uni- and multireceptor concepts of hormone multiple effects on cell metabolism were briefly defined. PMID- 2999761 TI - [Changes in the concentration of cyclic nucleotides in the blood of pulmonary tuberculosis patients during chemotherapy]. PMID- 2999762 TI - Dexamethasone suppression tests in psychiatry: is there a place for an integrated hypothesis? AB - The abnormal performance of the DST in depressive illness has been shown to be one of the most reproducible findings in biological psychiatry. Initial claims of its very high diagnostic specificity for the diagnosis of endogenous depression have not been substantiated: an abnormal response appears to reflect a biological dysfunction that cuts across the clinically established boundaries of psychiatric nosology. This lack of diagnostic utility does not reduce its prognostic value and abnormal DST response may indicate or reflect a versatile component in psychiatric disturbance and could serve therefore to predict or monitor the effects of physical and psychological intervention. Contributory factors to abnormal DST response are explored: factors such as stress, nutrition and age are reviewed and discussed. Concepts of biogenetic (neurohumoral) and psychological (psychodynamic and psychosocial) vulnerability and initiation/promotion are invoked and an integrated hypothesis is suggested: emotional strain provokes neurohumoral and neuroendocrine changes; these changes lead to vegetative disturbances including loss of appetite and weight with subsequent nutritional deficiencies that promote/reverse their neurohumoral and neuroendocrine changes. The role of 5-hydroxytryptamine is emphasized. Supportive evidence for aspects of this hypothesis is provided including animal studies and studies of the clinical and biological correlates of abnormal DST response. PMID- 2999763 TI - Cholera toxin inhibits chemotaxis by a cAMP-independent mechanism. AB - Cholera toxin inhibits chemotaxis of the RAW264 mouse macrophage cell line. The degree of inhibition by cholera toxin increases upon incubation with the cells, suggesting that the entry of the toxin is required for inhibition of chemotaxis. In the absence of guanine nucleotides, cholera toxin catalyzes the [32P]ADP ribosylation of RAW264 cell membrane proteins of Mr 41,000, Mr 45,000, and a doublet of Mr 48,000-50,000. GTP increases the labeling of the Mr 45,000 protein and the Mr 48,000-50,000 doublet, and it decreases the labeling of the Mr 41,000 protein. Experiments with cholera toxin treatment of intact cells indicate that the Mr 45,000 protein is the major membrane protein ADP-ribosylated by the toxin in vivo. Cholera toxin increases cAMP levels in RAW264 cells, but increased cAMP levels do not correlate with inhibition of chemotaxis, because isoproterenol and forskolin, which also increase cAMP levels, have no effect on chemotaxis. PMID- 2999764 TI - Differences in the side-chain metabolism of vitamin D3 between chickens and rats. AB - In vitro metabolism of 25-hydroxy-24-oxovitamin D3 was studied in kidney homogenates from vitamin D-supplemented chickens and rats. In chicken homogenates, 25-hydroxy-24-oxovitamin D3 was converted predominantly to 23,25 dihydroxy-24-oxovitamin D3, 24,25-dihydroxyvitamin D3, and 23,24,25 trihydroxyvitamin D3. In rat homogenates, 25-hydroxy-24-oxovitamin D3 was not converted to either 24,25-dihydroxyvitamin D3 or 23,24,25-trihydroxyvitamin D3, but it was converted to 23,25-dihydroxy-24-oxovitamin D3 and 23-hydroxy 24,25,26,27-tetranorvitamin D3. The latter metabolite was not produced by the chicken preparations. The stereochemical configuration at C-24 of the 24,25 dihydroxyvitamin D3 produced by chicken homogenates was determined to be S. This contrasts with the R configuration of 24,25-dihydroxyvitamin D3 produced by 24 hydroxylation of 25-hydroxyvitamin D3. These results suggest that chickens have an enzyme that can reduce the 24-oxo group to 24S-hydroxyl group, whereas rats do not. PMID- 2999765 TI - GTP-binding membrane protein of Escherichia coli with sequence homology to initiation factor 2 and elongation factors Tu and G. AB - The amino acid sequence of LepA protein, which has been shown to be cotranscribed with signal peptidase I in Escherichia coli, was compared with greater than 2000 known protein sequences. It was revealed that, of the 598 amino acid residues contained in LepA, an amino-terminal domain of 112 residues is homologous to a domain of similar size found in initiation factor 2, elongation factor Tu, and elongation factor G (IF2, EF-Tu, and EF-G), factors required for translation in E. coli. In this domain, 46 and 34 residues align perfectly with the corresponding regions of EF-G and EF-Tu, respectively. If functionally conserved residues within this domain (19 for EF-G and 17 for EF-Tu) are included, the overall resemblance is 58% and 46%, respectively, for EF-G and EF-Tu. A similar domain exists internally in IF2, where there is 42% overall resemblance with the domain of LepA. Immediately adjacent to this region is a small sequence of limited similarity that exists not only in EF-G, EF-Tu, and IF2 but also in the protooncogene c-Ha-ras-1 (from human bladder) and other GTP-binding proteins. Given these homologies, GTP-photoaffinity labeling and subcellular fractionation experiments were undertaken, and it was found that LepA is indeed a membrane bound GTP-binding protein. PMID- 2999766 TI - Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor. AB - In enteric bacteria the products of two nitrogen regulatory genes, ntrA and ntrC, activate transcription of glnA, the structural gene encoding glutamine synthetase, both in vivo and in vitro. The ntrC product (gpntrC) is a DNA-binding protein, which binds to five sites in the glnA promoter-regulatory region and appears to activate transcription initiation. Using as an assay the stimulation of glnA transcription in a coupled in vitro transcription-translation system, we have partially purified the ntrA gene product (gpntrA). The following evidence is consistent with the view that gpntrA is a sigma subunit for RNA polymerase: (i) The gpntrA activity copurifies with the sigma 70 holoenzyme (E sigma 70) and core (E) forms of RNA polymerase through several steps but can be separated from them by chromatography on heparin agarose. (ii) After further purification by molecular sieve chromatography, the partially purified gpntrA fraction allows transcription of glnA from the same startpoint used in vivo; transcription is dependent on gpntrC and on added E. The gpntrA fraction does not allow transcription from promoters that we have used as controls, including lacUV5. E sigma 70 has the reverse specificity. PMID- 2999767 TI - Genetic manipulation of membrane phospholipid composition in Escherichia coli: pgsA mutants defective in phosphatidylglycerol synthesis. AB - Unique mutants of Escherichia coli K-12, defective in phosphatidylglycerol synthesis, have been isolated from a temperature-sensitive strain incubated at its nonpermissive temperature. The parent strain had excess phosphatidylglycerol by harboring both the pss-1 allele [coding for a temperature-sensitive phosphatidylserine synthase (EC 2.7.8.8)] and the cls- allele (responsible for a defective cardiolipin synthase). The newly acquired mutations caused better growth at higher temperatures. One of the mutations (pgsA3) has been identified in the structural gene for phosphatidylglycerophosphate synthase [glycerophosphate phosphatidyltransferase (EC 2.7.8.5)]. Phospholipid compositions of these mutants were remarkable; phosphatidylethanolamine was the sole major lipid. In media with low osmotic pressures, these cells grew more slowly than the wild-type cells. They grew normally without recovering from the phospholipid abnormality in media appropriately supplemented with sucrose and MgCl2. Formation of cardiolipin and phosphoglycerol derivatives of membrane derived oligosaccharides was reduced in a pgsA3 mutant. E. coli strains having the pgsA3, pss-1, and cls- mutations, either individually or in combination, constitute an empirical system in which the molar ratio of three major membrane phospholipids can be variously altered. PMID- 2999768 TI - A human histone H4 gene exhibits cell cycle-dependent changes in chromatin structure that correlate with its expression. AB - By use of synchronized human HeLa S3 cells, a site sensitive to both DNase I and nuclease S1 was identified 50-150 base pairs upstream of the ATG codon of a cell cycle-dependent histone H4 gene. This site expanded to include a broad region of approximately equal to 300 base pairs sensitive to DNase I throughout S phase and then narrowed again to the original site after the completion of DNA replication. The level of nuclease S1 sensitivity was greatest during early S phase, when the gene is replicated and its transcription rate is maximal. The chromatin structure of the human beta-globin gene, which is not expressed in HeLa cells, was also analyzed throughout the cell cycle, and in no case was a sub-band seen as a result of DNase I or nuclease S1 digestion, nor were there any changes in nuclease sensitivity correlated with its replication. Thus the cell cycle dependent chromatin alterations in this histone H4 gene appear to be due to the coupled replication and expression of this gene rather than simply its replication. These results suggest that histone genes, as compared with developmentally regulated genes, exhibit an "intermediate" level of regulation whereby the gene is never in a completely inactive conformation, but changes in chromatin structure occur as a function of the cell cycle and expression. PMID- 2999769 TI - Incorporation of beef heart cytochrome c oxidase as a proton-motive force generating mechanism in bacterial membrane vesicles. AB - Membrane vesicles derived from the strictly fermentative lactic acid bacterium Streptococcus cremoris have been fused with proteoliposomes containing the beef heart mitochondrial cytochrome c oxidase by means of a freeze/thaw-sonication technique. Evidence that fusion has taken place was obtained by freeze-etch electron microscopy, showing a less-dense intramembranous particle distribution in the fused membranes than in the bacterial membranes, and by sucrose gradient centrifugation, indicating a buoyant density of the majority of the membranes after fusion that was between the buoyant densities of the starting membrane preparations. In the fused membranes, 55-60% of the cytochrome c oxidase molecules are oriented with the cytochrome c binding site at the outer surface of the membrane. With the electron-donor system ascorbate/N,N,N',N'-tetramethyl-p phenylenediamine/cytochrome c, a high proton-motive force (greater than 130 mV), inside negative and alkaline, can be generated in the fused membrane, and this proton-motive force can drive secondary transport of several amino acids. The procedure described can be used for incorporating a proton-motive force generating system in isolated membrane vesicles from bacterial or eukaryotic origin that lack a suitable primary proton pump. PMID- 2999770 TI - Inhibition of the Mg(II).ATP-dependent phosphoprotein phosphatase by the regulatory subunit of cAMP-dependent protein kinase. AB - We report potent inhibition of the Mg(II).ATP-dependent protein phosphatase, Fc.M, by the regulatory subunit dimer of type II cAMP-dependent protein kinase, RII2. The protein kinase catalytic subunit has no effect on phosphatase activity and is unable to substitute for kinase FA in the kinase FA- and Mg(II).ATP mediated phosphatase activation reaction. Phosphatase inhibition was investigated as a function of RII2 concentration. The results suggest that RII2 both inhibits the active phosphatase and inhibits phosphatase activation. The inhibition is shown to be noncompetitive with respect to substrate (phosphorylase a). The potential physiological significance of this inhibition is discussed in terms of phosphorylation/dephosphorylation cascade systems involving this kinase and phosphatase. PMID- 2999771 TI - A defined system for the DNA strand-transfer reaction at the initiation of bacteriophage Mu transposition: protein and DNA substrate requirements. AB - An early step in the transposition of bacteriophage Mu DNA in vitro is a DNA strand-transfer reaction that generates an intermediate DNA structure in which the Mu donor DNA and the target DNA are covalently joined. DNA replication, initiated at the DNA forks in this intermediate, generates a cointegrate product; simple insert products can also be formed from the same intermediate by degradation of a specific segment of the structure, followed by gap repair. This DNA strand-transfer reaction requires ATP, magnesium, the Mu A and Mu B proteins, and a factor supplied by an Escherichia coli cell extract. We have now shown that the host protein factor requirement can be satisfied by purified protein HU. The defined system has been used to determine the DNA substrate requirements for the reaction. The reaction requires the two Mu ends, located on the same DNA molecule, in the same relative orientation to one another as in the phage Mu genome. To participate in the strand-transfer reaction efficiently the mini-Mu plasmid, used as the transposon donor, must be supercoiled; the target DNA molecule may be supercoiled, relaxed circular, or linear. PMID- 2999772 TI - High-affinity receptors for peptides of the bombesin family in Swiss 3T3 cells. AB - Gastrin-releasing peptide (GRP) labeled with 125I at tyrosine-15 (125I-GRP) binds to intact quiescent Swiss 3T3 cells in a specific and saturable manner. Scatchard analysis indicates the presence of a single class of high-affinity binding sites of Kd = 0.5 X 10(-9) M and a value for the number of sites per cell of about 100,000. 125I-GRP binding was not inhibited by other mitogens for these cells, and cell lines that are mitogenically unresponsive to GRP do not exhibit specific GRP binding. Structure-activity relationships show a close parallel between the ability of a range of GRP-related peptides to both inhibit GRP binding and to stimulate mitogenesis. Further, GRP binding is selectively blocked in a competitive fashion by a novel bombesin antagonist, [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P. In addition, this compound selectively inhibits GRP and bombesin-induced mitogenesis. These results demonstrate that the mitogenic response of Swiss 3T3 cells to peptides of the bombesin family is mediated by a class of receptors distinct from those of other mitogens for these cells. PMID- 2999773 TI - Tumor necrosis factor: specific binding and internalization in sensitive and resistant cells. AB - Highly purified, Escherichia coli-derived recombinant human tumor necrosis factor (TNF) was labeled with 125I and employed to determine receptor binding, internalization, and intracellular degradation in murine L929 cells (highly sensitive to the cytotoxic action of TNF) and in diploid human FS-4 cells (resistant to TNF cytotoxicity). 125I-labeled TNF bound specifically to high affinity receptors on both L929 and FS-4 cells. Scatchard analysis of the binding data indicated the presence of 2200 binding sites per L929 cell and 7500 binding sites per FS-4 cell. The calculated dissociation constants are 6.1 X 10(-10) M and 3.2 X 10(-10) M for L929 and FS-4 cells, respectively. In both L929 and FS-4 cells, incubation at 37 degrees C resulted in a rapid internalization of the bulk of the cell-bound TNF, followed by the appearance of trichloroacetic acid-soluble 125I radioactivity in the tissue culture medium, due to degradation of TNF. Degradation but not cellular uptake of TNF was inhibited in the presence of chloroquine (an inhibitor of lysosomal proteases) in both L929 and FS-4 cells, suggesting that degradation occurs intracellularly, probably within lysosomes. These results show that resistance of FS-4 cells to TNF cytotoxicity is not due to a lack of receptors or their inability to internalize and degrade TNF. PMID- 2999774 TI - Polymorphism and evolution of Alu sequences in the human low density lipoprotein receptor gene. AB - Two clusters of Alu sequences in the human low density lipoprotein (LDL) receptor gene have been analyzed in detail. One Alu cluster is present within the intron separating exons 15 and 16 of the gene and contains a polymorphic Pvu II site. The presence or absence of this site gives rise to two allelic fragments of 14 and 16.5 kilobases, respectively, in genomic Southern blots using cloned cDNA probes. This DNA polymorphic site is caused by a single adenine to guanine transition within an Alu repetitive element. The second cluster of Alu sequences is located in exon 18 of the LDL receptor gene. Southern blotting of primate DNAs suggests that this cluster became associated with the gene about 30 million years ago. Comparison of bovine DNA sequences, which lack this Alu cluster, with those of the human indicates that the Alu sequences inserted in exon 18 in two independent events. PMID- 2999775 TI - Rapid change in mutation rate in a local population of Drosophila melanogaster. AB - The lethal and detrimental loads per second chromosome rapidly increased from 1968 to 1970 in a local population of Drosophila melanogaster in Japan (lethal load, from about 0.16 to 0.38; detrimental load, from 0.125 to 0.231 [Watanabe, T. K., Watanabe, T. & Oshima, C. (1976) Evolution 30, 109-118]). When the homozygous loads were measured in 1983, the lethal load had decreased to approximately the original amount (0.19) but the detrimental load had stayed high (0.241). The rise and fall of the lethal load can be accounted for by a P-type element that invaded a population with M cytotype, producing a high mutation rate. The mutation rate fell back to the earlier value after the cytotype became P. That the detrimental load did not decrease can be explained by assuming a longer persistence for detrimental mutations in the population. Evidence for a P type mutator factor is that the mutation rate of the wild-type chromosomes differs between the different cytoplasmic and chromosomal backgrounds, being lower in the background from which the chromosomes were taken. PMID- 2999776 TI - Primary structure of phage mu transposase: homology to mu repressor. AB - The phage Mu transposase is essential for integration, replication-transposition, and excision of Mu DNA. We present the complete nucleotide and derived amino acid sequence of the transposase and analyze implications for transposase/DNA interaction. The NH2 terminus of the Mu transposase has considerable sequence homology with the Mu repressor and with the NH2 terminus of the transposase of the Mu-like phage D108. These three proteins are known to share binding sites on DNA. The protein sequence and predicted secondary structural similarities at the NH2 termini of the three proteins suggest a common DNA-binding region similar to the regions found in proteins of known structure. An internal sequence in the Mu A protein also shares these features. We anticipate that these regions will be involved in DNA recognition during transposition. PMID- 2999778 TI - Identification of the porcine intestinal 1,25-dihydroxyvitamin D3 receptor on sodium dodecyl sulfate/polyacrylamide gels by renaturation and immunoblotting. AB - Identification of the porcine 1,25-dihydroxyvitamin D3 receptor protein on NaDodSO4/polyacrylamide slab gels was accomplished by two separate techniques: (i) assay of the specific binding activity of tritiated 1,25-dihydroxyvitamin D3 to protein eluted from NaDodSO4/polyacrylamide gels and renatured and (ii) immunoblotting of the partially purified receptor using two anti-receptor monoclonal antibodies. The porcine receptor preparation used in these studies was isolated from a crude nuclear extract of intestinal mucosa followed by chromatography on DNA-cellulose, ammonium sulfate precipitation, gel filtration HPLC, and DEAE-Sepharose chromatography. These receptor fractions were then electrophoresed on NaDodSO4/polyacrylamide gels. The receptor was eluted from the gel, renatured, and assayed for its ability to bind tritiated 1,25 dihydroxyvitamin D3. The renatured receptor appears as a single peak of specific tritiated 1,25-dihydroxyvitamin D3 binding activity. This binding activity corresponds to a band on a silver-stained gel that correlates with the receptor peak eluted from the DEAE-Sepharose column. It also corresponds to the highest molecular weight species identified on an immunoblot with anti-receptor monoclonal antibodies. The 1,25-dihydroxyvitamin D3 receptor protein has a molecular weight of 55,000 as deduced from its migration on NaDodSO4/polyacrylamide gels. PMID- 2999777 TI - Effects of intravenous and intraventricular injection of antisera directed against corticotropin-releasing factor on the secretion of anterior pituitary hormones. AB - To determine the physiological significance of corticotropin-releasing factor (CRF) in the control of pituitary hormone secretion, highly specific antibodies directed against the peptide were injected either intravenously or intraventricularly (third ventricle) and the effect on plasma levels of pituitary hormones was determined before and after application of ether stress for 1 min. The intravenous injection of CRF antiserum (0.5 ml) did not significantly alter basal corticotropin (ACTH) levels in freely moving ovariectomized rats but largely blocked the increase in plasma ACTH resulting from ether stress. These antibodies had no effect on the ether-induced decline in plasma growth hormone (GH), and they failed to modify plasma luteinizing hormone levels. In a second experiment, CRF antiserum (3 microliter) or normal rabbit serum was injected into the third ventricle. A blood sample was drawn 24 hr later and immediately thereafter another injection of CRF antiserum or normal rabbit serum was made. There was no modification in the level of any of the hormones 24 hr after the first injections, and they were similar in CRF antiserum and normal rabbit serum injected animals. After imposition of ether stress, the response of plasma ACTH was nearly completely blocked by the intraventricular CRF antiserum, but the degree of blockade was slightly less than that obtained by intravenous injection. The decline in plasma GH after ether stress was blocked by the intraventricular CRF antiserum. There was no effect of the intraventricular injection of the antiserum on the levels of the other pituitary hormones. The results with intravenous injection of the antisera indicate that CRF plays an extremely important but probably not completely indispensable role in the release of ACTH after ether stress. The results of the intraventricular injection of the antiserum suggest strongly that endogenous CRF may also modify its own release in response to stress, augmenting it by a positive ultrashort loop feedback, and that the antisera against the peptide blocked this action; however, an action at the pituitary of these intraventricularly injected antibodies cannot be completely ruled out. The blockade of the stress-induced suppression of GH release by the CRF antibodies suggests that CRF released intrahypothalamically during ether stress brings about an alteration in the hypothalamic control of GH secretion such that the stress-induced inhibition of GH release is blocked. PMID- 2999779 TI - Reconstitution of the GTP-dependent adenylate cyclase from products of the yeast CYR1 and RAS2 genes in Escherichia coli. AB - Plasmids carrying the CYR1 gene of yeast Saccharomyces cerevisiae, which encodes adenylate cyclase, were introduced into the cya mutant strain of Escherichia coli. The transformants had a GTP-independent adenylate cyclase activity but did not produce cAMP. The E. coli transformant carrying the yeast RAS2 or RAS2val19 gene had no adenylate cyclase activity. Transformant cells carrying both CYR1 and RAS2 produced GTP-dependent adenylate cyclase and cAMP, and those carrying CYR1 and RAS2val19 produced GTP-independent adenylate cyclase and a large amount of cAMP. Production of cAMP in the transformant carrying CYR1 and either RAS2 or RAS2val19 was confirmed by staining colonies on maltose-MacConkey plates and by measuring induction of beta-galactosidase by isopropyl beta-D thiogalactopyranoside. Mixing a crude extract from the E. coli transformant carrying CYR1 with a crude extract from cells carrying RAS2 reconstituted the GTP dependent adenylate cyclase. Reconstitution of the GTP-dependent adenylate cyclase was observed by mixing the plasma membrane fraction of yeast CYR1 ras1 ras2 bcy1 mutant and a crude extract from the E. coli transformant carrying RAS2 or by mixing a crude extract from the E. coli transformant carrying CYR1 and the membrane fraction of yeast cyr1 RAS1 RAS2 BCY1 mutant. The data suggest that the yeast GTP-dependent adenylate cyclase consists of catalytic and regulatory subunits encoded by the CYR1 and RAS2 genes, respectively. PMID- 2999780 TI - Expression of murine 21-hydroxylase in mouse adrenal glands and in transfected Y1 adrenocortical tumor cells. AB - The S region of the murine major histocompatibility complex contains two structurally related genes (21-OHase A and 21-OHase B) that encode 21-hydroxylase (21-OHase), an enzyme essential for the synthesis of adrenal steroids. Expression of these two genes has been analyzed by using oligonucleotide probes specific for the 21-OHase A and B genes and by DNA-mediated gene transfer. Hybridization of the oligonucleotides to blots of BALB/c adrenal RNA demonstrated that all 21 OHase mRNA is derived from the 21-OHase A gene. Cosmids bearing either the 21 OHase A or B gene were introduced into Y1 adrenocortical tumor cells by cotransfection with pSV2-neo. Cells transfected with the 21-OHase A gene expressed 21-OHase as determined by steroid metabolism and by RNA blot hybridization; 21-OHase transcripts were not detected in parent Y1 cells or in cells transfected with the 21-OHase B gene. Treatment of 21-OHase A transfectants with adrenocorticotropin increased 21-OHase mRNA levels by up to 10-fold, thus mimicking the observed effect of this hormone on 21-OHase levels in primary adrenal cultures. The regulated expression of the 21-OHase A gene in transfected Y1 cells should provide a useful system for the investigation of factors controlling the adrenal-specific regulation of 21-OHase activity. PMID- 2999782 TI - Lysine residue 121 in the proposed ATP-binding site of the v-mos protein is required for transformation. AB - The transforming gene product encoded by Moloney murine sarcoma virus clone 124, p37mos, contains a lysine residue (lysine-121) that is conserved among all members of the protein kinase family. This lysine has been shown to be part of a conserved ATP-binding site in both the catalytic subunit of the cAMP-dependent protein kinase and p60v-src. We wished to determine whether this lysine is required for the transforming activity of p37mos. Two site-specific mutations were therefore constructed, which result in the substitution of an aspartic acid or arginine codon in place of the codon for lysine-121. Both mutations abolished the ability of the mos gene to transform cells. These results show that lysine 121 is required for the ability of p37mos to transform cells and provide evidence for an ATP-binding site in p37mos. Furthermore, these results suggest that the conserved lysine residue is specifically involved in the catalytic activity of protein kinases in general. PMID- 2999781 TI - Identification and some biochemical properties of the major XBL gene product of bovine leukemia virus. AB - Using a rabbit antiserum directed against a synthetic oligopeptide whose sequence was deduced from the nucleotide sequence of the XBL gene of bovine leukemia virus, we detected a 38-kDa protein in virus-producing cell lines. In vitro translation of hybrid-selected RNA unequivocally demonstrates that this protein, designated p38(XBL), is indeed encoded by the XBL gene. Unlike the other virus encoded proteins, however, p38(XBL) resides within the cells without being incorporated into virions. It undergoes no gross post-translational modifications and has a relatively short half-life (5-6 hr) in vivo. Furthermore, cell fractionation combined with pulse-chase experiment reveals that a significant fraction (more than half) of the p38(XBL) localizes to the nucleus of the infected cell after synthesis. We conclude that the XBL gene of bovine leukemia virus is a functional gene encoding a nonvirion protein p38(XBL), which possibly functions within the nucleus of the infected cell to regulate viral or cellular gene expression. p38(XBL) is presumably translated from a doubly spliced, bicistronic mRNA that has the capability to encode another small polypeptide in a different reading frame. PMID- 2999783 TI - cDNA cloning of a human autoimmune nuclear ribonucleoprotein antigen. AB - Sera from patients with systemic lupus erythematosus and other autoimmune disorders contain antibodies against nuclear proteins. One such autoantibody system, known as Sm, reacts with antigens associated with small nuclear RNA molecules. In this paper we report the use of Sm autoantibodies to isolate a cDNA clone for the mRNA of one of these nuclear antigens. A HeLa cell cDNA library was screened by message selection followed by autoantibody reaction of cell-free translation products. This led to the identification of a cDNA clone, p281, containing sequences complementary to mRNA for an Sm autoantibody-reactive, 11,000 Mr protein. This cloned Sm antigen comigrated with the small nuclear RNA associated protein known as "E" and reacted with four out of four Sm autoantibodies that precipitate E protein from total mRNA translation products. RNA gel blot hybridization with clone p281 DNA revealed a poly(A)+ mRNA of approximately equal to 600 nucleotides in human and marmoset (New World primate) cells. Southern blot hybridization of HeLa cell and human lymphocyte DNA indicated the presence of 6-10 copies of p281-homologous sequences. Similar copy numbers were observed with genomic DNA from baboon, cat, and mouse, indicating that the Sm antigen mRNA sequence represented in p281 is conserved across three classes of the Mammalia (primates, carnivores, and rodents). However, no cross hybridization of p281 was observed with frog or Drosophila DNA. In light of existing evidence that the mammalian Sm antigen E is a weaker autoantigen than other small nuclear RNA-associated proteins, these results suggest a possible correlation between a protein's capacity to serve as an autoantigen during breakdown of the host's immunological tolerance and its extent of evolutionary conservation, whereas the inverse relationship applies to conventional immunity. We suspect, as have others, that this is a clue to the mechanism of autoimmunity. PMID- 2999784 TI - Direct mapping of adeno-associated virus capsid proteins B and C: a possible ACG initiation codon. AB - The three major capsid proteins of adeno-associated virus type 2 (AAV2) virions are designated A, B, and C and have molecular sizes of 90, 72, and 60 kDa, respectively. These proteins are related, and genetic studies have shown they are encoded by a long open reading frame located in the right half of the genome. The coding capacity distal to the first ATG in this reading frame is only 503 amino acids (i.e., a protein about the size of protein C), but an open frame sequence devoid of ATG codons extends upstream for an additional 184 codons. Although the amino terminus of the C capsid protein is blocked, partial amino acid sequence analyses of peptides from C have confirmed that it is encoded within the portion of the reading frame distal to the first ATG at nucleotide (nt) location 2810. The amino terminus of the B capsid protein is not blocked, and its sequence begins with alanine. The triplet encoding this alanine lies 64 codons upstream from the initiation site for protein C and is immediately preceded by the threonine codon, ACG, at nt 2615. This ACG codon lies in the most favorable sequence context for protein synthesis initiation. All three AAV2 capsid proteins are labeled in vitro with formyl[35S]methionyl-tRNAf, indicating that synthesis of each protein is initiated independently. Our data suggest that the nt 2615 ACG codon directs the methionyl-tRNA-dependent initiation of the AAV2 B capsid protein. Proteins B and C may be synthesized from the same mRNA species and their relative abundance could be determined by the efficiencies of their respective initiation codons. PMID- 2999785 TI - A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase II. AB - Purified eukaryotic nuclear RNA polymerase II consists of three subspecies that differ in the apparent molecular masses of their largest subunit, designated IIo, IIa, and IIb for polymerase species IIO, IIA, and IIB, respectively. Subunits IIo, IIa, and IIb are the products of a single gene. We present here the amino acid composition of calf thymus subunits IIa and IIb and the C-terminal amino acid sequence of subunit IIa (IIo) inferred from the nucleotide sequence of part of the mouse gene encoding this RNA polymerase subunit. The calculated amino acid composition of the peptide unique to subunit IIa indicates that subunit IIa contains a domain rich in serine, proline, threonine, and tyrosine. The sequence at the 3' end of the mouse RNA polymerase II largest subunit gene reveals that the C-terminal domain consists of 52 repeats of a seven amino acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This sequence is also unusual in that it contains a high percentage of potential phosphorylation sites. PMID- 2999786 TI - Binding of pea cytochrome f to the inner membrane of Escherichia coli requires the bacterial secA gene product. AB - Various sequences from the 5' end of the pea chloroplast gene for cytochrome f have been fused in the correct reading frame with lacZ, and the cellular location of the hybrid polypeptides in Escherichia coli has been examined. Hybrid polypeptides containing N-terminal parts of cytochrome f are located in the cytoplasmic membrane of E. coli. Membrane localization is most efficient when the intact signal sequence of cytochrome f is present at the N-terminal end of the fusion proteins. Fusion within the signal sequence, so that the processing site is absent, reduces the efficiency of membrane binding. Membrane insertion of fusion proteins containing signal sequences is prevented in a temperature sensitive secA strain at the nonpermissive temperature and the hybrid proteins accumulate in the cytoplasm. This indicates that specific recognition of the chloroplast signal sequence occurs in the bacterial secretory pathway. PMID- 2999787 TI - Sequence and mapping analyses of the herpes simplex virus DNA polymerase gene predict a C-terminal substrate binding domain. AB - The herpes simplex virus DNA polymerase provides an excellent model for studies of eukaryotic replicative polymerases. We report here the nucleotide sequence of the gene which encodes this enzyme. The gene includes a 3705-base-pair major open reading frame capable of encoding a Mr 136,519 polypeptide, in rough agreement with previous estimates of the size of the major polypeptide found in partially purified viral polymerase preparations. The predicted polymerase polypeptide shares extensive sequence homology with the Epstein-Barr virus open frame predicted to encode DNA polymerase and with a 13-amino acid segment of adenovirus 2 DNA polymerase. Mutations conferring altered sensitivity to antiviral deoxynucleoside triphosphate analogs, pyrophosphate analogs, or aphidicolin from eight different mutants map within the region encoding the carboxyl-terminal portion of the predicted polymerase polypeptide. Two of these are separated by a distance corresponding to at least 228 amino acids. We propose that this region of the gene encodes a polypeptide domain that contains the binding sites for deoxynucleoside triphosphates and pyrophosphate. PMID- 2999788 TI - Expression of a cDNA encoding a functional 241-kilodalton vesicular stomatitis virus RNA polymerase. AB - The large gene, L, of vesicular stomatitis virus (VSV), which codes for the multifunctional RNA-dependent RNA polymerase, was assembled from five overlapping cDNA clones. The sequence of the 6.4-kilobase gene of the final construct was identical to the consensus sequence reported earlier. The gene was inserted into the simian virus 40 transient expression vector pJC119. Antibodies directed against synthetic peptides corresponding to the amino and carboxyl termini of the L protein were raised in rabbits. Both antibodies specifically immunostained the cytoplasm of COS cells that had been transfected with the vector DNA. The expressed L protein was immunoprecipitated from cell extracts and it was identical in size to the L protein of the virion (241 kilodaltons). Most importantly, COS cells that expressed the recombinant L protein transcribed, replicated, and consequently complemented and rescued temperature-sensitive RNA polymerase mutants of VSV at the nonpermissive temperature. The kinetics of virus release were similar to those of a wild-type VSV infection. We conclude that the recombinant RNA polymerase protein L is indistinguishable in its size and its functions from the VSV polymerase. PMID- 2999789 TI - Homologies between the Salmonella typhimurium CheY protein and proteins involved in the regulation of chemotaxis, membrane protein synthesis, and sporulation. AB - Chemotactic receptors at the bacterial cell surface communicate with flagellar basal structures to elicit appropriate motor behavior in response to extracellular stimuli. Genetic and physiological studies indicate that the product of the cheY gene interacts directly with components of the flagellar motor to control swimming behavior. We have purified and characterized the Salmonella typhimurium CheY protein and have determined the nucleotide sequence of the cheY gene. Amino acid sequence comparisons showed CheY to be homologous over its entire length (129 residues) to the N-terminal regulatory domain of another protein involved in chemotaxis, the CheB methyl esterase. The entire CheY protein and the regulatory domain of CheB also homologous to the N-terminal portions of the Escherichia coli OmpR and Dye proteins and the Bacillus subtilis Spo0A protein. These homologies suggest an evolutionary and functional relationship between the chemotaxis system and systems that are thought to regulate gene expression in response to changing environmental conditions. PMID- 2999790 TI - Activation of Na+/H+ exchange in cultured fibroblasts: synergism and antagonism between phorbol ester, Ca2+ ionophore, and growth factors. AB - The effects of phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C, on Na+ influx were investigated in cultured human foreskin fibroblasts (HSWP cells). We report here that in serum-deprived HSWP cells the addition of PMA alone has no significant effect on Na+ influx. However, the addition of PMA to cells whose Na+/H+ exchanger is partially activated with a submaximal dose of the Ca2+ ionophore A23187 leads to a larger stimulation than seen with A23187 alone. These data suggest that although stimulation of protein kinase C is not a sufficient signal to activate the Na+/H+ exchanger in HSWP cells or in another human foreskin line (Jackson fibroblasts) studied, there are some cooperative effects of protein kinase C activation with a rise in Ca2+ to stimulate Na+/H+ exchange. In addition, we found that PMA actually inhibits the mitogen-induced stimulation of Na+ influx in HSWP and Jackson fibroblasts. This observation strengthens the argument that in these cells activation of protein kinase C is not sufficient to activate Na+/H+ exchange and suggests that there is a negative feedback control via protein kinase C that inhibits some signal that is necessary for activating Na+/H+ exchange. However, in contrast to observations in HSWP cells, we were able to activate the Na+/H+ exchanger in mouse 3T3 and human WI-38 cells with PMA alone, suggesting that there is some diversity in the mechanism for activation of Na+/H+ exchange in different types of fibroblasts. PMID- 2999791 TI - Cellular and viral DNA hypomethylation associated with induction of Epstein-Barr virus lytic cycle. AB - Epstein-Barr virus (EBV) producer and nonproducer cell lines have been treated with a combination of phorbol 12-myristate 13-acetate and n-butyrate (sodium salt). These inducers caused a massive hypomethylation of the EBV producer line P3HR-1 DNA (about 30%) at the time when DNA replication was inhibited. The viral DNA in these cells is heavily methylated as judged by digestion with Hpa II and probing with the Bam HI H fragment of EBV. However, upon induction with phorbol 12-myristate 13-acetate and n-butyrate, total hypomethylation of this viral DNA region was observed within 24 hr. This hypomethylation preceded EBV amplification, which became apparent only 32-36 hr after induction. When induction was carried out in the presence of retinoic acid, hypomethylation of cellular and viral DNA, viral DNA amplification, and production of the viral early antigen and viral capsid antigen were substantially inhibited. EBV DNA in another producer line (Jijoye nude) and in the nonproducer line Raji was hypomethylated and did not undergo further hypomethylation in response to induction. The observed hypomethylation of P3HR-1 and EBV DNA in the absence of DNA replication suggests that it is achieved by an active demethylation mechanism. This changes our perception of the DNA methylation phenomenon, since it has been generally accepted that hypomethylation of DNA takes place by a passive mechanism that involves DNA replication in the absence of methylation. PMID- 2999792 TI - Polymorphic restriction endonuclease sites linked to the HLA-DR alpha gene: localization and use as genetic markers of insulin-dependent diabetes. AB - Polymorphic restriction endonuclease sites within the HLA-DR alpha gene have been defined, localized, and used as genetic markers in the analysis of susceptibility to insulin-dependent diabetes mellitus (IDDM). Hybridization of Bgl II-digested human genomic DNA with a cDNA clone for the HLA-DR alpha chain (pDR alpha-1) has revealed three allelic restriction fragment lengths: 3.8 kilobase pairs (kb), 4.2 kb, and 4.5 kb. Hybridization of EcoRV-digested human genomic DNA with the same probe has revealed two allelic polymorphic restriction fragment lengths: 9.2 kb and 13.0 kb. By analysis of double digests of genomic DNA from individuals homozygous for each of the allelic variants, the polymorphic restriction sites were found to be clustered near the 3' end of the HLA-DR alpha gene. The observed correlations of DR alpha Bgl II restriction site variants with serologically determined DR specificities suggest linkage disequilibrium between the DR alpha and DR beta loci. The 3.8-kb fragment is correlated with the DR1 type (Pc = 4.4 X 10(-4)); and the 4.2-kb fragment, with a subset (B8,DR3) of the DR3 type (Pc = 5.1 X 10(-4)) and with the DR6 type. The segregation pattern of HLA-DR alpha polymorphic Bgl II restriction fragments was analyzed in six IDDM families. The observed association of IDDM with the Bgl II 4.2-kb DR alpha restriction variant is higher than with existing serological markers and supports the utility of this approach in elucidating IDDM inheritance. PMID- 2999793 TI - Transposase titration in Drosophila melanogaster: a model of cytotype in the P-M system of hybrid dysgenesis. AB - In the P-M system of hybrid dysgenesis of Drosophila melanogaster, some M strains possess chromosomal P elements. The chromosomes from one of these pseudo-M strains reduced the instability of a P element-insertion mutation of the singed bristle locus. We suggest that this reduction is an indication of competition for a transposase that binds to the P elements on the chromosomes from the pseudo-M strain as well as to the P elements at the singed locus; the pseudo-M strain's P elements might therefore be said to titrate the transposase, reducing its availability to interact with the P elements at the singed locus. We hypothesize that a similar mechanism regulates the movement of P elements in the P strains of D. melanogaster, although in this case we propose that the titrating elements are extrachromosomal and that they are generated by the action of the transposase itself. PMID- 2999794 TI - TnphoA: a transposon probe for protein export signals. AB - We constructed a derivative of transposon Tn5 that permits the generation of hybrid proteins composed of alkaline phosphatase (EC 3.1.3.1) lacking its signal peptide fused to amino-terminal sequences of other proteins. Such a hybrid gives alkaline phosphatase activity if the protein fused to alkaline phosphatase contributes sequences that promote export and thus compensate for the missing alkaline phosphatase signal peptide. Fusions to both a secreted periplasmic protein and a complex cytoplasmic membrane protein led to alkaline phosphatase activity. TnphoA fusions should help localize export signals within the structure of a protein, such as a transmembrane protein, as well as identify new chromosomal genes for secreted and transmembrane proteins. PMID- 2999795 TI - Identification of a polymorphic variant associated with HLA-DQw3 and characterized by specific restriction sites within the DQ beta-chain gene. AB - Restriction endonuclease digestion of genomic DNA from 24 lymphoblastoid cell lines homozygous for the HLA class II specificity DQw3, followed by hybridization with a DQ beta-chain cDNA probe, identified a genomic polymorphism with variable BamHI and HindIII recognition sites. This restriction fragment pattern was found for several haplotypes associated with the DQw3 specificity, including some haplotypes positive for the HLA-DR specificities DR4, DR5, DRw8, and DRw12. The variant fragment pattern corresponds precisely with the reactivity of a monoclonal antibody, A-10-83, previously shown to define a serologic split of DQw3. Serologic detection of the specific DQw3.1 genomic polymorphism indicated that the corresponding DQ beta-chain variants are expressed. This polymorphic restriction fragment pattern, then, represents a selective marker for DQ beta chain genes that appear to define a DQ beta-chain-associated specificity, here called DQw3.1. PMID- 2999796 TI - Intracellular class II HLA antigens are accessible to transferrin-neuraminidase conjugates internalized by receptor-mediated endocytosis. AB - Newly synthesized class II HLA antigens being transported to the surface of human B-lymphoblastoid cell lines (B-LCL) interact with transferrin-neuraminidase conjugates internalized by means of receptor-mediated endocytosis. Class II antigens, isolated from [35S]methionine-labeled B-LCL after incubation with the conjugates at 37 degrees C, showed extensive desialylation of associated invariant chain and detectable loss of beta-subunit sialic acid on analysis by two-dimensional gel electrophoresis. An equal amount of unconjugated neuraminidase had no effect, and desialylation of class II antigen components was blocked when access of transferrin-neuraminidase conjugates to the B-LCL transferrin receptors was competitively inhibited by the addition of excess iron saturated transferrin. The conjugates were shown to cycle through the cells in the same way as unconjugated transferrin, being first internalized and then rapidly secreted in an undegraded form. The data suggest that the exocytic pathway taken by class II antigens intersects the route followed by recycling transferrin receptors and that the interaction occurs prior to the dissociation of the invariant chain from the class II antigen complex. Similar intracellular interactions between class II molecules and foreign proteins internalized by antigen-presenting cells may be important in class II antigen-restricted recognition by helper T lymphocytes. PMID- 2999797 TI - Influence of the human T-lymphotropic virus/lymphadenopathy-associated virus on functions of human lymphocytes: evidence for immunosuppressive effects and polyclonal B-cell activation by banded viral preparations. AB - The etiologic agent for the acquired immunodeficiency syndrome (AIDS) is now firmly established as the retrovirus termed the human T-lymphotropic virus type III (HTLV-III) or the lymphadenopathy-associated virus, LAV. The disease is characterized by profound and progressive loss of immunity, but molecular evidence indicates that only a few cells in peripheral blood are being productively infected with this virus. In the present study we have investigated a disrupted HTLV-III viral preparation for biologic effects on normal lymphoid cells. Relatively dilute concentrations of this preparation were found to stimulate immunoglobulin secretion by peripheral blood lymphocytes; at the same dosages, the preparation was inhibitory for the B-cell differentiation responses that are induced with other known polyclonal B-cell activators, pokeweed mitogen, Staphylococcus aureus, and Epstein-Barr virus. This preparation was also inhibitory at high concentrations for T-lymphocyte proliferative responses to phytomitogens and antigens and resulted in a reduced expression of Tac antigen on phytohemagglutinin-activated lymphocytes. Paradoxically, incubation of lymphocytes of certain healthy donors with the HTLV-III preparation alone resulted in increased expression of Tac and Leu-12 antigens. These findings show that a disrupted preparation of HTLV-III virus can mimic many of the immunologic abnormalities present in patients with HTLV-III infection. Nonviable viral proteins may be responsible for some of the immunologic perturbations that occur in HTLV-III-infected states. PMID- 2999798 TI - The putative transforming protein of S13 avian erythroblastosis virus is a transmembrane glycoprotein with an associated protein kinase activity. AB - S13 is an avian retrovirus that transforms both fibroblasts and erythroblasts. The gene product responsible for the oncogenic effects of S13 is the env-related glycoprotein gp155. In this report we show that gp155 is a transmembrane protein with a 55-kDa cytoplasmic domain. Pulse-chase analysis shows that gp155 was cleaved posttranslationally into two glycosylated proteins, gp85 and gp70. In addition, we show that a tyrosine protein kinase activity is associated only with the gp70 protein in microsomes and in immune complexes. PMID- 2999800 TI - Supplementation of the diet with eicosapentaenoic acid: a possible approach to the treatment of thrombosis and inflammation. PMID- 2999799 TI - Corticotropin-releasing factor-induced adrenocorticotropin hormone release and synthesis is blocked by incorporation of the inhibitor of cyclic AMP-dependent protein kinase into anterior pituitary tumor cells by liposomes. AB - Corticotropin-releasing factor (CRF) is the most potent and effective natural stimulant of corticotropin (ACTH) secretion. In a tumor cell line of the mouse anterior pituitary (AtT-20/D16-16) consisting of a homogeneous population of corticotrophs, CRF is known to increase adenylate cyclase and cAMP-dependent protein kinase activities as well as to release ACTH. To determine whether activation of cAMP-dependent protein kinase is essential for CRF to evoke the secretion of ACTH, an inhibitor (PKI) of this kinase was inserted into AtT-20 cells. This was accomplished by first encapsulating PKI into liposomes and then covalently coupling them to protein A for binding to antibodies directed against an AtT-20 cell surface antigen, N-CAM (neural cell adhesion molecule). The binding of the liposomes to the anti-N-CAM antibodies led to the internalization of the PKI into the tumor cells. The PKI treatment greatly attenuated CRF stimulated ACTH release as well as the secretory response to beta-adrenergic agonists. However, ACTH release in response to caerulein, an agonist of cholecystokinin 8 receptors, was not altered by the PKI treatment. CRF treatment also increased the levels of mRNA for proopiomelanocortin (POMC), the precursor for ACTH in AtT-20 cells. Application of liposomes containing PKI to AtT-20 cells blocked the ability of CRF and 8-bromo-cAMP, but not phorbol ester, to increase POMC mRNA levels. The results revealed an essential role for cAMP in mediating the effect of CRF on ACTH release and POMC gene expression. PMID- 2999801 TI - Temperature responses in restrained and unrestrained rats to the selective mu opioid agonist, DAGO. PMID- 2999802 TI - Comparison of mu opioid agonists in vitro. PMID- 2999803 TI - Comparison of the antinociceptive and antitransit effects of DAGO and PLO17 in mice. PMID- 2999804 TI - Beta-adrenergic responses of facial vein from cold acclimated rabbits. PMID- 2999806 TI - Opiate agonist activity of various substituted 3-amino-2,2-dimethyltetralins in the guinea pig ileum. PMID- 2999805 TI - GABAergic involvement in the antinociceptive effects of morphine at the level of the periaqueductal gray matter in the rat. PMID- 2999807 TI - Voltage dependent changes in opiate inhibition of electrically-driven guinea pig ileum longitudinal muscle. PMID- 2999809 TI - Calmodulin inhibitors depress neuromuscular transmission. PMID- 2999808 TI - Effects of dietary lipids on cardiovascular parameters in the conscious rat and adrenergic responses in isolated perfused organ systems. PMID- 2999810 TI - The nature of opiate dependence. PMID- 2999811 TI - Site-specific promoter methylations and gene inactivation. PMID- 2999812 TI - Methylation of the PGK promoter region and an enhancer way-station model for X chromosome inactivation. PMID- 2999813 TI - In vitro methylation of B-DNA and Z-DNA. PMID- 2999814 TI - Human DNA methylation: methylated DNA-binding protein, differentiation and cancer. PMID- 2999815 TI - Methylation directed strand discrimination in mismatch repair. PMID- 2999816 TI - A. N. Richards lecture. Leukotrienes: possible mediators of disease. PMID- 2999817 TI - Leukotrienes in myocardial ischemia. PMID- 2999818 TI - Mast cells and mast cell mediators in models of airway disease. PMID- 2999819 TI - Modulation of the generation and release of leukotrienes from leukocytes. PMID- 2999820 TI - Leukotriene receptors. PMID- 2999822 TI - Endothelial cells metabolize but do not synthesize leukotrienes. PMID- 2999821 TI - Role of complement anaphylatoxins in neutrophil arachidonic acid metabolism. PMID- 2999823 TI - Pulmonary responses to exogenous leukotrienes. PMID- 2999824 TI - Effects of exogeneous leukotrienes on the pulmonary circulation. AB - This review illustrates the diverse effects of LTB4 and the peptidoleukotrienes (LTC4 and LTD4) on pulmonary hemodynamics and lung fluid balance. LTB4 had a lesser effect on pulmonary hemodynamics compared to the peptidoleukotrienes. The small increases in pulmonary artery pressure and pulmonary vascular resistance were the result of precapillary constriction associated with increases in effluent thromboxane concentration. On the other hand, peptidoleukotrienes resulted in marked increases in thromboxane concentrations, pulmonary artery pressure, pulmonary capillary pressure, and pulmonary vascular resistance. Increased thromboxane generation probably contributes to the noted hemodynamic alterations following peptidoleukotriene administration. Moreover, in the isolated perfused guinea pig lung, there was also a direct effect of LTD4 on pulmonary hemodynamics which was not blocked by cyclooxygenase inhibition. This latter, resistant vasoconstrictor response is analogous to the LTD4-mediated pulmonary vasoconstriction in some species which is not dependent on cyclooxygenase (Kadowitz et al., 1984). The primary site of vasoconstriction with LTD4 was in the postcapillary vessels in contrast to the LTB4 response. LTB4 increased lung vascular permeability directly (perhaps by a direct endothelial contracture) and may also contribute to increase in permeability via neutrophil activation. However, the neutrophil dependent permeability-increasing response could not be demonstrated in the isolated perfused guinea pig lung; thus, this response requires further study. In contrast, LTC4 and LTD4 had no effect on lung vascular permeability in the awake sheep, isolated perfused guinea pig lung, and the cultured bovine pulmonary endothelial monolayer system. PMID- 2999825 TI - Measurement of leukotrienes. PMID- 2999826 TI - Interactions between leukotrienes and other eicosanoids. PMID- 2999827 TI - On the inotropic effects of leukotrienes in the isolated urinary bladder of guinea pigs and rats. AB - Leukotrienes (LTs) B4, C4 and D4 were tested on the motility of rings isolated from the urinary bladder of guinea pigs and rats. LTB4 did not evoke inotropic influences in any of the preparations, whereas LTC4 and LTD4 augmented the magnitudes of tonic and phasic contractions in the guinea pig but not in the rat detrusor muscle, LTC4 evoking a greater enhancement than LTD4. On molar bases, acetylcholine induced smaller positive inotropic effects. In the presence of 10( 4) acetylsalicylic acid (ASA), cumulative dose-response curves of phasic and tonic contractions for LTC4 were shifted to the right of controls, whereas curves of the phasic motility for LTD4 remained unaltered. However, ASA shifted to the left the dose-response curve of tonic contractions for LTD4 and in addition evoked an augmentation of the absolute developed tension. The initial (postequilibrium) contractile levels of basal phasic contractions did not differ between controls and preparations incubated with ASA (10(-4) M), nordihydroguaiaretic acid (10(-7) M) or FPL-55712 (3 X 10(-6) M). The findings suggest that some musculo-active prostanoid(s) could be modulating, in opposite directions, the smooth muscle contractile reactivity of the guinea pig urinary bladder when challenged in vitro with LTC4 and LTD4. Our data reported hereing also suggest that the role, if any, of endogenous prostanoids and leukotrienes for the normal basal contractile functioning of the guinea pig urinary bladder, remains obscure. PMID- 2999828 TI - Effects of gamma-linolenic acid, dihomo-gamma-linolenic acid and ethanol on cultured human mammary carcinoma cells. AB - A number of fatty acids have been shown to inhibit the growth of malignant cells in vitro. In particular, gamma-linolenic acid (GLA) has been proposed to act as a precursor for the production of prostanoids especially prostaglandin E1 (PGE1). To test this hypothesis, the effects of GLA on cultured human breast carcinoma cells were compared with those of dihomo-gamma-linolenic acid (DGLA) the metabolite of GLA and the immediate precursor of PGE1. The influence of ethanol (which has been shown to enhance conversion of DGLA to PGE1) on the actions of each of the fatty acids was also investigated. In contrast to the inhibitory effects observed with all concentrations of GLA cell growth was promoted by the presence of 50 micrograms DGLA. Ethanol reduced the action of both GLA and DGLA possibly due to some physicochemical reaction between the alcohol and the fatty acids. The fact that the actions of GLA were not mimicked by DGLA which is the next step towards PG production casts doubt upon the role of PGE1 as mediator of the effects which have been observed with GLA in malignant cells. PMID- 2999829 TI - Modulation of growth, prostaglandin synthesis, and prolactin-binding in two cultured rat mammary carcinoma cell lines by flurbiprofen. AB - The effect of treatment with flurbiprofen (FB), a non-steroidal anti-inflammatory drug, on growth, prostaglandin synthesis, and prolactin receptor levels was examined in two established rat mammary carcinoma cell lines. Growth of NMU cells was suppressed with a concentration of 1 microgram FB/ml culture medium (4 x 10( 5) M); RBA cells, in contrast, were less sensitive, being inhibited only by a 100 micrograms/ml (4 x 10(-4) M) concentration of the drug. Prostaglandin (PG) synthesis by both cells lines, as indicated by decreased release of PGE2 and PGF2 alpha into the culture medium, was inhibited by 0.1, 1 and 10 micrograms/ml of FB. Both carcinoma cell lines exhibited high levels of specific prolactin receptors (PRLR) (9-11,000 sites/cell); binding was diminished in cells exposed to 1 microgram/ml (4 x 10(-6) M), and abolished completely by 10 micrograms/ml (4 x 10(-5) M) of FB. In marked contrast to the results at higher concentrations, at 0.1 microgram/ml (4 x 10(-7) M), the drug caused a significant increase in the prolactin binding capacity of RBA cells and a diminution in PG production, but in the absence of any measurable effect on cell proliferation. A similar, but less pronounced trend was seen in the NMU cell line. When NMU cells were cultured in the presence of 10 micrograms/ml FB for 4 days, and then in inhibitor-free medium for a further 3 days, recovery of growth was demonstrated, together with the reappearance of prolactin-binding capacity. The effect of FB on RBA cell PRLR expression was also reversible, though concomitant changes in cell growth were less obvious. Hence, the inhibitory effect of FB on PG synthesis, and the associated decrease in prolactin binding capacity, was specific and reversible and not the result of a generalized toxic effect. PMID- 2999830 TI - Operantly conditioned running: effects on brain catecholamine concentrations and receptor densities in the rat. AB - It was hypothesized that endurance exercise results in an alteration in the brain monoamine systems. Rats were trained to run for food reinforcement on a variable ratio schedule in running wheels. Yoked control rats were also allowed to run but were not specifically reinforced for running. The animals ran 5 days per week for 8 weeks and were sacrificed 48 hours after the last endurance training session. The brains were assayed for norepinephrine and dopamine concentrations and beta adrenergic (3H-dihydroalprenolol binding) and dopaminergic (3H-spiroperidol binding) receptor densities. Changes in norepinephrine concentration and beta adrenergic receptor densities were not significantly different between reinforced running and yoked control groups. Dopamine concentrations were significantly higher while dopamine receptor densities were significantly lower in the reinforced running group. These results suggest that chronic running elevates dopamine secretion and consequently produces a compensatory down-regulation of dopaminergic receptor sites. The relationship of these changes to motor activity and to the antidepressant effects of exercise are discussed. PMID- 2999831 TI - Effect of progesterone upon adenylate cyclase activity and cAMP levels on brain areas. AB - Changes induced by progesterone on adenylate cyclase activity and cAMP levels were determined in brain areas involved in the integration of sexual behavior in ovariectomized estradiol benzoate primed rats. Adenylate cyclase and cAMP concentrations were assayed in: preoptic area, ventromedial hypothalamus and cerebral cortex. Our results show a significant increase in both enzyme activity and cAMP levels at 2 and 4 hours after progesterone administration in all brain areas studied. These data suggest that the facilitatory effect of progesterone on sexual behavior involves an activating mechanism of this steroid upon the adenylate cyclase enzyme which results in an intraneuronal cAMP enhancement. PMID- 2999832 TI - One-way generalization of clonidine to the discriminative stimulus produced by cocaine. AB - Rats were trained to discriminate the stimulus properties of either cocaine or clonidine using a food reinforced two-lever choice paradigm. After training, cocaine was generalized to the cocaine lever in a dose-dependent manner, and clonidine was generalized to the clonidine lever in a dose-dependent manner. Yohimbine, an alpha-2 antagonist, blocked the clonidine stimulus but not the cocaine stimulus. Cocaine was not generalized to the clonidine stimulus; however, clonidine was generalized to the cocaine stimulus, and this generalization was blocked by yohimbine. The one-way generalization of clonidine to cocaine suggests that clonidine has at least two discrete stimulus components: a major component that is not cocaine-like, and a minor component that can be detected by cocaine trained subjects. In addition, the yohimbine blockade data suggest that both components of the clonidine stimulus are mediated via alpha-2 receptors. PMID- 2999833 TI - Discriminative stimulus properties and brain distribution of phencyclidine in rats following administration by injection and smoke inhalation. AB - Four male Sprague-Dawley rats were trained to discriminate IP injections of 3.0 mg/kg phencyclidine (PCP) from saline under a 2-lever fixed-ratio 32 schedule of food presentation. After reliable discriminative control of lever choice was established, other doses of injected PCP were tested resulting in dose-dependent increases in PCP-lever selection and dose-dependent decreases in rates of responding. When doses of PCP were administered by exposure to smoke from cigarettes containing PCP, a dose-dependent increase in PCP-lever responding was also observed. delta 9-Tetrahydrocannabinol administered via smoke exposure, up to doses which markedly suppressed response rates, did not result in PCP appropriate responding, demonstrating the specificity of the PCP stimulus by the inhalation route. Brain levels and distribution of 3H-PCP were determined in rats administered doses calculated to result in 50% generalization by the IP injection or smoke inhalation routes. By both routes of administration roughly equivalent brain levels were attained and the distribution was relatively even across the seven brain areas analyzed. These results demonstrate the validity of using the injection route of administration when studying PCP experimentally, in spite of the fact that PCP is abused primarily by smoking. PMID- 2999834 TI - Electroencephalographic investigations in rabbits of drugs acting at GABA benzodiazepine-barbiturate/picrotoxin receptors complex. AB - This paper describes the EEG profiles, observed in rabbits, of drugs which affect GABA synaptic activity at GBB complex. Drugs which enhance GABA synaptic activity induce sedation associated with EEG synchronization. However, muscimol, THIP, GHB and baclofen induce signs of CNS stimulation (light tremors of the forelimbs, chewing, light nystagmus and hyperpnea) associated with EEG spikes. Signs of light stimulation (chewing and jerks of the head) also occur after BDZs and barbiturates, and are associated with the presence of 12-24 and 20-25 Hz waves, respectively. Drugs which reduce GABA synaptic activity (bicuculline, inverse BDZ agonists, PTZ, picrotoxin and Ro 5-3663) induce three dose-dependent stages of EEG changes: trains of slow waves, trains of spike-and-wave complexes and paroxysmal activity in the rostral encephalic structures without apparent changes of the electrical activity in the spinal cord. The first two stages are associated with a behavioral state of alert and the third stage with tonico clonic convulsions. Among the inverse BDZ agonists, DMCM and beta-CCM elicit all three stages, whereas FG 7142 and beta-CCE induce only the first two and CGS 8216 only the first. The BDZ antagonists Ro 15-1788 and Ro 15-3505 (0.2-30 mg/kg IV) do not significantly affect the EEG pattern. However, they selectively inhibit the effects of diazepam and of the inverse BDZ agonists. In both cases, the inhibition is observed with doses as low as 0.2 mg/kg IV and leads to an EEG desynchronization. The possible involvement of the modifications of GABA synaptic activity in the etiology of both petit mal and grand mal epilepsies is discussed. PMID- 2999835 TI - Interaction of benzodiazepine receptor agonists and inverse agonists with the GABA benzodiazepine receptor complex. AB - The effects of the benzodiazepine receptor agonists, antagonists and inverse agonists on the in vitro binding of several ligands which label different recognition sites of the GABA benzodiazepine receptor complex are summarized. Also, results with a novel biochemical in vitro functional model of the GABA benzodiazepine receptor complex are presented. They are compatible with the concept that drugs which act on benzodiazepine receptors can lead to a bidirectional modulation of the gain of GABAergic neurotransmission. PMID- 2999836 TI - Drug-induced membrane modifications differentially affect prolactin and insulin binding in the mouse liver. AB - Modifications in prolactin and insulin specific binding in the mouse liver induced by repeated administrations of ovine prolactin (oPRL) or indomethacin were studied. oPRL induced a dose-dependent and reversible increase of prolactin binding capacity. No change was observed in dissociation constant values. Conversely, insulin binding capacity to the same liver membranes was not modified by the treatment with oPRL. The increase in the binding induced by oPRL was not influenced by cycloheximide and it is therefore not dependent on protein synthesis. On the other hand, indomethacin caused a dose-dependent inhibition of prolactin binding capacity, without changes in dissociation constant values. The inhibitory effect was specific since at the doses used insulin binding to the same membranes was not affected. When indomethacin treatment was associated to oPRL administration it was able to counteract the increase in binding capacity caused by oPRL, even at doses ineffective in reducing the binding in non oPRL treated mice. Our results suggest that drug-induced membrane modifications can selectively affect prolactin receptors and could consequently modify the ability of the target cell to respond to different physiological or pharmacological situations. PMID- 2999837 TI - Effect of alkaline cations on cyclic 3' 5'-AMP stimulated lipolysis in rat adipocytes. AB - In Krebs-Ringer phosphate medium, cyclic AMP had little effect on production of free fatty acids by fat cells in vitro, whereas dibutyryl cyclic AMP or epinephrine stimulated the production of free fatty acids. On the other hand, although under non-physiological condition, cyclic AMP was found to stimulate the lipolysis in simple KCl-Tris medium. The stimulation level was similar to that elicited by dibutyryl cyclic AMP. Cyclic AMP also stimulated lipolysis in LiCl Tris or RbCl-Tris medium, but not in NaCl-Tris medium. In KCl-Tris medium, addition of divalent alkaline cations (Mg2+, Ca2+ and Sr2+, at 40 mM) completely inhibited the stimulation by cyclic AMP, but not those by dibutyryl cyclic AMP and epinephrine. No cyclic AMP-induced lipolysis was observed in homogenized or freeze-thawed cells. PMID- 2999839 TI - D-Baclofen is an antagonist at baclofen receptors mediating antinociception in the spinal cord. AB - The antagonistic action of D-baclofen at baclofen receptors mediating antinociception in the spinal cord was examined. Drugs were administered intrathecally to rats and effects on nociceptive threshold evaluated in the tail flick test. L-Baclofen, D-baclofen and the racemate produced dose-related increases in tail flick latency, with L-baclofen being twice as potent as the racemate and approximately 100 times more potent than D-baclofen. When D-baclofen was injected 15 min prior to L-baclofen, it produced a dose-related inhibition of the effect of L-baclofen. Concomitant administration produced a more ambiguous effect. Antagonism appeared specific for baclofen receptors because analogues with full and partial agonist activity as well as an agonist dose of D-baclofen, but not morphine or noradrenaline, were inhibited by pretreatment with D baclofen. gamma-Aminobutyric acid (GABA) did not increase tail flick latency either alone or following pretreatment with an uptake inhibitor or a GABA transaminase inhibitor. Antinociception produced by intrathecal administration of Baclofen appears to result from activation of a receptor which is stereoselective for the L-isomer and can be blocked by D-baclofen in doses which have initial agonist activity. This receptor may not be a GABA subtype because GABA does not mimic the effect of baclofen and the rank order of potency of analogues differs from established GABAB systems. PMID- 2999840 TI - Acebutolol-induced decrease of mononuclear leukocyte beta-adrenoceptors in hypertension. AB - The regulatory action exerted on receptors by acebutolol, a cardioselective beta blocker containing intrinsic sympathomimetic activity, has been investigated. beta-Adrenoceptor affinity and density of human mononuclear leukocytes were assayed in hypertensive patients before and after treatment with 400 mg/day acebutolol. While receptor affinity showed no changes between pre- and post treatment values, a statistically significant decrease has been demonstrated in receptor density following treatment. Blood pressure and heart rate were also measured in order to test the efficacy of the administered drug. All these parameters showed a fall in the post-treatment values. It is concluded that the partial agonist acebutolol, in spite of the fact that it acts clinically as a beta-blocker, has the regulatory mechanism characteristic of agonists. PMID- 2999842 TI - Energy balance and play in juvenile rats. AB - These experiments systematically analyzed the relationship between energy balance and play in the juvenile rat. As expected, depriving pups of food for 24 hours or more resulted in a reliable reduction in levels of play, with a single meal being sufficient to return play to baseline levels. Consumption of saccharin did not reverse deprivation-induced reductions of play. It is proposed that the food induced restoration of play in food-deprived pups is perhaps reflective of normal satiety and may be useful in screening putative "satiety agents." Naloxone, cholecystokinin and bombesin, all putative satiety agents, were tested for their ability to reverse deprivation-induced reduction of play. Only bombesin was marginally effective in increasing play of food deprived rats. PMID- 2999838 TI - Effect of (Asu1,7)E-CT, synthetic analogue of eel-calcitonin, on nociceptive transmission. AB - The effect of (Asu1,7)E-CT a deaminodicarba-analogue of the synthetic eel calcitonin on the nociceptive transmission has been studied in mice. The analogue was intracerebroventricularly (0.0.5-0.01-0.02 U.I./kg) or intravenously (0.02 0.04-0.05 -0.1 U.I./kg) injected. This synthetic derivative of eel-calcitonin increased the antinociceptive effect also after peripheral administration. Moreover, preliminary studies on the time-course of this analogue showed that the dose of 0.1 U.I./kg i.v. injected, was able to elicit antinociceptive effect, already 5 min after the administration. PMID- 2999841 TI - The effects of paraventricular hypothalamic lesions on maternal behavior in rats. AB - The present study was undertaken to determine whether the disruptive effects of knife cuts which sever the lateral connections of the medial preoptic area (MPOA) on maternal behavior are mediated by interfering with the output of the paraventricular hypothalamic nucleus (PVN). Postpartum rats received one of the following: Knife cuts severing the lateral connections of the MPOA; knife cuts severing the lateral connections of the PVN; radiofrequency lesions of the PVN; sham lesions or knife cuts. Only females that received knife cuts severing the lateral connections of the MPOA showed severe deficits in maternal behavior. These results indicate that the influence of the MPOA on maternal behavior is not mediated by the output of the PVN. Since the PVN is the major source of oxytocin input to other brain regions, these results also suggest that oxytocinergic neural pathways are not critical for postpartum maternal behavior. Another important finding was that females with MPOA knife cuts that did not retrieve their young were capable of hoarding candy, suggesting that the retrieval deficit was not the result of a general oral motor deficit. PMID- 2999843 TI - Sensory preconditioning in the rabbit following ACTH injections. AB - The effects of ACTH elevation on sensory preconditioning were examined using the rabbit NM response. Sensory preconditioning is an associative form of learning which is presumed to rely on a "stimulus map" provided by the hippocampus. Elevation of levels of ACTH was found to have no significant effect on SPC. This finding was interpreted as evidence that not all deficits found in hippocampal lesioned animals may be attributed to hormonal mediation. It was suggested that ACTH elevation may selectively mimic those deficits ascribed to a "neural model" process involving the hippocampus. PMID- 2999844 TI - Elicitation of conspecific attack or defense in the male rat by intraventricular injection of a GABA agonist or antagonist. AB - The involvement of central GABAergic mechanisms in the control over offensive and defensive behaviours in the rat was studied using intracerebroventricular injections (5 microliter) of a GABA agonist (THIP) or a GABA antagonist (bicuculline methiodide). Intracerebroventricular injections of THIP (1.25 and 2.5 micrograms) induced attacks and offensive sideways towards an untreated partner, in animals placed in a neutral area where no aggressive reactions occur in controls. Social approach behaviours (partner investigation, allogrooming) were also increased in both attacking and non-attacking animals, whereas individual behaviours (cage exploration, autogrooming, immobile posture) were decreased. Inversely, intracerebroventricular injections of bicuculline methiodide (62.5 and 125 ng) suppressed offensive items (attacks, offensive sideways, upright postures) in resident animals confronted with untreated intruders and increased occurrence of defensive sideways. This treatment also decreased reactions oriented towards the partner (investigation, allogrooming and crawl under/over), while increasing individual behaviours (cage exploration, immobile posture). These data demonstrate that activation of central GABA receptors elicits intraspecific offensive behaviours in the rat. On the contrary, blockage of these receptors induces defensive reactions and suppresses offensive behaviours. The involvement of these receptors in the neural control over aggressive behaviour in the rat is discussed. PMID- 2999845 TI - Testosterone is not required for the enhancement of sexual motivation by yohimbine. AB - Yohimbine HCL (2 mg/kg, 20 min prior to testing) administration was followed by significant decreases in the latencies to initial mount, intromission and ejaculation in castrated male rats bearing 2 mm testosterone-containing Silastic capsules 51 days after castration. Further, yohimbine stimulated copulatory activity in castrated, nonhormone-treated male rats up to 91 days after castration. Finally, yohimbine induced mounting in intact, nonreceptive female rats. These observations indicate that testosterone is not required for the enhancement of sexual motivation by yohimbine and support the suggestion that alpha 2-adrenoceptors are involved in the modulation of sexual arousal. PMID- 2999846 TI - The effects of putative anxiogenic compounds (FG 7142, CGS 8216 and Ro 15-1788) on the rat corticosterone response. AB - The effects of FG 7142, CGS 8216 and Ro 15-1788, three compounds that are believed to produce anxiety by an action at benzodiazepine receptors in the CNS, are investigated on the plasma corticosterone concentrations in the rat both in the home cage and after exposure to novelty stress. FG 7142 (5 mg/kg) and CGS 8216 (10 mg/kg), but not Ro 15-1788 (4 or 10 mg/kg) increased basal corticosterone levels in the home cage, and all three compounds potentiated the increase in corticosterone concentrations observed after exposure to a novel environment. The relationship between the effects of drugs on corticosterone concentrations and on anxiety is considered in the light of these results. PMID- 2999847 TI - Adrenalectomy fails to alter radial maze performance of rats at retention intervals of 24 hours or less. AB - Evidence suggests that the behavioral actions of adrenalectomy and glucocorticoid replacement therapy result from changes in the binding of corticosterone to specific receptors in the hippocampus. Efficient foraging for bait on the radial maze has been demonstrated to require a functionally intact hippocampal system. The present experiment examined the effect of adrenalectomy on the ability of rats to locate unexplored arms in the radial maze after various retention intervals midway through completion of the maze. Rats were very proficient at locating the unexplored arms after retention intervals of 3 hours or less; significantly more trials were required to locate all of the previously unvisited arms at retention intervals of 8 and 24 hours. However, adrenalectomy failed to alter maze performance at any retention interval tested. It was concluded that the neural substrates involved in the performance of this spatial/working memory task are not dependent upon the neuromodulatory effects of physiological levels of adrenal corticosteroids. The results are discussed in terms of behavior/contingency conflicts and innate versus learned responses as factors which may be important for revealing behavioral actions of adrenalectomy or corticosteroid therapy. PMID- 2999848 TI - A 1100-bp sequence of mitochondrial DNA is involved in senescence process in Podospora: study of senescent and mutant cultures. AB - In Podospora, senescence is assumed to be caused by the amplification of short sequences of the mitochondrial genome (sen-DNAs). We have characterized a 1100-bp long mitochondrial DNA sequence which could be directly involved in the phenomenon. Indeed, by hybridization experiments, we show that this sequence is both present in all the sen-DNA molecules which originate from the beta region of the mitochondrial chromosome and rearranged in the mitochondrial genome of two mitochondrial mutants selected as resistant to senescence. PMID- 2999849 TI - Specific-purpose broad-host-range vectors. AB - Several plasmid derivatives of broad-host-range Inc P4 plasmid RSF1010 were constructed and characterized. Vector pAYC30 was constructed by insertion in vivo into the genome of RSF1010 the Hgr transposon Tn501, originating from the plasmid pVS1 of Pseudomonas aeruginosa. Plasmids with inserts of PstI or SacI fragments may be selected by inactivation of genes sul and aph, respectively. The cloning at unique site SalGI leads to the appearance of HgCl2--sensitive transformants. Versatile cloning vector pAYC1 consists of two replicons, RSF1010 and plasmid pMZ7, a derivative of R6K. The constructed plasmid is 16.9 kb in length and determines resistance to five drugs. Two promoter-probe broad-host-range vectors, pAYC36 and pAYC37, were obtained by replacing a small segment from the DNA sequence of the aph gene promoter of previously described plasmid pAYC32 with the polylinker from plasmid pUC19. Therefore, vector plasmids retained the intact gene aph (Smr); however, they have Sms phenotype because of the insertional inactivation of the promoter. The genetic structure of promoter-probe vectors allows one to select clones, containing hybrid plasmids with an active promoter for gene aph expression. PMID- 2999850 TI - Isolation and characterization of an extrachromosomal element from Nocardia mediterranei. AB - Strain LBG A3136 of Nocardia mediterranei (ETH Collection) was found to contain a low-copy-number covalently closed circular extrachromosomal element, pMEA 100, which could only be isolated from mycelium grown on agar plates. pMEA 100 could not be isolated from the closely related strain ATCC 13685. Hybridization experiments showed that pMEA 100 is present in strain LBG A3136 in the free as well as in the integrated form whereas in strain ATCC 13685 only an integrated form was detected. Excision and reintegration in strain LBG A3136 seemed to be site specific. pMEA100 was found to be self-transmissible, eliciting the lethal zygosis phenotype, and is possibly involved in fertility in N. mediterranei. PMID- 2999852 TI - Transfer of transposable drug-resistance elements Tn5, Tn7, and Tn76 to Azotobacter beijerinckii: use of plasmid RP4::Tn76 as a suicide vector. AB - Transposable elements Tn5, Tn7, and Tn76 were transferred to Azotobacter beijerinckii. Evidence was obtained for the transposition of Tn5 but cells of the majority of presumptive transposition isolates had abnormal morphologies and rapidly lost viability when subcultured. Data are presented that indicate that plasmid RP4::Tn76 behaves as a suicide vector upon transfer to this host, allowing the isolation of A. beijerinckii::Tn76 isolates at a high frequency. Nitrogen-fixing mutants and leucine and adenine auxotrophs were isolated from cultures in which the transposition of Tn76 occurred. PMID- 2999851 TI - A versatile multiple- and single-copy vector system for the in vitro construction of transcriptional fusions to lacZ. AB - A multiple-copy (plasmid) vector and a single-copy (lambda) vector were constructed for the in vitro formation of transcriptional fusions to lacZ. In both vectors the transcription of lacZ is dependent upon the attachment of a promoter upstream, but beta-galactosidase is independently translated from the hybrid mRNA. These vectors are based on the W205 trp-lac deletion, but most of the trpBA DNA has been removed. Promoters are fused to the 3' end of trpA rather than the HindIII site at the 5' end of trpB used in other vectors containing the W205 deletion. This modification avoids the polar effects encountered when either an untranslated sequence, or a sequence translated in a different reading frame is fused to trpB. Hence the level of beta-galactosidase synthesized by fusions in these new vectors accurately reflects the frequency of transcription from the attached promoter. A polyrestriction site linker precedes the lacZ gene in both vectors and allows the direct ligation of promoter containing DNA fragments produced by a large collection of restriction endonucleases. PMID- 2999853 TI - One-kilobase direct repeats of plasmid pSa. AB - One-kilobase, direct repeats were found on either side of the chloramphenicol resistance gene of plasmid pSa. The right repeat corresponded to the region coding for sulfanilamide resistance. The repeats were not identical as judged by distances between restriction enzyme sites, hybridization, and by the ability to confer resistance to sulfanilamide. PMID- 2999854 TI - Physical and genetic structure of the IncN plasmid R15. AB - Restriction sites for seven hexanucleotide-specific endonucleases were located on the map of the conjugative IncN plasmid R15 (SmrSurHgr, 62.3 kb). The distribution of the cleavage sites is strongly asymmetric. Twenty-eight of thirty four sites for BamHI, EcoRI, HindIII, SalI, SmaI, and PstI were located close to or within the sequences of an IS5-like element and the transposons Tn2353 and Tn2354. By analysis of R15::Tn1756 deletion derivatives and recombinant plasmids harboring R15 fragments, the genetic determinants for the streptomycin, sulfonamide, and mercury resistances were mapped, as well as the regions necessary for EcoRII restriction-modification and for plasmid replication and conjugation. The features of physical and genetic structures of the plasmid R15 and other IncN plasmids are discussed. PMID- 2999855 TI - Use of a pedicled parascapular flap for anterior shoulder and arm reconstruction. AB - We present a case of soft-tissue reconstruction of the shoulder and upper arm utilizing a pedicled parascapular flap and discuss its potential as a flap for regional reconstruction. PMID- 2999856 TI - [Response of the hypoglossal and phrenic nerves to the inhibition and stimulation of central chemoreceptors]. PMID- 2999857 TI - Health risk appraisal in primary care. AB - Preventive medicine focuses on the identification of factors that will lead to disability or disease and then the discovery of ways to eliminate or at least minimize these risk factors. In this article, the authors outline a guide for primary health care practitioners to practice preventive medicine using a computerized individual risk appraisal. PMID- 2999858 TI - Susceptibility of different leukemic cell lines to the anticellular and antiviral effects of interferons. AB - A total of 18 different types of human leukemic cell lines were tested for their susceptibility to the anticellular and antiviral effects of interferons (IFNs) alpha, beta and gamma. In general, only the three myelogenous leukemic cell lines U937, KG-1 and HL-60 were found to be highly susceptible to the anticellular effect of the different IFNs while cells of the other lineages were relatively resistant. In order to determine whether the cell lines were sensitive to the antiviral effects of IFNs, the cells were first screened for their ability to support the replication of vesicular stomatitis virus (VSV), sindbis virus (SBV) and semliki forest virus (SFV). Unexpectedly, only three cell lines--Raji, K562 and U937 were highly susceptible to SFV while other cell lines were relatively refractory to all three viruses. Using SFV as indicator virus, the antiviral activity of all IFNs could be detected in all three cell lines and their relative efficiency was in the order of alpha greater than beta greater than gamma. The significance of these results were discussed. PMID- 2999859 TI - Disturbed cortisol secretion in man: contrasting Cushing's disease and endogenous depression. AB - A disturbed regulation of cortisol secretion is the principal pathology of Cushing's disease and is also the most widely reported neuroendocrine dysfunction in endogenous depression. Because additional clinical signs in both diseases indicated a hypothetical common pathway, we examined 17 patients suffering from Cushing's disease, following a protocol identical to that used in depressed patients (e.g., Hamilton Rating Scale for Depression, self-rating scales, and a clinical interview). Affective disorders, frequently observed in patients with Cushing's disease, were undetectable after surgical treatment (adrenalectomy or microadenomectomy of hypercortisolism). This was an unexpected result, since we found that recovered patients were still characterized by a disturbance of glucocorticoid feedback regulation, probably acting at the hypothalamic level. Our results, as well as numerous reports from others, failed to support the hypothesis that an impaired regulation of cortisol is directly linked to depressive illness. PMID- 2999861 TI - Repair of radiation-induced DNA damage in rat epidermis as a function of age. AB - The rate of repair of radiation-induced DNA damage in proliferating rat epidermal cells diminished progressively with increasing age of the animal. The dorsal skin was irradiated with 1200 rad of 0.8 MeV electrons at various ages, and the amount of DNA damage was determined as a function of time after irradiation by the method of alkaline unwinding followed by S1 nuclease digestion. The amount of DNA damage immediately after irradiation was not age dependent, while the rate of damage removal from the DNA decreased with increasing age. By fitting an exponential function to the relative amount of undamaged DNA as a function of time after irradiation, DNA repair halftimes of 20, 27, 69 and 107 min were obtained for 28, 100-, 200-, and 400-day-old animals, respectively. PMID- 2999860 TI - Magnetic resonance studies of the pathophysiology of murine malaria. PMID- 2999863 TI - Differential effects of hyperthermia on the Na+,K+-ATPase of Chinese hamster ovary cells. AB - The effect of hyperthermia in the 41-50 degrees C range on the Na+,K+-ATPase of CHO cells has been investigated. Three separate activities of the enzyme, namely, ATP hydrolysis, K+ uptake, and binding of the specific inhibitor, ouabain, have been measured independently. The results can be summarized as follows: (1) The ouabain binding capacity of intact cells decreased with increasing temperature and with increasing time at any one temperature. (2) The loss of ouabain sensitive K+ uptake after treating cells at 45 degrees C was very similar to the loss of ouabain binding capacity. In contrast, the ATP hydrolyzing activity was much more resistant to heat. (3) The ouabain binding capacity was more resistant to hyperthermia at 45 degrees C if the cells were given a prior exposure at 45 degrees C 12 hr earlier. In contrast, the ATP hydrolyzing activity did not display this tolerance to heat. PMID- 2999862 TI - Influence of hyperthermia and gamma radiation on ADP-ribosyl transferase, NAD+, and ATP pools in human mononuclear leukocytes. AB - Effects of hyperthermia (42.5 degrees C) and gamma radiation (30 Gy) on ADP ribosyl transferase, NAD+, and ATP pools in human mononuclear leukocytes have been investigated. It was found that the gamma-ray activation level of the enzyme was not influenced by this hyperthermia for 45 min. Following deprivation of ATP synthesis by 2,4-dinitrophenol, an uncoupler of the oxidative phosphorylation, and omitting glucose from the culture medium, the NAD+ pool was reduced to about 60% of control value. The potentiation of ATP production by exogenously supplied adenosine was reduced after a combined treatment of the cells with hyperthermia and gamma radiation. Mitochondrial and endoplasmic changes within the mononuclear leukocytes were also observed. Based on these findings a model for the hyperthermia effect is proposed. PMID- 2999864 TI - DNA polymerase I is crucial for the repair of potentially lethal damage caused by the indirect effects of X irradiation in Escherichia coli. AB - The radiosensitivity of an Escherichia coli mutant deficient in DNA polymerase I was measured in the presence of OH radical scavengers. The extreme X-ray sensitivity of the mutant could be abolished by OH radical scavengers if a sufficiently high level of radioprotector was present. There was a direct correlation between the OH radical scavenging activity of the chemicals tested (NO2-, n-butanol, glycerol, t-amyl alcohol, and t-butanol) and their protective ability. I interpret the data as showing that the indirect actions of X rays (primarily OH radicals) result in major damage to the bacterial DNA which in large part consists of potentially lethal lesions. This potentially lethal damage is repaired through an enzymatic pathway requiring DNA polymerase I. In the mutant lacking DNA polymerase I, these potentially lethal lesions are expressed as cell lethality. PMID- 2999865 TI - [Radiation sensitivity of membrane-bound glucose-6-phosphatase]. AB - A comparative study was made of the effect of X-radiation on the membrane-bound glucoso-6-phosphatase of the nuclear membrane and microsomal fraction of calf thymus cells. Dose- and concentration-dependencies of inactivation of glucoso-6 phosphatase are indicative of a higher radiosensitivity of glucoso-6-phosphatase of nuclear membranes than that of microsomes. This difference in radiosensitivity is associated with the peculiarities of the composition and structural organization of these two membrane systems of a cell. PMID- 2999866 TI - [Pneumonitis and fibrosis in combination therapy (polychemotherapy, radiotherapy) of small cell bronchial cancer]. PMID- 2999868 TI - Left ventricular function evaluation using radionuclide methods in the intensive coronary care unit. AB - We describe an adapted first-transit (FT) technique to perform left ventricular ejection fraction (LVEF) measurements on patients with Swan-Ganz catheters in the intensive cardiac care unit (ICCU). The radionuclide is introduced directly into the right pulmonary artery through the catheter. High-quality images of the left ventricle are obtained owing to minimal activity in the right ventricle and left lung. LVEF measurements obtained by FT compared well with measurements obtained from gated blood pool studies (r = 0.91) but gave consistently lower values. The adapted FT method improves LVEF determination and left-ventricular wall motion evaluation in the ICCU patient. PMID- 2999867 TI - [Current in vivo and in vitro diagnosis of thyroid diseases]. AB - A single method which is sufficient to meet all diagnostic requirements in patients with thyroid disorders does not exist. In selecting a test combination one should always consider, that differentiation of patients with hyper- or hypothyroidism from those with euthyroidism should involve the least effort. A meaningful step-by-step diagnostic work-up should always start with individually selected in vitro tests, followed by in vivo methods such as ultrasonography, radionuclide scanning and X-ray examination. This accords with the recommendations for diagnosis of disturbed thyroid function and thyroid disorders published recently by the thyroid section of the German Association of Endocrinology. PMID- 2999869 TI - Chemotherapy for brain metastases. PMID- 2999870 TI - Polyneuropathy induced by cisplatin. PMID- 2999871 TI - Corticotropin releasing factor: basic studies and clinical applications. AB - Corticotropin releasing factor (CRF) is a newly sequenced peptide first isolated from sheep hypothalami and thought to be an important modulator of both the pituitary-adrenal axis and the sympathetic nervous system. We administered intravenous, intramuscular, and intracerebroventricular CRH to non-human primates and measured plasma ACTH, beta endorphin, cortisol, GH and PRL responses to CRF. In addition, we determined the pharmacokinetic properties of I125 in these primates. We administered CRF as an intravenous bolus or as a continuous infusion to normal volunteers and as an intravenous bolus to patients with disorders of the hypothalamic-pituitary-adrenal axis, such as Cushing's syndrome and adrenal insufficiency, and patients with endogenous depression and mild hypercortisolism, and assessed their plasma ACTH, cortisol, GH and PRL responses. In addition, we determined the pharmacokinetic properties of CRF in man by measuring CRF immunoreactivity in plasma. CRF given intravenously to primates or man is a slowly metabolized, long-acting, secretagogue of ACTH, beta-endorphin and cortisol. When given intracerebroventricularly to primates it stimulates the hypothalamic-pituitary-adrenal axis without escaping into the plasma and it is actively cleared in the CNS. It does not cross the blood brain barrier appreciably when given intravenously. CRF given to primates and men as an intravenous continuous infusion has only mild ACTH stimulating effects and this may be due to an intact cortisol negative feedback system. Finally, CRF causes characteristic plasma hormone responses in patients with Cushing's disease, adrenal insufficiency and depression. PMID- 2999872 TI - Acute and chronic effects of dextropropoxyphene on behaviour and central inhibitory neurotransmission in the rat. AB - There was no overt evidence of the development of physical dependence, as shown by a decrease in the body weight of rats following the abrupt withdrawal of dextropropoxyphene after two weeks administration. The ambulation and rearing scores in the 'open field' apparatus were increased after chronic, but not acute drug administration and returned to control values two days following drug withdrawal. GABA turnover, determined from the rise in GABA concentrations following GABA-transaminase inhibition, was reduced in the frontal and amygdaloid cortex after acute and chronic drug administration; a compensatory rise in GABA turnover in the amygdaloid cortex occurred two days after drug withdrawal. Na+, K+, ATP'ase activity, determined in a synaptosomal fraction from the mid-brain and hippocampus, was decreased in the latter region only during drug administration; a compensatory increase in the activity of this enzyme was found two days after drug withdrawal. These results support the view that chronically administered dextropropoxyphene may cause changes in inhibitory transmission and central neurotransmitter transport. PMID- 2999873 TI - [A genetic disease with a defect in biochemical sulfation--biochemical studies on Lowe's syndrome]. PMID- 2999875 TI - [Phosphodiesterase-phosphomonoesterase from a phytopathogenic fungus]. PMID- 2999874 TI - [Cellular biochemistry and partial structure of protein-membrane fusion and protein topogenesis]. PMID- 2999876 TI - [Evolutional relationship between copia-like transposable genetic elements in Drosophila and retroviruses in vertebrate]. PMID- 2999878 TI - [Evolution of sulfate respiration and cytochrome]. PMID- 2999877 TI - [Hepatocarcinoma and molecular biology of hepatitis B virus]. PMID- 2999879 TI - [Structural, functional and evolutional aspects of Nitrobacter agilis cytochrome c]. PMID- 2999880 TI - Magnetic resonance imaging (MRI) of primary liver cancer--MRI-pathologic correlation. AB - In seven primary liver cancers (HCC 5, CCC 1, mixed 1), MR images (0.35 Tesla super-conducting) were compared with macroscopic appearances, and relaxation times (T1 and T2) with microscopic characteristics. MRI was able to reveal the gross appearance of five nodular lesions, but did not reveal one diffuse HCC and one nodular HCC with marked extracapsular extension. T2-weighted SE images could not demonstrate fibrous capsules around the tumor in four nodular HCCs. The T1 and T2 values of the tumors were longer than those of the surrounding liver parenchyma, and the T1 elongation corresponded roughly to the degree of necrosis and fibrosis within the tumors. PMID- 2999881 TI - The biological relevance of HPLC-purified vasoactive intestinal polypeptide monoiodinated at tyrosine 10 or tyrosine 22. AB - Three major forms of monoiodinated VIP (M125I-VIP) were isolated after chloramine T iodination and HPLC purification. The iodinated tyrosine residue was located in each form of M125I-VIP using arginase C and trypsin digestion for obtaining defined fragments containing only one tyrosine residue. The HPLC isolated iodinated fragments thus obtained were used for HPLC comigration studies with iodinated synthetic C and N terminal VIP fragments and for amino acid analysis. The first two eluting peaks 1 and 2 are (M125I-Tyr10-VIP); peak 1 has an oxidized methionine; peak 3 is a (M125I-Tyr22-VIP) which also has an oxidized methionine. A reduced counterpart of peak 3 named peak 4 was isolated by further HPLC analysis. The ability of the different species of M125I-VIP to stimulate adenosine cyclic 3',5'-phosphate (cAMP) production in transformed colonic cells in culture (HT-29) was compared to that of native VIP. The mean potencies of the M125I-VIP species expressed as a percentage relative to the potency of native VIP were, peak (1): 0.98; (2): 0.84; (3): 1.38; (4): 1.48, in the range of concentrations tested (2-60 pM). The M125I-Tyr22-VIP are significantly more active than native VIP (P less than 0.01). Oxidation of methionine or iodination of tyrosine 10 does not significantly modify the biological activity of VIP. We conclude that iodination of Tyr-22 located in the apolar helical COOH-terminal of VIP increases the effectiveness of VIP interaction with its receptors. Thus the tyrosyl residue and the localized hydrophobic features of VIP are critically involved in the function of this neurotransmitter. PMID- 2999882 TI - Comparative effects of vasoactive intestinal peptide and secretin on exocrine pancreatic secretion in the cat. AB - The stimulatory effect of vasoactive intestinal peptide (VIP) and secretin has been compared on exocrine pancreatic secretion in anaesthetized cats. Both peptides were given by bolus intravenous injection and continuous intravenous infusion. After bolus injection, VIP stimulated pancreatic secretion only weakly. On the contrary, during intravenous infusion, the maximal effect of VIP did not differ significantly from that of secretin. Therefore, while the potency of VIP is always lower than that of secretin, its efficacy appears to be strictly dependent on the mode of administration. PMID- 2999883 TI - Binding of vasoactive intestinal polypeptide (VIP) by human blood monocytes: demonstration of specific binding sites. AB - Vasoactive intestinal polypeptide (VIP) interaction with a 94% pure preparation of monocytes isolated from human peripheral blood was studied by direct binding technique using 3-[125I]tyrosyl-VIP as a tracer ligand. Scatchard analysis of binding data was compatible with two classes of binding sites, one with Kd = 0.25 nM and maximal binding capacity of 16 fmol/10(6) cells, and another one with Kd = 25 nM and maximal binding capacity of 180 fmol/10(6) cells. The binding was time , temperature-, and pH-dependent and was saturable, reversible, and specific. This study has demonstrated that human monocytes have high affinity/low capacity as well as low affinity/high capacity binding sites for VIP. No specific VIP binding was found in pure preparations of human granulocytes, platelets or erythrocytes. PMID- 2999884 TI - [Diagnostic imaging by radioisotopes. Application (XI). Some considerations on radionuclide images of the liver]. PMID- 2999886 TI - [Intracranial arteritis with circulating immune complexes]. AB - Thirty-eight patients below the age of 50 years were investigated and in twelve, circulating immune complexes were found. Eight of these had angiographic evidence of an arteritis of the intracranial vessels. The causative factors, the role of the circulating immune complexes and the angiographic appearances in intracranial arteritis are discussed. PMID- 2999887 TI - [Nuclear spin tomography in brain tumors. Evaluation of 40 confirmed cases]. AB - Forty histologically confirmed primary and secondary cerebral tumours are described. These contained fifteen primary tumours, five adenomas of the hypophysis, four hamartomas or lipomas and craniopharyngiomas, three meningiomas and three vascular malformations. One malignant lymphoma, one chordoma and eight cerebral metastases were also included. In our series, the accuracy of CT and MR were the same, particularly since most patients coming for MR had abnormal CT findings. MR was superior in demonstrating vascular malformations and sometimes in the differential diagnosis of tumour versus infarct. MR is also more accurate in demonstrating the extent of a tumour and of infiltration, since it is possible to obtain images in several planes. Disadvantages of MR are related to the characterisation of primary brain tumours, since the signals from these tumours failed to show characteristic differences. Calcification cannot be recognised. In sixteen out of 36 patients, MR proved superior, whereas CT was superior in five. Tumour-related oedema was present in 18 cases and demonstrated by MR in seventeen. CT failed to differentiate tumour from oedema on one patient. PMID- 2999885 TI - [Magnetic resonance and computerized tomography in the study of hypophyseal adenomas. Comparative results]. AB - Magnetic resonance evaluation of 28 cases of pituitary adenomas has shown remarkable accuracy. Compared with HR-CT, MR gives comparable results in tumour identification. MR better demonstrates the suprasellar extension of macroadenomas and their relationship to the visual pathway and is more effective in showing direct and indirect signs of microadenomas. HR-CT however better recognized bone abnormalities of the sella turcica, due to adenomas. A typical increased signal intensity has been demonstrated in most of the adenomas studied. PMID- 2999888 TI - [Possibility of erroneous diagnosis with digital subtraction angiography and its causes]. AB - In digital subtraction angiography (DSA) motion artifacts, artifacts due to overshoot, high contrast enhancement, and long exposure time may result in erroneous evaluation of the vessels. Examples are given to demonstrate and analyse the causes of possible misdiagnoses. PMID- 2999889 TI - [Contrast medium concentration for intra-arterial digital subtraction angiography of vessels in the extremities]. AB - The influence of iodine concentrations and different volumes of a conventional ionic contrast media and a contrast media with low osmolarity on the quality of the results of intra-arterial digital subtraction angiography of the lower extremities in patients who underwent percutaneous transluminal angioplasty (n = 52) are reported. According to our results, intra-arterial injections of a conventional ionic contrast medium diluted with 0.9% sodium chloride solution, resulting in an iodine concentration of 190 mg/ml injectable solution, reveals the best results when injected in an amount of 6 ml per injection. The use of low osmolarity contrast-medium showed no advantages, the quality of the resulting pictures was inferior to that revealed with the ionic compound. Probably this is because the iodine concentration in the diluted low-osmolarity compound was less. PMID- 2999890 TI - [Occlusion or stenosis of the splenoportal axis. Supplementary information with duplex sonography?]. AB - In 21 patients with sonographic signs of occlusions or stenoses of the splenoportal axis additionally pulsed Doppler measurements in the region of the splenic and portal veins were conducted with a Duplex system. Examinations using the pulsed Doppler Duplex system yield functional data in respect of blood flow in addition to the analysis of morphological changes. It is possible to differentiate between cystic structures and collaterals as well as to assess residual perfusions or cavernous transformations in vascular occlusions. Besides qualitative information on blood flow in a vessel and determination of the direction of blood flow, quantitative data on the acceleration of blood flow in stenosis are also possible. PMID- 2999891 TI - [Technical problems in percutaneous transhepatic biliary drainage]. AB - Technical problems are repeatedly encountered during percutaneous transhepatic drainage by catheter or endo-prosthesis which are caused by the position, extent and tightness of the stenosis in the biliary ducts. All means of overcoming these problems must be used, otherwise technical failure or complications become inevitable. The methods to be adopted if catheters break and endo-prostheses become displaced or occluded, and for haemorrhage, are described, as well as methods for draining multiple, proximal or intrahepatic stenoses. This is based on an experience with 74 patients on whom 130 drainage procedures have been performed (80 catheters, 50 endo-prostheses). All complications and mortality (five patients, 3.8%) were due to problems which could not be solved. Internal biliary drainage by means of an endoprosthesis is markedly superior to catheter drainage. PMID- 2999893 TI - [Dosimetry in computerized tomography]. AB - During CT, the surface dose increases towards the centre of the axis of rotation. Measurements on 35 patients and theoretical considerations indicate a hyperbolic relationship between the axis of rotation and the surface dose. PMID- 2999892 TI - [Value of computerized tomography for the assessment of the parametrium in cervical cancer]. AB - The clinical and CT appearances of the parametrium in 79 patients with carcinoma of the cervix were compared with the histological findings obtained at surgery. The accuracy of CT in stages T1/T2a was 46.6%, in category T2b 64.9% and in category T3b 66.6%. Clinical examination showed fewer errors. CT does not contribute to the evaluation of infiltration of the parametrium from carcinoma of the cervix, nor can it correct the findings on palpation. Tumour infiltration and operability are not demonstrated with sufficient accuracy by CT. PMID- 2999894 TI - [Therapeutic results of ultrasonically-guided kidney cyst punctures]. AB - The behaviour of renal cysts following puncture was studied in 62 patients on whom a simple diagnostic puncture had been performed and on fourteen patients who, in addition, had some of their own blood injected into the cyst. Twenty-one patients (34%) of the first group and eight patients (57%) of the second group showed definite reduction in the size of the renal cyst after an average period of observation of fifteen months. In 7%, the cyst disappeared completely following puncture. Parapelvic cysts showed much less tendency to disappear than did cortical cysts. The results of injecting autologous blood are comparable with those of injecting lipid soluble contrast media as a sclerosing agent. Before surgical removal of a symptomatic renal cyst is contemplated, puncture and evacuation of the cyst with autologous blood injection is recommended. PMID- 2999895 TI - [Catheter embolization: parenchyma-preserving therapy of renal bleeding of nonmalignant etiology]. AB - 9 patients with life-threatening renal bleeding of non-malignant origin, including trauma, AV fistulas, pseudoaneurysms and polycystic kidneys, were embolised after angiographic demonstration of the leakage. In all cases, the bleeding was stopped and in one case only nephrectomy was necessary 3 days after the initial embolisation procedure. Transcatheter renal embolisation should be performed as selectively as possible. With this technique most of the renal parenchyma can be saved. Embolisation is a safe and inexpensive procedure which also can be performed in critically ill patients. PMID- 2999896 TI - Herniography in anterior abdominal wall hernia. AB - The clinical diagnosis of anterior abdominal wall hernia is difficult in patients with a negative or inconclusive physical examination. These hernias are often of an interparietal type which hampers their detection. Herniography may contribute to the clinical workup in patients with Spigelian, incisional, and umbilical hernias. As the clinical presentation may be spurious, herniography should be used on wide indications. The herniographic appearance and differential diagnosis of these hernias are reported. The additional use of ultrasonography in this setting is illustrated and discussed. PMID- 2999897 TI - [Fibrous bone dysplasia and ossifying bone fibroma in the orbital and periorbital region with special reference to CT]. AB - Fibro-osseous conditions affecting the craniofacial bones pose a complex diagnostic problem. Differentiation between monostotic fibrous dysplasia (FD) and ossifying fibroma (OF) is only possible by correlation of clinical, radiographical and histopathological features. CT was superior to conventional radiography/polytomography in defining exact extent and site of lesions and additional lesions, in verifying aetiology of secondary complications, as well as in depicting lesions and tandem-lesions simulating FD and OF. Density of fibro osseous conditions was variable due to the ratio of fibrous stroma and metaplastic bone present. Density measurements in FD were 32-695 HU, in immature types of OF, consisting mainly of fibrous and osteoid tissue, 30-250 HU and could reach 690 HU in mature OF, but were definitively lower than normal bone in all our cases. Focal intrinsic nonhomogeneity was more significant in mixed types of FD and immature OF. PMID- 2999898 TI - [Inflammatory changes in shoulder arthrography]. AB - Arthrography of the shoulder joint in so-called periarthritis humeroscapularis often shows nodular contrast defects, irregular defects in the bursa and contrast filling of irregular lymphatics; these are characteristic signs of a synovitis. Arthrography can provide an exact diagnosis, not only in suspected post-traumatic rotator cuff, tears, but also in other painful conditions that limit movement. It is superior to CT in demonstrating inflammatory changes. PMID- 2999899 TI - [Radiation-induced scars in mammography]. AB - Six patients with radiation scars are described. In each case the diagnosis was confirmed histologically in five cases corresponding mammograms were available. The histological appearances of radiation scars are described and the radiological features are presented. These lesions can be diagnosed mammographically in vivo. Macroscopically differentiation from a scirrhous carcinoma is not possible and therefore a radiation scar must always be excised; this also leads to definitive cure. On mammographic screening the incidence is 0.5 to 0.9 per thousand. The significance of radiation scars depends on the fact that they are pre-cancerous and therefore are equivalent to the early diagnosis of a carcinoma with the possibility of a complete cure. PMID- 2999900 TI - [A new mathematical method for the radiological determination of total lung capacity]. AB - Various radiological methods for estimating lung volume depend on anatomical correlation with the volume of the thorax. It is not possible to derive general factors for correcting these methods and the accuracy and reliability can be improved only by introducing individual corrections. It must also be remembered that radiometric calculations of gas volume and lung capacity depend on the assumption that there is a fixed relationship between these. This, however, is untrue for a whole series of patho-physiological circumstances. Because of these problems, we describe our own radiometric method. PMID- 2999901 TI - [The effect of periodic object movements on MRT image quality]. AB - Object movement influences the images in MRT, producing phantom images and blurred contours, while also affecting the intensity of the signal. Phantom images and blurring of contours can be largely prevented by suitable signal triggering in case of rhythmic movements caused, for example, by cardiac pulsation or respiration. However, the intensity of the signal follows a different pattern. Periodic movement in the image plane (sagittal plane) has no effect on signal intensity if the exposures are triggered. Object movement across the image plane causes changes in signal intensity, even with triggered exposures. PMID- 2999902 TI - [Calcification of the inferior vena cava imitates an aortic aneurysm]. PMID- 2999903 TI - [Unusual catheter embolism of the pulmonary artery and percutaneous transluminal extraction]. PMID- 2999904 TI - [The significance of narrowed intervertebral disks for the diagnosis of spinal tuberculosis]. PMID- 2999906 TI - [Case report of a rectal sliding hernia. Rare complication following partial abdominosacral rectum resection]. PMID- 2999905 TI - [Cerebral venous and sinus thrombosis in ulcerative colitis]. PMID- 2999907 TI - [Sonographic diagnosis of cystic degeneration of the adventitia]. PMID- 2999909 TI - [Primary hyperoxaluria in a 6-month-old infant: a radiological and sonographic diagnosis?]. PMID- 2999908 TI - [Radiological demonstration of pneumatosis intestinalis in 2 children with leukemia]. PMID- 2999910 TI - [Massive increase in calcification in mesenchymal chondrosarcoma as an effect of therapy following irradiation]. PMID- 2999911 TI - Percoll reversibly inhibits superoxide dismutase. AB - Incubation of pea leaf extracts (Pisum sativum L.) at 6 degrees C in isoosmotic media containing different Percoll concentrations significantly represses the total superoxide dismutase (SOD) activity in a concentration- and time-dependent manner. After 24 h incubation at 6 degrees C, 30-45% Percoll concentrations bring about an inhibition of Mn-SOD activity of more than 50%. Isozyme Cu,Zn-SOD II is affected to a lesser extent, with a maximum inhibition of 36% at high Percoll concentrations, whereas isozyme Cu,Zn-SOD I undergoes only slight variations. However, dilution of the samples followed by electrophoresis completely removes the Percoll inhibitory action. Results suggest that superoxide dismutases could be adsorbed onto the Percoll surface through electrostatic interactions. PMID- 2999912 TI - Peptide hormone production in primary lung tumors. PMID- 2999913 TI - Morphological growth characteristics and hormone secretion of small cell carcinoma of the lung in vitro. PMID- 2999914 TI - The serum-free establishment and in vitro growth properties of classic and variant small cell lung cancer cell lines. PMID- 2999915 TI - Expression of peptide and other markers in lung cancer cell lines. PMID- 2999916 TI - Neurotensin in human small cell lung carcinoma. PMID- 2999918 TI - Clinical evaluation of the neurophysins as tumor markers in small cell lung cancer. PMID- 2999917 TI - Peptide hormones in patients with lung cancer. PMID- 2999919 TI - Prospective multicenter study of hormone markers in small cell lung cancer. PMID- 2999921 TI - ACTH and related peptides in lung cancer. PMID- 2999920 TI - Oncogene expression in human small cell lung carcinoma. PMID- 2999922 TI - Physalaemin-like immunoreactivity from human lung small cell carcinoma: isocratic reversed-phase HPLC analysis of the chemically modified peptide. PMID- 2999923 TI - Calcitonin in human malignancies. PMID- 2999924 TI - Calcitonin in lung cancer. PMID- 2999925 TI - Parathyroid hormone and PTHmRNA in a human small cell lung cancer. PMID- 2999927 TI - Differentiation of reference strains of leptospires of the Pomona serogroup by cross-agglutination absorption and restriction endonuclease analysis. AB - The serological classification of all reference strains that have been described as representing separate serovars of Leptospira interrogans within the Pomona serogroup was investigated using cross-agglutination absorption and bacterial restriction endonuclease analysis (BRENDA). Comparative cross-agglutination absorption studies indicated that cornelli CB, monjakov Monjakov and kennewicki LT1026 were homologous with pomona Pomona, and dania K1 and tsaratsova B81/7 were homologous with mozdok 5621. BRENDA confirmed these results, except that pomona Pomona and monjakov Monjakov showed a difference in the high molecular weight region. It is proposed that four serovars be currently recognised within the Pomona serogroup: pomona, mozdok, proechimys and tropica. The relative merits of the use of cross-agglutination absorption and BRENDA with respect to identification of Pomona serogroup isolates are discussed. PMID- 2999926 TI - The pathogenesis of hormone-producing tumors of the lung. PMID- 2999929 TI - Transmission of capripoxvirus. AB - The transmission of capripoxvirus to sheep, using an aerosol suspension of a Yemen isolate of the virus, was demonstrated. Capripoxvirus was also transmitted by contact to sheep and goats kept with animals infected with virus isolates from the Yemen, Sudan, India and Nigeria. The incubation period for capripoxvirus infection in sheep and goats was approximately eight to 12 days. Animals that had well developed clinical signs transmitted capripoxvirus more rapidly than animals which died of peracute disease or animals that had only mild clinical signs. PMID- 2999928 TI - Identification by cross-agglutination absorption and restriction endonuclease analysis of leptospires of the Pomona serogroup isolated in the United Kingdom. AB - Five strains of Leptospira interrogans isolated in the United Kingdom and belonging to the Pomona serogroup were subjected to cross-agglutination absorption and bacterial restriction endonuclease DNA analysis (BRENDA) for their identification. British isolates were compared with reference strains representing the known serovars in the Pomona serogroup and also with isolates of the Pomona serogroup obtained from other countries. Three strains isolated from wildlife in England produced equivocal results when the cross-agglutination absorption and BRENDA results were compared. According to the World Health Organisation definition of a serovar the three English strains represented two new serovars, whereas by BRENDA all three had DNA electrophoresis patterns indistinguishable from serovar mozdok. Serovar pomona has not as yet been isolated in Great Britain and the epidemiology of the Pomona serogroup infections that have been detected by serology suggests that a serovar such as mozdok, maintained by wildlife, may be the causal agent. Two strains isolated in Northern Ireland were identified as pomona by the cross-agglutination absorption test. Further studies are needed to investigate the homogeneity of field and reference strains that are designated as pomona using the cross-agglutination absorption test. PMID- 2999930 TI - Guinea pig protection test as indicator of potency of oil emulsion foot-and-mouth disease vaccines. AB - A vaccine potency test is described involving virus challenge to six groups of 10 guinea pigs at five weeks after vaccination. Sixteen oil emulsion foot-and-mouth disease vaccines were so tested and nine retested after storage at 4 degrees C for up to 28.3 months. The results were compared with those of the routinely used oil emulsion vaccine potency test (protection afforded to eight pigs challenged 21 days after vaccination). When guinea pig estimates of 3 log2 PD50 or more were obtained, then, with one exception, the batches protected all or almost all pigs from challenge, but when the guinea pig estimates were less than 1 log2 PD50, the vaccines failed to protect five out of eight pigs. The sensitivity and reproducibility of the guinea pig method, established by repeated tests on two vaccine batches, seemed acceptable. The results suggested that guinea pig estimates might provide a suitable substitute for pig challenge potency tests because they reflected the potency of the vaccines, were likely to involve smaller standard errors and caused less discomfort to animals. PMID- 2999931 TI - Bronchoalveolar lavage in sarcoidosis. AB - Bronchoalveolar lavage (BAL) was performed in 1,188 patients suffering from sarcoidosis. After technical considerations, the authors analyze the results of BAL from a practical point of view concerning its value for the diagnosis of sarcoidosis and its prognostic value and its value for the selection of therapy, particularly for the decision as to steroid treatment. BAL helps in the diagnosis of sarcoidosis, but is not specific enough to provide this diagnosis on its own. The persistence of high alveolar lymphocytosis within the first year of evolution is strongly correlated with nonrecovery from pulmonary sarcoidosis at 2 years and thus with the evolution towards a chronic phase of the disease. On the other hand, BAL can provide basic information for a better understanding of the disease and permits immunocompetent cells and soluble factors to be recovered from the lung, which is useful for immunological studies. PMID- 2999932 TI - Prognostic value of chest radiograph, serum-angiotensin-converting enzyme and T helper cell count in blood and in bronchoalveolar lavage of patients with pulmonary sarcoidosis. AB - We studied the prognostic value of the initial radiologic stage, the serum angiotensin-converting enzyme (SACE) and T helper cells in blood (OKT-4-Bl) and in bronchoalveolar lavage (OKT-4-BAL) for patients with biopsy-proven pulmonary sarcoidosis. Thirty-seven patients without prior treatment were followed up for a period of 2 years. A BAL was performed in 22 of them as part of the diagnostic workup. Clinical examination, chest radiographs, vital capacity, diffusion capacity for CO, airway resistance and PaO2 at rest and during exercise were determined initially and after 6, 12 and 24 months. According to these results, patients were classified as having progressive or nonprogressive disease. The radiologic stage and the initial SACE (38.3 +/- 10.2 vs. 43.7 +/- 13.0 nmol/ml/min) could not discriminate between the two groups. Patients with progressive disease had significantly fewer OKT-4-Bl cells (403.3/microliter +/- 146.7/microliter vs. 842.0/microliter +/- 430.1/microliter) and more OKT-4-BAL cells (24.5 +/- 15.4% vs. 7.0 +/- 2.6%) than patients with stable disease (p less than 0.01). A negative correlation between OKT-4-Bl and OKT-4-BAL cells was shown (Rs = -0.79; p less than 0.001). We conclude that the number of OKT-4-Bl and OKT 4-BAL cells can be used as prognostic parameter for patients with sarcoidosis. PMID- 2999933 TI - Isolation and characterization of a lambda bacteriophage mutant able to grow on an Escherichia coli nusA-1 host. PMID- 2999934 TI - [A case of hypophosphatemic vitamin D-resistant rickets in which spontaneous dental abscesses were the first evidence]. PMID- 2999935 TI - [Anticancer food factors]. PMID- 2999936 TI - [Receptors in bronchial asthma]. PMID- 2999937 TI - [Lambert-Eaton pseudomyasthenic paraneoplastic syndrome in small-cell anaplastic bronchopulmonary cancer]. PMID- 2999939 TI - [The circulation of enteroviruses in Iasi Province]. PMID- 2999938 TI - [Comparative efficacy of disinfectants on the poliovirus]. PMID- 2999941 TI - Lack of a disulfide bond in the large subunit of (Na, K)ATPase. AB - Subunit distribution of disulfide bonds in the dog kidney (Na, K)ATPase was examined. It was estimated that there were no disulfide bonds in the alpha subunit and possibly one in the beta subunit. The disulfide bond located in the beta subunit would be a good probe for elucidation of the role of the beta subunit. Our previous overestimation was presumably due to the phospholipid attached to the subunits. PMID- 2999940 TI - [Effect of neuroendocrine and immune factors in the experimental induction of lipid metabolic and cardiovascular disorders]. PMID- 2999943 TI - Retrograde labeling of dorsal root ganglion neurons after injection of tritiated amino acids in the spinal cord of rats and cats. AB - The present experiments are based upon evidence that neurons may selectively take up at their terminals, and retrogradely transport, the same chemical they use as a neurotransmitter or its analogues. In an attempt to identify dorsal root ganglion (DRG) neurons that use glutamic acid as a neurotransmitter, [3H]D aspartate ([3H]D-Asp) was chosen as a marker, since it is a metabolically inert amino acid known to be taken up by the same affinity mechanism as L-aspartate and L-glutamate. Adult rats and cats received injections of 50 nl to 1.5 microliter of [3H]D-Asp (500 microCi/microliter) in the dorsal horn of cervical segments (C3 to C6). At 9 to 48 hr after injection, all animals were perfused with 5% glutaraldehyde. After sections were processed for autoradiography, the DRG neurons situated most closely to the injection site were chosen from representative cases, and the number and cross-sectional area of labeled and unlabeled perikarya with a nucleolus in the plane of the section were calculated. In rats, about 4% of the sampled DRG neurons were autoradiographically labeled, and the mean perikaryal area of these neurons was about twice that of unlabeled perikarya. In cats, the percentage of labeled perikarya ranged between 6.5% and 13.27% of the sampled population. The ratio of the mean perikaryal area of labeled neurons to that of unlabeled neurons ranged between 1.6 and 2.5. In a control cat injected with [3H]proline at C7, all perikarya in the C7 DRG were autoradiographically labeled. However, with injection of [3H]gamma-aminobutyric acid ([3H]GABA) selective retrograde labeling was observed. Quantitative data in rat showed that perikarya labeled at the C6 level after injection of this amino acid constituted about 8% of the sample population in C6 DRG. The ratio of the size of labeled to unlabeled perikarya was 2.02. In one cat injected with [3H]GABA at caudal C3, the largest number of labeled perikarya were in C4 DRG and comprised up to 5.32% of the sampled population. The ratio of the size of labeled to unlabeled perikarya was 1.57. The results in cases of injection with [3H]D-Asp may be interpreted as consistent with the idea that a fraction of DRG neurons use glutamate and/or aspartate as neurotransmitter(s).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 2999942 TI - Sympathetic activation of A-delta nociceptors. AB - Primary afferent units in the saphenous nerve of cats, functionally identified as A-delta myelinated nociceptors, were tested for their responses to stimulation of the sympathetic trunk. The units were subdivided functionally into A-mechano-heat receptors (AMHs), which respond to both noxious heat and pressure, and high threshold mechanoreceptors (HTMs), which respond only to pressure. No units of either subdivision were activated by sympathetic stimulation (SS) prior to noxious heating of their receptive fields. However, six of the seven AMH units with the highest mechanical thresholds (greater than 5 g von Frey) were activated by SS alone (10 Hz) after they had been sensitized by noxious heating of their receptive fields. Sensitized AMH units with lower mechanical thresholds (less than 5 g) were generally not activated by SS alone (1 of 22 units), and their responses to warming of their receptive fields were not altered by SS. The excitatory sympathetic action on AMH units was abolished by alpha- but not beta andrenergic blockade in the two units tested. HTMs were unresponsive to SS even after repeated noxious heating of their receptive fields (15 units tested). The results of this study indicate that relatively high rates of sympathetic efferent activity (10 Hz) can induce firing in a small population of AMH receptors in damaged skin, specifically those units with high mechanical thresholds. This sympathetically evoked activity might trigger or exacerbate pain associated with skin damage; however, functional conclusions are difficult to draw, because of the scarcity of such units and the fact that the responses in some were brief and of low firing rates. PMID- 2999946 TI - Mechanisms of action of antituberculosis drugs in standard and short-course chemotherapy regimens. PMID- 2999944 TI - Antigens of Chlamydia trachomatis. AB - Chlamydia trachomatis is an obligate intracellular parasite that elaborates antigens on its surface. These antigens are divided into genus-, species-, subspecies-, and serovar-specific determinants. The genus, or group antigen(s), are lipopolysaccharides similar to those found in gram-negative bacteria and a glycolipid that is secreted by infected cell cultures. Species-specific antigens differentiate Chlamydia trachomatis from Chlamydia psittaci and are expressed on the outer membrane. These proteins range in molecular weight from 155,000 to approximately 40,000. Monoclonal antibodies to outer-membrane proteins have demonstrated the presence of subspecies-reactive antigenic determinants. Type specific antigens are associated with the major outer-membrane protein and are secreted from infected cells as well. The molecular weights of these proteins range from 30,000 to 40,000. These antigens may participate in the binding of the organism to target cells and in an enzymatic process of some type that initiates endocytosis. The significance of the soluble antigens detected in the microenvironment in vitro may suggest immune-complex formation, a process that could contribute to the immunopathology of the disease. PMID- 2999945 TI - Bacteriological bases of short-course chemotherapy. PMID- 2999947 TI - Clinical and bacteriological studies on the comparative efficacy of short-course regimens starting with daily or twice-weekly phases. PMID- 2999948 TI - Modern short-course regimens in the treatment of tuberculosis. PMID- 2999949 TI - Efficiency of short-course tuberculosis chemotherapy in controlled clinical trials and in programme conditions in Romania. PMID- 2999950 TI - Report from the application of 9 months short-course regimens to 575 patients suffering from pulmonary tuberculosis. PMID- 2999951 TI - The short-course treatment of tuberculosis in Spain. PMID- 2999952 TI - Preliminary results of a short-course antituberculosis regimen including Sinerdol EH and applied under operational conditions. PMID- 2999953 TI - The chemotherapy of tuberculosis in Bulgaria today. PMID- 2999954 TI - Situation of TB control programme in Turkey. PMID- 2999958 TI - Cost of tuberculosis chemotherapy. PMID- 2999955 TI - Cost of tuberculosis chemotherapy short-course regimens and care. PMID- 2999957 TI - Reflections on short-course chemotherapy in routine conditions in Morocco. PMID- 2999956 TI - Cost of antituberculosis drug regimens in Morocco. PMID- 2999959 TI - Adverse reactions in the conditions of an ab initio intermittent (2/7) administration of antituberculous regimens. PMID- 2999961 TI - Evaluation of short-course chemotherapy in Romania. PMID- 2999960 TI - The surveillance of tuberculosis including the evaluation of chemotherapy programmes. PMID- 2999962 TI - Chemotherapy of tuberculosis under routine conditions in Czechoslovakia. PMID- 2999963 TI - Organization aspects of delivery of short-course chemotherapy within integrated tuberculosis programmes. PMID- 2999964 TI - Problems of short-course chemotherapy still awaiting solution. PMID- 2999965 TI - Hepatic binding of triglyceride-rich lipoproteins in humans. AB - Human plasma chylomicrons (Sf greater than 200), which equals large chylomicron remnants and VLDL remnants, were obtained from a subject with hypertriglyceridaemia. These plasma chylomicrons were radioactively labelled and incubated with human liver membranes. Plasma chylomicrons bound to the membranes in lipoprotein particle form, and binding occurred to the same degree after plasma chylomicrons had been further degraded in vitro by the enzyme lipoprotein lipase. Plasma chylomicron binding to human liver membranes contained a Ca2+ dependent and a Ca2+ independent part. The Ca2+ dependent binding was saturable at 37 degrees C. A 20-fold excess of LDL only displaced 20% of plasma chylomicron binding to liver membranes, indicating binding of the two lipoproteins to different sites. PMID- 2999966 TI - Thyroid hormone receptors in fetal and hormone resistant tissues. PMID- 2999967 TI - Blood hormone and metabolite levels during graded cycle ergometer exercise. AB - To study the effect of the intensity of physical exercise on plasma hormone and metabolite levels, a group of 11 well-conditioned males participated in cycle ergometer exercise. The subjects pedalled at three different work loads, corresponding to 63% of VO2max (duration 10 min), 86% of VO2max (duration 10 min) and maximal work load (tolerated 5-7 min) The increases in blood adrenaline, noradrenaline, growth hormone, cyclic AMP, glycerol and lactate concentrations were remarkably similar and exponentially related to the work load. The concentrations of blood glucose, cortisol and glucagon increased only at maximal work load. Many of these changes in blood metabolite and hormone concentrations seem to be related to the increased sympathetic activity during graded exercise. PMID- 2999968 TI - X-ray microanalysis of urinary stones, a comparison with other methods. AB - A previous study of urinary stones by a combined electron microscopy demonstrated the potential of scanning electron microscopy and X-ray analysis as an analytic tool for urinary stones. Electron diffraction was chosen for the final confirmation of crystals in the study. Although electron diffraction is highly accurate for this purpose, it is desirable to establish the sensitivity of X-ray analysis for the identification of stone components relative to the more commonly used methods. Eighty six consecutive urinary stones were analyzed by X-ray analysis and the findings were compared with those of X-ray diffraction, infrared spectrometry and chemical analysis. The results indicate that X-ray analysis exceeds X-ray diffraction and infrared spectroscopy in its sensitivity for the identification of stone components several fold. This was largely due to the inability of the latter methods to detect apatite in more than half of the apatite containing stones. The findings in X-ray analysis had the best correlation with chemical analysis, which was applied mainly to the detection of apatite. X-ray analysis is particularly suited for the detection of rare and minor inorganic components of urinary stones such as silica and gypsum, and is obviously one of the most powerful tools for the analysis of urinary stones. Further application of X-ray analysis to urinary stone is likely to discern rare inorganic components of urinary stones overlooked by other methods. PMID- 2999969 TI - Occupational toxic neuropathies--an update. AB - Peripheral neuropathy is the commonest neurological syndrome produced by industrial and agricultural chemicals. A number of chemicals have been recently found to be neurotoxins; in addition some known occupational neurotoxic hazards more often produce subtle alterations in the peripheral nervous system. There have been problems of both evaluating neurotoxic effects and identifying toxic causes. This article reviews and discusses the current research and progress covering neurophysiological, neuropathological, biochemical, and epidemiologic approaches in clinical and experimental neurotoxicology, as related to the assessment and pathogenesis of occupational toxic neuropathies. PMID- 2999970 TI - [Endobronchial granular myoblastoma: therapeutic stance]. AB - The case of a 34-year-old diabetic patient with an endobronchial granular myoblastoma is presented. This tumor was discovered during an episode of acute pneumonia on the same side as the neoplasm. Endobronchial granular myoblastoma is rare; it is sometimes multiple, with some degree of mucosal infiltration. The treatment (monitoring only, endoscopic or surgical resection) is not clearly defined in the literature: we suggest a therapeutic approach taking into account the characteristic features of the tumor (size, location and number) and of the patient (other diseases, age). PMID- 2999971 TI - The molecular basis of communication within the cell. PMID- 2999972 TI - The immune system in AIDS. PMID- 2999974 TI - Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. AB - A novel potential cell surface receptor of the tyrosine kinase gene family has been identified and characterized by molecular cloning. Its primary sequence is very similar to that of the human epidermal growth factor receptor and the v-erbB oncogene product; the chromosomal location of the gene for this protein is coincident with the neu oncogene, which suggests that the two genes may be identical. PMID- 2999973 TI - Enhanced transcription of c-myc in bursal lymphoma cells requires continuous protein synthesis. AB - In several bursal lymphoma cell lines in which c-myc transcription is regulated by avian leukosis virus (ALV) long terminal repeat (LTR) sequences, protein synthesis inhibition decreases the transcriptional activity of c-myc as well as other LTR driven viral genes. This decrease in transcription is associated with a change in the chromatin structure of c-myc, as measured by deoxyribonuclease I (DNase I) hypersensitivity, and a shift of transcription from the LTR to the normal c-myc promoter. In contrast, cycloheximide had little or no effect on the transcription of LTR driven genes in infected chicken embryo fibroblasts treated with the drug. These results suggest that a labile, cell type-specific protein may interact with the retroviral LTR and regulate transcription of genes under LTR control. Further, the results demonstrate that the increase in intracellular concentration of c-myc RNA induced by cycloheximide treatment of normal cells is the result of stabilization of this message. PMID- 2999975 TI - Politics and science clash on African AIDS. PMID- 2999976 TI - Africa and the origin of AIDS. PMID- 2999977 TI - Alpha 2-adrenergic mechanisms in prefrontal cortex associated with cognitive decline in aged nonhuman primates. AB - This study provides evidence that the alpha 2-adrenergic receptor agonist clonidine ameliorates the cognitive deficits exhibited by aged nonhuman primates through drug actions at alpha 2 receptors. Furthermore, pharmacological profiles in animals with lesions restricted to the dorsolateral prefrontal cortex indicate that this area may be the site of action for some of clonidine's beneficial effects. These results demonstrate that alpha-adrenergic systems contribute to cognitive function and suggest a new strategy for treating memory disorders in aged humans. PMID- 2999978 TI - The human gene encoding GM-CSF is at 5q21-q32, the chromosome region deleted in the 5q- anomaly. AB - Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 22,000 dalton glycoprotein that stimulates the growth of myeloid progenitor cells and acts directly on mature neutrophils. A full-length complementary DNA clone encoding human GM-CSF was used as a probe to screen a human genomic library and isolate the gene encoding human GM-CSF. The human GM-CSF gene is approximately 2.5 kilobase pairs in length with at least three intervening sequences. The GM CSF gene was localized by somatic cell hybrid analysis and in situ hybridization to human chromosome region 5q21-5q32, which is involved in interstitial deletions in the 5q- syndrome and acute myelogenous leukemia. An established, human promyelocytic leukemia cell line, HL60, contains a rearranged, partially deleted GM-CSF allele and a candidate 5q- marker chromosome, indicating that the truncated GM-CSF allele may reside at the rejoining point for the interstitial deletion on the HL60 marker chromosome. PMID- 2999979 TI - Direct evidence that endogenous GM1 ganglioside can mediate thymocyte proliferation. AB - The B subunit of cholera toxin, which is multivalent and binds exclusively to a specific ganglioside, GM1, was mitogenic for rat thymocytes. When exposed to the B subunit, the cells proliferated, as measured by 3H-labeled thymidine incorporation. Mitogenesis depended on the direct interaction of the B subunit with GM1 on the surface of the cells. This demonstrates that endogenous plasma membrane gangliosides can mediate proliferation in lymphocytes. PMID- 2999980 TI - Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. AB - Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA. PMID- 2999981 TI - AIDS therapy: new push for clinical trials. PMID- 2999982 TI - New sickle cell test. PMID- 2999983 TI - Promoter region of the human Harvey ras proto-oncogene: similarity to the EGF receptor proto-oncogene promoter. AB - Regulation of transcription of members of the ras gene family undoubtably plays an important role in controlling cellular growth. Examination of this level of regulation requires identification of the promoter regions of the ras proto oncogenes. Four major transcriptional start sites were detected in the human Harvey ras 1 proto-oncogene. The promoter region contains neither a TATA box nor a CAAT box in their characteristic upstream positions, has an extremely high G+C content (80 percent), and contains multiple GC boxes including seven CCGCCC repeats and three repeats of the inverted complement, GGGCGG. This region has strong promoter activity when placed upstream from the chloramphenicol acetyl transferase gene and transfected into monkey CV1 cells. In these ways the Harvey ras 1 proto-oncogene promoter resembles the promoter of the gene encoding the epidermal growth factor (EGF) receptor. The similarity between the two proto oncogene promoters may be relevant to the mechanism by which the expression of such "growth control" genes is regulated. PMID- 2999984 TI - Repression of the immunoglobulin heavy chain enhancer by the adenovirus-2 E1A products. AB - The products of the adenovirus-2 (Ad2) immortalizing oncogene E1A repress the activity of the SV40, polyoma virus and E1A enhancers. Evidence is presented that Ad2 infection of MPC11 plasmocytoma cells results in an inhibition of transcription of both the gamma 2b heavy chain (IgH) and the kappa light chain immunoglobulin genes. This inhibition is caused by the Ad2 E1A products. Furthermore, the Ad2 E1A products repress transcription activated by the immunoglobulin heavy chain enhancer in chimeric recombinants, which are either stably integrated in the genome of lymphoid cells or are present as episomes. The implications of negative regulation of cellular enhancers are discussed. PMID- 2999985 TI - Gene expression in mice after high efficiency retroviral-mediated gene transfer. AB - A retroviral expression vector (N2) containing the selectable gene, neoR, has been used to determine the optimal conditions for infecting murine hematopoietic progenitor cells at high efficiency. After infected bone marrow cells were introduced into lethally irradiated mice, the presence, stability, and expression of the vector DNA sequences were analyzed either in individual spleen foci 10 days later or in the blood, bone marrow, and spleens of mice 4 months later. When bone marrow cells were cultured in medium containing virus with titers of more than 10(6) colony-forming units per milliliter in the presence of purified murine interleukin-3, more than 85 percent of the resulting foci contained vector DNA. This proviral vector DNA was intact. Efficient expression of the neoR gene was demonstrated in most of the DNA-positive foci examined. The spleens of reconstituted animals (over a long term) contained intact "vector DNA" and the blood and bone marrow expressed the neoR gene in some animals. Thus, a retroviral vector can be used to introduce intact exogenous DNA sequences into hematopoietic stem cells with high efficiency and with substantial expression. PMID- 2999986 TI - A human Y-linked DNA polymorphism and its potential for estimating genetic and evolutionary distance. AB - A human DNA sequence (p12f2), derived from a partial Y-chromosome genomic library and showing homology with the X and Y chromosomes and with an undetermined number of autosomes, detected two Y-specific restriction fragment length variants on male DNA that had been digested with Taq I and Eco RI. These variants may have been generated through a deletion-insertion mechanism and their pattern of holoandric transmission indicates that they represent a two-allele Y-linked polymorphism (RFLP). By means of DNA from patients with inborn deletions in chromosome Y, this polymorphic DNA site was mapped to the interval Yq11.1 Yq11.22. The frequency of the rarest allele was about 35 percent in Algerian and Sardinian human males, whereas it was only 4 percent among Northern Europeans. The p12f2 probe also detected Y-specific DNA fragments in the gorilla and chimpanzee. In view of the monosomy of the Y chromosome in mammalian species, Y linked RFLP's may prove to be more useful than autosomal or X-linked markers in estimating genetic distances within and between species. PMID- 2999987 TI - Synthesis of rheumatoid factor in vitro: implications for the pathogenesis of rheumatoid arthritis. PMID- 2999989 TI - [Follow-up of the patient with CAPD in the office]. PMID- 2999988 TI - Central nervous system manifestations of chicken pox--a report of two cases. PMID- 2999990 TI - Cervical chlamydial infections: diagnostic accuracy of the Papanicolaou smear. AB - To assess the reliability and accuracy of the Papanicolaou smear for the detection of cervical Chlamydia trachomatis infections, we obtained chlamydial cultures and cervical smears from 252 patients attending a gynecologic service during an eight-month time period. In 12 of these patients (4.8%), the cervical culture was positive for chlamydiae but the cytologic smear was negative for chlamydial inclusions. Five smears (2.2%) were cytologically positive for chlamydiae but were negative by culture. In none of the patients were both culture and smear positive for chlamydiae. In our experience the smear was a completely unreliable test for the diagnosis of cervical C trachomatis infections. The cytologic smear should not replace culture for the detection of chlamydial infections. PMID- 2999991 TI - Spontaneous rupture of hepatoma: historical perspectives. PMID- 2999992 TI - Multicentric mullerian squamous neoplasia. AB - Pure squamous neoplasia of the uterine corpus and uterine tube is explained by two possible mechanisms termed horizontal spread and vertical proliferation. We have reported a case of epidermoid carcinoma in situ of the uterine tube exemplifying the latter. PMID- 2999994 TI - Metastatic oat cell carcinoma of the lung producing extrahepatic bile duct obstruction. AB - Although carcinoma of the lung metastatic to the pancreas is not unusual in autopsy series, it is rare as a cause of extra-hepatic bile duct obstruction among the living. We have reported two cases of oat cell carcinoma of the lung in which the first sign of metastatic disease was extrahepatic bile duct obstruction. Ultrasonography provides a quick, direct, and noninvasive means of evaluating such obstruction, allowing early diagnosis and surgical palliation. PMID- 2999993 TI - Peripheral neuropathy with hypophosphatemia in a patient receiving intravenous hyperalimentation. AB - We have described a patient receiving intravenous hyperalimentation in whom hypophosphatemia resulted in a neurologic syndrome characterized by ophthalmoplegia, ataxia, and areflexic paralysis of all extremities, requiring respiratory assistance. We have discussed pertinent literature related to neurologic dysfunction as a result of hypophosphatemia. PMID- 2999996 TI - The effect of interferon-alpha A on two cases of Japanese encephalitis in Thailand. AB - Two Japanese encephalitis cases with serious comatous symptoms were treated with the Human Recombinant Interferon-alpha A. The clinical responses to IFN were found to be satisfactory. The first case showed improvement on the 5th day of IFN treatment and the general condition slowly improved. The second case recovered from the comatous stage on the 6th day of IFN, followed by quick improvement of general symptoms in the 2nd week and complete recovery without any mental sequelae. Leukopenia and neutropenia occurred during the first week of administration of IFN, but were only temporary. Slight elevation of SGOT and SGPT was observed in the first case. No other side effects including general toxicity, neurotoxicity or allergy, or any abnormal hematological and blood chemistry changes were observed in these 2 cases. Two other JE cases (the 3rd and 4th consecutive JE cases) were not treated with IFN, but received the usual regimens of symptomatic and supportive drugs. Both patients died on the 7th-9th day of illness. This study suggests that the Human Recombinant Leukocyte A Interferon possibly is an effective and promising agent in the treatment of Japanese encephalitis in Thailand. More studies to treat JE cases with this IFN are being performed in order to assess the efficacy, tolerance and safety of rIFN-alpha A on Japanese encephalitis in Thailand. PMID- 2999995 TI - Intense transmission of Japanese encephalitis virus to pigs in a region free of epidemic encephalitis. AB - Epidemic Japanese encephalitis recurs annually in the northern provinces of Thailand, but in the southern provinces cases of human encephalitis are rare. We investigated transmission of Japanese encephalitis virus (JEV) to pigs in southern Thailand. Blood specimens from one hundred young pigs at abattoirs in three southern provinces were tested for JEV hemagglutination inhibiting (HAI) antibodies. Seventy-four percent were positive. Ten seronegative sentinel pigs were placed at five locations in one southern province. Seven of the ten pigs developed JEV HAI and JEV IgM ELISA antibodies within two weeks of placement. JEV was isolated from all seven seroconverting sentinel pigs from blood specimens collected 3 to 11 days after placement. Fifteen light-trap mosquito collections at the five locations all included known JEV vectors, some in large numbers. We conclude that there is intense transmission of JEV to pigs in southern Thailand despite the rare occurrence of human encephalitis in the same region. PMID- 2999997 TI - [Clinical variants of lesions of the peripheral nervous system in chronic renal insufficiency]. PMID- 2999998 TI - [Hyperparathyroid polyneuropathy]. PMID- 2999999 TI - [The glucagonoma syndrome]. PMID- 3000000 TI - Defective DNA topoisomerase I activity in a DNAts mutant of Balb/3T3 cells. AB - Cell and polyomavirus DNA synthesis in ts20, a temperature-sensitive mutant derived from Balb/3T3 cells, is inhibited at an early step in chain elongation in vivo and in vitro. Virus DNA synthesized under restrictive conditions, when analyzed by gel electrophoresis and fluorography, contained a series of equally spaced bands migrating between form I and form II. If restrictive conditions were prolonged, the relative amount of these less-supercoiled topoisomers increased while the overall amount of virus DNA decreased. DNA topoisomerase I activity was lower and more heat-labile when prepared from mutant cells compared to wild-type and revertant cells. An assay in which extracts from wild-type cells corrected defective cell DNA synthesis in lysed mutant cells was applied to purification of the active factor from such extracts. Salt fractionation and three cycles of column chromatography resulted in the isolation of the activity in a fraction containing 10 major polypeptides. The specific activity in the final preparation was increased fivefold and was accompanied by the activity of DNA topoisomerase I. Our results provide evidence that DNA topoisomerase I functions at an early step in chain elongation of cell and polyomavirus DNA synthesis and that the enzyme activity may be decreased as a result of the mutation in ts20. PMID- 3000001 TI - Variant of A431 cells isolated by ricin A-conjugated monoclonal antibody directed to EGF receptor: phosphorylation of EGF receptor and phosphatidylinositol. AB - A monoclonal antibody specific for human EGF receptors was cross-linked to subunit A of toxic ricin. Using this conjugate, we isolated a variant of A431 cells, designated C1-B7, with approximately 40 times less EGF binding capacity. Unlike the parental cells, the C1-B7 variant was resistant to EGF-induced suppression of cell growth. The EGF receptors retained in this variant were of high-affinity type and susceptible to EGF-induced autophosphorylation. Membrane prepared from C1-B7 cells was highly phosphorylated in the presence of 2 microM [gamma-32P]-ATP, primarily on the lipid components shown as phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. This same level of lipid phosphorylation was observed on A431 membrane only in the presence of higher ATP concentrations. After addition of EGF to A431 membrane, phosphatidylinositol phosphorylation was significantly decreased with a concomitant increase in EGF dependent protein phosphorylation. Thus, the EGF-dependent receptor-mediated protein phosphorylation precedes phosphatidylinositol phosphorylation. These observations support the idea that the growth inhibitory effect of EGF on A431 cells is caused by high ATP consumption due to the EGF-induced protein phosphorylation and reduction of phosphatidylinositol turnover. PMID- 3000002 TI - Hotspots for spontaneous and mutagen-induced lesions in regulatory subunit of cyclic AMP-dependent protein kinase in S49 mouse lymphoma cells. AB - From an S49 mouse lymphoma cell subline that carries an electrophoretic marker mutation in one allele for a regulatory (R) subunit of cyclic AMP-dependent protein kinase, 130 cyclic AMP-resistant mutants were isolated and characterized. Of the 77 independent spontaneous and mutagen-induced isolates identified, 74 had kinases with increased apparent activation constants (KaS) for cyclic AMP dependent activation. The "Ka" phenotype was invariably correlated with an apparent structural lesion in one R subunit allele. "Charge-shift" lesions in 43 independent isolates were mapped to small regions within the R subunit by two dimensional gel analysis of partial proteolysis peptides. Nine Ka mutations were distinguished by differences in charge or peptide maps of mutant R subunits, and the mutations were clustered in two regions associated with the cyclic AMP binding sites of the R subunit. The relative frequencies of different mutations differed among spontaneous, ethyl methanesulfonate-induced, and N-methyl-N'-nitro N-nitrosoguanidine-induced isolates. Mutation frequencies were also markedly different for the two R subunit alleles; this allele preference was strongest for mutagen-induced lesions in the more carboxy terminal cyclic AMP-binding site. PMID- 3000003 TI - Studies on gene transfer and reversion to UV resistance in xeroderma pigmentosum cells. AB - We have examined several parameters which address the feasibility of complementing the UV-sensitive phenotype of xeroderma pigmentosum (XP) fibroblasts by gene transfer. We present a comparative study which demonstrates that, relative to immortalized cells, human diploid cells are poor recipients for gene transfer. As measured by both transient and stable expression assays, diploid fibroblasts were completely refractory to DNA transfer by calcium phosphate coprecipitation and exhibited substantially reduced levels of expression following gene transfer by fusion with E. coli protoplasts. We also examined the significance of reversion of the phenotype of UV sensitivity in SV40 immortalized XP-A cell lines. In addition to confirming a previous report of reversion to wild-type levels of UV resistance at a frequency of approximately 10(-7), we have attempted to facilitate the identification of XP-A cells complemented with genomic DNA by employing less stringent selection schemes and cotransfection of a selectable marker. Under these conditions, we observed an increased frequency of reversion and were unable to identify true transfectants. PMID- 3000004 TI - The antiemetic effect of Cannabis sativa during cytotoxic therapy. PMID- 3000005 TI - [Interaction of fibrinogen with platelets. Review]. PMID- 3000006 TI - [Practical use of the platelet peroxidase reaction at the ultrastructural level in the characterization of an apparently undifferentiated blast cell population]. PMID- 3000007 TI - Solid malignancies in children and adolescents. AB - The cure rate in childhood cancer has improved markedly during the past 20 years. In the 1960s the cure rate was about 20 to 30 per cent, but today more than 50 per cent of children and adolescents with cancer are being cured. This improvement is principally due to multidisciplinary teamwork in diagnosing, staging, and treating children with cancer; newer and more chemotherapeutic agents; and a recognition that combination therapy consisting of surgery, radiotherapy, and chemotherapy is frequently indicated. PMID- 3000008 TI - Role of the surgeon in the treatment of children's cancer. AB - The management of children's tumors has changed significantly in the past several years. New techniques and combined surgical, chemotherapeutic, and radiation approaches are responsible for improved survival in most instances. Cooperation of the surgeon with the specialists in separate disciplines is imperative to continued advancements in neoplastic disease of childhood. PMID- 3000009 TI - Abdominal masses. AB - The more common abdominal masses of the pediatric age group are discussed along with current thoughts regarding preoperative imaging modalities. A careful physical examination is indispensable, and masses should be evaluated with consideration given to mobility, location, consistency, contour, and site. A rectal examination is essential. The more unusual clinical presentations are discussed in appropriate sections. PMID- 3000010 TI - The evaluation of postoperative function of the adrenal gland. AB - The cosyntropin (Cortrosyn) stimulation test has been adapted for use in the postoperative period. The normal adrenal gland response to 200 micrograms of cosyntropin given intravenously has been quantified at six, 12, 24, 48 and 72 hours after extensive general surgical procedures. The value of the test in quickly and accurately diagnosing postoperative acute insufficiency of the adrenal gland remains to be established. PMID- 3000011 TI - Epidermal growth factor receptors in normal and neoplastic thyroid tissue. AB - Epidermal growth factor (EGF) stimulates DNA synthesis and proliferation of thyroid cells in culture and may have an important role in the regulation of normal and neoplastic thyroid cell growth. We therefore studied paired normal and neoplastic thyroid tissue from eight patients for the presence of EGF receptors using a radioreceptor assay. 125I EGF binds to a particulate membrane fraction from both normal and neoplastic thyroid tissue with high affinity (dissociation constant ranged from 0.5 to 16.7 nmol/L). The binding is saturable, and maximal binding is achieved within 40 minutes at 37 degrees C and pH 7.5. This EGF binding is specific since it is competitively inhibited by unlabeled EGF but not by other hormones (thyrotropin, insulin, glucagon, and transferrin). The binding of EGF to thyroid neoplasms is higher than the binding to normal thyroid tissue (p less than 0.05). Thyroid tumors with a poorer prognosis appear to have higher EGF binding compared with adjacent normal thyroid tissue than have tumors with a better prognosis. EGF may have a role in the regulation of normal and neoplastic thyroid cell growth. Characterization of EGF receptors may help predict the clinical course of patients with malignant thyroid neoplasms. PMID- 3000012 TI - Morphologic and functional studies of a rat hypercalcemia-associated testicular tumor maintained in cell culture. AB - The Rice H500 tumor is a transplantable nonmetastasizing testicular tumor of Fischer rats associated with hypercalcemia and increased urine cyclic adenosine monophosphate (AMP) excretion, features similar to those of the clinical syndrome of humoral hypercalcemia of malignancy. Tumor cells can be maintained in tissue culture; one million cells grown in culture reinoculated in Fischer rats reproduce the syndrome of tumor growth and lethal hypercalcemia. Infusion of concentrated, serum-free cell culture supernatant into parathyroidectomized rats produced an increase in urine cyclic AMP similar to that produced by an infusion of bovine parathyroid hormone. Light and electron microscopic appearance of the H500 tumor in vivo and in vitro is similar to previous descriptions of a hypercalcemia-associated rat testicular tumor believed to be of Leydig cell origin. Ultrastructural characteristics of microvilli, intracellular glandlike lumina, and cell-cell attachments, however, suggest an epithelial origin. Absence of smooth endoplasmic reticulum typical of steroid-secreting Leydig cells suggest these cells are not actively involved in steroid synthesis and secretion. The ultrastructure of this tumor is sufficiently different from that of normal Leydig cells that the cell of origin is unclear. Nonetheless, this tumor provides a useful model of hormonally mediated tumor-associated hypercalcemia. PMID- 3000013 TI - Current status of adrenalectomy for Cushing's disease. AB - To evaluate the current use of adrenalectomy in the treatment of Cushing's disease, we reviewed seven consecutive patients who have undergone adrenalectomy for Cushing's disease at this medical center during 1983 to 1984. Seventy-one percent (5/7) had pituitary, or type I, Cushing's disease, while 29% (2/7) had adrenal, or type II, Cushing's disease from either an adenoma or an adrenocortical carcinoma. Presenting signs and symptoms, either initially or at the time of recurrence, were typical of Cushing's syndrome. Four of five patients with type I disease had recurrent disease after transphenoidal hypophysectomy, bilateral adrenalectomy, or unilateral adrenalectomy. In three of five patients, medical therapy of hypercortisolism was abandoned because of adverse side effects. Preoperative evaluation in all patients included cortisol and ACTH levels, dexamethasone suppression tests, and computerized tomography (both abdominal and head). In patients with a prior history of adrenalectomy, radiocholesterol scans were also performed and were useful. Angiographic procedures were not required in these patients. In patients with type I disease, posterior operative approaches were used. In patients with type II disease, an anterolateral approach was used. Posterolateral incisions are preferred over Hugh Young incisions and provide better exposure with a reduced risk of poor wound healing. Morbidity and mortality included one death and three nonhealing wounds. In the six surviving patients, symptoms resolved with variable frequency. Findings suggestive of Nelson's syndrome (hyperpigmentation) have occurred in two patients; serial computerized tomographic scans fail to reveal evidence of pituitary tumors. We conclude that adrenalectomy remains an essential form of therapy for patients with Cushing's syndrome caused by adrenal tumors or recurrence after previous surgery. The response to the operation is generally good, but long-term surveillance is required for the development of Nelson's syndrome. PMID- 3000014 TI - [Drug information. New principle in the treatment of elevated blood pressure]. PMID- 3000015 TI - [Radioisotope synovectomy]. PMID- 3000016 TI - [Exposure to solvents: clinical and biological criteria]. PMID- 3000017 TI - An intracellular pool of the procoagulant thromboplastin in human monocytes. AB - Human monocytes cell extracts have been analysed by density gradient centrifugation in Percoll gradients. Two peaks of activity of the plasma membrane marker enzyme 5' nucleotidase were detected. The main peak was at a density of 1.040 and a secondary one at a lower density. Thromboplastin activity was recovered associated with the main peak of 5' nucleotidase activity in control cells. In NH4Cl treated cells, thromboplastin activity is found at both densities. In agreement with this, labelling of the cell surface with 125I shows a reduction in availability of proteins for labelling between control and NH4Cl treatment. The availability of thromboplastin in monocytes for attack by exogenous trypsin or for initiation of coagulation is also reduced by cultivation with amines. We suggest that the low density peak of 5'nucleotidase and thromboplastin represents an intracellular compartment. PMID- 3000018 TI - [Small cell carcinoma of the lung. Combined therapy with cytostatics]. PMID- 3000019 TI - [Induction therapy in small cell lung carcinoma]. PMID- 3000020 TI - [Relative amounts of beta 1 and beta 2 adrenoreceptor subtypes in various parts of the myocardium]. PMID- 3000021 TI - [The occurrence of Salmonellae in organic fertilizer]. AB - Out of 24 samples of organic and organic-chemical fertilizers which were bought in retail stores ("Bio-Fertilizers") in 25% salmonellas and in 12.5% Clostridium perfringens were isolated. In each of the fertilizers, from which these pathogens were cultivated, products from animal origin were part of the ingredients. In the majority of the cases these were animal fecal residues but also blood meal and horn shavings. The epidemiological importance of these findings is discussed. It is appealed to the legislature to bring the ordinance on fertilizers to bear which demands "rendering to hygienically safe products". It is further necessary to lay down relevant executive orders for the hygienic control of the fertilizers and the supervision of the production plants. This is especially important in the context of the aim to check salmonellosis of man and animals. PMID- 3000023 TI - Immunomodulation in offspring mice after neonatal immunostimulation of mothers or newborn mice. AB - Mothers of offspring Balb/c mice were stimulated after birth by two substances, a bacterial lysate (LAB) and a chemical, diethyldithiocarbamate (DETC). Anti-sheep red blood cell (SRBC) antibodies were studied after immunization of stimulated mothers or offspring. An increase of anti-SRBC was observed in LAB-stimulated mothers, but these antibodies were decreased in their offspring before weaning. Sometimes, these antibodies were increased in LAB-stimulated newborn mice. DETC stimulation of mothers induced an elevation of antibody response in mothers and newborns. The same results were obtained in previous investigations where the pregnant mother was stimulated with the same agents. PMID- 3000024 TI - Distribution pattern of liver matrix proteins, fibronectin and type I collagen, in DAB-induced hepatoma of rat. AB - Specific antibodies to fibronectin and type I collagen in rat livers were used to demonstrate these matrix proteins with direct immunoperoxidase method in paraffin sections of normal liver, DAB-induced hepatoma and CCl4-induced fibrotic liver. In normal livers, the immunoreactive products of both matrix proteins were found in the periportal regions, while the hepatocytes and most of the interstitial matrix remained unstained. The specimens obtained from DAB-treated liver showed a more intense reaction with fibronectin antibody in the perisinusoidal space including proliferated cholangiolar cells, as compared to no reaction with type I collagen antibody. In CCl4-fibrotic liver, apparent reactions were also found for both matrix proteins in the periportal interstitium and in progressing fibrotic area with severe fatty metamorphosis. These findings suggest that these matrix proteins have an advantage in the attempt to distinguish different patterns of neoplastic alteration in experimental rat livers. PMID- 3000025 TI - A human atypical rotavirus found in an Ecuador infant with diarrhea. AB - One strain of atypical rotavirus, morphologically indistinguishable from known rotaviruses but lacking the common group antigen of typical rotavirus detectable by an enzyme-linked immunosorbent assay (ELISA), was isolated from one of 152 Ecuadorian infants with acute rotavirus gastroenteritis. The genome analysis by polyacrylamide gel electrophoresis revealed a unique RNA pattern clearly distinct from that of typical rotaviruses. PMID- 3000022 TI - [Vaccination against mouse pox]. AB - Attenuated MVA-strain of vaccinia virus has been efficient in the control of enzootic mousepox and in prophylactic vaccination. The virus has been used as a live vaccine for prophylactic and emergency vaccinations as well as for sanitation of populations. More than 100 000 vaccinations were carried out safely. Even after suspension of the obligatory vaccination of humans against smallpox the MVA-vaccine can be employed without risk and danger. PMID- 3000026 TI - Clinical significance of respiratory infection caused by Branhamella catarrhalis with special reference to beta-lactamase producing strains. AB - I found the recent increase during the past eight years of the incidence of respiratory infections caused by Branhamella catarrhalis. Namely, I experienced 74 cases (93 episodes) of the respiratory infections; 5 pneumonia, 14 acute bronchitis, 1 lung abscess, 36 chronic bronchitis, 7 chronic bronchiolitis, 21 bronchiectasis and 9 chronic pulmonary emphysema with infection. In 65 of 93 infectious episodes, Branhamella catarrhalis was isolated as a pure culture and in 28 episodes it was associated with other organisms, 13 Haemophilus influenzae etc. In all the cases, a positive correlation was found between beneficial clinical results and disappearance of the organism from the sputum. Minimum inhibitory concentrations of the representative beta-lactam and other antibiotics against 104 strains were determined. All of these strains were obtained during last four years from 1980 to 1983 from the purulent sputa as the main pathogen. Annually, this organism has significantly acquired resistance to beta-lactams. By 1983, 74% of Branhamella catarrhalis isolated from the purulent sputa became a beta-lactamase producers. And the failure cases of Branhamella catarrhalis infections treated with beta-lactams have increased during the last two years. These results have clearly showed also the importance of Branhamella catarrhalis as the common pathogen for respiratory organ. PMID- 3000027 TI - The oxidative disposition of potassium cyanide in mice. AB - The role of oxidative metabolism in the disposition of potassium cyanide (KCN), was investigated in mice administered KCN, (4.6 mg/kg, s.c.) containing 4.5 microCi [14C]KCN. The expired pulmonary metabolites, [14C]hydrocyanic acid (HCN) and 14CO2, were collected and analyzed. Approximately 1% and 2% of the KCN dose was expired as [14C]HCN and 14CO2, respectively. Expiration of the pulmonary metabolites was decreased following pretreatment with sodium nitrite, sodium thiosulfate, oxygen, or a combination of cyanide antidotes. Treatment with hydrogen peroxide lowered the amount of [14C]HCN expired and did not alter the expiration of 14CO2. Treatment with 3-amino-1,2,4-triazole (catalase inhibitor), superoxide dismutase, or diethyldithiocarbamic acid (superoxide dismutase inhibitor) did not change the amount of [14C]HCN expired. However, superoxide dismutase significantly increased the amount of 14CO2 expired, whereas diethyldithiocarbamic acid decreased 14CO2 expiration. The results from these studies suggest that in vivo cyanide can be oxidized to CO2 and treatment with agents that alter the availability of endogenous superoxide and/or hydrogen peroxide can alter the rate of cyanide oxidation. PMID- 3000028 TI - Inhibition of mitochondrial respiration by asteltoxin, a respiratory toxin from Emericella variecolor. AB - Asteltoxin, a respiratory toxin from Emericella variecolor, was examined for an inhibitory effect on mitochondrial function. Asteltoxin strongly inhibited state 3 respiration, which was released by an uncoupling agent, 2,4-dinitrophenol (DNP), indicating that the action site of asteltoxin is localized in the energy transfer system of mitochondria. Asteltoxin strongly depressed Mg2+-ATPase activity in mitochondria and only slightly affected Na+, K+-activated ATPase in microsomes at the concentration range for inhibition of mitochondrial respiration. PMID- 3000029 TI - Interaction of silica with plasma proteins: in vivo studies. AB - Silicic acid has been shown to be the main pathogenic factor in the toxicity of silicate dusts. Our earlier findings have shown the interaction of silica (silicic acid) with rat plasma and lung proteins in vitro. In the present communication, we report the binding of silica with plasma protein in vivo. The significance of in vivo silica-protein interaction is discussed in relation to its toxicity and clearance from the body. PMID- 3000030 TI - Yohimbine attenuates the delayed lethality induced in mice by amitraz, a formamidine pesticide. AB - We have found that a single dose of amitraz, a formamidine pesticide, produces death in mice 2-5 days after dosing. To further examine this phenomenon, adult albino mice of both sexes were treated with either yohimbine (10 mg/kg, i.p.) or deionized water (6 ml/kg, i.p.), immediately before an injection of amitraz (600 mg/kg, i.p.) and twice daily thereafter for 8 days. Male mice treated with water were more susceptible than water-treated females to the lethal effects of amitraz. In addition, yohimbine treatment significantly decreased the number of deaths in both sexes. These data suggest that an alpha 2-adrenergic mechanism is involved in the delayed lethality produced by amitraz. PMID- 3000031 TI - Effect of treatment with cyclosporine versus azathioprine on incidence and severity of cytomegalovirus infection posttransplantation. AB - The incidence and severity of cytomegalovirus (CMV) infection were evaluated in 24 renal transplant patients treated with steroids and cyclosporine and compared with 40 patients treated with steroids and azathioprine: 58% of patients receiving azathioprine and 33% of patients receiving cyclosporine required additional therapy with antithymocyte globulin (ATG) to treat steroid-resistant rejections. CMV antibody titers and cultures of urine and saliva were determined monthly for 4-6 months following transplant in all patients. Both the frequency of CMV infection (occurring in 58% of patients on steroids and cyclosporine and in 48% of patients on steroids and azathioprine) and its severity (21% of cyclosporine-treated patients and 22% of azathioprine-treated patients with symptoms) were similar in both groups. Use of ATG was associated with an increased incidence of CMV disease, especially for patients in the azathioprine group. Both the incidence of CMV disease, and the number of patients with symptoms in the azathioprine group were significantly lower when patients who had received ATG were excluded from analysis. When results were analyzed in just the cadaveric recipients in each group, the incidence and severity of CMV infection tended to be higher in azathioprine-treated patients compared with those maintained on cyclosporine. This could have been explained by the more frequent use of ATG in 84% of azathioprine maintained patients compared with 35% of cyclosporine-treated patients (P less than 0.002) since other factors, such as risk for CMV infection and Solumedrol dose for rejection were similar in both groups. The data demonstrate that ATG has a deleterious influence on the incidence and severity of CMV infection in renal transplant patients, even when the dosage of other immunosuppressive drugs is decreased during ATG therapy. Since patients treated with steroids and azathioprine tend to require ATG to treat steroid-resistant rejection more frequently than do patients on cyclosporine, this effect of ATG must be taken into account when evaluating CMV infection in patients on these two drug regimens. PMID- 3000032 TI - T cell lines and clones preferentially recognizing kidney-associated antigens in end-stage renal disease. AB - The specificity in the mixed lymphocyte kidney culture (MLKC) of T cell lines and clones derived from human end-stage renal disease (ESRD) kidneys was studied using collagenase dispersed kidney cells compared with lymphocytes as stimulating cells. These experiments were performed because of previous studies in which infiltrating lymphocytes freshly isolated from ESRD kidney tissue at nephrectomy (as well as autologous splenic T cells) were seen to directly generate this lymphoproliferative MLKC response when stimulated with autologous renal cortical cells. In the current studies, histopathologic staining of tissues and suspensions of infiltrating kidney lymphocytes showed predominance of OKT4 labeled phenotypes, and the stimulation indices in MLKC in general showed a direct relationship with the percentage of helper cells seen in the infiltrates. When T cell lines and clones derived from lymphocytes infiltrating the ESRD kidneys were tested in MLKC, there was evidence of kidney-associated, as opposed to lymphocyte-associated (MLC) reactivity using (3H) thymidine uptake as a reflection of a lymphoproliferative response. Several cell lines and clones derived from these T lymphocytes exhibited a dual reactivity. They served as responding cells in the MLKC reaction and completely suppressed a non-specific allogeneic MLC when added as third-party cells. Quantitatively, some clones suppressed when third-party x-irradiated cells were only 5% of the responding cell number in coculture. In addition to the dual reactivity, phenotypic analysis of these same cell lines and clones employing monoclonal antibodies revealed that individual cells expressed both OKT4 and OKT8 determinants. However, approximately 90% of the cells in the MLC enhancing line were labeled with OKT4. These results indicate that there is a complexity in the autologous MLKC response in that cells with both helper/inducer and suppressor/cytotoxic function take part in the reaction. Although delayed-type hypersensitivity to kidney-associated antigens is inferred as a result of these in vitro assays, nonspecific suppression of other Ia-dependent reactions can simultaneously occur. PMID- 3000033 TI - [Insertion of the bacterial gene for dihydrofolate reductase into colony-forming cells of mouse bone marrow]. AB - Introduction of the plasmid containing the methotrexate-resistant (Mtx-r) bacterial gene of dihydrofolate reductase (DHFR) under the control of the early promoter of SV 40 into the donor bone cells of the mouse with subsequent transplantation of the cells into lethally irradiated mice results in the increase in the life span of mice under conditions of methotrexate selection. It is due to the stable transformation of the bone marrow colony-forming cells with the plasmic DNA and the synthesis of the bacterial Mtx-r DHFR in the spleen and bone marrow of the recipient mouse. PMID- 3000034 TI - [Na,K-ATPase function and cellular proliferative activity in culture]. AB - A study was made of the dependence of ATP hydrolysis intensity upon different ratios of sodium and potassium ions in plasma membrane of L cells and of cells of clone Lebr 625, sensitive and resistant to ethidium bromide, and of the distribution of cells according to cell cycle phases in dense and sparse cultures. In dense cultures, the cell growth is arrested on G1 phase, the hydrolytic activity of (Na+ + K+)-ATPase decreases, and the Na+, K+ ratio for maximum activity of (Na+ + K+)-ATPase changes. The higher proliferative activity of Lebr 625 cells in dense culture corresponds to the higher hydrolytic activity of (Na+ + K+)-ATPase. PMID- 3000035 TI - [Kinetics of thermoinactivation of kidney Na+-,K+-ATPase]. AB - The studies of Na+,K+-ATPase (the fraction of microsomes and highly active preparation) thermoinactivation in kidneys at 50, 55 and 60 degrees C under conditions of the different ionic composition of the medium has shown that 20 mM K+ protects the enzymic complex from the thermal denaturation more effectively than 182 mM Na+ does. An increase in the Mg2+ concentration in the medium from 1 to 5 mM decreases Na+,K+-ATPase resistance to thermoinactivation. The enzyme half life becomes almost thrice as low in this case. The results obtained are discussed from the standpoint of specificity of ion-induced conformational states of Na+,K+-ATPase and regulatory role of cations in the process of its functioning. PMID- 3000036 TI - [Effect of phospholipases A2 from bee and cobra venom on the phospholipid composition and Na+-,K+-ATPase activity of synaptosomes]. AB - The bee and cobra venom phospholipases A2 as well as partially acetylated cobra venom phospholipase A2 are studied for their effect on phospholipid composition of synaptosomes and their Mg2+- and Na+,K+-ATPase activity. It is established that these phospholipases induce the splitting of phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine, inhibition of the Na+,K+-ATPase activity and activation of Mg2+-ATPase. Bee venom phospholipase A2 is more effective than cobra venom phospholipase A2, the both phospholipases splitting phosphatidylethanolamine most intensively. The ATPase activity may be partially or completely restored by exogenic phosphatidylcholine and phosphatidylserine; exogenic phosphatidylethanolamine is not efficient in this respect. PMID- 3000037 TI - [NADH-vanadate-oxidoreductase in emotional-pain stress and its possible role in the regulation of Na+-K+-ATPase activity]. AB - It is established that NADH-vanadate-oxidoreductase in brain and heart changes its activity depending on the period of the emotional pain stress (EPS) development. In early acute period of EPS (to 48 hours) NADH-vanadate oxidoreductase is activated in the brain, while in the heart its activity lowers in comparison with normal animals. In the later period after EPS the stimulation of NADH-vanadate-oxidoreductase activity in the both organs in observed. This dynamics creates possibilities for different accumulation of such Na+,K+-ATPase inhibitor as vanadate (VO3-) in the heart and brain. An increase of pO2 of the blood, probably, influences the enhancement of the VO3- concentration in the blood and organs. PMID- 3000040 TI - [Effect of amiloride on the activity of membrane-bound and soluble Na+-,K+-ATPase preparations]. AB - Amiloride (8 X 10(-4), an inhibitor of sodium channels of nonexcited membranes, inhibits the activity of Na+,K+-ATPase in the kidney cortex homogenate as well as that of the partially purified membrane-bound and lubrol-soluble Na+,K+-ATPase preparations from the cattle brain. Inhibition of Na+,K+-ATPase from different organs of various animals by amiloride, a blocker of sodium channels, indicates similarity of the molecular organization of the Na+-recognizing component both of sodium channels and sodium centres of Na+,K+-ATPase. PMID- 3000038 TI - [Effect of selenium on the system of superoxide anion radical formation and detoxication in hepatocarcinogenesis]. AB - It is established that the introduction of selenium in combination with diethylnitrosamine into rat organisms has a preventive influence on the tumour formation. The intensity of superoxide radicals formation by the liver cell microsomes in this case decreases, while the activity of superoxide dismutase, glutathione peroxidase I, glutathione reductase and concentration of selenium in microsomes increases. The anticarcinogenic action of selenium is considered as a result of an increase in the activity of superoxide dismutase, glutathione peroxidase I and glutathione reductase. This increase induces detoxication of superoxide radicals forming in considerable amounts in rat liver cells under the effect of carcinogen. PMID- 3000042 TI - [Reye's syndrome and adenovirus pneumonia with extrapulmonary manifestations. A comparison]. PMID- 3000041 TI - [Characteristics of nitrogen metabolism in chickens with gout]. AB - It is shown that the intensity of ammonium genesis, purines biosynthesis and redox processes increases in the organism of chicken with visceral gout. Sodium bicarbonate-correction of the acid-alkaline state of chickens is performed in order to normalize disturbances observed under gout. PMID- 3000039 TI - [Effect of the catalytic subunit of cAMP-dependent protein kinase on the electrical activity of the rat heart in isadrine myocarditis]. AB - The content of cAMP in the rat heart under neoepinephrine myocarditis does not differ from the control values and less increases relative to the control at the adrenalin concentrations of 5 X 10(-5) and 5 X 10(-4) M in the in vitro experiments (control: myocardium of healthy animals). Under these conditions dissociation of holoenzyme of cAMP-dependent protein kinase is disturbed in the presence of endogene-developed nucleotide and the phosphorylating activity decreases, respectively. The injection of the catalytic subunit of cAMP-dependent protein kinase encapsulated into neutral liposomes increases the duration of the myocarditis action potential for animals with the metabolic myocardial insufficiency. PMID- 3000044 TI - [Poland's syndrome]. PMID- 3000043 TI - [Malignant degeneration in mixed salivary gland tumors]. PMID- 3000045 TI - [Granular cell tumor of the breast]. PMID- 3000046 TI - Calcification and ossification in a congenital mesoblastic nephroma. AB - Calcification is uncommon in any of the intrarenal tumors of childhood. A 6-month old girl with multiple dense intrarenal calcifications was found to have congenital mesoblastic nephroma. PMID- 3000047 TI - Increasing incidence of testicular cancer in blacks. AB - Retrospective review of two consecutive five-year periods at a hospital with a large black patient population reveals an increasing incidence of testicular tumors in blacks. PMID- 3000048 TI - Ureteral obstruction in postmenopausal woman with endometriosis. AB - A case of ureteral obstruction occurring in a postmenopausal woman treated with long-standing unopposed estrogen is reported. PMID- 3000049 TI - Hematogenous metastasis of nonseminomatous germ cell testicular cancer. AB - A case is reported of a twenty-five-year-old man in whom paraplegia developed due to spinal metastasis from a nonseminomatous germ cell tumor immediately after retroperitoneal node dissection in which all nodes were found to be negative. This case emphasizes deficiencies in knowledge concerning the routes of metastasis of testicular tumor and points out that retroperitoneal lymphadenectomy is not an infallible staging procedure in patients with this disease. PMID- 3000050 TI - [Histological structure and tumor size as prognostic factors in the surgical treatment of patients with peripheral lung cancer]. AB - An analysis of long-term (5 years) results of treatment of 444 patients with peripheral lung cancer has shown that in adenocarcinoma the tumor size is of no significance for prognosis of the disease while in squamous cell carcinoma of more than 5 cm diameter the prognosis is significantly worse (almost 2 times). Since the squamous cell carcinoma is known to be the less biologically active form of lung carcinomas the authors consider that the main task is the organization of measures for early detection of this kind of tumors less than 5 cm in diameter. PMID- 3000051 TI - [Chemodectoma of the mediastinum]. PMID- 3000052 TI - Viral respiratory diseases. AB - An overview of the more commonly encountered viral diseases of the dog and cat is presented. The reader is acquainted with the principles of antiviral therapy and the drugs that have been studied for use in animal viral respiratory diseases. An update on vaccination principles and guidelines is provided. PMID- 3000053 TI - Serological survey of the incidence of infectious bursal disease serotypes 1 and 2 in chicken and turkey flocks in England. PMID- 3000054 TI - Parvovirus vaccination. PMID- 3000055 TI - Bovine virus diarrhoea vaccine. PMID- 3000056 TI - Parvovirus vaccination. PMID- 3000057 TI - Paramyxovirus infections of dogs. PMID- 3000058 TI - Disease in a dairy herd associated with the introduction and spread of bovine virus diarrhoea virus. AB - In January 1982 an outbreak of diarrhoea among adult dairy cows in a closed herd of approximately 200 milking animals was shown to be caused by the introduction of bovine virus diarrhoea virus (BVDV). Affected animals showed a significant reduction in milk yield. One animal died and four were culled. Eight cows aborted and one weak calf was born. Of 121 calves born that year, 26 died, mostly from pneumonia, but five aged from three weeks to five months had enteric lesions of mucosal disease. Subsequent investigations of the whole herd in 1983 and 1984 showed that virus spread among the adults was slow and that BVDV continued to make a major contribution to calf losses. Again the greatest cause of loss was suppurative or fibrinous pneumonia. Overall, BVDV was isolated from 36 animals. Isolation of virus from a wide range of tissues of individual animals confirmed that they were viraemic at death. Viruses from calves dying of pneumonia and from aborted fetuses were non-cytopathic in tissue culture. Isolates showing varying degrees of cytopathogenicity were obtained only from tissues of one calf with a congenital neurological defect and the seven animals with enteric lesions consistent with a diagnosis of mucosal disease. Blood from all 89 BVDV antibody free animals older than three months was tested for the presence of BVDV. Altogether, 12 calves were identified as persistently viraemic and all were apparently healthy when bled. Only two matured normally, four grew poorly and six died.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000059 TI - Excretion of louping-ill virus in ewes' milk. PMID- 3000060 TI - Chronic eosinophilic leukaemia in blast crisis in a cat negative for feline leukaemia virus. PMID- 3000062 TI - Parvovirus vaccination. PMID- 3000061 TI - Duration of the latent state in feline leukaemia virus infections. PMID- 3000063 TI - Immunoperoxidase study of adenovirus pneumonia in dogs. AB - The pathology of adenovirus pneumonia in 16 dogs is described. Clinically, these dogs had been severely ill, with severe dyspnoea and listlessness, but only faint coughing. Histopathological lesions could be associated directly with the presence of adenovirus antigens in the lungs of these dogs by using an unlabelled immunoperoxidase technique on paraffin tissue sections. The lesions were focal and located in alveoli and bronchioles. Infected cells were mostly alveolar macrophages and less frequently type 1 and 2 pneumocytes and bronchiolar epithelial cells. Infiltrating neutrophils and lymphocytes were not observed to be infected. This type of pneumonia appears to be a fairly well defined clinical and pathological entity in kennel dogs. PMID- 3000064 TI - [Therapeutic embolization of the hepatic arteries in Wilms' tumor]. PMID- 3000065 TI - [Metastatic liver disease]. PMID- 3000067 TI - Presence of HBsAg and anti-HBs in patients with primary liver cancer or hepatic metastases. AB - The presence of HBsAg was detected in the serum of 1 out of 13 patients with chronic hepatitis, in 2 out of the 7 patients with liver metastases, but in none of the 10 patients with hepatocellular carcinoma (HCC) investigated. HBsAg could be visualized by orcein staining in the liver cells of 66.6% of the HCC patients, in 50% of the patients with liver metastases and in 40% of the patients with liver cirrhosis. The prevalence of anti-HBs was low both in HCC patients and in the control groups. The possible significance of these findings is discussed. PMID- 3000066 TI - Persistence of viruses in the nasopharynx of apparently healthy children aged 0-5 years. Results of investigations performed in 1982-83. AB - Virus isolation attempts were made with nasopharyngeal secretions from 400 apparently healthy 0-5-year-old children of a semi-closed community. Repeated virus isolations were achieved in 25 cases subjected to periodic investigations in 1982-83. One-two reisolation(s), 1-5 months apart, of the initially detected viral agents could be obtained in 15 out of the 25 children. The following viruses were reisolated (in decreasing order of frequency): adenoviruses, respiratory syncytial virus, Coxsackie B1 virus, herpes virus type 1, parainfluenza virus type 3. The problems raised by the carriage of respiratory viruses in the nasopharynx of apparently healthy children are discussed. PMID- 3000068 TI - Structural particularities of parainfluenza type 1 (Sendai) virus progens obtained by cultivation in the presence of ceruloplasmin. AB - Sendai virus multiplication in the presence of ceruloplasmin resulted in the appearance of qualitatively modified progens. There was an increase in the proportion of incomplete virus particles and a decrease in hemagglutinin and neuraminidase activities, in virus infectivity and antigenicity (with the exception of subviral fractions with predominant hemagglutinating activity). Virus progens obtained in the presence of ceruloplasmin had a higher sensitivity to detergent treatment; virus ghosts formed by re-aggregation of envelope fragments could be observed in the electron microscope. PMID- 3000069 TI - The genomes of attenuated and virulent poliovirus strains differ in their in vitro translation efficiencies. AB - In mRNA-dependent extracts of Krebs-2 cells, RNAs from attenuated strains of poliovirus type 1 and type 3 exhibited diminished template activity as compared to RNAs from the respective virulent counterparts. This defect appeared to be due to the impaired initiation of viral polyprotein synthesis as evidenced by a relatively low level of accumulation of polypeptide 1a (which corresponds to an NH2-terminal region of the polyprotein) in samples programmed with RNAs from attenuated strains. In reticulocyte lysates, where poliovirus RNA is translated predominantly from abnormal (internal) sites [Dorner et al. (1984) J. Virol. 50, 507-514], this difference in the overall template activity of the attenuated and virulent poliovirus genomes was less pronounced, but the correct initiation (as judged by polypeptide 1a accumulation) was again more efficient on RNAs from virulent strains. It is suggested that template deficiency is a factor contributing to the attenuated phenotype of poliovirus strains studied. A possible involvement of nucleotide sequences located far upstream from the initiator codon in the control of translation of poliovirus genome is briefly discussed. PMID- 3000070 TI - P53-transformation-related protein: kinetics of synthesis and accumulation in SV40-infected primary mouse kidney cell cultures. AB - During abortive infection of Go/G1-arrested primary baby mouse kidney (BMK) cell cultures with simian virus 40 (SV40), expression of the viral large T antigen is followed by a mitotic host response including the stimulation of host macromolecular synthesis and induction into the cell cycle of Go/G1-arrested cells. We performed an extensive study of the sequential events taking place after SV40 infection of confluent BMK cell cultures. This study comprised a detailed kinetic analysis of transcription, synthesis, and accumulation of p53, in conjunction with the time course of large T antigen synthesis and SV40-induced cellular DNA replication. The monoclonal antibodies used for specifically recognizing mouse p53 were PAb 421, PAb 122, PAb 246, PAb 248, and RA3-2C2. Our results consistently show that under our experimental conditions, the stimulation of p53 synthesis and the accumulation of p53 occur well after the onset of T antigen-induced cellular DNA replication. This relatively late activation of p53 expression appears to be controlled at a level other than transcription. In conclusion, we suggest that, at least in certain cases, T antigen's mitogenic potential is not dependent on its interaction with p53. PMID- 3000071 TI - Nucleotide sequence of bacteriophage T4 uvsY gene. AB - We have determined the nucleotide sequence of a 1001-bp region comprising the uvsY gene of bacteriophage T4. An open reading frame of 420 base pairs was found to encode the uvsY gene product. The uvsY gene comprised a 140-amino acid protein with ATG (methionine) as the initiation codon, which is consistent with the molecular weight determined by SDS-polyacrylamide gel electrophoresis. The uvsY gene was oriented in the direction of the early genes and a sequence common to the middle promoter consensus was found in the 5'-upstream region. PMID- 3000072 TI - Physical mapping and DNA sequence analysis of the rifampicin resistance locus in vaccinia virus. AB - Rifampicin has been shown to inhibit the maturation of poxviruses at a discrete step in envelope formation (Moss et al., 1969; Pennington et al., 1970; Nagayama et al., 1970; Grimley et al., 1970). A rifampicin-resistant vaccinia virus mutant (RifR) was selected for its ability to grow in the presence of 100 micrograms/ml of rifampicin. Utilizing intact DNA or endonuclease restricted cloned DNA subfragments derived from the RifR mutant virus, the locus specifying rifampicin resistance was physically mapped by marker rescue analysis leftward of the unique XhoI site within the HindIII D fragment. DNA sequencing of a 445 bp fragment encompassing this region revealed an AT to GC transition when compared with the equivalent wild-type DNA fragment. Analysis of the six potential open reading frames within the 445-bp fragment indicated only one available open reading frame. On this basis, the rifampicin-resistant vaccinia virus mutant was shown to have a codon transition from asparagine to aspartic acid. PMID- 3000073 TI - Bacteriophage L: chromosome physical map and structural proteins. AB - Restriction endonuclease cleavage site mapping was used to locate the regions of highest sequence homology in the chromosomes of Salmonella typhimurium bacteriophages L and P22. These lie in the DNA packaging, tail, early transcription antitermination, and perhaps integration "gene modules." Other regions of the two genomes are substantially less closely related. Phage L, which has no functional immunity I region, lacks approximately 1300 bp of DNA when compared to P22 in this section of the chromosome. At least some of the virion structural proteins are interchangeable between the two phages, which suggests that the two phage structural protein genes are very closely related. In addition, the apparent molecular weights of most P22 and L phage structural proteins are very similar. However, the phage L virion contains about 140 molecules of a 15K capsid protein which apparently has no P22 analog. PMID- 3000074 TI - Association of the pX gene product of human T-cell leukemia virus type-I with nucleus. AB - Human T-cell leukemia virus type I (HTLV-I) contains a unique gene pX coding for p40 chi, and this protein was suggested to activate the transcription from the LTR of HTLV. By a similar mechanism, this viral function might be involved in immortalization of T-cells and leukemogenesis in adult T-cell leukemia induced by HTLV-I. In this communication, a part of the p40 chi was found to be tightly associated with nuclei in infected cell lines by subcellular fractionation and immunofluorescence staining. PMID- 3000075 TI - Leukemogenicity of avian oncovirus S13. AB - Avian oncovirus S13 induces erythroblastic and granulocytic leukemias in line 6 and Spafas chickens. It also causes anemia, sarcomas, and endothelial proliferation. The leukemic cells contain the transformation-specific protein of S13, gp155. PMID- 3000076 TI - The electroretinogram, standing potential, and light peak of the perfused cat eye during acid-base changes. AB - DC recordings of light-evoked responses were made in the isolated, arterially perfused cat eye during four acid-base changes designed to alter intracellular pH (pHi) without appreciably altering extracellular pH (pH0). Two acid-base changes were designed to decrease pHi: substitution of high pCO2, high [HCO3-] perfusate for control perfusate and injection of NaHCO3 solution (pH 7.4) into the control perfusate. The initial effects of these two changes were similar: standing potential decreased, the b-wave amplitude decreased, and the c-wave amplitude increased. Subsequent effects, which included rebounds, were complex. The two other acid-base changes were designed to increase pHi: substitution of low pCO2, low [HCO3-] perfusate for the control perfusate and injection of NH4Cl solution into the control perfusate. The initial effects of these two changes were similar; the effects were opposite to those described above for acid-base changes (i) and (ii). The effects of all four acid-base changes were reversible. From these and previously published findings on the effects of pH0, we conclude that during acid-base changes, the initial change in the standing potential varies directly with pHi/pH0, the initial change in b-wave amplitude varies directly with pHi, and the initial change in c-wave amplitude varies inversely with pHi. We also studied the effects of the four acid-base changes on the light peak, a slow voltage response to light generated by the retinal pigment epithelium. Under acid-base changes (i), (ii), and (iii) the light peak was severely depressed. Injection of 2 mM NH4Cl, acid-base change (iv), had little effect on the light peak; however, injection of 5-10 mM NH4Cl did depress the light peak. These results may be interpreted in several ways, for example, the light peak may be sensitive to changes in [HCO-3]0 or to pHi. In any case, we conclude that pH0 is a relatively minor factor influencing the amplitude of the light peak. PMID- 3000077 TI - [Clinico-electroneuromyographic evaluation of the efficacy of decimeter-wave therapy in patients with neurologic syndromes of lumbar osteochondrosis]. PMID- 3000078 TI - [Ultrastructure of the gastric mucosa in chronic gastritis patients and its changes as affected by combined treatment at the Morshin health resort]. PMID- 3000079 TI - [Use of the sensitized latex agglutination reaction for diagnosing viral infections]. PMID- 3000081 TI - [Discovery of the sequences of herpes simplex virus type 1 in the genomes of normal and transformed mouse and hamster cell lines]. AB - Sequences capable of hybridization with cloned fragments of herpes simplex virus type I (HSVI) DNA were found in genome DNA of Syrian hamster fibroblast lines transformed either by intact HSVI genome (cell line 14.012.81) or this virus DNA fragments (cell line EH/A44). The method of pinpoint hybridization demonstrated the presence in DNA of the cell lines under study of sequences capable of hybridization both with unique areas and with HSVI genome replicas. A correlation was established between the tumorigenic properties of the transformed lines and the number of HSVI sequences present in genomes of these lines. The genome of mouse cell line BaLB/3T3 was found to contain sequences homologous to HSVI replica areas, their number exceeding that of virus sequences present in the genome of nontransformed hamster cells (BHK/21 line). PMID- 3000080 TI - [3 cases of isolating the influenza A virus with human hemagglutinin Hsw1 in 1983 in Alma-Ata]. AB - In the interepidemic period of late 1983 in Alma-Ata, influenza viruses with hemagglutinin of the swine subtype and neuraminidases N1 and N2 were isolated from humans. One of the strains, A/Alma-Ata/1044/83 (Hsw1N1), was isolated from the lungs of a man of 65 with the diagnosis of influenza and disseminated intravascular syndrome who had had communal and occupational contacts with swine. In paired sera of a sick child, and in seroepidemiological screening for influenza antibodies to swine influenza virus in low titres were detected in 34% of cases in subjects under 40. Radioimmunoassay of blood clots from subjects of the same focus revealed in one specimen the presence of swine influenza antigen. PMID- 3000082 TI - [Monkey B-lymphocyte subpopulations transformed by baboon herpes virus in vivo and in tissue cultures]. AB - Baboon herpes virus (BHV) similarly to human Epstein-Barr virus shows tropism to B cell subpopulation. In lymphoblastoid cultures transformed in vitro by BHV no E RFC (T cells) are found but there are B cells with surface and cytoplasmic immunoglobulins. In a number of cultures IgM could be detected only in the cell cytoplasm. E-RFC are detectable in cultures in early passages but not after 17 passages which may be due to their elimination. The time course of cell-mediated immunity values was studied in Macaca arctoides monkeys inoculated with BHV. An increase in the relative and absolute number of B cells with surface IgM and IgG in the peripheral blood was noted. Two months after inoculation the values returned to the initial level. The number of E-RFC did not change. Changes in the number of B cells may be associated with the development of immune response, or BHV influence on the process of B cell circulation in the host. PMID- 3000084 TI - [Ts mutants of the Eastern equine encephalomyelitis virus. Their generation, isolation and preliminary characterization]. AB - Chick embryo fibroblast cultures of the C/O phenotype (leukemia--free) infected with eastern equine encephalomyelitis (EEE) virus were incubated in the presence of 15 micrograms/ml N-methyl-N-nitro-N-nitrosoguanidine in the culture medium. Seven (5%) temperature-sensitive mutants were isolated from cell homogenates only in those cases where cell cultures before infection had been treated with actinomycin D. The recovered ts mutants are characterized by the marked ts- phenotype and genetic stability. The method of obtaining EEE virus ts mutants under the effect of N-methyl-N-nitro-N-nitrosoguanidine in C/O phenotype (leukemia-free) chick embryo fibroblast cultures treated before virus inoculation with actinomycin D is discussed. PMID- 3000083 TI - [Persistence of the tick-borne encephalitis and Langat viruses in mouse thymocytes in experimental infection against a background of cyclophosphane induced immunodepression]. AB - The intensity of infection of immunocompetent organ cells with tick-borne encephalitis and Langat viruses in mice with temporary immunodeficiency induced by cyclophosphane is characterized. Using the method of infectious centres it was shown that at various stages of the infectious process splenocytes, bone marrow cells and thymocytes may be target cells for both viruses tested. The viruses persist for over 6 weeks in brain and thymocytes of clinically normal mice if at the time of inoculation the animals had a temporary immunodeficiency after a single administration of 200 mg/kg cyclophosphane). The asymptomatic persistence of the viruses, however, was 2-3 times more frequently detected in thymus cells than in the central nervous system. By the set of surface markers, the infected thymocytes were classified as a population of T lymphocytes. We failed to demonstrate the presence on the surface of the infected thymus cells of virus specific antigens detectable by the complement-dependent immune cytolysis test. The role of virus-infected thymocytes as a reservoir of the agent in chronic forms of flavivirus infections is discussed. PMID- 3000085 TI - [Morphological characteristics of the brain lesions in mice infected with a homogenate of L cells latently infected with the scrapie agent]. AB - Degeneration of neurons of the Ammon horn lower branch both in the early and terminal stages of the disease of mice infected with the homogenate of L cells latently infected with the scrapie agent (the L-S system) was frequently detected alongside with brain lesions typical of slow infections (vacuolation). Examinations of chromosomes in metaphase plates of L-S cells carried out by several methods including the TAC system for texture analysis of the image (Leutz, BRD) revealed three marker chromosomes new for continuous L cells, the appearance of true chromatid translocations as well as significant changes in chromosome numbers. Besides, ultrastructural features of L-S cells at later stages of cultivation were revealed. It is assumed that the active effect of the scrapie agent on L cells infected with it resulted in the emergency of a new antigen capable to induce selective affection of the neurons of the Ammon horn lower branch in susceptible mice. PMID- 3000086 TI - [Isolation of viruses from water using porous silica]. AB - The possibility of isolation of different viruses, hepatitis A virus among them, from water by means of adsorption chromatography on porous silica was demonstrated. Adsorption of viruses on the surface of silica depended on the kind of sorbent, water pH, and the rate of water flow through the column. The maximum elution of the adsorbed virus was observed using buffer solution with pH at least 11.0 and containing 1-2M NaCl. These conditions for adsorption-elution were found to be universal for different enterovirus types. In the field, the developed method was used for testing water specimens from various water bodies (sewage, river water, tap water). No viruses were found in 11 native specimens. Eluates of 4 out of 11 specimens were found to contain enteroviruses, hepatitis A virus among them. PMID- 3000087 TI - [Mathematical planning of an experiment for assessing the combined action of new interferon inducers]. PMID- 3000088 TI - [Relation between blood levels of immunoglobulins A, M and G and anti-insulin hormones in diabetics]. AB - Changes in immunoglobulins and secretion of contrainsular hormones were established in patients with diabetes mellitus according to literature data as well as on the base of the author's own material. On the other hand, the hormones are one of the factors with an influence on immunogenesis. With a view to that fact, the interrelations between them were, for the first time, studied in diabetics. A low correlation dependence was established between the indices studied. PMID- 3000089 TI - Disseminated cryptococcal infection, AIDS, Kaposi's sarcoma, chronic diarrhea, hepatitis B carrier, and cytomegalovirus. PMID- 3000090 TI - [AIDS--general characteristics and new clues in the studies of etiopathogenesis of the syndrome--the role of HTLV]. PMID- 3000091 TI - [Male breast cancer]. AB - Between 1974 and 1982 inclusive 18 male patients were treated for breast carcinoma. 12 patients had postoperative radiotherapy whereas 4 were referred for treatment of recurrent or metastatic disease. One patient showed signs of inflammatory breast cancer and was treated with chemo-radiotherapy and one was being followed up in our department after radiotherapy for prostatic cancer in 1970. Median overall survival was 52 months and the median disease-free interval was 21 months. PMID- 3000092 TI - Peroxidase-catalysed binding of [U-14C]phenol to DNA. AB - 14C-Phenol binds irreversibly to calf-thymus DNA in the presence of horseradish peroxidase and hydrogen peroxide, approximately 65% of the added phenol was bound to DNA. Binding was maximal at an equimolar concentration of hydrogen peroxide. Binding also occurred to homopolyribonucleotides polyadenylic acid, polyguanylic acid, polycytidylic acid and polyuridylic acid, and suggests that binding is relatively non-specific with respect to the nucleotide bases. p,p'-Biphenol, p,p' biphenoquinone, o,o'-biphenol and two unidentified products were formed by the oxidation of phenol, in the presence and in absence of DNA. DNA accelerated phenol oxidation four fold and prevented the polymerization of oxidized phenol products, but was found to have no effect on the range of ethyl acetate extractable products. Phenol accelerated the metabolism of o,o'-biphenol but had no effect on p,p'-biphenol metabolism. The mechanism of phenol activation is not clear, but p,p'-biphenoquinone binds to protein and not to DNA. DNA binding was prevented by glutathione, N-acetyl-cysteine and ascorbate, and the mechanism was shown to involve reduction of the activated phenol intermediates and the formation of conjugates with glutathione and N-acetyl-cysteine. DNA binding was not inhibited by lysine and proline. PMID- 3000093 TI - [AIDS--selective disease or public epidemic]. PMID- 3000094 TI - [Significance of rheobase determination in detection of threatened premature labor]. AB - In a prospective study of 176 pregnant patients it could be proved that the rheobase measurement during the whole course of pregnancy plays a significant role in recognizing the danger of premature labour. Taking a pathological limit from a number of rheobase determinations at a level of 3,4 mA, we had a sensitivity of 81% and a specificity of 50% in forecasting a premature delivery. The rheobase levels did not correlate significantly with the pelvic score (Bishop) and no correlation was found with the number of contractions measured tocometrically, nevertheless there was a correlation with the number of painful contractions between the 26th and 32nd week of gestation. Furthermore, we were able to certify the influence of psychic factors on the rheobase levels. As a result of our tests we consider the rheobase determination as being an independent, valid method for indicating premature labour. For this reason we included this procedure in our screening programme for recognition of threatened prematurity (Kubli u. Arabin, 1984). PMID- 3000095 TI - [Metastasizing soft tissue tumor of light damaged skin: atypical fibroxanthoma or malignant fibrous histiocytoma?]. AB - A 86-year-old female patient presented with a ca. walnut-sized, centrally ulcerated preauricular tumor on the right side as well as a firm submandibular lymph node. Histology disclosed a soft-tissue tumor breaking into the lymph node consisting partly of fibroblast-like and partly of histiocyte-like cells. Clinical and histological criteria speak for both an atypical fibroxanthoma (AFX) as well as a malignant fibrous histiocytoma (MFH). A differentiation into two entities does not appear appropriate in our opinion. As the term "AFX" is classified as a benign tumor, "metastasizing AFX" (mAFX) should be considered a special form of MFH or be diagnosed as such, respectively. PMID- 3000097 TI - [All blood donors must be tested for AIDS]. PMID- 3000098 TI - [Various dermatoses in workers in the chrysotile-asbestos industry]. PMID- 3000096 TI - [Current concepts concerning certain non-traditional neuroendocrine mechanisms of stress]. PMID- 3000099 TI - Infection of human epithelial cells by Epstein-Barr virus (EBV). II. Biochemical characterization of EBV-determined proteins synthesized in epithelial cells. AB - Primary cultures of epithelial cells were grown from tonsils of patients with diseases not related to EBV. The cells were implanted with EBV receptors and exposed to EBV of the transforming (B95-8, AG-876) and nontransforming (P3HR-1) strains. The EBV-infected and control cells were pulsed with [35S]methionine at 18-24 h after infection, and cell extracts were prepared for immunoprecipitation with anti-EBV sera and analysis by gel electrophoresis and autoradiography. About 20 EBV-determined proteins ranging from 22 to 185 kDa were detected in P3HR-1 virus-infected epithelial cells. Only a few polypeptides were detected in extracts of cells infected with AG-876 virus while no EBV-specific proteins were immunoprecipitated from extracts of B95-8 virus-infected cells. These results demonstrate that the system of EBV receptor-implanted normal human epithelial cells can be used for direct biochemical analysis of EBV infection in the epithelial tissue. PMID- 3000100 TI - In vivo and in vitro models of demyelinating diseases. XII. Persistence and expression of corona JHM virus functions in RN2-2 Schwannoma cells during latency. AB - The coronavirus JHMV persistently infects rat Schwannoma cells RN2-2 at 32.5 degrees C and enters a host-imposed reversible, latent state at 39.5 degrees C. JHMV can remain up to 20 days in the latent state and about 14 days before the cultures lose the capacity to resume virus production upon return to 32.5 degrees C. Although persistently and latently infected RN2-2 cells display resistance to superinfection by a heterologous agent VSV, these cells do not release detectable soluble mediators (e.g., interferon) of the antiviral state. Nevertheless, RN2-2 cells are competent to synthesize and release interferon when treated with the appropriate inducers. These observations suggest that interferon does not play any role or may not be the major factor in the control of latency in the Schwannoma cell. Hybridization with virus-specific cDNAs shows that all viral mRNAs are present during latency and that viral mRNAs are present in the polysomes of infected cells at 39.5 degrees C. Western immunoblotting with hybridoma antibodies demonstrates that viral specific proteins are produced at the restrictive temperature. These results suggest that despite the absence of production of infectious virus at 39.5 degrees C, there is active transcription and translation into virus-specified products. PMID- 3000101 TI - Coxsackievirus B3: primary structure of the 5' non-coding and capsid protein coding regions of the genome. AB - The nucleotide sequence from the 5' terminus to nucleotide 3822 has been determined for the genome of the enterovirus, coxsackievirus B3 (CB3). This region encompasses the 5'-terminal 738 nucleotide non-coding sequence and the region which codes for the four viral capsid proteins and for the initial products of the P2 region. Regions in the 5' non-translated RNA sequence which may be involved with a structural and/or regulatory role are discussed. PMID- 3000102 TI - On the predictive recognition of signal peptide sequences. AB - DNA sequence analysis of the short unique regions in the genomes of herpes simplex virus (HSV), types 1 and 2, has previously shown that within this region there are four genes, designated US2, US4, US5 and US7, whose functions are unknown but whose predicted amino sequences exhibit hydrophobic N-termini (D.J. McGeoch et al., 1985, J. Mol. Biol. 181, 1-13; D.J. McGeoch, H.W.M. Rixon and D. McNab, unpublished data). In this paper, the possibility was investigated that these hydrophobic sequences might be signal sequences associated with membrane bound translation of the proteins, and subsequent secretion or insertion into membranes. By using reference sets of protein sequences known to be translated either on membrane-bound or on free ribosomes, criteria were developed to distinguish between these two classes. These criteria comprised: length and net charge of the immediately N-terminal region which often precedes the hydrophobic stretch in membrane-translated proteins; length of the uncharged (hydrophobic) region; and degree of hydrophobicity of the 8-residue maximal hydrophobic region. The latter two parameters were found to be particularly effective when combined as a two dimensional plot, which clearly distinguished 96% of membrane-translated proteins from other classes. When the uncharacterized, predicted HSV protein sequences were judged by these tests, it was found that the products of genes US4, US5 and US7 were convincingly classified as membrane-translated, while the US2 product gave a less definitive result. In conclusion, the US4, US5 and US7 gene products were considered probably to be previously unrecognized, virion membrane-inserted glycoproteins. PMID- 3000103 TI - Expression of the herpes simplex virus type-2 major DNA-binding protein (ICP8) gene in COS-1 cells. AB - By the use of an SV40 origin of replication plasmid vector and the COS-1 cell system, expression of the gene encoding the herpes simplex virus type-2 (HSV-2) major DNA binding protein (ICP8) has been achieved. The HSV-2 4.5 kb BglII 0 DNA fragment containing the ICP8 coding region was inserted into the plasmid vector pSVOd containing the SV40 origin of replication. Transfection of COS-1 cells by the resultant recombinant plasmid (pSVOd20), and subsequent multiplication of this plasmid, led to the production of the HSV-2 major DNA binding protein in sufficient quantities to allow its detection with either monoclonal or polyclonal antibodies as well as sera taken from HSV-2 patients. PMID- 3000104 TI - Possible relationship between antigenic properties and isolation history of HSV-1 strains. AB - 29 monoclonal anti-herpes simplex virus (HSV)-1 antibodies were produced and characterized with regard to virus neutralizing activity, intracellular or cell surface location of viral antigens and, where possible, molecular weight of the viral protein recognized. 13 antibodies recognized viral antigens expressed on the surface of infected cells and 16 were directed to intracellular viral components. Only two antibodies exhibited virus neutralizing activity. Application of these antibodies to an antigenic comparison of standard laboratory HSV-1 strains F, HFEM, mP, Glasgow-17 and MAC revealed unique antigenic differences among these strains. The antibodies were further used in an antigenic comparison of 45 human HSV-1 isolates with defined isolation history. Except for two paired isolates from left and right trigeminal ganglia of two human cadavers, the antibody panel revealed antigenic differences among all isolates, including paired isolates from three additional cadavers. Overall, isolates from different human donors showed greater antigenic dissimilarity from each other in cell surface associated than in intracellular antigens. The data suggest the possibility of a correlation between antigenic and biologic properties of HSV-1. PMID- 3000105 TI - Human parainfluenza virus 3: purification and characterization of subviral components, viral proteins and viral RNA. AB - A simple method was established that allowed large quantities of human parainfluenza 3 (PF3) virions to be isolated from tissue culture cells. The purity of the virus was sufficient for biochemical analysis of virion proteins. The density of PF3 virions was 1.18-1.20. Purified virions contained seven viral proteins with estimated molecular weights of: L, 180 000; P, 83 000; HN, 69 000; NP, 66 000; F0, 60 000; F1, 51 000; and M, 38 000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. There were three phosphoproteins, P, NP and M, and two glycoproteins, HN and F (includes F0 and F1). F1.2, the activated, cleaved, fusion glycoprotein (60 000 Da), consisting of two disulfide-linked subunits, F1 and F2, was seen only under nonreducing conditions. Because of its small size (approximately 9000 Da) F2 could be seen only on gels with high acrylamide concentrations. As in other enveloped viruses, cellular actin (43 000 Da) was present in purified virions. Several minor bands migrating between NP and M represented breakdown products of NP. Solubilization of the virion membrane in low salt buffer with non-ionic detergent resulted in the loss of HN and F. In high salt buffer, the M protein was also removed. Nucleocapsids isolated by CsCl centrifugation contained L, P, NP and small amounts of M. Nucleocapsids isolated in the presence of the ionic detergent, sarcosyl, contained only the NP protein. The density of nucleocapsids was 1.29-1.30. Genomic 50S RNA isolated from nucleocapsids had an estimated molecular weight of 5 X 10(6). PMID- 3000106 TI - Nucleotide sequence of the entire protein coding region of canine distemper virus polymerase-associated (P) protein mRNA. AB - The entire coding region of the polymerase-associated (P) protein gene of canine distemper virus has been sequenced. A single cDNA clone which represents 98% of the mRNA encoding this protein was used to determine the nucleotide sequence. The sequence predicts a major protein of 507 amino acids and a molecular weight of 54 936. There is also a second, overlapping, open reading frame with a start signal 21 bases downstream of the first AUG which could code for a protein of 174 amino acids with a predicted molecular weight of 20 292. This arrangement of the genome for the P protein of canine distemper virus is exactly analogous to that published recently for the P gene of measles virus (Bellini, W.J. et al., 1985, J. Virol. 53, 908-919). When the sequences are aligned at the first AUG, considerable homology is seen at both the nucleotide and protein sequence level. PMID- 3000108 TI - [Nerve cells of Helix pomatia during hypercapnia]. AB - Hypercapnia leading initially to activation of synaptic activity and of pace maker mechanism is accompanied later by inactivation of the electro-excitable membrane and is completed by loss of chemosensitivity. Restoration of the properties of the neurone following transition to normal atmospheric air develops in reverse order. All hypercapnic changes of the properties of the electroexcitable and chemosensitive membrane take place at a constant level of the membrane potential which points to stability of K-Na-pump. PMID- 3000109 TI - [Shape of the auto- and cross-correlation histograms of the spike trains of 2 neurons with a common monosynaptic input]. AB - Form of auto- and cross-correlation histograms of impulse trains of two neurones with a common monosynaptic input from the third one were studied by methods of modelling of neuronal interaction--biomathematical (computer controlled experiment on molluscs neurones) and mathematical--in wide physiological ranges of values of parameters characterizing properties and conditions of functioning of neurones and synapses. In conditions typical of the central nervous system of mammals (but not invertebrates), when neurones are subjected to intensive random afferent synaptic bombardment and reveal no pace-maker properties, each of the possible type of common monosynaptic inputs to two neurones--excitatory, inhibitory or inhibitory-excitatory--is manifestated in cross-correlation histogram of their impulse trains in a specific way. PMID- 3000107 TI - Multiple sites of recombination within the RNA genome of foot-and-mouth disease virus. AB - Recombinant foot-and-mouth disease viruses were isolated from cells infected with a mixture of temperature-sensitive (ts) mutants belonging to different subtype strains. In order to select for recombination events in many different regions of the genome, crosses were performed between various pairs of mutants, with ts mutations in different regions of the genome. ts+ progeny were analysed by electrofocusing virus-induced proteins and RNase T1 fingerprinting of their RNA. All but 5 out of 43 independent isolates, from nine crosses, proved to have recombinant RNA genomes. Maps of these genomes, based on a knowledge of the locations of the unique oligonucleotides, were constructed. Most could be interpreted as being the products of single genetic cross-overs, although three recombinants were formed by two cross-overs each. Cross-overs in at least twelve distinct regions of the genome were identified. This evidence of a large number of recombination sites suggests that RNA recombination in picornaviruses is a general, as opposed to a site-specific, phenomenon. PMID- 3000110 TI - [Behavioral and neurochemical changes in pups prenatally treated with methamphetamine]. AB - Changes in behavior and central neurotransmitters were examined in pups prenatally treated with methamphetamine (MAP). Compared with the control pups, the following results were obtained in MAP pups : 1) No change was found in the development of the circadian rhythm of spontaneous locomotor activity; 2) Spontaneous locomotor activity was decreased, while vertical activity was increased significantly in the postnatal period from the 24th to 27th days; 3) No sensitization to the test dose of MAP was found regardless of the prenatal repeated MAP treatment; 4) Dopamine concentration and the number of specific 3H spiperone binding sites decreased in the frontal cortex. These findings indicate that prenatal treatment with MAP does not produce a lasting sensitization to MAP, whereas it does produce changes in behavior coupled with a neurochemical change in the frontal cortex. PMID- 3000111 TI - [AIDS fatalities in Hamburg (status: February 1985)--legal medicine aspects]. AB - The registration of patients with acquired immune deficiency syndrome (AIDS) and AIDS deaths is centralized for medical research. A short review of the epidemiological state of AIDS in Hamburg is given (situation as of February 1985), and the autopsy results of seven postmortem examinations are referred to. Kaposi's sarcoma was seen in four patients and opportunistic infections in all cases, especially as a result of Pneumocystis carinii and cytomegalovirus. Due to the constellation of groups with an increased risk of acquiring AIDS--homosexual males, intravenous drug abusers and prostitutes--forensic implications have to be expected. PMID- 3000113 TI - The demonstration of cell-mediated immunity in chickens vaccinated with fowlpox virus. PMID- 3000112 TI - Rapid detection of bovine leukaemia virus infection in a large cattle population with an ELISA performed on pooled sera grouped by herd. PMID- 3000114 TI - Investigations on lentivirus infections in Italian caprine population. PMID- 3000115 TI - [Features of the temporal structure of the respiratory cycle in the Tourette syndrome as a possible reflection of intensified dopaminergic processes]. AB - The effect of dopamine blockers (chlorpromazine and haloperidol) and the dopaminomimetic piribedil on the structure of the breathing cycle was studied under conditions of the clinical employment of the drugs. It was found that piribedil increased the inspiration fraction (IF) in the respiratory cycle whereas neuroleptics diminished it. Twenty-six children with Tourette's syndrome (TS) and 24 with a temporal form of epilepsy were examined. Patients with TS showed two specific features of respiration regulation: a greater value of IF and a greater variability of respiratory parameters. Taking into account the pharmacological data, an increase in IF is considered as physiological evidence of dopaminergic hyperfunction in TS whereas a greater variability of respiratory parameters may reflect the paroxysmal activity of deep cerebral structures. PMID- 3000119 TI - Cushing's syndrome presenting the coexistence of a pituitary corticotrophic cell hyperplasia and a unilateral functional adrenal adenoma. AB - A very unusual case of Cushing's syndrome is presented. Most of the preoperative biochemical and radiological examinations were compatible with Cushing's syndrome owing to a right adrenal adenoma. Exceptional findings include normal concentrations of adrenocorticotrophin (ACTH) in plasma as well as a disturbance of its circadian rhythmicity and a significant adrenocortical responsiveness to exogenous ACTH. Secretory patterns of ACTH did not change even after right adrenalectomy. Studies in vitro revealed that the adenoma itself, but not the surrounding normal adrenal, was the source of cortisol secreted in response to ACTH. Post mortem examinations disclosed unexpectedly a hormonally inactive left adrenal adenoma and a focal hyperplastic lesion of the anterior pituitary with an ACTH concentration 53 times higher than that of the remaining tissue of the gland. It is a possibility that this case may have represented a transition between pituitary-dependent adrenocortical hyperplasia and adrenal adenoma to this date reported in only one similar case. PMID- 3000116 TI - In vitro insulin action on different ATPases of erythrocyte membranes in normal and diabetic rats. AB - The in vitro effect of porcine insulin on Na+ + K+, Ca2+- and Mg2+-ATPases of the rat erythrocyte membrane of normal and alloxan-induced diabetic rats was investigated. Na+ + K+- and Ca2+-stimulated enzyme activities were significantly decreased in diabetic rats in comparison to normal animals. The specific activities of both these ATPases in the latter group were markedly reduced on pre incubating the ghosts with insulin. Similar treatment of the erythrocyte membranes of diabetic animals, however, resulted in a significant increase of these activities. These qualitatively different effects of the hormone in the two groups increased progressively with hormone concentration and duration of pre incubation. Mg2+-stimulated ATPase activity was not significantly affected in diabetes or by insulin. PMID- 3000118 TI - Cyclic Cushing's syndrome combined with cortisol suppressible, dexamethasone non suppressible ACTH secretion: a new variant of Cushing's syndrome. AB - A 55 year old woman with an unusual form of Cushing's disease was studied. During several periods (periods lasting up to 84 days) evidence of cortisol hypersecretion with cycles occurring every 6 days was found. Suppression of plasma cortisol through orally administered dexamethasone (up to 32 mg per day) could not be achieved either during periods of cyclic cortisol hypersecretion or during apparent remission with normal cortisol secretion. Marked suppression of plasma ACTH was measured in response to an iv infusion of 50 mg cortisol over a period of 55 min whereas a similar test with 2 mg dexamethasone (iv bolus) did not suppress ACTH secretion. Transsphenoidal exploration of the sella revealed a tumour surrounding the anterior pituitary. Examination of the pituitary showed a few tiny tumour structures embedded in normal tissue which could not be removed, when the tumour was resected selectively under preservation of normal appearing tissue. Post-operatively, clinical and chemical remission (normal response to 1 mg dexamethasone) was observed for about 4 months. Thereafter, cortisol hypersecretion occurred again necessitating bilateral adrenalectomy. Our results are compatible with the assumption that normal hypothalamic-pituitary-adrenal suppressibility with cortisol, but not with dexamethasone, was caused by the loss of feedback receptors for dexamethasone in the presence of cortisol receptors in the cells which secrete ACTH or CRF. The combination of cyclic hypercortisolism with dexamethasone non-suppressible Cushing's syndrome has not been reported before and thus represents a new variant of Cushing's syndrome. PMID- 3000117 TI - Effect of aging on growth hormone, ACTH and cortisol response to insulin-induced hypoglycemia in type I diabetes. AB - The influence of age on plasma growth hormone (GH) response to i.v. insulin (0.2 U/kg body weight) was evaluated in clinically stable type I (insulin-dependent) diabetics divided into four age groups (range 18.80 years). ACTH and cortisol were also assayed in two groups of diabetics under and over 50 years of age. A significant reduction with aging in GH response to insulin was observed. On the contrary, the glucose fall was similar in all the groups. ACTH and cortisol responses to insulin were slightly decreased in the older diabetics. Since insulin-induced hypoglycemia was similar in all the age groups, the progressive decline with aging in the GH response to insulin may be attributed to age-related changes of the pituitary gland. The data on ACTH and cortisol are less striking. Our data, as a whole, confirm that growth hormone response to insulin-induced glucose fall is not critical in acute glucose counterregulation in insulin dependent diabetics. In fact, in spite of a 4-fold difference in GH levels, there was no difference in the 2-h glycemic course after 0.2 U/kg of i.v. insulin between young and aged patients. When a group of 26 type I diabetics with proliferative retinopathy was compared with a group of age-matched type I diabetics without retinopathy and with 30 age-matched normal subjects (injected i.v. with 0.1 U/kg body weight of insulin), no difference was found in GH response to insulin, indicating that GH hypersecretion is not a characteristic finding of diabetic retinopathy. PMID- 3000120 TI - The effects of forskolin and LH on cAMP changes and maturation in the follicle enclosed oocytes of hamsters. AB - Graafian follicles from mature pro-oestrous hamsters were incubated with LH, various concentrations of forskolin, or forskolin plus LH. The incubations were either terminated at different time periods for analysis of follicular or oocyte cAMP levels or incubated for the entire 6 h and the oocytes examined to determine maturational status. Incubations with LH (1 microgram/ml) produced a short transient rise in follicular and oocyte cAMP concentrations, while forskolin (60 microM, 20 microM and 10 microM) produced cAMP values which remained elevated for longer periods of time. The 1 microM concentration of forskolin initiated oocyte maturation (28%) but at a level which was significantly below that stimulated by LH (74%). When LH was included with forskolin, a dramatic rise in follicular cAMP occurred which was approximately 2 times greater than levels seen with LH alone. A significant percentage of oocytes matured when 100 nM forskolin (45%) was included with LH (1 microgram/ml) but not with any other concentration of forskolin tested. Maturation percentages for follicle-enclosed oocytes exposed to 1 microM forskolin plus 1 microgram/ml of LH (3.8%) were not different from the controls (7%). However, when 1 microM forskolin was combined with 100 ng/ml of LH a significant percentage of oocytes matured (47%). While continuous incubations with forskolin did not stimulate a high percentage of oocytes to mature, oocytes from follicles exposed to forskolin (60 microM and 20 microM) for short periods (5 min-30 min) with a change to plain medium did mature. The results of these studies indicate that, in the hamster, long term exposure to forskolin inhibits maturation in follicle-enclosed oocytes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000121 TI - [Modulation of Tac antigen on non-T cell line cell membrane by anti-Tac antibody]. PMID- 3000122 TI - Poly(A)-polymerase activity in chronic lymphocytic leukemia of the B cell type. AB - Poly(A)-polymerase enzymic activity was biochemically determined in lymphocytic extracts from 40 patients with chronic lymphocytic leukemia of the B cell type. The enzymic activities of patients with stage A, B and C disease were (U/mg of protein): 4.9 +/- 5.5, 12.5 +/- 7.5 and 20.9 +/- 18.9, respectively. The difference in the enzyme level between stage A and C patients was statistically significant (p less than 0.05). Comparison of the enzyme activity level in relation to the pattern of bone marrow involvement revealed that patients with a diffuse pattern of infiltration had a significantly higher enzyme level (17.9 +/- 15.5 U/mg of protein) than patients with interstitial or mixed infiltration patterns (5.9 +/- 6.6 and 7.9 +/- 7.0 U/mg of protein; p less than 0.025). Finally, patients who required treatment for their disease also had a significantly higher poly(A)-polymerase activity level (14.5 +/- 13.9 U/mg of protein) than patients with stable disease (4.9 +/- 5.5 U/mg of protein; p less than 0.05). Our results indicate that the enzyme poly(A)-polymerase may be used as a biological marker in patients with chronic lymphocytic leukemia. PMID- 3000123 TI - A possible association between HTLV-I and B-cell chronic lymphocytic leukemia in Jamaica. AB - Cell surface markers were determined in 10 patients with chronic lymphocytic leukemia (CLL) in Jamaica, an area endemic for the human T-cell leukemia/lymphoma virus (HTLV-I) and with a high positivity for HTLV-I antibody titers in CLL patients. The results demonstrated that the predominant cell phenotype in all patients was of B-cell origin. When surveyed for HTLV-I antibody, 3 out of 5 patients with B-CLL from this series were found positive. A possible association between HTLV-I and B-CLL is discussed. PMID- 3000124 TI - [Two cases of Krukenberg tumors associated with ovarian dermoid cyst]. PMID- 3000125 TI - The effects of naloxone on central hemodynamics and myocardial metabolism in experimental propoxyphene-induced circulatory shock. AB - The courses of the hemodynamic and cardiometabolic effects of naloxone were evaluated in propoxyphene-induced shock in eight pentobarbital-anesthetized pigs. Circulatory shock was induced by an infusion of propoxyphene chloride 15 mg . min 1 i.v. At shock, i.e. MAP less than 60 mmHg and/or CI less than 2.0 l . min-1 . m 2, naloxone was administered at 0.75, 1.5 and 3.0 mg . kg-1 with an interval between increments of 8 min. The propoxyphene infusion of 15 mg . min-1 was continued throughout the study. Following the injection of naloxone 0.75 mg . kg 1, increases were observed (% of baseline value) in MAP (41%), i.e. deficit to baseline 59%, HR (66%), CI (67%) and SVI (108%), whereas MPAP and MPAOP were unchanged. dP/dt increased (34%). In the coronary circulation naloxone initiated the following changes: CSF increased (69%) as did MVO2 (48%) with unchanged MO2 extraction, but CVR decreased further (36%). The maximum effects of naloxone were registered 2-3 min after 0.75 mg . kg-1. Following 1.5 and 3.0 mg . kg-1, no changes in hemodynamics were observed other than those caused by progressing propoxyphene intoxication. PMID- 3000126 TI - Electron-microscopic observation of the nucleus basilis of Meynert in human autopsy cases. AB - The large neurons of the nucleus basalis of Meynert (nbM) were examined with the electron microscope in 13 autopsied human adults. The neurons were characterized by a prominent Nissl substance and accumulation of lipofuscin granules. Lamellar bodies were often observed among the Nissl substance. Many of the lipofuscin granules were large and had a characteristic pronounced mosaic pattern of pale areas within gray zones. Menbranous structures within the nucleus and periodic transverse processes in the cristae of the mitochondria were regarded as postmortem alterations. Alzheimer's neurofibrillary tangles (NFT) were observed in two cases. Intranuclear fibrillary bundles were identified in four cases. Crystalloid formation in rough endoplasmic reticulum was identified in two cases. Hirano body was observed in a case of parkinsonism with dementia. Axonal swelling was seen in three cases and interpreted as axonal dystrophy, an age-related phenomenon. A basal body, which is unusual in neurons of the central nervous system (CNS), was observed in one case. Lewy bodies were observed in a case of parkinsonism. PMID- 3000129 TI - Pressures recorded in ulnar neuropathy. AB - The pressure between the ulnar nerve and the arcade bridging the two heads of the flexor carpi ulnaris muscle was recorded peroperatively in ten patients with electrophysiologically confirmed ulnar neuropathy at the elbow. At rest, with the elbow extended, pressures ranged from 0 to 19 mm Hg but increased in flexion and during isometric contraction of the flexor carpi ulnaris muscle to maximal values above 200 mm Hg. PMID- 3000128 TI - Effect of oxygen free radical products on rabbit iris vascular permeability. AB - Oxygen free radicals and their products are known to be elaborated by inflammatory cells during the 'respiratory burst'. Xanthine oxidase combined with hypoxanthine was injected into the anterior chamber of rabbit eyes. This combination is known to result in the production of oxygen free radicals. Iris fluorescein angiography performed 2 h and 24 h following injection of xanthine oxidase and hypoxanthine into the anterior chamber resulted in increased iris vascular permeability. The increased permeability was not modified by either of the prostaglandin inhibitors naproxen or aspirin nor by the free radical scavenger D-penicillamine. This study demonstrates that iris vascular permeability, and possibly blood-aqueous barrier permeability is increased following exposure to chemically generated oxygen free radicals. It is possible that the increased iris vascular permeability that occurs during ocular inflammatory processes may in part be mediated by oxygen free radical products. This model may be useful in developing therapeutic modalities directed at preventing the damaging effect of oxygen free radical products, and this may be of benefit in reducing the untoward effects of ocular inflammatory disorders. PMID- 3000127 TI - Accumulation of amyloid in pituitary adenomas. AB - The accumulation of amyloid in pituitary adenomas was examined in relation to the types of adenoma and the effect of bromocriptine treatment. Amyloid had accumulated in 34 of 48 adenomas (71%). The occurrence in prolactin-secreting adenomas and growth hormone-secreting adenomas was 79%, respectively, while that in non-functioning adenomas was 50%. Treatment with bromocriptine enhanced the occurrence and extent of the amyloid accumulation in prolactin- or growth hormone secreting adenomas. Electron microscopy revealed the initial appearance of the amyloid fibrils in the smooth endoplasmic reticulum and a possible sequential process of their release from the cells. The presence of secretory granules in vesicles containing amyloid fibrils and their simultaneous release with amyloid fibrils suggested that degradation of secretory granules was involved in the formation of amyloid. PMID- 3000130 TI - Small cell carcinoma of the skin "non-Merkel cell type". AB - An 81-year-old Japanese woman developed small cell carcinoma of the skin, which was different from trabecular carcinoma or neuroendocrine carcinoma of the skin. The tumor was composed of spindle-shaped or fusiform cells with scanty cytoplasm and numerous mitoses. The tumor cells were arranged in a streaming pattern and not in anastomosing trabecular fashion at all. No granules were detected by Grimelius' stain either. Immunoperoxidase staining for neuron specific enolase (NSE) did not reveal any activity. Ultrastructural study showed scanty organelles in the cytoplasm which contained a few round mitochondria, rough endoplasmic reticulum, and free polysomes. Occasionally, filamentous bundles, desmosomes, and intracytoplasmic canaliculi were recognized in the cytoplasm, but neurosecretory granules were not found throughout the cytoplasm. Electron microscopic features suggest that this tumor originated from the embryonal stratum germinativum. The present tumor can be distinguished from trabecular carcinoma or neuroendocrine carcinoma of the skin, and may be regarded as "small cell carcinoma" of the skin. PMID- 3000131 TI - Immunoelectron microscopic study on the cross-reactivity of antibodies to adult T cell leukemia-associated antigens in human and monkey sera. AB - The cross-reactivity of antibodies to adult T-cell leukemia (ATL)-associated antigens (ATLA) in human and monkey sera was investigated by indirect immunoperoxidase and immunoferritin methods using a human cell line (MT-2) carrying a type C virus (HTLV), two monkey cell lines (Si-1 and Si-3) carrying HTLV, and a monkey cell line (Si-2) carrying a type C virus isolated from an anti ATLA-positive monkey. Anti-ATLA-positive but not-negative human and monkey sera gave positive immunoperoxidase reaction with all four virus-positive cell lines when studied by light microscopy. Electron microscopic findings revealed ferritin or peroxidase labeling of virus particles and plasma membranes of these four cell lines with antibody-positive but not-negative human and monkey sera. These results clearly indicate the cross-reactivity of anti-ATLA antibodies in human and monkey sera at light and electron microscopic levels, and the presence of antigenic determinants common to the surface of type C virus particles of human and monkey origin. PMID- 3000133 TI - Malignant fibrous histiocytoma of the lung. AB - A 75-year-old woman was admitted to a hospital for diagnosis of pulmonary infarction and died during treatment. An autopsy revealed a tumor 5 cm in diameter in the hilus of the left lung, spreading into the posterior mediastinum, and a metastasis was also found in the right lung. Histologically, this tumor consisted of two kinds of cells; one of fibroblast-like cells and the other of histiocyte-like cells, showing a storiform pattern. Furthermore, a positive staining for alpha 1-antitrypsin, but negative for CEA, keratin or S-100 protein was seen. Therefore, it was diagnosed as malignant fibrous histiocytoma (MFH) originating in the hilus of the left lung. In addition, many foci of hemorrhagic infarction due to metastasis and infiltration of the tumor into the pulmonary arteries were observed in the right lung. MFH is one of the rarest primary tumors of the lung. PMID- 3000132 TI - Non-functioning adrenocortical adenoma in culture. Quantitative and morphological observations. AB - This report describes the morphological responses of unstimulated and stimulated non-functioning adrenocortical adenoma in culture. The removed adrenocortical adenoma was composed mainly of clear-type cells and partially had a small area of cholesterol granuloma. These adenoma cells had many lipid droplets and round to long rod-shaped mitochondria with tubular or tubulo-lamellar cristae which were similar to those in Cushing's adenoma. The non-functioning adrenocortical adenoma cells which were incubated in vitro under ACTH (10 mIU/ml) and angiotensin II (10(-6) M/ml) stimulation, were examined by phase contrast microscopy, transmission and scanning electron microscopy, and the content of cortisol and aldosterone in the culture medium was measured by radioimmunoassay. As a result of exposure of ACTH, the cultured cells revealed the retraction response and production of cortisol and aldosterone. After administration of ACTH for many days, the cultured cells showed characteristic changes in sER and mitochondria. The sER were markedly developed and packed tightly into a network of dilated tubules. Mitochondria were larger and more numerous than in the unstimulated cells. The mitochondria appeared to be entwined by the tubules of the sER. Lipid droplets decreased in number. PMID- 3000134 TI - Aleutian disease of mink. Virology and immunology. PMID- 3000135 TI - [Effect on the beta adrenoceptor agonist, isoprenaline, on cAMP levels in the mouse uterus]. PMID- 3000136 TI - A novel 8-phenyl-substituted xanthine derivative is a selective antagonist at adenosine A1-receptors in vivo. PMID- 3000137 TI - Psychiatric effects of cannabis use. AB - That cannabis use may provoke mental disturbances is well known to Scandinavian psychiatrists today. A review of the psychiatric aspects of cannabis use is given, and the clinical signs of 70 cases of cannabis psychoses collected in Sweden are described. The bluntness and "amotivation" following chronic cannabis use are discussed. Anxiety reactions, flashbacks, dysphoric reactions and an abstinence syndrome are all sequels of cannabis use. Three risk groups begin to emerge: a) Young teenage cannabis users who lose some of their capacity to learn complex functions and who flee from reality to a world of dreams. With its sedative effect, cannabis could modify such emotions as anger and anxiety and slow down the liberation process of adolescence. b) Heavy daily users, often persons who cannot cope with depression or their life circumstances. c) Psychiatric patients whose resistance to relapses into psychotic reactions might be diminished according to the psychotropic effects of cannabis. PMID- 3000138 TI - ACTH 4-9 analogue (Org 2766) in depressed elderly patients. I. Effect on depressed mood. AB - Sixty-two moderately depressed elderly in- and out-patients were administered 80 mg ACTH 4-9 analogue (Org 2766) or placebo daily for 4 weeks in a double-blind interindividual comparison. Patients were rated on days 0, 15 and 29 with the Hamilton Psychiatric Depression Rating Scale (HPDRS), The Hamilton Anxiety Scale (HAS) and the Sandoz Clinical Assessment Geriatric Scale (SCAG). After 4 weeks 23 patients crossed over to the opposite treatment for a further 2 weeks. A statistically significant reduction in the severity of depression and anxiety was found in both treatment groups. No statistically significant differences were found between the treatment groups, when comparing scores for each item and total scores of HPDRS, HAS and SCAG on day 0, 15 or 29. Nor were there any statistically significant differences after the cross-over. Somatic examination and laboratory screenings before and at the end of the study did not reveal any pathological changes. No side effects were recorded. PMID- 3000139 TI - ACTH 4-9 analogue (Org 2766) in depressed elderly patients. II. Effect on memory and vigilance. AB - In a double-blind interindividual comparison 80 mg of an ACTH 4-9 analogue (Org 2766) or placebo was administered daily to 49 elderly depressed in- and out patients for 4 weeks. 20 patients then changed to the opposite treatment in a cross-over study for a further 2 weeks. All patients were tested for memory on days 0 and 29 with a battery consisting of the 30 World-Pair Test, the 30 Figure Test and the 30 Personal-Facta Test. Three scores were obtained from each test, immediate memory score (IMS), delayed memory score (DMS) and their difference, forgetting score (FS). Cross-over patients were tested for vigilance on days 0, 29 and 43 in an apparatus testing ability to rapidly detect and react to specific minor changes at random intervals. Org 2766 had no better effect than placebo on learning, consolidation, or retrieval of memorized material in elderly depressed patients. Statistically significant fewer target misses in the test of vigilance suggest a higher degree of sustained attention after treatment with Org 2766. PMID- 3000140 TI - Radionuclide angiography and scintigraphy in hepatocellular carcinoma. AB - Among 1 257 patients subjected to liver-spleen (RES) scintigraphy and radionuclide angiography (RNA), there were 13 cases of histologically confirmed hepatocellular carcinoma (1%). All 13 patients had scintigraphic findings indicating cirrhosis. Histologically, cirrhosis was present in only 9 out of 11 cases in which liver parenchyma was available for examination. One patient had hemochromatosis without evidence of cirrhosis. In 11 cases, the tumor was clearly demonstrated as a defect in the static scintigram. However, in 2 cases with cirrhosis and poorly differentiated hepatocellular carcinoma, the tumor nodules were hardly discernible. In RNA, the tumor displayed high activity in the arterial phase and decreasing activity during the portal phase, ending up as a defect. At RNA, the lesion was clearly outlined in 12 cases, including the 2 patients in whom the scintigraphic findings were inconclusive; in one case with severe cirrhosis and a well differentiated hepatocellular carcinoma, the tumor was barely detectable. We conclude that a combination of RNA and scintigraphy is a valuable screening which usually provides a correct diagnosis in hepatocellular carcinoma. PMID- 3000141 TI - Veno-occlusive disease and peliosis of the liver complicating the course of Wilms' tumour. AB - Veno-occlusive disease (VOD) of the liver was diagnosed in 8 patients with Wilms' tumour and peliosis hepatis (PH) in one. Fever of obscure origin, vague abdominal pain, hepatomegaly or hepatosplenomegaly, severe anaemia or sudden, unexplained drop in haemoglobin, thrombocytopenia, increasing serum transaminase levels, jaundice and ascites recorded within the first weeks or months of tumour diagnosis should arise suspicion of non-metastatic vascular hepatopathy. General or focal decreased accumulation of isotope at liver scintigraphy belong to the early radiologic findings. Sonography and CT may show a generalized irregular echogenicity or attenuation but no unequivocal metastases. One patient with PH had multiple low attenuating foci in both liver lobes and angiographically abnormal pooling of contrast medium in the liver. It is important to recognize these conditions as alternatives to suspected liver metastases, which as a rule develop much later yet on occasions may have very similar radiologic appearances. Therefore the relation in time between tumour diagnosis, initial operation and development of obscure hepatic manifestations is of critical significance for the recognition of VOD or PH. In these patients chemotherapy and irradiation must be discontinued without delay. If the disorders are adequately treated the prognosis may be considered fair. PMID- 3000142 TI - Responses of plasma cyclic AMP, serum immunoreactive insulin, C-peptide immunoreactivity and blood sugar levels to glucagon in patients with liver diseases. AB - Levels of plasma cyclic AMP, serum immunoreactive insulin (IRI), serum c-peptide immunoreactivity (CPR) and blood sugar (BS) were determined 0, 15, 30, 45 and 60 min after a glucagon injection (0.01 mg per kg body weight) in normal controls, patients with acute hepatitis and liver cirrhosis. Plasma cyclic AMP responses to glucagon in liver disease patients varied widely in peak value, and only in patients with fulminant hepatitis and decompensated liver cirrhosis with poor prognosis was the response suppressed. The peak response of BS was found significantly later in liver cirrhosis patients than in normal controls. IRI and CPR responses to glucagon were lower in acute hepatitis patients than in normal controls and liver cirrhosis patients. IRI levels and their sum were also lower in acute hepatitis patients, although CPR levels were not significantly different. Thus, the ratio of the sum of CPR from 0 to 60 min to that of IRI was significantly higher in acute hepatitis, indicating impaired pancreatic secretion of insulin to glucagon stimulation as well as increased uptake of insulin by the liver in acute hepatitis. PMID- 3000143 TI - Heat-stable regulatory factors are associated with polycation-modulable phosphatases. AB - Several protein phosphatases which we designated PCM-I, PCM-II and PCM-IId were identified in preparations from aortic smooth muscle. A unique feature of these enzymes is that phosphatase activities expressed against different substrates are subject to modulation by polycationic effectors such as polylysine and lysine rich histone-H1. They can be distinguished from each other by virtue of significant quantitative differences regarding (a) relative substrate specificities exhibited against phosphorylated myosin light chains, phosphorylase, and inhibitor-1, (b) apparent molecular weights as determined by sucrose density centrifugation, and (c) differential susceptibility to polylysine mediated modulation of phosphatase activities. Surprisingly, gel filtration of the very same PCM-phosphatase preparation yields either of two apparently different enzymes: namely PCM-II, or PCM-IId. The enzymes appear similar in that low concentrations of polylysine (0.03-0.13 microM) inhibit dephosphorylation of light chains by either enzyme and high concentrations inhibit dephosphorylation of phosphorylase a. However, PCM-II exhibits higher basal phosphatase activity against myosin light chains (480 U/mg) than against phosphorylase a (175 U/mg). In contrast, PCM-IId is more effective in dephosphorylating phosphorylase a (180 U/mg) than in dephosphorylating the light chains (88 U/mg). Moreover, phosphorylase phosphatase activity of PCM-II is biphasically stimulable by low concentrations of polylysine (0.01-0.5 microM), but no stimulation is seen with PCM-IId. In addition, earlier studies with PCM-I showed that, unlike either PCM II or PCM-IId polylysine biphasically stimulated the dephosphorylation of both phosphorylase a and the myosin light chains. Nevertheless, in spite of these obvious differences between the enzymes other data suggests that the PCM phosphatases may be related to each other. Incubation of PCM-II at 90 degrees C for 10 min apparently releases heat-stable regulatory proteins which reverse the modulatory effects of polylysine on light chain and phosphorylase phosphatase activities expressed by either PCM-II or PCM-I. Similarly, heat-stable proteins released from PCM-I also reverse polylysine-mediated modulation of both PCM-I and PCM-II. These findings are consistent with a working hypothesis suggesting that PCM-phosphatase may consist of a modulatory domain containing several regulatory proteins and a catalytic domain.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3000144 TI - Cytomegalovirus DNA polymerase inhibition and kinetics. AB - Cytomegalovirus (CMV) DNA polymerase has immunologic specificity in relation to the virus specific polymerases of other herpesviruses. During the early phase of CMV infection in vitro, the virus DNA polymerase is rapidly induced. Due to the lack of virus specific thymidine kinase, cytomegalovirus is resistant to nucleosides which require herpesvirus thymidine kinases for activation. Cytomegalovirus DNA polymerase is therefore the known possible target for antiviral drugs. Several pyrophosphate analogs have been assayed for their inhibitory effects on this enzyme. The most active compound is phosphonoformate (PFA). PFA also effectively inhibits other partially purified herpesvirus DNA polymerases as well as the multiplication of all human herpesviruses, at concentrations which do not affect cellular DNA polymerases or normal cell growth. The mechanism of inhibition is a noncompetitive inhibition of CMV DNA polymerase activity with respect to the four deoxyribonucleoside triphosphates, and an uncompetitive inhibition with respect to the template used. In cell culture, PFA inhibits the formation of late CMV polypeptides, but not the synthesis of early CMV polypeptides. The CMV specific polymerase persists in the presence of PFA, as measured by immunological methods, and enzyme activity can be demonstrated after removal of PFA. The inhibition of CMV replication is reversible even after long exposure to PFA. Our interpretation is that the CMV genome is highly resistant to the cellular metabolism also in non-producing cells. A new rapid CMV neutralization test was established, based on the appearance of early CMV-induced antigens. PMID- 3000145 TI - Temporal organization of the phosphofructokinase/fructose-1,6-biphosphatase cycle. AB - The dynamic and functional organization of the fructose-6-phosphate/fructose-1,6 bisphosphate cycle has been investigated in an open and homogeneous reconstituted enzyme system containing phosphofructokinase, fructose-1,6-biphosphatase, pyruvate kinase, adenylate kinase and glucose 6-phosphate isomerase. The properties of this system were analyzed by a model based on the kinetic properties of the individual enzymes. It could be shown that in a broad parameter region sustained oscillations arise. At low maximum activities of phosphofructokinase a domain of multiple stationary states occurs, in which stable stationary states can coexist with a stable oscillatory or with an alternate stable stationary state. The occurrence of oscillations and the emergence of alternate stationary motions are caused mainly by the reciprocal effect of the allosteric effectors AMP and fructose-2,6-bisphosphatase must be involved in the reaction network. The study of bisphosphatase. The attained states can either be glycolytic or gluconeogenic, their metabolic efficiencies depend mainly on the maximum activities of phosphofructokinase and fructose-1,6 bisphosphatase as well as on the supply of fructose-6-phosphate and fructose-1,6 bisphosphate. Efficient metabolic states arise only when both the enzyme concentrations and the rates of substrate supply favor either the glycolytic or the gluconeogenic mode of action. At medium maximum concentrations of the enzymes oscillations occur, in which glycolytic and gluconeogenic states are consecutively passed. A high rate of substrate cycling is observed only at the transitions between the functionally antagonistic phases of the periodicities. By this temporal organization the mean efficiency of the states is increased. The integration of fructose-2,6-bisphosphate as very sensitively acting activator of phosphofructokinase and inhibitor of fructose-1,6-bisphosphatase gives rise either to emergence of oscillations or of their extinction. Generally, the glycolytic mode is favored by this effector because of its stimulatory action on the phosphofructokinase activity. PMID- 3000146 TI - Regulation of 2-5 A phosphodiesterase activity by cAMP-dependent phosphorylation: mechanism and biological role. AB - The results of the present study permit the explanation of one of the mechanisms of the interconnection between the regulatory systems of cAMP and 2-5A. cAMP dependent regulation of 2'-PDE was found to involve phosphorylation of the specific protein inhibitor. Originally, a similar way of regulation of the enzyme activity was discovered for protein phosphatase I. This enzyme has a specific protein inhibitor type 1, which is phosphorylated by cAMP-dependent protein kinase and is activated by phosphorylation (18). It is interesting that the molecular weights of 2'-PDE protein inhibitor and of the inhibitor type 1 of protein phosphatase I are essentially the same. There is also a certain similarity between the above described mechanism and phosphorylation of the regulatory subunit of cAMP-dependent protein kinase type 2. The regulatory subunit can also act as a protein inhibitor of the enzyme and change its properties as a result of phosphorylation (19). The results obtained permit as well a more detailed explanation for cAMP-dependent inhibition of cell proliferation. Evidently, cAMP elevation causes activation of cAMP-dependent phosphorylation which, in turn, leads to the induction of 2-5A synthetase and inhibition of 2'-PDE. As a result of variations in the activities of these enzymes, the level of 2-5A rises. The latter brings about the changes characteristic of the resting state. They involve activation of RNase L and the succeeding acceleration of RNA hydrolysis, inhibition of protein synthesis and cell proliferation. The resting state is characterized by a rapid turnover of macromolecules due to their intensive degradation (20). The above described scheme suggested that the rapid turnover of RNA during inhibition of cell proliferation can be partially accounted for by activation of 2-5A-dependent RNase L. Thus, it can be thought that at least one of the mechanisms of the antiproliferative effect of cAMP-dependent phosphorylation of proteins involves cAMP-dependent elevation of intracellular 2-5A. Evidently, a number of properties of the resting cells are determined by the elevated content of 2-5A. Finally, it should be noted that the interconnection between the systems of cAMP and 2-5A is a multiple process. We have earlier demonstrated (12) that 2-5A activates cAMP phosphodiesterase in NIH 3T3 cell homogenates. These data suggest that the mutual regulation of cAMP and 2-5A levels involves the negative feedback mechanism (Fig. 8). PMID- 3000147 TI - Immune responses of cattle and mice to the G glycoprotein of vesicular stomatitis virus. AB - A subunit vaccine for vesicular stomatitis was developed from a purified vesicular stomatitis virus preparation by selectively removing the immunogenic G glycoprotein of the virus with the dialyzable, nonionic detergent, beta-D octylglucoside. Cattle immunized intramuscularly with a single dose of 112 micrograms of G glycoprotein preparation in complete Freund's adjuvant did not develop vesicular disease following challenge by intralingual inoculation of 400 times the infectious dose of the virus. Similarly, mice vaccinated subcutaneously with a single dose of 10 micrograms of G glycoprotein preparation, with or without complete Freund's adjuvant, were protected from lethal encephalitis caused by vesicular stomatitis virus. A subunit vaccine for vesicular stomatitis of cattle, horses, and swine avoids the hazards associated with attenuated and inactivated vaccines, such as vaccine breaks, reversion to virulence, or introduction of virus into potential wild reservoirs or arthropod hosts. Further, it is possible to distinguish serologically animals vaccinated with the subunit preparation from those that have had the clinical disease or that have been vaccinated with whole virus. This is an essential consideration both for epidemiological studies and for disease control or establishment of quarantine programs. PMID- 3000148 TI - Genetic approaches to study Pseudomonas aeruginosa protein antigens. AB - Pseudomonas aeruginosa produces a large number of extracellular products which may play a role in pathogenesis. We have used genetic techniques to elucidate the relative contribution of these proteins to virulence, and as a method of producing safe toxoids. A mutant has been isolated which produces an immunologically reactive nontoxic form of toxin A, the most toxic extracellular protein produced by P. aeruginosa. Although there are difficulties in production of sufficient quantities of this CRM toxoid, these are likely to be solved by further genetic manipulation. Protection studies with toxin A antibody and studies of mutants deficient in toxin A have confirmed that toxin A plays a role in pathogenesis while clearly showing that toxin A alone cannot totally account for the virulence of P. aeruginosa. Studies of mutants specifically altered in three other products, exoenzyme S, and the two major proteases of P. aeruginosa, elastase and alkaline protease, have clarified the contribution of these products to virulence. Demonstration by genetic studies that exoenzyme S was a major factor in the virulence for one P. aeruginosa strain allowed us to correctly predict that antibody to this product would be protective against infection with that strain. PMID- 3000149 TI - The structural proteins of the autonomous parvovirus feline panleukopenia virus. AB - Approximately 80% of the genome of feline panleukopenia virus was cloned into the plasmid pBR322. The entire 3943 nucleotide sequence of the cloned portion of FPV was determined. This DNA includes the gene which codes for the structural proteins of the virus. Portions of this gene were expressed in E. coli as fusion proteins with bacterial proteins. Some of the fusion proteins were capable of raising neutralizing antibodies in guinea pigs. Through the use of deletion mapping, monoclonal antibodies, and synthetic peptides, attempts were made to localize the portion of the protein responsible for raising these antibodies. PMID- 3000150 TI - Studies of TGEV spike protein gp195 expressed in E. coli and by a TGE-vaccinia virus recombinant. AB - The gene coding for the surface spike protein gp195 of TGEV has been cloned and expressed in E. coli in the form of fusion proteins. These proteins were isolated and used to immunize laboratory animals. All animals developed antibodies cross reacting with the TGEV virion but failed to neutralize the virus. The entire gp195 gene was also inserted into vaccinia to generate a TGEV-vaccinia recombinant virus (vTGE) that expressed TGEV gp195. Animals vaccinated with vTGE produced neutralizing antibodies against both TGEV and vaccinia. These results suggest the potential use of the recombinant vTGE as a vaccine against TGEV infection. PMID- 3000152 TI - [Analysis of human retinoblastoma for human adenovirus and JC virus genomes]. PMID- 3000151 TI - [3H]Adenosine binding to rat mast cells--pharmacologic and functional characterization. AB - Rat serosal mast cell adenosine receptors were characterized by [3H]adenosine binding to cell membrane particulates, and functional changes in mast cell mediator release and cyclic AMP levels were assessed, utilizing various adenosine analogs. [3H]adenosine binding to sonicated mast cell membrane preparations at 0 degrees C in the presence of deoxycoformycin is linear with initial cell number, rapid and reversible. The cells display 16,400 +/- 1600 high affinity [3H]adenosine binding sites/cell, equivalent to 118 fmol bound/mg protein, with an equilibrium dissociation constant of 27.97 +/- 3.0 nM. Competition studies reveal that adenosine greater than 2-chloroadenosine greater than NECA greater than L-PIA greater than D-PIA in competing for [3H]adenosine binding sites and that aminophylline and cromolyn sodium also bind to the putative receptor. Adenosine and its analogs, NECA, and L-PIA, appear to activate adenylate cyclase in resting mast cells by raising cyclic AMP, suggesting an Ra cell surface adenosine receptor subtype; these same analogs potentiate mast cell B hexosaminidase release stimulated by specific antigen. The identification of rat mast cell [3H]adenosine binding sites whose stimulation augments resting cell cyclic AMP levels and antigen-induced mediator release suggests that these receptors may be important in the biochemical mechanisms of allergic diseases. The ability to assess the number and affinity of mast cell adenosine receptors will enable one to monitor receptor alterations during pharmacologic manipulation and in disease states. PMID- 3000153 TI - Male mammography. AB - Over a 7-year period, 94 mammograms were performed for male breast disease. Indications included breast enlargement, tenderness, mass, and previous mastectomy. Fatty enlargement was easily diagnosed. Gynecomastia was unilateral in 28 of 40 cases and easily differentiated from malignancy in all but one patient in whom nipple retraction was present. In cases of bilateral gynecomastia, eight showed asymmetric involvement. Three carcinomas were studied, two of which were clinically obvious. One asymptomatic cancer was detected on routine follow-up in a man with previous mastectomy. PMID- 3000155 TI - Clear cell sarcoma of the kidney: a renal tumor of childhood that metastasizes to bone. PMID- 3000156 TI - MR recognition of supratentorial tumors. AB - Eighty patients with intrinsic tumors of the cerebral hemispheres and thalami were studied with a 0.5 T superconducting system and third- or fourth-generation computed tomographic (CT) scanners. Twenty-eight patients had histologically verified gliomas, 34 were presumed to have primary brain tumors on clinical grounds, 13 had metastases, and five were postoperative. Lesions shown on CT were equally well demonstrated on magnetic resonance (MR) imaging; more metastases were seen on MR than on CT images. MR revealed abnormal signals in 10 cases in which CT findings were equivocal. It was not possible to differentiate edema from tumor in many cases using the MR imaging sequences currently available. The histologic types of the tumors could not be determined from the MR appearances. PMID- 3000157 TI - AIDS. Part I: The Huntsville Hospital experience, epidemiology, etiology, immunology and pathogenesis. PMID- 3000154 TI - Breast cancer metastasis to intramammary lymph nodes. AB - Metastatic disease to the intramammary lymph nodes from breast cancer may be seen mammographically. In the four cases reviewed, the affected intramammary lymph nodes were enlarged (1 cm or greater in diameter), homogeneous, and well circumscribed. All lacked the lucent center or hilar notch characteristic of benign intramammary nodes. Differentiation of malignant from benign causes of intramammary lymph node enlargement, such as inflammation or hyperplasia, is impossible by mammography. Biopsy is recommended for all intramammary lymph nodes of 1 cm or greater that are not fat infiltrated unless the patient clearly has an associated dermatitis or mastitis. Metastatic disease to the intramammary lymph nodes may be the first clinical and/or mammographic sign of breast cancer and may significantly affect prognosis. PMID- 3000158 TI - Angiotensin converting enzyme activity, prostaglandin F2 alpha- and immunoglobulin E level in childhood asthma bronchiale. AB - Angiotensin converting enzyme activity, prostaglandin F2 alpha and immunoglobulin E level have been studied in 35 asthmatic and 20 control children. The patients were divided in three groups: sever, mid-severe and moderate according to the severity of asthma. Angiotensin converting enzyme activity was the lowest in the severe and mid-severe groups. Prostaglandin F2 alpha was high in all three asthmatic groups. Immunoglobulin E level was highest in the severe group, but in the mid-severe and moderate groups it was high too. There was a significant negative correlation between prostaglandin F2 alpha level and angiotensin converting enzyme activity. Neither we found a correlation between the levels of prostaglandin F2 alpha and immunoglobulin E, nor between the immunoglobulin E level and angiotensin converting enzyme activity. These data suggest that angiotensin converting enzyme and prostaglandin F2 alpha may play a primary or secondary role in control of vascular tone of asthma bronchiale. PMID- 3000160 TI - Systemic hypertension after cardiac transplantation: effect of cyclosporine on the renin-angiotensin-aldosterone system. AB - Fifteen patients who had undergone cardiac transplantation and who had hypertension (164 +/- 14/112 +/- 13 mm Hg), aged 16 to 57 years (mean 39), were treated with cyclosporine, 8 +/- 3 mg/kg/day, and prednisolone, 0.27 +/- 0.1 mg/kg/day, for 63 to 788 days (mean 288) after transplantation. They were not given antihypertensive drugs. Before treatment, the mean urinary sodium level was 104 +/- 48 mEq/day. Two discrete abnormalities accompanied their high blood pressure (BP): an increase in serum creatinine levels (p less than 0.05) to values exceeding those measured just before transplantation (2.1 +/- 1.0 vs 1.35 +/- 0.54 mg/dl) with low creatinine clearance (61 +/- 28 ml/min X 1.73 m2), and a 15% increase in plasma volume (+445 +/- 686 ml, p less than 0.02). Urinary excretion of vanilmandelic acid and total metanephrines was normal. Supine plasma renin activity was also normal (0.78 +/- 0.32 nmol/ml/hour). The stimulation of renin release after acute inhibition of converting enzyme by captopril was less marked than is usual in hypertensive subjects (0.86 +/- 0.54 nmol/liter/hour). Captopril induced a smaller drop in BP than nifedipine (-8 +/- 13/-6 +/- 10 mm Hg vs -14 +/- 11/-15 +/- 10 mm Hg). Levels of plasma aldosterone, angiotensinogen and converting enzyme activity were all normal, 308 +/- 147 pmol/liter, 712 +/- 164 nmol/ml and 30 +/- 6 mU/ml, respectively. It is concluded that hypertension is common in cardiac transplantation patients treated with cyclosporine, since 13 of our 15 subjects were normotensive before transplant.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000159 TI - Anti-ischemic actions of a new thromboxane receptor antagonist during acute myocardial ischemia in cats. AB - Thromboxane A2 (TxA2) production increases significantly during acute myocardial ischemia. Since TxA2 induces platelet aggregation, coronary vasoconstriction, and has a direct cytolytic effect, thromboxane receptor antagonism would be expected to be beneficial in acute myocardial ischemia. Thirty minutes after ligation of the left anterior descending coronary artery (LAD) in anesthetized cats, the TxA2 receptor antagonist BM-13,177 or its vehicle was given as a bolus injection at 20 mg/kg, followed by continuous infusion of 20 mg/kg/hr for 4.5 hours. ST segment elevation declined significantly (p less than 0.02) after BM-13,177 treatment, suggesting a reduction in cellular ischemia. The loss in myocardial creatine kinase (CK) activity and in free amino-nitrogen concentration in the ischemic area was also significantly reduced (p less than 0.01). No significant changes in blood pressure or heart rate were seen with BM-13,177 during myocardial ischemia or in nonischemic control cats. Blood levels of BM-13,177 were sufficient to inhibit ex vivo platelet aggregation induced by the prostaglandin endoperoxide analog, U-46,619. Data from isolated cat coronary arteries suggest that BM-13, 177 antagonizes the thromboxane/endoperoxide receptor in coronary vascular smooth muscle. These experiments indicate that TxA2 plays a significant role in propagating the extension of ischemic damage, and that thromboxane receptor antagonism is an effective means of reducing the damage provoked by TxA2 in acute myocardial ischemia. PMID- 3000161 TI - Hemodynamic attenuation and the nitrate-free interval: alternative dosing strategies for transdermal nitroglycerin. AB - Various dosing strategies to determine therapeutic effects of nitroglycerin (NTG) preparations are reviewed. The importance of individual patient titration in establishing an effective NTG dosage is emphasized by reviewing a nitroglycerin ointment study and a crossover study. Studies reporting the development of hemodynamic attenuation ("tolerance") with longterm nitrate therapy are also discussed. The results of these and other studies suggest that the magnitude of the hemodynamic response to NTG or isosorbide dinitrate diminishes over time, with acute or first-dose effects far exceeding those obtained during long-term therapy. However, patients on long-term therapy continue to respond to sublingual NTG, which suggests that this phenomenon is not true NTG tolerance. The effect of a nitrate-free interval as a mechanism for avoiding hemodynamic attenuation of NTG therapy is reviewed. The results of 4 studies discussed found that intermittent nitrate protocols were not associated with the attenuated hemodynamic effect observed during chronic therapy. Two possible mechanisms for the vasodilatory effects of nitroglycerin are discussed. The first relates to the production of cyclic guanosine monophosphate in the smooth muscle cells of arteries and veins; the second to the synthesis of prostaglandin I2 by vascular endothelial cells. A mechanism by which nitrate receptors could be manipulated to increase vascular responsiveness is theorized, as well as a means by which a nitrate-free interval might avoid the development of hemodynamic attenuation in terms of cellular mechanisms and receptors. PMID- 3000162 TI - Pathologic and immunologic characterization of malignant lymphoma in Taiwan. With special reference to retrovirus-associated adult T-cell lymphoma/leukemia. AB - One hundred four cases of malignant lymphomas, including 90 cases of non Hodgkin's lymphoma, 5 cases of histiocytic malignancy, and 9 cases of Hodgkin's disease were analyzed pathologically and immunologically using a panel of monoclonal and conventional antibodies for T-, B-, histiocyte, and Hodgkin's neoplastic cells. Our results revealed a high frequency of T-cell lymphoma (42.3%), a low percentage of follicular lymphoma (10.5%), and Hodgkin's disease (8.7%) in Taiwan. More than half of the malignant lymphomas belonged to the high risk unfavorable group. Peripheral T-cell lymphomas (33 cases) showed characteristic clinical and histologic features, which can sometimes be confused with Hodgkin's disease. Monoclonal antibodies Leu-M1 and 2H9 were an important aid for their differential diagnosis. Five of the 33 peripheral T-cell lymphomas were positive for antibody to adult T-cell lymphoma/leukemia (ATL) virus associated antigen (ATLA). Four patients were from the northeast coast of Taiwan, I-Lan county. Five (4.8%) were diagnosed as true histiocytic malignancies, including two true histiocytic lymphoma and three malignant histiocytosis. Two cases each of large cell lymphoma and immunoblastic lymphoma showed no identifiable marker expression. The distribution of lymphoproliferative disorders in Taiwan is similar to that in Japan but much different from western countries. PMID- 3000163 TI - Rotazyme assay in neonates without diarrhea. Results of screening survey and preliminary analysis of false positive specimens. AB - One hundred forty-seven stool specimens from 93 infants younger than four months of age in a Neonatal Intensive Care Unit were tested for rotavirus by the Rotazyme ELISA method (Abbott Laboratories, North Chicago, IL). None of the infants had diarrhea at the time of the testing. Ten of 147 (6.8%) specimens were either low or suspect positive. None had rotavirus by electron microscopy. Excluding the suspect positives, which were negative on retesting, the false positive rate was only 6 of 147 (4.1%). Of five specimens with sufficient material and repeatedly positive tests, heat to 56 degrees C for one-half hour eliminated the binding to the Rotazyme bead but had no effect on the rotavirus positive control. One patient was found to have an extremely high positive Rotazyme test, independently of the survey. No virus was found in this specimen by electron microscopy, and the material responsible for the false positive result was not removed by centrifugation (100,000 X g for one hour), heating to 56 degrees C for one-half hour, trypsin, ether/beta-mercaptoethanol, or dialysis. Thus, false positives were encountered, but the overall rate was acceptably low. Such false positives are likely to result from more than one cause and, depending on results of further study, may be confirmed as false positives by loss of reactivity at 56 degrees C for one-half hour and perhaps lack of binding to a control bead. PMID- 3000164 TI - Immunocytochemical demonstration of growth hormone-releasing factor in gastrointestinal and pancreatic endocrine tumors. AB - Growth hormone-releasing factor (GRF), a linear peptide that exists in a number of different molecular forms (GRF-44, -40, -37, and-31) has been shown to be responsible for the acromegaly associated with certain endocrine tumors of the pancreas and other foregut-derived structures. With the use of two anti-sera (#1A850 and G59/901) directed against different segments of the GRF molecule, a series of 24 pancreatic and 35 gastrointestinal endocrine tumors, not associated with acromegaly, were surveyed systematically for immunocytochemical localization of GRF in the tumor cells. Strong immunoreactivity for GRF was encountered in 10 tumors (6 pancreatic and 4 gastrointestinal). While all ten tumors were immunoreactive against G59/901, which recognizes GRF-44, -40, and -37, two jejunal carcinoids showed additional immunostaining with 1A850 that is specific for GRF-44. Seven of these ten tumors were also immunoreactive for a variety of other regulatory peptides and neurotransmitters, including gastrin, insulin, glucagon, serotonin, substance P, somatostatin, pancreatic polypeptide, vasoactive intestinal peptide (VIP), and adrenocorticotropic hormone (ACTH). No consistent pattern of association between GRF and the other regulatory substances was evident. These findings indicate that, even in the absence of associated acromegaly, up to 17% of endocrine tumors of the gastro-entero-pancreatic (GEP) axis show immunoreactivity for GRF and that such reactivity is associated more frequently with pancreatic (25%) than with gastrointestinal (11%) endocrine tumors. PMID- 3000166 TI - Synchronous triple malignant tumors of the lung. A case report of bronchial carcinoid, small cell carcinoma, and adenocarcinoma of the right lung. AB - The authors report a case in which a highly unusual, simultaneous occurrence of a peripheral small cell carcinoma and a central bronchial carcinoid in the right upper lobe and a peripheral adenocarcinoma in the right middle lobe was observed. This is the fourth case of triple lung cancer reported in the literature. The role of computerized tomography in disclosing multiple lung carcinomas and the significance of the concurrence of pulmonary small cell carcinoma and bronchial carcinoid are discussed. PMID- 3000165 TI - Spontaneous liver cell adenoma in children. AB - The clinical and pathologic features observed in five children with spontaneous liver cell adenoma are described. All tumors showed benign biologic behavior. Two patients had untreated tumors that were followed for 5 years and 26 years, respectively, and showed no evidence of malignant transformation; the latter tumor unprecedentedly showed signs of spontaneous regression over time. Unusual physical findings associated with the liver cell adenomas included generalized osteoporosis, urticaria, and koilonychia. Microscopically, the liver cell adenomas were composed of cords, rarely sheets, of neoplastic hepatocytes with negligible pleomorphism and rare to absent mitoses; bile ducts, portal tracts, and central veins were absent. A fibrous capsule was present in all cases. Previously undescribed histologic features of this tumor type included focal extramedullary hematopoiesis and multinucleate giant cell formation in association with tumor necrosis. Benign neoplasms of the type described in this and previous studies probably should be managed conservatively. PMID- 3000167 TI - The effect of etorphine on the human esophagus: evidence for subtypes of opioid receptors in the esophagus. PMID- 3000168 TI - Esophageal granular cell tumor removed by endoscopic polypectomy. AB - Granular cell tumors are rare neoplasms that are generally benign. They are usually found in the tongue, subcutaneous tissues, and breast. Since Abrikossof first described the histology of this tumor in 1926, there have been 86 cases involving the esophagus discussed in the literature. There is a low likelihood of these tumors becoming malignant and recurrence after resection is rare. However, these tumors can be confused with more malignant lesions. We report an additional case involving the esophagus and offer new management guidelines in the era of fiberoptic endoscopy. PMID- 3000169 TI - Enhancement by human bile of the binding of free and intrinsic factor-bound cobalamin (vitamin B12) to small bowel epithelial cell receptors. AB - The effect of human bile on the binding of free cobalamin (Cbl) and of intrinsic factor-Cbl (IF-Cbl) complexes to intestine receptors was examined in in vitro receptor extracts made from guinea pig small bowel epithelial cells. Cbl and IF Cbl complex binding to the receptors was measured in the effluent from a Sephadex G-200 column. Intrinsic factor-Cbl binding to the receptors in the control samples was 4.32 +/- 0.07 ng/animal (mean +/- SEM); addition of bile, saturated with excess 57Co-Cbl enhanced binding to 5.26 +/- 0.16 ng/animal (p less than 0.001). These data suggest that bile enhances binding of IF-Cbl complex onto ileal receptors. Whether bile also serves as "releasing factor" by releasing Cbl from intrinsic factor in vivo remains to be determined. PMID- 3000170 TI - Occurrence of three cases of carcinoma in individuals with Crohn's disease treated with metronidazole. AB - Controversy exists regarding the safety of metronidazole. Experimental studies have suggested both a carcinogenic and mutagenic effect in animals. In women treated with metronidazole for trichomoniasis which involves low dosages and short time periods, no carcinogenic effect was noted. Metronidazole is also used in the treatment of Crohn's disease which involves larger dosages over longer periods of time. The authors have recently encountered three individuals with Crohn's disease who were treated with large doses of metronidazole and who developed a malignancy [breast (two) and cholangiocarcinoma] at a rather young age (32, 31, and 27 years, respectively). Whereas this association based on three cases is not per se incriminatory or even suggestive, nevertheless, the cases are unusual and we urge prospective and long-term follow-up studies on individuals being treated with large doses of metronidazole over prolonged periods of time. Metronidazole (Flagyl) was introduced into The United States in the 1960's for the treatment of trichomoniasis (1). The conditions for which this drug is indicated and has been used have expanded to include such diverse entities as amebiasis (2), brain abscess (3), and Crohn's disease (4). A controversy exists regarding the safety of this drug (5, 6). Experimental studies have shown that metronidazole is both carcinogenic and mutagenic (7-9). However, a recent study (10) cited lack of evidence for a carcinogenic effect with the use of metronidazole in women treated for trichomoniasis. In the past 1 1/2 years we have encountered three unusual cases involving individuals who were treated with large doses of metronidazole for Crohn's disease and who developed a malignant neoplasm at a relatively young age. The features of these three cases are the subject of this report. PMID- 3000171 TI - Seroepidemiologic study of antibody to adult T-cell leukemia virus in Okinawa, Japan. AB - From 1980 to 1984, a total of 3,978 serum samples were collected from healthy subjects in the Yaeyama District of Okinawa, Japan. These serum samples were tested for presence of antibody to adult T-cell leukemia-associated antigen (anti ATLA) by the enzyme-linked immunosorbent assay (ELISA) method. Overall prevalence of anti-ATLA was 15.3%. Standardized prevalence differed from island to island: Hateruma Island had the highest (21.2%) and Taketomi Island the lowest (6.2%). Prevalence of anti-ATLA increased with age and was significantly higher in females (18.1%) than in males (12.2%) (p less than 0.001). For subjects younger than 20 years of age, the rate of anti-ATLA in males was slightly higher than that in females. For subjects 20 years of age and over, prevalence was higher in females than in males. In all age groups 40 years and over, prevalence was significantly higher in females than in males. PMID- 3000172 TI - Occupational health reporting systems--USA. AB - The three-fold purpose of this paper is to (1) describe the occupational hazard and health effect information systems used by the National Institute for Occupational Safety and Health (NIOSH), (2) highlight the parts of these data systems that are relevant to the topic of this dermatologic disease and chronic trauma workshop, and (3) to note the inadequacies of existing data systems in the United States. PMID- 3000173 TI - Clinical features to stage alveolitis in asbestos workers. AB - To analyze the clinical features of asbestos-induced alveolitis and stage its activity, we evaluated 217 asbestos workers by the usual clinical, radiological, and functional parameters and computerized gallium 67(Ga) lung scan; we obtained bronchoalveolar lavage (BAL) in 33 and lung biopsy in 6. In addition, we scored the profusion of lung rales and correlated it with other parameters of severity of asbestosis. In the 55 workers without asbestosis and normal 67Ga scan, BAL analyses were comparable to those of controls. Of the 56 without asbestosis but increased 67Ga lung uptake, BAL analyses in 8 documented a predominantly macrophagic alveolitis (confirmed on lung biopsy in 3), with the highest levels of BAL fibronectin. In the 106 workers with asbestosis, 67Ga lung uptake was increased in 75; BAL in 17 demonstrated a macrophagic and neutrophilic alveolitis with elevated fibronectin levels. Lung biopsy in 3 of the latter workers documented peribronchiolar fibrosing alveolitis. Rale scores in all workers or in those without asbestosis did not correlate with 67Ga scores; they correlated fairly well with profusion of parenchymal opacities (Rs = 0.42) and rigidity of the lung pressure-volume curve (Rs = 0.39). Thus, 67Ga lung uptake is an early indicator of chronic macrophagic alveolitis in asbestos workers, which usually progresses to asbestosis. In the disease, profusion of lung rales constitutes a simple clinical mode of assessment of disease severity that correlates better with radiological and functional parameters than with parameters of alveolitis. PMID- 3000174 TI - Asbestos exposure, smoking habits, and cancer incidence among production and maintenance workers in an electrochemical plant. AB - The incidence of cancer was studied in a cohort of 287 men who were exposed to asbestos at a nitric acid production plant from 1928 onwards. During the observation period from 1953 through 1980 all cancer cases among the cohort members were identified in The Cancer Registry. For the whole cohort 42 cases of cancer were observed versus 30.6 expected. The figures for cancer of the lungs and pleura combined were 17 observed versus 3.7 expected. The corresponding figures for a heavily exposed subcohort were 11 observed and 1.2 expected. In that group there was also an increased incidence of colon cancer with 3 cases observed against 0.8 cases expected. Within the whole cohort four cases of pleural and one case of peritoneal malignant mesothelioma were found. There was also an increased incidence of malignant melanoma of the skin with 3 cases observed against 0.6 expected. For cancer cases that were registered as of unknown origin there were 7 cases observed and 1.4 expected. There was no increased rate ratio for cancer at any site before 20 years after the first asbestos exposure. The smoking habits of all cohort members were recorded and the relative rates for lung cancer were calculated in relation to smoking habits. In common with previous studies the results indicate a multiplicative model for the interaction between asbestos exposure and smoking in regard to lung cancer risk. PMID- 3000175 TI - Recent advances in the therapy of diabetic peripheral neuropathy by means of an aldose reductase inhibitor. AB - Nerve conduction slowing, a hallmark of both experimental and human diabetic neuropathy, is improved or corrected by administration of aldose reductase inhibitors such as sorbinil. Recent experiments in animals attribute acutely reversible nerve conduction slowing in diabetes to a myo-inositol-related defect in nerve sodium-potassium adenosinetriphosphatase, which generates the transmembrane sodium and potassium potentials necessary for nerve impulse conduction and the sodium gradient necessary for sodium-dependent uptake of substrates. This myo-inositol-related abnormality in sodium-potassium adenosinetriphosphatase function is currently viewed as a cyclic metabolic defect involving sequential alteration of sodium-dependent myo-inositol uptake, myo inositol content, myo-inositol incorporation into membrane phospholipids, and phospholipid-dependent sodium-potassium adenosinetriphosphatase function in peripheral nerve. Aldose reductase inhibitors have been shown to normalize both nerve myo-inositol content and nerve sodium-potassium adenosinetriphosphatase activity. These observations suggest that the acute effects of aldose reductase inhibitors on nerve conduction in both animals and humans with diabetes may be mediated by correction of an underlying myo-inositol-related nerve sodium potassium adenosinetriphosphatase defect. Furthermore, this sorbinil-corrected sodium-potassium adenosinetriphosphatase defect in diabetic nerve may contribute to other biochemical, functional, and structural abnormalities present in diabetic peripheral neuropathy. PMID- 3000177 TI - Rapid adrenocorticotropic hormone test in practice. Retrospective review. AB - Retrospective analysis of the rapid adrenocorticotropic hormone (ACTH) test in a large adult population shows a marked interdependence of the basal cortisol concentration, peak cortisol concentration, and increase in cortisol concentration. Repetition of the rapid ACTH test in the same patient does not improve diagnostic accuracy. A significant number of falsely abnormal rapid ACTH test results were observed (in comparison to continuous ACTH infusion as a reference test). This supports the use of the rapid ACTH test as a screening test, but not as a diagnostic test for adrenocortical failure. It is proposed that a peak cortisol level greater than or equal to 20 micrograms/dl (550 nmol/liter) is a sufficient single criterion for normal adrenal function as assessed by the rapid ACTH test. PMID- 3000178 TI - Testosterone-secreting adrenal adenoma containing crystalloids characteristic of Leydig cells. AB - A 49-year-old woman with virilization demonstrated biochemical features traditionally ascribed to virilizing ovarian tumors: marked elevation of serum testosterone level with normal urinary excretion of 17-ketosteroids and normal serum dehydroepiandrosterone level. An adrenal cortical adenoma containing neoplastic cells indistinguishable from Leydig cells, including the demonstration of characteristic crystalloids of Reinke, was shown to be the source of the elevated testosterone level. A review of the literature revealed 13 cases with a similar biochemical profile, and of these, two were reported to contain crystalloids of Reinke. Taking cognizance of the fact that these characteristic crystalloids are only found in 40 percent of Leydig cell tumors of the testis, it is concluded that Leydig cells may be present in, and may be active participants in, the pathophysiology of a number of testosterone-secreting adrenal tumors. PMID- 3000176 TI - Effects of sorbinil therapy in diabetic patients with painful peripheral neuropathy and autonomic neuropathy. AB - Clinical investigations with the aldose reductase inhibitor sorbinil in patients with peripheral neuropathy due to diabetes are described. After an improvement in motor and sensory nerve conduction velocities was demonstrated in asymptomatic diabetic patients taking sorbinil (compared with velocities during a placebo period), 11 patients with painful diabetic neuropathy were treated with sorbinil for three weeks without alterations in diabetic management or control. Therapy was placebo-controlled in a single-blind fashion in eight patients. Pain (assessed by or on a zero to 20 rating scale), which had been constant for many months before entry into the study and unresponsive to numerous medications, improved from a mean score of 16 to 8 and returned when the drug was discontinued. Objective improvement in sensation and strength were observed in some cases. Improvements in nerve conduction velocity and cardiac autonomic function were also documented. Cardiac autonomic neuropathy was studied in 36 patients in a double-blind, placebo-controlled, randomized, noncrossover trial. Patients received one 250-mg sorbinil tablet or one placebo tablet daily for six weeks, after a one-week baseline period. Glycemic control did not change during the study period, as indicated by unaltered glycohemoglobin levels. Response was assessed by expiration-inspiration ratios, obtained on electrocardiography during six cycles per minute respiration, and by resting minimal heart rate, both measures of vagal function. In the sorbinil-treated group, expiration-inspiration ratios improved from 1.074 +/- 0.012 to 1.096 +/- 0.020 (p less than 0.03). There was a slight decrease in the ratios in the placebo-treated group, from 1.112 +/- 0.023 to 1.105 +/- 0.023 (not significant). The difference between the Week 0 to Week 6 changes in each group was significant (p less than 0.01). Resting minimal heart rate decreased in the sorbinil-treated group from 76.4 +/- 2.3 to 66.8 +/- 2.8 +/- 2.4 beats per minute (p less than 0.001), with a mean change of 10 +/- 2. In the placebo-treated group, heart rate was unchanged (77.9 +/- 3.9 to 77.5 +/- 3.3 beats per minute). The two-sample t test of the within-group differences was also significant (p less than 0.001). The changes in both expiration-inspiration ratios and resting minimal heart rate are consistent with a sorbinil-related improvement in cardiac parasympathetic nerve function. Several isolated cases of apparent sorbinil-related improvements in autonomic symptoms have been observed.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3000179 TI - Steroid secretion by a lipoid cell tumor causing virilization and its diagnosis with computerized tomography. AB - Presented is the case of a virilized woman with a lipoid cell tumor of the ovary localized by computerized tomography. The major secretory products of the tumor were testosterone and estradiol; the production of androgens was responsible for the clinical features and hindered the effects of estrogens. Elevated levels of estradiol indicated important ovarian aromatase activity as reflected by large ovarian-peripheral venous gradients. PMID- 3000181 TI - Asymptomatic herpesvirus genital excretion during labor. PMID- 3000180 TI - Variations in red blood cell proton T1 relaxation times that correspond to menstrual cycle changes. AB - A study was made of the relationship of proton T1 relaxation times and stage of the menstrual cycle. Preliminary data show that these indicators of red blood cell hydration status may be useful as objective markers of premenstrual tension. PMID- 3000182 TI - Assessment of the contemporary management of germ cell malignancies of the ovary. AB - Fortunately germ cell cancers of the ovary are infrequent. Modern therapy has changed an almost universally fatal disease to one that is highly curable. Conservative surgical procedures and intensive short-term chemotherapy appears to be the treatment of choice. In this young population subsequent pregnancies are a reality. Contemporary management is discussed. PMID- 3000184 TI - Granular cell myoblastoma of the vulva in a 6-year-old girl. AB - Granular cell myoblastomas are rare neoplasms thought to arise in neural tissue resembling Schwann cells. About 7% of these tumors are located on the vulva. This is the second reported case in a prepubertal girl with both cases having been found in 6 year olds. None of the vulvar lesions have been malignant. The treatment is wide surgical excision and observation for recurrence. Extragenital sites should be evaluated since multiple tumors are found in a number of patients. PMID- 3000183 TI - Adenylate cyclase in human ovarian cancers: sensitivity to gonadotropins and nonhormonal activators. AB - Adenylate cyclase activity in particulate preparations of ovarian tumors from 47 women was determined by measuring the conversion of phosphorus 32-labeled adenosine triphosphate to phosphorus 32-labeled cyclic adenosine monophosphate. Ovarian cancers typically exhibited an active adenylate cyclase which was stimulated by 50 mumol/L 5'-guanylylimidodiphosphate and 10 mmol/L of sodium fluoride. This activity was comparable to that in particulates of normal postmenopausal ovaries and was independent of the class of tumor. There was no significant increase in adenylate cyclase activity in any epithelial or germinal tumor in the presence of either 250 nmol/L of human chorionic gonadotropin or 333 nmol/L human follicle-stimulating hormone. However, cyclic adenosine monophosphate production by two sex cord stromal tumors was stimulated by follicle-stimulating hormone, but not by human chorionic gonadotropin. Follicle stimulating hormone stimulated a threefold increase in activity in the granulosa theca cell tumor, with an activation constant (57 nmol/L) similar to that in follicle-stimulating hormone-responsive rat ovaries. Prostaglandin E1 (50 mumol/L) increased cyclic adenosine monophosphate production by epithelial tumors more than twofold. These data suggest that sex cord stromal tumors, unlike the more common epithelial tumors, can be modulated directly by gonadotropin. PMID- 3000185 TI - Follicular development: lessons learned from human in vitro fertilization. AB - In vitro fertilization has offered new insights into our understanding of ovulation induction, folliculogenesis, and luteal phase events. This new information is provided by the ability to precisely study these cycles in a frequent and sequential fashion through the use of peripheral blood markers, ultrasound evaluation, and follicular fluid constituents and cell culture techniques, as well as direct observation of the oocyte, fertilization, and cleavage. In these stimulated cycles the follicular phase serum estradiol levels in conjunction with ultrasound were evaluated; a poor correlation was shown between follicle size and number and estrogen production. This distinct dyssynchrony suggests the recruitment of a number of cohorts of follicles in each stimulated cycle. From the biochemical markers in follicular fluid, cyclic adenosine monophosphate has a distinct predictive value in regard to pregnancy in in vitro fertilization-embryo transfer cycles. In the luteal phase, the mass effect of aspiration of great numbers of granulosa cells, the effect of supplemental progesterone, and the influence of high follicular phase estradiol levels remain controversial and, therefore, a less clear cut pattern emerges. Variations in the protocol have not greatly improved the major problems of folliculogenesis associated with ovulation induction and an in vitro fertilization-embryo transfer program, that is, follicular asynchrony and luteal phase deficiency. PMID- 3000186 TI - The nonexpansile, equilibrated concentration of perfluoropropane gas in the eye. AB - Bubbles of 100% perfluoropropane (0.4 ml) were injected into the vitreous cavities of 31 New Zealand White rabbits. Gas bubbles were aspirated from eyes at six and 12 hours and at one, two, three, four, five, seven, nine, and 14 days, and were analyzed by gas chromatography. The nonexpansile concentration of perfluoropropane (found at maximum expansion of the gas bubble at four days) was approximately 12%. The gas bubble concentration equilibrated at seven days with a perfluoropropane concentration of approximately 10% with little change through 14 days. Using a similar protocol, we injected 0.56-ml bubbles of 100% perfluoropropane into the vitreous cavities of three owl monkeys. When sampled on day 4 for analysis by gas chromatography, results were comparable to the rabbit data. A 12% concentration of perfluoropropane should approximate the ideal composition of a gas mixture for a total fluid-gas exchange. This would achieve complete retinal tamponade without later compromise of intraocular pressure by further expansion. PMID- 3000187 TI - Skull base surgery for glomus jugulare tumors. AB - Thirty-six patients with glomus jugulare tumors have been managed over a 13-year period using various combinations of skull base surgery and irradiation therapy. The data resulting from this study are presented; the techniques of diagnosis and treatment are reviewed. We conclude that irradiation therapy alone is a satisfactory form of treatment for elderly and poor-risk patients; preoperative x ray therapy followed by skull base surgery is an effective treatment for younger patients. PMID- 3000188 TI - Rare tumors of the skull base and temporal bone. AB - Skull base surgery has advanced significantly in the last decade. Neuro otologists and neurosurgeons are working together to apply their combined expertise to totally remove skull base lesions with minimal additional neurologic deficit. Standard approaches have been developed for the more common lesions of the skull base, such as the glomus jugular tumor. Rare tumors of the skull base can be removed using the standard skull base surgery techniques. However, there are specific problems with some of these tumors. This article will describe four types of unusual skull base tumors: hyalinized chemodactoma, giant cell tumor of the bone, papillary adenoma of the middle ear, and ganglioneuroma. The unique properties of these tumors and the surgical approach to their removal will be presented and illustrated by case reports. PMID- 3000190 TI - Effect of urinary alkalinization on the intrarenal formation of kinins. AB - Alterations in the urinary excretion rate of kallikrein (UKKV) have frequently been assumed to reflect alterations in the intrarenal generation of kinins. Since other factors such as urinary pH and the activity of renal kininases may affect the intrarenal concentration of kinins, we investigated the effect of urinary alkalinization on kinin excretion in the presence and absence of kininase II inhibition. Urine was collected from the ureters of rats during control (ctl) (0.15 M NaCl infusion) and experimental (exp) periods. During exp periods, group I (time control) was infused with 0.15 M NaCl, group II with 0.3 M NaCl, and groups III and IV with 0.3 M NaHCO3, all at 0.12 ml/min. Prior to the ctl period, group IV was pretreated with the kininase II inhibitor captopril (40 mg/kg). During the exp period, urinary kinin excretion (UKiV) increased significantly in all groups ([exp - ctl] UKiV = 21 +/- 7, 27 +/- 9, 52 +/- 14, and 70 +/- 9 pg X min-1 X kg-1 in groups I, II, III, and IV, respectively). Urinary pH increased significantly in groups II, III, and IV. The increases in UKiV and pH were significantly greater in the bicarbonate-infused rats than in control. Partial correlation coefficients show that UKiV correlates with a high degree of significance only with urinary pH (r = 0.6). During the ctl period, UKiV in the captopril-pretreated rats was not different from that in other groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000189 TI - Urate transport in the proximal tubule: in vivo and vesicle studies. AB - The transport of urate in the mammalian nephron is largely confined to the proximal tubule. Depending on the species, net reabsorption or net secretion is observed. The rat, like the human and the mongrel dog, demonstrates net reabsorption of urate and has been the most extensively studied species. The unidirectional reabsorption and secretion of urate in the rat proximal tubule occur via a passive and presumably paracellular route and by a mediated transcellular route. The reabsorption of urate, and possibly its secretion, can occur against an electrochemical gradient. A variety of drugs and other compounds affect the reabsorption and secretion of urate. The effects of these agents depend on their site of application (luminal or blood), concentration, and occasionally their participation in transport processes that do not have affinity for urate. Recent studies with renal brush border and basolateral membrane vesicles from the rat and brush border vesicles from the dog have determined the mechanisms for urate transport across the luminal and antiluminal membranes of the proximal tubule cell. Brush border membrane vesicles contain an anion exchanger with affinity for urate, hydroxyl ion, bicarbonate, chloride, lactate, p-aminohippurate (PAH), and a variety of other organic anions. Basolateral membrane vesicles contain an anion exchanger with affinity for urate and chloride but not for PAH. Both membrane vesicle preparations also permit urate translocation by simple diffusion. A model for the transcellular reabsorption and secretion of urate in the rat proximal tubule is proposed. This model is based on the vesicle studies, and it can potentially explain the majority of urate transport data obtained with in vivo techniques. PMID- 3000191 TI - Na-K-ATPase in nephron segments of rats developing spontaneous hypertension. AB - Na-K-ATPase activity was determined in seven specific nephron segments of 5- and 12-wk-old spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) controls. The enzyme activity in proximal convoluted tubule (PCT) and proximal straight tubule (PST) was significantly higher in 5-wk-old SHR than in WKY. However, Na-K-ATPase activity in medullary thick ascending limb (MTAL), cortical thick ascending limb (CTAL), and distal convoluted tubule (DCT) was significantly lower in 5-wk-old SHR than in WKY. There were no significant differences in the enzyme activity in PCT, PST, MTAL, CTAL, and DCT in 12-wk-old SHR and WKY. Furthermore, there were no significant differences in Na-K-ATPase activity in collecting duct segments of 5- or 12-wk-old SHR and age-matched WKY. The possible role of the abnormal pattern of Na-K-ATPase activity in PCT, PST, MTAL, CTAL, and DCT in 5-wk-old SHR in generation of hypertension in this strain remains to be determined. PMID- 3000192 TI - Mechanism of fluid secretion in isolated shark renal proximal tubules. AB - Renal proximal tubules from the glomerular Squalus acanthias were studied for evidence of fluid secretion by closing one end of isolated tubules and leaving the other end open so that secreted fluid could be collected. When tubules were bathed in shark Ringer, fluid secretion rate was 27.6 +/- 3.9 (SE) pl X min-1 X mm-1 (21 tubules). Dibutyryl cAMP stimulated fluid secretion 50% (P less than 0.02, n = 14), furosemide inhibited fluid secretion 50% (P less than 0.01, n = 6), and metabolic inhibitors blocked fluid secretion nearly 100%. Secreted fluid was slightly hyperosmotic to peritubular bath (P less than 0.01, n = 7) but Na, Cl, S, K, and Ca concentrations were not significantly different from bath concentrations (wavelength-dispersive spectroscopy, electron probe analysis). cAMP had no effect on secreted fluid composition, and in some tubules cAMP did not stimulate fluid secretion. In conjunction with previous data we propose that spontaneous fluid secretion is driven by secretion of NaCl. However, finding the mechanism of NaCl and fluid secretion in glomerular renal tubules offers new perspectives of some previously inexplicable phenomena in the renal physiology of fish. PMID- 3000193 TI - Intracellular K+ and Na+ activities under hypoxia, acidosis, and no glucose in dog hearts. AB - To gain a better understanding of the ionic mechanisms responsible for the electrophysiological disturbances occurring during myocardial ischemia, transmembrane potentials and intracellular potassium (aiK) and sodium (aiNa) ion activities were measured under individual and combined conditions of hypoxia (Po2 less than 50 mmHg), respiratory acidosis (pH 6.6), and no glucose in isolated epicardial ventricular muscle preparations of the canine heart using conventional and ion-selective microelectrode techniques. After 30 min superfusion with hypoxic, acidic, and glucose-free solution under 2-Hz stimulation, resting membrane potential (RMP) was significantly reduced from -85.5 +/- 0.6 to -69.5 +/ 1.0 mV, accompanied by decreases in action potential amplitude, maximum upstroke velocity of phase O, and action potential duration. aiK was significantly decreased from 101.0 +/- 5.6 to 79.5 +/- 5.8 mM, whereas aiNa was not significantly altered by the combined condition. RMP and aiK were moderately decreased by hypoxia alone, slightly decreased by acidosis, and hardly affected by the glucose-free condition. The extent of depolarization was well correlated with the decrease in aiK. These results suggest that Na+-K+ pump inhibition may not be a major cause of K+ efflux from myocardial cells under the hypoxic, acidic, and glucose-free condition and that hypoxia is the most important factor affecting aiK and RMP among these conditions. PMID- 3000194 TI - Effect of chronic surgical denervation of dog heart on myocardial Na+-K+ pump. AB - The effect of chronic denervation of the myocardium on myocardial Na+-K+ pump activity and numbers of pump sites was studied in dogs. Right ventricular biopsies were taken immediately before and 4 wk after chronic surgical denervation, when depletion of norepinephrine is complete. Na+-K+ pump activity was assayed by ouabain-sensitive 86Rb uptake into ventricular tissue slices. The number of pump sites was measured by [3H]ouabain binding in ventricular tissue slices. Ouabain-sensitive Rb uptake was increased after denervation by an average of 147%. [3H]-ouabain binding at a concentration of 1.25 X 10(-6) M increased by an average of 27.6% after denervation. Acutely applied norepinephrine (2 X 10(-7) M) stimulated ouabain-sensitive Rb uptake both before and after denervation. The stimulation was greater after denervation, indicating supersensitivity. The cause of these changes is not known. PMID- 3000195 TI - Superoxide anion selectively attenuates catecholamine-induced contractile tension in isolated rabbit aorta. AB - Xanthine oxidase-derived oxygen metabolites caused a selective loss of norepinephrine-induced contractile tension in rings and helical strips from rabbit aorta. Phenylephrine-induced tension was not affected. The relaxation was selectively and completely blocked by superoxide dismutase but not by catalase. Isoproterenol-induced relaxation was also reversed by xanthine oxidase-derived oxygen metabolites. These observations are consistent with the chemical reaction of superoxide anion with catecholamines and suggest that the reaction may have significance at physiological concentrations of norepinephrine. The time course of the effects of superoxide generation on contractile tension was consistent with the properties of the chemical reaction (measured spectrophotometrically) and with the dependence of tone on norepinephrine concentration. These results indicate that superoxide anion, in situations at which submicromolar concentrations of this reduced oxygen metabolite are present, will selectively oxidize catecholamines, which may attenuate local adrenergic regulation. PMID- 3000196 TI - Adenosine is unimportant in controlling coronary blood flow in unstressed dog hearts. AB - The adenosine hypothesis of local metabolic control of coronary blood flow was tested in the unstressed heart with adenosine deaminase, which converts adenosine to nonvasoactive inosine. If adenosine is normally an important physiological regulator, then adenosine deaminase should lower coronary blood flow. The left main coronary artery was perfused at constant pressure in anesthetized, closed chest dogs. Adenosine deaminase was deposited in one region of the left ventricle by selective infusion into a branch of the left coronary artery. Coronary blood flow measured with radioactive microspheres was not lower in the region treated with adenosine deaminase than flow measured simultaneously in an untreated control region of the same heart. This finding is contrary to the prediction of the adenosine hypothesis. Coronary vasodilation elicited by intracoronary adenosine infusion was inhibited in the adenosine deaminase-treated region compared with the control region, indicating that adenosine deaminase lowered adenosine concentration at the vascular adenosine receptor. Inhibition of exogenous adenosine vasodilation was fully reversed by intracoronary infusion of a specific inhibitor of adenosine deaminase. Measurement of adenosine deaminase activity in cardiac lymph provided evidence that adenosine deaminase reached the myocardial interstitial space. These results demonstrate that introducing adenosine deaminase into the interstitial space of the unstressed heart did not lower coronary blood flow. This finding indicates that adenosine is normally below the vasoactive threshold and therefore is not important in mediating local metabolic control of blood flow in the unstressed heart. PMID- 3000197 TI - Biochemical changes accompanying enhanced cardiac contractility by ionophore A23187. AB - We have reported that the divalent cation ionophore A23187, like the beta adrenergic agonist isoproterenol, increased the force of contraction and rate of relaxation and shortened the duration of contraction of papillary muscles isolated from guinea pigs. A23187 produced a fall in resting tension and decreased the contracture tension of K +/- depolarized muscles, as did isoproterenol. In the present studies, isoproterenol produced a concentration dependent, rapid, and sustained increase in the cyclic AMP (cAMP) content of papillary muscle. In contrast, A23187 had no detectable effect on cAMP levels, even in the presence of the phosphodiesterase inhibitor, papaverine. Neither drug, at concentrations maximal for contractile effects, altered cyclic GMP (cGMP). Isoproterenol increased the cAMP-dependent protein kinase activity ratio, whereas A23187 did not change the activity of this enzyme. However, both A23187 and isoproterenol produced a concentration-dependent increase in phosphorylase activity. Concentrations of A23187 or isoproterenol that enhanced contractility maximally increased the alkali-labile phosphate (by ca. 35%) but were without effect on the acid-labile, alkali-stable phosphate in the total acid precipitable protein. Contractile effects of isoproterenol, which reflect activated Ca2+ uptake, and the increase in phosphorylase activity produced by this agent are believed to be due to an increase in cAMP with subsequent activation of cAMP dependent protein kinases and phosphorylation of proteins. A23187 may produce similar contractile effects without an increase in cAMP or cAMP-dependent protein kinase activity by activating other protein kinases and/or inhibiting phosphoprotein phosphatases, most likely by its effects on intracellular calcium. PMID- 3000198 TI - Low concentrations of A23187 increase calcium uptake by cardiac sarcoplasmic reticulum. AB - The divalent cation ionophore A23187 at a concentration of 1 nM produced an increased rate of oxalate-supported calcium uptake by isolated cardiac sarcoplasmic reticulum as determined by absorbance changes of the calcium sensitive dye murexide. Addition of a higher concentration of A23187 (0.1 microM) produced a decreased rate of calcium uptake. Measurement of the time during which ATPase was activated by calcium addition also suggested an increased rate of calcium uptake in the presence of 1 nM A23187 and an inhibition of calcium uptake at a higher concentration of the ionophore (0.1 microM). Ca2+-stimulated ATPase activity and incorporation of 32Pi from [gamma-32P]ATP into sarcoplasmic reticular proteins were increased by A23187 at concentrations of 1 nM or greater. An increased coupling of calcium uptake to ATP hydrolysis was observed at 1 nM A23187, while concentrations of the ionophore greater than or equal to 10 nM produced a decreased coupling. Addition of an inhibitor of cyclic AMP-dependent protein kinase decreased the rate of calcium uptake, and this inhibition was reversed in a concentration-dependent manner by 0.01-1 nM A23187. These data suggest that A23187 can activate a mechanism involving the calcium-dependent phosphorylation of protein that may regulate the activity of the calcium uptake system of the sarcoplasmic reticulum. These observations appear to provide an explanation for some of the contractile effects of A23187 in intact cardiac muscle that suggest that treatment with the ionophore results in an increased sequestration of calcium from the cytoplasm. PMID- 3000199 TI - Effects of octanol on canine subendocardial Purkinje-to-ventricular transmission. AB - The effects of 0.2 mM octanol on action potential propagation were investigated using in vitro preparations of canine papillary muscles. In these preparations an action potential initiated in the superficial Purkinje (P) layer propagates across specific Purkinje-ventricular junction (PVJ) sites into the underlying ventricular (V) layer. The conduction delay at PVJ sites increased from 4.85 +/- 1.55 to 8.85 +/- 3.34 (mean +/- SD) ms (n = 10, P less than 0.005), an 82% increase. However, propagation within the V syncytium was much less affected, with a decrease of conduction velocity by only 10% and a decrease in the maximal rate of rise of the action potential of 23%. The results indicate that octanol, which has previously been shown to increase gap junctional resistance, has a preferential effect on PVJ sites, as predicted by the hypothesis that there is a restricted pathway for intracellular current flow from P cells to V cells at these sites. PMID- 3000201 TI - Platelet alpha 2-adrenoreceptor binding in elderly depressed patients. AB - Specific binding of 3H-clonidine to platelet membranes was measured in depressed elderly patients and in an elderly control group. Maximum specific binding was significantly higher in depressed patients than in the control group, whereas the binding affinity was not significantly different. PMID- 3000200 TI - Guanosine diphosphate binding to brown adipose tissue mitochondria is increased after single meal. AB - A single meal results in an increased thermic activity of brown adipose tissue (BAT). The purpose of the present studies was threefold: 1) to identify major metabolic origins involved in this thermic response, 2) to determine the effect of meal composition on it, and 3) to determine time changes in postprandial brown fat thermogenesis. Wistar rats were trained to eat during 2 feeding sessions/day. On the days of the experiment, rats received a test meal for 2 h, and respective control rats were simultaneously meal deprived. The animals were killed at one or more time points after meal onset, and their BAT was removed for determination of mitochondrial guanosine diphosphate (GDP) binding to indicate rate of uncoupled respiration (expts 1 and 3) or Na+-K+-ATPase activity representing coupled respiration (expt 2). Meal taking was followed by an 85% increase in GDP binding (P less than 0.001). In contrast, Na+-K+-ATPase activity was not altered by a test meal of a similar composition. The largest meal-induced rise in mitochondrial GDP binding was evident during the early postprandial hours, and it was greatly reduced by 10 h after meal onset. Expressed per total interscapular brown fat depot, a high-carbohydrate meal caused a greater increase in GDP binding than an equicaloric high-fat meal. Our data indicate that the BAT proton conductance pathway is activated by a single meal. PMID- 3000202 TI - Lithium and bulimia: the role of the dopaminergic and opiatergic systems. PMID- 3000204 TI - The pathology of malignant fibrous histiocytoma of bone. A study of 130 patients. AB - Since 1927, 130 patients with well-documented malignant fibrous histiocytoma of bone have been diagnosed and treated at Memorial Hospital for Cancer and Allied Diseases. This sarcoma is 10 times less frequent than osteogenic sarcoma in this hospital. It most commonly occurred spontaneously (72%), whereas in the rest (28%) it followed previous radiation or various pre-existent osseous conditions, most often Paget's disease. The appendicular skeleton was the commonest site of involvement. The majority of the patients were middle-aged or older adults with a mean of 40.5 years of age; only 21.5% were 21 years or younger. Histologically, the lesions were subclassified as fibrous (62%), histiocytic or xanthomatous (30%), and malignant giant cell tumor (8%) variants. Older patients were more likely to have a secondary malignant fibrous histiocytoma, especially following radiation or Paget's disease. Overall survival estimates at 2 years and 5 years were 71% and 53%, respectively. Survival was not dependent on the histologic subtype of the lesion, but was strongly influenced by the histologic grade of malignancy. Important prognostic factors were the age of the patients and whether the lesions were primary de novo or secondary sarcomas: the older patients and those with secondary lesions did substantially worse. PMID- 3000203 TI - Benign fibrous histiocytoma of bone. AB - The clinical, radiologic, and pathological features of eight cases of fibrohistiocytic bone lesions histologically identical to the nonossifying fibroma of childhood are presented. They differed from the childhood lesion in their clinical and radiological features. They occurred in adults, and were frequently associated with pain in the absence of complicating fracture. They were not confined to the metaphysis of long bones. When metaphyseal, the lesions also frequently showed a tendency to involve the epiphysis. Others occurred in the diaphysis of long bones, in the pelvis, and in a rib. Three recurred locally, but none has metastasized. Other fibrohistiocytic and fibroblastic tumours of bone, including malignant fibrous histiocytoma, giant cell tumour, fibrosarcoma, and desmoplastic fibroma can be differentiated on radiological and histological features, and hyperparathyroidism may need to be excluded by biochemical investigations. PMID- 3000205 TI - Aortic intimal sarcoma with embolic metastases. AB - A 46-year-old woman died from massive bowel infarction. At autopsy, a primary sarcoma was found growing along the intimal surface of the aorta at the level of the celiac axis. Tumor emboli were found in distal aortic branches and most abdominal organs. Immunoperoxidase for Factor VIII and electron microscopy (EM) did not support an endothelial origin. EM showed myofibroblastic differentiation. Review of the literature yields an array of diagnostic histologic terms for these tumors, hampering case comparison. The literature does suggest, however, that the clinical presentation of these rare neoplasms correlates nicely with the location and gross morphology of the lesion. We therefore propose a clinicopathologic classification, categorizing the lesions as intimal (obstructive and nonobstructive) and mural. The former are typically pleomorphic sarcomas and are probably of myofibroblastic origin, whereas the latter are usually leiomyosarcomas or fibrosarcomas that probably originate in the media or adventitia. PMID- 3000207 TI - Germ cell tumors of hematologic diseases. PMID- 3000206 TI - Identification of desmosomes in the granular cell tumor. Implications in histologic diagnosis and histogenesis. AB - Electron-microscopic examination of a malignant granular cell tumor revealed cells with abundant granular and glycogen-containing cytoplasm and eccentric nuclei. Numerous junctional structures including desmosomes were identified between tumor cells which, moreover, displayed a pattern of gland formation with the presence of short microvilli in one pole of the cell. The presence of junctional structures may provide a feature for positive identification of this tumor by electron microscopy. The findings may also have implications to further our understanding of the histogenesis of this tumor. This case further raises the question of familiar occurrence of this tumor. PMID- 3000208 TI - Characteristics of breast cancer in women over 80 years of age. AB - The records of 198 women over 80 years of age with breast cancer were reviewed to identify characteristics of breast cancer and to determine the effect of age in management of the disease in elderly women. Twenty percent of the patients were not staged at diagnosis. Axillary lymph node status was undetermined in 40 percent of the patients because operation was limited to lumpectomy or mastectomy (either simple or total). Complications were related to the operative wound. Breast cancer and cardiovascular disease accounted for 115 of 146 deaths and the 5 year survival rate was 35.6 percent. We have concluded that breast cancer in elderly women can be treated with appropriate surgical therapy and should not be limited because of age. PMID- 3000209 TI - [Postcoital and postimplantation contraception]. PMID- 3000210 TI - Significance of enzyme markers as a part of multiple marker analysis in leukemia research. AB - The multidisciplinary approach of leukemia phenotyping, called multiple marker analysis, led to changes in the classification systems of normal hematopoiesis and leukemic cells, and introduced the use of a biological and functional definition of leukemia, rather than merely morphological-cytochemical descriptions. Two major conclusions can be drawn from the findings of multiple marker analysis: 1) differentiation of leukemia is not abnormal but blocked ("maturation arrest"), and leukemic cells retain normal maturation-linked markers; and 2) no leukemia specific marker could be detected so far. Although leukemic cells show general qualitative features in common with normal cells, some quantitative characteristics of these similar attributes are peculiar to leukemic blasts. Qualitative and quantitative enzymological characteristics help to identify the cell lineage involved and to determine the developmental point at which maturation arrest occurs. The expression of isoenzymes is often linked to the presumptive sequence of developmental stages. Subsets within ALL subtypes showed pronounced modifications in their isoenzyme patterns associated with increasing maturity. Thus, enzyme markers can provide refined definitions of subgroups by biochemical criteria. Based on recent observations using the enzyme markers TdT, adenosine deaminase, 5'-nucleotidase, purine nucleoside phosphorylase, acid phosphatase, and hexosaminidase, a scheme of enzymological expression in the various commonly accepted subtypes of acute lymphoid leukemia and acute nonlymphoid leukemia is presented. Enzyme marker analysis represents a useful tool as an adjunctive method in multiple marker analysis for assessing diagnosis, prognosis, and the evolutionary and pathogenetic mechanisms underlying the spectrum of leukemia subtypes. Furthermore, enzyme marker analysis may provide further insight into certain aspects of the pathobiology of leukemia which might not be elucidated by other methods.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000211 TI - Effects of cyclic GMP-agonists on cyclosporin-induced suppression of human lymphokine production. AB - Cyclosporin (Cs) inhibits the elaboration of the lymphokine leukocyte migration inhibitory factor (LIF) from human blood mononuclear cells (MNC) stimulated with recall antigen. This inhibition was counteracted by 3 X 10(-5) M dibutyryl-cyclic GMP and 8-bromo-cyclic GMP and by the cyclic GMP-agonists, sodium nitroprusside (NaNPr), ascorbic acid (As A), sodium azide (NaN3) and carbacholine. Using 5 X 10(-5) M NaNPr, 1 X 10(-3) M NaN3, or 3 X 10(-3) M As A, 25-50-, 4-8- and 2-3 fold elevations of MNC and T-lymphocyte cyclic GMP-levels were obtained independently of the presence of Cs. NaNPr was the most potent of these three cyclic GMP-agonists in counteracting the effect of Cs. The results indicate that intracellular cyclic GMP is a major factor involved in the reversal of Cs-induced inhibition of LIF-production. None of the cyclic GMP-analogues or -agonists by themselves possessed Interleukin 1-like activity, measured by their ability to induce LIF-production by macrophage-depleted T-lymphocytes challenged by recall antigen. PMID- 3000213 TI - Two-dimensional electrophoretic display of restriction fragments from genomic DNA. AB - We have developed a procedure for the resolution of restriction enzyme digests of mammalian genomic DNA in two dimensions. Fragments from a first digestion are separated on a column of purified agarose containing a second restriction enzyme in the absence of the divalent cation required for enzyme activity. After enzyme activation and digestion, the fragments are resolved on an agarose slab gel. We have digested rat genomic DNA and found in the ethidium-stained pattern a variety of features which have not been described previously. PMID- 3000212 TI - Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: purification to high specific activity by a two-step affinity chromatography procedure. AB - This report describes a two-column scheme for purifying a pyrimidine nucleoside monophosphate kinase from rat bone marrow cells. Purification was achieved by affinity chromatography on Blue Sepharose and cellulose phosphate, with selective elution of the enzyme by substrates (UMP, ATP). The enzyme preparation appeared to be about 90% pure upon polyacrylamide gel electrophoresis, exhibited an exceptionally high specific activity (greater than 600 mumol/min/mg protein), and was obtained with 30-36% recovery of enzyme activity. It was concluded that UMP, dUMP, and CMP serve as phosphate acceptors for the enzyme, based on the parallel behavior displayed by enzyme activity with these substrates both during the purification process and during other procedures. The purified enzyme preparation did not display dTMP kinase activity. This report also describes a simplified radiotracer assay for pyrimidine nucleoside monophosphate kinases. Thin-layer chromatography on polyethyleneimine-cellulose is used to resolve residual substrates and products. Because both nucleoside di- and triphosphates remain at the origin, the assay is insensitive to the action of nucleoside diphosphate kinases and does not require the use of marker compounds. A variety of radiolabeled substrates can be used with this assay, including UMP, dUMP, CMP, and dTMP. PMID- 3000214 TI - Selective adsorption of 2'-O-anthraniloyl-AMP on DEAE-Sephadex: the basis of a direct, fluorescent assay for cyclic nucleotide phosphodiesterase. AB - The fluorescent, 2'-O-anthraniloyl derivative of AMP was selectively absorbed onto DEAE-Sephadex in the presence of zirconyl chloride in citrate buffer. Under these conditions 2'-O-anthraniloyl-cAMP was eluted from the column. The selective adsorption of the AMP derivative onto DEAE-Sephadex, in the presence of zirconyl chloride, was adapted to the direct discontinuous assay of cyclic nucleotide phosphodiesterase. In this assay the enzyme is incubated for 4 min with 2'-O anthraniloyl-cAMP; after quenching of the reaction by boiling, zirconyl chloride is added and the product (2'-O-anthraniloyl-AMP) is separated from the substrate on a column (0.6 ml) of DEAE-Sephadex. 2'-O-Anthraniloyl-AMP is then eluted with NaCl (2 M) and quantitated spectrofluorometrically. Under the conditions employed, 2'-O-anthraniloyl-AMP concentrations as low as 0.1 nmol can be detected. In the present study, this assay has been used to estimate Km and Vmax values for 2'-O-anthraniloyl-cAMP hydrolysis catalyzed by highly purified, as well as crude, preparations of cyclic nucleotide phosphodiesterase from bovine brain. PMID- 3000216 TI - Amperometric assays of total and free cholesterols in serum by the combined use of immobilized cholesterol esterase and cholesterol oxidase reactors and peroxidase electrode in a flow injection system. AB - A flow injection system for assays of total cholesterol and free cholesterol was described. The total cholesterol assay system included an amperometric peroxidase electrode to measure hexacyanoferrate(III) converted from hydrogen peroxide, which was generated by injecting a 2-microliter sample into the packed-bed reactors of immobilized cholesterol esterase and cholesterol oxidase covalently bound to silica. The free cholesterol was assayed with the same system without the cholesterol esterase reactor. The peak current was linearly related to cholesterol in the range 2-160 mg/dl and to total cholesterol in the range 3-300 mg/dl; the assay speed was about 80 samples/h for free cholesterol and 40 samples/h for total cholesterol. Reliable results were obtained in the assays of free cholesterol and total cholesterol in human sera. Both the reactors and the peroxidase electrode retained over 90% of their original activities, even after repetitive use for 4 and 2 months, respectively. PMID- 3000215 TI - Ion-pair reverse-phase high-performance liquid chromatography. Application to the study of chicken liver NAD+ kinase. AB - An ion-pair, reverse-phase, high-performance liquid chromatography method of assay was developed and used in a series of rate studies carried out with the enzyme chicken liver NAD+ kinase (ATP:NAD+ 2'-phosphotransferase, EC 2.7.1.23). Complete separation of all products and reactants was achieved within 15 min. ATP, NAD+, ADP, and NADP+ were monitored at 260 nm as they eluted from a Zorbax (Dupont) ODS (4.6 X 250-mm) column using an acetonitrile and 0.01 mM NH4(H2PO4)/0.005 M tetrabutylammonium phosphate (pH 7.0) gradient. The enzyme shows a marked preference for ATP (and dATP) and Mg2+ (or Mn2+) relative to other trinucleotides and divalent metal ions. It exhibits residual adenylate kinase and ATPase activity, but no NADH kinase activity. When polyphosphate replaced ATP, NADP+ production dropped to 2.5%. The addition of Ca2+ and/or bovine brain calmodulin did not significantly enhance the rate of NADP+ production. PMID- 3000218 TI - Precipitation of phenyl and phenoxypenicillin from solutions using ammonium sulfate. AB - An easy, rapid, and available method for separating 6-aminopenicillanic acid (6 APA), benzylpenicillin (penicillin G), and other related molecules from aqueous solutions or complex industrial broths is described. A high concentration of ammonium sulphate induces partially or totally the precipitation of the penicillin present in the solutions, while 6-APA, phenylacetic, and phenoxyacetic acid always remain in the supernatant. The filtration through No. 4 Pyrex glass fiber filter or Whatman 3MM paper permits the separation of the compounds present in the supernatant from the other ones precipitated. The precipitated product was identified, in all cases, as ammonium penicillin. This method is described here for the first time. PMID- 3000217 TI - A direct radioimmunoassay for human epidermal growth factor receptor using 32P autophosphorylated receptor. AB - A simple and reproducible radioimmunoassay of the epidermal growth factor (EGF) receptor which uses 32P-labeled EGF receptor and anti-receptor monoclonal antibodies is reported. In vitro phosphorylation of A431 cell membranes with [gamma-32P]ATP in the presence of 20% dimethyl sulfoxide (which stimulates autophosphorylation of the EGF receptor) and 10 microM Na3VO4 (a potent inhibitor of phosphotyrosyl protein phosphatase) provides radiolabeled EGF receptor for radioimmunoassay without further purification. The most selective phosphorylation of the EGF receptor is achieved at ATP concentrations of 0.1-0.2 microM, which corresponds to the reported Km value for the autophosphorylation reaction of the EGF receptor (W. Weber, P.J. Bertics, and G.N. Gill, 1984, J. Biol. Chem. 259, 14631-14939). The incorporation of 32P into EGF receptors increases in proportion to the increase of ATP concentration up to 6 mol of labeled phosphate at 2.0 microM ATP. The label is entirely on tyrosine residues. The cell membranes can be stored at -70 degrees C for 3 months without loss of immunoreactivity and autophosphorylating activity. Standard curves for the radioimmunoassay were constructed employing either A431 cell membranes or whole cell homogenates containing a known amount of EGF receptor. The assay can detect 7 X 10(10) EGF receptor molecules or 20 ng of the receptor protein, and can quantitatively distinguish the difference in EGF receptor numbers between A431 cells and 29E2 and KB cells with 10-fold and 15-fold fewer receptors than A431 cells, respectively. 29E2 cells and KB cells express twofold more immunoreactive EGF receptors than EGF-binding sites. In contrast, A431 cells possess the same number of immunoreactive sites and receptor sites for EGF binding. To assess total EGF receptor expression, it is necessary to use a method which detects EGF receptors regardless of their intrinsic kinase activity, or capacity to bind EGF. This radioimmunoassay detects immunoreactive receptor molecules, even those which do not bind EGF. PMID- 3000219 TI - Enzymatic synthesis of [beta-32P]ADP using adenylate kinase and [gamma-32P]ATP. AB - An enzymatic method for the synthesis of [beta-32P]ADP from [gamma-32P]ATP is described. This substrate is required for the assay of ADPase and is not commercially available. The method described results in a preparation of [beta 32P]ADP of high purity with a yield of approximately 40% the theoretical obtainable. PMID- 3000220 TI - Graphite furnace atomic absorption spectrometry with nitric acid deproteinization for determination of manganese in human plasma. PMID- 3000221 TI - Gas-phase ionization of selected neutral analytes during thermospray liquid chromatography/mass spectrometry. PMID- 3000222 TI - An ultrastructural morphometric analysis of the adenohypophysis of lactating rats. AB - A morphometric analysis of the adenohypophysis (pars distalis) of lactating rats was carried out by a semi-automated method at the ultrastructural level. The cellular elements were identified by their ultrastructural morphology. The following values were considered for the morphometric study: numerical density of cells/mm3 of tissue and the percentage of parenchymal volume occupied by every cell type. Mammotropes (PRL cells) numbered 624 X 10(3)/mm3 and occupied 59.9% of the parenchymal volume (p.v.). Somatotropes (GH cells) numbered 206 X 10(3)/mm3 and occupied 15.0% of the p.v. Folliculo-stellate cells (FS cells) numbered 128 X 10(3)/mm3 and occupied 8.1% of the p.v. Gonadotropes (GN cells) numbered 47 X 10(3)/mm3 and occupied 6.0% of the p.v. Adrenocorticotropes (ACTH cells) numbered 45 X 10(3)/mm3 and occupied 3.8% of the p.v. Thyrotropes (TSH cells) numbered 36 X 10(3)/mm3 and occupied 3.4% of the p.v. PRL cells were characterized by aspects compatible with intense hormone production. GH cells did not show differences with those of nonlactating animals. Folliculo-stellate elements appeared hypertrophic with abundant cytoplasm, enlarged Golgi complex, and dilation of the follicular lumina. GN cells had abundant cytoplasm with a well-developed and dilated ergastoplasm, particularly in type II GN cells. ACTH cells did not show differences with those of nonlactating animals. TSH cells showed moderate nucleocytoplasmic activation. These fine structural morphometric findings are discussed in relation to other studies regarding nonlactating adenohypophysis and hormone changes during lactation. PMID- 3000224 TI - Functional units in rainbow trout (Salmo gairdneri) liver: I. Arrangement and histochemical properties of hepatocytes. AB - The architectural arrangement and selected histochemical properties of hepatocytes in the rainbow trout (Salmo gairdneri Richardson) were examined. Light and transmission electron microscopic (TEM) examination following fixation by portal venous perfusion revealed a tubular arrangement of hepatocytes. Lobules, as defined in the adult mammal, were absent. Biliary epithelial cells associated with bile preductules and ductules were a prominent feature of trout liver. Patterns and location of reaction products for glucose-6-phosphatase (G-6 Pase), glucose-6-phosphate dehydrogenase (G-6-PDH), and magnesium-dependent adenosine triphosphatase (ATPase), enzymes preferentially distributed in mammalian liver, were demonstrated in trout liver. A slightly heavier staining pattern for G-6-Pase was seen around presumptive portal venules but all other enzyme reaction patterns were uniform throughout the liver parenchyma. Following ATPase localization, four sizes of biliary passageways (canaliculi, bile preductules, ductules, and ducts) were visualized. Maximum glycogen retention was achieved with freeze-drying and glycolmethacrylate embedding and with this method intense, uniform glycogen staining was observed in all areas of the liver. Companion TEM examinations revealed large depots of glycogen within hepatocytes. The results are important for interpretation and description of the effects of toxic/carcinogenic alteration on trout liver. PMID- 3000223 TI - A functional and structural heterogeneity is formed among fetal rat hepatocytes during culture. AB - Fetal rat hepatocytes (22-day-old, full-term) in vivo were homogeneous in ultrastructure and glucose 6-phosphatase (G6Pase) distribution throughout the liver acinus. These cells were isolated and cultured for 17 days in Williams medium E containing 10% fetal calf serum, dexamethasone, insulin, and glucagon. Heterogeneity among hepatocytes appeared progressively during culture. There were variations in numbers of binucleated cells, cytoplasmic ultrastructure (e.g., in the distribution of glycogen and the quantity of ultrastructure (e.g., in the distribution of glycogen and the quantity of mitochondria), and intensity of histochemical reactivity for G6Pase. The cells could be classified generally into two types: type I cells had some characteristics of periportal hepatocytes and type II cells those of centrolobular hepatocytes in in vivo adult liver. The results show that a functional and structural heterogeneity can be formed among fetal hepatocytes in monolayer culture without differences in supply of oxygen, nutrients, and hormones. A hidden heterogeneity might already exist among hepatocytes in full-term fetuses even though it cannot be detected ultrastructurally or cytochemically. PMID- 3000225 TI - [Glomus tumors of the fingers: clinical cases]. PMID- 3000228 TI - [Postoperative herpetic encephalitis. Value of Klapper's diagnostic method]. AB - A case of a 51 year old female with herpes encephalitis is reported. She underwent surgery for chronic pancreatitis with pseudocyst formation. On the third postoperative day, she developed a severe vesicular nasolabial eruption associated with a deep stupor. Anti-Herpes simplex viral (HSV) antibodies were found in both blood and cerebrospinal fluid. Klapper's anti-HSV antibody ratio was calculated and agreed with the hypothesis of herpes encephalitis. The patient was given nucleoside analogues. She rapidly improved and was discharged from the intensive care unit without any sequelae. The frequency of HSV infection in intensive care patients and the use of Klapper's index for the diagnosis of herpes encephalitis in these patients are then discussed. PMID- 3000226 TI - Viral contamination of intradermal skin test syringes. AB - Intradermal skin tests are often performed using a common syringe with multiple needles. Bacterial contamination of intradermal skin test syringes can occur as a result of apparent siphoning caused by needle changing. The bacterial contamination of the syringe can be prevented by flushing the contaminated needle prior to changing. In this study, two different needle changing techniques were examined using a polio virus contaminant. Viral contamination of the syringe was not prevented by flushing the infected needle prior to removal. All syringes were contaminated with virus regardless of needle changing technique. We, therefore, cannot recommend the continued use of a common syringe for intradermal skin tests between patients regardless of needle changing technique. PMID- 3000230 TI - Detection of viral antigens in bluetongue virus-infected ovine tissues, using the peroxidase-antiperoxidase technique. AB - An indirect immunoperoxidase procedure was developed to detect viral antigens in bluetongue virus (BTV)-infected tissues. Embryonating chicken eggs were infected with BTV serotypes 10, 11, 13, or 17, and the chorioallantoic membranes were subsequently fixed in formalin and embedded in paraffin. The peroxidase antiperoxidase (PAP) system was used to examine the infected membranes for the presence of viral antigens. Sheep antisera raised against BTV serotypes 10, 11, 13, and 17 served as the primary antibodies in the PAP procedure. Specific staining was observed when each of these antisera was applied to membranes expressing antigens of homologous and heterologous BTV serotypes. The PAP method was rapid, reliable, and specific in its detection of BTV. PMID- 3000227 TI - [Isoenzymes of blast cells in malignant hemopathies: present state and prospectives]. AB - Numerous isoenzymes are used as markers in the course of malignant haemopathies. These are notably the isoenzymes of lactic dehydrogenase, hexosaminidase, esterases, acid phosphatases, and thymidine kinase. Their study already permits a finer and more rigorous classification of leukaemias and makes it possible to entertain serious hopes in at least three spheres: a better choice of treatment, surveillance of therapeutic efficacy and of remissions, and the development of new modes of therapeutic action by means of selective inhibitors. PMID- 3000229 TI - Effects of multiple intramuscular injections and doses of dexamethasone on plasma cortisol concentrations and adrenal responses to ACTH in horses. AB - Adrenocortical function was assessed in horses given multiple IM doses of dexamethasone to determine the duration of adrenocortical suppression and insufficiency caused by 2 commonly used dosages of dexamethasone (0.044 and 0.088 mg/kg of body weight). Dexamethasone was administered at 5-day intervals for a total of 6 injections. Daily blood samples were collected. The plasma was frozen and later assayed for cortisol. An ACTH response test was determined 2 days before the first injection of dexamethasone and again 8 days after the last dexamethasone injection. Maximum suppression of plasma cortisol was observed in horses given both dosages of dexamethasone (0.044 and 0.088 mg/kg). Plasma cortisol concentrations returned to base-line values in all horses by 4 days after dexamethasone injection. Normal ACTH responses observed after 6 dexamethasone injections given at 5-day intervals indicated that measurable adrenal atrophy did not develop under the conditions of this study. PMID- 3000231 TI - Erythrogram and red cell distribution width of Equidae with experimentally induced anemia. AB - The erythrogram (erythrocyte histogram) and red cell distribution width (RDW) were evaluated in 5 purebred horses and 1 pony of mixed breeding with experimentally induced anemia. Four horses were studied for 6 weeks after 20% of their estimated blood volume was removed on each of 2 consecutive days (40% total blood loss; acute blood-loss group). Two horses were given acetylphenyl hydrazine IV daily, until acute Heinz body hemolytic anemia was induced; the 2 horses were then evaluated for 6 weeks. One horse and the pony had 20% of their estimated blood volume removed via phlebotomy once each week for 8 weeks to induce iron deficiency anemia (chronic blood-loss group); the horse had been partially depleted of iron before the study began. Weekly blood samples were examined for changes in the erythrogram, RDW, mean cell volume (MCV), and erythrocyte glucose 6-phosphate dehydrogenase activity. Fourteen days after acute blood loss, mild increases were seen in the MCV, which persisted to day 42. The RDW was increased at day 14 and remained increased until day 42; however, the percentage increase was double that of the MCV at days 14, 21, and 28. Erythrograms had mild extensions of the right slope at days 14 to 28. Mean erythrocyte glucose-6 phosphate dehydrogenase activity increased in all 3 groups, but individual concentrations were erratic. In the 2 horses with acute hemolytic anemia, modest increases of similar magnitude were seen in RDW and MCV.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000232 TI - Frequency of persistent bovine viral diarrhea virus infection in selected cattle herds. AB - Sera and blood buffy coat samples were obtained from 3,157 cattle in 66 selected herds. Antibodies to bovine viral diarrhea (BVD) virus were detected in 89% of the serum samples by immunoprecipitation or virus-neutralization tests. Cytopathic or noncytopathic BVD viruses were isolated from blood buffy coat samples from 60 cattle in 6 herds. A second blood buffy coat sample was obtained from 54 of the 60 cattle 2 months after the initial sampling, and BVD virus was isolated again from each cow. The 54 cattle were considered persistently infected with BVD virus. The frequency of persistent infection was 1.7%. PMID- 3000233 TI - The effect of procaterol treatment on beta-adrenergic bronchodilation and polymorphonuclear leukocyte responsiveness. AB - Procaterol is a new and effective beta-adrenergic bronchodilator. To determine if procaterol administration could cause tachyphylaxis, airway and leukocyte beta adrenergic function were monitored in 10 patients with asthma during two 4-wk, double-blind treatment periods, each preceded by a 2-wk beta-agonist washout. Treatment periods were randomized to placebo or procaterol (2 wk, 0.1 mg/day; 2 wk, 0.2 mg/day). At each 7 biweekly evaluations, the patient's cumulative bronchodilator dose-response to inhaled isoproterenol (0.1 to 0.64%) was measured, and venous blood was collected to quantitate, in vitro, the polymorphonuclear leukocyte (PMN) beta-adrenergic receptor's 125Iodo cyanopindolol (125I-CYP) ligand binding and the PMN cyclic AMP response to isoproterenol and procaterol. Neither the airway nor leukocyte beta-adrenergic characteristics were changed during placebo treatment. Procaterol treatment reduced (p less than 0.05) the maximal 125I-CYP binding to PMN membranes but only during the initial 2 wk at low dosage. The percent PMN cyclic AMP increase to procaterol (10(-5) M) was also significantly (p less than 0.05) less during active treatment (141 +/- 40%) than during washout (256 +/- 24%) or placebo (257 +/- 32%). In contrast, procaterol treatment did not alter the acute isoproterenol bronchodilation response as measured by either the percent improvement in FEV1 or the dose required to produce 50% maximal bronchodilation. The duration of bronchodilation was not measured. Therefore, although procaterol therapy of asthma is associated with decreased PMN beta-adrenergic function, airway smooth muscle function appears not to be altered. PMID- 3000234 TI - The effects of a fish-oil-enriched diet on pulmonary mechanics during anaphylaxis. AB - The pulmonary mechanical responses observed after antigen challenge in 2 groups of sensitized, mepyramine-treated, mechanically ventilated guinea pigs were compared: one group was fed a diet rich in fish oil and the other a control diet enriched with beef tallow. The lung tissue of animals fed a fish-oil-enriched diet for 9 to 10 wk incorporated eicosapentaenoic acid (EPA) and docosahexaenoic acid, which constituted 8 to 9% of the total fatty acid content, whereas these alternative fatty acids constituted less than 1% of total fatty acid content of the lung tissue of animals receiving a diet supplemented with beef tallow. With mepyramine pretreatment, animals receiving a fish oil diet exhibited a significantly greater decrease in dynamic compliance from 1.5 through 4.5 min after antigen challenge than did animals receiving a beef fat diet, whereas the decrements in pulmonary conductance were comparable. The combination of indomethacin and mepyramine markedly augmented the antigen-induced decrease in pulmonary mechanics in animals receiving a beef fat diet but not in those receiving a fish oil diet, such that the overall responses of the 2 groups were similar. These findings indicate that the fish oil diet and the indomethacin pretreatment of animals receiving the beef fat diet each facilitates the nonhistamine-mediated bronchoconstrictor response in pulmonary anaphylaxis. PMID- 3000236 TI - The lipid interstitial cell of the pulmonary alveolus. Age and species differences. AB - We have previously shown that alveoli of neonatal rat lungs contain 2 morphologically distinct fibroblasts; 1 that contains lipid droplets and 1 that does not. We have named these lipid interstitial cells (LIC) and nonlipid interstitial cells (NLIC). In this study, using stereologic methods, we evaluated the distribution of LIC and NLIC in the alveoli of immature and mature rats, hamsters, and mice and quantitated cytoplasmic lipid in the LIC. We found that fibroblasts account for approximately 50% of resident alveolar wall cells in immature and 20% in mature rodent lungs. At each age LIC accounted for half of the fibroblasts. There were age and species differences in the amount of LIC lipid. The volume density of cytoplasmic lipid was higher in immature rats (0.285) than in mice (0.205) or hamsters (0.010) and was higher in mature mice (0.176) than in hamsters (0.051) or rats (0.019). Possible explanations for these differences are discussed. Bundles of characteristic cytoplasmic myofilaments established that the LIC was identical to the previously described contractile interstitial cell of the lung. These studies confirm the presence of 2 distinct fibroblasts in the alveoli of immature and mature rodents of several species. PMID- 3000237 TI - Renal subcapsular islet cell transplantation. AB - This study was directed towards improving islet cell allotransplantation (ICTx) by testing the renal subcapsular region (RSC) as an alternative site for graft placement. In addition, a simplified, non-collagenase method was assessed for preparation of the allograft from the donor pancreas. Five groups of pancreatectomized mongrel dogs were followed. Group I (n = 10) were not transplanted and survived 5.0 +/- 2.92 days (M +/- SD). Five of six animals in Group II (n = 6) which received an ICTx prepared without collagenase in the RSC survived until allograft removal by nephrectomy at greater than 90 days (P less than .0005). No immunosuppression was given to this group. Recipients in Group III (n = 11) were transplanted as in Group II, but were given minimal immunosuppression with azathioprine. They also demonstrated excellent graft function until removal of the graft by nephrectomy at between 3 weeks and greater than 90 days. Animals in Group IV (n = 6) received an intrasplenic (IS) ICTx prepared without collagenase and survived only 4.16 +/- 1.16 days. In Group V (n = 6) poor survival was also noted (7.66 +/- 4.58 days) after IS-ICTx of a collagenase prepared allograft. All animals in Group IV and V received minimal immunosuppression as in Group III. These results indicate the potential for utilization of the RSC as an alternative site for ICTx. In addition, the collagenase-free method was satisfactory for ICTx preparation. PMID- 3000235 TI - Neutral endopeptidase in serum samples from patients with adult respiratory distress syndrome. Comparison with angiotensin-converting enzyme. AB - The activities of 2 peptidases, angiotensin-I converting enzyme (ACE) and neutral metalloendopeptidase (NEP), were measured in serum from patients with adult respiratory distress syndrome (ARDS). As noted by others, we found that the specific activity of serum ACE was reduced in patients with severe alveolocapillary damage caused by ARDS. In addition, patients who were severely ill with chronic obstructive lung disease had lower serum ACE than did normal ambulatory control subjects. Patients with cardiogenic pulmonary edema, however, had no consistent loss of the enzyme. The most striking changes occurred in the serum levels of NEP. Whereas the specific activity of this enzyme was very low in the normal subjects, it was elevated as much as 50- to 60-fold in serum samples from patients with ARDS. Serum from patients with cardiogenic pulmonary edema had high levels of NEP, and it was elevated also in a subset of patients with chronic obstructive lung disease. On the assumption that patients with ARDS sustain significant damage at the alveolar-capillary level, these changes in ACE and NEP could signal endothelial damage; ACE loss could result from direct injury to the vascular lumen, and NEP from subendothelial tissues could enter the bloodstream through damaged endothelium. Alternatively, NEP might be released from leukocytes sequestered in the lung and leak into the bloodstream. PMID- 3000238 TI - [AIDS in children]. PMID- 3000240 TI - Transmission of the acquired immunodeficiency syndrome through heterosexual activity. PMID- 3000241 TI - Human fibroblast interferon and acyclovir in cytomegalovirus pneumonia. PMID- 3000239 TI - [Fetal rhabdomyomatous nephroblastoma: study of 2 cases and review of the literature]. AB - Report of two cases of Fetal Rhabdomyomatous Nephroblastoma, a cytodifferentiated variant of Wilms' tumor. The incidence of this rare variant is of 2.10% in our material (88 nephroblastomas in a sixteen years period). The patients were ten months and two years old. Bilateral tumors were discovered in both patients. Preoperative radiotherapy was given but no reduction of the tumors size was obtained. Microscopically, benign looking striated muscle is the predominant component of the tumors, with scare foci of epithelial elements. The low age of presentation, high incidence of bilaterality and the peculiar intrapelvic renal growth are three relevant characteristics in the 35 cases previously reported as well as in these two new cases, that distinguish Fetal Rhabdomyomatous Nephroblastoma from conventional Wilms' tumor. PMID- 3000243 TI - Acute myelofibrosis and infection with the lymphadenopathy-associated virus/human T-lymphotropic virus type III. PMID- 3000242 TI - Possible genetic susceptibility to the acquired immunodeficiency syndrome in hemophiliacs. PMID- 3000244 TI - Lymphadenopathy-associated virus/human T-lymphotropic virus type III in allogeneic bone marrow transplantation. PMID- 3000246 TI - Retinal lesions in cytomegalovirus infection. PMID- 3000245 TI - Antibodies to human T-cell leukemia virus types I and III in blood donors from Calabar, Nigeria. PMID- 3000247 TI - Recommendations for viral hepatitis. PMID- 3000248 TI - Effect of 9-(1,3-dihydroxy-2-propoxymethyl) guanine on serious cytomegalovirus disease in eight immunosuppressed homosexual men. AB - Eight immunosuppressed homosexual men with cytomegalovirus viremia--seven with serious bilateral retinitis, one with colitis in addition to retinitis, and one with pneumonitis only--were treated with a new acyclovir derivative, 9-(1,3 dihydroxy-2-propoxymethyl) guanine, which has excellent in-vitro activity against cytomegalovirus. All patients had virologic and clinical improvement, but substantial leukopenia developed in three patients. Both clinical relapses and viral relapses occurred frequently, usually within 30 days after cessation of treatment. 9-(1,3-Dihydroxy-2-propoxymethyl) guanine represents the first clinically and virologically effective agent for the treatment of cytomegalovirus disease, but more effective and less toxic therapeutic regimens for both acute and chronic use must be developed. PMID- 3000249 TI - Antibodies to human T-lymphotropic virus type III and development of the acquired immunodeficiency syndrome in homosexual men presenting with immune thrombocytopenia. AB - In 35 homosexual men with isolated thrombocytopenia at initial presentation, who were evaluated and treated between 1982 and 1984, hematologic studies showed immune destruction. In contrast to findings in other autoimmune conditions, T lymphocyte subsets in these patients were reversed, with a mean helper to suppressor ratio of 0.4 and with an absolute depletion of helper cells to 390/mm3. An enzyme-linked immunosorbent assay detected antibodies to human T lymphotropic virus type III in 21 of 25 patients tested; Western blot analysis confirmed seropositivity in the other 4 patients, who had borderline findings. Although 19 of 24 patients treated with steroids responded, only 2 achieved sustained normal platelet counts. Ten of fifteen patients who had splenectomy achieved remissions. Three patients treated with steroids or splenectomy developed diagnoses compatible with the acquired immunodeficiency syndrome 16 to 34 months after their initial presentation with thrombocytopenia. These findings indicate that immune thrombocytopenia is part of the clinical spectrum of the acquired immunodeficiency syndrome. PMID- 3000250 TI - Acalculous cholecystitis and cytomegalovirus infection in the acquired immunodeficiency syndrome. PMID- 3000251 TI - [Vitamin D: metabolism and biological properties]. PMID- 3000252 TI - [Vitamin D and its therapeutic use]. PMID- 3000253 TI - Purine metabolism in leukemia. PMID- 3000255 TI - Regulation of mononuclear leukocyte function by transmethylation reactions. PMID- 3000254 TI - Ectoenzyme action on purine nucleotides in macrophages and subsequent reactions. PMID- 3000257 TI - Role of adenosine deaminase in human monocyte differentiation and tumor cell cytotoxicity. PMID- 3000258 TI - Purine catabolism as a source of superoxide in macrophages. PMID- 3000256 TI - Roles of alternative synthetic and catabolic purine pathways in T lymphocyte differentiation. PMID- 3000259 TI - Adenosine modulates the generation of superoxide anion by stimulated human neutrophils via interaction with a specific cell surface receptor. PMID- 3000260 TI - Human placental nucleoside kinase activities. AB - Our studies have indicated that normal human placental cytosol contains a complex mixture of nucleoside kinase enzymes, some of which conform to previously characterized activities. Deoxyadenosine is phosphorylated by deoxycytidine kinase, adenosine kinase, and two as yet uncharacterized activities. Deoxyguanosine phosphorylation is associated with deoxycytidine kinase. More complete and detailed studies will be necessary to characterize these enzymes fully. PMID- 3000261 TI - Genetic engineering and site-specific mutagenesis. Strategy of interplay. PMID- 3000262 TI - The photobiology of vitamin D and its consequences for humans. PMID- 3000264 TI - The industrial use of solvents and risk of neurotoxicity. PMID- 3000263 TI - Lipoproteins of special significance in atherosclerosis. Insights provided by studies of type III hyperlipoproteinemia. AB - In summary, the study of type III hyperlipoproteinemia has provided important insights into lipoprotein metabolism that have helped to elucidate several functional roles for apo E and have provided a better understanding of the mechanisms whereby specific lipoproteins may be atherogenic or anti-atherogenic. The molecular defect in type III hyperlipoproteinemia and dysbetalipoproteinemia is the presence of a mutant form of apo E, usually apo E2, that is defective in binding to both apo B,E(LDL) and apo E receptors. The receptor-defective apo E results in an impaired clearance of remnant lipoproteins (beta-VLDL). In addition, the abnormal apo E may impair the lipolytic processing of hepatic beta VLDL through its involvement in lipid transfer or exchange processes. The accumulation of beta-VLDL may provide the most direct mechanism responsible for the accelerated atherosclerosis observed in type III hyperlipoproteinemia, a mechanism that involves the receptor mediated uptake of beta-VLDL by macrophages, which are then converted to arterial foam cells. Alterations in the HDL of patients with type III hyperlipoproteinemia further support the concept that HDL are anti-atherogenic. The increase in HDL-with apo E provides insight into the role of these cholesterol-enriched HDL in reverse cholesterol transport and in the cellular redistribution of cholesterol, processes whereby cholesterol deposition may be reversed. It should be stressed that both the accumulation of beta-VLDL and alterations in HDL (reduction in typical HDL and an increase in HDL with apo E) are associated with accelerated atherogenesis in animals fed high levels of fat and cholesterol. Although valuable information has been gained concerning the mechanisms involved in type III hyperlipoproteinemia by the study of the disease, the clinical expression of this disorder is variable, ranging from hypocholesterolemia to marked hypercholesterolemia in subjects with the same molecular defect (E2/2). This variability in expression is more easily understood when one considers the various factors that can promote the hyperlipoproteinemia and when one considers the mechanisms of action whereby these factors may exacerbate the effects of the presence of an abnormal apo E. In most cases, development of type III hyperlipoproteinemia requires that a second event (a predisposing environmental factor or a second genetic defect) be associated with the primary genetic defect (an abnormal form of apo E). PMID- 3000265 TI - A comparison of the analyses of respirable quartz by infra-red spectrophotometry at HSE field and headquarters laboratories. PMID- 3000266 TI - Neuroendocrine markers in paragangliomas of the head and neck. AB - Eighteen paragangliomas of the head and neck (11 carotid body tumors, four glomus tympanicum tumors, three glomus jugulare tumors) were studied retrospectively. Tissue from each of these tumors was immunostained for the presence of serotonin, a variety of neuropeptide hormones, and the enzyme neuron-specific enolase (NSE). Seven tumors were studied by electron microscopy. The clinical and laboratory records were reviewed for evidence of endocrine activity or metabolic imbalance. All tumors displayed diffuse and intense immunostaining for NSE. In addition, a wide variety of hormonal substances could be identified. Those most frequently demonstrated were serotonin and leu-enkephalin. Ten of the 11 carotid body tumors demonstrated immunoreactivity for multiple hormones. By electron microscopy all tumors contained a heterogeneous population of membrane-bound neurosecretory granules. None of these tumors was associated with a clinically apparent endocrine syndrome. We conclude that paragangliomas of the head and neck are neuroendocrine tumors that are capable of synthesizing a variety of hormonal substances. These hormonal substances rarely elicit a clinically apparent endocrine or metabolic imbalance. All of the tumors demonstrated immunostaining for NSE. Future studies on serum levels of NSE may provide useful diagnostic and follow-up data. PMID- 3000268 TI - A simple colorimetric method for spot blood glucose estimation. PMID- 3000267 TI - Is it possible to diagnose pituitary-dependent Cushing's disease? PMID- 3000269 TI - Small cell carcinoma of lung: a prospective clinical study. AB - Seventeen patients with small cell carcinoma of the lung diagnosed on sputum cytology, bronchial biopsy/aspirate or lymph node biopsy, were prospectively followed up for 33 months. Four patients who had no or inadequate treatment survived an average of 10.5 months. Ten treated patients survived 10.6 months. Three patients are still alive receiving chemotherapy with no local irradiation. All patients were Chinese; all smoked cigarettes; two patients were women; all patients were older than 50 years. Four patients had no chest complaints but presented with Superior Vena Caval obstruction (two cases), dermatomyositis and the Eaton Lambert syndrome. Two patients had the syndrome of inappropriate antidiuretic hormone secretion. None was hypercalcaemic. Twelve patients had right sided lung lesions. The majority of patients underwent combined radiotherapy and chemotherapy, the latter consisting of three weekly cyclical methotrexate, adriamycin, cyclophosphamide and CCNU (MACC regime). PMID- 3000270 TI - New perspectives in therapy of small cell lung cancer. AB - Small cell lung cancer (SCLC) displays many unique features which are not present in the other subtypes of lung cancer. The clinical presentation with its unique paraneoplastic syndrome and early metastatic spread make SCLC a protean diagnostic problem. Until recently, SCLC was considered to have the worst prognosis in lung cancer. However the natural history of this cancer has been altered substantially over the past ten years with the application of combined modality therapy using combination chemotherapy and radiation therapy. Numerous clinical trials have demonstrated the four to five fold improvement in survival for patients with limited disease treated with combination chemotherapy and radiation therapy. There are several active chemotherapeutic agents such as cyclophosphamide, doxorubicin, etoposide, vincristine, methotrexate and cisplatinum which are currently used in various combination regimens. The role of radiation therapy is undergoing changes with an improvement in results when used in combination with chemotherapy. It no longer plays a primary role but remains a valuable adjunct in the management of small cell lung cancer. There is a diminishing role for surgery in this type of lung cancer. The therapeutic progress in SCLC has reached a plateau in the past four to five years. However much progress has been made in understanding the biology of SCLC. Armed with a better understanding of the biology of SCLC, we can hopefully formulate better treatment regimens and ultimately further improve therapeutic results. PMID- 3000271 TI - The role of leukotrienes in asthma. AB - Recent advances in biochemistry and cell biology have allowed the determination of the structure and biosynthetic pathways of the leukotriene constituents of slow-reacting substance of anaphylaxis. The sulphidopeptide leukotrienes have potent biological actions including effects on smooth muscle, mucus secretion and vascular permeability whereas leukotriene B3 is a powerful chemoattractant for neutrophils. It seems likely that the biological activities of the leukotrienes make a substantial contribution to the pathogenesis of asthma and that research directed towards the development of antagonists or inhibitors of leukotriene synthesis may lead to a major therapeutic advance. PMID- 3000273 TI - Differences in methylation on the active and inactive human X chromosomes. AB - Methylation of CCGG sites was examined in four regions of the X chromosome with four X-chromosome clones, three obtained by cloning random segments and one encoding a structural gene. In DNA from human peripheral blood cells unmethylated sites correlating with the inactive X chromosome were detected in the vicinity of two of the random clones and also in the vicinity of a cloned sequence of the X linked phosphoglycerate kinase gene (PGK). The third random clone covered a region whose methylation pattern was unchanged between the active and inactive X chromosomes. Differential methylation at the sites detected appears to have no functional role in the maintenance of the inactive X chromosome since both active and inactive X chromosomes were found to be undermethylated in DNA from human lymphoblastoid cells. PMID- 3000272 TI - Human myosin heavy chain genes assigned to chromosome 17 using a human cDNA clone as probe. AB - A cDNA clone complementary to the mRNA encoding human myosin heavy chain has been isolated from a human fetal skeletal muscle cDNA library. A 600 base pair fragment of the inserted human cDNA has been used as probe in the Southern analysis of DNA from panels of rat/human and mouse/human somatic cell hybrids. All the sequences detected by this probe have been mapped to chromosome 17 in the region 17pter----17p11. There was no evidence for MHC sequences on any other chromosome. PMID- 3000274 TI - Linkage between the loci for peptidase D and apolipoprotein CII on chromosome 19. AB - Families segregating for PEPD were investigated for linkage between PEPD and APOC2. The results provide evidence for close linkage between PEPD and APOC2 in males. PMID- 3000275 TI - The structural gene for aldolase B (ALDB) maps to 9q13----32. AB - We used a cloned cDNA probe for the B subunit of human aldolase (ALDB) and Southern blotting techniques to analyse DNA from a series of rodent X human somatic cell hybrids for the presence of specific ALDB-related sequences. Our results provide evidence for the assignment of the gene for ALDB to chromosome 9. Moreover, by direct gene dosage determination in two patients with chromosome 9 unbalanced rearrangements and by in situ hybridization we refined the regional chromosomal assignment to 9q13----q32 and most probably to 9q21.3----9q22.2. PMID- 3000277 TI - A cytochrome P-450 gene family mapped to human chromosome 19. AB - We have recently isolated a cloned cDNA coding for a cytochrome P-450 of human liver microsomal membranes, which corresponds to a major phenobarbital-inducible cytochrome P-450 of rat liver. This human cytochrome P-450 is encoded by a member of a multigene family. DNA extracted from a panel of 12 independent human-rodent somatic cell hybrids was analysed by Southern blot hybridization with the cloned cDNA. The results indicate that all components of this cytochrome P-450 gene family are located on chromosome 19. Evidence from hybrids derived from an individual carrying a balanced translocation suggests a regional localization of 19p13.2----qter. Analysis of human metaphase chromosomes by in situ hybridization localizes this cytochrome P-450 gene family further to the long arm of chromosome 19 in the region q13.1----qter. We propose the designation P450PB for this locus. PMID- 3000276 TI - Isolation of a cDNA clone for the human muscle specific carbonic anhydrase, CAIII. AB - The molecular cloning of cDNA for the human muscle specific carbonic anhydrase CAIII is described. The recombinant was isolated from a human muscle cDNA library prepared in the expression vector lambda gt11, and was characterized by hybridization selection and immunoprecipitation. A comparison of insert cDNA and mRNA sizes suggests that the cDNA is full length and includes extensive untranslated sequences. Preliminary sequence data have confirmed the authenticity of this clone and Southern blotting of human and rodent DNA indicates that it will be a useful probe in the analysis of somatic cell hybrids. PMID- 3000278 TI - Acute improvement in exacerbating multiple sclerosis produced by intravenous administration of mannitol. AB - The mode of action of adrenocorticotropic hormone (ACTH) treatment in exacerbating multiple sclerosis was studied by short-term infusions of agents that mimic specific and limited pharmacological actions of ACTH and observing for temporally phase-locked clinical changes. The study was double blinded, and agents were administered while the patients were being treated with a standard course of 10-day intramuscular ACTH therapy (40 U twice daily). Antiedema, alkalotic-hypocalcemic, extraadrenal, and sodium-retaining actions were studied using infusions of mannitol, sodium bicarbonate, ACTH, and sodium chloride, respectively. Seven of 8 patients receiving placebo infusions (2.5% glucose) showed no significant clinical change and 1 exhibited an equivocal improvement. Five of 9 patients receiving mannitol showed definite signs of clinical improvement phase-locked to drug administration, with subsequent gradual reversal to baseline. Similar improvements occurred with infusions of NaHCO3 in 5 of 8 patients and of ACTH in 4 of 8 patients. Three of 7 patients given NaCl infusion showed possible mild improvements. The results indicate that mannitol and NaHCO3 induced transient acute improvement in signs at the 95% confidence level in patients with exacerbating multiple sclerosis, with ACTH having a similar effect at the 90% confidence level. These agents mimic some of the known effects of ACTH, which may be important in the therapeutic action of ACTH in multiple sclerosis. A possible role for mannitol and high-dose ACTH in the treatment of demyelinating disease warrants further study. PMID- 3000279 TI - Progressive multifocal leukoencephalopathy: investigation of three cases using in situ hybridization with JC virus biotinylated DNA probe. AB - Using the technique of in situ DNA-to-DNA hybridization, a JC virus biotinylated DNA probe was developed and applied to formalin-fixed, paraffin-embedded, or fixed, frozen sections of brain tissue from three subjects with progressive multifocal leukoencephalopathy (PML). Light microscopy was carried out to correlate the presence of JC virus DNA with the selective infection of oligodendrocytes and astrocytes in PML. Oligodendrocytes (lytically infected) showed the greatest evidence of viral DNA. More astrocytes showing bizarre morphological changes had evidence of viral DNA than did astrocytes that were simply reactive. Viral DNA was not evident in vascular endothelial cells using this technique. Viral DNA replication may be an important initial step which produces the bizarre "transformed" astrocytes of PML. Findings in this study do not support the hypothesis that vascular endothelial replication is important in the pathogenesis of JC virus-induced PML. In situ hybridization with biotinylated JC virus probe may be useful in the diagnosis of PML on brain biopsy specimens. PMID- 3000280 TI - Dopaminergic and cholinergic lesions in progressive supranuclear palsy. AB - In 9 patients with progressive supranuclear palsy and in 27 controls, dopamine and homovanillic acid concentrations, choline acetyltransferase (CAT) activity, and the number of [3H]spiperone and [3H]quinuclidinyl benzilate binding sites were measured post mortem in the striatum (caudate nucleus, putamen, and nucleus accumbens), substantia innominata, and frontal cortex. Dopamine and homovanillic acid concentrations were reduced in the caudate nucleus and putamen but not in the nucleus accumbens or frontal cortex, indicating that the nigrostriatal dopaminergic system is lesioned in patients with progressive supranuclear palsy (as in those with Parkinson's disease) but not the mesocortical and mesolimbic dopaminergic systems, which are lesioned in parkinsonian patients. CAT activity and [3H]spiperone binding decreased in parallel fashion in all the structures. In the striatum, this suggests that the cholinergic neurons, which are target cells of the nigrostriatal system, also degenerate in this disease. This might explain the decrease in the number of dopamine receptors as well as the inefficacy of levodopa or anticholinergic therapy in these patients. The decrease in CAT activity in the substantia innominata and the frontal cortex indicates that the innominatocortical cholinergic system is lesioned in patients with progressive supranuclear palsy and may play a role in the intellectual deterioration observed. This lesion is also found in demented patients with Alzheimer's and Parkinson's diseases. PMID- 3000281 TI - Neurological complications in infants and children with acquired immune deficiency syndrome. AB - Neurological complications occurred in 6 children, aged 6 months to 5 years, with acquired immune deficiency syndrome who were followed for 14 months. The most frequent manifestations included encephalopathies, acquired microcephaly, and pyramidal tract signs. Computed tomographic examinations showed variable degrees of cortical atrophy with ventricular dilatation and calcification. Electrophysiological abnormalities were demonstrated. Two children had documented central nervous system infections. Neurological deterioration resulted in dementia in 3 children. Cognitive impairment and developmental delays were evident in the other 3. Postmortem examination of the 3 children who died showed subacute cytomegalovirus encephalitis in 1; nonspecific hemispheric white matter changes, calcific vasopathy of the basal ganglia, and striking bilateral corticospinal tract degeneration in the second; and extensive calcific vasopathy of the basal ganglia and frontal centrum semiovale, and bilateral attenuation of the frontopontine and corticospinal tracts in the third. PMID- 3000283 TI - Corticospinal tract conduction time in multiple sclerosis. AB - Anodal shocks of 400 to 700 V from a low-output impedance stimulator applied percutaneously over the motor cortex evoke muscle action potentials in partially voluntarily activated contralateral muscles. Cathodal shocks from the same device applied to the cervical spinal cord produce maximal ipsilateral muscle action potentials in a relaxed limb. This technique was used to study the central motor pathway in 15 healthy subjects and 8 patients with clinically definite multiple sclerosis. As stimuli were applied in the axilla, over the C7 vertebral level, and over the arm area of the motor cortex, recordings were made of muscle action potentials of forearm flexor muscles. In controls, cord-to-axilla conduction time was 4.1 +/- 0.61 ms, and cortex-to-cord time was 4.4 +/- 0.75 ms. In patients, cord-to-axilla conduction times were normal, while central conduction times were either markedly prolonged (6.4 to 31 ms) or absent. This technique is a potentially powerful tool for the investigation of central motor pathways in healthy subjects and patients with neurological disease. PMID- 3000284 TI - Clinical neurophysiology of conduction in central motor pathways. PMID- 3000282 TI - Japanese encephalitis: immunocytochemical studies of viral antigen and inflammatory cells in fatal cases. AB - The distribution of virus and the composition of the mononuclear inflammatory response were studied in the brains of 7 children who died with Japanese encephalitis. Viral antigen was localized to neurons, with greatest involvement in the thalamus and brainstem. Quantitation of perivascular inflammatory responses showed a preponderance of T cells, but only 7 to 30% of these cells were T suppressor/cytotoxic cells. Inflammatory cells invading the parenchyma were predominantly macrophages with small numbers of T cells. B cells remained localized to perivascular cuffs. Viral antigen was progressively cleared in patients with survival of 6 days or more. PMID- 3000285 TI - Recovery of herpesviruses from cerebrospinal fluid of immunodeficient homosexual men. AB - Over a one-year period the cerebrospinal fluid (CSF) obtained from a series of homosexual men immunocompromised with either Hodgkin's disease or acquired immune deficiency syndrome (AIDS) was cultured to assess the frequency with which infectious viruses could be recovered. Of 58 patients examined, 4 (6.9%) had CSF cultures that showed a cytopathology consistent with a virus infection. All isolates proved to be herpesviruses. Cytomegalovirus (CMV) and varicella-zoster virus were isolated from CSF obtained from 2 patients with neurological features consistent with a subacute encephalitis common among AIDS patients. CMV was also recovered from the CSF of an AIDS patient who developed an ascending myelitis of herpesvirus origin. Finally, a CSF sample obtained from an immunodeficient homosexual man who showed no detectable neurological abnormalities consistently yielded herpes simplex virus type 1 in culture. These results suggest that seeding of the CSF with infectious virus is an uncommon event in this patient population. However, our experience should not dissuade attempts to culture viruses from CSF in similar cases. Successful isolations may prove beneficial in the diagnosis of an accompanying neurological illness and facilitate treatment with antiviral therapy when indicated. PMID- 3000286 TI - Sea-blue histiocytosis associated with progressive anterior horn cell and axonal degeneration. PMID- 3000287 TI - Specificity of nerve changes in sea-blue histiocytosis. PMID- 3000288 TI - Synergistic effect of human leukocyte interferon and nonoxynol 9 against herpes simplex virus type 2. AB - The nonionic surfactant nonoxynol 9 (NP9), in combination with human alpha interferon, synergistically reduced the titer of herpes simplex virus type 2 (HSV 2) in vitro. The degree of synergy was highest at an interferon concentration of 10(3) IU/ml and an NP9 dilution of 1:1,500. We postulate that NP9 inactivates extracellular HSV-2, whereas interferon inhibits HSV-2 replication at the intracellular level. PMID- 3000289 TI - Variability of IncHI1 plasmids from Salmonella typhi with special reference to Peruvian plasmids encoding resistance to trimethoprim and other antibiotics. AB - In spite of extensive DNA homology among IncHI1 plasmids, ApaI and XbaI restriction digests of plasmids from Peruvian Salmonella typhi varied considerably from other IncHI1 plasmids isolated previously. IncHI1 plasmids appear to be undergoing a process of modular evolution, probably by sequential acquisition of resistance determinants. PMID- 3000290 TI - Effect of pyrimidinone treatment on lethal and immunosuppressive murine cytomegalovirus infection. AB - The 2-amino-5-halo-pyrimidinones, which are potent interferon inducers and antiviral agents, were found to be protective against lethal cytomegalovirus (CMV) challenge in weanling or neonatal mice when administered before virus challenge. This protection was dependent upon the dosage of pyrimidinone administered. Weanling mice infected with a sublethal challenge of CMV exhibited moderate to severe immunosuppression as measured by reduced splenic cell blastogenic responses in vitro to the mitogen concanavalin A. Treatment of mice with pyrimidinones during the course of CMV immunosuppression resulted in substantial augmentation of splenic cell blastogenic responses. The degree of augmentation appeared to be dependent on the severity of CMV-induced immunosuppression. PMID- 3000293 TI - Coyote control and taste aversion. AB - Studies in which conditioned taste aversion was used as a non-lethal method to suppress coyote predation are reviewed in light of the controversy that surrounds such research. It is concluded that the negative results obtained to date may have been due to theoretical and methodological problems in the studies. Uncritical acceptance of those results has slowed progress on an effective and inexpensive method of coyote management. PMID- 3000294 TI - Research on forms of conditioned avoidance in coyotes. PMID- 3000291 TI - Antiviral activity of 5-ethyl-2'-deoxyuridine against herpes simplex viruses in cell culture, mice, and guinea pigs. AB - The susceptibility of 3 laboratory strains and 24 clinical isolates of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) to 5-ethyl-2'-deoxyuridine was determined in plaque reduction assays in Vero cells. The median effective doses were 8.6 and 7.8 microM, respectively. The drug was less potent than acyclovir and other related antiviral drugs, but it had a high therapeutic index against both HSV-1 and HSV-2. Drug-resistant viruses were readily produced in cell culture. These variants were cross-resistant to acyclovir, 2'-fluoro-5 iodoaracytosine, and 2'-fluoro-5-methylarauracil but were susceptible to vidarabine or phosphonoformate. These findings confirm that the selective antiviral activity of 5-ethyl-2'-deoxyuridine is mediated by the virus-induced thymidine kinase. Oral or intraperitoneal administration of the drug at nontoxic doses was ineffective in protecting mice against intracerebral challenge with virus. Using implanted osmotic minipumps or coadministering the drug with dimethyl sulfoxide failed to decrease the mortality rate. In guinea pigs infected genitally with HSV-2, topical drug treatment was more effective than placebo in reducing lesion severity and other clinical and virological variables. These effects were noted whether the drug treatment was initiated 3 or 24 h after infection (ascertained serologically). Drug-treated animals had a significantly lower herpes antibody titer than did placebo-treated guinea pigs, suggesting that the drug can also reduce the viral antigen load. In this model, the drug appeared to be as effective as topical phosphonoformate or acyclovir. PMID- 3000292 TI - The fluoroquinolones: structures, mechanisms of action and resistance, and spectra of activity in vitro. PMID- 3000295 TI - Brief comments on "coyote control and taste aversion". PMID- 3000296 TI - Coyote control and taste aversion: a predation problem or a people problem? AB - Failures to suppress coyote predation on domestic livestock using the conditioned taste aversion paradigm may be due to such factors as poor livestock management procedures and overestimated coyote predation data, in addition to theoretical and methodological problems as indicated by Forthman Quick, Gustavson and Rusiniak. PMID- 3000297 TI - A comment on "coyote control and taste aversion". PMID- 3000298 TI - [The effect of lithium carbonate against leukopenia during systemic chemotherapy in patients with small cell carcinoma of the lung]. AB - To investigate whether lithium carbonate ameliorates the leukopenia and infectious complication that accompany systemic chemotherapy, we studied 19 patients with small cell carcinoma of the lung receiving combination chemotherapy. Eight patients received systemic chemotherapy and lithium carbonate and 11 patients received systemic chemotherapy alone. The mean leukocyte count nadir during chemotherapy was significantly higher in the patients of the lithium group than in the patients of the control group (p less than 0.05). Percentage of infectious complication related to leukopenia was lower in the lithium group than in the control group, although there was no significant difference between these two groups. There was almost no significant side effect except for liver dysfunction in one patient. We therefore believe that lithium carbonate is an effective and safe drug against leukopenia during cytotoxic chemotherapy. PMID- 3000299 TI - [A phase II study of etoposide (NK 171) in small cell lung cancer--comparison of results between intravenous administration and oral administration]. AB - A phase II study of Etoposide (NK 171) was carried out in 13 institutions of the National Chest Hospital Lung Cancer Cooperative Study Group. Twenty-two patients (pts.) were treated by intravenous (i.v.) administration of etoposide, 80 mg/m2/day, for 5 consecutive days, and 25 pts. by oral administration of the same drug, 130 mg/m2/day, for 5 consecutive days. Eight (36.4%) out of 22 evaluable pts. given i.v. etoposide showed partial response (PR) while 7 (28%) out of 25 evaluable pts. given oral etoposide showed PR. Thirteen (41%) out of 32 previously untreated pts. were responders, but only 2 (13%) out of 15 previously treated pts. responded. The average total dose of i.v. etoposide was 664 (368 1552) mg/m2 while that of oral etoposide was 1320 mg/m2, or about double the dose of i.v. etoposide. The major dose-limiting factor was leukopenia (less than 3000/mm3). being observed in 63.6% of the i.v. treated pts. and 31.8% of the orally-treated pts. The oral and i.v. etoposide provided equivalent results. Despite the advantage of the reduced myelotoxicity of oral etoposide, we may recommend that all pts. are treated parenterally at present until the problem of erratic absorption of the oral drug is resolved. PMID- 3000300 TI - [Clinical phase II study of 5'-DFUR for cancer of the digestive organs by a cooperative study group]. AB - Phase II study of a new 5-fluorouracil derivative, 5'-deoxy-5-fluorouridine (5' DFUR), was performed with oral administration. Forty-nine patients with advanced cancer of the digestive organs, lung and breast were entered, and 8 institutions in Hokkaido were involved. 5'-DFUR was administered three or four times a day at a daily dosage of 600 to 1200 mg. Partial response was observed in three gastric cancer cases and two breast cancer cases out of 39 evaluable cases, and minor response was observed in one colorectal cancer case. Overall response rate was 12.8%, 15.8% in gastric cancer and 66.7% in breast cancer. Side effects were observed in 15 cases out of 45 (33.3%), which mainly consisted of gastro intestinal disturbances such as diarrhea. PMID- 3000301 TI - [AIDS in the female]. PMID- 3000302 TI - [Polyneuropathy and renal carcinoma]. PMID- 3000304 TI - Electron microscopy of Leishmania donovani in splenic aspirates from patients with visceral leishmaniasis during treatment with sodium stibogluconate. AB - Studies were made of the ultrastructure of amastigotes of Leishmania donovani before and during treatment of patients with sodium stibogluconate. The most consistent effects of treatment on the amastigotes were a reduction in average size, greater irregularity of the cell outline, and a moderate increase in the electron density of the cytoplasm associated with a greater concentration of ribosomes. It is suggested that the drug affects active transport functions or permeability of the plasma membrane. PMID- 3000303 TI - Prognosis of breast cancer patients after mastectomy and dissection of internal mammary nodes. AB - The results of the analysis carried out on data on 1119 patients with operable breast cancer treated at the National Cancer Institute of Milan from 1965 to 1979 with enlarged mastectomy are reported. Metastases to internal mammary chain were found to be significantly associated with the maximum diameter of primary (16.1% for tumors less than 2 cm and 24.5% for larger tumors, p = 0.007), the age of the patients (27.6% in patients younger than 40 years, 19.7% in patients between 41 50 years, and 15.6% in patients older than 50 years, p = 0.01). The site of origin of the cancer had no impact on internal mammary node metastases. Patients with positive axillary nodes showed metastases to internal mammary nodes in 29.1% of the cases, while 9.1% of patients with axillary negative nodes had positive retrosternal nodes. Survival was significantly affected by the presence of positive internal mammary nodes: the percentage of 10-year survival varied from 80.4% in patients with axillary and internal mammary negative nodes to 30.0% in patients with both nodal basins involved. Intermediate survival rates (54.6% and 53.0%) were found when one or the other of the nodal stations (axillary and internal mammary) was separately affected. Maximum diameter of the primary significantly affected the survival of each group identified by the status of both axillary and internal mammary nodes. In conclusion, the information on the presence or absence of internal mammary node metastases would be of great importance in formulating the prognosis of breast cancer patients. To obtain this information, a biopsy at the first intercostal space may be reasonable in selected patients (age, maximum diameter, and axillary node involvement being the basis for selection) as long as noninvasive methods of diagnosis are available. PMID- 3000305 TI - Smoldering HTLV-associated T-cell leukemia. AB - Human T-cell lymphotropic virus type I-associated adult T-cell leukemia/lymphoma is a newly described clinical entity characterized by the abrupt onset of cutaneous manifestations, hypercalcemia, lymphadenopathy, and pleomorphic lobulated T cells found in the peripheral blood. The vast majority of cases reported in the United States have emphasized the rapid onset and fulminant course of the disease, which is unresponsive to conventional chemotherapeutic regimens. A smoldering form of this disease characterized by long duration of skin involvement has recently been described primarily in Japan. We describe a case of "smoldering" human T-cell lymphotropic virus type I disease in a patient from the United States. PMID- 3000306 TI - [Significant results of Riems cattle leukosis research since 1962]. PMID- 3000308 TI - [An optical method for the quantitative detection of antibodies to foot-and-mouth disease virus--initial results]. PMID- 3000307 TI - [Evaluation of the syncytial inhibition test for the detection of antibodies to the virus of enzootic cattle leukosis]. PMID- 3000309 TI - [Production of picornavirus concentrates using ultrafiltration]. PMID- 3000310 TI - [Effect of the virus content per vaccination dose on the effectiveness of the immune prevention of Marek's disease in general practice]. PMID- 3000312 TI - [Behavior of foot-and-mouth disease virus in various density gradient media]. PMID- 3000311 TI - [Intradermal test for the evaluation of reactivity of BLV infected cattle against tumor tissue]. PMID- 3000314 TI - [Flaccid paraplegia caused by spinal cord compression disclosing Wilms' tumor in a 12-year-old child]. AB - Flaccid paralysis with spinal cord compression led to discovery of Wilm's tumor with multiple subcutaneous and bone metastases in a 12 year-old child. Intraspinal seeding of Wilm's tumor by hematogenous route or direct extension is extremely rare and usually appears late in the course of therapy. Bone metastases are also rare and are frequently seen in the sarcomatous form of the tumor which involves the vertebral column and differential diagnosis with bone metastasizing renal tumor of childhood (BMRTC) should be considered. PMID- 3000313 TI - [Cholesterol pneumopathy in children. Apropos of 3 cases. Possible role of chronic Epstein-Barr virus infection]. AB - Three cases of cholesterol interstitial pneumonia in patients 3, 9 and 10 years of age respectively are reported. All three were born in Island of Reunion. Two were sisters. All had failure to thrive, dyspnea on rest and clubbing. Respiratory symptoms had appeared early in infancy. Open pulmonary biopsy was diagnostic. Prognosis was poor the boy dying at 4 years of age and severe respiratory insufficiency at 9 or 10 years in the two girls. Current etiological investigations were non contributory. However a profile of chronic infection with Epstein Barr virus (EBV) was found in each case while serological profiles ruled out infection with a virus of the herpes group virus (cytomegalovirus, herpes simplex). The possible role of EBV as an etiological agent of cholesterol pneumonia is discussed and genetic or environmental factors as well. PMID- 3000315 TI - [Intestinal pseudo-obstruction and cytomegalovirus infection of myenteric plexuses]. AB - The authors report an intestinal pseudo-obstruction syndrome occurring in a 2 month-old boy, with acquired major and persistent abdominal distension, leading to total parenteral nutrition. Rectal biopsy revealed hypoganglionosis with thickening of nerve processes and intranuclear inclusions in some neurons. Suspected cytomegalovirus infection was confirmed by viruria and specific IgM and IgG antibodies. PMID- 3000316 TI - Exaggerated orthostatic responsivity of plasma norepinephrine in depression. AB - An orthostatic challenge paradigm was used to assess noradrenergic regulation in depressive disorders. Plasma norepinephrine (NE) concentrations and concurrent blood pressure and pulse were measured at rest and after five minutes of standing in groups of bipolar (N = 22) and unipolar (N = 19) depressives and in 12 partially age-matched healthy female volunteers. Supine plasma NE levels were significantly lower in bipolar patients than in either unipolar depressives or normal volunteers. Following the orthostatic challenge, the fractional NE increase in both patient groups--particularly the bipolar group--was greatly exaggerated, exceeding that in the controls by approximately 100%. Nonetheless, the postural cardiovascular changes--elevations of diastolic blood pressure and heart rate--failed to distinguish the three subject groups. Noradrenergic dysregulation in depression thus is characterized by inefficient hyperreactivity to physiologic stress. PMID- 3000317 TI - The dexamethasone suppression test for diagnosis and prognosis in psychiatry. Commentary and review. AB - A modified dexamethasone suppression test (DST) has had unprecedented evaluation among biologic tests proposed for clinical use in psychiatry. It has not proved to reflect pathophysiologic changes at the level of the central nervous system or pituitary, and tissue availability of dexamethasone itself may contribute to test outcome. The sensitivity of the DST in major depression is limited (about 44% in over 5,000 cases) but is higher in psychotic affective disorders and mixed manic depressive states (67% to 78%). The high specificity of the DST vs control subjects (over 90%) is not maintained vs other psychiatric disorders (77% specificity overall), and acute "distress" may contribute to nonsuppression of cortisol. The test may have power in differentiating severe melancholic depression, mania, or acute psychosis from chronic psychosis (87% specificity) or dysthymia (77% specificity). The DST status adds about 11% to the prediction of short-term antidepressant response. Suggestions that failure to maintain normal suppression of cortisol predicts poor outcome are not secure. Uncritical enthusiasm or excessive skepticism regarding the DST are unwarranted. PMID- 3000318 TI - Guillain-Barre syndrome with cytomegalovirus infection of peripheral nerves. AB - Cytomegalovirus (CMV) infection involving multiple organ systems is a common finding in the acquired immunodeficiency syndrome. Acute CMV neuritis was a complication in two patients with acquired immunodeficiency syndrome. The diagnosis was made in both patients at autopsy, where typical CMV inclusions in the lumbar dorsal roots were noted in one patient and the same inclusions were found in the retroperitoneal peripheral nerves in a second patient. Electron microscopy confirmed the presence of viral particles in affected nerve segments. Patient 1 was hospitalized for ascending motor paralysis that remained unexplained at the time of death. In patient 2 the finding of CMV neuritis was incidental. Although CMV infection has been cited as an event antecedent to acute inflammatory polyradiculopathy (Guillain-Barre syndrome), to our knowledge morphologic evidence of the presence of virus has not been documented in this disease previously. PMID- 3000319 TI - Mixed tumor of the mediastinum. AB - A 36-year-old asymptomatic man was found to have a large middle mediastinal mass on a chest x-ray film. At surgery the tumor was located adjacent to the carina and beneath the aortic arch. It measured 7.0 X 5.0 X 4.0 cm and was well circumscribed and soft, with mucoid areas. The histologic features were those of a benign pleomorphic adenoma of salivary gland origin. This is, to our knowledge, the first reported case of primary pleomorphic adenoma of the mediastinum. We propose an origin from the ectopic salivary gland tissue. We also describe an additional patient in whom ectopic benign salivary gland tissue was found within mediastinal lymph nodes to support our hypothesis. PMID- 3000320 TI - [Relation between 15N excretion in feces following oral 15N urea administration and blood urea concentration in relation to raw fiber intake in swine]. AB - 9 pigs of a live weight at the beginning of the experiment of 33 kg received in 3 consecutive series of experiments (3 animals/group) a basic barley ration of 1.0 1.2 kg per animal and day. In groups 1 to 9 the following supplements were given: 1 = without N-supplement, 2 = 10.5 g urea, 3 = 79 g dried skim milk, 4 = 11 g urea, 5 = without N-supplement, 6 = 110 g horse bean coarse meal, 7 = without N supplement, 8 = 95 g dried skim milk, 9 = 120 g horse bean coarse meal. In groups 1-6 the ration was supplemented with 150-165 g DM partly hydrolysed straw meal per animal and day. After 20 days the animals received a single dosis of 0.5 g/kg0.75 15N-urea (72.1 atom-% 15N-excess) with the morning meal of the first day of the experiment. During the four days of the experiment groups 1-6, due to the straw meal supplement, excreted significantly higher N-amounts than the corresponding groups 7-9. In comparison with the first day of the experiment (1 h after the morning meal) urea concentration in the blood decreased to the following percentage in the sequence 1-9: 64; 65; 77; 54; 64; 73; 82; 88; 84 on the second day of the experiment (1 h before the evening meal. Between the excretion of 15N-excess in faeces (y = mg) during the four days of the experiment and the concentration of urea in the blood (x = mmol/l) there was the following significant negative correlation: y = -40.1 X +340.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000322 TI - [Cytomorphological diagnosis of nonepithelial tumors of the uterus]. AB - Retrospective histocytological correlations are performed of the material from 38 patients with non-epithelial uterine tumours. Cytomorphological features of highly, moderately and poorly differentiated leiomyosarcoma, leiomyoblastoma, endometrial stromal sarcoma, mixed mesodermal tumours and botryoid sarcoma are established. PMID- 3000321 TI - [Pathogenetic and pathomorphological problems of viral-bacterial associations in children dying of meningococcal infection]. AB - The information on 63 children dying from hypertoxic forms of meningococcal infection is presented. Four groups of brain damage by respiratory viruses (RV) are distinguished on the basis of the results of morphological and virological examination: 1) with a recent RV generalization (26 cases); 2) with dissemination of an etiological agent but without clear-cut structural changes (6 cases); 3) with an isolated affection of the brain (13 cases); 4) without clear-cut brain damage. Experimental influenza-meningococcal infection was reproduced in 260 white rats. Enhancement of the animal death rate, multiplication of virus and the degree of brain damage in cases of combined action of both etiological agents is demonstrated. The ability of influenza virus, when inoculated intranasally together with meningococcus, to penetrate and to multiply in the brain provoking meningitis and choroiditis is shown virologically, histologically and electron microscopically. PMID- 3000323 TI - [Malignant fibrous histiocytoma of the soft tissues]. AB - Clinico-morphological characterization of the malignant fibrous histiocytoma (MFH) of soft tissues in 142 patients is presented. According to the authors' data MFH is the most frequent tumour among soft tissue malignant neoplasms (15,7%) in adult patients. The age of patients is predominantly from 40 to 70, the most frequent site is a lower limb (thigh). A typical morphological variant of MFH clearly dominates (73%): there is a tendency to the development of an inflammatory variant at the retroperitoneal tumour site. Recurrences in all group of patients (104) who were followed-up were in 68,3%, metastasis in 45,2%, the 5 year survival in 59,6% of cases. A clear-cut dependence of these indices upon the depth of tumour location soft tissues, node size, tumour site is noted. In view of pronounced domination of a MFH typical variant the final conclusion on the feasibility of separation of other variants can be only made on the basis of a much greater number of cases. PMID- 3000324 TI - The effect of alloxan diabetes on prolyl and lysyl hydroxylase activity in uninflamed and inflamed rat gingiva. AB - The influence of diabetes on gingival inflammation was studied through its effect on prolyl and lysyl-hydroxylase activities and on tissue-collagen content. Inflammation induced for 7 days with either endotoxin or antigen-antibody complexes reduced the activity of both enzymes by about 45 per cent, and decreased the concentration of soluble and insoluble collagens. Diabetes alone decreased the enzyme activity by more than 50 per cent but prevented the loss of the soluble and insoluble collagens which occurs during inflammation. The complex interaction of diabetes and inflammation on collagen metabolism in gingival tissue may be explained in part by abnormalities of collagen synthesis; alterations in collagen maturation and degradation, and in leukocyte function, may also occur. PMID- 3000326 TI - Pathologic examination of ciliary body melanoma treated with proton beam irradiation. AB - Proton beam irradiation is one of the radiotherapeutic techniques currently used to manage uveal melanomas. Although this therapeutic modality has been in use for a decade and although nearly 500 patients have been so treated, there are only two published reports of the pathologic examination of these eyes. Key features found on pathologic examination of our patient's enucleated eye included vascular alterations in the tumor's blood supply, lymphocytic infiltrates, and lipoidal degeneration of tumor cells. The interval from therapy to enucleation in our patient was much longer than in the several previously reported cases, a factor allowing for the development of a more cumulative radiation effect on the tumor. The extent and degree of radiation-induced necrosis in our patient's tumor were more profound than in previous reports. PMID- 3000325 TI - Stimulation of glucose oxidation in rat submandibular gland cells in vitro by analogues of cyclic AMP. AB - Secretion from salivary glands, following autonomic stimulation, is energy dependent. Rat submandibular gland cells, when treated in vitro with both alpha- and beta-adrenergic agonists, showed increased glucose oxidation. The effects of alpha-adrenergic agents on glucose metabolism can be mimicked by non-receptor mobilization of Ca2+. Analogues of cyclic AMP were capable of elevating glucose metabolism to nearly the same extent as beta-adrenergic agonists. PMID- 3000328 TI - Pathologic quiz case 2. Granular cell myoblastoma. PMID- 3000327 TI - Proton beam irradiation and hyperthermia. Effects on experimental choroidal melanoma. AB - Ultrasonically induced hyperthermia (4.75 MHz) and proton irradiation (160 meV) were evaluated alone and combined to treat experimental choroidal melanoma in 58 rabbit eyes. Threshold tumoricidal doses were established for each modality. Therapy was performed combining subthreshold doses of heat and radiation. Focused ultrasonic energy via an external beam was found to deliver well-localized heat to an intraocular tumor. Ectopic temperature elevations due to soft-tissue-bone interfaces were alleviated by modifying beam alignment. The results indicate that hyperthermia (43 degrees C for one hour) potentiated the tumoricidal effects of radiation, while sparing normal ocular structures. Therefore, we believe that experimental hyperthermia is suitable as an adjuvant treatment modality. This shows that ultrasound hyperthermia has the potential to increase the efficacy of proton irradiation by lowering radiation doses and thus decreasing posttreatment ocular morbidity in human intraocular malignancies. PMID- 3000329 TI - Hematoporphyrin derivative therapy of papillomas. Experimental study. AB - Hematoporphyrin derivative has been shown to selectively localize in malignant tissues and chemically induced animal papillomas. It is a powerful photo sensitizing agent that can cause destruction and death of tissues in which it has localized by the generation of singlet oxygen when activated by light of the appropriate wavelength (photodynamic therapy [PDT]). Laryngeal papillomas are rapidly growing benign epithelial neoplasms with a clinical course marked by multiple recurrences after surgical removal. Before considering PDT as a therapeutic modality in the treatment of this debilitating disease we developed an animal model for experimentation. Using the cottontail rabbit papillomavirus, sometimes referred to as the Shope papillomavirus, large cutaneous papillomas were induced on the backs of Dutch Belted rabbits. Following intravenous administration of hematoporphyrin derivative, PDT was delivered with white light. Marked regression of the papillomas was noted with replacement by normal hair bearing skin. The clinical implications of this work to the control of laryngeal papillomatosis is discussed. PMID- 3000330 TI - Adenoviral bronchopneumonia of guinea pigs. PMID- 3000331 TI - Technetium pertechnetate imaging in apparent solitary thyroid nodule. PMID- 3000332 TI - Marek's disease in Japanese quails (Coturnix coturnix japonica): a study of natural cases. AB - Marek's disease was observed in quails. Gross lesions were confined mostly to the spleen and liver. Microscopic lesions were commonly seen in spleen, proventriculus, liver, and duodenum. Skin, peripheral nerves, and other visceral organs were also involved. Of 123 quails examined, 39 had serum antibodies against Marek's disease. These antibodies were detected from 11 to 17 weeks of age; the highest incidence was recorded at 15 weeks. Feather follicular antigen detected in 30 of the 95 quails was comparable to that of chicken. The disease was experimentally reproduced in susceptible quails. Marek's-disease-tumor associated surface antigens (MATSA) were demonstrated in the peripheral leukocytes and spleen cells of affected quails. The possible source of infection and its epidemiological importance are discussed. PMID- 3000333 TI - Immunoelectrophoresis of avian viral proteins in a phosphate-buffered system. AB - Avian influenza and hemorrhagic enteritis viral preparations were immunoelectrophoresed in a phosphate-buffered system. Excellent separation and resolution of viral proteins were achieved. Reasons are given why this method might be preferred over the conventional method employing a veronal (barbital) buffered system. PMID- 3000334 TI - Quantitation of intestinal D-xylose absorption in normal broilers and in broilers with pale-bird syndrome. AB - A micromethod was used in order to quantitate intestinal D-xylose absorption in young and extremely small birds. This test was performed in broilers collected from two farms from which birds were extremely uneven in body size and were passing poorly digested or undigested feed. A similar syndrome had been seen on all grow-outs during the 6 months before this investigation. Broilers were also collected and tested from three farms where no clinical signs of disease were seen. D-Xylose absorption peaks and curves for normal broilers closely resembled those observed in normal humans. Mean plasma D-xylose concentrations for virus infected broilers and for broilers with pale-bird syndrome were consistently lower than concentrations for normal broilers (P = 0.009). Reoviruses, small coronavirus-like particles, small round virus particles, and abundant bacterial flagellar fragments were seen in fecal samples from broilers with pale-bird syndrome. Production performance was lowest on farms showing clinical signs of this syndrome. PMID- 3000335 TI - Comparison of a kinetic-based enzyme-linked immunosorbent assay (KELISA) and virus-neutralization test for infectious bursal disease virus. I. Quantitation of antibody in white Leghorn hens. AB - The kinetics of the enzyme-substrate reaction served to evaluate a single-serum dilution indirect enzyme-linked immunosorbent assay (ELISA) for quantitating antibody in white leghorn hens inoculated with infectious bursal disease virus (IBDV) vaccines. The antibody profiles of primary and secondary responses induced by two IBDV immunization regimens were compared using a kinetic-based ELISA (KELISA) and the virus-neutralization (VN) test. KELISA was standardized with an IBDV-infected VERO cell suspension. Antigen was capable of binding minute quantities of sample (5 microliter) without requiring dilutions. Conjugate consisted of immunoglobulin G fraction of goat antiserum against chicken IgG bound to horseradish peroxidase. Neither test revealed a difference in antibody profiles between the two immunized groups. The KELISA was as efficient as the VN test in detecting antibody after vaccination and one log 2 unit more sensitive. The KELISA was suitable for testing large numbers of samples (n = 60) in a microplate compared with a conventional ELISA (n = 8). PMID- 3000337 TI - Enteric viruses in diarrheic turkey poults. AB - Thirty-three intestinal samples from 10-to-21-day-old diarrheic turkey poults were examined for the presence of enteric viruses by electron microscopy. Samples originated from 32 flocks in six commercial operations located in six states. Mortality in these flocks ranged from 3 to 15%, and birds from recovered flocks varied greatly in size. Rotavirus-like agents (RVLA) were the most common viruses associated with diarrhea outbreaks in the flocks examined, occurring in five out of six operations. Other viruses detected either singly or in combination, in order of prevalence, were astroviruses, reoviruses, rotaviruses, enteroviruses, and adenoviruses. With the exception of RVLA and rotaviruses, the other viruses were identified solely on the basis of morphology. Salmonellae were isolated from only one of the intestinal samples. By electron microscopy, RVLA were morphologically indistinguishable from rotaviruses, occurring as both 55-nm single-shelled and 70-nm double-shelled particles. However, immune electron microscopy was useful for antigenic differentiation of these two viruses. Turkey rotaviruses reacted with antisera to porcine and bovine rotaviruses, whereas turkey RVLA did not. Neither turkey rotaviruses nor RVLA reacted with antisera to porcine para-rotavirus or an antigenically distinct bovine rotavirus (bovine rotavirus-like agent). Similarly, convalescent anti-turkey RVLA serum (from recovered specific-pathogen-free poults) reacted with homologous virus but did not react with mammalian or avian rotaviruses or reoviruses. Further, RVLA were found to possess RNA electrophoretic migration patterns unlike those of conventional rotaviruses or reoviruses. This trait was used as an additional means of differentiating these viruses. PMID- 3000336 TI - Cell-culture virus-neutralization test and enzyme-linked immunosorbent assay for evaluation of immunity in chickens against fowlpox. AB - Two serological tests--the virus-neutralization (VN) test in chicken embryo fibroblasts (CEF) using a cell-culture-adapted virus, and the enzyme-linked immunosorbent assay (ELISA)--were used for evaluating the immune response in chickens against fowlpox virus. The VN test was conducted in 96-well tissue culture plates using a fowlpox virus that was adapted to induce cytopathic effects (CPE) in CEF in 48 hr. The ELISA was carried out with an antigen prepared by precipitation of a cell-culture-propagated virus suspension with ammonium sulfate and concentration by centrifugation. A 0.1 M acetate buffer, pH 5, was used as the sensitizing solution for maximum specific binding of the antigen to the microplate plastic well. No antibodies were detected by the VN test in 228 serum samples taken from chickens at irregular intervals between 1 and 39 weeks of age, even though the birds were vaccinated against fowlpox at 13 weeks of age. However, in sera collected 4 weeks after a sample of laying hens was challenged with fowlpox virus, VN titers of 1/10 to 1/40 were detectable. On the other hand, significant antibody reactions were detected by the ELISA on sera from chickens during the growing period, following vaccination and challenge. Although no maternal antibodies were found at 1 week of age, a continuous increase in the mean ELISA titers to fowlpox was demonstrated during the entire experimental period. This study showed that the ELISA was considerably more sensitive and practical than the VN test. PMID- 3000338 TI - Field outbreaks of colibacillosis of turkeys associated with hemorrhagic enteritis virus. AB - In a study of field material and a survey conducted by the authors, typical signs of colibacillosis of 6-to-12-week-old poults included sudden onset, listlessness, rales, and high mortality. Signs persisted for about 2 weeks and were often followed by a low incidence of lameness caused by Escherichia coli. Gross lesions included enlarged and congested spleens and livers, and dilated discolored black or purple duodenal loops. Microscopic lesions included splenic and hepatic congestion. In some birds (freshly killed and fixed immediately), the epithelium at the tips of the duodenal villi was sloughing, but in other birds the villi were intact and normal in appearance. Splenic enlargement, the presence of intranuclear splenic inclusions similar to those found in hemorrhagic enteritis (HE), and the isolation of HE virus from some of the field spleens all indicated that inapparent HE infection often occurs at approximately the same time as this type of colibacillosis. It is therefore believed that HE infection often exacerbates colibacillosis of older poults. PMID- 3000339 TI - Venereal pox in breeder turkeys in Minnesota. AB - Venereal pox was identified in four flocks of breeder turkeys following semen collection and artificial insemination. Proliferative fungate lesions were confined to the vent, cloaca, and, rarely, the oviduct. It was concluded that semen collection and artificial insemination may facilitate mechanical transmission of poxvirus. PMID- 3000340 TI - Correlation of serum antibody titer for avian encephalomyelitis virus (AEV) in hens with the resistance of progeny embryos to AEV. AB - Serum antibody titers for avian encephalomyelitis virus of vaccinated breeding hens were correlated with the resistance of the hens' progeny embryos to viral challenge. All of the embryos from hens that had enzyme-linked immunosorbent assay titers greater than 400 were resistant to challenge, and two-thirds of the embryos from hens that had titers greater than 300 were resistant. About one-half of the embryos from hens that had titers from 100 to 300 were immune to challenge. PMID- 3000341 TI - Immunization against psittacine pox. AB - Pox virus isolated from psittacine birds was used as a vaccine in trials with love birds (Agapornis roseicollis). The vaccine was applied by wing-web puncture using single- and double-needle applicators. Immunity was effective against challenge with virulent psittacine pox virus administered via the feather follicle/thigh. When unvaccinated contact control birds were placed with the vaccinated individuals immediately post-vaccination, virus spread was evident. However, susceptible birds placed with vaccinated ones at 27 days postvaccination remained uninfected for 11 weeks. The importance of a high vaccine virus titer was observed. PMID- 3000342 TI - The nutrition concept. PMID- 3000343 TI - The role of endothelium in the control of vascular tone. AB - In the last few years, experimental evidence has accumulated which suggests a substantial role for the endothelium in the control of vascular tone. Endothelium dependent dilatations have been demonstrated in various arteries of numerous mammalian species including man. Among the stimuli which elicit endothelium dependent dilatation are such varying stimuli as increases in blood flow and hypoxia, as well as endogenous (acetylcholine, ATP, ADP, bradykinin, substance P) and pharmacological agents (calcium ionophore A 23187, ergometrine, hydralazine, melittin). The functional importance of endothelium-dependent dilatation is emphasized by the fact that the direct vasoconstrictor effects of some of these substances (acetylcholine, histamine, norepinephrine, serotonin) on vascular smooth muscle is attenuated or even reversed by their simultaneous stimulatory effect on endothelial cells, resulting in the release of a vasodilator signal. Bioassay experiments have shown that a humoral vasodilator agent with a biological half-life in the range of seconds is released from the endothelium (native or cultured) during stimulation with acetylcholine, ATP and calcium ionophore. Experimental data are presented, which suggest that EDRF may act by direct stimulation of guanylate cyclase, resulting in smooth muscle relaxation due to increased smooth muscle cyclic GMP levels. The chemical nature of this nonprostaglandin endothelium-derived relaxant factor (EDRF) is still not known. The possible physiological and pathophysiological significance of endothelium dependent dilatation in situ is discussed. Special attention is paid in this context to the potential role of EDRF activity in coronary vasomotor control. PMID- 3000346 TI - [Pathogenesis and immunology of virus-induced neonatal diarrhea]. PMID- 3000347 TI - Effect of HLA on the cellular immune response to varicella-zoster virus. AB - The effect of HLA on varicella-zoster virus (VZV)-specific lymphocyte transformation (LTF) was studied in 100 normal immune adults and 64 children who were immunized with live attenuated varicella vaccine. In the normal adults, a statistically significant association was observed between low responsiveness and the presence of A2 (p less than 0.025), and also between high responsiveness and the presence of Aw24 (p less than 0.05). A similar but clearer association, i.e. low responsiveness with A2 (p less than 0.005) and high responsiveness with Aw24 (p less than 0.025), was observed in the vaccinated children. In these children, Aw31 was also found to be related to low responsiveness (p less than 0.05). These results suggest that the VZV-specific cellular immune response is in some way influenced by HLA. PMID- 3000345 TI - The coronary endothelium: a highly active metabolic barrier for adenosine. AB - Cultured coronary endothelial cells and the coronary endothelium of isolated perfused guinea-pig hearts are characterized by a very active adenosine and adenine nucleotide metabolism. Adenosine applied to the endothelium at low concentrations is avidly metabolized and preferentially incorporated into different nucleotide pools--only a minor amount is degraded to uric acid. Physiologically, the coronary endothelium therefore functions as an impermeable metabolic barrier for interstitially or intravascularly accumulating adenosine. Only at concentrations greater than or equal to 10(-6) M adenosine can pass the endothelial barrier. As a consequence, the vasodilatory action of adenosine formed in or administered into the coronary system cannot be induced by a direct association of the nucleoside with the putative adenosine receptor of the arteriolar smooth muscle cells, but must be mediated by the endothelium. High molecular weight derivatives of adenosine, clearly confined to the coronary system, can also induce a coronary dilation. The endothelium-mediated smooth muscle relaxation is therefore obviously due to triggering of an extracellular adenosine receptor at the luminal surface of the endothelium. Since this process is accompanied by a rapid and pronounced activation of the adenylate cyclase system, the endothelial receptor conforms to an A2-type. According to our results it is necessary to reconsider qualitative and quantitative facets of the adenosine hypothesis of metabolic regulation of coronary blood flow, which--in its original formulation--exclusively centers on the cardiomyocyte metabolism. With respect to the vasoactivity of adenosine one obviously has to distinguish between its action from the interstitial space directly via the myocyte receptors of the vessel wall, and/or its action from the intracoronary space via the newly detected endothelial A2-receptor. More information is needed to determine the extent to which both receptor populations actually participate in the metabolic regulation of coronary flow under physiological and pathophysiological conditions. PMID- 3000348 TI - Limited in vivo susceptibility of the South American monkey -Cebus apella- to type 1 poliovirus: physical and histopathological observations after intraspinal inoculation. AB - Type 1 polioviruses (an attenuated strain, Sabin 1 LSc 2ab and a virulent strain, Mahoney) were inoculated intraspinally into the South American Cebus monkey Cebus apella. Neither physical symptoms nor histological changes in the central nervous system were observed after inoculation of attenuated Sabin strain. But the virulent Mahoney strain caused flaccid paralysis in two of three monkeys. In these two paralyzed monkeys, definite specific histological changes were observed with spreading of the lesions to places far from the inoculation site, i.e., the cervical cord and brain. These results suggest that Cebus apella has limited susceptibility to type 1 poliovirus. PMID- 3000349 TI - Cell fusion and cell engineering. PMID- 3000344 TI - Prostaglandins, other eicosanoids and endothelial cells. AB - Endothelial cells are an important source of eicosanoid formation in the cardiovascular systems. All major pathways of eicosanoid production have been demonstrated in endothelial cells, yielding significant amounts of prostacyclin (PGI2), PGE2, PGF2 alpha, thromboxane A2, leukotrienes and a number of hydroxy fatty acids. The regulation of eicosanoid formation by endothelial cells is poorly understood. There is evidence that precursors, such as arachidonic acid or prostaglandin endoperoxides, may also be provided by other cell types. Endothelial cell-derived eicosanoids are involved in the regulation of local vessel tone, intravascular platelet activation, cell locomotion and, eventually, cell proliferation. Most of the available information considers PGI2. This compound is the quantitatively dominating eicosanoid in endothelial cells. Major actions of PGI2 include inhibition of platelet activation and aggregation, relaxation of arterial vessels and inhibition of growth-factor release. There is probably a tight interaction with other biologically active mediators which needs further evaluation. This also applies to the clinical significance of eicosanoid related pathways for the mechanism of action of cardiovascular drugs, such as organic nitrates or acetylsalicylic acid. The unique property of the eicosanoid system to become activated only in response to stimulation, the local nature of this reaction, the multiplicity of products formed and the short half-time of most of them are currently the most significant obstacles to define the role of endothelial cell-derived eicosanoids in clinical practice. PMID- 3000350 TI - Three-year survey of the epidemiology of rotavirus, enteric adenovirus, and some small spherical viruses including "Osaka-agent" associated with infantile diarrhea. AB - Studies were made by electron microscopy (EM) on the viruses associated with diarrhea of outpatients at a pediatric clinic in Osaka Prefecture during the three year period from 1980 through 1982. The viruses detected by EM by negative staining with phosphotungstic acid (PTA) were classified morphologically into 6 groups: rotavirus, adenovirus and four kinds of small spherical viruses, calicivirus, astrovirus, picornavirus/parvovirus (P/P)-like agent and Osaka agent. Osaka-agent seems to be a newly identified small virus. It is 35-40 nm in diameter with a fringe of spike-like structures on its surface. Viruses were detected in 181 of the 395 cases of diarrhea (45.8%). Rotavirus was detected in 122 (30.9%) of the total cases and in 67.4% of the virus-positive cases, while other viruses were detected in 15% of the total cases; adenovirus in 23 (6%) and small agents in 36 (9%). Rotavirus infection showed a distinctive seasonal variation, being mainly restricted to cooler months, but infections with other viruses did not show any seasonal variation. The age distribution of patients suggested that infants of 0 to 2 years old are very susceptible to all viruses. Attempts to cultivate these viruses in vitro were successful with only two isolates of adenovirus type 5. PMID- 3000352 TI - Purification of a metalloproteinase inhibitor from human rheumatoid synovial fluid. AB - A metalloproteinase inhibitor present in human rheumatoid synovial fluid was purified by a combination of heparin-Sepharose chromatography, concanavalin A Sepharose chromatography, ion-exchange chromatography and gel filtration. The Mr of the purified inhibitor was 28000 by SDS/polyacrylamide-gel electrophoresis and 30000 by gel filtration. The inhibitor blocked the activity of the metalloproteinases collagenase, gelatinase and proteoglycanase, but not thermolysin or bacterial collagenase. The serine proteinase trypsin was not inhibited. The inhibitory activity was lost after treatment with trypsin (0.5 micrograms/ml) at 37 degrees C for 30 min, 4-aminophenylmercuric acetate (1 mM) at 37 degrees C for 3 h, after incubation for 30 min at 90 degrees C and by reduction and alkylation. These properties suggest that the inhibitor closely resembles the tissue inhibitor of metalloproteinases ('TIMP') recently purified from connective-tissue culture medium. PMID- 3000351 TI - Dependence on Ca2+ of the activities of phosphatidylinositol 4,5-bisphosphate phosphodiesterase and inositol 1,4,5-trisphosphate phosphatase in smooth muscles of the porcine coronary artery. AB - The activities of phosphatidylinositol 4,5-bisphosphate (PIP2) phosphodiesterase (PDE) and inositol 1,4,5,-trisphosphate (IP3) phosphatase in the particulate and cytosol fractions prepared from porcine coronary artery smooth muscles were examined using 32P-labelled PIP2 and IP3 as substrates, respectively. The activity of PIP2 PDE, as assessed from the production of IP3, in the cytosol fraction was about 10-fold higher than that in the particulate fraction. In the absence of MgCl2, the activity of PIP2 PDE in both fractions showed no causal relation to the free Ca2+ concentration in the physiological range of 10(-7)-10( 5) M, but was enhanced remarkably by 10(-4) M free Ca2+. The addition of 1 mM MgCl2 to the assay medium markedly inhibited the activity of PIP2 PDE in both fractions in the presence of free Ca2+ (10(-8)-10(-5) M). In the absence of MgCl2, 10(-5)M-acetylcholine (ACh) produced IP3, and this action was blocked by 3 X 10(-6) M-atropine. The ACh-induced activation of PIP2 PDE ceased in the presence of 1 mM-MgCl2; however, the reactivation occurring on the addition of 10 microM-guanosine 5'-[gamma-thio]triphosphate did not depend on the free Ca2+ concentrations (10(-7)-10(-5)M). The activities of IP3 phosphatase, determined from decrease in the amount of IP3 in the particulate and cytosol fractions, had much the same potency in both fractions. The activity of IP3 phosphatase in the cytosol fraction was enhanced by MgCl2 in a concentration-dependent manner, the maximal value occurring at 1 mM-MgCl2, and was also enhanced in the presence of physiological concentrations of free Ca2+ (10(-7)-10(-6) M). These findings suggest that the activation of PIP2 PDE which occurs with application of ACh in the presence of guanine nucleotides and 1 mM-MgCl2 is independent of the free Ca2+ concentration, and that the hydrolysis of IP3 by phosphatase increases, depending on the concentration of free Ca2+. PMID- 3000353 TI - Genomic organization of human lactate dehydrogenase-A gene. AB - A human genomic clone containing the lactate dehydrogenase-A (LDH-A) gene of approx. 12 kilobases in length was isolated and characterized. The protein-coding sequence is interrupted by six introns, and the positions of these introns are at the random coil regions or near the ends of secondary structures located on the surface of the LDH-A molecule. An additional intron is present at 24 nucleotides 5' to the translation initiation codon ATG, while the 3' untranslated sequence of 565 nucleotides is not interrupted. The genomic blot analysis of human placenta DNA indicates the presence of multiple LDH-A gene-related sequences. PMID- 3000354 TI - Isolation of ATPase I, the proton pump of chromaffin-granule membranes. AB - Chromaffin-granule membranes contain two ATPases, which can be separated by (NH4)2SO4 fractionation after solubilization with detergents, or by phase segregation in Triton X-114. ATPase I (Mr 400000) is inhibited by trialkyltin, quercetin and alkylating agents, and hydrolyses both ATP and ITP. It contains up to five types of subunit, including a low-Mr hydrophobic polypeptide that reacts with dicyclohexylcarbodi-imide; these subunits are unrelated to those of mitochondrial F1F0-ATPase, as judged by size and reaction with antibodies. ATPase II (Mr 140000) is inhibited by vanadate, and is specific for ATP; it has not been extensively purified. Proton translocation by resealed chromaffin-granule 'ghosts', measured by uptake of methylamine or by quenching of the fluorescence of 9-amino-6-chloro-2-methoxyacridine, is supported by the hydrolysis of ATP or ITP, and inhibited by quercetin or alkylating agents, but not by vanadate. ATPase I must therefore be the proton translocator involved in the uptake of catecholamines and possibly of other components of the chromaffin-granule matrix, whereas ATPase II does not translocate protons. PMID- 3000357 TI - An active twenty-amino-acid-residue peptide derived from the inhibitor protein of the cyclic AMP-dependent protein kinase. AB - Digestion with Staphylococcus aureus V8 proteinase of the inhibitor protein of the cyclic AMP-dependent protein kinase results in the sequential formation of three active inhibitory peptides. The smallest active peptide has the sequence Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- His-Asp . This 20-amino-acid-residue peptide has 20-40% of the activity of the native molecule and a Ki of 0.2 nM. Inhibition, as a minimum, appears to be based upon the inhibitor protein containing the recognition sequences that dictate protein substrate-specificity. This inhibitory peptide also has sequence homology with the phosphorylation site for a protein kinase other than the cyclic AMP-dependent enzyme. PMID- 3000355 TI - Characterization of the effects of Ca2+ on the intramitochondrial Ca2+-sensitive enzymes from rat liver and within intact rat liver mitochondria. AB - The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent 'State 3.5' respiration condition. Ca2+ had no effect on NAD(P)H formation induced by beta hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed. PMID- 3000356 TI - Redistribution and characterization of (H+ + K+)-ATPase membranes from resting and stimulated gastric parietal cells. AB - When isolated from resting parietal cells, the majority of the (H+ + K+)-ATPase activity was recovered in the microsomal fraction. These microsomal vesicles demonstrated a low K+ permeability, such that the addition of valinomycin resulted in marked stimulation of (H+ + K+)-ATPase activity, and proton accumulation. When isolated from stimulated parietal cells, the (H+ + K+)-ATPase was redistributed to larger, denser vesicles: stimulation-associated (s.a.) vesicles. S.a. vesicles showed an increased K+ permeability, such that maximal (H+ + K+)-ATPase and proton accumulation activities were observed in low K+ concentrations and no enhancement of activities occurred on the addition of valinomycin. The change in subcellular distribution of (H+ + K+)-ATPase correlated with morphological changes observed with stimulation of parietal cells, the microsomes and s.a. vesicles derived from the intracellular tubulovesicles and the apical plasma membrane, respectively. Total (H+ + K+) ATPase activity recoverable from stimulated gastric mucosa was 64% of that from resting tissue. Therefore, we tested for latent activity in s.a. vesicles. Permeabilization of s.a. vesicles with octyl glucoside increased (H+ + K+)-ATPase activity by greater than 2-fold. Latent (H+ + K+)-ATPase activity was resistant to highly tryptic conditions (which inactivated all activity in gastric microsomes). About 20% of the non-latent (H+ + K+)-ATPase activity was also resistant to trypsin digestion. We interpret these results as indicating that, of the s.a. vesicles, approx. 55% have a right-side-out orientation and are impermeable to ATP, 10% right-side-out and permeable to ATP, and 35% have an inside-out orientation. PMID- 3000359 TI - The effect of polar head group substitution on phospholipid methylation and the beta-adrenergic response in C6 glial cells. AB - The membranes of intact C6 cells were enriched with phosphatidyldimethylethanolamine or phosphatidylmonomethylethanolamine. These cells showed enhanced rates of phospholipid methylation but this was not accompanied by an increased beta-adrenergic response. We conclude that phospholipid methylation is not coupled to the activation of adenylate cyclase in the beta-adrenergic response of C6 glial cells. PMID- 3000358 TI - Molecular cloning and characterization of the complementary DNA and gene coding for the B-chain of subcomponent C1q of the human complement system. AB - Plasmid clones containing cDNA coding for the B-chain of human Clq were isolated from a liver cDNA library. The longest cDNA insert isolated contained all the coding sequence for amino acid residues B1 to B226 plus a 3' non-translated region of 264 nucleotides that extended into the poly(A) tail, thus accounting for 950 nucleotides of the mRNA. The B-chain mRNA was estimated by Northern-blot analysis to be 1.46 kb (kilobases) long, which indicated that approx. 500 bases were not accounted for in the cDNA clone. A cosmid clone containing the C1q-B chain gene was isolated from a human genomic DNA library. The precise 5' limit of gene was not established, but from the data available it appears that the gene is approx. 2.6 kb long. The coding sequence for residues B1 to B226 in the gene is interrupted by one intron, of 1.1 kb, which is located within the codon coding for glycine at position B36. This glycine residue is located in the middle of the triple-helical regions found in C1q at exactly the position where there is an unusual structural feature, i.e. a bend in each of the helical regions brought about by the interruption of the Gly-Xaa-Yaa repeating triplet sequences in the A and C-chains and the presence of an 'extra' triplet in the B-chain. Nucleotide sequencing of the 5' end of the gene indicates the presence of a predominantly hydrophobic stretch of 29 amino acids, immediately before residue B1, which could serve as a signal peptide. PMID- 3000361 TI - Polyphosphoinositides are present in plant tissue culture cells. AB - Polyphosphoinositides have been isolated from wild carrot cells grown in suspension culture. This is the first report of polyphosphoinositides in plant cells. The phospholipids were identified by comigration with known standards on thin-layer plates. After overnight labeling of the cells with myo-[2-3H] inositol, the phosphoinositides as percent recovered inositol were 93% phosphatidylinositol., 3.7% lysophosphatidylinositol, 1.7% phosphatidylinositol monophosphate, 0.8% phosphatidylinositol bisphosphate. PMID- 3000360 TI - P2-purinergic control of liver glycogenolysis. AB - Purinergic agonists cause a dose-dependent activation of glycogen phosphorylase in isolated rat hepatocytes. Half-maximally effective concentrations are 5 X 10( 7)M for ATP, 2 X 10(-6)M for ADP, and about 5 X 10(-5) M for AMP and adenosine. This potency series indicates the presence of P2-purinergic receptors. The mode of action of ATP appears to be identical with that of the Ca2+-dependent glycogenolytic hormones angiotensin, vasopressin and alpha 1-adrenergic agonists. (1) They all require Ca2+ for phosphorylase activation; (2) they do not increase cyclic AMP levels; (3) they are susceptible to heterologous desensitization by vasopressin and phenylephrine; (4) they lower cyclic AMP concentrations in hepatocytes stimulated by glucagon, most probably mediated by an enhanced phosphodiesterase activity. PMID- 3000362 TI - Influence of adrenoceptors on thrombin-induced phosphoinositide metabolism in rat platelets. AB - Stimulation of washed rat platelets with thrombin resulted in an increased turnover of phosphoinositides. Adrenaline and isoproterenol both inhibited thrombin-induced phosphatidic acid formation in a dose-dependent manner. Inhibitory responses of both compounds were blocked by a beta-adrenoceptor antagonist. However, isoproterenol was a more potent inhibitor than adrenaline. Addition of a selective alpha2-adrenoceptor antagonist potentiated the inhibitory effect of adrenaline up to the level observed with isoproterenol. Prestimulation of beta-adrenoceptors with isoproterenol, followed by addition of adrenaline (or noradrenaline) markedly diminished the inhibitory effect induced by the full beta adrenoceptor agonist. Our results indicate that, in rat platelets, catecholamines are able to counteract, via alpha2-receptors, the beta-adrenoceptor-mediated inhibition of thrombin-induced phosphatidic acid formation. This suggests that catecholamines, by controlling cAMP level, may modulate phospholipase C activity and thereby platelet reactivity. PMID- 3000363 TI - Functional receptors for vasoactive intestinal peptide in cultured vascular smooth muscle cells from rat aorta. AB - Specific binding sites for vasoactive intestinal peptide (VIP), a potent vasodilatory polypeptide, and its effect on formation of intracellular cyclic AMP levels were studied in cultured vascular smooth muscle cells (VSMC) from rat aorta. Specific binding of 125I-labeled-VIP to cultured VSMCs was time- and temperature-dependent. Scatchard analysis of binding studies suggested the presence of two classes of high and low affinity binding sites for VIP; the apparent Kd and the number of maximal binding capacity were approximately 8 X 10( 9) M and 60,000 sites/cell (high-affinity sites) and approximately 4 X 10(-8) M and 140,000 sites/cell (low-affinity sites), respectively. Unlabeled VIP competitively inhibited the binding of 125I-labeled-VIP to its binding sites, whereas neither peptides structurally related to VIP, nor other vasoactive substances affected the binding. VIP stimulated formation of intracellular cyclic AMP in cultured VSMCs in a dose-dependent manner; the stimulatory effect of VIP on cyclic AMP formation was not blocked by propranolol and was additive with isoproterenol. The present study first demonstrates the presence of specific receptors for VIP in VSMCs functionally coupled to adenylate cyclase system. It is suggested that VIP exerts its vasodilatory effect through its specific receptors distinct from beta-adrenergic receptors. PMID- 3000364 TI - Ribonucleotide reductase in ascites tumour cells detected by electron paramagnetic resonance spectroscopy. AB - Tyrosine radicals localized in the M2 subunits of ribonucleotide reductase have been detected by electron paramagnetic resonance (EPR) in ordinary ascites tumour cells. The intensity of its doublet EPR spectrum is higher in rapidly proliferating cells. Hydroxyurea, a specific inhibitor of this enzyme, decreases the concentration of the tyrosine radical. Whereas in different ascites tumours the doublet EPR spectrum dominates at g = 2.004, in solid tumours another more intense EPR spectrum from nitrosyl-hemoproteins appears. In conclusion, EPR spectroscopy can be used to monitor the content and variations of active M2 subunits of ribonucleotide reductase in intact ascites tumour cells. PMID- 3000365 TI - Purification of cytochrome b from complex III of beef heart mitochondria. AB - Two cytochrome b preparations have been prepared from Complex III of beef heart mitochondria, by detergent-exchange chromatography on a butyl-Toyopearl column. One was eluted from the column with buffer containing Tween 20 after most of other subunits of Complex III were eluted with buffer containing guanidine-HCl, and the other was eluted from the column with buffer containing sodium dodecyl sulfate. The former is consisted of a single polypeptide (subunit III) and contained 37.5 nmol of heme b/mg of protein, and the latter consisted of subunits III and IX and contained 19.5 nmol of heme b/mg of protein. The former was labile when it was reduced by dithionite, whereas the latter was stable. Subunit IX in the latter is associated with cytochrome b even after gel filtration and density gradient centrifugation. These results suggest that subunit IX plays a role in stabilizing cytochrome b. PMID- 3000366 TI - NAD+-dependent conversion of 20-OH-LTB4 to 20-COOH-LTB4 by a cell-free system of human polymorphonuclear leukocytes. AB - Human polymorphonuclear leukocytes, but not mononuclear leukocytes, platelets, or erythrocytes, oxidized 20-hydroxy-leukotriene B4 (20-OH-LTB4) to 20-carboxy-LTB4 (20-COOH-LTB4). 20-OH-LTB4 was quantitatively converted to 20-COOH-LTB4 by the sonicate of polymorphonuclear leukocytes in the presence of NAD+, with an optimal pH of about 7.9. NADP+ could not replace NAD+. The conversion was not inhibited by 2 mM pyrazole, a potent inhibitor of alcohol dehydrogenase. When LTB4 was incubated with the sonicate in the presence of NADPH and NAD+, 20-OH-LTB4 and 20 COOH-LTB4 appeared concomitantly with the disappearance of LTB4. PMID- 3000367 TI - Activation by gamma interferon of human macrophage capability to produce toxic oxygen molecules is accompanied by decreased Km of the superoxide-generating NADPH oxidase. AB - Capability to release superoxide anion in response to phorbol myristate acetate by intact cells has been compared with Kinetic properties of NADPH oxidase by lysates of human monocytes and monocyte-derived macrophages. Maturation of monocytes in vitro is accompanied by substantial decrease of the capability to release superoxide anion in response to phorbol myristate acetate. Exposure of mature macrophages to recombinant interferon gamma enhances respiratory burst activity up to 3-4 fold. Modifications of NADPH oxidase accompany changes in the ability to release superoxide anion. The affinity of the oxidase for its substrate is higher in monocytes and gamma interferon treated macrophages, while Vmax is not changed. PMID- 3000368 TI - Hydrodynamic properties of a thromboxane A2/prostaglandin H2 antagonist binding site solubilized from human platelets. AB - The hydrodynamic properties of a binding site for the thromboxane A2/prostaglandin H2 receptor antagonist 9,11-dimethylmethano-11, 12-methano-16-(3 iodo-4-hydroxyphenyl)-13, 14-dihydro-13-aza-15 alpha beta-omega-tetranor thromboxane A2 (I-PTA-OH) were determined in solubilized membrane proteins from human platelets using the detergent 3-[(3-cholamidopropyl)-dimethylammonio] 1 propane-sulfonate (CHAPS). Gel filtration revealed a Stokes radius of 5.25 +/- 0.37 nm (n=9). Molecular weight determined by gel filtration assuming a spherical protein was 180,000-220,000 Daltons. Sedimentation through sucrose or glycerol gradients revealed a sedimentation coefficient of 6.3 +/- 0.2 Svedberg units (n=5). The molecular weight calculated using the Stokes radius and sedimentation coefficient was 140,000 Daltons. The frictional ratio f/fo was 1.4, corresponding to an axial ratio of 7:1. PMID- 3000369 TI - Enhanced phosphatidylinositol kinase activity is associated with early stages of hepatocarcinogenesis and hepatocellular carcinoma. AB - Liver phosphatidylinositol (PI) kinase activity was determined in rats exposed to two different hepatocarcinogenic regimens. In contrast to partial treatment regimens the complete Solt and Farber hepatocarcinogenic regimen caused a significant increase in liver PI kinase activity at day 11 after partial hepatectomy. PI kinase activity in hepatocarcinomas removed 15 1/2 months after initiation of the complete Solt and Farber regimen was 2-fold higher than normal liver tissue surrounding the tumors. Compared to a choline supplemented diet a hepatocarcinogenic regimen consisting of a diet deficient in choline and methionine significantly increased liver PI kinase activity after 26 days. These data demonstrate that liver PI kinase activity is selectively elevated during hepatocarcinogenesis. PMID- 3000370 TI - Inhibition of insulin and epidermal growth factor (EGF) receptor autophosphorylation by a human polyclonal IgG. AB - The immunoglobulin G of a polyclonal antiserum (pIgG) from a patient with insulin resistance and hypoglycemia was tested for its ability to inhibit insulin binding and to affect the autophosphorylation of partially-purified insulin receptors extracted from rat liver membranes. pIgG, when added 4 hr prior to insulin, inhibited subsequent insulin binding by 50% at 30 micrograms added protein; however, insulin previously bound to the receptor could not be displaced by a 4 hr subsequent exposure of up to 70 micrograms pIgG. pIgG, independent of its effect on insulin binding, inhibited both basal and insulin-stimulated autophosphorylation of the insulin receptor in a dose-dependent manner with a half maximal effect at 3.3 to 7 micrograms protein. Furthermore, pIgG also reduced basal autophosphorylation of the EGF receptor. The effect of pIgG to inhibit basal autophosphorylation of insulin and EGF receptors, together with its ability to reduce autophosphorylation of insulin receptors fully occupied by insulin, imply that the effect of pIgG on receptor autophosphorylation is largely independent of its effect on ligand binding. Moreover, these findings suggest that pIgG may inhibit autophosphorylation by acting on domains which are similar in the insulin and EGF receptors. PMID- 3000371 TI - FAD-dependent malate dehydrogenase from Mycobacterium smegmatis: activation of the lipid-depleted enzyme by incorporation into cardiolipin liposome. AB - The lipid-depleted, enzymatically inactive malate dehydrogenase isolated from Mycobacterium smegmatis membrane was found to be incorporated spontaneously into cardiolipin liposome, but not into phosphatidylcholine liposome, as was revealed by electron spin resonance spectra with the use of 5-doxylstearic acid as a spin probe in the phospholipid liposomes. In addition, sucrose density gradient centrifugation in 0.5 M KCl showed hydrophobic interaction of the enzyme with cardiolipin liposome and further proved that the enzyme thus interacted hydrophobically with cardiolipin liposome became enzymatically active. From the results obtained above, it was concluded that the lipid-depleted, enzymatically inactive malate dehydrogenase isolated from M. smegmatis membrane was found to be activated by incorporating into the hydrophobic region of cardiolipin liposome. PMID- 3000372 TI - How can iron salts mediate the degradation of nucleos(t)ides by elliptinium acetate via free-radicals? AB - Elliptinium acetate (NSC 264137) is an antineoplastic agent currently used in anticancer chemotherapy. We report here the first evidence that this drug is able to modify DNA models via a redox process with iron salts. In presence of iron (III) salts, EDTA and H2O2, 9-OH-NME degrades deoxyguanosine. The two main products are guanine and 8-hydroxydeoxyguanosine which result from the formation of hydroxyl radicals. The biological implications of this phenomenon are briefly discussed. PMID- 3000374 TI - Phosphatidylinositol 4-phosphate kinase is associated with the membrane skeleton in human erythrocytes. AB - Phosphatidylinositol 4-phosphate kinase was eluted from human erythrocyte stroma by three separate and distinct techniques which are known to disrupt the membrane skeleton. In addition, this kinase was found to be associated with the intact skeletons prepared by Triton X-100 extraction of stroma. Phosphatidylinositol 4 phosphate kinase which has been extracted from the membrane is a freely soluble protein with poor enzymatic activity toward added phosphatidylinositol-4 phosphate; however, the enzyme was shown to reassociate with skeleton-depleted stroma and then regain full enzymatic activity toward stromal bound substrate. PMID- 3000373 TI - Calcium mobilization in fluoride activated human neutrophils. AB - Fluoride ion, at concentrations above 10 mM, was found to elicit a rise in intracellular calcium levels in neutrophils, as monitored by changes in Quin 2 fluorescence intensity. The calcium mobilization response was characterized by a lag period of 4 to 10 min. and a prolonged duration of action (greater than 20 min.). In contrast, the chemotactic peptide, formylmethionyl-leucyl phenylalanine, induced a rise in intracellular calcium concentrations which peaked within 1 min. Preincubation of the cells with 1 microgram/ml pertussis toxin resulted in inhibition of the formylmethionyl-leucyl-phenylalanine induced response, but not that mediated by fluoride. Recent evidence suggests that the formylmethionyl-leucyl-phenylalanine receptor is coupled to phospholipase C and phosphoinositide degradation through a guanine nucleotide binding protein susceptible to inhibition by pertussis toxin. Present results suggest that fluoride ion may serve to activate this protein in a manner resistant to inhibition by pertussis toxin. PMID- 3000375 TI - Investigations of the biological activity of leukotriene A4 in human polymorphonuclear leukocytes. AB - An investigation was undertaken to compare the responses of human neutrophils to the epoxide leukotriene A4 with those elicited by its stable product leukotriene B4 under identical IN VITRO conditions. LTA4 evokes neutrophil responses similar in nature to those induced by LTB4 but at much higher concentrations. Evidence suggests that LTA4 is important primarily for its role as an intermediate rather than for inherent activity. PMID- 3000377 TI - Neurotoxic insecticides inhibit GABA-dependent chloride uptake by mouse brain vesicles. AB - The neurotoxic insecticides endrin, dieldrin, aldrin, lindane (gamma-1,2,3,4,5,6 hexachlorocyclohexane) and deltamethrin inhibited gamma-aminobutyric acid dependent 36Cl- uptake by mouse brain vesicles. Of the insecticides examined, the chlorinated cyclodienes endrin and dieldrin were the most potent, producing 50% inhibition at 2.8 and 13.9 microM, respectively. Lindane and deltamethrin were less effective, and with deltamethrin the effect was incompletely stereospecific. These results demonstrate the disruption of gamma-aminobutyric acid receptor chloride ionophore function in mammalian brain by neurotoxic insecticides and provide evidence that this complex is the principal site of cyclodiene action. PMID- 3000376 TI - Regulation of cyclic AMP and cyclic GMP levels by adrenocorticotropic hormone in cultured neurons. AB - The effect of adrenocorticotropic hormone (ACTH) on the intracellular concentration of cyclic nucleotides was studied in cultures of neurons from embryonic chick cerebral hemispheres. Incubation of neurons with ACTH(1-24) in the presence of phosphodiesterase inhibitor isobutylmethylxanthine resulted in a sustained increase in cyclic AMP while rise in cyclic GMP level was transient. The values obtained for half-maximal stimulation were 0.5 microM and 0.03 nM for cyclic AMP and cyclic GMP respectively. Concomitantly, ACTH(1-24) stimulated guanylate cyclase activity (half-maximal stimulation at 0.02 nM). These results suggest the existence of two distinct populations of ACTH receptors in neurons and provide the first evidence that cyclic GMP does mediate the action of ACTH in neurons. PMID- 3000379 TI - Protein kinase activities in mammalian blood fluid. AB - Two protein kinase activities, one specific for phosvitin and another specific for histone, were detected in serum and plasma of calf as well as of human blood after precipitation with ammonium sulfate (40%) and chromatography on DEAE Sephacel. The enzymes were separated by chromatography on phosphocellulose. The histone kinase is not related to the cyclic AMP-dependent protein kinase; it may derive at least partly from damaged cells. The phosvitin kinase activity carries characteristics of the so called casein kinase type II similar to that present at the surface of cells including blood cells. PMID- 3000378 TI - NMB: a human neuroblastoma cell line with specific opiate binding sites. AB - The human neuroblastoma cell line designated NMB (Brodeur et al., 1977, Cancer 40: 2256) has been shown to have specific opiate binding sites. These sites are highly stereospecific. Two characteristic delta specific peptides, D-Ala2-D-Leu5 enkephalin and D-Thr2-D-Thr6 enkephalin, have high affinity for the binding sites. Morphine binds specifically but with a much lower affinity. Dextrorphan and the mu specific peptide morphiceptin (Tyr-Pro-Phe-Pro-CO-NH2) do not bind to the site. The binding sites are heat and trypsin sensitive. Sodium ions specifically lower agonist binding to the sites. Approximately 14,000 binding sites per cell are found. The binding characteristics of these sites are very similar to those of the delta sites characterized on mouse neuroblastoma cell lines. PMID- 3000380 TI - DNA topoisomerases as targets for cancer therapy. PMID- 3000381 TI - Characterization of the Na+, K+-ATPase activity of basolateral plasma membranes of kidney proximal tubular cells from young and old rats. AB - Several characteristics of the Na+, K+-ATPase activity of basolateral plasma membranes of kidney proximal tubular cells from young (3 months) and old (24 months) rats were studied. In both cases, the ATPase activity reached optimum values under the following conditions: Mg2+:ATP concentrations (mM) 5:5 (apparent Km 0.5 mM); Na+ concentration 50 mM (apparent Km 18 mM); K+ concentration 20 mM (apparent Km 2.5 mM); pH 7.2; temperature 52 degrees. The values of the apparent energy of activation of the system were similar for young and old rats in the temperature range 20-52 degrees but were 55% higher for the old rats in the temperature range 10-20 degrees. PMID- 3000382 TI - Initial rate of sodium taurocholate uptake in isolated elutriated hepatocytes from untreated and phenobarbital-treated rats. AB - The initial rate of sodium taurocholate uptake was measured in rat hepatocytes separated by centrifugal elutriation into five cell fractions whose difference in size was verified by flow cytometry. The hepatocytes were prepared from untreated and phenobarbital-treated rats. For untreated animals, the initial rate of taurocholate uptake at concentrations of 5 or 50 microM was the same for hepatocytes prior to fractionation and for each of the five elutriated fractions. Treatment of the animals with phenobarbital was associated with a significant increase in hepatocyte size in all fractions and caused a significant increase in the initial uptake rate. The extent of the rate increase in hepatocytes prior to fractionation was similar to that observed for each of the five hepatocyte subpopulations. Our observation indicates that phenobarbital causes a significant increase in the initial rate of sodium taurocholate uptake and suggests that large and small hepatocytes possess no inherent differences controlling the initial uptake process. PMID- 3000383 TI - Identification and characterization of leukotriene D4 receptors in adult and fetal human lung. AB - Leukotriene D4 (LTD4) receptors were identified and characterized in adult and fetal human lung membranes. Macroscopically normal adult lung tissue was selected from seventeen surgical biopsy specimens, and twenty-seven fetal lung samples were obtained from therapeutic abortions. Binding assays were performed using pooled adult or fetal human lung membranes at 30 degrees under conditions which prevented metabolism of [3H]LTD4. Specific binding reached equilibrium within 30 min, remained constant for 60 min, was enhanced by Mg2+, and was inhibited by Na+ and guanyl-5'-yl-imidodiphosphate. Computer-assisted analyses of saturation binding data showed a single class of binding sites with similar apparent Kd (0.15 +/- 0.09 and 0.12 +/- 0.003 nM) and Bmax (68 +/- 29 and 62 +/- 14 fmoles/mg protein) values for adult and fetal samples respectively. Competition binding studies with [3H]LTD4 showed the same rank order potency for adult and fetal lung receptors (5S, 6R-LTD4 greater than 5S, 6R-LTD1 greater than 5R, 6S-LTD4 greater than 5S,6R-LTE4 greater than FPL 55712). A comparison of the receptor binding affinities of these compounds with their smooth muscle contractile agonist (pD2) and antagonist (-log[KB]) activities in guinea pig lung and trachea showed a good correlation (r = 0.88), suggesting that the saturable, high-affinity, stereoselective [3H]LTD4 specific binding sites identified in human lung may be physiologically relevant receptor moieties. PMID- 3000384 TI - Characterisation of a new transposon-mediated trimethoprim-resistant dihydrofolate reductase. PMID- 3000386 TI - Inhibition of hydrogen production in drug-resistant and susceptible Trichomonas vaginalis strains by a range of nitroimidazole derivatives. PMID- 3000385 TI - The crucial role of low steady state oxygen partial pressures in haloalkane free radical-mediated lipid peroxidation. Possible implications in haloalkane liver injury. PMID- 3000387 TI - [New plasmid vectors for cloning and the expression of genes]. AB - Multicopy plasmids of pMCR series have been constructed from pRRN2 (a pBR322 derivative) including plasmids carrying transcription terminators downstream the tet gene and those with opposite orientation of the tet and RNAI genes. The plasmids permit cloning and high expression of genes with strong promoters. PMID- 3000389 TI - Antihypertensive activity of alacepril, an orally active angiotensin converting enzyme inhibitor, in renal hypertensive rats and dogs. AB - Alacepril (1-[(S)-3-acetylthio-2-methylpropanoyl]-L-prolyl-L-phenylalanine, DU 1219) showed a dose related and long lasting antihypertensive effect in renal hypertensive rats (two-kidney, one-clip), a typical renin dependent hypertensive model. The maximum hypotensive potency of alacepril (1-30 mg/kg) after single oral administration was slightly weaker than that of captopril (1-30 mg/kg). Judging from the AOC (area over the antihypertensive curve) value, the overall antihypertensive activity of alacepril was 3 times more potent than that of captopril on a weight basis. The long lasting antihypertensive effect of alacepril in renal hypertensive rats was also confirmed by once daily successive oral administration (1-2 mg/kg/d). In renal hypertensive dogs, alacepril (3 mg/kg) showed a stable and sustained hypotensive effect, and its duration of action was longer than that of captopril. Although alacepril did not possess a significant in vitro angiotensin converting enzyme (ACE) inhibitory activity, orally given alacepril (5.6-56.1 mg/kg) produced a potent and prolonged in vivo ACE inhibition which was estimated by suppression on angiotensin-I (310 ng/kg i.v.) induced pressor response in conscious normotensive rats. The prolonged in vivo ACE inhibitory activity of alacepril (5.6 mg/kg) was also observed in conscious normotensive dogs. These results suggest that the disposition and metabolism of orally given alacepril are responsible for the prolonged ACE inhibition and, concomitantly, for exerting the long lasting antihypertensive effect. Consequently, alacepril is a novel orally active ACE inhibitor having a potent and prolonged antihypertensive activity, and these properties suggest that alacepril is favorable for the treatment of hypertension. PMID- 3000388 TI - [Specific binding of N-allylnormetazocine (SKF 10047), a ligand of sigma-opioid receptors, with liver membranes]. AB - A sigma-opioid receptor ligand, N-allylnormetazocine (SKF 10047), binds specifically and reversibly to rat liver membranes. The rat liver binding sites for SKF 10047 are similar to sigma-opioid CNS receptors. They fail to interact with classical opiates (morphine, naloxone) and opioid peptides but bind with high affinity benzomorphans (bremazocine, SKF 10047) and various psychotropic drugs (haloperidol, imipramine, phencyclidine etc). PMID- 3000390 TI - Antihypertensive activity of alacepril in spontaneously hypertensive rats and deoxycorticosterone acetate-salt hypertensive rats and dogs. AB - Antihypertensive activity of alacepril (1-[(S)-3-acetylthio-2-methylpropanoyl]-L prolyl-L-phenylalanine, DU-1219), an orally active angiotensin converting enzyme (ACE) inhibitor, was investigated in hypertensive models with normal or low plasma renin activity (PRA). After single oral administration in spontaneously hypertensive rats (SHR), alacepril (1-30 mg/kg) showed a dose related antihypertensive effect with a gradual onset and long lasting action. The maximum hypotensive effect was about 3 times more potent than that of captopril (3-100 mg/kg) on a weight basis. When comparing the AOC (area over the antihypertensive curve) values, the overall antihypertensive activity of alacepril was 8 times stronger than that of captopril. In deoxycorticosterone acetate-salt (DOCA-salt) hypertensive rats, alacepril (10-100 mg/kg) produced a significant and sustained hypotensive effect. The maximum hypotensive potency and the overall antihypertensive activity of alacepril were remarkably stronger than those of captopril (30, 100 mg/kg). During once daily successive oral administration for 10 days in SHR, alacepril (3-10 mg/kg/d) reduced dose relatedly the daily starting blood pressure. In DOCA-salt hypertensive rats and dogs, alacepril (30 mg/kg/d) produced a significant antihypertensive effect, while captopril (30 mg/kg/d) did not reduce daily starting blood pressure. Therefore, it may be expected that alacepril is a more effective antihypertensive agent than captopril in various hypertensions of different etiology. PMID- 3000391 TI - Interleukin 2: a review. AB - Interleukin 2 (IL 2) is a central mediator of the growth and functional activity of B- and T-cells, and cytotoxic cells, including Natural Killer and Lymphokine Activated Killer cells. Significant defects in the production of, and response to, IL 2 have been described in a variety of congenital and acquired immunodeficiency states. IL 2 has demonstrated major anti-tumor activity in animal models. The biochemistry and molecular biology of IL 2 and its gene are reviewed, along with data regarding the IL 2 receptor, normal T-cell activation, abnormalities in IL 2 production and response in immunodeficiency states and leukemia, and initial explorations of IL 2 in the treatment of human cancer. PMID- 3000392 TI - General pharmacological properties of cilostazol, a new antithrombotic drug. Part II: Effect on the peripheral organs. AB - The pharmacological effects of the new antithrombotic drug, cilostazol (6-(4-(1 cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-qui nolinone, OPC-13013) on the peripheral nervous system and miscellaneous organs were studied. Cilostazol produced a very slight increase in beating rate of the isolated atrium and a very slight increase in contraction of the papillary muscle of guinea pigs induced by cilostazol compared with that of isoproterenol (isoprenaline). The beating rate increasing effect was not antagonized by propranolol and it augmented isoproterenol's effect. When administered intravenously in anesthetized dogs, cilostazol increased blood flow in the coronary, internal carotid, vertebral and femoral arteries and transiently decreased blood flow in the renal and superior mesenteric arteries probably because of blood pressure fall. In anesthetized dogs, cilostazol decreased blood pressure by reducing the resistance in the peripheral blood vessels. An increase in heart rate, cardiac contractile force, myocardial oxygen consumption and respiration rate were also observed. In conscious rats, the drug increased heart rate. Cilostazol produced a slight relaxation of the smooth muscle of all organs except for blood vessels and slightly inhibited spontaneous motility of the isolated uterus of pregnant rats, the isolated ileum of rabbits and the ileum of rats in situ. It was considered that cilostazol had no specific effects against norepinephrine, serotonin, acetylcholine or histamine based on the results that the drug was only slightly antagonistic against the contraction of rabbit aorta induced by norepinephrine and serotonin, the contraction of isolated guinea-pig ileum induced by acetylcholine, histamine and barium chloride and the contraction of the isolated uterus of non-pregnant rats induced by oxytocin. The drug had little effect on the contraction of the nictitating membrane induced by stimulation of the preganglionic sympathetic nerve in the cat. These results suggest that cilostazol had little effect on the autonomic nervous system. Cilostazol slightly inhibited edema induced by carrageenin, but showed no diuretic effect and had little effect on neuromuscular transmission or the secretion of gastric juice, bile and pancreatic juice, and therefore it was considered to have no appreciable effect on the peripheral nervous system or organs except for its vasodilating and cardiac effects. PMID- 3000393 TI - A radioimmunoassay for the angiotensin converting enzyme inhibitor ramipril and its active metabolite. AB - A radioimmunoassay (RIA) has been developed for the measurement of 2-[N-[(S)-1 ethoxycarbonyl-3-phenylpropyl]-L-alanyl]-(1S,3S,5S)- 2-azabicyclo[3.3.0]octane-3 carboxylic acid (ramipril, Hoe 498) and its active metabolite (diacid: hydrolysis product of ramipril) in serum or plasma. Antibodies to the diacid were raised in rabbits against a lysine analogue conjugated to bovine serum albumin. A 125I ramipril-diacid derivative was used as radioligand. Separation of the antibody bound and free radiolabeled ligand was achieved by employing a polyethylene glycol solution. The RIA is specific for the active metabolite (diacid). Studies on specificity showed less than 0.6% cross-reactivity with intact ramipril or with dioxopiperazine metabolites. The levels for intact ramipril were obtained by prior enzymatic hydrolysis to the diacid. The sensitivity of the assay was 0.1 and 0.5 ng/ml for diacid and ramipril, respectively. Intra- and inter-assay coefficients of variation ranged from 3 to 14% at 1 to 30 ng diacid per ml. A recovery experiment yielded an average of 103%. The assay has been used to investigate the pharmacokinetic profile of both ramipril and its pharmacologically active metabolite. PMID- 3000394 TI - Antiviral effect of 1-morpholinomethyl-tetrahydro-2(1H)-pyrimidinone (DD-13) in experimental alphaviral infections in white mice. AB - 1-Morpholinomethyl-tetrahydro-2(1H)-pyrimidinone (DD-13), a selective inhibitor of the alphaviral reproduction in vitro, manifests a pronounced antiviral activity in experimental infections with Semliki forest virus (SFV) and Sindbis virus in white mice (intraperitoneally inoculated with 10-10 000 LD50). Introduced subcutaneously in mice infected with SFV the compound was effective within the dose range of 4.7-300 mg/kg. The effective dose (ED)50 value of DD-13 is about 18.7 mg/kg and the maximum effect is reached with a 150-300 mg/kg dose. The protection index reached 80% and the mean survival time from approximately 7 days in the placebo group was lengthened to 26 days. This high antiviral effect is distinguished by its high selectivity, the selectivity ratio (LD50/ED50) being 385 (LD50 = 7200 mg/kg) and is manifested in infections with massive viral inocula (100-1000 LD50). The effective treatment schedule was determined: two divided daily doses of 37.5-150 mg/kg, beginning on the 3rd day after infection to the 8th day. After intravenous administration of DD-13 in mice infected with SFV a high protective effect was also observed, which was equal to that of the subcutaneous application, but with doses several times lower: in the 3-40 mg/kg. ED50 is about 3.5 mg/kg and the optimal effective dose is 10-20 mg/kg, i.e. 1/12 1/6 of LD50 (116 mg/kg). The selectivity ratio is about 33. The most effective treatment course is accomplished by a 10-20 mg/kg daily dose (in two applications) starting on the 2nd day after the virus inoculation up to the 8th day.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000396 TI - Germ cell tumours. AB - Germ cell tumours form an important group of gonadal neoplasms and are also found in a number of extragonadal sites like the mediastinum, para-pineal and sacrococcygeal regions and retroperitoneum. Although there are considerable differences between germ cell tumours occurring in different anatomical locations they exhibit a remarkable homology, and are considered as a group. In this review germ cell tumours are discussed as a group emphasizing some of the recent developments in this field. In the testis germ cell tumours form the most common group of neoplasms comprising 90% of all testicular tumours and 99% of them are malignant. In the ovary germ cell tumours comprise approximately 20% of ovarian neoplasms, and more than 90% are mature cystic teratomas and are benign. Malignant testicular neoplasms are 10 times more common than their ovarian and 20 times more common than their extragonadal counterparts. Malignant germ cell tumours have a specific age incidence and occur mainly in children and young adults. Due to this they represent one of the most important groups of neoplasms in this age group. Testicular germ cell tumours show marked racial and geographical differences occurring much more frequently in Western Europe, especially in Scandinavia, as compared with Southern and Eastern Europe. They are rare in Africa and are very uncommon in Blacks as compared to Whites. These remarkable differences are not observed in ovarian or extragonadal germ cell tumours. It is now accepted that histogenetically all the tumours in this group are of germ cell origin, and that germ cell tumours are capable of somatic (embryonal) and extra-embryonal differentiation (fig. 1). The occurrence of extragonadal germ cell tumours in anatomical locations in the midline of the body is explained on the basis of migration of the primitive germ cells during embryonic life from the wall of the yolk sac to the primitive gonad. An all embracing classification of germ cell neoplasms based on the WHO classifications of ovarian and testicular tumours is presented. The importance of careful and thorough examination of germ cell tumours is emphasized, especially in view of the recent advances in the therapy of malignant germ cell neoplasms. The value of tumour markers like alphafoetoprotein (AFP) and human chorionic gonadotropin (HCG) produced by endodermal sinus tumour (EST) and some embryonal carcinomas and choriocarcinoma and syncytiotrophoblastic giant cells respectively in diagnosis, monitoring the progress of the disease, and the efficacy of therapy, as well, as for early detection of metastases and recurrences, is strongly emphasized.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3000397 TI - [Aschoff's center of proliferation. Experience of the Gustave Roussy Institute]. AB - "Le Centre de Proliferation d'Aschoff" (CPA) has many synonyms: radial scar, benign sclerosing ductal proliferation, non encapsulated sclerosing lesions, fibroelastic center, fibroadenosis with fibroelastic core etc... In a retrospective study from the files of the Gustave-Roussy Institute, 88 cases were found: 18 cases between 1955 and 1959 and 70 cases more recently, between 1976 and 1981. CPA is an unusual lesion, observed in 2% of benign breast lesions, and in less of 1% of malignant breast lesions. Macroscopically, when CPA is not associated to a carcinoma, it has either a pseudotumoral feature (46% of the cases), or only a microscopic appearance (54% of the cases). In these last cases, it is always found by chance at the time of the examination of specimens of fibrocystic disease. When it is associated to a carcinoma, these two features are less well individualized. Mammographically and macroscopically, they are often problematical. They cause considerable diagnostic problems because of their pseudotumoral features, being similar to small stellate carcinomas. Two cases were misinterpreted at the time of the extemporaneous examination for a carcinoma and in 36 other cases, the diagnosis was uncertain and the definitive report was delayed. For the non-pseudotumoral form, a relationship was found in this series between previous cytological aspirations and CPA. The evolution of this lesion was difficult to evaluate because of the great number of the patients lost to follow up in this series. Although a slight trend was observed for patients to subsequently develop a breast carcinoma, no statistically significant difference was observed. From this study, close follow-up of these patients is considered necessary. PMID- 3000395 TI - Blood pressure- and lipid-lowering effect of mackerel and herring diet in patients with mild essential hypertension. AB - Fourteen male patients with mild essential hypertension were put on a mackerel and herring diet within a prescribed isocaloric regimen in a cross-over design for 2 weeks. After mackerel diet eicosapentaenoic acid (EPA-C20:5, n-3) appeared more in cholesterol esters (1.7-11.0%), whereas docosahexaenoic acid (DHA-C22:6, n-3) was predominantly incorporated into serum triglycerides (1.0-8.3%). After herring diet, which contained half as much EPA and DHA, their increase was of minor degree. After mackerel diet serum triglycerides, total cholesterol, LDL cholesterol and lecithin cholesterol acyl transferase (LCAT) activity were significantly decreased (by 28%, 9%, 14% and 14%, respectively), returning to the initial levels 3 months later. On the contrary, HDL cholesterol appeared significantly increased (by 12%). After herring diet the differences were not significant. Serum sodium was significantly lower (by 2%) at the end of the mackerel diet as compared to the initial values. On the other hand, uric acid in serum appeared transiently increased (by 24%) at the end of both dietary periods. A significant decrease (by 8%) in casual systolic blood pressure, measured in recumbent position, could be observed only at the end of the mackerel period. Moreover, the level of systolic and diastolic blood pressure before and during a standardized psychophysiological stress test was significantly lower after mackerel diet. Nevertheless, the increments after stress were similar. Plasma renin activity was increased (by 64%) after mackerel diet.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000398 TI - [Pulmonary lesions following bone marrow graft. Study of 35 cases]. AB - Lung biopsy of 35 patients with interstitial pneumonitis following bone marrow transplantation (BMT) have been studied histologically, ultrastructurally and by immunofluorescence. Among infectious diseases, cytomegaloviruses (CMV) are the more frequently found, whereas Pneumocystis carinii infections are more frequently found in immunocompromised hosts without BMT. CMV infections are related to severe chronic graft-versus-host disease in allogenic or mismatched BMT. Hemorrhagic pulmonary oedema and vascular damage might be the consequence of high doses of cyclosporin A or of disseminated intravascular coagulation. Granulomatous and fibrosing lesions corresponded in 2 cases to an eosinophilic pneumonitis and in 11 cases to an "idiopathic" diffuse interstitial pneumonitis. 2 patients had concomitant diffuse lung fibrosis, sclerotic plaques of the skin and Sjogren-like syndrome. The pulmonary and cutaneous scleroses had common features in the types of collagen and in the composition of the infiltrate. Both fibroses might result from a common pathogenic mechanism related to an immunologic conflict between the lymphocytes of the graft and the cells from the host tissues. PMID- 3000400 TI - [Mixed tumor of the breast (pleomorphic adenoma)]. AB - Mixed tumor (pleomorphic adenoma) of the breast is a rare distinctive primary neoplasm. Its prognosis is comparable to the corresponding salivary neoplasm. Differential diagnosis can be difficult with metaplastic carcinoma containing bone and cartilage, specially on small biopsies as emphasized by the present observation. PMID- 3000399 TI - [Adrenomyeloneuropathy. Morphometric and ultrastructural study of the peripheral nerves]. AB - A sural nerve biopsy was performed in a case of adrenomyeloneuropathy which was confirmed by biochemical investigations. Morphometric study of the nerve revealed a loss of large myelinated fibers with signs of demyelination, and normal unmyelinated fibers. Electron microscopic study showed typical lipid inclusions in some histiocytes and Schwann cells. The interest of nerve biopsy in histopathological diagnosis of adrenomyeloneuropathy is emphasized. PMID- 3000401 TI - Prevalence of cytomegalovirus antibodies in Thai blood donors. AB - Antibodies to cytomegalovirus (CMV) were determined in Thai blood donors using the complement fixation (CF) test and enzyme-linked immunosorbent assay (ELISA). A total of 203 voluntary blood donors, 181 males and 22 females, who came to the Blood Bank at Siriraj Hospital during February 1985, were investigated. Their ages ranged from 17 to 53 years (mean 24.3 +/- 6.9). Seventy-three out of 156 (46.8%) and 171 out of 203 (84.2%) sera were positive for CMV antibodies as detected by the CF test and ELISA respectively. The result of ELISA showed that 95.5 per cent of the female blood donors and 82.9 per cent of the males possessed CMV antibodies. No difference in the geometric mean titres of either sex was noted. The findings indicated that ELISA was more sensitive than the CF test for detecting CMV antibodies. The high percentage of CMV-seropositive blood donors indicates that CMV infection is common in this country. Therefore, it might be necessary to test blood donors for CMV antibodies when they are giving blood for use by certain patients, especially immunocompromised ones; the same observation applies with regard to organ donors before transplantation is carried out. PMID- 3000402 TI - Different patterns of transposable elements in the vicinity of tRNA genes in yeast: a possible clue to transcriptional modulation. AB - We have extended the catalogue of yeast tRNA genes that are found associated with repetitive (transposable) elements. We determined the nucleotide sequences of loci containing the genes for a tRNAGln and a tRNASer2 (pY66), a tRNAGlu3 (pY80), and a tRNALys1 (pY109). Our analyses revealed that complex patterns exist in which different types of elements (Ty, delta, sigma, and tau) are involved. We could further demonstrate that in several there are alleles of which one contains a particular element and the other lacks it; such differences are also found when comparing hybridization patterns of DNA from a diploid and a haploid yeast strain. In order to investigate a possible functional role of the elements in conjunction with the tRNA genes, we compared the transcriptional activities of several tRNA genes by microinjection into Xenopus oocyte nuclei. The observed differences in expression may be attributed to the presence or absence of different elements in the vicinity of the tRNA genes. PMID- 3000403 TI - [Congenital cytomegalovirus infections in a neonatal pathology unit]. AB - In order to evaluate the incidence of congenital CMV infections we checked 200 babies in the special care baby unit of an obstetric clinic in Milan. In the same period 3779 babies were born in that clinic. Congenital CMV infection was diagnosed by virus isolation from urine specimens collected in the first week of life in two babies. One of them was asymptomatic, the other had the classical CID. Virological confirmation of a further CID observed in an infant hospitalized at that time in the unit was obtained. Different serological methods of congenital infections diagnosis, specific IgM, total IgM and IgA, rheumatoid factor determinations, were employed. Only the CMV-IgM ELA method didn't give false positive reactions and picked up 2 congenitally infected babies out of 3. From our data, we infer that the incidence of congenital CMV infection in the general milanese population might lie between 0.5-1% of live births. The level of seropositivity for CMV antibodies in the same population, as judged from the complement fixing antibodies determinations in the group of the mothers, was 87%. PMID- 3000404 TI - A prospective etiological and clinical study on gastroenteritis in Italian children. AB - Stool cultures of 188 children hospitalized for gastroenteritis in a two-year period (1981-1982) yielded Salmonella in 25.5%, Campylobacter in 16.0%, and Y. enterocolitica in 3.7% of cases. Rotavirus was identified in 22.3% of cases. Out of 82 lactose-positive microorganisms isolated from as many cases, three (one E. coli and two Klebsiella) produced heat-labile enterotoxin and two E. coli strains a "cytotoxic" toxin (in an HEp-2 in vitro model); two other E. coli strains possessed adhesive properties for HEp-2 cells in vitro; none revealed enteroinvasive for HEp-2 cells. Two out of 70 E. coli strains were EPEC. From stools of 643 childhood out-patients Salmonella was isolated in 9.6% of cases; Campylobacter and Y. enterocolitica in 9.0% and in 0.6% of cases respectively. Rotavirus was not looked for. Shigella strains were not isolated. Among 622 children without gastrointestinal symptoms, five (0.8%) excreted campylobacters and one (0.16%) salmonella. Children of 18-24 months of age were significantly more often infected with Campylobacter. Gross blood in feces, body temperature greater than 38 degrees C, and peripheral leukocytosis were significantly more often associated with Salmonella infection; vomiting and absence of blood in stools and of leukocytosis with rotavirus infection. Other features were not significantly associated with the etiological agent of the illness. Except for Salmonella infections, the enteritis cases did not show any pronounced seasonal pattern. PMID- 3000405 TI - [Mixed cryoglobulinemia with peripheral neuropathy in a case of HBsAg-positive chronic active hepatitis]. AB - We have observed a patient who developed a severe progressive peripheral neuropathy in the course of HBsAg positive chronic active hepatitis in whom high serum levels of cryoproteins was detectable, as described just once in the past. The role of HB virus in the pathogenesis of cryoglobulinemia and in the development of peripheral neuropathy was discussed. PMID- 3000407 TI - Isolation and growth of human endothelial cells from peripheral blood by PVP silica density gradients. PMID- 3000406 TI - Increasing prevalence of HTLV-III infection in intravenous drug users in Liguria, Italy. AB - Anti-HTLV III prevalence has been investigated in serum samples of 638 intravenous drug users collected over May 1981 - March 1985 and stored at -20 degrees C. Separately, in a prospective way, we have studied 68 IV drug abusers (53 Genoese and 15 of Sanremo area) of whom we have collected, at least, one serum specimen for each year, starting with 1981. We have also tested for anti HTLV III presence serum samples of: 91 subjects of Hospital staff (Infectious Diseases Department and Laboratory workers); 32 workers in Therapeutic Communities for drug users and 24 family contacts of anti-HTLV III positive drug users. And then serum samples of two groups of general population collected for other seroepidemiological investigations in 1982 (256 subjects) and 1984 (538 subjects) were tested. No IV drug user was positive in 1981 whilst from 1982 up to 1984 there was a strong rising of the prevalence of anti-HTLV III positive subjects: 2, 22, and 39 per cent, respectively. The prevalence remained about 40% in the first months of 1985. The investigations carried out also show that HTLV III spread in Sanremo area slightly before than in Genoa and neighbourhood. No subject positive for anti-HTLV III has been detected among the Hospital staff and in workers of Therapeutic communities who have more probability to get in contact with infected subjects or their blood, as well as in the general population. A positive case has been discovered in a family contact (the wife of a positive for anti-HTLV III IV drug user). Some epidemiological and public health questions linked to the situation observed, at present, in Liguria, are discussed. PMID- 3000408 TI - [Effects of atropine on synaptic transmission in human sympathetic ganglia]. PMID- 3000409 TI - Effects of decreased red cell deformability on the diabetic microcirculation, studied by means of measuring radioactivity levels in the feet after 99mTc erythrocyte injection. PMID- 3000410 TI - Development of cortical afferents and cortico-tectal efferents of the mammalian (rat) primary visual cortex. AB - At the time when the fibres from the striate cortex (area 17) begin to innervate the superficial layers of the superior colliculus of the young rat (postnatal days 4 and 5) a high degree of specificity in the organization of this newly formed cortico-tectal projection is already apparent. Thus, in young rats, as in adult mammals of virtually all species studied so far, the somata of cortico tectal neurones are confined to lamina V of the ipsilateral cortex. However, this high degree of laminar (radial) specificity in young animals is accompanied by a substantial degree of exuberance as indicated by a tangential distribution of the cortico-tectal cells which is wider than that in the adult. The exuberant projections are pruned during the second postnatal week. The cortico-cortical associational and commissural fibres start to enter the grey matter of the rat striate cortex after postnatal day 7. Again a high degree of specificity in the laminar distribution of those newly established projections is apparent. However, the cortico-cortical projection, at the time when cortico-cortical fibres enter the cortical laminae, is clearly exuberant since the tangential spread of cortical cells projecting to the striate cortex is wider than that in the adult. Pruning of these excessive projections takes place some time after postnatal day 14. It is believed that understanding the mechanism(s) underlying the development of connections of the rat visual cortex might be of general importance in understanding developmental abnormalities in the pattern of interconnections of the visual cortices of other mammalian orders. PMID- 3000411 TI - [Early diagnosis of herpes simplex virus encephalitis in childhood]. PMID- 3000412 TI - [Evaluation of ACNU alone and combined with tegafur as additions to radiotherapy of the treatment of malignant gliomas--a cooperative clinical trial]. AB - Controlled, prospective, randomized studies were performed to evaluate the effects of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3- (2-chloroethyl)-3 nitrosourea hydrochloride (ACNU) and ACNU plus tegafur as additions to radiotherapy for the treatment of malignant gliomas. In the first trial, 105 patients with glioblastoma or anaplastic astrocytoma were randomly divided into two groups after surgery and received radiotherapy (RT, 40 to 60 Gy to the whole brain), or radiotherapy plus concomitant chemotherapy with ACNU (100 mg/m2 on day 1 and 42). Effects of the treatment were compared in 82 evaluable patients from results of CT scans taken before and one month after the completion of radiotherapy. The regression rates more than 50% of the tumor size were observed in 15.0% of patients treated with RT alone and in 47.6% of patients treated with RT plus ACNU. The difference was statistically significant (p less than 0.005). In the second trial, 87 patients were randomly divided into two groups and received RT plus ACNU, or RT plus combined chemotherapy with ACNU and tegafur (400 mg/m2, daily for 8 weeks). Sixty-nine patients were within the valid study group. The regression rates more than 50% of the tumor size were observed in 34.2% of patients treated with RT plus ACNU: and in 41.2% treated with RT, ACNU plus tegafur. No statistical difference was noted in the response rate between the groups. These results indicate that ACNU is an effective agent in conjunction with radiotherapy for patients with malignant gliomas, and that tegafur does not enhance the effectiveness of ACNU. PMID- 3000414 TI - [Immunohistochemical and ultrastructural studies on tactile-like corpuscles in neurofibromas]. AB - Morphological characteristics of tactile-like corpuscles (tactoid bodies, pseudomeissnerian corpuscles) which are occasionally present in neurofibromas have already been detailed. However, there has yet been controversy on the cytogenesis of these lamellated structures. Probably, one of the most important problems is whether tactile-like corpuscles are composed of Schwann cells or of perineurial cells. In this study, seven cases of neurofibromas with tactile-like corpuscles were examined by rabbit or mouse polyclonal antisera to S-100 protein which could be regarded, at least in the peripheral nervous system, as a specific marker for Schwann cells. Since recent extensive studies have revealed that S-100 protein is a mixture of two predominant dimeric components with S-100 alpha and S 100 beta subunits, and the S-100 alpha subunit is absent from Schwann cells, tactile-like corpuscles were also examined by a mouse monoclonal antibody (ASA-1) specific for the S-100 alpha subunit. For immunohistochemical analysis, peripheral nerves, Meissner corpuscles and epidermal melanocytes were also examined in parallel. By the immunoperoxidase method using rabbit or mouse polyclonal antisera to S-100 protein, tactile-like corpuscles were intensely stained in sections from all seven cases. Both the flattened cell bodies and the eccentrically located nuclei of their constituent cells were stained. In the background neurofibroma tissue surrounding the tactile-like corpuscles, spindle shaped cells were stained variably. In three of the seven cases, numerous melanin containing cells which often surrounded the tactile-like corpuscles were also stained. In normal peripheral nerves, positive staining was confined to Schwann cells and their processes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000413 TI - [Brain function in patients with plateau waves studied by compressed spectral array in long-term EEG monitoring]. AB - The intracranial pressure, systemic blood pressure and compressed spectral array in EEG monitoring were studied in three patients with typical plateau waves in continuous intracranial pressure recordings. Two patients with brain-tumor and one patient with aqueductal stenosis were included. The intracranial pressure was recorded through an indwelling ventricular catheter attached to a pressure transducer. The systemic blood pressure was recorded through an intraarterial catheter placed in the femoral artery or the dorsalis pedis artery. To obtain continuous and compressed spectral array in the EEG, Berg-Fourier Analyzer by the OET-Biomedica Company of Italy was used. Simultaneous recordings of the intracranial pressure, systemic blood pressure and EEG spectral analysis were made for 180 minutes in each patient. During the plateau waves, the systemic blood pressure did not rise in spite of a marked increase in intracranial pressure, resulting in a marked decrease in the cerebral perfusion pressure. The patients, however, showed no clinical symptoms resulting from ischemia of the brain, such as vasopressor response and impairment of consciousness, but complained only headache. The spectrograms in these patients were characterized by a constant and predominant activity in the alpha or high frequencies. This structural aspect was retained throughout the continuation of plateau waves. The power of low frequencies only appeared transiently. The results suggest that some mechanisms producing and maintaining the fast and/or alpha wave activity may participate in the recurrent appearance of plateau waves. PMID- 3000415 TI - Silica, silicosis, and progressive systemic sclerosis. AB - An inquiry into the relation between exposure to silica dust, the presence of silicosis, and progressive systemic sclerosis was conducted in white South African gold miners by means of a case-control study. Seventy nine cases of progressive systemic sclerosis were matched by year of birth with an equal number of control miners selected randomly but bearing in mind the administrative channel through which the case had come to be identified. Analysis showed no association between silicosis and PSS but did show that the cumulative life time silica exposure was significantly higher in the cases compared with controls. This difference was due to a difference in the intensity of exposure to silica during mining service rather than a difference in duration of service. The results are discussed in the context of current thought on the aetiology of progressive systemic sclerosis, particularly in relation to autoimmune and genetic factors. PMID- 3000416 TI - Efficient solubilization and partial purification of sea urchin histone genes as chromatin. AB - Soluble chromatin fragments are rapidly and efficiently produced when nuclei are digested with restriction endonucleases in buffers containing very low concentrations of magnesium. Under these conditions, the sequence specificity of the restriction endonucleases is maintained, resulting in release of specific genes as fragments with discrete molecular weights that can be fractionated by size on glycerol gradients. Gradient fractions can be chosen to be significantly enriched in specific genes and their associated proteins. For instance, we can achieve a 16-fold enrichment of the chromatin containing the early histone genes of sea urchin. The enrichments produced by these methods are useful as a first step in techniques to purify specific genes as chromatin. Glycerol gradient analyses can also be used to test whether putative gene-specific proteins are actually bound to the same sequences in vivo. PMID- 3000418 TI - Differential modulation by spermidine of reactions catalyzed by type 1 prokaryotic and eukaryotic topoisomerases. AB - In the absence of DNA aggregation, spermidine inhibited the relaxation of negatively supercoiled DNA by Escherichia coli topoisomerase I at concentrations of the polyamine normally found intracellularly. Spermidine also curtailed the cleavage of negatively supercoiled ColE1 DNA by the enzyme in the absence of Mg2+. On the contrary, knotting of M13 single-stranded DNA circles catalyzed by topoisomerase I was stimulated by the polyamine. Relaxation of supercoiled DNA by eukaryotic type 1 topoisomerases, such as calf thymus topoisomerase I and wheat germ topoisomerase, was significantly stimulated by spermidine in the same range of concentrations that inhibited the prokaryotic enzyme. In reactions catalyzed by S1 nuclease, the polyamine enhanced the digestion of single-stranded DNA and inhibited the nicking of negatively supercoiled DNA. These results suggest that spermidine modifies the supercoiled duplex substrate in these reactions by modulating the degree of single strandedness. PMID- 3000417 TI - Characterization of the fusogenic properties of Sendai virus: kinetics of fusion with erythrocyte membranes. AB - A novel fluorescence assay [Hoekstra, D., De Boer, T., Klappe, K., & Wilschut, J. (1984) Biochemistry 23, 5675-5681] has been used to characterize the fusogenic properties of Sendai virus, using erythrocyte ghosts and liposomes as target membranes. This assay involves the incorporation of the "fusion-reporting" probe in the viral membrane, allowing continuous monitoring of the fusion process in a very sensitive manner. Fusion was inhibited upon pretreatment of Sendai virus with trypsin. Low concentrations of the reducing agent dithiothreitol (1 mM) almost completely abolished viral fusion activity, whereas virus binding was reduced by ca. 50%, indicating that the fusogenic properties of Sendai virus are strongly dependent on the integrity of intramolecular disulfide bonds in the fusion (F) protein. Pretreatment of erythrocyte ghosts with nonlabeled Sendai virus inhibited subsequent fusion of fluorophore-labeled virus irrespective of the removal of nonbound virus, thus suggesting that the initial binding of the virus to the target membrane is largely irreversible. As a function of pH, Sendai virus displayed optimal fusion activity around pH 7.5-8.0. Preincubation of the virus at suboptimal pH values resulted in an irreversible diminishment of its fusion capacity. Since virus binding was not affected by the pH, the results are consistent with a pH-induced irreversible conformational change in the molecular structure of the F protein, occurring under mild acidic and alkaline conditions. In contrast to virus binding, fusion appeared to be strongly dependent on temperature, increasing ca. 25-fold when the temperature was raised from 23 to 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000419 TI - Resonance raman spectra of CN--bound cytochrome oxidase: spectral isolation of cytochromes a2+, a3(2+), and a3(2+)(CN-). AB - Reduced cyanide-bound cytochrome oxidase in the absence of any oxygen gives a resonance Raman spectrum consistent with that expected for low-spin heme a. Thus, in contrast to prior reports, ligand binding of cytochrome a3 to form a six coordinate low-spin ferrous heme does not result in any unusual electronic structure, hydrogen bonding, environment, or conformation of the formyl group. It appears unlikely that there are any changes in this group in cytochrome a3 that control the ligand affinity or redox potential in physiological forms of the ferrous enzyme. With the use of our difference spectrometer and by appropriately selecting the laser excitation frequency, we are able to isolate spectrally cytochromes a2+, a3(2+), and a3(2+)(CN-). The addition of a small amount of oxygen to a preparation of the cyanide-bound reduced enzyme results in a complex with the same Raman spectrum as that previously reported to originate from the cyanide-bound reduced complex. Any oxygen present in the sample leads to enzyme turnover resulting in a mixed valence state [a2+a3(3+)(CN-)]. The comparison between the data on the cyanide-bound reduced enzyme and the data on the CO-bound reduced enzyme illustrates that cyanide binding affects only the modes that respond to the spin state of the ferrous iron, while CO binding affects vibrational modes that respond to a pi-electron density change as well. PMID- 3000420 TI - Ultraviolet resonance Raman spectra of cytochrome c conformational states. AB - Ultraviolet resonance Raman (UV RR) spectra are reported for ferricytochrome c from tuna and horse heart at pH 1.6, 7, 10, and 13, representing distinct conformational states of the protein (states II, III, IV, and V, respectively). The spectra were obtained with pulsed laser excitation at 200 and 218 nm, via H2 Raman shifting the fourth harmonic output of a pulsed YAG laser. At these deep UV wavelengths, strong enhancement is observed for vibrational modes associated with tryptophan, tyrosine, and phenylalanine side chains and with the amide groups of the polypeptide backbone. The amide I peak frequency is consistent with a dominant contribution from alpha-helical regions, although a broad high-frequency tail reflects a variety of unordered conformations. The peak frequency is 12 cm-1 higher for cytochrome c from tuna than from horse, suggesting a less tightly wound structure, which is consistent with the lower denaturation temperature previously reported for the tuna protein. The amide I peak broadens when native protein (state III) is converted to the low- or high-pH forms (states II and IV), reflecting some disordering of the polypeptide chain, but the peak frequencies are unshifted, establishing that the alpha-helical segments are not completely unfolded in these states. Raising the pH to 13 (state V), however, does produce a frequency upshift, reflecting helix unfolding. The amide II and III frequencies are likewise consistent with a dominant alpha-helix contribution in the native proteins; they gain intensity, and amide III is shifted to a lower frequency, in states II and IV, consistent with partial disordering.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000421 TI - Nonenzymic adenosine 5'-diphosphate ribosylation of poly(adenosine diphosphate ribose). AB - Poly(adenosine 5'-diphosphate ribose) [poly(ADP-ribose]) is spontaneously ADP ribosylated when it is incubated with nicotinamide adenine dinucleotide, especially in 0.5 M NaCl and at an alkaline pH. The ADP-ribose residues are monomeric and are attached to the middle of polymer chains. The linkage is similar to, and may be identical with, that of the branch points that are created in cells. RNA is also spontaneously ADP-ribosylated, but not DNA. PMID- 3000422 TI - Chemical synthesis and expression of a calmodulin gene designed for site-specific mutagenesis. AB - A gene coding for a calmodulin was synthesized and expressed in Escherichia coli. The gene was produced by the enzymatic ligation of 61 chemically synthesized DNA fragments. The gene possesses 27 unique, regularly spaced, restriction endonuclease cleavage sites to facilitate gene mutagenesis by the replacement of specific gene segments with synthetic double-stranded DNA. An expression vector containing the calmodulin gene was used to transform E. coli. Purification and characterization of calmodulin (VU-1 calmodulin) expressed by these transformants showed that it lacks two posttranslational modifications: an amino-terminal blocking group and N epsilon, N epsilon, N epsilon-trimethyllysine at position 115. The cyclic nucleotide phosphodiesterase activator properties of VU-1, higher plant, and vertebrate calmodulins were not statistically different. However, VU-1 calmodulin was found to activate nicotinamide adenine dinucleotide (NAD) kinase to a maximal level that was at least 3-fold higher than that found with higher plant and vertebrate calmodulins. This higher level of activation is also characteristic of calmodulins from Dictyostelium discoideum and Chlamydomonas reinhardtii [Roberts, D. M., Burgess, W. H., & Watterson, D. M. (1984) Plant Physiol. 75, 796-798; Marshak, D. R., Clarke, M., Roberts, D. M., & Watterson, D. M. (1984) Biochemistry 23, 2891-2899]. The only common feature among Dictyostelium, Chlamydomonas, and VU-1 calmodulins not found in higher plant and vertebrate calmodulins is an unmethylated lysine at position 115. The results indicate that the lack of methylation of lysine-115 may contribute to the maximal level of NAD kinase activation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000423 TI - Carbon isotope effect on dehydration of bicarbonate ion catalyzed by carbonic anhydrase. AB - The carbon-13 kinetic isotope effect on the dehydration of HCO3- by bovine carbonic anhydrase has been measured. To accomplish this, bicarbonate was added to a buffer solution at pH 8 containing carbonic anhydrase under conditions where purging of the product CO2 from the solution is rapid. Measurement of the isotopic composition of the purged CO2 as a function of the concentration of carbonic anhydrase permits calculation of the isotope effect on the enzymic reaction. The isotope effect on the dehydration is k12/k13 = 1.0101 +/- 0.0004. This effect is most consistent with a ping-pong mechanism for carbonic anhydrase action, in which proton transfer to or from the enzyme occurs in a step separate from the dehydration step. Substrate and product dissociation steps are at least 2-3-fold faster than the hydration/dehydration step. PMID- 3000425 TI - Identification of thyroid hormone receptors in rat liver nuclei by photoaffinity labeling with L-thyroxine and triiodo-L-thyronine. AB - Photoaffinity labeling of rat liver nuclear extract with underivatized thyroid hormones was performed after incubation with 1 nM [3',5'-125I]thyroxine ([125I]T4) or [3'-125I]triiodothyronine [( 125I]T3) by irradiation with light above 300 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the covalently photolabeled nuclear extract revealed four distinct hormone binding proteins of molecular masses 96, 56, 45, and 35 kilodaltons (kDa), respectively. Distribution of the hormone among these proteins was similar for T4 and T3. The 56- and 45-kDa proteins were the most prominently labeled. The specificity of the photoattachment of thyroid hormones to these nuclear proteins was verified by the irradiation of eight randomly chosen proteins and two proteins known to have thyroid hormone binding sites, human thyroxine binding globulin and bovine serum albumin. Only the latter two were photolabeled with [125I]T4. Competition studies performed by incubating nuclear extracts with [125I]T4 or [125I]T3 in the presence of increasing amounts of the corresponding unlabeled hormone (10-, 100-, and 1000-fold molar excess) demonstrated that (1) photoattachment of labeled T3 or T4 to the 56- and 45-kDa proteins was inhibited by 67-78% and 73-85%, respectively, after incubation with a 1000-fold molar excess of unlabeled hormone, (2) in the presence of lower molar excesses of the corresponding competitor (10- and 100-fold), photoattachment of labeled T3 or T4 to the 56- and 45-kDa receptors was gradually inhibited to a similar extent on both proteins, and (3) the 35- and 96-kDa proteins, although having thyroid hormone binding sites, display lower binding activities since the inhibition of photoattachment of labeled T3 or T4 by a 1000-fold molar excess of unlabeled hormone did not exceed 30-42% and 26-49%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000424 TI - Comparison of cytosolic and nuclear poly(A) polymerases from rat liver and a hepatoma: structural and immunological properties and response to NI-type protein kinases. AB - Poly(A) polymerases were purified from the cytosol fraction of rat liver and Morris hepatoma 3924A and compared to previously purified nuclear poly(A) polymerases. Chromatographic fractionation of the hepatoma cytosol on a DEAE Sephadex column yielded approximately 5 times as much poly(A) polymerase as was obtained from fractionation of the liver cytosol. Hepatoma cytosol contained a single poly(A) polymerase species [48 kilodaltons (kDa)] which was indistinguishable from the hepatoma nuclear enzyme (48 kDa) on the basis of CNBr cleavage maps. Liver cytosol contained two poly(A) polymerase species (40 and 48 kDa). The CNBr cleavage patterns of these two enzymes were distinct from each other. However, the cleavage pattern of the 40-kDa enzyme was similar to that of the major liver nuclear poly(A) polymerase (36 kDa), and approximately three fourths of the peptide fragments derived from the 48-kDa species were identical with those from the hepatoma enzymes (48 kDa). NI-type protein kinases from liver or hepatoma stimulated hepatoma nuclear and cytosolic poly(A) polymerases 4-6 fold. In contrast, the liver cytosolic 40- and 48-kDa poly(A) polymerases were stimulated only slightly or inhibited by similar units of the protein kinases. Antibodies produced in rabbits against purified hepatoma nuclear poly(A) polymerase reacted equally well with hepatoma nuclear and cytosolic enzyme but only 80% as well with the liver cytosolic 48-kDa poly(A) polymerase and not at all with liver cytosolic 40-kDa or nuclear 36-kDa enzymes. Anti-poly(A) polymerase antibodies present in the serum of a hepatoma-bearing rat reacted with hepatoma nuclear and cytosolic poly(A) polymerases to the same extent but only 40% as well with the liver cytosolic 48-kDa enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000426 TI - Comparative characterization of thyroid hormone receptors and binding proteins in rat liver nucleus, plasma membrane, and cytosol by photoaffinity labeling with L thyroxine. AB - Photoaffinity labeling with underivatized thyroxine (T4) was used to identify and compare the T4 binding proteins in rat liver cytosol, nuclear extract, and purified plasma membrane. When these subcellular fractions were incubated with a tracer concentration of [125I]T4, irradiated with light above 300 nm, and individually analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the radioactivity profiles revealed the presence of T4 binding proteins of molecular masses of 70, 52, 43, 37, 30, and 26 kilodaltons (kDa) in cytosol, of 96, 56, 45, and 35 kDa in nuclear extract, and of 70, 44, and 30 kDa in plasma membrane. Competition experiments performed in the presence of a 1000 fold excess of unlabeled T4 demonstrated that these binding proteins display different hormone binding activities. The similar electrophoretic mobilities of some binding proteins present in the different subcellular fractions, i.e., the 70-, 43-45-, and 30-kDa proteins, suggested that these proteins might be identical. However, double-labeling experiments in which plasma membrane, nuclear extract, and cytosol were photolabeled with either [125I] or [131I]T4 and mixed, two at a time, in all possible combinations showed that from one cellular fraction to another, the radioactivity peaks corresponding to the approximately 70-, 43-45-, and 30-kDa proteins were not superimposed. Their relative positions on the gel differed by one or two slices, which indicated differences in molecular mass of 1.9-3.6 kDa. Moreover, enzymatic digestion with Staphylococcus aureus V8 protease of these three proteins, prepared from each subcellular fraction, yielded dissimilar peptide patterns.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000427 TI - Different forms of the epidermal growth factor receptor kinase have different autophosphorylation sites. AB - Limited proteolysis converts the native (Mr 170 000) epidermal growth factor (EGF) receptor to the Mr 150 000 form of the receptor. Calcium-activated, neutral protease (purified to homogeneity from beef lung), chymotrypsin, and elastase were all similarly effective in generating the 150-kilodalton (150-kDa) form of the receptor in detergent-solubilized, membrane vesicles shed from A-431 cells. The rate of autophosphorylation with [gamma-32P]ATP of the 150-kDa form was only 10% of the rate with the native receptor. This decreased rate was not due to loss of kinase activity, since the phosphorylation of angiotensin was virtually unchanged after limited proteolysis of the native receptor kinase. However, maps of elastase-produced peptides from 170-kDa forms and elastase-generated 150-kDa forms of the EGF receptor showed that the major autophosphorylation sites in these two forms were totally different. Confirming this difference in autophosphorylation sites was the finding that the 32P label in the autophosphorylated native receptor could not be recovered in the 150-kDa form following proteolysis. This label was quantitatively recovered in 30-15-kDa peptide fragments generated simultaneously with the 150-kDa form of the receptor. Therefore, the decreased autophosphorylation of the 150-kDa form results from the loss of preferred autophosphorylation sites on the native receptor. Only 1-3% of the phosphate incorporated in the native receptor during autophosphorylation could be found on the 150-kDa autophosphorylation sites. Hence, autophosphorylation of the tyrosine sites in the 150-kDa form of the EGF receptor is markedly enhanced by removing the major sites autophosphorylated on the native form of the receptor. PMID- 3000428 TI - Characterization of membrane lipids of a general fatty acid auxotrophic bacterium by electron spin resonance spectroscopy and differential scanning calorimetry. AB - Lipids in the plasma membrane of the general fatty acid auxotroph Butyrivibrio S2 pack as a bilayer that is characterized by a high order and high motional anisotropy and a low membrane fluidity compared to mammalian plasma membranes. Lipid packing as determined by the electron spin resonance (ESR) order parameter and membrane fluidity as measured by ESR correlation times are, however, comparable to those of other bacterial membranes. Membranes of the organism grown with saturated fatty acids of well-defined hydrocarbon chain length undergo a broad reversible endothermic phase transition, the peak temperature of which is well below the growth temperature; the end-point temperature of this thermal transition approximately coincides with the minimum temperature supporting significant growth of the organism. The lipid phase transition is also reflected in the temperature dependence of various ESR parameters, whereby the transition temperature thus derived is higher than the peak temperature of the endothermic transition but still lower than the growth temperature. ESR and calorimetry evidence taken together suggest that the endothermic transition is a gel to liquid-crystal transition and that, at the growth temperature, the plasma membrane of Butyrivibrio S2 is in the liquid-crystalline state. Similar values were measured for the order parameter of cell membranes of Butyrivibrio S2 regardless of whether the organism was grown on myristic, palmitic, or stearic acid. Butyrivibrio S2 has a mechanism enabling it to maintain membrane packing and fluidity at a fairly constant level.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000429 TI - Ca2+-dependent cyclic nucleotide phosphodiesterase is activated by poly(L aspartic acid). AB - Ca2+-dependent cyclic nucleotide phosphodiesterase (Ca2+-PDE) activity was stimulated by poly(L-aspartic acid) but not by poly(L-glutamic acid), poly(L arginine), poly(L-lysine), and poly(L-proline). This activation was Ca2+ independent and did not further enhance the activation of Ca2+-PDE by Ca2+ calmodulin (CaM). Poly(L-aspartic acid) produced an increase in the Vmax of the phosphodiesterase, associated with a decrease in the apparent Km for the substrate, such being similar to results obtained with Ca2+-CaM. Poly(L-aspartic acid) did not significantly stimulate myosin light chain kinase and other types of cyclic nucleotide phosphodiesterase. CaM antagonists such as N-(6-aminohexyl) 5-chloro-1-naphthalenesulfonamide (W-7), trifluoperazine, and chlorpromazine selectively antagonized activation of the enzyme by poly(L-aspartic acid). Kinetic analysis of W-7-induced inhibition of activation of phosphodiesterase by poly(L-aspartic acid) was in a competitive fashion, and the Ki value was 0.19 mM. On the other hand, prenylamine, another type of calmodulin antagonist that binds to CaM at sites different from the W-7 binding sites, did not inhibit the poly(L aspartic acid)-induced activation of Ca2+-dependent cyclic nucleotide phosphodiesterase. These results imply that poly(L-aspartic acid) is a calcium independent activator of Ca2+-dependent phosphodiesterase and that aspartic acids in the CaM molecule may play an important role in the activation of Ca2+-PDE. PMID- 3000430 TI - Two-dimensional 1H NMR studies of cytochrome c. AB - Two-dimensional nuclear magnetic resonance techniques were used to assign the NH, C alpha H, and C beta H protons of over 60 of the 104 amino acid residues in the 1H NMR spectrum of horse ferrocytochrome c. The majority of these amino acids were completely assigned. Assignments were based on the analysis of two dimensional J-correlated (COSY), nuclear Overhauser effect (NOESY), and relayed COSY spectra and on comparisons of the J-correlated spectra of various cytochrome c species. Spin diffusion is not a problem with monomeric proteins the size of cytochrome c. Here these advances are illustrated with data that lead to the assignment of the heme-associated residues cysteine-14 and tryptophan-59, the axial ligands methionine-80 and histidine-18, the entire N-terminal helix, and several other amino acid spin systems. With these approaches, structure, structure change, the internal dynamics of cytochrome c, and the interaction of these with function are being studied, especially by observation of the hydrogen exchange behavior of essentially all the H-bonded amides and some side chain protons in both the reduced and oxidized proteins. PMID- 3000431 TI - Coordination scheme and stereochemical configuration of manganese(II) adenosine 5'-diphosphate at the active site of 3-phosphoglycerate kinase. AB - The structure of the MnIIADP complex at the active site of 3-phosphoglycerate kinase from yeast has been investigated by electron paramagnetic resonance (EPR) spectroscopy. Inhomogeneous broadening in the EPR signals for Mn(II) resulting from unresolved superhyperfine coupling to 17O regiospecifically incorporated into ADP shows that Mn(II) is coordinated to the alpha- and beta-phosphate groups of ADP at the active site of the enzyme. The EPR pattern for the enzyme-MnIIADP complex is characteristic of a predominantly axially symmetric zero-field splitting tensor. The symmetry and magnitude of the zero-field splitting interaction suggest that there is an additional negatively charged oxygen ligand in the coordination sphere of Mn(II). EPR measurements for solutions of the enzyme-MnIIADP complex in 17O-enriched water indicate that there are also two or three water molecules in the coordination sphere of the metal ion. EPR data for complexes with the two epimers of [alpha-17O]ADP have been used to determine the stereochemical configuration of the MnIIADP complex at the active site. EPR spectra for Mn(II) in the enzymic complex with (Rp)-[alpha-17O]ADP show an inhomogeneous broadening due to superhyperfine coupling with 17O whereas spectra for (Sp)-[alpha-17O]ADP complexes are indistinguishable from those for matched samples with unlabeled ADP. These results show that 3-phosphoglycerate kinase selectivity binds the alpha configuration of the alpha, beta chelate of MnIIADP. Addition of 3-phosphoglycerate to form the dead-end complex (enzyme-MnIIADP-3 phosphoglycerate) does not alter the EPR spectrum, but addition of vanadate to this complex causes marked changes in the spectral parameters.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000432 TI - Comparison of the active sites of atropinesterase and some serine proteases by spin-labeling. AB - The side chain of the serine residue in the active center of atropinesterase (AtrE), alpha-chymotrypsin (Chymo), and subtilisin A (Sub) was labeled with two paramagnetic reporter groups of different size (label I or II, respectively) by sulfonylation with N-[3-(fluorosulfonyl)phenyl]-1-oxy-2,2,5,5-tetramethyl pyrroline-3 -carboxamide or N-[6-(fluorosulfonyl)-2-naphthyl]-1-oxy-2,2,5,5 tetramethylpyrroline+ ++-3 -carboxamide. ESR spectra of labeled enzymes in 10 mM phosphate buffer, pH 7.4, were measured at temperatures between 133 and 298 K by using a home-built spectrometer operating in the absorption mode at 10-kHz field modulation. The spectra, in particular those at 276-298 K, were analyzed by computer simulation of the overall line shape according to the methods developed by Freed and co-workers, based on eigenfunction expansion. In the case of AtrE for both labels, the best agreement between experimental and simulated solution spectra was obtained with only one mobility component showing anisotropic, axially symmetric reorientation according to the Egelstaff jump-diffusion model. The axis of preferential reorientation was found to lie in the XZ plane at a polar angle of about 30 degrees with the X axis. The corresponding rotational correlation time (tau parallel) did not show appreciable viscosity/temperature (eta/T) dependence but had a constant value of 4.4 and 2.2 ns for labels I and II, respectively. The rotational correlation time associated with rotation around the axes perpendicular to that of preferential reorientation (tau perpendicular) showed the usual eta/T dependence and had a value of 22.0 ns at 276 K for both labels. The above results strongly suggest that in AtrE both nonpolar reporter groups reside in a pocket near the active serine. Contrary to the situation in AtrE, the overall mobility of the -N-O. fragments in Chymo and Sub was found to result from contributions of at least two distinct motional states, strongly and weakly immobilized. In going from label I to label II, the relative contribution of the latter state increases at the expense of that of the former. This is ascribed to an equilibrium between a relatively free state of the aromatic cores and a firmly bound position in the specificity pocket of these proteases. The apparently more rigid embedding of the spin-labels in the enzyme structure of AtrE suggests that the size of the nonpolar binding pocket in the active center region of this esterase allows a deeper penetration of the aromatic portions of the labels than is possible for the specificity pocket of Chymo or Sub. PMID- 3000433 TI - Protein and lipid structural transitions in cytochrome c oxidase dimyristoylphosphatidylcholine reconstitutions. AB - The thermotropic behavior of the mitochondrial enzyme cytochrome c oxidase (EC 1.9.3.1) reconstituted in dimyristoylphosphatidylcholine (DMPC) vesicles has been studied by using high-sensitivity differential scanning calorimetry and fluorescence spectroscopy. The incorporation of cytochrome c oxidase into the phospholipid bilayer perturbs the thermodynamic parameters associated with the lipid phase transition in a manner analogous to other integral membrane proteins: it reduces the enthalpy change, lowers the transition temperature, and reduces the cooperative behavior of the phospholipid molecules. Analysis of the dependence of the enthalpy change on the protein:lipid molar ratio indicates that cytochrome c oxidase prevents 99 +/- 5 lipid molecules from participating in the main gel-liquid-crystalline transition. These phospholipid molecules presumably remain in the same physical state below and above the transition temperature of the bulk lipid, thus providing a more or less constant microenvironment to the protein molecule. The effect of the phospholipid bilayer matrix on the thermodynamic stability of the cytochrome c oxidase complex was examined by high sensitivity differential scanning calorimetry. Detergent (Tween 80)-solubilized cytochrome c oxidase undergoes a complex, irreversible thermal denaturation process centered at 56 degrees C and characterized by an enthalpy change of 550 +/- 50 kcal/mol of enzyme complex. Reconstitution of the cytochrome c oxidase complex into DMPC vesicles shifts the transition temperature upward to 63 degrees C, indicating that the phospholipid bilayer moiety stabilizes the native conformation of the enzyme. The lipid bilayer environment contributes approximately 10 kcal/mol to the free energy of stabilization of the enzyme complex. The thermal unfolding of cytochrome c oxidase is not a two-state process.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000434 TI - Modulation of the receptor-coupled adenylate cyclase system in HeLa cells by sodium butyrate. AB - Exposure of HeLa cells to 5 mM sodium butyrate, but not 0.6 mM, resulted in a more efficient coupling between their beta-adrenergic receptors and the guanine nucleotide binding stimulatory (Ns) component of adenylate cyclase. Both concentrations of the fatty acid, however, caused an increase in receptor number. beta receptors from control and butyrate-treated cells had the same affinity for isoproterenol. Modulation of this affinity by GTP was greatly enhanced, however, in cells treated with 5 mM butyrate compared to untreated and 0.6 mM butyrate treated cells. The concentration of isoproterenol required to half-maximally stimulate adenylate cyclase (Kact) was reduced in cells treated with 5 mM butyrate. In addition, the Kact for GTP in the presence, but not the absence, of isoproterenol was reduced. The effect of butyrate on the coupling between beta receptors and Ns was analyzed in detail by monitoring the activation of Ns by guanine 5'-O-(3-thiotriphosphate) (GTP gamma S) in a two-step assay. In the absence of isoproterenol, Ns from control and 5 mM butyrate treated cells was activated to the same extent with the same time course and Kact for GTP gamma S. In the presence of isoproterenol, Ns from 5 mM butyrate treated cells was activated more rapidly and extensively than Ns from control cells. The Kact for both GTP gamma S and isoproterenol also was reduced. The rate of agonist-mediated activation of Ns was strongly dependent on temperature, which accentuated the differences between 5 mM butyrate treated and control cells. At 4 degrees C, the difference in rate was 8.8-fold.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000435 TI - cGMP- and phosphodiesterase-dependent light-scattering changes in rod disk membrane vesicles: relationship to disk vesicle-disk vesicle aggregation. AB - Visible light activates a large guanosine cyclic 3',5'-phosphate (cGMP)- and phosphodiesterase (PDE)-dependent infrared light-scattering change in suspensions of photoreceptor disk membranes. Reconstitution experiments show that this signal requires bleached rhodopsin, G protein (three polypeptide subunits of Mr 39 000, 37 000, and 6000 which comprise the GTPase), phosphodiesterase, cGMP, and GTP. The lowest light intensity which elicits the light-scattering signal bleaches 0.002% rhodopsin. cGMP and GTP hydrolysis occurs more slowly than the initial phase of the scattering signal, and the kinetics of nucleotide hydrolysis do not correlate with any phase of the signal. Hydrolysis-resistant analogues of cGMP and GTP support the initial decreasing phase of the signal. Thus, the signal apparently depends upon nucleotide binding rather than hydrolysis. Microscopic observations made under the same conditions as light-scattering experiments show that vesicle-vesicle aggregation and disaggregation occur. The data suggest that light and nucleotide activations of the cyclic nucleotide cascade enzymes are responsible for the vesicle aggregation process and nucleotide hydrolysis for vesicle disaggregation. The vesicle aggregation-disaggregation phenomenon appears likely to be the physical basis of the cGMP- and PDE-dependent changes in infrared transmission. PMID- 3000436 TI - Inhibition of (Na+,K+)-ATPase by dicyclohexylcarbodiimide. Evidence for two carboxyl groups that are essential for enzymatic activity. AB - N,N'-Dicyclohexylcarbodiimide (DCCD), a reagent that reacts with carboxyl groups under mild conditions, irreversibly inhibits (Na+,K+)-ATPase activity (measured by using 1 mM ATP) with a pseudo-first-order rate constant of 0.084 min-1 (0.25 mM DCCD and 37 degrees C). The partial activities of the enzyme, including (Na+,K+)-ATPase at 1 microM ATP, Na+-ATPase, and the formation of enzyme-acyl phosphate (E-P), decayed at about one-third the rate at which (Na+,K+)-ATPase at 1 mM ATP was lost. The formation of E-P from inorganic phosphate was unaffected by DCCD while K+-phosphatase activity decayed at the same rate as (Na+,K+)-ATPase measured at 1 mM ATP. The enzyme's substrates (i.e., sodium, potassium, magnesium, and ATP) all decreased the rate of DCCD inactivation of (Na+,K+) ATPase activity measured at either 1 mM or 1 microM ATP. The concentration dependence of the protection afforded by each substrate is consistent with its binding at a catalytically relevant site. DCCD also causes cross-linking of the enzyme into species of very high molecular weight. This process occurs at about one-tenth the rate at which (Na+,K+)-ATPase activity measured at 1 mM ATP is lost, too slowly to be related to the loss of enzymatic activity. Labeling of the enzyme with [14C]DCCD shows the incorporation of approximately 1 mol of DCCD per mole of large subunit; however, the incorporation is independent of the loss of enzymatic activity. The results presented here suggest that (Na+,K+)-ATPase contains two carboxyl groups that are essential for catalytic activity, in addition to the previously known aspartate residue which is involved in formation of E-P.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000437 TI - Multiple ion-dependent and substrate-dependent Na+/K+-ATPase conformational states. Transient and steady-state kinetic studies. AB - The hydrolysis of beta-(2-furyl)acryloyl phosphate (FAP), catalyzed by the Na+/K+ ATPase, is faster than the catalyzed hydrolysis of ATP. This is due to catalyzed hydrolysis of the pseudosubstrate by K+-dependent states of the enzyme, thus bypassing the Na+-dependent enzyme states that are required and are rate limiting in ATP hydrolysis. Unlike ATP, FAP is a positive effector of the E2 state. A study of FAP hydrolysis permits a detailed analysis of later steps in the overall ion translocation-ATP hydrolysis pathway. During the steady state of FAP hydrolysis in the presence of K+, substantial phosphoryl-enzyme is formed, as is indicated by the covalent incorporation of 32P from [32P]FAP. A comparison of the phosphoryl-enzyme yield with the rate of overall hydrolysis reveals that at 25 degrees C the phosphoryl-enzyme formed is all kinetically competent. Both the yield of phosphoryl-enzyme and the rate of overall hydrolysis of FAP are [K+] dependent. The transition E1 in equilibrium E2 is also [K+] dependent, but the rate of transition is differently affected by [K+] than are the above-mentioned two processes. Two distinct roles for K+ are indicated, as an effector of the E1 E2 equilibrium and as a "catalyst" in the hydrolysis of the E2-P. In contrast to the results at 25 degrees C, a virtually stoichiometric yield of phosphoryl enzyme occurs at 0 degree C in the presence of Na+ and the absence of K+. At lower concentrations of K+ and in the presence of Na+, the hydrolysis of FAP at 0 degree C proceeds substantially through the E1-E2 pathway characteristic of ATP hydrolysis. The selectivity of FAP for the E2-K+-dependent pathway is due to the thermal inactivation of E1 at 25 degrees C in the absence of ATP or ATP analogues, even at high concentrations of Na+. These results emphasize the existence of multiple functional "E1" and "E2" states in the overall ATPase-ion translocation pathway. PMID- 3000438 TI - Studies of the structure of fructose-6-phosphate 2-kinase:fructose-2,6 bisphosphatase. AB - Some physicochemical properties of a homogeneous preparation of a bifunctional enzyme, fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase, were determined. The molecular weight of the enzyme is 101 000 as determined by high speed sedimentation equilibrium. The molecular weight of dissociated enzyme is 55 000 in 6 M guanidinium chloride by sedimentation equilibrium and in sodium dodecyl sulfate by polyacrylamide gel electrophoresis. A value of 4.7 was observed for the isoelectric point. Tryptic peptide maps and high-performance liquid chromatography of the trypsin-digested enzyme revealed approximately 60 peptides. Amino acid analysis of the enzyme shows that it contains 27 lysine and 36 arginine residues per 55 000 daltons. No free N-terminal amino acid residue was detectable, suggesting that it is blocked. Hydrolysis of the enzyme by carboxypeptidases A and B releases tyrosine followed by histidine and arginine, indicating that the amino acid sequence at the carboxyl terminus is probably -Arg His-Tyr. Tryptic digestion of [32P]phosphofructose-6-phosphate 2-kinase:fructose 2,6-bisphosphatase yields a 32P-labeled peptide detected by tryptic peptide mapping and high-performance liquid chromatography. Thermolysin digestion of CNBr cleaved 32P-enzyme also yields a single 32P-peptide. These results indicate that fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase is a dimer of 55 000 daltons and the subunits are very similar, if not identical. PMID- 3000439 TI - Protein carboxyl methylation-demethylation system in developing rat livers. AB - Protein carboxyl methyltransferase and protein methylesterase activity was assayed in various cell fractions prepared from rat livers. Significant amounts of protein carboxyl methyltransferase were detected in the cytosol and nucleoplasm. The cellular concentration of this enzyme paralleled development, activity being highest in the liver from young animals. If methylation was inhibited at any point during the reaction with S-adenosylhomocysteine, protein methylesterase activity was evident by a rapid decrease in carboxyl-methylated proteins. Protein methylesterase activity could be assessed by measuring the amount of [3H]methanol present in reaction filtrates. After a 10-min lag, the rate of demethylation was equivalent to the rate of methylation. The turnover of methyl groups was primarily enzymatic, since little or no methanol was generated when adrenocorticotropic hormone was incubated with purified protein carboxyl methyltransferase. Assessment of protein methylesterase activity as a function of the amount of methanol in the reaction filtrates represents minimal values, since the resultant [3H]methanol was metabolized rapidly via an alcohol dehydrogenase and/or oxidase. The rapid turnover of the protein methyl esters makes it difficult to assess the endogenous methyl acceptor proteins. Protein methyl esters were not detectable in any significant amounts in hepatic cell fractions in vivo; however, the nuclei contained measurable amounts of carboxyl-methylated proteins in vitro. These proteins are firmly bound to DNA but are not an integral part of the nucleosome. Analysis of the proteins, after fractionation on hydroxylapatite and sodium dodecyl sulfate-acrylamide gel electrophoresis, revealed that several non-histone chromosomal proteins were carboxyl methylated. The approximate molecular weights of these proteins were 172K, 106K, 98K, 81K, 66K, 62K, 52K, and 38K.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000440 TI - Characterization of the bovine prothrombin gene. AB - The bovine prothrombin gene was characterized by Southern blot analysis of bovine genomic DNA using bovine prothrombin cDNA fragments as hybridization probes. These analyses suggested that the bovine genome contains a single prothrombin gene that is at least 10 kilobase pairs (kbp) in size. To characterize the gene more thoroughly, two bovine genomic phage libraries were screened by using prothrombin cDNAs as hybridization probes. Heteroduplex analysis of the cloned genomic DNA and cDNA showed that the prothrombin gene is 14.9 kbp in size and contains at least 14 exons interrupted by 13 introns. The exons vary in size from 28 to 317 base pairs (bp), while the introns vary in size from less than 100 to 6940 bp. Regions of self-complementarity were observed within some of the introns, suggesting the presence of inverted repeat sequences. The bovine prothrombin gene shows similarities in structure to both the human prothrombin gene and the human factor IX gene. PMID- 3000441 TI - Molecular structure of the beta-adrenergic receptor. AB - The beta-adrenergic receptor from several tissues has been purified to homogeneity or photoaffinity radiolabeled and its subunit molecular weight determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. In this study we have examined the oligomeric structure of nondenatured beta 1- and beta 2-adrenergic receptor proteins, as solubilized with the detergent digitonin. Model systems used were frog and turkey red blood cell as well as rat, rabbit, and bovine lung plasma membrane preparations. To correct for the effects of detergent binding, sedimentation equilibrium analysis in various solvents, as adapted for the air-driven ultracentrifuge, was used. With this approach an estimate of 6 g of digitonin/g of protein binding was determined, corresponding to a ratio of 180 mol of digitonin/mol of protein. Protein molecular weights estimated by this method were 43 500 for the turkey red blood cell beta 1 receptor and 54 000 for the frog red blood cell beta 2 receptor. Molecular weights of 60 000-65 000 were estimated for beta 1 and beta 2 receptors present in mammalian lungs. These values agree with estimates of subunit molecular weight obtained by SDS gel electrophoresis of purified or photoradiolabeled preparations and suggest beta-adrenergic receptors to be digitonin solubilized from the membrane as single polypeptide chains. PMID- 3000442 TI - Mode of reversible binding of neocarzinostatin chromophore to DNA: evidence for binding via the minor groove. AB - Two general approaches have been taken to understand the mechanism of the reversible binding of the nonprotein chromophore of neocarzinostatin to DNA: (1) measurement of the relative affinity of the chromophore for various DNAs that have one or both grooves blocked by bulky groups and (2) studies on the influence of adenine-thymine residue-specific, minor groove binding agents such as the antibiotics netropsin and distamycin on the chromophore-DNA interaction. Experiments using synthetic DNAs containing halogen group (Br, I) substituents in the major groove or natural DNAs with glucosyl moieties projecting into the major groove show that obstruction of the major groove does not decrease the binding stoichiometry or the binding constant for the DNA-chromophore interaction. Chemical methylation of bases in both grooves of calf thymus DNA, resulting in 13% methylation of N-7 of guanine in the major groove and 7% methylation of N-3 of adenine in the minor groove, decreases the binding affinity and increases the size of the binding site for neocarzinostatin chromophore. Similar results were obtained whether binding parameters were determined directly by spectroscopic measurements or indirectly by measuring the ability of the DNA to protect the chromophore against degradation. On the other hand, netropsin and distamycin compete with neocarzinostatin chromophore for binding to the minor groove of DNA, as shown by their decrease in the ability of poly(dA-dT) to protect the chromophore against degradation and their reduction in chromophore-induced DNA damage as measured by thymine release.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000443 TI - Apolipoprotein E: phospholipid binding studies with synthetic peptides containing the putative receptor binding region. AB - To define the lipid and receptor binding regions of apolipoprotein E (apoE), we have synthesized four peptides beginning at residue 169 and continuing through the putative receptor binding region and ending at residue 129 so as to include a proposed lipid binding domain. The peptides were synthesized by solid-phase techniques, cleaved with anhydrous HF, and purified by ion-exchange and semipreparative reversed-phase high-performance liquid chromatography (HPLC). The peptides had the correct amino acid composition and were greater than 99% pure by analytical reversed-phase HPLC. The circular dichroic spectrum of each peptide was recorded before and after mixing with dimyristoylphosphatidylcholine. With apoE (148-169), apoE (144-169), and apoE (139-169), no changes were observed in the ellipticity at 222 nm. However, with apoE (129-169), an increase in alpha helicity to approximately 42% was observed. Density gradient ultracentrifugation of the lipid-peptide mixture permitted isolation of a complex with apoE (129-169) with a molar ratio of lipid to peptide of 125:1, which was stable to recentrifugation. The alpha-helicity of the peptide in the complex was estimated to be 56%. No complexes were isolated from the gradients of the shorter peptides. Therefore, we conclude that the amphipathic helix formed by residues 130-150 contains one of the lipid binding regions of apoE. PMID- 3000444 TI - Direct evidence for an ADP-sensitive phosphointermediate of (K+ + H+)-ATPase. AB - Direct evidence for the occurrence of an ADP-sensitive phosphoenzyme of (K+ + H+) ATPase, the proton-pumping system of the gastric parietal cell is presented. The enzyme is phosphorylated with 5 microM [gamma-32P]ATP in 50 mM imidazole-HCl (pH 7.0) and in the presence of 7-15 microM Mg2+. Addition of 5 mM ADP to this preparation greatly accelerates its hydrolysis. We have been able to establish this by stopping the phosphorylation with radioactive ATP, by adding 1 mM non radioactive ATP, which leads to a slow monoexponential process of dephosphorylation of 32P-labeled enzyme. The relative proportion of the ADP sensitive phosphoenzyme is 22% of the total phosphoenzyme. Values for the rate constants of breakdown and interconversion of the two phosphoenzyme forms have been determined. PMID- 3000445 TI - A pH titration study on the ionic bridging within lipopolysaccharide aggregates. AB - The packing of lipopolysaccharide aggregates from rough strains of Escherichia coli was examined at different pH values. Lipopolysaccharide head-group motion, measured with an electron spin resonance probe, was found to be dependent on pH, and indicated the existence of multiple ionizable groups. Lipopolysaccharide from a rough (Ra) and a heptose-less (Re) mutant were more rigid at pH 5 than at pH 10.5. In addition, head-group mobility of the magnesium salt of Ra lipopolysaccharide was substantially less than that of the sodium salt at pH 7.0, whereas at high pH (pH 12) the two salts were equally fluid. Changes in head group packing were also reflected in pH-dependent changes in the phase transition measured with differential scanning calorimetry. The enthalpy of the transition, delta Ht, for the sodium salt of Re lipopolysaccharide was greatest at pH 7.5 and approached zero in both the acidic and the basic pH ranges. We propose that fixed charges in the core and lipid A regions significantly influence lipopolysaccharide head-group motion and the lipopolysaccharide aggregation state. Furthermore, ionic bridging among phosphate groups dramatically rigidifies head group interactions in the neutral to acidic pH ranges. PMID- 3000446 TI - ESR studies of the erythrocyte membrane skeletal protein network: influence of the state of aggregation of spectrin on the physical state of membrane proteins, bilayer lipids, and cell surface carbohydrates. AB - The stability of the human erythrocyte membrane skeletal network is reported to be dependent on the state of aggregation of spectrin and decreased or increased by polyphosphate anions or the polyamine, spermine, respectively. We have employed polyacrylamide gel electrophoresis and electron spin resonance (ESR) utilizing spin labels specific for membrane proteins, bilayer lipids, or cell surface sialic acid in order to gain insight into these observations and into the reliability of the ESR spectra of the protein-specific spin label used to correctly report the interactions of the skeletal protein network. The major findings are: (1) We confirm previous reports that the preferred state of spectrin aggregation in the skeletal network is tetrameric and that spectrin can be reversibly transformed to dimeric spectrin and back to tetrameric spectrin on the membrane. (2) The ESR spectra of the protein specific maleimide spin label employed accurately reflect the state of aggregation of spectrin. (3) As dimeric spectrin is increased on the membrane or when 2,3-bisphosphoglycerate was added to spin-labeled membranes, increased segmental motion of protein spin label binding sites reflecting decreased protein-protein interactions in the skeletal network is observed (P less than 0.002 and P less than 0.005, respectively). (4) Conversely, as protein-protein interactions between skeletal proteins or between skeletal proteins and the bilayer are increased by spermine (reflected in the total inability to extract spectrin from the membrane in contrast to control membranes), highly decreased segmental motion of the protein specific spin label binding site is observed (P less than 0.005). (5) The dimeric-tetrameric state of spectrin aggregation on the membrane does not have influence on the order or motion of bilayer lipids nor on the rotational rate of spin-labeled, cell-surface sialic acid, a result also observed when protein-protein interactions were decreased by 2,3-bisphosphoglycerate. In contrast, increased protein-protein interactions by addition of spermine produced a small, but significant, increase in order and decrease in motion of bilayer lipids near the membrane surface as well as a nearly 40% decrease in the apparent rotational correlation time of spin labeled, cell surface sialic acid (P less than 0.002). These latter observations are discussed with reference to possible associations of phospholipids and the major, transmembrane sialoglycoprotein with the skeletal protein network. PMID- 3000447 TI - Visualization of lactotransferrin brush-border receptors by ligand-blotting. AB - The uptake of iron (III) mediated by lactotransferrin to human biopsies from upper intestine has suggested the presence of specific receptors for human lactotransferrin at the brush border (Cox, T., Mazurier, J., Spik, G., Montreuil, J. and Peters, T.J. (1979) Biochim. Biophys. Acta 588, 120-128). In the present data, using 125I-radiolabeled transferrins, we have demonstrated that a preparation of microvillous membrane vesicles, from rabbit jejunal brush-border specifically binds human lactotransferrin. This binding is specific, saturable and calcium dependent. Scatchard plots analysis of lactotransferrin binding indicates 1.5 X 10(13) sites per mg of membrane proteins with an equilibrium constant of 1.2 X 10(6) M-1. Sodium dodecyl sulfate solubilization of the brush border proteins allows the lactotransferrin receptor to retain its binding activity. Moreover, the ligand blotting of the detergent solubilized membrane proteins on nitrocellulose sheet and after incubation with 125I-labeled lactotransferrin, has shown that the receptor is a protein of about 100 kDa. In the same experimental conditions, the rabbit microvillous membrane vesicles do not specifically bind rabbit serotransferrin indicating the absence of serotransferrin receptors at the brush border. PMID- 3000448 TI - Expression-linked demethylation of 5-methylcytosines in the chicken vitellogenin gene region. AB - We have studied the methylation status of the estradiol-controlled chicken vitellogenin (Vtg) gene, which is expressed in the liver. A 30-kb region was investigated, containing 17 HpaII and 18 HhaI sites, of which 21 are in the 22-kb gene. Of these 21 sites, 9 were found to be demethylated in laying-hen liver relative to immature chicken liver. Outside the transcribed region, only one site was found to be relatively undermethylated in laying-hen liver. This site, at 0.6 kb in front of the gene, is, as shown earlier, also demethylated in rooster or immature chicken liver upon primary hormone stimulation, as well as in the non expressing estradiol target organ oviduct. In this respect, this site sharply contrasts with those in the transcribed region, which appear to become demethylated only upon prolonged transcription of the gene. PMID- 3000449 TI - Comparison of the 5' regions of human and mouse carbonic anhydrase II genes and identification of possible regulatory elements. AB - The nucleotide sequence of the 5' region of the human carbonic anhydrase II gene has been determined. This sequence begins 643 base pairs upstream from the ATG start site and continues through exon 1, intron 1, exon 2 and the adjoining 125 nucleotides of intron 2. The human sequence is compared with homologous regions of the mouse (YBR strain) carbonic anhydrase II gene by aligning the two sequences for optimal homology. In addition to a TATA box and a putative CCAAT box (CCACC in human and CCACT in mouse), three conserved tandem-repeat elements in mouse and two in human (consensus: cCNGTCACCTCCgC) are located 15 and 22 base pairs upstream, respectively, from the CCAAT boxes in the human and mouse sequences. This repeat element is similar to a tandem repeat sequence located at about the same position in mammalian beta-globin genes, and may represent regulatory elements common to both the carbonic anhydrase and beta-globin genes. The regions surrounding exon 1 are extremely G + C-rich in both human and mouse genes. In addition, several CCGCCC or GGGCGG sequences which may be important for transcriptional efficiency are found in the 5' flanking regions of the human and mouse genes. PMID- 3000450 TI - A new restriction endonuclease Eco31I recognizing a non-palindromic sequence. AB - A restriction endonuclease with a novel site-specificity has been isolated from the Escherichia coli strain RFL31. The nucleotide sequences around a single Eco31I cut on pBR322 DNA and two cuts of lambda DNA have been compared. A common 5'GAGACC 3'CTCTGG sequence occurs near each cleavage site. Precise mapping of the cleavages in both DNA strands places the cuts five nucleotides to the left of the upper sequence and one nucleotide to the left of the lower sequence. This enabled us to deduce the following recognition and cleavage specificity of Eco31I: 5' GGTCTCN decreases 3' CCAGAGN NNNN increases. PMID- 3000451 TI - Increased labelling of polyphosphoinositide in chemically transformed cell line C3H10T1/2 CL8. AB - The effect of malignant transformation of cells on phosphatidylinositol metabolism was investigated using C3H10T1/2 cells and its chemically transformed cell line, MCA CL-16 cells. We found that incorporation of [32P]Pi into polyphosphoinositide was greatly increased in the transformed cells. A similar tendency was observed when myo-[2-3H]inositol was used as a labelling reagent. It is also observed that influx of labelled inorganic phosphate is enhanced 2-fold by the cell transformation. Therefore, promotion of polyphosphoinositide labelling in the transformed cell might be caused not only by the enhanced metabolism of phosphatidylinositol but also by the increased membrane permeability for radioactive labelling reagents. PMID- 3000453 TI - Choline kinase activity in Plasmodium-infected erythrocytes: characterization and utilization as a parasite-specific marker in malarial fractionation studies. AB - Choline kinase (EC 2.7.1.32) was investigated in plasmodium falciparum-infected erythrocytes. Disrupted infected erythrocytes had a choline kinase activity of 1.9 +/- 0.2 nmol phosphorylcholine/10(7) infected cells per h, whereas the activity in normal uninfected erythrocytes was less than 6 pmol/10(7) cells per h. A broad alkaline optimal pH (7.9-9.2) was observed. The Km values for choline and ATP were 79 +/- 20 microM, and 1.3 +/- 0.3 mM, respectively. ATP concentrations higher than 12 mM inhibited choline kinase. Maximal activity was registered with a Mg2+ concentration of 10 mM, whereas its replacement by Mn2+, or other divalent cations, involved a decrease in choline kinase activity of at least 75%. Inhibition by products of the reaction, such as phosphorylcholine and ADP was investigated. In plasmodium knowlesi-infected erythrocytes, choline kinase had similar properties, but with a much higher specific activity of 16.4 +/- 2.1 nmol/10(7) infected cells per h. Subcellular fractionation of P. knowlesi infected erythrocyte suspensions revealed that choline kinase was located exclusively in the cytosol of the parasite. We show that this enzyme is a useful index of parasite cytosolic content leakage, when infected erythrocytes are fractionated by saponin lysis or nitrogen decompression. PMID- 3000452 TI - Lipoxygenase in trout gill tissue acting on arachidonic, eicosapentaenoic and docosahexaenoic acids. AB - Lipoxygenase activity was characterized in the gill tissue of fresh-water trout. Incubation of arachidonic acid with gill preparations yielded 12 hydroxyeicosatetraenoic acid as the major product, suggesting a 12-lipoxygenase. Eicosapentaenoic acid was similarly converted to the 12-hydroxyeicosapentaenoic acid. Both arachidonic acid and docosahexaenoic acid were converted with equal apparent velocities and affinities into single monohydroxy derivatives. Analyses of the hydroxy product of docosahexaenoic acid were consistent with 14 hydroxydocosahexaenoic acid. This enzyme activity was localized to the cytosolic fraction and displayed a broad pH optimum around pH 7. The enzyme was insensitive to the cyclooxygenase inhibitors indomethacin and aspirin but activity was strongly inhibited in the presence of the lipoxygenase inhibitors, SnCl2 (5 mM), esculetin (10 microM) and eicosatetraynoic acid (100 microM). PMID- 3000454 TI - Discrimination between subclasses of human high-density lipoproteins by the HDL binding sites of bovine liver. AB - The binding of human 125I-labeled HDL3 (high-density lipoproteins, rho 1.125 1.210 g/cm3) to a crude membrane fraction prepared from bovine liver closely fit the paradigm expected of a ligand binding to a single class of identical and independent sites, as demonstrated by computer-assisted binding analysis. The dissociation constant (Kd), at both 37 and 4 degrees C, was 2.9 micrograms protein/ml (approx. 2.9 X 10(-8) M); the capacity of the binding sites was 490 ng HDL3 (approx. 4.9 pmol) per mg membrane protein at 37 degrees C and 115 at 4 degrees C. Human low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) also bound to these sites (Kd = 41 micrograms protein/ml, approx. 6.7 X 10(-8) M for LDL, and Kd = 5.7 micrograms protein/ml, approx. 7.0 X 10(-9) M for VLDL), but this observation must be considered in light of the fact that the normal circulating concentrations of these lipoproteins are much lower than those of HDL. The binding of 125I-labeled HDL3 to these sites was inhibited only slightly by 1 M NaCl, suggesting the presence of primarily hydrophobic interactions at the recognition site. The binding was not dependent on divalent cations and was not displaceable by heparin; the binding sites were sensitive to both trypsin and pronase. Of exceptional note was the finding that various subclasses of human HDL (including subclasses of immunoaffinity-isolated HDL) displaced 125I-labeled HDL3 from the hepatic HDL binding sites with different apparent affinities, indicating that these sites are capable of recognizing highly specific structural features of ligands. In particular, apolipoprotein A-I containing lipoproteins with prebeta electrophoretic mobility bound to these sites with a strikingly lower affinity (Kd = 130 micrograms protein/ml) than did the other subclasses of HDL. PMID- 3000455 TI - [Interaction of monovalent cations with phospholipid liposomes]. AB - Action of monovalent cations on phospholipid liposome surface properties was studied using hydrophobic spin labels. Addition of monovalent cations to liposome incubation media led to an increase of the partition coefficient of negatively charged I1,14 spin label in the membrane, but produced an opposite effect in the partition coefficient of positively charged CAT12 spin label. The effect increased with cation concentration in series of NH4+ greater than Li+ greater than Na+ greater than K+ greater than or equal to Rb+ greater than or equal to Cs+. The result is explained by the thermodynamics of cation hydration and quantum effects of cation interaction with specifically ordered ligands. PMID- 3000456 TI - [Effect of hydration on the molecular dynamic properties of human serum albumin at low temperatures]. AB - The effect of temperature and hydration on phosphorescence of chromatophores and on saturation curves of ESR spectra of spin labels covalently bound to human serum albumin was studied. It has been shown that at 90-260 degrees K albumin hydration results in intensification of motions of hydrophobic parts with low frequencies (vc less than or equal to 10(3) s-1) and does not affect the motions of hydrophobic and surfacial parts with high frequency. PMID- 3000457 TI - Post-translational processing and activation of insulin and EGF proreceptors. AB - We have investigated the role of glycosylation on the post-translational processing of the insulin, and EGF proreceptor polypeptides. Following translation of the insulin proreceptor, by 3T3-L1 adipocytes, about 1.5 h are required for its conversion into active receptor; an additional 1.5 h are needed for the active receptor to reach the plasma membrane. During this 3-hour period the proreceptor undergoes a complex series of processing events, glycosylation being an essential processing step. Thus, treatment of 3T3-L1 adipocytes with tunicamycin caused the loss of cellular insulin binding activity and the accumulation of an inactive aglyco-proreceptor. Similarly, it was demonstrated in human A431 epidermoid carcinoma cells that the initial EGF-proreceptor (160 kDa) translation product undergoes a slow (t 1/2 = 30 min) processing step by which ligand (EGF) binding activity was acquired. It was shown that N-linked core oligosaccharide addition is essential for this critical processing step and the acquisition of EGF binding activity. This was found not to require the conversion of high mannose chains to complex chains which have been capped with fucose and sialic acid. Possible explanations for this activation in terms of translocation of intermediates and/or formation of disulfide bonds are discussed. To investigate post-translational processing of normal insulin proreceptor and the role of glycosylation in active receptor formation, metabolic labeling experiments were conducted. The first 35S-methionine-labeled intermediate detected is a 190 kDa polypeptide (proreceptor) which is rapidly (t 1/2 = 15 min) processed into a 210 kDa species. Both polypeptides contain N-linked core oligosaccharide chains, but in the latter case these chains appear to contain terminal N-acetylglucosamine. The 210 kDa precursor is converted slowly (t 1/2 = 2 h) by proteolytic processing into a 125 kDa (alpha') and 83 kDa (beta') species. Immediately prior to insertion into the plasma membrane, 3 h after its synthesis, the alpha' and beta' precursors are converted to mature receptor comprised of alpha-(135 kDa) and beta-(95 kDa) subunits. The 125 kDa alpha'- and 83 kDa beta'-subunit precursors are endoglycosidase H-sensitive and their oligosaccharide chains do not contain terminal sialic acid. Just prior to insertion into the plasma membrane the alpha' and beta' precursors are sialylated, apparently in the Golgi apparatus, giving rise to the 135 kDa alpha and 95 kDa beta receptor subunits and become Endo H-resistant and neuraminidase sensitive. A proposed sequence of post-translational processing events for the insulin proreceptor is shown in Figure 10.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3000458 TI - Regulation of insulin receptor kinase by multisite phosphorylation. AB - The regulation of the insulin receptor kinase by phosphorylation and dephosphorylation has been examined. Under in vitro conditions, the tyrosine kinase activity of the insulin receptor toward histone is markedly activated when the receptor either undergoes autophosphorylation or is phosphorylated by a purified preparation of src tyrosine kinase on tyrosine residues of its beta subunit. The elevated kinase activity of the phosphorylated insulin receptor is readily reversed when the receptor is dephosphorylated with alkaline phosphatase. Analysis of tryptic digests of phosphorylated insulin receptor using reverse phase high pressure liquid chromatography suggests that phosphorylation of a specific tyrosine site on the receptor beta subunit may be involved in the mechanism of the receptor kinase activation. Further studies indicate that tyrosine phosphorylation-mediated increase in insulin receptor activity also occurs in intact cells. Thus, when the histone kinase activities of insulin receptor from control and insulin-treated H-35 hepatoma cells are assayed in vitro following the purification of the receptors under conditions which preserve the phosphorylation state of the receptors, the insulin receptors extracted from insulin-treated cells exhibit histone kinase activities 100% higher than those from control cells. The elevated receptor kinase activity from insulin-treated cells appears to result from the increase in phosphotyrosine content of the receptor. Taken together, these results indicate that tyrosine phosphorylation of the insulin receptor beta subunit exerts a major stimulatory effect on the kinase activity of the receptor. Insulin receptor partially purified by specific immunoprecipitation from detergent extracts of control and isoproterenol-treated cells have similar basal but diminished insulin-stimulated beta subunit autophosphorylation activities when incubated with [gamma-32 P]ATP. Similarly, the ability of insulin to stimulate the receptor beta subunit phosphorylation in intact isoproterenol-treated adipocytes is greatly attenuated, whereas, the basal phosphorylation of the insulin receptor is slightly increased by the beta catecholamine. These data indicate that in rat adipocytes, a cyclic AMP-mediated mechanism, possibly through serine and threonine phosphorylation of the receptor or its regulatory components, may uncouple the receptor tyrosine kinase activity from activation by insulin. Treatment of 32P-labeled H-35 hepatoma cells with phorbol myristate acetate (PMA) results in a marked increase in serine phosphorylation of the insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3000459 TI - Synthetic peptide approach to the analysis of kinase activities of avian EGF receptor and v-erbB protein. AB - Analysis of the structure and function of a protein such as the epidermal growth factor receptor is facilitated by the use of antibodies directed against discrete portions of the protein. Here, we describe the characterization and use of antibodies directed against synthetic peptides corresponding to specific portions of the epidermal growth factor receptor and/or v-erbB protein. In particular, one useful antiserum has allowed us to compare the protein kinase activities of the epidermal growth factor receptor and the v-erbB proteins and to conclude that the v-erbB protein is a protein-tyrosine specific kinase as is its homologue the avian epidermal growth factor receptor. PMID- 3000460 TI - The insulin receptor kinase. AB - The insulin receptor appears as a tetrameric glycoprotein consisting of two Mr 130,000 subunits (alpha), and two Mr 95,000 subunits (beta) in a disulfide-linked complex. Insulin bound to its specific cell surface receptors in its target cells leads to a complex array of molecular events resulting in insulin effects. It is now generally believed that protein phosphorylation-dephosphorylation reactions represent an important mechanism by which a variety of extracellular stimuli regulate cellular functions. Insulin mediates such reactions, but it is not known whether these are the biochemical link between the binding of insulin to its receptor and its final cellular effects. In search of initial post-binding events which might play a role in insulin action, we looked for phosphorylation of insulin receptors. We show that the insulin receptor displays two functional domains, an insulin binding alpha-subunit, and an insulin responsive protein kinase contained in the beta-subunit. We envisage the insulin receptor as an integrated system for transmembrane signal transmission in which hormone binding to the alpha-subunit leads to activation of the beta-subunit via conformational changes. PMID- 3000461 TI - Epidermal growth factor, but not nerve growth factor, stimulates tyrosine specific protein-kinase activity in pheochromocytoma (PC12) plasma membranes. AB - Rat pheochromocytoma (PC12) cells contain specific plasma membrane receptors for both epidermal growth factor (EGF) and nerve growth factor (NGF). Whereas EGF addition to PC12 cells causes a persistent enhancement of proliferation. NGF addition induces a transient stimulation of growth, followed by growth arrest and neuronal differentiation. Despite these differences in biological response, EGF and NGF share a number of early receptor-mediated responses, which are likely te be related to their effect on cell proliferation. In this paper we show that EGF, but not NGF, is able to stimulate the phosphorylation of membrane proteins. In addition, EGF was able to stimulate phosphorylation of a synthetic peptide (RR SRC) by PC12 membranes in a concentration-dependent manner. Kinetic analysis of the phosphorylation reaction indicated that EGF increased the Vmax from 13 to 70 pmoles/min/mg protein, while no change was observed in Km. Furthermore, EGF was able to stimulate tyrosine phosphorylation of angiotensin I and II, to the same extent as RR-SRC. In contrast no effects of NGF on peptide phosphorylation by PC12 membranes were observed. Cross-linking experiments demonstrated the presence of receptors for both NGF and EGF in PC12 membranes. These different effects of NGF and EGF on activation of membrane-associated protein-kinase activity demonstrate that NGF might be able to stimulate growth transiently without stimulating protein kinase activity. PMID- 3000463 TI - [Mechanism of increase in Ca2+ level in platelet cytoplasm induced by aggregating factors]. AB - Using the fluorescent dye, quin-2, it was shown that addition of ADP, thrombin and the so-called platelet activation factor (PAF) to a human platelet suspension results in a 5-20 increase in the concentration of free Ca2+ (Ca2+in) in the cytoplasm. No ADP and PAF-stimulated increase in Ca2+in occurs, when the concentration of extracellular Ca2+ sharply diminishes prior to addition of these agents; the value of the thrombin-stimulated increase in Ca2+in decreases by one order of magnitude under these conditions. Verapamil (10(-4) M) completely blocks the effect of PAF on Ca2+in concentration and decreases 2-3 times the response to ADP; in the case of thrombin verapamil increases the time necessary for the full response without changing its maximum value. Depolarization of the platelet plasma membrane after removal of the K+ diffusion potential does not affect Ca2+ content in the cytoplasm. No significant effect of ADP, thrombin or PAF on the electric potential of the platelet membrane followed by the intensity of diS-C3 (5) fluorescence was found. The elevation of the cAMP content in platelets after addition of prostaglandin E1, forskolin or papaverine leads to a removal of the PAF and ADP effects on the Ca2+in level. The data obtained suggest that the increase in Ca2+ content in platelet cytoplasm caused by the above-mentioned aggregation factors is due to the opening of the receptor-sensitive channels. PMID- 3000462 TI - [Evaluation of structuro-functional heterogeneity of isolated mitochondria from the normal and the ischemic myocardium]. AB - The structural and functional heterogeneity of mitochondria isolated from intact and ischemic (after 60 min exposure at 37 degrees C) rabbit myocardium was evaluated. In the presence of cytochrome c. a relatively high (260 +/- 26 ng at O/min . mg of protein) rate of rotenone-sensitive NADH oxidation was observed, which was increased in ischemia. Cytochrome c stimulated the increase of NADH oxidation in mitochondria of normal and ischemic myocardium by the factors of 3.5 and 3.4, respectively. Succinate oxidation in the presence of bromthymol blue in normal and ischemic myocardium mitochondria was activated by cytochrome c 3.3- and 2.9-fold, respectively. The percentage of mitochondria with both structurally damaged membranes was 15% and 25% in normal and ischemic myocardium preparations, respectively. In the absence of ADP, cytochrome c contributed to the increase of the succinate oxidase activity in ischemic mitochondria; that in the 3rd state was inhibited in ischemia and normalized by cytochrome c. A principle was proposed for estimating the percentage of mitochondria with damaged outer membranes, the indices being equal to 34% in control and to 56% in ischemic myocardium. Evidence was obtained suggesting that this mitochondrial fraction was characterized by lowered coupling and absence of rotenone-sensitive NADH: oxidase activity. The percentage of intact mitochondria, in which succinate oxidation is inhibited by bromthymol blue and does not need exogenous cytochrome c, is 51% in control and 19% in ischemic myocardium mitochondria. PMID- 3000464 TI - Cholinergic inhibition of follicle-stimulating hormone-induced progestin production by cultured rat granulosa cells. AB - The influence of cholinomimetics on follicle-stimulating hormone (FSH)-induced progestin production was studied in a primary culture of rat granulosa cells. Cells were cultured for 2 days with FSH and delta 4-androstenedione in the presence or absence of increasing concentrations of cholinergic agonists. Although ineffective as stimulators of steroidogenesis by themselves, the three nicotinic receptor-selective agonists lobeline, dimethylphenylpiperazinium iodide (DMPP), and phenyltrimethylammonium iodide (PTMA) inhibited FSH-induced progesterone and 20 alpha-hydroxypregn-4-en-3-one production in dose-dependent fashions. The rank order of inhibitory potencies was lobeline greater than DMPP greater than PTMA with IC50 values of 2 X 10(-6) M, 3 X 10(-5) M, and 3 X 10(-4) M, respectively. In contrast, the muscarinic receptor-selective agonists muscarine and bethanechol failed to inhibit steroid production. The inhibitory effect of lobeline on the time course of FSH-induced induced steroid production indicated an immediate inhibitory action; however, this inhibition was readily reversed upon removal of the drug. Further studies demonstrated that the FSH stimulated increase in intracellular cAMP levels, as well as progesterone production induced by cholera toxin and forskolin (agents that stimulate cAMP production) and by dibutyryl cAMP (a cAMP analog), were also suppressed by lobeline. The present observations indicate that nicotinic, but not muscarinic, cholinergic agonists inhibit progesterone biosynthesis in cultured granulosa cells and suggest that endogenous acetylcholine may play a modulatory role in ovarian steroidogenesis. PMID- 3000465 TI - Specific destruction of Leydig cells in mature rats after in vivo administration of ethane dimethyl sulfonate. AB - Effects of ethane dimethyl sulfonate (EDS) on Leydig cells have been studied using the following parameters: morphology, histochemistry of 3 beta hydroxysteroid dehydrogenase (3 beta-HSD) and esterase, quantitative activity of esterase, testosterone concentrations in plasma, and steroid production by isolated interstitial cells in vitro. Degenerating Leydig cells were observed within 16 h after the injection of mature rats with EDS (75 mg/kg body weight). At that time the testosterone concentration in plasma and the specific activity of esterase in testis tissue were decreased to approximately 35% and 60% of the control value, respectively. At 48 h after EDS only a few normal Leydig cells were left and the plasma testosterone concentration was less than 5% of the control value. The specific activity of esterase in total testis tissue was similar to the activity of dissected tubules from untreated rats. At 72 h no Leydig cells could be detected and no 3 beta-HSD and esterase-positive cells were present. At that time macrophages were still present in the interstitium and the appearance of the spermatogenic epithelium was normal, but 1 wk after EDS the elongation of spermatids was disturbed, probably due to a lack of testosterone. In some of the animals the cytotoxic effects of EDS on Leydig cells could be partly inhibited by human chorionic gonadotropin treatment. The basal steroid production by interstitial cells from mature rats 72 h after EDS was not significant and no stimulation by LH was observed, whereas no effect of EDS could be detected on steroid production by interstitial cells isolated from immature rats and mice 72 h after treatment. Other compounds with similar structures, such as butane dimethyl sulfonate (busulfan) and ethane methyl sulfonate (EMS) had no effect on Leydig cells from mature rats. It is concluded that EDS specifically destroys Leydig cells in mature rats. PMID- 3000466 TI - Content of K+ and Na+ in seminiferous tubule and rete testis fluids from Sertoli cell-enriched testes. AB - Seminiferous tubules of rats exposed to x-irradiation before birth were subjected to micropuncture in situ at 50 days of age to obtain samples of fluid 4 h after ligation of efferent ducts. The concentrations of cations in this fluid were: potassium, 39.7 +/- 1.2 mM, and sodium, 136.3 +/- 1.2 mM (means and standard errors, n = 5). Histologic examination revealed that germ cells constitute less than 1% of the cell population within the seminiferous tubules of these rats; the remaining cells were all Sertoli cells. Sertoli cells showed efflux of 86Rb+ with t1/2 of approximately 11 min and an active ATPase in plasma membranes. These activities were similar to those of Sertoli cells from normal rats. Germ cells from normal rats showed less rapid efflux of 86Rb+ (t1/2 greater than 60 min) and less active Na+/K+ ATPase in plasma membranes. It is concluded that Sertoli cells are responsible for the high concentration of potassium in seminiferous tubule fluid and that plasma membranes of these cells contain an active K+ pump that is not inhibited by ouabain (1 mM). PMID- 3000467 TI - A method for analysing case-control studies with ordinal exposure variables. AB - A method is proposed for analysing case-control studies with ordinal or continuous, but unruly, exposure levels. A test is proposed which is an appropriate linear combination of stratum-specific Wilcoxon tests. An estimator for the mean percentile shift between the case and control exposure levels is given. Methods for assessing heterogeneity of the mean percentile shift across strata are also discussed. The performance of the test as a function of the number of controls per case is studied for both local and distant alternatives. The asymptotic relative efficiency compared to the best parametric test with the same number of controls is evaluated and its performance is compared to tests based on a dichotomized exposure variable. Finally, the method is illustrated by two numerical examples. PMID- 3000469 TI - [Regional changes in the activity of the angiotensin-converting enzyme in the brain of rats with developing hereditarily induced hypertension]. AB - Angiotensin-converting enzyme (ACE) activity in the pituitary zone and 7 other brain areas has been studied in rats with developing spontaneous hereditary hypertension. ACE activity was significantly different in normotensive and spontaneously hypertensive rats, with the differences most prominent in pituitary body, cerebellum, striatum and medulla oblongata. Age-dependent variability in ACE activity was demonstrated. PMID- 3000468 TI - [Effect of the antioxidant dibunol on the function of the adrenal cortex, the thyroid and the adenohypophysis in adult and old rats]. AB - The effect of a single antioxidant (dibunol-D) injection (100 mg/kg body weight) on the functional activity of adrenal cortex (AC), thyroid gland (TG) and tropic hormone production by adenohypophysis (AH) has been studied in old and adult rats. For 48 hours following D administration two-phase changes in adrenocorticotropic function of AH and steroidogenesis were detected in the AC: activation during the first hours was followed by suppression 24 hours later, and recovery 48 hours later. Thyrotropic AH function and thyroidogenesis were found to be decreased during the first hours of D effect. Thyroidogenesis recovery by the end of the first day was delayed as compared to the recovery of AH thyrotropic function. It is suggested that the mechanisms of D action are based on its effects mediated by changes in the functional activity of endocrine glands and associated with resetting of endocrine regulation of body functions. PMID- 3000470 TI - [Monoacylglycerol phosphatide accumulation and the alteration of the properties of benzodiazepine receptors in the brain synaptosomes]. AB - Phospholipase A2-induced changes in the affinity of benzodiazepine receptors are due to accumulation of monoacylglycerol phosphatides, and not fatty acids, in the membranes. The data show the regulatory role of membrane phospholipids in ligand receptor interaction. PMID- 3000471 TI - [3H-diazepam binding to brain synaptic membranes during the development of generalized epileptic activity]. AB - The development of bemegride-induced generalized epileptic activity in rats was shown to reduce the constant (CB). of specific 3H-diazepam binding with synaptic membranes from 0.23 nM-1 to 0.15 nM-1 and to increase the maximum number of membrane binding sites (Bmax) from 410 fmol/mg protein to 550 fmol/mg protein. It is assumed that the changes of benzodiazepine receptor properties are due to alteration in physico-chemical characteristics of synaptic membrane lipids resulting from the activation of lipid peroxidation. PMID- 3000472 TI - [Modulating effect of cerulein on benzodiazepine receptors]. AB - Subcutaneous administration of caerulein (100-500 micrograms/kg) significantly reduced the development of picrotoxin (8 mg/kg) seizures in male mice. The same doses of caerulein inhibited 3H-flunitrazepam binding in in vivo experiments. Proglumide, an antagonist of cholecystokinin receptors, in low dose (5 mg/kg) potentiated the effects of caerulein (100 micrograms/kg), whereas the administration of proglumide in high dose (25 mg/kg) reduced the action of caerulein on 3H-flunitrazepam binding and picrotoxin seizures. Caerulein (5-1000 nM) decreased 3H-flunitrazepam binding in in vitro experiments only after supplementation of the binding medium with 120 mM NaCl and 5mM KCl. The results suggest the possible interaction of caerulein with chloride ionophor. It seems probable that the direct interaction of caerulein with chloride ionophor in involved in the inhibitory effect of caerulein on picrotoxin seizures and 3H flunitrazepam binding. PMID- 3000473 TI - [Effect of dalargin on the content of endorphins, leucine-enkephalin ACTH and corticosterone of the blood of stressed rats]. AB - Stress caused by acute cysteamine duodenal ulcer was induced in Wistar male rats. All the endogenous opioides under study were involved in the stress-reaction mechanism. Protective dalargin (synthetic enkephalin analogue) administration revealed a tendency towards normalization of endorphin, L-enkephalin and ACTH blood levels. PMID- 3000474 TI - High molecular weight kininogen: localization in the unstimulated and activated platelet and activation by a platelet calpain(s). AB - High mol wt kininogen (HMWK), the major cofactor-substrate of the contact phase of coagulation, is contained within and secreted by platelets. Studies have been performed to localize platelet HMWK in both the unstimulated and activated platelet and to ascertain the effect of platelet enzymes on HMWK itself. On platelet subcellular fractionation, platelet HMWK was localized to alpha granules, and platelets from a patient with a deficiency of these granules (gray platelet syndrome) had 28% normal platelet HMWK. Platelet HMWK, in addition to being secreted from the platelet, was also localized to the surface of the platelet when activated. Using a competitive enzyme-linked immunosorbent assay for HMWK as an indirect antibody consumption assay, the external membrane of thrombin-activated platelets as well as the releasate from these stimulated platelets had 17 ng HMWK antigen/10(8) platelets available, whereas unstimulated platelets and their supernatant had only 4.9 and 4.2 ng HMWK/10(8) platelets present, respectively. The anti-HMWK antibody consumption by activated normal platelets was specific for membrane-expressed platelet HMWK, since activated platelets from a patient with total kininogen deficiency did not adsorb the anti HMWK antibody. Enzymes in the cytosolic fraction of platelets cleaved 125I-HMWK (mol wt 120,000) into a mol wt 100,000 polypeptide as well as smaller products at mol wt 74,000, mol wt 62,000, mol wt 47,000, and a few components below mol wt 45,000. No cleavage products were observed when DFP and leupeptin were present. The cleavage of HMWK was specifically prevented by inhibitors of calcium activated cysteine proteases (leupeptin, N-ethylmaleimide, iodoacetamide, and EDTA) but not by inhibitors of serine proteases (DFP, benzamidine, soybean trypsin inhibitor, or aprotinin). Platelet cytosol increased the coagulant activity of exogenous purified HMWK with maximum HMWK coagulant activity (35 fold) occurring within ten minutes of exposure to platelet cytosol. Treatment of platelet cytosol with leupeptin prevented the increase in the coagulant activity of exogenous HMWK. These studies indicate that activated platelets express platelet HMWK on their external membrane and platelet enzymes can cleave and increase the coagulant activity of exogenous HMWK. PMID- 3000475 TI - HTLV-III infection after bone marrow transplantation. AB - We prospectively documented the development of a fatal, secondarily acquired severe immunodeficiency in a 19-year-old man who underwent uncomplicated bone marrow transplantation. He had no graft v host disease (GVHD) and had normal recovery of his immune system as determined by lymphocyte phenotyping, mitogenic responses of his peripheral blood lymphocytes, and his ability to secrete immunoglobulin. This alteration in immunity was associated with the acquisition of antibody to HTLV-III. His only risk factor for the development of HTLV-III infection was the transfusions he had received during the transplant and recovery period. Two of his 54 transfusions were from an asymptomatic individual at high risk for acquired immunodeficiency syndrome (AIDS), who was subsequently found to be seropositive for anti-HTLV-III and from whom HTLV-III was isolated. The loss of immunocompetence in patients without chronic GVHD disease is unusual, and our data support the view that this patient's immunodeficiency was due to HTLV-III. When bone marrow transplant recipients without chronic GVHD develop late opportunistic infections, consideration should be given to transfusion-associated AIDS. PMID- 3000476 TI - Modulation of human neutrophil effector functions by monoclonal antibodies against surface membrane molecules of 94,000 and 180,000 molecular weight. AB - Function-related antigens on the neutrophil (PMN) surface were identified using two newly developed PMN-specific mouse monoclonal antibodies. These IgG antibodies, designated Ab 1-14 and Ab 1-15, were selected for detailed study after initial testing revealed their significant inhibition of PMN superoxide generation in response to N-formyl-Met-Leu-Phe (FMLP) (64% for 1-14 and 64% for 1 15; P less than .05). In further experiments, Ab 1-14 augmented PMN adhesion (by 111%; P less than .01) and degranulation (by 52%; P less than .05) in response to FMLP, while Ab 1-15 inhibited these responses by 42% and 29%, respectively (P less than .05). Ab 1-14 reduced PMN chemotaxis in response to FMLP by 37% (P less than .02), and unlike Ab 1-15, Ab 1-14 significantly reduced unstimulated PMN binding of complement-coated sheep red blood cells. Ab 1-14 and Ab 1-15 significantly reduced PMN superoxide production in response to phorbol myristate acetate (PMA) (14% and 23%, respectively; P less than .05). Whereas 1-14 was found to increase PMA-induced cell degranulation significantly (175%), Ab 1-15 did not alter degranulation response to PMA. Immunoprecipitation showed that Ab 1 14 and Ab 1-15 recognized respective surface antigens of 94,000 mol wt and 130,000 to 180,000 mol wt. Our findings suggest that the surface molecules identified by these two monoclonal antibodies play a significant role in neutrophil activation by both FMLP and PMA. PMID- 3000478 TI - Platelet glycoproteins IIb and IIIa associated with blood monocytes are derived from platelets. AB - Platelet glycoprotein IIb/IIIa (GP IIb/IIIa), the receptor complex for fibrinogen, has been regarded as a megakaryocyte/platelet lineage-restricted antigen. Recently, however, it has been reported that GP IIb/IIIa is expressed in blood monocytes. Studies were performed to establish the origin and immunological characteristics of monocyte-associated glycoproteins IIb and IIIa (GPs IIb and IIIa). Preparations of blood monocytes containing varying platelet-monocyte ratios were metabolically labeled with [35S]methionine with the expectation that any newly synthesized GPs IIb and IIIa would be monocyte-derived, since platelets have only rudimentary protein synthetic apparatuses. Analyses of sodium dodecyl sulfate (SDS) gels of homogenates of cell preparations containing from 200 to 5:1 platelet-monocyte ratios revealed that unlabeled GPs IIb and IIIa were readily immunoisolated using protein A-Sepharose immunobeads. However, fluorographic analyses of the same cell preparations pulse-labeled with [35S]methionine failed to demonstrate synthesis of GP IIb or IIIa. Additionally, no GP IIb or IIIa was detected when immunoisolation was carried out in pure preparations of monocytes containing less than 1:100 platelet-monocyte ratios and SDS acrylamide gels were stained by the sensitive silver stain method. Furthermore, heterologous polyspecific antisera and two monoclonal antibody preparations against GPs IIb and IIIa, which bound to platelets, failed to bind to monocyte membranes. Thus, evidence was presented that indicated that monocytes do not synthesize platelet GPs IIb and IIIa and that detection of these molecules in blood monocyte preparations reflects platelet contamination. PMID- 3000477 TI - Partial characterization of a binding site for von Willebrand factor on glycocalicin. AB - The binding of von Willebrand factor (vWF) to platelet membrane glycoprotein Ib (GpIb) facilitates platelet adhesion to vascular subendothelium. In this study, we provide evidence that the vWF binding site is on glycocalicin (GC), a proteolytic fragment of GpIb, and we examine the role of the carbohydrate portion of GC on that binding. The binding to platelets of 6D1, a monoclonal antibody that recognizes an epitope on GpIb and blocks ristocetin-induced vWF binding to platelets, was inhibited by purified GC. In addition, purified GC inhibited ristocetin-dependent binding of 125I-labeled vWF to platelets. Since GC contains 60% carbohydrate by weight, we assessed the role of carbohydrate sequences on its interaction with antibody 6D1 and vWF. Based on the known sequence of the major oligosaccharide chain of GC--N-acetyl neuraminic acid, galactose, N-acetyl glucosamine, N-acetyl galactosamine--we treated GC sequentially with neuraminidase, beta-galactosidase, and beta-N-acetylglucosaminidase. Removal of sialic acid and galactose residues did not affect GC binding. Removal of N-acetyl glucosamine residues did not affect GC binding to 6D1 but did decrease the ability of GC to inhibit vWF binding to platelets, increasing the concentration needed to inhibit binding by 50% (IC50) 40-fold. This suggests that a portion of the oligosaccharide chains on GC contributes to the vWF binding activity of this molecule. PMID- 3000479 TI - Sulfhydryl reducing agents and shape regulation in human erythrocytes. AB - Metabolic crenation of red cells is reversible; on addition of nutrients, echinocytes recover the normal discoid shape. When the shape recovery takes place in the presence of reducing agents such as dithiothreitol (DTT), morphological change continues until the cells are stomatocytic. The degree of stomatocytosis varies, depending on the cell morphology when the nutrients and reducing agent are added. DTT has minimal effect on the shape of normal discocytes, but in its presence, mildly echinocytic cells become slightly cupped and advanced-stage echinocytes become severely stomatocytic. DTT must be present continuously for development and retention of stomatocytosis; echinocytes preincubated with or metabolically depleted in DTT do not become stomatocytic when supplemented in the absence of DTT, and DTT-induced stomatocytes revert to discocytes when the reducing agent is removed. DTT has no effect on adenosine triphosphate synthesis or equilibrium cell glutathione levels, and the induced stomatocytosis is not inhibited by excluding oxygen from cells during depletion. Spectrin phosphorylation and phosphate turnover are not affected by DTT. The echinocyte-to discocyte transformation coincides with phosphorylation of membrane inner monolayer lipids (diacylglycerol to phosphatidic acid and phosphatidylinositol to phosphatidylinositol-4,5-bisphosphate). Overphosphorylation of these phospholipids is not responsible for the exaggerated shape recovery seen with reducing agents; phosphorylation of inner monolayer lipids proceeds identically in the presence and absence of DTT. PMID- 3000481 TI - Epstein-Barr genomes. PMID- 3000480 TI - Platelet modulation of polymorphonuclear leukocyte shear induced aggregation. AB - A cone and plate viscometer and Coulter Counter were used to study platelet modulation of polymorphonuclear leukocyte (PMNL) aggregation caused by controlled shear stress. As an index of aggregation, the large-particle percentage (LPP) was calculated. This represents the ratio of aggregated cell count to total cell count. PMNL suspensions in buffer (1.0 X 10(7) cells per milliliter, final concentration) did not show any aggregate formation at shear stresses below 150 dynes/cm2 for one minute exposure time (LPP less than 3%). However, there was PMNL aggregation in mixed PMNL and platelet-rich plasma suspensions in this shear stress range. Supernatant plasma from sheared platelets initiated PMNL aggregation at moderate shear stress (150 dynes/cm2 for one minute; LPP, 20.3% +/ 2.5%). In contrast, platelet release factors, such as adenosine diphosphate (2 mumol/L) and serotonin (2 mumol/L) did not cause PMNL aggregation (LPP, 2.9% +/- 1.2% and 3.3% +/- 0.8%, respectively). The use of a cyclo-oxygenase inhibitor (acetylsalicylic acid, 50 mumol/L) did not suppress the aggregation of PMNLs after shear (LPP, 20.1% +/- 2.4%). However, preincubation with nordihydroguaiaretic acid (10 mumol/L), an inhibitor of C-5 and C-12 lipoxygenase, and 6,9-deepoxy-6,9-(phenylimino)-6,8-prostaglandin I1 (U-60257, 10 mumol/L), an inhibitor of C-5 lipoxygenase in human leukocytes, suppressed this aggregation (LPP, 9.1% +/- 2.5% and 10.4% +/- 3.2%, respectively). Also, the formation of lipoxygenase products (5-HETE, 12-HETE, 15-HETE, and LTB4) activated by shear stress was documented by reversed phase-high-performance liquid chromatography (RP-HPLC). These data support the possibility of a cooperation between platelets and leukocytes in shear-induced PMNL aggregation that is dependent on C-12 or C-5 lipoxygenase activity, or both. PMID- 3000482 TI - Anti-common acute lymphoblastic leukemia antibody (CALLA) (J5) reactivity by small cell lung cancer (SCLC) cells. PMID- 3000484 TI - [Mesenchymal breast sarcomas. Apropos of 25 cases]. AB - Breast sarcoma are rare, representing 1% of all malignant breast tumours. This is a retrospective study of 25 patients with a breast sarcoma, treated at Institut Gustave Roussy from 1954 to 1981. Thirty six per cent of these arose in a cystosarcoma phyllodes. A variety of histologies were found, the main one being malignant fibrohistiocytoma (44%). Nodal involvement was rare (4%) and, as in other sarcoma, hematogenous spread of metastases was more usual. Local recurrence occurred in 44% of cases and distant metastases (usually pulmonary) in 24%. The 3 year disease-free survival was 60% and the major prognostic factor was the mitotic index. Surgery is the treatment of choice of these tumours, supplemented by local irradiation in those cases where only a tumorectomy has been performed. The role of adjuvant chemotherapy remains undefined. PMID- 3000483 TI - Biosynthesis and secretion of factor VII, protein C, protein S, and the Protein C inhibitor from a human hepatoma cell line. AB - Using specific radioimmunoassays, 8 day cultures of Hep G2 cells were shown to contain in their supernatants 16, 74, and 828 ng/mL and in their cell lysates, 8, 55, and 48 ng/2 X 10(8) cells of factor VII, protein C, and protein S, respectively. These proteins and the protein C inhibitor were functionally active, and each of these activities was neutralized by their respective polyclonal antibodies. Although vitamin K had a modest effect, warfarin decreased the activity of secreted factor VII, protein C, and protein S by 50% to 90%. Protein C and protein S antigens were reduced three- to fourfold by warfarin. The protein C inhibitor antigen and activity were unaffected by vitamin K or warfarin treatment. Intrinsic labeling and immunoprecipitation indicated that factor VII, protein S, and the protein C inhibitor were secreted as 52,000, 77,000, and 58,000 molecular weight (mol wt) proteins, respectively. Protein C was secreted as a single-chain protein of about 65,000 mol wt, indicating that all of the vitamin K-dependent proteins are translated and secreted as single-chain molecules. Each of the four proteins studied represented their plasma protein counterparts structurally, functionally, and immunochemically. Thus, all of the known soluble components of the protein C pathway are produced by liver parenchymal cells. PMID- 3000485 TI - [Prognostic significance of hormone receptors in the localization of hepatic metastases of breast adenocarcinomas]. PMID- 3000486 TI - [Steroid hormone receptors in soft-tissue sarcomas]. PMID- 3000488 TI - Growth and photosynthetic response of a freshwater alga, Selenastrum capricornutum, to an oil shale by-product water. PMID- 3000487 TI - Loss of the herbicide triallate from a clay soil containing aged and freshly applied residues. PMID- 3000489 TI - Lung in acquired immune deficiency syndrome: infectious and immunological status assessed by bronchoalveolar lavage. AB - Bronchoalveolar lavage (BAL) has been performed in 63 patients with acquired immune deficiency syndrome (AIDS) and 20 patients with chronic generalized lymphadenopathy (CGL) for the diagnosis of lung opportunistic infections and analysis of immune effector cells of the lower respiratory tract. In patients with AIDS, Pneumocystis carinii was found in 63%. Cytomegalovirus (CMV) pneumonia was assessed by viral cultures of BAL fluid and microscopic examination: CMV was found in 62% and 39% respectively. Mycobacteria were encountered in 22% of cases. Altogether BAL yielded at least one opportunistic agent in 94% of patients who presented with clinical and/or radiographic pulmonary involvement, and in 80% of patients who presented with fever only. Conversely BAL was negative in all patients with CGL, except one positive CMV culture. Analysis of BAL cells revealed an increased cellularity in AIDS and CGL patients with normal numbers of alveolar macrophages. Alveolar lymphocytes were surprisingly increased in most patients with AIDS (mean 26.1 +/- 21.9%; range 1-76%) and CGL (mean 26.6 +/- 22.6%; range 3-76%) with criteria of activation contrasting with the blood lymphopenia. Evaluation of lung lymphocyte phenotypes revealed a marked decrease in T4 cell percentages, specially in AIDS, whereas the large majority of alveolar lymphocytes expressed the T8 phenotype. We conclude that BAL is a very reliable means for diagnosis of opportunistic lung infections and give interesting prospects to study local immunity in patients with AIDS and CGL. PMID- 3000490 TI - Chiari's pelvic osteotomy in the treatment of Legg-Calve-Perthes disease. AB - Results of Chiari's pelvic osteotomy in Legg-Calve-Perthes disease were analyzed in 37 hips of 36 patients. The mean age at surgery was nine years. The results were assessed according to both the quality of sphericity and containment of the femoral head. The results were satisfactory in 26 hips (70%), acceptable in 10 hips (27%), and poor in only one hip (3%). In cases where preoperative arthrography revealed a saddle-shaped distortion of the protruding and flattened epiphysis, the results were less satisfactory. The authors conclude that Chiari's osteotomy is needed where other kinds of containment cannot be expected to be successful. The indications are smooth flattenings of severely protruding epiphyses in patients aged seven years or more, and severe epiphyseal protrusions without flattening in patients 10 years or older. PMID- 3000491 TI - Dr. Michael S. Burman. Pioneer in the field of arthroscopy. PMID- 3000492 TI - Arthroscopy of the hip. Present status. PMID- 3000493 TI - Results of total knee replacement using an uncemented tibial component. AB - Fourteen patients (16 knees) underwent total knee arthroplasty with a noncemented tibial component. Clinical evaluation at one year and two years showed a level of function and pain relief comparable to that reported in studies of cemented prostheses. Radiographic studies demonstrated evidence of active bone implant consolidation. PMID- 3000494 TI - The use of hypnosis in the management of preoperative anxiety and postoperative pain in a patient undergoing laminectomy. AB - Patients undergoing laminectomy face a variety of concerns both pre- and postoperatively which may affect their emotional state and increase surgical risk. A case study of a laminectomy patient who was taught hypnosis for the control of preoperative anxiety and postoperative pain is presented. The benefits of such hypnotic intervention, as well as the long-term effects of hypnotic intervention on a patient who is in a crisis period are discussed. PMID- 3000495 TI - The medial head of the gastrocnemius. A review of the basis for partial rupture and for intermittent claudication. AB - Under physical stress, partial or complete tears of the muscle fibers of the medial head of the gastrocnemius may occur. Radiographs made by soft-tissue technique can be especially helpful in diagnosing partial ruptures, which are sometimes difficult to detect. Intermittent claudication can be caused by an abnormal position of the medial head of the gastrocnemius, resulting in compression of the popliteal vessels. Angiography or computerized tomography will usually disclose the site of local pressure. Surgical intervention may be necessary to eliminate the compression. PMID- 3000496 TI - Silicone-implant replacement arthroplasty in fractures of the radial head. A follow-up report. AB - A follow-up of the results of silicone replacement arthroplasty in radial head fractures in 51 patients after 3-11 years is presented. Although the clinical picture remained unchanged, radiographs revealed varying degrees of wear on the implants, down to complete fragmentation. The author concludes that silicone implants are well tolerated but not sufficiently resistant to wear. PMID- 3000497 TI - Multifocal Paget's sarcoma. PMID- 3000498 TI - Current trends in Legg-Calve-Perthes disease with special respect to older children. PMID- 3000499 TI - Actions of ATP and alpha, beta-methylene ATP on neuromuscular transmission and smooth muscle membrane of the rabbit and guinea-pig mesenteric arteries. AB - In the rabbit mesenteric artery, adenosine triphosphate (ATP), showed two actions on the membrane potential of muscle cells: low concentrations (1-10 microM) hyperpolarized and high concentrations (greater than or equal to 50 microM) depolarized the membrane. Both changes in the potential were accompanied by increases in ionic conductance. In the rabbit mesenteric artery, alpha, beta methylene ATP (MeATP), (greater than or equal to 30 nM) depolarized the muscle membrane at a lower concentration than ATP (greater than or equal to 50 microM), and increased the ionic conductance of the membrane. The depolarization induced by ATP was prevented by low concentrations of MeATP, but the hyperpolarization was retained. Furthermore, the hyperpolarization was not affected by theophylline (10 microM). In the guinea-pig mesenteric artery, ATP and MeATP depolarized and increased the ionic conductance of muscle membrane, but to depolarize the membrane, higher concentrations of both agents were required, compared to those in the rabbit mesenteric artery. In the mesenteric arteries from both species, perivascular nerve stimulation evoked excitatory junction potentials (e.j.ps). In both tissues, MeATP inhibited the amplitude of e.j.ps at lower concentrations than did ATP, and both agents had more potent inhibitory actions on rabbit than on guinea-pig. The inhibition of e.j.p. induced by low concentrations of these agents showed no relationship to depolarization, but the inhibition induced by high concentrations was paralleled by depolarization and increase in ionic conductance of the membrane. In the rabbit mesenteric artery, overflow of noradrenaline (NA) and its metabolite (3,4-dihydroxyphenylglycol; DOPEG) produced by perivascular nerve stimulation was examined. ATP (0.1 mM) but not MeATP (0.1 microM) reduced the overflow of NA, whereas both agents had no effect on the overflow of DOPEG. Exogenously applied high concentrations of NA (greater than or equal to 3 microM) depolarized the muscle membrane in both species. These NA induced depolarizations were not affected by treatment with ATP or MeATP. It is concluded that, in the rabbit mesenteric artery, ATP is more likely to be involved in generation of e.j.ps than is NA. A similar interpretation in the guinea-pig mesenteric artery is complicated by the depolarization produced by high concentrations of ATP or MeATP. PMID- 3000500 TI - Effects of diltiazem on electrical responses evoked spontaneously or by electrical stimulation in the antrum smooth muscle cells of the guinea-pig stomach. AB - In circular smooth muscle cells of the guinea-pig stomach (antrum), diltiazem (10(-6)-10(-5)M) blocked the overshooting spike potential generated either spontaneously or by electrical stimulation in the presence of 2 mM tetraethylammonium chloride, but did not block the slow wave and the abortive spike potential. The membrane was depolarized by high concentrations of diltiazem (more than 3 X 10(-6)M), and this depolarization was associated with an increase in the membrane resistance. The interval between slow waves was shortened to about 0.90 times the control (14.7s) by 10(-5)M diltiazem. Transmural nerve stimulation evoked an inhibitory junction potential (i.j.p.) and enhanced the subsequently generated slow wave. Tetrodotoxin (3 X 10(-7)M) blocked both responses but atropine (10(-6)M) blocked only the latter. Diltiazem (more than 10(-6)M) increased the amplitude of the i.j.p. and depressed the enhancement of the slow wave produced by transmural nerve stimulation, presumably due to depolarization of the membrane. The latency for the i.j.p. remained the same in the presence of diltiazem (10(-5)M). It is concluded that in the guinea-pig stomach, diltiazem blocks Ca-influx during the generation of the overshooting spike potential, but not the Ca-influx related to generation of the abortive spike potential or the slow wave. The cholinergic excitatory and the non adrenergic, non-cholinergic inhibitory transmission may not be much affected by diltiazem. PMID- 3000503 TI - AIDS antibody testing and counselling. PMID- 3000502 TI - The pathology of AIDS. PMID- 3000501 TI - Determination of the receptor selectivity of opioid agonists in the guinea-pig ileum and mouse vas deferens by use of beta-funaltrexamine. AB - The irreversible inhibitor of mu-opioid receptor-mediated effects, beta funaltrexamine (beta-FNA), was used to investigate the selectivity of various opioid agonists at mu-opioid receptors in the electrically stimulated guinea-pig ileum and mouse vas deferens preparations in vitro. In the guinea-pig ileum, pretreatment with beta-FNA (3 X 10(-8) - 3 X 10(-6)M) produced a concentration dependent antagonism of the inhibitory effect produced by the mu-opioid receptor agonist [D-Ala2, MePhe4, Gly(ol)5]enkephalin (DAGO). High concentrations of beta FNA (3 X 10(-6) - 1 X 10(-5)M) also antagonized the inhibitory effects of the kappa-opioid agonist U50488. Pretreatment of guinea-pig ileum with beta-FNA at 1 X 10(-6)M resulted in blockade of the effect of some opioid agonists. The compounds which showed the largest rightward shifts in their concentration response curves, and hence the greatest mu/kappa opioid receptor selectivity, were nalbuphine, [D-Ser2, Leu5]enkephalinyl-Thr6(DSLET), morphine, DAGO and normorphine. Responses to tifluadom, Mr 2034, ethylketocyclazocine, butorphanol, nalorphine, proxorphan and U50488 were not inhibited by beta-FNA. In the mouse vas deferens, pre-treatment with beta-FNA (1 X 10(-6)M) produced a similar shift in the dose-response curves for normorphine as in the guinea-pig ileum. The concentration-response curves for the delta-receptor agonists [D-Ala2, D-Leu5] enkephalin (DADLE) and DSLET were, however, also shifted, indicating that beta FNA will also block delta-opioid receptors. Since beta-FNA does not block kappa opioid receptor-mediated effects, it can be used in the guinea-pig ileum preparation as a selective mu-receptor inhibitor. However, its lack of selectivity between mu- and delta-opioid receptors should be taken into account in many other isolated tissues and experiments in vivo. PMID- 3000504 TI - Cooperation between the prison medical service and the NHS: a conversation. PMID- 3000505 TI - Alteration of alpha and muscarinic receptors in rat brain and heart following chronic nicotine treatment. AB - Adrenergic and muscarinic binding sites in 4 brain regions (cerebral cortex, corpus striatum, hypothalamus/thalamus and brainstem) and in heart ventricles were measured in rats chronically treated with nicotine added to the drinking water in doses ranging from 6 to 8 mg/kg/day, for 4 weeks. Control rats received only tap water. The nicotine treatment led to increases in the specific binding of both [3H]prazosin and [3H]clonidine in the cerebral cortex. An increase in [3H]prazosin binding was also observed in the hypothalamus/thalamus of nicotine treated rats. These changes were all due to an increase of about 23% in Bmax. In the brainstem and heart left ventricle, respectively, an increase and a decrease in the affinity of [3H]quinuclidinyl benzilate binding were observed. There were no changes of the binding parameters for the 3 radioligands in other regions tested, and no alteration of [3H]dihydroalprenolol binding was detected in any region examined. These results indicate that chronic administration of nicotine causes an increase in the density of alpha 1- and alpha 2-binding sites in some brain regions and reciprocal changes of the affinity of muscarinic binding sites in the brain and in the heart. PMID- 3000506 TI - Behavioral activities of axotomized abducens nucleus motoneurons in the alert cat. AB - The activity of identified control and axotomized abducens motoneurons has been recorded during spontaneous eye movements in the alert cat. Axotomized motoneurons showed a quick fatigability during eye fixations not observed in controls. Discharge rate of axotomized motoneurons during on direction saccades was lower and shorter than in controls and started usually after the beginning of the saccade. Axotomized motoneurons could be antidromically invaded only in the presence of synaptic facilitation. Electrophysiological explanations for the behavior of axotomized motoneurons in the alert cat are discussed in the text. PMID- 3000508 TI - Inhibition of dorsal column nuclei by stimulation of trigeminal afferents in decerebrate-decerebellate cats. AB - In decerebrate-decerebellate cats, stimulation of trigeminal afferents inhibited neurons in dorsal column (DC) nuclei driven by activation of DC input and produced primary afferent depolarization in DC primary afferent terminals. This inhibition was most likely mediated by a trigeminal-brainstem-DC nuclear loop. PMID- 3000507 TI - Cobalt ion enhancement of 2-chloro[3H]adenosine binding to a novel class of adenosine receptors in brain: antagonism by calcium. AB - We have recently reported the identification of a novel class of micromolar affinity adenosine binding sites in rat brain membranes using the adenosine agonist 2-chloro[3H]adenosine (C1[3H]Ado). These binding sites are distinguishable from the A1 and A2 adenosine receptors by a number of pharmacological criteria, and we have designated this new class of binding sites as the A3 adenosine binding sites. In the present study, the effects of a wide range of divalent and trivalent cations on micromolar C1[3H]Ado binding to brain membranes were examined. Co2+, Ni2+ and La3+ markedly stimulated specific C1[3H]Ado binding by 45-150% above control when tested at concentrations of 1-10 mM. Ca2+ had no significant effect on binding except at high concentrations where it depressed binding slightly. Ca2+, however, completely prevented the stimulation of C1[3H]Ado binding by Co2+. These findings further distinguish the A3 class of adenosine binding sites from the previously characterized adenosine receptors and suggest that the A3 binding sites are associated with calcium systems in brain. PMID- 3000509 TI - Postnatal development of GABA- and glycine-mediated inhibition of feline retinal ganglion cells in the area centralis. AB - Intraretinal iontophoresis in the optically intact eye of adult cats (18-22 weeks of age) and kittens (7-9 weeks of age) under pentobarbitone anaesthesia was performed. Studies were concentrated on retinal ganglion cells of the sustained (X) type in the area centralis under photopic conditions. In both the adult and kitten, gamma-aminobutyric acid (GABA) and muscimol inhibited the visually induced excitation, and bicuculline blocked the visually induced inhibition of on cells. On the other hand, glycine inhibited the excitation and strychnine blocked the inhibition of off-cells. However, a greater current of GABA (muscimol) and glycine was required to produce total inhibition in kitten's on- and off-cells respectively when compared with the adult's. Furthermore, a smaller current of bicuculline and strychnine was needed to abolish the visually induced inhibition of kitten on- and off-cells respectively when compared with the adult's. In the adult, GABA and glycine did not affect the responses of off- and on-cells respectively, but in the kitten GABA inhibited off-cells and glycine inhibited on cells to some extent. In neither the adult nor the kitten did bicuculline have any effect upon off-cells or strychnine any effect upon on-cells. Thus, the sustained on- and off-cells in the kitten area centralis exhibit: a reduced selectivity to inhibitory transmitters; a reduced sensitivity to exogenously applied inhibitory transmitter agonists; but a greater sensitivity to inhibitory transmitter antagonists, in comparison with the sustained on- and off-cells in the adult area centralis. The observed differences between the kitten and adult cat in transmitter actions on retinal ganglion cells appear to be analogous to those found in the postnatal development of functional synapses at the neuromuscular junction and sympathetic ganglia. PMID- 3000510 TI - Comparison of [3H]baclofen binding to GABAB receptors in spontaneously hypertensive and normotensive rats. AB - Baclofen-sensitive GABAB receptor binding to various brain regions was compared in age-matched spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rat. The specific [3H]baclofen binding was significantly higher in SHR cerebellum, hippocampus and nucleus tractus solitarii (NTS), but lower in medulla, when compared with WKY. Scatchard analysis of the binding isotherms indicated a higher Bmax of the high affinity sites in SHR cerebellum and hippocampus and a lower Bmax of the same sites in SHR medulla. Guanyl nucleotide (GTP) decreased [3H]baclofen binding in brain regions of SHR and WKY, although the percentage inhibition of binding did not differ between SHR and WKY. The basal adenylate cyclase activity in SHR cerebellum was lower, in contrast to higher [3H]baclofen binding, whereas lower baclofen binding in SHR medulla was accompanied with higher adenylate cyclase activity. (+/-)Baclofen inhibited adenylate cyclase activity in both SHR and WKY rat brain. Among various regions, the percentage inhibition of cyclase activity in SHR cerebellum was significant lower, as compared to WKY cerebellum. These results suggest that activity of GABABergic mechanisms may be different in SHR and WKY rat brain. PMID- 3000511 TI - Identification of extrasynaptic binding sites for [3H]GABA in peripheral nerve. AB - The binding of radiolabelled gamma-aminobutyric acid ( [3H]GABA) to extrasynaptic sites in peripheral nerve was examined in order to characterize the receptor species mediating the depolarizing action of GABA on myelinated axons. Binding of [3H]GABA was carried out under Na+-free conditions with fresh homogenates of amphibian sciatic nerve. The kinetics of association of the radioligand suggested the presence of a rapidly associating, reversible binding site, and a slowly associating, apparently irreversible one. Scatchard plots of the reversible component of binding were linear and yielded a mean affinity (Kd) of 22 nM. This stands in contrast with the ED50 of 90 microM for GABA-evoked depolarization of frog nerve; and therefore the high affinity binding sites are unlikely to be involved in such depolarization. The rank order of potency of agonists that competed with [3H]GABA was muscimol greater than GABA much greater than delta aminovaleric acid greater than beta-alanine. However, the GABAA analogues 3 aminopropanesulfonic acid, beta-guanidinopropionic acid, and guanidoacetic acid as well as the GABAB agonist baclofen all failed to displace the ligand. Bicuculline methiodide, picrotoxin, and nipecotic acid also did not compete. The bicuculline-insensitive, baclofen-insensitive high affinity binding sites identified here appear to be unique, as they are distinctly different from the classical GABAA and GABAB receptors. PMID- 3000512 TI - Opposing alpha- and beta-adrenergic mechanisms mediate dose-dependent actions of noradrenaline on supraoptic vasopressin neurones in vivo. AB - The effects of pressure-applied noradrenaline (NA) on the activity of neurosecretory cells of the supraoptic nucleus (SON) were examined in anaesthetized male rats. Spontaneously active, antidromically identified neurosecretory cells were classified as vasopressin (VP)-secreting on the basis of activity patterns and responsiveness to baroreceptor activation. The probability of encountering VP units was enhanced by confining electrode penetrations to the caudal aspect of the SON. Application of low concentrations of NA (50-150 microM) excited 75% of putative VP neurones tested (n = 45), while very high concentrations (1-100 mM) were inhibitory (79%, n tested = 14). The excitatory effects of NA were blocked by the alpha 1 antagonist prazosin (0.1-5 microM, n = 9) and mimicked by application of the alpha 1 agonist methoxamine (300 microM-1 mM, n = 29). The alpha 2 agonist clonidine (800 microM-1 mM) also frequently elicited mild excitations (92%, n tested = 13); however, this was commonly followed by an extended period of quiescence. Neither the alpha 2 antagonist yohimbine (5 microM, n = 4) nor the beta-adrenoreceptor antagonist timolol (5-20 microM, n = 6) blocked NA-induced excitations. The inhibitory effects of high concentrations of NA, however, were blocked by the application of timolol (5-20 microM, n = 5). It is suggested that the excitatory effect of low concentrations of NA on VP neurones reflects the actions of this substance when endogenously secreted at normal sites of release within the SON. PMID- 3000513 TI - The action of norepinephrine in the dentate gyrus: beta-mediated facilitation of evoked potentials in vitro. AB - The effects of superfusion of norepinephrine (NE) on perforant path (PP) evoked potentials in the dentate gyrus were evaluated in the rat hippocampal slice preparation. Superfusion of NE (10 microM) produced a facilitation of the PP evoked responses. Facilitation of the synaptically-evoked responses was expressed in the field potential as an increase in extracellular excitatory postsynaptic potential (EPSP) (117% of control), a decrease in population spike onset latency (94% of control) and an increase in population spike amplitude (131% of control). In 24% of the slices the facilitation of the population spike amplitude lasted longer than 30 min. Isoproterenol, a beta-agonist, mimicked NE effects while timolol, a beta-antagonist, blocked them. Facilitation of the population spike amplitude by NE could not be accounted for solely by the increase in EPSP slope also produced by NE. Superfusion of NE did not produce facilitation of the antidromically evoked field potentials, but in 4 of 8 slices produced a small decrease. NE effects were activity-independent, since the subsequently evoked PP responses were facilitated even when the PP was not concurrently stimulated during superfusion with NE. PMID- 3000514 TI - Dopaminergic modulation of sensory responses of striatal neurons: single unit studies. AB - The extracellular single unit responses of striatal neurons to repetitive stimulation of the sciatic nerve were recorded in the urethane anesthetized rat. Changes in the magnitude of these responses after pharmacological manipulation of dopamine (DA) neurotransmission were evaluated. While the intravenous administration of 0.25 mg/kg i.v. amphetamine (AMPH) had no significant effect on baseline firing rates as compared to saline controls, the magnitudes of excitatory and inhibitory evoked single unit responses were significantly decreased by 68% by AMPH. Further, this reduction in response magnitude produced by AMPH was completely blocked by pretreatment with 0.5 mg/kg i.v. haloperidol or by intrastriatal 6-hydroxydopamine lesions. This indicates that the observed effect is mediated by dopamine located in nerve terminals within the striatum. These results suggest that DA functions to modulate the responsiveness of striatal neurons to afferent signals. PMID- 3000515 TI - Neostriatal evoked inhibition and effects of dopamine on globus pallidal neurons in rat slice preparations. AB - Spontaneous unit discharges were recorded extracellularly from globus pallidal (GP) neurons in rat slice preparations. The firing rates of GP neurons ranged from 2.0 to 24.0 spikes/s and their firing patterns were predominantly of two types: regular and irregular. Stimulation of the neostriatum evoked two distinct types of inhibition which were dependent on GP neuronal firing patterns, a brief inhibition (about 75 ms) followed by resetting rhythmic neuronal activities and a relatively long-term inhibition (about 100 ms). These inhibitions evoked by neostriatal stimulation were attenuated or completely blocked by bath application of either bicuculline or strychnine (2 X 10(-5)-10(-4) M) but not by naloxone. Bath application of dopamine (10(-4)-10(-3) M) produced slow increases in the firing rates by 30-65% in about a half of GP neurons tested. Iontophoretic application of dopamine (10-20 nA) attenuated inhibition in GP neurons by 40-55% induced by either iontophoretically applied GABA (5-30 nA) or neostriatal stimulation without affecting their spontaneous firings. These results suggest that dopamine may produce change in the firing patterns of GP neurons by either acting directly or attenuating GABAergic inhibitory transmission from the neostriatum. PMID- 3000516 TI - Beta-adrenoceptor subtypes in the human brain: autoradiographic localization. AB - The distribution and characteristics of beta-adrenoceptors in postmortem human brain was studied using quantitative autoradiographic techniques. 125I Cyanopindolol was used as a ligand. High densities of beta-adrenoceptors were found in the caudate, putamen, different cortical areas and layers and the hippocampal formation. Low densities were present in other areas such as the thalamus, hypothalamus, midbrain and cerebellar cortex. Specific beta 1 and beta 2 antagonists were used to visualize and quantify separately the two subtypes of beta-adrenoceptors. Computer analysis of the competition curves obtained revealed that the putamen was enriched in beta 1 sites while the cerebellum contained predominantly beta 2 adrenoceptors. The regional distribution of beta adrenoceptor subtypes was found to be similar to that seen in the rat brain. PMID- 3000517 TI - The effect of lectins on desensitisation of locust muscle glutamate receptors. AB - At the excitatory neuromuscular junction of the locust, Schistocerca gregaria, desensitisation to L-glutamate is blocked by the lectin concanavalin A (Con A). In this study a range of lectins has been used to assess the influence of simple sugar binding specificity on desensitisation block. Pea and lentil lectins have similar simple sugar specificities (mannose/glucose) to Con A and block desensitisation in a similar manner. Soybean and wheatgerm lectins have other simple sugar specificities and do not block desensitisation of the locust muscle glutamate receptor. Native Con A is a tetramer at pH 7 but at lower pH or following succinylation (S-Con A) it becomes a dimer, with reduced biological activity. S-Con A does not block desensitisation but it does bind to locust muscle and protects the glutamate receptor from the desensitisation block caused by Con A. When Con A is applied to a desensitised neuromuscular junction the ongoing desensitisation is not blocked. It appears that desensitisation block by Con A, pea and lentil lectins is dependent on lectin binding to mannose or glucose moieties on or in the region of the glutamate receptor, and that these moieties are masked from lectin when the receptor is in its desensitised state. PMID- 3000518 TI - The effect of high-frequency electrical stimulation and norepinephrine on cyclic AMP levels in normal versus norepinephrine-depleted rat hippocampal slices. AB - Cyclic 3',5'-adenosine monophosphate (cAMP) generation by neuronal activity and norepinephrine (NE) was studied in rat hippocampal slices. High-frequency perforant path stimulation increased cAMP levels 2.5-fold in the dentate gyrus 1 min, but not 30 min, post-stimulation. This increase was abolished by depletion of NE with 6-hydroxydopamine. NE (50 microM) also caused a 3-fold rise in cAMP in whole slices and this stimulation was not altered by NE depletion. These results are consistent with our previous data suggesting that cAMP production is involved in the expression of long-term potentiation and NE-induced long-lasting potentiation in the dentate gyrus. PMID- 3000519 TI - Neurotensin-like immunoreactivity and neurotensin receptors in the rat hypothalamus and in the neurointermediate lobe of the pituitary gland. AB - In the rat hypothalamus, cell bodies containing neurotensin-like immunoreactivity were mainly found in the medial preoptic area, the periventricular nucleus, the paraventricular nucleus, the supraoptic nucleus and the arcuate nucleus. [3H]neurotensin binding sites were observed throughout the hypothalamus with a dense accumulation of silver grains over the paraventricular nucleus, the arcuate nucleus and the median eminence region. By radioimmunoassay neurotensin-like immunoreactivity was also found in the neurointermediate lobe of the pituitary gland of various mammalian species and in human postmortem posterior pituitary glands. In the rat studies involving pituitary stalk transections and the neurotoxin monosodium glutamate indicated the presence of a neurotensinergic pathway from the arcuate nucleus to the neurointermediate lobe of the pituitary gland. [3H]neurotensin binding sites were found to be concentrated over the intermediate lobe of the pituitary gland and their presence was not affected by pituitary stalk transection, indicating their localization on endocrine cells of the intermediate lobe of the pituitary gland. PMID- 3000520 TI - Effects of status epilepticus on extracellular amino acids in the hippocampus. AB - Extracellular amino acids were followed in the hippocampus during sustained seizures induced by systemic administration of kainic acid (KA) or bicuculline (BC). KA epilepsy was associated with marked increases in phosphoethanolamine (PEA) and taurine. Alanine and ethanolamine were moderately raised while other amino acids were unaffected. BC seizures encompassed a slightly different pattern of alterations. In contrast to KA seizures, BC epilepsy had no effect on taurine. Significant increments were observed for PEA and alanine while elevations of ethanolamine were subtle. In both types of seizures, glutamate and GABA remained unaffected extracellularly, probably due to efficient recapture mechanisms. PMID- 3000521 TI - Irreversible autonomic actions by lophotoxin suggest utility as a probe for both C6 and C10 nicotinic receptors. AB - The marine natural product lophotoxin has produced a non-reversible antagonism of parasympathetic and sympathetic functions that are known to be mediated by C6 sub type nicotinic receptors. Transmission through anuran paravertebral ganglia was eliminated in 20-40 min by 10-30-min treatments with 16-32 microM lophotoxin, in a time course resembling the onset of block of C10 sub-type nicotinic receptors at the neuromuscular junction and in cultured BC3H-1 cells. The action persisted through 16 h of washout. Nerve conduction was unaffected. Somewhat longer treatments (80 min) of in vitro ileal sections resulted in loss of sensitivity to nicotine, but not to acetylcholine, for at least 5 h. These data indicate that lophotoxin can serve as a more universal nicotinic receptor probe than the alpha neurotoxins, which may bind to both C6 and C10 sub-types, but block only the C10. PMID- 3000522 TI - Increase of dopamine turnover in bilateral striata after unilateral injection of haloperidol into substantia nigra of unrestrained rats. AB - In order to investigate self-regulation of dopamine (DA) neurons, the effects of intranigrally administered haloperidol (Hal), a DA receptor antagonist, on nigrostriatal DA systems were examined using differential pulse voltammetry with carbon fiber electrode. The measurements were achieved in the bilateral caudate putamen (CP) of behaving rats, in the region of which DA or 3,4 dihydroxyphenylacetic acid made an oxidative current peak (P2) spontaneously. Unilateral injection of Hal (5 micrograms in 1 microliter) into the substantia nigra of rat increased P2 in a time-dependent manner. This phenomenon was observed in both CP, but a more significant increase was in the ipsilateral side (156 +/- 2% of spontaneous height 2.75 h after injection) than in the contralateral side (129 +/- 7%). These effects enlarged in a dose-dependent manner. The same results were found in tissue homogenates determined by high performance liquid chromatography with electrochemical detection. In the latter case, however, no significant difference was observed between the left and right sides. The present results suggest that Hal, attaching nigral autoreceptors on the cell bodies and dendrites, blocks inhibitory influence of endogenous DA and then activates the nigrostriatal DA neurons, while the contribution of non dopaminergic neurons is also possible. PMID- 3000523 TI - Analgesic effects of mu antagonists after naloxone non-reversible stress-induced analgesia. AB - Three antagonists at the mu opiate receptor site: naloxone, naltrexone and diprenorphine, and one agonist-antagonist compound nalorphine, at doses usually not analgesic elicited analgesia in rats when administered after non-naloxone reversible shock-induced analgesia had disappeared. The chi receptor antagonist, MR 2266, and the delta antagonist, ICI 154129, were all ineffective. This effect was no longer present when non-naloxone-reversible shock-induced analgesia was inhibited by the administration of the chi receptor antagonist, MR 2266. These results suggest that the mu opiate receptor may change its conformation under particular conditions such as continuous inescapable shock. PMID- 3000524 TI - A potent transient outward current regulates excitability of dorsal raphe neurons. AB - Activity of neurons in the dorsal raphe nucleus of the rat was recorded intracellularly in a brainstem slice. The neurons had a high input resistance, a linear I-V curve in the hyperpolarizing direction and a long membrane time constant. Broad action potentials were followed by a large afterhyperpolarization (AHP). This AHP removes partial inactivation of a potent transient outward rectifier that is activated by a subsequent depolarization of the neuron; it clamps the cell membrane at a potential slightly below firing level and blocks generation of action potentials for up to 100 ms. The transient rectification was sensitive to 4-aminopyridine (4-AP) but not to tetraethylammonium (TEA). It appears to share similar properties with those of IA seen elsewhere and to function in the regulation of interspike intervals of dorsal raphe (DR) neurons in vitro. PMID- 3000525 TI - Benzodiazepine receptors modulate circulating plasma vasopressin concentration. AB - Chlordiazepoxide pretreatment decreased basal levels of plasma arginine vasopressin (AVP) and attenuated picrotoxin-induced increases in plasma AVP and blood pressure compared to saline-pretreated spinal animals. Prior administration of RO 15-1788 blocked the effects of chlordiazepoxide on basal plasma AVP as well as picrotoxin-evoked changes in plasma AVP and blood pressure. Thus, interactions at the benzodiazepine receptor may influence basal and evoked changes in plasma AVP concentration. PMID- 3000527 TI - Adrenocorticotropin and alpha-melanotropin in the myenteric plexus of the rat duodenum: an electron microscopic study. AB - Adrenocorticotropin (ACTH) immunoreactivity was localized at the ultrastructural level as positive 'cores' within large dense-cored vesicles (LDVs) of axons and dendrites of the rat duodenum. The immunostained vesicle 'cores' were 35-50 nm in mean diameter, corresponding to 'cores' of LDVs with a mean diameter of 80-90 nm. alpha-melanotropin (alpha-MSH) was detected also within LDVs, expressing the same mean diameter as ACTH-stained vesicles. alpha-MSH and ACTH were localized only within structures belonging to the enteric nervous system of the rat duodenum. alpha-MSH and ACTH, as detected by immunostaining, were absent in endocrine cells of the rat duodenum. These findings suggest the possibility that these peptides may have important physiological roles in the rat duodenum. PMID- 3000526 TI - Modulation of terminal excitability of mesolimbic dopaminergic neurons by D amphetamine and haloperidol. AB - Electrophysiological techniques were used to study the changes in the terminal excitability of mesolimbic DA and non-DA neurons following the infusion of D amphetamine (D-AMP) and haloperidol (HAL) into the nucleus accumbens (NAc) of rats. The amount of current needed to evoke antidromic spikes by electrical stimulation of the NAc was used as an index of the excitability of axon terminals of these neurons. The excitability of DA neurons was decreased by D-AMP and increased by HAL. In addition, the effect produced by D-AMP was reversed by HAL. By contrast, these drugs either induced an opposite effect or were ineffective in inducing changes on the excitability of nerve terminals of mesolimbic non-DA neurons. Infusion of the vehicle or saline produced no effect. D-AMP and HAL were still effective in modulating the excitability of mesolimbic DA nerve terminals after the destruction of NAc neurons by ibotenic acid. The results suggest that the effects seen after D-AMP and HAL are mediated primarily by DA autoreceptors. It is likely that the increase in the current needed for evoking antidromic spikes after infusion of D-AMP into the terminal region is the consequence of DA autoreceptor-mediated hyperpolarization of terminal membranes. On the other hand, HAL could exert its actions by blocking autoreceptor-mediated hyperpolarization. PMID- 3000529 TI - Autoradiographic distribution of mu1 and mu2 opioid binding in the mouse central nervous system. AB - Several types of opioid binding sites have been differentiated using biochemical and pharmacological criteria. We have used quantitative in vitro autoradiography to compare the levels of mu1 and mu2 opioid binding in the mouse central nervous system. Mu1 sites have a high affinity for all labeled opioids studied to date and have been associated with their analgesic effects, whereas mu2 sites have a high affinity only for opiate alkaloids and have been associated with their respiratory depressant effects. We used [3H]dihydromorphine (DHM) to visualize total mu sites (mu1 and mu2) and [3H]DHM plus a low concentration of [D-Ala2-D Leu5]enkephalin (DADL) to visualize mu2 sites. Levels of mu1 binding were determined by subtracting mu2 binding from total mu binding. This mu1 distribution was confirmed in selected regions by an alternate method using [3H]DADL. High ratios of mu1 to mu2 binding were noted in frontal cortex, nucleus accumbens, rostral striatum, ventral pallidum, ventral periaqueductal gray matter, and laminae I and II of the spinal cord. The observation of high densities of mu1 binding in certain pain processing areas correlates with behavioral and pharmacological studies suggesting that analgesia from opiates and opioids is mediated primarily by mu1 sites. In other areas, such as the limbic system, dorsal nucleus of the vagus nerve, and nucleus of the solitary tract, either a low ratio of mu1 to mu2 binding or no mu1 binding was observed. This differential regional localization of mu1 and mu2 binding provides further evidence for the distinctness of these sites. PMID- 3000528 TI - Autoradiographic analysis of mu1, mu2, and delta opioid binding in the central nervous system of C57BL/6BY and CXBK (opioid receptor-deficient) mice. AB - The recent development of in vitro autoradiography techniques has enabled investigators to determine the distribution and relative levels of multiple ligand binding sites in discrete anatomical areas. In this study we used semi quantitative in vitro autoradiography to compare the levels of binding to central mu1, mu2, and delta opioid sites in two strains of mice, C57BL/6BY and CXBK. The CXBK strain is known to be deficient in whole brain opioid binding sites and to be less sensitive than the C57 strain to the analgesic and locomotor stimulatory effects of opiates and opioids. Delta sites were visualized using [3H](D-Ala2-D Leu5]-enkephalin (DADL) plus a low concentration of morphine, total mu sites (mu1 and mu2) were visualized using [3H] dihydromorphine (DHM), and mu2 sites were visualized using [3H]DHM plus a low concentration of DADL. Binding to mu1 sites was determined by subtracting mu2 binding from total mu binding. We found that the two strains did not consistently differ in the levels of delta site; in some areas the CXBKs had lower levels but in many areas they had levels equal to or greater than those for the C57s. The CXBK strain, however, either had less or the same amount of mu binding as the C57 strain in all areas studied. The CXBK strain was especially deficient in mu1 binding, particularly in areas involved in pain processing. PMID- 3000531 TI - Functional plasticity in two afferent systems of the granule cells in the rat dentate area: frequency-related changes, long-term potentiation and heterosynaptic depression. AB - Monosynaptic evoked field potentials (MEFP) were recorded in the dentate gyrus of male Wistar rats upon stimulation of either the perforant path or the commissural system. While the perforant path potential exhibited in acute experiments a clear reversal point of the field excitatory postsynaptic potential (EPSP) and population spike when protruding the registration electrode from the hippocampal fissura to the hilus of the dentate gyrus, the simultaneously registered commissural potential elicited by stimulation of the contralateral hilus showed no reversal of the negative monophasic wave but merely an amplitude maximum 40 microns above the reversal point of the perforant path potential. Frequency related changes of the MEFPs during short tetanic stimulation with 15 Hz both in acute and chronic experiments, revealed differences in the properties of the input systems in that the commissural potential exhibited a clear frequency potentiation whereas the perforant path potential showed frequency depression. Pronounced long-term potentiation of the perforant path potential induced by 4 trains of tetanizing stimuli and lasting up to 72 h was accompanied by a long term heterosynaptic depression of the commissural potential for up to 7 days after tetanization. Both the different frequency-related changes of the inputs and the extremely long duration of the heterosynaptic depression are discussed with respect to their proposed functions in the mechanisms of functional plasticity. PMID- 3000530 TI - Non-invasive in vivo spectrophotometric monitoring of brain cytochrome aa3 revisited. AB - Cytochrome aa3 (cyt aa3) is the main catalyst of cellular oxygen consumption. The properties of cyt aa3 will define the tissue oxygen requirements and provide an insight into energy supply and demand. Currently dual-wavelength (605-590 or 605 620 nm) reflectance spectrophotometry is used to monitor cyt aa3 redox state in vivo. We have experimentally demonstrated that the compensation for blood contamination in the surveyed tissue by these wavelength pairs is less than optimal. An alternative approach, similar to spectrophotometric analysis of multicomponent systems used in vitro, is presented in the triple wavelength equation as follows: delta cyt aa3 = 1.000 (delta A605) - 0.662 (delta A586.1) + 0.316 (delta A580) Based on a series of experiments performed in cuvette in vitro, isolated perfused rat head in situ, and living rat head in vivo, we have demonstrated that the cyt aa3 equation fully compensates for changes in cerebral blood volume and saturation. This non-invasive method of in vivo monitoring of cyt aa3 can provide the means to reliably and accurately determine tissue oxygen delivery under physiological conditions. PMID- 3000532 TI - Botulinum A neurotoxin inhibits non-cholinergic synaptic transmission in mouse spinal cord neurons in culture. AB - The effects of botulinum A neurotoxin and tetanus toxin were studied in cultured mouse spinal cord neurons. In approximately 60% of the neurons (n = 150), botulinum A neurotoxin caused paroxysmal depolarizing events. In two cells hyperpolarizing shifts were observed. The pattern of the burst-like activity varied in shape and frequency in individual cells. Between the paroxysmal events, ongoing synaptic activity could be recorded. The other 40% of the treated neurons did not develop a characteristic pattern of bursts, but there was a decrease in frequency of synaptically generated events. In contrast to botulinum A neurotoxin, tetanus toxin invariably produced well organized paroxysmal events without any synaptic activity between them. At later stages botulinum A neurotoxin and tetanus toxin blocked inhibitory and excitatory postsynaptic potentials in all neurons studied. These results have demonstrated, for the first time using electrophysiological techniques, that botulinum A neurotoxin blocks both excitatory and inhibitory synaptic transmission in the mammalian central nervous system. There are however differences between these effects of botulinum A neurotoxin and the actions of tetanus toxin on these cells. It is suggested that at the femtomolar range tetanus toxin blocks selectively central inhibitory systems and botulinum A neurotoxin the motor endplate. At the picomolar range both toxins affect many if not all, transmitter systems. PMID- 3000533 TI - Low doses of ethanol inhibit the firing of neurons in the substantia nigra, pars reticulata: a GABAergic effect? AB - The intravenous administration of relatively low doses of ethanol (0.25-2.00 g/kg) produced a dose-dependent inhibition of the firing rate of the neurons located in the substantia nigra, pars reticulata (PR neurons). This effect was eliminated both by picrotoxin and bicuculline, two blockers of gamma-aminobutyric acid (GABA) transmission, and potentiated by muscimol (a direct GABA agonist) and diazepam (a representative of the benzodiazepine class which facilitate GABA transmission). The specific benzodiazepine antagonist, Ro 15-1788, blocked the potentiating effect of diazepam on the ethanol effect but failed to antagonize ethanol-induced inhibition of the firing rate of the neurons. These results indicate that ethanol might inhibit the firing of PR neurons through a GABAergic mechanism. Moreover, since PR neurons are thought to exert an inhibitory control on nigral dopaminergic neurons, it is suggested that the depression of the activity of such inhibitory interneurons may be responsible for ethanol-induced stimulation of dopaminergic activity. PMID- 3000534 TI - Modification of dental pain and cutaneous thermal sensitivity by physical exercise in man. AB - The effect of physical exercise on dental pain thresholds, the release of pituitary stress hormones and thermal sensitivity of skin was tested in healthy human subjects. Different levels of exercise (100-300 W) at different pedal frequencies were produced by a cycle ergometer. Thermal limen (the interval between warm and cool thresholds) determined from glabrous hand, hairy forearm and leg was used as a parameter of thermal sensitivity. In all subjects the heart rate and blood pressure were increased with increasing work load. Dental pain thresholds were elevated at high work loads with a concomitant activation of pituitary stress hormone (especially growth hormone) release. Thermal limens at all 3 sites were increased work load, too, independent of the pedal frequency. The increase of thermal limen was most marked in the leg and least in the glabrous hand. The results indicate that physical exercise produces a non segmental, load-dependent decrease of pain and thermal sensitivity with a concomitant activation of pituitary stress mechanisms. The magnitude of modification varies with skin region. Activation of inhibitory mechanisms at spinal levels via muscle and proprioceptive afferents, in a way suggested by the gate control theory of pain mechanisms, seems to have only a minor, if any, contribution to the present findings, since a higher pedal frequency did not produce a more marked decrease of sensitivity. PMID- 3000535 TI - Neuroblastoma X glioma NG108-15 hybrid cells cultured in a serum-free chemically defined medium: effects on acute and chronic opiate regulation of adenylate cyclase activity. AB - Neuroblastoma X glioma NG108-15 hybrid cells cultured in a chemically defined medium within 3 cell passages, exhibited viability, growth rate and morphology similar to those of cells grown in medium supplemented with 5% fetal calf serum. Hybrid cells cultured in the chemically defined medium within these periods of time also did not exhibit a difference in basal adenylate cyclase activity nor in the enzymatic activities stimulated by adenosine, forskolin, NaF, GppNHp or Mn2+. Furthermore, opiate receptor density in chemically defined medium cultured cells remained identical to that in cells cultured in 5% fetal calf serum. The acute and chronic effects of opiates on adenylate cyclase were similar for cells grown under either set of conditions. PMID- 3000536 TI - Distribution and quantification of 5-HT nerve cell bodies in the nucleus raphe dorsalis area of C57BL and BALBc mice. Relationship between anatomy and biochemistry. AB - BALBc and C57BL mice have been shown to have a different 5-HT metabolism. The present study compares the number and the distribution of 5-HT cell bodies in the nucleus raphe dorsalis area (B7 + B6) of these strains. By using 5-HT immunohistochemistry, we found a higher number of 5-HT neurons in the most caudal part of NRD (B6) of BALBc mice compared to C57BL. This difference may be correlated with a higher level of endogenous 5-HT, a higher uptake capacity toward exogenous [3H]5-HT, and a lower release of the amine in this same area of BALBc mice compared to C57BL. It could also imply a significant participation of the nerve cell bodies in the regulation of 5-HT transmission inside 5-HT nuclei. PMID- 3000537 TI - The sex hormone-dependent development of opiate receptors in the rat medial preoptic area. AB - The opiate receptor content of the sexually dimorphic medial preoptic area (MPOA) was examined in newborn and 5-day old (D6) male and female rats. A significant increase of [3H]naloxone binding was observed in and around the sexually dimorphic nucleus of the preoptic area (SDN-POA) in D6 female rats, relative to newborn females. Opiate receptor labeling did not increase over this period in males, nor was labeling different between males and females at birth. This dramatic alteration of MPOA opiate receptor content was observed to occur in either sex in the absence of testosterone postnatally; that is, neonatally castrated males exhibited the same increase of labeling by D6 as did normal females. Conversely, daily postnatal testosterone treatment of females from birth to D6 resulted in the development of male-like MPOA opiate receptor pattern. The sex hormone-dependence of MPOA opiate receptor development is discussed in relation to the sex hormone-dependent ontogeny of SDN-POA structure. The overlap of critical periods for the development of these structural and chemical sexual dimorphisms suggests a role for endogenous opioids in modulating MPOA development. PMID- 3000538 TI - Kindling with rapidly recurring hippocampal seizures. AB - Bipolar electrodes, stereotactically implanted in the hippocampus of adult rats, were used to deliver 10 s trains of suprathreshold tetanic electrical stimuli every few minutes. As indices of seizure intensity, durations of the afterdischarges triggered by these stimuli were measured, and the accompanying behaviors were scored on a 5-point scale. After 2-3 h, prolonged afterdischarges appeared in conjunction with severe limbic seizures, separated by periods of approximately 60 min. After 3-9 h, the stimulation was withheld until the following day. Upon reinstitution of the stimuli, intense seizures were seen at the onset, and the cycle time between them was shortened. Enhanced responsiveness to a fixed stimulus persisted for several months, the longest period tested. In addition, the enhanced epileptogenicity showed transference and was not stimulus specific. These studies, using stimuli with low intertrain frequency and short interstimulus intervals, establish a robust and rapidly-developing model of epileptogenesis in the hippocampus that is comparable to traditional kindling. PMID- 3000539 TI - [Changes of the expression of histocompatibility (HLA) antigens in human fibroblasts transformed by the avian leukosis-sarcoma virus]. AB - Monoclonal monomorphic antibodies anti-HLA class I and II antigens, were used to evaluate the expression of these molecules on normal and RSV-transformed human fibroblasts. The results indicate that the human diploid fibroblasts transformed in vitro by RSV present a reduced HLA-class I antigens expression as compared to the uninfected fibroblasts of the same donor. In parallel, it is demonstrated that the class II molecules absent on normal cells are expressed after transformation. PMID- 3000540 TI - [Phosphatidylethanolamine methylase and cyclic nucleotide phosphodiesterase activities in human B lymphoid hemopathies]. AB - Phospholipid methylase and cyclic nucleotide phosphodiesterase activities were studied in human B lymphoid hemopathies (51 patients: acute lymphoblastic leukemia, B lymphoma, chronic lymphocytic leukemia, hairy cell leukemia) and compared with activities in lymphoblastid and Burkitt lymphoma cell lines and with normal B lymphocytes: methylase activity proved to be lower in ALL and high grade lymphoma and inversely related to the percent of cells in S phase state; the A/G ratio of phosphodiesterases was low in ALL and CLL and high in hairy cell leukemia and it was related to the percent of cells in S phase state. PMID- 3000541 TI - [Ultrastructural alterations of rat myocardial cells in cell culture of the beating heart infected with coxsackie B-2 virus]. PMID- 3000542 TI - [Determination of serum angiotensin converting enzyme activity in normal children and in children with pulmonary disease]. PMID- 3000544 TI - Rotaviral and coronaviral diarrhea. AB - A number of different viruses can be primary pathogens in the neonatal calf diarrhea complex. By far the most common viruses causing calfhood diarrhea found throughout the world are rotaviruses and coronaviruses. Primary infection of newborn calves with either one of these viruses can cause severe intestinal alterations and diarrhea. Rotaviruses can produce high-morbidity outbreaks of diarrhea in calves under 10 days of age. Morality is variable mainly owing to secondary bacterial infections and electrolyte imbalances. Rotavirus infection of the small intestinal mucosa leads to loss of enterocytes of the upper third of the intestinal villi with subsequent villous atrophy and malabsorption. There is growing evidence that different rotavirus serotypes of different pathogenicity exist. Coronavirus infections can produce high-morbidity outbreaks of diarrhea in calves under 20 days of age, with variable mortality due to secondary complications. Coronaviruses affect not only the small intestinal mucosa, producing significant villous atrophy, but also the colon, causing a very severe intestinal damage that can lead to death due to subsequent electrolyte disturbances. All coronaviruses associated with neonatal calf diarrhea appear to be of the same serotype. The etiologic diagnosis of viral diarrheas of calves requires the support of the laboratory. One of the most useful diagnostic methods is the examination of fecal extracts for the presence of virus particles by electron microscopy. Other antigen-detection procedures like enzyme immunoassays have been found to be useful in the diagnosis of rotaviral diarrheas. The sample of choice for these diagnostic tests is a fresh fecal sample collected directly from the calf as close as possible to the onset of diarrhea. Samples from more than one calf during the outbreak enhance the laboratory ability to establish a proper viral diagnosis. PMID- 3000543 TI - Neurologic diseases. AB - The responses of apparently healthy newborn foals to neurologic testing differ significantly from those of adult horses. These responses and the diagnostic techniques pertinent to neurologic problems are reviewed as a basis for evaluation of the compromised neonatal foal. The more frequently encountered neurologic diseases are discussed in a problem-oriented format. These clinical problems include behavioral abnormalities, convulsions, changes in consciousness, blindness, ataxia without loss of strength, ataxia with weakness and paralysis, and the floppy foal. PMID- 3000545 TI - An in situ assay system to measure ornithine decarboxylase activity in primary cultures of chicken osteoblasts: effects of bone-seeking hormones. AB - We present a rapid and uncomplicated in situ assay for measuring ornithine decarboxylase activity in small cell quantities. This method is more economic than the in situ methods described by others. In addition, our system is faster and less complicated since it avoids manipulation of the CO2-trapping paper. Applying this method we demonstrate that parathyroid hormone, PGE1, and other inducers of intracellular cAMP levels, like IBMX and forskolin can induce ODC activity in primary cultures of chicken osteoblasts. Salmon calcitonin does not induce ODC activity, and 1.25 (OH)2D3 at higher concentrations can even give an inhibition of ODC activity. We confirm the recent findings that ODC activity is also dependent on calcium. PMID- 3000546 TI - Reconstitution of lymphocyte 5'-nucleotidase in lipid bilayers: behaviour and interaction with concanavalin A. AB - Pure 5'-nucleotidase (EC 3.1.3.5) and a membrane glycoprotein fraction (partially purified 5'-nucleotidase) were isolated from pig lymphocyte plasma membrane by affinity chromatography techniques, using the cationic detergent dodecyltrimethylammonium bromide as a solubilizing agent. A detergent-dialysis technique was used to reconstitute both partially purified and pure enzyme into large unilamellar phospholipid vesicles, where it remains functional. 5' Nucleotidase is relatively unstable in detergent solutions, but is highly stable once reconstituted into lipid vesicles. Arrhenius plots of the enzyme in bilayers of dimyristoyl phosphatidylcholine show a break point at 22-23 degrees C, with a different activation energy above and below the phospholipid gel-to-liquid crystalline phase transition. 5'-Nucleotidase in intact plasma membrane is inhibited more than 95% by concanavalin A in a positively cooperative fashion (Hill coefficient = 2.1), as is partially purified reconstituted enzyme. Purification of the enzyme before reconstitution results in less than 50% inhibition by concanavalin A and a complete loss of positive cooperativity (Hill coefficient less than 1.0). The inhibition properties of the enzyme can be fully restored by co-reconstituting pure 5'-nucleotidase with the remaining lymphocyte glycoproteins. PMID- 3000547 TI - Thermodynamic characterization of the partially denatured states of ribonuclease A in calcium chloride and lithium chloride. AB - The denaturations of ribonuclease A by calcium chloride and lithium chloride were studied by circular dichroism measurements in the far-ultraviolet region. The temperature dependence of the equilibrium constant for the unfolding of the protein by calcium chloride and lithium chloride gave values of 46 and 52 kcal mol-1 (1 cal = 4.1868 J) for the enthalpy of denaturation at 25 degrees C and pH 7.0, respectively. Thermodynamic parameters for the denaturation by calcium chloride and lithium chloride are compared with those for the heat and guanidine hydrochloride denaturation. It has been observed that the thermodynamic quantity, be it free energy, entropy, or enthalpy, cannot be related quantitatively to the extent of unfolding measured by various conformational properties of the protein. PMID- 3000548 TI - Polyphosphoinositide metabolism in aging human erythrocytes. AB - Normal human erythrocytes were fractionated in a density gradient. Capacity to metabolize polyphosphoinositides was compared in young (least dense) and old (most dense) cells. Polyphosphoinositide synthesis was assessed by following the incorporation of radioactivity from [gamma-32P]ATP into the 1-(3-sn-phosphatidyl) D-myo-inositol 4-phosphate (PtdIns4P) and 1-(3-sn-phosphatidyl)-D-myo-inositol 4,5-bisphosphate (PtdIns(4,5)P2) of isolated membranes. There was no significant age-dependent change in the ability to synthesize PtdIns4P and PtdIns(4,5)P2 or in the response of the PtdIns and PtdIns4P kinases to Mg2+. The cytosolic Mg2+ dependent PtdIns(4,5)P2 phosphatase was also unaffected by age. The membrane cation-independent PtdIns4P phosphatase activity declined slightly (12%). Therefore, the capacity to catalyse the interconversion among the three phosphoinositides in the membrane is retained throughout the life of the erythrocyte. The Ca2+-dependent polyphosphoinositide phosphodiesterase activity in the membranes was reduced in old cells (57%) to the same extent as the glutamate-oxaloacetate transaminase activity used as an index of cell age. Thus, irreversible loss of polyphosphoinositide from the membrane by the action of this diesterase (prevented in healthy cells by the active maintenance of a very low intracellular Ca2+ concentration) is not very likely even in senescent cells when Ca2+ homeostasis begins to fail. PMID- 3000550 TI - Inability to experimentally produce a polyneuropathy in dogs given chronic oral low level lead. AB - Electromyographic examinations were performed at various times over a 40 week period in four mature dogs receiving chronic oral low doses of lead acetate and a control dog receiving sodium acetate. Blood lead levels in the four dogs were elevated (mean values 1.15, 2.18, 1.13 and 1.72 mumol/liter). No clinical signs of lead intoxication were present. Two dogs had evidence of a nonregenerative anemia. Neither needle electromyographic nor nerve conduction velocity studies showed evidence of a polyneuropathy. Teased nerve fiber preparations of proximal and distal segments of the ulnar and tibial nerves and muscle biopsies of distal appendicular muscles were normal in all dogs. Light microscopic examination of the brain, kidneys and liver revealed no abnormalities in the two dogs necropsied. In conclusion, a polyneuropathy was not produced experimentally in dogs ingesting low doses of inorganic lead for up to 40 weeks. PMID- 3000549 TI - Site-directed cleavage of immunoglobulin gene segments by lymphoid cell extracts. AB - To study the enzyme(s) involved in the site-specific recombination of immunoglobulin (Ig) gene segments, we designed an assay to detect V-J joining in vitro. The DNA from a hybrid phage (lambda VJCK) containing the VK41 gene segment separated by a 6-kilobase spacer region from the entire J-CK sequence was incubated with lymphoid cell extracts and packaged in vitro. Phages carrying genomic deletions were selected by screening for ethylenediaminetetraacetic acid resistance. Although no site-specific V-J fusion events were detected, the packaging efficiency of lambda VJCK DNA was 10(2)- to 10(3)-fold lower than that of lambda DNA. This suggested the presence in the cell extracts of an endonucleolytic activity with a specificity for the mouse DNA sequences. To detect the endonuclease cleavage products, plasmids containing VK or JK gene segments were used as a DNA substrate and the products of the in vitro reaction were visualized by autoradiography in Southern blots. Double-stranded cleavages were observed to occur near the 5' end of each one of the five JK gene segments and near the 3' end of a VK gene segment. A plasmid containing the mouse I-A beta gene was found to be resistant to cleavage, thus confirming the specificity of the endonucleolytic activity for sequences associated with the mouse Ig gene segments. PMID- 3000551 TI - Epizootiological survey of parainfluenza-3, reovirus-3, respiratory syncytial and infectious bovine rhinotracheitis viral antibodies in sheep and goat flocks in Quebec. AB - A serological survey was conducted in an attempt to detect antibodies against bovine respiratory viruses in sheep and goats from seven geographical areas of Quebec. Sera from 10% of the animals in 182 sheep flocks and 40 goat flocks were collected and specific antibodies against parainfluenza-3, reovirus type 3, respiratory syncytial and infectious bovine rhinotracheitis viruses were detected by hemagglutination-inhibition tests for the former viruses and complement fixation and seroneutralization assays for the latter viruses. Results showed prevalence rates of serological reaction to parainfluenza-3, reovirus type 3 and respiratory syncytial viruses of 28, 72 and 35% in sheep and 26, 64 and 36% in goats, respectively. No antibodies in infectious bovine rhinotracheitis virus were detected in sheep or goats tested. Prevalence rates varied according to the geographical area. No relationships were detected between age, sex, breed, size of flock and prevalence rates of different antibodies except that parainfluenza-3 antibodies were more common in large goat flocks and in sheep flocks with total confinement housing. A relationship between presence of clinical signs in the flocks and prevalence rates of antibodies was only demonstrated for parainfluenza 3 infection in goat flocks. PMID- 3000552 TI - Relationship between levels and uptake of serotonin and high affinity [3H]imipramine recognition sites in the rat brain. AB - High affinity [3H]imipramine binding, endogenous levels of serotonin and noradrenaline, and serotonin uptake were determined in brain regions of rats with selective destruction of serotonergic neurons by 5,7-dihydroxytryptamine (5,7 DHT), of adrenergic neurons by 6-hydroxydopamine (6-OHDA), and of rats treated with reserpine. Neonatal treatment with 5,7-DHT resulted in a significant decrease of both serotonin levels and density (Bmax) of high affinity [3H]imipramine binding sites in the hippocampus. In contrast, an elevation of serotonin levels and an increase in Bmax of [3H]imipramine binding were noted in the pons--medulla region. No changes were observed in the noradrenaline content in either of these regions. Intracerebral 6-OHDA lesion produced a drastic suppression of noradrenaline levels in cerebral cortex but failed to alter the binding affinity (KD) or density (Bmax) of [3H]imipramine recognition sites. A single injection of reserpine (2.5 mg/kg) resulted in marked depletion of both serotonin (by 57%) and noradrenaline (by 86%) content and serotonin uptake (by 87%) in the cerebral cortex but had no significant influence of the parameters of high affinity [3H]imipramine binding in this brain region. The results suggest that high affinity [3H]imipramine binding in the brain is directly related to the integrity of serotonergic neurons but not to the magnitude of the uptake or the endogenous levels of the transmitter, and is not affected by damage to noradrenergic neurons or by low levels of noradrenaline. PMID- 3000554 TI - Effect of chronic ethanol and food deprivation on intestinal villus morphology and brush border membrane content of lipid and marker enzymes. AB - Brush border membranes (BBM) were isolated from the jejunum and ileum of control, ad libitum (CAL); control, food-restricted (CFR); control, weight gain (CWG); and ethanol-fed (EF) rabbits. Jejunal alkaline phosphatase activity was similar among control groups, but higher in CAL than EF animals. Sucrase activity was higher in EF and CWG animals than in CAL and CFR. The alkaline phosphatase/sucrase ratio was lower in EF than control animals. Ileal enzyme marker activity was similar among EF and control animals. Sucrase (S) activity was lower in the ileum than in the jejunum. Jejunal free fatty acid and phospholipid/cholesterol (PL/C) were lower in EF than control animals, whereas ileal lipid content was generally similar among all animal groups. Total phospholipid content was similar between sites, but the cholesterol and free fatty acid content were lower in the ileum than the jejunum. The phospholipid/cholesterol ratio was increased only in the ileum of EF animals. The amount of lecithin was decreased in the jejunal BBM of EF animals resulting in a decreased choline/amine phospholipid ratio as compared with control animals. The ileal phospholipid composition was similar among all groups. A large increase in villus height is observed in the jejunum of EF animals. Villus surface area and mucosal surface area are altered with ethanol feeding and food deprivation. Thus, (i) there is a gradient of S and cholesterol between the BBM of jejunum and ileum; (ii) changes in food intake are associated with changes in the morphology as well as the enzyme marker and lipid content of BBM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000553 TI - Effects of 6-mercaptopurine treatment on the membrane potentials of rat skeletal muscle fibers. AB - 6-Mercaptopurine (6-MP), injected daily (2 mg/kg s.c.) into Sprague-Dawley rats during the first 3 weeks of life, causes atrophy in muscles of the hindquarters beginning at 4 months of age. The extensor digitorium longus (EDL) muscles from 24 rats injected with 6-MP and 23 saline-injected controls, 6-18 months of age, were studied. Electron microscopy showed a number of abnormalities in the EDL muscle of 6-MP-treated rats, such as myocytes with atypical ultrastructure (including disorganized myofibrils) adjacent to structurally normal cells. Membrane potentials (Em) were measured in the isolated EDL and in the caudofemoralis (CF) muscle in situ. The mean Em of fibers in the EDL of 6-MP treated rats (-61.1 +/- 0.7 (SE) mV) was lower than that of the control rats ( 69.7 +/- 0.6 mV). The same was true for the fibers of the CF muscle (-64.9 +/- 1.5 mV for 6-MP-treated fibers vs. -71.6 +/- 1.3 mV for controls). The contribution of the electrogenic pump potential to Em (+/- ouabain) was similar in 6-MP-treated and control rats, and therefore could not account for the depolarization observed in 6-MP-treated rats. This depolarization was not due to a decreased intracellular K+ concentration. The Na+:K+ permeability ratio (PNa/PK) was higher in the 6-MP-treated rats and could account for the decrease in Em.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000555 TI - The influence of temperature and frequency of stimulation on the impairment of excitability of frog skeletal muscle by local anesthetics and alkyl amphipathic agents. AB - The potency of various types of alkyl amphipathic (cationic, anionic, and neutral) as well as tertiary amine local anesthetics in impairing the excitability of frog skeletal muscle was markedly enhanced by an increase in temperature from 20 to 30 degrees C. Enhancement of the local anesthetic effects of all types of agents was also produced by a decrease in temperature to 5 degrees C, but this effect was found to be frequency dependent. With abrupt increase or decrease in temperature, changes in excitability were rapid and unlikely to be the result of changes in the partition of the apolar portions of these molecules into the hydrophobic regions of the sarcolemma. These results are interpreted as indicating that both the presence of local anesthetics and alterations in temperature can influence the rates of potential-dependent changes in the conformation of membrane proteins that control the permeability of excitable sodium channels, possibly by modifying the fluidity of specific portions of their hydrophobic components or their immediate lipid environment. The accumulation of inactivation as the result of incomplete recovery from the effects of preceding depolarizations appears sufficient to explain the frequency dependent effects produced by these agents. PMID- 3000556 TI - Renal sodium transport in vitamin D resistant hypophosphatemic rickets. AB - To investigate the possible role of a Na transport defect in the pathogenesis of the phosphaturia in vitamin D resistant rickets, we studied the activity of the Na-K ATPase activity along the microdissected segments of the nephron in normal (N) and hypophosphatemic mice (Hyp), the Na uptake by renal brush border membrane (BBM), as well as the interrelationship between Na and phosphate transport through this membrane. In N mice, Na-K ATPase activity was present in decreasing order, in the distal tubule, the ascending branch of the loop of Henle, the proximal tubule, and the collecting tubule. In Hyp mice, the Na-K ATPase activity was comparable to that measured in N mice, except in the granular segment of the distal tubule where a 256% of the control activity was reproducibly observed. In N mice, Na initial uptake by BBM vesicles increased with Na concentration in the incubation medium, according to two kinetic components: one saturable, evident at low substrate concentrations and the other, nonsaturable, corresponding to a passive diffusion. The addition of 5 mM PO4 in the incubation medium did not significantly influence Na transport. In contrast, Na concentration in the incubation medium largely modified the kinetics of PO4 uptake: increasing Na concentration enhanced PO4 uptake and decreased the apparent Km. In Hyp mice, Na uptake by BBM was identical to that observed in N mice, but PO4 uptake was decreased by half. Na concentration in the incubation medium similarly influenced PO4 uptake in N and Hyp mice, and the Km values at each concentration of Na were comparable in the two series of animals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000557 TI - Renal brush border membrane phosphorylation: influence of pH, cAMP and ATP concentrations, parathyroid hormone status, and dietary phosphate. AB - It is known that parathyroidectomy, administration of parathyroid hormone (PTH), and dietary phosphate depletion or excess result in variations in phosphaturia and in phosphate transport through brush border membrane vesicles isolated from the kidneys of various animals. Parathyroid hormone has been shown to ultimately phosphorylate some brush border membrane proteins and it has been postulated that the resulting phosphaturia is related to this phosphorylation. However, it is not known whether the regulation of phosphate transport by the diet is affected through similar pathways. Our experiments were designed to study the phosphorylation of brush border membrane with [gamma-32P]ATP using the intrinsic protein kinase of the membranes. Five groups of rats were used: normal, phosphate loaded, phosphate depleted, and thyroparathyroidectomized and acutely loaded with parathyroid hormone. In each series of animals, the proteins whose phosphorylation was cAMP dependent were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and their phosphorylation with various concentrations of ATP, in the presence or absence of cAMP in the incubation medium, was quantified. In the normal rat, 17 proteins were phosphorylated, the phosphorylation of two of them (Mr, 71 000 and 84 000) being cAMP dependent. Maximal response to cAMP for these two proteins was obtained with 10 microM cAMP. The peaks of phosphorylation were observed at pH 7 for protein 71 000 and pH 10 for protein 84 000. When brush border membranes from normal rats were incubated with 10-100 microM ATP, cAMP-dependent phosphorylation increased to reach a maximal phosphorylation of 4.44 +/- 0.90 pmol/mg protein for protein 71 000 and 1.32 +/- 0.15 pmol/mg protein for protein 84 000.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000558 TI - Changes in pituitary responses to synthetic ovine corticotrophin releasing factor in fetal sheep. AB - The rise in cortisol in fetal sheep during late pregnancy has been related to increased responsiveness of the adrenal to ACTH. Most reports have suggested that plasma ACTH concentrations rise coincident with or after the prepartum increase in cortisol. To reexamine the relationship of cortisol with basal immunoreactive ACTH (IR-ACTH) throughout the last 40 days of pregnancy and to determine changes in fetal pituitary responsiveness during this time, we measured basal and synthetic ovine corticotrophin-releasing factor (oCRF) (10 ng-10 micrograms) induced rises in ACTH and cortisol in fetal sheep at days 110-115, 125-130, and 135-140 of pregnancy. The fetuses were catheterized on day 105-120 and entered spontaneous labour at greater than 140 days. Basal IR-ACTH (picograms per millilitre +/- SEM) rose from 16.7 +/- 2.9 pg/mL at day 110-115 to 34.8 +/- 8.7 pg/mL at day 141-145. There was a significant effect of time on basal ACTH concentrations with a mean increase of approximately 5 pg ACTH per millilitre of plasma per 5-day sampling interval. Plasma cortisol changed gradually between day 110 and 125 of gestation and then more rapidly to term. At day 110-115 of gestation there was no significant change in plasma ACTH after 10 or 100 ng oCRF, but there was a significant increase in ACTH after 1 microgram of oCRF. Plasma cortisol did not change after any CRF injection. The change in IR-ACTH after oCRF at day 125-130 of gestation was significantly greater than that at day 110-115. Plasma cortisol concentrations were elevated following 1- and 10-micrograms injections of oCRF.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000559 TI - Antagonist actions of bicuculline methiodide and picrotoxin on extrasynaptic gamma-aminobutyric acid receptors. AB - The effects of picrotoxin and bicuculline methiodide to block depolarizing responses of extrasynaptic receptors for gamma-aminobutyric acid (GABA) are compared using excitability testing of myelinated axons in amphibian peripheral nerve. The actions of the antagonists appear both complex and dissimilar. Picrotoxin (10-1000 microM) produces large reversible depressions of the maximal response to GABA (0.01-10mM) and increases the EC50 from 0.33 to 12.6 mM. With high concentrations of agonist and antagonist an insensitive component is apparent. The action of picrotoxin is not classically noncompetitive: it may represent a mixed antagonism (competitive and noncompetitive) or a noncompetitive one, masked by the presence of receptor reserve and (or) secondary depolarizing influences (e.g., GABA-evoked [K+] o accumulation). Bicuculline methiodide (10 200 microM) shifts the GABA concentration-response curve to the right; maximal responses persist and are even enhanced. The impression that bicuculline methiodide has a competitive action is supported by analysis of its inhibition of responses to low concentrations of the agonist. It is suggested that the enhancement of GABA responses by bicuculline methiodide and their apparent resistance to block by picrotoxin may be due to a common secondary effect of the antagonists such as a decrease in membrane conductance to K+ and (or) block of transmitter uptake. PMID- 3000560 TI - Isolation and characterization of calcineurin from bovine brain. AB - Calcineurin was isolated from bovine cerebrum extracts by sequential chromatography on Affi-Gel blue and calmodulin affinity columns. Calcineurin so isolated was approximately 90% pure and was composed of equimolar amounts of subunit A (Mr = 61 000-63 000) and subunit B (Mr = 15 000-17 000) when examined by sodium dodecyl sulfate gel electrophoresis. A polypeptide (less than 10%) with Mr = 71 000 whose function and role remains to be investigated, was routinely detected in the calcineurin preparation. Both inhibitory activity (towards calmodulin-dependent cAMP phosphodiesterase) and phosphatase activity (with 32P labelled myelin basic protein as substrate) were associated with calcineurin as evidenced by (i) coelution from Affi-Gel blue, Affi-Gel calmodulin, diethythaminoethyl-Sepharose, and Sephacryl S-200 chromatography columns; (ii) association with the same protein band on nondenaturing gels; (iii) similar stability upon storage at 4 degrees C and with repeated freezing and thawing; and (iv) parallel heat inactivation. Phosphatase activity of calcineurin was maximal with 32P-labelled myelin basic protein as the substrate. Using this substrate, enzyme activity was generally stimulated 5- to 10-fold in the presence of Ca2+ and calmodulin; half-maximal activation (A0.5) was observed with 25 nM calmodulin. Calmodulin increased the Vmax of the reaction without affecting the Km for the substrate. Optimum temperature and pH for the reaction were 45 degrees C and 7, respectively, in both the absence and presence of Ca2+ and calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000561 TI - Limitation of theophylline elimination by reduced oxygen availability in mouse hepatocytes and rat isolated livers. AB - The effect of oxygen availability on theophylline metabolism by mouse hepatocytes and rat isolated livers was examined. The elimination of theophylline by mouse hepatocytes and the metabolism of theophylline to dimethyluric acid by isolated, perfused rat livers was seriously impaired when the gas mixture supplied contained less than 28% oxygen. The correlation coefficients relating oxygen supply and the concentration of theophylline remaining in mouse hepatocyte suspensions were -0.74 to -0.84. In the isolated, perfused rat liver experiments, the correlation coefficient relating oxygen availability and dimethyluric acid production was 0.87. These observations are interpreted as supporting the hypothesis that oxygen availability per se is an important factor in determining the rate of theophylline metabolism. PMID- 3000562 TI - A novel cyclic GMP-lowering agent, LY83583, blocks carbachol-induced cyclic GMP elevation in rabbit atrial strips without blocking the negative inotropic effects of carbachol. AB - A novel cyclic GMP-lowering agent, LY83583(6-anilino-5,8-quinolinedione), was used to investigate the possibility that increases in myocardial cyclic GMP levels are responsible for the negative inotropic effects of cholinergic agonists. Concentrations of carbachol from 0.3 to 3 microM elevated cyclic GMP levels in electrically paced rabbit atrial strips by 75 to 200% and decreased contractile force in the strips by 30 to 60%. Pretreatment of the muscles for 10 min with 10 microM LY83583 significantly lowered resting cyclic GMP levels and completely blocked the elevation of cyclic GMP by these concentrations of carbachol. However, the negative inotropic effects of carbachol were not blocked by the LY83583. These results indicate that the negative inotropic effects of carbachol in rabbit atrium are not mediated by increases in tissue levels of cyclic GMP. PMID- 3000563 TI - Inactivation of the beta-adrenergic receptor in cardiac muscle by dithiols. AB - The effect of sulfhydryl reagents on binding of the beta-adrenergic antagonist ( )-[3H]dihydroalprenolol hydrochloride [-)-[3H]DHA) to a microsomal fraction of rabbit ventricular muscle was studied. Incubation with the disulfide reducing agents dithiothreitol (DTT), 2-mercaptoethanol, and reduced glutathione resulted in loss of (-)-[3H]DHA binding. At 500 microM DTT, less than 50% of specific binding activity remained; at 100 mM, binding was completely eliminated. 2 Mercaptoethanol and reduced glutathione were less effective than DTT at inhibiting binding activity. The total binding capacity (Bmax) decreased from 155.4 fmol mg-1 of protein, in the absence of DTT, to 92.4 and 77.5 fmol mg-1 at 0.25 and 0.7 mM DTT, respectively. The equilibrium dissociation constant (KD) increased from 7.6 nM, in the absence of DTT, to 10.3 nM at 0.25 mM DTT and to 20.8 nM at 0.7 mM DTT. Thus, DTT-induced decline in (-)-[3H]DHA binding results from a decrease in both the number and affinity of membrane binding sites for the tracer. Receptors could be protected from DTT inactivation by preincubation with beta-adrenergic ligands. Oxidants could not reverse inactivation, with the exception of o-iodosobenzoate which was only partially effective. Thus, the beta adrenergic receptor of rabbit ventricular muscle contains essential disulfide moietie(s) which can be inactivated by reducing thiols. PMID- 3000564 TI - Adenosine receptors in smooth muscle: structure-activity studies and the question of adenylate cyclase involvement in control of relaxation. AB - Structure-activity studies with a number of adenosine derivatives and analogs, measuring their relaxant effects in a variety of smooth muscle systems, were conducted in the hope of obtaining indications of the possible involvement of adenylate cyclase in their mechanism of action. While it was confirmed that a C6 aminofunction is of importance for agonist activity, several compounds, in particular the relatively potent N6-hydroxylaminopurine ribonucleoside, were not antagonized by 8-p-sulfophenyltheophylline, indicating that some nucleosides cause smooth muscle relaxation by a mechanism other than adenosine receptor stimulation. Nucleosides not bearing a C6 aminofunction were essentially inactive in rabbit intestine but showed weak relaxant effects in bovine coronary artery; this may indicate a difference between the adenosine receptor systems in these tissues and the intracellular mechanisms of relaxation. Comparing the relative potencies of compounds such as adenosine, 2-chloroadenosine, 5'-(N ethylcarboxamido)adenosine, and (-)N6-(R-phenylisopropyl)adenosine, which have been used widely to classify adenylate cyclase-coupled adenosine receptors, no uniform pattern became apparent among different smooth muscle systems used in this study and reported in the recent literature. Thus, we conclude that a classification of smooth muscle adenosine receptors according to criteria established for cyclase-coupled receptors may be inappropriate or misleading, particularly with respect to implications of adenylate cyclase involvement in the relaxant effects of adenosine and related nucleosides. PMID- 3000565 TI - Regulation of renal vitamin D hydroxylase activity in vitamin D deficient rats. AB - The regulation of renal mitochondrial 1-hydroxylase activity in chronic vitamin D deficiency was studied in male rats. These rats were born of mothers who had been raised from weaning (21 days) on a vitamin D deficient diet and who had no detectable serum 1,25-dihydroxycholecalciferol (1,25-(OH)2D) at the time their offspring were weaned (28 days). In the pups, renal mitochondrial 1-hydroxylase activity was undetectable before the 3rd week of life even though the animals were severely hypocalcemic from birth. The 1-hydroxylase activity first became detectable at 26 days of age, rapidly reached a maximum at day 34, then decreased to become undetectable again by 65 days. Throughout this time serum calcium concentration was less than 5.0 mg/dL and serum parathyroid hormone (PTH) concentration, measured by a midmolecule radioimmunoassay, was two- to five-fold greater than that found in vitamin D replete rats. 1-Hydroxylase activity could be restored in the +65-day-old animals by administration of a single dose of 2.5 micrograms vitamin D3. Enzyme activity was detected within 24 h, was maximal at 72 h, and returned to undetectable levels by 96 h after administration of the vitamin. Serum 1,25-(OH)2D which was undetectable before administration of the vitamin D3, was 108 and 458 pg/mL at 16 and 40 h, respectively, after the injection. The serum concentration of this metabolite then decreased progressively to 80 pg/mL by 6 days. 24-Hydroxylase activity first became detectable 48 h after vitamin D administration, increased to a maximum at 96 h, and thereafter decreased to become undetectable by 7 days.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000566 TI - Effect of relative humidity, atmospheric temperature, and suspending medium on the airborne survival of human rotavirus. AB - The Wa strain of human rotavirus, grown in MA-104 cells, was suspended either in tryptose phosphate broth or feces from a case of rotaviral diarrhea. It was then aerosolized into a rotating drum using a Collison nebulizer. The drum air was sampled using an all-glass impinger containing tryptose phosphate broth as collecting fluid. At 20 +/- 1 degree C, the virus aerosolized from tryptose phosphate broth was found to survive best at 50 +/- 5% relative humidity, where its half-life was 44.2 +/- 6.3 h. At 30 +/- 5% and 80 +/- 5% relative humidity, the half-life of the virus was 24.5 +/- 3.5 and 3.8 +/- 1.0 h, respectively. At 6 +/- 1 degree C, the airborne survival of the virus at the mid and low relative humidity levels was further enhanced, but at the high relative humidity it remained very similar to that seen at 20 +/- 1 degree C. When aerosols of fecally suspended human rotavirus were held at 20 +/- 1 degree C with 50 +/- 5% relative humidity, nearly 80% of the airborne virus particles remained infectious even at the aerosol age of 24 h. These findings may help in our understanding of the epidemiology of rotaviral infections. PMID- 3000567 TI - Characterization of incompatibility group HI1 plasmids from Salmonella typhi by restriction endonuclease digestion and hybridization of DNA probes for Tn3, Tn9, and Tn10. AB - Chloramphenicol resistance in Salmonella typhi is medicated by plasmids of the incompatibility group H, subgroup 1 (IncHI1). Eight IncHI1 plasmids from S. typhi strains originating in Mexico, Vietnam, Thailand, and India were examined by restriction enzyme digestion. The restriction enzymes, Apal, Xbal, and PstI were found to be most useful for comparison of plasmid DNAs. Four plasmids from S. typhi isolated in Mexico, Vietnam, and Thailand between 1972 and 1974 had identical restriction patterns with all three enzymes. The other IncHI1 plasmids showed only minor differences. However, some significant differences were noted between these IncHI1 plasmids and the prototype IncHI1 plasmid R27, which was isolated from S. typhimurium in 1961 and for which a restriction map has been constructed. Southern transfer hybridization with a nick-translated HI1 plasmid as a probe confirmed that there is a great deal of sequence homology among the IncHI1 plasmids. DNA probes were used to locate DNA sequences for ampicillin resistance (Tn3), chloramphenicol resistance (Tn9), tetracycline resistance (Tn10), and the one-way incompatibility between IncHI1 plasmids and the F factor, a characteristic property of IncHI1 plasmids. The results demonstrate that IncHI1 plasmids isolated from S. typhi from widely different geographic sources are very similar. Comparisons between the S. typhi plasmids and R27 indicated that conserved regions of DNA were those involved in conjugative transfer. PMID- 3000568 TI - Impact on surgery of preoperative localization of parathyroid lesions with dual radionuclide subtraction scanning. AB - In an effort to localize parathyroid lesions preoperatively, scanning with radioactive thallium and technetium was performed in 20 patients considered clinically to have hyperparathyroidism. In the 11 found at surgery to have single parathyroid adenomas, scanning correctly localized the lesion in 10; in the other patient the lesion was in the unscanned mediastinum. Preoperative scanning was not as rewarding in the seven patients with parathyroid hyperplasia. A thyroid lesion was the source of an abnormality seen on the parathyroid scan in one patient, while neck scanning and surgical exploration were negative in another. Comparison of the patients who had parathyroid adenomas localized in the neck with a control group of similar patients who did not undergo preoperative scanning showed that the average surgical time was reduced by 50% with preoperative localization and there was a decrease in the number of nonparathyroid tissue biopsies. PMID- 3000569 TI - Pancreatic polypeptide-secreting islet cell tumor. A follow-up report. AB - A case of documented pancreatic polypeptide (PP)-secreting islet cell tumor was followed for 3 years and 8 months until death due to multiple metastases. The patient initially presented with extremely high serum PP levels without clinical symptoms. After resection of the PP-secreting islet cell tumor, serum PP levels gradually decreased to normal levels. Serum PP levels started to elevate 10 months after the surgery, when liver metastases were verified by open biopsy. The patient was treated with streptozotocin (STZ), and normal serum PP levels returned. However, multiple liver and bone metastases were detected 32 months after resection of the tumor, which led to death. The recurrent tumor obtained at autopsy contained very little immunoreactive PP. The effect of STZ on PP secretion by the islet cell tumor is discussed. PMID- 3000570 TI - A case of hepatocellular carcinoma associated with ossification. A case report. AB - A 54-year-old man with hepatocellular carcinoma (HCC) of the typical trabecular pattern exhibiting unusual histologic features, both osteoid and bone formations, is reported. The tumor was associated with well-developed cirrhosis; thus, this may be different from mixed hepatic tumors of adults with bone formations because the latter are characterized by their prevalence in noncirrhotic livers and by the formation of myxoid connective tissue. Computed tomography of the current case revealed calcification within HCC. PMID- 3000571 TI - Patterns of recurrence in malignant mixed mullerian tumor of the uterus. AB - Of 120 patients treated definitively at University of Texas System Cancer Center, M. D. Anderson Hospital for malignant mixed mullerian tumor of the uterus, 67 had their tumors recur. These patients were analyzed for recurrence pattern and response of recurrence to treatment. The most frequent sites of recurrence were the lung and abdomen, accounting for 66% of all first-site recurrences and 80% of all first-site distant metastases. Locoregional recurrences were much less frequent, accounting for 18% of recurrences. There was no apparent change in recurrence pattern with the addition of adjuvant chemotherapy. The effect of adjuvant radiation could not be assessed, because 95% of patients treated surgically received adjunctive radiation. The survival after treatment of recurrence was shortest for patients with bone, brain, or liver metastases (median, 4 months). There were four long-term survivors (more than 36 months). Patients with resectable recurrences had the highest response rates. Response to chemotherapy alone was uniformly poor (6% response rate). PMID- 3000572 TI - Diagnostic application of a monoclonal antibody against small cell lung cancer. AB - A monoclonal antibody (MOC-1) directed against an antigen present in small cell lung cancer (SCLC) was used for diagnostic purposes. After screening of biopsy specimens of lung tumors, MOC-1 was found to react with SCLC (n = 10) and adenocarcinoma of the lung (4 of 9 cases). Except for a few cells in a poorly differentiated tumor, the reaction with squamous cell cancer was negative (n = 6). Staining with MOC-1 by an immunoperoxidase technique on imprints of biopsy specimens procured by rigid bronchoscopy was found to be a reliable and rapid method for diagnosing SCLC (16 of 17 positive). All cytologically proven bone marrow and pleural metastases of SCLC were found by staining on a cytospin preparation with MOC-1. Moreover, in three cytologically negative cases, MOC-1 positive cells were detected. PMID- 3000573 TI - Estrogen receptors in hepatocellular carcinoma. AB - Estrogen receptors (ER) were assayed on hepatocellular carcinoma (HCC) and surrounding liver tissue in 30 adult patients. All specimens were obtained at the time of surgery. Cirrhosis of the liver was associated with 28 patients and chronic hepatitis in 2 patients. ERs were detected in 12 of 30 HCCs. The value ranged from 1.4 to 9.2 fmol/mg cytosol protein with the dissociation constant (Kd) value less than 1 nanomol. On the other hand, 13 of 28 cirrhotic livers had measurable amounts of the receptors that ranged from 1.5 to 4.1 fmol/mg cytosol protein. Two livers with chronic hepatitis did not have detectable amounts of ERs. The receptors were not detected in both the tumor and liver in ten patients. The ER titers in HCC did not have any correlation with serum levels of alpha fetoprotein or carcinoembryonic antigen, hepatitis B virus profiles, and histologic types of the tumor. In the light of the current results, it would be of great interest whether hormone therapy can be used or not as a treatment of naturally occurring HCC in humans. PMID- 3000575 TI - Tiazofurin metabolism in human lymphoblastoid cells: evidence for phosphorylation by adenosine kinase and 5'-nucleotidase. AB - The exact route of metabolism of tiazofurin, a novel nucleoside with antitumor activity, is controversial. Using human cell lines severely deficient in salvage nucleotide enzymes, we were able to identify the route of activation in tiazofurin metabolism. With loss of adenosine kinase activity by mutation in two lymphoblastoid cell lines, CCRF-CEM and WI-L2, the growth sensitivity to tiazofurin decreased by 6- and 3-fold, respectively. In contrast, the mutant lines were about 3000- to 1500- and 16- to 4-fold more resistant to the structurally similar tiazofurin analogues pyrazofurin and ribavirin, respectively. Other mutants with defective deoxycytidine or uridine kinase activity showed normal sensitivity to all three analogues. Both cell lines with defective adenosine kinase activity accumulated about 50% wild-type levels of tiazofurin-5'-monophosphate and thiazole-4-carboxamide adenine dinucleotide analogue of tiazofurin at cytotoxic concentrations of the drug. Extracts of wild type lymphoblasts catalyzed the phosphorylation of tiazofurin in the presence of adenosine 5'-triphosphate and Mg2+. Loss of adenosine kinase activity in the mutant extract eliminated this phosphorylating activity for tiazofurin consistent with the notion that adenosine kinase catalyzes phosphorylation of tiazofurin. However, an enzyme activity that catalyzed the phosphorylation of tiazofurin in the presence of inosine-5'-monophosphate as donor and Mg2+ was detected in the extracts of both wild-type cells and adenosine kinase-deficient mutants. The monophosphate donor specificity, divalent metal, high salt requirement, and nucleoside acceptor specificity of this enzyme activity paralleled that of a 5' nucleotidase (EC 3.1.3.5) which catalyzes inosine phosphorylation. In addition, tiazofurin phosphorylation was competitively inhibited by inosine and the apparent Ki value was similar to the apparent Km value for inosine phosphorylation. These results indicate that two enzymes, adenosine kinase and a cytoplasmic 5'-nucleotidase, are functionally important anabolizing enzymes for tiazofurin in human cells. PMID- 3000577 TI - 2-Chloroethyl (methylsulfonyl)methanesulfonate (NSC-338947), a more selective DNA alkylating agent than the chloroethylnitrosoureas. AB - The novel chloroethylating agent 2-chloroethyl (methylsulfonyl)methanesulfonate (CIEtSoSo) has been shown to act like the chloroethylnitrosoureas (CIEtNu's) in its DNA damaging and cytotoxic effects in human cell lines and has similar activity to the CIEtNu's in the National Cancer Institute antitumor screening tests. Its simpler chemistry, however, suggests that it may alkylate DNA more selectively than do the CIEtNu's. The DNA base adducts produced in calf thymus DNA by CIEtSoSo have been compared to a representative, non-carbamoylating CIEtNu, 1-(2-chloroethyl)-3-(cis-2-OH)cyclohexyl-1-nitrosourea, using high pressure liquid chromatography. Two major modified base peaks were observed from the nitrosourea treated sample which have been subsequently identified by high pressure liquid chromatography comparison of synthesized standards and by electron impact gas chromatography/mass spectrometric analysis to be 7 hydroxyethylguanine and 7-chloroethylguanine. In contrast only 7 chloroethylguanine was obtained from the CIEtSoSo treated DNA at equimolar doses. Thus CIEtSoSo was found to be more specific in its reaction with DNA in that it produced less variety of products than the nitrosourea, with no apparent generation of hydroxyethyl products, which are major side reactions of the CIEtNu's. PMID- 3000574 TI - DNA topoisomerase II as a target of antineoplastic drug therapy. AB - A major goal of cancer therapy research is identification of critical biochemical targets that mediate the ability of effective cancer chemotherapy to kill tumor cells while allowing the maintenance of normal cell function. A candidate for such a target is DNA topoisomerase II, a ubiquitous enzyme that alters three dimensional conformation of supercoiled DNA. DNA intercalating agents and epipodophyllotoxins stabilize a DNA and topoisomerase II complex. The process of stabilization probably represents the poisoning of an intermediate state in the normal functioning of the enzyme. This stabilized intermediate state can be measured in whole cells using the filter elution method of Kohn to quantify protein-associated DNA cleavage produced when the cells are exposed to intercalators or epipodophyllotoxins. By altering cell populations in quantifiable ways, four factors appear to influence the magnitude of drug induced, topoisomerase II-mediated DNA cleavage and cytotoxicity: the proliferative state of the cell (proliferating cells are more sensitive than quiescent ones); the cell cycle state (cells pharmacologically recruited into G1 S are more sensitive than asynchronously growing cells); the chromatin conformation (DNA methylation, polyamine depletion, and other chromosomal changes can alter the magnitude of topoisomerase II-mediated effects); the cellular phenotype (in an as yet uncharacterized manner, malignant cells apparently are more sensitive to topoisomerase II-mediated events than normal cells). These data suggest that the biochemical basis of the therapeutic index of drugs such as the intercalating agents or epipodophyllotoxins may be the intrinsic hypersensitivity of the topoisomerase II in malignant cells to poisoning by these drugs. PMID- 3000576 TI - Deficiency in the catalase activity of xeroderma pigmentosum cell and simian virus 40-transformed human cell extracts. AB - It has been previously shown that skin biopsies isolated from various xeroderma pigmentosum (XP) patients present a permanent decline in catalase activity from the onset of the disease to the tumor formation. We report here that cultured XP cell strains are also markedly deficient in the catalase activity with about only 25% of the activity measured in normal human cells. No direct correlation between catalatic activity and excision repair ability has been found, since a XP variant line is as deficient as an XP-C strain. The exact cause of the catalase deficiency is still unknown but could be due to the synthesis of a modified enzyme or to an abnormal regulation leading to a limited enzyme synthesis. Furthermore, simian virus 40 transformation of normal and radiosensitive cells (XP, ataxia telangiectasia) provokes a decrease in catalase activity of about 80% compared to the control derivatives. Mathematical analysis performed on our data shows a clearcut distinction between XP and normal cells while some of the XP heterozygote cells exhibit an intermediate behavior. Although most of the XP syndrome could be explained by the impairment in the excision repair ability, the decrease in catalase activity leading to a probable increase in intracellular H2O2 concentration and/or to a higher sensitivity to any oxygen-activated species could represent an additive effect in inducing the carcinogenic process. PMID- 3000578 TI - Up regulation of the phorbol ester receptor-protein kinase C in HL-60 variant cells. AB - The human promyelocytic leukemia cell line HL-60 can be induced to differentiate into macrophage-like cells by nanomolar concentrations of phorbol esters. A phorbol ester-resistant variant R1B6 obtained by culturing HL-60 cells with increasing concentrations of 12-O-tetradecanoylphorbol-13-acetate, is reversibly resistant. These cells have been growing continuously in the presence of phorbol esters for more than 1 yr, but when the phorbol ester is removed, the cells gradually regain their sensitivity and express characteristics of macrophage-like cells upon readdition of phorbol ester. The concentration of phorbol ester receptors in R1B6 is about one-third that in the parental HL-60 cells. The reversion of the variants to sensitivity to phorbol esters is associated with the up regulation of the cytosol and membrane phorbol ester receptors. When partially purified, these receptor populations contain protein kinase C activity, in support of the identity of protein kinase C and the receptor. This study demonstrates that a phenotypic change in a clonal cell population correlates with the up regulation of the phorbol ester receptor-calcium-activated phospholipid dependent protein kinase. This variant cell line is a useful model for analyzing the relationship between phorbol ester binding and protein kinase C during differentiation of HL-60 cells. PMID- 3000579 TI - Periodate-oxidized adenosine induction of murine thymidine kinase: role of DNA methylation in the generation of tumor cell heterogeneity. AB - We previously reported that thymidine kinase (TK) activity in a spontaneously TK deficient (TK-) murine tumor cell line (called L61-M) could be partially restored following brief treatment of the cells in vitro with the potent DNA hypomethylating agent 5-azacytidine. We now show here that similar results may be obtained by exposing cells in vitro to periodate-oxidized adenosine, a potent inactivator of the S-adenosylhomocysteine hydrolase enzyme. The ability of periodate-oxidized adenosine to induce TK activity within the L61-M cell line was dependent upon the concentration of drug used and the treatment period. Inhibiting DNA synthesis completely prevented the effects of periodate-oxidized adenosine from being observed. Periodate-oxidized adenosine had no obvious mutagenic effect upon the L61-M cell line and had a slight but significant inhibitory effect upon the methylation of the cytosine nucleotides which were incorporated into DNA during the treatment period. These results suggest that during tumor development, alterations in the relative levels of S adenosylhomocysteine and S-adenosylmethionine may lead to the inhibition of DNA methylation, resulting in the activation of previously quiescent genes, thereby promoting the phenotypic diversification of tumor cell populations as well as their progression from a relatively benign to a highly malignant state. PMID- 3000580 TI - Enhancement by 1 alpha,25-dihydroxyvitamin D3 of chemically induced transformation of BALB 3T3 cells without induction of ornithine decarboxylase or activation of protein kinase C1. AB - We reported previously that 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], a hormonally active form of vitamin D3, markedly enhanced methylcholanthrene induced transformation of BALB 3T3 A31-1-1 cells. When the cells were treated with methylcholanthrene (1 microgram/ml) for 72 h and then with 1 alpha,25(OH)2D3 (5 ng/ml) for 2 wk, the transformation frequency was 1.95 +/- 0.73 (SD) foci/dish in 8 independent experiments, which was about 20 times that in cultures treated with methylcholanthrene only. Even at a physiological concentration in plasma, i.e., 0.05 ng/ml, 1 alpha,25(OH)2D3 enhanced the transformation frequency significantly (P less than 0.001). 1 alpha,25(OH)2D3 was not cytotoxic but slightly inhibited growth of the cells. Cells treated with 1 alpha,25(OH)2D3 were thin and became arranged in a meshwork with wide intercellular spaces. These morphological changes were reversible. 1 alpha,25(OH)2D3 induced DNA synthesis in quiescent BALB 3T3 cells dose and time dependently, but this effect was less than that of 12-O-tetradecanoylphorbol-13-acetate. Unlike 12-O-tetradecanoylphorbol-13 acetate, 1 alpha,25(OH)2D3 did not interfere with the binding of epidermal growth factor or phorbol dibutyrate. 1 alpha,25(OH)2D3 did not induce ornithine decarboxylase. Moreover, it did not activate protein kinase C in quiescent BALB 3T3 cells or this enzyme isolated from mouse brain. BALB 3T3 cells and their transformants contain a specific cytosol receptor for 1 alpha,25(OH)2D3, but the binding sites of the transformants were fewer and had lower affinity than those of untransformed BALB 3T3 cells. These effects of 1 alpha,25(OH)2D3 were specific, because other derivatives of vitamin D3 induced the same effects only at 200 times or more higher concentrations. PMID- 3000581 TI - Reduced formation of protein-associated DNA strand breaks in Chinese hamster cells resistant to topoisomerase II inhibitors. AB - DNA intercalating drugs and the epipodophyllotoxins etoposide and teniposide interfere with the action of mammalian DNA topoisomerase II by trapping an intermediate complex of the enzyme covalently linked to the 5'-termini of DNA breaks. This effect can be observed in intact cells by alkaline elution measurement of protein-associated DNA strand breaks. To assess the cytotoxic role of this effect, we have studied a subline of DC3F Chinese hamster lung cells selected for resistance to the intercalating agent 9-hydroxyellipticine. This subline (DC3F/9-OHE) was cross-resistant to other intercalators as well as to etoposide. Resistance to Adriamycin was associated with reduced uptake. However, resistance to 4'-(9-acridinylamino)methanesulfon-m-aniside and 2-methyl-9 hydroxyellipticinium was observed in the absence of changes in drug uptake, suggesting a second mode of resistance. DC3F/9-OHE cells formed fewer protein associated DNA strand breaks in response to 4'-(9-acridinylamino)methanesulfon-m aniside, 2-methyl-9-hydroxyellipticinium, or etoposide than did the sensitive parental cells. The same was true for isolated nuclei from these cells, which is consistent with a mode of resistance unrelated to drug uptake through the plasma membrane. These data suggest that resistance to DNA topoisomerase II inhibitors exhibited by DC3F/9-OHE cells is due in part to a modification of topoisomerase II activity. PMID- 3000582 TI - Increased levels of several retinoid binding proteins resulting from retinoic acid-induced differentiation of F9 cells. AB - The embryonal carcinoma cell line F9 is known to differentiate when exposed to retinoic acid. We have examined the quantities of two intracellular retinoid binding proteins in undifferentiated and differentiated F9 cells. The existence of a cell surface receptor that recognizes the plasma retinol-binding protein was also explored. It was shown that undifferentiated F9 cells contain low concentrations of the two retinoid-binding proteins. The cellular retinoic acid binding protein was present in approximately 3-fold molar excess over the cellular retinol-binding protein. Upon culture in the presence of retinoic acid, F9 cells display elevated concentrations of both cellular retinol-binding protein and cellular retinoic acid-binding protein. Since the levels of beta 2 microglobulin, a marker of the differentiated state with no known involvement in the metabolism of vitamin A, increased in parallel with the retinoid-binding proteins, it seems unlikely that retinoic acid selectively increased the levels of the two retinoid-binding proteins. The differentiated, in contrast to the undifferentiated cells, can accumulate retinol from plasma retinol-binding protein and display a cell surface receptor for this protein. Despite the fact that retinoic acid-induced differentiation of F9 cells promotes increased levels of several proteins involved in the normal metabolism of vitamin A, no evidence was obtained to suggest that the cells were dependent on retinoids to maintain their differentiated state. PMID- 3000583 TI - Progestin regulation of epidermal growth factor receptor in human mammary carcinoma cells. AB - Epidermal growth factor (EGF) receptors are present in human breast cancer and probably mediate the effects of EGF and the autocrine effects of alpha transforming growth factors, produced by breast cancer cells. Steroid hormones influence the growth of some human cancers, and both direct and indirect effects on cell proliferation have been proposed. One potential indirect effect of steroids would be to augment sensitivity to other endocrine and autocrine factors by up-regulation of their receptors. We therefore investigated the effects of various steroids on EGF receptor expression in T-47D, MCF-7, and BT 20 human mammary carcinoma cells in culture. Preincubation of T-47D cells for 24 h with a series of androgens, estrogens, glucocorticoids, and progestins resulted in a significant enhancement of specific 125I-EGF binding in the presence of progestins only. Increased binding of EGF was associated with neither a change in cell number nor changes in the specific binding of concanavalin A, insulin, or calcitonin but was accompanied by an increase in lactogenic receptor expression. When assayed at 20 degrees C, increased EGF binding was due to an increase in receptor number (33,380 +/- 7,410 sites/cell in control cultures; 67,460 +/- 20,330 sites/cell in cultures treated with 1 nM medroxyprogesterone acetate for 24 h; P less than 0.05) without a change in receptor affinity. Two- to 3-fold increases in receptor number were also apparent when binding was measured at 4 degrees C, indicating that the effect was due to an increase in expression of receptor at the cell surface rather than progestin effects on internalization and degradation. These data illustrate that the expression of EGF receptor in some breast cancer cells is regulated in part by mechanisms mediated via the progesterone receptor, since the effect was confined to progestins, potency among a series of progestins was correlated with their affinities for progesterone receptor, and sensitivity among the three cell lines studied was related to the presence and concentration of cellular progesterone receptor. PMID- 3000584 TI - Cholecystokinin inhibition of tumor growth and gastrin-stimulated cyclic adenosine 3':5'-monophosphate metabolism in human gastric carcinoma in nude mice. AB - This study deals with the effect of four types of COOH-terminal cholecystokinin (CCK) fragments on the growth of xenotransplantable human gastric cancer (SC-6 JCK, a poorly differentiated adenocarcinoma) whose growth has been promoted by pentagastrin. The growth of the tumor was inhibited using daily s.c. injections of CCK-octapeptide (CCK-8) and glutaryl-CCK-8 at a dose of 500 micrograms/kg body weight. After 30 days of treatment with CCK-8 or glutaryl-CCK-8, a significant decrease was observed in the tumor weight (P less than 0.05) and the tumor size P less than 0.01) in comparison with those of the control. But treatment with CCK 12 and pyroglutamyl-CCK-8 did not produce inhibition of tumor growth. Furthermore the correlation between the effect of CCK-8 on the normal rise in tumor cyclic adenosine 3':5'-monophosphate (cAMP) levels caused by pentagastrin injection and tumor growth was studied. The increase of cAMP by a single i.p. injection of pentagastrin at a dose of 20 micrograms/mouse was significantly inhibited by pretreatment with CCK-8 at concentrations equimolar to pentagastrin (P less than 0.05), while cAMP in the tumor was slightly elevated by a single i.p. injection of CCK-8 alone. Also in the in vitro study, CCK-8 inhibited the increase of cAMP and the activation of cAMP-dependent protein kinase which was stimulated by pentagastrin. These results suggest that proliferation of gastrin-dependent human gastric cancers may be suppressed by CCK in competition with gastrin. PMID- 3000585 TI - Emergence of differentiated subclones from a human salivary adenocarcinoma cell clone in culture after treatment with sodium butyrate. AB - A human salivary gland adenocarcinoma cell line, which has ultrastructure and biological markers specific to the intercalated duct cells of human salivary glands, was cultured in 5 mM sodium butyrate for 12 days; then the cells were trypsinized, subcultured for an additional 16 days, and then transferred to growth medium without sodium butyrate. Morphological changes appeared about 1 wk after return to growth medium without sodium butyrate; cells being spindle or stellate in shape appeared in the treated cells, whereas the untreated cells were polygonal in shape. This morphologically altered phenotype persists after more than 14 mo of culture in growth medium without sodium butyrate. Of 40 subclones isolated, 2 clonal cell lines were established from the subculture and characterized. The other 38 subclones were accompanied by cell death during the subcultures. The clonal lines exhibited a phenotype similar to myoepithelial cells such as myosin, beta-chain of S-100 protein, myofilaments, and oxytocin receptor in addition to decreased tumorigenicity and anchorage-independent growth. These findings indicate that commitment to differentiation into myoepithelial cells and conversion from malignant to normal phenotype occur in a human salivary gland adenocarcinoma cell line following the sodium butyrate treatment. PMID- 3000586 TI - Biosynthesis of procalcitonin in small cell carcinoma of the lung. AB - Immunoreactive calcitonin (CT) secreted by DMS 53, a cell line derived from human small cell carcinoma of the lung, consists almost entirely of molecular species larger than the mature hormone (Mr 3,420). Messenger RNA isolated from DMS 53 cells and nude mouse tumors was translated in wheat germ systems, and the products were precipitated with CT-specific antisera. Analyses of the translation products by electrophoresis on 15% polyacrylamide-sodium dodecyl sulfate gels indicated synthesis of a Mr 16,500 preprohormone that was reduced to Mr 14,500 by cotranslation with microsomal membranes. Immunoprecipitation of CT from media from pulse-labeled cultures revealed two major products (Mr 16,500 and Mr 14,500) and up to three minor secreted polypeptides (Mr 9,400, 8,400, and 6,800). Intracellular CT from cell homogenates appeared almost entirely as a single major product (Mr 14,500) and possibly 3-4 minor components (Mr 16,500; 9,200, 8,400, and 6,800). No glycosylated forms of CT were demonstrable by lectin binding methods or labeling attempts with tritiated sugars. The presence of multiple CT species in DMS 53 cells suggests significant post-translational processing of the larger precursor molecules and the accumulation and secretion by small cell carcinoma of the lung of several intermediate immunoreactive forms via a glycosylation-independent secretory pathway. PMID- 3000587 TI - Serial culture of single adult human prostatic epithelial cells in serum-free medium containing low calcium and a new growth factor from bovine brain. AB - Primary cultures of epithelial cells from human prostate acini proliferate in defined medium. However, the limited availability of human tissue and the lack of knowledge of the conditions required for clonal growth and serial culture of epithelial cells have limited progress in the study of human prostate cell biology. Here we report conditions that permit the proliferation of single epithelial cells from normal, benign hyperplastic, and carcinomatous prostate through three to four serial passages, which represents at least seven to nine cumulative doublings of the cell populations. Primary cultures were prepared from prostatic acini. Monolayers resulting from the outgrowth of epithelial cells from acini were harvested and dissociated into suspensions of single cells which gave rise to discrete colonies in subsequent culture. The requirements for successful serial culture were (a) a low calcium concentration, (b) the presence of a growth factor that is concentrated in bovine neural tissue, (c), detachment of the epithelial cells with collagenase, and (d) harvest of cells before the cell concentration reached 6000 cells/cm2 of culture surface. Suspensions of single cells were successfully stored between subcultures in 10% dimethylsulfoxide with 5% fetal bovine serum and revived after storage for up to 2 months in liquid nitrogen. PMID- 3000588 TI - Stimulation of cell proliferation and inhibition of differentiation expression by tumor-promoting phorbol esters in dog thyroid cells in primary culture. AB - 12-O-Tetradecanoylphorbol-13-acetate (TPA) and 4 beta-phorbol 12, 13-dibutyrate (PDBU) are potent tumor promoters and share several biological activities of epidermal growth factor (EGF). We have shown previously that EGF stimulates DNA synthesis and proliferation and inhibits TSH-induced markers of differentiation in dog thyroid follicle-derived primary cultures. Using this system, we have examined the biological action of TPA and PDBU in reference to that of EGF. Low concentrations (1.6-16 nM) and to a lesser extent higher concentrations (greater than 1.6 microM) of TPA and PDBU stimulated cell proliferation in a 1% serum, hormone-supplemented medium and triggered the DNA synthesis revealed by autoradiography in cells which were quiescent before stimulation in serum-free conditions. EGF, TSH, and dibutyryl cyclic adenosine 3':5'-monophosphate separately also induce DNA synthesis, but they produce little if any effects additive to those of TPA. In fact, TPA appeared to inhibit the mitogenic effects of EGF. Moreover like EGF, phorbol esters strongly inhibited in 2 days the morphological effects of TSH and basal and TSH-stimulated iodide transport capacity and thyroglobulin messenger RNA accumulation, two markers of thyroid differentiation. TPA also inhibited the expression of differentiation stimulated by dibutyryl cyclic adenosine 3':5'-monophosphate indicating a post-cyclic adenosine 3':5'-monophosphate site of action. TPA and EGF shared long-term morphological effects such as the induction of an elongated fusiform shape, but not acute effects. The thyroid cells progressively and spontaneously escaped both the mitogenic and differentiation-inhibiting effects of TPA and PDBU, while, as shown previously, these parameters are stably modified by continuous culture with EGF. This suggests specific desensitization processes to phorbol esters. As evidence is accumulating that phorbol esters act at least partly by stimulating the calcium-activated, phospholipid-dependent protein kinase C, our results shed light on the possible key role of this kinase in carcinogenesis and in the normal control of proliferation and expression of differentiation in the thyroid gland. Additionally they suggest that complex interactions occur between the mechanisms of action of EGF and of phorbol esters in the thyroid cell. PMID- 3000589 TI - Partial purification of transforming growth factors from human milk. AB - Crude, delipidated milk and the acid:ethanol extracts of primary human breast tumors contain several activities that biologically resemble transforming growth factors (TGFs) in that they promote the anchorage-independent growth of normal rat kidney and Mm5mt/c1 mouse mammary tumor cells in soft agar. Three major TGF species with isoelectric points (pl) of about 4.0, 6.0-6.5, and 7.0 have been detected in both tumors and milk. The pl 4.0 species from milk has been purified about 10,000-fold by isoelectric focusing and high-performance liquid chromatography. This species, designated milk-derived growth factor II (MDGFII), coelutes from gel filtration columns with an authentic human epidermal growth factor standard when using a low ionic strength eluting buffer. However, on the same column, MDGFII is completely resolved from human epidermal growth factor with high ionic strength eluting buffers. Nevertheless, MDGFII purified by the latter technique still competes with 125I-epidermal growth factor for receptor binding to A431 cell membranes. Additionally the TGF activity of MDGFII present in the pl 4.0 fraction of milk is markedly inhibited by anti-epidermal growth factor receptor antibody preparations. Consequently MDGFII appears to be an alpha TGF. MDGFII is a pepsin-sensitive, disulfide reducing agent-sensitive, heat stable protein that may be physiologically important for the mammary gland or the neonate. PMID- 3000590 TI - A case-control study of hepatocellular carcinoma and the hepatitis B virus, cigarette smoking, and alcohol consumption. AB - A case-control study was undertaken to evaluate the roles of the hepatitis B virus (HBV), cigarette smoking, and alcohol use in the etiology of hepatocellular carcinoma (HCC). A major purpose of the study was to evaluate the effect of cigarette smoking on HCC among hepatitis B surface antigen (HBsAg)-negative persons, since it had been suggested that the relative effect of cigarette smoking on HCC was higher among HBsAg-negative persons than among HBsAg-positive persons. Eighty-six cases and 161 hospital controls were included in the study. This study confirmed the strong relationship between the HBV and HCC. Twelve of 67 cases and none of 63 controls were chronically infected with HBV as evidenced by serum HBsAg. The study also found a moderately strong relationship between alcohol use and HCC. The results of the present study do not support the hypothesis that cigarette smoking is a risk factor for HCC. Among all subjects, the relative rate of HCC for cigarette smokers compared with nonsmokers after adjustment for alcohol consumption was 1.0 with 95% confidence limits, 0.5 to 1.8. Among HBsAg-negative subjects, the relative rate was 1.1 with 95% confidence limits, 0.5 to 2.4. There was also no consistent dose-response relationship between quantity smoked and HCC in this study. PMID- 3000591 TI - Activation of tumoricidal properties in peripheral blood monocytes of patients with colorectal carcinoma. AB - The purpose of these studies was to determine whether blood monocytes of patients with different stages of colorectal carcinoma could be activated by various immunomodulators to become tumor cytolytic. Monocytes obtained from 12 colorectal carcinoma patients and 8 normal donors were incubated in vitro with free or liposome-encapsulated agents. The cytotoxic properties of the monocytes were determined subsequent to interaction with radioactively labeled allogeneic colon carcinoma cells, melanoma cells, glioblastoma cells, and allogeneic nontumorigenic skin cells. Blood monocytes from normal donors and all colorectal carcinoma patients were activated in vitro to become tumoricidal by immunomodulators in free form or entrapped within liposomes; i.e., the monocytes recognized and lysed tumorigenic cells but not nontumorigenic cells. The tumoricidal activity of monocytes was observed in blood monocytes obtained from patients even after multiple doses of radiotherapy and chemotherapy, and that fact suggests that the in vivo activation of macrophages may be feasible. PMID- 3000592 TI - Disturbing central nervous system complications following combination chemotherapy and prophylactic whole-brain irradiation in patients with small cell lung cancer. PMID- 3000593 TI - Phase II trial of mitoxantrone in refractory germ cell tumors: a trial of the Southeastern Cancer Study Group. PMID- 3000594 TI - Polysaccharides and food processing. AB - The role of polysaccharides during processing and for the quality of foods is discussed. Starch is the most important energy source for man. Most other polysaccharides are not metabolized for energy, but play an important role as dietary fibres. Pectins, alginates, carrageenans, and galactomannans are discussed as functional food additives in relation to their structure and their rheological behaviour, stability and interactions. Endogenous polysaccharides of fruits and vegetables and in products derived from them are responsible for such phenomena as texture (changes), press yields, ease of filtration and clarification, cloud stability, and mouth feel. To achieve desirable properties, the action of endogenous enzymes on polysaccharides must be inactivated and/or exogenous enzymes added as processing aids. This is also true for overcoming haze phenomena in clear juices or to break down undesirable microbial polysaccharides. Dough properties for bread baking can be improved by enzymic breakdown of a restrictive pentoglycan network. Network formation may come about by oxidative coupling of phenol rings of ferulic acid bound to hemicelluloses by ester links. Gels may be made by inducing oxidative coupling in natural or synthetic systems. Stagnation in development of new polysaccharide food additives is ascribed to difficulties in obtaining government approval for food use. PMID- 3000595 TI - Hypertrophic cardiomyopathy characterised by beta-adrenoceptor density, relative amount of beta-adrenoceptor subtypes and adenylate cyclase activity. AB - The total quantity of beta-adrenoceptors and the relative amount of beta 1 and beta 2 receptor subtypes were determined in heart biopsies of 10 patients with various heart diseases and 5 patients suffering from hypertrophic cardiomyopathy (HOCM). In membrane particle preparations from the same patients we also examined the activity of the adenylate cyclase (AC), and its response to isoprenaline, terbutaline, histamine and sodium fluoride (NaF). The high affinity ligand [125I] (1)-cyanopindolol (CYP) was used in the binding assays, and the highly beta 2 selective antagonist ICI 118 551 for the determination of beta-adrenoceptor subtypes. No differences were found in total beta-adrenoceptor density between patients with HOCM and "controls" (27.6 +/- 14.2 vs 26.5 +/- 10.7 fmol . mg-1 protein). The relative amounts of beta 1 and beta 2 receptor subtypes were similar, patients with HOCM had 82.1 +/- 4.9% of beta 1 and 14.4 +/- 3.9% of the beta 2 receptor subtype, compared with 76.3 +/- 11.5% of beta 1 and 20.7 +/- 11.0% of the beta 2 subtype in the "control" patients. Both absolute activity of AC (pmol . mg-1 protein . min) as well as the relative responses to the various stimulators were not significantly different between the two groups. Thus, this study does not support the hypothesis that HOCM is a disorder with altered beta adrenoceptor number or adenylate cyclase response to adrenergic agonists. Furthermore, HOCM is not associated with altered response of the AC system to histamine or NaF. PMID- 3000596 TI - Human leukocyte gamma interferon in the treatment of epilepsy due to cytomegalovirus infection. AB - Two case reports are presented of epilepsy due to congenital cytomegalovirus treated with human leukocyte gamma interferon. Complete remission of grand mal and petit mal seizures was achieved, and the patients, a 7-year-old boy and a 2 year-old girl, have had no evidence of recurrent symptoms. No adverse effects of interferon were observed in these two patients. PMID- 3000597 TI - A chemoarchitectural study of telencephalon and diencephalon of a microchiropteran bat (Taphozous melanopogon Temminck). PMID- 3000598 TI - IS10 transposition is regulated by DNA adenine methylation. AB - We show that dam- mutants are a major class of E. coli mutants with increased IS10 activity. IS10 has two dam methylation sites, one within the transposase promoter and one within the inner terminus where transposase presumably binds. Absence of methylation results in increased activity of both promoter and terminus, and completely accounts for increased transposition in dam- strains. Transposition of Tn903 and Tn5 are also increased in dam- strains, probably for analogous reasons. Transposition is also increased when IS10 is hemimethylated. One hemimethylated species is much more active than the other and is estimated to be at least 1000 times more active than a fully methylated element. Evidence is presented that the promoter and inner terminus of IS10 are coordinately activated in a dam-dependent fashion, presumably because they are hemimethylated at the same time. Thus, in dam+ strains, IS10 will transpose preferentially when DNA is hemimethylated. We suggest specifically that IS10 transposition may preferentially occur immediately after passage of a chromosomal replication fork. PMID- 3000599 TI - Specific and stable intron-factor interactions are established early during in vitro pre-mRNA splicing. AB - Biochemical components (splicing factors) interact with specific intron regions during pre-mRNA splicing in vitro. The pre-mRNA specifically associates with factors at both the branch point and the 5' splice site and these RNA-factor interactions are maintained in the intron-containing RNA processing products. The first detectable event, the ATP-dependent association of a factor (or factors) with the branch point, is mediated by at least one factor containing an essential nucleic acid component. Mutant RNA substrates that lack either the 5' splice site or the vast majority of exon sequences can still associate with the branch point binding factor(s). However, this branch point-factor interaction does not occur with a mutant RNA substrate that contains the branch point but that lacks the 3' splice site consensus sequence. These results suggest that selection of the 3' splice site accompanied by the association of a factor with the branch point may be the initial step in mammalian pre-mRNA splicing. PMID- 3000600 TI - Conformational changes in a replication origin induced by an initiator protein. AB - The replication initiator protein of the plasmid R6K binds to seven contiguous 22 bp direct repeats that form an indispensable part of the three replication origins alpha, beta, and gamma. Binding of the initiator to the direct repeats induced a marked bending of the region of gamma replication origin. Binding of the initiator also promoted unwinding of the origin DNA by at least two turns. Distamycin appeared to antagonize the binding of the initiator to the seven 22 bp direct repeats. At the appropriate DNA and protein concentrations the initiator enhanced topoisomerase-induced catenation of the origin containing supercoiled DNA but not of DNA lacking the origin sequence. Thus, the initiator protein caused significant changes in the secondary and tertiary structures of the replication origin. PMID- 3000601 TI - c-myc gene expression is stimulated by agents that activate protein kinase C and does not account for the mitogenic effect of PDGF. AB - The role of the phosphoinositide turnover-protein kinase C pathway in mediating PDGF-stimulated c-myc expression and cell proliferation was studied. Both direct activators of kinase C (e.g. phorbol ester analogues) and hormones that activate kinase C via receptor-mediated phosphoinositide turnover (e.g. PDGF, bradykinin, or vasopressin) elicited a rapid increase in c-myc mRNA expression. Desensitization of the kinase C pathway by prolonged exposure to phorbol abolished the induction of c-myc by subsequent phorbol challenge and attenuated c myc induction by PDGF and bradykinin, but did not affect PDGF-stimulated mitogenesis. Bradykinin and phorbol esters stimulated the same magnitude of c-myc expression as PDGF but elicited less than one-tenth the PDGF-induced mitogenic response. We conclude that stimulation of c-myc expression is a common response to a diverse group of agents that elicit phosphoinositide turnover and activate protein kinase C, and that neither activation of protein kinase C nor enhanced c myc expression is sufficient for the mitogenic action of PDGF. PMID- 3000602 TI - Hierarchical down-modulation of hemopoietic growth factor receptors. AB - Granulocytes and macrophages can be produced in vitro when progenitor cells from mouse bone marrow are stimulated by any of four distinct colony stimulating factors, Multi-CSF (IL-3), GM-CSF, G-CSF, and M-CSF (CSF-1). At 0 degrees C the four CSFs do not cross-compete for binding to bone marrow cells, indicating that each has a specific cell surface receptor. However, at 21 degrees C or 37 degrees C, Multi-CSF inhibits binding of the other three CSFs and GM-CSF inhibits binding of G-CSF and M-CSF. Rather than competing directly for receptor binding, the binding of Multi-CSF, GM-CSF, or G-CSF to their own receptor induces the down modulation (and thus activation) of other CSF receptors at 37 degrees C. The pattern and potency of down-modulation activity exhibited by each type of CSF parallels the pattern and potency of its biological activity. We propose a model in which the biological interactions of the four CSFs are explained by their ability to down-modulate and activate lineage-specific receptors. PMID- 3000603 TI - Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation. AB - Sialyltransferase (Gal beta 1,4GlcNAc alpha 2,6 sialyltransferase) was localized by immunoelectron microscopy in rat liver hepatocytes using affinity-purified antibodies. Immunoreactivity for sialyltransferase was found in the Golgi apparatus, where it was restricted to an interconnected system consisting of the trans-cisternae and the trans-tubular network. This region of the Golgi apparatus exhibited both TPPase and CMPase activity and was the intracellular site where sialic acid residues bound to glycoprotein were detected using the Limax flavus lectin. Sialyltransferase and sialic acid residues were not detected in medial and cis-cisternae of the Golgi apparatus. These findings suggest that in rat hepatocytes sialylation of N-linked glycoproteins occurs in the complex formed by the trans-cisternae and the trans-tubular network of Golgi apparatus. PMID- 3000604 TI - Chemoattractant-elicited increases in myosin phosphorylation in Dictyostelium. AB - Cyclic AMP stimulation of chemotactically competent Dictyostelium amebas labeled with [32P]orthophosphate transiently increases phosphorylation in the heavy chain and the 18,000 dalton light chain of myosin. Immediately before the increase, heavy chain phosphorylation transiently decreases. These phosphorylation changes also occur when cAMP-induced activation of adenylate cyclase is blocked by pretreatment of amebas with caffeine. The time course of these phosphorylation responses correlates with the shape changes induced in amebas exposed to a temporal increase in cAMP concentration. The dose dependence of the phosphorylation responses is the same as that previously determined for chemotaxis. The phosphorylation responses exhibit adaptation properties in common with those of the shape change response and chemotaxis. Increases in the rate of myosin heavy chain and light chain phosphorylation can be observed in vitro by stimulating unlabeled amebas with cAMP and then lysing the cells into a gamma [32P]ATP-containing reaction mixture. PMID- 3000605 TI - Localization of the fushi tarazu protein during Drosophila embryogenesis. AB - The fushi tarazu (ftz) gene of Drosophila acts early in embryogenesis to regulate body segmentation. The localization of the ftz protein product in embryos was examined using indirect immunofluorescence microscopy. Antibodies were prepared against a beta-galactosidase-ftz hybrid protein made in E. coli. The ftz protein was first detectable in blastoderm-stage embryos as seven stripes of nuclei encircling the embryos transversely. The stripes persist through the early events of gastrulation, but disappear before overt segmentation is visible. The ftz protein is expressed a second time in some nuclei of the developing nervous system. In contrast to the early pattern, at the later stage, ftz is expressed in each of fifteen metameric subunits of the embryo. PMID- 3000606 TI - The granulocyte-macrophage colony stimulating factors. PMID- 3000607 TI - Homeo box gene complex on mouse chromosome 11: molecular cloning, expression in embryogenesis, and homology to a human homeo box locus. AB - The homeo box is a 180 bp protein-coding domain found within homeotic genes of Drosophila and conserved in a variety of invertebrate and vertebrate species. It has been suggested that the mammalian homeo box sequences may play a role in controlling pattern formation during embryogenesis. We report findings that support this hypothesis. We have cloned three overlapping recombinant phage clones that cover a region of mouse chromosome 11 that contains a cluster of four homeo boxes (the Hox-2 locus). This locus encodes multiple transcripts that are expressed during embryogenesis. Forty kilobases of the Hox-2 region is devoid of repetitive elements and shows extensive homology with the human Hox-2 locus. These results provide direct evidence for genetic expression during embryonic development, a conserved organization in comparison to the cognate human locus, and a complexity of organization and transcript expression similar to that found in Drosophila. PMID- 3000608 TI - The stability of bacteriophage T4 gene 32 mRNA: a 5' leader sequence that can stabilize mRNA transcripts. AB - In T4-infected cells, the gene 32 monocistronic mRNA is very stable. To study the molecular basis for this stability, we have constructed chimeric plasmids containing the monocistronic promoter and the gene 32 translation initiation sequence fused to either most of the E. coli lac operon or only a segment of the lacZ gene, followed by the gene 32 transcription terminator. The resulting hybrid transcripts are unstable in uninfected cells. In phage-infected cells, however, the hybrid mRNAs are at least as stable as gene 32 mRNA itself. Analysis of other plasmid constructs indicates that the sequences on the gene 32 mRNA from its 5' end to slightly beyond the initiation codon suffice to stabilize these hybrids. Studies with a series of deletions of the gene 32 leader sequence suggest that an RNA sequence near the gene 32 initiation codon is involved. Various models to explain this mRNA stabilization are discussed. PMID- 3000609 TI - The Sex-lethal gene of Drosophila: DNA alterations associated with sex-specific lethal mutations. AB - Genomic DNA encoding Sex-lethal, a developmental switch gene in Drosophila melanogaster that regulates sex determination and dosage compensation has been isolated. Wild-type DNA sequence organization of the gene has been compared at the restriction level with those of 17 female-specific, loss-of-function and five male-specific, gain-of-function mutant alleles. DNA lesions associated with 12 of these mutations delimit an 11 kb DNA region that is necessary for proper Sex lethal function in females. Males who are deleted for this region are both viable and fertile. Loss-of-function alleles are associated with gross DNA alterations as well as true point mutations; the former are located throughout the region. In contrast, all five gain-of-function alleles are associated with DNA insertions that are clustered within a 1 kb portion of the Sxl gene region. PMID- 3000610 TI - Polyoma regulatory region: a potential probe for mouse cell differentiation. PMID- 3000611 TI - lin-12, a nematode homeotic gene, is homologous to a set of mammalian proteins that includes epidermal growth factor. AB - The lin-12 gene of the nematode Caenorhabditis elegans controls certain binary decisions during development. The lin-12 locus was cloned by means of Tc1 transposon tagging: spontaneous lin-12 null alleles were isolated in a genetic background permissive for Tc1 transposition, and seven independently isolated mutations were found to be associated with Tc1 insertion events. All of these Tc1 induced mutations mapped to a single 2.9 kb restriction fragment within a 50 kb region examined. The DNA sequence of this fragment revealed that, although it does not contain the entire gene, it does include three complete exons. These three exons together encode 11 peptide units that are homologous to one another. The repeated peptide motif is also homologous to a set of mammalian proteins that includes epidermal growth factor. PMID- 3000612 TI - High frequency of homologous recombination in mammalian cells between endogenous and introduced SV40 genomes. AB - We have detected a high frequency of homologous recombination between introduced and chromosomal DNA in mammalian cells. Linear enhancerless SV40 DNA has been transfected into monkey cells that have either one (COS1 cells) or five to seven (COS7 cells) copies of the SV40 early region stably integrated into their genome. Enhancer-containing wild-type SV40 DNA is formed as a result of homologous recombination of the introduced DNA with chromosomal DNA. Up to 25% of the successfully transfected cells produce wild-type virus within 48 hr after transfection. The highest levels of wild-type virus were produced from transfections of molecules that contained a double-strand break at positions of uninterrupted homology with the chromosomal template. This SV40/COS cell system provides a rapid assay for recombination between introduced and genomic DNA. PMID- 3000613 TI - The internally located telomeric sequences in the germ-line chromosomes of Tetrahymena are at the ends of transposon-like elements. AB - The germ-line micronuclear genome of the ciliate Tetrahymena thermophila contains approximately 10(2) chromosome-internal blocks of tandemly repeated C4A2 sequences (mic C4A2). This repeated sequence is the telomeric sequence in the somatic macronucleus. Each of six cloned micC4A2 was found to be adjacent to a conserved 30 bp sequence, which we propose is the terminal inverted repeat of a family of DNA elements (the Tel-1 family). This 30 bp sequence contains a site for the infrequently cutting restriction enzyme Bst XI, which allows full-length Tel-1 elements to be cut out of the micronuclear genome. BAL 31 exonuclease digestion of Bst XI-cut micronuclear DNA showed the majority of micC4A2 blocks to be associated with the ends of the Tel-1 family. We propose that Tel-1 elements are transposable and suggest a novel mechanism to account for the origin of micC4A2, in which telomeric repeats are added to the ends of free linear forms of the transposable elements prior to reintegration. PMID- 3000614 TI - Mobile elements bounded by C4A4 telomeric repeats in Oxytricha fallax. AB - A novel family of micronuclear elements termed telomere-bearing elements (TBEs) is described. All 1900 family members are eliminated during macronuclear development. We conclude that they are transposons, first because the members are moderately conserved in sequence and probably dispersed in the genome. Second, in two cases, sequence comparison of the termini and flanks of the element with the corresponding empty site indicate that elements cause 3 bp target duplications (AAT) upon insertion; the 3 bp are part of the 5 bp target sequence, AATGA. Lastly, both elements carry 77 or 78 bp inverted terminal repeats. The tip of each inverted terminal repeat is the 17 bp telomere-like sequence 5' C1A4C4A4C4. At least half of the elements have these 17 bp or an extremely similar sequence. One possible pathway for transposition into new micronuclear sites starts in the developing macronucleus with excision to create a free linear form to which telomeres are added, followed by a low frequency of movement to the micronucleus, and insertion into the germ-line micronuclear DNA. PMID- 3000616 TI - High frequency germline acquisition of ecotropic MuLV proviruses in SWR/J-RF/J hybrid mice. AB - RF/J mice carry three endogenous ecotropic murine leukemia proviruses designated Emv-1, Emv-16, and Emv-17. Two of these proviruses, Emv-16 and Emv-17, are tightly linked and segregate with the high viremia phenotype in backcrossed mice. During the derivation of an SWR/J strain congenic for Emv-16 and Emv-17, we found that many of the progeny derived from female virus carriers acquired new germline ecotropic proviruses. Additional genetic crosses suggested that these proviruses are acquired early in development by virus infection and that this strain combination is particularly susceptible to these events. The frequency of proviral acquisition was only about 10-fold lower than the frequency of P element acquisition in dysgenic crosses of Drosophila melanogaster. Since virus integration in these hybrids occurs at many different sites, these types of hybrids may ultimately be useful for generating virally induced mutations that are amenable to study at the molecular level. PMID- 3000615 TI - Platelet-derived growth factor and double-stranded ribonucleic acids stimulate expression of the same genes in 3T3 cells. AB - Platelet-derived growth factor (PDGF) stimulates expression of a "competence" gene family in Balb/c-3T3 cells. The competence family contains the c-myc and c fos genes together with several functionally uncharacterized genes (JE, KC, and r fos) that have been isolated as cDNA clones. We show that double-stranded ribonucleic acid is a potent inducer of the competence gene family. Infection with vesicular stomatitis virus also induces expression of this gene family. Conversely, PDGF stimulates expression of genes hitherto characterized as responsive to double-stranded ribonucleic acids, including the beta-fibroblast interferon and (2'-5')-oligoadenylate synthetase genes. These PDGF-inducible genes could conceivably function in a feedback loop to control 3T3 cell growth. Some of the genes, such as c-fos and c-myc, are induced quickly by PDGF and may initiate a round of cell division. Others, such as beta-fibroblast interferon and (2'-5')-oligoadenylate synthetase, are induced more slowly and may function as feedback inhibitors of the growth response to PDGF. PMID- 3000617 TI - In vitro stimulation of specific RNA polymerase II-mediated transcription by the pseudorabies virus immediate early protein. AB - Nuclear extracts from human cells infected with pseudorabies virus (PRV) exhibited higher levels of accurate transcription of RNA polymerase II genes than did control extracts from mock-infected cells. Stimulation was maximal at low DNA concentrations and was not gene-specific. It was heat sensitive in extracts from cells infected with a virus containing a temperature sensitive mutation in the immediate early (IE) gene. The stimulatory activity copurified from the IE protein and was also heat sensitive when purified with cells infected with tsG, further indicating that the IE protein was responsible for this stimulation. These results thus demonstrate an in vitro system that mimics, at least in part, the in vivo stimulatory action of the PRV IE protein. They further imply that the IE protein acts not by increasing the amounts of cellular transcription factors, but rather by directly or indirectly altering their activities. PMID- 3000618 TI - An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells. AB - Epstein-Barr virus expresses a cytoplasmic and plasma membrane protein (LMP) in latently infected growth transformed lymphocytes. The gene specifying LMP has now been expressed in NIH3T3 and Rat-1 cells. Expression of the gene in these cells resulted in altered cell morphology and some resistance to the growth inhibiting effect of medium containing low serum. In Rat-1 cells, LMP expression often led to loss of contact inhibition and anchorage-independent growth in soft agar. Rat 1 cells expressing LMP were uniformly tumorigenic in nude mice. Thus, LMP is a transforming gene which is likely to account for many aspects of EBV induced cell transformations. This is the first demonstration of a transforming gene in Epstein-Barr virus, a ubiquitous human pathogen associated with neoplasia. PMID- 3000619 TI - Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus. AB - Mx+ mice are much more resistant to influenza virus than Mx- strains. The resistance is mediated by interferon (IFN) alpha/beta. After IFN treatment, Mx+ but not Mx- cells accumulate Mx protein and become specifically resistant to orthomyxoviruses. cDNA encoding Mx protein was cloned and sequenced. Southern analyses indicate that Mx- alleles derive from their Mx+ counterpart by deletions. IFN-treated Mx+ cells contained a 3.5 kb Mx mRNA, while Mx- cells showed only traces of shorter Mx RNA. Mx- cells transformed with Mx cDNA expressed Mx protein constitutively to varying extents; resistance of individual cells to influenza virus correlated with Mx protein expression. Thus, specific resistance to influenza virus in vivo may be attributed to Mx protein expression and is independent of other IFN-mediated effects. PMID- 3000620 TI - Chicken myeloid stem cells infected by retroviruses carrying the v-fps oncogene do not require exogenous growth factors to differentiate in vitro. AB - To determine the function of c-fps in chicken macrophages and granulocytic cells we have infected chicken bone marrow cells with retroviruses containing the v-fps oncogene. Normal chicken macrophage progenitors, M-CFCs, give rise to macrophage colonies in semisolid cultures when macrophage colony stimulating factor (M-CSF) is added into the culture medium. Upon infection with v-fps bearing retroviruses, we observed that M-CFCs were induced to develop macrophage colonies in vitro without exogenous M-CSF. This activation results from a direct effect of v-fps on the M-CFCs. No leukemic transformation was observed in the infected colonies. By comparing the effects of several retroviruses, we showed that the induction of M CFC development is specific to v-fps containing viruses and mediated by the v-fps protein. These observations support the hypothesis that the c-fps gene is involved in the control of proliferation and/or differentiation of myeloid cells. PMID- 3000621 TI - New approaches to a genetic analysis of mitosis. PMID- 3000622 TI - Tissue specificity of Drosophila P element transposition is regulated at the level of mRNA splicing. AB - We show that the germline specificity of P element transposition is controlled at the level of mRNA splicing and not at the level of transcription. In the major P element RNA transcript, isolated from somatic cells, the first three open reading frames are joined by the removal of two introns. Using in vitro mutagenesis and genetic analysis we demonstrate the existence of a third intron whose removal is required for transposase production. We propose that this intron is only removed in the germline and that its removal is the sole basis for the germline restriction of P element transposition. PMID- 3000623 TI - Nuclear location signals in polyoma virus large-T. AB - We have found two mutually independent sequence elements that contribute to the nuclear location of polyoma virus large-T. The first sequence (pro lys lys282 ala arg glu asp) resembles the SV40 large-T nuclear signal (pro lys lys128 lys arg lys val) and occurs at a corresponding position within polyoma large-T. The second sequence (val ser arg lys192 arg pro arg) may be structurally related to the SV40 signal, although it has little sequence homology and falls in a region of the protein that has no counterpart in SV40 large-T. The data suggest that nuclear location signals with characteristics similar to the SV40 large-T prototype may be a more general feature of nuclear proteins, and that several such signals in a given protein can exert cooperative effects. PMID- 3000624 TI - Neural induction and in vitro initial expression of neurofilament and tetanus toxin binding site molecules in amphibians. AB - Tetanus toxin (Tt) binding site and neurofilament (NIF), the intermediate-sized filaments, are neuronal markers essentially described in mammals and birds; are these molecular markers present in urodela neuronal cells and are they expressed immediately after neural induction? Our findings are based on immunofluorescent localization of NIF and Tt proteins using three previously characterized antisera against 200 kDa and 70 kDa neurofilament components and against fragment IIc derived from purified tetanus toxin. Embryonic undifferentiated neuronal cells from Pleurodeles waltlii neural plate and/or neural fold (early neurula stage) are cultured isolated in vitro without further chordamesodermal influence. At the beginning of the culture none of the undifferentiated neuronal precursors bind antibodies against NIF or Tt components. The binding is detected when phenotypical differentiation takes place (2/3-day cultures). Both the cell bodies and the cell processes are stained. After 2-3 weeks, immunostaining of the neurones is very distinctive and bright; the non-neuronal cultured cells do not exhibit any labelling. These observations indicate the early acquisition of NIF and Tt binding site expression by neuronal precursor cells (late gastrula stage). PMID- 3000625 TI - Interleukin 2 receptor blockade by anti-Tac antibody inhibits IFN-gamma induction. AB - Anti-Tac antibody, which binds to the interleukin 2 (IL-2) receptor and thus blocks IL-2 binding to and activation of T lymphocytes, was used to investigate the role of IL-2 in interferon-gamma (IFN-gamma) production. Three T-cell mitogens (phytohemagglutinin, concanavalin A, and the pan-T monoclonal antibody OKT3) were used as IFN-gamma inducers. In each case, anti-Tac antibody clearly inhibited IFN-gamma production. This occurred even under conditions where cellular proliferation (as measured by incorporation of [3H]thymidine) was only slightly inhibited. The inhibitory effects of anti-Tac were reversed by the addition of purified IL-2. Therefore, endogenous production of IL-2 and its binding to the IL-2 receptor are needed for maximum IFN-gamma production. PMID- 3000626 TI - A T lymphoblastoid cell line from a patient with AIDS-related complex (ARC). PMID- 3000628 TI - Inhibition of type I collagen film degradation by tumour cells using a specific antibody to collagenase and the specific tissue inhibitor of metalloproteinases (TIMP). AB - Rabbit VX2 tumour cells in culture produced a collagenolytic activity which was shown to be immunologically identical to collagenase from rabbit articular chondrocytes and bone. VX2 cells degraded type I collagen films spontaneously and did not produce detectable levels of the tissue inhibitor of metalloproteinases (TIMP). Chondrocytes, however, required both stimulation of collagenase synthesis and activation to effect lysis and were observed to make appreciable amounts of TIMP. The degradation of type I collagen films by VX2 tumour cells was significantly inhibited by both a specific antibody to rabbit collagenase and by purified TIMP, thus demonstrating the unequivocal role of collagenase in this model system. PMID- 3000629 TI - Expression of a type 2 pneumocyte-specific antigen by a cell strain from normal adult mouse lung. AB - The mouse lung epithelial cell strain NAL 1A has been confirmed as type 2 pneumocyte-related by immunostaining with a type 2 pneumocyte-specific antiserum. Cytoplasmic immunoreactivity with the antiserum paralleled the appearance of phospholipid-containing osmiophilic cytoplasmic inclusions, which were much more abundant in confluent cell cultures at low passage number than in exponentially growing cultures. However, phospholipid analysis indicated that NAL 1A cells were impoverished in phosphatidylglycerol as compared to lung type 2 pneumocytes. Confluent cultures of the neoplastic cell lines NAL 1AM and NUL 1 did not reveal any reactivity with the specific antiserum. PMID- 3000630 TI - [Diagnostic significance of the complement fixation reaction in poliovirus infections]. PMID- 3000627 TI - Influence of calcium and calcium-modulating agents on differentiation of murine erythroleukaemia cells. AB - Since the involvement of calcium ions in the regulation of cell division and differentiation has been proposed, in this study we have examined the effect of extracellular calcium and of calcium-modulating agents on the DMSO-induced differentiation of murine erythroleukaemia cells. Neither proliferation nor differentiation of these cells was affected by calcium deprivation in the culture medium. Moreover, calcium-chelating agents or agents blocking intracellular calcium uptake induced a marked inhibition of cell differentiation. Intracellular calcium antagonists induced inhibition when cells were grown in a calcium deprived medium. In contrast, murine erythroleukaemia cell differentiation was unaffected by agents that increased intracellular concentration of calcium. Our results indicate that a mobilization of calcium is indispensable for eliciting full cellular response, but the increase in intracellular level of this cation is not sufficient for complete signal transduction. It is likely that a marked alteration of the intracellular calcium system and availability could be responsible for the independence of our cell system from calcium modulation. PMID- 3000631 TI - The interaction of bisulfite and its free radical analog-nitroxyldisulfonate with periodic acid-oxidized lymphocytes and their effect on blastogenesis. AB - Nitroxyldisulfonate [Fremy's salt; (KSO3)2NO.] and bisulfite (NaHSO3) have abolished periodic acid (H5IO6)-induced blastogenesis of human peripheral blood lymphocytes (HPBL), but only inhibited the blastogenic response of H5IO6-oxidized rat and mouse lymphocytes, as determined by the rates of nucleic acids synthesis, BrdUrd incorporation and by cell numbers in S + G2 + M phases of the cell cycle. The viability of the intact human, rat and mouse lymphocytes remained essentially unimpaired by 30 min pulses of 1 mM Fremy's salt or bisulfite. The marked inhibition of periodic acid-induced blastogenesis, exerted by Fremy's salt and by bisulfite, was attributed to the effect of the corresponding carbonyl addition derivatives formed in situ of the oxidized cell membranes. Consequently, it is concluded that Fremy's salt like bisulfite possibly forms addition derivatives with membrane carbonyls of viable target cells. PMID- 3000633 TI - Inhibition of the Fenton reaction by the protein caeruloplasmin and other copper complexes. Assessment of ferroxidase and radical scavenging activities. AB - The copper-containing protein caeruloplasmin is an important biological extracellular protein. By catalysing the oxidation of ferrous ions to the ferric state (ferroxidase activity) it can inhibit lipid peroxidation and the Fenton reaction. This activity is readily destroyed by heat-denaturation. When a ferric EDTA complex is added to hydrogen peroxide, OH X radicals are formed in a reaction inhibitable by superoxide dismutase (SOD). This reaction is also inhibited by caeruloplasmin both before and after heat-denaturation, suggesting a non-catalytic scavenging role for the protein. A combination of ferroxidase and radical scavenging activities in fluids containing iron complexes and hydrogen peroxide, but no SOD or catalase, would make caeruloplasmin an important extracellular antioxidant. PMID- 3000632 TI - Activation of chloroform and related trihalomethanes to free radical intermediates in isolated hepatocytes and in the rat in vivo as detected by the ESR-spin trapping technique. AB - When hepatocytes isolated from phenobarbital-induced rats were incubated with chloroform and the spin trap phenyl-t-butyl nitrone (PBN) under anaerobic conditions, a free radical-spin trap adduct was detectable by ESR spectroscopy. A similar incubation of hepatocytes in the presence of air resulted in an ESR signal that was eight times less intense than that seen under anaerobic conditions; incubation mixtures exposed to pure oxygen had no detectable adduct signal. A significant reduction in the signal intensity was also produced by the addition of cytochrome P-450 inhibitors such as SKF-525A, metyrapone and carbon monoxide, indicating that free radical formation depended upon the reductive metabolism of chloroform mediated by the mixed oxidase system. The origin of the CHCl3-derived free radical has been confirmed by using [13C]CHCl3, while the comparison between the ESR spectra obtained in the presence of deuterated chloroform (CDCl3) and bromodichloro-methane (CHBrCl2) suggests that the free radical derived from CHCl3 may be CHCl2. Free radical intermediates were also detected during the aerobic and anaerobic incubation of isolated hepatocytes with bromoform (CHBr3), and iodoform (CHI3). The intensity of the ESR signal obtained with the various trihalomethanes increases in the order CHCl3 less than CHBrCl2 less than CHBr3 less than CHI3. The formation of PBN-free radical adducts has also been observed in phenobarbital-induced rats in vivo when intoxicated with chloroform, bromoform or iodoform, suggesting that the reductive metabolism of trihalomethanes might be of relevance to their established toxicity in the whole animal. PMID- 3000634 TI - Effect of particulates on metabolism and mutagenicity of benzo[a]pyrene. AB - Mechanisms of co-carcinogenicity of particulates, such as iron oxide and asbestos, and benzo[a]pyrene (B[a]P) are not completely understood. Particulates dramatically alter rates of uptake of B[a]P into membranes, a factor which could account for co-carcinogenicity. However, B[a]P must be activated to reactive forms to be carcinogenic and mutagenic so alterations in metabolism of B[a]P by particulates also could result in co-carcinogenesis. To elucidate mechanisms of particulate-B[a]P co-carcinogenesis, we have correlated rates of uptake of B[a]P into microsomes with metabolism of B[a]P and with mutagenicity of B[a]P in the Ames test. In general, aryl hydrocarbon hydroxylase (AHH) activity paralleled rates of uptake of B[a]P, though some inhibition of AHH activity by particulates which was not attributable to availability of B[a]P was evident. This inhibition was studied further by assaying separately mixed function oxidase and epoxide hydrase activities in the presence of particulates. Both chrysotile and iron oxide inhibited O-deethylation of 7-ethoxyresorufin and hydration of B[a]P-4,5 oxide. To determine effects of this inhibition on activation of B[a]P to reactive forms, we studied profiles of metabolites of B[a]P and mutagenicity of B[a]P. The only alteration in profiles of B[a]P metabolites produced by particulates was that due to effects on rates of uptake. Similarly, mutagenicity of B[a]P was positively correlated with rates of uptake into microsomes. We conclude that the predominant effects of chrysotile and iron oxide are in altering rates of uptake of particle-adsorbed B[a]P. Changes in uptake rates then result in alterations of B[a]P metabolism and mutagenicity. PMID- 3000635 TI - The hydrolytic autoxidation of 1,4-naphthoquinone-2-potassium sulphonate: implications for 1,4-naphthoquinone-2-potassium sulphonate-induced oxidative stress in the red blood cell. AB - 1,4-Naphthoquinone-2-potassium sulphonate (NQKS) undergoes an autoxidation reaction in aqueous solution under physiological conditions to produce 3-hydroxy 1,4-naphthoquinone-2-potassium sulphonate (NQKS-OH). Intermediates of dioxygen reduction, superoxide radicals, hydrogen peroxide and hydroxyl radicals, are also detected. The kinetics of the autoxidation of NQKS show a first order dependence on NQKS and hydroxide ion concentration and a zeroth order dependence on oxygen concentration. For the rate equation r = -d[O2]/dt = kappa obs., [NQKS] [-OH], kappa obs. = (6.4 +/- 0.6) X 10(2) M-1s-1 at 37 degrees C. Hydroxide ion attack on NQKS appears to be the rate determining step. The reaction may be conveniently described as a 'hydrolytic autoxidation'. The hydrolytic autoxidation of NQKS occurs in NQKS-treated red blood cells; the hydroxylated quinone NQKS-OH is produced and hydroxyl radical formation is stimulated. The importance of this reaction in NQKS-induced oxidative stress in red blood cells is discussed. The hydrolytic autoxidation of quinones bearing one or more unsubstituted (hydrogen) positions on the quinone centre is a novel mechanism by which such quinones may induce oxidative stress in cellular systems. PMID- 3000636 TI - Alkali lability and rapid initiation of excision repair following photoaffinity damage by ethidium azide. AB - DNA damage and repair provoked by ethidium azide (EA) photoaffinity labeling in mouse leukemia cells was studied by measuring sedimentation properties of nucleoids in neutral sucrose gradients, and it was found that the strand opening step was faster than that which followed damage of cells by ultraviolet (UV) light. The two insults were compared at levels of damage which gave the same overall rates of repair synthesis in intact cells and which required the same length of time to complete repair, as judged by the restoration of supercoiling of the isolated nucleoids. In the case of UV, single-strand breaks in DNA were detectable at 30 min, maximum at 2 h, and the superhelical properties restored at 21 h. With photoaffinity labeling, single-strand breaks were prominent immediately, even when photolabeling of cells was done on ice, but restoration of DNA supercoiling still required 21 h. Photolabeling of isolated nucleoids or isolated viral DNA with EA failed to introduce DNA strand breaks. However, it was discovered that photoaffinity labeling of DNA with EA resulted in alkali labile sites shown by single strand breaks produced on alkaline sucrose sedimentation or by alkali exposure followed by sedimentation on neutral formamide gradients. These results suggest that the drug attachment sites should be identifiable by the location of such single strand breaks. PMID- 3000639 TI - [Biochemical study of rotavirus strains isolated in New Caledonia between 1980 and 1983. Electrophoretypes]. AB - Between 1980 and 1983, 150 Rotavirus strains have been identified by ELISA test. RNA was extracted from 90 strains. We used a very simple and rapid extraction technique. Polyacrylamide gel electrophoresis permits the separation of double stranded RNA segments. The system of classification we retained, allows to recognize 8 classes of electrophoretypes. This study confirms the great variability of human rotaviruses. PMID- 3000637 TI - Peroxidase catalysed oxygen activation by arylamine carcinogens and phenol. AB - Peroxidase catalysed the formation of active oxygen in the presence of NADH or GSH and traces of H2O2 and arylamine or phenolic substrates. Some oxygen activation occurred with some arylamines even in the absence of NADH or GSH. Oxygen consumption was proportional to the NADH oxidized or GSSG formed. Approximately 0.80 and 0.40 mol of oxygen were consumed per mole of NADH or GSH oxidized respectively. The requirement for trace amounts of hydrogen peroxide and arylamine or phenolic substrates suggest that redox cycling resulted in H2O2 formation. It is proposed that initially formed phenoxy radicals or arylamine cation radicals oxidize NADH or GSH to radicals which react with oxygen to form superoxide radicals and H2O2. PMID- 3000638 TI - [The other face of oxygen (1)]. AB - The other face this two-faced Janus, the oxygen, allows us, by now, to see dimly is the bad, aggressive one shown by its free radicals, should they escape the respiratory machine, or, rather, should they follow the respiratory explosion of leucocytes and macrophage, or the same compromission of the cellular structure. The antioxidative mechanisms, although articulate and quibbled, appear inadequate, also because the oxygen radicals are formed on the outer side of the cellular membrane, and, just in the intercellular space, the defensive protections are extremely poor. If lung deserves a particular status, being the usual point of aggression by the oxygen radicals: here, these active metabolites are produced by its macrophage, but also by its endothelial cells, especially in conditions of hyperoxy; all organs and segments are their targets. Besides the immediate results, which are also caused by oxidizers having a long half-life, there are mediate results, essentially rotating around the arachidonic acid, a sort of multiplicating pin, with its products of the cyclooxygenase line (thromboxane A2, endoperoxides, prostacyclins) and the lipoxygenase line (the wide range of leukotrienes). But not even this "free body", in a position to escape, provided it wants so, the enzymatic control, seems inclined to free itself, what attenuates its bad face, from a basic rule in animal biology, that is, "negative feedback". PMID- 3000640 TI - The interaction of endogenous opiates with autonomic circulatory control in the dog. AB - The intravenous injection of either methionine or leucine enkephalin sharply reduces blood pressure, peak left ventricular pressure, and peak LV dP/dt in anesthetized dogs. The magnitude of the hypotensive response increases in proportion to the severity of the preceding surgical stress. The peptides are relatively ineffective after only simple surgical procedures but become highly effective when the autonomic balance is shifted toward sympathetic dependence after more complicated procedures or following bilateral carotid occlusion. The greater the animal's dependence upon sympathetic outflow to maintain blood pressure, the more effective is the opiate peptide. This suggests that the peripherally administered opiates may act by opposing existing adrenergic tone. Such antagonism of adrenergic tone during circulatory shock may help to explain some of the benefit of opiate receptor blockade in this condition. The rapid decline in blood pressure can be demonstrated in response to a variety of the proenkephalin-A derived peptides expected to circulate during physiological stresses. Based on a comparison of the responses to a series of peptides, the hypotensive effect is most likely mediated through activating opiate receptors of the delta subtype. PMID- 3000641 TI - Beta-adrenergic stimulation of myocardial cyclic AMP in endotoxic rats. AB - Sympathetic nervous system activity and myocardial response to adrenergic stimulation were studied in the rat during endotoxicosis. Plasma glucose concentration, adrenal norepinephrine (NE) and epinephrine (E) content, spleen NE content, and myocardial NE and cAMP content were analyzed in fed rats given saline or endotoxin (ETOX, 16.7 mg/kg). Values were determined at various times during a 6-hour period or at the agonal stage of shock. Myocardial cAMP content was reduced by 1 hour, recovered at 4 hours, and was again depressed at the agonal stage. In addition, isoproterenol-stimulated cAMP production in ventricular slices from ETOX rats was attenuated by 3 hours after administration. These data suggest that myocardial beta-adrenergic receptor mechanisms are altered during endotoxicosis, which may account for reports of decreased functional responsiveness to catecholamines under similar conditions. At 1 hour after ETOX, adrenal NE did not change, but E was depleted and remained low throughout the period. By 2 hours, spleen NE was also found depleted. Myocardial NE did not change until the agonal stage, when it was severely depressed. This implies that there may be nonuniform activation (duration and/or intensity) of sympathetic pathways during endotoxicosis. PMID- 3000642 TI - Enhancement by norepinephrine of automaticity in sheep cardiac Purkinje fibers exposed to hypoxic glucose-free Tyrode's solution: a role for alpha adrenoceptors? AB - A period of drive in the presence of norepinephrine (NE) may be followed by the induction or acceleration of spontaneous activity. Experiments were carried out in sheep cardiac Purkinje fibers to determine whether the effects of NE on automaticity were modified during superfusion with hypoxic glucose-free Tyrode's solution and to assess the possible contribution of alpha-adrenergic influences on automaticity under these conditions. The following results were obtained: Low concentrations of NE (10(-7) and 3 X 10(-7)M) were able to induce automaticity after a period of drive in normal oxygenated (97% O2, 3% CO2) Tyrode's solution. Superfusion with hypoxic (97% N2, 3% CO2) glucose-free Tyrode's solution enhanced NE-induced automaticity. Practolol, in concentrations able to block the effects of NE in normal oxygenated solution, did not counteract the effects of NE in hypoxic glucose-free solution. Yohimbine, but not prazosin, antagonized the effects of NE in hypoxic glucose-free solution. At the same concentration, yohimbine did not affect transmembrane potentials or automaticity induced by isoproterenol. It is concluded that alpha-adrenergic responsiveness appears to be enhanced during superfusion in vitro with hypoxic glucose-free solution, and that alpha-adrenoceptors belonging to the alpha 2-subtype in sheep cardiac Purkinje fibers might influence abnormal automaticity, possibly through an effect on oscillatory potentials. PMID- 3000643 TI - Biological variance of cholinesterase and 5'-nucleotidase in serum of healthy persons. AB - We measured cholinesterase (EC 3.1.1.8) and 5'-nucleotidase (EC 3.1.3.5) activities in serum of 24 healthy laboratory staff during 12 months. Overall mean activities ranged from 5.3 to 13.4 kU/L for cholinesterase and 5.4 to 9.8 U/L for 5'-nucleotidase. Cholinesterase activity was significantly (p less than 0.01) higher for men than for women. 5'-Nucleotidase activity was significantly (p = 0.01) higher for subjects 40 years or older than for those younger than 40, but was not different with respect to sex or time of year. Average intra- and interindividual variances (SD2) were 0.38 and 2.69 for cholinesterase and 1.41 and 0.97 for 5'-nucleotidase, respectively. Intra- to interindividual standard deviation ratios were 0.38 for cholinesterase and 1.21 for 5'-nucleotidase. Average within-run analytical variances were 0.13 and 0.3 (4% and 13% of total variance) for cholinesterase and 5'-nucleotidase, respectively. The importance of these findings in regards to diagnostic interpretation of serum cholinesterase and 5'-nucleotidase results is discussed. PMID- 3000644 TI - Pediatric reference intervals for normetanephrine/metanephrine. PMID- 3000645 TI - Comments on the uricase/peroxidase--phenol--4--aminoantipyrine reaction. PMID- 3000646 TI - Direct homogeneous phosphoroimmunoassay for carbamazepine in serum. AB - In this "phosphoroimmunoassay," a phosphorescent label is used: erythrosin (tetraiodofluorescein). Its long-lived phosphorescence was detected after pulsed excitation in a time-resolved luminescence spectrometer. To achieve convenient open-atmosphere phosphorimetry in liquid solution at room temperature, we used sodium sulfite as an "in situ" chemical deoxygenator, to eliminate oxygen quenching of the excited triplet state. Time-resolved detection allows for complete rejection of short-lived background signals, including those from fluorescent or light-scattering components of biological fluids that can interfere in fluoroimmunoassays. We chose the antiepileptic drug carbamazepine (CBZ) and its active metabolite CBZ-10, 11-epoxide (CBZE) to demonstrate the development and validation of nonseparation assays based on quenching the phosphorescence of erythrosin-labeled drug upon binding to antibody. These two compounds cross reacted equally with the antiserum used; hence, we measured the activity of both in patients' sera. Alternatively, simple pre-treatment of the sample with acid destroys CBZE immunoreactivity and enables specific assay of CBZ. PMID- 3000647 TI - Differential investigation of the capacity of succinate oxidation in human skeletal muscle. AB - Procedures are described for the estimation of the succinate:ubiquinone oxidoreductase and succinate:phenazine methosulfate oxidoreductase activities in post-nuclear supernatants of human skeletal muscle homogenates using 2,6 dichlorophenol indophenol as the terminal electron acceptor. The influence of ionic strength and of sucrose upon these assays and upon the succinate:cytochrome c oxidoreductase activity has been investigated. Sucrose markedly interferes with the activation of the succinate dehydrogenase complex. Succinate:cytochrome c oxidoreductase activity and succinate:phenazine methosulfate oxidoreductase activity are inhibited by increasing concentrations of ions and of sucrose. Our results lead us to propose the existence of a single acceptor site for phenazine methosulfate at the succinate dehydrogenase complex, not involved in the physiological electron flux across ubiquinone. Estimation of the enzymatic activities mentioned above allows differential investigation of the functional integrity of a large part of the respiratory chain in patients suspected of suffering from a neuromuscular disorder. PMID- 3000648 TI - Brainstem auditory evoked responses in Lafora disease. AB - Brainstem auditory-evoked responses (BAERs) have been studied in five patients suffering from Lafora-type of progressive myoclonus epilepsy proven by skin biopsy and in ten healthy volunteers. At the time of examination the patients were not taking benzodiazepines and showed myoclonic jerks. In all patients the central conduction time (interpeak latencies I-V, I-III, III-V) and the amplitude (amplitude ratio I/V) of BAERs were within the +/- 2 S.D. limits of the normal values. Since Lafora disease is a neuropathologically prevalent grey-matter illness (typical inclusion bodies are stored intraneuronally) these data indicate that diseases primarily affecting the brainstem grey-matter are usually associated with normal BAERs. PMID- 3000649 TI - On the stability in vitro of bioactive human adrenocorticotrophin in blood and plasma. AB - The disappearance in vitro of ACTH from whole human blood and plasma held at 22 degrees C has been monitored using a dispersed adrenal cell bioassay and an unextracted radioimmunoassay. In three normal subjects after a metyrapone test and two patients with Addison's disease, endogenous bioactive ACTH levels were unchanged for at least 1 h in blood and 2 h in plasma. Moreover greater than 50% of the bioactive plasma ACTH was still present in the plasma samples from the patients with Addison's disease after 24 h incubation at ambient temperatures. Human pituitary ACTH (1-39), spiked into plasma from dexamethasone suppressed subjects to give a concentration of 250 ng/l, was stable by bioassay for at least 2 h. No loss of biological activity was observed on subjecting plasma from a patient with Addison's disease or spiked plasma to two cycles of thawing at 37 degrees C and freezing at -70 degrees C or thawing at 20 degrees C and freezing at -20 degrees C. Some loss of bioactivity (20%) occurred on subjecting the patient's, but not ACTH-spiked plasma to four cycles of thawing at 20 degrees C/freezing at -20 degrees C. We conclude that bioactive ACTH (endogenous or exogenous) may be more stable in vitro in human blood and plasma than has been previously thought. If our studies can be confirmed in a larger series then it may be that conditions for handling blood specimens for ACTH assays could be reappraised. PMID- 3000650 TI - The geographical distribution of thyrotoxicosis in England according to the presence or absence of TSH-receptor antibodies. AB - In a prospective study of the incidence of thyrotoxicosis sera from 216 thyrotoxic patients in seven English towns were assayed for TSH-receptor antibodies. The incidence of antibody negative thyrotoxicosis correlated closely with the previous prevalence of endemic goitre in the towns (r = 0.9) indicating a high current incidence of toxic nodular goitre in previously goitrous towns. Antibody positive thyrotoxicosis, an indicator of Graves' disease, showed no correlation with goitre although there was statistically significant geographical variation in incidence. The percentage of all thyrotoxic patients who were antibody positive varied between towns, from 35% to 92%. PMID- 3000652 TI - Suppression of parathyroid secretion after administration of WR-2721 in a patient with parathyroid carcinoma. AB - WR-2721 is an organic thiophosphate, known as a radioprotective agent, which also inhibits parathyroid hormone (PTH) secretion and reduces the plasma calcium level in euparathyroid animals and human subjects. In this study we present evidence that WR-2721 is also able to reduce an abnormal PTH secretion in a case of parathyroid carcinoma. The intravenous infusion of a single dose (750 mg/m2) of WR-2721 was followed within 3 h by a reduction in the plasma concentration of PTH by 60%, and of Ca by about 0.45 mmol/l. These changes were associated with a diminution in urinary cAMP excretion. Based on this observation it appears that WR-2721 merits further investigation as a possible agent for the medical treatment of hyperparathyroidism. PMID- 3000651 TI - Ovine corticotrophin releasing factor stimulates ACTH release from human corticotrophinoma cells in culture; interaction with hydrocortisone and arginine vasopressin. AB - We have investigated the effects of ovine corticotrophin releasing factor (oCRF) and its interaction with hydrocortisone (HC), and arginine vasopressin (AVP) on ACTH release from human corticotrophinoma cells in culture. Tumour tissue was obtained from six patients (three with active Cushing's disease and three with Nelson's syndrome). Cultures were maintained for periods of up to six months. Ovine CRF (21 nmol) significantly (P less than 0.01) stimulated ACTH release from all tumours. Dose response (21 pmol-21 nmol) effects were observed for the three tumours investigated over 2 and 4 h. Cortisol (20 mumol) significantly (P less than 0.01) inhibited basal ACTH release from one tumour (Nelson's syndrome) by 75% over 4 h, and completely prevented the stimulatory effects of oCRF. AVP directly stimulated ACTH release from two tumours (Nelson's syndrome), and also potentiated the stimulatory action of oCRF during 30 min incubations. These data show corticotrophinoma cells from subjects with Cushing's disease and Nelson's syndrome can be directly stimulated by hypothalamic oCRF and may be potentiated by AVP. Cortisol and oCRF have been shown in one tumour to have antagonistic actions at the pituitary level. PMID- 3000654 TI - Enalapril in the treatment of renovascular hypertension. AB - Enalapril, an angiotensin converting enzyme (ACE) inhibitor, was given to 12 patients with renovascular hypertension: To five of them as a single drug after discontinuing other medications, and to seven patients as a substitute for one of their previous medications. The drug proved effective in controlling hypertension in all patients. Flushing and palpitations occurred in two of them, one of whom also showed a rise in creatinine and mild hyperkalemia. Two patients who had developed side effects while on captopril (renal deterioration in one, and severe rash in the other) tolerated enalapril well. Enalapril effectively reduced the blood pressure in the one patient with bilateral renal artery stenosis without causing renal failure. PMID- 3000653 TI - The effects of ACTH on the renin-aldosterone system in normotensive man. AB - The present study examined the effects of low dose ACTH administration (0.1 mg/day for 2 days) on plasma renin concentration, (PRC), activity (PRA) and substrate (PRS), cortisol and aldosterone in man. Six healthy male volunteers on a diet calculated to contain 150 mmol Na/day received an infusion of 5% dextrose (6 ml/h) for 24 hours, then ACTH (Synacthen, Ciba-Geigy) was added to the infusion at the rate of 100 micrograms per day, for 48 h. Blood samples were taken four hourly for determination of plasma cortisol, aldosterone, PRC, PRA and PRS. There was a highly significant increase in plasma cortisol and aldosterone concentrations during ACTH infusion compared with dextrose infusion, but no significant increase in active or inactive PRC, PRA or PRS. In a separate study of 15 healthy male volunteers, dexamethasone (1 mg at 2300 h) suppressed plasma cortisol but had no effect on PRC. These results do not support the view that stimulation of aldosterone by ACTH is mediated through the renin angiotensin system. PMID- 3000655 TI - Abnormal relationship between dietary sodium intake and red cell sodium transport in salt-sensitive patients with essential hypertension. AB - The effects of high sodium intake on erythrocyte 22Na efflux rate constants were studied in 25 patients with essential hypertension and 9 normal subjects. With changes in sodium intake from 100 mEq to 300 mEq/day, both total and ouabain sensitive 22Na efflux rate constants decreased significantly (p less than 0.001) in "salt-sensitive" patients (-0.031 +/- 0.005 and -0.035 +/- 0.006 /hr, respectively), but these responses were variable in "nonsalt-sensitive" patients and in normal subjects. The "salt-sensitive" patients showed a significant increase in their body weight, while intraerythrocyte sodium contents remained unchanged in the both groups. These results suggest that the abnormal change in membrane Na-K-ATPase activity may, at least in part, be involved in the mechanism of sodium susceptibility in patients with essential hypertension. PMID- 3000656 TI - New class of inhibitors specific for human renin. AB - Seven active tetrapeptide amides characterized by a C-terminal phenylalanyl aminoadamantane (PheNHAd) sequence, were identified by selective testing for human renin inhibitory activity among compounds with adjacent hydrophobic groups and molecular size equivalent to 3-5 amino acid residues. The new inhibitors were compared with known renin inhibitors (RIP, pepstatin, H-77) and opioid analgesic agents (Met-enkephalin, morphine), with the following results: The new inhibitors were active against human renin (IC50 approximately 10-5M), but inactive against rat renin and pepsin. Although active in opiate receptor binding studies (IC50 approximately 10(-7)M), they were, with few exceptions, inactive in the mouse writhing and hot plate tests for analgesia. SAR studies suggested a separation of the renin inhibitory from the analgesic activity of enkephalin analogs. Preliminary experiments with sodium-depleted rhesus monkeys indicated hypotensive activity for three of the new inhibitors at 3 mg/kg i.v., and RIP at 1 mg/kg. The recently reported clinical hypotensive properties of RIP (Zusman et al., Trans. Assoc. Am. Physicians 96:365, 1983) along with the present comparative studies suggest that the new inhibitors may lead to clinically useful agents. PMID- 3000657 TI - Renin-like activity in vascular tissue of DOC-salt hypertensive rats. AB - The presence of isorenin active at the physiological pH was investigated in DOC salt hypertensive rats. The effect of chronic captopril treatment was analyzed in similarly treated animals and in control rats. The levels of the plasma (PRA) and vascular enzyme (VRLA) were compared with those of untreated control animals. DOC salt administration was maintained during 4 or 8 weeks. Captopril was given in the drinking fluid beginning 4 days before DOC-salt treatment. Renin-like activity was measured in the aorta and mesenteric arteries. DOC-salt treatment reduced PRA to almost undetectable levels while aorta renin-like enzyme only decreased to 50% at 4 weeks and was not changed at 8 weeks. Isorenin levels in the mesenteric artery did not show any significant variation. Captopril did not prevent the increase in blood pressure due to DOC-salt administration and it induced a significant increase in PRA and in VRLA in control rats whereas it did not increase either - enzyme in DOC-salt treated rats. In summary these results confirm the existence of a vascular isorenin and suggest that both binding and local synthesis of the enzyme could take place in the arterial wall. PMID- 3000658 TI - The systemic significance of posturally poor foot position in the infant and child. AB - The cases and conditions reported in this article are but a minute sampling of the multitude of systemic conditions in which foot shape and position are valuable clues to associated serious illnesses in children. The podiatrist can contribute greatly by establishing important early diagnoses in these cases. Many times the podiatrist is the first-contact physician because of the early foot involvement. The podiatrist can make a valuable contribution to the health of children by recognizing early symptoms. PMID- 3000659 TI - Electroneurophysiological abnormalities in porphyria cutanea tarda. PMID- 3000660 TI - Subsets of T lymphocytes in relation to T lymphocyte function in multiple sclerosis. AB - T lymphocyte control of Epstein-Barr virus (EBV) infection of autologous B lymphocytes was examined in parallel to the enumeration of subpopulations of mononuclear cells in 22 multiple sclerosis (MS) patients and in 22 healthy individuals. All were seropositive for EBV. The incidence of lack of T cell control was significantly higher in patients than in controls, confirming previous published work. In the present study, we have shown in addition a significantly reduced proportion of OKT8+ cells and a significantly increased ratio of OKT4/OKT8 cells in the group of patients with lack of control. The findings point to abnormal immunoregulation in MS. PMID- 3000661 TI - Transmission of maternal cytomegalovirus-specific immunity in the guinea pig. AB - The prenatal and perinatal transmission of maternal cytomegalovirus (CMV) specific immunity was studied in the guinea pig by cross-fostering newborns of seropositive and seronegative mothers. Resistance to CMV was assessed by challenging the newborn and adult offspring with sublethal doses of CMV and following the course of their acute CMV disease. Newborns were found to acquire detectable CMV antibody via the placental route but not the breast milk route. When compared to neonates with no acquisition of CMV immunity, neonatal offspring born to or foster fed on seropositive mothers were protected against generalized CMV infection to various degrees. The immunity transferred by pregnant mothers that had seroconverted prior to pregnancy was short lived and was more protective in newborns with detectable CMV antibody. Mothers that experienced primary CMV infection during pregnancy conferred on their progeny the most significant and long-lasting resistance against CMV challenge. This study demonstrates that newborn guinea pigs may acquire CMV-specific immunity from seropositive mothers; the degree of protection against CMV infection depends on the route of acquisition of immunity and on the time of maternal seroconversion. PMID- 3000662 TI - Virus-associated deficiencies in the mitogen reactivity in celebes black macaques (Macaca nigra). AB - Celebes black macaques (Macaca nigra) with a history of diabetes mellitus, recurrent bacterial and protozoal infections, diarrhea, anemia, weight loss, anorexia, and a high mortality were studied to determine their immune status. Two groups of monkeys, healthy and unhealthy, were formed on the basis of a clinical assessment. The proliferative response and the pokeweed-mitogen-induced polyclonal IgG response of peripheral blood mononuclear cells of unhealthy monkeys were significantly less than the responses of healthy monkeys. The percentage of HLA-DR+ cells varied greatly in unhealthy monkeys. The OKT4/OKT8 ratios of unhealthy monkeys were generally greater than the ratios of healthy monkeys. Unhealthy monkeys usually had smaller percentages of OKT8+ cells than did healthy monkeys. The two groups of monkeys were examined for the presence of a syncytial forming retrovirus by a coculture assay involving Raji cells, a human B lymphoblastoid cell line. A type D retrovirus was detected in the unhealthy group but not in the healthy group. Retroperitoneal fibromatosis was detected in several monkeys in the unhealthy group. PMID- 3000664 TI - Modulation of E-receptor expression on activated T lymphocytes. AB - Our study indicated that the newly synthesized E-receptor, as measured with 125I labeled monoclonal anti-E receptor antibody, on activated T lymphocytes were responsible for forming stable E-rosettes at 37 degrees C. Maximum induction of new E-receptor expression required at least 50 hr of culture with polyclonal T cell activators, phytohemagglutinin, or phorbol myristate acetate. Polyclonal B cell activator, lipopolysaccharide were not able to induce new E-receptor expression on the surface of T lymphocytes. The expression of the new E-receptor paralleled with the induction of Tac antigen expression. Interleukins 1 and 2 or Interferon-gamma were not able to initiate the induction of new E-receptors. However, a neuropeptide, endorphin exhibited biphasic effect on modulating the E receptor expression, in the absence of polyclonal activators. As is the case with Tac antigen expression, induction of new E-receptor antigen may be a marker for activated T lymphocytes. PMID- 3000663 TI - Increase in proliferation and cytotoxic cell development in human mixed lymphocyte cultures in the presence of very low concentrations of LPS: role of IL 1 and prostaglandin E2. AB - Lipopolysaccharide (LPS) added to human allogeneic mixed lymphocyte cultures (MLC), even at very low concentrations, increased the level of specific cytotoxicity that developed. Proliferation was also increased by LPS in MLC but no increase was detectable when the allogeneic stimulus was absent. LPS enhanced only low cytotoxic responses while having little effect on naturally high responses. Significant enhancement in cytotoxic response was found within the picogram-nanogram per milliliter range of concentrations of LPS and only when it was added at the initiation of cultures. This early action of low concentrations of LPS suggested that IL-1 was involved. Indeed, a supernatant from silica treated human mononuclear cells containing IL-1 activity also enhanced cytotoxic and proliferative responses. Aside from increasing IL-1 secretion we also found that LPS significantly increased synthesis and secretion of PGE2 which had a selective inhibitory effect. Namely, addition of indomethacin or flurbiprofen to MLC further enhanced the cytotoxicity of LPS-treated but not that of untreated cultures without increasing the proliferative response. These results suggest a key role for macrophage-derived IL-1 and PGE2 in the regulation of proliferative and cytotoxic responses of T cells. They also suggest that very low amount of LPS may reach the immune system and contribute to the expression of cell-mediated immune responses. PMID- 3000666 TI - The nephropathy associated with male pseudohermaphroditism and Wilms' tumor (Drash syndrome): a distinctive glomerular lesion--report of 10 cases. AB - We report on 10 children, less than 2 years of age, who presented with a genuine type of glomerulopathy: diffuse mesangial sclerosis. In 5, the nephropathy was associated with male pseudohermaphroditism (MPH) and Wilms' tumor (WT); in 3 with MPH and in 2 with WT. The nephropathy was characterized by its very early onset, between the age of 2 weeks and 18 months. Eight patients presented with a nephrotic syndrome with (7 cases) or without (1 case) hypertension. All, but one, who is in advanced RF at 11 years of age, progressed to chronic or end-stage renal failure (ESRF) within a few months to 2 years from the onset. One additional child presented with advanced renal failure at the age of 8 months and the last one, who was hypertensive, developed an anuria related to thrombosis of renal veins at 1 year of age. Drash syndrome is characterized by the association of a "nephron disorder" with MPH and WT. We propose, on the basis of our histological findings, to extend the concept of Drash syndrome to patients who, in addition to the nephropathy, have either WT or MPH and to consider the distinctive glomerular lesions presented by all these patients as their common denominator. The pathogenesis of this glomerulopathy is obscure. Its early onset, its association with a dysembryoplastic tumor and/or with gonadal dysgenesis both suggest an antenatal dysgenetic process. PMID- 3000665 TI - Study of activated T cells in man. II. Interleukin 2 receptor and transferrin receptor expression on T cells and production of interleukin 2 in patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex. AB - Because the expression of interleukin 2 (IL-2) receptor and transferrin receptor is essential for the proliferation of T cells to mitogens and antigens, we examined the expression of monoclonal antibody defined IL-2 receptor (Tac antigen) and transferrin receptor on unstimulated as well as on phytohemagglutinin (PHA)-activated highly enriched T cells from patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex (ARC). A trend of increased proportion of unstimulated T cells with Tac antigen and transferrin receptor was observed in patients with AIDS and ARC when compared to healthy heterosexual controls, but the differences were not significantly (P greater than 0.1). The proportions of Tac+ PHA-activated T cells were, however, significantly decreased in AIDS (P less than 0.001). ARC (P less than 0.001), and asymptomatic homosexuals (P less than 0.01) when compared to healthy heterosexuals. The proportions of transferrin receptor positive PHA-activated T cells were not significantly different among various groups. A significantly (P less than 0.01) decreased production of IL-2 was observed in AIDS. This study suggests that the poor proliferative responses of T cells may be due to several defects in lymphocyte-cytokine cascade and the deficiency of Tac antigen expression and of the production of IL-2 could be a few of several abnormalities contributing to poor T-cell functions in AIDS. PMID- 3000667 TI - Does cytomegalovirus play a role in community-acquired pneumonia? AB - Cytomegalovirus (CMV) is recognized as an important pathogen in the immuno suppressed patient. Sporadic case reports of cytomegalovirus community-acquired pneumonia have appeared. We studied 443 patients with community-acquired pneumonia requiring hospitalization to define the role of cytomegalovirus in this illness. Four patients (0.9%) had good evidence that cytomegalovirus caused their pneumonia: 2 had the virus isolated from pulmonary tissue and 2 had cytomegalovirus inclusion bodies visualized in this tissue. An additional 14 patients had serologic evidence (a fourfold rise in the complement fixation tests) of cytomegalovirus infection. Analysis of these 18 patients suggest, that cytomegalovirus plays a role in community-acquired pneumonia. Six (33%) of the patients were immunosuppressed. Six others had concomitant infections: Chlamydia trachomatis (3); Epstein-Barr virus and M. pneumoniae (1); and bacteremia with Group B streptococcus and Bacteroides fragilis plus Eubacterium lentum (1 each). Seven patients (39%) required assisted ventilation, four of whom developed secondary bacterial pneumonia. Five (28%) died. Only two patients had a clinical and radiographic picture suggestive of a viral illness as a cause of the pneumonia. Three patients had atypical lymphocytes in their peripheral blood film. We found that the prevalence of complement fixing antibody to cytomegalovirus increased with age. Such antibody was lacking among those in the 16-20 year group while it peaked at 65% for males and at 78% for females ages 91 100 years. Despite the fact that 42.2% of the adults lacked antibody to cytomegalovirus, community-acquired pneumonia due to this virus is uncommon and does not justify routine serological testing for such infection among patients with community-acquired pneumonia. PMID- 3000668 TI - Ovarian cancer: histogenetic classification, histologic grading, diagnosis, staging, and epidemiology. PMID- 3000669 TI - Malignant germ-cell tumors of the ovary. PMID- 3000670 TI - Sex cord stromal tumors. PMID- 3000671 TI - [A case of perineuritis]. PMID- 3000672 TI - [Z-line alteration in plasmocid-induced myopathy]. PMID- 3000673 TI - [Klippel-Trenaunay-Weber syndrome associated with spinal arteriovenous malformation--a case report]. PMID- 3000674 TI - Discordant findings in technetium-99m pertechnetate and iodine-123 thyroid imaging caused by vascular retention in the superior vena cava. AB - This report demonstrates superior vena cava (SVC) retention of Tc-99m pertechnetate as a cause of discordant findings in thyroid imaging performed with Tc-99m pertechnetate and I-123. PMID- 3000675 TI - Intense myocardial uptake of gallium-67 citrate and technetium-99m pyrophosphate in a uremic patient. PMID- 3000676 TI - Free technetium simulating a solitary hepatic defect on technetium-99m liver scans. PMID- 3000678 TI - Utility of bladder lavage in Meckel's scanning. AB - Accumulation of radioactive urine in a bladder diverticulum on a Tc-99m pertechnetate scan simulated uptake in a Meckel's diverticulum. The abnormal activity was present even following bladder catheterization and drainage. However, saline lavage washed the residual activity out of the bladder, and prevented a false-positive diagnosis. In addition to preventing false-positive studies, bladder lavage may increase the sensitivity of the test by allowing the detection of Meckel's diverticuli that are obscured by residual bladder activity. PMID- 3000677 TI - The radionuclide diagnosis of horseshoe kidney. AB - The authors present an interesting case of horseshoe kidney diagnosed by nuclear imaging. PMID- 3000680 TI - Congenital absence of the mammary gland. AB - Tc-99m pertechnetate is known for its uptake and excretion by the mammary gland. Generally, lactating women are not advised to have any radioisotopic studies. If it is necessary to have one, it is recommended that they do not breast-feed their children for at least 24 hours. Congenital absence of the mammary gland is a rare condition. This case was accidently discovered. Nuclear medicine physicians should be aware of the value of clinical examination of patients, and that not every absent or asymmetric breast shadow is due to surgical resection. PMID- 3000679 TI - Demonstration of pericardium by radionuclide angiocardiography in the presence of pericardial and pleural effusions. PMID- 3000682 TI - Imaging of head and neck tumors with technetium(V)-99m DMSA. A new tumor-seeking agent. AB - Tumor scintigraphy, using Tc(V)-99m DMSA was performed on 76 patients with head and neck tumors. In 32 cases, SPECT also was performed. Tc(V)-99m DMSA was found to have a sensitivity of 75% (56 cases), a specificity of 85% (20 cases) and an accuracy of 78% on planar imaging. ECT studies showed accumulation of Tc(V)-99m DMSA in all 25 malignant cases studied. However, in benign tumors, four of seven cases (57%) showed radionuclide uptake. Tc(V)-99m DMSA has superior physical properties to Ga-67 and could be of use in the diagnosis of head and neck tumors. PMID- 3000683 TI - Altered distribution of technetium-99m sodium pertechnetate associated with antimicrobial therapy. AB - Three patients underwent brain scanning for evaluation of central nervous system disease and were simultaneously treated for infectious diseases unrelated to the central nervous process. All revealed intense vascular pooling on their brain images. The imaging studies had been performed following the administration of Tc 99m pertechnetate. None of the patients had prior nuclear medicine examinations to suggest the causal effect of stannous ion as a source of interference. All of the patients were on combination antimicrobial drugs: two on sulfamethoxazole and trimethoprim, and one on isoniazid and ethambutol. One patient revealed 75% Tc 99m red cell tagging. Another patient's repeat brain scan with Tc-99m DTPA revealed normal distribution. Our findings suggest that patients on antimicrobial combination drug regimens who require brain scans should be imaged routinely with agents other than Tc-99m. PMID- 3000681 TI - Sequential uptake patterns of technetium-99m pyrophosphate in hepatoma. AB - Sequential liver scintiphotography with Tc-99m pyrophosphate (PYP) was used to prospectively evaluate its uptake patterns in hepatoma. The scintiphotos and time activity curves of 40 cases were analyzed. Two distinct patterns of tumor activity were noted: gradual but complete extraction and trapping of Tc-99m PYP in hepatoma in 38% of the patients (group 1), and absence of subsequent Tc-99m PYP uptake in hepatoma after initial blood pool activity in 62% of the patients (group 2). Since extraction and trapping of Tc-99m PYP occur approximately in two fifths of the patients with hepatoma, we conclude that Tc-99m PYP liver scintigraphy is not worthwhile supplementing the conventional radionuclide studies for diagnosing hepatoma, even in the selected patients in the countries where the prevalence of hepatoma is high. PMID- 3000685 TI - Ultrasonographic studies in hepatic neoplasms: patterns and comparisons with radiological contrast studies. AB - A prospective blind study of 100 patients with hepatic neoplasms was performed using the grey-scale linear-array ultrasonographic technique. Seventy-four patients had hepatocellular carcinoma, eight had cholangiocarcinoma, 17 had metastases and one had haemangiosarcoma. The overall diagnostic accuracy was 96%, comparable to the 98% diagnostic accuracy of other radiological studies which were performed in 70 patients. The sites and extent of the lesions and the state of the portal system were identical in both the ultrasonographic and radiological contrast studies. No false positives were found in 254 patients who had ultrasonography of the liver for non-maligant disorders during the period of study. We conclude that linear-array ultrasonography is a simple, sensitive, specific and non-invasive procedure for the investigation of suspected hepatic neoplasms and is useful in assessing operability. PMID- 3000684 TI - False-negative delayed imaging in Meckel's diverticulum. PMID- 3000686 TI - Clinical prediction of the adult respiratory distress syndrome. AB - The use of clinical, physiologic, and laboratory parameters in the prediction or early detection of ARDS has been reviewed. From both a clinical and research standpoint, the ability to identify patients at risk is extremely important. The selection of patients according to predisposing clinical events has been the most successful thus far. The use of physiologic variables and gauges of injury severity have been of limited value, particularly for assessing ARDS risk in the individual patient. Only a handful of the proposed mediators or markers of acute lung injury have been studied prospectively in patients at risk. Of these, factor VIII antigen, lactoferrin, and phospholipase A2 appear the most promising as laboratory tests for selecting patients at risk. In the future it may be possible by using sophisticated statistical analysis techniques to combine important clinical, physiologic, and laboratory information into a numerical ARDS risk index, essentially assigning a probability of ARDS in individual patients. PMID- 3000688 TI - Avfail in color avoidance learning by starlings (Sturnus vulgaris) and red-winged blackbirds (Agelaius phoeniceus). AB - Certain unconditioned stimuli (UCS) in flavor avoidance learning sometimes become ineffective after pairings with relatively stronger UCS. This failure of avoidance learning (avfail) has been demonstrated only with rodents. The present investigations were conducted to determine whether avfail might also occur with avian species, the food selection of which is guided primarily by visual cues. In Experiment 1, starlings were given pairings of methiocarb (a relatively weak UCS) and LiCl (a relatively strong UCS). In Experiment 2, red-winged blackbirds were given pairings of two concentrations of methiocarb (relatively weak and relatively strong UCS, respectively). Pairings were followed by a conditioning trial (UCS gavage in the presence of a color cue) and two-choice tests. Conditioned avoidance was always observed except when methiocarb preceded LiCl and when the low preceded the high methiocarb dose in preconditioning pairings. Experiment 3 demonstrated that UCS habituation could not account for the results of Experiments 1 and 2. The data reflect avfail in the visual modality, and a biological implication of the results is that birds may not learn strong avoidance of aposematic prey containing varied levels of toxicant. PMID- 3000687 TI - Epstein-Barr virus in rheumatoid arthritis. PMID- 3000689 TI - Vaginal contraceptive activity of aryl 4-guanidinobenzoates (acrosin inhibitors) in rabbits. AB - Nine aryl 4-guanidinobenzoates were synthesized as inhibitors of the sperm enzyme acrosin. These esters were prepared from 4-guanidinobenzoic acid and a number of phenols which had been approved by the FDA for clinical use. The vaginal contraceptive activity of the inhibitors was evaluated in the rabbit at nonspermicidal concentrations (0.1 mg/ml). All the inhibitors except the 2' carboxamidophenyl and the 2'-isopropyl-5'-methylphenyl 4-guanidinobenzoates caused significant reductions in fertilization compared to the controls. Several of the aryl 4-guanidinobenzoates appeared to be particularly effective. Nonoxynol 9, under the same conditions but at 10- and 100-fold higher concentrations, also showed an antifertility effect. However, even at these increased dose levels, the contraceptive efficacy of nonoxynol-9 was no higher than that of most of the inhibitors and was less consistent than that of the most active aryl 4 guanidinobenzoates. The relatively high in vivo antifertility activity exhibited by several of the aryl 4-guanidinobenzoates encourages their further evaluation as vaginal contraceptive agents. PMID- 3000690 TI - A comparison of the effects of nonoxynol-9 and chlorhexidine on sperm motility. AB - The effects of Nonoxynol-9 and chlorhexidine on sperm motility were compared using the objective Transmembrane Migration Ratio method. These agents were found to be of similar potency in inhibiting sperm motility. The concentrations which reduced sperm motility by 50% (EC50) were 0.205 mg/ml for Nonoxynol-9 and 0.215 mg/ml for chlorhexidine. The implications of these findings in relation to the comparison of the effects of drugs on sperm motility and the development of new contraceptive agents are discussed. PMID- 3000692 TI - An open study of oat bran meal biscuits ('Lejfibre') in the treatment of constipation in the elderly. AB - Fifty elderly patients consulting their general practitioner with the complaint of constipation were entered into an open trial to assess the benefit on their symptoms of adding bran biscuits ('Lejfibre') twice daily to their diet. Patients were followed-up over 12 weeks. Treatment caused a marked improvement in bowel frequency, stool consistency and pain on defaecation and no patients complained of side-effects. The biscuits were well tolerated and compliance with therapy was good. In addition to the improvement in bowel symptoms, it was noted that mean body weight was significantly reduced at the end of the study. PMID- 3000691 TI - Acid dissociation constants and pH values for standard "bes" and "tricine" buffer solutions in 30, 40, and 50 mass% dimethyl sulfoxide/water between 25 and -25 degrees C. AB - Information is given concerning two standard buffer solutions suitable as pH references in 30, 40, and 50 mass% dimethyl sulfoxide (DMSO)/H2O mixed solvents at subzero temperatures from -20 to 0 degrees C, with the intention of establishing a pH (designated pH*) scale. The two buffers selected were the ampholytes N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid ("bes") and N tris(hydroxymethyl)methylglycine ("tricine"), and the reference standard consisted of equal molal quantities of the buffer and its respective sodium salt. The assignment of pH* values was based on measurements of the emf of cells without liquid junction of the type: Pt;H2(g,l atm) /Bes, Na Besate, NaCl / AgCl;Ag and Pt;H2(g,l atm) /Tricine, Na Tricinate, NaCl /AgCl;Ag and the pH* was derived from a determination of K2, the equilibrium constant for the dissociation process (Buffer)+/- in equilibrium with(Buffer)- + H+. PMID- 3000693 TI - From membrane to molecule with the lac permease of Escherichia coli. PMID- 3000694 TI - The cellular entry of EGF and transferrin: a problem in intracellular sorting. PMID- 3000695 TI - Change in NAD+/NADH content of S-adenosyl-L-homocysteine hydrolase upon NAD+ reversible inactivation by cAMP and 2'-deoxyadenosine. PMID- 3000696 TI - Receptor-mediated transport of acid hydrolases to lysosomes. AB - Lysosomal enzymes are the products of 40-50 unlinked genes in the nucleus. Like membrane and secretory proteins, they are synthesized on membrane-bound ribosomes in the rough endoplasmic reticulum. They receive high-mannose oligosaccharide chains from lipid-linked intermediates on asparagine residues. They must be sorted from other proteins present in the lumen of the endoplasmic reticulum and delivered to the lysosomes. The best understood mechanism for this sorting and delivery involves the Man 6-P recognition system. The newly synthesized acid hydrolases acquire Man 6-P residues by a two-step reaction. First, GlcNAc 1-P is transferred to the C-6 position of the mannose residues which are present on the asparagine-linked high-mannose oligosaccharides. Then, N-acetylglucosamine residues are removed by the N-acetylglucosaminyl phosphoglycosidase to generate the Man 6-P monoester, which is capable of binding the Man 6-P receptor. Phosphorylated enzymes can then bind to Man 6-P receptors which collect into vesicles and bud off for delivery of enzymes to lysosomes. The region of the Golgi apparatus where the receptors containing newly synthesized enzymes bud off is not yet clear. Enzymes which fail to bind receptors are secreted. Some cell types express on their cell surfaces receptors which are capable of recapturing phosphorylated enzyme by receptor-mediated endocytosis. This secretion - recapture pathway provides an alternate route to lysosomes. Following delivery of enzyme to lysosomes, the enzymes undergo post-lysosomal processing by acid phosphotases, which remove the phosphomonoester groups, and acid proteases which reduce their size and trim off excess polypeptides. Although the evidence is very persuasive that enzymes can reach lysosomes by pathways that do not depend on the Man 6-P receptor, the mechanisms of Man 6-P receptor-independent segregation of acid hydrolases to lysosomes are totally unclear. In addition to this question, there are two other significant questions that remain to be answered. One of these is the precise intracellular route of newly synthesized enzyme. Where does the enzyme first bind receptor, and where does receptor actually bud off the Golgi apparatus to effect sorting? The second major question is really the central question of the mechanism of sorting of acid hydrolases. Although we know now that the sorting is effected through an enzyme which phosphorylates acid hydrolases, the question remains: How does the processing phosphotransferase distinguish acid hydrolases from other glycoproteins in the endoplasmic reticulum?(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3000697 TI - Inorganic pyrophosphate and polyphosphates as sources of energy. PMID- 3000698 TI - Mechanisms of exogenous purine nucleotide utilization in Bacillus cereus. PMID- 3000699 TI - Platelet-derived growth factor: structure, function, and roles in normal and transformed cells. PMID- 3000700 TI - Hormonal secretion and enzyme activity of cultured roe-deer (Capreolus capreolus L.) Leydig cells, as measured by radio-immunological and histochemical assays. AB - Leydig cells from roe-deer collected according to Steinberger's (1975) technique were cultured as monolayers in Leighton tubes for 10 days. Cultures were grown in medium 199 supplemented with 10% calf serum. Androgen and oestrogen secretion by Leydig cells into the culture medium was measured using appropriate radio immunoassays. Using histochemical tests the activity of the following oxydoreductive enzymes in cultured Leydig cells was shown: delta 5, 3 beta hydroxysteroid dehydrogenase (delta 5, 3 beta-HSD), 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), succinate and lactate dehydrogenases (SDH and LDH). Strong activity of the enzymes investigated during the first 4 days of culture was observed. The androgen level was high throughout the second and fourth day of culture. A decrease in hormone secretion after day 4 occurred, and this was closely correlated with enzyme activity. The oestrogen level was very low during culture. The direct effect of the luteinizing hormone (LH) added into the culture medium caused an increase in not only enzyme activity but also androgen and oestrogen levels. PMID- 3000701 TI - Moving right atrial mass associated with hepatoma. Two cases detected by echocardiography. AB - Hepatoma with right atrial (RA) metastasis is rare, and to our knowledge, the echocardiographic description of a RA mass associated with hepatoma has never been described. Herein we report two cases of hepatoma, whose two-dimensional echocardiograms demonstrated a RA mass protruding into right ventricle during diastole, mimicking RA myxoma. However, in contrast to RA myxoma, these RA masses could be traced to the inferior venae cavae, which were dilated and filled with tumor echoes. Since prompt diagnosis and removal of these RA masses might prevent sudden cardiac death, we advocate performing echocardiographic examination in patients with hepatoma, who have cardiac symptoms or signs. PMID- 3000702 TI - [Possibilities of plastic surgical reconstruction in limb-sparing resection of malignant soft tissue tumors of the extremities]. PMID- 3000703 TI - Nerve growth factor. AB - In contrast to all other molecules which are labelled 'growth factor', NGF is not a mitogen. It is a neurotrophic molecule essential for the development and maintenance of function of specific populations of peripheral and possibly also central neurons. The availability of NGF in large quantities from exocrine glands (e.g. male mouse submandibular gland), where NGF does not play a neurotrophic role, has allowed the purification of NGF, the production of specific antibodies, the determination of its amino acid sequence and finally the molecular cloning of NGF leading to the elucidation of its precursor structure and its genomic organization. Comparison of the biological activities and the immunological properties of NGF isolated from different sources demonstrated that the active centre of the molecule has been highly conserved during evolution, whereas other parts of the molecule determining immunological properties have undergone considerable changes. After a survey of the essential biological actions of NGF, this paper concentrates on two actual questions of NGF research, namely the regulation of NGF synthesis in the target tissues of NGF-responsive neurons, and the molecular mechanism(s) of action of NGF on these neurons. PMID- 3000704 TI - Haemopoietic growth factors: structure and receptor interactions. AB - The proteins which regulate the production of blood cells appear to have overlapping functions. There are several forms of the haemopoietic growth factors (HGFs). Although a few have been purified, the functions of the different growth factors have not yet been clarified. The amino acid sequence of murine granulocyte-macrophage colonystimulating factor (GM-CSF) has been determined from a cDNA clone and several molecular forms of the molecule have been purified. Although there is no extensive homology with other haemopoietic growth factors, the mRNA for GM-CSF suggests two possible functions for this molecule. Radioiodination of GM-CSF to high specific activity has permitted the detection of two classes of specific GM-CSF receptors on myeloid cells. Although the different haemopoietic growth factors do not compete directly for binding to their specific receptors, GM-CSF and interleukin 3 (IL-3) can modulate the availability of other HGF receptors. PMID- 3000706 TI - Ectopic peptides released by a human melanoma cell line that modulate the transformed phenotype. AB - Cells derived from a human melanoma strain by low serum selection in monolayer were found to be capable of growth in semi-solid medium, forming colonies ranging from tight, to loose, or dispersed. These phenotypes were found to be stable on cloning and retesting. Examination of the serum-free media conditioned by these clones indicates that the clones release activities capable of inducing agar colonies, in an indicator cell line (NRK, 49F), that express phenotypes similar to those of the melanoma clones used to produce the serum-free conditioned media (SF-CM). These SF-CM contained a transforming growth factor (TGF) beta (apparent Mr 14 700) and a single, apparently high Mr TGF-alpha species. Co-eluting with the TGF-alpha at an Mr of approximately 22 500 was a previously undescribed activity capable of modulating the phenotype of the NRK agar colonies induced by the combination of TFGs alpha and beta. This new activity can modulate the usual tight colony to express a loose or dispersed phenotype characteristic of the producer cells. The possible role these ectopic peptides play in the expression of the transformed phenotype by the tumour cells producing them, and the possible correlation between the 22 500 Mr EGF-like peptide (TGF-alpha) released by this particular tumour line and high Mr EGF-like peptides found in the urine of cancer patients, are both discussed. PMID- 3000705 TI - Protein phosphorylation and growth control. AB - Many growth factor receptors and retroviral transforming proteins share the property of phosphorylating proteins on tyrosine. Several substrates for both types of protein-tyrosine kinase have been identified. Treatment of quiescent cells with growth factors such as EGF and PDGF, whose receptors have ligand stimulated protein-tyrosine kinase activities, induces tyrosine phosphorylation of three proteins, p45, p42 and p41. Two phosphorylated forms of p42 are found, the more basic of which is present in some but not all cells transformed by viral protein-tyrosine kinases. p42 is rapidly (as early as 1 min) but transiently (decreased to baseline by 2h) phosphorylated following PGDF or EGF treatment of quiescent fibroblasts. At saturating levels of mitogen the stoichiometry of p42 phosphorylation is greater than 50%. p42 is a highly conserved, rare (0.002% of total cell protein), soluble cytoplasmic protein. IGF I and insulin, whose receptors also have ligand-stimulated protein-tyrosine kinase activity, induce p42 phosphorylation in appropriate cells. In the case of insulin this effect has been observed in cells with large numbers of insulin receptors. p42 is also phosphorylated in response to mitogens whose receptors lack protein-tyrosine kinase activity, for example 12-O-tetradecanoylphorbol-13-acetate (TPA) and thrombin. For TPA there is evidence that this is an indirect effect due to the activation of a protein-serine/threonine kinase. On the basis of the highly conserved nature of this response and its generality, it seems likely that tyrosine phosphorylation of p42 is important for at least early responses to mitogens. PMID- 3000707 TI - The EGF receptor kinase: evidence for allosteric activation and intramolecular self-phosphorylation. AB - The membrane receptor for epidermal growth factor is a transmembrane protein composed of an EGF-binding domain and a cytoplasmic kinase domain, connected by a single hydrophobic stretch. The binding of EGF to the extracellular domain activates the cytoplasmic kinase function even in highly purified preparations of EGF receptor, suggesting that the activation occurs exclusively within the EGF receptor moiety. The experiments presented indicate that self-phosphorylation of the EGF receptor is dependent on the concentration of the receptor and that antibodies which cross-link the receptor molecules stimulate self-phosphorylation and increase the affinity of EGF towards the receptor. Moreover, immobilization of the EGF receptor on various solid matrices prevents EGF from activating the kinase function. These results are compatible with an intermolecular activation of the tyrosine kinase followed by an intramolecular self-phosphorylation process. An allosteric aggregation model is formulated as a framework to these and other regulatory responses attributed to the EGF receptor complex. PMID- 3000708 TI - Role of growth factors in oncogenesis: growth factor-proto-oncogene pathways of mitogenesis. AB - Cellular genes which encode proteins involved in the response of cells to stimulation by growth factors may be potential oncogenes. The factors involved in the signal transmission from growth factor-receptor interaction to DNA synthesis constitute a cascade system which we call the 'growth factor-proto-oncogene pathway(s) of mitogenesis'. For each growth factor, all the responsive cells, regardless of cell types and tissue source, have specific growth factor receptors which are similar, if not identical, in molecular weight and biological activity. Thus, we believe that the growth factor-proto-oncogene pathway(s) functions in the same manner in all responsive cells. Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and brain-derived growth factor (BDGF) are major growth factors for connective tissue cells and do not share a common pathway in mitogenesis in responsive cells. The gene product of c-myc may be involved in the cellular response of cells stimulated by PDGF or FGF, but not directly in the signal transmission which leads to DNA synthesis. PMID- 3000710 TI - Platelet-derived growth factor: its potential roles in wound healing, atherosclerosis, neoplasia, and growth and development. AB - Platelet-derived growth factor (PDGF) has been found to be derived not only from platelets that have been induced to release their contents, but also from a number of transformed cells (including cells transformed by both DNA and RNA viruses, spontaneous transformation, and cells from various human tumours), from activated macrophages, from embryonic rat aortic smooth muscle cells, and from rat aortic smooth muscle cells derived from experimentally induced intimal proliferative lesions. The studies discussed demonstrate the potential role of platelets and macrophages in atherosclerosis and in wound repair, and indicate the ability of anti-PDGF IgG to inhibit proliferative responses in vitro. With the demonstration that the transforming protein derived from the oncogene from the simian sarcoma virus is highly homologous with PDGF, it was possible to show that a number of transformed cells secrete PDGF and show markedly decreased binding of PDGF. The same is true for embryonic rat aortic smooth muscle cells and for cells from experimentally induced proliferative lesions in the rat carotid artery. All these findings point to the role of PDGF in the formation of these lesions and can be correlated with the capacity of the cells noted above, as well as injured endothelial cells, to secrete PDGF or PDGF-like molecules. The biological significance of these observations is discussed and a model for atherogenesis is proposed. PMID- 3000711 TI - [Physiopathology and treatment of tardive dyskinesia]. PMID- 3000709 TI - Signalling mitogenesis in 3T3 cells: role of Ca2+-sensitive, phospholipid dependent protein kinase. AB - Understanding the molecular mechanisms that control cell proliferation requires the identification of the early signals important for initiating a mitogenic response. In this context, the activation of Ca2+-sensitive, phospholipid dependent protein kinase (protein kinase C), which is stimulated by diacylglycerols and serves as a major phorbol ester receptor, may play an important part in signalling mitogenesis. This conclusion is based on two main lines of evidence. Firstly, activation of protein kinase C in intact quiescent fibroblasts is one of the earliest events elicited by a variety of growth promoting agents including serum, platelet-derived growth factor (PDGF), vasopressin and bombesin, as judged by the increase in the phosphorylation of a cellular protein characterized by an Mr of 80 000 and a pI of 5. Secondly, the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, which directly competes with [3H]phorbol dibutyrate for binding to specific receptors in intact 3T3 cells and rapidly stimulates protein kinase C in these cells, is a potent mitogen for Swiss 3T3 cells, acting synergistically with other growth factors. We propose that activation of protein kinase C may be one of the early signals that mediate the mitogenic effects of a variety of growth factors and peptide hormones in quiescent fibroblastic cells. PMID- 3000712 TI - [The effect of the natural inhalation of quartz dust on the lipid content of bronchoalveolar lavage in the rat]. PMID- 3000713 TI - An update on the acquired immunodeficiency syndrome (AIDS). Associated disorders of the alimentary tract. AB - The pandemic, acquired immunodeficiency syndrome (AIDS) has been described in 40 nations throughout the world. This paper describes the wide spectrum of gastrointestinal tract manifestations seen in this syndrome, with particular attention to the epidemiology, etiology, and measurement of these problems. Discussion of candidiasis, herpes simplex, "hairy" leukoplakia, Kaposi's sarcoma, cytomegalovirus, anal warts and carcinoma, chlamydial proctitis (LGV), coccidiosis, and mycobacterial diarrhea, as well as "gay bowel syndrome," demonstrates the complex management problems associated with this condition. PMID- 3000714 TI - A fiber-rich diet for the treatment of diabetic patients with chronic renal failure. PMID- 3000716 TI - High fiber diets: how and why. PMID- 3000715 TI - Cloning and isolation of human cytochrome P-450 cDNAs homologous to dioxin inducible rabbit mRNAs encoding P-450 4 and P-450 6. AB - Human cytochrome P-450s structurally related to 2,3,7,8-tetrachloro-p-dioxin (TCDD)-inducible rabbit P-450 4 and 6 have been cloned from a human liver cDNA library. The human P-450 4 cDNA clone, hpP-450 4, and the human P-450 6 cDNA clone, hpP-450 6, were identified by hybridization to rabbit P-450 4 and P-450 6 cDNAs, respectively. DNA sequence analysis demonstrates that hpP-450 4 is 83% and 75% homologous to rabbit P-450 4 and P-450 6 mRNAs, respectively, whereas hpP-450 6 is 79% and 72% homologous to rabbit P-450 6 and P-450 4, respectively. A comparison of DNA sequence of the two human cDNA clones shows they are 80% homologous. This is similar to the homology found between the cDNA sequences of rabbit P-450 4 and P-450 6. Northern blot analysis has shown that the human P-450 4 mRNA is approximately 3000 bases, while the human P-450 6 mRNA is 2600 bases in length. Clone hpP-450 4 preferentially hybridizes to TCDD-inducible rabbit P-450 4 and mouse P3-450 mRNAs, whereas hpP-450 6 preferentially hybridizes to TCDD inducible rabbit P-450 6 and mouse P1-450 mRNAs. Both hpP-450 4 and hpP-450 6 recognize different genomic fragments, indicating that each is encoded by different genes. These results indicate the existence of at least two P-450 genes in humans that are highly homologous to the TCDD-inducible P-450s in rabbits and mice. PMID- 3000717 TI - Marijuana and diabetes. PMID- 3000718 TI - [Sequence of 3372 RNA nucleotide links in the hepatitis A virus coding for capsid VP4-VP1 and nonstructural proteins]. PMID- 3000721 TI - [Malignant fibrous histiocytoma. Prognostic value of the histological findings]. AB - Surgical specimens after excision of malignant fibrous histiocytomas from 41 patients were classified histologically according to subtype; semiquantitatively by cell content, mitosis count, necrosis tendency and cell polymorphism; and "subjective grading". There was no significant difference in survival time between storiform-pleomorphic and myxoid subtypes. The marked scatter in the storiform-pleomorphic subtype reflects the inhomogeneity of this group. Analysis of the individual semiquantitative criteria gave better results. Cases with many mitoses, many necroses, marked cell polymorphism and high cell content had significantly shorter survival times than those with less marked changes. "Subjective grading" was also of prognostic value. However, overlapping of prognostic factors and limitations due to tumour biology allow of only a rough orientating prediction based on histological assessment of malignancy grade. PMID- 3000720 TI - [Unaimed biopsy in gastroscopy?]. PMID- 3000719 TI - Pseudo-Cushing's syndrome: an example of alcohol-induced central disorder in corticotropin-releasing factor-ACTH release? AB - Six chronic alcoholics with stigmata of Cushing's syndrome were studied before and after a period of alcohol abstinence. In all of them, after a minimum period of 3-4 weeks, a marked clinical and laboratory improvement was noted. The authors suggest that damage at brain level, with neurotransmitters' disturbance, caused by chronic alcoholism underlies the Pseudo-Cushing's Syndrome. The primum movens could be a disorder of the pituitary-adrenal axis secondary to a dysfunction of neurotransmitters with stimulation of ACTH-secreting cells of the adenohypophysis by the certicotropin-releasing factor (CRF). PMID- 3000723 TI - [Successful eradication of enzootic bovine leukosis in Yugoslavia]. PMID- 3000722 TI - [Observations and results of a program evaluated in Italy for the control of enzootic leukosis in cattle]. PMID- 3000724 TI - [Pathologic anatomy and bacteriologic findings in a field infection with IBR/IPV virus]. PMID- 3000725 TI - [Restriction fragment length polymorphism--our diagnostic genetic fingerprints?]. PMID- 3000726 TI - [Diagnosis of Duchenne's muscular dystrophy using recombinant DNA technics]. PMID- 3000727 TI - Induction of growth hormone (GH) receptors in adipocytes of hypophysectomized rats by GH. AB - The ability of GH to regulate its own receptor in adipocytes of hormone substituted hypophysectomized rats was studied. Male rats (130-150 g) were hypophysectomized and substituted with T4 and cortisone. A fixed dose of GH (bovine GH, human GH, or ovine GH), was administered for 5-6 days in three different ways: 1) two injections/day, 2) four injections/day, or 3) by osmotic minipumps. GH binding was measured in cell aliquots using [125I]human GH. In unsubstituted hypophysectomized animals, GH binding was decreased and was approximately 25% of the binding observed in adipocytes of normal rats. With T4 and cortisone replacement, GH binding was partially restored. When GH was administered in four daily injections or via osmotic minipumps, a further increase in GH binding was observed. This increase was observed if the animals were killed up to 6 h but not 12 h after the last GH injection. GH given in two daily injections had no effect on GH binding even when studied at different time periods after the last GH injection. The GH receptor induced by frequent or continuous administration of GH was mainly somatogenic, since an excess of unlabeled ovine PRL inhibited GH binding only to a minor extent. There was no difference in accumulated body weight gain between the GH-treated groups during the treatment period. The results show that GH regulates its own receptor in adipocytes and that the mode of administration of the hormone is of importance for this effect of GH. PMID- 3000728 TI - Induction of epidermal growth factor-related polypeptides by 17 beta-estradiol in MCF-7 human breast cancer cells. AB - MCF-7 human breast cancer cells release polypeptides related to insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) into serum-free culture medium (CM). Treatment of MCF-7 cells with 17 beta-estradiol, which is required in vivo for MCF-7 tumor growth in the nude mouse and stimulates the MCF-7 growth rate in vitro, resulted in selectively enhanced growth factor activities in CM. Autostimulatory growth-promoting activity was elevated at least 2-fold, and EGF like polypeptides were elevated 5-fold but IGF-I immunoreactivity was not elevated. Several species of estrogen-induced receptor-reactive EGF-like polypeptides, suggestive of high molecular weight transforming growth factor alpha, were detected after gel exclusion chromatography of CM extracted with 1 M acetic acid. A 30,000 mol wt peak of EGF receptor competing activity comigrated with a peak of autostimulatory and fibroblast-transforming activity. It is possible that estradiol stimulation of MCF-7 growth and/or tumor formation may depend on induction of EGF-related polypeptide growth factors. EGF-I- and EGF related polypeptides may act together as autocrine or paracrine growth factors in breast cancer. PMID- 3000729 TI - Microheterogeneous forms of radioiodinated bovine thyrotropin: discrimination of different receptor-active components by gel permeation chromatography. AB - The products of the radioiodination and subsequent receptor adsorption of bovine TSH (bTSH) radiolabeled by the lactoperoxidase method have been further investigated. After receptor adsorption, [125I]bTSH was resolved by gel permeation chromatography on Sephadex G-100 (superfine) under low ionic strength conditions into three peaks of radioactivity (tracers 2a, 2b, and 2c, respectively). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions demonstrated that each tracer component was radiolabeled on both the alpha- and beta-subunits. Analysis of the three tracers by TSH radioreceptor assay (under different radioreceptor assay conditions) showed that tracers 2b and 2c exhibited saturable rebinding to crude thyroid membranes containing functional TSH receptors. However, tracer 2c exhibited a maximum binding 2-fold greater than tracer 2b. This difference has been attributed to the abundance of an apparently low affinity binding component in tracer 2c. Rechromatography of tracers 2b and 2c on Sephadex G-100 (superfine) under high ionic strength conditions yielded tracer profiles that were coincident, demonstrating that the initial separation under low ionic strength conditions was not based on differences in molecular volume. The data indicate that radioiodination of highly purified bTSH yields multiple tracer components. Further, receptor adsorption, commonly used to purify freshly iodinated bTSH before radioreceptor assay, purifies at least two species of receptor-active [125I] bTSH. PMID- 3000730 TI - Corticotropin-releasing factor stimulates the release of adrenocorticotropin from domestic fowl pituitary cells. AB - The effect of synthetic ovine CRF on ACTH secretion of dispersed, domestic fowl pituitary cells was investigated. Cells preincubated for 2 h, 16 h, or after 48-h culture were incubated briefly with CRF (up to 4 h). ACTH was bioassayed using isolated rat adrenocortical cells; ACTH-(1-24) served as the standard for expressing the data. Results with 16-h preincubated cells were as follows: CRF induced ACTH secretion in a concentration-dependent manner: ED50 and maximal stimulatory concentrations were 1.0 nM and 5.0 nM, respectively. CRF (10 nM) induced significant ACTH secretion within 10 min of incubation; maximal secretion (370% over basal value) was attained at 2 h. Dexamethasone (DEX) inhibited basal and CRF-induced ACTH secretion in a concentration-dependent manner; half-maximal inhibitory and maximal inhibitory concentrations were approximately 10 nM and 1 microM, respectively. In addition, DEX (10 microM) acutely (within 2 h) inhibited maximal CRF-induced ACTH secretion by 46%. 8-Bromo-cAMP (1 mM) also induced ACTH secretion, and DEX inhibited this secretion with a potency equivalent to that for CRF-induced ACTH secretion. In contrast to the effect of CRF, high concentrations (100 nM) of ovine LHRH, TRH, and synthetic human pancreatic GH-releasing factor (1-32) failed to induce significant ACTH secretion, thus suggesting that the effect of CRF was peptide specific. Domestic fowl pituitary cells cultured for 48 h before treatment also responded to CRF but not to any greater extent than that of 16-h preincubated cells. In contrast to 16-h preincubated cells or 48-h cultured cells, 2-h preincubated cells had high basal values of ACTH secretion that may have partially diminished or masked the actions of CRF. These data suggest that 1) CRF is a potent and specific stimulator of ACTH secretion by domestic fowl pituitary cells and 2) 16-h preincubated cells or 48-h cultured cells are amenable for other in vitro investigations on the regulation of avian ACTH secretion. PMID- 3000731 TI - Somatomedin-C as an amplifier of follicle-stimulating hormone action: enhanced accumulation of adenosine 3',5'-monophosphate. AB - Somatomedin-C (Sm-C) has recently been found to amplify the FSH-mediated acquisition of granulosa cell progestin biosynthetic capacity, aromatase activity, and LH receptors, an effect distinct from its established replicative property. To further characterize the cellular mechanism(s) underlying the synergistic interaction of Sm-C with FSH, we have set out to evaluate the intermediary role of cAMP in this regard. Isolated granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were cultured for up to 3 days under serum-free conditions. The basal extracellular accumulation of cAMP remained unchanged in response to treatment with highly purified Sm-C (50 ng/ml). However, concurrent treatment with increasing concentrations (0.3-50 ng/ml) of Sm C, produced dose- and time-dependent increments in the FSH-stimulated accumulation of cAMP, with an apparent median effective dose (ED50; mean +/- SE) of 5.1 +/- 0.6 ng/ml, a maximal response 8.8-fold greater than that induced by FSH alone, and a minimal time requirement of 1-2 days. Given increasing concentrations of FSH, treatment with a constant concentration (50 ng/ml) of Sm-C resulted in 1.7-, 5.8-, and 4.3-fold increases in cAMP accumulation for 10, 30, and 100 ng/ml FSH, respectively. The ability of Sm-C to augment FSH-stimulated cAMP accumulation was evident and, in fact, enhanced by ZK62711 (Rolipram; 3 X 10(-6) M)-induced blockade of cAMP-phosphodiesterase activity. Decreasing dilutions (1:64,000 to 1:1,000) of a monoclonal antibody raised against Sm-C (sm 1.2) produced progressive and complete immunoneutralization of the synergistic interaction of Sm-C with FSH, suggesting specificity of action. Taken together, these findings suggest that Sm-C, acting at nanomolar concentrations compatible with its granulosa cell receptor binding affinity (0.6-2.0 nM), is capable of amplifying FSH-stimulated cAMP accumulation in a time- and dose-dependent manner. These observations suggest that the synergistic action of Sm-C is exerted, at least in part, at a site(s) proximal to cAMP generation. PMID- 3000732 TI - Effects of thyrotropin-releasing hormone on phosphoinositides and cytoplasmic free calcium in thyrotropic pituitary cells. AB - We have previously demonstrated differences in several cellular responses to TRH in mouse thyrotropic pituitary (TtT) cells and in rat mammotropic pituitary (GH3) cells. In this report, we further explore the mechanism of TRH action in TtT cells by measuring its effects on phosphoinositides and on cytoplasmic free Ca2+ concentration [( Ca2+]i). We demonstrate that TRH stimulates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by a phospholipase C and elevates [Ca2+]i. Furthermore, we present evidence that hydrolysis of PtdIns(4,5)P2 is not secondary to the elevation of [Ca2+]i. TRH caused a rapid decrease in the level of PtdIns(4,5)P2 to 57% of control and stimulated an increase in inositoltriphosphate, the unique product of phospholipase C-mediated hydrolysis of PtdIns(4,5)P2, to a peak of 280% of control. In control cells, resting [Ca2+]i was 106 +/- (SE) 27 nM, and TRH stimulated a rapid elevation to 700 +/- 210 nM. In experiments performed to determine whether PtdIns(4,5)P2 hydrolysis induced by TRH may have been caused by the elevation of [Ca2+]i, the following results were obtained: the effect of TRH to decrease the level of PtdIns(4,5)P2 was not reproduced by the calcium ionophore A23187 or by membrane depolarization with 50 mM K+; the calcium antagonist TMB-8 did not inhibit the TRH-induced decrease in PtdIns(4,5)P2; and, most importantly, inhibition by EGTA of the elevation of [Ca2+]i did not inhibit the TRH-induced decrease in PtdIns(4,5)P2. We suggest that phospholipase C-mediated hydrolysis of PtdIns(4,5)P2 to yield inositoltriphosphate may be the initial event in TRH action in TtT cells, as in GH3 cells, that leads to elevation of [Ca2+]i and to TSH secretion. PMID- 3000733 TI - Effects of angiotensin II, adrenocorticotropin, and potassium on aldosterone production in adrenal zona glomerulosa cells from streptozotocin-induced diabetic rats. AB - Hyporeninemic hypoaldosteronism has been shown to occur in streptozotocin-induced chronic diabetic rats with normokalemia. To test the nature of the aldosterone deficiency, we investigated the responses of aldosterone production to angiotensin II (AII), ACTH, and potassium in adrenal zona glomerulosa cells from diabetic rats at 6 weeks after an injection of streptozotocin compared with those in the cells from control rats. In diabetic rats, plasma glucose was high and plasma immunoreactive insulin was low. Diabetic rats also had low levels of PRA and plasma AII, low levels of plasma aldosterone, and normal levels of plasma corticosterone and plasma potassium. The zona glomerulosa width was narrower in diabetic rats than in control rats. Basal aldosterone production, when corrected to an uniform number of cells per group, was similar in the cells from control and diabetic rats. Cells from diabetic rats showed a less sensitive and lower response of aldosterone production to AII, increases in the threshold and the ED50, and a decrease in the maximal AII-stimulated aldosterone level. ACTH, however, caused a similar effect on aldosterone production in the cells from control and diabetic rats. Cells from diabetic rats exhibited a less sensitive response of aldosterone production to potassium and a tendency to be low in the maximal potassium-stimulated aldosterone level, presumably attributable to the impairment of adrenal zona glomerulosa cells to AII. We conclude that the hypoaldosteronism observed in our diabetic rats may be secondary to the deficiency of AII. PMID- 3000734 TI - Involvement of protein kinase C in the regulation of adrenocorticotropin release from rat anterior pituitary cells. AB - The involvement of protein kinase C in normal corticotroph function was studied by analysis of the effects of the phorbol ester derivative phorbol 12-myristate 13-acetate (PMA) and the synthetic diacylglycerol dioctanoylglycerol (DOG) on basal and stimulated ACTH release in cultured rat anterior pituitary cells. Incubation of rat pituitary cells with increasing concentrations of PMA or DOG caused dose-related increases in ACTH release up to 13.4 +/- 2.1- and 10.1 +/- 0.9-fold, respectively, similar to that caused by CRF (9.8 +/- 1.6-fold). Also, stimulation of endogenous diglyceride formation by phospholipase C (100 mU/ml) stimulated ACTH release by 2.5 +/- 0.1-fold. In cells incubated with maximum stimulatory concentrations of CRF (10 nM) or 8-bromo-cAMP (8-Br-cAMP; 5 mM), addition of either 100 microM DOG or 100 nM PMA caused significantly higher ACTH responses than those obtained with CRF, 8-Br-cAMP, DOG, or PMA alone. 8-Br-cAMP (5 mM) and 10 nM CRF significantly increased the effect of 100 nM PMA by 1.4 +/- 0.2- and 1.5 +/- 0.1-fold, respectively. Combinations of 10 nM CRF with either vasopressin (VP) or angiotensin II (AII) increased ACTH secretion to values higher than those produced by CRF, VP, or AII alone. However, addition of maximal stimulatory concentrations of VP or AII (10 nM) did not further increase the effects of either PMA alone or PMA/CRF combinations, indicating that their mechanisms of action may be similar to that of PMA. These results indicate that in addition to the established cAMP-dependent mechanism, stimulation of ACTH release in normal pituitary cells may be elicited by activation of protein kinase C. The evidence also suggests that protein kinase C is involved during stimulation of ACTH release by the cAMP-independent regulators VP and AII and in the synergistic effects of VP and AII with CRF on the corticotroph. PMID- 3000735 TI - Arginine vasopressin as an intragonadal hormone in Brattleboro rats: presence of a testicular vasopressin-like peptide and functional vasopressin receptors. AB - We have previously demonstrated the presence of an arginine vasopressin (AVP) like peptide and AVP receptors in rat testis. We have also shown a direct inhibitory effect of AVP on androgen biosynthesis by cultured testicular cells. This study examined the presence of testicular AVP-like peptides and AVP receptors in homozygous (di/di) Brattleboro rats, a genetic mutant known to be deficient in hypothalamic, pituitary, and circulating AVP. The supernatant of homogenized 0.1 N acetic acid-extracted testis from adult homozygous Brattleboro rats was chromatographed on a Sephadex G-25 column. The elution profile of AVP immunoreactivity, as measured by a specific RIA, showed three distinct peaks. The first peak eluted close to the column void volume, a second peak eluted at the column total volume, while a third peak coeluted with synthetic AVP. The third peak of immunoreactive material (375 pg/g tissue) behaved similarly to authentic AVP on octadecylsilica adsorption chromatography, showed a competition curve parallel to that of AVP in the RIA, and comigrated with AVP on Sephadex G-25 and reverse phase TLC. The first, but not the second, immunoreactive peak contained enzyme activity that degraded labeled AVP in a time-dependent manner. Additional studies investigated the presence of AVP receptors in testes from Brattleboro rats. Saturable and specific [3H]AVP-binding sites were present in an enriched testicular interstitial cell preparation from these animals. Scatchard analysis indicated a Kd of 5.6 X 10(-10) M and a binding capacity of 9.7 fmol AVP bound/10(6) cells. These receptors were of the vasopressor (V1) subtype, as indicated by the potencies of selective AVP analogs for competition of [3H]AVP binding. The functionality of these receptors was shown by AVP inhibition of gonadotropin-induced androgen biosynthesis in cultured testicular cells derived from Brattleboro rats. Thus, testes from Brattleboro rats contain a high amount of an AVP-like peptide even though these animals lack hypothalamic, pituitary, and circulating AVP. Also, an AVP-degrading enzyme and AVP receptors with a Kd and binding capacity similar to those of Sprague-Dawley rats are present in the testes of Brattleboro rats. These findings add further support to the hypothesis that locally produced AVP acts as an intratesticular modulator of androgen biosynthesis. PMID- 3000736 TI - Angiotensin peptides stimulate phosphoinositide breakdown and prolactin release in anterior pituitary cells in culture. AB - We investigated the effects of angiotensin peptides on the breakdown of specific membrane phospholipids, the inositol lipids, in anterior pituitary cells in culture, measuring the water-soluble products (inositol phosphates) produced during the cleavage of phosphoinositides by phospholipase C. Both angiotensin II and angiotensin I in the presence of 10 mM LiCl potently increased, in a concentration-dependent manner, total [3H]inositol phosphate and PRL release in cultured rat anterior pituitary cells. The release of LH, TSH, or GH was not significantly enhanced by the peptides. The effect on inositol phosphate accumulation was significant at 0.01 nM, and maximal stimulation (approximately 5 fold increase) occurred at 10 nM, with an ED50 of about 0.3 nM. The stimulatory effects of both angiotensin II and angiotensin I were antagonized by the receptor antagonists saralasin and Sar1,Ile8-angiotensin II. Moreover, 1 microM captopril, an inhibitor of angiotensin-converting enzyme, antagonized the effects of 0.1 and 1 nM angiotensin I, suggesting that the effect of angiotensin I on phosphoinositide breakdown and PRL release is dependent on prior conversion of angiotensin I to angiotensin II. The effect of angiotensin II was very rapid. Fractionation of the water-soluble inositol phosphates showed that angiotensin II significantly increased inositol bisphosphate and inositol triphosphate at 10 sec, whereas inositol monophosphate was increased only after 40 sec. These data indicate that in the pituitary, and presumably in the lactotroph, the binding of angiotensin II to specific membrane receptors provokes increased polyphosphoinositide hydrolysis, leading to increased production of intracellular messengers, i.e. inositol triphosphate and 1,2-diacylglycerol, responsible for the stimulation of PRL release. PMID- 3000737 TI - The effects of 1,25-dihydroxyvitamin D3 and dexamethasone on rat osteoblast-like primary cell cultures: receptor occupancy and functional expression patterns for three different bioresponses. AB - The effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and dexamethasone to regulate collagen and osteocalcin synthesis and induction of 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) activity were studied in rat osteoblast-like cell primary cultures. In this culture system, the basal levels of collagen and osteocalcin synthesis increased with rising cell density in culture. At maximal doses, both 1,25-(OH)2D3 (8.1 nM) and dexamethasone (130 nM) reduced collagen synthesis to about 50% of the control levels, 1,25-(OH)2D3 affected osteocalcin synthesis in a biphasic manner: stimulatory at low doses, which peaked near 0.33 nM to reach 3- to 5-fold the basal level, followed by a gradual return to the basal level at higher concentrations. Dexamethasone had only a slight stimulatory effect on osteocalcin. 1,25-(OH)2D3 also induced 24-hydroxylase activity in rat osteoblast-like cells, while dexamethasone had no effect on the enzyme. Induction of enzyme activity achieved a 4- to 6-fold rise, but required higher concentrations of 1,25-(OH)2D3 to achieve maximal levels (16 nM). The half maximal doses (ED50) of 1,25-(OH)2D3 required for each bioresponse were different. The approximate ED50 for the inhibition of collagen synthesis was near the Kin (0.4 nM; apparent dissociation constant of receptor nuclear internalization), while the ED50 for osteocalcin synthesis (0.08 nM) was below the Kin, and the ED50 for 24-hydroxylase induction (20 nM) was greater than the Kin. The ED50 for dexamethasone on collagen synthesis (20 nM) was about 5-fold higher than the Kin (4 nM) of dexamethasone receptor binding. The potencies of various vitamin D3 metabolites in all three functional responses followed their abilities to compete for the 1,25-(OH)2D3 receptor, indicating that these actions were 1,25-(OH)2D3 receptor mediated. In summary, these studies explored bone cell bioresponses to 1,25-(OH)2D3 and dexamethasone and examined the relationship between receptor occupancy and functional expression. Each action exhibited a different dose-response pattern, implying that different levels of control are required for each individual response. PMID- 3000738 TI - GABAergic regulation of melanocyte-stimulating hormone secretion from the pars intermedia of Xenopus laevis: immunocytochemical and physiological evidence. AB - alpha-MSH secretion from the amphibian pars intermedia is under inhibitory hypothalamic control, and the catecholamine dopamine is thought to be the physiological MSH release-inhibiting factor. In the present study we evaluated the possible role of the neurotransmitter gamma-aminobutyric acid (GABA) in the regulation of the pars intermedia of Xenopus laevis. Immunocytochemical staining with antibodies to glutamic acid decarboxylase showed the presence of a rich GABAergic network in the intermediate lobe of the pituitary gland. Administration of GABA to superfused neurointermediate lobes caused a rapid and dose-dependent inhibition of basal release of MSH and immunoreactive endorphin. Pulse-chase experiments revealed that GABA gave a coordinate inhibition of the release of all peptides derived from proopiomelanocortin. In vivo administration of GABA resulted in almost complete pigment aggregation in dermal melanophores of both adults and larvae. Altogether, our results indicate that GABA is a physiologically important factor for regulation of the pars intermedia in Xenopus laevis. PMID- 3000739 TI - Forskolin enhances basal and potassium-evoked hormone release from normal and malignant pituitary tissue: the role of calcium. AB - The release of immunoreactive ACTH (IR-ACTH) from AtT-20 pituitary tumor cells was transiently increased by exposure to an elevated concentration of potassium ion in an osmotically balanced extracellular medium. With the calcium-sensitive dye Quin 2, the concentration of free cytosolic calcium (Cai) in the AtT-20 tumor was determined to be 115 nM. Challenge of these cells with 60 mM potassium in an osmotically balanced salt solution raised the concentration of Cai to 246 nM. This is in accord with the view that agents promoting calcium entry into pituitary cells trigger hormone secretion. Addition of forskolin to the extracellular medium caused a sustained release of IR-ACTH from AtT-20 tumor cells. Challenge with forskolin (10 microM) increased the concentration of Cai to 149 nM. This observation is also in accord with the view that calcium entry is a necessary and sufficient stimulus to trigger hormone secretion from the anterior pituitary lobe. Exposure of cells to forskolin (10 microM) before a potassium challenge increased the quantity of IR-ACTH released in response to potassium, but did not alter the minute by minute time course of the response to this ion. Forskolin pretreatment did not alter the potassium-evoked rise in Cai concentration. This observation suggests that the magnitude of the secretory response of the pituitary gland can be enhanced by agents other than those promoting an increase in Cai. After exposure of the tumor cells to potassium for a sufficient time to permit the rate of release of hormone to return to the basal value, forskolin could still stimulate the release of hormone from the tumor cells. Under these circumstances, forskolin did not increase the concentration of Cai. This observation suggests that pituitary hormone secretion can be initiated by a factor(s) other than an acute change in the Cai concentration. Both forskolin and 8-bromo-cAMP stimulated hormone secretion from dispersed melanotrophs and potentiated the potassium-evoked secretory response of these cells. Neither compound affected the apparent time course of the response to potassium. These observations suggest that the effects of forskolin and potassium on the AtT-20 tumor cell may use mechanisms occurring in normal pituitary cells. PMID- 3000740 TI - Unique properties of the follicle-stimulating hormone- and cholera toxin sensitive adenylyl cyclase of immature granulosa cells. AB - Previous studies have shown that the adenylyl cyclase of intact granulosa cells from immature porcine follicles has a uniquely prolonged responsiveness to FSH and an atypically poor responsiveness to cholera toxin relative to other cells. The present studies were designed to determine if these characteristics were due to unique regulation of the adenylyl cyclase activation process by Mg2+ and guanine nucleotides. GTP and Mg2+ enhanced adenylyl cyclase activity in the absence and presence of FSH in a manner similar to that in other systems. For example, GTP and Mg2+ increased the maximal velocity rather than the sensitivity of the cyclase to GTP and Mg2+. However, several unique properties distinguish the FSH-sensitive adenylyl cyclase from other hormonally activated adenylyl cyclases. First, FSH did not increase the sensitivity of the enzyme to Mg2+; thus the apparent Km for Mg2+ remained nonphysiologically high in the presence of FSH. Second, FSH was only one fourth as effective as NaF in activating the enzyme. Third, maximal activity attained in the presence of NaF was very low relative to that in other cells. Fourth, even in the presence of exogenous NAD+, cholera toxin activated adenylyl cyclase only as well as FSH rather than as well as NaF. Fifth, instead of causing maximal activation in the absence of stimulator, guanyl 5'-ylimidodiphosphate enhanced by almost 2-fold basal, FSH-activated, and cholera toxin-activated adenylyl cyclase. The inability of either guanyl-5' ylimidodiphosphate or cholera toxin alone to cause maximal activation of the guanine nucleotide-binding protein, as reflected by cyclase activity, indicates that guanine nucleotide binding in the absence of hormone or cholera toxin is limiting. PMID- 3000741 TI - Superior efficacy of pulsatile versus continuous hormone exposure on hepatic glucose production in vitro. AB - To elucidate the potency of continuous vs. intermittent exposure to hormonal stimuli, hepatic glucose production of isolated perfused rat livers was monitored in response to glucagon and insulin infusion. Using a nonrecirculating perfusion system, continuous exposure to glucagon (35 pM) induced a rise in hepatic glucose production from basal 0.33 +/- 0.03 mmol/(96 min X 100 g BW) to 0.65 +/- 0.02 mmol/(96 min X 100 g BW), while intermittent exposure (3 min on/off intervals; total dose 50%) to the same glucagon concentration elicited an almost identical rise in hepatic glucose production to 0.59 +/- 0.12 mmol/(96 in X 100 g BW). Insulin (100 mU/liter) given continuously and intermittently (3 min on/off intervals) inhibited glucagon-stimulated (70 pM) hepatic glucose production to the same extent, i.e. by 37.4% and 41.1%, respectively. Doubling the off period to 6 min and thereby reducing the total hormone dose to 33% did not diminish insulin's suppressive effect on glucagon-stimulated hepatic glucose release (34.6%). When the latter infusion protocol was applied with insulin at 300 mU/liter, hepatic glucose production during the first 40 min of glucagon infusion was more restrained (P less than 0.01) than during continuous delivery of 100 mU/liter, although the same amount of insulin was infused per period of time. In parallel, glucagon-stimulated cAMP release was similarly suppressed by insulin in all experiments. From this we conclude that the effect on hepatic glucose production of pulsatile administration of glucagon as well as of insulin, depending on the applied time interval of hormone exposure, is equipotent or even superior to the respective hormones' continuous infusion even if the hormone load is significantly reduced. PMID- 3000742 TI - Proopiomelanocortin-derived peptides in testicular interstitial fluid: characterization and changes in secretion after human chorionic gonadotropin or luteinizing hormone-releasing hormone analog treatment. AB - Recent studies indicate that proopiomelanocortin (POMC)-derived peptides are present in the testis and may be involved in the regulation of gonadal function, although their precise role still remains obscure. In the present study, we investigated in the rat whether testicular interstitial fluid (TIF), a functionally important compartment of the testis, contains POMC-derived peptides. Adult rats were used for all the experiments, and TIF was collected from each individual testis and analyzed for the presence of ACTH and beta-endorphin-like immunoreactivity (beta end-LI). Initial studies indicated that both ACTH and beta end-LI can be readily detected in TIF from intact rats, and that the concentrations of these peptides are severalfold higher than those in the peripheral plasma of the same animals. Similar studies in rats 3-5 days after hypophysectomy indicate that although ACTH and beta end-LI levels in plasma were undetectable, high levels of both peptides were measured in TIF. Moreover, serial dilution curves of TIF showed parallelism with the respective standard curves in the beta end and ACTH assays. Additional studies indicated that levels of POMC peptides in TIF were modified by treatments that affect the endocrine function of the testis. In that respect, hCG given sc to hypophysectomized rats increased testosterone levels in TIF and plasma, and similarly increased beta end-LI concentrations in TIF. A LHRH analog (LHRH-A; [D-Ala6,des-Gly10]LHRH-ethylamide) given sc to hypophysectomized rats resulted in increased testosterone production, but decreased beta end-LI in TIF, suggesting that the effects of hCG and LHRH-A on testicular beta end-LI secretion are not directly coupled with testosterone production. Prolonged treatment of intact rats with the LHRH-A for 2-10 days resulted in progressive declines in testicular weight and testosterone levels in TIF and in plasma, and a significant suppression of beta end-LI levels in TIF as early as 2 days after analog treatment. On the other hand, acute intratesticular injection of LHRH-A to intact rats (5 ng into the right testis) produced a short lived increase in beta end-LI in TIF 2 h after injection, coincident with a temporary decrease in TIF volume. These results indicate that the POMC-derived peptides ACTH and beta end are present in TIF and are secreted locally into this compartment, supporting previous reports demonstrating the presence and local synthesis of POMC peptides in testicular tissue.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3000743 TI - Regulation of pituitary gonadotropin-releasing hormone (GnRH) receptors by pulsatile GnRH in female rats: effects of estradiol and prolactin. AB - GnRH has been shown to modulate the concentration of its own pituitary receptors (GnRH-R), and changes in GnRH-R during the rat estrous cycle may reflect changes in GnRH secretion. To examine the relationship between GnRH and GnRH-R in female rats, we measured GnRH-R and serum gonadotropin responses to pulsatile GnRH in restrained ovariectomized (OVX) and ovariectomized estradiol-implanted (OVX-E2) rats. In addition, we examined the effects of suppression of serum PRL. Pulsatile injections of GnRH (10-250 ng/pulse) given every 30 min for 24 or 48 h did not increase GnRH-R in OVX or OVX-E2 rats compared to that in saline controls (246 +/ 27 fmol/mg). Bromocriptine treatment (2 mg/day) had no effect on GnRH-R in OVX animals. In contrast, OVX-E2 rats treated with bromocriptine showed significantly increased GnRH-R (500 +/- 43 fmol/mg) in response to GnRH injections. When ovine PRL was administered to bromocriptine-treated OVX-E2 rats, the GnRH induced rise in GnRH-R was abolished. Gonadotropin responses to GnRH were not correlated with changes in GnRH-R. In OVX animals, LH was only elevated in response to 250-ng pulses of GnRH. In OVX-E2 animals, basal LH was increased by all doses of GnRH, and acute responses to 50- and 250-ng pulses were observed. Bromocriptine treatment resulted in increased LH sensitivity to GnRH in OVX rats, but did not further enhance the responses in OVX-E2 animals. We conclude that in female rats, the presence of both E2 and a low serum PRL level is necessary for GnRH to increase GnRH-R, and the interaction of these factors may be involved in the regulation of GnRH-R during the estrous cycle. PMID- 3000744 TI - Regulation of pituitary gonadotropin-releasing hormone receptors by androgens in the male rabbit. AB - The regulation of pituitary GnRH receptors was studied in adult male rabbits after castration and androgen replacement with testosterone (T) or 7 alpha-methyl 19-nortestosterone acetate (U-15,614; T analog) supplied by Silastic capsules implanted sc. Castration increased pituitary GnRH receptors significantly, from 99.3 to 329.5 fmol/mg protein within 4 weeks, without a change in the equilibrium association constant. Serum LH concentrations increased from 0.45 to maximum levels of 2.6 ng/ml by day 8 after orchiectomy; these levels persisted throughout the 4 weeks of study. Serum FSH reached maximum levels of 33.6 ng/ml 5 days after castration. T replacement with 250, 500, and 1000 micrograms/kg X day, prevented a postcastration rise in both pituitary GnRH receptor concentrations and gonadotropin secretion, while 100 micrograms/kg X day prevented an increase in GnRH receptors, but did not completely inhibit hypersecretion of gonadotropins. Administration of T analog at doses of 6.25 and 12.5 micrograms/kg X day partially suppressed the castration-induced increase in pituitary GnRH receptor concentrations, while 25, 50, and 100 micrograms/kg X day suppressed GnRH-binding sites to the levels found in intact controls in 15 of 16 rabbits. By contrast, none of the T analog doses was able to prevent completely LH and FSH hypersecretion. The fact that both T and T analog induced dose-dependent stimulation of prostate and seminal vesicle weights indicates that there are tissue-specific differences in the sensitivity to androgens. We conclude that in the male rabbit 1) pituitary GnRH receptors significantly increase after castration; 2) this increase may partially mediate the postcastration hypersecretion of LH and FSH; 3) castration-induced effects can be prevented by androgen replacement. These results are similar to those obtained in rats, where castration increases LHRH receptors, but contrast with results in mice and hamsters, where castration either reduces or does not change receptor levels. This indicates significant species differences in the response of pituitary GnRH receptor concentrations to elimination of the negative feedback effects of androgens. PMID- 3000745 TI - Developmental changes in the responsiveness of rat kidney to vitamin D metabolites. AB - Kidneys from both normal and vitamin D-deficient rats were found to show changes in responsiveness to vitamin D metabolites during postnatal development, correlated with the concentrations of the specific receptor for 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] or the specific binding protein for 24R,25 dihydroxyvitamin D3 [24,25(OH)2D3]. Cytosol preparations from kidneys of vitamin D-deficient rats, in the second week of life, contained specific binding proteins for 24,25-(OH)2D3. From the fourth week of life, specific receptors for 1,25(OH)2D3 were predominant. In the third week after birth, both the receptor for 1,25(OH)2D3 and the 24,25(OH)2D3 binding protein were present. We have used a sensitive parameter for vitamin D action, the stimulation of creatine kinase BB (CKBB) activity, to measure the response of kidneys from vitamin D-deficient or normal rats. In the first days of life of vitamin D-deficient rats, the kidneys did not respond to either vitamin D metabolite; in the second week of life, there was stimulation of renal CKBB only by 24R,25(OH)2D3; beginning in the fourth week of life, only 1,25(OH)2D3 stimulated renal CKBB. However, during the third week of life, CKBB activity was increased by both metabolites. In normal animals, which showed a lower CK activity at all ages, the response was similar to that in vitamin D-deficient animals but the peak was achieved a few days later. The stimulation of CKBB by vitamin D metabolites occurred in all the zones of the kidneys. An increase in renal CKBB by 1,25(OH)2D3 was also detected immunohistochemically. The increase of CKBB activity caused by the two vitamin D metabolites at different stages of development, closely correlated with changes in the presence of the 1,25(OH)2D3 receptor or the 24,25(OH)2D3 binding protein, suggests a specific role for each metabolite during renal development. PMID- 3000747 TI - Inhibitory effect of adrenocorticotropin on corticotropin- releasing factor release from rat hypothalamus in vitro. AB - Effects of ACTH and ACTH fragments on immunoreactive corticotropin-releasing factor (I-CRF) release were examined by utilizing rat hypothalamic perifusion system and a rat CRF RIA. ACTH-(1-39) had a dose-related inhibitory effect on I CRF release. Mean percent inhibition of I-CRF release was 52, 55, 49, 30 and less than 5 percent by ACTH-(1-39), ACTH-(1-24), alpha-MSH and ACTH-(18-39) at 2.2 nM concentrations, respectively. These results suggest the presence of a negative short-loop feedback mechanism, and also that the active core is contained within the ACTH-(1-17) structure. PMID- 3000746 TI - Low serum somatomedin-C in insulin-dependent diabetes: evidence for a postreceptor mechanism. AB - In insulin-dependent diabetic rats, plasma somatomedin (Sm) levels are low and are not corrected by GH treatment, suggesting GH resistance. To define the mechanism of this GH-resistant state, the number and affinity constant of bovine liver GH-binding sites and the serum Sm-C responses to injections of bovine GH were determined in control (diluent-injected) and diabetic (streptozotocin injected; 40 mg/kg BW) hypophysectomized rats. The affinity constants (Ka) of the GH-binding sites of control (0.92 +/- 0.07 X 10(9) M-1) and diabetic animals (0.68 +/- 0.04 X 10(9) M-1) were not significantly different (P less than 0.1). Likewise, there were no significant differences in the liver GH-binding capacities between control and diabetic hypophysectomized rats, whether these capacities were expressed as picomoles per liver (26.99 +/- 3.43 vs. 22.27 +/- 2.55, controls vs. diabetics), picomoles per mg DNA (1.26 +/- 0.15 vs. 1.10 +/- 0.12), or femtomoles per mg protein (30.95 +/- 4.08 vs. 29.98 +/- 2.70). Despite the absence of alterations in liver GH-binding sites, the Sm-C responses 24 h after sc injections of graded doses of bovine GH were severely blunted in the diabetic animals. The maximal serum Sm-C response in the controls was 0.81 +/- 0.12 U/ml, but was only 0.09 +/- 0.01 U/ml in the diabetics (P less than 0.01). The dose of GH required to achieve the half-maximal Sm-C response (ED50) was similar in diabetic and nondiabetic rats (700-900 micrograms). The absence of significant alterations in liver GH binding and the decreased maximal serum Sm-C response without changes in the ED50 suggest that the GH-resistant state in insulin-dependent diabetes is due to a postreceptor defect. PMID- 3000748 TI - Glucocorticoid modulation of corticotropin-releasing factor desensitization in cultured rat anterior pituitary cells. AB - The ability of ovine corticotropin-releasing factor (CRF) to stimulate both ACTH release and intracellular cAMP accumulation is rapidly and reversibly desensitized when rat anterior pituitary cells are cultured in the absence of added glucocorticoids. Since this desensitization has not been readily apparent in vivo, where initial CRF exposure results in high levels of ambient glucocorticoids, we examined the effect of glucocorticoids on the desensitization process in vitro. Rat anterior pituitary cells were cultured for 3-5 days in the absence of added steroid hormones. Dexamethasone was then added to some culture wells 24 h before the desensitization experiment began. Desensitization of CRF was achieved by preincubating the cells for 4 h with varying concentrations (10( 11)-10(-7) M) of ovine CRF, washing the cells with medium alone, and then reexposing the cells to CRF. In the absence of glucocorticoid, the ED50 for CRF desensitization (the preincubation dose causing 50% desensitization of subsequent ACTH release) was 3 X 10(-10) M, but cells that had been preexposed to dexamethasone desensitized less readily. With concentrations of dexamethasone of 10(-8) M or greater, no desensitization occurred. When cells were incubated in the absence of added glucocorticoids, CRF-stimulated intracellular cAMP accumulation was diminished by prior exposure to CRF. No decrease in intracellular cAMP accumulation was seen in those cells that had been preincubated with dexamethasone, however. Similar changes in CRF desensitization of ACTH release were observed when cells were incubated with corticosterone, but not with 10(-8) M testosterone, progesterone, aldosterone, or estradiol. These data demonstrate that glucocorticoids profoundly alter the development of CRF desensitization in vitro and suggest that high ambient glucocorticoid concentrations prevent the development of substantial CRF desensitization in vivo. PMID- 3000749 TI - Androstenedione metabolism in the late gestation sheep fetus. AB - We have determined metabolic parameters for androstenedione (A) in chronically catheterized late gestation (day 130) sheep fetuses. The MCR (MCRA) was 3210 +/- 229 (SEM, n = 12) ml/min, the fetal arterial whole blood concentration of A [A] was 65 +/- 5 pg/ml, and the blood production rate (PRA) was 204 +/- 20 ng/min. Pulsatile administration of ACTH in amounts that raised fetal arterial plasma cortisol concentrations by 5- to 7-fold increased [A] to 154 +/- 20 pg/ml and PRA to 471 +/- 31 ng/min with no change in MCRA. In the presence of metopirone to block fetal adrenal cortisol output, ACTH treatment still provoked elevations in [A] (to 198 +/- 23 pg/ml) and PRA (539 +/- 158 ng/min), without altering MCRA. The major radiolabeled product in blood of infused [3H]A was [3H]testosterone; smaller amounts of phenolic steroids were formed. Extensive metabolism of [3H]A occurred in whole blood in vitro. The major product was [3H]testosterone; the 17 oxidoreductase activity was associated with the red blood cells. Umbilical vein [A] was greater than umbilical artery [A]; ACTH treatment increased [A] in both vessels. Concomitant metopirone abolished the arteriovenous difference by eliminating the ACTH-induced increase in venous [A], although arterial [A] rose significantly. The venous [A] and the arteriovenous gradient were restored with exogenous glucocorticoid treatment to the fetus. Collagenase-dispersed fetal adrenal cells secreted A. Adrenal cells from fetuses pretreated with ACTH in vivo had higher basal and ACTH-induced output of A in vitro than cells from fetuses pretreated with saline in vivo. We conclude that the MCRA in fetal sheep is extremely high, in part due to conversion of A to testosterone in fetal blood. The elevated PRA after ACTH plus metopirone and the lack of an umbilical arteriovenous gradient of [A] in this, but not other groups of fetuses, suggests a source of A production independent of the cortisol-induced changes in the placenta. Direct evidence is provided for fetal adrenal secretion of A which is enhanced by ACTH pretreatment of the fetus in vivo and for the utilization of circulating A in the fetus as a precursor for estrogen in both fetal and maternal compartments. PMID- 3000750 TI - Differential responsiveness of the somatotroph to growth hormone-releasing factor during early neonatal development in the rat. AB - The effects of human pancreatic GH-releasing factor-40 (hpGRF-40; 0.01-100 nM) and (Bu)2cAMP (0.015-1.5 mM) on GH release from primary monolayer cultures of pituitary cells were evaluated in rats of three age groups: postnatal days 2 and 12, and young adult males (3-4 months). Both hpGRF-40 and (Bu)2cAMP elicited a dose-related increase in GH release in cell cultures from each age group. However, the magnitude of the fractional increase over basal release was markedly age dependent. hpGRF-40-stimulated GH release (expressed as a percentage of control values) was greater in cultured cells of 2-day-old than of 12-day-old rats, which was, in turn, significantly greater than in cells of adult rats (P less than 0.001). Maximum hpGRF-40-stimulated GH release was 1058 +/- 50% of control values (+/- SE) in 2-day-old, 617 +/- 21% of control values in 12-day old, and 405 +/- 6% of control values in adult pituitary cell cultures. The slopes of the dose-response curves differed significantly among the three age groups (P less than 0.001) and varied inversely with increasing age. GH release induced by (Bu)2cAMP was similarly age dependent; maximal stimulated release was 1073 +/- 20%, 414 +/- 4%, and 259 +/- 7% of control values in cultured cells of 2 day-old, 12-day-old, and adult rats, respectively (P less than 0.001 for age effect at each dose). As with hpGRF-40, the slopes of the dose-response curves for (Bu)2cAMP decreased with advancing age (P less than 0.001). Intracellular GH storage during culture, basal release of GH, and serum GH were also age dependent. Pooled serum GH was consistently elevated in 2-day-old rats (139 +/- 2 ng ml-1), became lower and more variable in 12-day-old rats (62 +/- 14 ng ml-1), and was even more variable in adult male rats (79 +/- 23 ng ml-1), owing to random sampling during spontaneous secretory pulses. These results indicate that the stimulatory effects of GRF and (Bu)2cAMP on GH secretion from cultured rat pituitaries vary with age; pituitary cells of newborn rats are relatively more sensitive to these secretagogues than those of adult rats. This increased responsiveness of the neonatal somatotroph to GRF may contribute to the elevation of the plasma GH concentration which is characteristic of the perinatal period in the rat. PMID- 3000751 TI - Evaluation of the pituitary--adrenal function in patients with anorexia nervosa. PMID- 3000752 TI - The effect of hormones on erythrocyte acid phosphatase after cortisol treatment. PMID- 3000753 TI - [Corticoliberin--structure, occurrence, properties and clinical use]. PMID- 3000755 TI - Steroid hormone receptors and the nucleus. PMID- 3000754 TI - Evidence that 1,25-dihydroxyvitamin D3 is the physiologically active metabolite of vitamin D3. PMID- 3000756 TI - Local regulators of skeletal growth: a perspective. PMID- 3000758 TI - cAMP dependent actin phosphorylation in developing rat lung and type II epithelial cells. AB - Cyclic adenosine monophosphate (cAMP) increased in vitro phosphorylation of protein of 43,000 daltons in cytosolic fractions of rat lung and type II epithelial cells. The phosphoprotein was identified as 32P-actin by means of migration in one- and two-dimensional SDS-polyacrylamide gel electrophoresis and by phosphopeptide mapping followed by immunoperoxidase staining of peptides with anti-actin monoclonal antibody. Phosphorylation of actin in lung and type II cell cytosol was entirely cAMP dependent and the phosphorylated amino acid was identified as 32P-serine. Actin phosphorylation increased during the perinatal period of development, was barely detectable between 17 and 20 days gestation, increased prior to birth, and increased dramatically during the first week of life. Actin was the major substrate of cAMP-dependent protein kinase in lung cytosol from postnatal rats. Changes in actin phosphorylation that occur during development were not due to changes in cytosolic actin content or cAMP-dependent protein kinase activity but appeared to be related to the presence of factors inhibiting cAMP-dependent actin phosphorylation in fetal lung cytosol. Actin was also the major cAMP-dependent phosphoprotein identified in cytosolic fractions of purified type II epithelial cells. cAMP-dependent phosphorylation of pulmonary actin is developmentally regulated, occurring in association with other aspects of type II epithelial cell maturation during the perinatal period. PMID- 3000757 TI - Pulmonary macrophages: alveolar and interstitial populations. AB - The sizes of the alveolar macrophage (AM) and interstitial macrophage (IM) populations in the lungs of adult Fischer-344 rats were determined during steady state. AM labeled with opsonized erythrocytes during an in situ phagocytic assay were lavaged from excised lungs. The lungs were then dispersed into single-cell suspensions with collagenase and mechanical agitation, and the remaining mononuclear phagocytes were identified following a second labeling step. The size of the AM population was 1.3 X 10(7) cells, or approximately equal to 3% of the total lung cell population. The AM were negative for cytoplasmic myeloperoxidase granules. The size of the IM population was 8 X 10(6) cells, or approximately equal to 2% of the total lung cell population. IM were also negative for myeloperoxidase and, like AM, demonstrated marked Fc gamma R-mediated phagocytic activity. The high cell yields (approximately equal to 4 X 10(8) cells/lung; viability, greater than 85%) and the percentages of type II cells (11%) and ciliated epithelial cells (less than 0.5%) indicated the enzymatic dispersion method resulted in a highly efficient and representative sampling of the lung parenchyma. The collagenase method used in this study to disperse the lung cells into single-cell suspensions, in conjunction with additional cell separation techniques, may be of potential use for isolating enriched populations of IM, as well as other lung cell types, for in vitro study. PMID- 3000760 TI - Aminophylline and CGS 8216 reverse the protective action of diazepam against electroconvulsions in mice. AB - Aminophylline (50 and 100 mg/kg) and CGS 8216 (20 and 40 mg/kg) decreased the anticonvulsant potency of diazepam (5 and 10 mg/kg) against electroshock-induced seizures. It should be emphasized that aminophylline moderately affected the protective action of the benzodiazepine at a dose of 5 mg/kg, whereas it was equipotent with CGS 8216 with regard to diazepam at a dose of 10 mg/kg. Consequently, participation of a purinergic component in the anticonvulsant action of diazepam is suggested. On the other hand, the use of aminophylline in epileptic patients suffering from asthma seems unjustified. PMID- 3000759 TI - Comparison of progabide with other antiepileptic and GABAergic drugs. AB - The effect of the experimental antiepileptic gamma-aminobutyric acid (GABA) agonist drug progabide, [alpha-(chloro-4-phenyl)fluor-5-hydroxy-2 benzilideneamino]-4-buty ramide, on the trigeminal complex of cats was compared with the effect of established antiepileptic drugs and with the effect of various GABA agonists and antagonists. Intravenous administration of 10-40 mg/kg progabide depressed excitatory transmission and descending periventricular inhibition, similar to carbamazepine and phenytoin. However, progabide depressed, rather than facilitated, segmental inhibition. The serum levels of progabide were comparable with those in patients receiving long-term treatment with progabide. The GABA antagonist bicuculline had the opposite effect of progabide on our experimental model, but the other GABA agonists THIP (4,5,6,7-tetrahydroisoxazolo 5,4-C-pyridine-3-ol) and muscimol did not have the same effects as progabide. THIP had no effect on excitatory transmission, periventricular inhibition, or segmental inhibition, whereas muscimol facilitated periventricular inhibition and sometimes segmental inhibition and had no effect on excitatory transmission. Our experiments thus indicate that progabide, but not THIP or muscimol, should have antiepileptic properties, in agreement with the clinical experiences that have been reported. The reason for the differential effect of these three GABA agonists remains to be elucidated. PMID- 3000762 TI - Sequences of papillomavirus DNA in equine sarcoids. AB - DNA was extracted from 14 equine sarcoids, electrophoresed and hybridised with a radioactively labelled probe of bovine papillomavirus type I (BPV 1) DNA under conditions of low stringency. Twelve sarcoids contained sequences of DNA that hybridised with the probe and that comigrated with BPV 2 DNA. The viral DNAs in four of these sarcoids differed from BPV 1 and BPV 2 DNA on restriction endonuclease analysis. One of four cell lines derived from sarcoids also contained BPV 1 related DNA. The results confirm the frequent presence in equine sarcoids of unintegrated papillomaviral DNA and suggest a role for papillomavirus infection in this disease. PMID- 3000761 TI - Potential risks to human respiratory health from "acid fog": evidence from experimental studies of volunteers. AB - Observations of high acidity (pH as low as 1.7) in fogwater collected in polluted areas have provoked concern for public health. Effects of exposure to acidic pollutants have not been studied under foggy conditions; thus there is no directly relevant information from which to estimate the health risk. Indirectly relevant information is available from numerous studies of volunteers exposed to "acid fog precursors" under controlled conditions at less than 100% relative humidity. The effect of fog in modifying responses to inhaled acidic pollutants is difficult to predict: depending on circumstances, fog droplets might either increase or decrease the effective dose of pollutants to the lower respiratory tract. Fog inhalation per se may have unfavorable effects in some individuals. Sulfur dioxide is known to exacerbate airway constriction in exercising asthmatics, at exposure concentrations attainable in ambient air. Nitrogen dioxide has shown little untoward respiratory effect at ambient concentrations in most studies, although it has been suggested to increase bronchial reactivity. Sulfuric acid aerosol has shown no clear effects at concentrations within the ambient range. At somewhat higher levels, increased bronchial reactivity and change in mucociliary clearance have been suggested. Almost no information is available concerning nitric acid. PMID- 3000763 TI - Tissue-specific regulation of a chicken delta-crystallin gene in mouse cells: involvement of the 5' end region. AB - A cloned delta-crystallin gene of the chicken is preferentially expressed in lens cells after introduction into various mouse tissues. The level of expression in the lens epithelium is 20 times higher than in fibroblasts. Taking advantage of this system, we attempted to define regulatory regions of the delta-crystallin gene using a variety of deletion and substitution mutants. The results indicate that tissue-specific regulation of the delta-crystallin gene is mediated by the 5' end region of the gene; sequences upstream from -93 are not required for expression and sequences downstream from +58 are not involved in tissue specificity. The high expression in lens cells requires 5' flanking sequences of 80-bp long from the cap site, whereas the low expression in fibroblasts requires an additional 12 bp upstream sequence. Expression of both types is lost in a mutant with only 51 bp of the 5' flanking sequence. Thus, fine deletion analysis demonstrated that expression in lens cells and expression in fibroblasts are distinct not only in level but in regulation. PMID- 3000764 TI - Heterogeneity of T-cell beta-chain gene rearrangements in human leukaemias and lymphomas. AB - The state of T-cell receptor beta-chain gene rearrangement in human T-cell leukaemias has been analysed. All forms of leukaemia tested (T-CLL, ALL, PLL, Sezary syndrome and ATL) exhibit rearrangements of C beta genes confirming the clonality of these neoplasias. However we find no evidence for common gene rearrangements nor for restricted rearrangement patterns within this type of neoplasia. We find evidence of T-cells with C beta 1 and C beta 2 rearrangements, sometimes associated with Igh JH rearrangements, but several cases of T-cell leukaemia with a marker inversion of chromosome 14 (q11;q32) do not have Igh JH rearrangements. The results suggest that TCR beta gene rearrangement occurs early in T-cell ontogeny but that this rearrangement is most often irrelevant to leukaemogenesis. PMID- 3000765 TI - The human HLA class II alpha chain gene DZ alpha is distinct from genes in the DP, DQ and DR subregions. AB - A new human HLA class II alpha gene DZ alpha was sequenced. The structure and organisation of the gene was similar to other alpha chain genes except for a particularly small intron (95 bp) after the exon encoding the alpha 2 domain, and the position of the stop codon, which was on a different exon to that encoding the cytoplasmic portion of the molecule. Comparison of the DZ alpha sequence with other class II genes showed that the gene is about as distantly related to alpha chain genes in the DP, DQ and DR subregions as they are to each other. The DZ alpha gene results in an unusually large mRNA transcript of greater than 3.0 kb, detected on Northern blots of B cell lines. From the sequence, there are no obvious features that would render DZ alpha a pseudogene, except for an unusual poly(A)+ addition signal, ACTAAA. Analysis of Northern blots shows that sequences downstream (3') of this signal are present in mature mRNA. The large transcripts are probably due to defects in the signals for processing of the mRNA transcript at the 3' end. PMID- 3000766 TI - Human proto-oncogene c-mos maps to 8q11. AB - The c-mos proto-oncogene is the cellular counterpart of the viral oncogene v-mos isolated from Moloney murine sarcoma virus. The c-mos gene locus has previously been assigned to human chromosome 8. By both in situ hybridization and molecular hydridization to sorted chromosome DNA (using a c-mos probe) we have localized the c-mos gene to band 8q11. This regional localization is at variance with the one previously reported at 8q22 and may explain why no rearrangement of c-mos has been found in acute leukaemia with the chromosomal translocation t(8;21)(q22;q22). PMID- 3000767 TI - A linkage map of three anonymous human DNA fragments and SOD-1 on chromosome 21. AB - Using DNA polymorphisms adjacent to single-copy genomic fragments derived from human chromosome 21, we initiated the construction of a linkage map of human chromosome 21. The probes were genomic EcoRI fragments pW228C, pW236B, pW231C and a portion of the superoxide dismutase gene (SOD-1). DNA polymorphisms adjacent to each of the probes were used as markers in informative families to perform classical linkage analysis. No crossing-over was observed between the polymorphic sites adjacent to genomic fragments pW228C and pW236B in 31 chances for recombination. Therefore, these fragments are closely linked to one another (theta = 0.00, lod score = 6.91, 95% confidence limits = 0-10 cM) and can be treated as one 'locus' with four high-frequency markers. There is a high degree of non-random association of markers adjacent to each of these two probes which suggests that they are physically very close to one another in the genome. The pW228C - pW236B 'locus' was also linked to the SOD-1 gene (theta = 0.07, lod score = 4.33, 95% confidence limits = 1-20 cM). On the other hand, no evidence for linkage was found between the pW228C-pW236B 'locus' and the genomic fragment pW231C (theta = 0.5, lod score = 0.00). Based on the fact that pW231C maps to 21q22.3 and SOD-1 to 21q22.1, we suggest that the pW228C-pW236B 'locus' lies in the proximal long arm of chromosome 21. These data provide the outline of a linkage map for the long arm of chromosome 21, and indicate that the pW228C pW236B 'locus' is a useful marker system to differentiate various chromosome 21s in a population. PMID- 3000768 TI - Visualization of RNA polymerase II ternary transcription complexes formed in vitro on a Xenopus laevis vitellogenin gene. AB - Stable ternary transcription complexes assembled in vitro, using a HeLa whole cell extract, have been isolated and visualized by electron microscopy. The formation of these stable complexes on the DNA fragment used as template, the 5' end region of the Xenopus laevis vitellogenin gene B2, depends on factors present in the whole-cell extract, RNA polymerase II and at least two nucleotides. Interestingly, bending in the DNA fragment was frequently observed at the binding site of RNA polymerase II. Dinucleotides that can prime initiation within a short sequence of approximately 10 contiguous nucleotides centered around the initiation site used in vivo, also favour the formation of stable complexes. In addition, pre-initiation complexes were isolated and it was shown that factors in the extract involved in their formation are more abundant than the RNA polymerase II molecules available for binding. The possible implication of this observation relative to the in vivo situation is discussed. PMID- 3000769 TI - Bombyx mori 28S ribosomal genes contain insertion elements similar to the Type I and II elements of Drosophila melanogaster. AB - We have examined the 28S ribosomal genes of the silkmoth, Bombyx mori, for the presence of insertion sequences. Two types of insertion sequences were found, each approximately 5 kb in length, which do not share sequence homology. Comparison of the nucleotide sequences of the junction regions with the uninserted gene reveals that one type of insertion has resulted in a 14 bp duplication of the 28S coding region at the insertion site. The location of this insertion and the 14 bp duplication are identical to that found in the Type I ribosomal insertion element of Drosophila melanogaster. The second type of insertion element is located at a site corresponding to approximately 75 bp upstream of the first type. The location of this insertion, the variability detected at its 5' junction, and a short region of sequence homology at its 3' junction suggest that it is related to the Type II element of D. melanogaster. This is the first example of a Type II-like rDNA insertion outside of sibling species of D. melanogaster, and the first example of a Type I-like rDNA insertion outside of the higher Diptera. PMID- 3000770 TI - Sequence of gene malG in E. coli K12: homologies between integral membrane components from binding protein-dependent transport systems. AB - The MalG protein is needed for the transport of maltose in Escherichia coli K12. We present the sequence of gene malG. The deduced amino acid sequence corresponds to a protein of 296 amino acid residues (mol. wt. = 32 188 daltons). This protein is largely hydrophobic (hydrophobic index = 0.83) and is thus presumably an integral inner membrane protein which could span the membrane through six hydrophobic segments. We provide direct evidence from fusion proteins for the translation frame and we also identified the in vitro made MalG protein. We have found a sequence which is highly conserved between MalG and MalF, the other integral inner membrane protein of the maltose transport system. This conserved sequence is also present in all known integral membrane proteins of binding protein-dependent transport systems, always at the same distance (approximately 90 residues) from their COOH terminus. We discuss briefly this finding. PMID- 3000771 TI - A single amino acid alteration in the initiation protein is responsible for the DNA overproduction phenotype of copy number mutants of plasmid R6K. AB - A novel type of high copy-number (cop) mutants of a mini-R6K plasmid were isolated. The mutations were mapped in the pir gene which encodes the pi initiation protein for plasmid R6K DNA replication. They resulted in an alteration by substitution of a single amino acid: threonine to isoleucine at the 108th position for the cop41, and proline to serine at the 113th position for the cop50, of the 305 amino acid pi protein. The cop41 mutation in the pi protein was found to be trans-dominant over the wild-type allele in the copy control of plasmid R6K. Moreover, it was shown that the altered pi protein was not overproduced in maxicells carrying this mutant plasmid and had a higher affinity to the repeated sequence which is present in the pir promoter region. Most likely the mutated pi protein also interacts more efficiently with the same repeated sequences, a target of pi, in the replication origin region and increases the frequency of the initiation event per cell division. PMID- 3000773 TI - The receptor specificity of bacteriophages can be determined by a tail fiber modifying protein. AB - T-Even type bacteriophages recognize their cellular receptors with the distal ends of their long tail fibers. The distal part of these fibers consists of a dimer of gene product (gp) 37. The assembly of this gp to a functional dimer requires the action of two other proteins, gp57 and gp38. Genes (g) 38 have been cloned from five T-even type phages which use the Escherichia coli outer membrane protein OmpA as a receptor. The phages used differ in their ability to infect a series of ompA mutants producing altered OmpA proteins, i.e., each phage has a specific host range for these mutants. The cloned genes 38 complemented g38 amber mutants of phage T2, which uses the outer membrane protein OmpF as a receptor. The complemented phages had become phenotypically OmpA-dependent and, with one exception, OmpF-independent, but regained the host range of T2 upon growth in a host lacking the cloned g38. The host range of the complemented phages, as determined on the ompA mutants, was identical to, similar to, or different from that of the phage, from which the cloned g38 originated. The results presented show that gp38 from one phage can phenotypically 'imprint', in a finely-tuned manner, a host range onto gp37 of another phage with a different host specificity. In view of the extreme diversity of host ranges observed, it is suggested that gp38 of T2 and of the OmpA-specific phages may remain attached to gp37 in the phage particle and in cooperation with gp37 determine the host range. PMID- 3000774 TI - Mechanism of control of cytochrome oxidase activity by the electrochemical potential gradient. AB - Cytochrome c oxidation by bovine cytochrome oxidase embedded into liposomal vesicles with high respiratory control ratio (RCR = 6-10) has been studied by rapid-mixing experiments in the presence and absence of different ionophores. Kinetic analysis of the reaction indicates a linkage between the intrinsic activity of the enzyme, the efficiency of coupling and the electrochemical potential across the membrane. A simple model, based on two allosteric states with different catalytic properties in rapid equilibrium, is presented and successfully applied in the simulation of the observed time-course. PMID- 3000775 TI - The uncoupling protein from brown fat mitochondria is related to the mitochondrial ADP/ATP carrier. Analysis of sequence homologies and of folding of the protein in the membrane. AB - We report here, for the first time, the primary structure of uncoupling protein as established by amino acid sequencing. Like the ADP/ATP carrier, this protein has a tripartite structure comprising three similar sequences of approximately 100 residues each. These six 'repeats' exhibit striking conservation of several residues, in particular glycine and proline, at possible structurally strategic positions. Although the two proteins differ strongly in their amino acid composition, their sequences are distantly homologous. Three membrane-spanning alpha-helices can be deduced from hydropathy plots. A modified plot accounting for amphiphilic helices indicates 5-6 such alpha-segments. In addition an amphiphilic beta-strand of membrane-spanning length can be discerned. The tripartite sequence structure is also distinctly reflected in the hydropathy distribution. Based on the membrane disposition of the segments of the ADP/ATP carrier, a model for the transmembrane folding path of the polypeptide chain of the uncoupling protein is proposed. PMID- 3000776 TI - Activation of glycolysis by insulin with a sequential increase of the 6 phosphofructo-2-kinase activity, fructose-2,6-bisphosphate level and pyruvate kinase activity in cultured rat hepatocytes. AB - The involvement of 6-phosphofructo-2-kinase, fructose 2,6-bisphosphate [Fru(2,6)P2] and pyruvate kinase in the insulin-dependent short-term activation of glycolysis was studied in primary cultures of rat hepatocytes. The short-term influence of insulin on these parameters was dependent on the insulin concentration used for the long-term culture. Cells were cultured either with 10 nM or 0.1 nM insulin for 48 h, and are referred to as 'insulin cells' and 'control cells', respectively. Insulin cells exhibited a high level of Fru(2,6)P2. Addition of insulin to insulin cells led to an immediate stimulation of glycolysis (two-fold) and activation of pyruvate kinase. The concentration of Fru(2,6)P2 and activity of 6-phosphofructo-2-kinase remained constant. Control cells exhibited a very low level of Fru(2,6)P2 and low activity of 6 phosphofructo-2-kinase directly after the medium change. However, both parameters increased during a 1-2-h incubation in the absence of insulin. Although the level of Fru(2,6)P2 thus changed up to tenfold the glycolytic rate remained at a constant value. Addition of insulin to control cells led to a 5-8-fold stimulation of glycolysis but only after a 30-90-min lag phase. During this lag period insulin strongly increased sequentially the 6-phosphofructo-2-kinase, the level of Fru(2,6)P2 and the pyruvate kinase activity. The activation of the latter enzyme slightly preceded the onset of the insulin-stimulated glycolysis. Addition of insulin to control cells, which were preincubated for 3 h in the absence of insulin and in which the Fru(2,6)P2 level had risen insulin independently, led to an immediate increase in glycolysis without a lag phase. It is concluded that in this insulin-sensitive cell system: the changes of glycolytic flux did not correlate with changes in the level of total Fru(2,6)P2 either in insulin or in control cells; an increase in the Fru(2,6)P2 concentration was not obligatory for the insulin-dependent stimulation of glycolysis in insulin cells; activation of pyruvate kinase and thus glycolysis by insulin did not proceed unless the Fru(2,6)P2 level had been elevated above a threshold level. The lack of correlation between total Fru(2,6)P2 levels and the glycolytic flux and the apparent existence of a threshold concentration for Fru(2,6)P2 suggest a permissive action for this effector in enzyme interconversion. PMID- 3000778 TI - Nitrous oxide reductase from denitrifying Pseudomonas perfectomarina. Purification and properties of a novel multicopper enzyme. AB - Nitrous oxide reductase from the denitrifying bacterium Pseudomonas perfectomarina has been isolated and purified to homogeneity. The enzyme contained about eight copper atoms/120 kDa and was composed of two presumably identical subunits. The isoelectric point was 5.1. Several spectroscopically distinct forms of the enzyme were identified. A 'pink' form of the enzyme was obtained when the purification was done aerobically. The specific activity of this species was around 30 nkat/mg protein as measured by the nitrous-oxide dependent oxidation of photochemically reduced benzyl viologen. A 'purple' form of the enzyme, whose catalytic activity was 2-5-fold higher, was obtained when the purification was done anaerobically. The activity of both forms of the enzyme was substantially increased by dialyzing the protein against 2-(N cyclohexylamino)ethanesulfonate buffer at pH approximately equal to 10. A maximal activity of 1000 nkat/mg protein has been obtained for the purple form using this procedure. A 'blue', enzymatically inactive form of the enzyme resulted when either the pink or the purple species was exposed to excess dithionite or ascorbate. Anaerobic, potentiometric titrations of both the purple and the pink form of the enzyme gave a Nernst factor, n540, of 0.95 and a midpoint potential, E'0,540 of +260 mV (vs SHE, 25 degrees C, Tris/HCl buffer, pH 7.5). Electron paramagnetic resonance (EPR) and optical spectra of N2O reductase suggested the presence of an unusual type 1 copper center. Type 2 copper was absent. The hyperfine splitting in the g parallel region consisted of a seven-line pattern. In the presence of excess of reductant, a broad EPR signal with g values at 2.18 and 2.06 was observed. The EPR spectra of the pink and purple forms of the enzyme were similar; however, the spectrum of the purple form was better resolved with g parallel = 2.18 (A parallel = 3.83 mT) and g perpendicular = 2.03 (A perpendicular = 2.8 mT). Most of the copper in N2O reductase was removed by anaerobic dialysis against KCN. Reaction of the apoprotein with Cu(en)2SO4 partially regenerated the optical and EPR spectra of the holoprotein; the resulting protein was enzymatically inactive. Monospecific antibodies against the copper protein strongly inhibited the N2O reductase activity of purified samples and cell-free extracts. PMID- 3000777 TI - Modification of nuclear matrix proteins by ADP-ribosylation. Association of nuclear ADP-ribosyltransferase with the nuclear matrix. AB - Nuclear matrices were isolated by treatment of isolated HeLa cell nuclei with high DNase I, pancreatic RNase and salt concentrations. ADP-ribosylated nuclear matrix proteins were identified by electrophoresis, blotting and autoradiography. In one experimental approach nuclear matrix proteins were labeled by exposure of permeabilized cells to the labeled precursor [32P]NAD. Alternatively, the cellular proteins were prelabeled with [35S]methionine and the ADP-ribosylated nuclear matrix proteins separated by aminophenyl boronate column chromatography. By both methods bands of modified proteins, though with differing intensities, were detected at 41, 43, 46, 51, 60, 64, 69, 73, 116, 140, 220 and 300 kDa. Approximately 2% of the total nuclear ADP-ribosyltransferase activity, but only 0.07% of the nuclear DNA, was tightly associated with the isolated nuclear matrix. The matrix-associated enzyme catalyzes the incorporation of [32P]ADP ribose into acid-insoluble products of molecular mass 116 kDa and above, in a 3 aminobenzamide-inhibited, time-dependent reaction. The possible function of ADP ribosylation of nuclear matrix proteins and of the attachment of ADP ribosyltransferase to the nuclear matrix in the regulation of matrix-associated biochemical processes is discussed. PMID- 3000772 TI - The release of growth arrest by microinjection of adenovirus E1A DNA. AB - The induction of DNA synthesis in growth-arrested mouse fibroblasts (NIH 3T3) was studied by microinjection of different constructs of adenovirus DNA using SV40 DNA and plasmid DNA as positive and negative controls. The E1A region of adenovirus types 2 and 12 appears to be sufficient to induce cellular DNA synthesis after growth arrest in approximately 30% of the cells and both 13S and 12S cDNA constructs mediate this effect. The presence of the E1A protein products as assayed by immunofluorescence does not strictly correlate with the induction of DNA synthesis in microinjected cells in contrast to the SV40 large T-antigen. Microinjection of truncated fragments of the Ad12 E1A region suggests, however, that the protein products of 12S and 13S may be involved in the induction process. A sequence comparison of the SV40 T-antigen and the adenovirus E1A products identified a region of significant homology providing a basis for a hypothesis concerning the evolution of T-antigen genes in DNA viruses. PMID- 3000779 TI - A novel, highly phosphorylated protein, of the high-mobility group type, present in a variety of proliferating and non-proliferating mammalian cells. AB - The present work describes a perchloric-acid-soluble high-mobility-group (HMG) like protein present in HeLa and Ehrlich ascites cells, rat and calf liver. The protein is designated P1 and has, depending on the source, a molecular mass 48-53 kDa and an amino acid composition which, like the HMG proteins, is characterized by a high content of acidic and basic residues and of proline. The protein contains about 10 mol serine/100 mol amino acid residues, is highly phosphorylated and has, in contrast to the known HMG proteins, an acidic isoelectric point of 5.0. An estimate suggests that protein P1 in HeLa interphase cells contains 25-30 residues of phosphate. Like HMG 1 and 2 it is distributed between the nucleus and the cytoplasm. In HeLa metaphase cells P1 is further modified, resulting in an increase in apparent molecular mass from 53 kDa to 56 kDa. PMID- 3000781 TI - Comparison of asymmetric forms of acetylcholinesterase from the electric organ of Narke japonica and Torpedo californica. AB - The asymmetric forms of acetylcholinesterase were purified from the electric organs of the electric rays Narke japonica and Torpedo californica, and their properties were compared. Asymmetric acetylcholinesterase was purified by immunoaffinity chromatography with a monoclonal antibody (Nj-601) to acetylcholinesterase. The MgCl2 extracts of these electric organs were applied to a column of Nj-601-Sepharose, and the bound acetylcholinesterase was eluted by lowering the pH of the eluent to 2.8. The purified asymmetric acetylcholinesterases gave peaks of 17 S (A12) and 13 S (A8) on sucrose density gradients. The enzyme from N. japonica contained more A8 than A12, while that of T. californica contained more A12. After treatment with collagenase, the enzymes gave three peaks on sedimentation; 20 S, 16 S and 11 S for N. japonica, and 19 S, 15 S and 11 S for T. californica, indicating the presence of collagen-like tails. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the asymmetric acetylcholinesterase from N. japonica gave bands of Mr 140 000, 100 000, 70 000 and 60 000, while that from T. californica gave bands of Mr 140 000, 100 000, 70 000 and 55 000. The bands of Mr 70 000 and 140 000 were monomers and non reducible dimers, respectively, of the catalytic subunits. The bands of Mr 60 000 and 55 000 were the tail subunits, since collagenase treatment of the purified enzymes markedly decreased the amounts of these components. The Mr 100 000 subunit constituted less than 3% of the total asymmetric acetylcholinesterase from N. japonica but 18% of that from T. californica. The tail subunits constituted 6-8% of the two preparations. The catalytic subunits and the Mr 100 000 subunits bound concanavalin A, indicating that they are glycoproteins. The amino acid compositions of the enzymes from N. japonica and T. californica were very similar. Both contained hydroxyproline and hydroxylysine, characteristic of the collagen-like tails. The enzyme required divalent metal ions for activity, but only Mn2+, Mg2+ and Ca2+ were effective. Mn2+ was effective at the lowest concentrations, while Mg2+ gave the highest activity. PMID- 3000780 TI - Interaction of human alpha-thrombin and gamma-thrombin with antithrombin III, protein C and thrombomodulin. AB - Conversion of human alpha-thrombin to gamma-thrombin by limited proteolysis resulted in a decrease in the inactivation rate of the enzyme by antithrombin III. The second-order rate constants were similar but significantly different: 11 +/- 1.7 X 10(3) and 7 +/- 0.5 X 10(3) M-1 s-1 for alpha- and gamma-thrombin respectively. This difference is probably related to a slight change in reactivity of the catalytic site, rather than to a structural alteration of the recognition site for antithrombin III. The rate of protein C activation, measured in the absence of thrombomodulin, was greatly reduced by conversion of alpha thrombin to gamma-thrombin. In addition, gamma-thrombin failed to displace alpha thrombin from its complex with thrombomodulin, as demonstrated by measuring either the rate of protein C activation by thrombin-thrombomodulin, or the fibrinogen clotting activity of thrombin-thrombomodulin, in the presence of competing diisopropylphospho-thrombin. It is concluded that the recognition sites involved in protein-C-thrombin and thrombomodulin-thrombin interactions are both dramatically affected by the loss of peptide material occurring during the conversion of alpha-thrombin to gamma-thrombin and/or by the resulting conformational changes. PMID- 3000782 TI - Rat epidermal growth factor: complete amino acid sequence. Homology with the corresponding murine and human proteins; isolation of a form truncated at both ends with full in vitro biological activity. AB - Epidermal growth factor (EGF) isolated from the submaxillary gland of the rat (rEGF) is missing the COOH-terminal five residues present in both mouse and human EGF. rEGF competes for the binding of 125I-labelled mEGF to human carcinoma cells with the same affinity as mEGF. rEGF and mEGF have identical mitogenic activities on mouse 3T3 fibroblasts, thus the C-terminal region of the sequence is not necessary for the in vitro activity of EGF. Using reversed-phase high-performance liquid chromatography, four molecular forms of EGF have been extracted from rat submaxillary glands. These forms represent rEGF, rEGF(2-48), rEGF(3-48) and rEGF(4-48); all forms appear to be equipotent in both the receptor binding and mitogenic assays. The isoelectric points of these rEGFs are in the range of pH 5.1 to 5.2. The primary structure of rEGF was determined from approximately 10 micrograms protein by sequence analysis of the intact molecule and fragments obtained from the reduced and alkylated protein by chemical cleavage with CNBr and enzymic cleavage with chymotrypsin and a proline-specific endopeptidase. Subnanomole amounts of generated peptides were purified to homogeneity by reversed-phase microbore high-performance liquid chromatography and analysed by automated Edman degradation in a gas-phase sequencer. There are 48 amino acid residues in the complete polypeptide chain which lacks alanine, phenylalanine, lysine and tryptophan. The amino acid sequence of rat epidermal growth factor is: Asn-Ser-Asn-Thr-Gly-Cys-Pro-Pro-Ser-Tyr-Asp-Gly-Tyr-Cys-Leu-Asn- Gly-Gly-Val-Cys Met-Tyr-Val-Glu-Ser-Val-Asp-Arg-Tyr-Val-Cys-Asn-Cys -Val-Ile-Gly-Tyr-Ile-Gly-Glu Arg-Cys-Gln-His-Arg-Asp-Leu-Arg. The calculated relative molecular mass from the sequence analysis is 5377. PMID- 3000783 TI - Scintigraphic evidence that the right ventricular myocardium tolerates ischaemia better than the left ventricular myocardium. AB - To study the incidence of right ventricular infarction and the effect of intracoronary thrombolysis on the ischaemic right ventricular myocardium, we performed intracoronary myocardial thallium scintigraphy in 18 patients with complete occlusion of the right coronary artery who underwent intracoronary thrombolysis. In 15 of these patients, intracoronary thallium-201 and technetium 99 m pyrophosphate scintigrams were performed simultaneously. All 18 patients had a right ventricular thallium defect before thrombolysis, and all had new thallium uptake after thrombolysis. 17 out of 18 patients had a left ventricular thallium defect before thrombolysis, but only 10 of them showed new thallium uptake after thrombolysis. 14 out of 15 patients had a left ventricular technetium-99 m pyrophosphate spot after thrombolysis and some diffuse pyrophosphate accumulation in the area of the right ventricle. In one patient pyrophosphate accumulation was found only in the area of the right ventricle. Thus, right ventricular thallium defects were detected by intracoronary thallium scintigraphy in the majority of patients with inferior acute myocardial infarction due to right coronary artery occlusion. Right ventricular thallium defects were always reversible in contrast to left ventricular thallium defects in the same patients, suggesting that right ventricular myocardium tolerates ischaemia better than left ventricular myocardium. PMID- 3000785 TI - Spin label study of red blood cell membranes in Huntington's disease. AB - Huntington's disease (HD) is an inherited degenerative disorder of the central nervous system. Various studies of peripheral tissues from HD patients have led to the assumption that this disease could be the expression of a generalized membrane defect. We have been unable to reproduce fundamental results on which this theory is based, namely differences between electron spin resonance spectra for erythrocytes from diseased and healthy persons. Contradictory results have been published for other methods as well so that it is necessary to rethink the membrane defect theory. PMID- 3000786 TI - Combined therapy in advanced breast cancer. PMID- 3000784 TI - Nuclear medicine in the diagnosis of cardiac contusion. AB - In 16 patients with blunt trauma to the chest, the role of cardiovascular nuclear medicine was evaluated using anterior chest flow assessment, with first-pass ejection fraction of left and right ventricles and 99mTc-pyrophosphate scintigraphy. The radiopharmaceutical used was pyrophosphate, labelled with approximately 20 mCi 99mTc. The anterior chest flow and first-pass ejection fractions were initially obtained during the injection of 99mTc-pyrophosphate and were followed up 3 h later by anterior, LAO 45 degrees, and left lateral views of the chest, using an LFOV gamma camera with a data processor. The results were compared with serial cardiac enzymes studies, electrocardiograms and echocardiograms. Of the patients, 77% showed scintigraphic evidence of cardiac contusion. The intensity of activity varied from grades I to II; five patients had abnormal echocardiographic findings. Only two had abnormal ejection fractions, and one patient had evidence of left ventricular aneurysm along with poor ventricular performance. Cardiac enzymes were found to be the least helpful. Electrocardiograms, though non-specific for myocardial damage, were abnormal in 62% of the patients. Eleven of our patients had both abnormal ECG and increased PYP uptake. Even though there is no agreement as to which noninvasive parameter is more sensitive in the diagnosis of myocardial contusion, 99mTc-pyrophosphate scintigraphy, in conjunction with ECG, seems promising in this respect. PMID- 3000787 TI - Detection of virus antigen in male breast cancer. PMID- 3000788 TI - Current status of research into small cell carcinoma of the lung: summary of the Second Workshop of the International Association for the Study of Lung Cancer (IASLC). PMID- 3000789 TI - The concept of priming. PMID- 3000790 TI - Variable bioavailability following repeated oral doses of etoposide. AB - Following oral administration considerable variation in the bioavailability of etoposide has been reported between patients and with different formulations of the drug. The variation within patients following repeated doses is unknown and has therefore been studied in seven patients receiving therapy on three successive days for relapsed small cell lung carcinoma. Etoposide was administered at a dose of 400 mg orally and plasma concentrations were measured using high-performance liquid chromatography. Within-patient coefficients of variation over three successive days ranged over 19-45% for peak plasma concentrations and 16-53% for the area under the plasma concentration-time curve. There was no evidence of a trend to suggest improving or worsening absorption and accumulation did not occur. Urinary excretion was less than 25% and showed no increase over the 3 days. These data indicate that etoposide bioavailability is not constant and oral therapy may lead to unsuspected underdosing or unexpected toxicity in schedules extending over several days. Monitoring blood concentrations for a single day following oral therapy may give a misleading idea of the total bioavailability of etoposide during a course of therapy. Studies of the relationship between the pharmacokinetics of prolonged schedules of etoposide and disease outcome may lead to unreliable conclusions unless intravenous etoposide is used. PMID- 3000791 TI - Cytosolic free calcium, a key second messenger in cellular activation. PMID- 3000792 TI - Antibodies to human T-cell lymphotropic virus type III in promiscuous healthy homosexual men. Relation to immunological and clinical findings. AB - Antibodies to human T-cell lymphotropic virus type III (HTLV-III Ab) were present in twenty-one out of sixty-four asymptomatic promiscuous homosexual men from Copenhagen. The presence of HTLV-III Ab was associated with lymphadenopathy (P less than 0.0005), cytomegalovirus isolation (P less than 0.01), low skin test reactivity (P less than 0.01) and episodes of fever within the 2 month period prior to investigation (P less than 0.05). No significant differences occurred in the total number of T-cells, T-suppressor cytotoxic cells, T-helper cells or helper to suppressor ratio (H/S ratio) between HTLV-III Ab positive and negative homosexuals. An H/S ratio less than or equal to 1.0 was significantly more frequent in homosexual men who both had HTLV-III Ab and excreted cytomegalovirus (P less than 0.01). The H/S ratio of HTLV-III negative homosexuals were significantly lower than that of the controls suggesting that a non-HTLV-III related immunosuppression occurs among homosexuals. Within 2 years after the investigation AIDS or the AIDS related complex developed in three of the men, who at the first investigation all had HTLV-III Ab, alterations in T-lymphocyte subsets and cutaneous anergy. It is suggested that a combination of T-cell subset determination and determination of HTLV-III Ab may provide more valuable prognostic information than isolated determination of HTLV-III Ab. PMID- 3000793 TI - Receptor binding of propranolol is the missing link between plasma concentration kinetics and the effect-time course in man. AB - In a double-blind, placebo-controlled study in 6 healthy volunteers, the correlation between beta-adrenoceptor binding, the time course of the effect and plasma concentration kinetics was investigated from 0 to 48 h after a single oral dose of propranolol 240 mg. First, the in vitro beta-adrenoceptor interaction of propranolol was investigated. Propranolol inhibited beta-adrenoceptor binding to rat parotid (beta 1) and reticulocyte (beta 2) membranes in the presence of pooled human plasma with a Ki of about 8 ng/ml plasma. After oral administration of 240 mg propranolol, concentration kinetics in plasma could be described by a Bateman function with a fictive concentration at time 0 of 275 ng/ml plasma, and a mean elimination half-life of 3.5 h. Using the concentration kinetics of propranolol in plasma together with its in vitro beta-adrenoceptor binding characteristics in the presence of placebo plasma from each individual, the time course of antagonism against beta-adrenoceptor mediated effects was predicted. The latter was in agreement with the time course of propranolol-induced inhibition of tachycardia due to orthostasis. After bicycle ergometry, however, the time course of inhibition of tachycardia was shorter than was predicted. Plasma sampled at various times after propranolol administration inhibited beta adrenoceptor binding of the radioligand 3H-CGP 12177 to rat reticulocyte membranes in a fashion reflecting the time course of inhibition of exercise tachycardia observed in the volunteers. A direct, linear relation was shown between the in vitro inhibition of beta-adrenoceptor binding by the plasma samples withdrawn after propranolol administration and the inhibition of exercise tachycardia observed in parallel. The results show that the concentrations of antagonist present in plasma are representative of the concentrations in the effect compartment. Deep compartments of drug distribution appear irrelevant to the effects of the drugs. The relation between the plasma concentration of propranolol and the reduction in heart rate at various levels of physical effort shows no significant inhibition at rest and increasing IC50-values from orthostasis to 2 min and to 4 min of ergometry. IC50-values after orthostasis are in the range of the Ki-values from in vitro receptor binding studies, whereas the IC50-values after exercise are shifted 2- to 3-fold to the right relative to the Ki-values. This finding is in agreement with increased beta-adrenoceptor stimulation with increasing effort (release of endogenous noradrenaline), which shifts the antagonist concentration-effect curve to the right.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3000794 TI - Long-term low dose ticlopidine treatment in rheumatoid arthritis: effects on serum sulphydryl levels, technetium index, erythrocyte sedimentation rate, and clinical disease activity. AB - In 22 patients with rheumatoid arthritis oral ticlopidine 250 mg/day for 18 months induced clinical improvement, confirmed by a significant decrease in the counts of involved joint. A significant decrease was observed in the technetium index (Tc-index) and the erythrocyte sedimentation rate (ESR), and a significant increase occurred in the serum sulphydryl (SH) levels. The long-term changes in serum SH and Tc-index produced by ticlopidine may represent a specific antirheumatic activity of this platelet-inhibiting drug. PMID- 3000795 TI - Stimulation of aldosterone secretion by metoclopramide is not affected by chronic converting enzyme inhibition. AB - To assess if dopaminergic control of aldosterone secretion is mediated by the renin-angiotensin system, the effect of chronic angiotensin converting enzyme inhibition by enalapril on the aldosterone response to metoclopramide has been studied in 10 patients with mild to moderate essential hypertension. Enalapril reduced supine blood pressure and increased the heart rate significantly. Plasma renin activity and urinary sodium excretion rose significantly. PRA was not changed by metoclopramide, neither during placebo nor during enalapril treatment. Metoclopramide induced a two-fold increase in plasma aldosterone, the peak response being reached within 15 min. Enalapril treatment did not alter the aldosterone response to metoclopramide. Dopaminergic control of aldosterone secretion appears to be independent of the renin-angiotensin system. PMID- 3000796 TI - Penbutolol: beta-adrenoceptor interaction and the time course of plasma concentrations explain its prolonged duration of action in man. AB - Beta-adrenoceptor binding of (-) penbutolol and its active metabolite 4-hydroxy penbutolol to rat reticulocyte membranes was shown in the presence of native human plasma. Due to the high plasma protein binding (approximately 99%) the apparent Ki-values of penbutolol were shifted 100-fold to the right after inclusion of plasma in the assay; the Ki was approximately 40-70 ng/ml. That value is comparable to the IC50-values calculated from clinical studies. The interaction of 4-hydroxy-penbutolol with beta-adrenoceptors was not affected to the same extent by inclusion of plasma protein binding approximately 80%, apparent Ki-value approximately 7 ng/ml. Thus, the active metabolite of penbutolol displays higher potency at beta-adrenoceptors in vitro due to its lesser degree of plasma protein binding. A prediction procedure for antagonist activity after penbutolol administration using beta-adrenoceptor interaction and plasma concentration kinetics suggests that, in addition to a rapid elimination process from human plasma, a slow elimination phase of penbutolol (or an active metabolite) is necessary to explain the long duration of action observed in clinical studies after a single oral dose. Inhibition in vitro of beta adrenoceptor binding by plasma samples obtained after oral administration of 40 mg penbutolol to 3 healthy volunteers indicated a biphasic concentration-time profile of the antagonist in plasma and was in accordance with the time course of the reported reduction in exercise tachycardia. Finally, plasma concentrations of penbutolol equivalents derived from the receptor assay were in the range of penbutolol concentrations detected by physico-chemical methods.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000797 TI - The effect of cimetidine on the pharmacokinetics, pharmacodynamics and alpha 1 adrenoceptor responsiveness of trimazosin in man. AB - The effect of cimetidine treatment on the pharmacokinetics and pharmacodynamics of single doses of trimazosin was studied in 6 normotensive volunteers. Co administration of cimetidine did not significantly affect the overall magnitude of the hypotensive effect of trimazosin. However, the time profile of the blood pressure response was significantly modified particularly with attenuation of the delayed component. Co-administration of cimetidine did not alter alpha 1 adrenoceptor antagonism by trimazosin. There was no significant change in the clearance and volume of distribution of trimazosin but there was a significant reduction in the area under the concentration-time curve for the metabolite, 1 hydroxy-trimazosin. The reduction in the AUC of 1-hydroxy-trimazosin corresponds in time with the attenuation of the delayed hypotensive response. This is consistent with the suggestion that the delayed hypotensive response is related to an active metabolite, probably 1-hydroxytrimazosin. PMID- 3000798 TI - Inhibition by amiloride of both adenylate cyclase activity and the Na+/H+ antiporter in fish erythrocytes. AB - In fish erythrocytes isoproterenol stimulates cellular accumulation of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and produces a large increase in sodium permeability which corresponds to the activation of Na+/H+ exchanges and chloride-dependent sodium uptake. The stimulation of sodium transport by isoproterenol was reproduced by adding cyclic AMP or forskolin to the medium and was blocked by propranolol. This increase in sodium permeability was completely inhibited by amiloride at the relatively high levels (0.1-1 mM) of the diuretic required to inhibit the activity of the Na+/H+ exchanger under physiological conditions in various biological systems. It was shown that amiloride inhibited cyclic AMP accumulation. This effect, which was reversible and dose-dependent (ED50 6 X 10(-6) M-maximal effect 0.5 mM), resulted from the inhibition of the catalytic unit of adenylate cyclase. Amiloride also directly inhibited the sodium entry system but the Na transporter was less sensitive than adenylate cyclase to amiloride (ED50 6 X 10(-5) M). It appears from the data presented in this report that the inhibition of sodium permeability observed in fish erythrocytes in the presence of amiloride can result either from the effect of the diuretic on the adenylate cyclase system or from the effect on the sodium transport system, depending on the conditions in which amiloride is used. Thus, caution is required when interpreting amiloride action in terms of inhibition of specific transport processes. PMID- 3000799 TI - Light microscopic autoradiography of the distribution of [3H]rauwolscine binding to alpha 2-adrenoceptors in rat kidney. AB - The localization of [3H]rauwolscine binding to microscope slide mounted sections of rat kidney has been examined using the technique of in vitro labelling and autoradiography. Binding to sections equilibrated within 60 min and was reversible following the addition of 10 microM phentolamine. Saturation studies revealed a single population of high affinity (KD 4.27 nM) non-interacting sites (nH 0.97) with a density of 11.1 fmol/section. Stereoselectivity was observed with respect to the isomers of noradrenaline and the relative affinity of a series of alpha-adrenoceptor antagonists suggested binding to alpha 2 adrenoceptors. Autoradiographic studies using 3H-Ultrofilm showed that the binding is largely confined to the renal cortex. More detailed studies using emulsion coated coverslips indicates that the major concentration of binding sites is over the proximal tubules. This study provides evidence that alpha 2 adrenoceptors, known to be coupled in an inhibitory fashion to renal adenylate cyclase, are highly localized to particular structures in the kidney. PMID- 3000800 TI - Functional evidence for the existence of adenosine receptors in the human heart. AB - Adenosine added to isolated electrically driven preparations of human ventricular heart muscle antagonized the positive inotropic effect of isoprenaline (mean EC50 19 mumol 1(-1), n = 9). Similar effects were observed with the adenosine receptor agonist (-)-N6-phenylisopropyladenosine (mean EC50 0.5 mumol 1(-1), n = 7). These data provide functional evidence for the existence of adenosine receptors in the human myocardium which may modulate the force of contraction during beta adrenergic stimulation and thus could be involved in the autoregulation of myocardial contractility. PMID- 3000801 TI - Alpha 2-adrenoceptors in rat hypothalamus and cerebral cortex: functional evidence for pharmacologically distinct subpopulations. AB - The presynaptic alpha 2-adrenoceptors regulating, respectively, [3H]noradrenaline and [3H]5-hydroxytryptamine release were compared in experiments with noradrenaline and clonidine as agonists and the two enantiomers of mianserin as antagonists in rat hypothalamic and cortical synaptosomes depolarized with 15 mM KCl. The affinity of clonidine was 10 times higher at the alpha 2-autoreceptors than at the alpha 2-heteroreceptors. (-)Mianserin antagonized noradrenaline at the heteroreceptors but not at the autoreceptors. In contrast, (+)mianserin did not discriminate between the two receptors. The results support the existence in the rat brain of subtypes of alpha 2-adrenoceptors having different neuronal location, function and pharmacological properties. PMID- 3000803 TI - Allosteric effects of diprobutine on acetylcholine receptors. AB - The nicotinic effects of a novel antiparkinsonian compound, diprobutine were investigated on the acetylcholine receptor (AChR) from Torpedo marmorata electric organ and on rat brain membranes by a variety of techniques including stopped flow measurements. On the nicotinic AChR from Torpedo, diprobutine behaved as a typical noncompetitive blocker: it inhibited the agonist-regulated 22Na+ efflux from excitable microsacs; it shifted in the ms-s time-range the conformation of the AChR towards a high affinity state for agonists; it competed with [3H]PCP bound to its high affinity 'allosteric' site. On rat brain membrane, it displaced [3H]PCP bound to its high affinity site. The pharmacological properties of diprobutine are discussed in the context of its biochemical effects. PMID- 3000802 TI - Behavioral effects of microinjections of SR 95103, a new GABA-A antagonist, into the medial hypothalamus or the mesencephalic central gray. AB - The behavioral effects of unilateral microinjections of SR 95103, a new GABA-A receptor antagonist, into periventricular structures were studied. When injected into the medial hypothalamus (MH) or into the dorsal part of the mesencephalic central gray (CG), SR 95103 produced a dose-dependent behavioral activation together with jumps. However, the characteristics of this behavioral activation differed according to whether SR 95103 was injected into the MH or into the CG. The behavioral activation was found to be attenuated by pretreatment with THIP, a GABA receptor agonist. When injected into the CG or into the deep layers of the superior colliculus, SR 95103 proved to affect the rat's reactivity to tactile stimuli as evidenced by ipsilateral 'neglect' combined with contralateral hyperreactivity expressed as withdrawal reactions and jumping. Similar results were obtained following microinjections of bicuculline methiodide at the same sites. These data confirm that in both the MH and the CG, GABA-A receptors are involved in the neural control of the generation and/or expression of aversive effects. The data further suggest that at the level of the CG and the deep layers of the superior colliculus, GABA is also involved in the gating of sensory information towards the substrate underlying the generation of such aversive effects. PMID- 3000804 TI - Benzodiazepine anticonvulsants accelerate and beta-carboline convulsants decelerate the kinetics of [35S]TBPS binding at the chloride ionophore. AB - The effect of flunitrazepam and DMCM was examined on the kinetics of [35S]t butylbicyclophosphorothionate (TBPS) binding. The enhancing effect of flunitrazepam and the decreasing effect of DMCM on TBPS binding was due to non equilibrium conditions. The anticonvulsant benzodiazepine increased both the association and dissociation rate constants. The convulsant beta-carboline had the opposite effect. Benzodiazepine receptor ligands might affect the convulsant TBPS sites parallel with opening/closing of the chloride ionophores. PMID- 3000805 TI - Central and peripheral involvement of mu receptors in gastric secretory effects of opioids in the dog. AB - The effects of dermorphin and morphine on gastric acid secretion were studied in conscious dogs with both gastric fistulas (GF) and Heidenhain pouches (HP). Under basal conditions dermorphin and morphine, infused systemically at graded doses, produced a significant increase in acid secretion from both GF and HP. This increase was significantly inhibited by naloxone, naltrexone methylbromide and N methyl-levallorphan methanesulphonate. Dermorphin did not modify the acid output stimulated by 2-deoxy-D-glucose from GF, while morphine significantly inhibited it; on the contrary acid secretion from HP was increased in this test by both dermorphin and morphine. Acid secretion from GF stimulated by pentagastrin was unaffected by morphine and significantly enhanced by dermorphin. Under these conditions a significant increase in acid secretion from HP was recorded with dermorphin and morphine. Naloxone and N-methyl-levallorphan methanesulphonate, given during pentagastrin-stimulated secretion, significantly inhibited acid output 'per se' from GF and HP and prevented the stimulatory effect of dermorphin and morphine. Bethanechol-induced secretion from GF and HP was significantly increased by both dermorphin and morphine. The present results demonstrate that opioids have simultaneous yet opposite effects on acid secretion in the dog and that mu receptors are involved in both the excitatory and inhibitory effects. Excitatory effects do not seem to be mediated via a vagal pathway (peripheral ?), in contrast to the inhibitory effects (central ?). The inhibitory effects of opiate antagonists on pentagastrin-stimulated secretion suggest a physiological role of peripheral opioid receptors in gastric acid secretion. PMID- 3000807 TI - 2-Amino-7-phosphonoheptanoic acid depresses gamma-motoneurons and polysynaptic reflexes in the cat spinal cord. AB - The effects of 2-amino-7-phosphonoheptanoic acid (APH), a selective antagonist of N-methyl-D-aspartate (NMDA) receptors, were studied on spinal cord functions in unanaesthetized spinal cats. APH (10 mg/kg i.v.) depressed spontaneous activity of gamma-motoneurons and segmental polysynaptic ventral root reflexes (VRRs) without affecting monosynaptic VRRs. The NMDA-induced enhancement of polysynaptic VRRs and activation of gamma-motoneurons were antagonized by APH. The results support the hypothesis that NMDA receptors are involved in the polysynaptic excitation of motoneurons, including gamma-motoneurons, and thus participate in motor functions of the spinal cord. PMID- 3000806 TI - Modulation of cholinergic neurotransmission in canine airways by thromboxane mimetic U46619. AB - We studied the effect of a thromboxane A2-mimetic, U46619, on the contractile responses of canine bronchial smooth muscle to cholinergic stimulation in vitro. U46619 (3 X 10(-10) M), at a concentration that did not cause contraction, enhanced the effect of electrical field stimulation at all frequencies; histamine (10(-7) M) and prostaglandin F2 alpha (10(-7) M) did not. U46619 (3 X 10(-10) M) did not affect methacholine-induced contractions. U46619 may therefore increase the prejunctional release of acetylcholine. PMID- 3000808 TI - Cisplatin and etoposide alternating with vincristine, doxorubicin and cyclophosphamide in patients with small cell lung cancer. AB - Fifty-seven patients with small cell lung cancer were treated with cisplatin and etoposide alternating with vincristine, doxorubicin and cyclophosphamide. Seven patients were withdrawn because of drug toxicity. Of 50 evaluable patients 21 had limited and 29 extensive disease of whom 19 and 21 respectively achieved an objective response. Seven patients survived for longer than 2 years of whom 5 remained alive and tumour free 121-167 weeks from the start of treatment. Patients with limited disease and a complete response had a median survival of 72 weeks and the median survival of the whole group was 43 weeks. Seven out of 22 (32%) of patients who obtained a complete response relapsed and died with solitary evidence of cerebral metastases. This indicates the need for reassessment of the role of prophylactic cranial irradiation in patients achieving a complete response with effective systemic chemotherapy. PMID- 3000809 TI - Alteration of electrical correlates of sensory processing by tetrahydrocannabinol. AB - The action of tetrahydrocannabinol on the function of four multimodality responsive neocortical areas was assessed in the cat. Cats, anesthetized with alpha-chloralose, were subjected to auditory, somatosensory, and two types of visual stimulation. Simultaneous evoked field potentials and multiple-unit activity were recorded. The drug produced a significant reduction in evoked field responses at the three most rostral cortical areas, whereas responses at the posterior suprasylvian gyrus were spared. There were no significant differences between sensory modalities. In addition to being the first pharmacological study to demonstrate differential drug sensitivity between these polysensory areas, these studies localized an effect of tetrahydrocannabinol. Sensory evoked multiple-unit activity showed a pattern of change after THC consistent with a reduction in activity of pyramidal output cells and a concomitant increase in interneuron activity. The action of tetrahydrocannabinol on polysensory systems is important in attention and orientation. PMID- 3000811 TI - Stress by restraining potentiates morphine catalepsy in rats. AB - Restraint-induced stress potentiated morphine catalepsy in rats. This potentiation was partially antagonized by pharmacologic treatments decreasing central serotonin, acetylcholine, prostaglandins and by naloxone. Selective increase in central dopamine also inhibited the potentiation. PMID- 3000810 TI - Leishmania donovani: surface membrane acid phosphatase blocks neutrophil oxidative metabolite production. AB - We show that a purified preparation of the prominent tartrate-resistant acid phosphatase (E.C.3.1.3.2), isolated from the external surface of the intracellular parasite Leishmania donovani (promastigote form), inhibits toxic oxidative metabolite production of neutrophils. Preincubation of a neutrophil suspension (2.5 X 10(6) cells/ml) for 15 min at 37 C with 250 units (1 unit equals 1 nmole of 4-methylumbelliferyl phosphate cleaved per hr at pH 5.5) of the acid phosphatase in Krebs-Ringer phosphate buffer (pH 7.4) decreased O2 consumption, O2- production, and H2O2 production of N-formyl-methionyl-leucyl phenylalanine (fMet-Leu-Phe)-stimulated neutrophils to 15-25% of control values. The acid phosphatase also affected concanavalin A-stimulated O2-production by neutrophils, but had no effect on the rate of phorbol myristic acetate-stimulated O2- production, chemotactic peptide binding, degranulation, or membrane depolarization. Addition of an acid phosphatase inhibitor (Complex E; (NH4)6[P2Mo18O62] X 9H2O) to suspensions of opsonized promastigotes and neutrophils resulted in a threefold or greater enhancement of O2- production. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes. PMID- 3000813 TI - Biliverdin as an electron transfer catalyst for superoxide ion in aqueous medium. AB - Stopped flow experiments gave evidence of the formation of a biliverdin superoxide complex and/or a biliverdin radical anion by reaction of aqueous O2- with biliverdin. Such transient species are likely intermediates both in the bleaching of biliverdin, during exposure to the aerobic xanthine oxidase reaction, and in the reduction of ferricytochrome c under the same conditions. PMID- 3000812 TI - Beta-adrenergic-stimulated adenylate cyclase activity in normal and EBV transformed lymphocytes. AB - Beta-adrenergic-associated cyclic AMP accumulation was studied in intact lymphocytes before and after transformation with Epstein-Barr virus into immortal cell lines. Although a marked reduction in isoproterenol-stimulated cyclic AMP synthesis was observed in transformed cells, forskolin-stimulated cyclic AMP accumulation was preserved. A parallel loss of 125-iodocyanopindolol binding sites suggests that the reduction in beta-adrenergic-stimulated AMP synthesis is due to receptor down-regulation. PMID- 3000814 TI - Effect of pyrophosphate and orotidine monophosphate on cytosine deaminase regulatory properties. AB - The maximal velocity of the reaction (Vmax) and the half-saturation constant (K0.5) values of the S. typhimurium cytosine deaminase were altered in the presence of its effectors, pyrophosphate and orotidine monophosphate. From the kinetics of orotidine monophosphate inhibition of cytosine deaminase, it was characterized as a mixed-type noncompetitive inhibitor. PMID- 3000816 TI - SV40 transformed fibroblasts recognize the same 140 kD fibronectin chemotactic fragment as non-transformed cells. AB - SV40-virus-transformed human embryonal fibroblasts show an enhanced chemotactic response to the glycoprotein fibronectin. However, they recognize the same chemotactic active region as non-transformed fibroblasts. The result suggests that an enhancement of chemotaxis by fibroblasts which have been transformed with Simian Virus 40 is due not to the utilization of further chemotactic domains in the molecule, but to an increased sensitivity of the cells to the chemoattractant. PMID- 3000815 TI - TRH analogue with C-terminal thioamide group. Synthesis, receptor binding, TSH releasing activity and alpha-MSH-releasing activity. AB - A new TRH analogue containing a C-terminal thioamide group was synthesized. This peptide was shown to have receptor-binding affinity, and TSH- as well as alpha MSH-releasing activities very similar to native TRH. PMID- 3000817 TI - [Use of a magnetic field for guided transport of curare-like substances]. AB - Magnetically carried microspheres were used to investigate whether the curare like drugs can be selectively transported to the muscles of one of the limbs of the cat. Pyrocurine and diadonium incorporated in such microspheres primarily suppressed neuromuscular transmission in the limb placed in magnetic field as compared with the control placed outside the field. The use of magnetic microspheres cointaining pyrocurine and diadonium produced no changes in systemic arterial blood pressure, local blood flow, EEG or ECG. The method also diminished the respiratory depression produced by the curare-like substances when they are routinely used for body muscle relaxation. PMID- 3000819 TI - [Synthesis of virus-specific and cellular proteins in the presence of bonafton]. AB - Inhibition of the synthesis of virus-specific proteins of influenza A/WSN/33 virus in the cells of chick embryo fibroblasts and in continuous cells of dog embryonal kidney was discovered as was a negligible inhibition of the synthesis of cell proteins in the presence of an original synthetic antivirus drug bonaphthon. Experiments were made to attain selective inhibition of individual virus-specific proteins of influenza virus in these cultures. In model experiments in vitro in a cell-free protein-synthesizing system, bonaphthon was demonstrated to inhibit translation of RNA-virus of encephalomyocarditis on the virus template without affecting translation on the cell template of hemoglobin mRNA. PMID- 3000818 TI - [Effect of prostaglandin E2 and its derivatives on the reaction of isolated mollusk neurons to acetylcholine]. AB - A study was made of the effect of PGE2, ethyl ether and a carbohydrate derivative of PGE2 on the sensitivity to acetylcholine (ACh) of identified neurons of the mollusk pond snail. The response recorded in the mode of membrane potential fixation diminished in the presence of PGE2 and its derivatives. The rate of the decrease and the degree of the response blockade depended on the substance concentration. Washing of PGE2 made the neuronal sensitivity to ACh return to normal. With the use of PGE2 derivatives the sensitivity recovery was incomplete. The radioimmunochemical technique has demonstrated PGE2 to raise the cAMP level in the pond snail brain. PMID- 3000820 TI - The rate-limiting step and nonhyperbolic kinetics in the oxidation of ferrocytochrome c catalyzed by cytochrome c oxidase. AB - The level of reduction of cytochrome a and CuA during the oxidation of ferrocytochrome c has been determined in stopped-flow experiments. Both components are partially reduced but become progressively more oxidized as the reaction proceeds. When all cytochrome c has been oxidized, CuA is also completely oxidized, whereas cytochrome a is still partially reduced. These results can be simulated on the basis of a model which requires that the intramolecular electron transfer from cytochrome a and CuA to cytochrome a3-CuB is a two-electron process and, in addition, that the binding of oxidized cytochrome c to the electron- transfer site decreases the rate constants for intramolecular electron transfer from cytochrome a. The first requirement is related to the function of the oxidase as a proton pump. Product dissociation is not by itself rate-limiting, making it less likely that the source of the nonhyperbolic substrate kinetics is an effect on this step from electrostatic interaction with ferricytochrome c bound to a second site. It is pointed out that nonhyperbolic kinetics is, in fact, an intrinsic property of ion pumps. PMID- 3000821 TI - A calcium-protease activator associated with brain microsomal-insoluble elements. AB - A factor which markedly activates Ca2+-dependent thiol protease (calpain) is associated with Triton X-100-insoluble materials, presumably structural elements such as cytoskeletons, of bovine brain microsomal fraction. This factor is extracted with 0.6 M KC1, and purified partially by sucrose density gradient centrifugation and hydroxyapatite column chromatography. The factor appears to be a heat-stable protein with an approximate Mr of 15 000. With casein as substrate this factor activates both calpain I and calpain II several-fold up to more than 10- fold without alteration of their affinity to Ca2+. Calmodulin is unable to substitute for this factor. A similar factor is associated with human platelet insoluble materials. PMID- 3000822 TI - Internally transposed signal sequence of carp preproinsulin retains its functions with the signal recognition particle. AB - It is shown that the signal sequence of carp preproinsulin is functional with the dog pancreatic signal recognition particle (SRP) both when present at its normal location at the amino-terminus of the protein or when engineered to an internal location. Inhibition of translation by SRP in the absence of microsomal membranes, reconstitution by SRP of the translocation competence of high-salt inactivated microsomes and signal peptide cleavage all occur with the signal sequence being preceded by a highly charged peptide segment of 39 amino acid residues (the distance from the amino-terminus to the cleavage site of the signal peptidase is increased to 56 residues). PMID- 3000823 TI - Evidence for a mobile semiquinone in the redox cycle of the mammalian cytochrome bc1 complex. AB - Experimental evidence is presented to demonstrate that cytochromes b of the mammalian cytochrome bc1 complex may be rapidly oxidised by a pulse of oxidising equivalents which react with cytochrome c1, even when all cytochrome b is fully reduced before the pulse. The oxidation is sensitive both to antimycin and to myxothiazol. Such behaviour is inconsistent with models in which only the fully oxidised ubiquinone may move between the centres 'o' and 'i' of the complex. It is proposed that the charged semiquinone (Q-) may move between these centres, which may constitute separate reaction domains of a single ubiquinone-binding site. The bearing of this on the mechanism of electron, proton and charge transfer in the complex is discussed. PMID- 3000825 TI - 13C and proton NMR studies of horse cytochrome c. Assignment and temperature dependence of methyl resonances. AB - The 13C and proton chemical shifts of the 55 methyl groups of horse cytochrome c have been determined over a range of temperatures both in the diamagnetic ferrocytochrome and in the paramagnetic ferricytochrome. Specific assignments of many proton resonances have been published previously and all of the remaining methyl proton resonances are now specifically assigned. The corresponding 13C assignments follow directly, including those of contact shifted 13C resonances which are reported for the first time. PMID- 3000824 TI - Kinetics of 125I-ubiquitin conjugation with and liberation from rabbit reticulocyte stroma. AB - The breakdown of mitochondria-containing stroma of rabbit reticulocytes is an ATP and ubiquitin-dependent process and there is no evidence for an ATP-dependent but ubiquitin-independent proteolysis in these cells. The ubiquitin conjugate formation with heat-denatured stroma proteins is about one-fifth of that with native stroma. In reticulocytes there exist two mechanisms of ubiquitin liberation from its conjugates with stroma proteins: an ATP-dependent and hemin resistant release of ubiquitin, which is assumed to be the first step in the degradation of ubiquitin conjugates by the protease system, and a release of ubiquitin catalyzed by an isopeptidase activity. PMID- 3000826 TI - Amino acid sequence of a region on the glycogen-binding subunit of protein phosphatase-1 phosphorylated by cyclic AMP-dependent protein kinase. AB - The amino acid sequence of a region on the glycogen-binding (G)-subunit of protein phosphatase-1G that is phosphorylated by cyclic AMP-dependent protein kinase has been determined. The sequence is: (formula see text) This finding will facilitate studies of the effects of hormones on the phosphorylation state of the G-subunit in vivo. PMID- 3000827 TI - The heparin-binding site(s) of histidine-rich glycoprotein as suggested by sequence homology with antithrombin III. AB - A high degree of sequence homology has been found between the N-terminal region of histidine-rich glycoprotein (HRG) and that of antithrombin III (AT III) where the putative heparin-binding site of AT III is located. The amino acid residue at the position corresponding to Arg-47 of AT III that is essential for the heparin binding was also arginine (Arg 23 and 78) in the homologous sequences of HRG. These observations strongly suggest that the heparin-binding sites of HRG and AT III are evolutionarily related. There was no apparent sequence similarity between the remaining about 70% portions of the two proteins. PMID- 3000828 TI - Gene structure of calcium-dependent protease retains the ancestral organization of the calcium-binding protein gene. AB - The gene structure of calcium-dependent protease (Ca2+-protease) was determined. It comprises at least 21 exons, and these were assigned to the 4 functional domains of the protease. The protease domain does not show clear correlation between exons and functional units, but the calmodulin-like calcium-binding domain shows strong correlation. Each of the 4 consecutive calcium-binding regions in the C-terminal part of Ca2+-protease is encoded by one exon. This gene structure supports the idea that the 4 calcium-binding regions of calcium-binding proteins such as calmodulin arose by 2 steps of gene duplication. PMID- 3000829 TI - Poliovirus-encoded proteinase 3C: a possible evolutionary link between cellular serine and cysteine proteinase families. AB - Here we demonstrate significant similarities between the amino acid sequences of trypsin (a serine protease) and the N-terminal piece of a specific fragment of the poliovirus polyprotein encompassing the sequence of the viral proteinase 3C, and also between cathepsin H (a cysteine protease) and the C-terminal piece of the same fragment. A coherent alignment of the sequences of the 3 proteases was obtained, in which the principal catalytically active residues occupy identical positions. A hypothesis is proposed that the viral enzyme may provide an evolutionary link between serine and cysteine protease families. PMID- 3000830 TI - TSH-induced cyclic AMP production in an ovine thyroid cell line: OVNIS 5H. AB - The TSH-induced cyclic AMP response was studied using a 3-year-old ovine thyroid cell line TSH-independent for growth: OVNIS 5H. The kinetics of cyclic AMP production was followed both in cell layers and in cell culture media, with or without phosphodiesterase inhibitor. It is noteworthy that following the first wave in cyclic AMP obtained within minutes, we observed later a sustained exponential increase in cyclic AMP during the 5 days following TSH stimulation. A bioassay of TSH was derived allowing measurement of 1 microU/ml TSH from a crude bTSH preparation. PMID- 3000831 TI - Cell nuclei generate DNA-nicking superoxide radicals. AB - Rat liver nuclei generate superoxide radicals in the presence of NADPH. Active oxygen species induced nicks in nuclear DNA. This was prevented by superoxide dismutase and catalase as well as by anaerobiosis. EDTA-Fe3+ dramatically increased the active oxygen-dependent DNA nicking. PMID- 3000832 TI - cDNA clones coding for the beta-subunit of human liver alcohol dehydrogenase have differently sized 3'-non-coding regions. AB - Three different size classes of cDNA clones coding for the beta 1-subunit of human alcohol dehydrogenase (ADH) were characterized from a human liver cDNA library. Clones were identified by hybridization with synthetic oligodeoxyribonucleotides. A total of 2530 nucleotides were determined, covering an ADH-coding region of 1122 nucleotides, a preceding 72-nucleotide segment and 3 types of 3'-non-coding region. The coding nucleotide sequence is in full agreement with the amino acid sequence of the beta 1-subunit. Of 8 clones identified, 6 had a short, 213-nucleotide 3'-non-coding region; 1 an intermediate, 590-nucleotide 3'-region; and 1 a long, 1330-nucleotide 3'-region. In addition, 2 unused polyadenylation signals were found. These results suggest that human liver beta-ADH mRNAs occur in several size classes, and that in addition to the consensus sequence AATAAA further signals are important for 3' end formation. PMID- 3000833 TI - Transport and metabolism of vitamins. AB - Although the biochemical roles of most vitamins in the body are reasonably well understood, our knowledge of how the body transports and metabolizes the vitamins is incomplete. This paper summarizes the information available on riboflavin, vitamin B-6, biotin, vitamin D, vitamin C, and pantothenic acid. As might be expected on the basis of the diverse chemistry and biology of these substrates, the body has quite unique mechanisms for handling each of them. PMID- 3000834 TI - Physiological and molecular correlates of age-related changes in the human beta adrenergic receptor system. AB - Aging decreases hormone responsiveness in several receptor systems. In this article I consider both physiological and biochemical studies supporting the hypothesis that beta-adrenergic receptor responsiveness is reduced with aging in humans. Reduced chronotropic and vasodilator responses to the beta-receptor agonists isoproterenol and metaproterenol have been demonstrated. In human leukocytes a reduction in adenylate cyclase (EC 4.6.1.1) activity occurs with aging. More recently it has been suggested that this reduction in beta-adrenergic responsiveness with aging may be caused by an uncoupling of the beta receptor from the catalytic component. PMID- 3000835 TI - Decreased beta-adrenergic responsiveness during senescence. AB - The capacity of the myocardium to respond to catecholamines is diminished in senescence. The intrinsic inotropic and chronotropic responses of the contractile elements to direct stimulation are unaltered with age, which suggests that the mechanism for diminished responsiveness lies in the sequence of events between the beta-adrenergic receptor and effector site. In a series of investigations of rat myocardial, rat lung, and human lymphocyte alpha- and beta-adrenergic function with age, it has been demonstrated that 1) there are no changes in either alpha 2- or beta-adrenergic receptor density or receptor antagonist affinity, 2) there is decreased beta-adrenergic receptor-agonist affinity with a corresponding decrease in the ability to form the high-affinity binding complex, 3) there is no change in alpha 2-adrenergic receptor agonist affinity, 4) there is decreased NaF- and hormone-stimulated adenylate cyclase activity, 5) there is decreased nucleotide regulatory protein function in the rat myocardium, 6) there is decreased catalytic unit function, and 7) there is decreased translocation of membrane-bound protein kinase. These changes in the beta-adrenergic stimulatory pathway may account for the diminished myocardial catecholamine responsiveness associated with aging. PMID- 3000836 TI - Regulation of brain adrenergic receptors during aging. AB - Recent studies in our laboratory suggest that the synthesis of alpha- and beta adrenergic receptors in certain tissues from brain and pineal gland may be impaired with age. This decreased ability of the aged brain to synthesize adrenergic receptors may explain the loss of these receptors in selected brain regions during the aging process, as well as the reduced capacity of aged brain tissue to increase or up-regulate the density of these receptors in response to reduced noradrenergic activation of the tissues or to reduced estrogen levels. The reduced adaptability of brain adrenergic receptors, in turn, may account for the decreased ability of aged individuals to adjust their physiological responses to a changing environment. PMID- 3000837 TI - Effects of aging on mechanisms of alpha-adrenergic and dopaminergic action. AB - Mammalian parotid glands have provided excellent model systems for studying adrenergic control of defined biochemical and physiological processes during exocrine secretion. The findings point to a deficiency in a key coupling step (postulated here to exist just distal to the alpha 1-adrenergic receptor yet proximal to the phospholipid turnover/Ca2+ mobilization steps) required for alpha adrenergic-mediated fluid and electrolyte secretion from the aging rat exocrine parotid gland. Because the steps involved in this process are not fully elucidated, natural perturbation of rat parotid gland function should prove to be of particular value as a model toward clarification of the mechanisms of alpha adrenergic signal transduction. It is now generally agreed that dopamine receptors are lost from the corpus striatum during aging in a variety of species, including humans. Most studies of age changes in striatal dopamine receptors have detected no alterations in binding affinity or dissociation constant (Kd). Only reduction in concentration (Bmax) with increasing age is apparent. Such receptor loss appears to be at least partially responsible for decreased dopamine stimulation of certain stereotypic behavioral responses and decreased neurotransmitter release. In addition, dopamine-sensitive adenylate cyclase (EC 4.6.1.1) decreases during senescence. PMID- 3000840 TI - Alkalosis as a potential complication of air polishing systems. A pilot study. PMID- 3000838 TI - [Evidence for a role of calmodulin in cellular cAMP production in response to vasopressin, prostaglandin E2 and forskolin in cultured rat renal papillary collecting tubule cells]. AB - In the present study, the role of calmodulin in the cellular action of arginine vasopressin (AVP), prostaglandin (PG) E2 and forskolin on adenosine-3', 5' monophosphate (cAMP) production was examined in cultured rat renal papillary collecting tubule cells. In the presence of the phosphodiesterase inhibitor, submaximal concentrations of 10(-9) M AVP, 2 X 10(-8) M PGE2 and 2.4 X 10(-7) M forskolin significantly increased cellular cAMP accumulation by 2.3, 6.0 and 8.4 fold, respectively. Two chemically dissimilar inhibitors of calmodulin, namely trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), attenuated the cellular production of cAMP in a dose-related manner in response to all three stimuli. A dose which inhibited the cellular production of cAMP by 50% (ID50) ranged from 1.6 X 10(-5) to 2.8 X 10(-5) M for trifluoperazine and from 3.5 X 10(-5) to 4.4 X 10(-5) M for W-7. Basal accumulation of cellular cAMP was also decreased by treatment with either trifluoperazine or W-7, but an effective dose was relatively higher than that which inhibited agents-stimulated cellular cAMP production. Since forskolin is recognized as activating adenylate cyclase at one step of the catalytic component and the cellular action of AVP to activate adenylate cyclase is mediated through receptor-catalytic component, the present study indicates calmodulin regulation of basal, AVP-, PGE2- and forskolin activated adenylate cyclase in the papillary collecting tubule cells. Further study demonstrated the inhibition of AVP- or PGE2-induced cellular cAMP production by treatment with either a calcium-free medium or verapamil, a blocker of cellular calcium uptake, before and during the experiment. These findings suggest that an increase in cytosolic calcium, which interacts with calmodulin to form an active complex, is, at least in part, due to the increased cellular influx of calcium from the extracellular space. PMID- 3000839 TI - Clinical use of high-fiber diets. PMID- 3000842 TI - [Focal epithelial hyperplasia: first cases in Switzerland and review of the literature]. AB - Three cases of 'focal epithelial hyperplasia' (FEH) of the oral mucosa observed for the first time in Switzerland are reported. The patients were of Turkish and North African extraction. The lesions of FEH were multiple, painless, located at various sites of the oral mucosa including the tongue, in the form of either soft papules or hard nodules. Evidence of a human papilloma virus origin was ascertained. Among the 1,067 cases reported in the literature and reviewed for this study, this condition has been described to occur among American Indians, Eskimos and Cape Coloureds; also in Israeli and European cases the disorder was often reported in individuals of Turkish or North African extraction. PMID- 3000841 TI - Neoplastic proliferation in a case of epidermotropic high-grade malignant lymphoma associated with hypercalcemia. AB - We report a case of epidermotropic high-grade malignant lymphoma associated with hypercalcemia occurring in a European woman aged 30 years. The clinical, histological, and biological presentation of the patient could enter the spectrum of HOTS (hypercalcemia, osteolysis, T cell lymphoma syndrome). The proliferation of neoplastic lymphocytes was very high in patch, plaques, and nodules of the skin. PMID- 3000843 TI - Survival and rehabilitation after total pancreatectomy. A follow-up of 36 patients. AB - Thirty-six totally depancreatectomized patients were followed up for 4-124 months. Pancreatectomy had been performed because of fulminant pancreatitis (in 10), chronic hyperalgic otherwise untractable pancreatitis (in 7), exocrine carcinoma of the pancreas (in 16), cystadenocarcinoma of the pancreas (in 2) and insulinoma (in 1). The longest survival duration was in chronic pancreatitis patients: 57 +/- 17 months. A normal socio-professional reinsertion was obtained in 16 patients, mainly those with non-malignant pancreotopathies. At the end of the survey, ten of the carcinoma patients had died, versus none in the other groups. Diabetes mellitus was characterized by the absence of ketonuria, and the frequent occurrence of hypoglycemia (in 15 patients) and infection (in 6). Malabsorption caused osteomalacia in one patient. PMID- 3000844 TI - Detection of a pregnancy-associated protein. AB - Monospecific antiserum was produced with the use of a placental extract showing high activity of a proteinase which hydrolyses a synthetic substrate, N-benzoyl D,L-arginine p-nitroanilide, used for measuring tryptic activity. An immunological study showed that antibodies were not generated against the component with this activity but against an antigen which was not related to several well-known pregnancy-associated proteins. Pregnancy sera contained an antigen which immunologically was completely identical to the antigen of placental origin. Single radial immunodiffusion showed an elevated level of reactive antigen in pregnant subjects: 70% of 333 cases were positive. Cross reacting antigen was also detected in sera as well as ascitic fluid from cases of advanced ovarian cancer. Sephadex G-200 gel filtration indicated that the antigen has an apparent molecular weight of 94000, and immunoelectrophoresis showed the molecule to be of beta-mobility. These properties suggest that the substance may represent an additional pregnancy-associated protein entity. Partial purification and some immunological properties of this antigen are described. PMID- 3000845 TI - Partial purification and characterization of cytochrome P-450 from human placenta. AB - Two isozymes of cytochrome P-450 were partially purified to specific contents of 7.0 and 0.5 nmol/mg of protein, respectively, from placenta of non-smoking women by chromatography on octyl Sepharose, hydroxylapatite, DEAE-cellulose and CM cellulose. NADPH-cytochrome P-450 reductase was purified from phenobarbital induced mouse liver and from human placenta and was combined with cytochrome P 450 and dilauroylphosphatidylcholine to reconstitute the cytochrome P-450 monooxygenase system. Substrates investigated were benzo[a]pyrene, 7 ethoxycoumarin and delta 4-androstene-3,17-dione. PMID- 3000846 TI - Presence of adenine phosphoribosyltransferase and adenosine kinase in chloroplasts of spinach leaves. AB - Activity of adenine phosphoribosyltransferase and adenosine kinase was detected in purified spinach chloroplasts by using differential centrifugation and discontinuous Percoll density gradients. This is the first report of purine salvage enzymes being located in chloroplasts. The role of adenine and adenosine salvage in chloroplasts is discussed. PMID- 3000848 TI - A mechanism in the control of intracellular cAMP level: the activation of a calmodulin-sensitive phosphodiesterase by a rise of intracellular free calcium. PMID- 3000847 TI - Interactions of 1,25-dihydroxyvitamin D3 and the immune system. AB - A series of recent discoveries indicate that the hormonal form of vitamin D3, namely, 1,25(OH)2D3 plays a role in the regulation of the immune system. Cells of the monocyte/macrophage lineage possess receptors for 1,25(OH)2D3 regardless of their activation stage; cells of the lymphoid lineage also express these receptors but only at certain stages of their differentiation pathway and upon activation. Further, 1,25(OH)2D3 promotes the differentiation of monocyte precursors towards monocyte/macrophages and enhances monocyte function in antigen presentation. In addition 1,25(OH)2D3 is a potent inhibitor of interleukin-2 (IL 2) and suppresses effector functions of both T and B lymphocytes via IL-2 dependent as well as via IL-2-independent mechanisms. The theoretical and clinical implications of these discoveries are discussed. PMID- 3000849 TI - Secretion of anti-Mullerian hormone by immature bovine Sertoli cells in primary culture, studied by a competition-type radioimmunoassay: lack of modulation by either FSH or testosterone. AB - Secretion of anti-Mullerian hormone (AMH) by immature bovine Sertoli cells in primary culture was studied through a competition-type RIA employing a polyclonal antibody and 125I-labelled purified AMH. This RIA is approximately 10 times more sensitive than the solid-phase two-site monoclonal antibody-based RIA described previously. Biosynthesis and secretion of AMH by cultured Sertoli cells require approximately 48 h, are not influenced by FSH or testosterone and steadily decrease over a one-week period of culture. Cyclic AMP response to FSH stimulation is normal in cultured cells. Whether the factors responsible for the extinction of AMH production in vitro are in any way related to those operating during normal maturation, which lead to repression of AMH biosynthesis in adult Sertoli cells, is not known at the present time and deserves further study. PMID- 3000850 TI - Use of GH4C1 cell variants to demonstrate a non-spare receptor model for thyrotropin-releasing hormone action. AB - Thyrotropin-releasing hormone (TRH) stimulates maximally both the release of previously synthesized prolactin and the de novo synthesis of prolactin by GH4C1 rat pituitary cells at concentrations less than those necessary to fully occupy the TRH receptor at equilibrium. We have examined the dependency of maximal TRH enhanced prolactin release and synthesis on receptor number using GH4C1 cell variants with different numbers of TRH receptors. GH4C1 cell variants with increased and decreased numbers of TRH receptors were selected by using a morphological response known as stretching which renders the cells more adherent to the tissue culture substrate. We found that maximal TRH-enhancement of prolactin release or synthesis increased proportionally to the number of TRH receptors per cell, indicating that spare receptors do not exist for TRH on these GH4C1 cells. We also found that occupancy of the TRH receptor by the analogue, N3im-methyl-TRH (MeTRH), in contrast to TRH, closely paralleled stimulated prolactin release in a manner consistent with Clark's receptor-occupancy model. We conclude that differences between apparent Kd and ED50 for TRH do not necessarily result from spare receptors in GH4C1 cells. PMID- 3000851 TI - Protein kinase C in adrenal cells: possible role in regulation of steroid synthesis. AB - Y-1 adrenal tumor cells and rat fasciculata cells were shown to possess an enzyme with the properties of protein kinase C. Activity was stimulated by Ca2+ and phospholipid (specifically phosphatidylserine). Enzyme activity was stimulated by addition of phorbol ester to a cell homogenate (ED50 10 nM) and inhibited by trifluoperazine (ID50 10 microM). ACTH and cyclic AMP added to Y-1 cells increased the activity of protein kinase C. Dose-response curves with ACTH showed that the hormone was effective in stimulating protein kinase C at lower concentrations than those required to increase steroid synthesis. When phorbol ester was added to Y-1 cells, total kinase C activity was diminished. Neither phorbol ester nor ACTH causes redistribution of protein kinase C between membranes and cytosol. Phorbol ester also stimulates steroid production by Y-1 cells. Protein kinase C phosphorylates 5 proteins in Y-1 cells (67, 61, 32, 16 and less than 14.4 kDa). Puromycin and cycloheximide increase the activity of protein kinase C in adrenal cells. It is concluded that protein kinase C may play an ancillary role in regulation of adrenal steroid synthesis but does not mediate the classical steroidogenic response that results from activation of adenylate cyclase by ACTH. PMID- 3000852 TI - Hormonal regulation of proteins secreted by cultured pig Sertoli cells: characterization by two-dimensional polyacrylamide gel electrophoresis. AB - Using a primary culture of immature porcine Sertoli cells, we studied the effect of porcine FSH (pFSH), testosterone and retinoic acid on the labelled secreted protein. Cells were cultured in a chemically defined medium for 3 days and, on day 3, they were incubated for different times in another medium containing labelled amino acids either in the presence or absence of pFSH (50 ng/ml or 2 micrograms/ml), or testosterone (10(-6) M), or retinoic acid (10(-7) M), either alone or in several combinations. After 4 and 8 h of incubation, 20 and 30 secreted peptides were detected respectively by a two-dimensional polyacrylamide gel electrophoresis of the radiolabelled secreted proteins. During these 2 periods, the effect of pFSH was negligible. After 25 h, about 84 spots (pI in the range of 5-8) were identified on the autoradiograms. pFSH (2 micrograms/ml) induced an increase of 14 spots, and a decrease of 7, but at 50 ng/ml only 5 spots were increased and one decreased. Retinoic acid alone induced the increase of one peptide, while testosterone alone or in combination with pFSH (50 ng/ml) did not modify the electrophoretic pattern. When pig Sertoli cells were treated with retinoic acid, testosterone and pFSH (50 ng/ml), the effects on secreted proteins were higher than those induced by pFSH (50 ng/ml) alone. PMID- 3000853 TI - Thermodynamics and kinetics of mouse prolactin-hepatic receptor interaction. AB - Kinetic and thermodynamic parameters associated with the binding of secreted mouse prolactin (smPRL) to mouse hepatic receptors were investigated. When the reaction temperature was increased from 8 degrees C to 37 degrees C, the association rate constant k+1, increased approximately 5-fold, from 2.3 X 10(4) M 1 . S-1 to 12.6 X 10(4) M-1 . S-1. An Arrhenius plot indicated that there was a linear relationship between ln (k+1) and 1/T. When the reaction temperature was increased from 8 degrees C to 37 degrees C, the equilibrium binding constant, Ka, decreased approximately 1.5-fold, from 2.8 X 10(8) M-1 to 1.9 X 10(8) M-1. When the pH of the binding reaction was lowered from 9.0 to 6.2, Ka decreased approximately 3-fold, from 2.6 X 10(8) M-1 to 0.9 X 10(8) M-1. Transition state thermodynamic parameters for the formation of the smPRL-receptor complex, represented by delta G+', delta H+' and delta S+', were +45.7 kJ/mol, +41.2 kJ/mol and -15.1 J/(mol . K), respectively. Parameters for the equilibrium reaction, described by delta G0', delta H0' and delta S0', were -47.6 kJ/mol, 10.6 kJ/mol and +124 J/(mol . K), respectively. Over the temperature range studied, a Van't Hoff plot of the binding constants demonstrated a linear relationship between ln (ka) and 1/T, indicating that changes in enthalpy for the binding reaction were temperature independent. The binding reaction was largely entropically driven (delta S0' greater than 0), suggesting that hydrophobic interactions are involved in forming the smPRL-receptor complex. PMID- 3000854 TI - Introduction of cloned DNA into sea urchin egg cytoplasm: replication and persistence during embryogenesis. AB - Cloned DNA sequences were introduced into the cytoplasm of unfertilized sea urchin eggs by a simple microinjection technique. Sperm was then added, and development allowed to proceed. If linearized plasmids are injected they form random concatenates, and during the early development of the embryos replicate repeatedly. Eukaryotic sequences are not required for replication of the exogenous DNA. Injected supercoiled DNAs neither ligate nor replicate. Both forms of exogenous DNA persist in the embryo through pluteus stage. PMID- 3000855 TI - Persistence and integration of cloned DNA in postembryonic sea urchins. AB - Cloned DNA was injected into the cytoplasm of unfertilized sea urchin eggs which were then fertilized and cultured in the laboratory through metamorphosis. The exogenous DNA replicated manyfold and persisted for weeks in a majority of growing larvae, as shown by hydridizing "dot blots" of the DNA of single individuals with appropriate labeled probes. After metamorphosis 5-15% of the juvenile sea urchins retained the exogenous sequences. Genomic integration of the exogenous sequence was observed in the DNA of a postmetamorphosis juvenile. PMID- 3000856 TI - Evidence that cholecystokinin interacts with specific receptors and regulates insulin release in isolated rat islets of Langerhans. AB - To determine the nature of the pancreatic islet cell cholecystokinin (CCK) receptor, we studied CCK receptor binding and biologic activity in isolated rat pancreatic islets. Binding of 70 pM 125I-CCK to collagenase-prepared isolated rat pancreatic islets at 24 degrees C was one-half maximal after 5 min and maximal at 60 min. At 60 min, specific binding was 12% of total radioactivity per 100 micrograms islet protein; nonspecific binding (in the presence of 1 microM CCK 8) was less than 2% of total radioactivity. Unlabeled CCK 33 inhibited labeled hormone binding one-half maximally at 2 nM; Scatchard analysis showed one binding site (Kd, 2.3 +/- 0.4 nM; Bmax, 8.1 pmol/mg protein). The agonist selectivity of this binding site was: CCK 8 = CCK 33 greater than desulfated-CCK 8 greater than CCK 4. Two CCK antagonists were studied; N-carbobenzoxy-L-tryptophan was more potent than dibutyryl-cGMP. When the effect of CCK on insulin release from the islets was studied, the order of potency of CCK agonists and antagonists on insulin secretion was the same as the order of their ability to inhibit 125I-CCK binding. The effect of CCK on insulin secretion was dependent on the glucose concentration in the media. CCK had no effect at 5.6 mM glucose and was fully effective at 11.0 mM glucose. These data, therefore, indicate that: specific binding sites for CCK are present in rat pancreatic beta cells; and CCK acts in concert with glucose to stimulate insulin secretion. PMID- 3000857 TI - Chlorpropamide raises fructose-2,6-bisphosphate concentration and inhibits gluconeogenesis in isolated rat hepatocytes. AB - The addition of chlorpropamide to hepatocytes isolated from fed rats raised the cellular concentration of fructose-2,6-bisphosphate (F-2,6-P2), a regulatory metabolite that plays a relevant role in the control of hepatic glucose metabolism. The effect of chlorpropamide was dose dependent; a statistically significant increase was already seen at 0.2 mM of the sulfonylurea. The accumulation of F-2,6-P2 caused by chlorpropamide (1 mM) was parallel to the stimulation of L-lactate production (36.6 +/- 4.8 versus 26.1 +/- 2.6 mumol of lactate/g of cells X 20 min; N = 5, P less than 0.05) and to the inhibition of gluconeogenesis (0.57 +/- 0.1 versus 0.94 +/- 0.09 mumol of [U-14C]pyruvate converted to glucose/g of cells X 20 min; N = 5, P less than 0.05). In addition, chlorpropamide enhanced the inhibitory action evoked by insulin on glucagon stimulated gluconeogenesis. This combined effect of chlorpropamide and insulin seems to be correlated with the synergistic accumulation of F-2,6-P2 provoked by the simultaneous action of these two agents on glucagon-treated hepatocytes. Finally, neither 6-phosphofructo-2-kinase activity nor hepatocyte cyclic AMP levels were significantly changed by the presence of the sulfonylurea in the incubation medium. Our results support the concept that chlorpropamide, by a cyclic AMP-independent mechanism, increases the hepatic content of F-2,6-P2 and, in this way, enhances the glycolytic flux and inhibits glucose output by the liver. PMID- 3000859 TI - Infection control guidelines for home respiratory therapy. PMID- 3000858 TI - Royal Alexandra develops AIDS infection control protocol. PMID- 3000860 TI - Chronic allyl chloride poisoning. An epidemiology, clinical, toxicological and neuropathological study. AB - It was previously reported that chronic exposure to allyl chloride resulted in liver and kidney damage. No neurotoxic effect of allyl chloride had been noticed until two outbreaks of polyneuropathy without liver and kidney dysfunction due to exposure to allyl chloride in China in the early 1970's. Epidemiological and clinical studies done within 1973-1982 revealed that the main risk of industrial exposure to allyl chloride is damage to the peripheral nervous system. Polyneuropathy is thought to be the main clinical manifestation of chronic allyl chloride poisoning. Electroneuromyography is essential and valuable for early diagnosis and biological monitoring. Toxicological and neuropathological studies in rabbits and mice have given the evidence of a pattern of central-peripheral distal axonopathy in peripheral nervous system which has further confirmed the neurotoxicity of allyl chloride found in man. Based on the above results, the maximum allowable concentration of allyl chloride and diagnostic criteria for chronic allyl chloride poisoning are proposed. PMID- 3000861 TI - beta-Endorphin and enkephalins stimulate duodenal mucosal alkaline secretion in the rat in vivo. AB - Secretion of HCO3- by duodenum just distal to the Brunner's glands area and devoid of pancreatic HCO3- was titrated in situ in anesthetized rats. Secretion increased significantly after intravenous injection of small amounts (10-20 ng/kg) of the opioid peptides beta-endorphin, methionine-enkephalin, and leucine enkephalin. Maximum (approximately twofold) stimulation by beta-endorphin and leucine-enkephalin occurred at 20 ng/kg. Morphine (50 micrograms/kg) caused a similar stimulation and the mu-selective opiate antagonist naloxone prevented the stimulation by beta-endorphin and morphine. The synthetic analogue [D-Ala2,D Leu5]-enkephalin (500 ng/kg), which is an agonist primarily at delta-opiate receptors, had no effect, further suggesting that the stimulation of duodenal HCO3- secretion is mediated by mu-receptors. Naloxone alone did not affect basal HCO3- secretion but reduced the duration of the rise in secretion in response to a 5-min exposure to luminal acid (pH 2.00). Endogenous opioid peptides may thus have a role in the humoral or neural control, or both, of duodenal surface epithelial HCO3- secretion and mucosal protection. PMID- 3000862 TI - More on germ cell tumors. PMID- 3000863 TI - A gonadotropin-like thermostable peptide, prepared from bovine anterior pituitary, inducing spermiation in the frog Rana esculenta (L.) and binding to a rat ovarian FSH receptor. AB - A thermostable peptide, inducing sperm release in Rana esculenta L. and having immunological properties which resemble those of the gonadotropins but nonidentical with any one of the gonadotropins or their subunits, has been prepared from pars distalis of bovine anterior pituitaries by means of acetic acid extraction, heating, ethanol precipitation, and reverse-phase high performance liquid chromatography (HPLC). During the purification, the biological activity was regularly followed by means of the potency of collected fractions to induce sperm release in frogs. Using HPLC (5 micron C18 silica), the activity was eluted with 38% of acetonitrile:water:trifluoroacetic acid (80:19.5:0.5) and 62% of 0.5% trifluoroacetic acid, pH 3.3. The active principle was also found to bind to a rat ovarian follitropin (FSH) receptor. In contrast, bovine FSH and LH treated identically to the peptide lost all such affinity, indicating that the peptide is not created from either of these during the purification procedure. Further, the peptide was, like FSH, also shown to stimulate aromatase activity in intact Sertoli cells from immature male rats in vitro. PMID- 3000864 TI - The effects of castration and/or methimazole feeding on the pituitary response to temperature extremes by cockerels. AB - The pituitary of goitrogen-treated White Leghorn cockerels is smaller in size than control birds and the pituitaries of castrated cockerels is nearly twice the size of control birds. The pituitary cells generated by these treatments may not be functional thyrotrophs or gonadotrophs and may not be able to respond to their usual stimuli. Low ambient temperature is a well-known stimulus to the thyroid gland acting through pituitary TSH. Cyclic-AMP-dependent protein kinase activity levels are used here as an index of cellular activity in the pituitary and thyroid glands. Castrated cockerels with or without methimazole treatment do not have an increased pituitary cAMP-dependent protein kinase activity in cold. Methimazole-treated birds have an exaggerated pituitary protein kinase response to cold stress when compared with controls. Pituitary cAMP-dependent protein kinase activity is paralleled by a similar activity increase in the thyroid gland of methimazole-treated cockerels and no increase in the thyroid of castrated birds. Castrated birds at all temperatures have an elevated thyroid cAMP dependent protein kinase activity ratio which is interpreted as the result of removal of testosterone inhibition. PMID- 3000865 TI - Thyroid hormone binding to isolated trout (Salmo gairdneri) liver nuclei in vitro: binding affinity, capacity, and chemical specificity. AB - Isolated nuclei from trout hepatocytes demonstrated in vitro high-affinity and low-capacity binding for 3,5,3'-tri-iodo-L-thyronine (T3). Binding was reversible; the dissociation rate was 5.7 X 10(-3) min-1 at 15 degrees C. Linear Scatchard plots suggested a single class of noncooperative sites (Kd = 1.4 +/- 0.10 X 10(-10) M; maximum binding capacity (MBC) = 62 +/- 10 fmol/mg DNA). After correction for site degradation, site occupancy by endogenous T3, and the dissociation rate of endogenous bound T3, the MBC was 106 +/- 16 fmol/mg DNA. The T3 affinity exceeded slightly that of the hepatocyte nucleus of the rat; the MBC was lower than for most other vertebrates. The relative binding affinities of thyroid hormone (TH) analogs for the T3 site were: TRIPROP greater than TRIAC greater than methyl-bridged T3 greater than TETRAPROP greater than T3 greater than TETRAC greater than TRIFORM greater than 3,5-dibromo, 3'-isopropyl thyronine greater than L-thyroxine (T4) greater than DL-T4 greater than 3'-isopropyl 3',5' dimethyl thyronine greater than reverse T3 greater than 3,5-T2. MIT and DIT did not bind at all. This structure-affinity profile was similar but not identical to that of rat liver, indicating considerable but not complete evolutionary conservation of site structure. Parallel studies of T4 binding also indicate a single class of noncooperative sites (Kd = 7.2 +/- 2.4 X 10(-10) M). Both the MBC and the structure-affinity profile for T4 corresponded to those for T3. These observations, combined with the ability of excess T3 or T4 to completely displace both labeled T3 or T4, support a previous suggestion that in teleosts T3 and T4 bind to the same class of nuclear sites. These sites probably represent TH receptors. PMID- 3000866 TI - Molecular genetic characterization of a locus that contains duplicate Adh genes in Drosophila mojavensis and related species. AB - Drosophila mojavensis and other species of the mulleri subgroup contain a duplicate gene encoding the enzyme alcohol dehydrogenase (ADH). Studies on the genetic relationship of the two genes using electrophoretic variants show them to be closely linked. We have cloned a 13.5-kb fragment of D. mojavensis DNA into the lambda vector, Charon 30. This fragment contains both Adh genes separated by approximately 2 kb of DNA. The clone hybridized to a single position on chromosome 3 in D. mojavensis following in situ hybridization. It is likely that the genes are tandemly arranged in the genome. One of the two genes shows a complexity in its structure that suggests the close linkage of a pseudogene or part of a gene. The structure of the Adh locus in five species of the mulleri subgroup have been compared by constructing restriction maps of genomic DNA. Two of these species D. arizonensis and D. mojavensis express Adh-1 in the ovaries; the others do not. In comparing these species it is evident that there has been one or two insertions into the region between the Adh genes. It is possible that one of these structural changes is related to the change in Adh tissue-specific expression that has occurred during the evolution of these species. PMID- 3000868 TI - The evolution of self-regulated transposition of transposable elements. AB - This paper examines the conditions under which self-regulated rates of transposition can evolve in populations of transposable elements infecting sexually reproducing hosts. Models of the evolution of both cis-acting regulation (transposition immunity) and trans-acting regulation (transposition repression) are analyzed. The potential selective advantage to regulation is assumed to be derived from the deleterious effects of mutations associated with the insertion of newly replicated elements. It is shown that both types of regulation can easily evolve in hosts with low rates of genetic recombination per generation, such as bacteria or bacterial plasmids. Conditions are much more restrictive in organisms with relatively free recombination. In haploids, the main selective force promoting regulation is the induction of lethal or sterile mutations by transposition; in diploids, a sufficiently high frequency of dominant lethal or sterile mutations associated with transpositions is required. Data from Drosophila and maize suggest that this requirement can sometimes be met. Coupling of regulatory effects across different families of elements would also aid the evolution of regulation. The selective advantages of restricting transposition to the germ line and of excising elements from somatic cells are discussed. PMID- 3000867 TI - Molecular genetic analysis of the dilute-short ear (d-se) region of the mouse. AB - Genes of the dilute-short ear (d-se) region of mouse chromosome 9 comprise an array of loci important to the normal development of the animal. Over 200 spontaneous, chemically induced and radiation-induced mutations at these loci have been identified, making it one of the most genetically well-characterized regions of the mouse. Molecular analysis of this region has recently become feasible by the identification of a dilute mutation that was induced by integration of an ecotropic murine leukemia virus genome. Several unique sequence cellular DNA probes flanking this provirus have now been identified and used to investigate the organization of wild-type chromosomes and chromosomes with radiation-induced d-se region mutations. As expected, several of these mutations are associated with deletions, and, in general, the molecular and genetic complementation maps of these mutants are concordant. Furthermore, a deletion breakpoint fusion fragment has been identified and has been used to orient the physical map of the d-se region with respect to the genetic complementation map. These experiments provide important initial steps for analyzing this developmentally important region at the molecular level, as well as for studying in detail how a diverse group of mutagens acts on the mammalian germline. PMID- 3000869 TI - A model for DNA sequence evolution within transposable element families. AB - A quantitative model is proposed for the expected degree of relationship between copies of a family of transposable elements in a finite population of hosts. Special cases of the model (in which the process of homogenization of element copies either is or is not limited by transposition rate) are presented and illustrated, using data on mobile sequences from different species. It is shown that transposition will be expected, in large populations, to result in only a rather distant relationship between transposable elements at different genomic sites. Possible inadequacies of the model are suggested and quantified. PMID- 3000870 TI - [The role of te transposition of Tn1000 from Flac+-plasmid into chromosome of Erwinia chrysanthemi in the formation of Hfr-type donors]. AB - Based on the data of stability of the donor state of Hfr-like strain Erwinia chrysanthemi VY1-10 in RecA+ and RecA- cells, it can be suggested that the donor properties of the strain are mediated by the presence of the genetic homology region which occurred as a result of transposition of the Tn1000 from the Flac+ plasmid into the chromosome of E. chrysanthemi. Tn1000 may be transposed into several sites on the chromosome of E. chrysanthemi ENA49. This leads to the appearance of donors transferring their chromosome from several fixed points oriT and in opposite directions. The location of these points and the direction of transfer are determined by Tn1000 insertion sites and their orientation. PMID- 3000871 TI - [Restriction analysis of cDNA for cow alpha s1-, beta- and kappa-caseins]. AB - By means of in situ hybridization to cloned cDNA fragments coding for cow alpha s1-, beta- and kappa-caseins, screening of the library of clones containing the cDNA complementary to mRNA of lactating cow mammary gland was carried out. The clones containing the sequences of alpha s1-, beta- and kappa-casein cDNAs were shown to constitute 4.0, 3.2 and 0.7% of all the colonies, respectively. The analysis of the data on cross-hybridization points to the absence of extensive regions of homology between the above-mentioned cDNAs. The restriction analysis of cDNAs of the selected clones was carried out and the restriction maps of cDNAs of these three caseins were constructed. The restriction analysis data and determination of the nucleotide sequence of 5'-termini of the studied cDNAs indicated that the cloned sequences were the full-length mRNA copies of alpha s1 , beta- and kappa-caseins. The data obtained on restriction analysis are utilized in mapping the corresponding natural genes of cow caseins. PMID- 3000872 TI - A rapid method of gene detection using DNA bound to Sephacryl. AB - A rapid method of gene detection has been developed utilising DNA fragments immobilized on resins and a sandwich hybridization assay. This method permits the detection of restriction fragment length polymorphisms (RFLPs) without the need to immobilize sample DNA. The method is based on the use of two non-overlapping DNA restriction fragments, one of which is attached to a resin (fragment A) and the other 32P-labelled (fragment B). Fragments A and B will not hybridize to each other unless there is a DNA or RNA fragment capable of hybridizing to both A and B present in the same reaction. Hybridization in this instance will result in the resin being radioactively labelled. The RFLP associated with the mutation causing sickle-cell anaemia was used as a model to develop the method. The resin Sephacryl S-500 appeared most suited to our method for two reasons: (i) DNA immobilization experiments using two coupling procedures and four resins indicated that Sephacryl S-500 bound the most DNA with very little non-covalent coupling. (ii) Hybridization experiments with DNA bound to a number of resins showed that DNA bound to Sephacryl S-500 hybridized most efficiently with a low level of nonspecific hybridization. Using optimum hybridization conditions 5 X 10(-18) mol of beta-globin DNA could be detected. The method has been used to distinguish between DNA from sickle, heterozygote and normal patients. PMID- 3000873 TI - Alterations upstream from the Shine-Dalgarno region and their effect on bacterial gene expression. AB - A vector containing the leftward promoter (pL) as transcription initiation signal and a synthetic, easily adaptable translation initiation region have been constructed. We have used the expression system to assess the relevance of sequences upstream from the Shine-Dalgarno (SD) region in the translational initiation process. To this end, a series of structural variants of the prototype ribosome-binding site were used to direct the synthesis of both mature human fibroblast interferon and beta-galactosidase (beta-gal). It was found that alterations 5' to the SD element can considerably affect the rate of mRNA translation. The observation that the relative efficiency of the various 5' untranslated regions depends on the downstream coding information implies that secondary (and/or tertiary) structure formation is of major importance in the initiation process. But an mRNA folding, in which the SD and ATG determinant are set free in single-stranded regions, does not unconditionally guarantee an efficient initiation of translation. PMID- 3000874 TI - The RAD2 gene of Saccharomyces cerevisiae: nucleotide sequence and transcript mapping. AB - We have determined the nucleotide (nt) sequence of a segment of the yeast chromosome carrying the RAD2 gene. The coding region consists of 2925 bp and could encode a protein of 975 amino acids with a calculated Mr of 111 100. A major transcriptional start point was mapped to a position of approx. 22 bp upstream from the first ATG codon. A number of minor transcriptional start points were also identified in this region, all of them 5' to the putative translational start codon. We noted a number of consensus nt sequences in the 5' and 3' non coding regions of the RAD2, RAD1 and RAD3 genes of Saccharomyces cerevisiae. In addition, three regions of amino acid sequence homology in the putative RAD1 and RAD2 polypeptides were observed. PMID- 3000875 TI - Transformation of Rhodosporidium toruloides. AB - Rhodosporidium toruloides protoplasts could be transformed, in the presence of polyethylene glycol (PEG), at frequencies of approx. 1 X 10(3) transformants/micrograms of DNA. The plasmid used, pHG2, which contains the phenylalanine ammonia-lyase (PAL)-coding gene (PAL) of R. toruloides, could replicate as an unstable plasmid in the yeast, or could integrate at the PAL locus to give stable transformants. Plasmids that function in R. toruloides were constructed using either the PAL gene or LEU2 gene of Saccharomyces cerevisiae as dominant selectable markers. R. toruloides transformed with pHG8, which contains both genes, coinherited the two markers. It is also shown that the 2mu replicon of S. cerevisiae does not function in R. toruloides; neither is the PAL gene expressed in S. cerevisiae. PMID- 3000876 TI - The nucleotide sequence of the essential cell-division gene ftsZ of Escherichia coli. AB - The nucleotide sequence of a 1.8-kb fragment of Escherichia coli DNA containing the essential cell division gene ftsZ is reported. The FtsZ protein has an Mr of 40294 and has 23% charged residues with a calculated isoelectric point of 4.9. The codon usage of the ftsZ gene reflects that of a highly expressed gene. Also located on this DNA fragment is the 3' end of the ftsA gene and the 5' end of the envA gene. These designations were confirmed by locating Tn5 insertions within the ends of these genes that inactivate each of these genes. A potential promoter for ftsZ overlapped the 3' end of the ftsA gene. A Tn5 insertion was located within the 3' end of the ftsA and within this potential promoter. No transcription terminators were evident between ftsA and ftsZ or between ftsZ and envA. PMID- 3000877 TI - Structural organization of rat ribosomal RNA genes: interspersed sequences and their putative role in the alignment of nucleosomes. AB - We have observed four regions containing highly repetitive interspersed sequences in the nontranscribed spacer (NTS) of the rat rRNA genes. Two of them (A and B) are located at a distance of 3-5 kb upstream from the transcription start point and two others (C and D) at a distance of 2-5 kb downstream from the 3' end of the 28S rRNA gene. These repetitive sequences are widely dispersed in the genome and are included both in small-copy regions and in the families of extended reiterated sequences. The sequences of three fragments were determined: one from the C2 region, 1100 bp in length and two from A and C1 regions, 110-120 bp long. These regions are characterized by the presence of 'simple' sequences, such as (AC)n, (ACC)n, (GAG)n, (GGGA)n, (TAAG)n, and also of long blocks, (G)n and (A)n. In the C2 region two palindromes, 16 and 14 nt long, were found, one of them including a XhoI site. Mobile element B2 was observed in regions B and C. All four regions, A, B, C and D, contain sets of simple sequences, among which some common elements have been found. Theoretical prediction of the nucleosomal disposition in the C region indicates that the combination of simple sequences existing in the given area secures fixed positions of the nucleosomes, one of the nucleosomes being formed on the B2 element. Moreover, a striking periodicity, with the repeat length close to that of the rat nucleosomal DNA, has been observed. A hypothesis is put forward that the simple sequences can dictate the location of nucleosomes on the adjoining DNA sequences, thereby regulating the gene activity. PMID- 3000878 TI - Putative regulatory sequences for the transcription of mini-exons in Trypanosoma brucei as revealed by S1 sensitivity. AB - The 35-nucleotide (nt) mini-exon found at the 5' end of most Trypanosoma brucei mRNAs is encoded as part of a tandem 1.35-kb repeat in genomic DNA. We cloned this DNA and identified an S1-sensitive site in supercoiled plasmids containing mini-exon repeats. This site is situated on a poly(dA-dT) stretch that is variable in length in different copies of repeat. Poly(dA-dT) is capable of forming abnormal DNA helix configurations, some of which are induced by supercoiling. The S1 site may have a role in regulation of mini-exon transcription. PMID- 3000879 TI - Immunological detection of cauliflower mosaic virus gene V protein produced in engineered bacteria or infected plants. AB - Antiserum was prepared against a synthetic peptide corresponding to the C terminal 25 amino acids (aa) of the protein encoded by cauliflower mosaic virus (CaMV) gene V, which is thought to be a reverse transcriptase involved in viral DNA replication. This antiserum was used to detect the expression of CaMV gene V either in Escherichia coli JM103 transformed by an expression vector containing CaMV gene V or in CaMV-infected plants. In both cases, an 80-kDal protein has been detected. PMID- 3000880 TI - Insertion of a long KpnI family member within a mitochondrial-DNA-like sequence present in the human nuclear genome. AB - Structural analysis of a phage lambda Charon 4A clone carrying one of the human nuclear mitochondrial(mut)-DNA-like sequences revealed that a KpnI-family member (KpnI 5.5-kb DNA) is inserted within this sequence. The inserted KpnI 5.5-kb DNA contains several possible polyadenylation signal sequences followed by an A-rich sequence at its 3' end and is flanked by perfect 13-bp direct repeats of the duplicated mtDNA-like sequences. These structures strongly suggest that the KpnI 5.5-kb DNA is a mobile element. Comparison of the 5' terminal sequences of the KpnI 5.5-kb DNA and four other long KpnI-family DNAs so far examined, using the predicted general promoter sequence for eukaryotic tRNAs, indicates that they contain the consensus sequences for the split internal RNA polymerase III control region. PMID- 3000881 TI - Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta. AB - Bacillus thuringiensis subsp. kurstaki HD-73 produces a crystal protein which is lethal to many lepidopteran larvae. The gene encoding this crystal protein has been isolated from a 75-kb plasmid and engineered into a recombinant Escherichia coli plasmid for analysis. The complete nucleotide sequences of the coding region and 387-bp 5' and 376-bp 3' to the coding region have been determined. The 3537 bp of the coding region specify a protein of Mr 133 330. The full-length gene and several 3' -truncated derivatives of the gene were examined in both E. coli and in an E. coli minicell-expression system to determine if the carboxy end of the protein is essential for toxicity. The results presented here provide the primary structure of the crystal protein gene and show that the N-terminal 68-kDal peptide is toxic, but at a lower level than the full-length gene product. PMID- 3000882 TI - Expression of a mouse U1b gene in mouse L cells. AB - A 6.9-kb DNA fragment containing two mouse U1b genes was introduced by cotransfection with the Herpes simplex virus (HSV) thymidine kinase (TK) gene into tk- mouse L cells. The parent Ltk- cells produce primarily U1a RNA and only small amounts of U1b RNA. However, after introduction of exogenous U1b genes by cotransfection these cells express large amounts of U1b RNA. Subcloned DNA fragments containing a single U1b gene and varying amounts of DNA flanking the 5' -end of the gene were introduced into Ltk- cells. All of the constructs containing 400 bp or more upstream of the U1b gene were efficiently expressed. By comparison, a subclone containing only 150 bp of DNA flanking the 5' -end of the U1b gene was not efficiently expressed. The U1b transcripts synthesized in transfected cells were identified by hybrid selection and by precipitation of ribonucleoproteins (RNP) containing U1 RNA with anti-RNP antibody. PMID- 3000883 TI - Development of a high-frequency transforming vector for Aspergillus nidulans. AB - The pyr4 gene of Neurospora crassa, which codes for orotidine-5'-phosphate decarboxylase, is capable of transforming an Aspergillus nidulans pyrG mutant by chromosomal integration, despite low homology between the transforming DNA and the recipient genome. Integration of pFB6, a plasmid carrying pyr4 and capable of replication in Escherichia coli, was not observed at the pyrG locus. The efficiency of transformation was considerably enhanced (50-100 fold) by inclusion in the transforming vector of a 3.5-kb A.nidulans chromosomal sequence, ans1. Although this sequence was isolated on the basis of replicating activity in Saccharomyces cerevisiae, there was no evidence for such activity in A.nidulans. Part of the ans1 fragment appears to be reiterated in the A.nidulans genome, though it is not yet clear whether this is directly responsible for the high transformation frequency. The efficiency of transformation of A.nidulans by plasmids bearing ans1, using an improved protocol, was approx. 5 X 10(3) stable transformants per microgram of plasmid DNA. PMID- 3000884 TI - Analysis of the inducible MEL1 gene of Saccharomyces carlsbergensis and its secreted product, alpha-galactosidase (melibiase). AB - We have determined both the nucleotide sequence of the MEL1 gene of Saccharomyces carlsbergensis and the N-terminal amino acid (aa) sequence of its extracellular gene product, alpha-galactosidase (melibiase) (alpha-Gal). The predicted translation product of MEL1 is a pre-alpha-Gal protein containing an 18 aa N terminal signal sequence for secretion. The purified enzyme is a dimer consisting of two 50-kDal polypeptides, each of which is glycosylated with no more than eight side chains. The 5'-flank of the MEL1 gene contains a region (UASm) having certain areas of sequence homology to similar sites found upstream of the structural genes GAL1, GAL7 and GAL10, which are also regulated by the action of the products of genes GAL4 and GAL80. There are three TATA boxes between UASm and the initiation codon of pre-alpha-Gal, as well as a typical yeast cleavage/polyadenylation sequence in the 3'-flank of the gene. PMID- 3000886 TI - Homology between the photoreactivation genes of Saccharomyces cerevisiae and Escherichia coli. AB - A cloned fragment of Saccharomyces cerevisiae chromosomal DNA carrying the photoreactivation gene (PHR) has been sequenced. The fragment contains a 1695-bp intronless open reading frame (ORF) coding for a polypeptide of 564 amino acids (aa). The phr gene of Escherichia coli was also sequenced, and the sequence is in agreement with the published data. The yeast PHR gene has a G + C content of 36.2%, whereas 53.7% was found for the E. coli gene. Despite the difference in G + C content there is a 35% homology between the deduced aa sequences. This homology suggests that both genes have originated from a common ancestral gene. PMID- 3000885 TI - Inverted terminal repeats and terminal proteins of the genomes of pneumococcal phages. AB - The nucleotide (nt) sequence at the ends of the genomes of the Streptococcus pneumoniae phages Cp-5 and Cp-7 has been determined and compared with the corresponding sequence of phage Cp-1. The genomes of phages Cp-5 and Cp-7 have inverted terminal repeats (ITRs) 343 and 347 bp long, respectively. In Cp-1 DNA the ITR is 236 bp long and the following 116 bp are 93% homologous. Some regions within the ITRs are conserved in the three genomes although the complete sequence of the ITRs is no more conserved than the rest of their genomes. The chromatographic behavior of their tryptic peptides suggests that the terminal proteins (TPs) of at least two of the phages are similar and that the TPs of the three pneumococcal phages differ markedly from that of the Bacillus subtilis phage psi 29. PMID- 3000887 TI - Sequence comparison of human and murine erythrocyte alpha-spectrin cDNA. AB - The results of hybridization analyses using cDNA probes for mouse and human alpha spectrin mRNA indicate that a single gene encodes the alpha-subunit of erythrocyte spectrin. Sequencing of the cDNA clones showed that they code for 370 amino acids (aa) covering three repeat domains close to the C terminus of alpha spectrin. The cloned cDNAs will now permit the isolation of the alpha-spectrin gene and should lead to the characterization of the genetic aspects in human hereditary anemias in which alpha-spectrin has been characterized as the site of the molecular defect. PMID- 3000888 TI - Restriction endonuclease EcoO109 from Escherichia coli H709c with heptanucleotide recognition site 5'-PuG/GNCCPy. AB - A new restriction endonuclease, EcoO109, has been isolated from Escherichia coli H709c by polyethyleneimine (PEI) precipitation, DEAE-cellulose chromatography and heparin agarose chromatography. The yield was high, more than 3000 units/g of wet cells. The EcoO109 endonuclease recognizes and cleaves a nucleotide sequence of (formula: see text), in the presence of 10 mM Mg2+. The enzyme will be useful for structural analysis and molecular cloning of DNA because of the stability, high yield and easy handling of the producer strain. PMID- 3000889 TI - The structure of a cloned mouse gamma-actin processed pseudogene. AB - The complete nucleotide (nt) sequence of a gamma-actin-like pseudogene (M gamma A psi 1), isolated from a mouse genomic library in phage lambda, was determined. The pseudogene was shown to be of the processed type by the fact that it lacked introns, ended in a poly(dA) region, and was flanked by direct repeats. There were ten differences in the predicted amino acid (aa) sequence from that of the authentic nonmuscle gamma-actin. An unusual feature of M gamma A-psi 1 was the complete absence of DNA corresponding to the 5' end of the mRNA up to the nt preceding the Ala codon at aa position 7. This suggests that M gamma A-psi 1 originated from a truncated mRNA or from an incomplete reverse transcript. PMID- 3000890 TI - Identification of a mutation that relieves gamma-glutamyl kinase from allosteric feedback inhibition by proline. AB - A 1.75-kb DNA fragment containing the entire Escherichia coli proB+ gene has been sequenced. The proB locus encodes the structural gene for gamma-glutamyl kinase (GK), the enzyme responsible for the first step in proline biosynthesis, and the primary regulatory point of the pathway. We have previously reported the nucleotide (nt) sequence of a mutant proB gene isolated from an E. coli strain resistant to the toxic analog of proline, 3,4-dehydro-DL-proline (DHP). This mutant gene encodes a GK which is refractory to allosteric feedback inhibition by proline (DHPR). Comparison of the proB+ and DHPR proB sequences revealed a single base difference, an A-T to C-G transversion localized at nt position 428 within the amino acid (aa) coding region of proB. This mutation predicts an aa change from glutamic acid in the wild-type (wt) enzyme to alanine in the DHPR enzyme. PMID- 3000891 TI - Isolation and characterization of the Streptomyces cattleya temperate phage TG1. AB - A temperate actinophage, TG1, was isolated from soil by growth on Streptomyces cattleya and has been shown to be potentially useful for the cloning of DNA in this organism and other streptomycetes. It forms stable lysogens by integration at a unique site on the chromosome. The phage genome consists of 41 kb of double stranded DNA with cohesive ends. It has unique sites for ClaI, NdeI, PstI, SmaI, and XbaI. The PstI site has been shown to be in a dispensable region of the phage genome. Deletions (2 kb in length) were obtained which retain this site and should be useful for the cloning of DNA. PMID- 3000892 TI - Restriction endonucleases in Azospirillum. AB - Azospirillum brasilense, A. amazonense, and A. lipoferum strains were screened for restriction endonucleases using phage lambda DNA. The extract of A. brasilense 29711 cleaved lambda DNA into specific fragments. It was concluded that this strain possesses a class II restriction endonuclease which was named AbrI. AbrI has a single recognition site on lambda DNA at position of approx. 33 500 bp. AbrI was characterized as an isoschizomer of XhoI, which cuts lambda DNA at 33 498 bp and cleaves double-stranded DNA at the sequence 5'-C TCGAG-3'. From other Azospirilla strains only A. amazonense QRZ42 extracts (AamI activity) cleaved DNA into specific fragments under certain conditions. PMID- 3000893 TI - Cloning of mini-mu bacteriophage in cosmids: in vivo packaging into phage lambda heads. AB - A technique has been developed that permits the packaging of mini-Mu-carrying cosmids into phage lambda heads. This procedure has several advantages over packaging into Mu helper capsids: the amounts of DNA to be packaged can be increased, the packaging efficiency is improved, and the stability of transducing lysates is high. PMID- 3000894 TI - A method for efficient gene isolation from phage lambda gt11 libraries: use of antisera to denatured, acetone-precipitated proteins. AB - Experience with cloning pseudorabies virus (PRV) DNA in the lambda gt11 phage vector has shown that there are special requirements for the antisera used in screening the libraries, in addition to the requirement that the antisera recognize proteins on a Western blot. Initial screening of a lambda gt11 library of sheared PRV DNA fragments in Escherichia coli for expression of PRV antigens using PRV hyperimmune antisera was unsuccessful. It was only after screening the library with antisera raised against PRV proteins eluted from sodium dodecyl sulfate (SDS)-polyacrylamide (PA) gels that positive results were obtained. These "gel-slice" antisera (GSA) were equivalent in potency to hyperimmune antisera in standard immunoassays (including ELISA, immunoprecipitation, Western blots, and neutralization of virus), but only the GSA could recognize PRV fusion proteins expressed by recombinant lambda gt11 phage. This difference was seen despite the fact that hyperimmune antisera performed satisfactorily on Western blots of denatured PRV-infected cell extracts. These results show that the efficiency of screening expression libraries in E. coli can be improved if antibodies are raised against denatured proteins. PMID- 3000895 TI - [Tubal reanastomosis in the rabbit with different suture materials]. PMID- 3000896 TI - [Effect of fibrogenous dust on oxygen consumption by the peritoneal macrophages (electron paramagnetic resonance study]. PMID- 3000897 TI - [Working conditions of the workers of the "Sel'khozkhimiia" warehouses]. PMID- 3000898 TI - [Disorders of creatine metabolism in vibration disease]. PMID- 3000899 TI - [Use of the interpolation method to evaluate working conditions in high-frequency plasma technology]. PMID- 3000901 TI - [Prostaglandins in the cardio-vascular system]. PMID- 3000902 TI - [Malignant fibrous histiocytoma of the paranasal sinuses]. PMID- 3000900 TI - Membrane receptors for fibrinogen and factor VIII and abnormalities of platelet function. A review. PMID- 3000903 TI - [The viral hypothesis of the etiology of multiple sclerosis]. PMID- 3000904 TI - Fish and heart disease. PMID- 3000905 TI - Asymptomatic decreased activities of hepatic glucose-6-phosphatase and glycogen phosphorylase in a number of children with chronic liver disease. AB - The evaluation of hepatic degradation of glycogen in patients with different chronic liver diseases was carried out on the basis of: a) specific activities of hepatic enzymes involved in catabolism of glycogen; b) level of glycogen in liver biopsies; c) concentration of glucose and cAMP in serum after the intravenous administration of glucagon. In 13 out of 35 patients investigated the activity of glucose-6-phosphatase was decreased to 14-50% of the control value. In the livers of 3 patients glycogen phosphorylase activity was decreased to 10% of the control value. In patients with the significantly low activities of hepatic glucose-6 phosphatase and phosphorylase a, however, normal catabolism of glycogen in the liver was observed, neither hypoglycemia nor abnormal glycogen storage in liver biopsies nor abnormal response to glucagon being found. In the group of patients with decreased and normal activities of glucose-6-phosphatase and phosphorylase a, biochemical parameters in the serum (i.e. markers of liver damage) were not detectable. Possible causes of the selective and asymptomatic decrease in the activities of glucose-6-phosphatase and phosphorylase a are discussed. PMID- 3000906 TI - [Analysis of Epstein-Barr virus transformation of human lymphocytes: susceptibility of B lymphocyte subpopulations and differentiation stage of the transformed cells]. AB - The subpopulations of B lymphocytes in human adult peripheral blood which are susceptible to infection and transformation by Epstein-Barr virus (EBV), and also the stage of EBV-transformed cells in B cell differentiation lineage were identified. The EBV susceptibility were analyzed by two-color immunofluorescence and colony formation in semisolid agar. Surface Ig-bearing lymphocytes of 3 major Ig classes, IgM, IgG, and IgA, in peripheral blood expressed viral nuclear antigen (EBNA) after exposure to the virus, indicating that all these subpopulations are susceptible to EBV infection. In contrast, cytoplasmic Ig positive lymphocytes were totally resistant to infection. The Ig-positive cells of either Ig class proliferated equally after EBV infection and formed colonies in semisolid agar, thereby showing that these subpopulations are also susceptible to transformation by EBV. Analysis of clonally transformed cells originating from each target cell by using a series of newly developed monoclonal antibodies to B cell differentiation antigens showed that EBV-transformed cells corresponded to the immunoblast stage of B cell differentiation. These data suggest that EBV infects and transforms mature B lymphocytes, and makes them differentiated to the more advanced immunoblast stage within the terminal B cell differentiation process. PMID- 3000907 TI - In-vitro studies of the development of pituitary and testicular functions in diabetes (C57Bl/KsJ-db/db) mutant mice. AB - The hypothesis that male diabetes mutant mice (C57Bl/KsJ-db/db) are suffering from impairment of testicular steroidogenic function and pituitary LH release was tested. A smaller postpubertal increase of testicular weight and a reduction of plasma testosterone and androstenedione levels by 65% at 17 weeks of age were most obvious from the comparison to homozygous lean controls. The ability of constant amounts of Leydig cells, either in crude interstitial cell or in purified Leydig cell suspensions, to respond to maximal doses of hCG or cyclic AMP-was reduced by at least 40% in adult diabetes mice. This defect could be attributed to a 40% decrease of steroid-17 alpha-monooxygenase activity as compared to lean mice. No differences occurred, however, if Leydig cells were submaximally stimulated. GnRH-stimulated pituitary LH release was not significantly changed. The impairment of testicular steroidogenic function in diabetes mutant mice may represent a further aspect of infertility of these animals and of diabetes mellitus. PMID- 3000908 TI - Specific epidermal growth factor receptors on porcine and human thyroid membranes. AB - Specific, high affinity, saturable receptors for epidermal growth factor (EGF) have been demonstrated both on porcine and on human thyroid membranes. The binding affinities of porcine (Ka 3.0 X 10(-9) M) and human thyroid EGF receptors (Ka 1.75 X 10(-9) M) are very similar. TSH does not inhibit the binding of 125I EGF to either membrane. These results suggest the possibility that EGF may be involved in the regulation of human as well as porcine thyroid follicular cell growth and function. PMID- 3000909 TI - ACTH-provoked cortisol peaks during sleep and their effect on the endogenous secretory activity. AB - The effects of low physiological doses of ACTH on nocturnal plasma cortisol patterns were investigated in six male subjects. Concomitant sleep EEG recordings were analysed in relation to the cortisol level. 250 ng ACTH1-24 (Synacthene), injected through an indwelling catheter at a period of low adreno-cortical activity (2400 h), induced a cortisol peak followed by a four to five-hour period without any cortisol secretion. The same dose of ACTH injected at 0430 h, when cortisol secretory activity was high, did not entirely abolish endogenous secretion, which was diminished for a shorter time (2.5 hr). The ACTH-provoked cortisol peaks of comparable size to endogenous secretory peaks, can suppress cortisol secretion for several hours. This suppressive capacity depends on timing in relation to high or low secretory activity periods. However, spontaneous cortisol peaks have no appreciable effect on further secretory episodes. This difference in suppressive capacities suggests that the 24 hr cortisol rhythm is regulated independently of such feedback mechanisms. PMID- 3000910 TI - Autoradiographical portrayal of TSH receptors in human thyroid gland tissues. PMID- 3000911 TI - Some properties of the plasma hGH activity in patients with Laron-type dwarfism determined by a radioreceptor assay using human liver tissue. AB - A radioreceptor assay for human growth hormone (hGH) using the 100,000-g pellet of human liver tissue homogenates obtained from a 13-year-old male donor of a kidney transplantation is described. The dilution curves of the plasma hGH of 6 patients with Laron-type dwarfism (LTD) as well as those of the plasma hGH from 1 normal child and 2 acromegalic patients were all found to be parallel to the standard curve, suggesting that in the LTD syndrome the circulating hGH is biologically active. PMID- 3000912 TI - ACTH stimulation of adrenal epinephrine and norepinephrine release. AB - Epinephrine (E) and norepinephrine (NE) levels were measured simultaneously in the adrenal veins of 6 patients before and after stimulation with 0.25 mg beta 1 24 ACTH. In 1 patient with Cushing's syndrome, E and NE were also measured before and 30 min after dexamethasone. There was a significant increase in NE and E secretion (p less than 0.002) from both adrenal glands after ACTH stimulation. In the patient with Cushing's syndrome, there was also a slight increase in plasma E levels after dexamethasone. It is postulated that ACTH stimulated NE and E secretion by augmenting blood flow through the adrenals and by induction of tyrosine hydroxylase and dopamine beta-hydroxylase, although a direct effect of ACTH on NE and E secretion cannot be excluded. It is also possible that the increase in adrenal catecholamine secretion after ACTH may be due to ACTH augmentation of catecholamine secretion by endogenous opioids such as beta endorphin. PMID- 3000913 TI - Cushing's syndrome due to unilateral adrenal nodular hyperplasia with incomplete inhibition of the contralateral gland. AB - A 57-year-old woman was demonstrated to be affected by adrenocorticotropic hormone (ACTH)-independent Cushing's syndrome. Computed-axial tomography of the abdomen demonstrated an expansion of the left adrenal. In apparent contrast with these findings, adrenal scintigraphy demonstrated radiocholesterol uptake also by the right gland. At surgery, the left adrenal was found to be hard and enlarged and was excised, while the right gland was found of normal appearance and left in place. Histologic examination of the excised gland demonstrated nodular hyperplasia. Early after surgery, plasma cortisol returned to normal values with a normal circadian rhythm and complete inhibition by low dose dexamethasone; the response of plasma cortisol to ACTH was normal. The patient represents a rare case of unilateral adrenal nodular hyperplasia. Radiocholesterol uptake by the contralateral gland and early recovery from adrenal atrophy after surgery are exceptional findings and suggest incomplete inhibition of endogenous ACTH. PMID- 3000914 TI - AIDS: a time bomb at hospitals' door. PMID- 3000915 TI - Telomeric association in a malignant fibrous histiocytoma. AB - In a malignant soft-tissue fibrous histiocytoma 50-56 chromosomes were found in the majority of the metaphases. The most frequent numerical aberrations were one or two extra copies of chromosomes 4, 5, 18, 20, 22, and a missing chromosome 15. Structural rearrangements encountered were 11p+ and 1-5 unidentifiable markers. The most conspicuous feature was pairs of chromosomes intimately associated or fused at their telomeres, observed in 20 out of 22 metaphases. Although the telomeres of 6p, 11p, 16q, 20q, and 21p were involved most frequently, no preferential pattern of associations was detectable. This peculiar chromosomal behavior is compared to similar observations recently reported in a case of a B cell lymphoid leukemia. PMID- 3000916 TI - Human ferritin light chain gene sequences mapped to several sorted chromosomes. AB - The iron storage ferritin light-chain gene exhibits multiple restriction enzyme fragments which have been mapped by analyzing sorted human chromosomes. A dual laser chromosome sorter was used to construct spot-blot filter panels representing 22 chromosome fractions. Hybridization of radiolabeled human ferritin-L gene probe to spot-blot panels revealed the ferritin-L gene on more than one chromosome. Miniaturized restriction enzyme analysis was used to map each of the ferritin-L restriction fragments uniquely to one of three chromosomes. This combination of sorted chromosome analyses provides a rapid method to map homologous DNA sequences located on more than one chromosome. PMID- 3000918 TI - Regulatory studies on inducible catechol 1,2-dioxygenase in Pseudomonas solanacearum. PMID- 3000917 TI - Adrenocorticotrophic influence on activity of the human kidneys. PMID- 3000919 TI - [Current status of bone marrow transplantation]. PMID- 3000920 TI - [Epidemiology, etiology and laboratory diagnosis of infectious diarrhea diseases in the tropics]. AB - Diarrhoeal diseases belong to the leading causes of morbidity and mortality in tropical countries, especially in infants and small children. About one billion episodes are estimated for this group of age with 4.6 million fatalities. Many causes are discussed to explain the high incidence: bottle feeding of infants, protein malnutrition, unsafe drinking water and unsafe disposal of excrements and sewage, unsufficient consciousness of personal and domestic hygiene, lack of knowledge on the origin of disease, and inadequate food hygiene. Among the viral infectious agents rotavirus is isolated from 30-40% of enteritis cases in infants and small children; Norwalk virus, adenovirus and human calicivirus, too, appear to occur worldwide. Enteropathogenic (EPEC) and, especially, enterotoxigenic Escherichia coli (ETEC) are of primary importance as bacterial pathogens. Campylobacter jejuni and C. coli primarily cause disease in infants and small children. Shigellae are important whereas salmonellae are less frequently identified as a cause for diarrhoeal illness. Yersinia enterocolitica is rare in tropical countries. Cholera and infections with other vibrionaceae (V. cholerae non-01, V. mimicus, V. parahaemolyticus, aeromonas, Plesiomonas shigelloides) mainly occur in coastal areas. Clostridium perfringens type C is the causal agent of Enteritis necroticans in several countries, especially New Guinea. C. difficile appears to be of minor importance. Among the parasites Giardia lamblia and Entamoeba histolytica are the most important organisms causing diarrhoeal disease. Macroscopic (visible blood) and microscopic stool examination (faecal leukocytes) may be helpful in dysenteric disease to distinguish between shigella and E. histolytica infections; it is, however, of limited usefulness to discriminate between organisms causing watery diarrhoea. PMID- 3000921 TI - [Modification of the incidence and course of CMV infections following kidney transplantation by passive immunization--initial experiences with a hyperimmunoglobulin]. AB - In 45 recipients of a renal transplant the CMV-antibody titer was measured preoperatively (ELISA method). If possible, the donors were examined likewise. All seronegative recipients of grafts of a seropositive donor were immunized passively with a CMV-hyperimmunoglobulin for 6 months (2 ml/kg bw in 3 weeks intervals). 7 patients showed that constellation, and they were treated. 4 of them demonstrated neither serological nor clinical signs of a CMV-infection at any time. In 3 patients an infection was found serologically, but only 2 showed concomitant clinical symptoms. No serious complications (pneumonia etc.) were seen. One year later all patients are doing well with a functioning graft. For this reason we think the passive immunization of a seronegative recipient of a graft from a seropositive or unexamined donor to be advisable. PMID- 3000922 TI - [Frequency of infection with cytomegaloviruses (CMV) following kidney transplantation and initial experiences with CMV gamma globulin prevention]. AB - In a retrospective study we investigated 364 patients who had received cadaveric kidney transplants between 1978 and 1984 as to cytomegalovirus (CMV) infections using complement-fixation test and indirect immunofluorescent test (IgM, IgG). Before transplantation, 194/364 patients were seropositive and 170/364 were seronegative. After transplantation, seroconversion or an increase in titre (greater than or equal to 4 fold) was found in 26% of seropositive recipients and in 30% of seronegative recipients. In contrast to the secondary infections (approximately 40% asymptomatic or oligosymptomatic), the primary infections were regularly connected with a clinical symptomatology. As to the recipient's and donor's pre-transplant antibody status the highest infection rate (62%) was seen in the group of seronegative recipients of kidneys from seropositive donors. Preformed CMV antibodies seem to be useful in preventing severe CMV-diseases. Therefore, in April 1985 we started a randomized study in order to investigate the efficacy of an i.v. cytomegalovirus-immunoglobulin (CMV-Polyglobin/Cutter). There were no side effects. Already 4 h after infusion we were able to detect CMV IgG-antibodies (IFT) in peripheral blood. This passive immunization, however, was not capable to prevent a CMV infection in each case; there were 5/8 seroconversions and 4/8 symptomatic CMV infections. Nevertheless, we think the application of CMV immunoglobulin has a beneficial effect. PMID- 3000923 TI - [Use of a polyvalent intravenous immunoglobulin or specific cytomegalovirus hyperimmunoglobulin for modification of cytomegalovirus infections and prevention of interstitial pneumonias following bone marrow transplantation]. AB - The effects of prophylactic, polyvalent intravenous immune globulin on cytomegalovirus infection and interstitial pneumonia in allogenic marrow transplants were evaluated in an ongoing, randomized controlled trial. Thirty eight patients were given weekly doses (20 cc/kg) of polyvalent intravenous immune globulin before and after transplantation, and 37 patients were controls. Both symptomatic cytomegalovirus infection (17 of 37 or 46% vs. 8 of 38 or 21%, p = 0.04) and interstitial pneumonia (17 of 37 or 46% vs. 7 of 38 or 18%, p = 0.02) occurred less frequently in the recipients of polyvalent intravenous immune globulin. In separate kinetic studies, a 5 cc/kg dose of a cytomegalovirus specific hyperimmune globulin produced cytomegalovirus antibody titers in patients equivalent to those achieved after the 20 cc/kg dose of polyvalent intravenous immune globulin. All immune globulin preparations were well tolerated. These preliminary results suggest that intravenous immune globulin can modify the severity of cytomegalovirus infection and prevent interstitial pneumonia in marrow transplants. Additional trials are now needed to define the minimal effective dose of intravenous immune globulin and to compare the effectiveness of different intravenous immune globulin formulations. PMID- 3000924 TI - [Risk factors for the occurrence of pneumonia caused by cytomegaloviruses in patients with bone marrow transplants during prevention with cytomegalovirus hyperimmune globulin]. AB - After bone marrow transplantation (BMT) transient combined immunodeficiency occurs which promotes the occurrence of severe cytomegalovirus (CMV) infections, the most frequent lethal complication at present. 28 patients received a CMV-IgG hyperimmunoglobulin (CMV-IG) intravenously as CMV-prophylaxis. The efficacy of this treatment and the risk factors for the occurrence of interstitial pneumonia (IP) caused by CMV were analyzed. Risk factors promoting a CMV-IP were: immunosuppression after BMT, CMV-seropositivity of recipient and donor, granulocyte transfusions and HLA-mismatched BMT. In this study graft versus host disease did not influence the occurrence of CMV-IP. PMID- 3000925 TI - [Cytomegalovirus hyperimmune globulin prophylaxis in bone marrow transplants in children with various diseases]. AB - The effect of hyperimmune globulin for the prevention of cytomegalovirus (CMV) infections following bone marrow transplantation (BMT) was evaluated in 21 children with various diseases and compared to a historical group of 23 children without prophylaxis. A higher incidence of interstitial pneumonia (19%) as well as CMV-associated infections with or without pneumonitis (29%) could be demonstrated in patients with CMV-prophylaxis as against the rate of interstitial pneumonia (4%) and CMV-infections (8%) in children without prophylaxis. This surprising observation was very likely due to selection of patients with high risk features and higher incidence of GVHD in the prophylaxis group. The analysis of patients with prophylaxis failure shows a low CMV-infection rate in initially seronegative marrow recipients and a high risk for CMV-infections in seropositive patients. Therefore, even though CMV-infections could not be completely abrogated, hyperimmune globulin administration might have reduced CMV complications in seronegative patients. In CMV-seropositive marrow recipients, however, this type of prophylaxis remains unsatisfactory in preventing severe CMV infections caused by virus reactivation. PMID- 3000927 TI - Oxygen radical release by adherent cell populations during the initial stages of a lethal rodent malarial infection. AB - A series of experiments was carried out to assess the levels of reactive oxygen intermediates (ROI) released by macrophages and monocytes during an acute malarial infection, and to consider the importance of oxidant-induced parasite killing in host protection. Adherent cell populations were removed from the peritoneum and spleen of BALB/c and B10/D2/n mice between Days 0-5 of a Plasmodium yoelii nigeriensis infection. These cell populations were quantified, characterized and their ROI-releasing capacity was measured by following ferricytochrome c reduction upon stimulation with phorbol myristate acetate (PMA). Both strains of mice displayed higher numbers of macrophages and macrophage precursors as the infection progressed; this rise was more marked and accompanied by splenomegaly in BALB/c mice. A concurrent decrease in peritoneal cell numbers was observed. Splenic adherent cell populations released much lower levels of ROI than peritoneal macrophages upon triggering. The levels of ROI released from BALB/c splenic adherent cells rose gradually until Day 3, when the parasitaemia was slightly decreased. In contrast, splenic populations from B10 mice had a decreased capacity to release ROI, particularly after Day 3, when the parasitaemia rose sharply. In further studies, electron microscopy was used to detect H2O2 release during the in vitro interaction of peritoneal macrophages and parasitized erythrocytes. Cerium chloride staining techniques demonstrated that H2O production was not dependent on phagocytosis or the presence of immune serum, although levels were increased by the presence of the latter. PMID- 3000930 TI - [Histochemical and immunohistochemical study of mixed tumors with unusual histologic aspects]. PMID- 3000929 TI - [Plantar ulcer caused by motor and sensory radicular neuropathy]. PMID- 3000926 TI - Cyclosporine does not inhibit mitogen-induced inositol phospholipid degradation in mouse lymphocytes. AB - The degradation of phosphatidylinositol bisphosphate (PIP2) to diacylglycerol and inositol trisphosphate is elicited by ligand-receptor interactions in many cell types, and may be involved in the induction of cell growth. The mitogens concanavalin A and anti-immunoglobulin antibodies have previously been shown to induce degradation of PIP2 in mouse thymocytes and B cells, respectively. We have now investigated the effects of the immunosuppressive peptide cyclosporine (CS) on this response, since CS appears to inhibit an early step in lymphocyte activation by mitogens that induce PIP2 degradation and Ca2+ mobilization. We found that CS, at doses that completely abrogated the proliferative responses, did not affect the degradation of inositol phospholipids in either thymocytes or B cells. We therefore conclude that if PIP2 degradation is implicated in lymphocyte activation, then CS does not interfere with the second messenger production initiated by PIP2 breakdown, but rather with a later event(s) elicited by this pathway. PMID- 3000928 TI - Cytotoxic T lymphocytes and natural killer cell activity in the course of mengo virus infection of mice. AB - Inbred C57BL/6 mice were inoculated intraperitoneally (i.p.) with mengo virus. The activity of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells were measured during the first 22 days following infection. The CTL response began 7 days after virus inoculation, persisted for at least 22 days and was related to the dose of the virus inoculated. NK cell activity was elevated within 24 hr, reached its peak level on the fourth day and declined to normal levels on the eleventh day after exposure to the virus. These results suggest that NK cells represent the first cellular immune response to restrict mengo virus spread while specific CTL appear later and are probably responsible for further restriction, elimination and prevention of the viral disease. PMID- 3000931 TI - Effect of Geriforte in vivo and in vitro on age-related enzyme changes in liver and brain of rats. PMID- 3000932 TI - Increased levels of lipids, NAD-isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase and phosphatases in mated cultures of Blakeslea trispora. PMID- 3000933 TI - Delta-9-tetrahydrocannabinol induced changes in rat intestinal brush border enzymes. PMID- 3000936 TI - Urinary pyrophosphate excretion in stone formers. PMID- 3000934 TI - Evidence for chromatin structure as a regulatory determinant in HLA-DR alpha gene expression. AB - We examined the possibility that one mechanism for controlling HLA-DR alpha gene expression involves the alteration of chromatin structure. Chromatin structure was analyzed by measuring the susceptibility of DR alpha genes in intact nuclei to nuclease treatment. We first examined a somatic cell hybrid of a T lymphoblastoid cell line (LCL) and a B-LCL, since the DR alpha gene, which is inactive in the T-LCL parent, is expressed in the hybrid, thus providing a system to study DR alpha gene induction. The hybrid line 174 X CEM.T1 contains and expresses solely the DR alpha gene from the T-LCL parent, since the DR alpha gene from the B-LCL parent, 174, is deleted. Using cytoplasmic dot blot analysis and RNA-DNA Northern hybridization, we detected DR alpha-specific transcripts in the hybrid, but not in the parental lines, indicating activation of the DR alpha gene in the hybrid. The transcribed DR alpha gene from the hybrid was compared with the untranscribed gene from the T-LCL parental line, and an association between DR alpha gene expression and increased sensitivity to DNase I was observed. A switch in the chromatin structure of the DR alpha gene from a closed to an open configuration apparently occurred in this hybrid. Such a change is associated with DR alpha gene expression. Comparison of a DR-positive B-LCL and an isogenic DR-negative T-LCL also showed that the chromatin of the former is more sensitive to DNase I digestion. There were no restriction enzyme fragment length differences between the DR alpha genes from 174 X CEM.T1 and CEMR, indicating that the process of somatic cell hybridization did not result in DNA rearrangement or translocation. PMID- 3000935 TI - DNA polymorphism related to HLA-DR2 Dw2 in patients with narcolepsy. PMID- 3000937 TI - Role of angiotensin II in renal wrap hypertension. AB - The role of angiotensin II in the development of renal wrap hypertension was studied in rabbits that underwent either bilateral renal cellophane wrap or sham operation. In half the rabbits, angiotensin II production was blocked by continuous administration of enalapril. Four weeks after renal wrapping, mean arterial pressure had risen by 48 +/- 5 mm Hg in untreated rabbits, but by only 25 +/- 4 mm Hg in enalapril-treated rabbits (p less than 0.01). Similar differences were also measured 6 weeks after wrapping. In untreated rabbits, plasma renin activity had increased fourfold 4 and 6 weeks after renal wrapping. There were no significant changes in blood pressure or plasma renin activity following sham operation. Compared with that in sham-operated rabbits, renal blood flow was reduced by 60% in the untreated rabbits 4 weeks after wrapping but by only 30% in the enalapril-treated wrapped rabbits (p less than 0.05). Renal vascular resistances were 5.5 +/- 1.7 mm Hg . ml-1 . min-1 and 1.2 +/- 0.1 mm Hg . min . ml-1 in the untreated wrapped and sham-operated rabbits respectively and 1.9 +/- 0.4 mm Hg . min . ml-1 and 0.8 +/- 1 mm Hg . min . ml-1 in the enalapril treated wrapped and sham-operated rabbits. Renal wrapping did not alter filtration fraction in untreated rabbits, but markedly reduced it in enalapril treated rabbits. These results suggest that angiotensin II had two major effects in rabbits after bilateral renal wrapping: it contributed substantially to the increase in blood pressure and caused renal vasoconstriction, primarily at a postglomerular site. PMID- 3000939 TI - Abnormalities of platelet function in hypertension and diabetes. AB - The increased frequency of hypertension in diabetes and of abnormalities of carbohydrate metabolism in hypertension are now well established. It is conceivable that the high coincidence of the two diseases is based on a common metabolic defect. Studies of platelets permit the evaluation of the stimulatory, phosphoinositol-linked and the inhibitory, cyclic adenosine 3',5'-monophosphate dependent pathways of cell activation. Furthermore, platelets may be relevant for the development of angiopathy through their contents of growth factors. Abnormalities of platelet aggregation have been demonstrated in hypertension and diabetes. They are accompanied by exaggerated stimulation of adenylate cyclase in hypertension and abnormal activity of cyclic guanosine 3',5'-monophosphate phosphodiesterase in diabetes. Defective function of platelets is also observed in patients and animals when the two diseases are present at the same time. Both increased and decreased aggregation have been described in these two diseases in the literature. The apparent discrepancies may be due to different types of platelet preparation, evaluation of aggregation, evolution of defect with age, and form of the disease. Integrated studies of biochemical mechanisms responsible for cell activation are needed to characterize the exact defect present in diabetes and hypertension in platelets. PMID- 3000938 TI - Effect of dietary sodium on platelet alpha 2-adrenergic receptors in essential hypertension. AB - To study the aggregation, adhesion, and specific binding of an alpha 2 antagonist, [3H]rauwolscine, to the platelet membrane fractions, platelets were obtained from 30 patients with essential hypertension and nine normotensive subjects fed a high sodium diet (NaCl, 16-18 g/day) for 7 days and thereafter a low sodium diet (NaCl, 1-3 g/day) for 7 days. The patients with essential hypertension were classified as either salt responders (all those who had greater than 7% decrease in mean arterial pressure from the high to low sodium period) or salt nonresponders (all others). In salt responders, the number of alpha 2 adrenergic receptors on platelet membrane fraction was increased from 523.4 +/- 55.4 fmol/mg of protein in the high sodium period to 669.4 +/- 84.0 fmol/mg of protein in the low sodium period (p less than 0.01), whereas it did not change in salt nonresponders. In contrast, the epinephrine-induced platelet aggregation through alpha 2-adrenergic receptors was decreased in nonresponders, from 47.3 +/ 7.4% in the high sodium period to 24.5 +/- 9.3% in the low sodium period (p less than 0.05), while it did not change in responders. No significant change in the number of alpha 2-adrenergic receptors or epinephrine-induced platelet aggregation was observed in the normotensive subjects. PMID- 3000941 TI - Differential effects of monoHETEs (monohydroxyeicosatetraenoic acids) on arachidonic acid metabolism in glycogen-elicited rat polymorphonuclear leukocytes. AB - We investigated the effects of monohydroxyeicosatetraenoic acids (monoHETEs) on lipoxygenase- and cyclooxygenase-catalyzed reactions in glycogen-elicited rat PMNs challenged with A23187 and exogenous [14C]arachidonic acid. A23187 (10 microM) stimulated a 10-, 4-, 1.7- and 1.8-fold increase in the synthesis of radiolabeled 5-HETE, LTB4, TxB2, and PGE2 by rat PMNs. Addition of 5-HETE, 5 lactone-HETE, 12-HETE, and 15-HETE led to a dose-related reduction in [14C]5-HETE and [14C]LTB4 synthesis by these cells. These monoHETEs also inhibited [14C]TxB2 synthesis, but only 5-HETE and 5 lactone-HETE inhibited the synthesis of [14C]PGE2. Both 12-HETE and 15-HETE failed to reduce the formation of [14C]PGE2. These results suggest that monoHETEs differ significantly in their effects on arachidonic acid metabolism in rat PMNs and may play a role in modulating the synthesis of both lipoxygenase and cyclooxygenase products. PMID- 3000940 TI - NADPH and "cocktails" containing polyarginine reactivate superoxide generation in leukocytes lysed by membrane-damaging agents. AB - Human blood leukocytes generated large amounts of superoxide (O2-) following stimulation by certain "cocktails" of soluble agents consisting of poly-L arginine (PARG), phytohemagglutinin, the chemotactic peptide formyl-methionyl leucyl-phenylalanine and polyanethole sulfanote (liquoid). A variety of cytochalasins, which markedly boosted O2- generation by the soluble cocktails, markedly depressed luminol-dependent chemiluminescence (LDCL) which had been induced either by opsonized streptococci or by soluble agents. Glutathione, which totally reversed the inhibition of LDCL induced by cytochalasin A, failed to reverse the inhibition of LDCL induced by cytochalasin B. Generation of O2- by all the soluble agents employed, except PMA, was strongly inhibited either by the omission of extracellular calcium and magnesium or by treatment with the calcium blocker TMB-8. Generation of O2- was enhanced following stimulation of leukocytes with soluble agents if the cells had been exposed to slightly hypotonic buffers. Leukocytes, which had been preincubated for short periods (5 min) with PARG, saponin, digitonin, or lysolecithin (LL) and which lost their viability, and their O2- and LDCL-generating capacities following stimulation by soluble agents containing cytochalasin B, nevertheless regained these activities by the addition of NADPH. It is suggested that the lytic agents induced the leakage out of NADPH rather than acting as inactivators of the oxidase in the leukocyte membranes. Prolonged incubation of leukocytes with lytic agents failed to allow restoration, by NADPH, of the generation of SOD-inhibitable O2- generation. Since PARG acted both as a cytolytic agent and as a inducer of O2- generation, we postulate that lytic agents might also act as "primers" of the nascent membrane oxidase which could, however, be further potentiated and activated by soluble agents acting in "multiple hits," PARG could be totally replaced either by LL or by digitonin in the generation of O2- provided that both PHA and cytochalasin B were present in the reaction mixtures. We suggest that the various ingredients of the soluble "cocktails" may help to assemble components of the NADPH oxidase. Such an assembly and regulations are prerequisite for stimulation of the NADPH oxidase and the generation of oxygen radicals in leukocytes. PMID- 3000942 TI - Slow exponential decay of rate of superoxide production in phorbol ester activated human neutrophils. AB - Proper quantification of the superoxide (O2-) respiratory burst induced in human neutrophils is important for better understanding of the mechanism of action of stimulators and inhibitors. Reexamination of the reaction triggered by phorbol myristate acetate (PMA) indicated that it was a persistent process which lasted over 60 min. Plots of rates versus time show that rates of O2- release decayed logarithmically with a mean half-life (T1/2) of 21 +/- 6 min (SD), N = 12). Calculations of areas under curves indicate an average O2- yield of 217 +/- 99 nmol/10(6) cells. The inclusion of catalase in incubation mixtures did not alter the T1/2 or O2- yield, nor was the latter value affected by the quantitive scavenging of O2- by cytochrome c. Under certain conditions--the presence of excess dimethyl sulfoxide, the substitution of a less potent phorbol ester or activation of cells at high densities--the initial rate was either increased or decreased but a complementary alteration in the T1/2 resulted in little or no change in the total O2- yield. Retinol and retinol acetate decreased the initial rate, but retinoic acid enhanced it. By comparison, total O2- production was markedly reduced by all three agents with the following order of potency: retinoic acid greater than retinol greater than retinol acetate. In contrast, the serine protease inhibitor, TPCK, suppressed both the O2- yield and initial rate to a similar extent. On the basis of present observations, it is proposed that under normal conditions of PMA cellular activation, the logarithmic decay of the rate of O2- release was not due to autoinactivation of the O2--generating system, but rather to another factor, a possibility being the depletion of intracellular NADPH. PMID- 3000943 TI - Neutrophils may directly synthesize both H2O2 and O2- since surface stimuli induce their release in stimulus-specific ratios. AB - Many stimuli induce neutrophils to undergo an oxidative burst and generate toxic oxygen metabolites. The major products are O2- and H2O2, the latter being presumed to arise by spontaneous dismutation of the former. If H2O2 were indeed derived exclusively from released O2- according to the equation 2O2- + 2H+--- H2O2 + O2, one would expect that relationship to be reflected in the ratio of the two metabolites detectable in the extracellular mileu of stimulated neutrophils. A second corollary is that H2O2 should not form when cytochrome c is present to scavenge O2- before it can dismutate. Although H2O2 cannot be measured directly in the presence of cytochrome c because it is consumed in reoxidizing reduced cytochrome c, its presence can be detected indirectly by the ability of catalase to improve the apparent yield of reduced cytochrome c. We found that the relative amounts of extracellular H2O2 and O2- that could be measured in the environment of stimulated neutrophils varied with the stimulus and that catalase protected reduced cytochrome c from H2O2 oxidation when some stimuli were used but not with others. For example, the ratio of O2- to H2O2 produced by neutrophils exposed to PMA was about 2:1, the expected result if H2O2 were derived from O2-. However when cytochalasin B was added to the cells before the stimulus, the yield of H2O2 was reduced but not the yield of O2-. When cells were allowed to settle and spread on tissue culture plastic they produced equimolar amounts of O2- and H2O2. Coating the plastic with IgG doubled cytochrome c reduction without effecting H2O2. In contrast, coating with albumin reduced H2O2 without effecting cytochrome c reduction. Soluble IgG aggregates induced production of mostly O2- whereas immune complexes resulted in release of both metabolites. FMLP and A23187 were similar to the soluble IgG aggregates in their effects and induced release of proportionately more O2- than H2O2. The addition of catalase to the cytochrome c solution improved the yield of reduced cytochrome c when PMA or IgG was used to stimulate the cells but not when FMLP was used. These and other data suggest that H2O2 release is not a linear function of the amount of O2- generated and that either a variable fraction of O2- spontaneously dismutates to H2O2 or the neutrophil NADPH oxidase, in a manner analogous to xanthine oxidase, is capable, under some circumstances, of producing H2O2 as well as O2-.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3000946 TI - Collagenolytic activity of Coccidioides immitis. AB - Coccidioides immitis appears to be unable to digest particulate collagen when cultured on collagen-containing semisolid culture media. However, all C. immitis strains solubilized collagen when the fungus was grown in liquid suspension cultures. Moreover, sterile culture filtrates were collagenolytic in collagen buffer-agar plate assays. PMID- 3000944 TI - Eicosanoid modulation of stress fibers in cultured bovine aortic endothelial cells. AB - Leukotrienes (LT) B4, LTD4, and thromboxanes (TX) are cytotoxic, and increase microvascular permeability. Because endothelial stress fibers are theorized to be part of the cytoskeletal mechanism by which microvessels maintain their barrier function, the effect of these eicosanoids on stress fibers in bovine aortic endothelial cells was tested. LTB4, LTD4, and TXB2 each decreased stress fiber numbers by 93%, 62%, and 66%, respectively, when compared to controls (P less than 0.01). In contrast, endothelial cells treated with prostacyclin (PGI2) at 10(-7) M, produced a significant increase (P greater than 0.01) in stress fiber numbers, 211% to that of controls. Azaprostanoic acid (13-APA) and FPL55712, receptor antagonists to TX and slow-reacting substance of anaphylaxis (SRS) receptors, respectively, the cyclooxygenase inhibitor ibuprofen, and the TX synthase inhibitors imidazole and ketoconazole were used to test for possible endothelial cell receptor mediation and de novo prostanoid synthesis associated with inflammatory eicosanoid-induced disassembly of stress fibers. Each pharmacologic agent inhibited the LTD4- and TXB2-induced decreases in stress fibers; LTB4-stimulated stress fiber decreases were inhibited only by pretreatment with TX synthase blockers. These data suggest that increased permeability associated with inflammatory eicosanoid metabolites may have as a common target stress fiber disassembly, and the mechanism may be receptor mediated. That cyclooxygenase and TX synthase blockers inhibited the eicosanoid action suggests that endogenous TX synthesis may be a step in the mechanism. PGI2 enhancement of the microvascular barrier may be related to the effect of PGI2 on promoting stress fiber assembly. In summary, endothelial cell synthesized autocoids derived from arachidonic acid may help to regulate microvascular permeability by way of their action on stress fiber assembly/disassembly, and unbalanced prostanoid secretion by way of LT stimulation may result in a loss of the microvascular barrier and increased permeability. PMID- 3000945 TI - Alteration of alveolar macrophage functions after aerosol infection with bovine herpesvirus type 1. AB - Calves were aerosol challenged with bovine herpesvirus type I, and bronchoalveolar cells were subsequently retrieved by lavage from days 1 to 8 postinfection. Alveolar macrophages (AM), which were depleted of contaminating cells, were characterized with respect to phenotypic markers and functional activities. In most aspects, the changes suggested a stimulation of the AM. With variations in kinetics the percentage of AM expressing an MHC II antigen and Fc (immunoglobulin G)-mediated phagocytosis increased, as did the activity level of two ectoenzymes and the lysosomal hydrolase beta-glucoronidase. The generation of prostaglandin E2 by the AM also rose significantly. However, selective suppression of cellular cytotoxicity and interleukin-1 generation was observed. These findings may have important implications for understanding the events involved in the virus-bacterial interaction in respiratory diseases. PMID- 3000948 TI - Response of rat and guinea-pig subcutaneous tissue to implanted human roots containing collagen/hydroxyapatite preparations. PMID- 3000947 TI - Legionella pneumophila pneumonia associated with reactivation of cytomegalovirus infection. AB - Two patients with Legionella pneumophila infection (serogroup 1) associated with a reactivated cytomegalovirus infection are described. Predisposing underlying factors were not evident. PMID- 3000949 TI - Role of leukotrienes in rat reversed passive Arthus pleurisy and the effect of AA 861, a 5-lipoxygenase inhibitor. AB - In studies of the role of leukotrienes in inflammatory reactions, the induction of rat reversed passive Arthus pleurisy (a type III allergic reaction) was employed. Increases of exudate volume, vascular permeability, and migration of inflammatory cells in the pleural cavity were observed. The vascular permeability was enhanced biphasically during 0-30 min (early response) and during 3-6 h (late response) after induction of the pleurisy. The infiltration of inflammatory cells, mainly polymorphonuclear leukocytes, into the cavity increased and reached a maximum 6 h after the pleurisy was induced. Leukotriene B4 (LTB4), 5 monohydroxyeicosatetraenoic acid (5-HETE), and slow-reacting substance of anaphylaxis (SRS-A), consisting of LTC4, LTD4 and LTE4, were detected in the exudate by reversed-phase high-performance liquid chromatography during the early response. The contents of LTC4 reached a maximum 10 min after the challenge, followed by a rapid decrease within 1 h. The rise and decay of LTC4 correlated with the increase in vascular permeability during the early phase. AA-861, a 5 lipoxygenase inhibitor, given intrapleurally inhibited the increase in vascular permeability, cell migration, and generation of leukotrienes during the early phase of the pleurisy. These results indicate that products of the 5-lipoxygenase pathway, such as LTC4 and LTB4, may play an important role as chemical mediators in the inflammatory reaction. PMID- 3000950 TI - Autonomous proliferation of MeWo human melanoma cell lines in serum-free medium: secretion of growth-stimulating activities. AB - The growth properties of a human melanoma cell line (MeWo) and of a variant (MeWo LC1) endowed with higher metastatic potential in nude mice were compared using hormonally-defined serum-free media. The two cell lines failed to arrest in G0 following serum deprivation, and responded to INS and MSA but not to EGF, PDGF or FGF. Only MeWo-LC1 cells divided persistently in completely serum-free medium, and formed a high percentage of spheroids in agarose supplemented or not with serum or individual growth factors. The conditioned media of serum-free cultures of MeWo and MeWo-LC1 cells exhibited mitogenic activities. These were detected without prior concentration, fractionation or acid treatment. They stimulated DNA replication into sparse monolayers of autologous (MeWo, MeWo-LC1) or homologous (SK-MEL28 melanoma) cells and into NRK-49F normal rat fibroblasts, and acted in synergy with INS in a dose-dependent manner. Over a period of 5 days in culture, MeWo-LC1 cells produced bioactive material at a 2 to 3-fold higher rate than MeWo cells, in both the absence and presence of INS. MeWo-LC1-conditioned medium promoted or enhanced colony formation of MeWo and NRK-49F cells plated in serum free (+/- INS) agarose. The two cell lines expressed the same amount of NGF and EGF receptors. On the basis of these results we suggest that: (i) MeWo and MeWo LC1 melanoma cells have obviated allocrine stimulation of the division process characteristic of normal cells by responding to their own mitogens; (ii) some of these mitogens are akin to TGFs; (iii) the less efficient synthesis of autostimulatory factors by MeWo cells may account for their weaker proliferative capacity in vitro, and possibly in vivo. PMID- 3000951 TI - Genetic and epigenetic factors that influence the occurrence of spontaneous lymphoid tumors in crosses of mice of high- and low-incidence strains. AB - AKR mice, which spontaneously develop greater than 90% incidence of lymphocytic leukemia (LL), crossed with SJL mice, which show greater than 80% incidence of Hodgkin's-like reticulum-cell sarcoma (RCS), produced F1 progeny showing incidences of 30% LL and 0% RCS. Thus, each strain possesses one or more dominant genes capable of interfering with the emergence of the tumor type typical of the other strain. Although mice of reciprocal F1 crosses showed a profound difference in expression of endogenous ecotropic murine leukemia virus (E-MuLV) due to a maternal resistance factor transmitted by SJL females but not males, the two populations did not differ detectably in LL incidence. Like AKR mice, mice of 5 other strains studied (C58, DBA/2, PL, RF and ST/b) possessed one or more genes conferring resistance to RCS in F1 crosses with SJL. Analysis of LL incidences in F1 generations of all possible crosses among these 7 strains revealed 4 different categories of strains with respect to susceptibility/resistance to LL; only ST/b mice, which show no significant incidence of spontaneous LL, lacked genes that could suppress the disease in crosses with high- or moderate-incidence strains. SJL mice treated topically with 3-methylcholanthrene (MCA) developed a 50% incidence of LL, mostly before one year of age; treated mice surviving after one year of age developed a high incidence of RCS. PMID- 3000952 TI - Actin filaments and tumorigenicity in a Fischer rat embryo fibroblast cell line (3Y1) transformed by ultraviolet-irradiated HSV. AB - The characteristics of the cytoskeleton of a Fischer rat embryo fibroblast cell line (3Y1) transformed by ultraviolet (UV)-irradiated HSV were studied by indirect immunofluorescence using anti-actin IgG. Parental 3Y1 cells possessed well-developed actin filaments, while 3Y1 cells transformed by UV-irradiated HSV also retained well-developed actin filaments. Transformed cells were divided into 2 groups according to tumorigenicity in newborn Fischer rats; one had a strongly tumorigenic potential and the other a weakly tumorigenic potential. Tumor-derived cell lines exhibited a highly tumorigenic potential, and were also divided into 2 groups, one with well-developed actin filaments and the other without well developed actin filaments. Our results suggested that transformation or tumor formation by HSV is a multi-step process and that morphological loss of actin filaments in the cells is not essential to the tumorigenic potential of the cells transformed by HSV. PMID- 3000953 TI - Antigens related to three core proteins of HTLV-I (p24, p19 and p15) and their intracellular localizations, as defined by monoclonal antibodies. AB - Three distinct monoclonal antibodies (MAbs) specific for human T-cell leukemia virus type-I (HTLV-I) core proteins with molecular weights of 24 kDa (p24), p19 or p15 were produced, characterized and compared. These antibodies were named NOR 1 (anti-p24, IgG2a), GIN-7 (anti-p19, IgG2b) and FR-45 (anti-p15, IgG2a). Immunofluorescence assay showed that they reacted specifically with methanol fixed cells of virus-bearing cell lines, and that only GIN-7 bound, albeit weakly, to the surface of a small percentage of viable cells. Like natural antibodies to HTLV-I in human serum, GIN-7 stained the fixed cells brightly and diffusely, and gave more intense fluorescence than NOR-1 and FR-45, which stained restricted areas of the cells. NOR-1, GIN-7 and FR-45 specifically precipitated core proteins p24, p19 and p15, respectively, from a lysate of HTLV-IMT-2 labelled with 35S-cysteine. NOR-1 precipitated p53, p36, and p24, GIN-7 precipitated p53, p32, p28 and p19, and FR-45 precipitated p53, p36, and p15 from a lysate of 35S-cysteine-labelled MT-2 cells. GIN-7 also precipitated p32, p28 and p19 from a lysate of MT-2 cells, labelled by surface iodination, but NOR-1 and FR-45 did not detect any proteins in this lysate. GIN-7 also detected p28 in 3H-glucosamine-labelled MT-2 cells. Antibody binding competition assay showed that the sera of ATL patients significantly interfered with the binding of NOR-1 and GIN-7 but not with that of FR-45, to antigens of disrupted virus of MT-2 cells. This complete set of MAbs against the HTLV-I gag gene products is useful for biological and functional studies of the HTLV-I core proteins. PMID- 3000954 TI - Human papillomavirus types 16 and 18 in carcinomas of the penis from Brazil. AB - Human papillomavirus (HPV) type-16 DNA sequences were found in 26/53 (49%) and HPV type-18 in 5/53 (9%) of penile cancers. In only one specimen was HPV18 found on its own. HPV16 sequences were integrated into the host cell chromosomes although some monomeric and oligomeric free forms of DNA were detected in a few tissues. HPV18 was, as far as detectable, free and unintegrated in all tissues tested. HPV16 DNA was also detected in 40% of cases of carcinoma of the cervix in 19 women from the same social groups as the males. The viral DNA in the female cases was a mixture of integrated and free forms. The DNA binding protein (ICSP 11/12) of herpes simplex type 2 (HSV2) was not detected in 10 penile cancers tested with a monoclonal antibody. PMID- 3000955 TI - Molecular cloning and characterization of the DNA of a new human papillomavirus (HPV 30) from a laryngeal carcinoma. AB - DNA from a human laryngeal carcinoma was molecularly cloned in Lambda L47. The gene library was screened for human papillomavirus (HPV)-related sequences by hybridization analysis with 32P-labelled HPV 16 DNA at conditions of low stringency (Tm -40 degrees C). One of the clones (4-5) with an insert of 7.8 kb showed cross-hybridization with most of the known HPV types (Tm -40 degrees C), and with several of them even under more stringent conditions (Tm -30 degrees C). No signal was detected under high-stringency conditions (Tm -20 degrees C). The co-linear alignment of clone 4-5 with HPV 16 DNA could be demonstrated by hybridization experiments and also by partial DNA sequence analysis. We conclude that clone 4-5 represents a new HPV type tentatively designated HPV 30. HPV 30 DNA was also detected in 2 genital lesions but not in 41 laryngeal carcinomas analyzed so far. Its presence in other tumor DNA is now under investigation. PMID- 3000956 TI - Hydrocortisone and some other hormones enhance the expression of HTLV-III. AB - The ability to productively infect fresh normal human peripheral blood mononuclear leukocytes with HTLV-III was improved by supplementing cell culture medium with either the gonadal steroid, chorionic gonadotropin, or insulin, and more substantially with the adrenocortical steroid, hydrocortisone. Several other sex hormones and another corticosteroid, dexamethasone, had no significant effect. In addition, the isolation of HTLV-III from lymphocytes established in culture from patients with the acquired immunodeficiency syndrome (AIDS), AIDS related complex (ARC), and healthy, at-risk donors was greatly facilitated by the inclusion of hydrocortisone in cell culture media. In 13/20 primary cell cultures tested from AIDS and ARC patients from whom virus was isolated, the amount of virus produced was elevated from low to easily detectable levels in those containing hydrocortisone. In 3/20 specimens tested, virus was detected and isolated from cell cultures supplemented with hydrocortisone but was undetectable in those lacking the hormone. These experiments demonstrate that hydrocortisone, a readily available, inexpensive supplement to cell culture media, can facilitate the detection and isolation of HTLV-III. These studies, furthermore, suggest a role for corticosteroids and possibly gonadal steroids in the modulation of virus expression and/or release and suggest that the viral inductive capacity of these and other compounds should be considered as they are evaluated for clinical use. PMID- 3000957 TI - Estrogen and progesterone receptors in benign breast tumors and lesions: relationship with histological and cytological features. AB - The pattern of estrogen (ER) and progesterone receptors (PR) and their relationship to histo- and cyto-pathological parameters has been studied in 97 cases of benign breast disease and benign phyllode tumors (95 women, of whom 76 were premenopausal, and 2 men). Total (cytosolic + nuclear) ER and PR were assayed by a single-saturating dose method using a tris-KCl buffer. The cut-off between positive and negative ER and PR assay was 100 femtomoles/g tissue. All specimens were processed for histological examination: epithelial and fibroblastic proliferation, epithelial/stromal ratio and presence of focal or diffuse hyalinosis. In 33% of the 46 cases of fibrocystic disease one receptor at least was present (13% ER+, 31% PR+). All the 8 cases in which infiltrating epitheliosis was present were PR+ and 4 of them were also ER+. In 72% of the 31 fibroadenomas one receptor at least was present (19% ER+, 71% PR+). In all these cases levels of receptors were lower than in malignant tumors. An inverse correlation between PR + prevalence and fibrohyalinosis was observed; on the other hand a positive relationship between PR + and fibroblastic (p less than 0.001) or epithelial (p less than 0.01) proliferation was found. In all 5 benign phyllode tumors examined PR + were present at a very high level, almost as high as in malignant tumors. Of the 15 other benign breast lesions, all but one (1 hamartoma) were ER- and PR-. PMID- 3000958 TI - Aftercare in drug abuse treatment. AB - Preventing relapse among treated drug abusers is the primary goal of aftercare. This paper identifies posttreatment factors associated with relapse and links these findings to known aftercare approaches. The literature on the effectiveness of these approaches is summarized and limitations in this literature are noted. Promising directions for policy, program development, and research (R&D) in aftercare are described. PMID- 3000959 TI - Drug abuse treatment process: a review of the literature. AB - The literature on drug abuse treatment is reviewed in two sections. The first of these briefly describes the three major treatment modalities (outpatient methadone, residential, and outpatient drug-free). The objectives and approaches of each modality are outlined, and a number of issues surrounding each modality are sketched. The second section of the review covers general studies of drug abuse treatment. In particular, studies of retention, counselor characteristics, program policies and goals, the nature and extent of services received by clients, the context in which treatment is administered, methadone dosage levels, and other such variables are reviewed. Various typologies of treatment are presented and discussed. Conclusions and implications for future research are also discussed. PMID- 3000961 TI - Morphine and methadone impact on human phagocytic physiology. AB - Human subjects submitted to treatment with morphine show a severe depression of phagocytosis, killing properties and superoxide production both of their polymorphonuclear leukocytes and monocytes. Polymorphonuclear leukocyte adherence, chemotaxis, random migration, myeloperoxidase content, lysozyme content and lymphocyte Rosette E formation were poorly influenced. Methadone treated subjects show a similar effect at phagocytic level but far less evident. These results confirm those previously found in animals and reinforce the evidence of a depressive role of morphine on phagocytic physiology. PMID- 3000960 TI - Synergistic inhibition of complement induced granulocyte margination by BW755C and calcium channel blockers. AB - Intravenous infusion of granulocyte (PMNL) chemotactic factors including C5ades Arg present in zymosan activated plasma (ZAP), induces granulocytopenia due to PMNL margination. Since some PMNL responses are dependent on Ca++ ions and lipoxygenation of arachidonic acid, we evaluated the effects of a lipoxygenase (and cyclooxygenase) inhibitor, BW755C and Ca++ channel blocking agents, verapamil and nifedipine, on chemotactic factor induced granulocytopenia and margination in rabbits. BW755C (20 mg/kg i.v.) treatment significantly attenuated ZAP induced granulocytopenia. Verapamil or nifedipine alone were without effect. However, combined treatment with BW755C and verapamil or nifedipine (250 micrograms/kg) completely prevented ZAP-induced granulocytopenia. Ibuprofen, a cyclooxygenase inhibitor, was without effect either by itself or in combination with the calcium channel blockers. In striking contrast to the effect on ZAP induced granulocytopenia, BW755C plus verapamil or nifedipine had virtually no effect on f-met-leu-phe, platelet activating factor or leukotriene B4 induced granulocytopenia. PMNL aggregation in vitro in response to all of the above chemotactic factors was inhibited by BW775C to similar degrees (56-75%) and was not influenced by simultaneous treatment with verapamil. We conclude that: (a) inhibitors of the lipoxygenase pathway may synergize with Ca++ channel blocking agents in inhibiting PMNL responses to complement derived chemotactic factors in vivo; (b) that in vivo PMNL margination to other chemotactic factors may be less dependent on endogenous lipoxygenation and/or Ca++ fluxes; and (c) there is a poor correlation between pharmacological inhibition of PMNL aggregation in vitro and PMNL margination in vivo in this system. PMID- 3000962 TI - The effect of cyclosporine on nucleotide content of rat lymphocytes. AB - In vivo administration of cyclosporin A (CyA) was found to determine some variations in nucleotide content of rat lymphocytes. ATP levels were reduced by CyA treatment, and the effect was more evident in peripheral blood than in spleen lymphocytes. In contrast, cAMP values were increased upon pharmacologic treatment with the same major evidence at the blood lymphocyte level. Intralymphocytic phosphodiesterase enzyme activity became detectable during CyA administration, whereas the intracellular redox state (NAD+/NADH ratios) did not vary significantly. These results were amplified by increasing CyA concentration. PMID- 3000963 TI - Dose-dependent suppression by the synthetic retinoid, 4-hydroxyphenyl retinamide, of streptococcal cell wall-induced arthritis in rats. AB - We studied the effects of oral administration of the retinoid, 4-hydroxyphenyl retinamide (4-HPR), on group A streptococcal cell wall-induced polyarthritis in the rat, a model characterized initially by exudative inflammation of peripheral joints followed by chronic proliferative/erosive synovitis. Experimental arthritis was induced in female LEW/N rats by i.p. injection of streptococcal cell walls in saline (15 micrograms/g body weight). Depending upon the experiment, continuous daily oral administration of the retinoid was begun either 14 days prior to induction of the disease, at the time of cell wall administration and/or 11 days and 31 days after cell wall injection. Dosage was either 1 or 2 mmol 4-HPR/kg of chow. During the course of the disease, severity of clinical illness was assessed by determination of clinical severity index, by histological or radiologic examination, and by measurement of production in vitro of collagenase and prostaglandin E2 by excised synovial tissue. In rats fed the retinoid prior to cell wall injection, both the acute and the chronic responses were suppressed. In rats given the retinoid at the time of cell wall injection, the acute inflammatory response was only partially suppressed on the diet containing 2 mmol 4-HPR/kg chow, but the chronic disease was impressively inhibited in a dose dependent manner. Similarly, in animals with established disease, the drug was also effective; however, the more advanced the illness, the less effective the drug. Clinical observations were paralleled by the histological, radiographical and biochemical analyses. Treated animals showed far less synovial proliferation and joint destruction, and synovial tissues taken from these rats produced lesser amounts of collagenase and prostaglandin E2. No significant toxicity of the retinoid was noted. We conclude that oral administration of 4-HPR suppresses, in a dose and time dependent manner, both the acute and chronic stages of streptococcal cell wall-induced arthritis in rats without apparent significant toxicity. Our data suggest that studies of the effects of this retinoid on patients with chronic inflammatory synovitis are warranted. PMID- 3000964 TI - Drug preventive education and rehabilitation programmes in Singapore. AB - The drug situation in Singapore is considered to be under control now because of the successful adoption of its two-pronged strategy of both supply and demand reduction. This paper only discusses the latter which is carried out through preventive education, treatment and rehabilitation, and aftercare services. Drug preventive education and publicity aimed at the public and the students are mainly done by the Singapore Anti-Narcotics Association (SANA). How various methods and ways have been organized to heighten the awareness of the dangers of drug abuse is discussed. Drug abusers perceived as patients are sent directly, when arrested, to Drug Rehabilitation Centres for treatment and rehabilitation. Discussion is concentrated on both the 5-stage programme and the day release scheme. Also included in the paper is the aftercare service provided to the ex drug addicts after completing the programme or the scheme by the SANA's voluntary aftercare officers either from the religious/secular groups or at the constituency level. PMID- 3000966 TI - Effects of hyperthermia and nicotinamide on DNA repair synthesis, ADP-ribosyl transferase activity, NAD+ and ATP pools, and cytotoxicity in gamma-irradiated human mononuclear leukocytes. AB - Effects of hyperthermia and nicotinamide on ADP-ribosyl transferase activity (ADPRT), unscheduled DNA synthesis (UDS), NAD+- and ATP-pools and cytotoxicity were investigated in gamma-irradiated human mononuclear leukocytes. A significant decrease in radiation-induced UDS after heat treatment for 45 min was found. Nicotinamide increased the UDS levels in irradiated cells, but no effect of hyperthermia on these increased UDS values was observed. In the presence of 2 mM nicotinamide radiation-induced ADPRT activity was reduced to about 50 per cent. However, hyperthermia for 45 min was found to have no effect on the enzyme activity for temperatures below 46 degrees C. Nicotinamide increased the NAD+ pool in unirradiated cells. Damaging the cells with gamma-radiation leads to a severe depletion of the NAD+ pool. The NAD+ pool is restored, however, if the cells repair for 5 h at 37 degrees C. When radiation-damaged cells were treated with hyperthermia, exogenously supplied nicotinamide could not be converted to NAD+ in sufficient amounts to prevent NAD+ depletion. These data indicate that the radiosensitizing effect of heat and nicotinamide could both be explained by effects on the enzyme ADPRT, i.e. nicotinamide by directly blocking the enzyme and hyperthermia by limiting the co-substrate (NAD+). PMID- 3000965 TI - The radiolysis of pyrimidines in aqueous solutions: an updating review. PMID- 3000969 TI - Free radical-induced cross-linking of polydeoxythymidylic acid in deoxygenated aqueous solution. AB - Radiation-generated hydroxyl radicals and hydrogen atoms were shown to induce the cross-linking of polydeoxythymidylic acid (mol X wt approximately 170 000) in N2O saturated acqueous solution. The irradiated samples were hydrolysed with formic acid and then analysed by high performance liquid chromatography. Products were isolated and subsequently characterized by capillary gas chromatography-mass spectrometry. The presence of previously described monomeric thymine products was also shown. Yields were determined and mechanisms of formation were described for the products. PMID- 3000968 TI - Hydroxyl radical-induced strand break formation of poly(U) in the presence of oxygen: comparison of the rates as determined by conductivity, e.s.r. and rapid mix experiments with a thiol. AB - The rate of OH radical-induced strand break formation of single-stranded poly(U) in N2O/O2-saturated aqueous solution was studied by measuring the time-dependence of the electrical conductivity following pulse radiolysis. The first half-life of the total conductivity increase depends slightly on pH and the molecular weight and on the dose per pulse. The activation parameters for strand break formation were found to be EA = 52 kJ mol-1 and A = 5 X 10(8) s-1. Similar first half-lives were observed when the decay of peroxyl radicals of poly(U) was measured by e.s.r. under various conditions. This indicates that poly(U)-peroxyl radicals are involved in the rate-determining step of strand break formation. After pulse radiolysis, strand break formation can be inhibited by the addition of dithiothreitol (DTT) in a rapid-mix apparatus. It is postulated that peroxyl radicals of poly(U) react with DTT by formation of hydroperoxides, thereby preventing strand breakage. PMID- 3000967 TI - Reaction of the hydrated electron with histone H1 and related compounds studied by e.s.r. and spin-trapping. AB - The reactions of the hydrated electron with histone H1, protamine and related compounds (poly-L-lysine, poly-L-arginine and poly-D,L-alanine) were investigated by the spin-trapping technique. In order to identify the radical structure of the spin-adducts originating from macromolecules, the usual spin-trapping technique was developed as follows: N2-saturated aqueous solutions of proteins containing sodium formate were X-irradiated (4.5 kGy) in the presence of 2-methyl-2 nitrosopropane (MNP) as a spin-trap. The side-products due to the self trapping of MNP radicals were then removed from the spin-adducts of the proteins by a Sephadex G-25 column. Finally the spin-adducts were enzymatically digested to transform the broad e.s.r. signals due to slow tumbling of nitroxyl radicals to identifiable ones. The e.s.r. spectra obtained for all samples showed that the deaminated radical, R--CH--CO--NH--(R:amino acid side chain), was produced. Furthermore, polyacrylamide gel electrophoresis of the irradiated protamine and histone H1 indicated reduction of molecular size. These results confirm that hydrated electrons react with proteins and induce the deamination reaction which leads to main-chain scission. PMID- 3000970 TI - Pathogenesis of buffalo-pox virus in buffalo calves. AB - Pathogenesis of buffalo-pox virus (BP4 strain) in buffalo calves following intradermal inoculation revealed bimodal thermal reaction. The prominent symptoms were lacrimation, mucoprulent nasal discharge and diarrhoea. The typical pook lesions produced in the skin were passed through reseolar, papular, vesicular, pustular and desquamative stages of infection followed with a second rash, between day 6-8 on the lips, tongue, neck, perinium region and around the nostrils and eyes. After the eclipse phase of 10 hours, the concentration of the virus started increasing logarithmically. Thereafter, the virus was subsequently detected in the regional lymphnode, blood stream and central organs viz., lung, liver & spleen on 2nd, 4th and 5th day, respectively. In blood stream the virus was found to be associated with white blood cells. Secondary viremia was again on day 6 post-inoculation. Gross and microscopic changes were observed in these organs. The presence of virus along with pathologic changes were also detected in stomach and intestine. The disease ran a course of 13 to 15 days. PMID- 3000972 TI - [Hepatitis DNA viruses and their correlation to hepatocellular cancer]. PMID- 3000973 TI - [Endoscopic photodynamic diagnosis and therapy with the laser in esophageal, stomach and lung cancer]. PMID- 3000971 TI - Cell biology of the asialoglycoprotein receptor system: a model of receptor mediated endocytosis. AB - Substantial information about the ASGP-R has accumulated in the 10 years following the initial studies of this receptor by Ashwell and Morell. Many of its biochemical properties, its structure, and its orientation within the plasma membrane are now known. The pathways of ASGP ligand and receptor, with the CURL organelle being a central component, are summarized in Fig. 18. The major pathway of the ligand through the cell, beginning with binding at the cell surface and ending with degradation in lysosomes, has been investigated in detail. Recently, alternate routes of the ligand such as the ligand recycling pathway have been observed. With regard to the itinerary of the receptor, there is now biochemical, kinetic, and morphological evidence to support receptor recycling. The new concept of CURL as an important intracellular organelle has originated from studies of ASGP-R recycling. Its importance in the dissociation and segregation of ligand and receptor as well as in receptor recycling is now evident. In addition, there has been a concurrent investigation of other receptor systems that participate in receptor-mediated endocytosis, providing parallels and contrasts to the ASGP-R of hepatocytes. Many critical issues still exist in the cell biology of the ASGP-R. What are the structural requirements of the receptor for ligand binding and subsequent endocytosis of the receptor-ligand complex? Very little is known about the interactions between the receptor and the lipid bilayer in which it resides. How does the receptor move laterally in the plasma membrane? Are there proteins or glycolipids closely associated with the ASGP-R and, if so, what is their function? What is the mechanism that causes receptor clustering into coated pits? Although the existence of a pathway for ligand recycling has been demonstrated, there are still many issues to be addressed. What signals a particular ligand molecule for recycling? Is it a stochastic process? What is the function of this route of ligand movement? How are the various ligand pathways coordinated and regulated? In addition, there are many unanswered questions regarding the receptor pathway. How does CURL mediate the sorting of ASGP-R from ligand? How are receptors with different destinations (e.g., ASGP-R and IgA receptor) sorted in CURL? What is the mechanism of ASGP-R degradation and how is it regulated? Finally, how does the Golgi function in the ASGP system and what is the relationship between the Golgi and CURL? Future investigation of these issues will require further observations with existing techniques as well as new approaches.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3000974 TI - H2O2-modification of Na,K-ATPase. Alterations in external Na+ and K+ stimulation of K+ influx. AB - Studies, at steady state, of the Na,K-ATPase dependent influx of K+ into bovine lenses in organ culture are used to characterize further the H2O2-modification of the Na+ pump. Control lenses display constants for interaction with external Na+ and K+ similar to those obtained for the erythrocyte. H2O2 treatment of the bovine lens leads to total loss of external Na+ stimulation and alteration of external K+ stimulation. PMID- 3000975 TI - Spread of HSV and establishment of latency after corneal infection in inbred mice. AB - The spread of herpes simplex virus (HSV) through neural tissues was studied in three inbred mouse strains that differ in susceptibility to HSV stromal keratitis. The left eyes of BALB/c, C57BL/6, and DBA/2 mice were inoculated topically with HSV type 1. The optic and trigeminal nerves, trigeminal ganglia, and eyes were assayed for infectious virus on days 1, 2, 3, 4, 7, 9, 11 and 14 after inoculation. At 2-4 months post-inoculation, eyes and trigeminal ganglia were assayed for latent virus. Up to 7 days post-inoculation, infectious virus was present at a similar frequency in the inoculated eyes of mice from all three strains. The quantity of virus recovered, however, was mouse strain-dependent: DBA mice yielded the most virus; C57BL/6, the least. The frequency of virus recovery and the quantity of virus recovered from trigeminal nerves and ganglia also varied according to mouse strain. Infectious virus was recovered from the uninoculated right eye of some DBA and C57BL/6 mice 1 wk after inoculation. The overall incidence of latency differed among inbred mouse strains. However, in mice that developed ocular disease (blepharitis, dendritic keratitis, or stromal keratitis), there was no host strain-related difference in the incidence of latency. These results support the hypothesis that host genetic factors play a role in controlling HSV replication and the spread of virus to neural tissues after ocular HSV inoculation. This control may influence the development and severity of disease. However, once infection occurs, latency is established in both susceptible and resistant mouse strains. PMID- 3000976 TI - A tissue culture assay of corneal epithelial wound closure. AB - Experimental assays have been developed using cultured tissue derived from rabbit corneal epithelium to study migration of epithelial sheets during wound closure and cell-substrate adhesion. To study wound closure, epithelial defects, 6 mm in diameter, were produced in vitro in 24 well multiplates by a local freezing technique, and the size of the remaining defect was quantitated over time by staining. To study adhesion, cultured cells were labeled with 3H-leucine, suspended, and added to fresh culture plates. At various times, adherent cells were lysed and the radioactivity of the lysate was determined. Serum enhances the closure of experimental defects, but laminin and fibronectin have no effect. Agents which alter mitotic rate, such as epidermal growth factor and 5 fluorouracil, do not influence the rate of wound closure in this assay. Compounds which elevate intracellular levels of cyclic AMP inhibit wound closure but promote cell-substrate adhesion. Thus, cultured corneal epithelial cells may be used to assay for influences on the migratory events governing closure of superficial epithelial wounds. PMID- 3000977 TI - Liposomes as carriers of iodolipid radiocontrast agents for CT scanning of the liver. AB - We studied the potential of liposomes to deliver oil soluble radiocontrast agents to the liver and have developed a new preparation for CT liver scanning. The preparation consists of Ethiodol with a large amount of phospholipids. Nuclear magnetic resonance (NMR) spectroscopy was done for this lipid preparation, and the spectra show that the lipids are in the bilayer liposome configuration. Electron microscopy provides direct visualization of the liposomes. X-ray fluorescence measurements suggest that the Ethiodol is incorporated in the liposomes, and since no other particulate configurations are observed, we conclude that the Ethiodol is contained within the hydrophobic region of the liposomes. We used a GE CTT-8800 scanner and a rabbit model to study the liver uptake of the iodine from the Ethiodol. The iodine uptake in the liver was rapid and significant, and an increase in HU number of more than 40 was observed within 20 minutes after i.v. injection of 50 mg I/kg of body weight. Significant image enhancement was obtained. The iodine from the Ethiodol remained in the liver for several hours. Studies in rabbits with hepatic implants of the VX2-carcinoma show that while normal liver concentrates, areas of tumor do not concentrate the Ethiodol liposomes. Tumors not visible on ordinary scans become visible after administration of this combination. The advantages of this liposomal mode of radiocontrast agent administration are small particle size, rapid uptake in the liver, long retention times, a large increase in HU number and low iodine dose. PMID- 3000978 TI - Flaviviridae. AB - The family Flaviviridae comprises the genus Flavivirus, which contains 65 related species and two possible members. They are small, enveloped RNA viruses (diameter 45 nm) with peplomers comprising a single glycoprotein E. Other structural proteins are designated C (core) and M (membrane-like). The single strand of RNA is infectious and has a molecular weight of about 4 X 10(6) and an m7G 'cap' at the 5' end but no poly(A) tract at the 3' end; it functions as the sole messenger. The gene sequence commences 5'-C-M-E.... The replication strategy and the mode of morphogenesis are distinct from those of the Togaviridae which are slightly larger and morphologically similar in some respects. Flaviviruses infect a wide range of vertebrates, and many are transmitted by arthropods. PMID- 3000979 TI - Junin virus-induced chromosomal aberrations in the guinea pig. Synergism between the attenuated strain XJ-clone 3 and caffeine. AB - The frequency of chromosomal aberrations in bone marrow cells of guinea pigs inoculated with the pathogenic XJ strain of Junin virus increased significantly at 6, 9, and 11 days postinoculation (p.i.). Animals inoculated with the attenuated XJ-clone 3 strain only showed significant increments of achromatic lesions (gaps) at 9 days p.i. Guinea pigs inoculated with the XJ-clone 3 strain and then treated with two doses of caffeine 24 and 12 h before killing at 9 days p.i. exhibited a significant increase of chromatid breaks and a parallel decrease of gaps. Because caffeine acts as an inhibitor of repair mechanisms of genetic damage, these results suggest a mutagenic effect of the attenuated strain. PMID- 3000980 TI - Construction of rat cell lines that contain potential morphologically transforming regions of the herpes simplex virus type 2 genome. AB - Hybrid recombinant plasmids were constructed; they were composed of the herpes simplex virus type 2 (HSV2) thymidine kinase (tk) gene and DNA sequences of HSV2 that have been reported to induce morphological and/or oncogenic transformation of rodent cells in culture. Several plasmids were made in two versions, with or without the simian virus 40 enhancer sequences. These plasmids were employed to transfect contact-inhibited Rat-2 tk- cells. It was demonstrated that cell lines with stably integrated, morphologically transforming regions (MTRs) of HSV2 could be isolated at a low frequency. In addition, many cell lines were isolated that had lost the MTR moiety of the transforming plasmids. None of the isolated tk positive cell lines exhibited significantly altered growth properties when compared to control cell lines. Transient expression of transfecting enhancer plasmids was detectable 40 h posttransfection, but no transformed cell lines were obtained from these experiments. This conflicted with some versions of the 'hit and-run' hypothesis for HSV-mediated cell transformation. PMID- 3000981 TI - Fusion from without induced by herpes simplex virus type 1. AB - Strains ANG and ANG path of herpes simplex virus type 1 (HSV1) produced fusion from without (FFWO) of cells in culture. FFWO required 45 min to become complete. In contrast, fusion from within (FFWI) was not detected until 3-4 h after infection, depending on the cell type. FFWO was temperature dependent: at 0 degrees no fusion could be observed, but increase of temperature increased the degree of fusion. The pH optimum for FFWO was 7.8-8.5. The FFWO activity of the virus was found to be slightly more heat stable at 46 degrees than was infectivity. FFWO was produced in Vero, CV-1 and BSC1 cells, but not in BHK clone 13 or in primary or secondary rabbit kidney cells. FFWO was linked to the presence of virus particles and perhaps to other sedimentable, infected-cell material but not to soluble factors. Actinomycin D, cycloheximide, and UV irradiation did not block this activity, indicating no direct activity of the HSV1 genome for FFWO. PMID- 3000982 TI - Recurrent genital herpes simplex virus infection in guinea pigs. AB - After recovery from initial genital herpes simplex virus (HSV) infections, female guinea pigs developed spontaneous recurrent infections characterized by discrete erythematous or vesicular herpetic lesions on the external genital skin. HSV type 2 (HSV2) caused significantly more recurrent infections in guinea pigs than did HSV type 1 (HSV1). HSV2-infected animals demonstrated a significant decline in frequency of recurrences over time. The viral nature of the recurrent lesions was confirmed by recovery of infectious HSV, detection of HSV antigen, and histologic examination. Latent HSV2 could be demonstrated in dorsal root ganglia and external genital skin after recovery from the primary infection. Recurrent genital HSV infection in the guinea pig shares many features with recurrent genital herpes in humans and provides a model for studying the relationship between latency and recurrences and for exploring methods for control of recurrent disease. PMID- 3000983 TI - 1983-84 Irish and British dietary guidelines and their implications. PMID- 3000984 TI - Infections in renal transplant recipients in Israel. AB - A 5-year retrospective survey of infections following 258 renal transplants in 233 patients is reported from a large medical center in Israel. The most common sites of infection were the urinary tract, the surgical incision, and the lung. We recorded 157 episodes of bacteriuria, 75% during the first month following transplantation. In 24 patients, 25 episodes of bacteremia were documented, with gram-negative bacteria being the most commonly involved organism. Pneumonia was diagnosed in 36 patients and was associated with relatively high mortality. Cytomegalovirus was the most common single organism responsible for infection. Fatal rhinocerebral mucormycosis was observed in three patients and was the most common invasive fungal infection. Other serious opportunistic infections were seen only rarely. Infectious diseases were the most frequent cause of death (51.1%) among these patients. PMID- 3000986 TI - Deposition rates of Rn progeny in houses. AB - Deposition rates and velocities of indoor Rn progeny for both attached and unattached species are estimated from simultaneous measurements performed in 20 houses during 12 months. Fitting the values of working level ratio (the "equilibrium fraction"), the ratio between track densities of filtered and bare nuclear track detectors, section the air exchange rate, and the concentration of condensation nuclei to a theoretical model enabled us to calculate the average deposition rates for the houses in the study. This approach avoids the need to measure deposition rates on different types of surfaces in a house, and yields average values for houses with similar features (e.g. room sizes, heating, and air conditioning). Deposition rates were found to lie within about a factor of two of the average during the year, and exhibit higher values in the winter and lower values in the summer. Average deposition rates for the houses in the study were calculated to be 8 hr-1 and 1 hr-1 for unattached and attached radon daughters, respectively. These values correspond to deposition velocities of about 0.1 cm/sec and 0.015 cm/sec. PMID- 3000985 TI - Pulmonary resections in patients over 70 years of age. AB - Fifty-one men and 7 women greater than 70 years of age (mean 72.3) underwent pulmonary resection over a 5-year period. Fifty-two of these patients had a malignancy, of which 48 were primary lung neoplasms, including 42 cases of non small-cell lung cancer. The overall operative mortality rate was 10.3%. Mortality was correlated with several preoperative factors, including pulmonary function tests, arterial blood gas tension, age, extent of surgery, stage of disease, and additional systemic diseases. Only the extent of surgery (mortality of 36.4% for pneumonectomies compared with 4.3% for less extensive resections, P less than 0.01) and FEV1/VC (the ratio of forced expiratory volume in 1 sec to vital capacity) (P less than 0.05) had statistical significance. The 5-year survival rate for patients with primary lung malignancies was 40%. In view of the aggressive nature of lung tumors in any age-group, the life expectancy of greater than 10 years at age 70, and the reasonable operative risk compared with the high mortality rate in patients not operated upon, we advocate surgical treatment for most patients greater than 70 years of age with a curable disease. PMID- 3000987 TI - Time variation of 222Rn progeny concentration in rainwater. PMID- 3000988 TI - Comments on the use of radiation-induced, long-lived free radicals for dose measurement following a radiation accident. PMID- 3000989 TI - Hemoglobin H disease in two Turkish females and one Iranian newborn. PMID- 3000990 TI - The alpha alpha alpha anti-3.7 globin haplotype with an additional Bgl II site mutation (alpha alpha alpha anti-3.7 Bgl II(-)). PMID- 3000991 TI - 6,7-Dimethoxy-1-veratrylisoquinoline alters the oxygen dissociation properties of human hemoglobin: an ESR study. PMID- 3000992 TI - Acquired immunodeficiency syndrome (AIDS) in pediatric patients. Case report and review. AB - The first pediatric case of acquired immunodeficiency syndrome (AIDS) observed in Switzerland is described. The 3-year-old African/Swiss patient was most probably vertically infected from her asymptomatic, HTLV-III antibody positive Zairian mother. Clinical symptomatology started at 14 months of age, and diagnosis was made at 22 months when medical care and comprehensive investigation were initiated at this clinic. Review of the literature revealed 125 pediatric patients with AIDS. Based on these data, relevant and practically orientated information is given concerning definition, epidemiology, clinical presentation, laboratory findings, management, and prognosis of this newly recognized entity. PMID- 3000993 TI - Dietary zinc intake of pre-menopausal women. AB - Assessment of the diets of 73 pre-menopausal women completing dietary frequency questionnaires suggests that 11 per cent consume a daily average of less than 7.5 mg of zinc. A further 26 per cent consume less than 10 mg of zinc daily, an amount considered to approximate the average daily requirement. Dietary records from a second group of 18 working women (aged less than 35 y), covering periods from 6 to 76 days are also analysed. Of these, the zinc intake of 8 women averages less than 7.5 mg/day. The apparent zinc intake of all women shows large day-to-day fluctuations. The presence of such wide variability within individual eating patterns suggests that short-term estimates of dietary zinc intake cannot provide a reliable estimate of overall zinc nutriture. PMID- 3000996 TI - [New contraceptive agent: various questions on p-sponges]. PMID- 3000995 TI - [High-resolution real-time sonography in salivary gland diseases. II: Salivary gland tumors]. AB - 74 non-selected patients with suspected tumors of the major salivary glands were examined by high-resolution real-time sonography (7 MHz). The different tumors presented with characteristic but nonspecific echomorphological features. Cysts of the salivary glands, intraglandular lymphadenitis, and paraglandular lesions can be distinguished from real tumors of the salivary glands. In case of uncertain palpatory findings the demonstration of an intact echographic texture pattern proves the absence of a tumor. No false negative ultrasound tumor diagnoses were encountered (sensitivity 100%). In salivary-gland neoplasms high resolution real-time sonography completes the clinical findings and provides important informations about size, number and type of space occupying lesions. PMID- 3000994 TI - The dithizone, Timm's sulphide silver and the selenium methods demonstrate a chelatable pool of zinc in CNS. A proton activation (PIXE) analysis of carbon tetrachloride extracts from rat brains and spinal cords intravitally treated with dithizone. AB - From rats intravitally treated with dithizone (diphenyl-thiocarbazone) brains and spinal cords were removed and freeze-dried. The dithizonates present in the CNS tissue were extracted with carbon tetrachloride and subjected to a multielement analysis (proton activation, PIXE). It was found that the extract contained two metals. Most of the metal was zinc, but small traces of copper were also detected. Because prior treatment with the chelating agent, dithizone, can block both the Timm and the selenium metal staining methods, it is suggested that the three techniques label predominantly zinc in the neuropil (DTS-zinc). PMID- 3000997 TI - Evaluation of adrenal function in psittacine birds, using the ACTH stimulation test. AB - The effect of ACTH (16 units) on plasma cortisol and corticosterone concentrations in healthy psittacine birds was evaluated. Plasma corticosterone significantly increased (P less than 0.01) from a mean (+/- SD) basal concentration of 3.25 +/ 3.6 ng/ml to 26.47 +/- 9.25 (one hour after ACTH administration) and 25.69 +/- 13.23 ng/ml (2 hours after ACTH administration). For maximal increase in plasma corticosterone as measured by radioimmunoassay (RIA), heat denaturation was necessary to release corticosteroids from steroid binding proteins. As measured by RIA, plasma cortisol concentrations did not increase, whether or not the heat denaturation step was included. Addition of cortisol to avian plasma did not prevent accurate quantification of cortisol as measured by RIA. Plasma corticosterone concentrations in cockatoos, macaws, Amazon parrots, conures, and lorikeets before and after ACTH administration indicated that the ACTH stimulation test could be used to evaluate adrenal secretory capacity in psittacine birds. PMID- 3000998 TI - Endocrine responses of healthy parrots to ACTH and thyroid stimulating hormone. AB - Effects of exogenous ACTH on plasma corticosterone and cortisol concentrations and the effects of thyroid stimulating hormone (TSH) on plasma triiodothyronine (T3) and thyroxine (T4) were determined in the following 3 species of parrots: red-lored Amazon (group 1), blue-fronted Amazon (group 2), and African gray (group 3). Each bird was given ACTH (0.125 mg/bird) IM, except for 3 to 4 birds in each group, which were given saline solution (controls). Blood samples were collected before and 90 minutes after ACTH stimulation. In group 1 (n = 12), mean plasma corticosterone concentrations increased significantly (P less than 0.001) from 1.06 microgram/dl (before ACTH) to 4.89 micrograms/dl (after ACTH); mean corticosterone concentrations increased in the control birds from 1.06 microgram/dl to 1.84 microgram/dl; and mean cortisol concentrations increased only slightly from 0.228 microgram/dl to 0.266 microgram/dl. In group 2 (n = 12), mean corticosterone concentrations increased significantly (P less than 0.001) from 2.09 micrograms/dl to 10.58 micrograms/dl; control mean corticosterone concentrations decreased slightly from 2.09 micrograms/dl to 1.77 microgram/dl; and mean cortisol concentrations increased from less than or equal to 0.16 microgram/dl to 0.266 microgram/dl. In group 3 (n = 12), mean plasma corticosterone concentrations increased significantly (P less than or equal to 0.001) from 2.33 micrograms/dl to 4.67 micrograms/dl; mean control plasma corticosterone concentrations decreased from 2.33 micrograms/dl to 1.68 microgram/dl; and plasma corticol concentrations were not detectable. Each bird was given TSH, IM (1 U/bird). Blood samples were collected before and 6 hours after TSH administration. Saline solution was not administered as controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3000999 TI - Effect of ACTH on plasma corticosterone and cortisol in eagles and condors. AB - The effect of ACTH on plasma corticosterone and cortisol was determined in 12 eagles (Haliaeetus leucocephalus) and in 6 Andean condors (Vultur gryphus). In all raptors, the concentration of plasma corticosterone was substantially greater than that of cortisol. After ACTH administration, the eagles had a marked increase (P less than 0.001) in plasma corticosterone concentrations, but not in plasma cortisol. Administration of saline solution did not induce increased plasma corticosterone concentrations in the eagles. The condors had a smaller increase (P less than 0.002) in plasma corticosterone concentrations after ACTH administration, as compared with that of the eagles. However, administration of saline solution in 2 condors resulted in an increase in corticosterone similar to the increase after ACTH administration. In the condor, a stress-related release of endogenous ACTH may have an effect similar to that induced by exogenously administered ACTH. Plasma cortisol concentrations did not increase significantly after administration of ACTH or saline solution in either raptor species. PMID- 3001000 TI - Serologic evaluation of vaccinated American river otters. AB - The Oklahoma Department of Wildlife Conservation acquired 20 American river otters (Lutra canadensis) between 1984 and 1985 for reintroduction into Oklahoma waterways. In 1985, 10 otters were evaluated for serum antibody titers after vaccination with canine distemper virus, canine adenovirus type 2, canine parvovirus (CPV), feline panleukopenia virus (FPV), feline rhinotracheitis virus (FRV), and feline calicivirus. Prevaccination serum-virus neutralization (SVN) antibody to feline rhinotracheitis virus was found in 2 otters and to feline calicivirus in 1 otter. Using an indirect fluorescent antibody (IFA) assay, prevaccination antibody to CPV and FPV was found in 2 otters. A significant increase in SVN antibody titers was found after vaccination of otters with canine adenovirus type 2 (6 of 8 animals) and feline calicivirus (1 of 8 animals). One of 8 otters developed significant antibody titers to CPV and FPV, as measured by IFA assay. Otters did not develop SVN antibody titers to canine distemper virus after vaccination. Antigens of feline leukemia virus, using ELISA, or antibodies to feline infectious peritonitis, using IFA assay, were not found in the 20 otters. PMID- 3001001 TI - Epornitic of avian pox in a raptor rehabilitation center. PMID- 3001002 TI - Metastatic cholangiocarcinoma in a Florida sandhill crane. PMID- 3001003 TI - Adenovirus-like infection in a boa constrictor. PMID- 3001004 TI - Equine herpesvirus type 1 abortion in an onager and suspected herpesvirus myelitis in a zebra. PMID- 3001005 TI - Perinatal bluetongue viral infection in exotic ruminants. PMID- 3001006 TI - Polycythemia in a New Zealand White rabbit with an embryonal nephroma. PMID- 3001007 TI - Hepatic neoplasia in two polar bears. PMID- 3001008 TI - T-cell lymphoma associated with immunologic evidence of retrovirus infection in a lowland gorilla. PMID- 3001009 TI - Adult T-cell leukemia-like disease in monkey naturally infected with simian retrovirus related to human T-cell leukemia virus type I. AB - Spontaneous T-cell leukemia similar to human adult T-cell leukemia (ATL) was found in an African green monkey naturally infected with simian retrovirus closely related to human T-cell leukemia virus type I (HTLV-I). Monoclonal integration of the simian retrovirus was detected in the primary leukemic cells, suggesting an association of the retrovirus with ATL-like leukemia in the monkey. PMID- 3001010 TI - The cervical tumor-associated antigen (ICP-10/AG-4) is encoded by the transforming region of the genome of herpes simplex virus type 2. AB - BglII fragment C mapping between 0.416 and 0.580 map units (mu) on the herpes simplex virus type 2 (HSV-2) genome was used for in vitro translation to identify proteins encoded on this fragment. RNA homologous to the BglII C fragment directs the synthesis of three proteins with approximate molecular weights of 144,000, 52,000 and 27,000. The 27,000 dalton protein is encoded by sequences within the EcoRI/HindIII AE fragment (0.419-0.525 mu) that overlap the immortalizing sequences within BglII C. The 144,000 (144K) and 52,000 dalton proteins are encoded by sequences within the BamHI "e" fragment of HSV-2 DNA (0.535-0.585 mu). The 144K protein is the only species translated in vitro from mRNA hybrid selected from cells arrested in the "early" (beta) phase of viral protein synthesis. It is precipitated by anti-ICP-10 serum and by monoclonal antibody 48S (previously shown to precipitate the HSV-induced ribonucleotide reductase). The 48S antibody competes with the anti-ICP-10 serum for the 144K protein. Furthermore the in vitro translated 144K protein is structurally similar to ICP 10, an HSV-2-infected cell protein that is antigenically identical to AG-4, the cervical tumor-associated antigen. PMID- 3001011 TI - Establishment of mutant FM3A murine mammary carcinoma cell strains transformed with the herpes simplex virus type 1 thymidine kinase gene. AB - To establish cell systems appropriate for investigating the mode of action of anti-herpetic nucleoside analogs, mutants were constructed from murine FM3A mammary carcinoma cells, which were deficient in both thymidine kinase (EC 2.7.1.21) and thymidylate synthase (EC 2.1.1.45), but were transformed with a recombinant plasmid DNA containing the herpes simplex virus type 1 thymidine kinase gene. The transformed cells expressed viral thymidine kinase activity and showed unusually high sensitivity to the cytostatic action of the antiherpetic agent (E)-5-(2-iodovinyl)-2'-deoxyuridine, which was only weakly inhibitory to the growth of the parent cells. PMID- 3001012 TI - Effects of chloramphenicol on the long term trophic action of ACTH on rat adrenocortical cells: a combined stereological and enzymological study. AB - Chronic chloramphenicol administration was found to block the ACTH-induced increase in both the surface area of mitochondrial cristae and the activity of 11 beta-hydroxylase in zona fasciculata cells of the rat adrenal cortex. The surface area of agranular endoplasmic reticulum membranes and the activity of 3 beta hydroxysteroid dehydrogenase were not affected by treatment with chloramphenicol. These findings suggest that the mechanism of the ACTH-induced enhancement of the growth and steroidogenic capacity of rat adrenocortical cells involves stimulation of mitochondrial DNA-dependent protein synthesis. PMID- 3001013 TI - The effect of delta 9-tetrahydrocannabinol in utero exposure on rat offspring fertility and ventral prostate gland morphology. AB - Male rats exposed in utero to delta 9-tetrahydrocannabinol (delta 9-THC) had lower levels of testosterone (T) and luteinizing hormone (LH) prior to puberty (P less than 0.01). At puberty, the levels returned to within the normal range. Ultrastructural examination of the ventral prostate gland at puberty revealed alterations suggestive of degenerative changes. A drastic reduction in secretory granules and acini reflected depressed androgen production and function during the developmental period. The fertility of the F1 and F2 male offspring was decreased by 30 to 40%. It is concluded that THC exposure in utero caused a permanent reduction in fertility and altered ventral prostate gland morphology. PMID- 3001014 TI - 5'-Nucleotidase inhibitory activity of nucleoticidin, melanocidin A and melanocidin B. Structure-activity relationships. AB - Nucleoticidin and melanocidins A and B exhibited potent inhibitory activity against 5'-nucleotidases from rat liver membrane and snake venom. These inhibitors are polysaccharides with highly branched side chains having at least disaccharide units. This conclusion was supported by the results with polysaccharides of known chemical structures. The inhibitors showed non competitive inhibition with respect to AMP, and urea-treatment caused a marked decrease or a disappearance of the 5'-nucleotidase inhibitory activity. Therefore, it is concluded that steric factors also play an important role in their inhibitory activity. PMID- 3001015 TI - Sulbactam/ampicillin: effects on glucose metabolism in diabetics with soft tissue infection. AB - Rats and dogs chronically treated with high doses of sulbactam are known to sequester protein-bound glycogen in their hepatocytes. As a result, previous UK studies of sulbactam/ampicillin excluded patients suffering from diabetes mellitus. This study examined the effects of sulbactam/ampicillin compared to flucloxacillin/ampicillin on diabetic control, the ability to mobilize glycogen and the pancreatic beta cell response to glucagon, in diabetic patients suffering from soft tissue infection. There was no significant effect between treatment groups on any of these parameters. Sulbactam/ampicillin is unlikely to have an adverse effect on diabetic control in clinical practice when used short term in the doses employed in this study. PMID- 3001016 TI - Pulmonary neutrophil kinetics in sheep: effects of altered hemodynamics. AB - We investigated the effect of elevated left atrial pressure and reduced cardiac output on pulmonary neutrophil kinetics in the sheep. Sheep neutrophils were isolated, labeled with 111In-oxine, and reinfused. Erythrocytes were labeled with [99mTc]pertechnetate. A gamma camera measured the lung activities of the labeled neutrophils and erythrocytes. The results indicated that 38.5% of the total injected neutrophils marginated in the lung. Pulmonary hemodynamics were altered by inflating a left atrial balloon three times in each sheep for 15-30 min to achieve 5- to 25-mmHg increments in pulmonary arterial wedge pressure. At least a 30-min recovery period was allowed between inflations. After each left atrial balloon inflation, neutrophil uptake remained unchanged from base line, despite decreased mean cardiac output to 0.67 +/- 0.24 (+/- SD) 1/min and increased pulmonary blood volume. The absence of pulmonary neutrophil uptake was confirmed by arterial-venous measurements. Increased pulmonary blood volume had little effect on lung neutrophil uptake, suggesting that most of the pulmonary neutrophils are marginated. We conclude that the lungs have a large marginated neutrophil pool compared with the circulating pool and that reduced cardiac output and elevated left atrial pressure have no effect on pulmonary neutrophil kinetics in the sheep. PMID- 3001017 TI - Leukotriene B4 induces airway hyperresponsiveness in dogs. AB - We studied the effect of leukotriene B4 aerosols on airway responsiveness to inhaled acetylcholine aerosols and on the cellular components and cyclooxygenase metabolites in bronchoalveolar lavage fluid in dogs. Inhalation of leukotriene B4 aerosols had no effect on resting total pulmonary resistance but increased airway responsiveness, an effect that was maximum in 3 h and that returned to control levels within 1 wk. Three hours after leukotriene B4, the number of neutrophils and the concentration of thromboxane B2 recovered in lavage fluid increased markedly. Pretreatment with the thromboxane synthase inhibitor OKY-046 prevented the increases in airway responsiveness and in thromboxane B2 but did not alter neutrophil chemotaxis. Thus we speculate that leukotriene B4 causes neutrophil chemotaxis and release of thromboxane B2, which increases airway responsiveness. PMID- 3001018 TI - Lung myeloperoxidase as a measure of pulmonary leukostasis in rabbits. AB - Pulmonary leukostasis can be associated with acute lung injury. We studied lung peroxidase activity using myeloperoxidase (MPO) as a granulocyte marker to quantitate pulmonary leukostasis in rabbits. Lungs were homogenized in detergent, freeze-thawed, sonified, and centrifuged, and supernatants were assayed for MPO. Seven extractions were performed, and greater than 80% of cumulative MPO was found in the first three extractions. By use of a three-extraction procedure, the mean lung MPO (delta A X min-1 X g tissue-1) was determined in normal [20.9 +/- 5.2 (SE)], granulocyte-depleted (6.5 +/- 2.0), saline-injected (22.2 +/- 5.6), and pneumococcus (PNC)-challenged (69.7 +/- 10.6) animals. Lung MPO was significantly decreased in granulocyte-depleted compared with normal animals (P less than 0.005) and significantly increased in PNC-challenged compared with saline-injected animals (P less than 0.001). MPO extracted from granulocytes and lungs from normal as well as PNC-challenged animals were all biochemically identical. Lung extract did not inhibit MPO, and no MPO was detected in bronchoalveolar lavage fluid obtained from leukostatic lungs. Lung MPO significantly (P less than 0.01) correlated with intravascular intrapulmonary granulocytes. Determination of lung MPO is a relatively simple quantitative method that can be used to detect pulmonary leukostasis. PMID- 3001019 TI - Dimethylthiourea consumption reflects H2O2 concentrations and severity of acute lung injury. AB - Even though dimethylthiourea (DMTU) effectively scavenges O2 metabolites in vitro, it is often unclear if scavenging of O2 metabolites is the mechanism by which DMTU decreases tissue injury in biological models. Since DMTU not only scavenges O2 metabolites but is also consumed in a dose-response manner following reaction with hydrogen peroxide (H2O2) in vitro, we wondered whether DMTU would also be consumed by O2 metabolites in biological systems and if DMTU consumption would then reflect O2 metabolite concentrations and O2 metabolite-mediated injury. Our results supported this possibility. We found that selected nonprotecting concentrations of DMTU were consumed in isolated rat lungs perfused with H2O2 and that the amounts of DMTU consumed reflected both the added amounts of H2O2 and the corresponding degrees of H2O2-induced acute edematous injury. DMTU consumption was relatively specific for reaction with H2O2 occurring in isolated lungs that were injured by H2O2 but not lungs injured by elastase, oleic acid, histamine, or a venous pressure challenge. Our results suggest that measurement of DMTU consumption may be useful for assessing the presence and toxicity of O2 metabolites and the specificity of the protective effects of DMTU in biological systems. PMID- 3001020 TI - Selection of transformed cells in serum-free media. AB - NIH3T3 cells grow in a serum-free basal nutrient medium supplemented with fibronectin, transferrin, insulin, epidermal growth factor (EGF) and high density lipoprotein (HDL). The individual omission from the serum-free medium of insulin, EGF, or HDL results in greatly reduced cell growth. These growth-restrictive conditions can be used to select for cells transformed with SV40, the polyomavirus middle T antigen gene, the activated human ras gene, and the mouse c myc gene. PMID- 3001021 TI - fii, a bacterial locus required for filamentous phage infection and its relation to colicin-tolerant tolA and tolB. AB - We describe mutations in a new bacterial locus, designated fii, which do not allow the filamentous bacteriophage f1 to infect bacteria harboring the F plasmid. Mutations at this locus do not affect the ability of F plasmid containing bacteria to undergo conjugation or be infected by the F plasmid specific RNA phage f2. The filamentous phage can still adsorb to the F sex pilus, but the DNA is unable to enter the bacteria. All fii mutants become tolerant to colicins E1, E2, and E3. Strains with amber mutations in fii also are unable to plaque P1, even though they can be infected with this phage. Mutations in fii also prevent infection of bacteria harboring the N plasmid by the filamentous bacteriophage IKe. The fii locus maps adjacent to tolA, mutants of which demonstrate tolerance to high levels of the E and K colicins. The three genes tolA, tolB, and fii are shown to reside on a 4.3-kilobase fragment of the Escherichia coli chromosome. Each gene has been cloned into a chimeric plasmid and shown to complement, in trans, mutations at the corresponding chromosomal locus. Studies in maxicells show that the product of fii appears to be a 24 kilodalton protein which copurifies with the cell envelope. The product of tolA has been identified tentatively as a 51-kilodalton protein. Data from cloning, Tn5 mutagenesis, and P1 transduction studies are consistent with the gene order sucA-fii-tolA-tolB-aroG near 17 min on the E. coli map. PMID- 3001023 TI - Mycoplasma restriction: identification of a new type of restriction specificity for DNA containing 5-methylcytosine. AB - Mycoplasma bacteriophage L51 single-stranded DNA and L2 double-stranded DNA are host cell modified and restricted when they transfect Acholeplasma laidlawii JA1 and K2 cells. The L51 genome has a single restriction endonuclease MboI site (recognition sequence GATC), which contains 5-methylcytosine when the DNA is isolated from L51 phage grown in K2 cells but is unmethylated when the DNA is from phage grown in JA1 cells. This GATC sequence is nonessential, since an L51 mutant in which the MboI site was deleted was still viable. DNA from this deletion mutant phage was not restricted during transfection of either strain K2 or JA1. Therefore, strain K2 restricts DNA containing the sequence GATC, and strain JA1 restricts DNA containing the sequence GAT 5-methylcytosine. We conclude that K2 cells have a restriction system specific for DNA containing the sequence GATC and protect their DNA by methylating cytosine in this sequence. In contrast, JA1 cells (which contain no methylated DNA bases) have a newly discovered type of restriction-modification system. From results of studies of the restriction of specifically methylated DNAs, we conclude that JA1 cells restrict DNA containing 5-methylcytosine, regardless of the nucleotide sequence containing 5-methylcytosine. This is the first report of a DNA restriction activity specific for a single (methylated) base. Modification in this system is the absence of cytosine methylating activity. A restriction-deficient variant of strain JA1, which retains the JA1 modification phenotype, was isolated, indicating that JA1 cells have a gene product with restriction specificity for DNA containing 5-methylcytosine. PMID- 3001022 TI - Isolation and characterization of lon mutants in Salmonella typhimurium. AB - In this paper we report the isolation and characterization of lon mutants in Salmonella typhimurium. The mutants were isolated by using positive selection by chlorpromazine resistance. The physiological and biochemical properties of the lon mutants in S. typhimurium are very similar to those of Escherichia coli lon mutants. Mutants altered at this locus contain little or no activity of the ATP dependent protease La and show a number of pleiotropic phenotypes, including increased production of capsular polysaccharides, increased sensitivity to UV light and other DNA-damaging agents, and a decreased ability to degrade abnormal proteins. PMID- 3001025 TI - Nucleotide sequence of the transcription unit containing the aroL and aroM genes from Escherichia coli K-12. AB - The nucleotide sequence of 2,021 base pairs (bp) of DNA containing the Escherichia coli aroLM operon was determined, and the coding regions of both aroL and aroM were identified. The 501-bp intercistronic region between aroL and aroM contains an open reading frame which might encode a 63-residue protein. Northern blots with RNA from strains carrying multicopy aroL+ plasmids detected one longer (2,000-base) and two shorter (950- and 1,100-base) transcripts which contained aroL. It was concluded that the longest transcript, which was not abundant, spanned the entire operon and that the shorter transcripts resulted from either termination or posttranscriptional processing in the intercistronic region. The DNA upstream of aroL contains a number of imperfect palindromes which are closely homologous to known sites of regulation by the TyrR protein in other operons. PMID- 3001024 TI - Genetic and molecular analysis of aroL, the gene for shikimate kinase II in Escherichia coli K-12. AB - The gene aroL in Escherichia coli K-12, specifying shikimate kinase II, was contransduced with proC at a frequency of 99%. The gene order is lac proC aroL. A 2.7-kilobase BamHI fragment containing aroL+ was cloned into pBR322. This plasmid conferred highly elevated levels of shikimate kinase synthesis which were subject to repression control by tyrR. The aroL gene was localized within a 730-base-pair region by both subcloning and insertional mutagenesis with Tn1000. A second gene, designated aroM and encoding a protein of molecular weight 26,000, is cotranscribed with aroL. Transcription proceeds in the order aroL aroM in a clockwise direction on the chromosome. The function of aroM remains unknown. PMID- 3001026 TI - Two different parasporal inclusions are produced by Bacillus thuringiensis subsp. finitimus. AB - Bacillus thuringiensis subsp. finitimus produced at least two parasporal inclusions. One inclusion was formed within the exosporium and remained with the spore after mother cell lysis. A second inclusion formed somewhat later exterior to the exosporium. Each inclusion contained a major polypeptide of about 135,000 daltons with unique antigenic determinants. This subspecies contained only two plasmids, of 98 and 77 megadaltons (MDa). Strains cured of these plasmids produced only the free inclusion. Since the plasmid-cured strains did not contain DNA sequences homologous to plasmid DNA, the gene for the free-inclusion protein must be encoded in the chromosome. In contrast, the enclosed parasporal inclusion was produced only when the plasmid of 98 MDa was present. In addition, transfer of the 98-MDa plasmid to Bacillus cereus resulted in transcipients that produced small inclusions enclosed within the exosporium, and the protein extracted from these inclusions reacted with antibody specific for enclosed inclusion protein of B. thuringiensis subsp. finitimus. Genes in both the chromosome and a plasmid function in the synthesis of distinct parasporal proteins in this subspecies. PMID- 3001027 TI - Identification of the tip-encoded receptor in bacterial sensing. AB - A chemotaxis gene encoding a protein with receptorlike properties has been identified in Salmonella typhimurium and termed tip for taxis-involved protein. Based on the stringency of DNA hybridization, the tip gene has about 75% homology with a region of the tar gene encoding the cytoplasmic domain of the aspartate receptor. Introduction of the tip gene into a smooth-swimming Escherichia coli receptor mutant (tar tsr tap) restored both chemotaxis ability on soft-agar tryptone plates and a wild-type swimming phenotype. We have shown, by overexpressing the CheY protein, that shifting of the mutant swimming bias in the absence of receptors is insufficient to restore chemotaxis ability. This suggests that in addition to resetting the swimming bias, the tip gene product functions as a receptor. By functional criteria, we found that Tip is not a duplicate aspartate (Tar) or serine (Tsr) receptor gene. Based on behavioral properties, the S. typhimurium Tip receptor provides functional features similar to those of the E. coli Tap receptor. PMID- 3001028 TI - RNase H is not involved in the induction of stable DNA replication in Escherichia coli. AB - rnh mutations of Escherichia coli inactivating RNase H activity allow the initiation of rounds of DNA replication in the absence of protein synthesis (stable DNA replication). However, levels of RNase H did not change during or after the induction of stable DNA replication in rnh+ strains by incubation with nalidixic acid or UV irradiation. PMID- 3001029 TI - Purification and properties of shikimate kinase II from Escherichia coli K-12. AB - Shikimate kinase II was purified to near homogeneity from an Escherichia coli strain which overproduced the enzyme. The apparent Km of this isoenzyme for shikimate was 200 microM, and for ATP it was 160 microM. The Km for shikimate is approximately 100-fold lower than the Km of shikimate kinase I, suggesting that shikimate kinase II is the isoenzyme normally functioning in aromatic biosynthesis. Shikimate kinase II is dependent on metal ions for activity. PMID- 3001031 TI - Analysis of cloned DNA from Leptospira biflexa serovar patoc which complements a deletion of the Escherichia coli trpE gene. AB - To analyze the cloned region of the chromosome of the spirochete Leptospira biflexa serovar patoc which complemented a defect in the trpE gene of Escherichia coli, we performed a series of experiments involving subcloning, transposon mutagenesis, and maxicells. By subcloning into pBR322 we were able to isolate the Leptospira genes on a 9.7-kilobase pair plasmid (pYC6). Transposon mutagenesis with Tn5 identified a 2.8-kilobase pair region of this plasmid as being necessary to complement a trpE deletion mutation in E. coli. Transformation of plasmid pYC6 into E. coli cells deleted for trpE and the proximal end of trpD showed that the Leptospira DNA complemented both defects. A maxicell analysis of various transposon-induced mutations of the plasmid revealed that three proteins (53.5, 23.6, and 22 kilodaltons) were encoded by the 2.8-kilobase pair region of the Leptospira genome. Two different promoters controlled the production of these three proteins. PMID- 3001030 TI - Conjugative transfer of the naturally occurring plasmids of Acetobacter xylinum by IncP-plasmid-mediated mobilization. AB - Broad-host-range plasmids and cloning vectors were conjugatively transferred to Acetobacter xylinum. One of the plasmids, RP4::Mu cts61, was used for the insertion of Tn1 into the 16-, 44-, and 64-kilobase-pair plasmids of A. xylinum. The Tn1-labeled plasmids could be mobilized by a helper plasmid. Many of the Tn1 insertions affected the copy number of the plasmids. PMID- 3001032 TI - PPi-dependent phosphofructotransferase (phosphofructokinase) activity in the mollicutes (mycoplasma) Acholeplasma laidlawii. AB - A PPi-dependent phosphofructotransferase (PPi-fructose 6-phosphate 1 phosphotransferase, EC 2.7.1.90) which catalyzes the conversion of fructose 6 phosphate (F-6-P) to fructose 1,6-bisphosphate (F-1, 6-P2) was isolated from a cytoplasmic fraction of Acholeplasma laidlawii B-PG9 and partially purified (430 fold). PPi was required as the phosphate donor. ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, dUTP, ITP, TTP, ADP, or Pi could not substitute for PPi. The PPi-dependent reaction (2.0 mM PPi) was not altered in the presence of any of these nucleotides (2.0 mM) or in the presence of smaller (less than or equal to 300 microM) amounts of fructose 2,6-bisphosphate, (NH4)2SO4, AMP, citrate, GDP, or phosphoenolpyruvate. Mg2+ and a pH of 7.4 were required for maximum activity. The partially purified enzyme in sucrose density gradient experiments had an approximate molecular weight of 74,000 and a sedimentation coefficient of 6.7. A second form of the enzyme (molecular weight, 37,000) was detected, although in relatively smaller amounts, by using Blue Sepharose matrix when performing electrophoresis experiments. The back reaction, F-1, 6-P2 to F-6-P, required Pi; arsenate could substitute for Pi, but not PPi or any other nucleotide tested. The computer-derived kinetic constants (+/- standard deviation) for the reaction in the PPi-driven direction of F-1, 6-P2 were as follows: v, 38.9 +/- 0.48 mM min-1; Ka(PPi), 0.11 +/- 0.04 mM; Kb(F-6-P), 0.65 +/- 0.15 mM; and Kia(PPi), 0.39 +/- 0.11 mM. A. laidlawii B-PG9 required PPi not only for the PPi phosphofructotransferase reaction which we describe but also for purine nucleoside kinase activity. a dependency unknown in any other organism. In A. laidlawii B-PG9, the PPi requirement may be met by reactions in this organism already known to synthesize PPi (e.g., dUTPase and purine nucleobase phosphoribosyltransferases). In almost all other cells, the conversion of F-6-P to F-1,6-P2 is ATP dependent, and the reaction is generally considered to be the rate-limiting step of glycolysis. The ability of A. laidlawii B-PG9 and one other acholeplasma to use PPi instead of ATP as an energy source may offer these cytochrome-deficient organisms some metabolic advantage and may represent a conserved metabolic remnant of an earlier evolutionary process. PMID- 3001033 TI - Isolation and characterization of ack and pta mutations in Azotobacter vinelandii affecting acetate-glucose diauxie. AB - Azotobacter vinelandii mutants defective for acetate utilization that were resistant to fluoroacetate (FA) were isolated. FA-resistant mutant AM6 failed to transport [14C]acetate and lacked enzymatic activity for both acetate kinase and phosphotransacetylase. Growth of wild-type A. vinelandii was sensitive to 10 mM glycine; however, all FA-resistant strains were resistant to glycine toxicity. Isolated mutants that were spontaneously resistant to glycine were also resistant to FA and lacked both acetate kinase and phosphotransacetylase activity. The glycine-resistant mutant AM3, unlike mutant AM6, was capable of growth on acetate. The mutant strain AM6 was unable to growth under acetate-glucose diauxie conditions. Glucose utilization in this mutant, unlike that in wild-type A. vinelandii, was permanently arrested in the presence of acetate. Revertants of strain AM6 were selected on plates with acetate or acetate-glucose. Two classes of revertants were isolated. Class I revertant mutants AM31 and AM35 were positive for both acetate kinase and phosphotransacetylase activities. These revertants were also sensitive to both FA and glycine. Class II revertant strains AM32 and AM34 still lacked acetate kinase and phophotransacetylase activity. Both of these revertants remained resistant to FA and glycine. PMID- 3001034 TI - SOS-independent coupling between DNA replication and cell division in Escherichia coli. AB - Inhibition of DNA synthesis in Escherichia coli mutants in which the SOS dependent division inhibitors SfiA and SfiC were unable to operate led to a partial arrest of cell division. This SOS-independent mechanism coupling DNA replication and cell division was characterized with respect to residual division, particle number, and DNA content. Whether DNA replication was blocked in the initiation or the elongation step, numerous normal-sized anucleate cells were produced (not minicells or filaments). Their production was used to evaluate the efficiency of this coupling mechanism, which seems to involve the cell division protein FtsZ (SulB), also known to be the target of the division inhibitors SfiA and SfiC. In the absence of DNA synthesis, the efficiency of coupling was modulated by the cyclic-AMP-cyclic-AMP receptor protein complex, which was required for anucleate cell production. PMID- 3001035 TI - Characterization of three genomic loci encoding Rhizobium sp. strain ORS571 N2 fixation genes. AB - Sixty-five independent, N2 fixation-defective (Nif-) vector insertion (Vi) mutants were selected, cloned, and mapped to the ORS571 genome. The recombinant Nif::Vi plasmids obtained in this way were used as DNA hybridization probes to isolate homologous phages from a genomic library of ORS571 constructed in lambda EMBL3. Genomic maps were drawn for three ORS571 Nif gene loci. Forty-five Nif::Vi mutants in genomic Nif locus 1 defined two gene clusters separated by 8 kilobase pairs (kb) of DNA. In the first cluster, 36 Nif::Vi mutants mapped to a 7-kb DNA segment that showed DNA homology with Klebsiella pneumoniae nifHDKE and encoded at least two Nif operons. In the other cluster, nine Nif::Vi mutants mapped to a 1.5-kb DNA segment that showed homology with K. pneumoniae and Rhizobium meliloti nifA; this DNA segment encoded a separate Nif operon. Fifteen Nif::Vi mutants mapped to a 3.5-kb DNA segment defined as Nif locus 2 and showed DNA homology with the R. meliloti P2 fixABC operon. Nif locus 2 carries a second nifH (nifH2) gene. Four Nif::Vi mutants mapped to a 2-kb DNA segment defined as Nif locus 3 and showed DNA homology with K. pneumoniae nifB. DNA from lambda Nif phages comprising all three genomic Nif loci was subcloned in plasmid vectors able to stably replicate in ORS571. These plasmid subclones were introduced into ORS571 strains carrying physically mapped Nif::Vi insertions, and genetic complementations were conducted. With the exception of certain mutants mapping to the nifDK genes, all mutants could be complemented to Nif+ when they carried plasmid subclones of defined genomic DNA regions. Conversely, most nifDK mutants behaved as pseudodominant alleles. PMID- 3001036 TI - Insufficient mobilization of calcium by early breakdown of phosphatidylinositol 4,5-bisphosphate for aggregation of human platelets by collagen. AB - When human platelets (5 X 10(8)/ml) were stimulated by a threshold concentration of collagen (2 micrograms/ml), a lag period of about 60 s was seen before the initiation of release reaction and aggregation. Breakdown of [32P]phosphatidylinositol 4,5-bisphosphate was seen within 10 s after the addition of collagen. The concentration of intracellular free Ca2+ (monitored by Quin II) rose from 80 nM to 145 nM within 10 s after stimulation by collagen. However, a lag period of about 50 s remained. The rise was not blocked by indomethacin. It was supposed that the initial Ca2+ mobilization by myo-inositol 1,4,5-trisphosphate was too small to cause aggregation. Thromboxane A2 was gradually accumulated during the lag period and then abruptly increased in parallel with aggregation. These events were completely inhibited by 10 microM indomethacin. Thus, aggregation appeared to be dependent on the generation of thromboxane A2. Addition of 25 nM A23187 at 10 s after stimulation by collagen shortened the lag period before initiation of the abrupt thromboxane A2 generation, secretion and aggregation, whereas 25 nM A23187 could not cause these reactions in the absence of collagen. Accordingly, the lag period is assumed to be required for accumulation of free Ca2+ to the threshold for aggregation of platelets. It is considered that thromboxane A2 plays a central role in Ca2+ mobilization during stimulation of human platelets by collagen. PMID- 3001037 TI - Development of myokinase mRNA during embryogenesis of the chick. AB - During chick embryogenesis, the mRNA for myokinase, as determined by in vitro translation, appears on the 16th day. The mRNA levels do not change drastically in succeeding stages and increase immediately after hatching. In contrast, myokinase activity and its protein gradually increase from the 16th day. Thus, myokinase mRNA that is translatable in vitro seems to accumulate transiently in the late stage of embryogenesis. In sucrose density gradient centrifugation, the mRNA sedimented as one peak with a coefficient of 10S. PMID- 3001038 TI - Purification and properties of nucleotide pyrophosphatase from human placenta. AB - Nucleotide pyrophosphatase was purified from human placenta to near homogeneity with a specific activity of about 500-fold over the Triton extract of the homogenate. Purification was achieved most effectively by successive chromatographic steps with AMP-agarose and ADP-agarose columns, based on the affinity of the enzyme towards 5'-adenylate and adenosine 3',5'-diphosphate, and a lectin-Sepharose column, based on the glycoprotein nature of the enzyme. The purified enzyme was found to be essentially homogeneous on SDS-polyacrylamide gel electrophoresis with a mobility corresponding to 130K. The purified enzyme was found to hydrolyze a wide variety of nucleotides, i.e. 3'-phosphoadenosine 5' phosphosulfate (PAPS), adenosine 5'-phosphosulfate (APS), NADH, ATP, nucleotide sugars, oligonucleotides, and p-nitrophenyl-thymidine 5'-phosphate (PNTP). From the oligonucleotides, the enzyme produced 5'-phosphates. Mg2+ was required for full activity. Glycine and sulfhydryl compounds such as 2-mercaptoethanol and 2,3 dimercapto-1-propanol were inhibitory. Most of these properties are common to nucleotide pyrophosphatases [EC 3.6.1.9] and type I (5'-phosphate forming) phosphodiesterases [EC 3.1.4.1] from various sources. The relevance of this enzyme to a unique genetic disease, Lowe's syndrome, is discussed. PMID- 3001039 TI - Disaggregation-induced changes of developmentally regulated proteins in Dictyostelium discoideum. AB - By means of two-dimensional gel electrophoresis, we analyzed proteins present in a slug-shaped tissue mass of D. discoideum and examined the changes in their amounts after disaggregation of the slugs. Of approximately one hundred polypeptides, six were found to decrease in amount after disaggregation. The decreases of four polypeptides were inhibited by the presence of 1 mM cAMP or 250 micrograms/ml cycloheximide. The decreases of the two other proteins were not suppressed by cAMP or cycloheximide. The patterns of proteins present in vegetative and aggregative cells were also examined. None of the six proteins which showed a decrease after slug disaggregation was found in vegetative or preaggregative cells. These results indicate that both synthesis and degradation of these proteins are controlled by cell-cell contact. PMID- 3001041 TI - Two isozymes of chicken muscle acylphosphatase: purification and properties. AB - Two acylphosphatases, named Ch1 and Ch2, have been purified from chicken skeletal muscle. The molecular weights were determined to be 11,900 and 12,000 for Ch1 and Ch2, respectively, by sedimentation equilibrium. In the amino acid compositions of Ch1 and Ch2, two residues of histidine were contained in Ch2, but none in Ch1, and one residue of cysteine was contained in Ch1, but none in Ch2. There were 11 lysines and 6 arginines in Ch1, whereas there were 6 lysines and 11 arginines in Ch2. In addition, the contents of methionine, serine, glutamic acid and glutamine, and alanine were considerably different between Ch1 and Ch2. There were also differences in the peptide maps and carboxyl-terminal amino acid sequences (-Ser-Thr-Arg-Tyr-COOH for Ch1, and -Phe-Thr-Ile-Arg-Lys-COOH for Ch2). In the double immunodiffusion, Ch2 did not form a precipitin line with the rabbit anti-Ch1 antiserum. These results indicate that Ch1 and Ch2 are different, genetically specified isozymes of acylphosphatase of chicken skeletal muscle. Ch2 is considered to be a new type of acylphosphatase from skeletal muscle. PMID- 3001040 TI - Sulfation of chondroitin sulfate secreted by baby hamster kidney cells and their polyoma virus-transformed counterparts. AB - Sulfation of glycosaminoglycans (GAGs) secreted by baby hamster kidney (BHK) cells and the polyoma virus-transformants (PY-BHK) was investigated. It has been reported that chondroitin sulfate (CS) of cell membranes from PY-BHK cells is undersulfated compared to that from BHK cells (Cancer Res. 43, 2712-2717, 1983). In the first series of experiments of the present study, cells were incubated with [3H]glucosamine and [35S]sulfate, and GAGs isolated from the culture medium were examined. GAG composition was comparable between the BHK and PY-BHK cultures. Disaccharide analysis of the chondroitinase ACII digests of the hyaluronate lyase-resistant materials showed a high proportion (68% for BHK and 47% for PY-BHK) of delta Di-0S, with delta Di-4S (32% for BHK and 53% for PY-BHK) as the major sulfated disaccharide on the basis of 3H-radioactivities. The beta-D xyloside treatment did not alter the degree of undersulfation of the CS of either culture. In the second series of experiments, disaccharide analysis of the chondroitinase ABC digests of unlabeled GAGs demonstrated similar disaccharide composition for the two cell types. The BHK and PY-BHK preparations showed 28 and 17% (mol percent) of delta Di-0S, 58 and 72% of delta Di-4S, and 14 and 11% of delta Di-6S, respectively. These results indicate a considerable degree of undersulfation of secretory CS from both cells, and a slightly higher degree, if any, of under-sulfation of secretory CS from BHK cells if compared between the two cell types, which is in contrast to the results reported for membrane CS. PMID- 3001042 TI - Aluminum ions are required for stabilization and inhibition of hepatic microsomal glucose-6-phosphatase by sodium fluoride. AB - Stabilization and inhibition of hepatic microsomal glucose-6-P phosphohydrolase (EC 3.1.3.9) by F- requires the presence of Al3+ ions. At millimolar concentrations, reagent grade NaF inhibited glucose-6-P hydrolysis and protected the enzyme against inactivation induced by heat in the presence of 0.025% (w/v) Triton X-100 or by reaction of the catalytic site with the histidine-specific reagent, diethyl pyrocarbonate. The presence of millimolar EDTA in all test systems abolished the effectiveness of NaF, yet EDTA by itself was without significant influence on the kinetics of phosphohydrolase reaction, the thermal stability of the enzyme or its reactivity with diethyl pyrocarbonate. Although ultrapure NaF was ineffectual in all test systems, its potency as a competitive inhibitor or protective agent was markedly increased by micromolar AlCl3 or when assays were carried out in flint glass test tubes. The latter response is explained by the well documented ability of fluoride solutions to extract Al3+ from glass at neutral pH. Our analysis indicates that the effectiveness of fluoride in all test systems derives from the formation of a specific complex with Al3+, most likely Al(F)4-. The apparent dissociation constant for interaction of the enzyme and Al(F)4- is 0.1 microM. The combination of NaF and AlCl3 holds promise as an unusually effective and versatile means to stabilize this notoriously labile enzyme during efforts to purify it. PMID- 3001043 TI - Nucleotide sequence analysis of TL-DNA of Agrobacterium rhizogenes agropine type plasmid. Identification of open reading frames. AB - We have determined the nucleotide sequence of the Ri TL-DNA region from an Agrobacterium rhizogenes agropine-type plasmid using subcloned regions from the essentially identical Ri TL-DNAs from strains A4 and HRI. This sequenced region of 21,126 base pairs (bp) contains the complete TL-DNA region of the Ri plasmid as determined by analysis of TL-DNA borders in the genome of infected, clonal, Convolvulus arvensis plants. The left and right borders of the TL-DNA are flanked by 25-bp sequences which match the 25-bp terminal sequences found near the borders of T-DNA regions of Agrobacterium tumefaciens Ti plasmids. Other DNA sequences similar to these 25-bp terminal sequences are found within the TL region, and some of these sequences appear to be associated with Ri TL-DNA structures found in transformed tobacco plants. The TL-DNA region contains 18 open reading frames, many of which have 5' and 3' regulatory elements similar to those found in eukaryotic genes. In many cases, CCAAT and TATA elements were found upstream from putative transcriptional initiation codons, and poly(A) addition (AATAAA) elements were observed in presumed 3'-noncoding regions. Comparison of Ri TL-DNA coding and noncoding sequence regions with T-DNA sequence regions from octopine type Ti plasmid pTi15955 reveals no extensive sequence homologies. PMID- 3001044 TI - Isolation and characterization of the inositol cyclic phosphate products of phosphoinositide cleavage by phospholipase C. Metabolism in cell-free extracts. AB - The phosphoinositides are metabolized by phospholipase C in response to hormone or agonist stimulation in many cell types to produce diglyceride and water soluble inositol phosphates. We have recently shown that the phospholipase C reaction products include cyclic phosphate esters of inositol. One of these, inositol 1, 2-cyclic 4,5-trisphosphate, is active in promoting Ca2+ mobilization in platelets and in inducing changes in conductance in Limulus photoreceptors similar to those produced by light (Wilson, D. B., Connolly, T. M., Bross, T. E., Majerus, P. W., Sherman, W. R., Tyler, A., Rubin, L. J., and Brown, J. E. (1985) J. Biol. Chem. 260, 13496-13501. In the current study, we have examined the metabolism of the inositol phosphates. We find that both cyclic and non-cyclic inositol trisphosphates are metabolized by inositol 1,4,5-trisphosphate 5 phosphomonoesterase, to inositol 1,2-cyclic bisphosphate and inositol 1,4 bisphosphate, respectively. However, the apparent Km of the enzyme for the cyclic substrate is approximately 10-fold higher than for the non-cyclic substrate. These inositol bisphosphates are more slowly degraded to inositol 1,2-cyclic phosphate and inositol 1-phosphate, respectively. Inositol 1,2-cyclic phosphate is then hydrolyzed to inositol 1-phosphate, which in turn is degraded to inositol and inorganic phosphate by inositol 1-phosphate phosphatase. The human platelet inositol 1,2-cyclic phosphate hydrolase enzyme and a similar rat kidney hydrolase do not utilize the cyclic polyphosphate esters of inositol as substrates. These results suggest that the inositol cyclic phosphates and the non-cyclic inositol phosphates are metabolized separately by phosphatases to cyclic and non-cyclic inositol monophosphates. The cyclic monophosphate is then converted to inositol 1 phosphate by a cyclic hydrolase. We suggest that the enzymes that metabolize the inositol phosphates may serve to regulate cellular responses to these compounds. PMID- 3001045 TI - Transport of long-chain fatty acids in Escherichia coli. Evidence for role of fadL gene product as long-chain fatty acid receptor. AB - Transport of long-chain fatty acids (LCFA) across the cytoplasmic membrane of Escherichia coli requires functional fadL and fadD genes. The fadD gene codes for an acyl-CoA synthetase (fatty acid: CoA ligase (AMP forming] which has broad chain length specificity and is loosely bound to the cytoplasmic membrane. The fadL gene codes for a 43,000-dalton cytoplasmic membrane protein which, acting by an unknown mechanism, is needed specifically for LCFA transport. As a first step to define the role of the fadL gene product, studies were performed to determine if it functions as a LCFA receptor. The LCFA-binding activity was quantitated in intact cells in the absence of LCFA transport by comparing the binding of LCFA in fadD fadL and fadD fadL+ strains. These studies revealed that (i) fadD fadL+ strains bind 6-fold more LCFA than fadD fadL strains; (ii) fadD fadL strains harboring a plasmid containing the fadL gene bind 16-fold more LCFA than fadD fadL strains harboring only the plasmid vector; and (iii) the fadL-specific LCFA binding activity is regulated by the fadR gene and catabolite repression. Studies with fadL strains harboring fadL plasmids containing in vitro constructed deletions indicate that mutations which alter the physical properties of the 43,000-dalton fadL gene product also affect fadL gene product-specific LCFA binding activity. Overall, these studies suggest that one role of the fadL gene product in the LCFA transport process is to sequester LCFA at sites in the cell membrane for transport. PMID- 3001046 TI - Ouabain binding sites and (Na+,K+)-ATPase activity in rat cardiac hypertrophy. Expression of the neonatal forms. AB - The adaptation of the myocardium to mechanical overload which results in cardiac hypertrophy involves several membrane functions. The digitalis receptor in sarcolemma vesicles from hypertrophied rat hearts is characterized by binding of [3H]ouabain and ouabain-induced inhibition of (Na+,K+)-ATPase. The results show the existence of two families of ouabain binding sites with apparent dissociation constants (Kd) of 1.8-3.2 X 10(-8) M and 1-8 X 10(-6) M, respectively, which are similar to those found in normal hearts. The presence of the high affinity receptor in hypertrophied rat heart is correlated to a detectable inhibition of the (Na+,K+)-ATPase (IC50 = 1-3 X 10(-8) M). However, the high and low affinity sites in hypertrophied hearts bind and release ouabain at 4-5-fold slower rates than the corresponding sites in normal hearts. These properties are similar to that we observed in newborn rat cardiac preparations. Taken together with the expression of myosin isoforms (Schwartz, K., Lompre, A.M., Bouveret, P., Wisnewsky, C., and Whalen, R.G. (1982) J. Biol. Chem. 23, 14412-14418), our data show that the physiological adaptation of the heart also involves the resurgence of the neonatal forms of the digitalis receptor. PMID- 3001047 TI - Complex formation and electron transfer between mitochondrial cytochrome c and flavocytochrome c552 from Chromatium vinosum. AB - Flavocytochrome c552 from Chromatium vinosum catalyzes the oxidation of sulfide to sulfur using a soluble c-type cytochrome as an electron acceptor. Mitochondrial cytochrome c forms a stable complex with flavocytochrome c552 and may function as an alternative electron acceptor in vitro. The recognition site for flavocytochrome c552 on equine cytochrome c has been deduced by differential chemical modification of cytochrome c in the presence and absence of flavocytochrome c552 and by kinetic analysis of the sulfide:cytochrome c oxidoreductase activity of m-trifluoromethylphenylcarbamoyl-lysine derivatives of cytochrome c. As with mitochondrial redox partners, interaction occurs around the exposed heme edge at the "front face" of cytochrome c. However, the domain recognized by flavocytochrome c552 seems to extend to the right of the heme edge, whereas the site of interaction with mitochondrial cytochrome c oxidase and reductase is more to the left. Km but not Vmax of the electron transfer reaction with mitochondrial cytochrome c increases with increasing ionic strength. The correlation of chemical modification and ionic strength dependence data indicates that the electrostatic interaction between the two hemoproteins involves fewer ionic bonds than that with other redox partners of cytochrome c. PMID- 3001048 TI - Characterization of the cell surface receptor for a multi-lineage colony stimulating factor (CSF-2 alpha). AB - 125I-Labeled colony-stimulating factor (CSF) 2 alpha (interleukin 3, multi-CSF, and mast cell growth factor) was used to characterize receptors specific for this lymphokine on the cell surface of the factor-dependent cell line FDC-P2. CSF-2 alpha binding to these cells was specific and saturable. Among a panel of lymphokines and growth factors, only unlabeled CSF-2 alpha was able to compete for the binding of 125I-labeled CSF-2 alpha to cells. Equilibrium binding studies revealed that CSF-2 alpha bound to 434 +/- 281 receptors/cell with a Ka of 8.7 +/ 3.9 X 10(9) M-1. Affinity cross-linking experiments with the homobifunctional cross-linking reagents disuccinimidyl suberate, disuccinimidyl tartrate, and dithiobis(succinimidyl propionate) produced a radiolabeled band of Mr = 97,000 on intact cells and in purified cell membranes, while an additional band of Mr = 138,000 was produced upon cross-linking to intact cells only. The relationship between these two bands is discussed. The results indicate that the receptor for CSF-2 alpha on FDC-P2 cells consists at a minimum of a subunit of Mr = 72,500. PMID- 3001049 TI - Cyclic nucleotide-dependent phosphorylation of proteins in rod outer segments in frog retina. Characteristics of the phosphorylated proteins and their dephosphorylation. AB - To clarify the function of cyclic nucleotides in rod outer segments (ROS) of frog retinas, we studied cyclic nucleotide-dependent phosphorylation and dephosphorylation of protein. cGMP or cAMP with [gamma-32P]ATP in the dark enhanced the phosphorylation of two ROS proteins with Mr = 10,500 (Band 1) and 8,500 (Band 2) according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The phosphorylation was maximally enhanced at 2.0 mM cGMP and cAMP in the presence of Mg2+. The cGMP-activated protein kinase showed near-optimal activity between pH 6.5 and 8.0. GMP, GDP, GTP, AMP, and ADP did not enhance the phosphorylation. The stoichiometry of the phosphate incorporated into Bands 1 and 2 could not be calculated because the amount of Bands 1 and 2 was too small to measure. Both 32P-phosphorylated Bands 1 and 2 (32P-Bands 1 and 2) were solubilized during preparation and the molecular weight of each, in the native preparation, was 19,000. Their isoelectric point was 5.2. The sites of phosphorylation were the serine residue(s). DEAE-Sephadex A-50 column chromatography gave a good separation of Bands 1 and 2 from other 32P phosphoproteins at 60 mM NaCl. Dephosphorylation of 32P-Bands 1 and 2 in dark adapted ROS suspension required Mn2+ or Mg2+; the former was more effective than the latter at concentrations below 0.5 mM. Both phosphorylation and dephosphorylation were inhibited by Zn2+. PMID- 3001050 TI - Monoacylglycerol acyltransferase. Evidence that the activities from rat intestine and suckling liver are tissue-specific isoenzymes. AB - The monoglycerol acyltransferase (EC 2.3.1.22) (recommended name acylglycerol palmitoltransferase) activities from rat intestinal mucosa and suckling liver microsomes were compared in order to determine why substrate specificities differed in the two tissues. Suckling liver monoacylglycerol acyltransferase activity was highly specific for sn-2-mono-C18:1 glycerol and acylated rac-1-mono C18:1 glycerol and 1- and 2-mono-C18:1 glycerol ethers poorly. In contrast, the substrate specificity of intestinal monoacylglycerol acyltransferase activity was broad. 1-Acyl- and 1- and 2-alkylglycerols were acylated at rates that were 45 78% of the rate observed with the preferred substrate sn-2-mono-C18:1 glycerol. Partial heat inactivation did not alter these relative specific activities, making it unlikely that intestinal microsomes contained a second acyltransferase capable of acylating the alternate substrates. The hypothesis that intestine and liver contain non-identical monoacylglycerol acyltransferase activities was further tested. Intestinal mucosa monoacylglycerol acyltransferase was much more thermolabile than the liver activity. Incubation with 50 microM diethylpyrocarbonate inactivated liver monoacylglycerol acyltransferase activity 84% but had little effect on the intestinal activity. Hydroxylamine completely reversed diethylpyrocarbonate inactivation, suggesting that critical histidine residues were more accessible in liver monoacylglycerol acyltransferase. 2,4,6 Trinitrobenzene sulfonic acid inactivated hepatic monoacylglycerol acyltransferase more than the intestinal activity, suggesting that critical lysine residues were more accessible. The intestinal and liver activities were also differently affected by acetone, detergents, MgCl2, phospholipids, and bovine serum albumin. Taken as a whole, the data strongly suggest that rat intestinal mucosa and suckling liver contain tissue-specific monoacylglycerol acyltransferase isoenzymes. PMID- 3001051 TI - Glucocorticoids regulate the expression of a rat growth hormone gene lacking 5' flanking sequences. AB - Rat growth hormone (rGH) gene expression is regulated by glucocorticoids in vivo and in cultured pituitary cells. After the co-transfer of a plasmid containing the rGH gene into mouse L-cells (with or without the simian virus 40 enhancer), little or no normal rGH mRNA is produced. Instead, the predominant rGH gene transcripts are about 0.75 kilobase pairs (kb); these lack the first two exons of the rGH but possess a 3' end that terminates accurately. Nevertheless, the levels of these transcripts are increased by glucocorticoids. When all of the rat sequences 5' to an Xho1 site located 7 nucleotides downstream from the rGH gene physiological cap site are deleted and the mutant gene introduced into L-cells, the transfectant cell lines still produce the 0.75-kb transcripts; however, in addition, these cells produce a more abundant 1.1-kb mRNA that has a 3' terminus similar to that of rGH mRNA, but whose 5' termini begin in the region of, but not at, the initiation site used in pituitary cells. Both of these transcripts are increased 3- to 5-fold by 1 microM concentration of the glucocorticoid dexamethasone. These data indicate that 1) deletion of all of the rGH gene 5' flanking sequences allows formation of approximately full length transcripts and 2) sequences containing information for regulation of rGH gene expression in L cells by glucocorticoids are contained in the structural portion of the gene and/or the 3' flanking sequence. PMID- 3001052 TI - cGMP influences guanine nucleotide binding to frog photoreceptor G-protein. AB - A rapid light-induced decrease in cGMP is thought to play a role in regulating the permeability or light sensitivity of photoreceptor membranes. Photo-excited rhodopsin activates a guanine nucleotide-binding protein (G-protein) by catalyzing the exchange of bound GDP for GTP. This G-protein X GTP complex activates the phosphodiesterase resulting in a decrease in cGMP concentration. We have observed two processes in vitro which may be relevant for the regulation of G-protein activation. First, we have found that free GDP binds to G-protein with an affinity similar to that of GTP. These two nucleotides appear to compete for a common site. Since G-protein X GDP does not activate phosphodiesterase, light induced changes in the GTP/GDP ratio known to occur on illumination may serve to reduce G-protein activation and hence reduce phosphodiesterase activation. Second, addition of cGMP in the presence of equimolar GTP and GDP causes GTP binding to G-protein to be enhanced compared to GDP binding. This effect increases as the cGMP concentration is increased from 0.05 to 2 mM. Thus, light induced decreases in cGMP concentration may also act as a feedback control in reducing G-protein activation. One or both of these processes may be involved in the desensitization (light adaptation) of rod photoreceptors. PMID- 3001053 TI - Chromatin structure of the telomeric region and 3'-nontranscribed spacer of Tetrahymena ribosomal RNA genes. AB - The chromatin structure of the 3'-nontranscribed spacer of the linear rRNA gene molecules of Tetrahymena thermophila was examined. This region includes the transcription termination site, two sets of recently identified conserved spacer repeats (Type IV and V repeats (Challoner, P. B., Amin, A. A., Pearlman, R. E., and Blackburn, E. H. (1985) Nucleic Acids Res. 13, 2661-2680], and the terminus of the molecule. Using sensitivity to nucleases as a probe, a unique chromatin structure was found in this rDNA region. Proceeding from the end of the rDNA molecule, the telomeric repeated sequence, (CCCCAA)n, was packaged in a non nucleosomal complex adjacent to three phased nucleosomes. This nucleosomal structure was disrupted at the Type V repeat region, which, compared with the neighboring nucleosomal region, was more accessible to nucleases and, from both micrococcal nuclease and DNase I digestion results, was packaged in chromatin differently from the sequences flanking it on both sides. The region between the Type V repeats and adjacent to the transcription termination site was in yet another distinguishable chromatin structure as judged by its sensitivity to nucleases. It includes sites protected in chromatin and sites which were cleaved in chromatin but not detectably digested in DNA controls, suggesting that specific proteins are also associated with this region. PMID- 3001054 TI - Bacteriophage P1 Cre-loxP site-specific recombination. Site-specific DNA topoisomerase activity of the Cre recombination protein. AB - Site-specific recombination in bacteriophage P1 occurs between two loxP sites in the presence of the Cre recombination protein. The structure of the 34-base pair loxP site consists of two 13-base pair inverted repeats separated by an 8-base pair spacer region. A mutation in the loxP site has been constructed which deletes one of the internal bases of the spacer region at the axis of dyad symmetry. This mutant loxP site shows a 10-fold reduction in recombination activity with a wild-type site both in vivo and in vitro. This low level of intramolecular recombination between a wild-type loxP site and the mutant loxP501 site is observed in vitro only when the DNA substrate is supercoiled. The majority of the supercoiled substrate is relaxed by the Cre protein, and on longer incubations, single-stranded nicks accumulate in the DNA. We have determined that these nicks occur in both the wild-type and the mutant sites. The positions of these nicks correspond to the positions of cleavage found during recombination of two wild-type sites, suggesting that the Cre protein is attempting to carry out recombination with the mutant site but most of the time this reaction is abortive. We have determined that the Cre protein relaxes a supercoiled topoisomer of a DNA substrate containing one wild-type site and one mutant site to yield a distribution of topoisomers whose linking numbers differ by steps of one, indicating that Cre can act as a type I topoisomerase. PMID- 3001055 TI - Structural features in the 3'-terminal region of polyribosome-bound rabbit globin messenger RNAs. AB - A nuclease S1 mapping procedure was used to identify sites accessible to nucleases in the 3'-noncoding region of the rabbit globin mRNAs. A complex structure was evident in the alpha-globin species, with one highly accessible single-stranded site, large portions in an accessible double-stranded configuration, and a portion not accessible to any of the nucleases. In the beta globin mRNA, the region was more uniformly accessible to RNase T1 and to a cobra venom enzyme specific for double-stranded RNA, but it had only a single site highly accessible to a bulkier Neurospora endonuclease. The patterns of cleavage were nearly identical in the deproteinized mRNAs and in the mRNAs associated with polyribosomes in reticulocyte extracts. In both species, a zone of secondary structure occurred around the poly(A) junction. In each species, virtually all the molecules had a poly(A) sequence of at least 20-25 AMP residues. A periodicity in poly(A) size distribution was observed. These results indicate that the beginning of this sequence is well protected against degradation inside the cell and that zones of partial protection occur at measured intervals. In crude extracts, where the poly(A) is covered with proteins, this sequence was protected against nuclease digestion. PMID- 3001056 TI - Alterations in amino acid transport in Na,K-ATPase amplified HeLa cells. AB - Amino acid transport was studied in C1 cells which contain amplified levels of sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase), in C4 cells which are ouabain-sensitive revertants, and in parental HeLa S3. Sodium dependent uptake of aminoisobutyric acid and alanine was increased 2-fold in the amplified C1 cells. After a 6 h amino acid starvation period, the rate of sodium dependent uptake of methylaminoisobutyric acid was 70-90% greater for C1 than for C4 and HeLa. This uptake was inhibitable by ouabain and the apparent Km values for high affinity uptake were similar in all three lines. Overall, neutral amino acid uptake through Systems A, ASC, and L was 2-fold higher in the Na,K-ATPase amplified C1 cells relative to C4 or HeLa. The induction of System A uptake of methylaminoisobutyric acid after starvation was more rapid in both the amplified C1 cells and the revertant C4 when compared to HeLa, which suggests that the selection for amplification of the Na,K-ATPase produced membrane alterations affecting the adaptive regulation of System A. PMID- 3001057 TI - Structure of the Escherichia coli K12 regulatory gene tyrR. Nucleotide sequence and sites of initiation of transcription and translation. AB - The nucleotide sequence of 1964 base pairs of the Escherichia coli K12 chromosome containing the autogenously regulated regulatory gene tyrR has been determined. The site of initiation of transcription of tyrR has been mapped by primer extension analysis, and the initiation codon has been identified by site-specific deletion mutagenesis. The nucleotide sequence predicts a subunit molecular weight of 53,099 for the TyrR protein. Codon usage in the tyrR structural gene shows a bias toward those synonymic codons which are used rarely in efficiently expressed E. coli genes. The nucleotide sequence of a 22-base pair region adjacent to the promoter and distal to the structural gene exhibits considerable identity with corresponding regions of other genes regulated by tyrR. It is proposed that this is a site for repression by the TyrR protein. PMID- 3001058 TI - Human placental cytoplasmic 5'-nucleotidase. Kinetic properties and inhibition. AB - The kinetic properties of highly purified human placental cytoplasmic 5' nucleotidase were investigated. Initial velocity studies gave Michaelis constants for AMP, IMP, and CMP of 18, 30, and 2.2 microM, respectively. The enzyme shows the following relative Vmax values: CMP greater than UMP greater than dUMP greater than GMP greater than AMP greater than dCMP greater than IMP. The activity was magnesium-dependent, and this cation binds sequentially with a Km of 14 microM for AMP and an apparent Km of 6 mM for magnesium. A large variety of purine, pyrimidine, and pyridine compounds exert an inhibitory effect on enzyme activity. IMP, GMP, and NADH produce almost 100% inhibition at 1.0 mM. Nucleoside di- and triphosphates are potent inhibitors. ATP and ADP are competitive inhibitors with respect to AMP and IMP as substrates with Ki values of 100 and 15 microM, respectively. Inorganic phosphate is a noncompetitive inhibitor with Ki values of 19 and 43 mM. Nucleosides and other compounds studied produce only a modest decrease of enzyme activity at 1 mM. Our findings suggest that the enzyme is regulated under physiological conditions by the concentrations of magnesium, nucleoside 5'-monophosphates, and nucleoside di- and triphosphates. The nucleotide pool concentration regulates the enzyme possibly by a mechanism of heterogeneous metabolic pool inhibition. These properties of human placental cytoplasmic 5'-nucleotidase may be related to the control of nucleotide degradation in vivo. PMID- 3001059 TI - Differential phosphorylation of the progesterone receptor by insulin, epidermal growth factor, and platelet-derived growth factor receptor tyrosine protein kinases. AB - Purified preparations of insulin, epidermal growth factor (EGF), and platelet derived growth factor (PDGF) receptors were compared for their abilities to phosphorylate purified hen oviduct progesterone receptors. The specific activities of all three peptide hormone-induced receptor kinases were first defined using a synthetic tridecapeptide tyrosine protein kinase substrate. Next, equivalent ligand-activated activities of the three receptor kinases were tested for their abilities to phosphorylate hen oviduct progesterone receptor. Both the insulin and EGF receptors phosphorylated progesterone receptor at high affinity, exclusively at tyrosine residues and with maximal stoichiometries that were near unity. In contrast, the PDGF receptor did not recognize progesterone receptor as a substrate. Insulin decreased the Km of the insulin receptor for progesterone receptor subunits as substrates, but had no significant effect on Vmax values. On the other hand, EGF increased the Vmax of the EGF receptor for progesterone receptor subunits as substrates. Phosphorylation of progesterone receptor by the insulin and EGF receptor kinases differed in two additional ways. 1) EGF activated receptor phosphorylated the 80- and 105-kDa progesterone receptor subunits to an equal extent, whereas insulin-activated receptor preferentially phosphorylated the 80-kDa subunit. 2) Phosphopeptide fingerprinting analyses revealed that while insulin and EGF receptors phosphorylated one identical major site on both progesterone receptor subunits, they differed in their specificities for other sites. PMID- 3001060 TI - Analysis of Z-DNA in fixed polytene chromosomes with monoclonal antibodies that show base sequence-dependent selectivity in reactions with supercoiled plasmids and polynucleotides. AB - Five monoclonal anti-Z-DNA antibodies were characterized with respect to their binding of synthetic nucleic acid polymers and of supercoiled circular plasmid DNA. All of the antibodies reacted only with DNA in the Z-conformation; however, they fell into two classes on the basis of sequence specificity. One class, with broad specificity, reacted well with all sequences in the Z-form, including poly(dG-dC), poly(dG-dm5C), and poly (dG-dBr5C) in linear polymers and poly(dG dC)n and poly[(dC-dA)n.(dT-dG)n] sequences in supercoiled plasmids. The other class bound only Z-DNA formed by poly(dG-dC). Binding of the monoclonal antibodies specifically to inserts of Z-DNA-forming sequences in plasmids was mapped directly by cross-linking of antibody to the DNA, digestion with restriction nuclease, and electrophoretic analysis of both the unbound fragments and the bound fragments recovered from immune complexes. The monoclonal antibodies were used for indirect immunofluorescence staining of Drosophila polytene chromosomes fixed by two procedures. One procedure yielded chromosomes with Z-specific antibody binding in many interbands, a few specific bands, and parts of some puffs. On chromosomes fixed by the second procedure, antibody staining appeared to follow the DNA concentration, staining all bands brightly. For each fixation procedure, chromosomes showed the same staining pattern with each of the broad specificity monoclonal antibodies that had been seen with polyclonal antibodies. The antibodies that reacted only with poly(dG-dC) and poly (dG-dC)n plasmid inserts did not stain chromosomes fixed by either protocol. We conclude that stretches of poly(dG-dC)n sequences do not contribute significantly to the presence of Z-DNA in fixed polytene chromosomes of Drosophila melanogaster. PMID- 3001061 TI - Nucleotide sequence of cDNA containing the complete coding sequence for human lysosomal glucocerebrosidase. AB - Complementary DNA clones for human glucocerebrosidase were isolated from a human hepatoma library in lambda gt11. The complete nucleotide sequence of the 1805 base pair cDNA insert has been determined. In addition to 5' and 3' untranslated regions (51 and 206 base pairs, respectively), the cDNA insert contains 1548 base pairs that completely encode human glucocerebrosidase. All possible N-linked glycosylation sites are identified. Examination of the 19 amino acids of the leader polypeptide beginning with the ATG at position 52 revealed a hydrophobic core and a carboxyl-terminal glycine at the peptidase cleavage site, features consistent with the leader sequences described for other human translocated proteins. The Mr of 57,000 calculated from the 516 amino acids deduced from cDNA sequence is in good agreement with that identified by immunoprecipitation following in vitro translation of human placental mRNA. PMID- 3001062 TI - Selective oxidation and reduction of methionine residues in peptides and proteins by oxygen exchange between sulfoxide and sulfide. AB - Treatment of amino acids, peptides, and proteins with aqueous solution of dimethyl sulfoxide (Me2SO) and hydrochloric acid (HCl) resulted in the oxidation of methionine to methionine sulfoxide. In addition to methionine, SH groups are also oxidized, but this reaction proceeds after a lag period of 2 h. Other amino acids are not modified by aqueous Me2SO/HCl. The reaction is strongly pH dependent. Optimal conditions are 1.0 M HCl, 0.1 M Me2SO, at 22 degrees C. The reaction exhibits pseudo-first order kinetics with Kobs = 0.23 +/- 0.015 M-1 min 1 at 22 degrees C. Incubation of methionine sulfoxide with dimethyl sulfide and HCl resulted in the conversion of methionine sulfoxide to methionine. This reaction is fast (t1/2 = 4 min at room temperature) and quantitative at relatively anhydrous condition (i.e. at H2O:concentrated HCl:dimethyl sulfide ratio of 2:20:1). Quantitative conversions of methionine sulfoxide back to methionine are obtained in peptides and proteins as well, with no observable other side reactions in amino acids and proteins. The wide applications of this selective oxidation and reduction of methionine residues are demonstrated and discussed. PMID- 3001063 TI - Molecular cloning and sequence of the B880 holochrome gene from Rhodospirillum rubrum. AB - Restriction fragments of genomic Rhodospirillum rubrum DNA were selected according to size by electrophoresis followed by hybridization with [32P]mRNA encoding the two B880 holochrome polypeptides. The fragments were cloned into Escherchia coli C600 with plasmid pBR327 as a vector. The clones were selected by colony hybridization with 32P-holochrome-mRNA and counterselected by hybridization with Rs. rubrum ribosomal RNA, a minor contaminant of the mRNA preparation. Chimeric plasmid pRR22 was shown to contain the B880 genes by hybrid selection of B880 holochrome-mRNA. We report a restriction map of its 2.2 kilobase insert and the sequence of a 430 base pair fragment thereof. Genes alpha and beta are nearly contiguous, indicating that they are transcribed as a single operon. The predicted amino acid sequences coincide with the sequences of the alpha and beta polypeptides established in other laboratories, except for additional C-terminal tails of 10 and 13 amino acid residues, respectively. We suggest that these tail sequences may serve, during membrane assembly, to give these intrinsic membrane proteins their peculiar orientation with their C terminus facing the periplasm and their N terminus facing the cytoplasm. Intraspecific sequence homology between the alpha and beta genes of R. rubrum is low, showing no evolutionary relatedness. This is in contrast to the high interspecific homology between the corresponding sequences of Rs. rubrum and Rhodopseudomonas capsulata B880 genes. PMID- 3001065 TI - Lycorine: a eukaryotic termination inhibitor? AB - The effect of the alkaloid lycorine on viral protein synthesis was studied in poliovirus-infected HeLa cells. The incorporation of [3H]leucine was inhibited by lycorine in a dose-dependent way, although lycorine never completely abolished translation. Using polyacrylamide gel electrophoresis, the viral proteins were identified as derived from the P1 (5' terminal), P2 (middle), or P3 (3' terminal) region of the poliovirus translation unit. The residual labeling of viral proteins in the presence of lycorine was mainly due to synthesis of P1 proteins and slightly less to P2 proteins, while virtually no P3-derived proteins were made. It is suggested that lycorine may act at the level of termination. PMID- 3001064 TI - The relationship of hormone-sensitive and hormone-insensitive phosphatidylinositol to phosphatidylinositol 4,5-bisphosphate in the WRK-1 cell. AB - We have previously characterized two distinct pools of phosphatidylinositol (PI) in the WRK-1 rat mammary tumor cell, one whose metabolism is enhanced in response to vasopressin and another which is insensitive to hormonal manipulation. The purpose of the present study was to examine the relationship between cellular phosphatidylinositol 4,5-bisphosphate (PIP2) and each of the two PI pools. We have found that in WRK-1 cells, vasopressin induces the rapid loss of PIP2 and the accumulation of inositol phosphates. By making use of kinetic differences in 32Pi uptake into the two pools of PI and assessing radioactivity levels in the 1 phosphate of PIP2, we have determined that hormone-sensitive PI is the precursor of approximately 60% of the cellular PIP2; the remainder is synthesized from the hormone-insensitive pool. Additional data indicate that PIP2 derived from hormone sensitive PI is likewise hormone-sensitive, while that synthesized from hormone insensitive PI remains stable over a long period of time and is not affected by the presence of vasopressin. PMID- 3001066 TI - Secretion of rat serum albumin by COS cells transfected with a spliced cDNA gene. A system to study protein sorting. AB - Certain structural features of secreted proteins may function as "sorting signals" to direct the various steps required in the secretory pathway. In order to identify and study the function of these signals we have cloned a complete cDNA gene encoding rat serum albumin (RSA) and expressed this gene in COS-1 cells via an SV40-plasmid shuttle vector. The gene was constructed by splicing together a segment of genomic DNA and three cDNA fragments excised from recombinant plasmids. DNA endonuclease digestion and ligation at restriction sites common to overlapping regions of these four RSA DNA fragments assured the maintenance of the translation reading frame during the construction of this gene. COS-1 cells transfected with the recombinant vector containing the full-length RSA gene (pSV2rsa) synthesize and secrete RSA immunoreactive material into the culture medium. This mammalian expression system provides a means to study the signals and processes involved in intracellular transport of secreted proteins. PMID- 3001067 TI - Cytoplasmic pH regulation in normal and abnormal neutrophils. Role of superoxide generation and Na+/H+ exchange. AB - The cytoplasmic pH of human neutrophils was determined fluorometrically using carboxylated fluorescein derivatives. When normal neutrophils were activated by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) in Na+-containing medium, the cytoplasmic pH initially decreased but then returned to near normal values. In Na+-free media or in Na+ medium containing amiloride, TPA induced a marked monophasic intracellular acidification. The cytoplasmic acidification is associated with net H+ equivalent efflux, suggesting metabolic acid generation. The metabolic pathways responsible for the acidification were investigated by comparing normal to chronic granulomatous disease neutrophils. These cells are unable to oxidize NADPH and generate superoxide. When treated with TPA in Na+ free or amiloride-containing media, chronic granulomatous disease cells did not display a cytoplasmic acidification. This suggests that in normal cells NADPH oxidation and/or the accompanying activation of the hexose monophosphate shunt are linked to the acidification. Unlike normal neutrophils, chronic granulomatous disease cells treated with TPA in Na+-containing medium displayed a significant cytoplasmic alkalinization. The alkalinization was Na+-dependent and amiloride sensitive, indicating activation of Na+/H+ exchange. Thus, the Na+/H+ antiport, which can be indirectly stimulated by the metabolic cytoplasmic acidification, is also directly activated by the phorbol ester. PMID- 3001068 TI - Regulation of Na,K-ATPase biosynthesis in developing Artemia salina. AB - Regulation of the biosynthesis of the sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase) (EC 3.6.1.3) was studied in the developing brine shrimp, Artemia salina. Measurement of levels of the subunits of the Na,K-ATPase by radioimmunoassay indicated the presence of both alpha and beta subunits in undeveloped cysts and developing embryos prior to the appearance of enzymatic activity. The quantity of each subunit increased dramatically between 8 and 24 h of development and then reached a plateau at about 32 h. The quantities of translationally active mRNA alpha and mRNA beta were also determined. Undeveloped cysts contained mRNA alpha and mRNA beta, and the amounts increased 9- and 3 fold, respectively, during the first 24 h of development. The data suggest that the increase in Na,K-ATPase activity was at least in part due to increases in protein synthesis related to changes in mRNA levels. The data also suggest involvement of additional regulatory mechanisms. The alpha-subunit has been detected as two molecular weight forms (alpha 1 and alpha 2) which demonstrate changes in relative amounts during development (Peterson, G. L., Churchill, L., Fisher, J. A., and Hokin, L. E. (1982) J. Exp. Zool. 221, 295-308). We show here that this was not due to changes in mRNA alpha 1 and mRNA alpha 2. PMID- 3001069 TI - Phorbol esters inhibit alpha 1-adrenergic effects and decrease the affinity of liver cell alpha 1-adrenergic receptors for (-)-epinephrine. AB - 4 beta-Phorbol 12-myristate 13-acetate (PMA) modified the metabolic actions of three calcium-dependent hormones in different ways. The stimulations of glycogenolysis ureogenesis and phosphatidylinositol labeling produced by alpha 1 adrenergic agonist was blocked by the phorbol ester. In contrast, PMA slightly increased the stimulation of ureogenesis produced by low concentration of angiotensin II without modifying the maximal response. No effect of PMA was observed on the stimulation of ureogenesis induced by vasopressin. The stimulation of phosphatidylinositol labeling induced by vasopressin was decreased by PMA, whereas that induced by angiotensin II was not affected. In intact freshly isolated hepatocytes, [3H]prazosin binds with high affinity to a site which displays the characteristics of alpha 1-adrenergic receptor. Competitive inhibition studies with (-)-epinephrine reveal two different sites for this agonist: a high affinity site (Kd 9 nM) and a low affinity site (Kd 2 microM). In the presence of phorbol esters, (-)-epinephrine binding data now show the presence of a single class of low affinity sites, with similar affinity to those present in control cells. Thus, the inhibition of hepatocyte alpha 1-adrenergic action by PMA may be related to the loss of high affinity binding sites caused by the tumor promoter. PMID- 3001071 TI - The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane. AB - We have investigated the subcellular localization of the peripheral-type benzodiazepine receptor in rat adrenal gland using the high affinity ligand 3H labeled 1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide ([3H]PK11195). The autoradiographic pattern of [3H]PK11195 binding sites in tissue sections of adrenal gland is similar to the histochemical distribution of the mitochondrial marker enzymes, cytochrome oxidase and monoamine oxidase, which are present in high concentrations only in the cortex. Subcellular fractionation studies of homogenates of adrenal gland indicate that the recovery and enrichment of [3H]PK11195 binding sites in the nuclear, mitochondrial, microsomal, and soluble fractions correlate closely with cytochrome oxidase activity, but not with markers for the nuclei, lysosomes, peroxysomes, endoplasmic reticulum, plasma membrane, or cytoplasm, indicating an association of the peripheral-type benzodiazepine receptor with the mitochondrial compartment. Titration of isolated mitochondria with digitonin results in the simultaneous release of the peripheral type benzodiazepine receptor and of monoamine oxidase, but not cytochrome oxidase, indicating association of the peripheral-type benzodiazepine receptor with the mitochondrial outer membrane. Scatchard analysis and drug displacement studies of the binding of [3H] PK11195 to intact mitochondria and to the outer membrane-enriched digitonin extract further confirm the localization of the peripheral-type benzodiazepine receptor to the mitochondrial outer membrane. PMID- 3001070 TI - Ionic and GTP regulation of binding of platelet-activating factor to receptors and platelet-activating factor-induced activation of GTPase in rabbit platelet membranes. AB - Specific binding of 3H-labeled platelet-activating factor (PAF) to rabbit platelet membranes was found to be regulated by monovalent and divalent cations and GTP. At 0 degrees C, inhibition of [3H]PAF binding by sodium is specific, with an ED50 of 6 mM, while Li+ is 25-fold less effective. On the contrary, K+, Cs+, and Rb+ enhance the binding. The divalent cations, Mg2+, Ca2+, and Mn2+ enhance the specific binding 8-10-fold. From both Scatchard and Klotz analyses, the inhibitory effect of Na+ is apparently due to an increase in the equilibrium dissociation constant (KD) of PAF binding to its receptors. However, the Mg2+ induced enhancement of the PAF specific binding may be attributed to an increased affinity of the receptor and an increased availability of the receptor sites. In the presence of Na+, PAF receptor affinity decreased with increasing temperature with a 100-fold sharp discontinuous decrease in receptor affinity at 24 degrees C. In contrast, the Mg2+-induced increase is independent of temperature suggesting that the Mg2+ regulatory site is different from Na+ regulatory site. [3H]PAF binding is also specifically inhibited by GTP; other nucleotides have little effect. PAF also stimulates hydrolysis of [gamma-32P]GTP with an ED50 of 0.7 nM, whereas 3-O-hexadecyl-2-O-acetyl-sn-glyceryl-1-phosphorylcholine showed no activity even at 10 microM. Moreover, such stimulatory effect of PAF is dependent on Na+ and can be abolished by the PAF-specific receptor antagonist, kadsurenone, but not by an inactive analog, kadsurin B. These results suggest that the PAF receptor may be coupled with the adenylate cyclase system via an inhibitory guanine nucleotide regulatory protein. PMID- 3001072 TI - Discrete catalytic sites for quinone in the ubiquinol-cytochrome c2 oxidoreductase of Rhodopseudomonas capsulata. Evidence from a mutant defective in ubiquinol oxidation. AB - A non-photosynthetic mutant (Ps-) of Rhodopseudomonas capsulata, designated R126, was analyzed for a defect in the cyclic electron transfer system. Compared to a Ps+ strain MR126, the mutant was shown to have a full complement of electron transfer components (reaction centers, ubiquinone-10, cytochromes b, c1, and c2, the Rieske 2-iron, 2-sulfur (Rieske FeS) center, and the antimycin-sensitive semiquinone). Functionally, mutant R126 failed to catalyze complete cytochrome c1 + c2 re-reduction or cytochrome b reduction following a short (10 microseconds) flash of actinic light. Evidence (from flash-induced carotenoid band shift) was characteristic of inhibition of electron transfer proximal to cytochrome c1 of the ubiquinol-cytochrome c2 oxidoreductase. Three lines of evidence indicate that the lesion of R126 disrupts electron transfer from quinol to Rieske FeS: 1) the degree of cytochrome c1 + c2 re-reduction following a flash is indicative of electron transfer from Rieske FeS to cytochrome c1 + c2 without redox equilibration with an additional electron from a quinol; 2) inhibitors that act at the Qz site and raise the Rieske FeS midpoint redox potential (Em), namely 5 undecyl-6-hydroxy-4,7-dioxobenzothiazole or 3-alkyl-2-hydroxy-1,4-napthoquinone, have no effect on cytochrome c1 + c2 oxidation in R126; 3) the Rieske FeS center, although it exhibits normal redox behavior, is unable to report the redox state of the quinone pool, as metered by its EPR line shape properties. Flash-induced proton binding in R126 is indicative of normal functional primary (QA) and secondary (QB) electron acceptor activity of the photosynthetic reaction center. The Qc functional site of cytochrome bc1 is intact in R126 as measured by the existence of antimycin-sensitive, flash-induced cytochrome b reduction. PMID- 3001073 TI - 5-Methyltryptamine decreases net accumulation of 32P into the polyphosphoinositides from [gamma-32P]ATP in a cell-free system from blowfly salivary glands. Activation of breakdown of the newly synthesized [32P]polyphosphoinositides. AB - Incubation of blowfly salivary gland homogenates with 30 microM [gamma-32P]ATP resulted in a rapid, Mg2+-dependent, synthesis of [32P]polyphosphoinositides and [32P]phosphatidic acid. 5-Methyltryptamine, in the presence of 10 microM guanosine 5'-(3-O-thio)trisphosphate, reduced the net accumulation of 32P label into phosphatidylinositol-4,5-P2 and phosphatidylinositol-4-P by 35 and 20%, respectively. 5-Methyltryptamine did not affect synthesis of [32P]phosphatidic acid. Phosphorylation of polyphosphoinositides was not affected by 5 methyltryptamine. In membranes labeled in vitro with [gamma-32P]ATP, 5 methyltryptamine stimulated a rapid breakdown of the [32P]polyphosphoinositides. These results indicate that in blowfly salivary gland homogenates, hormone stimulates breakdown of the newly synthesized polyphosphoinositides. In the presence of hormone, the rate of polyphosphoinositide synthesis does not compensate for the rate of polyphosphoinositide degradation. PMID- 3001074 TI - Platelet-activating factor-mediated vasoconstriction and glycogenolysis in the perfused rat liver. AB - Infusion of platelet-activating factor (alkyl acetylglycerophosphocholine (AGEPC] into isolated perfused rat livers caused a dose-dependent, transient increase in portal vein pressure, indicative of constriction of the hepatic vasculature. A close correlation was observed between the changes in portal pressure and concomitant transient increases in hepatic glucose output. The two processes displayed similar dose dependence and were attenuated to a similar extent by reducing the perfusate calcium concentration. Reducing the perfusate free calcium concentration to 1 nM by co-infusion of EGTA did not abolish completely the hepatic responses to AGEPC. Verapamil inhibited both the hemodynamic and glycogenolytic responses to AGEPC in a dose-dependent fashion; the IC50 was approximately 10 microM at an AGEPC concentration of 6.6 X 10(-11) M. Also, both responses displayed similar degrees of tachyphylaxis in response to repeated short infusions of AGEPC. Measurement of glycogen phosphorylase a in extracts from freeze-clamped livers demonstrated a rapid increase in phosphorylase a in response to infusion of AGEPC. A small but significant increase in whole tissue ADP was found in response to AGEPC (2 X 10(-8) M); cAMP levels were not changed by AGEPC infusion. It is concluded that glycogenolysis in the perfused liver in response to AGEPC may be a result of the hemodynamic effects of AGEPC, rather than a direct effect of the phospholipid mediator on the hepatocyte. PMID- 3001075 TI - Control of glycogen synthase by insulin and isoproterenol in rat adipocytes. Changes in the distribution of phosphate in the synthase subunit in response to insulin and beta-adrenergic receptor activation. AB - Rat adipocytes were incubated with [32P]phosphate to label glycogen synthase, which was rapidly immunoprecipitated from cellular extracts and cleaved using either CNBr or trypsin. All of the [32P]phosphate in synthase was recovered in two CNBr fragments, denoted CB-1 and CB-2. Isoproterenol (1 microM) rapidly decreased the synthase activity ratio (-glucose-6-P/+glucose-6-P) and stimulated the phosphorylation of both CB-1 and CB-2 by approximately 30%. Insulin opposed the decrease in activity ratio and blocked the stimulation of phosphorylation by isoproterenol. Incubating cells with insulin alone changed the 32P content of neither CB-1 nor CB-2. Trypsin fragments were separated by reverse phase liquid chromatography and divided into peak fractions, denoted F-I-F-VII in order of increasing hydrophobicity. F-V contained almost half of the [32P]phosphate and was phosphorylated when synthase was immunoprecipitated from unlabeled fat cells and incubated with [gamma-32P]ATP and the cAMP-independent protein kinase, FA/GSK 3. That F-V also had the same retention time as the skeletal muscle synthase fragment containing sites 3(a + b + c) suggests that it contains sites 3. Muscle sites 1a, 5, 1b, and 2 eluted with F-I, F-II, F-VI, and F-VII, respectively. F-V was increased approximately 25% by isoproterenol, but the largest relative increases were observed in F-I (4-fold), F-III (4-fold), and F-VI (2-fold). These results indicate that beta-adrenergic receptor activation results in increased phosphorylation of multiple sites on glycogen synthase. Insulin plus glucose decreased the overall 32P content of synthase by approximately 30%, with the largest decrease (40%) occurring in F-V. Without glucose, insulin decreased the [32P]phosphate in F-V by 17%, an effect which was balanced by increases in F-I, F II, and F-III so that no net change in the total 32P contents of the fractions was observed. Thus, activation of glycogen synthase by the glucose transport independent pathway seems to involve a redistribution of phosphate in the synthase subunit. PMID- 3001076 TI - Cation/proton antiport systems in Escherichia coli. Solubilization and reconstitution of delta pH-driven sodium/proton and calcium/proton antiporters. AB - Uptake of 22Na+ and 45Ca2+ into everted membrane vesicles from Escherichia coli was measured with imposed transmembrane pH gradients, acid interior, as driving force. Vesicles loaded with 0.5 M KCl were diluted into 0.5 M choline chloride to create a potassium gradient. Addition of nigericin to produce K+/H+ exchange resulted in formation of a pH gradient. This imposed gradient was capable of driving 45Ca2+ accumulation. In another method vesicles loaded with 0.5 M NH4Cl were diluted into 0.5 M choline chloride, creating an ammonium diffusion potential. A gradient of H+ was produced by passive efflux of NH3. With an ammonium gradient as driving force, everted vesicles accumulated both 45Ca2+ and 22Na+. The data suggest that 22Na+ uptake was via the sodium/proton antiporter and 45Ca2+ via the calcium/proton antiporter. Uptake of both cations required alkaline pHout. A minimum pH gradient of 0.9 unit was needed for transport of either ion, suggesting gating of the antiporters. Octyl glucoside extracts of inner membrane were reconstituted with E. coli phospholipids in 0.5 M NH4Cl. NH4+ loaded proteoliposomes accumulated both 22Na+ and 45Ca2+, demonstrating that the sodium/proton and calcium/proton antiporters could be solubilized and reconstituted in a functional form. PMID- 3001077 TI - Characterization of a cyclic AMP-activated Cl-transport pathway in the apical membrane of a human colonic epithelial cell line. AB - This report describes a Cl- transport pathway in confluent monolayer cultures of the T84 human colonic carcinoma cell line which is: 1) activated by vasoactive intestinal polypeptide, or other agents which induce or mimic cAMP; 2) independent of extracellular Na+ or K+; 3) refractory to inhibition by 0.1 mM bumetanide and 1 mM 4-acetamido-4'-isothiocyanostilbene-2,-2'-disulfonic acid; 4) competitively inhibited by NO3-, I-, SCN-, and Br-; 5) inhibited in a noncompetitive-complex manner by the putative Cl- channel-blocking agent, N phenylanthranilic acid; and 6) localized to the apical membrane of confluent monolayers. This Cl- transport system is, therefore, distinct from the bumetanide sensitive, basolateral membrane-localized, Na+, K+, Cl- cotransport system previously described in these cells (Dharmsathaphorn, K., Mandel, K., Masui, H., and McRoberts, J.A. (1985) J. Clin. Invest. 75, 462-471). Kinetic studies revealed that Cl- transport by this pathway fit simple Michaelis-Menten kinetics with an apparent Km for Cl- of about 6 mM. Activation by vasoactive intestinal polypeptide increased the Vmax but did not alter the apparent Km. We discuss the possibility that this transport system is a Cl- channel which is intimately involved in hormonally mediated, electrogenic Cl- secretion across T84 cell monolayers. PMID- 3001078 TI - Lack of association of epidermal growth factor-, insulin-, and serum-induced mitogenesis with stimulation of phosphoinositide degradation in BALB/c 3T3 fibroblasts. AB - The hypothesis that inositol phospholipid degradation is a step in the mechanism by which epidermal growth factor (EGF) stimulates mitogenesis in confluent monolayers of quiescent BALB/c 3T3 fibroblasts was tested. The maximum mitogenic response (a nearly 30-fold increase in incorporation of [3H]thymidine) occurred at 1 ng/ml EGF (0.16 nM). This degree of stimulation corresponded to 60% of that elicited by 10% serum. To determine whether EGF stimulated formation of inositol phosphates via degradation of polyphosphoinositides, the intracellular levels of [3H] inositol phosphates and [3H]phosphoinositides were determined after EGF addition to BALB/c 3T3 fibroblasts prelabeled with [3H]inositol. These experiments were performed under conditions designed to mimic exactly those conditions used to study mitogenesis. The results demonstrated that 10% serum or 10 ng/ml of platelet-derived growth factor, but not as much as 50 ng/ml EGF or 10 micrograms/ml insulin, increased the levels of inositol phosphates via degradation of phosphoinositides in the presence of 10 mM Li+. The serum-induced effects occurred in 30 s, the earliest time investigated. Phorbol dibutyrate (100 nM), alone or in conjunction with EGF (10 ng/ml), failed to stimulate inositol phospholipid degradation. However, phorbol dibutyrate inhibited the serum-induced stimulation. Finally, fetal bovine serum dialyzed so as to retain peptide mitogens lost almost 70% of the capacity to stimulate degradation of inositol phospholipids while remaining as mitogenic as the control serum. Thus, stimulation of inositol phospholipid degradation is an unlikely component in the mechanism by which EGF and probably insulin and serum stimulate mitogenesis in BALB/c 3T3 fibroblasts. PMID- 3001079 TI - Heterogeneity of N-acetylglucosamine 1-phosphotransferase within mucolipidosis III. AB - The primary defect responsible for mucolipidosis III is a deficiency of UDP-N acetylglucosamine:lysosomal enzyme N-acetylglucosamine 1-phosphotransferase activity (GlcNAc phosphotransferase). Genetic complementation analysis of cultured fibroblasts derived from 12 patients with mucolipidosis III identified complementation groups A, B, and C (Honey, N. K., Mueller, O. T., Little, L. E., Miller, A. L., and Shows, T. B. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7420 7424). The GlcNAc phosphotransferase activity present in the cell lines comprising the complementation groups was characterized with respect to endogenous substrates and two exogenous acceptors, alpha-methyl-D-mannoside and high mannose glycopeptides. All group C cell lines and one group A cell line were found to have normal GlcNAc phosphotransferase activity levels at 37 degrees C when screened with these exogenous acceptors. The enzyme activity in group A cell lines was within normal range when assayed at 23 degrees C. Inhibition of the phosphorylation of alpha-methyl-D-mannoside in the presence of increasing amounts of endogenous substrate N-acetyl-beta-D-hexosaminidase B was demonstrated in normal cell lines at 23 and 37 degrees C and in group A cells at 23 degrees C. However, group C cell lines did not show any inhibition at either temperature. This suggests that the alteration of the GlcNAc phosphotransferase from individuals in group C affects the recognition site for the protein portion of lysosomal enzymes, whereas group A individuals have mutations which result in a temperature-sensitive enzyme. PMID- 3001080 TI - Modified platelet responses to thrombin. Evidence for two types of receptors or coupling mechanisms. AB - The normally quick response of platelets to alpha-thrombin is delayed under two conditions. After pretreatment of platelets with chymotrypsin or certain other proteases, aggregation and secretion induced by alpha-thrombin begins after a delay of 30 s or more compared to less than 5 s for control platelets, and control platelets are activated by gamma-thrombin with a similar delay (Tam, S. W., Fenton, J. W., II, and Detwiler, T. C. (1980) J. Biol. Chem. 255, 6626-6632). Under these conditions, thrombin-induced inhibition of adenylate cyclase was blocked. The impaired regulation of adenylate cyclase and delayed secretion had in common: (i) partial correction with high concentrations of thrombin; (ii) similar thrombin dose-response relationships; (iii) similar concentration dependence for chymotrypsin during pretreatment; and (iv) specificity for thrombin. Other parameters of thrombin-induced platelet activation were also analyzed under these conditions. Thrombin-induced hydrolysis of arachidonyl esters and synthesis of prostanoids were similar to regulation of adenylate cyclase; they were blocked, with partial correction at high concentrations of thrombin. In contrast, thrombin-induced synthesis of phosphatidic acid and phosphorylation of a 20- and a 40-kDa protein were similar to secretion; they occurred after a delay but to a normal extent. The thrombin-induced increase in cytosolic calcium ion activity was slightly slower in chymotrypsin-treated platelets or in response to gamma-thrombin, but it was complete prior to initiation of the delayed responses. It is concluded that platelets have at least two types of thrombin receptors or coupling mechanisms, one of which is sensitive to chymotrypsin, unresponsive to gamma-thrombin, and coupled to inhibition of adenylate cyclase and activation of prostanoid synthesis. PMID- 3001081 TI - Regulation of epidermal growth factor receptor number and phosphorylation by fasting in rat liver. AB - The binding of 125I-epidermal growth factor (EGF) to microsomal membrane preparations from the livers of rats fasted for 72 h or fed control or high carbohydrate diets was examined to determine whether alterations in nutrient intake could affect the EGF receptor system. Fasted rats had 40-50% less membrane binding than did control or carbohydrate-fed rats. Scatchard analysis of the binding data indicated that the decrease in EGF binding in fasted rats was due to a decrease in receptor number with no change in receptor affinity. Cross-linking of 125I-EGF to EGF receptors with disuccinimidyl suberate revealed specific binding of a Mr 170,000 protein, which was diminished by approximately 75% in fasting, and a Mr = 150,000 protein, which accounted for 40-50% of the total labeling in the control and carbohydrate-fed rats and which was relatively unchanged by fasting. The sum of the labeling of the 2 bands was reduced by approximately 40% in fasting and is consistent with the reduction in EGF binding detected by Scatchard analysis. EGF stimulated a 1.5-3-fold increase in 32P incorporation into one major protein of 170 kDa in all 3 groups. Basal and EGF stimulated autophosphorylation of 170 kDa, when normalized for protein, was 75% lower in membranes from fasted animals, compared to those from control or carbohydrate-fed rats. The comparable reduction of 125I-EGF binding to, and 32P incorporation into, the 170-kDa EGF receptor protein suggested that kinase activity/receptor was unaffected by fasting. Moreover, EGF receptor kinase activity in the 3 groups was comparable for an exogenous substrate, as judged by equal basal and EGF-stimulated phosphorylation of Val5-angiotensin II, when normalized for total EGF-binding capacity. These results suggest that fasting regulates EGF receptor kinase activity primarily by regulation of the number of hepatic EGF receptors. The possibility exists that some in vivo effects of fasting may be mediated by a reduction in EGF receptor levels. PMID- 3001082 TI - Klebsiella pneumoniae nifM gene product is required for stabilization and activation of nitrogenase iron protein in Escherichia coli. AB - A series of plasmids encoding various Klebsiella pneumoniae nif (nitrogen fixation) genes were constructed to determine which were required to produce active iron (Fe) protein in Escherichia coli, a species which does not normally fix nitrogen. The greatest success was achieved with binary plasmid systems that produced nifA regulatory protein under the control of a tac promoter on one plasmid, which then induced synthesis of nifH and nifM proteins from their native promoter sites on a second plasmid. nifH protein, the monomeric subunit of Fe protein, produced in the presence of nifM constituted nearly 10% of the whole cell protein and exhibited the corresponding amount of C2H2-reducing activity in nitrogenase assays conducted in vitro. nifH protein formed in the absence of nifM constituted 4.7% of the whole cell protein and exhibited no detectable activity in assays of whole cell extracts. The plasmid-encoded Fe protein was purified to homogeneity and was found to be indistinguishable from that isolated from derepressed wild type K. pneumoniae, having a similar specific activity, approximately 4 Fe/dimer of 68 kDa, and similar epr features. Although these experiments do not exclude the participation of other E. coli gene products in the maturation of nifH protein, they limit the nif-specific genes required for active Fe protein production to nifA, nifH, and nifM. Since nifA is thought to be the required activator protein involved in nif operon transcription, the simplest explanation for these observations is that nifH codes for the peptide of the Fe protein, while nifM acts to convert this nifH peptide to the functioning Fe protein of nitrogenase. In the absence of nifM, only an inactive nifH polypeptide is produced. PMID- 3001083 TI - Cloning of the gene coding for the outer membrane receptor protein for ferric pseudobactin, a siderophore from a plant growth-promoting Pseudomonas strain. AB - Plant growth-promoting Pseudomonas B10 produces its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin under iron-limiting conditions. A structural gene encoding the 85,000-Da putative outer membrane receptor protein for ferric pseudobactin was identified in a gene bank from Pseudomonas B10 prepared with the broad host-range conjugative cosmid cloning vector pLAFR1. Transposon Tn5 mutagenesis of recombinant plasmid pJLM300 localized the functional gene to a region of approximately 2.4 kilobases consistent with the apparent molecular weight of the receptor protein. Mobilization of pJLM300 into Pseudomonas A124 and A225, whose growth was inhibited by Pseudomonas B10 or pseudobactin, rendered these strains no longer susceptible to iron starvation by pseudobactin because they were now able to transport ferric pseudobactin. Pseudobactin biosynthetic genes flanked this receptor gene on both sides and were on separate operons. Transposon Tn5 insertion mutants of Pseudomonas B10 lacking this receptor protein were generated by a marker exchange technique and were defective in ferric pseudobactin transport. Such mutants could be complemented in trans by pJLM300. The production of pseudobactin, the receptor protein, and four other outer membrane proteins in Pseudomonas B10 was coordinately regulated by the level of intracellular iron. PMID- 3001084 TI - Isolation and nucleotide sequence of the rabbit globin gene cluster psi zeta alpha 1-psi alpha. Absence of a pair of alpha-globin genes evolving in concert. AB - A cloned 13.3-kilobase (kb) region of rabbit genomic DNA contains a cluster of alpha-like globin genes arranged 5'-psi zeta-(3.6 kb)-alpha 1-(2.2 kb)-psi alpha 3'. Genomic blot hybridization data show that this is the major alpha-like globin gene cluster in rabbits, although a second alpha-globin gene (alpha 2) is also detected. Repetitive sequences from the C family of short repeats flank the psi zeta gene, and a new repetitive element, the F repeat is located 3' to psi alpha. The sequence was determined for a 4024-base pair (bp) segment that extends from 149 bp 5' to the cap site of alpha 1 to 207 bp 3' to psi alpha. Gene alpha 1 is functional and encodes one of the major allelic variants of rabbit alpha-globin. This gene has very short introns (77 bp in intron 1 and 83 bp in intron 2) and an unusual ATA box in the 5' flanking region (CTTAAA), which does function to promote transcription by RNA polymerase II in a cell-free system. Short (4 or 9 bp) G + C-rich repeats are interspersed throughout the flanking regions and the introns. Gene psi alpha cannot encode a globin polypeptide, and it is probably inactive. This is shown by the absence of a normal globin gene promoter, the replacement of the 5' untranslated sequence by tandem repeats of the sequence GCCCGCCGC, frameshift deletions in exon 2, and the modification of the polyadenylation signal to AGTAAA. The intergenic region between alpha 1 and psi alpha is very G + C rich (65.7% G + C) and contains many short, tandem repeats. Gene psi zeta hybridizes specifically to a human zeta-globin gene probe, but a partial sequence reveals frameshift mutations that probably make psi zeta a pseudogene. Mammalian alpha-globin gene clusters vary in the presence or absence of pseudogenes, and if present, the position of the pseudogene differs in various gene clusters. Among the mammalian alpha-like gene clusters so far analyzed, the rabbit gene cluster is unique in the absence of duplicated alpha-globin genes that are undergoing concerted evolution. PMID- 3001085 TI - Nuclear genes for cytochrome c oxidase subunits of Neurospora crassa. Isolation and characterization of cDNA clones for subunits IV, V, VI, and possibly VII. AB - We obtained cDNA clones for cytochrome oxidase subunits IV, V, VI, and possibly VII by constructing a lambda gt11 library of Neurospora crassa cDNA and probing it with antiserum directed against Neurospora cytochrome oxidase holoenzyme. Positive clones were further characterized with antisera directed against individual cytochrome oxidase subunits and subsequently by DNA sequencing. The clones for subunits IV and V encode proteins with regions matching the known N terminal amino acid sequences of purified Neurospora cytochrome oxidase subunits IV and V, respectively. The sequences of these clones provide the first evidence that cytochrome oxidase subunits IV and V are made as precursors with N-terminal extensions in Neurospora. The N-terminal extensions encoded by these clones share homology, and are rich in arginine, as are signal sequences of other mitochondrially destined proteins. The subunit VI clone codes for the carboxyl terminus of a protein homologous to the carboxy termini of yeast cytochrome oxidase subunit VI and bovine cytochrome oxidase subunit Va. The subunit VII clone contains an open reading frame for a 47-residue protein, the expected size for subunit VII. However, the protein coded by this clone has an unusual amino acid composition. Whether this clone represents an authentic cytochrome oxidase subunit is not established. PMID- 3001086 TI - A human gene family with sequence homology to Drosophila melanogaster Hsp70 heat shock genes. AB - A growing literature describes the structure and regulation of prokaryotic and eukaryotic heat shock genes. We here report the isolation of several members of a human heat shock protein 70 (hsp 70) multigene family which contains at least 10 different genes and/or pseudogenes exhibiting sequence homology to the hsp70 gene of Drosophila melanogaster. Eight nonoverlapping recombinant lambda phages from a lambda-Charon4A human genomic library were studied by restriction mapping. One lambda clone was sequenced and characterized as a hsp70 pseudogene inserted into a rearranged human HindIII 1.9-kilobase repeated DNA sequence. This pseudogene is probably located on the X chromosome. Its predicted amino acid sequence shows extensive homology to those of Drosophila hsp70, trout hsp70, Xenopus hsp70, yeast hsp70, and some homology to the heat-inducible dnaK gene product of Escherichia coli. Amino acid homology is clustered, suggesting evolutionary conservation of domains critical to the function of this protein in both prokaryotic and eukaryotic cells. PMID- 3001087 TI - Monoclonal antibodies against 5'-nucleotidase from chicken gizzard. Evidence for species and tissue specific differences of 5'-nucleotidase. AB - 5'-Nucleotidase from chicken gizzard smooth muscle was purified to homogeneity and used as immunogen for generating monoclonal antibodies. From about 150 positive clones nine IgG producing hybridoma cell lines have been selected for further characterization and antibody preparation. The resulting antibodies bind 5'-nucleotidase from chicken smooth muscle, chicken skeletal muscle, and chicken heart muscle but not the enzyme from chicken liver or rat liver. It could clearly be demonstrated that the nine antibodies recognize different antigenic determinants. Four of these antibodies are strong inhibitors of the AMPase activity of 5'-nucleotidase. One antibody is a weak inhibitor and four other antibodies have no effect on its enzymic activity. One of the monoclonal antibodies was used for immunoaffinity purification of 5'-nucleotidase from chicken heart muscle and chicken skeletal muscle. Pure and active enzymes could be isolated from detergent extracts in one step with a 10 to 20-fold higher yield compared to classical purification procedures. The subcellular distribution of 5' nucleotidase in chicken gizzard was investigated using indirect immunofluorescence. We found a staining of the plasma membrane of smooth muscle cells and endothelial cells by all of the nine antibodies with variations in the staining intensity. PMID- 3001088 TI - Phosphorylation of receptors for insulin and insulin-like growth factor I. Effects of hormones and phorbol esters. AB - The phosphorylation of receptors for insulin and insulin-like growth factor I was studied by phosphoamino acid analysis and tryptic phosphopeptide maps in an attempt to determine if protein kinase C is involved in their phosphorylation in response to insulin and insulin-like growth factor I, respectively. Two cell lines were utilized, Hep G2 and IM-9 cells. sn-1,2-Dioctanoylglycerol and 12-O tetradecanoylphorbol 13-acetate (TPA), agents known to activate protein kinase C, stimulated the phosphorylation of the beta subunits of both receptors, as did their hormones. In unstimulated cells, phosphorylation of the insulin receptor occurred on seryl and to a lesser extent on threonyl residues. TPA stimulated seryl and threonyl phosphorylation that resulted in the appearance of four major phosphoserine-containing phosphopeptides which were not detected in the basal state and an increase in phosphorylation of a phosphothreonine-containing peptide which was present in the basal state. Insulin treatment resulted in the appearance of three major phosphotyrosine-containing tryptic peptides. In IM-9 cells, insulin also increased the phosphoserine and possibly the phosphothreonine content of the beta subunit. In both cells, the major phosphoserine-containing peptides that were stimulated by TPA were not detected following treatment with insulin. Very similar results, including similar peptide maps, were obtained for the insulin-like growth factor I receptor from cells treated with TPA and insulin like growth factor I. Although not entirely conclusive, these results suggest that the insulin- and insulin-like growth factor I-stimulated phosphorylation of their receptors does not result from activation of protein kinase C. PMID- 3001089 TI - Complete glycosylation of the insulin and insulin-like growth factor I receptors is not necessary for their biosynthesis and function. Use of swainsonine as an inhibitor in IM-9 cells. AB - Swainsonine, an indolizidine alkaloid which is a potent inhibitor of the Golgi enzyme, mannosidase II, leads to the production of incompletely processed glycoproteins lacking complex type oligosaccharides. This inhibitor has been used to examine the importance of terminal sugar groups in the biosynthesis and function of both the insulin receptor and the insulin-like growth factor I receptor. IM-9 cells were metabolically labeled using [35S]methionine and the two receptors were independently immunoprecipitated using specific monoclonal antibodies. The incompletely processed receptors have slightly lower molecular weights and contain hybrid rather than complex type oligosaccharides as indicated by their sensitivity to endoglycosidase H and neuraminidase. Both receptors made in the presence of swainsonine are still autophosphorylated in the presence of the respective hormone. The insulin receptor made in the presence of the inhibitor can be affinity labeled at the cell surface using 125I-insulin and disuccinimidyl suberate cross-linking; there is also no significant difference in its affinity for insulin. These results suggest that for the insulin and insulin like growth factor I receptors to be synthesized, processed, and function normally, they do not require all of the sugars which are normally added in the terminal stages of glycosylation. PMID- 3001090 TI - The structure of bone apatite surfaces. AB - This is a review of the surface chemistry of bone mineral and its synthetic counterpart hydroxyapatite. Small-angle x-ray scattering and low-temperature nitrogen adsorption measurements show bone mineral surfaces range from 100 to 200 m2/g. The heats of adsorption of small molecules on bone and apatite surfaces show that these materials have polarizing surfaces which form strong bonds with polar and polarizable molecules. Water is hydrogen bonded to these surfaces with energies ranging from 23 Kcal/mol, for low coverage, to 11 Kcal/mol after two full layers; the latter value shows that after two monolayers the water is bonded as strongly to the solution as it is to the apatite surface. Stearic acid in cyclohexane adsorbs on bone and apatite surfaces in a closed-packed manner with the straight-chain molecules in parallel array with the end carboxyl groups hydrogen bonded to surface electronegative ions. Synthetic hydroxyapatite has long been used in chromatography because of the bonding capacity apatite surface has for certain proteins and polynucleotides. The metabolic interplay between bone mineral and the body results from the high magnitude and high reactivity of the mineral surface. PMID- 3001092 TI - In vivo corrosion of sodium silicate glasses. AB - Square-shaped implants of various sodium silicate glasses were weighed and implanted intraperitoneally in rat for periods ranging from 8 to about 60 days. The implants were then removed and their aspect was compared to their aspect before exposure to physiological environment. The corrosion products were studied by x-ray diffraction and electron microprobe analysis. Weight changes were also measured to calculate a biodegradation rate. The glass 66 SO (66.6 SiO2-33.3 Na2O) was strongly corroded, as early as after the first week. The nonsoluble degradation products formed a cocoon encapsulating the now smaller specimen. The analysis of the cocoon showed that it was made of a silica-rich layer containing also calcium and phosphorus. In this layer the ratio Ca/P could correspond to that of an apatite. The biodegradation rate reached 71 x 10(-4) g . cm-2 . day-1. The glass 75 SO (75 SiO2-25 Na2O) was not so quickly corroded: Cracks appeared at the surface and progressively reached the center of the implants. There was no removable shell but a white deposit, adherent to the surface. This deposit contained silica and also calcium and phosphorus at the periphery. The biodegradation rate was only 2.6 x 10(-4) g . cm-2 . day-1. PMID- 3001091 TI - Bioglass coatings and bioglass composites as implant materials. AB - This article addresses three aspects of the mechanical and biological properties of bioreactive glasses. First, it describes the composition and the relationship between the composition and bonding and its influence on the bone bonding mechanism and the rate of bond formation. Second, the mechanical properties of bioglass are dealt with. It is shown that the approaches to use bioglass in highly stressed applications, have met with various degrees of success. Third, the issue of the effect of loading on the glass properties and, above all, on the glass bonding properties is discussed. PMID- 3001093 TI - Influence of 4-methacryloxyethyl trimellitic anhydride on composites subjected to hygrothermal cycling. AB - Pastes were made by spatulating a hydroxyapatite powder into triethylene glycol dimethacrylate (TEGDMA) that contained benzoyl peroxide. For comparison, similar pastes included 5 wt-% 4-methacryloxyethyl trimellitic anhydride (4-META) in the TEGDMA. Composites were made by heating at 120 degrees C under pressure. Inclusion of 4-META reduced the rate of diffusion of water by about one-third, at 27 degrees C. Also after thermal cycling in water, inclusion of 4-META decreased the fractional drop in compressive strength by about one-half. Examination of fracture surfaces indicated that inclusion of 4-META improved the wetting of filler particles and gave more coherent composites. All these findings are consistent with the view that 4-META acts as a coupling agent. PMID- 3001095 TI - Hypophosphatemic osteomalacia secondary to neoplasia. PMID- 3001096 TI - An electromyographer's view of the ulnar nerve. PMID- 3001094 TI - A new glass-ceramic for bone replacement: evaluation of its bonding to bone tissue. AB - Glass powders (350 mesh) of the composition MgO, 4.6; CaO, 44.9; SiO, 34.2; PO, 16.3; CaF, 0.5 in weight ratio were compacted, heated to 1050 degrees C at a rate of 5 degrees C/min and kept at 1050 degrees C for 2 h. The resultant glass ceramic having oxyapatite, fluoroapatite, and wollastonite crystals showed high bending and compressive strengths of 157 and 1060 MPa, respectively. The biocompatibility and bonding ability of this new glass-ceramic to the bone tissue was evaluated using rabbit tibial bones, and the failure load to break the bonding of several ceramics (the new glass-ceramic, dense hydroxyapatite, 45S5 Bioglass, alumina-ceramic) to bone tissues was measured. The new glass-ceramic showed tight bonding to bone comparable with dense hydroxyapatite, and in 25 weeks its load was 70% of that of bone tissue. PMID- 3001097 TI - Soft tissue sarcoma as second malignant lesion after therapy for Hodgkin's disease. Report of two cases and review of the literature. AB - Two cases of soft tissue sarcoma following treatment of nodular sclerosing Hodgkin's disease are described. One patient developed a malignant schwannoma after radiotherapy, the other was diagnosed as having a malignant fibrous histiocytoma after treatment with radiation and chemotherapy. Both secondary malignancies arose within the irradiated field after a latent period ranging from 9.5 years in the first to 21 years in the second case. A review of the pertinent literature is given and previous reports of malignant tumors and leukemias following therapy for Hodgkin's disease are summarized. PMID- 3001099 TI - Immuno-identification of Ca2+-induced conformational changes in human gelsolin and brevin. AB - Gelsolin is a 90,000-mol-wt protein with two actin and two high affinity calcium binding sites that can form complexes with Ca2+ ions and monomeric actin. These complexes will nucleate filament growth and cap the barbed end of filaments, but will not fragment F-actin. Uncomplexed gelsolin severs F-actin. (Bryan, J., and L. M. Coluccio, 1985, J. Cell Biol., 101:1236-1244). These associations with actin are modulated by Ca2+. We have purified and characterized monoclonal antibodies that recognize Ca2+-induced conformational changes in human platelet gelsolin (G) and human plasma brevin (B), a closely related protein. Two hybridomas, 8G5 and 4F8, were adapted to growth in serum-free medium. 8G5 was found to secrete an IgG; 4F8 secretes an IgA. On immunoblots, both antibodies gave a strong reaction if Ca2+ was present, but gave barely detectable reactions if EGTA was used. 8G5 IgG-Sepharose columns retained gelsolin (as GCa2) or brevin (as BCa2) in 0.1 mM CaCl2 containing buffers, but released these molecules when eluted with 4 mM EGTA. 8G5 IgG-Sepharose columns also retained gelsolin-actin Ca2+ complexes, as GA1Ca2 or higher oligomers from platelet extracts containing 0.1 mM CaCl2. Elution with 4 mM EGTA released material that gel filtration showed to be the EGTA-stable 130,000-mol-wt gelsolin-actin complex, GA1Ca1. The results demonstrate that the 8G5 IgG recognizes a conformation of gelsolin or brevin induced by binding of an easily exchangeable Ca2+ ion. Actin is not required for this conformational change, and the antibody discriminates, for example, GCa2 from G and GCa1. A 4F8 IgA-Sepharose column retained brevin or gelsolin in 0.1 mM CaCl2-containing buffers, but, like the 8G5 IgG, released these molecules when eluted with 4 mM EGTA. The 4F8 IgA column also retained gelsolin or brevin-actin Ca2+ complexes, for example, as BA1Ca2, or higher oligomers, in 0.1 mM CaCl2. No protein was recovered, however, upon elution with 4 mM EGTA, but elution with 0.1 M glycine-HCl, pH 2.8, released bound brevin or gelsolin and actin. Similarly, preformed brevin-actin-Ca2+ complex, equilibrated with EGTA, was retained by 4F8 IgA-Sepharose. The results demonstrate that the 4F8 IgA recognizes a conformation of gelsolin or brevin that is maintained and presumably induced by binding of a nonexchangeable Ca2+ ion that is trapped in the complex. PMID- 3001098 TI - Alternatively spliced mRNAs code for different polypeptide chains of the chicken neural cell adhesion molecule (N-CAM). AB - Rabbit polyclonal antibodies directed against the chicken neural cell adhesion molecule (N-CAM) were used to isolate four overlapping cDNA clones from a chicken cDNA expression library in bacteriophage gamma gt11. These clones collectively accounted for 3.8 kilobases of N-CAM mRNA sequence and hybridized specifically to two 6-7-kilobase brain polyadenylated RNA species that co-migrated with previously identified N-CAM mRNAs. DNA fragments derived from an internal region of the cloned cDNA sequences hybridized to the larger but not to the smaller N CAM mRNA species, while fragments on either side of this region hybridized to both mRNAs. A cDNA fragment that recognized only the larger mRNA was subcloned into gamma gt11, and the expressed fusion protein was used to affinity-purify rabbit polyclonal antibodies; the antibodies recognized only the larger of the two structurally related N-CAM polypeptides. In contrast, when several cDNA clones that recognized both mRNAs were used to purify antibodies, the antibodies recognized both polypeptides. The results, in conjunction with other data indicating that there is one gene specifying N-CAM, suggest that different N-CAM polypeptides are synthesized from multiple N-CAM messages generated by alternative splicing of transcripts from a single N-CAM gene. PMID- 3001100 TI - A re-evaluation of cytoplasmic gelsolin localization. AB - Gelsolin is a 90,000-mol-wt Ca2+-binding, actin-associated protein that can nucleate actin filament growth, sever filaments, and cap barbed filament ends. Brevin is a closely related 92,000-mol-wt plasma protein with similar properties. Gelsolin has been reported to be localized on actin filaments in stress fibers, in cardiac and skeletal muscle I-bands, and in cellular regions where actin filaments are known to be concentrated. Previous localization studies have used sera or antibody preparations that contain brevin. Using purified brevin-free IgG and IgA monoclonal antibodies or affinity-purified polyclonal antibodies for gelsolin and brevin, we find no preferential stress fiber staining in cultured human fibroblasts or I-band staining in isolated rabbit skeletal muscle sarcomeres. Cardiac muscle frozen sections show no pronounced I-band staining, except in local areas where brevin may have penetrated from adjacent blood vessels. Spreading platelets show endogenous gelsolin localized at the cell periphery, in the central cytoplasmic mass and on thin fibers that radiate from the central cytoplasm. Addition of 3-30 micrograms/ml of brevin to the antibodies restores intense stress fiber and I-band staining. We see no evidence for large scale severing and removal of filaments in stress fibers in formaldehyde-fixed, acetone-permeabilized cells even at brevin concentrations of 30 micrograms/ml. The added brevin or brevin antibody complex binds to actin filaments and is detected by the fluorescently tagged secondary antibody. Brevin binding occurs in either Ca2+ or EGTA, but is slightly more intense in EGTA suggesting some severing and filament removal may occur in Ca2+. The I-band staining is limited to the region where actin and myosin do not overlap. In addition, brevin does not appear to bind at the Z-line. A comparison of cells double-labeled with fluorescein-phallotoxin, exogenous brevin, and a monoclonal antibody, detected with a rhodamine-labeled secondary antibody, shows almost complete co localization of F-actin with the brevin-gelsolin-binding sites. A major exception is in the area of the adhesion plaque. A quantitative comparison of the fluorescein-rhodamine fluorescence intensities along a stress fiber and into the adhesion plaque shows that the fluorescein signal, associated with F-actin, increases while the rhodamine signal decreases. We infer that exogenous brevin or endogenous gelsolin can bind to and potentially sever most actin filaments, but that actin-associated proteins in the adhesion plaque can prevent binding and severing.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3001102 TI - Breakdown of self/nonself recognition in cannibalistic strains of the predatory slime mold, Dictyostelium caveatum. AB - Dictyostelium caveatum amebas feed upon both bacteria and the amebas of other cellular slime molds. The capacity to feed extensively upon other cellular slime molds is unique to D. caveatum amebas. They are able to phagocytose amebas larger than themselves by nibbling pieces of the cells until they are small enough to ingest. Here we report the isolation from previously cloned stock cultures of stable, cannibalistic strains of D. caveatum in which self/nonself recognition has broken down. Because of the extensive cannibalism, amebas of these strains do not complete multicellular development, and instead wander about for long periods while feeding upon each other. Although the cannibalistic behavior resembles that exhibited by the presumably diploid giant cells in the sexual cycle of other cellular slime molds, these strains are haploid and do not form macrocysts. PMID- 3001101 TI - Receptor-mediated endocytosis of epidermal growth factor by rat hepatocytes: receptor pathway. AB - Substantial amounts of epidermal growth factor (EGF) are cleared from the circulation by hepatocytes via receptor-mediated endocytosis and subsequently degraded within lysosomes. We have used a combined biochemical and morphological approach to examine the fate of the receptor after exposure to EGF. Polyclonal antibodies were prepared against the purified receptor and their specificity established by immunoprecipitation and immunoblotting techniques. The EGF receptor was then localized by immunofluorescence and immunoperoxidase techniques and quantified on immunoblots. In untreated livers, EGF receptor was restricted to the sinusoidal and lateral surfaces of hepatocytes. 2-4 min after exposure of cells to EGF, the receptor was found in small vesicles (i.e., coated vesicles) as well as larger vesicles and tubules at the cell periphery. By 15 min the receptor was found in multivesicular endosomes located near bile canaliculi. Exposure of hepatocytes to EGF also resulted in a rapid loss of receptor protein from total liver homogenates and a decrease in its half-life from 8.7 h in control livers to 2.5 h. This EGF-induced loss of receptors was not observed when lysosomal proteinases were inhibited by leupeptin or when endosome/lysosome fusion was prevented by low temperature (16 degrees C). In the presence of leupeptin, receptor could be detected in structures identified as lysosomes using acid phosphatase cytochemistry. All these results suggested rapid internalization of EGF receptors in response to ligand and degradation within lysosomes. However, four times more ligand was degraded at 8 h than the number of high-affinity (Kd of 8-15 nM) EGF-binding sites lost, suggesting either (a) high-affinity receptors were recycled, and/or (b) more than 300,000 receptors were available for EGF uptake. We identified and characterized a latent pool of approximately 300,000 low-affinity receptors (Kd approximately 200 nM) that could be separated on sucrose gradients from the plasma membrane pool of approximately 300,000 high affinity receptors (Kd of 8-15 nM). Despite the differences in their binding affinities, the high- and low-affinity receptors appeared to be structurally identical and were both EGF-dependent protein kinases. In addition, the dynamics of the low-affinity receptors were consistent with a functional role in EGF uptake and delivery to lysosomes. PMID- 3001103 TI - Routing of internalized ricin and ricin conjugates to the Golgi complex. AB - Receptor-mediated endocytosis and intracellular routing of native ricin, and of ricin conjugated to colloidal gold (Ri-Au) and to horseradish peroxidase (Ri HRP), have been studied in cultured MCF-7 and Vero cells by electron microscopical techniques including serial section analysis. Both native ricin, as demonstrated by immunoperoxidase cytochemistry, and the ricin conjugates were internalized via a common coated pit-coated vesicle pathway to reach vacuolar and tubulo-vesicular portions of the endosomal system. In addition, native ricin and a purified monovalent fraction of Ri-HRP reached distinct Golgi cisterns, whereas Ri-Au and polyvalent Ri-HRP did not. The results delineate intracellular routing of native ricin and compare it with the routing of different ricin conjugates. Moreover, our study shows that conjugates of a particular ligand (ricin) and various probes (e.g., gold and peroxidase), may be handled differently by cells. Sorting apparently takes place in the endosomal system, allowing some but not other molecules to reach Golgi elements. This sorting seems to depend on the valency of the ricin conjugate. PMID- 3001104 TI - Regulation of cortical vesicle exocytosis in sea urchin eggs by inositol 1,4,5 trisphosphate and GTP-binding protein. AB - To investigate the roles of inositol 1,4,5-trisphosphate (InsP3) and guanyl nucleotide binding proteins (G-proteins) in the transduction mechanism coupling fertilization and exocytosis of cortical vesicles in sea urchin eggs, we microinjected InsP3 and guanyl nucleotide analogs into eggs of Lytechinus variegatus. Injection of 28 nM InsP3 caused exocytosis. However, if the egg was first injected with EGTA ([Cai] less than or equal to 0.1 microM; EGTA = 1.6 mM), InsP3 injection did not cause exocytosis, supporting the hypothesis that InsP3 acts by causing a rise in intracellular free calcium. Injection of 28 microM guanosine-5'-0-(3-thiotriphosphate) (GTP-gamma-S), a hydrolysis-resistant analog of GTP, caused exocytosis, but exocytosis did not occur if the egg was pre injected with EGTA. Injection of 3 mM guanosine-5'-0-(2-thiodiphosphate) (GDP beta-S), a metabolically stable analog of GDP, prevented sperm from stimulating exocytosis. However, injection of GDP-beta-S did not prevent the stimulation of exocytosis by InsP3. These results suggested the following sequence of events. The sperm activates a G-protein, which stimulates production of InsP3. InsP3 elevates intracellular free calcium, which causes exocytosis. PMID- 3001105 TI - Rates of membrane-associated reactions: reduction of dimensionality revisited. AB - The hypothesis that reactions associated with intracellular membranes enjoy a kinetic advantage from a reduced dimensionality for diffusion is inconsistent with available data on lateral diffusion rates, membrane-substrate affinities, and endogenous concentrations of enzymes and their aqueous substrates. PMID- 3001107 TI - Interaction of the insulin receptor kinase with serine/threonine kinases in vitro. AB - Insulin causes rapid phosphorylation of the beta subunit (Mr = 95,000) of its receptor in broken cell preparations. This occurs on tyrosine residues and is due to activation of a protein kinase which is contained in the receptor itself. In the intact cell, insulin also stimulates the phosphorylation of the receptor and other cellular proteins on serine and threonine residues. In an attempt to find a protein that might link the receptor tyrosine kinase to these serine/threonine phosphorylation reactions, we have studied the interaction of a partially purified preparation of insulin receptor with purified preparations of serine/threonine kinases known to phosphorylate glycogen synthase. No insulin dependent phosphorylation was observed when casein kinases I and II, phosphorylase kinase, or glycogen synthase kinase 3 was incubated in vitro with the insulin receptor. These kinases also failed to phosphorylate the receptor. By contrast, the insulin receptor kinase catalyzed the phosphorylation of the calmodulin-dependent kinase and addition of insulin in vitro resulted in a 40% increase in this phosphorylation. In the presence of calmodulin-dependent kinase and the insulin receptor kinase, insulin also stimulated the phosphorylation of calmodulin. Phosphoamino acid analysis showed an increase of phosphotyrosine content in both calmodulin and calmodulin-dependent protein kinase. These data suggest that the insulin receptor kinase may interact directly and specifically with the calmodulin-dependent kinase and calmodulin. Further studies will be required to determine if these phosphorylations modify the action of these regulatory proteins. PMID- 3001106 TI - Receptors for insulin and epidermal growth factor: interaction with organomercurial agarose. AB - The receptor for both insulin and epidermal growth factor (EGF) from human placental membranes, after crosslink labeling with 125I-labeled insulin and EGF, can be absorbed to an organomercurial-agarose derivative (Affi-Gel 501) and can be recovered from the gel by elution with dithiothreitol (DTT). Pretreatment of crosslink-labeled membranes with N-ethylmaleimide (NEM) blocks the ability of the receptor to react with the organomercurial column. NEM also abolishes the protein kinase activity of both receptors. Under appropriate conditions, insulin can promote the reaction of the insulin receptor with the organomercurial-agarose derivative. For both the insulin and EGF receptors, our results provide an avenue for the isolation of the sulfhydryl-containing receptor domains that may play a role in the control of receptor function. PMID- 3001108 TI - Recent advances in research on fibronectin and other cell attachment proteins. PMID- 3001110 TI - Regulation of the epidermal growth factor receptor by phosphorylation. AB - The receptor for epidermal growth factor (EGF) is a glycosylated transmembrane phosphoprotein that exhibits EGF-stimulable protein tyrosine kinase activity. On EGF stimulation, the receptor undergoes a self-phosphorylation reaction at tyrosine residues located primarily in the extreme carboxyl-terminal region of the protein. Using enzymatically active EGF receptor purified by immunoaffinity chromatography from A431 human epidermoid carcinoma cells, the self phosphorylation reaction has been characterized as a rapid, intramolecular process which is maximal at 30-37 degrees C and exhibits a very low Km for ATP (0.2 microM). When phosphorylation of exogenous peptide substrates was measured as a function of receptor self-phosphorylation, tyrosine kinase activity was found to be enhanced two to threefold at 1-2 mol of phosphate per mol of receptor. Analysis of the dependence of the tyrosine kinase activity on ATP concentration yielded hyperbolic kinetics when plotted in double-reciprocal fashion, indicating that ATP can serve as an activator of the enzyme. Higher concentrations of peptide substrates were found to inhibit both the self- and peptide phosphorylation, but this inhibition could be overcome by first self phosphorylating the enzyme. These results suggest that self-phosphorylation can remove a competitive/inhibitory constraint so that certain exogenous substrates can have greater access to the enzyme active site. In addition to self phosphorylation, the EGF receptor can be phosphorylated on threonine residues by the calcium- and phospholipid-dependent protein kinase C. The sites on the EGF receptor phosphorylated in vitro by protein kinase C are identical to the sites phosphorylated on the receptor isolated from A431 cells exposed to the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. This phosphorylation of the EGF receptor results in a suppression of its tyrosine kinase and EGF binding activities both in vivo and in vitro. The EGF receptor can thus be variably regulated by phosphorylation: self-phosphorylation can enhance tyrosine kinase activity whereas protein kinase C-catalyzed phosphorylation can depress enzyme activity. Because these two phosphorylations account for only a fraction of the phosphate present in the EGF receptor in vivo, other protein kinases can apparently phosphorylate the receptor and these may exert additional controls on EGF receptor/kinase function. PMID- 3001109 TI - Structure and chromosomal localization of the human lymphotoxin gene. AB - We have isolated, sequenced, and determined the chromosomal localization of the gene encoding human lymphotoxin (LT). The single copy gene was isolated from a human genomic library using a 32P-labeled 116 bp synthetic DNA fragment whose sequence was based on the NH2-terminal amino acid sequence of LT. The gene spans 3 kb of DNA and is interrupted by three intervening sequences. The LT gene is located on human chromosome 6, as determined by Southern blot analysis of human murine hybrid DNA. Putative transcriptional control regions and areas of homology with the promoters of interferon and other genes are identified. PMID- 3001111 TI - Comparative analysis of p21 proteins from various cell types by two-dimensional gel electrophoresis. AB - The products of the ras gene family are related proteins at a molecular weight of 21 kDa, designated p21. In the present study we used two-dimensional gel electrophoresis to compare p21 proteins from five different normal and malignant cell lines. Using a known protein (3H-labeled translation initiation factor [eIF 4D]) as a standard internal marker for isoelectric point (pI), we show that p21 proteins from various cells differ only slightly in molecular weight (21-24 kDa) but express a wide variety in charge (pI 4.8 to 7) that could only be detected by the use of two-dimensional gel electrophoresis. p21 in NIH/3T3 cells was expressed as a single protein, which migrated at 21 kDa and pI 5.1. This peptide, which is probably the product of the normal cellular ras gene, was also detected in normal human lymphocytes. The synthesis of this peptide was not elevated in the transformed cells. However, transformation of NIH/3T3 fibroblasts and of human leukocytes was found to be associated with expression of qualitatively different forms of p21 peptides. Four additional p21-associated peptides of identical molecular weight (23 kDa), but multiple charge forms, were detected selectively in Kirsten murine sarcoma virus-transformed NIH/3T3 cells. Transformation of cells with Harvey murine sarcoma virus was found to be associated with prominent expression of two major pairs of p21-associated proteins, one at 21 kDa (pI, 5.2 and 5.3) and the other at 23 kDa (pI, 5.1 and 5.2). In HL-60 leukemic cells there was an additional, more acidic form (pI 5.0) of p21, which appeared to be absent or reduced in normal human lymphocytes. These results indicate that p21 from viral origin or cellular origin might be expressed in the cells in multiple charge forms. The capability to distinguish multiple forms of p21 and slight charge modifications associated with malignancy should call for the use of 2-D gel electrophoresis as an important tool in future studies involving p21 proteins. PMID- 3001112 TI - Studies on lectins. LVIII. Sugar-binding properties, as determined by affinity electrophoresis, of alpha-D-galactosidases from Vicia faba seeds possessing erythroagglutinating activity. AB - The interaction of alpha-D-galactosidases from Vicia faba seeds with saccharides was studied by means of affinity electrophoresis on polyacrylamide gel in an acidic buffer system. For the preparation of affinity gels, water-soluble O glycosyl polyacrylamide copolymers and polysaccharides were used. alpha-D Galactosidases interact with immobilized O-alpha-D-galactosyl residues and glycogen, but no interaction was observed with immobilized O-alpha-D-mannosyl residues. On the basis of the results of affinity electrophoresis performed in the presence of various free sugars, dissociation constants of the various alpha D-galactosidase-free sugar complexes were calculated. PMID- 3001113 TI - Rapid solid-phase radioimmunoassay for detection of equine infectious anemia viral antigen and antibodies: parameters involved in standardization. AB - Solid-phase radioimmunoassays (SPRIA) are described for the detection of equine infectious anemia (EIA) viral antigen and antibodies. Protein-antigen P29 currently used in the agar-gel immunodiffusion (AGID) test was used as antigen in the SPRIA. Rabbit sera selected from positive AGID test data were used to standardize the method. Briefly, wells of flexible microtitre plates coated with antigen were incubated with antiserum followed by a secondary labelled antibody. The radioactivity remaining in the wells after washing provided a measure of the amount of specific antibodies in the serum. When testing a group of rabbit sera, negative for EIA virus antibodies by the AGID test, in the SPRIA a range of positive reactivities was noted. The specificity of the reaction was assessed by inhibition with the antigen. The reaction of immune serum against EIA-virus antigen adsorbed to the wells, was completely inhibited by the antigen in solution. This property was applied in an indirect competitive SPRIA for the detection of viral protein P29. The detection threshold of the SPRIA for EIA virus protein was about 5 ng and about 1 ng of antibody can be detected. The assay is rapid, specific and sensitive and allows the testing of multiple serum samples with the advantage of employing a single secondary labelled antibody. PMID- 3001114 TI - In situ hybridization with biotinylated DNA probes: a rapid diagnostic test for adenovirus upper respiratory infections. AB - Adenovirus DNA was detected in cells from nasopharyngeal secretions of children with acute respiratory infections by in situ hybridization with biotinylated probes. The technique was easy to perform, giving rapid results which were well correlated with those of immunofluorescence assays of the same samples. Adenoviruses of subgroups B, C and E were detected equally well by probes prepared either from purified adenovirus type 5 or from a plasmid (A1) carrying a cloned insertion of BamH1 fragments C and D of adenovirus type 2 in pAT 153. The use of stable non-radioactive probes makes in situ hybridization a feasible assay for use in clinical laboratories with moderate resources. PMID- 3001116 TI - The estimation of virus density in isopycnic cesium chloride gradients. AB - Methods for estimating the density of virus particles after isopycnic centrifugation in cesium chloride gradients were examined. If solutions were prepared in different buffers, the standard tables relating refractive index and density, based on solutions prepared in water, did not give consistent estimates even if corrections were made for the greater refractive indices of the buffers. Depending on the method and buffer used, estimates of the density of cricket paralysis virus, an insect picornavirus, differed by as much as 0.05 g/ml. However, consistent estimates were obtained if separate regression equations were derived to relate refractive index and density for each different buffer solution of cesium chloride. This has wider implications and could account for density differences or variations in estimates reported for many other viruses. PMID- 3001115 TI - Development of a sensitive protein A-gold immunoelectron microscopy method for detecting viral antigens in fluid specimens. AB - Protein A-colloidal gold immunoelectron microscopy (PAG IEM) has been employed to specifically detect rotavirus and enterovirus antigen in negatively stained fluid specimens. Unlike other IEM methods, PAG IEM can detect not only viral antigen associated with morphologically recognizable particles but also viral antigens of unrecognizable ultrastructure. This rapid and sensitive immunoassay was found to be applicable to virus-infected stool specimens as well as partially purified virus preparations. The sensitivity of viral antigen detection by PAG IEM was 2- to 40-fold greater than direct IEM and 200- to 1,000-fold greater than direct electron microscopy. In addition, PAG IEM appears to offer a more reliable and sensitive alternative to standard IEM for detection and quantitation of viral antibody. PMID- 3001117 TI - Use of immunosorbent electron microscopy for detection of rota- and hepatitis A virus in sucrose solutions. AB - Immunosorbent electron microscopy was used to demonstrate rotavirus in solutions of varying sucrose concentrations after 18, 42 and 66 h of incubation. About 50% of adsorption of virus particles to the grid was achieved after 18 h incubation and nearly 100% after 42 h when compared to trapping of virus from sucrose free solutions. Hepatitis A virus was purified in a 10-30% sucrose gradient and each fraction was examined by immunosorbent electron microscopy, direct electron microscopy, immune electron microscopy and radioimmunoassay. The sensitivities of immunosorbent electron microscopy and radioimmunoassay were essentially similar and considerably greater than direct electron microscopy and conventional immune electron microscopy. PMID- 3001118 TI - Typing of herpes simplex virus isolates with monoclonal antibodies and by nucleic acid spot hybridization. AB - Fifty-one clinical isolates of herpes simplex virus (HSV) were typed by an enzyme immunoassay (EIA) using mouse monoclonal antibodies, by DNA spot hybridization, and by restriction enzyme analysis using restriction endonuclease Eco RI. Extracts of VERO cells infected with the isolates were used for coating microtitre plates or denatured and spotted onto nitrocellulose filters. Viral antigens passively adsorbed to microtitre plates were detected by an indirect EIA using mouse monoclonal antibodies specific for HSV type 1 (HSV-1) or HSV type 2 (HSV-2). Spotted DNA was hybridized with 32P-labeled probes containing Hind III/Sal I-fragments of either HSV-1 or HSV-2 DNA and bound radioactivity was detected by autoradiography and counted in a liquid scintillation counter. All the three methods gave identical results for the 51 isolates studied. Twenty-six isolates were identified as HSV-1 and 25 as HSV-2. An additional 30 specimens were tested only by EIA and hybridization. Results by both techniques were in complete agreement. PMID- 3001119 TI - A simple method for increased recovery of purified paramyxovirus virions. AB - A simple method involving vigorous agitation of infected cell monolayers prior to collection of culture medium is described to greatly increase the recovery of purified virions of measles virus, respiratory syncytial virus and human parainfluenza virus. Vigorous agitation of the flasks containing monolayers of infected cells increased the recovery of purified virions by at least 3- to 10 fold as judged by the intensity of [35S]methionine labeled viral proteins on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). These protein profiles also indicated that these virions were as clean as those purified from gently collected medium. Analysis of titers of infectious virus recovered from medium of agitated and non-agitated flasks showed similar increases. These results suggest that the cell associated nature of these viruses may be at least partly due to either partially budded virions or mature virions sticking to the cell membrane, since these both might be expected to be freed from the cell by mechanical shearing. PMID- 3001120 TI - A modified immunofluorescence test for Epstein-Barr virus-specific IgM antibody. AB - The fluorescent antibody (FA) test for Epstein-Barr virus (EBV)-specific IgM antibody was improved by the use of sodium butyrate to induce a higher level of EBV antigen expression in P3HR-1 slide preparations and by removal of rheumatoid factor (RF) and IgG antibodies from test sera by means of adsorption with suspensions of Sepharose-IgG and Streptococcus pyogenes strain AR1. This method was compared with the Paul-Bunnell test (PB) on 1106 sera submitted to a routine virus diagnostic laboratory for infectious mononucleosis serology and 96.4% of sera showed concordant results. Thus the EBV-IgM-FA method was suitable for routine diagnostic use. However, it proved helpful to test EBV-IgM positive sera by PB to assist in the detection of cross-reacting IgM antibodies sometimes present. PMID- 3001121 TI - Detection of antibodies to Aujeszky's disease virus in whole blood by Elisadisc. AB - A rapid method for the detection of antibodies in whole blood by ELISA was developed. A punched filter paper disc configuration, the Elisadisc, was loaded with blood, dried, and applied directly to the Aujeszky's disease ELISA, with minimal pretreatment. Rapidity, throughput and sensitivity were equal to that of the serum ELISA. No loss of activity could be detected in the discs after storage at +4 degrees C for almost 1 yr. PMID- 3001122 TI - Radioimmunoassay and enzyme-linked immunoassay of antibodies directed against lymphadenopathy-associated virus (LAV) proteins larger than the core protein (P24). AB - The acquired immunodeficiency syndrome (AIDS) may be transmitted by blood transfusions and by blood products from donors who have been infected with the lymphadenopathy-associated virus (LAV). Such donors may generally be identified on the basis of a positive test for antibodies-against LAV proteins. We have already described an anti-LAV assay based on the use of crude virus-infected tissue culture medium, which avoids elaborate, expensive and potentially hazardous virus purification steps. This test was operationally specific for antibodies to the approximately 24 kD core protein of the virus (P24; Neurath et al. J. Virol. Methods 11, 75, 1985). Molecular exclusion chromatography of crude LAV antigen preparations allows separation of most of P24 from larger proteins of LAV (PL). PL and 125I- or beta-lactamase-labeled anti-LAV were used as reagents for radioimmunoassay (RIA)--or enzyme-linked immunoassay (ELISA)--inhibition tests to detect antibodies directed predominantly against PL (anti-PL). Among 257 individuals belonging to groups at high risk of developing AIDS, 117 (45.5%) were positive for anti-PL and 108 (42%) for anti-P24, respectively. The 2 individuals among 600 random blood donors found to be anti-P24-positive in the preceding study also had anti-PL in their serum. Sera from 500 additional blood donors were screened for anti-PL and 1 of these was positive. The implication of these findings for screening of blood donors is discussed. PMID- 3001124 TI - Characterization of corticotropin receptors in human adrenocortical cells. AB - [125I-Tyr23,Phe2,Nle4]ACTH-(1-38) ([125I]ACTH analog), which is equipotent with ACTH, was used to characterize ACTH receptors in human adrenocortical cells. Adrenals were obtained from brain-dead patients at the time of renal harvest with permission. Binding of [125I]ACTH analog to human adrenocortical cells was highly specific, rapid, reversible, and saturable. Analysis of the inhibition of binding of [125I]ACTH analog by ACTH was compatible with a single class of binding sites with an apparent dissociation constant (Kd) of 1.6 nM and a mean binding capacity of 3560 sites/cell. Concentration-response curves for cAMP and cortisol production were shifted to the left of the binding curve, with ACTH concentrations required for half-maximal stimulation being 20- and 720-fold less, respectively, than those for binding. Extracellular calcium was essential for binding and stimulation of cAMP production. These results indicate that human adrenocortical cells contain a single class of ACTH receptors which are highly similar in affinity, capacity, and calcium requirement to those of rat adrenocortical cells, but differ from the rat receptors in the concentration of ACTH needed for cAMP generation. PMID- 3001123 TI - Modulation of lactogenic receptors by progestins in cultured human breast cancer cells. AB - Progesterone receptors (PgR) are present in many breast cancers, but few specific actions of progestins in breast cancer cells have been reported. We now report that progestins specifically modulate lactogenic receptor expression in cultured T-47D and MCF-7 human mammary carcinoma cells. When T-47D cells were preincubated for 24 h with 1 nM medroxyprogesterone acetate (17 alpha-acetoxy-6 alpha-methyl-4 pregnene-3,20-dione), specific binding of [125I]human GH ([125I]hGH) and [125I]human PRL was increased to 205 +/- 22% (+/- SE) (P less than 0.01) and 175 +/- 32% (P less than 0.05), respectively, of that in control cultures. There was no significant effect on cell number and no significant enhancement of specific binding of [125I]porcine insulin, [125I]salmon calcitonin, [125I]human transferrin, or [3H] Concanavalin A. Lactogenic receptor number was increased from 6,490 +/- 500 (n = 12) to 13,180 +/- 3,270 (n = 7; P less than 0.01) sites/cell, with no significant change in affinity for hGH. Progesterone, which is readily metabolized by these cells, was less potent than the synthetic progestins (medroxyprogesterone acetate, R 5020 (17 alpha, 21-dimethyl-19 norpregn-4,9-diene-3,20-dione), and ORG 2058 (16 alpha-ethyl-21-hydroxy-19 norpregn-4-en-3,20-dione), but physiological concentrations of progesterone (1 nM) significantly enhanced specific binding of [125I]hGH to 153 +/- 23% of the control value (n = 6; P less than 0.05). Physiological concentrations of androgens, estrogens, and glucocorticoids had no significant effect. MCF-7 cells were considerably less sensitive to these effects of progestins than T-47D cells, probably due to the lower PgR concentration in MCF-7 cells. These observations, which indicate that lactogenic receptor expression is controlled, at least in part, by progestins in these mammary carcinoma cell lines, may have important implications in the management of human breast cancer, where high levels of this receptor may reflect a functional PgR and a highly hormone-dependent phenotype. PMID- 3001126 TI - Ovine corticotropin-releasing hormone stimulation test in normal children. AB - We administered ovine corticotropin-releasing hormone (CRH) as a bolus iv injection (1 microgram/kg) to 21 normal boys and girls, aged 6-15 yr. CRH stimulated release of immunoreactive ACTH and cortisol in all children. The peak plasma ACTH and cortisol levels after CRH were 15.7 +/- 9.4 (SD) pg/ml and 14.3 +/- 3.6 micrograms/dl, respectively, in the girls, and 20.7 +/- 9.7 pg/ml and 16.6 +/- 3.3 micrograms/dl, respectively, in the boys. Plasma ACTH and cortisol responsiveness to CRH did not differ between girls or boys, or between children and adults. Cortisol-binding globulin concentrations in plasma did not change with age. We conclude that CRH provides a safe means of stimulating the pituitary adrenal axis in children. PMID- 3001125 TI - The responses of plasma adrenocorticotropin and cortisol to corticotropin releasing hormone (CRH) and cerebrospinal fluid immunoreactive CRH in anorexia nervosa patients. AB - Pituitary-adrenocortical responses to the iv injection of 100 micrograms synthetic ovine corticotropin-releasing hormone (CRH) were studied in 13 patients with anorexia nervosa, and the concentrations of immunoreactive CRH in cerebrospinal fluid were measured in 7 of them. Mean basal levels of plasma ACTH and cortisol were 32 +/- 5 pg/ml (+/- SEM) and 21.1 +/- 1.5 micrograms/dl, respectively. The latter value was significantly higher than that in age-matched normal women (P less than 0.005). The mean increments of plasma ACTH and cortisol in response to CRH injection in those 13 patients were 21 +/- 5 pg/ml and 5.3 +/- 1.7 micrograms/dl, respectively, significantly lower than those in normal women (58 +/- 6 pg/ml and 15.3 +/- 7.7 micrograms/dl, respectively; P less than 0.005). When 4 patients were reexamined after weight gains of between 3 and 22 kg, their responses to the CRH injection increased. The mean concentration of immunoreactive CRH in the cerebrospinal fluid of seven patients was 30.8 +/- 3.9 pg/ml (+/- SEM), which was higher than the value of 18.4 +/- 1.1 pg/ml (P less than 0.005) in control subjects with cervical spondylosis. These findings suggest the possibility that hypersecretion of CRH may occur in patients with anorexia nervosa. PMID- 3001127 TI - Multiple pre- and postreceptor defects in pseudohypoparathyroidism (a multicenter study with twenty four patients). AB - Three different pathophysiological mechanisms are probably responsible for hereditary pseudohypoparathyroidism: 1) a defect at the prereceptor-level, 2) a defective membrane N-protein accounting for diminished second messenger production, and 3) a defect in the cytosolic response to the hormone. In a cooperative, study 24 patients (mean age, 13 yr; range, 3-23 yr, 8 girls, 16 boys) receiving vitamin D metabolites (5,000-80,000 U/day) were examined and compared to a control group of 36 normal children. Immunoreactive N-terminal PTH (N-PTH), mid-C-regional PTH (mid-C-PTH), intact PTH and bio-PTH, vitamin D metabolites, and serum calcium and phosphate, alkaline phosphatase activity, and the N-protein activity of erythrocyte membranes were measured in each subject. By clinical and biochemical criteria three groups were differentiated. Eight patients had the completely expressed features of Albright's Hereditary Osteodystrophy (AHO+), including brachydactyly and/or sc calcifications, and increased N-PTH, mid-C-PTH, and alkaline phosphatase activity. Bio-PTH, intact PTH, and N-protein were normal. Nine additional patients with complete (AHO+) had elevated levels of bio-PTH, N-PTH, and mid-C PTH, normal hydroxylation of vitamin D, but decreased N-protein activity. Seven patients with pseudohypoparathyroidism had no features of AHO (AHO-), no increase of urinary cAMP excretion after exogenous PTH, normal PTH peptide levels and N-protein activity, but elevated 25 hydroxyvitamin D and decreased 1,25-dihydroxyvitamin D concentrations. In conclusion, we identified three subpopulations of PsHP: group a had a dissociation of N-PTH and bio-PTH suggesting a defective N-PTH causing renal resistance, whereas their bones respond to PTH. Group b had defective N-protein causing generalized PTH resistance. Group c was characterized by high 25 hydroxyvitamin D and relatively low 1,25-dihydroxyvitamin D levels, thus providing evidence for a defect in the cytosolic interaction of the two different second messengers for PTH, cAMP, and calcium. PMID- 3001128 TI - Synthesis of prostaglandins and cyclic AMP by cultured embryonic palate mesenchyme at various population densities. AB - During embryonic development, facial and palate mesenchymal cells exhibit differential growth rates. Normal palatal growth is regulated in part by hormones and growth factors. Because hormonal responsiveness of some cells correlates with their cell density, we have investigated the relationship between embryonic palate mesenchymal cell population density and their ability to synthesize prostaglandins (PGs) and cyclic AMP. Primary cultures of palate mesenchymal cells exhibited typical lag, log, and stationary phases of growth with a doubling time of 32-34 hrs. The ability of cells to produce PGE2 in response to a calcium ionophore (A23187), an activator of phospholipase A2 (melittin), arachidonic acid, or serum was maximal during the period of early exponential growth. Prostaglandin F2 alpha synthesis in response to A23187 or arachidonic acid showed a similar transient increase also corresponding temporally to the period of early exponential growth. The ability to synthesize PGF2 alpha in response to melittin, however, failed to diminish after early exponential growth. The pattern of cAMP synthesis in response to isoproterenol and PGE1 was different from that seen for induced prostaglandin synthesis. A transient increase in sensitivity to isoproterenol and PGE1 was seen that corresponded temporally to the period of late exponential growth just prior to attainment of confluency. Decreased sensitivity to stimulation of either prostaglandin or cAMP production as the cells became confluent was shown to be a density-dependent phenomenon; confluent cultures that were subcultured to reestablish logarithmic growth exhibited density-dependent hormonal responses identical to those seen in primary cultures. The ability of palate mesenchymal cells to synthesize both prostaglandins and cAMP, thought to be critical for proper palatal development, might thus be related to local differential craniofacial growth rates. PMID- 3001129 TI - Virus-specific polymeric immunoglobulin A antibodies in serum from patients with rubella, measles, varicella, and herpes zoster virus infections. AB - More than 85% of the immunoglobulin A (IgA) antibodies in normal adult serum are monomeric (m-IgA). By contrast, virus-specific IgA is mainly polymeric (p-IgA) in sera from patients with rubella, measles, and varicella. Specific m-IgA antibodies only reach quantitative significance in late convalescence. In patients with herpes zoster, on the other hand, a varying response was observed: in three of six sera, specific IgA was absent or at a very low titer, whereas in the remaining three cases, a high titer of both p-IgA and m-IgA was noted. These results suggest that in the initial response to rubella, measles, and varicella zoster viruses, specific IgA first appears as p-IgA and only later becomes, or is replaced by, m-IgA. PMID- 3001130 TI - Prevalence of antibody to human calicivirus in Japan and Southeast Asia determined by radioimmunoassay. AB - Three hundred ninety single sera from adults in Japan, Indonesia, Singapore, and Papua New Guinea were tested for antibodies to human calicivirus (HCV) by a radioimmunoassay blocking test. A high prevalence of antibodies was observed in samples collected in Japan and Southeast Asia. Of 240 serum specimens collected from five districts in Japan, 209 (87.1%) were positive for HCV antibodies. No striking difference in the prevalence of the antibody was seen among those districts. In sera collected in Southeast Asia, 84.0% (126 of 150) had antibodies to HCV (70% [35 of 50] in Singapore, 88% [44 of 50] in Indonesia, and 94% [47 of 50] in Papua New Guinea). These results indicate that HCV is a common infectious agent in Japan and Southeast Asia. PMID- 3001131 TI - Production of monoclonal antibodies against parainfluenza 3 virus and their use in diagnosis by immunofluorescence. AB - Monoclonal antibodies were produced against parainfluenza virus type 3 (PI-3) and used to identify PI-3 clinical isolates in cell culture and PI-3 antigen in cells obtained from nasopharyngeal (NP) washes of patients. Two (2E9 and 4G5) of the three monoclonal antibodies characterized reacted by immunoblotting with a 67,000 dalton PI-3 protein, and one antibody (4E5) reacted with two viral proteins in the range of 29,000 to 31,000 daltons. The three monoclonal antibodies did not cross-react by indirect immunofluorescence (IFA) with PI-1 or PI-2 and identified by IFA 18 isolates of PI-3 in cell culture. The 2E9 antibody reacted with PI-3 antigen in cells of 8 NP wash specimens that also yielded PI-3 in cell culture. Cells from 12 specimens reactive by IFA for respiratory syncytial virus, 1 specimen yielding adenovirus in cell culture, and 5 specimens yielding influenza virus were not reactive. PMID- 3001132 TI - Recent vesicular stomatitis virus infection detected by immunoglobulin M antibody capture enzyme-linked immunosorbent assay. AB - We developed an enzyme-linked immunosorbent assay (ELISA) that was capable of detecting immunoglobulin M (IgM) antibody to vesicular stomatitis virus (VSV) in the sera of experimentally and naturally infected cattle and horses. The detection of IgM in the sera of these animals permitted an estimate of the recency of infection by VSV serotype New Jersey. A VSV serotype New Jersey epizootic strain isolated from a horse and passed once in an Aedes albopictus cell line was used to infect a horse and a calf. Sera from these animals were used to standardize the ELISA. This assay was used to test sera from cattle and horses involved in the 1982 VSV epizootic. Comparative antibody titrations were performed by three systems: the serum-dilution plaque-reduction neutralization, complement fixation, and indirect immunofluorescent tests. The antibody titers by neutralization and the ELISA were comparable for the period that IgM was present; when IgM ELISA titers diminished, the neutralization titers remained high. The complement fixation and indirect immunofluorescent antibody titers followed closely the IgM pattern determined by the ELISA. The capture IgM ELISA is applicable for the rapid detection of IgM antibody to VSV in cattle and horses and is a useful assay of recent infection. PMID- 3001133 TI - Immunoradiometric assay of a recombinant human alpha-2 interferon (SCH 30500). AB - A sensitive immunoradiometric assay for recombinant alpha-2 interferon (IFN-alpha 2; SCH 30500) was developed by using the monoclonal antibody NK2. A polystyrene bead with polyclonal sheep anti-human IFN-alpha (Hu IFN-alpha) antibody adsorbed to it formed the solid phase of the assay. The monoclonal antibody, labeled with 125I, reacted with the IFN-alpha 2 which bound to the polyclonal antibody on the bead. The assay was sensitive, capable of measuring less than 5 IU/ml, and it was precise. Coefficients of variation were less than 10%, often less than 5%. Interassay coefficients of variation for the same sample were also less than 10%. Specificity of the monoclonal antibody for IFN-alpha 2 was greater than for Hu IFN-alpha produced by leukocytes. In specificity studies it was necessary to add at least 200 IU of Hu IFN-alpha per ml to 25 IU of IFN-alpha 2 per ml before the Hu IFN-alpha affected the accuracy of the assay of IFN-alpha 2. Accuracy as determined by correlation studies with the antiviral cytopathic effect assay was very high. Results of the two assays usually agreed within 15%. This correlation was maintained in studies of IFN-alpha 2 after storage under accelerated degradation conditions, when the IFN was cleaved into smaller sequences of amino acids by cyanogen bromide and when the IFN was carboxymethylated. PMID- 3001134 TI - Development of a DNA probe to detect Salmonella typhi. AB - This study was undertaken to identify a DNA sequence that could be used to facilitate the diagnostic identification of Salmonella typhi, the causative agent of typhoid fever. All virulent S. typhi strains encode a relatively unique capsular antigen termed the virulence (Vi) antigen. Two distinct genetic loci, viaA and viaB, are involved in the synthesis of this antigen. The structural genes, located at viaB, were considered as a possible specific DNA probe. The viaB locus, contained in a recombinant cosmid, was subcloned to various plasmid vectors for this purpose. Selected viaB-region DNA fragments were then analyzed for specificity in DNA colony hybridization reactions with more than 170 strains representing a variety of enteric bacteria. An 8.6-kilobase EcoRI fragment was highly specific for the viaB gene region and was considered a good hybridization probe. This DNA probe should prove useful in rapid diagnostic assays set up to detect S. typhi in mixed bacterial samples (e.g., stools) within a few hours of specimen collection. PMID- 3001135 TI - Rapid and simplified protocol for isolation and characterization of leptospiral chromosomal DNA for taxonomy and diagnosis. AB - We have developed a rapid method for the isolation of leptospiral chromosomal DNA which yields DNA of a purity suitable for restriction endonuclease analysis. A small volume (15 to 20 ml) of an exponentially growing culture of leptospires yielded 2 to 4 micrograms of chromosomal DNA. In a 1-day protocol, the DNA was isolated, restricted with endonucleases, and fractionated on an agarose gel. Chromosomal DNA from dinger zones (visible subsurface zones of leptospiral growth) of first semisolid subcultures of field isolates was also isolated and characterized, thus greatly speeding up the diagnostic process. PMID- 3001136 TI - Detection of herpes simplex virus type 2-specific antibody with glycoprotein G. AB - A recently described herpes simplex virus (HSV) type 2 (HSV-2)-specific glycoprotein (gG-2) was purified on an immunoaffinity column prepared with monoclonal antibody. This purified antigen was used in an immunodot enzymatic assay on nitrocellulose paper for the detection of HSV-2 antibodies in human serum. The test was very sensitive in that HSV-2 antibodies were detected in the convalescent sera of 132 of 134 patients with recurrent genital infections in which HSV-2 had been isolated earlier. Antibodies to gG-2 were detected in 17% of sera obtained within 10 days after the onset of a primary HSV infection and in 95% of sera obtained more than 10 days after onset. The specificity of the immunodot assay was demonstrated by testing sera from 245 HSV-seronegative adults, 344 children, 29 nuns, and 13 patients with primary genital HSV-1 infections. None of these 631 sera was reactive with the gG-2 antigen. When compared with a microneutralization test, the immunodot assay was found to be more specific in detecting HSV-2 antibodies. Reproducibility of the gG-2 assay, obtained by retesting 391 sera, was 95%. Thus, this assay has the sensitivity, specificity, and reproducibility necessary for the measurement of HSV-2 antibodies in seroepidemiological studies. PMID- 3001137 TI - Rapid detection of herpes simplex virus DNA in human brain tissue by in situ hybridization. AB - A commercial system for detection of herpes simplex virus (HSV) DNA by in situ hybridization gave positive results on 16 of 17 stored human brain specimens that were positive for HSV on initial testing by virus isolation and immunofluorescence staining, and the hybridization system gave negative results on 13 brain specimens that showed no evidence of HSV by isolation or immunofluorescence staining. PMID- 3001139 TI - Physiologic regulation of atrial natriuretic peptide receptors in rat renal glomeruli. AB - Isolated rat renal glomeruli and cultured glomerular mesangial and epithelial cells were examined for atrial natriuretic peptide (ANP) receptors, and for ANP stimulated cyclic guanosine monophosphate (cGMP) generation. In glomeruli from normal rats, human (1-28) 125I-ANP bound to a single population of high affinity receptors with a mean equilibrium dissociation constant of 0.46 nM. Human (1-28) ANP markedly stimulated cGMP generation, but not cAMP generation in normal rat glomeruli. Analogues of ANP that bound to the glomerular ANP receptor with high affinity stimulated cGMP accumulation, whereas the (13-28) ANP fragment, which failed to bind to the receptor, was devoid of functional activity. Cell surface receptors for ANP were expressed on cultured glomerular mesangial but not epithelial cells, and appreciable ANP-stimulated cGMP accumulation was elicited only in mesangial cells. Approximately 12,000 ANP receptor sites were present per mesangial cell, with an average value for the equilibrium dissociation constant of 0.22 nM. Feeding of a low-salt diet to rats for 2 wk resulted in marked up regulation of the glomerular ANP receptor density to a mean of 426 fmol/mg protein, compared with 116 fmol/mg in rats given a high-salt diet. A modest reduction in the affinity of glomerular ANP receptors was also observed in rats fed the low-salt diet. ANP-stimulated cGMP generation in glomeruli did not change with alterations in salt intake. We conclude that high salt feeding in the rat results in reduced glomerular ANP receptor density relative to values in salt restricted rats. Furthermore, the mesangial cell is a principal target for ANP binding in the glomerulus. PMID- 3001140 TI - Self-perpetuating mechanisms of immunoglobulin G aggregation in rheumatoid inflammation. AB - When human IgG is exposed to free radical generating systems such as ultraviolet irradiation, peroxidizing lipids, or activated human neutrophils, characteristic auto-fluorescent monomeric and polymeric IgG is formed (excitation [Ex], 360 nm, emission [Em], 454 nm). 1 h ultraviolet irradiation of IgG results in the following reductions in constituent amino acids; cysteine (37.0%), tryptophan (17.0%), tyrosine (10.5%), and lysine (3.6%). The fluorescent IgG complexes, when produced in vitro, can stimulate the release of superoxide from normal human neutrophils. In the presence of excess unaltered IgG, further fluorescent damage to IgG occurs. Measurement and isolation of fluorescent monomeric and polymeric IgG by high performance liquid chromatography, from in vitro systems and from fresh rheumatoid sera and synovial fluid, indicates that identical complexes are present in vivo; all these fluorescent complexes share the property of enhancing free radical production from neutrophils. The results described in this study support the hypothesis that fluorescent monomeric and aggregated IgG may be formed in vivo by oxygen-centered free radicals derived from neutrophils, and that in rheumatoid inflammation this reaction may be self-perpetuating within the inflamed joint. PMID- 3001138 TI - Biologically active atrial peptides. PMID- 3001141 TI - Ischemia induces partial loss of surface membrane polarity and accumulation of putative calcium ionophores. AB - To determine if ischemia induces alterations in renal proximal tubule surface membranes, brush border (BBM) and basolateral membranes (BLM) were isolated simultaneously from the same cortical homogenate after 50 min of renal pedicle clamping. Ischemia caused a selective decrease in the specific activity of BBM marker enzymes leucine aminopeptidase and alkaline phosphatase, but did not effect enrichment (15 times). Neither specific activity nor enrichment (10 times) of BLM NaK-ATPase was altered by ischemia. Contamination of BBM by intracellular organelles was also unchanged, but there was an increase in the specific activity (41.1 vs. 60.0, P less than 0.01) and enrichment (2.3 vs. 4.3, P less than 0.01) of NaK-ATPase in the ischemic BBM fraction. Ischemia increased BLM lysophosphatidylcholine (1.3 vs. 2.5%, P less than 0.05) and phosphatidic acid (0.4 vs. 1.3%, P less than 0.05). Ischemia also decreased BBM sphingomyelin (38.5 vs. 29.6%, P less than 0.01) and phosphatidylserine (16.1 vs. 11.4%, P less than 0.01), and increased phosphatidylcholine (17.2 vs. 29.7%, P less than 0.01), phosphatidylinositol (1.8 vs. 4.6%, P less than 0.01), and lysophosphatidylcholine (1.0 vs. 1.8%, P less than 0.05). The large changes in BBM phospholipids did not result from new phospholipid synthesis, since the specific activity (32P dpm/nmol Pi) of prelabeled individual and total phospholipids was unaltered by ischemia. We next evaluated if these changes were due to inability of ischemic cells to maintain surface membrane polarity. Cytochemical evaluation showed that while NaK-ATPase could be detected only in control BLM, specific deposits of reaction product were present in the BBM of ischemic kidneys. Furthermore, using continuous sucrose gradients, the enzymatic profile of ischemic BBM NaK-ATPase shifted away from ischemic BLM NaK-ATPase and toward the BBM enzymatic marker leucine aminopeptidase. Taken together, these data suggest that NaK-ATPase activity determined enzymatically and cytochemically was located within ischemic BBM. We propose that ischemia impairs the ability of cells to maintain surface membrane polarity, and also results in the accumulation of putative calcium ionophores. PMID- 3001142 TI - Biosynthesis of the transferrin receptor in rabbit reticulocytes. AB - These studies were performed to determine whether the reticulocyte can synthesize its own transferrin receptor and, if so, whether synthesis is subject to translational control by intracellular heme. Reticulocytosis (20-35%) was produced by bleeding rabbits and the washed cells were incubated for 1-4 h at 37 degrees C in buffered nutritional medium containing L-[35S]methionine. After washing and detergent lysis in the presence of protease inhibitors, supernatant reticulocyte extracts were analyzed for transferrin receptors by immunoprecipitation with specific ovine receptor antibody raised against denatured rabbit transferrin receptor. Immunoprecipitates were analyzed by SDS gel electrophoresis and fluorography. Antibody, but not preimmune sheep immunoglobin, consistently precipitated a 35S-labeled protein with an Mr of 90,000 (reduced), coincident with bona fide receptor subunits purified by ligand affinity chromatography. Incorporation of radioactive methionine was exclusively associated with receptor in reticulocyte stroma, and nascent receptor was not detected on free polyribosomes. Incorporation of radioactivity in the receptor moiety accounted for 0.1-0.2% of total incorporation into TCA insoluble cell protein. Treatment of the cells with 40 micrograms/ml cycloheximide markedly inhibited amino acid incorporation into the receptor, thus indicating de novo synthesis of receptor protein. On treatment of reticulocytes with 4,6 dioxoheptanoate to induce heme deficiency by diminishing the formation of intracellular heme, synthesis of the receptor was inhibited by greater than 50%; synthesis was restored to control rates on addition of 50 microM exogenous hemin. These findings indicate that the reticulocyte retains receptor mRNA and that synthesis of the receptor in erythroid cells is subject to translational regulation by intracellular heme. PMID- 3001143 TI - An intragenic deletion of the factor IX gene in a family with hemophilia B. AB - A family of seven patients severely afflicted with hemophilia B has been studied for their factor IX genes through the use of factor IX cDNA and genomic DNA probes. The patients had detectable (less than 10% of normal) factor IX antigen in urine and no detectable inhibitors in sera to factor IX protein. Based on the DNA hybridization analysis, these patients showed a partial intragenic deletion in their factor IX gene. The deletion included two exons (exons V and VI) coding for the amino acid sequence from number 85 to 195 of the factor IX protein. The deleted portion of the gene contained the entire factor IX activation peptide. The length of the deletion was estimated to be 10 +/- 0.3 kilobase pairs. This specific gene has been named FIXSeattle. In this family both the deletion and a Taq 1 restriction fragment length polymorphism can be used as a useful marker for accurate detection of female carriers of the deficient factor IX gene. PMID- 3001144 TI - Thrombomodulin is present in human plasma and urine. AB - Thrombomodulin is an endothelial cell membrane protein that is a cofactor required for the rapid activation of plasma protein C. We now report that plasma and urine of normal subjects contains a modified form of thrombomodulin that is soluble. The levels measured by radioimmunoassay were 292 +/- 60 ng thrombomodulin/ml plasma and 102 +/- 38 ng thrombomodulin/ml urine. Thrombomodulin was isolated from both plasma and urine by immunoaffinity chromatography using a polyclonal anti-human thrombomodulin IgG column. The apparent molecular weight of soluble thrombomodulin was estimated by immunoblot analysis using 125I-monoclonal anti-thrombomodulin IgG. When run without 2 mercaptoethanol, soluble thrombomodulin appeared as two polypeptides, Mr = 63,000 and 54,000, while samples run with 2-mercaptoethanol migrated mainly at Mr = 85,000. These results imply that the soluble form of thrombomodulin is smaller than the cellular form, presumably because of a lack of the membrane-binding domain. Soluble thrombomodulin is similar to cellular thrombomodulin in its intrinsic protein C-activating cofactor activity as measured by antibody neutralization. The apparent Km for protein C was the same for cellular and soluble thrombomodulin, while the soluble form requires a higher concentration of thrombin (three- to fivefold) for one-half maximal activity than the cellular form. Thrombomodulin functional activity cannot be directly measured in plasma because of some inhibitory substance(s). The physiological significance of circulating and urinary thrombomodulin is presently obscure. PMID- 3001145 TI - DNA binding to human leukocytes. Evidence for a receptor-mediated association, internalization, and degradation of DNA. AB - Previous studies have indicated that white blood cells possess DNA on their outer membranes. In this study we set out to determine whether exogenous DNA bound to cells in a fashion compatible with a ligand receptor union. Purified populations of white blood cells; neutrophils (polymorphonuclear leukocytes, PMN), adherent mononuclear cells (ADMC), rosetting lymphocytes (E+ cells), and nonrosetting lymphocytes (E- cells) were incubated with radiolabeled lambda phage DNA in increasing concentrations. Binding of [3H]DNA was a saturable process and was inhibited by excess cold DNA and prior trypsinization of the cells. Rate zonal density centrifugation of purified cell membrane preparations confirmed that DNA was binding to the outer cell surface. The dissociation constant for all four cell types was approximately 10(-9) M, and from 0.81 X 10(3) to 2.6 X 10(3) molecules of lambda phage DNA bound to each cell depending upon cell type. Binding was not competitively inhibited by RNA, polydeoxyadenylic acid polydeoxythymidylic acid (poly [d(A).d(T)]), or mononucleotides. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE)-separated proteins from PMN, ADMC, E+, and E- cells were electrophoretically blotted onto nitrocellulose sheets; a probe of biotin-labeled DNA indicated a single species of DNA-binding molecule migrating in a position consistent with a molecular weight of 30,000. Isotopic and immunofluorescent studies indicate that DNA is internalized and degraded to oligonucleotides; this process is inhibited by cycloheximide. These results support the notion that there is a common binding site for DNA on white blood cells, that the stoichiometry of the association is compatible with a ligand receptor relationship, and that this apparent receptor is responsible for the endocytosis and degradation of exogenous DNA. PMID- 3001146 TI - Lysosomal enzyme phosphorylation in human fibroblasts. Kinetic parameters offer a biochemical rationale for two distinct defects in the uridine diphospho-N acetylglucosamine:lysosomal enzyme precursor N-acetylglucosamine-1 phosphotransferase. AB - The primary genetic defect in the lysosomal storage disease mucolipidosis III (ML III) is in the enzyme uridine diphospho-N-acetylglucosamine:lysosomal enzyme N acetylglucosamine-1-phosphotransferase. This enzyme has two well-defined functions: specific recognition of lysosomal enzymes (recognition function) and phosphorylation of their oligosaccharides (catalytic function). Using fibroblasts from patients with ML III as the source of enzyme, and alpha-methylmannoside and two lysosomal enzymes as the substrates, we have identified defects in both of these functions. In one group of fibroblasts, the catalytic activity of the N acetylglucosaminylphosphotransferase is decreased while the ability to recognize lysosomal enzymes as specific substrates remains intact. In the second group of fibroblasts, the ability to recognize lysosomal enzymes is impaired while the catalytic activity of the enzyme is normal. These data provide a biochemical rationale for the previously described genetic heterogeneity among patients with ML III (Honey, N. K., O. T. Mueller, L. E. Little, A. L. Miller, and T. B. Shows, 1982, Proc. Natl. Acad. Sci. USA., 79:7420-7424). PMID- 3001147 TI - Loss of high affinity cardiac beta adrenergic receptors in dogs with heart failure. AB - We studied the alterations in myocardial beta-adrenergic receptor-adenylate cyclase activity and muscarinic receptor density in a canine model of left ventricular (LV) failure. LV failure was characterized by a doubling of LV weight/body weight ratio (3.3 +/- 0.1 to 6.9 +/- 0.4 g/kg) and an elevation of LV end-diastolic pressure, 32 +/- 4.5 mmHg, compared with 7.7 +/- 0.6 mmHg in normal dogs. Despite a 44% increase in receptor density as measured by antagonist binding studies with [3H]dihydroalprenolol, there was a twofold decrease in receptor affinity, i.e., an increase in the dissociation constant (Kd) (5.6 +/- 0.7 to 12 +/- 1.6 nM) in heart failure. Agonist displacement of [3H]dihydroalprenolol binding with isoproterenol in the presence and absence of 5'-guanylylimidodiphosphate [Gpp(NH)p] demonstrated a striking loss of high affinity binding sites in heart failure (51 +/- 16 to 11 +/- 5%). Beta-Adrenergic receptor-mediated stimulation of adenylate cyclase and maximal stimulation with Gpp(NH)p or sodium fluoride was reduced in heart failure. There was a concomitant marked, P less than 0.01, reduction in muscarinic receptor density (242 +/- 19 vs. 111 +/- 20 fmol/mg). Thus, while muscarinic receptor density fell, beta adrenergic receptor density actually increased in LV failure. However, a larger portion of the beta-adrenergic receptors are not functionally coupled to the GTP stimulatory protein (Ns), as evidenced by a decrease in the fraction of receptors that bind agonist with high affinity. PMID- 3001150 TI - Malignant optic nerve gliomas in adults. AB - Two patients with malignant optic nerve gliomas are described. The clinical courses and radiographic appearances are discussed, with emphasis on the fact that this condition should be considered in the differential diagnosis of rapid visual failure. It can now be strongly suspected preoperatively on the basis of computed tomographic scan appearances. PMID- 3001148 TI - Structural requirements for parathyroid hormone action in mature bone. Effects on release of cyclic adenosine monophosphate and bone gamma-carboxyglutamic acid containing protein from perfused rat hindquarters. AB - To determine the structural requirements for parathyroid hormone (PTH) activity in mature bone, we perfused the surgically isolated hindquarters of adult male rats with either native bovine PTH-(1-84) [bPTH-(1-84)] or the synthetic amino terminal fragment, bovine PTH-(1-34) [bPTH-(1-34)]. Changes in the release of cyclic AMP (cAMP) and bone Gla protein (BGP) were monitored as evidence of bone specific response to PTH; tissue specificity of the cAMP response was confirmed through in vitro examination on nonskeletal tissue response to PTH. Biologically active, monoiodinated 125I-bPTH-(1-84) was administered to determine if mature murine bone cleaves native hormone. We found that perfused rat bone continuously releases BGP, and that both bPTH-(1-84) and bPTH-(1-34) acutely suppress this release. In addition, both hormones stimulate cAMP release from perfused rat hindquarters. When examined on a molar basis, the magnitude of the cAMP response was dose-dependent and similar for both hormones, with doses yielding half maximal cAMP responses. The response for bPTH-(1-34) was 0.5 nmol and for bPTH-(1 84) was 0.7 nmol. Moreover, biologically active 125I-bPTH-(1-84) was not metabolized in our hindquarter perfusion system. These findings indicate that PTH (1-84) does not require extraskeletal or skeletal cleavage to an amino-terminal fragment in order to stimulate cAMP generation in, or suppress BGP release from, mature rat bone. PMID- 3001151 TI - Mesoblastic nephroma: prenatal sonographic detection. PMID- 3001149 TI - Metabolism and effects on platelet function of the purified eicosapentaenoic and docosahexaenoic acids in humans. AB - Metabolism and effects on platelet function of 6 g/d for 6 d of either eicosapentaenoic acid (EPA, C20:5 omega-3) or docosahexaenoic acid (DCHA, C22:6 omega-3) in volunteers were compared in a randomized crossover study. Incorporation kinetics revealed that EPA appeared in plasma free fatty acids and plasma phospholipids after 4 h, but was not incorporated into platelet phosphatidylcholine and -ethanolamine until day 6. This indicates that platelet fatty acid composition does not immediately reflect that of the surrounding plasma milieu, but rather may be determined during megakaryocyte maturation. Importantly, EPA was not incorporated into platelet phosphatidylinositol or serine in vivo, thus reflecting selective biosynthesis of platelet phospholipids. After dietary EPA, C22:5 omega-3 increased in plasma and platelet phospholipids. In contrast, DCHA-levels were unaltered. After DCHA-ingestion, C20:5 omega-3 concentrations rose in plasma phospholipids, implying that retroconversion took place. These findings indicate that dietary DCHA can serve as a source of EPA. During this short-term study, ingestion of both EPA and DCHA resulted in reduced platelet aggregation in response to collagen. The response to ADP was lowered significantly only by DCHA. After either EPA or DCHA, thromboxane formation was unchanged in serum derived from clotted whole blood as was total in vivo synthesis measured by excretion of immunoreactive 2,3-dinor thromboxane B2/3. We conclude that DCHA reduces platelet responsiveness, contributing to the antithrombotic effects of omega-3 fatty acid-rich fish oil ingestion, of which DCHA is a major component. PMID- 3001152 TI - Improving detection of "Viridans streptococcus" bacteraemia by adding sodium polyanethol sulphonate to blood cultures. AB - To detect streptococcal bacteraemia in patients undergoing dental extraction blood cultures containing glucose broth with 0.05% sodium polyanethol sulphonate (Liquoid) were compared with identical cultures without Liquoid. PMID- 3001154 TI - Detection of rotavirus by latex agglutination. PMID- 3001153 TI - Observer variability in reporting of breast lesions. AB - The consistency of histological diagnosis of breast lesions by members of the panel was studied using a simplified six point classification system that covered the range from normal tissue to invasive carcinoma. In a representative set of 40 sections of the range of breast disease included in the Trial for Early Detection of Breast Carcinoma (the consecutive series), there was, overall, 82% agreement between the diagnoses submitted by members of the panel: diagnosis was virtually consistent (99% agreement) for frankly invasive carcinoma, but there was much greater variability in the diagnosis of borderline lesions. Diagnostic consistency was greatly improved when two categories only (benign and malignant) were considered (94% agreement). The diagnostic consistency of individual pathologists was studied by recirculating the sections: the overall agreement was 78% and, again, most of the inconsistencies occurred when borderline lesions were diagnosed. Additional studies were undertaken on borderline lesions that had been flagged during the first two years of the trial (the borderline series). Unsurprisingly, there was less agreement among the pathologists when all six diagnostic categories were used (70% and 77% for the specimens from the first and second years, respectively), but consistency was greatly improved when classification was simplified to either benign or malignant (86% and 91%, respectively). The diagnoses submitted by individual pathologists were found to deviate from the "majority" diagnosis to a relatively minor extent: each pathologist was generally consistent in either under-diagnosing or overdiagnosing in both the consecutive and borderline series. PMID- 3001155 TI - A papular eruption associated with human T cell lymphotropic virus type III disease. AB - The clinical spectrum of human T cell lymphotropic virus type III (HTLV-III) disease is associated with myriad cutaneous findings, commonly of infectious origin. A clinically characteristic, yet histologically nonspecific, papular eruption was observed in seven of thirty-five patients followed up for HTLV-III disease (acquired immunodeficiency syndrome and the related complex). Noncoalescing 2- to 5-mm skin-colored papules of the head, neck, and upper trunk typify the lesions. Histologically, a chronic perivascular infiltrate of mononuclear cells was regularly present. The eruption was often, but not always, pruritic. The clinical course was chronic. Many patients had persistent lesions for more than 9 months; however, the number of papules tended to wax and wane with time. Although the cause of this eruption is unknown, it is sufficiently distinct and frequent to be recognized by clinicians as a cutaneous sign of human retrovirus infection. PMID- 3001156 TI - Severe molluscum contagiosum infection in a patient with human T cell lymphotrophic (HTLV-III) disease. PMID- 3001158 TI - Extramammary Paget's disease: prognosis and relationship to internal malignancy. AB - Extramammary Paget's disease is a rare cutaneous adenocarcinoma, usually of epidermal origin and glandular differentiation and frequently associated with an underlying adnexal carcinoma and perhaps with underlying internal malignancy. One hundred ninety-seven cases of extramammary Paget's disease (196 cases reported in the English literature from 1962 to 1982 and one case of my own) are reviewed. It remains a rare cutaneous malignancy that occurs primarily in elderly people. It is seen more frequently in women than in men and occurs predominantly in vulvar and perianal locations. Twenty-six percent of patients with this disease will ultimately die of it or an associated internal malignancy. Twenty-four percent of patients with the disease have an associated underlying cutaneous adnexal adenocarcinoma. These patients have a higher mortality rate--46%--than patients with extramammary Paget's disease without underlying cutaneous adnexal adenocarcinoma. Twelve percent of patients with extramammary Paget's disease have an associated concurrent underlying internal malignancy. The location of the underlying internal malignancy appears to be closely related to the location of the extramammary Paget's disease--i.e., a perianal location is associated with adenocarcinoma of the digestive system, a penile-scrotal-groin location with genitourinary malignancy, etc. A directed internal malignancy search may be of benefit in patients who are diagnosed as having extramammary Paget's disease. PMID- 3001157 TI - Unusual cutaneous lesions associated with acquired immunodeficiency syndrome. AB - We review a spectrum of unusual infectious, nutritional, and malignant processes that have been observed in the skin of patients with acquired immunodeficiency syndrome. PMID- 3001159 TI - Extramammary Paget's disease and occult hypernephroma. PMID- 3001160 TI - Effect of filler content and size on properties of composites. AB - Two series of dental composites, along with the unfilled resin matrix, were examined to determine the effects of filler level and size on selected properties. Both series were prepared by incorporating a silanated barium borosilicate filler into a visible-light-activated polyphenylene polymethacrylate resin matrix. One series had a filler particle size of 2 microns, with filler levels of 20, 40, 45, 50, and 53% (vol). The second series contained a 15-microns filler in amounts of 20, 40, 50, 60, and 65% (vol). Tests conducted included: depth of cure as evaluated by hardness, water sorption, compressive strength, stress-strain behavior under slow compression, toothbrush abrasion, and wear by hydroxyapatite. Analysis of the data indicated that increased filler levels resulted in increased hardness, compressive strength and stiffness, and decreased water sorption. Also, there was a slight trend toward improved depth of cure. Incorporation of the 2-microns filler decreased the abrasion resistance of the resins to toothbrushing as compared with the unfilled resin, while addition of the 15-microns filler improved resistance. All filled resins exhibited a significant improvement in resistance to wear by hydroxyapatite as compared with the unfilled resin. There was a trend for increased wear with increased filler level. The particle size of the filler appeared to have a moderate influence on the properties. When compared with 15-microns filled resins of the same filler levels, the 2-micron filled series appeared to have inferior properties in terms of depth of cure, compressive strength, water sorption, and resistance to toothbrush abrasion. Properties which were less affected by particle size were hardness, stiffness, and wear resistance to hydroxyapatite. PMID- 3001161 TI - Adsorption of zirconyl salts and their acids on hydroxyapatite: use of the salts as coupling agents to dental polymer composites. AB - Zirconyl methacrylate (I) and zirconyl-2-ethylhexanoate (II) were synthesized, and their adsorption isotherms from solutions onto synthetic hydroxyapatite were studied. The isotherms of methacrylic and 2-ethylhexanoic acids were also determined from the same solvents. The adsorption of I was irreversible from methylene chloride, and that of II was irreversible from cyclohexane. The adsorption in both cases was constant from solutions above a certain concentration, and exhaustive below this threshold concentration. Both compounds rendered the dried apatite powder extremely hydrophobic; however, the adsorbate was slowly washed off by excess water. The configuration of the adsorbate molecules, deduced from the maximum adsorption and other adsorption characteristics of the two compounds, indicated that: (i) in both cases the adsorbate may be held to the surface by concerted hydrogen bonding of the carboxylate and zirconyl oxygen atoms; and (ii) the hydrocarbon moieties in both adsorbates expose themselves toward the solution, thereby making the dried surface hydrophobic. The adsorptive behavior of the respective acids was similar to that of the salts. Polymer, filled with synthetic hydroxyapatite covered with irreversibly adsorbed I, had a diametral tensile strength about 50% greater than that of the polymer filled with untreated apatite. The strength of the composite was not affected by treatment of the apatite with II or with the acids. PMID- 3001162 TI - Pharmacological studies on the anti-inflammatory action of phenolic compounds. AB - The mechanism of the anti-inflammatory action of phenolic compounds was examined using neutrophil chemotaxis. Chemotactic activity of guinea pig peritoneal neutrophils to N-formylmethionyl-leucylphenylalanine (FMLP) was suppressed in a concentration-dependent manner. The order of drug potency in inhibiting the neutrophil chemotaxis was eugenol much greater than thymol greater than guaiacol much greater than phenol. The concentrations of phenolic compounds used in these experiments did not induce lactate dehydrogenase (LDH) release and did not affect neutrophil viability. There was a consistent positive relation between the ID50 of superoxide anion generation in neutrophils and the inhibitory dose for neutrophil chemotaxis by phenolic compounds. A free phenolic hydroxyl group is essential for scavenging oxygen free-radicals and is also essential for inhibiting leukocyte chemotaxis, as was demonstrated in these experiments. These findings suggest that inhibition of leukocyte chemotaxis may be involved in the anti-inflammatory action of phenolic compounds, and that one of the anti inflammatory actions of phenolic compounds is the prevention of the production of oxygen free-radicals by leukocytes. PMID- 3001163 TI - [Binding centers of adrenoreceptors. Topography of erythrocyte beta adrenoreceptors in the turkey]. PMID- 3001164 TI - Tritiated imipramine binding to platelets in manic subjects. AB - We studied 21 patients with bipolar affective disorder and 25 healthy controls in order to determine if tritiated imipramine binding to platelets distinguished the manic from the depressed phase of bipolar disorder. Depressed patients had a significantly lower mean Bmax value (754 +/- 149 fmole/mg protein) than the manic and control groups (1112 +/- 248 and 1237 +/- 201 fmole/mg protein, respectively), which did not differ from each other. These differences could not be attributed to differences in age, sex, menopausal status, the presence of psychotic features or medication history among the subject groups. These findings confirm that decreased imipramine binding to platelets is a state marker for bipolar depression and not a trait marker of bipolar disorder. PMID- 3001165 TI - Neuropeptides, behavior, and aging. PMID- 3001166 TI - Toxicity of aromatic disulphides. I. Generation of superoxide radical and hydrogen peroxide by aromatic disulphides in vitro. AB - The aromatic thiol, thiophenol, is readily autoxidized at neutral pH in a reaction which generates superoxide radical and hydrogen peroxide. The oxidation product, diphenyl disulphide, may be reduced back to thiophenol by glutathione and in the presence of an excess of the latter thiol a reduction/autoxidation cycle for generation of 'active oxygen' species is established. The autoxidation reaction is strongly catalysed by haematin; haemoglobin is also an effective mediator of 'active oxygen' generation from the diphenyl disulphide/glutathione couple, being oxidized to methaemoglobin in the process. Certain derivatives of diphenyl disulphide also generate superoxide radical and hydrogen peroxide in the presence of glutathione, although the rate of the reaction is strongly influenced by the nature of the substituent groups. Among the ring-substituted derivatives of diphenyl disulphide investigated, the rate of 'active oxygen' production decreased in the order 4-amino greater than 2-amino greater than 4-methyl greater than unsubstituted greater than 4-nitro greater than 2-carboxyl; little reaction was detected with the homologous compound, dibenzyl disulphide. PMID- 3001168 TI - The influence of gonadotropic hormones on the EGF receptor regulation in the rat ovary. AB - Ovaries of immature rats are endowed with only a small number of EGF binding sites (59.2 X 10(-15) mol/mg protein). During development from day 20 to 28, the binding capacity increases 2.5 times, while the growth of the ovaries remains small. Treatment with FSH on day 20 advances the EGF binding capacity with a 4 fold increase in the number of binding sites within the first two days. HCG has no or only a slight effect on the EGF binding capacity. The EGF receptor formation after FSH treatment takes place before the ovaries begin to grow rapidly. Accelerated ovarian growth is associated with a decrease of EGF binding capacity reaching values below those of the control animals during the subsequent days. The dissociation constants (KD) of the FSH stimulated receptors and of receptors present during development are found in the order of 2.7 and 8.7 X 10( 9) M. The physiological function of the EGF receptor for ovarian cell proliferation and follicular growth is discussed. PMID- 3001167 TI - Evidence for ectopic ACTH production years after bilateral adrenalectomy for Cushing's syndrome: in vivo and in vitro studies. AB - Three patients presented with hyperpigmentation and high plasma levels of ACTH 4 5 years after bilateral adrenalectomy for Cushing's "disease". X-rays and ct-scan of the lungs showed a small pulmonary mass in all. ACTH levels (1100-2000 pg/ml) were not suppressible by high doses hydrocortisone. A carcinoid tumor was removed in two cases and a chemodectoma in the third. Evidence for ACTH secretion by these tumors was provided by both decrease of ACTH levels after surgery and/or in vitro studies. Both carcinoid cultures showed a basal ACTH production, which was clearly increased by LVP (10(-7) M) and CRF (10(-7) M). Our findings in vitro studies suggest the presence of specific receptor sites for physiological stimuli (LVP and CRF) in tumor cell membranes. PMID- 3001169 TI - Sexual differentiation of prolactin responsiveness to thyrotropin releasing hormone (TRH) in the rat. Effects of postnatal testosterone on adenohypophyseal TRH receptor ontogenesis in male rats. AB - The influence of postnatal testosterone on thyrotropin releasing hormone (TRH) induced prolactin (PRL) response was tested in male rats. Under urethane anesthesia following estradiol pretreatment, neonatally castrated males showed a female-like plasma PRL response 4-fold greater than in adult-castrated males. Substitution of testosterone reversed the NC effect. However, postnatal age dependent difference was observed in the effect of testosterone. Testosterone produced a marked inhibition on PRL response when given at 2 or 4 weeks of life but not at earlier days. Determinations of adenohypophyseal PRL concentration and TRH receptor density revealed that testosterone inhibition occurred via its detrimental influence not on PRL concentration but on TRH receptor density. These results indicate that the sex-related difference in the rat PRL response to TRH ensues at least partially through inhibition by postnatal testosterone on the adenohypophyseal TRH receptor ontogenesis in male rats, and that there might be a functional dissociation in testosterone-dependent temporal development between two hypothalamic centers which independently regulate cyclic secretability and secretory reserve of PRL. PMID- 3001171 TI - [Obstetrical consequences of female circumcision. Study in 71 circumcised African women]. AB - The delivery of 71 African women who had been circumcised was studied against a control of 781 women who were not circumcised. We have not been able to find any difference as far as caesarean section, forceps delivery or fetal distress in the two groups that were studied. The only difference is a higher frequency of perineal injury in the group of circumcised women. PMID- 3001173 TI - Immunohistochemical localization of ACTH in the adult and fetal sheep pituitary. AB - Light microscope peroxidase-antiperoxidase immunohistochemistry has been applied to the pituitary of adult and fetal sheep from 40 to 145 days of gestation. In the adult, immunoreactive ACTH cells were darkly stained and angular with cytoplasmic processes surrounding neighbouring unstained cells. In the fetus, cells which stained for ACTH were observed in the pars distalis at 40 days. From approximately 90 days, two morphologically distinct ACTH-positive cell types were clearly discernible. The predominant type was large and variably stained. These cells usually occurred in clusters and were often arranged in palisades. The other type resembled ACTH-positive cells in the adult. After 130 days the population of large cells declined and completely disappeared before term in most fetuses. The pars intermedia showed a different pattern of staining. In the fetus, ACTH-positive cells were observed in this region after 60 days gestation and by 90 days almost all the pars intermedia cells were strongly stained. By contrast, the cells in the adult pars intermedia were only lightly stained. PMID- 3001172 TI - Changes in carnitine-palmitoyl-transferase and carnitine-acetyl-transferase activity in rat kidney during development; effects of fasting. AB - Developmental changes in the activities of two enzymes catalysing transfer of fatty acid across the mitochondrial membrane (carnitine-palmitoyl-transferase and carnitine-acetyl-transferase) were studied in the kidneys of developing rats from late fetal life to 10 days post-partum and were compared to cortical adult value. The activities of carnitine-palmitoyl-transferase and carnitine-acetyl transferase increased after birth to reach a maximal value on day 5. Thereafter both activities decreased to reach adult cortical value. The cytochrome c oxidase activity (index of mitochondrial activity) increases continuously from late fetal age to adult. In kidneys of fetuses from starved mothers the carnitine-palmitoyl transferase activity is higher than that of controls while carnitine-acetyl transferase activity is not changed. In postmature fetuses (23 days post-coitum) carnitine-palmitoyl-transferase activity is the same as in 21 days post-coitum old fetuses. These results are discussed in relation to variations in nutritional and hormonal changes occurring during the perinatal period. PMID- 3001175 TI - An unusual etiology of trigger finger: a case report. AB - A case of triggering of the right long finger presented in a patient with obvious recurrence of a cavernous hemangioma (unrelated to the triggering). A rare cause of triggering, a giant cell tumor of tendon sheath in the carpal canal, was found at operation. PMID- 3001174 TI - Primary malignant tumors of the hand. AB - Three cases of primary malignant tumors seen at the Hand Rehabilitation Clinics of the Lagos University Teaching Hospital from 1980 to 1983 are reported. The primary diagnoses were malignant synovioma, trabecular carcinoma of the sweat glands, and alveolar rhabdomyosarcoma. These three cases occurred in young Nigerian females and constituted 3.3% of 90 hand tumors treated at the clinic during this period. PMID- 3001176 TI - Congenital upper limb anomalies: an etiologic grouping of clinical, genetic, and epidemiologic data from 387 patients with "absence" defects, constriction bands, polydactylies, and syndactylies. PMID- 3001170 TI - Thyroid autoregulation. PMID- 3001177 TI - Detection of viral DNA and RNA by in situ hybridization. AB - Using cloned restriction endonuclease fragments of Herpes simplex virus (HSV), human papillomavirus (HPV), and cytomegalovirus (CMV) DNA as probes, viral DNA and RNA sequences have been detected in human tissues. The probes were labeled either with a radioactive isotope, for subsequent detection by autoradiography, or with biotin. This latter technique has been successfully used to visualize HPV DNA in tissues that have been fixed in formalin and embedded in paraffin, and is therefore of value in retrospective studies of histological specimens. HPV DNA was detected under non-stringent conditions (Tm = -42 degrees C) with heterologous probes in plantar and common warts, laryngeal papillomas, and anogenital condylomas. The specific type of HPV was established using stringent hybridization conditions (Tm = - 17 degrees C). Results from these and from malignant tissues show the distribution and localization of HSV and HPV RNA and DNA sequences in malignancies of squamous cell origin in the anogenital region. Both HSV and HPV DNA sequences have occasionally been detected in the same tumor, providing a further impetus to test the hypothesis that an initiator-promoter relationship might involve these common human viruses in the development of some tumors. PMID- 3001179 TI - Activation of cation transport by lymphokines in B cells without induction of DNA synthesis or immunoglobulin gene transcription. AB - We report on an experimental model that permitted us to evaluate the biologic relevance of membrane-associated biochemical events with respect to cell proliferation and maturation, each induced by distinct sets of signals. Antigen affinity-enriched murine B cells cultured in the presence of a proliferative signal induced by LPS showed activation of Na+/K+ ATPase and enhanced the uptake of proline, followed by RNA, protein, and DNA synthesis, without the generation of antibody. Stimulation with both the proliferative signal(s) and the maturation signal(s) derived from lymphokines of an EL-4 thymoma induced B cells to proliferate and synthesize mRNA encoding mu-chain of IgM and to mature into IgM secreting cells. Most important, the secretory product of EL-4, in the absence of LPS, activated Na+/K+ ATPase but failed to stimulate uptake of proline and synthesis of DNA or mu-specific mRNA. A similar response was observed in splenocytes depleted of T cells and in unfractionated spleen cells. Thus a component secreted by EL-4 can induce some of the early molecular events characteristic of the proliferative response but lacks the ability to initiate blast transformation and DNA synthesis. PMID- 3001178 TI - A transferrin receptor antibody represents one signal for the induction of IL 2 production by a human T cell line. AB - We previously demonstrated a two-signal requirement for the activation of the human T cell lines Jurkat and HUT 78. Interleukin 2 (IL 2) production by these lines can be induced by phytohemagglutinin (PHA), T3 antibodies, or calcium ionophores, but only in combination with phorbol myristate acetate (PMA). To obtain further information about surface structures involved in T cell activation, we produced a monoclonal antibody that could substitute for PMA in the activation of HUT 78. This antibody, designated J64, induced IL 2 secretion by HUT 78 in combination with PHA, T3 antibodies, or calcium ionophores, however not by itself. J64 also had other PMA-like effects on HUT 78, such as an increase in IL 2 receptor expression and an inhibition of cell growth. J64 was shown to immunoprecipitate the transferrin receptor (TfR). However, it bound to an epitope different from those recognized by other TfR antibodies and different from the transferrin-binding site. In addition, other previously described TfR antibodies did not, like J64, function as activating stimuli for HUT 78. Possible mechanisms for activation signaling in T cells involving the TfR are discussed. PMID- 3001180 TI - A phosphatidylinositol kinase in rat mast cell granules. AB - Intact granules were isolated from sonicated purified rat serosal mast cells on a Percoll gradient. The granules were shown to contain a highly active phosphatidylinositol kinase that catalyzes the formation of diphosphoinositide from endogenous phosphatidylinositol in the granule membrane. The enzyme requires ATP and Mg2+ or Mn2+ for activity; Ca2+, fluoride and cyclic AMP are inhibitory. The Km for ATP is 25 microM. The initial reaction is rapid, but the response ceases within a few minutes. A comparison of the rate of phosphorylation of intact and broken membrane granules suggests that the phosphorylation occurs on the outer (cytoplasmic) surface of the granules. PMID- 3001181 TI - Generation of leukotrienes by human monocytes pretreated with cytochalasin B and stimulated with formyl-methionyl-leucyl-phenylalanine. AB - The N-formylated tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) initiated the generation of immunoreactive C-6 sulfidopeptide leukotrienes and of leukotriene B4 (LTB4) in a dose-dependent manner from monolayers of human monocytes pretreated for 10 min with 5 micrograms/ml of cytochalasin B. The EC50 for the immunoreactive C-6 sulfidopeptide leukotrienes was 10(-8) M FMLP and for immunoreactive LTB4 was 5 X 10(-8) M FMLP. The maximal response to FMLP occurred within 10 min, and the sum of the two classes of leukotrienes generated was about 1/6 that obtained from monocytes stimulated with calcium ionophore A23187. The requirement for cytochalasin B in order for FMLP, but not the calcium ionophore, to stimulate leukotriene generation is compatible with the ability of cytochalasin B to augment in other cells certain stimulus-specific transmembrane responses that are not dependent on the integrity of the cytoskeleton. Resolution by reverse phase high performance liquid chromatography of the products released from monocytes pretreated with cytochalasin B and stimulated with FMLP or calcium ionophore yielded a single peak of immunoreactive LTB4 eluting at the same retention time as the synthetic standard; immunoreactive C-6 sulfidopeptide leukotrienes eluted at the retention times of leukotriene C4 (LTC4) and leukotriene D4 (LTD4). [3H]LTB4 was not metabolically altered by monocytes pretreated with cytochalasin B and activated with FMLP in comparison with cells treated with buffer alone, whereas [3H]LTC4 was partially converted to [3H]LTD4. The leukotriene-generating response of monolayers of human monocytes pretreated with cytochalasin B to FMLP is receptor-mediated, as indicated by the inactivity of the structural analog N-acetyl-methionyl-leucyl-phenylalanine and by the capacity of the FMLP receptor antagonist carbobenzoxyphenylalanyl-methionine to inhibit the agonist action of FMLP in a dose-response fashion. PMID- 3001182 TI - Targeting of drug loaded immunoliposomes to herpes simplex virus infected corneal cells: an effective means of inhibiting virus replication in vitro. AB - The goal of our studies was to develop liposomes containing antiviral drugs and targeted with antiviral antibody (immunoliposomes) that would be effective at inhibiting replication of herpes simplex virus (HSV) in vitro. To achieve this, a monoclonal antibody to glycoprotein D of HSV was derivatized with palmitic acid and was incorporated into the lamellae of dehydration-rehydration vesicles. The gD containing immunoliposomes were shown to bind specifically to HSV-infected rabbit corneal cells in vitro, whereas control immunoliposomes prepared with a monoclonal antibody of the same class as the anti-gD failed to preferentially bind to virus-infected cells. The gD immunoliposome binding was inhibitable by pretreatment with rabbit anti-HSV serum but not by aggregated normal serum. Thus liposome binding was judged to represent an antigen-antibody reaction not binding to Fc receptors expressed by cells infected with HSV. Immunoliposomes loaded with iododeoxyuridine (IUDR) leaked drug rapidly at 37 degrees C, whereas acyclovir (ACV)-loaded liposomes still contained 48% of drug after 24 hr at 37 degrees C. The ACV-liposomes retained 44% of drug after 14 days at 4 degrees C. The ability of immunoliposomes to inhibit virus replication was compared with that of untargeted and empty liposomes by means of virus yield assays in vitro, Immunoliposomes loaded with either IUDR or ACV inhibited virus replication, although ACV-containing immunoliposomes were the most efficacious. The implications of our in vitro results for the development of immunoliposomes suitable for the treatment of ocular herpes infection are briefly discussed. PMID- 3001183 TI - Neuroendocrine hormones suppress macrophage-mediated lysis of herpes simplex virus-infected cells. AB - Herpes simplex viruses (HSV) remain latent in sensory and peripheral ganglia and can be reactivated to cause recurrent HSV infections. Recent evidence has suggested that stress can induce an immunosuppressive state and increase the frequency and severity of recurrent herpes infections. Because macrophages play a central role in the host defense against HSV, the effects of stress-related neuroendocrine hormones on macrophage-HSV interactions were examined. Norepinephrine and epinephrine blocked the capacity of recombinant interferon gamma (IFN-gamma) to activate murine macrophages to a cytotoxic state capable of selectively killing HSV-infected cells. In contrast, ACTH, dopamine, serotonin, and beta-endorphin had no effect. The suppression of IFN-gamma-induced, macrophage-mediated lysis of HSV-infected cells occurred concomitantly with a marked increase in macrophage intracellular cyclic AMP levels. Moreover, exogenous administration of dibutyryl cyclic AMP blocked induction of macrophage mediated cytotoxicity, suggesting that the neurohormones were modulating macrophage function via an adrenergic receptor-mediated system. These findings demonstrate that selective stress-related neurohormones modify the cytolytic activity of macrophages against virus-infected cells and suggest a possible neuroendocrine-immunologic basis for the recurrence of HSV infection. PMID- 3001184 TI - Colchicine inhibits ionophore-induced formation of leukotriene B4 by human neutrophils: the role of microtubules. AB - Neutrophils which ingest particles (serum-treated zymosan, monosodium urate crystals) or are exposed to calcium ionophore A23187 generate leukotriene B4 (LTB4). Earlier work has shown that cells exposed to colchicine before exposure to monosodium urate crystals produce less LTB4; the formation of 5-HETE is unaffected. To determine whether inhibition by colchicine of LTB4 generation was stimulus-specific and was mediated by microtubule integrity, the effects of colchicine (10 microM, 60 min) on the release of lipoxygenase products from neutrophils exposed to ionophore A23187 (10 microM, 5 min) were examined. In the presence of exogenous arachidonic acid (100 microM, 15 min), colchicine decreased LTB4 to 48% +/- 11.7 of control and 5-HETE to 60.5% +/- 5.7 of control (mean +/- SEM); 15-HETE was also decreased to 61% +/- 10.3 of control. In the absence of exogenous arachidonate, LTB4 was decreased to 22.2% +/- 11.7 of control and 5 HETE to 13% +/- 4.8 of control. Lumicolchicine did not significantly affect formation of 5-HETE or LTB4. However, vinblastine sulfate (20 microM, 60 min), another microtubule-disruptive agent, decreased the formation of both 5 lipoxygenase products. The effects of colchicine and vinblastine were not due to impairment of cell viability because the release of cytoplasmic lactic dehydrogenase was unaffected. Ultrastructural analysis of centriolar microtubules showed that decrements in microtubule numbers of colchicine- and vinblastine treated cells paralleled decrements in 5-lipoxygenase products. These pharmacologic manipulations suggested that functional microtubules might be required for optimal lipoxygenase activity. Consequently, we prepared neutrophil derived cytoplasts, devoid of an intact microtubule system. No significant decreases in the 5- or 15-lipoxygenase products were found when cytoplasts were exposed to colchicine in the presence of exogenous arachidonate and A23187. The data show that colchicine inhibits the formation of lipoxygenase products from neutrophils stimulated with A23187, most likely via its effect on microtubules, the integrity of which appears necessary for full expression of 5- and 15 lipoxygenases. PMID- 3001185 TI - Lipid composition of alveolar macrophage plasma membrane during postnatal development. AB - This study was undertaken to define the age-related alterations in lipid composition that resident rabbit alveolar macrophages (AM) undergo during postnatal development. The eventual goal is to correlate these changes with the functional maturation of these cells. The number of AM recorded from total lung lavages rose markedly during the first 14 days of life, from 4.9 X 10(5) to 1.1 X 10(7). Adult lungs yielded 1.1 X 10(8) AM. A gradual but significant increase in fluorescence polarization (P) was observed during development when purified AM plasma membranes were tagged with the probe 1,6-diphenyl-1,3,5 hexatriene trimethyl ammonium. The rise ranged from a mean P value of less than or equal to 0.22 to 0.24 (p less than 0.001) for AM plasma membranes from rabbits 1- or 7-day old to 30- or 150-day-old rabbits, respectively. This finding suggests that the fluidity of the AM plasma membrane decreased during postnatal development. Palmitic, stearic, oleic, and linoleic acids were the most prevalent fatty acids found in the neutral lipid fraction of the AM plasma membrane throughout development. The content of stearic acid rose from 10 to 16%, arachidonic acid rose from 2.8 to 9%, myristic acid decreased from 3.2 to 1.3%, palmitic acid decreased from 42 to 36%, and oleic and linoleic acids changed relatively little during the first 30 days of life. The levels of docosatetraenoic and docosapentaenoic increased gradually during the first 14 days of life, and by 30 days of life the levels declined to that observed at birth. The sum of these changes resulted in an increase in the ratio of unsaturated to saturated fatty acids (1 to 1.15) in the neutral lipid fraction. During the first month of life, the neutral lipid fatty acid pool in the total lipid fraction of AM plasma membrane increased from 12 to 18 mole %, cholesterol increased from 7 to 14 mole %, and total phospholipids decreased from 81 to 67 mole %. These changes resulted in increasing the cholesterol to phospholipid ratio from 0.09 at birth to 0.23 by 150 days of life. The levels of all three major lipid fractions were comparable at 30 days and 150 days of life. Adult levels of choline phosphoglycerides, the predominant phospholipid, were observed by 7 days of life to have decreased from 47 to 34.5 mole %, and the levels of ethanolamine phosphoglycerides and sphingomyelin increased from 17.5 to 25 mole % and from 9 to 13 mole %, respectively. Adult levels of lyso-bis-phosphatidic acid were reached by 30 days of life increasing from 8.2 to 17.8 mole %.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3001186 TI - Immunity to herpes simplex virus type 2: viral antigen-presenting capacity of epidermal cells and its impairment by ultraviolet irradiation. AB - Ia+ epidermal cells (EC) had accessory cell function for herpes simplex virus type 2 (HSV-2)-induced T cell proliferation of immune lymph node cells (LNC). The EC-mediated virus-induced proliferative response of immune LNC was inhibited by ultraviolet B (UVB) irradiation. When EC were pulsed with viral antigen before UVB irradiation, the response was partially restored by addition of epidermal cell-derived thymocyte-activating factor (ETAF). Although normal EC secreted prostaglandin E2, the levels secreted by UVB-irradiated EC were significantly reduced, and the UVB-induced suppression of the proliferative response of immune LNC was not corrected by indomethacin. Soluble factor(s) that suppresses proliferation was generated in supernatants from cultures containing UVB irradiated but not nonirradiated EC. Sephadex chromatography revealed the presence of factors differentially modulating the proliferative response of HSV stimulated immune LNC and concanavalin A-stimulated normal lymphoid cells. PMID- 3001187 TI - Molecular mapping of murine I region recombinants: crossing over in the E beta gene. AB - The parental origin of genomic DNA from two independently derived murine I-region recombinants, B10.ASR7 [as3] and B10.BASR1 [as4], was determined by Southern blot hybridization by using DNA probes corresponding to A beta, A alpha, 5'-E beta, 3' E beta, and A alpha genes. New E beta gene probes were specifically constructed to make analysis of the E beta gene region definitive. Although the immune response phenotypes of the recombinants had suggested an I-A subregion cross over, a number of restriction fragment length polymorphisms distinguishing the k and the s haplotypes showed that both recombinations mapped within a 7-kb segment of the E beta gene. The validity of these results was tested by analysis of two other H-2k/s recombinants. One of them, B10.S(8R) [as1], mapped within the same 7 kb region of the E beta gene, whereas the other, B10.BASR2 [as5], mapped outside the I-region as expected. Including those studied here, there are a dozen I region recombinants whose cross-over positions have been determined at a molecular genetic level, and all of the cross-overs occurred within the E beta gene. PMID- 3001188 TI - Subpopulations of neutrophils with increased oxidative product formation in blood of patients with infection. AB - Stimulated human polymorphonuclear leukocytes (PMNL) have a marked increase in oxidative metabolism, producing reduced oxygen species (e.g., H2O2) that mediate bacterial killing. Previously, quantitation of metabolic responses of PMNL from patients with acute infections employed assays that measure mean activity of the entire PMNL population; such studies reported a modest and highly variable increase in oxidative metabolic responses of such "toxic" PMNL compared with normal cells. To assess metabolic capability of PMNL from 51 patients with acute bacterial infection, we employed a quantitative flow cytometric assay of H2O2 dependent oxidative product formation, the intracellular oxidation of 2',7' dichlorofluorescin (DCFH). After stimulation by phorbol myristate acetate, the PMNL of patients demonstrated an increase in mean DCFH oxidation (315 +/- 14 and 180 +/- 4.5 amol/cell, patients and controls). Hexose monophosphate shunt activation was similarly increased in stimulated PMNL from bacteremic patients. These data are comparable with previous studies of mean metabolic activities of toxic PMNL. However, these mean values underestimate the quantitative responses of the hyperresponsive ("primed") PMNL within a mixture of normal and primed PMNL in the patients' blood. The flow cytometric assay demonstrated that the PMNL of the patients were composed of two populations. One population of PMNL had normal oxidative responses; the other "primed" population had up to 4.6 times the oxidative product formation of normal cells. Similar priming of circulating PMNL was caused by infection with gram-positive or gram-negative staining bacteria or by Candida species. The proportion and oxidative ability of the primed PMNL occurred independently of the number of juvenile neutrophil forms and independently of "toxic" morphologic changes of Wright's-stained PMNL. On the average, 40% of the PMNL of patients were primed, but the size of the primed PMNL population varied widely between patients (range 0 to 80%). This variable subpopulation may explain the variability of mean responsiveness of the PMNL of patients reported previously. Moreover, the marked increase in oxidative metabolic capability of the primed PMNL may be a significant component of the host response to acute infection. It could also contribute to the damage to host tissues such as pulmonary vascular endothelium during bacteremia. PMID- 3001189 TI - Congenital macrodactyly. PMID- 3001190 TI - Prevalence of antibodies to Epstein-Barr virus and cytomegalovirus in sera from a group of children in the People's Republic of China. PMID- 3001191 TI - Seroepidemiology of varicella. PMID- 3001192 TI - The hepatitis B carrier state and the development of primary hepatocellular carcinoma. PMID- 3001194 TI - Detection of herpes simplex virus type 1-IgM immune complexes in the brain of a patient with prolonged herpes encephalitis. AB - A 56-year-old man with herpes encephalitis died 5.5 months after disease onset. Herpes simplex virus was isolated from minced temporal lobe after one month of cocultivation with green monkey kidney cells. The virus was identified as type 1 by neutralization and the fingerprinting pattern on restriction enzyme digestion. By immunofluorescence, IgM deposition was seen in the cerebral vascular walls. After dissociation of IgM with 3.0 M NaSCN, viral antigen was noted at the same loci as the IgM deposition. Histopathology showed marked perivascular cuffings, approximately 80% of which consisted of T cells and widely distributed necrotic foci. When the patient's serum and spinal fluid were analyzed by the immunoblotting method, the spinal fluid contained antibodies that were reactive with two very-high-molecular-weight viral polypeptides. PMID- 3001193 TI - Killing of human cytomegalovirus-infected fibroblasts by antiviral antibody and complement. AB - Complement-dependent cytolytic antibodies (CyAb) to cytomegalovirus (CMV) infected fibroblasts were detectable in acute- and convalescent-phase sera from renal allograft recipients (n = 44) and nonimmunocompromised patients (n = 14) with symptomatic CMV infection but not in sera from control donors (n = 75; P less than .001 by Wilcoxon rank sum test). Renal allograft recipients with secondary CMV infection had the highest levels of CyAb activity. Activity closely correlated with the serum antibody titer to CMV membrane antigens (r = .9106 by linear regression analysis) and was present in both the IgM and IgG fractions of human sera. IgG F(ab)2 fragments were inactive, thus implicating the classical pathway of complement activation. Maximal CMV-specific lysis was obtained with target cells expressing CMV late membrane antigens (greater than or equal to 72 hr after inoculation) irrespective of the CMV strain used. Adsorption and cold target inhibition studies indicated that the target antigens for the CyAb response are specific for the plasma membrane of CMV-infected cells and may only partly be shared by the virion envelope. PMID- 3001196 TI - Histoid leprosy with histoid leproma in peripheral nerves--a case report. PMID- 3001195 TI - Serum angiotensin-converting enzyme in leprosy. AB - Serum angiotensin-converting enzyme activity was measured in 91 adult healthy and lepromatous armadillos before inoculation with M. leprae and at necropsises. Mean ACE values were significantly elevated in armadillos with leprosy and the degree of elevation was roughly proportional to the extent of infection. There was also significant difference in the serum ACE levels between Florida and Louisiana armadillos. The dapsone treatment resulted in bringing these levels to normal. Serial assays of serum, ACE provided information on the response of armadillos to dapsone therapy. PMID- 3001197 TI - [Therapy of non-seminomatous germ cell tumors in the mediastinum]. PMID- 3001198 TI - [Immunohistochemical study on trophoblast subpopulations in normal pregnancy and trophoblastic disease]. AB - Trophoblast subpopulations were analyzed by their antigen expressions in normal pregnancy and trophoblastic disease. Samples were taken from uteri which were removed surgically. Frozen sections were stained by an avidin-biotin-peroxidase complex method, using monoclonal antibodies to HLA antigens and anti-Trop 1 antibody. Troma 1, a rat monoclonal antibody, was used for the identification of trophoblasts. The results were as follows: Troma 1 reacted with all trophoblasts specifically and could be used as a trophoblast marker in sections. HLA-A,B,C was negative on syncytiotrophoblast and villous cytotrophoblast but positive on nonvillous cytotrophoblast in normal pregnancy and hydatidiform mole. Some choriocarcinoma cells were positive for HLA-A,B,C and they were intermediate to large cytotrophoblast-like cells. No trophoblasts were positive for HLA-DR. Trop 1 was positive on villous cytotrophoblast in normal pregnancy and hydatidiform mole. Some choriocarcinoma cells were positive for Trop 1 and they were exclusively negative for HLA-A,B,C. From these immunohistochemical findings, trophoblasts of normal pregnancy and hydatidiform mole were divided into syncytiotrophoblast, villous cytotrophoblast, and nonvillous cytotrophoblast. Choriocarcinoma cells were classified into syncytiotrophoblast-like cells and three kinds of cytotrophoblast-like cells. They were analogous to those of normal pregnancy and hydatidiform mole to some extent, but a choriocarcinoma-specific subpopulation which was negative for HLA-A,B,C, HLA-DR, and Trop 1 was recognized. PMID- 3001199 TI - [Study on the direct inhibitory action of luteinizing hormone-releasing hormone on the steroidogenesis of cultured human corpus luteum cells]. AB - To evaluate the direct inhibitory action of luteinizing hormone-releasing hormone (LH-RH) on the steroidogenesis of the human ovary, the primary cultured human corpus luteum cells were investigated. The following were the effects of the addition of LH-RH: Estradiol (E2) and progesterone were produced and secreted in the cultured corpus luteum cells. In the cytoplasm of the cultured corpus luteum cells, E2 and P-antibody complexes were observed as fine granules by the immunohistochemical staining method. The progesterone production of these cells was not inhibited in the cells cultured with LH-RH 10(-8) Mol alone. The progesterone production of the cells was stimulated in the cells, cultured with gonadotropins (LH, HCG and HMG). The gonadotropin stimulated progesterone production was inhibited by LH-RH administration in the cells. In the short term incubation of the human corpus luteum cell suspension, the cyclic adenosine monophosphate (c-AMP) accumulation of the cells incubated with LH-RH alone did not change, but the gonadotropin-stimulated c-AMP accumulation of the corpus luteum cells was significantly inhibited by LH-RH. Concerning these results, it is concluded that LH-RH inhibits the gonadotropin stimulated progesterone production directly in vitro. It is suggested that the mechanisms of these inhibitory actions of the LH-RH are related to the gonadotropin receptor-adenyl cyclase systems, c-AMP metabolizing enzyme and/or progesterone metabolizing enzyme. PMID- 3001200 TI - Hypolipidemic effects of catecholestrogen and 2-methoxy-catecholestrogen. PMID- 3001201 TI - [Clinical significances of tissue polypeptide antigen in patients with gynecological malignancies]. AB - Recently, tissue polypeptide antigen (TPA) has become of general interest as one of the new tumor-related antigens. In this study, TPA was measured mainly in patients with various gynecological malignancies by radioimmunoassay, and serum TPA levels above 110u/l were considered pathological. The serum TPA level in normal women was 66 +/- 21 u/l (mean +/- S.D.) and the positive rate was 0 percent. Serum TPA levels in patients with cervical cancer, endometrial cancer, ovarian cancer and uterine sarcoma (181 +/- 262, 117 +/- 49, 217 +/- 215, 142 +/- 49u/l) and positive rates (55, 40, 53, 75 percent) were significantly higher than those in normal women. Serum TPA levels in patients with advanced or recurrent diseases had a tendency to show highly positive. In all of 13 patients with positive TPA, serum TPA levels were significantly reduced and turned negative after the appropriate treatments. However, among our follow-up patients, serum TPA levels were often positive even when there was no clinical evidence of recurrence. Conclusion; serum TPA measurement could be a useful subordinate tumor marker in many patients with gynecological malignancies, even though careful judgement is necessary to interpret positive TPA. PMID- 3001202 TI - [Clinical evaluation of ferromagnetic microembolization in the treatment of hepatoma]. PMID- 3001203 TI - [A treatment-planning procedure for proton beam therapy]. PMID- 3001204 TI - [Effect of intra-arterial injection of a lipiodol-adriamycin suspension combined with arterial embolization on hepatocellular carcinoma]. PMID- 3001205 TI - [A case report of isolated inappropriate secretion of adrenocorticotropic hormone (ACTH) associated with primary aldosteronism]. PMID- 3001206 TI - [Adult T-cell leukemia/lymphoma]. PMID- 3001207 TI - [A case of mucin-producing adenocarcinoma of the thyroid presented with cardiac tamponade as an initial manifestation]. PMID- 3001209 TI - Effect of temperature on production of hypochlorous acid by stimulated human neutrophils. AB - Under natural conditions or because of therapy with heat or cold, neutrophils may function at times in the human body at temperatures other than 37 degrees C. Therefore, we evaluated the effects of temperature on several functions of these cells. Phagocytosis and superoxide production by stimulated neutrophils were optimal at 37 degrees C and attained at least 70% of this peak value at 42 degrees C. In contrast, production of hypochlorous acid (as measured by an assay using the chlorination of taurine) by stimulated neutrophils was optimal at temperatures less than 37 degrees C and attained only 13% to 15% of this peak value at 42 degrees C. During a 2-hour incubation, the major suppressive effects of the higher temperature occurred during the second hour. This result was not explainable by factors related to the hypochlorous acid assay system or by loss of cell viability or myeloperoxidase activity in the cell supernatants, but rather appeared to be caused by reduced generation of hydrogen peroxide at the higher temperatures. Because the extracellular release of a strong oxidant such as hypochlorous acid might result in significant tissue injury, suppression of the release of this oxidant by elevated temperatures may explain why the application of local heat sometimes benefits certain inflammatory conditions. PMID- 3001208 TI - [A case of isolated adrenocorticotropic hormone (ACTH) deficiency with aseptic necrosis of femoral head complicating the long-term corticosteroid replacement therapy]. PMID- 3001210 TI - Simple radioimmunoassay for transferrin using insolubilized antitransferrin antibodies: its application to cultured cells. AB - A radioimmunoassay (RIA) for transferrin is described. The assay uses antitransferrin antibodies covalently coupled to the particulate support Matrex Pel 102, and is simple, sensitive, reproducible, and rapid. Transferrin measurement with this assay is independent of the degree of iron saturation of the protein. The RIA was applied to the measurement of transferrin concentrations in a variety of cultured human cells. Each of 11 cell lines studied contained endogenous transferrin, but the greatest concentration was found in Chang liver cells. Red blood cells were used as a negative control. PMID- 3001211 TI - Relation to chemotactic factor gradients to neutrophil migration and orientation under agarose. AB - Chemotactic substances confer a migratory pattern for neutrophil granulocytes under agarose that is characteristic for each agent. To analyse the cause of such differences, we have studied neutrophil migration and orientation with f-Met-Leu Phe (fMLP), leukotriene B4 (LTB4), and serum as chemoattractants. When these agents were used at optimal concentrations, it was observed that cells stimulated by LTB4 did not start migration as fast and did not migrate as far as those exposed to fMLP, but they maintained a higher degree of orientation. This delay in initiation of migration and maximal degree of orientation was even more marked when serum was the chemoattractant. These migration variables were related to the generation of gradients in the agarose of fML[3H]P, arachidonic-[3H]acid (AA, of which LTB4 is a metabolite), and fluorescein. The curvilinear AA gradient was flatter and more stable than those of fML[3H]P and fluorescein, which were linear. Thus, differences in the development and shape of the gradient of chemoattractant may contribute to differences in migration kinetics. PMID- 3001213 TI - Bovine alveolar macrophages: phenotypic and functional properties of subpopulations obtained by Percoll density gradient centrifugation. AB - Bronchoalveolar cells retrieved from conventionally raised, healthy calves were separated into four fractions on a discontinuous Percoll density gradient. The alveolar macrophage (AM) subpopulations and nonseparated AM were assayed for such phenotypic markers as la-antigen, ectoenzymes, and immune receptors, as well as for functional activity in antibody-dependent cellular cytotoxicity (ADCC) against virus-infected cells, superoxide anion generation, and their influence on lectin-induced lymphocyte proliferation. The low-density fraction was composed of large cells with low Ia-antigen expression, low ADCC activity, and high ecto enzyme and C3b-receptor activity. In contrast the high-density fraction contained mainly small, monocytelike cells, with high Ia expression and low-level expression of most other markers and functions. Two fractions of intermediary density overlapped in most of the characteristics, but could be distinguished on the basis of ADCC activity, interleukin-1 generation, and the level of leucine amino peptidase activity. PMID- 3001212 TI - Release of interleukin-1 (IL-1) and IL-1-like factors from rabbit macrophages with silica. AB - Oil-elicited rabbit macrophages stimulated by endotoxin were found to release increased amounts of interleukin 1 (IL-1) when incubated with silica. The assays used to determine the amount of IL-1 released were uptake of thymidine by mouse thymocytes, fever in rabbits, and neutrophilia in rats. All three assays showed that endotoxin-stimulated macrophages released five to 10 times more IL-1 when incubated with silica. Most of the IL-1 had a molecular weight (MW) of about 14,000 with a smaller amount at a MW of approximately 35,000. All of the neutrophilia-producing activity had an isoelectric point (pl) near 7. Thymocyte proliferation was promoted about equally by activities near pH 5 and 7. Fever was found not only at these two isoelectric points but also at a pl above 8 which had neither of the other two activities. PMID- 3001214 TI - Japanese encephalitis in Thailand. PMID- 3001216 TI - Hypothalamo-pituitary-adrenal axis in iatrogenic Cushing's syndrome. PMID- 3001215 TI - Macrophage plasma membrane and secretory properties in murine malaria. Effects of Plasmodium yoelii blood-stage infection on macrophages in liver, spleen, and blood. AB - We have studied the effect of infection with the blood-stage of Plasmodium yoelii 17X, a nonlethal parasite, on plasma membrane antigens, receptors, and secretory properties of macrophages (M phi) in murine liver, spleen, and blood. mAb F4/80 (M phi specific), F7/4 (a marker for immature and immunologically activated M phi, as well as neutrophils), and Mac-1, which binds to the type 3 complement receptor, were used to measure the distribution and total content of antigens in situ and to assay surface expression of antigens on M phi isolated by collagenase perfusion-digestion and adherence. We also examined respiratory burst activity after stimulation with PMA, FcR activity, Ia antigen expression, and binding of 125I-mannose-BSA and unopsonized sheep erythrocytes by isolated M phi. In the normal animal, spleen M phi expressed Mac-1 and F7/4 antigens and relatively high levels of respiratory burst activity, in contrast to Kupffer cells in liver, where all three features were virtually absent. The introduction of parasitized erythrocytes into the circulation resulted in a large influx of F4/80+ M phi into the blood, liver, and spleen, where local M phi proliferation could also contribute. Liver M phi during malaria infection showed increased Mac-1 and 7/4 antigen and an increased respiratory burst potential compared with uninfected controls. Increases in total, but not specific activity of FcR, Ia antigen, and binding of unopsonized sheep erythrocytes were found in spleen and liver M phi populations after infection. In both populations, there was an early but persistent marked reduction in specific binding and uptake of 125I-mannose-BSA. These results confirm and extend observations that normal Kupffer cells are relatively homogeneous in morphology, surface markers, and anatomical location, in contrast to M phi in normal spleen, and that both of these populations differ from resident M phi elsewhere, including the peritoneal cavity. In the course of infection by P. yoelii, M phi with high levels of opsonic receptors (CR3, FcR) and respiratory burst potential are mobilized in large numbers at specific sites such as liver and spleen, in accordance with an important role for M phi in the clearance of parasitized erythrocytes from blood. PMID- 3001217 TI - Basolateral Na-H exchange in the rabbit cortical collecting tubule. AB - We used the intracellular absorbance spectrum of the dye 4',5'-dimethyl-5- (and 6-) carboxyfluorescein (Me2CF) to measure intracellular pH (pHi) in the isolated, perfused cortical collecting tubule (CCT) of the rabbit nephron. The incident spot of light was generally 10 micron in diameter, large enough to illuminate from two to six cells. No attempt was made to distinguish principal from intercalated cells. All experiments were carried out in HCO3- -free Ringer to minimize HCO3- transport. When cells were acid-loaded by briefly exposing them to Ringer containing NH+4 and then withdrawing the NH+4, pHi spontaneously recovered from the acid load. The pHi recovery was best fit by the sum of two exponentials. When the acid loading was performed in the absence of Na+, the more rapid of the two phases of pHi recovery was absent. The remaining slow phase never returned pHi to normal and was sometimes absent. Returning Na+ to the lumen had only a slight effect on the pHi recovery. However, when Na+ was returned to the basolateral (i.e., blood-side) solution, pHi recovered rapidly and completely. The apparent Km for basolateral Na+ was 27.3 +/- 4.5 mM. The basolateral Na dependent pHi recovery was reversibly inhibited by amiloride. We conclude that the mechanism responsible for the rapid phase of pHi recovery is an Na-H exchanger confined primarily, if not exclusively, to the basolateral membrane of the CCT. PMID- 3001218 TI - Excitation of skinned muscle fibers by imposed ion gradients. I. Stimulation of 45Ca efflux at constant [K][Cl] product. AB - 45Ca efflux from skinned muscle fibers is stimulated transiently, by a highly Ca2+-dependent mechanism, by KCl replacement of K propionate. In the present studies, Cl replaced the much less permeant anion methanesulfonate (Mes) either (a) at constant [K], in which increased [K][Cl] permits net KCl and water flux across internal membranes, or (b) at constant [K][Cl] (choline substitution), in which the imposed gradients and diffusion potentials should dissipate slowly. 45Ca efflux and isometric force were measured simultaneously on segments of frog semitendinosus fibers skinned by microdissection. EGTA was applied to chelate released 45Ca either (a) shortly after high [Cl] (interrupted response), to minimize reaccumulation, (b) before high [Cl] (pretreated response), to evaluate Ca2+ dependence, or (c) under control conditions in KMes. KCl replacement of KMes stimulated release of 65% fiber 45Ca within 1 min in interrupted responses; EGTA pretreatment was only moderately inhibitory with substantial residual stimulation. In contrast, choline Cl replacement of KMes induced release of 26 35% fiber 45Ca in interrupted responses; EGTA pretreatment was strongly inhibitory, but release significantly exceeded control with a small, sustained increase in Ca2+-insensitive efflux. These differences in 45Ca release and EGTA inhibition suggest that Cl replacement of Mes at constant [K] stimulates efflux by osmotic effects as well as imposed diffusion potentials; at least half the stimulated 45Ca loss (above control) in interrupted KCl responses is attributable to an osmotic component with low Ca2+ sensitivity. In the highly Ca2+-sensitive stimulation at constant [K][Cl], 45Ca release (above control) in interrupted responses correlated well with that in the pretreated responses of segments from the same fiber, with a slope of 8.4. This relationship suggests that imposed diffusion potentials stimulate a small Ca2+-insensitive component that gradates a much larger Ca2+-dependent efflux. The Ca2+-insensitive component apparently reflects intermediate steps in the excitation-contraction coupling that require positive feedback to result in sufficient Ca release for contraction. PMID- 3001219 TI - Changing seroepidemiology of hepatitis A virus infection in Taiwan. AB - Hepatitis A antibody (anti-HAV) in serum was studied from June to October, 1984, by radioimmunoassay in 647 male and 553 female apparently healthy children under 15 years of age in Taipei City. The prevalence rate of anti-HAV was 27.0% in infants, decreased to around 1% during the preschool age, then increased and remained around 5% until 11-12 years of age, when another increase was noted, and reached 13.6% among the early teenagers. The age-specific prevalence of anti-HAV increased with age but differed in three age ranges, which reflected three apparently different calculated annual incidences. Compared with previous studies in Taipei, the results showed a significant reduction in the prevalence of anti HAV in almost every age group from 3 to 14 years. This fact probably reflects the marked improvement of hygienic conditions and progress in health education in recent years, which reduced the exposure to HAV infection among young children. The age of primary infection in the children was older than in previous studies, and it is expected that the susceptibility of HAV will extend to early adulthood. PMID- 3001220 TI - Human immune response to cytomegalovirus structural polypeptides studied by immunoblotting. AB - To define better the human immune response to individual structural proteins of human cytomegalovirus (HCMV), 55 human sera with different IgG and IgM titres were studied for their reactivity with HCMV structural polypeptides separated by SDS-PAGE and electrotransferred to nitrocellulose paper. The results obtained showed that antibody titres detected by immunoassay correlate with the intensity and the number of polypeptides reacting by immunoblotting (IB). The IB profiles of HCMV polypeptides reacting with different sera having the same antibody titres show considerable variation. Sera with high levels of IgG antibody and that are IgM-positive frequently react with 155, 149, 82.5, 74.5, 67, 57, 55, 38.5, and 28 kD polypeptides; all these sera react with 155, 67, 57, 55, 38.5 kD polypeptides. Sera with high levels of IgG antibody but that are IgM negative frequently react with all these polypeptides, with the exception of 149 and 74.5. Only 155 and 28 kD polypeptides were recognized by all sera of this group. The sera with moderate levels of IgG antibody preferentially recognize 155, 110, 82.5, 62, 55, 38.5 and 28 kD polypeptides. The sera with low levels of antibody reacted especially with 155 and 62 kD polypeptides. IgM antibody seems to recognize preferentially 155, 110, 67, 57, 55, 38.5 kD polypeptides. PMID- 3001221 TI - Identification of genital tract papillomaviruses HPV-6 and HPV-16 in warts of the oral cavity. AB - Warty lesions of the oral cavity were examined for etiologic association with genital tract papillomaviruses HPV-6, HPV-11, and HPV-16. DNAs extracted from ten oral biopsies were screened for HPV genomic sequences by Southern transfer hybridization with 32P-labeled viral DNA probes. Nonstringent hybridization with an HPV-6 probe revealed papillomavirus DNA sequences in four of seven tissues with histologic evidence of papillomatosis, in none of two tissues without histologic evidence of papillomatosis, and in one tissue that was not examined by histology. Stringent hybridization tests with HPV-6 and HPV-16 probes identified the genome in one tissue as being HPV-16, in a second tissue as being HPV-6 subtype a, and in a third tissue as HPV-6 (subtype unidentified); papillomavirus DNA sequences in two tissues are as yet not identified. An additional case of HPV 6 or HPV-11 related oral cavity lesion was diagnosed by in situ hybridization of paraffin sections with a 35S-labeled, mixed HPV-6 + HPV-11 probe. The hybridization in the positive section was extensive and confined to epithelial nuclei. The oral lesions associated with genital tract papillomaviruses were asymptomatic, multiple or single, and were located in different parts of the oral cavity, for example, on the gingivae, on the tongue, on the lip, on the tonsillar pillar, and on the floor of the mouth. PMID- 3001223 TI - A survey of respiratory syncytial virus and parainfluenza virus type 3 neutralising and immunoprecipitating antibodies in relation to Paget disease. AB - The aetiology of Paget disease of bone has not been established but certain features have suggested involvement of a parainfluenzalike virus. To seek further evidence of the possible role of paramyxoviruses in Paget disease we have surveyed the presence of neutralising and immunoprecipitating antibodies to both respiratory syncytial virus and parainfluenza virus type 3 in the sera of patients attending a bone disease clinic. These two viruses were implicated by the sporadic observation of viral antigen in individual nuclei of osteoclasts in Paget disease bone lesions. A total of 315 samples were obtained from 177 patients attending the clinic during 1 year. Thirty-six of the patients had confirmed Paget disease and the remainder other conditions. All sera possessed neutralising activity to both viruses. The mean titres for each virus were similar in patients with Paget disease and those with other conditions whether matched or not. In the case of respiratory syncytial virus the neutralising titres were distributed closer to the mean in the Paget group and showed little variation in repeat samples taken over periods of up to 1 year in contrast to the greater variability of the control group. The antigenic specificity of 20 age- and sex-matched sera from each group was examined by immunoprecipitation. No significant differences were observed between Paget and non-Paget patients. These results do not provide confirmation of involvement of either virus in Paget disease, but the serological data suggest that persistent infection with respiratory syncytial virus can occur. PMID- 3001222 TI - Cellular immune responses in mice challenged with an amyocarditic variant of Coxsackievirus B3. AB - An amyocarditic variant of a temperature-sensitive (ts) mutant derived from the parent myocarditic variant Coxsackievirus B3 (CVB3m) was studied in a murine model of CVB3m-induced myocarditis to assess virus-induced antigens and their possible role in the disease process. Amyocarditic variant ts5R induced a heart tissue antigen(s), extractable by hypertonic KC1, which inhibited migration of peritoneal exudate cells from CVB3-inoculated myocarditic mice in an agarose droplet cell-migration-inhibition assay. The ts5R variant was amyocarditic at inoculum doses of 10(3) to 10(8) plaque-forming units per mouse, but in cyclophosphamide-immunosuppressed mice, ts5R induced myocarditis. Viable ts5R served as a vaccine and protected mice against CVB3m-induced myocarditis. Murine neonatal skin fibroblasts (MNSF) infected with either virus served as in vitro targets and were lysed by splenic cytotoxic T lymphocytes from mice inoculated with either virus variant. ts5R and CVB3m replicated to similar titers in murine neonatal skin fibroblasts (MNSF) at 24 hr postinoculation (pi), but differences in titers were found by 72 hr pi. Levels of natural killer cell activities in spleens of ts5R-inoculated mice were slightly lower than in spleens of CVB3m inoculated mice at 7 days pi. The data suggest that viral induction of new antigens on target cells and viral induction of specific cytotoxic T lymphocytes that recognize these antigenic changes do not always result in induction of myocarditis. PMID- 3001224 TI - Apparent absence of a translocase in the cerebral glucose-6-phosphatase system. AB - In the hepatocyte endoplasmic reticulum, a substrate transporter could provide a means of regulating hydrolysis of glucose-6-phosphate by specifically modulating access of the substrate to the hydrolase. Several characteristics of the cerebral microsomal enzyme suggest that such an hypothesis is untenable in the brain. These are: (a) the inability of the enzyme in either untreated or detergent disrupted brain microsomes to distinguish between glucose-6-phosphate and mannose 6-phosphate; (b) the close agreement of the apparent Km values for either substrate in intact or disrupted microsomal preparations; (c) the constancy of the latency toward both substrates over a wide concentration range; (d) the inability of nonpenetrating, covalently-linking reagents [e.g., 4,4' diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)] to affect the accessibility of the hydrolase to its substrate; (e) the absence of a putative transporter polypeptide, such as that of the liver, in experiments where tritiated H2DIDS, polyacrylamide gel electrophoresis, and radioautography are applied to brain microsomes. PMID- 3001225 TI - Oxygen free-radical reduction of brain capillary rubidium uptake. AB - Free radicals are proposed to play a role in the injury following cerebral ischemia in which cerebral edema is a prominent feature. To determine whether free radicals might alter the movement of ions and water across the blood-brain barrier, we examined their effect on brain capillary transport. Rat brain capillaries were isolated, incubated with a system that generates free radicals, and various capillary transport systems were studied. Rubidium uptake was reduced 74% whereas rubidium efflux, glucose transport, and capillary water space were unchanged. The results following the addition of radical scavengers indicated that hydrogen peroxide or a related free radical was the toxic species. These data suggest that free radicals can impair capillary endothelial cell mechanisms that help maintain homeostasis of electrolytes and water in brain. PMID- 3001226 TI - Relation of cellular phospholipid composition to oligodendroglial differentiation in C-6 glial cells. AB - The relation of the polar head group composition of cellular phospholipids to a biochemical expression of oligodendroglial differentiation was studied in cultured C-6 glial cells. Induction of the oligodendroglial enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), was determined after alteration of the polar head group composition of phospholipids by exposure of the cells to choline analogues, especially N,N'-dimethylethanolamine. To accomplish the phospholipid alteration, cells were grown in the presence of the analogue in medium free of exogenous lipid, i.e., first for 24 h in 10% delipidated serum and then for 48 h in serum-free medium. The 48-h exposure to serum-free medium resulted in untreated C-6 cells in a several fold increase in CNP activity, but in cells treated with 2.5 mM N,N'-dimethylethanolamine, total inhibition of this induction was observed. A graded, concentration-dependent inhibitory effect of the analogue on the induction of CNP was defined. The effect of the analogue was relatively specific, e.g., the activity of another plasma membrane enzyme of C-6 cells, (Na+ + K+)-activated ATPase, was not affected. Morever, there was no evidence of a toxic effect of the analogue; thus, total protein synthesis and cell growth were not altered, and the induction of CNP in serum-free medium recurred after removal of the analogue. N,N'-Dimethylethanolamine was shown to be incorporated into cellular phospholipids, primarily at the expense of phosphatidylcholine. The data define an important role for the polar head group composition of membrane phospholipids in oligodendroglial differentiation in this model system. PMID- 3001227 TI - H2 histamine receptors on the epithelial cells of choroid plexus. AB - A major site of cerebrospinal fluid production in vertebrates is the choroid plexus. The epithelial cells of the choroid plexus accumulate intracellular cyclic AMP in response to several effectors, including histamine. Since histamine is known to regulate fluid secretion in the stomach via H2 histamine receptors, we asked whether H2 receptors might also be present on epithelial cells of bovine choroid plexus. Using agonists and antagonists of histamine, we show that an agonist and antagonist pair specific for the H2 subtype were clearly more effective than an H1 agonist and antagonist pair in mimicking or inhibiting histamine stimulation of cellular cyclic AMP. Analysis by Schild plot allowed assignment of an apparent dissociation constant to the H2 antagonist metiamide which was 34-fold lower than that of its H1 counterpart, diphenhydramine. These results indicate that epithelial cells of the choroid plexus possess H2 histamine receptors. PMID- 3001228 TI - Purification and characterization of a soluble cyclic nucleotide-independent Ca2+ calmodulin-sensitive protein kinase from rat brain. AB - Following partial purification, the characteristics of a cytosol protein kinase were investigated. The protein kinase was purified by ammonium sulfate precipitation and diethylaminoethyl-cellulose, ATP-agarose, and hydroxyapatite chromatography. Analysis of the purified protein kinase preparation by polyacrylamide gel electrophoresis revealed three major protein bands. The cytosol protein kinase was purified approximately 442-fold, as calculated from the cyclic nucleotide independent protein kinase activity in the 40,000 g supernatant. The activity of the kinase was found to be independent of either cyclic AMP or cyclic GMP. Moreover, the kinase activity was unaffected by the addition of the endogenous protein kinase inhibitor, or the regulatory subunit from the type II cyclic AMP-dependent protein kinase from bovine heart. The molecular weight of the enzyme was determined to be 95,000 by Sephadex G-200 gel filtration. The activity of the kinase was increased approximately twofold in the presence of 10 microM Ca+2 and calmodulin. This increase was reversed by the addition of EGTA. The subcellular distribution of the protein kinase was also examined. The soluble fraction from nerve terminal was found to have the highest concentration of the kinase activity. PMID- 3001229 TI - Differential effect of denervation on free-radical scavenging enzymes in slow and fast muscle of rat. AB - To determine the effect of denervation on the free-radical scavenging systems in relation to the mitochondrial oxidative metabolism in the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles, the sciatic nerve of the rat was crushed in the midthigh region and the muscle tissue levels of five enzymes were studied 2 and 5 weeks following crush. Recently developed radioimmunoassays were utilized for the selective measurement of cuprozinc (cytosolic) and mangano (mitochondrial) superoxide dismutases. Total tissue content of cuprozinc superoxide dismutase showed a mild decrease after denervation in slow but not in fast muscle. Manganosuperoxide dismutase and fumarase decreased markedly at 2 weeks and returned toward control levels by 5 weeks, the changes appearing to be greater in slow than in fast muscle. At 2 weeks, cytochrome c oxidase decreased significantly in slow, but not in fast muscle. GSH-peroxidase at baseline was 10 fold higher in slow than in fast muscle, markedly decreased at 2 weeks in slow muscle, and returned toward control levels at 5 weeks, whereas the total enzyme activity in fast muscle did not change through 5 weeks. These data represent the first systematic report of free radical scavenging systems in slow and fast muscles in response to denervation. Selective modification of cuprozinc and manganosuperoxide dismutases and differential regulation of GSH-peroxidase was demonstrated in slow and fast muscle. PMID- 3001231 TI - Arginine-vasopressin stimulates inositol phospholipid metabolism in rat hippocampus. AB - The hippocampal vasopressin receptors have been characterised by measuring the stimulated accumulation of inositol monophosphate in the presence of 10 mM LiCl after hippocampal slices were prelabelled with [3H]inositol. Arginine-vasopressin caused a dose-dependent increase in inositol monophosphate accumulation (ED50 = 7.1 nM). The response was unchanged in the absence of Ca2+ and significantly reduced in the presence of a V1-receptor antagonist. Equimolar oxytocin was ineffective as a stimulus. This suggests that the hippocampal receptors are of the V1 type. PMID- 3001230 TI - Receptor binding activities of biotinylated derivatives of beta-nerve growth factor. AB - beta-nerve growth factor (NGF) was modified by biotinylation via carboxyl group substitution (C-bio-NGF) using biotin hydrazide and the coupling reagent 1-ethyl 3-(3-dimethylaminopropyl)-carbodiimide, under reaction conditions that yielded an average of 3 biotin additions per NGF subunit. NGF was also biotinylated through amino group substitution, using N-hydroxysuccinimidyl biotin, to produce derivatives with ratios of one, two, and four biotin moieties per NGF subunit (N bio-NGF). The various biotinylated NGF derivatives were compared with native NGF for their capacity to compete with 125I-NGF for binding to NGF receptors on rat pheochromocytoma (PC12) cells at 4 degrees C. On the basis of such radioreceptor assays, C-bio-NGF was as effective as native NGF in binding to NGF receptors. C bio-NGF was also as effective as native NGF in promoting neurite outgrowth from PC12 cells. In contrast, N-bio-NGF containing one biotin per NGF subunit was only 28% as active in binding as native NGF. Increasing the biotin:NGF ratio to 2 to 4 further decreased receptor binding to 13% and 6%, respectively, as compared to native NGF. Once bound to cells, C-bio-NGF had the capacity to mediate the specific binding of 125I-streptavidin to PC12 cells. This binding of streptavidin was prevented by excess native NGF and by antiserum to NGF, but not by RNase A, insulin, cytochrome c, or nonimmune serum. In addition, a variant PC12 line lacking functional NGF receptors was not labeled by 125I-streptavidin after prior incubation with C-bio-NGF. PMID- 3001233 TI - Effect of naltrexone on senile dementia of the Alzheimer type. AB - Some reports have suggested that naloxone, a short-acting opiate receptor blocker given intravenously, has a beneficial effect on the symptoms of senile dementia of the Alzheimer type. We have performed a double-blind, crossover trial of naltrexone, an orally active, long acting opiate antagonist, in 17 Alzheimer-type dementia patients. None showed any improvement in assessments of day-to-day living skills or on a battery of neuropsychological tests. No side effects were noted. In the dosage used, naltrexone appears not to be useful in Alzheimer-type dementia. PMID- 3001232 TI - Verapamil-induced changes in central conduction in patients with multiple sclerosis. AB - The electrophysiological characteristics of demyelinated axons are sensitive to changes in plasma calcium concentration. This study investigated the effect of verapamil, a calcium antagonist drug, on brainstem auditory, visual, and somatosensory evoked potentials in multiple sclerosis patients. Eight clinically stable patients with abnormal visual and/or brainstem auditory evoked potentials and four normal volunteers were studied. During intravenous infusions of verapamil (mean plasma concentration = 130.0 +/- 56.4 ng/ml), the latencies of peaks III and V were shortened (p less than 0.05) in multiple sclerosis patients with abnormally prolonged BAEPs. The I-III (delta = 0.08 ms), III-V (delta = 0.46 ms), and I-V (delta = 0.53 ms) interpeak intervals, and the P100 latency (delta = 10.15 ms) of the visual evoked potential were similarly affected in these patients. In contrast, normal evoked potentials of both multiple sclerosis patients and control subjects were not altered compared to baseline recordings obtained 24 hours earlier. Intravenous verapamil, therefore, alters the BAEPs and VEPs of some multiple sclerosis patients with demyelinated auditory and visual pathways by shortening pathologically prolonged latencies toward normal. The present study suggests pharmacological manipulation of calcium-dependent processes, possibly at the level of the demyelinated axon, can acutely facilitate central conduction of electrical impulses in some patients with clinically stable multiple sclerosis. PMID- 3001234 TI - Peripheral neuropathy after high-dose cytosine arabinoside, daunorubicin, and asparaginase consolidation for acute nonlymphocytic leukemia. AB - Two patients with acute nonlymphocytic leukemia (ANLL) developed peripheral motor and sensory neuropathies after consolidation chemotherapy with high-dose cytosine arabinoside (ara-C), daunorubicin, and asparaginase. Evidence for ara-C and daunorubicin-induced peripheral neuropathies is reported. Despite the frequent use of these agents, only two cases of peripheral neuropathy after systemic therapy have been previously described; neurotoxic effects may be potentiated and become clinically important when the three drugs are used in combination. PMID- 3001236 TI - Abnormal neuronal excitability in hippocampal slices from kindled rats. AB - To determine if electrophysiological properties of hippocampal pathways are altered in kindled rats, extracellular recordings were made from hippocampal slices of rats kindled in the lateral entorhinal cortex and compared with those from implanted but unstimulated controls. Studies were made either 24 h or 28 days after the last kindled seizure and done in normal (3.5 mM) or elevated (7 mM) K+. The preparation of slices, data accumulation, and data analyses were done blind. One day or 28 days after the last kindled seizure, the proportion of slices with spontaneous epileptiform bursts recorded from the CA2/3 region in elevated K+ was significantly (P less than 0.001) increased in the kindled animals. The frequency of spontaneous burst firing was also increased and reached significance (P less than 0.02) at 28 days following the last kindling stimulus. One day after the last kindling stimulus, paired-pulse (GABAergic) inhibition in the CA1 region was decreased (P less than 0.001). Several measures suggested an increased synaptic inhibition in the dentate gyrus of slices from the kindled groups 1 day after kindling. Paired-pulse inhibition was increased (P less than 0.01), the current required to evoke a near-threshold population spike was increased (P less than 0.05), and the population spike amplitude was reduced for a given field excitatory postsynaptic potential (EPSP) (P less than 0.01). Twenty eight days after the last kindling stimulus, however, paired-pulse inhibition in the dentate was slightly less in slices from kindled rats (P less than 0.005). In other respects the CA1 and dentate regions did not differ between kindled and control groups within 24 h of the last stage V seizure. Thus the maximum amplitudes of presynaptic fiber volley, population spike, and field-excitatory postsynaptic potential (EPSP) slope, and the number of population spikes evoked by a near-maximally effective afferent stimulus, were unchanged. In the CA1 region the input-output curve of field EPSP versus population spike, and the current intensity required to evoke a near-threshold population spike were also unchanged. In addition, no spontaneous bursts were recorded from CA1 in 3.5 mM K+. We conclude that either synapses or neurons intrinsic to the hippocampus are altered by kindling stimuli applied outside this brain area. The transient increase in inhibition in the dentate gyrus suggests that it may reflect a compensatory reaction to kindled seizures. In contrast, the long-lasting (at least 28 days) increase in burst firing in CA2/3 may represent a mechanism for the initiation or propagation of kindled seizures.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3001235 TI - Effects of single intracortical microstimuli in motor cortex on activity of identified forearm motor units in behaving monkeys. AB - We examined the magnitude and extent of output effects elicited from focal cortical sites on the activity of individual motor units (MUs) by delivering single-pulse intracortical microstimuli (S-ICMS) (5-15 microA) during isometric wrist activity. Stimulation sites in the precentral gyrus (area 4) were chosen for study if stimulus-triggered averages (stimulus-TAs) of multiunit electromyograms (EMGs) revealed poststimulus facilitation (PStimF) of EMG activity in any of the coactivated wrist muscles. Single MUs were then isolated in the facilitated muscles with a remotely controlled tripolar microelectrode. MUs were identified by their signatures in their parent muscles (from MU triggered averages of EMGs) and by their firing pattern during ramp-and-hold wrist responses. One objective was to quantify the magnitude and time course of the effects on single MUs by compiling peristimulus histograms of MU firing. The cross-correlation histograms between S-ICMS and MU action potentials showed peaks with onset latencies of 8.8 +/- 1.7 ms (mean +/- SD, n = 64) and durations of 1.8 +/- 1.2 ms (n = 104). The cumulative sums of the correlogram peaks resembled the rising phase of corticomotoneuronal excitatory postsynaptic potentials previously recorded in forelimb motoneurons. Comparison of correlogram peaks with stimulus TAs of MU potentials suggests that the duration of PStimF of multiunit EMG can be accounted for, in approximately equal proportions, by l) the variation in firing time of single MUs (i.e., the width of the MU correlogram peaks), 2) the width of single MU potentials, and 3) the contribution of different MUs at different latencies. The sizes of the correlogram peaks relative to base line were larger than the PStimF of multiunit EMGs, and increased more rapidly with stimulus intensity, indicating appreciable cancellation in the multiunit records. A second objective was to determine whether S-ICMS affected all the MUs of a facilitated muscle, or only a particular subset. Of 104 MUs sampled in facilitated muscles, 99 (95%) were found to be individually facilitated (P less than 0.05). MU firing patterns during isometric ramp-and-hold torque responses were characterized as phasic, phasic-tonic, tonic, or decrementing; stimulation at a given cortical site was found to facilitate all four types of MUs. When more than one muscle showed PStimF from a site, MUs belonging to each of the facilitated muscles were facilitated individually by S-ICMS at that site.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3001237 TI - Presynaptic inhibition in the crayfish CNS: pathways and synaptic mechanisms. AB - I studied the pathways that produce primary afferent depolarization (PAD) and presynaptic inhibition during crayfish escape behavior. Simultaneous intracellular recordings were obtained from interneurons and primary afferent axons in the neuropil of the sixth abdominal ganglion. In several experiments, a sucrose-gap recording of PAD accompanied the intracellular impalements. I have identified PAD-producing inhibitory interneurons (PADIs) that are fired by a single impulse in the lateral (LG) or medial (MG) giant, escape-command axons; the PADIs appear to be directly responsible for presynaptic inhibition of primary afferent input to identified mechanosensory interneurons. PADI spikes, elicited by injection of depolarizing current, produced unitary PAD with constant short latency (mean = 0.97 +/- 0.12 SD ms). The unitary PADs were capable of following PADI impulses one for one at frequencies greater than 100 Hz, and the amplitude of unitary PAD was increased by injection of chloride into the afferent terminals. Therefore, the PADIs appear to directly produce an increase in chloride conductance in the primary afferent terminals. Intracellular injections of Lucifer yellow or horseradish peroxidase (HRP) revealed three morphological types of PADI. Their axonal branches and terminals are bilateral and overlap extensively with the innervation fields of all 10 sensory roots of the sixth ganglion. The three morphological types of PADI were physiologically indistinguishable. In several cases, the impaled PADI was shown to produce unitary PAD in more than one afferent of a given root as well as in afferents of adjacent roots. Therefore, the PADIs appear to diverge widely and contact many afferents in all of the sixth-ganglion sensory roots. Stimulation, caudal to the fifth ganglion, of an MG that had been interrupted rostral to the fifth ganglion produced no PAD in sixth-ganglion afferents. Also, stimulation of an MG or an LG in a surgically isolated sixth abdominal ganglion failed to produce PAD. Therefore, the pathway between the MGs and PADIs is activated exclusively within the rostral abdominal ganglia. Direct stimulation in the second and third abdominal ganglia of the segmental giants (SGs) produced a polysynaptic, suprathreshold response in the PADIs. This response was compound and was not due to the activity of the identified corollary discharge interneurons, CDI-2 and CDI 3, that are fired by the SGs. Therefore, the primary input to the PADIs must come from other, unidentified CDIs that are driven by the SGs. PADIs were not fired by shocks to the sensory portions of any peripheral roots even though these shocks produced PAD.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3001238 TI - Beta-receptor-stimulated and cyclic adenosine 3',5'-monophosphate-mediated taurine release from LRM55 glial cells. AB - Adrenergic stimulation of LRM55 glial cells results in the release of the neuroactive amino acid taurine. The present study characterizes the receptors involved in taurine release and shows that taurine release is mediated by cyclic adenosine 3',5'-monophosphate (cAMP). beta-Receptors in LRM55 cells were first characterized by [125I]iodohydroxybenzylpindolol binding. Binding was stereospecific and saturable with time and ligand concentration. Kinetic analysis of equilibrium binding at 37 degrees C revealed a single component of high affinity (Km = 113 pm; Bmax = 52.1 +/- 5.0 fmol/mg of protein). The pharmacologies of the stimulation of cAMP accumulation and taurine release were similar. The agonists isoproterenol (IPR), epinephrine (E) and norepinephrine (NE) showed a rank order of potency characteristic of a beta-adrenergic system (IPR greater than E greater than or equal to NE). The beta-antagonists alprenolol and propranolol inhibited the IPR stimulation of both processes; the alpha antagonist phentolamine did not. The dependence of taurine release on cAMP was further suggested by the similarity of the two time courses and was demonstrated by the stimulation of taurine release by the cAMP analogue dibutyryl cAMP. Thus, one physiological response of glial cells to beta-adrenergic stimulation is the release of taurine. Receptor-activated release of taurine from glia represents a previously undescribed neuronal-glial interaction by which glia may actively regulate neuronal excitability. PMID- 3001239 TI - Corticotropin-releasing factor receptors are widely distributed within the rat central nervous system: an autoradiographic study. AB - Corticotropin-releasing factor (CRF) receptor-binding sites have been localized and quantified in the rat central nervous system (CNS) by autoradiography with an iodine-125-labeled analogue of ovine CRF substituted with norleucine and tyrosine at amino acid residues 21 and 32, respectively. High affinity and pharmacologically specific receptor-binding sites for CRF were found in discrete areas within the rat CNS. CRF receptors were highly concentrated in laminae 1 and 4 throughout the neocortex, the external plexiform layer of the olfactory bulb, the external layer of the median eminence, several cranial nerve nuclei in the brainstem including the facial, oculomotor, trochlear, vestibulocochlear, and trigeminal nuclei, the deep cerebellar nuclei, and the cerebellar cortex. Moderate concentrations of CRF receptors were present in the olfactory tubercle, caudate-putamen, claustrum, nucleus accumbens, nucleus of the diagonal band, basolateral nucleus of the amygdala, paraventricular nucleus of the hypothalamus, mammillary peduncle, inferior and superior olives, medullary reticular formation, inferior colliculus, and brainstem nuclei including tegmental, parabrachial, hypoglossal, pontine, cuneate, and gracilis nuclei, and in spinal cord. Lower densities of CRF binding were found in the bed nucleus of the stria terminalis, central and medial amygdaloid nuclei, and regions of the thalamus, hypothalamus, hippocampus, and brainstem. The distribution of CRF-binding sites generally correlates with the immunocytochemical distribution of CRF pathways and with the pharmacological sites of action of CRF. These data strongly support a physiological role for endogenous CRF in regulating and integrating functions in the CNS. PMID- 3001240 TI - The circadian pacemaker in the Aplysia eye sends axons throughout the central nervous system. AB - Each eye of Aplysia contains a population of electrically coupled pacemaker neurons whose synchronous activity can be recorded from the optic nerve as a compound action potential (CAP). The CAP frequency continues to show a circadian rhythm even when the eye is isolated from the animal and maintained in constant conditions, and thus it contains an autonomous circadian pacemaker, which may reside in the pacemaker neurons. The pacemaker neurons, along with retinal photoreceptors, send axons out of the optic nerve, which connects to the cerebral ganglion of the central nervous system (CNS). Pacemaker neurons, but not photoreceptors, may contain an aminergic transmitter, possibly dopamine (DA). We describe the central projections of optic nerve fibers using horseradish peroxidase filling of the cut optic nerve, and transport of radiolabeled macromolecules after selective exposure of the eye to [3H] leucine, which labels both pacemaker neurons and photoreceptors. We were able to determine the projections of pacemaker axons by exposing the eye to [3H]-3,4 dihydroxyphenylalanine [( 3H] DOPA and [3H]DA, which is preferentially taken up and transported by the pacemaker neurons. Pacemaker axons project bilaterally to the cerebral, pedal, and pleural ganglia and may extend as far as the abdominal ganglion. We corroborate this anatomical evidence by recording an orthodromic CAP in the optic nerve that had originated in the eye and subsequently recording the CAP in the CNS connectives and nerves that contained [3H]DOPA-labeled fibers. These results suggest that circadian pacemaker information from the eye is widely distributed throughout the CNS, including neural structures known from studies by others to mediate circadian-regulated behaviors, such as locomotion. Thus, Aplysia can now be used as a model system to examine the influence of the central projections of an identified circadian pacemaker on behavior, such as locomotion, at the level of identified central neurons. PMID- 3001241 TI - Map formation in the developing Xenopus retinotectal system: an examination of ganglion cell terminal arborizations. AB - Single axonal arbors of retinal ganglion cells have been stained by injecting cobalt extracellularly into the retinae of Xenopus embryos and tadpoles. The axonal endings of the earliest retinal axons to arrive in the midbrain were usually simple in appearance, often ended in growth cones, and terminated in tectal regions appropriate to their location in the eye. Thus, a topographic projection exists very early in the development (stages 37 to 39) of the projection, before the elaboration of complex axonal arbors. Retinal axons began acquiring more mature features, exemplified by the elaboration of terminal arbors, by stage 39. The arbors of most ganglion cells were elongated in the rostral-to-caudal dimension during early larval life (stages 40 to 45) and covered a large portion of tectal neuropil. During mid-larval stages (stages 46 to 50), arbors covered a relatively smaller proportion of the tectal neuropil. A quantitative analysis of this change suggests that the apparent decrease in size of the arbors, with respect to the tectum, is due to rapid growth of tectal neuropil and not due to retraction of an initially diffuse arbor. Thus, the refinement in targeting of axonal arbors during development is a phenomenon distinct from that seen during regeneration. PMID- 3001242 TI - Intrinsic connections of macaque striate cortex: afferent and efferent connections of lamina 4C. AB - We have studied the intrinsic organization of macaque striate cortex by tracing the pattern of horseradish peroxidase (HRP)-labeled axons and cell bodies produced by microinjections of HRP into single cortical laminae. Both anterograde and retrograde transport results were used to examine: (1) the pattern of projections from lamina 4C to the superficial layers; (2) the projection from lamina 4C to deeper cortical layers; and (3) the projections to lamina 4C from other cortical laminae. Laminae 4C alpha and 4C beta differ in their pattern of projections to the superficial layers of striate cortex. Axons from neurons in lamina 4C beta ascend through lamina 4B without giving off collaterals and terminate in lamina 4A and in the base of lamina 3. By contrast, axons from neurons in lamina 4C alpha terminate in lamina 4B and less densely in the 4A/3B region. The projection from lamina 4C beta to lamina 4A is particularly dense and is distributed in a patchy fashion immediately above each injection site. The projection from lamina 4C beta to lamina 3B appears less dense and more widespread; we estimate that individual 4C beta axons may spread laterally for more than 400 micron. Furthermore, the pattern of HRP-labeled cell bodies in lamina 4C beta following injections into laminae 4A and 3B provides evidence for a subdivision within 4C beta. These injections always produce a large number of labeled neurons in the upper part of lamina 4C beta, whereas the lower portion contains few labeled neurons that are located immediately under the center of the injection site. Both lamina 4C alpha and lamina 4C beta also contribute less dense projections to the deeper layers of cortex. Lamina 4C beta projects mainly to lamina 6, whereas lamina 4C alpha contributes axon terminals to both lamina 5A and lamina 6. Neurons in lamina 6 provide the bulk of the intracortical projections to lamina 4C. The axons of these neurons are fine in caliber and have a delicate side-spine morphology that is quite distinct from lateral geniculate axon arbors. Neurons in lamina 5A also project onto lamina 4C, but the projections of these neurons appear concentrated in lamina 4C alpha. These results confirm or refine many conclusions about intrinsic connections of striate cortex drawn from Golgi material and suggest new patterns of connections not suspected from previous work. PMID- 3001243 TI - Intrinsic connections of macaque striate cortex: axonal projections of cells outside lamina 4C. AB - We have exploited a technique for making small injections of horseradish peroxidase into single cortical laminae in order to study axonal projections in macaque striate cortex. In the preceding paper (Fitzpatrick, D., J. S. Lund, and G. G. Blasdel (1985) J. Neurosci. 5: 3329-3349) we examined the projections of cells in lamina 4C--cells that receive most of their input from the lateral geniculate nucleus. The present paper deals with the projections of neurons that lie outside of lamina 4C. Among our findings are several projections that previously had not been described in the monkey. These include: a strong and precise (point-to-point) projection from lamina 4B to lamina 2/3A, a reciprocal projection from 2/3A back to 4B, a definite projection from lamina 4B to 5B, as well as a prominent input to lamina 6 from 5B. In many cases, we find it possible to trace the flow of visual information through several "circuits" in striate cortex that have, as their output, projections to extrastriate cortex or to the brainstem. Our results offer additional insights in this regard since we are able, in many cases, to compare the lateral spreads of particular projections. These vary and can be separated into at least three categories: those that terminate in a precise, point-to-point, fashion, those that spread widely, and those that terminate in a laterally periodic fashion. In several cases we find evidence for a correlation between specific patterns of projection and known physiological differences between the topographies of laminae that are connected. In cases where two laminae possess similar topographies (for example, where both contain orderly maps for orientation) their interconnections appear precise, with little diffuse spread. In cases where two laminae are characterized by strikingly different topographies (where, for example, one contains an orderly map for orientation and the other a precise map for retinotopic position, but no specificity for orientation), the connections appear more diffuse. PMID- 3001244 TI - Identification of protein phosphatase 1 in synaptic junctions: dephosphorylation of endogenous calmodulin-dependent kinase II and synapse-enriched phosphoproteins. AB - A calcium/calmodulin-dependent protein kinase termed CaM-kinase II is a major component of synaptic junctions from forebrain and constitutes approximately 12% of total synaptic junction protein. CaM-kinase II phosphorylates at least seven polypeptides that are enriched in synaptic junctions, of which two represent the 50- and 60-kilodalton subunits of the protein kinase. In this report the nature of endogenous protein phosphatases which dephosphorylate each of the seven synaptic junction phosphoproteins was examined. Assays of synaptic junctions and other subcellular fractions from rat forebrain for type-1 and type-2 protein phosphatases revealed that protein phosphatase 1 (PrP-1) was specifically enriched in synaptic junctions with respect to cytosolic fractions. The activity of type-2 protein phosphatases was very low in synaptic junctions. Homogeneous PrP-1 from rabbit skeletal muscle was found to dephosphorylate each of the seven phosphoproteins in synaptic junctions. Inhibitors-1 and -2 were found to inhibit endogenous protein phosphatase activity by 70 to 80%. Since inhibitors-1 and -2 are specific inhibitors of PrP-1, these results indicate that this enzyme accounts for the majority of endogenous protein phosphatase activity in synaptic junctions. Approximately 15% of the protein phosphatase activity in synaptic junctions was type 2A, whereas PrP-2B and PrP-2C accounted for little, if any, of the activity toward endogenous or exogenous phosphoproteins. These results indicate that PrP-1 may be important in controlling the state of phosphorylation of synaptic junction proteins. PMID- 3001245 TI - Some properties of 2,3-bisphosphoglycerate phosphatase from rabbit masseters. PMID- 3001246 TI - Pleomorphic adenoma of the lower lip: a case and review of the Japanese literature. PMID- 3001247 TI - In vitro killing of human glioblastoma by interleukin-2-activated autologous lymphocytes. AB - Culture of peripheral blood lymphocytes (PBL) from brain-tumor patients with recombinant interleukin-2 (IL-2) results in the activation of lymphokine activated killer cells (LAK) with the capacity to lyse autologous and allogeneic glioblastoma. In this study, PBL obtained from brain-tumor patients were cultured with or without IL-2 for 3 to 7 days and then tested for their ability to lyse target cells in a 4-hour chromium release cytotoxicity assay. The PBL were drawn 1 to 2 weeks following operative tumor debulking. Cells used as targets included fresh brain-tumor cells obtained at the time of craniotomy, fresh brain-tumor cells grown from 1 to 3 weeks in tissue culture, fresh autologous PBL, and allogeneic glioblastoma cells grown in tissue culture. Peripheral blood lymphocytes from brain-tumor patients that were cultured without IL-2 did not significantly lyse autologous or allogeneic glioblastoma. However, when these PBL were cultured with IL-2, LAK were generated which produced marked lysis of autologous as well as allogeneic tissue-culture glioblastoma in all of eight cases. Significant lysis of autologous fresh tumor by patient LAK was observed in four of five experiments. By contrast, patient LAK did not kill autologous normal PBL. The ability to generate LAK was not influenced by the patient's age, previous therapy, or the administration of steroids. PMID- 3001248 TI - Development of cyclooxygenase and lipoxygenase metabolites of arachidonic acid after transient cerebral ischemia. AB - Vasoactive arachidonic acid metabolites are postulated to play a role in the pathogenesis of cerebral ischemia. In order to characterize the local generation of cyclooxygenase and lipoxygenase metabolites of arachidonic acid in transient ischemia with reperfusion, Mongolian gerbils were studied for regional cerebral blood flow (CBF), using the hydrogen clearance technique, and for cerebral levels of the thromboxane metabolite TXB2, and prostaglandins 6-keto-PGF1 alpha and PGE2, as well as the leukotriene LTB4. The gerbils were anesthetized with pentobarbital, and half of the animals were pretreated with the cyclooxygenase inhibitor indomethacin. All received 10 or 20 minutes of dense forebrain ischemia followed by reperfusion of 10 minutes, 50 minutes, or 100 minutes. A separate control group received no ischemic lesion. Regional CBF decreased significantly from 23.7 +/- 2.6 to 4.3 +/- 1.7 cc/100 gm/min during ischemia (p less than 0.01). Reperfusion resulted in initially normal flows (22.5 +/- 5.1 cc/100 gm/min) followed by a progressive hypoperfusion (11.3 +/- 2.7 cc/100 gm/min). All metabolites showed parallel significant (p less than 0.05) increases after transient ischemia and reperfusion compared to baseline levels (values (in pg/mg protein) were: TXB2 45.5 +/- 7.1 vs 23.3 +/- 3.6; 6-keto-PGF1 alpha 262.8 +/- 47.9 vs 175.8 +/- 26.8; PGE2 256.5 +/- 35.6 vs 112.5 +/- 11.2; and LTB4 37.8 +/- 4.6 vs 24.6 +/- 6). These levels were all significantly decreased (p less than 0.05) by pretreatment with indomethacin except for the leukotriene LTB4, which was increased. Transient cerebral ischemia results in a reperfusion abnormality and the local generation of cyclooxygenase products, which are reduced by pretreatment with indomethacin; however, cyclooxygenase inhibition may result in increased substrate availability for the lipoxygenase system. Studies of such an interaction may lead to new understandings of the pharmacological modification of detrimental vascular changes after transient cerebral ischemia. PMID- 3001249 TI - Faculty governance: a key to professional autonomy. AB - Faculty governance can play a central role in modeling professional autonomy to student nurses during their socialization processes. Nurse educators, however, may be more familiar with bureaucratic models of governance than with collegial models of governance. The collegial or shared governance model offers nursing access to enhanced academic maturity, and deserves nursings' close attention and study. PMID- 3001250 TI - Experiential learning and changing leadership style. AB - One of the many problems facing the nursing profession today is the lack of preparedness of its leaders. Nursing educators, collaborating with nursing service, can teach baccalaureate students leadership skills and to develop leadership styles. Experiential real-world management tasks selected by faculty and head nurses can serve as learning opportunities. Students can learn leadership ability and change style. Utilizing t-test, the before and after course mean scores on the standardized Leadership Ability Evaluation instrument were statistically analyzed. Significant differences and style changes were identified. Students in the total class became more effective leaders as did the students in both the traditional and experiential groups. Traditional students (lecture only) became less autocratic-submissive and more democratic. The experiential group significantly became less autocratic-aggressive, less laissez faire and more democratic. PMID- 3001251 TI - Malpractice and nurse educators: defining legal responsibilities. AB - This article discusses the malpractice crisis in the health professions as it threatens to affect nurse educators. There is a concise explanation and review of immunity and liability doctrines for supervisory personnel. The most common conditions for patient injury are examined, and pertinent legal decisions are cited. Examples are provided for the problem areas of negligence and several practical recommendations are presented for the nurse educator who wishes to do everything possible to minimize exposure to claims of malpractice. PMID- 3001252 TI - Promoting enthusiasm for research among undergraduate students. AB - Methods for increasing the undergraduate nursing student's awareness and appreciation of nursing research are presented. Faculty members are asked to examine their own attitudes towards research as well as the factors contributing to the students' developing attitudes. Ways of assisting students to identify researchable problems are discussed and suggestions made for the involvement of the students in ongoing research. PMID- 3001253 TI - Ethical dilemmas in nursing: the role of the nurse and perceptions of autonomy. AB - This study investigated decision making in ethical dilemmas and attitudes toward professional autonomy. It was based on Murphy's identification of three nurse patient relationship models. The model identification was the result of Murphy's investigation of the levels of moral reasoning of nurse practitioners, from Kohlberg's theory of moral development. Autonomy is necessary for patient advocacy in Murphy's highest order model of nurse-patient relationship. 109 freshmen, 103 seniors, and 82 graduates (baccalaureate nursing) were examined for model selection, risk-taking, restrictions, and anxiety in the decision-making process in specific situations. Autonomy was measured independently. The most significant results indicated that freshmen were less likely to select the autonomous model of relationship, had lower attitudes toward professional nursing autonomy, and were less willing to take risks. Graduates were lower than either student group in their perceptions of restrictions and anxiety. The responses to each dilemma itself varied by situation in relation to the model preferred. PMID- 3001254 TI - Expanding horizons: innovations in community health nursing education. AB - Application of this learning process (problem assessment, program planning, intervention, and evaluation) at the aggregate level, was a creative, enjoyable, growth producing experience for the senior nursing students. It is a process that is not only useful in the local community, but also prepares nurses for working at county, state, and national levels. They learn to make valid observations and firm decisions, to carry out actions, to overcome obstacles, to alter behavior, and to evaluate results. It does not replace other practices and former services of community health nursing, but complements them. There exists a tremendous potential for nurses in planning health care, already being realized in many settings. Certainly grass roots communities, rural populations and urban neighborhoods are in the highest need of creative, effective health programs that take into account the total population. Given such creativity, it is possible by the year 2000, that the Community Health Nurse may become a combination medical advisor, health instructor, community leader, playwright, photographer, author and television director; certainly a captivating career for people of the New World. PMID- 3001256 TI - Concept analysis as a strategy for promoting critical thinking. PMID- 3001255 TI - Graduate students' use of group work with adolescents for primary prevention. PMID- 3001257 TI - Helping students write. PMID- 3001259 TI - A new look at an old shibboleth. PMID- 3001258 TI - Advisory committees in schools of nursing. PMID- 3001260 TI - Hydroxylapatite alveolar ridge reconstruction: clinical experiences, complications, and technical modifications. AB - Results of the reconstruction of 228 deficient alveolar ridges (208 patients) using hydroxylapatite with or without autogenous cancellous bone over a six-year period are reported. Complications included erosion, mental nerve neuropathy, migration and displacement of particles, overfill, and loose material. Modified techniques are presented that minimize the occurrence of these complications in Class III and IV ridge-deficient patients. PMID- 3001261 TI - Use of an open splint in ridge augmentation with hydroxylapatite. PMID- 3001262 TI - [Cell biology and biochemistry of VX2 cancer cell motility--relevance to its invasiveness and metastatic potential]. PMID- 3001263 TI - Sexual dimorphism and electrophoretic variation in the form I cyclic nucleotide phosphodiesterase from Drosophila melanogaster. AB - We have investigated the form I cyclic nucleotide phosphodiesterase (PDE) from Drosophila melanogaster and shown that whereas heads and male thoraces and abdomens contain high levels of Ca2+-stimulated enzyme, female thoraces and abdomens contain little Ca2+-stimulated activity. The electrophoretic patterns of form I PDE from these 3 sources have also been studied and reveal that heads, and male thoraces and abdomens, produce two bands of form I PDE both of which are stimulated by Ca2+. Extracts of female thoraces and abdomens, on the other hand, show only a single, faster running band of PDE activity which is only marginally stimulated by Ca2+, if at all. Surveying wild-type strains of Drosophila has revealed that one strain, Swedish, shows altered electrophoretic mobility of the PDE band from female thoraces and abdomens. The alteration is such that the Swedish PDE band runs more anodally than the Oregon-R and Canton-S PDE activities. Mixing experiments, using co-homogenization of heads with female thoraces and abdomens, yield a single faster running band on electrophoresis. This band contains only Ca2+-insensitive PDE. Attempts to reconstruct this loss of Ca2+-sensitive PDE without electrophoresis have failed. The Swedish electrophoretic variation of the PDE from female thoraces and abdomens has been found to be recessive with respect to the Canton-S phenotype, but the variation is observed to re-emerge and segregate with the third chromosome in the F2 generation. The results indicate that electrophoretic variation in the form I PDE is, by itself, insufficient to allow the location of the structural gene for this enzyme. PMID- 3001264 TI - Localization of cloned unique DNA to three different regions of chromosome 19: screen for linkage probes for myotonic dystrophy. AB - Screening polymorphic DNA probes for linkage to myotonic dystrophy (DM) and to other reported chromosome 19 (CH19) genes will develop a linkage map for human CH19. We report here the assignment of 3 cloned unique DNA sequences to 3 distinct regions of CH19. The novel use of 35S-labeled probes facilitated the rapid localization of the gene for the third complement factor (C3) to 19p13.2 by in situ hybridization. Metaphase chromosomes were from normal peripheral lymphocytes as well as from a fibroblast line containing a 15;19 translocation which permitted clear identification of CH19 regions of localization. Two random clones isolated from a plasmid library of human F-group enriched chromosomal DNA (D19S5 and D19S6) were in like manner assigned to 19p1.2 and 19q13.2 to 19qter, respectively. PMID- 3001265 TI - Development of a biochemical profile for gingival crevicular fluid. Methodological considerations and evaluation of collagen-degrading and ground substance-degrading enzyme activity during experimental gingivitis. AB - The potential application of gingival crevicular fluid (GCF) analysis to periodontal diagnosis has been examined for more than 25 years. Unfortunately, the information available has not provided the clinician with a more sensitive means of diagnosing periodontal disease or an effective means of monitoring periodontal therapy. A careful review of the literature on GCF, however, suggests that discrepancies occur in the method of GCF collection, the use of GCF for analysis from pooled or isolated crevicular locations, the method of analyzing the samples and the way in which the data is reported. Studies in our laboratory have suggested a technique for GCF analysis that collects GCF from individual crevices with a filter paper strip inserted for a standard time, determines the volume of GCF collected with a calibrated electronic meter and elutes the material into a larger volume of diluent. This approach allows for detection of site-to-site and patient-to-patient differences in GCF volume while providing sufficient samples to analyze GCF for multiple constituents. We have used this approach to evaluate GCF for vertebrate forms of the enzymes collagenase (latent and active forms), beta-glucuronidase and arylsulfatase during the development of experimental gingivitis in man. Interproximal and midradicular areas were studied. Our results indicate that during the 4 weeks of the gingivitis, the absolute amount of active collagenase in GCF increased 550% at the interproximal sites and 190% in the midradicular sites, and the per cent of active collagenase increased from 15 to 71% at the interproximal sites, and from 16 to 36% at the midradicular sites.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001266 TI - Tetracyclines inhibit tissue collagenases. Effects of ingested low-dose and local delivery systems. AB - In a series of experiments, Golub et al. demonstrated that tetracyclines, but not other antibiotics, can inhibit mammalian collagenases and proposed that this property could be useful in treating diseases, such as periodontal disease (but also included certain medical conditions, e.g., corneal ulcers) characterized by excessive collagen degradation (J Periodont Res 1983, 1984 and 1985; Experientia 1984; Cornea 1984). One effect was the dramatic reduction of tissue collagenase activity within the gingival crevicular fluid (GCF) of periodontal pockets after administering a standard regimen of a tetracycline (e.g., 200 mg minocycline or 1000 mg tetracycline/day). The preliminary studies described below determined the effect of (1) low-dose (LD; 40-80 mg/day) orally administered minocycline on GCF collagenase activity and on the subgingival microflora (Exp. I), and (2) tetracycline-loaded monolithic fibers (TF) on collagenase activity in vitro (Exp. II). In Exp. I, GCF collagenase activity was reduced by 45 to 80% 2 weeks after initiating LD minocycline therapy, an effect that lasted for at least several weeks after stopping drug treatment. No consistent change in the relative proportions of G(+), G(-) and motile subgingival microorganisms was detected as a result of LD treatment suggesting that the reduction in GCF collagenase activity was a direct inhibition of the enzyme by the drug. In Exp. II, 3- and 6-mm lengths of TF in vitro established tetracycline concentrations in 250 microliters of 132 micrograms/ml, from 3-mm lengths, and 265 micrograms/ml, from 6-mm lengths, after an 18-hour incubation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001267 TI - Effects of dopamine, (+/-)-dobutamine and the (+)- and (-)-enantiomers of dobutamine on cardiac function in pithed rats. AB - The effects of dopamine, (+/-)-dobutamine (racemic mixture) and the (+)- and (-) enantiomers of dobutamine on myocardial function were evaluated in pithed rats. Dopamine and (+/-)-dobutamine produced effects on cardiac function in pithed rats that were qualitatively similar to those reported for these compounds in humans. The increase in cardiac output produced by dopamine and (+/-)-dobutamine was due mainly to an increase in stroke volume, with increases in heart rate contributing to a significant but lesser degree. For both dopamine and (+/-)-dobutamine, the increase in stroke volume appears to result from an increase in myocardial contractility as assessed by increases in left ventricular (LV) dp/dt. Dopamine produced a marked increase in mean arterial blood pressure, whereas (+/-) dobutamine only modestly increased blood pressure. The (-)-enantiomer of dobutamine, which possesses mainly alpha-1 adrenoceptor agonist activity, produced dose-dependent increases in cardiac output, stroke volume, LVdp/dt and mean arterial blood pressure, but did not significantly increase heart rate except at high doses. Thus, the increase in cardiac output produced by (-) dobutamine was derived almost exclusively from an augmentation in stroke volume resulting from an increase in myocardial contractility. In contrast, (+) dobutamine, which possesses predominantly beta-1 and beta-2 adrenoceptor agonist activity, elicited only a modest increase in cardiac output which was due both to an increase in heart rate and stroke volume. Mean arterial blood pressure was not significantly affected by (+)-dobutamine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001268 TI - Differential diazepam-antagonist effects of the benzodiazepine receptor ligand CGS 9895 in rodents. AB - CGS 9895, a pyrazoloquinolone benzodiazepine receptor ligand, was administered alone and concomitantly with diazepam in order to assess its agonist and diazepam antagonist properties on various behaviors in rodents. In mice, CGS 9895 neither potentiated nor blocked the convulsant effects of pentylenetetrazole. However, doses of 3.0 and 10 mg/kg of CGS 9895 i.p. produced dose-related antagonism of the anticonvulsant effects of diazepam against pentylenetetrazole (80 mg/kg i.p.). In rats, diazepam produced dose-related increases in ataxia as measured on the rotarod. CGS 9895 (0.3-10 mg/kg i.p.) was without effect on performance on the rotarod, but produced dose-related parallel shifts to the right in the diazepam dose-effect curve. Also in rats, behavior was maintained under a multiple schedule where in one component every 20th response resulted in water presentation (unpunished behavior) and in a second component every 20th response resulted in both shock and water presentation (punished behavior). CGS 9895 (0.3 30 mg/kg i.p.) was without significant effect on either punished or unpunished responding. Increasing doses of diazepam (0.1-10 mg/kg p.o.) first increased and then decreased rates of punished responding but only decreased rates of unpunished responding. CGS 9895 (3.0 mg/kg i.p.) neither potentiated nor antagonized diazepam. In another group of rats, behavior was maintained under a multiple fixed-interval 5 min fixed-ratio 20 response schedule of water presentation. CGS 9895 (0.3-30 mg/kg i.p.) did not affect performance under this schedule. Diazepam (0.3-30 mg/kg p.o.) primarily decreased rates under the fixed ratio schedule, but increasing doses first increased and then decreased rates under the fixed-interval schedule.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001269 TI - Measurement of thermodynamic parameters for norepinephrine contraction of isolated rabbit thoracic aorta. AB - The thermodynamic quantities of change in free energy (delta G degree'), change in enthalpy (delta H degree') and change in entropy (delta S degree') were determined for the interaction of norepinephrine with the alpha-1 adrenoceptor of vascular smooth muscle. Specifically, a standard isolated rabbit thoracic-aorta preparation was used to examine the effect of temperature on norepinephrine induced isometric tension development. Dissociation constants (KA) for norepinephrine were determined at several temperatures over the range 25-40 degrees C from equiactive concentrations obtained before (A) and after (A') partial irreversible receptor blockade by phenoxybenzamine, plotted as 1/A against 1/A' (KA = (slope-1)/intercept). The values of KA increased with temperature over the range 25-40 degrees C, indicating that the affinity of norepinephrine for the alpha-1 adrenoceptor is an inverse function of temperature over this range. From these results, the thermodynamic quantities delta H degree' and delta S degree' were determined from a van't Hoff plot of In (KA) against 1/T. The relative magnitudes of the change in enthalpy (delta H degree' = -25.58 kcal mol-1) and the change in entropy (delta S degree' = -0.052 kcal mol-1 deg-1) suggest that the reaction between norepinephrine and the alpha-1 adrenoceptor (delta G degree' = -9.15 kcal mol-1) is enthalpy driven, which is consistent with radioligand binding studies of other adrenoceptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001270 TI - Relationship between Ah receptor-mediated polychlorinated biphenyl (PCB)-induced humoral immunosuppression and thymic atrophy. AB - Thymic atrophy and humoral immunosuppression by certain polychlorinated biphenyls is associated with the aromatic hydrocarbon (Ah) receptor in mice. We examined the relationship between these two toxic effects. 3,3',4,4'-Tetrachlorobiphenyl (TCB), which causes immunosuppression and thymic atrophy, and 2,3,3',4,4',5 hexachlorobiphenyl, which causes immunosuppression without thymic atrophy, were administered i.p. to C57BL/6 mice at 0, 35 and 350 mumol/kg b.wt. 2 days before i.v. immunization with 10 micrograms of Escherichia coli lipopolysaccharide. Both congeners caused significant suppression of the day 4 anti-lipopolysaccharide plaque-forming cell response/spleen (less than or equal to 46% of control). TCB (350 mumol/kg) was also administered 2 days before either a primary or secondary i.p. immunization with sheep erythrocytes. TCB treatment before primary immunization had no effects on the day 5 secondary response, whereas treatment before the secondary immunization significantly inhibited both day 5 immunoglobulin M and immunoglobulin G plaque-forming cells (less than 10 and less than 2% of control, respectively) and decreased serum antibody. TCB administered either 8 or 2 days before or 2 or 4 days after immunization with sheep erythrocytes demonstrated that significant suppression of both plaque-forming cells and serum antibody could occur without thymic atrophy. Immunity was most impaired when TCB was given 2 days before immunization. These results demonstrate that thymic atrophy does not always accompany the severe immunosuppression caused by Ah receptor ligands and suggests that it may not be a sensitive measure of Ah receptor-mediated immunosuppression. The data also suggests that differentiation of B lymphocytes into antibody producing cells is impaired during Ah receptor mediated gene activation. PMID- 3001271 TI - Graded regional vasodilation with converting enzyme inhibitors in conscious spontaneously hypertensive rats. AB - The present study was designed to examine the effects of two different converting enzyme inhibitors on regional hemodynamics in conscious spontaneously hypertensive rats. Rats were chronically instrumented with miniaturized pulsed Doppler flow probes for measurement of renal, mesenteric and hindquarters blood flow. Equidepressor doses of captopril (10 mg/kg) or a potent new converting enzyme inhibitor, Wy-44,221 [(-)-(S)-2,3-dihydro-1-[(S)-3-mercapto-2-methyl-1 oxypropyl]-1 H-indoline-2-carboxylic acid] (2 mg/kg) were administered by i.a. bolus injection. The converting enzyme inhibitors caused a reduction in mean arterial pressure, which was accompanied by a tachycardia. Renal blood flow was significantly increased by approximately 30 to 37% within 5 min after administration of the converting enzyme inhibitors, and renal vascular resistance was reduced. The renal hemodynamic effects were sustained for the 45-min duration of the experiment. Pretreatment with an angiotensin II receptor antagonist markedly attenuated the renal vasodilator effects of Wy-44,221, whereas antagonism of kinin or prostaglandin synthesis failed to diminish the renal effects of Wy-44,221. Both converting enzyme inhibitors also caused a significant but transient reduction in mesenteric vascular resistance, but had no significant effect on hindquarter hemodynamics. These data indicated that the converting enzyme inhibitors in conscious spontaneously hypertensive rats caused a prolonged increase in renal blood flow as a result of removing the renal vasoconstrictor effects of angiotensin II. These data further suggest that converting enzyme inhibitors exerted graded actions on regional vascular resistance with renal greater than mesenteric greater than hindquarters dilation. PMID- 3001272 TI - Proenkephalin A fragments exhibit spinal and supraspinal opioid activity in vivo. AB - The inhibition of reflex urinary bladder contractions, recorded isometrically in the urethane-anesthesized rat, was used as an index of central opioid activity. Inhibition of bladder activity has been shown to be mediated both spinally and supraspinally by mu and delta opioid receptors. Using this model a number of neuropeptide fragments of the proenkephalin A molecule (peptide F, peptide E, BAM 22P, BAM 12P, Met5-enkephalin-Arg6,Phe7,Leu8, Met5-enkephalin-Arg6,Phe7, Met enkephalin, Leu-enkephalin) were tested for in vivo activity. Each of the fragments inhibited reflex bladder contractions when administered by bolus microinjection into a lateral ventricle (i.c.v.) or intrathecally into the spinal subarachnoid space (between L3 and L4 vertebra). The larger molecular weight peptides produced more prolonged inhibition of bladder activity than the smaller molecular weight ones, with the rank order of activity being similar at spinal and supraspinal sites: peptide F greater than or equal to peptide E = BAM 22P greater than BAM 12P greater than Met5-enkephalin-Arg6,Phe7,Leu8 = Met5 enkephalin-Arg6,Phe7 greater than or equal to Met-enkephalin = Leu-enkephalin. The relative receptor selectivity of each fragment was further determined using the opioid antagonists naloxone (mu receptors) and ICI 174,864 (N, N-diallyl-Tyr Aib-Aib-Phe-Leu-OH: Aib = alpha-aminoisobutyric acid) (delta receptors). The activity (Ke) of each antagonist against equieffective doses of the highly selective opioid receptor ligands [D-Ala2,Me-Phe4,Gly(ol)5]enkephalin (mu receptors) and [D-Pen2,D-Pen5]enkephalin (delta receptors) was compared with that against the proenkephalin A fragments.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001273 TI - Beta adrenergic-mediated vasodilator response to insulin in the human forearm. AB - Insulin, in doses sufficient to cause systemic hypoglycemia, decreases vascular resistance; however, a vasoactive effect of insulin in humans, in the absence of hypoglycemia, is unknown. Venous occlusion strain gauge plethysmography was utilized to determine forearm blood flow (FBF) and forearm vascular resistance (FVR) during incremental intra-arterial infusion of insulin (0.1, 0.5 and 1.0 mU/kg/min) in seven normal subjects. With the increasing doses of insulin, FBF increased 55, 76 and 145% and FVR decreased 30, 42 and 58%, respectively. Systemic glucose concentration decreased only during the highest dose insulin infusion. In eight subjects who received insulin during euglycemic glucose clamping, FVR also decreased. Therefore, it was demonstrated that insulin dilates the forearm vasculature in the absence of hypoglycemia. In order to determine whether the vasodilatory effect of insulin was mediated by the sympathetic nervous system, plasma levels of epinephrine and norepinephrine were measured. During the incremental insulin infusions, norepinephrine levels did not change, but epinephrine concentration increased during the 1.0 mU/kg/min infusion when hypoglycemia developed. Furthermore, 21 subjects were pretreated with intra arterial phentolamine, propranolol or the combination of phentolamine and propranolol. Phentolamine did not potentiate the fall in FVR during insulin administration; however, propranolol attenuated or completely inhibited the changes in FBF and FVR. During combined alpha and beta adrenergic blockade, insulin increased FBF and decreased FVR only during the higher dose insulin infusions. It is concluded that insulin causes forearm vasodilation primarily via a beta adrenergic mechanism. In addition, at the higher doses used in this study, insulin may decrease FVR directly or by a mechanism other than adrenergic stimulation. PMID- 3001274 TI - Thyroid status and adrenergic receptor subtypes in the rat: comparison of receptor density and responsiveness. AB - The density and functional responsiveness of adrenergic receptor subtypes were determined in tissues from control, hyperthyroid and hypothyroid rats. There was a decrease in sensitivity to isoproterenol in spontaneously beating right atria, electrically driven left atria and field-stimulated vas deferens associated with hypothyroidism, with no change in maximum response. Hyperthyroidism increased the potency of isoproterenol in right atria, but not in left atria or vas deferens. The maximal response to isoproterenol was greatly reduced in hyperthyroid left atria. The potency of procaterol, a partial agonist at beta adrenergic receptors in right atria, was unaltered in hyper- or hypothyroidism, although the maximum stimulation by procaterol was increased in hyperthyroidism. Scatchard analysis of specific [125I]pindolol binding showed that beta adrenergic receptor density was greater in hyperthyroidism than in hypothyroidism in left atria, right atria, ventricles, vas deferens and cerebral cortex, although the proportions of beta-1 and beta-2 adrenergic receptor subtypes did not change. There was no change in the responsiveness of alpha-1 adrenergic receptors mediating contraction of caudal artery and vas deferens or mediating [3H]inositol phosphate accumulation in cerebral cortex in hyperthyroid or hypothyroid rats, although the maximal contraction of caudal artery was significantly reduced in hyperthyroidism. Scatchard analysis of specific [125I]BE 2254 binding showed that alpha-1 adrenergic receptor density was significantly decreased in the ventricles from hyperthyroid rats and increased in the ventricles of hypothyroid rats, but was unchanged in vas deferens, caudal artery and cerebral cortex. Alpha-2 adrenergic receptor density in cerebral cortex, determined by Scatchard analysis of specific [3H] rauwolscine binding, was not altered in hyperthyroid or hypothyroid rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001275 TI - Binding of a thromboxane A2/prostaglandin H2 receptor antagonist to washed human platelets. AB - A binding site for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist [125I]9,11-dimethylmethano-11, 12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14 dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2 to washed human platelets was studied. 9,11-Dimethylmethano-11, 12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14 dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2 competitively antagonized aggregation of washed human platelets induced by the TXA2/PGH2 mimetic U46619. A Schild analysis of the pharmacologic study revealed a pA2 of 8.08 and a slope of 1.12. The pA2 value yielded a Kd of 8 nM. The association rate constant (k1) for [125I]PTA-OH was 6.6 X 10(6)M-1 min-1 and the dissociation rate constant (k-1) was 1.82 X 10(-1), yielding a kinetically determined Kd (k-1/k1) of 27 nM. Scatchard analysis of [125I]PTA-OH binding to washed human platelets revealed one class of binding sites with a Kd of 21 +/- 5 nM and maximum binding of 42 +/- 6.4 fmol/10(7) platelets (N = 5) (2530 +/- 380 binding sites/platelet). Several TXA2/PGH2 receptor agonists and antagonists competed with [125I]PTA-OH for binding. For the four antagonists used in this study, the rank order of potency for displacing the ligand from its binding site correlated (r = 0.93) with the rank order of potency for their ability to inhibit U46619-induced aggregation in human platelet-rich plasma. The antiaggregatory prostaglandins prostaglandin F2 alpha, prostaglandin D2, and Iloprost also displaced the ligand, but only at concentrations considerably higher than that required to produce their pharmacologic effects.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001276 TI - Presynaptic inhibitory dopamine receptors on noradrenergic nerve terminals: analysis of biphasic actions of dopamine and apomorphine on the release of endogenous norepinephrine in rat hypothalamic slices. AB - Electrical field stimulation (5 Hz)- or high K+ (20 mM)-evoked release of endogenous norepinephrine from superfused rat hypothalamic slices in the presence of cocaine (20 microM) was measured by high-performance liquid chromatography with an electrochemical detector. Apomorphine (10-1000 nM) dose-dependently facilitated the electrically evoked release. Apomorphine (1 microM)-induced facilitation was abolished by pretreatment with yohimbine (100 nM), was converted to inhibition by yohimbine (1 microM), but was not antagonized by propranolol (300 nM). Epinephrine (100 nM) decreased the electrically evoked release and the decrease was antagonized by yohimbine (100 nM) and by apomorphine (100 nM), but not by S-sulpiride (100 nM). In the presence of yohimbine (1 microM), apomorphine (10-1000 nM) dose-dependently inhibited the electrically evoked release. Furthermore, in the presence of tetrodotoxin (300 nM), apomorphine (100 nM) also decreased the high K+-evoked release and this decrease was antagonized by S sulpiride (100 nM). Dopamine produced biphasic actions on the electrically evoked release, a dose-dependent decrease at 30 and 100 nM and an increase at 300 and 1000 nM. Dopamine (300 nM)-induced increase was antagonized by propranolol (300 nM) but not by yohimbine (100 nM). The dopamine (100 nM)-induced decrease was antagonized by S-sulpiride (1 nM), but not by the R-isomer. S-sulpiride (10 to 100 nM) alone dose-dependently increased the electrically evoked release, whereas the R-isomer had no effect. Haloperidol (100 nM) also increased the electrically evoked release.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001277 TI - Predominance of postsynaptic mechanism in vagal suppression of sympathetic tachycardia in the dog. AB - The amplitude of reduction in heart rate induced by vagal stimulation is greater when the background level of sympathetic tone is increased (accentuated antagonism). Both pre- and postsynaptic muscarinic mechanisms have been proposed to account for this phenomenon. We attempted to clarify the relative importance of each mechanism by comparing the magnitude of vagal bradycardia during neurally induced tachycardia with that during nonneurally induced tachycardia in the anesthetized dog. Graded tachycardia was induced by raising the stimulus frequency of cardiac sympathetic nerve stimulation stepwise from 1 to 3 and 5 and 10 Hz, and it was also induced by norepinephrine infusion (0.3-10 micrograms/min into the right coronary artery), isoproterenol infusion (0.1 and 0.3 micrograms/kg/min i.v.) and glucagon injection (3-30 micrograms/kg i.v.). The magnitude of the bradycardia produced by vagal nerve stimulation at 3 Hz was determined during the resting state and during the tachycardic state produced by cardiac nerve stimulation and by drug administration. The magnitude of vagal bradycardia was greater during tachycardic state than during the resting state regardless of the means through which tachycardia was produced. Vagal bradycardia during norepinephrine or isoproterenol infusion was of the same magnitude as that during the cardiac sympathetic nerve stimulation when they were compared at the same heart rate. Vagal bradycardia during the glucagon-induced tachycardia was greater than that which occurred during sympathetic tachycardia. Temporary bradycardia resulting from a single-pulse vagal nerve stimulation was augmented markedly during the cardiac sympathetic nerve stimulation, whereas a 2-sec interruption of the cardiac sympathetic nerve stimulation did not alter the sustained tachycardia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001278 TI - Effect of netilmicin on the phospholipid composition of subcellular fractions of rat renal cortex. AB - The purpose of this study was to determine the subcellular site(s) of the renal cortical phospholipidosis induced by aminoglycosides. For this purpose we injected male Sprague-Dawley rats s.c. with netilmicin, containing tracer quantities of [3H]netilmicin, at 100 mg/kg/day for 2 days; control rats were injected with saline. Twenty-four hours after the second injection of drug the rats were sacrificed and the renal cortex was fractionated by differential ultracentrifugation and Percoll gradient density techniques to obtain purified lysosomes, mitochondria, microsomes, brush border membranes and basolateral membranes. The total phospholipid content of the renal cortex was 300 +/- 5 nmol/mg of protein in control rats and 340 +/- 5 nmol/mg of protein in netilmicin injected rats. The total phospholipid content of the lysosomal fraction of netilmicin rats, which was enriched in myeloid bodies and [3H]netilmicin, was 91% greater than that of control rats and reflected significant increases of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. This pattern is identical to that reported previously for the rat renal cortical phospholipidosis induced by aminoglycosides. The total phospholipid contents of the mitochondrial, microsomal, brush border membrane and basolateral membrane fractions of netilmicin-injected rats were higher by approximately 10% than the respective fractions of control rats and each fraction exhibited a significant increase of one or more of the four phospholipids elevated in the renal cortical homogenate and in the lysosomal fraction. The data indicate that the myeloid body is the primary source of the lysosomal phospholipidosis induced by netilmicin which provides support for the hypothesis that the lysosomal phospholipidosis is secondary to aminoglycoside-induced inhibition of phospholipid degradation. In addition the findings of increased phospholipid content and altered phospholipid composition of the other subcellular fractions raise the possibility that aminoglycoside antibiotics cause a more generalized disturbance of phospholipid metabolism characterized by altered synthesis as well as degradation in renal proximal tubular cells. PMID- 3001280 TI - [3H]leukotriene B4 binding to the guinea-pig spleen membrane preparation: a rich tissue source for a high-affinity leukotriene B4 receptor site. AB - Intact human granulocytes contain a leukotriene (LT) B4 receptor binding site, but the limited supply of these cells could adversely affect further progress of the study of this receptor. To select a tissue homogenate rich for this site, we have characterized the binding of highly specific [3H]LTB4 to guinea-pig spleen membranes and we have determined the ability of LTB4 to compete with [3H]LTB4 for binding sites in the membranes of 10 nonspleen tissues. In the spleen membrane, MgCl2 and CaCl2 enhanced [3H]LTB4 binding, but NaCl and KCl decreased it. Spleen [3H] LTB4 binding was a function of protein concentration and was rapid, reversible, stereoselective and saturable. Kinetic analyses showed that the rate constant for association and dissociation at 25 degrees C was 0.47 nM-1 min-1 and 0.099 min-1, respectively. A Scatchard plot of the data of the equilibrium experiment resulted a straight line with a dissociation constant of 1.8 nM and a density of 274 fmol/mg of protein. Moreover, the LTB4/[3H]LTB4 competition study performed at 4 or 25 degrees C revealed the inhibitory constant (Ki) of LTB4 to be in the nanomolar range. The rank order of agents competing for spleen [3H]LTB4 binding was: LTB4 (Ki = 2.8 nM) greater than 20-hydroxy-LTB4 (23 nM) greater than LTA4 (48 nM) greater than LTA4 methyl ester (0.13 microM) greater than 20-carboxy LTB4 (greater than 6.6 microM) greater than or equal to arachidonic acid (0.15mM) = FPL-55,712 and FPL-57,231 (0.1-0.2 mM). Competition studies further indicated that felodipine, a 1,4-dihyropyridine Ca++ channel blocker, exhibited micromolar inhibition of spleen [3H]LTB4 binding.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001279 TI - Comparative cardiorespiratory effects produced by taurine and glycine applied to the ventral surface of the medulla. AB - The purpose of our study was to determine whether taurine, like other naturally occurring central nervous system amino acid neurotransmitters (e.g., gamma aminobutyric acid and glycine), act at the ventral surface of the medulla to influence cardiorespiratory activity. This was accomplished by monitoring cardiorespiratory activity. This was accomplished by monitoring cardiorespiratory activity in chloralose-anesthetized cats and then applying several doses of taurine locally to the ventral medullary surface chemosensitive areas. We found that 2 and 4 mumol of taurine applied to the intermediate area of the ventral surface produced cardiorespiratory depression, whereas taurine, in a similar dose range, produced only respiratory depression when applied to the rostral area. In contrast, taurine applied to the caudal area had no cardiorespiratory effects. Similar experiments with glycine revealed that this inhibitory amino acid elicited a similar pattern of cardiorespiratory depression as taurine. Furthermore, strychnine, an antagonist of glycine, counteracted the cardiorespiratory depressant effects of both taurine and glycine effectively. Pretreatment with strychnine prevented most of the cardiorespiratory depressant effects of taurine and glycine. 6-Aminomethyl-3-methyl-4H-1, 2,4-benzothiadiazine 1,1-dixoide, a putative antagonist of taurine, had antagonistic effects similar to those of strychnine in both treatment and pretreatment studies. 6-Aminomethyl 3-methyl-4H-1,2,4-benzothiadiazine-1,1-dioxide, per se, increased tidal volume when applied to the intermediate area; strychnine had no effect. These results indicate that taurine acts at the chemosensitive areas on the ventral surface of the medulla to produce cardiorespiratory depression, and these effects are due to an interaction of taurine with receptors similar to, but probably not identical with, glycine receptors. PMID- 3001281 TI - Mianserin attenuates naloxone-precipitated withdrawal signs in rats acutely or chronically dependent upon morphine. AB - The effects of the atypical antidepressant and serotonin antagonist mianserin on the expression of opiate withdrawal was examined using an acute and a chronic model of morphine dependence. In the first experiment, rats, trained to perform a food-reinforced, autoshaped lever touch response, were injected with naloxone (5 mg/kg) 4 hr after treatment with a single moderate dose of morphine (15 mg/kg). Mianserin (0.25, 1.0 and 2.5 mg/kg) attenuated the naloxone-induced suppression of autoshaped responding. Colonic temperatures were also monitored. Morphine treatment resulted in significant hyperthermia, while precipitation of withdrawal by naloxone produced hypothermia. Mianserin also attenuated the naloxone-induced hypothermia. In the second experiment, rats were implanted s.c. with a single 75 mg morphine or placebo pellet. Withdrawal was precipitated with naloxone (5 mg/kg) 24, 48 and 120 hr post implantation. Mianserin (2.5 mg/kg) blocked or attenuated signs of withdrawal precipitated by naloxone. Naloxone-precipitated weight loss was also attenuated 48 and 120 hr post implantation. At 120 hr post implantation, rats were decapitated 1 hr after the administration of naloxone and trunk blood was collected. Mianserin did not block the naloxone-induced rise in plasma corticosterone levels. Thus, several signs of withdrawal (e.g., behavioral effects, weight loss and hypothermia) seem to involve serotonergic mediation and can be blocked by mianserin, while others (e.g., rise in plasma corticosterone), which may be unaffected by mianserin, may be a reflection of a compensatory response to withdrawal stress, rather than a mediator of maladaptive consequences of withdrawal that are not mediated by serotonin. PMID- 3001283 TI - Effects of caffeine and 8-phenyltheophylline on the actions of purines and opiates in the guinea-pig ileum. AB - The antagonist effects of 8-phenyltheophylline (8-PT) and caffeine against the actions of adenosine, (-)-N6-(R-phenyl-isopropyl)-adenosine (PIA), morphine and nalorphine on the guinea pig ileum preparation were examined. Antagonism of both adenosine and PIA by caffeine and 8-PT was concentration dependent. The slopes of Schild plots for both alkylxanthines vs. adenosine were significantly less than 1 unless the adenosine reuptake blocker dipyridamole (0.1 microM) was include in the tissue bath. Under these conditions, the 95% confidence intervals of the Schild plot slopes embraced theoretical unity, suggesting competitive antagonism. The antagonism of PIA by the alkylxanthine was also competitive. Dipyridamole had no effect on the potency of PIA. The pA2 value for caffeine-adenosine was not different from that for caffeine-PIA, and the pA2 values of 8-PT-adenosine and 8 PT-PIA were similar, suggesting that these two agonists interacted with similar receptors. The pA2 values using 8-PT were approximately 1.5-fold higher than those employing caffeine, suggesting higher affinity for 8-PT at these receptors. Caffeine significantly antagonized morphine at all concentrations used (0.5-1.0 mM), but only antagonized nalorphine at the two highest concentrations. After treating ilea with the mu-specific irreversible antagonist beta-FNA (beta funaltrexamine) (resulting in a preparation with relatively pure kappa receptor population), the antagonist effect of caffeine against morphine was reduced such that only a concentration of 1 mM resulted in significant antagonism, while the effects of caffeine against nalorphine were unchanged. 8-PT did not antagonize morphine or nalorphine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001282 TI - Oxytocin causes endothelium-dependent relaxations of canine basilar arteries by activating V1-vasopressinergic receptors. AB - Experiments were designed to study the effects of oxytocin on canine basilar and femoral arteries and to compare these with the effects of vasopressin. Rings of the arteries were suspended in physiological salt solution for isometric tension recording. Oxytocin and vasopressin caused endothelium-dependent relaxation of basilar arteries contracted with prostaglandin F2 alpha. Vasopressin was more potent than oxytocin. In the femoral artery, the two hormones caused endothelium independent contractions with the same order of potency. The relaxations of the basilar artery occurred at lower concentrations of each substance than the contractions of the femoral artery. The relaxations in response to both agonists were inhibited competitively, and the contractions noncompetitively, by the V1 vasopressinergic antagonist d(CH2)5Tyr(Me)AVP; the antagonist did not affect endothelium-dependent relaxations in response to bradykinin. Thus, both oxytocin and vasopressin cause endothelium-dependent relaxation of the basilar artery by activating V1-vasopressinergic receptors; the contractions of femoral arteries that they cause also may be mediated in part by V1-vasopressinergic receptors. PMID- 3001284 TI - Stimulation of cardiac slow Ca++ current by high concentration of cimetidine. AB - Electrophysiological studies on the cardiac effects of a H2-antagonist, cimetidine, were examined in three kinds of preparations: guinea-pig papillary muscles, rat left atria and perfused chick hearts. Cimetidine (10(-5) to 10(-4) M) depressed or abolished the slow action potentials (APs) induced by histamine (10(-5) to 10(-4) M) in hearts whose fast Na+ channels had been inactivated by 25 mM K+. Higher concentrations of cimetidine (10(-3) to 5 X 10(-3) M) increased myocardial cyclic AMP level and allowed the generation of slow APs in such inexcitable tissues. These cimetidine-induced slow APs were not prevented by propranolol (10(-6) to 10(-5) M) or pretreatment with 6-hydroxydopamine (50 mg/kg). These results suggest that cimetidine, in doses higher than that required to block cardiac H2-receptors, may have a cardio-stimulating action mediated through increase of inward Ca++ current. PMID- 3001285 TI - Cardiac and coronary effects of prazosin and phenoxybenzamine during coronary hypotension. AB - Left coronary perfusion pressure was reduced to 50 mm Hg in alpha-chloralose anesthetized open chest dogs, causing significant reductions in coronary blood flow and left ventricular oxygen consumption (P less than .05). Infusion of the specific alpha-1 adrenergic antagonist prazosin (0.1 mg/min i.c.) during coronary hypotension significantly increased coronary flow and oxygen consumption within 6 to 8 min. These effects of prazosin were not attenuated by beta adrenergic blockade with propranolol. In the absence of propranolol, infusion of the nonspecific alpha adrenergic antagonist phenoxybenzamine (0.37 mg/min i.c.) increased coronary flow and oxygen consumption within 10 to 15 min, and these increases were significantly greater than with prazosin. In the presence of propranolol, phenoxybenzamine caused increases in coronary flow and oxygen consumption that were not different from those caused by prazosin. The results indicate that 1) prazosin increases coronary flow and oxygen delivery by abolition of a coronary constrictor tone mediated by postsynaptic alpha adrenoceptors; 2) the increases in coronary flow and oxygen consumption caused by phenoxybenzamine in the absence of beta adrenergic blockade were due to antagonism of pre- and postsynaptic alpha adrenoceptors; and 3) the increases in coronary flow and oxygen consumption caused by phenoxybenzamine after beta adrenergic blockade were due entirely to antagonism of coronary postsynaptic alpha-1 adrenoceptors. PMID- 3001286 TI - Alpha adrenoceptor subtype mediating sympathetic mobilization of blood from the hepatic venous system in anesthetized cats. AB - Previous studies have suggested that sympathetic effects on hepatic blood volume may be mediated through alpha-2 adrenoceptors in anesthetized cats as prazosin did not block whereas phentolamine and phenoxybenzamine impaired these responses markedly. In this study we have shown that yohimbine blocks hepatic volume responses to both nerve stimulation and norepinephrine infusions. In comparison with norepinephrine, phenylephrine had much weaker effects on hepatic blood volume than on arterial and portal pressures. Clonidine produced a slow weak contraction of the hepatic venous bed which was blocked by yohimbine but not by prazosin. alpha-Methylnorepinephrine produced a norepinephrine-like contraction of the hepatic venous bed which was blocked by yohimbine but not by prazosin. Taken together, these data suggest that hepatic blood volume responses to both sympathetic nerve stimulation and infusions of catecholamines are mediated through alpha-2 adrenoceptors, whereas portal pressure responses are mediated through both alpha-1 and alpha-2 adrenoceptors. PMID- 3001287 TI - Noradrenergic modulation of cortical acetylcholine release is both direct and gamma-aminobutyric acid-mediated. AB - The effect of norepinephrine (NE) on the outflow of gamma-aminobutyric acid (GABA) as well as the influence of endogenous GABA on NE-induced inhibition of acetylcholine (ACh) release have been investigated in guinea-pig cortical slices, in order to clarify the role of this amino acid in the noradrenergic control of the cholinergic signal. NE in the range of 9 to 30 microM increased GABA outflow from unstimulated slices, doubling it at 30 microM. This effect was antagonized by prazosin (0.1 microM) and by tetrodotoxin (0.5 microM), suggesting an involvement of alpha-1 receptors and of sodium-dependent mechanisms. 2,4 diaminobutyric acid (DABA), an inhibitor of neuronal GABA uptake, at 60 microM steadily doubled the amino acid outflow without affecting ACh release However, DABA increased the GABA-induced inhibition of electrically evoked ACh release by a factor of about 10 and the NE-induced inhibition by a factor of 2.8. Prazosin (1 microM) reduced the effects of NE (60 microM) on the spontaneous and evoked ACh release in normal slices and the effects of NE (30 microM) in DABA-treated slices. Thus, an alpha-1 GABA-mediated component of NE influence on ACh can be demonstrated even in vitro (beside the well-known alpha-2 direct inhibition), provided high NE concentrations are used or the GABA availability is enhanced. On the other hand, bicuculline and picrotoxin antagonized not only GABA but also NE, whereas yohimbine counteracted not only NE but also GABA inhibition of ACh release, suggesting a close assembly of alpha-2 and GABAA receptors on the cholinergic axons.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001289 TI - Sulfide and sulfoxide derivatives of substituted benzimidazoles inhibit acid formation in isolated gastric glands by different mechanisms. AB - The sulfoxide and sulfide forms of three pairs of substituted benzimidazoles, including omeprazole, were investigated using isolated gastric glands and microsomal membranes containing H+,K+-adenosine Triphosphatase (ATPase). The sulfoxides inhibited stimulated aminopyrine (AP) uptake giving IC50 values between 0.47 and 1.1 microM, whereas the sulfides were less potent and showed a greater variation in IC50 values, 9.5 to 170 microM. The decrease in stimulated oxygen consumption induced by the sulfoxides was parallel to their inhibition of AP-uptake, with IC50 values of 0.40 to 3.7 microM, whereas the sulfides were virtually without effect, IC50 greater than 100 microM. The permeable buffers imidazole and 2,6-dimethylpyridine mimiced the effect of the sulfides on both AP accumulation and oxygen consumption. When tested on H+, K+-ATPase, an enzyme suggested to be the proton pump of the gastric mucosa, the sulfoxides inhibited the ATPase activity with IC50 values between 0.25 to 2.8 microM, in contrast to the sulfides, which were without inhibitory action. The sulfoxide-induced inhibition of AP-uptake in gastric glands and H+, K+-ATPase activity was prevented by the addition of beta-mercaptoethanol, whereas the mercaptane was without effect on sulfide-induced inhibition of AP-accumulation. When tested on vesicles containing H+, K+-ATPase, both the sulfide and sulfoxide derivatives dissipated the proton gradient generated by the enzyme, but only the sulfoxide induced inhibition was prevented by the addition of beta mercaptoethanol.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001288 TI - Genetic influence on the regulation of beta adrenergic receptors in mice. AB - The regulation of beta adrenergic receptors was investigated in inbred mouse strains in which previous studies revealed differences in the regulation of dopamine receptors. The density of beta adrenergic receptors in the cerebral cortex of BALB/J mice was about one-third of that in CBA/J and C57BL/6J mice. Strain differences in the binding of [125I]iodohydroxypindolol to beta adrenergic receptors were due to changes in the density of beta-1 adrenergic receptors. Chronic administration of propranolol did not result in an increase in the density of beta adrenergic receptors receptors in cortices of C57BL/6J and BALB/cJ mice were observed. In contrast, pretreatment with 6-hydroxydopamine resulted in increases in the density of beta adrenergic receptors in the cerebral cortex of all three strains. Analysis of the effects of these treatments on the subtypes of beta adrenergic receptors revealed that the changes were restricted to changes in the density of beta-1 receptors. The failure to observe a response to propranolol in CBA/J mice expands the extent of deficits reported previously in this strain for striatal dopamine receptor supersensitivity after chronic treatment with haloperidol (Severson et al., Brain Res. 210: 201-215, 1981). CBA/J mice may be a useful model for genetic analysis of mechanisms for the control of receptor sensitivity and to investigate the impairments of the regulation of catecholaminergic receptors observed in aged rodents. PMID- 3001290 TI - Autoradiographic localization of benzodiazepine receptor downregulation. AB - Regional differences in downregulation of brain benzodiazepine receptors were studied using a quantitative autoradiographic method. Rats were given a 4-week flurazepam treatment known to cause tolerance and receptor downregulation. A second group of rats was given a similar treatment, but for only 1 week. A third group was given a single acute dose of diazepam to produce a brain benzodiazepine like activity equivalent to that found after the chronic treatment. Areas studied included hippocampal formation, cerebral cortex, superior colliculus, substantia nigra, dorsal geniculate nucleus, lateral amygdala and lateral hypothalamus. There was a regional variation in the degree of downregulation after 1 week of flurazepam treatment, ranging from 12% to 25%. Extending the flurazepam treatment to 4 weeks caused little further downregulation in those areas studied, except for the pars reticulata of the substantia nigra, which showed a 13% reduction in [3H]flunitrazepam binding after 1 week and a 40% reduction after 4 weeks of treatment. In a few areas, such as the lateral hypothalamus, no significant change in binding was found after 4 weeks. Acute diazepam treatment caused no change in binding. This latter finding as well as results obtained during the development of the methodology show that downregulation was not an artifact due to residual drug content of brain slices. The regional variations in degree and rate of downregulation suggest areas that may be most important for benzodiazepine tolerance and dependence and may be related to the varying time courses for tolerance to different benzodiazepine actions. PMID- 3001292 TI - In vivo dephosphorylation of WR-2721 monitored by 31P NMR spectroscopy. AB - The in vivo dephosphorylation of the radioprotective agent S-2-[3 (aminopropylamino)]ethylphosphorothioic acid (WR-2721) in male CD2F1 mice was measured by 31P NMR spectroscopy after i.p. injection. The disappearance of the WR-2721 phosphate NMR signal with time was concurrent with an increase and splitting of the inorganic phosphate NMR signal. The more acidic inorganic phosphate resonance is shown to be attributed to phosphate (inorganic phosphate) in the urine. Using regression first-order kinetic analysis of data, after i.p. injection of 600 mg/kg, the half-life of WR-2721 was determined to be 40.9 +/- 5.9 (S.D.) min (n = 10). PMID- 3001291 TI - Dissimilarities between methylene blue and cyanide on relaxation and cyclic GMP formation in endothelium-intact intrapulmonary artery caused by nitrogen oxide containing vasodilators and acetylcholine. AB - The objective of the present study was to ascertain whether cyanide shares the properties of methylene blue as a selective inhibitor of vascular smooth muscle relaxation elicited by agents that stimulate the formation of cyclic GMP. Experiments were performed with endothelium-intact rings prepared from bovine intrapulmonary artery. Methylene blue, a good inhibitor of soluble guanylate cyclase, antagonized both arterial relaxation and cyclic GMP accumulation in response to sodium nitroprusside, glyceryl trinitrate, S-nitroso-N acetylpenicillamine and acetylcholine. In contrast, cyanide inhibited only the responses to sodium nitroprusside. Increasing concentrations of methylene blue depressed resting arterial levels of cyclic GMP and caused slowly developing but marked contractions whereas cyanide was without effect. Contractile responses to phenylephrine, potassium and U46619 were potentiated by methylene blue but not by cyanide. Preincubation of dilute solutions of cyanide containing sodium nitroprusside in oxygenated Krebs' buffer at 37 degrees C for 15 min before addition to bath chambers depressed relaxation and cyclic GMP accumulation caused by sodium nitroprusside markedly. Similar treatment of glyceryl trinitrate, however, failed to alter its effects in arterial rings. A chemical inactivation of sodium nitroprusside by cyanide appears to account for the specific inhibitory action of cyanide on arterial responses to sodium nitroprusside. This study indicates clearly that cyanide does not share the properties of methylene blue as an inhibitor of arterial relaxation elicited by vasodilators that stimulate cyclic GMP formation. PMID- 3001293 TI - Kinetic definition of agonist efficacy at a 5-hydroxytryptamine (5-HT2) receptor in the isolated rabbit aorta. AB - The contractile response of the isolated rabbit aorta elicited by 5 hydroxytryptamine (5-HT) and five partial agonists acting on the 5-HT2 receptor were separated into a phasic and a tonic response by altering the [Ca++] in the buffer. A kinetic analysis of the two responses yields parameters that provide a mechanistic insight into the different nature of these responses. The kinetic parameters of the phasic contraction indicate that the onset of this response depends on the access of the drug to the receptor and that its decay is independent of the nature and the concentration of the agonist. The observed rate constant of the onset of the tonic response, kobs, is saturable with increasing drug concentration, suggesting that the rate determining step is the activation of an effector by the preformed drug-receptor complex. These kinetic characteristics of the 5-HT2-mediated response are similar to those observed previously by us for the alpha-1 adrenergic receptor-mediated response in the rabbit aorta, suggesting that these receptors activate similar mechanisms related to the mobilization of Ca++. Furthermore, it is shown that the maximal values of kobs for the 5-HT2 agonists follow the rank order of maximal amplitudes of the phasic responses and the maximal steady-state levels of the tonic response. It is suggested that the maximal value of kobs may serve as a kinetic measure of drug efficacy. PMID- 3001294 TI - Effect of age on beta adrenergic relaxation of the rat jugular vein. AB - Using the Fisher 344 rat model and blood vessel ring segments in vitro, age related changes in vascular beta adrenergic relaxation were investigated. In the pulmonary artery and aorta, maximum isoproterenol-induced relaxation and sensitivity to isoproterenol declined from 1 to 3 months of age confirming previous reports. In animals 6 months of age, these vessels no longer relaxed to isoproterenol. In the jugular vein, in which beta adrenergic mechanisms predominate, there was no change in maximum relaxation to isoproterenol or in EC50 values in animals 3 to 27 months of age. Furthermore, determination of propranolol dissociation constants (KB) showed no change in affinity up to 27 months of age. Thus, in venous smooth muscle, in contrast to arteries, beta adrenergic relaxation is well maintained through senescence. PMID- 3001296 TI - Evidence for the ordered release of rubidium ions occluded within the Na,K-ATPase of mammalian kidney. AB - When Na,K-ATPase containing occluded rubidium ions is exposed to orthophosphate, in the presence of magnesium ions, there is a rapid release of half or all of the occluded ions. This behaviour is observed irrespective of whether the occluded rubidium form of the enzyme is generated by putting the unphosphorylated enzyme in a sodium-free medium containing rubidium ions, or by allowing rubidium ions to catalyse the hydrolysis of phosphoenzyme made by adding ATP to enzyme suspended in a medium containing sodium and magnesium ions. The release of occluded rubidium ions by orthophosphate requires the presence of magnesium, presumably because phosphorylation is necessary. Whether the addition of orthophosphate causes the rapid release of all or of half of the occluded rubidium depends on whether free rubidium (or potassium, thallium or (probably) caesium ions) are present in the medium at the time the orthophosphate is added. In the absence of free ions of these species, all of the occluded rubidium is released. In their presence (in adequate concentration), only half of the occluded rubidium is released. The relative effectiveness of the different potassium congeners in preventing the rapid release of 50% of the occluded rubidium when orthophosphate is added is: thallium greater than rubidium greater than potassium greater than caesium. Lithium and sodium are ineffective even at high concentrations, and sodium ions strongly antagonize the effect of free rubidium ions. In a sodium free, Tris medium, the concentration of free rubidium ions necessary for a half maximal effect is about 30 microM. In a medium containing 250 microM-free rubidium, the concentration of sodium necessary to reduce the effect of free rubidium by 50% is about 500 microM. These figures are compatible with the hypothesis that the free rubidium or other ions act at the potassium-loading sites at the extracellular face of the pump. By starting with enzyme occluding unlabelled rubidium, and using 86Rb-labelled free rubidium, it is possible to show that the free ions that prevent the rapid release of half of the occluded ions themselves become occluded. These experiments are significant in two ways. First, they provide direct evidence for the existence of a second route for the release of occluded rubidium (and therefore presumably of occluded potassium) ions. Secondly, they seem to require that the release of occluded ions by this route occurs in an ordered fashion. PMID- 3001295 TI - Adaptation in the input-output relation of the synapse made by the barnacle's photoreceptor. AB - A study was made of synaptic transmission between the four median photoreceptors of the giant barnacle (Balanus nubilus) and their post-synaptic cells (I-cells). Simultaneous intracellular recordings were made from the presynaptic terminal region of a photoreceptor and from the soma of an I-cell. The photoreceptor's membrane potential provided feed-back to bath electrodes that passed current into the receptors' axons, permitting the voltage to be controlled at the point of arborization of their presynaptic terminals. Simultaneous recordings from a second photoreceptor showed that its voltage tracked the first. Step depolarizations of the receptors from their dark resting potential (about -60 mV) caused hyperpolarizations of the I-cell that reached a peak, then decayed to a plateau value. The amplitude of the I-cell's response grew with presynaptic depolarizations, saturating at presynaptic values 10-20 mV depolarized from dark rest. Step hyperpolarizations of the receptors from dark rest evoked depolarizations of the I-cell consisting of an initial peak, which varied greatly in amplitude and wave form from preparation to preparation, followed by a plateau. The presence of this post-synaptic response indicates that transmitter is released continuously from the receptors at their dark resting potential. An input-output relation of the synapse was obtained by presenting step depolarizations from a holding potential of -80 mV, where steady-state transmitter release is shut off. The relation is sigmoidal; in the exponentially rising phase of the curve, a 5-11 mV presynaptic change produces a 10-fold change in post-synaptic response. When the presynaptic holding potential was set at values ranging from -80 to -40 mV, the relation between the I-cell's response and the absolute potential to which the receptor was stepped shifted along the presynaptic voltage axis. The slopes of the input-output relations were roughly parallel or increased as the photoreceptors were held more depolarized. This observation limits the possible mechanisms of the shift. PMID- 3001297 TI - Differential effects of carbon dioxide and pH on central chemoreceptors in the rat in vitro. AB - The brain stem, cervical cord and attached phrenic nerve were excised from neonatal rats and superfused in vitro. Respiratory activity was recorded from the phrenic nerve following transection of all the cranial nerves and dorsal roots. The frequency of spontaneous periodic activity recorded from the phrenic nerve was 6-14/min during superfusion with a saline solution equilibrated with 5% CO2 in O2 at 25 degrees C (pH 7.3). The magnitude of respiration was estimated from the peak value of phrenic activity integrated for each 0.1 s period. When the pH of the superfusion fluid was altered by changing the HCO3-concentration at constant PCO2, respiratory activity increased in low pH and decreased in high pH. These changes were maintained as long as a given pH was held. Respiratory changes observed under these conditions were characterized by alterations in both respiratory frequency and magnitude. When the CO2 level of the superfusion fluid was altered, maintaining constant pH by modified HCO3-concentrations, respiratory activity increased at high PCO2 and decreased at low PCO2. These changes were transient and lasted only for a few minutes after exposure to a new level of PCO2. Respiratory changes observed under these conditions were characterized by alterations in magnitude but not in frequency. At constant PCO2 an increase in the HCO3-concentration occasionally enhanced the magnitude of respiration before respiratory activity was depressed by the increased pH. This suggests that HCO3- may act independently as a stimulus to the central chemoreceptor. It is concluded that the mammalian central chemoreceptor for respiratory control is responsive independently to H+ and CO2 and that H+ and CO2 exert differential effects on the respiratory centre in terms of frequency and magnitude. It is suggested that frequency modulation and magnitude (tidal volume) modulation for respiratory control are triggered at different regions in the respiratory centre and/or rely on different mechanisms. PMID- 3001298 TI - Caring for the patient who has oat cell lung cancer. PMID- 3001299 TI - Thyroid function and urine-concentrating ability during lithium treatment. AB - It has been suggested that adenylate cyclase inhibition may be important in the development of both nephrogenic diabetes insipidus and hypothyroidism during lithium treatment. We measured serum thyroxine and urine-concentrating ability (Umax) in response to desmopressin (DDAVP) in 85 patients receiving lithium. Hypothyroidism developed in eight patients while they were taking lithium. Impaired Umax was found in both euthyroid and hypothyroid patients while some hypothyroid patients concentrated their urine well. It is concluded that the dominant mechanisms by which lithium exerts these two effects are different. PMID- 3001300 TI - Platelet alpha 2-adrenoceptors in major depression: relationship with urinary 4 hydroxy-3-methoxyphenylglycol and age at onset. AB - Platelet alpha 2-adrenoceptor number and affinity were measured in 31 drug-free patients with major depressive illness utilizing 3H-clonidine as ligand. A significant negative correlation was found between number of alpha 2 adrenoceptors, baseline urinary 4-hydroxy-3-methoxyphenylglycol (MHPG) excretion, present age and age at onset of the disease. Kd did not correlate with any of these variables not with the Bmax of platelet alpha 2-adrenergic binding. Multiple regression analysis, with MHPG and age at onset as independent variables, explained variance for alpha 2-adrenoceptor density better than single regression (from 19% for MHPG and 30% for age at onset to 40%), with the addition of both these variables being significant. PMID- 3001301 TI - Imaging of [11C]-labelled Ro 15-1788 binding to benzodiazepine receptors in the human brain by positron emission tomography. AB - The benzodiazepine antagonist Ro 15-1788 was labelled with [11C] and examined for possible use as ligand for PET scan studies on benzodiazepine receptors in the brain of cynomolgus monkeys and human subjects. [11C] Ro 15-1788 allowed the in vivo visualization of benzodiazepine receptor binding in cerebral and cerebellar cortical areas as well as in basal brain nuclei in PET scan images. [11C] Ro 15 1788 exhibited a high ratio of specific benzodiazepine receptor binding (cerebral cortex) to non-specific binding (pons) and the kinetics of binding should be satisfactory for quantitative clinical PET scan studies using [11C]. The in vivo binding of [11C] Ro 15-1788 in the cerebral cortex of cynomolgus monkeys and healthy human subjects was reduced by approximately 90% within 10 min after the intravenous injection of a high dose of unlabelled Ro 15-1788 (0.5 mg/kg i.v.). Different areas of the healthy human brain showed an approximately 10-fold variation in maximal [11C] Ro 15-1788 binding that corresponded to the previously known distribution of benzodiazepine receptors in these regions. The highest degree of binding was obtained in the medial occipital cerebral cortex followed by frontal cortex, cerebellum, thalamus, striatum and pons. Two psychiatric patients with anxiety syndromes who had been treated for a long time with high doses of benzodiazepines had roughly the same degree of maximal [11C] Ro 15-1788 binding in brain regions as the healthy subjects but the rate of decline of [11C] Ro 15-1788 in the brain was higher. This indicates that there is measurable competition between [11C] Ro 15-1788 binding and clinical benzodiazepine concentrations in the body fluids of psychiatric patients. The results demonstrate that [11C] Ro 15-1788 should be a valuable tool for quantitative analyses of benzodiazepine receptor characteristics and receptor occupancy in the brain of patients with neuropsychiatric disorders. PMID- 3001302 TI - Diverticular disease treated with corticotrophin. AB - Since 1968 the inflammatory stage of diverticular disease (acute and chronic diverticulitis) has been treated with tetracosactrin in one practice. This paper reviews 100 episodes treated in this way and compares these with 50 episodes treated with rest in bed and dietary measures. Abatement of pyrexia, swelling and tenderness, as well as relief of the symptoms of pain and malaise, were usually found to occur within 24 hours of the administration of tetracosactrin zinc (1 mg) intramuscularly. No complications directly attributable to this therapy have been observed, while the duration of the clinical illness has been reduced by more than half. In neither group were antibiotics found to influence the outcome. PMID- 3001303 TI - Parvovirus infection causing arthralgia. PMID- 3001304 TI - Stereoselective syntheses of the trans-decahydroquinoline-5-carboxylic acid epimers. Diastereomeric zwitterionic probes of gamma-aminobutyric acid related biological properties in vitro and in vivo. AB - The syntheses of the 5 beta and 5 alpha epimers of trans-(4a alpha,8a beta) decahydroquinoline-5-carboxylic acids (3 and 4) from vinylogous bicyclic imide 10 are described. The reduction of trans-5-(1,3-dithian-2-ylidene)octahydro-2(1H) quinolinone (13) to afford the 5 alpha-(1,3-dithian-2-yl) compound 16 was a key step in the synthesis of trans-4 while hydroboration-H2O2 treatment of phenylmethyl trans-octahydro-5-methylene-1(2H)-quinolinecarboxylate (21) to afford the 5 beta-hydroxymethyl compound 22 was a key step in the synthesis of 3. These trans diastereomers 3 and 4 and the previously prepared cis analogues 1 and 2 were investigated for their ability to interact with GABAA and GABAB receptors and picrotoxin binding sites as well as with neuronal GABA transport systems in brain tissue. Like 1 and 2, tonic-clonic seizures were induced when trans-3 or -4 were administered to mice intracerebroventricularly. Only trans-4 weakly inhibited [3H]GABA binding to GABAA and GABAB receptors in vitro. Large doses (10 mg/kg) of diazepam reversed the convulsant activity of both trans-3 and trans-4. Although trans-3 is the more potent convulsant, trans-4 may have GABA antagonist activity in vivo. However, none of the decahydroquinoline diastereomers have a pronounced effect on GABA receptors that can currently be studied in vitro. Results obtained in vivo lead us to propose that these diastereoisomers may serve as unique conformational probes relating certain zwitterionic topographies to stimulatory activity in the central nervous system. PMID- 3001305 TI - [p-[(Thienylcarbonyl)amino]phenoxy]propanolamines derivatives as diuretic and beta-adrenergic receptor blocking agents. AB - The synthesis of [[(thienylcarbonyl)amino]phenoxy]propanolamines and their beta adrenergic blocking and diuretic activity are described. Structure-activity relationships demonstrated that ortho substitution of the phenoxy ring with an hydrogen or an ester function leads to compounds possessing both activities. Ethyl 2-[3-[(1,1-dimethylethyl) amino]-2-hydroxypropoxy]-5-[(2 thienylcarbonyl)amino]benzoate (3d) was selected as the most active compound for further investigation. PMID- 3001307 TI - Correspondence analysis applied to steroid receptor binding. AB - The relative binding affinities of 48 steroids for four classes of hormone receptor (progestin, PR; androgen, AR; glucocorticoid, GR; mineralocorticoid, MR) have been analyzed by correspondence analysis. The steroids were, for the most part, derivatives of nortestosterone, differing by their degree of unsaturation, by the presence or absence of a 17 alpha-ethynyl group, and by the length of the C-13 alkyl substituent. Derivatives of norprogesterone were included as reference compounds. Distribution maps visualizing the results of the mathematical analysis revealed that the majority of the test steroids were within the zone of influence of AR and PR and had limited affinity for GR and MR. Overall lack of specificity and enhanced affinity for GR and MR were induced by increasing unsaturation and by the presence of a C-13 ethyl group. The general and specific conclusions of the analysis confirm and extend previous intuitive and partial interpretations of the data. Correspondence analysis, however, has the advantage of taking into account the sum total of the available information, without any preconceived notion of the relative importance of a specific structural feature or biological parameter and, furthermore, enables simultaneous representation on a single graph of the receptor and steroid fields. The present example demonstrates the use of this type of methodology in processing routine screening data involving multiple parameters. PMID- 3001306 TI - Nucleosides. 136. Synthesis and antiviral effects of several 1-(2-deoxy-2-fluoro beta-D-arabinofuranosyl)-5-alkyluracils. Some structure-activity relationships. AB - In order to study structure-activity relationships between antiherpetic activity and the size of the C-5 alkyl substituents of 2'-fluoro-ara-U derivatives, six new nucleosides (1c-h) were synthesized. The 5-allyl analogue 1c was prepared by a Pd(II)-catalyzed reaction of 5-(chloromercuri)-1-(2-deoxy-2-fluoro-beta-D arabinofuranosyl)uracil with allyl chloride. Partial hydrogenation of 1c afforded the 5-n-propyl derivative 1d (FPAU). Nucleosides 1e-h were obtained by condensation of 3-O-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-D-arabinosyl bromide with the corresponding 5-substituted uracils. Preliminary in vitro data show that, as the alkyl side chain is increased by one carbon unit, the antiherpetic potency is decreased by approximately 1 log order. The cytotoxicity also diminishes as the size of the 5-substituent is increased. FPAU exerts good activity against HSV-1 and HSV-2. FiPAU still shows good therapeutic indices, whereas the higher alkyl analogues are essentially inactive. PMID- 3001308 TI - Synthesis and antiviral activity of the carbocyclic analogues of 5-ethyl-2' deoxyuridine and of 5-ethynyl-2'-deoxyuridine. AB - The carbocyclic analogue of the antiviral agent 5-ethyl-2'-deoxyuridine (EDU) was synthesized by two routes. The pivotal step in the first route is the reaction of lithium dimethylcuprate with the carbocyclic analogue of 5-(bromomethyl)-2' deoxyuridine dibenzoate (6). The second route is based on the synthesis of the carbocyclic analogue of 5-ethynyl-2'-deoxyuridine (12) by a coupling reaction catalyzed by bis(triphenylphosphine)palladium(II) chloride and copper(I) iodide, a method reported recently (Robins and Barr) for the synthesis of the true nucleoside 5-ethynyl-2'-deoxyuridine (1b). The carbocyclic analogue of EDU inhibits the replication of type 1 and type 2 herpes simplex viruses in Vero cells. The carbocyclic analogue of 5-ethynyl-2'-deoxyuridine has modest activity against herpes simplex virus, types 1 and 2. PMID- 3001309 TI - Genetics and biochemical variability of variants of 21 hydroxylase deficiency. AB - In a population and family study we have examined the relationship between HLA types, classical congenital adrenal hyperplasia (CAH), and variants of 21 hydroxylase (21 OH) deficiency detected by increased blood levels of 17 hydroxyprogesterone (17 PO) in response to ACTH after overnight suppression with dexamethasone ('short Synacthen test'). In a non-CAH population, 7.7% of subjects were found to have raised 17 PO response suggesting reduced activity of 21 OH. Such subjects with raised 17 PO levels were designated simply as type 2 responders because the relationship with genotype was unknown. Post-ACTH levels of 17 PO were significantly greater in type 2 responders than in obligate carriers of CAH. A total of 2.5% of the population studied also had raised progesterone (PO) levels in the Synacthen test. HLA-A28 and B14 (in linkage disequilibrium) were significantly increased in frequency and HLA-B12 decreased in the type 2 responders. HLA-Bw47, which is known to be associated with CAH, was found only among obligate carriers of classical CAH. Because type 2 response and classical CAH are linked to HLA but are associated with different antigens, it is likely that they are determined by two (or more) alleles. PMID- 3001311 TI - Genetic linkage between Huntington's disease and the DNA polymorphism G8 in South Wales families. AB - Analysis of the polymorphism shown by the DNA probe G8 in eight South Wales families with Huntington's disease has confirmed close genetic linkage between this marker and the disorder, the most likely genetic distance being two centimorgans (cM). The closeness of the linkage suggests that G8 may have clinical applications in genetic prediction for this condition. PMID- 3001310 TI - Prenatal diagnosis of the common haemoglobin disorders. AB - New techniques of DNA analysis have been applied to the prenatal diagnosis of the common haemoglobin disorders. Currently, it is possible to provide a comprehensive programme for the prevention of these conditions, although this entails the use of several different techniques including globin chain synthesis analysis, direct identification of mutations with restriction enzymes, linkage analysis of restriction fragment length polymorphisms, and the use of oligonucleotide probes. At present, the best combination of these approaches has to be worked out for individual populations, but as the techniques of chorion villus sampling and DNA analysis improve it should be possible to rationalise these prenatal diagnosis programmes and thus make them simpler and less expensive. PMID- 3001312 TI - Prenatal diagnosis of ornithine carbamoyl transferase deficiency using a gene specific probe. AB - A gene specific DNA probe has been used to predict the genotype of two fetuses in families at risk for ornithine carbamoyl transferase deficiency. Although the probe does not detect the mutation directly, prediction was possible by examining restriction fragment length polymorphisms of the parents and sibs to identify the X chromosome carrying the mutation. It is suggested that in all pregnancies, regardless of the predicted outcome, the biochemical status of carrier mothers should be monitored because hyperammonaemia and arginine deficiency may have a deleterious effect on the fetus. PMID- 3001314 TI - On the sequence determinants and flexibility of the kinetoplast DNA fragment with abnormal gel electrophoretic mobilities. AB - A 410 base-pair (bp) Sau3A restriction fragment derived from a Leishmania tarentolae kinetoplast DNA minicircle, which is known to have slower than expected electrophoretic mobilities in polyacrylamide gels, has been cloned in a plasmid and deletions from one end of the cloned segment have been constructed. Analysis of the gel electrophoretic mobility data of a large number of restriction fragments derived from the kinetoplast DNA clone and its deletion subclones has led to the conclusion that two sequences, one in the region bp 100 to 170 and the other bp 190 to 250, both numbered from one end of the 410 bp kinetoplast DNA segment, are important for the abnormal gel electrophoretic behavior of the kinetoplast DNA fragment. One common feature of these sequences is the periodic presence of short runs of A residues (3 to 6 As in each); auto correlation analysis of these runs of A residues shows a strong harmonic component with a period around 11 bp. These results support and extend the previous analysis of Wu & Crothers (1984). The abnormal electrophoretic behavior is accentuated at low temperature and by the addition of Mg2+ to the electrophoresis buffer; addition of Na+ has the opposite effect. Insertion of sequences derived from the kinetoplast DNA fragment into nicked circular DNA causes no unexpected change in its electrophoretic mobility in agarose gel, suggesting that the 410 bp sequence, or segments of it, has no significant spatial writhe. Abnormal shifts in agarose gel mobilities are observed, however, when certain segments of the kinetoplast DNA are inserted into positively or negatively supercoiled DNA topoisomers. These results are consistent with a bent structure of the kinetoplast DNA in which the bend has zero writhe in its undistorted form but is easily distorted. PMID- 3001313 TI - Collagen genes and proteins in osteogenesis imperfecta. AB - Type I collagen is a heteropolymer of alpha 1(I) and alpha 2(I) chains, each of which is a separate product of genes localised to chromosomes 17 and 7 respectively. Molecular defects of type I collagen produce a group of inherited disorders of connective tissue primarily affecting bones, which are easily broken and collagen depleted (osteogenesis imperfecta). Sillence classifies these diseases into four groups, two of which are autosomal dominant and relatively mild, the others being either genetic lethals or responsible for very severe progressive disease. Here we describe two specific molecular abnormalities of type I collagen. One, a cysteine substitution in alpha 1(I) collagen, causes a mild Sillence type I disease, the other, a four base deletion in the C terminal extension of alpha 2(I) collagen, causes progressive Sillence type III disease in the homozygously affected patient and mild premature osteoporosis in his clinically symptomless parents. We have briefly reviewed a variety of other similar mutations causing various OI syndromes, which are tabulated, including various helical and non-helical deletions and a variety of structural protein changes. Several restriction fragment length polymorphisms for alpha 2(I) and alpha 1(II) collagens have also been described, and 5' EcoRI and 3' MspI polymorphisms for alpha 2(I) collagen segregate with Sillence type IV OI. PMID- 3001315 TI - On the mechanism of amplification of satellite II DNA sequences of the domestic goat Capra hircus. AB - A restriction enzyme analysis of the satellite II DNAs of the domestic goat Capra hircus, sheep Ovis aries and ox Bos taurus (p = 1.720, 1.723 and 1.722 g/cm3, respectively) has been carried out and shows that, although all three are composed of repeat units of 700 base-pairs, goat satellite II is present in the genome primarily in the form of 2100 base-pair trimers. Unequal crossing-over between repeat units has produced an oligomer series, whose oligomers gradually decrease in copy number the further they are in molecular weight from the trimer. The trimer population is much more uniform than the monomer population, as most trimers have similar restriction patterns, whereas their component monomers differ considerably in their restriction properties. This heterogeneity was confirmed by cross-hybridization of the different monomers of a cloned trimer. Here, heterologous hybrids were much less stable than the homologous hybrids. Attempts were made to simulate such an oligomer series by computer, using a longitudinal (saltatory), and two horizontal (unequal crossover and drive) models. Simulations of both the saltatory and drive mechanisms could produce the oligomer series in approximately the observed ratios, but only the former could simultaneously produce other restriction properties of this sequence family. This was because the other restriction sites were in a different (monomer) register, and it is difficult for a drive model promoting traits in only one register to fix properties in different registers. The unequal crossover model proposed by Smith (1973, 1976) generally produced homogeneous arrays from heterogeneous arrays, but higher-order repeat structures could be produced when the efficiency of crossing-over between different monomer types was reduced. In most of these arrays, the dimer was the predominant oligomer, but in approximately 10% the trimer was predominant. Since the unequal crossover model produces dimeric arrays with high frequency given appropriate conditions, it is an attractive model for explaining the production of satellite DNAs whose structure has evolved through a series of doublings, such as mouse major satellite DNA and the "alphoid" satellite sequences of primates. Other factors necessary for this model to work are generally considered to be natural components of the speciation process. It is therefore suggested that, although the saltatory model conforms most closely to the observed structure of goat satellite II, this particular satellite DNA may represent one of the few cases when the unequal crossover mechanism does not give rise to a dimeric structure. PMID- 3001316 TI - Human alphoid family of tandemly repeated DNA. Sequence of cloned tetrameric fragments and analysis of familial divergence. AB - Three tetramers of the 170 base-pair monomer repeat unit of human centromeric DNA (alphoid DNA) have been cloned and sequenced. Adjacent subunits differed in sequence by 30 to 45%, while dimers varied by 13 to 20% whether adjacent or not. Divergence was distributed unevenly across the monomeric sequence, such that two highly conserved segments adjoined clusters of insertions/deletions. Divergence, calculated from the cloned sequences or measured in uncloned DNA by thermal destabilization of mismatched reassociated duplexes, was far greater than previously estimated for the total human alphoid family. The population of these repeats within the human genome was not uniformly diverged, however, since restriction subsets of alphoid DNA contained as little as one-tenth the overall level of divergence. This indicates a hierarchical or familial structure of the genomic repeat population. Using cloned probes, human alphoid DNA was shown to be highly methylated and transcriptionally inactive. These data, along with evidence of conserved segments and periodicities (dimeric and higher-order) overlying considerable sequence diversity, support a structural role for this DNA family. PMID- 3001317 TI - EcoA and EcoE: alternatives to the EcoK family of type I restriction and modification systems of Escherichia coli. AB - The genes (hsd A) encoding EcoA, a restriction and modification system first identified in Escherichia coli 15T-, behave in genetic crosses as alleles of the genes (hsd K) encoding the archetypal type I restriction and modification system of E. coli K12. Nevertheless, molecular experiments have failed to detect relatedness between the A and K systems. We have cloned the hsd A genes and have identified, on the basis of DNA homology, related genes (hsd E) conferring a new specificity to a natural isolate of E. coli. We show that the overall organization of the genes encoding EcoA and EcoE closely parallels that for EcoK. Each enzyme is encoded by three genes, of which only one, hsdS, confers the specificity of DNA interaction. The three genes are in the same order as those encoding EcoK, i.e. hsdR, hsdM and hsdS and, similarly, they include a promoter between hsdR and hsdM from which the M and S genes can be transcribed. The evidence indicates that EcoA and EcoE are type I restriction and modification enzymes, but they appear to identify an alternative family to EcoK. For both families, the hsdR polypeptide is by far the largest, but the sizes of the other two polypeptides are reversed, with the smallest polypeptide of EcoK being the product of hsd S, and the smallest for the EcoA family being the product of hsdM. Physiologically, the A restriction and modification system differs from that of K and its relatives, in that A-specific methylation of unmodified DNA is particularly effective. PMID- 3001318 TI - EcoA: the first member of a new family of type I restriction modification systems. Gene organization and enzymatic activities. AB - The characterization of the EcoA restriction-modification enzymes from Escherichia coli 15T- is described. The reactions catalysed by these enzymes are very similar to those catalysed by the classical type I restriction and modification enzymes, a family of genetically related proteins. The detailed mechanisms, particularly for DNA modification, differ. The genetic and transcriptional organizations are also very similar to those of the classical systems, despite the fact that EcoA is not allelic to the others. We demonstrate that the expression of the EcoA genes is controlled following conjugative transfer to other strains in such a way that no lethality is observed, probably because the recipient chromosome is completely modified before restriction activity is expressed. PMID- 3001319 TI - The sodium/hydrogen exchange system in cardiac cells: its biochemical and pharmacological properties and its role in regulating internal concentrations of sodium and internal pH. AB - This paper describes the properties of the amiloride-sensitive Na+/H+ antiporter in chick cardiac cells, compares them with those known in other cellular systems and analyzes the role of the Na+/H+ exchanger in the regulation of internal Na+ concentrations and internal pH. Among the different properties which have been studied one can mention: (i) The external Na+ concentration [( Na+]o) dependence: the activity increases when [Na+]o increases (KNa+ = 20 mM); (ii) The external pH (pHo) dependence: the activity of the exchanger increases when pHo increases (pHmo = 7.05 and Hill coefficient = 1); (iii) The internal pH (pHi) dependence; the activity of the exchanger increases in a cooperative way when internal pH (pHi) decreases (pHmi = 7.35 and Hill coefficient = 3); (iv) There are derivatives of amiloride which are 200 times more potent than amiloride itself (Kethylisopropylamiloride = 30 nM) and which are selective on the Na+/H+ exchange system v. other Na+ transporting system including the Na+/Ca2+ exchange system. Under physiological conditions, the Na+/H+ exchange system contributes little to the regulation of the internal pH of chick cardiac cells. The antiporter then serves as an uptake system for Na+ using the H+ gradient created by other pHi regulatory mechanisms. Treatment of cardiac cells with ouabain inhibits Na+ efflux and produced an increase in intracellular Na+ activity. Ethylisopropylamiloride was used to show that the Na+/H+ exchange system is the main pathway for Na+ entry and accumulation in digitalis action. As expected amiloride derivatives which block Na+ entry via the Na+/H+ antiporter were found to antagonize ouabain action on cardiac cells. When the internal pH of cardiac cells is lowered, the Na+/H+ exchanger becomes the major pHi regulating system. It is the essential system by which cardiac cells recover from cellular acidosis. The situation is due both to an increased activity of the exchanger at acidic pHi and to a decreased activity of other pHi regulatory systems. We propose in this paper that the Na+/H+ exchange system plays a key role in Na+ accumulation followed by Ca2+ accumulation which is observed when ischemic hearts are reperfused. PMID- 3001320 TI - Influence of protein kinase phosphorylation on isolated sarcolemmal membranes. AB - A cardiac muscle sarcolemmal preparation, enriched in adenylate cyclase, Na+, K+ ATPase, beta, muscarinic and ouabain receptors, also contained endogenous protein kinase activity. Phosphorylation of sarcolemmal membrane proteins by the endogenous protein kinase occurred mainly on 22 000 and 12 000 Mr proteins. To determine the effect of this phosphorylation on sarcolemmal properties, sarcolemmal vesicles were preincubated under conditions for optimal phosphorylation while control vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both control and phosphorylated vesicles were centrifuged, resuspended in 10 mM Tris-Cl (pH 7.4) and subsequently assayed for ATPase activities and for binding of ouabain, dihydroalprenolol and quinuclidinyl benzilate to the membranes. Sarcolemmal phosphorylation was associated with an increase in Ca2+ -ATPase activity but had no effect on Mg2+ ATPase or Na+, K+ -ATPase activity or on ouabain binding. Muscarinic receptor and beta-adrenoreceptor binding also appeared to be unaffected. PMID- 3001322 TI - Strophanthidin and potassium on intracellular sodium activity in sheep heart. AB - Double-barrelled sodium-selective microelectrodes were used to measure intracellular Na+ activity (aiNa) in sheep cardiac Purkinje fibres. A single rubber membrane gap voltage clamp controlled transmembrane potential (Vm) in some experiments. In quiescent preparations, the mean (+/- S.E.M.) aiNa was 8.9 +/- 0.3 mM (n = 46, Vm = -74.7 +/- 0.6 mV) at an extracellular potassium concentration [( K]o) of 5.4 mM and 7.5 +/- 0.4 mM (n = 37, Vm = -63.3 +/- 0.6 mV) at 10.8 mM [K]o. Brief strophanthidin exposures (5 X 10(-6) M for 6 to 10 mins) reversibly increased aiNa by 2 to 18 mM. The magnitude of this increase (delta aiNa) was similar in quiescent and paced (1 Hz) preparations but was inversely related to [K]o in the range studied (2.7, 5.4 and 10.8 mM). Voltage clamp experiments at a constant [K]o (5.4 or 10.8 mM) demonstrated a 27% lower delta aiNa at a Vm of -64 mV compared to -76 mV. In contrast, delta aiNa decreased by 48% when [K]o was increased from 5.4 to 10.8 mM with Vm held constant at either -64 or -76 mV. Hence, the observed antagonism between K+ ions and strophanthidin appeared to be due to a voltage-dependent effect and, to a greater extent, a specific ion-dependent effect. This latter finding is consistent with competitive behaviour between K+ ions and strophanthidin at the Na/K ATPase receptor. PMID- 3001321 TI - On the cooperativity of ouabain-binding to intact myocardium. AB - A theoretical concept is presented which proposes that binding of ouabain to intact myocardium should be positive cooperative. It is based on the assumption that the myocardial Na/K-ATPases expose the ouabain-binding site only at a particular conformation adopted during a turnover cycle. The turnover rate and thus the ouabain-binding properties are regulated by the cytosolic Na-ion concentration Nai. Any occupation of cellular Na/K-ATPases should affect the ouabain-binding properties of the unoccupied Na/K-ATPases, because their turnover rate is increased via an elevated Nai. A computer model which takes into account the interrelationships of the Na/K-ATPases both with Nai and with the ouabain concentration predicts that ouabain-binding should proceed in a concentration proportional fashion as long as the Na-load can be counterbalanced by non occupied Na/K-ATPase molecules. The concentration-proportional binding reflects a positive cooperativity. Experimental results reveal that (3H)ouabain-binding to Na/K-ATPase of electrically stimulated guinea-pig left atria was in fact concentration-proportional under certain experimental conditions. The biological significance of the proposed concept remains to be elucidated. PMID- 3001323 TI - Diffusion-controlled receptor occupancy determines the rate of inotropic action of some cardioactive steroids. AB - In guinea-pig myocardium the rates of onset and offset of the inotropic effects of digitoxigenin-monodigitoxoside, digitoxin, ouabain, digoxin, digoxigenin, and dihydroouabain correlated negatively, and independently of the lipophilicity of the steroids, with their intropic and Na-K-ATPase inhibitory potencies. The intropic potency of ouabain was considerably higher than that of dihydroouabain whereas its inotropic effect developed much more slowly (T50 17 min v. 3 min). By contrast, the inhibition of sarcolemmal Na-K-ATPase by equieffective concentrations of these steroids appeared at similar rates within a few minutes. The homogeneity of the effects, as shown by parallel concentration-effect curves with similar order of potencies of the inotropic and the Na-K-ATPase inhibitory effects, excluded the different activation of inhomogeneous receptor populations. The correlation between the geometry of the papillary muscles and the rates of onset of the inotropic ouabain effect or tissue/medium ratios of 3H-ouabain indicated the relevance of diffusion. The discrepancy between the rates of action in sarcolemmal preparations and in papillary muscles could thus be related to the different diffusion distances. The apparently contradictory results in which receptor occupation (dependence of T50 on EC50) appeared as the rate-limiting step are explained by diffusion-controlled receptor occupancy: the reduced rate of diffusion due to an affinity-dependent reduction of the free drug concentration (receptor occupancy as a temporal 'site of loss') accounted for the slow onset of the inotropic effect of the highly potent cardioactive steroids. PMID- 3001324 TI - Nuclease S1 analysis of eubacterial 5S rRNA secondary structure. AB - Single-strand-specific nuclease S1 was employed as a structural probe to confirm locations of unpaired nucleotide bases in 5S rRNAs purified from prokaryotic species of rRNA superfamily I. Limited nuclease S1 digests of 3'- and 5'-end labeled [32P]5S rRNAs were electrophoresed in parallel with reference endoribonuclease digests on thin sequencing gels. Nuclease S1 primary hydrolysis patterns were comparable for 5S rRNAs prepared from all 11 species examined in this study. The locations of base-paired regions determined by enzymatic analysis corroborate the general features of the proposed universal five-helix model for prokaryotic 5S rRNA, although the results of this study suggest a significant difference between prokaryotic and eukaryotic 5S rRNAs in the evolution of helix IV. Furthermore, the extent of base-pairing predicted by helix IV needs to be reevaluated for eubacterial species. Clipping patterns in helices II and IV appear to be consistent with a secondary structural model that undergoes a conformational rearrangement between two (or more) structures. Primary clipping patterns in the helix II region, obtained by S1 analysis, may provide useful information concerning the tertiary structure of the 5S rRNA molecule. PMID- 3001326 TI - Update in cancer chemotherapy, Part IV. Lung cancer, Part 2. AB - An update of cancer chemotherapy in 1985 that deals with the various therapies of lung cancer is described. At present, the stage of the disease and cell type are the major factors that determine the treatment. Important differences in the biological behavior and response to treatment exist between small cell and non small cell cancers. Treatment of the small cell type, which is sensitive to many of the cancer chemotherapeutic agents, is discussed in this paper. Treatment of the non-small cell types was described in the October issue of the Journal. PMID- 3001327 TI - Effect of chlordane on influenza type A virus and herpes simplex type 1 virus replication in vitro. AB - The effect of chlordane on the susceptibility of Madin-Darby canine kidney cells and African Green monkey kidney cells to infection with influenza type A/PR/8/34 (HON1) virus and herpes simplex type 1 virus was determined. Exposure of both cell lines to various concentrations of chlordane for 24 h at 37 degrees C (acute exposure) effected a marked reduction in the efficiency of influenza type A virus infection, except at a dose of 0.025 ppm. Acute exposure of the monkey cells did not alter their susceptibility to herpes simplex virus infection. Viral adsorption studies at 4 and 37 degrees C revealed a marked reduction in the attachment of influenza type A virus to both cell lines following acute exposure to 10 ppm chlordane. Viral inactivation studies carried out at 4 and 37 degrees C failed to reveal differences in the level of influenza type A virus inactivation in the presence or absence of chlordane. Madin-Darby canine kidney cells exposed to 10 ppm chlordane for 60 d (chronic exposure) manifested a decrease in the efficiency of influenza type A virus infection, whereas cells chronically exposed to 0.025 ppm chlordane manifested an increase in the efficiency of influenza type A virus infection relative to mock-treated control cells. When chronically exposed cells were passaged six times in the absence of chlordane, these effects were reversed. Viral adsorption studies carried out at 4 and 37 degrees C on cells chronically exposed to 10 ppm chlordane revealed a decrease in the adsorption of influenza type A virus. Quantitation of the levels of cell-surface sialic acid, the essential terminal sugar on the receptor for influenza type A virus, indicated that the reduced adsorption of influenza type A virus to Madin Darby canine kidney cells was not due to a loss of cell-surface sialic acid. Our findings indicate that chlordane alters the susceptibility of cells to infection with influenza type A virus but not to herpes simplex type 1 virus. PMID- 3001325 TI - The mitochondrial DNA molecular of Drosophila yakuba: nucleotide sequence, gene organization, and genetic code. AB - The sequence of the 16,019 nucleotide-pair mitochondrial DNA (mtDNA) molecule of Drosophila yakuba is presented. This molecule contains the genes for two rRNAs, 22 tRNAs, six identified proteins [cytochrome b, cytochrome c oxidase subunits I, II, and III (COI-III), and ATPase subunits 6 and 8] and seven presumptive proteins (URF1-6 and URF4L). Replication originates within a region of 1077 nucleotides that is 92.8% A + T and lacks any open reading frame larger than 123 nucleotides. An equivalent to the sequence found in all mammalian mtCDNAs that is associated with initiation of second-strand DNA synthesis is not present in D. yakuba mtDNA. Introns are absent from D. yakuba mitochondrial genes and there are few (0-31) intergenic nucleotides. The genes found in D. yakuba and mammalian mtDNAs are the same, but there are differences in their arrangement and in the relative proportions of the complementary strands of the molecule that serve as templates for transcription. Although the D. yakuba small and large mitochondrial rRNA genes are exceptionally low in G and C and are shorter than any other metazoan rRNA genes reported, they can be folded into secondary structures remarkably similar to the secondary structures proposed for mammalian mitochondrial rRNAs. D. yakuba mitochondrial tRNA genes, like their mammalian counterparts, are more variable in sequence than nonorganelle tRNAs. In mitochondrial protein genes ATG, ATT, ATA, and in one case (COI) ATAA appear to be used as translation initiation codons. The only termination codon found in these genes is TAA. In the D. yakuba mitochondrial genetic code, AGA, ATA, and TGA specify serine, isoleucine, and tryptophan, respectively. Fifty-nine types of sense condon are used in the D. yakuba mitochondrial protein genes, but 93.8% of all codons end in A or T. Codon-anticodon interactions may include both G-A and C A pairing in the wobble position. Evidence is summarized that supports the hypothesis that A and T nucleotides are favored at all locations in the D. yakuba mtDNA molecule where these nucleotides are compatible with function. PMID- 3001328 TI - Procedures to enhance withdrawal of xenobiotics from chickens. AB - Egg- and meat-type chickens were fed diets containing 1 or 10 ppm of fireMaster (FF-1), a commercial mixture of polybrominated biphenyls (PBBs), predominantly 2,4,5,2',4',5'-hexabromobiphenyl (6BB-4) and 2,3,4,5,2',4',5'-heptabromobiphenyl (7BB-8). These congeners account for approximately 65 and 14%, respectively, of the total mix. In three experiments, colestipol hydrochloride, a bile acid binding resin, and mineral oil, alone or in combination with restricted feeding, were examined as procedures to enhance the removal of the PBBs. The combination of 50% dietary restriction plus a 10% dietary concentration of colestipol or mineral oil reduced body burdens of PBBs about 70% within 21 d for chickens previously fed 10 ppm PBBs. The use of feed restriction, colestipol, or mineral oil produced only borderline effects for hastening withdrawal. Chickens not treated showed only a 3% loss of PBBs during the withdrawal period. Colestipol at 2.5% in the diet in combination with feed restriction was not consistently effective in removing body burdens of PBB from chickens previously fed 1 or 10 ppm PBBs, indicating a dose-response effect. Chickens previously fed 1 ppm eliminated up to 40% of the PBBs in 21 d without any treatment, as compared to the 3% loss occurring after feeding with 10-ppm concentrations. PMID- 3001329 TI - A model for the treatment of accidental severe hypothermia. AB - Central to the controversy that surrounds the treatment of accidental severe hypothermia is the question of how the method of rewarming affects myocardial performance, and therefore survival. We induced severe hypothermia and cardiac arrest in 15 mongrel dogs. Each dog was rewarmed by one of three methods: partial cardiac bypass (Group I); peritoneal dialysis (Group II); or external rewarming with a fluid-circulated blanket (Group III). The cardiac arrest state was supported by partial cardiac bypass in Group I and by standard mechanical cardiopulmonary resuscitation (CPR) in Groups II and III. In all dogs, the hypothermically depressed myocardial performance returned to normal upon rewarming. Groups I and II had similar rewarming times and required similar volumes of crystalloid and bicarbonate solutions to maintain adequate cardiac filling pressures and arterial pH. However, Group III had a significantly slower rewarming time and required significantly greater volumes of crystalloid and bicarbonate solutions. The sole procedural death occurred in Group III. Our results show that partial cardiac bypass, peritoneal dialysis, and the fluid circulated blanket are equally effective in rewarming severely hypothemic dogs with cardiac arrest, provided that the cardiac arrest is relieved by partial cardiac bypass or standard mechanical CPR and that physiologic levels of intravascular volume, oxygenation, and pH are maintained. PMID- 3001332 TI - Electron dense mitochondrial inclusions in a pituitary oncocytoma. AB - A 65-year-old man had a pituitary adenoma with a large expansion of the sella turcica; computerized tomography demonstrated suprasellar extension. The tumor, removed surgically, was diagnosed as nonsecretory partly chromophobic, partly acidophilic adenoma by light microscopy and immunocytology. Electron microscopic investigation revealed a pituitary oncocytoma with an unusual mitochondrial abnormality. Some mitochondria harbored single or multiple electron dense bodies which were also found in the cytoplasm as well as in the extracellular space. It appears that these bodies were originally formed in the mitochondria, then extruded into the cytoplasm and subsequently to the extracellular space. PMID- 3001330 TI - Mitochondrial cytochemistry in experimental myopathies. AB - In this paper, we have described mitochondrial cytochemistry (NADH oxidase, cytochrome c oxidase), in the light and electron microscopic studies of the experimental mitochondrial myopathies. DNP, oleate and crotoxin were employed to produce mitochondrial changes in the rat skeletal muscle. The DNP and oleic acid lesions showed strong NADH oxidase and cytochrome c oxidase activities in mitochondria--both at the light microscopic as well as at ultrastructural level. However, the crotoxin lesions showed marked reduction of both the enzyme activities in histochemistry and electron microscopy. Muscle necrosis was seen only in the crotoxin lesion. On the basis of these data, we propose that DNP and oleic acid treatment produced mitochondrial myopathies in which the mitochondrial structure was altered but they were enzymatically active, while the crotoxin treatment produced the structural and enzymatic alterations of the mitochondria. PMID- 3001331 TI - An ultrastructural study of adipocyte precursors from epididymal fat pads of adult rats in culture. AB - It has been demonstrated that cells with poor or no lipid content, isolated from epididymal fat pads of young rats, develop fully into mature adipocytes in vitro. The ultrastructural feature of these cells during such a development have been studied recently. In the present work the electron microscopic aspects of similarly obtained cells from adult rats have been investigated. It has been found that no morphological differences are present in the cells immediately after isolation between young and adult rats. During culture the cells from both adult and young rats show the same maturative process, but in adult rats this process is generally slower and not homogeneous, suggesting the presence of two populations of adipocyte precursors. The former accumulate lipids quickly and have electron microscopic features very similar to those observed for young rat precursors, however maturation is still faster in the latter cells. The latter have electron microscopic aspects of less differentiated cells (some fibroblast like features) during the first days of culture; however they show a similar development when longer culture periods are studied. These data suggest that the fibroblast-like cells seen after a period of time in culture probably represent an early stage of adipocyte precursor differentiation. Thus it is suggested that in adult mammals, there may be a population of adipocyte precursors present within the adipose tissue which upon stimulation by as yet unknown factors would develop fully into adipocytes. PMID- 3001333 TI - A serial section study of nuclear pockets containing nuclear material. AB - A serial section study of leukaemic lymphocytes was carried out to elucidate the three dimensional morphology of nuclear pockets containing nuclear material and their mode of genesis. From this and a previous study on nuclear pockets containing cytoplasmic material we conclude that these are two distinct lesions each with its own mode of genesis and one does not evolve from the other. Hence we propose that the common nuclear pocket containing cytoplasmic material be designated 'type I nuclear pocket' and the nuclear pocket containing nuclear material as the 'type 2 nuclear pocket'. Our serial section study also showed that some type 2 nuclear pockets are open, (i.e. there is continuity between the material in the pocket and the contents of the nucleus) while others are closed (i.e. the material in the pocket is completely separated from the material in the nucleus). At times one sees profiles suggesting that small satellite nuclei occur in leukaemic cells. Our serial section studies show that such profiles can be produced by fortuitous sections through type 2 nuclear pockets. PMID- 3001334 TI - High-resolution electron microscope and computed images of human tooth enamel crystals. AB - The structure of human enamel crystallites has been studied at a near atomic level by high-resolution electron microscopy. Electron micrographs have been obtained from crystallites present in human enamel with a structure resolution of 0.2 nm in the [0001], [1210], [1213], [1100] and [4510] zone axes directions. In most cases it was possible to match the experimental images with images calculated using the atomic positions of mineral hydroxyapatite. However, in some cases a discrepancy between calculated and experimental image detail was observed in the c direction of the [1210] and the [1100] images. This shows: (i) a structural heterogeneity of the crystals, and (ii) a loss of hexagonal symmetry of the structure. The resolution required to distinguish individual atomic sites in the different zones has been determined, and this will provide a useful basis for future work. As the determination of the "real structure" of biological crystals is of prime importance for the study of calcification mechanisms (crystal growth), biological properties and destructive phenomena of calcified tissues (i.e., dental caries and bone resorption). PMID- 3001335 TI - E5 open reading frame of bovine papillomavirus type 1 encodes a transforming gene. AB - We have previously shown that the early region of the bovine papillomavirus type 1 genome contains two nonoverlapping segments that can independently induce the morphological transformation of cultured cells. The transforming gene from the 5' end of the early region is encoded by the E6 open reading frame. The second transforming segment was previously localized to a 2.3-kilobase fragment (2.3T) from the 3' end of the early region. To determine which of the four open reading frames (E2, E3, E4, and E5) located within 2.3T encodes a transforming gene, we have now introduced a series of insertion and deletion mutations into a clone (pHLB1) in which 2.3T is activated by the Harvey viral long terminal repeat, and we tested the mutants for their ability to induce focal transformation. Our results indicate that the E5 open reading frame, which could encode a low molecular-weight hydrophobic peptide, is required for pHLB1-induced transformation of NIH 3T3 cells, but that the E2, E3, and E4 open reading frames are not. PMID- 3001336 TI - Analysis of reassortment of genome segments in mice mixedly infected with rotaviruses SA11 and RRV. AB - Seven-day-old CD-1 mice born to seronegative dams were orally inoculated with a mixture of wild-type simian rotavirus SA11 and wild-type rhesus rotavirus RRV. At various times postinfection, progeny clones were randomly isolated from intestinal homogenates by limiting dilution. Analysis of genome RNAs by polyacrylamide gel electrophoresis was used to identify and genotype reassortant progeny. Reassortment of genome segments was observed in 252 of 662 (38%) clones analyzed from in vivo mixed infections. Kinetic studies indicated that reassortment was an early event in the in vivo infectious cycle; more than 25% of the progeny clones were reassortant by 12 h postinfection. The frequency of reassortant progeny increased to 80 to 100% by 72 to 96 h postinfection. A few reassortants with specific constellations of SA11 and RRV genome segments were repeatedly isolated from different litters or different animals within single litters, suggesting that these genotypes were independently and specifically selected in vivo. Analysis of segregation of individual genome segments among the 252 reassortant progeny revealed that, although most segments segregated randomly, segments 3 and 5 nonrandomly segregated from the SA11 parent. The possible selective pressures active during in vivo reassortment of rotavirus genome segments are discussed. PMID- 3001337 TI - Modification of Epstein-Barr virus replication by tunicamycin. AB - The effect of tunicamycin, which inhibits N-linked glycosylation, on the replication of Epstein-Barr virus was examined. Tunicamycin markedly reduced the yield of virus from producing cells. At concentrations of 1 to 2 micrograms of tunicamycin per ml, there was a buildup of intracellular virus in P3HR1-Cl13 cells but not in MCUV5 cells; at a concentration of 5 micrograms of tunicamycin per ml in P3HR1-Cl13 cells, viral DNA synthesis was inhibited as well. Viral glycoproteins lacking N-linked sugars were apparently inserted into the cell membrane, and the small amount of virus made in the presence of drug was able to bind specifically to its receptor on B cells. However, the ability of the virus to induce immunoglobulin secretion by fresh human lymphocytes was impaired. This implies a role for viral glycoproteins in the penetration as well as the attachment of virus. PMID- 3001339 TI - Repression of adenovirus early gene expression by coinfection with a temperature sensitive mutant in the immediate-early gene of pseudorabies virus. AB - Wild-type adenovirus was coinfected with a mutant temperature sensitive for the immediate-early gene of pseudorabies virus. At the nonpermissive temperature, this mutant, tsG, strongly inhibited the transcription of all adenovirus early genes, including E1A. This inhibition was not observed with wild-type pseudorabies virus coinfection or with tsG coinfection at the permissive temperature. The level of repression was dependent upon the ratio of tsG to adenovirus in the infection. The results suggest that the temperature-sensitive protein may be interacting with transcription factors on the viral DNA or with the DNA itself to inhibit adenovirus transcription. PMID- 3001338 TI - Effects of the adenovirus 2 late promoter on simian virus 40 transcription and replication. AB - A 100-base-pair fragment of adenovirus 2 (Ad2) DNA encompassing the major late transcriptional promoter was inserted into the simian virus 40 (SV40) late promoter region at SV40 nucleotide 294 to study the effects of a strong TATA box containing promoter on SV40 late transcription. pSVAdE contains the insert in an orientation such that it would promote transcription towards the origin and early region of SV40, while the insert is in the opposite orientation in pSVAdL. Nuclease S1 analysis with 5'-end-labeled probes showed that in cells transfected with pSVAdE, the late mRNA initiation sites are essentially the same as in wild type, demonstrating that an insert of 100 base pairs can have no effect on utilization of the SV40 late start sites. In pSVAdL-transfected cells, however, the major late viral initiation site is now in the insert at +1 with respect to the Ad2 major late cap site. However, all of the SV40 initiation sites are still utilized and with the same efficiency relative to each other as in wild type. Thus, it appears that the Ad2 late promoter and the SV40 late promoter can function independently on the same DNA molecule, even when one promoter is embedded within the other. By using cytosine arabinoside to block DNA replication and thereby inhibit the onset of late expression, it has been shown that both the Ad2 late promoter and the SV40 late promoter have similar requirement for DNA replication in this context. In addition, pSVAdL showed dramatically diminished virus viability and VPI expression compared with both wildtype and pSVAdE. Possible explanations for this unexpected finding are discussed. PMID- 3001340 TI - Functional interactions of the simian virus 40 core origin of replication with flanking regulatory sequences. AB - We constructed a matched set of plasmids to investigate the interactions of essential core sequences of the simian virus 40 replication origin with flanking regulatory sequences. Deletions of either T-antigen-binding region I or the 21 base-pair repeated promoter elements reduced replication to 50 to 70% of wild type levels. The simultaneous deletion of both regions decreased replication to less than 5% of wild-type levels. Thus, the double deletion greatly amplified the defects of the single deletions. We conclude that region I and the 21-base-pair repeats have related rather than independent functions in DNA synthesis. Insertion of a synthetic region I or the adenovirus 2 major late promoter at the late side of isolated core sequences in place of the 21-base-pair repeats failed to restore replication. In contrast, insertion of a single 72-base-pair enhancer element stimulated replication of the core origin more than fivefold. Thus, three distinct regulatory elements appear to facilitate core DNA replication by related mechanisms. Flanking sequences have only a small direct effect on T-antigen binding to naked core DNA. Possible mechanisms of action include the regulation of transcription or of chromatin structure. PMID- 3001342 TI - Mutation in the polyomavirus genome that activates the properties of large T associated with neoplastic transformation. AB - We have constructed a polyomavirus mutant genome which exhibits an increased immortalization potential when transfected into primary rat embryo fibroblasts. The mutation is a 30-base-pair deletion (nucleotides 1367 through 1396) that inactivates the transforming potential of middle T but activates some of the properties of large T associated with neoplastic transformation. Unlike the wild type large T, the mutant large T can fully complement polyoma middle T in the tumorigenic process in vivo as well as in the transformation of primary cells in vitro. The activity of the mutant can be explained by its inability to replicate in cells and, hence, its inability to exert a cytopathic effect after gene transfer at high multiplicity. A recombinant which encodes the middle and small T antigens, but not the large T antigen, can also elicit a fully transformed phenotype when introduced into primary rat fibroblasts. These results confirm previous observations from this laboratory indicating that two, and not three, viral gene functions are required for polyomavirus-mediated oncogenic transformation. PMID- 3001341 TI - Varicella-zoster virus p32/p36 complex is present in both the viral capsid and the nuclear matrix of the infected cell. AB - Varicella-zoster virus (VZV) directs the synthesis of numerous glycosylated and nonglycosylated infected-cell-specific proteins, many of which are later incorporated into the virion as structural components. In this study, we characterized a nonglycosylated polypeptide complex with the aid of a VZV specific murine monoclonal antibody clone, 251D9. As detected by indirect immunofluorescence, the antibody bound mainly to antigens located within the nuclei of infected cells and did not attach to an uninfected cell substrate. The polypeptide specificity of the monoclonal antibody was determined by immunoblot analysis of electrophoretically separated infected cell extracts to react with a 32,000-molecular-weight VZV-specific protein (p32); in addition, the antibody also bound to a 36,000-molecular-weight polypeptide. The synthesis of these antigens was unaffected by inhibitors of glycosylation. Nonionic or ionic detergents were only marginally effective in solubilization of the p32-p36 complex, and relatively small amounts were eluted from nuclei by high salt concentrations (2 M NaCl). The same proteins remained associated with the nuclear matrix of VZV-infected cells. We also demonstrated that the protein complex was a major component of purified VZV nucleocapsids; p32 was especially prominent in both full and empty capsids. Immunoblot analysis of the nucleocapsid preparation revealed two additional species (p34 and p38) in the p32-p36 complex. Phosphorylation was a distinctive feature of some of the constituents. In summary, these results indicate that the p32-p36 complex represents a family of structural proteins closely associated with the assembly of VZV nucleocapsids and the encapsidation of viral DNA. PMID- 3001343 TI - In vitro transcription of human pararotavirus. AB - Purified human pararotavirus obtained from stool samples from a 6-month-old infant was characterized. Electron microscopy of the viral particles subjected to different treatments suggested that the protein shells differed from those described for rotavirus. Treatment with both EDTA or ethylene glycol-bis(beta aminoethyl ether)-N,N,N',N'-tetraacetic acid in the presence or absence of Mg2+ seemed to convert the virions into core particles by removal of both the outer and inner shells, and no particles equivalent to single-shelled rotavirus were observed. Different procedures were used to activate the human pararotavirus associated RNA-dependent RNA polymerase. The enzyme was not activated by chelating agents or by thermal shock as in rotavirus. Activation by thermal shock occurred only in the presence of the four ribonucleoside triphosphates and Mg2+. However, the polymerase of pararotavirus was found to be similar to those described for rotaviruses. When in vitro transcripts were analyzed, 11 RNA species having a migration pattern similar to that of the original genomic RNA were detected. PMID- 3001344 TI - Proteins specified by the short unique region of the genome of pseudorabies virus play a role in the release of virions from certain cells. AB - Two pseudorabies virus vaccine strains (Bartha and Norden) that have a similar deletion in the short unique (Us) region of the genome have been identified previously (B. Lomniczi, M. L. Blankenship, and T. Ben-Porat, J. Virol. 49:970 979, 1984). These strains do not code for the glycoprotein gI, a glycoprotein that has been mapped on the wild type virus genome by T. C. Mettenleiter, N. Lukacs, and H. J. Rziha (J. Virol. 53:52-57, 1985) to the sequences deleted from the vaccine strain. Restoration of these deleted sequences to the Bartha strain genome restores to the virus the ability to specify the gI glycoprotein. The Bartha vaccine strain grows as well as wild-type virus in pig kidney and in rabbit kidney (RK) cells, but is not released efficiently from and forms small plaques in RK cells. The rescued Bartha 43/25a strain (which has an intact Us) is released considerably more efficiently than the Bartha vaccine strain, but less efficiently than wild-type virus from RK cells; it also forms larger plaques on RK cells than does the parental Bartha vaccine strain. The Norden vaccine strain, which has a deletion in the Us, is released better from RK cells than is the Bartha strain, but not as well as is wild-type virus. We conclude that whereas the sequences in the Us that are deleted from the Bartha and Norden strain genomes specify functions that play a role in the release of virions from some cell types, at least one other function (which is defective in the Bartha strain but not in the Norden strain) also affects release of virus from these cells. Since restoration to the Bartha strain of an intact Us restores to the virus both the ability to grow in chicken brains (B. Lomniczi, S. Watanabe, T. Ben-Porat, and A. S. Kaplan, J. Virol. 52:198-205, 1984) and to be released from RK cells, the possibility that the lack of virulence of the Bartha vaccine strain may be related to its limited release from some target cells is discussed. PMID- 3001345 TI - Superinfection rescue of an integrated defective polyomavirus genome. AB - We have characterized the viral sequences integrated in a polyomavirus transformed mouse cell line, Py-3T3 (clone Py-6), and followed their excision and packaging upon superinfection. The polyomavirus sequences contained in Py-6 cells are present as a single insert of nonidentical tandem copies which includes, in addition to a normal middle T-antigen-coding region, some very rearranged sequences. Infection of Py-6 cells with polyomavirus strains encoding a normal large T antigen leads to the reproducible recovery in the resulting viral stock of specific defective viral genomes. The defective genomes contain a wild-type coding region for middle and small T antigens and intact viral origin and enhancer sequences. The remainder of the viral genome is rearranged or lost, so that there is no capacity to code for large T antigen or viral capsid proteins. The recovered defective sequences are also found integrated in Py-6 genomic DNA. Presumably, in infections of Py-6 cells, large T antigen, provided by the superinfecting virus, amplifies and excises the integrated viral sequences. The superinfecting helper virus must also produce viral capsids for packaging of the defective viral DNA and thus provides a means to shuttle the defective sequences from the mouse cells into other hosts, such as rat cells. In the latter host, the defective sequences are able to induce transformation. PMID- 3001346 TI - Properties of cells transformed by the middle T-antigen-coding region of polyomavirus. AB - A series of 10 Fischer rat transformed clonal cell lines were independently obtained in infections with a defective polyomavirus containing a scrambled genome except for an intact middle and small T-antigen-coding region. These cells synthesize middle and small T antigens; no fragment of large T antigen can be detected in any of them. The transformed phenotype of this set of cell lines (designated LT-) has been studied with respect to serum dependence, saturation density, and anchorage independence and compared with the phenotype of a set of six transformants (designated LT+) which synthesize detectable to high levels of shortened or normal-sized large T antigen. Both the LT+ and the LT- groups of polyomavirus transformants display a range of transformed phenotypes. These ranges overlap, and the variations within each group are larger than the variations between the two groups. Thus, the results suggest that, for established Fischer rat fibroblasts, the maintenance of any of the three phenotypes tested and, in particular, of serum independence is not necessarily correlated with the levels of large T antigen or fragments thereof. PMID- 3001347 TI - Direct method for quantitation of extreme polymerase error frequencies at selected single base sites in viral RNA. AB - Methods are described which allow direct quantitation and sequence analysis of base substitution levels at predetermined single nucleotide positions in cloned pools of an RNA virus genome or in its RNA transcripts in vitro. Base substitution frequencies for vesicular stomatitis virus (VSV) at one highly conserved site examined were reproducible and extremely high, averaging between 10(-4) and 4 X 10(-4) substitutions per base incorporated at this single site. If polymerase error frequencies averaged as high at all other sites in the 11 kilobase VSV genome, then every member of a cloned VSV population would differ from most other genomes in that clone at a number of different nucleotide positions. The preservation of a consensus sequence in such variable RNA virus genomes then could only result from strong biological selection (in a single host or multihost environment) for the most fit and competitive representatives of extremely heterogeneous virus populations. PMID- 3001348 TI - Significance of the gastrin homology and surrounding sequences in polyomavirus middle T antigen for cell transformation. AB - Deletion of residues 305 to 327 of polyomavirus middle T antigen, including the (Glu)6-Tyr-315 sequence that is a preferred site of phosphorylation in vitro by pp60c-src, markedly altered viral transformation of rat cells. The efficiency of transformation by the deletion mutant depended on how it was introduced into cells, and the resulting transformants displayed limited growth rates in monolayer and in suspension. Substitution of the polyomavirus residues 305 to 327 with a homologous region (containing [Glu]5-Ala-Tyr) from porcine gastrin did not restore wild-type transforming activity. These mutant middle T antigens interacted with pp60c-src and were phosphorylated in vitro. Thus, although a sequence of consecutive glutamic acid residues followed by a tyrosine is a dominant structural element which strongly influences the physical properties of middle T antigen, its presence did not ensure the biological activity of the protein. Other elements in this region of middle T antigen also contributed substantially to the transforming capacity of polyomavirus. PMID- 3001349 TI - Development of a helper-independent human adenovirus vector and its use in the transfer of the herpes simplex virus thymidine kinase gene. AB - Approximately 2 kilobases (kb) of additional DNA can be packaged into wild-type virions of human adenovirus type 5 (Ad5). To extend this limit, a helper independent Ad5 cloning vector was constructed by deleting most of early region 3 (E3) from map coordinates 78.5 to 84.7 and essentially all of early region 1 (E1) from coordinates 1.0 to 10.6. E3 is nonessential for adenovirus replication in cultured cells, and E1 is nonessential when the virus is propagated in 293 cells which constitutively express the E1 gene products. The resulting new virus, dlE1,3 is about 5.5 kb shorter than wild-type Ad5 and therefore should be able to accept up to 7.5 kb in foreign DNA. To test the usefulness of this vector, the herpes simplex virus type 1 (HSV-1) thymidine kinase gene (tk) along with its regulatory sequences was inserted into the unique XbaI site of dlE1,3 (at map position 78.5/84.7). The resulting recombinant virus, Adtk, expressed the HSV tk at a low level (as compared with HSV-1) in infected cells; however, tk expression was markedly enhanced when Adtk-infected cells were superinfected with a tk- mutant of HSV. Furthermore, the Adtk virus efficiently transformed tk- mouse cells (line LTA) to the tk+ phenotype. At a low efficiency, it was also possible to transform tk- human cells (line 143), and tk+ transformants of both mouse and human origin have been established as permanent lines. PMID- 3001350 TI - Toward an in vitro system for picornavirus assembly: purification of mengovirus 14S capsid precursor particles. AB - Mengovirus 14S subviral protein particles generated in infected L cells and in a cell-free translation system primed with mengovirus RNA were purified by sucrose gradient centrifugation and immunoaffinity chromatography. The preparations from both sources contained essentially pure proteins epsilon, alpha, and gamma, as was demonstrated in terms of virus-specific proteins (by autoradiography) and total protein content (by silver staining of sodium dodecyl sulfate polyacrylamide electrophoresis gels). These purified proteins sedimented as discrete particles at the 14S position when recentrifuged in sucrose gradients. Although their assembly properties have not yet been studied in detail, preliminary results indicate that during incubation with virion RNA the 14S particles purified from infected cells can form a structure cosedimenting with mature mengovirus. PMID- 3001351 TI - Patterns of proviral insertion and deletion in avian leukosis virus-induced lymphomas. AB - Sixty-eight lymphomas induced by eight different avian leukosis viruses have been analyzed on Southern blots for virus-induced mutations in the chicken c-myc gene. Sixty-six of the lymphomas exhibited a proviral insertion in c-myc, whereas one exhibited a new transduction of c-myc. Sixty-four of the proviral insertions were in the same transcriptional orientation as c-myc. Two were in the opposite transcriptional orientation. All of the insertions were upstream of the protein coding sequences of c-myc, with most residing in the first exon or the first intron of c-myc. All of the lymphoma-inducing proviruses had deletions that included either sequences near the 5' long terminal repeat (LTR) or an LTR. The most frequent lymphoma-inducing provirus appeared to have retained both of its LTRs, but had lost sequences near its 5' LTR. The second and third most frequent lymphoma-inducing proviruses consisted of solo LTRs or of proviruses that had lost the 5' LTR as well as some internal sequences. Twenty-four insertions were mapped in c-myc. Each of these mapped to within 150 base pairs of one of the five DNase I-hypersensitive sites that occur in a 3-kilobase region immediately 5' to the protein-coding sequences of c-myc. One lymphoma contained a new c-myc transducing virus. This virus, MYC-3475, caused rapid-onset myelocytomatosis. PMID- 3001352 TI - Monoclonal antibodies against Aleutian disease virus distinguish virus strains and differentiate sites of virus replication from sites of viral antigen sequestration. AB - Monoclonal antibodies (mAbs) were used to study antigenic differences among strains of Aleutian disease virus (ADV) and to characterize viral proteins in vitro and in vivo. A number of ADV field strains could be discriminated, and highly virulent Utah I ADV was clearly delineated from the tissue culture-adapted avirulent ADV-G strain. This specificity could be demonstrated by indirect immunofluorescence against infected cultures of Crandell feline kidney cells or against tissues of Utah I ADV-infected mink. Viral antigens were demonstrated in both the nuclei and the cytoplasm of infected tissue culture cells. However, in mink mesenteric lymph node, spleen, and liver, viral antigen was observed only in the cytoplasm. Absence of nuclear fluorescence suggested that the detected antigen represented phagocytized viral antigens rather than replicating virus. This conclusion was supported by the finding that mAbs reactive only against low molecular-weight polypeptides derived from intact viral proteins gave the same pattern of in vivo fluorescence as mAbs with broad reactivity for large or small (or both) viral polypeptides. The distribution of infected cells was the same as that described for macrophages in these tissues and suggested that cells of the reticuloendothelial system had sequestered viral antigens. PMID- 3001355 TI - Detection of pseudorabies virus DNA in the inner ear of intranasally infected BALB/c mice with nucleic acid hybridization in situ. AB - Evidence for the pathogenicity of pseudorabies virus for the auditory and vestibular organs of experimentally infected mice is presented. We demonstrate viral genomes in cells of the peripheral sensory organs, the nerve structures, and the affected areas of the brain in single sections from an entire cranium of an adult mouse. The data were obtained by an in situ hybridization technique adapted for use with fixed, plastic-embedded materials. In contrast to conventional methods which use frozen sections, we were able to analyze cartilaginous and bony materials with high resolution. PMID- 3001353 TI - Temperature-sensitive splicing defect of ts110 Moloney murine sarcoma virus is virus encoded. AB - ts110 Moloney murine sarcoma virus (Mo-MuSV)-nonproductively infected cells (6m2) have a transformed phenotype at 28 to 33 degrees C and a normal phenotype at 39 degrees C. At temperatures permissive for transformation, 6m2 cells contain P58gag produced from the 4.0-kilobase (kb) viral RNA genome and P85gag-mos translated from a 3.5-kb spliced mRNA. At 39 degrees C, only the 4.0-kb RNA and its product P58gag are detected. Two temperature-sensitive defects have been observed in ts110-infected 6m2 cells: (i) the splicing of the 4.0-kb RNA to the 3.5-kb RNA; and (ii) the thermolability of P85gag-mos and its kinase activity relative to the wild-type revertant protein, termed P100gag-mos (R.B. Arlinghaus, J. Gen. Virol. 66:1845-1853, 1985). In the present study, we examined the mos gene products of two cell lines (204-2F6 and 204-2F8) obtained by infection of normal rat kidney cells with ts110 Mo-MuSV as a simian sarcoma-associated virus pseudotype to see whether the temperature-sensitive splicing defect could be transferred by viral infection. Southern blot analysis of these two cell lines showed that viral DNAs containing restriction fragments from cellular DNA are different from those in 6m2 cells, indicating that 204-2F6 and 204-2F8 cells have different ts110 provirus integration sites from those of 6m2 cells. Northern blots, S1 mapping analyses, and immunoprecipitation experiments showed unequivocally that the splicing defect of ts110 Mo-MuSV is virus encoded and is independent of host cell factors. PMID- 3001354 TI - Molecular basis underlying phenotypic revertants of Moloney murine sarcoma virus MuSVts110. AB - We examined the molecular basis for phenotypic reversion in cells infected with a transformation mutant of murine sarcoma virus, MuSVts110. In MuSVts110-infected NRK cells (6m2 cells), the manifestation of the transformed phenotype at 33 degrees C and the normal phenotype at 39 degrees C is governed by thermosensitive splicing of the MuSVts110 primary transcript, a 4.0-kilobase (kb) RNA which contains the gag and mos genes joined out of frame. At 33 degrees C, selectively, the 4.0-kb RNA is processed to a spliced 3.5-kb RNA in which the gag and mos genes are rejoined in a continuous open reading frame, thus allowing synthesis of the P85gag-mos-transforming protein. In contrast, the MuSVts110 revertant cell lines (designated 54-5A4 and 204-3) appear transformed at all growth temperatures from 33 to 39 degrees C and express a P100gag-mos-transforming protein from an apparently unprocessed 4.0-kb viral RNA. In the current study we established both by S1 nuclease analysis and primer extension sequencing that the revertant 54-5A4 and 204-3 4.0-kb viral RNAs suffered a 5-base deletion at the intron-exon border of the 3' splice site. The effect of this deletion is twofold. First, because of the damage to the 3' splice site, the revertant viral 4.0-kb RNAs cannot be processed to the spliced 3.5-kb RNA and, consequently, cannot be translated to P85gag-mos. Second, the 5-base deletion excises an in-frame stop codon positioned at the intron-exon border in the parental RNA and restores the original mos gene reading frame. The net effect is to produce a continuous open reading frame from the gag, alternate mos, and authentic mos gene reading frames which are fused together in the revertant 4.0-kb RNA. This continuous open reading frame can be translated into the P100gag-mos-transforming protein at any growth temperature. PMID- 3001356 TI - Analysis of integrated proviral DNA sequences with an octadecanucleotide probe designed for specific identification of spleen focus-forming virus in the mouse genome. AB - The Friend spleen focus-forming virus (SFFV) is an envelope gene recombinant between the ecotropic Friend murine leukemia virus and the endogenous xenotropic mink cell focus-forming retroviral sequences. We synthesized an octadecanucleotide complementary to the 3' end of the SFFV env gene designed for discriminating the SFFV proviruses from the xenotropic mink cell focus-forming virus and ecotropic exogenous or endogenous viral sequences. Under appropriate hybridization conditions this probe allowed the identification, in addition to few endogenous DNA fragments, of multiple SFFV proviruses integrated in the genome of Friend malignant cells. Therefore this probe should be of interest in further characterizing the SFFV integration sites and possibly the SFFV ancestor gene. PMID- 3001357 TI - Isolation and characterization of a novel human papillomavirus type 6 DNA from an invasive vulvar carcinoma. AB - Human papillomavirus type 6 (HPV-6) DNA was detected in a rapidly growing vulvar verrucous carcinoma and two recurrent tumor samples. The viral DNA (HPV-6vc) was molecularly cloned and found to have a high degree of DNA sequence homology to HPV-6b DNA. Comparison of restriction endonuclease cleavage patterns between HPV 6b and HPV-6vc genomes and DNA sequencing analysis demonstrated an additional 106 bases in the HPV-6vc genome. These additional nucleotides were located in the noncoding region of the viral genome which contains the putative viral DNA replication and early gene transcriptional control elements. Seventy-four of the additional 106 nucleotides were found as one insert in the purine-thymidine-rich region 3' to the end of the L1 open reading frame. This 74-base-pair addition had homology with viral sequences immediately upstream to it and to poly(dG-dT) sequences found in the human genome including the conserved repeated sequences in human DNA (EC1) and in the human cardiac muscle actin gene. Two smaller inserts, 19 and 15 nucleotides, were found upstream from the transcriptional control elements and demonstrate homology with regions of human alpha and gamma interferon genes. PMID- 3001358 TI - Truncated forms of the polyomavirus middle T antigen can substitute for the small T antigen in lytic infection. AB - Cloned polyomavirus genomes encoding the small T antigen or truncated forms of the middle T antigen facilitated the growth of genomes encoding only the large T antigen in mouse 3T6 cells. We conclude that an N-terminal domain of the middle T antigen, in the appropriate cellular location, can substitute for the small T antigen during lytic infection. PMID- 3001359 TI - Identification of the two rotavirus genes determining neutralization specificities. AB - Bovine rotavirus NCDV and simian rotavirus SA-11 represent two distinct rotavirus serotypes. A genetic approach was used to determine which viral gene segments segregated with serotype-specific viral neutralization. There were 16 reassortant rotaviruses derived by coinfection of MA-104 cells in vitro with the SA-11 and NCDV strains. The parental origin of reassortant rotavirus double-stranded RNA segments was determined by gene segment mobility in polyacrylamide gels and by hybridization with radioactively labeled parental viral transcripts. We found that two rotavirus gene segments found previously to code for outer capsid proteins vp3 and vp7 cosegregated with virus neutralization specificities. PMID- 3001361 TI - Analysis of two strains of Friend murine leukemia viruses differing in ability to induce early splenomegaly: lack of relationship with generation of recombinant mink cell focus-forming viruses. AB - Friend murine leukemia helper viruses (F-MuLV) 57 and B3 were indistinguishable by genomic structural analyses with RNase T1-resistant oligonucleotide fingerprinting and by antigenic reactivity with a panel of 31 monoclonal antibodies directed against murine leukemia viruses. Nevertheless, F-MuLV 57 and B3 had strikingly different virulences. Approximately 2 months after inoculation, IRW and NFS/N mice inoculated as newborns with F-MuLV 57 had gross splenomegaly caused by erythroid proliferation. In contrast, an equivalent dose of F-MuLV B3 induced spleen or lymph node enlargement 4 to 13 months after inoculation. Although most cases of spleen enlargement in F-MuLV B3-inoculated mice were due to erythroid proliferation, lymphoid or myeloid proliferation was also frequently observed. The replication of both F-MuLV 57 and B3 was equally efficient, and both viruses generated recombinant dual-tropic mink cell focus-forming (MCF) viruses with the same kinetics and efficiency. Moreover, MCF viruses induced by F MuLV 57 and B3 had the same antigenic patterns. Therefore, the ability of F-MuLV to induce early splenomegaly did not correlate with the generation of recombinant MCF viruses. PMID- 3001360 TI - Construction of recombinant murine retroviruses that express the human T-cell leukemia virus type II and human T-cell lymphotropic virus type III trans activator genes. AB - Recombinant retroviruses containing the trans activator genes of human T-cell leukemia virus (HTLV) type II and human T-cell lymphotropic virus type III were constructed. The trans activator genes tat II and tat III were inserted into the murine retroviral vector pZIPNEOSV(X)1. Recombinant plasmids were transfected into the psi 2 and psi AM packaging cell lines that produce murine leukemia virions containing no retroviral RNA. Functional tat II and tat III gene products were expressed as demonstrated by trans activation of HTLV type I and II and human T-cell lymphotropic virus type III long terminal repeat-directed gene expression in the respective infected cells. Use of these recombinant vectors permits high-efficiency gene transfer into a wide variety of cells, thereby providing the opportunity to study the biochemical effects associated with tat II and tat III gene expression. PMID- 3001362 TI - Efficient and accurate in vitro processing of simian virus 40-associated small RNA. AB - Nuclei were isolated from simian virus 40 (SV40)-infected cells with a hypotonic, detergent-free buffer and incubated in vitro in a high-ionic-strength buffer containing [alpha-32P]UTP. The labeled viral RNAs produced were analyzed by gel electrophoresis together with 3-h-labeled viral RNAs extracted from SV40-infected cells. The in vitro-synthesized RNA contained a major RNA species of 62 to 64 nucleotides that appeared on the gel at the same position as in vivo-synthesized SV40-associated small RNA (SAS-RNA). Analyses of the in vitro-synthesized 62- to 64-nucleotide RNA by hybridization to restriction fragments and by the use of an SAS-RNA deletion mutant clearly identified it as SAS-RNA. The intensity of the band of the in vitro-synthesized SAS-RNA increased with an increase in the labeling time or when a short pulse was followed by a chase. Moreover, the SAS RNA band disappeared when ITP replaced GTP in the transcription reaction mixture. These results indicate that SAS-RNA is processed from a precursor molecule and that an RNA secondary structure could be an element recognized by the processing enzyme. PMID- 3001363 TI - Cloned long-term cytolytic T-lymphocyte line with specificity for an immediate early membrane antigen of murine cytomegalovirus. AB - Long-term cytolytic T-lymphocyte (CTL) lines that are specific for distinct antigens associated with different phases of the replicative cycle of the murine cytomegalovirus (MCMV) were established by cloning of CTL lines derived from lymph nodes of latently infected BALB/c mice. Two CTL clones were characterized in detail. Both displayed the Lyt-2+, L3T4- surface phenotype, and the recognition of their respective target antigens was class I (DLd) major histocompatibility complex antigen restricted. Clone S1 was specific for a structural antigen of MCMV, and clone IE1 detected an MCMV-specified immediate early (IE) membrane antigen. Clone IE1 retained lytic activity, antigen specificity, and self-restriction after prolonged propagation in the presence of recombinant human interleukin-2 without restimulation by antigen. This interleukin-2-dependent line of the clone IE1, line IE1-IL, can serve as a reference line for the definition of the antigenic determinant IE1 of an IE membrane antigen. PMID- 3001364 TI - Molecular basis of rotavirus virulence: role of gene segment 4. AB - Bovine rotavirus NCDV and simian rotavirus SA-11 exhibited markedly different patterns of gastrointestinal tract disease when inoculated orally into newborn mice. A genetic approach was used to define the molecular basis of these differences. The SA-11 strain of rotavirus was more virulent than the NCDV strain when inoculated orally into newborn mice; the dose of SA-11 required to cause diarrhea in 50% of infant mice was 50-fold less than that required for NCDV. Nineteen reassortant viruses were derived by coinfection of MA-104 cells in vitro with the SA-11 and NCDV strains. The parental origin of reassortant virus double stranded RNA segments was determined by gene segment migration differences in polyacrylamide gels and hybridization with radioactively labeled parental viral transcripts. The neutralization antigen phenotype of reassortant viruses was determined by plaque reduction neutralization. We found that the dose of SA-11 and NCDV rotavirus required to induce gastroenteritis in newborn mice was determined by gene segment 4. The results suggest that rotavirus virulence may be manipulated by modification or reassortment of gene segment 4. PMID- 3001365 TI - Simian virus 40 origin DNA-binding domain on large T antigen. AB - Fifty variant forms of simian virus 40 (SV40) large T antigen bearing point, multiple point, deletion, or termination mutations within a region of the protein thought to be involved in DNA binding were tested for their ability to bind to SV40 origin DNA. A number of the mutant large T species including some with point mutations were unable to bind, whereas many were wild type in this activity. The clustering of the mutations that are defective in origin DNA binding both reported here and by others suggests a DNA-binding domain on large T maps between residues 139 and approximately 220, with a particularly sensitive sequence between amino acids 147 and 166. The results indicate that the domain is involved in binding to both site I and site II on SV40 DNA, but it remains unclear whether it is responsible for binding to cellular DNA. Since all the mutants retain the ability to transform Rat-1 cells, we conclude that the ability of large T to bind to SV40 origin DNA is not a prerequisite for its transforming activity. PMID- 3001366 TI - Isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses. AB - Reciprocal competition binding assays have previously demonstrated that 20 of 24 human rhinovirus serotypes tested compete for a single cellular receptor. These studies suggested that the vast majority of rhinovirus serotypes utilize a single cellular receptor. With HeLa cells as an immunogen, a mouse monoclonal antibody was isolated which had the precise specificity predicted by the competition binding study. The receptor antibody was shown to protect HeLa cells from infection by 78 of 88 human rhinovirus serotypes assayed. In addition, the receptor antibody protects HeLa cells from infection by three coxsackievirus A serotypes. The receptor antibody was unable to protect cells from infection by a wide range of other RNA and DNA viruses. Using the receptor antibody and human rhinovirus type 15, we determined that the cellular receptor utilized by the vast number of human rhinovirus serotypes is present only on cells of human origin, with the exception of chimpanzee-derived cells. The receptor antibody has a strong affinity for the cellular receptor as evidenced by its rapid binding kinetics and ability to displace previously bound human rhinovirus virions from receptors. No viral variants were identified which could bypass the receptor blockage. PMID- 3001368 TI - Expression of the late gene of simian virus 40 under the control of the simian virus 40 early-region promoter in monkey and mouse cells. AB - We constructed a recombinant plasmid (pVNR4) with the simian virus 40 (SV40) early promoter positioned 30 nucleotides upstream from the major SV40 late transcription initiation site at residue 325. After transfection of the recombinant plasmid DNA into COS and mouse L cells, the transcripts of the SV40 late region were analyzed by S1 nuclease and primer extension analysis. The following are the principal findings. (i) The 16S and 19S late RNAs used the characteristic wild-type splice; no detectable levels of 19S unspliced RNA were observed. (ii) The majority of the late RNAs were heterogeneous and initiated in the early region (upstream and downstream from the Hogness-Goldberg sequence), and a minor population initiated at residue 325, the principal 5' terminus of the wild-type late RNA. (iii) During SV40 lytic infection there was a shift in initiation sites used to transcribe the early region from sites that are downstream to sites which are upstream (up RNA) of the origin of DNA replication. We observed that unlike lytic infection, T antigen and viral DNA replication were not needed for the appearance of up RNA in mouse L cells. (iv) In mouse L cells late RNAs were made, and the residue 325 5' end was utilized in the absence of T antigen or DNA replication. (v) In COS cells we found down RNA and up RNA transcribed from the extrachromosomally replicating plasmid but only down RNA produced by the integrated SV40 genome. PMID- 3001369 TI - An adrenocorticotropic hormone-producing pheochromocytoma: diagnostic and immunohistochemical studies. AB - A patient is described with Cushing's syndrome owing to a pheochromocytoma that was producing adrenocorticotropic hormone. Preoperative diagnosis was suggested by finding bilateral adrenocortical hyperplasia plus a separate unilateral adrenal medullary mass and was confirmed laboratory studies. Proper preoperative preparation was followed by a unilateral adrenalectomy and a clinical cure of both conditions. Immunohistochemical studies confirmed the ectopic production of adrenocorticotropic hormone and its related peptides more thoroughly than previous reports. The hormone production appeared clinically and immunocytochemically to resemble pituitary Cushing's disease more closely than ectopic production of adrenocorticotropic hormone by other tumors. The clinical aspects of this case illustrate the importance of proper preoperative recognition to reduce the high known percentage of morbidity and mortality. PMID- 3001371 TI - The varied radiographic manifestations of retroperitoneal malignant fibrous histiocytoma revealed through 27 cases. AB - Malignant fibrous histiocytoma, a pleomorphic sarcoma, represents the most common soft tissue sarcoma of late adult life. Approximately 15 per cent of the cases of malignant fibrous histiocytoma arise within the abdominal cavity or retroperitoneum. Although tumors in this series arose in many different sites, their proximity to the kidney necessitated a nephrectomy in 44 per cent of the cases (12 of 27). In 3 cases calcification was noted, which varied from a few small speckled areas to extensive large, coarse densities. The masses tended to compress or displace adjacent structures and vessels radiographically. Renal, duodenal and cecal invasion occasionally occurred. Ultrasonically, most tumors were hypoechoic (9 cases), with a few having a mixed pattern (2). On computerized tomography the masses were well circumscribed, with computerized tomography numbers in the range of solid tissue except for a few small areas of decreased density. The vascularity was variable, with 8 tumors being hypovascular and 8 showing a moderate to hypervascular pattern. Interestingly, the tumor derived a part of its blood supply from the renal vasculature in 12 cases (44 per cent). Even though malignant fibrous histiocytoma often was diagnosed incorrectly as an intrarenal or adrenal neoplasm, a cleavage plane usually could be identified between the tumor and the renal parenchyma in 1 of the studies, especially on the angiogram. This finding suggested its extrarenal origin. Knowing the tendency of malignant fibrous histiocytoma to arise from or be physically near the kidney and/or adrenal gland in elderly men may be of some diagnostic value. PMID- 3001370 TI - Renal function and (NA+ + K+)-ATPase in chronic unilateral hydronephrosis in dogs. AB - Recovery of various parameters of kidney function after varying periods of complete unilateral ureteral obstruction was studied in dogs under hydropenic conditions. Changes of PAH and inulin clearance appeared to be parallel. After one week of obstruction, renal clearances of PAH and inulin were decreased to seven per cent of values measured before the obstruction period, after two weeks to four per cent and after three or four weeks to two per cent. Within 10 to 28 days after release of obstruction by cutaneous ureterostomy, PAH and inulin clearance increased to 66 per cent after one week, to 50 per cent after two weeks, to 10 per cent after three weeks with no change after four weeks of obstruction. Na+ content in the hydronephrotic kidney differed from contralateral kidneys only in the inner medulla. The affinity for ouabain (dissociation constant, KD, normal = 3.85 X 10(-9) M; hydronephrotic = 3.05 X 10(-9) M; contralateral = 7.05 X 10(-9) M) was significantly higher only in the outer medulla of contralateral kidneys. Turnover number (normal = 3.6; hydronephrotic = 5.1; contralateral = 3.4 X 10(3) min.-1) in hydronephrotic or contralateral outer medulla was not significantly different from normal. Changes in kinetic constants (association rate constant, k+1, normal = 4.49 X 10(4) M-1 sec.-1; hydronephrotic = 4.13 X 10(4) M-1 sec.-1; contralateral = 5.97 X 10(4) M-1 sec.-1; dissociation rate constant, k-1, normal = 1.03 X 10(-4) sec.-1; hydronephrotic = 1.29 X 10( 4)sec.-1; contralateral = 1.39 X 10(-4) sec.-1) were considered to be too small to be relevant. Similar changes of KD, k+1 and k-1 were observed in the renal cortex. The osmotic concentrating capacity correlated well with (Na+ + K+)-ATPase activity (r = 0.85) and number of 3H-ouabain binding sites (r = 0.89) in renal outer medulla. The results indicate that recovery of osmotic concentrating capacity depends on the length of obstruction, and that a reduction of (Na+ + K+) ATPase molecules in the thick ascending limb of the loop of Henle is a primary factor in the decrease in the concentrating capacity of chronic unilateral hydronephrotic kidneys. PMID- 3001367 TI - Rapid emergence of novel antigenic and genetic variants of equine infectious anemia virus during persistent infection. AB - Previous results from our laboratory have demonstrated that equine infectious anemia virus displays structural variations in its surface glycoproteins and RNA genome during passage and chronic infections in experimentally infected Shetland ponies (Montelaro et al., J. Biol. Chem. 259:10539-10544, 1984; Payne et al., J. Gen. Virol. 65:1395-1399, 1984). The present study was undertaken to obtain an antigenic and biochemical characterization of equine infectious anemia virus isolates recovered from an experimentally infected pony during sequential disease episodes, each separated by intervals of only 4 to 8 weeks. The virus isolates could be distinguished antigenically by neutralization assays with serum from the infected pony and by Western blot analysis with a monoclonal antibody against the major surface glycoprotein gp90, thus demonstrating that novel antigenic variants of equine infectious anemia virus predominate during each clinical episode. The respective virion glycoproteins displayed different electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels, indicating structural variation. Tryptic peptide and glycopeptide maps of the viral proteins of each virus isolate revealed biochemical alterations involving amino acid sequence and glycosylation patterns in the virion surface glycoproteins gp90 and gp45. In contrast, no structural variation was observed in the internal viral proteins pp15, p26, and p9 from any of the four virus isolates. Oligonucleotide mapping experiments revealed similar but unique RNase T1-resistant oligonucleotide fingerprints of the RNA genomes of each of the virus isolates. Localization of altered oligonucleotides for one virus isolate placed two of three unique oligonucleotides within the predicted env gene region of the genome, perhaps correlating with the structural variation observed in the envelope glycoproteins. Thus these results support the concept that equine infectious anemia virus is indeed capable of relatively rapid genomic variations during replication, some of which result in altered glycoprotein structures and antigenic variants which are responsible for the unique periodic disease nature observed in persistently infected animals. The findings of envelope specific differences in isolates of visna virus and of human T-cell lymphotropic virus III (acquired immune deficiency syndrome-related virus) suggest that this variation may be a common characteristic of the subfamily Lentivirinae. PMID- 3001372 TI - Endocrine and metabolic responses of the collared peccary (Tayassu tajacu) to immobilization with ketamine hydrochloride. AB - Serial physiological responses were examined for 150 min from captive collared peccaries during immobilization with ketamine hydrochloride. Rectal temperatures decreased significantly (P less than 0.01) during anesthesia. Serum concentrations of total proteins, albumin, cholesterol, alanine aminotransferase, and calcium declined significantly (P less than 0.05) during the first 45 min post-immobilization before stabilizing. Concentrations of lactate dehydrogenase and alkaline phosphatase in sera showed similar but nonsignificant (P greater than 0.05) trends. Inorganic phosphorus and aspartate aminotransferase concentrations increased significantly (P less than 0.05) throughout the trial. Concentrations of serum glucose and glucocorticoid during the immobilization period were highly variable between individuals. Serum electrolytes, urea nitrogen, creatinine, gammaglutamyl transferase and progesterone were not significantly (P greater than 0.05) affected by immobilization. Elevations in serum testosterone were noted. Results indicated appropriate sampling times relative to immobilization for assay of particular serum biochemicals and steroid hormones during investigations of the physiology of the collared peccary. PMID- 3001373 TI - Serologic survey of canine coronavirus in wild coyotes in the western United States, 1972-1982. PMID- 3001374 TI - Spontaneous poxviral dermatitis and keratoconjunctivitis in free-ranging mule deer (Odocoileus hemionus) in Wyoming. PMID- 3001376 TI - Toxic shock syndrome and the vaginal contraceptive sponge. AB - Thirteen confirmed cases of toxic shock syndrome temporally related to use of the vaginal contraceptive sponge have been reported. The observed risk of toxic shock syndrome in sponge users may be elevated above estimated background rates, but this risk remains very low. Traumatic manipulation of the sponge, use during menstruation or the puerperium, and prolonged retention of the sponge may additionally increase toxic shock syndrome risk. As with all contraceptives, risks must be balanced against benefits. PMID- 3001375 TI - HTLV-III/LAV antibody and immune status of household contacts and sexual partners of persons with hemophilia. AB - We evaluated the human T-cell lymphotropic virus type III/lymphadenopathy associated virus (HTLV-III/LAV) antibody and immune status of 88 persons living with and/or sexual partners of 43 hemophiliacs, 12 of whom had AIDS, five of whom had AIDS-related complex (ARC), 17 of whom were clinically well but HTLV-III/LAV antibody positive, and nine of whom were well and HTLV-III/LAV antibody negative. No nonhemophilic household contacts (0/50) of healthy hemophiliacs were HTLV III/LAV antibody positive; two of 33 nonhemophilic AIDS/ARC contacts were positive. One was a spouse and one a sexual partner of a hemophiliac. One of these antibody-positive contacts herself had AIDS, and one had ARC. Antibody negative, nonhemophilic contacts of AIDS/ARC and of antibody-positive hemophiliacs had significantly lower numbers of lymphocytes, T helper lymphocytes, and T suppressor lymphocytes than did contacts of antibody-negative hemophiliacs. We conclude that risk of HTLV-III/LAV transmission may exist for spouses and/or sexual contacts of hemophiliacs with AIDS/ARC, but we cannot now determine the risk for contacts of asymptomatic hemophiliacs. PMID- 3001377 TI - Mapping cold virus structure may expand therapeutic strategies. PMID- 3001378 TI - Investigators seek shared virus antigens. PMID- 3001380 TI - Pathogens in fecal samples from apparently healthy children. PMID- 3001379 TI - Immunoaugmentative therapy. A primer on the perils of unproved treatments. AB - Immunoaugmentative therapy is an unproved cancer treatment that until recently was offered to patients by zoologist Lawrence Burton, PhD, at a facility in Freeport, Bahamas. The therapy consists of serum measurements of certain "immune deficiencies" and purportedly restores immune function by injection of products variously derived from tumor tissue and blood from individuals with cancer and healthy volunteers. Immunoaugmentative therapy represents a potentially serious public health risk, since it is capable of transmitting hepatitis B and the presumed etiologic agent for the acquired immunodeficiency syndrome. Physicians and health officials who learn of patients receiving this therapy are advised that its efficacy remains unproved and that the risk of receiving contaminated blood products is considerable. PMID- 3001381 TI - Significance of E. coli and rotavirus in infantile diarrhoea. PMID- 3001383 TI - [A case of male breast cancer with special reference to the hormonal environment during chemoendocrine therapy]. AB - Chemoendocrine therapy was performed on a man with advanced breast cancer, and partial response was observed for 11 months. Estrogen receptor in cancer tissue was detected before and after the therapy. Progesterone receptor, however, was not detected after the treatment, whether it had been there or not prior to it. The serum estrone level was continuously high during the treatment, but the serum testosterone level was obviously decreased after that. PMID- 3001384 TI - [Producibilities and properties of beta-lactamase and superoxide dismutase produced from Legionella species]. PMID- 3001382 TI - [Objective for clinical use of particle radiations with high LET]. AB - Radiation therapy has made a steady progress in local control of cancer following introduction of megavoltage radiations. Nevertheless, the fact that the doses delivered to the target volume are strongly dependent on the radiation tolerance of the surrounding normal tissues still remains a critical problem. Particle radiations have offered an impact in radiation therapy to improve further local control of cancer. Clinical experiences performed with fast neutrons and protons indicate that the features of high LET radiations will be realized by use of radiations with excellent dose distributions. Heavy ions characterized by Bragg peak with high LET will be introduced with an emphasis to confirm the doses delivered to the target volume. PMID- 3001385 TI - [Colloid carcinoma of gastrointestinal tract. I. Stomach]. PMID- 3001386 TI - [Luminol-dependent chemiluminescence of polymorphonuclear leukocytes from patients with acute gouty arthritis]. PMID- 3001387 TI - [Colloid carcinoma of gastrointestinal tract. II. Large intestine]. PMID- 3001388 TI - [Demonstration of transferrin receptor by enzyme-labelled antibody method on blood smears using monoclonal antibody-OKT 9]. PMID- 3001389 TI - [Membrane fluidity of B-16 melanoma cells exposed to ultraviolet light UV-B]. PMID- 3001390 TI - [An attempt to apply nuclear DNA cytofluorometry using endoscopically biopsied materials of the stomach]. PMID- 3001391 TI - [New therapy of hepatocellular carcinoma--topical infusion of ethanol guided by ultrasonics]. PMID- 3001392 TI - Modulation of cell communication and carcinogenesis. AB - In this paper, recent studies on the role of cell communication in cancer induction, particularly in two-stage carcinogenesis, were reviewed. Cell communication has been proposed to play an important role in cell growth and differentiation since its discovery. The recent finding that tumor promoters inhibit cell communication supports this possibility. The inhibition of cell communication by phorbol ester tumor promoters was also shown to correlate with enhancement of in vitro carcinogenesis in Balb/c 3T3 cells. This strongly suggests that the blocked cell communication may play a crucial causative role in the process of carcinogenesis. Accumulated evidence indicates that phorbol ester may induce blockage of cell communication through binding to its membrane receptor which is presumably Ca2+/phospholipid-dependent kinase. cAMP enhances cell communication and protects its inhibition by phorbol ester, presumably through activating cAMP-dependent kinase. This indicates the possibility that the two kinases may be key elements for physiological regulation of cell communication. It is proposed that the disturbance of the kinase systems by endogenous and exogenous factors may be responsible for the promotion phase of cancer induction. However, the true physiological role of cell communication in carcinogenesis remains to be demonstrated more directly. Especially, what kinds of molecules can pass through the gap junction and regulate cell functions in a cell community must be challenged in future. Some such molecules were speculatively described in this review. PMID- 3001393 TI - Regulatory mechanism of cell pH in the renal proximal tubule of bullfrog nephron. AB - To examine the cellular mechanism of urinary acidification in detail, micropuncture studies were performed on the in situ bullfrog proximal tubule with nigericin-based pH microelectrodes. Pencil-type double-barreled antimony microelectrodes were also used for monitoring pHs of the tubular fluids. Luminal perfusion of 10(-3) M cyanide caused a biphasic change in cell pH (pHi): i.e., early acidification by 0.04 pH unit in 2 min and later alkalinization by 0.04. A profound depolarization of 30-35 mV was observed in the peritubular membrane potential (EM Peri), although the tubular fluid pH (pHTF) was elevated by 0.11 unit. Luminal substitution of 100 mM Na+ by Li+ acidified the cell by 0.06 pH unit with a depolarization of EM Peri by 8 mV and an alkalinization of pHTF by 0.10 unit. It is a fact that cellular acidification and luminal alkalinization are in good agreement with the depression of luminal H+ secretory mechanism. Perfusion of 10(-4) M SITS from the peritubular side caused a rise in pHi by 0.04 without appreciable changes in EM Peri in the short period application. Peritubular perfusion of 10(-4) M ouabain lowered the pHi by 0.07 with a resulting depolarization of EM Peri by 15.4 mV, meanwhile, the pHTF, while initially lowered by 0.07 unit, was elevated 4 min later by 0.12. Inhibitions of the peritubular ion transport mechanism caused some pH changes in the same direction, both in the cell interior and the tubular fluid. Further, from the ouabain experiment, it is inferred that some linkages, mediated by Na+ and H+(or HCO3-), would exist between the peritubular and luminal membranes. PMID- 3001394 TI - Tension development in frog skeletal muscle induced by silver ions. AB - In single fibers of frog toe muscles placed in a Cl- free MOPS solution containing 1.8 mM Ca2+, tension developed slowly in the presence of very low concentrations of Ag+. This tension was not blocked by the administration of Co2+ or Ni2+. On the other hand, two types of transient tensions developed with the application of 5 microM Ag+, in fibers pretreated with 0-Ca2+ MOPS solution, containing either 2 mM Co2+ or 1mM Ni2+, for 10 min. In the presence of divalent cations or TTX, the first repetitive twitch-like contraction disappeared, indicating this tension is induced by action potentials repeatedly generated by the lack of divalent cations. The 2nd subsequent transient tension was caused by 5 microM Ag+ in the presence of various kinds of divalent cations, or TTX. After reversion to the resting tension, the fiber was contracted by adding more than 0.1 mM of Ca2+ or 25 mM caffeine to the external medium. Even when placed in a Ca2+-free solution containing 3 mM EGTA and 3 mM Mg2+ for 30 min, the fiber still developed an appreciable tension in response to 5 microM Ag+. These findings suggest that a transient development of the Ag+-induced tension does not require the presence of external Ca2+. A specific sulfhydryl reagent, pCMPS, did not contract the muscle fiber. Therefore, Ag+ may develop tension by mediating unknown chemical reaction(s) other than the sulfhydryl group on T-tubular membrane proteins. PMID- 3001395 TI - [A study of surface morphology and function of human pulmonary alveolar macrophages]. PMID- 3001396 TI - [Accessory cardiac bronchus found coincidentally in a patient with lung cancer]. PMID- 3001397 TI - [Tracheobronchial reconstruction in bronchial gland carcinoma]. PMID- 3001399 TI - Onset of neoplastic phenotype in an epithelial cell strain from adult BALB/c mouse lung alveolus. AB - Prolonged culture of NAL1A in 1 microM dexamethasone (CAS: 50-02-2) at low passage numbers resulted in the emergence of a morphologically altered and malignant cell strain, NAL1AM. (NAL1A was derived from lungs of normal adult female inbred BALB/c mice and exhibits several characteristics of epithelial cells.) A clone of NAL1A, B5, was shown to undergo a spontaneous morphologic alteration during culture in normal medium to resemble NAL1AM and cells of NUL1, a strain cultured directly from urethane (CAS: 51-79-6)-induced adenomas of mouse lung. Whereas NAL1A did not form colonies in soft agar, the clone B5 of NAL1A as well as NAL1AM and NUL1 showed quite high anchorage-independent growth (colony forming efficiency, 6.3-7.8%). Compared with NAL1A cells, clone B5, NAL1AM, and NUL1 each exhibited a ninefold-reduced level of cellular binding of epidermal growth factor. PMID- 3001398 TI - Comparative pathology of cardiac neoplasms in humans and in laboratory rodents: a review. AB - This review deals with the frequency, heredity, and morphology of 19 different histologic types of cardiac tumors that may affect humans, rat, mouse, guinea pig, and mastomys. Chemicals, durable fibrous materials, viruses, and probably irradiation have induced cardiac tumors in rodents. Apart from the involvement of asbestos in the induction of pericardial mesothelioma, no carcinogens have been identified in induction or promotion of cardiac tumors in humans. A hereditary tendency for the occurrence of myxoma and aortic paraganglioma has been indicated in humans as well as strain-specific heredity for rhabdomyoma in the guinea pig and for atriocaval node tumor in the rat. PMID- 3001401 TI - Influence of immunosuppressive therapy with azathioprine and prednisolone on serum-immunoglobulin concentration in renal transplanted patients. AB - Serological diagnosis of infectious diseases are based on the assumption that a change in virus-specific antibody - titer reflects the response to a certain viral infection due to changes in the concentration of the respective virus - specific antibodies. On the other hand immunosuppressive medication interacts with that system responsible in producing antigen-specific antibodies. This study was outlined therefore to follow the variation of the concentration of serum immunoglobulins of classes IgG and IgM with regard to a better evaluation of virus-specific antibody titers especially for those viruses that remain persistent after a primary infection and an reactivate. The study followed ten patients after allogenic cadaver kidney transplantation under immunosuppressive medication with azathioprine and corticosteroids. Concentration of serum-IgG and IgM protein was continuously measured for 6 months after transplantation along with measurement of virus-specific antibody-titers with enzyme immunoassay (Elisa) especially for cytomegalovirus. The results show a drastic decrease in serum immunoglobulins IgG and IgM - the lowest concentration being reached 25-50 days after transplantation. The concentration of IgG increased thereafter if no severe infectious diseases occurred during the post-transplant period. The concentration of IgM seems to react more sensitively upon infectious processes. In general, virus-specific antibody-titers (IgG) follow the sometimes drastic variation in the respective immunoglobulin class. It therefore reveals that antigen-specific antibody-titers in those patients should be controlled continuously during the time after transplantation for better evaluation of titer variations that eventually occur in correlation to the absolute concentration of the immunoglobulin class. PMID- 3001403 TI - [Radionuclide methods of examination in the diagnosis of lymphedema]. PMID- 3001404 TI - Basalt pneumoconiosis. PMID- 3001400 TI - [Alpha-adrenoceptors in the myocardium: incidence and functional significance]. AB - Alpha-adrenoceptors mediating positive inotropic effects are well established in the heart of various species including human heart. The mechanism by which alpha adrenoceptor stimulation increases force of contraction is not known. cAMP is unlikely to be involved as a mediator. Evidence has been presented that an increase in magnitude and duration of the slow Ca++ inward current may be partly responsible for the positive inotropic effect. In addition, stimulation of alpha adrenoceptors may increase Ca++ sensitivity of the contractile proteins. Stimulation of alpha-adrenoceptors by endogenous catecholamines may serve as a reserve mechanism under various conditions of impaired beta-adrenergic influence, e.g. hypothyroidism, bradycardia or ischemia. Furthermore, alpha-adrenoceptors may be involved in the genesis of reperfusion arrhythmias in ischemic heart. PMID- 3001405 TI - Bacterial clearance and granulocyte response in experimental peritonitis. AB - Clearance kinetics of Escherichia coli and Bacteroides fragilis from the peritoneal cavity and functional properties of peritoneal and blood polymorphonuclear leukocytes (PMNL) were studied in pigs. The bacteria, approximately 10(10) colony forming units (CFU) of each species, were intraperitoneally injected. Blood and peritoneal exudate were sampled regularly during 24 hr for bacterial quantification and PMNL analysis. The peritoneal concentration of both species fell rapidly in the first 3 hr and then stabilized. Approximately 10(2) CFU/ml remained after 24 hr. Chemotaxis, phagocytosis, and metabolic activation of the peritoneal PMNL showed little change. In some pigs a second bacterial dose was injected 6 hr after the first dose and gave an identical clearance pattern. Exhaustion of the local defence capacity thus was excluded as cause of the impaired bacterial elimination. PMID- 3001402 TI - [The liver and environmental poisons]. AB - For almost a century now numerous examples of acute and subacute hepatic injury from exposure to toxic agents in the occupational or non-occupational environment have been extensively studied and are well documented, but such events are comparatively rare. In contrast, epidemiological data associating exposure to environmental chemicals with chronic liver disease or primary hepatic malignancies in the human is scarce as compared with the vast body of literature concerning chronic pulmonary disease as a consequence of exposure at the workplace. Large-scale industrial production of many newly synthesized organic chemicals began during the period 1930-1940 but it was not until the 1960s that the output increased exponentially. Consequently, the spectrum of environmental influences is gaining increasing complexity since simultaneous or sequential exposure to a variety of pollutants is becoming the rule rather than the exception. Possible interaction or synergism of environmental agents--even of those which in themselves or for their low dosage level may be considered "harmless" - and particularly latency periods of more than one decade further complicate preventive strategies. The liver, as the central site for the biotransformation of xenobiotics, deserves special attention when new chemicals which are to be introduced into the environment are being tested for their potential toxicity, especially since many hepatotoxic agents have been shown to undergo bioactivation in the liver. Currently available information on hepatic injury due to environmental agents is briefly reviewed and comprises solvents and degreasing agents, pesticides, polyhalogenated biphenyls, dioxins and dibenzofuranes, epoxy resin hardeners, vinyl chloride, naturally occurring hepatotoxins in plants and fungi, herbal medicines and traditional remedies and a side-light on the Reye syndrome and the Spanish "toxic oil syndrome". PMID- 3001406 TI - Improved graft patency associated with altered platelet function induced by marine fatty acids in dogs. AB - Female mongrel dogs fed a marine fish diet rich in long-chain polyenoic fatty acids had improved patency of small-diameter arterial prosthetic grafts as compared to controls. Also, in vivo platelet function as measured by bleeding times was significantly prolonged. Eicosapentaenoic acid, not found in the serum of control animals, was present in relatively high concentrations in both the serum and a platelet-rich fraction of the marine oil-fed group. Eicosapentaenoic acid, unlike arachidonic acid, does not induce platelet aggregation and this phenomonon may account for the altered platelet function demonstrated in our animals and hence the improved graft patency. These data lend further support to the role of platelets in determining the patency of vascular grafts. PMID- 3001407 TI - Sex differences in adrenocortical structure and function. XX. The effects of gonadectomy and testosterone or estradiol replacement on cholesterol content and distribution in the gland. AB - Sex differences in the distribution of free and esterified cholesterol in the mitochondria and lipid droplets of the rat adrenal glands, as well as their dependence on gonadal hormones were studied. For these purposes intact, gonadectomized and gonadectomized-testosterone or estradiol replaced rats were employed. The concentration of free cholesterol [FC] as well as esterified cholesterol [EC] in full homogenates of decapsulated glands was higher in male than in female rats. Neither orchiectomy nor testosterone replacement had an effect on FC and EC concentration. Ovariectomy increased FC and EC concentration in full adrenal homogenates, an effect reversed by estradiol replacement. Similar changes were found in the lipid droplets. The concentration of FC and EC in the adrenal mitochondria was higher in male than in female rats. Orchiectomy increased the concentration of FC and decreased the concentration of EC. Testosterone administration resulted in further increase in FC concentration and restored EC concentration to the level of the control group. On the other hand ovariectomy increased FC and EC concentration, an effect reversed by estradiol replacement. Results obtained clearly showed that sex differences in FC and EC concentration in rat adrenal gland depend mainly on estradiol which lowers FC and EC concentration in the adrenal lipid droplets and mitochondria. PMID- 3001408 TI - Catecholestrogen binding sites in breast cancer. AB - The binding of 2-hydroxyestrone (2OH E1), a catecholestrogen which is the main end product of the 2-hydroxylation of estrogen, was investigated in breast cancers. 2OH E1-specific bindings were found in the cytosol (Kd = 0.54 +/- 0.10 nM) and in the endoplasmic reticulum (Kd = 3.36 +/- 1.32 nM). The dissociation rate constants of complexes between [3H]2OH E1 and cytosol or membrane binding sites were 3.30 h-1 and 8.30 h-1 respectively. Qualitative analysis of [3H]2OH E1 cytosolic complexes demonstrated a specific binding component with a mol. wt of 330,000 Daltons. Specificity experiments showed that nonestrogenic hormones were unable to compete with 2OH E1 for its binding sites, whereas triphenylethylene derivatives and catecholamines were potent 2OH E1 competitors. The presence of 2OH E1 specific bindings suggests a potential role of catecholestrogen in breast cancer. PMID- 3001409 TI - The adrenal secretion of progesterone stimulates testicular steroidogenesis in the rat in vitro. AB - We have used a multicolumn isolated cell superfusion system to investigate the interaction between isolated rat adrenal and testicular cells in the secretion of sex steroids. The steroid responses of a mixed population of adrenal and testicular cells to the administration of ACTH 100 pg/ml was compared with the responses of the separate cell types. Steroid responses from 5 separate experiments were analysed. We have demonstrated increased secretion of 17 alpha hydroxyprogesterone (P less than 0.0005), androstenedione (P less than 0.05) and testosterone (P less than 0.0005) by a mixture of cells when compared with the responses of either cell type alone. In contrast, the secretion of progesterone (P less than 0.0005) and corticosterone (P less than 0.005) was reduced in the mixed cell population, suggesting that progesterone is preferentially converted by testicular cells to 17 alpha-hydroxyprogesterone, androstenedione and testosterone. This is confirmed by increased secretion of 17 alpha hydroxyprogesterone, androstenedione and testosterone but not corticosterone by a mixed population following the administration of progesterone 10 ng/ml. These results suggest that the adrenal secretion of progesterone can stimulate testicular steroidogenesis in the male rat in vitro. PMID- 3001410 TI - Demonstration and characterization of a 1 alpha,25-(OH)2D3 receptor-like macromolecule in cultured rat pituitary cells. AB - Recent studies have demonstrated the importance of prolactin (PRL) and growth hormone (GH) in the regulation of 25-hydroxycholecalciferol-1 alpha-hydroxylase activity. We have previously shown that 1 alpha,25-dihydroxycholecalciferol (1 alpha,25-(OH)2D3) reduces PRL and GH production by a clonal strain of rat pituitary tumour cells (GH3). The biologically active form of vitamin D3, 1 alpha,25-(OH)2D3, acts via an initial binding to cytoplasmic receptor proteins in target cells, and we demonstrate in this study the presence of specific receptors for 1 alpha,25-(OH)2D3 in the GH3 cells. GH3 cell cytosol was incubated with [3H]1 alpha,25-(OH)2D3 at 0-4 degrees C. Maximal binding was obtained between 2 and 6 h, and Scatchard analysis showed one single class of binding sites with Kd of 0.33 +/- 0.05 nM (mean + SD) and a Bmax of 103 +/- 26 fmol/mg cytosol protein. Competitive binding experiments revealed the following potency order: 1 alpha,25 (OH)2D3 greater than 25-OHD3 greater than 1 alpha-OHD3, 24,25-(OH)2D3. In contrast, corticosterone, testosterone, progesterone and oestradiol showed negligible ability to displace [3H]1 alpha,25-(OH)2D3 from its receptor. Sucrose gradient ultracentrifugation in high salt concentration revealed that GH3 cell cytosol possessed at 3.7S [3H]1 alpha,25-(OH)2D3 receptor protein which was inactivated by heating and protease treatment, but not after incubation with DNase or RNase. The receptor protein aggregated in salt-free sucrose gradients since the 3.7S complex was shifted reversibly to a approximately 6S form. Isoelectric focussing localized most of the [3H]1 alpha,25-(OH)2D3 to a protein peak with an isoelectric point of approximately 6 (pI 5.8-6.2). Since this 1 alpha,25-(OH)2D3 receptor protein has similar properties as the corresponding 1 alpha,25-(OH)2D3 receptors found in normal rat tissues, we suggest that lactotropes and somatotropes represent true target cells for 1 alpha,25-(OH)2D3. PMID- 3001411 TI - Effect of dehydroepiandrosterone on human erythrocytes redox metabolism: inhibition of glucose-6-phosphate dehydrogenase activity in vivo and in vitro. AB - In order to elucidate the role of G6PD inhibition by DHEA on erythrocyte redox metabolism, we measured: (1) G6PD activity in erythrocytes collected at different times after injection of synthetic ACTH in 13 normal subjects; (2) G6PD activity and GSH levels in erythrocytes incubated in the presence of different DHEA concentrations; (3) DHEA distribution and metabolic clearance in red cells, together with G6PD activity; (4) GSH regeneration after hydroperoxide oxidative stress in red cells preincubated in the presence of DHEA. In vivo DHEA increase elicits a clear-cut inhibition of red cell G6PD activity. Decreasing DHEA goes in step with the recovery of enzyme activity. In vitro G6PD inhibition by DHEA reaches its maximum within 10-15 min (20-25% inhibition at 10(-7) M DHEA concentration) and the recovery time is dose-dependent. More than 2/3 of DHEA is concentrated in the red cell after 5 min incubation. GSH levels change in step with G6PD activity. After oxidative stress by BHP, DHEA-treated cells fail to restore normal GSH concentrations. These results show that DHEA inhibits human erythrocyte G6PD activity at concentrations usually observed after ACTH plasma increase and increases red cell sensitivity to oxidant agents. Moreover, it is possible that the high DHEA concentrations present in target tissues may interfere with metabolic pathways in which NADPH is the cofactor. PMID- 3001412 TI - GnRH receptors and actions in the control of reproductive function. AB - The hypothalamic control of reproductive function is expressed through the receptor-mediated actions of GnRH on the pituitary gonadotroph. GnRH receptors in the pituitary gland exhibit prominent variations in number during the ovarian cycle and after changes in steroid feedback, and are modulated by the rate of GnRH secretion from the hypothalamus. In cultured pituitary cells, GnRH receptors undergo down-regulation during exposure to GnRH agonists, followed by a subsequent elevation of sites that is dependent on protein synthesis. GnRH antagonists do not cause receptor down-regulation, but high-affinity antagonist analogs bind for extended periods to cause receptor occlusion and prolonged inhibition of GnRH action. Analysis of the rat pituitary GnRH receptor by photoaffinity labeling reveals two binding subunits of mol. wt 53,000 and 42,000. The receptor-activated processes leading to gonadotropin secretion are highly calcium-dependent, and are initiated by rapid phospholipid hydrolysis with production of arachidonic acid metabolites, diacylglycerol, and inositol phosphates. The role of protein kinase C in gonadotropin secretion is indicated by the ability of phorbol esters and synthetic diacylglycerols to stimulate LH release, the inhibition of protein kinase C and LH release by retinal, and the redistribution of protein kinase C between cytosol and membrane fractions during stimulation of pituitary gonadotrophs by GnRH. It is likely that the effects of arachidonate metabolites are integrated with those of calcium-calmodulin and calcium, phospholipid-dependent protein kinases during the immediate and sustained phases of GnRH-induced gonadotropin secretion. PMID- 3001413 TI - Gonadotrophin releasing hormone receptor regulation in relationship to gonadotrophin secretion. AB - The relationship between pituitary GnRH receptors (GnRH-R) and LH responsiveness to GnRH stimulation is not straightforward. In some circumstances, e.g. post gonadectomy of rats, in lactating rats, during the rat, hamster and monkey oestrous cycles there appears to be a good positive correlation between GnRH-R, basal serum LH values and LH responses to exogenous GnRH. However, in mice following gonadectomy GnRH-R fall by 50% while serum LH levels rise by 10-fold, and in cultured pituitary cells, GnRH exposure increases GnRH-R yet desensitizes cellular responsiveness to subsequent GnRH stimulation. Thus, our original hypothesis that GnRH-R regulation was closely coupled to gonadotroph secretory function does not always hold. Further, we and others, using the rat as an experimental model, hypothesised that the pituitary GnRH receptor content reflected the level of previous pituitary exposure to endogenous GnRH. This view is supported with studies in the GnRH deficient hypogonadotrophic hypogonadal (hpg) mouse in which exogenous GnRH rapidly normalises GnRH-R from very low levels, and is accompanied by rapid activation of pituitary FSH synthesis. However, the post-castration fall in GnRH-R in mice, which is opposite to that in rats, does not appear to be so closely related to endogenous GnRH secretion and cannot be reversed by exogenous GnRH. Using the ovariectomised mouse as an experimental model, evidence has been obtained that estradiol, in addition to GnRH, is essential for maintenance of pituitary GnRH-R in this species. Exogenous estradiol stimulates GnRH-R in OVX mice while it reduces the high values in OVX rats. In female mice estradiol and GnRH have additive stimulatory effects on GnRH R. Thus, there is species variability in the predominant hormonal regulation of GnRH receptors. In rat pituitary cells in vitro up-regulation of GnRH-R can be effected by several agents which stimulate LH release (GnRH, KCl, DbCAMP) as well as some which do not (Ca ionophore at low concentrations). Receptor up-regulation requires Ca2+ mobilisation and protein synthesis. The data obtained from several in vivo and in vitro model systems supports the conclusion that GnRH receptor changes represent another, medium-term, consequence of GnRH action on the gonadotroph and are not always a locus for the modulation of gonadotrophin secretion and synthesis. PMID- 3001414 TI - Molecular mechanism of gonadotropin releasing hormone action. AB - This work summarizes some of our studies related to the mechanism of action of gonadotropin releasing hormone including those leading to identification of the three steps of the gonadotropin releasing process: receptor binding, mobilization of extracellular calcium, and granule exocytosis. Evidence is also presented to suggest how these steps are integrated one to another and how they are integrated to other actions of the releasing hormone such as regulation of target cell sensitivity, and receptor regulation. PMID- 3001415 TI - Mechanism of action of gonadotropin releasing hormone: role of lipoxygenase products of arachidonic acid in luteinizing hormone release. AB - The mechanism of action of gonadotropin-releasing hormone (GnRH) upon pituitary luteinizing hormone (LH) secretion has not yet been elucidated, but recent evidence has suggested that arachidonic acid or its metabolites are involved in GnRH action. In cultured rat pituitary cells, arachidonic acid and 5-hydroxy 6,8,11,14-eicosatetraenoic acid (5-HETE) elicited concentration-dependent release of LH with EC50 of about 12 microM. Other lipoxygenase derivatives including 11-, 12- and 15-HETE, had no consistent effect on LH release, and leukotrienes (B4 and C4) exerted only minor stimulatory actions on LH release. The lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), and 3-amino-1-(3-trifluoromethyl phenyl)-2-pyrazoline hydrochloride (BW 755C) caused dose-dependent inhibition of GnRH-induced LH release, with IC50 values of 5, 8.5, and 175 microM, respectively. In contrast, the cyclooxygenase inhibitor, indomethacin, had a biphasic action on GnRH-stimulated LH release, with potentiation of GnRH action at low doses (up to 25 microM) and no effect at higher concentrations. These findings are consistent with the potential role of a 5-lipoxygenase product of arachidonic acid in the mechanism of action of GnRH on pituitary gonadotropin release. PMID- 3001416 TI - In the central role of GnRH in the regulation of reproductive functions: mechanism of action and practical results. AB - The regulation of reproductive functions is influenced by several hormones. The hypothalamic hormone GnRH has a central role in reproduction. Several superactive and inhibitory analogs of GnRH synthesized in our laboratory showed use in animal breeding. GnRH was suggested to be degraded by the hypothalamus itself and also by the pituitary. We have used cultured fetal hypothalamic cells, and cultured pituitary cells to study the storage, the release and the action of this neuropeptide. It was found that neither the cultured hypothalamic nor the pituitary cells degrade GnRH, and pretreatment of these cultured cells with steroids do not induce GnRH degradation. The basal GnRH secretion from the cultured hypothalamic cells was increased by steroid pretreatment while the neurotransmitter induced GnRH release did not change. At the pituitary level it was found that GnRH regulates its own effect by priming or desensitizing gonadotropin release depending on the pretreatment time or the concentration of GnRH. The self-regulatory action of GnRH follows a similar pattern for both LH and FSH release. PMID- 3001417 TI - Fetal Leydig cell culture--an in vitro system for the study of trophic hormone and GnRH receptors and actions. AB - In fetal and neonatal rat Leydig cells cultured in the presence of LH, gonadotropin and GnRH receptors and acute testosterone responses to hCG, were maintained for up to 78 days. Addition of GnRH agonists markedly inhibited steroid production in LH-treated cultures, and abolished the acute testosterone response to hCG. GnRH receptors were not detectable in fetal testes but were present post-natally and increased markedly with age. In cultured fetal testis, GnRH receptors were detected on the third day, and were increased by exposure to GnRH agonists. In LH-treated cultures, GnRH sites were reduced by about 50% and did not increase during incubation with GnRH agonists. LH supported 17 alpha hydroxylase/17-20 desmolase activities and pregnenolone formation were observed in short (1-4 days) and long-term cultures. Also, LH stimulation of 3 beta hydroxysteroid dehydrogenase was observed by histochemical studies at 7 days of culture. GnRH agonists inhibited LH dependent steroid production in a dose dependent fashion and abolished the acute testosterone response to human chorionic gonadotropin. The major component of the steroid inhibitory effect of GnRH agonist occurs beyond cAMP production. A distal lesion of the microsomal enzymes of the androgen pathway is largely responsible for the GnRH-induced decreases in LH-supported androgen production. The expression of functional GnRH receptors during culture and their suppression by LH suggest that pituitary gonadotropins exert a tonic inhibitory effect upon testicular GnRH receptors. The presence of functional GnRH receptors and inhibitory actions in cultured fetal and neonatal Leydig cells indicates that GnRH-related peptides can influence the actions of gonadotropins on the fetal Leydig cell population. PMID- 3001419 TI - Gonadotropin-releasing hormone as a modulator of ovarian function. AB - GnRH and its agonist analogs exert direct inhibitory and stimulatory effects on the ovaries of animals from several species. In the immature follicle, GnRH inhibits the actions of FSH on an integrated array of biochemical responses that lead to follicular development and corpus luteum formation. GnRH also suppresses gonadotropin action in mature follicles, and stimulates certain ovarian processes such as steroidogenesis and oocyte maturation. The inhibitory and stimulatory effects of GnRH are mediated through the binding of the peptide to high-affinity receptors in granulosa and thecal cells. Recent studies have shown that GnRH action in the ovary is dependent upon calcium mobilization and probably operates through stimulation of phospholipid turnover and activation of protein kinase C. PMID- 3001418 TI - Antigonadotropic actions of GnRH agonist on ovarian cells in vivo and in vitro. AB - GnRH and its agonists have recently been shown to inhibit a variety of reproductive functions, in addition to its well-known gonadotropin releasing action in the pituitary. In order to determine the inhibitory mechanism and the site of action of these peptides in ovary, the direct actions of an agonistic analogue [D-Leu6, des-Gly-NH2] GnRH ethylamide on hypophysectomized immature rat ovaries in vivo and on rat luteal cells as well as porcine granulosa cells incubated in vitro were investigated. 125I-Labeled GnRH agonist, when injected to immature female rats, bound specifically not only to pituitary but also to ovaries. GnRH agonist inhibited hCG stimulation of progesterone production and ovarian weight augmentation in hypophysectomized immature female rats in vivo. FSH-stimulated induction of ovarian LH/hCG receptors and ovarian weight gain in diethylstilbestrol (DES)-treated hypophysectomized immature female rats were also suppressed by GnRH agonist. Moreover, treatment with GnRH agonist inhibited hCG stimulated progesterone production by rat luteal cells incubated in vitro. In short time incubation of porcine granulosa cells obtained from medium follicles, the capacity and affinity of LH/hCG receptors were not affected by GnRH agonist. However, in long term culture of porcine granulosa cells obtained from small follicles, concomitant treatment with GnRH agonist markedly inhibited the induction of LH/hCG receptors stimulated by FSH and insulin. It may be possible that GnRH agonist prevents the differentiation of granulosa cells and subsequent acquisition of LH/hCG receptors, but has no effect on the LH/hCG receptors already induced. On the other hand, GnRH agonist delayed hCG-stimulated accumulation of cyclic AMP in porcine granulosa cells obtained from medium follicles. This delay of cyclic AMP accumulation may be responsible for the inhibition of progesterone production by ovarian cells. These findings suggest that GnRH agonist acts directly on ovarian cells and inhibits the action of FSH and LH/hCG, independently. PMID- 3001420 TI - Comparisons of the potential utility of LHRH agonists and antagonists for fertility control. AB - Prospects for the use of LHRH analogs for human fertility control have been reviewed with particular reference to two highly potent representatives. Nafarelin acetate, the LHRH agonist, has a potency about 200 X that of LHRH and is consistently effective in suppressing gonadal function in females through a desensitization of LHRH receptors in the pituitary. Such agents show promise as ovulation inhibitors for women although concern has been expressed over the dangers of unopposed estrogen or alternatively hypoestrogenemia. Although early studies indicated luteolysis in women and interceptive action in baboons it is now clear that the LHRH agonists will not be useful clinically to terminate pregnancy. Wide species differences in the male response to LHRH agonists exist. Unfortunately azoospermia has not been achieved in men. The LHRH antagonists, typified by [N-Ac-D-Nal(2)1, D-pCl-Phe2, D-Trp3, D-hArg(Et2)6, D-Ala10]LHRH, require high doses to competitively inhibit responses to endogenous LHRH. Their advantages include a rapid induction of the hypogonadal state with apparently little species or sexual variation in response. Based on animal studies, preferable utility of the antagonists would lie in male contraception and pregnancy interception. PMID- 3001422 TI - Spontaneous rupture of hepatocellular carcinoma. AB - Spontaneous rupture of a primary hepatocellular carcinoma is an extremely rare event in the western hemisphere. Only a handful of single-case reports have been authored in the continental United States. A ruptured hepatoma carries a dismal prognosis and is usually beyond a "resection for cure" stage. In this report, two cases of spontaneously ruptured primary hepatocellular carcinoma are described. Both cases involved cirrhotic livers, and the tumor in each case was resected to attempt cure. One patient survived nearly 2 years; the other is alive and well at this time. PMID- 3001421 TI - Inhibition of sex-steroid hormone induced ornithine decarboxylase and poly(A) polymerase activities by a GnRH agonist in the rat kidney. AB - Recently GnRH and its synthetic agonists were shown to exert extrapituitary actions and inhibit the growth-promoting effects of testosterone and estradiol. In the present study, the long-term effect of GnRH agonist [des-Gly10, D-Ala6 GnRH ethylamide GnRHa] on the steroid-induced renotropic effect was investigated. Gonadectomy of 30-day old male and female rats resulted in the drastic reduction of kidney ornithine decarboxylase (ODC) activity. Injection of testosterone (75 micrograms/rat) or estradiol (1 microgram/rat) to castrated rats significantly increased the enzyme activity at 24 h. Treatment with the GnRH agonist, two doses of 100 micrograms each, caused significant inhibition of the hormone induced ODC activity. Similarly injection of dihydrotestosterone to castrated adult male rats significantly increased poly(A)-polymerase activity at 24 h. Two doses of 100 micrograms of GnRHa caused significant inhibition of DHT induced poly(A) polymerase activity. These results show that chronic exposure to potent GnRH analogs may have anti-steroidal activity in the kidney. PMID- 3001423 TI - The response to further chemotherapy in patients with carcinoma of the breast who progressed while receiving adjuvant therapy. AB - The effect of known chemotherapeutic programs in patients with breast cancer who developed metastasis during or after adjuvant chemotherapy with L-PAM plus 5-FU (PF) were studied. Thirteen patients failed while receiving adjuvant therapy at 3 22 months from the time therapy started. Three patients failed 6-28 months after 2 years of therapy. Thirteen patients received PF followed by cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) at the time of progression on PF. Of these, ten received Adriamycin and vincristine (AV) at the time of progression on CMF. The response to CMF was 7%. No patient responded to AV after progression on CMF. Two of three patients treated with AV after failure on PF did respond, but both for only 6 months. This suggests that adjuvant therapy may render the tumor less responsive to further chemotherapy. Since most patients failed while on adjuvant therapy, this may indicate a poor prognosis regardless of the agents used. PMID- 3001424 TI - Hepatic tumors and oral contraceptives: surgical management. AB - The clinical and pathological features of 14 patients with benign liver tumors are reviewed. There were two males and 12 females in this series of cases. All but one of the females had been on contraceptive steroid therapy for an average of 7.8 years. Abdominal pain was the presenting complaint in 75% of cases, a palpable abdominal mass was present in 22%, while 12.5% of the patients presented with acute hemorrhagic shock due to rupture of a liver cell adenoma. Liver cell adenomas (LCA) were found in 87.5% of the cases and a diagnosis of focal nodular hyperplasia (FNA) was made at histologic examination of the resected tumors in 12.5% of cases. Surgical resection of the liver tumors was performed successfully in 89% of the cases. Hepatic lobectomy was accomplished in four patients, hepatic segmentectomy was possible in three cases, while local wedge resection or focal excision were indicated on seven occasions. There was no operative mortality in this series, but one patient required reoperation for drainage of a complicating subphrenic abscess. PMID- 3001426 TI - Pathological evaluation of hepatic dearterialization in encapsulated hepatocellular carcinoma. AB - Specimens of encapsulated hepatocellular carcinoma from 48 patients who had received no previous treatment and from six patients previously treated by hepatic arterial interruption by embolization or ligation were obtained by resection and examined for incidence and extent of necrosis of the tumor. Among the 48 specimens from the non-pre-treated patients, 25 tumors were smaller than 5 cm and showed no notable necrosis; of the 23 remaining tumors--which were larger than 5 cm--12 (52%) showed spontaneous central necrosis. The necrosis rate of the tumor ranged from 10% to 40%. In contrast, in six specimens from pretreated cases, more than 60% of the tumors were found to be necrotic. Two tumors smaller than 5 cm were completely necrotized by embolization. The findings indicated the significance of hepatic dearterialization for the management of encapsulated hepatocellular carcinoma. Daughter nodules, however, were found to have escaped necrosis. PMID- 3001425 TI - Paget's disease of the breast presenting as a cutaneous horn. AB - A 67-year-old woman developed a cutaneous horn on the nipple of her right breast. Biopsy of the skin underlying the horn disclosed Paget's disease of the breast. An intraductal adenocarcinoma of the same breast was found on mastectomy. High index of suspicion is mandatory in evaluating all nipple lesions. PMID- 3001427 TI - Unusual faces of lung cancer. AB - Lung cancer is the leading cause of death from malignancy in men; and prediction indicates that in women it will soon surpass breast carcinoma in incidence and mortality. Four unusual modes of presentation of lung cancer are described, namely: ascites, gynecomastia, rectal bleeding, and sudden death. An illustrative case for each characteristic, along with a brief literature review, is reported. PMID- 3001428 TI - Adenoid cystic carcinoma of Bartholin's gland: review of the literature and report of two cases. AB - Two patients with adenoid cystic carcinoma of Bartholin's gland and a review of the relevant literature are presented. With the inclusion of these two patients, there are now 24 cases reported. Both patients had large vulvar masses with a short clinical history, and several local tumor recurrences within the first 21/2 years after radical vulvectomy. The characteristic cribriform pattern and perineural involvement in addition to vascular invasion were present in the pathological material. No metastases were found in the inguino-femoral lymph nodes removed. Both patients are alive, without evidence of local recurrence but with lung metastases. A chemotherapeutic treatment scheme (adriamycin and cyclophosphamide) is underway but it is too early to evaluate its results. PMID- 3001429 TI - Advances in the diagnosis and treatment of EBV-associated lymphoproliferative diseases in immunocompromised hosts. AB - The clinical, immunopathologic, and virologic features of the lymphoproliferative diseases occurring after renal transplantation have been characterized. Clinically, patients may present with an infectious mononucleosis-like illness or with localized solid tumor masses. These lymphoproliferative diseases have unique histologic features that can be classified as polymorphic diffuse B-cell hyperplasia (PDBH) or polymorphic B-cell lymphoma (PBL). Immunologic cell-typing studies have shown that the majority are polyclonal B-cell proliferations, but monoclonal B-cell tumors have also been documented. These B-cell proliferations may, however, evolve from a benign polyclonal B-cell hyperplasia to a monoclonal malignant lymphoma. The Epstein-Barr virus (EBV) has been implicated as the cause of these disorders. Serologic studies frequently demonstrate evidence of a primary or reactivation infection, touch imprints from involved tissue may stain for the presence of EBNA (Epstein-Barr nuclear antigen), and EBV DNA hybridization studies demonstrate the presence of EBV-specific DNA sequences within tumor cells. Since EBV induces a polyclonal B-cell proliferation in vitro and in vivo, the polyclonality of these diseases also implicates EBV. Acyclovir, a new synthetic antiviral agent that inhibits EBV DNA replication may be effective in some patients during the polyclonal growth phase but is ineffective once the tumor evolves into a monoclonal lymphoma. We have identified several factors that may be useful in predicting responsiveness to acyclovir therapy. PMID- 3001430 TI - Wilms' tumor--treatment and results: a five-decade experience. AB - During the past 53 years, 105 patients with Wilms' Tumor have been treated at the Children's Hospital of Buffalo. Prospective and retrospective staging of these cases has allowed comparison with other reported series. All-stage survival improved from 37% with operation alone to 46% with operation and planned radiotherapy. During the past 22 years, with the addition of adjunctive chemotherapy, all-stage survival is now 80% with lesser stage survival approaching 100%. Participation in the cooperative National Wilms' Tumor Study has brought the most effective proven treatment to the patient in the shortest period of time. PMID- 3001431 TI - The treatment of primary tumors of the femur with chemotherapy (if indicated), resection and reconstruction with an endoprosthesis. AB - The treatment protocol of 15 patients with a primary tumor of the femur, including osteosarcoma, malignant fibrous histiocytoma and chondrosarcoma is presented. All patients had been selected for resection and reconstruction with an endoprosthesis. An endoprosthesis was implanted in 12 patients. The results of this type of treatment appear to be satisfactory. In eight osteosarcoma cases resection and reconstruction with an endoprosthesis combined with preoperative and postoperative chemotherapy, according to Rosen, were performed. Follow-up in all 15 patients, varying from 1.4 to 6.0 years, showed no evidence of disease in 12 patients. Three patients had died. Function of the involved leg was satisfactory in most cases. The advantage and disadvantages of the use of an endoprosthesis are discussed as well as complications in this series of patients. PMID- 3001432 TI - [Enkephalinergic regulation of cardiovascular centers]. PMID- 3001434 TI - Nucleotide distribution and the recognition of coding regions in DNA sequences: an information theory approach. AB - A method for the recognition of coding-regions along DNA sequences is described. The method is based on the observation, made in several cases, that nucleotide distribution at the third position of the codon is more biased (less random) than that in the other two positions. It is suggested that since nucleotide distribution at the third position is only weakly influenced by the amino acid distribution in the coded protein, there must be some constraints at the DNA level which bias the nucleotide distribution at the third position. The distinction between DNA-level constraints and protein-level constraints is discussed in the frame of Information Theory, and the analysis of the Mitochondrial gene coding for subunit-1 of the yeast cytochrome oxidase is presented. PMID- 3001433 TI - The biological significance of G-T/G-U mispairing in nucleic acid secondary structure. AB - We have computed the expected distribution of the potential for hairpin-like secondary structures with small loops (3-20 bases) and uninterrupted stems and compared that to the distribution observed in the complete genomes of seven DNA viruses from animals, plants and bacteria, as well as a bacterial plasmid. The formation of G-T mismatches in the stems of these structures was allowed. Furthermore we have analyzed the distribution of the potential for such structures along the genetic maps of these genomes, specifically around the start sites of known genes. Our data reveal that the potential for mismatch containing structures with stem length exceeding eight base pairs is over-represented and non-randomly distributed, but to a much lesser degree than that for perfect structures of equal size. Moreover, the potential for both types of structures is preferentially located near functional start codons. From this we deduce that in general G-T/G-U containing nucleic acid secondary structures are biologically relevant, though possibly less significant than perfect ones. PMID- 3001435 TI - HTLV-I infection of T and B cells of a patient with adult T-cell leukemia lymphoma (ATLL) and transmission of HTLV-I from B cells to normal T cells. AB - We analysed the DNA of different tissues of a patient (HS) with adult T-cell leukemia/lymphoma virus (HTLV-I). We detected viral sequences in fresh specimens from spleen, thymus, liver, skin and peripheral blood neoplastic lymphocytes. The pattern of HTLV-I integration is identical in the leukemic cells and in all other tissues analysed, but the signal intensity is strongest in the leukemic cells, indicating the source of HTLV-I proviral sequences was the leukemic T-cells which had infiltrated these tissues. In fact, the cultured skin fibroblasts of the patient did not contain HTLV-I sequence. However, cultured lymphocytes of this patient was consistently an immortalized B-cell line containing HTLV-I sequences in a manner indicative of a polyclonal infection. This cell line was also infected with the Epstein-Barr virus (EBV). In order to determine whether HTLV-I alone was sufficient for B-cell immortalization, we obtained single cell clones by limiting dilution. The DNA of all the cell clones that we analysed contained both the HTLV-I and EBV genomes, suggesting that immortalization of the B-cell was more likely due to the EBV rather than HTLV-I. Infectious HTLV-I viruses produced by the B-cell line still had the propensity to infect and transform T lymphocytes in normal human umbilical cord blood. Unlike the parental B cells, the transformed T lymphocytes were clonally selected. Our results indicate that although the predominant infected cell population of the patient was his leukemic T lymphocytes, some of his EBV-positive B-lymphocytes were also polyclonally infected. The latter had a growth advantage in culture over the T lymphocytes but the virus produced by these immortalized B cells has not been adapted and has maintained its tropism for T cells. PMID- 3001436 TI - Reversal of feline retroviral suppression by indomethacin. AB - The immunosuppressive effect of feline leukemia virus (FeLV) and its 15,000 dalton envelope protein (p15E) were studied to determine if the mechanism of action was due to an increase in prostaglandin production. We examined the effects of exogenous PGE1 and PGE2 on the normal Con A response of feline peripheral blood lymphocytes (PBL) and found them to be inhibitory. The addition of the cyclooxygenase inhibitor indomethacin to cells incubated with FeLV or FeLV p15E and Con A completely abrogated the viral suppressive effects. This reversal was titratable and time-dependent. Other non-steroidal anti-inflammatory (NSAI) drugs were found to have similar actions. Indomethacin was also able to increase the suppressed Con A response of PBL from FeLV-infected cats. Upon measurement of PGE2 levels from PBL cultured with FeLV, we found a decrease in PGE2 accumulation associated with FeLV presence during the first 24 h of culture. These findings indicate that FeLV does not cause its immunosuppressive effects by increasing PG production and suggests that indomethacin and the other tested NSAI drugs do not produce their effect by PG inhibition. PMID- 3001437 TI - Further evidence for the existence of 'homing' receptors on murine leukemia cells which mediate adherence to normal bone marrow stromal cells. AB - A significant proportion of 131IUDR-labelled cells from murine leukemia cell lines L1210 and P388, but not the L5178Y lymphoma cell line, are retained in the bone marrow (B.M.) following i.v. injection into syngeneic mice. Following this, L1210 and P388 cells grow and rapidly replace the normal hematopoietic cells of the B.M. L1210 and P388 cells, but not several lymphoma cell lines, also bind avidly to monolayers of B.M. stromal cells (Dexter cultures) and soon overgrow the cultures following rapid cell proliferation. P388 cells bound equally well to confluent monolayers of B.M., whole mouse embryo and newborn mouse kidney while L1210 cells bound well to B.M. and whole mouse embryo but showed little binding to newborn kidney monolayers. The accumulation of the two leukemia cell lines in the B.M. was constant and indistinguishable over a 48-h period. In contrast, in both spleen and liver the number of L1210 cells decreased during the same period while P388 cells were retained at a constant level. Generally there was a lack of correlation of B.M. metastasis of a cell line and its metastasis to other organs although P388 cells, but not L1210 cells, demonstrated a tremendous capacity for metastatic growth in both spleen and liver. Normal B.M. cells were fused with the syngeneic SP2/0 murine myeloma fusor line and 10 hybridomas plus the SP2/0 parent were tested for in-vitro adherence to B.M. monolayers and in-vivo metastatic behavior. The same 3 (out of 10) hybridomas showed a high level of adherence to B.M. monolayers, high levels of retention of cells in the B.M. following i.v. injection, and rapid growth and takeover of the normal B.M. In marked contrast, neither the SP2/0 parent nor the remaining 7 hybridomas show significant adherence, B.M. retention or growth in the B.M. A distinct lack of correlation of B.M. vs liver or spleen metastasis was once again noted for the hybridomas although all of the hybridomas showed much less metastatic growth in the liver than the SP2/0 parent. Seven out of 10 hybridomas also showed less metastatic growth in the spleen including all 3 of the hybridomas which showed preferential growth in the B.M. Our data are consistent with the existence of cell surface 'homing' receptors on leukemia cells for normal B.M. stromal cells which function to retain the leukemia cells in the B.M. Such receptors might serve as functional markers for cell differentiation and leukemia classification. PMID- 3001438 TI - Laboratory diagnosis of hypopituitarism. AB - Hypopituitarism can be caused by failure or loss of one or more of the eight identifiable hormones in the anterior lobe of the pituitary gland. The endocrine manifestations of hypopituitarism are related to the type and degree of hormonal deficiency and the stage in life during which the deficiency occurs. In patients with suspected hypopituitarism, the diagnostic approach consists of determining the extent and the cause of the hormonal loss. Specific provocative tests for the diagnosis of hypopituitarism are reviewed in detail in this article. PMID- 3001439 TI - Cushing's syndrome: update of diagnosis and management. AB - Although considerable advances have been made in the understanding of Cushing's syndrome in the recent past, many difficulties persist in the diagnosis and management of patients with hypercortisolism. Precision in the diagnosis of Cushing's syndrome and the differentiation of its various forms have gradually improved, but a substantial number of cases have laboratory or radiologic findings that can be misleading or at least difficult to interpret. Furthermore, other conditions may mimic Cushing's syndrome and add to the diagnostic difficulties. Surgical extirpation of primary adrenal lesions that cause the hypercortisolism or of the neoplasms responsible for the ectopic production of adrenocorticotropic hormone remains the treatment of choice for these problems. Currently, transsphenoidal surgical exploration is the treatment of choice for Cushing's disease. PMID- 3001440 TI - Decrease of lymphocyte (Na+,K+)ATP-ase activity in aged people. AB - Activity of (Na+,K+)ATP-ase measured by means of influx of 86Rb was compared in lymphocytes from young, middle-age and old people. It was found that the (sensitive to ouabain) activity of (Na+,K+)ATPase was significantly diminished in the lymphocytes from aged subjects. PMID- 3001442 TI - [Peripheral neuropathy associated with polycythemia vera]. PMID- 3001441 TI - [Acquired immune deficiency syndrome in Spain. Is the alarm justified?]. PMID- 3001443 TI - [Hepatic cholangiocarcinoma and angiosarcoma after the administration of thorotrast]. PMID- 3001445 TI - [Converting enzyme inhibitors in the treatment of essential hypertension]. PMID- 3001444 TI - [Severe thrombocytopenia secondary to a cytomegalovirus infection. Presentation of a case]. PMID- 3001447 TI - [Undifferentiated primary small cell carcinoma of the cardia]. PMID- 3001446 TI - [Hormonal diagnosis of insulinomas]. PMID- 3001448 TI - [Cytochemical study of 5'-nucleotidase in various lymphoproliferative syndromes]. PMID- 3001450 TI - [Isotopic localization of hepatic metastasis of medullary carcinoma of the thyroid with Tc-99 pyrophosphate]. PMID- 3001449 TI - [Possibilities of tissue cultures in the study of tumors]. PMID- 3001451 TI - Kaposi's sarcoma and AIDS. AB - Mucocutaneous lesions are often a prominent manifestation of the acquired immune deficiency syndrome (AIDS). Patients with this syndrome are susceptible to a number of opportunistic skin infections as well as an aggressive form of Kaposi's sarcoma. The diagnosis, the clinical setting, and the treatment of these diseases are discussed. PMID- 3001452 TI - Myeloma, paraproteinemias, and the skin. AB - This review focuses on those systemic diseases or syndromes associated with monoclonal plasma cell disorders that may present with important cutaneous manifestations. Amyloidosis, POEMS syndrome, cutaneous plasmacytoma, xanthomas, benign hypergammaglobulinemic purpura of Waldenstrom, and scleromyxedema are emphasized. PMID- 3001454 TI - Silymarin: potent inhibitor of cyclic AMP phosphodiesterase. AB - Silymarin, the active principle of the Milk Thistle (Silybum marianum Gaertner), is a very potent inhibitor of cyclic AMP breakdown in vitro by a commercial beef heart phosphodiesterase preparation. Its main constituents, silybin, silydianin and silychristin, are 12.66 to 52.06 times more active than theophylline and 0.77 to 3.17 times more active than papaverine in this respect. Using a novel HPLC technique, the enzyme kinetical analysis can be performed much faster than by the classical methods. PMID- 3001453 TI - Differential responses of plasma aldosterone, cortisol and adrenocorticotropin to two dopamine receptor antagonists. AB - The response of plasma aldosterone, cortisol and adrenocorticotropin (ACTH) to the dopamine antagonists metoclopramide and domperidone, administered intravenously in a dose of 1 mg/kg, was investigated in healthy volunteers. Within 15 min after metoclopramide administration, plasma aldosterone (+ 99%), cortisol (+ 75%) and ACTH (+ 55%) increased (p less than 0.001), whereas the plasma levels of these hormones were not altered after domperidone. The differential responses of plasma aldosterone and cortisol to high doses of metoclopramide and domperidone are therefore, at least partially, mediated via the enhanced adrenal stimulation by ACTH after metoclopramide. PMID- 3001455 TI - [The mechanism of action of insulin is an unsolved problem--new discoveries can explain the effects]. PMID- 3001456 TI - [Malignant fibrous histiocytoma of the larynx]. AB - Report on two patients with (malignant) fibrous histiocytomas of the larynx. Fibrous histiocytomas of the larynx, malignant or benign are extremely rare. Because of the variety of the tissue the histiological diagnosis of these tumours -especially with regard to the tumour status--can cause great difficulties. Basing on our cases and on the few previous publications on this subject, the biological behaviour and therapy of laryngeal histiocytomas, as well as differential diagnoses of other neoplasms, are discussed. Since the (malignant) fibrous histiocytomas often recur locally, even tumours without unequivocal histological malignancy must be treated by extensive excision. PMID- 3001457 TI - [Kaposi sarcoma in acquired immune deficiency syndrome (AIDS). II: Light and electron microscopic and immunohistochemical peculiarities including the cytoskeleton]. AB - AIDS-related lymphoadenopathy is characterised by follicular hyperplasia with irregularities in the dendritic network, a loss of the follicular mantle zone and an enhanced influx of T-suppressor cells into the germinal centres. The histological features of AIDS-associated epidemic Kaposi's sarcoma are spindle like tumour cells and a source vascularisation, which is of clinical relevance for the biopsy. Since immunohistochemical markers specific for blood vessel endothelium can not be detected within the tumour cells an origin from this cell type gets unlikely. An origin from lymphatic endothelium however can still be discussed. PMID- 3001458 TI - The application of magnetic resonance imaging in otolaryngology. AB - Magnetic resonance imaging is a new diagnostic technique that is being applied to study disease processes that involve the upper aerodigestive tract and cranial nerves of interest to otolaryngologists. As with all imaging techniques, an in depth knowledge of the normal anatomy of a region is a prerequisite for the appreciation of disease states. In addition, magnetic resonance imaging requires a familiarity with the signal intensity produced by the different structures and the relationship of that signal to various radiofrequency pulse sequences that may be used. This report briefly reviews the techniques of magnetic resonance imaging and the normal anatomy of the cervical region and presents examples of pathologic processes that have been studied with this powerful diagnostic technique. PMID- 3001459 TI - [Endocavitary radiotherapy in an after-loading technic in malignant stenoses of the upper gastrointestinal tract and bile ducts]. AB - Palliative brachytherapy with 192iridium in high dose rate technique was applied until August 1985 in 44 patients: 40 suffering from an inoperable malignant stenoses of the esophagus or cardia, and 4 from bile duct carcinomas located in the upper part of the duct. The patients with malignant stenoses of the upper gastrointestinal tract underwent laser therapy until an endoscope could be passed beyond this stenoses. Then an after-loading tube was placed endoscopically and the stenoses irradiated endocavitarily (7/Gray/session in 1 cm distance). In the patients with malignant bile duct obstruction a PTCD-catheter served to guide the iridium wire up to the stenotic region. PMID- 3001460 TI - Leu-enkephalin actions on avoidance conditioning are mediated by a peripheral opioid mechanism. AB - Leu-enkephalin (100 micrograms/kg, i.p.) administered to mice 5 min before training in a one way active avoidance task significantly reduced the number of avoidances observed in the peptide treated animals. This impairing action of Leu enkephalin was partially attenuated by methylnaloxonium (naloxonium), a quarternary form of naloxone with a limited ability to penetrate the blood brain barrier. Passive immunization (i.v.) of mice with a Leu-enkephalin antiserum 4 hrs before training produced an effect on avoidance conditioning that was the opposite to that observed with Leu-enkephalin alone. That is, passive immunization increased the number of avoidances observed in the treated mice. The results suggest that Leu-enkephalin actions on avoidance conditioning are mediated by a peripheral opioid mechanism, that leu-enkephalin may have a primary site of action outside the blood brain barrier, and that peripheral Leu enkephalin systems may normally operate to influence conditioned avoidance behavior. PMID- 3001461 TI - Phorbol ester effects on alpha 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured vascular smooth muscle cells. AB - Tumor promoting phorbol esters stimulate Ca++ phospholipid-dependent protein kinase C. It has been suggested that this enzyme regulates the functional properties of different cell membrane receptors. In this study we investigated the effect of phorbol esters on alpha 1-adrenoceptor binding and phosphatidylinositol metabolism in cultured smooth muscle cells derived from rabbit aorta. Treatment of these cells with biologically active phorbol esters for 15 min. to 2 hours caused a marked decrease of norepinephrine stimulation of inositol phospholipid metabolism and a 3 fold decrease in agonist affinity for 125I-HEAT binding to alpha 1-adrenoceptors in the intact smooth muscle cells. The ability of phorbol esters to modulate alpha 1-adrenoceptor responsiveness suggests that activation of protein kinase C may represent an important mechanism regulating alpha 1-adrenergic receptor functional properties. PMID- 3001462 TI - Conditioned media from cultured cystic fibrosis fibroblasts inhibits Na/K ATPase activity. AB - Conditioned culture media taken from fibroblast cell lines derived from skin biopsies of control or of patients with Cystic Fibrosis (CF) were incubated with membranes of rat submandibular glands. The Na/K - ATPase activity of these membranes was inhibited when treated with CF-media, including both ouabain sensitive and insensitive activities. However, the membrane associated Mg-ATPase, Ca-ATPase, and both basal and hormone-stimulated adenylate cyclase activities were relatively unaffected. Thus, a factor or factors produced by CF-fibroblasts was shown to be active in a cell-free system derived from an exocrine gland. PMID- 3001463 TI - [3H] Boc [Nle28, 31]CCK27-33, a new highly labelled ligand for CCK receptors: binding on brain and on pancreas. AB - Preliminary results on the binding of [3H]Boc[Nle28, 31]CCK27-33, designated [3H]Boc[diNle]CCK7, on mouse brain and rat pancreas membranes are presented. This new ligand for CCK receptors possesses a high specific activity (144 Ci/mmole), and binds in a saturable manner to mouse brain (Kd = 0.49 nM, Bmax = 49 fmoles/mg protein) and rat pancreas (Kd = 4.4 nM, Bmax = 696 fmoles/mg protein). Unlabelled Boc[diNle]CCK7 displaces [125I]CCK8 from its binding sites on mouse brain membranes with a high affinity, slightly superior to that of CCK8. The order of potencies to displace [3H]Boc[diNle]CCK7 from its binding sites was the same in mouse brain and rat pancreas: [3H]Boc[diNle]CCK7 greater than CCK8, Boc-CCK7 greater than non-sulfated CCK8, the pancreas binding sites being more discriminative than the brain binding sites. Thus, [3H]Boc[diNle]CCK7 is a very promising new probe for the characterization of CCK receptors and their interaction with different CCK fragments. PMID- 3001464 TI - Attenuating effect of diazepam on stress-induced increases in noradrenaline turnover in specific brain regions of rats: antagonism by Ro 15-1788. AB - One-hour immobilization stress increased levels of the major metabolite of brain noradrenaline (NA), 3-methoxy-4-hydroxyphenyl-ethyleneglycol sulfate (MHPG-SO4), in nine brain regions of rats. Diazepam at 5 mg/kg attenuated the stress-induced increases in MHPG-SO4 levels in the hypothalamus, amygdala, hippocampus, cerebral cortex and locus coeruleus (LC) region, but not in the thalamus, pons plus medulla oblongata excluding the LC region and basal ganglia. The attenuating effects of the drug on stress-induced increases in metabolite levels in the above regions were completely antagonized by pretreatment with Ro 15-1788 at 5 or 10 mg/kg, a potent and specific benzodiazepine (BDZ) receptor antagonist. When given alone, Ro 15-1788 did not affect the increases in MHPG-SO4 levels. Behavioral changes observed during immobilization stress such as vocalization and defecation, were also attenuated by diazepam at 5 mg/kg and this action of diazepam was antagonized by Ro 15-1788 at 10 mg/kg, which by itself had no effects on these behavioral measurements. These findings suggest: (1) that diazepam acts via BDZ receptors to attenuate stress-induced increases in NA turnover selectively in the hypothalamus, amygdala, hippocampus, cerebral cortex and LC region and (2) that this decreased noradrenergic activity might be closely related to relief of distress-evoked hyperemotionality, i.e., fear and/or anxiety in animals. PMID- 3001465 TI - Dexamethasone suppresses pituitary-adrenal but not behavioral effects of centrally administered CRF. AB - Intracerebral ventricular (icv) administration of corticotropin-releasing factor (CRF) significantly enhances the expression of stress-related behaviors in the rat and also activates the pituitary-adrenal system. The pituitary-adrenal response can be blocked by pretreatment of animals with dexamethasone. The behavioral effects (motor activation, increased grooming and decreased eating) on the other hand are resistant to suppression by dexamethasone. The independence of the behavioral effects from activation of the pituitary-adrenal axis suggests that stress-induced release of CRF could contribute to behavioral alterations even in the presence of high concentrations of endogenous steroids. PMID- 3001466 TI - Antagonism of receptor-activated biological effects mediated by second messenger pathways. AB - It is generally held that many biologically active compounds produce their effects through a sequence of events that are initiated when the substances combine with selective receptors located on the cell surface membrane. Activation of these receptors produces a stimulus that is somehow transmitted intracellularly. The transduction between the stimulus and the response is now known to be mediated, in many systems, by an intracellular intermediary or second messenger. A model describing the relation of agonist concentration, receptor occupation, and biological response in such a system is herein extended to include antagonist binding at two target sites the cell-surface receptor and the receptor for the second messenger. It is demonstrated that the shift of an agonist's dose-response curve is characteristic of the site of antagonism and that analysis of this shift can reveal the existence of a second messenger pathway or, if this is known, the site of action of the antagonist. PMID- 3001467 TI - The viral hepatitis surveillance system in the State of Maryland. PMID- 3001468 TI - Comprehensive rehabilitation of head injured adults. PMID- 3001469 TI - Center for living: trauma aftercare and outcomes. PMID- 3001470 TI - [Comprehensive radionuclide diagnosis of pulmonary circulation disorders in children with acute pyo-destructive pneumonia]. AB - A comprehensive radionuclide study of pulmonary circulation disorders in 55 children with acute pyo-destructive pneumonia (at the age of 1 month to 13 years) revealed the degree of vascular network reduction, pulmonary regional specific activity and changes in the time of the blood flow in the pulmonary circulation microcirculatory channel. These indices of disturbed pulmonary hemodynamics in combination with the results of total perfusion deficiency can serve as diagnostic markers of pulmonary circulation pathology and define a tendency to the generalization of a pyo-destructive process or enhancement of the blood flow of compensatory nature as well as prognosis of a course of disease. PMID- 3001472 TI - [Clinical x-ray and scintigraphic evaluation of the state of the sacroiliac joints in patients with chronic monarthritis]. AB - The paper is concerned with some comparative data on the frequency of the detection of sacroileitis using clinicoroentgenological and scintigraphic studies in 57 patients with chronic monarthritis of the knee joint of different genesis. On clinical examination 14 patients (24.7%) only had indirect signs of the affected sacroiliac joints, in 30 patients (52.6%) sacroileitis was diagnosed by x-ray. Scintigraphy with 99mTc-pyrophosphate proved to be most effective for the detection of early signs of sacroileitis because lesions in the sacroiliac joints were found in 35 patients (61.4%). PMID- 3001471 TI - [Potentialities of gamma-scintigraphy in the diagnosis of Meckel's diverticulum in children]. AB - Altogether 84 children aged 1 to 15 were examined. The index of 99mTc pertechnetate relative accumulation in the ectopic gastric mucosa was proposed to be used to objectify and improve the quality of diagnosis of Meckel's diverticulum. The index over 42% was shown to be typical of Meckel's diverticulum and it can be used for automated diagnosis of the disease. The use of the above quantitative index made it possible to reject 20% of false positive results obtained at visual interpretation of scintigrams. PMID- 3001474 TI - [Radionuclide diagnosis of acute pyelonephritis]. AB - Nephroscintigraphy using a 67Ga-citrate complex and 99mTc-pyrophosphate was performed in 88 patients with acute pyelonephritis. Nuclide hyperfixation was revealed in 97.8% of the cases. Three groups of patients were singled out on the basis of the intensity of incorporation and nature of the distribution of the radiopharmaceuticals (RP) in the kidneys. In the 1st group the RP incorporation was insignificant but higher than normal values; the RP distribution in the affected kidney was diffuse-inhomogenous. These changes were considered to be typical of acute serous pyelonephritis. In the 2nd and 3rd groups a sharp rise of the RP accumulation was noted, being typical of acute purulent pyelonephritis. One could distinguish between diffuse and focal lesions by the picture of the RP distribution in the renal parenchyma. Diuresis stimulation made it possible to differentiate an actual nuclide fixation during inflammation from nuclide mechanical retention as a result of urine outflow disorder. According to the authors, both radiopharmaceuticals could be applied for the diagnosis of acute pyelonephritis as well as for differential diagnosis of various forms of the disease. PMID- 3001473 TI - [Radionuclide scanning and radioscintigraphy of the salivary glands]. PMID- 3001475 TI - Pedigree analysis of the 5' flanking region of the insulin gene in familial diabetes mellitus. AB - Non-insulin-dependent diabetes mellitus (NIDDM) has been reported to be associated with an insertion polymorphism in the 5' flanking region of the human insulin gene. We have attempted to examine linkage of this polymorphism to the phenotype of NIDDM by studying multiple pedigrees. We evaluated 142 individuals (120 white, 22 black), 80 of whom were from 7 pedigrees (5 white, 2 black) ranging in size from 4 to 37 members. Of these, 52 subjects had NIDDM, 10 had insulin-dependent diabetes mellitus (IDDM), and 80 were nondiabetics (ND). DNA was extracted from leukocytes and after digestion with Sst 1, and electrophoresis, the DNA was blotted to nitrocellulose filters and hybridized to a alpha 32P-labeled insulin gene probe. Two alleles of 6.0 and 7.6 kb in size were detected, the latter corresponding to the common previously described insertion polymorphism. In these families, the 7.6 kb allele occurred in 32 of 57 ND, 3 of 5 IDDM, and 10 of 18 NIDDM (P = 0.98). When sibships were analyzed the 7.6 kb allele occurred in 6 of 13 ND, 3 of 5 IDDM, and 8 of 12 NIDDM (P = 0.58). In examining 72 unrelated subjects, including 12 spouses from the pedigrees, the 7.6 kb allele was documented in 16 of 36 ND, 1 of 5 IDDM, and 16 of 31 NIDDM (P = 0.59). In these individuals and in the multiple families studied the insertion polymorphism flanking the insulin gene showed Mendelian inheritance and assorted independently of the phenotype of diabetes mellitus. PMID- 3001476 TI - Modulation of myocardial L-triiodothyronine receptors in normal, hypothyroid, and hyperthyroid rats. AB - To characterize the nuclear receptor believed to mediate the thyroid hormones' actions on the heart, binding of L-[125I]T3 to extracts of myocardial cell nuclei from normal, propylthiouracil, and T4-treated rats has been investigated. Analysis of in vitro iodothyronine binding to this solubilized nuclear preparation revealed multiple saturable, specific binding sites for T3. High affinity binding for T3 (Kd = 4.2 +/- 1.0 X 10(-10) mol/L), and lower affinity (Kd approximately 10(-8) mol/L) binding activity appeared to be independent (Hill plot slope = 1). The mean maximal binding capacity of the high affinity binding site for T3 in euthyroid rats (84 +/- 8 fmol/mg DNA) was increased by approximately 50% in hypothyroidism (120 +/- 6 fmol/mg DNA) and unchanged in hyperthyroidism (88 +/- 25 fmol/mg DNA). The molecular weight of this T3 binding site is estimated to be 50,000-55,000 daltons. The properties of this solubilized binding activity from rat myocardial nuclei are consistent with its putative role as the biologic thyroid hormone receptor. The increase in binding capacity with hypothyroidism suggest regulation by thyroid hormone of its nuclear receptor in myocardium. PMID- 3001478 TI - A systematic method for aligning double-focusing mirrors. PMID- 3001477 TI - Nuclear thyroid hormone receptors in cultured bone cells. AB - Thyroid hormones influence bone metabolism, but a direct interaction of triiodothyronine with nuclear T3 receptors in bone cells has not yet been reported. We investigated 125I-T3 binding to nuclei isolated from the cloned osteoblastlike rat osteosarcoma cells ROS 17/2.8. At 37 degrees C, saturable 125I T3 binding to isolated nuclei reached equilibrium by 30 minutes and was completely displaced upon the addition of 500 nmol/L unlabeled T3. Nonsaturable binding represented about 0.5% of the radioactivity added (20% of the total binding). Thyroxine and 3,3',5'triiodothyronine competed with 125I-T3 with a 20 fold and 400-fold lower affinity than T3, respectively. Analysis of equilibrium competition experiments revealed the presence of a single class of homogeneous binding sites with an association constant of 5.0 +/- 0.3 X 10(9) mol/L-1 and a maximum nuclear binding capacity of 0.13 +/- 0.02 ng/mg DNA. A twofold increase of bone Gla protein (BGP) secretion was observed with T3 treatment suggesting that these T3 nuclear receptors are coupled with a biological response. PMID- 3001479 TI - Application of transient electric birefringence to the study of biopolymer structure. PMID- 3001480 TI - Ligand binding to proteins by equilibrium gel penetration. PMID- 3001481 TI - Protein chromatography on hydroxyapatite columns. PMID- 3001482 TI - Effects of medrogestone and conjugated oestrogens on serum lipid and lipoprotein concentrations. AB - Twenty patients, aged 30-60 yr, who had undergone bilateral ovariectomy, were treated orally with 5 mg medrogestone (6,17-dimethylpregna-4,6-diene-3,20-dione) and 1.25 mg conjugated oestrogens per day, according to a constant dosage pattern during the cycle (22 + 6 days). The lipids and lipoproteins were determined twice before the start of therapy and 3, 6 and 12 mth thereafter. The lipids were quantified enzymatically and the lipoproteins by quantitative lipoprotein electrophoresis. Whilst cholesterol and triglyceride concentrations showed no detectable change, a slight but significant increase was seen in the high-density alphalipoprotein (HDL) cholesterol concentrations. The low-density beta lipoprotein (LDL) cholesterol level showed a moderate fall. There was a resultant reduction in the beta/alpha-lipoprotein ratio. Accordingly, the apoprotein A1 concentrations were found to be elevated, while apoprotein B tended to fall to lower levels during therapy. When these changes are measured by the lipid metabolism risk criterion for the occurrence of coronary heart disease applicable to post-menopausal patients, the effects of the above-mentioned combination may be regarded as entirely favourable. PMID- 3001483 TI - Serum hormone levels in men exposed to vaginal oestrogen cream: a preliminary report. PMID- 3001484 TI - The role of NK cell activity in age-dependent resistance of mice to murine cytomegalovirus infection. AB - The resistance of mice to cell culture passaged murine cytomegalovirus (CC-MCMV) infection developed with age. In parallel with this finding, augmentation of the splenic NK cell activity in older mice was always higher than that of younger mice. The splenic NK cell activity reached the maximum level at 6 day post infection (PI) in 2-4-week-old mice while in 6-8-week-old mice it peaked at 4 days PI. When the dose of CC-MCMV was increased, the NK cell activity was potentiated accordingly. However, it was decreased on the infection with increased doses of the salivary gland passaged MCMV (SG-MCMV). NK cells augmented by MCMV infection actually inhibited in vitro replication of MCMV when they were added to mouse embryonic fibroblast (MEF) monolayers infected with CC-MCMV. Splenic and peritoneal macrophages inhibited in vitro replication of MCMV, but their activities were less potent than those of NK cells. PMID- 3001485 TI - Kunjin virus encephalomyelitis. AB - Kunjin virus encephalomyelitis is described in a 49-year-old man who presented with an episode of acute encephalitis. He developed profound bulbar, truncal and proximal muscle weakness suggestive of damage to cranial motor nuclei and anterior horn cells. This resolved very slowly. A marked rise in antibody titres to Kunjin virus was shown by haemagglutination inhibition tests. Specific IgM antibodies were detected only against Kunjin virus. PMID- 3001486 TI - [Epidemiological study on the incidence of malignant tumors in children in the province of Vercelli in the years 1975-1979]. PMID- 3001487 TI - [Poland syndrome. Case contribution and critical review of the literature]. PMID- 3001488 TI - Pathologic analysis of primary brain tumors. AB - Diffuse astrocytomas of the cerebrum, cerebellum, brain stem, and spinal cord are classified into three groups according to the degree of tumor anaplasia. These groups are the astrocytoma, anaplastic astrocytoma, and glioblastoma multiforme. Juvenile pilocytic astrocytomas have a better prognosis and are clinically and biologically distinct from the diffuse, fibrillary astrocytomas. The prognosis of astrocytomas depends not only on histologic characteristics, but also age of the patient, location of the tumor, and extent of surgical resection. The pattern of invasion into surrounding brain distinguishes gliomas from metastatic carcinomas and sarcomas. Topographic correlations have shown that malignant gliomas may invade the brain for distances of up to several centimeters from the enhancing rim seen on CT scan. However, the junction between glioblastoma and adjacent brain may also be fairly abrupt, with a peripheral margin of less than 1 mm. Recurrent glioblastomas are more widely invasive and often extend into areas that appear normal on CT scan. The optimal site for tumor biopsy corresponds to areas of contrast enhancement. Primitive neuroepithelial tumors are malignant neoplasms with a poor prognosis. They tend to recur locally and metastasize throughout the neuraxis via the CSF. It remains controversial whether these tumors should be classified as a single entity with the potential for differentiation along different cell lines, or whether the categories of neuroblastoma, spongioblastoma, ependymoblastoma, pineoblastoma, and medulloblastoma should be retained as specific entities. The medulloblastoma is the most common of these neoplasms, its clinicopathologic features are well characterized, and the current 5-year survivals of 50 to 60 per cent are better than for other "primitive" neoplasms. Glial fibrillary acidic protein is a specific marker for immature, reactive, and neoplastic astrocytes and ependymal cells. Although the absence of GFAP in a neoplasm does not exclude an astrocytic origin, the presence of GFAP indicates astrocytic or ependymal differentiation. This has important diagnostic applications. The expression of GFAP is used to distinguish astrocytic neoplasms from epithelial or mesenchymal tumors that may on occasion mimic a glioma. The detection of GFAP is also useful in the investigation of tumor histogenesis and differentiation both in vivo and in vitro. Although meningiomas exhibit a wide variety of histologic patterns, most tumors exhibit similar biologic and clinical behavior regardless of the histologic subtype.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3001489 TI - DNA content and chromosomal composition of malignant human gliomas. AB - A short review is given on DNA aberrations and chromosomal composition of malignant human gliomas. By flow cytometric DNA analysis, a wide range of different ploidies has been reported in biopsied gliomas, from diploid to strongly aneuploid nuclear DNA. However, with the preparation and analysis methods used so far, no clear relationship between the type of ploidy and histology or prognosis has been established. A high proportion of glioblastomas is near-diploid, indicating a high degree of biologic malignancy is not necessarily connected to aberration of the nuclear DNA content. It is possible that improved methods giving a higher degree of resolution will allow separation of the near-diploid populations of malignant human gliomas from normal diploid cells and permit the detection of subpopulations with small differences from the dominant DNA mode. Chromosomal studies of malignant gliomas have confirmed that the majority of them have near-diploid stemlines. These populations are seldom normal diploid, however, as both numerical and structural abnormalities are usually present. In addition, chromosomal analyses have shown that when gliomas are bimodal, the polyploid populations are usually doubled versions of the near diploid ones. In contrast to the near-diploid populations that characterize biopsied malignant gliomas, both FCM studies and karyotyping have demonstrated that permanent cultured cell lines derived from malignant gliomas are usually near-triploid or near-tetraploid. Sequential karyotypic studies of these tumors from biopsy through establishment in vitro have shown an evolutionary pattern consisting of doubling of the original stemline, followed by gains or losses of individual chromosomes with new marker formation in late culture. Evaluation of biopsied malignant gliomas by karyotyping has also demonstrated that subgroups of them are characterized by specific numerical and structural deviations. These groupings may prove useful in predicting prognosis or responsiveness to specific therapeutic regimens. The specific chromosomal abnormalities observed in malignant human glioma may provide clues as to the genes important in glial transformation. As chromosomal loci for production of structural proteins and enzymes involved in glial metabolism are mapped and patterns of oncogene activation and amplification are determined for human gliomas, the meaning of the nonrandom chromosomal changes seen in these tumors may become clear. PMID- 3001490 TI - Growth factors and oncogenes in human malignant glioma. AB - Normal cell replication is regulated by growth factors such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) that act through binding to specific surface receptors on target cells. Oncogenes may exert their transforming activity by encoding proteins that mimic the function of the normal regulatory factors along the mitogenic pathway, growth factors, their receptors or elements along the postreceptor signaling system. This may be exemplified by the human malignant glioma, in which the sis gene (encoding a growth factor homologous to PDGF) and the erb B gene (encoding a membrane protein homologous to the EGF receptor) have been implicated. PMID- 3001491 TI - Chemotherapy of primary brain tumors. AB - This article covers chemotherapy of malignant astrocytomas, ependymoma, and medulloblastoma. The future direction of anticancer drugs is discussed. PMID- 3001493 TI - Stereotaxic interstitial irradiation of malignant brain tumors. AB - The authors discuss the feasibility of treatment of malignant tumors with brachytherapy. The history of brain tumor brachytherapy, its present day use, and future directions are detailed. PMID- 3001492 TI - Conventional external beam radiotherapy for central nervous system malignancies. AB - Fractionated external beam photon radiotherapy is an important component of the clinical management of malignant disease of the central nervous system. The practicing neurologist or neurosurgeon frequently relies on the consultative and treatment skills of a radiotherapist. This article provides a review for the nonradiotherapist of the place of conventional external beam radiotherapy in neuro-oncology. PMID- 3001494 TI - Treatment of malignant gliomas with neutron radiation. AB - The author contends that neutron radiation therapy coupled with radiation therapy or other forms of particle radiation therapy may play a role in the management of malignant gliomas. Improved survival, particularly for the patients with the diagnosis of glioblastoma, is discussed. PMID- 3001495 TI - Need for malaria prophylaxis by travelers to areas with chloroquine-resistant Plasmodium falciparum. PMID- 3001496 TI - Forskolin potentiation of cholera toxin-stimulated cyclic AMP accumulation in intact C6-2B cells. Evidence for enhanced Gs-C coupling. AB - Forskolin directly stimulates adenylate cyclase activity and acts synergistically with receptor-mediated agonists which stimulate cyclic AMP production. We have previously observed that a 3-hr incubation of C6-2B rat astrocytoma cells with 6 nM cholera toxin in the presence of 1 microM forskolin results in cyclic AMP accumulation 9-fold greater than in the absence of forskolin. Since the action of cholera toxin is mediated by the stimulatory guanine nucleotide-binding regulatory component (GS) of the adenylate cyclase complex, we proposed that the mechanism by which forskolin augments hormone responses involves an enhanced coupling of GS with the adenylate cyclase catalytic component (C). In the present communication, we report the detailed characterization of the synergistic interaction between forskolin and cholera toxin as effectors of cyclic AMP accumulation in intact C6-2B cells. After a 3-hr incubation, maximal cholera toxin-stimulated cyclic AMP accumulation was 990 +/- 34 pmol/mg of protein. In the presence of 1 microM forskolin, the response to cholera toxin increased to 13,137 +/- 1,595 pmol of cyclic AMP/mg of protein. The half-maximally effective cholera toxin concentrations estimated by nonlinear least squares regression analysis determined in the absence or presence of 0.1 mM forskolin were 56.6 and 57.5 pM, respectively. The highly reproducible lag in forskolin-stimulated cyclic AMP accumulation in C6-2B cells was abolished by cholera toxin pretreatment, indicating a possible role for GS-associated GTPase in the mechanism of forskolin action. Cholera toxin treatment markedly augmented forskolin-stimulated cyclic AMP accumulation and shifted the forskolin concentration-response curve to the left approximately 1.5 log units. When C6-2B cells were treated for 1 min with 10 nM cholera toxin, the response to forskolin was significantly potentiated by 10 min. No significant increase in cellular cyclic AMP content in the absence of a forskolin challenge was apparent for up to 45 min. It appears that prior promotion of GS-C coupling by cholera toxin treatment enhances the ability of forskolin to stimulate cyclic AMP accumulation. Whether or not forskolin interacts (i.e., binds) exclusively to C remains to be proven. However, the actions of forskolin to stimulate cyclic AMP formation and potentiate agonist stimulated cyclic AMP formation are modulated by the activity state of GS, and at least part of the response to forskolin is mediated by GS. PMID- 3001497 TI - Angiotensin-converting enzyme inhibitors potentiate the analgesic activity of [Met]-enkephalin-Arg6-Phe7 by inhibiting its degradation in mouse brain. AB - The proteolytic degradation of the enkephalin-containing heptapeptide Tyr-Gly-Gly Phe-Met-Arg-Phe (YGGFMRF) was investigated by incubating the peptide with synaptic membranes from mouse whole brain and characterizing the formed products. The degradation products were derivatized with 4-dimethylaminoazobenzene-4' isothiocyanate and then analyzed by high pressure liquid chromatography and by amino-terminal analysis. The incubation of YGGFMRF with synaptic membranes yielded YGGFM and RF as the degradation products. The angiotensin-converting enzyme (ACE) inhibitors, MK-422 and captopril, potently inhibited the formation of YGGFM and RF with IC50 values of 8 nM and 95 nM, respectively. The "enkephalinase A" inhibitor, thiorphan, weakly inhibited this dipeptidyl carboxypeptidase activity with an IC50 greater than 1 microM. YGGFMRF, MK-422, captopril, and thiorphan all produced a dose-dependent analgesic response in the mouse hot plate test when administered intracerebroventricularly. However, when subanalgesic doses of inhibitors were co-administered with a subanalgesic dose of YGGFMRF, only the ACE inhibitors, MK-422 and captopril, potentiated the analgesic response of the peptide. These data provide in vitro and in vivo evidence that ACE is the primary enzyme involved in the proteolytic degradation of YGGFMRF in the mouse brain. PMID- 3001498 TI - Arachidonic acid stimulates phosphoinositide hydrolysis and human placental lactogen release in an enriched fraction of placental cells. AB - Previous investigations in this laboratory have indicated that arachidonic acid stimulates a rapid, dose-dependent, and reversible increase in human placental lactogen (hPL) release which is not dependent on cyclooxygenase or lipoxygenase metabolism. To investigate further the mechanism by which arachidonic acid stimulates the release of hPL, the effects of arachidonic acid on phosphoinositide hydrolysis were examined in an enriched cell culture population of term human syncytiotrophoblast. Phosphoinositide hydrolysis was assayed by three methods: the release of 3H from perfused cells prelabeled with [3H]myoinositol, the measurement of inositol phosphate accumulation, and the distribution of radioactivity in phospholipids separated by two-dimensional thin layer chromatography after exposure of 32P-labeled placental cells to arachidonic acid. Arachidonic acid stimulated a concentration-dependent, rapid, and reversible increase in the release of both [3H]myoinositol and hPL from perfused placental cells. This effect was not inhibited by prior incubation of cells with indomethacin (20 microM). In contrast, palmitic acid and oleic acid stimulated phosphoinositide hydrolysis only at a high concentration (100 microM). Arachidonic acid also stimulated the rapid appearance of inositol monophosphate in placental cells. The effect of arachidonic acid was specific for hydrolysis of phosphoinositides and phosphatidylserine and did not involve other phospholipids. Since phosphoinositide hydrolysis is associated with hormone release in a variety of secretory systems, these results suggest that the stimulation of hPL release by arachidonic acid may be mediated, at least in part, by the activation of phospholipase C. PMID- 3001499 TI - Highly selective cytostatic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine derivatives for murine mammary carcinoma (FM3A) cells transformed with the herpes simplex virus type 1 thymidine kinase gene. AB - (E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU) and various structurally related analogues thereof, i.e., (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2 bromovinyl)-2'-deoxycytidine (BVDC), and the carbocyclic analogues of BVDU, IVDU, and BVDC, were evaluated for their inhibitory effects on the growth of murine mammary carcinoma FM3A cells, deficient in thymidine kinase (TK) activity but transformed with the herpes simplex virus type 1 (HSV-1) TK gene (designated FM3A/TK-/HSV-1 TK+). BVDU and its congeners were much more inhibitory to the growth of FM3A/TK-/HSV-1 TK+ than to the growth of the wild type (FM3A/0) cells. For BVDU, for example, the 50% inhibitory dose for the FM3A/TK-/HSV-1 TK+ cells was 0.5 ng/ml, as compared to 11 micrograms/ml for the FM3A/0 cells. Evidently, BVDU and its congeners required phosphorylation by the HSV-1 TK to exert their cytostatic action. In attempts to evaluate further the mechanism of this cytostatic action, BVDU, IVDU, and their carbocyclic analogues were evaluated for their inhibitory effects on thymidylate synthetase (TS) and their incorporation into DNA. TS was identified as one, but not the sole, target in the cytostatic activity of BVDU and its derivatives. With [125I]IVDU and its carbocyclic analogue C-[125I]IVDU, clear evidence was obtained for the incorporation of these radiolabeled analogues into DNA of the FM3A/TK-/HSV-1 TK+ cell line and a TS deficient mutant thereof, FM3A/TK-/HSV-1 TK+/TS-. No incorporation was detected with [125I]IVDU or C-[125I]IVDU into DNA of FM3A/0 and FM3A/TS- cells. To what extent the incorporation of [125I]IVDU and C-[125I]IVDU contributed to their cytostatic action against FM3A/TK-/HSV-1 TK+ cells remains the subject of further study. PMID- 3001501 TI - The chloroplast genome of Carthamus tinctorius. AB - A physical map of safflower (Carthamus tinctorius L.) chloroplast DNA has been generated using SalI, PstI, KpnI and HindIII restriction endonucleases. Southern blots to single and double digests by these enzymes were hybridized with 32P-dCTP nick-translated KpnI probes, which were individually isolated from agarose gels. The plastid genome was found to be circular (151 kbp), to contain a repeated sequence of about 25 kbp, and to have small and large single copy regions of approximately 20 and 81 kbp, respectively. Heterologous probes from spinach and Euglena containing psbA, rbcL, atpA or rrnA structural genes were also hybridized with such single and double restriction enzyme digests and mapped on this circular chloroplast genome. The genetic map was found to be co-linear with that of spinach and many other higher plants. PMID- 3001500 TI - Evidence for calcium enhanced phosphorylation of pyruvate kinase by pancreatic islets. AB - Pancreatic islet cytosol contains a calcium-calmodulin dependent protein kinase that can mediate the phosphorylation of an endogenous protein that has an Mr of 57 000, as well as exogenous muscle pyruvate kinase (subunit Mr, 57 000). EGTA and trifluoperazine decreased the phosphorylation. Alkaline inactivation of pyruvate kinase made it a better substrate for the kinase. As in rat islet cytosol, rabbit islet cytosol catalyzed the phosphorylation of a 57 000 Mr protein in the presence of calcium and calmodulin. This phosphoprotein was immunoprecipitated with anti-pyruvate kinase antibody. This is consistent with the idea that the 57 000 Mr phosphoprotein in islet cytosol is the subunit of pyruvate kinase. The paper following this paper shows that the kinetic and immunologic properties of the islet pyruvate kinase indicate it is the M2 isoenzyme and that its phosphorylation does not affect its catalytic activity. PMID- 3001502 TI - Purification and characterization of DNA polymerase alpha of Chinese hamster ovary cells. AB - The major pol alpha activity of CHO cells was purified 2 800-fold to near homogeneity and was characterized with respect to its physical and catalytic properties. The purified enzyme, upon analysis in denaturing 'activity' gels, displayed a major, 120 kilodalton, catalytically active core and two minor, catalytically inactive components of 180 and 135 kilodaltons. The native form of the enzyme behaved in velocity sedimentation and gel permeation experiments as an asymmetric protein of an apparent Mr. of 515 kilodaltons. The purified enzyme displayed catalytic behavior and inhibitor sensitivity typical of that displayed by other mammalian pol alphas. Specifically, the enzyme: was sensitive to n ethylmaleimide and the pol alpha-specific inhibitors, BuPdGTP and aphidicolin; was subject to neutralization by specific monoclonal antibodies raised against human pol alpha; was devoid of detectable 3' to 5' exonuclease activity, and displayed a ribonucleotide-dependent DNA primase activity. PMID- 3001503 TI - Nucleoside phosphotransferase in animal tissues. Tissue distribution and kinetic properties. AB - Amphibian, avian and mammal tissues contain a nucleoside phosphotransferase clearly different from those previously described in vegetables and bacteria. Whatever the animal source, the enzyme showed many similar characteristics as far as substrate specificity, dependence upon Mg2+, instability at 37 degrees C, and the protecting effect of nucleotides were concerned. Moreover, when submitted to gel filtration, the enzyme behaved in all cases as a dissociable high molecular weight protein, whose degree of association was controlled by nucleotides. In amphibian and avian tissues multiple forms of the enzyme seem to be present which differ for the substrate concentration at half-maximal velocity (S0.5); the concentration of nucleotide effector which affords half-maximal protection at 37 degrees C (P0.5); and the Hill coefficient for monophosphate donor. Within each single species, the higher the interaction coefficient was, the lower S0.5 and P0.5 values were. In mammalian tissues one form of nucleoside phosphotransferase seems to prevail where cooperative interactions are almost absent and whose S0.5 as well as P0.5 values do not vary significantly from one tissue to another. PMID- 3001505 TI - Interaction of 9-(1,3-dihydroxy-2-propoxymethyl)guanine with cytosol and mitochondrial deoxyguanosine kinases: possible role in anti-cytomegalovirus activity. AB - The acyclic nucleoside 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) is a potent inhibitor of human cytomegalovirus in vitro and in vivo. In order to investigate the phosphorylation of DHPG to the monophosphate and identify the enzyme responsible, attempts were made to isolate DHPG kinase from calf thymus and from human cytomegalovirus-infected lung cells. From calf thymus, a mitochondrial deoxyguanosine kinase was partially purified which co-migrated with DHPG phosphorylating activity on DEAE-cellulose, and had the same mobility by electrophoresis. DHPG triphosphate and DHPG kinase were elevated in cytomegalovirus-infected cells, but not enough enzyme activity was recovered to identify the kinase. However, DHPG was found to inhibit a cytosol deoxyguanosine kinase induced in these infected cells. The role of mitochondrial and cytosol deoxyguanosine kinases is discussed relative to the anti-cytomegalovirus activity of DHPG. PMID- 3001506 TI - [Effectiveness of expression of the chloramphenicol acetyltransferase gene controlled by foreign regulatory regions in Escherichia coli cells. I. Construction of vectors for the cloning of transcription regulatory elements]. AB - New plasmids pML2.1 and pML4 were constructed for cloning the transcription regulatory regions. In the pML2.1 the structural part of chloramphenicol acetyltransferase gene of the pBR325 is under control of the lacUV5-promotor. Because the unique BamH1 cleavage site is in the joint region, one may use it for cloning transcription termination regions and selecting recombinant clones with the AprCms phenotype. As for the pML4, the foreign fragment integration is carried directly before the structural part of cat-gene and it is expressed only if the promotor regions are present. The plasmids were sequenced and their restriction maps were established. Small molecular weight (about 2,0 MDa, AprCmr) or only Apr intact genes and convenient disposition of many unique cleavage sites by restriction endonucleases make these plasmids useful for different genetic engineering experiments. PMID- 3001504 TI - Metabolism of leukotrienes. AB - The in vitro metabolism of leukotriene B4 is initiated by omega-hydroxylation. This reaction is followed by oxidation of the omega-hydroxyl group to a carboxyl group. In vivo extensive beta-oxidation occurs and the main excreted products after administration of leukotriene B4 are water and carbon dioxide. Experiments performed in vitro and in vivo have demonstrated that a major pathway of metabolism of the glutathione containing leukotrienes involves modifications of the tripeptide substituent. The metabolic alterations are initiated by enzymatic elimination of the N-terminal gamma-glutamyl residue, catalyzed by the enzyme gamma-glutamyl transferase. This reaction is followed by hydrolysis of the remaining peptide bond resulting in elimination of the C-terminal glycine residue. The enzyme catalyzing the latter reaction is a membrane bound dipeptidase which occurs in kidney and other tissues. The product formed by these reactions, leukotriene E4, has been tentatively identified as a urinary metabolite in man following intravenous administration of leukotriene C4. In rats, the two major fecal metabolities of leukotriene C4 were characterized as being N-acetyl leukotriene E4 and N-acetyl 11-trans leukotriene E4. These compounds are formed in reactions between leukotriene E4 or 11-trans leukotriene E4 and acetyl coenzyme A. The reactions are catalyzed by a membrane bound enzyme present in liver, kidney and other tissues. PMID- 3001509 TI - [Study of dynamic properties of water in poly(A) and poly(U) solutions by proton magnetic resonance]. AB - Proton magnetic relaxation in aqueous solutions of polyadenylic and polyuridylic acids in the temperature range (10-80 degrees C) and acidities (pH 3-9.7) has been investigated. Activation energies of water molecule diffusion and proton exchange, as well as the velocities of these processes have been determined. It is established that from the point of view of magnetic relaxation, the state of single helices resulted from the thermal conformation transition, are not equal to the state obtained by the change of the pH of solution; it refers both to the secondary structure of the chains and the dynamical behaviour of the biopolymer hydrate layers. PMID- 3001507 TI - [Immunity to repeated transposition of the insertion sequence IS21]. AB - The ability of pBR325 derivatives carrying a copy of IS21-element to accept the second copy of this element from plasmid pRP19.6, a temperature-sensitive for replication mutant of RPI containing the duplicated IS21 was studied. It was shown that the frequency of IS21 transposition into plasmids pBR32S::IS21 differing by localization IS21 was lower by two orders of magnitude as compared to that of pBR325. The restriction endonuclease analysis revealed that the insertion of the second copy of IS21 resulted in the formation of pBR325 derivatives carrying the tandem repeated copies of IS21. It was also shown that the plasmids pBR325::IS21 were capable of increasing the frequency of pRP19.6 insertion into the bacterial chromosome from 3-9 to 200-300 times depending on IS21 localization. On the basis of the results obtained and literature data the possible mechanism of the transposition immunity is discussed. PMID- 3001508 TI - [Deletion of late genes of the prophage lambda]. AB - The deletions in tandem prophage lambda appear with high frequency (to 10%) in rec A- strain of Escherichia coli. The deletions were shown by marker rescue and hybridization of fragments of DNA on nitrocellulose filters with nick-translated phage lambda DNA localized only in prophage area. Right and left att sites are not involved. The majority of defective lysogens had all regulatory regions and deletions of late structural genes. These strains may be used for construction of the host-vector systems with the strongest promoter p'R of phage lambda. PMID- 3001510 TI - Quantitation of the immunogenic potential of protein antigens. AB - A procedure to quantitate the immunogenic potential of protein antigens is presented. It is hypothesized that the intrinsic immunogenicity of an accessible region on the protein arises from the overall structural effect of the presence of the particular assemblage of amino acid residues in the given region. Structural parameters previously derived by Grantham [Science 185, 862-864 (1974)] to differentiate the various amino acids are assigned to each residue position. At each point chosen on the molecule, an av. value is computed due to all residues within 8.5 A of this point; it is proposed that this local average is proportional to the immunogenic potential of the region centered at this point. The method can be used to locate the immunodominant regions of a molecule and to compare the antigenicity of related molecules. Test calculations on hen egg-white lysozyme, sperm whale myoglobin and horse cytochrome c show that segments in these molecules, that have been shown in immunochemical studies to possess antigenic activity, are predicted by this method to be immunodominant. PMID- 3001511 TI - [Clinical picture of infectious mononucleosis in childhood]. AB - Infectious mononucleosis, described for the first time many years ago, is still nowadays a frequent disease in children. History, etiology, pathogenesis and immunologic response to infectious mononucleosis are described. The value and limitations of diagnostic tests are discussed. The differential diagnosis, prognosis and possible complications are outlined. Data from the literature are compared with results of our own experience with 35 serologically proven infections in children of different ages. PMID- 3001512 TI - [Persistent (chronic active) Epstein-Barr virus infection and arthritis in childhood]. AB - Between 1981 und 1983 some 1300 patients with the primary diagnosis of juvenile chronic arthritis were admitted. In 9 of them a persistent EBV-infection was simultaneously evident. The course of disease was primarily systemic with the characteristic criteria: high septic intermittent fever, rheumatic exanthema, hepatosplenomegaly and lymphadenopathy. It is still unclear whether a replication of virus in the synovia or a precipiation of immune complexes is involved. A common cause for the persistent EBV-infection and arthritis via an immune suppressive agent is possible. PMID- 3001513 TI - [Function of breast milk macrophages]. AB - Human milk is a suspension of viable cells. Macrophages are the most abundant cells, comprising 40-80% of the total cell count. The present study was initiated to examine the principal cell functions of phagocytic milk macrophages: adherence, chemotaxis and phagocytosis-associated bactericidal oxidative metabolism. Adherence of milk macrophages to nylon wool was significantly decreased when compared with blood monocytes. In addition chemotaxis of macrophages in response to C 5a or a synthetic chemotactic peptide was also decreased. However, macrophages produced oxygen intermediates (O2-), H2O2) to a similar extent as blood monocytes after stimulation of the "oxidative burst" with phorbol myristate acetate or opsonized Candida particles. Macrophages cultured in vitro in endotoxin-free medium without serum lost the ability to produce oxygen metabolites over the course of a few days. Partial restoration of the oxygen radical production could be detected in macrophages exposed to bacterial lipopolysaccharide, i.e. endotoxin (LPS, 10 ng/ml). Endotoxin, which is present in the gut even of newborns might provide enough stimulation to maintain macrophages in the "primed" state. We conclude that oxygen metabolites released from milk macrophages are highly reactive and could contribute essentially to the protection of neonates against microbial infections. PMID- 3001514 TI - Amphicrine cells in the peritumoral gastric mucosa. AB - Electron microscopic researches pointed out the existence of amphicrine cells in the peritumoral mucosa on specimens of surgically resected gastric cancer. According to the ultrastructural features two cell subtypes were distinguished: mucoenterochromaffine (EC1, EC2) and empty clear mucoforming cells, corresponding to the mucoargentaffine and mucoargyrophobic types, respectively as the literature showed. Our results showed the amphicrine cells may be interpreted as transformed pathological endocrine cells under gastric cancer conditions. PMID- 3001516 TI - Effects of arachidonic acid and indomethacin on sister-chromatid exchange induction by polycyclic aromatic hydrocarbons in mammalian cell lines. AB - Arachidonic acid (AA), a prostaglandin precursor, significantly potentiated sister-chromatid exchange (SCE) induction in vitro by benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) in the aryl hydrocarbon hydroxylase (AHH) inducible human hepatoma C-HC-4 cells, and to a lesser extent in the non inducible rat tumor AH66-B and R1 and Chinese hamster Don-6 cells, all of which were less sensitive to these compounds than C-HC-4 cells. Indomethacin (IM), an inhibitor of prostaglandin endoperoxide synthetase (PES), moderately suppressed SCE induction by BP or DMBA in AH66-B and R1 cells, but it exerted no such effect in C-HC-4 and Don-6 cells. In C-HC-4 cells, however, IM completely eliminated the potentiating effect of AA on SCE induction by both BP and DMBA. The above findings suggest that PES in prostaglandin biosynthesis may also be involved in the metabolic activation of polycyclic aromatic hydrocarbons to genotoxic forms capable of inducing SCEs, in addition to AHH system. PMID- 3001515 TI - The role of the (6-4) photoproduct in ultraviolet light-induced transition mutations in E. coli. AB - Available evidence rules out the possibility that cyclobutane dimers are the major premutagenic lesions responsible for point mutations at sites of adjacent pyrimidine residues in the experiment systems examined to date in sufficient detail, that is, UV-induced mutations in chromosome loci in E. coli and UV induced mutations in the cI gene of phage lambda. However, it is likely that the major cytotoxic effects of UV irradiation can be attributed to the cyclobutane pyrimidine dimer, as these lesions occur at 10 times the frequency of other UV induced photoproducts in the dose range of 0.1-100 J/m2. The evidence also suggests that cyclobutane pyrimidine dimers are the major lesions responsible for induction of the SOS response and that as such they play an important, though indirect role, in the formation of mutations in irradiated DNA. Cyclobutane dimers may also be the major lesions responsible for other types of UV-light induced mutations such as deletions. None of the available evidence rules out (6 4) photoproducts as a major premutagenic lesion induced by UV irradiation using these experimental systems. On the contrary, the mutation spectrum induced both in the lacI gene and the cI gene of phage lambda is that predicted for mutations induced by (6-4) photoproducts. The observation that neither the premutagenic lesions nor the (6-4) photoproduct is subject to enzymatic photoreactivation also implies that the (6-4) photoproducts are premutagenic. As reviewed above, neither the photosensitization experiments nor the action spectrum of the (6-4) photoproducts rules out such a role. Might a lesion other than the (6-4) photoproduct be the major premutagenic lesion responsible for point mutations in these experimental systems? It cannot be ruled out that another as yet undefined minor photoproduct that occurs with the same sequence distribution specificity as that of the (6-4) photoproduct and that is also not subject to the reactivating treatments is more mutagenic than the (6-4) photoproduct itself. Candidates for such a lesion might include a photohydrate of the (6-4) photoproduct itself or as yet undefined photoproducts. However, we believe these alternative possibilities to be remote.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3001517 TI - Use of T-cell receptor gene probes to quantify the in vivo hprt mutations in human T-lymphocytes. AB - T-cell receptor (Ti) gene restriction fragment patterns (RFPs) were determined by Southern blots of genomic DNA obtained from T-lymphocyte colonies isolated from a single normal individual. 4 wild-type colonies and 11 in vivo derived 6 thioguanine-resistant mutant colonies with previously characterized hprt gene structural alterations were studied. Among the hprt mutants, 10 of the 11 showed unique Ti RFPs indicating their origins in different in vivo progenitors. Unique Ti RFPs were also seen among the wild-type T-cell colonies. One, however, shared its Ti RFP with a mutant. These results suggest that mutation in vivo of the hprt gene in human T-lymphocytes occurred after thymic maturation and that the 11 recovered hprt mutants probably resulted from 11 independent mutational events. PMID- 3001518 TI - Prophylactic efficacy of intranasal alpha 2-interferon against rhinovirus infections in the family setting. AB - In a double-blind evaluation of alpha 2-interferon as prophylaxis against naturally acquired respiratory infections, 120 adult members of 46 Australian families used 325 courses of intranasal spray during a six-month period, applying 5 million IU to the anterior nasal mucosa daily for seven days when respiratory symptoms developed in another member of the family. Used in this way, the alpha 2 interferon was well tolerated, and the rate of minor nasal bleeding (12 percent) did not increase with repeated courses. By comparison with the control group of 109 members of 49 families who used 319 seven-day courses of placebo spray, the users of alpha 2-interferon experienced 33 percent fewer days with nasal symptoms and 41 percent fewer episodes of "definite" respiratory illness. The users of alpha 2-interferon who were exposed to rhinovirus infections experienced 76 percent fewer days with symptoms and 86 percent fewer "definite" illnesses than their counterparts who used placebo. All of the observed clinical benefits, which suggested prevention of 6.8 "definite" respiratory illnesses per 100 courses of medication used, could be explained by a protective effect against illness associated with rhinoviruses that was not demonstrated for influenza A or B or coronavirus 229E. PMID- 3001519 TI - Prevention of natural colds by contact prophylaxis with intranasal alpha 2 interferon. AB - We conducted a randomized, placebo-controlled, double-blind study to determine whether intranasal alpha 2-interferon could prevent respiratory illnesses in healthy contacts of ill family members. Beginning within 48 hours of the onset of illness in a family member, contacts self-administered interferon (5 X 10(6) IU) or placebo spray once daily for seven days. Respiratory illness developed during the eight-day period, starting with the second day of spraying, in 52 of 222 persons in the placebo group as compared with 32 of 226 in the interferon group (P = 0.02; efficacy, 39 percent). Among persons exposed to laboratory-documented rhinovirus colds, illness developed in 2 of 27 interferon recipients as compared with 12 of 34 placebo recipients (P = 0.02; efficacy, 79 percent). During the two week period during and after spraying, rhinovirus colds developed in 1.3 percent of those spraying with interferon and in 15.1 percent of those spraying with placebo (P = 0.003; efficacy, 88 percent). Blood-tinged mucus or nasal mucosal bleeding or both were detected in 7.7 percent of placebo and 13.6 percent of interferon users (P = 0.04), but no evidence of cumulative nasal toxicity was found. We conclude that postexposure prophylaxis with intranasal interferon may in some cases provide an effective strategy for controlling the spread of natural colds, especially those caused by rhinoviruses. PMID- 3001521 TI - Smoking. PMID- 3001520 TI - Vidarabine versus acyclovir therapy in herpes simplex encephalitis. AB - We randomly assigned 208 patients who underwent brain biopsy for presumptive herpes simplex encephalitis to receive either vidarabine (15 mg per kilogram of body weight per day) or acyclovir (30 mg per kilogram per day) for 10 days. Sixty nine patients (33 percent) had biopsy-proved disease; 37 received vidarabine, and 32 acyclovir. The mortality in the vidarabine recipients was 54 percent, as compared with 28 percent in the acyclovir recipients (P = 0.008). Six-month mortality varied according to the Glasgow coma score at the onset of therapy. For scores of greater than 10, 7 to 10, and less than or equal to 6, mortality was 42, 46, and 67 percent in the patients treated with vidarabine, as compared with 0, 25, and 25 percent in those treated with acyclovir. A six-month morbidity assessment using an adapted scoring system revealed that 5 of 37 patients receiving vidarabine (14 percent) as compared with 12 of 32 receiving acyclovir (38 percent) were functioning normally (P = 0.021). Eight vidarabine-treated patients (22 percent) and three acyclovir-treated patients (9 percent) had moderate debility. Patients under 30 years of age and with a Glasgow coma score above 10 had the best outcome with acyclovir treatment. We conclude that acyclovir is currently the treatment of choice for biopsy-proved herpes simplex encephalitis. PMID- 3001522 TI - Response of ACTH and cortisol to corticotropin-releasing hormone in anorexia nervosa. PMID- 3001523 TI - Treatment of varicella-zoster virus infection in severely immunocompromised patients. A randomized comparison of acyclovir and vidarabine. AB - In a prospective, randomized trial, we compared intravenous acyclovir and vidarabine in the treatment of varicella-zoster virus infection in severely immunocompromised patients who presented within 72 hours of onset of the infection. Eleven patients were treated in each group. Cutaneous dissemination of infection occurred in none of the 10 acyclovir recipients and in 5 of the 10 vidarabine recipients who had presented with localized dermatomal disease (P = 0.016). As compared with vidarabine, acyclovir treatment shortened the median periods during which cultures were positive for the virus (four vs. seven days, P = 0.004) and new lesions formed (three vs. six days, P = 0.03). Acyclovir also shortened the median interval until the first decrease in pain (4 vs. 7 days, P = 0.005), the pustulation of all lesions (4 vs. 7 days, P = 0.0004), the crusting of all lesions (7 vs. 17 days, P = 0.0003), and the complete healing of lesions (17 vs. 28 days, P = 0.003). In addition, acyclovir reduced the incidence of fever (two vs. eight patients, P = 0.015). We conclude that acyclovir is better than vidarabine for the treatment of varicella-zoster infection in immunocompromised patients. PMID- 3001524 TI - Cellular sodium transport in essential hypertension. PMID- 3001525 TI - Cyclic adenosine 3',5' monophosphate (cAMP) and dimorphism in the pathogenic fungus Paracoccidioides brasiliensis. AB - Exogenous cAMP or its analogs inhibit the mycelium transformation of yeast and induce bulging of the apex of mycelia. But intracellular cAMP levels of yeast and mycelial cells are not significantly different. PMID- 3001526 TI - Consequences of perinatal drug administration on brain development: striatal opiate receptors and dopamine. PMID- 3001527 TI - Correlating behavior with neural activity: an approach to study the action of drugs in the behaving animal. PMID- 3001528 TI - Epidermal growth factor receptor occupancy inhibits vaccinia virus infection. AB - Vaccinia virus encodes VGF, an early protein of relative molecular mass 19,000 (19K) which, from amino-acid residues 45 to 85, is homologous in 19 residues to epidermal growth factor (EGF), and transforming growth factor-alpha (TGF-alpha). The conserved sequence includes a region of high homology (6 out of 10 amino acids) from residues 71 to 80, corresponding to the third disulphide loop of both EGF and TGF-alpha. This region has recently been shown to contain a binding region of TGF-alpha for the EGF receptor, and this raises the question of whether vaccinia virus utilizes the EGF receptor in order to bind to and infect cells. We now show that occupancy of the EGF receptor inhibits vaccinia virus infection. Inhibition is observed in a dose-dependent fashion by pre-treatment with either EGF or synthetic decapeptide antagonists of EGF's mitogenic activity which correspond to the sequence of the third disulphide loop of VGF or TGF-alpha. The relative ability of the peptides to inhibit vaccinia virus infection parallels their binding affinity to the EGF receptor. PMID- 3001529 TI - Characterization of receptors for human tumour necrosis factor and their regulation by gamma-interferon. AB - Tumour necrosis factors, TNF-alpha and TNF-beta (previously called lymphotoxin), are the products of activated monocytes and lymphocytes, respectively, and both have recently been purified, sequenced and cloned by recombinant DNA methods, revealing 35% identity and 50% homology in the amino-acid sequence. Both proteins have been found to be specifically toxic to many tumour cells. Furthermore, it has been reported that various interferons are synergistic with TNF for anti tumour effects in vitro, while activities attributed to the two proteins have also been shown to necrotize various tumours in vivo. We have now prepared 125I labelled highly purified recombinant human TNF-alpha to study in detail its binding to the human cervical carcinoma cell line ME-180. Our results indicate that there is a single class of specific high-affinity receptors for TNF on this cell line which has a Kd of about 0.2 nM and an average of 2,000 receptor sites per cell. The binding of labelled TNF-alpha to these cells can be inhibited by both TNF-alpha and TNF-beta but not by gamma-interferon (IFN-gamma). However, preincubation of cells with IFN-gamma increases the total number of TNF receptors two to threefold without any significant change in the affinity constant. This is the first report that TNF-alpha and -beta share a common receptor and that the receptors can be up-regulated by interferon. Our results may explain previous observations regarding similar biological activities observed for these two cytotoxic proteins and also their synergistic action with interferons. PMID- 3001530 TI - Cloning of the breakpoint of an X;21 translocation associated with Duchenne muscular dystrophy. AB - Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder which affects approximately 1 in 3,300 males, making it the most common of the neuromuscular dystrophies. The biochemical basis of the disease is unknown and as yet no effective treatment is available. A small number of females are also affected with the disease, and these have been found to carry X; autosome translocations involving variable autosomal sites but always with a breakpoint within band Xp21 of the X chromosome (implicated by other kinds of genetic evidence as the site of the DMD lesion). In these female patients the normal X chromosome is preferentially inactivated, which it is assumed silences their one normal DMD gene, leading to expression of the disease. In one such affected female the autosomal breakpoint lies in the middle of the short arm of chromosome 21, within a cluster of ribosomal RNA genes. Here we have used rRNA sequences as probes to clone the region spanning the translocation breakpoint. A sequence derived from the X-chromosomal portion of the clone detects a restriction fragment length polymorphism (RFLP) which is closely linked to the DMD gene and uncovers chromosomal deletions in some male DMD patients. PMID- 3001531 TI - Simian virus 40-mediated cis induction of the Xenopus beta-globin DNase I hypersensitive site. AB - Regions in chromatin which are hypersensitive to the action of DNase I appear to be associated with sites of genetic activity; the association between DNase I hypersensitivity and transcriptional activation is well known. In the case of the chicken beta-globin gene the establishment of a DNase I hypersensitive site is dependent on tissue-specific trans-acting factors. Such factors have also been implicated in the action of viral and cellular enhancers, which are themselves hypersensitive to DNase I. Enhancers have been defined operationally as DNA sequences which act in cis to potentiate transcription from their own, heterologous or cryptic promoters. This activity is essentially unaffected by changes in the orientation, position (5' or 3') or distance of the enhancer element with respect to its cognate promoter. We demonstrate here that the transcriptional rescue of the Xenopus laevis beta-globin gene by simian virus 40 (SV40) sequences including the enhancer coincides with the conferment of DNase I hypersensitivity upon that gene, and that this occurs in the absence of any change in the complement of trans-acting factors. These results suggest that a propensity to form sites hypersensitive to the action of DNase I is encoded in the primary sequence of DNA, and that this predilection is aggravated by SV40 sequences, perhaps through a mechanism dependent on supercoiling. PMID- 3001532 TI - Assembly of microtubules from nucleotide-depleted tubulin. AB - In vitro assembly of microtubules from tubulin is considered to have an absolute requirement for added GTP (or a non-hydrolysable GTP-analogue) involving binding at the E(exchangeable)-site located on the beta-subunit of the tubulin dimer. By contrast, GDP inhibits assembly. Nucleotide hydrolysis has been implicated in the dynamic properties of microtubules, treadmilling and mechanical coupling. Here we demonstrate that assembly is not necessarily dependent on the presence of GTP at the E-site; microtubules can be formed efficiently in the absence of GTP in the presence of pyrophosphate. These microtubules, which have normal morphology and lability at cold temperatures, contain N(non-exchangeable)-site GTP and a significant proportion of E-site GDP. This demonstrates the possibility of direct incorporation of GDP-containing tubulin dimer during assembly which probably derives from microtubule-associated protein (MAP)-containing oligomers. This finding has important implications for the mechanism of microtubule elongation. The effects of pyrophosphate suggest that charge neutralization by the bidentate ligand is an essential step in promoting microtubule assembly, and that this interaction involves only a minimal conformational change in the protein. PMID- 3001533 TI - Approaches to AIDS therapy. PMID- 3001534 TI - Further anxieties about AIDS. PMID- 3001535 TI - Requirement of stereospecific alignments for initiation from the simian virus 40 early promoter. AB - The distance between the simian virus 40 early promoter elements has been altered by inserting either odd or even multiples of half a DNA turn. There are marked differences in the in vivo effects of these two types of insertions on initiation of transcription from this promoter. PMID- 3001537 TI - Importance of helical periodicity. PMID- 3001536 TI - Functional corticotropin releasing factor receptors in the primate peripheral sympathetic nervous system. AB - Corticotropin releasing factor (CRF) is a key hormone in the integrated response to stress, acting both as the major regulator of pituitary adrenocorticotropic hormone (ACTH) release and as a neuropeptide in the brain. The actions of CRF are mediated by specific plasma membrane receptors in the anterior pituitary gland and in discrete brain areas including the cerebral cortex and several regions related to the limbic system. In addition to the pituitary and central actions of CRF, systemic administration of the peptide in the rat, dog, monkey and man causes hypotension and tachycardia because of a decrease in peripheral vascular resistance. These observations, in conjunction with the finding of immunoreactive and bioactive CRF in peripheral tissues, suggest that the peptide is locally released in tissues to act as a neurotransmitter or paracrine hormone. As CRF is present in the adrenal medulla and the peptide is known to modulate the central activity of the autonomic nervous system, we investigated the possibility that CRF is involved in the regulation of the peripheral autonomic nervous system. Such an action of CRF is supported by our demonstration of specific CRF receptors in the monkey adrenal medulla and sympathetic ganglia. In the adrenal medulla, these receptors are coupled to adenylate cyclase and can stimulate the secretion of catecholamines and Met-enkephalin. PMID- 3001538 TI - Magnocellular axons in passage through the median eminence release vasopressin. AB - Vasopressin (arginine vasopressin, AVP) is present in two types of nerve fibres in the median eminence (ME). First, it is found in nerve terminals that originate in the parvicellular neurones of the hypothalamic paraventricular nucleus (PVN) and abut on the pericapillary space surrounding the fenestrated capillaries of the primary pituitary portal plexus in the external zone (EZ) of the ME. These neurones also synthesize corticotropin-releasing factor (CRF), which acts synergetically with vasopressin to stimulate release of adrenocorticotropin (ACTH) from the pituitary gland (see ref. 7). Second, vasopressinergic axons of the magnocellular neurosecretory system pass through the internal zone (IZ) of the ME to terminate in the neurohaemal contact zone of the neurohypophysis. The involvement of vasopressinergic magnocellular neurones in the control of ACTH secretion is much debated. Of particular interest in this context is the origin of the vasopressin found in pituitary portal blood. Although it has been demonstrated that vasopressin and CRF are present in the same neurosecretory granules of EZ fibres, parallel determinations of vasopressin and CRF in pituitary portal blood have shown alterations of the concentration of vasopressin without a concomitant change in that of CRF. Such a dissociation suggests that either differential release of vasopressin and CRF can occur from a single population of nerve endings, or there are fibres in the pituitary-stalk ME which release vasopressin but not CRF. Here we present evidence for the latter. Our results indicate that stimuli causing depolarization of the axonal membrane in vitro elicit release of vasopressin from nerve fibres in the external and internal zones of the ME. PMID- 3001540 TI - Aspirin and Reye syndrome: a saga unfolds. PMID- 3001539 TI - Disease-specific and tissue-specific production of unintegrated feline leukaemia virus variant DNA in feline AIDS. AB - Feline leukaemia viruses (FeLVs) have long been known to be associated with induction of proliferative and anti-proliferative diseases of domestic cats. Strains of FeLV have been recognized which specifically induce lymphosarcoma, aplastic anaemia, myelodysplastic anaemia, and, recently, feline AIDS (acquired immune deficiency syndrome), a naturally occurring immunosuppressive syndrome strikingly similar to human AIDS which is lethal in 100% of inoculated and viraemic specific-pathogen-free (SPF) cats. Here, we have analysed FeLV DNA in tissues of 22 SPF cats that had been inoculated with the feline AIDS strain (FeLV FAIDS) and we find two classes of viral DNA--a monotypic common form which is detectable in bone marrow regardless of disease state, and variant forms, recognizable by restriction site differences, whose appearance correlates with onset of disease symptoms and persists throughout the course of the disease. FeLV FAIDS variant DNA is detected at high concentration (10-50 copies per cell) and principally as unintegrated viral DNA (UVD) in bone marrow of cats with feline AIDS. In marked contrast high levels of UVD were not present in cats in the terminal-stages of T-cell lymphosarcoma, aplastic anaemia, or myelodysplastic anaemia induced by other FeLV strains. These results parallel recent observations in humans, where high levels of UVD were sometimes found in cells derived from AIDS patients infected with human T-lymphotropic virus type III (HTLV-III)/lymph adenopathy-associated virus (LAV), and suggest that persistence of unintegrated variant viral DNA is a crucial indicator of retrovirus-induced cytopathic disease syndromes such as AIDS. PMID- 3001541 TI - [Breast cancer in a family]. PMID- 3001542 TI - [Subcutaneous mastectomy; indications and results]. PMID- 3001543 TI - [Current developments in the field of diabetic neuropathies]. PMID- 3001544 TI - [Initial manifestation of acquired immunodeficiency syndrome (AIDS) in the nervous system]. PMID- 3001545 TI - [Treatment termination in psychotherapy]. PMID- 3001547 TI - AIDS in The Netherlands. Clinical and microbiological data on 36 cases. PMID- 3001546 TI - Cardiac glycoside interactions at 'silent' and at active receptors. Putative biochemical backgrounds and possible clinical relevance. PMID- 3001548 TI - Acute renal failure due to bilateral ureteral obstruction by metastases from breast cancer. AB - Two female patients with metastatic breast carcinoma had acute renal failure secondary to metastatic ureteral obstruction. Retrograde pyelography showed bilateral segmental constriction and dilatation of the ureters with hydronephrosis. Drainage procedures reversed the renal failure in both patients. A review of the literature indicates that ureteral involvement is frequent in patients with malignancies. PMID- 3001549 TI - Serum factor from patients with chronic renal failure enhances polymorphonuclear leukocyte oxidative metabolism. AB - Sera from patients with chronic renal failure (CRF) contain a factor(s) which enhances the oxidative metabolism of polymorphonuclear leukocytes (PMN) as assessed by chemiluminescence (CL), superoxide anion generation, and hexose monophosphate shunt activity. PMN oxidative metabolic activity was higher in CRF sera than in sera from hospitalized patients with normal renal function or in sera from normal healthy subjects. The enhancement occurred regardless of whether PMN were unstimulated or were stimulated by a nonspecific soluble membrane stimulant (phorbol myristate acetate), or by opsonized Candida albicans. The enhanced CL was significantly reduced in their sera after normal renal function was restored with successful renal transplantation. This CL-enhancing factor was also detected in dialysate fluids from CRF patients and in urine from normal healthy subjects. When serum, urine, dialysate fluids of these CRF patients were fractionated by Sephadex G-25 column chromatography, the specific fraction responsible for enhanced CL was found in the molecular weight range less than 1,000 daltons, and is an ethanol extractable substance with natural fluorescence. Our findings suggest that the enhanced PMN stimulatory activity in CRF serum is specifically associated with renal dysfunction and can be useful, along with other conventional parameters, for monitoring the progression of CRF. PMID- 3001550 TI - Xylene induced feeding and drinking behavior and central adrenergic receptor binding. AB - Mice were exposed to 1600 ppm m-xylene 4 hours a day, 5 days a week in 7 weeks. At the end of exposure binding of 3H-clonidine to four brain regions was measured, and it was found that the binding was significantly decreased in the hypothalamus region but not altered in diencephalon, cortex and cerebellum. During the m-xylene exposure the mice ate and drank more than the control group, which resulted in a loss of weight for the controls compared to the exposed mice, when weighed before and after the 4 hours exposure. The results show that m xylene can induce eating and drinking response in mice, and it is suggested that there is a connection between this phenomenon and the decrease in alpha-receptor binding in the hypothalamus region. PMID- 3001551 TI - Perinatal caffeine treatment: behavioral and biochemical effects in rats before weaning. AB - Administration of drinking water containing 0, 0.02%, 0.04% and 0.08% of caffeine to female rats throughout gestation and lactation affects several behavioral parameters in the offspring. Righting reflexes, swimming ability development, motor coordination and muscle tone were affected. The activity of these animals, as measured with an open-field test at weaning (i.e., at the end of the treatment), was reduced. The effects observed were dose-dependent. However, for righting reflexes the dose-dependency was direct (the highest dose producing maximal effects), whereas in all the other tests, the dose-dependency was inverse, the lowest dose producing maximal effects and the highest dose producing no effects. This might reflect the presence of subclasses of receptors having different affinities for adenosine, mediating opposite effects and antagonized by caffeine. On the other hand, perinatal caffeine effects are certainly not mediated by blockade of phosphodiesterases, since cAMP levels at the end of the treatment were dose-dependently reduced. This study shows therefore that administration of caffeine to rat dams is able to influence the neurobehavioral development of the offspring. Moreover, all the doses utilized and corresponding to 27, 58 and 108 mg/kg, were able to produce all or some of the mentioned effects, indicating that further testing with doses lower than 27 mg/kg is required to find a dose which does not affect the offspring. PMID- 3001552 TI - Prenatal ethanol exposure and hyperactivity in rats: effects of d-amphetamine and alpha-methyl-p-tyrosine. AB - Pregnant Wistar rats were exposed to either a liquid diet containing ethanol, pair-fed an identical diet with sucrose substituted for ethanol, or received ad lib chow and water, during days 6-19 of gestation. The activity of their offspring was tested at 10, 16, 22 or 28 days of age. Pups exposed to alcohol prenatally were more active than controls at 16 and 22 days of age. Thirty min after being placed in the activity chamber, the pups were injected with either 0, 0.5, 1.0 or 4.0 mg/kg of d-amphetamine sulphate, and returned to the chamber for a further 120 min. Amphetamine brought about a dose-related increase in activity at 10, 22 and 28 days. At 16 days, peak activity was associated with the 0.5 mg/kg dose, activity declining at the higher doses. In a second study, pups received injections of 0, 25, 50 or 100 mg/kg of alpha-methyl-p-tyrosine (AMPT). Activity was unaffected by AMPT at 10, 22 or 28 days of age, but was increased at 16 days of age by the 25 and 50 mg/kg doses. Importantly, the time courses of the effects on activity of both d-amphetamine and AMPT were the same regardless of the treatment received during gestation. These data indicate that the hyperactivity associated with fetal alcohol exposure is not a result of alterations in the ontogeny of the catecholamine systems involved in arousal. PMID- 3001553 TI - [Mechanism of action of aldosterone on the toad bladder: correlation between biological response and occupation of the receptor sites]. AB - The mechanism of aldosterone uptake in the epithelial cells of toad bladder was studied using mathematical modeling. It seems that two types of receptors are necessary to obtain a complete action of aldosterone. If this observation is confirmed, the traditional notion of type I receptors i.e. mineralocorticoids receptors and type II receptors i.e. glucocorticoids receptors has to be reviewed. PMID- 3001554 TI - [Site and mechanism of tubular Na-K-ATPase regulation by aldosterone]. AB - Effects of adrenalectomy and acute administration of physiologic doses of aldosterone on the activity and the number of catalytic units of Na-K-ATPase, the biochemical equivalent of the sodium pump, were evaluated on single microdissected nephron segments. After adrenalectomy, Na-K-ATPase is markedly decreased in all rabbit nephron segments, except the proximal tubule. However, aldosterone administration restores Na-K-ATPase exclusively in the collecting tubule, where mineralocorticoid receptors have been localized. Aldosterone induces both the activity and the appearance of new catalytic units with a kinetics similar to that of the antinatriuretic action of the hormone. These results suggest that Na-K-ATPase might be a primary target for aldosterone action. PMID- 3001556 TI - [Hypertension induced by adrenocorticotropin (ACTH)]. AB - Studies of ACTH-induced hypertension in sheep have enabled the hypertensinogenic actions of steroid hormones to be separated from their classical glucocorticoid and mineralocorticoid actions. In man ACTH produces systolic hypertension which can be reproduced by infusion of cortisol, but not deoxycorticosterone, at rates appropriate for conditions of ACTH stimulation. Whether steroids raise blood pressure in man by a hypertensinogenic action distinct from their glucocorticoid and mineralocorticoid activities remains to be determined. PMID- 3001555 TI - [The renin-angiotensin system and renal adaptation to sodium restriction]. AB - The renin angiotensin aldosterone system (RAAS) is activated during the early phase of renal adaptation to sodium restriction. The effect of the converting enzyme inhibitor (CEI) MK 421 (MK) was studied when administered 3 days before and 6 days after sodium restriction. Mean arterial pressure, variations of urinary aldosterone, renal blood flow and sodium balance were studied. The results are the following; (Formula: see text) Those results demonstrate that CEI is associated with a renal inadaptation to sodium restriction. Other studies demonstrate that this disequilibrium persists during the second week of treatment in the rat. Those results may be related to an inhibition of aldosterone effect during the sodium restriction, a renal vasodilatation or an accumulation of kinin and endogenous prostaglandins. In summary, a functional RAAS is necessary during the functional adaptation to a sodium restriction. The renal prostaglandins stimulation may contribute to the disequilibrium balance in association with the CEI administration. PMID- 3001557 TI - Simultaneous expression of glial fibrillary acidic (GFA) protein and neuron specific enolase (NSE) by the same reactive or neoplastic astrocytes. AB - In normal cells of the central nervous system (CNS), glial fibrillary acidic (GFA) protein is demonstrable by immunohistochemistry in fibrillated astrocytes, and neuron-specific enolase (NSE) in neurons and their processes. However, it has been shown that NSE may also be expressed in reactive astrocytes and in various neoplastic cells of non-neuronal origin, including those of astrocytomas and glioblastomas. In the present study, a double-labelling technique using immunoperoxidase (PAP) and immunofluorescence (FITC) was employed to determine whether GFA protein and NSE could be expressed simultaneously by the same cell. This was found to be the case in some, but not in all reactive astrocytes in the human brain. In glial tumours, many of the neoplastic cells in the glioblastomas and astrocytomas examined demonstrated either GFA protein or NSE, but usually not both. However, occasional neoplastic cells in those gliomas were found to show both proteins. Because of the relatively low sensitivity of the FITC technique, the percentage of cells expressing both proteins could not be determined, but it is clearly possible for a single reactive or neoplastic astrocyte to demonstrate both GFA protein and NSE. PMID- 3001558 TI - Primary demyelination in the central nervous system of cats. AB - Primary demyelinating lesions have been observed in the central nervous system of 16 (approximately 7%) of a total of 235 clinically normal cats. The size of the lesions varied from small perivascular lesions in white matter to a large lesion occupying the diameter of an optic nerve. Intracytoplasmic inclusions consisting of tubular structures were common to all lesions examined by electron microscopy. The features of these feline lesions are briefly compared with those seen in multiple sclerosis. PMID- 3001559 TI - Effects of chronic exposure to cigarette smoke on amine levels and turnover in various hypothalamic catecholamine nerve terminal systems and on the secretion of pituitary hormones in the male rat. AB - Male rats were exposed to the smoke from 2 cigarettes every morning for a total period of 9 days. The next day they were decapitated immediately after the exposure to the smoke from 4 cigarettes (Kentucky reference IR-1 type) burned at 30-min intervals. Control animals were exposed to air alone or to nicotine-free cigarette smoke (Cambridge glass fibre filters). In contrast to chronic exposure to filtered smoke, exposure to unfiltered smoke resulted in a 10% increase in catecholamine (CA) levels (quantitative histofluorimetry) within the lateral palisade zone, the posterior periventricular hypothalamic nucleus and within the dorsomedial hypothalamic nucleus. There was also an increase in amine turnover (tyrosine hydroxylase inhibition by alpha-methyl-dl-p-tyrosine methylester; alpha MT) in the dopamine (DA) systems of the medial and lateral palisade zones and in the periventricular noradrenaline (NA) hypothalamic systems. Chronic exposure to unfiltered cigarette smoke resulted in reductions of prolactin, LH and FSH levels (radioimmunoassay). Following alpha MT treatment chronic exposure to unfiltered cigarette smoke still led to reduced prolactin serum levels. In addition an increased vasopressin serum concentration was found. The effects of chronic exposure to cigarette smoke on neuroendocrine function and on hypothalamic CA systems are suggested to be mediated via nicotine. Combined with the results from a previous study the present results indicate that tolerance does not develop with regard to the inhibitory effects of exposure to cigarette smoke on prolactin, LH and FSH secretions. The same is true for the stimulatory effects on the tubero-infundibular DA neurons and the periventricular NA systems. But chronic exposure to cigarette smoke seemed to induce tolerance with regard to its stimulatory effects on subependymal, dorsomedial and paraventricular hypothalamic NA systems and on corticosterone release. PMID- 3001560 TI - Seasonal changes in the sensitivity of ovine pineal beta-adrenoceptors to steroids. AB - Immunoneutralization of endogenous gonadal steroids has recently been shown to modify pineal beta-adrenoceptor function in intact Merino ewes. In the current study, interactions between gonadal steroids and these receptors have been further investigated. beta-Adrenoceptor density and binding affinity both showed time-related changes in ewes; the significance of these changes requires further study. Two observations, firstly modification of beta-adrenoceptor function by androstenedione and 17 beta-estradiol in ovariectomized, but not in intact ewes, and secondly that steroid-mediated effects on receptor density and binding affinity in the pineal of ovariectomized Merino ewes could be demonstrated during anestrus, but not during the breeding season for intact ewes, indicate that gonadal steroids may regulate pineal beta-adrenoceptor variables in Merino ewes. It is suggested that gonadal steroids may regulate ovine pineal function in ewes, and that the seasonal differences in sensitivity of luteinizing hormone release to steroid feedback may be mediated in part via effects on the pineal gland. PMID- 3001561 TI - Corticotropin-releasing activity of the renin-angiotensin system peptides in rat and in man. AB - In this study we investigated in the rat the binding and corticotropin-releasing factor (CRF) activity of various constituents of the renin-angiotensin system and the possible angiotensin II receptor changes following procedures known to alter plasma renin activity. We investigated also the CRF activity of angiotensin II in vitro and in vivo in humans. The CRF activity of peptides was studied by their ability to stimulate ACTH release from pituitary cells. Deleting amino acids from the N-terminus of angiotensin II resulted in decreased CRF activity; while the ED50 for angiotensin II was 2 nM, it increased to about 10 nM for the (2-8) heptapeptide. Angiotensin I had a weak CRF activity, whereas the substrate angiotensinogen had no stimulatory effect even at a concentration of 100 nM. There was a strong correlation between the activation and binding properties of all peptides tested. Dietary salt load or depletion as well as dexamethasone treatment did not affect the number nor the affinity of pituitary angiotensin II receptors. Angiotensin II had a CRF activity on human pituitary cells in vitro. However, peripherally injected agiotensin II at a pressive dose of 7 ng/kg/min did not produce any ACTH release in normal male volunteers. These data suggest that angiotensin II may play a modulatory role in the physiological regulation of ACTH secretion, but this role might be attributed to the endogenous brain angiotensin II as it is not closely dependent on the angiotensin II plasma levels. PMID- 3001562 TI - Evidence against a self-inhibition of ACTH secretion in man. AB - To ascertain the physiological relevance of an autoregulation of adrenocorticotropin hormone(ACTH) secretion in man, we studied the effect of alsactide (beta-Ala1, Lys17-ACTH1-17-4-amino-N-butylamide), a synthetic ACTH analogue with potent steroidogenic activity but not recognized in the endogenous ACTH immunoassay, on plasma ACTH pattern in patients with Addison's disease. Three experimental models were employed as follows: (a) in 6 patients, whose steroid replacement therapy had been discontinued 36 h previously, we compared the effect of alsactide, administered at two dose levels (10 or 100 micrograms i.v. als bolus followed by the same dose infused over 2 h, and of placebo, on the plasma ACTH pattern; (b) the previous experiment was repeated in 4 patients in whom cortone replacement therapy was substituted for 3 days with dexamethasone, 0.5-1.5 mg daily p.o., so as to lower plasma ACTH levels to within the high normal range; (c) in 4 patients off therapy for 36 h, we evaluated the ACTH response to synthetic ovine corticotropin-releasing factor, 1 microgram/kg body weight injected intravenously, occurring during concomitant administration of alsactide, 100 micrograms i.v. as bolus plus 100 micrograms infused over 2 h, or placebo. Compared to placebo, alsactide did not significantly affect the pattern of ACTH under any of the experimental conditions investigated. Collectively, our findings, although they have to be interpreted with caution, do not support the idea that a self-regulation mechanism plays an important role in the control of ACTH secretion in man. PMID- 3001563 TI - Intravenous application of ovine and human corticotropin releasing factor (CRF): ACTH, cortisol and CRF levels. AB - Synthetic ovine corticotropin releasing factor (oCRF) and human CRF were given as an intravenous bolus to 6 healthy volunteers in 4 different dosages (oCRF: 25, 50, 100 and 200 micrograms) respectively, 2 different dosages (hCRF: 50 and 100 micrograms). ACTH and cortisol were measured over a 2-hour period after CRF injection. CRF immunoreactivity was measured with a newly developed homologous radioimmunoassay for both oCRF and hCRF. All CRF dosages tested led to a definite ACTH response compared to placebo control. No dose dependency of side effects was observed. Furthermore, there was no clear-cut dose response relationship between the injected CRF dosages and the maximal ACTH- and cortisol levels. However, when the area under the ACTH and cortisol-response curves after oCRF were compared a dose-response relationship emerged, which could not be demonstrated in the same way for the two hCRF dosages. Serum CRF immunoreactivity correlated to the injected dosage. The halftime of serum disappearance for oCRF was 18 min, and hCRF, 9 min. The maximal ACTH responses after injection of oCRF and hCRF were not significantly different. PMID- 3001564 TI - Increased number of angiotensin II binding sites determined by autoradiography in anterior pituitary of water-deprived and Brattleboro rats. AB - Pituitary angiotensin II (ANG) binding sites were characterized by autoradiography in individual male Long Evans (LE) rats, heterozygous Brattleboro (HZ) rats, and homozygous Brattleboro (DI) rats which were water satiated or water deprived. An additional group of DI rats was treated with arginine vasopressin for 1 week. The technique utilized 8-micron pituitary sections which were incubated with 125I-[Sar1]-ANG in concentrations ranging from 25 pM to 10 nM. Angiotensin binding in the anterior pituitary of water-satiated LE rats was characterized by a single class of high-affinity, saturable sites with a Bmax of 1.309 +/- 119 fmol/mg of protein and a Ka of 0.51 +/- 0.03 X 10(9) M-1. Compared to LE rats the density of ANG-binding sites in anterior pituitary was higher in DI rats (+84.2%), with HZ having an intermediate concentration (+41.6). Dehydration increased the ANG-binding site density in all groups. Five days of water deprivation increased the number of ANG-binding sites by 86.5% and by 36.9% in LE and HZ rats, respectively, when compared to their water-satiated controls. After 1 day of water deprivation, the ANG-binding site density increased by 19.4% in DI rats. No changes in ANG-binding sites occurred after hormonal replacement with arginine vasopressin in water-satiated DI rats. The binding affinity constant of the agonist for the ANG-binding sites compared with LE rats was decreased in both HZ and DI rats (-27.5 and -19.4%) and was also decreased after 5 days of dehydration in LE rats (-34%) when compared to the water-satiated state.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001565 TI - Opioid kappa receptors and the secretion of prolactin (PRL) and growth hormone (GH) in the rat. I. Effects of opioid kappa receptor agonists bremazocine and U 50,488 on secretion of PRL and GH: comparison with morphine. AB - The effects of bremazocine and U-50,488, two selective opioid kappa receptor agonists, and the preferential mu receptor agonist morphine on the secretion of PRL and GH were compared in conscious male rats bearing permanent right atrial cannulae for serial blood sampling and drug delivery. All three opioids stimulated PRL secretion in a dose-related manner, but the kappa agonists differed from morphine in several respects. They were considerably more potent than morphine in triggering a PRL response, but were unable to elevate PRL levels to more than 100 ng/ml, whereas morphine, at the highest dose (4.5 mg/kg), induced an almost twice larger response. Also their PRL-releasing effect was inhibited more strongly by the preferential kappa receptor antagonist Mr-2266 than by naloxone, whereas Mr-2266 and naloxone, which are equipotent as antagonists of the mu receptors, were equipotent in suppressing the PRL stimulating effect of morphine, a mu agonist. In a complete contrast to morphine, which effectively stimulated GH secretion, the kappa agonists had no effect on GH release at lower doses and suppressed it at higher doses. It is concluded that the PRL-releasing effect of the kappa agonists is mediated by the kappa receptors which may participate with the mu receptors in regulation of PRL secretion by opioids. The GH-inhibiting effect of the kappa agonists requires further clarification. PMID- 3001566 TI - Opioid kappa receptors and the secretion of prolactin (PRL) and growth hormone (GH) in the rat. II. GH and PRL release-inhibiting effects of the opioid kappa receptor agonists bremazocine and U-50,488. AB - An analysis of the GH release-inhibiting action of the opioid kappa receptor agonists bremazocine and U-50,488, established earlier, was attempted by testing the agonists against activation of GH secretion by morphine or clonidine in male rats bearing right atrial cannulae for serial blood sampling and drug delivery. Both kappa agonists inhibited the effect of subsequent administration of clonidine in a dose-related manner. Bremazocine was approximately ten times more potent than U-50,488, a ratio corresponding to the known affinities of the two compounds for the kappa receptors. The inhibiting action of bremazocine was more strongly reversed by the preferential kappa receptor antagonist Mr-2266 than by naloxone, neither of which interfered with the GH-stimulating effect of clonidine. Bremazocine, however, did not alter the activation of GH secretion by exogenous growth hormone releasing factor. Thus, the inhibiting effect of bremazocine and probably U-50,488 seems to be derived from stimulation of the kappa receptors which in turn activates a GH release inhibiting mechanism of unknown identify which, however, does not involve release of somatostatin. Both kappa agonists also inhibited the effect of morphine, but in this case U-50,488 was approximately hundred times less effective than bremazocine. Since bremazocine and U-50,488 are antagonists of the delta receptors, which seem to mediate the GH-releasing effect to morphine, their inhibiting effect in this instance may be related to this property rather than to an action on the kappa receptors. Bremazocine, but not U-50,488, was also highly effective in inhibiting stimulation of PRL secretion by morphine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001567 TI - Peripheral nerve involvement in children with chronic cholestasis and vitamin E deficiency. A clinical, electrophysiological and morphological study. AB - Seven children with early onset cholestasis who developed signs of peripheral neuropathy were investigated before and after one and a half to three years of treatment by vitamin E. This neuropathy appeared to be due to neuronoaxonal degeneration, but unusual Schwann cell inclusions were also observed. Although the treatment stabilized or improved the condition of all patients, no striking changes were noted neither in the EMG nor in the second nerve biopsy of a patient treated for three years. As the pathological process concerned it is probably both a developmental disorder and a degenerative phenomenon, substitutive vitamin E treatment should be proposed very early in life. PMID- 3001568 TI - A case of childhood multiple sclerosis with peripheral neuropathy. AB - A twelve-year-old girl with multiple sclerosis and peripheral neuropathy is reported. When nine years old, she was diagnosed as having Devic disease (optic atrophy and transverse myelitis). During the three years after onset of her illness, she suffered from three relapses and remissions of her multiple sclerosis. On the third occasion, neurological examination revealed signs of cerebellar dysfunction including ataxic gait, nystagmus and dysmetria, and absence of all tendon reflexes with muscle weakness especially on the left side. Markedly slowed conduction velocity in her ulnar nerve especially on the left and elevated CSF protein were noted. Biopsied sural nerve showed decreased density of myelinated fibers and a selective loss of large diameter fibers. Electron microscopy disclosed onion-bulb formation, myelin debris within Schwann cell cytoplasm and demyelinated axons. These findings showed demyelination and remyelination of the peripheral nervous system in this patient with multiple sclerosis. We discuss the relation of multiple sclerosis and peripheral neuropathy. PMID- 3001569 TI - The opioid receptors in the hamster vas deferens are of the delta-type. AB - The motor responses of the isolated vas deferens of the hamster were unaffected by opioid-receptor agonists which are selective for the mu- or kappa-receptor, while agonists which show degrees of selectivity for the delta-opioid receptor caused dose-related inhibition of the stimulation-evoked contractions. The agonist action of the enkephalins and their congeners was only apparent when various inhibitors of tissue peptidases were present. Responses to opioid agonists were antagonised in a competitive manner by naloxone and by the selective delta-receptor antagonist, ICI 174864. It is noteworthy that the benzomorphans bremazocine, ethylketocyclazocine and Mr 2034, which are agonists at kappa-receptors in other tissues, are antagonists at the delta-receptor of the hamster vas deferens. Thus, the vas deferens of the hamster contains opioid receptors only of the delta-type and may therefore provide a simple and specific test for the assay of activity at the delta-opioid receptor. PMID- 3001570 TI - Adenosine A1 receptors mediate the inhibitory effects of exogenous adenosine in the rat olfactory cortex slice. AB - A study has been undertaken to identify the category of receptors mediating the inhibitory effects of adenosine on evoked activity in slices of olfactory cortex in the rat. The approach has been to measure the relative potencies of adenosine and a range of structural analogues [2-chloroadenosine, 2' deoxyadenosine, cyclohexyladenosine, (-)-5'N-ethyl-carboxamide adenosine and N6(L-2 phenylisopropyl)adenosine] required to: inhibit excitatory transmission at the lateral olfactory tract-pyramidal cell synapse; inhibit the specific binding of [3H]cyclohexyladenosine to membrane preparations and evoke formation of cyclic AMP. In contrast to the relative concentrations of the analogues necessary to increase levels of cyclic AMP, those required to inhibit synaptic transmission were characteristic of a selectivity for adenosine A1 receptors. The presence of adenosine A1 receptors has been demonstrated directly by characterizing the binding of [3H]cyclohexyladenosine to membranes prepared from slices of olfactory cortex. It is concluded that inhibition of transmission at the lateral olfactory tract-pyramical cell synapse by adenosine is mediated by receptors of the A1 category. PMID- 3001572 TI - Facilitation and inhibition of nicotinic transmission by eserine in the sympathetic ganglia of the rabbit. AB - The effects of eserine on neurons and on ganglionic transmission of the isolated superior cervical ganglia of the rabbit were investigated by means of intracellular recording techniques. At the concentration of 10 microM or less, eserine reversibly increased the amplitude and duration of the fast excitatory postsynaptic potential (f-epsp) induced by preganglionic nerve stimulation and of the membrane depolarization evoked by iontophoretically-applied acetylcholine (ACh), but not carbachol. At the concentration of 50 microM or more, eserine consistently and reversibly depressed the fast excitatory postsynaptic potential as well as the depolarization induced by iontophoretic application of either ACh or carbachol. Furthermore, depolarization by ACh evoked in a low Ca/high Mg solution, which blocked the liberation of transmitter was similarly reduced by eserine in greater concentrations. The passive membrane properties of the sympathetic neurons were not significantly altered by eserine in the majority of neurons studied. The results indicate that the facilitatory action of eserine on ganglionic transmission may be explained by its anticholinesterase activity, whereas eserine-induced block of transmission appears to be related to a direct interaction between the compound and the postsynaptic ACh receptor-channel complex. PMID- 3001571 TI - 2-Amino-6-trifluoromethoxy benzothiazole, a possible antagonist of excitatory amino acid neurotransmission--II. Biochemical properties. AB - Two models have been chosen to study the effect of 2-amino-6-trifluoromethoxy benzothiazole (PK 26124) on excitatory amino acid neurotransmission: the pool of cyclic guanosine monophosphate (cGMP) in the cerebellum and the release of acetylcholine in the striatum and olfactory tubercles. The release of acetylcholine induced by N-methyl-DL-aspartate in the striatum and olfactory tubercles was antagonized by PK 26124 which was less potent on the release of acetylcholine induced electrically. The increase in levels of cGMP in the cerebellum induced by excitatory amino acids such as glutamate and quisqualate was antagonized by PK 26124, but the drug was inactive against N-methyl-DL aspartate, L-aspartate, kainate and cysteine sulphinate. In vivo it antagonized the increases of cGMP in the cerebellum elicited by all these excitatory compounds. All these results are compatible with a possible antagonism by PK 26124 of the excitatory amino acid neurotransmission and may explain its anticonvulsant properties. PMID- 3001573 TI - Evidence for down-regulation of 3H-nitrendipine recognition sites in mouse brain after long-term treatment with nifedipine or verapamil. AB - Mice were fed powdered food which contained nifedipine, verapamil or diltiazem for 28 days. Long lasting treatment with nifedipine (0.28 mg/g b.w./day) or verapamil (0.27 mg/g b.w./day), but not with diltiazem (0.38 mg/g b.w./day) reduced the number of 3H-nitrendipine recognition sites in membranes prepared from cerebral cortex, caudate nucleus, and hippocampus. In addition, the veratridine-elicited stimulation of 45Ca-uptake in slices of the same brain areas was decreased in mice which were fed nifedipine or verapamil for 28 days. PMID- 3001574 TI - Activation of acetylcholinesterase by vanadate. AB - Vanadate activated acetylcholinesterase in rat ventricular strips. This effect of probably due to the action of vanadate (V) form rather than to vanadyl (IV). Vanadate also activated purified acetylcholinesterase from the electric eel and also erythrocytes, suggesting a direct effect upon this enzyme system. It is possible that the increase in the activity of acetylcholinesterase produced by vanadate may contribute, at least in part, to the positive inotropic effect of vanadate. PMID- 3001575 TI - Preparation of rat brain membranes highly enriched with opiate kappa binding sites using site-directed acylating agents: optimization of assay conditions. AB - The goal of this study was to determine optimal conditions with which to measure opiate kappa binding sites in rat brain. Membranes were pretreated with mu selective (BIT) and delta-selective (FIT) site-directed acylating agents (Rice et al., Science 220, 314-316), and the binding of [3H]bremazocine to the residual binding sites was defined as the kappa binding site. The binding of [3H]bremazocine to BIT/FIT-treated membranes was greatly increased by conducting the assay at 0 degrees C in the presence of 0.4 M NaCl. Using this 0 degrees C/NaCl assay condition, the binding of [3H]bremazocine was best described by a one-site binding model with a KD of 0.45 nM and a Bmax of 378 fmol/mg protein. Autoradiographic studies demonstrated that, using this assay condition, [3H]bremazocine densely labeled the deep layers of guinea pig cortex, an area known to be enriched with kappa binding sites. These and additional data suggest that the binding of [3H]bremazocine to the kappa binding site of rat brain is optimally assayed at 0 degrees C in the presence of 0.4 M NaCl using BIT/FIT treated membranes and that rat brain is endowed with a high level of kappa binding sites. PMID- 3001576 TI - Characterisation of the delta-opioid receptor on the hamster vas deferens. AB - It has recently been reported that the hamster vas deferens contains only delta opioid receptors. We have demonstrated that three delta-receptor agonists [D Ala2, D-Leu5]enkephalin (DADLE), [D-Pen2, D-Pen5]enkephalin (DPDPE) and [D-Thr2, Leu5, Thr6] enkephalin (DTLET) appear to mediate their effects via the delta receptor since they are readily reversed by the selective delta-receptor antagonist ICI 174864. In addition, a number of classical mu and k receptor compounds were devoid of activity in this preparation. However it was observed that some compounds such as etorphine and MR 2034 reported to possess delta receptor affinity in other assay systems were weak or inactive on the hamster vas deferens. PMID- 3001578 TI - Quantitative anatomy of the thoracolumbar epidural space. AB - Quantitative measurements of the epidural space between T-7 and L-4 were made in the sagittal and coronal planes utilizing x-ray films made after the injection of iodized oil into the epidural space in the low thoracic and upper lumbar areas. These data reveal a 1-mm ventral epidural space and a 2-mm lateral epidural space, with a sawtooth shape to the dorsal epidural space measuring between 1.1 and 2.9 mm at the rostral lamina and between 3.8 and 6.5 mm at the caudal lamina. Additionally, five patients with chronic pain were studied by computed tomography of T-8 to T-12, with confirmation of the sawtooth shape of the dorsal epidural space. Computed tomography showed the measurements of the epidural space at the rostral lamina to vary between 1.3 and 1.6 mm and those at the caudal lamina/interlaminar space to range from 6.9 to 9.1 mm. PMID- 3001577 TI - Naloxone reverses the effects of enalapril and enalaprilic acid on the pressor responses to afferent vagal stimulation. AB - The effects of intracisternal injection of enalapril (MK 421) or its bioactive form enalaprilic acid (MK 422) (0.075 mg/kg) on the pressor responses elicited by afferent stimulation of the vagus were investigated in the urethane-anaesthetized dog. The angiotensin converting enzyme inhibitors (ACEI)-induced decrease in the systolo-diastolic pressor responses to vagal stimulation was reversed by naloxone (0.1 mg/kg iv). These data support evidence for an additional central site of action of These data support evidence for an additional central site of action of ACEI and suggest the involvement of central opioid mechanisms in the hypotensive properties of ACEI. PMID- 3001579 TI - Evidence for excitatory actions of histamine on supraoptic neurons in vitro: mediation by an H1-type receptor. AB - The effects of histamine on the firing of supraoptic neurosecretory neurons in the rat were examined in vitro using acutely prepared, hypothalamo neurohypophysial explants perifused with an artificial cerebrospinal fluid. Extracellular action potentials meeting the criteria of antidromic invasion from neurohypophysial stalk stimulation were recorded from 135 neurons in the tuberal portion of the supraoptic nucleus, which lies superficially along the tuber cinereum and consists of mostly vasopressin-containing neurons. Units could be classified as slow/silent (76.3%), phasic (21.5%) or continuous (2.2%) on the basis of their spontaneous activity. Histamine applied briefly to the perifusate excited approximately one-third of the slow silent neurons and approximately two thirds of the phasic neurons, with a wide range (10(-3)-10(-9)) in the effective concentration across neurons. The H1-receptor agonists 2-pyridylethylamine and 2 thiazolylethylamine mimicked these excitations in 10 of 12 and 3 of 6 neurons tested, respectively. The H2-receptor agonists dimaprit (4 neurons) and impromidine (5 neurons) failed to excite any of the tested neurons previously excited by histamine. The H1-receptor antagonist promethazine antagonized histamine's excitatory effect in 8 of 9 cells, while the H2-receptor antagonist cimetidine had little effect on the 9 cells tested. Histamine also modified bursts of activity induced in some slow/silent neurons by antidromic stimulation without having an observable effect in the absence of an antidromic burst. In 10 of 18 neurons histamine produced an elongation of burst duration and a modest increase in intraburst firing rate when applied during an antidromically evoked burst. In an additional 5 of 17 neurons, which had neither previously responded to histamine nor shown an antidromically-evoked burst, the pairing of histamine application and antidromic shocks resulted in an antidromically evoked burst. The effects of histamine on evoked bursts also appeared to be mediated by an H1 receptor. Histamine's excitation of supraoptic neurons is thus dependent on the electrical activity expressed by the neuron at the time of testing. Conductances activated by depolarization of the neuron may be modified by histamine or this compound may alter the threshold for burst generation. Considered with data showing H1-receptor localization and histamine-immunoreactive fibers within the supraoptic nucleus, the present results, as well as those showing the potency of centrally applied histamine in releasing vasopressin, suggest histamine may act physiologically by altering the electrical activity of vasopressin-secreting neurons. PMID- 3001580 TI - Behavioral analysis of opiate-mediated inhibition in the early chick embryo. AB - Morphine (opiate agonist) produced a dose-dependent decrease in the spontaneous motility of 5- and 9-day chick embryos. Naloxone (opiate antagonist) appeared to reverse competitively the inhibition of motility caused by morphine. The effects of morphine on spontaneous motility in 5-day embryos were also reversed stereospecifically by the opiate antagonist pairs WIN 44441-3/WIN 44441-2 and levallorphan/dextrallorphan. Levorphanol (opiate agonist) also produced a dose dependent decrease in the motility of 5-day embryos while its inactive (+) isomer, dextrophan, was not effective. Etorphine (opiate agonist) was more than 1000-fold more effective than morphine in inhibiting the motility of 5-day embryos. The effectiveness of several opiate agonists and antagonists on the spontaneous motility of 5-day embryos was similar to their effectiveness in radioligand-binding studies on isolated membrane receptors from either adult mammalian brain or ileum. Levorphanol was more effective than dextrophan and etorphine was substantially more effective than morphine in decreasing the spontaneous motility of 4-day embryos. WIN 44441-3 was more effective than WIN 44441-2 in reversing the inhibition of motility in 4-day embryos caused by morphine. Morphine inhibited spontaneous hind-limb motility in both thoracic spinal and sham-operated 7-day embryos; the inhibition of motility caused by morphine was reversed by WIN 44441-3 in both thoracic spinal and sham-operated 7 day embryos. [Leu5]enkephalin-like immunoreactivity in the lumbar spinal cord was concentrated in the superficial laminae of the dorsal horn and along the midline rostral to the central canal. A lesser concentration of immunoreactive processes occurred in the medial and lateral motor columns where labelled varicosities appeared to contact motoneurons. Opiate receptors appear to be present at least as early as day 5 (and perhaps as early as day 4) in the chick embryo. Opiate receptors appear to be present in the lumbar spinal cord of the chick embryo at least as early as day 7. The structural requirements for ligand binding to opiate receptors in the 5-day chick embryo are similar to the requirements for ligand binding to opiate receptors in the adult. PMID- 3001582 TI - Presynaptic autoinhibition during rest and sodium-pump inhibition in isolated rat portal vein preparation. AB - In the presence of cocaine and corticosterone low-frequency (2 Hz) nerve stimulation evoked release of [3H]noradrenaline measured from isolated rat portal vein preparation. In normal Krebs solution exogenously applied l-noradrenaline (3 X 10(-8)-10(-6) M) significantly reduced the nerve-evoked [3H]noradrenaline release. The IC50 value of L-noradrenaline proved to be 1.8 X 10(-7) M. Yohimbine (3 X 10(-7) M) maximally blocked the alpha 2-adrenoceptors and enhanced nerve evoked [3H]noradrenaline release. In the presence of 5.9 mM external K+, ouabain up to 10(-4) M did not affect either the resting or the stimulation-evoked release of radioactivity from tissues. In the absence of external K+ both the resting and the nerve-evoked release of [3H]noradrenaline increased markedly. When K+ was readmitted to preparations which had been kept in K+-free solution both the resting and the stimulation-evoked [3H]noradrenaline release were greatly reduced temporarily. In K+-free solution L-noradrenaline (10(-6) M) and yohimbine (3 X 10(-7) M) failed to significantly alter the nerve-evoked release. However, 3 X 10(-6) M yohimbine in K+-free solution significantly increased the stimulation-evoked release of [3H]noradrenaline. It is concluded that presynaptic alpha 2-adrenoceptor-mediated "negative feed-back" is present in rat portal vein preparations which can be inhibited by the preferential alpha 2-adrenoceptor blocker, yohimbine. However, if the Na+-pump is inhibited (which by itself enhanced the transmitter release), presynaptic autoinhibition is more pronounced, since a high concentration of yohimbine is required to block it. PMID- 3001581 TI - mu-Opioid receptors and alpha 2-adrenoceptors coexist on myenteric but not on submucous neurones. AB - Intracellular recordings were made from neurones in the myenteric and submucous plexuses of the guinea-pig ileum. All myenteric neurones that were hyperpolarized by [Met5]enkephalin (or normorphine) were also hyperpolarized by noradrenaline (or clonidine); neurones unaffected by opioids were unaffected by noradrenaline. The hyperpolarizations resulted from an increase in potassium conductance of the membrane and were blocked by the respective antagonists naloxone and idazoxan. Neurones of the submucous plexus were hyperpolarized by noradrenaline but not by normorphine. The results suggest that myenteric neurones possesses both mu-opioid receptors and alpha 2-adrenoceptors whereas submucous neurones have alpha 2 adrenoceptors but not mu-opioid receptors. PMID- 3001583 TI - Frequency-dependent action potential prolongation in Aplysia pleural sensory neurones. AB - The effects of repetitive activity on action-potential shape in Aplysia californica pleural sensory cells are described. Action potentials were evoked by intracellular current injection at frequencies between 7.41 and 0.2 Hz. In contrast to other molluscan neurons having brief action potentials, it was found that at these firing rates the normally brief action potential develops a prominent shoulder or plateau during the repolarization phase. Higher stimulus rates broaden the action potential more rapidly and to a greater extent than lower stimulus rates. Inactivation is slow relative to activation; effects of 3-s 6-Hz trains are detectable after 1 min rest. The amplitude of the plateau voltage reaches a maximum of 50-70 mV at the highest stimulus rates tested. Frequency dependent increases in action-potential duration measured at half-amplitude normally range between 6 and 15 ms. Cadmium, at concentrations between 0.05 and 0.5 mM, antagonizes frequency-dependent broadening. The increases in duration induced by repetitive activity are more sensitive to cadmium than are the increases in plateau amplitude. Tetraethylammonium, at concentrations between 0.5 and 10 mM, slightly increases the duration and amplitude of single action potentials. During repetitive activity at high stimulus rates the maximum duration and rate of broadening are both increased but the amplitude of the plateau potential is not affected by these tetraethylammonium concentrations. Above 10 mM, tetraethylammonium greatly increases the duration and amplitude of single action potentials as well as the rates of action-potential duration and amplitude increase during repetitive activity. These high tetraethylammonium concentrations also cause the normally smoothly increasing duration and amplitude to reach a maximum value early in a train and then decline slowly during the remainder of the train. The consequences of frequency-dependent spike broadening in these neurons have not yet been investigated but it is clear from these data that repetitive activity in these cells will augment calcium entry and that this increased calcium entry has a complex but predictable dependence on the duration of, and firing rate within, an afferent volley. Because these cells are involved in important adaptive behaviour it is inferred that these behaviours will be complexly affected by the intensity and duration of the stimulation of the receptive fields of the pleural sensory neurons. PMID- 3001584 TI - Different effects of 4-aminopyridine on sensory and motor fibers: pathogenesis of paresthesias. AB - Mammalian motor and sensory fibers respond differently to the potassium channel blocking agent, 4-aminopyridine (4-AP). The action potentials of the motor fibers increase in duration after 4-AP, while the sensory fibers respond with bursts of action potentials after a single stimulus. These differences may account for the paresthesias reported by patients with multiple sclerosis following treatment with 4-AP. PMID- 3001585 TI - Progressive dementia and epilepsy in a young adult: unusual intraneuronal inclusions. AB - A 30-year-old woman had a syndrome of dementia, dystonia, myoclonus, and intention tremor. Brain biopsy showed PAS-positive inclusions of Lafora's disease, but electronmicroscopy demonstrated that the inclusions differed from previously reported Lafora bodies. This may represent a previously undescribed disorder. PMID- 3001586 TI - [AIDS and blood transfusions. International symposium held in Turin, 13-15 June 1985]. PMID- 3001587 TI - [Value and limitations of scintigraphy of the hand in rheumatoid arthritis]. AB - 99mTc-polyphosphate joint imaging of the hand has been performed in 18 patients, with evidence of inflammatory joint disease, but without any significant radiographic lesions, which might be related to rheumatoid arthritis. The hand scans were compared to clinical and radiographic data. An year after, the same subjects were re-examined, with both the radionuclide imaging and radiography. Scintigraphy has been shown to be significantly more sensitive for detecting inflammatory joint disease than x-ray, especially in the early stage of rheumatoid arthritis. Although radionuclide imaging is non specific (activity is increased also in osteoarthritis, trauma, metabolic bone disease, infarction, etc.). Radiography is highly specific but relatively non sensitive. PMID- 3001588 TI - [Infiltrating comedocarcinoma of the breast]. PMID- 3001589 TI - [Arrhenoblastoma of the ovary. Clinico-pathologic synthesis and description of a case]. PMID- 3001590 TI - Autoradiographic localization of binding sites for the gamma-aminobutyric acid analogues 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP)isoguvacine and baclofen on cultured neurons of rat cerebellum and spinal cord. AB - By means of light microscopic autoradiography, binding sites for the gamma aminobutyric acid (GABA) analogues, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3 ol ([7-3H]THIP), [3H]isoguvacine and [3H]baclofen were found on many cultured cerebellar and spinal neurons of fetal and newborn rats. The number of neurons labelled by [3H]THIP was considerably smaller than that by the other two radioligands. Unlabelled THIP, GABA and bicuculline methiodide inhibited binding of [3H]THIP and [3H]isoguvacine, whereas binding of [3H]baclofen was inhibited by unlabelled GABA and baclofen but not by bicuculline methiodide. Our results indicate that cultured cerebellar and spinal neurons possess both GABAA and GABAB binding sites and that [3H]THIP possibly binds to a subclass of GABAA receptors. PMID- 3001592 TI - Striatal and septal influence on hippocampal theta and spikes in the cat. AB - The experiments studied the modulation exerted by the septum and the caudate nucleus on hippocampal activity in the cat. Injections (i.v.) of sodium penicillin were performed in order to obtain a steady interictal epileptic activity. Hippocampal slow rhythmic activity showed a marked decrease either in duration or in frequency following penicillin activation. Both septal and caudate electrical stimulation inhibited spike frequency through a theta eliciting mechanism. Caudate stimulation failed to determine any sort of effect after medial septum lesions. The importance of the septum as modulation station between basal ganglia and hippocampus is emphasized. PMID- 3001591 TI - Reconstitution of the purified gamma-aminobutyric acid-benzodiazepine receptor complex from bovine cerebral cortex into phospholipid vesicles. AB - The purified gamma-aminobutyric acid-benzodiazepine receptor complex from bovine cerebral cortex has been reconstituted into phospholipid vesicles by a cholate dialysis procedure. The reconstituted receptor bound [3H]flunitrazepam at a single class of sites with dissociation constant Kd = 2.9 +/- 0.3 nM, an increase in affinity to the membrane level from the 4-fold weakening found in detergent solution. It also bound [3H]muscimol with a Kd for the high-affinity sites of approximately 50 nM. [35S]tert.-Butyl-bicyclophosphorothionate, for which there is evidence in membranes for binding to a channel gating site on this receptor, showed similar binding to the reconstituted receptor. PMID- 3001593 TI - Kindling of dopaminergic pathways modifies the effect of substantia nigra lesions on rotational behaviour. AB - Recent studies in rats have implicated a crossed nigrostriatal (NS) pathway in the behavioural recovery that may follow unilateral lesions of the substantia nigra pars compacta (SNc). In the present study, cerebral dominance was determined from the rat's preferred direction of amphetamine-induced rotation. The ventral mesencephalic area giving rise to the crossed nigrostriatal pathway was then kindled either in the dominant hemisphere (in half of the rats), or in the non-dominant hemisphere (in the rest). All rats were then given 6 hydroxydopamine lesions in the lateral portion of the SNc on the dominant side. In control groups, the kindling procedure or electrode implantation was omitted. Neither kindling alone nor the lesion alone was found to alter the direction of amphetamine-induced rotation. In combination, however, kindling in the dominant hemisphere followed by lesioning of the lateral SNc in the dominant hemisphere caused a significant shift in the direction of rotation. This finding can be interpreted in terms of a long-term potentiation of the crossed nigrostriatal pathway after kindling. It is suggested that partial deafferentation of the neostriatum in the dominant hemisphere, combined with enhanced cross-innervation of the contralateral system, led to a shift of cerebral dominance to the intact side. PMID- 3001594 TI - Numerical estimation of gamma-aminobutyric acid (GABA)-containing neurons in three thalamic nuclei of the cat: direct GABA immunocytochemistry. AB - The percentage of neurons that are immunoreactive for the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) was determined within: (1) the lateral geniculate nucleus (LGN), (2) the ventrobasal complex (VB) and (3) the antero-ventral nucleus (AV) of the thalamus in the cat. An antiserum to GABA was used to stain GABA-containing perikarya in 1.0 micrometer thick Araldite-embedded sections. Immunostained somata in all three nuclei were invariably smaller than the immuno-negative nerve cells. 27% of all neurons in the LGN, 33% in the VB and 25% in the AV were immunoreactive for GABA. PMID- 3001595 TI - Immunohistochemical localization of GABAergic cells in the developing human dorsal lateral geniculate nucleus. AB - GABAergic neurons were demonstrated in the dorsal lateral geniculate nucleus (dLGN) of human foetuses of 21 weeks gestation using unlabelled antibodies against gamma-aminobutyric acid (GABA). These inhibitory neurons are thus cytochemically identifiable prior to appearance of lamination, at a period when there is reportedly segregation of retinal afferents from both eyes and formation of morphologically defined synapses. Their presence in the prenatal period indicates that the morphological substrate responsible, in part, for the orientation sensitivity of the dLGN neurons exists before birth in man. PMID- 3001596 TI - Opioid activity of pro-enkephalin-derived peptides in mouse vas deferens and guinea pig ileum. AB - The inhibitory activity of several pro-enkephalin A-derived opioid peptides, containing the sequence of Met-enkephalin, was evaluated in two isolated organ preparations sensitive to opioids, the guinea pig ileum (GPI) and the mouse vas deferens (MVD). All peptides tested were able to inhibit the electrically stimulated contraction in both tissues by interacting with specific receptors sensitive to the opioid antagonist naloxone. The shorter peptides in this family (Met-enkephalin, Met5-enkephalin-Arg6-Phe7, Met5-enkephalin-Arg6-Gly7-Leu8) displayed their highest potency in MVD. In contrast, the larger bovine adrenal medulla (BAM) peptides (BAM 12P, BAM 22P and peptide E) were more potent in the GPI system compared with the MVD assay. Therefore, the larger peptides seem to bind better to the mu receptor, whereas the shorter ones prefer the delta type. PMID- 3001597 TI - Differences in the properties of bovine brain benzodiazepine receptors in the cerebellum and hippocampus revealed after reduction of disulphide bonds. AB - Treatment of bovine brain cell membranes with dithiothreitol decreases the affinity without changing binding capacity for [3H]flunitrazepam in the cerebellum, hippocampus and frontal cortex. Specific binding of beta [3H]carboline-3-carboxylic acid ethyl ester in the cerebellum and frontal cortex was influenced by dithiothreitol in a similar manner. However, in the hippocampus the binding of this inverse agonist was modified by dithiothreitol in another way: a significant (25%) decrease of binding capacity, or (and) appearance of heterogeneity of binding sites was detected. Sensitivity of benzodiazepine receptors to gamma-aminobutyric acid (GABA) stimulation was not changed by dithiothreitol. PMID- 3001598 TI - Noradrenaline mediates slow excitatory synaptic potentials in rat dorsal raphe neurons in vitro. AB - Repetitive focal stimulation to the slice surface within the region of the dorsal raphe (DR) nucleus of rat brain elicited a slow excitatory synaptic potential (slow EPSP), which followed a slow inhibitory synaptic potential (slow IPSP) in a majority of the DR neurons. The slow EPSPs were associated with either an increase of a decrease in membrane resistance. Noradrenaline (NA) application caused a membrane depolarization in most of the DR neurons. The NA-induced depolarization was also accompanied by either an increase or a decrease in membrane resistance. Both the slow EPSP and NA-induced depolarization were inhibited by phentolamine and prazosin but not by yohimbine and propranolol. The result suggests that slow EPSPs in rat DR neurons are mediated by NA interacting with an alpha 1-adrenoceptor. PMID- 3001600 TI - Retinoic acid regulates 1,25-dihydroxyvitamin D3-binding sites in rat osteosarcoma cells. PMID- 3001599 TI - Dysfunction of central angiotensinergic aminopeptidase activity in spontaneously hypertensive rats. AB - Alert spontaneously hypertensive (SH) rats, prepared with indwelling carotid artery catheters, demonstrated heightened and prolonged blood pressure (BP) responses to intracerebroventricular (i.c.v.) injections of 10 and 100 pmol angiotensin II and III (AII and AIII) as compared with Wistar-Kyoto (WKY) and Sprague-Dawley normotensive animals. Pretreatment with the aminopeptidase B inhibitor bestatin (10 nmol, i.c.v.) potentiated and prolonged the heightened pressor response to AIII (100 pmol, i.c.v.) in SH rats. These results suggest that dysfunction of angiotensin peptidase activity may be contributing to the progressive and sustained elevations in blood pressure noted to occur in the SH rat model of human essential hypertension. PMID- 3001602 TI - Functional role of phosphodiesterase in gecko photoreceptors. PMID- 3001603 TI - A possible amacrine cell substrate for the detection of stimulus motion. PMID- 3001601 TI - Dopamine and thermoregulation: an evaluation with special reference to dopaminergic pathways. AB - The complex role of dopamine (DA) in the diencephalic mechanisms involved in the control of body temperature is reviewed and evaluated. In the context of the monoamine theory of thermoregulation, catecholaminergic synapses in the anterior hypothalamic pre-optic area, are proposed mediate the pathways in the brain-stem which subserve heat dissipation. Within this theoretical framework, hypothalamic DA is considered to underlie a portion of the functional component of the heat loss system. This deduction is based on pharmacological studies in which both the catecholamine and receptor antagonists have been infused directly into the hypothalamus. In view of the action of DA applied to the substantia nigra and other subcortical structures, the unique anatomical circuitry of the central dopaminergic projections has also been analyzed in terms of specific connections within critical morphological regions related to thermal functions. In particular, the nigro-striatal pathway could be involved in the mediation of one or more of the different aspects of the thermoregulatory system integrating both autonomic and behavioral responses. Finally, an anatomical schema which portrays the suggested mechanisms of DA activity is presented. PMID- 3001604 TI - Glomus tumor of the clitoris. PMID- 3001605 TI - Enalapril maleate and atenolol combined with hydrochlorothiazide in moderate to severe essential hypertension. AB - This open randomised parallel trial compared the antihypertensive efficacy of enalapril and atenolol given alone once a day or with hydrochlorothiazide in 20 patients with moderate to severe hypertension. Active treatment was over a 26 week period, consisting of an initial titration phase followed by a fixed dose phase. Both treatment regimes effectively lowered systolic and diastolic blood pressures. All patients on enalapril reached normotension (supine diastolic blood pressure less than or equal to 90 mmHg) compared with 78% on atenolol. Pulse rate was not appreciably changed by enalapril, but was significantly reduced by atenolol. No serious adverse reactions or significant changes in laboratory values were noted in either group. The commonest adverse reaction with enalapril was dizziness which occurred in two cases and resolved on dosage reduction. Enalapril with hydrochlorothiazide given once daily may provide a useful combination in the treatment of moderate to severe hypertension. PMID- 3001606 TI - Assessment of HTLV III screening tests in Christchurch. AB - The Christchurch Hospital blood bank has assessed commercial kits designed to test blood donations for the presence of antibody to the retrovirus responsible for acquired immune deficiency syndrome (AIDS). Twenty of 1002 blood donations have been found to be repeatably, positive by one or more of these commercial kits. None was positive by the Western blot technique. Certain patient groups were found to have an elevated number of false positive reactions. Extreme caution is advised when interpreting screening test kit results from donor populations at low risk of AIDS. PMID- 3001608 TI - [Role of self-help groups in the network of health care--exemplified by cancer after-care]. PMID- 3001607 TI - NIOSH blames the workplace for too many of society's problems. PMID- 3001609 TI - [Theses on the role of public health offices in dealing with AIDS]. PMID- 3001611 TI - Cholinergic muscarinic receptor in the nasal mucosa of guinea pigs with bronchial asthma with and without hypersensitive nasal symptoms. AB - Using [3H]-quinuclidinyl benzilate (QNB) as a radioligand, receptor binding assay was performed to evaluate the changes of the muscarinic receptor in the nasal mucosa of guinea pigs sensitized with toluene diisocyanate (TDI), which showed hypersensitive airway symptoms both in the lower airway and in the nose. The same study was performed in guinea pigs sensitized with bacterial crystalline alpha amylase (BCA) which showed typical symptoms of bronchial asthma but without hypersensitive nasal symptoms. In the nasal mucosa of guinea pigs sensitized with TDI, an increased density of the muscarinic receptor was confirmed with no change in affinity of the receptor. However, in the nasal mucosa of guinea pigs sensitized with BCA, no changes were observed either in the density of the muscarinic receptor of the nasal mucosa or in its affinity. The increased density of the muscarinic receptor observed in subjects with nasal allergy, which was reported earlier, is not specifically related to IgE-mediated nasal allergy and is not induced by systemic sensitization itself but is assumed to be induced by some pathophysiological changes in the nasal mucosa including antigen-antibody reaction with development of hypersensitive nasal symptoms. PMID- 3001612 TI - Quantitative measurement of smooth pursuit using the continuously changing sinusoidal wave in neurological patients. AB - In a previous paper [Ohashi, Watanabe, Kobayashi, Mizukoshi: ORL 47: 49-56] we developed a new sinusoidal wave with 20 continuously changing sinusoidal waves from 0.1 to 2.0 Hz and reported that in normal subjects the limit of smooth pursuit was 1.2 Hz, retinal error velocity (REV) was the most reliable parameter as input of smooth pursuit and amplitude gain was the most reliable parameter as output. In the present paper, we analyzed smooth pursuit system by the same method and discussed the relation between input and output of smooth pursuit in patients with neurological disorders. Our results indicated that the relationship between input and output of the smooth pursuit was particularly abnormal in patients with cerebellar disorders. PMID- 3001610 TI - Oral acyclovir in the therapy of acute herpes zoster ophthalmicus. An interim report. AB - A prospective, randomized, double-masked, placebo-controlled clinical trial was conducted to study the effects of oral acyclovir on 55 patients with acute herpes zoster ophthalmicus. Treatment with oral acyclovir resulted in more prompt resolution of signs and symptoms, particularly in patients treated within 72 hours after onset of skin rash (P less than 0.05), and shortened the duration of viral shedding (P = 0.02). Vesicular skin lesions involving other dermatomes (microdissemination) occurred in five (19%) placebo-treated patients but in no acyclovir-treated patients (P = 0.03). Interim analysis of this longitudinal study suggests that the incidence and severity of secondary ocular inflammatory disease was reduced by acyclovir. Prolonged observation of these patients is ongoing to determine if oral acyclovir reduces post-herpes zoster neuralgia or the late ocular complications of ophthalmic zoster. PMID- 3001613 TI - [Theoretical and clinical significance of hormone receptors]. PMID- 3001614 TI - [Questions about retroperitoneal lymphadenectomy in the management of childhood testicular germ cell tumors]. PMID- 3001615 TI - [The beta adrenergic receptor function of lymphocytes in bronchial asthma]. PMID- 3001616 TI - Sex steroid metabolism and follicular development in the ovary. PMID- 3001617 TI - Stress and population regulation in small mammals. PMID- 3001618 TI - Recombinant DNA technology in prenatal diagnosis. PMID- 3001619 TI - [Vitamin D deficiency rickets: single-dose therapy versus continuous therapy]. AB - This is a comparative study of massive single dose treatment (400 000 I. U. vitamin D3, divided in 2 X 200 000 I. U. on day 1 and day 3, orally) versus continuous treatment (9600 I. U. vitamin D3-drops for 18 days) of vitamin D deficiency rickets (VDR). To estimate the effectiveness of these two kinds of treatment the serum concentrations of calcium (Ca), phosphorus (P), alkaline phosphatase (aP), calcitriol (25-OH-CC), parathyroid hormone (PTH) and calcitonin (Ct) before and during treatment (on day 3, 7, 14, 21) were studied in 10 patients, 5 in each group, with VDR. There was no difference in the course of serum concentrations of Ca, P and aP in these two treatment-groups. The elevated PTH returned more quickly to normal range in the single dose study group. Ct serum concentrations showed too different courses in the studied patients, therefore no comparison could be made. 25-OH-CC showed a very quick increase in the single dose-group and a slower one in the continuous treatment group the climax in both groups reached a value far over the tentative range. According to these results an unequivocal better effect of one of these two compared VDR treatments could not be seen, what means that both treatments can be recommended. PMID- 3001620 TI - [Steroidogenic action of 2 analogs of ovine LH on cells isolated from ovine corpus luteum]. AB - Lysine residues appear to play an important role in the biological activity of luteinizing hormone or lutropin (LH). Some derivatives obtained by chemical modification such as N-methylated LH exhibit the same hormonal activity than LH in the different steps of the mechanism of steroidogenesis. Some others, on the contrary, preserve the hormonal activity only at some steps. In this work was investigated the action of ovine LH and some derivatives on isolated cells prepared from ovine corpora lutea. Guanidinated LH (which is able to bind to LH receptors in Leydig cells but whose steroidogenic potency is very low) exhibits no binding or steroidogenic activity in the female (sheep or rat). As a consequence guanidyl LH can act as an inhibitor of LH action in the male (Leydig cells). PMID- 3001621 TI - Fine-needle aspiration biopsy cytology of hepatic tumors in adults. PMID- 3001622 TI - Human papillomavirus (HPV) infections of the female genital tract and their associations with intraepithelial neoplasia and squamous cell carcinoma. AB - Human papillomaviruses (HPV) consist of a heterogenic group of viruses (32 different HPV types currently recognized) known to induce a variety of squamous cell tumors (papillomas and warts) in the skin, and on mucous membranes of respiratory, gastrointestinal, and genitourinary tracts. The most familiar HPV manifestation in the genital tract is the venereal wart (condyloma acuminatum) recognized since antiquity, and shown to be a sexually transmitted disease (STD). In 1976, two other morphologically distinct HPV lesions were described in the uterine cervix, currently known as a flat and an inverted condyloma. Subsequently, these new HPV lesions were also shown to be an STD, and in addition, they frequently seem to occur concomitantly with CIN, CIS, and occasionally with invasive cervical carcinomas as well. These morphologic findings substantiated by the recent reports on malignant transformation of HPV lesions, as well as data from animal experiments and epidemiologic surveys, have lent support to the concept that HPV might be involved in the development of cervical (and other) human squamous cell carcinomas. Further evidence has been provided by the recent discoveries of HPV structural proteins (viral antigens) and HPV type 11 DNA in lesions of CIN, and HPV 16 and 18 DNA predominantly in invasive cervical carcinomas. So far, HPV 16 and HPV 18 seem to be the only HPV types with DNA capable of existing integrated in the host cell DNA. At the moment, cervical (and other) HPV lesions are the subject of intense study utilizing epidemiologic, morphologic, immunohistochemical, biochemical, and molecular biologic methods (recombinant gene technology) to provide further evidence of the suggested causal relationship between HPV and cancer. Prospective follow-up studies are also in progress to explore the natural history of cervical HPV lesions as well as the factors (e.g., immunologic, epidemiologic synergistic actions,) modifying them. In the light of present understanding, the factors linking HPV to cervical squamous cell carcinogenesis can be summarized as follows: (1) HPV infection in the uterine cervix is a sexually transmitted disease; (2) HPV lesions in the uterine cervix seem to be equivalent to CIN in their clinical behavior, i.e., possess the potential to progress towards CIS; (3) malignant transformation seems to depend on HPV type, being conditioned by integration of HPV DNA with the host cell DNA; (4) malignant transformation most probably requires synergistic effects between the virus and chemical or physical carcinogens, or other infectious agents; (5) genetic disposition (data available on animals only) significantly contributes to the process PMID- 3001623 TI - Russell bodies in New World cutaneous leishmaniasis. PMID- 3001624 TI - Pure apocrine carcinoma of the female breast presenting as a cyst: a case report. PMID- 3001625 TI - [Malignant mixed tumors of the gallbladder. Report of a case and review of the literature]. PMID- 3001626 TI - Special types of seizures. PMID- 3001628 TI - Neutrophil myeloperoxidase concentration: changes with development and during bacterial infection. AB - In experimental animals, the quantity of myeloperoxidase (MPO) in a volume of whole blood, and the neutrophil concentration in that same volume, were determined and the results expressed as units of MPO (10(-7))/neutrophil. Two situations are reported in which the concentration of MPO/neutrophil was found to change; during the growth and development of animals and during bacterial infection. Premature and newborn rats had only 25% of the MPO/neutrophil found in adults. One to three wk olds had 50% of the adult enzyme concentration. During fatal bacterial infection, MPO/neutrophil fell rapidly, often to undetectable levels, but during sublethal infections, following a 24-h lag period in adults and a 48-h lag in neonates, the concentration increased to twice normal. PMID- 3001627 TI - Effect of age and malnutrition on rotavirus infection in mice. AB - Litters of malnourished and normally nourished suckling mice of various ages were fed mouse rotavirus (10(5) ID50). Infection was monitored by immunofluorescent staining of isolated epithelial enterocytes 24, 28, and 72 h after virus ingestion; enzyme linked immunosorbent assay of daily fecal samples; and determining the severity of diarrhea by noting the color and consistency of the feces and the presence or absence of staining. In malnourished mice 5 to 9 days old, all the parameters of infection were significantly greater than in their normally nourished counterparts. However, in mice 10 to 14 days old the differences in extent and severity of infection in malnourished and normally nourished animals was not significant. After 15 days of age there were low levels of virus shedding and antigen-positive enterocytes in both malnourished and normally nourished animals, but there was no diarrhea in either group. Therefore it appears that nutritional manipulation, resulting in malnutrition, does not alter the state of refractoriness to symptomatic rotavirus infection in mice greater than 15 days old. PMID- 3001630 TI - [Role of Coxsackie B viruses in kidney diseases and their association with diseases of the digestive organs in children]. PMID- 3001629 TI - [Cortisol and ACTH levels in the blood of children with glomerulonephritis]. PMID- 3001631 TI - [Case of secondary chronic adrenal insufficiency]. PMID- 3001633 TI - [Burkitt's lymphoma-Epstein-Barr virus association in western Algeria. Clinical, cytological and serological aspects apropos of 34 pediatric cases]. AB - Burkitt's lymphoma (BL) and naso-pharyngeal carcinoma (NPC) are tumors generally believed to be associated with Epstein-Barr virus (EBV). Algerian population could be considered at high risk for BL-EBV association. In a series of 34 BL patients observed by us between 1982 and 1984 characteristics were: 70% of children were under 5 years age, abdominal location in 90% of cases (n = 30), 13% only isolated, massive tumor burden (70% stage III after Murphy, 30% stage IV after Murphy and Duque-Hammershaimb), EBV serology titers above or equal to 640 in 60% of cases (anti-EBNA and anti-VCA) and 50% (anti-EA) respectively, presence of EBNA antigen in tumor cells found in 63% of cases, no complete correlation between serology positivity and EBNA presence. PMID- 3001632 TI - [Burkitt's lymphoma in 1985]. AB - Burkitt Lymphoma is a model for either clinical research and fundamental research. Initially described in Africa, it is the most frequent of all childhood Lymphoma. Burkitt Lymphoma allowed in the laboratory to study the virus cancer relationship, the chromosome cancer relationship, the gene and cancer relationship and the oncogene and cancer relationship. This study is made considerably more easy because of the numerous cell lines which were established from malignant cells at different stages of the disease. Patients who have a complete remission of more than 8 months could be considered as cure of the disease. Then, B.L. is also a model for clinical study because we can conclude rapidly the efficacy or not of a new protocol. Since 1980, overall survival in France has moved from 40 to 80%. Chemotherapy alone is responsible for these results either by conventional protocol or in a small % cases by massive therapy followed by autologous bone marrow transplantation. PMID- 3001634 TI - [Giant axonal neuropathy. Apropos of a case]. AB - A new case of giant axonal neuropathy is reported about an 11 years old boy born with the Robin anomalad. The diagnosis is based on a peripheral nerve biopsy. This new observation can be added to 12 others ones which have already been published to study the characteristics of this peripheral neuropathy. The etiology and physiopathology are unknown. PMID- 3001636 TI - [Integrated treatment planning system of proton therapy for deeply seated tumors]. PMID- 3001635 TI - cAMP increases the basolateral Cl- -conductance in the isolated perfused medullary thick ascending limb of Henle's loop of the mouse. AB - The effect of cAMP on transepithelial and transmembrane potential differences and resistances was examined in isolated in vitro perfused mouse medullary thick ascending limbs of Henle's loop (mTAL). The effects of furosemide and barium were tested. Stimulation of NaCl transport by ADH 10(-9) + dbcAMP 4 X 10(-4) + forskolin 10(-6) mol X l-1 (paired experiments) resulted in: a) an increase in transepithelial potential difference, referenced to the grounded bath, from +6.7 +/- 0.3 mV to +12.0 +/- 0.4 mV (n = 47); b) a decrease in transepithelial resistance from 25 +/- 1 omega cm2 to 20 +/- 1 omega cm2 (n = 47); c) a depolarization of the basolateral membrane by 12 mV and of the apical membrane by 7 mV (n = 36); d) a decrease in the fractional resistance of the basolateral membrane from 0.27 +/- 0.05 to 0.15 +/- 0.06 (n = 12). Furosemide (10(-4) mol X l 1) abolished the active transepithelial transport potential and hyperpolarized the basolateral membrane potential to values which were similar in both control and cAMP treated mTAL segments. Barium increased the transepithelial resistance and depolarized PDb1 to similar values in both functional states. An increase in the fractional conductance of the basolateral membrane was also seen, if, prior to the cAMP treatment, the luminal Na+2Cl-K+ cotransport was inhibited by furosemide. Thus, we propose that stimulation of active NaCl reabsorption in the mTAL segment of the mouse by ADH, mediated via cAMP, increases primarily the basolateral chloride conductance. PMID- 3001637 TI - Isolation and buoyant density of Pneumocystis carinii in Percoll gradients. AB - A new gradient material, Percoll, a collodial suspension of silica particles covered with polyvinylpyrolidon, has been used to isolate Pneumocystis carinii from infected rat and human lung tissues. The specific gravity of Pneumocystis carinii was shown to be within the range of 1.018-1.062 g/ml with most pneumocysts within 1.033-1.049 g/ml. PMID- 3001638 TI - Insertion of an Alu SINE in the human homologue of the Mlvi-2 locus. AB - Fifty-nine human DNA samples derived from either normal tissues or hematopoietic neoplasias were examined for rearrangements in the Mlvi-2 locus, a putative oncogene. The rearranged Mlvi-2 sequences in one of them, a B cell lymphoma, were shown to result from the insertion of an approximately 300 bp DNA fragment that hybridized to a human Alu probe. DNA sequence analysis of both the rearranged and the nonrearranged allele around the site of the insertion revealed the following: a) the insert was 88.4% homologous to the consensus sequence of the Alu family of repeats and 75% homologous to the Alu related sequence in the human 7SL RNA; b) similar to other sequenced SINES, a poly(d.A) tract was present at the 3' end of this element; c) an 8 bp direct repeat was present at both ends of the inserted element; d) this repeat was present as a single copy in the unrearranged allele. We conclude from these findings that: Alu sequences can transpose and that the direct repeats flanking certain Alu SINES may be generated by the duplication of single copy cellular sequences at the site of the insertion. Furthermore the recent nature of the Alu insertion in the Mlvi-2 locus coupled to the low degree of homology of the inserted Alu to the Alu related sequence in the 7SL RNA suggest that this event did not occur via reverse transcription and reintegration of the 7SL RNA. PMID- 3001639 TI - Nucleotide sequence of the PaeR7 restriction/modification system and partial characterization of its protein products. AB - Bal31 deletion experiments on clones of the PaeR7 restriction-modification system from Pseudomonas aeruginosa demonstrate that it is arranged as an operon, with the methylase gene preceding the endonuclease gene. The DNA sequence of this operon agrees with in vitro transcription-translation assays which predict proteins of 532 amino acids, Mr = 59,260 daltons, and 246 amino acids, Mr = 27,280 daltons, coincident with the methylase and endonuclease genes, respectively. These predicted values coincide with the measured molecular weights of the purified, denatured PaeR7 endonuclease and methylase proteins. The first twenty amino acids from the amino-terminus of the purified endonuclease exactly match those predicted from the DNA sequence. Finally, potential regulatory mechanisms for the expression of phage restriction are described based on the properties of several PaeR7 subclones. PMID- 3001640 TI - The yeast alpha-factor receptor: structural properties deduced from the sequence of the STE2 gene. AB - The STE2 gene of the yeast Saccharomyces cerevisiae encodes a component of the receptor for the oligopeptide pheromone alpha-factor. We have cloned and determined the nucleotide sequence of the STE2 gene. A sequence involved in the control of cell-type expression of the STE2 gene was found 5' of an open reading frame that could encode a protein of 431 amino acids. The predicted STE2 protein contains seven hydrophobic segments, suggesting that the alpha-factor receptor is an integral membrane protein. No extensive homology at the primary sequence level was detected between the predicted STE2 protein and other available protein sequences. PMID- 3001641 TI - Human tumor cell lines with EGF receptor gene amplification in the absence of aberrant sized mRNAs. AB - A survey of human tumor cell lines for increased PDGF or EGF receptors identified 5 lines which bind from 6 to 13 times more EGF than normal human fibroblasts. Immunoprecipitation analysis links the elevated binding activity to increased quantities of the EGF receptor protein. EGF receptor gene amplification was detected in 2 of the cell lines. No evidence for EGF receptor gene rearrangements was found at the level of DNA or RNA structure. The results suggest that elevated levels of EGF receptor can be associated with at least three distinct mechanisms. These include gene amplification accompanied by rearrangement, gene amplification without accompanied alteration of mRNA transcripts, and extensive expression without gene amplification. PMID- 3001642 TI - Analysis of cloned cDNA and genomic sequences for phytochrome: complete amino acid sequences for two gene products expressed in etiolated Avena. AB - Cloned cDNA and genomic sequences have been analyzed to deduce the amino acid sequence of phytochrome from etiolated Avena. Restriction endonuclease site polymorphism between clones indicates that at least four phytochrome genes are expressed in this tissue. Sequence analysis of two complete and one partial coding region shows approximately 98% homology at both the nucleotide and amino acid levels, with the majority of amino acid changes being conservative. High sequence homology is also found in the 5'-untranslated region but significant divergence occurs in the 3'-untranslated region. The phytochrome polypeptides are 1128 amino acid residues long corresponding to a molecular mass of 125 kdaltons. The known protein sequence at the chromophore attachment site occurs only once in the polypeptide, establishing that phytochrome has a single chromophore per monomer covalently linked to Cys-321. Computer analyses of the amino acid sequences have provided predictions regarding a number of structural features of the phytochrome molecule. PMID- 3001643 TI - EcoK selection vectors for shotgun cloning into M13 and deletion mutagenesis. AB - For shotgun cloning into M13 vectors, a double-stranded cassette of synthetic oligonucleotides containing a SmaI site within the two halves of an EcoK site, has been introduced into the vector M13mp8. Cloning of blunt end DNA into the SmaI site destroys the EcoK site, and recombinants are therefore preferentially selected on transfection into a K strain of E.coli. For deletion mutagenesis using synthetic oligonucleotides, an M13 vector with four copies of the EcoK cassette has been made to facilitate the joining of lacZ or a Factor Xa cleavage site to any protein reading frame. PMID- 3001644 TI - SV40 vector with early gene replacement efficient in transducing exogenous DNA into mammalian cells. AB - An early replacement SV40 vector, SV40-Mu beta, was constructed by replacing part of the early genes of the virus with mouse interferon-beta (IFN-beta) cDNA. Upon transfection of COS-7 cells with this DNA, transducing viral particles were produced, which could infect various cells and cause efficient production of mouse IFN-beta. The viral stock contained no detectable wild-type SV40. The IFN production after the virus infection was very high in monkey kidney cells, but less so in human epithelial cells, and low in mouse, pig, hamster cells and in human lymphocytes. The efficiency of introduction of the DNA to monkey kidney cells was compared with that by the calcium phosphate precipitation method, and the viral vector was found to be more efficient by a factor of several tens to hundreds. PMID- 3001646 TI - Rsa1 polymorphism at the insulin receptor locus (INSR) on chromosome 19. PMID- 3001645 TI - Nucleotide sequence and transcriptional mapping of the yeast pet56-his3-ded1 gene region. AB - Genes of the baker's yeast Saccharomyces cerevisiae are densely clustered on 16 linear chromosomes. Here, I characterize a 1.8 kb region of chromosome XV containing the entire structural gene for the histidine biosynthetic enzyme imidazoleglycerolphosphate (IGP) dehydratase (his3) as well as the promoter sequences and 5'-proximal mRNA coding regions for the adjacent genes. The his3 gene encodes several mRNA species averaging 820 bases in length, all of which contain an open reading frame of 219 codons. The location of this open reading frame coincides with the his3 gene as defined by functional criteria, suggesting that the primary translation product of yeast IGP dehydratase has a molecular weight of 23,850. Phenotypic analysis of mutations constructed in vitro indicate that one of the adjacent genes (pet56) is required for mitochondrial function, whereas the other gene (ded1) is essential for cell viability. The pet56 and his3 genes are transcribed divergently from initiation sites that are separated by only 192 bp. Transcription of the ded1 gene is initiated only 130 bp beyond the 3'-end of the his3 mRNA coding region. These results suggest that these unrelated genes are located extremely close together and that the spacer regions between them consist largely of promoter and terminator sequences. PMID- 3001647 TI - AccIII, a new restriction endonuclease from Acinetobacter calcoaceticus. AB - A new site-specific restriction endonuclease, AccIII, was isolated from Acinetobacter calcoaceticus. AccIII recognizes T/CCGGA and cleaves at the position shown by the arrow. AccIII activity was inhibited by adenine methylation at the overlapping dam methylase recognition sequence. PMID- 3001648 TI - A wheat HMW glutenin subunit gene reveals a highly repeated structure. AB - A wheat genomic library was screened with two synthetic oligonucleotides (24 and 25 bases in length) complementary to a partial cDNA clone encoding a glutenin gene [Thompson et al. (1983) Theor. Appl. Genet. 67, 87-96]. Glutenins are large molecular weight aggregated proteins of grain endosperm, and major determinants of bread making quality of wheat. Of the two clones obtained one was fully characterized. It contained the sequence of the high molecular weight subunit of glutenin. The amino acid sequence derived from the gene sequence reveals a mature protein (817 amino acids) with a highly repeated structure of two different motifs corresponding to the high glutamine (35.7%), glycine (20.1%) and proline (13.1%) content. The gene does not contain an intron, and possesses a typical eukaryotic promoter; the RNA initiation site is 25-30 bases downstream. PMID- 3001649 TI - The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA. AB - The RF IV form of M13 DNA was synthesized enzymatically in vitro, using the viral (+)strand as template, to contain phosphorothioate-modified internucleotidic linkages of the Rp configuration on the 5' side of every base of a particular type in the newly-synthesized (-)strand. Twenty nine restriction enzymes were then tested for their reactions with the appropriate modified DNA types having a phosphorothioate linkage placed exactly at the cleavage site(s) of these enzymes in the (-)strand. Eleven of the seventeen restriction enzymes tested that had recognition sequences of five bases or more could be used to convert the phosphorothioate DNA entirely into the nicked form, either by simply allowing the reaction to go to completion with excess enzyme (Ava I, Ava II, Ban II, Hind II, Nci I, Pst I or Pvu I) or by stopping the reaction at the appropriate time before the nicked DNA is linearized (Bam HI, Bgl I, Eco RI or Hind III). Only modification of the exact cleavage site in the (-)strand could block linearization by the first class of enzymes. The results presented imply that the restriction enzyme-directed nicking of phosphorothioate M13 DNA occurs exclusively in the (+)strand. PMID- 3001650 TI - The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. AB - M13 RF IV DNA may be prepared in vitro to contain phosphorothioate-modified internucleotidic linkages in the (-)strand only. Certain restriction enzymes react with this modified DNA to hydrolyze the (+)strand exclusively when a phosphorothioate linkage occurs at the normal cleavage point in the (-)strand. The reaction of Pvu I with M13mp2 RF IV DNA containing dCMPS residues in the ( )strand is of this type, and is exploited to allow subsequent digestion with exonuclease III of a portion of the (+)strand opposite different mutagenic mismatched oligonucleotide primers. Two methods are described by which this approach has been used to produce mutations in M13mp2 phage DNA with high efficiency as a result of simple and rapid in vitro manipulations. Plaques containing mutant phage in a genetically-pure form are obtained at a frequency of 40-66%, allowing their characterisation directly by sequence analysis without prior screening and plaque purification. The wide applicability of this approach is discussed. PMID- 3001651 TI - In vitro transcription initiation of the spinach chloroplast 16S rRNA gene at two tandem promoters. AB - Two potential prokaryotic promoters, P1 and P2, are characterized 164 and 114 bp upstream of the spinach chloroplast 16S rRNA gene. The strengths of these promoters, calculated according to an homology score established for E. coli RNA polymerase, are identical. Experiments performed with a Taq I-DNA fragment, containing 16 bp of the 16S rDNA and 243 bp upstream of the gene, give evidence that in vitro, E. coli RNA-polymerase starts transcription at these two promoters. These results are based on both the size of the transcripts and their nucleotide sequences. A possible regulation by differential control of these dual promoters is suggested. S1 mapping with RNAs extracted either from green or from etiolated spinach plants, indicates that, at these two steps of plastid development, transcription in vivo starts at P1. Surprisingly only P2 appears to be conserved in the homologous sequences reported for maize, mustard and Spirodela. PMID- 3001652 TI - Structure of the human gene encoding the invariant gamma-chain of class II histocompatibility antigens. AB - The primary structures of a cDNA and the genomic DNA of a gene selectively expressed in chronic lymphocytic leukemia were determined. A computer search of the nucleotide sequence data bank identified this gene as the invariant gamma chain associated with class II histocompatibility antigens. The invariant gamma chain genomic sequence spans about 11 kilobases, with eight exons and seven introns. Three of the introns contain members of the Alu repeat family. A putative cap site and promoter sequence were identified at the 5' end of the gene. One or two copies of the gene is present in each haploid genome, and no evidence for amplification or polymorphism was obtained. PMID- 3001653 TI - Cloning, sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis. AB - The gene encoding subtilisin Carlsberg from Bacillus licheniformis has been isolated by molecular cloning using a mixture of synthetic oligonucleotides. The entire nucleotide sequence of the coding sequence as well as 5' and 3' flanking sequences have been determined. The deduced amino acid sequence reveals an N terminal signal peptide consisting of 29 residues, a pro-peptide of 76 residues followed by the mature protein comprising 274 residues. The ATG initiator codon is preceded by two putative overlapping ribosomal binding sequences. A palindromic sequence typical for transcription termination is found downstream from the TAA stop codon. Structural comparisons between different known subtilisin genes reveal extensive homology, particularly in the parts coding for the pro-region and the mature protein. Expression studies in Bacillus subtilis show that the cloned fragment produces a functional enzyme when inserted after a B. subtilis promoter. PMID- 3001655 TI - Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. V. Study of single-strand cleavages. AB - Concatemer DNA duplexes which contain at the EcoRII restriction endonuclease cleavage sites (formula; see text) phosphodiester, phosphoamide or pyrophosphate internucleotide bonds have been synthesized. It has been shown that this enzyme did not cleave the substrate at phosphoamide bond. EcoRII endonuclease catalyzes single-strand cleavages both in dA- and dT-containing strands of the recognition site if the cleavage of the other strand has been blocked by modification of scissile bond or if the other strand has been cleaved. This enzyme interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of another one. Nucleotide sequences flanking the EcoRII site on both sides are necessary for effective cleavage of the substrate. PMID- 3001654 TI - Integration site preferences of the Alu family and similar repetitive DNA sequences. AB - Numerous flanking nucleotide sequences from two primate interspersed repetitive DNA families have been aligned to determine the integration site preferences of each repetitive family. This analysis indicates that both the human Alu and galago Monomer families were preferentially inserted into short d(A+T)-rich regions. Moreover, both primate repeat families demonstrated an orientation specific integration with respect to dA-rich sequences within the flanking direct repeats. These observations suggest that a common mechanism exists for the insertion of many repetitive DNA families into new genomic sites. A modified mechanism for site-specific integration of primate repetitive DNA sequences is provided which requires insertion into dA-rich sequences in the genome. This model is consistent with the observed relationship between galago Type II subfamilies suggesting that they have arisen not by mere mutation but by independent integration events. PMID- 3001656 TI - Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. VI. The binding and cleavage of substrates containing nucleotide analogs. AB - The present study deals with the binding and cleavage by EcoRII endonuclease of concatemer DNA duplexes containing EcoRII recognition sites (formula; see text) in which dT is replaced by dU or 5-bromodeoxyuridine, or 5'-terminal dC in the dT containing strand is methylated at position 5. The enzyme molecule is found to interact with the methyl group of the dT residue of the DNA recognition site and to be at least in proximity to the H5 atom of the 5'-terminal dC residue in dT containing strand of this site. Modification of any of these positions exerts an equal effects on the cleavage of both DNA strands. Endonuclease EcoRII was found to bind the substrate specifically. At the same time modification of the bases in recognized sequence may result in the formation of unproductive, though stable, enzyme-substrate complexes. PMID- 3001657 TI - Diet and cancer: value of different types of epidemiological studies. AB - Diet and nutrition are increasingly recognized as likely to be major determinants of cancer, notably cancers of the gastrointestinal tract, breast, endometrium, ovary, and prostate. Dietary factors may collectively account for a greater proportion of all cancers that occur in contemporary Western society than does any other category of environmental exposure (1). With the development of knowledge of the protective properties of certain components of food, links with diet have been suggested for other cancer sites (2). The epidemiological evidence for the association of diet and cancer is, however, not uniformly convincing; also, the likely biological pathways are not always clear. In this paper, we comment on some current hypotheses in this area and examine the best epidemiological methods to test them. PMID- 3001658 TI - Natural history of left ventricular function in neonatal Coxsackie myocarditis. AB - Three neonates are described who had severe congestive cardiac failure following Coxsackie-B virus infection. Overall left ventricular function was depressed and accompanied by regional differences in wall motion. Recovery has been gradual, and after 40 or more months of follow-up, all three infants still have evidence of myocardial damage. This provides further evidence linking myocarditis with dilated cardiomyopathy. PMID- 3001659 TI - Dose-response study of RIT 4237 oral rotavirus vaccine in breast-fed and formula fed infants. AB - The RIT 4237 live attenuated bovine rotavirus vaccine was given orally at three dose levels to 75 breast-fed, 40 formula-fed and 24 fasting infants ages 4 to 6 months. Vaccine of 10(8.3) (50% tissue culture-infective doses) (TCID50) per dose gave a neutralizing antibody response in 14 of 14 (100%) formula-fed, in 18 of 26 (69%) breast-fed and in 5 of 8 (63%) fasting infants, or an overall response rate of 77% (37 of 48). The overall response rate to a vaccine of 10(7.2) TCID50 per dose was 61% (33 of 49), or slightly but not significantly lower than that at the higher dose level. On the other hand a vaccine of 10(6.3) TCID50 per dose gave a significantly (P less than 0.01) lower composite response rate of 33% (14 of 42). The overall serologic response rate to the vaccine at the two higher doses was somewhat better (24 of 28, 86%) in formula-fed infants than in breast-fed infants (37 of 52, 71%). However, the response rate of the breast-fed infants can also be considered satisfactory. Thus the current recommendation for use of the RIT 4237 vaccine would be administration of a dose of at least 10(8) TCID50 after feeding with either formula or breast milk. PMID- 3001660 TI - Diverse serologic response to rotavirus infection of infants in a single epidemic. AB - Eight infants with onset of gastroenteritis occurring within a 32-day period were examined for stool rotavirus antigen and serum antibody response to rotavirus. Each infant presented evidence of rotavirus infection but no single test for rotavirus-specific antigen or antibody was positive for every infected individual. Rotavirus was identified in the stools of five infants; in the others rotavirus etiology was determined by a specific immune response only. The neutralizing antibody response to each of four human rotavirus serotypes and to bovine rotavirus Nebraska calf diarrhea virus ranged from totally negative to a specific response to up to four serotypes. Three originally seropositive infants each showed an increase in neutralizing antibody titer to two or more serotypes. However, two of five originally seronegative infants also developed antibody to two or more serotypes. No consistent pattern of responses to different serotypes was detected. PMID- 3001661 TI - Rotavirus RNA variation during chronic infection of immunocompromised children. AB - Polyacrylamide gel electrophoresis was performed on RNA from rotaviruses isolated from two immunocompromised patients with prolonged rotavirus diarrhea. Extensive variations were observed in the genomes of rotaviruses isolated from each patient during a 16-week period of their illness. An animal model of rotavirus infection was used to help evaluate the results found in our patients. Individual mouse rotavirus strains were found to be stable during serial passage, but individual animals could be coinfected with more than one viral strain. The changes found in the rotavirus RNA from our patients possibly represented reinfection by different viral strains. Appropriate precautions should be observed to prevent such reinfections in immunocompromised patients. PMID- 3001662 TI - Ribavirin: beginning the blitz on respiratory viruses? PMID- 3001663 TI - Centers for Disease Control (CDC) case definition for Kawasaki syndrome. PMID- 3001664 TI - ACTH 4-9 effects on the human visual event-related potential. AB - Three oral doses (5, 10 and 20 mg) of an analog of ACTH 4-9 were compared with a vehicle control and d-amphetamine (10 mg). In a double-blind procedure, five men and five women were tested at weekly intervals with each treatment. In each session, four visual event-related potentials (ERPs) were recorded at hourly intervals. Visual ERPs were averaged from the electroencephalogram recorded from the left and right hemisphere. Dosage, time after administration, hemisphere of the brain and sex of the subject influenced the ERP. The ACTH 4-9 analog decreased amplitude of P100 but increased integrated activity of the ERP. This effect peaked at 60 minutes then "recovered." The effects of the peptide were more pronounced with doses of 5 and 10 mg, in the right hemisphere of men and in the left hemisphere of women. The findings indicated that the ACTH 4-9 analog influenced components of ERP commonly related to the perceptual/attentional state of the organism in a sexually dimorphic manner. PMID- 3001665 TI - An investigation on pro-opiomelanocortin and processed peptides from the teleost fish Prochilodus platensis. AB - Acid extracts of carefully dissected proadenohypophysis (PA) and metaadenohypophysis (MA) of the teleost Prochilodus platensis were subjected to chromatography in Sephadex G-50 after which several pro-opiomelanocortin (POMC) peptides were detected by means of three heterologous RIA systems: alpha-MSH, ACTH and beta-endorphin. Parallelism among extracts displacement curves ranged from 26% to 95% of those of the standard curves for the different systems employed. In PA chromatograms, peaks of ACTH immunoreactivity (IR) were detected at the positions of 30 kilodalton (K), 20K, 9K, a large 4.5K peak and 2K. Only one peak of beta-endorphin IR was detected at 30K. In MA chromatograms, ACTH IR detected similar peaks as in PA runs, but 4.5K peak was much smaller, whereas a large 2K peak roughly coincided with all alpha-MSH detected in the chromatograms. beta-Endorphin IR was detected mainly as a large peak coinciding with synthetic beta-endorphin in MA runs. Bioactivity was detected in both PA and MA 4.5K ACTH peaks, whereas little activity could be demonstrated associated with the 30K, 20K and 9K ACTH IR peaks. Prochilodus PAs and MAs were incubated with tritiated aminoacids and the extracts immunoprecipitated with ACTH, beta-endorphin and N terminal POMC (N-POMC) antisera. The dissociated complexes were run in SDS polyacrylamide slab gel electrophoresis. The tritiated bands detected confirmed the results obtained with Sephadex chromatography. N-POMC immunoprecipitated peptides were located at 28K, 18K and 9K positions. The first two probably accounted for POMC and the N-POMC/ACTH intermediate respectively; the third corresponded to the mammalian 1-76N-POMC.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3001666 TI - FMRFamide suppresses kappa opiate induced feeding in the mouse. AB - Intracerebroventricular administration of 0.01-1.0 micrograms of the peptide FMRFamide (Phe-Met-Arg-Phe-NH2) to mice suppressed feeding induced by the specific kappa opiate agonist, U-50, 488H. This suggests that FMRFamide, or FMRFamide-like neuropeptides, may have a role in the control of kappa opioid mediated feeding in the mouse. PMID- 3001667 TI - Relationship of CRF-immunostained cells and magnocellular neurons in the paraventricular nucleus of rat hypothalamus. AB - The distribution of corticotropin-releasing factor (CRF), vasopressin (VP) and oxytocin (OXY) containing neurons within the magnocellular and parvocellular divisions in the paraventricular nucleus (PVN) of rat hypothalamus is described in brains from normal untreated, colchicine treated and adrenalectomized animals. Double immunostained preparations using glucose oxidase-antiglucose oxidase (GAG) complex combined with PAP complex to visualize two antigens with contrasting colors in the same tissue sections were employed. Separate and distinct populations of cells containing the immunoreactive (ir) elements were seen. Immunostained CRF neurons present in the ventral medial portion of the posterior magnocellular division were juxtaposed to oxytocin-ir perikarya in colchicine treated and adrenalectomized animals. CRF-ir cells were for the most part concentrated in the medial parvocellular component of PVN. An intimate anatomical proximity between CRF-ir and VP-ir perikarya was evident in this medial parvocellular division in brains of adrenalectomized animals; this area is normally VP-ir poor except in the adrenalectomized rats. This extension of VP-ir cells into this CRF rich region and the very close approximation between the two cell bodies suggests potential cell to cell communication following perturbation of the brain-pituitary-adrenal axis. No evidence for the co-existence of two peptidergic systems in the same neuron was apparent in the present study. PMID- 3001668 TI - Relationship of ACTH1-39-immunostained fibers and magnocellular neurons in the paraventricular nucleus of rat hypothalamus. AB - Dual antigen immunocytochemical staining procedures were used in the same tissue section to determine the distribution of ACTH immunostained fibers and varicosities within the magnocellular and parvocellular divisions in the paraventricular nucleus (PVN) of rat hypothalamus and elucidate its anatomical relationship to vasopressin (VP) and oxytocin (OXY)-containing neurons. Double immunostained preparations using glucose oxidase-antiglucose oxidase complex combined with PAP complex to visualize two antigens with contrasting colors in the same tissue section were employed. ACTH-immunoreactive (ir) fibers were distributed throughout the periventricular stratum and the parvocellular component of the PVN; in the latter area fibers were particularly dense in the ventral medial portion of the medial parvocellular division. Dual immunostained sections revealed a close anatomical association between opiocortin fibers and oxytocin and vasopressin parvocellular neurons. ACTH immunostained fibers were present in the anterior and medial magnocellular component of PVN and in the ventral medial portion of the posterior magnocellular division; these immunoreactive fibers were in intimate proximity to oxytocin-ir perikarya. The very close approximation between the ACTH-ir fibers and oxytocin-containing cell bodies suggests potential cell to cell communication between the two peptidergic systems in PVN. Few ACTH immunostained fibers were seen in the dorsal lateral portion of the posterior magnocellular division in which vasopressinergic neurons predominate. The present anatomical study supports pharmacological and physiological studies which indicate that opioids can influence the activity of magnocellular PV neurons. This study also elucidates an anatomical relationship between opiocortins (ACTH1-39) and parvocellular PV neurons which suggests that the opiocortin system may play a role in the regulation of both the neuroendocrine and autonomic activities of specific PV neurons. PMID- 3001669 TI - Pregnancy related changes of opiate receptors identified in rat uterine membranes by 3H-naloxone binding. AB - 3H-Naloxone was used to demonstrate the presence of specific opiate binding sites in uterine membrane preparations of rats. 3H-Naloxone binding (0.41-27 nM) was found to be rapid, saturable and reversible showing two populations of binding sites with the characteristic of high (KD 2.2 nM; Bmax 46.6 fmol/mg prot.) and low (KD 18.1 nM; Bmax 143.7 fmol/mg prot.) affinity. The number and affinity of the binding sites labelled by 3H-naloxone in the uterus were measured in the rat at mid (14 days), late (21 days) pregnancy and at parturition. The high and low affinity recognition sites labelled by 3H-naloxone showed a consistent reduction during pregnancy and at parturition without changes in the affinity constant. We concluded that pregnancy and parturition are associated with significant changes in the number of the opiate receptors bound in the uterus by 3H-naloxone. This phenomenon which seems to be linked with the several pregnancy-related changes in the levels of endogenous peptides and hormones could be relevant to further explain the pregnancy related changes in pain perception and maternal behavior. PMID- 3001670 TI - Autoradiographic distribution of 125I calcitonin gene-related peptide binding sites in the rat central nervous system. AB - Using autoradiographic method and 125I-Tyro rat CGRP as a ligand, receptor binding sites were demonstrated in the rat central nervous system. Saturation studies and Scatchard analysis of CGRP-binding to slide mounted tissue sections containing primarily cerebellum showed a single class of receptors with a dissociation constant of 0.96 nM and a Bmax of 76.4 fmol/mg protein. 125I-Tyro rat CGRP binding sites were demonstrated throughout the rat central nervous system. Dense binding was observed in the telencephalon (medial prefrontal, insular and outer layers of the temporal cortex, nucleus accumbens, fundus striatum, central and inferior lateral amygdaloid nuclei, most caudal caudate putamen, organum vasculosum laminae terminalis, subfornical organ), the diencephalon (anterior hypothalamic, suprachiasmatic, arcuate, paraventricular, dorsomedial, periventricular, reuniens, rhomboid, lateral thalamic pretectalis and habenula nuclei, zona incerta), in the mesencephalon (superficial layers of the superior colliculus, central nucleus of the geniculate body, inferior colliculus, nucleus of the fifth nerve, locus coeruleus, nucleus of the mesencephalic tract, the dorsal tegmental nucleus, superior olive), in the molecular layer of the cerebellum, in the medulla oblongata (inferior olive, nucleus tractus solitarii, nucleus commissuralis, nuclei of the tenth and twelfth nerves, the prepositus hypoglossal and the gracilis nuclei, dorsomedial part of the spinal trigeminal tract), in the dorsal gray matter of the spinal cord (laminae I-VI) and the confines of the central canal. Moderate receptor densities were found in the septal area, the "head" of the anterior caudate nucleus, medial amygdaloid and bed nucleus of the stria terminalis, the pyramidal layers of the hippocampus and dentate gyri, medial preoptic area, ventromedial nucleus, lateral hypothalamic and ventrolateral thalamic area, central gray, reticular part of the substantia nigra, parvocellular reticular nucleus. Purkinje cell layer of the cerebellum, nucleus of the spinal trigeminal tract and gracile fasciculus of the spinal cord. The discrete distribution of CGRP-like binding sites in a variety of sensory systems of the brain and spinal cord as well as in thalamic and hypothalamic areas suggests a widespread involvement of CGRP in a variety of brain functions. PMID- 3001672 TI - ACTH peptides as organizers of neuronal patterns in development: maturation of the rat neuromuscular junction as seen by scanning electron microscopy. AB - SEM was used to visualize the normal postnatal development of the neonatal rat neuromuscular junction (nmj). Maturational changes evoked by ACTH/MSH 4-10 (10 micrograms/kg/day IP) or ACTH/MSH 4-9 (Org 2766) (0.01 microgram/kg/day IP) were compared to controls and to pups treated with nicotine during prenatal and postnatal life, or only during the gestation period. Pregnant females received 0.25 mg/kg 2X daily IP; neonates 0.05 mg/kg/day SC. The Desaki and Uehara and Fahim et al. methods revealed the nmj on the extensor digitorum muscle to be covered by a delicate drapery of postjunctional folds that surround the immature endplate region. By the second week of postnatal life, these folds become more complex and cover a larger area. Upon maturation the folds descend and invaginate into the muscle fiber. Peptide treatment with either ACTH/MSH 4-10 or ACTH/MSH 4 9 accelerates maturation of the endplate as demonstrated by the increased convolutions of the folds. Similar effects follow nicotine administration. The observed changes in morphology of the developing nmj subjected to nicotine may be mediated through nicotine-evoked ACTH release. PMID- 3001671 TI - Clarification of the behavioral functions of peripheral and central cholecystokinin: two separate peptide pools. AB - Cholecystokinin is localized in two distinct systems, the gastrointestinal and the central nervous systems. At both locations, cholecystokinin (CCK) is synthesized, stored, and released, and high affinity binding sites for CCK have been identified. The blood-brain barrier is thought to completely block passage of CCK from the gut to the brain compartment, although CCK can pass from the cerebrospinal fluid out into the blood. A variety of behavioral actions of CCK have been described. Evidence that the actions of CCK on feeding and behavioral sedation are mediated through the peripheral site is reviewed. Recent studies demonstrating behavioral effects of CCK administered in nanogram doses directly into brain nuclei are reviewed. Caveats against administration of microgram doses of CCK into the cerebral ventricles are raised, emphasizing the interpretational difficulties inherent in this approach. PMID- 3001673 TI - Postnatal development of ACTH and alpha-MSH in the medulla oblongata of rat: alpha-MSH is the predominant peptide. AB - ACTH and alpha-MSH levels were measured by radioimmunoassays in extracts of the caudal medulla oblongata of developing rats on postnatal (P) days 1-42 at 7 day intervals, and in adult rats. From P1 to adulthood, ACTH increased greater than 11-fold from 7.2 +/- 1.9 fmol to 82.4 +/- 12.6 fmol per medulla section (mean +/- S.E.M.). In comparison, alpha-MSH increased greater than 7-fold from 68.75 +/- 11.0 fmol to 491 +/- 97.8 fmol during this time period. ACTH/microgram of soluble protein decreased during postnatal development from 0.006 +/- 0.01 to 0.005 +/- 0.001 fmol/microgram of protein and alpha-MSH increased from 0.06 +/- 0.01 fmol/microgram of protein to 0.11 +/- 0.009 fmol/microgram of protein between P1 and P7, decreased to 0.015 +/- 0.003 fmol/microgram of protein by P42 and increased to 0.03 +/- 0.006 fmol/protein per unit protein by adulthood. These data indicate a significant shift in the levels of alpha-MSH detected during development with a decrease in the concentration of material occurring from early postnatal development (P1-P7) to adulthood, which does not appear to be solely related to a regional increase in protein. These studies, as well as radioimmunoassays for ACTH and alpha-MSH in combination with sizing chromatography of pooled extracts at P1, P7 and the adult, demonstrated the predominance of alpha-MSH at all ages. PMID- 3001674 TI - Substance K: vascular and cardiac effects in rat and pig. AB - The effects of substance K (SK), a newly discovered tachykinin, on the cardiovascular and sympathetic system were evaluated in the pithed rat preparation and in the in situ domestic pig heart. In pithed rats, SK (10 nmol/kg, IV) produced a triphasic mean blood pressure (MAP) response: short depressor, short pressor (+11 +/- 1 mmHg), and prolonged depressor phase (-9 +/- 1 mmHg, n = 9-24, p less than 0.001). Neither effect was significantly affected by pretreatment with propranolol (2 mg/kg) or phentolamine (1 mg/kg). The pressor response was accompanied by increased heart rate (HR): 41 +/- 4 beats/min, while lower doses produced a decrease: -8 +/- 2 beats/min (p less than 0.01). Propranolol abolished the increase in HR. SK inhibited the pressor response evoked by electrical stimulation of the spinal cord (SCS) and by Arg8-vasopressin (AVP). SK increased circulating levels of epinephrine and norepinephrine but did not change release of catecholamines evoked by SCS. Direct intracoronary injections of SK (0.3-100 nmol, intact pig heart) increased coronary blood flow; higher doses decreased MAP and increased HR. These results indicate that: SK can produce pressor and depressor effects in the rat and is a potent coronary dilator in the pig. In the pithed rat SK causes catecholamine release which mediates its cardiac accelerator effect and it antagonizes adrenergic and non-adrenergic pressor stimuli. PMID- 3001675 TI - The opioid system and central cardiovascular control: analysis of controversies. AB - Opiates, like morphine, were long known to produce changes in blood pressure and cardiac functions. However, the nature of these changes are a subject of continuous controversy. The substantial differences in the opiate effects on the cardiovascular system is also apparent in more recent studies using enkephalins, beta-endorphin and dynorphins. The present review is aimed to indicate the source of the variations in the experimental data and analyze the relative contribution of different experimental factors to the observed effects of opiates and opioid peptides on the cardiovascular system. The major factors which contribute to the nature of the opioid effect on the cardiovascular system are: anesthesia, species, dose, site of action in the brain, respiratory changes and receptor specificity. However, the cardiovascular status per se is an important determinant of the opiates and opioid peptide effects on hemodynamic functions as indicated in states of hypertension and shock. A newly described factor is the plasticity of the opioid receptor system which changes its level and distribution pattern in different physiological and pathophysiological states. This review emphasizes the importance of utilization of highly specific ligands to opiate receptors administered to discrete brain areas in the conscious animal. PMID- 3001676 TI - Vasopressin and ACTH analogs affect orienting but not activity in rabbits. AB - Two experiments were conducted in which albino rabbits were tested for cardiac Orienting Reflexes to novel tones and open field locomotor activity in a novel environment. In Experiment I subcutaneous doses of desglycinamide-arginine8 vasopressin (DGAVP; 25 micrograms/kg) and ACTH fragment 4-10 (250 micrograms/kg) altered tonic heart rate but did not affect bradycardiac Orienting Reflexes, activity, habituation or retention of habituation. In Experiment II a lower dose of DGAVP (5 micrograms/kg) enhanced the magnitude of cardiac orienting reflexes when administered 60 min before an initial orienting test. Both DGAVP and a lower dose of ACTH4-10 (50 micrograms/kg) administered before the first orienting test enhanced cardiac orienting and delayed habituation during a second (retention) test conducted following saline treatment; neither peptide had any effect on orienting when administered before the retention test. The lower doses of the peptides also failed to affect activity, habituation of activity or retention of habituation. These data suggest that low doses of DGAVP and ACTH4-10 affect stimulus processing and attention, but not more generalized responses to environmental novelty, in rabbits. PMID- 3001677 TI - [Role of the active forms of oxygen in the metabolism of neutrophils]. PMID- 3001678 TI - Immunopathology of AIDS (acquired immune deficiency syndrome) observations in 75 patients with pre-AIDS and AIDS. AB - Sequential changes in the morphology of immunocompetent and other organs, in the accompanying immunocytological and immunovirological measurements are described in 75 patients with lymphadenopathy syndrome and with AIDS. The data presented demonstrate a stepwise development of the disease from a hyperimmunization syndrome to T-cell immune deficiency. Excessive antigenic stimulation by a large number of infectious organisms or by transfusion of blood and blood products account for antigenic overloading and hyperimmunization. Among such infectious organisms are certain viruses which per se interfere with cells of the immune system as for instance Epstein-Barr virus, cytomegalovirus, and HTLV3. Developing immunological incompetence will favor the persistence of these and other infectious organisms enhancing the damage of the immune reactivity and finally allow lethal infections or malignant tumors to occur. PMID- 3001679 TI - Benign fibrous histiocytoma of the skin. An immunohistochemical analysis of 30 cases. AB - In this study the immunohistochemical analysis of distinct morphologic variants of benign fibrous histiocytoma (BFH) of the skin was performed with immunoperoxidase technique for both lysozyme and alpha-1-antitrypsin (A1AT). Thirty cases including cellular, fibrous and xanthomatous variants of BFH were selected. Out of the total 6 cases (20%) showed positive staining only for A1AT, 3 cases (10%) only for lysozyme and 10 (33.3%) for both markers. Thus, 19 cases (63.3%) showed positive staining for one of both markers. Positive staining was higher in the cellular variant than the fibrous and xanthomatous types. Negative staining of tumors of definite histiocytic morphology may be interpreted as a variable enzymatic expression of different histiocytic activation and/or undetectable enzymatic content by the current techniques. These results are in accordance with our previous evolutional hypothesis of BFH which considered the cellular tumors as functionally more active variants evolving to less cellular, more fibrous and less active types. Current histogenetic concepts about this controversial group of skin neoplasms are discussed. PMID- 3001680 TI - Immunohistochemical demonstration of carcinoembryonic antigen (CEA) in 120 mammary carcinomas and its correlation with tumor type, grading, staging plasma CEA, and biochemical receptor status. AB - Antisera to CEA were used for the immunohistochemical localization and quantification of this antigen in 120 Bouin-fixed, paraffin embedded mammary carcinomas. These results were compared to tumor type, grading, staging, biochemical receptor status, cytosolic CEA-levels of the same tumors, and preoperative plasma CEA-levels. Mammary carcinomas were usually characterized by a low percentage of CEA-positive tumor cells: 50.9% of the cases contained more than 5% CEA-positive tumor cells and were therefore defined as being CEA histopositive in this study. A relation could be shown between CEA histopositivity and the histologic tumor type. The majority of invasive lobular carcinomas, tubular, and cribriform carcinomas was CEA-negative (72%). Conversely, 70% of invasive ductal carcinomas were CEA-positive. There was a significantly higher percentage of CEA-histopositivity in grade III tumors than in grade I/II carcinomas. The results obtained by quantification of the immunohistochemical staining of CEA were positively correlated with the results obtained by cytosolic CEA-assay. The overall concordance between tissue and plasma determinations of CEA was found to be 57.1%. A positive trend could be found between CEA-positivity and staging. However, no correlation was observed between CEA-positivity and estrogen receptor status. PMID- 3001681 TI - Clinical pathological correlation. PMID- 3001682 TI - [Can left-handed Z-DNA perform a biological function?]. PMID- 3001683 TI - [Barbara McClintock--Nobel laureate in the physiologic and medical sciences]. PMID- 3001684 TI - Abnormal facial movements. Guide to differential diagnosis. AB - Spontaneous facial movements are disturbing to those who have them, yet some such movements are benign and cause no more than cosmetic embarrassment. Other abnormal facial movements, however, are more serious and can be associated with neurologic disorders such as multiple sclerosis, brainstem tumor, peripheral neuropathy, and Guillain-Barre syndrome. Occasionally, an abnormal movement of the face is the first sign of such an underlying disorder. Accurate differential diagnosis of these perplexing movement disorders is imperative in determining prognosis. PMID- 3001686 TI - [A new method of functional assay of des-gamma-carboxyprothrombin using staphylocoagulase. Application to the diagnosis of hepatocellular carcinoma]. AB - A new method to assay des-gamma-carboxyprothrombin (DCP) activity is described, using staphylocoagulase on undiluted citrated plasma after adsorption with bentonite (to remove fibrinogen), then with aluminium hydroxide. The thrombin coagulase which is formed is measured by following the increase in optical density per minute of a chromogenic substrate. The results are expressed in milliunits (m.U.). The new method is sensitive and specific. It confirms the usefulness of the DCP assay for the diagnosis of hepatocellular carcinoma (Liebman et al.). Ninety-six normal subjects had levels of DCP ranging from 0 to 12 m.U. Out of 42 patients with hepatocarcinoma, 30 (71%) had DCP levels higher than 15 m.U. The increased DCP appears to be complementary to alpha-foetoprotein, since one or the other marker were positive in 37 out of 40 cases (92.5%) of hepatocellular carcinoma. Chronic hepatitis or cirrhosis (36 cases) and non hepatocellular liver cancer (9 cases) gave normal DCP values. PMID- 3001685 TI - Characterization of the opioid receptor binding and animal pharmacology of meptazinol. AB - Meptazinol is a unique opioid analgesic. Binding studies suggest a relative selectivity for mu 1 sites, as opposed to the other opioid receptor binding sites. This binding selectivity is consistent with meptazinol's supraspinal site of action and its sensitivity to the mu 1-selective opiate antagonist naloxonazine. Furthermore, at equianalgesic doses, morphine depressed respiration to a greater degree than meptazinol. PMID- 3001687 TI - [Vascular purpura caused by human parvoviruses]. PMID- 3001688 TI - [Immunohistochemical study of S 100 protein in granular cell tumors. Argument for their schwannian origin]. PMID- 3001689 TI - [Bioassay of circulating human parathyroid hormone]. PMID- 3001690 TI - [Inhibition of angiotensin converting enzyme in human pregnancy. 15 cases]. AB - Angiotensin converting-enzyme inhibitors cross the placenta and modify the maternal, foetal and utero-placental renin-angiotensin system. Eight cases of pregnancy in women taking captopril have been published, 7 other cases being reported in this review paper. There were one spontaneous and 2 therapeutic abortions, one of which disclosed a malformation of uncertain diagnosis and imputation. One intrauterine death at 28 weeks was probably due to the severity of the maternal disease. Two children born to mothers also treated with frusemide died of neonatal anuria. Delivery or caesarean section occurred before term in 8 cases, and there were 3 cases of neonatal respiratory distress with a favourable outcome. Finally, one mother gave birth at term to twins of normal weight. The cases with respiratory distress can be attributed to the mother's hypertension, to prematurity and/or to concomitant treatment with beta-blockers, while the cases with anuria seem to be due to inhibition of the effects of angiotensin on renal haemodynamics, with salt depression as a possible aggravating factor. Treatment with angiotensin converting enzyme inhibitors does not seem to warrant therapeutic abortion. However, these drugs are contra-indicated in pregnancy and should only be given to women wishing to become pregnant if they present with resistant and dangerous arterial hypertension. A programme of pharmacovigilance is being set up to follow up such pregnancies. PMID- 3001691 TI - [The cytomegalovirus in perinatal medicine]. AB - The epidemiology of the cytomegalovirus and the characteristics of mother-to foetus transmission indicate that most neonatal infections (0.5 to 2% of live births) are due to reactivation of a latent virus during pregnancy. These infections are, and probably remain, subclinical. Even in case of primary infection, there seems to be little risk for the foetus. However, the patent forms of the disease are so serious that an effective and safe immunization method is highly desirable. PMID- 3001692 TI - Salivary buffer systems and the Dentobuff test. PMID- 3001693 TI - Semisynthesis of horse heart cytochrome c analogues from two or three fragments. AB - Horse heart cytochrome c was cleaved with cyanogen bromide. The largest fragment, [Hse65]cytochrome c-(1-65)-pentahexacontapeptide lactone, was used in condensations involving four analogues of the complementary cytochrome c-(66-104) nonatriacontapeptide. Two of the latter compounds were obtained from a semisynthesis starting with a partially protected fragment N epsilon 86-88,99,100 penta(methylsulfonylethyloxycarbonyl)cytochrome c-(81-104)-tetracosapeptide (also arising from a cyanogen bromide-mediated degradation) and analogues of the middle part, cytochrome c-(66-80)-pentadecapeptide, which were prepared by organochemical synthesis. Two other analogues of the cytochrome c-(66-104) nonatriacontapeptide were prepared entirely by organochemical synthesis. Each of the covalently recombined analogous cytochromes c could retain an electron in the presence of oxygen and transfer it to cytochrome c oxidase, although with different reaction rates and Michaelis constants. Their redox potentials varied over a broad range. The exchanges Tyr67----Phe(F) and Thr78----Val gave rise to analogues with a lower redox potential than native cytochrome c, while the exchange Phe82----Leu or Tyr97----Leu led to analogues with the same and a higher redox potential, respectively. PMID- 3001694 TI - Analysis of the transcript encoding the latent Epstein-Barr virus nuclear antigen I: a potentially polycistronic message generated by long-range splicing of several exons. AB - The Epstein-Barr virus nuclear antigen (EBNA I) present in latently infected cells is encoded in a 2-kilobase exon contained in the BamHI K viral genomic fragment. This exon is, however, found within a 3.7-kilobase mRNA transcript. The origin of the remaining 1.7 kilobases is unknown, although it is not derived from adjacent Epstein-Barr virus DNA sequences. A 1.1-kilobase cDNA clone generated by primer extension using an oligonucleotide complimentary to a sequence 245 base pairs 3' to the putative initiation codon for EBNA I in the BamHI K fragment has been isolated. This clone contains seven exons (from the BamHI W, Y, U, E, and K viral genomic fragments), which are spread over approximately 70 kilobases of the viral genome. However, this clone does not appear to contain the complete 5' end of the transcript. In addition to the open reading frame in the exon encoding EBNA I, three other open reading frames are found in this transcript that potentially encode other viral antigens present in latently infected cells. PMID- 3001696 TI - A long and complex enhancer activates transcription of the gene coding for the highly abundant immediate early mRNA in murine cytomegalovirus. AB - Using the simian virus 40 "enhancer trap" approach, we have identified a transcription enhancer located just upstream of the major immediate early gene of murine cytomegalovirus. This enhancer has several striking properties. (i) Together with the enhancer of human cytomegalovirus, it is the strongest transcription enhancer found to date. (ii) It is an extremely long enhancer, spanning greater than 700 base pairs. (iii) It consists of a rather complex pattern of sequence repeats, the longest of which is 181 base pairs. Also, several types of short sequence motifs are scattered throughout the enhancer in monomeric, heterodimeric, or homodimeric (palindromic) form. These motifs have been identified to be components of other enhancers and promoters, and they are presumably binding sites for specific nuclear factors. Our analysis suggests that enhancers are composed of a modular arrangement of short conserved sequence motifs and that enhancer strength is correlated with the redundancy of these motifs. PMID- 3001695 TI - Effects of phospholipids and ADP-ribosylation on GTP hydrolysis by Escherichia coli-synthesized Ha-ras-encoded p21. AB - The Ha-ras protooncogene product p21, which may be involved in control of cellular growth, is a membrane protein that binds guanine nucleotides and hydrolyzes GTP. p21 GTPase activity is stimulated by lysophosphatidylcholine; a delay in activation was observed unless p21 was incubated with the phospholipid prior to assay. Maximal activation by the phospholipid was observed over a narrow concentration range; the presence in the assay mixture of lysophosphatidylcholine at concentrations above this optimum markedly inhibited p21 GTPase. GTP hydrolysis was also stimulated, but to a lesser degree, by phosphatidylcholine. Phosphatidylinositol and phosphatidylserine did not significantly enhance GTPase activity. The stimulatory effect of phospholipid was mimicked, in part, by nonionic detergents. p21 may be related to other GTPases, the regulatory guanine nucleotide-binding G proteins of the hormone-sensitive adenylate cyclase complex and transducin of the retinal light-activated phosphodiesterase system. The G proteins and transducin are heterotrimers; the alpha subunits possess GTPase activity and the beta gamma subunit complex along with agonist-receptor complex or light-activated rhodopsin enhance GTP hydrolysis. p21 GTPase activity was slightly stimulated by rhodopsin, but, in contrast to the GTPase activity of transducin, stimulation was not light-dependent. GTP hydrolysis was enhanced somewhat by beta gamma subunit complex in the absence, but not in the presence, of rhodopsin. Like the G proteins and transducin, activity of p21 was altered by ADP-ribosylation. Modification of p21 catalyzed by an NAD: arginine ADP ribosyltransferase purified from turkey erythrocytes decreased both GTPase activity and guanine nucleotide binding activity. PMID- 3001697 TI - Human apolipoprotein B-100: cloning, analysis of liver mRNA, and assignment of the gene to chromosome 2. AB - Human apolipoprotein B-100 (apo B-100) is the major apolipoprotein of low density lipoproteins and the principal ligand for interaction with the low density lipoprotein receptor. The human apo B-100 gene has been inserted into a lambda gt 11 expression vector, and the apo B-100 cDNA clones have been identified by screening with a monospecific apo B-100 antiserum, by screening with synthetic oligonucleotides based on the amino acid sequence of peptides isolated from apo B 100, and by immunoblot analysis of the expressed protein with a monoclonal antibody to apo B-100. The complete nucleotide and derived-amino acid sequence of a 1.7-kilobase cDNA clone of apo B-100 was determined. The 560-amino acid residues of apo B-100 contain no unique linear or repeating sequences of amino acids. The computer-predicted conformation of the apo B-100 protein contains segments of helical structure; however, a large portion of the protein is organized into beta-structure. The beta-structure may be important in lipid-apo B 100 interactions in low density lipoprotein and may contribute to the insolubility of delipidated apo B-100 in aqueous buffers. RNA blot hybridization analysis of liver mRNA utilizing a Nco I/HindIII apo B-100 cDNA probe revealed that the apo B-100 mRNA is 15-18 kilobases long, which is of sufficient size to code for a 250-387 kDa apolipoprotein, the proposed molecular size of delipidated plasma apo B-100. The gene for human apo B-100 has been localized to chromosome 2 by filter hybridization of human-mouse somatic cell hybrids utilizing a 400-base pair Nco I/HindIII apo B-100 cDNA probe. This location is in contrast to the low density lipoprotein receptor that has been localized to chromosome 19. The cloning of human apo B-100 has provided new insights into the structure and physicochemical properties of apo B-100 and will facilitate studies on the factors modulating apo B-100 biosynthesis and the expression of the apo B-100 gene in patients with dyslipoproteinemias. PMID- 3001698 TI - Exonuclease III recognizes urea residues in oxidized DNA. AB - Escherichia coli exonuclease III was found to be associated with an activity that recognizes urea residues in DNA but not thymine glycol residues from which the urea residues were prepared. This activity was not due to a contaminating activity such as endonuclease III since urea-containing DNA was a competitive inhibitor of exonuclease III when apurinic DNA was used as a substrate and vice versa. The apparent kinetic constants for both the substrate and inhibitor were determined. Like its apurinic activity, exonuclease III activity against urea residues was endonucleolytic, nicking on the 5' side of the damage and having an optimal Mg2+ concentration between 2 and 10 mM. Also, the enzyme recognized alkali-stable damages produced in DNA by H2O2 in vitro. We suggest that it may be this activity of exonuclease III that accounts for its biological role in vivo. PMID- 3001699 TI - p27x-III and p21x-III, proteins encoded by the pX sequence of human T-cell leukemia virus type I. AB - Human T-cell leukemia virus type I (HTLV-I) is an etiological agent of adult T cell leukemia and has a unique sequence, pX, that contains four possible open reading frames, I-IV. p40x was previously identified as the gene product of frame IV (x-lor) and was suggested to mediate transcriptional trans-activation of the viral long terminal repeats. We have identified two pX gene products, p27x-III and p21x-III, encoded by frame III, which mostly overlapped frame IV. These proteins were detected with rabbit antiserum against the synthetic peptide predicted from the 3' end of frame III. p27x-III is phosphorylated in cultured cells, and the phosphoprotein (pp27x-III) is localized in nuclei; some pp27x-III was tightly bound to nuclear components. p27x-III was detected in a number of cell lines that express other viral antigens, including a cell line previously reported to express only p40x as a viral protein. The function(s) of p27x-III and p21x-III is not known, but the tight binding of pp27x-III to nuclear components suggests that it is associated with regulation of viral gene expression. PMID- 3001700 TI - Epidermal growth factor regulates the expression of its own receptor. AB - The epidermal growth factor (EGF) receptor gene is the cellular homolog of the avian erythroblastosis virus erbB oncogene. Control of EGF receptor expression determines cellular responsiveness to EGF and might play an important role in neoplastic development. Using RNA blot hybridization, we have found that exposure of human KB carcinoma cells to EGF results in elevated levels of EGF receptor mRNA. The phorbol ester 4 beta-phorbol 12-myristate 13-acetate also stimulates EGF receptor RNA accumulation. Immunoprecipitation of metabolically labeled (30 min) EGF receptor protein revealed that synthesis of new EGF receptor follows the increase in receptor RNA. Addition of cycloheximide together with EGF further enhances EGF receptor RNA accumulation. Results of nuclear runoff-transcription experiments suggest that the stimulatory effects of EGF and cycloheximide are most likely due to a posttranscriptional control mechanism. PMID- 3001701 TI - Properties of the Syrian hamster phosphomannosyl receptor: an aggregate of low molecular weight proteins. AB - Phosphomannosyl receptor (PMR) isolated from Syrian hamster liver was purified to apparent homogeneity by affinity chromatography and one-dimensional PAGE. On one dimensional PAGE, the receptor migrated with a Mr approximately equal to 215,000 as detected by a silver-staining reagent or by immunoblotting [utilizing antiPMR generated against purified hamster PMR (Mr 215,000) sequentially purified by affinity chromatography and NaDodSO4/PAGE]. On two-dimensional PAGE the receptor was partially dissociated into low-molecular weight components. The protein distribution on immunoblots of two-dimensional gels of hamster liver homogenates was nearly identical to that observed for purified hamster liver PMR. When liver homogenates were subjected to one-dimensional PAGE and immunoblotted under nonreducing conditions, an intensely labeled band that migrated with an apparent Mr of 43,000-49,000 was observed; under reducing conditions a single band with a Mr of 49,000 was observed. The low molecular weight compound was present in the soluble 130,000 X g supernatant but not in the particulate fraction of liver homogenates. An immunoreactive component of similar molecular weight was also present in hamster serum and plasma. These results suggest that PMR is either an aggregate comprised of small molecular weight components or, alternatively, that a number of small molecular weight components are tightly associated with PMR. PMID- 3001702 TI - Specific regions of the intervening sequences of beta-globin RNA are resistant to nuclease in 50S heterogeneous nuclear RNA-protein complexes. AB - The specific assembly of heterogeneous nuclear RNA-protein complexes (hnRNPs) containing precursor beta-globin RNA was investigated by using the 50S hnRNP released from chicken reticulocyte nuclei by endogenous nuclease. The nuclease resistant regions were mapped on adult beta-globin intervening sequences (IVS) at the resolution of nucleotides with an RNA mapping method [Patton, J. R. and Chae, C.-B. (1983) J. Biol. Chem. 258, 3991-3995]. We found that there is one 28 nucleotide-long nuclease-resistant region in the first IVS and there are four nuclease-resistant regions in the second IVS. Of particular interest is the presence in 50S hnRNP of a nuclease-resistant region (24-28 nucleotides long) in both IVS immediately upstream from the putative lariat branch site in an RNA splicing intermediate. Our results demonstrate that hnRNPs containing precursor beta-globin RNA are, like those containing mature beta-globin RNA, assembled in a site-specific manner. PMID- 3001703 TI - In vitro synthesis of infectious poliovirus RNA. AB - Replication of the infectious RNA genome of poliovirus is accomplished in cells by the viral RNA polymerase through negative-strand RNA intermediates. Full length negative-strand poliovirus RNA was synthesized in vitro by transcription of infectious poliovirus cDNA with bacteriophage SP6 DNA-dependent RNA polymerase. When provided with this negative-strand RNA as template, the poliovirus RNA-dependent RNA polymerase synthesized full-length positive-strand molecules. The positive-strand RNAs synthesized in vitro were infectious when transfected into HeLa cells. In contrast, positive-strand copies of poliovirus RNA synthesized in vitro by SP6 polymerase, using a poliovirus cDNA template, were not infectious. Production of infectious positive-strand RNA by the poliovirus polymerase was not observed when magnesium or negative-strand RNA template was omitted from the reaction mixture. Infectivity of the product RNA was not destroyed by DNase treatment. The specific infectivity in HeLa cells of in vitro-synthesized positive-strand RNA was 4 X 10(4) plaque-forming units/micrograms of RNA. PMID- 3001704 TI - Identification of a novel receptor in Drosophila for both epidermal growth factor and insulin. AB - The notable amino acid homology among mammalian growth factor receptors with tyrosine-specific protein kinase activity has led to speculation that these receptors derived from a common evolutionary precursor. We report the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100-kDa protein is also related antigenically to the mammalian receptors for EGF and possibly insulin but may not correspond to the mammalian counterpart of either receptor in Drosophila. The Drosophila protein is recognized by antisera directed against the mammalian receptor for EGF in immunoblot hybridizations. It can be affinity labeled with either 125I-labeled insulin or 125I-labeled EGF after immunoprecipitation with anti-EGF receptor antiserum. Excess unlabeled EGF or insulin will block the affinity labeling with either growth factor, suggesting that both EGF and insulin share a common binding site on the 100-kDa Drosophila receptor. This Drosophila protein, therefore, may be closely related to an evolutionary precursor of the mammalian receptors for insulin and EGF. PMID- 3001705 TI - Multistep transformation by defined fragments of herpes simplex virus type 2 DNA: oncogenic region and its gene product. AB - Diploid Syrian hamster embryo cells transfected with Bgl II C fragment of herpes simplex virus type 2 DNA acquired a neoplastic phenotype. Cultures transfected with its left-hand 64% subclone EcoRI/HindIII fragment AE (0.419-0.525 map unit) grew into established but nontumorigenic lines. Transfection of EcoRI/HindIII AE immortalized cells with a 4.4-kilobase Sac I/BamHI subfragment within BamHI E (0.554-0.584 map unit; overlaps the right-hand 16% of Bgl II C) converted them to tumorigenicity. The 4.4-kilobase subfragment encodes a 144-kDa protein immunologically and structurally similar to an infected cell protein designated ICP 10. DNA extracted from cells transformed with the 4.4-kilobase subfragment exhibited discrete hybridizing bands homologous to BamHI E fragment. Monoclonal antibody to ICP 10 precipitated a 144-kDa protein from the transformed cells and stained them in immunofluorescence. A tumor derivative established with the transformed cells did not stain with this antibody, but approximately equal to 25% of the cells stained with a monoclonal antibody to c-myc protooncogene products. PMID- 3001706 TI - Isolation of renin-producing human cells by transfection with three simian virus 40 mutants. AB - A human juxtaglomerular cell (JGC) tumor was used for the immortalization of renin-secreting cells. The transfection of primary JGC with three different simian virus 40 (SV40) mutants resulted in the continuous production of renin secreting cells. The most efficient renin-producing cells (producing about 400 pg of renin per 24 hr per ml of culture medium) were those transfected with the PAS SV40 mutant. The renin production was stable and the cell cultures have been maintained for greater than 1 year. Two types of cells were cultured together and could not be separated: round and birefringent cells, which exhibited features of mast cells, and elongated cells containing myofilaments and secretory granules. Immunocytochemical staining showed the presence of renin in this latter cell type. The renin produced by the transfected cells was not stored within the cells but was released rapidly into the medium. More than 95% of the renin produced was prorenin, which, after activation, had characteristics similar to those of pure human standard renin as to its enzymatic, immunologic, and biochemical properties, except that it was less glycosylated. These stable JGC tumoral cell lines provide a unique system for studying human renin biosynthesis and its regulation in vitro. PMID- 3001707 TI - Inhibition of gonadotropin-induced granulosa cell differentiation by activation of protein kinase C. AB - The induction of granulosa cell differentiation by follicle-stimulating hormone (FSH) is characterized by cellular aggregation, expression of luteinizing hormone (LH) receptors, and biosynthesis of steroidogenic enzymes. These actions of FSH are mediated by activation of adenylate cyclase and cAMP-dependent protein kinase and can be mimicked by choleragen, forskolin, and cAMP analogs. Gonadotropin releasing hormone (GnRH) agonists inhibit these maturation responses in a calcium dependent manner and promote phosphoinositide turnover. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also prevented FSH-induced cell aggregation and suppressed cAMP formation, LH receptor expression, and progesterone production, with an ID50 of 0.2 nM. In FSH-treated cells, PMA did not reduce the initial increase in cAMP formation during the first 24 hr of culture but prevented its secondary increase from 24 to 48 hr. PMA also inhibited LH receptor induction by cholera toxin, forskolin, and 8-bromo-cAMP, but it did not impair cAMP responses to the former two agents, indicating that the site of action of the phorbol ester is distal to adenylate cyclase. The early stimulation of cAMP-dependent protein kinase activity by FSH was also unaffected by PMA, consistent with its lack of effect on the initial cAMP response to FSH. However, PMA caused a marked decrease in cytosolic protein kinase C activity within 1 min of its addition to the cells. The permeant diacylglycerols, 1-oleoyl-2-acetoyl-sn-glycerol and sn-1,2 dioctanoyl glycerol, also inhibited LH receptor formation, while the nonpermeant diacylglycerol, diolein, was inactive. These results indicate that in situ activation of protein kinase C by PMA or permeant diacylglycerols inhibits cAMP dependent granulosa cell differentiation, and suggest that the inhibitory actions of GnRH agonists on granulosa cell maturation are also mediated by protein kinase C. PMID- 3001708 TI - Type beta transforming growth factor controls the adipogenic differentiation of 3T3 fibroblasts. AB - Differentiating mouse 3T3-L1 preadipocytes have been used as a model system to study the ability of type beta transforming growth factor (TGF-beta) to modulate cell development. We find that TGF-beta inhibits potently (ID50 approximately equal to 25 pM) the adipogenic conversion of 3T3-L1 cells. Inhibition is observed only when cells are exposed to TGF-beta before they become committed to differentiation. Even a transient (4 hr) exposure to TGF-beta immediately before the commitment point is sufficient to prevent differentiation. This point coincides with the time point immediately preceding the onset of coordinate expression of differentiation-specific proteins in 3T3-L1 cells. TGF-beta interacts with cell surface receptors in 3T3-L1 cells that have structural and binding properties similar to TGF-beta receptors in other cell types in which TGF beta acts as a growth activator or a growth inhibitor. However, TGF-beta does not markedly alter differentiation-related mitosis in 3T3-L1 cells. The action of TGF beta on 3T3-L1 cells does not involve changes in cAMP or prostaglandin E levels. These results suggest that TGF-beta is a unique modulator of adipogenic differentiation of fibroblasts. PMID- 3001709 TI - Each of three "TATA elements" specifies a subset of the transcription initiation sites at the CYC-1 promoter of Saccharomyces cerevisiae. AB - Transcription initiation of the yeast iso-1-cytochrome c gene (CYC-1) occurs in six major clusters at positions +1, +10, +16, +25, +34, and +43. Potential "TATA elements" lie upstream at positions -154, -106, -52, and -22. Analysis of the TATA region suggests that three of these TATA sequences are functional and contribute to initiation at CYC-1, with the -106 TATA promoting initiation at +1, +10, and +16; the -52 TATA, at +16, +25, +34, and +43; and the -22 TATA, at +34 and +43. Deletions changing the spacing between the TATA sequences and the region of transcription initiation do not change the location of the CYC-1 transcription start points. This finding suggests that at least part of the information determining mRNA initiation sites is encoded within the DNA sequence at the site of transcription initiation. Analysis of 18 yeast RNA polymerase II promoters suggests that two classes of DNA sequences serve as preferred sites for transcription initiation. To test this possibility, we replaced some of the normal CYC-1 start sites with one of these sequences, TCGA, and found that transcription initiates at this newly introduced sequence. These results are in contrast to those from higher eukaryotes, where RNA polymerase II typically initiates transcription a fixed distance downstream from the TATA element. The presence of multiple, functional TATA sequences at CYC-1 is inconsistent with the idea that RNA polymerase or another transcription factor attaches to the template at an upstream activation site and scans for the nearest TATA element. PMID- 3001710 TI - Molecular analysis of chromosome 11 deletions in aniridia-Wilms tumor syndrome. AB - We describe five individuals who have constitutional deletions of the short arm of one chromosome 11, including all or part of the band p13. All of these individuals suffer from aniridia; two have had a Wilms tumor removed. We have established lymphoblastoid cell lines from these and in three cases constructed somatic cell hybrids containing the deleted chromosome 11. Analysis of DNA from the cell lines and hybrids with a cloned cDNA probe has shown that the catalase gene is deleted in four of five patients. The catalase locus must be proximal to the Wilms and aniridia-related loci. We have not detected a deletion of the beta globin or calcitonin genes in any of these individuals; we conclude these genes are likely to be outside the region 11p12-11p15.4. In addition, we have used monoclonal antibodies in fluorescence-activated cell sorting analysis to measure expression in the hybrids of two cell surface markers encoded by genes that map to the short arm of chromosome 11. The genes for both of these are deleted in two individuals but are present in the individual with the smallest deletion. PMID- 3001712 TI - HLA-B locus polymorphism: studies with a specific hybridization probe. AB - The large number of class I histocompatibility genes (HLA) and their extensive homology has made it difficult to assign bands on genomic Southern blots to known genes. Therefore, we have tried to obtain nucleic acid probes for class I genes that are locus specific or have restricted locus specificity. Computer sequence homology analysis was used to compare the nucleic acid sequences of two genomic clones, one coding for the HLA-B7 antigen (JY150) and one containing a class I pseudogene (pHLA12.4). A sequence in the 3' untranslated region with very low homology was identified. This sequence from the HLA-B7 gene was subcloned into M13 phage. This fragment, JY150/C5, hybridized with two genomic bands in DNA from human HLA homozygotes--presumably the HLA-B locus gene and a closely related gene. The probe was used to assess restriction fragment polymorphism at the HLA-B locus in homozygous consanguineous cell lines. This analysis permitted the association of certain polymorphic restriction enzyme fragments with some alleles of this locus. However, many HLA-B alleles have identical restriction fragments produced by a number of restriction endonucleases. PMID- 3001711 TI - The lacI shuttle: rapid analysis of the mutagenic specificity of ultraviolet light in human cells. AB - A system has been devised that allows the effect of mutagens acting in human cells to be readily analyzed at the DNA sequence level. The bacterial gene lacI, carried on a shuttle vector, is introduced into human tissue culture cells by transfection and allowed to replicate in the cell nucleus. Twenty-four to 48 hr after transfection, the cells are exposed to a mutagen. After 1-2 days of further replication, vector DNA is purified and transfected back into Escherichia coli for scoring and analysis of mutations in lacI. The nucleotide sequence changes for 53 UV light-induced mutations have been deduced in this way. Most of the mutations are transitions and occur at pyrimidine-pyrimidine sequences. The mutagenic specificity observed closely resembles that of UV light in E. coli, suggesting that human and bacterial cells respond similarly to damage from UV light. Use of the lacI shuttle in this way should permit determination of the mutagenic specificity of a wide range of mutagens and carcinogens in human cells. PMID- 3001713 TI - Murine plasma cell membrane antigen PC-1: molecular cloning of cDNA and analysis of expression. AB - PC-1 is a membrane glycoprotein expressed selectively on murine antibody secreting plasma cells. Previously, we have obtained partial amino acid sequence data for this protein. Here, we describe the use of these data in the isolation of PC-1 cDNA clones. To avoid the "3'-bias" of conventional cDNA libraries we constructed a cDNA library in lambda gt10 by priming the first strand of cDNA synthesis with random hexadeoxynucleotides. The library was screened with oligonucleotide probes, 17 nucleotides in length, the design of which was based on the amino acid sequence data. Two cDNA clones of 1.0 and 0.9 kilobase pairs (lambda RR3 and lambda RR20, respectively) were isolated. Both contained a sequence encoding the 15-residue tryptic peptide that was used to derive the sequences of the oligonucleotide probes. lambda RR3 cDNA hybridized to an approximately equal to 3.5-kilobase mRNA that was present in plasmacytomas, spleen, and liver but not in other cell types screened. We were unable to detect PC-1 mRNA or protein in mouse brain. In the spleens of mice chronically infected with Mesocestoides corti, PC-1 mRNA was present at 2.5-fold higher levels than found in normal mouse spleens, whereas immunoglobulin mRNA levels were 15-fold higher. Southern blot analyses revealed the presence of only one gene copy per haploid mouse genome. Restriction fragment length polymorphisms were detected in genomic DNA from mice bearing different PC-1 alleles. A related gene is present in rat genomic DNA. PMID- 3001715 TI - Human T-cell growth factor (interleukin 2) and gamma-interferon genes: expression in human T-lymphotropic virus type III- and type I-infected cells. AB - Acquired immune deficiency syndrome (AIDS) is characterized by severe depletion of OKT4+ T lymphocytes and leukemia is associated with abnormal proliferation of maturation-arrested lymphocytes. Human T-lymphotropic virus type III (HTLV-III) or lymphoadenopathy virus (LAV) and type I (HTLV-I) are etiologically linked to AIDS and adult T-cell leukemia/lymphoma, respectively. T-cell growth factor (TCGF, also known as interleukin 2) is required for the growth of activated T cells, which play an important role in immune regulation. gamma-Interferon (IFN gamma) is also implicated in immune modulation. It was possible that T-cell depletion in acquired immune deficiency syndrome could be due to an impairment of TCGF synthesis and that adult T-cell leukemia could be due to unregulated production of TCGF. The results reported here show that the transcription of the TCGF gene was not impaired in cultured HTLV-III-infected cells. Paradoxically, the TCGF gene in HTLV-I-infected cells was transcriptionally inactive. The reverse was the case for the gamma-interferon gene--it was actively transcribed in HTLV-I-infected cells but not in the HTLV-III-infected and virus-producing H9 and H4 cell line. No evidence was obtained suggesting abnormal regulation of the TCGF or of the IFN-gamma gene consequent to HTLV-III infection. It thus appears that in both HTLV-III and HTLV-I infection, growth control and immune regulatory mechanisms may bypass a modulatory role of TCGF or of IFN-gamma. PMID- 3001714 TI - A pertussis toxin-sensitive GTP-binding protein in the human neutrophil regulates multiple receptors, calcium mobilization, and lectin-induced capping. AB - Human neutrophils treated with pertussis toxin had decreased functional responses to several agents including zymosan-treated serum, heat-aggregated immunoglobulin, platelet-activating factor, and fMet-Leu-Phe. Responses affected include superoxide generation and release of lysozyme. The degree and type of inhibition was dependent on the individual receptor and the cellular response studied. Measurement of intracellular calcium levels with quin-2 showed that both fMet-Leu-Phe- and platelet-activating factor-mediated increases in quin-2 fluorescence were diminished as a result of pertussis toxin treatment. fMet-Leu Phe-mediated calcium uptake was also inhibited. However, under conditions where fMet-Leu-Phe-mediated effects on cell function were completely abolished, only a partial inhibition of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8) sensitive calcium uptake was observed. A study of the linked reactions of chemotaxis, capping, and shape change revealed that chemotaxis was inhibited regardless of the chemoattractant utilized (zymosan-treated serum, fMet-Leu-Phe, and platelet-activating factor) and the associated reactions of Con A capping and fMet-Leu-Phe- or Con A-mediated shape change were reduced in pertussis toxin treated cells. Our results suggest that multiple mediators of inflammation act through a pertussis toxin-sensitive GTP-binding protein that regulates the mobilization of internal calcium as well as calcium uptake and is, in addition, a key control element of shape change, capping, and chemotaxis. PMID- 3001716 TI - Independent segregation of two antigenic specificities (VP3 and VP7) involved in neutralization of rotavirus infectivity. AB - Antiserum prepared against the M37 strain of rotavirus, recovered from an asymptomatic newborn infant in Venezuela, neutralized two prototype human rotaviruses that define two separate serotypes: serotype 1 (Wa) and serotype 4 (ST3). Thus, the M37 strain is a naturally occurring intertypic rotavirus. Analysis of reassortant viruses produced during coinfection in vitro indicated that the observed dual serotype specificity of M37 resulted from sharing a related outer capsid protein, VP3, with the ST3 virus and another related outer capsid protein, VP7, with the Wa virus. Analysis of single (VP3)-gene substitution reassortants indicated that VP3 was as potent an immunogen as VP7. In addition, direct evidence was obtained that the serotype specificity of neutralizing antibody elicited by VP3 can differ from the serotype specificity of neutralizing antibody elicited by VP7, indicating the need for a dual system of rotavirus classification in which the neutralization specificity of both VP3 and VP7 outer capsid proteins are identified. PMID- 3001717 TI - Cloning, sequencing, and chromosomal localization of human term placental alkaline phosphatase cDNA. AB - A human term (third trimester) placental alkaline phosphatase (PLAP; EC 3.1.3.1) cDNA was isolated from a human placental lambda gt11 cDNA library. The expression library was screened by using rabbit antibodies against PLAP and oligonucleotide probes. DNA sequence analysis of a positive clone with an insert of 2.7 kilobase pairs allowed us to predict the complete amino acid sequence of PLAP (530 residues), which coincided with the reported 42 N-terminal amino acid sequence of PLAP except at position 3. Contrary to the previous supposition that there was no amino acid sequence homology between PLAP and Escherichia coli alkaline phosphatase (471 residues), we found 30% overall homology, with regions of strong homology including the putative active site and the metal-binding sites. The 44 residue C-terminal extension of PLAP has a stretch of 17 hydrophobic amino acids, which presumably anchors the protein to the plasma membrane, a change perhaps necessary for the transition from a bacterial periplasmic enzyme to a mammalian membrane-associated enzyme. We have also localized PLAP-related DNA sequences mainly on chromosome 2 and to a lesser degree on chromosome 17. It seems likely therefore that the PLAP gene resides on chromosome 2 and other member(s) of the alkaline phosphatase family may exist (on this chromosome and) on chromosome 17. PMID- 3001718 TI - Kinetic properties of a sex pheromone-degrading enzyme: the sensillar esterase of Antheraea polyphemus. AB - Behavioral and electrophysiological evidence has suggested that sex pheromone is rapidly inactivated within the sensory hairs soon after initiation of the action potential spike. We report the isolation and characterization of a sex-pheromone degrading enzyme from the sensory hairs of the silkmoth Antheraea polyphemus. In the presence of this enzyme at physiological concentration, the pheromone [(6E,11Z)-hexadecadienyl acetate] has an estimated half-life of 15 msec. Our findings suggest a molecular model for pheromone reception in which a previously reported pheromone-binding protein acts as a pheromone carrier, and an enzyme acts as a rapid pheromone inactivator, maintaining a low stimulus noise level within the sensory hairs. PMID- 3001719 TI - The gene for human p53 cellular tumor antigen is located on chromosome 17 short arm (17p13). AB - A clone that cross-hybridizes with a mouse p53 probe has been isolated from a cDNA library of simian virus 40-transformed human fibroblasts. This cloned human p53 cDNA was used as a probe to examine DNAs obtained from human-rodent somatic cell hybrids that have segregated human chromosomes. The results show that the human p53 gene is located on chromosome 17. In addition, Southern analysis of hybrids prepared from human cells containing a chromosome 17 translocation allowed regional localization of the human p53 gene to the most distal band on the short arm of this chromosome (17p13). Localization of the p53 gene to 17p13 was confirmed by in situ hybridization of metaphase spreads with the human p53 probe. PMID- 3001720 TI - Construction and properties of Tn917-lac, a transposon derivative that mediates transcriptional gene fusions in Bacillus subtilis. AB - A derivative of Tn917 was constructed, referred to as Tn917-lac, which is capable of generating fusions that connect the transcripts of Bacillus subtilis chromosomal genes to the coding sequence of the lacZ gene of Escherichia coli. Two independent insertions of Tn917-lac into the gltA gene and one insertion into the trpE gene (in the trpEDCFBA operon) of B. subtilis were studied in detail, and the results confirmed that Tn917-lac-mediated transcriptional fusions produce levels of beta-galactosidase that reflect accurately the regulated expression of interrupted genes. To facilitate these studies, a procedure was developed that permits the analysis of Tn917-lac-mediated fusions in partial diploids where insertional mutations are complemented by an intact copy of the interrupted genes. Tn917 is known to function efficiently in bacteria representing three quite different Gram-positive genera (Streptococcus, Bacillus, and Staphylococcus) and is known to display a relatively high degree of randomness in its insertions into bacterial genomes, making it likely that Tn917-lac will be useful for the identification and study of many kinds of regulated genes in a wide range of Gram-positive species. PMID- 3001721 TI - Two types of immunoglobulin-negative Abelson murine leukemia virus-transformed cells: implications for B-lymphocyte differentiation. AB - Both alleles of immunoglobulin (Ig) heavy-chain joining region (JH) genes in three Ig-negative Abelson murine leukemia virus (Ab-MuLV)-transformed cell lines were characterized by DNA cloning and nucleotide sequence determination. These studies unambiguously identified two distinct types of Ig-negative B-lineage cells. The first type of cell (e.g., R8) is an "immature pre-B cell," and it contains at least one intermediate recombinant structure containing heavy-chain diversity (DH) and JH sequences but no variable region (VH) sequence. This type of cell, which has also been characterized by other investigators, generates mu positive sublines during subsequent culturing of cells and represents a precursor stage to pre-B cells. The second type of cell (e.g., RAW253) is an "abortive pre B cell," in that both JH alleles contain nonfunctional VH-DH-JH structures. The nucleotide sequence determinations in this study demonstrated that these nonfunctional V-D-J structures were generated by nonproductive somatic recombinations, involving either out-of-phase joining events, or the formation of termination codons in the DH coding sequences. The identification of abortive pre B cells suggests that the recombinational joining of Ig VH, DH, and JH segments is not actively regulated by a putative recombinase to preserve the translational reading frame. This in turn implies that a large portion of precursor cells at the early stage of B-cell differentiation are abortive and possibly blocked to further differentiation. PMID- 3001722 TI - Characterization and distribution of receptors for the atrial natriuretic peptides in mammalian brain. AB - Both rat 125I-labeled atrial natriuretic polypeptide [125I-ANP or atrial natriuretic factor fragment ANF-(99-126)] and human 125I-ANP [125I-alpha-ANP or human ANF-(99-126)] bind with high specificity and affinity (Kd = 20-80 pM) to an apparent single class of sites in guinea pig brain. The ligand selectivity pattern demonstrates that ANF-(101-126) greater than ANF-(99-126) greater than ANF-(103-125) greater than ANF-(103-123) on 125I-alpha-ANP binding sites. [International nomenclature starting at the end of the signal peptide of the recently sequenced prepropeptide is used; thus, ANF-(101-126) corresponds to the earlier designation ANF-(8-33), ANF-(103-123) to rat atriopeptin I, and ANF-(103 125) to rat atriopeptin II.] Similar results have been reported in peripheral tissues, which indicate that central and peripheral ANP binding sites have fairly similar structural requirements. In vitro receptor autoradiography shows that in the guinea pig brain, 125I-ANP binding sites are highly concentrated in the external plexiform layer of the olfactory bulb, subfornical organ, various thalamic nuclei, medial geniculate nucleus, and cerebellum. Lower densities are found in the central nucleus of the amygdala, dentate gyrus, hippocampus, and area postrema. Most remaining regions contain much lower densities of sites. In rat brain, 125I-ANP binding sites are differentially distributed, with high densities in the subfornical organ, area postrema, and linings of ventricles but low densities in the thalamus and cerebellum. In monkey brain, 125I-ANP binding sites are concentrated in the cerebellum. The presence of high densities of 125I ANP binding sites in various brain regions strongly suggests the existence of a family of brain-heart peptides, in analogy to the well-known brain-gut peptides. Moreover, the extensive distribution of 125I-ANP binding sites in mammalian brain suggests that the possible roles of ANP/ANF-like peptides in brain are not restricted to the central regulation of cardiovascular parameters. PMID- 3001723 TI - Two prohormones for gastrin-releasing peptide are encoded by two mRNAs differing by 19 nucleotides. AB - In our studies on the molecular biology of human gastrin-releasing peptide (GRP), we have discovered an example of a change in translational reading frame apparently produced through alternative RNA splicing. Complementary DNAs prepared from a pulmonary carcinoid tumor rich in GRP immunoreactivity had one of two different-sized internal DNA fragments after digestion with the restriction enzyme Pvu II. Nucleotide sequences of the two DNA fragments were identical except for 19 additional nucleotides present in the larger fragment. The region of the mRNA containing the 19 nucleotides corresponded to the carboxyl-terminal region of the human GRP precursor. The resulting shift in reading frame causes a difference of 10 amino acids in size and an overall sequence difference of 27 amino acids between the two GRP prohormones so formed. The change in reading frame described here is unusual in eukaryotes and is yet another mechanism to produce diversity in the generation of biological peptides. PMID- 3001724 TI - Molecular analysis of a deletion mutant provirus of type I human T-cell lymphotropic virus: evidence for a doubly spliced x-lor mRNA. AB - The genome of the human T-cell leukemia/lymphotropic virus type I (HTLV-I) contains a functional gene denominated x-lor that may be important in HTLV-I transformation of human T cells. To study the role of x-lor and other HTLV-I genes in cellular transformation, we obtained a transformed nonproducer human T cell line containing a single defective HTLV-I provirus (HTLV-I 55/PL). This 7 kilobase provirus had undergone a deletion involving the entire envelope gene and the nonconserved region. The point of the deletion corresponded to the junction of a donor splice site, located between the polymerase gene and the envelope gene (nucleotide 5183), and the acceptor site for the mRNA of the x-lor gene (nucleotide 7302). The juxtaposition of nucleotides 5182 and 7302 brings the initiating methionine codon of the envelope gene immediately 5' to the x-lor region, leaving the DNA sequence in frame for expression of a protein product. This finding suggests that a double splicing mechanism is used to express the x lor gene, and that the defective provirus 55/PL was generated through the reverse transcription of a partially spliced mRNA. Analysis of the x-lor mRNA of other HTLV-I-transformed cell lines revealed that a double splicing process is commonly used. Furthermore, since 55/PL can be faithfully transmitted and is able to immortalize recipient T cells, we can conclude that the envelope gene is not necessary for in vitro transformation by HTLV-I. PMID- 3001726 TI - Characterization of solubilized human and rat brain beta-endorphin-receptor complex. AB - Opioid receptors have been solubilized from human striatal and rat whole-brain membranes by use of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Tritiated human beta-endorphin (3H-beta h-EP) binding revealed high affinity competition by morphine, naloxone, and various beta-EP analogues, suggesting predominantly mu-type binding. Lack of high-affinity competition by (+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneaceta mide methanesulfonate (U50-488, Upjohn) indicated that kappa sites were not labeled by 3H-beta h-EP under these conditions. Affinities were similar in both soluble and membrane preparations except for [Met]enkephalin, which appears to be rapidly degraded by the solubilized extract. Size differences between human and rat solubilized 3H-beta h-EP-receptor complexes were revealed by exclusion chromatography. PMID- 3001725 TI - An arachidonate metabolite is involved in the conversion from alpha 1- to beta adrenergic glycogenolysis in isolated rat liver cells. AB - In vitro incubation of isolated rat liver cells in a serum-free buffer leads to the suppression of the glycogenolytic effect of phenylephrine and the simultaneous emergence of a glycogenolytic response to isoproterenol within 4 hr. This time-dependent conversion of the adrenergic receptor response from alpha 1 to beta type is prevented by the presence in the incubation medium of 0.5% fatty acid-free, but not regular, bovine serum albumin. A 20-min exposure of freshly isolated liver cells to arachidonic acid (10 micrograms/ml), but not to stearic or palmitic acid, causes an acute shift in the receptor response from alpha 1 to mixed alpha 1/beta type, similar in direction to that seen after prolonged incubation of the cells. This effect of arachidonic acid is prevented by 0.2 microM ibuprofen but not by the same concentration of nordihydroguaiaretic acid. Ibuprofen (1 microM) or indomethacin (1 microM) also inhibits the time-dependent shift in the receptor response. Actinomycin D inhibits the change in receptor response that is caused by prolonged incubation but not the change that is caused by exogenous arachidonic acid. It is proposed that the time-dependent conversion from alpha 1- to beta-adrenergic receptor-mediated glycogenolysis in isolated rat liver cells is related to a parallel increase in the phospholipase-mediated release of arachidonic acid and the subsequent formation of a key cyclooxygenase metabolite. A protein factor appears to be involved in the regulation of the release of arachidonic acid but not in the action of its metabolite. A possible mechanism by which this metabolite may regulate inverse changes in the coupling of alpha 1- and beta-receptors to postreceptor pathways is discussed. PMID- 3001727 TI - Phosphorylation and inactivation of protein phosphatase 1 by pp60v-src. AB - Protein phosphatase 1, one of four major protein phosphatases involved in cellular regulation, was phosphorylated in vitro by pp60v-src, the transforming gene product of Rous sarcoma virus. Phosphorylation was accompanied by a loss of protein phosphatase activity. The inactivation of protein phosphatase 1 was time dependent and the extent of inactivation correlated closely with the stoichiometry of phosphorylation. Under optimal conditions, 0.34 +/- 0.01 mol of phosphate were incorporated per mol of protein phosphatase and the activity of the enzyme was decreased by 39 +/- 2%. The inactivation required the presence of both MgATP and pp60v-src. There was no loss of activity when adenosine 5'-[beta gamma-imido]triphosphate was used in place of ATP. Phosphorylation of protein phosphatase 1 occurred exclusively on tyrosine residues and was blocked by specific antibodies to pp60v-src. During preincubation of pp60v-src at 41 degrees C, its protein kinase activity towards casein was lost rapidly. The ability of pp60v-src to phosphorylate and inactivate protein phosphatase 1 declined in parallel with the loss of casein kinase activity. Limited chymotryptic digestion of 32P-labeled protein phosphatase 1 (Mr 37,000) resulted in its quantitative conversion to a Mr 33,000 species. Conversion to this species was accompanied by the loss of 32P-labeling and by reactivation of the protein phosphatase. When various concentrations of chymotrypsin were used in the digestion, there was a close correlation between conversion to the Mr 33,000 species and the restoration of protein phosphatase activity. pp60v-src was unable to phosphorylate or inactivate a partially proteolyzed species of protein phosphatase 1 (Mr 33,000/34,000). PMID- 3001728 TI - Evidence against use of bacterial amino acid sequence data for construction of all-inclusive phylogenetic trees. AB - It has been proposed that phylogenetic trees, intended to show divergence of eukaryotic protein and nucleic acid sequences, be extended to include those from bacteria. However, we have compared the amino acid sequences of 18 of the most divergent mitochondrial cytochromes c with those of 18 bacterial cytochromes c2 and have found that the average percentage difference between these mitochondrial cytochromes c and cytochromes c2 was not significantly greater than that among the cytochromes c2 alone. The large discontinuities in physical-chemical properties recognized between the prokaryote and eukaryote cytochromes render it highly improbable that members of the two classes should be no more different from one another than members of either class alone, assuming that sequence differences can accurately reveal evolutionary divergence. Instead, we propose that divergent amino acid sequences approach a limit of change considerably less than for comparison of random sequences. This limit of change presumably is determined by the structure/function relationship. When two homologous protein sequences have reached such a limit, convergence or back-mutations and parallel mutations become as frequent as divergent mutations. As two diverging proteins approach this steady-state condition, sequence differences no longer reflect the numbers of mutations resulting in amino acid substitution and therefore species cannot be positioned on a phylogenetic tree. Insertions and deletions are less reversible than are amino acid substitutions and, provided they are well documented, might be more reliable indicators of bacterial relationships. Nevertheless, we suggest that data available on bacterial protein sequences do not permit construction of all-inclusive phylogenetic trees. Comparisons of protein and rRNA trees suggest that similar restrictions apply to use of rRNA sequence data. PMID- 3001729 TI - Transcriptional control of herpesvirus gene expression: gene functions required for positive and negative regulation. AB - We have used an in vitro nuclear run-off assay to measure the levels of transcription of specific herpes simplex virus genes at different times during a lytic infection. We analyzed the effects of inhibition of DNA replication and of defects in two herpes simplex virus regulatory proteins on the transcription of these genes. We present evidence that the transcription of the alpha ICP4 gene is negatively regulated during a lytic infection. The regulation of ICP4 gene transcription requires the beta protein ICP8 (where ICP = infected cell polypeptide). Transcription of the beta ICP8, gamma 1 ICP5, and gamma 2 glycoprotein C (gC) genes was dependent on ICP4, and transcription of the gamma 2gC gene was strongly inhibited when DNA replication was blocked. Defects in ICP8 also resulted in increased levels of transcription of the ICP4, ICP8, ICP5, and gC genes from parental viral genomes. Our results suggest that ICP8 may be important in maintaining the highly ordered cascade of viral gene expression. PMID- 3001730 TI - Molecular cloning of the neu gene: absence of gross structural alteration in oncogenic alleles. AB - The neu gene is distantly related to the erbB gene and encodes a cell surface protein that appears to function as a growth factor receptor. To study the mechanisms that caused the conversion of the normal neu gene to an oncogenic allele, we have isolated molecular clones of the neu oncogene as well as a clone of the corresponding protooncogene. The transforming neu oncogene and the proto neu gene clones exhibit identical restriction enzyme patterns. Amplification of the proto-neu gene in NIH 3T3 cells by means of cotransfection with a dihydrofolate reductase gene resulted in methotrexate-resistant colonies that produce high levels of normal neu-encoded p185 protein. In contrast to cells carrying low levels of the oncogene-encoded protein, these cells appeared normal. The results suggest that the lesion that led to activation of the neu gene is a minor change in DNA sequence and is apparently located in the protein-encoding region of the gene. PMID- 3001731 TI - Variable stoichiometry of phosphate-linked anion exchange in Streptococcus lactis: implications for the mechanism of sugar phosphate transport by bacteria. AB - Phosphate/2-deoxyglucose 6-phosphate antiport in Streptococcus lactis showed an exchange stoichiometry that varied over a 2-fold range when assay pH was shifted between pH 8.2 and pH 5.2. At pH 7.0 and above, 2 mol of phosphate moved per mol of sugar phosphate; at pH 6.1 the ratio was 1.5:1, while at pH 5.2 the overall stoichiometry fell to 1.1:1. This pattern was not affected by valinomycin in potassium-based media, nor could variable stoichiometry be attributed to altered hydrolysis of the sugar phosphate substrate. In kinetic studies at pH 7.0 or pH 5.2, sugar 6-phosphate was a competitive inhibitor of phosphate transport, indicating operation of a single system. Parallel tests showed that the affinity of antiport for its sugar 6-phosphate substrate was insensitive to pH in this range. Overall, such results suggest a neutral exchange that has specificity for monovalent phosphate but that selects randomly among the available mono- and divalent sugar 6-phosphates. A simple model that shows this behavior suggests a mechanistic role for anion exchange in bacterial transport of sugar phosphate or other organic anions. PMID- 3001732 TI - Epidermal growth factor-nonresponsive 3T3 variants do not contain epidermal growth factor receptor-related antigens or mRNA. AB - We have previously isolated three independent variants of Swiss 3T3 cells that are unable to generate a mitogenic response to epidermal growth factor (EGF). Each of the variants is unable to bind 125I-labeled EGF; each lacks a functional EGF receptor. We used an antiserum to murine EGF receptor to look for an EGF receptor gene product in wild-type 3T3 cells and in the three EGF-nonresponsive variants. No cross-reactive material could be detected in any of the three variants, either in 125I-labeled cell extracts or in [35S]methionine metabolically labeled cells. 3T3 cells contained mRNA molecules homologous to a cDNA probe for the human EGF-receptor coding region. In contrast, no homologous RNA could be detected in any of the three variants. Analysis of genomic Southern blots of the DNA from 3T3 cells and the three EGF-nonresponsive variants indicated sequences from the EGF-receptor gene are present in the DNA of all four cell lines. These EGF-nonresponsive lines, which demonstrate proliferative responses to a variety of mitogens, will be ideal recipients for structure function studies of the EGF receptor by transfection of the cloned gene. PMID- 3001733 TI - Evidence for a tyrosine protonation change during the primary phototransition of bacteriorhodopsin at low temperature. AB - Isotopically labeled tyrosines have been selectively incorporated into bacteriorhodopsin (bR). A comparison of the low-temperature bR570 to K Fourier transform infrared-difference spectra of these samples and normal bR provides information about the role of tyrosine in the primary phototransition. Several tyrosine contributions to the difference spectrum are found. These results and comparison with the spectra of model compounds suggest that a tyrosinate group protonates during the bR570 to K transition. This conclusion is strongly supported by the results of UV difference spectroscopy. PMID- 3001734 TI - Homology between nucleotide sequences of promoter regions of nah and sal operons of NAH7 plasmid of Pseudomonas putida. AB - The in vivo transcription start sites of the nah and sal operons of the NAH7 plasmid were determined by S1 nuclease mapping and the nucleotide sequence surrounding these transcription start sites was determined. Since expression of both of these operons is coordinately controlled by the product of the transcriptional activator gene nahR, the sequences were compared to locate potential sites involved in common regulation. In the 100-base-pair region preceding transcription start sites of both operons, three regions of extensive homology were found and may be involved in nahR-mediated transcriptional control: between -80 and -60 with 81% homology; between -40 and -28 with 75% homology; between -1 and +15 with 70% homology. Comparison of the promoter sequences of nah and sal with the analogous sequences of the xylABC and xylDEFG operons of the TOL plasmid showed little homology between the 5' regions of these two sets of positively regulated hydrocarbon degradation operons. In addition, the transcription start site of the nahR regulatory gene was located and its promoter sequence was determined. The nahR promoter overlapped at the -35 position of the sal promoter; however, the nahR gene is transcribed in the opposite direction. Sequences similar to the consensus sequences of Escherichia coli promoters (at 35 and -10) were found in nah, sal, and nahR at the appropriate positions. PMID- 3001735 TI - Molecular characterization of the Drosophila vermilion locus and its suppressible alleles. AB - We have cloned vermilion (v), one of the genes required for brown eye pigment synthesis in Drosophila, using a mutant (vH2a) "tagged" with the transposon P factor. Mutations that disrupt v gene expression are clustered within approximately 2 kilobases of DNA. A 1.4-kilobase transcript, homologous to this same region, is present in v+ RNA but absent in RNA from several v mutants. The spontaneous v alleles that are suppressed by the suppressor of sable [su(s)] are apparently identical insertions of 412, a copia-like transposable element. Preliminary evidence suggests that su(s)-suppressible alleles at other loci may also be 412 insertions. PMID- 3001736 TI - Sequence evolution within populations under multiple types of mutation. AB - DNA sequence and restriction map data from natural populations can be used to estimate the phylogenetic history of observed sequences. Studies of this sort usually examine large regions of DNA where many evolutionary events have taken place. From such data, detailed phylogenies can be constructed and qualitatively different kinds of mutational and substitutional processes can be studied. The value of investigating more than one mutational type is in the power of comparing relative rates of these distinct substitutional processes. In this paper, we construct a neutral model to describe the frequencies of sequence haplotypes according to the haplotypes from which they arose. This theory of the frequency of haplotypes (incorporating their historical past) is applied to data from the alcohol dehydrogenase gene region of Drosophila melanogaster. The observed patterns of change associated with transposable elements around the Adh locus are not in accord with a neutral model. Values for the mutation rates cannot be found that will bring the observed data into agreement with a simple neutral model, but with the addition of mildly deleterious selection, the model can explain these patterns of change. These results suggest that transposable elements are deleterious to the organisms carrying them, but at levels only several times their rate of transpositional insertion. Similar analyses of small deletions in the Adh region suggest that they may experience mildly deleterious selection. PMID- 3001737 TI - Evidence for rearrangement, amplification, and expression of c-myc in a human glioblastoma. AB - Amplified cellular genes are frequently manifested in one of two cytologically recognizable forms, double minutes or homogeneously staining regions. Additionally, evidence is accumulating that aberrant expression of cellular genes (including oncogenes) may be mediated by gene amplification. We now describe the amplification and expression of the cellular oncogene c-myc in double-minute containing cells from a patient with glioblastoma multiforme, and we have shown that the amplification is associated with rearrangement of the c-myc gene. This finding further supports the common association of the myc gene family in neurogenic tumors and provides evidence of myc gene amplification in human brain cancer. PMID- 3001739 TI - Influence of dietary fiber on the intestinal environment. PMID- 3001740 TI - Dietary fiber and the glycemic response. AB - Addition of purified fiber to carbohydrate test meals has been shown to flatten the glycemic response in both normal and diabetic volunteers, reduce the insulin requirement in patients on the artificial pancreas and in the longer term reduce urinary glucose loss and improve diabetes control. In the context of high fiber high carbohydrate diets these findings have had a major impact in influencing recommendations for the dietary management of diabetes internationally. The mechanism of action appears in part to be due to the effect of fiber in slowing absorption rather than by increasing colonic losses of carbohydrate. Consequently postprandial GIP and insulin levels are reduced and the more viscous purified fibers (e.g., guar and pectin) appear most effective. In addition it has been suggested that colonic fermentation products of fiber may enhance glucose utilization. More recently it has become clear that many aspects of carbohydrate foods (food form, antinutrients, etc.) in addition to fiber may influence the rate of digestion and has led to a classification especially of starchy foods in terms of glycemic index to define the degree to which equicarbohydrate portions of different foods raise the blood glucose. Use of such data may maximize the effectiveness of high carbohydrate and high fiber diets in the management of diabetes and related disorders. PMID- 3001738 TI - Optical measurements of intracellular pH in single LLC-PK1 cells: demonstration of Cl-HCO3 exchange. AB - An optical method was used to continuously monitor intracellular pH (pHi) in single cultured LLC-PK1 cells. Rapidly growing or quiescent cells, attached to coverslips, were loaded with the pH-sensitive dye 4',5'-dimethyl-5(and -6) carboxyfluorescein by exposing them to the dye's permeant precursor. pHi was calculated from the intracellular absorbance spectrum of the dye in a single cell. For cells incubated in HCO3(-)-free Ringer's solution, pHi recovered exponentially from acid loads applied by NH+4 prepulsing. Because the recovery was Na+-dependent and amiloride-sensitive, it was probably caused by Na-H exchange at the plasma membrane. In HCO3- Ringer's solution, external Cl- removal caused pHi to reversibly increase by approximately equal to 0.3. This pHi increase was substantially reduced by 50 microM 4,4'-diisothiocyanostilbene-2,2' disulfonate (DIDS) or by conducting the Cl- removal in the nominal absence of HCO3-. Reducing [HCO3-]o from 25 to 5 mM at constant pCO2 (lowering pHo from 7.4 to 6.7) caused pHi to reversibly fall by approximately equal to 0.2. This pHi change was greatly diminished by DIDS, by removal of extracellular Cl-, or by performing the same pHo shift in the nominal absence of HCO3-. The pHi changes induced by altering [Cl-]o or pHo were not inhibited by Na+ removal. Our data indicate that LLC-PK1 cells possess a Na-independent Cl-HCO3 exchanger and that this transporter may be as important as the Na-H exchanger in determining pHi. PMID- 3001742 TI - Dietary fiber and lipid metabolism. PMID- 3001741 TI - Dietary fibers and absorption of nutrients. PMID- 3001743 TI - Lymphocyte protein kinase activity in cells from young and elderly men and women. AB - Protein kinase activity of lymphocytes isolated from human subjects was assayed using histone as substrate. The activity was stimulated about twofold by cyclic AMP and total enzyme activity, determined in the presence of cyclic AMP, was inhibited by 65% by the specific heat-stable inhibitor of cyclic AMP-dependent protein kinase. Histone phosphorylation was not stimulated by cyclic GMP in the presence of the inhibitor. Cyclic AMP-dependent protein kinase could be activated in vitro by incubating intact cells with isoproterenol or with forskolin and was reflected by a significant (P less than 0.05) increase in the protein kinase activity ratio. In contrast to these well-characterized adenylate cyclase activators, incubating cells for up to 2 hr in vitro in the presence of the specific beta-blocker propranolol had no significant effect on the amount of cyclic AMP-dependent protein kinase that was in the activated state. When compared in subjects between the ages of 21 and 74 years, lymphocyte protein kinase activity was unaltered by age or gender. These results indicate that cyclic nucleotide-dependent protein kinase is of the cyclic AMP-dependent variety in the human lymphocyte. A low amount of the cyclic AMP-dependent activity (about 15%) is in the already activated state in freshly isolated cells, and this is not further reduced by incubation in vitro or by beta-blockade. In contrast to previously reported changes in the capacity to synthesize cyclic AMP, lymphocyte protein kinase is unaltered by gender or age in human subjects. PMID- 3001744 TI - A new heat-stable regulatory factor is associated with aortic polycation modulated (PCM)-phosphatase. AB - An aortic phosphatase which dephosphorylates several proteins including phosphorylase a and the 20-kDa myosin light chains is subject to modulation in vitro by polycationic effectors such as lysine-rich histone-H1 and polylysine. This study was based on the hypothesis that polycationic modulation of expressed enzymic activity involves interactions between the effectors and a regulatory site associated with the polycation-modulated (PCM)-phosphatase. Basal PCM phosphatase activity expressed against myocardial myosin light chains (MLC, 1258 nmole/min/mg) was about eightfold greater than activity expressed against phosphorylase a (149 nmole/min/mg). However, dephosphorylation of phosphorylase a was stimulated four- to sevenfold by low concentrations of polylysine (Mr = 13,000; 0.01-0.1 microM), whereas MLC phosphatase activity was virtually abolished. Higher concentrations of polylysine inhibited dephosphorylation of either substrate. Interestingly, a heat-stable fraction prepared from the PCM phosphatase reversed the stimulatory effect of polylysine on phosphorylase phosphatase activity and the inhibitory effect on dephosphorylation of MLC. No reversal of the modulatory effects of polylysine occurred when protein phosphatase inhibitor 1 or inhibitor 2 was substituted for the heat-stable factor derived from the PCM-phosphatase. Sucrose density centrifugation of the enzyme yielded a single peak (Mr = 63,000) exhibiting polycation-modulated activity against phosphorylase a and MLC. Moreover, heating each of the gradient fractions showed the presence of a heat-stable factor which reversed the modulatory effects of polylysine on dephosphorylation of either phosphorylase a or MLC. These results show that a specific heat-stable factor, which differs from both inhibitor 1 and 2, is associated with the PCM-phosphatase. The results suggest that polycationic modulation of expressed PCM-phosphatase activity may involve interactions between the polycationic effector and the enzyme-associated regulatory factor. PMID- 3001745 TI - Effect of contractile activity on rat skeletal muscle beta-adrenoceptor properties. AB - The effect of fiber type and endurance exercise training on skeletal muscle beta adrenoceptor properties were assessed using a direct radioligand binding technique. Six separate muscles, composed of a variety of different fiber types, were examined in treadmill trained and sedentary rats. In trained animals, sarcolemmal preparations from heart and slow twitch soleus muscle exhibited a significantly greater receptor concentration than membranes from white fast twitch glycolytic fibers of the vastus lateralis. No significant changes were observed between trained and sedentary rat muscle beta-adrenoceptor density (beta max, fmole/mg protein) or affinity (Kd, nM) within each muscle type, despite significantly increased myocardial/body weight ratios and skeletal muscle enzyme adaptations associated with the exercise program. These results suggest that muscle beta-adrenoceptor properties may be influenced in part by the motor nerve innervation to that muscle, and are further discussed with respect to a possible relationship between exercise intensity and receptor regulation. PMID- 3001746 TI - Cholecystokinin-dopamine receptor interactions as studied with cholecystokinin receptor antagonists. AB - It has previously been shown that cholecystokinin octapeptide (CCK-8) can modulate the binding of dopaminergic agonists and antagonists to brain homogenates in vitro. The present study was designed to more carefully address the possibility that CCK-8 could modulate the number of affinity of rat striatal D2 receptors. It was found that (1) unsulfated CCK-8 will antagonize the binding of tritiated spiperone to D2 receptors, but sulfated CCK-8 is considerably less potent; (2) the putative CCK antagonists benzotript, proglumide, and the phenoxyacetyl derivative of proglumide were without effect upon tritiated spiperone binding to D2 receptors up to concentrations of 100 microM; (3) the phenoxyacetyl derivative of proglumide, a potent CCK antagonist in the periphery, is only modestly more potent than proglumide in CCK receptor binding assays using 125-I-(Bolton-Hunter)-CCK-8. It is suggested that the modulatory actions of CCK upon D2 receptors do not appear to proceed through a recognition site corresponding to the conventional pharmacological definition of a neuronal CCK receptor. PMID- 3001747 TI - Central nervous system cholecystokinin and the control of feeding behavior in sheep. AB - There is increasing evidence that brain cholecystokinin (CCK) peptides function as neuropeptide signals of satiety. Injection of as little as femtomole amounts of CCK-8 into the cerebral ventricles selectively decreased feeding in sheep, and other species (chicken, pig, hamster and rat) also decrease feeding in response to CNS injections of CCK. As would be expected with a physiological satiety agent, CCK-8-induced suppression of feeding interacts in a corrective manner with the energy deficit of the animal, in that the longer the fasting period, the greater the amount of CCK-8 required to suppress feeding. Although it has not been possible to measure changes in concentration of CCK in cerebrospinal fluid (CSF) with feeding, neutralization of endogenous CCK by administration of CCK antisera into the CSF delayed satiety in sheep. This and other evidence not only supports a physiological role for endogenous brain CCK in feeding behavior, but also suggest that CSF transports CCK to site(s) of action. Little is known of CCK's mechanisms of action, but because CNS CCK also causes changes in both gut motility and secretion of insulin and glucagon, the satiety could result directly from effects on behavior, and indirectly through metabolic changes. Evidence of selective uptake of CCK from CSF suggested involvement of hypothalamic periventricular areas in these functions. Recent findings in sheep of a 60% decrease in CCK content in anterior hypothalamus two hrs after a meal also supports this hypothesis. PMID- 3001748 TI - Endogenous inhibitors of [3H] Ro 5-4864 binding to "peripheral-type" binding sites for benzodiazepines are present in peripheral tissues and brain. AB - Recent observations have shown that "peripheral-type" binding sites for benzodiazepines (PBS) are under neural and/or hormonal control in the pineal gland, olfactory bulb, and kidney. These studies resulted in a search for endogenous substances which might physiologically subserve PBS. Acidified methanol or trichloroacetic acid extraction of both peripheral tissues and brain followed by ultrafiltration and/or gel filtration and high performance liquid chromatography revealed the presence of both high (Mr greater than 10,000) and low (Mr less than 500) molecular weight substances which inhibit the binding of [3H] Ro 5-4864 to PBS while only slightly inhibiting the binding of [3H] diazepam to classical "brain-type" benzodiazepine receptors. PMID- 3001749 TI - Endocoids: prospects and strategies for their discovery. PMID- 3001750 TI - Pharmacology of peripheral type benzodiazepine receptors in the heart. AB - RO5-4864 decreased in a dose-dependent manner the duration of intra cellular action potential and the contractility in the guinea pig papillary muscle. Diazepam was less active and clonazepam inactive. The effects of RO5-4864 were blocked by PK 11195 but not by RO15-1788. These results indicate that peripheral type benzodiazepine binding sites are pharmacological receptors. PK 11195 antagonized the increase and the decrease in the duration of intracellular action potential induced by BAY K-8644 and calcium channel blockers (nitrendipine, diltiazem, verapamil) respectively but not the increase induced by tetraethylammonium which blocks potassium channel. Moreover the decrease in contractility provoked by RO5-4864 was antagonized by 4 mM Ca2+. Thus peripheral type BZ receptors are coupled with Ca2+ channels in the heart. PMID- 3001751 TI - Specific high-affinity [3H]Ro5-4864 Ro5-4864 benzodiazepine binding sites in the brain and periphery. PMID- 3001753 TI - Endogenous sodium-potassium pump inhibitor in low renin hypertension. AB - In 1978, we reported that plasma supernate from acutely volume expanded animals reduces Na+-K+ pump activity when applied to blood vessels from another animal and then in 1980 we reported that the same is the case for plasma from an animal model of low renin hypertension. Since then, we and a number of other investigators have described Na+-K+ pump inhibitory activity in the plasma of animals and humans with hypertension, particularly of the low renin variety. The activity results from a heat stable small molecule, probably the putative natriuretic hormone. We here review these and other studies. PMID- 3001752 TI - Are "peripheral-type" binding sites for benzodiazepines in brain related to the convulsant actions of Ro 5-4864? AB - Previous studies have shown that Ro 5-4864 is a potent convulsant. Investigation of a series of compounds structurally related to Ro-4864 revealed a good correlation (r = .93, p less than 0.01) between their potencies as convulsants and their abilities to displace [35S]t-butylbicyclophosphorothionate from sites associated with the chloride ionophore. In contrast, there appears to be no direct relationship between the convulsant potencies of these compounds and their affinities for "peripheral-type" binding sites for benzodiazepines. These data suggest that "peripheral-type" binding sites for benzodiazepines are not directly involved in the convulsant actions of Ro 5-4864 and related compounds nonetheless, several lines of evidence suggest that "peripheral-type" binding sites for benzodiazepines may be indirectly involved in the convulsant properties of Ro 5-4864. PMID- 3001754 TI - Natriuretic peptides derived from pro-opiocortin. AB - Natriuresis in response to hypertonicity, volume loads, or high sodium diets has been proposed to be mediated by the release of a humoral factor(s), and controlled by the central nervous system. We have determined that the natriuretic activity of ACTH or alpha MSH resides in a common sequence, ACTH(4-10)/alpha MSH(4-10). Steric restriction of this peptide by replacement of an L-Phe residue with a D-Phe produced a superagonist. Gamma-2 MSH is a peptide derived from the 16K N-terminus of pro-opiocortin, which contains an ACTH(4-10)-like sequence. The natriuretic effect of gamma-2 MSH was maximal between 0.64 pmole to 64 pmole. Doses greater than 128 pmole produced no significant effects on renal sodium excretion. The regulated release of gamma MSH peptides may contribute to CNS regulation of renal sodium excretion. PMID- 3001755 TI - Progesterone derivatives that bind to the digitalis receptor. AB - Examination of mammalian steroid hormones, their metabolites and semi-synthetic analogs in a radioreceptor binding assay for cardiac glycosides led to the identification of active derivatives of hydroxyprogesterone. The active compounds are potent inhibitors of purified sodium, potassium-ATPase and of the sodium pump in isolated tissues. Metabolism of progesterone offers a mechanism for the generation of endogenous substances that resemble, both structurally and biologically, the digitalis cardioactive steroids. PMID- 3001756 TI - Quantitative autoradiography of angiotensin II receptors in brain and kidney: focus on cardiovascular implications. AB - Quantitative techniques of receptor autoradiography have been applied to localize [125I]-angiotensin II binding sites in brain and kidney. High densities of autoradiographic grains, indicating the presence of angiotensin II receptors, have been localized to several rat brain nuclei including the dorsal motor nucleus of the vagus, nucleus of the solitary tract, anterior pituitary, locus coeruleus and several hypothalamic nuclei. Cat thoracic spinal cord exhibited a high density of sites over the intermedio-lateral cell column. In sections of rat kidney, angiotensin II receptors were detected in the glomerulus, vasa recta and ureter. The cardiovascular implications of these results are apparent and relate angiotensin II to hypertensive mechanisms. Thus, angiotensin II represents an endocoid which is involved in control of blood pressure through its effects on peripheral organs as well as the central nervous system. PMID- 3001757 TI - Opiate receptor mediated effects of IFN-alpha and lymphocyte derived endorphin like peptides. AB - Viral infection of lymphocytes induces the synthesis of interferon (IFN)-alpha and immunoreactive corticotropin (irACTH). We have previously shown the irACTH to be antigenically, structurally, and functionally related (if not identical) to pituitary ACTH. Virus infected lymphocytes also synthesize in endorphin as measured by immunofluorescence. The endorphin-like activity was found to be associated directly with IFN-alpha as well as other, lower molecular weight peptide(s) having no antiviral activity. Crude IFN-alpha at 1000 U/ml exhibited opiate receptor binding activity by inhibiting 47.2% of 3H dihydromorphine (4 X 10(-9) M) binding to mouse brain tissue. Homogeneous HuIFN-alpha (10(8.3) U/mg) also bound to opiate receptors but required 10 times the antiviral activity than crude IFN-alpha for a 50% inhibition of dihydromorphine binding. When injected intracerebrally both crude and homogeneous IFN-alpha induced transient analgesia in mice that was reversible and preventable by the opiate antagonist naloxone. The low molecular weight (less than 10 Kd) ir endorphins purified from the IFN alpha had no antiviral activity, but may represent the majority of the opiate receptor binding material in the infected lymphocyte culture fluid. These data seem to indicate that the opiate-like side effects of exogenous IFN administration may be due to the IFN-alpha molecule binding to opiate receptors and also may be due to the associated low molecular weight endorphin-like moieties synthesized by lymphocytes. PMID- 3001758 TI - Brain endocoid identification as a strategy for the development of a new generation of psychoactive drugs. PMID- 3001759 TI - Novel analgesic peptides. AB - Several different classes of peptides have been found to confer antinociception upon treated animals. The now-classical enkephalins, endorphins and dynorphin have been much studied. This paper reviews enkephalins of atypical structure as well as a group of peptides which evoke antinociception apparently by stimulating release of opioid peptides. Among the latter are kyotorphin, substance P, bradykinin and neurotensin. PMID- 3001760 TI - Opiate receptor activity of 17-alpha-estradiol and related steroids. AB - Of a large number of steroid hormones, semisynthetic analogues, and metabolites examined for activity in an opiate radioreceptor binding assay, only 17 alpha estradiol (E-17 alpha) and closely related 17 alpha-dihydroequilin (DHE1) and 17 alpha-dihydroequilenin (DHE2) were active. 17 beta-estradiol (E-17 beta) was much weaker than E-17 alpha, particularly in the presence of Na+. On the basis of the effects of Na+ and Mn+2 on binding potency, E-17 alpha is an agonist/antagonist and DHE1 and DHE2 pure antagonists. There is evidence for diverse biological activities of 17-alpha estranes, some of which may be mediated through opiate receptors. PMID- 3001761 TI - Presence of endogenous morphine in toad skin. AB - Morphine was identified by immunological, pharmacological and physical criteria to be present in the skin of toad, rat and rabbit. PMID- 3001762 TI - Tribulin: an endogenous monoamine oxidase inhibitor/benzodiazepine receptor ligand. AB - Tribulin is a low molecular weight inhibitor both of monoamine oxidase and of benzodiazepine receptor binding. It has been highly purified from human urine and has also been isolated from human plasma and animal brain. Its structure is still unknown but its properties do not appear to correspond with any known monoamine or benzodiazepine receptor binding inhibitor. Tribulin output has been found to be increased in a variety of states associated with stress and anxiety, including lactate-induced panic attacks, alcohol or benzodiazepine withdrawal and generalized anxiety disorder. PMID- 3001763 TI - Serine phospholipids and aging brain. AB - For many years phospholipids have been studied mainly for their possible use as membrane models or cell-like carriers. Recently, on the basis of the new knowledge on the phospholipid membrane structure and function, a novel interest has arisen on the pharmacological properties of these compounds particularly at cerebral level. In this context particular attention has been devoted to phosphatidylserine (BC-PS), an anionic phospholipid extracted and purified from bovine brain, which in young animals has been shown to modify a number of brain electrophysiological and biochemical parameters. PMID- 3001764 TI - Characterization of the endocoid for imipramine recognition sites. PMID- 3001765 TI - 5-methoxytryptoline and close analogs as candidates for the endogenous ligand of the 3H-imipramine recognition site. AB - 3H-Imipramine labels with high affinity a site associated with the macromolecular complex of the serotonin transporter in brain and platelets. There is a good correlation between the potencies of drugs at inhibiting 3H-5HT uptake and at inhibiting 3H-imipramine binding. Dissociation experiments indicate that the site labelled by 3H-imipramine is not identical with the substrate recognition site of the serotonin transporter and thus, it appears that 3H-imipramine labels a modulatory site for the 5HT transport system. On this basis, the existence of an endacoid acting on the 3H-imipramine recognition site to modulate 5HT uptake is discussed. The possibility that 5-methoxytryptoline or a closely related analog may be the endogenous ligand for the 3H-imipramine recognition site is analysed on the basis of the fact that 5-methoxytryptoline is a potent inhibitor of 3H imipramine binding that also inhibits 3H-5HT uptake. PMID- 3001766 TI - Characterization of endogenous inhibitors of [3H]-imipramine binding and [3H] serotonin uptake from rat serum. AB - The effects of rat serum extracts on the uptake of [3H]-serotonin and the displacement of [3H]-imipramine binding in rat forebrain synaptosomes and human platelets was studied. Deproteinated rat serum markedly inhibited synaptosomal [3H]-serotonin uptake in a dose-dependent and reversible manner. The crude extract was fractionated by C18-reverse phase HPLC. Three major peaks of inhibitory activity were found. One of the peaks was identified as serotonin and was significantly reduced after chronic reserpinization. The second major peak inhibited both [3H]-serotonin uptake and [3H]-imipramine binding in synaptosomes and platelets. This fraction had a minimal effect on the uptake of [3H] norepinephrine, [3H]-dopamine or [3H]-GABA and was less effective in inhibiting [3H]-desipramine binding than [3H]-imipramine binding. PMID- 3001767 TI - Demonstration of an imipramine endacoid in rat brain. AB - The partial purification and characterization of an imipramine endacoid from rat brain is reported. High affinity 3H-imipramine binding as well as 3H-serotonin uptake in both brain and platelet preparations is inhibited in a dose-dependent fashion by the endogenous substance. The effect of the endogenous factor appears to be specific since it does not affect the binding of other drugs to their membrane receptors. The substance is resistant to proteolytic enzymes and is unevenly distributed in rat brain being highly concentrated in the hypothalamus and striatum. Its concentration in rat brain is not changed after repeated electroconvulsive shock treatment. PMID- 3001768 TI - On the brain endocoid for benzodiazepine recognition sites. AB - Human and rat brain contain a neuropeptide with 105 amino acid residues which inhibits the binding of 3H-diazepam and other specific benzodiazepine recognition site ligands to crude brain synaptic membranes. DBI injected intracerebroventricularly in thirsty rats which are subjected to a conflict test (Corda et al., 1983), lowers the threshold for behavioral suppression by punishment. In this test DBI acts like an anxiogenic endocoid for the benzodiazepine recognition sites. The large abundance of lysine and arginine residues in the DBI molecule suggest that this polypeptide functions as a precursor of a putative endocoid which regulates anxiety levels. This endocoid acts as an agonist for the benzodiazepine recognition site. Because of the anxiogenic properties of this endocoid, it is proposed that anxiolytic benzodiazepine should be classified as an antagonist and beta-carboline as an agonist at the receptor level. PMID- 3001769 TI - Isolation and purification of an endogenous brain ligand for benzodiazepine receptor(s). AB - An endogenous brain ligand which competes with 3H-flunitrazepine for the binding to benzodiazepine receptor has been isolated and purified to homogeneity. The purification procedures involve the liberation of the ligand into the high speed supernatant followed by ultrafiltration through a PM10 membrane (exclusion limit: 10,000-dalton) column chromatographies on Sephadex G-50, Bio-Rad P2 and finally with three times reversed phase HPLC using C18 columns. The purified endogenous ligand is heat stable, insensitive to DNAase or RNAase and contains about 5-10% amino acid residues. It has an absorption maximum at 220 nm and a minor peak at 313 nm. The exact chemical structure is unknown. PMID- 3001770 TI - Evidence for an endogenous peptide ligand and antagonist for PCP receptors. AB - alpha-Endopsychosin, an endogenous ligand for the phencyclidine receptor, has been isolated from porcine brain. The endogenous ligand is a peptide and has similar actions to PCP and is selectively distributed in the brain. A PCP analogue, Metaphit, has also been found to be an effective PCP antagonist. This antagonist may be useful for evaluating the role of the brain's alpha endopsychosin system and may have a number of important clinical uses. PMID- 3001771 TI - Historical perspectives on the isolation and characterization of neuropeptides. PMID- 3001772 TI - ACTH1-24 effects on d-amphetamine self-administration and the dynamics of brain dopamine in rats. AB - The present experiments investigated the role of ACTH fragments in d-amphetamine self-administration in rats. Presession injections of 20 or 40 ug/80 ul ACTH1-24 (but not 10 ug/80 ul ACTH1-24 nor any dose of ACTH4-10) significantly reduced rates of d-amphetamine self-infusion for 2 days. Neurochemical experiments revealed that ACTH1-24, followed 24hr later by haloperidol, attenuated haloperidol-induced increases in HVA and DOPAC in both the caudate and nucleus accumbens. It was tentatively concluded that the corticosteroid mediated neuromodulatory action of ACTH1-24 on dopaminergic neurons might increase the rewarding quality of d-amphetamine, thus rendering control levels of self infusion superfluous. PMID- 3001773 TI - Genetic thyroid diseases. PMID- 3001775 TI - 21-Hydroxylase deficiency: HLA genotypes and hormonal phenotypes in the families of 32 Italian patients. PMID- 3001774 TI - Hereditary defects of adrenal cortical steroid biosynthesis. PMID- 3001776 TI - The Perlman syndrome: clinical and biological aspects. PMID- 3001777 TI - Genetic disorders of the anterior pituitary gland. AB - This survey deals with disorders caused by genetically disturbed function of the anterior pituitary gland. Genetic Dwarfism may be caused by isolated growth hormone deficiency (IGHD) or panpituitary diseases, such as congenital absence of the pituitary or familial panhypopituitarism. Genetic disturbances of isolated pituitary hormone secretion without dwarfism may occur as isolated gonadotropin deficiency (IGD), isolated luteinizing hormone deficiency ("fertile eunuch"), Kallmann syndrome (olfactogenital dysplasia), isolated thyrotropin deficiency (ITD) and isolated corticotropin deficiency (ICD). Pituitary dysfunction may also be associated with other genetic disease entities. PMID- 3001778 TI - Double-blind trial of suloctidil versus placebo in moderate to severe mental deterioration. AB - A double-blind, parallel group, placebo-controlled study was carried out in 30 elderly patients with moderate to severe mental deterioration to assess the effect of suloctidil on their mental condition. A battery of clinical and psychometric evaluations failed to demonstrate any significant differences between the two groups at the end of a 6-month treatment period during which patients received either 200 mg suloctidil or placebo 3-times daily. However, further analysis of the results showed that in the sub-group of patients with moderate mental deterioration on entry, suloctidil treatment produced significant improvement from baseline in the Rey 15 words test and the results were significantly different from those in patients with severe mental deterioration. No consistent changes were observed in the placebo group. PMID- 3001779 TI - The effects of PK 11195, a ligand for benzodiazepine binding sites, in animal tests of anxiety and stress. AB - PK 11195 (1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinolinecarboxami de, a potent ligand for peripheral benzodiazepine binding sites, was unable to reverse the anxiogenic effects of Ro 5-4864 (chlorodiazepam) in the social interaction test or in the punished drinking test. However, at 90 mg/kg PK 11195 also reduced social interaction, indicating an anxiogenic effect. Both PK 11195 (30-120 mg/kg) and Ro 5-4864 (20-60 mg/kg) significantly increased the plasma corticosterone concentrations of rats left in their home cages after injection, and in those placed in novel apparatus. Because both drugs had effects in the same direction and because the doses were far higher than those needed to saturate the peripheral receptor, it is unlikely that these behavioural actions are mediated by peripheral benzodiazepine binding sites. It is suggested that the effect of PK 11195 could even be mediated by the classical CNS benzodiazepine binding sites. PMID- 3001780 TI - Acute effects of marihuana on luteinizing hormone in menopausal women. AB - Plasma luteinizing hormone (LH) levels were determined under double blind crossover conditions in 10 healthy menopausal adult females prior to and following smoking of a 1-g marihuana cigarette containing 1.83% delta 9 tetrahydrocannabinol (THC) and a 1-g marihuana placebo cigarette. A significant increase in pulse rate and levels of intoxication occurred after marihuana smoking but not after smoking placebo cigarettes. LH levels determined before administration of marihuana and placebo cigarettes were not significantly different and were within the range of normal values for healthy menopausal women. No significant differences were found between LH levels following marihuana and placebo smoking. PMID- 3001782 TI - Opiate dependence and withdrawal--a new synthesis? AB - There is a growing body of evidence to suggest that adrenocorticotropin (ACTH) may have a physiological role as an endogenous contra-opioid agonist. In addition to having appreciable affinity for opiate receptors and inducing many behavioural and intracellular effects opposite to those observed following opioid administration, ACTH may interact with endorphins in a mutually antagonistic manner. On the basis of these data a model of opiate dependence is proposed whereby several aspects of the opiate abstinence syndrome may be attributed to the excitatory actions of ACTH acting at opiate receptors. Thus, it may be predicted that opiate antagonist administration during primary abstinence should significantly attenuate many aspects of this behavioural syndrome. The present study was conducted in order to investigate this hypothesis. Results indicated that whilst naloxone (1.5 mg/kg) exerted little influence in non-dependent animals, it significantly attenuated abstinence-exacerbated grooming, body shaking, teeth chattering and sneezing, in addition to completely antagonizing withdrawal hyperalgesia in post-dependent animals. These data are consistent with the proposed existence of an endogenous contra-opioid ligand, the antagonism of which markedly reduces the severity of the morphine withdrawal syndrome. PMID- 3001781 TI - Electric footshock-induced changes in behavior and opioid receptor function. AB - The present electric shock (ES) schedule produced significant behavioral changes, such as analgesia and motor suppression, and functional changes in binding capacities for opioid agonist and antagonist. In the naloxone (5 mg/kg, SC 15 min before ES application) pretreated rats, these behavioral and biochemical changes were blocked. In addition, when preincubation (37 degrees C, 30 min) was not carried out in the process of preparation of synaptic membrane, the ES-induced functional changes in hibh affinity binding sites were not observed. Moreover, the present data indicated that preincubation may produce the destruction of [3H] D-ala2,L-met5-enkephalinamide ([3H]-DAMEA) specific binding sites with the forced dissociation of endogenous delta-type opioid peptides from delta opioid receptors. In addition, the significant decrease of [3H]-DAMEA specific binding in the ES membrane suggested that delta-type opioid peptides were released more than steady state level by ES application and bound to the delta opioid receptors. Therefore, these results suggest that ES-induced behavioral and biochemical changes were mediated by opioid peptides which were released by ES application. In addition, the ES-induced analgesia may be mediated by high affinity delta opioid receptor. PMID- 3001783 TI - Mu and kappa opiate receptor involvement in agonistic behaviour in mice. AB - The influence of opioid drugs on agonistic behaviour is reviewed and an experiment is reported that examines the impact of U-50488 (a kappa agonist) and DAGO (a mu agonist) on the social encounters of male and female mice interacting with anosmic male partners. Although DAGO did not significantly influence inter male social encounters, U-50488 decreased social investigation, increased timid/defensive behaviour and potently suppressed aggression. In contrast, U 50488 did not significantly influence the behaviour of timid female mice, whereas DAGO decreased social and timid/defensive behaviour. It was concluded that a kappa mechanism increases whereas a mu mechanism decreases submissive behaviour. PMID- 3001784 TI - The effects of Org 2766 on the performance of sham, neocortical, and hippocampal lesioned rats in a food search task. AB - The behavioral effects of an ACTH4-9 variant, Org 2766, given for one week postoperatively at a dose of 1 microgram/rat daily, were evaluated in animals given hippocampal, neocortical, or "sham" lesions. After the week during which the injections were given, the animals were tested for 5 days in a food-search task in which food was hidden in two recessed holes in the floor. On the next day the ability of the rats to find food in these same two baited holes was tested in the presence of 14 additional holes that were not baited. On the following day, the animals were tested again, this time with all 16 holes baited. To assess the long-term effects of Org 2766 treatment, the animals were tested once again 2-3 months later in the same apparatus with 16 empty holes. In general, rats with lesions restricted to the neocortex were severely impaired in the task and were unaffected by prior treatment with Org 2766. Animals with hippocampal damage quickly learned the task and were hyperactive. During the test session with 16 baited holes they showed differential behavioral changes suggesting attentional deficits not seen in "sham" operated rats. These deficits were attenuated by prior Org 2766 treatment, whereas the lesion-induced hyperactivity was not. Treatment with Org 2766 impaired all aspects of performance of "sham" operated animals. PMID- 3001785 TI - Delta-9-tetrahydrocannabinol potentiates the disruptive effects of phencyclidine on repeated acquisition in monkeys. AB - Patas monkeys acquired a different four-response chain each session by responding sequentially on three keys or levers in the presence of four discriminative stimuli (geometric forms or numerals). The response chain was maintained by food presentation under a fixed-ratio schedule. Errors produced a brief timeout but did not reset the chain. Each day there were four 15-min sessions, with a 10-min intersession interval. Cumulative dose-effect curves for phencyclidine were obtained by giving an IM injection before each of the four sessions; successive injections increased the cumulative dose by 1/4 log-unit steps. When phencyclidine was administered alone, overall response rate decreased and percent errors increased with increasing doses. When delta-9-tetrahydrocannabinol (THC) was administered PO before the first session at a dose that was ineffective when given alone, the phencyclidine dose-effect curves for both rate and accuracy tended to shift to the left. After pretreatment with a higher dose of THC, which decreased rate in one of three subjects without affecting accuracy when given alone, the rate-decreasing and error-increasing effects of phencyclidine were generally even more pronounced. The results indicate that THC potentiates the disruptive effects of phencyclidine on complex operant behavior in monkeys. PMID- 3001786 TI - Morphine dependence and protracted abstinence: regional alterations in CNS radioligand binding. AB - Rats (Fisher F-344) were given free access to a 10% sucrose solution containing 0.5 mg/ml morphine sulfate (controls received the sucrose vehicle only) as their sole source of fluid. Daily morphine intake averaged 119 +/- 21 mg/kg, an amount sufficient to induce physical dependence. After 18 days on this regimen, the control and dependent subjects were sacrificed. A protracted abstinence group was weaned from morphine by reducing its concentration in the vehicle by 20% over the next 5 days, followed by a 5-week drug-free period before sacrifice concurrent with the other groups. These subjects showed no signs of an abstinence syndrome. Binding assays for alpha-2 adrenergic sites (3H-clonidine), beta-1/beta-2 adrenergic sites (3H-dihydroalprenolol), and dopaminergic (D2)/serotonergic (5 HT2) sites (3H-spiroperidol) were performed on tissue from frontal cortex, hippocampus, striatum, and brainstem. No alterations in 3H-clonidine or 3H dihydroalprenolol binding were observed in dependence or protracted abstinence, suggesting that noradrenergic systems are well-regulated both during dependence and in protracted abstinence. 3H-spiroperidol binding was significantly elevated in the striatum (D2 sites) and hippocampus (5-HT2 sites) during dependence. Hippocampal 3H-spiroperidol binding returned to control levels in protracted abstinence, reflecting a morphine-induced change in 5-HT2 binding sites which had normalized by 5 weeks post-drug. Striatal 3H-spiroperidol binding was significantly decreased below control levels after withdrawal, suggesting that alterations of D2 sites in this structure may play a role in protracted abstinence. PMID- 3001787 TI - [3H]-Flunitrazepam binding in the presence of beta-phenylethylamine and its metabolites. AB - It has recently been reported that the concentration of beta-phenylethylamine (PEA) was elevated in the plasma of an individual experiencing convulsions because of an overdose of tranylcypromine. Also, high concentrations of PEA, injected into mice, were reported to induce convulsions. This convulsive effect was prevented by pretreatment with the benzodiazepines diazepam and chlordiazepoxide. In this study, PEA in concentrations from 0.5 to 100 microM failed to alter the binding of [3H]-flunitrazepam ([3H]-FLU) in membrane preparations from mouse rostral forebrain. The metabolites of PEA: phenylacetic acid, phenylethanolamine, octopamine and tyramine, also failed to affect [3H]-FLU binding. This suggests that although there are substances that act as convulsants by interacting with the benzodiazepine receptor sites, the convulsant effect of PEA and its metabolites is mediated elsewhere. PMID- 3001788 TI - An ethological analysis of the effects of tifluadom on social encounters in male albino mice. AB - The effects of treatment with saline, 0.5 or 1.0 mg/kg of tifluadom were assessed 30 min after injection on the behaviors shown by isolated Alderly Park strain mice in their home cages in the presence of an anosmic 'standard opponent' mouse. Tests involved videotaping encounters and examining the incidences of 45 behavioral elements and their sequences (by producing 'dendrograms'). The kappa agonist appeared to stimulate olfactory exploration of the substrate at the expense of other forms of non-social exploration; it suppressed olfactory investigation of the 'standard opponent'; reduced some aggressive elements and increased immobility (at reportedly non-sedative doses) and fearful activity. The 'dendrograms' revealed that tifluadom greatly altered the relationships between some elements. The higher dose of the kappa agonist resulted in self-grooming and digging (displacement?) being associated with the agonistic items suggesting that these animals evidenced increased timidity in social encounters. PMID- 3001789 TI - Some effects of orthovanadate on smooth muscle cells of guinea-pig taenia caeci. AB - The effect of orthovanadate on the response caused by the beta-adrenergic receptor agonist isoprenaline and on the ATP response were investigated by measuring potential changes and changes in the contractile state of smooth muscle cells of guinea-pig taenia caeci at 35 degrees C using the sucrose-gap method. The contraction evoked by carbachol (10(-7) M) or potassium (15 mM) was not modified by orthovanadate (1.0 mM). The hyperpolarization evoked in smooth muscle cells by ATP (0.4 mM) was affected to some extent by orthovanadate (1.0 mM), but was not modified in the absence of extracellular calcium. The hyperpolarization caused by isoprenaline (3 X 10(-6) M), however, was inhibited completely both in the presence and absence of calcium. These results are consistent with the view that isoprenaline activates calcium extrusion from the smooth muscle cells, a process being electrogenic in nature. PMID- 3001790 TI - Cellular transformation by the herpesviruses and antiviral drugs. PMID- 3001792 TI - A proposed method for in vivo determination of lithium in human brain. AB - A method for measuring Li in vivo in human brain is presented. The technique is based on the measurement of tritium gas exhaled by the subject following neutron irradiation of the organ of interest. The gas collection facility used to separate minute amounts of tritium from the breath is described. Methods for reducing the background levels of tritium were investigated. The limit of detection of the system is estimated to be 350 micrograms of Li for the whole brain for a dose of 10 mSv. This detection limit is sufficient for the study of patients treated with lithium compounds, but is too high to study 'normal' brain lithium content. The rate of elimination of tritium gas from the body was also investigated in animal studies. The method also appears suitable for the measurement of lithium levels in the kidney. PMID- 3001791 TI - Developmental implications of ocular pharmacology. PMID- 3001794 TI - Electronic aspects of serine protease catalysis. AB - A new electrostatic approach is applied to serine protease catalysis. It is is based upon the demonstration that the polarities, or partial charges, of the atomic components of the molecules involved in the reaction alternate in sign. When the atomic components of opposite polarities of the enzyme and substrate approach close to each other during the catalysis, the electrostatic interactions between them increase in intensity. These increasing interactions are related to the decrease in the energy barrier. When the serine protease--catalyzed reaction is followed from this perspective, it is shown to result in a marked simplification of the catalytic mechanism. A number of concerted proton transfers and electron density displacements around the active site are indicated. This approach is not inconsistent with other electrostatic methods, and is supported by independent partial charge calculations. PMID- 3001793 TI - Sodium transport and distribution of electrolytes in frog skin. AB - The objective of this study on frog skin was to examine correlations between transepidermal active Na-transport and intracellular [Na]c, [K]c, [Cl]c homeostasis. Isolated, whole skins, and "split skins" were used in measurements of short-circuit current (SCC) and open skin potential (PD). Water and ion contents were estimated on split skins. Absolute [Na]c and [K]c varied over the range of 18 to 46, and 113 to 80 mM, respectively (Figure 7), but a complementary relationship existed between Na and K, such that [Na]c + [K]c remained approximately equal to 129 mM. Average values for [Na]c and [K]c were approximately equal to 31 and approximately equal to 96 mM, respectively. [Cl]c remained constant at approximately equal to 38 mM. This complementary relationship does not seem to be an artifact, caused by collagenase, used in the preparation of split skins. Whole skins and split skins in Ringer's solution, when treated with fluoroacetate (FAc), ouabain (Ou), or vanadate (Va) over wide ranges of concentrations, showed that FAc greatly depressed the SCC and the PD, without changing [Na]c, [K]c, [Cl]c. FAc acted only from the corium side of the skin. The decreasing SCC remained a Na-current, as in control skins. By comparison, such a separation of cellular functions could not be established with Ou, or Va. These inhibitors either affected SCC, PD, and cellular ion concentration, or they had no effect on any of these parameters. The complementary relationship between [Na]c and [K]c, with [Cl]c remaining again at approximately equal to 38 mM, was also found in tissues exposed to inhibitors. These results indicate that transcellular active Na transport and electrolyte homeostasis are not always rigidly coupled, suggesting that these processes may not be uniformly distributed within the epithelial cells, or among the interconnected cell layers of the frog skin epidermis. PMID- 3001795 TI - Nitroxide-doped liposomes containing entrapped oxidant: an approach to the "reduction problem" of nitroxides as MRI contrast agents. AB - The purpose of this investigation is to test the feasibility of a nitroxide regeneration system involving liposomes as an approach toward solving the "reduction problem" when nitroxides are used as contrast enhancing agents in MRI applications. It is shown that the inclusion of an entrapped oxidant (K3Fe(CN)6) in the aqueous compartment of nitroxide-doped liposomes causes a 4-5-fold increase in the duration of the nitroxide ESR signal in the presence of the external reductant sodium ascorbate. Confirmation was obtained by monitoring the concentration of the internalized Fe(CN)6(3-) ion versus time by visible spectroscopy at 410 nm. Trans bilayer (flip-flop) motion of the long chain nitroxide ester is the likely mechanism of this nitroxide regeneration system. PMID- 3001796 TI - Erythropoietin binding to the red cell membranes. AB - Some characteristics of Epo binding to the red cell membranes were investigated in a system in which the hormone was added to a suspension of cells in a mixture of normal serum phosphate buffered saline (1:7 (V/V)), testing the unbound Epo. Epo binding shows a gradual decrease during the maturation of red cells (0.15 U/ml packed erythrocytes, 0.285 U/ml reticulocytes and 0.375 U/ml erythroblasts from a total of 1.4 U/ml). The binding is also time- and pH-dependent. The amounts of Epo bound within 60 min are independent of temperature, but after 120 min a dissociation of the hormone-receptor complex occurred at 23 and 37 degrees; at 4 degrees C the binding continue, although at a very low rate. RBC membranes bound the hormone at pH 7.8-8 and release it at pH 6.5. The "in vivo" stimulation of beta receptors with isoproterenol increases the erythropoietic response caused by Epo, suggesting a possible relation with the adenylcyclase system. PMID- 3001797 TI - Functional response of the hyperthyroid patients with beta adrenoreceptor blockade to exercise. AB - The submaximal test of triangular type was used for studying the adaptability to exercise, on an ergometric bicycle. It was performed, starting from O W, with progressive increasing load of 10 W/min until the maximum optimum heart rate (fho) was reached. The heart rate, arterial blood pressure, electrocardiogram, CO2 output and O2 consumption. Computed O2 consumption (VO2 max, ml/kg/min, Astrand) were determined, also taking into account the subjective sensation linked to exercise, measured on a modified Borg scale. The adaptability capacity to exercise was expressed by the adaptability coefficient (AC), according to the formula: AC = W'/2 + (W'-f'). 20 hyperthyroid patients, without cardiothyreosis were examined, both before the uptake of propranolol and after, at a certain interval of time. A decrease of the exercise capacity before the treatment and its increase after it, was noticed in the hyperthyroid patients. PMID- 3001798 TI - Comparisons of the influence of morphine sulphate, morphine-3-glucuronide and tifluadom on social encounters in mice. AB - The influences of morphine sulphate, morphine-3-glucuronide and tifluadom on social encounters were compared in male and female mice that were allowed to interact with an anosmic male partner. The drugs were compared, as morphine sulphate is thought to act via mu, and tifluadom via kappa opiate receptors; morphine-3-glucuronide is the breakdown product of morphine. Neither morphine sulphate nor morphine-3-glucuronide significantly influenced inter-male social encounters. However, tifluadom increased non-social and decreased social behaviour, while increasing timid/defensive activities. Tifluadom did not significantly influence the behaviour of female mice in this social encounter: in contrast both morphine sulphate and morphine-3-glucuronide decreased timid/defensive behaviour and stimulated non-social behaviour. It was concluded that the results could be explained by suggesting that morphine sulphate decreases whereas tifluadom increases timidity. PMID- 3001799 TI - Self-stimulation of the lateral hypothalamus and ventrolateral tegmentum: excitability characteristics of the directly stimulated substrates. AB - Recovery from refractoriness in the neural substrate for self-stimulation of the ventrolateral tegmentum (VLT) was estimated using psychophysical techniques, and compared to recovery in the substrate for self-stimulation of the lateral hypothalamus (LH). A computer-controlled testing setup was developed to minimize experimenter error and testing time. In order to assess the performance of this setup, refractory period estimates obtained in the newly designed, computer controlled equipment were compared to those obtained in the hand-operated equipment used in previous experiments of this type. No meaningful differences were found. Thus, the more convenient computer-controlled setup was used to collect refractory period estimates from the VLT and LH. A comparison of these values revealed differences in the slopes of the recovery curves, and in the C-T intervals bracketing their rising portions. The VLT curves rose more gradually, and both began to rise and levelled off at longer C-T intervals than the LH curves. One explanation of these results is that the distribution of excitability in the LH substrate is shifted towards higher values than the VLT distribution. The overlapping portions of the recovery curves could reflect the contribution of a common bundle of reward-related fibers. PMID- 3001800 TI - The role of the midbrain reticular formation in the expression of two opposing nigral denervation syndromes. AB - The present study investigated the role of the midbrain reticular formation (MRF) in the expression of opposing locomotor asymmetries elicited from the medial and lateral substantia nigra pars compacta (SNC). It was found that unilateral MRF lesions produced ipsiversive circling that was potentiated by amphetamine. Lateral SNC lesions produced contraversive circling while medial SNC lesions caused ipsiversive circling. When SNC lesions were combined with MRF lesions animals circled ipsiversively as they did with MRF lesions alone regardless of whether the SNC lesion was in the medial or lateral part of the SNC. Taken together, the results are consistent with the notion that a striato-nigral-MRF system is an output path for circling derived from both the medial and lateral SNC. PMID- 3001801 TI - Antiviral properties of garlic: in vitro effects on influenza B, herpes simplex and coxsackie viruses. PMID- 3001802 TI - Involvement of spinal serotonergic pathways in nociception but not in avoidance learning. AB - The effects of selective lesions of the descending serotonergic (5-HT) pathways on analgesia and avoidance deficit induced by the 5-HT releasing compound p chloroamphetamine (PCA, 2.5 mg/kg) were investigated in male rats. Intrathecal injection of 5,6-DHT (20 micrograms/rat) reduced the uptake of labelled 5-HT into spinal synaptosomes by approximately 85% but did not significantly affect the uptake of noradrenaline. The lesions produced a significant hyperalgesia and strongly attenuated the analgesic effect of PCA in the hot-plate test. In the flinch-jump test 5,6-DHT lesioned rats receiving PCA did not differ from the saline control group. Spinal lesioning did not, however, affect one-way active avoidance performance and did not prevent the marked impairment of avoidance performance induced by PCA. Thus, the avoidance deficit caused by PCA is independent of the descending serotonergic pathways and of the analgesia induced by PCA. These results support the view of a differential involvement of the ascending and descending serotonergic projections in behavioural processes controlled by aversive stimuli. PMID- 3001803 TI - Cholinergic modulation of memory in rats. AB - Central cholinergic systems have long been implicated in the modulation of learning and memory processes in animals and man. Drugs that affect the central cholinergic system have been found either to enhance or to hinder performance in tests of learning and memory. Few studies have evaluated the effects of different cholinergic drugs within a single experimental paradigm and with a relatively wide dose range. The studies reported here investigated the effects of cholinergic drugs with diverse modes of action on the retention of a passive avoidance response. Physostigmine, arecoline, oxotremorine, nicotine, and 4 aminopyridine were administered IP immediately following the acquisition of a one trial passive avoidance task. All of the drugs were found to enhance 72-h retention of passive avoidance; however, the effective doses were different for each of the drugs studied. PMID- 3001804 TI - Stereoselective behavioral effects of N6-phenylisopropyl-adenosine and antagonism by caffeine. AB - Effects of the (-)- and (+)-isomers of N6-(phenylisopropyl)-adenosine (PIA) were studied in rats trained to respond under fixed-interval and fixed-ratio schedules of food reinforcement. Both isomers of PIA decreased response rates; however, the (-)-isomer decreased response rates at doses as low as 0.1 microM/kg and was 100 300 times more potent than the (+)-isomer. The potency differences suggest that the effects observed were due to actions at A1-adenosine receptors. Caffeine, an adenosine-receptor antagonist, when administered alone in doses of 10-154 microM/kg, increased response rates under the fixed-interval schedule and did not affect rates of responding under the fixed-ratio schedule. Higher doses decreased response rates under both schedules. Caffeine shifted the (-)-PIA dose-effect curve to the right. At a low dose of caffeine (25.7 microM/kg), which alone modestly increased response rates under the 5-min fixed-interval schedule, the disruptions in rates and patterns of responding produced by (-)-PIA were restored to resemble control performances. The higher dose of caffeine (77.2 microM/kg), which alone produced larger increases in rates of responding under the fixed interval schedule, restored overall response rates to control levels when administered in combination with (-)-PIA. However, patterns of responding after the combination of doses remained disrupted. These effects suggest that some of the behavioral effects of caffeine are a result of mechanisms other than adenosine-receptor blockade. PMID- 3001805 TI - The effects of aging on day-night rhythms of kappa opiate-mediated feeding in the mouse. AB - Day-night rhythms in feeding behavior and response to the specific kappa opioid agonist U-50,488H (0.10-10. mg/kg) were measured in young (1-2 months), mature (8 12 months) and old (24-30 months) male CF-1 mice. All the mice consumed more food at night than in the day-time, though this nocturnal peak was markedly reduced in old and mature animals. Young mice also displayed a significant, dose-related, nocturnal enhancement in U-50,488H-stimulated feeding. This day-night rhythm was reduced in mature animals and absent in old mice. In old mice, U-50,488H significantly stimulated feeding only after the high dose of 10 mg/kg. Additionally, old animals did not show the dose-dependent latency to initiation of feeding after administration which was observed in young mice and to a lesser extent in mature animals. PMID- 3001806 TI - Tolerance to the benzodiazepine diazepam in an animal model of anxiolytic activity. AB - The antipunishment properties of diazepam (DZP) were investigated in mice treated acutely, or following nine daily treatments with either DZP (5 mg/kg, PO) or its vehicle. Acutely, or following chronic vehicle treatment, DZP produced a dose related increase in activity punished by footshock. Following chronic DZP, test doses of DZP given 24 or 48 h following the last chronic treatment were no longer, or less effective in enhancing punished activity. Effects on unpunished activity were unaffected. In a study of the time course of tolerance development, tolerance was not seen after one or three daily treatments but was present after 6 days. Following establishment of tolerance by 9 days' treatment, the antipunishment activity of DZP reappeared after 8 days' withdrawal and was restored to acute levels after 16 days. Tolerance was not associated with changes in benzodiazepine (BZ) receptor affinity or numbers, but the ability of GABA to enhance BZ binding was increased. There was no change in the ability of DZP or the convulsant beta-carboline DMCM to modulate 35S-TBPS binding. The mechanism of tolerance to the antipunishment properties of DZP therefore remains unknown. PMID- 3001808 TI - The effects of pimozide on the establishment of conditioned reinforcement as a function of the amount of conditioning. AB - In an attempt to understand some inconsistent findings, the present experiment investigated the effects of pimozide, a dopamine (DA) receptor blocker, on the establishment of conditioned reinforcement as a function of the amount of conditioning. In Experiment 1, rats received three phases of training in a two lever box. The pre-exposure phase measured the operant rates of pressing the levers; one produced a 3-s tone and the other turned the lights off for 3 s. In the conditioning phase, with the levers absent, the light-off stimulus was paired with food for two or four sessions. The test phase again measured the rate of pressing the levers. Conditioned reinforcement was shown by a relative increase in responding on the light lever during the test. Of the groups receiving four conditioning sessions, pimozide (0.5, 1.0, 2.0 and 4.0 mg/kg) produced a dose dependent attenuation of conditioned reinforcement, those rats treated with 4.0 mg/kg failing to demonstrate a significant effect. When 2 conditioning days were employed, pimozide treatment also produced a dose-dependent attenuation; however, in these less conditioned animals 2.0 mg/kg blocked the effect. The possibility that pimozide produced a conditioned taste aversion to the food was ruled out in Experiment 2. These data suggest that DA transmission may be necessary for the establishment of conditioned reinforcement and that the effects of receptor blockade may be related to the amount of conditioning. PMID- 3001807 TI - Cocaine: effects on acoustic startle and startle elicited electrically from the cochlear nucleus. AB - Startle-like responses can be elicited by single pulse electrical stimulation of nuclei within the acoustic startle pathway. Compared with acoustically-elicited startle, this technique provides a method for localizing the ultimate sites of action of a drug that affects the acoustic startle response. Strychnine (1 mg/kg) increased both acoustically-elicited startle and startle elicited from the ventral cochlear nucleus (VCN), the first central nucleus in the acoustic startle pathway. In contrast, cocaine (10 mg/kg) increased acoustically-elicited startle but depressed VCN-elicited startle. These results suggest that cocaine increases startle by acting on sensory rather than final motor systems and are discussed in relation to the putative effect of cocaine on dopamine neurotransmission and the involvement of dopamine in sensorimotor reactivity. PMID- 3001809 TI - Inhibition by naloxone of the rise in hypothalamic dopamine and serum prolactin induced by ethanol. AB - We investigated the effect of naloxone on the concentration of dopamine in the hypothalamus and on the concentration of prolactin in serum and anterior pituitary of male rats acutely treated with ethanol. Acute ethanol administration increased serum prolactin levels and hypothalamic dopamine concentration. Pituitary prolactin was not modified by this treatment. Naloxone administered 15 min before the animals were sacrificed decreased serum prolactin levels and hypothalamic dopamine concentration in ethanol-treated rats. These results suggest that ethanol increases prolactin secretion because it decreases the release of dopamine by the hypothalamus. Naloxone decreases prolactin release probably because it antagonizes the inhibitory action of opioids on dopaminergic neurons. PMID- 3001811 TI - The effect of dinitrophenol on magnesium transport across an isolated preparation of sheep rumen epithelium. AB - Mg transport across an isolated preparation of sheep rumen epithelium was studied in vitro. The efflux (flux from the mucosal side to serosal side) and the influx (flux with serosal to mucosal side) were estimated with 28Mg. Under physiological conditions a significant net efflux of Mg was observed, in which the unidirectional efflux was seven to eight times that of the influx. In experiments where ATP synthesis was blocked by the addition of dinitrophenol, Mg efflux was reduced to that of the influx. These observations support the conclusion that Mg absorption across the rumen wall of the sheep is an active process. PMID- 3001812 TI - Hepatocellular carcinoma: one of the world's most common malignancies. PMID- 3001810 TI - The effect of chronic in vivo infusion of forskolin on noradrenergic receptor sensitivity. AB - Forskolin, a diterpene isolated from the plant Coleus forskolii, activates the catalytic subunit of adenylate cyclase, resulting in a hormone receptor independent increase in the intracellular production of cyclic AMP. This study was undertaken to assess the effect of chronic in vivo infusion of forskolin on noradrenergic neuronal activity. Forskolin was infused into the right lateral ventricle of male Sprague Dawley rats via Alzet osmotic minipumps (model 2001) for 7 days. Chronic infusion of forskolin resulted in a decrease in norepinephrine-stimulated cyclic AMP accumulation in the limbic forebrain. Chronic infusion of forskolin also resulted in a decrease in the Bmax for 3H dihydroalprenolol (3H-DHA) binding to beta-adrenergic receptors in the cerebral cortex and hippocampus, with no apparent change in the Kd values. These data suggest the possibility of a novel therapeutic approach to modulating receptor sensitivity, and that chronic infusion of forskolin may be a useful model for studying the role of cyclic AMP in the control of neuronal activity. PMID- 3001813 TI - A case of hepatocellular carcinoma complicating hepatitis B infection in a nine year-old boy. AB - A 9-year-old boy born of Chinese parents in England, and adopted by English parents at an early age, presented with primary hepatocellular carcinoma in a non cirrhotic liver. His serum contained hepatitis B surface antigen and 'e' antibody, a probable result of perinatal infection from an HBsAg carrier mother. The management of infants at risk of perinatal hepatitis B virus transmission should now include active and passive immunisation. PMID- 3001814 TI - Centrifugal fractionation of human erythrocytes according to age: comparison between Ficoll and Percoll density gradients. PMID- 3001815 TI - In vitro cell transformations induced by 31 MeV protons. AB - Experimental data are presented on the frequencies of transformations in C3H10T1/2 cells exposed to 31 MeV protons (LET = 1.83 +/- 0.02 keV/mum in tissue) in a dose interval between 0.25 and 7.0 Gy. The transformation frequency per surviving cell curve showed a marked change in slope over the dose range used. At higher doses, above about 2 Gy, it steepened very sharply in comparison with the lower dose range. If fitted to a power of the dose, the power in the higher range was about five times that in the lower range. PMID- 3001816 TI - Behavioral toxicity and efficacy of WR-2721 as a radioprotectant. AB - S-2-(3-Aminopropylamino)ethylphosphorothioic acid (WR-2721) is a promising protectant for radiation-induced lethality. However, treatment with WR-2721 also produces nausea, vomiting, diarrhea, and hypotension, which implies severe functional consequences. Three studies were conducted to assess the effects of WR 2721 on rat motor performance and weight and to assess the ability of WR-2721 to mitigate the early performance decrement (PD) produced by ionizing radiation. In the first study, rats trained on the accelerod motor performance task were give 200, 300, or 400 mg/kg WR-2721 intraperitoneally (ip). The highest dose used referenced the maximum tolerated dose in the rat, which is two-thirds the median lethal dose (590 mg/kg). The subjects were tested immediately after treatment, at 30-min intervals for 3 h, and again at 24 h. All groups (N = 6/group) demonstrated a significant decrease in accelerod performance compared to control levels across the eight test trials, which ranged from 24 to 44% in the 200 and 400 mg/kg dose groups, respectively. Performance returned to baseline levels at 24 h. Some deaths occurred at all dose levels. In the second study, motor performance was measured after exposure to radiation alone or a drug/radiation combination (N = 8/group). WR-2721 was administered 30 min before exposure to 130 Gy of gamma radiation from a 60Co source at a dose rate of 20 Gy/min. Rats were tested on the accelerod immediately after WR-2721 treatment and at 10, 15, 30, 60, and 120 min and 24 h following radiation. Performance was significantly depressed compared to control throughout the 24 h following radiation exposure, with and without WR-2721. The decrement produced by WR-2721 and radiation alone appeared to add up to the combined drug/radiation decrement found over the 15- to 120-min test periods. In the third study assessing the effects of WR-2721 on weight, untrained rats treated with 200 or 400 mg/kg WR-2721 exhibited significant weight loss that lasted up to 3 days. Weight returned to pretreatment levels in 15 days, and no deaths occurred. In summary, the data suggest that in the rat (1) WR-2721 is behaviorally toxic at doses relevant to radioprotection, (2) WR-2721 treatment along with the stress of motor performance may combine to lower the level at which lethalities occur, (3) WR-2721 does not protect for radiation-induced PD, and (4) WR-2721 combined with radiation disrupts performance more severely than either radiation or WR-2721 alone. PMID- 3001817 TI - DNA-mediated gene transfer efficiency is enhanced by ionizing and ultraviolet irradiation of rodent cells in vitro. I. Kinetics of enhancement. AB - The enhancement effects of ionizing and ultraviolet radiation on the efficiency of DNA-mediated gene transfer were studied. The established cell line, Rat-2, consists of cells that are density-dependent contact-inhibited and produce flat monolayers in vitro. When these cells are infected with SV40 virus, a small fraction of cells becomes morphologically "transformed" due to the stable expression of the viral A-gene. Rat-2 cells are competent for DNA-mediated gene transfer, deficient in thymidine kinase activity (TK-), and will die in HAT selective media. Confluent Rat-2 cells were transfected with purified SV40 viral DNA (via calcium phosphate precipitation), irradiated with either X rays or ultraviolet, trypsinized, plated, and assayed for the formation of foci on Rat-2 monolayers. Both ionizing and ultraviolet radiation enhanced the frequency of A gene transformants/survivor compared to unirradiated transfected cells. These enhancements were nonlinear and dose dependent. A recombinant plasmid, pOT-TK5, was constructed that contained the SV40 virus A-gene and the Herpes Simplex virus (HSV) thymidine kinase (TK) gene. Confluent Rat-2 cells transfected with pOT-TK5 DNA and then immediately irradiated with either X rays or 330 MeV/amu argon particles at the Berkeley BEVALAC showed a higher frequency of HAT+ colonies/survivor than unirradiated transfected cells. In both cases the enhancement contained a linear and a higher order component in dose, but the argon ions were at least twice more efficient than X rays in producing enhancement per unit dose. Rat-2 cells transfected with pOT-TK5, X-irradiated, and assayed for either TK transformation or A-gene transformation showed the same dose dependence for radiation enhancement. Rat-2 cells transfected with the plasmid, pTK2, containing only the HSV TK-gene were enhanced for TK transformation by both X rays and ultraviolet radiation. SV40 A-gene products are not necessary for the radiation enhancement of the efficiency of gene transfer. This in vitro system will be used to study the lesions produced by ionizing radiation on mammalian cell DNA that may act as substrates for integration of exogenously introduced plasmid DNA. PMID- 3001819 TI - An immunochemical probe for 8,5'-cycloadenosine-5'-monophosphate and its deoxy analog in irradiated nucleic acids. AB - Polyclonal antisera specific for 8,5'-cycloadenosine-5'-monophosphate (8,5'-cyclo AMP) or its deoxy analog (8,5'-cyclo-dAMP) were elicited by immunizing rabbits with a conjugate prepared by the method of Johnston et al. [Biochemistry 22, 3453 3460 (1983)]. A competitive enzyme-linked immunosorbent assay (ELISA) was developed and used to detect the formation of these products in irradiated solutions of polyadenylic acid [poly(A)] or DNA which were saturated with nitrous oxide, nitrogen, or oxygen. The 8,5'-cyclo-AMP or 8,5'-cyclo-dAMP moieties could be detected in poly(A) at 1.0 krad and in native DNA at 10 krad, respectively. The yield of 8,5'-cyclo-dAMP was found to be two to three times higher in irradiated double-stranded DNA than in single-stranded DNA. The hydroxyl radical appears to initiate 8,5'-cyclonucleotide formation in irradiated nucleic acids, as demonstrated by the inhibition of 8,5'-cyclo-AMP formation in irradiated poly(A) by dimethyl sulfoxide. However, irradiation under nitrous oxide, particularly at low doses, does not lead to the expected increases in the yield of the 8,5'-cyclonucleotide. PMID- 3001818 TI - Radiation chemistry of high-energy carbon, neon, and argon ions: hydroxyl radical yields. AB - Chemical yields of H2O2 have been measured from aerated aqueous solutions of bromide and of formate irradiated at various pHs and solute concentrations with high-energy ions from the Berkeley Bevalac. Hydroxyl radical yields have been deduced from these data as a function of beam penetration into the solutions. By taking fragmentation of the primary ions into account, estimates of the instantaneous Gi OH values for the primary beams as a function of their energy have been made. PMID- 3001820 TI - Radiation-induced changes in the cell membrane of cultured human endothelial cells. AB - We investigated the effect of irradiation on the kinetic characteristics of amino acid and glucose transport, and the effect on the activity of the cell membrane bound enzyme 5'-nucleotidase and on the receptor-mediated stimulation of cyclic adenosine monophosphate synthesis by prostaglandin E1. Irradiation inhibited the sodium-dependent amino acid transport by a reduced binding of the amino acid to the transport unit. The transport of glucose, which appeared to be a sodium independent process, was temporarily stimulated by increased maximal velocity of the transport. No effect was found on the binding to the transport unit. Irradiation increased the 5'-nucleotidase activity and decreased the prostaglandin E1-stimulated cyclic adenosine monophosphate synthesis 48 h after exposure to 20 Gy. It is concluded that irradiation decreases sodium-dependent transport by impairment of the transport unit, does not impair a sodium independent process, and has opposite effects on membrane-bound enzyme activity and a receptor-mediated process. PMID- 3001821 TI - [Effect of ionizing radiation on the kinetic parameters of rat cerebral cortex Na, K-ATPase]. AB - A study was made of the effect of ionizing radiation of 10.3 and 180.6 mC/kg on kinetic parameters of the processes of activation of Na,K-ATPase of rat brain cortex by Mg-ATP-substrate and Na+ and K+ ions. The obtained results prompt an assumption that a conformational rearrangement occurs under the effect of ionizing radiation which is not identical after relatively small and lethal radiation doses. PMID- 3001822 TI - [Rapid freezing technique for electron paramagnetic resonance spectroscopy]. PMID- 3001823 TI - [Electron spin resonance]. PMID- 3001824 TI - [Time-resolved spin-label ESR method]. PMID- 3001825 TI - [Excitation and adaptation mechanisms of vertebrate photoreceptors]. PMID- 3001826 TI - [Herpes simplex virus and its DNA polymerase]. PMID- 3001827 TI - [Expression vectors for mammalian cells]. PMID- 3001828 TI - [Nuclease P1]. PMID- 3001829 TI - Cyclic adenosine 3'5' monophosphate stimulates prostaglandin E production by human adherent synovial cells. AB - Production of prostaglandin E (PGE) by rheumatoid synovium appears important to regulation of the pathologic process in rheumatoid arthritis. Cells derived from human synovium by proteolytic digestion produce large amounts of PGE which in turn can elevate synovial cell cAMP levels and inhibit cell proliferation. Data presented here indicate that cAMP can further increase production of PGE from adherent synovial cells (ASC). PGE production occurs over 12-72 hr and is not due to the ability of cAMP to inhibit cell proliferation. Exposure of cells to cAMP results in increased release of 3H arachidonic acid from precursors but not in activation of the cyclooxygenase enzyme. This phenomenon suggests the presence in adherent synovial cells of a mechanism for amplifying PGE production. PMID- 3001830 TI - Involvement of 5-lipoxygenase metabolites in ACTH-stimulated corticosteroidogenesis in rat adrenal glands. AB - Various lipoxygenase (LO) products of arachidonic acid (AA) have been found to have potent biological activities and modulate physiological processes in various cells including endocrine cells. However, no studies concerning LO products in adrenocortical cells have been reported. The present study was performed to investigate LO products in rat adrenocortical cells and its role in ACTH stimulated adrenal steroidogenesis. LO metabolites produced in ACTH-stimulated rat adrenocortical cells prelabeled with [3H]AA was analyzed by reverse phase and straight phase HPLC and two 5-LO products, 5-hydroxyeicosatetraenoic acid (5 HETE) and leukotriene B4 (LTB4) were identified. ACTH-induced 5-HETE and LTB4 production in adrenal cells was dose dependently inhibited by AA861, a specific inhibitor of 5-LO. AA861 reduced ACTH-stimulated corticosteroid production without any change in cyclic AMP formation, while indomethacin did not affect both corticosteroid and cyclic AMP production. Reduced steroidogenesis by AA861 was reversed by the addition of 5-hydroperoxyeicosatetraenoic acid (5-HPETE). Also exogenously added 5-HPETE dose dependently augmented ACTH-stimulated corticosteroid production without any concomitant change in cyclic AMP production. However, 5-HETE and LTB4 had no such effect. These results indicate that 5-LO pathway is present in rat adrenocortical cells and its metabolites, most likely 5-HPETE, may play an important role in adrenal steroidogenesis. PMID- 3001831 TI - Intraperitoneal injection of zymosan in mice induces pain, inflammation and the synthesis of peptidoleukotrienes and prostaglandin E2. AB - Intraperitoneal injection of zymosan in mice induced rapid extravasation and accumulation of plasma protein in the peritoneal cavity. Neutrophils began to appear in the peritoneal cavity after a lag period of approximately 3 hours. The injected mice exhibited a pain response (writhing) during the first 30 minutes after injection, but writhing ceased before protein or cell accumulation had reached maximum levels. The injection of zymosan induced synthesis of PGE2 (measured by RIA) which reached maximum levels at 30 minutes, then declined slowly. Peptido-leukotriene levels (detected by bioassay, RIA and HPLC) increased rapidly after injection, reached a peak within an hour of injection and declined to undetectable levels within 4 hours. The early peptido-LT was predominantly LTC4, while later, LTE4 was the major component. LTD4 levels remained low throughout and no LTB4 was detected at any time. Indomethacin treatment elevated levels of peptido-LTs, reduced PGE2 levels and inhibited writhing. Phenidone reduced peptido-LT levels. In vitro studies demonstrated that zymosan stimulates LTC4 synthesis by peritoneal cells whereas LTE4, LTD4, LTB4 or monoHETES were not detectable (using HPLC methods). The source of enzymes responsible for the in vivo metabolism of LTC4 to LTD4 and LTE4 could not be identified. PMID- 3001832 TI - Leukotriene B4 produces hyperalgesia in humans. AB - During inflammation, pain receptors are sensitized by inflammatory mediators causing hyperalgesia. Leukotriene B4 (LTB4) is a potent chemoattractant for polymorphonuclear leukocytes in humans in vivo. In the present study we have demonstrated a reduction of the pain threshold in humans after intracutaneous deposition of LTB4. The pain threshold was quantitated by the "Marstock" method in which the testsubject reversed the direction of the temperature change of a thermostimulator whenever a painful temperature was reached. This enabled a quantitative description of the persons pain sensibility. After intracutaneous infiltration of LTB4 (10 microM) a highly significant decrease in the pain threshold could be detected as compared to control with a maximum effect between 6 and 24 h. The decrease in the pain threshold amounted to less than 15% when compared to control and corresponds with the published kinetics of the influx of polymorphonuclear leukocytes. These results support the hypothesis that LTB4 produces hyperalgesia indirectly through recruitment of polymorphonuclear leukocytes and not via a direct effect on the pain receptors. Inhibition of the lipoxygenase might be an advantageous adjunct to the effect of the Non Steroidal Anti-Inflammatory Drugs. PMID- 3001833 TI - Arachidonic acid metabolites induce beta-adrenoceptor desensitization in rat lung in vitro. AB - The possible involvement of arachidonic acid (AA) or its metabolites in beta adrenoceptor desensitization has been studied in rat lung parenchyma both from a functional and a biochemical point of view. In vitro perfusion of rat lungs with AA (3 X 10(-5)M for 20 min) reduced the relaxant effect of isoproterenol (ISO) on lung parenchymal strips, shown by a shift to the right of ISO dose-response curve, similar to that obtained using desensitizing concentration of specific beta-agonist. Moreover, AA treatment reduced the capacity of ISO to stimulate adenylate-cyclase activity, whereas the number of beta-receptor binding sites was not significantly modified. Inhibition of cyclo-oxygenase pathway by indomethacin (INDO) (1.5 X 10(-5)M) prevented both the loss of ISO-relaxing capacity and the decrease of adenylate-cyclase activity induced by AA treatment. In order to support the role of eicosanoids in beta-adrenoceptor desensitization, changes of endogenous free AA levels have also been studied in lung homogenates. Perfusion of rat lung with ISO (10(-6)M for 20 min) decreased by about 50% the levels of free AA and the pretreatment with BW755C (9 X 10(-5)M), a lipo- and cyclo oxygenase inhibitor, prevented this phenomenon. On the basis of these results, we suggest that the activation of AA cascade is actually involved in beta adrenoceptor desensitization in lung tissues with a possible interference at the site beyond the drug-receptor interaction. PMID- 3001834 TI - Relationship of thromboxane generation to the aggregation of platelets from humans: effects of eicosapentaenoic acid. AB - A non-linear relationship between the percent aggregation of human platelets and the amount of TXB2 generated requires investigators to use caution when using the data to assess antiplatelet regimens. The relationship approximates a hyperbola with a roughly linear relationship from 0 to 70% aggregation and 0 to 50 ng TXB2 per ml of platelet-rich plasma. Above these values, the amount of TXB2 produced may increase up to 500 ng per ml of platelet-rich plasma with no clear relationship to the observed platelet function of aggregation. Also, appreciable inhibition of TXB2 formation can occur at high TXB2 levels with no detectable decrease in aggregation. Thus, assessment of antiplatelet regimens using TXB2 formation alone are unlikely to be interpretable without reference to this non linear property of platelet function. We applied this concept when evaluating a study of forty subjects with dietary supplements of 1.8 g or 2.7 g of ethyl eicosapentaenoate (20:5n-3) for four weeks. There was a moderate, but statistically significant decrease in average values for the percent aggregation (60 +/- 15 to 45 +/- 30) and thromboxane production (51 +/- 30 to 33 +/- 31 ng/ml). Although the differences in mean values were slight relative to the overall standard deviations, reductions of platelet function were clearly evident in 31 of 40 subjects when paired results were examined relative to the recognized hyperbolic relationship. PMID- 3001835 TI - [Occurrence of anti-respiratory virus antibodies in the sera of patients with respiratory tract infections 1981-1983]. PMID- 3001836 TI - [Isolation of new strains of tick-borne encephalitis virus in the Gdansk region. Characteristics of their biological properties]. PMID- 3001837 TI - [Effect of immunomodulators on the production of interleukin 2]. PMID- 3001838 TI - Radiation protection of rat parenchymal hepatocytes with S-2-(3 aminopropylamino)ethylphosphorothioic acid. AB - The ability to protect parenchymal hepatocytes from ionizing radiation damage with S-2-(3-aminopropylamino)-ethylphosphorothioic acid (WR-2721) was investigated. The clonogenic survival of irradiated hepatocytes was determined with an in vivo transplantation assay system. Injection of WR-2721 (400 mg/kg) immediately after irradiation was without protective effect. In contrast, an intraperitoneal (IP) injection of WR-2721 30 min prior to 60Co irradiation resulted in a significant protection of parenchymal hepatocytes. The mean lethal dose (Do) and extrapolation number (n) of the survival curve for unprotected hepatocytes as 2.9 Gy and 1.8, respectively. The Do and n values for hepatocytes protected with WR-2721 were 5.7 Gy and 1.8, respectively. Thus, WR-2721 at a concentration of 400 mg/kg acted as a dose modifying agent with a dose modifying factor of approximately 2.0. Decreasing the concentration of WR-2721 to 200 mg/kg did not significantly reduce the radioprotective effectiveness of the drug. However, at concentrations less than 200 mg/kg, the radioprotective effect decreased in a dose-response manner with a concentration of 150 mg/kg providing 50% of the maximum effect observed. PMID- 3001839 TI - Preoperative radiation therapy in advanced carcinoma of the ovary. AB - Preoperative radiotherapy in advanced ovarian carcinoma was evaluated. The overall 5-year survival rate in the irradiated group was 27%. When tumour mass remaining after operation was less than 2 cm in diameter, this figure rose to 52%. Comparison was made between those in whom operation became feasible only after preoperative irradiation and patients in advanced stages who were primarily successfully operated to less than 2 cm and with a 5-year survival rate of 44%. Selection of the cases for preoperative radiotherapy is obviously necessary. Fixed, bulky tumours in the pelvis, with or without metastases, may be suitable for preoperative radiotherapy. PMID- 3001840 TI - Social consequences of brain or liver relapse in small cell carcinoma of the bronchus. AB - The time spent in hospital when cerebral metastases occur as the first site of relapse in patients with small cell carcinoma of the lung (SCCL) has been analysed and compared to the consequences of relapse in the liver. In the course of a clinical trial with 370 patients, 50 patients relapsed initially in the brain and 20 in the liver. The 2 groups were comparable with respect to performance status at diagnosis and the amount of home support available. Patients who relapsed in the brain suffered a greater deterioration in performance status, and spent a greater proportion of their remaining life in hospital than did patients whose initial relapse was in the liver. This difference was most marked in patients who died soon after relapse. Radiotherapy (20 Gy in 5 fractions over one week or 30 Gy in 10 fractions over 2 weeks) and dexamethasone were not very effective treatments for brain relapse though subjective responses were common. The substantial morbidity and lengthy hospitalisation resulting from brain relapse compared with relapse at another site is a important factor to be considered in assessing whether prophylactic cranial irradiation should routinely be offered to patients with SCCL. PMID- 3001841 TI - Analysis of the dual mechanism of ACTH release by arginine vasopressin and its analogs in conscious rats. AB - Plasma ACTH and/or corticosterone levels were measured in conscious rats 30 min after subcutaneous administration of arginine vasopressin (AVP), oxytocin (OT) and various analogs with a large range of activity on the vasopressor (V1), antidiuretic (V2) or oxytocic receptors. The comparison of their dose-response curves indicated that two different mechanisms are involved in the release of ACTH by neurohypophysial peptides and their analogs. AVP itself and a specific vasopressor agonist (Phe2, Orn8, OT) displayed a similar, high slope dose response curve. Non-vasopressor analogs, such as dDAVP were characterized by a low slope dose-response curve. Furthermore, dDAVP potentiated CRF and neither its own ACTH-releasing action nor its potentiation of CRF were sensitive to previous VI- or V2-receptor blockade. These results, together with other available data, are interpreted as indicative of the existence of two mechanisms of action for ACTH release by AVP and its analogs in vivo: an indirect action via endogenous CRF release, mediated by a VI receptor mechanism, and a direct action on the pituitary, shared by dDAVP and other non-vasopressor analogs, with receptor characteristics different to both the V1 and the V2 classical types. PMID- 3001842 TI - Vasoactive intestinal peptide effects on GH3 pituitary tumor cells: high affinity binding, affinity labeling, and adenylate cyclase stimulation. Comparison with peptide histidine isoleucine and growth hormone-releasing factor. AB - The vasoactive intestinal polypeptide (VIP) receptor was characterized on the GH3 rat pituitary tumor cell line using competitive binding studies with peptides having sequence homology with VIP. Further studies investigated receptor coupling to the adenylate cyclase complex by measurement of cAMP levels. Finally, the molecular weight of the receptor was estimated by affinity labeling techniques. Studies using 125I-VIP and unlabeled competing peptides revealed a single class of high affinity binding sites with a dissociation constant (KD) of 17 +/- 2 nM (mean +/- S.E.M.) for VIP, 275 +/- 46 nM for peptide histidine isoleucine (PHI), and 1380 +/- 800 nM for human pancreatic growth hormone releasing factor (GHRF). VIP and PHI each stimulated intracellular cAMP accumulation in a dose-dependent manner; both peptides demonstrated synergism with forskolin. In contrast, GHRF neither stimulated accumulation of cAMP nor demonstrated synergism with forskolin. VIP plus PHI (1 microM each) caused no significant increase in cAMP over either VIP or PHI alone, implying that the two peptides act through the same receptor. Covalent crosslinking of 125I-VIP to its binding site using either disuccinimidyl suberate (DSS) or ethylene glycol bis(succinimidyl succinate) (EGS) was followed by SDS-PAGE and autoradiography. The result is consistent with an Mr 47 000 VIP-binding subunit comprising or being associated with the VIP receptor of GH3 pituitary tumor cells. PMID- 3001843 TI - Involvement of thyrotropin-releasing hormone (TRH) neural system of the brain in pentylenetetrazol-induced seizures. AB - In order to study the relationship between pentylenetetrazol (PTZ)-induced seizures and the thyrotropin-releasing hormone (TRH) neural system, immunoreactive TRH (IR-TRH) and TRH receptor binding activity were determined in discrete regions of the rat brain before as well as 40 s (immediately before seizures), 150 s (during seizures) and 24 h after an intraperitoneal injection of PTZ (75 mg/kg). IR-TRH markedly increased in the septum 40 and 150 s after the injection, and also in the hippocampus and the thalamus-midbrain region 40 and 150 s after the injection, respectively. However, no significant changes were observed in the TRH receptor binding before, during or after the seizures, suggesting that the increased IR-TRH was not released into the synaptic cleft. This speculation was supported by the dose-dependent inhibition of PTZ-induced generalized seizures by the pre-treatment with TRH or its analogue DN-1417 into the cerebral ventricle. PMID- 3001845 TI - [EPR and TL measurements of BeO "Termalox 995" irradiated with photons]. AB - Beryllium oxide dosimeters (Termalox 995) have been irradiated (at 60-Co facility) with graduated doses from 3 X 10(-1) to 1 X 10(2) Gy (30 to 10.000 rad). EPR measurements show a very good linear correlation between the amplitude of signal and the absorbed dose. This method has the advantage that the stored information into dosimeter doesn't regress "after reading procedure". PMID- 3001844 TI - [Meningeal prophylaxis with radiocolloids in childhood leukemia and non-Hodgkin's lymphoma]. AB - Experiences in 13 children treated with radiocolloids (198Au and 32P) applied intrathecally are presented. This treatment may replace the external radiation therapy in prophylaxis or therapy of central nervous system (CNS) involvement in childhood leukemia and non Hodgkin lymphoma. The follow-up median value was 18 months. Ten children were treated for prophylactic aim. Two out of 10 presented meningeal failure 1 month after. Three patients treated for meningosis died for bone marrow relapse without evidence of blasts in CSF sediment. PMID- 3001846 TI - [Effects of intravenous perfusion of glucagon on plasma concentrations of glucose, insulin and cyclic AMP in patients with hepatic cirrhosis]. PMID- 3001847 TI - [Mediastinal tumor, esophageal neoplasm, osteosarcoma and neoplasm of the breast in 3 members of a family]. PMID- 3001848 TI - [Primary immunodeficiencies and purine metabolism]. PMID- 3001849 TI - [Eosinophilic gastroenteritis with extraintestinal involvement and cancer of the colon]. PMID- 3001850 TI - [Magnetic resonance tomography in syringomyelia]. AB - Thirteen patients with a clinical diagnosis of syringomyelia were examined by nuclear tomography (0.35 T magnet) in the spin-echo mode. In all thirteen patients, the T1 images (SE 400/35) showed a longitudinal cavity with a signal intensity of CSF. The shape and extent of the syrinx could be adequately demonstrated in 12 of the 13 examinations. Downward displacement of the cerebellar tonsils was seen in eight cases. The examination took between half and one hour. Advantages of magnetic resonance tomography (nuclear tomography) include the absence of artifacts, images in the line of the lesion and its non invasiveness. PMID- 3001851 TI - [Proton spin tomography in diseases in the petrous bone region]. AB - Magnetic resonance tomography (MRI = magnetic resonance imaging) can provide sectional images of the petrous bones in all planes. It is complementary to computed tomography (CT) and can demonstrate inflammatory changes in the petrous bones and mastoids, and cholesteatomas, with accuracy and without artifacts. MRI is inferior to CT for the demonstration of bone destruction and this this limits its use in the diagnosis of glomus tumours. The use of special surface coils combined with thin sections and a gradient-zoom technique provides high resolution images which approach the quality of modern CT examinations. This opens new possibilities for the diagnosis of small acoustic neuromas; because of the excellent tissue contrast, they can be well demonstrated without using intravenous contrast media or intrathecal air (CT-air meatography). PMID- 3001852 TI - [Diagnosis and differential diagnosis of intracranial vascular malformations using proton spin tomography]. AB - Eleven patients are described, nine of whom had angiomas and two/had aneurysms in the brain. Magnetic resonance tomography produced characteristic findings in all these patients. Angiomas typically show irregular margins with little or no surrounding oedema. Thrombosis is common and can be recognised by a reduction of T1 and prolongation of T2. Zones with high blood flow show reduced signal strength. In typical cases the lesion resembles a cauliflower. Enlarged draining veins could be recognised in five of the nine angiomas. Aneurysms, on the other hand, have a smooth configuration and it may be possible to demonstrate their connection to the vessels; signals arising from them are more homogeneous and less speckled. Their signal may be increased, reduced or absent, depending on blood flow. Angiomas and aneurysms may be associated with intracerebral bleeding. Magnetic resonance tomography is markedly superior to CT in diagnosing these lesions. Unlike CT, magnetic resonance tomography does not require the use of contrast media. PMID- 3001853 TI - [Real-time sonography in the diagnosis and follow-up of malignant tongue tumors. I. Anatomic basis]. AB - Normal structures of the tongue and floor of the mouth were studied using anatomic sections cut with a stainless steel band saw. The sections were performed on the same planes as used in US and CT scanning. The ultrasound studies were carried out with 20 young and healthy volunteers. CT images were obtained from head/neck preparations that were subsequently used for anatomic sectioning. On comparing these sections to US and CT images, normal structures including intrinsic and extrinsic tongue muscles, vessels and salivary glands were identified. Knowledge of the anatomic landmarks is mandatory for optimal US image reading. PMID- 3001854 TI - [Catheter embolization of the thoracic arteries in the treatment of lung hemorrhage]. AB - In cases of acute, life-threatening pulmonary haemorrhage, immediate angiographic localisation of the source of haemorrhage is mandatory. Instead of a surgical intervention, transcatheter angiographic therapeutic embolisation may be performed. Embolisation is an effective method with only minimal complications, in favourable contrast to the 23 to 30% mortality encountered with surgical procedures. Embolisation, therefore, should be the first procedure in patients presenting with acute pulmonary haemorrhage. It must be emphasised that in cases of chronic pleuro-pulmonary diseases bleeding occurs not only from bronchial arteries. In 64% of these cases, the bleeding artery originates from a thoracic or diaphragmatic artery. The authors were able to treat 26 of 34 pulmonary haemorrhages (76%) by transcatheter embolisation. In 4 of these patients recurrent haemorrhage was treated by transcatheter embolisation. Severe complications were not noted in any case. PMID- 3001855 TI - [Quantification of vascular stenoses using digital subtraction angiography]. AB - Experimental studies using vessel phantoms with varying stenoses have been carried out and a method has been developed for quantitative analysis of vascular stenoses using DSA. The digital information provides quantitative measurements of the amount of contrast within a vessel. The relative contrast density in the region of interest is measured by video densitometry and is proportional to the transverse area of the vessel. The relative densities in and proximal to the stenosis provides a quantitative measure of the relative degree of stenosis. The standard deviation does not exceed 7.5%. PMID- 3001857 TI - [Indications for the intra-arterial infusion of urokinase in the treatment of acute intestinal ischemia in patients with heart disease]. AB - The poor prognosis of acute mesenteric artery occlusion can be improved by reaching a rapid angiographic diagnosis and by instituting treatment at an early stage. In addition to operative embolectomy, success may be expected from the use of urokinase infused super-selectively into the superior mesenteric artery. This treatment is only likely to be successful if it is carried out within ten hours of the onset of clinical signs and symptoms. In patients with heart disease, angiography is recommended as soon as there is any suspicion of mesenteric occlusion, in order to confirm the diagnosis, localise the embolus and decide on the form of treatment. Urokinase treatment can be successful for embolic occlusion of the main branches or peripheral branches of the superior mesenteric artery. However, complete occlusion of the main superior mesenteric artery should be treated operatively. A contra-indication to urokinase therapy is occlusion due to infected emboli from an endocarditis. PMID- 3001856 TI - [Quantitative flow measurement of the carotid artery using a combination of intra arterial digital subtraction angiography and ultrasound]. AB - Arterial blood flow was estimated using digital subtraction roentgen-densitometry in a model of the cervical arteries. The best results were obtained with two sampling windows calculating the mean passage time using the line of gravity of the contrast bolus. Mechanical contrast injection created a systematic error by acceleration. This procedure was then used for examining five patients. Quantitative blood flow measurements required knowledge of the transverse vessel area. This was determined by high resolution sonography. Blood flow was calculated as the product of cross-sectional area and mainstream velocity. PMID- 3001858 TI - [Computerized tomography and sonography in the diagnosis of recurrent colorectal tumors]. AB - CT and ultrasound of the abdomen were retrospectively evaluated in 44 patients operated on colorectal cancers (18 colon, 26 rectal carcinomas) in detecting a recurrence suspected on biochemical, endoscopic or clinical grounds. CT is superior to ultrasound in detecting a recurrence (metastases of liver and lymph nodes, local recurrence, intra-abdominal recurrence of another localisation). CT is recommended as a routine examination in the follow-up of patients operated on colorectal cancers. PMID- 3001859 TI - [Computerized tomography of the elbow joint]. AB - An essential pre-condition for the interpretation of computer tomographs of the elbow joint is a knowledge of the normal anatomy. The complexity of the joint requires a definite protocol for the examination, since only in this way is it possible to distinguish all anatomical structures and pathological processes. A clinical example is used to show that CT of the elbow joint is superior to all other radiographic methods for demonstrating osteochondritis dissecans. It is therefore an important additional method for demonstrating the elbow joint. PMID- 3001860 TI - Bone scanning in patients with breast carcinoma. Should it be a routine examination in every stage group? AB - Skeletal imaging using radionuclides has proved to be a sensitive method for the detection of early bony metastases from breast carcinoma. Recent studies have found a relatively low rate of abnormal scans in patients with Stage I and II breast cancers, and therefore it is open to question whether bone scanning should be part of the preoperative evaluation of any patient prior to breast surgery. We reviewed our experience with bone scans in 329 patients out of 406 histologically proven breast cancer patients to determine if any or all patients should have this procedure done routinely prior to breast surgery. PMID- 3001861 TI - [Data processing in radiology: summary and prospects]. AB - The technical aspects of radiology are particularly suitable for electronic data processing. In addition to automation of radiological apparatus and tumour registration, there are three areas in radiology particularly suitable for electronic data processing: treatment planning, dose calculations and supervision of radiotherapy techniques in radio-oncology. It can be used for word processing in the office and for documentation, both in diagnostic and therapeutic radiology, and digital techniques can be employed for image transmission, storage and manipulation. Computers for treatment planning and dose calculation are standard techniques and suitable computers allow one to spot occasional and systematic errors during radiation treatment and to eliminate these. They also provide for the automatic generation of the required protocols. Word processors have proved particularly valuable in private practice. They are valuable for composing reports from their basic elements, but less valuable for texts that are stereotypes. The most important developments are in digital imaging, image storage and image transmission. The storage of images on video discs, transmission through fibre-optic cables and computer manipulation of images are described and the consequences and problems, which may affect the radiologist, are discussed. PMID- 3001862 TI - [CT in radiotherapy. Its value in computer-supported 3D-irradiation planning for the breast]. AB - The use and value of CT-directed radiation planning in three planes is discussed. Various modifications for simulation are proposed. In particular, reconstruction for the horizontal position of the sternum is considered. PMID- 3001863 TI - [Experimental studies on the accuracy of mineral content assessment in spongiosa bone using quantitative CT (single energy measurement)]. AB - The mineral content of 42 lumbar vertebral bodies (using a phantom to simulate living conditions) and of 30 calcanea was measured by quantitative CT. The bones were subsequently ashed and the calcium and fat content determined chemically. The mineral concentration (calibrated against a K2HPO4 solution) showed good correlation with the calcium content (for the lumbar vertebrae, r = 0.963 and for the calcanea r = 0.94. The fat content of the bone marrow leads to a systematic under-estimation of the mineral content, if one uses single energy CT measurements. The experimental findings can be satisfactorily treated in a quantitative way with the help of a model which contains the three components of spongy bone (mineral, fat and fat-free connective tissue. PMID- 3001864 TI - [The effect of beam hardening on CT values]. AB - The investigation has shown that the effect of beam hardening and the nature of surrounding structures must not be neglected when carrying out quantitative measurements. This is particularly true in the presence of strongly attenuating, and therefore strongly beam hardening structures such as bone, in regions where there are wide variations in CT values and when using varying kilo-voltages. Within the range of 0 to 200 HU, the effect of beam hardening is less important when compared with other statistical errors. PMID- 3001865 TI - [In vivo dosimetry in whole body irradiation]. AB - The results of measurements on an Alderson phantom, as well as in-vivo measurements, on five patients during total body irradiation are described. They were compared with computer-predicted values using the lateral A.P./P.A. irradiation, as used in Tubingen; measurements were carried out by a TLD method. Particular attention was paid to the critical areas such as the head, the neck and the lungs. The results in general agreed to within 5% and were in good accord with international findings. PMID- 3001866 TI - [Experimental studies of the scatter behavior of fast electrons in the use of electron applicators in radiotherapy]. AB - When using high energy electrons in radiotherapy, there is frequently undesirable electron scatter, which is derived from the applicators used to delimit the field. In order to improve our understanding of the factors which influence scatter, this was studied using fast electrons and a variety of materials to reduce this scatter. The geometry and angulation of the scattering plate and the electron energies were varied during these experiments. PMID- 3001867 TI - [Dose distribution of electrons obtained from linear accelerators equipped with a beam scanning system]. AB - Homogeneous distribution of electrons used for therapeutic purposes and obtained from accelerators, is achieved by means of Potter-Bucky diaphragms or by repeated, staggered, sawtooth-shaped sweeping movements of the electron beam (scanning) over the radiation field. The repetition of the scanning process (number of scans) can result in long measurement times for achieving a sufficiently homogeneous, dosimetrically adequate distribution of the electrons. This "time problem" makes it imperative to achieve good homogeneity while keeping the number of scans as low as possible. To solve the problem, the scanning movement of the electron beam is simulated by a computer programme and the interdependence of the homogeneity of the irradiation field and number of scans is investigated. Since changing the ratio of the two deflection rates exercises a significant influence, it is mandatory in dosimetry to pay close attention to a strict observance of the deflection rates. PMID- 3001868 TI - [Supralinearity of radiothermoluminescent dosimetry]. AB - The authors studied how supralinearity influences individual "deep trap" terms (or levels) of a glow curve that represents the luminous flux emitted by heated LiF rods as a function of energy required for heating. It is assumed that a Gaussian function can be used for glow curve analysis, developing measured glow curves to represent individual "deep trap" terms. Analysis reveals that supralinearity occurs much earlier and more markedly with high temperature peaks than with low ones. This has a bearing on practice. PMID- 3001869 TI - [Coagulation disorder following angiography with a nonionic tri-iodized contrast medium? Effect due to contrast medium or to heparin?]. PMID- 3001870 TI - A case of lower limb ischaemia following aortography with cutaneous lesions but with preservation of the peripheral pulses. PMID- 3001871 TI - [Angiodysplasia of the liver and gastrointestinal system in Rendu-Osler-Weber disease]. PMID- 3001872 TI - [E. coli sepsis with systemic air embolism]. PMID- 3001873 TI - [Thoracic radiographic findings in mucocutaneous lymph node syndrome]. PMID- 3001875 TI - [Brushings and bronchial biopsies in small-cell cancer. Comparison of results]. AB - The typing of bronchial carcinoma is important for therapeutic decisions and may be made on bronchial brushings. Sixty six small cell and poorly differentiated squamous cancers were studied. Our personal method of preparing brushing is suggested. The cytological criteria which characterise the two tumour types were analysed. The results from the biopsies and particularly the brushings were related to the quality of the technique and the experience of the Pathologist. There is a good correlation between biopsy and brushing such that brushings may be considered reliable. In cases where the biopsy was not feasible or uninterpretable, brushings confirmed a cancer in all the cases and the type of cancer in eleven. The examination is inexpensive and avoids the need for ultrastructural studies in the majority of cases. PMID- 3001874 TI - [Case report of osteochondrosis dissecans of the navicular bone]. PMID- 3001876 TI - [Bronchio-alveolar carcinoma: diagnostic possibility with transbronchial pulmonary biopsy]. PMID- 3001877 TI - Properties of an ecto-5'-nucleotidase of the renal brush border. AB - A decrease of glomerular filtration rate can be observed during accelerated catabolism of ATP in kidney. It has been proposed that this effect is due to the increase in the renal production of adenosine from ATP. The last reaction in the pathway concerned is the conversion of 5'-AMP to adenosine. We found that brush border membranes purified from homogenates of the rat renal cortex carry out this reaction. The enzyme involved in the hydrolysis has the characteristic properties of ecto-5'-nucleotidases: It is inhibited by ATP, ADP, and by alpha, beta methyleneadenosine-5'-diphosphate, and it is not stimulated by magnesium. All catalytic sites are accessible from the outside of the vesicles. The Km of the enzyme for 5'-AMP is 5.77 microM. The enrichment of the 5'-AMP-hydrolyzing activity in the brush border fraction as compared to the homogenate is 9.2 +/- 1.5 times. Histochemical staining of kidney sections reveals hydrolysis of 5'-AMP only at the brush border of the proximal tubule. We conclude that the brush border of the proximal tubule of the rat kidney possesses an ecto-5'-nucleotidase which has the same properties as the ecto-5'-nucleotidases in other tissues. PMID- 3001878 TI - The physiology of corticotropin-releasing factor (CRF). AB - Corticotropin-releasing factor (CRF) was characterized, purified and synthesized in 1981 by Vale. The composition of this 41 amino-acid peptide varies slightly from one species to another. Its principal origin is the paraventricular nucleus of the hypothalamus and, by the portal vessels, it reaches the adenohypophysis where it stimulates the corticotrophs. The effects of CRF have been analysed by in vitro and in vivo experiments. Some actions on the autonomic nervous system have been described. The action of CRF on adrenocorticotrophic hormone (ACTH) secretion is predominant, but it acts in relation with arginine-vasopressin and accessorily with angiotensin and catecholamines. Adrenal steroids act on feedback control of CRF secretion in the hypothalamus and also on pituitary corticotrophs. The CRF function starts between the 16th and 19th day in the fetal rat. The placenta is able to produce CRF. PMID- 3001879 TI - Heterogeneity of adrenocorticotrophin in the pituitary gland and plasma during the perinatal period. AB - Three immunoreactive forms of ACTH are characterized by their apparent molecular weight (MW) and bioactivity when acid extracts of the anterior or of the neurointermediate lobe of fetal and newborn rat pituitary glands are subjected to gel filtration on Sephadex G50 fine columns. The "big" ACTH form which shows an apparent MW close to 44 000 hardly stimulates the in vitro release of corticosterone by perifused fetal adrenals. In contrast, the "intermediate" (MW: congruent to 13 000) and "little" (MW: congruent to 4 500) forms show high biological activity by eliciting corticosterone secretion which is log-dose dependent. During the perinatal period, the relative proportions between these different molecular forms of ACTH change in both the anterior and neurointermediate lobes. "Big" ACTH is the main form in the neurointermediate lobe of the fetal rat pituitary. After birth, the "big" ACTH/total ACTH ratio regularly decreases until postpartum week 4; it is not very different then from that of pregnant adult females. The three immunoreactive forms of ACTH are present in the anterior lobe throughout the perinatal period. The gradual increase of the "intermediate" and "little" forms is accompanied by a correlative decrease in the "big" form. The anterior lobes of 17, 19 and 21-day old fetuses, stimulated in vitro by an acid extract of adult hypothalamus, release the three immunoreactive forms of ACTH in the same proportions as those observed in corresponding extracts of anterior lobes. On days 17, 19 and 21 of gestation, fetal plasma contains all the immunoreactive forms of ACTH previously observed in pituitary glands. The proportion of the "little" ACTH form gradually increases, whereas that of the "big" form decreases as gestation progresses. At term, the relative proportion of "little" ACTH is greater in the plasma than in the fetal pituitary. Controlled trypsic digestion of pituitary "big" ACTH results in a conversion to the "intermediate" and "little" immunoreactive forms. When "intermediate" ACTH is submitted to tryptic digestion under the same conditions, there is continuous loss of immunoreactivity but no change of hormonal form. These findings strengthen the hypothesis that "big" ACTH is a precursor of "intermediate" and "little" ACTH; in contrast, the "intermediate" form is not a precursor of the "little" one. The high molecular-weight form of ACTH might be converted endogenously into lower MW forms in the fetal circulation at term as well as in newborns. In fetal plasma, immunoreactive ACTH levels reach peak values on day 19 of gestation and decrease thereafter until day 21.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3001880 TI - Biochemical modifications involved in the maturation of the ovine fetal adrenal gland in late gestation: their modalities and regulation. AB - During the last month of intra-uterine life, the steroidogenic response of the ovine fetal adrenal glands to ACTH increases and becomes maximal at the time of birth. This development involves modifications at different steps of the adrenocorticotropic hormone (ACTH) action mechanism. It has been shown that the enhanced capacity of the cells to produce cAMP is related to at least three factors: an increased number of ACTH receptors, increased activity of the Ns subunit of adenylate cyclase, and enhancement of guanosine 5'-triphosphate (GTP) availability. The ability to produce pregnenolone and the activity of both 3 beta hydroxy steroid dehydrogenase/isomerase and 17 alpha-hydroxylase are mainly enhanced in the steroidogenic pathway. The infusion of ACTH for 5 days into 115 to 120-day old fetuses results in the development of most of these biochemical process. Similarly, ACTH can induce maturation of cultured fetal adrenal cells and some other proopiomelamocortin (POMC)-derived peptides can potentiate its acute steroidogenic activity in vitro. However, even in the absence of ACTH, the adenylate cyclase system and the steroidogenic potency of cultured cells increase but to a lesser extent than when ACTH is present in the culture medium. It is suggested that ACTH is the main trophic hormone of the ovine fetal adrenal during the last month of gestation, even if other stimulatory factors may also be important. The in vivo maturation of ovine fetal adrenal is blocked by the presence of some unknown inhibitory factors in the fetal circulation which are of likely extrapituitary origin. PMID- 3001881 TI - Control of aldosterone secretion in domestic mammals during the perinatal period. AB - Aldosterone in ovine fetal circulation is mainly of fetal origin. Three of the renin-angiotensin system (RAS) parameters (renin, renin substrate and angiotensin II) originate in the fetus. The fetal RAS responds to stimulation (furosemide administration, hemorrhage, hypoxemia) or inhibition (phenylephrine or angiotensin II infusion) in a manner similar to that of the adult. The response of the fetal adrenal gland to angiotensin II is age-dependent and the near-term adrenal gland is not fully sensitive to angiotensin II and relatively insensitive to potassium (K+) as well as to adrenocorticotrophin hormone (ACTH) stimuli. In the newborn, the RAS is fully operative since it responds to diuretic stimulation in the lamb and the calf or to inhibition by angiotensin II administration in the lamb. Angiotensin II infusion, or angiotensin-converting enzyme blockade in the newborn lamb is followed by changes in plasma renin activity (PRA) or plasma aldosterone concentration (PAC) that suggest that angiotensin II regulates aldosterone secretion and that there is a negative feed back loop between angiotensin II and renin release. The newborn lamb adrenal gland is sensitive to potassium ions while in the newborn calf, ACTH stimulates cortisol secretion but fails to induce any change in PAC, suggesting that, in this species, the zona glomerulosa of the adrenal cortex is insensitive to ACTH. PMID- 3001882 TI - Effects of heterocyclic sulfonamides on bone metabolism. AB - Sulfonamide inhibitors of carbonic anhydrase attenuate parathyroid hormone and vitamin D3 induced bone resorption. Parathyroid hormone stimulates bone cell accumulation of cAMP in the presence of concentrations (10(-7) - 10(-3) M) of acetazolamide (an inhibitor of carbonic anhydrase) that are antagonistic to bone resorption. Bone cell acid phosphatase activity and lactic acid production are stimulated by parathyroid hormone or vitamin D3. These cellular responses are not influenced by the presence of acetazolamide (10(-4) M). Bone cells incubated in the presence of parathyroid hormone reveal an increase in carbonic anhydrase like activity. This activity is lost upon heating a supernatant of the bone cell population or upon treatment of the supernatant with appropriate concentrations of acetazolamide. PMID- 3001885 TI - Effects of certain CNS depressants and verapamil on cGMP in the cerebellum of rats. AB - The influence, individually and in combination, of certain general CNS depressants, Ca+2, A23187, and verapamil on rat cerebellar cGMP was studied. When Ca+2 was given to rats treated with ethanol, ether, or pentobarbital a minor increase in cGMP resulted. However, A23187 alone, or with Ca+2 did not produce a rise in cGMP levels in the presence of those drugs. A similar effect was seen with verapamil. Increasing doses of pentobarbital with verapamil enhanced the inhibition of cGMP. PMID- 3001883 TI - Medroxyprogesterone acetate and phenobarbital induce NADPH producing enzyme activities in rats with a chemical liver injury. AB - The administration of medroxyprogesterone acetate (MPA) and phenobarbital (PB) improves liver function in rats with liver damage. This was seen here as increased aryl hydrocarbon hydroxylase (AHH) activity after therapy with MPA or PB in rats with a chemical liver injury, produced by dimethylnitrosamine (DMN). Hepatic glucose-6-phosphatase (G6Pase) activity, an index of glucose metabolism was also normalized in the MPA treated rats. The present study further shows that MPA induced hepatic malic enzyme (ME) and isocitrate dehydrogenase (ICDH) activities and PB enhanced glucose-6-phosphate dehydrogenase (G6PDH), 6 phosphogluconate dehydrogenase (6PGDH) and ME activities in the DMN pretreated rats. This suggests that MPA and PB enhanced the capacity of altered liver tissue to generate NADPH, a cofactor in the monooxygenase system, which may, in part, enhance the restoration of drug hydroxylation in the rats. Since G6PDH, 6PGDH and ME participate in glucose metabolism, the finding that the compounds influenced these enzymes in distinct ways, may explain the different effects of MPA and PB on the restoration of glucose metabolism. PMID- 3001884 TI - Reduction of specific arginine-vasopressin binding sites of renal medulla membranes in lithium treated rats. AB - To elucidate the early effects of lithium treatment on the kidney, arginine vasopressin (AVP) binding sites in the renal medulla were measured by radiolabeled receptor assay (RRA) in rats injected intraperitoneally with lithium chloride (4 mmol/kg body weight) for 7 days. After the 8th day, urine volume of rats injected lithium (Li group) increased and was significantly larger than that of the control rats that were injected physiological saline (control group). On the same day, the AVP binding capacities of renal medulla membranes of the Li group (Bmax = 28.9 +/- 7.2 fmol/100 micrograms protein) were significantly lower than that of the control group (Bmax = 50.0 +/- 6.3 fmol/100 micrograms protein), but there was no difference in the dissociation constant between the two groups. Our findings suggested that lithium-induced diabetes insipidus was due to a reduction in the number of AVP binding sites in the kidney. PMID- 3001886 TI - In vitro binding of platinum compounds to human transferrin. AB - The binding of the antineoplastic drug cis-dichlorodiammineplatinum II (CDDP) and a related complex, potassium tetrachloroplatinite (K2PtCl4), to human transferrin in buffered anion solutions was studied over a period of two weeks. Separation of bound and free platinum was accomplished by gel chromatography with platinum analysis by atomic absorption spectrophotometry. The results demonstrated that K2PtCl4 was bound to a greater degree than CDDP in this system with 3-5 and 1-2 platinum atoms respectively, bound per transferrin molecule. However, it appears this interaction, which may involve the specific cation binding sites, does not play a significant role in the pharmacology of CDDP in vivo. PMID- 3001887 TI - Biochemical identification of renin in human pheochromocytoma. AB - A high activity of renin was demonstrated in human pheochromocytoma tissue. This activity was inhibited by specific antibody raised against human renal renin, indicating that it was not due to the nonspecific actions of proteases such as cathepsin D. The specific renin shared some biochemical features with well-known kidney renin, such as molecular weight (47,000 daltons), optimum pH (6.0), the presence of trypsin-activatable inactive renin, and glycoprotein nature. However, the isoelectrofocusing pattern of renin from the pheochromocytoma differed from that of kidney and plasma renins hitherto reported, a discrepancy which could be interpreted as evidence for endogenous synthesis of the enzyme. Furthermore, angiotensin converting enzyme activity was found in the tissue. Since pheochromocytoma is considered to be of neural crest origin, these results provide biochemical and immunological evidence for the presence of the renin angiotensin cycle within human neuronal cells. PMID- 3001888 TI - [Mediastinal neoplasm, diagnosis and treatment]. PMID- 3001889 TI - [Adult T-cell leukemia/lymphoma]. PMID- 3001891 TI - [Adult T-cell leukemia/lymphoma]. PMID- 3001892 TI - [The etiologic agent of AIDS and its relation to the virus causing adult T-cell leukemia/lymphoma]. PMID- 3001890 TI - [Dazoxiben blocks the increase of angiotensin converting enzyme in bronchoalveolar lavage induced by free fatty acids]. PMID- 3001894 TI - [Mononucleosis syndromes]. PMID- 3001893 TI - [Hepatitis caused by suloctidil]. PMID- 3001895 TI - [Intravascular bronchioloalveolar tumor or I.V.B.A.T. Apropos of a case and review of the literature]. AB - Intravascular bronchioloalveolar tumor is a rare tumour of the lung. Having observed a case detected during systematic examination in a 44-year old woman, the authors compare the radiological, clinical, histological and evolutive signs of the disease with those reported in the 40 cases published since the first description by Dail and Liebow, in 1975. PMID- 3001896 TI - [Endometrial hyperplasia]. AB - Atypical and severe hyperplasia of the endometrium is often associated with a carcinoma and, on their own, present a malignant potential which requires a specific treatment, involving an extension of the indications for hysterectomy after the menopause. PMID- 3001897 TI - [Evaluation of 3 immuno-enzyme kits for the detection of anti-LAV antibodies. Comparison with confirmation tests. Study Subgroup of the National Blood Transfusion Society]. AB - Three enzymo immuno assay kits for the detection of anti-LAV/HTLV III antibodies were evaluated: Diagnostics Pasteur (Elavia) Abbott Laboratories (Abbott/HTLV III) and Organon (Vironostika). A total of 6017 blood donors (5980 unselected and 37 prisoners) and a panel of 55 sero negative or sero positive samples were tested. Very weak differences were noted between these 3 kits: Abbott-HTLV III seems the least specific and the most sensitive and Vironostika the most specific and the least sensitive. ELISA quantitative results are given and compared to the results obtained by indirect immunofluorescence, Western-Blot and radio immuno precipitation. PMID- 3001898 TI - [L forms of Mycobacterium tuberculosis and their clinico-epidemiological significance]. PMID- 3001900 TI - [Current modalities of the onset and manifestation of pulmonary tuberculosis]. PMID- 3001901 TI - [The late impact on young people of prophylactic measures aimed at children and adolescents]. PMID- 3001899 TI - [Special actions for preventing and combating tuberculosis in children in Tulcea County]. PMID- 3001903 TI - [Risk factors in chronic bronchitis]. PMID- 3001902 TI - [Integral mass chest x-ray of the population over 45. The experience of a region with 2 rural medical dispensaries]. PMID- 3001904 TI - [Role of constitutional factors in chronic suppurative bronchopneumonias]. PMID- 3001905 TI - [Changes in the cytology of the sputum of workers exposed to carcinogenic hazards]. PMID- 3001906 TI - [Tracheobronchomegaly]. PMID- 3001907 TI - [Test of peripheral mononuclear adherence in the diagnosis and prognosis of pleurisy]. PMID- 3001908 TI - [Diagnosis and therapy of necrotizing pneumonia in adults]. PMID- 3001909 TI - [Observations of cases of the posttuberculosis syndrome hospitalized over the last 10 years at the Constanta TB Hospital]. PMID- 3001911 TI - [Peripheral neuropathies]. PMID- 3001910 TI - [Contribution of Iuliu Hatieganu to combating tuberculosis in Romania]. PMID- 3001915 TI - The fine structure of adult Onchocerca volvulus recovered by collagenase digestion. AB - Very young, middle-aged and old macrofilariae of Onchocerca volvulus were isolated alive or intact from onchocercomata using the collagenase digestion technique and studied by transmission electron microscopy. The body wall and the internal organs showed a well preserved morphology and no differences were detected compared with worms which had not been in contact with collagenase solution. PMID- 3001917 TI - [Clinical picture of bacterial pneumonias and their differential diagnosis]. AB - Under the influence of antibiotic therapy, bacterial pneumonias have undergone a remarkable change in the last few decades. Individual forms of pneumonia can be distinguished morphologically by their localization, the way in which they spread, their limitations, and their course. Clinically, opportunistic bacterial infections predominate. Increasingly, secondary pneumonias are observed in poststenotic areas, areas of infarction, in hypostatic areas, after aspiration, and in previously damaged lobes. Radiologic criteria for differentiating from atypical pneumonias (viruses, mycoplasmas and chlamydia) are discussed. PMID- 3001913 TI - [Neurophysiologic acquisitions of neuronal aggregates. Biological evolution from neuron to brain]. AB - The great operative possibilities of human brain have developed parallel to the aggregation of neurons in always more complex coacervations making possible the development of new neurophysiologic properties, such as convergence and divergence, facilities and inhibition. And it is just from the above mentioned properties that adaptability rises. PMID- 3001918 TI - [Effects of alcoholism on the peripheral nervous system and striated muscle]. PMID- 3001916 TI - [Computer tomography in bone metastases]. AB - A case is presented which bone metastasis could be proved by means of CT examination. This was in spite of normal bone scanning and normal X-ray findings for the lumbar column. The merits of the individual examination techniques are briefly discussed. PMID- 3001914 TI - Cytofluorometry as a method for the differentiation of trypanosomes. AB - The DNA binding guanine specific antibiotic, chromomycin A3, has been evaluated for fluorescence intensity measurements of T. cruzi, T. brucei brucei and T. musculi. Optimal fixation and staining conditions have been determined. The fluorometry was performed with a microscope photometer equipped with electronic systems for short time excitation of 7 milliseconds and operation control. The trypomastigote bloodstream forms of these species have a different chromomycin specific DNA content. The total DNA content of T. cruzi was 2.1-fold higher than for T.b. brucei and 2.3-fold higher than for T. musculi. The nuclear DNA content also was higher in T. cruzi. The nuclear DNA values were recorded to be 1.6-fold greater than in T.b. brucei and 2.0-fold greater than in T. musculi. The amount of the kinetoplast DNA of T. cruzi was shown to be 3.2-fold higher than in T. musculi and 11.7-fold higher than in T.b. brucei. The higher total DNA of T.b. brucei in relation to T. musculi was based on the nuclear values because the content of the kinetoplast DNA of T.b. brucei was 3.7-fold smaller than of T. musculi. The kDNA comprised 25% in T. cruzi, 18% in T. musculi and only 4% in T.b. brucei of the total amount of the chromomycin specific DNA. The chromomycin fluorescence intensities of the DNA of trypanosomes were subjected to a statistical model of discriminant analysis. It was possible to get perfect separation of the three trypanosome species. The hit rate was 100%. PMID- 3001912 TI - Virology and immunology of the common cold. AB - The common cold is a complex infectious syndrome caused by any one of a large number of antigenitically distinct viruses found in four groups. These groups are the myxo- and paramyxoviruses, the adenoviruses, the rhinoviruses and the coronaviruses. The members of the different groups differ in their physical, biochemical, and immunologic characteristics. With currently available methods, it is possible to determine the cause of 60-70% of colds. The large rhinovirus group is the most important of the known common cold viruses, accounting for approximately 30% of colds. These small RNA viruses have a genome of 7000 nucleotides, which shares considerable homology with poliovirus. The capsid of the rhinovirus is loosely packed, resulting in a relative acid sensitivity compared to the enteroviruses. Although there are at least 89 different antigenic types, all rhinoviruses attach to either one of two cellular receptors. Immunity to rhinovirus is type-specific and associated with neutralizing antibody in nasal secretions and serum. There is a steady acquisition of antibody to the rhinoviruses during childhood and adolescence. The rhinoviruses may be undergoing slow antigenic drift. PMID- 3001919 TI - [From familial forms of the Parsonage-Turner syndrome to tomaculous hereditary neuropathies of the brachial plexus]. PMID- 3001920 TI - [A new case of pseudo-sarcomatous bone metastasis]. PMID- 3001922 TI - [Activation of T lymphocytes in the blood and joints of patients with rheumatoid polyarthritis]. AB - The proportion of T Lymphocytes with receptors for interleukin 2 is increased in the blood, the synovial fluid and, in particular, the synovial membrane in rheumatoid arthritis. However, there is no correlation between the percentage in blood and the percentage in the joints. The helper T lymphocytes are proportionately more activated than the suppressor T lymphocytes. PMID- 3001921 TI - [Cauda equina syndrome with plantar perforating ulcer and neurogenic osteoarthropathies in a case of rheumatismal pelvospondylitis]. AB - The authors report a case of ankylosing spondylitis complicated by a cauda equina syndrome presenting with a plantar perforating ulcer and lesions of neuropathic arthropathy of the foot. On the basis of an analysis of 54 cases published in the literature they review the principal aspects of this complication of ankylosing spondylitis. Neurological manifestations occur late after several decades of progression of the disease. Urinary sphincter problems are common whilst trophic disturbances remain rare. Myelography and, above all, lumbar CAT scan reveal characteristic and stereotyped anatomical lesions: widening of the dural sac in its posterior part with meningoceles eroding the posterior vertebral arch. The pathogenic mechanisms are unknown. The authors suggest that lesions may start with posterior epidural inflammation rather than a primary process of arachnoiditis as has been mentioned in earlier publications. PMID- 3001923 TI - [Efficacy of the Sabin vaccine in malnourished children of the Amazonian region]. PMID- 3001924 TI - A new method for rapid technetium-99m labelling of leucocytes: functional cell studies in vitro. AB - The aim of this study was to assess the cytotoxicity of a new leucocyte-labelling method, which may be used clinically to localize inflammatory and immune reactions. Human blood leucocytes, their mononuclear sub-population, and mouse mononuclear bone marrow cells were labelled with 99mTc for 30-45 min, washed once, and then evaluated in various functional assays. The new procedure includes [99mTc]-labelling with a bisalt method, in the presence of dihydroxybenzoic acid as an intermediate antioxidant-complexing stabilizer, and a carboxylic acid salt of stannous ions as a reducing agent. To challenge the method, cells were labelled about two orders of magnitude more heavily in these initial methodological studies than in on-going clinical trials. Labelled leucocytes ingested latex beads as readily as the controls, but migrated chemotactically and randomly somewhat slower than the control cells. The lymphocytes were triggered by PHA and Con A in a normal way. However, lymphocytes and haemopoietic progenitor cells exposed to radiation for several days, were killed by the isotope doses used, of which about 2% (i.e. 20 MBq) were bound per million cells. All deleterious effects were apparently due to irradiation, and the labelling procedure itself did not damage the cells. PMID- 3001926 TI - Liver disease in alpha 1-antitrypsin deficiency. Aspects of incidence and prognosis. PMID- 3001925 TI - The hydrogen (H2) breath test. Sampling methods and the influence of dietary fibre on fasting level. AB - Three end-expiratory breath hydrogen (H2) sampling methods were compared in a patient group (n = 12) and a laboratory staff group (n = 12) on two separate occasions. H2 samples obtained with each method showed significantly different concentrations (p less than 0.001) but no significant differences in coefficient of variation when individual triplicate samples were evaluated. There was a high correlation between the breath H2 concentrations obtained by the three methods (r = 0.93-0.96). Fasting breath H2 values after an overnight fast and an unrestricted diet the day before the investigation were compared with values obtained after an overnight fast and a low-fibre diet the day before the test in two patient groups (n = 39 and 39) with a comparable distribution of diagnoses and in one group of healthy volunteers (n = 17). Fasting breath H2 concentrations were significantly lower after a low-fibre diet in the patient groups (p less than 0.005) and in healthy volunteers (p less than 0.02). We conclude that each of the three end-expiratory sampling methods can be chosen for use in H2 breath tests depending on suitability and convenience and that a low-fibre diet the day before the H2 breath test lowers fasting breath H2 concentration. PMID- 3001927 TI - Antibodies against HTLV-III in Danish haemophiliacs: relation to source of factor VIII used in treatment and immunological parameters. AB - 18 out of 40 healthy Danish type A haemophiliacs had antibodies against HTLV-III as measured by an enzyme linked immunosorbent assay (ELISA). The overall seropositivity was 45%. A significant positive correlation was found between seropositivity and total lifetime dose of factor VIII and the age of the patients. 63% and 79% of the patients predominantly treated with commercial American and European preparations, respectively, had antibodies, compared with 11% among patients predominantly treated with Danish cryoprecipitate. Patients exclusively treated with preparations from a single source in the year prior to investigation showed 69% seropositivity when treated with American and European preparations. None of the patients treated with Danish cryoprecipitates prepared from voluntary Danish donors had antibodies. No difference between seropositive and seronegative groups was found in total lymphocyte count, leu 2+ cells, leu 3+ cells and leu 2+/leu 3+ ratio but the seropositive group had significantly higher total IgG and lower skin test score. It is concluded that treatment with local European preparation carries less risk of HTLV-III infection compared with commercial preparations from either the USA or Europe. The results also suggested that T-cell subset alterations among haemophiliacs are not primarily due to HTLV III infection. PMID- 3001928 TI - Epstein-Barr virus-transformed B cell lines present M. leprae antigens to T cells. AB - We have investigated whether or not Epstein-Barr virus-transformed lymphoblastoid B cell lines (EBV-BLCL) are able to present Mycobacterium leprae (M. leprae) to antigen-reactive T cell lines and clones. Such EBV-BLCL would provide us with a homogeneous and unlimited source of antigen-presenting cells. Antigen-triggered proliferation of T cells has been studied with co-cultures either with autologous or allogeneic irradiated EBV-BLCL. Our results show that EBV-BLCL are able to present M. leprae as efficiently as peripheral blood mononuclear cells, and that they also function in an HLA-DR-restricted fashion. Apart from their possible in vivo relevance, these results have important practical implications, in particular for the generation and study of M. leprae-reactive T-cell clones. PMID- 3001929 TI - A method for combined quantitative pertechnetate and bone scintigraphy of the sacro-iliac joints. AB - A technique for combined quantitative bone and pertechnetate (perfusion) scintigraphy of the sacro-iliac joints is described. The method is based on localization of the joints in the image obtained after the injection of a small dose of bone-seeking technetiated methyldiphosphonate. This image is later subtracted from the image obtained after the injection of pertechnetate. This combination of methods is believed to constitute a more accurate way of localizing the sacro-iliac joints than the previously used method, based on fluoroscopy. A minor comparison between these two ways of localising the joints is reported too. It is possible that the combination of quantitative bone and pertechnetate scintigraphy will improve the overall sensitivity of the scintigraphic method. PMID- 3001930 TI - [Low early mortality after allogeneic bone marrow transplantation thanks to rigorous sterility measures, modified whole body irradiation, cyclosporin A and antiviral precautions]. AB - A heterogeneous group of 20 consecutive patients was treated according to a standardized allogeneic marrow transplantation protocol designed to reduce early posttransplant lethality. In spite of the fact that 13 patients had at least one (10 of them 2-3) risk factors related to high early lethality, only 2 early transplantation-associated deaths occurred (1 GvH, 1 int. pneumonia). The low incidence of GvH II-IV and interstitial pneumonia, as well as the absence of lethal infections observed in our small patient group, is presumed to be due to the rigorous isolation/decontamination conditions, the reduction of radiation toxicity and GvH prophylaxis by cyclosporin A. PMID- 3001932 TI - AIDS priority fight goes to court. PMID- 3001931 TI - [Rotavirus and pararotavirus infections in adults. Analysis of a nosocomial infection and some sporadic cases]. AB - A nosocomial infection in adults due to rotavirus is described. The identity of the infecting rotavirus strains was demonstrated by genomic dsRNA electrophoresis. At an interval of 19 days another patient in the same hospital developed a pararotavirus infection. Pararotaviruses or rotavirus-like agents are antigenetically distinct from rotaviruses and thus cannot be detected at present by the available antibody mediated antigen detection systems. The current prevalence of pararotavirus infections in adults is unknown in Switzerland. The question whether there is a relationship between the small nosocomial rotavirus epidemic and the sporadic pararotavirus infection remains speculative. We also performed microbiological workups of 394 fecal samples from outpatients with diarrhoea. During the study period from January to April 1984 rotaviruses were the third most frequent aetiological agent, after salmonella and campylobacter, in acute adult gastroenteritis. Selecting the bacteriologically negative fecal samples with a watery and non-bloody aspect, rotaviruses were found in 30%. PMID- 3001933 TI - Reye's data to be turned over to company. PMID- 3001934 TI - Binding of HTLV-III/LAV to T4+ T cells by a complex of the 110K viral protein and the T4 molecule. AB - Human T-lymphotropic virus type III (HTLV-III) or lymphadenopathy-associated virus (LAV) is tropic for human T cells with the helper-inducer phenotype, as defined by reactivity with monoclonal antibodies specific for the T4 molecule. Treatment of T4+ T cells with monoclonal antibodies to T4 antigen blocks HTLV III/LAV binding, syncytia formation, and infectivity. Thus, it has been inferred that the T4 molecule itself is a virus receptor. In the present studies, the surfaces of T4+ T cells were labeled radioactively, and then the cells were exposed to virus. After the cells were lysed, HTLV-III/LAV antibodies were found to precipitate a surface protein with a molecular weight of 58,000 (58K). By blocking and absorption experiments, this 58K protein was identified as the T4 molecule. No cell-surface structures other than the T4 molecule were involved in the antibody-antigen complex formation. Two monoclonal antibodies, each reactive with a separate epitope of the T4 molecule, were tested for their binding capacities in the presence of HTLV-III/LAV. When HTLV-III/LAV was bound to T4+ T cells, the virus blocked the binding of one of the monoclonal antibodies, T4A (OKT4A), but not of the other, T4 (OKT4). When HTLV-III/LAV was internally radiolabeled and bound to T4+ T cells which were then lysed, a viral glycoprotein of 110K (gp110) coprecipitated with the T4 molecule. The binding of gp110 to the T4 molecule may thus be a major factor in HTLV-III/LAV tropism and may prove useful in developing therapeutic or preventive measures for the acquired immune deficiency syndrome. PMID- 3001936 TI - ras-transformed cells: altered levels of phosphatidylinositol-4,5-bisphosphate and catabolites. AB - Steady-state cellular levels of phosphatidylinositol-4,5-bisphosphate (PIP2), 1,2 diacylglycerol (DAG), and inositol phosphates have been measured in two different fibroblast cell lines (NIH 3T3 and NRK cells) before and after transformation with three different ras genes. At high cell density the ratio of DAG to PIP2 was 2.5- to 3-fold higher in the ras-transformed cells than in their untransformed counterparts. The sum of the water-soluble breakdown products of the polyphosphoinositides, inositol-1,4-bisphosphate and inositol-1,4,5 trisphosphate, was also elevated in ras-transformed NRK cells compared with nontransformed NRK cells. These findings suggest that the ras (p21) protein may act by affecting these levels, possibly as a regulatory element in the PIP2 breakdown pathway. PMID- 3001935 TI - Prostacyclin stimulation of the activation of blood coagulation factor X by platelets. AB - When platelets were incubated with prostacyclin, prostaglandin E1, or prostaglandin D2 at concentrations insufficient to increase the level of adenosine 3',5'-monophosphate (cyclic AMP), coagulation factor X was activated by a platelet cysteine protease. Prostacyclin or prostaglandin E1 at higher concentrations increased the cyclic AMP level and inhibited the activation of factor X by platelets. Inhibition of platelet adenylate cyclase by 2',5' dideoxyadenosine allowed the activation of the protease at higher concentrations of the autocoids. Prostaglandins A1, A2, B1, B2, E2, F2 alpha, 6-keto prostaglandin F1 alpha, and thromboxane B2, which do not affect platelet cyclic AMP level, did not stimulate the protease. PMID- 3001937 TI - The slow, insidious natures of the HTLV's. PMID- 3001939 TI - The sympathochromaffin system and the pituitary-adrenocortical response to hypoglycemia. PMID- 3001938 TI - Irreversible block of the mycelial-to-yeast phase transition of Histoplasma capsulatum. AB - p-Chloromercuriphenylsulfonic acid (PCMS), a sulfhydryl inhibitor, prevented the mycelial-to-yeast transition of the dimorphic fungal pathogen, Histoplasma capsulatum. The effect of PCMS was specific for the mycelial-to-yeast transformation; it had no effect on growth of either the yeast or mycelial forms or on the yeast-to-mycelial transition. The failure of PCMS-treated mycelia to transform to yeast was permanent and irreversible. PCMS-treated mycelia could not infect mice but could stimulate resistance to infection by a pathogenic strain of Histoplasma capsulatum. These results suggest a new general strategy for vaccine development in diseases caused by dimorphic pathogens. PMID- 3001940 TI - Pathogenesis of osteoarthritis and use of diclofenac in the treatment of experimental models. PMID- 3001942 TI - Chemotherapy of small-cell lung cancer. AB - Combination chemotherapy has become the cornerstone of management for patients with small-cell lung cancer. Although the majority of patients are palliated, most continue to die of their disease. Studies designed to improve treatment results and to understand the basic biology of this neoplasm are continuing. Several strategies are now being investigated, including the use of non-cross resistant chemotherapy, dose intensification, manipulation of the duration of therapy, combined modality therapy, and salvage chemotherapy. Studies in these areas have not yet yielded results that have definitively improved response rates or survival. It appears that further therapy after remission induction has not yet proven beneficial. This paper will review selected areas of therapeutic investigation. PMID- 3001941 TI - Prostaglandins, thromboxanes, and leukotrienes in inflammation. PMID- 3001943 TI - Liver rejection and its differentiation from other causes of graft dysfunction. AB - Numerous causes can lead to hepatic dysfunction following orthotopic liver transplantation. The most common cause is rejection, which is usually nonpreventable. The clinical presentation, time of onset, and even treatment are variable. Other causes, such as perioperative ischemic injury, vascular thrombosis, and complications of bile duct reconstruction may be preventable with good surgical technique. Infections can also be minimized by careful adjustment of immunotherapy, avoidance overimmunosuppression, and the judicious use of antibiotics. Hepatic dysfunction following orthotopic liver transplantation requires rapid assessment and proper treatment in order to prevent serious and possibly fatal complications. PMID- 3001944 TI - [New viral infections and the acquired immunodeficiency syndrome]. PMID- 3001945 TI - The effects of chemonucleolysis on the mechanical properties of the canine lumbar disc. AB - One hundred and twenty-two lumbar intervertebral discs from 43 mongrel dogs were used to study the effect of chemonucleolysis on the flexion, torsion, and lateral bending flexibilities of the disc. The dogs were killed 2, 4, 12, 26, and 52 weeks following injection with 0.1-0.15 ml of either crude collagenase, semipurified collagenase, or chymopapain. Controls consisted of saline-injected and uninjected discs. The bending and torsional properties of each disc were determined by applying incremental moments up to 0.8 Nm and measuring the resultant rotations 60 seconds after each load increment was applied. The discs were then sectioned for morphologic evaluation. Increases in disc flexibilities ranging from 1.4 to 5.8-fold were found 2 weeks after injection with all three enzymes. The largest increase was noted in flexion in discs injected with chymopapain. By 3 months, all lateral bending flexibilities had returned to control values. In general, however, flexion and torsion flexibilities did not return to control values 6 months following chemonucleolysis. The extent of the gross morphologic changes produced by each of the three enzyme preparations did not correlate with the acute increases in disc flexibilities. Chymopapain and semipurified collagenase had similar morphologic and mechanical effects. The temporary increases in flexibility appeared to be due to decreases in the overall compression, tensile and shear stiffness of the annulus caused by the enzymes. PMID- 3001946 TI - Effects of collagenase on nerve tissue. An experimental study on acute and long term effects in rabbits. AB - Intradiscal injection of proteolytic enzymes implies a potential risk for exposure of nervous tissue to the enzyme solution. In the present study, the effects on peripheral nerve tissue of application of a solution containing a clinically recommended concentration of collagenase have been evaluated. Acute experiments showed that the enzyme induces transient swelling at the site of application and edema in the epineurium. There were, however, no detectable effects on the permeability of the perineurium or the endoneurial microvascular bed. Four and 8 weeks later, there was a slight fibrosis in the epineurium, but neurophysiologic tests did not reveal any impairment of the nerve function. The results are discussed in terms of the clinical use of collagenase for dissolution of herniated intervertebral discs. PMID- 3001947 TI - Differences in postprandial acidity and rates of gastric emptying in duodenal ulcer patients and healthy subjects after the addition of guar gum to meals. PMID- 3001948 TI - Anti-HTLV-III testing--a practical solution for blood transfusion services? AB - Screening blood donations for anti-HTLV-III with a modification to a commercially available test kit has so far been found satisfactory--the reagent cost is less than R1.00 a donation. Should all transfusion services adopt this method, South Africa's total blood donations could be tested at a cost of about R650 000, instead of R2.7 million or more, a year. PMID- 3001949 TI - Gastric carcinoma at Tygerberg Hospital, 1979-1983. A retrospective study. AB - A retrospective study was carried out on patients with histologically proven gastric carcinoma diagnosed at the Gastro-intestinal Clinic, Tygerberg Hospital, over a 5-year period--1979-1983. Fifty per cent of patients were coloured men. The overall median age was 65 years but the coloured patients were significantly younger than the white. The main symptoms were loss of appetite and weight, abdominal pain and vomiting. The median duration of symptoms in all patients was 3 months. An abdominal mass, anaemia and obvious weight loss were the most important physical signs. A normocytic, normochromic anaemia, an elevated erythrocyte sedimentation rate, raised liver enzyme levels and hypo-albuminaemia were the most important laboratory findings. In 96% of the 149 patients gastroscopy yielded a positive diagnosis of gastric carcinoma and barium meal examination showed abnormalities in 87%. In the majority of cases the carcinoma was poorly differentiated. PMID- 3001950 TI - Crystalline arrays and hepatitis viruses. PMID- 3001951 TI - Resectable pulmonary nephroblastoma metastases presenting as chest wall lesions on radiography. A report of 2 cases. AB - Two children with pulmonary metastases from nephroblastomas showed the radiological features of chest wall tumour involvement. Diagnostic surgical exploration subsequently revealed pedunculated and fully resectable peripheral lung metastases. The mechanism by which these pedunculated lesions mimicked the classic 'D sign' of an extrapulmonary (pleural or chest wall) tumour is explained. PMID- 3001952 TI - Risk of recurrence after treatment of first-episode genital herpes with intravenous acyclovir. AB - To determine whether intravenous acyclovir treatment for a first episode of genital herpes could prevent or reduce subsequent recurrences, we combined and analyzed the results of two independently conducted, randomized, double-blind, placebo-controlled studies. Sixty-one patients were enrolled in the two trials; 30 received the drug, and 31 received placebo. At entry the demographic, epidemiologic, and clinical features of acyclovir- and placebo-treated patients from the two centers showed no significant differences. The median time to the first recurrence and the frequency of recurrences showed no significant differences when acyclovir and placebo recipients infected with either herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2) were compared. However, irrespective of treatment, the median time to the first recurrence was significantly longer (293 days vs. 69 days; P less than .02) and the frequency of recurrence significantly less (0.11 recurrences per month vs. 0.43 recurrences per month; P less than .01) among patients with HSV-1 infection as compared with those who had HSV-2. It is concluded that in patients with first attack genital herpes, the type of HSV is the most important determinant of subsequent recurrences and that intravenous acyclovir has little effect on subsequent recurrences. PMID- 3001953 TI - Association of cytomegalovirus and herpes simplex virus infections of the cervix in four clinic populations. AB - The rates of isolation of cervical cytomegalovirus (CMV) and herpes simplex virus (HSV) were compared for populations of four different clinics attended by a total of 1,755 women. The prevalence of CMV infection could be predicted by the prevalence of HSV infection, with CMV being 2.5 times as prevalent as HSV in each population. The overall infection rates for CMV and HSV were 4.1% and 1.7%, respectively. The 252 women attending the Sexually Transmitted Disease Clinic had significantly higher rates of CMV and HSV infection (12.5% and 5.6%, respectively) than populations attending the other clinics. A strong relationship between marital status and CMV infection was observed. The estimated relative risk for single compared to married women was 2.9. These data verify the importance of the sexual route of transmission in the epidemiology of cervical infection with CMV. PMID- 3001955 TI - Endocrine tumors of the pancreas and their clinical syndromes. PMID- 3001954 TI - [Silica urolithiasis. Review of the literature and presentation of a new case]. PMID- 3001956 TI - The acquired immune deficiency syndrome. AB - Although the etiologic agent of AIDS has been identified, clinicians are still left with treating the complications of this immune deficiency, that is, the unusual neoplasms such as KS and the myriad of opportunistic infections. Efforts at immune reconstitution, including bone marrow transplantation from a normal identical twin to his brother with AIDS, have been unsuccessful. The transplanted lymphocytes are as likely to become infected as the patient's own. Therefore, future efforts must be directed at defining the natural history and infectivity of individuals showing evidence of HTLV III or LAV infection and toward specific antiviral therapy for those with infection and a vaccine for those at high risk for HTLV III infection and, consequently, AIDS. PMID- 3001957 TI - A new surgical approach for the management of carcinoma of the junction of the main hepatic ducts. AB - In the present study we discuss our early results and some technical aspects of a new surgical approach in dealing with high-lying malignant biliary obstruction. After transection of the common bile duct we proceed through a cephalad reflection of its proximal end in a meticulous dissection of the intrahepatic biliary tree. During dissection we follow the intrahepatic course of the right and left portal vein, which leads us to follow the respective course of the main right and left hepatic ducts and to reach the levels of their subsequent consecutive segmental bifurcations. By transecting at high levels, we excise completely or partially the tumor with the confluence of common hepatic duct. We reconstruct the biliary tree by multiple intrahepatic cholangiojejunostomies by use of a Roux-Y jejunal loop and a varied number of segmental hepatic ducts. Transanastomotic tubes were used only temporarily. Fifteen patients underwent operations by the described surgical approach without mortality. All were relieved of their jaundice completely and in all the tumor was either completely (5) or partially (10) removed. All patients had a good quality of postoperative life without being obliged to carry transanastomotic tubes and the complications and problems associated with them. PMID- 3001958 TI - Malignant fibrous histiocytoma arising from the meninges of the posterior fossa. AB - A case of malignant fibrous histiocytoma presenting as a cerebellar mass in a young man is described. The tumor was found to be arising from the dura mater and infiltrating the cerebellum. The ultrastructural and immunocytochemical studies confirmed the mesenchymal origin of the neoplasm. Focal reactive astrocytomalike areas were observed in the midst of this tumor. PMID- 3001959 TI - Increased vascular contraction and sensitivity to norepinephrine after endothelial denudation is inhibited by prazosin. AB - The vasomotor function of rabbit aorta was examined after deendothelialization with a Fogarty balloon catheter and during the subsequent development of intimal hyperplasia. Helical strips of injured lower abdominal aortic tissue showed an increase in norepinephrine-induced contraction when compared with control strips from normal upper abdominal aorta. This increase was 235% +/- 40% of control immediately after injury and 341% +/- 51% at 28 days after injury. Standardized dose-response curves demonstrated that the injured tissue was increasingly sensitive over time to norepinephrine and that this was more marked at physiologic levels of norepinephrine. Contraction was blocked by prazosin hydrochloride but not by yohimbine or propranolol. Furthermore, a single intramuscular dose of prazosin hydrochloride 2 hours before injury significantly (p = 0.001) reduced the maximal contraction and sensitivity. These results imply an increase in vasomotor reactivity after deendothelialization mediated through the alpha 1-adrenergic receptor. These functional changes are related to the pertinent morphologic observations. PMID- 3001961 TI - [Drugs active against herpesvirus]. PMID- 3001960 TI - Cowden's disease: a further indication for prophylactic mastectomy. AB - Cowden's disease (multiple hamartoma syndrome) is a syndrome involving abnormalities of multiple organ systems. Transmitted in an autosomal dominant pattern, it carries a high frequency of mammary carcinoma in early middle age in affected women. The hyperkeratotic cutaneous and gingival markers of the disease are its principal overt manifestations. Prophylactic bilateral total mastectomy with optional immediate reconstruction is recommended for women Cowden's disease. An illustrative family with the disease is presented in which one affected young woman was found to have invasive mammary carcinoma with regional metastasis at the time of prophylactic mastectomy. PMID- 3001962 TI - [Effect of balneological methods on the functional state and endocrine regulation of the stomach and duodenum in patients with duodenal ulcer]. AB - A study of 102 patients with duodenal ulcers showed a salubrious clinical effect of mud therapy on a course of the disease, alkalizing duodenal function, indices of thyroid hormones, gastrin and blood cyclic nucleotides in 62.7% of cases. PMID- 3001963 TI - [Viral myocarditis (the etiologic, clinical, diagnostic and treatment problems)]. AB - To elucidate the etiology of respiratory infection and pharyngitis associated myocarditis a serological study was made of 201 patients who were successively admitted with a clinical diagnosis of myocarditis. Coxsackie viral infection of group B, influenza A and B, para-influenza and adenoviral infection and beta hemolytical streptococcus of group A were determined. Preceding Coxsackie infection was established in 38,3% of the patients, influenza A and B in 27.5%, adenoviral infection in 3.6% and para-influenza in 1.7%. beta-hemolytical streptococcus as the cause of myocarditis was detected in 4.9% of the patients only. In view of the viral etiology of most cases of myocarditis the authors discussed the problems of its pathogenesis, clinical course and therapy. PMID- 3001964 TI - Studies of alpha 2-adrenergic receptors of intact and functional washed human platelets by binding of 3H-dihydroergocryptine and 3H-yohimbine--correlation of 3H-yohimbine binding with the potentiation by adrenaline of ADP-induced aggregation. AB - The binding of 3H-dihydroergocryptine and 3H-yohimbine to intact, discoid, functional, washed human platelets resuspended in Tyrode's buffer containing Ca2+, Mg2+, human albumin and apyrase, was studied at 37 degrees C. The binding of 3H-dihydroergocryptine was rapid, reversible and saturable (KD = 19.3 +/- 4.2 nM, Bmax = 2590 +/- 670 sites per platelet). The results were difficult to interpret because the bound ligand was not easily dissociated. In contrast, 3H yohimbine bound in a rapid, reversible and saturable fashion to one class of sites (KD = 8.1 +/- 1 nM, Bmax = 395 +/- 35 sites/platelet) with the characteristics of alpha 2-adrenergic receptors. Adrenaline alone did not aggregate intact platelets but potentiated ADP-induced aggregation. This effect of adrenaline was specifically inhibited by alpha 2-antagonists such as yohimbine. The inhibition of 3H-yohimbine binding and the inhibition of the synergistic effect of adrenaline on ADP-induced aggregation by 16 different alpha and beta-adrenergic compounds was significantly correlated (p less than 0.001). Thus, intact and functional washed human platelets can be used as a simple pharmacological model to screen alpha-adrenergic antagonists by measuring the inhibition of the potentiation of ADP-induced aggregation by adrenaline which is a direct reflection of the physiological effect of adrenaline on human platelet alpha 2-adrenergic receptors. The inhibition constant derived from aggregation studies expresses the affinity of the ligand for its receptor as measured by more cumbersome binding studies with radioactive adrenergic antagonists such as 3H yohimbine. PMID- 3001965 TI - Carrier detection of haemophilia B by using an intragenic restriction-fragment length polymorphism. AB - We analysed DNA from individuals of five families with haemophilia B, including nineteen potential carriers. A gene-specific probe was used to reveal a TaqI restriction-fragment length polymorphism. Segregation analysis of the polymorphic marker and the deleterious mutation within families allowed diagnosis at the gene level for 16 out of the 19 potential carriers, two proving to be carriers and 14 non-carriers. The obvious advantage is that lyonisation, which is a limiting factor when gene product (clotting factor IX) measurements are used for carrier detection, does not interfere with this procedure and that the result is a definitive diagnosis instead of a risk estimate. The method also permits prenatal diagnosis on chorionic villi in the first trimester of pregnancy. Restriction fragment length analysis, based upon the probe and restriction enzyme used in this study, will be informative for approximately 45% of the individuals at risk of carrying or transmitting the haemophilia B mutation. PMID- 3001966 TI - Anti-LAV and concentrate consumption in Italian hemophiliacs. PMID- 3001967 TI - Effects of arachidonic acid on the metabolism of eicosapentaenoic acid in washed human platelets. AB - We examined effects of arachidonic acid (AA) on eicosapentaenoic acid (EPA) metabolism in washed human platelets. Although human platelets had been considered to metabolize scarcely EPA, a simultaneous addition of EPA and AA to washed platelet suspensions stimulated markedly EPA metabolism. In addition, the stimulatory effect was more potent over the formation of thromboxane (TX) B3 than that of 12-hydroxy-5,8,10,14,17-eicosapentaenoic acid (HEPE). The stimulation by AA can be due to AA itself and/or AA metabolites. Indomethacin decreased the stimulatory effect of AA on the HEPE formation, suggesting that cyclooxygenase product(s) of AA stimulated the HEPE formation. Among the metabolites of AA investigated, prostaglandin (PG)G2 and 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid had the stimulatory effect on both TXB2 and HEPE formations, whereas PGH2, PGD2, TXB2 and 12-hydroxy-5, 8,10,14-eicosatetraenoic acid were ineffective. PMID- 3001969 TI - Thin-layer chromatography of polyphosphoinositides from platelet extracts: interference by an unknown phospholipid. AB - Different ratios of radioactive polyphosphoinositides in platelets pulse-labelled with 32p-orthophosphate have been reported by various laboratories. We studied whether these differences originate from differences in methodology. Extracts of 32p-Pi labelled human platelets were prepared at various times after gel filtration and phosphatidylinositol (PI)-, mono (PIP)- and bisphosphate (PIP2) were separated by thin-layer chromatography using four different solvent systems. The 32p-levels in PIP and PIP2 remained constant during one hour after gel filtration, whereas 32p-PI increased continuously and more than doubled within the first h. In two of the systems PIP co-chromatographed with a radioactive compound which separated well from PIP in the two other systems. This unknown compound was also labelled with 3H-glycerol, 3H-inositol and 3H-arachidonic acid, but it was metabolically and functionally different from the polyphosphoinositides. Both the co-chromatography of this unknown phospholipid and the increase in 32p-PI in gel-filtered platelets can explain the difference in 32p-labelling in phosphoinositides reported in the literature. PMID- 3001968 TI - Heparin does not interfere with prostacyclin and prostaglandin D2 binding to platelets. AB - Heparin has been reported to antagonize the platelet antiaggregating activity of prostacyclin (PGI2) and prostaglandin D2 (PGD2). To investigate whether heparin interferes with PGI2 and/or PGD2 binding to the specific membrane platelet receptors, the number and the affinity of binding sites for tritiated PGI2 and PGD2 were determined in 8 healthy subjects (aged 25-32; 4 for PGI2 and 4 for PGD2 studies) in the absence and in the presence of heparin. Preliminary aggregation tests indicated that the heparin concentration tested (2 I.U./ml) reduced the antiaggregating activity of PGI2 and PGD2. Heparin, when preincubated with platelet suspension, did not induce any significant change in the number/platelet (bs/plt) and affinity (Kd) of PGI2 and PGD2 binding sites (bs) in comparison to control values (PGI2 high affinity bs: bs/plt = 106 +/- 12 vs 107 +/- 17 - Kd = 9.7 +/- 3.1 vs 10.0 +/- 2.3 nM; PGI2 low affinity bs: bs/plt = 3551 +/- 233 vs 3670 +/- 465 - Kd = 877 +/- 125 +/- 125 vs 833 +/- 104 nM; PGD2 bs: bs/plt = 228 +/- 14 vs 234 +/- 24 - Kd = 68 +/- 10 vs 73 +/- 7 nM; p greater than 0.4 for all the differences). No significant binding modification was also observed when heparin was preincubated with 3H-PGI2 and 3H-PGD2. The present demonstration that heparin does not interfere with PGI2 and PGD2 binding to platelets is consistent with the hypothesis that heparin reduces the antiaggregating effects of PGI2 and PGD2 by directly potentiating platelet aggregation. PMID- 3001970 TI - [Feline infectious peritonitis]. AB - This paper gives a summary of our present-day knowledge concerning etiology, clinical aspects, diagnosis, pathology and pathogenesis of feline infectious peritonitis. Special emphasis is given to the participation of the immune system in the development of lesions. A therapy protocol is proposed and an extensive list of original literature for further study is given. PMID- 3001971 TI - Purification and characterization of the specific binding protein for platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) from human platelets. AB - The binding protein to platelet activating factor (PAF) was solubilized with 2% Triton X-100 from human platelet membranes and purified approximately 23-fold by sequential chromatography on DEAE-cellulose, CM-cellulose and Sephadex G-200. The final preparation migrated as a single protein band corresponding to a radioactive peak of [3H]PAF bound on polyacrylamide disc gel electrophoresis. The molecular weight of the native form of the binding protein was estimated to be approximately 160,000 daltons by Sephadex G-200 column chromatography, and its isoelectric point was near 8.0. Exposure of the platelet membranes to an excess of unlabeled PAF diminished the binding of [3H]PAF. PMID- 3001972 TI - Histological evidence for rescue of radiation dermatitis in an ultraviolet model system. AB - Rescue of acute radiation dermatitis has already been shown feasible with cytochrome c(Sci. Rep. Res. Inst. Tohoku Univ.-C 29: 1, 1982). Interest in the probable reduction of inflammatory sequelae led to histological studies in an ultraviolet model system in the rabbit. The relative dermal thickness in the field of ultraviolet irradiation was 1.70 +/- 0.30 (5) in the untreated control and 1.31 +/- 0.07 (5) in the cytochrome c group respectively (p less than 0.05). Usefulness of the principle cannot be overemphasized in the strategies of radiotherapy, and in the treatment and/or prevention of actinic radiation dermatitis. PMID- 3001973 TI - Morphological and biochemical studies on minocycline-induced black thyroid in rats. AB - The thyroid glands of the rats treated with 100 mg/kg/day of minocycline for 35 days showed black discoloration. In these thyroids, brown pigment granules were seen in the follicle epithelial cells, and they histochemically stained in the same manner as melanin. By electron microscopy, the deposition of the electron dense material first occurred in lysosome-like granules and, with further administration, was observed also in the rough endoplasmic reticulum in some cells. Thin-layer chromatography showed that the Rf value of the extract from minocycline-treated rat thyroid was the same as that of the black substance obtained by mixing minocycline and 3% hydrogen peroxide but different from that of melanin. In minocycline-treated rats, the release of thyroxine (T4) from perifused thyroids was significantly less than that from control, and the analysis of iodoamino acids showed an increased monoiodotyrosine fraction. Thus, the pigment of thyroid in minocycline-treated rats was demonstrated to be a metabolic derivative of minocycline itself and minocycline may interfere with thyroid function in high-dose, long-term treatment. PMID- 3001974 TI - Placental transfer and developmental toxicity of 2,2',4,4',5,5'-hexabromobiphenyl in B6C3F1 mice. AB - The 2,2',4,4',5,5' congener of hexabromobiphenyl (HBB) is the major constituent (55%) of a polybrominated biphenyl mixture that accidentally entered the human food chain through livestock feed. The embryotoxicity of HBB in B6C3F1 conceptuses was examined by adding 0, 100, 300, 500, 750, or 1000 ppm of HBB to the diet of pregnant C57BL/6J mice impregnated by C3H males. The females consumed HBB feed from Gestation Day (gd) 6 through 15. Pregnancy rates of plug-positive mice were adversely affected at greater than 300 ppm HBB (0 ppm = 77%, 100 = 73%, 300 = 55%, 500 = 49%, 750 = 42%, 1000 = 33%). Some pregnant dams died at dosages of 750 ppm (2 of 8). Body weight gain among the 300-ppm dams was reduced only on gd 15, indicating maternal toxicity, but it was indistinguishable from controls on gd 17. In the conceptuses HBB caused cleft palate and a cystic lesion in the region of the cerebellum at concentrations greater than or equal to 300 ppm. The nature of the cystic abnormality observed grossly could not be accurately defined, but it was probably in the meninges and was not associated with the brain ventricular system. In some HBB-exposed fetuses there were minor abnormalities of brain development, which were probably indicative of developmental delay rather than direct toxic effects on differentiation of specific regions of the brain. Thus, HBB affected embryonal development adversely only at exposure concentrations which caused toxicity in dams. PMID- 3001975 TI - [Serological and cytological diagnosis of herpetic stomatitis]. PMID- 3001976 TI - Proton therapy at Harvard. AB - Fractionated precision high-dose proton radiotherapy has been carried out at the Harvard Cyclotron Laboratory (HCL) since 1973, in a collaborative effort with the Radiation Medicine Department of Massachusetts General Hospital (MGH) and the Retina Service of the Massachusetts Eye and Ear Infirmary (MEEI). This paper will discuss proton treatment in general, treatment planning procedures, and results to date in major patient categories. 846 patients have been treated with fractionated proton therapy at the Harvard Cyclotron, with normal tissue and tumor responses consistent with an RBE of 1.1 for the proton beam. Proton beam therapy is the treatment of choice for patients with uveal melanomas, and chordomas and chondrosarcomas involving the skull base and cervical spine. Improved dose distribution possible with protons have allowed greater doses than are given conventionally to be delivered to patients with prostatic carcinoma, head and neck malignancies, ano-rectal cancers, and retroperitoneal tumors. Doses employed have been usually 10 to 20% greater than normally would be delivered in our department to such tumors. Generally, local control rates have been good. PMID- 3001977 TI - Proton radiotherapy with the Uppsala cyclotron. Experience and plans. AB - From 1957 to 1968, the 230-cm synchrocyclotron at the Gustaf Werner Institute was used for clinical tests with a 185 MeV proton beam. The radiotherapeutic research was part of an extensive research programme in physics, chemistry, biology and medicine. Only a small series of patients were treated. A brief review of the early development and clinical experience of the cyclotron activities at Uppsala from 1957 to 1968 is given. The former accelerator is now being converted. Beams are expected to be available in the new radiotherapy treatment rooms in 1986. Plans for the new facilities with special reference to alternative methods of proton acceleration and beam transport, i.e. fixed beams or an gantry system are presented. The corresponding activities at the Institute of Theoretical and Experimental Physics (ITEP) in Moscow are also referred to thanks to a bilateral research programme which has existed in the past and from which the Uppsala group has benefited greatly. PMID- 3001978 TI - Thyroidal regulation of rat thymic and splenic (Na+ + K+)-adenosine triphosphatase. AB - The present studies concern the effects of triiodothyronine (T3) on rat thymic and splenic NaK-ATPase activities. When hypothyroid rats were given a single injection of T3 (250 micrograms/100 g body weight), thymic NaK-ATPase activity increased by 18% at 24 h (p less than 0.005), and remained at this peak at 72 h. Injection of T3 (250 micrograms/100 g body weight, on alternate days X 3) significantly augmented NaK-ATPase activity from the thymus and spleen of hypothyroid rats by 19 and 49%, respectively. Two weeks of daily injections of T3 (1 microgram/100 g body weight) to hypothyroid rats significantly increased NaK ATPase activity in the thymus and the spleen by 14 and 28%, respectively. Reverse T3 had no significant effect of thymic and splenic NaK-ATPase activities. Mg ATPase and 5'-nucleotidase activities were not significantly affected by T3 in either tissue under different thyroid states. The lack of response of thymic and splenic NaK-ATPase to reverse T3, and Mg-ATPase and 5'-nucleotidase to T3, substantiates the specificity of the hormone action on NaK-ATPase. To investigate whether the T3-dependent increase in NaK-ATPase activity is a result of an increase in the number of enzyme units, the effect of T3 on the quantity of phosphorylated intermediate of NaK-ATPase was assessed in the partially purified membrane fraction. In the euthyroid state, NaK-ATPase activity and phosphorylated intermediate units were 33 and 43% higher, respectively than in the hypothyroid state, implying that the T3-dependent increase in NaK-ATPase activity is a result of an increase in the number of NaK-ATPase units. PMID- 3001979 TI - Absence of porcine parvovirus transmission to man. PMID- 3001980 TI - Evaluation of the use of high-dose cytoreduction with autologous marrow rescue in various malignancies. PMID- 3001981 TI - Anti-Rho(D) in two Rh-positive patients receiving kidney grafts from an Rh immunized donor. AB - Kidneys from an Rh-negative cadaver donor were transplanted to two Rh-positive patients. The donor's serum contained anti-Rho (D). In both patients, anti-Rho (D) was detected in their serum and on their red cells 3.5 weeks after transplantation. One patient had hemolysis. The antibodies persisted for nearly six months, despite graft rejection and nephrectomy in one case. These antibodies presumably arose from passenger B lymphocytes in the grafts from the Rh-immunized donor. PMID- 3001982 TI - Ultrastructural localization of 5'nucleotidase in human normal and malignant lymphoid cells. AB - 5'Nucleotidase (EC 3.1.3.5), a purine pathway enzyme, occurs as a cellular ectoenzyme. Widely differing 5'N activity has been reported in different types of lymphoid cells and appears to be related to lymphocyte function, differentiation and immunological subtype. This paper reports a study on the ultrastructural distribution of 5'N in in different types of lymphoid leukemias and non-Hodgkin's lymphomas. In lymphoid leukemias, only cALL cells showed strong 5'N staining. In CLL, PLL and HCL the intensity of staining was less than in normal cells. In the NHL group, the reaction pattern was mixed. Infiltrating neoplastic cells, specially the large lymphoid cells, consistently showed very mild activity of the enzyme, whereas follicular center cells and other mature lymphocytes were characterized by moderately strong to strong 5'N activity. These results support the view that 5'N is a marker of lymphocyte cell differentiation. PMID- 3001983 TI - Osteoclastoma-like giant-cell tumor of the liver. Case report. PMID- 3001984 TI - Crystals and alpha-1-antitrypsin-reactive globoid inclusions in an islet cell tumor of the pancreas. AB - An islet cell tumor of the pancreas with unusual light microscopic, ultrastructural, and immunocytochemical features is reported. In addition to secretory granules and positive immunostaining for pancreatic polypeptide, the tumor contained globoid intracytoplasmic inclusions by light and electron microscopy, which correlated with a positive immunoreaction for alpha-1 antitrypsin, and Reinke-like crystals. PMID- 3001985 TI - Paragangliomas of the head and neck: ultrastructural and immunohistochemical analysis. AB - Eighteen head and neck paragangliomas were studied by light microscopy and light microscopic immunohistochemistry by the peroxidase technique for the presence of NSE (neuron-specific enolase), serotonin, and a battery of neuropeptides. Seven of these tumors were also studied by electron microscopy. All 18 cases demonstrated immunostaining for NSE; 10 of the 11 carotid body tumors had immunostaining for multiple hormones. Considering all 18 cases, the most frequently demonstrated hormonal substances were in order: serotonin, leu enkephalin, gastrin, substance P, vasoactive intestinal polypeptide (VIP), somatostatin, bombesin, calcitonin, and alpha MSH. In several tumors, adjacent step sections stained for different hormonal substances strongly suggested reactivity for more than one hormone in given tumor cells. By electron microscopy, all 7 cases studied displayed considerable heterogeneity of the neurosecretory granules with respect to size, shape, and electron density. This demonstrated that branchiomeric paragangliomas are capable of producing a spectrum of neuropeptides in addition to their known amine content. The presence of immunoreactive serotonin in most of these neoplasms was confirmed. In addition to these findings, neurofibrils within the substance of carotid body paragangliomas demonstrated immunoreactivity for somatostatin and a gastrinlike neuropeptide. The significance of the neuropeptides in these neoplasms and their possible presence and role in normal and hyperplastic paraganglia remain to be defined. PMID- 3001986 TI - Spindle cell tumor of the ovary. PMID- 3001988 TI - [Human papilloma virus (HPV) infection of the uterine cervix. I. Historical development, nomenclature and diagnosis]. PMID- 3001987 TI - Cytoplasmic structures in endothelial cells of the choroid. PMID- 3001989 TI - [Human papilloma virus (HPV) infection of the uterine cervix. II. A prospective colposcopic and cytological examination]. PMID- 3001990 TI - [Human papilloma virus (HPV) infection of the uterine cervix. III. A prospective study of women referred because of abnormal smears]. PMID- 3001991 TI - [Human papilloma virus. Relation to cervical neoplasia]. PMID- 3001992 TI - Increasing demands on today's blood donors. AB - Recently in Northern Ireland there has been a rapid increase in demand for a variety of blood components. To meet this need a large proportion of routine blood donations must be processed at the Transfusion Centre. In addition, several blood components are collected direct from donors by apheresis techniques. Apheresis is currently restricted to the collection of components from highly selected donors, but in future this method is likely to be employed for collection of some routine components. This changing pattern is placing increasing demands on many of our blood donors. PMID- 3001993 TI - Lipid emulsions as contrast agents for hepatic sonography: an experimental study in rabbits. AB - An ultrasound contrast agent capable of increasing hepatic echogenicity would be useful for the detection of hepatic tumors and metastases. Fatty liver is known to produce increased liver echogenicity. Intravenously administered lipid emulsions are phagocytosed by cells of the reticuloendothelial system the liver with transient hepatic lipid accumulation. We examined the effectiveness of three lipid emulsions of differing particle size as potential ultrasound contrast agents using a rabbit liver model. None of the tested emulsions showed any consistent ability to alter liver echogenicity at maximum tolerable doses. Lipid emulsions do not appear to have potential as contrast agents for ultrasound examination of the liver. PMID- 3001994 TI - Treatment of malakoplakia of bladder with intravesical neosporin irrigation. PMID- 3001995 TI - Hormonal agents and treatment of cancer. AB - Hormonal control of tumor growth has shifted from what was basically an empirical discipline twenty-five years ago to what is now primarily a pharmacologic and biochemical science. Early on, medical management of malignancy relied heavily on the knowledge that deprivation of steroids produced by the sex glands could result in the regression of tumors in breast cancer and prostatic cancer. Today's approach to management is focused on the way in which different steroids feed the cancer cell itself. As a result, many new hormonal agents have been developed on the basis of the knowledge that it is possible to interfere with the process of cancer growth directly at the cellular level. The biochemical rationale for the development of many of these agents is discussed. PMID- 3001996 TI - Pathogenesis of canine parvovirus enteritis: sequential virus distribution and passive immunization studies. AB - After oral inoculation, the sequential distribution of canine parvovirus was studied in 14 nine-week-old seronegative beagle dogs. Two or three dogs were necropsied on days 1 through 6 after inoculation. Tissues were collected for virus isolation, immunofluorescence testing, and light microscopy. Virus was isolated from, and fluorescent cells were seen in the tonsil, retropharyngeal and mesenteric lymph nodes one and two days after inoculation. Virus infection of systemic and intestinal lymphoid tissues occurred as early as three days after inoculation and was associated with viremia. Intestinal epithelial infection was first seen four days after oral inoculation. All dogs were viremic before intestinal epithelial infection was found. Fecal virus excretion first occurred four days after oral virus inoculation. Intestinal virus infection and lesions became progressively more severe between four and six days after inoculation. The severity of intestinal lesions was variable and related to the severity of systemic lymphoid tissue lesions and the magnitude and duration of viremia. Four littermates of virus-infected dogs were passively immunized against canine parvovirus with convalescent canine serum 24 hours after oral virus inoculation. Neither clinical signs, lymphopenia, nor fecal virus excretion occurred in passively immunized dogs. Intestinal epithelial infection was not demonstrable by immunofluorescence testing when passively immunized dogs were necropsied four, five, and six days after virus inoculation. PMID- 3001997 TI - Reversal of xylazine in red deer. PMID- 3001998 TI - Parvovirus vaccination. PMID- 3001999 TI - Porcine parvovirus: a serological survey in the United Kingdom January 1984 to January 1985. PMID- 3002000 TI - Outbreaks of nephritis in pheasants (phasianus colchicus) with a possible coronavirus aetiology. PMID- 3002001 TI - Immune response of sheep to bluetongue virus: in vitro induced lymphocyte blastogenesis. AB - Sheep were inoculated with 2 ml of 10(7) plaque forming units per ml of purified prototypes of the four United States serotypes (10, 11, 13 and 17) of bluetongue virus. Nine weeks following the initial inoculation, a challenge inoculation with homologous virus was done. Animals were followed for virus isolation and evidence of cell-mediated immunity by weekly lymphocyte stimulation tests (LST). Two dilutions (10 micrograms/ml and 1 microgram/ml) of pure virus from each of the purified serotypes were used as antigen as were the phytomitogens phytohemagglutinin, Concanavalin A, and pokeweed mitogen. LST data were analyzed by the analysis of variance method and reported as counts per minute and stimulation index (SI). Significant SI were observed following primary and secondary challenge with both homologous and heterologous virus. There was evidence of lymphocyte perturbations characterized by a sharp decrease in response to mitogens following primary and secondary challenge lasting for one week followed by a significant increase in blastogenesis three to four weeks after inoculation of virus. These results provide evidence that cell-mediated immunity is evident in bluetongue infection, that there is cross reactivity between viral serotypes and that BTV infection leads to perturbations in lymphocyte function including suppression of responses. An increase in the blastogenic response to phytomitogens correlated with viral clearance. PMID- 3002003 TI - [Electron microscopic research on an experimental infection in chicks caused by the infectious bursal virus]. AB - A total of 18 birds at the age of 25 days were infected intraocularly with the pathogenic strain 52/70 of the infectious bursitis virus. The bursa of Fabricius and the thymus were sampled for electron-microscopic investigations. The ultrastructural changes in the infected cells consisted in pycnosis of the lymphocyte and macrophage nuclei, vacuolization of the cytoplasm, and the production of lipid droplets as well as of inclusion bodies of varying density, myelin figures, multivericular bodies, and other irregular structures in the cytoplasm. These changes were slightly visible at the 24th hour, were characteristic at the 48th hour, and were well manifested at the 72nd hour following infection. Beside the degenerative changes in the cytoplasm of the affected cells (mainly lymphocytes) there were at the 24th hour of infection tiny granular matter of fibre-like structure and particles within it of varying electron density. In the later phases (at the 48 h and the 72nd hour) viral particles that were either free or in the vacuoles were predominantly seen in the cytoplasm of the macrophages, having a dia of 50-60 nm and a hexagonal form, with crystalographic arrangement. It was also demonstrated that the replication of the virus of the infectious bursal infection took place in lymphocytes, and in the later stages of infection--chiefly in macrophages. These were the two main types of cells in which virus replication was seen to take place. PMID- 3002002 TI - Immunosuppression in experimental trypanosomiasis: effects of Trypanosoma equiperdum on the pathogenesis of influenza virus infection in deer mice (Peromyscus maniculatus). AB - The effects of Trypanosoma equiperdum infections on the immunological and pathological responses of deer mice (Peromyscus maniculatus) to influenza virus exposure were investigated. Mice carrying a 5 week trypanosome infection along with an age- and sex-matched trypanosome-free control group were simultaneously exposed to influenza Ao (WSN) virus. T. equiperdum infection significantly (P less than 0.01) converted a sub-lethal virus attack into a fatal pneumonic process in a small proportion of animals. In addition, the trypanosome caused a reduction (p less than 0.1) in virus replication on PID 1 and 2, accompanied later by a tendency towards virus persistence in the lungs of affected mice. This tendency was manifested by a log reduction in virus titres between PID 2 and 4 and PID 4 and 6 in the lungs of trypanosome-infected mice, compared to 2 log drops over the same periods in the lungs of control mice. T. equiperdum infection also significantly (p less than 0.001) depressed serum and pulmonary neutralizing antibody titres to influenza virus. PMID- 3002004 TI - [Demonstration of a transmissible gastroenteritis virus by contrast immunofluorescence]. AB - Tested were a number of procedures to employ the immunofluorescence method for diagnosing transmissive gastroenteritis in pigs--impression preparations, frozen cross-sectioned material, and infected cell cultures of the established cell line SPEV. It was found that immunofluorescence microscopy was a dependable method for the express diagnosis of transmissive gastroenteritis. It could be employed in its three variants. The use of impression preparations, however, did not prove as dependable as the remaining ways of applying the method, and this drawback had to be compensated for with the study of a greater number of impression preparations taken from more pigs that had contracted the disease. It was also established that most promising and effective was to apply the method with the use of cell cultures infected with suspensions of organs. Cell cultures of the established SPEV cell line infected with material that contained the virus could produce dependable positive results in immunofluorescence investigations at the 24th hour following inoculation. This method could be employed for the express diagnosis of transmissive gastroenteritis. PMID- 3002005 TI - [Creation of an avirulent immunogenic mutant of the transmissible gastroenteritis virus and its use as a vaccine]. AB - The serial cultivation of a virulent strain of the virus of transmissive gastroenteritis in the presence of the antimetabolite 5-bromurazil has led after a definite number of passages to the production of a virus mutant of new biologic properties. It was established that the mutant is resistant to the antimetabolite, and it retains its infectious titer. It is avirulent to newborn and growing pigs, and confers solid immunity in the vaccinated animals which do not become virus carriers. It is stable with regard to its newly-obtained biologic properties. PMID- 3002006 TI - [Demonstration of coronavirus infection in buffaloes]. AB - The hemagglutination-inhibition test and a bovine corona virus antigen were used to investigate a total of 293 samples of buffalo sera, of which 152 were taken from buffalo calves at the age of up to 12 months and 141 were taken from buffalo cows at the age of between 18 and 30 months. Corona virus antibodies were proven in 52.6 per cent of the sera of buffaloes originating from 3 cooperative farms and a number of private farms. The antibodies in the sera of buffalo calves had an average geometric titer of 182, and those in the sera of buffalo cows--74. The parallel study of 40 of the sera revealed a correlation between the hemagglutination inhibiting and the neutralizing antibodies. Twenty-nine fecal samples from buffalo calves with diarrhea, aged 5 to 30 days, were also studied via hemagglutination-inhibition on the same public farms. Corona virus of high titers was demonstrated in 7 of the samples, the hemagglutination activity of which had been suppressed by a bovine corona virus antiserum. It was concluded that a hemagglutinating coronavirus was circulating among the buffaloes. It seemed to be analogous to the bovine coronavirus and to participate in the etiology of part of the enteritis cases in growing buffalo calves. PMID- 3002007 TI - [Serotyping of cytopathic rotavirus strains isolated from calves]. AB - Antistrain sera were obtained against cytopathic rotavirus strains isolated from calves as well as against reference rotavirus strains, belonging to I and II serotype of the bovine rotaviruses with which cross antigenic studies were carried out. It was found that the isolated DS 39/82 and TR 248/82 strains belonged to I serotype of the bovine rotaviruses. Data was obtained speaking of some minor antigenic differences between strain TR 248/82 and strain DS 39/82 as well as between strain TR 248/82 and the reference rotavirus strain Lincoln (I serotype), which showed that there existed serologic variants between the strains of this serotype. PMID- 3002008 TI - [Development of a method for the quantitative determination of the immunizing antigen (140S) of the foot-and-mouth disease virus]. AB - Attempts were made to work out a method for measuring the amount of the 140 S antigen in virus suspensions. Early postinfection sera were obtained from guinea pigs against the productional strains of the foot-and-mouth disease virus which were used in the radial immunodiffusion test. The investigated virus suspensions were concentrated 50 to 200 times and were placed in a CsCl gradient for gradient centrifugation. The 140 S antigen fractions obtained were titrated in a radial immunodiffusion test. The size of the resulting precipitation circles were defined for the individual dilutions of the antigen, and the method of least squares was employed to establish the standard straight lines for each of the investigated productional strains of the FMD virus. The new method makes it possible to fix the content of the 140 S antigen in productional series of the FMD virus intended for vaccine production. PMID- 3002009 TI - [Cultivation of bovine pestivirus in organ culture]. AB - Attempts were made to culture strains of the bovine pestivirus in organ cultures of tracheal rings. Contrary to other scientists the authors used roller organ cultures of calf tracheal rings. The development of the virus in the cultures was judged by its presence in the maintaining medium through ELISA or by titration in cell cultures of fetal calf kidney. It was found that the two strains tested could be replicated in organ cultures of calf trachea, the intensity of multiplication being strongest between the third and the sixth day as demonstrated by ELISA. It was also established that the various batches of tracheal tissue had varying susceptibility. PMID- 3002010 TI - [Associated vaccination of poultry against infectious bronchitis, Newcastle disease and infectious bursitis]. AB - The effectiveness of immunity was studied following a mixed vaccination with live vaccines against infectious bronchitis (strains H120 and H52), Newcastle disease (strain La Sota), and infectious bursitis (strain Th75Vn82). The three vaccines were applied simultaneously via the drinking water, through the spray method, and nasally. Experiments were carried out with a total of 31,466 birds: broilers, growing layers, broiler parents--all without preliminary treatment with biopreparations. Immunity against Newcastle disease was followed up through the hemagglutination-inhibition test and challenging with a virulent virus; against infectious bursitis--through immunodiffusion in agar gel after Ouchterlony; and against infectious bronchitis--through virus-neutralization with strain Beaudette. The birds were treated with mixed vaccines in the following combinations: infectious bronchitis--Newcastle disease; infectious bursitis- Gumboro; infectious bronchitis, Newcastle disease, Gumboro. The simultaneous application of live vaccines against infectious bronchitis, Newcastle disease, and infectious bursitis was shown to be well tolerated with no harmful aftereffects whatever. The immunity built up with the simultaneous use of the three vaccines was not inferior in effectiveness to that conferred with the use of two vaccines or only one of them. PMID- 3002011 TI - [Diagnosis of transmissible gastroenteritis in swine by the agar gel immunodiffusion test]. AB - A transmissive gastroenteritis antigen was obtained from intestinal content of pigs with symptoms of the disease, the presence of which was demonstrated by the agar gel immunodiffusion test. It was shown that the antigen was rough and lacking in purity, but it proved to be sensitive and specific. In the conditions of agar gel immunodiffusion in the presence of a reconvalescent serum the antigen produced a clearly expressed precipitation line for 24 to 48 hours. A correlation was also found with 17 out of 25 reconvalescent sera of pigs, investigated via virus-neutralization and agar gel immunodiffusion. Four sera were positive through virus-neutralization only, and four--through agar gel immunodiffusion, while the controls were negative. The agar gel immunodiffusion test was useful in the demonstration of both antibodies in reconvalescent sera and virus in the intestinal content of pigs either suffering or died of transmissive gastroenteritis. PMID- 3002012 TI - Immunohistochemical localization of alpha 1-antitrypsin and alpha 1 antichymotrypsin in salivary pleomorphic adenomas. AB - Immunohistochemical identification of alpha 1-antitrypsin (alpha 1-AT) and alpha 1-antichymotrypsin (alpha 1-ACh) in pleomorphic adenomas of salivary glands is reported in order to compare their distribution profiles with those of lysozyme and lactoferrin, already described elsewhere. Normal salivary glands indicated positive alpha 1-AT staining in ductal segments and had no alpha 1-ACh in any glandular cell. Pleomorphic adenomas displayed moderate positivity to alpha 1-AT staining in duct-like, tubular and glandular epithelia which was particularly intense in luminal cells. The limited number of tumour cells which showed duct like structures with a single cellular layer arrangement, displayed the highest staining to alpha 1-ACh. Strongly alpha 1-AT positive tumour cells located on the inner side of luminal cavities were also markedly positive to alpha 1-ACh. Spindle shaped tumour cells existed outside tubular and ductal structures and were negative to alpha 1-AT and alpha 1-ACh. Distribution of alpha 1-AT in salivary glands was similar to that of lysozyme as is usual in ductal segments or their transformed cells, and occurrence of alpha 1-ACh localization rather resembled that of lactoferrin, with occurrence in acinar compartments and changed epithelia within acini. The biological role of a specific immunohistochemical distribution of alpha 1-AT and alpha 1-ACh in pleomorphic adenomas may be associated with a self regulating mechanism which inhibits degradation by tissue proteinases. PMID- 3002014 TI - Familial congestive cardiomyopathy with nemaline rods in heart and skeletal muscle. AB - Primary familial cardiomyopathy, once exclusively associated with hypertrophic disorders, is now recognized to occur in a dilated or congestive form. In some instances, characteristic myocellular inclusions of varying morphologies have been identified. Nemaline rods are inclusions which typically have been linked with a rather benign and nonprogressive congenital myopathy. We report finding myocellular inclusions consistent with nemaline rods in two brother who died with congestive cardiomyopathy. Although there was no history or clinical evidence of a myopathy, characteristic nemaline rod inclusions were also identified in the skeletal muscle of one sibling. PMID- 3002013 TI - Light and electron microscopic localization of the N-terminal fragment of human pro-opiomelanocortin in the human pituitary gland and in neoplasms. AB - Immunohistochemical localization of the N-terminal fragment (1-76) (NTF) of human pro-opiomelanocortin (POMC) was studied in human adult and fetal pituitary glands, as well as in pituitary adenomas associated with Cushing's syndrome and in ectopic ACTH-producing tumors. Comparison of localization between NTF and ACTH was performed using mirror sections. Our results indicated concomitant localization of NTF and ACTH in the same cells, not only in normal adult and fetal pituitaries but also in pituitary adenomas and ectopic ACTH producing tumours. Specificity of the NTF staining was confirmed by immunoabsorption. Negative staining of the bovine pituitary gland indicated the immunohistochemical localization of N-terminal (1-45) of human POMC as there is a known species difference in the sequence 1-45 between human and the bovine N-terminal fragment. Presence of NTF in cisterna of rough endoplasmic reticulum indicates its production by small cell carcinoma. These findings, together with the previous studies, suggest that the complete form of POMC is produced in the tumours as well as in normal pituitaries. PMID- 3002015 TI - Modification of simian virus 40 large tumor antigen by glycosylation. AB - The SV40-encoded transforming protein, large tumor antigen (T-ag), is multifunctional. Chemical modifications of the T-ag polypeptide may be important for its multifunctional capacity. T-ag is additionally modified by glycosylation. T-ag was metabolically labeled in SV40-infected cells with tritiated galactose or glucosamine, but not with mannose or fucose. The identity of glycosylated T-ag was established by immunoprecipitation with a variety of T-ag-specific antisera, including monoclonal antibodies. Incorporation of labeled sugar into T-ag was inhibited in the presence of excess unlabeled sugars, but not in the presence of excess unlabeled amino acids. Labeled monosaccharides could be preferentially removed from T-ag with a mixture of glycosidic enzymes. In addition, galactose was removed from purified T-ag by acid hydrolysis and identified as such by thin layer chromatography. T-ag oligosaccharides were resistant to treatment with EndoH, and glycosylation was not inhibited by tunicamycin. Together, these data strongly suggest that T-ag is glycosylated. Several characteristics, including lack of mannose labeling, EndoH resistance, and tunicamycin resistance, suggest that T-ag is not an N-linked glycoprotein. Rather, these properties are more consistent with the identification of T-ag as an O-linked glycoprotein. PMID- 3002016 TI - A putative transforming gene of Jijoye virus differs from that of Epstein-Barr virus prototypes. AB - The P3HR-1 strain of Epstein-Barr virus (EBV), a nontransforming clonal derivative of Jijoye (EBV), is characterized by a deletion of 6.6 kb involving part of the BamHI-W repeats and the adjacent region including the NotI repeats. In the transforming parental Jijoye virus this region differs from the corresponding regions in B95-8 or M-ABA virus. The HindIII-B fragments which carry this region from both Jijoye and prototype M-ABA (EBV) viruses have been cloned and subclones have been constructed which contain the left-hand part of HindIII-B from the HindIII to the BglII site (BglII-delta C fragment). By restriction enzyme analysis the inserts were found to be of equal size (6.3 kb) but to differ in their restriction enzyme pattern. Heteroduplexes formed under stringent conditions in the presence of T4 gene 32 protein revealed a substitution loop of 1750 +/- 200 nucleotides. Heteroduplex formation under nonstringent conditions showed that the substituted sequences are partially homologous to each other, with the regions of nonhomology confined to three distinct areas of 100 to 200 nucleotides. The partial homology observed between both regions indicates that they have evolved from a common ancestor. By hybridization of a Jijoye virus subclone containing only sequences of the substituted region to Northern blots a 2.8-kb polyadenylated transcript was detected indicating that the substituted region is expressed in Jijoye cells. PMID- 3002017 TI - Analysis of the cellular proto-oncogene mht/raf: relationship to the 5' sequences of v-mht in avian carcinoma virus MH2 and v-raf in murine sarcoma virus 3611. AB - The avian carcinoma virus MH2 contains a hybrid gene delta gag-mht with a contiguous open reading frame of 2682 base pairs as well as v-myc and avian helper virus-related sequences. delta gag is a partial retroviral core protein gene while v-mht and v-myc are cell-drived sequences. The v-mht sequence can be divided into two regions: the v-raf-related region at its 3' end contains 969 nucleotides which are 94% related as amino acid sequence to the onc-specific v raf sequence of murine sarcoma virus 3611 (MSV 3611), and the v-mht-specific region at its 5' end contains 173 nucleotides which are unrelated to either MSV 3611 or avian helper virus sequences. To study the origin of the v-mht-specific sequences, the 5' region of the proto-mht/raf gene was molecularly cloned from a phage lambda library containing genomic chicken sequences. Nucleic acid hybridization, heteroduplex and DNA sequence analyses indicate that the v-mht specific sequences are encoded in three exons. The first and second exons are separated by a 3.4-kb intron while the second and third exons are separated by a 90-bp intron. The last 14 bp of the third exon are shared with v-raf and thus represent the start of v-raf-related sequences. The junction between v-mht unrelated and related cellular sequences occurs within the first exon. There is no homology between the v-mht-unrelated sequences and the retroviral helper sequences indicating that the viral transduction of the proto-mht/raf sequences occurred through illegitimate recombination. The predominant v-mht-related messenger RNA (4.0 kb) hybridizes to several noncontiguous regions on the molecularly cloned cellular proto-mht/raf DNA indicating that the proto-mht/raf gene is distributed over at least 10 kb of DNA in the chicken genome. Thus the v mht oncogene is a subset of its normal cellular homolog in that it lacks intervening sequences and possibly lacks 5'-coding sequences. PMID- 3002018 TI - Effect of myelocytic maturation of HL60 cells on replication of influenza and polioviruses. PMID- 3002019 TI - Characterization of virus obtained from MDBK cells persistently infected with a variant of herpes simplex virus type 1 strain MP [HSV-1(MP)]. AB - Virus clones which express glycoprotein gC (gC+) were obtained from two persistently infected (p.i.) MDBK cell lines which had been independently established by infection with HSV-1(MP)10311, a gC- syncytial (syn) variant of herpes simplex virus type 1 strain MP [HSV-1(MP)]. The gC+ revertants were syn in MDBK, HEp-2, and Vero cell lines and in primary human fibroblasts; this offers further evidence that glycoprotein gC does not inhibit cell fusion. The gC+ revertants represented from 70 to 100 percent of the virions present in the virus populations examined, thus suggesting a possible selective advantage of the gC+ revertants in this system of persistent infection. PMID- 3002021 TI - Characterization of ecotropic murine leukemia viruses in SJL/J mice. AB - Backcross mice assorting for the two ecotropic murine leukemia virus loci of SJL/J mice (Emv-9 and Emv-10) were tested for production of infectious leukemia viruses. It was found that tail tissue from mice carrying Emv-9 could not be induced to produce ecotropic MuLV whereas tail tissue from mice carrying Emv-10 were inducible. No mice showed detectable levels of spontaneous virus production. Preliminary restriction enzyme analyses indicated Emv-9 contains a deletion within the polymerase region. PMID- 3002020 TI - Detection of visna virus antigens and RNA in glial cells in foci of demyelination. AB - Visna is a slow virus infection of sheep in which the characteristic pathological change is demyelination in foci of inflammation. The latter is thought to be the result of an immunopathological process directed against cellular and antigenic targets that have been difficult to define because of restricted viral gene expression. A new simultaneous detection assay is used to demonstrate viral RNA in cells identified unambiguously as oligodendrocytes and astrocytes. These cells were found in inflammatory foci. With a new strain of virus that causes a rapid form of visna in Icelandic sheep, viral antigens were demonstrated in cells in the inflammatory lesions. These findings are consistent with the postulated immunopathological mechanism of demyelination: cells that maintain intact myelin sheaths in the central nervous system are destroyed by the inflammatory response to viral antigens expressed in these cells. PMID- 3002023 TI - Microinjected ras family oncogenes stimulate DNA synthesis in quiescent mammalian cells. AB - Oncogenes of the ras family stimulate DNA synthesis when microinjected into quiescent mouse and hamster fibroblasts, as detected by in situ autoradiography. The molecularly cloned genomes of Harvey and Kirsten sarcoma viruses, the cloned Harvey ras gene, and the product of the v-ras gene, the p21v-rasH protein, stimulate DNA synthesis in quiescent cells. This stimulation is comparable to the stimulatory activity of the microinjected SV40 T-antigen-coding gene. The demonstration that these oncogenes can stimulate transient DNA synthesis in quiescent cells is relevant to understanding the mechanism by which these genes are able to transform cells in vitro and induce tumors in animals. PMID- 3002022 TI - Molecular cloning and characterization of feline cellular genetic sequences homologous to the oncogene of the McDonough strain of feline sarcoma virus. AB - The organization within the cat genome of cellular genetic sequences homologous to the viral oncogene v-fms of the McDonough strain of feline sarcoma virus (SM FeSV) was determined. Four cosmid clones containing overlapping v-fms homologous cellular DNA inserts representing a contiguous region of cellular DNA of approximately 80 kbp in length have been isolated from a feline cosmid gene library. Within this region of the cat genome, the c-fms genetic sequences are dispersed over a region of around 30 kbp and are interspersed with at least three intervening sequences. PMID- 3002024 TI - Induction of clonogenic and erythroleukemic cells by different helper virus pseudotypes of Friend spleen focus-forming virus. AB - The properties of clonogenic and leukemic cells, derived from mice infected with different helper virus pseudotypes of the polycythemic strain of Friend spleen focus-forming virus (SFFVp), have been analyzed. Four different replication competent murine leukemia viruses (MuLVs) were used as helpers for the defective SFFVp genome: the Friend MuLVs, Moloney MuLV, and an amphotropic MuLV. Three different biological parameters were measured: (i) the kinetics of emergence of clonogenic cells characteristic of the late stages of Friend erythroleukemia; (ii) the ability of cells in these colonies to give rise to secondary colonies (self-renewal capacity); and (iii) the capacity of cell lines derived from these colonies to respond to inducers of erythroid differentiation. The properties of these cells was found to be independent of the helper virus used, suggesting that it is the SFFVp genome, not the helper virus, that plays a determinant role in the late stages of erythroleukemia. PMID- 3002025 TI - Differential expression of Epstein-Barr virus membrane antigens defined with monoclonal antibodies. AB - Four different mouse hybrid cell lines producing IgG2b monoclonal antibodies against Epstein-Barr virus (EBV) membrane antigens (MA) were analyzed. The antigens defined by these antibodies, MA-2, MA-4, MA-5, and MA-7, were expressed only on EBV-producing cells. The antigens were induced on P3HR-1 cells by treatment with tumor-promoting agent (TPA) plus n-butyrate, and this induction was inhibited by treatment with phosphonoacetic acid (PAA) or acyclovir. Most MA monoclonal antibodies neutralized the infectivity of EBV in vitro in the presence of complement. The monoclonal antibody MA-4 precipitated two polypeptides with mol wt of 340K and 240K, while other monoclonal antibodies MA-2, MA-5, and MA-7, did only 340K peptide. The frequency of positive cells in MA-induced cells varied for each monoclonal antibody tested. It was also found that MA-4 (anti-340K and 240K) antibody reacted on both chemically induced cells and EBV-superinfected cells, but others did only on chemically induced cells. It was suggested that MA had a different pattern of expression between chemically induced cells and EBV superinfected cells. PMID- 3002026 TI - Structure of viral DNA in a rat cell line, GY1, transformed by Ad12 HindIII fragment-G. AB - The cell line GY1, established by transformation of a rat cell line 3Y1 with the Ad12 HindIII fragment-G (leftmost 6.8%, nucleotide 1 to 2322), contains more than 100 viral copies per haploid genome. The viral DNAs in this cell line were cloned into a phage vector, lambda gtWES lambda B, and recloned into pBR322 with their flanking cellular DNAs. Independently isolated 39 clones were analyzed by restriction enzyme cleavage and Southern blot hybridization experiment and divided into 11 classes. Some of classes contained multiple identical clones, at maximum 16 clones. It may be interpreted that amplification of some of the recombined sequences had occurred after the multiple integrations of transfected DNAs within cells. Using five clones from different classes the sequences of recombination sites were determined. Viral DNAs deleted with varying degrees at both ends were flanked by quite different cellular sequences in different clones and no common sequences were revealed around viral-cellular junctions. Tandemly repeated viral DNAs were found in one of the clones to be integrated in a head to tail manner into cellular DNA. The linkage of these two viral DNAs had occurred at the site where parental viral DNAs shared 2 bp. Palindrome structures could be constructed around viral-cellular and viral-viral junction sites and around the regions of parental viral DNAs corresponding to the junction sites in all of the cases investigated. PMID- 3002027 TI - Polyomavirus-transformed FR 3T3 rat cells are able to form metastases in syngeneic rats. AB - A series of polyomavirus-transformed FR 3T3 rat cell lines were tested for their tumorigenic and metastatic properties after subcutaneous inoculation of syngeneic Fisher rats. All of them grew into tumors, which appeared with variable latency periods; the TD50 varied from cell line to cell line. Eight of the 18 transformants that were inoculated gave rise to metastases, always localized in the lung. The capacity to form metastases, though at a low frequency, was also conferred on FR 3T3 cells upon transformation with a recombinant plasmid encoding only the middle-T protein. Fibroblast-like cells were predominantly observed upon histological examination of the metastases. Culture cell lines were derived from independent tumors and metastases induced by two transformants with low and high metastatic potentials, respectively. Metastasis-derived cell lines exhibited metastatic potentials similar to those of the respective original transformants. All the tumor- and metastasis-derived cell lines synthesized the same early viral polypeptides as the respective original transformants; in contrast, the viral DNA integrations evolved during tumor and metastasis formation. PMID- 3002028 TI - A new sensitive target-bound DNA binding assay for SV40 large T antigen. AB - We have developed a new sensitive target-bound DNA binding assay (TB assay) for SV40 large T antigen (large T). The major advantage of this assay is that in contrast to commonly used DNA binding assays, DNA binding is not performed in large T extracts, but instead is performed with immunopurified target-bound large T. Thereby interference of cellular components present in large T extracts is avoided. Thus the TB assay allows DNA binding analysis of large T from different sources (extracts, cell lines) under standardized conditions. Large T is first immunopurified with an anti-T monoclonal antibody not interfering with DNA binding and protein A-Sepharose. Then SV40 DNA is added to the large T immune complex. For analysis of bound DNA and large T, we developed a two-step elution procedure by which bound DNA and large T in the immune complex can be analyzed separately and which allows the determination of the actual amounts of bound DNA and large T. Binding data obtained with the TB assay allowed us to determine an equilibrium dissociation constant (Kd). As a further application of this assay, we analyzed the ORI binding of SVR9D mutant large T which has been reported to exhibit no ORI binding activity. We found that a small percentage of SVR9D large T binds specifically to the SV40 ORI. PMID- 3002029 TI - Isolation and characterisation of herpes simplex virus type 1 mutants which fail to induce dUTPase activity. AB - The herpes simplex virus (HSV) type 1 dUTPase gene was inactivated by insertion of HindIII oligonucleotide linker sequences into the KpnI site within the coding region of the cloned gene. The mutated gene was introduced into wild type herpes simplex virus by marker rescue and the recombinants were identified by the acquisition of a HindIII site within genome map coordinates 0.69 to 0.70 and the failure to induce virus-specific dUTPase activity. A spontaneous dUTPase deficient mutant, which had an identical restriction endonuclease DNA pattern to wild type virus, was also isolated from this transfection experiment. Both types of dUTPase-negative mutants failed to induce a virus-specific 39,000 mol wt polypeptide. Cells infected with the insertional mutant contained instead a novel polypeptide about 40,000 mol wt. No abnormal virus specific polypeptide was detected in cells infected with the spontaneous mutant. We conclude that the 39,000 mol wt polypeptide induced by wild type HSV-1 is the virus-coded dUTPase. Since both types of mutants grew well in exponentially growing and serum-starved tissue culture cells in the absence of wild type helper virus, the dUTPase is not required for virus replication under these conditions. Thymidine kinase deficient, dUTPase deficient double mutants were constructed by recombination of a thymidine kinase insertional mutation into dUTPase deficient virus. These mutants also grew as well as wild type virus both in normal tissue culture cells and cells lacking the cellular thymidine kinase. PMID- 3002030 TI - JC virus T protein during productive infection in human fetal brain and kidney cells. AB - We have examined the synthesis of the T protein of the human polyomavirus, JCV, during productive infection in primary cultures of human fetal glial and kidney cells. Immunoprecipitation of protein extracts from virus infected cells revealed that the JCV large T protein from both the prototype Mad and HEK adapted strains migrated as a 94-kDa protein in denaturing polyacrylamide gels. Resolution of the JCV T protein in brain cells could best be achieved following alkylation of the immunoprecipitated proteins prior to gel electrophoresis. The small t protein of either strain of JCV, however, could not be detected. In comparative experiments, the large T protein of the simian polyomavirus, SV40, was also identified as a 94 kDa protein in immortalized human fetal glial and kidney cultures. There were also protein complexes between p53 and SV40 T protein in the human glial and kidney cell lines. No evidence for a similar protein complex could be detected in JC virus infected human fetal brain or kidney cells. PMID- 3002031 TI - Transactivation induced by human T-lymphotropic virus type III (HTLV III) maps to a viral sequence encoding 58 amino acids and lacks tissue specificity. AB - The acquired immune deficiency syndrome (AIDS) retrovirus, HTLV-III/LAV, encodes a transacting factor which directly or indirectly stimulates the expression of genes linked to its LTR. To further dissect this phenomenon, we have cotransfected a biologically active molecular clone of HTLV-III and a recombinant plasmid containing an indicator gene, the bacterial gene for chloramphenicol acetyltransferase (CAT), under the control of the HTLV-III LTR. Amplified CAT activity was detected in both lymphoid cells and fibroblasts from a number of species in the presence of the proviral DNA. Deletion experiments confirm the previous assignment of the gene required for transactivation to a region immediately 5' to the envelope gene, and further narrow down the critical functional domain to a coding sequence of 58 codons. PMID- 3002032 TI - Purification and characterization of the feline sarcoma virus tyrosine-specific kinase pp85gag-fes. AB - The transforming phosphoprotein pp85gag-fes of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV) was purified in a form which exhibits tyrosine specific kinase activity. Cell lysates of ST-FeSV-transformed mink nonproducer cells were applied to a column of DEAE-Sephacel and eluted with a linear gradient of NaCl in phosphate buffer. Kinase activity was found uncomplexed (pp85gag-fes) in the flow-through and was eluted with 200 mM NaCl in a complex with pp50 and pp90. The flow-through material was further fractionated by chromatography on hydroxylapatite, followed by phosphocellulose and a final gel filtration step using Sephacryl S200. The final preparation was specifically enriched 1400-fold, and tyrosine-specific kinase activity was increased by about 300-fold as determined by autophosphorylation or phosphorylation of casein. Pp85gag-fes kinase activity was inhibited by nicotinamide adenosine dinucleotide (NAD) and slightly inhibited by NADH, while quercetin, a strong inhibitor for pp60src associated tyrosine kinase activity, had no inhibiting effect. PMID- 3002033 TI - Restriction endonuclease map of endogenous mouse mammary tumor virus loci in GR, DBA, and NFS mice. AB - Mouse mammary tumor virus (MMTV) is integrated in the genome of most mice as an endogenous provirus. Two of these MMTV proviral loci (Mtv-1 and Mtv-2) are associated with virus expression and tumorigenicity. We prepared restriction endonuclease maps of the endogenous MMTV proviruses in two strains, DBA and GR, which contain the Mtv-1 and Mtv-2 loci, plus a third strain, NFS, which has a low mammary tumor incidence. We find that all these mouse strains have certain MMTV loci in common even though their origins are widely divergent. We also find that some integrated MMTV proviruses appear to have undergone alterations or deletions when compared with MMTV exogenous proviral DNA. We have thus made a comprehensive characterization of MMTV loci in these mouse strains which could serve as a basis for the study of their differences in expression. PMID- 3002034 TI - Secondary structure of poliovirus RNA: correlation of computer-predicted with electron microscopically observed structure. AB - A secondary structure map of poliovirus 1, strain Mahoney, RNA was determined by psoralen crosslinking the (+) strand and visualizing the structures in the electron microscope. Hairpins and looped hairpins were observed, and the size and distribution were measured. To orient map features the 3' end of the RNA was linked to polybromodeoxyuridine [poly(BUdR)]SV40 and histograms were constructed from these measurements. Secondary structure maps of the RNA were likewise constructed from the results of computer prediction programs for secondary structure. The programs used were those of M. Zuker (RNA2 and FOLD) which calculate a minimal global energy for a given sequence. Many single hairpins predicted by both RNA2 and FOLD showed a correlation with the histograms of hairpin structures of RNA crosslinked with psoralen. A secondary structure map was also constructed for the entire 7433 bases using the option in FOLD which allows multi-branch loops by folding uniformly stepped overlapping segments. Any structure that occurred at or greater than a given frequency was selected and mapped with respect to genome position. A correlation in structured regions was seen between psoralen-derived and computer-predicted maps of secondary structure. Furthermore, a region of large loops from base position 681 to 3899 was noted that corresponded to frequently observed large loop(s) in electron micrographs of psoralen preparations of RNA. Agreement between the two methods of determining secondary structure strengthens the credibility of the computer-aided methods used for predicting secondary structure and allows us to suggest an overall secondary structure map for poliovirus RNA. PMID- 3002035 TI - Kinetics of nuclear transport and oligomerization of simian virus 40 large T antigen. AB - The kinetics of nuclear transport and of oligomerization of simian virus 40 (SV40) large T antigen in lytically infected cells were investigated by pulse chase experiments, cell fractionation, and sedimentation analyses in sucrose gradients. After synthesis, large T was rapidly translocated to the nucleus. Within 10 min, half of the pulse-labeled molecules had entered the nucleus and after an additional 30 min, nuclear accumulation of large T reached a constant plateau of about 95%. Within that time, the majority of large T was in monomeric form suggesting that nuclear transport takes place in this state. In the nucleus, conversion to tetramers proceeded slowly and steadily. By 60 min half of the molecules had formed tetramers and by 6 hr a steady-state ratio between tetramers and monomers of 4:1 was observed. A small fraction of large T remaining in the cytoplasm oligomerized considerably faster than large T in the nuclear fraction. This phenomenon of accelerated oligomerization was also observed with a mutant of large T defective for nuclear transport. Perhaps, the nuclear envelope is a barrier for the complex forms of large T which prevents premature oligomers in the cytoplasm from entering the nucleus and oligomers in the nucleus from migrating back to the cytoplasm. PMID- 3002037 TI - Modulation of the expression of poliovirus proteins in reticulocyte lysates. AB - The translation of poliovirus RNA into specific viral proteins in mRNA-dependent reticulocyte lysates (MDLs) was found to be highly dependent on individual lysate preparations. Under optimal conditions, the first polypeptide detected was always P3-1b (formerly NCVP 1b), the product of the 3' portion of the poliovirus genome; the formation of P1-1a (formerly NCVP 1a) followed as shown by time-course and pulse-chase experiments. However, some lysates synthesized little or no P1-1a despite their ability to synthesize P3-1b and to translate normally other cellular and viral mRNAs. When an MDL competent in synthesizing P1-1a was diluted ca. twofold, while maintaining optimal concentrations of salts, tRNA, DTT, creatine phosphate, and amino acids, P1-1a formation was virtually eliminated, while the synthesis of P3-1b, presumably as a consequence of a more downstream initiation, was maintained. The synthesis of P1-1a in a diluted MDL was restored, and P3-1b synthesis suppressed, by the addition of a S10 fraction prepared from uninfected or virus-infected HeLa cells. Nuclease treatment and dialysis of the S10 fraction did not inhibit its activity. These findings indicate that individual MDLs either possess limiting quantities of, or occasionally are deficient in, a factor(s) that promotes the utilization of the presumed 5' proximal initiation site (the AUG at nucleotide position 781-783) and that a homologous factor(s) exists in HeLa cells. The implication of these findings for the strategy of poliovirus replication is discussed. PMID- 3002038 TI - Herpes simplex virus expressing Epstein-Barr virus nuclear antigen 1. AB - DNA fragments containing an open reading frame known to encode most or all of the EBNA1 protein of Epstein-Barr virus (EBV) were fused in the proper transcriptional orientation to the promoter regulatory domain, capping site, and a portion of the 5' transcribed noncoding sequences of the HSV-1 alpha 4 gene of herpes simplex virus 1 (HSV-1). In these constructs 20, 130, or 385 bp of EBV DNA and 28 bp of HSV-1 DNA separated the alpha 4 cap site from a putative initiator codon of the EBNA1 gene. The chimeric alpha 4-EBNA1 genes were introduced into L cells or recombined into the viral genome using the thymidine kinase selection system. The three chimeric gene constructs resident in the L cell clones expressed a protein indistinguishable from authentic EBNA1 with respect to electrophoretic and immunologic properties indicating that the ATG at the beginning of the EBV open reading frame initiated translation of the bonafide EBNA1 protein. The chimeric alpha 4-EBNA1 genes resident in L cells were induced by HSV-1 infection and were regulated as alpha genes. The chimeric alpha 4-EBNA1 gene recombined into the viral genome was also regulated as an alpha gene. The recombinant viruses were stable and expressed 50- to 100-fold more EBNA1 than is ordinarily expressed in human lymphocytes carrying the EBV genome. EBNA1 did not alter the program of HSV-1 protein expression. The utility of the vector is discussed. PMID- 3002036 TI - Purification of a polypeptide complex (p52) belonging to the D-subspecificites of Epstein-Barr virus-induced early antigens. AB - A two-dimensional immunoblot analysis of chemically induced EBV DNA carrying Burkitt's lymphoma cell lines shows besides a large number of minor components at least two major groups of polypeptides: the most prominent group of polypeptides is observed in the range of 48 to 58 kDa (pI 4.5 to 8.5) and another group at 38/36 kDa (pI 4.4). A polypeptide complex (p52) belonging to the Epstein-Barr virus (EBV)-induced early antigen complex (EA) has been isolated from IdU-induced Raji and B95-8 cells as well as from n-butyrate-induced P3HR-1 cells. The p52 polypeptides have been purified by chromatography on Blue-, DEAE-, CM-, and Phenyl-Sepharose. The purification of these components of the EA complex was monitored by ELISA and by two-dimensional immunoblots using a well-characterized high anti-EBV positive human serum. The isolated polypeptides have an apparent mol wt of about 52,000 Da as determined under nondenaturing conditions by gel filtration chromatography on Sephacryl S-300. One- and two-dimensional immunoblots show a major group of polypeptides of 52 kDa (pI 8.5 to 5.5) with EA activity and some minor components with smaller size up to 40 kDa. The latter seem to be generated by limited proteolysis of p52 polypeptides. The EA activity of the isolated polypeptides could be confirmed by their reaction with IgG anti EA positive as well as IgA anti-EA positive sera by ELISA. The purified polypeptide complex did not react with anti-EA-D negative, anti-EA-R positive sera obtained from patients with African Burkitt's lymphoma, suggesting that these polypeptides belong to the EA-D complex. The monoclonal antibody R3 reacted with the isolated 52 kDa components of EA suggesting a common epitope present on these polypeptides, the same result was obtained with three rabbit sera produced against the isolated polypeptide complex. PMID- 3002039 TI - A common mouse mammary tumor virus integration site in chemically induced precancerous mammary hyperplasias. AB - Mammary carcinomas can be induced by chemical and hormonal as well as viral carcinogens. Irrespective of the class of inducer, these tumors develop in discrete stages, of which alveolar hyperplasia is one of the earliest identifiable. Since carcinogenesis by the mammary tumor virus is now thought to involve proviral activation of adjacent cell genes at specific loci, we sought to determine if a similar mechanism also played a role in chemical and hormonal carcinogenesis and if its role was stage specific. Three high-tumor-incidence BALB/c hyperplastic alveolar nodule outgrowths of two different etiologies were found to have exogenous mouse mammary tumor virus proviruses integrated at the same site in the genome. This common site of integration is not within the bounds of the int-1 and int-2 loci into which proviruses detected at these loci are clustered in MMTV-induced mammary tumors. All three HANs are commonly impaired in end-point differentiation. We propose that mouse mammary tumor virus integration at this site is responsible for a specific abnormality in differentiation associated with the preneoplastic phenotype. PMID- 3002040 TI - Human T-cell leukemia virus type I is a member of the African subtype of simian viruses (STLV). AB - The nucleotide sequences of the long terminal repeat (LTR) of simian retroviruses (STLV), which are closely related to human T-cell leukemia virus type I (HTLV-I) were found to be of at least two subgroups: an Asian subtype in macaques and an African subtype in African green monkeys and chimpanzee. The nucleotide sequence of HTLV-I was found to be included within the divergence among STLV, but showed closer homology (95%) to African subtype STLV than to Asian subtype STLV (90%). PMID- 3002041 TI - [Immunological aspects of Kaposi's disease]. AB - A dynamic complex evaluation of immunologic vigor was carried out in 17 cases of Kaposi's sarcoma. Immunologic disorders were found to be in a correlation with clinical course. Three groups were identified: (1) patients with favourable prognosis (OKT4+/OKT8+ ratio--1.46 +/- 0.09), (12) slowly advancing disease (OKT4+/OKT8+ ratio--0.87 +/- 0.13) and (3) unfavorable clinical course (OKT4+/OKT8+ ratio--0.47 +/- 0.04). The said variations are comparable to those of cyclic nucleotide level in blood lymphocytes. PMID- 3002042 TI - [Patterns in the metastasis of malignant ovarian tumors based on autopsy data]. AB - The results of post-mortem examination of 532 cases of ovarian malignancies are presented. A certain pattern of metastatic spread was established. Tumors most frequently disseminated into the parietal and visceral peritoneum (93.9%), greater omentum (73.6), lymph nodes (48.2), pleura (31.5) and liver (25.4%). Implantation was shown to be the most common mode of spread. Lymphogenous and hematogenous metastases were of low clinical significance since they did not develop so fast as implantation metastases did which advanced rapidly and determined the clinical course. PMID- 3002043 TI - [Proliferative response of HeLa and Hep2 cell lines to exogenous nucleotides]. AB - Nucleotides cAMP, cUTP and beta NAD were tested for antiproliferative activity in HeLa and HEP2 cell cultures. The tests conducted at different stages of cell culturing established a relationship between the proliferative response of cells, on the one hand, and the dosage and time of injection of nucleotide into cell culture, on the other. In the cAMP series, the injection of a minimal dose at the time of inoculation was followed by inhibition of cell growth, as shown by the proliferative index. However, the maximum concentration of cAMP produced a pronounced stimulating effect on the cell growth of both lines. Neither cUTP nor beta NAD injected in the dose used in the study changed the proliferative activity of cells, as compared with control. PMID- 3002044 TI - Human T-cell lymphotropic virus antibody screening: data survey on 33,603 German blood donors correlated to confirmatory tests. PMID- 3002045 TI - [Balneologic sanatorium "Siniak"]. PMID- 3002046 TI - [Detection of HTLV-III antibodies using ELISA and the western blot]. AB - Three commercially available ELISA test kits were used for the detection of antibodies to HTLV-III, the cause of AIDS. Positive results were confirmed by means of the Western Blot. In the course of our routine diagnostic studies, a total of 112 infections with HTLV-III were found among persons in high-risk groups or in blood donors, of whom only 13 presented the clinical picture of AIDS. About 20% of all seropositive persons were intravenous drug addicts. Upon comparison of the ELISA kits of Abbott, Organon, and Sorin, no conclusive indication of a false negative result has been obtained to date with any test kit. Differences were, however, found as regards the number of repeatedly positive samples that could not be confirmed in the Western Blot. The highest rate of these false positive results was found with the Abbott-ELISA, followed by Sorin, while the Organon test did not yield a single false positive result. The lack of agreement in a certain percentage of the tested samples is probably caused by differences in the cut-off values of the various ELISAs used. This value is chosen in such a manner that false positive results are avoided, while even low levels of antibodies are still detected. Since there was no evidence of false negative results, all three kits are suitable for HTLV-III screening of sera. Our results show the need for confirmation of positive results with ELISA by means of the Western Blot. PMID- 3002048 TI - [Modulation of alveolar macrophage activity by ambroxol, bromhexine and exogenous arachidonic acid]. AB - The increased generation of reactive oxygen metabolites (ROM) (O2-, H2O2, 1O2, X OH, OX-) by alveolar macrophages (AM) and neutrophils has been proposed as an important step in the pathogenesis of many acute and chronic lung disorders. With regard to a possible therapeutic application in this context the effect of bromhexine and ambroxol on the activity of AM of of guinea-pigs and of patients with lung diseases was investigated. The activity of AM, which were isolated by bronchoalveolar lavage, was determined by means of chemiluminescence (CL) measuring. Ambroxol and bromhexine cause a depression of the spontaneous as well as of the induced CL [yeast cell walls, yeast glucan, exogenous arachidonic acid (AA)] of AM. AM from guinea-pigs and in some cases also from patients show primarily an increase of the CL-signal under the influence of bromhexine. These results suggest that ambroxol and bromhexine reduce the production of ROM by AM. The mechanism is possibly due to the activation of acyl-CoA-lysophosphatide acyltransferase. The increase of this enzyme activity lowers the intracellular concentration of the free AA, whereby the generation of ROM is diminished too. The investigations exhibit a new possibility of influence of respiratory burst and thus indirectly of AA-liberation from phospholipids. The probable reduction of AA-liberation by these drugs could also be of importance for other lung cells especially concerning the biosynthesis of eicosanoids (AA-metabolites), which play an important part as pathogenetic mediators in different lung diseases. PMID- 3002047 TI - Review of the clinical manifestations, laboratory findings, and complications of infectious mononucleosis. PMID- 3002050 TI - [Controversies in gastroenterology. Treatment of chronic constipation with wheat bran. Pro]. PMID- 3002051 TI - [Controversies in gastroenterology. Treatment of chronic constipation with wheat bran. Contra]. PMID- 3002049 TI - [Gonadal and adrenocortical hormone changes in myocardial infarct]. AB - On a male group of patients investigations of the behaviour of the testosterone, LH, cortisol and ACTH serum concentrations in the hospital phase of the acute myocardial infarction were carried out. Apart from an early activation of the adreno-cortico-pituitary axis a decrease of the testosterone concentrations at consistent LH levels was observed. On healthy test persons in the ACTH and hydrocortisone tolerance test possible interactions between adrenocortical and gonadal hormone system were controlled. PMID- 3002052 TI - [Sexual transmissibility of papillomaviruses]. AB - Papillomavirus infections in the genitoanal region are detectable by the demonstration of type-specific HPVDNA (DNA-DNA hybridization, in situ hybridization on cell smears) or by a peroxidase-antiperoxidase assay using formalin-fixed paraffin sections for demonstration of common structural antigens of papillomaviruses. Using these methods an epidemiological study on sexual partners with genitoanal papillomavirus infections has been initiated. PMID- 3002054 TI - [Heck's disease]. AB - The clinical, microbiological, histological and electron microscopical findings in cases of oral papillomatosis (Heck's disease, focal epithelial hyperplasia), as well as differential diagnosis and therapy are presented. Electron microscopic investigations confirmed the presence of human papilloma virus (HPV) in the lesions of Heck's disease. PMID- 3002053 TI - [Type-specific morphology of viral papillomas (electron microscopic findings in HPV-1 and HPV-4 warts)]. AB - Electron microscopical investigations of cutaneous warts induced by HPV-1 and HPV 4 did not show any differences regarding the ultrastructural morphology of infected cells. There are, however, differences in respect to the degree of the cellular disturbance: Cells rich in particles revealed greater damages than those only showing small amounts of viral particles. PMID- 3002055 TI - [Rapid electron optical diagnosis of skin viral diseases]. AB - By means of electron of microscopy and the negative staining technique, virus particles of herpes, pox and papillomaviruses from skin lesions may be demonstrated in a rather simple and quick way. Advantages and limitations of this method are presented. PMID- 3002056 TI - [Recent diagnostic methods in herpes simplex]. AB - Monoclonal antibodies do not only allow a rapid identification of herpes simplex viruses (HSV) on smear specimens, but also a differentiation between HSV type 1 and type 2. Their specificity and sensitivity correspond to conventional molecular biologic methods. Newer insights into epidemiology and recurrency mechanisms are gained by restriction enzyme analysis of the viral genome, which allows accurate determination of types and strains. Finally, hybridization techniques are utilized for further investigations concerning the latency and oncogenic potency of HSV. Thus the spectrum of diagnostic possibilities of HSV infections has considerably been broadened during recent years. PMID- 3002057 TI - [The infantile acrodermatitis syndrome and Epstein-Barr virus infection]. AB - Six cases of APVS, associated with Epstein-Barr-Virus-infection are reported, the clinical picture, however, is not diagnosed as it is in acrodermatitis papulosa infantum (API). For this reason, we suggest to look for the signs of a primary viral infection in all cases of acro-localized papulo-vesicular skin manifestations. In contrast of APVS, API is a primary hepatitis B virus infection. PMID- 3002058 TI - [Concentrations of external noradrenergic axonal networks in the area of type-III neuronal aggregates and dense capillary networks of the external submucosal plexus (Schabadasch's) in the pig small intestine]. PMID- 3002059 TI - Phosphorylation of coformycin and 2'-deoxycoformycin, and substrate and inhibitor properties of the nucleosides and nucleotides in several enzyme systems. AB - Under conditions where 2'-deoxycoformycin is enzymatically phosphorylated by wheat shoot phosphotransferase to the 5'-phosphate in 15-20% yield, coformycin is a relatively poor substrate, and is phosphorylated only to the extent of less than or equal to 5%. However, chemical phosphorylation of coformycin by modifications of the Yoshikawa procedure led to isolation of coformycin-5' phosphate in 20% overall yield. Coformycin-5'-phosphate was characterized by various criteria, including 1H NMR spectroscopy. Comparison of the spectrum with that of the parent nucleoside indicated that the nucleotide is predominantly, although not exclusively, in the conformation anti about the glycosidic bond. Like 2'-deoxycoformycin-5'-phosphate, coformycin-5'-phosphate was a feeble substrate of snake venom 5'-nucleotidase, and is hydrolyzed, quantitatively, at only 2% the rate for 5'-AMP. With 5'-AMP analogues as substrate, the 5' phosphates of both coformycin and deoxycoformycin were poor inhibitors of the enzyme, with Ki values greater than 0.3 mM. The 5'-phosphates of both coformycin and deoxycoformycin do not significantly inhibit adenosine deaminase (Ki greater than 0.2 mM), but are potent inhibitors of adenylate deaminase (Ki less than or equal to 10(-9) M). Neither coformycin nor deoxycoformycin are inhibitors of mammalian purine nucleoside phosphorylase. The stabilities of coformycin, deoxycoformycin, and their 5'-phosphates, have been examined as a function of pH, and nature of the buffer medium. In particular, all exhibit instability in acid and neutral media, but are relatively stable in the vicinity of pH 9. Some biological aspects of the overall results are presented. PMID- 3002061 TI - Immunogenicity of bluetongue virus inactivated by gamma irradiation. AB - The immunogenicity of vaccines prepared from bluetongue viruses inactivated by gamma irradiation was tested by their ability to stimulate the production of neutralizing antibody in mice. Antibody titres induced by monovalent and polyvalent vaccines, vaccines prepared from virus produced in mice and cell cultures and vaccines containing virus exposed to six, eight and ten megarads of irradiation indicated that immunogenicity was not adversely affected by the inactivation procedure. The results suggest that gamma irradiation would be an effective means of inactivating bluetongue virus without affecting antigenic determinants for neutralizing antibody. PMID- 3002060 TI - Quantitative assay for the transforming DNA activity in cell culture products for human use. AB - DNA isolates from a number of established cell lines have been tested for their transforming capacity in the NIH3T3 transformation assay. It has been found that this assay can be used to quantify the amount of transforming DNA at a submicrogram to milligram level. This assay system has been applied to validate the reduction of transforming DNA in various purification steps of cell culture products. The NIH3T3 assay system has been shown useful for in-process control and release of cell culture derived vaccines or therapeutics. PMID- 3002062 TI - [Genetic control of recombination processes associated with transposons]. PMID- 3002063 TI - [Structural properties of toxins and the molecular mechanism of their interaction with the eucaryotic cell]. PMID- 3002064 TI - [Effect of polycarbacin on the skin and mucous membranes]. PMID- 3002066 TI - Expression of herpes simplex virus glycoprotein B gene in yeast. AB - The DNA sequence coding for herpes simplex virus type 1 glycoprotein B was placed under control of the acid phosphatase promoter of the yeast Saccharomyces cerevisiae in a plasmid capable of replicating in both yeast and Escherichia coli. Yeast transformed by the plasmid synthesized immunologically active glycoprotein B polypeptide. PMID- 3002068 TI - Identification of a 65 000 dalton virion envelope protein of human cytomegalovirus. AB - Human cytomegalovirus (CMV) specific monoclonal antibodies were shown to react with a broadly migrating 65-52 kDa virion envelope protein. These antibodies efficiently neutralized CMV in vitro (greater than 98% plaque reduction), suggesting that this envelope protein was an important target of neutralizing antibodies. This virion envelope protein was shown to be antigenically and electrophoretically distinct from a more abundant 68 kDa virion protein. The relationship between these virion proteins is discussed. PMID- 3002065 TI - Mouse cells surviving polyoma virus infection generally retain the whole viral genome. AB - Permissive mouse 3T6 cells were exposed to polyoma virus--either wild-type or early mutant--at high multiplicities of infection. From colonies arising from surviving cells, so-called lines and clones were derived under conditions precluding superinfection. These lines and clones were examined for the presence of viral genetic information, using a variety of techniques. Two salient findings were made: most lines or clones analyzed had retained viral genetic material; generally, this material was nondefective, as evidenced by the production of virus and/or viral DNA molecules of genomic size. These findings indicate that mouse cells can survive for many generations while carrying a complete, infectious, and potentially cytocidal polyoma virus genome. PMID- 3002067 TI - Production and characterization of monoclonal antibodies directed against pseudorabies virus. AB - Hybridoma cell lines producing monoclonal antibodies to pseudorabies virus (PRV) were established. The monoclonal antibodies were characterized with respect to their antigenic specifications and biological activities. Two monoclonal antibodies immunoprecipitated the 50 kDa PRV glycoprotein (gp50) and two immunoprecipitated the 82 kDa glycoprotein (gp82). The monoclonal antibodies were used to analyze the biological roles of these two glycoproteins. One monoclonal antibody directed against each glycoprotein did not require complement for in vitro viral neutralization while the other monoclonal antibody directed against the glycoprotein required complement for neutralization. The monoclonal antibodies against gp50 were shown to be directed against different epitopes within the glycoprotein. In contrast, the monoclonal antibodies against gp82 were shown to be directed against the same antigenic site on the glycoprotein. In vivo passive immunity studies in mice showed that monoclonal antibodies directed against either gp50 or gp82 could be protective. PMID- 3002069 TI - Site of suppression of Banzi viral replication by an antiviral factor released from Aedes albopictus cells persistently infected with Banzi virus. AB - The ability of the antiviral factor present in culture medium of Aedes albopictus cells persistently infected with the flavivirus, Banzi virus, to inhibit the replication of Banzi virus was examined. The anti-Banzi viral factor did not inhibit the uncoating of the virion. Levels of viral RNA were markedly reduced in mosquito cells treated with the antiviral factor. Syntheses of negative-strand and of positive-strand viral RNA species were inhibited to approximately the same extent. This inhibition was virus-specific in that the anti-Banzi viral factor had no effect on the synthesis of viral RNA in mosquito cells infected with either Japanese encephalitis or Eastern equine encephalitis viruses. The anti Banzi viral factor inhibited the in vitro Banzi viral RNA synthesis but not that of Eastern equine encephalitis virus or of Japanese encephalitis virus. PMID- 3002070 TI - Variation among hepatitis A virus strains. I. Genomic variation detected by T1 oligonucleotide mapping. AB - The genomes of eight hepatitis A virus (HAV) strains originating from far distant geographic regions such as Europe, North Africa, Middle and North America, Australia and The People's Republic of China were compared by RNase T1 oligonucleotide mapping. For this purpose, the viruses were propagated in cell cultures and viral RNA was isolated from highly purified mature virions. It could be shown that variation in nucleotide sequence is common among HAV isolates, but is in the order of magnitude reported for other picornaviruses. For viruses isolated in cell culture directly from stool samples of diseased individuals, changes usually amounted to 1-4% of RNA genome sites. Genomic differences between two virus strains derived from one fecal sample but replicating at either 32 or 37 degrees C were in the same order of magnitude. Thereby, the number of consecutive in vitro passages proved to have only limited influence on the development of genetic variation. For two HAV strains, however, adaptation to and passage in marmosets evidently had imposed highly selective conditions which had favored the appearance of viral genomes differing in up to 75% of their large oligonucleotides (about 10% in sequence) from the oligonucleotide map of a reference HAV strain. PMID- 3002071 TI - Oriented synthesis and cloning of influenza virus nucleoprotein cDNA that leads to its expression in mammalian cells. AB - The influenza virus nucleoprotein gene has been cloned by a procedure that involves direct cDNA synthesis onto the primer-vector pBSV9, a pBR322-SV40 recombinant plasmid. dT-tailed pBSV9 was used to prime the synthesis of cDNA on a template of in vitro synthesized viral mRNA. The synthesis of ds-cDNA was initiated by a specific oligodeoxynucleotide and the resulting recombinant was circularized by intramolecular ligation. Recombinant pSVa963 contained the viral nucleoprotein gene directly fused to the SV40 early promoter region included in pBSV9 and followed by a dA:dT tail and the SV40 polyadenylation signal. When pSVa963 was used to transfect COS-1 cells, the presence of three NP-specific mRNAs of 1600, 1900 and 2500 nucleotides in length could be detected. Pulse labelling experiments of COS-1 transfected cells and immunobinding to a nucleoprotein monoclonal antibody indicated the synthesis of nucleoprotein. This nucleoprotein accumulated in the nucleus of transfected cells at a level similar to that found in infected cells. The vector and method described may be useful for the specific cloning and expression of any mRNA for which a 5'-terminal sequence is known. PMID- 3002073 TI - [Benign fibrous histiocytoma of the breast]. PMID- 3002072 TI - [Serologic diagnosis of primary cytomegalovirus infections in patients following kidney transplantation using the indirect immunofluorescence technic and the complement-fixation test]. AB - Sera of 22 recipients of kidney transplants with clinically and serologically (complement binding reaction) ascertained cytomegalovirus infection were retrospectively examined for virus-specific antibodies of the class IgM by means of the indirect immunofluorescence test. Already two to five days after the appearance of the adequate clinical symptoms (leucopenia, fever, T lymphocytopenia, increase of creatinine and thrombocytopenia) CMV-IgM-antibodies could be proved. On average a positive complement binding reaction was found only two weeks later. In other 13 patients of the actual control programme the CMV-IgM antibody findings first obtained were compared with the results of the complement fixing reaction obtained later. Neither falsely positive nor falsely negative results could be found. PMID- 3002075 TI - [Significance of intracervical cytology in the postmenopause]. AB - The carcinomas of the menopause were numbered in relation to all carcinomas and precancerous lesions diagnosed in our laboratory in the last two years. The results were discussed in connection with statistical establishments on the effectivity of gynecological screening. PMID- 3002074 TI - [Reconstruction of the pulmonary artery in parenchyma-preserving lung resections for bronchial cancer]. AB - Parenchyma-preserving resection techniques with broncho- and angioplastic methods contribute to avoid palliative pneumonectomy in cases of extended bronchial carcinoma (greater than T2; greater than N1). 18 cases are analysed. PMID- 3002076 TI - Degradation of azlocillin in human serum. AB - We developed a rapid and precise high performance liquid chromatographic method (HPLC) for the determination of azlocillin and its metabolites penicilloate and penilloate in serum and tissue as well as in vivo as in vitro. The linear relationship (r greater than 0.99) of determination ranged between 0.05 and 10 micrograms. No interference with other serum components was observed. The metabolism of azlocillin in vitro was analysed in serum. Within 24 h the amount of penicilloate increased from 1.5% up to 18% and from penilloate up to 1% respectively. Buffer controls revealed a significant lower metabolism (4.7% penicilloate, 0.9% penilloate). These data suggest that the degradation of azlocillin does not only depend on bacterial beta-lactamase activity but is influenced by enzymatic activities within serum and human tissue. PMID- 3002077 TI - [Renogenic neurologic disorders]. AB - A total of 137 patients with chronic diseases of the kidneys were examined, including 34 without and 103 with chronic renal insufficiency. The neurologic syndromes under study included encephalomyelopathy with a predominant damage to the coordination systems, polyneuropathy and myopathy. These neurological changes were expressed irrespective of chronic renal failure, while their degree directly correlated with its severity. Stabilography and tremorography proved adequate and objective methods of assessing coordination disorders and made it possible to detect the above changes at the preclinical stage. PMID- 3002078 TI - [Myasthenic syndromes linked to mediator secretion disorders]. AB - On the basis of clinical and electrophysiological examinations of 23 patients with an impaired secretion of the mediator, the following 5 groups of patients with myasthenic syndromes (MS) characterized by disturbances of the neuromuscular transision were identified: patients with bronchogenic carcinoma of the lung; males with a clinical picture closely resembling the one described in bronchogenic cancer of the lung; young males and females with an abnormal thyroid gland; patients with a mild subcortical syndrome and with ataxia; patients with chronic botulinic intoxication. Correlation of the results of electrophysiological examination did not reveal any specificity for any of the identified groups of patients. PMID- 3002079 TI - [Strategy for using glucocorticoid preparations in neuromuscular diseases]. AB - A total of 1630 patients with various neuromuscular diseases of autoimmune genesis were treated. Glucocorticoid drugs taken every other day were shown to be highly effective. On the basis of a large experience the authors propose recommendations for the management of patients in relation to the form, severity and course of the disease with the objective of achieving the maximum therapeutic effect. PMID- 3002080 TI - Nonaspiration-needle smear preparations of pulmonary lesions. A comparison of cytology and histology. AB - Material for cytologic smears was obtained from pulmonary lesions in 146 patients at the Ohio State University between 1979 and 1984 using Rotex or Lee screw needles. Corresponding histologic specimens were available for comparison in 77 of these cases. Diagnoses of malignant neoplasms made by cytologic evaluation (55 cases) were confirmed by the corresponding histologic specimens in 93% of those cases. Possible explanations for the cytologic false-positive diagnoses of malignancy are presented. Correlations between the cytologic and histologic diagnoses of the morphologic type of tumor were 100% for adenocarcinoma, 75% for squamous-cell carcinoma and 20% for large-cell undifferentiated carcinoma. The correlation was 100% for small-cell carcinoma when the histology specimen represented the tumor. Nonneoplastic benign lesions diagnosed cytologically had corresponding benign histologic diagnoses in 94% of the cases. These results compare favorably with those reported for other fine needle aspiration studies of pulmonary lesions. The advantages of using Rotex needles as compared to fine needle aspiration are discussed. PMID- 3002081 TI - Fine needle aspiration cytology of a trabecular adenoma of the parotid gland. AB - What is believed to be the first description of the cytologic presentation of a trabecular adenoma of the parotid gland in a fine needle aspirate is reported in the case of a 52-year-old woman. The cytologic findings included translucent spherical bodies surrounded by small round cells with a thin rim of cytoplasm and round-to-oval nuclei in Papanicolaou-stained specimens. The cells, which varied little in size, formed densely packed clusters. In May-Grunwald-Giemsa-stained preparations, mucoid globules were seen surrounded by palisading tumor cells. Nuclear anaplasia was not seen. The aspiration cytologic and even the histologic appearance of trabecular adenoma can closely mimic that of adenoid cystic carcinoma, which was the preoperative diagnosis in this case. Because the treatment of these two entities is different, it is important that trabecular adenoma be considered in the differential diagnosis. The observations in this case indicate that tinctorial properties of the intercellular substance may aid in differentiating between these two neoplasms in fine needle aspirates. PMID- 3002082 TI - Application of radio-detoxified endotoxin as adjuvant for experimental foot-and mouth disease vaccine. AB - The immunity enhancing adjuvant activity of radiodetoxified endotoxin (RD-LPS) on the potency of "C" type foot-and-mouth (FMD) vaccine was tested in different animal species. The suitable quantity of RD-LPS (20 micrograms per mouse) adjuvated a small than a high dose of FMD antigen. In cattle and sheep the adjuvant effect of oil + RD + LPS surpassed only slightly that of oil alone. The effect of RD-LPS in the pig was very pronounced when applied in small doses but further studies in larger animal populations have to confirm this result. PMID- 3002083 TI - In vitro analysis of BCNU-sensitivity in human malignant gliomas. I. A model study with alkylating, cross-linking and carbamoylating agents in anaplastic astrocytomas of pediatric age. AB - Like all chloroethyl-nitrosoureas of major clinical use, 1,3 bis-(2-chloroethyl) 1-nitrosourea (BCNU) - which is one of the most effective chemotherapeutic agents for CNS malignancies - biologically degrades into active alkylating and carbamoylating moieties. Using a human brain tumor stem cell assay, we analyzed a series of anaplastic astrocytomas of pediatric age, characterized by different degrees of BCNU-resistance. Early (2-4) passage cultures from these tumors were treated in vitro with model drugs for alkylation (BCNU, CHLZ (2-[3-(2 chloroethyl)-3-nitrosoureido]-2-deoxy-D-glucopyranose), ENU (N-ethyl-N nitrosourea), cross-linking (BCNU, CHLZ) and carbamoylation BHCNU (1,3 bis (trans 4-hydrocyclohexyl)-1-nitrosourea): dose-schedules were compatible with clinically achievable levels. Results of chemosensitivity tests confirmed that - as previously reported in malignant gliomas of the adult - cellular resistance to BCNU was closely related to the cross-linking activity of alkylating species. However, in pediatric gliomas the levels of cell kill after treatment with the purely carbamoylating agent BHCNU, even at the highest doses tested, were lower than expected. PMID- 3002084 TI - Indications of the occurrence of inflammatory reactions in the clinical improvement phase in multiple sclerosis patients. AB - In patients with multiple sclerosis (MS) the spontaneous burst activity (BA) of peripheral blood mononuclear cells was related to the clinical course of the disease. In five patients clinical improvement was found while the BA was significantly increased (more than 300% of the controls). During the appearance of new or deteriorating signs and in the period without clinical changes, the BA was not at all or not markedly increased. In two patients without clinical improvement the BA did not reach levels above 300% of the controls. Our findings suggest that inflammatory reactions represented by the BA occur in the phase of clinical improvement. Since burst-stimulating activity was found in the serum of MS patients, cytokines produced by activated immuno-competent cells are assumed to cause the increased BA. A possible role of prednisone and ACTH on the BA of peripheral mononuclear leucocytes is discussed. PMID- 3002085 TI - Neurological and cardiac complications of carcinoma of the breast. Case report. AB - A case is presented of a young female with a paraneoplastic subacute cerebellar degeneration, parkinsonian syndrome, autonomic disturbance, profound depression, myopathy and cardiomyopathy. Her paraneoplastic affection preceded the actual detection of carcinoma of the breast by nine months. Block dissection of the carcinoma resulted in alleviation of her muscle weakness and a return of the electrocardiogram to normal. PMID- 3002086 TI - Congenital neuropathy with prevailing axonal changes. A clinical and histological report. AB - A case of congenital neuropathy associated with an unclassified chronic brain disorder is described. Morphological findings differ from the reported congenital neuropathies where primary myelin change have been usually found. In contrast, sural nerve biopsy showed marked signs of active or past axonal degeneration; at the teasing and morphometric analysis there was also some evidence of segmental demyelination. Atypical onion bulb formations (Evans and Murray type) and a very poor demyelination activity stressed the prevailing axonal involvement with possibly secondary segmental demyelination. PMID- 3002088 TI - 27. meeting of Nordic Ophthalmologists. Proceedings. Arhus, Denmark, June 2-5, 1985. PMID- 3002089 TI - Treatment of senile macular degeneration by laser photocoagulation. AB - 117 eyes of 103 patients among 476 patients with senile macular degeneration fulfilled a criterion for treatment with blue-green Argon (n = 20 eyes), green Argon (n = 15 eyes) Krypton-red (n = 58 eyes) and combined treatment (24 eyes). In 50 consecutive treated eyes (= 50 patients) with an observation time exceeding one year a visual acuity greater than or equal to 6/18 was preserved in 34 eyes. This post-laser course differs from the spontaneous course of the disease reported in literature indicating that about 70% of eyes with perifoveal neovascular lesion will develop legal blindness within 2 years. Even though the spontaneous course in this material is unknown, and the comparability to patient material in literature is questionable, it might be concluded that a considerable part of the patients with senile macular degeneration with neovascular lesions might benefit from laser-treatment. PMID- 3002090 TI - The blood-retinal barrier permeability to fluorescein in juvenile diabetics treated with continuous subcutaneous insulin infusion. AB - To determine possible quantitative changes of the blood-retinal barrier permeability in juvenile diabetics treated with continuous subcutaneous insulin infusion (CSII), we studied seven patients (three females and four males, mean age 36 years) with a mean duration of the disease of 19 years. The pump treatment was continued for seven to eight days and during the treatment mean blood glucose level decreased to near-normal values (before 13.7 mmol per liter - during 6.2 mmol per liter). There was no changes in retinal appearance during treatment. Determination of the blood-retinal barrier permeability showed no quantitative changes during the one week treatment with CSII (mean permeability before 7.6 10 divided by 7 cm/sec - mean permeability during 7.8 10 divided by 7 cm/sec). In order to quantitate possible long-term reversibility of break-down of the blood retinal barrier we have design to extend the treatment period. PMID- 3002087 TI - Herpes simplex virus antigen detection in human acute encephalitis: an immunohistochemical study using avidin-biotin-peroxidase complex method. AB - Autopsy specimens from six patients with clinically diagnosed herpes simplex virus (HSV) encephalitis were studied. Since immunocytochemistry has been reported to be a more reliable and successful method to identify HSV as the etiologic agent, antitype 1 HSV (HSV1) and antitype 2 HSV (HSV2) and the avidin biotin-peroxidase complex (ABC) method were applied to brain paraffin sections. Positive immunostaining was observed in front-orbital, mediobasal temporal lobes, cingulate gyrus, and insula. The staining was bilateral but predominant on one side. In neurons, the cytoplasmic staining was prominent in perikarya and processes, less often observed in nuclei, rarely seen in nuclear inclusions. The positive staining was intense in oligodendrocytes and macrophages, in both nuclei and cytoplasm. In two cases astrocytic processes were stained strongly. Perivascular lymphocytes were always negative. Positive reactions were obtained with both anti-HSV1 and anti-HSV2 but weaker with anti-HSV2. This results suggests that, because of its high sensitivity, ABC method permits viral antigen detection not feasible with other methods. However, this method lacks of accuracy for HSV typing mainly because of probable antigens changes resulting from tissue processing. PMID- 3002091 TI - Evidence for efferent projections from the brain to the retina of the Mongolian gerbil (Meriones unguiculatus). A horseradish peroxidase tracing study. AB - Mongolian gerbils were injected into the vitreous body of the left eye with either horseradish peroxidase or horseradish peroxidase coupled to wheat germ agglutinin. After survival times ranging from 6 to 48 hours, the animals were sacrificed and perfused with glutaraldehyde. Cryostat sections were cut through the brains and reacted for peroxidatic activity. Labelled perikarya were observed in the dorsal nucleus of the lateral geniculate body, the medial and lateral nuclei of the optic tract, the pretectal nuclei and the superior colliculus. These results indicate the presence of an efferent innervation of the retina from the brain via the optic nerve in the Mongolian gerbil. PMID- 3002092 TI - The effect of ocular pigmentation on intraocular pressure response to timolol. AB - The one-hour intraocular pressure (IOP) response to topical ocularly applied timolol was studied in darkly and lightly pigmented eyes. One drop of 0.25% 1 timolol was applied to one eye of ten subjects with brown and ten subjects with blue iris color. In the treated and untreated eyes with blue iris color the IOP fell significantly from the baseline value, whereas no pressure reduction was obtained either in the treated or untreated eyes with brown iris color. Beta blocking agents are known to accumulate in pigment-containing ocular structures. The lack of a one-hour pressure response to timolol in darkly pigmented eyes observed in our study may refer to the reduction of 1-timolol in the biophase due to excessive pigment binding. PMID- 3002093 TI - A statistical trap in the evaluation of visual field decay. AB - Computerized perimetry has made it possible to evaluate quantitatively the visual field decay in glaucoma and other cases. The performance fluctuates with considerable amplitude and therefore the heart of the matter concerns the detection of a signal in presence of noise. How insufficient number of observations and uncautious sampling may result in false conclusions as to the effect of therapy etc is described in the present paper. PMID- 3002094 TI - Electrophysiology in pigment epithelial changes. AB - The c-wave of the ERG and the slow SP variations reflect mainly the activity of the pigment epithelium. However, both potentials are dependent upon the photoreceptors and/or the inner retina as well. In pigment epithelial abnormalities the c-wave is reduced or abolished, and the slow SP variations, d.c. recorded directly or investigated with the EOG, reduced or abolished as well. PMID- 3002095 TI - Prostaglandin E2 in tears. AB - Prostaglandin E2 (PGE2) concentration in tears was measured before and following cataract extraction, during acute anterior uveitis and in patients with chronic conjunctival complaints. Following cataract extraction a significant increased PGE2 level in tears was observed, returning towards normal level within two weeks. In patients with acute anterior uveitis low levels of PGE2 was found in tears. Half of the patients with chronic conjunctival complaints exhibited high levels of PGE2. Conjunctiva is proposed as a likely source of PGE2 in tears. PMID- 3002096 TI - Eye lesions induced by mustard gas. PMID- 3002097 TI - Tear pH after instillation of buffer in vivo. AB - The pH of the conjunctival fluid was measured with a micro-electrode at the middle of the inferior conjunctival fornix of 160 eyes (86 subjects). After instillation of buffer of pH 5 (in 22 eyes pH 9) the linear course of pH changes was followed until the starting value had been regained. The coefficient of regression K was computed (pH-units/min.). In normal eyes the K-value declines with increasing age from 1.0 to 0.5, corresponding to a return to the starting value after 2 to 5 minutes. The K-value is reduced significantly in keratoconjunctivitis sicca (K = 0.21, pemphigoid (0.36), during local anaesthesia (0.38 +/- 0.03 against 1.04 +/- 0.10 prior to 0.4% oxibuprocain), in the presence of a cotton plug in the inferior fornix, and in wearers of soft contact lenses. The K-value is normal in cases of lacrimal occlusion (owing to overflow), in exophthalmos and endothelial dystrophy, following corneal grafting, in conjunctivitis and keratitis, and in wearers of hard contact lenses. In cadaveric eyes we find incomplete, irregular neutralization. CONCLUSION: Tear dilution is the most important factor in the elimination of buffered eye drops in vivo, compared with the proper buffering capacities of the tears and the tissue. PMID- 3002098 TI - Juvenile vitreoretinal degeneration and retinal detachment. AB - The proband was a 10-year-old boy with bilateral retinal detachment. His mother had unilateral retinal detachment at age of 12. The eye disease occurred in eight family members, both men and women, in four consecutive generations suggesting autosomal dominant inheritance. The eye findings were seen at the age of 4 to 12 and included syneresis and posterior vitreous detachment; spotty areas of whitish, glistening degeneration, spotty hyperpigmentation, and greyish-white patches with pigmentations suggestive of healed retinochoroiditis in the peripheral retina. Retinal detachment with holes in the peripheral retina occurred in four family members, and three other family members had unilateral blindness since early childhood. In eyes without retinal detachment the visual acuity, visual fields, dark adaptation, and colour vision were normal. PMID- 3002099 TI - Macular recovery time recorded by nyctometry--a screening method for selection of patients who are at risk of developing proliferative diabetic retinopathy. Results of a 5-year follow-up. AB - A 5-year study on the predictive value of the macular recovery time, as recorded by nyctometry, in the development of proliferative diabetic retinopathy in insulin-dependent diabetes mellitus has been completed. Seventy-seven patients with a median age of 30,8 years and a median duration of the disease of 15,8 years participated. In the follow-up period, 16 out of 20 patients initially showing abnormally prolonged macular recovery time had developed proliferative diabetic retinopathy the median duration from the initial investigation to the diagnosis of retinal neovascularization being 34 months. Contrary to this finding, only 4 out of 57 patients initially showing normal macular recovery had advanced into proliferative retinopathy, and the median duration until diagnosis of this condition was 45 months. It is concluded that nyctometry can serve as an easily performed screening method in selecting those at risk of developing proliferative retinopathy within a few years. PMID- 3002101 TI - Blood pressure and retinopathy in insulin treated diabetic patients with early onset. An epidemiological study. PMID- 3002100 TI - The retinal photoreceptors and the pigment epithelium. Structure and function. Transduction. A brief review. PMID- 3002102 TI - Single binocular vision. Clinical aspects. PMID- 3002104 TI - Von Hippel-Lindau's angiomatosis--a disease with multiple manifestations. AB - We describe a Finnish family, in which two sisters, 54 and 57 years of age, have bilateral retinal hemangiomas, cerebral/cerebellar symptoms, renal adenocarcinomas and renal cysts. Two daughters of the younger sister have bilateral retinal angiomas; three daughters and a son are unaffected. PMID- 3002103 TI - A preliminary report on the denervation hypersensitivity of the pupil. PMID- 3002105 TI - Idiopathic uniocular juxtafoveolar retinal telangiectasis in a female patient. AB - This article describes a female patient with uniocular juxtafoveolar telangiectasis which previously has been reported in male patients only. PMID- 3002106 TI - Ophthalmologic prognosis in benign intracranial hypertension. AB - In a prospective study twenty patients with benign intracranial hypertension, 15 females and five males with mean age 34 years (range 12-61 years), were followed up from 12 to 61 months (mean 22 months). Initially all patients showed marked papilledema, normal visual acuity, considerable enlargement of the blind spot area, and significantly delayed pattern reversal visual evoked potentials. During medical treatment eleven patients showed within 3-6 months a rapidly regression and normalization of papilledema, blind spot area, and visual evoked potentials. Eight patients continued in showing papilledema including disc gliosis, enlargement of blind spot area, and pathological visual evoked potentials. One patient developed optic nerve atrophy. The results indicated that repeated examinations of visual evoked potentials, when suspect of threatening visual loss has come up (papilledema, increased blind spot area, and field defects), might increase the change of diagnose of an optic nerve atrophy as early as possible. PMID- 3002107 TI - The initial stage of the exfoliation syndrome. AB - Exfoliation of devitalized tissue and pigment from the neuroepithelium of the uveal tract constitute the earliest stage of the exfoliation syndrome according to a biomicroscopic study. With an immuno-histochemical technique exfoliative material from lenses with the classic exfoliation syndrome, demonstrated the same staining characteristics as the pigmentary epithelium of the uvea. PMID- 3002108 TI - 24 cases of carotid cavernosus fistulas: frequency, symptoms, diagnosis and treatment. PMID- 3002109 TI - The treatment of presumed non-infective uveitis with cyclosporin A. PMID- 3002110 TI - Intracapsular cataract extraction with and without implantation of an anterior chamber lens. A comparative study. AB - Intracapsular cataract extraction with anterior lens implantation was performed in 60 eyes and matched to a control group of 55 eyes, which were operated without implantation. Per- and immediately postoperatively 8 eyes in each group had minor complications without consequences for the final visual outcome. 76.7% of the implanted eyes obtained a final visual acuity of 0.5 or better versus 70.9% in the non-implanted eyes. In both groups senile maculopathy was the main cause of permanent visual reduction. At the time of follow-up (between 12 and 29 months after surgery) maculopathy was seen in 12 eyes of the implanted group versus 14 eyes of the non-implanted group. 91% of the eyes in the implanted group gave a subjectively satisfactory result to the patient, in the non-implanted group 63% gave a satisfactory result. It is concluded that anterior chamber lens implantation in this material has been a safe procedure with no more short term complications than cataract extraction alone, and that the patients obtain a high degree of visual comfort. PMID- 3002112 TI - Neodymium YAG-laser for posterior capsulotomy. PMID- 3002111 TI - Out-patient cataract surgery. First experiences from a large hospital department. AB - Out-patient cataract surgery was recently introduced as a routine procedure in our department. Background population, selection criteria and practical administrative procedures for the first 33 patients are described. 19 females and 14 males, age range 45 to 90 years, were offered ambulatory surgery; 19 had extracapsular surgery and 27 intraocular lenses. Insignificant complications occurred in four cases, one of them related to the out-patient procedure. It definitely seems possible to offer this modality to selected patients without compromising medical ethics, and the results have been met with unanimous satisfaction by the patients. PMID- 3002113 TI - Bacterial growth in the conjunctival sac and the local defense of the outer eye. AB - 25 patients admitted for cataract surgery were subjected to conjunctival bacterial culturings preoperatively and during the postoperative observation period. Simultaneously lactoferrin (LF), lysozyme (LY) and secretory immunoglobulin A (s-IgA) were measured in tears. The preoperative flora disclosed the growth of Staphylococcus albus (SA) and diphtheroids. Other species were only sporadically present. There was a significant rise in number of patients affected by SA and diphtheroids postoperatively (from 60 to 80%), whereas other bacteria were not present to any significant extent. LF, LY and s-IgA concentration decreased to about 50% of the preoperative level in the early postoperative period gradually returning towards their initial concentration. Correlating an antibacterial score with bacterial score we found a significant inverse relationship between the two (P less than 0.05). PMID- 3002114 TI - Some physiological and psychological characteristics of myopic and non-myopic young men. AB - As a part of a research project on the health and functional capacity of men at different ages a comparison of selected physiological and psychological characteristics of myopic and non-myopic 31-35 year-old men was made. The random sample studied consisted of 31 myopic and 100 non-myopic men. It was found that the body mass index and fat content were lower among the myopic than among the non-myopic. No significant differences were found in the elastic properties of skin, in blood pressure or in haematological assays studied between the groups. With respect to physical performance it was observed that the myopic had a higher aerobic capacity whereas there were no significant differences in muscular strength between the groups. In the psychological functions the myopic had a higher level in certain tests of intelligence. In the ophthalmological examination there were no significant differences between the groups in corrected vision, in eye tension and in dark adaptation. The amplitude of accommodation was 0.6-0.9 D. wider among the myopic. The results indicated some differences in physical and psychological characteristics between myopic and non-myopic young men. The differences could mainly be explained by differences in education and in physical activity during leisure. PMID- 3002115 TI - The retinal pigment epithelium controls the potassium activity in the subretinal space. AB - The potassium transport of the isolated retinal pigment epithelium from bullfrogs was studied using micropuncture with double barrelled ionselective microelectrodes. Transient changes in intracellular values of electrical potential and potassium activity were monitored in response to abrupt changes in the K+ concentration on the retinal side of the tissue. It is shown, that the transport of potassium in the pigment epithelium depends on the K+ concentration on the retinal side of the tissue in such a way as to keep variations in this concentration at a minimum. PMID- 3002116 TI - A comparative analysis of the collagen type and distribution in the trabecular meshwork, sclera, lamina cribrosa and the optic nerve in the human eye. AB - A theory has been advanced (Tengroth et al 1984) that one common factor might be involved in the pathogenesis of chronic open angle glaucoma. The mechanical properties of the collagen could be one such factor. To characterize the collagen composition of the critical structures in cases with chronic open angle glaucoma i.e. the trabecular meshwork and the lamina cribrosa in the human eye, the following investigations were performed. Using immunoperoxidase technique and type specific antibodies to the genetically distinct collagen types I, III, IV and procollagen I along with the major non collagen proteins in the extracellular matrix laminin and fibronectin a light microscopic study was undertaken on the trabecular meshwork, cornea, sclera, lamina cribrosa and the optic nerve of the human eye. Furthermore biochemical analysis was performed on the collagen associated aminoacids hydroxyproline (Hyp), hydroxylysine (Hyl) and prolin (Pro) from microdissected samples of trabecular meshwork, sclera, lamina cribrosa and optic nerve. Both the immunohistological and the biochemical findings suggested similarities in the collagen composition between the trabecular meshwork and the lamina cribrosa. The immunohistochemical findings showed stronger staining of type III and IV in the trabecular meshwork and lamina cribrosa as compared to sclera or cornea while the opposite was true for type I and procollagen I. Fibronectin and laminin were present in both the trabecular meshwork and lamina cribrosa. These findings were in agreement with the amino acid analysis when the Hyp/Hyl ratio was calculated for each sample.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002117 TI - Photocoagulation of degeneratio maculae senilis. PMID- 3002118 TI - Senile macular degeneration. Results of laser treatment. A preliminary report. AB - A preliminary report on laser treatment of senile macular degeneration is presented. Over a one year period 234 patients with symptoms and signs of macular degeneration were examined with fluorescein angiography. 32 patients (14%) showed neovascular membranes accessible to treatment with blue/green argon laser. At the end of the observation period (mean 8.7 months) one half of the treated group had unchanged or better visual acuity, whereas the other half showed deterioration of the visual acuity. It is not known whether this result differs from the average spontaneous course. A controlled trial is necessary in order to evaluate the possible beneficial effect of laser treatment of senile macular degeneration in our population. PMID- 3002119 TI - Collagenase activity in middle ear effusions. AB - Collagenolytic enzyme activity has been demonstrated in middle ear effusions from patients suffering from otitis media with effusion. Collagenase activity was characterized using different biochemical techniques. Various chemical and physical parameters were titrated for optimal enzyme activity. Bivalent cations activated the enzyme with Ca2+ as the most potent activator. Chelators such as EDTA reduced the enzyme at low concentrations. The enzyme was found to have a higher specific activity in mucoid effusions than in serous and had characteristics similar to granulocyte derived collagenase from human leukocytes. Possible relationships between the presence of collagenase activity and tissue destruction in the middle ear after otitis media with effusion are discussed. The collagenase activity showed wide ranges within different categories of middle ear effusions. PMID- 3002120 TI - Comparative study of abnormality in glycogen storing capacity and other histochemical phenotypic changes in carcinogen-induced hepatocellular preneoplastic lesions in rats. AB - A sequential comparison was made between abnormal glycogen storage and other histochemical phenotypic changes in hepatocellular precancerous lesions (altered foci and neoplastic nodules) during various stages in the process of development of cancer in rat liver. N-2-fluorenylacetamide was fed to male rats for 8 weeks and groups of rats were killed at the end of carcinogen feeding and at 12 and 24 weeks on control diet. Foci rich in glycogen storage accounted for a majority of all foci over the course of experiment, while foci devoid of glycogen storage, which were absent at the end of carcinogen feeding, gradually increased in number during maintenance. Glycogen-deficient lesions that might appear to arise from glycogen-rich lesions displayed hyperbasophilia demonstrated by toluidine blue reaction, but often lacked gamma-glutamyl transpeptidase activity. Resistance to iron accumulation was consistently shown in all precursor lesions for hepatocellular carcinoma in the siderotic liver regardless of abundance or absence of cellular glycogen. It was suggested that properties such as loss of glycogen storing capacity, hyperbasophilia, and some cellular atypicality resembling those of carcinoma cells might be essential elements for malignant progression. PMID- 3002121 TI - [The pharmacological effect of ligustrazine on human platelets]. PMID- 3002122 TI - [Effect of human leukocyte interferon on Coxsackie B-2 virus-infected rat beating heart cells in culture]. PMID- 3002123 TI - [Cultured human hepatoma cells (BEL-7404) for anticancer drugs screening]. PMID- 3002124 TI - [Comparisons of effects of Hoe-498 and enalapril in isolated guinea pig hearts and rat myocardium]. PMID- 3002125 TI - Effect of glyceryltrinitrate and 8-Br-cGMP on tension and phosphorylase a activity in vascular smooth muscle. AB - The aim of the present study was to examine the effect of glyceryltrinitrate (GTN) and 8-Br-cGMP on tension and cytosolic calcium concentration in pre contracted bovine mesenteric arteries (BMA). The activity of glycogen phosphorylase a was used as a measure of the cytosolic calcium concentration. The activity of this enzyme is regulated by the cytosolic calcium concentration and/or cAMP. Since the cAMP level was not found to be affected by GTN-treatment, the use of phosphorylase a activity to monitor changes in the cytosolic calcium concentration can be justified. The vessels were contracted with phenylephrine (10 microM) or 100 mM K+-depolarization, which caused an increase in phosphorylase a activity. Addition of 1 microM GTN to the phenylephrine contracted vessels resulted in a 3-4-fold rise in intracellular cGMP level, which was accompanied by a large decrease in tension and phosphorylase a activity. The K+-depolarized vessels, on the other hand, were largely resistant to the relaxant action of GTN, and there was only a slight reduction of the phosphorylase a activity. In phenylephrine-contracted vessels, made tolerant to GTN by incubation at elevated pH in the presence of GTN (0.44 mM), no changes in tension and phosphorylase a activity were seen after stimulation with a test dose of GTN (1 microM). The cGMP response was also markedly blunted in the tolerant vessels. Relaxation of phenylephrine-contracted BMA induced by 8-Br-cGMP (0.5 mM) was also accompanied by a reduction in phosphorylase a activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002126 TI - Distribution of 64Cu in foetal and adult tissues in mice: influence of sodium diethyldithiocarbamate treatment. AB - 64Cu (as 64CuCl2) was given intravenously to male C57BL mice and to pregnant C57BL mice at various stages of gestation. The disposition of the 64Cu in the adult animals and in the foetuses was studied by autoradiography and gamma spectrometry. The effects of treatments with diethyldithiocarbamate (DEDTC) on the disposition of the 64Cu in the animals were also examined. In addition, the ability of Cu to affect chondrogenesis was studied in an embryonic limb bud culture system. The results showed a strong uptake of 64Cu in the liver of the adult animals at all intervals (5 min.-24 hrs). At short survival intervals, there was also an uptake in the kidney cortex, the gastrointestinal mucosa, the adrenal, the pancreas, and the erythrocytes. Exposure to Cu may cause liver and kidney injuries, which may be related to the strong accumulation in these organs. 64Cu passed the placenta to the foetuses at all stages of gestation, although this occurred at a relatively slow rate. Within the foetuses the highest concentrations were found in the liver. Cu was observed to be toxic in the chick limb bud mesenchymal spot culture system although at relatively high concentrations. Foetal malformations and embryotoxicity may therefore be interpreted as a result of direct action of Cu on embryonic structures, although placental and/or maternal influence cannot be excluded. Pre- or posttreatment of the animals with DEDTC, which is a chelating agent, caused a very marked increase in the concentration of 64Cu in most tissues of the adult animals and also an increased foetal uptake of the metal.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002127 TI - Actions of bronchodilator drugs, glucocorticoid, and their combinations on airways in rats and guinea pigs. AB - The principal aim of the present investigation was to examine in detail individual and combined actions on airways of four antiasthmatic drugs with different mechanisms of action. They were theophylline (THEO), the beta 2 adrenoceptor agonist terbutaline (TER), the antimuscarinic ipratropium bromide (IPRA), and the glucocorticoid betamethasone (BM). The respiratory measurements (intratracheal pressure in rats and lung resistance and dynamic lung compliance in guinea pigs) were performed in anaesthetized, mechanically ventilated animals. Mild, moderate, and submaximal obstruction of mainly large or small airways were induced by a cumulative administration of either methacholine (MeCh) or leukotriene D4 (LTD4), both i.v. The antiasthmatic drugs were given acutely i.v., but THEO and BM also as intraperitoneal pretreatments. Moreover, actions of the antiasthmatic drugs were studied ex vivo on lung beta-adrenoceptors in rats and guinea pigs. 3H-dihydroalprenolol was used as a ligand in the beta-receptor binding assay. A detailed evaluation of the lung resistance and dynamic lung compliance responses to MeCh and LTD4 suggested that MeCh induced obstruction in more proximal airways than LTD4. THEO attenuated both airway challenges, being about 2-4 times more efficient on LTD4 than on MeCh. TER inhibited both MeCh- and LTD4-induced airway obstruction about equally, whereas IPRA was effective on the MeCh-induced obstruction only. In most cases, treatment with THEO+TER or THEO+IPRA attenuated the MeCh-induced mild or moderate obstruction of large airways additively, and the submaximal airway response to MeCh synergistically. The relatively good effect of THEO on small airway obstruction caused by MeCh was not augmented by TER or IPRA. In contrast to these results, combination of subefficient doses of THEO and TER, but not of THEO and IPRA, resulted in an antagonistic interaction on the MeCh challenge in rats and guinea pigs. The combined action of THEO+TER was at its best additive on large and small airways obstruction caused by LTD4. Addition of IPRA to THEO treatment did not modify the effects on LTD4 challenge. Neither TER nor IPRA enhanced the cardiovascular effects of THEO in rats or guinea pigs. THEO administration to rats and guinea pigs resulted in plasma concentrations (7.5-32 micrograms/ml) that were roughly comparable to clinically relevant values. The respective lung tissue concentrations were 53-81% of the plasma values. In rats, but not in guinea pigs, combined treatment with THEO+TER increased the THEO concentration in lung tissue.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3002128 TI - Effects of halothane on the transducer and potential activated currents of the crustacean stretch receptor. AB - Halothane was applied to the stretch receptor neuron of the crayfish (Astacus astacus) and the effects on the transducer properties and the potential activated currents were studied with potential clamp technique using two microelectrodes. Exposure to halothane reduced the frequency of action potentials during stretch. This was shown to be due to effects both on the action potential generating currents and the transducer current. Halothane partially blocked the TTX sensitive fast inward current in a dose-dependent manner (Apparent KD = 3 mM). Halothane also reduced the outward current produced by a positive potential step. Both the fast and the slow component were affected, although the fast outward current seemed to be most sensitive. There was little or no change in the currents resulting from negative potential steps. The peak of the receptor potential and the receptor current were very little affected by halothane. The amplitude of the static phase of the receptor potential was reduced to a greater degree than the static phase of the receptor current (cells treated with TTX). A change in reversal potential of about--13 mV was observed for the peak and the static phase of the receptor current in four cells indicating an increased cord conductance for the transducer channel. PMID- 3002129 TI - Dual effects of adenosine and adenosine analogues on motor activity of the human fallopian tube. AB - Effects of adenosine and adenosine analogues on spontaneous contractility of the human fallopian tube during different phases of the menstrual cycle were studied. In isthmic preparations, a stimulatory effect by L-N6-phenylisopropyladenosine (L PIA), with preference for adenosine A1-receptors, was seen mainly during the proliferative phase. In ampullary preparations, stimulation by L-PIA was seen both in the secretory and proliferative phases. Adenosine and 2-chloroadenosine exerted similar stimulatory effects. 5'-N-ethylcarboxamide-adenosine (NECA), with selectivity for adenosine A2-receptors, or D-PIA never showed a stimulatory effect. At concentrations above those needed for stimulation, adenosine, 2 chloroadenosine and L-PIA inhibited spontaneous contractions, in common with NECA and D-PIA. Here, NECA was more potent than L-PIA. The D-PIA and L-PIA were equipotent. The inhibition was seen during the whole menstrual cycle. The competitive adenosine antagonist 8-p-sulphophenyltheophylline (PSoT) reversibly antagonized the stimulatory and inhibitory effects elicited by adenosine and the analogues. The PSoT alone could exert a stimulatory or an inhibitory action on spontaneous contractility. We suggest that adenosine can modulate contractile activity in the human fallopian tube via stimulatory A1-and inhibitory A2 receptors. These receptors are located on the smooth muscle cells, and might act via cAMP. The relative receptor dominance may be influenced by cyclic hormonal changes. PMID- 3002130 TI - ATP-induced cation influx in myotubes is additive to cholinergic agonist action. AB - Using biochemical methods, ATP was shown to induce an inward flux of 86Rb into cultured chick myotubes. A biphasic dose-response curve was observed, the first part of which was saturable and had an EC50 value of approximately 10 microM. beta, gamma-imido ATP inhibited the ATP-induced uptake. ADP, AMP and adenosine were less potent than ATP in producing influx. ATP-induced influx of 86Rb was found to be additive to carbachol-induced influx and, in contrast to the latter, not blocked by the nicotinic acetylcholine receptor antagonist alpha bungarotoxin. The results suggest the presence of an ATP recognition-site on myotubes, triggering ion-permeation. PMID- 3002131 TI - Effects of adrenaline infusion on cAMP and glycogen phosphorylase in fast-twitch and slow-twitch rat muscles. AB - This study investigated the effect of adrenaline infusion on the cAMP content, glycogen phosphorylase activity and the rate of glycogen breakdown in rat extensor digitorum longus (EDL) and soleus muscles. Adrenaline was constantly infused in a dose of 0.15 micrograms kg-1 body wt min-1. The cAMP content increased approximately 2.8-fold in both muscles after 2 min of infusion. Phosphorylase a + b activity was six times higher in fast-twitch muscle (EDL) than in slow-twitch (soleus) and remained unchanged during the infusion. Phosphorylase a activity increased by 8.4-fold in EDL and 2.4-fold in soleus muscles during the infusion period. Glycogen content decreased in EDL muscle by 10% whereas no change was observed in soleus. It is concluded that beta adrenergic stimulation by adrenaline results in a similar cAMP increase in both muscles. The low rate of glycogen breakdown in EDL and the unchanged content of glycogen in soleus muscle suggest that cAMP mediated transformation of phosphorylase b to a in itself is not adequate for a rapid glycogenolysis in muscle. PMID- 3002132 TI - Effects of electroconvulsive therapy on neuropsychological function and circulating levels of ACTH, cortisol, prolactin, and TSH in patients with major depressive illness. AB - Thirteen patients with major depressive illness received unilateral electroconvulsive therapy (ECT). Memory and some other neuropsychological functions were studied concomitantly with changes in clinical symptoms. ACTH in plasma and cortisol, prolactin (PRL) and TSH in serum were measured 30 min before and 1, 5, 15, 30 and 60 min after treatment. Memory functions, impaired after the ECT series, were completely regained 1 month later. ACTH, cortisol, PRL and TSH were significantly increased by ECT. The maximum hormone level after ECT was lower at the last ECT in the series as compared with the first. After the last treatment, nonverbal memory performance was negatively associated with the maximum ACTH level after ECT and verbal learning was negatively correlated to the maximum cortisol level. The reason for these relationships is not known. Since both the ACTH secretion and memory function may be dependent upon the intracerebral catecholamines, the present findings may reflect variations in central monoaminergic receptor function. PMID- 3002134 TI - Validity of certified causes of death in breast carcinoma patients. AB - The accuracy of the cause of death certification was surveyed in 802 breast carcinoma patients reported to the Swedish Cancer Registry from Stockholm county during 1961-1963. A total of 502 deaths occurred during the follow-up period of which 484 were eligible for the analysis. In 7 per cent the officially recorded underlying cause of death failed to accurately reflect the cancer status at death, which was mostly due to diagnostic errors or errors in the coding of the death certificate data. There seemed to be a net overestimation of intercurrent deaths in the official mortality statistics and a corresponding net underestimation of breast carcinoma deaths. Breast carcinoma was mentioned as a contributory cause of death in only 52 per cent of those certified to have died from intercurrent causes but who had recurrent disease at death. Among those with breast carcinoma as a certified contributory cause, 52 per cent did not have recurrent disease according to the clinical records. It is therefore concluded that official data on breast carcinoma as a contributory cause of death are of limited use in descriptive epidemiology. PMID- 3002133 TI - Proton relaxation in a multicompartmental system with exchange, simulating human tissue. AB - The proton magnetic resonance behaviour of human tissue was simulated with a numerical model in which the resonating nuclei were assumed to belong to five different compartments of defined relative sizes and with distinct relaxation properties. Effects of various proton exchange rates between the compartments were included in the model. The time evolution of longitudinal and transverse macroscopic magnetizations was calculated. Applications of this model in magnetic resonance imaging are discussed. PMID- 3002135 TI - Chemotherapy in advanced breast carcinoma. Comparison between doxorubicin cyclophosphamide and cyclophosphamide-methotrexate-5-fluorouracil-vincristine prednisone. AB - Two chemotherapeutic regimens were compared in 102 patients with metastatic breast carcinoma, randomized to either doxorubicin-cyclophosphamide (DC) or cyclophosphamide-methotrexate-5-fluorouracil-vincristine-prednisone (CMFVP). The objective response rates were 32 per cent in the former and 35 per cent in the latter group, the complete response rates 6.4 and 21.8 per cent, and mean duration of the remission 7.7 and 11.2 months, respectively. Most responders had multiple metastases and had received previous hormonal treatment. The DC regimen was found to be slightly more toxic than CMFVP. PMID- 3002136 TI - Temporary ischaemia of a large bowel segment induced by degradable starch microspheres in man. AB - Degradable starch microspheres were used to induce temporary ischaemia measured by electromagnetic flowmetry in colon segments during bowel resection. Severe reversible ischaemia free from unwanted side effects was induced in eight of nine patients. In one of the five patients where the tumour was included in the microsphere-embolised tissue volume no reduction in blood flow was seen. Segmental enteric ischaemia induced by degradable starch microspheres in man seems safe and may be useful for radioprotective purposes in clinical radiation therapy. PMID- 3002137 TI - The protective effect of intra-arterial vasopressin injections on the small bowel during fractionated abdominal irradiation. A study in pigs. AB - The radioprotective effect of vasopressin-induced intestinal ischemia was investigated in pigs. A bolus injection in the cranial mesenteric artery, of a vasopressin solution, 0.05 IU/kg body weight, was followed by irradiation of the small intestine with 6 MV roentgen rays. Three different fraction schedules were used. Weight changes in the animals and the histologic appearance of their intestines were recorded two weeks after irradiation and compared with the findings in untreated animals. Intra-arterial vasopressin was very effective in protecting the intestine. Vasopressin treatment given before 2 fractions with relatively high radiation doses in a 6-fraction regimen was so effective that it may form the basis of a treatment model applicable in man. PMID- 3002138 TI - Variation in the lung inhomogeneity correction factor with beam energy. Clinical implications. AB - In order to determine the magnitude of the dosimetry error introduced by failing to correct for increased transmission through lung tissue in treating thoracic malignancies, measurements in a phantom were taken using different field sizes, inhomogeneity thicknesses and photon qualities. The results indicate that the error introduced by neglecting the inhomogeneity correction is greatest at lower photon energies, smaller field sizes and greater thickness of inhomogeneity. Correction factors to account for the lung inhomogeneity were obtained from phantom measurements and were compared with those calculated using the tissue-air ratio and Batho-Young algorithms; correlation coefficients describing the relationship between measured and calculated values exceeded 0.995. The calculated values tended to overestimate the correction factor and differed most from the measured correction factors at lower energies, smaller field sizes, and greater inhomogeneity thicknesses. The importance of these results in clinical radiation therapy is discussed. PMID- 3002139 TI - Radiation sensitivity of human malignant lymphocytes. AB - A simple and rapid in vitro technique to assess the sensitivity of human malignant lymphocytes to roentgen irradiation is described. A variety of established malignant lymphocyte cell lines were cloned in microwells and clone survival was used as the end-point. The survival of the clonogenic malignant lymphocyte down to a fraction of approximately 0.001 could be measured accurately. Except for a T-cell line, the radiation sensitivities of the cell lines were similar to that of normal T-lymphocytes. PMID- 3002140 TI - Peripheral blood lymphocyte subsets in radiologists exposed to ionizing radiation. AB - To investigate whether low dose ionizing radiation caused perturbation of peripheral blood lymphocytes in radiology unit staff, the T-helper/suppressor ratio was examined in eight radiologists exposed to low dose radiation over a period of 6 to 27 years (mean 12 years). No significant difference was noted in the T-cell subsets between exposed radiologists and non-exposed control subjects. The effect of low dose ionizing radiation on peripheral blood lymphocyte subsets seems to be virtually negligible. Further, measurement of the T-helper/suppressor ratio is not a reliable way of demonstrating any damage to bone marrow caused by low dose ionizing radiation. PMID- 3002142 TI - Chemical modification of adrenal response to external whole body irradiation in mice. AB - Adult Swiss albino mice were exposed to whole body gamma irradiation with 1.5, 3.0, 6.0 and 9.0 Gy in the presence or absence of the protective drugs MPG and WR 2721. The changes in the total cell population, pyknotic nuclei and necrotic cells, and binucleate cells were observed at various post-irradiation times. The degree of damage increased with the radiation dose. The number of total cells decreased with a corresponding increase in pyknotic nuclei, necrotic cells and binucleate cells. Both drugs provided protection against radiation injury, and reduced early post-irradiation cell damage and enhanced recovery after sublethal exposures. After higher doses, especially after lethal doses, the drugs became less effective in protecting the tissue. PMID- 3002141 TI - Protection against radiation induced chromosome injury by sulfhydryl compounds. AB - The radiation induced changes in a critically radiation-sensitive tissue, bone marrow of Swiss albino mice and its modification by two sulfhydryl compounds, MPG and WR-2721, were studied cytogenetically after whole body exposure to 0.5, 1.5, 3.0, 4.5 and 6.0 Gy of 60Co gamma radiation. The changes in the structural arrangements of chromosomes in the bone marrow were studied at various post irradiation times from 1 to 28 days. Both the control (irradiated) and experimental (drug + irradiation) animals showed qualitatively similar types of aberrations; the severity of lesions increased with the radiation dose. The maximum damage in all the groups was seen on day 1, post-irradiation. Both the drugs afforded better protection at lower doses of exposure, their effectiveness decreasing with an increasing radiation dose. Of the two drugs, WR-2721 was more effective than MPG against initial aberration yield but the former, at the present drug dose, showed some toxic effect at the later post-irradiation intervals (2 weeks onward) as manifested by an increase in the chromatid breaks and polyploidy. PMID- 3002143 TI - Dose-response studies of single dose ionizing radiation on the ciliated epithelium of the trachea of the rabbit. A physiologic and ultrastructural investigation. AB - The effects of ionizing radiation on the ciliated epithelium of the rabbit's trachea were studied by ultrastructural and physiologic examinations during 10 days after single doses of 2 to 30 Gy. Low doses (2 and 2.5 Gy) caused during the first days after irradiation increased ciliary activity which during the following days returned to normal. Ultrastructurally blebs were observed on the cilia. Five and 10 Gy caused slight increment of the ciliary activity, also more marked structural effects with swollen ciliary tips, bent tips and some cilia broken and adhering to each other. Larger single doses (15-30 Gy) caused reduced and irregular ciliary activity; ultrastructurally, most cilia were broken and adhered to each other, but some cilia seemed to be unaffected. The goblet cells increased in number already after 2 and 2.5 Gy. Mucinous granula could be observed on the ciliary surface and after 30 Gy there was an explosive emptying of the goblet cells. PMID- 3002144 TI - Response of rat brain tissue for the extraction of 125I antipyrine after single and fractionated roentgen ray doses. A comparison of fractionation models applied to the central nervous system. AB - Changes in the cerebral blood flow in rats were studied 9 months after irradiation with single and fractionated doses of 250 kV roentgen rays. The single doses used were 11.9, 14.5, 17.0, 19.6 and 22.1 Gy. Corresponding fractionated doses (FD) were calculated by the formula: FD = single dose X N0.42, where N is the number of fractions. The fractionated doses were given as 4, 7 and 10 dose fractions in the same overall treatment time of 3 weeks. The blood flow changes were estimated by the 125I antipyrine extraction technique. In the non irradiated control group the extraction in the brain was 0.93 per cent of the injected dose of 125I antipyrine per g of tissue. In the irradiated rats the corresponding extraction was on average 20.4 per cent higher than that in the control group. The extraction was significantly increased in 7 of the 20 irradiated groups of animals. The fractionation model used in these experimental studies is compared with other published fractionation models for radiation tolerance of the rat and human brain and spinal cord. PMID- 3002145 TI - Effects of irradiation and inhibition of adenosine diphosphate ribosyl transferase in radiation sensitive and resistant mouse lymphoma (L5178Y) cells. AB - The effects of treatment with benzamide, an inhibitor of adenosine diphosphate (ADP) ribosyl transferase, were studied in two strains of L5178Y lymphoma cells of differential sensitivity to ionizing radiation. Continuous 2 mmol/l benzamide treatment enhanced the killing effect of roentgen irradiation in the sensitive cells, but not in the resistent cells. Three hours' treatment with 2 to 15 mmol/l benzamide sensitized irradiated cells of both strains to a similar extent. Rejoining of DNA breaks was delayed by 2 mmol/l benzamide in sensitive cells more than in resistant cells. PMID- 3002146 TI - Effects of dietary supplementation with selenomethionine on the teratogenic effect of ionizing radiation in mice. AB - Female C3H mice were fed a standard pellet diet (containing 0.2 ppm Se and 30 ppm vit. E) or the same diet supplemented with 0.8 ppm (low dose) or 3.4 ppm (high dose) of selenomethionine for 10 weeks. After mating with males receiving the standard diet the mice were subjected, on the 9th day of pregnancy, to whole body roentgen irradiation of 1.75 Gy. On day 18 of gestation the frequency of resorptions, mortality and the incidence of fetal malformations were studied. Supplementation with Se-methionine resulted in a significant but dose-independent decrease (p less than 0.005) of the number of malformed fetuses from 62 per cent in the irradiated controls to 47 per cent in the low Se-group and high Se-group, respectively. In addition, the number of total malformations as well as fetal resorptions were significantly decreased in a dose-independent manner in the supplemented groups. The decrease in fetal malformations occurred proportionally for all the major malformations observed, i.e. short or kinked tail, rib and vertebral malformations, coloboma and deformation of retina and iris. Glutathione peroxidase activity in whole blood of Se-methionine fed mice was significantly increased. Thus, Se-rich diet may result in scavenaging of radiation-induced hydroperoxides. PMID- 3002147 TI - Primary cytomegalovirus infection following open heart surgery. AB - Among 674 patients undergoing open heart surgery in 1981-82, 86 (13%) were cytomegalovirus (CMV) antibody-negative when tested by an enzyme-linked immunosorbent assay prior to operation. At follow-up, 54 (67%) of 80 patients restudied had seroconverted after the operation, and 35 of the 54 seroconvertants had been ill with fever and elevated liver enzymes. Among the latter 35 patients, 26 demonstrated a significant rise in CMV antibody titre, most often detected in the third week following the onset of illness. The older patients were more susceptible to illness and seroconversion, and there was a positive correlation between age and the number of blood units given. Thus, at least one third of the seronegative patients developed symptomatic CMV illness after open heart surgery. This is a much higher incidence than earlier reported. PMID- 3002148 TI - Studies of SV40-infected Werner syndrome fibroblasts. AB - Skin fibroblasts from patients with the Werner syndrome (WS) and fibroblasts from normal donors were infected with SV40 of a wild type strain (777) or of a temperature-sensitive mutant strain (tsA900). The infected WS cells and the infected normal cells proliferated at permissive temperatures, and then entered into "crisis" in which state they ceased proliferation. In their proliferative phase, both WS and normal cells infected with tsA900 proliferated rapidly at 34 degrees C, but their proliferation slowed down at 39 degrees C. Some lines of infected WS cultures accrued more population doublings before they entered into "crisis" than uninfected WS cells. Among 13 series of infected cultures, one line, which had been infected with 777, passed through "crisis" and proliferated indefinitely. This line (PSV811) was from a 29-year-old female WS patient. We compared a cloned line of PSV811 with a permanent line of SV40-infected WI-38 normal human fibroblasts (VA13 2RA). Cells of both lines, PSV811 and VA13, were epithelioid, did not shed viruses into the culture medium, and their chromosome number distributed broadly across the diploid range. An autoradiographic study suggested that both lines are capable of unscheduled DNA synthesis. In both cultures, the rate of proliferation depended on the concentration of fetal bovine serum in culture medium. However, the PSV811 cells grew slower, and produced colonies with fewer cells than did VA13 cells. The frequency of sister chromatid exchange was about 70% higher in PSV811 than in VA13. In an adjunct paper, Murata and others report that the percentage of hyaluronic acid in total acidic glycosaminoglycans released into culture medium was larger in PSV811 than in VA13. We concluded from these results that PSV811 is not a contaminant of VA13, that an SV40 infection partly modify the proliferative characteristics of WS cells, and that SV40-infected WS cells and permanent lines obtained therefrom, are useful in furthering the study of WS. PMID- 3002149 TI - Experimental studies on Werner's syndrome fibroblasts. PMID- 3002150 TI - Cellular mechanisms of aging in the Werner syndrome. PMID- 3002151 TI - Extrachromosomal circular DNA and aging cells. AB - A DNA sequence situated in the human genome between Alu-repeat clusters ("Inter Alu" DNA) is progressively amplified in extrachromosomal DNA, including covalently closed DNA circles, during serial passage of diploid fibroblasts. A single size-class of Inter-Alu circles is also amplified in lymphocytes from 16 of 24 old donors and yet is not detected in cells from 18 young donors. PMID- 3002152 TI - Acidic glycosaminoglycans of SV40-transformed Werner's syndrome cells. AB - Acidic glycosaminoglycans (AGAGs) were isolated from the culture fluids of a SV40 transformed permanent line (PSV811) of the Werner syndrome skin cells and of a SV40-transformed permanent line (VA13) of WI-38 normal lung cells. The relative composition of component AGAGs was estimated by acetate membrane electrophoresis followed by Alcian blue staining and desitometry. Hyaluronic acid (HA) was the major AGAG component in all the preparations tested. The relative content of HA fluctuated considerably among the preparations obtained in independent experiments and among the results using three different buffer systems. However, the proportion of HA was persistently greater in PSV811 culture fluid than in VA13 culture fluid. VA13 cells released an appreciable amount of heparan sulfate as the second major AGAG component, but PSV811 released only a small amount. These results show a difference between the two cell lines, and suggest that PSV811 may possibly reflect the association of the Werner syndrome with abnormal HA metabolism. PMID- 3002153 TI - Glycosaminoglycan synthesis in untransformed and transformed Werner syndrome fibroblasts: a preliminary report. AB - Glycosaminoglycan (GAG) synthesis was studied in untransformed and transformed normal and Werner syndrome (WS) fibroblasts, because WS manifests pleiotropic abnormalities in connective tissue. Continuous labelling of cells with [3H] glucosamine and [35S] sulfate for 48 hours revealed enhanced synthesis of cellular GAG, more rapid transfer of these into the pericellular fraction, and more accumulation of GAG in the medium in cultures of untransformed WS fibroblasts compared with cultures of normal diploid cells. Total GAG in the 24 hour medium from confluent cultures was composed of 80-90% hyaluronic acid (HA) and 10-20% sulfated GAG (S-GAGs) in both untransformed normal and WS fibroblasts, whereas it was approximately 50% each HA and S-GAG in transformed normal and WS cells. The proportional enhancement of [35S] GAG synthesis in response to exogenous beta-D-xylopyranosides was similar in normal and WS cells, although transformed cells demonstrated only approximately one-half the enhancement observed in non-transformed cells. Thus, the overall activity of GAG synthesis is not grossly altered by the WS gene mutation. Enhanced synthesis and accumulation of HA and dermatan sulfate (DS) in the medium was characteristic of untransformed WS fibroblasts, but appeared to be normalized in an SV40-transformed WS cell line (PSV811), as in transformed normal cells (WI38CT-1). We need more experiments to determine whether aberrant GAG metabolism in WS cells is a direct or indirect expression of the primary gene defect. PMID- 3002154 TI - Viral hepatitis: implications to pediatric practice. AB - A major frontier is the development of effective therapy for patients with chronic type B hepatitis, which currently remains refractory to all known antiviral agents and is associated with severe long-term consequences. Future research should focus on the relationship of HBV to hepatocellular carcinogenesis. This might answer the question as to when integration of viral DNA into the host cell occurs and whether this is an irreversible step in the progression toward hepatocellular carcinoma. The exciting work regarding vaccines against human hepatitis will continue in view of the worldwide impact of elimination of these viruses. Chemically synthesized vaccines may be safer, cheaper, and more effective, and also produce more persistent protective immunity. This technology requires determination of immunogenic protein molecules, identification and in vivo synthesis of the amino acid sequences important for cellular recognition, and the application of recombinant DNA technology to clone and propagate the critical antigenic determinants by incorporation into other hosts such as vaccinia. The potential of newer forms of hepatitis B vaccine has been demonstrated by the study of Scolnick et al., in which HBsAg, produced by a recombinant strain of yeast, was given to 37 healthy low-risk volunteers as a means of vaccination. The dose and schedule were the same as that used for the serum HBsAg-derived vaccine. The results were encouraging in that an 80%-100% anti-HBs positivity was documented at three months in these individuals. These experiences also suggest that a similar sequence of events, namely definition, elucidation of the molecular biology, and development of immunologic prevention of non-A, non-B viruses is at hand. PMID- 3002156 TI - Prostacyclin acts in rat gastric (fundic) mucosa via the intracellular 'second messenger system'. AB - Using a specific radioimmunoassay technique it seems that Prostacyclin (PG-I2) has a strong effect on the cyclic nucleotide content of the rat gastric (fundic) mucosa. 1 min after an intragastric application of 100 micrograms/kg PG-I2 the cAMP-content and in the 5th min the cGMP-content showed a highly significant decrease. It seems that the basic mechanism of action of PG-I2 is a typical 'hit and run' effect, acting on the intracellular 'second messenger system'. PMID- 3002155 TI - Hypocalcemia from deficiency of and resistance to parathyroid hormone. PMID- 3002158 TI - [Calpain and calpastatin in pig retina]. PMID- 3002159 TI - [Acquired immunodeficiency syndrome (AIDS) and stomatology]. PMID- 3002157 TI - Studies on the effects of cyclo-oxygenase and lipoxygenase inhibitors on the macrophage stimulated synthesis of collagenase by rabbit chondrocytes. AB - Recent work from this laboratory has shown that macrophages in culture synthesize and secrete a soluble factor(s) that induces the synthesis of collagenase in primary cultures of rabbit chondrocytes (Arth. Rheum. 23, 448, 1980). The current studies were undertaken to determine the role of arachidonate metabolism in this process. Incubation of chondrocytes with MCM (Macrophage Conditioned Medium) and low doses of indomethacin (1-10 microM) had no effect on collagenase synthesis. The lipoxygenase inhibitor NDGA, indomethacin at high doses (50 microM), diethylcarbamazine and the phospholipase inhibitor dibromoacetophenone, inhibited the MCM dependent synthesis of collagenase in chondrocytes. These inhibitors did not affect collagenase activity nor did they interfere with the activation of latent collagenase. Our data indicate that although cyclooxygenase plays no role in the MCM dependent induction of collagenase in chondrocytes, lipoxygenase activity may be essential. PMID- 3002160 TI - [Renal function study by 99mTc-DMSA renal scintigraphy in each treatment of staghorn calculi]. AB - From 1976 through 1984, 94 staghorn calculi of 86 patients were treated in this department. Kidney function was assessed by Tc-DMSA renal scintigraphy consisting of renal cortical imaging and DMSA renal uptake rate, in 84 kidneys preoperatively and 43 kidneys pre- and postoperatively. There was an increase in the postoperative DMSA renal uptake in the operated kidney, in 3 out of 14 kidneys in which pyelolithotomy was performed and in one out of 10 kidneys in which nephrolithotomy was done. It was still impossible to answer the question of which mode of operation should be chosen only from consideration of kidney function study. But it was suggested by the statistical investigation that nephrectomy seemed to be selected in the case of severely decreased renal function. It was reasonable that pyelolithotomy was the best method from the point of predicting the postoperative recovery of renal function. But in the near future, advances in endoscopical stone surgery and extracorporeal procedures, might reduce the damage of the renal function caused by conventional stone surgery. PMID- 3002161 TI - [Condyloma acuminatum of the urethral in a child: a case report and review of the literature]. AB - A rare case of condyloma acuminatum of the urethral meatus in a male child is reported. The patient was five years old and developed a warty mass on the glans penis. He underwent successful resection with a Nd-YAG laser. Excised specimen showed typical characteristics of condyloma acuminatum on histology. Although the disease is thought to develop by a contact infection of human papilloma virus (HPV), the present case had no history suggesting this pathogenesis. At 4 months after operation, no disease recurrence was seen. A short review of the disease found in younger persons is also presented. PMID- 3002162 TI - Effects of sugars on indices of glucose tolerance in humans. AB - Ten men and nine women were studied to determine whether replacement of utilizable complex carbohydrate by sugars (mono- and disaccharides) in a high fiber, low-saturated fat diet would affect indices of glucose tolerance. Diets differed in that the 50% of calories derived from carbohydrate was either 35% complex and 15% sugars (low-sugar) on 15% complex and 35% sugars (high-sugar). Summation of glucose responses 30-180 min following an oral glucose tolerance test was significantly higher in men, but not women, after they consumed the high sugar diet. Corresponding insulin responses were significantly higher in men consuming the high-sugar compared to the low-sugar diet. Insulin binding was significantly lower during the base line period and after the high-sugar diet compared to the low-sugar diet. Results indicate that sugars adversely affect indices of glucose tolerance when they replace complex carbohydrates even in a high-fiber, low-saturated fat diet. PMID- 3002163 TI - To what extent does increased dietary fiber improve glucose and lipid metabolism in patients with noninsulin-dependent diabetes mellitus (NIDDM)? AB - The present study assesses the impact of variations in the amount of fiber in high carbohydrate diets on carbohydrate and lipid metabolism in NIDDM. The amount and source of carbohydrate, and source of dietary fiber, were held constant. Two 4-wk diet periods were randomly assigned and all subjects completed both dietary periods. Diets were identical in the proportion of carbohydrate, fat, protein, P/S ratio, and cholesterol. The normal fiber diet contained 11 g/1000 kcal, while the high fiber diet contained 27 g/1000 kcal. The results showed no significant difference in fasting plasma glucose and insulin, day-long glucose and insulin, fasting hemoglobin AIc, or 24 h urinary glucose. Fasting plasma triglyceride and VLDL-triglyceride, as well as fasting plasma cholesterol, LDL-cholesterol, and HDL-cholesterol were also unchanged. In conclusion, an increase in the fiber content from 11 to 27 g/1000 kcal did not lead to measurable improvements in overall plasma glucose, insulin, or lipid metabolism. PMID- 3002164 TI - Metabolic effect of pre-cooked instant preparations of bean and potato in normal and in diabetic subjects. AB - The effect of a 50 g starch meal prepared with pre-cooked instant bean flakes on glucose and insulin plasma levels and on glucose oxidation rate as measured by continuous indirect calorimetry was assessed in six healthy human volunteers during 4 h following the meal. Comparison was done with the same load of starch as potato flakes to which the fiber and protein content of the bean meal had been added. Thirty minutes after ingestion, plasma glucose and insulin rise was less after beans than after potatoes (0.4 +/- 0.2 vs 2.0 +/- 0.3 mmol . l-1), p less than 0.01, and 8 +/- 2 vs 38 +/- 5 microU . ml-1, p less than 0.001). The elevation of glucose oxidation was markedly less during the 2 h following the ingestion of beans when compared to potatoes. The bean flakes meal gave rise to only moderate elevation of plasma glucose and insulin levels and of glucose oxidation rate when given to four noninsulin-dependent-diabetes mellitus patients. It is suggested that the slow carbohydrate property of legumes is not due to their fiber content, but might be related to the histological structure of the seed. PMID- 3002165 TI - Phase II evaluation of aclacinomycin-A in patients with adenocarcinoma and large cell carcinoma of the lung. AB - Aclacinomycin-A (ACLA-A), the new anthracycline antibiotic that produces substantially less cardiotoxicity relative to doxorubicin, was evaluated in a phase II trial for advanced large cell and adenocarcinoma of the lung patients. Twenty-three patients with measurable disease were entered into the trial and received ACLA-A in doses of a weekly infusion of 65 mg/m2 and 85 mg/m2. Eighteen patients were evaluable for response and toxicity. Two patients were evaluable for toxicity only, one died before completion of a full course of therapy, and two did not receive the drug. There were no complete or partial remissions in this study. Three patients had disease stabilization for a median of 10 weeks (range 6-17). Toxicity was mainly hematologic. Nausea and vomiting were moderate. ACLA-A, in the dose schedules used, appears to have no activity in large cell and adenocarcinoma of the lung. PMID- 3002166 TI - Relapsed Wilms' tumor. Factors affecting survival and cure. AB - To identify factors contributing to extended survival among patients with relapsed Wilms' tumor, we assessed 10 clinical and biologic variables thought to have predictive value. With a median follow-up of 6 years, 32 (20%) of 156 patients who achieved complete remission have relapsed. Twenty-four have died with recurrent tumor, and eight are surviving for 2 to 12 years from diagnosis. Only time to relapse, or length of initial complete remission, had a significant influence on survival. Of 11 patients with complete remissions lasting longer than 12 months, six have died--compared with seven of 10 having remissions of 6 to 12 months and 11 of 11 with shorter remissions (p = 0.014). Surgery alone was the curative therapy in three of the eight surviving patients. Until more effective chemotherapy regimens are developed, an aggressive surgical approach may be indicated in selected patients with relapsed Wilms' tumor. PMID- 3002167 TI - A cross-over comparison of nabilone and prochlorperazine for emesis induced by cancer chemotherapy. AB - An anti-emetic drug, nabilone, a synthetic cannabinoid, has been compared with prochlorperazine in 24 lung cancer patients receiving cancer chemotherapy. Each of the drugs studied was given orally every 12 hours, starting the night before chemotherapy, during one of two consecutive identical chemotherapy cycles in accordance with a double-blind cross-over random order assignment. Single doses were 2 mg of nabilone, or 15 mg of prochlorperazine. The chemotherapeutic regimens given included the following drugs in various combinations: cis platinum, vincristine, cyclophosphamide, adriamycin, vindesine, and etoposide (VP16). Nabilone was significantly superior to prochlorperazine in the reduction of vomiting episodes. Side effects, mainly vertigo, were evident in nearly half of the patients after nabilone, and three patients were withdrawn from the study due to decreased coordination and hallucinations after nabilone. Side effects from prochlorperazine were limited to mild drowsiness in one patient. Two-thirds of the patients preferred nabilone to prochlorperazine. We conclude that nabilone is a moderately effective anti-emetic drug, but that the unpredictability of its side effects call for careful patient information, especially with elderly outpatients. We recommend that at least after the first dose of nabilone, the patient should be kept under close observation during 4 hours. PMID- 3002168 TI - Multiagent chemotherapy, prophylactic neuraxis irradiation, and consolidative irradiation for small cell carcinoma of the lung. AB - Between 6/81 and 6/83, 73 patients with small cell carcinoma of the lung were treated according to a prospective protocol in which cyclophosphamide, doxorubicin, and vincristine (CAV) were given concurrently with prophylactic craniocervical irradiation to the level of C5. Both limited and extensive disease patients with normal computed tomography of the brain received 25 Gy in 10 fractions in 2 weeks. Complete responders to CAV received consolidative thoracic irradiation (CTI) to the local-regional primary (37.5 Gy in 15 fractions in 3 weeks), the first 25 Gy in 10 fractions serving as prophylaxis of the C6 to T12 spinal cord. The neuraxis from L1 to S2 then received 25 Gy in 10 fractions in 2 weeks. Consolidative irradiation of localizable metastatic sites was given in extensive disease patients. Partial and nonresponders to CAV received 50-60 Gy in 5-6 weeks to local-regional disease. With a median followup of 29 months, survival was significantly better (p less than .01) in patients receiving CTI to the chest after complete response to CAV (both limited disease and extensive disease) than without CTI. Of 41 patients completing the protocol and without central nervous system (CNS) involvement at presentation, four (9%) failed initially in the CNS (two brain, two spinal axis); CNS failure was the cause of death in all four patients with no other sites of metastases at death in two of these. Failure to complete protocol treatment was due to disease progression during chemotherapy in 25/73 (34%) and chemotherapy related complications (three sepsis, one gastrointestinal bleed) in four of 73 (5.5%) patients. CTI and prophylactic neuraxis irradiation did not increase morbidity or result in mortality in the sequence utilized; prophylactic neuraxis irradiation appears to reduce the CNS relapse rate, and CTI benefits survival. PMID- 3002170 TI - Cholangiocarcinoma associated with liver fluke infection: a preventable source of morbidity in Asian immigrants. AB - In the Far East infection with the liver flukes Clonorchis sinensis and Opisthorchis viverrini is the most frequently documented cause of cholangiocarcinoma. Liver fluke infection in the United States remains a health problem for more than 500,000 Southeast Asian refugees who have immigrated to this country since 1975. Recent surveys have revealed that up to 26% of Asian immigrants have an active liver fluke infection. However, the common clinical manifestations of this condition, as well as the possibility of developing such long-term sequelae as cholangiocarcinoma, remain unknown to many physicians providing care for this population. This report describes a clinically unsuspected C. sinensis infection associated with cholangiocarcinoma in an elderly Chinese immigrant, and emphasizes the importance of early diagnosis and treatment of all liver fluke infections in the prevention of bile duct neoplasms in high risk populations. PMID- 3002169 TI - Parainfluenza virus bronchiolitis. Epidemiology and pathogenesis. AB - An investigation of the epidemiology and pathogenesis of bronchiolitis due to parainfluenza virus (PV) was carried out. Bronchiolitis due to PV occurred most commonly in non-Caucasian males. Breast-fed infants exhibited a reduced risk of developing bronchiolitis. Once an episode of PV bronchiolitis occurred, both exposure to cigarette smoke and bottle feeding were associated with an increased frequency of recurrent wheezing, and subsequent infection with respiratory virus almost uniformly resulted in wheezing. Cell-mediated immune responses to PV antigen and titers of PV-specific IgE were greater among patients with bronchiolitis than among patients with upper respiratory tract infection. The epidemiology and pathogenesis of bronchiolitis due to PV is similar to that of respiratory syncytial virus. Lower respiratory tract infection may predispose to episodes of bronchoconstriction on subsequent exposure to cigarette smoke or other viral infections. PMID- 3002171 TI - Regional localization of the human transferrin receptor gene to 3q26.2----qter. AB - Transport of iron across the cell membrane is mediated by the iron-binding serum protein, transferrin, and its cell-surface receptor. Transferrin receptor is required for cell proliferation and may play a functional role in the pathogenesis of iron-storage disorders and some neoplasias. To better understand the possible involvement of transferrin receptor in such disorders, we have determined the chromosomal locus of the receptor gene by in situ hybridization. The human transferrin receptor gene was thus mapped to 3q26.2----qter, a region of chromosome 3 that appears to be involved in metal transport and that is subject to nonrandom structural rearrangements associated with neoplasia. PMID- 3002174 TI - Compatibility and availability of sodium bicarbonate in total parenteral nutrient solutions. PMID- 3002172 TI - A multigene deletion within the immunoglobulin heavy-chain region. AB - The immunoglobulin heavy-chain genes are located in a cluster on chromosome 14. The simultaneous absence of the human IgG1, IgG2, IgG4, and IgA1 subclasses was previously reported in a healthy Tunisian Berber and was later shown to be due to a multigene deletion. We now describe a serological and molecular study of a different deletion observed in a healthy Tunisian. Blot hybridization analysis of the proband's DNA using gamma, epsilon, alpha, and mu switch probes showed that the deletion involves a large region of the immunoglobulin heavy-chain gene cluster: C psi epsilon, C alpha 1, C psi gamma, C gamma 2, and C gamma 4. Incidentally, we showed that the restriction enzyme EcoRI alone can be used with the alpha probe to differentiate A2m types. The deletion described, present in a person homozygous for GM-Am haplotypes (Gm1,17;..;5,14,11,13,10 A2m2), is consistent with previous location, by association analysis, of C psi gamma between C alpha 1 and C gamma 2. There is evidence to suggest that deletions may be more common in the Mediterranean region than in North American Caucasians. PMID- 3002173 TI - Thymidylate synthase-deficient Chinese hamster cells: a selection system for human chromosome 18 and experimental system for the study of thymidylate synthase regulation and fragile X expression. AB - Chinese hamster lung (CHL) V79 cells already deficient in hypoxanthine phosphoribosyltransferase were exposed to uv light and selected for mutations causing deficiency of thymidylate synthase (TS) by their resistance to aminopterin in the presence of thymidine and limiting amounts of methyl tetrahydrofolate. Three of seven colonies chosen for initial study were shown to be thymidylate synthase deficient (TS-) by enzyme assay, thymidine auxotrophy, and their inability to incorporate labeled deoxyuridine into their DNA in vivo. Complementation analysis of human X TS- hamster hybrids revealed that TS activity segregated with human chromosome 18. Southern analysis of a panel of 14 human X hamster hybrids probed with complementary DNA from mouse TS confirmed the chromosome assignment of TS to human chromosome 18; quantitative Southern blotting using unbalanced human cell lines further localized the gene to 18q21.31 ---qter. Another hybrid was generated that contained a human X chromosome with the Xq28 folate-dependent fragile site as its only human chromosome in a hamster TS- background. The fragile site could be easily and reproducibly expressed in this hybrid without the use of antimetabolites simply by removing exogenous thymidine from the medium. These TS-deficient cells are useful for: somatic cell genetics as a unique selectable marker for human chromosome 18, studies on regulation of the TS gene, and analysis of the fragile (X) chromosome and other folate-dependent fragile sites. PMID- 3002175 TI - Effects of methylxanthines on airway mucociliary function. AB - Mucociliary interaction and hence mucus clearance in the airways is governed by ciliary activity and the depth and rheologic properties of periciliary fluid and mucus. Therefore, a defect in one or more of these component functions must be responsible for the impairment of mucociliary clearance in patients with a variety of airway diseases. Methylxanthines stimulate ciliary beat frequency, augment net ion secretion with a substantial increase in water flux toward the lumen, and promote mucus secretion in the lower airways. The net result is an enhancement of mucociliary clearance. This beneficial action of methylxanthines on mucociliary function may complement their effects on bronchoconstriction and respiratory muscle dysfunction in patients with chronic bronchitis, cystic fibrosis, and bronchial asthma. PMID- 3002176 TI - The mast cell and theophylline in asthma. AB - Mast cells are central to the development of bronchial inflammation and thus to bronchial hyperreactivity, the cardinal feature of asthma. Inflammation is due to the concerted action of mast cell-dependent vasoactive/spasmogenic mediators, chemotactic factors, and enzymes. Adenosine, a newly synthesized mast cell mediator (from adenosine triphosphate), is one of the important inflammatory mediators capable of causing bronchospasm and, by interacting with mast cell membrane receptors, of augmenting mediator release induced by antigen. These inflammatory and pro-asthmatic actions of adenosine can be inhibited by concentrations of theophylline achievable in humans that are insufficient to alter cyclic adenosine monophosphate metabolism. Thus, a new therapeutic consideration in the use of xanthine drugs is their ability to inhibit adenosine binding to cell surface receptors and thereby inhibit the effects of this purine nucleoside. PMID- 3002177 TI - Treatment of cytomegalovirus retinitis with dihydroxy propoxymethyl guanine. AB - Eight patients with cytomegalovirus retinitis in one or both eyes were treated with 9-(1,3 dihydroxy 2-propoxymethyl) guanine. Of the 14 eyes with retinitis, eight demonstrated more than 90% resolution, four had partial improvement, one failed to respond, and one could not be evaluated. Two of the eight patients had chemotherapy-induced immunosuppression and had ocular remissions for at least several months. The remaining six patients had AIDS or probable AIDS and relapsed in five weeks or less after discontinuation of therapy. Dihydroxy propoxymethyl guanine appears to be effective biologically in treating human cytomegalovirus retinitis without the development of unacceptable side effects but a single course of therapy is not capable of eradicating the virus. PMID- 3002179 TI - Vasoactive intestinal peptide inhibits insulin-stimulated glucose transport in rat adipocytes. AB - Patients with tumors secreting vasoactive intestinal peptide (VIP) often develop hyperglycemia and glucose intolerance. Although VIP has been reported to increase glucose output by the liver, the concentration required for this effect greatly exceeds that observed clinically. We therefore investigated the effects of VIP on insulin-stimulated glucose transport in isolated adipocytes. Inhibition of insulin action was observed at a concentration of 1 ng/ml VIP with half-maximal inhibition at approximately 20 ng/ml. 125I-VIP bound to specific high-affinity sites on the adipocytes. Fifty percent inhibition of binding occurred at a concentration of unlabeled VIP of approximately 10 ng/ml and was not affected by insulin, glucagon, or growth hormone. As we have observed previously with glucagon and catecholamines, inhibition of insulin action by VIP was observed only when accumulation of adenosine in the incubation medium was prevented by addition of adenosine deaminase. Under these conditions VIP markedly increased cellular cAMP levels. A good correlation was observed among VIP binding, inhibition of insulin-stimulated glucose transport, and cellular concentrations of cAMP. The results suggest that inhibition of insulin action in adipose tissue contributes to the hyperglycemic effect of VIP. Together, with our published findings on glucagon and catecholamines, these results support the hypothesis that counterregulatory hormones inhibit insulin action by increasing cellular concentrations of cAMP. PMID- 3002180 TI - Renal cAMP and 1,25(OH)2D3 synthesis in estrogen-treated chick embryos and hens. AB - Onset of sexual maturity in female chickens or administration of estrogen to mature males or to juveniles of either sex results in increased parathyroid hormone (PTH)-dependent adenylate cyclase activity and increased 25 hydroxyvitamin D3-1-hydroxylase activity in kidney. The relationship between estrogen-mediated alterations of these two enzyme systems was investigated in embryonic and mature, egg-laying chickens treated in vivo with 17 beta-estradiol (E2). Basal and PTH- and forskolin-stimulated adenylate cyclase activity in kidney plasma membrane preparations was not affected by E2 treatment of 19-day old chick embryos or of 41-wk-old egg-laying females. High, possibly maximal, levels of catalytic activity in control embryos and hens may have precluded further stimulation by E2. In contrast, E2 significantly enhanced 25 hydroxyvitamin D3-1-hydroxylase activity of embryonic kidney up to 10-fold (P less than 0.005). In mature females, E2 caused cessation of egg laying accompanied by a significant reduction (P less than 0.005) of 25-hydroxyvitamin D3-1-hydroxylase activity. These results indicate that the PTH-dependent adenylate cyclase and the 25-hydroxyvitamin D3-1-hydroxylase systems of avian kidney can be regulated independently and suggest that factors in addition to estrogen are involved in their regulation. PMID- 3002181 TI - Pituitary and adrenal hormone responses to naloxone in euhydrated and dehydrated dogs. AB - To assess the role of endogenous opioids in the secretion of pituitary and adrenal hormones, we injected intravenously the antagonist naloxone (1 mg/kg) into six dogs, euhydrated or dehydrated. Plasma renin activity (PRA), osmolality, and concentrations of adrenocorticotropic hormone (ACTH), cortisol, aldosterone, vasopressin, Na+, and K+ were measured. Dehydration elevated (P less than 0.05) PRA, vasopressin, osmolality, and Na+. Thirty minutes after injection of naloxone, osmolality, Na+, K+, hematocrit, and plasma protein were not altered. Naloxone-induced elevations of ACTH (25 +/- 10 and 22 +/- 4 pg/ml) and cortisol (4.8 +/- 1.0 and 5.1 +/- 1.0 micrograms/dl) were similar during euhydration and dehydration, respectively. The increase in aldosterone due to naloxone was greater after euhydration (7.7 +/- 3 ng/dl) than during dehydration (2.3 +/- 0.8 ng/dl). Naloxone increased vasopressin by (5.3 +/- 2.8 microU/ml) during dehydration but not during euhydration. Intravenous hypertonic saline infusions showed that naloxone potentiates the osmotic release of vasopressin. Our results indicated that dehydration did not alter the inhibitory role of opioids in regulation of ACTH and cortisol but suppressed the inhibition of aldosterone secretion. Our findings also showed that opioids inhibit secretion of vasopressin during dehydration by decreased responsiveness to osmotic stimulation. PMID- 3002178 TI - Inbred guinea pig model of intrauterine infection with cytomegalovirus. AB - Outbred guinea pigs have previously been utilized in an experimental model for the study of congenital infection with cytomegalovirus (CMV). Development of an inbred model of intrauterine CMV infection would allow analysis of the cells involved in CMV immunity, studies of transplacental CMV transfer, and investigation of the cellular immune factors that participate in intrauterine CMV infections. This study was therefore designed to assess the inbred guinea pig as a model for the study of congenital CMV infection. Intrauterine fetal and placental infection with CMV was demonstrated in inbred Strain 2 guinea pigs, and the maternal factors influencing transplacental transmission of CMV were evaluated. Infectious virus was recovered from placentas and offspring of mothers that experienced primary CMV infection during pregnancy, but not from placentas and offspring of mothers that were inoculated with CMV prior to pregnancy. However, histologic lesions consisting of focal necrosis and inflammation were seen in tissues of offspring from both groups of mothers. Inoculation of seronegative pregnant Strain 2 animals with low doses of virus (2.5 to 3.5 log10 TCID50) resulted in both placental and fetal CMV infection without significant maternal death. Infection of placentas and offspring occurred in utero regardless of the stage of pregnancy. In addition, infectious virus was detectable in fetal tissues at the time of maternal viremia but also later during the course of maternal infection, ie, 4 weeks after inoculation. These findings indicate that the inbred guinea pig model can be used to investigate the pathogenesis of intrauterine CMV infections. PMID- 3002182 TI - Canine gastric emptying of fiber meals: influence of meal viscosity and antroduodenal motility. AB - Dietary fibers such as psyllium and guar gum have been shown to delay the gastric emptying of liquids and solids, presumably due to an increase in meal viscosity. For liquid test meals containing fats, delayed gastric emptying is associated with a reversal of the usual antral-to-duodenal contractile gradient. The present studies were performed to determine whether the gastric emptying of increasingly viscous psyllium and guar gum meals was associated with antroduodenal motility changes. Dogs were surgically fitted with mid-duodenal cannulas for the measurement of gastric emptying. Strain-gauge force transducers were used to monitor antral and duodenal contractile responses to the test meals. Low viscosity fiber meals emptied from the stomach rapidly (E 1/2 approximately 10 min) compared with the high-viscosity meals (E 1/2 approximately 40 min). None of the test meals stimulated antral or duodenal motility despite differences in gastric emptying time. Other motor parameters such as the time of reappearance and the duration of the burst interval were also unchanged. We conclude a) as test meals' fiber content and viscosity increase, gastric emptying is slowed; and b) viscosity-related delays in gastric emptying are not due to an effect on postprandial antroduodenal motility. PMID- 3002183 TI - D-Glucose transport in piglet jejunal brush-border membranes: insights from a disease model. AB - We measured glucose transport in jejunal brush-border membrane vesicles isolated from piglets with acute viral diarrhea, comparing our results with those from control animals. Characterization of membranes from both study groups demonstrated comparable purity and integrity. In the presence of an inwardly directed Na SCN gradient, D-glucose accumulated in control vesicles to a concentration several times the 60-min equilibrium level. "Overshooting" uptake was much lower and more gradual in vesicles from 40-h transmissible gastroenteritis (TGE)-infected pigs compared with control pigs. Equilibrium kinetic studies, in which gramicidin was used to clamp membrane potential at zero, demonstrated a pattern of Na-dependent D-glucose transport in 40-h TGE infected membranes that differed greatly from the control pattern. From an Eadie Hofstee plot of stereospecific Na-dependent D-glucose uptake into control vesicles, a pattern suggesting two carrier populations emerged: one with a low affinity, apparent Km equaling 52.63 +/- 13.81 mM and the other a high-affinity apparent Km equaling 3.92 +/- 0.24 mM for D-glucose. In 40-h TGE-infected membranes, the pattern conformed to a single line, suggesting a homogeneous population of low-affinity carriers, (Km = 37.03 +/- 1.92 mM), which did not differ from the low-affinity carriers seen in control animals. We conclude that the absence of the high-affinity D-glucose carriers in jejunal brush-border membrane is an important determinant of the defective glucose transport that characterizes viral diarrhea. Because previous studies have strongly suggested that in acute TGE diarrhea the epithelium is composed of relatively undifferentiated crypt-type cells, we speculate that high-affinity D-glucose carriers are lacking in normal crypt epithelial cells and that they are incorporated into brush-border membranes of jejunal enterocytes as the cells differentiate in the course of their migration from crypt to villus. PMID- 3002184 TI - Gastrin receptors in normal and malignant gastrointestinal mucosa: age-associated changes. AB - Synthetic human gastrin 17-I (MG) and an analogue, [Leu15]gastrin-17-I (LG), were radiolabeled with Na125I by Iodo-Gen, EnzymoBead, and chloramine-T methods, and the characteristics of the radiolabeled peptides were determined. When 125I-MG was iodinated by chloramine-T, its biological activity and its binding activity were almost abolished, whereas the biological activity of 125I-LG, iodinated by either of the methods, and of 125I-MG, iodinated by Iodo-Gen and EnzymoBeads, was not significantly affected. The kinetics, affinity, and specificity of binding of 125I-MG, iodinated by Iodo-Gen, to crude and purified membranes from rat fundic mucosa were examined and found to be similar to that for 125I-LG. Age-associated changes in the number and affinity of gastrin receptors (GR) on the crude membranes of gastrointestinal mucosa of rats was also examined. Significantly fewer GR were observed on the crude membranes of fundic mucosa of aged (24 mo old) compared with young (3- and 6-mo-old) rats. In addition, specific gastrin binding sites (4.7 +/- 0.9 fmol/mg prot) with low affinity (Kd = 3.7 +/- 1.2 nM) were observed in the antrum of aged rats, the significance of which is not understood. There were, however, no differences in the number and characteristics of GR in other regions of the intestine of old and young rats. The presence of GR was additionally assessed in cell lines of gastrointestinal cancers from humans, mice, and hamsters.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002185 TI - Complete response to vasopressin requires epithelial organization in A6 cells in culture. AB - Arginine vasopressin (AVP) stimulates adenylate cyclase activity in A6 epithelia grown on filters but not in A6 epithelia grown on plastic culture dishes. When A6 cells are subcultured from culture dishes and seeded at high density on filters, stimulation of adenylate cyclase by AVP is not evident for approximately 3 days. Peak stimulation by AVP occurs between 5 and 10 days. The time course for development of responsiveness to AVP corresponds to the development of an ordered epithelium with polarized cells, tight junctions, and a high transepithelial resistance. A similar correlation is seen when mature filter-grown epithelia that respond to AVP are dissociated by chelation of calcium and subcultured as single cells onto another filter. Stimulation of adenylate cyclase is not evident until an epithelium with significant electrical resistance is reformed. Filters seeded at 1.6 X 10(6) cells/cm2 require 1-2 days to show AVP sensitivity and electrical resistance; filters seeded at 0.4 X 10(6) cells/cm2 require 4 days. Experiments to test the function of the subunits of adenylate cyclase with forskolin (catalytic subunit) and with cholera toxin (Ns, guanine nucleotide-binding subunit) indicate that they are functional during the entire time and that it is the receptor for AVP that is not functional until an ordered epithelium has been formed. PMID- 3002186 TI - Isolation and characterization of plasma membranes from bovine carotid arteries. AB - A plasma membrane fraction from bovine carotid arteries has been isolated by extraction of a crude microsomal fraction with a low-ionic-strength buffer containing ATP and Ca2+. This step was followed by sucrose-density-gradient centrifugation in the presence of 0.6 M KCl. The plasma membrane vesicles were enriched 60- to 80-fold in Na+-K+-adenosinetriphosphatase, 5'-nucleotidase, and phosphodiesterase I activities. The final yields of these marker enzymes were 12 18% of the total activities in the postnuclear supernatant, and the protein yield was 100-120 micrograms/g wet wt of carotid arteries. Contamination of the plasma membrane fraction by mitochondria and sarcoplasmic reticulum was low as judged by low activities of succinate--cytochrome-c reductase and NADPH--cytochrome-c reductase, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with smooth muscle-specific actin antibodies showed that the plasma membrane fraction was substantially free from myosin and actin contamination. The plasma membrane vesicles accumulated Ca2+ in the presence of ATP, and the accumulation was increased by calmodulin. Ca2+ accumulated in the presence or absence of calmodulin could be released almost completely from the vesicles by the addition of the Ca2+ ionophore A23187 but not by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid, indicating that Ca2+ uptake in the presence of ATP is intravesicular. The effects of phosphate and oxalate on Ca2+ uptake in the plasma membranes were different from one another. Phosphate increased Ca2+ uptake in a concentration- and time dependent manner, and the increase in Ca2+ uptake could be observed as early as 1 min. On the other hand, oxalate at concentrations up to 5 mM did not increase Ca2+ uptake significantly during the 30-min incubation. These plasma membranes can prove useful for the study of ion transport across plasma membranes, hormone binding, characterization of calcium channels, and preparation of antibodies against plasma membrane proteins. PMID- 3002187 TI - Sodium retention after adrenal enucleation. AB - Adrenal enucleation (removal of the adrenal gland, leaving the capsule intact) results in regeneration of the adrenal cortex. During the first 1-2 wk of adrenal regeneration, marked renal sodium avidity and positive sodium balance are noted. This renal sodium avidity appears mediated via adrenocorticotropin-stimulated secretion of a potent mineralocorticoid by the regenerating adrenal cortex. In this review, we have examined relationships between the histology and ultrastructure of the regenerating adrenal cortex, renal sodium handling, and adrenal steroid production at various times after the initiation of adrenal regeneration. Plasma levels of known mineralocorticoids are subnormal during the period of most intense sodium avidity, while urinary excretion of a potent mineralocorticoid, 19-nordeoxycorticosterone, has been found to be increased in rats with regenerating adrenals during this period of most intense sodium avidity. This hormone, however, is not elevated in rats with regenerating adrenals after resolution of the period of sodium avidity. In this article, we review the experimental evidence regarding the potency of this mineralocorticoid and its likely role in the sodium retention after adrenal enucleation. PMID- 3002188 TI - cAMP augmentation of secretagogue-induced luteinizing hormone secretion. AB - Whether adenosine 3',5'-cyclic monophosphate (cAMP) acts as a mediator for luteinizing hormone-releasing hormone (LHRH) in either its immediate LH release action or in its self-priming action was investigated. Pituitary pieces from either proestrous or estrous rats were superfused in vitro in the presence of dibutyryl cAMP [(Bu)2cAMP], 8-bromo-cAMP (8BrcAMP), forskolin, or control for 2-3 h. For proestrous but not estrous pituitary pieces, a slight increase in base line LH secretion rate occurred at approximately 70 min of exposure to elevated cAMP; in the same system LHRH caused an increase in LH secretory rate within 2 min in either proestrous or estrous tissue. In contrast to its ineffectiveness as a secretagogue, cAMP elevation resulted in a severalfold augmentation of both LHRH- and elevated K+-induced LH secretion from proestrous but not estrous pituitary pieces; for these experiments, superfusion with a cAMP analogue or forskolin for varying times preceded a 10-min pulse of either 8 nM LHRH or 47 mM K+. Augmentation was evident after 30 min of cAMP elevation; longer exposures were coincident with greater potentiation. Cycloheximide prevented (Bu)2cAMP augmentation of LHRH-induced secretion. These data show that cAMP does not mediate the immediate LH release action of LHRH, but cAMP does augment LHRH- or K+-induced LH secretion with characteristics in common with the self-priming action of LHRH. PMID- 3002189 TI - Effect of rate of hemorrhage on sympathoadrenal catecholamine release in cats. AB - The effect of rate of blood loss on catecholamine release and cardiovascular compensation to hemorrhage (H) was assessed in alpha-chloralose-urethane anesthetized cats. Arterial blood was withdrawn at a rapid rate (10% measured blood vol/min) or at a slow rate (2%/min), and responses were compared across three volumes of hemorrhage (10, 20, or 30% of total blood vol). Plasma epinephrine did not increase after 10% H, but increased modestly after rapid (199 +/- 25 pg/ml) or slow 20% H (359 +/- 92 pg/ml). In contrast, rapid 30% H evoked a significantly (P less than 0.01) greater increase in epinephrine than slow 30% H (1,827 +/- 274 vs. 630 +/- 165 pg/ml). The rate of hemorrhage had no differential effect on hemorrhage-evoked plasma norepinephrine, but a graded increase was seen with increasing volume of hemorrhage. Similarly, hemorrhage-evoked plasma glucose was proportional to the volume of hemorrhage and was not affected by rate of hemorrhage. Volume of hemorrhage was a good indicator of norepinephrine release after rapid or slow rate of hemorrhage, whereas epinephrine release was well correlated with the mean changes in blood pressure during the posthemorrhage sampling period after rapid or slow rate of hemorrhage. The data indicate that hemorrhage-evoked release of epinephrine depends on the rate of blood loss, but only after large (30%) volumes of hemorrhage. Norepinephrine released into the peripheral circulation demonstrates no dependency on rate of hemorrhage, but is well correlated with volume of hemorrhage.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002190 TI - Effect of rate of hemorrhage on release of ACTH in cats. AB - The effect of rate of blood loss by acute hemorrhage (H) on adrenocorticotropin (ACTH) and cortisol plasma concentrations was assessed in anesthetized cats. Arterial blood was withdrawn at a rapid rate (10% blood vol/min) or at a slow rate (2%/min), and responses were compared across three volumes of H (10, 20, and 30% of blood vol). After rapid rate of H, ACTH increased in proportion to volume of H (r = 0.669, P less than 0.001) with a mean elevation of 124 +/- 27, 267 +/- 102, and 950 +/- 195 pg/ml for 10, 20, and 30% H, respectively. Slow rate of H evoked a significant increase in ACTH that was not proportional to volume of H (r = 0.314, P greater than 0.10), and the mean change during the post-H sampling period was 611 +/- 166, 828 +/- 302, and 1,070 +/- 239 pg/ml for 10, 20, and 30% H, respectively. Control animals showed no change in ACTH to the repeated sampling paradigm. Rapid H evoked an immediate decrease in mean arterial pressure (MAP) with a post-H recovery of MAP inversely proportionate to volume of H (r = 0.552, P less than 0.01). Slow H caused a progressive decrease in MAP with no significant post-H recovery of MAP at any volume of H. Cortisol concentration increased in proportion to volume of H after rapid H (r = 0.515, P less than 0.025), but not after slow H.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002191 TI - Pituitary-adrenal function after transplantation in rats: dependence on age of the adrenal graft. AB - Comparisons of resting plasma adrenocorticotropin (ACTH) and corticosterone in the morning and afternoon were made among adult rats bearing regenerated adult adrenal grafts, neonatal (day 1) adrenal grafts, adult adrenal capsule grafts, or intact adrenals. In the morning plasma ACTH and corticosterone were similar in all rats. In the afternoon, plasma ACTH was elevated in rats bearing neonatal adrenal grafts or adult adrenal capsule grafts, but not in rats bearing whole adult adrenal grafts. There was no difference in afternoon plasma corticosterone among rats bearing transplanted adrenals, although afternoon plasma corticosterone was decreased in rats bearing transplants compared with rats with intact adrenals. Thus the increased plasma ACTH after adrenal transplantation cannot be explained entirely by decreases in resting plasma corticosterone. Adrenal responsiveness to ACTH was tested at 5 wk after transplantation in the afternoon by measuring the plasma corticosterone response to submaximal doses of ACTH. The responsiveness was decreased in rats bearing transplants. In addition, responsiveness was inversely related to the age of the grafted adrenal tissue. Adrenals regenerated from adult adrenals were more responsive than adrenals regenerated either from neonatal adrenals or from adult adrenal capsules. The findings suggest that following adrenal transplantation reestablishment of normal pituitary-adrenocortical function does not occur in rats bearing adrenals regenerated from immature adrenal cells. In addition, comparable alterations occur after regeneration of adrenal tissue from neonatal adrenal cells and adult adrenal capsular cells. Elevated plasma ACTH associated with adequate plasma corticosterone in rats bearing adrenals regenerated from immature adrenal cells may result from chronic alteration in responsiveness to steroid feedback.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002192 TI - Na-H exchange in rat liver basolateral but not canalicular plasma membrane vesicles. AB - Na+-stimulated H+ movement and H+-stimulated Na+ uptake were studied in basolateral (blLPM) and canalicular (cLPM) rat liver membrane vesicles. H+ movement was monitored with the fluorescent amine acridine orange; 22Na uptake was assayed by a rapid Millipore filtration technique. In blLPM, inwardly directed Na+ gradients stimulated H+ efflux and outwardly directed Na+ gradients stimulated proton influx. Outwardly directed proton gradients (pH in 5.9/pH out 7.9) stimulated initial 22Na uptake rates 5- to 10-fold over pH-equilibrated conditions (pH in 7.9/pH out 7.9). Conversely, inwardly directed proton gradients (pH in 7.9/pH out 5.9) inhibited 22Na uptake. pH-dependent 22Na uptake was inhibited by amiloride and harmaline but not by other transport inhibitors, bumetanide, furosemide, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, 4 acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and acetazolamide. Lithium also inhibited H+-stimulated 22Na uptake. Although a component of pH stimulated 22Na uptake appeared to be dependent on membrane potential, this electrogenic component was amiloride insensitive. Proton gradient-stimulated 22Na uptake in blLPM was saturable, with a Km of 5.4 mM and a Vmax of 14 nmol . min-1 . mg prot-1. In contrast, in cLPM, no Na+ gradient-stimulated proton movement and no pH-dependent Na+ uptake occurred. These findings establish an electroneutral Na-H antiport in blLPM but not cLPM in rat liver. The polarity of this exchanger supports a model of bile formation that is dependent, in part, on canalicular HCO 3 and/or OH- excretion. PMID- 3002193 TI - Alpha 2-adrenoceptor stimulation and cellular cAMP levels in microdissected rat glomeruli. AB - A functional role for the numerically predominant glomerular alpha 2 adrenoceptors is unknown. In other tissues, activation of alpha 2-adrenoceptors inhibits adenylate cyclase activity. We therefore examined the effect of alpha 2 adrenoceptor stimulation with (-)-epinephrine (E) on the cellular cAMP concentration in glomeruli isolated by microdissection. Parathyroid hormone (1-34 PTH), prostaglandin E2 (PGE2), histamine, serotonin, or adenosine, in the presence of 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor) and propranolol, was used to activate adenylate cyclase in single intact rat glomeruli. alpha 2-Adrenoceptors were activated with varying concentrations of E (37 degrees C, 2 min). In the presence of PTH-stimulated cAMP production, alpha 2 adrenoceptor activation with E (5 X 10(-7) to 5 X 10(-6) M) suppressed cellular cAMP levels in a dose-dependent fashion with the maximum at 30%. This suppression by E was inhibited by 5 X 10(-6) M yohimbine but not by 5 X 10(-6) M prazosin, confirming alpha 2-adrenoceptor mediation of this effect of E. Consistent with the above findings, the specific alpha 2-adrenoceptor agonist BHT933 inhibited PTH-stimulated cAMP accumulation. E also inhibited cAMP accumulation stimulated by serotonin. However, E did not suppress the PGE2-, histamine-, or adenosine stimulated increase in cellular cAMP in the glomerulus. Activation of alpha 2 adrenoceptors inhibits cAMP formation stimulated by PTH or serotonin but not by PGE2, histamine, or adenosine in the rat glomerulus. Thus, the ability of alpha 2 adrenoceptors to inhibit adenylate cyclase appears to be hormone and probably function specific. PMID- 3002194 TI - Atriopeptin III increases cGMP in glomeruli but not in proximal tubules of dog kidney. AB - Atriopeptin III is natriuretic, diuretic, and phosphaturic in the intact dog and relaxes vascular and nonvascular smooth muscle in vitro. The mechanism of the renal effects of atriopeptin remains ill defined but may include hemodynamic alterations and/or direct tubular effects. Since many peptide hormones act through changes in cyclic nucleotide formation, we examined the effect of synthetic atriopeptins on cAMP and cGMP accumulation in isolated glomeruli and proximal tubules from normal dog kidney. Synthetic atriopeptin produced a dose related increase in cGMP in glomeruli with a Kact of 200 +/- 19 nM and a maximal activity of 64.1 +/- 6.3 pmol cGMP X mg prot-1 X 5 min-1. In the presence of 4 mM Mn2+ Kact was reduced to 19 +/- 2 nM with no change in maximal cGMP production (66.2 +/- 11.8 pmol cGMP X mg prot-1 X 5 min-1). Atriopeptin (1 microM) did not increase cGMP accumulation above basal levels in proximal tubule segments. The cAMP production of neither glomeruli nor proximal tubules was stimulated by atriopeptin. These studies demonstrate a dose-dependent stimulation of guanylate cyclase by atriopeptin in glomeruli but not in proximal tubule segments. In addition, atriopeptin neither stimulates nor inhibits cAMP accumulation in glomeruli or proximal tubules. These data suggest a major role for the renal effects of atriopeptin at the glomerular level, possibly through hemodynamic effects or changes in the glomerular ultrafiltration coefficient.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002195 TI - 5-Hydroxytryptamine delays relaxation time of norepinephrine-induced vasoconstriction. AB - The effect of low concentration of 5-hydroxytryptamine (5-HT; 0.03-0.3 microM) on cellular and extracellular Ca2+ -dependent norepinephrine (NE; 5 microM)-induced vasoconstriction was assessed in isolated perfused rabbit ear artery. The most remarkable effect of 5-HT is a concentration-dependent enhancement of the duration of the extracellular Ca2+ -dependent NE-induced vasoconstriction. This effect of 5-HT is still present in 6-hydroxydopamine-pretreated arteries and in the presence of deoxycorticosterone and is not mimicked by the K+ channel blocker tetraethylammonium. Administration of nifedipine (0.3 microM) and EDTA (1 mM) failed to antagonize 5-HT-induced prolongation of relaxation kinetics of extracellular Ca2+ -dependent NE-induced vasoconstriction. On the other hand, this phenomenon was abolished by caffeine (15 mM) and markedly reduced by dibutyryl cyclic AMP (3 mM). 5-HT (0.3 microM), but not NE (30-50 nM), enhanced the vasoconstriction elicited by caffeine (15 mM) that was procaine sensitive and unaffected by nifedipine. These results suggest that 5-HT prolonged relaxation kinetics of NE-induced vasoconstriction through an impairment of cellular mechanisms that decrease the myoplasmic level of free Ca2+. A similar mechanism could be of relevance for 5-HT enhancement of vascular smooth muscle responsiveness to vasoactive agents. PMID- 3002197 TI - Naloxone treatment of L-dopa-induced dyskinesias in Parkinson's disease. PMID- 3002198 TI - Lymph node metastases in adenoid cystic carcinoma. AB - The clinical characteristics of 76 patients with adenoid cystic carcinoma seen during a 22-year period are described. Lymph node metastases at presentation or later occurred almost exclusively in men, and the five-year rate of "node-free" survival was 62 per cent for men and 95 per cent for women. Node metastases were more common in poorly differentiated tumors, but site and stage of the primary disease did not affect the metastatic rate. Surgery scarcely improved the rate of survival of patients with nodal metastases. The authors were unable to confirm previous observations that embolic nodal metastases at a distance from the primary tumor do not occur. PMID- 3002196 TI - Beta-adrenoceptor-mediated release of angiotensin II from mesenteric arteries. AB - Essential components of the renin-angiotensin system such as renin enzymes, angiotensinogen, converting enzyme, and angiotensin receptors have been found in vascular tissues. Locally generated angiotensin (ANG) II may regulate vascular tone by contracting vascular smooth muscle or potentiating sympathetic activity. Recently it was suggested that beta-adrenoceptor-induced enhancement of noradrenergic neurotransmission is mediated by the vascular renin-angiotensin system. The present study was designated to obtain direct evidence for the release of ANG II from the vasculature by beta-adrenoceptor activation. Isolated rat mesenteric arteries were perfused in vitro with Krebs-Ringer solution, and released ANG II was concentrated in a Sep-Pak C-18 cartridge connected to the perfusion system. High-pressure liquid chromatography combined with radioimmunoassay clearly demonstrated the presence of ANG I, II, and a small amount of ANG III in the perfusate. Isoproterenol (10(-9) - 10(-6) M) induced the enhancement of pressor responses to nerve stimulation. This effect was markedly suppressed by propranolol (5 X 10(-7) M), captopril (2 X 10(-6) M), or [Sar1 Ile8]ANG II (10(-6) M). Isoproterenol (10(-9) - 10(-6) M) caused increase in the release of ANG II from mesenteric arteries. The increase in ANG II release during isoproterenol (10(-6) M) infusion was blocked by propranolol (10(-6) M). Captopril (2 X 10(-6) M) also inhibited the increase in ANG II induced by isoproterenol. These results indicate that locally generated ANG II is released from isolated perfused rat mesenteric arteries and its release is mediated by beta-adrenoceptors. PMID- 3002199 TI - [Role of androgens in the physiology of the reproductive function of women]. PMID- 3002200 TI - A rare case of Aicardi syndrome with severe brain malformation and hepatoblastoma. AB - A 2-month-old Japanese girl exhibited tonic seizure, agenesis of the corpus callosum, lacunar chorioretinopathy, vertebral anomalies, electroencephalographic abnormalities and a malignant tumor. Autopsy revealed a hepatoblastoma and severe brain malformations consisting of callosal agenesis, arhinencephaly, marked polymicrogyria and optic nerve anomalies. It was thought that the pathogenic factor in this case may exert its effect during the fourth or fifth week of intrauterine life, and then may continue until the beginning of neuronal migration (about 3 months). This is the first reported case of Aicardi syndrome associated with hepatoblastoma, and may provide a link between teratogenicity and oncogenicity. PMID- 3002201 TI - [Cancer of the lung as a second primary neoplasm in patients treated for laryngeal cancer]. PMID- 3002202 TI - [Ceruminomas of the external auditory canal. Apropos of a case]. PMID- 3002203 TI - [A new model of adrenergic nerve termination]. PMID- 3002204 TI - High-flow-rate hydroxylapatites. AB - We have established a novel and simple method for preparing rigidly crystallized or granulated hydroxylapatites, which showed flow rates one order of magnitude higher in column chromatography than those of the Tiselius specimen (1). The essential difference in the procedures from the Tiselius method is that calcium phosphate, the precursor of hydroxylapatite, is generated not at room temperature but at either 45 or 95 degrees C. The capacities of the two hydroxylapatites for binding and fractionating protein, RNA, and DNA upon column chromatography were similar to those of Tiselius's hydroxylapatite. The 95 degrees C specimen can be dried with no change in the properties. Because of the reproducibly observed properties, including the extremely high flow rate with ease in handling as well as low cost and simple procedures with no additives and with no special equipment in preparation, our hydroxylapatites seem to be best so far for routine laboratory use. PMID- 3002205 TI - A radiolabeled-ligand-binding technique for the characterization of opioid receptors in the intact mouse vas deferens. AB - The mouse vas deferens has served as a useful bioassay for examining the properties of opiate receptor subtypes. However, recent data indicate that the response of the vas deferens to opiates may be mediated by one or more of the several opiate receptors found in this preparation. Although a number of techniques can be utilized to assess the relative contribution of these receptors to the response of the mouse vas deferens to opiates (e.g., selective tolerance and naloxone antagonism studies), a radiolabeled-binding technique would provide an independent means of more completely characterizing the opiate receptor profiles in this preparation. Up to the present, however, there has been only limited success in developing a binding assay utilizing crude membrane fractions of the mouse vas deferens. To circumvent these problems, we have developed a binding technique utilizing the intact vas deferens. In contrast to results obtained with membrane fractions, we found highly specific (90-95%) and saturable binding of D-[2-3H]alanine, 5-D-leucine enkephalin, a ligand selective for delta opiate receptors, to the intact vas. Scatchard analyses indicated a single class of binding sites with an apparent Kd of 1.5 nM and a Bmax of approximately 12 pmol/2 vas. The selectivity of binding was also examined. Naltrexone was 40 times less potent than unlabeled 2-D-alanine, 5-D-leucine enkephalin in displacing binding, whereas morphine and ethylketocyclazocine were 300 and 500 times less effective, respectively. This technique, coupled with the mouse vas deferens bioassay, should provide a more complete characterization of opioid receptor populations than has heretofore been possible. PMID- 3002206 TI - Ultracytochemical study of the stria vascularis of the guinea pig cochlea. AB - The localization of the K-NPPase, ALPase and Mg-ATPase activities in the stria vascularis of the guinea pig cochlea was ultracytochemically studied. The K NPPase reflecting the Na-K-ATPase activity was localized on the cytoplasmic side of the deeply infolded basolateral plasma membrane of the marginal cells. On the other hand, activities of ALPase and Mg-ATPase were found in the intercellular spaces between the marginal and the intermediate or the basal cells. The surface of the stria vascularis facing to endolymph showed only the Mg-ATPase activity. The ALPase activity localized also around the capillaries. The mechanism of the K and Na transport was discussed. PMID- 3002207 TI - The cerebellar nucleo-olivary and olivo-cerebellar nuclear projections in the cat as studied with anterograde and retrograde transport in the same animal after implantation of crystalline WGA-HRP. II. The fastigial nucleus. AB - The bidirectional connections between the inferior olive and the fastigial nucleus were studied by means of anterograde and retrograde transport after implantation of crystalline wheat germ agglutinin-horseradish peroxidase (WGA HRP) complex into the fastigial nucleus. The fastigio-olivary fibres terminate in the caudal half of the medial accessory olive, nucleus beta and the dorsal cap, and the olivo-fastigial projection has its origin within the same olivary regions. A topical arrangement is indicated for both pathways. The lateral part of the medial accessory olive appears to be connected with the lateral part of the fastigial nucleus, while the medial part of the medial accessory olive appears to be connected with more medial fastigial regions. Retrogradely labelled olivo-fastigial neurons were often located within the terminal field of anterogradely labelled fastigio-olivary fibres, indicating that the olivo fastigial and fastigio-olivary projections are at least in part reciprocally organized. The findings are discussed and related to previous studies on the olivo-cerebellar nuclear and cerebellar nucleo-olivary pathways. Some methodological considerations are made, and comments are made concerning the active area for uptake and transport from the stained area at the WGA-HRP injection site. PMID- 3002209 TI - Do hypothalamo-cerebellar fibres terminate in all layers of the cerebellar cortex? AB - The terminal distribution of hypothalamo-cerebellar fibres has been studied with anterograde transport of the wheat germ agglutinin-horseradish peroxidase complex in the cat and rat. The hypothalamo-cerebellar fibres appear to enter all three layers of the cerebellar cortex. Anterogradely labelled branching axons were found in the granular layer and near the Purkinje cell perikarya. In addition, anterogradely filled hypothalamo-cerebellar axons could be traced into the molecular layer, where they ramified. The branches ran parallel to the long axis of the folia, resembling parallel fibres. Our findings give evidence that the hypothalamo-cerebellar fibres are neither mossy fibres nor climbing fibres, but represent a third type of cerebellar cortical afferents. Fibres of this third category are tentatively called multi-layered fibres. It appears from the literature that some other cerebellar afferent projections show the same general pattern of cortical terminal distribution. PMID- 3002208 TI - The projection from the superior colliculus to the lateral reticular nucleus in the cat as studied with retrograde transport of WGA-HRP. AB - Medium-sized and large superior collicular neurons were retrogradely labelled after small ejections of the wheat germ agglutinin-horseradish peroxidase complex in the lateral reticular nucleus of the feline medulla. The projection from the superior colliculus to the lateral reticular nucleus is bilateral with a contralateral predominance. It originates mainly from the intermediate, but also from the deep gray layer of the superior colliculus. Our observations provide evidence that the lateral reticular nucleus is an important target of tectal efferents. The findings are discussed in relation to the organization of other fiber connections of the superior colliculus. PMID- 3002210 TI - Inhibition of superoxide production and Ca2+ mobilization in human neutrophils by halothane, enflurane, and isoflurane. AB - The inhibitory effects of three inhalation anesthetics, i.e., halothane, enflurane, and isoflurane, on superoxide production and the intracellular mobilization of calcium in human neutrophils were studied. The superoxide production induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP) was inhibited by the anesthetics, but the binding of FML[3H]P to the cells and the superoxide-forming NADPH oxidase of the phagocytic vesicles were not inhibited. The inhibition of the cellular superoxide production was partially reversed by the addition of a calcium ionophore, A23187. The increase in intracellular free calcium monitored by a calcium-sensitive fluorescent probe, quin-2 and the release of calcium from hydrophobic environment monitored by chlortetracycline were inhibited dose dependently by the anesthetics. These observations suggest that decreased mobilization of intracellular Ca2+ is one of the mechanisms by which the anesthetics inhibited the superoxide production of human neutrophils stimulated by FMLP. PMID- 3002211 TI - Cost-effective application of the Centers for Disease Control Guideline for Isolation Precautions in Hospitals. AB - Isolation, as practiced in many hospitals today, is associated with ineffective, costly rituals. The isolation process can be monitored and critically examined to determine illogical practices. When these practices and their associated costs are known, specific strategies can be implemented to reduce the costs associated with isolation. PMID- 3002212 TI - Cost-effective application of the Centers for Disease Control Guideline for Prevention of Catheter-associated Urinary Tract Infections. AB - To decrease UTIc in a cost-efficient manner, efforts should be concentrated in areas that have been shown to be beneficial. These include the following measures: have an established infection control program; catheterize patients only when necessary, by using aseptic technique, sterile equipment, and trained personnel; maintain a closed sterile drainage system; do not disconnect the catheter and drainage tubing unless absolutely necessary; remove the catheter as soon as possible; and follow and reinforce good handwashing technique. Changing indwelling catheters at arbitrary fixed intervals and regular bacteriologic monitoring of catheterized patients are not cost-effective practices and should not be performed. Other measures should be avoided until further data are available. New products that appear to be questionable or gimmicky probably are and should also be avoided. PMID- 3002213 TI - Cost-effective application of the Centers for Disease Control Guideline for Prevention of Intravascular Infections. AB - Implementation of the recommendations in the CDC Guideline for Prevention of Intravascular Infections pertaining to MDVs and 48-hour administration set changes are cost-effective. Application of antibiotic ointment to cut-down sites is also cost-effective. Although not stated in the guideline, reserving antibiotic ointment only for IV lines inserted in one site more than 3 days would appear to be cost-effective. Finally, the cost-effectiveness of using IV in-line filters cannot be determined on the basis of current existing data. More information is needed pertaining to the effect of filtration on infection rates and the cost of filtration versus the cost of a case of phlebitis. PMID- 3002214 TI - Response of cattle persistently infected with noncytopathic bovine viral diarrhea virus to vaccination for bovine viral diarrhea and to subsequent challenge exposure with cytopathic bovine viral diarrhea virus. AB - Nine steers persistently infected with noncytopathic bovine viral diarrhea (BVD) virus were allotted into 3 groups (3 cattle/group). Cattle in group A were vaccinated with a modified-live BVD virus vaccine of porcine cell origin, cattle in group B with a modified-live BVD virus vaccine of bovine cell origin, and cattle in group C with a killed BVD virus vaccine of bovine cell origin. Detrimental effects due to vaccination were not seen. Six weeks after vaccination, the steers were challenge exposed with a cytopathic BVD virus. All steers developed mucosal disease after challenge exposure, produced antibodies that neutralized various isolates of BVD virus, and remained persistently infected until death. Steers given killed virus vaccine had a minimal neutralizing-antibody response and developed mucosal disease as quickly as reported for challenge-exposed, nonvaccinated, persistently infected cattle. Steers given modified-live virus vaccines had higher neutralizing-antibody response and longer intervals from challenge exposure to development of mucosal disease. The specificity of the neutralizing-antibody response differed between groups of vaccinated cattle. PMID- 3002215 TI - Goat papillomatosis. AB - The present study indicates that the development of caprine mammary gland warts seems to depend on several factors--namely, nonpigmented skin, adult age, excessive exposure to sunlight, and contact with a yet undefined infective agent. Three types of goat papillomas are described: mammary, cutaneous, other than mammary, and genital. Warts on animals lacking pigmented skin are more frequent in adult animals that live in areas where there is abundant sunlight. Mammary gland papillomas are the most numerous and occur in different stages: ie, goats with mammary gland papillomas that regress, never to recur, goats with papillomas that regress in the winter and reoccur in summer, goats with persistent papillomas of which some are from the group that had previous winter time regression, and goats that have progression of persistent papilloma to carcinoma. Saanen, Saanen crossbreeds, and goats of other breeds that lack pigmented skin and live in sunbelt areas are at high risk for papillomatosis. An infective agent was not defined even after electron microscopic, immunocytochemical, and DNA-DNA hybridization studies. Yet, there is high probability that an infective agent is involved, because mammary gland papillomas usually occur in the susceptible herd 4 to 6 months after an affected goat is introduced. PMID- 3002216 TI - Experimentally induced pneumonic pasteurellosis: dose-response relationships and protection against natural reinfection in calves. AB - Calves were inoculated intranasally with 2 X 10(6.2) tissue culture infective doses of infectious bovine rhinotracheitis virus, followed in 7 days by intratracheal inoculations with 1 of 4 challenge doses of pathogenic Pasteurella haemolytica. Severity and duration of the ensuing clinical signs of respiratory tract disease were correlated with the challenge dose of bacteria. Calves given 1 X 10(6) colony-forming units (CFU) of bacteria did not develop reliable clinical evidence of disease, whereas those given 1 X 10(8) CFU or 1 X 10(10) CFU of bacteria developed clinical signs of pneumonic pasteurellosis within 12 to 24 hours of bacterial challenge. Severity of clinical signs was equal at the 10(8) and 10(10) doses of bacteria, but duration of clinical signs was greater in calves given the 10(10) dose. Calves given 1 X 10(12) CFU of bacteria developed relatively severe respiratory tract disease in excess of what was necessary for positive clinical detection. Positive correlations were found between the bacterial challenge dose and the height and duration of increased rectal temperature, amount and duration of increases in ocular and nasal discharges, and the subjective evaluation of depressed attitude and appetite. Correlations were not found between challenge dose and respiratory rate or character, or between challenge dose and complete blood cell count. Convalescent calves were resistant to naturally occurring pneumonic pasteurellosis, which caused severe disease in nontreated calves. Adverse effects of P haemolytica were not observed after the first 4 to 15 days after bacterial administration; however, the bacteria were isolated from nasal secretions of convalescent calves 89 to 116 days after bacterial inoculation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002218 TI - CDC issues AIDS guidelines for health workers. PMID- 3002217 TI - Seroepidemiologic survey for antibodies to selected viruses in the respiratory tract of lambs. AB - A serologic study was conducted to determine the prevalence of antibodies to, and infection rate of, Mastadenovirus ovi 5, M ovi 6, parainfluenza-3 (PI-3) virus, bovine herpesvirus-1 (BHV-1), respiratory syncytial virus (RSV), bovine viral diarrhea (BVD) virus, and ovine progressive pneumonia (OPP) virus in lambs at a ram lamb growth-rate test station. For 2 consecutive years, serum samples were prepared from blood collected from 1- to 2-month-old ram lambs as they entered the test station (1st sample) and again 2 months later (2nd sample). The 1st year, 59 producers submitted 237 lambs; the 2nd year, 65 producers submitted 253 lambs. Microtitration serum virus-neutralization tests were used to determine antibody titers for M ovi 5, M ovi 6, PI-3 virus, BHV-1, and BVD virus. Antibodies to RSV and OPP virus were determined, using indirect hemagglutination and agar-gel immunodiffusion, respectively. Based on results of the 1st blood samples collected, the mean prevalence for both years was as follows: 95% of the lambs were seropositive for M ovi 5; 87.2% for PI-3 virus; 84.5% for RSV; 41.7% for M ovi 6; 8.7% for BVD virus; 5.4% for BHV-1; and 3.3% for OPP virus. Based on the 2-year mean, M ovi 6 had the highest infection rate (207 of 484 [42.8%]) as determined by the number of lambs evaluated having a greater than or equal to 4 fold increase in serum antibody titer from the 1st to the 2nd sampling. Infection rates of the other viruses were: 31.0% for M ovi 5; 15.3% for PI-3 virus; 5.6% for RSV; 0.6% for BVD virus; and 0.4% for BHV-1. One lamb became seropositive for OPP virus the 2nd year. PMID- 3002219 TI - [Arsenous anhydride poisoning. Peripheral neuropathy and changes in cognitive functions]. AB - The authors report the evolution over an 11 months period of a case of subacute intoxication with arsenic in a 30 years old woman. In addition to the classical peripheral neuropathy, we observed impairment of the superior neurological functions which improved together with the neuropathy. These malfunctions are rarely described in the literature although arsenic seems to cross the blood brain barrier easily. There is no other explanation, and we believe arsenic to be responsible for these disturbances. We suggest systematic testing of the superior neurological functions in cases of arsenic intoxication. PMID- 3002220 TI - Calcium-activated proteolysis of intermediate filaments. PMID- 3002221 TI - Intermediate filament proteins in the study of tumor heterogeneity: an in-depth study of tumors of the urinary and respiratory tracts. PMID- 3002222 TI - [Effect of proteolytic enzymes on the penetration of Trypanosoma cruzi in peritoneal macrophages of mice]. AB - The present study deals on the modifications produced by three proteolytic enzymes in the interiorization of cultured metacyclic forms of Trypanosoma cruzi in peritoneal macrophages of non-stimulated mice. The proteolytic enzymes used were trypsin, papain and collagenase. Pretreatment of metacyclic forms or macrophages with the enzymes resulted in lower penetration ratios with respect to controls. On the other hand, percentages of parasitization were maintained throughout the periods of time assayed. PMID- 3002224 TI - [Superior vena cava compression syndrome. Our experience apropos of 26 cases]. PMID- 3002223 TI - [Inhibition of Plasmodium berghei in rats infested with Strongyloides ratti or Trichinella spiralis; role of high blood corticosterone in reaction to the development of helminths]. AB - The plasma corticosterone induced in the rat by the development of Strongyloides ratti or Trichinella spiralis reaches a sufficient level of intensity to determine reticulocytopenia. The latter is linked chronologically to the inhibition of parasitemia in Plasmodium berghei, which occurs when this protozoa develops at the same time as the Nematodes, and seems to be the causal factor. This hypothesis may be verified by replacing the helminths with the corticotropic action of A.C.T.H. which causes a decrease in the number of reticulocytes. PMID- 3002225 TI - Results of surgical treatment of synchronous multiple primary lung carcinomas. AB - Four male patients with synchronous multiple lung carcinoma were operated on. In one patient both tumours were adenocarcinomas; both were bronchioloalveolar carcinomas in a second patient. In a third patient the tumours were adenocarcinoma and bronchioloalveolar carcinoma while in the fourth patient an adenocarcinoma and a squamous cell carcinoma were present. One patient was asymptomatic and free from carcinoma at follow-up examination 11 years and 7 months after the operation. The other three died of pulmonary carcinoma 11 months, 1 year 3 months and 9 years 8 months postoperatively. PMID- 3002226 TI - Tumour growth rate and its relationship to prognosis in bronchiolo-alveolar and pulmonary adenocarcinoma. AB - Tumour growth rates and their effects on the survival of 26 patients with bronchioloalveolar or pulmonary adenocarcinomas were analysed following surgery. Twelve of the tumours were classified as bronchioloalveolar carcinomas, 7 were classed as mixed forms of bronchioloalveolar carcinoma and 7 were classed as adenocarcinomas. The mean doubling time was 300.1 days for the bronchioloalveolar carcinomas, 288.4 days for the mixed forms of bronchioloalveolar carcinoma and 224.6 days for the adenocarcinomas. The mixed forms of bronchioloalveolar carcinoma had poorer prognoses than bronchioloalveolar carcinomas. They also differed from adenocarcinomas in that metastases were more frequent. No correlation between tumour doubling times and patient survival was established in the present series, nor did tumour doubling times correlate with the occurrence of metastases in regional lymph nodes. The actual survival times were similar to, or shorter than, predicted survival times in most of the patients who died from their pulmonary carcinoma. In contrast, they were considerably longer than predicted survival times in long-term survivors and in half of the patients who did not die from pulmonary carcinoma. This finding indicates that predicted survival times could allow more objective evaluation of the results of treatment of pulmonary carcinoma. PMID- 3002227 TI - [Radioautographic localization of neuropeptide receptors in the central nervous system]. AB - The first step of any physiological effect of a neuropeptide (NP) is its recognition by specific receptor sites. The very organization of the central nervous system (CNS) does not permit a precise localization of these binding sites by conventional binding assays. The aim of the present paper is to describe in detail a recently developed in vitro methodology for the localization, visualization and quantitation of specific binding sites for various NP such as TRH, neurotensin and vasoactive intestinal peptide (VIP) in the rat CNS. The combination of this autoradiographic technique with radioimmunological measurements of NP, reveals that the endogenous distribution of THR, for example, in various brain regions, is not correlated with the presence of its binding sites. In vitro autoradiography may also be used to study the neurotransmitter/neuromodulatory role of NP in the CNS. This point will be illustrated by the effect of VIP on serotonin binding sites in both rat suprachiasmatic nucleus and hippocampal formation. Besides, the importance of the endocrine environment of the target tissue for NP action will also be discussed. PMID- 3002228 TI - [Vasopressin and oxytocin receptors in the central nervous system of the rat]. AB - Synaptic plasma membranes containing binding sites for (3H) oxytocin and (3H) arginine vasopressin were isolated from rat amygdala, olfactory bulb and hippocampus. In the hippocampus, two specific binding sites have been characterized: an "oxytocic" binding site, which has a high affinity for oxytocin, arginine vasopressin and arginine vasotocin, and a "vasopressic" binding site, which has a high affinity for arginine vasopressin, arginine vasotocin and a low affinity for oxytocin. The specificity of these binding sites were tested in competition experiments. The affinity of different antidiuretic and vasopressic analogues for the vasopressic site was similar to that observed for the V1 type of vasopressin receptors present in the hepatocytes and vascular smooth muscle cells. The affinity of several analogues for the oxytocic site shows some similarities with their corresponding relative activities in increasing the firing rate of non pyramidal neurones in hippocampal slices. Arginine vasopressin and oxytocin did not change the activity of adenylate cyclase present in the hippocampal synaptic plasma membranes. The properties of these specific binding sites for the neurohypophyseal hormones are compared with the receptors present on the peripheral targets. PMID- 3002229 TI - [Neurosecretory synapses, yesterday and today]. AB - The concept of peptidergic neurotransmission in the central nervous system ranks high in contemporary neurobiology. It was first formulated by J. Barry when reporting, in 1954-55, his observation with light microscopy of the existence of "Gomori-positive" synapses in sections of the mammalian brain. However, this important discovery had virtually no impact on the scientific community for about twenty years because of an extremely restrictive definition of "neurosecretion" during this period by the practitioners of the new field known as neuroendocrinology. The rejection of Barry's finding raises numerous epistemological questions; it provides another example of a "premature discovery", such as those of A.A. Berthold (testicular transplant studies) and of J.G. Mendel (artificial plant hybridization) during the last century. PMID- 3002230 TI - [Intervention of the calcium-calmodulin system in the release of growth hormone induced by somatocrinin]. AB - This study was carried out to evaluate the role of the calcium-calmodulin system in cellular mediation of somatocrinin action by cells producing growth hormone. We compared GH release to intra- and extracellular cAMP levels, after incubation of pituitary cells in monolayer culture with somatocrinin alone or somatocrinin associated with exogenous calmodulin, or specific calmodulin inhibitor (W13). Calmodulin (10(-6) M) decreases somatocrinin - induced cAMP levels, maintaining elevated GH levels. W13 (10(-5) M) increases somatocrinin - induced cAMP and GH levels. Thus GH release induced by somatocrinin is modulated by two interrelated systems, adenylcyclase - cAMP system and Ca++-calmodulin system. PMID- 3002231 TI - [Imaging of hepatoma. Review of 50 cases]. AB - The authors present 50 cases of hepatoma involving 32 cirrhotic and 18 healthy livers. Only 6 cases were amenable to exeresis completed by peripheral (4 cases) or intra-arterial (2 cases) chemotherapy. In addition to a semeiological study, the usefulness of various imaging techniques (ultrasound, CT, angiography) is discussed. Ultrasound (50 cases), the basic examination, is often sufficient for diagnosis (28 cases of puncture under ultrasound guidance); in certain cases it suffices to rule out surgery (multiple forms, vascular involvement). CT scans (40 cases) are obtained in different manners depending on whether the aim is to detect a hepatoma not seen on sonograms (as in the case of a high alpha foetoproteinemia) or to obtain more precise anatomical data for an already discovered lesion. At present, angiography (13 cases) appears reserved for cases in which the other two techniques do not reveal any contraindications for surgery. PMID- 3002232 TI - [Surgery of insulinomas using an artificial pancreas]. AB - The risks of failure of conventional treatment of insulinomas should be reduced, it seems, when the surgical operation is carried out using an artificial pancreas. The Biostator, used with feed back controlled glucose infusion in 32 cases of which two personal effectively allows one firstly to operate in complete security since it is programmed to maintain a pre-determined glycaemia during the various stages of the operation; it also allows one to put into evidence an occult tumour in a segment rendered suspect due to the sudden hypoglycaemia observed during its mobilisation; it finally allows one by the appearance of a post-excision hyperglycaemia peak to find out whether the procedure has been complete. PMID- 3002234 TI - Leukotrienes as mediators of immediate hypersensitivity. PMID- 3002233 TI - [Various presentations of bran in therapeutics. Their respective properties]. PMID- 3002235 TI - Do leukotrienes fully account for immediate hypersensitivity? PMID- 3002236 TI - Leukotrienes as mediators of immediate hypersensitivity. PMID- 3002237 TI - Structural relationship between the genes encoding 3'-aminoglycoside phosphotransferases in Campylobacter and in gram-positive cocci. AB - Campylobacter coli strain BM2509 resistant to ampicillin, chloramphenicol, erythromycin, kanamycin, spectinomycin, streptomycin and tetracycline was isolated from a patient with hospital-acquired diarrhoea. Resistance to kanamycin has not been thus far described in Campylobacter. Phosphocellulose paper-binding assay indicated that resistance to kanamycin and structurally related antibiotics in strain BM2509 was due to synthesis of a 3'-aminoglycoside phosphotransferase of type III (APH(3')-III), an enzyme so far confined to Gram-positive cocci. The kanamycin and tetracycline resistances were transferable en bloc by conjugation to C. fetus but not to Escherichia coli. Analysis by agarose gel electrophoresis of crude bacterial lysates revealed the presence, in BM2509 and in the transconjugants, of a plasmid, pIP1433, with a size of 47.2 kilobases (Kb). Strain BM2509 also harboured a 4.5-Kb cryptic plasmid. DNA annealing studies indicated a close structural relationship between the kanamycin resistance gene of C. coli BM2509 and that representative of this type of resistance determinant in Gram-positive cocci. These results indicate that emergence of resistance to kanamycin in Campylobacter is due to acquisition in vivo of a gene or a plasmid from Gram-positive bacteria. PMID- 3002238 TI - Restriction endonuclease mapping and cloning of Mycobacterium fortuitum var. fortuitum plasmid pAL5000. AB - A restriction map of Mycobacterium fortuitum var. fortuitum plasmid pAL5000 was established. The unique sites for ApaI, BamHI, BglII, BstEII, ClaI, EcoRI, EcoRV, HpaI, KpnI and NarI were located on the 5.0-Kb plasmid. The plasmid had no sites for AhaIII, BclI, HindIII, PstI, SphI and XbaI. pAL5000 was cloned into pBR322 and propagated in Escherichia coli. Three hybrid pAL5000-pBR322 plasmids carrying the complete pAL5000 sequence were constructed by joining the plasmids at their BamHI, EcoRI or EcoRV sites. We also cloned into these plasmids a 1489-bp DNA fragment conferring resistance to kanamycin and originating from the streptococcal plasmid pJH1. The construction of these plasmids will facilitate the analysis and manipulation of pAL5000, and may allow the development of a vector system for genetic analysis in mycobacteria. PMID- 3002239 TI - Vasoactive drugs produce selective changes in flow to experimental brain tumors. AB - Most vasoactive drugs do not readily penetrate the blood-brain barrier and do not affect cerebral blood flow. We tested the hypothesis that vasoactive drugs may alter blood flow to brain tumors in which the blood-brain barrier is abnormal. Blood flow was measured with microspheres in dogs with brain tumors induced by avian sarcoma virus. Intravenously administered adenosine increased blood flow to tumor more than twofold but did not alter flow to normal brain. Intravenously administered norepinephrine decreased blood flow to tumor but not to normal brain. Thus, vasoactive drugs, which have little effect on blood flow to normal cerebrum, produce large changes in blood flow to brain tumors. We also examined responses to systemic hypercapnia. Hypercapnia increased blood flow to normal cerebrum more than twofold but failed to increase flow to tumors. Impaired vasodilator responses to hypercapnia in brain tumors, which cannot be explained primarily by an abnormality of the blood-brain barrier, probably reflect another fundamental difference between vessels in normal brain and brain tumors. The finding that vasoactive drugs have selective effects on blood flow to brain tumors has important implications for delivery of lipid-soluble chemotherapeutic drugs to the tumors. PMID- 3002240 TI - Maize Adh1. PMID- 3002241 TI - In vitro mutagenesis. PMID- 3002242 TI - Recent progress in reovirus research. PMID- 3002243 TI - Population genetics. PMID- 3002244 TI - Semiautomated assessment of in vitro activity of potential antileishmanial drugs. AB - We have compared the in vitro activity of six agents against macrophage-contained Leishmania tropica amastigotes determined by the conventional Giemsa staining procedure, with the activity determined by the semiautomated assessment of incorporation of radiolabeled uracil into the nucleic acid of the organisms. Although the mean 50% effective dose of Pentostam by Giemsa staining (4.1 micrograms/ml) was somewhat higher than that by uracil incorporation (2.8 micrograms/ml), the ED50S for the other two clinical agents (pentamidine, 0.035 versus 0.037 micrograms/ml; amphotericin B, 0.67 versus 0.70 micrograms/ml) and for three promising experimental agents (ketoconazole, 11.3 versus 11.3 micrograms/ml; the 8-aminoquinoline WR 6026, 1.6 versus 1.5 micrograms/ml formycin B, 0.018 versus 0.017 micrograms/ml) were virtually identical. The radiolabeling technique has several advantages over the Giemsa staining procedure. These include the need for relatively few macrophages, rapid and objective data generation, and viability of the test organism being measured. The successful application of the radiolabeling technique to at least six different chemical classes of compounds suggests that it would be useful for the routine assessment of antileishmanial activity in vitro. PMID- 3002245 TI - Drug resistance patterns of herpes simplex virus isolates from patients treated with acyclovir. AB - A decrease in the in vitro sensitivity to acyclovir (ACV) was observed in successive isolates of herpes simplex virus type 1 from three immunocompromised patients during intravenous therapy with this drug. The ACV-resistant isolate from patient 1 was cross-resistant to dihydroxypropoxymethylguanine and bromovinyldeoxyuridine, but still susceptible to three fluoro-substituted pyrimidines, 2'-fluoro-5-iodo-1-beta-D-arabinofuranosylcytosine (FIAC), 2'-fluoro 5-iodo-1-beta-D-arabinofuranosyluracil (FIAU), and 2'-fluoro-5-iodo-1-beta-D arabinofuranosylthymine (FMAU). The thymidine kinase (TK) from the resistant isolate showed a 50-fold or greater reduction in affinity for thymidine, FIAU, FMAU, and ACV, but the total enzyme activity was similar to that of the sensitive isolate. The ACV-resistant isolate from patient 2 was also resistant to dihydroxypropoxymethylguanine, bromovinyldeoxyuridine, and the fluoro-substituted compounds; TK activity for this isolate was less than 1% of the patient's pretherapy isolate. An isolate obtained during a subsequent recurrence in patient 2 was susceptible to ACV and the other TK-dependent agents. The ACV-resistant isolate from patient 3 was partially resistant to FIAC and FIAU but still susceptible to FMAU; the viral TK had a 10-fold-lower affinity for ACV, FIAU, and FMAU than did the sensitive pretherapy isolate, while the level of TK activity detected was reduced to 6%. In none of the isolates studied was a change in sensitivity to phosphonoformic acid observed. Compared with the corresponding pretherapy ACV-sensitive isolates, there was a 30-fold decrease in neurovirulence for mice of the two drug-resistant isolates with diminished levels of thymidine phosphorylating activity and no change in virulence for the third isolate. These findings indicate that mixed patterns of drug-resistance to TK-dependent antiviral compounds can occur in clinical isolates, resulting from changes in either the amount or the affinity of viral TK activity. PMID- 3002246 TI - Efficacy of UK-49,858 (fluconazole) against Candida albicans experimental infections in mice. AB - UK-49,858 (fluconazole), a new, orally absorbed bis-triazole derivative, has been evaluated against systemic infections with Candida albicans in normal and immunosuppressed mice and against an intestinal infection with C. albicans in immunosuppressed mice. Orally administered ketoconazole was used as a comparison agent throughout, and orally administered amphotericin B was included for comparative in the experimental intestinal infection. In a 10-day dosage regimen, UK-49,858 was far more active than ketoconazole against systemic infections with C. albicans in normal and immunosuppressed mice. In normal mice, extension of UK 49,858 dosing to 30 days resulted in prolongation of survival to over 90 days, and up to 60% of treated animals had no detectable C. albicans in their kidneys. In addition, over 90% of mice with intestinal candidiasis had culture-negative feces after a 3-day treatment with UK-49,858, but only 62 and 23% of mice gave this response after amphotericin B and ketoconazole therapy, respectively. These data suggest that UK-49,858 may be of value in the treatment of systemic and gastrointestinal infections due to C. albicans in humans. PMID- 3002247 TI - Pharmacokinetic and therapeutic trial of sultamicillin in acute sinusitis. AB - Sultamicillin, an antibiotic combining ampicillin and the beta-lactamase inhibitor sulbactam, was administered to 13 patients diagnosed as having acute sinusitis. Specimens from sinus were obtained for all 13 patients by transantral puncture. Pharmacokinetics, bacteriology, and therapeutic efficacy were assessed. Eighty-five percent (11 of 13) were cured; two treatment failures were subsequently shown to have chronic (rather than acute) sinusitis during surgical exploration. Diarrhea was frequently encountered, and Clostridium difficile associated enteritis was documented for one patient. Beta-lactamase-producing organisms were not encountered in this study; however, this study provides impetus for further controlled clinical trials. PMID- 3002248 TI - Basic biochemical and pharmacological aspects of antiviral agents. PMID- 3002249 TI - Rapid detection of herpes simplex- and varicella-zoster virus antigens from clinical specimens by enzyme immunoassay. PMID- 3002250 TI - Problems of herpes simplex virus latency. PMID- 3002251 TI - Effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine on replication of Epstein-Barr virus in human lymphoblastoid cell lines. AB - The effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) on replication of Epstein-Barr virus (EBV) was investigated and compared with Acyclovir (ACV). Both drugs inhibited EBV replication very rapidly. However, the inhibitory effect of ACV was readily reversed after removal of the drug, in contrast to the more prolonged effect exerted by BVDU, which persisted for more than 21 days. The 50% inhibitory doses of BVDU for virus replication (ED50) and lymphoblastoid cell growth (ID50) were 0.06 microM and 390 microM, respectively; the in vitro therapeutic index (ID50/ED50) was 6,500. Synthesis of EBV-induced polypeptides with molecular weights of 145K, 140K and 110K was partially reduced by BVDU. When superinfected Raji cells were exposed to 125I-labeled (E)-5-(2-iodovinyl)-2' deoxyuridine (IVDU), larger amounts of (125I)IVDU were incorporated into viral DNA than cellular DNA. PMID- 3002252 TI - Cytomegalovirus infections in renal transplant recipients. Correlation between clinical disease and results of a specific ELISA. PMID- 3002254 TI - A comparative study of the biological assay and radioimmunoassay of interferon: their usefulness in clinical studies. PMID- 3002253 TI - Involvement of cytotoxins in the immune response to viral infection. AB - A human cytotoxin (CTX) with an Mr of 17,500 was purified to homogeneity from cytokine preparations by the use of a monoclonal antibody against that protein. Sendai virus and, to a lesser extent, the lectin phytohemagglutinin were found to induce effective production of that CTX in cultures of human peripheral-blood mononuclear cells, the first - by stimulating monocytes to produce the proteins, and the latter - by stimulating T cells. With both kinds of inducers, CTX production correlated to a marked increase in the cellular levels of mRNA for CTX, as quantitated by translation of that mRNA, to biologically active CTX, in microinjected Xenopus oocytes. Crude CTX preparations, as well as purified CTX, were found to be selectively cytotoxic to metabolically depressed and to virus infected target cells; they effectively killed cells which were treated with inhibitors of macromolecule synthesis, such as cycloheximide, or infected by viruses, such as VSV, but failed to exert a cytotoxic effect in the absence of such sensitizing treatments. IFN, most notably IFN-gamma, further potentiated destruction of virus-infected cells by the purified CTX, when applied on these cells at subantiviral concentrations, while uninfected cells remained resistant to CTX following treatment with IFN. Formation of the 17.5K CTX in response to viral infection and the selective cytotoxic effect of that protein on cells infected by viruses, indicate a role of CTX in the defense against viral infections. PMID- 3002255 TI - Efficacy of consensus interferon alpha against HSV-2 infections. PMID- 3002256 TI - 5-(2-Chloroethyl)-2'-deoxyuridine: a potent and selective inhibitor of herpes viruses in vitro and in vivo. PMID- 3002257 TI - Bovine interferon-alphaI 1 is an effective inhibitor of bovine herpes virus-1 induced respiratory disease. PMID- 3002258 TI - Synergistic interaction between interferon-alpha and acyclovir in the treatment of herpes simplex virus type 1 infection in mice. AB - Hairless mice were infected intracutaneously with HSV-1 and treated with rHuIFN alpha A/D, a recombinant DNA-derived hybrid human interferon-alpha that is active on mouse cells in vitro and in vivo. When given alone (1 or 2 X 10(5) units/dose) at times soon after infection, interferon showed some efficacy, reducing disease severity by 20-30% compared to control. Oral acyclovir was also effective in reducing disease severity in a dose-dependent manner, even when treatment was begun 72 h post-infection after herpetic vesicles had become apparent. When used in combination with acyclovir (400 mg/kg/day beginning 72 h post-infection), rHuIFN-alpha A/D (beginning 4 h post-infection) greatly enhanced the therapeutic effect of the nucleoside, giving a 64% reduction in disease severity score relative to control (compared to 14% for acyclovir alone). Furthermore, although interferon treatment alone was ineffective if begun after disease was apparent, it nonetheless potentiated the activity of acyclovir when co-administered with the nucleoside beginning 72 h post-infection. Combination therapy markedly reduced disease severity, limited the progression of the infection to the vesicular stage in 50% of recipient mice and promoted a more rapid onset of healing than was obtained by treatment with acyclovir alone. PMID- 3002259 TI - Treatment and prevention of virus infections in immunosuppressed patients. PMID- 3002260 TI - Herpes simplex virus vaccines. PMID- 3002261 TI - Booster vaccination of healthy adults with VZV antibody but without a VZV specific cell-mediated immune response. PMID- 3002263 TI - Rotavirus infections and their prevention by vaccination. PMID- 3002262 TI - Antiherpes activity of 5-(2-bromoethyl)-2'-deoxyuridine. PMID- 3002264 TI - Therapy of varicella-zoster virus infection--mechanism of action of (E)-5-(2 bromovinyl)-2'-deoxyuridine. PMID- 3002265 TI - Differential metabolism of (E)-5-(2-bromovinyl)-2'-deoxyuridine in wild-type and drug-resistant herpes simplex virus-infected cells. PMID- 3002266 TI - Incorporation of carbocyclic (E)-5-(2-iodovinyl)-2'-deoxyuridine (C-IVDU) into DNA of herpes simplex virus-infected cells. AB - C-IVDU, the carbocyclic analogue of IVDU [(E)-5-(2-iodovinyl)-2'-deoxyuridine], in which the sugar moiety is replaced by a cyclopentane ring, is, like its parent compound, a very potent and selective inhibitor of herpes simplex virus type 1 (HSV-1) replication. Both C-IVDU and IVDU are effectively phosphorylated by HSV-1 infected cells and incorporated into viral and cellular DNA of these cells. PMID- 3002267 TI - Antiviral agents against picornaviruses. PMID- 3002268 TI - Comparison of arildone and 3-methylquercetin as stabilizers of poliovirus. PMID- 3002269 TI - Isolation and partial characterization of plasmid DNA from Streptococcus thermophilus. AB - Of 23 strains of Streptococcus thermophilus examined, 5 were found to contain a single small cryptic plasmid, designated pHM1 through pHM5. Through analysis by restriction endonuclease mapping and DNA-DNA hybridization, the five plasmids were found to be closely related. They were present in 4 to 18 copies per cell and ranged in size from 1.4 to 2.2 megadaltons. Plasmids pHM1 and pHM5, as well as pHM2 and pHM4, were found to be identical. Single restriction endonuclease sites were observed on each plasmid for PvuII and MboI. Plasmids pHM2 and pHM3 each had an additional single site for HhaI, and pHM1 had an additional single site for HindIII. The characteristics of these plasmids may make them useful for the development of cloning vectors for use in S. thermophilus. PMID- 3002270 TI - Streptococcus cremoris M12R transconjugants carrying the conjugal plasmid pTR2030 are insensitive to attack by lytic bacteriophages. AB - Conjugal transfer of lactose-fermenting ability (Lac+), nisin resistance (Nisr), and phage resistance (Hsp+) was demonstrated in matings between Streptococcus lactis ME2 (donor) and Streptococcus cremoris M43a (recipient), a derivative of M12R. Transconjugants were detected by transfer of Lac+ and were found to exhibit Nisr and harbor a 40-megadalton plasmid (pTR1040). Fifty-six percent of Lac+ transconjugants were resistant to the S. cremoris M12R lytic phage. Efficiency of plaquing for phage m12r . M12 on a phage-resistant transconjugant, T2r-M43a, was less than 4.3 X 10(-10). Five additional phages which were virulent for S. cremoris M12R and isolated from industrial sources failed to plaque on S. cremoris T2r-M43a. Mating experiments with T2r-M43a revealed that phage resistance was accompanied by high-frequency conjugation ability (Tra+) and the appearance of both pTR1040 and pTR2030 encoding Lac+ Nisr and Tra+ Hsp+, respectively, in transconjugants of S. lactis LM2302. Phage-sensitive Lac+ transconjugants of S. cremoris M43a (T2s-M43a) showed no conjugal ability. These observations confirmed that pTR2030 was present and responsible for the phage resistance and conjugal ability exhibited by the S. cremoris transconjugant T2r M43a. Unlike the S. lactis LM2302 transconjugant carrying pTR2030, resistance of T2r-M43a to phage was not affected at high temperatures (35 to 40 degrees C) or destabilized in repeated transfers through a starter culture activity test. These results demonstrated that phage resistance conferred by pTR2030 in the S. cremoris transconjugant was effective against industrially significant phages under fermentation conditions normally encountered during cheese manufacture. PMID- 3002271 TI - Single-site chromosomal Tn5 insertions affect the export of pectolytic and cellulolytic enzymes in Erwinia chrysanthemi EC16. AB - Exponentially growing cells of Erwinia chrysanthemi EC16 usually export about 98% of their pectate lyase (PL) and protease, about 40% of their polygalacturonase (PG), and about 60% of their cellulase (endoglucanase or carboxymethyl cellulase; CL). By using the R plasmid, pJB4JI (pPH1JI::Mu::Tn5), three independent Tn5 insertion mutants were obtained that exported normal levels of protease but 10% or less of PL, PG, and CL. Physical analysis revealed that single copies of Tn5 had inserted into the E. chrysanthemi chromosome, producing a similar export defective (Out-) phenotype. The synthesis of PL, PG, and CL was not affected by the Tn5 insertions. These enzymes were released from the mutants on spheroplast formation, indicating that they were located in the periplasmic space. Tn5 insertions caused the loss of a 35-kilodalton periplasmic protein, but did not alter the outer membrane protein composition. The findings are discussed with respect to the current knowledge on protein export in gram-negative bacteria. PMID- 3002273 TI - Reactions of fish to microorganisms in wastewater. AB - Fish were inoculated with various microorganisms present in wastewater. A threshold concentration was determined over which these microorganisms were recovered from the muscles. The threshold concentrations were different for bacteria, bacteriophages, and polio 1 LSc virus. The threshold values were lower when fish were inoculated than when they were immersed in water containing these organisms. Depuration experiments were efficient when the fish did not contain high concentrations of bacteria in their muscles. As the threshold concentrations are an expression of the capability of the immune system of the fish, these values can be useful for the design and management of fishponds in which treated wastewater is used. PMID- 3002272 TI - Micro-lipid-droplet encapsulation of Bacillus thuringiensis subsp. israelensis delta-endotoxin for control of mosquito larvae. AB - The crystal delta-endotoxin of Bacillus thuringiensis subsp. israelensis is less toxic to larvae of Anopheles freeborni than to larvae of Aedes aegypti. However, when solubilized crystal was used, larvae from both species showed similar sensitivities. This effect presumably was due to the differences in feeding behavior between the two mosquito larvae when crystal preparations are used. A procedure is described whereby both crystal and solubilized B. thuringiensis subsp. israelensis toxin were emulsified with Freund incomplete adjuvant, with retention of toxicity. The use of Freund incomplete adjuvant also allowed one to assay the solubilized toxin at a low nanogram level. Furthermore, coating the toxin with lipophilic material altered the buoyancy of the toxin and reversed the sensitivities of the two mosquito larvae toward the B. thuringiensis subsp. israelensis toxin. This difference in buoyancy was determined by using an enzyme linked immunosorbent assay that was specific for the toxic peptides. These data indicate that economically feasible buoyant formulations for the B. thuringiensis subsp. israelensis crystal can be developed. PMID- 3002274 TI - Effects of t-butyl hydroperoxide on NADPH, glutathione, and the respiratory burst of rat alveolar macrophages. AB - The effects of t-butyl hydroperoxide on glutathione and NADPH and the respiratory burst (an NADPH-dependent function) in rat alveolar macrophages was investigated. Alveolar macrophages were exposed for 15 min to t-butyl hydroperoxide in the presence or absence of added glucose. Cells were then assayed for concanavalin A stimulated O2 production or for NADPH, NADP, reduced glutathione, glutathione disulfide, glutathione released into the medium and glutathione mixed disulfides. Exposure of rat alveolar macrophages to 1 X 10(-5) M t-butyl hydroperoxide causes a loss of concanavalin A-stimulated superoxide production (the respiratory burst) that can be prevented or reversed by added glucose. Cells incubated without glucose had a higher oxidation state of the NADPH/NADP couple than cells incubated with glucose. With t-butyl hydroperoxide, NADP rose to almost 100% of the NADP + NADPH pool; however, addition of glucose prevented this alteration of the NADPH oxidation state. Cells exposed to 1 X 10(-5) M t-butyl hydroperoxide in the absence of glucose showed a significant increase in the percentage GSSG in the GSH + GSSG pool and increased glutathione mixed disulfides. These changes in glutathione distribution could also be prevented or reversed by glucose. With 1 X 10(-4) M t-butyl hydroperoxide, changes in glutathione oxidation were not prevented by glucose and cells were irreversibly damaged. We conclude that drastic alteration of the NADPH/NADP ratio does not itself reflect toxicity and that significant alteration of glutathione distribution can also be tolerated; however, when oxidative stress exceeds the ability of glucose to prevent alterations in oxidation state, irreversible damage to cell function and structure may occur. PMID- 3002275 TI - Immunochemical study on the mannans of Candida albicans NIH A-207, NIH B-792, and J-1012 strains prepared by fractional precipitation with cetyltrimethylammonium bromide. AB - The mannans of Candida albicans NIH A-207 (A strain, serotype A), C. albicans NIH B-792 (B strain, serotype B), and C. albicans J-1012 (J strain, serotype C) prepared by fractional precipitation with cetyltrimethylammonium bromide (Cetavlon) were investigated for their immunochemical properties. Upon treatment with 10 mM HCl at 100 degrees C for 60 min, the mannans of A and B strains each released a mixture of manno-oligosaccharides ranging from hexaose to mannose together with (for each one) an acid-modified mannan, while J-strain mannan released lower oligosaccharides, tetraose to mannose. The acid-modified mannan of B strain did not show antibody-precipitating activity against homologous antiserum, whereas acid-modified A- and J-strain mannans retained most of this activity. The acid-released oligosaccharides were assumed to consist of beta-1,2 linked D-mannopyranosyl residues from the results of specific rotation and proton magnetic resonance studies. PMID- 3002276 TI - Interaction of PGBx and peroxides with cytochrome c and inhibition of lipid peroxidation. AB - PGBx, a derivative of prostaglandin B1, stimulated the oxidation of cytochrome c in the presence of H2O2. Although the reaction was nonenzymatic, the apparent activation energies of 12 and 4.9 kcal above and below the transition at 21.5 degrees C were similar to those for oxidation by cytochrome oxidase. Depletion of H2O2 and oxidation of cytochrome c followed similar time courses, suggesting that H2O2 was consumed in the reaction. PGBx was a specific requirement, but organic hydroperoxides (ethyl and T-butyl) could replace H2O2. Low concentrations of ethyl or t-butyl hydroperoxide initially stimulated the oxidation of cytochrome c; this stimulation disappeared before completion of the oxidation, but was restored when the hydroperoxide concentration was renewed, suggesting that these hydroperoxides were probably also consumed in the reaction. The concentration of PGBx (8.9 microM) required for half-maximum stimulation of the oxidation was similar to the apparent Kd for its dissociation from oxidized cytochrome c (6.8 microM). Binding data and CD spectra suggested that a 1:1 complex between cytochrome c and PGBx was formed, altering the conformation of the heme region. This conformational change caused a shift of the Soret absorption peak from 410 to 406 nm and may be responsible for the enhanced oxidizability of the cytochrome c by H2O2. Cytochrome c inhibited lipid peroxidation in microsomes, an effect enhanced by the addition of PGBx. In the absence of lipid peroxidation, cytochrome c and PGBx stimulated NADPH oxidation via NADPH-cytochrome c reductase. Thus the inhibition of lipid peroxidation by cytochrome c and PGBx may involve either the removal of hydroperoxides or deviation of electron transfer away from the pathway for lipid peroxidation. PMID- 3002277 TI - Synthetic peptide substrates for mammalian pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. AB - The specificities of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase were probed using synthetic peptides corresponding to the sequence around phosphorylation sites 1 and 2 on pyruvate dehydrogenase [Tyr-His-Gly-His Ser(P1)-Met-Ser-Asp-Pro-Gly-Val-Ser(P2)-Tyr-Arg]. The dephosphotetradecapeptide containing aspartic acid at position 8 was a better substrate for the kinase than was the tetradecapeptide containing asparagine at position 8. The apparent Km and V values for the two peptides were 0.43 and 6.1 mM and 2.7 and 2.4 nmol of 32P incorporated/min/mg, respectively. Methylation of the aspartic acid residue also increased the apparent Km of the tetradecapeptide about 14-fold. These results indicate that an acidic residue on the carboxyl-terminal side of phosphorylation site 1 is an important specificity determinant for the kinase. Phosphate was incorporated only into site 1 of the synthetic peptide by the kinase. The phosphatase exhibited an apparent Km of 0.28 mM and a V of 2.3 mumol of 32P released/min/mg for the phosphorylated tetradecapeptide containing aspartic acid. Methylation of the aspartic acid residue had no significant effect on dephosphorylation. The octapeptide and phosphooctapeptide produced by cleavage of the aspartyl-prolyl bond by formic acid were poorer substrates for the kinase and phosphatase than were the tetradecapeptide and phosphotetradecapeptide, respectively. Modification of the amino terminal by acetylation or lysine addition had only a slight effect on the kinase and phosphatase activities. PMID- 3002278 TI - Plastocyanin stimulation of whole chain and photosystem I electron transport in inside-out thylakoid vesicles. AB - Inside-out and right-side-out thylakoid vesicles were isolated from spinach chloroplasts by aqueous-polymer two-phase (dextran/polyethylene glycol) partitioning. Externally added plastocyanin stimulated the whole-chain and PSI electron transport rates in the inside-out thylakoid vesicles by about 500 and 350%, respectively, compared to about 50% stimulation for both assays in the fraction enriched in right-side-out vesicles. No apparent stimulation by plastocyanin was observed in unbroken Class II thylakoids. The electron transport between PSII and PSI in inside-out thylakoid vesicles appears to be interrupted due to plastocyanin release from the thylakoids by the Yeda press treatment, but it was restored by externally added plastocyanin. The P700 content of the inside out membrane preparations, measured by chemical and photochemical methods, was 1 P700 per 1100 to 1500 chlorophylls while it was about 1 P700 per 500 chlorophylls for the right-side-out vesicles. The data presented support the concept of lateral heterogeneity of PS I and II in thylakoid membranes, but does not support a virtual or total absence of PSI in the appressed grana partitions. Further, the heterogeneity does not appear to be as extreme as suggested earlier. Although PSI is somewhat depleted in the appressed grana membrane region, there is adequate photochemically active P700, when sufficient plastocyanin is available, to effectively couple PSI electron transfer with the preponderant PSII in linear electron transport. PMID- 3002279 TI - Characterization of electron transfer and proton translocation activities in trypsin-treated bovine heart mitochondrial cytochrome c oxidase. AB - Bovine heart mitochondrial cytochrome c oxidase has been treated with trypsin in order to investigate the role of components a, b, and c (nomenclature of Capaldi) in cytochrome c binding, electron transfer, and proton-pumping activities. Cytochrome c oxidase was dispersed in nondenaturing detergent solution (B. Ludwig, N. W. Downer, and R. A. Capaldi (1979) Biochemistry 18, 1401) and treated with trypsin. This treatment inhibited electron transfer activity by 9% when compared to a similarly treated control in a polarographic assay (493 s-1) and had no large effect on the high affinity (Km = 6.1 X 10(-8) M) or low affinity (Km = 2.2 X 10(-6) M) sites of cytochrome c interaction with cytochrome c oxidase. Direct thermodynamic binding experiments with cytochrome c showed that neither the high affinity (1.04 +/- 0.06 mol cytochrome c/mol cytochrome c oxidase) nor the high-plus-low affinity (2.21 +/- 0.15 mol cytochrome c/mol cytochrome c oxidase) binding sites of cytochrome c on the enzyme were perturbed by the trypsin treatment. Control and trypsin-treated enzyme incorporated into phospholipid vesicles (prepared by the cholate dialysis method) exhibited respiratory control ratios of 6.5 +/- 0.7 and 6.3 +/- 0.6, respectively. The vectorial proton translocation activity in the phospholipid vesicles was unaffected by trypsin treatment with proton translocated to electron transferred ratios being equivalent to the control. NaDodSO4-PAGE showed that components a, b, and c were completely removed by the trypsin treatment. [14C]Iodoacetamide labeling experiments showed that the content of component c in the enzyme was depleted by 85% and that greater than 50% of component a was cleaved upon the trypsin treatment. These results suggest that components a, b, and c are not required for maximum electron transfer and proton translocation activities in the isolated enzyme. PMID- 3002280 TI - Citrate effects on the Ca2+-loading capacity of isolated rat liver mitochondria: interaction of citrate and ATP. AB - The maximal amounts of Ca2+ being accumulated (delta Ca2+max) and H+ emitted (delta H+max) by Ca2+-loading mitochondria, with succinate (+rotenone) as respiratory substrate, were evaluated. delta Ca2+max was increased by providing either citrate or ATP to a Pi- and Mg2+-free medium. With citrate, delta H+max was only scarcely increased, so that the effect of the proton-carrying anion resulted essentially from an increase in the Ca2+/H+ ratio, i.e., from preservation of membrane potential. With ATP (+/- oligomycin), the Ca2+/H+ ratio was unaltered; i.e., the increase of delta Ca2+max was paralleled by a related increase in delta H+max. Mitochondria appeared to retain Ca at higher delta pH, i.e., at lower membrane potential, in the presence of ATP. With citrate and ATP together, both the Ca2+/H+ ratio and delta H+max were largely increased, and the product of these two terms, delta Ca2+max, was considerably enlarged. The effect of either citrate or ATP was markedly reinforced in the presence of the other anion. In addition to increasing the Ca2+/H+ ratio, citrate contributed to increasing delta H+max in the presence of ATP, i.e., apparently sensitized mitochondria to the action of ATP. A citrate-induced depression of Ca2+ cycling across the inner membrane, even though pronounced, did not account for the sensitization. Supraadditive effects of citrate and ATP persisted in the presence of MgCl2 and Pi, under conditions of massive Ca2+ loading, and may contribute to the high capacity of mitochondria, in situ, to retain calcium. PMID- 3002281 TI - The negative cooperativity in cytochrome c oxidase redox reactions: the electrostatic effect. AB - The electrostatic interaction of two hemes is calculated on the basis of the data on the cytochrome c oxidase structure. The interaction energy corresponds to the positive shift by approximately greater than 100 mV of the redox potential of one of the hemes when the other is oxidized. This effect seems to be the most likely reason of the negative cooperativity in the redox behavior of cytochrome c oxidase. PMID- 3002283 TI - [U.S.-Japan Cooperative Cancer Research Program--combined modality of chemotherapy & radiotherapy]. PMID- 3002282 TI - [A phase II study of etoposide (VP-16) injection in primary lung cancer by cooperative study group]. AB - A Phase II Study of VP-16 was performed in 58 patients with primary lung cancer. VP-16 was administered via infusion at a dosage of 60-100 mg/m2 daily for 5 days, and repeated every 3 to 4 weeks. Of the 58 patients who entered into the study, 38 were evaluable. Partial response was observed in 6 patients who had been diagnosed as having small cell carcinoma of the lung. Response rates were 15.8% for all evaluated patients and 33.3% for the patients with small cell carcinoma of the lung. As for dose-limiting toxicity, leukopenia (less than 3,000/mm3) was observed in 53.1% of cases. Other major adverse reactions were hematologic toxicities such as anemia and thrombocytopenia, gastrointestinal toxicities and alopecia, but these were all well tolerated. PMID- 3002284 TI - [Postoperative chemotherapy of pathological stage I non-small cell carcinoma of the lung]. AB - Between August 1982 and February 1985, 13 patients with pathological stage I non small cell lung cancer received postoperative chemotherapy with ACNU, ADM and 5 FU. Nine patients had squamous cell carcinoma, two adenocarcinoma, and two large cell carcinoma. All patients were alive in August, 1985, but four of them had recurrent disease. The site of recurrence was the brain in three and the contralateral lung in one. Of these four patients, three had t2n0(-) p0 squamous cell carcinoma and one t1n0(-) p1 large cell carcinoma. In a control group consisting of six squamous cell carcinoma and six adenocarcinoma, recurrence occurred in one patient with t1n0(-) p0 squamous cell carcinoma. Although the treated group had unfavorable conditions of cell type and tumor size, there was no evidence that this combination chemotherapy could control recurrence of the tumor under these conditions. PMID- 3002285 TI - [Bone and soft-tissue tumors]. AB - There have been great advances in recent years in the treatment of malignant bone and soft-tissue tumors thanks to advanced diagnostic techniques, improved chemotherapy and improved surgery involving extensive resection of the area involved, with bone and joint replacement. Here, we present details of the pathological findings, treatment and prognosis of cases of malignant bone and soft-tissue tumors, such as osteosarcoma, bone MFH, soft-tissue MFH, rhabdomyosarcoma and synovial sarcoma. PMID- 3002286 TI - [Combination chemotherapy and radiation therapy for small cell carcinoma of the lung]. AB - From January, 1982 to March, 1983, patients with small cell carcinoma of the lung were treated at Kyushu Cancer Center. Eleven patients received combination chemotherapy-radiotherapy and one patient chemotherapy alone. The chemotherapy regimen consisted of cyclophosphamide, adriamycin and vincristine which was repeated every 4 weeks for as long as possible. Radiotherapy was administered to the primary lesion and mediastinum following 2 cycles of induction chemotherapy. The overall response rate after receiving 2 cycles of chemotherapy was 75% with 4 complete (33%) and 5 partial responses (42%). After radiotherapy, response increased to 100% with 8 complete (73%) and 3 partial responses (27%). Complete response occurred in 6 of the 7 patients with limited disease and 2 of the 5 patients with extensive disease. Overall survival rate was 73% at 1 year, 36% at 2 years and 12% at 3 years with a median survival time of 21 months. Survival was better in patients with limited disease than in those with extensive disease (median survival time, 21.5 months vs. 14 months). In the 3-Year follow-up period, all patients had recurrences consisting of 4 distant, 2 local and 5 both. Myelosuppression was mild to moderate and there were no deaths related to the side effects of treatment. PMID- 3002288 TI - Effect of loxapine and amoxapine on oxidative metabolism of rat brain "in vitro". PMID- 3002287 TI - The effects of acute respiratory virus infection upon tracheal mucous transport. AB - Tracheal mucous velocity was measured in 13 healthy non-smokers using a radioisotope-labeled aerosol and a multidetector probe during respiratory virus infections. The movement of boluses of tracheal mucous were either absent or reduced in number in five subjects with myxovirus infection (four influenza and one respiratory syncytial virus) within 48 hr of the onset of symptoms and in four subjects 1 wk later. One subject with influenza still had reduced bolus formation 12-16 wk after infection. Frequent coughing was a feature of those subjects with absent tracheal boluses. In contrast, four subjects with rhinovirus infection had normal tracheal mucous velocity at 48 hr after the onset of symptoms (4.1 +/- 1.3 mm/min). Tracheal mucous velocity was also normal (4.6 +/- 1.1 mm/min) in four subjects in whom no specific viral agent could be defined but of respiratory viral infection. During health tracheal mucous velocity was (4.8 +/- 1.6 mm/min) in the eleven subjects who had measurements made. Disturbances in tracheal mucous transport during virus infection appear to depend upon the type of virus and are most severe in influenza A and respiratory syncytial virus infection. PMID- 3002289 TI - [Development of a method for the study of drugs which inhibit phospholipase A2]. PMID- 3002290 TI - Serum angiotensin-converting enzyme levels in sarcoid arthritis. AB - The serum angiotensin-converting enzyme (SACE) level is elevated in 75% of patients with sarcoidosis and often is associated with disease activity. In an attempt to correlate the SACE level with sarcoid arthritis, we did a retrospective chart review of 116 patients with sarcoidosis. Of the 24 patients who complained of arthritis, five were excluded from the study because they were receiving corticosteroids, SACE levels were not determined, or another cause for their arthritis was found. The mean SACE levels were 65 units/mL for the patients with acute arthritis and 51 units/mL for the patients with chronic arthritis. Levels of SACE may be helpful in the differential diagnosis of patients with "seronegative polyarthritis." PMID- 3002291 TI - [Biological and bone histomorphometric studies in hypophosphatemic vitamin resistant rickets treated with 1,25-(OH)2D and phosphorus]. AB - Five children aged 6 to 15 years were studied. They presented all the biological signs of hypophosphatemic vitamin resistant rickets: normal calcemia, hypophosphatemia, decrease in phosphorus Tm and normal PTH plasma level. Two children had never been given vitamin D treatment previously. The 1.25 (OH)2D has been given in 4 doses a day, to a total of 1 microgram/day, for one year. Phosphate was administered for the same time, doses varying from 40 to 150 mg/kg/day. Bone biopsies were performed at the onset and the end of treatment. Normal growth concerning bone age was observed in 3 cases. In the two others, growth remained disturbed. Radiological recovery was observed in all cases. Treatment induced and increase in serum phosphate. Tm PO4/FG remained lower than normal and even decreased during treatment. After one year of treatment, the osteoid volumes and surfaces decreased in all cases but did not always return to normal. The thickness index of osteoid and the speed of calcification were improved in 4 cases and worsened in the 5th. In two previously untreated patients an increase in plasma PTH and in the osteoclastic surface of resorption were observed on the bone biopsy during treatment. PMID- 3002292 TI - [Value of chlormethine in children with corticoid-dependent or partially corticoid-sensitive nephrosis, in steroid poisoning]. AB - Mechlorethamine was administered at a low dose (0.8 mg/kg) to 27 children presenting with steroid dependent or partially responsive nephrotic syndrome, with signs of steroid toxicity. This drug induced a fast decrease of proteinuria (average delay: 7 days). It led to long lasting remission (average follow-up 34 months) in 16 cases (59%). Relapse occurred in 11 children (41%) most often (9 of 11 cases) in the first 7 months. However the evolutive pattern was clearly improved in 5 of these cases. Altogether, mechlorethamine allowed to stop corticosteroid therapy or, at least, to reduce the given dose, with a decrease of the signs of steroid toxicity, in 78% of the cases. In 6 cases (22%) evolution was almost not improved. One may hope that this dosage of mechlorethamine will not be gonadotoxic. This should be checked later on. PMID- 3002293 TI - Biochemical and functional evidence of supersensitive platelet alpha 2 adrenoceptors in major affective disorder. Effect of long-term lithium carbonate treatment. AB - The hypothesis that depressive illness is related to supersensitive alpha 2 adrenoceptors in the brain has been tested indirectly in blood platelets. The binding of tritiated clonidine hydrochloride to platelet membranes, a ligand that labels only the high-affinity state of the alpha 2-adrenoceptor that is coupled with cell functions, and the aggregation response induced by epinephrine hydrochloride, which is the result of the activation of the high-affinity state, were measured and correlated in 13 patients with major affective disorder. Both the number of high-affinity binding sites and the aggregation response were increased in depressed patients. There was a negative and significant correlation between both measures in the same depressed patients. Treatment with lithium carbonate (Plenur [Spain]; Linthane, comparable US product) was associated with a decrease in the high-affinity state and with an increase in the aggregation response. Thus, major effective disorder may be related to a dysfunction of the high-affinity state of the alpha 2-adrenoceptor that recognizes agonists and mediates physiological effects. PMID- 3002294 TI - Factors influencing the biological behaviour of cervical human papillomavirus (HPV) infections in prospectively followed-up women. AB - To assess the natural history of Human papillomavirus (HPV) infections in uterine cervix, recently linked with cervical intraepithelial neoplasia (CIN), a long term prospective follow-up study was started at our clinic in 1981. At this writing, a total of 418 women have been followed-up for a mean of 20 +/- 15 (M SD) months. On each attendance (at six-month intervals), the patient is subjected to colposcopy accompanied either by Papanicolaou (PAP) smears or punch biopsies. The latter are analysed for the cytopathic changes of HPV, for concomitant CIN, as well as for HPV structural proteins with IP-PAP technique. The local immunocompetent cell (ICC) infiltrates are analysed using ANAE technique to define B cells, MPS cells and T cells and monoclonal antibodies (McAb) for T cells subsets, NK (natural killer) cells and Langerhans cells. The relative levels of B- and T lymphocytes and MPS cells did not correlate with the clinical course, i.e. regression (RE), persistence (PE) or progression (PR) of the HPV lesions. The same was true with the expression of HPV antigens, intensity of the ICC infiltrate, as well as with the relative levels of NK (HNK-1+) cells and Langerhans (OKT-6+) cells. The T helper/T suppressor cell ratio was subject to minor fluctuations only as determined in three subsequent biopsies, and did not bear any meaningful relationship to the natural history of the HPV lesions. During the follow-up, 24.0% of the HPV lesions regressed, 54.9% remained persistent, and 21.1% progressed, 26 (6.2%) having been coned due to progression into CIS. The clinical course was most significantly influenced by the grade of HPV-associated CIN, to which RE was inversely and PR directly related. The results clearly confirm that cervical HPV infections are capable of progressing into CIS and thus show a natural history equivalent to that of classical CIN. Therefore, cervical HPV infections should be regarded as truly precancerous lesions, which should be examined, treated and followed-up with the same concern as the classical CIN. PMID- 3002295 TI - Light and electron microscopic immunocytochemistry of the pituitary gland of the tortoise. AB - The pars distalis of the tortoise, Geoclemis reevesii, was studied immunocytochemically with regards to its fine structure and distribution of different cell types. The pars distalis consists of cephalic and caudal lobes and a zona tuberalis, each with distinct cellular components. The cephalic lobe contains ACTH cells and prolactin (PRL) cells, while the caudal lobe consists of STH (GH) cells, TSH cells and FSH/LH (GTH) cells. The zona tuberalis contains GTH cells and a small number of TSH cells. ACTH cells, found exclusively in the cephalic lobe, are amphophilic cells containing ACTH immunoreactive secretory granules 200-240 nm in diameter. PRL cells occur only in the cephalic lobe and are large oval or columnar cells containing large secretory granules 300-500 nm in diameter. The cells immunoreactive to anti-chicken LH serum are also reactive to anti-chicken FSH serum, and were identified as GTH cells. They are distributed in the caudal lobe and zona tuberalis, with a few in the pars tuberalis. The GTH cells contain numerous secretory granules of varying density and diameter 200-280 nm, and large globules. TSH cells are small rounded cells containing small secretory granules 180-240 nm in diameter, and distributed in the caudal lobe and zona tuberalis. After thyroidectomy, these cells are hypertrophied and develop to thyroidectomy cells containing large vacuoles and a few secretory granules. GH cells are typical orangeophiles distributed in the caudal lobe. They contain secretory granules 200-400 nm in diameter. The zona tuberalis, which covers the antero-lateral portion of the cephalic lobe, is closely similar in cellular constitution to that of caudal lobe. It may presumably be a structure extended from the caudal lobe and not a homologue of the mammalian zona tuberalis. PMID- 3002296 TI - [Chromatographic artefacts of drugs of the 3,5-pyrazolidinedione group. 2. "Post photoeffect" of irradiated silica gel plates]. PMID- 3002297 TI - Degranulation inhibition. A potential mechanism for control of neutrophil superoxide production in sepsis. AB - Previous studies with neutrophils from patients with intra-abdominal sepsis have provided convincing evidence of in vivo exposure to C5a. However, in contradistinction to normal cells pretreated with C5a, patient cells showed depressed superoxide response to N-formyl-methionyl-leucyl-phenyl-alanine (FMLP) and enhanced FMLP receptor affinity. To identify possible mechanisms responsible for these findings, we examined the effects of lysosomal alkalinization with the weak base clindamycin on normal neutrophils with and without C5a. Our results showed a specific suppression of FMLP-induced superoxide production and a loss of low-affinity FMLP receptors. These results occurred in the presence of clindamycin levels that did not interfere with other cellular processes. These findings suggest that regulation of neutrophil function during the course of intra-abdominal sepsis may be due to effectors active both at the cell surface (C5a) and within the lysosome. The clinical significance of our findings relates to a possible mechanism for specific pharmacologic suppression of oxide-radical production by neutrophils. Such oxide radicals are believed to be important in the capillary injury accompanying severe sepsis. PMID- 3002299 TI - Demonstration of non-degraded Aleutian disease virus (ADV) proteins in lung tissue from experimentally infected mink kits. Brief report. AB - Isolates of ADV replicate to rather high quantities in lungs from neonatally infected mink kits. The virus was analysed for polypeptide composition, and for the first time high molecular weight polypeptides have been observed in in vivo produced ADVs. These polypeptides are analogous to those of in vitro produced ADVs. The molecular weights of the structural polypeptides of the low virulence Pullman ADV and the highly virulent DK and Utah I isolates of ADV were found to be 88kD and 78kD and in vivo produced ADV-G polypeptides were found to be 85kD and 75kD, the same molecular weights as those described for in vitro produced ADV G. Presence of the ADV coded, non-structural polypeptide with the molecular weight of 71kD (p71) was also demonstrated in the lung tissue from mink kits. PMID- 3002298 TI - HSV infected RAJI-cells specify HSV specific immediate early and/or early DNA binding proteins. AB - Herpes Simplex Virus specified DNA-binding proteins were purified from virus infected VERO and RAJI cells. The expression of the proteins differed depending on the type of the host cell. Polypeptides corresponding to HSV-1 specific ICP 8 and HSV-2 specific ICSP 11/12 were the major products of HSV-infected VERO cells. In RAJI cells polypeptides with a corresponding molecular weight, together with a cellular protein having almost similar mobility on SDS gels could be detected. Using immuno-blotting technique the HSV origin of these 135K molecular weight proteins synthesized in the HSV-1 and HSV-2-infected RAJI cells could be confirmed. PMID- 3002300 TI - Reactivity patterns to human rotavirus strains of a monoclonal antibody against VP2, a component of the inner capsid of rotavirus. Brief report. AB - A non-neutralizing monoclonal antibody (YO-60) against human rotavirus was found to be directed to VP2 (90,000-dalton protein), one of the two major components of the inner capsid. The reactivity patterns of the YO-60 antibody were very similar, though not identical, to those of subgroup II-specific YO-5 monoclonal antibody directed to VP6 (42,000-dalton protein), the other major component of the inner capsid. These results indicated the possible presence of a subgroup specific antigen on VP2 in addition to the one on VP6. PMID- 3002303 TI - [Experimental studies on toluene-diisocyanate (TDI) specific IgG antibodies. (I). Detection in the serum from toluene-diisocyanate skin-sensitized mice]. PMID- 3002301 TI - Persistent infection of rat insulinoma cells with Coxsackie B4 virus. Brief report. AB - Plaque purified Coxsackie B4 virus (CB4) caused a persistent infection of rat insulinoma (RIN) cells which lasted for the 70 day observation period. Infectious virus was produced and no cytopathic effect was observed. Indirect immunofluorescence and infectious center assays demonstrated that a majority of RIN cells were infected. Defective interfering particles were not demonstrated. Despite persistent infection, insulin synthesis by RIN cells was not altered. PMID- 3002302 TI - Increased sugar transport in BHK cells infected with Semliki Forest virus or with herpes simplex virus. AB - Infection of BHK cells by SFV increases the rate of uptake of [3H]MeGlc and of [3H]dGlc at approximately 2 hours p.i. Infection by HSV increases the uptake of [3H]MeGlc and [3H]dGlc at approximately 10 hours p.i.; the increased uptake is prevented by acyclovir. It is concluded that an increased sugar uptake by infected cells reflects an increased rate of transport across the plasma membrane and is the result of cellular changes caused by virus infection. PMID- 3002304 TI - [Studies on changes in histamine levels in the blood of infants and children. Part 7. The time studies of histamine, lipoxygenase metabolites, thromboxe B2 and cyclic AMP release from human platelets and leucocytes on treadmill exercise in asthmatic children]. PMID- 3002305 TI - The role of presynaptic beta-receptors in Raynaud's disease. AB - This report concerns the results of a study carried out to show the presence of presynaptic Beta adrenergic receptor supersensitivity in RD patients and the possibility to prevent or at least decrease this supersensitivity with Beta blocker treatment. It is shown that the paradoxical vasoconstriction response to ISP infusion can be prevented in RD patients by the acute administration of Beta blocker, such as Atenolol or DL Propranolol. Moreover, chronic treatment with the correct Beta-blocker was seen to prevent angiospastic crisis in these patients. PMID- 3002307 TI - Cyclic nucleotides and growth regulation of the mandibular condylar cartilage of the rat in vitro. AB - By exogenous interference in the intracellular level of cAMP and cGMP during growth in vitro without and with compression, an indication was obtained for the mediatorial involvement of these cyclic nucleotides in the major growth-processes in the mandibular condylar cartilage of 4-day-old rats. Raising the intracellular level of cAMP reduced proliferative activity in the prechondroblast zone, did not affect matrix synthesis by the functional chondroblasts and stimulated the process of hypertrophy. Intracellular elevation of cGMP had an antagonistic effect; it stimulated proliferation as well as matrix synthesis, but had no effect on hypertrophy. In this specific type of cartilage, cGMP can be considered as a major secondary intracellular messenger for proliferation-stimulating continuous biomechanical stimuli. PMID- 3002306 TI - Improved lipid preservation by malachite green-glutaraldehyde fixation in rat incisor predentine and dentine. AB - A network of granules and filaments stained with malachite green-aldehyde (MGA) was observed in predentine in the spaces between the collagen fibres and many small granules were associated with these fibres in peripheral and circumpulpal dentine. The granules were present throughout the entire thickness of the dentine, especially at the periphery of the calcospherites, although scarce or absent at their centre. This MGA-stainable material (i) resisted demineralization in acetic acid and EDTA solutions, and (ii) differed from non-collagenous material and crystal ghosts which stained on control sections after prior demineralization. In predentine, transport and diffusion of MGA-stainable material occurred in the direction of the dentine in which this material was associated with periodical striation of mineralized collagen fibres. This suggests that true matrix-associated lipids might play a role in dentine mineralization. PMID- 3002308 TI - Mixed mesodermal tumor of the uterine cervix--a trial search for intermediate forms between carcinoma and sarcoma. PMID- 3002309 TI - Preterm labour: a rational approach to improve management. PMID- 3002310 TI - Evaluation of intracellular killing of bacteria by enriched populations of mouse peritoneal exudate neutrophils. AB - Elicited, mouse peritoneal exudate cells were fractionated by centrifugation on discontinuous Percoll density gradients. Two subpopulations of neutrophils, each of greater than 90% purity, were isolated at discontinuous density gradient interfaces different from the region of mononuclear cell enrichment (i.e., 1.0694 1.0871 and 1.0872-1 X 1002 g/ml for neutrophils and less than 1.0694 g/ml for mononuclear cells). Peritoneal exudate cells were mixed with Proteus mirabilis in the presence of 1% normal mouse serum for 30 min. The mixtures were fractionated on gradients of Percoll diluted with a calcium-free medium. Populations of cells banding at densities greater than 1.0693 g/ml were washed free of gradient material, and neutrophil suspensions containing intracellular bacteria and which were relatively free of extracellular bacteria were isolated. Less than 7% of the total bacteria present was extracellular. The continuing extracellular presence of a heat-labile component of normal mouse serum was essential for maximal intracellular kill of P. mirabilis by mouse peritoneal neutrophils. PMID- 3002311 TI - Molecular epidemiology and pathogenesis of ruminant herpesviruses including bovine, buffalo and caprine herpesviruses l and bovine encephalitis herpesvirus. AB - Restriction endonuclease DNA fingerprints of herpesviruses isolated from 3 unrelated epidemics of bovine encephalitis are similar to each other and totally different from bovine herpesvirus 1 (BHV1). Herpesviruses, antigenically related to BHV1, isolated from goats and buffalo have distinct DNA fingerprints. We propose that bovine encephalitis herpesvirus is prototypic of a new bovine herpesvirus type and that alpha herpes viruses from individual ruminant species are species specific. PMID- 3002312 TI - Isolation of equine herpesvirus 1 from the brain of a horse affected with paresis. PMID- 3002313 TI - Congenital and perinatal cytomegalovirus infections. PMID- 3002314 TI - The incidence of cytomegalic inclusion disease (CID) in an obstetric teaching hospital, 1975-1984. AB - A low incidence (1 in 4,000) of neonatal CID was found in 47,320 consecutive live births at an obstetric hospital over a decade. The mortality was high, 5 of 16 neonates died in hospital and, of those discharged, 7 were left with severe cerebral and/or ophthalmic handicaps. Minor, remediable conditions were also found in 7 infants. Previous studies have indicated that CMV infection occurs in 1-2% of all pregnancies and 10% of the infected neonates have signs of CID at birth. A highly significant increase in the incidence of antenatal complications was found in mothers delivered of cytomegalovirus (CMV)-infected infants which may have compounded the effects of the viral disease on the fetus. A CMV-specific defect in the maternal and neonatal immune systems is discussed. PMID- 3002315 TI - Endocrine correlates of susceptibility to motion sickness. AB - Motion sickness releases ACTH, epinephrine, and norepinephrine. We are interested in endocrine responses to motion sickness, in adaptive responses leading to the resolution of the syndrome, and in how antimotion-sickness drugs influence the endocrine responses. Susceptible or insusceptible subjects were administered antimotion-sickness drugs prior to stressful stimulation. Insusceptible subjects displayed more pronounced elevations of ACTH, epinephrine, and norepinephrine after stressful motion. Predrug levels of ACTH were higher in insusceptible subjects (p less than 0.01). Acute blockade of hormone responses to stressful motion or alteration of levels of ACTH by drugs was not correlated with individual susceptibility. No correlation was apparent between epinephrine and ACTH release. These endocrine differences may represent neurochemical markers for susceptibility to motion, stress, or general adaptability, and it may be that the chronic modulation of their levels might be more effective in preventing motion sickness than the acute blockade or stimulation of specific receptors. PMID- 3002316 TI - Sex-dependent effects of neonatally administered morphiceptin and D-ala-D-leu enkephalin on maze learning in rats. AB - Newborn rats were injected (ip) with morphiceptin [72.7 micrograms/kg (a mu-type opioid receptor agonist)], D-alanine2-D-leucine5-enkephalin [79.4 micrograms/kg (a delta-receptor agonist)], or saline for 7 days after birth. Testing on a complex maze on Day 25 revealed a significant sex-dependent facilitation of performance by the opioid peptides. Peptide-treated females performed better than the males on the first day of training as measured by errors. Opioid treatment increased mortality, as three times as many peptide-treated animals died in comparison to the saline control group. Peptide treatment did not affect locomotor activity measured in an open field. Weight at Day 24 was also affected by the peptide treatment, females and males injected with the opioids being lighter and heavier, respectively, than the control group. PMID- 3002317 TI - Effect of beta-funaltrexamine on retention of passive-avoidance conditioning. AB - Rats received administration of an opiate antagonist immediately following single trial passive-avoidance training. Retention of passive-avoidance conditioning was assessed 1 week after training. Compared to noninjected and vehicle-injected control groups, post-training naloxone (2.0 mg/kg) administration significantly increased retention. A comparable facilitation of retention was also observed when animals received post-training administration of beta-funaltrexamine (40 mg/kg). These data provide additional support for mu opiate receptor activity in the regulation of memory processes. PMID- 3002318 TI - Effects of preexposure flavor concentration on conditioned aversion and neophobia. AB - In Experiment 1, 128 experimentally naive, water-deprived rats (Rattus norvegicus) received pretraining access to either 0.25 or 1.5% saccharin, distilled water, or 2.0% saline, followed either by a pairing of 0.25 or 1.5% saccharin with an intraperitoneal injection of 0.15 M lithium chloride (LiCl) or by a pairing of distilled water with LiCl. Preexposure to either saccharin concentration reliably reduced conditioned aversion effects to 0.25% saccharin, relative to that for preexposure to distilled water or saline. But only preexposure to 1.5% saccharin reduced aversion effects to that concentration. In Experiment 2, 48 naive, water-deprived rats received preexposure procedures as in Experiment 1. Afterwards, the rats were tested for neophobia to 0.25 or 1.5% saccharin. Neophobia was reliably greater to the 1.5% concentration. However, preexposure to either saccharin concentration obliterated evidence for neophobia to saccharin, relative to that following preexposure to distilled water or saline. PMID- 3002319 TI - Human antigen-specific T hybridomas. AB - Production of antigen-specific hybridomas has been achieved by fusion of HGPRT negative mutant of Jurkat and antigen-specific TH cell lines. Selection of clones for specificity was best accomplished by the binding of radiolabeled hybrid clones to antigen-pulsed monolayers. Functional lymphokines such as IL-2 and BCGF were induced by antigenic stimulation in the presence of AC. AC function of EBV transformed autologous B cells was as efficient on a per cell basis as autologous monocytes. Loss of specificity and function was observed in the first 5-6 months of culture which was coincident with chromosome loss. Recovery of specific functional hybrids was accomplished by repeated subcloning during this period. After this time, hybrids have maintained function for at least one year. This methodology allows the study of certain T cell functions without the requirement of periodic restimulation with autologous monocytes. Since EBV-B cell lines can serve as AC to induce lymphokines, large quantities of material can be prepared for biochemical studies. PMID- 3002320 TI - Specific B lymphocytes efficiently pick up, process and present antigen to T cells. AB - To study the nature of the antigen bridge between specific B and T lymphocytes we have isolated from the same donor clones of human T cells and EBV-transformed B cells specific for tetanus toxoid (TT). We found that TT-specific B cells are extremely efficient in presenting TT (but not an unrelated antigen) to T cells, as they can trigger T cell proliferation in the presence of only 10(-12) M TT, a concentration that is approximately 10(4) times lower than that required for presentation by conventional antigen-presenting cells such as macrophages or non specific B cells. The high avidity of the interaction between specific B and T cells is reflected by the resistance to inhibition by antibodies against the T4 antigen. Experiments with fixed and chloroquine-treated B cells show that the role of surface antibodies is limited to the uptake of antigen and that in order to obtain presentation, the antigen has to be internalized, processed and subsequently displayed on the cell surface, where it becomes available for T cell recognition in an MHC-restricted fashion. PMID- 3002321 TI - Diet and cancer: a whirlwind odyssey through a sea of inconsistency. PMID- 3002322 TI - Dietary fiber in the Polish diet. PMID- 3002323 TI - Bovine mitochondrial DNA polymorphism in restriction endonuclease cleavage patterns and the location of the polymorphic sites. AB - Cleavage patterns of mitochondrial DNA (mtDNA) by restriction endonuclease analysis were examined in four Japanese Black cows, three Japanese Shorthorn cows, and six Holstein cows. Seventeen restriction enzymes which recognize six base pairs and two restriction enzymes which recognize four base pairs were used in this study. Polymorphism was observed with three restriction enzymes, HindIII, TaqI, and MspI, and was detected within the breeds. Nucleotide substitution was determined in the HindIII polymorphic site by DNA cloning and sequencing; this is C----T at position 10126 of the URF-3 region. Furthermore, the MspI and TaqI polymorphic sites were located on the physical map. PMID- 3002324 TI - Upstream activation of ribosomal RNA biosynthesis in Saccharomyces cerevisiae. AB - Yeast was transformed with eight recombinants that contained an rRNA minigene and upstream elements of rDNA in different orientations in the multi-copy yeast Escherichia coli shuttle vector, pJDB207. The effect of these elements of upstream rDNA on the initiation of transcription of the minigene at the site for rRNA biosynthesis was determined by using an S1 nuclease mapping procedure to measure the abundance of the minigene transcript in RNA from the yeast transformants. Transcription of the minigene was enhanced 3-fold by DNA within a 2.2 kb element more than 1.5 kb upstream from the initiation site. Inversion of the 2.2 kb element decreased expression of the minigene by 40%. This 2.2 kb element contained approx. 500 bp from the 25S rRNA coding region at the 3' end of the preceding rRNA gene and 1 kb of adjacent nontranscribed spacer rDNA. The enhancing activity was independent of interference from readthrough that might have contributed to the 7-fold decrease in minigene expression caused by removing all rDNA upstream from -209 bp. PMID- 3002325 TI - ADP-ribosyltransferase in isolated nuclei during the cell cycle of Physarum polycephalum. AB - ADP-ribosyltransferase was measured in isolated nuclei of Physarum polycephalum. Activity was determined with and without exogenous DNA and histones. During the synchronous cell cycle the activity measured with exogenous substrates exhibited a typical peak enzyme pattern with a maximum of activity in S-phase, whereas activity measured without exogenous substrates displayed a step enzyme pattern. Both activities doubled in each cell cycle. PMID- 3002326 TI - Inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate formation in Ca2+ mobilizing-hormone-activated cells. AB - The inositol trisphosphate liberated on stimulation of guinea-pig hepatocytes, pancreatic acinar cells and dimethyl sulphoxide-differentiated human myelomonocytic HL-60 leukaemia cells is composed of two isomers, the 1,4,5 trisphosphate and the 1,3,4-trisphosphate. Inositol 1,4,5-trisphosphate was released rapidly, with no measurable latency on hormone stimulation, and, consistent with its proposed role as an intracellular messenger for Ca2+ mobilization, there was good temporal correlation between its formation and Ca2+ mediated events in these tissues. There was a definite latency before an increase in the formation of inositol 1,3,4-trisphosphate could be detected. In all of these tissues, however, it formed a substantial proportion of the total inositol trisphosphate by 1 min of stimulation. In guinea-pig hepatocytes, where inositol trisphosphate increases for at least 30 min after hormone application, inositol 1,3,4-trisphosphate made up about 90% of the total inositol trisphosphate by 5-10 min. In pancreatic acinar cells, pretreatment with 20 mM-Li+ caused an increase in hormone-induced inositol trisphosphate accumulation. This increase was accounted for by a rise in inositol 1,3,4-trisphosphate; inositol 1,4,5 trisphosphate was unaffected. This finding is consistent with the observation that Li+ has no effect on Ca2+-mediated responses in these cells. The role, if any, of inositol 1,3,4-trisphosphate in cellular function is unknown. PMID- 3002327 TI - Inhibition of platelet-activating-factor-induced human platelet activation by prostaglandin D2. Differential sensitivity of platelet transduction processes and functional responses to inhibition by cyclic AMP. AB - It has been proposed that cyclic AMP inhibits platelet reactivity: by preventing agonist-induced phosphoinositide hydrolysis and the resultant formation of 1,2 diacylglycerol and elevation of cytosolic free Ca2+ concentration [( Ca2+]i); by promoting Ca2+ sequestration and/or extrusion; and by suppressing reactions stimulated by (1,2-diacylglycerol-dependent) protein kinase C and/or Ca2+ calmodulin-dependent protein kinase. We used the adenylate cyclase stimulant prostaglandin D2 to compare the sensitivity to cyclic AMP of the transduction processes (phosphoinositide hydrolysis and elevation of [Ca2+]i) and functional responses (shape change, aggregation and ATP secretion) that are initiated after agonist-receptor combination on human platelets. Prostaglandin D2 elicited a concentration-dependent elevation of platelet cyclic AMP content and inhibited platelet-activating-factor(PAF)-induced ATP secretion [I50 (concn. causing 50% inhibition) approximately 2 nM], aggregation (I50 approximately 3 nM), shape change (I50 approximately 30 nM), elevation of [Ca2+]i (I50 approximately 30 nM) and phosphoinositide hydrolysis (I50 approximately 10 nM). A 2-fold increase in cyclic AMP content resulted in abolition of PAF-induced aggregation and ATP secretion, whereas maximal inhibition of shape change, phosphoinositide hydrolysis and elevation of [Ca2+]i required a greater than 10-fold elevation of the cyclic AMP content. This differential sensitivity of the various responses to inhibition by cyclic AMP suggests that the mechanisms underlying PAF-induced aggregation and ATP secretion differ from those underlying shape change. Thus a major component of the cyclic AMP-dependent inhibition of PAF-induced platelet aggregation and ATP secretion is mediated by suppression of certain components of the activation process that occur distal to the formation of DAG or elevation of [Ca2+]i. PMID- 3002328 TI - Acylphosphatase from human skeletal muscle: purification, some properties and levels in normal and myopathic muscles. AB - Human skeletal muscle acylphosphatase was purified by immunoaffinity chromatography using anti-horse muscle acylphosphatase antibodies. The three forms of the enzyme present in human muscle are very similar to those found in muscles of other animal species. The two main forms, Hu 1 and Hu 3, were also characterized with respect to molecular weight and some kinetic properties. Levels of acylphosphatase activity were measured in specimens of muscle from normals and from patients with various forms of muscular dystrophies and other myopathies. Acylphosphatase activity appears to be lower in all myopathic forms considered than in controls, and seems to be correlated with percentage of Ca2+ activation of (Ca2+ + Mg2+)-ATPase. PMID- 3002329 TI - [Chemical synthesis, isolation and sequencing of tetradecadeoxyribonucleotides containing the modified bases 5-fluorouracil and 5-methylcytosine]. AB - It is reported on phosphate-triester synthesis, isolation and sequencing of five tetradecadeoxyribonucleotides containing the modified bases 5-fluorouracil and 5 methylcytosine. The modified nucleotides can be exactly determined in the oligonucleotide chain by solid-phase MAXAM/GILBERT-sequencing. By combination of the oligonucleotides new hemi-modified DNA-duplexes were constructed which serve as model compounds for investigating the interaction with the EcoRII restriction and modification enzymes. PMID- 3002330 TI - Proteolysis during in vitro-maturation of rabbit reticulocytes. AB - We investigated the ATP-dependent proteolysis, cytochrome oxidase and the succinate-cytochrome c-oxidoreductase in reticulocytes under different conditions of incubation in vitro at 37 degrees C. Under standard conditions the proteolysis virtually stops after 4 h. The degradation of the stroma proteins amounted to 30 65% depending on the percentage of reticulocytes. The decrease of the cytochrome oxidase amounted to 70% after 20 h of incubation. Inhibition of the cytosolic reticulocyte lipoxygenase (LOX) by the reversible inhibitor salicylhydroxamate (SHAM) leads to an inhibition of both proteolysis and cytochrome oxidase activity by about two-thirds after 20 h of incubation. In the presence of a Ca++-ionophore the rate of proteolysis was increased by 33%, while the cytochrome oxidase and succinate-cytochrome c-oxidoreductase activities both decreased more rapidly than in the control experiments. PMID- 3002332 TI - Microviscosity of water-containing heterogeneous systems. An ESR spin probe study. AB - The microstructure of maltodextrin gels was studied by ESR spin probe technique. From investigations of the time dependence of the gelling process and from the Arrhenius plot of the rotational correlation time, important information about the phase behaviour of the system is obtained. The microviscosity decreases with swelling time, whereas the macroviscosity increases. The conclusion is drawn that the maltodextrin gel is a two-phase system and can be characterized in the nomenclature of Papkov as a gel of the second type. PMID- 3002331 TI - The use of progress curves for the estimation of inactivation rate constants of enzymes. AB - Progress curve analysis is applied to the estimation of inactivation rate constants. The heat inactivation of inorganic pyrophosphatase from baker's yeast was studied assuming a simple one-substrate mechanism containing two irreversible inactivation steps for the free enzyme and the enzyme-substrate complex, respectively. Four kinetic parameters including Km simultaneously may be detected from one progress curve. The temperature dependences derived from the estimated rate constants for several inactivating temperatures agree well with that obtained from preincubation measurements. PMID- 3002333 TI - Restriction endonuclease cleavage map of the DNA of hamster papovavirus. AB - A detailed restriction map of hamster papovavirus (HaPV)-DNA has been constructed on the basis of the complete nucleotide sequence of the viral genome. The HaPV DNA restriction map is totally divergent from those of all other recently known papovaviruses. This map will be useful for future functional studies of the HaPV genome. PMID- 3002334 TI - Biosynthesis of beta-endorphin in pancreatic islets of neonatal Wistar rats. AB - Pancreatic islets of neonatal Wistar rats were isolated by collagenase digestion and incubated with [14C]-L-prolin. Using a specific beta-endorphin antiserum radioactivity was found in the antibody-antigen-complex. The radioactivity was displaced by unlabelled beta-endorphin. These findings give the first evidence for the biosynthesis of beta-endorphin or beta-endorphin-like peptides in mammalian pancreatic islets. PMID- 3002335 TI - Significance of Cys-153 for the phosphatase activity of glyceraldehyde-3 phosphate dehydrogenase. AB - After oxidation of the catalytically active sulfhydryl group of Cys-149, glyceraldehyde-3 phosphate dehydrogenase is known to acquire an acylphosphatase activity. Modification of one cysteine residue, obviously Cys-153, with p mercuribenzoate or N-ethylmaleimide fully inactivates the phosphatase activity of the oxidized enzyme suggesting that this amino acid residue is involved in the reaction. An acyl transfer between Cys-153 and the sulfenic acid derivative of Cys-149 in the mechanism of the phosphatase reaction could explain the data obtained. Compounds which contain heteroatoms at two adjacent carbon atoms such as alpha-amino acids, peptides, EDTA and o-phenanthroline are shown to inhibit the phosphatase reaction of glyceraldehyde-3-phosphate dehydrogenase. These compounds bind in the active centre region close to Cys-153 and may interfere with the acyl transfer reaction thus inhibing the overall phosphatase reaction. PMID- 3002336 TI - Action of (D-Pro4)-beta-casomorphin1-5 on processes of synaptic transmission. AB - The peptide (D-Pro4)-beta-casomorphin1-5 is a potent and long acting analgesic. Furthermore it is able to antagonize apomorphine-induced behavioral patterns, which are preferentially used as screening methods to detect dopaminolytic or neuroleptic properties. Because all of these tests do not exclude interaction of drugs with transmission systems other than the dopaminergic, biochemical studies were undertaken to estimate possible influences of the opioid peptide on processes of dopaminergic, serotonergic, and cholinergic transmission systems. In lower concentrations (D-Pro4)-beta-casomorphin1-5 enhances the potassium stimulated release of acetylcholine from hippocampal slices and the basal overflow of dopamine from striatal slices. In high concentrations an augmentation of the potassium evoked release of dopamine and a reduction of the binding of [3H]spiperone on dopaminergic and serotonergic striatal receptors could be observed. These biochemical findings are discussed with regard to the behavioral patterns induced by this opioid peptide. PMID- 3002338 TI - The N-terminal portion of an erythrotropin-like peptide from fetal bovine serum has sequence homology with the LDL-receptor and two proteins of the Epstein-Barr virus. AB - An erythrotropin-like peptide (ELP) and bovine serum erythrotropin (ET) coeluted after more than four chromatographic steps. ELP was separated from ET on gel permeation high pressure liquid chromatography due to its large molecular weight. The effect of ELP on the stimulation of [3H] Thymidine incorporation in cell cultures of fetal bovine liver was much lower than the effect of ET. The N terminal amino acid sequence of ELP indicated that it has a group of amino acids identical to portions of the LDL-receptor and the hypothetical proteins BHLF1 and BOLF1 deducted from the DNA sequence of the Epstein-Barr virus. These results are consistent with the idea that cell surface proteins have structural homologies with secreted proteins or some growth factor-related peptides. PMID- 3002337 TI - Unique pyrimidine 2D-COSY aromatic cross-peaks as monitors of pyrimidine environments and mobility in oligo- and polynucleotides. AB - Only cytosine contains two adjacent aromatic protons that give rise to cross peaks in the aromatic region of 2D-COSY spectra of oligodeoxynucleotides. In two GC-containing sequences several such cross-peaks were resolved. The intensity of these cross-peaks is a sensitive monitor of local mobility, and upon the addition of the intercalating drug daunomycin selective intensity losses were observed, indicating binding to specific GC base pairs. We have also monitored the 2D-COSY cross-peaks from mobile pyrimidine bases in tRNA (Phe) as a function of temperature. PMID- 3002340 TI - Synthesis and in vitro activity of a hormone-diphtheria toxin fragment A hybrid. AB - The synthesis and in vitro biological activity of a hybrid protein composed of intact human chorionic gonadotropin and fragment A of diphtheria toxin in a disulfide conjugate is reported. This hybrid retained greater than 90% of the binding ability of uncoupled hCG and was shown to be specifically toxic to a mouse Leydig cell tumor which binds hCG while being non-toxic towards cells which lack receptors for hCG. PMID- 3002341 TI - Solubilization of L-triiodothyronine binding site from human erythrocyte membrane. AB - A thyroid binding peripheral membrane protein(s) has been characterized in human red cell. Two classes of affinity sites for triiodothyronine have been demonstrated. The high affinity, low capacity site showed values for dissociation constant of 2 X 10(-10)M. The binding activity depended on the presence of free SH group and showed a high stereospecificity for L-triiodothyronine, L-thyroxine was less potent (about 1,000-fold) than L-triiodothyronine in competing for this site. The results are discussed with respect to their cellular significance. PMID- 3002339 TI - Structural features involved in the biological activity of insulin and the insulin-like growth factors: A27 insulin/BIGF-I. AB - A synthetic insulin-like compound consisting of the A-chain of insulin extended at its carboxyl terminus with the hexapeptide "D-domain" of insulin-like Growth Factor II, linked via disulfide bonds to a B-chain corresponding to the "B domain" of insulin-like Growth Factor I, has been examined for insulin-like metabolic activity and for mitogenic activity. The synthetic material (A27 insulin/BIGF-I) is less potent than insulin in metabolic assays, and less potent than both insulin and IGF-I in mitogenic assays. It is proposed that neither the "D-domain" nor the "B-domain" of the IGFs is a major contributor to mitogenic activity. Their presence in the same molecule does not result in significant growth-promoting activity. PMID- 3002342 TI - Gonadotropin releasing hormone activation is mediated by dimerization of occupied receptors. AB - A dinitrophenyl (DNP)-derivative of a gonadotropin releasing hormone (GnRH) antagonist was prepared by chemical modification of the epsilon amino group in position 6 of [D-pGlu1,D-Phe2,D-Trp3,D-Lys6]GnRH with 1-fluoro-2, 4 dinitrobenzene. The DNP-antagonist D-pGlu-D-Phe-D-Trp-Ser-Tyr-D-Lys(N epsilon DNP)-Leu-Arg-Pro-Gly-NH2, retained high affinity binding to the GnRH receptor of pituitary membrane preparations and exhibited antagonistic activity when assayed in cultured pituitary cells. Both antibodies against DNP and their Fab fragments were able to bind the DNP-antagonist. However, only the addition of bivalent antibodies (and not the Fab fragments) converted the DNP-antagonist to an agonist. These results suggest that divalency is a critical factor in GnRH action. PMID- 3002343 TI - Uterine changes on removal of submaxillary glands in rats (histological changes/uterine peroxidase/blood estradiol level). AB - Surgical removal of submaxillary gland in immature rats causes a large increase in size and about three to four fold increase in dry and wet weight of uterus compared to that of the sham operated animals of the same age group. Histological examination reveals a significant increase in the diameter of the uterus with considerable elongation of the luminal epithelium from cubical to columnar in the experimental group. Biochemical studies show that the uterine peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7), a marker enzyme for uterine growth, increases by ten to fifteen fold on submaxillariectomy and returns almost to the normal level on administration of submaxillary gland extract (105,000 X g supernatant) to the submaxillariectomized animals. Estrogen estimation by radioimmunoassay shows a similar increase of three to four fold on removal of submaxillary glands and decrease almost to the normal value on administration of the submaxillary extract. PMID- 3002344 TI - Differential actions of phorbol ester and diacylglycerol on inhibition of granulosa cell maturation. AB - Hormonal induction of granulosa cell maturation is inhibited by phorbol esters and permeant synthetic diacylglycerols, but these activators of protein kinase C differ in their effects on cAMP production and actions. Both agents prevented the induction of luteinizing hormone receptors and progesterone biosynthesis by follicle-stimulating hormone, choleragen, and forskolin, but only diacylglycerol abolished the cAMP responses to these stimuli. Granulosa cell aggregation and aromatase activity were inhibited by phorbol ester but not completely by diacylglycerol. In intact granulosa cells, cytosolic C kinase activity was rapidly decreased by phorbol ester but unaffected by diacylglycerol. Although diacylglycerol has a marked inhibitory action on cAMP production, the more prominent suppression of granulosa cell differentiation by phorbol ester may be related to its rapid and prolonged action on kinase C. PMID- 3002345 TI - Cryptic urokinase binding sites on human foreskin fibroblasts. AB - Human foreskin cells possess sites on their surfaces that specifically bind both active and diisopropylphosphofluoridate-inactivated 2 chain 54 K Da [125I] urokinase, but do not bind the 54 K Da single chain form of urokinase. 125I urokinase bound to these sites is not internalized and is very slow to dissociate. There are about 40,000 available binding sites per cell. Brief incubation with pH 2.5 buffer at 5 degrees C unmasks another two to six fold more sites and also extracts plasminogen activator that, based on its accessibility to trypsin, appears to be at the cell surface. This suggests that the cryptic urokinase binding sites could be sites occupied with endogenous plasminogen activator. PMID- 3002346 TI - A new synthetic inhibitor of mammalian tissue collagenase inhibits bone resorption in culture. AB - A specific and potent synthetic inhibitor of mammalian tissue collagenase and related metallo-proteinases inhibits the collagen matrix resorption induced by parathyroid hormone (PTH) in cultured embryonic mouse calvaria. The inhibition is reversible, dose-dependent and virtually complete at 50 microM inhibitor concentration whereas that due to a less potent stereoisomer is much weaker. The PTH-enhanced secretion of calvarial lysosomal enzymes and the small spontaneous leakage of lactate dehydrogenase are not affected by the inhibitor. These results suggest that collagenase plays a critical role in bone resorption. Its role is discussed in relation to that of cysteine-proteinases that have also been implicated in this process. PMID- 3002347 TI - Sarcolemmal Na+-Ca2+ exchange during the development of genetically determined cardiomyopathy. AB - The UM-X7.1 myopathic and control hamsters at 40, 120 and 280 days of age were employed for the examination of heart sarcolemmal Ca2+-transport activities. Na+ dependent Ca2+ uptake activities were significantly depressed in myopathic animals at 120 and 280 days of age in comparison to the control values. No difference in Na+-induced Ca2+ release activities was found between control and experimental sarcolemmal vesicles. ATP-dependent Ca2+ binding and Ca2+ stimulated, Mg2+ ATPase activities were depressed in the experimental animals at 120 and 280 days of age. Similar alterations in the sarcolemmal Na+-dependent Ca2+ exchange and Ca2+-pump activities were seen upon treating the control hamsters with 40 mg/kg isoproterenol for 24 hr. It is suggested that a depression in the sarcolemmal Ca2+ transport activities may contribute to the development of intracellular Ca2+ overload in the genetically determined cardiomyopathy in hamsters and such a defect may be due to excessive amount circulating catecholamines in these animals. PMID- 3002348 TI - Core histone variants and ubiquitinated histones 2A and 2B of rat cerebral cortex neurons. AB - The pattern of non-allelic variants of core histones was investigated in terminally differentiated rat cerebral cortex neurons. At 30 days two major H2A variants are present, H2A.1 and .2, together with two minor components, .x and .z. H2B has two variants, H2B.1 and .2, and H3 presents three variants, H3.1, .2 and .3. The ubiquitinated adducts of all H2A and H2B variants can be recognised on two-dimensional electrophoresis as forming a pattern similar to that of the unmodified species. uH2A amounts to 12-14% of total H2A. All H2A variants appear to be equally modified. uH2B amounts to 1-2% of total H2B. PMID- 3002349 TI - Amino acid sequence similarity between spinach chloroplast and mammalian gluconeogenic fructose-1,6-bisphosphatase. AB - Chloroplast fructose-1,6-bisphosphatase is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars and the properties of this enzyme are clearly distinct from cytosolic gluconeogenic fructose-1,6 bisphosphatase. Light-dependent activation via a ferredoxin/thioredoxin system and insensitivity to inhibition by AMP are unique characteristics of the chloroplast enzyme. In the present study, purified spinach chloroplast fructose 1,6-bisphosphatase was reduced, S-carboxymethylated with iodoacetic acid, and cleaved with either cyanogen bromide or trypsin. The resulting peptides were purified by reversed-phase high performance liquid chromatography. Automated Edman degradation of some of the purified peptides showed amino acid sequences highly homologous to residues 72-86, 180-199, and 277-319 of pig kidney fructose 1,6-bisphosphatase. These findings suggest a common evolutionary origin for mammalian gluconeogenic and chloroplast fructose-1,6-bisphosphatase, enzymes catalyzing the same reaction but having different functions and modes of regulation. PMID- 3002350 TI - Atrial natriuretic peptide secretion: synergistic effect of phorbol ester and A23187. AB - To investigate the role of inositol phospholipid turnover in the atrial natriuretic peptide (ANP) secretion, the secretory responses from isolated perfused rat hearts to the ionophore, A23187, and the phorbol ester, 12-O tetradecanoylphorbol-13-acetate (TPA), alone or in combination, were studied. A23187 induced a sharp increase in ANP secretion, whereas TPA caused a slowly progressive increase in secretion rate. 4 alpha-phorbol-12,13-didecanoate, which lacks the ability to activate protein kinase C, had no effect on ANP secretion. The combination of A23187 and TPA stimulated ANP secretion higher than the calculated additive value for each agent. The synergistic effect of the agents suggests a role of calcium-activated protein kinase C in ANP secretion from atrial cardiocytes. PMID- 3002351 TI - The role of the carbohydrate moiety in thyrotropin action. AB - The relative binding affinity of deglycosylated human TSH was 6-fold higher than that of native TSH. Although deglycosylated human TSH significantly stimulated adenylate cyclase, it was less effective than the native hormone. When deglycosylated human TSH was added with bovine TSH, however, a dose-dependent antagonism was observed. In particular, submaximal and maximal concentrations of bovine TSH and deglycosylated human TSH resulted in cAMP values much lower than the sum of activities of the individual hormones. The data suggest that although the effects of TSH deglycosylation are not as dramatic as with the gonadotropins, the carbohydrates of TSH appear to be required for maximal activation of adenylate cyclase by the hormone. PMID- 3002352 TI - Regulation of the glucose enhanced insulin pool. AB - Exposure of islets to high levels of glucose at a critical time leads to enhanced insulin release when later stimulated by glucose. Newly synthesized insulin is preferentially released with subsequent stimulation, implying the creation or enlargement of a separately regulated pool of insulin in response to the initial stimulus. Epinephrine via beta adrenergic receptors can trigger the discharge of the enhanced insulin pool via a beta adrenergic receptor response. Raising intracellular cAMP levels or stimulation by arginine also discharge the marked pool. The enhanced pool is accessible by several independent mechanisms. PMID- 3002353 TI - Differential effects of phorbol ester on the beta-adrenergic response of normal and ras-transformed NIH3T3 cells. AB - The basal and isoproterenol stimulated levels of cyclic AMP in NIH3T3 and H-ras transformed NIH3T3 cells were equivalent. In exponentially growing cells, the phorbol ester 12-O-tetradecanoyl-13-acetate (TPA) inhibited the beta-adrenergic response of NIH3T3 cells, but not of the ras-transformed line. Another line of NIH3T3 cells transformed by a non-ras gene exhibited the normal loss of beta adrenergic response by the tumor promoter. These results are consistent with a role for p21 in signal transduction related to the effects of TPA. PMID- 3002354 TI - Collagenase synthesis in clonal rabbit odontoblast-like cells (RP cells). AB - Post-confluent cultures of cloned rabbit odontoblast-like cells (RP: Rabbit Pulp cells) produced latent collagenase, which was isolated from serum-containing culture media by heparin-Sepharose affinity chromatography. RP collagenase was purified 4,420-fold from serum-containing medium to a specific activity of 1,313 units/mg with a yield of 14% by heparin-Sepharose affinity chromatography, carboxymethyl-cellulose ion-exchange chromatography, and by immunoadsorption chromatography. The RP cell culture appears to be a suitable model to use for studying collagenase synthesis in mineralized tissue-forming cells and for elucidating the mechanism of collagen degradation in pulp tissue and dentin matrix. PMID- 3002356 TI - Receptors for insulin-like growth factor-I (IGF-I) in myocardial capillary endothelium of the intact perfused heart. AB - Isolated intact, beating hearts were perfused with HPLC-pure [125]-IGF-I (1 ng/ml) alone or [125]-IGF-I (1 ng/ml) plus varying concentrations of unlabeled IGF-I (10-3,000 ng/ml) or unlabeled insulin (1,000-100,000 ng/ml). After 1 min of perfusion with peptides, the hearts were rapidly fixed, sectioned and analyzed for radioautographic [125I] grain counts. Greater than 90% of [125I] grains were shown to represent intact [125I]-IGF. Maximal grain counts over capillaries occurred after perfusion with [125I]-IGF-I alone and decreased in a dose dependent manner when unlabeled IGF-I was coperfused. Coperfusion of [125I]-IGF-I with unlabeled insulin also decreased 125I grains over capillaries but less potently than unlabeled IGF-I. EM radioautography demonstrated that [125I]-IGF-I grains were localized over capillary endothelial cells. Thus, specific IGF-I receptors are present in the capillary endothelium of the intact heart and have properties similar to IGF-I receptors in cultured capillary endothelial cells. PMID- 3002355 TI - Calcium/phospholipid-dependent protein kinase and its relationship to antidiuretic hormone in toad urinary bladder epithelium. AB - The hydro-osmotic response of the toad urinary bladder to antidiuretic hormone (ADH) and cyclic AMP was inhibited by phorbol myristate acetate (PMA) and 4 beta- phorbol dideconate (4 beta-PDD), activators of protein kinase C (PKC). The inactive epimer of 4 beta-PDD, had no effect on the ADH response. The osmotic transfer of water in the absence of ADH was unaffected by PMA. PKC activity, localized in the soluble fraction of isolated toad bladder cells, was activated by PMA. ADH initially inhibited and subsequently stimulated 32Pi incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI). Carbachol, which inhibits ADH-induced water flow, also stimulated 32P incorporation into PA and PI. It is suggested that phosphoinositide breakdown to diacylglycerol may activate PKC which functions to attenuate the hormone-mediated permeability response. PMID- 3002357 TI - Cyclosporin A and rabbit mammary prolactin receptors. AB - The binding of cyclosporin A and ovine prolactin to rabbit mammary gland membranes was determined. CsA bound with a Kd of 2.2 X 10(-6)M whereas prolactin bound with a Kd of 2 X 10(-10)M. The binding of each ligand was an independent event and neither ligand influenced the binding of the other ligand showing that CsA does not inhibit the binding of prolactin to its specific receptor in this system. PMID- 3002358 TI - Cigarette tar causes single-strand breaks in DNA. AB - The results of this study demonstrate, for the first time, that cigarette tar causes DNA damage. Incubation in vitro of phage PM2 DNA with aqueous extracts of cigarette tar results in the introduction of DNA single-strand breaks. The effects of protective enzymes and radical scavengers indicate the involvement of active oxygen species. Although the semiquinone components of tar reduce dioxygen forming superoxide radicals and hydrogen peroxide, our results suggest that hydroxyl radicals formed via metal catalyzed decomposition of hydrogen peroxide are ultimately responsible for the DNA lesions. Our results also suggest that the metals in tar are reduced by the semiquinone components of tar and by superoxide at comparable rates. PMID- 3002359 TI - High resolution proton nuclear magnetic resonance spectroscopy of lactoperoxidase. AB - The first high resolution proton nuclear magnetic resonance spectra are reported for the native ferric and ferric cyano complexes of bovine lactoperoxidase. The spectrum of the native species exhibits broad heme signals in a far downfield region characteristic of the high-spin ferric state. The low-spin cyano complex yields a proton nuclear magnetic resonance spectrum with signals as far as 68.5 ppm downfield and as far as -28 ppm upfield of the tetramethylsilane reference. These peak positions are anomalous with respect to those seen only as far as 35 ppm downfield in other cyano hemoprotein complexes. An extreme asymmetry in the unpaired spin delocalization pattern of the iron porphyrin is suggested. The unusual proton nuclear magnetic resonance properties parallel distinctive optical spectral properties and the exceptional resistance to heme displacement from the enzyme. Lactoperoxidase utilized in these studies was isolated from raw milk and purified by an improved, rapid chromatographic procedure. PMID- 3002360 TI - The inhibition of progesterone secretion and the regulation of cyclic nucleotides by atrial natriuretic factor in gonadotropin responsive murine Leydig tumor cells. AB - We have found that atrial natriuretic factor (ANF) has a profound effect on testicular cells in altering intracellular cyclic nucleotide levels as well as progesterone secretion. Using clonal cultured Leydig tumor cells we found that 1 X 10(-8)M ANF caused a two thousand-fold elevation in the accumulation of cellular cGMP and inhibited cAMP in treated cells by more than 90% as compared to the controls. ANF (1 X 10(-8)M) also significantly inhibited gonadotropin stimulated accumulation of cAMP in response to bovine luteinizing hormone (bLH) or human chorionic gonadotropin (hCG). Gonadotropin-stimulated progesterone secretion was inhibited by ANF (1 X 10(-10) - 1 X 10(-9)M) in these cultured Leydig tumor cells. Approximately 50% inhibition of progesterone secretion was observed at the peptide concentration of 1 X 10(-9) M. PMID- 3002361 TI - Resistance to mutagenesis of cells biochemically transformed by herpesvirus DNA fragments. AB - LM(TK-) mouse fibroblast cells that were biochemically transformed to the dThd kinase-positive phenotype by restriction nuclease fragments of herpes simplex virus or marmoset herpesvirus DNA, all of which contained the virus dThd kinase coding region, or by HeLa S3 DNA were more resistant to mutagenesis by N-methyl N'-nitro-N-nitrosoguanidine or 5-bromodeoxyuridine than were dThd kinase-positive LM and LM(TK-) cells. Measurements of dNTP pool sizes did not reveal relative imbalances for representative cell lines under several conditions of growth. PMID- 3002362 TI - Estrogen 1,2-epoxides or estrogen quinones/semiquinones. AB - Metabolic activation of estradiol leading to the formation of catechol estrogens is a prerequisite for its genotoxic activity. Both estrogen-o quinones/semiquinones and estrogen 1,2-epoxides have been proposed to be responsible for this activity. Incubations of [3H]estradiol and [3H]1 alpha,2 alpha-epoxy-4-estrene-3-one-17 beta-ol (ketotautomer of estradiol 1,2-epoxide) with rat liver microsomal and cytosol preparations were carried out in the presence of SKF 525A, ascorbic acid, glutathione and cysteine. Ascorbic acid decreased binding to proteins and aqueous-soluble fraction with both [3H] estradiol and [3H]epoxyestrenolone in incubations with microsomes but no effect with cytosol fraction. Incubations of microsomes with thiols gave water-soluble metabolites which were characterized as 1(4)-thioether derivatives of 2 hydroxyestradiol and incubations of [3H]epoxyestrenolone with cytosol and thiols gave estradiol-2-thioether. Incubations with ascorbic acid and thiols resulted in decreased formation of water-soluble metabolites in microsomal incubations but not in cytosol incubations. These studies indicate that the major pathway for irreversible binding of estrogens to macromolecules involves estrogen-o quinones/semiquinones and not estrogen 1, 2-epoxide. PMID- 3002363 TI - Spin trapping of a free radical intermediate formed during microsomal metabolism of hydrazine. AB - A radical formed during oxidative metabolism of hydrazine in rat liver microsomes was spin-trapped with alpha-phenyl-t-butylnitrone. The trapped species was identified as hydrazine radical by comparison of its ESR parameters and mass spectrum with those of the adduct formed during CuCl2 catalyzed oxidation of hydrazine. The requirement for oxygen and NADPH in the microsomal oxidation and the occurrence of a typical binding spectrum by difference spectroscopy suggest the involvement of the participation of the cytochrome P-450 enzyme system in the formation of hydrazine radical which must be a precursor of diimide during microsomal oxidation of hydrazine. PMID- 3002364 TI - The effect of nitrite on cytochrome oxidase. AB - Nitrite inhibits the oxygen uptake by the system ferrocytochrome c-cytochrome oxidase with Ki = 1.5 mM. In the absence of ferrocytochrome c the oxygen uptake by cytochrome oxidase in the presence of nitrite was observed indicating that the enzyme has some nitrite oxidase activity. Nitrite induces changes in optical difference spectra of cytochrome oxidase and, in particular, the formation of the transient band at 607 nm. The reciprocal relation was observed between the intensity of this band and the rate of the oxygen uptake by cytochrome oxidase. This means that the form of the enzyme with this band does not involved in the nitrite oxidase activity. It is suggested that the nitrite oxidase activity relates to the oxygen binding site rather than the cytochrome c binding site of the enzyme. PMID- 3002365 TI - Bacterial vectors which confer resistance to kanamycin. AB - On the base of plasmid pLD720 (a deletion derivative of the cosmid vector pHC79) a number of hybrid plasmids which confer in Escherichia coli cells the kanamycin resistance was constructed. All hybrid plasmids contain the promoterless part of kanamycin resistance gene (which codes for aminoglycoside 3'-phosphotransferase II) from transposon Tn5. The Km gene expression is driven by a promoters situated on pLD720. The hybrid plasmids pLD723, pLD724 and pLD728 contain a complete DNA sequences of plasmids pC194 or pE194 from Staphylococcus aureus that permits them to replicate into Bacillus subtilis as well. However, no expression of the Km gene in Bacillus subtilis was observed. There is a unical Bgl II site on pLD728 is front of the beginning of a Km gene structural part. This property of pLD728 may be useful when cloning in this plasmid a promoter sequences of different species. PMID- 3002366 TI - The lack of a solvent accessible hydroxide or water ligand to iron at the 3Fe center of Azotobacter vinelandii ferredoxin I. AB - The X-ray crystal structure of Azotobacter vinelandii ferredoxin I (FdI) describes a planar 3Fe-3S center in which one of the iron atoms is ligated to a solvent accessible oxo ligand, presumably from water or hydroxide (Ghosh et al., (1982) J. Mol. Biol. 158, 73-109). Efforts to displace the proposed oxo ligand with cyanide were unsuccessful, even in 80% dimethylsulfoxide. In addition, comparison of the electron spin echo envelopes for H2O- and D2O-equilibrated samples of FdI showed only a slight deuterium modulation, far less than would be expected were water to be bound as an iron ligand. These results do not support the presence of a solvent accessible oxo ligand to the 3Fe center as described in the X-ray crystal structure. PMID- 3002367 TI - Measurement of the potential activity of the type-1 and type-2 protein phosphatases in the crude tissue extract. AB - Although activation and conversion of the inactive/latent high-molecular-weight phosphatases to the Mr = approximately 35,000 catalytic subunits can be achieved by several procedures including freezing/thawing in 0.2 M 2-mercaptoethanol and digestion by proteolytic enzymes, strong indications have been obtained that they are distinct stimulating processes. For instance, when pig brain extracts were treated with trypsin, the type-1 phosphatase could be stimulated approximately 40 fold while the type-2 phosphatase was not slightly modified. Conversely, when the same crude extracts were treated with freezing/thawing in 2-mercaptoethanol, the type-2 phosphatase could be stimulated approximately 10 fold but surprisingly the type-1 species was not slightly influenced. It is suggested that hormonal study on the activity changes of the type-1 and type-2 phosphatase in a crude extract can be made possible by the selective using of these two triggers. PMID- 3002368 TI - Concanavalin A and phorbol ester cause opposite subcellular redistribution of protein kinase C. AB - Concanavalin A and phorbol ester induce human blood monocytes to produce superoxide. We tested whether activation of human monocytes by these agents is accompanied by a subcellular redistribution of protein kinase C. Phorbol ester predictably caused a profound shift of the enzyme from the cytosol to the particulate fraction. In contrast concanavalin A induced a shift of the enzyme from the particulate fraction to the cytosol. The opposite effect of these agents on kinase C translocation was observed also by analysis of the phosphorylation of cytosolic proteins. Kinase C is either not involved in monocyte activation or does so by distinct pathways determined by the activating agent. PMID- 3002369 TI - Structural difference of the insulin receptors from circulating monocytes and erythrocytes. AB - We compared insulin receptors obtained from cells widely used in human studies, the circulating monocytes and erythrocytes. Biochemically, these receptors possess both binding (alpha-subunit) and tyrosine kinase (beta-subunit) activities similar to insulin receptors from other sources. Subtle differences in molecular weight, however, were detected between the alpha-subunits of these two cell types when analyzed by NaDodSO4-PAGE. Crosslinked [125I]insulin-labeled alpha-subunit of the monocyte insulin receptor was of higher apparent molecular weight than the alpha-subunit derived from red cells. Neuraminidase treatment of the alpha-subunits from each cell type indicated more sialic acid residues were present on the monocyte than the red cell alpha-subunit. The structural properties of the insulin receptors of human circulating cells are similar but not identical to insulin receptors of other characterized systems. PMID- 3002370 TI - Calmodulin regulation of cholinergic muscarinic receptor: effects of calcium and phosphorylating states. AB - Calmodulin (CaM) regulation of cholinergic muscarinic receptor was investigated using synaptic membrane isolated from rat brains and [3H]-QNB as a binding ligand. CaM exerts a biphasic effect on receptor binding showing both a Ca2+ dependent receptor loss and an increase depending on the state of membrane phosphorylation. Calcineurin, a CaM-dependent protein phosphatase, mimicked the stimulatory effect of CaM in a dose-dependent manner. CaM-antagonists, W-7 and TFP reversed the stimulatory effect by CaM. A mechanism of protein phosphorylation and dephosphorylation of the cholinergic muscarinic receptors regulated by CaM-Ca2+ was proposed. PMID- 3002371 TI - Solubilization and characterization of a membrane 3, 3', 5-triiodo-L-thyronine binding protein from rat pituitary tumor GH3 cells. AB - To understand the mechanism by which T3 enters cells and carries out its biological functions membrane binding sites for 3, 3', 5-triiodo-L-thyronine were solubilized from rat pituitary tumor GH3 cells by detergents. Among three detergents tested, CHAPS is the best in preserving hormonal binding affinity and specificity. Least square analysis of the binding data show one class of binding site with a Kd of (6.35 +/- 1.27) nM and Bmax of (0.84 +/- 0.056) pmoles/50 micrograms protein. Hormone binding activity is lost by heating, pronase digestion and in the absence of NaCl. The pH optimum for binding is 7.0 and the binding activity is enhanced by dithiothreitol. The solubilization of membrane associated thyroid hormone binding proteins will facilitate further characterization and exploration of their biological functions. PMID- 3002372 TI - Stimulation of cell proliferation and polyphosphoinositide metabolism in Saccharomyces cerevisiae GL7 by ergosterol. AB - The effect of ergosterol on cell division and phospholipid metabolism was investigated in Saccharomyces cerevisiae strain GL7, a sterol and unsaturated fatty acid auxotroph. Cells growing poorly on cholesterol were stimulated to grow more rapidly by supplementing the medium with 100 ng of ergosterol per ml. Within 10 min after ergosterol addition to cells prelabeled with 32Pi or [3H]inositol the isotope content of the polyphosphoinositides increases markedly followed by an equally striking and rapid decrease. Subsequently upon continuous labeling, 32P incorporation into phosphatidylinositol and, to a lesser degree, other phospholipids increased. Finally 3h after ergosterol addition the growth rate increased. Only stimulation of the first process, i.e. polyphosphoinositide metabolism, upon ergosterol addition is resistant to inhibition by cycloheximide. PMID- 3002373 TI - Specificity of the heat-stable protein inhibitor of the branched-chain alpha-keto acid dehydrogenase phosphatase. AB - A potent, heat-stable protein inhibitor of branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase has been identified and purified to near homogeneity from bovine kidney mitochondria (Damuni, Z., Humphreys, J. S., and Reed, L. J., Proc. Natl. Acad. Sci. U.S.A., in press). This protein is a noncompetitive inhibitor of BCKDH phosphatase, with a Ki about 0.13 nM. By contrast, this protein inhibitor did not affect the activity of the cytosolic protein phosphatase-1 and phosphatase-2A or the mitochondrial pyruvate dehydrogenase (PDH) phosphatase at concentrations up to 10 nM. The cytosolic protein phosphatase inhibitor-1 and inhibitor-2 had no effect on the activity of BCKDH phosphatase or PDH phosphatase at concentrations up to 50 and 300 nM respectively. These results, together with previous evidence, demonstrate that BCKDH phosphatase and its inhibitor protein are distinct from the cytosolic protein phosphatase-1 and phosphatase-2A and from protein phosphatase inhibitor-1 and inhibitor-2, respectively. PMID- 3002374 TI - Interaction of phosphorylase kinase with polymyxins. AB - Nonactivated rabbit skeletal muscle phosphorylase kinase is inhibited by the polymyxins A, B, D and E when assayed at pH 8.6. Polymyxin B is the most effective inhibitor, causing 50% inhibition at 0.3 mM. Following the effect of polymyxin B on the kinase activity toward troponin, no inhibition was observed. In contrast, polymyxin B was found to greatly stimulate the autophosphorylation of phosphorylase kinase. About 10 mol of phosphate per tetramer (alpha beta nu delta) were incorporated in presence of polymyxin B (full autophosphorylation). This incorporation was about 6-fold higher than that observed without polymyxin. The stimulation of autophosphorylation by polymyxin B was accompanied with enhancement of the rate of autoactivation at pH 6.8. PMID- 3002376 TI - Partial purification of a 5.7K glycoprotein from bovine vitreous which inhibits both angiogenesis and collagenase activity. AB - An inhibitor of angiogenic activity similar to that described by Raymond and Jacobson (1) has been partially purified and shown to be a glycoprotein of molecular mass 5,700. This inhibitor gave rise to an avascular zone on the chick vitelline plexus and also negated the action of a low Mr angiogenic factor. When the angiogenic inhibitor was incubated with mammalian collagenase it inhibited the enzyme activity by nearly 70%. The relevance of these findings to the role of collagenase in angiogenesis is discussed. PMID- 3002375 TI - Differential effects of tumor promoters on cAMP production: inhibition of receptor-mediated and potentiation of cholera toxin-mediated stimulation. AB - Tetradecanoylphorbol-acetate and other tumor promoters inhibit prostaglandin E2 and isoproterenol-induced cAMP accumulation in mouse thymocytes but markedly potentiate cAMP production induced by cholera toxin. Cholera toxin is known to stimulate cAMP production by inducing ADP-ribosylation of the alpha-subunit of a guanine nucleotide-binding regulatory (G) protein, resulting in activation of the catalytic unit of adenylate cyclase. G proteins have been implicated as plasma membrane transducers for a variety of additional signals. It is possible that the growth promoting and co-mitogenic properties of tumor promoters are related to their effects on G proteins. PMID- 3002377 TI - Isolation and partial characterization of distinct species of phosphotyrosyl protein phosphatases from rat spleen. AB - Three phosphotyrosine protein phosphatases (PTP-I PTP-II and PTP-III) inhibited by Zn2+ and active on the phosphotyrosyl residues included into the acidic co polymer poly (Glu, Tyr) 4:1 previously phosphorylated by a spleen tyrosyl kinase have been resolved and partially purified from rat spleen cytosol by DEAE cellulose chromatography followed by phosphocellulose chromatography and/or Ultrogel AcA 44 gel filtration. PTP-I (Mr 65,000 by gel filtration) was purified about 1000 folds. It is stimulated by EDTA and unaffected by either vanadate (50 microM) or molybdate (up to 25 microM). PTP-II (Mr 30,000) and PTP-III (Mr 50,000) are insensitive to EDTA and inhibited by molybdate. In addition PTP-III is also inhibited by vanadate. PMID- 3002378 TI - Activation of endogenous retroviral sequences in human leukemia. AB - Endogenous retroviral sequences have been identified in the genomes of several species including humans. Proteins similar to those of primate endogenous viruses have been found on the surface of various malignant cells in man. To further define this role, we have used a primate retrovirus DNA from the Baboon Endogenous Virus to probe a human genomic library for related sequences. A total of 45 clones homologous to BaEV gag-pol were isolated under low stringency hybridization. Of these, several were found to contain DNA which was expressed as RNA at higher levels in human lymphoid leukemic cells than in normal lymphocytes. PMID- 3002379 TI - Epinephrine decreases low density lipoprotein processing and lipid synthesis in cultured human fibroblasts. AB - The effect of epinephrine on 125I-low density lipoprotein (LDL) uptake and cholesterol metabolism was investigated after a 24 hours pretreatment of cultured human fibroblasts. Epinephrine decreased LDL uptake (binding + internalization) and degradation in a dose-dependent manner. Cholesterol synthesis from 14C sodium acetate and cholesterol esterification measured by 14C oleic acid incorporation into cholesteryl esters were also decreased. These results are in agreement with the general view that epinephrine increases cyclic AMP intracellular level, as it was previously demonstrated that dibutyryl cyclic AMP or isoproterenol treatment of cultured fibroblasts had similar effect on these pathways. The decrease in LDL processing induced by epinephrine could be involved in the worsening effect of epinephrine on atherosclerosis. PMID- 3002380 TI - Urokinase binding sites on human foreskin cells. Evidence for occupancy with endogenous urokinase. AB - In cultures of human foreskin fibroblasts most of the cell surface binding sites for 2-chain urokinase are masked and can be exposed by 10 min. incubation on ice at pH 2.5 (A. Bajpai and J.B. Baker (1985), Biochem. Biophys. Res. Commun.133, 475-482). Here we show that incubation on ice at pH 2.5 also releases from the cell surface a plasminogen activator that is similar to 2-chain urokinase in terms of its electrophoretic mobility, chromatographic behavior on concanavalin A Sepharose or p-amino-benzamidine-Sepharose, and sensitivity to anti-urokinase antibody. Two observations suggest that the masked binding sites are sites occupied by this cell surface urokinase. First, glucocorticoid-treated cells, which lack cell surface urokinase, have a large number of urokinase binding sites but none that are masked. Second, the extraction of surface urokinase and the exposure of urokinase binding sites exhibit similar pH dependence. Both are complete at about pH 4.0. PMID- 3002381 TI - Dipyridamole-induced interferon production in mouse peritoneal leukocytes. AB - The in vitro explanted mouse peritoneal leukocytes were used for the optimization of the dipyridamole-induced interferon production. After 90-120 min incubation of cells with 30-100 microM dipyridamole, the production of interferon reached 6.4 X 10(4) IU/ml. It was demonstrated that the interferon production phase is preceded by the dipyridamole-dependent increase in cAMP concentration. The possibility of cAMP involvement in the mechanism of interferon production is being discussed. PMID- 3002383 TI - Leukotrienes as mediators of ischemia and shock. AB - Leukotrienes have been implicated as mediators of ischemia and shock. Recent evidence has been obtained supporting the four major criteria of acceptance of leukotrienes as mediators of shock, namely (a) increased concentration in body fluids during shock states, (b) ability to exert significant pathophysiologic effects which aggravate ischemia and shock, (c) amelioration of the shock state by leukotriene synthesis inhibitors and leukotriene receptor antagonists, and (d) production of a shock-like state by exogenous administration of leukotrienes. In conclusion, both LTB4 and the peptide leukotrienes (e.g. LTC4, LTD4 and LTE4) also known as the slow reacting substance of anaphylaxis (SRS-A) can be considered as mediators of ischemia and shock. Although difficulties exist with measuring leukotrienes in circulating blood and in obtaining long lasting selective blockers of leukotriene synthesis, innovative experiments measuring leukotrienes in bile and other body fluids and in employing specific leukotriene receptor antagonists have helped in assessing the significance of the leukotrienes in shock states. Additional studies are necessary to evaluate these findings in perspective, and to compare and contrast the role of leukotrienes to that of other vascular mediators including prostaglandins and thromboxanes, as well as non-eicosanoids including serotonin, histamine, angiotensin II and vasopressin, all of which can play a mediator role in ischemia and shock states. Further clarification of these issues promises to open exciting new chapters in shock research. PMID- 3002382 TI - Coexistence of two ATP sites on the ouabain-complexed (Na+ + K+)-ATPase. AB - When the effects of varying concentrations of ATP on the dissociation rate of the ouabain-enzyme complex were studied, the dissociation rate constant increased with increasing ATP concentrations up to 1 mM, and then decreased with further rise in ATP; indicating that ATP binds to two distinct sites on the complex. ADP and AMP-PNP had similar biphasic effects. GTP, CTP, UTP, and AMP-PCP reduced the dissociation rate. AMP and Pi had no effects. Increase in dissociation rate caused by 0.5 mM ATP was not abolished by saturating CTP, indicating the binding of CTP to only one of the two ATP sites. The data suggest the existence of separate catalytic and regulatory sites, with different affinities and nucleotide specificities. PMID- 3002384 TI - Cytochrome P-450 induction by 3-methylcholanthrene and its antagonism by 2,2 dimethyl-5-t-butyl-1,3-benzodioxole. AB - Previous studies in this laboratory have shown 2,2-dimethyl-5-t-butyl-1,3 benzodioxole (DBBD) to antagonize 3-methylcholanthrene induction of cytochrome P 450 in Dub:ICR mice yet have no effect on phenobarbital induction. In the present experiments, C57BL/6 mice, an Ah responsive strain, produced a similar response under the same experimental conditions. The hypothesis that DBBD, although not a cytochrome P-450 inducer, competes with 3-methylcholanthrene for binding to the Ah receptor was tested. Using sucrose density gradients, the Ah receptor was measured in hepatic cytosol from Dub:ICR and C57BL/6 male mice. DBBD was unable to displace either 2,3,7,8-tetra-chlorodibenzo-p-dioxin or 3-methylcholanthrene from the Ah receptor, in vitro. However, in in vivo experiments, DBBD treatment of Dub:ICR mice caused Ah receptor depression at 6 and 24 hr with complete recovery in between, while 3-methylcholanthrene treatment caused a 2-fold Ah receptor reduction at 2 hr followed by complete recovery after 12 hr. When 3 methylcholanthrene and DBBD were coadministered, the depression of the Ah receptor was additive. DBBD-pretreated mice had a 2.25-fold reduction in Ah receptor level, effectively blocking the ability of 3-methylcholanthrene to increase the cytochrome P-450 content and either benzo[a]pyrene hydroxylase or ethoxyresorufin O-deethylase activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that 3-methylcholanthrene induction of cytochrome P 450 was inhibited by DBBD pretreatment. Hence, although DBBD does not displace 3 methylcholanthrene from the Ah receptor in vitro, it does antagonize 3 methylcholanthrene induction of cytochrome P-450 and also reduces the amount of available receptor in vivo. This interaction may be due either to antagonism or to downregulation of the Ah receptor. PMID- 3002385 TI - Release of leukotrienes into the perfusate of calcium-ionophore stimulated rabbit lungs. Influence of 5-lipoxygenase inhibitors. AB - Arachidonic acid and the calcium ionophore A23187 are known to provoke a pulmonary artery pressor response, edema formation and release of thromboxane B2 (TxB2) and 6-keto prostaglandin-F1 alpha (6-keto PGFl alpha) into the recirculating perfusion fluid of isolated blood-free perfused rabbit lungs. Here we investigated the release of leukotrienes (LTs) by repetitive 0.1 microM A23187 challenge in the presence or absence of cyclooxygenase and 5-lipoxygenase inhibitors. RP-HPLC analysis of perfusion fluid extracts persistently showed peaks with retention times of authentic LTC4, -D4, -E4 and -B4. Fractionated RP HPLC eluate subjected to radioimmunoassay (RIA) with LTC4 and LTB4 antibodies showed two major peaks of immunoreactivity corresponding to those compounds and minor immunoreactivity with LTD4 and LTE4 in accordance with the stated cross reactivities of the LTC4 antibody. Good correlation for both LTB4 and LTC4 levels measured by RP-HPLC versus RIA of collected HPLC peaks was found. Five to ten min after A23187 challenge, LTC4, -D4 and -B4 levels ranged from 800 to 1600 pg/ml perfusate. LTC4 reached a maximum level at 20 min whereas LTB4 slightly increased over a 35 min period. Upon repeated A23187 challenge, interrupted by rinsing phases with fresh perfusion fluid, the LT release was reproducible several times with increasing reaction strength. This performed in presence of increasing concentration of the 5-lipoxygenase inhibitors AA-861 or U-60,257 caused a dose dependent inhibition of the release of all LTs with an IC50 of approximately 10( 8) to 10(-7) M and 10(-6) M, respectively. Cyclooxygenase inhibition with acetylsalicylic acid at doses completely suppressing the A23187 induced pressor response did not inhibit the peptidoleukotriene release and only slightly depressed LTB4 release. CONCLUSION: using a rapid and sensitive extraction and RP HPLC method isolated lungs are found to release nanomolar amounts of LTs into the perfusate upon repetitive A23187 challenge, suppressed by 5-lipoxygenase inhibition. PMID- 3002386 TI - Characterization of peripheral benzodiazepine binding sites in human term placenta. AB - Peripheral benzodiazepine binding sites were characterized in human term placental membranes using [3H]PK 11195, which is a ligand specific for peripheral benzodiazepine binding sites. Binding of [3H]PK 11195 to human term placental membranes was found to be saturable. Scatchard analysis revealed a single population of binding sites (r = 0.98). Equilibrium dissociation constant (KD) was 2.1 +/- 0.3 nM, and density of binding sites (Bmax) was 920 +/- 105 fmol/mg protein. The KD value calculated from kinetic experiments was 3.6 +/- 0.2 nM. The ability of various drugs to displace [3H]PK 11195 from human term placental binding sites was tested: the inhibition constants (KI) for PK 11195, Ro 5-4864, and diazepam were 2.9, 11.8, and 177 nM, respectively, whereas clonazepam, methyl beta-carboline-3-carboxylate, Ro 15-1788, chlordiazepoxide, atropine, and estradiol were inefficient in displacing [3H]PK 11195 (KI greater than 10(-5) M). PMID- 3002387 TI - Flavonoid impairment of neutrophil response. AB - Flavonoids are a class of phenolic plant pigments which impair the oxidative burst of neutrophils to an extent dependent on their hydrophobicity. The distribution of quercetin and of morin in nitrogen-cavitated neutrophils paralleled their respective hydrophobic characteristics and respiratory burst inhibition. While both flavonoids were localized primarily in the specific granule membrane of neutrophils, the amount of quercetin was considerably greater than that of morin. We here demonstrate inhibition of the initial stimulation response, depolarization of the membrane potential as monitored by fluorescence of the membrane probe diS-C3-(5), and of the respiratory burst, monitored by following the destruction of diS-C3-(5), a reaction mediated by the H2O2 produced in the burst. The flavonoids kaempferol, morin, quercetin, or fisetin were preincubated with human neutrophils at a concentration of 100 microM per 2 X 10(6) cells/ml for 2-3 min and subsequently stimulated with 1 microgram/ml of the tumor promoter phorbol myristate acetate (PMA) or with 60 micrograms/ml of immune complex. The effect of each compound differed, i.e. depolarization was enhanced by some and inhibited by others, while H2O2 generation was inhibited by each, supporting our previous findings that membrane potential depolarization and the respiratory burst are dissociable events. Concentration-response experiments, performed at flavonoid concentrations between 12.5 and 500 microM to determine the IC50 values of these compounds for depolarization and burst activation, indicated that none of the flavonoids affected the resting potential, while all perturbed the stimulus-coupled response, the direction and extent of the perturbation depending upon the stimulus, and the function assessed. These data show that the effects of flavonoids on human neutrophils are complex and suggest several sites of action depending upon the flavonoid's subcellular distribution and pathway of stimulation. PMID- 3002388 TI - Inhibition of human platelet cyclic AMP phosphodiesterase and of platelet aggregation by a hemisynthetic flavonoid, amentoflavone hexaacetate. AB - Amentoflavone hexaacetate (AmAc) was synthesized from natural amentoflavone (Am), a biflavonoid extracted from Viburnum lantana L. Am does not inhibit aggregation of intact platelets up to a concentration of 100 microM but inhibits human platelet cAMP phosphodiesterase (IC50 = 22.0 microM). AmAc is a potent inhibitor of the aggregation of washed human platelets induced by ADP (IC50 = 2.3 microM) or collagen (IC50 = 4.7 microM). AmAc inhibits crude (IC50 = 8.6 microM) or partially purified (IC50 = 42.2 microM) human platelet cAMP phosphodiesterase. In the presence of prostaglandin E1, AmAc (10 microM) induces a 3.7-fold increase in total platelet cAMP. The characteristics of this action suggest a role for cAMP in the mechanism of action of AmAc. The incubation of AmAc with intact platelets for 5 min is necessary for its activity. PMID- 3002389 TI - Elevation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) polychlorinated biphenyls. Structure-activity relationships. AB - Administration of the commercial polychlorinated biphenyl (PCB) Aroclor 1254 to immature male Wistar rats resulted in increased levels (80-110%) of the 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) hepatic cytosolic receptor protein which remained elevated for 14 days. The effects of structure on the activity of individual PCB congeners to modulate hepatic cytosolic receptor levels were compared to the structure-activity relationships (SARs) which have been developed previously for PCBs as inducers of hepatic microsomal monooxygenases. 3,3',4,4' Tetra- and 3,3',4,4',5-pentachlorobiphenyl induced the cytochrome P-448-dependent monooxygenase, ethoxyresorufin O-deethylase (EROD), and resembled 3 methylcholanthrene in their mode of monooxygenase enzyme induction. These congeners also bound to the receptor protein; however, neither compound increased hepatic cytosolic receptor protein levels. Several PCB congeners which exhibit low binding affinities for the cytosolic receptor protein resembled phenobarbitone (PB) in their mode of monooxygenase enzyme induction and, like PB, elevated cytosolic receptor protein levels. Nevertheless, a comparison of the time course of monooxygenase enzyme induction and receptor protein elevation by 2,2',4,4',5,5'-hexachlorobiphenyl and PB illustrated significant differences in their activities. PB-mediated elevation of receptor levels was maximized 24 hr after the last dose, and 48 hr later the receptor levels decreased to control values. In contrast, 5 days after administration of a single dose of 2,2',4,4',5,5'-hexachlorobiphenyl (300 mumoles/kg) the receptor levels were elevated significantly, and these increased levels (205-127% increases over control) persisted for 14 days. There was no correlation between increased levels of hepatic receptor protein and the induction of the cytochrome P-450-dependent monooxygenases, aldrin epoxidase or 4-dimethylaminoantipyrine N-demethylase. Two PCBs, 2,3,3',4,4',5- and 2,2',3,4,4',5-hexachlorobiphenyl, which resembled Aroclor 1254 in their mode of monooxygenase enzyme induction, also elevated hepatic receptor protein levels but were less active than the PB-type inducers. Thus, the SARs developed for PCBs which elevate cytosolic receptor levels demonstrate that the most active compounds exhibit the lowest affinity for the receptor protein and do not induce EROD. In contrast, the more toxic PCB congeners which are approximate isostereomers of 2,3,7,8-TCDD both induced EROD and bound with high affinity to the receptor protein but did not increase hepatic cytosolic receptor protein levels. PMID- 3002390 TI - Effect of superoxide dismutase on glomerular nephritis. AB - The antiinflammatory effect of superoxide dismutase was studied in rats with kidney intoxication induced by the injection of nephrotoxic serum. The urinary excretion of protein was increased significantly by the administration of an intravenous injection of nephrotoxic serum. The daily injection of superoxide dismutase significantly suppressed the increase in urinary excretion of protein. The injection of nephrotoxic serum increased the renal malondialdehyde level by 3 fold compared to the level in the control rats. The daily injection of superoxide dismutase also significantly suppressed the renal malondialdehyde concentration. The change of serum Cu,Zn-superoxide dismutase level and tissue distribution after the intramuscular injection of this enzyme was investigated by immunoassay. Human Cu,Zn-superoxide dismutase localized in kidney exclusively and reached maximum concentration at about 3 hr after the injection. From these results, we propose that superoxide dismutase, which is a scavenger of superoxide anion radicals, inhibits lipid hydroperoxidation in the kidney induced by activated oxygen, and thus protects the renal cells from the damage induced by the injection of nephrotoxic serum. PMID- 3002391 TI - [Primary structure of the alpha subunit of Na+,K+-ATPase. I. Analysis of hydrophilic fragments of the polypeptide chain]. AB - The selective tryptic digestion of the native membrane-bound enzyme was carried out under conditions that provide the extensive hydrolysis of hydrophilic regions of the alpha-subunit into small fragments and allow to preserve the integrity of the beta-subunit. Twenty-seven water-soluble peptides comprising approximately 40% of the total polypeptide chain were isolated by HPLC and their complete or partial amino acid sequence was determined. It led to general outline of the structural organisation of the alpha-subunit hydrophilic regions exposed from membrane. The information thus obtained was used in synthesis of specific oligonucleotide probes. PMID- 3002392 TI - [Problems associated with the use of highly degenerated oligonucleotide probes. Identification of mRNA and cDNA clones corresponding to the gene for the alpha subunit of Na+,K+-ATPase]. AB - Oligonucleotides deduced from the amino acid sequence of a hexapeptide Lys-Asp Phe-Ala-Glu-Asn were synthesized and used as probes to screen a pig kidney cDNA library for a specific DNA sequence coding for the alpha-subunit of Na+, K+ ATPase. It was shown that the mixed oligoprobe, consisting of 64 heptadecamers, could be only suitable for mRNA blot analysis. To identify the clones with specific cDNA inserts, mixed oligoprobes were fractionated by HPLC technique. For the same purpose a new set of oligonucleotides, synthesized as four groups of 16 different heptadecamers each, was used. PMID- 3002393 TI - [Highly effective liquid chromatography of cytochrome oxidase polypeptides]. AB - HPLC of the beef heart cytochrome oxidase subunits on TSKgel-column has been studied. It was found that the resolution of the subunits depends on the sodium dodecyl sulphate and buffer concentration. Strong interaction of subunits Va, VIc and VIIb with hydrophobic polypeptide chains was observed. PMID- 3002394 TI - [Primary structure of the crossover region in the genome of two intertype poliovirus recombinant]. AB - The nucleotide sequence of the crossover region on genomes of two intertypic (type 3/type 1) poliovirus recombinant, has been determined by the primer extension method. No deletions, insertions or rearrangements have been observed. Identical contiguous sequences, 7 or 11 nucleotides in length, respectively, have been found in two regions of the parental genomes, involved in the recombination. PMID- 3002395 TI - [A new method of studying DNA conformation by chemical modification]. AB - A new method of discrimination of double-stranded (ds) and single-stranded (ss) regions in DNA molecules has been developed. It makes use of two alkylating reagents, a voluminous and a small-sized, the former being sensitive to the DNA conformation. A bulky reagent, N,N,N'-tri(beta-chloroethyl)-N'-(p-formylphenyl) propylendiamine-1,3 (TFP), was used to detect the hairpin structure in the palindrome-containing DNA fragment 373 nucleotides long prepared from the ds EcoRI-BamHI fragment of the plasmid pBR322. The fragment was modified by TFP and cleaved by piperidine at the alkylated guanine residues according to the Maxam Gilbert procedure. Guanine residues in the hairpin formed by palindrome were protected from the TFP action, while dimethylsulfate modified all guanines. Application of the method for the identification of loops, stem-and-loop structures, and unwinded regions of DNA is discussed. PMID- 3002396 TI - Peripheral inflammatory vascular disease in Sjogren's syndrome. Association with nervous system complications. AB - Two histopathologic types of inflammatory vascular disease (IVD) occur in Sjogren's syndrome (SS): mononuclear IVD (MIVD) and neutrophilic IVD (NIVD). We describe 50 SS patients with IVD (30 with NIVD and 20 with MIVD). Thirty-three (66%) of the SS patients with biopsy-documented IVD had nervous system disease unattributable to other causes. Nineteen patients (58%) had involvement of both the central and peripheral nervous systems, while 9 had peripheral and 5 had central nervous system dysfunction alone. Patients with both histopathologic types of IVD were at risk for the development of nervous system abnormalities (57% of NIVD patients and 80% of MIVD patients). Indirect evidence is presented which suggests that IVD may play a role in the immunopathogenesis of nervous system disease, at least in a subset of SS patients. PMID- 3002397 TI - Effects of gold sodium thiomalate on functional correlates of human monocyte maturation. AB - The mechanism of action of gold salts in the treatment of rheumatoid arthritis is unknown. Effects of gold on monocyte-macrophage function could be due to inhibition of maturation and differentiation. We found that 3 markers of monocyte differentiation, loss of peroxidase activity, spontaneous synthesis of C2, and spontaneous cytotoxicity for chicken erythrocytes, were all inhibited by gold treatment. This was not a general toxic effect since phorbol myristate acetate could still induce gold-treated monocytes to lyse chicken erythrocytes. Also, phorbol myristate acetate-stimulated superoxide production, a monocyte function not requiring further differentiation, was not inhibited by incubation with gold. Lymphokine-stimulated cytotoxicity for nucleated target cells, another function of monocytes, was inhibited only partially for certain target cells and not at all for others. These data suggest that gold has the capacity to selectively inhibit some monocyte functions which are associated with macrophage differentiation. PMID- 3002398 TI - Interaction of piracetam with several neurotransmitter receptors in the central nervous system. Relative specificity for 3H-glutamate sites. AB - The influence of the nootropic drug piracetam (Normabrain) on specific ligand binding to several neurotransmitter and drug receptors was investigated using established receptor binding procedures. Even at concentrations of 20 mmol/l of piracetam no or only marginal inhibition was observed for the dopamine- and muscarinic cholinergic receptors and for the peripheral benzodiazepine binding site, while for the benzodiazepine receptor, the GABA receptor, the opiate receptor, and the serotonin receptor half-maximal inhibitory concentrations between 20 and 50 mmol/l could be determined. In contrast to these effects seen only at relatively high piracetam concentrations, piracetam is considerably more active at the L-glutamate receptor with a half-maximal inhibitory concentration of about 1 mmol/l. This relatively specific effect of piracetam at the L glutamate receptor could also take place under therapeutic conditions in vivo, since brain levels of piracetam in man may range between 0.1 and 1 mmol/l. It is concluded that effects within the glutaminergic system of the brain could contribute to the therapeutical effects of piracetam in man. PMID- 3002399 TI - [Mechanism of action of the analgesic flupirtine]. AB - To answer the questions of mode and site of action partly supplementary, partly new investigations with flupirtine (Katadolon) were carried out which are described below. The investigation for opiate receptor affinity of flupirtine in rat brain homogenate did not show any reduction in 3He-etorphine binding up to the highest concentration of flupirtine of 10(-5) mol/1. This result suggests that flupirtine either has a very low opiate receptor affinity or lacks it fully. Therefore the analgesic activity of flupirtine is not based on opiate mechanism. The intracerebroventricular and intrathecal administration of flupirtine and the other analgesics tested showed dose dependent analgesic activity in doses which, when applied systemically, did not cause any analgesia in rats. Thus these substances show cerebral or spinal analgesic activity. In relation to the effective doses (ED50 in micrograms/rat) flupirtine was of the same efficacy in both kinds of administration. Pethidine tested comparatively was found to be less potent by intrathecal than by intracerebroventricular application. On the other hand, morphine was weaker by intracerebroventricular than by intrathecal application. As in the experiments by oral administration, naloxone did not show any effect on the analgesic activity of flupirtine, neither by intracerebroventricular nor by intrathecal application. On the other hand, the analgesic effects of pethidine and morphine were completely suppressed by naloxone. These results demonstrate that the analgesic activity of flupirtine is not caused by the opiate mechanism.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002400 TI - Influence of disulfiram, diethyldithiocarbamate and carbon disulfide on the metabolic formation of trifluoroacetic acid from halothane in the rat. AB - Halothane (H), 2-bromo-2-chloro-1,1,1-trifluoroethane, was metabolized to trifluoroacetic acid (TFAA) over a very long time (several days) in rats after a 1 h exposure to the inhalational anesthetic. H inhibited its own metabolism as long as anesthetically active concentrations existed in the serum and tissue. That fraction of H which was not exhaled rapidly by the lung after the anesthesia was metabolized mainly over the time range from 5-48 h: 68% of the total amount of TFAA eliminated in the urine during 144 h (about 4 mg) were found in this time period. Disulfiram (D) dose dependently inhibited the formation of TFAA from H in vivo and in vitro, in liver microsomes of phenobarbital treated rats. Diethyldithiocarbamate (DDTC) and carbon disulfide (CS2), two metabolites of D, given to rats immediately after the H-anesthesia also reduced the metabolic formation of TFAA. However, if DDTC and CS2 were given 24 h before the anesthesia they caused only a small decrease in serum TFAA concentration. This finding indicates that both metabolites of D have only short-lasting inhibitory effects. In contrast, the inhibition of the oxidative metabolism of H by D seems to persist over a long time, since a small but significant decrease in the serum TFAA concentration was found even if D was given 72 h before the H-anesthesia. It was concluded that in vivo CS2 is really the active inhibitor. The short-lasting inhibitory effect of DDTC may be explained by its fast metabolic transformation into CS2, whereas the long-lasting effect of D is caused by its delayed degradation into this metabolite. PMID- 3002401 TI - The effect of treatment with defined spleen dialysate on plasma levels of 21 deoxycortisol and 17-hydroxyprogesterone in women with polycystic ovary disease. AB - Based upon the use of a specific radioimmunoassay method, the plasma levels of 21 deoxycortisol (21-DF) as well as of 17-hydroxyprogesterone (17- OHP) were examined in women with polycystic ovary disease - in normal conditions and after stimulation with corticotropin and chorionic gonadotrophin. It was shown that there was an increased level of 21-DF in women with PCOD in comparison with the control group, both in normal conditions and after ACTH stimulation. Besides there was a pathological increase in the level of 21-DF after HCG stimulation, which was not observed in the control group. These results suggest an enzymatic defect ("11-aberrant hydroxylation") in PCOD, which is due to a disturbance of steroidogenesis. Based upon the positive experiences with a defined spleen dialysate ("DSD", Solcosplen) in the treatment of climacteric discomforts and its known influence on steroidogenesis, we examined possible effects on the levels of 21-DF in females with PCOD. The decrease in the level of this steroid was associated with bleeding in women with amenorrhea. This shows the importance of 21-DF in the pathogenesis of PCOD and proves the efficacy of the spleen dialysate in women suffering from this disease. PMID- 3002402 TI - Modification of the United States' diet to effect changes in blood lipids and lipoprotein distribution. AB - Twenty men, 19 premenopausal and 14 postmenopausal women consumed a diet for 13 weeks that supplied 35% of the calories from fat, 50% from carbohydrate, and 15% from protein. The diet was low in cholesterol, saturated fat, and salt, and high in complex carbohydrate and fiber. The 7-day menu was composed of common well accepted foods prepared in a simple attractive manner. Plasma total cholesterol, LDL cholesterol, and VLDL cholesterol were reduced, but triglyceride levels were not different than after self-selected diets. When 20% of the complex carbohydrate was replaced by simple carbohydrate and other diet components remained optimal, triglyceride and VLDL cholesterol levels increased in men and premenopausal women and total cholesterol increased in premenopausal women. These results suggest that beneficial effects on the blood lipids and lipoprotein distribution of men and women may be obtained by minimal modification of a typical U.S. diet. PMID- 3002403 TI - The effects of alcohol exposure in utero on acetylcholinesterase, Na/K-ATPase and Ca-ATPase activities in six regions of rat brain. AB - The neural membrane-bound enzymes, acetylcholinesterase, Na/K-ATPase and Ca ATPase were examined in six brain areas in perinatal rats at 5, 15 and 25 days of age following alcohol exposure in utero. Female Wistar rats were mated with adult male Wistar rats and maintained on either a liquid diet containing ethanol (5% w/v) or on a control liquid diet for various periods during gestation and the perinatal period. These periods were: before and during gestation only (until birth of the offspring); before and during gestation and following birth; during only a period of gestation and continuing postnatally. Enzyme activity was determined by spectrophotometric methods in the following areas of the brain in each offspring: telencephalon, cerebellum, hippocampus, septal area, preoptic area, and medial basal hypothalamic area. Analysis of the data indicates that alcohol exposure in utero will heterogeneously decrease enzyme activity among the six brain areas when compared to enzyme activity in the same brain areas in animals which were not exposed to alcohol in utero. The activity of the three enzymes were most significantly reduced on day 5 of age, compared to levels in the control animals. Enzyme activity in each brain area was generally most significantly affected in animals exposed to alcohol both pre- and postnatally. The results indicate that fetal alcohol exposure reduces neuronal enzyme activity relative to the period of ethanol exposure in utero; the longer the period of time that alcohol was consumed by the mothers pre- and postnatally, the greater the effect of alcohol on the enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002404 TI - Prostaglandins, thromboxane, leukotrienes and the cerebral circulation in health and disease. PMID- 3002406 TI - Production of poultry viral vaccines in embryonated eggs. AB - The review was written in attempt to summarize some aspects of current poultry viral vaccine production. Problems associated with the production of safe and effective viral vaccines were described. Embryonated eggs or CEF are used in veterinary medical research and in the production of viral vaccines owing to their advantages as a suitable substrate for a wide range of viruses. PMID- 3002405 TI - Hyperalgesia mediated by peripheral opiate receptors in the rat. AB - Behavioural experiments were undertaken to investigate the possible functional significance of opiate receptors located at peripheral endings of primary sensory neurons. The responses of animals to noxious chemical stimuli applied to the ear (ear scratch test) were measured after local pretreatment of these areas with etorphine. Local etorphine administration produced a low dose hyperalgesia and high dose analgesia. Local as opposed to systemic effects of etorphine were inferred from the absence of effects on the contralateral vehicle-treated ear. Systemic administration of naloxone or of a quaternary opiate antagonist (MRZ 2663-BR), which is relatively ineffective in crossing the blood-brain barrier, blocked the low dose hyperalgesic effect of etorphine in the ear scratch test. As a test for the putative hyperalgesic function of peripheral sensory nerve opiate receptors, neonatal rats were treated with capsaicin (50 mg/kg s.c.) to destroy specifically the subpopulation of primary sensory neurons on which the peripheral opiate receptors are thought to be located, without markedly altering pain thresholds. As adults, these neonatally treated rats showed potentiated analgesic responses to systemic morphine, as would be predicted by central 'analgesic' opiate receptors now acting without opposition from peripheral 'hyperalgesic' opiate receptors. These findings suggest that opiate receptors on primary sensory neurons may mediate hyperalgesic functions and that endogenous opioids might normally play a role in the peripheral induction of irritation, inflammation and pain reactions. PMID- 3002408 TI - Pathologic quiz case 2. Adenoid cystic carcinoma of the nasopharynx. PMID- 3002407 TI - Management of lacrimal fossa masses. AB - Mass lesions of the lacrimal fossa are evenly divided between epithelial and nonepithelial diseases. Nonepithelial lesions include pseudotumor, benign lymphoid hyperplasia, lymphoma, and sarcoid. Epithelial lesions are evenly divided between benign and malignant neoplasia, and are similar to those of the salivary glands. Benign mixed tumors compose 25% of lacrimal gland tumors, and are very slow-growing, painless lesions that should be removed en bloc without a biopsy to avoid seeding the orbit. Adenoid cystic and malignant mixed carcinomas are the most common malignancies, and present with rapid growth and pain. These require incisional biopsy to establish the diagnosis, and then a radical exenteration and postoperative radiation therapy. PMID- 3002409 TI - Pathologic quiz case 2. Benign mixed tumor. PMID- 3002410 TI - Blood test for the AIDS virus. PMID- 3002411 TI - Attempted acceleration of the onset of action of pancuronium. Effects of divided doses in infants and children. AB - The study was designed to determine whether the onset of action of pancuronium in infants and children could be accelerated by its administration in divided doses. Sixty paediatric patients (0-1 yr (n = 20); 1-3 yr (n = 20); 3-10 yr (n = 20)) were studied during nitrous oxide-oxygen-halothane anaesthesia using train-of four stimulation, and the results were compared with data obtained previously in adults. The time to onset correlated with the patient's age, and an additional, small acceleration was produced following divided doses which did not alter the duration or pattern of neuromuscular blockade. It was concluded that, in children, divided doses of pancuronium are unlikely to offer important clinical advantages. PMID- 3002412 TI - Etomidate and adrenocortical synthesis in man. PMID- 3002413 TI - Opioid receptor binding and its significance. PMID- 3002414 TI - Alopecia in mice infected with murine cytomegalovirus (MCMV). AB - When mouse cytomegalovirus was injected subcutaneously into 4-12 day old CDI mice there was infection of dermal cells and the dermal papillae of hair follicles. Infected cells were never seen in the epidermis nor in the epithelium of hair follicles. When larger doses of virus (5 X 10(4) pfu) were given, dermal infection led to gross necrosis of the skin, ulceration, scabbing and healing with alopecia. Smaller doses (10(4) pfu) did not cause gross necrosis but damage to follicles resulted in alopecia or sparse hair growth. Skin lesions were not seen after infection of 4-8 week old mice, even when the inoculated skin area had been epilated, or when hyaluronidase was mixed with the virus inoculum. These experiments show that cytomegalovirus, in contrast to herpes simplex and varicella-zoster viruses, infects dermal but not epidermal cells, and that dermal tropism is age-restricted. PMID- 3002415 TI - Correlations between enzyme histochemical reactions and respective enzyme activities in global ischaemic rat hearts. AB - NADH-diaphorase, succinate dehydrogenase (SDH), beta-hydroxybutyrate dehydrogenase (beta-HBDH), malate dehydrogenase (MDH) and cytochrome oxidase (CytO) were demonstrated histochemically in isolated perfused rat hearts during global ischaemia from 0 to 12 hours. The corresponding enzyme activities were measured when possible. The histochemically demonstrable activities of NADH diaphorase and MDH decreased during the first hour of ischaemia. The time course of inactivation of biochemically detectable NADH-ferricyanide oxidoreductase was much the same as that of NADH-diaphorase. Both histochemically and biochemically detectable beta-HBDH gradually decreased by about 6 h of ischaemia. NADH diaphorase but not MDH itself proved to be the rate-limiting factor when demonstrating MDH histochemically with nitroblue tetrazolium (NBT), whereas in the case of beta-HBDH the situation was probably the reverse. CytO and SDH activities did not change during the experimental period. Histochemistry clearly demonstrated ischaemic cellular injury, even though no significant diagnostic changes of ischaemia were visible by light microscopy. Even though this shows that enzyme-histochemical methods can be sensitive indicators of early ischaemic injury, in practice the time between the onset of injury and death as well as between death and autopsy must be taken into consideration when interpreting the results. PMID- 3002416 TI - Arachidonic acid metabolism by polymorphonuclear leukocytes in psoriasis. AB - The metabolism of endogenous arachidonic acid by human polymorphonuclear leukocytes (PMNL) isolated from peripheral blood has been studied in 19 patients with chronic plaque psoriasis and 19 healthy controls. Using calcium ionophore A23187 as a stimulus, the PMNL synthesized leukotriene B4 (LTB4), 6-trans leukotriene B4, 12-epi-6-trans-LTB4, and 5-hydroxy-6,8,11,14-eicosatetraenoic acid. There was no significant difference in the amounts of the products formed between the psoriatic and control groups. The elevated levels of LTB4 that have been described in psoriatic skin may therefore be due to the PMNL infiltrate or to enhanced synthesis by another cell type. The reported increase in activity of the circulating PMNL in psoriasis does not appear to be due to increased 5 lipoxygenase activity in these cells. PMID- 3002418 TI - AIDS and HTLV-III/LAV infection: consequences for obstetrics and perinatal medicine. PMID- 3002417 TI - Transferrin modulation of colony stimulating factor elaboration by adherent blood and bone marrow cells. AB - When added to cultured normal, adherent blood and marrow cells at concentrations of 25-100 micrograms/ml (3 X 10(-7) to 1.3 X 10(-6) M), human transferrin enhanced colony-stimulating factor (CSF) elaboration. Fe saturated and relatively unsaturated Tf were equally effective in increasing CSF release, but soluble ferric nitriloacetate was ineffective. Dose-dependent increases in CSF release by adherent blood and marrow cells occurred in serum-free media and the continuous presence of Tf was necessary for this effect. Using a monoclonal anti-Tf receptor antibody, normal blood mononuclear cells contained no detectable Tf receptor positive cells. However, after 5 d culture in serum-free medium, Tf receptor positive cells were identified among normal adherent blood cells, and the per cent receptor positive cells increased with Tf concentration. We conclude Tf modulates CSF production from normal, adherent blood and marrow cells in vitro. These findings indicate a possible role for Tf in intramedullary regulation of normal granulopoiesis. PMID- 3002419 TI - Effects of dydrogesterone on the oestrogenized postmenopausal endometrium. AB - Postmenopausal women receiving conjugated oestrogens 1.25 mg daily continuously were also given dydrogesterone either 5, 10 or 20 mg daily for the first 12 days of each calendar month. Endometrial tissue obtained on the sixth day of combined therapy in the third or subsequent treatment cycle was subjected to histological, ultrastructural and biochemical assessments. Dydrogesterone provoked secretory histological and ultrastructural changes within the endometrium in a dose dependent manner. A daily dose of 5 mg produced sub-optimal responses but 10 and 20 mg daily produced effects similar to those observed in the secretory phase of the ovulatory cycle. Dydrogesterone 10 mg and 20 mg daily reduced epithelial DNA synthesis and nuclear oestradiol receptor levels to values within the secretory phase range. A dose-response relation was seen in the induction of oestradiol-17 beta and isocitrate dehydrogenase activities; hyperphysiological values were observed with 20 mg of dydrogesterone daily. This study has demonstrated that dydrogesterone exerts potent anti-oestrogenic and progestational effects on the human endometrium which are dose-related. The 10 and 20 mg doses induced responses equal to or greater than those observed in the secretory phase of the ovulatory cycle and both dosages can be recommended for use in combination with exogenous oestrogens in postmenopausal women: and they may also have a role in the management of anovulatory dysfunctional uterine bleeding. PMID- 3002420 TI - Developments in chemotherapy for medium- and high-risk patients with gestational trophoblastic tumours (1979-1984). AB - Identification of various prognostic factors at the start of chemotherapy allows patients with gestational trophoblastic tumours to be categorized into low-, medium- and high-risk groups so that they can be given the minimum treatment necessary to eliminate their disease. Most patients in the low-risk category can be treated with minimal toxicity using a methotrexate/folinic acid regimen and these patients are not considered in this report. Before 1979 patients in the medium-risk category were treated with a sequence of drugs which included, hydroxyurea, methotrexate, 6-mercaptopurine, actinomycin D, vincristine and cyclophosphamide. Since 1979 etoposide has been substituted for vincristine and cyclophosphamide. The 76 patients treated between 1979 and 1983 are all alive and in remission 1.1.85. Three patients (4%) relapsed and required retreatment and all are in remission. Fifty-six patients in the high-risk group, most at risk of developing drug resistance, were treated with a regimen incorporating etoposide with methotrexate, actinomycin D (EMA) and vincristine and cyclophosphamide (CO). EMA and CO were given on alternate weeks, resulting in an overall survival rate of 84%. Patients who had received prior chemotherapy had a survival rate of 74% and a relapse rate of 19% compared with 93% survival and 3% relapse rate in those who had not received prior chemotherapy. Toxicity with the EMA/CO regimen was significantly less than with an earlier regimen (CHAMOCA) used in the high-risk group. PMID- 3002421 TI - Mapping labeled sites in Escherichia coli ribosomal RNA: distribution of methyl groups and identification of a photoaffinity-labeled RNA region putatively at the peptidyltransferase center. AB - We have developed a method for the rapid localization of sites of ribosomal RNA labeling to limited regions (approximately 200 bases). The method is based on the formation and polyacrylamide gel electrophoretic separation of hybrids between restriction fragments of rrnB DNA and isotopically labeled rRNA and the subsequent determination of radioactivity across the gel. Using [3H]adenine labeled rRNA as a control sample, we optimized experimental conditions with respect to a number of variables, including rRNA:DNA stoichiometric ratio, temperature of the annealing step, and levels of nucleases. An important result is that different rRNA X DNA hybrid fragments are obtained in different yields. The method was then applied to analyses of C3H3-labeled rRNA, giving results in good accord with known and proposed sites of rRNA methylation, and of rRNA that has been photoaffinity-labeled with 5-azido-2-nitrobenzoyl-[3H]Phe-tRNAPhe, a probe directed toward the peptidyltransferase center. The latter study showed a single major site of RNA labeling, falling within bases 2445-2668 of 23S rRNA. The extent of labeling was shown to be dependent on light-induced formation of a reactive intermediate and to be decreased in the absence of poly(uridylic acid) or in the presence of puromycin. The location of this major site of labeling is consistent with recent results obtained with an analogous tRNA photoaffinity label [Barta, A., Steiner, G., Brosius, J., Noller, H. F., & Kuechler, E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3607-3611] and with related genetic and biochemical studies of antibiotic interaction with ribosomes suggesting that the peptidyltransferase center falls within region V (bases 2043-2625) of 23S rRNA. PMID- 3002422 TI - Transcription from bacteriophage T7 and SP6 RNA polymerase promoters in the presence of 3'-deoxyribonucleoside 5'-triphosphate chain terminators. AB - RNA synthesis by T7 RNA polymerase or SP6 RNA polymerase is 100-1000 times more sensitive to the presence of the 3'-deoxyribonucleoside 5'-triphosphate chain terminators than is RNA synthesis by Escherichia coli RNA polymerase or Q beta replicase. These ribonucleotide analogues do not alter the specificity of each polymerase for its own promoters nor do they alter the site at which synthesis is initiated. Transcription by T7 RNA polymerase or SP6 RNA polymerase in the presence of relatively low concentrations of these chain terminators offers a useful route for determining the nucleotide sequence of any DNA segment that is inserted immediately downstream from a homologous bacteriophage promoter. This sequencing procedure was used to explore the effects that different dinucleotides have on the specificity of initiation at two different T7 RNA polymerase promoters. PMID- 3002423 TI - Single-strand-specific degradation of DNA during isolation of rat liver nuclei. AB - We have investigated structural change in rat liver DNA produced by different isolation procedures and specifically compared the integrity of DNA derived by phenol extraction from isolated and purified nuclei with preparations extracted immediately from a crude liver homogenate containing intact nuclei. As indicated by stepwise elution from benzoylated DEAE-cellulose, most structural change in DNA was evident following nuclei isolation. Damage principally involved generation of single-stranded regions in otherwise double-stranded DNA fragments; totally single-stranded DNA was not detected by hydroxylapatite chromatography. Caffeine gradient elution suggested formation of single-stranded regions extending for up to several kilobases. In neutral sucrose gradients, differences in sedimentation rates of respective DNA samples consequent upon S1 nuclease digestion could be detected after isolation of nuclei, though not in other circumstances. The observed single-strand-specific nuclease digestion of DNA could apparently be reduced if steps were taken to reduce autodigestion during nuclei isolation by reduction of temperature and covalent cation concentration. The results are discussed in terms of the use of exogenous and endogenous nucleases in chromatin fractionation studies involving isolated nuclei and possible artifactual findings that may be generated by single-strand-specific autodigestion. PMID- 3002424 TI - Expression of the cytochrome b-URF6-URF5 region of the mouse mitochondrial genome. AB - The nature of RNA coded by the only light-strand (L-strand) open-reading frame unidentified reading frame 6 (URF6) was studied by using a variety of single- and double-strand DNA subclones derived from the 3.6-kilobase (kb) cytochrome b (cyt b)-URF5 coding region of the mouse mitochondrial genome. Northern blot experiments using single-strand-specific M13 clones indicate that both the heavy (H) and L strands of this genomic region are symmetrically transcribed and processed into poly(adenylic acid) [poly(A)] RNAs of comparable size. The 1.2- and 2.4-kb RNAs coded by the H strand, putative mRNAs for cyt b and URF5 reading frames, respectively, are derived from a common precursor of 3.6-kb RNA. The L strand-coded 1.15-kb RNA, on the other hand, is derived from a short-lived precursor of 3.6-kb RNA by a multiple-step processing involving a 2.4-kb intermediate RNA. The S1 nuclease protection experiments using both the 3'- or 5' end-labeled DNA probes and also affinity-purified 32P-labeled RNA probes indicate that the 1.15-kb RNA maps between the start of the URF6 reading frame (3' end) and a region 590-600 nucleotides to the 5' end of this reading frame. The 1.15-kb RNA thus contains the entire URF6 coding sequence and an about 590-nucleotide long 3' untranslated region. The molar abundance of the three mRNAs in the steady state mitochondrial RNA varies markedly. The 1.15-kb URF6 mRNA is only one-tenth the level of 1.2-kb cyt b mRNA, although it is nearly as abundant as the 2.4-kb URF5 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002425 TI - Fine structure-activity analysis of mutations at position 51 of tyrosyl-tRNA synthetase. AB - Residue Thr-51 at the active site of tyrosyl-tRNA synthetase (Bacillus stearothermophilus) has been replaced with all the smaller amino acids by protein engineering to investigate direct and indirect effects of mutation on substrate binding and catalysis. The gamma-hydroxyl group of Thr-51 was thought to be 0.5 A too far from the ribose ring oxygen of ATP to form a hydrogen bond. Consistent with this, it is found that mutation of Thr-51----Cys-51, which should place the gamma-thiol group within its correct distance for hydrogen bonding, increases the affinity of the enzyme for ATP. Other mutations (Ser-51, Ala-51, and Gly-51) show the contributions to binding of the other atoms in the side chain of Thr-51. A family of enzymes has been produced, TyrTS(Thr-51) (wild type), TyrTS(Ala-51), TyrTS(Cys-51), and TyrTS(Pro-51), in which the value of kcat/KM for ATP in aminoacylation increases along the series. This is achieved by the value of KM decreasing significantly (2.5, 1.25, 0.29, and 0.019 mM, respectively) while there are smaller decreases in kcat (4.7, 4.0, 2.9, and 1.8 s-1, respectively). These variations cause each one of the enzymes to be more active than the others at particular concentrations of ATP. For example, at concentrations of ATP greater than 5.9 mM, TyrTS(Thr-51) is the most active, while TyrTS(Ala-51), TyrTS(Cys-51), and TyrTS(Pro-51) are the most active at 5.9-2.2, 2.2-0.42, and less than 0.42 mM ATP, respectively. Interestingly, position 51 shows variation in tyrosyl-tRNA synthetases isolated from different organisms. PMID- 3002426 TI - Transforming growth factors from a human tumor cell: characterization of transforming growth factor beta and identification of high molecular weight transforming growth factor alpha. AB - Intracellular transforming growth factors (TGFs) were extracted from a human rhabdomyosarcoma cell line and purified to apparent homogeneity by using gel filtration, cation-exchange, and high-performance liquid chromatography. Two types of transforming growth factor activities, TGF-alpha and TGF-beta, were detected. The intracellular polypeptides which belonged to the TGF-alpha family required TGF-beta for full activity in inducing nonneoplastic normal rat kidney fibroblasts to grow in soft agar. These peptides also bound to the membrane receptor for epidermal growth factor. As determined by sodium dodecyl sulfate polyacrylamide gels, the apparent molecular weight of these intracellular TGF alpha's was 18 000. Intracellular TGF-beta required either epidermal growth factor or TGF-alpha for stimulation of soft agar growth. The intracellular TGF beta was purified to homogeneity as judged by a single peak after reverse-phase high-performance liquid chromatography and a single band on a sodium dodecyl sulfate-polyacrylamide gel. The intracellular TGF-beta from the human tumor cell line was similar in all respects tested (migration on sodium dodecyl sulfate polyacrylamide gels, stimulation of soft agar growth, binding to the membrane receptor for TGF-beta, and amino acid composition) to intracellular TGF-beta from normal human placenta. PMID- 3002427 TI - Papain fragmentation of the (Na+,K+)-ATPase beta subunit reveals multiple membrane-bound domains. AB - Purified dog kidney (Na+,K+)-ATPase was reacted with tritiated sodium borohydride after treatment with neuraminidase and galactose oxidase. This procedure did not affect the ATPase activity of the enzyme, and all of the covalently bound radioactivity was found in the beta subunit (Mr 54 000). Papain digestion of the tritiated enzyme produced two labeled fragments of Mr 40 000 and 16 000. Further proteolysis generated an Mr 31 000 peptide from the larger fragment. Unlike the tryptic and chymotryptic sites of the alpha subunit, the sites of papain hydrolysis were insensitive to conformations of the (Na+,K+)-ATPase. Determination of the NH2-terminal sequences was used to arrange the fragments within the linear map of the beta chain. Finally, none of the labeled peptides was released from the membrane under nondenaturing conditions. These results are consistent with a model of the beta subunit containing a 40 000-dalton NH2 terminal piece and a 16 000-dalton COOH-terminal piece. Both fragments have extracellularly exposed carbohydrate and at least one membrane-bound domain. PMID- 3002428 TI - Fluorescent derivatives of ganglioside GM1 function as receptors for cholera toxin. AB - A fluorescent derivative of ganglioside GM1 was prepared by oxidation of the sialic acid residue with sodium periodate and reaction of the resulting aldehyde with Lucifer yellow CH. The biological activity of the fluorescent derivative was compared with that of native GM1 using GM1-deficient rat glioma C6 cells. When the cells were exposed to either native or fluorescent GM1, their ability to bind 125I-labeled cholera toxin was increased to a similar extent. This increase in binding was directly proportional to the amount of ganglioside added to the medium. The affinity of the toxin for cells treated with either native or fluorescent GM1 also was similar. More importantly, the fluorescent GM1 was as effective as native GM1 in enhancing the responsiveness of the cells to cholera toxin. Thus, the ganglioside-treated cells exhibited a 9-fold increase in toxin stimulated cyclic AMP production over cells not exposed to GM1. There was a similar increase in iodotoxin binding and toxin-stimulated cyclic AMP accumulation in cells treated with other GM1 derivatives containing rhodaminyl or dinitrophenyl groups. On the basis of these results, it is clear that these modified gangliosides retain the ability to function as receptors for cholera toxin. Consequently, fluorescent gangliosides are likely to be useful as probes for investigating the dynamics and function of these membrane components. PMID- 3002429 TI - Characterization of the oligosaccharides of prolyl hydroxylase, a microsomal glycoprotein. AB - Prolyl hydroxylase is a tetrameric glycoprotein that catalyzes a vital posttranslational modification in the biosynthesis of collagen. The enzyme purified from whole chick embryos (WCE) possesses two nonidentical subunits, alpha and beta, and has been shown by several techniques to reside in the endoplasmic reticulum of chick embryo fibroblasts. The studies described here demonstrate that the larger of the two subunits (alpha) exists in two forms in chick embryo fibroblasts (CEF); these two forms differ in carbohydrate content. The larger alpha subunit, alpha', contains two N-linked high mannose oligosaccharides, each containing eight mannose units; the smaller subunit, alpha, contains a single seven-mannose N-linked oligosaccharide. Both oligosaccharides could be cleaved by endo-beta-N-acetylglucosaminidase H and completely digested with alpha-mannosidase to yield mannosyl-N-acetylglucosamine. PMID- 3002431 TI - Primary structure of tyrosinase from Streptomyces glaucescens. AB - The complete amino acid sequence of Streptomyces glaucescens tyrosinase is reported. The molecule consists of 273 amino acids and has a Mr of 30 900 including two copper atoms. The primary structure was determined by a combination of amino acid and DNA sequence analysis. Peptide sequence information was derived from the cyanogen bromide, tryptic, and thermolytic fragments of apotyrosinase by automated Edman degradation and aminopeptidase M and carboxypeptidase C digestions. The nucleotide sequence of the tyrosinase gene cloned into the PvuII site of pBR322 was determined. The enzyme contains no apparent leader peptide despite the fact that it is secreted into the culture medium. As observed for a number of different Streptomyces genes, the tyrosinase gene shows a strong preference (97%) for codons ending in G or C. A comparison of the amino acid sequence of Streptomyces glaucescens tyrosinase with that of Neurospora crassa tyrosinase reveals an overall sequence homology of only 24.2%. However, the sequence homology is much higher in those regions thought to be involved in metal binding of the binuclear active site copper of this monooxygenase. PMID- 3002430 TI - Biosynthesis of prolyl hydroxylase: evidence for two separate dolichol-media pathways of glycosylation. AB - Prolyl hydroxylase is a glycoprotein containing two nonidentical subunits, alpha and beta. The alpha subunit of prolyl hydroxylase isolated from 13-day-old chick embryos contains a single high mannose oligosaccharide having seven mannosyl residues. Two forms of alpha subunit have been shown to exist in enzyme purified from tendon cells of 17-day-old chick embryos, one of which (alpha) appears to be identical in molecular weight and carbohydrate content with the single alpha of enzyme from 13-day-old chick embryos, as well as another form (alpha') that contains two oligosaccharides, each containing eight mannosyl units [see Kedersha, N. L., Tkacz, J. S., & Berg, R. A. (1985) Biochemistry (preceding paper in this issue)]. Biosynthetic labeling studies were performed with chick tendon cells using [2-3H]mannose, [6-3H]glucosamine, [14C(U)]mannose, and [14C(U)]glucose. Analysis of the labeled products using polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that only the oligosaccharides on alpha' incorporated measurable mannose or glucosamine isotopes; however, both alpha subunits incorporated 14C amino acid mix and [14C(U)]glucose [metabolically converted to [14C(U)]mannose] under similar conditions. Pulse-chase labeling studies using 14C amino acid mix demonstrated that both glycosylated polypeptide chains alpha and alpha' were synthesized simultaneously and that no precursor product relationship between alpha and alpha' was apparent. In the presence of tunicamycin, neither alpha nor alpha' was detected; a single polypeptide of greater mobility appeared instead. Incubation of the cells with inhibitory concentrations of glucosamine partially depressed the glycosylation of alpha' but allowed the glycosylation of alpha.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002432 TI - Fat cell beta 1-adrenergic receptor: structural evidence for existence of disulfide bridges essential for ligand binding. AB - Agents that react chemically with sulfhydryl groups of proteins modify the response of adenylate cyclase to stimulation by beta-adrenergic agonists. N Ethylmaleimide, an agent that alkylates sulfhydryl groups, inactivates both the catalytic moiety of adenylate cyclase and the stimulatory, regulatory guanine nucleotide binding protein Ns of rat fat cells but fails to affect binding of antagonists to the beta-adrenergic receptor [Malbon, C. C., Graziano, M. P., & Johnson, G. L. (1984) J. Biol. Chem. 259, 3254-3260]. Treating membranes of rat fat cells with dithiothreitol or beta-mercaptoethanol, agents that reduce disulfide bridges of proteins, results in a loss of binding of beta-adrenergic radioligands to the receptor. The specific binding of radioligands to beta adrenergic receptors that are solubilized in digitonin is affected similarly by treatment with disulfide bridge reducing agents. beta-Adrenergic receptor purified from rat fat cells and treated with beta-mercaptoethanol (10%) and then subjected to gel electrophoresis in the presence of sodium dodecyl sulfate migrates as a Mr 67 000 peptide [Cubero, A., & Malbon, C. C. (1984) J. Biol. Chem. 259, 1344-1350]. In the absence of disulfide bridge reducing agents, however, the purified receptor exhibits greater electrophoretic mobility, migrating as a peptide with Mr 54 000. Treating the native form of the purified receptor with beta-mercaptoethanol (0.1-10%) or dithiothreitol (0.1-10 mM) decreases the ability of the receptor to bind beta-adrenergic ligands, decreases the electrophoretic mobility of the receptor, and results in receptor peptides migrating with molecular weight ranging from 54 000 to 67 000 when subjected to gel electrophoresis in the presence of sodium dodecyl sulfate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002433 TI - Aldehyde and ketone substrate analogues inhibit the collagenase of Clostridium histolyticum. AB - The collagenase from Clostridium histolyticum is a mixture of several collagenases, all of which are zinc metalloproteases. This enzyme catalyzes the cleavage of the X-Gly peptide bond in the repeating sequence of collagen: -Gly Pro-X-Gly-Pro-X-. Thus the S3, S2, and S1 subsites on the enzyme appear to be occupied by the sequence -Gly-Pro-X- and the S1', S2', and S3' subsites also by Gly-Pro-X-. Short peptides up to and including N alpha-acyltetrapeptides containing the repeat sequence do not detectably inhibit the enzyme (IC50 greater than 10 mM). However, peptide aldehydes of the form aminoacyl-X-glycinal, presumably occupying the S1, S2, ..., Sn subsites, are inhibitors. The most potent of these was Pro6-Gly-Pro-glycinal, with an IC50 of 340 +/- 70 microM. The single peptide aldehyde investigated, which could occupy the S1' and S2' subsites, 4-oxobutanoyl-L-proline, did not inhibit collagenase (IC50 greater than 20 mM). The peptide ketone 5-benzamido-4-oxo-6-phenylhexanoyl-Pro-Ala (XXV), which could occupy the S1-S3' subsites, inhibits collagenase with an IC50 of 120 +/- 50 microM, over 80-fold more potently than its parent peptide analogue benzoyl-Phe-Gly-Pro-Ala (XXIII). The alcohol analogue of XXV, 5-benzamido-4 hydroxy-6-phenylhexanoyl-Pro-Ala (XXVI), is over 60-fold less potent with an IC50 of 8 +/- 2mM. Extending the peptide ketone XXV to occupy the S2-S3' subsites gave 5-(N alpha-carbobenzoxy-L-prolinamido)-4-oxo-6-phenylhexanoyl-Pro -Ala (XXVII). Surprisingly, XXVII had an IC50 of only 5.2 +/- 2 mM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002434 TI - Reaction of the antitumor antibiotic CC-1065 with DNA. Location of the site of thermally induced strand breakage and analysis of DNA sequence specificity. AB - CC-1065 is a unique antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of this drug are thought to be due to its ability to form a covalent adduct with DNA through N3 of adenine. Thermal treatment of CC 1065-DNA adducts leads to DNA strand breakage. We have shown that the CC-1065 structural modification of DNA that leads to DNA strand breakage is related to the primary alkylation site on DNA. The thermally induced DNA strand breakage occurs between the deoxyribose at the adenine covalent binding site and the phosphate on the 3' side. No residual modification of DNA is detected on the opposite strand around the CC-1065 lesion. Using the early promoter element of SV40 DNA as a target, we have examined the DNA sequence specificity of CC-1065. A consensus sequence analysis of CC-1065 binding sites on DNA reveals two distinct classes of sequences for which CC-1065 is highly specific, i.e., 5'PuNTTA and 5'AAAAA. The orientation of the DNA sequence specificity relative to the covalent binding site provides a basis for predicting the polarity of drug binding in the minor groove. Stereo drawings of the CC-1065-DNA adduct are proposed that are predictive of features of the CC-1065-DNA adduct elucidated in this investigation. PMID- 3002435 TI - Primary structure of the succinyl-CoA synthetase of Escherichia coli. AB - The primary structure of the succinyl-CoA synthetase of Escherichia coli has been deduced from the nucleotide sequence of a 2451-base-pair segment of DNA containing the corresponding sucC (beta subunit) and sucD (alpha subunit) genes. The genes are located at one end of a gene cluster that encodes several citric acid cycle enzymes: gltA-sdhCDAB-sucABCD; gltA, citrate synthase; sdh, succinate dehydrogenase; sucA and sucB, the dehydrogenase (E1) and succinyltransferase (E2) components of the 2-oxoglutarate dehydrogenase complex. The sucC and sucD genes are separated from the sucA and sucB genes by a 273-base-pair segment containing four palindromic units, but they appear to be expressed from a sucABCD read through transcript that extends from the suc promoter to a potential rho independent terminator at the distal end of sucD. The stop codon of the sucC gene overlaps the sucD initiation codon by a single nucleotide, indicating close translational coupling of the sucC and sucD genes. The sucC gene comprises 1161 base pairs (388 codons, excluding the stop codon), and it encodes a polypeptide of Mr 41 390 corresponding to the beta subunit of succinyl-CoA synthetase. The sucD gene comprises 864 base pairs (288 codons, excluding the start and stop codons), and it encodes a product of Mr 29 644, corresponding to the alpha subunit of succinyl-CoA synthetase. The alpha subunit contains a 12-residue amino acid sequence that is identical with the histidine peptide previously isolated from the phosphoenzyme. This sequence forms part of one of the two potential nucleotide binding sites detected in the alpha subunit. PMID- 3002437 TI - Isolation and partial characterization of genomic clones coding for a human pro alpha 1 (II) collagen chain and demonstration of restriction fragment length polymorphism at the 3' end of the gene. AB - We have isolated two clones containing 19 kilobases (kb) of the human gene coding for a pro-alpha 1 (II) collagen chain from human lambda genomic DNA libraries. A 3' clone, HC2A, was selected by cross-hybridization with a cDNA clone containing sequences coding for the carboxy propeptide of chick type II procollagen. A second clone, HC2B, was obtained by screening the library with the 5' part of HC2A. The sequence analysis of exon 3 corresponding to the C propeptide reveals the presence of stretches of conserved nucleotides between the human and the chick type II procollagen genes. On Northern blots, the human collagen clone hybridizes strongly to a 5.5-kb RNA for the rat type II procollagen chain. Finally, studies of genomic DNAs from normal individuals reveal the presence of a HindIII and a BamHI polymorphic site at the 3' end of the gene. PMID- 3002436 TI - Influence of detergent polar and apolar structure upon the temperature dependence of beef heart cytochrome c oxidase activity. AB - The temperature dependence of lipid-depleted beef heart cytochrome c oxidase activity was studied in a series of chemically homogeneous detergents. The detergents that were tested included C10 to C18 maltosides, C8 to C12 glucosides, C8 to C16 Zwittergents, and C12 poly(oxyethylene) ethers. The observed rates of electron transport were dependent upon the structure of the polar head group and the length of the hydrocarbon tail. Of the detergents tested, the alkyl maltosides were the best in terms of both high rates of electron transport and superior enzyme stability. With the maltosides, changing the length of the alkyl tail affected the activity of cytochrome c oxidase in a manner quite similar to that reported with synthetic phosphatidylcholines and phosphatidylethanolamines [Vik, S. B., & Capaldi, R. A. (1977) Biochemistry 16, 5755-5759], suggesting that the alkyl maltosides can mimic some of the features of the membrane environment. In each of the detergents, the activation enthalpy (determined from the slope of an Arrhenius plot) was nearly identical, suggesting that the same electron transfer step within cytochrome c oxidase is rate limiting. This result has been interpreted as evidence for the existence of two or more conformers of cytochrome c oxidase, one of which is significantly more active than the other(s). The enzyme turnover number, which changes by 2 orders of magnitude depending upon the structure of the bound detergent, may reflect the ability of each detergent to alter the equilibrium between the active and nearly inactive conformers. PMID- 3002438 TI - Isolation and characterization of the human cellular myc gene product. AB - Antibodies against the product of the human cellular myc gene (c-myc) were prepared against a bacterially expressed human c-myc protein by inserting the ClaI/BclI fragment of the human c-myc DNA clone in an expression vector derived from pPLc24. These antibodies cross-react with viral-coded myc (v-myc) proteins from MC29 and OK10 viruses. Furthermore, IgGs specific for synthetic peptides, corresponding to the 12 carboxy-terminal amino acids of the human c-myc gene and 16 internal amino acids, were isolated. By use of the various myc-specific antisera or IgGs, a protein of Mr 64 000 was detected in several human tumor cell lines including Colo320, small cell cancer of the lung (417d), HL60, Raji, and HeLa. This protein is larger than the corresponding v-myc or chicken c-myc proteins from avian virus transformed cells or avian bursa lymphoma cells (RP9), both of which are proteins of Mr 55 000. The human c-myc protein is located in the nucleus of Colo320 cells, exhibits a half-life of about 15 min, and is expressed at significantly lower levels than the viral protein. The human c-myc protein was enriched about 3000-fold from Colo320 cells using c-myc-specific IgG coupled to Sepharose beads. The protein binds to double-stranded DNA in vitro, a reaction that can be inhibited to more than 90% by c-myc specific IgG. PMID- 3002439 TI - Effects of DNA intercalating agents on topoisomerase II induced DNA strand cleavage in isolated mammalian cell nuclei. AB - Intercalator-induced DNA double-strand breaks (DSB) presumably represent topoisomerase II DNA cleavage sites in mammalian cells. Isolated L1210 cell nuclei were used to determine the saturability of this reaction at high drug concentrations. 4'-(9-Acridinylamino)methanesulfon-m-anisidide (m-AMSA) and 5 iminodaunorubicin (5-ID) both produced DSB in a concentration-dependent manner, and the production of these breaks leveled off above 10 microM. Addition of m AMSA to 5-ID-treated nuclei did not raise the plateau level. Thus, both drugs seemed to interact similarly on identical targets. The ellipticine derivative 2 methyl-9-hydroxyellipticinium (2-Me-9-OH-E+) had two effects on the production of DSB. Below 10 microM, 2-Me-9-OH-E+ produced DSB as did ellipticine, m-AMSA, or 5 ID. Above 10 microM, 2-Me-9-OH-E+ did not induce DSB and inhibited the DSB induced by m-AMSA, 5-ID, or ellipticine. 2-Me-9-OH-E+ and m-AMSA competed with each other to produce either double-strand break formation (m-AMSA-induced reaction) or double-strand break inhibition (2-Me-9-OH-E+-induced reaction at concentrations greater than 10 microM). Because these results were reproduced in experiments using DNA topoisomerase II isolated from L1210 nuclei, it is likely that the intercalator-induced protein-associated DNA breaks detected by alkaline elution in nuclei represent DNA topoisomerase II-DNA complexes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002440 TI - Effects of the DNA intercalators 4'-(9-acridinylamino)methanesulfon-m-anisidide and 2-methyl-9-hydroxyellipticinium on topoisomerase II mediated DNA strand cleavage and strand passage. AB - DNA topoisomerase II is believed to be the enzyme that produces the protein associated DNA strand breaks observed in mammalian cell nuclei treated with various intercalating agents. Two intercalators--4'-(9 acridinylamino)methanesulfon-m-anisidide (m-AMSA, amsacrine) and 2-methyl-9 hydroxyellipticinium (2-Me-9-OH-E+)--differ in their effects on protein associated double-strand breaks in isolated nuclei. m-AMSA stimulates their production at all concentrations, whereas 2-Me-9-OH-E+ stimulates at low concentrations and inhibits at high concentrations. We have reproduced these differential effects in experiments carried out in vitro with purified L1210 DNA topoisomerase II, and we have found that concentrations of 2-Me-9-OH-E+ above 5 microM prevent the trapping of DNA-topoisomerase II cleavable complexes irrespective of the presence of m-AMSA. It also stimulated topoisomerase II mediated DNA strand passage, again with or without inhibitory amounts of m-AMSA (this result suggests that extensive intercalation by 2-Me-9-OH-E+ destabilized the cleavable complexes). From these data, it is concluded that intercalator induced protein-associated DNA strand breaks observed in intact eukaryotic cells and isolated nuclei are generated by DNA topoisomerase II and that intercalators can affect mammalian DNA topoisomerase II in more than one way. They can trap cleavable complexes and inhibit DNA topoisomerase II mediated DNA relaxation (m AMSA and low concentrations of 2-Me-9-OH-E+) or destabilize cleavable complexes and stimulate DNA relaxation (high concentrations of 2-Me-9-OH-E+). PMID- 3002441 TI - Methyl transfer from methylcobalamin to thiols. A reinvestigation. AB - The methyl transfer from methylcobalamin to thiols has been reinvestigated. By use of methylcobalamin selectively enriched with 13C in the methyl moiety, the methyl transfer to thiols was followed by 13C NMR. The methyl transfer occurs in aqueous mildly alkaline (pH 8-12) solution, even in the complete absence of oxygen. 31P NMR and EPR studies demonstrate that cob(II)alamin is the final corrinoid product. However, the pH dependence of the methyl-transfer reaction from methylcobalamin to beta-mercaptoethanol is consistent only with a nucleophilic displacement of the methyl group by a thiolate anion, resulting in the heterolytic cleavage of the carbon-cobalt bond. Difference visible spectroscopic measurements of the reaction mixture suggest that cob(I)alamin is formed as an intermediate. PMID- 3002442 TI - all-trans-retinoids and dihydroretinoids as probes of the role of chromophore structure in rhodopsin activation. AB - The absorption of a photon of light by rhodopsin results in the cis to trans isomerization of the 11-cis-retinal Schiff base chromophore. In the studies reported here, an attempt is made to determine the mechanism of the energization of rhodopsin as it relates to the chemistry of the isomerization process and the geometrical state of the chromophore. Studies were performed with vitamin A analogues to probe this mechanism. Both 11-cis-7,8-dihydroretinal and 9-cis-7,8 dihydroretinal form bleachable pigments when combined with opsin. Photolysis of these pigments in the presence of G-protein results in the activation of the latter as revealed by its GTPase activity. Phosphodiesterase is also activated when it is included in the incubation. Therefore, the possibility that rhodopsin is energized by mechanisms involving photochemically induced charge transfer from the protonated Schiff base to the beta-ionone ring can be discarded. Further studies were conducted with all-trans-vitamin A derivatives to determine if these compounds can form the GTPase-activating state R*, a situation that is possible, in principle, by microscopic reversibility. Neither all-trans-retinal nor its oxime, when incubated with bovine opsin in the dark, caused activation of the GTPase, requiring at least a 5 kcal/mol energy gap between them. Furthermore, stoichiometric adducts of all-trans-retinoids and opsin were also unable to mediate activation of the GTPase. Since both all-trans-15,16-dihydroretinylopsin and all-trans-retinoylopsin possess an all-trans-retinoid permanently adducted to opsin, it can be concluded that the all-trans-retinoid chromophore-opsin linkage may be necessary but not sufficient to achieve activation of the visual pigment. PMID- 3002443 TI - Photoaffinity labeling of peptide binding sites of prolyl 4-hydroxylase with N-(4 azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5. AB - The synthesis is described of the photoaffinity label N-(4-azido-2 nitrophenyl)glycyl-(Pro-Pro-Gly)5 for the peptide binding site of prolyl 4 hydroxylase. The photoaffinity label is a good substrate and is capable of light induced inactivation of prolyl 4-hydroxylase activity. Inactivation depends on the concentration of photoaffinity label and is prevented by competition with excess (Pro-Pro-Gly)5. Two moles of photoaffinity label per mole of enzyme is needed for 100% inactivation of enzymic activity. Oxidative decarboxylation of 2 oxoglutarate measured in the absence of added peptide substrate is not affected by labeling. We conclude that the covalently bound nitreno derivative of N-(4 azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 acts by preventing the binding of peptide substrate to the catalytic site without interfering with the binding of the other substrates and cofactors 2-oxoglutarate, O2, Fe2+, and ascorbate. Labeling is specific for the alpha subunit of the tetrameric alpha 2 beta 2 enzyme. In addition to two catalytic binding sites that are blocked by the photoaffinity label, the enzyme contains binding subsites for peptide substrates, as judged from the capability of photoinactivated enzyme to bind to a poly(L proline) affinity column. These binding subsites may account for the rapidly increasing affinity for peptide substrates with increasing chain length. PMID- 3002445 TI - Complementary substrate specificities of class I and class II collagenases from Clostridium histolyticum. AB - The substrate specificities of three class I (beta, gamma, and eta) and three class II (sigma, epsilon, and zeta) collagenases from Clostridium histolyticum have been investigated by quantitating the kcat/KM values for the hydrolysis of 53 synthetic peptides with collagen-like sequences covering the P3 through P3 subsites of the substrate. For both classes of collagenases, there is a strong preference for Gly in subsites P1' and P3. All six enzymes also prefer substrates that contain Pro and Ala in subsites P2 and P2' and Hyp, Ala, or Arg in subsite P3'. This agrees well with the occupancies of these sites by these residues in type I collagen. However, peptides with Glu in subsites P2 or P2' are not good substrates, even though Glu occurs frequently in these positions in collagen. Conversely, all six enzymes prefer aromatic amino acids in subsite P1, even though such residues do not occur in this position in type I collagen. In general, the class II enzymes have a broader specificity than the class I enzymes. However, they are much less active toward sequences containing Hyp in subsites P1 and P3'. Thus, the two classes of collagenases have similar but complementary sequence specificities. This accounts for the ability of the two classes of enzymes to synergistically digest collagen. PMID- 3002444 TI - Inactivation of guanosine cyclic 3',5'-monophosphate dependent protein kinase from bovine lung by o-phthalaldehyde. AB - Guanosine cyclic 3',5'-monophosphate (cGMP) dependent protein kinase is inactivated by o-phthalaldehyde. The loss of phosphotransferase activity following treatment with o-phthalaldehyde was rapid, and the second-order rate constant at 25 degrees C and pH 7.3 was 35 M-1 s-1. The inactivation reaction did not follow saturation kinetics. The cGMP-dependent protein kinase was protected from inactivation by its substrates, MgATP and Ser-peptide. Fluorescence excitation and emission spectroscopic data showed that an isoindole derivative was formed following the reaction between cGMP-dependent protein kinase and o phthalaldehyde. Four moles of isoindole per mole of the cGMP-dependent protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. In the absence of cGMP, the protein kinase lost only 50% of its cGMP binding activity while there was almost a complete loss of its phosphotransferase activity. Studies in the presence of 20 microM cGMP, however, showed that about 2 mol of isoindole groups per mole of the protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. The second-order rate constant for inactivation of cGMP-dependent protein kinase by o-phthalaldehyde in the presence of 20 microM cGMP was 40 M-1 s-1. Fluorescence measurements of samples containing inactivated, iodoacetamide-modified, or 5'-[p-(fluorosulfonyl)benzoyl]adenosine modified, cGMP-dependent protein kinase and o-phthalaldehyde showed that the intensity of fluorescence in each case was about 50% of that obtained from unmodified, active cGMP-dependent protein kinase and o-phthalaldehyde.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002446 TI - Mode of hydrolysis of collagen-like peptides by class I and class II Clostridium histolyticum collagenases: evidence for both endopeptidase and tripeptidylcarboxypeptidase activities. AB - The action of three class I (beta, gamma, and eta) and three class II (delta, epsilon, and zeta) collagenases from Clostridium histolyticum on two series of peptides with collagen-like sequences has been examined. The peptides in the first series all contain 4-nitrophenylalanyl-Gly-Pro-Ala in subsites P1 through P3', but each is successively lengthened in the N-terminal direction by addition of an appropriate residue until subsite P5 is occupied. The second group of peptides all have cinnamoyl-Leu in subsites P2 and P1, respectively, but each is successively lengthened in the C-terminal direction by partial additions of the Gly-Pro-Leu triplet until subsite P6' is occupied. N-Terminal elongation causes the kcat/KM values to rise markedly and to level off after occupancy of subsite P6 for the class I enzymes and subsite P3 for the class II enzymes. C-Terminal elongation produces the best substrates for both classes of enzymes when subsites P3' or P4' are occupied by amino acids with free carboxyl groups. The kcat/KM values for the hydrolysis of both Leu-Gly bonds of cinnamoyl-Leu-Gly-Pro-Leu-Gly Pro-Leu have been measured for both classes of enzymes. Both rates are large, but both classes preferentially hydrolyze the Leu-Gly bond of the C-terminal triplet. Thus, both classes of enzymes exhibit both endopeptidase and tripeptidylcarboxypeptidase activities. PMID- 3002447 TI - Covalent complexes formed between plasma gelsolin and actin with a zero-length cross-linking compound. AB - Actin and plasma gelsolin were covalently cross-linked with the zero-length cross linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Two major intermolecularly linked products were identified on polyacrylamide gels. By use of 14C-labeled actin and 125I-labeled gelsolin, these were shown to be the 1:1 and 2:1 complexes of actin with gelsolin, respectively. The higher molecular weight complex predominated under all conditions tested including the presence and absence of Ca2+. In titration experiments in which actin at different concentrations was reacted with a fixed concentration of gelsolin, end points were obtained for the formation of both cross-linked species at about two actins per gelsolin, implying that a 2:1 noncovalent complex is cross-linked. In 0.1 mM Ca2+, the extent of cross-linking was independent of protein concentration down to 50 nM gelsolin. At low Ca2+ concentrations (less than 10(-8)M), the extent of cross-linking was very much reduced at micromolar gelsolin and fell to zero at about 100 nM gelsolin. The binding of actin to gelsolin to give a cross-linkable complex is therefore very strong at 0.1 mM Ca2+ but much weaker at low Ca2+ concentrations. PMID- 3002448 TI - pH-induced conformation changes and equilibrium unfolding in yeast iso-2 cytochrome c. AB - The relationship between pH-induced conformational changes in iso-2 cytochrome c from Saccharomyces cerevisiae and the guanidine hydrochloride induced unfolding transition has been investigated. Comparison of equilibrium unfolding transitions at acid, neutral, and alkaline pH shows that stability toward guanidine hydrochloride denaturation is decreased at low pH but increased at high pH. In the acid range the decrease in stability of the folded protein is correlated with changes in the visible spectrum, which indicate conversion to a high-spin heme state--probably involving the loss of heme ligands. The increase in stability at high pH is correlated with a pH-induced conformational change with an apparent pK near 8. As in the case of homologous cytochromes c, this transition involves the loss of the 695-nm absorbance band with only minor changes in other optical parameters. For the unfolded protein, optical spectroscopy and 1H NMR spectroscopy are consistent with a random coil unfolded state in which amino acid side chains serve as (low-spin) heme ligands at both neutral and alkaline pH. However, the paramagnetic region of the proton NMR spectrum of unfolded iso-2 cytochrome c indicates a change in the (low-spin) heme-ligand complex at high pH. Apparently, the folded and unfolded states of the (inactive) alkaline form differ from the corresponding states of the less stable native protein. PMID- 3002449 TI - Synthetic substrates of vertebrate collagenase. AB - The active site specificity of vertebrate collagenase was mapped with the synthesis of a variety of peptides, peptolides, and peptide esters. The enzyme was found to prefer very lipophilic sequences, and it was also found to be an esterase. The thio peptolide Ac-Pro-Leu-Gly-SCH[CH2CH(CH3)2]CO-Leu-Gly-OC2H5 was found to be an exceptional substrate. High-performance liquid chromatography and tandem mass spectrometry were used to unambiguously establish the cleavage site in several peptide substrates. PMID- 3002450 TI - Phospholipid spin probes measure the effects of ethanol on the molecular order of liver microsomes. AB - Ethanol, in vitro, is known to perturb the molecular order of the phospholipids in biological membranes, while chronic ethanol exposure, in vivo, leads to resistance to disordering. Such changes have usually been measured by electron spin resonance, utilizing fatty acid spin probes. The use of such probes is controversial, since their orientation in the membrane may not accurately represent that of individual phospholipids. We, therefore, compared ethanol induced structural perturbations in the membranes of rat hepatic microsomes measured with the spin probe 12-doxylstearic acid (SA 12) with those assayed with various phospholipid spin probes. With SA 12, the addition of increasing amounts of ethanol (50-250 mM) in vitro caused a progressive decrease in the membrane molecular order, as measured by electron spin resonance (ESR). By contrast, microsomes obtained from rats chronically fed ethanol were resistant to the disordering effect of ethanol. Microsomes labeled with the phospholipid spin probes 1-palmitoyl-2-(12-doxylstearoyl)phosphatidylcholine, phosphatidylethanolamine, or -phosphatidic acid also exhibited increased disordering with the addition of increasing amounts of ethanol. However, the effect noted with phospholipid spin probes was less than that observed with the fatty acid probe. Microsomes obtained from the livers of chronically intoxicated animals labeled with the phospholipid probes were also resistant to the disordering effects of ethanol in vitro. These results suggest that fatty acid spin probes are qualitatively valid for measuring membrane perturbations in biological membranes, ethanol affects all microsomal phospholipids, regardless of chemical dissimilarities (e.g., head-group structure), in a qualitatively similar fashion, and the fluidization of fatty acyl chains in microsomal membranes is comparable in different membrane phospholipids. PMID- 3002451 TI - Purification and properties of an O2-.-generating oxidase from bovine polymorphonuclear neutrophils. AB - A membrane-associated O2-.-generating oxidase has been purified from activated bovine polymorphonuclear neutrophils (PMN). The oxidase was extracted with Triton X-100 from a PMN membrane fraction largely devoid of lysosomal granules. The Triton extract was purified by a series of steps, including ion-exchange chromatography on DE-52 cellulose, gel filtration on Sephadex G-200, and isoelectric focusing. The O2-.-generating oxidase activity was assayed as a superoxide dismutase inhibitable cytochrome c reductase. The activity of the purified enzyme was strictly dependent on NADPH as electron donor. The purification factor with respect to the phorbol myristate acetate activated PMN was 75, and the recovery was about 6%. The reactivity of the purified oxidase was increased by 3-4-fold after incubation with asolectin. The minimum molecular weight of the oxidase, deduced from migration in sodium dodecyl sulfate polyacrylamide gel electrophoresis, was 65 000 +/- 3000. The optimum pH of the oxidase was 7.5, its KM,NADPH was congruent to 30 microM, and its isoelectric point was at pH 5.0. The enzyme was inhibited by low concentrations of mersalyl (half-inhibition congruent to 10 microM) and Cibacron Blue (half-inhibition less than 10 microM). It was insensitive to 1 mM cyanide. Rapid loss of activity occurred at 0-2 degrees C, concomitantly with a decrease in sensitivity to superoxide dismutase: both activity and sensitivity to superoxide dismutase could be restored by addition of asolectin. The purified oxidase contained no spectrophotometrically detectable cytochrome b, and enzymatic assay failed to detect FAD in oxidase preparations subjected to heat treatment or trypsin digestion. PMID- 3002452 TI - Comparison of physicochemical properties of purified mucus glycoproteins isolated from respiratory secretions of cystic fibrosis and asthmatic patients. AB - The major nonreduced mucus glycoproteins (mucins) from sputa of cystic fibrosis (CF) and asthmatic patients have been purified to electrophoretic homogeneity and subjected to physical and chemical characterization. The sputum specimens were solubilized in buffer containing 0.22 M KSCN and fractionated on Bio-Gel A-5m, followed by digestion with DNase, rechromatography on the same column, and chromatography on hydroxylapatite. Sodium dodecyl sulfate gel electrophoresis of purified mucins gave a single band. Carbohydrate analyses of the purified mucins showed no significant differences in the sugar components from the two mucins. However, the CF mucin contained substantially higher (11%) sulfate content than that observed for the asthmatic mucin (5.9%). Amino acid analyses indicated that the CF mucin had higher levels of serine plus threonine (35%) as compared to the asthmatic mucin (29%). In contrast, CF mucin contained a lower content of aspartate, glutamate, and glycine than that observed for the asthmatic mucin. Molecular weights of 3.8 X 10(6) and 3.5 X 10(6) were obtained for CF and asthmatic mucins, respectively, from light-scattering studies of mucins in the presence of 6 M guanidine hydrochloride. Reduction of the disulfide bonds of the two mucins did not alter their molecular weights. Liquid chromatographic studies on Sepharose CL2B showed that CF mucin forms aggregates sufficiently large to be excluded from the gel. As compared to the CF mucin, the asthmatic mucin formed fewer of these large aggregates under identical experimental conditions. Reduction and alkylation of the mucins resulted in their inability to form aggregates. The higher state of aggregation of CF mucin may influence the viscoelastic properties of the CF lung's mucus secretions. PMID- 3002453 TI - A stereochemical study of the mechanism of activation of donor oligonucleotides by RNA ligase from bacteriophage T4 infected Escherichia coli. AB - RNA ligase from bacteriophage T4 infected Escherichia coli catalyzes the activation of adenosine 3',5'-bisphosphate (representing the donor oligonucleotide) by adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate with retention of configuration at P alpha. Since single-step enzyme-catalyzed nucleotidyl transfer reactions proceed with inversion, this stereochemical result provides support for a double displacement mechanism involving an adenylyl-enzyme intermediate as proposed previously from isotope exchange experiments. PMID- 3002454 TI - Nucleoprotein hybridization: a method for isolating specific genes as high molecular weight chromatin. AB - We describe a new technique designed to isolate specific eukaryotic genes as native oligonucleosome fragments. The isolation method consists of hybridization of single-stranded termini of chromatin restriction fragments to complementary mercurated DNA probes, followed by isolation of the hybrids by sulfhydryl Sepharose chromatography. SV40 minichromosomes were used to test the effectiveness of the technique. About 80% of KpnI- or BamHI-restricted and lambda exonuclease treated SV40 minichromosomes hybridized to an appropriate DNA probe after a 12-h hybridization reaction under mild conditions (0.1 M aqueous salt, 37 degrees C, pH 8). When the restricted minichromosomes were mixed with a 15-fold excess of "background" chromatin from sea urchin embryos, nucleoprotein hybridization was able to reisolate the SV40 chromatin to 88% purity with a 63% yield. This represented a 115-fold enrichment of specific genes as chromatin. Results of electron microscopy and polyacrylamide gel electrophoresis indicate that the hybridized SV40 chromatin has not lost the major chromosomal proteins characteristic of SV40 nor acquired significant amounts of protein due to exchange with background chromatin. Our experimental results show that it is currently possible to isolate repeated genes from higher eukaryotes for structural and biochemical study of the proteins involved with gene regulation. PMID- 3002455 TI - The use of a water-soluble carbodiimide to study the interaction between Chromatium vinosum flavocytochrome c-552 and cytochrome c. AB - The interaction between horse heart cytochrome c and Chromatium vinosum flavocytochrome c-552 was studied using the water-soluble reagent 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide (EDC). Treatment of flavocytochrome c-552 with EDC was found to inhibit the sulfide: cytochrome c reductase activity of the enzyme. SDS gel electrophoresis studies revealed that EDC treatment led to modification of carboxyl groups in both the Mr 21 000 heme peptide and the Mr 46 000 flavin peptide, and also to the formation of a cross-linked heme peptide dimer with an Mr value of 42 000. Both the inhibition of sulfide: cytochrome c reductase activity and the formation of the heme peptide dimer were decreased when the EDC modification was carried out in the presence of cytochrome c. In addition, two new cross-linked species with Mr values of 34 000 and 59 000 were formed. These were identified as cross-linked cytochrome c-heme peptide and cytochrome c-flavin peptide species, respectively. Neither of these species were formed in the presence of a cytochrome c derivative in which all of the lysine amino groups had been dimethylated, demonstrating that EDC had cross-linked lysine amino groups on native cytochrome c to carboxyl groups on the heme and flavin peptides. A complex between cytochrome c and flavocytochrome c-552 was required for cross-linking to occur, since ionic strengths above 100 mM inhibited cross-linking. PMID- 3002456 TI - A novel, simple and rapid method for the isolation of mitochondria which exhibit respiratory control, from rat small intestinal mucosa. AB - A method is described for the isolation of functional mitochondria from rat intestinal mucosa. Its novel feature is the removal of mucus from the initial homogenate by treatment with DEAE-cellulose. The preparations exhibited acceptable ADP:O ratios, high State-3 respiration rates, and respiratory control ratios in excess of 3 when succinate, beta-hydroxybutyrate, glutamate/malate and glutamine were test substrates. PMID- 3002457 TI - Reversible and irreversible effects of alkaline pH on Photosystem II electron transfer reactions. AB - Incubation of highly active, O2-evolving PS II preparations at alkaline pH inhibits donor side electron-transfer reactions in two distinct fashions, one reversible the other irreversible. In both cases, O2 evolution is inhibited, with concomitant loss of the light-induced multiline and g = 4.1 EPR signals and an increased steady-state level of EPR Signal II induced by continuous illumination. However, the inhibition that is observed between pH 7.0 and 8.0 is readily reversible by resuspension at low pH, while above pH 8.0 the effect is irreversible. In addition, under repetitive flash conditions the ms decay kinetics remains largely unchanged at pH less than or equal to 8.0 but shows about a 2-fold increase in amplitude and is slowed at pH above 8.0. The irreversible component of inhibition most likely can be attributed to the loss of Mn and the 16, 24 and 33 kDa proteins. The reversible component may be mediated by displacement of Cl- from an anion-binding site by OH- or by titration of ionizable groups on the protein(s) associated with water-splitting. We propose that the reversible inhibition blocks electron transfer between the O2-evolving complex and an intermediate which serves as the direct donor to Signal II, while the irreversible inhibition blocks the reduction of Signal II by this intermediate donor species. PMID- 3002458 TI - Monoclonal antibody to human cytochrome c: effect on electron-transfer reactions. AB - A monoclonal antibody has been produced to an antigenic site on human cytochrome c which includes amino acid number 58 (isoleucine). This area is on the bottom back of the cytochrome, removed from the postulated binding/reaction sites for oxidase and reductase, but in the area of the molecule where an appreciable change in conformation is seen on oxidation-reduction. In spectrophotometric assays, where binding of cytochrome c to the oxidase or reductase is rate limiting, the antibody gave stimulation of the reductase reaction under some conditions, where the oxidase reaction was inhibited. Also variation of the pH of the reaction medium resulted in differential effects on the oxidase and reductase reactions. Different effects of the antibody were seen when the oxidase was assayed polarographically, as compared to the spectrophotometric measurements. The data show that the binding/reaction sites on cytochrome c for the oxidase and reductase must be different. They suggest that binding of antibody may affect conformational changes in the whole molecule, distorting the binding/reaction sites. Conformational changes may be involved as a control mechanism in cytochrome c-mediated electron-transfer reactions. PMID- 3002460 TI - Interaction of adriamycin with cardiolipin-containing vesicles. Evidence of an embedded site for the dihydroanthraquinone moiety. AB - In the presence of cardiolipin-containing small unilamellar vesicles, the antitumor compound adriamycin loses its ability to catalyse the flow of electrons from NADH to molecular oxygen through NADH dehydrogenase. The data strongly suggest that in the presence of cardiolipin the dihydroanthraquinone moiety is embedded in the phospholipid bilayer and thus inaccessible to the enzyme. PMID- 3002459 TI - The role of molecular conformation in ion capture by carboxylic ionophores: a circular dichroism study of narasin A in single-phase solvents and liposomes. AB - Conformational and thermodynamic aspects of cation binding by the carboxylic ionophore narasin A were studied by circular dichroism (CD). In single-phase solvents, dramatic increases in the maximum differential absorption (delta epsilon) of the C-11 carbonyl were observed upon the binding of K+, Na+ and protons to the free anionic form. These changes were associated with major shifts in the conformation equilibrium between extended and pseudocyclic conformers of narasin. Similar CD changes observed upon the binding of K+ to narasin A in dimyristoylphosphatidylcholine vesicles provided evidence that in the membrane environment, comparable conformation changes were associated with ion binding. Variation of the polar and protic properties of single-phase solvents was also found to influence the delta epsilon of the cation bound species of narasin A, supporting previous evidence for polarity-mediated modulation of conformation. Comparison of cation binding affinities indicated that in both single-phase solvents and liposomes, narasin had a marked equilibrium selectivity for K+ over Na+. PMID- 3002461 TI - Free protons do not substitute for sodium ions in buffer-mediated phosphorylation of (Na+ + K+)-ATPase. AB - In view of our recent finding of imidazole-activation of the phosphorylation of (Na+ + K+)-ATPase and the suggestion by others of an activating role of protons, in lieu of sodium ions, in the overall hydrolytic and phosphorylation processes of the enzyme, we have investigated the effect of pH on the phosphorylation process. No indication of proton activation is found. Rather, phosphorylation at low pH in the absence of Na+ is dependent on the buffer concentration. Imidazole H+ stimulated phosphorylation at pH 5 reaches the same maximal steady-state level as Na+-stimulated phosphorylation. Low pH also elicits Tris-H+ stimulated phosphorylation, but due to a simultaneous inhibitory effect of this buffer the maximal steady-state level is no more than 50% of the Na+-stimulated phosphorylation level. Protons inhibit rather than activate phosphorylation. Upon decreasing the pH from 7 to 5, we observe for all ligands, whether activating or inhibiting phosphorylation (ATP, Na+, protonated imidazole, Mg2+ and K+), a decrease in affinity (largest for Mg2+) and a decrease in the maximal steady state phosphorylation capacity. The effects of Na+ and imidazole-H+ on the phosphorylation step have been compared with those on the E2----E1 conformational change, which leads to the phosphorylation step. The different pH-dependence of the affinities for Na+ and protonated buffer in the E2----E1 transition suggests that there are separate activation sites with different pK values for Na+ and the buffer cation. The above findings rule out a role of free protons as a substitution for Na+ in the phosphorylation process. PMID- 3002462 TI - Changes in membrane lipid composition of human erythrocytes after dietary supplementation of (n-3) polyunsaturated fatty acids. Maintenance of membrane fluidity. AB - The effect of dietary (n-3) polyunsaturated fatty acids on erythrocyte membrane lipid composition, fluidity, and flexibility was studied in seven healthy subjects. An eight weeks daily supplementation of 3 g of the (n-3) fatty acids eicosapentaenoic and docosahexaenoic acid resulted in an increased unsaturation of erythrocyte phosphatidylcholine (PC) and phosphatidylethanolamine (PE). This change was accompanied by a slight decrease in PC and PE content (P less than 0.05) and an increase in sphingomyelin content (P less than 0.01). The erythrocyte membrane fluidity, measured with electron spin resonance of intact erythrocytes and with fluorescence polarization of erythrocyte ghosts did not change. No change was seen in the viscosity of erythrocyte suspensions of haematocrit = 0.80, measured at various shear rates. The supplementation caused a 42% decrease in plasma triacylglycerol levels. We suggest that the change in the erythrocyte membrane fatty acid composition induced by the dietary supplementation of (n-3) fatty acids might be counteracted by a change in the phospholipid class distribution, resulting in overall maintenance of membrane fluidity. PMID- 3002463 TI - One-dimensional crystals of (Na+ + K+)-ATPase dimers. AB - Preparations of purified (Na+ + K+)-ATPase contain both fragments of membranes and long and undulating cylindrical structures. These structures have been described as edgeways of membrane fragments. We have analyzed these structures using negative staining, thin sectioning and freeze-fracture-etch electron microscopy and describe their structure for the first time. Each cylinder is 12 19 nm in width and is comprised of an unstained core from which rows of distinct particles spaced 5-6 nm apart project on both sides. Each cylindrical structure was interpreted as a linear polymer of (alpha beta)2 dimers of (Na+ + K+)-ATPase molecules. Therefore, the particles that project from both sides are the cytoplasmic domains of the molecules of the enzyme, whereas the membrane-spanning domains form the unstained core of the cylinder. From considerations of the packing of the dimers in the cylinder we conclude that the cross-sectional area of the cytoplasmic domain should be larger than that of the membrane-spanning domain. Our results are consistent with the hypothesis that the (alpha beta) protomer is the native state of the enzyme. The (alpha beta)2 dimers observed in the fractions are the result of a secondary aggregation process occurring during the purification procedure. PMID- 3002464 TI - Large-scale purification of alpha 2-adrenergic receptor-enriched membranes from human platelets. Persistent association of guanine nucleotides with nonpurified membranes. AB - A simple large-scale purification of alpha 2-adrenergic receptor-enriched membranes from human platelets is described. Binding of the antagonist [3H]yohimbine is enriched 3-5-fold compared to a crude membrane fraction. Binding of low concentrations of the partial agonist 3-H-rho-aminoclonidine is increased 15-20-fold due to a higher binding affinity for the purified membranes. A soluble inhibitor of 3H-rho-aminoclonidine binding to purified membranes is found even in thrice-washed crude platelet membranes. The guanine nucleotides GDP and GTP are found to account for this inhibitory activity. Forskolin-stimulated adenylate cyclase activity is also enriched in the purified membrane fraction. Adenylate cyclase activity is inhibited by alpha 2-agonist to a comparable extent in all membrane fractions. This membrane preparation should prove useful in studies of alpha 2-adrenergic receptor mechanisms. PMID- 3002465 TI - Kinetic properties of the purified Ca2+-translocating ATPase from human erythrocyte plasma membrane. AB - The basic kinetic properties of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte membranes were studied. A complex interaction between the major ligands (i.e., Ca2+, Mg2+, H+, calmodulin and ATP) and the enzyme was found. The apparent affinity of the enzyme for Ca2+ was inversely proportional to the concentration of free Mg2+ and H+, both in the presence or absence of calmodulin. In addition, the apparent affinity of the enzyme for Ca2+ was significantly increased by the presence of calmodulin at high concentrations of MgCl2 (5 mM), while it was hardly affected at low concentrations of MgCl2 (2 mM or less). In addition, the ATPase activity was inhibited by free Mg2+ in the millimolar concentration range. Evidence for a high degree of positive cooperativity for Ca2+ activation of the enzyme (Hill coefficient near to 4) was found in the presence of calmodulin in the slightly alkaline pH range. The degree of cooperativity induced by Ca2+ in the presence of calmodulin was decreased strongly as the pH decreased to acid values (Hill coefficient below 2). In the absence of calmodulin, the Hill coefficient was 2 or slightly below over the whole pH range tested. Two binding affinities of the enzyme for ATP were found. The apparent affinity of the enzyme for calmodulin was around 6 nM and independent of the Mg2+ concentration. The degree of stimulation of the ATPase activity by calmodulin was dependent on the concentrations of both Ca2+ and Mg2+ in the assay system. PMID- 3002466 TI - The interaction of Sendai virus glycoprotein-bearing recombinant vesicles with cell surfaces. AB - Sendai virus glycoproteins HN and F were purified by immunoaffinity chromatography from virions disrupted by beta-D-octylglucoside. The purified glycoproteins were reconstituted in recombinant vesicles with phosphatidylcholine or phosphatidylethanolamine and phosphatidylserine. P815 or EL-4 cells treated with glycoprotein HN/F-phosphatidylcholine recombinant vesicles acquired the glycoproteins and retained them in the plasma membrane for 4 h as demonstrated by surface immunofluorescence specific for each protein. Cells treated with glycoprotein HN-phosphatidylcholine recombinant vesicles initially bore glycoprotein HN on the surface but the protein eluted within 2 h. Surfaces of cells treated with glycoprotein F-phosphatidylcholine recombinant vesicles did not acquire the glycoprotein. Cells treated with glycoprotein HN phosphatidylethanolamine: phosphatidylserine recombinant vesicles or glycoprotein F-phosphatidylethanolamine: phosphatidylserine recombinant vesicles in the presence of 5 mM Ca2+ acquired each protein for at least 2 h. Experiments showed that the acquired glycoproteins capped with antibody and that when glycoproteins HN and F were together on the surface they co-capped. Acquired viral glycoproteins did not co-cap with intrinsic H-2 glycoproteins. PMID- 3002467 TI - Ouabain-insensitive, halide-sensitive Tl+ uptake by canine iliac arteries. AB - Ouabain-insensitive Tl+ uptake by canine iliac arteries was studied. A significant fraction was halide-sensitive, with Br- substituting well for Cl-. The halide-sensitive component was inhibited by diuretics (MK196, bumetanide), PCMBS, low temperatures and external cations K+, Rb+. External but not internal Na+ was necessary for the uptake process. The process was not sensitive to disulphonic stilbenes. The halide-sensitive uptake appears to represent the operation of a (Na+/K+/Cl-)-cotransport process in arteries. PMID- 3002468 TI - (Na+ + K+)-ATPase in artificial lipid vesicles: influence of lipid structure on pumping rate. AB - (Na+ + K+)-ATPase from kidney outer medulla was incorporated into tightly-sealed, single-shelled lipid vesicles by a detergent-dialysis procedure. The rate of ATP driven potassium extrusion from vesicles formed from different phosphatidylcholines (PC) was measured optically, using a voltage-sensitive dye in the presence of valinomycin. High transport rates were observed for di(18:1)PC, di(20:1)PC and di(22:1)PC, whereas vesicles formed from di(14:1)PC and di(16:1)PC were virtually inactive. The variation of pumping activity with lipid structure mainly results from differences in the amount of enzyme incorporated with the correct orientation into the vesicle membrane, and to a lesser extent from lipid-dependent variations of the intrinsic turnover rate of the enzyme. The activation energy of ion transport decreases in the order di(16:1)PC, di(18:1)PC, di(20:1)PC approximately equal to di(22:1)PC. PMID- 3002469 TI - Association of gangliosides with the lymphocyte plasma membrane studied using radiolabels and spin labels. AB - Gangliosides are known to act as potent suppressors of lectin-stimulated lymphocyte activation when added to the culture medium. Since this effect may be mediated via ganglioside association with (or insertion into) the plasma membrane, we have used 3H- and spin-labelled derivatives of mixed gangliosides to probe the nature of this interaction. Gangliosides bind rapidly to the lymphocyte membrane and show no preference for association with either inside-out or right side-out membrane vesicles. Around 20% of the bound gangliosides can be removed by repetitive washing, and a further 22-28% by treatment with pronase for 1 h, suggesting that this fraction is tightly bound to membrane proteins at the cell surface. The ESR spectrum of membrane-bound gangliosides did not resemble the spin-exchanged spectrum of micellar spin-labelled gangliosides in aqueous solution, but was similar to that seen for 5 mol% ganglioside spin label in liposomes of egg phosphatidylcholine. This suggests that the bulk of the membrane bound gangliosides are inserted and molecular dispersed in the lymphocyte membrane. Binding of wheat-germ agglutinin to lymphocyte-associated gangliosides results in specific immobilization of the carbohydrate headgroup, while concanavalin A and other lectins have little or no effect on oligosaccharide mobility. Membrane-inserted gangliosides show a response to lectin binding which is qualitatively different from that seen for gangliosides in bilayers of phosphatidylcholine. PMID- 3002471 TI - Effects of very small amounts of cholesterol on gel-phase phosphatidylcholine membranes. AB - The mobility of 5-doxyl stearic acid spin label (5-SASL) in the gel phase of dipalmitoylphosphatidylcholine membranes between the main transition and subtransition temperatures was studied as a function of cholesterol content. Very small amounts of cholesterol (0.01-1 mol%) cause a dramatic increase in the mobility of 5-SASL. Temperature-drop experiments from 38 degrees C to 28 degrees C were made across the pretransition temperature and the rate of approach to equilibrium was measured. Cholesterol at low concentrations also affects this rate. The membrane reached equilibrium after 10 h in the absence of cholesterol, 3 h at 0.01 mol% cholesterol, and less than 10 min at 0.03 mol% cholesterol. PMID- 3002470 TI - Spin-label studies on phosphatidylcholine-cholesterol membranes: effects of alkyl chain length and unsaturation in the fluid phase. AB - Dynamic properties of phosphatidylcholine-cholesterol membranes in the fluid phase and water accessibility to the membranes have been studied as a function of phospholipid alkyl chain length, saturation, mole fraction of cholesterol, and temperature by using spin and fluorescence labelling methods. The results are the following: (1) The effect of cholesterol on motional freedom of 5-doxyl stearic acid spin label (5-SASL) and 16-doxyl stearic acid spin label (16-SASL) in saturated phosphatidylcholine membrane is significantly larger than the effects of alkyl chain length and introduction of unsaturation in the alkyl chain. (2) Variation of alkyl chain length of saturated phospholipids does not alter the effects of cholesterol except in the case of dilauroylphosphatidylcholine, which possesses the shortest alkyl chains (12 carbons) used in this work. (3) Unsaturation of the alkyl chains greatly reduces the ordering effect of cholesterol at C-5 and C-16 positions although unsaturation alone gives only minor fluidizing effects. (4) Introduction of 30 mol% cholesterol to dimyristoylphosphatidylcholine membranes decreases the lateral diffusion constants of lipids by a factor of four, while it causes only a slight decrease of lateral diffusion in dioleoylphosphatidylcholine membranes. (5) If compared at the same temperature, 5-SASL mobilities plotted as a function of mole fraction of cholesterol in the fluid phases of dimyristoylphosphatidylcholine-, dipalmitoylphosphatidylcholine- and distearoylphosphatidylcholine-cholesterol membranes are similar in wide ranges of temperature (45-82 degrees C) and cholesterol mole fraction (0-50%). (6) In isothermal experiments with saturated phosphatidylcholine membranes, 5-SASL is maximally immobilized at the phase boundary between Regions I and III reported by other workers (Recktenwald, D.J. and McConnell, H.M. (1981) Biochemistry 20, 4505-4510) and becomes more mobile away from the boundary in Regions I and III. (7) 5-SASL in unsaturated phosphatidylcholine membranes showed a gradual monotonic immobilization with increase of cholesterol mole fraction without showing any maximum in the range of cholesterol fractions studied. (8) By rigorously determining rigid-limit magnetic parameters of cholestane spin labels in membranes from Q-band second-derivative ESR spectra to monitor the dielectric environment around the nitroxide radical, it is concluded that cholesterol incorporation increases water accessibility in the hydrophilic loci of the membrane. In contrast, 12-(9-anthroyloxy)stearic acid fluorescence showed that water accessibility is decreased in the hydrophobic loci of the membrane. PMID- 3002472 TI - The lipid fluidity of rat colonic brush-border membrane vesicles modulates Na+-H+ exchange and osmotic water permeability. AB - Brush-border membrane vesicles were prepared from rat colonic epithelial cells. Steady-state fluorescence polarization techniques, using the fluorophores 1,6 diphenyl-1,3,5-hexatriene and DL-12-(9-anthroyl)stearic acid (12-AS), revealed that benzyl alcohol (25-75 mM) but not methyl alcohol (50-125 mM) significantly increased the fluidity of these vesicles. Benzyl alcohol (50 and 75 mM) but not methyl alcohol also increased amiloride-sensitive sodium-stimulated proton efflux from these vesicles at all concentrations of sodium tested (2.5-50.0 mM), as assessed by changes in the fluorescence of acridine orange. Benzyl alcohol, at 50 and 75 mM concentrations, increased the maximal velocity (Vmax) of this exchange process by approximately 58 and 75%, respectively. Neither concentration, however, altered the Km for sodium. Osmotic water flow, measured as rate constants of osmotic shrinkage of these vesicles using a stopped-flow nephelometric technique, was also increased by 75 mM benzyl alcohol but not by a similar concentration of methyl alcohol. The present data, therefore, demonstrate that the fluidity of rat colonic brush-border membranes can influence Na+-H+ exchange and osmotic water flow across these vesicles. PMID- 3002473 TI - Effects of chlorpromazine and other calmodulin antagonists on phosphatidylcholine induced vesiculation of platelet plasma membranes. AB - Dilauroylglycerophosphocholine (C12:0PC)-induced vesiculation of platelet plasma membranes (Kobayashi, T., Okamoto, H., Yamada, J.-I., Setaka, M. and Kwan, T. (1984) Biochim. Biophys. Acta 778, 210-218; Kobayashi, T., Yamada, J.-I., Satoh, N., Setaka, M. and Kwan, T. (1985) Biochim. Biophys. Acta 817, 307-312) was inhibited by chlorpromazine. Preincubation of platelets with chlorpromazine was required for inhibition but incorporation of chlorpromazine into C12:0PC liposomes was not necessary for it, indicating that the observed inhibition of vesiculation was mainly due to the effect of chlorpromazine on platelets and not that on liposomes. The change in platelet membrane fluidity caused by chlorpromazine was not the cause of inhibition of vesiculation. The inhibition of vesiculation by various other calmodulin antagonists was also observed. The inhibitory activities of these calmodulin antagonists and chlorpromazine correspond very well to their abilities to bind to calmodulin. N-(6-Aminohexyl)-5 chloro-1-naphthalenesulfonamide (W-7) inhibited vesiculation but a structural analogue of it, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), had no inhibitory activity. These results suggest the involvement of calmodulin in membrane vesiculation. PMID- 3002474 TI - Transverse tubules from frog skeletal muscle. Purification and properties of vesicles sealed with the inside-out orientation. AB - Transverse tubule vesicles were isolated from frog skeletal muscle by a procedure initially described by Rosemblatt et al. (J. Biol. Chem. 256, 8140-8148 (1981)) and later modified by Hidalgo et al. (J. Biol. Chem. 258, 13937-13945 (1983]. A large fraction of the isolated vesicles (80-90%) were sealed, as indicated by the detergent induced increase in (Na+ + K+)-ATPase activity and ATP-dependent ouabain binding. To determine the orientation of the sealed vesicles binding of digoxin, a lipid soluble derivative of ouabain, was measured. The same values of ATP-dependent digoxin binding were found with or without detergents, indicating that all the vesicles that are sealed have the ATP site accessible, and hence are sealed with the cytoplasmic side-out (inside-out orientation). The transverse tubule preparation isolated from frog muscle is highly purified, as indicated by its cholesterol content and its (Na+ + K+)-ATPase activity; negligible contamination with sarcoplasmic reticulum was observed, as indicated by the protein composition and the lack of measurable Ca2+-ATPase activity of the isolated transverse tubules. High initial rates of Mg2+-ATPase activity were found, with the peculiar property of being inhibited during the course of the reaction. Addition of lysophosphatidylcholine or saponin partially prevented the inhibition of Mg2+-ATPase activity during the reaction. PMID- 3002475 TI - Ion pathways in transverse tubules. Quantification of receptors in membranes isolated from frog and rabbit skeletal muscle. AB - The presence of four cation pathways in membrane vesicles isolated from transverse tubules of frog and rabbit skeletal muscle was studied by measuring binding of specific blockers. Transverse tubules purified from frog muscle have a maximal binding capacity for [3H]nitrendipine (a marker for voltage-dependent calcium channels) of 130 pmol/mg of protein; this binding is strongly dependent on temperature and, at 37 degrees C, on the presence of diltiazem. Receptors for [3H]ethylenediamine tetrodotoxin (a marker for voltage-dependent sodium channels) and for 125I-labeled alpha-bungarotoxin (a marker for acetylcholine-mediated channels) showed maximal binding values of about 5 pmol/mg. The number of sodium pumping sites in the isolated tubule vesicles, inferred from [3H]ouabain binding, was 215 pmol/mg. The high purity of this preparation makes feasible the use of these values as a criterion to judge the degree of purity of isolated preparations, and it allows investigation of transverse tubule contamination in other muscle membrane fractions. PMID- 3002476 TI - Muscle adenylate kinase catalyzes adenosine 5'-tetraphosphate synthesis from ATP and ADP. AB - Muscle adenylate kinases from rabbit and porcine sources were found by NMR to catalyze the formation of adenosine tetraphosphate (5'AdoP4) from adenosine triphosphate (ATP) and adenosine diphosphate (ADP). The reaction was completely reversible, with an equilibrium constant of approx. 0.1 at 30 degrees C. The synthesis of 5'AdoP4 from ATP and ADP occurred very slowly, taking over 12 h to reach equilibrium under the conditions used in this study. The sources of micromolar concentrations of 5'AdoP4 found in biological tissues is unknown: potentially, the slow adenylate-kinase-catalyzed breakdown and synthesis of 5'AdoP4 may serve as an important regulator of the steady-state concentration of 5'AdoP4 in muscle tissue. PMID- 3002478 TI - Studies on the spin-spin interaction between flavin and iron-sulfur cluster in an iron-sulfur flavoprotein. AB - When the di- or trimethylamine dehydrogenases (trimethylamine:(acceptor) oxidoreductase (demethylating), EC 1.5.99.7) of certain methylotrophic bacteria are reduced by two electrons with substrate unusual EPR signals arise at g = 2 and g = 4 (Steenkamp, D.J. and Beinert, H. (1982) Biochem. J. 207, 233-239; 241 252) indicative of spin-spin interaction between the FMN and iron-sulfur compounds of these enzymes. An attempt is made to understand, describe and simulate these spectra in terms of a triplet state with possible contributions from both dipolar and anisotropic exchange (J) interactions. No direct measurement of J is available, but various approaches to setting limits to J are outlined. According to these, J approximately 0.4 to 3 cm-1 or 15 to 50 cm-1. The spectra show, in the g = 2 region, a pair of rather sharp inner and a pair of broad outer lines; the latter broaden as well as move out from the center with increasing time (after substrate addition) and substrate concentration, while there is little change of g = 4. The best fits to such spectra were obtained by assuming distribution of D and E values, depending on substrate effects and arriving presumably from 'g-strain'. The fact that both shapes and intensities at g = 2 and g = 4 could be reproduced simultaneously at two frequencies indicates that the assumptions underlying our approaches and interpretations are permissible and reasonable, although we cannot claim their uniqueness. The distance between the centers of the spin densities of the flavin radical and the Fe-S cluster is thought to lie between the limits 3 to 5 A if the asymmetries in the spin-spin interaction are magnetic dipole-dipole in origin. Because there is an indication that the interaction is anisotropic exchange, the upper limit is less stringent. PMID- 3002477 TI - The effects of vanadium compounds on the activation of adenylate cyclase from rat adrenal membrane. AB - In rat adrenal membrane, vanadyl sulfate, but not vanadate, inhibits the nonhydrolyzable GTP analogs-, forskolin- and NaF-stimulated activation process of adenylate cyclase. In these reactions, the half-maximum concentration of vanadyl for inhibition was approx. 0.3 mM. The binding of [3H]guanyl-5'-yl imidodiphosphate to the membrane (Kd = 2 microM) was not affected by vanadyl sulfate under the conditions in which the vanadyl sulfate inhibits the activation process. Also, the binding of ACTH to its receptor was inhibited by neither vanadyl sulfate nor vanadate, and the catalytic unit of adenylate cyclase appears to be unaffected by vanadyl sulfate. When the activation by nonhydrolyzable GTP analog was enhanced by Ca2+, vanadyl sulfate strongly inhibited the activation of adenylate cyclase. PMID- 3002479 TI - Effect of temperature and low pH on structure and stability of matrix porin in micellar detergent solutions. AB - Thermal and low pH stabilities of matrix porin (Omp F) solubilized in the micellar solutions of ionic (SDS) and nonionic detergents were investigated by the methods of circular dichroism, intrinsic fluorescence, light scattering and sedimentation velocity. The stability of porin structure in solution is much higher in the presence of beta-octyl glucoside than with SDS. In the presence of SDS, sharp transitions were detected by all parameters measured, above 55 degrees C at neutral pH and below pH 4.5 at 20 degrees C. These transitions involve at least three concomitant processes: unfolding of protein, dissociation of trimers to monomers and the disruption of the protein-detergent micellar complexes, all events being irreversible in the presence of SDS. The nonionic detergent, beta octyl glucoside, increases the stability of porin in acidic conditions, since neither dissociation nor denaturation was observed in the pH region between 7.5 and 2.0. However, at pH less than 3.5, small, reversible changes in protein structure became evident. The thermal stability of porin is also increased by beta-octyl glucoside as evidenced by a transition temperature 15-20 degrees C higher as compared to SDS. A considerable degree of native porin structure was regained after heat treatment in the presence of beta-octyl glucoside, though the reconstituted trimers were not identical to the native ones. The addition of lipopolysaccharide and divalent cations (Ca2+, Mg2+) to the experimental system did not improve the thermal reversibility. PMID- 3002481 TI - Phosphatidate phosphatase activity in isolated rod outer segment from bovine retina. AB - Phosphatidate phosphohydrolase (EC 3.1.3.4) was detected in isolated bovine rod outer segments and its properties investigated. The enzyme activity was assayed using aqueously dispersed 1,2-diacyl-sn-[2-3H]glycerol 3-phosphate as substrate. The phosphatidic acid concentration was optimal at 1 mM and the estimated Km value was 6.7 X 10(-4) M. The activity was linear for 60 min with a protein concentration of 0.3 mg. A clear pH optimum was observed at 7.5. When the enzyme activity was measured using a substrate phosphatidic acid containing 15% lyso compound, the production of diacylglycerols was inhibited by about 70-75% at all concentrations studied. In rod outer segment preparations containing 0.2 mM Mg2+, further additions of the ion (0.2-2.5 mM) only produced a slight inhibition of the activity at 2.5 mM. F- (50 mM), Ca2+ (1 mM) and EDTA (50 mM) inhibited the dephosphorylation of the substrate by 80, 10 and 70%, respectively. The aqueously dispersed phosphatidic acid-dependent activity present in rod outer segments was stimulated by Triton X-100 and taurocholate. Similar values were observed in the enzyme activity of entire rod outer segments and in that of disks obtained from them, showing the enzyme to be associated with disk membrane. The [3H]diacylglycerol production from [3H]phosphatidic acid was analyzed in synaptosomal-mitochondrial and microsomal fractions and in a crude pigment epithelium preparation. The degree of dephosphorylation of phosphatidic acid in these subcellular fractions was as follows: microsomes greater than synaptosomal mitochondrial fraction greater than rod outer segment greater than crude pigment epithelium. The present report is the first evidence of phosphatidic acid phosphohydrolase activity associated with rod outer segment membranes. PMID- 3002480 TI - Studies of endogenous polyphosphoinositide hydrolysis in human platelet membranes. Evidence that polyphosphoinositides remain inaccessible to phosphodiesterase in the native membrane. AB - Human platelet plasma membranes incubated in the presence of [gamma-32P]ATP and 15 mM MgCl2 incorporated radioactivity mostly into phosphatidylinositol 4,5 bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP), which represented together over 90% of the total lipid radioactivity. After washing, reincubation of prelabelled membranes revealed some hydrolysis of the two compounds by phosphomonoesterase(s), as detected by the release of radioactive inorganic phosphate (Pi) from the two phospholipids. This degradation attained 40%/30 min for PIP in the presence of 2 mM calcium and cytosol. The effect of calcium was observed at concentrations equal to or greater than 10(-4) M. In no case did calcium alone facilitate the formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2). In contrast, simultaneous addition of 2 mM calcium and 2 mg/ml sodium deoxycholate promoted the formation of IP3 and IP2, indicating phosphodiesteratic cleavage of PIP2 and PIP. Phospholipase C activity was detected at calcium concentrations as low as 10(-7) M, in which case PIP2 hydrolysis was slightly more pronounced compared to PIP. Addition of cytosol increased to some extent the phospholipase C activity, suggesting that the low amount of enzyme remaining in the membrane is sufficient to promote submaximal degradation of PIP2 and PIP. We conclude that platelet polyphosphoinositides are present in the plasma membrane in a state where they remain inaccessible to phospholipase C, which is still fully active even at basal calcium concentrations, i.e., 10(-7) M. These results support the view that phosphodiesteratic cleavage of PIP2 promotes and thus precedes calcium mobilization brought about by IP3. The in vitro model presented here may prove very useful in future studies dealing with the mechanism rendering polyphosphoinositides accessible to phospholipase C attack upon agonist-receptor binding. PMID- 3002482 TI - Stimulation of the LDL receptor activity in the human hepatoma cell line Hep G2 by high-density serum fractions. AB - The regulation of the LDL receptor activity in the human hepatoma cell line Hep G2 was studied. In Hep G2 cells, in contrast with fibroblasts, the LDL receptor activity was increased 2.5-fold upon increasing the concentration of normal whole serum in the culture medium from 20 to 100% by volume. Incubation of the Hep G2 cells with physiological concentrations of LDL (up to 700 micrograms/ml) instead of incubation under serum-free conditions resulted in a maximum 2-fold decrease in LDL receptor activity (10-fold decrease in fibroblasts). Incubation with physiological concentrations of HDL with a density of between 1.16 and 1.20 g/ml (heavy HDL) resulted in an approximately 7-fold increase in LDL receptor activity (1.5-fold increase in fibroblasts). This increased LDL receptor activity is due to an increase in the number of LDL receptors. Furthermore, simultaneous incubation of Hep G2 cells with LDL and heavy HDL (both 200 micrograms/ml) resulted in a 3-fold stimulation of the LDL receptor activity as compared with incubation in serum-free medium. 3-Hydroxy-3-methylglutaryl-CoA reductase activity was also stimulated after incubation of Hep G2 with heavy HDL (up to 3 fold). The increased LDL receptor activity in Hep G2 cells after incubation with heavy HDL was independent of the action of lecithin:cholesterol acyltransferase during that incubation. However, previous modification of heavy HDL by lecithin:cholesterol acyltransferase resulted in an enhanced ability of heavy HDL to stimulate the LDL receptor activity. Our results indicate that in Hep G2 cells the heavy HDL-mediated stimulation of the LDL receptor activity overrules the LDL mediated down-regulation and raises the suggestion that in man the presence of heavy HDL and the action of lecithin:cholesterol acyltransferase in plasma may be of importance in receptor-mediated catabolism of LDL by the liver. PMID- 3002483 TI - Regulation of palmitoylcarnitine oxidation in isolated rat liver mitochondria. Role of the redox state of NAD(H). AB - The redox-mediated regulation of palmitoylcarnitine oxidation was studied in isolated rat liver mitochondria in which the mitochondrial free NADH/NAD+ ratio was controlled by graded concentrations of acetoacetate and ketomalonate in a rotenone and malonate-inhibited system in the presence of ADP. The NADH/NAD+ ratio was buffered kinetically by adjusting the concentrations of the hydrogen acceptor substances and determined by calibrated NAD(P)H fluorometry of the mitochondrial suspension. A two-fold variation in the beta-oxidation rate and a five-fold variation in the free NADH/NAD+ ratio was obtained in the presence of rotenone. A non-linear negative correlation was found between the acetyl-CoA concentration and the beta-oxidation rate and a negative correlation between the long-chain acyl-CoA concentration and the beta-oxidation rate. The data indicate that the redox state is a partial controller of the beta-oxidation rate in liver mitochondria. The contribution of acetyl-CoA, a putative regulator of beta oxidation at the acyl-CoA thiolase step is small under the conditions used. PMID- 3002484 TI - Eicosapentaenoic acid utilization by bovine aortic endothelial cells: effects on prostacyclin production. AB - We have investigated whether the presence of other fatty acids in physiologic amounts will influence the effects of eicosapentaenoic acid on cellular lipid metabolism and prostaglandin production. Eicosapentaenoic acid uptake by cultured bovine aortic endothelial cells was time and concentration dependent. At concentrations between 1 and 25 microM, most of the eicosapentaenoic acid was incorporated into phospholipids and of this, 60-90% was present in choline phosphoglycerides. Eicosapentaenoic acid inhibited arachidonic acid uptake and conversion to prostacyclin (prostaglandin I2) but was not itself converted to eicosanoids. Only small effects on the uptake of 10 microM eicosapentaenoic acid occurred when palmitic, stearic or oleic acids were added to the medium in concentrations up to 75 microM. In contrast, eicosapentaenoic acid uptake was reduced considerably by the presence of linoleic, n-6 eicosatrienoic, arachidonic or docosahexaenoic acids. Although a 100 microM mixture of palmitic, stearic, oleic and linoleic acid (25:10:50:15) had little effect on the uptake of 10 or 20 microM eicosapentaenoic acid, less of this acid was channeled into endothelial phospholipids. However, the fatty acid mixture did not prevent the inhibitory effect of eicosapentaenoic acid on prostaglandin I2 formation in response to either arachidonic acid or ionophore A23187. An 8 h exposure to eicosapentaenoic acid was required for the inhibition to become appreciable and, after 16 h, prostaglandin I2 production was reduced by as much as 60%. These findings indicate that the capacity of aortic endothelial cells to produce prostaglandin I2 is decreased by continuous exposure to eicosapentaenoic acid. Even if the eicosapentaenoic acid is present as a small percentage of a physiologic fatty acid mixture, it is still readily incorporated into endothelial phospholipids and retains its inhibitory effect against endothelial prostaglandin I2 formation. Therefore, these actions may be representative of the in vivo effects of eicosapentaenoic acid on the endothelium. PMID- 3002485 TI - Elongation of arachidonic and eicosapentaenoic acids limits their availability for thrombin-stimulated release from the glycerolipids of vascular endothelial cells. AB - This study has examined the thrombin-stimulated release of polyunsaturated fatty acids from endothelial glycerolipids. Human umbilical vein endothelial cells were incubated with 1.25 microM [14C]arachidonate or [14C]eicosapentaenoate and then exposed to thrombin in buffered saline plus albumin. After an incorporation period of 0.5 h, the thrombin-stimulated release of the two radiolabeled fatty acids was quite similar. By contrast, after 24 h of fatty acid incorporation, the thrombin-stimulated release of radiolabeled fatty acid from cells incubated with [14C]eicosapentaenoate was only 25-30% of that from cells with [14C]arachidonate. Analysis of cellular glycerolipids indicated that 23 and 72%, respectively, of the incorporated [14C]arachidonate and [14C]eicosapentaenoate had been elongated to 22-carbon fatty acids in 24 h. Both 20- and 22-carbon 14C-labeled fatty acids were released to albumin in the medium in control incubations. Addition of thrombin stimulated the release of [14C]arachidonate and [14C]eicosapentaenoate, but not of their respective elongation products. Furthermore, endothelial cells incorporated exogenous [14C]docosatetraenoate into cellular glycerolipids but did not release it in response to thrombin. Thus, the thrombin-stimulated release of polyunsaturated fatty acids from vascular endothelial cells is highly selective for arachidonate and eicosapentaenoate. These results suggest that the extensive elongation of eicosapentaenoate by these cells serves to remove n - 3 polyunsaturated fatty acids from the pool of cellular acyl groups which are released in response to thrombin and are thus made available for metabolism by cyclooxygenase and lipoxygenase enzymes. PMID- 3002486 TI - Fibronectin distribution in human aortic intima and atherosclerotic lesions: concentration of soluble and collagenase-releasable fractions. AB - Fibronectin is associated with cell attachment and migration and interacts with fibrin, collagen and glycosaminoglycans; thus, it may be a factor in the focal proliferation of smooth muscle cells and collagen in atherosclerosis. We have measured, by rocket immunoelectrophoresis, the concentrations of soluble and collagenase-releasable fibronectin in normal human aortic intima and different types of atherosclerotic lesions. Soluble fibronectin concentration showed no significant difference between normal intima and lesions, but was 6-8-times higher than expected on the basis of plasma concentration and molecular mass. The concentration free in the interstitial fluid was about 3-times the expected level, suggesting that it originates from local synthesis as well as plasma insudation. In tissue, about half the fibronectin appeared to be reversibly associated with tissue components. Incubation with collagenase released fibronectin equal to twice the soluble fraction from normal intima and early proliferative lesions. In more advanced plaques that were accumulating lipid, the amount released was significantly higher (P less than 0.05) and more than 3-times the soluble fraction, suggesting that it might be involved in lipid accumulation. However, there was no correlation between release of fibronectin and bound low density lipoprotein. PMID- 3002487 TI - Requirement of Ca2+ for the production and degradation of inositol 1,4,5 trisphosphate in macrophages. AB - The requirement of Ca2+ for the hydrolysis of phosphatidylinositol 4,5 bisphosphate (PtdInsP2) or the accumulation of inositol 1,4,5-trisphosphate (InsP3) in macrophages stimulated with fMet-Leu-Phe was examined using [32P]Pi or [3H]inositol-labeled cells. The dependence on Ca2+ of inositol-trisphosphate phosphatase was also examined. The application of 1 X 10(-8) M fMet-Leu-Phe caused a rapid decrease in the amount of PtdInsP2 to 70% of the control within 10 s, and the decrease was reverted to the control level by prolonged incubation. The decrease in the amount of PtdInsP2 accompanied the accumulation of phosphatidic acid and of InsP3, indicating that the loss of PtdInsP2 is due to phosphodiesteric breakdown. The dose-dependence of fMet-Leu-Phe or its analog on the hydrolysis of PtdInsP2 was much the same as that of the increase in intracellular free Ca2+ concentration in macrophages. The loss of PtdInsP2 as induced by fMet-Leu-Phe was similarly observed in macrophages treated with ionophore A23187 in the absence of external Ca2+ for 10 min. InsP3 was degraded by the particulate or cytosol fraction prepared from macrophages, and the activity of inositol-trisphosphate phosphatase in the particulate fraction was higher than that in the cytosol fraction. The enzyme in the cytosol fraction required Mg2+ for activity, and was activated by free Ca2+ concentrations ranging from 10(-7) to 10(-6) M in the presence of 1 mM MgCl2. PMID- 3002488 TI - Short-term stimulation by adenosine of basal and insulin-induced glycogen synthesis in rat adipose tissue. AB - The effects of adenosine on glycogen metabolism have been studied in isolated fat pads from epididymal adipose tissue. Adenosine caused a sustained short-term increase in the incorporation of [U-14C]glucose into glycogen, as well as a stimulation of both basal and insulin-induced [1-14C]glucose oxidation. Adenosine produced changes also in the activity of glycogen synthase and phosphorylase, these effects being apparent only when glucose was present in the incubation medium. The addition of adenosine prevented the depressed synthesis of glycogen observed in the presence of dibutyryl cyclic AMP. In the presence of adenosine deaminase, the stimulation by insulin of glycogen synthesis was markedly decreased. The results suggest that adenosine may have a regulatory role on glycogen synthesis by facilitating the glucose transport. PMID- 3002489 TI - Demonstration of the association of the cell-surface enzyme, 5'-nucleotidase, with microvillar microfilaments by phalloidin shift on velocity sedimentation gradients. AB - The cell-surface enzyme 5'-nucleotidase in microvilli from 13762 rat mammary adenocarcinoma cells remains largely associated with microfilament-containing high-speed pellets from Triton X-100 extracts of the microvilli. The fraction remaining with the insoluble portion is higher under ionic conditions which enhance microfilament stability. To minimize trapping and cosedimentation we have analyzed the distribution of microfilaments and 5'-nucleotidase activity on velocity sedimentation sucrose gradients of the microvillar extracts. A large fraction of the total enzyme activity is found in the filament fractions in the middle of the gradient. When phalloidin is included in the extraction buffer to stabilize the microfilaments, both the microfilaments and the bulk of the nucleotidase activity are shifted further into the gradients. Both the position of the filament fraction and the percentage of the total nucleotidase activity remaining with the filament fraction varies with extraction buffer composition and conditions. Nonetheless, under all conditions tested, a large percentage of the activity was shifted, along with the microfilaments, in the presence of phalloidin. These results are consistent with a specific association of 5' nucleotidase with microfilaments in the ascites tumor cell microvilli. PMID- 3002490 TI - Correlation of the internal microviscosity of human erythrocytes to the cell volume and the viscosity of hemoglobin solutions. AB - The microviscosity of the cytoplasm of human erythrocytes as well as of membrane free hemoglobin solutions was investigated measuring the rotation of the small spin-label molecule, Tempone. The dependence of the intracellular microviscosity on the extracellular pH and osmotic pressure which was varied by NaCl or sucrose was sufficiently explained on the basis of alterations of the red blood cell volume. The intracellular microviscosity depended exclusively on the hemoglobin concentration. It did not differ from that of comparable membrane-free hemoglobin solutions. It was not necessary to take into account long-range interactions between hemoglobin molecules. The conclusion therefore was that the intracellular viscosity is not modified by cytoplasmic structures or the cell membrane. Above a hemoglobin concentration of 6 mM the viscosity of hemoglobin solutions increased much faster than the microviscosity. From measurements obtained with different spin-labels it followed that also the charge of these molecules is of importance. PMID- 3002491 TI - The physical chemistry of cruciform structures in supercoiled DNA molecules. AB - Inverted repeat DNA sequences extrude cruciform structures when present in negatively supercoiled molecules, stabilised by the release of torsional stress brought about by the negative twist change. We have revealed the presence of cruciform structures by means of enzyme and chemical probing experiments and topological band shift methods. The geometry of cruciform structures has been studied from two points of view. The unpairing of bases in the loop region has been investigated using bisulphite modification, with the result that the central four nucleotides have single-stranded character, and the next pair have only partially single-stranded nature. Gel electrophoretic studies of a pseudo cruciform structure indicate that the cruciform junction introduces a pronounced bend into the molecule. The dependence of the formation of the ColE1 cruciform upon DNA supercoiling shows that it has a free energy of formation of 18.4 +/- 0.5 kcal mole-1. The kinetics of the extrusion process are complex. Most sequences extrude slowly with considerable temperature coefficients, but the detailed properties are strongly sequence-dependent. One synthetic inverted repeat sequence which we have studied in detail has an Arrhenius activation energy of 42.4 +/- 3.2 kcal mole-1. We discuss possible mechanistic pathways for the extrusion process. PMID- 3002492 TI - Synthetic oligodeoxyribonucleotides as tools in molecular genetics: the characterization of the CYC1 (iso-1-cytochrome c encoding) locus of Saccharomyces cerevisiae. AB - The use of synthetic oligodeoxyribonucleotides as tools for the isolation, characterization and mutagenesis of eukaryote genes has played a major role in the molecular definition of the CYC1 locus of Saccharomyces cerevisiae, which is the structural gene for the apoprotein of iso-1-cytochrome c. Thus, the possibility of using a synthetic oligodeoxyribonucleotide as a probe to identify and monitor the isolation of a specific gene was first established by model studies which defined the melting temperatures (TmS) for duplexes of oligonucleotides of different lengths and base compositions. This led to the isolation of the CYC1 locus using a synthetic 13 nucleotide probe. A more convenient strategy for determination of DNA sequences by the Sanger method was provided by using synthetic oligodeoxyribonucleotides as primers with denatured double-strand plasmid DNA as template. By this means, the sequence of the CYC1 locus was determined by "walking" along the gene without isolating restriction fragments of the DNA or separating DNA strands. Synthetic oligodeoxyribonucleotides, used as primers for reverse transcriptase with mRNA as template, were also used to precisely define the 5'- and 3'-ends of the iso-1 cytochrome c mRNA. Yet another application of synthetic oligodeoxyribonucleotides is their use as specific mutagens after in vitro incorporation into double stranded DNA. In the case of iso-1-cytochrome c, this mutagenic strategy is being used to define the role of conserved amino-acids in cytochrome function.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002494 TI - Total DNA synthesis and cloning in Escherichia coli of a gene coding for the human growth hormone releasing factor. AB - A DNA containing a sequence coding for the human growth hormone releasing factor (hGRF) has been obtained by enzymatic assembly of chemically synthesized DNA fragments. The synthetic gene consists of a 140 base-pair fragment containing initiation and termination signals for translation and appropriate protruding ends for cloning into a newly constructed plasmid vector (pULB1219). Eleven oligodeoxyribonucleotides, from 14 to 31 bases in length, sharing pairwise stretches of complementary regions of at least 13 bases were prepared by phosphotriester solid-phase synthesis. The DNA sequence was designed to take into account the optimal use of E. coli codons. Oligomers were annealed in one step and assembled by ligation. The DNA fragment of the expected size (140 bp) was recovered and cloned into the pULB1219 vector. The expected sequence was confirmed by DNA sequencing. PMID- 3002493 TI - Control of ribonucleic acid function by oligonucleoside methylphosphonates. AB - Oligodeoxyribonucleoside methylphosphonates contain nonionic 3'-5' linked methylphosphonate internucleotide bonds in place of the normal charged phosphodiester linkage of natural nucleic acids. These oligomers are resistant to nuclease hydrolysis, can pass through the membranes of mammalian cells in culture and can form stable hydrogen-bonded complexes with complementary nucleotide sequences of cellular RNAs such as mRNA. The oligomers are readily synthesized on insoluble polymer supports. Their chainlength and nucleotide sequence can be determined by chemical sequencing procedures. Oligonucleoside methylphosphonates which are complementary to the 5'-end, initiation codon region, or coding region of rabbit globin mRNA inhibit translation of the mRNA in rabbit reticulocyte lysates and globin synthesis in rabbit reticulocytes. This inhibition is due to the interaction of the oligomers with mRNA and the extent of inhibition is influenced by the secondary structure of the mRNA and the location of oligomer binding site on the mRNA. Oligomers complementary to the initiation codon regions of N, NS and G protein mRNAs of Vesicular stomatitis virus (VSV) inhibit virus protein synthesis in VSV-infected Mouse L-cells. These oligomers do not affect L cell protein synthesis or growth. Virus protein synthesis and growth can also be selectively inhibited by oligonucleoside methylphosphonates which are complementary to the donor or acceptor splice junctions of virus pre mRNA. An oligomer complementary to the donor splice junction of SV40 large T antigen mRNA inhibits T-antigen synthesis in SV40-infected African green monkey kidney cells but does not inhibit overall cellular protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002495 TI - Linker mutagenesis in the gene encoding the periplasmic maltose-binding protein of E. coli. AB - A plasmid carrying the malE gene, coding for the periplasmic maltose-binding protein of E. coli, was submitted to random mutagenesis by the insertion of a BamHI linker. About 25% of the clones recovered had acquired a BamHI site in the gene malE. Most of the linker insertions were accompanied by small deletions with an average size of 30 base pairs. Among 21 mutants synthesizing a stable maltose binding protein, 8 were still able to grow on maltose. A preliminary analysis of these mutants indicates that certain regions of the protein may not be essential for maltose transport. PMID- 3002496 TI - Transcription of integrated MMTV LTR DNA in fibroblast clones is not associated with DNA methylation but with the integration site. AB - By transfecting 3T3 TK- fibroblasts with a plasmid DNA containing the MMTV LTR promotor and the thymidine kinase gene, we have isolated TK+ clones that contain only one copy of the recombinant DNA. The newly acquired DNA is integrated at different sites in each TK+ clones and can be amplified as a tandem repeat in some cases. The basal level of expression of the newly acquired LTR sequence is variable among the TK+ clones studied. Among those producing detectable amounts of MMTV RNA, we found that transcription is initiated from the LTR promotor and that the synthesis is stimulated by a short dexamethasone treatment. In addition, the presence of tandem repeated LTR DNA does not amplify the MMTV RNA synthesis. Endogenous LTR sequences have been found to be methylated, whereas we show that the newly integrated sequences, both as single copy and tandem repeats, remain unmethylated. Thus, non-methylation of MMTV LTR DNA is necessary but not significant to allow its transcription. Our results suggest that the level of LTR promotor activity could be modulated by the nature of its integration site within the cellular genome. PMID- 3002498 TI - [Phencyclidine: behavioral effects and molecular targets]. PMID- 3002497 TI - Molecular recognition in noncovalent antitumor agent-DNA complexes: NMR studies of the base and sequence dependent recognition of the DNA minor groove by netropsin. AB - We have investigated intermolecular interactions and conformational features of the netropsin complexes with d(G1-G2-A3-A4-T5-T6-C7-C8) duplex (AATT 8-mer) and the d(G1-G2-T3-A4-T5-A6-C7-C8) duplex (TATA 8-mer) by one and two-dimensional NMR studies in solution. We have assigned the amide, pyrrole and methylene protons of netropsin and the base and sugar H1' protons of the nucleic acid from an analysis of the nuclear Overhauser effect (NOESY) and correlated (COSY) spectra of the complex at 25 degrees C. The directionality of the observed distance-dependent NOEs demonstrates that the 8-mer helices remain right-handed and that the arrangement of concave and convex face protons of netropsin are retained in the complexes. The observed changes in NOE patterns and chemical shift changes on complex formation suggest small conformational changes in the nucleic acid at the AATT and TATA antibiotic binding sites and possibly the flanking G.C base pairs. We observe intermolecular NOEs between all three amide and both pyrrole protons on the concave face of the antibiotic and the minor groove adenosine H2 proton of the two central A4.T5 base pairs of the AATT 8-mer and TATA 8-mer duplexes. The concave face pyrrole protons of the antibiotic also exhibit NOEs to the sugar H1' protons of residues 5 and 6 in the AATT and TATA 8-mer complexes. We also detect intermolecular NOEs between the guanidino and propioamidino methylene protons at either end of netropsin and the adenosine H2 proton of the two flanking A3.T6 base pairs in the AATT 8-mer and T3.A6 base pairs in the TATA 8-mer duplexes. These studies establish a set of nine contacts between the concave face of the antibiotic and the minor groove AATT segment and TATA segment of the 8-mer duplexes in solution. The observed magnitude of the NOEs require that there be no intervening water molecules sandwiched between the concave face of the antibiotic and the minor groove of the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation. The observed differences in the netropsin amide proton chemical shifts in the AATT 8-mer and TATA 8-mer complexes suggest differences in the strength and/or type of intermolecular hydrogen bonds at the AATT and TATA binding sites.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3002499 TI - Complete sequence of ovine alpha s2-casein messenger RNA. AB - The primary structure of mRNA coding for ovine alpha s2 casein has been determined by chemical sequencing of three cDNA clones and the primer extension products of the longest one. The mRNA was 1,024 nucleotides long, excluding the poly(A) tail. The length of the 5' noncoding, coding and 3' noncoding regions was 53, 669 and 302 nucleotides, respectively. A comparison of the nucleotide sequence of ovine alpha s2-casein and guinea-pig casein A mRNAs revealed an extensive homology in the 5' and 3' noncoding regions. The deduced amino acid sequence of ovine alpha s2-casein was compared with its bovine and guinea-pig counterparts. Moreover, an heterogeneity was evidenced in the mRNA population of the alpha s2-casein. PMID- 3002500 TI - Sequence analysis of the glyW region in Escherichia coli. AB - We have determined the DNA sequence of a 1-kilobase segment of the Escherichia coli chromosome. The segment contains glyW, a duplicate gene for tRNA3Gly, and its flanking regions. An insertion sequence, previously known to have occurred spontaneously within the sequenced fragment, was identified as IS1. Possible sites for initiation and termination of transcription were identified by comparing them with the sequences of model promoter regions and termination structures. The results suggest that the expression of glyW may depend upon the expression of the preceding gene, pgsA, by transcriptional or translational overlap, by cotranscription of these two genes, or both. PMID- 3002501 TI - Construction and identification of recombinant plasmids carrying cDNAs coding for ovine alpha S1-, alpha S2-, beta-, kappa-casein and beta-lactoglobulin. Nucleotide sequence of alpha S1-casein cDNA. AB - An ovine mammary cDNA library has been constructed from total poly(A)+ RNA isolated from the mammary gland of a lactating ewe, using a classical procedure. Blunt-ended double-stranded cDNAs prepared with reverse transcriptase and nuclease S1 were tailed with dCTP, inserted into the dGMP-tailed PstI site of plasmid pBR322 and cloned in E. coli. Five series of homologous clones representing abundant messenger RNAs (strong hybridization with a single-stranded cDNA probe generated from total poly(A)+ RNA) were selected using each time a different predominant cloned ds-cDNA as probe, then identified by positive hybridization-translation of the cognate mRNA and subsequent immunoprecipitation and electrophoresis of the protein. The lengths of alpha s1-, alpha s2-, beta-, kappa-casein and beta-lactoglobulin mRNAs are in the range of 1.2, 1.1, 1.25, 1.0 and 0.85 kb, respectively, as determined by Northern blotting analysis. Five homologous mRNAs of similar sizes were identified in the porcine species by dot blot hybridization and Northern analyses. The nucleotide sequence of alpha s1 casein mRNA was determined by sequencing, according to Maxam and Gilbert, both a 1080 bp long cloned ds-cDNA and a ss-cDNA (268 nucleotides) generated by 5' extension of a 5' terminal truncated radiolabeled fragment (83 bp) of the relevant ds-cDNA, used as primer for reverse transcription. The 3' non coding region (431 nucleotides, excluding the poly(A) tail) represents 70% of the length of the coding region (618 nucleotides) flanked by a 61 nucleotide 5' region. Comparison of sequences of ovine and bovine, rat and guinea-pig alpha s1-casein mRNAs has revealed a greater homology in the 3' and especially 5' non coding regions. In the reading frame, the conserved regions are essentially those corresponding to the signal peptide and phosphopeptide domains. The derived 206 amino acid sequence of ovine pre-alpha s1-casein differs from that of its bovine counterpart (genetic variant B) by 24 amino acid substitutions and a deletion of 8 amino acid residues occurring in the polypeptide chain of the mature protein. Such a variation (84% homology only) in two phylogenetically closely related species indicates a high rate of evolution of alpha s1-casein. PMID- 3002502 TI - In vivo and in vitro effect of mutations in tetA promoter from pSC101: insertion of poly(dA.dT) stretch in the spacer region does not inactivate the promoter. AB - Two mutants, mapping at the HindIII site (between the consensus sequences) of the pSC101 tetA promoter, were studied: MA2 corresponds to a 4 bp deletion between positions -12 and -15; B30 bears a 44 bp insertion C(TA)21 G at the HindIII site. Both mutants were assayed in vivo (ability of the plasmid to confer resistance to tetracycline, plasmid-directed protein synthesis, S1-mapping of mRNA) and in vitro (abortive initiation assay). Compared to w.t., MA2 is a poor promoter in vivo; RNA polymerase binding, complex activation and rate of initial oligonucleotide synthesis are strongly reduced in vitro; this is in keeping with the known effects of altering the consensus elements in E. coli promoters. In contrast, B30 shows in vivo a promoter activity only slightly reduced in comparison to that of the w.t. tetA promoter; both in vivo and in vitro, the transcription start site is outside and downstream the (TA)21 stretch, 5-7 bp upstream that found in the w.t. To adjust the behaviour of B30 and the claimed consensus distance between the E. coli promoter consensus sequences, some structural modification in the (TA)21 stretch -either spontaneous or induced by RNA polymerase- can be hypothesized. Unless the (TA)21 stretch itself plays the role of a relatively good promoter, the results suggest that promoter-specific elements may be distributed along the DNA sequences over distances longer, but seldom less, than the 17 +/- 2 bp consensus distance. PMID- 3002503 TI - Antianxiety effect of cannabis: involvement of central benzodiazepine receptors. AB - The present work, involving clinical, behavioral, and biochemical studies, was undertaken to elucidate the probable mechanism of the observed antianxiety effects of cannabis. The population for the clinical study consisted of 50 male chronic cannabis users who were otherwise healthy and 50 matched controls. When evaluated on Taylor's Manifest Anxiety Scale (TMA), these subjects had low anxiety scores as compared with the controls. To explore the possible interaction of cannabis with the benzodiazepine receptors, behavioral and biochemical studies in mice were devised, involving acute and chronic cannabis administration. Behavioral study revealed that mice under chronic cannabis treatment scored significantly higher on foot shock-induced aggression, but this was significantly blocked by benzodiazepine receptor antagonist. Furthermore, chronic cannabis treatment significantly (p less than 0.001) increased the frequency of licking response periodically punished by shocks. This confirms the antianxiety effect of cannabis, which also appears to be mediated through a benzodiazepine receptor, as it was reduced significantly (p less than 0.001) by a benzodiazepine receptor blocker. Specific 3H-diazepam binding was carried out in frontal cortex to assess both the population and affinity of benzodiazepine receptors. Our results indicate that acute cannabis treatment has no significant effect, whereas chronic cannabis treatment significantly increased 3H-diazepam binding as compared with controls. Scatchard analysis further reveals that increased affinity is responsible for increased binding to these receptors. It is therefore our contention that the antianxiety effect of cannabis is mediated through central benzodiazepine receptors. PMID- 3002504 TI - In vivo pretreatment with human chorionic gonadotropin fails to reverse the dysfunction of isolated Leydig cells from chronically uremic rats. AB - In order to further investigate the previously reported hypogonadal state of chronically uremic rats, we examined the effects of in vivo pretreatment with human chorionic gonadotropin (hCG) on in vivo and in vitro Leydig cell function, comparing paired intact rats with rats made chronically uremic by 5/6 nephrectomy. The in vitro testosterone (T) secretory responses to varying concentrations of hCG or dibutyryl cAMP and the number of gonadotropin receptors were determined following hemicastration. The rats were then treated with hCG for 3 days and the remaining testes were removed and studied as before. Compared with intact rats, the uremic rats had higher serum concentrations of urea nitrogen (P less than 0.001); serum T concentrations were lower in uremic rats before (P less than 0.001), but not after (P greater than 0.6) treatment. Treatment produced increases in serum T only in uremic rats (P less than 0.001). Serum LH was lower in uremic rats before treatment (P less than 0.001) and was reduced (P less than 0.001) to similar levels (P greater than 0.8) in both groups after treatment. Baseline in vitro T secretion was lower (P less than 0.001) from Leydig cells of uremic than intact rats both before and after treatment. Analysis of variance of dose-response curves showed pre- and post-treatment T secretory responses to hCG or dibutyryl cAMP in vitro to be less from Leydig cells of uremic rats (P less than 0.01). Before treatment, Leydig cell gonadotropin receptor number was lower in uremic than intact rats (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002505 TI - Recovery of pulsatile luteinizing hormone secretion following permanent disruption of the ascending noradrenergic fiber tract in the ovariectomized rat. AB - To determine whether noradrenergic (NE) support was essential for the generation of pulsatile luteinizing hormone (LH) secretion, we studied the effect of acute and chronic disruption of central noradrenergic pathways on patterns of LH release in the ovariectomized female rat. We argued that if NE pathways were essential for the generation of pulsatile LH secretion, then disruption of central NE activity should effect a blockade of LH pulses. To test this hypothesis we chronically disrupted NE activity by transecting the ascending NE pathway (ANP) and acutely disrupted NE activity by administering the alpha adrenergic receptor blocking agent phenoxybenzamine (PBZ); and we evaluated the effect of these treatments on the pattern of pulsatile LH secretion. Four weeks after the ANP transections, we found no differences in either LH pulse amplitude or frequency between ANP-cut animals (n = 6) and sham-cut controls (n = 8). In sham-cut animals (n = 7), PBZ reduced LH pulse frequency by 85% (P less than 0.002) and had no effect on pulse amplitude. In contrast, PBZ was significantly less effective in reducing LH pulse frequency in ANP- cut rats (n = 9); pulse frequency in these animals was 4 times greater than that found in PBZ-treated, sham-cut rats. We conclude from these data that, whereas noradrenergic pathways usually interact with LH regulatory mechanisms, they are not essential for the generation of normal gonadotropin-releasing hormone pulses. PMID- 3002506 TI - Localization of the antigotensin-converting enzyme activity in testis and epididymis. AB - Angiotensin-converting enzyme (ACE) has been studied in different reproductive organs of the male rat, in somatic cell lines clonally derived from both rat and mouse testes, and in isolated spermatogenic cells of the mouse. Among the various reproductive organs only testis and epididymis show high levels of enzyme activity. The testicular activity is found mainly in the isolated germinal cells and residual bodies, whereas somatic cell lines contain negligible levels of activity even after addition of hormones and growth factors. Testicular homogenates, spermatogenic cells, epididymal spermatozoa, and spermatozoan cytoplasmic droplets, when fractionated by anion exchange chromatography, contain one major and one minor activity peak, whereas epididymal homogenates and epididymal secretions reveal an additional major activity peak together with the minor peak. All forms of ACE have a similar response to different modifiers, and are equally sensitive to the specific inhibitor N-[(S)-1-(ethoxycarbonyl)-3 phenylpropyl]-L-alanyl-L-proline (Enalapril). The testicular enzyme could provide a useful marker for spermatogenic maturation and/or cytoplasmic processes both in testis and epididymis. The separate epididymal peak is a secretory enzyme that may be responsible for the processing of spermatozoan plasma membrane constituents during epididymal transit, or may have a role in attacking some biologically active compounds. PMID- 3002507 TI - Follicle stimulating hormone binding inhibitor produced by the bacteria Serratia interacts with receptor for follicle stimulating hormone in calf testis membranes. AB - Bacteria of the genus Serratia, including a strain of Serratia liquifaciens isolated from contaminated porcine follicular fluid, produced an inhibitor of 125I-human follicle-stimulating hormone (hFSH) binding to calf testis membranes in vitro. In order to evaluate its potential usefulness and significance, we undertook studies to identify the site of action of this inhibitor. Large quantities (grams) of inhibitor-containing material (SL-1) were obtained by enrichment culture techniques and its chemical composition was determined. Follicle-stimulating hormone-binding inhibitory activity (FSH-BI) in SL-1 was associated with a protein-containing substance of approximately 30,000 Mr and also with larger Mr (greater than 300,000) forms. Preincubation studies demonstrated that binding inhibition by SL-1 was due to effects on the membranes rather than effects on the radioligand (125I-hFSH). Kinetics studies indicated that FSH inhibition by SL-1 was a relatively slow process that reached steady state conditions between 23 and 25 h at 20 degrees C, in contrast to FSH, which reached steady-state conditions by 12 h at 20 degrees C. Estimates of FSH-BI activity, e.g., mass required to produce a 50% inhibition of 125I-hFSH binding, varied drastically when these kinetics differences were not taken into account. Our observations emphasize the need to establish steady-state conditions for each ligand before assessing mechanisms of action using Michaelis-Menton assumptions (e.g., competitive binding assays, Scatchard analyses).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002508 TI - The extraction of a tissue collagenase associated with ovulation in the rat. AB - A method has been developed to assay collagenase in ovarian extracts in the presence of tissue inhibitors. Rat ovarian tissue is first extracted with Triton X-100 and then heated to 60 degrees C in 50 mM Tris buffer containing 100 mM CaCl2. This extract contains collagenase activity and putative inhibitor(s). The inhibitory activity is removed by reduction with dithiothreitol and alkylation with iodoacetamide. Collagenase is then activated with aminophenylmercuric acetate and assayed using 3H-acetylated collagen from which the telopeptides have been removed. Identification of this activity as collagenase was performed by using the metalloprotease inhibitors EDTA and o-phenanthroline and by demonstration of the typical collagen cleavage fragments on sodium dodecyl sulfate-gel electrophoresis. To investigate the changes in collagenase activity associated with ovulation, immature rats received 20 IU of pregnant mare's serum gonadotropin and 52 h later 10 IU of human chorionic gonadotropin (hCG). After hCG administration, ovaries were removed at intervals from 0 to 20 h. Collagenase activity rose from 4.9 +/- 1.4% digestion of the 3H-collagen at 0 time to a maximum of 24.7 +/- 1.5% digestion at 8 h after hCG and remained high at 12 h (time of ovulation) and up to 20 h (18.7 +/- 1.9% and 16.1 +/- 1.6% digestion, respectively). These findings support a role of collagenase in the rupture of the follicle and they suggest a further role for this enzyme in the events following ovulation. PMID- 3002509 TI - The release of fluoride and other chemical species from a glass-ionomer cement. AB - The elution of fluoride, sodium and silica from a glass-ionomer cement was studied for 598 days. It was found that these species were still being released when the experiments were concluded, however, the rate of release was much diminished. The release of fluoride, sodium and silica was incongruent. Only fluoride associated with sodium appeared to be available for release. PMID- 3002511 TI - [Prevention of genito-anal and bucco-laryngo-esophageal cancers caused by sexually transmitted viruses]. AB - Whilst some viruses of the Papilloma family cause warts on the skin, others infect mucosal cells. The types called 6 and 11 produce benign papillomas, called condylomata acuminata, visible to the naked eye, not only on the vulva, vagina, penis (cockscomb), but also in the anus, and occasionally the larynx, mouth (tongue) and oesophagus. Types 16 and 18 cause cervical cancer (generally called in situ) and especially very small flat lesions that can only be seen through the colposcope in women and a lens in men. These flat micro-lesions can also be found on the vulva, vaginal walls and on the glans and, balano-preputial area and shaft in males, the distal urethra, anus, larynx (especially the vocal cords), the mouth and oesophagus. These flat micro-lesions are either early cancers (here the deoxyribonucleic acid (DNA) of the virus 16 and/or 18 is integrated into the cell genome), or precancerous lesion in which case the viral DNA is not integrated. Their malignant transformation is much more frequent at the junction of the glandular and squamous parts of the cervix, than in the vulva or vagina. Co carcinogenic factors appear to have an important role in the malignant transformation;--as for instance sexually transmissible infections including chlamydiae, bacteria that produce carcinogens such as nitrosamines, herpes virus which is known to cause mutations predisposing to the integration of the Papova viruses, chemical substances applied to the genitalia. The role of low hygiene standards in male sexual partners is the major cause (such men can carry simultaneously several sexually transmissible diseases (STD], who are never examined in search for flat lesions, who do not seek medical advice and have multiple sexual contacts with many women among whom some are more dangerous than prostitutes, especially since the wide use of hormone contraceptives and abortion that has multiplied the incidence of cervical cancer by 3 among the 20 year-old females, by 4 among the 25 year-old ones and by 2.5 among the 30 year-old ones, between 1961-65 and 1982-83. These changes in contraception have now made intra vaginal ejaculation the rule (this not only carries viruses and other micro organisms into the female genital tract, but also deposits sperm that contains some thirty factors that suppress local immunity). This with the rise of multiple partners, early sexual activity in particular in girls (hardly post-puberty) explains the increase of the frequency of cervical cancer in younger and younger women.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3002510 TI - [Inorganic pyrophosphates and parathormone in hypophosphatasia. Study of a family]. AB - The serum concentration of parathormone is usually normal in hypophosphatasia, a rare disease with a defect of bone mineralisation and low serum alkaline phosphatase activity. Nevertheless there are three cases in the literature presenting a hyperparathyroidism with or without hypercalcemia. No anomaly of parathyroid was found at autopsy. The authors describe the first cases of hypophosphatasia with low serum concentration of parathormone and raise the possibility of a trouble in the calcium-parathormone feed-back. They also emphasize the interest of the urinary pyrophosphate excretion. Its increase seem to be the most constant and the most specific biological disorder. PMID- 3002512 TI - Cyclic AMP response to vasoactive intestinal peptide and beta-adrenergic or cholinergic agonists in isolated epithelial cells of rat ventral prostate. AB - Vasoactive intestinal peptide (VIP) and the beta-adrenergic agonist isoproterenol stimulated cyclic AMP formation through independent receptors in isolated epithelial cells of rat ventral prostate. The specific beta-adrenergic antagonist propranolol inhibited the stimulatory effect of isoproterenol but not that of VIP. Besides small differences in the efficiency of both agents, results indicated that isoproterenol was 500 times less potent than VIP. Acetylcholine did not modify the basal cyclic AMP levels but inhibited the accumulation of the cyclic nucleotide in the presence of either VIP or isoproterenol. The inhibitory action of muscarinic receptors was calcium-dependent. The coexistence of receptors for cholinergic, adrenergic and peptidergic agents which can regulate cyclic AMP suggests that the functions of prostatic epithelium may be interdependently controlled by multiple neural effectors. PMID- 3002513 TI - [Peroxidase transport through the endotheliocytes of the microvasculature of the rat jejunal villi]. AB - The use of horseradish peroxidase as a macromolecular tracer showed partial differences in paracellular and transcellular pathways of the net transendothelial protein transport related to the segmental belonging of microvessels. The most likely pathway of transport through precapillary arteriolar endothelium is poorly expressed vesicular transfer. High endothelial permeability in subepithelial and marginal capillary and postcapillary venule of intestinal villi for horseradish peroxidase, is mostly due to vesicular transport and open junctions. Transendothelial channels and fenestrations in exchange microvessel endothelius were marked by horseradish peroxidase. PMID- 3002514 TI - [Enzymatic activity of peripheral blood cells in experimental chronic myocarditis caused by persistent Coxsackie A virus]. AB - Molecular mechanisms of metabolic modifications in blood cells were found to be associated with immunological tolerance to Coxsackie A virus in the modelling of chronic systemic diseases in rats. Blood cellular enzymatic activity correlated with morphological damage in internal organs. Cytochemical analysis is an informative test for investigating destructive and inflammatory changes in the affected organs. PMID- 3002515 TI - [Changes in the surface charge of plasma lipoproteins during their auto oxidation]. AB - Low density lipoproteins (LDL) and high density lipoproteins (HDL) surface potential (charge) changes were studied upon autooxidation, using positively charged spin probes. Lipid peroxidation product accumulation in LDL and HDL suspensions was found to be accompanied by a significant reduction in their surface area associated with a decreased negative surface charge, and probably, deposition of lipid peroxidation polar products and/or surface charge redistribution as a result of lipoprotein autooxidative modification. PMID- 3002516 TI - [Structural changes in erythrocyte membranes during cooling studied by a spin probe method]. AB - Peculiarities of structural changes in erythrocyte membranes during freezing (from -20 degrees to -50 degrees) were studied by electron paramagnetic resonance method using spin-labelled derivative of stearic acid-5-doxylstearate. It was established that membranes underwent a number of structural reconstructions due to the temperature decrease and water freezing-out. Differences were found in temperature dependences that characterize lipid ordering during probe insertion into membranes of native erythrocytes, white ghosts, and liposomes from total lipids of erythrocyte membranes. The data obtained indicate the impairment in the structure of lipid components and lipid-protein interactions in erythrocyte membranes during cooling. PMID- 3002517 TI - [Interaction of piracetam with 3H-imipramine binding sites and the GAMA benzodiazepine receptor complex of brain membranes]. AB - Piracetam at a concentration of 10(-6) M was shown to behave as a noncompetitive inhibitor of 3H-imipramine specific binding to rat brain membranes. At the same time piracetam failed to influence specific binding of 3H-mianserin to membranes of guinea-pig cerebellum, which is indicative of its inability to suppress histamine H1 receptors, a component of 3H-imipramine specific binding sites. At a concentration of 10(-4) M piracetam does not change specific binding of 3H flunitrazepam to rat hippocampal membranes in the absence of GABA, but in the presence of 5 X 10(-5) M GABA, like atypical tranquilizer mebicar, acts as a competitor of 3H-flunitrasepam binding. Though Ro-15 1788 did not suppress anxyolytic piracetam (and mebicar) effect, our results give evidence of a possible involvement of GABA-benzodiazepine supramolecular complex in the anxiolytic activity of piracetam. PMID- 3002518 TI - [Cerebrovascular effects of met- and leu-enkephalins]. AB - Met- and leu-enkephalines have a two-phase influence on the brain blood supply: initial short-term blood flow increase is replaced by the decrease of cerebral blood flow. Enkephalines are established to possess a pronounced depressive influence on neurogenic spasms of cerebral vessels and somatosympathetic and vasomotor reflex both under systemic administration and administration into brain lateral ventricles. Bicucullin has no effect on leuenkephaline action on cerebral circulation and its nervous control, while naloxone either removes or reduces the effects. Hence, opiate receptors take part in the realization of cerebrovascular effects of opioid peptides. The data obtained show the brain opioid system involvement in the regulation of brain circulation. PMID- 3002519 TI - [Opiate component in the realization of the vascular effects of clonidine]. AB - Systemic and regional hemodynamic changes (microsphere technique) were studied in urethan-chloralose anesthetized cats after intravenous clonidine and naloxone administration in different sequence. It was shown that antihypertensive effect of clonidine was caused by a decrease in the cardiac output and an increase in the total peripheral vascular conduction. The ability of clonidine to increase total peripheral conduction was completely blocked with naloxone preadministration. It was concluded that endogenous opiate receptors take part in the vasodilating clonidine effect in abdominal organs. PMID- 3002520 TI - [Effect of chronic psychogenic stress on a number of behavioral and neurochemical characteristics of rats]. AB - Fifteen-day chronic stress with randomized footshock reinforcement (group 4) causes profound steady disturbances in the animal behavior. With the use of regular aversive reinforcement in the shuttle-box the rats were divided into two groups: "escaping" (group 2) and "non-escaping" (group 3). Minor behavioral disturbances were observed in group 2, moderate in group 3 and maximum in group 4. In the latter group "down-regulation" of beta-adrenoceptors and benzodiazepine receptors was observed. The receptor changes appear to be rather the result of psychogenic factors caused by randomized reinforcement than of physical stress as such. PMID- 3002521 TI - [The role of mu- and delta-opiate receptors in the realization of the autonomic effects of opioid peptides]. AB - In the first series of experiments on the isolated mouse vas deferens and guinea pig ileum the capacity of 10 opioid peptides to activate mu- and delta-receptors was evaluated. [DAla2, DLeu5]-enkephalin (DADLE) and [DAla2, MePhe4, Gly5-ol] enkephalin (DAMPGE) were the most selective agonists of delta- and mu-opiate receptors, respectively. In the second series of experiments on urethan anesthetized rats it was shown, that intravenous administration of DADLE or DAMPGE (10(-7) M/kg each) elicited hypotension, bradycardia and expiratory apnoe. These effects disappeared both after naloxone injection and bilateral cervical vagotomy. A reflex nature of the vegetative effects of opioid peptides and the role of both mu- and delta-receptors in their realization are suggested. PMID- 3002522 TI - The molecular biology and functions of the granulocyte-macrophage colony stimulating factors. AB - Rapid progress has occurred recently in characterizing the molecular nature of the specific glycoprotein colony-stimulating factors (CSFs) controlling the proliferation; and some functional activities of granulocytes and monocyte macrophages. All four known murine CSFs have been purified, and cDNAs for two have been cloned and expressed by mammalian and bacterial cells. Similarly, three human CSFs have been purified, and cDNAs for two cloned and expressed. This work has opened up the exciting prospects of testing the effects of these recombinant CSFs on hematopoiesis in vivo. Each CSF has a broader range of hematopoietic target cells than previously suspected, and it is now clear that the CSFs are not simply proliferative stimuli but can also regulate the functional activity of mature cells. There are increasing reasons to believe that these CSFs will be useful therapeutic agents in stimulating hematopoietic regeneration in leukopenic states and the functional activity of granulocytes and monocytes in infections. PMID- 3002524 TI - Thrombomodulin, an endothelial anticoagulant protein, is absent from the human brain. AB - Protein C activation by thrombin is significantly accelerated by the endothelial cell cofactor, thrombomodulin. In this study, we have developed a radioimmunoassay for thrombomodulin and have measured the cofactor content in several human tissues. The assay method detects as little as 2 ng of thrombomodulin. The highest thrombomodulin content was found in lung and placenta, but the antigen was also detected in spleen, pancreas, liver, kidney, skin, heart, and aorta. Unexpectedly, thrombomodulin was absent from brain. Extracts from cerebral cortex, cerebellum, centrum semiovale, midbrain, basal ganglia, pons, and medulla were devoid of thrombomodulin. In contrast, thrombomodulin antigen is present in extracerebral intracranial vessels, including basilar and internal carotid arteries and choroid plexus, as well as in endothelium of the pia-arachnoid. PMID- 3002523 TI - Inhibition of neutrophil oxidative metabolism by lysosomotropic weak bases. AB - Maintenance of an acidic intralysosomal compartment may be relevant to multiple aspects of neutrophil function. The effect of lysosomal alkalinization on the neutrophil respiratory burst was studied by measuring cytochrome c reduction in response to soluble stimuli in the presence of lysosomotropic weak bases. The weak bases chloroquine, ammonium chloride, methylamine, and clindamycin all raised the intralysosomal pH and inhibited neutrophil oxidative metabolism at concentrations ranging from 0.1 to 100 mmol/L. Inhibition was dose dependent for each base and correlated significantly with the degree of lysosomal alkalinization. Concentrations that did not alkalinize the lysosome did not inhibit the respiratory burst. Inhibition by weak bases was seen when oxidative metabolism was stimulated by phorbol myristate acetate, calcium ionophore A23187, formyl-methionyl-leucyl-phenylalanine, opsonized zymosan, or sodium fluoride. Increasing the stimulus concentration (from 5 ng/mL to 5 micrograms/mL phorbol myristate acetate and from 0.5 to 1 mumol/L A23187) diminished or abolished inhibition by weak bases. Washing the cells after incubation with bases and before stimulation substantially reversed the inhibition. None of the bases impaired detection of superoxide in a cell-free xanthine-xanthine oxidase assay. Other indexes of oxidative metabolism, including oxygen consumption and hydrogen peroxide release, were also inhibited by weak bases. Analysis of particulate NADPH oxidase activity from neutrophils stimulated in the presence of bases suggested that these cells assemble a subnormal amount of an enzyme complex with normal kinetic characteristics. Lysosomotropic weak bases alkalinized the neutrophil lysosome and produced inhibition of oxidative metabolism that was dose related, was not stimulus specific, and was largely reversed by washing the cells before stimulation. A possible explanation would be altered assembly of the enzyme complex involved in respiratory burst activation as a consequence of impaired granule/plasma membrane fusion in the presence of diminished transmembrane pH gradients. PMID- 3002525 TI - Incorporation of platelet and leukocyte lipoxygenase metabolites by cultured vascular cells. AB - When arachidonic acid metabolism is studied during platelet-endothelial interactions in vitro, the predominant cyclooxygenase end products of each cell type (thromboxane B2 and 6-keto-prostaglandin-F1 alpha, respectively) are essentially completely recovered in the cell-free supernatants of these reactions. In contrast, 50% of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12 HETE), the major lipoxygenase metabolite from platelets, is released into the cell-free supernatant. In investigating the basis of this observation, we have found that platelet lipoxygenase metabolites were generated to the same extent during these coincubations but became rapidly incorporated into the endothelial cells. The endothelial cell-associated 12-HETE was present not only as free fatty acid, but was also incorporated into cellular phospholipids and triglycerides. When purified 3H-12-HETE, 3H-5-HETE (the major hydroxy acid lipoxygenase product of leukocytes), and 3H-arachidonic acid (the common precursor of these metabolites) were individually incubated with suspensions of cultured bovine aortic endothelial cells or smooth muscle cells, different patterns of intracellular lipid distribution were found. In endothelial cells, 12-HETE was incorporated equally into phospholipids and triglycerides, whereas 5-HETE was incorporated preferentially into triglycerides, and arachidonic acid was incorporated into phospholipids. In smooth muscle cells, both 12-HETE and 5-HETE showed more extensive incorporation into triglycerides. The rapid and characteristic incorporation and esterification of platelet and leukocyte monohydroxy fatty acid lipoxygenase products by endothelial and smooth muscle cells suggests a possible physiologic role for these processes in regulating vascular function. PMID- 3002527 TI - A novel mutation in the TATA box in a Japanese patient with beta +-thalassemia. AB - A single base substitution (A-G) at position -31 within the highly conserved proximal promoter element, the TATA box, was identified in the beta-globin gene cloned from a Japanese woman with beta +-thalassemia. It appears that she is homozygous for this specific allele, as determined by haplotype analysis using seven different polymorphic sites in the beta-globin gene cluster. Transient expression of the mutant gene in COS cells revealed a 45% reduction in beta globin RNA production, relative to normal. These results establish the functional significance of the second base of the TATA box for in vivo transcription of the human beta-globin gene. PMID- 3002526 TI - Clonal rearrangement and expression of the T cell receptor beta gene and involvement of the breakpoint cluster region in blast crisis of CGL. AB - A case of lymphoid blast crisis of Ph1-positive CGL is described in which the blast cells had an immature T cell phenotype, clonal rearrangement and expression of the T cell receptor beta gene, and a rearrangement of the breakpoint cluster region (bcr) on chromosome 22. This case therefore provides definite evidence for transformation involving a common myeloid-T lineage progenitor, penetrance of the Ph1 molecular alteration into the T cell lineage, and clonal selection in blast crisis at the level of a committed T lineage precursor. PMID- 3002528 TI - [Purification of membrane-bound Na+, K+-ATPase from the rat submandibular gland]. PMID- 3002529 TI - [A case of so-called myoepithelioma of the cheek]. PMID- 3002530 TI - Mathematical modeling of opiate binding to mouse brain membrane. PMID- 3002531 TI - On control of the function of the sodium-potassium pump in malignant cells. PMID- 3002532 TI - Sodium and potassium as nutritional factors in the genesis, treatment and prevention of hypertension. PMID- 3002533 TI - The influence of retinoic acid receptor (RAR) status of bladder tumours on the course of the disease. AB - Retinoids influence bladder tumour development in animals and tumour recurrence in man. Some human tumours contain intracellular receptor proteins for retinoids. The effect of the presence of such retinoic acid receptors (RAR) on tumour response to conventional therapy has been studied in 59 patients. Thirty-two of 36 RAR+ve and 11 of 23 RAR-ve tumour patients were alive after a mean follow-up period of 2.6 years. Higher stage and grade were more commonly associated with RAR-ve tumours. Independent of stage, recurrence was more common in RAR-ve tumours. These studies suggest that RAR-ve tumours are more likely to recur and more likely to become invasive. However, further studies are required to determine the clinical value of RAR receptor status as a prognostic indicator. PMID- 3002534 TI - Biochemical markers and skeletal metabolism in carcinoma of the prostate. Use of decision matrix theory and ROC analysis. AB - The discriminative ability of several skeletal and tumour markers was assessed in 102 patients with prostatic disease. These comprised serum acid and alkaline phosphatase, serum albumin and osteocalcin, urinary excretion of calcium, hydroxyproline and 6-oxo prostaglandin F1 alpha. None of the tests was of value in distinguishing patients with benign prostatic disease from those with tumour not involving the skeleton. Values of serum osteocalcin, urinary excretion of calcium and urinary 6-oxo prostaglandin F1 alpha failed to discriminate significantly between patients with or without metastases. The remaining four markers were compared by decision matrix analysis and receiver operating characteristic (ROC) curves. Serum alkaline phosphatase provided the most sensitive marker of skeletal metastases (80.5%), followed by serum acid phosphatase (80%), hydroxyproline (68%) and albumin (30%). ROC analysis suggested that alkaline phosphatase conformed most closely to the "ideal marker" with highest specificity and sensitivity. PMID- 3002535 TI - Surveillance or lymph node dissection in clinical stage I non-seminomatous germinal testis cancer? AB - Surveillance following orchiectomy alone has gained popularity in the management of clinical stage I non-seminomatous germ cell tumours (NSGCT) of the testis. However, long-term follow-up of the retroperitoneal nodes can be difficult. We analysed the results of 71 consecutive patients followed for more than 1 year. Fifty men (70.5%) remain disease-free and 21 (29.5%) have relapsed. Relapses occurred 2 to 36 months after orchiectomy (median 6 months). Retroperitoneal nodes were involved in 12 cases (17%). In only one patient were retroperitoneal metastases diagnosed when smaller than 2 cm and in four they were diagnosed when larger than 5 cm. Furthermore, the late relapses occurred in the retroperitoneal nodes. After treatment of metastases, 69 patients (97%) are alive, disease-free and off therapy. As retroperitoneal relapses do not occur after a properly executed retroperitoneal lymph node dissection (RPLND) and ejaculation problems can be avoided with unilateral RPLND, it is suggested that RPLND can be used for clinical stage I NSGCT of the testis in experienced surgical centres, with the advantage of an easier follow-up. PMID- 3002537 TI - Opiate toxicity after self poisoning with aspirin and codeine. PMID- 3002539 TI - The natural history of HTLV-III infection. PMID- 3002538 TI - Pneumonia associated with infection with pneumocystis, respiratory syncytial virus, chlamydia, mycoplasma, and cytomegalovirus in children in Papua New Guinea. AB - Paired serum samples were collected from 94 children with pneumonia admitted to Goroka Hospital, Papua New Guinea. All but three of the children were aged 1-24 months. Only nine children were malnourished, with weight for age less than 70% of the Harvard median (three had weight for age less than 60% of the Harvard median). Pneumocystis carinii antigen was detected in the serum of 23 children. Twenty two children had serological evidence of recent infection with respiratory syncytial virus. Five children were probably infected with Chlamydia trachomatis at the time of the study, and there was less convincing serological evidence of current infection in a further 11 children. Five children showed a fourfold rise in antibody to Mycoplasma pneumoniae. Although only one child showed a fourfold rise in antibody to cytomegalovirus, 86 children had this antibody. No child showed a fourfold rise in antibody to Ureaplasma urealyticum or Legionella pneumophila. P carinii, respiratory syncytial virus, C trachomatis, M pneumoniae, and cytomegalovirus may be important causes of pneumonia in children in developing countries. PMID- 3002536 TI - The natural history of human T lymphotropic virus-III infection: the cause of AIDS. PMID- 3002540 TI - Renal artery occlusion in patients with renovascular hypertension treated with captopril. PMID- 3002541 TI - Enhanced retinal and optic nerve excitability associated with demyelination in mice infected with Semliki Forest virus. AB - The physiological effects of Semliki Forest virus (SFV) induced demyelination on the visual system of mice have been examined by recording electroretinograms (ERGs) and the spike activity in the retinal ganglion cell axons in control and SFV-infected mice. The amplitudes of ERGs evoked by a single flash in the dark adapted eyes of the SFV infected mice were abnormally enhanced, whereas flicker ERGs were slightly depressed. This hyperactivity was also seen in the ganglion cell axonal discharge. Both visually evoked and spontaneous activity recorded from the optic tract fibres of the infected mice were characterized by rhythmic oscillatory bursts of firing. Similar patterns were seen only very rarely in the evoked activity recorded from control mice. We suggest that this oscillatory firing might be a functional correlate of the types of 'positive symptoms' reported by multiple sclerosis patients with optic neuritis. PMID- 3002542 TI - Increased numbers of neurons occur in the inferior colliculus of the young genetically epilepsy-prone rat. AB - To determine if the increase in the number of neurons observed in the inferior colliculus (IC) of the adult genetically epilepsy-prone rat (GEPR) as compared to the Sprague-Dawley rat was present in the young GEPRs prior to the time at which seizure activity commences, brains from both types of rats 4-10 days of age were studied. A statistically significant increase in the numbers of small neurons occurred in the IC of the young GEPR. At 4 days of age, a 55% increase in the number of small neurons was found in the GEPR as compared to the Sprague-Dawley rat and at 10 days of age this increase was 105%. The numbers of the medium and large neurons were similar in the older group of rats. These data suggest that the increase in cell number observed in the adult GEPR is not compensatory to the seizure activity, but is genetically programmed. PMID- 3002543 TI - Differential postnatal development of mu-, delta-and chi-opioid binding sites in mouse brain. AB - By selective labelling techniques together with analysis of saturation curves it is shown that the concentrations of the mu-, delta- and chi-binding sites increase during postnatal development particularly in the first few weeks after birth. There are little or no changes in affinity. The rate of development is different for each type of site. The most striking finding is that the development of mu- and chi-sites precedes that of delta-sites. PMID- 3002544 TI - Effects of 4-aminopyridine (4-AP) on rat neostriatal neurons in an in vitro slice preparation. AB - Effects of 4-aminopyridine (4-AP) on the rat neostriatal neuron were studied using the in vitro slice preparation. The intracellularly recorded neurons had resting membrane potentials of more than 50 mV and were capable of generating action potentials with the amplitude greater than 60 mV. Application of 4-AP in the superfusing media depolarizes the cell membrane and increases its input resistance. Local electrical stimulation induces excitatory postsynaptic potentials (EPSPs) overlapping with inhibitory postsynaptic potentials (IPSPs) in these neurons. 4-AP application enhances the amplitude and duration of the postsynaptic potentials. With application of higher concentration of 4-AP, local stimulation induces a second EPSP and a bicuculline sensitive long duration depolarization. These results indicate that 4-AP clearly has effects on local stimulation-induced postsynaptic responses of neostriatal neurons. Possible mechanisms underlying the 4-AP actions on neurotransmission in the neostriatal slice are discussed. PMID- 3002545 TI - Effect of single or repeated estrogen administration on tuberoinfundibular GABA neurons and anterior pituitary GABA receptors: biochemical and functional studies. AB - The effect of single or protracted administration of estradiol valerate on the hypothalamo-pituitary gamma-aminobutyric acid (GABA)ergic system and on plasma prolactin levels has been evaluated in female rats 2 months after the last (chronic treatment) or the single dose of the steroid. In the group of animals receiving one dose of estrogen, no modifications were detected in the activity of the tuberoinfundibular GABAergic neurons as implied by unchanged GABA accumulation either in the median eminence or the anterior pituitary after blockade of GABA catabolism with ethanolamine-O-sulphate. However, a complete disappearance of the low affinity population of GABA receptors in the anterior pituitary was observed. In this experimental condition, where baseline prolactin levels were 3-fold higher than in control rats, muscimol, a potent GABA agonist, was effective in significantly lowering plasma prolactin concentrations. Chronic estradiol valerate administration reduced GABA accumulation in the median eminence and the anterior pituitary at 4, but not at 2 h, after intracerebroventricular injection of ethanolamine-O-sulphate. Moreover, in this instance, a complete disappearance of the high affinity population of GABA receptors in the anterior pituitary was detected. Long-term estrogen administration induced also a 55-fold increase of plasma prolactin titers and muscimol was ineffective in reducing prolactin concentrations in plasma. The ability of muscimol to inhibit prolactin release only in single-estrogen-treated animals strongly suggests that the high affinity population of anterior pituitary GABA receptors is that involved in the mechanisms whereby GABA inhibits prolactin release from anterior pituitary. PMID- 3002546 TI - Rostral pontine and caudal mesencephalic control of arterial pressure and iliac, celiac and renal vascular resistance. I. Anatomic regions. AB - The rostral pons and caudal mesencephalon in 26 cats were electrically stimulated (greater than 2400 sites) while measuring arterial pressure and iliac, celiac and renal vascular resistance. Areas active in control of arterial pressure and iliac vasoconstriction were located in the marginal nucleus of the brachium conjunctivum (BCM) and in parts of the central tegmental fields (FTC) of the mesencephalon. Areas active in control of celiac and renal vasoconstriction were confined to the BCM. Areas active in control of iliac and celiac vasodilation were generally found ventral to the constrictor areas in the FTC of the mesencephalon. Stimulation of the caudal periaqueductal grey, locus caeruleus and underlying reticular formation elicited no change in any parameter measured. These findings suggest that multiple pathways for control of arterial pressure and vasoconstriction pass through or synapse in a discrete region of the dorsal rostral pons that is limited to the BCM. PMID- 3002547 TI - [3H] kainic acid binding sites in the synaptosomal-mitochondrial (P2) fraction from goldfish brain. AB - Binding of [3H]kainic acid to the synaptosomal-mitochondrial fraction (P2) of the goldfish brain was studied. Specific binding to this fraction represents about half of the total binding capability of the homogenate particulate material and is enriched in synaptic membranes; it is greater by about two orders of magnitude than those given for rat brain and pigeon optic tectum membranes. Association of the ligand-site complex has a time constant lower than 1 min and the same is true for the main component of the dissociation process. The binding equilibrium is apparently not affected by substances contained in the fraction material. The analysis of the dose-response data showed a main receptor population (B max = 139 pmol/mg protein) which displayed positive cooperativity (nH = 1.29). The same behaviour was shown by washed membranes from the same fraction but, in this case, the affinity for the ligand was lower (apparent affinity constants: K'D = 0.28 nM for the intact fraction and K'D = 0.38 nM for membranes). A smaller population of sites with higher affinity was also detected both in the intact fraction and in membranes. Among the substances tested as displacers of kainic acid from the synaptosomal sites, the most effective were quisqualate and L-glutamate. Folic acid and its dihydro and tetrahydro derivatives were half as potent as glutamate whereas methyltetrahydrofolic acid and folinic acid had a very weak action. The difference between these sites and those detected on rat brain membrane preparations is discussed. PMID- 3002548 TI - Basal forebrain neurons projecting to the rat frontoparietal cortex: electrophysiological and pharmacological properties. AB - Neurons located in the ventromedial globus pallidus (nucleus basalis) and substantia innominata, that were antidromically driven by electrical stimulation of the frontoparietal cortex, were recorded in the urethane anesthetized rat. The basalocortical neurons (BCNs) were antidromically driven with latencies of 1.1 13.5 ms, giving conduction velocities of 0.6-6.8 m/s. Many BCNs had regular patterns of spontaneous discharge (mean spontaneous activity: 20 impulses/s). Most BCNs were not responsive to non-noxious peripheral somatic stimulation. BCNs were readily excited by the iontophoretic application of glutamate and strongly inhibited by GABA. Eighty-five percent of the BCNs could be excited by acetylcholine. They could also be excited by cholinergic agonists. Muscarinic agonists excited a higher proportion of BCNs than nicotinic agonists. Excitatory responses to acetylcholine, carbachol and muscarinic agonists were abolished by atropine. PMID- 3002549 TI - Effects of age on kindling and kindled seizure-induced increase of benzodiazepine receptor binding. AB - We examined the effects of age on kindled seizure development, benzodiazepine receptor binding, and kindled seizure-induced increases of benzodiazepine receptor binding. The results disclosed that: development of kindling required greater numbers of stimulations in middle-aged than in young-adult animals; in comparison to young-adult animals, middle-aged animals exhibited increased benzodiazepine receptor binding in the dentate gyrus of hippocampal formation: and no age-related differences existed in the effects of seizures on benzodiazepine receptor binding. We suggest that senescence-related impairment of kindling development is due at least in part to alterations in the hippocampus, and that the increased benzodiazepine receptor binding in dentate gyrus may be one of the factors responsible for this impairment. PMID- 3002550 TI - Two brain cholecystokinin receptors: implications for behavioral actions. AB - The distribution and relative specificity of cholecystokinin (CCK) receptors in the rat brain was mapped by in vitro autoradiography with [125I]CCK-33. We identified two distinct binding patterns, suggesting two CCK receptor types. The first is widespread and relatively non-specific. The second, localized to a few subcortical nuclei, has the specificity demonstrated for pancreatic CCK receptors. Localization of this receptor type to the area postrema provides a possible entry site into brain for circulating CCK that would distinguish between CCK and gastrin and could mediate some of CCK's behavioral effects. PMID- 3002551 TI - Direct synaptic linkage of ventrolateral nucleus of thalamus terminal with cat fast pyramidal tract neuron. AB - An electron microscopic study on the synaptic connections between neurons of ventrolateral nucleus of thalamus (VL) and pyramidal tract neurons (PTNs) in cat motor cortex was conducted by means of the anterograde degenerating procedure coupled with horseradish peroxidase (HRP) intracellular staining. Following VL lesions, a large majority of the degenerating terminals were found to terminate on dendritic spines and a few on the dendritic shaft. An asymmetric type synapse formed by a VL degenerating terminal and the dendritic shaft of a branch of apical dendrite of a labeled fast pyramidal tract neuron was demonstrated. PMID- 3002552 TI - Pharmacological characterization of serotonin receptor induced by rat brain messenger RNA in Xenopus oocytes. AB - Serotonin receptors incorporated in the membrane of Xenopus oocytes injected with mRNA extracted from the rat brain was investigated by intracellular recording. Serotonin elicited the membrane depolarization accompanied with membrane potential fluctuations. This serotonin action was suppressed by serotonin antagonists such as methysergide, cyproheptadine and ketanserin. Dibutyryl cyclic AMP and papaverine depolarized the membrane as seen in applying serotonin. These observations indicate that serotonin actions might involve the cAMP system. PMID- 3002553 TI - Schwann cell responses to cyclic AMP: proliferation, change in shape, and appearance of surface galactocerebroside. AB - Surface galactocerebroside (galC) was induced on cultured Schwann cells by two analogues of cyclic adenosine 3',5'-monophosphate (cAMP), dibutyryl cAMP and 8 bromo cAMP (as previously reported by Sobue and Pleasure) and also by forskolin, a potent adenylate cyclase activator. These reagents also induced a morphological transition of many of the Schwann cells, from an elongated spindle shape to flattened cells extending fenestrated cytoplasmic sheets. Surface galC and these changes in Schwann cell shape were not elicited by raising the extracellular cAMP concentration, nor by many compounds known to promote the differentiation of other cell types, suggesting that intracellular cAMP is the unique signal for their induction. The cAMP analogues also induced Schwann cell proliferation (as previously reported by Raff et al.), as did forskolin. The concentrations of cAMP analogues and forskolin eliciting largest increases in numbers of Schwann cells in the cultures were 10-fold lower than the concentrations required for optimal induction of Schwann cell surface galC. PMID- 3002554 TI - Properties of antidromically identified neurons in the enkephalinergic magnocellular dorsal nucleus of the guinea pig hypothalamus. AB - In the guinea pig, immunocytochemical and neuroanatomical studies have demonstrated that enkephalin-containing neurons in the hypothalamic magnocellular dorsal nucleus (MDN) terminate in the lateral septum (LS). In the present investigation, 114 MDN neurons, studied with extracellular recording techniques, were identified by antidromic activation from the LS. Latencies of responses from ipsilateral and contralateral LS were 13.5 and 18.78 ms, respectively, corresponding to an axonal conduction velocity of 0.1 m/s. By using the reciprocal collision test, evidence is presented for bilateral projection of individual MDN neurons to the LS. Fifty-one (44.73%) MDN-LS neurons discharged in a slow irregular pattern. Interspike time histograms were very similar and had a mode of about 280 ms. Peristimulus time histograms were compiled from 15 active MDN-LS neurons. Stimulation which elicited antidromic spikes resulted in a brief silent period in the spontaneous activity which was related to the normal interspike interval pattern of the firing. Prolonged silent periods as well as silent period occurring after subthreshold stimulus and increasing with the stimulus intensity were attributed to inhibitory synaptic effects. On the other hand, some MDN-LS neurons displayed orthodromic excitatory responses following LS stimulation. These observations provide electrophysiological evidence of a direct MDN-LS pathway, in all likelihood of enkephalinergic nature, and indicate that some MDN-LS neurons receive inhibitory and excitatory afferents from the LS. PMID- 3002556 TI - Effects of ventro-medial mesencephalic tegmentum (VMT) stimulation on the spontaneous activity of nucleus accumbens neurones: influence of the dopamine system. AB - The effects of VMT-stimulation (100-500 microA, 0.6 ms; 1 Hz) on the spontaneous activity of neurones in the nucleus accumbens were analyzed in ketamine anaesthetized rats. On spontaneously active cells (firing greater than 0.5 spikes/s), 3 types of responses were observed: either inhibition (36%), excitation (5%) or a composite sequence of excitation followed by inhibition (12%). Moreover, 14% of silent nucleus accumbens neurones were excited by single pulse VMT-stimulation. Finally, 3% of nucleus accumbens neurones recorded were driven antidromically by VMT-stimulation. Destruction of dopamine (DA) projections by 6-hydroxydopamine prevented the inhibitory responses to VMT stimulation in the great majority of cells studied, without affecting the excitatory responses. After systemic administration of haloperidol or sulpiride, the inhibitory responses to VMT stimulation were attenuated markedly, whilst the excitatory responses were, however, maintained. These results suggest that the inhibitory, but not the excitatory, effects of VMT-stimulation on nucleus accumbens neurones may be mediated by an activation of the mesolimbic DA system. PMID- 3002555 TI - Do thalamic regions which project to rostral primate motor cortex receive spinothalamic input? AB - The purpose of this study was to examine whether spinal pathways terminate in regions of the primate thalamus which project directly to the motor cortex. In order to examine this issue we employed a 'double labeling' procedure which utilized transport of both anterograde and retrograde tracers in the same macaque. One tracer substance was injected into the cervical spinal cord and the other was injected into the rostral zone of primary motor cortex. We observed no overlap of spinothalamic terminations and thalamic regions which project to the motor cortex. Thus, our results provide no anatomical support for a direct thalamic relay of peripheral afferent input to the motor cortex of primates. PMID- 3002557 TI - Changes in growth hormone and prolactin release after superior cervical ganglionectomy of rats. AB - Superior cervical ganglionectomy (SCG X) decreased significantly serum growth hormone (GH) levels in rats 14-96 h after surgery, during and immediately after anterograde degeneration of regional sympathetic terminals. At later times (up to 28 days after SCG X) an increase in serum GH was observed. SCG X augments prolactin (PRL) release, but only at the earliest time examined (14 h after surgery). Injection of the alpha-adrenoceptor blocker, phenoxybenzamine, but not of the beta-blocker, propranolol, negated the depression in serum GH found in SCG X rats 14 h after surgery, without affecting PRL release. PMID- 3002558 TI - The modulation of transmitter release in motor nerve endings varies with the type of muscle fiber innervated. AB - Electrophysiological studies of synaptic transmission in mammalian muscles revealed substantial differences between neuromuscular synapses associated with fast versus slow-twitch motor units. Synapses of fast units exhibited much higher tetanic and post-tetanic potentiation of neurotransmitter release than those of slow units. On the other hand, the tetanic potentiation of slow units increased faster as a function of the frequency of activation than that of fast units. These differences suggest a possible specialization of neuromuscular synapses with the type of motor units they are associated with. PMID- 3002559 TI - Behavioral recovery following kainic acid lesions and fetal implants of the striatum occurs independent of dopaminergic mechanisms. AB - Transplantation of fetal striatal tissue into rats with kainic acid lesions of the striatum reversed the spontaneous locomotor abnormalities caused by the lesions, but had no effect on the lesion-induced hyperactivity that followed amphetamine or apomorphine injection. Conversely, transplants into intact (non lesioned) striatum led to abnormalities in spontaneous locomotion, but did not effect locomotion under amphetamine or apomorphine conditions. Dopamine autoradiography found a relative absence of dopamine receptors within the transplants. These results suggest that the mechanism which accounts for transplant-induced recovery of spontaneous locomotion is independent of striatal dopamine mechanisms. PMID- 3002561 TI - [Genetic aspects of dyslipoproteinemias]. PMID- 3002560 TI - Localization of [3H]cyclohexyladenosine and [3H]nitrobenzylthioinosine binding sites in rat striatum and superior colliculus. AB - The localization of adenosine receptors labelled with [3H]cyclohexyladenosine ([3H]CHA) and adenosine transport sites labelled with [3H]nitrobenzylthioinosine ([3H]NBI) was examined in striatum and superior colliculus (SC) using radioligand binding and lesioning methods. Striatal kainic acid lesions significantly reduced the number (Bmax) of a single class of high affinity binding sites for [3H]CHA by 50% and that for [3H]NBI by 15% without altering Kd values for either ligand. In SC, enucleations significantly reduced both high and low affinity [3H]CHA binding sites by about 60% while levels of [3H]NBI binding were unaffected. Thus, adenosine receptors are present on striatal interneurons and retinal projections to the SC and some [3H]NBI binding sites are located on striatal interneurons. PMID- 3002562 TI - [Cell receptors for lipoproteins and their regulation]. PMID- 3002563 TI - Radiation-induced malignant fibrous histiocytoma: a report of five cases including two occurring post whole brain irradiation. AB - Five patients who were successfully treated with external radiation therapy for a variety of histologically unrelated tumors subsequently developed malignant fibrous histiocytoma (MFH) within the irradiated field. It is suggested that therapeutic irradiation was a causative factor in the development of these tumors with latent periods ranging between 3 and 17 years. A review of the pertinent literature is presented. It is apparent that postradiation MFH is being recognized with increasing frequency and at an earlier age than when it appears de novo. PMID- 3002564 TI - Feline leukemia virus-and feline sarcoma virus-related polypeptides released by virus producer and nonproducer cells. AB - Polypeptides specific for feline leukemia virus (FeLV) have been identified in the media of cells that produce FeLV as well as in nonproducer cells transformed by feline sarcoma viruses (FeSV). Cat fibroblasts that were persistently infected with FELV release the major virus envelope glycoprotein, whereas cultured cat lymphoma cells shed both glycopeptides related to the virus core gene (gag) and glycopeptides related to the virus envelope gene (env). Mink cells and cat cells transformed by FeSV secrete polypeptides of a wide range of sizes that cross react with the major virus core protein p27. Differences in the classes of p27 related proteins produced may be related to the strain of virus and the cell type. Cat cells transformed by FeSV release a glycopeptide that appears to be processed differently from those identified in the media of FeSV-transformed mink cells. The possibility that such FeLV-related secretory proteins may interfere with the immune response of the host is discussed. PMID- 3002565 TI - The role of radiation therapy in the treatment of sarcoma of soft tissue. AB - The data presented indicate that the combination of function-preserving surgery and radiation therapy is of value in the treatment of soft tissue sarcomas of the extremity. Local control is obtained in approximately 85% of patients and with survival results comparable to those obtained in patients treated with radical surgery. The one randomized series of patients treated with conservative resection and radiation compared to amputation has shown no difference in overall survival. These local control results have been obtained while maintaining good functional results. Combined local resection and radiation is an appropriate treatment option in a large proportion of patients with soft tissue sarcomas. PMID- 3002566 TI - [Passive immunization with an anti-oCRF41 immune serum inhibits the circadian increase of plasma ACTH in rats]. AB - For 24 hrs. after i.v. injection of 1 ml of an undiluted immune serum raised against oCRF41, the diurnal surge of plasma ACTH dropped to a short-lived limited rise above baseline level. On the second day after injection, the ACTH level in treated rats rose to a subnormal level, although both plasma dilution of the immune serum and its binding capacity in the plasma remained unchanged throughout the experiment. Plasma corticosterone, on the contrary, displayed a normal circadian rhythm during the entire experiment. However, in animals given a second injection of 0.5 ml oCRF41 immune serum 32 hrs. after the first, both ACTH and corticosterone titers fell rapidly below their circadian minimal levels in controls. Concomitantly, the concentration of immune serum in the peripheral plasma, and its capacity to bind to oCRF, rose by 50%. The major role of CRF41 as a diurnal trigger of the circadian rhythm of ACTH is discussed, as well as the limits of passive immunization. PMID- 3002567 TI - [Expression of MMTV proviruses in lymphopoietic organs of C3H Bi mice]. AB - We show that transcription of MMTV proviruses takes place in lymphopoietic tissues (spleen, thymus) of C3H Bi mice, a strain with high incidence of tumors. This transcription is found in the spleen of 21 day old mice but is no longer detectable in the spleen of 4 month old mice. In the mammary tumor, the 3.8 kb RNA (24S) which encodes the viral envelope proteins is well transcribed whereas the 1.7 kb RNA (13 S) is barely detectable. In contrast, the 1.7 kb (13 S) is the major species present in the spleen of 21 day old mice. It appears that the 7.8 kb genomic RNA (35 S) is differentially spliced into a mature RNA of either 3.8 kb (24 S) or 1.7 kb (13 S). The function of the latter is still unknown, though it could encode a protein involved in the induction of carcinogenesis. PMID- 3002568 TI - [Evaluation of the inhibitory effect of circulating corticosteroids on circadian stimulated and stress induced secretion of ACTH in rats]. AB - Male rats were bilaterally adrenalectomized in order to measure the extent of inhibition exerted by endogenous corticosteroids on both basal ACTH secretion along its circadian rhythm and ether-stress induced ACTH secretion. In intact controls, plasma ACTH levels at the circadian maximum exceeded by 4 times the circadian minimum, and ACTH response 15 min after ether-stress surpassed the circadian minimum by 20 times. In adrenalectomized rats, the daily minimum was 8 times that of the controls. Nevertheless the circadian maximum was 3 times above the rhythm's minimum, while the maximal stress response (15 min) surpassed the circadian minimum by 8 times. In adrenalectomized rats supplemented with a solid source of corticosterone inducing a stable plasma corticosterone level equivalent to the controls' circadian minimum (3 micrograms/100 ml), the ACTH rhythm still fluctuated twice as high as in intact controls. The tonic feed-back inhibition exerted by endogenous corticosteroids on ACTH secretion appeared thus significantly stronger than the GABAergic inhibition to the corticotropic system which was previously studied under similar standard conditions. PMID- 3002569 TI - [Ultrastructural localization of pituitary hormones by immunocytochemical reactions in ultrathin sections of adenomas]. AB - Human pituitary adenomas were fixed in glutaraldehyde and embedded in epon. Ultrathin sections were incubated either with anti-hGH, anti-hPRL or anti-hLH. They were incubated with second step goat anti-rabbit immunoglobulins linked to gold particles. Two PRL secreting adenomas, one GH and PRL secreting adenoma, one ACTH secreting adenoma and two non secreting adenomas were studied. The specificity and the limits of the method were discussed in relation with the results obtained in light microscopy with the PAP method. PMID- 3002570 TI - [Inhibition of virus multiplication by immunoactive peptides]. AB - In the present study we show that peritoneal macrophages obtained from the mice treated with the immunoactive peptides inhibit the multiplication of Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), but not that of vesicular stomatitis virus (VSV), and that the intraperitoneal administration of the peptides suppresses the infection with HSV-1 in mice. PMID- 3002571 TI - Temperature-sensitive mutants in rfaI and rfaJ, genes for galactosyltransferase I and glucosyltransferase II, for synthesis of lipopolysaccharide in Salmonella typhimurium. AB - Certain rough mutants of Salmonella typhimurium LT2 were shown to be temperature sensitive for the production of lipopolysaccharide (LPS). When grown at the restrictive temperature (42 or 45 degrees C), the cells contained LPS deficient in O (somatic) side chains, based on phage-sensitivity data and gel electrophoresis of the LPS. Cells grown at the permissive temperature, 30 degrees C, made LPS resembling that of smooth cells. The mobility of the LPS in gels, the phage sensitivity patterns, and gas chromatographic analysis indicate that LPS of 45 degrees C-grown cells of SA126 (rfaJ3012) is of chemotype Rb2, with one glucose and two galactose units (and thus inferred to be due to a mutation in rfaJ), and LPS of 45 degrees C-grown cells of SA134 (rfa13020) is of chemotype Rb3, with one glucose and one galactose unit (inferred to be rfaI). These inferences were confirmed, for pKZ26 (pBR322-rfaGBIJ) and pKZ27 (pBR322-rfaGBI) both complement rfaI3020, but only pKZ26 complemented rfaJ3012. In addition, pKZ26 carrying a Tn5 insertion resulting in loss of complementation of a known rfaJ mutation, but not of rfaG, B, or I, also resulted in loss of rfaJ3012 complementation. Based on gel analysis, there is a small amount of the LPS containing smooth side chains in cells of SA126 grown at 45 degrees C; following a switch to 30 degrees C, the amount of LPS with O side chains gradually increased, and the amount of core LPS was reduced, though even after 3 h the LPS does not fully resemble that of smooth strains.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002573 TI - Isolation of HTLV-III/LAV from serum proteins for cancer therapy in the Bahamas. PMID- 3002572 TI - Photodynamic therapy: cavitary photoillumination of malignant cerebral tumours using a laser coupled inflatable balloon. AB - Interest in photodynamic therapy of malignant brain tumours has been growing in recent years as intra-operative laser applications become more available. We have developed an inflatable balloon which can be coupled to an argon dye pump laser in order to deliver light to a brain tumour cavity created by the subtotal resection of tumour. Eight patients with primary malignant brain tumors have been treated with photodynamic therapy (PDT) using this device. The 8 patients tolerated the treatment well; morbidity attributable to the PDT was acceptably low. PMID- 3002574 TI - Recommendations for preventing possible transmission of HTLV-III/LAV from tears. PMID- 3002575 TI - Laboratory reports of herpesvirus infections in Canada in 1984. PMID- 3002576 TI - More treatment centres responding to growing cocaine use. PMID- 3002577 TI - Pathologic findings from the National Surgical Adjuvant Breast Project (protocol 6). I. Intraductal carcinoma (DCIS). AB - Seventy-eight examples of intraductal carcinoma (DCIS) were identified after pathologic review of 2072 specimens obtained from National Surgical Adjuvant Breast Project protocol 6. This randomized clinical trial compares the therapeutic merit of total mastectomy (TM) with lumpectomy (L), with (LX) and without (LO) postoperative irradiation. All patients were subjected to axillary lymph node dissection. Seven (14%) of the 51 patients with DCIS treated by L exhibited breast recurrence within or close to the site of the initial lesion 4 to 53 months (average, 16 months) after L. Only 2 (7%) of these events occurred in the 29 women treated by LX, as opposed to 23% in the LO group. No pathologic features were noticed that might have been considered predictive of local breast recurrence. The three DCIS recurrences and the four invasive forms noted are considered to represent overlooked or incompletely excised foci of cancer because of the multifocality (not multicentricity) of some breast cancers. The possibility that DCIS may represent a marker of risk for the development of cancer rather than a precursor lesion per se is suggested. Despite apparent difficulties in the pathologic diagnosis of DCIS as well as uncertainty concerning its natural history, no evidence was found to indicate that it represents a more ominous disease than invasive cancer. Indeed, treatment failure occurred in only one patient treated by LX and a similar number subjected to TM (4% versus 2%). Although these observations are short term (average follow-up, 39 months), estimates of the probability of local recurrence or survival suggest that they will not be significantly altered after longer periods of surveillance. Thus, there are no compelling reasons why DCIS may not be treated in a cosmetically acceptable manner by LX. A randomized clinical trial addressing this issue is now in progress. PMID- 3002578 TI - Characterization of renal neoplasms with monoclonal antibodies to leukocyte differentiation antigens. AB - Several monoclonal antibodies against human leukocyte differentiation antigens have been shown to react with normal kidney. Four monoclonal antibodies with different patterns of reactivity on normal kidney were tested against 20 renal epithelial neoplasms and 5 Wilms' tumors. In general, the expression of these antigens on renal tumors was faithful to their presence on normal kidney; this finding suggested that most renal tumors may be derived from proximal tubule epithelium. There was, however, both intertumor and intratumor heterogeneity of expression of these antigens, but the different phenotypes encountered did not correlate in any simple way with histologic subclassification. In contrast, the five Wilms' tumors were phenotypically different from the epithelial neoplasms, consistent with an origin from a more primitive renal cell. PMID- 3002579 TI - The differentiation of carcinomas of teratomatous origin from embryonal carcinoma. A light and electron microscopic study. AB - Carcinomas derived from teratomatous epithelium occur rarely in the metastases of patients with testicular cancer. These carcinomas of teratomatous origin (CTO) are easily confused with residual embryonal carcinoma (EC). For that reason, we compared the light and electron microscopic appearances of 6 CTOs with those of 12 ECs. As seen by light microscopy, the CTOs formed glands and more frequently had well-defined cytoplasmic borders, eosinophilic cytoplasm, and nonoverlapping, regular nuclei with small or absent nucleoli and little chromatin clumping and clearing, compared with the ECs. Mucin was present in the cells or glandular lumina of three CTOs but was absent in all the ECs. The demonstration of cytoplasmic glycogen was of no differential aid. The most useful differentiating ultrastructural features were long tight junctions and telolysosomes, which occurred in all of the EC but which were absent in the six cases of CTO. Desmosomes with inserting tonofilaments were present in three cases of CTO but were much less developed in EC. Two cases of CTO had microvilli with anchoring rootlets; such anchoring rootlets were not observed among EC. The distinction of CTO from residual EC is important, because CTO will likely need to be treated in a different manner. PMID- 3002580 TI - Transformation of Hodgkin's disease into malignant fibrous histiocytoma. AB - The continuity that exists between Hodgkin's disease and the fibrohistiocytoses has only recently been recognized. Four cases are presented that illustrate this phenomenon. The fibrohistiocytic component of the Hodgkin's lesion may lag behind the lymphoreticular component, may be present in equal proportion, or may assume a dominant role, with the emergence of a frank fibrohistiocytosis or fibrous histiocytoma being evidenced as the lymphoreticular component recedes. PMID- 3002581 TI - Adenoid cystic carcinoma of salivary glands. A study of 61 cases with clinicopathologic correlation. AB - Sixty-one cases of adenoid cystic carcinoma of the head and neck region, excluding the ear canal, lacrimal glands, larynx, esophagus, and trachea, were studied, and their different clinicopathologic aspects were analyzed. Adenoid cystic carcinoma occurred more commonly in the minor salivary glands; the palate was affected in 31% of the cases. The fifth decade of life was the age at which patients were most commonly affected, and there was a slight predominance of white women. In most patients a mass was the main complaint; in 63% the duration of symptoms was 1 year or less. Forty-one patients had Stages 3 or 4 disease when first seen, and 51.7% of the patients died of disease, with a mean survival period of 35.4 months. Three basic patterns of growth, solid, cribriform, and tubular, were identified in the histopathologic examination of the cases. Other pathologic aspects analyzed were cellular pleomorphism, mitotic activity, necrosis, vascular invasion, and perineural infiltration. The study revealed a positive correlation between location of the tumor, clinical staging, duration of symptoms, and histologic pattern of growth with the prognosis of the lesion. Tumors located in the minor salivary glands, those in which the duration of symptoms was less than 1 year, and those that showed advanced clinical staging and a predominantly solid pattern of growth had an extremely poor prognosis. Surgery is the treatment of choice of adenoid cystic carcinoma, and microscopically free surgical lines of resection must be obtained. Radiation therapy, although not curative, plays an important role in prolonging survival. PMID- 3002582 TI - Small cell carcinoma (non-oat cell type) of the esophagus concomitant with invasive squamous cell carcinoma and carcinoma in situ. A case report. AB - A case of double primary invasive carcinoma of the esophagus, consisting of well differentiated squamous cell carcinoma and non-oat cell small cell carcinoma without squamous differentiation, is presented. This is the first reported case of a double or multiple primary invasive carcinoma of the esophagus in which one component is small cell carcinoma (oat cell or non-oat cell). Furthermore, the mucosal epithelium around the non-oat cell small cell carcinoma revealed multiple dysplasia and carcinoma in situ. These lesions were definitely separated from the invasive carcinoma and from each other. The results suggest that pure non-oat cell small cell carcinoma of the esophagus without squamous differentiation is derived from the esophageal squamous epithelium, and is a variant of squamous cell carcinoma. PMID- 3002583 TI - Adenoid cystic salivary gland carcinoma. A histopathologic review of treatment failure patterns. AB - Seventy-one cases of adenoid cystic salivary gland carcinoma were reviewed according to treatment modality and clinical course. Thirty-six patients (51%) were treated by combined surgery and radiation therapy. The tumors were classified by their histologic patterns into tubular, cribriform, and solid forms. Distant metastases, in 52%, were the most frequent and ominous sources of failure. In 35% of cases, distant metastases developed despite local control at the primary site. In this group, the disease had a more fulminant course with shorter survival. Histopathologically, the cribriform subtype was associated with multiple local recurrences, greater local aggressiveness, and a poorer salvage rate as compared with the tubular subtype. Late onset of local recurrences and distant metastases was especially associated with the cribriform subtype. Overall prognosis in terms of distant metastases and survival was worst for the solid subtype. Control of local disease is best achieved with combined surgery and radiation therapy. The high incidence of distant metastases may not be affected by this regimen. The ultimate outcome of therapy is poorly predicted. Survival appears to be based on the pattern in which distant metastases develop. Overly aggressive and mutilating surgical approaches for these tumors are not recommended in many instances. The need for the development of new, more effective forms of therapy is emphasized. PMID- 3002584 TI - Krukenberg tumors of the ovary. Ultrastructural, histochemical and immunohistochemical studies of 15 cases. AB - The ultrastructural, histochemical, and immunohistochemical characteristics of 12 classical signet ring cell Krukenberg tumors (CKT) and three tubular Krukenberg tumors (TKT) were evaluated and related to their possible influence on the ovarian stroma. In CKT, single signet ring cells predominated over lumen-forming cells and contained ultrastructural and histochemical characteristics similar to goblet cells in colonic and ovarian mucinous adenocarcinomas. In TKT, lumen forming nonsecretory and secretory signet ring cells were prominent. Rare argentaffin cells were found in TKT but not in CKT. Cells in both CKT and TKT produced neutral and sialomucins. The stroma contained extracellular mucin, hypertrophied stromal fibroblasts and myofibroblasts, and in two cases stromal lutein cells with steroidogenic type ultrastructure. It appears that Krukenberg tumors are made up exclusively of intestinal type cells. Based on cell differentiation, TKT is better differentiated than CKT. Hypertrophy and hyperplasia of ovarian stromal cells may occur in response to malignant growth and/or the extracellular mucinous products of malignant cells and may play a role in the control of tumor invasiveness. None of the 15 cases were immunohistochemically positive for chorionic gonadotropin, placental lactogen, or luteinizing hormone. These hormones are suspected to be related to stromal luteinization in KT. PMID- 3002585 TI - Decreased methylation rates of DNA in SV40-transformed human fibroblasts. AB - The rates of methylation of total cellular DNA and newly synthesized DNA were measured in four unrelated SV40-transformed human fibroblast lines and in four control parent fibroblast lines. Rates of methylation of total cellular DNA were decreased by a factor of 1.8-2.3 in the transformed cells relative to control cells. Methylation was largely (75%-87%) restricted to newly synthesized DNA in control and transformed fibroblasts, and methylation rates of newly synthesized DNA were diminished in transformed cells by 12- to 19-fold relative to control cells. Growth rates were similar in the normal and transformed cells. The cellular uptake of methionine and conversion to S-adenosylmethionine were similar in the normal and transformed cells, suggesting no major differences between the normal and transformed cells in the cellular transport of methionine, methionine S-adenosyltransferase activity, or the intracellular concentrations of methionine and S-adenosylmethionine. The diminished rates of DNA methylation that we have observed suggest a possible mechanism for altered gene expression and growth control in transformed cells. PMID- 3002587 TI - Morphologic variations of small cell lung cancer. A histopathologic study of pretreatment and posttreatment specimens in 104 patients. AB - A consecutive series of 104 autopsies on patients treated in protocol for small cell carcinoma of the lung (SCCL) was studied with respect to metastatic pattern at autopsy in relation to pretreatment WHO 1981 classification, and extent and significance of non-SCCL tumor tissue at autopsy. The only significant difference in the metastatic pattern at autopsy between patients with pretreatment oat cell or intermediate subtype was metastases to the brain. Thus, the frequency of brain metastases was 17/35 (49%) in patients with oat cell type compared to 2/18 (11%) in patients with intermediate type (P less than 0.05). At autopsy 13 of 98 patients (13%) had non-SCCL tumor tissue in at least one site. These patients had a significantly shorter survival (P = 0.020) compared to patients with pure SCCL at autopsy. Furthermore, none of these 13 patients had obtained complete remission. Whether these morphologic variations had been present at the pretreatment stage of the disease or were due to the chemotherapeutic treatment could not be solved in the present study. However, the results might indicate that mixed SCCL/non-SCCL histology is a negative prognostic factor in the treatment of SCCL. Prospective studies including more extensive pretreatment tissue sampling seem to be required. PMID- 3002586 TI - Small cell carcinoma cell lines contain opioid peptides and receptors. AB - Two human small cell carcinoma cell lines were assayed for total opioid and beta endorphin-like immunoreactivity. Small cell carcinoma cell line NCI-H146 contained approximately 1.1 pmol/mg protein of total opioid immunoreactivity. This material was similar in size and immunoreactive determinants to C-terminally modified beta-endorphin. Small cell carcinoma cell line NCI-H187 contained approximately 0.2 pmol/mg protein total opioid immunoreactivity, which was of low molecular weight. NCI-H187 also contained approximately 1.2 pmol/mg protein of material similar in size and immunoreactive determinants to beta-lipotropin. The two small cell carcinoma cell lines were also examined for opioid receptors with the use of [3H]-etorphine as ligand. Both cell lines contained between 50 and 100 fmol/mg protein of specific, saturable, high-affinity opioid receptor binding sites. Together, these findings suggest a possible autocrine role for opioids in small cell carcinoma of the lung. PMID- 3002588 TI - Uncommon variants of cervical carcinoma treated with radical irradiation. A clinicopathologic study of 66 cases. AB - Although certain histologic types are uncommon in cervical carcinoma, these tumors as a group comprise almost one in five patients. The present study throws some light on the therapeutic approaches that are appropriate. From 1968 through 1978, 396 patients with carcinoma of the cervix were treated primarily with radiation therapy, at the University of Virginia Medical Center. The treatment policy remained consistent throughout the study interval. Diagnostic pathologic material was reviewed and uniformly classified in 365 cases (92.2%). Over 80% were invasive keratinizing or nonkeratinizing squamous cell carcinoma. There were 66 patients with uncommon histologic types including 24 adenocarcinomas (6.6%), 13 adenosquamous carcinomas (3.6%), 10 small cell carcinomas (2.7%), 6 papillary squamous carcinomas (1.6%), 5 glassy cell carcinomas (1.4%), and 8 miscellaneous types (2.2%). These 66 patients form the basis for this report. Five-year survival rates and causes of failure are presented along with management recommendations. PMID- 3002589 TI - Bowenoid papulosis. A clinicopathologic study with ultrastructural observations. AB - One hundred eight patients were studied who had anogenital lesions showing microscopic features as seen in bowenoid papulosis (BP), a recently described condition occurring most commonly in young adults. Patients typically show multiple papules, small nodules, or plaques that clinically mimic verrucae or nevocellular nevi. Although the lesions show microscopic cytologic atypia, a distinction from Bowen's disease, erythroplasia of Queyrat, and other forms of carcinoma in situ can usually be made on the basis of histologic and clinical criteria. The disorder responds to conservative treatment, although recurrences are not uncommon. Evolution of the lesions to invasive carcinoma was not observed. Mounting evidence links the development of BP to infection with human papilloma virus, but other viruses, as well as hormonal and immunologic factors, may also play a role. PMID- 3002590 TI - Superior vena caval obstruction syndrome in small cell lung cancer. AB - In a series of 643 patients with small cell lung cancer (SCLC), 55 patients (8.6%) had signs or symptoms of superior vena caval obstruction syndrome (SVCO). Relatively long intervals from the onset of the first symptoms of SVCO to the start of therapy were observed, and invasive diagnostic procedures were safely performed in most patients. The pretreatment characteristics of patients with SVCO were not significantly different from those of patients without signs of the syndrome, and survival was similar in both groups. Patients with SVCO were usually treated first with induction chemotherapy, and prompt resolution of signs and symptoms occurred in the majority. Radiation was effective in controlling SVCO at relapse or after failure of initial chemotherapy. It was concluded that SVCO in patients with SCLC should be treated initially with systemic chemotherapy, as for other presentations of this disease. The current data do not support the commonly held view that SVCO in SCLC should be approached as an oncologic emergency. PMID- 3002591 TI - Effect of dibutyryl cyclic AMP on morphologic features and biologic markers of a human salivary gland adenocarcinoma cell line in culture. AB - Dibutyryl cyclic AMP (dB-cAMP) has marked effects on the growth, morphologic features, and biologic markers of a human salivary gland adenocarcinoma cell line in culture. A cell line with ultrastructure and biologic markers specific to the intercalated duct cells of human salivary glands was cultivated in the presence of dB-cAMP. Major alterations, such as process formation and expression of myofilaments and oxytocin receptor in addition to myosin and S-100 protein, were observed in those cells with a phenotype similar to myoepithelial cells. Both the anchorage-independent and anchorage-dependent growths were markedly suppressed in the presence of dB-cAMP. After the removal of dB-cAMP from the culture, the treated cells returned rapidly to the phenotype of the untreated cells. These findings indicate that reversible differentiation into the myoepithelial cells of a human salivary gland adenocarcinoma cell line occurs in growth medium containing dB-cAMP. PMID- 3002592 TI - Carcinoembryonic antigen. A possible predictor of recurrence in cystosarcoma phyllodes. AB - Tissue carcinoembryonic antigen (CEA) and cytosolic estrogen and progesterone receptors were studied in 15 patients with cystosarcoma phyllodes (CSP) aiming at predicting recurrence of the tumor. Polyclonal (rabbit, monospecific) and monoclonal (mouse) antibodies anti-CEA were applied to formalin-fixed, paraffin embedded tissue sections using an indirect (PAP) immunoperoxidase method. Estrogen receptors (ER) and progesterone (PR) receptors were determined by a charcoal-dextran method. ER was detected in 4 of 15 primary CSP (mean level, 22 fmol/mg protein). CEA was demonstrated exclusively in the epithelial components of 12 of 15 tumors. Strong expression of CEA was verified in eight tumors, six of which recurred locally one or more times. None of the seven tumors negative or weakly reactive for CEA had recurrences. No correlation was found between expression of tissue CEA and steroid receptor status of the tumors. Our data indicate that strong CEA expression in CSP correlates with tumor recurrence. PMID- 3002594 TI - Vindesine and mitomycin C in chemotherapy: refractory advanced breast cancer. AB - Thirty-five unselected postmenopausal women with metastatic breast carcinoma, refractory to hormonal manipulation and/or chemotherapy, were treated with vindesine (3 mg/m2 day 1, then weekly for 6 weeks, then every other week) and mitomycin (12 mg/m2 day 1, then every 6 weeks). Thirty-one patients were evaluable for response. Two patients obtained a complete response (CR), three patients a partial response. Duration of the response in the two patients with CR was 24 and 16 months, respectively. Toxicity was mild, consisting of leukopenia and neurologic toxicity. Thrombocytopenia was not a significant clinical problem. Vindesine with mitomycin C, as administered in this study, is a safe, but marginally effective regimen for previously treated patients with metastatic breast cancer. PMID- 3002593 TI - Contralateral cancerous breast lesions in women with clinical invasive breast carcinoma. AB - Eighty-four consecutive autopsies of women with a clinical diagnosis of invasive breast carcinoma (BC) were examined by extensive histopathologic methods for malignant changes of the contralateral breast. Sixty-eight percent of the women were found to have primary contralateral BC, of which 33% were invasive and 35% in situ lesions. Another 16% had metastases to the breast. Only two women had had treatment for their contralateral BC. In eight cases a malignant lesion was diagnosed or suspected clinically, but in the remaining cases, the malignancies were identified only by histopathologic examination. No clinical data or histologic characteristics of the first BC had any predictive value for the risk of contralateral BC. In the contralateral breast, a significant coincidence was found between fibrocystic disease and the occurrence of primary malignant BC. The majority of the BC on both sides were of ductal type. Seventy-nine percent of the invasive contralateral BC were tumefacient, and 71% had axillary lymph node metastases. The mean survival time was comparable for women with and without contralateral primaries, but a significantly higher proportion of women with contralateral invasive BC died of disseminated BC. The frequency of contralateral malignancies is thus much higher than previously reported. The consequence of these findings may implicate a reevaluation of the treatment and control schedule regarding the contralateral breast in women with invasive BC. PMID- 3002596 TI - Sequential excision of residual thoracic and retroperitoneal masses after chemotherapy for stage III germ cell tumors. AB - Twenty-three patients with advanced (Stage III) mixed germ cell tumors underwent laparotomy and thoracotomy or neck dissection for excision of persistent radiographic masses after systemic chemotherapy. In those who received multidrug regimens incorporating high-dose cisplatin, 4 of 15 (27%) harbored persistent tumor in at least one site, 6 of 15 (40%) demonstrated necrotic tumor or fibrosis only in all sites examined, and the remaining 5 of 15 (33%) harbored mature teratoma in at least one area. In patients treated with high-dose platinum chemotherapy regimens 11 of 15 (73%) remain disease-free with a median follow-up period of 29 months (range, 1-58 months). Histologic comparison of tissues resected during thoracotomy and retroperitoneal node dissection indicated that patterns were dissimilar in 8 of 23 patients (35%). These data indicate the favorable impact of combined sequential chemotherapy and surgery in patients with advanced mixed germ cell tumors. In patients with Stage III tumors, persistent radiographic disease after cyclic cisplatin-based chemotherapy is appropriately managed by excision of both thoracic and retroperitoneal deposits. PMID- 3002595 TI - The significance of supraclavicular fossa node recurrence after radical mastectomy. AB - The clinical and pathologic features of 35 patients who had ipsilateral supraclavicular node (SCF) recurrence after radical mastectomy were reviewed. These were compared with the features of 70 patients who had a local skin recurrence after radical mastectomy, 48 of whom had a single nodule and 22 of whom had multiple skin nodules. There were no significant differences between age at diagnosis, disease-free interval, menstrual status, tumor type and grade, and extent and location of axillary node metastases in the three groups. Survival of the SCF group was intermediate between those of the single-nodule and multiple nodule groups. SCF recurrence is an almost invariable signal of micrometastatic disease; thus, such patients are ideal candidates for trials of adjuvant therapy. PMID- 3002598 TI - Mullerian adenosarcoma with ovarian sex cord-like differentiation. A light- and electron-microscopic study. AB - A Mullerian adenosarcoma in which the sarcomatous element showed ovarian sex-cord like differentiation, occurred as a polypoid growth in the uterine cervix of a 53 year-old woman. The tumor was composed of an admixture of benign neoplastic glands and a sarcomatous stroma, the latter containing in addition to endometrial stromal sarcoma, nests of lipid-rich cells resembling ovarian sex-cord elements. This is the first report of such differentiation in Mullerian adenosarcoma. Origin from a focus of cervical endometriosis is postulated. PMID- 3002597 TI - Lobular carcinoma of the breast in a patient with Klinefelter's syndrome. A case with bilateral, synchronous, histologically different breast tumors. AB - A case of bilateral breast cancer in a patient with a Klinefelter mosaic syndrome is presented. The tumor in the left breast was an infiltrating lobular carcinoma with characteristic in situ component. To the knowledge of the authors, this is the first case in the English literature of lobular carcinoma of the breast in a phenotypic man. In fact, it was the pathologic diagnosis which led to the study of the chromosomal abnormality. PMID- 3002599 TI - Serum neuron-specific enolase in children with neuroblastoma. Relationship to stage and disease course. AB - Serum neuron-specific enolase (NSE) was measured in 61 children at diagnosis with all stages of neuroblastoma. The median serum values for Stages I, II, III, IV, and IV-S were 13, 23, 40, 214, and 40 ng/ml, respectively. Mean serum levels were different between groups I versus IV, (P = 0.0004) II versus IV (P = 0.0001) and IV-S versus IV (P = 0.004). The prognostic value of serum NSE for disease-free survival was determined in 54 patients at risk for relapse 2 or more years after diagnosis. The disease-free survival rate of all patients with levels of less than 100 ng/ml was 27/34 (79%), whereas it was 2/20 (10%) for those with higher levels. In 28 patients with lower stage disease and a good prognosis (Stages I, II, and IV-S) NSE levels were not predictive of relapse. Only 1 of these 28 patients had a raised level (greater than 100 ng/ml) and survived without relapse, whereas 4 patients who relapsed had serum NSE less than 100 ng/ml at diagnosis. In patients with Stages III and IV disease, a raised serum NSE level was associated with poor outcome: only 1/19 (5%) survived with NSE levels greater than 100 ng/ml, whereas survival was 5/8 (63%) with values below 100 ng/ml. Serial samples were analyzed on 17 patients; all 8 patients with initial NSE levels greater than 100 ng/ml achieved near normal levels during remission (median, 21 ng/ml). However, in only 4/10 patients studied at time of relapse, did the levels rise coincident with relapse. The sera of 47 patients with other forms of cancer and 19 siblings of cancer patients were at or near the normal limits (0-15 ng/ml), with three exceptions: acute lymphoblastic leukemia (286 ng/ml), hepatoblastoma (176 ng/ml), and primitive neuroectodermal tumor (105 ng/ml). Serum NSE is a useful marker for patients with advanced neuroblastoma in whom elevated levels were associated with a poor outcome; the raised NSE levels returned to near normal after therapy. In patients with Stage IV-S disease serum NSE levels were significantly lower than those in Stage IV despite their extensive tumor burden. Serum NSE estimation may confirm Stage IV-S status and suggest a more benign clinical course. PMID- 3002600 TI - Nutrition and diet in the etiology of endometrial cancer. AB - The risk of endometrial cancer in relation to nutrition and frequency of consumption of a few selected dietary items was evaluated in a case-control study of 206 patients with endometrial cancer and 206 control subjects with acute conditions unrelated to any of the established or potential risk factors for endometrial cancer. Obesity was strongly and positively associated with the risk of endometrial cancer, and several conditions related to body weight, such as early menarche, diabetes mellitus, or hypertension were more common in cases. The risk of endometrial cancer was elevated in subjects reporting (on a subjective basis) greater fat (butter, margarine, and oil) intake (relative risk estimate for the higher compared to the lower scores equals 5.65, with 95% confidence interval of 2.76-11.55). Cases reported less frequent intake of green vegetables, fruit, and whole-grain foods: thus, the risk of endometrial cancer appeared inversely related to indices of beta-carotene and fiber intake. Furthermore, cases consumed milk, liver and fish less frequently than controls. No significant difference was noted between cases and controls in the frequency of intake of carrots, meat, eggs, ham, and cheese. Alcohol consumption was somewhat larger among the cases, but this trend in risk was not significant. Dietary information collected in this study probably is too limited and inconsistent to permit analysis of biologic correlates of these findings or discussion of their potential implications in terms of prevention on a public health scale. Nonetheless, the mere existence of differences in reported diet between endometrial cancer cases and controls is of interest, and may warrant further, more detailed investigation. PMID- 3002601 TI - Bilateral testicular germ cell tumors. Report of nine cases and review of the literature. AB - In a series of 181 patients with testicular germ cell tumors, the phenomenon of bilateral involvement of the testicles was observed nine times (5%). Two patients evidenced a simultaneous bilateral tumor, and, in seven others, a second tumor occurred after an interval ranging between 8 months and 6 years and 11 months. The incidence, histologic findings, therapy, and prognosis of bilateral testicular tumors are discussed on the basis of a survey of the literature. Emphasis is placed on the significance of a long-term follow-up period as well as on the important role of sonography in the early detection of a contralateral tumor. PMID- 3002602 TI - Chromosome changes in germ cell tumors of the testis. AB - Chromosome analysis was performed on short-term cultures established from samples of six tumors of the testis. Histologically, four tumors were embryonal cell carcinomas (three primary, one metastatic) and two of mixed histology with predominance of teratoma. The modal chromosome number was hypotriploid in four tumors, triploid in one, and hypertriploid in another. All tumors contained structurally abnormal chromosomes, ranging in number from 1 to 10 in different cases. A small metacentric marker chromosome, identified as an isochromosome of the short arm of chromosome #12 [i(12p)], was present in all tumors analyzed. Unlike other marker chromosomes, this one was invariably present in at least two copies per metaphase in all cases; all other chromosome markers were present in single copy in all tumors. Together with the previous reports on the presence of i(12p) in seminoma and teratoma of the testis, our findings suggest that this karyotypic abnormality is characteristic for all histologic varieties of germ cell tumors of the testis. PMID- 3002603 TI - Transformation of chromosome breakage syndrome fibroblasts by SV40 DNA transfection. AB - SV40 DNA was transfected into three Fanconi's anemia, two classical ataxia telangiectasia, two variant ataxia-telangiectasia, three Bloom's syndrome, and three normal fibroblast cultures in order to study T-antigen expression, growth transformation, and survival of the crisis phase. Although Fanconi's anemia cells exhibited an initially higher frequency of uptake of transfected DNA compared with all the other cell strains studied, they did not acquire the transformed phenotype any faster than the others. Classical ataxia-telangiectasia fibroblasts, on the other hand, showed approximately the same rate of initial uptake of SV40, but were significantly slower than all other cell strains in expressing the transformed phenotype. The uptake and growth transformation properties of Bloom's syndrome and variant ataxia-telangiectasia cells were similar to those of normals. Serial subculturing of transfected cells was found to accelerate the appearance of growth-transformed cells. Although a number of transformed sublines of Fanconi's anemia, classical and variant ataxia telangiectasia, and Bloom's syndrome strains apparently survived the crisis phase, no immortalized strains emerged from them. PMID- 3002604 TI - Chromosome abnormalities in bovine papillomavirus type 1-transformed Syrian hamster cells before and after tumor formation. AB - Syrian hamster embryonic fibroblasts transformed by infection with bovine papillomavirus type 1 cause tumors when inoculated into hamsters. Chromosome examinations revealed several abnormal clones in the transformed fibroblasts and a variety of additional markers in three tumors. Only one aberration, trisomy 11, was present in each cell. The extra chromosome #11, thus, is considered to be essential for tumor formation in this model system. PMID- 3002605 TI - The effect of RU 41.740 (Biostim) on the production of interleukin-1 by monocytes and enriched large granular lymphocytes in normals and patients with hepatocellular carcinoma. AB - The effect of RU 41.740 on natural killer (NK) cell cytotoxicity from normals and patients with advanced hepatocellular carcinoma (HCC) was studied. RU 41.740 treated normal enriched large granular lymphocytes (LGLs) showed increased cytotoxic activity against 51Cr-labelled K562 cells. Although the drug increased the cytotoxic activity of LGLs from HCC patients, these results were not statistically significant. As LGLs have been shown to release interleukin-1 (IL 1) after suitable stimulation the effects of RU 41.740 on production of this cytokine from both monocytes and LGLs was studied. The drug stimulated IL-1 release from normal cells but monocyte and LGL IL-1 production from HCC patients was not influenced by RU 41.740 by RU 41.740. It is concluded, therefore, that although RU 41.740 can stimulate natural cytotoxicity and IL-1 production by normal LGLs, it is unable to correct the defect which exists in these functions in patients with large mass tumours. PMID- 3002606 TI - Correlation of tumor specific delayed type hypersensitivity reaction and tumor protection to SV40-induced mKSA fibrosarcoma. AB - Mice immunized by excision of a primary, subcutaneously growing SV40-induced mKSA solid tumor which resisted challenge of homologous tumor cells administered at a contralateral site, were found to develop a specific DTH response to SV40 tumor associated transplantation antigens (TATA). In a two-way criss-cross experiment, this DTH response (assessed by direct challenge) was found to be one-way SV40 specific in that chemically induced, non SV40, MCA tumor failed to elicit a DTH response in mice primed by excision of mKSA tumor. These mice also showed a corresponding one-way specific protection against challenge with live homologous mKSA sarcoma cells. In contrast immunization and challenge of MCA-excised mice with either MCA or mKSA tumor cells, exhibited cross-reactivity in both DTH response and protection against either tumor. Unlike this cross-immunity by the direct challenge method, transfer of "immune" spleen cells from mKSA or MCA excision-primed mice demonstrated a specific DTH response and protection to the original immunizing, homologous but not heterologous tumor. Tumor resistant, DTH primed mice remained DTH reactive to the primary tumor cells over a period of 4 weeks. Characterization of the splenic T-DTH cells in mice primed by excision of mKSA tumor, indicated a Lyt 1+2+ phenotype of cells conferring both the DTH response and the immune protection against mKSA sarcoma in a local (Winn) adoptive transfer assay, thus reinforcing the correlation between the DTH response and the antitumor protection. PMID- 3002607 TI - Growth factors and cancer. AB - Growth factors, defined as polypeptides that stimulate cell proliferation, are major growth-regulatory molecules for cells in culture and probably also for cells in vivo. Nontransformed cells show an absolute requirement for growth factors for proliferation in culture and generally more than one growth factor is required. Under usual culture conditions, growth factors are more rapidly depleted than other media components and thus become rate limiting for proliferation. The loss of or decreased requirement for specific growth factors is a common occurrence in neoplastically transformed cells and may lead to a growth advantage, a cardinal feature of cancer cells. Recent work with transforming growth factors, the platelet-derived growth factor, and oncogenes has produced some insight into the mechanisms through which alterations in growth factor-receptor-response pathways could lead to a growth advantage. Evidence has been derived for autocrine secretion in which the cell produces its own growth factor. Many transformed mesenchymal cells produce PDGF (the product of the c-sis proto-oncogene) and certain transformed cells both produce and respond in a growth-stimulatory manner to TGF beta. With TGF beta, which is a growth inhibitor for certain epithelial and other cell types, the loss of the normal inhibitory response in transformed cells could have the same result as the activation of a growth-stimulatory response. Two proto-oncogenes, erbB and fms, encode growth factor receptors. In the erbB case, the viral erbB aberrant receptor produced is truncated and appears to be constitutively activated without the need for a growth factor. Recent studies suggest that the p21 product of the ras oncogene may be an obligatory intermediate in transducing the growth factor signal. Activation of ras may, therefore, activate the growth factor pathway without the need for either a growth factor or its receptor. The transcription of myc and fos is induced by growth factor stimulation of quiescent cells. The protein products of both are nuclear associated and conceivably could be involved in regulating other genes important in the control of cell proliferation. Activation or inappropriate expression of either myc or fos could produce the same end result as stimulation of a growth factor pathway leading to a growth advantage. Study of the molecular mechanism(s) of growth factor action has just begun. The excitement and attention focused on cellular oncogenes in recent years is now turning toward growth factors, not only as they concern the control of normal cell growth but also the involvement of growth factor-initiated pathways in the etiology of cancer.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3002608 TI - Role of epidermal growth factor in carcinogenesis. AB - For cell growth and division to occur, a large variety of metabolic processes must be carefully coordinated in the cell. Through evolutionary pressures, specific hormones and growth factors have acquired the ability to trigger a complex coordinated "pleiotropic growth response" in their target cells. This complex response is mediated by specific cellular receptors and intracellular messengers. Teleologically then, it makes sense that in oncogenesis this growth regulating network is utilized by the production of proteins which mimic growth factors, the activated form of their receptors or, the messengers themselves. Several lines of evidence indicate that the epidermal growth factor-stimulated growth regulatory system is involved in cellular proliferation, both normal and neoplastic. Some of the effects of epidermal growth factor in carcinogenesis are separable from its direct, growth stimulatory effects. Thus, the role of epidermal growth factor in carcinogenesis is more complex than is its role in stimulating growth. PMID- 3002609 TI - Role of proteases in red blood cell target cell destruction by cells transformed by Rous sarcoma virus mutants. AB - The tumor induced RBC cytotoxicity assay has been used to explore the mechanism by which Rous sarcoma virus mutant transformed chick embryo fibroblasts and mouse 3T3 cells cause the cytolysis of RBC in vitro. All Rous sarcoma virus and viral mutant transformed cells were cytolytic for RBC. Three mutant viruses, tsGl251, rASV1702, and rASV157, appeared to cause quantitatively less cytolysis after transformation of chick embryo fibroblasts than other virally transformed cells. This decreased cytolytic activity may be correlated with decreased in vivo tumorigenicity. Temperature sensitive mutant transformed chick embryo fibroblasts and mouse 3T3, which were phenotypically normal and which secrete little if any plasminogen activator at non-permissive temperatures, were cytolytic at non permissive temperatures. In addition, inhibitors of plasminogen activator and plasmin were ineffective inhibitors of cytotoxicity. The only effective inhibitor of cytotoxicity for both transformed chick embryo fibroblasts and mouse 3T3 cells was leupeptin. In Rous sarcoma virus transformed mouse 3T3 cells, the leupeptin inhibitable enzyme appears to be a plasma membrane enzyme. PMID- 3002610 TI - Production of transforming growth factors by human colon cancer lines. AB - Three human colon cancer lines (SW 480, SW 620, WIDR) were characterized as to their production of molecules with transforming growth factor (TGF)-like activity. Production of both TGF alpha-like and TGF beta-like activity was quantitated, as were cellular receptors for these molecules, and growth response in soft agar to exogenous epidermal growth factor (EGF) (as a substitute for TGF alpha) and TGF beta. Serum-free medium conditioned by these cells showed differing amounts of TGF alpha-like and TGF beta-like competing activity in EGF and TGF beta radioreceptor assays. Likewise the cells showed differing abilities to bind 125I-labeled EGF and TGF beta. SW 620 cells produced relatively large quantities of TGF alpha-like activity and had no detectable EGF receptors; specific TGF beta binding was observed. SW 480 cells produced the most TGF beta like activity and had no measurable TGF beta membrane receptors, but EGF receptors were detectable. WIDR cells had both EGF and TGF beta membrane receptors and produced relatively low levels of EGF and TGF beta receptor competing activity. All three of the cell lines grew spontaneously in soft agar (in medium containing 10% serum). In contrast to other carcinoma cell lines, exogenous EGF and TGF beta had no significant effect on soft agar growth of the colon carcinoma cells. The production of both TGF alpha-like and TGF beta-like polypeptides by colon carcinoma cell lines has been shown, yet involvement of these factors in autostimulatory activity could not be demonstrated. The possibility that these endogenous factors could be involved in paracrine stimulation of stromal cells remains to be explored. PMID- 3002611 TI - Alteration in insulin receptor expression accompanying differentiation of HL-60 leukemia cells. AB - Differentiation of leukemic cells in vitro is characterized by the sequential appearance of morphological, functional, and biochemical markers of maturation. The interaction of insulin with its receptor may be a regulator of growth and differentiation of leukemic cells. Human promyelocytic leukemia cells (HL-60) demonstrate specific reversible insulin binding consistent with properties of human insulin receptor. HL-60 cells treated with 500 microM N6,O2-dibutyryl adenosine 3',5'-cyclic monophosphate, 1 microM 1 alpha, 25-dihydroxyvitamin D3, or 41 nM phorbol-12-myristate-13-acetate expressed monocytic markers of differentiation and an increase in insulin receptor expression. The change in insulin receptor expression with 1 microM 1 alpha, 25-dihydroxyvitamin D3 and N6,O2-dibutyryl adenosine 3',5'-cyclic monophosphate induction was further characterized by Scatchard analysis. High affinity binding (Kd) constant was not altered, and the change in binding was attributed to receptor number. Commitment to increased insulin receptor expression was demonstrated after 1-h exposure to 1 microM 1 alpha, 25-dihydroxyvitamin D3. Agents which induced granulocytic differentiation, such as 160 mM dimethyl sulfoxide and 100 nM retinoic acid, significantly decreased insulin receptor expression compared to monocytic inducing agents. This difference in insulin receptor expression correlated with binding characteristics in normal human peripheral granulocyte and monocytes. The HL-60 cell line offers a model for the study of the molecular events which lead to the contrasting insulin receptor expression during myeloid and monocytoid hematopoiesis. PMID- 3002613 TI - Induction of liver tumors in F344 rats by crotonaldehyde. AB - The tumorigenic activities in F344 rats of crotonaldehyde, a representative alpha, beta-unsaturated carbonyl compound, and N-nitrosopyrrolidine, which could produce crotonaldehyde upon metabolism, were compared. Groups of rats were treated with either crotonaldehyde (0.6 mM or 6.0 mM) or N-nitrosopyrrolidine (0.6 mM) in their drinking water for 113 or 84 weeks, respectively. At the 0.6 mM dose, crotonaldehyde induced neoplastic lesions of the liver in 9 of 27 rats; 2 rats had hepatocellular carcinomas, and 9 rats had neoplastic nodules. It also caused altered liver cell foci in 23 of 27 rats. The incidences of tumors and foci were significantly higher than those of the control group. N Nitrosopyrrolidine induced hepatocellular carcinomas in 20 of 23 rats, liver neoplastic nodules in 16 of 23 rats, and altered liver cell foci in 23 of 23 rats. Thus, crotonaldehyde appears to be a weaker tumorigen than N nitrosopyrrolidine. At the 6.0 mM dose, crotonaldehyde treatment caused moderate to severe liver damage in 10 of 23 rats. No preneoplastic or neoplastic lesions were observed in these rats. The remaining 13 rats of this group developed altered liver cell foci. The tumorigenicity of crotonaldehyde suggests that alpha, beta-unsaturated carbonyl compounds, which are ubiquitous in the human environment and can be formed endogenously, may be an important class of potential carcinogens. PMID- 3002612 TI - Secretin/vasoactive intestinal peptide-stimulated secretion of bombesin/gastrin releasing peptide from human small cell carcinoma of the lung. AB - Bombesin/gastrin releasing peptide-like immunoreactivity (BLI) is found in the majority of small cell carcinoma of the lung (SCCL) cell lines examined. Because BLI is present in high concentration in SCCL we studied the mechanism of BLI secretion from several SCCL cell lines and in patients with SCCL. In cell line NCI-H345 the structurally related polypeptide hormones secretin, vasoactive intestinal peptide, and peptide histidine isoleucine as well as theophylline, a phosphodiesterase inhibitor, N6,O2'-dibutyryl cyclic adenosine 3':5' monophosphate, a cyclic nucleotide analogue, increased BLI release by 16-120% and cyclic adenosine 3':5'-monophosphate by 36-350%. Similar results were obtained in SCCL cell line NCI-H209. i.v. injection of secretin (2 units/kg) significantly increased plasma BLI in 2 patients with extrapulmonary SCCL. These data suggest that SCCL cells possess receptors for secretin/vasoactive intestinal peptide and that receptor occupation stimulates in vitro and in vivo BLI secretion. PMID- 3002615 TI - Collagenases in human breast carcinoma cell lines. AB - The intense stromal response to some human tumors is termed the desmoplastic reaction. It is found with most human breast carcinomas. Dissolution of this response, containing predominantly fibrous proteins such as collagen and elastin, can occur with treatment. We have undertaken a study of the collagenases of the breast tumor desmoplastic reaction using a tissue culture model composed of human breast tumor cell lines and various human fibroblasts. The breast tumor cells had the higher collagenase activity, particularly the ZR75-31A cell line. Activity was 10-fold higher than that of the stromal cells. The enzyme was secreted into the media and required trypsin pretreatment for activity to be manifest. Partial purification was achieved of the major collagenase species. The protein was a metalloprotease and, like other mammalian collagenases, had a relative molecular weight of 60,000. Classical 3/4 and 1/4 cleavage products of the triple helical collagen substrate were demonstrated, typical of most mammalian collagenases. Only types I and III collagens were suitable substrates for this enzyme, with no apparent preference between the two. The breast tumor collagenases were not responsive to hormones; however, stimulation of activity was apparent in the absence of proteolytic pretreatment. This may represent conversion of the procollagenases of the breast tumor cells to the active form by an estrogen sensitive plasminogen activator secreted by the same tumor cells. PMID- 3002614 TI - Epidermal growth factor and proliferation in rat hepatocytes in primary culture isolated at different times after partial hepatectomy. AB - Primary hepatocyte cultures have been prepared from normal adult rat liver and from rat liver at 4, 8, 12, 24, and 48 h following partial hepatectomy (removal of 70% of the liver). Cells were maintained in minimal essential medium alone or supplemented with hormones. Comparing DNA synthesis in normal adult rat hepatocytes with DNA synthesis in hepatocytes isolated from regenerating livers, we found with minimal essential medium alone little DNA synthesis in normal adult rat hepatocytes and in hepatocytes isolated 4, 8, or 12 h after 70% hepatectomy. In hepatocytes isolated 24 h after partial hepatectomy, however, the incorporation of [3H]thymidine was 3 times the rate of normal hepatocytes. The addition of insulin to minimal essential medium had minimal effect on DNA synthesis in all hepatocytes. Addition of epidermal growth factor alone or in combination with insulin resulted in a dramatic increase in DNA synthesis in hepatocytes from regenerating rat liver. Increased incorporation was detectable as early as 4 h after partial hepatectomy and reached a maximum at 24 h after the operation. Results obtained with [3H]thymidine incorporation were confirmed by autoradiography and by direct DNA determinations in hepatocyte cultures. Epidermal growth factor binding to the hepatocytes was determined and agreed with previously reported binding studies. Binding of epidermal growth factor in hepatocytes isolated at 4 h after partial hepatectomy was the same as in normal hepatocytes but was undetectable in hepatocytes isolated from rats at 12 and 24 h after partial hepatectomy. PMID- 3002616 TI - Detection of a ganglioside antigen associated with small cell lung carcinomas using monoclonal antibodies directed against fucosyl-GM1. AB - Monoclonal antibodies with an apparent specificity for fucosyl-GM1 (Fuc-GM1) were produced by the immunization of mice with Fuc-GM1 adsorbed to Salmonella minnesota bacteria and fusion of the spleen cells with the myeloma cell line Sp 2/0. The antibodies detected Fuc-GM1 with a unique ceramide composition containing 2-hydroxy fatty acids in 11 of 12 cases of small cell carcinoma of the lung. Trace amounts of Fuc-GM1 were detected in 1 of 11 squamous epithelial cell lung carcinomas. Fuc-GM1 was also detected in 1 of 7 pancreas carcinomas but was not detected in any of the other cancers analyzed. Small amounts of Fuc-GM1 without 2-hydroxy fatty acids were detected in normal adult pancreas, spleen, and brain but could not be detected in normal lung tissue. Fuc-GM1 with 2-hydroxy fatty acids is suggested to be a specific ganglioside associated with small cell lung carcinomas. The monoclonal antibodies directed against Fuc-GM1 may be useful for specific immunodiagnosis of small cell lung carcinomas and might also be useful for specific immunotherapy of these malignant tumors. PMID- 3002617 TI - 3' c-myc rearrangement in a human leukemic T-cell line. AB - The human leukemic T-cell line Hut 78, derived from a patient suffering from Sezary syndrome and expressing a mature postthymic membrane phenotype, shows a c myc rearrangement beginning within 500 base pairs immediately 3' to the c-myc exon 3. Chromosome analysis of the Hut 78 reveals the presence of a hyperdiploid karyotype with a large number of markers and rearrangements, though trisomy is the only cytogenetic anomaly involving chromosome 8. Moreover, as the abnormal c myc appears to be duplicated, a duplication of the chromosome 8 carrying the abnormal c-myc probably occurred. Unlike four other human leukemic T-cell lines tested, the Hut 78 cells express a high amount of c-myc transcript, suggesting that the 3' c-myc anomaly may cause a deregulation of the expression of this gene. PMID- 3002618 TI - Nerve growth factor receptors on dissociated neurofibroma Schwann-like cells. AB - Neurofibromatosis is a disorder which predominantly involves cellular elements of peripheral neural sheaths. Little is known about the regulation of differentiation and proliferation of cells comprising neurofibromas. Because nerve growth factor-like activity may be present in neurofibromas and the cells comprising neurofibromas are neural crest derivatives, we have investigated whether nerve growth factor (NGF) receptors are present on cells from dissociated dermal neurofibromas. Using 125I-NGF to measure binding to cultured cells in suspension and for autoradiography, we identified a population of cells having characteristics of Schwann cells which exhibited saturable 125I-NGF binding. This binding is characteristic of the "fast" (low affinity) NGF receptor, having a Kd of approximately 1 nM and a Bmax of at least 120 fmol/10(6) cells. Less than 20% of the bound 125I-NGF (5 ng/ml) is not displaced when transferred to 0 degrees C by an excess of unlabeled NGF (10 micrograms/ml) and is therefore bound to either "slow" (high affinity) sites or is rapidly internalized. NGF receptors with characteristics of fast sites have recently been reported on Schwann-like cells from chick dorsal root ganglia [Zimmerman, A., and Sutter, A. Beta nerve growth factor (beta NGF) receptors on glial cells. Cell-cell interaction between neurones and Schwann cells in culture of chick sensory ganglia. EMBO J., 2: 879 885, 1983]. The identification of NGF receptors on both fetal chick dorsal root ganglia and neurofibroma Schwann-like cells suggests that NGF may have a role in the regulation of Schwann cell function in both normal development and in neurofibromatosis. PMID- 3002619 TI - Differential expression of p21ras gene products among histological subtypes of fresh primary human lung tumors. AB - Activation of the ras oncogene has been associated with a number of human tumors. In this study, expression of p21ras in different histological types of fresh primary bronchogenic carcinomas was examined. p21ras products were detected in all lung tissues that were analyzed. Only 1 of 23 tumors demonstrated aberrant migration of p21ras. In contrast, 10 tumors had substantially elevated levels of p21ras products with respect to the adjacent normal lung tissues and with respect to the other lung tumors. There was no correlation between increased ras protein expression and tobacco exposure of the patient or extent of disease at the time of diagnosis. However, 9 of 11 tumors with a squamous component as opposed to only 1 of 12 tumors belonging to other histological classifications demonstrated increased p21ras. These data suggest that, in bronchogenic carcinomas, mutations associated with structural abnormalities and aberrant migration of p21ras occur infrequently as compared to quantitative changes in p21ras. Furthermore, differential expression of c-ras products in primary human lung tumors correlates with pathological cell type, and may be a common event in squamous cell carcinomas, but not adenocarcinomas or small cell carcinomas of the lung. PMID- 3002620 TI - Alternation of chemotherapy and radiotherapy in cancer management. III. Results in experimental solid tumor systems and their relationship to clinical studies. PMID- 3002621 TI - Contributions of nitrosoureas to cancer treatment. PMID- 3002622 TI - cis-Diammine-1, 1-cyclobutane dicarboxylate platinum II (carboplatin) in the treatment of testicular germ-cell tumours: a preliminary report. AB - Between 1982 and 1985 47 patients with metastatic testicular germ-cell tumours were treated with carboplatin alone or combined with bleomycin and etoposide and/or vinblastine. Of 14 untreated seminoma patients 13 (93%) are free from active disease at 7-38 months (median 12 months). Twenty patients with untreated advanced non-seminomatous testicular germ-cell tumours have been entered into a study of carboplatin, etoposide and bleomycin (CEB) as first-line treatment. Preliminary data show that of 15 men observed for 6-12 months (mean 7.5 months) 11 are disease-free. Toxicity with the single agent and combination has been mild. Of 12 patients receiving carboplatin alone or in combination for relapse after cisplatin chemotherapy 10 showed no response, one a transient complete response and one a partial response consolidated with radiotherapy. On the basis of these preliminary observations it is concluded that carboplatin is an active drug in testicular germ-cell tumours and that cross resistance with cisplatin may be a significant problem. PMID- 3002623 TI - Carboplatin: the clinical spectrum to date. AB - The existing literature data base on carboplatin updated to June, 1985 has been reviewed. The compound seems to retain the same spectrum of activity as cisplatin, and a definite set of efficacy data is available for ovarian cancer of epithelial origin, small cell carcinoma of the lung and epidermoid carcinoma of the head and neck. A yet unpublished toxicity data base on carboplatin suggests that the compound has an improved therapeutic index over the parent compound, cisplatin, and that it does not seem inferior to another platinum coordination compound currently in clinical trials, iproplatin. PMID- 3002624 TI - Carboplatin (JM8) as a single agent and in combination in the treatment of small cell lung cancer. AB - Carboplatin, a cisplatin analogue without significant nephrotoxicity, was used as a single agent in the treatment of 56 patients with small cell lung carcinoma at a dose of 300-400 mg/m2 i.v. monthly. Twenty-three patients (41%) achieved a response including 5 (9%) complete remissions. Eighteen (60%) of 30 previously untreated patients achieved a response. The drug was well tolerated with nausea or vomiting in only 43% of patients and no nephrotoxicity was seen. Myelosuppression was dose limiting and 39% of patients developed leukopenia. In a subsequent study carboplatin in a dose of 300 mg/m2 was used in combination with etoposide 100 mg/m2 i.v. days 1-3, repeating monthly for 4 courses. So far 32 (89%) of 36 evaluable patients have achieved a response. In patients with limited disease 20/23 patients (87%) have responded including 7 (30%) complete remissions. Leukopenia was dose limiting and occurred in 83% of patients. Carboplatin is a highly active new drug in the treatment of small cell lung cancer. PMID- 3002625 TI - Wilms' tumor with intracranial metastases presenting with intracranial hemorrhage. AB - Intracranial metastasis of Wilms' tumor is very rare. Furthermore, intracerebral hemorrhage is an unusual presentation of metastases. We report the case of a 4 year-old girl who had multiple intracranial metastatic lesions, initially presenting as an intracranial hemorrhage. Removal of the tumors and hematoma was followed by radiation therapy and chemotherapy. Thereafter, complete remission occurred. It is thought that this malignant tumor with metastases may be curable through combined therapy. PMID- 3002627 TI - [Receptors in illness and health]. PMID- 3002626 TI - Synthesis of glycosides of alpha-D-mannopyranose 6-(alpha-D-glucopyranosyl phosphate). The putative ligatin receptor. AB - p-Nitrophenyl alpha-D-mannopyranoside 6-(alpha-D-glucopyranosyl phosphate) (7) and 6-(alpha-D-galactopyranosyl phosphate) (8) were synthesized by condensation of the appropriate peracetylated glycosyl phosphate with p-nitrophenyl 2,3,4-tri O-benzoyl-alpha-D-mannopyranoside followed by alkaline deprotection. 1H-, 31P-, and 13C-n.m.r. spectroscopy were used to establish the structures of 7 and 8, and to examine the conformational preferences about the phosphoric diester linkage. PMID- 3002628 TI - [Sensitivity and response of beta 2-adrenergic receptors of isolated lymphocytes in essential hypertension]. PMID- 3002629 TI - In-vivo labeling of (3H)D-aspartate uptake sites in monkey retina. AB - Following prolonged topical application of (3H)D-aspartate in vivo, selective labeling of three distinct cell classes was observed in light-microscopic radioautographs from squirrel monkey retina. Muller (glial) cell bodies and their processes were intensely and consistently labeled in all preparations. Moderately labeled perikarya were occasionally detected in the area of bipolar cells, within the inner nuclear layer. These were particularly numerous in sections from the central retina where an intense diffuse labeling of the inner plexiform layer was also prominent. Finally, moderate to dense accumulations of label were observed over the cell bodies, internal segments and fiber processes of cone photoreceptors. These results strongly suggest that cones, as well as a sub population of bipolar cells, use glutamate and/or aspartate as neurotransmitter(s) in monkey retina. PMID- 3002630 TI - Muscle nerve sympathetic activity in migraine. Lack of abnormality. AB - Microelectrode recordings of muscle nerve sympathetic activity (MSA) in the peroneal nerve were performed in eight patients with common migraine, when they were free of headache and during a spontaneously occurring attack of migraine. During the migraine headache all subjects remained on the same level of MSA as in the control situation and the responses to manoeuvres (slow deep breathing, the Valsalva manoeuvre, sustained hand grip, immersion of one hand into ice water) showed no qualitative or quantitative change. Assessment of vagal influence on the heart showed no change from control situation to attack of migraine. The study provides direct evidence against the existence of any abnormality of MSA during ongoing migraine headache and does not support the assumption that migraine is a generalized vasomotor disorder. No conclusions about possible dysfunction in other parts of the sympathetic nervous system can be drawn. PMID- 3002631 TI - Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. AB - Introduction of torsional stress into active chromatin domains requires that linear DNA molecules be anchored in vivo to impede free rotation. While searching for these anchorage elements, we have localized a nuclear matrix association region (MAR) within the mouse immunoglobulin kappa gene that contains two topoisomerase II sites and is adjacent to the tissue-specific enhancer. The same matrix contact occurs when the kappa locus is in germ-line (inactive) or rear ranged (transcribed) configurations. This constitutive anchorage site partitions the gene into V-J and C region chromatin domains. We demonstrate that at least 10,000 similar and evolutionarily conserved MAR binding sites exist in the nucleus. We propose that these sites, in association with topoisomerase II and possibly in conjunction with enhancers, play fundamental roles in the functional organization of chromatin loop domains. PMID- 3002632 TI - Excess function hairy-wing mutations caused by gypsy and copia insertions within structural genes of the achaete-scute locus of Drosophila. AB - Hairy-wing (Hw) mutations cause the differentiation of extra chaetes on the cuticle of Drosophila. They are associated with modifications of the achaete scute complex that consist, in the mutants studied, of insertions of the transposable elements gypsy (Hw1, HwBS) or copia (HwUa). gypsy and copia are inserted in achaete and scute transcribed regions, respectively. Transcription of the insertion-split genes starts at the normal site but terminates within the transposable element sequences. The RNA truncated within gypsy is 5-20 times more abundant than its homolog in wild-type flies. The abundance is reduced in Hw1 revertants and Hw1 stocks carrying su(Hw) mutations. These and other data suggest that the excess function phenotypes of Hw mutations are generated by an increase in achaete or scute transcripts. PMID- 3002633 TI - Role of a ras homolog in the life cycle of Schizosaccharomyces pombe. AB - We have analyzed the function of the only ras homolog in S. pombe detectable by Southern blotting, ras1, which is homologous to mammalian ras genes and has been cloned. We have disrupted the ras1 gene and have replaced it with ras1Val17, which corresponds to a transforming variant of mammalian ras. Loss of ras1 activity by disruption results in the complete inability to mate. The cell body of a ras1- strain is extensively deformed, and a ras1-/ras1- diploid sporulates very poorly. Unlike RAS1 and RAS2 of S. cerevisiae, ras1 of S. pombe appears to have no effect on adenylate cyclase activity. This suggests that the target enzymes presumably modulated by ras proteins in signal transduction are not the same for all organisms. PMID- 3002634 TI - Abelson virus drives the differentiation of Harvey virus-infected erythroid cells. AB - Abelson murine leukemia virus (A-MuLV) and Harvey murine sarcoma virus (Ha-MSV) are retroviruses carrying unrelated onc genes. However, both of these viruses are capable of stimulating the growth and differentiation of erythroid precursor cells; the target cells for both appear at the same time during fetal development and follow a similar pattern throughout ontogeny. In addition, the colonies induced by each virus are morphologically similar and synthesize the adult form of hemoglobin. However, A-MuLV-infected cells are Epo-independent, whereas Ha-MSV infected cells are Epo-dependent. Superinfection of Ha-MSV-infected cells with A MuLV overrides their Epo-dependency. Thus, the consequences of the infection are determined by the interaction of the different onc gene products with identical or similar erythroid cells. PMID- 3002635 TI - Topoisomerase I interacts with transcribed regions in Drosophila cells. AB - The in vivo distribution of topoisomerase I on specific DNA sequences is determined at high resolution in Drosophila cells using a photo-crosslinking method. Topoisomerase I-DNA adducts are generated by irradiation of intact cells with UV light and then purified by immunoprecipitation with antibody to topoisomerase I. Analyses of the DNA sequences crosslinked to topoisomerase I by blot-hybridization with appropriate DNA probes indicate that topoisomerase I is concentrated on transcribed regions and not on nontranscribed flanking sequences. Like RNA polymerase II, topoisomerase I is recruited to heat-shock genes during the heat-shock response. However, topoisomerase I and RNA polymerase II can interact independently with the transcribed region because different ratios of topoisomerase I and RNA polymerase II are crosslinked to the highly transcribed hsp70 gene and the moderately transcribed copia genes. We hypothesize that topoisomerase I allows topological changes in DNA that are required for transcription. PMID- 3002636 TI - High frequency targeting of genes to specific sites in the mammalian genome. AB - We corrected a defective gene residing in the chromosome of a mammalian cell by injecting into the nucleus copies of the same gene carrying a different mutation. We determined how the number, the arrangement, and the chromosomal position of the integrated gene, as well as the number of injected molecules influence the gene-targeting frequency. Recombination between the newly introduced DNA and its chromosomal homolog occurred at a frequency of 1 in 10(3) cells receiving DNA. Correction events were mediated by either double reciprocal recombination or gene conversion. This resulted in sequences in the genome being replaced by sequences of the introduced DNA or, in separate experiments, sequences in the incoming DNA being replaced by chromosomal sequences. Both point mutations and deletion mutations were corrected; however, the nature of the mutation carried by the respective sequence influenced whether the integrated or injected sequence was corrected. PMID- 3002637 TI - Phosphoprotein phosphatase inhibits flagellar movement of Triton models of sea urchin spermatozoa. AB - Phosphoprotein phosphatase prepared from bovine cardiac muscle was used to study the roles of axonemal phosphoproteins in the flagellar motility of sea urchin spermatozoa. When isolated axonemes were incubated with cyclic AMP-dependent protein kinase, gamma-[32P]ATP and cyclic AMP, more than 15 polypeptides were phosphorylated. Most were dephosphorylated by treatment with phosphoprotein phosphatase. When Triton models of sea urchin spermatozoa were treated with phosphoprotein phosphatase followed by an addition of ATP, the flagellar motility of the models was drastically reduced in comparison with that of the untreated models. The motility of the phosphatase-treated Triton models was partially restored by an addition of cyclic AMP and cyclic AMP-dependent protein kinase. These data give strong support to the idea that the motility of eukaryotic flagella is controlled by a protein phosphorylation-dephosphorylation system. PMID- 3002638 TI - Changes in microsomal Na+, K+-, Mg2+- and Ca2+-ATPase activities during proliferation of Chinese hamster V-79 and human HeLaS-3 cells. AB - Changes in microsomal Na+, K+-, Mg2+- and Ca2+-ATPase activities during cell proliferation were examined in Chinese hamster V-79 (V-79) cells (normal cells) and human HeLaS-3 (HeLaS-3) cells (malignant cells). For V-79 cells, the Mg2+ ATPase activity per cell (pmol Pi/h/cell) in the confluent phase was higher than that in the logarithmically growing (log) phase. The amount of microsomal protein per cell was also high in the confluent phase. Specific activities (mumol Pi/h/mg protein) of Na+, K+-, Mg2+- and Ca2+-ATPase were significantly lower in the confluent phase than in the log phase. For HeLaS-3 cells, an increase in Ca2+ ATPase activity per cell was observed. The amount of microsomal protein per cell did not change between the log and confluent phase. The specific activity of Ca2+ ATPase in the confluent phase was also markedly higher than in the log phase. The relation between changes in ATPase activities and cell proliferation is discussed. PMID- 3002639 TI - [Interaction of drugs with receptors]. PMID- 3002640 TI - [Preparation of 4-(hydroxyanilino)- and 4-(alkoxyanilino) derivatives of 1,3 dimethyl-1H-pyrazolo[3,4-b]quinoline with a potential antiviral effect]. PMID- 3002641 TI - [Epidemiologic characteristics and virologic findings in women with precanceroses and carcinoma of the uterine cervix]. PMID- 3002642 TI - [Aminobutyric acid receptors (GABA)]. PMID- 3002644 TI - Generation of cytotoxic material to murine spleen cells by periodate oxidation and borohydride reduction of pectic substances. PMID- 3002643 TI - Possible involvement of calmodulin in the induction of phosphodiesterase by cyclic adenosine 3',5'-monophosphate in Dictyostelium discoideum. PMID- 3002645 TI - [Relationship between astrocytomas and their surrounding astrocytes]. PMID- 3002646 TI - [Influence of riboflavin upon the development of dimethylaminoazobenzene (DAB) induced liver cancer in rats]. PMID- 3002647 TI - [Budd-Chiari syndrome (with 8 autopsy cases)]. PMID- 3002648 TI - [The cultivation of Coruns officinalis]. PMID- 3002649 TI - [Use of the YAG laser in bronchology in the treatment of tracheobronchial cancers, benign tumors and iatrogenic stenoses: clinical experience involving 50 treatments]. PMID- 3002650 TI - Japanese encephalitis: current worldwide status. AB - The changing epidemiological and distribution patterns of Japanese encephalitis in various southern and east Asian countries are described. Immunization is considered to be the only practical way to control the infection. Several vaccines have been developed and two types of inactivated vaccine are now available for use in man. PMID- 3002651 TI - Can variola-like viruses be derived from monkeypox virus? An investigation based on DNA mapping. AB - The results are presented of a special study to determine whether variola-like "whitepox" viruses could arise as white pock variants of monkeypox virus after one or a few mutations. DNA mapping by cross-hybridization of restriction endonuclease DNA fragments was carried out on 18 orthopoxviruses relevant to this study, including variola and monkeypox viruses and white (non-haemorrhagic) pock producers recovered from chorioallantoic membranes infected with red (haemorrhagic) pock-producing monkeypox viruses. The distinctiveness of the DNA maps of true variola and monkeypox viruses indicated that spontaneous production of "whitepox" from monkeypox virus was genetically impossible. These and other observations led to the conclusion that the "whitepox" viruses recovered from monkeypox virus stocks had an exogenous origin. PMID- 3002653 TI - Comparison of the metabolism and DNA binding of the carcinogen 15,16-dihydro-11 methylcyclopenta[a]phenanthren-17-one by hamster embryo cells and the human hepatoma cell line HepG2. AB - Metabolism of 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one with hamster embryo cells and a human hepatoma cell line HepG2 is compared. Essentially, no metabolism was seen with hamster embryo cells but with the HepG2 cells, especially after induction with benzanthracene or Arochlor, this carcinogenic ketone was metabolized to give 1,2-dihydroxy-11-methyl-1,2,15, 16 tetrahydrocyclopenta[a]phenanthren-17-one, 3,4-dihydroxy-11-methyl-3,4,15,16 tetrahydrocyclopenta[a] phenanthren-17-one, 15,16-dihydro-15-hydroxy-11 methylcyclopenta[a]phenanthren-17-one, 15,16,-dihydro-16-hydroxy-11 methylcyclopenta[a]phenanthren-17- one, and three new metabolites, 16,17-dihydro 11-methyl-4,15,17-trihydroxy-15H-cyclopenta[a]phe phenanthrene, 15,16-dihydro 16,17-dihydroxy-11-methylcyclopenta[a]phenanthrene and 16,17-dihydro-11-methyl 15H-cyclopenta[a]phenanthren-17-ol. The reduction of the 17-ketone group and the formation of phenols with HepG2 cells appears to be the major difference between metabolism of 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one with HepG2 cells and rat liver microsomes. The metabolic products of this ketone also bind to DNA. These results suggest that the component of the aryl hydrocarbon hydroxylase enzyme system that is responsible for the activation of cyclopenta[a]phenanthrenes is specific and not the same as that needed for polycyclic aromatic hydrocarbons, and that HepG2 cells contain a reductase which is specific for the 17-ketone function in the cyclopenta[a]phenanthrenes. PMID- 3002652 TI - Prospective serological and clinical studies on infants born in Kuwait with an elevated IgM in cord blood. AB - Little is known about congenital viral and toxoplasmal infections in the developing countries. This study attempts to provide preliminary data on such infections among Arab mothers and infants in Kuwait. Babies born with an elevated IgM in cord blood were selected at birth and then studied prospectively during the first year of life for serological and clinical evidence of viral and toxoplasmal infections. There appeared to be a significant association between the elevation of total cord blood IgM and the selection of cases with cytomegalovirus infection. Demonstration of specific IgM in cord sera was attempted, whenever possible, to provide further evidence of congenital infection and to exclude early postnatal infections. In 18.5% of cases there was an association between serological evidence of infection and certain clinical abnormalities. Apart from cytomegalovirus and rubella virus, no evidence was found for congenital infection by other viruses or toxoplasma in this study. PMID- 3002654 TI - Expression of the E. coli O6-methylguanine-methylphosphotriester methyltransferase gene in mammalian cells. AB - Many prokaryotic and eukaryotic cells contain enzymes that repair damage introduced into their DNA following exposure to chemical, physical and biological agents. One such lesion that has received considerable attention is the potentially miscoding and mutagenic base O6-alkylguanine which is produced in varying amounts in DNA following reaction with monofunctional alkylating agents. As part of a study to assess the role of this lesion and its repair in the processes of cytotoxicity, mutagenicity and transformation, we have recently cloned the Escherichia coli gene which codes for the protein responsible for the repair of such damage in DNA. In the present study we describe the construction of a plasmid which allows the efficient expression of the bacterial gene in mammalian cells. PMID- 3002655 TI - Rat hypothalamic extract inhibits vasopressin-stimulated Na+-K+-ATPase activity in the rat renal medulla. AB - Arginine vasopressin stimulates Na+-K+-ATPase activity located in the rat thick ascending limb of Henle's loop. mammalian hypothalamus appears to produce a factor capable of inhibiting Na+-K+-ATPase activity in a variety of tissues. The effect of a purified rat hypothalamic extract with and without AVP on rat renal Na+-K+-ATPase activity was evaluated by a cytochemical technique. The hypothalamic extract alone failed to affect basal Na+-K+-ATPase activity throughout renal segments after 10 min exposure. Na+-K+-ATPase activity stimulated by AVP (1-10 fmol l-1) for 10 min was inhibited by rat hypothalamic extract over the concentration range 10(-7)-10(-3) U ml-1 in a dose-dependent manner. Complete inhibition of AVP-stimulated Na+-K+-ATPase activity occurred at a hypothalamic extract concentration of 10(-3) U ml-1. Only Na+-K+-ATPase activity located in the renal medullary thick ascending limb was influenced by the rat hypothalamic extract. PMID- 3002657 TI - In vitro trophic effects of ACTH. PMID- 3002656 TI - Investigation of the role of ubiquinone in rat liver subcellular compartments. AB - The role of ubiquinone in the Golgi apparatus is still unknown, even if it might be considered as a lipid marker of the Golgi compartment because of its high content in these subcellular fractions. In vivo modulation of ubiquinone with ethanol and in vitro pentane extraction show that ubiquinone is not required either for NADH-ferricyanide reductase, acetaldehyde dehydrogenase activity, or Ca2+ and Mg2+ stimulated ATPases. Since ubiquinone does not seem to be involved in these enzymic activities in Golgi compartments, other possible functions are discussed, related to a role in membrane fluidity or as a barrier to the propagation of free radicals. PMID- 3002658 TI - Adrenergic coronary tone during submaximal exercise in the dog is produced by circulating catecholamines. Evidence for adrenergic denervation supersensitivity in the myocardium but not in coronary vessels. AB - The goal of this study was to test the hypothesis that circulating catecholamines are primarily responsible for alpha-adrenergic coronary vasoconstriction during submaximal exercise. The experimental series consisted of chronic studies in which a regional left ventricular sympathectomy was performed with phenol. Myocardial perfusion to the innervated and sympathectomized left ventricular regions was measured in these animals during (1) a control period, (2) treadmill exercise, (3) exercise during beta-adrenergic blockade, and (4) exercise during combined alpha- + beta-adrenergic blockade. We found no differences in myocardial perfusion between the innervated and sympathectomized regions or the transmural distribution of perfusion during any of these interventions. Thus, there is no evidence for neurogenic alpha-adrenergic coronary vasoconstriction. However, during exercise in the presence of alpha- and beta-blockade, coronary resistance (mmHg X min X 100 g/ml) was significantly less in both the innervated (0.65 +/- 0.07) and sympathectomized (0.68 +/- 0.07) regions than during beta-blockade, 0.90 +/- 0.17 and 0.89 +/- 0.16, respectively. This suggests that coronary alpha adrenergic constriction was produced by circulating catecholamines. This concept of humorally mediated, alpha-adrenergic coronary vasoconstriction was strengthened by in vivo and in vitro studies that demonstrated that alpha adrenergic supersensitivity of the coronary vasculature was not present. Myocardial beta-adrenergic supersensitivity was observed in the phenol regional sympathectomy model; however, this effect was blocked by propranolol (1 mg/kg). This indicates that alpha-adrenergic vasoconstriction in both myocardial regions during submaximal exercise is produced by circulating catecholamines. The major conclusion of this study is that, during submaximal exercise in the canine, alpha adrenergic coronary vasoconstrictor tone is predominantly due to circulating catecholamines rather than direct neural effects. PMID- 3002659 TI - Adrenergic regulation of myosin adenosine triphosphatase activity. AB - The amount of inorganic phosphate liberated by the adenosine triphosphatase activity of myosin in a thin section of cardiac tissue can be measured quantitatively by precipitation with calcium in an alkaline medium under a defined set of conditions. Specificity of the procedure for myosin adenosine triphosphatase has been confirmed by the response to inhibitors and to different degrees of contractile filament overlap. Precise quantitation of adenosine triphosphatase activity has been demonstrated by (1) constant rate over time, (2) linearity with amount of enzyme, (3) correct values for the Km of adenosine triphosphate, and (4) a similar value for Vmax to those determined by more traditional procedures. Stimulation of the beta-adrenergic system by the release of catecholamines following injection of the animal with 6-hydroxydopamine causes a rise and then a fall of both calcium- and actin-activated adenosine triphosphatase in parallel with the changes in blood levels of the transmitter. Tyramine injection of rats produces a dose related increase in myosin adenosine triphosphatase. Perfusion of isolated hearts with isoproterenol increases myosin adenosine triphosphatase in dose-related manner. Addition of cyclic adenosine monophosphate and phosphodiesterase inhibitor to the solution bathing frozen, dried sections of heart increases both calcium- and actin-activated adenosine triphosphatase activity by almost 150%. The data show that the beta-adrenergic system, through cyclic adenosine monophosphatate, regulates the enzymatic activity of myosin, independent of the concentration of calcium. The possible role of this regulatory mechanism in the physiological modulation of cardiac contractility is discussed. PMID- 3002660 TI - Prognostic importance of serum sodium concentration and its modification by converting-enzyme inhibition in patients with severe chronic heart failure. AB - Although past reports have identified a variety of prognostic factors in patients with severe chronic heart failure, previous studies have not evaluated the interaction of prognostic variables and drug treatment. We analyzed the association of 30 clinical, hemodynamic, and biochemical variables with survival in 203 consecutive patients with severe heart failure; all variables were assessed just before initiation of treatment with a variety of vasodilator drugs, and all patients were subsequently followed for 6 to 94 months. By regression analysis, pretreatment serum sodium concentration was the most powerful predictor of cardiovascular mortality, with hyponatremic patients having a substantially shorter median survival than did patients with a normal serum sodium concentration (164 vs 373 days, p = .006). The unfavorable prognosis for hyponatremic patients appeared to be related to the marked elevation of plasma renin activity that we noted in these individuals (10.0 +/- 2.0 ng/ml/hr), since hyponatremic patients fared significantly better when treated with angiotensin converting-enzyme inhibitors than when treated with vasodilator drugs that did not interfere with angiotensin II biosynthesis (median survival 232 vs 108 days, p = .003). In contrast, there was no selective benefit of converting-enzyme inhibition on the survival of patients with a normal serum sodium concentration, in whom plasma renin activity was low (1.9 +/- 0.3 ng/ml/hr). This interaction between serum sodium concentration, drug treatment, and long-term outcome suggests that the renin-angiotensin system may exert a deleterious effect on the survival of some patients with chronic heart failure, which can be antagonized by converting enzyme inhibition, and provides a clinical counterpart for the similar prognostic role that has been postulated for angiotensin II in experimental preparations of heart failure. PMID- 3002662 TI - Inotropic effects of amrinone and milrinone on contraction and relaxation of isolated cardiac muscle. AB - The inotropic response to amrinone and milrinone in isolated cat papillary muscle is characterized by a dose-dependent increase in contractility, with milrinone about five times as potent as amrinone, no effect on load dependence of relaxation, no change in timing and duration of the contraction-relaxation cycle, and marked temperature dependence. This response necessitates, at least in part, the presence of a well-functioning sarcoplasmic reticulum (SR). Amrinone and milrinone are less active when the SR is poorly developed, as in frog myocardium, mammalian atrial myocardium, Purkinje fibers, and ventricular muscle from fetal and newborn animals; when the SR has been destroyed, as in single mammalian cardiac cells; and when the SR, for reasons still under investigation, has become inactive, as in isolated human ventricular myocardium. Amrinone and milrinone are also less active or may depress contractility under conditions in which the SR is known to function near maximal calcium saturation (as in rat ventricular myocardium) or to be overloaded with calcium (as during reoxygenation). This depressant action suggests concomitant desensitization of the contractile proteins to calcium. PMID- 3002663 TI - The inotropic effects of amrinone and milrinone on neonatal and young canine cardiac muscle. AB - Standard techniques were used to study developmental changes in the effects of amrinone and milrinone on contractile properties of isolated canine cardiac papillary and trabecular muscle. In contrast to milrinone, which induced a positive inotropic effect, amrinone had a negative inotropic effect on the neonatal canine muscles studied. For both drugs there was an age-dependent increase in contractility beyond the neonatal period. The negative inotropic effect of amrinone was not related to a change in phosphodiesterase inhibition, although developmental changes in phosphodiesterase inhibition did occur. These results highlight the differences in the mechanism of action of two similar molecules. They also suggest that use of amrinone as an inotropic agent in the early neonatal period should be viewed with caution. PMID- 3002661 TI - Differential inhibition of cardiac cyclic nucleotide phosphodiesterase isozymes by cardiotonic drugs. AB - Several new cardiotonic drugs are postulated to act as potent inhibitors of cyclic nucleotide phosphodiesterase activity. Unfortunately, the presence of multiple phosphodiesterase isozymes in cardiac muscle makes it difficult to determine whether any of these enzymes are specific targets for the cardiotonic agents. We have developed a method for rapid isolation and assay of bovine cardiac muscle phosphodiesterases using monoclonal antibodies that distinguish between isozymes without inhibiting catalytic activities. By this method, milrinone, amrinone, and MDL 17,043 were tested for potency as inhibitors of soluble bovine heart phosphodiesterases. All three drugs were highly selective for a low-Km, cyclic GMP (cGMP)-inhibited phosphodiesterase. IC50s (half-maximal inhibitory concentrations) for cGMP-inhibited phosphodiesterase were 0.5 microM (milrinone), 30 microM (amrinone), and 2 microM (MDL 17,043) when measured at 0.35 microM cyclic AMP (cAMP). Milrinone and MDL 17,043 had greater than 50-fold lower potencies for the other heart phosphodiesterases (and amrinone 20-fold). These data suggest the cGMP-inhibited phosphodiesterase as a probable site of action for these new cardiotonic drugs. In addition, the method described here should be useful for screening new drugs, and for studying physical and chemical properties of this phosphodiesterase/drug receptor in cardiac and other tissues. PMID- 3002664 TI - Effects of amrinone and milrinone on calcium influx into the myocardium. AB - We have examined the effects of milrinone on calcium influx into cardiac cells by testing its effects on three mechanisms of calcium entry: the slow inward calcium current, sodium-calcium exchange diffusion, and the passive calcium permeability of sarcolemmal membranes. Milrinone increased the magnitude of the slow inward calcium current in voltage-clamped calf cardiac Purkinje fiber. High concentrations of this agent also caused a small, inconsistent inhibition of calcium uptake mediated by sodium-calcium exchange in bovine cardiac sarcolemma membranes, but did not alter the time course of passive efflux of calcium from these vesicles. In conclusion, milrinone and its analogue amrinone appear to increase the entry of calcium into myocardial cells, primarily by increasing the influx of this cation during the slow inward calcium current. A small and sodium calcium exchange may also influence calcium influx, particularly at high drug concentrations. The relationship existing between these effects and the inotropic responses produced by amrinone and milrinone is discussed. PMID- 3002665 TI - Analytical variance and definition of a reference change as a function of calcium concentration. AB - Using data from the Centers for Disease Control (CDC) Proficiency Testing (PT) Surveys, we obtained estimates of repeatability (intralaboratory variability between results on the same material) and reproducibility (interlaboratory variability between results on the same material) for the Technicon SMA 6 (or 12/60) and SMAC 1 (or II) systems used with cresolphthalein complexone methodology to measure serum calcium. The two systems were comparable in terms of short-term (within-day) repeatability, long-term (three to six months) repeatability, short-term (one to two weeks) reproducibility, and long-term (three to six months) reproducibility. The long-term repeatability was essentially the same as the long-term reproducibility. Short-term repeatability, long-term repeatability, and long-term reproducibility increased linearly with increased calcium concentration over the range 1.75 to 2.95 mmol/L; short-term reproducibility showed no significant change over this range. The effect of analytical variance on the definition of a reference change in semiannual calcium measurements was demonstrated. PMID- 3002666 TI - Serum angiotensin converting enzyme activity in cigarette smokers. AB - Low activity of angiotensin converting enzyme (ACE) has been reported in patients with smoking related diseases, such as chronic bronchitis, emphysaema and carcinoma of the lung [1] but this has not been reported in healthy, chronic smokers. Serum ACE was measured in 40 healthy cigarette smokers and in 42 healthy non-smokers. The mean value was significantly lower in the smokers. Hence a non smokers. The mean value was significantly lower in the smokers. Hence a patient's smoking habits should be taken into consideration when assessing the significance of his serum ACE levels. PMID- 3002667 TI - Short- and long-term evaluation of normal and abnormal human thyroid cells in monolayer culture. AB - We have investigated the TSH responsiveness of normal and abnormal human thyroid cells cultured in the short term with high serum concentrations and for up to 6 months in a low serum, chemically-defined, medium. Cells from normal human thyroid tissue (n = 9), multinodular goitre (n = 6), benign follicular adenomata (n = 6), and differentiated thyroid carcinoma (n = 3) formed confluent monolayers which were sensitive to bovine TSH (bTSH) in concentrations greater than 25 microU/ml when assessed by the intracellular response of cyclic AMP at 7 d of culture. Such sensitivity was less than that observed with a continuously proliferating thyroid cell line (FRTL-5) derived from Fisher rat thyroid and which responded to concentrations of bTSH as low as 5-10 microU/ml. Human cells derived from iodine/antithyroid drug treated Graves' thyroid tissue (n = 6) were less sensitive than normal cells requiring up to 500 microU/ml bTSH to increase intracellular cyclic AMP and poorly differentiated thyroid cancer cells (n = 3) failed to respond to bTSH. Long-term human thyroid cultures of normal and follicular adenoma cells in the chemically-defined medium used for the FRTL-5 cells had absent fibroblast growth and continued in monolayer form without significant follicle formation. These cells remained highly sensitive to bTSH stimulation when tested after 4, 13, and 26 weeks of continuous culture. All such cell preparations failed to proliferate under conditions which favoured the rapid growth of the rat thyroid cells. These data demonstrated that while thyroid cell culture conditions described in the literature do not permit proliferation of human thyroid cells, they do allow an assessment of their functional state in vitro which may lead to a further understanding of thyroid cell pathophysiology. PMID- 3002669 TI - Selective alpha 2 receptor blockade facilitates the insulin response to adrenaline but not to glucose in man. AB - The adrenergic nervous system plays an important role in the control of insulin release and animal work suggests that this is mediated by way of alpha 2 adrenoceptors. A specific alpha 2 adrenoceptor antagonist (idazoxan) is now available for use in man and we have studied its effects on insulin release in normal volunteers (a) during the infusion of adrenaline (0.05 micrograms/kg/min) and (b) after an intravenous dose of glucose (20 g). The infusion of adrenaline alone had no significant effect on insulin release while in the presence of idazoxan, insulin release was markedly stimulated by adrenaline. Despite this, adrenaline-induced hyperglycaemia was unaffected by pretreatment with idazoxan. In the second study, pretreatment with either idazoxan or a specific alpha 1 antagonist (prazosin) had no significant effect on either glucose tolerance, glucose-induced insulin release or glucose-induced suppression of glucagon. Intravenous glucose also had no significant effect on pancreatic polypeptide levels. Therefore the effect of adrenaline on insulin release is mediated by way of inhibitory alpha 2 adrenoceptors in the pancreas, while the release of insulin in response to glucose in a resting subject is independent of the alpha adrenergic system. PMID- 3002668 TI - Catecholamine, vasoactive intestinal peptide and thyrotrophin-dependent cAMP levels display a different sensitivity to iodothyronines in both normal and pathological human thyroid cells in culture. AB - As the interactions of iodothyronines on adrenergic and vipergic receptors are not clear, the effect of exogenous T3 and T4 on catecholamine- and VIP-induced cAMP accumulation in human normal thyroid cells after eight days of primary culture has been investigated. To evaluate the effect of endogenous iodothyronines, the response of the adenylate cyclase system to isoprenaline, adrenaline, VIP, and TSH was studied during a 10 d period. T3 and T4 were unable to modify the catecholamine- and VIP-induced cAMP accumulation in human normal thyroid cells after 6-8 days of culture, while the response to TSH was significantly inhibited. In cells cultured from thyrotoxic tissue, the response of the adenylate cyclase system to catecholamines and VIP, during a 10 d primary culture, showed a behaviour similar to controls. TSH responsiveness was negligible up to the fourth day of culture, while in normal cells a response to all the agonists was present from the beginning. In view of the lack of effect of iodothyronines on catecholamine- and VIP-induced cAMP accumulation, and of the superimposable behaviour of the response to catecholamines and VIP in normal and hyperthyroid cells during the first days of culture, we can conclude that iodothyronines do not directly modify the response of the adenylate cyclase system to adrenergic and vipergic stimulation in human thyroid follicular cells. The lack of responsiveness to TSH of cells obtained from hyperthyroid tissue during the first 4 d of culture, associated with normal responsiveness to catecholamines and VIP, points to a possible involvement of biogenic amines and neuropeptides in sustaining such hyperthyroid states. PMID- 3002670 TI - Factors affecting the secretion of 18-hydroxycortisol, a novel steroid of relevance to Conn's syndrome. AB - A recently developed radioimmunoassay for direct measurement of 18 hydroxycortisol (18-OH-F) in plasma and urine has been used to study the physiology of this newly described steroid in normal subjects. Plasma levels of 18-OH-F show a circadian variation similar to that of cortisol and are increased and suppressed by administration of ACTH and dexamethasone respectively. A clear increase was observed in response to sodium restriction but despite this, angiotensin II infusion failed to cause a rise in 18-OH-F levels and a possible explanation is discussed. The results are interpreted in terms of a proposed biosynthetic pathway involving 18-hydroxylation of cortisol during a second passage through the adrenal gland. PMID- 3002672 TI - Long-term enalapril--a new converting enzyme inhibitor--in the treatment of mild to moderate essential hypertension, results of a worldwide multiclinic study. Comparing two ways of analyzing data. AB - The antihypertensive effect of enalapril maleate, a new converting enzyme inhibitor, was evaluated in a multiclinic, double-blind, randomized study in patients with mild to moderate essential hypertension. The analyses were done in two ways, with patients who violated the entry criteria of the protocol excluded, and according to the intention to treat principle. Enalapril in dosages of 10 to 40 mg daily administered alone or concomitantly with hydrochlorothiazide was compared to propranolol (80 to 240 mg daily) alone or concomitantly with the diuretic. The study showed that enalapril significantly lowered both systolic and diastolic blood pressure. At each timepoint measured in the course of 26 weeks of therapy, the patients in the enalapril group consistently had greater decreases in blood pressure than patients in the propranolol group although not always significantly. The enalapril treatment group had a decrease in the mean arterial blood pressure of 22.2 mmHg compared to the propranolol group of 17.9 mmHg at the end of the study. These results were similarly independent of the way the data were analyzed. Fewer patients in the enalapril group required the addition of hydrochlorothiazide to maintain optimal control of blood pressure. Enalapril was found to be safe and well tolerated over the long-term of 48 weeks. Side effects such as leukopenia and taste perversions believed to be sulfhydryl-related were not encountered. The occurrence of rash and proteinuria was rare. Thiazide induced hypokalemia, hyperuricemia and hyperglycemia appeared to be attenuated by enalapril. The favorable efficacy and side-effect profile provide the basis for enalapril to be a drug of choice when initiating antihypertensive therapy. PMID- 3002671 TI - Accurate localization of insulinoma using percutaneous transhepatic portal venous sampling--usefulness of simultaneous measurement of plasma insulin and glucagon levels. AB - By means of percutaneous transhepatic portal venous sampling (PTVS), plasma insulin and glucagon levels were determined simultaneously at various sites of the hepatic portal venous system in two patients with insulinoma and four patients without. In the two cases of insulinoma, an obvious rise of insulin concentration was observed at the vicinity of the tumour, while glucagon levels were not elevated in the blood samples showing an insulin peak. In the remaining four cases without evidence of insulinoma, significant step-ups of plasma insulin occurred in the splenic vein as well, but were accompanied with concomitant elevation of plasma glucagon levels in the same blood samples. Thus, by measuring insulin alone, false positive data may frequently be obtained when PTVS is performed on patients with suspected insulinomas. The simultaneous measurement of insulin and glucagon might be helpful in avoiding such errors. PMID- 3002673 TI - Effects of alterations in cell phenotype and hypokalemia on sodium-potassium pump activity in rabbit vascular smooth muscle. AB - We investigated in cell culture, how alterations in phenotype accompanying proliferation of rabbit aortic smooth muscle and chronic hypokalemia could affect the Na,K pump. Total rubidium-86 uptake as well as ouabain and frusemide sensitive uptake into cells was measured in physiological salts solution (PSS), PSS containing 5% foetal calf serum and PSS containing foetal calf serum plus 15 microM monensin. In physiological salts solution 90% of the rubidium-86 uptake into contractile or synthetic state cells was frusemide-sensitive and less than 8% ouabain-sensitive. Total and frusemide-sensitive rubidium-86 uptakes, measured in PSS or PSS containing foetal calf serum were similar in cells cultured and maintained in the contractile phenotype, cells in the synthetic phenotype and those which had recently reverted from the synthetic to contractile phenotype. When cells were sodium loaded in the presence of monensin and foetal calf serum, ouabain-sensitive rubidium-86 uptake was 50% higher in cells which were maintained in culture in the contractile phenotype. Frusemide-sensitive rubidium 86 uptake was similar in each cell phenotype. To examine how cell culture in hypokalemic media would affect the Na,K pump, we determined ouabain-sensitive rubidium-86 uptake in the presence of monensin plus foetal calf serum in cells incubated for 24 hours in low and normal potassium containing culture media. Ouabain-sensitive uptake was 20% higher in cells cultured in a 0.76 mM potassium medium than in those cultured in 5.4 mM potassium medium. Frusemide-sensitive rubidium-86 uptake was unaffected. These results demonstrate that 'maximal' Na,K pump activity in sodium-loaded cells is reduced when cells change from the contractile to synthetic phenotype. This reduction appears only very slowly reversible when cells revert from the synthetic to contractile phenotype. Prolonged hypokalemia increases 'maximal' activity of the Na,K pump. PMID- 3002674 TI - Effect of atrial natriuretic factor [ANF (Arg 101--Tyr 126)] on kallikrein and cyclic GMP in the renovascular hypertensive rat. AB - The intravenous injection of an ED50 natriuretic dose (1 microgram) of synthetic ANF decreases blood pressure by 61 +/- 6 mmHg in 2-K, 1-C, and of 45 +/- 6 mmHg in 1-K, 1-C hypertensive rats, which was positively correlated with its initial level only in the 2-K, 1-C group. The hypotensive response lasted longer in the latter (greater than 40 min) than in normotensive sham-operated rats. No difference in duration was seen between 1-K, 1-C hypertensive and its uninephrectomized normotensive controls. The diuretic response to ANF was higher in 2-K, 1-C rats. No hematocrit changes were observed in any group. ANF induced a rise in urinary kallikrein in all groups but the 1-K, 1-C. Urinary kallikrein excretion was positively correlated with natriuresis in normotensive but not in hypertensive groups. ANF induced an increase in urinary cGMP excretion in all groups but the 1-K, 1-C, and an increase in plasma cGMP in the normotensive sham operated animals. Our results suggest that the fall in blood pressure induced by synthetic ANF could be due to vasodilatation, a drop in cardiac output cannot, however, be eliminated. Whether the hypotensive effect of ANF is mediated by cGMP remains to be demonstrated. PMID- 3002675 TI - The pituitary-adrenocortical axis and the immune system. AB - Recent findings indicate that interactions between the pituitary-adrenocortical axis and the immune system involve more than simply the effects of glucocorticoid hormones. This altered view has resulted from the observations that: 1) cells of the immune system have receptors for and are directly acted upon by ACTH and endorphins, and 2) the immune system is an important non-pituitary source of these peptide hormones. In this chapter, while we review in a cursory way the older findings with glucocorticoid hormones, we concentrate on the newer developments which suggest that leukocyte- and pituitary-derived ACTH and endorphins perform regulatory functions within and between the immune system and the pituitary-adrenocortical axis. PMID- 3002676 TI - Histopathology of the pituitary. AB - The cytoplasmic secretory granules of corticotrophs in the anterior pituitary are basophil in trichrome stains and periodic acid-Schiff positive in the histochemical stain for glycoprotein due to their content of the glycosylated 16 000 N-terminal fragment of the precursor protein proopiomelanocorticotrophin (POC). The granules show a positive immunocytochemical reaction to antibodies raised against ACTH, beta-endorphin and N-terminal fragments of POC. A small subset of corticotrophs contains immunoreactive alpha MSH in addition. Immunocytochemistry shows the corticotrophs to constitute about 15-20% of the anterior pituitary cells arranged both singly and in clumps. They are distributed in the median wedge and anteriorly, laterally and posteriorly adjacent to the pars nervosa which is often 'invaded' by corticotroph basophils. The alpha MSH subset is prominent in the rudimentary intermediate lobe and is scattered anteriorly in the pituitary of the human fetus. Crooke cell hyalinization is associated with pathologically maintained hypercortisolaemia and with glucosteroid therapy. The hyalinization is demonstrated in ultrastructure to be due to massive accumulation of intermediate cytoplasmic filaments 7-8 nm in diameter that are normally present in only small number. The change is associated with a varying degree of loss of secretory granules. In untreated Addison's disease there is a marked increase in the number of corticotrophs, many of which are arranged in distended alveoli to form micronodules. The vast majority of cases of pituitary-dependent Cushing's disease and all cases of Nelson's syndrome are associated with a basophil or chromophobe adenoma. These give a positive immunocytochemical reaction with anti-ACTH, beta-endorphin and N-terminal POC. In ultrastructure the cells of the chromophobe adenomas are seen to contain sparse secretory granules that are usually smaller than those in the chromophil adenomas. There are only very few reports of pituitary-dependent Cushing's disease found to be due to immunocytochemically confirmed corticotroph hyperplasia with or without a corticotroph adenoma. A few cases have been described in which the adenoma cells show Crooke's hyalinization, associated in one example with secretion of a big ACTH found more typically in ectopic ACTH secreting tumours. A group of cases due to corticotroph adenoma has been reported whose excessive ACTH secretion is reduced by treatment with the dopamine agonist bromocriptine, in which it is suggested that the tumour cells arise from a subset of corticotrophs of pars intermedia origin.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3002677 TI - Histopathology of the human adrenal cortex. AB - The morphological features of the adult human adrenal cortex are described with particular respect to changes induced by alterations in function of the hypothalamo-pituitary axis. The occurrence of nodules in the normal and hyperplastic cortex (Cushing's and Conn's syndromes) is discussed in relation to the diagnostic problems that they still pose. Explanations based on the normal mechanisms of functional zonation in the cortex are provided for the different cell types which comprise cortical neoplasms associated with various syndromes of hypercorticalism (Cushing's, adrenogenital and Conn's syndromes), together with morphological and functional criteria to distinguish adenomas from carcinomas. PMID- 3002678 TI - The CRFs and their control: chemistry, physiology and clinical implications. AB - The 41-amino acid CRF fulfils all the criteria for a corticotrophin releasing factor, although considerable evidence suggests that other factors, particularly VP, also play a physiologically significant role in controlling ACTH release. Although human CRF has now been identified as a 41-residue peptide, most studies to date have used oCRF-41 in their exploration of the physiology and pathology of the hypothalamic--pituitary--adrenal axis. Low doses of oCRF-41 appear to be safe, and for specific tests of the readily-releasable pool of ACTH and related peptides 100 micrograms is a practical dose for most purposes. Although serious side-effects have only been noted at doses above 100 micrograms, it is reasonable to monitor all patients administered CRF-41 with great care, and in particular to be alert to hypotension, especially in patients with corticosteroid deficiency. There is little doubt that, in combination with the standard insulin-tolerance test, the CRF test is a useful means of diagnosing hypothalamic or portal dysfunction in patients with secondary adrenal failure. However, in the diagnosis and differential diagnosis of Cushing's syndrome, the role of the CRF test remains unclear. In normal subjects, a high basal cortisol level usually inhibits the response to CRF, such that a greatly enhanced response is suggestive of pituitary-dependent Cushing's syndrome. In patients with diagnosed ACTH-dependent Cushing's syndrome, an absent response to CRF predisposes towards an ectopic source of ACTH. However, there are exceptions in all directions, and it is uncertain whether the CRF test will prove of greater value than the traditional procedures, such as the dexamethasone suppression test. The differential diagnosis of depression and Cushing's disease may be its greatest value. In terms of treatment, there are as yet few data on the usefulness of CRF in expediting recovery of the pituitary-adrenal axis following long-term suppression, such as in patients with Cushing's syndrome treated by removal of a unilateral adenoma or trans-sphenoidal microadenomectomy. It is possible that such treatment may eventually be a useful application of CRF, although data are not yet available. PMID- 3002679 TI - Cushing's syndrome. AB - Cushing's syndrome remains one of the most challenging problems in clinical endocrinology. Cushing's disease is caused in the majority of cases by basophil pituitary microadenomas which may be successfully treated by trans-sphenoidal hypophysectomy. Treatment with metyrapone or o,p'-DDD can always induce a clinical remission but not a cure, and neurotransmitter therapy may be effective in a minority of cases. Pituitary irradiation cures about half of cases in the long-term and may be used for surgical failures. Tumours producing ectopic ACTH are frequently benign, small and occult and may produce a syndrome clinically indistinguishable from Cushing's disease. Biochemical investigations cannot absolutely distinguish pituitary from ectopic sources of ACTH and therefore body CT scanning and percatheter venous sampling are essential diagnostic investigations. Tumour localization may result in resection and complete cure, although even small tumours may have a malignant potential. Adrenal tumours are readily diagnosed by plasma ACTH measurement and adrenal CT scanning. Adrenal adenomas are cured by adrenalectomy. Carcinomas may be treated by a combination of adrenalectomy, radiotherapy and o,p'-DDD, but long-term prognosis is poor. PMID- 3002680 TI - Adrenocortical insufficiency. AB - Adrenocortical insufficiency causes difficulty in diagnosis and morbidity out of proportion to its rarity, because of the non-specific, multi-system nature of the clinical features. Most of these are due to cortisol deficiency. Prominent features are well-known ones such as weight loss and asthenia, and hypoglycaemia. Less prominent in recent accounts are those due to failure of cellular sodium export and to vasopressin excess, which are frequent and clinically significant. For this reason, the clinical features of isolated ACTH deficiency, isolated glucocorticoid deficiency and Addison's disease overlap greatly. In addition, cortisol deficiency has secondary endocrine effects, e.g. glucocorticoid reversible hypothyroidism, hyperprolactinaemia and hypercalcaemia. Further overlap between the various steroid insufficiency syndromes occurs because of the association of various organ-specific autoimmune endocrinopathies with Addison's disease. Over 80% of Addison's disease is of the autoimmune type, though almost any systemic destructive process can cause similar steroid insufficiency. Demonstration of adrenal insufficiency requires various combinations of tetracosactrin adrenal stimulation tests, and hypoglycaemia or equivalent tests, if the cause is ACTH deficiency but the correct test can only be chosen to suit a firm clinical diagnosis. The treatment of adrenocortical insufficiency is described. PMID- 3002681 TI - Diagnosis and treatment of primary aldosteronism and isolated hypoaldosteronism. PMID- 3002683 TI - Colonic disease in the elderly. PMID- 3002682 TI - The adrenal cortex and virilization. AB - The physiological control of adrenal androgen secretion has not been definitively established. However, there is evidence to suggest that a dexamethasone suppressible factor other than ACTH may have a specific role to play. The majority of patients with idiopathic hirsutism (hirsutism associated with regular menstruation) have findings suggestive of adrenal androgen excess, including enhanced androgen responsiveness following administration of metyrapone, and respond to treatment with dexamethasone, 0.5 mg given each night. Patients with idiopathic hirsutism have elevated androgens but normal oestrogen and gonadotrophin levels. In contrast, while patients with polycystic ovary syndrome (PCOS) also demonstrate evidence of adrenal androgen excess, these patients have elevated oestrone levels and gonadotrophin secretion is abnormal. Approximately 50% of patients with PCOS treated with dexamethasone resume regular menstruation. Oestrone excess appears to be primary to the abnormal gonadotrophin secretion and to the development of PCOS. In non-obese patients with PCOS elevated oestrone appears to occur as a consequence of the availability of the excessive amounts of its immediate precursor, androstenedione, an androgen mainly of adrenal origin. Androstenedione is converted to oestrone in fat. Obese amenorrhoeic subjects have normal androstenedione values but elevated oestrone levels with abnormal gonadotrophin secretion as seen in PCOS. These findings indicate that abnormal gonadotrophin secretion is associated with elevated oestrone levels whether these occur as a consequence of excessive adrenal androgen secretion, or the excessive conversion of normal amounts of available androstenedione. Patients with idiopathic hirsutism and elevated androstenedione levels but normal oestrone values appeared to be protected against the development of PCOS by relatively poor conversion of androstenedione to oestrone. It is likely, therefore, that if patients with idiopathic hirsutism gain additional adipose tissue, elevated oestrone levels will result and PCOS will develop. These observations explain the frequent association of PCOS and obesity. There is a close clinical association between elevated androgen levels and hirsutism and between elevated oestrone levels and menstrual disturbances. However, some patients with amenorrhoea but without hirsutism may demonstrate marked elevations of androgens and oestrone, the correction of which leads to the resumption of regular ovulation. This presentation, 'amenorrhoea with cryptic hyperandrogenaemia', is probably explained by diminished sensitivity of androgen receptors.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3002684 TI - The effect of cytomegalovirus infection on T lymphocytes after allogeneic bone marrow transplantation. AB - The influence of cytomegalovirus (CMV) infection on peripheral T lymphocyte repopulation was studied in 59 bone marrow transplant (BMT) recipients who received either cyclosporin A (CyA) or methotrexate (MTX) as prophylaxis for acute graft-versus-host disease. We used monoclonal antibodies and single- or double-marker immunofluorescence for the quantitation of T4+, T8+ and HNK1+ T cell subpopulations. CMV infection was serologically diagnosed by an enzyme linked immunosorbent assay (ELISA), and by viral cultures and histological studies. Among the 52 patients who were evaluable for CMV infection, one had a primary infection and 24 had CMV reactivation/reinfection after BMT. In the latter patients, increases to supranormal levels were observed in T8+ T cells and HNK1+ T cells, both in patients on CyA and in patients on MTX. Double-marker immunofluorescence revealed that the two markers were largely expressed by the same cells, which therefore had the T8+ HNK1+ phenotype. In addition, the very small subset of T4+ HNK+ T cells was slightly, but consistently, increased in the patients with CMV reactivation/reinfection. CMV infection did not influence the numbers of T4+ HNK1- and T8+ HNK1- T cells. The long-lasting presence of large numbers of T8+ HNK1+ T cells in patients who had CMV reactivation/reinfection suggests a continuing interaction between the virus and the immune system of its host. PMID- 3002685 TI - Differentiated HL60 promyelocytic leukaemia cells have a deficient myeloperoxidase/halide killing system. AB - Following induction of differentiation by incubation with 1.25% dimethylsulfoxide (DMSO), cells of the HL60 promyelocytic leukaemia cell line acquire certain characteristics of the mature polymorphonuclear leucocyte (PMN) including the ability to produce oxygen radicals and to phagocytose opsonized bacteria. However, these cells are unable to fix 125I during phagocytosis and are only able to kill phagocytosed microorganisms (C. albicans and S. aureus) to a small degree compared to mature PMN. Further, release of myeloperoxidase (MPO) from cytoplasmic granules occurs to approximately 20% of control levels after 6 days culture with DMSO, and drops to negligible levels by 7 days. These data suggest an immature or inactive MPO/peroxide/halide killing system. Insensitivity to the cyclooxygenase pathway inhibitor indomethacin suggests that there may also be a defect in this pathway. PMID- 3002686 TI - Transferrin receptor bearing cells in the peripheral blood of patients with rheumatoid arthritis. AB - Activated, proliferating lymphocytes are a feature of rheumatoid arthritis. They are present both in the synovial membrane and in the peripheral circulation. The expression of transferrin receptors(TFR) is a good marker of cellular proliferation. This study shows increased levels of circulating TFR-bearing lymphocytes in patients with rheumatoid arthritis (RA). The TFR+ population contains a disproportionately large number of T4+ cells, leading to a high T4:T8 ratio (5:1 in the TFR+ population, compared to 2:1 in the total circulating pool of lymphocytes). This reflects the pattern found in the rheumatoid synovium and suggests that lymphocyte activation in RA may be an extra-articular phenomenon. The TFR+ population also contains a range of non-T cells, including B cells, and a population bearing phenotypic similarities to natural killer (NK) cells. PMID- 3002687 TI - A B-cell hybridoma product inhibiting antibody secretion via 5'-nucleotidase. AB - A mouse spleen cell/plasmacytoma fusion designed to generate hybridomas making monoclonal antibodies to human lymphocyte 5'-nucleotidase yielded three hybridomas secreting material which inhibited about 50% of human and mouse lymphocyte ecto 5'-nucleotidase activity. The inhibition proved not to be due to antibody but to material of molecular weight 44,000 +/- 7,000 which was not part of an immunoglobulin molecule, although it may be related to a B cell Fc gamma receptor. In a haemolytic plaque assay this material inhibited the production of IgG but not IgM antibody by spleen cells of mice immunized with dinitrophenylated keyhole limpit haemocyanin. By contrast, IgG production by pokeweed mitogen stimulated human tonsillar lymphocytes (assessed by reverse haemolytic plaque assay) was partially inhibited only by ascitic fluid of one of the hybridomas. The factor was called BAN (B cell anti 5'-nucleotidase). The suppressive action of BAN on IgG production was blocked by antibodies against 5'-nucleotidase suggesting that the lymphocyte enzyme may be acting as a BAN receptor. PMID- 3002688 TI - Cytomegalovirus (CMV): specific lymphokine production in congenital CMV infection. AB - The studies reported here were designed to examine cytomegalovirus (CMV)-specific lymphokine production in infants with congenital CMV infection to elucidate the role of the efferent limb of the immune response to this virus. Mononuclear cells (MNC) from adult control donors and congenitally infected infants were stimulated with mitogen (PHA) or antigens (CMV, mumps), and the supernatants were assayed for production of migration inhibitory factor (MIF) and interferon (IFN). Concordance was observed between lymphocyte proliferative responses to CMV antigen and production of MIF in both control donors and congenitally infected infants. PHA-stimulated cultures from both controls and patients resulted in immune-specific IFN-gamma production in all cases. CMV-stimulated MNC from controls produced IFN when lymphocyte proliferation responses to the antigen were present, whereas those with absent proliferation did not produce IFN. CMV-induced IFN was detected in several patients who had reduced lymphocyte proliferative responses to CMV. The predominant species of IFN stimulated by CMV was neutralized by anti-IFN-gamma, suggesting that CMV induced primarily immune specific IFN-gamma. In some cases, IFN activity was also reduced to a lesser degree by anti-IFN-alpha, suggesting either the presence of IFN-alpha or of cross reacting antigenic determinants shared by the two species of IFN. Mumps viral antigen induced primarily IFN-alpha regardless of the immunological status of the donor. We conclude that lymphokine production in congenital CMV reflects the state of activation and/or proliferation of CMV-specific T helper cells rather than an intrinsic defect in the efferent limb of the immune response. PMID- 3002689 TI - Cytomegalovirus infection in cardiac transplant recipients associated with chronic T cell subset ratio inversion with expansion of a Leu-7+ TS-C+ subset. AB - Lymphocyte subsets were analysed in 18 patients during the first 3 years after cardiac transplantation. The patients received Cyclosporin A and prednisolone for maintenance immunosuppression. Serological evidence of active cytomegalovirus (CMV) infection was found in 13 cases (72%), and in 12 of these an inversion usually of the T helper/T suppressor-cytotoxic ratio (TH/TS-C) was detected. T subset inversion usually preceded the diagnostic rise in CMV antibody titre. In 69% of patients with CMV the TH/TS-C ratio remained inverted throughout follow-up (245-951 days). Persistent T subset inversion was not found in all five patients who lacked serological evidence of active CMV. Chronic inversion consisted of an average increase in TS-C of 152% and an average decline in TH cells of 31% as compared to CMV negative patients. The proportion of lymphoid cells reacting with a phenotypic marker for natural killer (NK) cells (Leu-7) was increased by 83%. These alterations were also reflected in the absolute numbers of cells with these markers. Two-colour immunofluorescence analysis revealed that the expanded TS-C population present during chronic inversion was predominantly Leu-7+. As TS-C+ Leu-7+ cells in healthy persons may be hyporesponsive NK cells, a sustained increase in this cell type in allograft recipients could further reduce immunocompetence, thereby predisposing to superinfection or malignancy. PMID- 3002690 TI - Immunity to herpes simplex virus in children receiving treatment for acute lymphoblastic leukaemia (ALL). AB - Children receiving anti-leukaemic therapy often become susceptible to severe herpesvirus infection. To investigate specific immunity to herpes simplex virus (HSV) in patients with acute lymphoblastic leukaemia (ALL), in first remission and receiving maintenance chemotherapy, we measured their HSV specific T responder cell frequency (RCF) and their in vitro anti-HSV antibody production. Concurrently, we determined their serum anti-HSV antibody titres and their lymphocyte response to phytohaemagglutinin (PHA). Neither the presence of anti HSV antibody in serum, nor the HSV-RCF correlated with the in vitro anti-HSV antibody response or the PHA response. This suggests that antigen-specific immune responses in patients being treated for ALL may be impaired independently of mitogen-induced proliferative responses. PMID- 3002692 TI - Effects of phenoxybenzamine on responses to some receptor agonists and calcium in vitro. AB - Noradrenaline-induced contractions of the rabbit and rat isolated aorta and guinea-pig spleen strips were inhibited by concentrations of phenoxybenzamine which did not affect responses to calcium. This may suggest a specific action on alpha-adrenoceptors. However, analysis of noradrenaline concentration-effect curves in guinea-pig spleen indicated that 1 mumol/l phenoxybenzamine should have reduced the available receptor population to 6% of control, but data from radioligand binding experiments on the same tissues using [3H]-prazosin indicated a reduction of the receptor population to only 82% of control. The reduced responsiveness observed in the organ bath study after phenoxybenzamine pretreatment, whilst not apparently related to effects on voltage-dependent calcium channels, could be due to the actions of phenoxybenzamine on other (non receptor) processes such as receptor-operated calcium channels. Maximal contractile responses to histamine in rabbit isolated aorta but not those in guinea-pig isolated ileal preparations, were depressed by concentrations of phenoxybenzamine which depressed responses to calcium. Phenoxybenzamine produced parallel rightward shifts of curves to carbachol in guinea-pig ileal preparations but only depressed maximal responses to the agonist in higher concentrations which reduced responses to calcium. On the basis of the results obtained with calcium it is possible that the effects of phenoxybenzamine on receptor-mediated responses could be produced through the actions of this antagonist at less specific sites such as voltage-dependent calcium channels for histamine in rabbit aorta and carbachol in guinea-pig ileum. For alpha-receptor mediated responses in aortic and splenic strip preparations and for histamine-mediated responses in guinea-pig ileum, the actions of phenoxybenzamine may reflect an interaction of the antagonist with receptor-operated calcium channels. PMID- 3002691 TI - Analysis of T cell activation with a non-mitogenic anti CD3 antibody and the phorbol ester TPA. AB - We used a non mitogenic anti CD3 antibody, termed VIT3, to study the signals required for the activation of normal resting T lymphocytes. Besides being not mitogenic, this antibody completely inhibits mitogen induced proliferative responses. In the presence of the phorbol ester 12-O-tetradecanoylphorbol-13 acetate (TPA), however, VIT3 induces DNA replication and cell proliferation comparable to PHA responses. In addition, T cells cultured with VIT3 plus TPA but not with VIT3 or TPA alone express high levels of interleukin 2 (IL-2) receptors and transferrin receptors. This co-stimulation appears to be accessory cell independent. Purified T cells respond equally well to VIT3 plus TPA as do unseparated mononuclear cells and addition of non-T cells has no enhancing effect. We conclude that the IgM antibody VIT3, although non-mitogenic by itself, still delivers a first and for the activation essential signal. In resting T cells this signal does not induce demonstrable anti-Tac antibody binding nor does it lead to a fully developed proliferative response in the presence of recombinant IL-2. Together with a second signal provided by TPA it serves, however, as a potent inducer of T cell growth. PMID- 3002693 TI - Effects of opioids on the ACh-induced contraction of the toad rectus. AB - The inhibitory effect of morphine, nalorphine, oxymorphone, naloxone, naltrexone, N-methylnaloxone, levorphanol, levallophan, dextrorphan, dextrallophan, levo methadone, dextro-methadone, pethidine, leu-enkephalin and U-50 488 on the acetylcholine (ACh)-induced contraction of the toad rectus was investigated. The dose-response curves obtained in the absence and presence of increasing concentrations (0.1-3.2 or 10-320 mumol/l) of each opioid were subjected to three types of inhibition model discrimination, namely competitive, noncompetitive and uncompetitive. The results show that the opioids, except morphine, nalorphine and levorphanol, inhibit the ACh-induced contraction by multiple modes of action. Except for the morphinans, the opioids inhibit competitively indicating that they are able to compete with ACh for cholinergic receptors at the nicotinic site. The opioids also inhibit noncompetitively (except oxymorphone and N-methylnaloxone) and uncompetitively (except oxymorphone) indicating the possible existence of separate opioid binding sites and the combination of the opioids with the ACh receptor complex, respectively. The effect of morphine and levorphanol on the ACh induced contraction varies with the concentration of the two opioids. At concentrations below 80 mumol/l both opioids tend to inhibit the contraction while at concentrations of 80 mumol/l and above there is no apparent effect. Nalorphine has no effect on the contraction at the concentrations (10-320 mumol/l) employed in the study. An apparent structure-activity relationship between the phenanthrenes and the various binding sites is noticeable; so also is the difference in the activity between the dextro and levo-isomers of levorphanol and levallorphan indicating that the receptors exhibit stereospecificity. PMID- 3002694 TI - An investigation of the receptors involved in the coronary vasodilatory effect of adenosine analogues. AB - The coronary vasodilatory effect of stable adenosine analogues is mediated by adenosine A2 receptors. These coronary receptors differ from A2 receptors found in other tissues. PMID- 3002695 TI - Alpha 2-adrenoceptors in rat tail artery. PMID- 3002697 TI - A functional defect in the early phase of the immune response observed in patients with hemophilia A. AB - In search of a functional immunological defect in patients with hemophilia A, we investigated the early phase of the immune response and found a deficiency in the monocyte-T cell interaction after in vitro exposure to a bacterial antigen. T cell proliferation in response to Escherichia coli-pulsed autologous monocytes was significantly (P less than 0.025) reduced in hemophiliacs [mean dpm +/- SEM (n = 25): 9036 +/- 2600] as compared to healthy controls [mean dpm +/- SEM (n = 24): 17,812 +/- 2985]. This functional defect was expressed in a severe form (antigen-induced monocyte-T cell interaction less than or equal to 2000 dpm) in both HTLV III antibody-positive and -negative patient populations. Significantly decreased T-helper/T-suppressor-cell ratios (mean dpm +/- SEM: patients 1.47 +/- 0.13; controls 2.04 +/- 0.11; P less than 0.001)--but without a concomitant reduction in the absolute number of T-helper cells--could also be observed. PMID- 3002696 TI - Effect of herpesvirus infections on T-lymphocyte subpopulations and blastogenic responses in renal transplant recipients receiving cyclosporine. AB - The immunosuppressive effects of three herpesviruses--cytomegalovirus (CMV), Epstein-Barr virus (EBV), and herpes simplex virus (HSV)--were assessed in 29 renal transplant recipients treated with cyclosporine and prednisone. The ratios of Leu 3-positive ("helper-inducer") to Leu 2-positive ("suppressor-cytotoxic") T lymphocytes in peripheral blood were only moderately and transiently decreased during primary CMV infection, with or without concurrent reactivated EBV and HSV infections. This effect was due to an increase in absolute numbers of Leu 2 phenotypic and decrease in Leu 3-phenotypic T cells and was associated with symptomatic viral illness. Reactivated CMV infection alone or together with reactivated EBV and HSV infections resulted in less significant alterations in T cell subsets than did primary CMV infection. Lymphocyte blastogenesis was not significantly altered during the herpesvirus infections. The data suggest that cyclosporine treatment inhibits the activation of suppressor cells and depression of cellular immune function that have been associated with herpesvirus infections in renal transplant recipients undergoing conventional immunotherapy. PMID- 3002698 TI - Extramedullary hematopoiesis. Demonstration by transmission and emission computed tomography. AB - Extramedullary hematopoiesis occurring in the thorax is a well-known complication of a number of various blood dyscrasias, and its appearance with planar Tc-99m sulfur colloid marrow scintigraphy and transmission computed tomography (CT) has been described previously. The authors present a case of thoracic extramedullary hematopoiesis evaluated by CT and emission computed tomography (ECT). PMID- 3002699 TI - Calculation of relative glomerular filtration rate and correlation with delayed technetium-99m DMSA imaging. AB - The relative renal uptake of Tc-99m DMSA was compared with the relative glomerular filtration rate (GFR) in ten patients with serum creatinines ranging from 0.3 to 2.5 mg/dl. Relative GFR was based on the renal uptake of Tc-99m DTPA determined by two methods: 1) integrating the counts from 1 to 3 minutes postinjection and correcting for background. 2) Totalizing the individual renal counts in a single 15-second frame from 2:45 minutes to 3:00 minutes postinjection and correcting for background. The two methods of determining relative DTPA uptake showed excellent correlation, r = 0.98. Relative DMSA uptake determined at 24 hours post-injection using computer-assisted regions of interest showed excellent correlation with the relative GFR determined by either the integral or single-frame method, r = 0.98. The addition of background subtraction for the DMSA images at 24 hours did not improve the correlation. PMID- 3002700 TI - From "grass" roots to clinical utility. PMID- 3002701 TI - Changes in blood pressure and body fluid volumes during diuretic therapy in patients with essential hypertension who receive enalapril. AB - We evaluated the effect of additional chlorthalidone therapy on blood pressure and body fluid volumes in 10 patients with essential hypertension who did not respond to chronic converting enzyme inhibition with enalapril. Values assessed after 3 days and 6 weeks of combined enalapril and chlorthalidone therapy were compared with initial values during enalapril monotherapy. After 3 days the mean arterial pressure (MAP), plasma volume (PV), blood volume (BV), and extracellular fluid volume (ECFV) decreased. There was a positive correlation between the percentage decreases in MAP and BV. After 6 weeks the MAP decreased further, but the decreases in PV, BV, and ECFV were less pronounced. At this time there was a positive correlation between the percentage decreases in MAP and ECFV. Our results support the hypothesis that contraction of the ECFV is an antihypertensive mechanism of diuretics. The antihypertensive effect of diuretics is enhanced during converting enzyme inhibition, while the body remains protected against volume deficits, possibly by the lower blood pressure itself. PMID- 3002702 TI - The pharmacokinetic and pharmacodynamic profiles of the thromboxane A-2 receptor blocker BM 13.177. AB - The pharmacokinetics and pharmacodynamics of BM 13.177 were investigated in eight healthy men who received a single oral dose of 800 mg on the first day and seven equal doses in 8-hour intervals on the second to fourth days. Pharmacodynamic effects were measured ex vivo by the testing of platelet functions such as shape change, aggregation, and [3H]serotonin release. The maximum serum concentration of 6.6 or 6.7 mg/L was achieved within 1.6 hours after the first dose and within 1.5 hours after multiple doses, respectively. Afterwards, BM 13.177 was eliminated in urine with a terminal elimination t1/2 of 0.84 or 1.0 hours after single and multiple dosing, respectively. The inhibition of platelet function showed the same close correlation with the serum concentrations of BM 13.177 after single and after multiple doses. Apparently, BM 13.177 induces neither refractoriness to BM 13.177 nor desensitization of the platelet thromboxane receptor. Because BM 13.177 was also well tolerated without subjective or objective side effects, this drug appears to be useful in evaluating the clinical benefit of thromboxane receptor blockade. PMID- 3002703 TI - Mathematical modelling of stimulus-secretion coupling in the pancreatic B-cell. IV. Dissociated time course for the glucose-induced changes in distinct Ca2+ movements. AB - A mathematical model for the interaction between Ca and cyclic AMP in the process of glucose-stimulated insulin release from the pancreatic B-cell is presented. This model, which takes into account such features as the stratification of cytosolic Ca and the process of Ca-stimulated Ca release from the vacuolar system, entails a dissociated time course for the effect of glucose upon distinct Ca movements. Thus, whereas glucose is postulated to cause a rapid and sustained facilitation of Ca pumping into intracellular organelles, the glucose-induced stimulation of Ca influx into the B-cell is assumed to represent a delayed and discontinuous phenomenon. Such a dissociated time course allows for a fair simulation of both 45Ca efflux and insulin release from islets perifused in the absence or presence of Ca and exposed to a sudden increase in extracellular glucose concentration. PMID- 3002704 TI - Lipoprotein receptors and atherosclerosis. AB - Mammalian cholesterol metabolism is governed by two key features of the steroid nucleus: it is poorly soluble in plasma and it cannot be degraded in animal tissues. Cells which require the lipid import it through their cytoplasmic membranes in the form of solubilized lipid-protein complexes, and a similar export mechanism is essential in order to maintain cellular homoeostasis. The translocation is mediated by high affinity receptors which, under normal circumstances, operate in close synchrony. Changes in the nature of the lipoprotein ligand or of the cell itself may disturb this balance and lead to the pathological sterol accumulation which characterizes atherosclerotic lesions. All body tissues engage in this traffic in cholesterol but the liver is the single most important organ since it has the capacity both to synthesize large quantities of the lipid and to excrete it from the body via a variety of receptors designed to maintain peripheral sterol levels within safe limits. PMID- 3002705 TI - Elevation of sodium levels in the cerebral ventricles of anaesthetized dogs triggers the release of an inhibitor of ouabain-sensitive sodium, potassium ATPase into the circulation. AB - The present studies are designed to determine whether a ouabain-sensitive inhibitor of Na+,K+-stimulated ATPase is released into the circulation when the Na+ levels are elevated in the cerebral ventricles in pentobarbital anaesthetized dogs. Na+-pump activity was estimated in the plantar and dorsal branches of the lateral saphenous veins by using the 86Rb-uptake method. Infusion of the cerebral ventricles with the artificial cerebrospinal fluid (CSF) containing normal Na+ (0.15 mol/l) for over 120 min resulted in significant increases in only ouabain sensitive 86Rb-uptake. In contrast, when the ventricles were perfused with the CSF containing high Na+ (0.3 mol/l) for similar periods, there were significant reductions in the ouabain-sensitive as well as in ouabain-insensitive 86Rb-uptake by the blood vessels. Similar blood vessels from a separate group of dogs were incubated for 120 min in the plasma samples collected from the above groups in which normal or hypertonic Na+ solutions were infused. The data showed that ouabain-sensitive 86Rb-uptake (but not the insensitive component) was significantly inhibited only in those vessels which were incubated in the plasma samples that were obtained at 120 min after perfusion of the cerebral ventricles with CSF containing high Na+ (0.3 mol/l). These studies have provided direct evidence which would suggest that the elevation of Na+ levels in the cerebral ventricles would precipitate the release of an inhibitor of the ouabain-sensitive Na+-pump into the circulation, perhaps due to an activation of Na+-sensitive and/or osmo-sensitive sites in the circumventricular organs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002706 TI - Age-related differences in granulocyte chemotaxis and degranulation. AB - In the present study we have demonstrated that human peripheral blood granulocytes from elderly subjects exhibit reduced chemotaxis and degranulation in response to stimulation with fMet-Leu-Phe. Cyclic AMP levels in non-stimulated cells were not significantly different (mean +/- SEM): 4.8 +/- 0.6 and 3.9 +/- 0.4 pmol/10(7) cells for young and elderly respectively. Stimulation with fMet Leu-Phe (5 X 10(-7) mol/l) for 30s induced the production of cyclic AMP: 8.4 +/- 0.5 and 6.7 +/- 0.3 pmol/10(7) cells for young and elderly. These findings, together with those previously published by us, suggest that cyclic AMP production and cellular functions which this molecule participates in are reduced in cells from elderly individuals. PMID- 3002707 TI - Inhibition by omeprazole of adrenocortical response to ACTH: clinical studies and experiments on bovine adrenal cortex in vitro. AB - Omeprazole, a substituted benzimidazole, is a potent inhibitor of gastric acid secretion which is currently being evaluated in patients with peptic ulcer and Zollinger-Ellison syndrome. Drugs which possess an imidazole nucleus have previously been shown to inhibit cortisol release from the adrenal cortex, secondary to inhibition of mitochondrial cytochrome P-450 dependent hydroxylation reactions. In a double-blind placebo-controlled crossover study in healthy male volunteers, omeprazole (60 mg daily for 7 days) did not alter basal cortisol levels. The peak cortisol response to ACTH stimulation was significantly reduced. Cortisol levels 60 min after ACTH were 824 +/- 27 nmol/l on omeprazole (mean +/- SEM), and 929 +/- 35 on placebo (P less than 0.005). In vitro, omeprazole caused a concentration-dependent inhibition of ACTH-stimulated cortisol release from isolated bovine adrenal cells (ED50 = 20 micrograms/ml). This was associated with a decrease in deoxycortisol synthesis. Therefore, unlike some other imidazole containing drugs, the inhibitory effects of omeprazole are not entirely due to steroid 11 beta-hydroxylase inhibition. Substantial inhibition occurred at omeprazole concentrations which are higher than plasma levels normally achieved in clinical use. However, impairment of adrenocortical function may occur in patients on long-term high dose omeprazole treatment for Zollinger-Ellison syndrome. PMID- 3002709 TI - Simplified method for the isolation, identification, and characterization of Staphylococcus epidermidis in epidemiologic studies. AB - A simplified method for the isolation, identification, and characterization of Staphylococcus epidermidis from humans is described. Swabs of the nose and skin are cultured on mannitol salt agar. Isolated colonies not producing acid from mannitol (presumptive coagulase-negative staphylococci or micrococci) are then inoculated onto purple agar containing erythromycin and glycerol. All colonies growing on this medium are then replicated onto media that tests for the production of phosphatase, the production of acid from trehalose, and susceptibility to four antibiotics. All S. epidermidis sensu stricto are confirmed by the API Staph-Ident system. As a result, Staphylococcus aureus and all other coagulase-negative staphylococci are effectively identified and eliminated from further study and only strains of S. epidermidis are left for further characterization. Of the 252 isolates from 48 cultures of the nares and the fingers, 112 (44%) were eliminated during different stages of this isolation and identification procedure. The antibiotic susceptibility data further distinguished those isolates in the predominant API biochemical profile number. This scheme has applications in the early stages of either ecologic or epidemiologic studies of this important nosocomial pathogen. PMID- 3002708 TI - Cardiovascular effects of adenosine. AB - The results, briefly summarized above, indicate that adenosine could be a physiologically important modulator of several aspects of cardiovascular regulation. Most cells are equipped with adenosine receptors. These receptors are of at least two subtypes which can be defined by the relative agonist potency. At these adenosine receptors, methylxanthines, including caffeine and theophylline, act as competitive antagonists. The role of adenosine antagonism, as a mechanism behind the cardiovascular effects of these xanthines, was recently reviewed (Fredholm, 1984). The concentrations of adenosine are low during resting conditions, but may be raised substantially by, for example, hypoxia, ischaemia and increased mechanical or biochemical work. The adenosine levels can also be raised by drugs, including uptake inhibitors such as dipyridamole. Already the concentrations of adenosine that occur during basal conditions are sufficient to produce significant effects, for example, on blood-flow. When the concentrations are raised the importance of endogenous adenosine becomes even greater. Adenosine may not only be of physiological significance but may also be pharmacologically important. First, there are several drugs that may act by affecting the levels of adenosine or by influencing its receptors. Second, the possibility exists that adenosine itself could be used clinically. For example, adenosine may be an attractive alternative to sodium nitroprusside or nitroglycerin when controlled hypotension is to be achieved. Adenosine may also be used to preserve blood platelets during extracorporal circulation or to produce selective regional vasodilatation. Both the physiological and pharmacological aspects are subject to intense study in several laboratories. PMID- 3002710 TI - Rotavirus in nasopharyngeal secretions of children with upper respiratory tract infections. AB - Nasopharyngeal secretions from 30 infants and children presenting with respiratory tract infection, were tested for rotavirus antigen. Two of 30 children with signs and symptoms of seromucoid nasal secretions, cough, and low grade fever were positive for the antigen. Nasopharyngeal secretions may play a role in the spread of this infection. PMID- 3002711 TI - The carboxyl fragment released by bacterial collagenase from human type I procollagen: antibodies to the propeptide determinants. AB - A protocol is offered for the isolation of the carboxyterminal propeptides of human type I procollagen and the development of an antibody specific for these propeptides. Type I procollagen was harvested from the media of cultured human fibroblasts. Digestion with bacterial collagenase released a carboxyterminal fragment that was isolated by ion-exchange chromatography. The fragment contained telopeptides joined to propeptides and could be cleaved by a carboxyl procollagen peptidase. Rabbit antibodies raised to the collagenase-generated fragment were sequentially adsorbed on affinity columns of the reference antigen and human type I collagen. The antibody obtained was shown by sensitive radioimmunoassays to recognize conformational carboxyl propeptide determinants and not to react with triple helical and telopeptide determinants of human type I collagen. Indirect immunofluorescence and indirect immunoperoxidase staining of cultured fibroblasts localized the antigen in the cytoplasm, at the cell surface, and in the extracellular matrix. A radioimmunoassay with the same antibody has reported altered concentrations of the antigen in the sera of patients with diseases affecting collagen metabolism (Taubman, et al., 1976; Savolainen et al., 1984; Carey et al., 1985). PMID- 3002712 TI - Synthesis of PGE2, collagenase and tissue factor by fibroblast substrains: substrains are differentially activated for different metabolic products. AB - Metabolic heterogeneity has previously been demonstrated for cloned fibroblast populations. It is unclear whether a "high producer" or "activated" phenotype for one cell product reflects general metabolic activation or whether activated phenotypes for different metabolic markers segregate independently among cloned populations. We examined human foreskin fibroblast substrains, isolated by limiting dilution cloning, for heterogeneity in biosynthesis of PGE2 and collagenase (stimulated by Interleukin 1-containing mononclear cell supernates), and tissue factor. Synthesis of these products varied 5- to 10-fold among 16 substrains isolated from two parent lines (AT and WB). Metabolic phenotypes (high versus low levels of synthesis) were stable for the substrains over multiple weeks of culture. In neither group of substrains was there a correlation between levels of stimulated PGE synthesis and stimulated collagenase synthesis (r = 0.24 and 0.29 for AT and WB substrains, respectively). Similarly, tissue factor generation did not correlate with either PGE production (r = 0.12 and -0.38 for AT and WB substrains, respectively) or collagenase synthesis (r = 0.17 and 0.21 for AT and WB substrains, respectively). The discordance between high producer phenotypes for the different products was observed even when all three products were measured in the same culture. Activated phenotypes for different metabolic markers thus appear to segregate among cells independently. The process of connective tissue activation by immune cell-derived mediators may depend on the constituent makeup of the responding connective tissue cell population. PMID- 3002713 TI - Activation of macrophages. AB - The role of macrophages is essential in the development of a normal immune response. Not only are these cells involved in the initiation of this response by presenting antigens to lymphocytes and by producing Interleukin I, but they also participate in the various phenomena of cellular co-operation and regulation. It is also evident that macrophages can act as cytotoxic effector cells, especially against micro-organisms and tumor cells. This last function is restricted to activated macrophages. The aim of this review is to summarize our present knowledge concerning this "macrophage activation". PMID- 3002714 TI - Mechanisms and functions of the oxygen radicals producing respiration of phagocytes. AB - Respiratory burst is due to the activation of a membrane bound NADPH oxidase induced by perturbation of the plasma membrane during phagocytosis or following interaction between the cell surface and a number of environmental stimuli. It refers to the increase in the non-mitochondrial O2 consumption with a concomitant production of different reactive species (superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen ...). The effects of the respiratory burst depend on the intensity and combination of the different actions which are defensive, toxic, activatory and modulatory of the inflammatory process. PMID- 3002715 TI - Distribution of a COOH-terminal amino acid extension of liver fructose-1,6 bisphosphatase among rodent species. AB - We have recently established from sequence analysis that rat liver fructose-1,6 bisphosphatase contains a 24-26 residue extension beyond the COOH-terminal amino acid of other mammalian fructose-1,6-bisphosphatases that results in an increased subunit molecular weight (Rittenhouse et al. (1983) J. Biol. Chem. 258, 7648 7652). In the present work the distribution of the COOH-terminal extension of fructose-1,6-bisphosphatases was tested by subunit molecular weight analysis of the enzyme immunoprecipitated from liver extracts. Of all rodent species tested, including several Muridae other than Rattus; only the enzyme from animals of the genus Rattus was found to have the extension. Further studies on the distribution of the enzyme extension could provide a simple tool to study the phylogeny of the genus Rattus. PMID- 3002717 TI - 3'-nucleotidase activity in procyclic and bloodstream stages of Trypanosoma rhodesiense. AB - Both procyclic and bloodstream-derived trypanosomes of Trypanosoma rhodesiense exhibit a nucleotidase activity which is capable of hydrolyzing 3' ribonucleotides. Nucleotidase activity in assays employing 5'-nucleotide substrates is not detectable in preparations of either phenotype. The 3' nucleotidase activity in lysates of procyclic trypanosomes is pelletable at 100,000 g, whereas that activity from bloodstream forms is operationally soluble under the conditions employed. The 3'-nucleotidase activities of both stages share the following characteristics: pH optimum of 8.5, substrate preference for purine ribonucleotides, resistance to commonly used phosphatase inhibitors, including fluoride, tartrate and molybdate ions. Both activities are reversibly inhibited by EDTA and reactivated by Co2+ ions. As visualized by fine-structure cytochemical methods, 3'-nucleotidase activity was distributed over the entire surface of procyclic trypanosomes, but not bloodstream parasites bearing an intact surface coat. PMID- 3002716 TI - Activities and some properties of 5'nucleotidase in isolated hamster fat cells. AB - Kinetic parameters and the possible cellular location of 5'nucleotidase were studied in isolated hamster fat cells. Measurements of the enzyme were performed using 5'IMP instead of 5'AMP as substrate. The apparent Vmax and Km values obtained support the view that two forms of 5'nucleotidase, having different affinities, are present in hamster fat cells. The major form was located in the outer surface of plasma membrane and the other one inside the cell. The effects of ADP, adenosine and inosine on the enzyme were studied. ADP and adenosine were competitive inhibitors. The enzyme was not modified by inosine. PMID- 3002718 TI - Metastatic pancreatic islet cell carcinoma causing manifestations of glucagon and gastrin hypersecretion. PMID- 3002719 TI - ORF 13904, a new long-acting vaginal contraceptive. AB - A sulfonated polystyrene polymer, ORF 13904, was discovered in our laboratory to be a highly effective vaginal contraceptive in the rabbit model and to possess a unique mode of action. It was found to be non-spermicidal even at high concentrations, yet it greatly impeded sperm penetration of cervical mucus in vitro. When a gel formulation containing 5% ORF 13904 was administered to rabbits intravaginally, the mean number of fetal implants was reduced dramatically even when coitus was delayed for up to 8 hours or when multiple matings at different time intervals were permitted. The non-spermicidal qualities and extended duration of action of this compound represent major breakthroughs in the development of vaginal contraceptives. PMID- 3002720 TI - A comparative study of Neo Sampoon, Ortho Vaginal Tablets and Emko Vaginal Tablets in Accra, Ghana. AB - Neo Sampoon is an effervescent contraceptive vaginal tablet manufactured in Japan that contains 60 mg of the spermicide menfegol. Ortho Vaginal Tablets (OVT) and Emko Vaginal Tablets (EVT), both containing 100 mg of the spermicide nonoxynol-9, were manufactured in the USA. The three products were compared in a randomized clinical trial conducted at the family planning clinics of the Korle-Bu Teaching Hospital and the Kotobaabi Polyclinic in Accra, Ghana. Three-hundred volunteers participated. At 12 months, the life-table pregnancy rates were 9.6, 11.3 and 12.5 per 100 women in the Neo Sampoon, OVT and EVT groups, respectively (p greater than 0.10). More EVT than Neo Sampoon or OVT users discontinued because of discomfort as well as for other product-related reasons (p less than 0.01). The most common reason for discontinuation was the temporary absence of sexual partner, with more than 40% of the women overall terminating for this reason. The 12-month life-table continuation rates per 100 women were higher for the Neo Sampoon group (62.4) than the OVT group (48.6) or the EVT group (38.5) (p less than 0.01). The effectiveness of the three products seems to be similar, but Neo Sampoon and OVT appear to be more acceptable than EVT in this Ghanaian population. PMID- 3002722 TI - The PBB episode in Michigan: an overall appraisal. AB - Polybrominated biphenyls (PBB) were used as a fire retardant. In common with other halogenated hydrocarbons, PBBs are lipophilic and resistant to chemical and metabolic degradation. Cattle on about 25 Michigan farms were exposed to as much as 250 g per head of PBB when it was accidentally mixed in cattle feed in 1973 to 1974. Livestock exposures several orders of magnitude lower occurred on several hundred other farms because of carryover and equipment contamination in feed mills. Approximately 85% of the Michigan population received some exposure to PBB because dairy product marketing involves mixing milk from many farms. A few cases of high human exposure, which may have been as great as 10 g, occurred when residents of the more highly exposed farms consumed their own products. Although numerous clinical signs and pathological changes were reported in exposed cattle, only anorexia, lacrimation, emaciation, hyperkeratosis, and kidney damage were confirmed in controlled studies. The acute toxicity of PBB in laboratory animals is low, but a variety of subacute effects have been reported. Induction of microsomal enzymes, enlargement and histopathological changes of the liver, fetotoxicity, and immunosuppression are among the more significant. Epidemiological studies of exposed humans have revealed no pattern of clinical signs or symptoms that were related to PBB exposure. A complete evaluation of the human consequences of exposure to PBB await the conclusion of long-term epidemiological studies. PMID- 3002721 TI - Benign mixed tumor of salivary gland origin presenting as a mediastinal mass. AB - Benign mixed tumor is a common neoplasm of the salivary gland. It may rarely occur as an intrabronchial lesion. A case of a mixed tumor of salivary gland origin presenting as a mediastinal mass is described. PMID- 3002723 TI - Recognition of physiological receptors by anti-idiotypic antibodies: molecular mimicry of the ligand or cross-reactivity? PMID- 3002725 TI - 201Tl and 67Ga uptake in malignant fibrous histiocytoma of the heart. AB - A patient with malignant fibrous histiocytoma of the heart is described who was initially presented with a left atrial tumor. Subsequent 201Tl and 67Ga scintigraphy showed massive uptake of the tracers by the tumor and the pattern of uptake was thought to reflect underlying necrosis and hemorrhage within the tumor. PMID- 3002724 TI - Serum angiotensin-converting enzyme and lysosomal enzymes in tobacco workers. AB - The activities of angiotensin-converting enzyme (ACE) in people with extrinsic allergic alveolitis (EAA) have been both increased and decreased. These observations suggest that pulmonary macrophages or endothelial cells participate in the disease process. Exposure to molds in the tobacco industry has recently been suspected to be associated with chronic extrinsic allergic alveolitis. In the present study, we analyzed the serum activities of ACE and two lysosomal enzymes, beta-N-acetylglycosaminidase (NAG) and beta-glucuronidase (GLU), among 57 tobacco workers. The tobacco workers not exposed had serum ACE levels similar to those of the reference population workers not occupationally exposed to dust (N = 127). The tobacco workers' serum levels of NAG (16.0 +/- 2.0 units/L and GLU (2.4 +/- 0.7 units/L) were higher than among the referents (NAG, 9.1 +/- 2.0 units/L; GLU, 1.0 +/- 0.6 units/L; p less than 0.01). Fifteen tobacco workers with respiratory symptoms compatible with pulmonary diseases caused by organic dust had a trend toward increased ACE, NAG, and GLU levels. The mean level of ACE in serum was higher among the workers with (25.8 +/- 4.5 units/L) than among those without pulmonary fibrosis (20.7 +/- 7.5 units/L; p less than 0.025). The mean ACE level was also higher among workers with the highest exposure to molds (24.6 +/- 7.1 units/L) compared to those with the mildest exposure (18.3 +/- 5.7 units/L; p less than 0.05). Tobacco workers with or without antibodies against one or more microbes had similar mean levels of ACE, NAG, and GLU. All of these findings indicate that raw tobacco dust and its contaminants may cause allergic or toxic reactions or both, reflected by the serum levels of ACE, NAG, and GLU. PMID- 3002726 TI - [Breast cancer of the male. Analysis of our patient sample and the literature]. PMID- 3002727 TI - [Comparison of the physical parameters and handling properties of short-duration and middle-duration absorbable suture materials]. AB - In recent years the structure and coating of rapidly absorbable suture materials (RAS) have been changed: Dexon S----Dexon plus, Vicryl III----Vicryl IV. Medium fast absorbable sutures (MAS) have been introduced: Maxon and PDS. Being important clinical parameters, the knotting qualities were investigated, for instance knot holding capacity in dry and in NaCl or blood incubated sutures. The newest generations of RAS require more complicated configurations of knots than the initial products. After incubation in blood the knotting qualities improved only with Dexon plus. PMID- 3002728 TI - [Fibrolamellar cancer of the liver]. PMID- 3002729 TI - [Complex urogenital fistula surgically treated in the 2 University Hospital Centers of Abidjan. Value of viscero- and parietoplasties. Apropos of 1018 cases]. PMID- 3002730 TI - High resolution mapping of in situ hybridized biotinylated DNA to surface-spread Drosophila polytene chromosomes. AB - We describe a method of mapping genes or transcripts on polytene chromosomes by transmission electron microscopy. We present several applications which illustrate that, in favorable cases, the method has a resolution of ca. 10 kg, and that high resolution mapping of hybridization sites relative to bands and puffs can be achieved. We mapped sites of transcription for poly-(A) RNA and present evidence which shows that these sites are localized in some bands and puffs, but are also found in interbands. PMID- 3002732 TI - [Adenoid cystic carcinoma of the salivary gland: a ultrastructural and histochemical study]. PMID- 3002731 TI - Chromosomal localization of the Epstein-Barr virus (EBV) genome in Bloom's syndrome B-lymphoblastoid cell lines transformed with EBV. AB - The localization of the Epstein-Barr virus (EBV) genome in chromosomes of human B lymphoblastoid cell lines (LCLs) transformed with EBV, and the effect of EBV DNA on the level of sister chromatid exchange (SCE) in Bloom's syndrome (BS) B-LCLs, were examined with chromosomal in situ hybridization techniques using a 3H-EBV DNA probe. EBV DNA was detected in chromosomes 1-5 and 13-15 at specific G band regions in BS as well as in normal B-LCLs, regardless of SCE. Several chromosomal sites (1p31, 1q31, 4q22-24, 5q21, 13q21, 14q21) carrying EBV DNA seemed to be very characteristic in normal as well as in BS B-LCLs. There was no statistically significant difference in silver grain counts due to EBV DNA and their distribution in different chromosomes or groups among normal and BS B-LCLs with normal and high SCE. These findings strongly indicate that EBV infection did not introduce a correcting factor for BS SCE. PMID- 3002733 TI - [Current status of the treatment of endemic goiter and cretinism with oral iodized oil]. PMID- 3002734 TI - Bilateral adrenal hemorrhage during ACTH treatment of ulcerative colitis. Report of a case and review of the literature. AB - Adrenal hemorrhage is uncommon and usually associated with severe stress, sepsis, or anticoagulant therapy. The association of adrenal hemorrhage and acute ulcerative colitis is rare, and is probably related to exogenous therapy with ACTH. The case of a 29-year-old woman who was hospitalized with severe ulcerative colitis, treated with ACTH, and who developed bilateral adrenal hemorrhage is presented. The difficulties of diagnosis and management are discussed. A review of the relevant literature concerning the pathophysiology of adrenal hemorrhage is presented also. PMID- 3002735 TI - Sulbactam/ampicillin compared with cefoxitin for chemoprophylaxis in elective colorectal surgery. AB - In a prospective, randomized, comparative study, patients undergoing elective major colorectal surgery received four six-hour doses of either sulbactam (a beta lactamase inhibitor) with ampicillin (1 gm with 1 gm), or cefoxitin (2 gm) commencing at induction of anesthesia. The groups were well matched for age, sex, diagnosis, and surgical procedures. Three patients in the sulbactam group (N = 44), and four in the cefoxitin group (N = 48) developed significant wound sepsis. Minor wound sepsis occurred in an additional four sulbactam patients, and in five cefoxitin patients. There was no difference between the groups in deep sepsis or anastomotic leak rates (sulbactam, four patients; cefoxitin, seven patients). No serious side effects were recorded in either group. These results suggest that sulbactam combined with ampicillin provides a safe, effective alternative to cefoxitin for prophylaxis in colorectal surgery. PMID- 3002736 TI - Effect of exogenous mouse interferon on murine fulminant hepatitis induced by mouse hepatitis virus type 2. AB - We investigated the effect of exogenous mouse alpha- + beta-interferon produced by mouse L cells on the growth of mouse hepatitis virus type 2 (MHV-2) in the liver, the development of liver cell necrosis, and survival in murine fulminant hepatitis induced by MHV-2. Murine fulminant hepatitis was induced in 4-week-old male ICR mice by intraperitoneal inoculation of MHV-2. Mouse interferon (10(3) IU/mouse/day) was intraperitoneally injected every day. Exogenous mouse interferon suppressed both the growth of MHV-2 in the liver tissue and development of liver cell necrosis, and prolonged the survival. It was also found that the earlier mouse interferon was administered, the greater was the prolongation of survival. PMID- 3002738 TI - [Germ cell tumor of the testis--clinicopathologic analysis of 114 cases]. AB - The histologic features and clinical records of 114 cases of germ cell tumors of the testis proved by pathology in our hospital from 1951 to 1980 are reviewed. These cases were divided into six types: seminoma 49.1%, embryonal carcinoma 8.8%, yolk sac tumor 17.5%, choriocarcinoma 0.9%, teratoma 14.9% and mixed carcinoma 8.8%. The over-all incidence of metastasis observed on exploration was 41.2%, and in embryonal and mixed carcinoma it was the highest, each amounting to 80%. Site of metastasis was: inguinal and retroperitoneal lymph nodes 63.8%, mesenteric lymph nodes 6.4%, liver 12.8%, lung 8.5% and supraclavicular lymph nodes 8.5%. In the 24 followed cases, 13 were seminomas and 11 non-seminomatous germ cell tumors. 5 of the former and 9 of the latter died of the tumor. It is advised that, in addition to radical orchiectomy, supplementary radiotherapy to the original site in the testis and the retroperitoneal draining lymphatics be given to seminoma and retroperitoneal lymph node dissection be given to non seminomatous germ cell tumors except the pure choriocarcinoma to improve the result. PMID- 3002737 TI - Bile secretion in acute and chronic hypercalcemia in the cat. AB - The reported coincidence of primary hyperparathyroidism and cholelithiasis led us to investigate the effects of acute and chronic hypercalcemia on bile secretion in cats. Acute hypercalcemia (6-7 mmol/liter) was induced by an intravenous calcium infusion. Chronic hypercalcemia (3-4 mmol/liter) was induced and maintained for 8-10 weeks by treatment with subcutaneous vitamin D3, oral dihydrotachysterol, and feeding a calcium-rich diet. Bile secretion was then studied in acute experiments. We found that calcium concentrations in serum and hepatic bile were similar during all experimental normo- or hypercalcemic conditions (y = 1.12x - 0.85; r = 0.76). Biliary volume outputs were significantly decreased during both acute (P less than 0.002) and chronic (P less than 0.05) hypercalcemia compared with normocalcemic controls. Acute hypercalcemia also decreased total bile acid outputs (P less than 0.05), but had no effect on biliary bile acid concentrations. The inhibitory effect of acute hypercalcemia on biliary fluid and bile acid secretion was dose dependent and not antagonized by atropine. These findings suggest that calcium is secreted in hepatic bile at similar concentrations as present in the serum and that elevations of serum calcium concentration inhibit biliary volume and bile acid secretion in cats. Similar effects of hypercalcemia on bile composition in humans might promote calcium salt precipitation in bile. PMID- 3002739 TI - [Combination therapy of small cell anaplastic carcinoma of the lung and the role of prophylactic whole brain irradiation--report of 39 cases]. AB - A series of 39 cases of small cell anaplastic carcinoma of the lung treated by combination of chemotherapy and radiotherapy is reported. The role of prophylactic whole brain irradiation in this combination is emphasized. Apart from 2 cases whose treatment was interrupted, the chief symptoms of the other 37 were significantly improved by irradiation. The response rate was over 86% and the median survival was 8 months. The 2-year survival rates of the patients with or without brain irradiation (group A and B) were 26.7% and 9.1% respectively. None of the group A patients (15 cases) developed brain metastasis while 4 (18%) of the group B patients (22 cases) did. The results prove the validity of the prophylactic whole brain irradiation. In this series, the mortality of liver metastasis amounted to 38%. The authors believe that prophylactic liver irradiation may reduce the metastases into the liver and prolong the survival. PMID- 3002740 TI - [Effect of natural and recombinant interferons on the development of an antiviral state and on the activity of natural killers]. PMID- 3002741 TI - [3',5'-cyclic AMP-dependent protein kinase: the hybrid kinetic mechanism of the phosphotransferase reaction]. PMID- 3002742 TI - [Activation of Tn1 transposition in the functioning of the recF-dependent pathway of homologous recombination in Escherichia coli cells]. PMID- 3002743 TI - [cAMP-dependent mechanism of relaxation of vascular smooth muscle cells in hypoxia not associated with decrease in the Ca2+ level in myoplasma]. PMID- 3002744 TI - [Granular cell tumor of the common bile duct]. PMID- 3002745 TI - [HTLV-III and cardiopulmonary resuscitation]. PMID- 3002746 TI - [Serologic studies on the occurrence of the arteritis virus in the horse in West Germany]. PMID- 3002747 TI - [Malignant cell transformation in rats in vitro as affected by the variolovaccine virus]. AB - The capacity of the variolovaccine virus with low cytopathogenic activity to cause morphologic and malignant transformation of normal rat cells in vitro is detected. The transformed cells induced tumours in gamma-irradiated rats. PMID- 3002749 TI - Influence of cholesterol and unsaturated fatty acids on [125I]hCG binding to rat testicular membranes. AB - Incubation of testicular membranes with cholesteryl-hemisuccinate resulted in an increase of both the membrane microviscosity and [125I]hCG specific binding. 14C labelled cholesterol as well as fatty acids were effectively incorporated into membrane preparations. Insertion of unsaturated fatty acids (cis-vaccenic, linoleic, oleic, linolenic and arachidonic) into the membrane decreased its microviscosity under simultaneous disappearance of [125I]hCG specific binding. The latter phenomenon seems to be caused by an increase of nonspecific binding which was not due to the uptake of radio-activity from labelled gonadotropin by fatty acids or double bonds of fatty acids incorporated into testicular membranes or taken up by a resin Amberlite IRA-400. The disappearance of specific binding of [125I]hCG by the fluidizing action of unsaturated fatty acids can be reversed by the subsequent action of cholesterol. PMID- 3002748 TI - [Genome expression of endogenous type-B oncoviruses and carcinogenesis in the murine and human mammary gland]. AB - Data from literature and author's own experiments concerning conditions of the endogenous MuMTV genome expression and its influence on the organism are reviewed. Some findings on the expression in women of antigens related to the MuMTV antigens and antibodies to them are analyzed. PMID- 3002750 TI - TRH analogue with C-terminal thioamide group: rapid degradation by plasma and its biological effects. AB - L-pyroglutamyl-L-histidyl-L-proline thioamide--([Prot3]TRH), a new TRH analogue, has been previously found to have the same binding affinity to adenopituitary receptors as well as TSH and alpha-MSH releasing activities as native TRH. In this paper we report also the same time course of TSH response after i.p. injection of this compound (10 micrograms kg-1) to rats. Binding affinity to specific receptors in rat amygdala, cortex (frontal lobes), hypothalamus, striatum (order according to decreasing affinity) of both peptides was also similar. In contrast to TRH, however, [Prot3]TRH in doses 0.5 and 5 mg kg-1 i.p. did not affect sleeping time and breathing frequency in the rats during barbiturate anaesthesia. Surprisingly, human plasma degraded the new analogue much faster (T1/2 8.5 min) than native TRH (T1/2 30 min). [Prot3]TRH was also degraded faster in plasma of adult rats. Plasma of 6-day-old rat pups failed to degrade both peptides. It was concluded that the substitution of proline amide, for proline thioamide group in TRH molecule did not change binding affinity to receptors in the central nervous system, but decreased biological effectiveness in CNS and substantially decreased the resistance to degradation in human and rat plasma. PMID- 3002751 TI - Activity of 5'-nucleotidase, AMP deaminase, adenosine deaminase, acid and alkaline phosphatase and nucleotide pyrophosphatase in human thyroid. AB - The activity of 5'-nucleotidase, AMP deaminase, adenosine deaminase, acid phosphatase, alkaline phosphatase and nucleotide pyrophosphatase was assayed in human thyroid glands. The 5'-nucleotidase activity was higher than that of AMP deaminase which suggested that AMP undergoes degradation primarily as a result of dephosphorylation in thyroid tissue. A high acid phosphatase activity was noted as compared to that of alkaline phosphatase activity. In toxic goitre the increase in adenosine deaminase and acid phosphatase was observed together with the decrease in pyrophosphatase activity. PMID- 3002752 TI - Modulation of nuclear receptor affinity for 3,5,3'-triiodothyronine by nucleotide in vitro. AB - Nuclear non-histone protein fraction containing saturable receptors for thyroid hormone was prepared from rat liver by extraction with 0.3 mol l-1 NaCl. Inhibitory effect of ATP, UTP, GTP and CTP (1 mmol l-1 each) when co-incubated with the nuclear receptor and 3,5,3'-triiodothyronine was demonstrated. The experiments were conducted in the presence of 25 mmol l-1 Na2HPO4 in order to suppress the effect of the phosphate groups of nucleotide molecule by inorganic phosphate. All nucleotides inhibited (P less than 0.05 to 0.001) the specific binding of 3,5,3'-triiodothyronine to soluble receptors in the order ATP greater than CTP greater than GTP greater than UTP. In addition inhibitory effect of 0.5 mmol l-1 ATP on 3,5,3'-triiodothyronine binding to receptors was re-examined by Scatchard plot analysis. The data suggested that the diminution in the specific binding of receptors in the presence of ATP was due to an effect on the equilibrium association constant. The maximum binding capacity of receptors remained unchanged when the concentrations of ATP ranging from 0.1 to 1.0 mmol l 1 were used. PMID- 3002753 TI - Are endogenous glucocorticoids involved in short-term regulation of hepatic glycogen metabolism? AB - The release of endogenous corticosterone was stimulated by intraperitoneal administration of ACTH to conscious rats. Corticosterone had no reproducible stimulatory effect either on the activation of synthase or inactivation of phosphorylase. On the other hand, an induction of the synthesis of total synthase enzyme protein was observed which could be blocked by actinomycin D. In Sephadex filtered liver homogenates, a particularly impaired pattern of phosphorylase inactivation/synthase activation was observed during incubation at 20 degrees C in the ACTH-treated rats. PMID- 3002754 TI - Relaxin affects the shape of rat myometrial cells in culture. AB - The shape of rat myometrial cells in culture was altered by exposure to hormones. Oxytocin (107 microU/ml) significantly decreased mean cell length and area. Treatment of oxytocin-treated cells with relaxin (1.5 micrograms/ml), isoproterenol (10 microM) or (Bu)2 cAMP (1 mM) for 15 min resulted in a significant increase in cell length and area. The effect of relaxin was time dependent, with a significant increase in cell length by 1 min and in cell area by 3 min. Relaxin elicited a concentration-dependent increase in cell length and area between 0.1 and 2 micrograms/ml. The actions of relaxin correlate in time course and concentration dependence with its effects on cAMP elevation in the presence of 0.4 microM forskolin and on changes in myosin light chain kinase kinetic parameters in these cells. Myometrial cells in culture constitute a useful model system in which to correlate physical and biochemical effects of relaxin and other hormones. PMID- 3002755 TI - Erythropoietin production by fetal mouse liver cells in response to hypoxia and adenylate cyclase stimulation. AB - This study was done to investigate aspects of control of extrarenal erythropoietin (Ep) production. To this end we studied the effects of three stimuli of renal Ep production in the adult, i.e. hypoxia, cobalt, and activation of adenylate cyclase on Ep generation by cultured fetal mouse liver cells. The fetal liver was taken as a model for extrarenal Ep production because this organ is considered the predominant site of extrarenal Ep production. We found that Ep production by the cells increased as the oxygen concentration was decreased in the incubation atmosphere from 20% to 1%. Cobalt (10(-4)-10(-5) M) had no effect on Ep production. Activation of adenylate cyclase by forskolin (10(-5) M) or isoproterenol (10(-5) M) greatly enhanced Ep production. These findings indicate that the Ep-stimulating effect of cobalt is specific for the kidney. However, oxygen depletion and activation of adenylate cyclase seem to be more general stimuli in Ep-producing cells. Furthermore we found that Ep production in hypoxia correlated with lactate formation in the cultured liver cells. This finding suggests that Ep production in fetal livers under hypoxic conditions parallels the shift from aerobic to anaerobic cellular energy metabolism. PMID- 3002756 TI - The identification of a plasma membrane 3,3',5-triiodo-L-thyronine binding protein on the cultured Swarm rat chondrosarcoma chondrocyte and the lack of its up-regulation by insulin in vitro. AB - Primary cultures of Swarm rat chondrosarcoma chondrocytes were examined for the presence of T3 plasma membrane binding proteins and their possible regulation by insulin. Incubation of this tumor cell with [125I]T3 at 4 C yielded saturable and reversible binding of the radioligand. As assessed by LIGAND computer analysis, the binding data in one experiment revealed two classes of [125I]T3 binding sites with association constants of 2.2 X 10(9) M-1 and 4.8 X 10(6) M-1, and binding capacities of 4.9 X 10(3) and 1.9 X 10(6) sites per cell, respectively. Whole cells and a preparation enriched for plasma membrane were affinity labeled with N bromoacetyl-[125I]T3 (N-BrAc-[125I] T3), and the molecular weights of the radiolabeled proteins were analyzed both by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by Sephadex G-200 gel filtration chromatography. One major chondrosarcoma protein of 55,000 mol wt and two minor proteins of 47,000 and 33,000 mol wt bound N-BrAc-[125I]T3, suggesting that the mol wt of the T3 binding protein was 55,000. As assessed by isoelectric focusing, the 55,000 mol wt protein had an isoelectric point of 5.1. The radiolabeled 55,000 mol wt chondrosarcoma protein was immunoprecipitated with anti-T3 and anti-GH3 plasma membrane T3 antisera. The high degree of homology between this chondrocyte N-BrAc [125I]T3 binding protein and the protein on rat GH3 cells was demonstrated by a comparison of the peptide maps of Staphylococcus aureus V8 protease and elastase digested radiolabeled binding protein. Although culture of the chondrocytes in medium containing insulin resulted in an approximate 400% increase in plasma membrane [125I]insulin binding, no significant increase in [125I]T3 binding was observed. Thus, expression of the T3 plasma membrane binding protein was similar to that observed previously for the insulin-like growth factor-II receptor on these chondrosarcoma chondrocytes in not being influenced by the concentration of insulin in the culture medium. PMID- 3002757 TI - Down-regulation of parathyroid hormone (PTH) receptors in cultured bone cells is associated with agonist-specific intracellular processing of PTH-receptor complexes. AB - Exposure of cultured embryonic chicken bone cells to the PTH agonists bovine (b) PTH-(1-34) and [8Nle, 18Nle, 34Tyr]bPTH-(1-34)amide [bPTH-(1-34)A] reduces the subsequent cAMP response to the hormone and decreases the specific binding of 125I-labeled PTH to these cultures. To determine whether PTH receptor down regulation in cultured bone cells is mediated by cellular internalization of PTH receptor complexes, we measured the uptake of [125I]bPTH-(1-34) into an acid resistant compartment. Uptake of radioactivity into this compartment was inhibited by incubating cells at 4 C with phenylarsineoxide and unlabeled bPTH-(1 34). Tracer uptake into the acid-resistant compartment at any time was directly proportional to total cell binding at 22 C. Thus, it is likely that PTH-receptor complexes are internalized by bone cells. This mechanism may explain the loss of cell surface receptors after PTH pretreatment. To determine whether internalized PTH-receptor complexes are reinserted into the plasma membrane, we measured PTH binding and PTH stimulation of cAMP production after cells were exposed to monensin, a known inhibitor of receptor recycling. Monensin (25 microM) had no effect on PTH receptor number or affinity and did not alter PTH-stimulated cAMP accumulation. However, monensin (25 microM) incubated with cells pretreated with various concentrations of bPTH-(1-34) for 1 h potentiated the effect of the hormone to reduce subsequent [125I]bPTH-(1-34) binding and PTH-stimulated cAMP accumulation by more than 2 orders of magnitude. Chloroquine also potentiated PTH induced down-regulation of PTH receptors. By contrast, neither agent influenced PTH binding or PTH-stimulated cAMP production in cells pretreated with the antagonist bPTH-(3-34)A. Thus, monensin potentiated PTH receptor loss only in cells pretreated with PTH agonists, indicating that antagonist-occupied receptors may be processed differently from agonist-occupied receptors in bone cells. The data further suggest that the attenuation of PTH stimulation of cAMP production in treated bone cells may be, at least in part, due to receptor-mediated endocytosis of the hormone. PMID- 3002758 TI - The glycogenic effects of placental lactogen and growth hormone in ovine fetal liver are mediated through binding to specific fetal ovine placental lactogen receptors. AB - To examine the relative roles of placental lactogen (PL) and GH in fetal metabolism, we have examined the effects of ovine PL (oPL), ovine GH (oGH), and ovine PRL (oPRL) on glycogen metabolism in cultured ovine fetal hepatocytes and have examined the binding of these hormones to hepatic membranes from fetal and neonatal lambs. In ovine fetal hepatocytes, oPL (150 ng/ml-20 micrograms/ml) stimulated dose-dependent increases in [14C]glucose incorporation into glycogen (18-167%) and total cellular glycogen content (10-69%). oGH and oPRL also stimulated glycogen synthesis in fetal hepatocytes, but the potencies of these hormones were only 12% and 4% that of oPL. The dose-response curves of the three hormones were parallel, and their maximal effects were identical, suggesting a common mechanism of action. In hepatic membranes from fetal lambs, the maximal specific binding of [125I]oPL was 26.3% while the maximal specific binding of [125I]oGH was only 0.9-1.5%. The binding of [125I]oPL was saturable and reversible and varied with incubation time and temperature. Unlabeled oPL (1 ng/ml-5 micrograms/ml) caused a dose-dependent inhibition of the binding of [125I]oPL to fetal hepatic membranes, with half-maximal displacement of [125I]oPL by 5-7 ng unlabeled oPL/ml. oGH and oPRL caused parallel displacement of [125I]oPL, but with potencies only 2% and 0.1% that of oPL. Scatchard analysis of oPL dose-response curves indicated that the hormone bound to a single class of receptors with a dissociation constant of 1.1 X 10(-10) M. The maximal specific binding of [125I]oGH to hepatic membranes of neonatal lambs (20.1%) greatly exceeded the binding of oGH to fetal hepatic membranes. In addition, the potency of oGH in competing for [125I]oPL binding sites in neonatal liver greatly exceeded the potency of oGH in competing for [125I]oPL binding sites in fetal liver. Although the biological effects of both oPL and oGH in postnatal subprimate tissues may be mediated through binding to nonprimate GH receptors, the results of these studies suggest that the glycogenic effects of oPL in ovine fetal liver are mediated through binding to specific fetal oPL receptors. The relatively weak biological effects of oGH and oPRL in ovine fetal liver appear to be mediated through the binding of the hormones to fetal oPL receptors. The presence of specific, high affinity PL receptors in ovine fetal tissues provides a mechanism whereby oPL may function as a GH in the ovine fetus. PMID- 3002759 TI - Gonadotropin depression of adenosine triphosphate levels and interaction with adenosine in rat granulosa cells. AB - Cellular ATP levels were measured with the luceferin-luciferase enzyme method in incubated preovulatory granulosa cells in vitro from PMSG-treated immature rats. The ATP levels were depressed by both FSH and LH, FSH being the more effective. Adenosine enhanced the ATP levels about 3-fold, but the depressive effects of gonadotropins could not be overcome by the addition of adenosine. Uptake of adenosine in granulosa cells followed Michaelis-Menten kinetics, with a Km of 15.9 +/- 3.6 microM and a maximum velocity of 1.6 +/- 0.1 pmol/min X 10(5) cells. The half-time for uptake of adenosine was about 40 min. The maximal uptake of adenosine was lowered from 48 +/- 5 to 30 +/- 1 pmol/10(5) cells by FSH treatment of the cells. The basal secretions of cAMP and progesterone from the granulosa cells were slightly but significantly enhanced by adenosine alone. Adenosine markedly enhanced FSH-stimulated cAMP secretion, but not progesterone secretion. A nonmetabolizable adenosine analog, 2-chloro-adenosine, did not affect the ATP levels or the secretion of cAMP from granulosa cells. This study confirms previous observations that adenosine can increase ATP levels and amplify the response to gonadotropins in gonadal cells. A novel finding is that the levels of ATP in granulosa cells are markedly depressed by gonadotropins. It is speculated that this depression of ATP may be a factor in the metabolic control of granulosa cells. PMID- 3002760 TI - Low density lipoprotein receptor activity in the guinea pig adrenal cortex. I. Zonal characterization and response to adrenocorticotropin. AB - The binding of [125I]iodo-low density lipoprotein ([125I]iodo-LDL) to isolated membranes was determined for the outer (glomerulosa/fasciculata) and inner (reticularis) zones of the guinea pig adrenal cortex. Binding of [125I]iodo-LDL to membranes from both zones was found to be highly specific and dependent on the presence of divalent cations, such as calcium. Saturation analysis revealed that the maximum high affinity binding capacity was similar for both zones under control conditions (approximately 400 ng/mg protein). When ACTH was administered, however, the maximum high affinity binding capacity increased in the outer zone by at least 3-fold, while there was no significant change for the inner zone. It was also noted that dexamethasone administration decreased [125I]iodo-LDL high affinity binding activity to less than half the control level in the outer zone without altering the binding activity in the inner zone. The cholesterol content in the outer zone was 2-3 times greater than that in the inner zone. When ACTH was administered, the concentration of cholesterol decreased significantly in both zones. Thus, ACTH appeared to influence cholesterol metabolism in the outer and inner adrenocortical zones, but was able to modulate LDL receptor activity only in the outer zone. PMID- 3002762 TI - Evolution of prolactin and placental lactogen receptors in ewes during pregnancy and lactation. AB - The present paper describes a method of membrane preparation from ewe mammary gland using a sucrose cushion (1.3 M) to select smooth membranes; this results in a membrane preparation richer in PRL receptors than the microsomal preparation classically used. This method was used for the characterization and measurement of PRL and ovine placental lactogen (oPL) receptors in three organs of the ewe (mammary gland, liver, and adipose tissue). PRL receptors were measured by competition of iodinated human GH ([125I]hGH) with ovine PRL (oPRL). This hormone, which has both growth and lactogenic activities, appears to interact with PRL receptors with a higher affinity than oPRL itself and is a good probe for the determination of PRL receptors in the ewe. oPL receptors were measured by the specific binding of [125I]oPL. This hormone appears to bind exclusively to a somatogenic site in the ewe, since various GHs compete efficiently for binding, whereas oPRL is without effect. The evolution of PRL and oPL receptors was determined during pregnancy and lactation and at different periods after an estradiol and progesterone treatment, which provokes growth of the mammary gland and milk secretion. During pregnancy, PRL receptors increased in the mammary gland up to day 100. During the last trimester, receptor content remained stable, and a second increase occurred during early lactation. No additional significant changes were observed either for PRL receptors in liver or adipose tissue or for oPL receptors in any of the organs studied (mammary gland, liver, adipose tissue). Injections of large doses of estradiol and progesterone to nonpregnant ewes were able to reproduce effectively the pattern of PRL receptors observed during pregnancy, but had no effect on oPL receptor levels. These studies demonstrate the independence of PRL and PL receptor sites in the ewe and suggest a different hormonal regulation for each type of receptor. PMID- 3002761 TI - Influence of lactation upon pancreatic islet function. AB - The activity of adenylate cyclase, its responsiveness to NaF and forskolin, the activity of the protein kinases A and C, and the oxidation of exogenous D glucose, L-leucine, and L-glutamine were all higher in pancreatic islets removed from lactating, as distinct from nonlactating, rats. Yet, the release of insulin evoked by D-glucose or the association of L-leucine and L-glutamine was lower in the islets obtained from lactating animals. The lactation-induced decrease in secretory activity was not attributable to a change in the insulin content of the islets, was not corrected by exposure of the islets to theophylline or forskolin, and was also observed in response to stimulation by Ba2+. The rapidly exchangeable islet Ca pool, as estimated from the basal value for 45Ca net uptake, was severely decreased in lactation. Moreover, a hypoglycemic sulfonylurea, which stimulated islet 45Ca net uptake much more markedly in lactating than nonlactating animals, provoked, in association with 12-O tetradecanoylphorbol-13-acetate, a greater insulin output in lactating than nonlactating rats. It is speculated that the decreased secretory activity in islets removed from lactating rats may be accounted for, in part at least, by a decreased Ca content of the islet cells. PMID- 3002763 TI - Phosphorylation of three proteins in the plasma membrane of Y-1 adrenal cells by a membrane-bound adenosine 3',5'-monophosphate-dependent protein kinase. AB - Highly purified plasma membranes from Y-1 adrenal tumor cells were incubated with [gamma-32P]ATP with and without cAMP to determine whether endogenous protein substrates are phosphorylated by a cAMP-dependent protein kinase. Three membrane proteins (mol wt 270,000, 35,000, and 17,000) were phosphorylated without cAMP and, to a greater extent, with the nucleotide (0.05-20 microM). Phosphorylation was rapid (less than 60 sec), specific for cAMP, and occurred exclusively at serine residues. Two of the three proteins (35,000 and 17,000) were phosphorylated in whole cells under the influence of cAMP when the cells were incubated with [32P]orthophosphate. The cAMP-dependent protein kinase of these plasma membranes was not extracted by Triton X-100 (0.5% wt/vol) nor by KCl (0.4 M) but was almost completely extracted by the two agents together. By means of photoactivation of 8-azido-[32P]cAMP, it was found that both regulatory subunits RI and RII are present in the membranes. To the extent that the second messenger acts only by way of protein kinase enzymes, these changes in the three proteins are likely to be important in the responses of the plasma membranes of adrenal cells to ACTH. PMID- 3002764 TI - Ethylene dimethanesulfonate destroys Leydig cells in the rat testis. AB - Ultrastructural changes in the interstitial cells of the adult rat testis were studied up to 45 days after administration of a single dose (100 mg/kg) of the antifertility compound ethylene dimethanesulfonate (EDS). Most Leydig cells showed degenerative changes 12 h after treatment. Twenty-four and 48 h after injection, all Leydig cells observed showed gross degenerative changes. At 4 and 14 days, intact Leydig cells could not be identified in the interstitial spaces. Twenty-one days after treatment with EDS, small Leydig cells were visible, and at 45 days, Leydig cells appeared normal. The seminiferous epithelium appeared morphologically normal until 4 days after injection of EDS, when slight abnormalities were observed. At 14 and 21 days, the seminiferous epithelium was grossly abnormal, but at 48 days, spermatogenesis appeared normal. Twelve, 24, and 48 h after treatment, large quantities of material, presumably from dead Leydig cells, were observed within the macrophage cytoplasm. The predominant cell in the interstitial space 4 and 14 days after EDS was the macrophage. Inclusions from the dead Leydig cells within the cytoplasm of the macrophages had almost disappeared. LH receptors (hCG binding) in testicular homogenates were consistent with the cytological changes in Leydig cells. Receptor concentration was low at 24 h and was almost zero at 4 days. This change was accompanied by a decrease in serum testosterone to castrate levels by 2 days. The responses of the endocrine system to destruction of the Leydig cell by EDS, as monitored by serum FSH, LH, and testosterone, were slower than those after castration, indicating that the response to EDS reflects the time required to kill the Leydig cell rather than direct impairment of the steroidogenic pathway. These experiments demonstrate that Leydig cells can be specifically destroyed by a cytotoxic drug. The availability of a specific cytotoxic agent for Leydig cells offers further opportunities to study the interrelationships between the Leydig cell and the seminiferous tubule. PMID- 3002765 TI - Phorbol ester stimulates progesterone production by isolated bovine luteal cells. AB - The tumor promoter, phorbol 12-myristate-13-acetate (PMA), is known to modulate the response of several steroidogenic tissues presumably by activating a Ca++- and phospholipid-dependent protein kinase (protein kinase C). The presence of this kinase has been demonstrated in bovine corpus luteum, although its role in steroidogenesis by these cells is unknown. We report here the effects of PMA on progesterone production by the enzymically dispersed bovine luteal cells in vitro. PMA (1-50 nM) produced a dose- and time-related increase in progesterone production by the luteal cells. The maximum stimulation was achieved with 10 nM PMA. Higher concentrations of PMA led to a decline of steroidogenesis close to the basal level. A nonpromoting derivative, 4 alpha-phorbol 12,13-didecanoate had no effect. The PMA-induced stimulation of progesterone production was not associated with a change in the cAMP level. PMA added together with suboptimal doses of human CG, 8Br-cAMP, cholera toxin, or forskolin significantly increased the amount of progesterone produced. PMA as well as human CG-induced steroidogenesis was sensitive to cycloheximide inhibition. The conversion of exogenous pregnenolone or 25-hydroxycholesterol to progesterone was not altered by PMA. We conclude that PMA at nanomolar concentrations is able to stimulate progesterone production by bovine luteal cells and that the site of action of PMA is distal to the formation of cAMP but before the formation of pregnenolone. The observed effects of PMA in luteal cells are probably linked to its ability to activate protein kinase C, since a diacylglycerol could mimic the steroidogenic action of PMA. PMID- 3002766 TI - Prolactin (PRL), follicle-stimulating hormone, and luteinizing hormone are regulators of testicular PRL receptors in golden hamsters. AB - The ability of PRL, FSH, and LH to regulate testicular PRL receptors in golden hamsters was evaluated using a variety of experimental protocols. Exposure to a photoperiod of 5 h of light and 19 h of darkness (5L:19D) for 11 weeks precipitated a 94% reduction in content (femtomoles per testes) of testicular PRL receptors and, concomitantly, a decrease (P less than 0.05) in plasma PRL, but not LH or FSH. One pituitary gland under the kidney capsule in 5L:19D-housed hamsters increased (P less than 0.05) both the concentration (femtomoles per mg protein) and content of PRL receptors, as well as those of plasma PRL and FSH. Similar treatment in 14L:10D-housed hamsters produced comparable changes in plasma PRL and FSH without affecting PRL receptors. Injections of L-dopa for 7 days into hamsters housed in 5L:19D for 11 weeks significantly elevated serum FSH concentrations, had no measurable effect on serum PRL and LH, and induced a greater than 2-fold increase in PRL receptor levels. In a separate study, hamsters housed in 5L:19D for 12 weeks were injected for 3 days with 250 microgram ovine (o) PRL, 25 microgram oLH, or 5 microgram oFSH, and results were compared with vehicle-injected, 5L:19D- and 14L:10D-housed controls. Injections of oPRL and oLH increased (P less than 0.05) PRL receptor concentration and content, with PRL being more efficacious. No anti-oPRL antibodies were produced by oPRL injections. In this study, injections of oFSH were without effect on PRL receptors. To ascertain the effects of each hormone in the absence of other trophic influences, experiments were conducted in hypophysectomized hamsters injected daily for 3 days (2-4 days posthypophysectomy) with one of the following: 5 or 25 microgram oLH; 10, 50, or 250 microgram oPRL; or 1 or 5 microgram oFSH. Hypophysectomy reduced the concentration and content of PRL receptors by 85%, and treatment with 50 or 250 microgram oPRL increased (P less than 0.05) these low levels almost 3-fold. Again, no anti-oPRL antibodies were induced. Injection of 5 microgram oLH or 25 microgram oFSH also induced increases (P less than 0.05) in PRL receptors. Hypophysectomy reduced basal and hCG stimulated in vitro testicular testosterone production (nanograms per testes/4 h) to levels less than 20% of control values. None of the hormonal treatments affected (P less than 0.05) basal testosterone production in vitro.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3002767 TI - Maturation of the prolactin and proopiomelanocortin-derived peptide responses to ether stress and morphine: neurochemical analysis. AB - The hormonal and neurochemical responses to acute ether stress, morphine, and/or naloxone were analyzed in infantile (13-day-old) and prepubertal (36-day-old) male CD rats in an attempt to identify a possible neurochemical correlate(s) for the previously demonstrated requisite maturation of the PRL response to ether stress. Neuronal serotonin (5-HT), norepinephrine (NE), and dopamine (DA) activities were examined in the medial preoptic hypothalamic area (MPOH), medial basal hypothalamic area (MBH), and median eminence (ME). Ether stress increased plasma PRL, ACTH, and beta-endorphin-like immunoreactivity (beta end) as well as NE metabolism in the MPOH and MBH and neuronal 5-HT activity in the MBH, and decreased neuronal DA activity in the ME of prepubertal animals. Ether stress elicited similar changes in infantile animals, with the important exceptions that plasma PRL, neuronal 5-HT activity in the MBH, and neuronal DA synthesis in the ME were not affected at this earlier age. Morphine increased plasma PRL, ACTH, and beta end levels, elevated neuronal NE and 5-HT activities in the MPOH and MBH, and decreased DA synthesis in the ME in both infantile and prepubertal animals. Naloxone administration did not alter basal hormone concentrations or neuronal monoamine activity in any brain area, but did prevent all of the morphine-induced changes as well as the ether stress-induced changes in PRL, MBH neuronal 5-HT activity, and DA synthesis in the ME of prepubertal animals. In addition, naloxone augmented the ether stress-induced increases in ACTH and beta end in prepubertal rats. Indirect stimulation of 5-HT neurons by administration of the amino acid precursor of 5-HT, 5-hydroxytryptophan, resulted in decreased DA synthesis in the ME of infantile animals and increased plasma PRL levels in that age group, indicating that this portion of the neurochemical connection is already present in infantile animals. Furthermore, the 5-hydroxytryptophan induced increase in PRL was blocked by pretreatment with naloxone. The results demonstrate that both the ether stress- and morphine-induced increases in plasma PRL, but not in ACTH or beta end, are associated with increased neuronal 5-HT activity in the MBH and a decreased neuronal DA activity in the ME, that these are opiate receptor-mediated effects, and that infantile rats apparently lack a functional opiate-5-HT connection, which matures some time between days 13 and 36 postnatally. PMID- 3002768 TI - Antiidiotypic antibodies raised against anti-prolactin (PRL) antibodies recognize the PRL receptor. AB - Antiidiotypic antibodies capable of recognizing the PRL receptor have been raised against antibodies to ovine PRL (oPRL) and rat PRL (rPRL). Anti-oPRL or anti-rPRL antibodies were induced in rats or rabbits, respectively, and the immunoglobulin G (IgG) fractions were isolated by affinity chromatography on a Protein A Sepharose 4B column. Specific anti-oPRL antibodies were purified on an oPRL Sepharose 4B affinity column. The specific antibodies (0.5 mg/rabbit) in Freund's complete adjuvant were injected into rabbits at 2- to 3-week intervals for 3 months. Antiidiotypic antibodies against rat anti-oPRL specifically inhibited [125I]iodo-oPRL binding to the immunizing anti-oPRL antibody. Membrane binding of the antiidiotypic antibodies was determined by [125I]iodo-Protein A precipitation. It was found to be significantly higher toward membrane preparations rich in PRL receptors, such as liver membranes from pregnant mice or from estradiol-treated male rats or prostate membranes from adult rats. This membrane binding by the antiidiotypic antibody was competitively inhibited by the immunizing anti-oPRL antibody, suggesting that the idiotypic antibody may share common determinants with the PRL receptor. Recognition of the PRL receptor by the antiidiotypic antibodies was also assayed by indirect binding studies. After preincubation of the antiidiotypic antibodies with the membranes, the resulting complex was precipitated and the amount of free antiidiotypic antibody remaining in the supernatant estimated according to its ability to inhibit [125I]iodo-oPRL binding to anti-oPRL IgG. The lowest degree of inhibition of [125I]iodo-oPRL binding was achieved after incubation of the anti-idiotypic IgG with liver membranes from pregnant mice, while the inhibitory capacity was about 5-fold higher subsequent to parallel incubations with the same membranes, which had previously been heated for 30 min at 65 C, or with membranes of male rat liver, demonstrating a direct correlation between the binding of the antiidiotypic antibody to the membranes and the PRL receptor content of the membranes. Furthermore, significant and dose-dependent inhibition of [125I]iodo-oPRL binding to its receptors in various PRL receptor-rich membrane preparations from rats and rabbits was demonstrated with antiserum of rabbits immunized with rabbit anti rPRL IgG. These results suggest that effective and specific anti-PRL receptor antibodies can be produced using the antiidiotypic antibody procedure, thus avoiding the necessity of isolating and purifying the PRL receptor itself. PMID- 3002769 TI - Thyrotropin-independent mutant clones from FRTL5 rat thyroid cells: hormonal control mechanisms in differentiated cells. AB - Mutant cells varying in the pathways of their responses to hormonal stimulation are useful in defining the subcellular steps in the mechanisms of hormone action. FRTL5, a strain of normal and differentiated cells originally derived from adult rat thyroids, which depends on TSH for growth in vitro, was used to produce five TSH-independent mutants, after chemical mutagenesis and selection in medium lacking TSH. Their characterization and comparison with wild type cells demonstrated full retention of differentiated thyroid function markers such as thyroglobulin production and active iodide transport, and a slower growth rate. Characterization of cAMP metabolism in mutants revealed levels of basal cAMP and adenylate cyclase and phosphodiesterase activities similar to those of wild type cells kept in a nonproliferative state in medium lacking TSH. Adenylate cyclase responsiveness to very low doses of TSH (10(-12) M) was fully retained in all mutant clones, but the TSH-dependent cAMP elevation, although comparable to that reported in wild type cells, was not followed by significant growth stimulation in mutants. These findings demonstrate that the persistence of functional TSH receptors in these cells and that of growth regulation in them is independent of cAMP elevation. PMID- 3002771 TI - Gamma-MSH and its related peptides do not augment ACTH-induced steroidogenesis in isolated rat adrenal cells. AB - In a sensitive ACTH bioassay system using isolated rat adrenal cells, we tested the effect of gamma-MSH related peptides on ACTH-induced steroidogenesis. Peptides, including synthetic gamma1-, gamma2-, gamma3- and Lys-gamma3-MSH, exerted no effect in augmenting ACTH-induced steroidogenesis. None of the 16 kilodalton fragment of ACTH/beta-lipotropin precursor and its cleaved fragment had such an activity. The results are in contrast with previous reports concerning ACTH-potentiating activity of gamma-MSH related peptides and, therefore, indicate the necessity of further investigation of the principle involved in this unique biological activity. PMID- 3002770 TI - Thyroid hormone inhibition of phosphatidylinositol-specific phospholipase C in rat liver. AB - We investigated the effect of thyroid hormone on phosphatidylinositol-specific phospholipase C activity in rat liver. Thyroidectomy increased the activity of the enzyme. Thyroid hormone (T4, 40 micrograms) administration to thyroidectomized-rats decreased phospholipase C activity. The inhibition induced by thyroid hormone was of a non-competitive type. The higher concentration of Ca2+ strongly inhibited the activity of the enzyme obtained from thyroidectomized rats' liver in vitro. The diminished activity of the enzyme obtained from thyroxine-treated-thyroidectomized-rats was recovered by pretreatment of the enzyme with EGTA. The activity of the enzyme derived from thyroidectomized-rats was not affected by EGTA treatment. These results suggest that thyroid hormone decreases the activity of phosphatidylinositol-specific phospholipase C activity through the mobilization of Ca2+ in the intracellular space. PMID- 3002772 TI - Thyrotropin (TSH) binding activities in bovine thyroid tissue: possible role of adenosine 3',5'-monophosphate dependent phosphorylation of thyroid plasma membranes in TSH receptor degradation. AB - The relationship between thyroid plasma membrane phosphorylation and thyrotropin (TSH) receptor degradation was investigated by using bovine thyroid tissues. By fractionation of thyroid cytosol (105,000 X g supernatant of thyroid homogenate) in a continuous sucrose density gradient centrifugation, three different TSH binding activities were separated. During the incubation of thyroid plasma membranes, TSH binding activities were spontaneously released in vitro. By fractionation of the fraction containing released TSH binding activities in the same sucrose density gradient centrifugation, three different TSH binding activities were isolated. These peaks of TSH binding activity corresponded to the peaks of TSH binding activity obtained in cytosol fraction. Adenosine 3',5' monophosphate (cyclic AMP) enhanced the release of TSH binding activities from the plasma membranes in vitro. After fractionation on a sucrose density gradient centrifugation of the supernatant of the plasma membranes which were preincubated with cyclic AMP, three different peaks of TSH binding activity were identified. These peaks corresponded to the peaks obtained in spontaneously released TSH binding activity. In this case, however, the amount of small molecule TSH binding activities was predominant compared to that of large molecule TSH binding activity. During the incubation of the plasma membranes with [r-32P]-ATP and with cyclic AMP, phosphorylated soluble proteins were released. The profile of the phosphorylated soluble proteins in the sucrose density gradient centrifugation showed three different peaks which corresponded to the peaks of binding activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002773 TI - In vivo and in vitro ACTH response to ovine corticotropin-releasing factor in a bronchial carcinoid from a patient with ectopic ACTH syndrome. AB - ACTH response in vivo and in vitro to synthetic ovine corticotropin-releasing factor (o-CRF) was examined in a bronchial carcinoid from a patient with ectopic ACTH syndrome. o-CRF, 1 microgram/kg iv bolus, scarcely increased plasma ACTH or cortisol on one occasion, but they showed a low response on retesting. On the other hand, 10(-8) and 10(-7) M of o-CRF significantly stimulated ACTH release in cultured bronchial carcinoid cells. PMID- 3002774 TI - Formation and repair of psoralen-DNA adducts and pyrimidine dimers in human DNA and chromatin. AB - DNA damage and repair in human cells exposed to ultraviolet light (254 nm) or to psoralen derivatives plus 360 nm light were compared by means of a variety of analytic techniques. The two kinds of damage show considerable structural similarity; both involve cyclobutyl bonds to 5,6 positions of pyrimidines as major products and have various minor products. In purified DNA, pyrimidine dimers, but not psoralen adducts, cause structural distortions that are substances for digestion with single-strand-specific nucleases. Whereas pyrimidine dimers are randomly produced in chromatin, psoralen adducts, are concentrated approximately 2- to 4-fold in linker regions of chromatin at doses that are not highly lethal. Chromatin shows considerable mobility; assignment of DNA to linker or core regions is not permanent, and psoralen adducts initially concentrated in linker regions become randomized after 10 hr. Pyrimidine dimers and psoralen adducts are excised by normal cells but not by repair-deficient xeroderma pigmentosum cells. This repair process requires DNA polymerase alpha, but its rate in ultraviolet-damaged cells is twice that in psoralen-damaged cells. Conversion of monoadducts to DNA-DNA crosslinks reduces the rate of repair because of the increased complexity of the damaged site. PMID- 3002775 TI - Dose dependency of aflatoxin B1 binding on human high molecular weight DNA in the activation of proto-oncogene. AB - The binding of aflatoxin B1, AFB1, a potent hepatocarcinogen, to various high molecular weight (HMW) DNAs from human normal liver and two liver cancer cell lines, Alexander primary liver carcinoma (PLC) and Mahlavu hepatocellular carcinoma (hHC) and from NIH/3T3 cell have been investigated. The kinetics of AFB1 binding to these DNAs showed similar initial rates but the extents of binding to the PLC and hHC DNAs seemed to be slightly higher. Preferential AFB1 bindings were identified in both PLC and hHC DNAs compared to normal liver DNA when analyzed by restriction endonuclease digestions and agarose gel electrophoresis. A critical AFB1 binding dosage, ranging 100 to 460 fmole/microgram DNA, was found to activate the carcinogenic effect of the Mahlavu hHC HMW DNA, but not normal liver HMW DNA, rendering it capable of inducing focal transformation in NIH/3T3 cell. Excessive AFB1 binding on the hHC and PLC HMW DNAs resulted in an "over-kill" of both cell transformation capability and templating activity of the DNA. PMID- 3002776 TI - Biochemical and immunologic characterization of cotton bract extract and its effect on in vitro cyclic AMP production. I. Field-dried bract. AB - Field-dried cotton bract, a contaminant of cotton dust, has been implicated in byssinosis pathogenesis. The results from this study demonstrated that a standard bract extract (FDBE) could be prepared. FDBE was antigenic in rabbits; however, precipitating antibodies were not detectable in the serum of cotton textile mill workers. Although exposure of mononuclear leukocytes (MNL) to FDBE did not alter intracellular cyclic AMP levels, it did induce changes in the cyclic AMP response of MNL to isoproterenol and prostaglandin E1. These changes were FDBE dose dependent. The possible relationship of these findings to pathogenesis of byssinosis is discussed. PMID- 3002777 TI - Biochemical and immunologic characterization of cotton bract extract and its effect on in vitro cyclic AMP production. II. Green bract. AB - Cotton bract has been proposed as a probable byssinogenic agent. Mill workers are exposed to bract which has weathered in the field, but it is not known whether biologic effects of bract are due to intrinsic plant compound(s) or to contamination occurring during field weathering. Exposure of mononuclear leukocytes (MNL) to green bract extract (GBE) lowered basal cyclic AMP levels. Concomitant exposure of MNL to GBE plus agonist altered MNL cyclic AMP levels in a dose-dependent fashion. Differences between immunologic and pharmacologic test results of GBE and field-dried bract extract were mainly quantitative, suggesting the bioactive agent(s) in cotton bract is intrinsic to the plant. PMID- 3002778 TI - Growth changes of 3T3 cells in the presence of mineral fibers. AB - The relationship between exposure to asbestos fibers and the development of mesothelioma or bronchial carcinoma prompted many countries to ban its use from commercial products. The biological mechanism by which asbestos induces or promotes mesothelioma or carcinoma is still unknown. In order to study the influence of fibers on the cell surface, 3T3 fibroblasts were cultured in the presence of various mineral fibers. The acute cytotoxicity produced by mineral fibers was evaluated by the trypan blue dye exclusion method; growth of 3T3 cells was measured as well as the maximum cell density at saturation. We found that growth of 3T3 cells was slower in the presence of chrysotile while light microscopy revealed an increased cellular chromogenicity and a modification of the cell-cell arrangement in the presence of this fiber. We describe an assay in which chrysotile causes an increase in the maximum cell density at saturation. PMID- 3002779 TI - On the toxic effects of tetraethyl lead and its derivatives on the chrysophyte Poterioochromonas malhamensis. VI. Effects on lorica formation, mitosis, and cytokinesis. AB - Effects of tetraethyl lead (TEL) and derivatives triethyl lead (TriEL), diethyl lead (DiEL), and inorganic lead (Pb) on lorica formation of the unicellular alga Poterioochromonas malhamensis were investigated by light and electron microscopy. Lorica formation is microtubule (MT)--mediated and disturbed by agents interfering with MTs. TEL, largely ineffective as such, inhibited lorica formation of P. malhamensis when the lead compound was illuminated during or before the experiment. TriEL inhibited most; DiEL produced qualitatively effects similar to those of TriEL at about 10 times higher concentrations. Inorganic lead was even less toxic and did not selectively inhibit lorica formation of the algae. Low concentrations of TriEL (5 to 7.5 microM) selectively disturbed lorica formation, causing formation of numerous stalk-less loricae which exhibited gross and ultrastructural alterations like those induced by the antimitotic drug colchicine. The effects of TriEL on mitosis and cytokinesis of P. malhamensis were also investigated. The most sensitive mitotic phase was metaphase, which, however, accumulated only up to 5% after treatment of the cells with toxic concentrations (10 microM) of TriEL for 24 hr (control, 2%). On the other hand, up to 15% telophases (including binucleated cells) and even multinucleated cells with up to eight nuclei per cell were found, indicating that cytokinesis was considerably more effectively disturbed by TriEL than mitosis. In giant multinucleated algae, mitoses normally proceeded synchronously; some asynchronous mitoses were found. Beside normal-looking mitotic spindles in giant algae, multipolar spindles, disoriented spindles, and metaphase-like chromosome arrays completely lacking MTs were observed by electron microscopy. The effects of TriEL on cytokinesis of the algae were largely reversible. Giant cells spontaneously recovered and underwent cytokinesis after transferred into TriEL-free growth medium. Colchicine acted qualitatively identical to TriEL (accumulation of metaphases and telophases: 5 and 19%, respectively), but TriEL was about 600 times more toxic than colchicine. Unlike colchicine, its derivative, colchicine, which is known not to interfere with MTs, remained without any selective inhibitory influence on mitosis and cytokinesis of the algae, although much more toxic than the parent compound. From the inhibitory effects of TriEL and the close qualitative similarities to the effects of colchicine, it is concluded that TriEL selectively interferes with cytoplasmic and mitotic MTs of the algae, thereby causing the observed inhibitory effects on lorica formation, mitosis, and cytokinesis. PMID- 3002781 TI - Effects of chrysotile asbestos on coho salmon and green sunfish: evidence of behavioral and pathological stress. AB - The effects of chrysotile asbestos on larval coho salmon (Oncorhynchus kisutch) and juvenile green sunfish (Lepomis cyanellus) were investigated at levels approximating those reported in the Great Lakes basin (10(6) fibers/liter). Behavioral stress effects, such as loss of rheotaxic position and balance, were observed in salmon exposed at 3.0 X 10(6) fibers/liter and in sunfish exposed at 1.5 and 3.0 X 10(6) fibers/liter. Coho larvae at 1.5 X 10(6) fibers/liter were significantly more susceptible to an anesthetic stress test, becoming ataxic and losing equilibrium faster than control cohorts (P less than 0.001). Two of 106 larvae exposed at 3.0 X 10(6) fibers/liter developed tumorous swellings and three additional fish developed coelomic distentions. Cytological examination of ventral epidermal tissue revealed cellular histolysis, and evidence by transmission electron microscopy confirmed the presence of asbestos in the salmon larvae. Distortion of the lateral line region in asbestos-treated coho salmon was linked to behavioral and orientational aberrations. Differential mortality was not observed between control and treated groups of either test species. PMID- 3002780 TI - Effects of the inhalation of dusts from calcium silicate insulation materials in laboratory rats. AB - The effects of respirable dust from three commercially produced calcium silicate insulation materials were examined in laboratory rats by long-term inhalation and injection techniques. These calcium silicate products have been used as replacements for asbestos in the insulation of the engine rooms of ships, and the particle size distribution of the dust clouds generated for the experimental study closely matched those found in ships during the installation of this type of material. One year of exposure to a dust cloud of 10 mg/m3 of respirable dust had no discernible effect on the length of survival of treated animals compared to controls. No pulmonary lesions were found that appeared associated with the inhalation of calcium silicate per se, but one sample did contain significant amounts of quartz and this did produce a few small pulmonary nodules. While two small pulmonary neoplasms, one malignant and one benign, were found in dusted animals, neither was the cause of death, and the incidence was not significantly different from the control group where no tumors were found. One peritoneal mesothelioma was found in an animal from one of the inhalation groups, but this was considered to be a spontaneous tumor as none of over 100 animals injected intraperitoneally with 25 mg of calcium silicate developed these tumors. While the white blood cell count of dusted animals, compared to controls, was significantly raised in all treated groups at the end of the dusting period, these figures were within the published normal ranges for laboratory rats. It was concluded that the three tested calcium silicate products were harmless to the rats of this species at the doses tested. PMID- 3002782 TI - Cadmium-induced ultrastructural changes in Euglena cells. AB - The ultrastructure of Euglena gracilis grown in the presence of Cd showed only numerous myelin-like structures in mitochondria, chloroplasts altered in shape, and thylakoid arrangement and increase of osmiophilic plastoglobuli. These alterations indicate that respiratory processes are the initial target of Cd toxicity. PMID- 3002783 TI - Comparative in vivo and in vitro study of the influence of experimental diabetes on rat liver linoleic acid delta 6- and delta 5-desaturation. AB - Rats with experimental diabetes were administered in vivo a tracer dose of either [1-14C]-linoleic, [1-14C]-gamma-linolenic or [2-14C]-dihomo-gamma-linolenic acid and sacrificed 48 h later. With all three radioactive precursors, the radioactivity incorporated into arachidonic acid was lower in experimental diabetes, compared to nondiabetic rats similarly treated, while the weights of hepatic arachidonic acid were not significantly affected by the diabetic state. Streptozotocin-treated rats were administered moderate or excessive quantities of protamine-zinc-insulin. Streptozotocin diabetes inhibits rat liver homogenate [2 14C]-dihomo-gamma-linolenic acid delta 5-desaturation; only moderate injections of protamine-zinc-insulin restore the in vitro delta 5-desaturation. These results suggest that experimental diabetes, a reported inhibitor of delta 6 desaturation, also causes partial inhibition of delta 5-desaturation in rat liver; this suggests that dihomo-gamma-linolenic acid desaturation, a secondary regulatory step in linoleic acid metabolism, may be restored through an optimum insulin therapy. PMID- 3002785 TI - The clinic presentation of hepatocellular carcinoma by bone secondaries: a report of 25 cases. PMID- 3002784 TI - Validity of lymphoid cell line for enzymatic studies of GM2-gangliosidosis variant 0 (Sandhoff disease). AB - A lymphoid cell line established by Epstein-Barr virus (EBV)-transformation of peripheral blood B-lymphocytes from a patient with Sandhoff disease showed a severe deficiency of beta-N-acetylhexosaminidase activity (residual activity around 10% of that in lymphoid cell lines from normals or other lipidotic patients). This residual beta-N-acetylhexosaminidase was completely heat-labile in contrast to that of normals. The molecular forms of residual beta-N acetylhexosaminidase from Sandhoff lymphoid cell line were separated by Con A sepharose and electrofocusing. Their properties and electrofocusing profiles were compared to those from Sandhoff fibroblasts and from fetal brain: this comparison permitted to identify the residual molecular forms with Hex S and Hex C. The microheterogeneity of Hex S and Hex C, demonstrated by electrofocusing, was discussed. 2-Acetamido-2-deoxy-D-galactonolactone (GalNAcLone) showed a strong inhibitory effect on lysosomal Hex A, B and S, but only a very slight effect on Hex C. Studies of the inhibition type (competitive on Hex A, B and S and mixed on Hex C) gave some informations about the enzymatic site. Elsewhere, differences in affinity of GalNAcLone for the various isoenzymes could be utilized to define optimal assay conditions for specifically determining Hex C (standard assay containing 400 mumol/l of GalNAcLone). These results demonstrated that EBV transformed lymphoid cell lines represent an accurate model system for enzymatic studies of Sandhoff disease. PMID- 3002787 TI - Phosphorylation of cGMP-dependent protein kinase increases the affinity for cyclic AMP. AB - Cyclic-GMP-dependent protein kinase contains two binding sites for cGMP, which have different affinities for cGMP. Autophosphorylation of the enzyme affects mainly the binding of cGMP to the 'high'-affinity site (site 1). The enzyme binds cAMP and cAMP stimulates the phosphotransferase activity of the native enzyme half-maximally at 44 microM. Autophosphorylation of the enzyme decreases the apparent Ka value to 7 microM. Autophosphorylation does not affect the catalytic rate of the enzyme if measured at a saturating concentration of ATP. Tritiated cAMP apparently binds at 4 degrees C to one site with a Kd value of 3 microM. Binding to the second site is not measurable. Autophosphorylation of the enzyme increases the affinity of the high-affinity site for cAMP sixfold (Kd 0.46 microM) and allows the detection of a second site. In accordance with these data the dissociation rate of [3H]cAMP from the high-affinity site is decreased from 4.5 min-1 to 1.2 min-1 by autophosphorylation. Experiments in which unlabeled cAMP competes with [3H] cGMP for the two binding sites confirmed these results. Recalculation of the competition curves by a computer program for two binding sites indicated that autophosphorylation decreases the Kd value for binding of cAMP to the high-affinity site from 1.9 microM to 0.17 microM. Autophosphorylation does not affect significantly the affinity for the second site. Kd values for site 2 varied from 17 microM to 40 microM. These results suggest that autophosphorylation of cGMP-dependent protein kinase increases the affinity of the enzyme for cAMP by affecting mainly the properties of binding site 1. PMID- 3002786 TI - Endocrine response to surgery in children after premedication with midazolam or papaveretum. AB - The effect of two premedications on the sympatho-adrenal and endocrine stress response to minor surgery under halothane anaesthesia was investigated in 16 children. One group (n = 9) was premedicated with midazolam, 0.1 mg kg-1, and atropine 0.2-0.4 mg i.m. The other group (n = 7) received papaveretum 0.4 mg kg-1 and hyoscine 0.008 mg kg-1 i.m. Plasma concentrations of catecholamines, ACTH and cortisol were measured during undisturbed anaesthesia, during surgery and 15 min post-operatively. There were no differences in catecholamine concentrations between the groups. Prior to surgery, plasma ACTH was significantly lower (P less than 0.05) in the papaveretum group. During surgery, plasma cortisol and plasma ACTH were significantly lower after papaveretum premedication. Post-operatively there were no differences. End-tidal CO2 concentrations were similar in the two groups. It was concluded that the endocrine stress-response immediately after induction of anaesthesia and during surgery was lower after papaveretum than after midazolam premedication. PMID- 3002788 TI - Effect of benzoate on the metabolism of fructose 2,6-bisphosphate in yeast. AB - When benzoate (2 mM, pH 3.5) was added together with glucose (0.1 M) to a suspension of Saccharomyces cerevisiae in the stationary phase, it caused a relative increase in the concentration of glucose 6-phosphate and fructose 6 phosphate and a decrease in the concentration of fructose 1,6-bisphosphate. These effects are in confirmation of similar observations made by Krebs et al. [Biochem. J. 214, 657-663 (1983)] and are indicative of an inhibition of 6 phosphofructo-1-kinase. Benzoate also caused an about fourfold relative decrease in the concentration of fructose 2,6-bisphosphate, an increase in that of cyclic AMP with no change in that of ATP. It also greatly decreased the activation of 6 phosphofructo-2-kinase, but not that of trehalase, both of which normally occur upon addition of glucose to a yeast suspension. When added 10 min after glucose, benzoate caused a rapid (within 2-3 min) decrease in fructose 2,6-bisphosphate concentration and in 6-phosphofructo-2-kinase activity. In the presence of benzoate, there was also a parallel decrease in the concentration of fructose 2,6 bisphosphate and in the rate of ethanol production when the external pH was dropped from 5.0 to 2.5, with minimal change in the concentration of ATP. Purified 6-phosphofructo-2-kinase was inhibited by benzoate and also by an acid pH. Experiments with cell-free extracts did not provide an explanation for the rapid disappearance of fructose-2,6-bisphosphate or the inactivation of 6 phosphofructo-2-kinase in yeast upon addition of benzoate. PMID- 3002789 TI - Mitochondrial adenylate kinase (AK2) from bovine heart. The complete primary structure. AB - The sequence analysis of adenylate kinase isoenzyme 2 (AK2) was completed using a gas-phase sequencer constructed in our laboratory. The enzyme contains 238 amino acid residues in the following order: (sequence; see text) The four cysteine residues of AK2 were reinvestigated. Cys-41 and Cys-233 contain free thiols, which can be carboxymethylated in the intact protein without loss of enzymic activity. Chemical and model-building studies suggest that the pair Cys-43/Cys-93 forms a disulfide in native AK2. The relative molecular mass of AK2, as deduced from the sequence, is 26104. Other methods, including titration of -SH groups, sedimentation equilibrium ultracentrifugation and gel filtration yielded Mr values in the range from 26 000 to 31 500, each value depending on the respective method of determination. Bovine heart AK2 contains 44 residues more than the homologous isoenzyme AK1 (myokinase). As all but one single insertions and deletions cancel, the higher Mr of AK2 is due to 9 residues preceding the N terminus of AK1, a stretch of 30 residues in the middle of the molecule and 6 residues at the end. AK2 and AK1 are similar in their active-site geometry. In contrast, AK2 does not possess any of the three antigenic sites of AK1, which is consistent with the lack of immunological cross-reactivity between AK1 and AK2. PMID- 3002790 TI - Incorporation of sulphate into type III procollagen by cultured human fibroblasts. Identification of tyrosine O-sulphate. AB - Confluent cultures of normal human skin fibroblasts were labelled overnight with [35S]sulphate, and the incorporation of the isotope into type III procollagen, secreted into the medium, was verified by radioimmunoassay and immunoprecipitation after removing the heavily sulphated proteoglycans by anion exchange chromatography. Type III procollagen and its pro and pN alpha chains were visualized in fluorographs of the immunoprecipitates. The labelled procollagen could be isolated by a combination of ion-exchange chromatography and gel filtration and was found to contain tyrosine O-sulphate, which was identified by thin-layer electrophoresis after Ba(OH)2 hydrolysis. The regions sulphated in the type III procollagen molecule were susceptible to pepsin digestion. Digestion with purified bacterial collagenase at +37 degrees C produced a labelled fragment that was recognized by antibodies against the aminoterminal propeptide of type III procollagen, indicating that the sulphated tyrosine residues are located either in this propeptide or in the non-helical telopeptide region of the type III collagen molecule proper. Sulphation of tyrosine residues is a new post translational modification in procollagen, which could be involved in the regulation of the processing of type III procollagen into collagen and thus affect the formation of collagen fibres. PMID- 3002791 TI - Modulation of the interaction between the two halves of troponin C by the other troponin subunits. AB - The interactions between troponin subunits have been studied by intrinsic fluorescence and electron spin resonance (ESR) spectroscopy. The tryptophan fluorescence of troponin T (TnT) and troponin I (TnI) when complexed with troponin C (TnC) undergoes a Ca2+-dependent transition. The midpoints of such spectral changes occur at pCa approximately equal to 6, suggesting that the conformational change of TnT and TnI is induced by Ca2+ binding to the low affinity sites of TnC. When TnC is labelled at Cys-98 with a maleimide spin probe (MSL), the spin signal is sensitive to Ca2+ binding to both the high and the low affinity sites of TnC in the presence of either or both of the other two troponin subunits. Since Cys-98 is located in the vicinity of one of the high-affinity sites, these results are indicative of a long-range interaction between the two halves of the TnC molecule. Our earlier kinetic studies [Wang, C.-L. A., Leavis, P. C. & Gergely, J. (1983) J. Biol. Chem. 258, 9175-9177] have shown such interactions in TnC alone. Since the ESR spectral change associated with metal binding to the low-affinity sites is only observed when MSL-TnC is complexed with TnT and/or TnI, this long-range interaction within TnC appears to be mediated through the other troponin subunits. PMID- 3002792 TI - Interactions of plasma gelsolin with actin. Isolation and characterization of binary and ternary plasma-gelsolin-actin complexes. AB - We have studied the interactions between plasma gelsolin and actin: firstly the complex formation between both proteins, secondly the effects of gelsolin and its complexes on G-actin polymerization and F-actin fragmentation. Complex formation has been studied by high-performance gel permeation chromatography; plasma gelsolin alone elutes at an Mr of about 77000 and a Stokes radius of 3.7 nm; complex formation occurs in the presence of Ca2+: by chromatography in the presence of EGTA, a binary complex is obtained with an Mr of 134000 and a Stokes radius of 4.7 nm; and by chromatography in the presence of Ca2+, a ternary complex is obtained with an Mr of 173000 and a Stokes radius of 5.2 nm. The binary complex is EGTA-stable. In relation to this stability of the binary complex, the depolymerizing function of gelsolin is not reversed upon chelation of Ca2+. The effects of plasma gelsolin and its complexes on both G-actin polymerization and F-actin fragmentation, and their Ca2+ dependence have been examined by viscometry and electron microscopy. The main conclusions of these studies are the following: the fast processes are the formation of ternary complex, which acts as a heteronucleus for G-actin polymerization, and the severing function of gelsolin, these fast processes are Ca2+-dependent; the slow processes are related to the capping ability of gelsolin or its complexes and are Ca2+-independent. PMID- 3002793 TI - Interaction of guanidinium and guanidinium derivatives with the Na+/H+ exchange system. AB - Guanidinium, a small organic monovalent cation that is permeant through voltage dependent cationic channels cannot be transported by the cardiac Na+/H+ exchange system. Yet it recognizes the exchanger and is able to block its activity (K0.5 = 30 mM). Guanidinium derivatives that do not belong to the amiloride series and which possess potent antihypertensive properties also block the activity of the Na+/H+ exchange system in various cell types with a greater potency than unsubstituted guanidinium. The most potent compound found, guanochlor, has an affinity for the exchanger ranging between 0.5 microM and 6 microM in different systems and is more potent than amiloride in all systems studied. Guanochlor has the same action as amiloride derivatives on the cardiac cells; it prevents intracellular pH recovery in cardiac cells that have been acidified and also antagonizes the effect of ouabain on 45Ca2+ uptake by chick cardiac cells. Guanochlor does not compete with [3H]ethylpropylamiloride for its binding to the Na+/H+ exchange system of rabbit kidney brush border membrane. It is suggested that guanochlor recognizes a binding site on the Na+/H+ exchanger that is distinct from the amiloride binding site. PMID- 3002795 TI - Polyamines stimulate the phosphorylation of phosphatidylinositol in membranes from A431 cells. AB - The phosphorylation of phosphatidylinositol in plasma membranes from A431 cells was investigated using [gamma-32P]ATP as the substrate. Phosphatidylinositol 4 phosphate was found to be the major product after an incubation time of 5-10 min. Little, if any, phosphatidylinositol 4,5-bisphosphate was found under these conditions. Epidermal growth factor (EGF) had no effect on the formation of phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate. On the other hand, the polyamines spermidine and spermine stimulated the phosphatidylinositol kinase activity about eightfold yielding almost exclusively phosphatidylinositol 4-phosphate as the reaction product. Half-maximum stimulation by spermidine occurred under near physiological conditions (1.5 mM). Furthermore various proteins and amino acid polymers containing clustered basic amino acid residues (e.g. histones and polylysine) stimulated the formation of phosphatidylinositol 4-phosphate to a similar extent. Half-maximal concentrations for the activation were considerably lower ranging from 1.5 microM to 80 microM. The ATP specificity of the phosphatidylinositol kinase(s) was investigated with a small set of selected ATP derivatives. In the presence of spermidine the specificity changed significantly indicating that (a) spermidine acts on a kinase and not on a phosphatase, (b) this activity is distinct from the EGF-receptor protein kinase activity. The results do not suggest an involvement of the EGF receptor in the growth-factor-dependent formation of phosphatidylinositol phosphates. It is proposed that the phosphorylation of phosphatidylinositol by polyamines might be a mechanism to replenish the pool of inositolphospholipids. PMID- 3002794 TI - Trehalase activation in yeasts is mediated by an internal acidification. AB - It has been reported that the addition of glucose, uncouplers and nystatin to yeast cells grown in a sugarfree medium causes trehalase activation; it has been postulated that this activation might be mediated by the depolarization of the plasma membrane. In this article the values of membrane potential and pH gradient across the plasma membrane of Saccharomyces cerevisiae have been determined under the same conditions as those in which trehalase is activated. Membrane potential was evaluated from the distribution of triphenylmethylphosphonium, the pH gradient from the distribution of benzoic acid across the plasma membrane. When the effect of several agents on the two components of the electrochemical proton gradient across the plasma membrane of ethanol-grown yeast cells were studied, under trehalase activation conditions, the following observations were made. (a) The addition of glucose activated trehalase and caused internal acidification of the cells, but had practically no effect on the membrane potential. (b) The addition of 200 mM KCl depolarized the cell membrane but did not affect the internal pH, nor trehalase activity. (c) Although carbonyl cyanide m chlorophenylhydrazone depolarized the cells at external pH 6.0 and 7.0, it only activated trehalase at an external pH 6.0, leading to the acidification of the internal medium at this pH. (d) Nystatin caused an increase in the triphenylmethylphosphonium accumulation at external pH 6.0 and 7.0, but only activated trehalase at external pH 6.0, causing acidification of the cell interior at this pH. (e) Activation of trehalase was also observed when the internal acidification was caused by addition of a weak acid such as acetate. It is concluded that trehalase activation is mediated by an intracellular acidification and is independent of the membrane potential. PMID- 3002796 TI - Influence of 2-aminoadenosine, A', on the conformational behaviour of d(T-A'-T A'). A one-dimensional proton NMR study at 300 MHz and 500 MHz. AB - Proton NMR studies at 300 MHz and 500 MHz in aqueous solution were carried out on the modified self-complementary tetranucleoside trisphosphate d(T-A'-T-A'), in which d(A') represents 2-aminodeoxyadenosine. The NMR spectra were observed at two sample concentrations over the temperature range 2-70 degrees C. Assignments, based on homonuclear decoupling and nuclear Overhauser enhancement (NOE) experiments, are given. The concentration dependence of the chemical shift vs temperature profiles was used to extract information concerning duplex formation. The proton-proton and proton-phosphorus coupling constants were obtained at four temperatures and yielded accurate conformational data on the sugar ring and on the back-bone angles beta, gamma and delta. From the observed line broadening, shift profiles, NOEs, and from the observation of imino proton resonances, it is concluded that the compound exists as a miniduplex at low temperature. Comparison of these observations with similar observations on the parent compound d(T-A-T-A) indicate that substitution of dA by dA' increases the tendency towards duplex formation. At low temperature the compound adopts a stacked B-DNA type structure: destacking occurs on raising the temperature. The sequence-dependent sugar ring geometry [Mellema, J.-R., Pieters, J. M. L., van der Marel, G. A., van Boom, J. H., Haasnoot, C. A. G. & Altona, C. (1984) Eur. J. Biochem. 143, 285-301] is present in this molecule. The conformational parameters of d(T-A'-T-A') and d(T-A T-A) are quite similar, thus substitution of dA by dA' has no measurable influence on the geometry of the sugar ring or on the backbone angles beta, gamma and delta. PMID- 3002797 TI - The DNA sequence recognized by the EcoDXX1 restriction endonuclease. AB - EcoDXX1 is a type-I restriction enzyme coded for by the plasmid pDXX1. Like other type-I restriction endonucleases, the enzyme catalyses the modification of susceptible DNA. We have determined the DNA sequence recognised by EcoDXX1 to be: 5'TCANNNNNNNATTC-3' 3'-AGTNNNNNNNTAAG-5' where N can be any nucleotide. This sequence has an overall structure very similar to previously determined type-I sequences. PMID- 3002798 TI - Purification of a hexaheme cytochrome c552 from Escherichia coli K 12 and its properties as a nitrite reductase. AB - Anaerobic cytochrome c552 was purified to electrophoretic homogeneity by ion exchange chromatography and gel filtration from a mutant of Escherichia coli K 12 that synthesizes an increased amount of this pigment. Several molecular and enzymatic properties of the cytochrome were investigated. Its relative molecular mass was determined to be 69 000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. It was found to be an acidic protein that existed in the monomeric form in the native state. From its heme and iron contents, it was concluded to be a hexaheme protein containing six moles of heme c/mole protein. The amino-acid composition and other properties of the purified cytochrome c552 indicated its similarity to Desulfovibrio desulfuricans hexaheme cytochrome. The cytochrome c552 showed nitrite and hydroxylamine reductase activities with benzyl viologen as an artificial electron donor. It catalyzed the reduction of nitrite to ammonia in a six-electron transfer. FMN and FAD also served as electron donors for the nitrite reduction. The apparent Michaelis constants for nitrite and hydroxylamine were 110 microM and 18 mM, respectively. The nitrite reductase activity of the cytochrome c552 was inhibited effectively by cupric ion and cyanide. PMID- 3002799 TI - Complete amino acid sequence of rat L-type pyruvate kinase deduced from the cDNA sequence. AB - cDNA clones, containing the entire coding region of rat L-type pyruvate kinase, were isolated and their nucleotide sequences were determined by the dideoxy-chain termination method. The predicted coding region, which spans 543 amino acids, established the complete amino acid sequence of the L-type isozyme of pyruvate kinase for the first time. The deduced amino acid sequence of the L type has one phosphorylation site in its amino terminus and shows about 68% and 48% homologies with M1-type pyruvate kinase of chicken and yeast pyruvate kinase respectively. Domain A exhibits higher homology than domains B and C. The residues in the active site of the L-type enzyme of rats, lying between domains B and A2, are rather different from those of the M1-type enzyme of chickens, but other residues constituting the active site are identical with those of the chicken M1 type except for one amino acid substitution. PMID- 3002800 TI - Characterization of alpha-adrenoceptors in myometrium of preparturient rats. AB - We describe three methods for the quantitative analysis of the alpha-adrenoceptor subtypes in preparturient rat myometrial membrane fractions. A non-subtype selective antagonist radioligand. [3H]dihydroergocryptine ([3H]DHE), was used to label all of the alpha-receptors. [3H]DHE bound to both alpha 1- and alpha 2 receptors with indistinguishable affinity. Computer modelling of competition curves of unlabeled selective antagonists or agonists was then required in order to determine reliably alpha 1 and alpha 2 affinities and proportions: the alpha 1 receptors represent 45% and the alpha 2-receptors 55% of the entire alpha receptor population in rat uterus. The second approach involved the administration of phenoxybenzamine (POB) that irreversibly blocks the alpha 1 adrenoceptors. Myometrial membranes obtained from rats 1 h after the administration of varying amounts of POB showed a dose-dependent reduction in specific [3H]DHE binding. This reduction was accompanied by a progressive increase of the value of the dissociation constant. Our data indicate that a dose of 1 mg of POB left the alpha 2-receptors intact while entirely blocking the alpha 1-receptors in rat myometrium. The third approach utilized the selective radioligand antagonists [3H]prazosin ([3H]PRAZ) and [3H]rauwolscine ([3H]RAUW). The results obtained with these radioligands confirmed our observations on the alpha-adrenoceptor subtypes in experiments with [3H]DHE. The results obtained with the 3 methods are in good agreement. Each approach appears valid and applicable to the characterization of alpha 1- and alpha 2-adrenoceptor subtypes in rat uterus, but the method using [3H]PRAZ and [3H]RAUW demonstrates more directly the presence of the two receptor subtypes. PMID- 3002801 TI - Effects of enprofylline on A1 and A2 adenosine receptors. AB - The effects of enprofylline were studied on A1 adenosine receptors of rat fat cells and on A2 adenosine receptors of human platelets and of guinea-pig lung. Enprofylline antagonized the 5'-N-ethylcarboxamidoadenosine (NECA)-induced stimulation of platelet adenylate cyclase activity with a KB of 130 microM. In human platelets, enprofylline did not antagonize but potentiated the NECA-induced inhibition of aggregation. This potentiation was abolished in the presence of the phosphodiesterase inhibitor papaverine. An adenosine antagonistic effect of enprofylline could not be evaluated on A2 receptors of guinea-pig lung because the xanthine enhanced basal and NECA-stimulated cyclic AMP accumulation. Enprofylline antagonized the N6-R-(-)-phenylisopropyladenosine (R-PIA)-induced inhibition of rat fat cell adenylate cyclase with a KB of 32 microM. The Ki value for inhibition of [3H]PIA binding to fat cell membranes was 45 microM. Enprofylline inhibited cyclic AMP phosphodiesterase activity of human platelets, guinea-pig lung and rat fat cells with Ki values of 15, 130 and 110 microM, respectively. The results show that enprofylline was nearly equipotent as antagonist at A1 and A2 adenosine receptors. Mechanisms other than adenosine antagonism or phosphodiesterase inhibition may be involved in the pharmacological effects of enprofylline. PMID- 3002802 TI - p-Azidoclonidine: a photoaffinity label for the alpha 2-adrenoceptor. AB - We have synthesized and characterized p-azidoclonidine (AZC) as a putative alpha 2-adrenoceptor photoaffinity label. [3H]AZC demonstrated high affinity (KD = 11.8 +/- 2.5 nM), saturability (Bmax = 171 +/- 21 fmol/mg protein), stereo specificity, and rank order of potency expected of a specific alpha 2-receptor label when used as a reversible ligand in the rat cerebral cortex. The pharmacologic profile of AZC was similar to p-aminoclonidine (PAC), an established alpha 2-adrenoceptor partial agonist. Membranes covalently prelabeled with nonradioactive AZC showed a dose dependent decrease in the number of alpha 2 receptor sites subsequently detected by [3H]PAC and [3H]yohimbine. Specific covalent [3H]AZC binding to rat cerebral cortical alpha 2-receptors represented 35 +/- 7% of the total [3H]AZC bound. These data indicate that AZC is a selective alpha 2-adrenoceptor photoaffinity label which may be useful in the identification and purification of the alpha 2-Adrenoceptor. PMID- 3002803 TI - Antagonism of flurazepam and other effects of Ro15-1788, PK8165 and Ro5-4864 on the GABA-A receptor complex in rat cuneate nucleus. AB - In slices of rat cuneate nucleus, responses to the GABA-A receptor agonist muscimol were potentiated by flurazepam. The maximal potentiation by 1 microM flurazepam was antagonized by the neuronal benzodiazepine receptor ligand Ro15 1788 3 microM, by the quinoline derivative PK8165 0.1 microM and by the peripheral-type benzodiazepine receptor ligand Ro5-4864 0.1 microM. Another ligand for the peripheral-type benzodiazepine receptor, PK11195 10 microM, enhanced the potentiating effect of a submaximal concentration of flurazepam 0.1 microM and prevented the antagonism of flurazepam by Ro5-4864. At much higher concentrations of these drugs, additional effects were seen. Ro15-1788 30 microM and PK11195 30 microM each caused small potentiations of muscimol while Ro5-4864 30 microM caused a small antagonism. Ro15-1788 30 microM, PK8165 100 microM and Ro5-4864 30 microM all antagonized the potentiation of muscimol by pentobarbitone 10 microM; also, PK8165 and Ro5-4864 enhanced the potency of picrotoxin as an antagonist of muscimol. It is concluded that both Ro5-4864 and PK8165 have several distinct effects on the GABA-A receptor complex, all of which could result in reduced responses to muscimol. PMID- 3002804 TI - High and low affinity [3H]imipramine binding sites in control and parkinsonian brains. AB - [3H]Imipramine binding was studied in the prefrontal cortex and putamen of post mortem brains from control and Parkinsonian subjects. Saturation and inhibition curves showed both high affinity [3H]imipramine binding related to the serotonin uptake mechanism and low affinity binding which was sodium-independent and unrelated to serotonergic uptake. After subcellular fractionation, high affinity [3H]imipramine binding sites were enriched in synaptosomal fractions. In Parkinson's disease, where brain serotonin concentrations are decreased, there was a significant reduction in the density of the high affinity binding in the prefrontal cortex and putamen while the characteristics of the low affinity binding sites remained unchanged. After subcellular fractionation of the putamen of Parkinsonian patients, the decrease in [3H]imipramine binding was found predominantly in the synaptosomal fractions. These results are consistent with a relation between the high affinity [3H]imipramine binding sites and the neuronal serotonin uptake mechanism. Estimation of [3H]imipramine binding could be used as a specific marker for the study of serotonergic innervation in human post-mortem material. The reduction in the density of tricyclic antidepressant binding sites found in cortical and subcortical areas of Parkinsonian brains may be somehow implicated in the depression often seen in patients. PMID- 3002805 TI - Characterization of the 5-HT1B recognition site in rat brain: binding studies with (-)[125I]iodocyanopindolol. AB - (-)[125I]Iodocyanopindolol ([125I]CYP) labels rat brain membrane sites which display high affinity for several serotonergic and beta-adrenergic compounds. The binding of [125I]CYP to these serotonergic recognition sites was evaluated in the presence of 30 microM (-)isoprenaline in order to suppress binding to beta adrenoceptors. [125I]CYP binds in rat cortex membranes rapidly, reversibly and stereoselectively to a finite number of recognition sites: Bmax = 180 fmol/mg, KD = 230 pM. Similar affinity values of [125I]CYP were obtained in membranes from rat hippocampus and striatum. Kinetic, saturation and competition experiments suggest that under these conditions [125I]CYP binds to a single serotonergic recognition site named 5-HT1B. The pharmacological profile of 5-HT1B sites is characteristic of a 5-HT1 binding site and shows the following rank order of affinity for agonists: RU 24969, (5-methoxy-3-[1,2,3,6-tetrahydropyridin-4-yl]1H indole) greater than 5-CT, (5-carboxamidotryptamine) greater than 5-HT, (5 hydroxytryptamine, serotonin) greater than 5-OCH3-T, (5-methoxytryptamine) much greater than 2-CH3-5-HT, (2-methylserotonin) greater than 8-OH-DPAT, (8-hydroxy-2 (di-n-pro-pylamino)-tetralin). The rank order of affinity for antagonists is: (+/ )ICYP, ((+/- )-3-I-cyano-pindolol) greater than (-)21-009, (4-[3-ter-butyl-amino 2-hydroxy-propoxy]-indol-2-carbonic acid isopropyl ester) greater than (+)21-009 greater than (-)propranolol greater than metitepin greater than (-)pindolol much greater than ketanserin greater than spiroperidol greater than mesulergine. 5 HT1B recognition sites display low affinity for selective beta 1- and beta 2 adrenoceptor antagonists, e.g. atenolol, betaxolol, ICI 89-406 and ICI 118-551. The low affinity of 5-HT1B recognition sites for some 5-HT1A, 5-HT1C and 5-HT2 selective compounds (e.g. 8-OH-DPAT, mesulergine, ketanserin) suggests that 5 HT1B recognition sites are pharmacologically different from 5-HT1A, 5-HT1C and 5 HT2 recognition sites. PMID- 3002806 TI - Interaction of pitrazepin with the GABA/benzodiazepine receptor complex and with glycine receptors. AB - Pitrazepin (3-(piperazinyl-1)-9H-dibenz(c,f)triazolo(4,5-a)azepin) is a new GABAA receptor antagonist reported to antagonize electrophysiological effects of GABA. We have investigated in some detail the interaction of pitrazepin with the GABA/benzodiazepine receptor chloride channel complex. Pitrazepin was found to be a competitive inhibitor of the GABAA receptor which is coupled to [3H]diazepam and [35S]TBPS binding sites; the KI value obtained by Schild analyses was 80 nM. Although pitrazepin interacted weakly with BZ receptors the compound did not affect the chloride gating mechanism (labelled with [35S]TBPS or [3H]avermectin B1a). Further, pitrazepin was a non-selective GABA antagonist since glycine receptors, labelled with [3H]strychnine, were affected at low concentrations (the KI values in rat brain-stem were 71-110 nM). PMID- 3002807 TI - Endothelial alpha 2-adrenoceptors in canine pulmonary and systemic blood vessels. AB - The present study was designed to determine whether alpha 2-adrenoceptors mediate endothelium-dependent relaxations in veins and arteries of the pulmonary and systemic vasculature of the dog. Rings of either pulmonary or femoral artery and vein with and without endothelium were suspended for isometric force measurements in a buffered salt solution (37 degrees C; gassed with 95% O2 and 5% CO2). Removal of the endothelium caused a shift to the left of the concentration response curves to epinephrine, norepinephrine and the selective alpha 2 adrenoceptor agonist UK 14,304, but not of that to the selective alpha 1 adrenoceptor agonist phenylephrine. In the presence of alpha 1- and beta adrenoceptor blockers, norepinephrine and UK 14,304 initiated relaxations of previously contracted rings. These relaxations were inhibited by the alpha 2 adrenoceptor antagonist, rauwolscine, and were absent in rings without endothelium. In the pulmonary artery and femoral vein, the endothelium-dependent responses to UK 14,304 were masked in part by direct alpha 2-adrenergic activation of the vascular smooth muscle. PMID- 3002808 TI - Inotropic, chronotropic and coronary vasodilator potency of forskolin. AB - The cardiodynamic profile of forskolin, a direct activator of adenylate cyclase, was examined in an isovolumic left ventricular (LV) preparation of coronary perfused guinea-pig hearts. Forskolin consistently induced concentration dependent increases in LV systolic pressure development, increases in spontaneous beating frequency and decreases in coronary vascular resistance. However, the forskolin concentration required for one-half of the maximal effect (EC50) for coronary vasodilation (3.8 +/- 0.6 X 10(-8) M, n = 6) was 7- to 10-fold less than the EC50 for positive chronotropy (29.3 +/- 4.6 X 10(-8) M, P less than 0.01) and 17- to 20-fold less than the EC50 for positive inotropy (66.3 +/- 7.4 X 10(-8) M, P less than 0.001). The inotropic response to forskolin was not secondary to the concomitant increase in bearing frequency. Also, the coronary vasodilator response persisted in K+-depolarized non-beating hearts, and therefore did not depend on endogeneous coronary vascular autoregulation secondary to increased myocardial oxygen demand. We conclude that forskolin induces direct pharmacologic effects in ventricular muscle, pacemaker cells and the coronary vasculature, but add that the coronary vasculature is over one order of magnitude more sensitive to forskolin than is ventricular muscle. Perhaps adenylate cyclase systems in the heart exist as different subtypes with different affinity characteristics for forskolin-like drugs. PMID- 3002809 TI - Isosorbide 5-mononitrate effects on isolated rabbit aorta and vena cava: relationship between cyclic GMP and relaxation of vascular smooth muscle. AB - The effects of isosorbide 5-mononitrate (5-ISMN) on vascular smooth muscle tone and cyclic GMP levels in isolated rabbit aorta and vena cava were compared. 5 ISMN induced concentration-dependent relaxation of noradrenaline-contracted strips of aorta and vena cava. 5-ISMN was over 100 times more potent in eliciting relaxation in vena cava than in aorta. Unlike 5-ISMN, 8-bromo cyclic GMP produced concentration-dependent relaxation with similar potency in both strips. The relaxation induced in both strips by 5-ISMN but not by 8-bromo cyclic GMP was inhibited by pretreatment with 5 X 10(-5) M methylene blue. The 5-ISMN-induced changes in tone of both strips were closely associated with those in cyclic GMP levels. 5-ISMN increased the cyclic GMP levels of both strips in a concentration dependent manner. 5-ISMN was also considerably more potent in increasing cyclic GMP levels in vena cava than in aorta. This 5-ISMN-induced increases in cyclic GMP levels of both strips were inhibited by pretreatment with methylene blue. These results suggest that cyclic GMP could be involved in the 5-ISMN-induced relaxation of smooth muscle from both aorta and vena cava. Furthermore, the finding that 5-ISMN was more active on vena cava than on aorta both to cause relaxation and increase cyclic GMP levels indicates that cyclic GMP-mediated vasodilatation may be responsible for the pharmacological action of 5-ISMN in vivo. PMID- 3002810 TI - Estradiol induces oxytocin binding sites in rat hypothalamic ventromedial nucleus. PMID- 3002811 TI - Characteristics of P2 (nucleotide) receptors mediating contraction and relaxation of rat aortic strips: possible physiological relevance. AB - ATP and ADP relaxed rat aortic strips precontracted with noradrenaline by an endothelium-dependent mechanism. 5'-AMP was much less potent and adenosine was essentially without effect. The metabolically stable analogues alpha,beta methylene ATP and beta,gamma-methylene ATP further contracted precontracted aorta. Aortic strips, which had not been precontracted with noradrenaline, contracted when exposed to either ATP or alpha,beta-methylene ATP, the latter nucleotide being much more potent than the former. Removal of the endothelium increased the contractions to ATP. ANAPP3 had no effect on the endothelium dependent relaxations produced by ATP but it antagonized contractions produced by alpha,beta-methylene ATP. These results provide evidence for the possible existence of two subtypes of P2 receptors in rat aorta; a P2 receptor mediating contraction residing on smooth muscle which can be antagonized by ANAPP3 and where alpha,beta-methylene ATP is more potent than ATP, and a P2 receptor mediating relaxation located on the endothelium which cannot be antagonized by ANAPP3 and where ATP is much more potent than alpha,beta-methylene ATP. PMID- 3002812 TI - Modulation of alpha 1-and alpha 2-adrenoceptor binding sites in the brain of audiogenic seizure susceptible mice (DBA/2J). AB - The binding of [3H]dihydroalprenolol ([3H]DHA), [3H]prazosin and [3H]clonidine was assayed in whole brain and various brain regions of audiogenic seizure (AS) susceptible DBA/2J (D2) mice aged 10, 24 and 50 days (i.e. before, during and after their period of AS susceptibility, respectively) and in age-matched C57BL/6J (B6) controls. In whole brain, at 24 days, [3H]DHA binding was similar in the two strains, while the binding of [3H]prazosin and [3H]clonidine was significantly lowered in D2 mice. No difference could be detected in 10 and 50 day old mice with any of the ligands. Regional studies indicated an involvement of the cerebral cortex, the olfactory bulbs and the brain-stem. alpha- (but not beta-)adrenoceptor changes were concomitant with the AS susceptibility period. These changes were unevenly distributed in the brain of D2 mice; they suggest that alpha 1- and alpha 2-adrenoceptor subtypes might play different roles in the AS of the D2 mouse strain. PMID- 3002813 TI - Evidence for GABAB autoreceptors in median eminence. AB - The effect of the selective GABAB receptor agonist baclofen was examined on stimulus-induced release of [3H]GABA from crude synaptosomal preparations of median eminence (ME) and pituitary neurointermediate lobe (NI). Baclofen stereospecifically inhibited release of [3H]GABA in a concentration-dependent manner from ME but had no effect in NI. The effect of (+/-) baclofen was partly antagonized by the putative GABAB receptor antagonist delta-aminovaleric acid, but these experiments were complicated by a degree of heteroexchange. These results provide the first evidence for GABAB autoreceptors in the CNS. PMID- 3002814 TI - Antidepressant drugs given repeatedly increase binding to alpha 1-adrenoceptors in the rat cortex. AB - The effect of antidepressant drugs, administered repeatedly, on binding to alpha 1-adrenoceptors in the rat cerebral cortex was studied using [3H]prazosin as a ligand. Imipramine, amitriptyline, citalopram and mianserin increased the binding (Bmax) assessed at 8:00 h. At 20:00 h such an effect was produced by imipramine, citalopram and mianserin. [3H]Prazosin binding in control rats was higher at 20:00 h than at 8:00 h. An increase in the density of alpha 1-adrenoceptors may be associated with the therapeutic activity of antidepressant drugs. PMID- 3002815 TI - 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice is enhanced by pretreatment with diethyldithiocarbamate. PMID- 3002816 TI - Pertussis toxin separates two muscarinic receptor-effector mechanisms in the striatum. PMID- 3002817 TI - Evidence for functional alpha-adrenoceptors in rabbit basilar arteries. AB - The contractile response of rabbit basilar arteries to 1-norepinephrine was studied both in the absence and in the presence of prazosin (10 nM and 1 microM). Prazosin produced a parallel shift to the right of the norepinephrine, but not the histamine, dose-response curves indicating the presence of prazosin-sensitive alpha-adrenoceptors in this vessel. The alpha-adrenoceptor-mediated contractile responses to norepinephrine in rabbit basilar arteries were much more sensitive to the deleterious effects of storage (for 18 h at 4 degrees C) than those to histamine. It is suggested that the low number of alpha-adrenoceptors in the rabbit basilar artery combined with their low sensitivity to alpha-adrenoceptor agonists, and detrimental effects associated with tissue storage may explain some inconsistent reports in the literature regarding the presence of functional alpha adrenoceptors in rabbit basilar arteries, particularly ligand binding studies. PMID- 3002818 TI - Enhancement of GABA binding by the benzodiazepine partial agonist CGS9896. AB - Compounds have been reported that act on the benzodiazepine receptor as full agonists (diazepam and CL218872), full antagonists (CGS8216 and RO15-1788), on partial agonists (CGS9896). We examined the effect of these compounds on [3H]GABA binding to membrane fragments from rat brain. Incubations were performed at 37 degrees C in a buffer containing EGTA to reduce free calcium ion levels. Centrifugation was then used to separate bound from free [3H]GABA. Diazepam caused a 20-45% enhancement of [3H]GABA binding and this effect was inhibited by 5 mM CaCl2. The magnitude of the enhancement of [3H]GABA by CL218872 was similar to that of diazepam. In contrast, the benzodiazepine antagonists, RO15-1788 and CGS8216 caused little enhancement of [3H]GABA binding. Finally, the partial agonist CGS9896 was distinguishable from both the benzodiazepine antagonists and full agonists by an intermediate level of enhancement of [3H]GABA binding. The extent of enhancement of [3H]GABA binding appears to be predictive of the pharmacological efficacy of compounds acting at the benzodiazepine receptor. PMID- 3002819 TI - (Na+,K+)-ATPase and noradrenergic regulation: effects of cardiac glycoside treatment and noradrenergic manipulations. AB - We examined effects of treatment with cardiac glycosides, in combination with noradrenergic stimulation or depletion, on (Na+,K+)-ATPase activity in rat cerebral cortex, heart, and kidney. Treatment with digitoxin increased the apparent number of (Na+,K+)-ATPase sites in heart, cerebral cortex, and kidney. Ouabain, which crosses the blood-brain barrier poorly, did not affect enzyme in brain but was otherwise similar. Norepinephrine depletion prevented the increase in heart but not in cerebral cortex. Noradrenergic stimulation increased the number of sites in cerebral cortex and in heart. In rats treated with digitoxin, noradrenergic stimulation increased enzyme activity further in heart but not in cerebral cortex. Examination of effects on noradrenergic receptor binding and on norepinephrine metabolite concentrations suggested that, while in heart cardiac glycosides appeared to increase norepinephrine release, in brain there was no effect on release but there may have been appreciable inhibition of norepinephrine reuptake under stimulated conditions. PMID- 3002820 TI - Increased sensitivity to dopamine agonists following a single dose of morphine or levorphanol in mice. AB - Acute administration of an opiate has been suggested to result in the development of supersensitive dopamine receptors. This hypothesis was tested in mice by determining the effect of a single administration of morphine or levorphanol on dopamine agonist-induced stereotypic behaviors and [3H]spiroperidol binding. Administration of morphine (1.0 mg/kg s.c.), which itself had no significant effect on spontaneous locomotor activity 3 h following administration, significantly potentiated locomotor activity induced by 1.5 or 4.5 mg/kg of apomorphine (i.p.) administered 3 h later. Morphine (10 mg/kg, s.c.) or levorphanol (0.2 and 2.0 mg/kg, s.c.), but not dextrorphan (up to 20 mg/kg, s.c.), enhanced climbing behavior induced by apomorphine (i.p.) or (-)-N-n propylnorapomorphine (i.p.; NPA) administered 3 h later. An increase in whole brain and striatal [3H]spiroperidol binding sites was found 3 h after administration of 10 mg/kg of morphine. Concurrent administration of 5 mEq/kg of LiCl (i.p.) or 5 mg/kg of naloxone (i.p.; administered twice) attenuated both the potentiation of the climbing behavior induced by the two dopamine agonists and the increase in [3H]spiroperidol binding sites. These results suggest that a single administration of an opiate can induce the development of supersensitive dopamine receptors that is mediated by an interaction at opioid receptors. PMID- 3002821 TI - Revertants of Ha-MuSV-transformed MDCK cells express reduced levels of p21 and possess a more normal phenotype. AB - Four subclones of the originally cloned Harvey murine sarcoma virus-transformed Madin Darby canine kidney (MDCK) cells have been isolated. These subclones fall into two general classes. Two subclones have a fibroblastic morphology, have lost the growth requirement for prostaglandin E1 (PGE1), do not respond to glucagon or vasopressin, and, in general, appear transformed. Two other subclones have epithelioid morphologies, are growth-stimulated by PGE1, respond to vasopressin with an increase in intracellular cAMP. We propose that these cells represent revertants to a more non-transformed phenotype. Unlike normal cells, however, these revertants grow under anchorage-independent conditions, express detectable but reduced amounts of the transforming gene product, p21, and grow in nude mice. The appearance of such revertants may be one cause of the observed heterogeneity of tumor cells. PMID- 3002822 TI - Role of Ca2+ and Ca2+-activated protease in myoblast fusion. AB - In this report, we have examined the effects of a calcium chelator, EGTA, and a calcium ionophore, A23187, on fusion of a cloned muscle cell line, L6. Our results confirm that EGTA essentially blocks all myoblast fusion because the lateral alignment of presumptive myoblasts cannot occur in the absence of extracellular calcium. A23187, however, promotes the precocious fusion of myoblasts, apparently by facilitating Ca2+ transport into myoblasts. We have also demonstrated that a Ca2+-activated protease, CAF (mM), appears to relocate in response to the Ca2+ flux, changing from a random, dispersed distribution in proliferative myoblasts to a predominantly peripheral distribution in prefusion myoblasts. Coincident with the mM CAF relocation is an altered distribution of a surface glycoprotein, fibronectin. Extracellular fibronectin is seen in abundance in proliferating myoblasts, but is essentially absent from the surface of fusing myoblasts. We suggest that mM CAF when activated by Ca2+ influx may act to promote the release of fibronectin from the myoblast cell surface, thus providing a mechanism by which the membrane of the fusing myoblast may be rearranged to accommodate fusion. PMID- 3002823 TI - Development of differentiated characteristics in cultured kidney (thick ascending loop of Henle) cells. AB - This study describes the characterization of epithelial cells in culture following their isolation from the thick ascending limb of Henle's loop of rabbit kidney, by enzymatic digestion and subsequent purification using density gradient centrifugation. In culture, these cells expressed a variety of morphological, enzymatic and functional parameters expected of such cells in vivo. These cells were polarised, formed tight junctions and exhibited considerable lateral interdigitation between adjacent cells. They also developed characteristically high levels of activity of Na,K-ATPase, comparable to those seen in freshly isolated cells, and also expressed the functionally important Na,K,Cl-co transport system. The development of these systems in culture, however, was not coincident and their activities were reduced upon extended culture. The ability of these cells to develop and express differentiated characteristics in culture indicates that cells derived from defined kidney cell populations should provide valuable models for the study of the factors involved in the development and regulation of kidney cell type-specific characteristics. PMID- 3002824 TI - Efficient immortalization and morphological transformation of human fibroblasts by transfection with SV40 DNA linked to a dominant marker. AB - Immortal cell lines are essential for genetic and biochemical studies. Unlike rodent cells, which will form continuously growing cultures either spontaneously or after infection with an oncogenic virus (e.g., Simian Virus 40 (SV40)), human cells fail to form continuous cell lines spontaneously and in only rare cases from cell lines after oncogenic virus infection. We have used a plasmid (pSV3gpt) containing both the SV40 early region encoding T antigen and the bacterial gene xanthine-guanine phosphoribosyl transferase (gpt) to achieve high efficiency morphological transformation and immortalization of primary human skin fibroblasts. Transfection of this plasmid into primary human skin fibroblasts derived from a normal individual, two Cockayne's syndrome patients, and an immuno deficient patient and selection for the gpt gene resulted in an altered cell morphology and growth properties characteristic of previously described SV40 transformed cells. Transfected cultures subsequently senesced, entered crisis and in each case formed a rapidly growing culture. The high efficiency of immunortalization described here (four out of four cell strains) is in contrast to previously described procedures utilizing focal overgrowth. We suggest that the use of a dominant selectable marker linked to the SV40 early region increases the probability of establishing an immortal human cell line. PMID- 3002825 TI - Interaction of cAMP with the CDC25-mediated step in the cell cycle of budding yeast. AB - Addition of exogenous cAMP to cultures of the start mutant cdc25-1 of Saccharomyces cerevisiae shifted to restrictive temperature causes a partial reversion of the mutated phenotype, with a marked increase of the percentage of budded cells. This effect is coupled to a progression in the cell cycle, as demonstrated by DNA histograms obtained by flow cytometry. Moreover cdc25 cells have a high intracellular cAMP content also at restrictive temperature, and no change in the cAMP content was seen during a transition from restrictive to permissive temperature. These data suggest that CDC25 gene product allows cell proliferation by interacting with a cAMP-mediated mechanism. PMID- 3002826 TI - Subcellular localization of cryptic prolactin receptors in mammary tumor cells. AB - Primary cultures of carcinogen-induced rat mammary tumors incubated at 37 degrees C with 125I-labeled ovine prolactin (5 ng/ml) accumulate intact prolactin. A steady state is reached at 24--48 h and loss of accumulated prolactin is slow t1/2 24 h). Accumulated prolactin is rapidly released when cryptic prolactin receptors are unmasked by energy depletion, suggesting that accumulated prolactin and cryptic receptors reside in the same cellular compartment. Under normal conditions, the accumulated prolactin is released slowly and is partially degraded. Subcellular fractionation on discontinuous sucrose gradients indicates that cryptic receptors reside in vesicle fractions (p less than or equal to 1.16). After energy depletion, the unmasked receptors are in cell surface membrane fractions (p = 1.18-1.20). Prolactin accumulation within receptor containing vesicles in mammary tumor cells may account for their increased growth sensitivity (compared with normal mammary cells) to low physiologic levels of prolactin. PMID- 3002827 TI - Zinc as a second messenger of mitogenic induction. Effects on diadenosine tetraphosphate (Ap4A) and DNA synthesis. AB - DNA synthesis and adenosine(5')tetraphosphate(5')adenosine (Ap4A) levels decrease in cells treated with EDTA. The inhibitory effect of EDTA can be reversed with micromolar amounts of ZnCl2. ZnCl2 in micromolar concentrations also inhibits Ap4A hydrolase and stimulates amino acid-dependent Ap4A synthesis, suggesting that Zn2+ is modulating intracellular Ap4A pools. Serum addition to G1-arrested cells enhances uptake of Zn, whereas serum depletion leads to a fivefold decrease of the rates of zinc uptake. These results are discussed by regarding Zn2+ as a putative 'second messenger' of mitogenic induction and Ap4A as a possible 'third messenger' and trigger of DNA synthesis. PMID- 3002828 TI - Embryonic myosin heavy chain as a differentiation marker of developing human skeletal muscle and rhabdomyosarcoma. A monoclonal antibody study. AB - Hybridoma cell lines were obtained from the fusion of NS-O myeloma cells with spleen cells of mice immunized with bovine fetal skeletal myosin. A stable hybridoma clone, BF-G6, produced immunoglobulin G1 k antibodies reacting specifically with embryonic-type myosin heavy chains present in fetal but not in neonatal or adult human skeletal muscle, as determined by enzyme immunoassay and immunoblot analysis. Fetal but not adult skeletal muscle fibers were stained by this monoclonal antibody in indirect immunofluorescence assays; smooth muscle cells and cardiac muscle cells, as well as non-muscle cells were also unreactive. Solid tumors of infants and children were tested for reactivity with BF-G6 by immunofluorescence and immunoperoxidase staining. Embryonic myosin heavy chain was expressed in rhabdomyosarcomas but not in other types of tumor, except for Wilms' tumor. Rhabdomyosarcoma cells isolated from a bone marrow metastasis and grown in vitro for several months were also labelled by BF-G6. Embryonic myosin heavy chain can thus be used as a specific differentiation marker of normal and neoplastic skeletal muscle tissue. PMID- 3002829 TI - Reversible cAMP-dependent change in nuclear localization of microtubule associated protein-1 analogues. AB - Intranuclear immunofluorescent staining by monoclonal and polyclonal antibodies against microtubule-associated protein-1 (MAP-1) on SV-3Y1 cells disappeared when the cells were treated with 1 mM db-cAMP and 1 mM theophylline for 20-30 min at 37 degrees C. The nuclear dots of immunofluorescence disappeared and reappeared repeatedly on successive incubation of the cells with and without these drugs. The same phenomenon was induced by treatment of the cells with 6 mM theophylline or 6 mM papaverine which inhibits the cAMP-hydrolysing enzyme. The following results seem to support the hypothesis that cAMP-induced transfer of antigenic molecules from the nucleus to the cytoplasm is mediated by microtubules: Partial staining of the nucleus during the transitional period. Bright staining of the cytoplasm on treated cells in contrast to nuclear staining on control cells. Disappearance of the nuclear staining not only by the monoclonal antibody but also by the polyclonal antibody. Complete prevention of disappearance of nuclear dots induced by these drugs by pretreatment of the cells with colchicine (1 microgram/ml) or colcemid (1 microgram/ml). PMID- 3002830 TI - Growth stimulation by retinol apparently independent of EGF receptor. AB - A respiratory epithelial cell line M3E3/C3, derived from fetal Syrian hamster lung, grew very slowly in RPMI 1640 medium with serum concentrations as low as 1.5%. Under these conditions the simultaneous action of epidermal growth factor (EGF) and vitamin A (retinol) was apparently a combination of growth enhancement attributable to each compound. Both compounds also affected the morphological growth pattern considerably. Binding experiments with 125I-labelled EGF showed that retinol had only a small (if any) reducing effect on the numbers of EGF receptors. Nonetheless, retinol showed a marked growth stimulation. As far as we are able to judge from most of the other reports that retinoids inhibited growth of cells and increased the EGF receptor number, this type of growth regulation is unusual. PMID- 3002832 TI - A 135,000 molecular weight plasma membrane glycoprotein involved in fibronectin mediated cell adhesion. Immunofluorescence localization in normal and RSV transformed fibroblasts. AB - Monospecific antibodies to GP135, a plasma membrane glycoprotein involved in cell attachment and spreading on fibronectin-coated substratum (Giancotti et al., Exp cell res 156 (1985) 182), were affinity-purified from a broad-spectrum antiserum using the GP135 molecule immobilized on nitrocellulose filters. The purified antibodies specifically inhibited attachment and spreading of normal as well as RSV-transformed fibroblasts on fibronectin-coated dishes indicating that GP135 is involved in adhesion of both normal and transformed cells. Immunofluorescence experiments showed that GP135 has a discrete distribution on control 3T3 fibroblasts. On cells in the process of spreading GP135 was concentrated in 'close contact' sites, while on fully spread cells the glycoprotein was concentrated in 'adhesion plaques' and in streaks co-aligning with portions of stress fibres. In RSV-transformed cells the discrete distribution of GP135 was lost. The protein appeared in a diffuse distribution at the plasma membrane and was not concentrated in the dot- or rosette-shaped substratum contacts sites peculiar to these cells. It is concluded that GP135 mediates adhesion of both normal and RSV-transformed fibroblasts to fibronectin. While in normal cells this glycoprotein participates in the organization of specialized adhesion structures, in RSV-transformed fibroblasts recruiting of GP135 molecules within cell substratum contact sites is perturbed. PMID- 3002831 TI - Cellular compartmentation of calcium in mouse fibroblasts in vitro depends on cell density and transformation. AB - Cellular compartmentation of Ca has been investigated by kinetic analysis of 45Ca efflux from preloaded cells at various states of cell density-dependent proliferation of normal (3T3) and transformed (3T6 and SV40-3T3) mouse cells. Three pools of exchangeable calcium were separated on the basis of their differing exchange kinetics. For each of the cell lines tested, all three compartments decrease with cell density. Significant differences between normal and transformed cells are observed upon quiescence of the normal cells, where the slowly exchanging compartment of normal cells gradually increases, whereas that of the transformed cells continues to decrease (with increasing cell density). Free cytoplasmic Ca2+ concentration as determined by the Quin 2 method, was found to be significantly higher in transformed cells than in normal cells. These results indicate significant differences in Ca homeostasis between normal and transformed cells. PMID- 3002833 TI - Expression of CD11, CDw15, and transferrin receptor antigens on human hematopoietic progenitor cells. AB - The expression of transferrin receptor-associated antigens and of CD11 and CDw15 antigens was investigated on myeloid committed progenitor cells (CFU-GM day 10, CFU-GM day 7, and cluster-forming cells [CFC] day 4), on erythroid committed progenitor cells (BFU-E and CFU-E), and on multilineage progenitor cells (CFU GEMM). Both complement-dependent cytotoxicity and fluorescence-activated cell sorting assays were performed. Complement-dependent cytotoxicity appeared to be the more sensitive assay. Transferrin receptor-associated antigens appeared to be clearly present on all myeloid and erythroid committed progenitor cells, but were found to be only weakly expressed on CFU-GEMM. CD11 antigens appeared to be strongly expressed only on mature granulocytes, monocytes, and certain lymphocytes, but not significantly on myeloid committed precursor cells. Surprisingly, CD11 antigens were weakly, but significantly, present on CFU-E. CDw15 antigens appeared to be restricted to myeloid differentiation and were increasingly expressed from CFU-GM day 10 to CFC day 4. Thus, antitransferrin receptor, CD11, and CDw15 antibodies can be used to separate hematopoietic progenitor cells and may be useful tools in the study of hematopoietic differentiation. PMID- 3002834 TI - Connections between pericruciate cortex and the medullary reticulospinal neurons in cat: an electrophysiological study. AB - The connections between the pericruciate cortex and the medullary reticulospinal (RS) neurons were studied in anesthetized cat. Intracellular recordings were made from reticulospinal neurons and the effects of stimulating different areas of the pericruciate cortex were compared. EPSPs were elicited in all the 93 neurons studied which were antidromically activated by spinal stimulation and had an IS SD notch on the ascending limb of their antidromic spikes. According to the conduction velocity (c.v.) of the axon and the minimal EPSP latency to cortical stimulation, the neurons could be divided into two groups, i.e. fast-conducting RS neurons (FRS neurons, c.v. greater than 45 m/s) and slow-conducting RS neurons (SRS neurons, c.v. less than 45 m/s). The minimal latencies of FRS neurons were equal to or shorter than 2 ms whereas those of SRS neurons were longer than 2 ms. EPSPs with short latency (less than 2 ms) could be evoked in FRS neurons by stimulating a relatively wide cortical area including the major part of precruciate area 4 and area 6, with a central area of strongest excitatory effect located in area 4 slightly medial to the tip of the cruciate sulcus. Stimulation of the postcruciate area 4 only produced long latency EPSPs. By extrapolation from the cortical and peduncular latencies and the conducting distances it was revealed that the earliest part of the minimal latency EPSPs were monosynaptically evoked in FRS neurons and were mediated by fast-conducting corticobulbar fibers. FRS neurons could be excited by stimuli applied to both ipsilateral and contralateral pericruciate cortex. The influence from the contralateral cortex was slightly stronger. PMID- 3002835 TI - GABAergic neurons comprise a major cell type in rodent visual relay nuclei: an immunocytochemical study of pretectal and accessory optic nuclei. AB - The enzyme glutamic acid decarboxylase (GAD) has been localized in sections of rodent brains (gerbil, rat) using conventional immunocytochemical techniques. Our findings demonstrate that large numbers of GAD-positive neurons and axon terminals (puncta) are present in the visual relay nuclei of the pretectum and the accessory optic system. The areas of highest density of these neurons are in the nucleus of the optic tract (NOT) of the pretectum, the dorsal and lateral terminal accessory optic nuclei (DTN, LTN), the ventral and dorsal subdivisions of the medial terminal accessory optic nucleus (MTNv, MTNd), and the interstitial nucleus of the posterior fibers of the superior fasciculus (inSFp). The findings indicate that 27% of the NOT neurons are GAD-positive and that these neurons are distributed over all of the NOT except the most superficial portion of the NOT caudally. The GAD-positive neurons of the NOT are statistically smaller (65.9 microns2) than the total population of neurons of the NOT (84.3 microns2) but are otherwise indistinguishable in shape from the total neuron population. The other visual relay nuclei that have been analyzed (DTN, LTN, MTNv, MTNd, inSFp) are similar in that from 21% to 31% of their neurons are GAD-positive; these neurons are smaller in diameter and are more spherical than the total populations of neurons. The data further show that a large proportion of the neurons in these visual relay nuclei are contacted by GAD-positive axon terminals. It is estimated that approximately one-half of the neurons of the NOT and the terminal accessory optic nuclei receive a strong GABAergic input and have been called "GAD-recipient neurons". Further, the morphology of the GAD-positive neurons combined with their similar distribution to the GAD-recipient neurons suggest that many of these neurons are acting as GABAergic, local circuit neurons. On the other hand, the large number of GAD-positive neurons in the NOT and MTN (20-30%) in relation to estimates of projection neurons (75%) presents the possibility that some may in fact be projection neurons. The overall findings provide morphological evidence which supports the general conclusion that GABAergic neurons play a significant role in modulating the output of the visually related NOT and terminal accessory optic nuclei. PMID- 3002836 TI - Cytochrome oxidase activity in the striate cortex and lateral geniculate nucleus of the newborn and adult macaque monkey. AB - The laminar location of cytochrome oxidase staining has been compared in the lateral geniculate nucleus and area 17 in newborn and adult macaque monkeys. In area 17 of the adult, the distribution of cytochrome oxidase activity confirmed published findings. In the newborn animals, the tissue reacted as strongly for cytochrome oxidase as in the adult but the pattern of labelling was different in two respects. Firstly in layer 1 activity was stronger and occupied a wider portion of this layer. Secondly, cytochrome oxidase staining in layer 4C occupied two separate bands, a small narrow band at the bottom of 4C beta and a wider one occupying the full width of 4C alpha and spilling over into 4B. The pattern of cytochrome oxidase activity did not appear to be influenced by eccentricity in the newborn whereas, in the adult, label in 4C was more intense in cortex subserving central vision. In the lateral geniculate nucleus of the adult, the magnocellular layers and the most dorsal parvocellular layer reacted most strongly for cytochrome oxidase. In the newborn, parvocellular layers were more uniformly labelled and the difference between parvo- and magnocellular layers more pronounced. These results are discussed in relationship to the development of thalamo-cortical projections in the monkey. PMID- 3002838 TI - Effects of hypoxia on rat brain metabolism: unilateral in vivo carotid infusion. AB - An in vivo brain perfusion technique was used to examine effects of hypoxia on cerebral cortical metabolism in barbiturate-anesthetized rats. Dulbecco's phosphate-buffered solution (PBS), or Dulbecco's PBS + 6 mM glucose, was infused into the right carotid circulation for 0 to 3 min, at a rate that reduced regional cerebral blood flow to the ipsilateral parietal lobe by more than 40% and O2 delivery by about 50%. The duration of infusion of either solution was correlated negatively with the ipsilateral parietal lobe concentrations of glucose, ATP, and phosphocreatine (PCr), and positively with parietal concentrations of lactate and cAMP. cGMP increased in relation to infusion duration of Dulbecco's PBS. Statistically significant elevations of brain lactate occurred after 1 min of infusion of Dulbecco's PBS; lactate was elevated and glucose was reduced after 2 min of infusion of either solution. Brain ATP, PCr, and glycogen concentrations decreased in relation to the elevation in brain lactate, and the [PCr]:[ATP] ratio declined. The results demonstrated that limited hypoxia stimulated cerebral glycolysis and produced a concurrent decrease in brain ATP and PCr. However, ATP was spared to a degree, at the expense of PCr. PMID- 3002837 TI - Direct excitatory and inhibitory synaptic inputs from the medial mesodiencephalic junction to motoneurons innervating extraocular oblique muscles in the cat. AB - This study investigated the nature of synaptic inputs from the Forel's field H (FFH) in the medial mesodiencephalic junction to inferior oblique (IO) motoneurons in the oculomotor nucleus and superior oblique (SO) motoneurons in the trochlear nucleus in anesthetized cats, using intracellular recording techniques. Stimulation of the FFH induced monosynaptic EPSPs in IO motoneurons on both sides. Paired stimulation of the ipsilateral FFH and contralateral vestibular nerve substantiated that the FFH-induced EPSPs were caused mainly by direct excitatory fibers from the FFH to IO motoneurons and partly by axon collaterals of excitatory neurons in the vestibular nuclei. Among parts of the FFH, the medial part was most effective for producing the EPSPs. Systematic tracking with the stimulating electrode in and around the FFH revealed that effective sites of stimulation inducing negative field potentials in the IO subdivision of the oculomotor nucleus, identified as extracellular counterparts of the EPSPs in IO motoneurons, were also located in the interstitial nucleus of Cajal, nearby reticular formation and posterior commissure, besides within and near the medial part of the FFH. Areas far rostral, dorsal and ventral to the FFH were ineffective. EPSP-IPSPs or EPSPs were mainly induced in SO motoneurons on both sides by FFH stimulation. Latencies of these EPSPs and IPSPs were close to those of the EPSPs in IO motoneurons, indicating their monosynaptic nature. Effective stimulation sites for inducing these synaptic potentials overlapped those for the EPSPs in IO motoneurons. Based on these results, it was suggested that excitatory and inhibitory premotor neurons directly controlling IO and SO motoneurons were located within and near the medial part of the FFH. PMID- 3002839 TI - Direct projection of type II vestibular neurons to eye movement-related pause neurons in the cat pontine reticular formation. AB - Brain stem pause neurons play an important role in the regulation of rapid eye movements. However, the input sources that drive pause neurons are uncertain. In the present study, horizontal canal type II neurons in the medial vestibular nucleus were antidromically activated by electrical stimulation of the pause neuron region. Systematic microstimulation tracks within that region showed an antidromic activation pattern of low-threshold sites separated by high-threshold sites consistent with axonal branching of type II neurons in the pause neuron region. Spike-triggered averaging of single spontaneously firing type II vestibular neuronal discharges in the pause neuron region resulted in short latency, positive field responses. These results supported the conclusion that horizontal canal type II neurons of the medial vestibular nucleus project to and inhibit pause neurons monosynaptically. PMID- 3002840 TI - Electrophysiologic identification of preganglionic neurons in rat dorsal motor nucleus and analysis of vagus afferent projections. AB - Electrophysiological studies on preganglionic neurons (PGNs) in the dorsal motor nucleus (DMN) of the vagus nerve has been hampered by technical limitations. Conventional electrical stimulation of the vagus nerve with cathodal square-wave pulses activates both preganglionic and afferent fibers. Thus, some PGNs cannot be identified because the anticipated antidromic responses would be blocked due to collision with those orthodromic responses evoked with shorter latencies by activation of fast-conducting afferent fibers. Projections of vagus afferent fibers to PGNs are difficult to analyse with conventional methods because a preceding antidromic response may affect an orthodromic response which has a slightly longer latency. A new stimulation method was designed, consisting of anodal triangular pulse stimulation and spontaneous-spike triggered stimulation. In chlorase-urethane-anesthetized rats, unitary responses of the DMN to electrical stimulation of the ipsilateral vagus nerve were recorded. When only an orthodromic response by a DMN neuron was recorded with conventional stimulation, application of anodal triangular pulse stimulation revealed an antidromic response, so that the cell in question could be identified as a PGN. Some neurons that produced only an antidromic response to conventional stimulation, revealed an orthodromic response on spontaneous-spike triggered stimulation, which blocked the antidromic response due to collision. With these procedures, orthodromic responses due to vagus afferent projections were recorded in 35% of the identified PGNs, mostly due to C and partly, A fiber activations. All these projections were polysynaptic in nature. In conclusion, one-third of the PGNs of the DMN are involved in vagovagal reflexes, which occur through multisynaptic pathways. PMID- 3002841 TI - Ethanol influence on insulin secretion from isolated rat islets. AB - This study was done to delineate the role of alpha- and beta-adrenergic receptors and cyclic AMP in the mechanism of ethanol effects on insulin release from isolated islets. Rats were given an alpha-adrenergic blocker, phentolamine, or a beta-adrenergic blocker, propranolol. In addition, ethanol 1 g/kg was given intragastrically 1 h prior to sacrifice. Glucose mediated insulin release from isolated islets was enhanced by phentolamine and decreased by propranolol. Ethanol treatment inhibited glucose-induced insulin release from isolated islets of control rats as well as those given phentolamine and/or propranolol. Insulin release from isolated islets in response to dibutyryl-cyclic AMP was attenuated by ethanol. Theophylline enhanced glucose mediated insulin release from control islets but ethanol treatment produced a significant inhibition of insulin response. The data suggest that the site of action of the deleterious effects of ethanol on insulin release from isolated islets in rat does not involve adrenergic system and cyclic AMP. PMID- 3002842 TI - Detection of pyrimidine 5'-nucleotidase deficiency using 1H- or 31P-nuclear magnetic resonance. AB - We describe here a further Japanese family with pyrimidine 5'-nucleotidase (P5'N) deficiency diagnosed using a nuclear magnetic resonance (NMR) spectrum, in Kumamoto prefecture where two families having the disease have been reported before. The specific spectra in 1H-NMR of P5'N deficient erythrocytes were due to three methyl protons of CDP-choline at 3.22 ppm and to H-2, H-8 and ribose-1' of pyrimidine nucleotide phosphate(s) in the lower fields (at 5.82 and 8.00 ppm). The other specificities in 31P-NMR spectra were due to CDP-choline, CDP ethanolamine and UDP-glucose. Those spectra were not detected in other types of hemolytic anemia. PMID- 3002843 TI - [Effect of phenobarbital on the kinetics of the change in the level of paramagnetic metalloprotein complexes in dinitrophenol poisoning]. AB - EPR spectroscopy was used to study phenobarbital influence on the kinetics of paramagnetic complexes variability of metal proteins--cytochrome P-450 and iron sulfur proteins of rat liver in acute oral poisoning with the dinitrophenol pesticides DNOK and dinoseb. It was proved experimentally that the barbiturate favoured marked prevention of the decrease of the content of cytochrome P-450 and iron-sulfur proteins, thereby protecting the detoxifying and energy liver system from dinitrophenol-induced injuries. It is discussed that phenobarbital may act both as an inducer of cytochrome P-450 and iron-sulfur proteins involved in electron transport of the mitochondria. PMID- 3002844 TI - [Pharmacological analysis of the participation of the catecholaminergic system in the development of the abstinence syndrome in rats]. AB - The behavioural activity of rats in "the open field" was studied. It was revealed that rats alcoholized for 8 months do not practically differ in their behavioural indicators from the intact ones. After the discontinuation of alcohol marked disturbances appear in their behaviour, that are arrested by apomorphine (0,1 mg/kg). In intact animals dopamine (50 mkg into the brain ventricles) induces behavioural disorders similar to those in rats during abstinence. Noradrenaline does not induce similar disorders. A conclusion is made on the dopaminergic nature of disorders in the behaviour of rats in the state of alcohol abstinence. PMID- 3002846 TI - [Delayed neurotoxic action of a new fungicide aphos]. AB - It has been established that administration of aphos to chickens within a wide dose range (3000-25 mg/kg) produces a retarded neurotoxic action that is manifested by the development of ataxia, pareses and paralyses; delay in the rate of the distribution of excitation via the peripheral axons, disorders of myoneural lability; degeneration of myelin fibers, focal granular degradation of myelin. The retarded neurotoxic action is not caused by impurities contained by the technical-grade preparation in view of the fact that such an action is also common to the chemically pure aphos. PMID- 3002845 TI - [Synergism of picrotoxin with the endogenous convulsants kynurenine and quinolinic acid and its antagonism with their antagonists]. AB - In experiments on mice the existence of common mechanisms of action was revealed in picrotoxin and kynurenine and quinolinic acid. Picrotoxin selectively potentiated convulsions induced by kynurenine and quinolinic acid and the antagonists of these endogenous convulsants--picolinic, kynurenic and xanthurenic acids--selectively antagonized picrotoxin convulsions. GABA-ergic preparations, effective in kynurenine convulsions and the anticonvulsants diazepam and phenobarbital, were highly effective against picrotoxin-induced convulsions also. Puphemid and trimethin indicated for treating petit mal epilepsy, proved ineffective. The obtained data suggest the possibility of action of kynurenine and quinolinic acid via picrotoxin-sensitive subunits of the benzodiazepine-GABA receptor-ionophore complex. PMID- 3002847 TI - [Action of alcoholic intoxication in rats during lactation on the function of the hypothalamo-hypophyseal-adrenal system in the progeny]. AB - The authors review the effect of alcoholic intoxication of mother's body during lactation on hypothalamo-hypophyseal-adrenocortical function (HHACF) of the offspring both at rest and during performance of muscle exercise of medium duration. During lactation, alcoholic intoxication disturbs the normal development of HHACF), with the disturbances being persistent and aggravated with age. The offspring of rats exposed to alcoholic intoxication responded to muscle exercise by a significant lowering of all the parameters under study. Of paramount importance are two concepts derived from the studies described. First, it is clearly seen that abnormalities are experienced both by the adrenal cortex and the central component of HHACF--the hypothalamic control of the corticotropic function of the hypophysis. Second, the data obtained enrich the concepts about the adverse action of alcohol on the offspring (alcohol intake by lactating mothers). PMID- 3002848 TI - Search for new Ca++ antagonists. Lipophilic oximes and phosphonates. AB - On the basis of the reported slow channel-inhibiting activity of oximes and phosphonates a series of compounds bearing such functions on lipophilic carriers were synthesized and studied. Among the compounds studied only (VI b) shows a slight cardiovascular activity. The surprising lack of activity of diethyl 3,4 dimethoxybenzyl-phosphonate (XIV), which is closely related to Fostedil, is discussed. PMID- 3002849 TI - Mobilization of intracellular calcium by glucagon and cyclic AMP analogues in isolated rat hepatocytes. AB - Separate or combined addition of cyclic AMP-dependent and Ca2+-linked hormones to isolated rat hepatocytes suspended in a low Ca2+ medium reduced the total cellular Ca. When the hormones were administered together, their effects were not additive. This suggests that both types of hormones mobilize Ca2+ from a common intracellular pool. In the presence of 1.8 mM extracellular Ca2+, the Ca2+ influx counterbalanced or even exceeded the hormone-induced Ca2+ loss, depending on the ability of the hormones to stimulate the Ca2+ influx. PMID- 3002850 TI - Incorporation of synthetic 1,2-diacylglycerol into platelet phosphatidylinositol is increased by cyclic AMP. AB - 1,2-Didecanoylglycerol (diC10) is taken up by human platelets and sequentially converted to 1,2-didecanoylphosphatidic acid (PA10) and 1,2 didecanoylphosphatidylinositol (PI10). Agents that increase cyclic AMP in platelets, such as prostacyclin and forskolin, sequentially convert diC10 to PA10 and PI10. They decrease formation of PA10 with a parallel accumulation of PI10. This might reflect an inhibition of phosphatidylinositol kinase. PMID- 3002851 TI - Organization of the human genes for insulin-like growth factors I and II. AB - Recently, we have reported the isolation of cDNAs encoding the precursors of insulin-like growth factors I and II (IGF-I and II) [(1983) Nature 306, 609-611; (1985) FEBS Lett. 179, 243-246. These cDNAs were employed as specific probes to detect and isolate the corresponding genes from human cosmid DNA libraries. Three cosmids were detected, together containing the entire cDNA sequence of IGF-I, and one cosmid containing the sequence of IGF-II cDNA. Southern blot hybridization, physical mapping and nucleotide sequence analysis of these cosmids revealed that the IGF-I and -II genes have a discontinous structure. The IGF-I gene contains at least four exons spanning a region of probably more that 45 kilobasepairs (kb), while the IGF-II gene consists of at least five exons, spanning a region of 16 kb. PMID- 3002853 TI - Evidence for genetic divergence in ribosomal RNA genes in mycobacteria. AB - DNA was isolated from Mycobacterium phlei and from M. smegmatis. Each DNA sample was restricted with endonucleases, the fragments were separated by agarose gel electrophoresis and transferred to nitrocellulose film. Fragments of DNA containing rRNA sequences were identified by means of 125I-labelled rRNA of M. phlei or of M. smegmatis. The distributions of restriction endonuclease sites within the rRNA gene(s) and flanking sequences were found to be characteristic for each of the two species. Hybridizations with heterologous probes indicate that although M. phlei rRNA and M. smegmatis rRNA share regions of sequence homology, they are probably not identical in primary structure. The results suggest that the rRNA genes might prove to be useful taxonomic markers for mycobacteria. PMID- 3002852 TI - Genomic organization of rDNA in Pseudomonas aeruginosa. AB - We have examined the number and organization of rRNA genes in Pseudomonas aeruginosa by hybridization of restriction nuclease digests of genomic DNA to 3' 32P-labelled 23 S, 16 S and 5 S rRNAs and corresponding labelled DNA from the rrnB operon of Escherichia coli. The immediate conclusion from these hybridization data is that there are 4 transcriptional units coding for rDNA in P. aeruginosa. We report here a putative model of the genomic organization of all 4 rDNA operons. PMID- 3002854 TI - Origin of sequence-specific recognition of DNA by non-intercalating anti-tumor antibiotics. AB - Partitioning of energy in the interaction of non-intercalating antibiotics (netropsin, netropsin without its cationic ends and two analogs of distamycin A) with different base sequences of B-DNA is studied here by the atom-atom potential technique and geometry optimization procedures. The results show that electrostatic forces contribute substantially to the stabilization energy as well as to the sequence specificity. The hydrogen-bonding term is also sequence specific and is significant in properly orienting the drug molecule. Relative roles of the hydrogen bonding and electrostatic interactions depend on the dielectric property of the medium. PMID- 3002855 TI - Effect of 2,4-dinitrofluorobenzene on the enzymatic properties of the b-c1 complex isolated from beef heart mitochondria. AB - A study is presented on the effect of 2,4-dinitrofluorobenzene (DFNB) on the enzymatic properties of mitochondrial b-c1 complex. The chemical modification by DNFB strongly inhibits the reductase activity of the complex, this being accompanied by labelling by [3H]DNFB of core protein I, the apoprotein of b cytochromes and the 12 kDa subunit. Chemical modification by DNFB appears to alter, in particular, the domain of heme b-562. PMID- 3002857 TI - Cloning and DNA sequence of the 5'-exonuclease gene of bacteriophage T5. AB - The nucleotide sequence of the BalI-PstI fragment of T5 DNA, 1347 bp in length, coding for 5'-exonuclease (D15 gene), has been determined. A coding region of the gene contains 873 bp and is preceded by a typical Shine-Dalgarno sequence. The D15 gene belongs to a cluster, consisting of at least 3 genes, in which a termination codon of a preceding gene overlaps an initiation codon of the following one. The sequence contains an open reading frame for 291 amino acid residues. The molecular mass of the 5'-exonuclease calculated from the predicted amino acid sequence is 33 400 Da. PMID- 3002856 TI - Alpha 1-adrenergic activation of brown adipocytes leads to an increased formation of inositol polyphosphates. AB - alpha 1-Adrenergic activation of isolated brown adipocytes causes a rapid mobilization of intracellular Ca2+. The cells also respond with an increased turnover of inositol lipids. The present work demonstrates that alpha 1 adrenergic stimulation of brown adipocytes results in phospholipase C-mediated breakdown of phosphatidylinositol bisphosphate to form inositol trisphosphate. The rate of appearance of inositol trisphosphate is sufficiently rapid for it to mediate or contribute to Ca2+ mobilization in these cells. PMID- 3002858 TI - The extracellular matrix proteins laminin and fibronectin modify the AMPase activity of 5'-nucleotidase from chicken gizzard smooth muscle. AB - Laminin and fibronectin, but not collagen, affect the AMPase activity of the purified transmembrane protein 5'-nucleotidase. Laminin stimulates whereas fibronectin inhibits the AMPase activity of this ectoenzyme. The AMPase modulating effects by these components of the extracellular matrix require a preincubation period of several hours when detergent-solubilized 5'-nucleotidase is employed, they can, however, instantaneously be elicited with liposome incorporated 5'-nucleotidase. PMID- 3002859 TI - Evidence for a diprotomeric structure of Na,K-ATPase. Accurate determination of protein concentration and quantitative end-group analysis. AB - Three methods were used to assess protein concentration in membrane-bound Na,K ATPase preparations: standard Lowry assay, Kjeldahl nitrogen determination and amino acid analysis. While the first two methods showed excellent agreement, the third one always gave a lower value which varied drastically depending on the condition of sample treatment before amino acid analysis. This result reinforces the Lowry method in assessing the true concentration of Na,K-ATPase protein and suggests 250 kDa to be a true estimate of the molecular mass of the smallest ligand-binding unit of the enzyme. The cyanate method reveals two NH2-terminal residues of the beta-subunit (NH2-Ala) and one such residue of the alpha-subunit (NH2-Gly) per ligand-binding unit. From the data on equimolarity of the alpha- and beta-subunits in Na,K-ATPase this suggests that the enzyme molecule is composed of two alpha beta-protomers, one possessing a modified (presumably an N blocked) alpha-subunit. PMID- 3002860 TI - The protein kinase C activator phorbol-12-myristate-13-acetate enhances cyclic AMP accumulation in pheochromocytoma cells. AB - The protein kinase C activator, phorbol-12-myristate-13-acetate (PMA), augments the cyclic AMP accumulation induced by forskolin in pheochromocytoma (PC 12) cells with an EC50 value of 14 nM, while having no effect on basal values. At a concentration of 100 nM PMA markedly augmented the magnitude of the forskolin response and, in addition, caused a slight increase in the potency of forskolin. PMA also enhanced the maximal cyclic AMP accumulation produced by 2 chloroadenosine, and caused a slight increase in potency of the adenosine analog. Since PMA mimics the effect of diacylglycerols that form during the turnover of the membrane lipid, phosphatidylinositol, the results suggest an interrelationship between the systems involved in phosphatidylinositol turnover and cyclic AMP generation in PC 12 cells. PMID- 3002862 TI - The activity of the Na+/H+ antiporter in cultured cardiac cells is dependent on the culture conditions used. AB - When chick cardiac cells are maintained in serum-free NCTC-135 medium, the pHi dependence of the Na+/H+ exchanger is much more acidic (pK = 7.0) than when a serum-free M199 medium is used (pK greater than 7.5). Phorbol esters activate the Na+/H+ exchanger and produce a cell alkalinization in cells maintained in NCTC 135 medium by shifting the pHi dependence of the system to more alkaline values. They have no action on cells maintained in M199 medium. The alkaline pHi dependence of the Na+/H+ exchanger and the loss of responsiveness of phorbol ester action in M199 medium correlate with increased rates of phosphatidylinositol turnover. PMID- 3002861 TI - Autophosphorylation of calmodulin kinase II: functional aspects. AB - Autophosphorylation of purified calmodulin kinase II dramatically inhibited protein kinase activity and enhanced substrate selectivity. Inhibition was observed over a wide range of calmodulin concentrations but calmodulin binding was unaffected. Autophosphorylation of calmodulin kinase II may be a mechanism for limiting phosphorylation to physiological substrates and terminating some of calcium's actions in synaptic events. PMID- 3002863 TI - Characterization and quantitative topographical distribution of salmon calcitonin binding sites in rat kidney sections. AB - Renal binding sites for labelled salmon calcitonin (sCT) were studied using cryostat sections and autoradiography. Increasing concentrations of unlabelled sCT inhibited 125I-sCT binding. 125I-sCT bound to a single site with a Kd of 2 nM and a number of sites of 220 fmol/mg protein. Mammalian calcitonins had low affinities and peptides unrelated to CT were devoid of any significant affinity for 125I-sCT receptors. Autoradiograms disclosed a high concentration of 125I-sCT receptors mainly located in the outer medulla and heterogeneously in the renal cortex. The distribution of specific binding sites is in agreement with the current concepts of renal action of calcitonin. PMID- 3002864 TI - Interaction amongst calcineurin subunits. Stimulatory and inhibitory effects of subunit B on calmodulin stimulation of subunit A phosphatase activity depend on Mn2+ exposure of the holoenzyme prior to its dissociation by urea. AB - Calcineurin was dissociated into subunits A and B by 6 M urea in the presence (method A) and absence (method B) of MnCl2 and dissociated subunits were isolated by gel filtration in urea in the absence (method B) or presence (method A) of MnCl2. Phosphatase activity was associated with the A subunit isolated by either method. The phosphatase activity (nmol/mg) of subunit A isolated by method A was greater (2-5-fold) than by method B. Mn2+ increased subunit A phosphatase and calmodulin further increased the enzyme activity. Subunit B isolated by method A or B increased Mn2+ + calmodulin stimulated subunit A phosphatase prepared by method B but interestingly and unexpectedly inhibited such stimulated activity of the subunit A prepared by method A. These results imply the tightly bound cation (in our case, most likely Mn2+) with subunit A dramatically and differentially influences the effects of two Ca2+-binding proteins, calmodulin and subunit B, on the subunit A phosphatase. PMID- 3002866 TI - [Relation between adrenocorticotropic and glucocorticoid responses in the dog]. AB - In chronic experiments on dogs, changes of adrenocorticotropic and glucocorticoid activities of blood plasma were studied under the effect of factors activating the hypothalamo-hypophyseal-adrenal system (morphine, electrical stimulation of the hypothalamus, surgical lesions). Similar as the general dynamics was, there was no complete analogy of the above two indices as the ACTH and cortisol (ACTH activity and 17-OCS in some experiments) attain the maximal concentrations not always simultaneously, the character of changes can be opposite while in similar changes their intensity can be rather different. The data obtained disagree with the concept of exclusive dependence of the glucocorticoids secretion on the adenohypophysis. Apart from the adrenocorticotropic hormone, additional factors of extrahypophyseal origin seem to exist and affect the glucocorticoid response. PMID- 3002865 TI - Nutritional factors in lung, colon, and prostate carcinogenesis in animal models. AB - Dietary factors are now considered to be among the most important environmental risk determinants for cancer. In addition to epidemiological studies, experimental animal studies are an important tool to investigate dietary modulation in carcinogenesis. Results of recent experimental studies on the effect of some nutrients indicate that vitamin A did show an inverse relation with the occurrence of preneoplastic respiratory lesions but not with respiratory tract tumors in benzo[a]pyrene-induced respiratory carcinogenesis. Dietary fat increases respiratory tract tumors and preneoplastic lesions. In colon carcinogenesis, a fat-fiber interrelation was noticed in 1,2-dimethyl-hydrazine- and N-methyl-N'-nitro-N-nitrosoguanidine-induced tumors. Preliminary results in prostate carcinogenesis indicate that dietary fat did not influence the incidence of prostate cancer in a recently developed rat model. Some possible mechanisms in colon and prostate carcinogenesis are discussed. PMID- 3002867 TI - [Adrenoreactivity of different portions of the vas deferens in the rat]. AB - Adrenoreactivity of rat vas deferens was shown to vary along its length. These variations are manifested in a greater reaction of epididymal part to adrenomimetic substances and transmural stimulation and are due to a higher sensitivity and greater number of alpha-adrenoreceptors in the epididymal part. Different populations of alpha-adrenoreceptors seem to exist in the rat vas deferens, with the population of receptors having a greater affinity for noradrenaline concentrated in the epididymal part. PMID- 3002868 TI - [Cyc1AMP levels and effect of calcium regulating hormones]. AB - Effects of calcitonin and parathyroid hormone on the functional activity of contractile cardiomyocytes were studied against the background of theophylline blockade of phosphodiesterase, as well as effects of calcitonin on activity of contractile and pace-maker cardiomyocytes and of the parathyroid hormone on activity of contractile myocytes in conditions of adrenaline stimulation of adenyl cyclase. The data obtained show a difference in effects of calcitonin and parathyroid hormone upon the myocardium under conditions of an increased intracellular level of cAMP induced with theophylline and adrenaline. PMID- 3002869 TI - [Distribution of adrenoreceptors in the microcirculatory components of the circulatory bed of the kidney in the cat]. AB - Topography of alpha- and beta-adrenoreceptors and their significance for smooth muscle cells of various links of the arterial division of the cat kidney blood channel were studied by means of alpha- and beta-adreno-stimulators and adrenoblockers administration under the control of general arterial pressure. The initial divisions of the major arteries and arterioles and their primary branches of the kidney fibrous capsule, distal divisions of afferent glomerulus vessels and their corresponding precapillary arterioles as well as the initial divisions of the straight arterioles of the kidney substance medullaris were shown to contain beta-adrenoreceptors. Alpha-adrenoreceptors are present in all other extra-parenchymatous and parenchymatous arteries and arterioles. PMID- 3002871 TI - [Role of the pituitary in modulating the effect of the dopaminergic and serotoninergic systems on the immune response]. AB - Dissection of the pituitary stalk prevented the changes of the immune response caused with drugs influencing the activity of dopaminergic and serotoninergic systems. Stimulating effect of the dopamine receptor agonist--apomorphine, and inhibitory effect of blocking agent for these receptors--haloperidol on the immune response were not manifest in animals after lesion of the hypothalamo- pituitary stalk. The inhibitory action of serotoninergic system on the immunity (administration of serotonin and its precursor--5-hydroxytryptophan) was also prevented by interrupting the link between the hypothalamus and the hypophysis. The data obtained suggest central action of dopaminergic and serotoninergic systems on the immunity and indicate the important role of hypophysis in neuroimmunomodulation. PMID- 3002870 TI - [Effect of gamma-aminobutyric acid on neurons of the spinal lymphatic center]. AB - In the frog isolated perfused 7-11th spinal cord segments with the lymphatic center, GABA inhibited both the activity of pace-maker neurons and the burst-type activity of lymphatic motoneurons. Picrotoxin, bicuculline, ammonia and furosemide reversibly blocked the inhibition induced with GABA. The data obtained suggest existence of GABA-ergic control of the lymphatic center activity in the 9 11th segments of the spinal cord. PMID- 3002873 TI - 5'-nucleotidase activity in the spinning gland cells of Diacrisis obliqua (Arctiidae: Lepidoptera). AB - 5'-nucleotidase activity in the spinning gland cells of an Indian moth, Diacrisia obliqua has been studied for the first time during prefunctional, functional and postfunctional phases of development. The enzyme seems to play an active role in the metabolism of silk secretion. Differential utilization of 5'-nucleotidase substrate in the growing and degenerating cells has been studied and discussed. PMID- 3002872 TI - The physiology of the major histocompatibility complex. AB - The term compound receptors (C.R.) is used here to describe reversible molecular complexes in the cell membrane which attain their final biologically active structure by rearrangement and assembly of several structural subunits. The C.R. to be discussed involve the participation of the glycoproteins belonging to the major histocompatibility complex (MHC), notably of class I molecules which are themselves reversible compounds of a heavy chain and a light chain (beta 2-m). The main thesis of this discussion is the postulate that the fundamental immunological phenomenon known as MHC restriction is due to the formation in the membrane of reversible C.R. with additional roles in the physiology of the cell. The interaction between MHC class I molecules and insulin receptor molecules will be mentioned as an illustration of the general hypothesis. PMID- 3002875 TI - [Enzyme immunoassay for serum 18-hydroxycorticosterone and its clinical application]. AB - An enzyme immunoassay for serum 18-hydroxycorticosterone was established using alkaline phosphatase as a label. The antiserum for 18-hydroxycorticosterone was produced by immunization of rabbits with 18-hydroxycorticosterone 3-(o carboxymethyl) oxime conjugated to bovine serum albumin. Sephadex LH-20 column chromatography was used to separate 18-hydroxycorticosterone from other steroids in serum samples. The minimal detectable amount of 18-hydroxycorticosterone was 50 pg/tube, and the measurable range was from 5 to 1000 ng/dl when a 1.0 ml serum sample was used. Intra- and inter-assay coefficients of variance were 5.0% (n = 6), and 5.8% (n = 6), respectively. In normal controls the serum 18 hydroxycorticosterone level was 4.8 approximately 34.0 ng/dl (mean +/- S.D. = 17.1 +/- 9.0 ng/dl) on an unrestricted diet. Seven out of 8 patients with aldosterone-producing adenoma had above-normal serum 18-hydroxycorticosterone levels. Serum 18-hydroxycorticosterone increased and decreased significantly following ACTH and dexamethasone administration, respectively. In essential hypertensive patients, serum 18-hydroxycorticosterone was high during a low sodium diet and was suppressed remarkably by captopril. These observations support the previous reports that adrenal 18-hydroxycorticosterone synthesis is dependent on both ACTH and the renin-angiotensin system. The present method is sufficiently sensitive and producible, avoids the use of radioisotopes and is quite satisfactory for clinical use. PMID- 3002874 TI - The morphology and histochemistry of adult rats neurocytes after BCNU administration. AB - Studies were performed on adult rats of Wistar strain given four 7-days-spaced intraperitoneal doses of BCNU with single dose resembling to used in clinical practice. The animals were sacrificed at the seventh day after the last dose of the drug. Morphological alterations were evaluated in H + E or cresyl violet stained sections. In frozen microtome sections histoenzymatic reactions were performed to detect enzymatic activity of some phosphatases and esterases. Karyo- and cytophotometric measurements of pyramidal cell nuclei in frontal and parietal cortex and of motor neurons in trigeminal nerve nucleus were performed in sections subjected to Feulgen reaction, using automatic microscopic image analyzer "Morphoquant" (VEB Carl Zeiss, Jena). The performed studies showed that administration of multiple therapeutic doses of BCNU lead to primary injury of vascular wall as oedema and proliferation of endothelium and small perivascular haemorrhages. The cytostatic drug induced a decrease in NsE and AlkP enzymatic activities, increased activity of AChE, ChE, AcP and ATPase and topographically variable changes in intensity of TPPase enzymatic reaction. Several karyo- and cytophotometric alterations were observed also in neurocyte cell nuclei which became elongated and acquired a more round shape. This was associated with a decrease in relative DNA content, loosening of nuclear chromatin structure and with shifting chromatin lumps toward periphery of cell nuclei. PMID- 3002876 TI - [The biocellular effect of thyroid hormone on functional differentiation of porcine granulosa cells in culture]. AB - To elucidate if the thyroid hormone acts directly on the ovary, the biocellular effect of L-thyroxine (T4) on porcine granulosa cells cultured in vitro was investigated. Monolayer cultures of porcine granulosa cells obtained from small (1 approximately 2 mm), medium (3 approximately 5 mm) or large (6 approximately 11 mm) follicles were carried out in the presence of porcine FSH (100 ng/ml). Concomitant treatment with T4 promoted FSH-dependent morphological luteinization, i.e. alteration of immature granulosa cells obtained from small follicles to epithelioid form. T4 also increased FSH-stimulated induction of hCG/LH receptor on immature granulosa cells. Furthermore, T4 augmented FSH-mediated production of progesterone and estradiol by immature granulosa cells cultured in vitro. The concentration of T4 to produce the maximal stimulatory effect was 10-7 M, demonstrating that optimal concentration of thyroid hormone is required for the expression of this stimulatory action. Since T4 alone demonstrated no effect on the differentiation of porcine granulosa cells and all the stimulatory effect of T4 seems to have a permissive action on FSH-induced granulosa cell luteinization. Although insulin showed a similar effect on porcine granulosa cells, no stimulation of estradiol production by porcine granulosa cells was observed with insulin in the culture system used in this study. These results suggest that the thyroid hormone acts directly on the ovary and plays an important role in modifying the FSH-dependent cellular differentiation of immature granulosa cells. PMID- 3002877 TI - [The role of Ca ions and cAMP in ACTH lipolysis]. AB - Ca ions and cAMP are known to be involved in the action of hormones. In this study, the actions of Ca ions and cAMP in ACTH lipolysis were investigated by comparing ACTH1-24, ACTH1-10 and ACTH11-24 with regard to: lipolytic activity, binding affinity to the fat cell membrane, changes in Ca ion binding to the fat cell membrane and cAMP formation. Rat adipocytes were used. Lipolytic activity was measured in terms of the amount of FFA released from fat cells into the medium after incubation with the ACTH analogs at 37 degrees C for 30 minutes. Binding affinity to the fat cell membrane was expressed as competitive binding affinity, determined by simultaneously incubating fat cell ghosts with 125I-ACTH1 24 and unlabelled ACTH analogs at 4 degrees for 40 minutes. Changes in Ca ion binding to the fat cell membrane was expressed as Ca binding capacity, measured by simultaneously incubating fat cell ghosts with 45Ca and ACTH analogs at 24 degrees C for 20 minutes. CAMP formation was measured by RIA as the amount of cAMP formed in fat cells incubated with 10(-6)M ACTH analogs at 37 degrees C for 0, 3, 5 and 10 minutes. FFA release after incubation with each of the ACTH analogs at 10(-6) M was 1.17 mEq/L/tube for ACTH1-24, 0.26 mEq/L/tube for ACTH1 10 and 0.25 mEq/L/tube for ACTH11-24, showing a high value only for ACTH1-24. The binding affinity to the fat cell membrane (high affinity constant) was 4.4 X 10( 8) M for ACTH1-24, 4.6 X 10(-8) M for ACTH1-10 and 4.6 X 10(-8) M for ACTH11-24. There were no significant differences among the binding affinities of these ACTH analogs. The binding capacity of 45Ca++ to the fat cell membrane (high affinity constant) was 3.64 X 10(-6) M for ACTH1-24, 4.49 X 10(-6) M for ACTH1-10 and 5.46 X 10(-6) M for ACTH11-24. There were no significant differences among these ACTH analogs. The increase in cAMP formation during the first 3 minutes of incubation was 35.5 fmol/mg dry weight of fat cells for ACTH1-24, 2.2 fmol/mg for ACTH1-10 and 1.8 fmol/mg for ACTH11-24. An increase was only observed for ACTH1-24. The fact that FFA release and cAMP formation were high only with ACTH1-24 among the ACTH analogs tested indicates that cAMP formation plays an important role in the lipolytic activity of ACTH in fat cells and that the three-dimensional structure of ACTH is important for cAMP formation. PMID- 3002878 TI - [Scrutinization of the direct assay method for plasma cyclic AMP and clinical applications of nephrogenous cyclic AMP]. AB - Several problems in the measurement of plasma cyclic AMP (PcAMP) and nephrogenous cyclic AMP were studied using a YAMASA RIA Kit (YAMASA Shoyu, Choshi, Japan). In this assay method, cAMP in plasma is directly succinylated without prior deproteinization, and then it is bound to antibody in an imidazole buffer. So far as the blood samples were obtained with EDTA-4Na at least 5.0 mM in the final concentration, PcAMP was not reduced until 16 hours after the blood samples were drawn. Even without EDTA, the reductions in PcAMP were not detected within 1 hour after the blood samples were drawn. This assay method for PcAMP showed parallelism in the dilution curve. Recovery was almost complete. Intra- and interassay variations were low. When plasma was incubated at 37 degrees C for 24 hours, PcAMP became negligible. Furthermore, the values of PcAMP measured with this direct assay system almost agreed with those obtained after the purification by deproteinization and Dowex column chromatography through an anion-exchange resin. The normal values of PcAMP were 13.6 +/- 3.62 pmol/ml [mean +/- SD, n = 43]. Nephrogenous cAMP expressed as a function of GFR never did show any negative values in various clinical situations. From the data of basal levels and the oral calcium tolerance test, nephrogenous cAMP appeared to be more useful than total urinary cAMP in the diagnosis of parathyroid disorders, especially hyperparathyroidism. PMID- 3002880 TI - Basement membrane structures in tumors of the ovary. AB - The location and amount of the basement membrane (BM) components collagen IV and laminin were studied in ovarian epithelial, sex cord-stromal and germ cell tumors. BM structures were found in the epithelial stromal interface of benign surface epithelial tumors and, though discontinuous, around well-differentiated tumor islets, being less well developed in invasive undifferentiated neoplasms. The stromal components in Mullerian mixed tumors had less distinct BM structures, a finding useful for the classification of these neoplasms. Thecomas and fibromas had scanty collagen IV and laminin; granulosa cell tumors contained large amounts of BM material. A fine diffuse BM-positive pattern occurred in dysgerminomas and endodermal sinus tumors; BM structures in cystic teratomas were distinct. Collagen IV and laminin were well-developed in benign and slow-growing tumors with epithelial components and in their metastases, but less distinct in stromal tumors and highly malignant undifferentiated tumors, showing the usefulness of this method for the clinical and biological classification of such tumors. PMID- 3002881 TI - Femoral neuropathy subsequent to abdominal hysterectomy. A comparative study. AB - In a prospective study of two 5-yr periods two groups of patients undergoing pelvic operative procedures were compared. Iatrogenic femoral neuropathy occurred in 282 patients who underwent pelvic abdominal surgery in the first group of women, an overall incidence of 7.45%. The neuropathy was associated with the use of self-retaining retractors during surgery. Femoral nerve neuropathy occurred only in two patients in the second group in whom no retractors were used during operation. No other contributing factors were found. The duration of complaints ranged from 3 to 90 days. Spontaneous recovery occurred in 265 patients, while in 17 there were residual mild symptoms for up to 116 days. No serious sequelae have been observed. Prevention of this mostly mild yet annoying syndrome may be achieved if no retractors are used in gynecological operations, or, if they are necessary, due to difficult conditions at surgery or if the operation is long, they should be used with care and loosened from time to time. It is estimated that the incidence of iatrogenic femoral nerve neuropathy is higher than reported, since in very mild cases the patient fails to mention her complaints. The prognosis is usually rather good. PMID- 3002879 TI - Moderate guar-gum addition to usual diet improves peripheral sensitivity to insulin and lipaemic profile in NIDDM. AB - The addition of vegetable fibres to the diabetic diet has been reported to ameliorate glycaemic and plasma lipid profiles, and Guar flour seems to obtain the best results. At its usual dose, Guar produces several gastro-intestinal side effects. A lower dose (4 + 4 g/day) was therefore employed in 10 non-insulin dependent diabetics (NIDD). The following parameters were measured at the end of treatment and after a control period: HbA1 levels, hepatic glucose production (3H Glucose infusion), peripheral sensitivity to insulin and insulin secretion (hyperglycaemic clamp), and specific insulin binding to isolated monocytes. The ultracentrifugal plasma lipid pattern was also measured. No significant body weight change was recorded during the study. A significant glycaemic and insulinaemic decrease in the fasting state was observed after Guar, together with a significant decrease of HbA1 levels (from 8.5 +/- 0.4 to 7.9 +/- 0.4%, p less than 0.05) and amelioration of peripheral sensitivity to insulin (M/I = 14.3 +/- 6.6 versus 24.3 +/- 8.8, p less than 0.025; 50% increase of insulin binding to circulating monocytes) without significant variation of the fasting hepatic glucose production. Decreased B-cell stimulation by flattening post-prandial glycaemic peaks may be an explanation of the reduction of insulin resistance via down-regulation mechanism. As far as the lipid profile is concerned, a significant reduction in total and LDL cholesterol (p less than 0.05 and p less than 0.01) and an increase in HDL-phospholipids (p less than 0.05) were recorded after Guar. These results suggest that Guar in low doses is well accepted and can contribute to a better glycaemic and lipaemic control in NIDDM. PMID- 3002882 TI - Study of alpha-methyldopa oxidation by tyrosinase. AB - The pathway for alpha-methyldopa oxidation to alpha-methyldopachrome, by mushroom tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometry determination have both been undertaken. The steps for alpha-methyldopa transformation into its aminochrome would be: alpha-methyldopa-- -o-alpha-methyldopaquinone-H+----o-alpha- methyldopaquinone----leuko-alpha methyldopachrome----alpha- methyldopachrome. The stoichiometry for this conversion corresponded to the equation: 2 o-alpha-methyldopaquinone-H+----alpha methyldopa + alpha-methyldopachrome. At very acid pH values, another route implying the addition of water to the quinonic ring, competes with the first one. Two chemical pathways can be proposed from alpha-methyldopaquinone-H+, the relative importance of which is determined by the pH. A theoretical and experimental kinetic approach was applied to this oxidative reaction. Rate constants and thermodynamic activation parameters of the chemical steps, have been evaluated. The results obtained confirmed that alpha-methyldopa oxidation by tyrosinase followed a scheme similar to that established for L-dopa and alpha methylnoradrenaline. PMID- 3002884 TI - Studies on the stimulation of the bacterial, collagenolytic enzyme clostridiopeptidase A by cobalt (II) ions. AB - Co(II) ions increase the Vmax of clostridiopeptidase A, producing a maximum stimulation of overall enzymic activity of 120%. Co(II) does not displace Zn(II) from the active site, nor Ca(II) from its binding site on the enzyme. There appears to be an additional transition metal-binding site on clostridiopeptidase A, accepting Zn(II), which is inhibitory (Ki = 550 microM), or Co(II), which is stimulatory (Kact = 200 microM). PMID- 3002883 TI - The concentrations of glucose 1,6-bisphosphate and other regulatory metabolites, and the activities of enzymes of the glycogen metabolism in the perfused rabbit psoas muscle. AB - The following parameters were determined in the rabbit psoas muscle after perfusion in the presence of either insulin, propranolol, or isoproterenol: Concentrations of cyclic AMP, glucose 1,6-bisphosphate, fructose 2,6 bisphosphate, glucose-1-phosphate, glucose 6-phosphate, and fructose-1,6 bisphosphate. Maximum and "regulatory" activities of the enzymes glycogen phosphorylase, glycogen synthase, phosphofructokinase, and histone phosphorylating protein kinase. PMID- 3002885 TI - Epithelial effects on limb chondrogenesis involve extracellular matrix and cell shape. AB - Collagen gel cultures of limb bud mesenchymal cells are normally permissive for chondrogenesis but become inhibitory for chondrogenesis when they are preconditioned by limb ectoderm. This inhibition is specific for cartilage differentiation, inasmuch as myoblast differentiation is unaffected and flattened, fibroblastic cells are more numerous on conditioned gels. The antichondrogenic effect of ectoderm-conditioned gels is not blocked by agents that elevate intracellular cyclic AMP levels and that promote chondrogenesis under other conditions. In contrast, the inhibitory effect of the ectoderm is alleviated when cultures are treated with cytochalasin D, a cytoskeleton disrupting agent that causes the cells to remain spherical. These results suggest that ectoderm-conditioned collagen gels inhibit chondrogenesis through an effect on cell shape. PMID- 3002886 TI - Collagenase inhibitor stimulates cleft formation during early morphogenesis of mouse salivary gland. AB - A collagenase inhibitor obtained from the culture medium of bovine dental pulp markedly enhanced the cleft formation of mouse embryonic salivary gland epithelium when the inhibitor was included in the culture medium for 12-day and 13-day salivary glands. Determination of collagenase activity using [3H]collagen as substrate indicated that there was a latent collagenase activity in 12-day glands. In addition, a highly purified Clostridial collagenase freed from protease and hyaluronidase activities, strongly inhibited initiation of the cleft formation of the 12-day epithelium. Scanning electron microscopic observation showed that abundant collagen-like fibrils were seen on the epithelium in the collagenase-inhibitor-treated glands compared to those in the control. These findings suggest that during early morphogenesis tissue collagenase may regulate the cleft formation in the epithelium. PMID- 3002887 TI - Variable relationship between peripheral somatic and autonomic neuropathy in patients with different syndromes of diabetic polyneuropathy. AB - The relationship between abnormal peripheral nerve electrophysiology and abnormal cardiovascular autonomic function has been studied in four groups of diabetic subjects, comparable with regard to age, duration, and type of diabetes. Thirty three had no symptoms of neuropathy, 28 had newly developed painful neuropathy, 24 had chronic painful neuropathy, and 21 had painless neuropathy with associated recurrent foot ulcers. In all three symptomatic groups, electrophysiology and autonomic function were more abnormal than in asymptomatic diabetic subjects. There was a significant overall relationship between peripheral nerve (electrophysiologic) and autonomic (cardiovascular reflex) dysfunction. However, when considered by groups, the degree of cardiovascular reflex abnormality was similar in the three symptomatic groups, whereas electrophysiology was appreciably worse in the foot ulcer group than in patients with painful neuropathy. Thus, patients with painful neuropathy had a higher ratio of autonomic (small fiber) abnormality to electrophysiologic (large fiber) abnormality. By contrast, foot ulceration was associated with the worst electrophysiologic (large fiber) abnormality. Heavier alcohol consumption and more severe retinopathy were also related to foot ulceration. In diabetic subjects with symmetrical sensory neuropathy, the relationship between large fiber and small fiber damage is not uniform. We conclude that there may be different etiologic influences on large and small fiber neuropathy in diabetic subjects and that the predominant type of fiber damage may determine the form of the presenting clinical syndrome. PMID- 3002888 TI - Protein kinase C agonists acutely normalize decreased ouabain-inhibitable respiration in diabetic rabbit nerve. Implications for (Na,K)-ATPase regulation and diabetic complications. AB - Diminished (Na,K)-ATPase activity in diabetic peripheral nerve is attributed to an underlying depletion of free myo-inositol, but no biochemical mechanism linking myo-inositol metabolism and (Na,K)-ATPase has emerged. Since inositol phospholipid turnover releases inositol-(1,4,5)-tris-phosphate and diacylglycerol, two putative "second messengers" that modulate protein kinase C, the effect of protein kinase C agonists on (Na,K)-ATPase activity was examined in diabetic nerve. Phorbol myristate acetate or the diacylglycerol sn-1,2 dioctanoylglycerol acutely normalized depressed ouabain-inhibitable respiration [a measure of (Na,K)-ATPase activity], suggesting that myo-inositol metabolism modulates (Na,K)-ATPase activity via protein kinase C, and that reduced myo inositol impairs (Na,K)-ATPase activity in diabetic nerve by this mechanism. PMID- 3002890 TI - Rat hepatic microsomal glucose-6-phosphatase protein levels are increased in streptozotocin-induced diabetes. AB - Hepatic microsomal glucose-6-phosphatase activity levels and the hepatic output of glucose are increased in diabetes. We have used protein chemistry and immunological techniques to determine the mechanism by which the activity levels of the glucose-6-phosphatase system are increased in streptozotocin-induced diabetic rats. In the streptozotocin-induced diabetic rats, the activity of the glucose-6-phosphatase enzyme increased four-fold without appreciably altering the transport capacity of the glucose-6-phosphatase system. The solubilized diabetic rat liver glucose-6-phosphatase enzyme appeared to be very similar to the solubilized enzyme from control rat liver microsomes. They exhibit the same Km, are labile at 30 degrees C, are stabilized by sodium fluoride and they migrate to the same position during density gradient centrifugation. Immunological studies demonstrated that a greater amount of hepatic microsomal glucose-6-phosphatase enzyme protein is present in diabetic rats than in control rats. Thus, we have determined for the first time that increased levels of the glucose-6-phosphatase protein are present in streptozotocin-induced diabetes. The significance of this finding in relation to the regulation of the hepatic microsomal glucose-6 phosphatase system is discussed. PMID- 3002889 TI - Ultrastructural detection of granulated cells in the autonomic ganglia of the rat pancreas. AB - Electron microscopic examination of the intrinsic autonomic ganglia of the rat pancreas revealed the presence of small cells, when compared to the principal ganglionic neurons, within a particular type of ganglia. The small cells were often located in clusters around fenestrated capillaries, but their most striking characteristic was the presence of catecholamine-like granules distributed throughout the cytoplasm. The possible implication of this new source of catecholamines, acting either as interneurons or as neuroendocrine cells, is discussed in the light of a local regulatory mechanism for islet secretion. PMID- 3002892 TI - [Negativity of myocardial scintigraphy with technetium-pyrophosphate in a case of primary cardiac amyloidosis]. AB - We describe herein the case of a 62-year-old man affected by congestive heart failure of obscure origin, in which the clinical history, the electrocardiographic and echocardiographic findings were compatible with the diagnosis of amyloid cardiomyopathy. Cardiac catheterization disclosed a restrictive physiology. Tc-99m-pyrophosphate myocardial scintiscan was negative despite the markedly increased left ventricular wall thickness on echocardiographic examination. A definite diagnosis was obtained by means of endomyocardial biopsy. PMID- 3002891 TI - [Update on AIDS: critical review]. PMID- 3002893 TI - [Giant esophageal ulcer in a cytomegalovirus infection in a patient with the acquired immunodeficiency syndrome]. PMID- 3002894 TI - [Hepatitis caused by suloctidil with antinuclear antibodies]. PMID- 3002895 TI - Fibrolamellar hepatocarcinoma: clinical, radiologic, and pathologic features. AB - Three new cases of an unusual subtype of hepatocellular carcinoma (HCC) referred to as fibrolamellar hepatocarcinoma (FLHC) recently seen at our institution are described. This report focuses on the clinical, radiologic, and pathologic features of this rare subset of HCC. All three patients were under 30 years of age with no previous history of hepatitis or cirrhosis. Each had had subacute symptoms for 5 months to 1 year before medical attention was sought and/or diagnosis of FLHC was established. There was no reliable correlation with oral contraceptive use in the 2 female patients. Serum alpha-1-fetoprotein levels were normal with only mild elevation of liver enzymes. The CT features, although not specific, were suggestive of an aggressive tumor with amorphous calcification in 2 of the 3 cases. Angiographically all tumors were hypervascular without any evidence of arterioportal shunting or venous invasion of major vessels. The clinical and radiologic recognition of these tumors is important since the surgical resectibility rate and 2- and 5-year survival rates are higher than those applicable to conventional HCC. PMID- 3002897 TI - Hepatocellular carcinoma metastatic to the oral cavity including the maxilla and the mandible: report of two cases and review of the literature. AB - Two cases of hepatocellular carcinoma metastatic to the oral cavity are presented. One patient had metastases to the maxilla and finally, to the mandible, and the other patient, to the mandible. Both cases histologically showed highly-differentiated trabecular hepatocellular carcinoma which had vascularized stroma, explaining the frequently observed oral hemorrhage. The clinical signs and symptoms described here suggested the existence of a tumor metastatic to the oral cavity, and might indicate an unusual manifestation of hepatocellular carcinoma. Reports of metastatic lesions of hepatocellular carcinoma to the oral cavity, including the mandible, maxilla and gingiva proper, are reviewed. PMID- 3002896 TI - Localization of hepatitis A virus in marmoset liver tissue during the acute phase of experimental infection. AB - Electron microscopic and virological studies of marmoset liver tissue with acute infection of hepatitis A virus (HAV), especially in the earlier stages of infection, were carried out to characterize the maturation process of HAV. Four marmosets were inoculated intravenously with HAV suspension and sacrificed 1 week, 2 weeks, 3 weeks and 4 weeks after inoculation respectively. Hepatitis A antigen (HAAg) in 10% liver homogenates of marmosets was examined by radioimmunoassay and a large amount of HAAg was detected in the liver homogenate of two marmosets sacrificed 2 weeks and 3 weeks after inoculation respectively. The histodiagnosis of the marmoset sacrificed 2 weeks after HAV inoculation was normal. However, many clusters of virus-like particles about 27 nm in diameter, in both "solid" and "empty" forms were found, mainly in vesicles of Kupffer cells by electron microscopy. In the animal that developed mild hepatitis 3 weeks after inoculation HAV-like particles were found in vesicles of hepatocytes by electron microscopy. By immune electron microscopy using peroxidase-conjugated anti hepatitis A antibody, HAAg was detected on the particles present within the cytoplasmic vesicles of Kupffer cells or hepatocytes and on the surrounding membrane of the vesicles which contained HAV-like particles. PMID- 3002898 TI - Large hepatitis B surface antigen polypeptides of Dane particles with the receptor for polymerized human serum albumin. AB - Large hepatitis B surface antigen polypeptides with apparent molecular sizes of 39,000 and 43,000 daltons (P39 and P43) were liberated from a purified preparation of Dane particles of subtype adr. They were tested for reactivity with monoclonal antibodies raised against three synthetic oligopeptides representing fundamental sequences of the pre-S region in deoxyribonucleic acid of hepatitis B virus (subtype adr), as well as with monoclonal antibody against the major surface antigen polypeptide (P22) coded for by the S gene. Both P39 and its glycosylated form P43 bound to all four monoclonal antibodies, thereby indicating that they were coded for by the sequence of 1200 nucleotides, from the second ATG codon in the pre-S region to the stop codon of the S gene. Both P39 and P43 bound to polymerized human and chimpanzee albumins, but not to polymerized albumin from species without susceptibility to hepatitis B virus. Due to their presence in Dane particles and the expression of a polyalbumin receptor, the immune responses against P39 and P43 may have significance in infection with hepatitis B virus and its immunoprophylaxis. PMID- 3002899 TI - Adrenoreceptors in hepatocellular carcinoma and liver. PMID- 3002900 TI - [Cytomegalovirus infection in leukemia]. PMID- 3002901 TI - [The functional state of neutrophils after kidney allotransplantation]. PMID- 3002902 TI - Changes in GABAergic system parameters after chronic administration of bicuculline. AB - Male Wistar rats were chronically treated with 2 mg/kg per day bicuculline i.p. during 10 days. Biochemical parameters related to the GABAergic system in CNS were analyzed. A 63% increase in glutamic acid decarboxylase activity was found in treated animals. Endogenous GABA levels were not modified. The treatment increased Bmax of GABA binding by 112% without changing the KD value. The treatment decreased Bmax of diazepam binding by 40% whereas it did not change the KD value. Diazepam binding was enhanced by in vitro addition of GABA at the same extent as in treated animals. PMID- 3002903 TI - Involvement of guanine nucleotide and sodium in regulation of yohimbine and clonidine binding sites in the HT29 human colon adenocarcinoma cell-line. AB - Sodium and GppNHp, a GTP analogue, decrease the specific binding of the alpha 2 adrenergic agonist [3H]clonidine on membranes from the HT29 cells; affinity of the ligand for the receptor is also decreased. The effects of the two agents are additive. By contrast, combination of sodium and GppNHp increases the number of binding sites for the alpha 2-adrenergic antagonist [3H]yohimbine. Sodium and GppNHp also decrease the potency of epinephrine and clonidine to displace [3H]yohimbine from the alpha 2-adrenoceptors of the HT29 cells PMID- 3002904 TI - 4-Aminopyridine-induced changes in the electrical and contractile activities of the gastric smooth muscle. AB - Effects of 4-aminopyridine (4-AP) on the electrical and contractile activities of the fundus and antrum of the cat stomach were studied using the sucrose-gap technique. In the fundus, low concentrations of 4-AP (up to 1 mmol/l) induced membrane depolarization and appearance of spike potentials and phasic contractions. After preliminary administration of atropine, 4-AP produced an opposite effect: hyperpolarization and relaxation. On the background of tetrodotoxin (TTX) plus antagonists of cholinergic and adrenergic receptors, high concentrations of 4-AP (greater than 5 mmol/l) caused membrane depolarization and appearance of spike potentials and phasic contractions. In the antrum, 4-AP in low concentrations (up to 1 mmol/l) decreased both the amplitude and the duration of the second component of the plateau-action potential, as well as those of the phasic contractions. This effect decreased in the presence of adrenergic receptor antagonists and was abolished by TTX. On this background, high concentrations of 4-AP (greater than 5 mmol/l) led to the appearance of spike potentials superimposed on the second component of the plateau-action potentials, and to a further increase in the phasic contraction amplitudes. The present data suggest that 4-AP exerts its effects via an increase in neurotransmitter release (low concentrations) and/or directly on the smooth muscle cell membrane (high concentrations). PMID- 3002905 TI - Polarography of cytochrome c in ammoniacal buffers containing cobalt ions. The effect of the protein conformation. AB - Catalytic currents yielded by cytochrome c in ammoniacal buffers containing cobalt ions at a dropping mercury electrode (Brdicka's catalytic currents) were investigated by means of direct current, differential pulse, normal pulse (NP) and phase-selective alternating current polarography. It was found that Brdicka's catalytic current of cytochrome c, (the more negative part of Brdicka's double wave, wave B) is influenced by the presence of cytochrome c denaturants in the background solution. The wave B rose with the increasing concentrations of urea and sodium perchlorate, and increased in parallel with absorbance changes at 409 and 695 nm measured for identical cytochrome c solutions. The latter absorbance changes reflect unfolding of cytochrome c molecules in the bulk of solution by these denaturants. The results of NP polarography (a technique working with large potential excursion during the drop lifetime) indicate that in Brdicka's solution cytochrome c could extensively be unfolded due to its adsorption at the mercury electrode, polarized to potentials around that of zero charge. PMID- 3002906 TI - Reconstitution in bilayer lipid membranes of the crab Potamon transcaspicum spider venom sensitive glutamate receptors. AB - Membrane proteins have been isolated from neuromuscular synapses of the crab Potamon transcaspicum using a specific blocker of glutamatergic synapses, the neurotoxin of the spider Argiope lobata. These membrane components have been shown to induce glutamate-sensitive conductance in bilayer lipid membranes (BLM) in the presence of sodium ions. As an agent blocking desensitization of glutamatergic synapses, concanavalin A was shown to enhance the conductance and to abolish desensitization. Diethylester of glutamic acid as a blocker of glutamatergic synapses inhibited glutamate-induced conductance. No similar change in conductance was seen when BLM was modified by a membrane proteins -neurotoxin complex. Conductance current fluctuations induced by these receptor protein components were monitored by the single-channel registration method. PMID- 3002907 TI - The Mu transposable elements of maize: evidence for transposition and copy number regulation during development. AB - The Mu transposon of maize exists in a highly mutagenic strain called Robertson's Mutator. Plants of this strain contain 10-50 copies of the Mu element, whereas most maize strains and other plants have none. When Mutator plants are crossed to plants of the inbred line 1S2P, which does not have copies of Mu, the progeny plants have approximately the same number of Mu sequences as did their Mutator parent. Approximately one-half of these copies have segregated from their parent and one-half have arisen by transposition and are integrated into new positions in the genome. This maintenance of copy number can be accounted for by an extremely high rate of transposition of the Mu elements (10-15 transpositions per gamete per generation). When Mutator plants are self-pollinated, the progeny double their Mu copy number in the first generation, but maintain a constant number of Mu sequences with subsequent self-pollinations. Transposition of Mu and the events that lead to copy number maintenance occur very late in the development of the germ cells but before fertilization. A larger version of the Mu element transposes but is not necessary for transposition of the Mu sequences. The progeny of crosses with a Mutator plant occasionally lack Mutator activity; these strains retain copies of the Mu element, but these elements no longer transpose. PMID- 3002908 TI - The use of restriction fragment length polymorphisms and DNA duplications to study the organization of the actin multigene family in Dictyostelium discoideum. AB - The techniques of restriction fragment length polymorphism analysis and examination of gene copy number in duplication-bearing Dictyostelium discoideum strains have been used to map four actin genes of the wild-type strain NC4 to specific linkage groups. In part, this was accomplished by identification of restriction fragments corresponding to particular cloned actin genes using gene specific probes from unique sequence 5' and 3' untranslated regions. Cloned gene Actin 8 (designation act-8) maps to linkage group I; Actins 12 (act-12) and M6 (actM6) to linkage group II. An uncloned gene (act-100) also maps to linkage group II in the same region as actM6, as defined by a chromosomal duplication. From analysis of other wild isolates of D. discoideum, it was determined that in these isolates at least two actin genes map to linkage group I and at least four map to linkage group II. These results demonstrate the utility of molecular techniques in genetic analysis of Dictyostelium, particularly for developmentally regulated genes that have been cloned but that have no identified mutant phenotypes. PMID- 3002909 TI - Persistence or rapid generation of DNA length polymorphism at the zeta-globin locus of humans. AB - Extensive restriction mapping of 76 human genomic DNAs defines multiple sites of length and point mutation near the zeta-globin locus, which codes for an embryonic alpha-like globin chain. There are two major sites of DNA length variation: one in the intergenic region with three alleles and one in the first intron of the zeta 1 gene with at least four alleles. Our mapping establishes that the intronic polymorphism is associated with a tandem array of short, repeated sequences. The length alleles occur in each of four human populations sampled, suggesting an ancient origin with persistence of several length alleles, or rapid regeneration of these particular variants. Four polymorphic restriction sites were also found; the frequency of polymorphic sites is comparable to that found in the human beta-globin gene region. Analysis of haplotypes indicates either that multiple recombinations have occurred near the 5' end of the zeta 1 gene or that this region is prone to recurrent length mutation. PMID- 3002910 TI - [Expression of Pseudomonas aeruginosa phage transposons in Pseudomonas putida PpG1 cells. II. Zygotic induction--a necessary condition for the formation of defective lysogens]. AB - The transfer of hybrid plasmid RP4::PT (where PT is the genome of a transposable phage specific for Pseudomonas aeruginosa) into recipient cells of P. putida strain PpG1 occurs with the same frequency as into P. aeruginosa, the homologous host for PT. Approximately 1/3 of all PpG1 exconjugants carrying RP4 markers lost the capability to produce viable PT phage. In contrast, in a cross with homologous recipient P. aeruginosa all exconjugant clones contained nondefective prophages in the hybrid plasmids. Zygotic induction is an obligatory condition for detection of PpG1 exconjugants with defective phages. The defective prophages in RP4::PT hybrid plasmids have deletions of different size; the other carry mutations indistinguishable from point mutations in an essential phage gene. Some of deletions also cover plasmid genes. At least some of the defective prophages, including deleted ones, have arisen in the recipient cells of P. putida after transfer of the hybrid plasmid. PMID- 3002911 TI - [Hygienic evaluation of mineral fertilizers]. PMID- 3002912 TI - [Use of cattle manure drainage on "sliding" tilled fields]. PMID- 3002913 TI - [Various questions concerning soil protection when using bedding-free cattle manure]. PMID- 3002914 TI - [Use of correlation-regression analysis in studying the effect of mineral fertilizers on the concentration of microelements in vegetables]. PMID- 3002915 TI - Human papilloma virus and the gynecologist. PMID- 3002916 TI - Ten year's experience with methotrexate and folinic acid as primary therapy for gestational trophoblastic disease. AB - Methotrexate and folinic acid was administered as primary therapy in 185 patients with gestational trophoblastic disease between 1974 and 1984. Methotrexate and folinic acid induced complete remission in 147 (90.2%) of 163 patients with nonmetastatic disease and in 15 (68.2%) of 22 patients with low-risk metastatic disease. Sustained remission was achieved in 132 (81.5%) patients following only one course of chemotherapy. All patients with methotrexate resistance subsequently achieved remission with Actinomycin D or combination chemotherapy. Methotrexate when administered with folinic acid was associated with granulocytopenia, thrombocytopenia, and hepatotoxicity in 11 (5.9%), 3 (1.6%), and 26 (14.1%) patients, respectively. The human chorionic gonadotropin (hCG) regression curve served as a reliable guide for the administration of chemotherapy and enabled the attainment of a high remission rate while limiting chemotherapy exposure. Methotrexate and folinic acid achieves an excellent therapeutic outcome with limited chemotherapy exposure and effectively limits systemic toxicity. PMID- 3002917 TI - Modified Bagshawe's regimen in high-risk gestational trophoblastic disease. AB - Fifty patients with high-risk gestation trophoblastic disease received primary multiagent chemotherapy (CHAMOMA). Forty-one patients (82%) achieved sustained completed biochemical remission. The importance of recognizing the high-risk factors and the effectiveness of primary multiagent chemotherapy are demonstrated. PMID- 3002919 TI - Adjuvant therapy in mixed mullerian tumors of the uterus. AB - We report 54 patients with mixed mullerian tumors of the uterus treated at Yale New Haven Hospital from 1962 to 1983. Seven had previous pelvic irradiation. Twenty-five neoplasms were homologous and 29 were heterologous. The mainstay of therapy was surgery and radiation. By FIGO criteria 9 patients had stage IA disease, 31 stage IB, 6 stage II, and 8 patients had clinical extrauterine disease. Ten of forty-six patients (23%) with FIGO stage I and II disease had extrauterine disease found at surgery. No patient with extrauterine disease had prolonged survival. The 2-year disease-free survival with stage IA was 66%, with stage IB surgically confirmed 32%, and for stage II 33%. Surgically advanced disease in clinical stage I and II patients and recurrence were associated more frequently with a heterologous histology (67%). The small uterus with a less than 10-cm cavity had a better prognosis. Among 29 surgically confirmed stage I and II patients, 83% of recurrences appeared within 2 years (mean 16 months +/- 7 months). Patients who received both intracavitary radiation and external beam developed only 17.6% pelvic recurrence but this reduction in local recurrence was not associated with significant improved long-term survival. Six of eight patients treated with cis-platinum, Adriamycin, and dimethyl triazeno imidazole carboximide for persistent disease or for recurrence showed response for 4 to 24 months, none complete. Five patients were treated by radiation, surgery, and adjuvant chemotherapy (4 with Adriamycin-Cytoxan, 1 with Adriamycin-platinum). Four of the five (80%) are disease free from 36 to 60 months. These data and the experience of others support the need for a clinical trial with adjuvant platinum and Adriamycin in this disease. PMID- 3002918 TI - Gestational trophoblastic diseases: the ratio of choriogonadotropin to specific pregnancy protein, hCG/SP1, provides useful diagnostic and prognostic evidence. AB - Serum levels of human chorionic gonadotropin (hCG), specific pregnancy protein (SP1), and hPL were measured in 675 samples from women with uneventful pregnancy, and serially from the time of presentation in 125 patients with hydatidiform mole (HM), 43 with invasive mole (IM), and 34 with choriocarcinoma (CC). In HM serum levels of hCG and SP1 declined steadily from presentation to remission; when gestational age at the time of molar evacuation was shorter than 11 weeks, hCG declined to the normal range later than SP1 (57% patients), and when the age was longer--at the same rate as SP1 (26% patients) or earlier (17% patients). Serum levels of either marker were higher in IM than in HM and tended to increase, and in CC were either lower or higher than in IM. Treatment was followed by parallel decline of either marker, although SP1 declined to the normal range later than hCG in 12% of patients with IM and in 10% with CC. The hCG/SP1 ratios in normal pregnancy declined exponentially between the beginning and 23rd week of gestation and stayed level thereafter. The ratios calculated for the gestational age at the time of initial evacuation of the uterus or delivery were close to those of normal pregnancy in 80%, slightly increased in 20% of patients with spontaneously regressing HM, and markedly increased in 70% of patients with IM and in 74% of patients with CC. The ratios tended to increase during chemotherapy. An increase in the hCG/SP1 ratio seemed to be a characteristic sign of malignant change when compared with this ratio in normal pregnancy and hydatidiform mole. Determination of SP1 for monitoring therapy seemed redundant, and hPL assay was useful for discrimination between relapse and pregnancy. PMID- 3002920 TI - Malignant mixed mullerian tumor arising in extragenital endometriosis: report of a case and review of the literature. AB - A primary malignant mixed Mullerian tumor (MMMT) was found in the wall of the rectum of a 67-year-old female. The uterus, fallopian tubes, and ovaries had been previously excised for benign disease. The tumor was associated with foci of benign endometriosis. A literature review of extragenital MMMTs indicates that these are extremely rare tumors. This case supports the theory that endometriosis may serve as a progenitor to extraendometrial malignancy. PMID- 3002921 TI - Presence of gamma-aminobutyric acid and its specific receptor binding sites in the human term placenta. AB - The concentration of gamma-aminobutyric acid (GABA), the activity of glutamate decarboxylase (GAD), and the presence of specific GABA receptor binding sites have been examined in human term placentas. The estimated values of GABA concentration and GAD activity in the placenta were about 50 nmol/g tissue and 1.1 nmol/mg protein/h, respectively. A remarkable density of high-affinity and specific GABA binding sites has also been demonstrated in membranes of the human term placenta. These binding sites showed the properties of a GABAA receptor. The present findings suggest a possible role of GABA in the function of human placenta. PMID- 3002922 TI - Replication of tick-borne encephalitis (TBE) virus in Ixodes ricinus ticks. AB - The influence of external factors on virus carriage of Ixodes ricinus ticks in laboratory and in nature was studied. In laboratory experiment, only one nymph was positive for the presence of virus on 120th day after metamorphosis. The virus titer was 10(2) mouse i.c. LD50/0.03 ml. Transmission experiments were negative. The nymphs were positive on 75th, 111th and 159th day after metamorphosis, always chilling in the field experiment. The titres of virus varied from the lowest detectable amount value to 10(3.6) mouse i.c. LD50/0.03 ml. The transmission of virus was positive in two cases. PMID- 3002923 TI - [Ouabain binding and the conformational change in Na+, K+ -ATPase]. AB - The addition of ouabain to the Na+, K+-ATPase [EC.3.6.1.3] of pig kidney modified with N-[p-(2-benzimidazolyl) phenyl] maleimide gradually increased the fluorescence and the amount of phosphoenzyme from Pi with the same time course in the presence of 0.43 mM Mg2+, 16 mM Na+, 27 microM ADP and 27 microM Pi. The extent of the increment of the fluorescence intensity was dependent on the concentration of ouabain. A Hill plot of the data showed that n (Hill coefficient) and K1/2 (apparent affinity) were equal to 0.27 and 0.84 microM, respectively. Addition of ouabain to give 93 microM increased the intensity to the highest level, similar to that of K+-sensitive phosphoenzyme (E2P), and increased the extent of phosphorylation to half the amount of E2P formed with Mg2, Na+ and ATP. ADP inhibited the phosphorylation from Pi without affecting the binding of ouabain. The extent of the fluorescence intensity induced by ouabain in the presence of 0.43 mM Mg2+, 16 mM Na+ and 27 microM ADP was the same irrespective of the presence of 27 microM Pi. Addition of inorganic phosphate to give 2.6 mM accelerated the rate of fluorescence increase and 27 microM ADP retarded it without affecting the extent of the increment. The addition of ouabain to the Na+-bound enzyme increased the fluorescence with time to a level similar to that of E2P. These results and those of others indicate that ouabain can bind to nonphosphorylated Na+, K+-ATPase, and the relative fluorescence intensity of ouabain bound Na+, K+-ATPase was similar to that of E2P irrespective of the phosphorylation. PMID- 3002924 TI - [Contribution of prostaglandins to the activity of guinea-pig phrenic and chicken esophageal parasympathetic nerves]. AB - In order to clarify the role of prostaglandins (PGs) in the activity of cholinergic neurones other than the ileal myenteric plexus, the effects of indomethacin (IND) and PGE2 on the contractile responses and the release of acetylcholine (ACh) induced by electrical stimulation were investigated in isolated guinea-pig phrenic nerve-diaphragm and chicken parasympathetically innervated oesophagus preparations. In the guinea-pig phrenic nerve-diaphragm preparations, IND at 56 microM did not affect the twitch responses induced by direct or indirect electrical stimulation. PGE2 at 1 microgram/ml augmented the twitch responses induced by direct or indirect stimulation of submaximal, but not of supramaximal intensity. The amounts of ACh released from the phrenic nerve by electrical stimulation were unaffected by IND (56 microM). PGE2 (0.01-1 microgram/ml) inhibited the ACh release by the nerve stimulation of high frequency (50 Hz) in a concentration-dependent manner. The inhibitory effect of PGE2 was less clear on the ACh release by lower frequency (1 Hz). Neither IND nor PGE2 affected the ACh release induced by 40 mM-K+. In the chicken parasympathetically innervated oesophagus preparation, IND (2.8-5.6 5.6 microM) augmented the twitch responses induced by the nerve stimulation at a frequency of 0.017 Hz, but not those produced by train pulse (5 sec at a frequency of 1 or 10 Hz). This augmentation was reversed by the application of PGE2 at 10 ng/ml. PGE2 at 10 ng/ml produced a transient inhibition of the twitch responses induced by 0.017 Hz or train pulses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002925 TI - [Effects of single and repeated oral administration of MK-421 and captopril on blood pressures in normotensive and experimental hypertensive rats]. AB - In the single dose study, the aortic blood pressure in conscious normotensive rats, 2-kidney, 1-clip renal hypertensive rats (2K-RHR), 1-kidney, 1-clip renal hypertensive rats (1K-RHR) or DOCA hypertensive rats was measured for 24 hr after the oral administration of angiotensin converting enzyme (ACE) inhibitors such as MK-421 or captopril. MK-421 at 3 mg/kg and captopril at 10 mg/kg markedly lowered the blood pressure of 2K-RHR. MK-421 at 10 mg/kg and captopril at 30 mg/kg only modestly lowered the blood pressure of 1K-RHR. In contrast, both ACE inhibitors failed to reduce blood pressure in DOCA and normotensive rats. In the repeated dose study, the systolic blood pressures in normotensive rats, 2K-RHR or spontaneously hypertensive rats (SHR) were measured twice a week for 3 weeks treatment of either MK-421 at 3 mg/kg or captopril at 10 mg/kg. Both ACE inhibitors produced significant antihypertensive effects in these model rats, and the effects were sustained throughout the treatment period. The antihypertensive effects in 2K-RHR were greater than those in SHR and normotensive rats. These results indicate that MK-421 and captopril cause the most significant antihypertensive effect in 2K-RHR in which the renin-angiotensin system played a dominant role in blood pressure regulation. The antihypertensive effect of MK-421 was approximately 3 times as potent as that of captopril in these hypertensive models. PMID- 3002926 TI - [Correlation between the inhibition of renin-angiotensin system and antihypertensive effect of MK-421 and captopril in 2-kidney, 1-clip renal hypertensive rats after single and repeated oral administration of MK-421 or captopril]. AB - The angiotensin converting enzyme (ACE) activity in tissues and plasma renin activity (PRA) were measured in 2-kidney, 1-clip renal hypertensive rats (2K-RHR) and normotensive rats after a single and 3-weeks oral administrations of ACE inhibitors such as MK-421 and captopril. In the single dose study, MK-421 (1 and 3 mg/kg) and captopril (3 and 10 mg/kg) inhibited the ACE activities in kidney, aorta and plasma in a dose-dependent fashion. The inhibition of ACE activity in kidney or aorta was observed for a longer time than that in plasma. PRA took a time course reversal to that of plasma ACE activity. In the 3-weeks repeated dose study, the ACE activity in kidney and aorta was strongly inhibited after the administration of each ACE inhibitor, while there was no significant change in lung ACE activity at any time point examined. The plasma ACE activity markedly elevated after the administration of each agent. PRA significantly increased after the administration of either agent, while the plasma angiotensin II level was significantly inhibited. These results indicate that the inhibition of the ACE activity in blood vessel or kidney correlate well with the antihypertensive activity in 2K-RHR after a single and repeated administration of both ACE inhibitors, but not well with the inhibition of plasma ACE activity. PMID- 3002927 TI - Transformation of Streptomyces granaticolor with natural and recombinant plasmid vectors. AB - Protoplasts of Streptomyces granaticolor were found to be transformable by the broad-host-range plasmid pIJ350 but no transformants were detected when the narrow-host-range plasmid pIJ2 or the shuttle vector pPM66 (pIJ350--pBR322) isolated from E. coli cells were used. The onset of blue colour granaticin production by S. granaticolor cells was used as a marker to prepare protoplasts with a high transformation capacity. The presence of a restriction system is discussed. PMID- 3002928 TI - [Horton's temporal arteritis. Clinical aspects, diagnosis and pathomechanisms]. PMID- 3002929 TI - [Practical useful information on the topic of AIDS. Concrete progress in immunology]. PMID- 3002930 TI - Determination of a new angiotensin converting enzyme inhibitor (CV-3317) and its metabolites in serum and urine by high-performance liquid chromatography. PMID- 3002931 TI - Modulation of insulin action by fasting: a study using a phosphodiesterase activation system in rat fat cells. AB - Fat cells from control and 72 h fasted rats were incubated with increasing concentrations of insulin at 37 degrees C for 10 min. A crude microsomal fraction from these cells was used for the determination of phosphodiesterase activity. Specific activities of the enzyme in fat cells from the fasted rats were higher at overall insulin concentrations. In the fasted rats the curve shifted to the left at the lower concentrations of insulin and the half-maximal dose was lower than in the controls. Specific binding of insulin to the receptor was increased at the lower concentrations of insulin in fat cells from the fasted rats and Scatchard analysis of the data revealed that the change was due to an increase in binding affinity rather than that in receptor number per cell. Therefore, it is feasible that there is a good correlation with alteration of insulin sensitivity and insulin binding. The net amount of maximal response to insulin assessed as enzyme activity per cell was markedly decreased with fasting, however, this seems to be due to a decrease in absolute amount of the enzyme per cell. Since the maximal activation of the enzyme expressed as a percent of the basal remained unchanged, the steps between insulin receptor and the phosphodiesterase may not be altered under these conditions. PMID- 3002932 TI - Inhibition of spontaneous ACTH release and improved response to CRF in sheep premedicated with the enkephalin analogue DAMME. AB - The effect of IV injections of an enkephalin analogue (DAMME) in sheep prior to the administration of CRF has been compared with chlorpromazine-morphine (CPZ-M) injections as well as a chlorpromazine-morphine-nembutal (CPZ-M-N) regime. The results showed that the administration of DAMME 60 minutes prior to CRF injections, gave more consistent and more sustained inhibition of spontaneous plasma ACTH and plasma cortisol secretion than either CPZ-M or CPZ-M-N pre treatment. Changes in plasma ACTH and plasma cortisol levels were more readily detectable in DAMME-treated sheep as compared with saline controls when 50 micrograms injections of CRF were given into the carotid artery. This suggests that DAMME may inhibit endogenous CRF release by an action at hypothalamic level or above. PMID- 3002933 TI - Cyclic AMP release from perfused dog thyroid lobes as a sensitive indicator of TSH activation of the secretion process. Evidence for two types of thyroid secretion latency. AB - It has been demonstrated in various types of thyroid tissue preparations that cyclic AMP (cAMP) released into the medium reflects the amount of cAMP in the cells. In the present study employing perfused dog thyroid lobes the dynamics of cAMP release were compared to those of thyroxine (T4) and triiodothyronine (T3) release. The experiments gave evidence that even the lowest concentrations of TSH which stimulate hormone release (in this study 1 microU/ml) also activate the cAMP system; the very high levels of cAMP obtained by stimulation with high concentrations of TSH (in this study 10,000 microU/ml) are not accompanied by corresponding high increases in hormone release. On the contrary the T4 and T3 release is lower than during stimulation with more moderate concentrations of TSH (100 microU/ml). Hence studies employing high concentrations of TSH and measurements of cAMP as indicator of activity of secretory processes should be interpreted very cautiously; the prolonged lag in thyroid hormone secretion observed after stimulation with low concentrations of TSH is accompanied by a corresponding lag in activation of the cAMP system. This pattern suggest that the duration of late secretory processes such as thyroglobulin pinocytosis and hydrolysis is independent of the degree of stimulation and not involved in the variations in secretion latency. PMID- 3002934 TI - Impaired pancreatic polypeptide response to insulin hypoglycemia in obese subjects. AB - We have studied the effect of insulin hypoglycemia on the secretion of pancreatic polypeptide (PP) in 14 obese subjects with normal glucose tolerance and in 6 normal controls. Infusion of insulin 0.1 U/kg/h in controls and 0.12 U/kg/h in the obese, for one hour, produced a progressive hypoglycemia, similar in both groups (nadir 2 mmol/l at 50 min). The secretion of PP was less in obese subjects than in controls (peak 116 mmol/l vs 184 pmol/l, P less than 0.01) (integrated secretion sigma delta PP 288 vs 472 pmol/l, P less than 0.01) and was also delayed in the obese subjects beginning at 50 min instead of 40 min. The secretion of glucagon and of C-peptide were not different in the two groups, but the integrated response of ACTH was higher in the obese (sigma delta ACTH 52 pmol/l vs 25 pmol/l, P less than 0.01). The secretory response of growth hormone (STH) was smaller in the obese group (peak 8.6 +/- 1.28 vs 21.4 +/- 6.4 ng/ml, P less than 0.01). The reduced secretion of PP in obese subjects could be due to impaired sensitivity to hypoglycemia of the central control mechanism for PP release. The similarity of the reductions in the secretion of both PP and STH support this hypothesis, although a reduction in the secretory capacity of pancreatic PP cells cannot be excluded. PMID- 3002935 TI - Glucocorticoid receptors in Epstein-Barr virus-transformed human lymphocytes. AB - Glucocorticoid receptors were studied in cultured human lymphocytes from normal donors after transformation with the Epstein-Barr Virus (EBV) and compared to those of circulating human mononuclear leukocytes. Both whole cell and cytosol fractions were examined for [3H]-dexamethasone binding. The concentration and absolute number of glucocorticoid binding sites were increased five-fold in the transformed cells when compared to the non-transformed human mononuclear leukocytes. However, the affinity (Kd) of the glucocorticoid receptor for dexamethasone was the same in both types of cells. The denatured glucocorticoid receptor, covalently labelled with [3H]-dexamethasone-21-mesylate, was identified by SDS polyacrylamide gel electrophoresis as a protein moiety with Mr approximately equal to 92,000, similar to that obtained from human non transformed mononuclear leukocytes. The pattern of the activation of the hormone receptor complexes, analyzed by phosphocellulose chromatography, was similar in both types of cells, and also the time-courses of loss of specific binding during thermal activation were similar. These results suggest that viral transformation is associated with increases in the concentration and the absolute number of glucocorticoid receptors whereas other qualitative receptor characteristics remain similar. Thus, transformation of cells with EB virus can provide a constant source of glucocorticoid receptors for study. PMID- 3002936 TI - Quantitative autoradiographic localization of neurotensin binding sites in lean and obese Zucker rats. PMID- 3002937 TI - Rise in plasma beta-endorphin and ACTH in response to hyperthermia in sauna. PMID- 3002938 TI - Low-set osmotic threshold for vasopressin release in a patient with prolactinoma. AB - This report describes a 38-year-old patient with prolactinoma, without adrenal or thyroid insufficiency, with syndrome of inappropriate secretion of vasopressin due to downward resetting of the hypothalamic osmoreceptors. Random measurements of plasma and urine osmolality revealed an inappropriately high urine osmolality for a given plasma osmolality. Simultaneous plasma vasopressin levels were within normal limits. Urine dilution after water load was normal. During infusion of hypertonic saline, the osmotic threshold was demonstrated at plasma osmolality of 267 mosmol/kg, which is markedly lower than the normal 287.3 +/- 3.3 mosmol/kg. Thirst sensation seemed to be intact. The defect in the osmoreceptor function might have been induced by the tumor mass or by chronic hyperprolactinemia. PMID- 3002941 TI - Ultrastructural analysis of salivary gland pleomorphic adenoma, with particular reference to myoepithelial cells. AB - Previous ultrastructural studies of pleomorphic adenoma have presented conflicting results with regard to the role of myoepithelial cells in the histogenesis of this tumour. In the present study specimens of ten major salivary gland pleomorphic adenomas were examined ultrastructurally and a number of cell types identified. The material was subjected to quantification using the stereological method of point counting. The results showed a wide spectrum of differentiation within these tumours in which typical myoepithelial cells were rarely encountered even in situations where they are reported to occur in routine histological preparations. Cells with some myoepithelial features were more numerous but duct cells accounted for the majority of tumour cells. The ultrastructural findings correlated well with previously reported immunocytochemical data and further support certain ideas about salivary gland tumour histogenesis. PMID- 3002939 TI - Hemagglutination and immunofluorescence studies on polymerized human serum albumin binding activity in chronic hepatitis B virus infection. AB - The binding activity of polymerized human serum albumin was determined in 202 HBsAg carriers. The presence of polymerized human serum albumin receptor sites was tested by hemagglutination and differentiated from antihuman albumin antibodies by immunofluorescence, isolation of IgG and IgM fractions and testing of HBsAg anti-HBs immune complexes. A granular pattern with anti-HBs was specific for polymerized human serum albumin receptor sites as demonstrated with purified HBsAg. In addition, a linear pattern with fluoresceinated antihuman immunoglobulins might suggest the presence of antihuman albumin antibodies (which was generally due to an IgG antibody). However, a granular pattern with fluoresceinated antihuman immunoglobulins may indicate the presence of HBsAg anti HBs immune complexes. A weak linear pattern was also observed simultaneously in these cases, probably due to IgM antihuman albumin antibodies or an antipolymerized human serum albumin receptor site antibody. Of 202 HBsAg-positive patients, 71 showed polymerized human serum albumin receptor sites activity. The highest percentage of polymerized human serum albumin receptor sites was found among patients showing HBeAg and hepatitis B virus DNA polymerase positivity (96%), followed by HBeAg positivity and hepatitis B virus DNA polymerase negativity (48%), and anti-HBe positivity and hepatitis B virus DNA polymerase negativity (17%). In addition, a significant correlation between polymerized human serum albumin titers and hepatitis B virus DNA polymerase was found (r = 0.573, p less than 0.01). However, at similar HBeAg titer, patients who were positive for hepatitis B virus DNA polymerase had a higher polymerized human serum albumin receptor sites titer than those who were negative for hepatitis B virus DNA polymerase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3002940 TI - Transport and intracellular distribution of copper in a human hepatoblastoma cell line, HepG2. AB - The uptake of radiocopper by HepG2 cells is a saturable, temperature-dependent and cellular energy-independent process with a Vmax of 7.1 +/- 0.2 pmoles min-1 mg protein-1 and an estimated Km of 3.3 +/- 0.5 microM. The rate of copper uptake is reduced at an equimolar concentration of albumin and is unaffected by zinc at a 10-fold molar excess. Approximately 70% of the newly incorporated radiocopper binds to membranes and organelles, while 30% is recovered in the cytosol. The soluble fraction can be resolved into two copper-binding protein peaks. Incubation of HepG2 with nonisotopic copper results in displacement of radiocopper associated with the proteins contained in the lower molecular weight peak. Exposure of the cells to cycloheximide inhibits the incorporation of the isotope into this fraction. PMID- 3002942 TI - Papillary Wilms' tumour with carcinoma-like foci and renal cell carcinoma in childhood. AB - Five cases of Wilms' tumour with prominent papillary formation and focal carcinoma-like epithelium are described. The morphology of these tumours is compared with a group of six renal cell carcinomas in childhood. A link between this type of nephroblastoma and renal carcinoma is suggested but pure renal carcinomas are considered to be a separate category. The biological behaviour of the papillary Wilms' tumours was found to be more aggressive than is generally believed. The course of the disease may be influenced by carcinoma-like structures although this fact is not definitely established. Staging seems to be more helpful in such patients as is the case with renal cell carcinomas. Further studies on this type of Wilms' tumour are needed to establish more consistent data. PMID- 3002943 TI - Treatment research and the NIMH re-reorganization. PMID- 3002944 TI - NIMH report. NIMH reorganization focuses on specific disorders, basic science, and research training. PMID- 3002945 TI - Hospital-sponsored chemical dependency self-help groups. PMID- 3002946 TI - Neuropsychiatric manifestations of AIDS-spectrum disorders. AB - Psychiatric symptoms among patients with acquired immune deficiency syndrome (AIDS) may be functional reactions to contracting a fatal and stigmatizing disease or may be secondary to malignancies and opportunistic infections in the central nervous system (CNS). More recent evidence indicates that HTLV-III, the virus that causes AIDS, directly infects the CNS and may cause psychiatric symptoms before signs of immunodeficiency, cognitive impairment, or neurological abnormalities emerge. AIDS-related organic mental syndromes may mimic functional disorders such as chronic mild depression and acute psychosis. Both of these common presentations are illustrated with detailed case reports, and diagnostic and management guidelines are provided. PMID- 3002947 TI - Community care. PMID- 3002950 TI - Pure mucinous carcinomas of breast: morphologic features and prognostic correlates. AB - Fifty-three pure mucinous carcinomas of breast were studied, with long-term follow-up evaluation (average, 15 years) for 37. The mean age of the patients was 63 years. Long-term survival (Kaplan-Meier method) was 68 per cent, excluding non tumor-related deaths. Median survival for the patients with fatal carcinomas was 11.3 years. Fatal and disseminated carcinomas were best predicted by the presence of axillary metastases and by tumor cellularity and size. Only five of the patients had axillary metastases at mastectomy, but in four of these five patients distant metastases developed later. The diameters of all ten of the fatal and disseminated carcinomas were at least 2.5 cm, while 17 of the 27 long term survivors had smaller tumors. All ten of the fatal and disseminated carcinomas had cellularity of more than 10 per cent, while 12 of 27 long-term survivors had less cellular tumors. Tumor cellularity did not correlate with tumor size; these features were entirely independent. Thus, in contrast to the findings of some prior studies with shorter follow-up periods, pure mucinous carcinomas were seen to be slowly growing, but fully malignant, tumors warranting standard breast carcinoma treatment. PMID- 3002949 TI - Chronic mononucleosis: pitfalls in the laboratory diagnosis. PMID- 3002948 TI - The human T-cell leukemias: clinical, cytomorphologic, immunophenotypic, and genotypic characteristics. AB - The distribution of the conventional lymphoid cell markers on T lymphocytes and the principal panels of monoclonal antibodies used to recognize distinctive T lymphocyte-associated differentiation antigens are discussed. These reagents have been used to probe the early and late stages of T-cell differentiation, and a hypothetical schema of T-cell differentiation has been constructed. Application of these reagents to the investigation of neoplastic T cells has resulted in the determination of the subset of origin and the stage of differentiation of the neoplastic cells in T-cell-derived lymphoproliferative malignancies. Recent advances in molecular biology have made possible the Southern blot hybridization analysis of DNA extracted from neoplastic T cells for patterns of T-cell-receptor gene rearrangements. Examination of these patterns in benign and malignant T and non-T cell has provided the basis for the use of T-cell-receptor gene rearrangements as specific genetic markers of T-cell lineage, clonality, and differentiation. These and other advances have resulted in the delineation of a new category of T-cell neoplasia, the adult T-cell leukemia/lymphoma syndrome. They have also demonstrated that the majority of clinically indolent neoplasms composed of large granular lymphocytes in so-called T gamma-lymphoproliferative disease are monoclonal proliferations. Further phenotypic, functional, and genotypic analyses of the T-cell malignancies should provide better understanding of T-lymphocyte differentiation and heterogeneity. Such studies should also lead to better clinicopathologic correlations and greater understanding of the basis for the clinical diversity of the T-cell-derived lymphoproliferative malignancies. PMID- 3002951 TI - Tetraploid partial hydatidiform moles: two cases with a triple paternal contribution and a 92,XXXY karyotype. AB - In the course of a systematic study of cytogenetics, morphology, and clinical follow-up of hydatidiform moles we encountered two unusual cases of partial hydatidiform moles each with a 92,XXXY karyotype. Previously reported cases of tetraploidy, of 92,XXXX or 92,XXYY karyotype, resulted from a failure of the first mitotic division of a normal zygote. This is to our knowledge the first report of tetraploidy with XXXY sex chromosomes. Study of chromosomal heteromorphisms, isozymes, and restriction fragment length polymorphisms reveal that both present cases resulted from a combination of a haploid ovum with three haploid sets of paternal chromosomes either by the mechanism of trispermy (involving three separate haploid spermatozoa) or through dispermy (involving one haploid and one diploid sperm). Both cases resembled closely partial moles in their morphology; one gave a highly typical clinical picture while the other was recognized at an early voluntary abortion. Partial moles are ordinarily triploids of nearly always diandric constitution that evince focal villous swelling with cistern formation and focal trophoblastic hyperplasia. The findings here presented point to an association of molar phenotype with an excess of paternal over maternal haploid sets. PMID- 3002953 TI - Confirmation of the close linkage between the loci for human apolipoproteins AI and AIV by the use of a cloned cDNA probe and two restriction site polymorphisms. AB - We have isolated a cDNA probe for human apolipoprotein AI (apoAI) and used two DNA polymorphisms detected by this probe to analyse the inheritance of the apoAI gene in families informative for apoAIV protein variants. We have thereby increased the lod score for this linkage from 3.01 to 6.32 at a recombination fraction of zero. PMID- 3002952 TI - Characterization of a set of X-linked sequences and of a panel of somatic cell hybrids useful for the regional mapping of the human X chromosome. AB - We have characterized 19 DNA fragments originating from the human X chromosome. Most of them have been isolated from an X chromosome genomic library (Davies et al. 1981) using a systematic screening procedure. These DNA probes have been used to search for restriction fragment length polymorphisms (RFLP). The frequency of restriction polymorphisms (1 per 350 bp analysed) was lower than expected from data obtained with autosomal fragments. The various probes have been mapped within 12 subchromosomal regions using a panel of human-rodent hybrid cell lines. The validity of the panel was established by hybridization experiments performed with 27 X-specific DNA probes, which yielded information on the relative position of translocation breakpoints on the X chromosome. The DNAs from the various hybrid lines are blotted onto a reusable support which allows one to quickly map any new X-specific DNA fragment. The probes already isolated should be of use to map unbalanced X chromosome aberrations or to characterize new somatic cell hybrid lines. The probes which detect RFLPs define new genetic markers which will help to construct a detailed linkage map of the human X chromosome, and might also serve for the diagnosis of carriers or prenatal diagnosis. PMID- 3002954 TI - Lysosomal alpha-galactosidase in endothelial cell cultures established from a Fabry hemizygous and normal umbilical veins. AB - An endothelial cell line has been established from the umbilical vein obtained after abortion of a male fetus suffering from Fabry disease. This X-linked inborn error of glycosphingolipid catabolism results from deficiency of the lysosomal hydrolase alpha-galactosidase A. The clinical manifestations of the disease are mainly caused by glycosphingolipid depositions in the endothelium of all vessels. The hemizygous cell line and eight endothelial cell lines originating from the umbilical cords of normal newborns were grown for more than 10 passages. They had a short generation time that allowed us to get sufficient cells for qualitative and quantitative investigations of alpha-galactosidase. The enzyme in normal endothelial cells had a similar thermostability and isoelectric focusing pattern as that in fibroblasts, but the activity was essentially higher in endothelial cells. The hemizygous endothelial cells were deficient in alpha-galactosidase A. It is concluded that endothelial cell lines are an important alternative to fibroblasts for in vitro studies of the lysosomal storage diseases. PMID- 3002955 TI - Restriction fragment length polymorphisms in the D7S1 region of human chromosome 7. AB - A restriction fragment length polymorphism in the D7S1 region of chromosome 7 is detected by hybridizing the recombinant plasmid pA2H3 to HindIII- or HinfI digested human DNA. Three HindIII and two HinfI alleles were detected, and it was found that all individuals carrying the variant HinfI allele also had the commoner of the two variant HindIII alleles. All variants seem to result from point mutation in restriction enzyme recognition sites. Restriction mapping of the D7S1 region revealed that the associated HindIII and HinfI alleles are defined by sites 300 base-pairs (bp) apart, and it is suggested that the close proximity of these sites is sufficient to account for the strong phenotype association observed. PMID- 3002956 TI - Four restriction fragment length polymorphisms revealed by probes from a single cosmid map to human chromosome 12q. AB - Human gene mapping would be greatly facilitated if marker loci with sufficient polymorphism information content were generally available. As a source of such markers, we have used cosmids from a human genomic library. We have used a rapid method for screening random cosmids to identify those homologous to genomic regions especially rich in restriction fragment length polymorphisms (Litt and White 1985). This method allows whole cosmids to be used as probes against Southern transfers of genomic DNA; regions of cosmid probes homologous to repeated genomic sequences are rendered unable to anneal with Southern transfers by prehybridization of the probes with a vast excess of non-radioactive genomic DNA. From one cosmid (C1-11) identified by this procedure, we have isolated four single-copy probes, each of which identifies a polymorphic locus. Despite the existence of some linkage disequilibrium in this system, the polymorphism information content was computed as 0.73. Using a somatic cell hybrid mapping panel, we have mapped probes from cosmid 1-11 to human chromosome 12q. Additionally, in situ hybridization of the whole cosmid to metaphase spreads allowed more precise assignment of the locus to the region 12cen----q13. The locus revealed by probes from cosmid 1-11 has been designated D12S6. PMID- 3002957 TI - Mothers of children with Down syndrome have higher herpes simplex virus type 2 (HSV-2) antibody levels. AB - The antibody response to herpes simplex virus (HSV) was studied in 53 mothers of children with Down syndrome (Ds) and compared with that in 154 controls, using sera sampled during pregnancy or at delivery. Conventional analysis of HSV complement fixing antibodies showed the same frequency of positivity for the two groups (70%). When the levels of IgG antibodies to an HSV-1 and an HSV-2 antigen preparation were determined by an enzyme-linked immunosorbent assay (ELISA) technique, it was found that the Ds and control mothers had similar levels of IgG antibodies to HSV-1, whereas the level of IgG antibodies to HSV-2 was significantly (P less than 0.001) higher in Ds mothers. The ratio of HSV-2 to HSV 1 ELISA IgG was calculated for each mother and the distribution of these ratios also differed significantly between the control and Ds mothers. The differences found were not due to differences in age distribution in the control and Ds groups. For comparison a third procedure, measurement of thymidine kinase blocking antibody (TK ab), was used. With this procedure the mothers were divided into groups estimated to be positive for HSV-1, HSV-2, or both. Statistical analyses showed a good correlation between the type found in TK ab analyses and the ratio found in the ELISA HSV test. The results clearly demonstrated an overrepresentation of HSV-2 antibody positivity among Ds mothers, though not of sufficient magnitude to imply that HSV-2 can be the major cause of Ds. It is discussed whether HSV-2 might be related to the recently increased birthrate of children with Ds among young mothers in Sweden or to localized geographical clustering of Ds births, or whether the increased HSV-2 antibody positivity merely indicates that factors following the same epidemiological pattern are involved in the aetiology of Ds. PMID- 3002958 TI - Mitochondrial DNA polymorphism in Japanese. II. Analysis with restriction enzymes of four or five base pair recognition. AB - Mitochondrial DNA (mtDNA) from 116 Japanese was analyzed with nine restriction enzymes that recognize a four or five base pair sequence. The sizes of the mtDNA fragments produced by digestion by each enzyme were compared after gel electrophoresis. Double digestion experiments indicated that, in the coding region from URF2 (unidentified reading frame) to tRNAAsn (bp 5274-5691), there is an insertion of about 60 base pairs (bp) compared with the published mtDNA sequence, which is common to all individuals in the present sample. A total of 95 different morphs were detected with the nine enzymes, 60 of which have not been documented previously. Based on a comparison of the cleavage maps of all individuals, 62 different combinations of restriction types were observed. By pairwise comparison of each restriction type, the average number of nucleotide substitutions per nucleotide site (delta) was estimated to be 0.0026. Phylogenetic analysis of the present data indicates that at least two distinct lineages exist in the Japanese population. PMID- 3002959 TI - Heterogeneity of haplotypes among patients with severe Cooley disease in Eastern Sicily. AB - There is a large variation of clinical severity among thalassemic patients in Sicily. A heterogeneous molecular basis has already been demonstrated among the patients presenting with thalassemia intermedia. The same approach, based mostly on linked haplotypes of the beta gene cluster polymorphisms and in some cases on the demonstration of the molecular defect itself, was used to investigate 55 patients presenting with severe Cooley anemia, all maintained under permanent transfusion regimen. A large heterogeneity was demonstrated in the observed haplotypes, and only a limited overlap with those already found in thalassemia intermedia. It has been noted that many of the patients are compound heterozygotes, the various observed associations making the antenatal diagnosis at the DNA level difficult in the near future. PMID- 3002960 TI - Assignment of the human tissue-type plasminogen activator gene (PLAT) to chromosome 8. AB - Using 1.2kb 3'-terminal Pst-I fragment of a full length tissue-type plasminogen activator (t-PA) cDNA clone (ptPA-8FL) and a set of rodent human somatic cell hybrids, the corresponding human gene PLAT was localized on chromosome 8. PMID- 3002962 TI - Sexual behaviour of women with human papillomavirus lesions of the uterine cervix. PMID- 3002961 TI - Two regimens of sultamicillin in treating uncomplicated gonorrhoea. AB - Sultamicillin is a covalent union of ampicillin and the beta lactamase inhibitor, sulbactam (CP-45,899). Two studies were conducted to assess its efficacy in treating uncomplicated gonorrhoea. In the first study treatment comprised sultamicillin 1.5 g and probenecid 1 g; 124 (89.2%) of 139 patients responded including seven of 11 patients harbouring beta lactamase (penicillinase) producing strains of Neisseria gonorrhoeae (PPNG). In the second study sultamicillin 2.25 g and probenecid 1 g were given; 122 (93.8%) of 130 patients responded. Only two of seven pharyngeal infections resolved, and if pharyngeal infections are excluded the overall cure rate rose to 95.3%. Thirteen of 14 patients infected with PPNG strains were cured by the larger dose. Side effects were mild and transitory. It may be concluded that sultamicillin 2.25 g plus probenecid 1 g is an effective regimen to treat uncomplicated rectal and genital gonorrhoea and is useful for treating infections with PPNG strains. Most beta lactamase resistant antimicrobials must be given parenterally; sultamicillin is given by mouth. PMID- 3002963 TI - Epidemiology of infections with cytomegalovirus (CMV) and herpes simplex virus in promiscuous women: absence of exogenous reinfection with CMV. AB - Herpes simplex virus (HSV) was shed from the genital tracts of 26 (15%) and cytomegalovirus (CMV) from 19 (11%) of 177 promiscuous women. Virus excretion was especially common (HSV 41% and CMV 19%) in the youngest age group (aged 19 or less). Episodic shedding of CMV was studied by restriction enzyme analysis of the viral DNAs prepared from repeat isolates. Repeat isolates from each woman were identical, which indicated that recurrent shedding of CMV resulted from endogenous reinfection. Even under conditions of high exposure no exogenous reinfection with CMV could be shown, which indicated that exogenous reinfection is rare. PMID- 3002964 TI - Effect of a single exposure to weak electromagnetic fields of very low frequency on parameters of the endocrine system. PMID- 3002965 TI - Influence of vitamin D3 metabolites on the production of interleukins 1,2 and 3. AB - We investigated the effect of vitamin D3 metabolites on the release of the three interleukins (IL) IL 1, IL 2 and IL 3 by mononuclear cells. Models for the production of these mediators were the release of IL 1 by the murine macrophage cell line P388D1, of IL2 by rat spleen cells, and of IL 3 by the murine WEHI-3 cell line. IL 1 production was significantly increased with 1 alpha,25 dihydroxyvitamin D3 (1,25(OH)2D3) at 10(-10)M and above. 1,25(OH)2D3 did not stimulate cell proliferation as assessed with [methyl-3H]thymidine (3H-TdR) incorporation. 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 25-hydroxyvitamin D3 (25(OH)D3) were about 1000 times less effective than 1,25(OH)2D3. IL 2 production, by cultured rat spleen cells stimulated with Concanavalin A, was decreased by increasing concentrations of 1,25(OH)2D3. The minimal effective dose varied between experiments and ranged from 10(-11) to 10(-8) M. Moreover, proliferation (3H-TdR incorporation) of mouse thymocytes treated with phytohemagglutinin and IL 1 was decreased in a dose-dependent fashion by 1,25(OH)2D3 starting at 10-11) M. This effect might be secondary to a decrease of endogenous IL 2 production. IL 3 release by WEHI-3 cells was significantly increased with 10(-11)-10(-9) M 1,25(OH)2D3, whereas higher concentrations were less effective or decreased IL 3 production. These results show that 1,25(OH)2D3 and, to a lesser extent, 24,25(OH)2D3 and 25(OH)D3 have selective effects on lymphokine production. It is tempting to speculate that the actions of 1,25(OH)2D3 on bone might in part be mediated by lymphokines. Moreover, we suggest that 1,25(OH)2D3 might not be an immunoregulator per se, but makes use of the immune system to exert its influence on one of its classical targets, namely the bone, and possibly on other connective tissues. PMID- 3002966 TI - Evidence that transferrin may function exclusively as an iron donor in promoting lymphocyte proliferation. AB - In order to distinguish between a requirement for iron and a possible additional requirement for the iron-binding protein transferrin per se, the ability of mouse lymphocytes to proliferate in response to concanavalin A has been investigated. Cells proliferated well when cultured in medium containing 5% fetal calf serum, but if iron-free mouse or human transferrins were added, proliferation was inhibited by greater than 80%, whereas the same transferrins saturated to 30% with iron enhanced proliferation by 40-70%. In serum-free medium, proliferation was greater in the presence of 30% iron-saturated transferrin than when the protein was saturated only to 10%. Addition of Mn3+ to the latter, to bring the total metal saturation to 30%, gave no improvement in proliferation. Lymphocytes took up iron preferentially when transferrin containing both iron and manganese was present in the culture medium. The degree of proliferation in serum-free medium in the presence of a variant of human transferrin with abnormal iron binding and receptor-binding properties was almost identical to that when normal human transferrin was used. Finally, when a monoclonal antibody to the mouse transferrin receptor and iron-nitriotriacetate were substituted for iron transferrin in serum-free medium, proliferation was reduced by greater than 95%. These results strongly suggest that transferrin promotes lymphocyte proliferation solely as a result of its iron-donating properties, and that an additional role such as the provision of a proliferation-inducing membrane signalling event following interaction with the transferrin receptor seems unlikely. PMID- 3002967 TI - Effects of anti-Tac antibody on the response of large granular lymphocytes to interleukin-2. AB - Large granular lymphocytes (LGL), which consist of a unique population comprising almost all of the natural killer (NK) cell activity, were separated from peripheral blood mononuclear cells by sequential depletion of monocytes and conventional discontinuous Percoll density gradient sedimentation. The LGL populations thus obtained exhibited significant levels of interferon-gamma (IFN gamma) production and proliferation, as well as augmentation of NK cell activity in response to interleukin-2 (IL-2). Among these IL-2-driven phenomena, only IFN gamma production was markedly inhibited by the monoclonal anti-Tac antibody, which presumably recognized the IL-2 receptor, whereas the proliferative response and the augmentation of NK cell activity were only minimally affected by the same antibody, even at the higher concentration. The dissociation of the effects of anti-Tac antibody on these IL-2-driven events gives us some insight on the role of IL-2 receptors involved in these immunologically interesting functions of IL 2. PMID- 3002968 TI - Vitamin D3, gamma interferon, and control of proliferation of Mycobacterium tuberculosis by human monocytes. AB - Previous studies have shown that recombinant interferon gamma (IFN-gamma), crude T cell supernatants, or appropriate T-cell lines can cause total inhibition of the growth of M. tuberculosis inside murine peritoneal macrophages. In similar experiments with human monocytes much smaller effects are seen. This could be due to the relative immaturity of these cells. Because dihydroxy vitamin D3 (1,25 (OH)2 D3) can cause phenotypic differentiation of immature leukemic lines into macrophage-like cells, we have explored the possibility that exposure to cholecalciferol metabolites in vitro might increase the ability of monocytes to control proliferation of M. tuberculosis, or cause monocytes to mature into cells able to respond appropriately to IFN-gamma. Incubation of monocytes with three cholecalciferol metabolites induced anti-tuberculosis activity to an extent that correlated with their binding affinities to the intracellular receptor protein for the derivatives. 1,25-(OH)2 D3 also primed monocytes for phorbol myristate acetate-triggered reduction of nitroblue tetrazolium. The effects were additive rather than synergistic with those of IFN-gamma. Monocytes incubated with IFN gamma developed 25-OH D3 1-hydroxylase activity, detected by conversion of tritiated 25-(OH) D3 to a more polar metabolite which coeluted with 1,25-(OH)2 D3 on straight and reverse-phase HPLC. The latter is a more active form in vivo. These findings help to explain claims for the efficacy of vitamin D in the treatment of some forms of tuberculosis, and also the occasional finding of raised serum calcium, and disturbed vitamin D metabolism in these patients. PMID- 3002969 TI - Production and characterization of an anti-idiotypic antibody specific for a monoclonal antibody to glycoprotein D of herpes simplex virus. AB - A monoclonal antibody specific for glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) was used to prepare an anti-idiotypic antibody in rabbits. After removal of antibody reactivity to constant region determinants by absorption with polyclonal mouse immunoglobulins and a monoclonal antibody of the same subclass as the anti-gD monoclonal, the anti-idiotypic (anti-id) antibody reacted specifically with anti-gD. Using an ELISA inhibition assay with immunoaffinity purified gD, the anti-id D reagent inhibited the binding of anti-gD to gD, suggesting that anti-id D mimics an epitope of gD by binding the antigen combining site of anti-gD. Immunization of mice with anti-id D could prime splenocytes in vivo to proliferate in response to HSV antigen stimulation in vitro. The possibility that anti-id D could act similarly to gD and stimulate an immune response to HSV when administered in vivo is discussed. PMID- 3002971 TI - Large-molecular-weight thymocyte-activating factors in extracellular matrix of SV40-transformed human embryo fibroblasts. AB - It has been reported that the major thymocyte-activating factors at 10-15 kDa exist in the culture medium of SV40-transformed human embryo fibroblasts. However, these factors could not be detected in the extracellular matrix (ECM) from the same transformed cells, but the 30-40 kDa factors were found. They showed a similar lectin requirement, charge heterogeneity and Concanavalin A binding property as compared with the factors reported previously. They were separated from fibroblast DNA synthetic activities in ECM and did not contain interleukin 2 activity. A possible relationship between 30-40 kDa factors in ECM and 10-15 kDa factors in the culture medium is discussed. PMID- 3002970 TI - The importance of the Fc receptors for IgA in the recognition of IgA by mouse liver cells: its comparison with carbohydrate and secretory component receptors. AB - The numbers of murine hepatocytes and Kupffer cells with receptors for human polymeric (pIgA) or monomeric IgA (mIgA) were determined by rosette formation with immunobeads coated with human pIgA or mIgA. About 63% of hepatocytes were found to express receptors for pIgA. Receptors for mIgA were also found in a significantly lower percentage of hepatocytes (50%). Only about 10% of Kupffer cells had this type of receptor. Blocking studies performed with purified human immunoglobulins suggested that both cells expressed a receptor that recognized the Fc portion of IgA (either pIgA or mIgA). In addition, murine multimeric MOPC 315 IgA strongly inhibited in a dose-dependent fashion the pIgA and mIgA rosette formation by both cells. When cells were incubated in the presence of a rabbit antiserum to secretory component (SC), only pIgA rosette-forming hepatocytes were partially inhibited. No such inhibition was seen when an antiserum to human IgG was used. Both galactose and mannose monosaccharides failed to inhibit the IgA rosette formation by either type of cell. These results show that murine hepatocytes, besides having SC receptors for polymeric IgA, also express receptors for the Fc portion of IgA. The carbohydrate receptors did not seem play any role in the recognition of IgA. The fact that Kupffer cells exclusively present Fc alpha R suggests that monomeric and polymeric IgA, either in form of immune complexes or not, are handled in a different manner by the liver cells. PMID- 3002972 TI - Direct activation of Sendai virus (HVJ)-specific cell-mediated immunity of mice for second set rejection of the virus-infected syngeneic tumor by noninfectious virus-sensitized spleen cells. AB - Syngeneic spleen cells (SPC) sensitized in vitro with noninfectious NVJ were shown to effectively stimulate mice to generate the HVJ-specific cell-mediated immunity for second set rejection (SSR) of virus-infected syngeneic leukemia cells. As few as 10(4) live but not disrupted SPC either infected with a temperature-sensitive mutant of HVJ (HVJts) or sensitized passively with ultraviolet (UV)-inactivated HVJts were active as immunogen. Syngeneic SPC as the carrier of virus could be replaced by allogeneic SPC or L cells, a fibroblast cell line, without reduction of the immunogenicity. Further study demonstrated that a special density of antigen on the surface of HVJts-sensitized SPC is required for high immunogenicity. It was suggested that live cells appropriately sensitized with noninfectious virus would serve as an excellent vaccine for virus specific cell-mediated immunity. PMID- 3002973 TI - Antibody enhanced viral growth in macrophages. AB - Antiviral antibody can promote viral entry into macrophages by pathways involving cellular receptors for the Fc portion of immunoglobulin or for complement components. Whether virus taken up through these routes is restricted or results in productive infection depends upon a balance between a number of variables. These include the virus strain and dose, the macrophage source and state of activation, the concentration, class and viral specificity of the antibody, and environmental factors such as time and temperature. Under appropriate conditions viral replication can be enhanced by antiviral antibodies. PMID- 3002974 TI - Genetic aspects of macrophage involvement in natural resistance to virus infections. AB - Macrophages are thought to constitute an important element in the body's natural defense against invasion and dissemination of viruses. Possible antiviral mechanisms of macrophages are defined and referred to as intrinsic, i.e. the ability of macrophages to serve as a nonpermissive barrier between the virus and susceptible cells and extrinsic, i.e. the ability of macrophages to affect the virus or virus replication in surrounding cells. Most studies on the role of macrophages in natural resistance to virus infections have been performed in animal models. An interesting aspect of many viral infections in animals is the finding of a genetically determined variation in natural resistance. Because of the availability of numerous inbred and congenic strains most studies on genetically determined resistance have been performed in mice. The classical examples are resistance to flaviviruses and susceptibility to mouse hepatitis virus, both of which are inherited as dominant, monogenic traits. With these viruses macrophage intrinsic restriction of virus replication has been found to express at the cellular level the genetics of resistance/susceptibility seen in the intact animal. Other examples, where macrophages have been implicated in genetically determined resistance include herpes simplex virus and influenza virus. The involvement of macrophages in natural resistance to these viruses is discussed in relation to other putative resistance determinants like interferon production and sensitivity and natural killer cell activity. PMID- 3002976 TI - A DR3-related DX alpha gene polymorphism strongly associates with insulin dependent diabetes mellitus. AB - HLA-DQ alpha and HLA-DX alpha gene polymorphisms were analyzed by Southern blot techniques in 78 Caucasoid insulin-dependent diabetes mellitus (IDDM) subjects and 55 control subjects. Five restriction fragment length polymorphisms of the HLA-DQ alpha gene correlated with HLA-DR typing. Two allelic DX alpha-related gene fragments, of 2.1 kb (U) and 1.9 kb (L) in size were identified. Genotype frequencies in the IDDM group for UU, UL, and LL were 54%, 38.5%, and 7.5%, respectively, whereas the corresponding frequencies in the control group were 24%, 40%, and 36% (P less than 0.00005 for differences in genotype frequencies). The U allele was associated particularly with IDDM patients who were DR3, with healthy controls who were DR3, as well as with IDDM patients who were not DR3. Thus, if this DX alpha U allele is not the DR3-associated IDDM susceptibility gene, it is the closest marker hitherto studied. PMID- 3002977 TI - Nitrates: epidemiological evidence. PMID- 3002975 TI - Genetic interaction between non-MHC T- and B-cell alloantigens in response to Rous sarcomas in chickens. AB - Chickens of Regional Poultry Research Laboratory (RPRL) inbred line 6(3) regress sarcomas induced by Bryan high-titer Rous sarcoma virus to a greater extent than chickens of line RPRL 100, although these lines are identical for the major histocompatibility B complex. They differ, however, at three independent autosomal loci: Ly-4 and Th-1 determine the surface alloantigens of partly overlapping subsets of T lymphocytes, and Bu-1 determines a surface alloantigen of B lymphocytes. The association of genotypes at these loci with quantitative variation in their ability to regress Rous sarcomas was tested in segregating F4 generation progeny derived from crosses of lines 100 and 6(3). The Ly-4 and Bu-1 genotypes showed association with Rous sarcoma regression, but the Th-1 genotype did not. Chickens of the Ly-4a/Ly-4a, Bu-1b/Bu-1b and Ly-4b/Ly-4b, Bu-1a/Bu-1a genotypes had a significantly higher regressor ability than the other two double homozygous genotypes. These results indicate that higher regression is associated with (1) interaction between the Ly-4 and Bu-1 loci, and (2) complementation between either the line 6 Ly-4a allele and the line 100 Bu-1b allele, or the line 100 Ly-4b allele and the line 6 Bu-1a allele. PMID- 3002978 TI - How does salt raise blood pressure? A hypothesis. AB - Existing data in the literature indicate that alpha 2-adrenergic receptor agonists have a profound hypotensive action, that sodium attenuates the affinity of alpha 2-adrenergic receptors for agonists, that the location of these receptors in the central nervous system is mainly at the sites of cardiovascular regulation, and that these sites exert a constant tonic inhibition of sympathetic vasoconstrictor tone. This article proposes the theory that sodium exerts its hypertensive action by decreasing the state of affinity of the alpha 2-adrenergic receptors of the central nervous system for locally occurring agonist neurotransmitters, which results in disinhibition of sympathoinhibitory neurons and leads to the hyperadrenergic state characteristic of salt-induced hypertension. PMID- 3002979 TI - Renal and systemic effects of enalapril in chronic one-kidney hypertension. AB - We have investigated the role of angiotensin II in the development of high blood pressure and in the maintenance of renal function during 2 weeks of one-kidney renal artery stenosis in conscious dogs. Responses to a fixed degree of inflation of a balloon cuff around the renal artery were compared in dogs with or without continuous enalapril (MK 421) treatment. In six untreated dogs, mean aortic pressure was increased by 17.1 +/- 2.0 mm Hg, due primarily to increases in total peripheral resistance with little change in cardiac output, while glomerular filtration rate, renal blood flow, renal artery pressure, and plasma renin activity were back to prestenosis levels. In seven enalapril-treated dogs mean aortic pressure was increased by 23.0 +/- 2.7 mm Hg and was not significantly different from that occurring in untreated dogs. This rise was due to increases in total peripheral resistance (10%) and cardiac output (12%). In the absence of angiotensin II, glomerular filtration rate remained low, at only 56 +/- 6% of prestenosis levels. Renal blood flow returned to normal, but the renal artery pressure remained 25% lower than control values. Thus, the main role of angiotensin II in chronic one-kidney Goldblatt hypertension does not appear to be through its pressor properties but rather through its actions in the kidney to preserve glomerular filtration. This effect on renal function persisted throughout the course of the hypertension, even when the plasma renin levels returned to normal. PMID- 3002980 TI - Central adrenergic receptor control of renal function in conscious hypertensive rats. AB - The role of central nervous system alpha-adrenergic and beta-adrenergic receptors in the increased renal sympathetic nerve activity and antinatriuresis resulting from environmental stress (air stress) in conscious spontaneously hypertensive rats (SHR) was examined. Intracerebroventricular administration of the alpha 2 adrenergic receptor agonist clonidine (1, 5, and 15 micrograms) prevented the effects of air stress on renal sympathetic nerve activity and urinary sodium excretion. Clonidine, 5 and 15 micrograms, lowered baseline mean arterial pressure and renal sympathetic nerve activity and increased baseline urine flow rate and urinary sodium excretion; clonidine, 1 micrograms, had no effect on these baseline levels. Intravenous administration of 5 micrograms, but not 1 microgram of clonidine, abolished the renal responses to air stress. Intracerebroventricular administration of alpha 2-adrenergic receptor antagonists (yohimbine, rauwolscine) reversed the effects of clonidine, alpha 2-adrenergic receptor blockade alone, alpha 1-adrenergic receptor blockade (20 micrograms prazosin), or combined alpha 1-adrenergic and alpha 2-adrenergic receptor blockade (30 micrograms phenoxybenzamine) had no effect on the renal sympathetic nerve activity or antinatriuretic responses to air stress. Intracerebroventricular, but not intravenous, administration of the beta 2 adrenergic receptor antagonist ICI 118551 (30 micrograms) prevented the increased renal sympathetic nerve activity and antinatriuretic responses to air stress. In contrast, intracerebroventricular administration of the beta 1-adrenergic receptor antagonist atenolol (30 micrograms) had no effect on the renal responses to air stress. These results indicate that the increased renal sympathetic nerve activity and antinatriuresis resulting from environmental stress in conscious SHR can be prevented by pharmacological stimulation of central alpha 2-adrenergic receptors or by blockade of central beta 2-adrenergic receptors. PMID- 3002982 TI - Syndrome of hypertension and hyperkalemia with normal glomerular filtration rate. PMID- 3002981 TI - Aldosterone excretion rates in children and adults during sleep. AB - The present study undertook to examine aldosterone excretion during sleep as an integrated measurement of aldosterone production. A 24-hour urine collection was divided into awake and sleep fractions. Urinary aldosterone and electrolyte excretion were measured in 26 healthy children (mean age, 8.9 +/- 1.9 [SD] years) and 28 adults (mean age, 29.9 +/- 9.5 years). Aldosterone excretion in children was 5.6 +/- 3.9 (SD) micrograms/g creatinine during the awake period, which was significantly different from the 3.9 +/- 4.1 micrograms/g creatinine value recorded during sleep (p less than 0.002). In adults, awake aldosterone excretion was significantly greater than that during sleep; 4.9 +/- 2.7 versus 3.2 +/- 1.6 micrograms/g creatinine (p less than 0.001). Sleep aldosterone excretion values were highly correlated with the corresponding 24-hour aldosterone excretion values (r = 0.85, p less than 0.001) in children and in adults (r = 0.64, p less than 0.001). Sleep aldosterone excretion was correlated with 24-hour potassium excretion (p less than 0.02) only in children. Sleep aldosterone excretion correlated with neither sleep nor 24-hour sodium excretion in children or adults. Sleep electrolyte excretion rates were highly correlated with 24-hour excretion rates in both children and adults. Dexamethasone, 1 mg, administered the night before to suppress the normally high morning levels of endogenous adrenocorticotropic hormone, had no discernible effect on sleep aldosterone excretion. These results indicate that measurement of aldosterone excretion in an easily collected sleep urine sample provides a reliable index of aldosterone production in children and adults. PMID- 3002983 TI - Isolation and characterization of a 60-kilodalton salivary glycoprotein with agglutinating activity against strains of Streptococcus mutans. AB - A bacterial agglutinin specific for strains of Streptococcus mutans was isolated from human saliva. Physiochemical analyses showed the agglutinin to be a glycoprotein with a molecular weight of 60,000. The agglutinin aggregated four of the eight strains of Streptococcus mutans tested but did not aggregate the strains of Streptococcus salivarius, Streptococcus sanguis, and Streptococcus mitis tested. Chemical modification of carbohydrate moieties of the agglutinin with sodium metaperiodate had no effect on aggregation, whereas modification of the polypeptide portion with trypsin abolished aggregating activity. A set of five murine hybridoma antibodies was employed to further analyze the agglutinin. Two carbohydrate-specific antibodies, directed against D-mannose and N acetylgalactosamine moieties, respectively, failed to block agglutinin- or whole saliva-mediated aggregation of S. mutans cells. In contrast, two antibodies directed against pronase-sensitive antigenic sites blocked both agglutinin- and saliva-mediated aggregation of S. mutans cells. Western blot analysis with the agglutinin-specific hybridoma antibodies demonstrated the agglutinin in whole saliva and in artificial tooth pellicles formed on hydroxyapatite beads incubated with saliva. These results suggest that a 60-kilodalton glycoprotein of human saliva is a bacterial agglutinin with specificity for certain strains of S. mutans. They further suggest that aggregation is mediated by polypeptide rather than carbohydrate determinants of the glycoprotein. PMID- 3002984 TI - Virulence genes regulated at the transcriptional level by Ca2+ in Yersinia pestis include structural genes for outer membrane proteins. AB - Yersinia pestis, the causative agent of plague, has a virulence determinant called the low-Ca2+ response (Lcr+ phenotype) that confers on the bacterium Ca2+ dependence for growth at 37 degrees C and expression of V antigen. This virulence determinant is common to the three species of Yersinia and is mediated by Lcr plasmids (called pCD in Y. pestis). In this study, we generated insertions of Mu dI1(Ap lac) in pCD1 of Y. pestis KIM, screened for cells showing transcriptional regulation by Ca2+, and obtained inserts that define at least four pCD1 genes. Their patterns of transcription under different growth conditions closely paralleled the pattern of expression of the V antigen. We tested for expression of Lcr-specific yersinial outer membrane proteins (Yops) by the pCD1::Mu dI1(Ap lac) plasmids. Four of the inserts each eliminated expression of a different Yop; one of these Yops was unique to Y. pestis. Two of the insertions affecting Yops caused avirulence, and one caused strongly decreased virulence of Y. pestis in mice. These data indicate that Yops, like the V antigen, are virulence attributes regulated in the low-Ca2+ response. PMID- 3002985 TI - Molecular alteration of the 140-megadalton plasmid associated with loss of virulence and Congo red binding activity in Shigella flexneri. AB - A plasmid of about 140 megadaltons has been associated with the invasiveness of Shigella flexneri. Upon subculturing in liquid media of fully virulent isolates of Shigella flexneri 2a YSH6000, which contains only a 230-kilobase-pair (kbp) plasmid in addition to 3.3- and 4.2-kbp cryptic plasmids characteristic to all S. flexneri strains, loss of invasiveness, loss of Congo red binding activity (Pcr), and complete loss of, or a deletion, or even a single-site IS insertion in the plasmid occurred simultaneously. This was ascribed to the fact that, once a noninvasive Pcr- cell has emerged, it overgrows the wild type as a consequence of its selective advantage in artificial media. A deletion map of the 230-kbp plasmid was made by analyzing SalI digests of 39 deletion derivatives plus 1 formed by insertion of an IS1-like element in independently isolated, noninvasive Pcr- mutants. Of 39 deletion derivatives, 16 belonged to a single type, and 6 belonged to another, suggesting deletion hot spots. The deletion map was confirmed and extended by analyzing 359 SalI-generated partial digests of the wild-type plasmid cloned into pBR322. Three copies of IS1-like elements were found on three different SalI fragments by Southern hybridization. Segments required for the Pcr+ phenotype seemed to occur at several different locations in the plasmid. Each of 28 representative Pcr- mutants were negative by the Sereny test. Hence, many, or possibly all, Pcr determinants were required for full virulence. PMID- 3002987 TI - Human antibody response to a group B serotype 2a meningococcal vaccine determined by immunoblotting. AB - The antibody response of 30 volunteers vaccinated with a complex of group B polysaccharide and outer membrane vesicles (OMV) from serotype 2a Neisseria meningitidis and of 3 individuals who received a placebo vaccine was determined by immunoblotting. OMV were separated by sodium dodecyl sulfate-gel electrophoresis and electrotransferred to nitrocellulose filters. Binding of immunoglobulin G (IgG), IgA, and IgM antibodies in the human sera to OMV components was detected with class-specific peroxidase-conjugated antibodies. The immunoblotting results were also related to the bactericidal activity of the sera and the meningococcal carrier status of the volunteers. Before vaccination weakly reactive bands in the molecular weight range of 140,000 to 10,000 were observed on the blots. Sera from carriers showed more marked bands. Individual patterns of increased reactivity were seen 6 weeks after vaccination. The main immunoreactive components of OMV corresponded to a molecular weight of 43,000 (class 1 protein), 30,000 (class 5 proteins), and 22,000. IgG antibodies in postvaccination sera of high bactericidal titers showed distinct binding to the 43,000-molecular-weight antigen. Meningococcal carriers had antibodies against an antigen of 22,000 molecular weight; in polyacrylamide gels this component did not stain with Coomassie brilliant blue or silver. The marked binding of IgG antibodies to the class 5 proteins decreased considerably between weeks 6 and 25 after vaccination. Periodate oxidation of OMV abolished the binding of IgG antibodies to the class 5 proteins, whereas the antigenicity of the 43,000-molecular-weight (class 1 protein) and 22,000-molecular-weight antigens was unaffected. PMID- 3002989 TI - Altered growth properties and cell surface changes in ras transformed mouse bladder epithelium. AB - Transfection of the c-Ha-ras-1 oncogene, cloned from EJ/T24 cells, into different mouse bladder epithelial cell lines resulted in the acquisition of tumorigenic potential and, in all but one cell line (MB33I), anchorage-independent growth. Sera from syngeneic mice bearing tumours immunoprecipitated an 18 kDa protein from ras-transfected urothelial cells which was not detectable in their parental counterparts. Screening of a limited panel of mouse cell lines showed this protein to be urothelium-specific and associated with the expression of an activated ras gene. Polyoma middle T and v-myc-transfected bladder epithelial cells did not express this 18 kDa protein. Localization of this protein to the cell surface was demonstrated by immunofluorescence and absorption studies. PMID- 3002986 TI - Mycobacterium leprae fails to stimulate phagocytic cell superoxide anion generation. AB - Mycobacterium leprae is an intracellular pathogen that is ingested by and proliferates within cells of the monocyte/macrophage series. Mechanisms by which intracellular pathogens resist destruction may involve failure to elicit a phagocyte "respiratory burst" or resistance to toxic oxygen derivatives and lysosomal enzymes. We have studied the ability of M. leprae and Mycobacterium bovis BCG to stimulate the generation of superoxide anion (O2-) in vitro by human blood neutrophils and monocytes and murine peritoneal macrophages. M. leprae bacteria failed to stimulate significant O2- release except at high bacteria-to cell ratios (greater than 50:1) whether or not they were pretreated with normal serum or serum from patients with lepromatous leprosy. Either viable or irradiated BCG; on the other hand, stimulated the three cell types to release significant amounts of O2- when challenged with as few as 10 organisms per cell. Serum pretreatment enhanced the release of O2- by the three cell types. Preincubation for 18 h with viable M. leprae did not inhibit the ability of monocytes to respond with an oxidative burst to phagocytic stimuli. The failure of M. leprae to stimulate phagocyte O2- generation may be an important factor in its pathogenicity. PMID- 3002988 TI - Computer-controlled large scale production of high specific activity [11C]RO 15 1788 for PET studies of benzodiazepine receptors. AB - Ethyl 8-fluoro-5,6-dihydro-5-[11C]methyl-6-oxo-4H-imidazo [1,5-a] [1,4]benzodiazepine-3-carboxylate ([11C]RO 15-1788) has been prepared automatically with high specific activity for in vivo visualization or quantitative analysis of brain benzodiazepine receptors. The yield, radiochemical yield, radiochemical purity and specific activity of the product ready for an i.v. injection were 276 +/- 76 mCi, 50.8 +/- 7.8%, 99.3 +/- 0.3% and 2.9 +/- 0.5 Ci/mumol, respectively, taking an average of the latest 3 runs. The time required was about 25 min. Each product was sufficient to carry out three successive clinical studies by positron emission tomography (PET). All the procedures other than evaporation and filtration at the final stage were carried out with specially designed equipment connected to a central control system for radioisotope production. PMID- 3002990 TI - Cloning and characterization of putative genes that specify sensitivity to neoplastic transformation by tumor promoters. AB - Two putative genes (termed pro 1 and pro 2) specifying sensitivity to induction of neoplastic transformation by TPA in mouse epidermal JB6 cells were cloned by sib selection from a size-selected genomic library of clonal cells sensitive to promotion of transformation. By restriction analysis, heteroduplex analysis, direct hybridization, and sequencing, the putative genes are different from and have no homology to known oncogenes. Both genes are independently and equally active as total DNA in the transfection assay. The transformation-promoting potential of these putative genes does not appear to result from gene amplification or detectable rearrangements, suggesting that small structural changes might confer the promoting activity. The mouse pro sequences are also found in monkey and human DNAs. The pro-1 sequence is homologous to middle repetitive elements in the mouse genome, namely the BAM 5 and B1 repeats. The sequence of pro-1 was determined and suggests that it contains the signals to be transcribed by RNA polymerase II and to encode a protein of 7.1 kDa. PMID- 3002991 TI - Correlation between quantitative expression of H-2K, H-2D and MuLV antigens on spontaneous AKR lymphomas. AB - Thymocytes of leukemic AKR mice were analysed with a fluorescence-activated cell sorter (FACS) using monoclonal antibodies (MAbs) reactive with H-2Kk and H-2Dk and conventional hetero-antibodies against MuLV gp-70 antigens. Comparison was made with thymocytes of the low-leukemia H-2k strain C3H, as well as with thymocytes of young AKR mice, late preleukemic AKR mice (6-9 months old) and mitogen-stimulated thymocytes of young AKR mice. Expression of H-2 antigens increased on cells of the majority of tumors, on thymocytes of some late preleukemic mice and on mitogen-stimulated thymocytes. An increase in H-2K and H 2D antigens was noted: imbalance of the K/D ratio in favor of H-2D was observed mostly in tumors with relatively lower total amounts of H-2K and D antigens; ratio shifts in favor of H-2K were also found, mostly in tumors with relatively higher amounts of H-2K and D antigens. Increased expression of MuLV antigens was found on cells of all tumors and on thymocytes of several late preleukemic mice. We show that in primary spontaneous AKR leukemias, in spite of large individual differences, the expression of high amounts of MuLV gp70 is not random, but is associated with high expression of H-2K and H-2D antigens. The same phenomenon is seen in thymocytes of preleukemic mice, but in mitogen-stimulated thymus cells increase of H-2 expression is not accompanied by increase of MuLV gp70. PMID- 3002993 TI - Angiotensin-converting enzyme and coronary haemodynamics in coronary artery disease. AB - Coronary sinus pacing was performed in 12 patients with and 12 patients without significant coronary artery disease as verified by angiography in order to elucidate a possible link between cardiac exchange of angiotensin-converting enzyme and the coronary circulation. The coronary sinus and arterial blood enzyme activity remained unchanged and were similar in the two groups, both at rest and during pacing, and the activity was not related to pacing-induced changes in oxygen consumption or coronary vascular resistance. Therefore it does not seem that global cardiac formation or depletion of angiotensin-converting enzyme is of regulatory importance in the coronary vascular bed in patients with and without coronary artery disease under these circumstances. PMID- 3002992 TI - Function of the autonomic nervous system in young, untreated hypertensive patients. AB - The reactivity of the sympathetic nervous system was studied in 10 young, adult patients with labile hypertension and compared with a normotensive age-matched control group. Upon graded exercise on a constant speed bicycle ergometer, the hypertensive subjects reacted with an exaggerated blood pressure response and a significantly greater increase in the plasma adrenaline and noradrenaline levels. The basal catecholamine levels, however, were similar in both observation groups. This was in spite of an intact baroreceptor reflex in the hypertensives as indicated by a normal hemodynamic response to angiotensin II. This apparent discrepancy may be explained by an enhanced uptake of adrenaline during stress into the neuron, where it acts as a cotransmitter and facilitates the release of noradrenaline via presynaptic beta 2-adrenoceptors. Similar blood pressure and heart rate responses to isoproterenol and atropine were observed in both groups. This indicates normal beta-adrenoceptor sensitivity and vagal nerve activity in the hypertensive subjects. PMID- 3002994 TI - Mucin production in defining mixed carcinoma of the uterine cervix: a clinicopathologic study. AB - Eighty-seven Stage 1 cervical carcinomas treated by radical hysterectomy between 1970 and 1979 were reviewed for histologic type, outcome, and factors predicting behavior. Initially, the cases were histologically classified by the Wentz and Reagan system and graded according to the Broders method. Stains for intracellular mucin were then examined in 69 cases and 39% were shown to contain intracellular mucin. Using intracellular mucin as an indicator of mixed carcinoma, this study showed a distribution of 35% keratinizing, 16% nonkeratinizing, 3% small cell, 16% adeno-, 3% undifferentiated, and 26% mixed carcinoma. The mixed carcinomas were derived from the traditional keratinizing, nonkeratinizing and small cell categories. Mixed carcinoma was the only histologic type that predicted lymph node metastasis (p = 0.009). The presence of lymph node metastasis predicted death due to disease or recurrence (p = 0.014) as did pure adenocarcinoma histology (p = 0.025). Overall 5 year survival was 92%. Survival at 5 years for adenocarcinoma was 85%, but one additional death occurred at 12 years and a first recurrence occurred at 7 years. An additional patient with a collision tumor (adenocarcinoma and squamous carcinoma) died at 8 years. Mixed carcinoma is relatively common and appears to be associated with a higher incidence of lymph node metastasis. Adenocarcinoma appears to have a poorer prognosis and a tendency for late recurrence in distant sites. PMID- 3002996 TI - Diffuse viral papillomatosis (condyloma) of the uterine cavity. AB - A case of viral papillomatosis (condyloma) of the uterine cervix is reported, which was followed for 13 years and in which the lesion spread to involve superficially the entire uterine cavity without any change in its histologic appearance. The presence of intranuclear papillomavirus antigen was demonstrated by using the peroxidase-antiperoxidase technique. PMID- 3002997 TI - Human retinoblastomas have binding sites for the COOH-terminal segment of human beta-endorphin. AB - Two human retinoblastoma cell lines (Y79 and McA) were evaluated for the presence of binding sites for human beta-endorphin (beta h-EP). Using tritiated beta h-EP (3H-beta h-EP) and synthetic beta-EP analogues, it was possible to demonstrate binding sites for 3H-beta h-EP with an ED50 of 3.5 nM in Y79 cells and 8 nM in McA cells respectively. The non-opioid segment [beta h-EP-(6-31)] retained about 20% relative potency in Y79 and 40% in McA in displacing the tritiated hormone when compared with beta h-EP. Camel beta-EP had a relative potency of less than 1% and beta h-EP-(1-27) was inactive in both cells in doses as high as 4 microM. Taken together with previous reports on similar binding sites in human neuroblastoma and glioblastoma cell lines, it appears that cell lines of neural origin have binding sites for the COOH-terminal of human beta-EP. PMID- 3002995 TI - DNA flow cytometry of ovarian tumors with correlation to histopathology. AB - Tissue samples from 49 women with seven benign and 42 malignant ovarian tumors were examined by means of DNA flow cytometry (FCM). The FCM data (DNA-ploidy and the number of S-phase fractions) were compared with the International Federation of Gynecology and Obstetrics (FIGO) stage, the histologic grade of differentiation and in some cases with the clinical outcome. The benign ovarian tumors were all diploid with a mean of 2.1% (+/- 1.66) S-phase fractions. Adenocarcinomas with the histologic grade 1 were diploid and had a mean of 2.1% (+/- 1.17) S-phase fractions. Grade 2 adenocarcinomas were aneuploid in eight of nine cases and revealed an increased proliferative activity (7.7% +/- 2.67 S phase fractions). A high number of aneuploid cases (nine of 13) and an increased DNA synthesis were found in grade 3 adenocarcinomas (12.0% +/- 5.72 S-phase fractions). Four of six malignant nonepithelial tumors also had high numbers of S phase fractions (9.7-14.5%). A significant correlation between the histologic grade and the DNA synthesis, FIGO stage, and DNA-ploidy was found. DNA-FCM may be used as an additional diagnostic tool supplementing routine histopathologic examination of ovarian tumors for better biological characterization, especially for those with uncertain grading, for grade 2 neoplasms, and for malignant nonepithelial tumors. PMID- 3002999 TI - The design of a pentavalent 99mTc-dimercaptosuccinate complex as a tumor imaging agent. AB - In our search for a technetium-tumor seeking agent, the chemical characteristic of polynuclear Ga-citrate attracted our attention. On this basis, we selected the ligand dimercaptosuccinic acid (DMS) and appropriate labeling conditions in the design of 99mTc(V)-DMS. Chemical characterization was performed by thin layer chromatography, electrophoresis, Sephadex column chromatography and spectrophotometric studies at the chemical concentration (99Tc). Biodistribution in mice bearing Ehrlich ascites tumor and scintigraphic images in VX-2 tumor bearing rabbit, indicated the great applicability of 99mTc(V)-DMS as a new tumor imaging agent. Its distinctive characteristic, different from the kidney imaging agent (99mTc-DMSA) is demonstrated and the biological implication of hydrolytic polynucleation of pentavalent technetium, through an anionic specie Tc(V)O3-(4), in the tumor cell is discussed. PMID- 3002998 TI - Comparison of 68Ga-EDTA, [1-11 C]alpha-aminoisobutyric acid, and [99mTc]sodium pertechnetate in an experimental blood-brain barrier lesion. AB - Unilateral opening of the blood-brain barrier (BBB) in rats, induced by infusion of a hyperosmotic solution of mannitol into the left internal carotid artery, was applied to compare 68Ga-EDTA, [1-11C]alpha-aminoisobutyric acid (using the long lived 14C-labeled analog), and [9mTc]sodium pertechnetate as agents for diagnosis of BBB disruption. Of the three agents and two time intervals studied, 68Ga-EDTA at 30 min postinjection gave the highest target-to-nontarget ratio. In addition, 68Ga-EDTA, unlike commonly used [99mTc]sodium pertechnetate, can be used in conjunction with positron emission tomography, which could make possible earlier and better assessment of BBB defects using 68Ga-EDTA. PMID- 3003000 TI - Liver cAMP levels in rats under inadequate protein nutrition. PMID- 3003001 TI - Sodium diethyldithiocarbamate influences the early immunological changes evoked by an acute non-antigenic inflammation process. AB - Calcium pyrophosphate (CaPp) produces an acute local inflammatory process when administered intra-pleurally. It is the simplest model of an inflammation uncomplicated by pharmacological properties of the agent or the presence of an antigen. Spleen or lymph-node T- or B-cell numbers and activities, including NK activity, are modified within 2 h under these conditions. A treatment with sodium diethyldithiocarbamate (lmuthiol), already known as a T-cell augmenting agent, restores towards normal values both the inflammatory reaction and the modified immune parameters induced in mice by CaPp pleurisy. The use of lmuthiol as a non toxic, non-steroidal antiinflammatory agent is therefore suggested. PMID- 3003002 TI - Pharmacology of free radical scavenging in inflammation. AB - Current available evidence suggesting a role for oxygen free radicals in inflammatory processes is reviewed. The effects of NSAID as scavengers or inhibitors of superoxide anion generation are described. The anti-inflammatory effect of the superoxide dismutase drug version named Orgotein is discussed in relation to its enzymic activity and pharmacokinetics profile. PMID- 3003005 TI - Lung cancer. Part I: Etiology, pathology, natural history, manifestations, and diagnostic techniques. PMID- 3003003 TI - Rhodopsin phosphorylation in developing normal and degenerative mouse retinas. AB - The developmental pattern of rhodopsin phosphorylation in degenerative (rdle homozygote) and normal (rd/+ heterozygote) mouse retina has been investigated. The results indicate that rhodopsin levels are comparable in the 2 retinas up to about 10 days of age but that rhodopsin phosphorylation is not. The phosphorylation of rhodopsin is substantially reduced in the degenerative retina during development. This abnormality may be an expression of the rd lesion. The rhodopsin kinase/phosphatase system, the G protein, and the visual pigment are all involved in the modulation of cGMP-phosphodiesterase activity in normal retinas. A defect in any of these components could account for the reduced level of cGMP-phosphodiesterase activity in rd retinas, resulting in cGMP accumulation and subsequent photoreceptor degeneration. PMID- 3003004 TI - Evaluation of a new drug 7-N-(p-hydroxyphenyl)-mitomycin C [KW 2083] against carcinoma of the lung by the human tumor clonogenic assay. AB - KW 2083, which is a new mitomycin C derivative currently under clinical investigation, was tested for its antitumor effect on the growth of human carcinoma of the lung by using an in vitro clonogenic assay system. The in vitro results in this study were as follows: We succeeded in producing clonal growth at a high rate in all histologic types of carcinoma of the lung in the assay system. That is, 61 of 81 specimens (75%) of either primary or metastatic tumors gave adequate growth for chemosensitivity testing. The in vitro responses to vindesine, adriamycin, mitomycin C and melphalan (substituted for cyclophosphamide in vitro), as standard anticancer drugs for chemotherapy of carcinoma of the lung were 18, 23, 18 and 19%, respectively, which are in good general agreement with the clinical responses to these drugs reported previously. Therefore, the clonogenic assay system might prove to be a very effective tool for an in vitro phase II study of new drugs. The rate of response to KW 2083 tested simultaneously in 51 cancer specimens was 22%, which was superior to that of mitomycin C. These results indicate that KW 2083 might be more useful than mitomycin C in clinical practice. PMID- 3003007 TI - Total extrinsic ophthalmoplegia as only paraneoplastic sign two years before X ray diagnosis of bronchial carcinoma. AB - An extrinsic total ophthalmoplegia developing two years before radiologic evidence of bronchial carcinoma and onset of Eaton-Lambert myasthenic syndrome is reported. Clinical and ENG data showed the neuromuscular location of the ophthalmoplegia, but repeated Tensilon and Prostigmine tests were negative. CT scan and CSF examinations revealed neither carcinomatous metastases nor inflammatory CNS disease. The case is an exceptional example of a paraneoplastic myasthenic syndrome long confined to the oculomotor muscles. PMID- 3003006 TI - Congenital varicella. PMID- 3003008 TI - Infantile spasms syndrome in monozygotic twins. A 7-year follow-up. AB - The Infantile Spasms Syndrome is a fairly common form of seizures in infancy. Many papers and several books have been published on this syndrome but several aspects are still obscure. In particular, there is some controversy about anticonvulsant treatments and on the question of improvements in mental status. An unusual case of 2 monozygotic twins with by this syndrome, both with clinical manifestations appearing within a few hours on the same day, at 6 months has been followed up for 7 years, giving us the opportunity to understand some aspects of the clinical course of the disease and long term treatment. PMID- 3003009 TI - Porphyric neuropathy: a clinical, neurophysiological and morphological study. AB - A case of neuropathy in the course of an attack of acute intermittent porphyria was studied from the neurophysiological and morphological points of view. The neurophysiological findings (acute neuropathy with almost complete denervation despite normal or slightly reduced conduction velocity) and the morphological findings (no segmental demyelination after teasing, conservation of the linear fiber diameter/internodal distance ratio, mainly axonal damage on ultrastructural study) seem to indicate that the disease process is chiefly an axonal neuropathy. PMID- 3003010 TI - The Kotz modular prosthesis in massive osteo-articular resections for bone tumours: preliminary results in 27 cases. AB - The authors report 27 cases in which neoplasms of the proximal or distal femur were treated by resection and replacement with a Kotz modular prosthesis. In 25 cases this was not cemented but in the other 2 cases cement had to be used. After describing the prosthesis and presenting the case material, the authors discuss the advantages and limitations of this surgical technique and compare the results obtained with those reported in the literature. PMID- 3003011 TI - [History of 2 cells in T cell lymphomas and leukemias: the cerebriform Lutzner cell in Sezary syndrome and the polylobulated or polypetaloid cell in acute T cell lymphoma/leukemia]. AB - In normal blood, there appears to be two similar but different subsets of T lymphocytes present: (1) the cerebriform or convoluted Sezary-syndrome Lutzner cell, which may give rise to cutaneous T-cell lymphoma; (2) the polypetaloid or lobated acute T-cell lymphoma/leukemia (ATLL) cell, which may give rise to ATLL. Both cell types can be differentiated by their characteristic nuclear shapes. PMID- 3003012 TI - Mitochondrial DNA variability in natural populations of Hawaiian Drosophila. I. Methods and levels of variability in D. silvestris and D. heteroneura populations. AB - We describe techniques by which mitochondrial DNA (mtDNA) restriction site information can be obtained for up to 16 different restriction endonucleases on individual Hawaiian Drosophila, in particular D. silvestris and D. heteroneura. We have constructed mtDNA restriction site maps for a total of forty-eight wild caught genomes of both species, from seven major collecting sites on the island of Hawaii. Levels of variability are, in general, high in D. silvestris (p of Ewens et al., 1981 = 0.0486) and lower in D. heteroneura (p = 0.0327). Measures of population subdivision using Nei's Gst indicate that about 50-60 per cent of the observed variability is due to interdemic subdivision. In accordance, populations within a species show a much lower level of variability, however some populations harbour individuals that are very divergent from the rest of their conspecifics at the same locality. We review two possible mechanisms that could explain the presence of these divergent individuals, hybridisation and the effects of stochastic branching processes. PMID- 3003013 TI - Mitochondrial DNA variability in natural populations of Hawaiian Drosophila. II. Genetic and phylogenetic relationships of natural populations of D. silvestris and D. heteroneura. AB - Restriction site mapping of mitochondrial DNA with 23 restriction endonucleases was used to examine the genetic and phylogenetic relationships of populations of D. silvestris and D. heteroneura from the island of Hawaii. Two morphological races of D. silvestris are known on the island of Hawaii. One has three bristle rows on the tibia of the foreleg and is found on the east side of the island. The other is found on the west side of the island and has the ancestral bristle row character of two rows on the tibia of the foreleg. All D. heteroneura have the ancestral bristle row character state. We demonstrate that mtDNA restriction site analysis can also differentiate the two D. silvestris races, and that the two bristle row D. silvestris are more closely related to D. heteroneura than they are to their three bristle row conspecifics using both distance and character state analysis. Our study (which uses six base recognition restriction endonucleases) is not sensitive enough to determine the phylogenetic relationships of populations within either of the D. silvestris lineages. PMID- 3003014 TI - Cylindroma of the nasopharynx: a chronic disease. AB - From March 1958 to October 1984, 10 patients with cylindroma (cystic adenoid epithelioma) arising in the nasopharynx were treated in our hospital. The presenting symptoms of these patients ranged in duration for 2 weeks to 8 years, with an average of 25 months. Eighty percent of the patients had destruction of the base of skull and 60% had cranial nerve involvement. Of these 10 patients, two had Stage II disease, two had Stage III, and six had Stage IV. Response was seen in this tumor at tissue doses between 5000-8000 rad. Of the seven patients at risk for 5 years, 86% survived, yet 36% of the patients died of tumor between the fifth to tenth year. Fifty percent of the patients lived beyond 10 years. Based on this experience, a dose of more than 8000 rad is advised for the primary lesion. For the neck region, radiation is indicated only when there are lymph node metastases; prophylactic radiation of the neck is not necessary. Local recurrence or single distant metastasis is amenable to radiation therapy. Among the six patients who failed in the course of follow-up, three had local recurrences, two had pulmonary metastases, and one died of cerebral vascular accident. PMID- 3003016 TI - Insulin-secreting tumor: diagnosis and medical and surgical management in 55 dogs. AB - Insulin-secreting tumor of the pancreas was diagnosed in 55 dogs. Diagnosis was based mainly on the increase of serum insulin concentrations in the presence of hypoglycemia. The use of the amended insulin/glucose ratio to diagnose the tumor, although providing less false-negative results than did increased serum insulin concentrations alone, appeared less specific and gave false-positive results in dogs without insulin-secreting tumors. Management of the disease included surgical intervention alone (26 dogs), surgery plus medical management with diazoxide (14 dogs), and medical management with diazoxide alone (4 dogs). Eleven dogs were euthanatized at the time of diagnosis. Diazoxide therapy controlled hypoglycemia in about 70% of the dogs. PMID- 3003015 TI - Breed-related risk factors for canine parvovirus enteritis. AB - Case records of 305 dogs with canine parvovirus (CPV) enteritis, seen at the Veterinary Hospital of the University of Pennsylvania from July 1, 1981 to Aug 31, 1982, were selected on the basis of admitting diagnoses or signs of diarrhea and vomiting. The case records were subdivided into 3 diagnostic categories, based on final diagnoses and laboratory test results. There were 96 dogs with definite CPV enteritis, 139 with possible CPV enteritis, and 70 with unlikely CPV enteritis. These cases were then stratified by animal's age (less than or equal to 6 months or greater than 6 months) and specific hospital service (medicine or emergency). A control group was selected from all canine case records from the Veterinary Hospital of the University of Pennsylvania for conditions other than the criteria used in selecting the case group. Approximately 2 hospital patients were selected for each CPV enteritis case by frequency matching for hospital service and age. The proportion of dogs with definite CPV enteritis that had each of the clinical signs that were studied was greater than that of dogs in the other CPV enteritis diagnostic categories. The overall survival rate for dogs with definite CPV enteritis was 64.0%; survival was not associated with any given clinical sign of disease. Odds ratios (OR) for the risk of CPV enteritis were calculated for breeds with 3 or more dogs with definite CPV enteritis. The Doberman Pinschers (OR = 3.1), Rottweilers (OR = 6.0), and English Springer Spaniels (OR = 8.1) had a significantly increased risk of CPV enteritis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003017 TI - Growth-promoting effect of gastrin on human gastric carcinoma cell line TMK-1. AB - A human gastric carcinoma cell line TMK-1 was established in vitro by the soft agar method from SC-6-JCK, a poorly differentiated adenocarcinoma xenotransplanted in nude mice. TMK-1 cells had a doubling time of approximately 35 hr and showed carcinoembryonic antigen (CEA), alpha 1-antitrypsin and secretory component immunoreactivity. Ultrastructurally, the tumor cells were characterized by numerous mitochondria, tubulovesicles and intracytoplasmic canaliculi filled with abundant microvilli. The growth of TMK-1 cells was promoted by 10nM human gastrin (G-17), 2 microM tetragastrin or 2 microM pentagastrin, among which human gastrin showed the most effective growth promotion. Moreover, incorporation of [3H]thymidine into TMK-1 cells was stimulated by gastrin in a dose-dependent manner. The content of cyclic adenosine 3',5'-monophosphate (cAMP) in TMK-1 cells was increased by gastrin treatment but decreased to the control level within 10 min. cAMP-dependent protein kinase was also activated by gastrin administration. PMID- 3003019 TI - The effect of loop diuretics upon summating potentials in the guinea pig. AB - Ototoxic diuretics, ethacrynic acid (50 mg/kg) and furosemide (80 mg/kg) were injected intravenously in guinea pigs. Cochlear microphonics (CM), summating potentials (SPs) and endocochlear potential (EP) were recorded with a microelectrode in scala media of the second turn. CM changes after the injection were comparable with changes in the EP: an initial decrease was followed by an increase which was significantly slower in the case of the ethacrynic acid. SP changes following either diuretic were different from the CM and EP changes. In the first phase, roughly corresponding with the EP decrease to negative values, all SPs irrespective of the original polarity, attained high positive values. The high positive SPs then decreased and 12-18 min after injection reversed polarity. In the late phase all sounds evoked high negative SPs. Approximately 90 min after injection of furosemide normal SPs were again recorded. The return of SPs to control values was very slow after ethacrynic acid; even 140 min after injection the SPs were abnormal. The observed changes in the SPs were compared with those found during asphyxia and anoxia and are considered to result from diuretic effects on the inner and outer hair cells. PMID- 3003018 TI - Dose and time relationships in the endocrine response of the irradiated adult rat testis. AB - The dose- and time-dependent responses for the interstitial and tubular compartments in irradiated adult rat testes are described. Leydig cell dysfunction, as indicated by increased serum LH (to a maximum of 385% of control after 5 Gy) and decreased serum T (to a minimum of 30% of control after 10 Gy), was observed at 8 weeks postirradiation. Subsequent recovery of Leydig cell function was then observed, so that after 9 months serum T was normal but LH was still marginally elevated. The dysfunction, with a threshold of about 4 to 5 Gy, was associated with a loss of Leydig cells from the testis. Spermatogenic damage was observed; after doses of 3 Gy and above a marked dose-response was recorded as assessed by counts of tubule cross sections exhibiting spermatogenesis. Reduced serum levels of androgen binding protein indicated Sertoli cell dysfunction at 8 weeks after 3 Gy and above, with values of less than one half of those seen in the controls. Serum FSH also was elevated to between 150% and 200% of control, and after 9 months closely reflected androgen binding protein changes. Unlike the Leydig cell, no recovery with time was observed for this aspect of Sertoli cell function. PMID- 3003020 TI - Beta-adrenoceptors and the regulation of blood pressure and plasma renin during exercise. AB - The relative role of beta 1- and beta 2-adrenoceptors in the regulation of blood pressure and plasma renin at rest and during exercise was studied in 17 normal male volunteers. They performed, in a randomized order and according to a double blind crossover study design, three graded and uninterrupted exercise tests until exhaustion after being pretreated during 3 consecutive days with a placebo, with a predominantly beta 1-blocker (atenolol, 50 mg once/day), or with a predominantly beta 2-blocker (ICI 118551, 20 mg 3 times/day). Both drugs caused a decrease of heart rate, but the reduction by ICI 118551 was less pronounced at rest and no additional decline occurred at exercise. ICI 118551 did not affect blood pressure at rest, but during exercise diastolic blood pressure was higher than after a placebo. Atenolol lowered systolic blood pressure at rest and suppressed the exercise-induced increase in systolic blood pressure. At rest and during exercise plasma renin activity was lowered by predominantly beta 1 blockade and unchanged during beta 2-antagonism. The exercise-induced increase in plasma renin was, however, not affected by the beta 1-blocker. After atenolol the urinary excretion of aldosterone was decreased but the plasma aldosterone concentration was not changed. ICI 118551 did not alter plasma or urinary aldosterone. Our results therefore provide further evidence that the adrenoceptors mediating the release of renin at rest and during exercise in humans are partially of the beta 1-subtype, whereas beta 2-adrenergic receptors probably play only a minor role in the control of renin secretion, especially at exercise.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003021 TI - Characterization of a growth-inhibiting protein present in rat serum that exerts a differential effect on in vitro growth of nonmalignant rat liver cells when compared with Rous sarcoma virus-transformed rat liver cells. AB - We have previously reported the transformation by Rous sarcoma virus of a cloned epithelial cell line (BRL) established from Buffalo rat liver by H. Coon. The nontransformed (BRL) and transformed (RSV-BRL) cells grew at comparable rates in culture, whereas only the transformed cells were tumorigenic in vivo. We report here on the existence in rat and mouse sera of a growth inhibitor for the nontransformed BRL cells. The transformed BRL cells (RSV-BRL) were insensitive to this inhibitor. The inhibitory activity was not prominent in sera from other species of animals tested except for rabbit; this serum inhibited the growth of RSV-BRL cells more strongly than that of BRL cells. The growth inhibitor was partially purified from rat serum. It is a protein free of lipid and has a molecular weight of about 220 000. The inhibitor could be separated into three components of pI 4.6, 5.2 (major) and 5.6 by isoelectric electrophoresis. PMID- 3003023 TI - Occurrence of small Hsd plasmids in Salmonella typhi, Shigella boydii, and Escherichia coli. AB - The natural occurrence of small Hsd (host specificity for DNA) plasmids was demonstrated in restriction endonuclease-producing strains of Salmonella typhi, Shigella boydii, and Escherichia coli. The five Hsd plasmids isolated were between 5.0 and 12.2 kilobases long. The copy number of all the Hsd plasmids was high (more than 10 copies per cell). Introduction of these small plasmids into E. coli strain 0 drastically lowered the efficiency of plating of the lambda.0 phages (the efficiency of plating was less than 5 X 10(-5) PFU-1). High restriction endonuclease activities were detected in the Hsd plasmid-positive strains because of the elevated copy numbers of the hsdR+ gene. The advantages of using E. coli strains containing the small Hsd plasmids for purification of type II restriction endonucleases are discussed. PMID- 3003024 TI - Additional DNA in methicillin-resistant Staphylococcus aureus and molecular cloning of mec-specific DNA. AB - Additional DNA was shown to be present in methicillin-resistant Staphylococcus aureus by one- and two-dimensional restriction endonuclease analyses of the chromosomal DNA. A 3.5-kilobase Bg/II fragment, which was present in methicillin resistant strains but not in the isogenic methicillin-sensitive parental strain, was cloned into newly constructed plasmid pWDB1 in Escherichia coli. Hybridization of this 3.5-kilobase Bg/II fragment with different methicillin sensitive and methicillin-resistant S. aureus clinical isolates indicated that the fragment represents part of the methicillin resistance determinant (mec). In addition, the fragment carries a sequence that is present in some large staphylococcal plasmids, as well as in penicillinase plasmid pI524. PMID- 3003022 TI - Site-specific recombinases: changing partners and doing the twist. PMID- 3003025 TI - Sensitivity of a Salmonella typhimurium aspC mutant to sulfometuron methyl, a potent inhibitor of acetolactate synthase II. AB - Sulfometuron methyl is a potent and specific inhibitor of acetolactate synthase II in Salmonella typhimurium. Mutant strains sensitive to sulfometuron methyl on minimal medium were isolated following mutagenesis with Tn10. A conditionally auxotrophic insertion mutant, strain SMS409, which required aspartate at high temperatures or in the presence of tyrosine, was found among the 15 mutants isolated. The Tn10 insertion in strain SMS409 was mapped by conjugation and transduction to the region between aroA and pncB at 20 min on the chromosome of S. typhimurium; this location is similar to the genetic location of aspC in Escherichia coli. The specific activity of the aspC product, aspartate aminotransferase, was severely reduced in strain SMS409. This indicated that the Tn10 insertion in strain SMS409 inactivated aspC. An aspC mutant of E. coli was also inhibited by either sulfometuron methyl or tyrosine. We present a hypothesis which relates the observed alpha-ketobutyrate accumulation in sulfometuron methyl inhibited cultures of strain SMS409 to aspartate starvation. PMID- 3003026 TI - Stage-specific DNA methylation in a fungal plant pathogen. AB - Significantly more 5-methylcytosine residues were found in the DNA from the dormant sclerotia of Phymatotrichum omnivorum than in the DNA from the metabolically active mycelia of the fungus, as shown by high-pressure liquid chromatography of acid-hydrolyzed DNA digests and by restriction of the DNA with the isoschizomers MspI and HpaII. N6-Methyladenine was not detected in GATC sequences in the DNA isolated from either stage. PMID- 3003027 TI - Partial purification and characterization of pyruvate, orthophosphate dikinase from Rhodospirillum rubrum. AB - We confirmed an earlier report (B. B. Buchanan, J. Bacteriol. 119:1066-1068, 1974) that the nonsulfur purple photosynthetic bacterium Rhodospirillum rubrum contains pyruvate, orthophosphate dikinase (EC 2.7.9.1) activity that is absolutely dependent upon all three substrates by performing enzyme assays in both the forward (phosphoenolpyruvate formation) and reverse (ATP formation) directions. Of the various carbon sources tested, photoheterotrophic growth on DL lactate plus bicarbonate proved to be best for the production of dikinase activity units. A four-step protocol, which included batch DEAE-cellulose processing, ammonium sulfate fractionation, and chromatography on hydroxylapatite and Blue A Dyematrex gels, was devised for partially purifying the enzyme from such cells. The protein was purified about 80-fold to an apparent electrophoretic purity of about 60% and a final specific activity of 3.6 U/mg of protein, with about a 35% overall recovery of activity units. Estimations of native and monomeric relative molecular weights by sucrose density gradient centrifugation, high-pressure liquid chromatography-based size exclusion chromatography, denaturing electrophoresis, and immunoblotting suggested that the holoenzyme was most likely a homodimer of 92.7-kilodalton subunits. The results are compared with related previous data on the nonphotosynthetic bacterial dikinase and the C4 mesophyll chloroplast enzyme. PMID- 3003028 TI - Lipopolysaccharide-free Escherichia coli OmpF and Pseudomonas aeruginosa protein P porins are functionally active in lipid bilayer membranes. AB - Escherichia coli porin OmpF and Pseudomonas aeruginosa porin protein P were eluted from sodium dodecyl sulfate-polyacrylamide gels. The resultant porin preparations were found to be devoid of detectable lipopolysaccharide (LPS) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining for LPS, direct enzyme-linked immunosorbent assays with LPS-specific monoclonal antibodies, and 2-keto-3-deoxyoctulosonic acid assays. The average conductances, ionic selectivities and incorporation rates of the electroeluted porins were identical to those of their conventionally purified counterparts. These data suggest that LPS is not required per se for porin function. PMID- 3003029 TI - Indigenous plasmids in Pseudomonas syringae pv. tomato: conjugative transfer and role in copper resistance. AB - Twenty strains of Pseudomonas syringae pv. tomato were examined for the presence of plasmid DNA. P. syringae pv. tomato plasmids were grouped into five size classes: class A ranged from 95 to 103 kilobases (kb); class B ranged from 71 to 83 kb; class C ranged from 59 to 67 kb; class D ranged from 37 to 39 kb; and class E was 29 kb. All strains contained at least two plasmids in classes A and B. The conjugative ability of P. syringae pv. tomato plasmids in three strains was demonstrated by mobilization of the nonconjugative plasmid RSF1010 into Pseudomonas syringae pv. syringae recipients. Plasmids from the three conjugative strains were labeled with Tn5. Four conjugative plasmids were identified by their repeated transfer to P. syringae pv. syringae recipients. P. syringae pv. tomato strains varied in sensitivity to copper sulfate (CuSO4): MICs were 0.4 to 0.6 mM for sensitive strains, 1.2 mM for moderately resistant strains, and 1.6 to 2.0 mM for very resistant strains. One very resistant strain, PT23, functioned as a donor of copper resistance. Recipient P. syringae pv. syringae strains PS51 and PS61 were inhibited by 0.1 mM CuSO4, whereas the CuSO4 MICs for transconjugant strains PS51(pPT23A) and PS61(pPT23C) were 1.8 and 2.6 mM, respectively. P. syringae pv. tomato strains PT12.2 and PT17.2 were inhibited by 0.6 mM copper sulfate, but their copper sulfate MICs were 2.6 and 1.8 mM, respectively, when they acquired pPT23C. Therefore, copper resistance in PT23 was controlled by two conjugative plasmids, designated pPT23A (101 kb) and pPT23C (67 kb). PMID- 3003030 TI - Amplification and deletion of the amyE+-tmrB+ gene region in a Bacillus subtilis recombinant-phage genome by the tmrA7 mutation. AB - A 22.4-kilobase DNA fragment containing the tmrA7-amyR2-amyE+-tmrB+-aroI+ region of the Bacillus subtilis N7 chromosomal DNA was cloned into a recombinant B. subtilis bacteriophage, p11-AA248. The amyE+-tmrB+ gene region, approximately 12.6 kilobases, in the phage genome was amplified in a tunicamycin-resistant (Tmr) Amy+ AroI+ transductant of B. subtilis by p11-AA248. On the other hand, the amyE+-tmrB+ region in the genomes of 80 to 90% of the phage particles was deleted when the phages were induced from the Tmr Amy+ AroI+ transductants by treatment with 1.0 micrograms of mitomycin C per ml. From analyses of the physical maps and DNA nucleotide sequences in the junction region of the deleted phage genome and the parental DNA fragments, it is suggested that the deletion occurred within a direct repeat sequence composed of 18 base pairs. The endpoints of the amplified gene region seemed to be closely related to both terminal regions of the deleted DNA. PMID- 3003031 TI - Cloning and expression of Acinetobacter calcoaceticus catBCDE genes in Pseudomonas putida and Escherichia coli. AB - This report describes the isolation and preliminary characterization of a 5.0 kilobase-pair (kbp) EcoRI DNA restriction fragment carrying the catBCDE genes from Acinetobacter calcoaceticus. The respective genes encode enzymes that catalyze four consecutive reactions in the catechol branch of the beta ketoadipate pathway: catB, muconate lactonizing enzyme (EC 5.5.1.1); catC, muconolactone isomerase (EC 5.3.3.4); catD, beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24); and catE, beta-ketoadipate succinyl-coenzyme A transferase (EC 2.8.3.6). In A. calcoaceticus, pcaDE genes encode products with the same enzyme activities as those encoded by the respective catDE genes. In Pseudomonas putida, the requirements for both catDE and pcaDE genes are met by a single set of genes, designated pcaDE. A P. putida mutant with a dysfunctional pcaE gene was used to select a recombinant pKT230 plasmid carrying the 5.0-kbp EcoRI restriction fragment containing the A. calcoaceticus catE structural gene. The recombinant plasmid, pAN1, complemented P. putida mutants with lesions in catB, catC, pcaD, and pcaE genes; the complemented activities were expressed constitutively in the recombinant P. putida strains. After introduction into Escherichia coli, the pAN1 plasmid expressed the activities constitutively but at much lower levels that those found in the P. putida transformants or in fully induced cultures of A. calcoaceticus or P. putida. When placed under the control of a lac promoter on a recombinant pUC13 plasmid in E. coli, the A. calcoaceticus restriction fragment expressed catBCDE activities at levels severalfold higher than those found in fully induced cultures of A. calcoaceticus. Thus there is no translational barrier to expression of the A. calcoaceticus genes at high levels in E. coli. The genetic origin of the cloned catBCDE genes was demonstrated by the fact that the 5.0-kbp EcoRI restriction fragment hybridized with a corresponding fragment from wild-type A. calcoaceticus DNA. This fragment was missing in DNA from an A. calcoaceticus mutant in which the cat genes had been removed by deletion. The properties of the cloned fragment demonstrate physical linkage of the catBCDE genes and suggest that they are coordinately transcribed. PMID- 3003032 TI - Siderophores and outer membrane proteins of antagonistic, plant-growth stimulating, root-colonizing Pseudomonas spp. AB - As an approach to understanding the molecular basis of the reduction in plant yield depression by root-colonizing Pseudomonas spp. and especially of the role of the bacterial cell surfaces in this process, we characterized 30 plant-root colonizing Pseudomonas spp. with respect to siderophore production, antagonistic activity, plasmid content, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis patterns of their cell envelope proteins. The results showed that all strains produce hydroxamate-type siderophores which, because of the correlation with Fe3+ limitation, are thought to be the major factor responsible for antagonistic activity. Siderophore-negative mutants of two strains had a strongly decreased antagonistic activity. Five strains maintained their antagonistic activity under conditions of iron excess. Analysis of cell envelope protein patterns of cells grown in excess Fe3+ showed that most strains differed from each other, although two classes of similar or identical strains were found. In one case such a class was subdivided on the basis of the patterns of proteins derepressed by iron limitation. Small plasmids were not detected in any of the strains, and only one of the four tested strains contained a large plasmid. Therefore, it is unlikely that the Fe3+ uptake system of the antagonistic strains is usually plasmid encoded. PMID- 3003034 TI - Complementation of an Escherichia coli pyrF mutant with DNA from Desulfovibrio vulgaris. AB - A PyrF- mutant of Escherichia coli (SK1108, pyrF::Tn5 Kanr) was complemented with the Desulfovibrio vulgaris (Hildenborough) structural gene for orotidine-5' phosphate decarboxylase (EC 4.1.1.23). Either orientation of a 1.6-kilobase-pair D. vulgaris DNA fragment (pLP3B or pLP3A) complemented the PyrF- strain suggesting that the D. vulgaris pyrF promoter was functional. The apparent product of the D. vulgaris pyrF gene was a single 26-kilodalton polypeptide. These results demonstrate the utility of E. coli cloning systems in studying metabolic and energetic pathways in sulfate-reducing bacteria. PMID- 3003033 TI - Cloning of the Bacillus subtilis DLG beta-1,4-glucanase gene and its expression in Escherichia coli and B. subtilis. AB - The gene encoding beta-1,4-glucanase in Bacillus subtilis DLG was cloned into both Escherichia coli C600SF8 and B. subtilis PSL1, which does not naturally produce beta-1,4-glucanase, with the shuttle vector pPL1202. This enzyme is capable of degrading both carboxymethyl cellulose and trinitrophenyl carboxymethyl cellulose, but not more crystalline cellulosic substrates (L. M. Robson and G. H. Chambliss, Appl. Environ. Microbiol. 47:1039-1046, 1984). The beta-1,4-glucanase gene was localized to a 2-kilobase (kb) EcoRI-HindIII fragment contained within a 3-kb EcoRI chromosomal DNA fragment of B. subtilis DLG. Recombinant plasmids pLG4000, pLG4001a, pLG4001b, and pLG4002, carrying this 2-kb DNA fragment, were stably maintained in both hosts, and the beta-1,4-glucanase gene was expressed in both. The 3-kb EcoRI fragment apparently contained the beta 1,4-glucanase gene promoter, since transformed strains of B. subtilis PSL1 produced the enzyme in the same temporal fashion as the natural host B. subtilis DLG. B. subtilis DLG produced a 35,200-dalton exocellular beta-1,4-glucanase; intracellular beta-1,4-glucanase was undetectable. E. coli C600SF8 transformants carrying any of the four recombinant plasmids produced two active forms of beta 1,4-glucanase, an intracellular form (51,000 +/- 900 daltons) and a cell associated form (39,000 +/- 400 daltons). Free exocellular enzyme was negligible. In contrast, B. subtilis PSL1 transformed with recombinant plasmid pLG4001b produced three distinct sizes of active exocellular beta-1,4-glucanase: approximately 36,000, approximately 35,200, and approximately 33,500 daltons. Additionally, B. subtilis PSL1(pLG4001b) transformants contained a small amount (5% or less) of active intracellular beta-1,4-glucanase of three distinct sizes: approximately 50,500, approximately 38,500 and approximately 36,000 daltons. The largest form of beta-1,4-glucanase seen in both transformants may be the primary, unprocessed translation product of the gene. PMID- 3003035 TI - Characterization of the OCT plasmid encoding alkane oxidation and mercury resistance in Pseudomonas putida. AB - Transformation of Pseudomonas putida and analysis for plasmid DNA revealed that both n-alkane oxidation and mercury resistance are encoded on a single 220 megadalton OCT plasmid molecule. Derivatives of OCT having lost the mercury resistance function could be readily isolated and contained a smaller plasmid estimated to be 170 megadaltons. The results show that segregation of the mercury resistance property occurs not by loss of a separate MER plasmid as previously thought but by a deletion in the OCT plasmid. PMID- 3003036 TI - Characterization of a Haemophilus ducreyi mobilizing plasmid. AB - The OriV site of Haemophilus ducreyi mobilizing plasmid pHD147, determined by replication in Escherichia coli polA, is located close to the OriT site. The OriT site, located by recombination-proficient and -deficient cells, and the OriV site map in a region of pHD147 homologous to the beta-lactamase-specifying plasmids of H. ducreyi and Neisseria gonorrhoeae. PMID- 3003038 TI - Potent inhibition of angiotensin-converting enzyme by [des-Pro3]-bradykinin or "converstatin" in comparison with Captopril. AB - The mechanism of bradykinin-potentiating activity of [des-Proline3]-bradykinin, a kinin originally generated from human plasma protein by trypsin, was studied in terms of its inhibitory actions on angiotensin-converting enzyme and kininase II prepared from rat lung. The results were compared with those obtained with Captopril. [Des-Pro3]-bradykinin was found to have a potent inhibitory action against angiotensin-converting enzyme with a K1 of 4.5 X 10(-12) M, which is approximately 7 times more potent than Captopril. It was also inhibitory to kininase II with a Ki of 4 X 10(-11) M, which is approximately 2,300-fold more potent than Captopril. The pattern of inhibition was purely competitive with increased apparent Km but no change in apparent Vmax for both angiotensin converting enzyme and kininase II. This is in contrast to Captopril, which showed a mixed competitive and non-competitive type of inhibition with increased apparent Km and decreased Vmax for both enzymes. Such a potent inhibitory activity of [des-Pro3]-bradykinin or Arg-Pro-Gly-Phe-Ser-Pro-Phe-Arg is noteworthy, and accordingly we propose the name "converstatin" for this peptide. PMID- 3003037 TI - Pharmacologic probes of neurotransmitter systems in tardive dyskinesia: implications for clinical management. AB - Despite the plethora of clinical drug trials in tardive dyskinesia, few consistent findings have emerged. One possible reason for this is that there have been no serious attempts to define the role of major neurotransmitter systems (dopamine, norepinephrine, acetylcholine, serotonin, GABA) in one specific population of tardive dyskinesia patients. This study reports a series of five controlled drug trials in a population of patients with persistent tardive dyskinesia; each drug probed one of four neurotransmitter systems. The intra- and interpatient responses are analyzed and the implications of the pharmacologic response profiles for the clinical management of tardive dyskinesia are discussed. PMID- 3003040 TI - Purification and characterization of 210,000-dalton inhibitor of calcium activated neutral protease from rabbit skeletal muscle and its relation to 50,000 dalton inhibitor. AB - An endogenous inhibitor of calcium-activated neutral protease (CANP), which was isolated from rabbit skeletal muscle with chemically drastic pretreatments, comprised major (high-molecular-weight form, HMW-inhibitor) and minor (low molecular-weight form, LMW-inhibitor) components. HMW-inhibitor was purified to homogeneity using FPLC and preparative electrophoresis. The purified inhibitor appeared as a single protein with a molecular weight of 110,000 on SDS polyacrylamide gel electrophoresis, and a molecular weight of 210,000 on gel filtration. It was therefore presumed that the inhibitor is a dimer protein under native conditions. It contained large amounts of glutamic acid, alanine, and proline, and small amounts of aromatic amino acids, showing an amino acid composition similar to that of LMW-inhibitor. HMW-inhibitor inhibited CANPs with both low (m-type) and high (mu-type) Ca2+-sensitivity but had no effect on any other proteases examined. It was demonstrated that the inhibition was due to the formation of a stoichiometric complex between rabbit mCANP and inhibitor subunit in the ratio of five to one. These results suggest that HMW-inhibitor might have several reactive sites per molecule and that LMW-inhibitor subunit might be a proteolytic fragment of HMW-inhibitor containing an active site. PMID- 3003039 TI - Phosphorylation of five histone H1 subtypes of L5178Y cells at the exponential growth and mitotic phases. AB - Histone H1 of cells of L5178Y, a mouse lympholeukemic cell line, consists of five molecular species designated as H1-I, II, III, IV, and V. The phosphorylation of these H1 subtypes was examined at the exponential growth phase and during mitosis, by BioRex 70 column chromatography and two-dimensional polyacrylamide gel electrophoresis. In exponentially growing cells, the degree of phosphorylation was different for each subtype. H1-II was the most highly phosphorylated, 1.8 phosphate residues per molecule, followed by H1-IV/V, 1.4, I, 0.8, and III, 0.5. In the mitotic phase, H1-II was also the most highly phosphorylated 6.0 phosphate residues per molecule, H1-IV/V, 3.5, I, 2.7, and III, 1.2. The phosphorylation started simultaneously among the subtypes after colcemid addition, and phosphorylated H1 subtypes accumulated linearly. The rate of incorporation of 32P into each H1 subtype was almost constant during colcemid treatment. During 4 h after colcemid addition, the phosphate residues incorporated into H1 did not dephosphorylated. The H1 kinase activities increased to six times higher during the colcemid treatment. PMID- 3003041 TI - Mechanisms of growth inhibition by propionate and restoration of the growth by sodium bicarbonate or acetate in Rhodopseudomonas sphaeroides S. AB - Mechanisms of growth inhibition by propionate on the growth of Rhodopseudomonas sphaeroides were studied. Partially purified pyruvate dehydrogenase complex (PDC) from R. sphaeroides was inhibited by propionyl-CoA, one of the metabolic intermediates of propionate, while propionate itself did not inhibit the enzyme. This suggests that the inhibitor of the growth in vivo is not propionate but propionyl-CoA. The inhibition by propionyl-CoA was competitive with respect to coenzyme A concentration. The K1 value for propionyl-CoA was 0.84 mM. Addition of NaHCO3, which restored the growth of this bacterium in the presence of propionate, increased the rate of propionate incorporation by 1.7-fold and decreased the intracellular level of propionyl-CoA by half. These findings suggest that HCO3-ion lowers the level of propionyl-CoA by accelerating its carboxylation reaction, which is catalyzed by propionyl-CoA carboxylase. Effects of NaHCO3 and acetate on the growth restoration were also studied by the use of propionyl-CoA carboxylase-deficient mutants. NaHCO3 did not restore the growth of the mutants, indicating an essential role of propionyl-CoA carboxylase on the restoration of growth by NaHCO3 as suggested above. Addition of acetate restores the growth of the mutants in the presence of propionate. Acetate probably restores the growth by supplying acetyl-CoA. PMID- 3003042 TI - Nucleotide sequence of the 5' flanking region responsible for the enhancement of the expression of yeast enolase 1 gene. AB - Several plasmids carrying different length of the 5' flanking region of yeast (Saccharomyces cerevisiae) enolase 1 gene (ENO1) which is fused in frame to the Escherichia coli lacZ gene were constructed by recombination in vitro. Promoter activity of ENO1 was assayed by measuring beta-galactosidase activity of the fused gene product. Comparison of the promoter activity of these plasmids suggests that the sequences required for a strong promoter activity lie within the DNA segment -724 to -353 base pairs (bp) upstream from the start of ENO1 coding sequence. The nucleotide sequence of this region was determined. PMID- 3003043 TI - Mannan-binding protein and conglutinin in bovine serum. AB - Conglutinin is a bovine plasma protein which mediates the agglutination of the sensitized erythrocyte-solid phase iC3b complex (conglutination). The serum mannan-binding protein (MBP) is a lectin specific for mannose and N acetylglucosamine. Since conglutination was shown to be inhibited specifically by N-acetylglucosamine [Leon, M.A. & Yokohari, R. (1964) Science 143, 1327-1328], the possibility was raised that conglutinin might be a bovine serum MBP. The present study, undertaken to solve this problem, revealed that bovine plasma contained an MBP besides conglutinin. These two proteins were very similar in their chemical and physicochemical properties as well as binding specificity. Both bound with high affinity (Kd = 10(-8) M) to glycoproteins terminated with mannose and/or N-acetylglucosamine residues in the presence of calcium, although conglutinin preferred N-acetylglucosamine rather than mannose. They were multimeric proteins of large molecular size (over 1,000,000 daltons, and approximately 600,000 daltons for conglutinin and MBP, respectively) and consisted of a single kind of subunit with molecular weight of around 45,000. The MBP was shown to have a collagen-like structure in the molecule, as was recently reported for conglutinin [Davis, A.E., III & Lachmann, P.J. (1984) Biochemistry 23, 2139-2144]. Despite these similarities, the MBP and conglutinin were immunochemically distinct, and the MBP did not show any conglutination activity. PMID- 3003044 TI - Dissociation of bovine cytochrome c1 subcomplex and the status of cysteine residues in the subunits. AB - Purified bovine heart two-band cytochrome c1 subcomplex was dissociated by treatment with p-chloromercuribenzoic acid (pCMB) into its heme subunit and a colorless subunit called hinge protein, which is essential for the formation of cytochrome c1-c complex. The subcomplex was found by titration to react with 4 mol of pCMB per mol of cytochrome c1. The contents of mercury of the dissociated heme subunit and the hinge protein were 3 and 1 mol per mol of polypeptide, respectively. These results, together with the sequence analysis, indicated that the three cysteine residues in cytochrome c1 heme subunit not involved in heme binding existed in free thiol form. One of the five cysteine residues in the hinge protein was in free form and four in two disulfide bonds. The dissociated hinge protein was digested with staphylococcal protease and the cysteine containing peptides were separated by reversed-phase high-performance liquid chromatography (HPLC). The content of mercury and the result of performic acid oxidation of cystine peptides revealed that Cys-30 existed in free thiol form and two disulfide bridges were formed between Cys-24 and Cys-68 and between Cys-40 and Cys-54. The conformation of the hinge protein was predicted to be composed largely of either two-alpha-helical or four-alpha-helical conformation with the amino (N)-terminal 20 residues being in a random structure. PMID- 3003046 TI - Effect of ubiquinone extraction on the reaction of the mitochondrial bc1 complex with ferricyanide. AB - Depletion of endogenous ubiquinone by pentane extraction of mitochondrial membranes lowered succinate-ferricyanide reductase activity, whereas quinone reincorporation restored the enzymatic activity as well as antimycin sensitivity. The oxidant-induced cytochrome b extrareduction, normally found upon ferricyanide pulse in intact mitochondria in the presence of antimycin, was lost in ubiquinone depleted membranes, even if cytochrome c was added. Readdition of ubiquinone-2 restored the oxidant-induced extrareduction with an apparent half saturation at 1 mol/mol bc1 complex saturating at about 5 mol/mol. These findings demonstrate a requirement for the ubiquinone pool of the cytochrome b extrareduction. Since the initial rates of cytochrome b reoxidation upon ferricyanide addition, in the presence of antimycin, did not saturate by any ferricyanide concentration in ubiquinone-depleted mitochondria, a direct chemical reaction between ferricyanide and reduced cytochrome b was postulated. The fact that such direct reaction is much faster in ubiquinone-depleted mitochondria may explain the lower antimycin sensitivity of the succinate ferricyanide reductase activity after removal of endogenous ubiquinone. PMID- 3003047 TI - Isolation and characterization of a Chinese hamster ovary cell line requiring ethanolamine or phosphatidylserine for growth and exhibiting defective phosphatidylserine synthase activity. AB - A mutant cell line (designated M.9.1.1) requiring ethanolamine for growth was derived from Chinese hamster ovary (CHO-K1) cells using 5-bromodeoxyuridine enrichment. The ethanolamine requirement was readily replaced by 20 microM phosphatidylserine and 10 microM lysophosphatidylethanolamine. When M.9.1.1 cells were supplemented with phosphatidyl[3H]serine it was rapidly taken up, and subsequently decarboxylated to form phosphatidyl[3H]ethanolamine. The incorporation of [3H]serine into phosphatidylserine in the mutant cells was 57% of that in the parental cells. Phosphatidylethanolamine synthesis from [3H]serine in the mutant cells was 35% of that in parental cells. When M.9.1.1 cells were deprived of ethanolamine for 48 h the level of phosphatidylserine decreased 34% and the level of phosphatidylethanolamine decreased 26% compared to parental cells. At the same time the rate of turnover of phosphatidylserine was reduced to half that found in parental cells. Examination of the enzymes of phosphatidylserine metabolism indicated defective phosphatidylserine synthase activity in the mutant. When exogenous phosphatidylcholine was used as the phospholipid substrate for the reaction the apparent kinetic constants were Vmax (mutant) = 5.7 pmol/min/mg protein and Vmax (parental) = 17.5 pmol/min/mg protein. Measurement of the back reaction (ATP-independent incorporation of choline into phospholipid) gave no detectable activity in the mutant cells. The data indicate that the phosphatidylcholine-dependent synthesis of phosphatidylserine is the primary lesion in M.9.1.1. PMID- 3003045 TI - Detection of antimycin-binding subunits of complex III by photoaffinity-labeling with an azido derivative of antimycin. AB - Deformamidoazidoantimycin A (DAA), a photoactive derivative of antimycin A containing an azido group substituting for the formamido group attached to the phenyl ring, was synthesized. The ultraviolet spectrum of DAA was almost identical to that of antimycin A, indicating little alteration of the electronic structure of the substituted phenyl ring by the azido substitution. However, the inhibitory effectiveness of DAA toward ubiquinol-cytochrome c reductase (Complex III) purified from bovine heart (Ki = ca. 0.5 microM) was considerably less than that of antimycin (Ki less than or equal to 3 pM), indicating a direct rather than a supporting role of the formamido group in the inhibitory activity of antimycin. Exposure of purified Complex III to [3H]DAA plus ultraviolet light caused a major labeling by tritium of SDS-PAGE band 7 (m = 13 kDa by SDS-PAGE) and lesser but significant labeling of bands 3, 6, 8, and 9. Pretreatment of Complex III with antimycin greatly suppressed the labeling of bands 5, 6, and 7 but caused an apparent increased labeling of bands 8 and 9 by [3H]DAA, respectively. The labeling of band 7 by [3H]DAA also was strongly suppressed by reduction of Complex III by either sodium borohydride or ascorbate. Based on magnitude of labeling by [3H]DAA and the degree of suppression of labeling by antimycin, the protein of band 7 qualified as the principal component for specific binding of antimycin with the protein of band 6 (m = 16 kDa) showing a lesser but significant amount of specific binding. PMID- 3003048 TI - The (Na+ + K+)-ATPase of chick sensory neurons. Studies on biosynthesis and intracellular transport. AB - The (Na+ + K+)-ATPase of cultured chick sensory neurons was studied with the aid of antibodies specific for this enzyme. Immunofluorescent labeling indicated the (Na+ + K+)-ATPase is evenly distributed on the neuronal cell surface; cell bodies, neurites, and growth cones were labeled with comparable intensity. Pulse chase experiments with [35S]methionine, followed by immunoprecipitation, indicated concurrent synthesis and rapid association of the alpha (Mr = 105,000) and beta (Mr = 47,000) subunits. The alpha subunit is oligosaccharide-free while the beta subunit contains three Asn-linked oligosaccharide chains attached to a core peptide of 32,000 molecular weight. The time required for oligosaccharide processing of the newly synthesized beta subunit to endoglycosidase H-resistance suggests the (Na+ + K+)-ATPase takes 45-60 min to move from the site of polypeptide synthesis to the Golgi apparatus. Significantly less time was required for transport through the Golgi apparatus and insertion in the plasma membrane. From 30% to 55% of the newly synthesized (Na+ + K+)-ATPase did not appear on the cell surface but accumulated intracellularly. When tunicamycin was used to inhibit glycosylation of the beta subunit, there was no effect upon subunit assembly, intracellular transport, or degradation rate (t1/2 = 40 h). PMID- 3003049 TI - Nucleotide sequence of lysC gene encoding the lysine-sensitive aspartokinase III of Escherichia coli K12. Evolutionary pathway leading to three isofunctional enzymes. AB - The lysC gene encoding the lysine-sensitive aspartokinase III of Escherichia coli K12 has been cloned and its nucleotide sequence determined. Analysis of the deduced protein sequence (449 amino acid residues) reveals that the entire sequence of aspartokinase III is homologous to the N-terminal part of the two iso and bifunctional aspartokinase-homoserine dehydrogenases I and II of E. coli. An evolutionary pathway leading to the three molecular species present in the same organism is proposed, and the possible involvement of a highly conserved region in subunit interactions is discussed. PMID- 3003050 TI - Covalent modification of substrate-binding sites of Escherichia coli ADP-glucose synthetase. Isolation and structural characterization of 8-azido-ADP-glucose incorporated peptides. AB - A photoaffinity substrate analogue, 8-azido-ADP-[14C]glucose, reacts specifically and covalently with Escherichia coli ADP-glucose synthetase. The site(s) of reaction of 8-azido-ADP-[14C]glucose with the enzyme was identified by isolation of tryptic peptides containing the labeled analogue by use of high performance liquid chromatography technique and subsequent NH2-terminal sequence analysis of the purified radioactive peptides. One major binding region of the azido analogue is a peptide segment composed of residues 107-114 of the enzyme's polypeptide chain. Lys 108 and Arg 114 become trypsin-resistant sites when the enzyme is photoinactivated by 8-azido-ADP-[14C] glucose, suggesting that the analogue binds at or near the vicinity of these 2 basic amino acid residues. Conformational analysis of this peptide segment (residues 107-114) shows a strong probability of a reverse beta-turn secondary structure, suggesting that this peptide segment is on the enzyme surface. Two minor reaction regions of the enzyme with the analogue were also identified by chemical characterization. One region was composed of residues 162-207. Lys 194 was previously suggested as the activator-binding site by chemical modification studies with pyridoxal phosphate (Parsons, T. F., and Preiss, J. (1978) J. Biol. Chem. 253, 7638-7645). Another minor region where the analogue binds the tryptic peptide composed of residues 380-385 is near the COOH terminal side of the enzyme. It is postulated that all these peptide segments are juxtaposed in tertiary structure. PMID- 3003051 TI - A human neuroblastoma cell line expresses mu and delta opioid receptor sites. AB - A series of neuroblastoma cell lines were screened for the presence of opioid receptor sites with the tracers [3H]diprenorphine (mu, delta, kappa ligand) and [3H]naloxone (mu-selective ligand). One human neuroblastoma cell line, SK-N-SH, displayed avid binding for both tracers. Binding experiments with multiple tracers revealed the presence of both mu and delta sites. These sites were stereospecific, saturable, and proteinaceous in character. Saturation binding experiments provided an estimate of 50,000 mu and 10,000 delta sites/cell. NaCl (100 mM) and guanine nucleotide, guanylyl imidodiphosphate (50 microM), reduced opioid agonist but not antagonist binding to these sites. Etorphine at 1 nM inhibited prostaglandin E1-stimulated cyclic AMP production by approximately 20%, which was reversible by naloxone. The opioid-binding sites on SK-N-SH cells closely resemble the previously reported mu and delta sites in human and rodent brain. Therefore, the SK-N-SH neuroblastoma cell line represents a useful tool to study the molecular functions of opioid receptors. PMID- 3003053 TI - Inhibitory effect of prostaglandin E2, forskolin, and dibutyryl cAMP on arachidonic acid release and inositol phospholipid metabolism in guinea pig neutrophils. AB - The effect of prostaglandin E2 (PGE2), forskolin, and dibutyryl cAMP on arachidonic acid release, inositol phospholipid metabolism, and Ca2+ mobilization was investigated. The chemotactic tripeptide (formylmethionyl-leucyl phenylalanine (fMLP))-induced arachidonic acid release in neutrophils was significantly inhibited by PGE2, forskolin, and dibutyryl cAMP. Among them, PGE2 was found to be the most potent inhibitor. However, when neutrophils were stimulated by Ca2+ ionophore A23187, such inhibitory effect by these agents was less marked. PGE2 also suppressed the enhanced incorporation of [32P]Pi into phosphatidic acid (PA) and phosphatidylinositol in a dose-dependent manner in fMLP-stimulated neutrophils. Also in this case, Ca2+ ionophore-induced alterations were hardly inhibited by PGE2. As well, PGE2 inhibited the fMLP induced decrease of [3H]arachidonic acid in phosphatidylcholine and phosphatidylinositol and the increase in PA very significantly. But the inhibitory effect by PGE2 was found to be weak in Ca2+ ionophore-stimulated neutrophils. These results suggest that a certain step from receptor activation to Ca2+ influx is mainly inhibited by PGE2. Concerning polyphosphoinositide breakdown, PGE2 did not affect the fMLP-induced decrease of [32P]phosphatidylinositol 4,5-bisphosphate which occurred within 10 s but inhibited the subsequent loss of [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol, suggesting that the compensatory resynthesis of phosphatidylinositol 4,5-bisphosphate was inhibited. On the other hand, fMLP induced diacylglycerol formation was suppressed for the early period until 1 min, but with further incubation, diacylglycerol formation was rather accelerated by PGE2. Moreover, the inhibition of PA formation by PGE2 became evident after a 30 s time lag, suggesting that the conversion of diacylglycerol to PA is inhibited by PGE2. The formation of water-soluble products of inositol phospholipid degradation by phospholipase C, such as inositol phosphate, inositol 1,4 bisphosphate, and inositol 1,4,5-trisphosphate, was also suppressed by PGE2 treatment. However, the inhibition was not so marked as that of arachidonic acid release and PA formation. Thus, PGE2 appeared to inhibit not only initial events such as polyphosphoinositide breakdown but also turnover of inositol phospholipids. PGE2, forskolin, and dibutyryl cAMP did not block the rapid elevation of intracellular Ca2+ which was observed within 10 s in fMLP-stimulated neutrophils. However, subsequent increase in intracellular Ca2+ which was caused from 10 s to 3 min after stimulation was inhibited by PGE2, forskolin, and dibutyryl cAMP.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3003052 TI - Acidification activity of human neutrophils. Tertiary granules as a site of ATP dependent acidification. AB - The acidification activity of human neutrophils, known to occur extracellularly and intraphagolysosomally, was studied in intact and in fractionated cells. The subcellular location of the acidification activity was investigated by rate zonal sedimentation of post-nuclear supernatants from resting cells on continuous sucrose gradients. The acidification measurements indicated a dominance of activity in gelatinase-rich tertiary granules. On the other hand, ATPase activities were located in plasma membrane and in the membranes of the cytoplasmic granules (specific, azurophilic, and tertiary). All of these activities were diminished by the inhibitors dicyclohexylcarbodiimide and diisothiocyanostilbene disulfonic acid; however, studies with other inhibitors, especially N-ethylmaleimide and duramycin, suggested ATPase enzymatic differences depending on location. The results taken together provide direct and strong indication of involvement of a proton pump ATPase in acidification inside neutrophils. Furthermore, the dominant location of acidification activity in tertiary granules that very readily degranulate presumably has significant implications for the importance of low pH in cidal events and the inflammatory process. PMID- 3003054 TI - Inactivation of the renal microvillus membrane Na+-H+ exchanger by histidine specific reagents. AB - We examined the effect of histidine-specific reagents on the transport activity of the Na+-H+ exchanger in microvillus (brush-border) membrane vesicles isolated from the rabbit renal cortex. Rose bengal-catalyzed photo-oxidation caused irreversible inhibition of the rate of Na+-H+ exchange but also caused significant loss of vesicle integrity. Treatment of the membrane vesicles with diethylpyrocarbonate caused inactivation of Na+-H+ exchange that could not be attributed to vesicle disruption or collapse of transmembrane H+ gradients. Inactivation of Na+-H+ exchange by diethylpyrocarbonate followed pseudo-first order kinetics to below 10% residual activity, could be reversed by hydroxylamine, was reflected by a decreased Vmax with no change in the Km for Na+, was dependent on external pH but not internal pH, was blocked by amiloride, and was enhanced by Na+. These data are consistent with the hypothesis that a diethylpyrocarbonate-sensitive imidazolium residue is the titratable group found in kinetic studies to bind H+ at the external transport site of the Na+-H+ exchanger. PMID- 3003056 TI - ADP- and K+-sensitive phosphorylated intermediate of Na,K-ATPase. AB - In the phosphoenzyme (EP) of the electric eel Na,K-ATPase, the sum of the ADP sensitive EP and the K+-sensitive EP exceeds 150% of EP in the presence of 100 mM Na+. This unusual phenomenon can be explained by the formation of three phosphoenzymes: ADP-sensitive K+-insensitive (E1P), K+-sensitive ADP-insensitive (E2P), and ADP- and K+-sensitive (E*P) phosphoenzymes, as proposed by Norby et al. (Norby, J. G., Klodos, I., and Christiansen, N. O. (1983) J. Gen. Physiol. 82, 725-757). By applying a simple approximation method for the assay of E1P, E*P, and E2P, it was found that the phosphorylation of the enzyme was much faster than the conversion among each EP and the phosphoenzyme changed as E1NaATP----E1P ---E*P----E2P. In the fragmental eel enzyme, the step of E*P to E2P was much slower than the step of E1P to E*P. In the steady state, the E1P was predominant above 400 mM Na+, whereas E*P and E2P were predominant between 60 and 300 mM Na+ and below 60 mM Na+, respectively. The characteristic difference of the eel enzyme from the beef brain enzyme and probably from the kidney enzyme seems to be that the dissociation constant of Na+ on the E1P-E*P equilibrium is higher than that on the E*P-E2P. The E*P and E1P both interacted with ADP to form ATP without formation of inorganic phosphate in the absence of free Mg2+. In the Na,K-ATPase proteoliposomes, the vesicle membrane interfered with the conversion of E1P to E2P, especially the change of E1P to E*P, and furthermore, the E1P content increased. This barrier effect was partially counteracted by monensin or carbonyl cyanide m-chlorophenylhydrazone. Oligomycin reacted with E1P and probably with E*P, therefore inhibiting their conversion to E2P and interaction with K+. PMID- 3003055 TI - Tetraheme cytochrome c-554 from Nitrosomonas europaea. Heme-heme interactions and ligand binding. AB - Cytochrome c-554 functions in the ammonia oxidizing system of Nitrosomonas europaea. We have investigated its molecular and ligand binding properties and studied the protein with optical, EPR, and Mossbauer spectroscopies in the pH range from 2 to 13. Amino acid, heme, and metal analyses show that the protein has Mr = 25,000 and that it contains four c-type hemes per molecule. Optical spectra reveal that the heme ligand structures are sensitive to the pH of the medium and that the hemes can bind small molecules such as CN-, CO, and NO under certain conditions. According to the Mossbauer and EPR studies of the ferric protein, the hemes are predominantly (75%) high spin at pH 2 and low spin (approximately equal to 100%) above pH 10. At neutral pH, Mossbauer data show that 75% of the heme is low spin and that the remainder is high spin. The EPR data, however, do not reveal any signals attributable to typical high spin or low spin species. Rather, a very complex and unusual spectrum with a main feature at g = 3.3 is observed at X-band, this feature shifts to approximately g = 3 at S band. The EPR and Mossbauer data show clearly that the hemes are magnetically interacting, by dipolar and exchange interactions. At pH 2, the EPR spectra reveal resonances at g = 6 and 2. The Mossbauer spectra prove that all hemes are magnetically coupled at this pH. Coupling is also borne out by the observation of a half-field EPR resonance near g = 12. PMID- 3003057 TI - Potentiation of free radical-induced lipid peroxidative injury to sarcolemmal membranes by lipid amphiphiles. AB - The effects of naturally occurring lipid amphiphiles on free radical-mediated peroxidative injury in isolated canine sarcolemma were studied. Highly enriched canine myocytic sarcolemmal membranes were preincubated for 10 min at 37 degrees C with or without different amphiphilic lipids before the addition of a free radical-generating system consisting of dihydroxyfumarate and Fe3+-ADP. Lipid peroxidation, assayed as malondialdehyde formation, was catalyzed linearly up to 40 min in the control samples. Pretreatment of the sarcolemma with palmitoyl-CoA, palmitoylcarnitine, or lysophosphatidylcholine accelerated the initial rates (20 min) of peroxidation in a concentration-dependent manner (10-100 microM) and achieved maximal stimulation (240%, 160%, and 210%, respectively, of controls) at 50 microM concentrations of each of these amphiphiles. However, free fatty acids, CoA, and carnitine were without effect. These promoting effects of the amphiphiles persisted over a wide pH range (pH 6.0-7.8) and exhibited additive effects when lower levels of different amphiphiles were combined together. Associated with the accelerated rates of peroxidation produced by palmitoyl-CoA and palmitoylcarnitine were greater losses in the activity of sarcolemmal (Na,K) ATPase. Since all three kinds of amphiphilic lipids accumulate during ischemia, this study suggests a novel mechanism of potentiation of sacolemmal membrane injury when free radicals are present. PMID- 3003058 TI - Nucleotide sequence of the Pseudomonas putida cytochrome P-450cam gene and its expression in Escherichia coli. AB - Cytochrome P-450cam catalyzes the stereospecific methylene hydroxylation of camphor to form 5-exohydroxycamphor and is encoded by the camC gene on the CAM plasmid of Pseudomonas putida, ATCC 17453. The cytochrome P-450cam structural gene has been cloned by mutant complementation in P. putida (Koga, H., Rauchfuss, B., and Gunsalus, I. C. (1985) Biochem. Biophys. Res. Commun. 130, 412-417). We report the complete nucleotide sequence of the camC gene along with 155 base pairs of 5' and 175 base pairs of 3' flanking sequence. Upon comparison of the amino acid sequence derived from the gene sequence to the one obtained from the purified protein (Haniu, M., Armes, L. G., Yasunobu, K. T., Shastry, B. A., and Gunsalus, I. C. (1982) J. Biol. Chem. 257, 12664-12671), five differences were found. The most significant was the addition of a Trp and a Thr residue between Val-54 and Arg-55, thereby increasing the amino acid numbering scheme by 2 after Val-54, bringing the total number of amino acids to 414. Other differences were: Gln-274----Glu-276, Ser-359----His-361, and Asn-405----Asp-407. N-terminal amino acid sequence analysis of the cloned cytochrome P-450cam enzyme expressed in Escherichia coli under the lac promoter showed a faithful translation of the hemo protein, with the N-terminal Met removed by processing as found in P. putida. Purification to homogeneity of the cloned protein was accomplished by the method used for the CAM plasmid-encoded enzyme of P. putida. The G + C content of the camC gene was found to be 59.0%, caused by a preferred usage of G and C terminated codons. The gene encoding putidaredoxin reductase, camA, was located 22 nucleotides downstream from the cytochrome P-450cam gene. The camA gene initiated with a novel GUG codon, the first such initiator documented in Pseudomonas. PMID- 3003059 TI - Delta endotoxin is a potent inhibitor of the (Na,K)-ATPase. AB - A 68-kDa protein, delta endotoxin, produced by Bacillus thuringiensis ssp. Kurstaki inhibits ion transport, (Na,K)-ATPase, and K+-p-nitrophenylphosphatase activity catalyzed by the Na+ pump. The Ki for inhibition of the K+-p nitrophenylphosphatase activity of purified dog kidney (Na,K)-ATPase was approximately 0.37 microM. Delta endotoxin had a similar Ki for inhibition of (Na,K)-ATPase activity when assayed at low Na+ concentration (10 mM) but the inhibition was reversed when high concentrations of Na+ (100 mM NaCl) were added to the assay. Phosphorylation of the active site aspartyl residue with 32PO3-4 was also blocked by delta endotoxin. Ouabain-sensitive 86Rb+ uptake into intact human red blood cells was not inhibited by externally added toxin; however, strophanthidin-inhibitable 22Na+ uptake into inside-out vesicles from red blood cells was completely blocked by delta endotoxin (Ki = 0.73 microM). These data suggest that delta endotoxin must enter the cell before it can inhibit the Na+ pump. PMID- 3003061 TI - Studies of cGMP analog specificity and function of the two intrasubunit binding sites of cGMP-dependent protein kinase. AB - The specificity of the two intrasubunit cGMP binding sites of cGMP-dependent protein kinase was determined by measuring the ability of 46 cGMP analogs to compete with [3H]cGMP. Both sites of the enzyme exhibited high specificity for the ribose cyclic phosphate moiety, and lower specificity for the guanine moiety. Effects of modifications in the ribose cyclic phosphate moiety suggested that cGMP is bound at both sites by three hydrogen bonds at 2'-OH, 3'-O, and 5'-O. A negative charge in the cyclic phosphate is apparently required. Modifications of the pyrimidine part of guanine, particularly at C-1, generally caused selectivity for the rapidly exchanging site while modifications of the imidazole part of guanine at C-7 and C-8 caused selectivity for the slowly exchanging site. These increases in selectivity for a site were mainly due to losses in affinity of the other site. There was an apparent requirement of the intact amino group at C-2, particularly for the slowly exchanging site. Comparison of the molecular interactions of cAMP and cGMP with their specific protein kinases showed that both nucleotides are bound by similar forces in the 2', 3' and 5' region, both bases may be bound in syn conformation, but that each base moiety is bound by different molecular interaction, thus leading to the selectivity of the two enzymes. cGMP analogs which possessed strong selectivity for the rapidly exchanging site, but not those selective for the slowly exchanging site, stimulated the binding of [3H]cGMP. Only a few cGMP analogs were more potent than cGMP in stimulating protein kinase activity. The potency of cGMP analogs as stimulators of kinase activity correlated better with the mean binding affinity for both binding sites than with the affinity for either site alone. Two analogs added in combination were synergistic in kinase activation, particularly if one analog was selective for the slowly exchanging site and the other for the rapidly exchanging site. These observations are suggestive that cGMP binding at the rapidly exchanging site stimulates cGMP binding at the slowly exchanging site and that both sites are involved in the activation process. PMID- 3003060 TI - Mechanism of the inhibition of catalase by ascorbate. Roles of active oxygen species, copper and semidehydroascorbate. AB - Ascorbate reversibly inhibits catalase, and this inhibition is enhanced and rendered irreversible by the prior addition of copper(II)-bishistidine. In the absence of copper, the inhibition was prevented and reversed by ethanol, but not by superoxide dismutase, benzoate, mannitol, thiourea, desferrioxamine, or DETAPAC. In the presence of the copper complex mannitol, benzoate, and superoxide dismutase still had no effect, but thiourea, desferrioxamine, DETAPAC, or additional histidine decreased the extent of inactivation to that seen in the absence of copper. In the presence of copper, ethanol protected at [ascorbate] less than 1 mM, but was ineffective at [ascorbate] greater than 2 mM, even in the absence of oxygen. Although in the absence of copper, complete removal of oxygen provided full protection against inactivation by ascorbate, this protection was not seen if the catalase was briefly preincubated with H2O2 prior to flushing with nitrogen, or if copper was present. In fact, if copper was present, inactivation was enhanced by the removal of oxygen. Increasing the concentration of oxygen from ambient to 100% slowed the inactivation, whether or not copper was present. It is concluded that the initial reversible inactivation involves reaction with H2O2 to form compound I, followed by one electron reduction of compound I to compound II. In the presence of added copper, the initial (reversible) inactivation allows H2O2 to accumulate sufficiently to permit irreversible inactivation. Since in the presence of copper oxygen is not required, and neither the reversible nor the irreversible inactivation was prevented by conventional scavengers of active forms of oxygen, the inactivation is likely mediated by semidehydroascorbate, and/or it may involve site-specific generation of the damaging intermediates. PMID- 3003062 TI - Sequence-dependent recognition of DNA duplexes. Netropsin complexation to the AATT site of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution. AB - We have investigated intermolecular interactions and conformational features of the netropsin X d(G-G-A-A-T-T-C-C) complex by one- and two-dimensional NMR studies in aqueous solution. Netropsin removes the 2-fold symmetry of the d(G-G-A A-T-T-C-C) duplex at the AATT binding site and to a lesser extent at adjacent dG X dC base pairs resulting in doubling of resonances for specific positions in the spectrum of the complex at 25 degrees C. We have assigned the amide, pyrrole, and CH2 protons of netropsin, and the base and sugar H1' protons of the nucleic acid from an analysis of the nuclear Overhauser effect (NOESY) and correlated (COSY) spectra of the complex at 25 degrees C. We observe intermolecular nuclear Overhauser effects (NOE) between all three amide and both pyrrole protons on the concave face of the antibiotic and the minor groove adenosine H2 proton of the two central A4 X T5 base pairs of the d(G1-G2-A3-A4-T5-T6-C7-C8) duplex. Weaker intermolecular NOEs are also observed between the pyrrole concave face protons and the sugar H1' protons of residues T5 and T6 in the AATT minor groove of the duplex. We also detect intermolecular NOEs between the guanidino CH2 protons at one end of netropsin and adenosine H2 proton of the two flanking A3 X T6 base pairs of the octanucleotide duplex. These studies establish a set of intermolecular contacts between the concave face of the antibiotic and the minor groove AATT segment of the d(G-G-A-A-T-T-C-C) duplex in solution. The magnitude of the NOEs require that there be no intervening water molecules sandwiched between the antibiotic and the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation. PMID- 3003063 TI - Signal recognition particle mediates the insertion of a transmembrane protein which has a cytoplasmic NH2 terminus. AB - The mechanism by which rat liver asialoglycoprotein receptor (rat hepatic lectin, RHL) is inserted into membranes has been investigated. RHL is a prototype for transmembrane proteins which are oriented with their NH2 termini in the cytoplasm and their COOH termini outside the cell. Such transmembrane proteins are synthesized without cleavable NH2-terminal signal sequences. An in vitro translation system has been developed in which RHL is translated from RNA produced in a bacteriophage SP6 in vitro transcription system. RHL produced in this way can be inserted cotranslationally in the correct orientation into dog pancreas microsomes. This insertion process has been shown to be dependent on the signal recognition particle and its receptor (the docking protein) in the microsomal membranes. A detailed mechanism for the insertion of this type of transmembrane protein into the lipid bilayer is proposed. PMID- 3003064 TI - Purification and characterization of prolactin receptors from rat ovary. AB - Prolactin receptors were purified to homogeneity by two affinity chromatography steps using concanavalin A-Sepharose and human growth hormone (hGH)-Sepharose. The purified receptors showed specificity and high affinity for lactogenic hormones and a binding capacity of 20 nmol/mg of protein. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis revealed that purified receptors were composed of two major protein bands of Mr = 41,000 and 88,000, which were identified as radioactive bands by binding of 125I-hGH to blotted renatured receptors and by autoradiogram of free and 125I-radiolabeled purified receptors. Autoradiographic analysis of SDS-polyacrylamide gel electrophoresis of cross-linked 125I-hGH-receptor or hGH-125I-iodinated receptor complexes showed two radioactive bands of Mr = 63,000 and 106,000. Analysis of the free receptors by high performance liquid chromatography using Superose 12 revealed two peaks of binding activity for 125I-hGH eluting in the positions of Mr approximately 150,000 and 250,000. After cross-linking with 125I-hGH, SDS-polyacrylamide gel electrophoresis analysis revealed that both peak fractions contained two binding species with Mr = 63,000 and 106,000. Chromatography of 125I-hGH-receptor complexes showed two radioactive fractions with approximate Mr approximately 180,000 and 300,000. The treatment of 125I-iodinated receptors with SDS and reductant resulted in the dissociation of the higher Mr form into the lower Mr form upon gel filtration. Chromatofocusing of free receptors showed three isoforms with pI 4.0, 5.0, and 5.3. These results indicate that detergent solubilized prolactin receptors appear to be aggregated forms of holoreceptor containing two binding species of Mr = 41,000 +/- 2,000 and 88,000 +/- 3,000. PMID- 3003065 TI - Structure and expression of the gene locus encoding the phosphatidylglycerophosphate synthase of Escherichia coli. AB - This paper presents definitive results which establishes a direct gene-protein product relationship between the pgsA gene and the phosphatidylglycerophosphate synthase of Escherichia coli. The predicted protein sequence derived from the determined DNA sequence of pgsA is in close agreement with the amino acid composition and partially determined amino acid sequence of the purified enzyme. The purified synthase has the same apparent molecular mass as the gene product made by a plasmid-directed transcription-translation system. The plasmid-borne copy of the pgsA gene is also capable of expressing enzymatically active synthase in vitro. The DNA sequence analysis has established the exact linear relationship between the uvrC, pgsA, and glyW loci and revealed that these three genes are transcribed in the same direction. The terminal coding regions of these three genes also share common sequences with transcriptional regulatory elements for the adjacent genes. PMID- 3003066 TI - The binding of the chromosomal protein HMG-2a to DNA regions of reduced stabilities. AB - The interaction of immunopurified high mobility group 2a protein (HMG-2a) with DNA was examined by the nitrocellulose filter binding assay. The relative binding activity of HMG-2a for synthetic polynucleotides was: (dI).(dC) greater than (dA dT).(dA-dT) greater than (dA).(dT) much greater than (dG).(dC) greater than (dG dC).(dG-dC). The protein also exhibited a marked preference for (A + T)-rich restriction fragments derived from rat and Drosophila satellites, yeast centromeres, phage lambda, and the ovalbumin gene and its 5' flanking sequences. These preferential DNA interactions occurred at ionic strengths and temperatures within the physiological range which argue for an in vivo role of DNA stability in dictating the genomic distribution of the large Mr HMG proteins. PMID- 3003067 TI - Characterization and photoaffinity labeling of receptor sites for the Ca2+ channel inhibitors d-cis-diltiazem, (+/-)-bepridil, desmethoxyverapamil, and (+) PN 200-110 in skeletal muscle transverse tubule membranes. AB - In order to further understand the molecular nature of the voltage-sensitive Ca2+ channel in skeletal muscle, we have performed classical radioligand binding studies and photoaffinity labeling with different types of tritiated inhibitors of the Ca2+ channel. The equilibrium dissociation constants (KD) for (-) [3H]desmethoxyverapamil, d-cis-[3H]diltiazem, and (+/-)-[3H]bepridil at their receptor sites in skeletal muscle transverse tubule membranes are: 1.5 +/- 0.5, 50 +/- 5, and 20 +/- 5 nM, respectively. Maximum binding capacities in picomoles/milligram of protein were: 70 +/- 10 for (-)-[3H]desmethoxyverapamil, 50 +/- 15 for d-cis-[3H]diltiazem, and 75 +/- 15 for (+/-)-[3H]bepridil. The kinetics of association at 10 degrees C for the three types of tritiated compounds were relatively slow (3 X 10(5) M-1 S-1 for (-) [3H]desmethoxyverapamil, 8 X 10(3) M-1 S-1 for d-cis-[3H]diltiazem, and 4.2 X 10(5) M-1 S-1 for (+/-)-[3H]bepridil). The dissociation of (-) [3H]desmethoxyverapamil and d-cis-[3H]diltiazem from their receptor sites was also a slow process with half-lives of dissociation of 33 and 36 min, respectively. Competition studies using the three tritiated ligands suggest that they bind to the same receptor site which appears to be in a 1:1 stoichiometry with the dihydropyridine receptor. Photoaffinity labeling with high intensity ultraviolet light in the presence of (+/-)-[3H]bepridil or d-cis[3H]diltiazem resulted in the specific covalent incorporation of radioactivity into a polypeptide of Mr 170,000 +/- 10,000. A polypeptide of Mr 170,000 was also specifically labeled in photoaffinity labeling experiments using the high affinity dihydropyridine derivative (+)-[3H]PN 200-100. PMID- 3003069 TI - Crystallization of the gastric H,K-ATPase. AB - Crystalline arrays of the gastric H,K-ATPase were obtained in membrane preparations from hog and rabbit gastric mucosa. The lattice was formed rapidly in a medium containing K+, vanadate, Mg2+, and dimethyl sulfoxide at pH 6.0-6.9 in imidazole buffer from 4 to 22 degrees C. The crystal lattice exhibited P2 symmetry, and the unit cell dimension (a = 5.6, b = 11, and c = 10 nm) could accommodate 2 polypeptides of mass 116-129 kDa. In addition, the isolated preparation contained previously undescribed long cylindrical structures 16 nm thick. These structures consisted of a central core 6-7 nm wide from which particles spaced 5.5 nm apart protruded symmetrically. PMID- 3003068 TI - Purification of nuclear factor I by DNA recognition site affinity chromatography. AB - Nuclear factor I (NF-I) is a cellular protein that enhances the initiation of adenovirus DNA replication in vitro. The enhancement of initiation correlates with the ability of NF-I to bind a specific nucleotide sequence within the viral origin of replication. We have developed a method for the purification of NF-I which is based upon the high affinity interaction between the protein and its recognition site. This approach may be generally applicable to the purification of other site-specific DNA binding proteins. The essential feature of the method is a two-step column chromatographic procedure in which proteins are first fractionated on an affinity matrix consisting of nonspecific (Escherichia coli) DNA and then on a matrix that is highly enriched in the specific DNA sequence that is recognized by NF-I. During the first step NF-I coelutes with proteins that have similar general affinity for DNA. During the second step NF-I elutes at a much higher ionic strength than the contaminating nonspecific DNA binding proteins. The DNA recognition site affinity matrix used in the second step is prepared from a plasmid (pKB67-88) that contains 88 copies of the NF-I binding site. This plasmid was constructed by means of a novel cloning strategy that generates concatenated NF-I binding sites arranged exclusively in a direct head to tail configuration. Using this purification scheme, we have obtained a 2400 fold purification of NF-I from crude HeLa nuclear extract with a 57% recovery of specific DNA binding activity. Throughout the purification procedure NF-I retained the ability to enhance the efficiency of initiation of adenovirus DNA replication in vitro. Electrophoresis of the purified fraction on sodium dodecyl sulfate-polyacrylamide gels revealed a population of related polypeptides that ranged in apparent molecular weight from 66,000 to 52,000. The native molecular weight of NF-I deduced from gel filtration and glycerol sedimentation studies is 55,000 and the frictional ratio is 1.3. These results suggest that NF-I exists as a globular monomer in solution. PMID- 3003070 TI - Chromatin structure of the human dihydrofolate reductase gene promoter. Multiple protein-binding sites. AB - The chromatin structure of the promoter region of the human dihydrofolate reductase gene was determined using a variety of nucleases including DNase I, micrococcal nuclease, several restriction endonucleases, exonuclease III, and Bal31. Two separate regions from -670 to -340 (the distal hypersensitive region) and from -170 to +150 (the proximal hypersensitive region) were shown to be essentially free of proteins as indicated by their accessibility to both endo- and exonucleases. Within the proximal hypersensitive region, one protein appears to be bound at the start site for transcription. A 170-base pair fragment between the two hypersensitive regions was highly resistant to all nucleases tested. Multiple barriers against exonuclease digestion and resistance to dissociation by high salt concentrations suggest that more than one protein is tightly bound to this region. The upstream sequence from -670 and the downstream sequence from +150 were shown to be packaged into nucleosomes. The selective accessibility of certain sites to micrococcal nuclease cutting indicates that the initial nucleosomes are phased upstream from the distal hypersensitive region. There appears to be a protein bound between the phased nucleosomes and the upstream boundary of the distal hypersensitive region. These results suggest that the normal nucleosome array is interrupted by about 900 base pairs of nucleosome-free DNA to which several nuclear proteins bind in a DNA sequence-specific manner. PMID- 3003071 TI - Evidence for differences in the sensitivity to ouabain of NaK-ATPase along the nephrons of rabbit kidney. AB - To determine the possible intrarenal site of action of an endogenous ouabain-like natriuretic factor, we searched for the presence of NaK-ATPase highly sensitive to ouabain in the kidney, an organ previously reported to display a low sensitivity to ouabain. For this purpose, the sensitivity of NaK-ATPase to ouabain was determined at the level of single, well defined segments of nephron microdissected from rabbit kidney. Results indicated that NaK-ATPase activity is 10- to 30-fold more sensitive to ouabain in the collecting tubule, where final adjustments of sodium excretion take place, than in more proximal segments of the nephron. [3H]Ouabain binding experiments confirmed this finding as the affinity for ouabain increases from the proximal tubule to the collecting tubule. These results suggest that endogenous natriuretic factor may control sodium transport in the collecting tubule preferentially. PMID- 3003073 TI - Inhibition of herpes simplex virus DNA polymerase by purine ribonucleoside monophosphates. AB - Purine ribonucleoside monophosphates were found to inhibit chain elongation catalyzed by herpes simplex virus (HSV) DNA polymerase when DNA template-primer concentrations were rate-limiting. Inhibition was fully competitive with DNA template-primer during chain elongation; however, DNA polymerase-associated exonuclease activity was inhibited noncompetitively with respect to DNA. Combinations of 5'-GMP and phosphonoformate were kinetically mutually exclusive in dual inhibitor studies. Pyrimidine nucleoside monophosphates and deoxynucleoside monophosphates were less inhibitory than purine riboside monophosphates. The monophosphates of 9-beta-D-arabinofuranosyladenine, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), 9-(2 hydroxyethoxymethyl)guanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine exerted little or no inhibition. In contrast to HSV DNA polymerase, human DNA polymerase alpha was not inhibited by purine ribonucleoside monophosphates. These studies suggest the possibility of a physiological role of purine ribonucleoside monophosphates as regulators of herpesvirus DNA synthesis and a new approach to developing selective anti-herpesvirus compounds. PMID- 3003072 TI - Trimethyllysine and protein function. Effect of methylation and mutagenesis of lysine 115 of calmodulin on NAD kinase activation. AB - Unmethylated calmodulins have been enzymatically methylated at lysine 115, and a direct effect of this methylation on NAD kinase activation has been shown. Similar to naturally occurring calmodulins with trimethyllysine 115, the enzymatically methylated calmodulins activated an NAD kinase preparation to a maximal level that was at least 3-fold lower than the level of activation obtained with the corresponding unmethylated calmodulins. Methylation did not alter the cyclic nucleotide phosphodiesterase activator properties of these calmodulins. A genetically engineered calmodulin containing an arginine at position 115 instead of a lysine was produced by site-specific mutagenesis of a cloned synthetic calmodulin gene. The arginine derivative retained the higher maximal NAD kinase activator properties of the unmethylated calmodulins but was no longer susceptible to the effects of the methyltransferase. The data indicate that the reduction in the level of NAD kinase activation is the direct result of trimethylation of lysine 115 of calmodulin, provide a precedent for a functional effect of trimethyllysine in a protein, and raise the possibility that some of calmodulin's physiological activities may be affected by lysine methylation. PMID- 3003074 TI - Affinity cross-linking of atrial natriuretic factor to its receptor in bovine adrenal zona glomerulosa. AB - 125I-Labeled atrial natriuretic factor (ANF) was covalently cross-linked to its binding sites in bovine adrenal zona glomerulosa membranes using the hydrophilic cross-linker bis(sulfosuccinimidyl) suberate. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol revealed that two protein bands with apparent Mr 68,000 and 114,000 were specifically labeled. The labeling of the two bands was prevented in a dose-dependent fashion by unlabeled ANF with a significant inhibition observed at 10(-10) M. High concentrations of angiotensin II and adrenocorticotropic hormone did not compete with 125I-ANF for binding and cross-linking. The dose-response curve for inhibition of covalent cross-linking of 125I-ANF by unlabeled ANF coincided with the dose-response curve for inhibition of binding to the receptor. No radioactive bands were observed in liver membranes. Experiments in which adrenal membranes were prepared and incubated in the presence of protease inhibitors showed no difference in the labeling pattern. Electrophoresis in the absence of reductant showed that the affinity-labeled species are not derived from larger disulfide linked components. The apparent molecular weight of the two labeled species was not affected by a 100-fold variation in cross-linker concentration, and the labeling of both species increased in parallel. Possible relationships between the two labeled species are discussed. PMID- 3003075 TI - ATP hydrolysis by the gelsolin-actin complex and at the pointed ends of gelsolin capped filaments. AB - To obtain kinetic information about the pointed ends of actin filaments, experiments were carried out in the presence of gelsolin which blocks all events at the kinetically dominant barbed ends. The 1:2 gelsolin-actin complex retains 1 mol/mol of actin-bound ATP, but it neither hydrolyzes the ATP nor exchanges it with ATP free in solution at a significant rate. On the other hand, the actin filaments with their barbed ends capped with gelsolin hydrolyze ATP relatively rapidly at steady state, apparently as a result of the continued interaction of ATP-G-actin with the pointed ends of the filaments. ATP hydrolysis during spontaneous polymerization of actin in the presence of relatively high concentrations of gelsolin lags behind filament elongation so that filaments consisting of as much as 50% ATP-actin subunits are transiently formed. Probably for this reason, during polymerization the actin monomer concentration transiently reaches a concentration lower than the final steady-state critical concentration of the pointed end. At steady state, however, there is no evidence for an ATP cap at the pointed ends of gelsolin-capped filaments, which differs from the barbed ends which do have an ATP cap in the absence of gelsolin. As there is no reason presently to think that gelsolin has any effect on events at the pointed ends of filaments, the properties of the pointed ends deduced from these experiments with gelsolin-capped filaments are presumably equally applicable to the pointed ends of filaments in which the barbed ends are free. PMID- 3003076 TI - Characterization of the oligosaccharide alditols from ovarian cyst mucin glycoproteins of blood group A using high pressure liquid chromatography (HPLC) and high field 1H NMR spectroscopy. AB - A combination of reverse phase and normal phase high pressure liquid chromatography has been used to separate the reduced oligosaccharides produced by alkaline borohydride degradation of a blood group A ovarian cyst mucin glycoproteins. Fourteen compounds, ranging in size from a monosaccharide to a decasaccharide, have been isolated preparatively using a Zorbax C-18 reverse phase column eluted with water and a MicroPak AX-5 normal phase column eluted with aqueous acetonitrile. The purity of the products and their structures were determined from the fully assigned high field proton NMR spectra. The resonances of exchangeable amide protons, observed by the Redfield selective pulse sequence in H2O, were assigned by decoupling to the resonances of H2 of the 2-acetamido sugars. Nuclear Overhauser effects were used to establish the relationship of the anomeric protons and those of the aglycone. In exception to earlier proposals that nuclear Overhauser effect on irradiation of the anomeric proton should always be observed at the proton attached to the aglycone carbon, we find that for the linkage of GalNAcp(1----3)Gal, nuclear Overhauser effect on irradiation of the alpha-anomeric proton resonance is observed not at H3 but at H4 of galactose. A combination of NMR methods and enzymatic degradation was employed to determine the structures of 13 different oligosaccharides of which seven have not previously been reported. These oligosaccharides, which terminate with beta-Gal, alpha-Fuc, beta-GlcNAc, and alpha-GalNAc, account for 75% of the total glycoprotein carbohydrate, the remainder being isolated as a mixture of glycopeptides and a high molecular weight polysaccharide whose NMR spectrum implies a simple repeating subunit structure closely related to that of the oligosaccharides. PMID- 3003077 TI - Reduction of 2,4,6-trinitrobenzenesulfonate by glutathione reductase and the effect of NADP+ on the electron transfer. AB - Glutathione reductase has been found to catalyze an NAD(P)H-dependent electron transfer to 2,4,6-trinitrobenzenesulfonate (TNBS). In the presence of oxygen TNBS is not consumed in the reaction, but is rapidly reoxidized with concomitant production of hydrogen peroxide. Cytochrome c can replace oxygen as the final electron acceptor, indicating that a one-electron transfer takes place. The rate is slightly higher in the absence than in the presence of oxygen, ruling out superoxide anion as an obligatory intermediate in cytochrome c reduction. In the absence of oxygen (or cytochrome c), TNBS limits the reaction and accepts a total of four electrons. The TNBS-dependent NADPH (or NADH) oxidation is markedly stimulated by NADP+, and to a smaller extent also by NAD+. The TNBS-dependent reactions are inhibited by excess of NADPH but not by NADH. The kinetics of these reactions are consistent with a branching reaction mechanism in which a pathway including a ternary complex between the two-electron reduced enzyme and NADP+ has the highest turnover. NADPH-dependent reductions of ferricyanide or 2,6 dichloroindophenol catalyzed by glutathione reductase are also markedly influenced by NADP+. Evidently NADP+ facilitates a shift of the catalyzed reaction from the normal two-electron reduction of glutathione disulfide to a more unspecific one-electron reduction of other acceptors. Spectral as well as kinetic data suggest that the rate of radical formation limits the reactions with the artificial electron acceptors and that NADP+ promotes this rate-limiting step. PMID- 3003078 TI - Inhibition of erythrocyte superoxide dismutase by diethyldithiocarbamate also results in oxyhemoglobin-catalyzed glutathione depletion and methemoglobin production. AB - N,N-Diethyldithiocarbamate (DDC), a copper-chelating agent, not only inhibits superoxide dismutase activity in the red cell, but also depletes glutathione and promotes the production of methemoglobin, sulfhemoglobin, and small amounts of lipid peroxidation products. DDC reacts with oxyhemoglobin to yield disulfiram, hydrogen peroxide, and methemoglobin. Disulfiram and hydrogen peroxide both convert GSH to GSSG, while DDC reduces methemoglobin to oxyhemoglobin. Although disulfiram also reacts with the hemoglobin sulfhydryl groups, this reaction does not play a role in the conversion of GSH to GSSG. Other hemoglobin derivatives, ferrous, and ferric ions do not catalyze the oxidation of GSH by DDC. These results support the conclusion that DDC reacts with the super-oxo-ferriheme complex of oxyhemoglobin to generate hydrogen peroxide and disulfiram and that the cyclic conversion of oxyhemoglobin to methemoglobin and DDC and disulfiram results in the net oxidation of GSH. Thus, damage to DDC-treated erythrocytes exposed to a putative superoxide-generating toxin, such as 1,4-naphthoquinone-2 sulfonate, may actually be due to diminished GSH concentration and hemoglobin oxidation rather than to superoxide radicals. Glucose added to the incubation medium of DDC-treated erythrocytes fully prevented glutathione depletion but not the oxidation of oxyhemoglobin to methemoglobin. Several other copper-chelating agents either failed to inhibit the activity of purified superoxide dismutase or when incubated with erythrocytes produced more extensive GSH depletion and hemoglobin oxidation than DDC. It is concluded that the interpretation of results with erythrocytes exposed to copper-chelating agents must consider their effects on GSH and hemoglobin as well as on superoxide dismutase inhibition. Moreover, one must be mindful of the interference by DDC in the analysis of GSH with 5,5' dithiobis-(2-nitrobenzoic acid) in the absence of sufficient quantities of metaphosphoric acid to destroy DDC and that contamination of DDC with trace quantities of disulfiram may be a significant problem. PMID- 3003079 TI - One- and two-electron oxidation of reduced glutathione by peroxidases. AB - The oxidation of glutathione by horseradish peroxidase forms a thiyl free radical as demonstrated with the spin trapping ESR technique. Reactions of this thiyl free radical result in oxygen consumption, which is inhibited by the radical trap 5,5-dimethyl-1-pyrroline-N-oxide. In contrast to L-cysteine oxidation, glutathione oxidation is highly hydrogen peroxide-dependent. The oxidation of glutathione by glutathione peroxidase forms glutathione disulfide without forming a thiyl radical intermediate, except in the presence of the thiyl radical generating horseradish peroxidase. PMID- 3003080 TI - Phosphorylation of prolactin. AB - Rat prolactin exhibits microheterogeneity when examined in electrophoretic systems, running as three isoforms having the same molecular weight but different net charges (prolactins 1, 2, and 3 with isoform 3 being the most acidic). As there is precedent for the phosphorylation of a pituitary hormone and phosphorylation is a common cause of microheterogeneity, we examined the possibility that rat prolactin existed in differentially phosphorylated forms. The investigation included examinations of rat prolactin phosphorylation both in vitro and in vivo. For the in vitro studies, purified rat prolactin was incubated with [gamma-32P]ATP and low levels of each of five purified protein kinases. Phosphorylated rat prolactin was identified by autoradiography of silver-stained one- and two-dimensional gels. For the in vivo studies, rat anterior pituitary cells in primary culture were incubated in the presence of H3 32PO4 for 2 or 12 h, after which time the proteins were extracted from the cells, cold acetone precipitated, or immunoprecipitated and run on two-dimensional gels. We report the in vitro phosphorylation of rat prolactin by cAMP-dependent protein kinase, casein kinase I, protease-activated kinase I, and the calcium/phospholipid dependent kinase, that phosphorylation with these kinases results in phosphate incorporation only into isoforms 2 and 3, and the phosphorylation of prolactin in rat pituitary cells in primary culture. PMID- 3003081 TI - Steady-state and transient-state kinetic studies on the oxidation of 3,4 dimethoxybenzyl alcohol catalyzed by the ligninase of Phanerocheate chrysosporium Burds. AB - Catalysis of the H2O2-dependent oxidation of 3,4-dimethoxybenzyl (veratryl) alcohol by the hemoprotein ligninase isolated from wood-decaying fungus, Phanerochaete chrysosporium Burds, is characterized. The reaction yields veratraldehyde and exhibits a stoichiometry of one H2O2 consumed per aldehyde formed. Ping-pong steady-state kinetics are observed for H2O2 (KM = 29 microM) and veratryl alcohol (KM = 72 microM) at pH 3.5. The magnitude of the turnover number varies from 2 to 3 s-1 at this pH, depending on the preparation of the enzyme. Each preparation of enzyme consists of a mixture of active and inactive enzyme. Extensive steady-state kinetic studies of several different preparations of enzyme, suggest a mechanism in which H2O2 reacts with enzyme to form an intermediate that subsequently reacts with the alcohol to return the enzyme to the resting state. The pH dependence of the overall reaction indicates that an ionization occurs having an apparent pK alpha approximately 3.1. The activity is, thus, nearly zero at pH 5 and increases to a maximum near pH approximately 2. However, the enzyme is unstable at this low pH. Transient-state kinetic studies reveal that, upon reaction of ligninase with H2O2, spectral changes occur in the Soret region, which, by analogy to previous studies of horseradish peroxidase, are consistent with formation of Compounds I and II. The active form of the enzyme appears to react rapidly with H2O2; we observed a positive correlation between the turnover number of the enzyme preparation and the extent of a rapid reaction between H2O2 and ligninase to form Compound I. Free radical cations derived from veratryl alcohol do not appear to be released from the enzyme during catalysis; however, other substrates are known to be converted to cation radicals (Kersten, P., Tien, M., Kalyanaraman, B., and Kirk, T.K. (1985) J. Biol. Chem. 260, 2609-2612). Our results are generally consistent with a classical peroxidase mechanism for the action of ligninase on lignin-like substrates. PMID- 3003082 TI - Uncoupling the red cell sodium pump by proteolysis. AB - In situ proteolysis of Na,K-ATPase was studied using inside-out red cell membrane vesicles. Proteolysis of the enzyme in its "E1" conformation with either trypsin or chymotrypsin inactivated cation translocation more than ATP hydrolysis. This was evident both in the absence of intravesicular alkali cations when Na-ATPase was compared to ATP-dependent 22Na+ influx, and in the presence of K+ when Na+/K+ exchange was compared to (Na+ + K+)-activated ATPase. This differential loss in pump versus hydrolysis was observed also when the activities of only intact, non leaky vesicles were compared and therefore reflects intramolecular uncoupling rather than nonspecific leakage. Although oligomycin and thimerosal, like trypsin and chymotrypsin, inhibit the enzyme's conformational step(s), neither effect uncoupling. It is concluded that specific cleavage(s) of Na,K-ATPase, at least as it exists in situ, alters the reaction sequence with respect to the normal ordered mechanism. Accordingly, cytoplasmic Na+ and extracellular K+ bind to the enzyme, stimulate phosphorylation (ATP + E1----E1P + ADP) and dephosphorylation (E2P----E2 + Pi), respectively, but each is then released to the same side from which it had bound; presumably release occurs prior to the conformational transitions of E1P to E2P and E2 to E1. This conclusion is supported by experiments showing that, ar micromolar ATP concentration, the hydrolytic activity (Na-ATPase) of the trypsinized but not the unmodified enzyme is stimulated by K+, consistent with earlier experiments (Hegyvary, C., and Post, R. L. (1971) J. Biol. Chem. 246, 5234-5240) showing that the K X E2 to K X E1 transition is slower than the E2 to E1 transition. PMID- 3003084 TI - Nucleotide sequence of the Escherichia coli dnaJ gene and purification of the gene product. AB - The dnaJ and dnaK genes are essential for replication of Escherichia coli DNA, and they constitute an operon, dnaJ being downstream from dnaK. The amount of the dnaJ protein in E. coli is substantially less than that of the dnaK protein, which is produced abundantly. In order to construct a system that over-produces the dnaJ protein, we started our study by determining the DNA sequence of the entire dnaJ gene, and an operon fusion was constructed by inserting the gene downstream of the lambda PL promoter of an expression vector plasmid, pPL-lambda. Cells containing the recombinant plasmid produced dnaJ protein amounting to 2% of the total cellular protein when cells were induced. The overproduced protein was purified, and Edman degradation of the protein indicated that the NH2-terminal methionine was found to be processed. From the DNA sequence of the dnaJ gene, the processed gene product is composed of 375 amino acid residues, and its molecular weight is calculated to be 40,975. PMID- 3003083 TI - Inhibition of homogeneous human eosinophil arylsulfatase B by sulfidopeptide leukotrienes. AB - Arylsulfatase B, purified to homogeneity from human eosinophils, is a tetrameric enzyme whose activity varied in accordance with the state of association of its monomeric subunits. The rate of dissociation of oligomeric forms was slow relative to the rate of the enzymatic reaction so that the kinetic properties of the enzyme depended on the concentration of the enzyme before assay. For concentrated enzyme solutions (14 micrograms/ml), Lineweaver-Burk analysis demonstrated substrate inhibition at greater than or equal to 20 mM substrate and revealed two distinct regions of activity at low and intermediate substrate concentrations. The addition of bovine serum albumin (60 micrograms/ml) or sucrose (0.25 M), which prevent subunit dissociation, yielded a linear relationship on Lineweaver-Burk analysis at non-inhibitory substrate concentrations. For dilute enzyme concentrations (4.7 micrograms/ml), inhibition occurred at greater than or equal to 2 mM substrate. Nanomolar amounts of leukotriene C4 (LTC4), relative to millimolar concentrations of substrate, inhibited eosinophil arylsulfatase B. On Lineweaver-Burk analysis, the pattern of inhibition of LTC4 with concentrated enzyme was compatible with competitive inhibition of only one oligomeric form of the enzyme, whereas at low enzyme concentrations the pattern of inhibition was apparently competitive. These findings suggest that LTC4 is a potent competitive inhibitor of a dissociated, possibly dimeric, form of the enzyme. Nanomolar concentrations of LTC4, leukotriene D4, and leukotriene E4 were equally inhibitory, whereas leukotriene B4 and isomeric 5,12-dihydroxyeicosatetraenoic acids had no inhibitory activity, indicating a requirement for a thiopeptide at C-6. Thiopeptide leukotriene analogs without an intact triene structure also lacked inhibitory activity. Sulfoxide analogs of LTC4 and leukotriene D4 were potent inhibitors, although two sulfone analogs of leukotriene D4 were not inhibitory. Arylsulfatase B did not inactivate the spasmogenic activity of sulfidopeptide leukotrienes. These findings indicate that sulfidopeptide leukotrienes and their sulfoxide derivatives may regulate by competitive inhibition the activity of oligomeric forms of the eosinophil lysosomal hydrolase, arylsulfatase B. PMID- 3003086 TI - Evidence for the existence of multiple alpha 1-acid glycoprotein genes in the mouse. AB - Three alpha 1-acid glycoprotein (AGP) cDNA clones have been isolated from a mouse liver library. Restriction enzyme mapping and sequencing of these cDNAs have shown that two, pMAGP2 and pMAGP3, are virtually identical, whereas the third, pMAGP4, differs significantly both in sequence and restriction enzyme sites. The sequences are 91% identical and differ from each other by single base differences exclusively. No frameshifts are observed, and no termination codons are generated by the single base differences. We interpret these data to indicate that there are at least two distinct AGP genes in the mouse and that two species of AGP mRNA are formed by the transcription of these genes in the liver. Based on the large number of single amino acid substitutions previously observed in human AGP (Schmid, K., Kaufmann, H., Isemura, S., Bauer, F., Emura, J., Motoyama, T., Ishiguro, M., and Nanno, S. (1973) Biochemistry 12, 2711-2724), we propose that at least two functional AGP genes may also exist in humans. PMID- 3003085 TI - The nucleotide sequence of the Escherichia coli K12 dnaJ+ gene. A gene that encodes a heat shock protein. AB - The Escherichia coli dnaJ gene product is required for bacteriophage lambda DNA replication at all temperatures. It is also essential for bacterial viability in at least some conditions, since mutations in it result in temperature-sensitive bacterial growth. We have previously cloned the dnaJ gene and shown that its product migrates as a Mr 37,000 polypeptide under denaturing conditions. Here we present the primary DNA sequence of the dnaJ gene. It codes for a processed basic protein (63 basic and 51 acidic amino acids) composed of 375 amino acids totaling Mr 40,973. The predicted NH2-terminal amino acid sequence, overall amino acid composition, and isoelectric point agree well with those of the purified protein. We present evidence that the rate of expression of the dnaJ protein is increased by heat shock under the control of the htpR (rpoH) gene product. PMID- 3003087 TI - Laminin increases the release of type IV collagenase from malignant cells. AB - We have studied the effect of laminin on type IV collagenolytic activity elaborated by malignant cells in culture. Laminin (at concentrations of 4-8 micrograms/ml) added to serum-free culture supernatants of subconfluent A2058 human melanoma cells significantly increased the release of the type IV collagenolytic activity (200-300%). The induction of type IV collagenase was more pronounced (580%) using a fragment of laminin which binds to the cell surface laminin receptor. A monoclonal antibody against the human laminin receptor blocked the effect of laminin on type IV collagenase, suggesting that occupation of the laminin receptor may be necessary for the effect. Increase in the type IV collagenolytic activity mediated by laminin was also demonstrated in two other malignant cell lines, HT fibrosarcoma (168%) and mouse melanoma (B16-F10) (271%). The increase in type IV collagenase was found to be specific for laminin because another cell-binding matrix protein, fibronectin, did not have any effect, and epidermal growth factor and transferrin actually decreased the type IV collagenase in human melanoma culture medium (epidermal growth factor, 50% at 20 ng/ml; and transferrin, 20% at 10 micrograms/ml). These studies suggest that tumor cell binding to laminin, which comprises the first step of basement membrane invasion, will induce the second step, namely the collagenolytic dissolution of the basement membrane. PMID- 3003089 TI - The induction of stress-related proteins by lead. AB - Differential inductive effects of lead on protein synthesis in rat fibroblasts and kidney epithelial cells were examined. The lead was administered as lead glutamate, a complex known to introduce lead into cells. Lead exposure induced the synthesis of three proteins which constitute two separate stress protein subgroups. Two of these proteins have been previously identified as the glucose regulated proteins because their synthesis is induced by reagents which perturb glucose utilization. The third protein is inducible by several sulfhydryl-binding reagents including lead. This third protein has been compared with a protein, p32/6.3, of very similar size and isoelectric point, which has been associated with lead-induced intranuclear inclusion bodies. However, several features, including one-dimensional peptide maps, indicated that the third protein and p32/6.3 are not identical. The three lead-induced proteins are distinguished from the major group of stress proteins by their relative insensitivity to heat stress. Lead glutamate, on the other hand, induces neither the heat shock protein 70 nor metallothionein, both of which are strongly induced by several metals including cadmium, zinc, and mercury. PMID- 3003088 TI - Purification and characterization of heparin-binding endothelial cell growth factors. AB - Thirteen endothelial cell growth factors have been purified to homogeneity by heparin affinity and reversed-phase high performance liquid chromatography, and their chromatographic and electrophoretic properties were compared. The amino acid compositions of 10 of these mitogens have also been determined. The results indicate that these heparin-binding growth factors (HBGFs) can be subdivided into two classes. Class 1 HBGFs are anionic mitogens of molecular weight 15,000-17,000 found in high levels in neural tissue and include acidic brain fibroblast growth factor and retina-derived growth factor. Class 2 HBGFs are cationic mitogens of molecular weight 18,000-20,000 found in a variety of normal tissues and are typified by pituitary fibroblast growth factor and cartilage-derived growth factor. Typical class 2 HBGFs have also been isolated from a rat chondrosarcoma, a human melanoma, and a human hepatoma, suggesting that tumors do not make a structurally distinct HBGF class. These results provide a sound basis for the evaluation of the HBGFs purified from a variety of tissues and species and for the delineation of their normal and pathological functions in vivo. PMID- 3003090 TI - The stereochemical course of phosphoryl transfer catalyzed by herpes simplex virus type I-induced thymidine kinase. AB - Herpes simplex virus type I (HSV-I)-induced thymidine kinase has been shown to catalyze phosphoryl transfer from adenosine 5'-[gamma-(S) 16O,17O,18O]triphosphate to thymidine with inversion of configuration at phosphorus. The simplest interpretation of this result is that phosphoryl transfer occurs by a single in-line group transfer between ATP and thymidine within the ternary enzyme complex. PMID- 3003091 TI - Biochemical and cross-resistance studies with HeLa cell mutants resistant to cardiac glycoside SC4453. Regulation of the resistant form of Na+/K+-ATPase in the mutant cells. AB - In HeLa cells, stable mutants which are between 25-to about 200-fold resistant to the cardiac glycoside derivative SC4453 (a digoxin analog which contains a pyridazine ring in place of a lactone ring in the C-17 position) have been isolated after a single step selection in the presence of the drug. Based on their cross-resistance pattern towards various cardiac glycosides, the mutants resistant to SC4453 (SCR mutants) appear to be of two different kinds and they differ from the two classes of ouabain-resistant mutants described previously (Gupta, R. S., and Chopra, A. (1985) J. Biol. Chem. 260, 6843-6850). One type of SCR mutants (designated as group C) exhibit a high degree of cross-resistance to all cardiac glycosides and their genins (viz. ouabain, digitoxin, digoxin, digoxigenin, convallatoxin, gitoxin, strophanthidin, and bufalin). In contrast, the second type of SCR mutant (group D) exhibit considerable resistance to only SC4453, digoxin, and digoxigenin, but showed very little or no cross-resistance to the other cardiac glycosides examined. The cross-resistance of the mutants towards cardiac glycosides was highly specific as they exhibited no cross resistance towards a large number of other structurally and functionally related compounds (viz. ethacrynic acid, sanguinarine nitrate, penicillic acid, methyl quinolizinum bromide, 5,5'-diphenylhydantoin, deoxycorticosterone, vanadium pentoxide, and adriamycin). The cellular uptake of 86Rb in the mutant cells was found to be resistant to specific cardiac glycosides. Studies on the sensitivity of plasma membrane Na+/K+-ATPase to cardiac glycosides show that about 10-15% of the enzymic activity in the mutant cells was highly resistant to inhibition by the specific drugs to which the mutants exhibit increased resistance. Very interestingly, when the mutant cells are grown in cardiac glycoside-containing medium, the resistant form of the enzyme accounts for about 50-60% of the total enzyme. These results show that both classes of SCR mutants are affected in Na+/K+-ATPase and that the amount of the resistant enzyme in the mutant cells is regulated in response to cardiac glycosides. PMID- 3003092 TI - Mitochondrial location of rat liver dinucleoside triphosphatase. AB - Rat liver dinucleoside triphosphatase (EC 3.6.1.29) is associated with sucrose gradient purified mitochondria and can be extracted by freeze and thaw treatment. The proportion of mitochondrial dinucleoside triphosphatase approaches 50% of total liver enzyme. Evidence is also presented that 10% of total liver bis(5' guanosyl)tetraphosphatase (EC 3.6.1.17) might be equally linked to mitochondria. Those data suggest that diadenosine 5',5'''-P1,P3-triphosphate, diadenosine 5',5'''-P1,P4-tetraphosphate, or other substrates of those enzymes, might be somehow related to mitochondria or mitochondrial function(s), although the occurrence of dinucleoside polyphosphates has not been reported in that organelle. PMID- 3003093 TI - Ligand binding to (Na,K)-ATPase labeled with 5-iodoacetamidofluorescein. AB - The equilibrium binding of sodium, potassium, and adenine nucleotides to dog kidney (Na,K)-ATPase was studied by measuring changes in the fluorescence of enzyme labeled with 5-iodoacetamidofluorescein (5-IAF). The intensity of the fluorescence emission at 520 nm of the bound fluorescein (excited at 490 nm) is increased by ATP, adenyl-5'-yl imidodiphosphate (AMP-PNP), ADP (but not AMP), and Na+, and decreased by K+, Rb+, NH+4, and LI+. Thus the fluorescence effects correlate with the ability of these groups of ligands to stabilize E1 and E2 conformations, respectively. The Na+-induced increase in fluorescence has two components: a slow, high-affinity increase of approximately 7% (K0.5 = 0.16 mM) with positive cooperativity; and a large (approximately 15%), rapid, low-affinity (K0.5 = 34 mM) increase that is not cooperative. The K0.5 for the high-affinity effect is decreased by oligomycin and increased by K+. ATP effects on the fluorescence follow Michaelis-Menten kinetics and are of high affinity (K0.5 = 0.12 microM); K+ increases the K0.5 for ATP, AMP-PNP, and ADP but does not induce cooperative behavior. K+ itself decreases the fluorescence signal by about 9%, with high affinity (K0.5 = 5 microM), showing Michaelis-menten behavior in the absence of other ligands, while with ATP, Na+, or Mg2+ present, K+ effects are cooperative and of lower affinity. PMID- 3003095 TI - Evolution of the fibronectin gene. Exon structure of cell attachment domain. AB - Genomic DNA coding for human fibronectin was identified from a human genomic library by screening with a cDNA clone that specifies the cell attachment domain in human fibronectin. Two clones which together provided more than 22 kilobase pairs of the fibronectin gene were isolated. The exons in this region correspond to approximately 40% of the coding region in the fibronectin gene. They code for the middle region of the polypeptide which consists of homologous repeating segments of about 90 amino acids called type III homologies. Nucleotide sequence of the portion of the gene corresponding to the cell attachment domain showed that the Arg-Gly-Asp-Ser cell attachment site is encoded within a 165-base pair exon. This exon, together with a 117-base pair exon codes for a homology unit. Analysis of the exon/intron organization in some of the neighboring homology units indicated a similar 2-exon structure. An exception to this pattern is that a single large exon codes for a type III homology unit that, due to alternative mRNA splicing, exists in some but not all fibronectin polypeptides. The introns separating the coding sequences for the type III homology units are located in conserved positions whereas the introns that interrupt the coding sequence within the units are in a variable position generating variations in the size of the homologous exons. This exon/intron organization suggests that the type III homology region of the fibronectin gene has evolved by a series of gene duplications of a primordial gene consisting of two exons. Specification of one of these homology units to the cell attachment domain has occurred within this exon/intron arrangement. PMID- 3003094 TI - 5-Iodoacetamidofluorescein-labeled (Na,K)-ATPase. Steady-state fluorescence during turnover. AB - The fluorescence of (Na,K)-ATPase labeled with 5-iodoacetamidofluorescein was studied under turnover conditions. At 4 degrees C the hydrolysis of ATP is slowed sufficiently to permit study of the effects of Na+, K+, and ATP on the steady state intermediates. With Na+ and Mg2+ (Na-ATPase conditions), addition of ATP produces a 7% drop in signal that reverts back to the initial, high fluorescence after a steady state of several minutes. K-sensitive phosphoenzyme is formed under these conditions, indicating that the fluorescence signal during the steady state is associated with E2P. Under (Na,K)-ATPase conditions (Na+, K+, Mg2+), micromolar ATP produces a steady-state signal that is 25% lower than the initial fluorescence, with no detectable phosphoenzyme formed. This low-fluorescence intermediate, which is also formed by adding K+ to enzyme in the Na-ATPase steady state described above, resembles the state produced by adding K+ directly to enzyme under equilibrium conditions, i.e. E2K. The K0.5(K+) for the fluorescence decrease and for keeping the enzyme dephosphorylated are nearly identical, indicating that the fluorescence change accompanies K+-dependent dephosphorylation. High ATP increases the steady-state fluorescence during the (Na,K)-ATPase reaction; while oligomycin produces still another steady-state fluorescent intermediate. These last two intermediates may be associated with the formation of E2P and E1P, respectively. PMID- 3003096 TI - Apparent translocation of glucose transport activity in rat epididymal adipocytes by insulin-like effects of high pH or hyperosmolarity. AB - The basal and plus insulin states of glucose transport activity in adipocytes are known to show different responses to changes in the pH or osmolarity of the incubation mixture. When the pH was raised from 7 to 8, the basal glucose transport activity (assessed from the rate of 3-O-methyl-D-glucose uptake) was increased approximately 3-fold while the plus insulin activity remained virtually unaffected. Likewise, when cells were exposed to 300 mM sorbitol, the basal glucose transport activity, but not the plus insulin activity, was considerably increased. In both cases, the change in the transport activity was ATP-dependent and was completed in approximately 60 min. The increase in the cellular glucose transport activity was accompanied, in both cases, by an increase in the glucose transport activity in the plasma membrane fraction and a decrease in the activity in the high-speed pellet fraction. The transport activity in the subcellular fractions was determined after reconstitution into egg lecithin liposomes. Both isotonic buffer at pH 8.0 and hypertonic buffer at pH 7.4 significantly stimulated membrane-bound cAMP phosphodiesterase in adipocytes. It is concluded that the above two experimental conditions may induce insulin-like effects in fat cells and may facilitate translocation of the glucose transport activity from an intracellular site to the plasma membrane. PMID- 3003097 TI - Hormone-stimulated polyphosphoinositide breakdown in rat liver plasma membranes. Roles of guanine nucleotides and calcium. AB - Calcium-sensitive inositide release in a purified rat liver plasma membrane preparation is increased by calcium-mobilizing hormones in the presence of guanine nucleotides. Vasopressin-stimulated inositide release is evident in the presence of GTP or its nonhydrolyzable analogs guanyl-5'-yl imidodiphosphate and guanosine 5'-(3-O-thio)triphosphate (GTP gamma S). The stimulation of inositide release by (-)-epinephrine (alpha 1), angiotensin II, or vasopressin in the presence of either 1 microM or 10 microM GTP gamma S correlates with the number of receptors present for each hormone. The guanine nucleotide and hormonal stimulation is evident on both inositol trisphosphate production and phosphatidylinositol bisphosphate degradation. Ethylene glycol bis(beta aminoethyl ether)-N,N,N',N'-tetraacetic acid (1 mM) completely abolishes stimulation by guanine nucleotides and hormone. Prior treatment of plasma membranes with cholera toxin or islet activating protein or prior injection of animals with islet activating protein does not affect stimulation of inositide release by GTP gamma S or GTP gamma S plus vasopressin. Stimulation by GTP gamma S is dependent upon magnesium and is inhibitable by guanosine 5'-(2-O-thio) diphosphate. Inositide release from the plasma membrane exhibits half-maximal stimulation by calcium at approximately 100 nM free calcium in the presence of 1.5 mM MgCl2 and at approximately 10 microM free calcium in the presence of 10 mM MgCl2. Addition of guanine nucleotides decreases the requirement for calcium and also increases the activity at saturating calcium. The results presented suggest that calcium-mobilizing hormones stimulate polyphosphoinositide breakdown in rat liver plasma membranes through a novel guanine nucleotide binding protein. PMID- 3003098 TI - Binding of 17O-labeled substrate and inhibitors to protocatechuate 4,5 dioxygenase-nitrosyl complex. Evidence for direct substrate binding to the active site Fe2+ of extradiol dioxygenases. AB - Pseudomonas testosteroni protocatechuate 4,5-dioxygenase catalyzes extradiol-type oxygenolytic cleavage of the aromatic ring of its substrate. The essential active site Fe2+ binds nitric oxide (NO) to produce an EPR active complex with an electronic spin of S = 3/2. Hyperfine broadening of the EPR resonances of the nitrosyl complex of the enzyme by protocatechuate (3,4-(OH)2-benzoate, PCA) enriched specifically with 17O (I = 5/2) in either the 3 or the 4 hydroxyl group shows that both groups can bind directly to the Fe2+ in the ternary complex. Analogous results are obtained for PCA binding to catechol 2,3-dioxygenase-NO complex suggesting that substrate binding by the Fe2+ may be a general property of extradiol dioxygenases. The protocatechuate 4,5-dioxygenase inhibitor, 4-17OH benzoate binds directly to the Fe of the nitrosyl adduct of the enzyme through the OH group. Since previous studies have shown that water also is bound to the Fe in this ternary complex, but not in the ternary complex with PCA, the data strongly imply that there are 3 sites in the Fe coordination which can be occupied by exogenous ligands. 3-17OH-benzoate is an inhibitor of the enzyme but does not elicit detectable hyperfine broadening in the EPR spectrum of the nitrosyl adduct suggesting that it binds to the enzyme, but not to the Fe. The EPR spectra of ternary enzyme-NO complexes with PCA or 4-OH-benzoate labeled with 17O exclusively in the carboxylate substituent are not broadened, suggesting that this moiety does not bind to the Fe. PMID- 3003099 TI - The cellular mechanism of glucocorticoid acceleration of fetal lung maturation. Fibroblast-pneumonocyte factor stimulates choline-phosphate cytidylyltransferase activity. AB - The cellular mechanism by which glucocorticoids stimulate phosphatidylcholine biosynthesis has been studied in the fetal rat lung in vivo and in cultured fetal rat lung cells of varying levels of complexity. Administration of dexamethasone to pregnant rats at 18 days gestation resulted in a significant increase in saturated phosphatidylcholine content in fetal lung 24 h after injection. Dexamethasone administration increased the activity of fetal lung choline phosphate cytidylyltransferase by 34%. It had no effect on the activities of fetal lung choline kinase and choline phosphotransferase. Exposure of fetal lung type II cells in organotypic cultures (which contain both type II cells and fibroblasts) to cortisol resulted in a 1.6-fold increase in the incorporation of [Me-3H]choline into saturated phosphatidylcholine. The activities of the enzymes in the choline pathway for the de novo biosynthesis of phosphatidylcholine were not significantly altered except for a 105% increase in choline-phosphate cytidylyltransferase activity. Treatment of monolayer cultures of fetal type II cells with cortisol-conditioned medium from fetal lung fibroblasts resulted in a 1.5-fold increase in saturated phosphatidylcholine production. This effect correlated with a doubling of choline-phosphate cytidylyltransferase activity. Additional evidence that this stimulatory action is mediated by fibroblast pneumonocyte factor, produced by fetal lung fibroblasts in response to cortisol, was obtained. The factor was partially purified from cortisol-conditioned medium of fetal lung fibroblasts by gel filtration and affinity chromatography. Based on biological activity, a 3000-fold purification was obtained. Stimulation of saturated phosphatidylcholine synthesis in type II cells by fibroblast pneumonocyte factor was maximal within 60 min of incubation. Pulse-chase experiments indicated that the stimulatory effect was correlated with an increased conversion of choline phosphate into CDP choline. Moreover, the enhanced phosphatidylcholine formation by fetal type II cells in response to fibroblast-pneumonocyte factor was accompanied by decreased levels of cellular choline phosphate. These findings further support the concept that glucocorticoid action on surfactant-associated phosphatidylcholine synthesis occurs ultimately at the level of the alveolar type II cell and involves fibroblast-pneumonocyte factor which stimulates the activity of choline-phosphate cytidylyltransferase. PMID- 3003100 TI - HepG2. A human hepatoblastoma cell line exhibiting defects in bile acid synthesis and conjugation. AB - We used capillary gas chromatography/mass spectrometry to demonstrate that a cell line derived from a well differentiated human hepatoblastoma, HepG2, synthesized and secreted the following bile acids (ng/10(7) cells/h): chenodeoxycholic acid (131.4), cholic acid (3.3), 3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid (DHCA; 4.5), and 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26 oic acid (THCA; 32.0). Deuterium from [7 beta-2H]7 alpha-hydroxycholesterol, which was added to the media, was incorporated into newly synthesized chenodeoxycholic acid, DHCA, and THCA, but not into cholic acid. Since THCA is a known precursor of cholic acid, these data suggest that HepG2 is specifically deficient in the side chain cleavage that transforms THCA into cholic acid. Greater than 90% of the bile acids synthesized and secreted by HepG2 were unconjugated. Conjugation could not be stimulated by the addition of glycine or taurine to the media. Approximately 30% of newly synthesized DHCA and THCA were sulfated. Chenodeoxycholic acid and cholic acid were not appreciably sulfated. In summary, cultured HepG2 cells synthesize bile acid, but in a pattern distinct from that of adult human liver. This cell line may be a model for studying pathways of human bile acid synthesis, conjugation, and sulfation. PMID- 3003101 TI - A novel prefusion complex formed during protein transport between Golgi cisternae in a cell-free system. AB - Examination of a cell-free reconstitution of intercompartmental transport through the Golgi apparatus has enabled detection of two intermediates in the pathway (Balch, W. E., Glick, B. S., and Rothman, J. E. (1984) Cell 39, 525-536). These intermediates are thought to represent stages in the budding and fusion reactions of transport vesicles mediating such a transport process. Here we describe a new transport intermediate that is interposed between the previously established primed donor formation and the N-ethylmaleimide (NEM)-resistant acceptor intermediates. Consumption of this intermediate requires much less cytosol than its formation, and thus it has been termed the "dilution-resistant" intermediate. The dilution-resistant intermediate only forms in the presence of donor and acceptor membranes, and its consumption is sensitive to NEM. The transition from this state to the later, NEM-resistant form of the prefusion complex requires ATP as well as cytosol and may represent a processing of transport vesicles to permit their fusion. PMID- 3003102 TI - Multiple cytosolic components promote intra-Golgi protein transport. Resolution of a protein acting at a late stage, prior to membrane fusion. AB - Cytosolic components are required to produce the "primed donor" and to consume the "dilution-resistant" intermediates of the intercompartmental protein transport pathway as elucidated in a cell-free system (Balch, W. E., Glick, B. S., and Rothman, J. E. (1984) Cell 39, 525-536, and Wattenberg, B. W., Balch, W. E., and Rothman, J. E. (1986) J. Biol. Chem. 261, 2202-2207). Widely different levels of crude cytosol are required for each of these steps, suggesting that different cytosolic components might mediate each step. Here, we fractionate cytosol and demonstrate that there are multiple transport-active components. Furthermore, we report the development of stage-specific functional assays which reveal that a distinct soluble component is required in the consumption of the dilution-resistant intermediate. This component, of about 25 kilodaltons in its apparent native molecular mass, is derived from calf brain cytosol. While this component mediates the consumption of the dilution-resistant intermediate, it is inactive in the priming stage. This stage-specific component seems likely to be involved in the processing of transport vesicles after the attachment of those vesicles to the target membranes. PMID- 3003104 TI - Polypeptide sequences involved in the cleavage of DNA by the restriction endonuclease EcoRI. AB - We have prepared a variety of fragments of the restriction endonuclease EcoRI by partial or total CNBr or acid cleavage of the protein. These fragments were isolated by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. They were analyzed in a qualitative manner for phosphodiesterase activity. Antibodies against these fragments were elicited in rats and tested for binding to native EcoRI in an enzyme-linked immunoassay. We conclude from these experiments that the DNA binding site of EcoRI is located in the COOH-terminal half of the molecule, close to and probably comprising amino acid residues 137 to 157. This conclusion is reinforced by the observation that this sequence shows homology to the sequences of the recognition helix of other gene-regulatory proteins. PMID- 3003103 TI - Biosynthesis and intracellular transport of rat liver 5'-nucleotidase. AB - To investigate biosynthesis and intracellular transport of 5'-nucleotidase, we purified this enzyme from rat liver and prepared antibodies. In immunoblot analysis, 5'-nucleotidase from the plasmalemmal, Golgi, and tritosomal fractions migrated as a single band of 72 kDa. The immunoreactive 68-kDa band was detected in the rough microsomal fraction and only the 72-kDa polypeptide contained sialic acids. The single polypeptide of 61 kDa immunoprecipitated from the translation products with the membrane-bound polysomal RNAs by the cell-free system converted to the 68-kDa form associated with membranes, when translated in the presence of microsomal vesicles. 5'-Nucleotidase from cultured primary hepatocytes pulse labeled with [35S]methionine for 20 min migrated as a 68-kDa polypeptide. Within 45 min of chase, the 68-kDa form was converted to the 72-kDa form. Upon treatment with tunicamycin, a new immunoreactive polypeptide of 59 kDa was obtained. These results suggest that 5'-nucleotidase is translated on the membrane-bound polysomes as a 61-kDa precursor and that the cleavage of the 2-kDa signal peptide and the core glycosylation co-translationally convert the precursor to the 68-kDa form, which is subsequently processed in the Golgi complex to the 72-kDa form. PMID- 3003105 TI - The use of circular dichroism to study conformational changes induced in Sendai virus envelope glycoproteins. A correlation with the viral fusogenic activity. AB - Fusion of Sendai virus envelopes with erythrocyte membranes or with phosphatidylcholine/cholesterol liposomes requires the presence of the two viral glycoproteins, namely the hemagglutinin/neuraminidase (HN) and the fusion (F) polypeptides. Membrane vesicles bearing only the HN or the F glycoprotein (HN or F vesicles) or a mixture of both do not possess fusogenic activity. These results clearly show that in order to be fusogenic, the two viral envelope glycoproteins must be present within the same membrane, thus indicating their mutual interaction. Circular dichroism studies revealed that the conformation of the viral glycoproteins in reconstituted viral envelopes or in HN-F vesicles (vesicles formed by co-reconstitution of the HN and F glycoproteins) is different from that of the conformation of these glycoproteins in either HN or F vesicles or in a mixture of both. It has been observed that the mean residue ellipticity as measured at 222 nm (theta 222) of the viral glycoproteins in reconstituted Sendai virus envelopes (RSVE) is lower by about 75% than the value observed for these glycoproteins in isolated HN or F vesicles. Treatment of RSVE or of HN-F vesicles with inhibitors of the viral fusogenic activity such as phenylmethylsulfonyl fluoride, proteolytic enzymes, or incubation at 70 degrees C caused a substantial conformational change in the viral glycoproteins. The theta 222 of unfusogenic RSVE or unfusogenic HN-F vesicles is very close to that observed for a mixture of HN and F vesicles. It is proposed here that in order to be fusogenic, the viral envelope glycoproteins must possess a certain conformation which exists only when they are present within the same membrane. PMID- 3003106 TI - Lipoprotein-28, a cytoplasmic membrane lipoprotein from Escherichia coli. Cloning, DNA sequence, and expression of its gene. AB - Escherichia coli contains several lipoproteins in addition to the major outer membrane lipoprotein (Ichihara, S., Hussain, M., and Mizushima, S. (1981) J. Biol. Chem. 256, 3125-3129). We cloned the gene for one of these new lipoproteins by using a synthetic 15-mer oligonucleotide probe identical to the DNA sequence at the signal peptide cleavage site of the major lipoprotein. The DNA sequence of the cloned gene revealed an open reading frame encoding a 272-amino acid protein with a signal peptide of 23 amino acid residues. The amino acid sequence of the putative cleavage site region of the signal peptide, -Leu-Leu-Ala-Gly-Cys-, is identical to that of the major lipoprotein. When the cloned gene was expressed in E. coli, a gene product with an apparent molecular weight of approximately 29,000 was identified which agrees well with the calculated molecular weight (27,800). The product was labeled with [3H]glycerol, and a precursor molecule of increased molecular weight was accumulated when cells were treated with globomycin, a specific inhibitor for prolipoprotein signal peptidase. We thus designed the gene product as lipoprotein-28. Unlike the major lipoprotein, lipoprotein-28 was found to be localized in the cytoplasmic membrane. A possible orientation of lipoprotein-28 in the E. coli envelope is discussed. PMID- 3003107 TI - Chronic lead intoxication causes a brain-specific nuclear protein to accumulate in the nuclei of cells lining kidney tubules. AB - A characteristic feature of chronic lead intoxication is the induction of intranuclear inclusion bodies in cells lining kidney proximal tubules. These are relatively insoluble lead- and protein-rich structures which may serve a protective function by sequestering lead. The most abundant protein of isolated inclusion bodies, p32/6.3, has been partially characterized by use of a monoclonal antibody. As predicted by biochemical analysis, p32/6.3 occurs in kidney only in conjunction with lead intoxication and inclusion body formation. It does not accumulate in other tissues as a result of lead exposure. Unexpectedly, p32/6.3 was found to be a constitutive protein of adult brain, occurring primarily in the cerebral cortex. Within this tissue, both neurons and astrocytes contained p32/6.3. The brain p32/6.3 was concentrated in the insoluble nuclear protein or matrix fraction, a localization reminiscent of the intranuclear inclusion bodies from lead-exposed kidney. Brain p32/6.3 was detected in rat, mouse, dog, man, and chicken. In rat brain, the appearance of p32/6.3 was developmentally regulated. Only traces were detected 3 days after birth but within 1-2 weeks adult levels were achieved. The presence in brain of a protein which is involved in a potentially protective response to lead suggests that the brain may also have such a protective mechanism. PMID- 3003108 TI - Effects of secA mutations on the synthesis and secretion of proteins in Escherichia coli. Evidence for a major export system for cell envelope proteins. AB - We have followed the synthesis and secretion of a number of periplasmic and outer membrane proteins in three strains of Escherichia coli, a secA amber mutant, a secA temperature-sensitive mutant, and a strain that blocks protein secretion due to a high level of expression of an export-defective hybrid protein between maltose-binding protein and beta-galactosidase (MalE-LacZ). Our results show that after several hours under nonpermissive conditions the specificity and extent of the export blocks in the secA temperature-sensitive mutant and the strain producing the MalE-LacZ hybrid protein are identical, affecting at least four major outer membrane proteins and most but not all periplasmic proteins. The secA gene product, therefore, appears to be an essential component of the major export pathway in E. coli which is used by many envelope proteins independent of whether they are cotranslationally or post-translationally secreted. In contrast, the synthesis of only a subset of these envelope proteins is reduced in the secA amber mutant after shift to the nonpermissive condition. These results indicate that the SecA protein serves roles both in the synthesis and the secretion of certain cell envelope proteins. PMID- 3003109 TI - alpha DNA in African green monkey cells is organized into extremely long tandem arrays. AB - We have determined the size of arrays formed by tandemly repeated monomers of alpha DNA in African green monkey cells. DNA fragments containing intact alpha DNA arrays were generated by digestion of genomic DNA with restriction endonuclease that do not have sites in the alpha DNA consensus sequence. Their size was determined by Southern analysis and by sedimentation through neutral sucrose gradients followed by probing of each fraction for alpha sequences. The restriction fragments varied in size with the most frequent being 78 kilobase pairs long. We have also shown that they contain very little non-alpha DNA sequences. This suggests a minimum array of 450 tandemly repeated alpha DNA monomers, which is more than an order of magnitude larger than previously supposed. PMID- 3003110 TI - Identification of a ternary complex between cAMP and a trimeric form of cAMP dependent protein kinase. AB - One of the intermediates involved in dissociation and reassociation of the subunits of the type II cAMP-dependent protein kinase has been characterized. This intermediate can be generated when the protein kinase is prepared from the isolated catalytic subunit (C) and the isolated regulatory subunit-[3H]cAMP complex (R2-[3H]cAMP4) by dialysis for 18 h followed by gel filtration. The intermediate, which could be separated from the holoenzyme and the isolated subunits by polyacrylamide gel electrophoresis, had an apparent molecular weight of 149,000, consistent with an R2C form. Following electrophoresis, measurements of R and bound nucleotide indicated that R2C was half-saturated with [3H]cAMP. The bound [3H]cAMP exhibited biphasic dissociation kinetics indicating that both types of cAMP binding sites were occupied. These findings suggested that the intermediate is R2C-cAMP2. This intermediate was not seen when the dialysis time was increased to 5 days, but could be observed when cAMP was added to the holoenzyme or when holoenzyme was mixed with R2cAMP4 and cAMP. The presence of two occupied cAMP binding sites on this intermediate suggests that there is minimal cooperativity between the two members of the regulatory subunit dimer, i.e. one member of the dimer binds 2 molecules of cAMP while the other binds C. PMID- 3003111 TI - Replication of kinetoplast DNA in isolated kinetoplasts from Crithidia fasciculata. Identification of minicircle DNA replication intermediates. AB - The kinetoplast DNA (kDNA) of trypanosomes is comprised of thousands of DNA minicircles and 20-50 maxicircles catenated into a single network. We show that kinetoplasts isolated from the trypanosomatid species Crithidia fasciculata incorporate labeled nucleotides and support minicircle DNA replication in a manner which mimics two characteristics of minicircle replication in vivo: 1) the minicircles are replicated as free molecules and subsequently reattached to the kDNA network, and 2) a replication intermediate having a structure consistent with a highly gapped minicircle species is generated. In addition, a class of minicircle DNA replication intermediates is observed containing discontinuities at specific sites within each of the newly synthesized DNA strands. By using a strain of C. fasciculata possessing nearly homogenous minicircles, we were able to map the discontinuities to two small regions situated 180 degrees apart on the minicircle. Each region has two sites at which a discontinuity can occur, one on each strand and separated by approximately 100 base pairs. These sites may represent origins of minicircle DNA replication. PMID- 3003113 TI - Ubiquitin-lysozyme conjugates. Purification and susceptibility to proteolysis. AB - To produce ubiquitinated substrates for studies on ATP-dependent proteolysis, 125I-lysozyme was incubated in hemin-inhibited rabbit reticulocyte lysates. A portion of the labeled molecules became linked to ubiquitin in large covalent complexes. When these were partially purified and returned to uninhibited lysates containing ATP, the conjugated lysozyme molecules were degraded 10 times faster than free lysozyme. Purification of covalently modified lysozyme from hemin inhibited lysates containing 125I-ubiquitin and 131I-lysozyme confirmed that both molecules were present in the complexes. The doubly labeled conjugates also permitted us to determine the fate of each molecule in uninhibited lysates. Besides degradation of lysozyme, there was a progressive release of intact lysozyme molecules from the complexes. This disassembly, which was the only fate of the complexes in the absence of ATP, proceeded through a series of smaller intermediates, several having molecular weights expected for ubiquitin-lysozyme conjugates, and eventually free lysozyme was regenerated. The behavior of labeled ubiquitin was similar, though not identical, to that of lysozyme. Even in lysates containing ATP ubiquitin emerged from the complex undegraded. Furthermore, ubiquitin was present in a greater number of species than was lysozyme. The demonstration that ubiquitin-lysozyme conjugates are rapidly degraded provides support for the hypothesis of Hershko, Rose, Ciechanover, and their colleagues that a key function of ubiquitin is to modify the proteolytic substrate. Further support for the hypothesis is presented in the following paper where we show that the conjugated lysozyme molecules are substrates for an ATP-dependent protease that does not degrade free lysozyme. PMID- 3003112 TI - Transient expression of type IV collagenolytic metalloproteinase by human mononuclear phagocytes. AB - A type IV collagenolytic metalloproteinase secreted by human monocytes/macrophages has been isolated and characterized. Monocytes isolated from peripheral blood and cultured in vitro exhibited a high type IV collagenolytic activity during the first and second day, but such activity declined markedly over subsequent days. Type IV collagenolytic activity was also transiently elaborated by macrophages isolated from (a) bronchioalveolar lavage of patients with pulmonary sarcoidosis, (b) primary human colostrum, and (c) peritoneal lavage of a patient with peritonitis. In contrast, macrophages isolated from the bronchioalveolar lavage of normal individuals, or from noninflammatory peritoneal fluids, failed to exhibit type IV collagenolytic activity. A type IV collagenolytic neutral proteinase was purified from macrophages isolated from inflammatory peritoneal fluid. The proteinase has a mass of 67 kDa on gel electrophoresis and is not altered in its migration under reducing conditions. It produces a characteristic 1/4-3/4 cleavage of type IV collagen, and its activity is abolished by treatment with EDTA but not phenylmethanesulfonyl fluoride. The isoelectric pH of the proteinase is 5.2 as judged by two-dimensional gel electrophoresis. The amino acid composition of the proteinase was notable for a high content of serine, glutamic acid, glycine, and alanine and no detectable hydroxyproline, cysteine, or methionine residues. The carbohydrate content of the proteinase was 11.2%, and galactose was the most abundant monosaccharide (8.7%) released following acid hydrolysis, followed by glucose (1.3%), mannose (1.2%), and trace amounts of fucose and galactosamine. Such a type IV collagenolytic protease may play an important role during the traversal of the vascular basement membrane by extravasating monocytes. The biochemical characteristics and biologic function of the macrophage proteinase may be similar or identical to the type IV collagenolytic proteinase identified in metastatic tumor cells. PMID- 3003114 TI - Ubiquitin-lysozyme conjugates. Identification and characterization of an ATP dependent protease from rabbit reticulocyte lysates. AB - Ubiquitin-lysozyme conjugates have been used as substrates to identify an ATP dependent protease from rabbit reticulocyte lysates. The enzyme, which has been partially purified by DEAE chromatography and glycerol gradient centrifugation, has an apparent molecular weight greater than 600,000 based on sedimentation and gel filtration. Whereas it degrades conjugated lysozyme molecules in the presence of ATP, the protease does not degrade free lysozyme molecules even upon addition of ubiquitin, lysozyme-ubiquitin conjugates, and ATP. Degradation of lysozyme conjugates is independent of added ubiquitin and occurs in fractions incapable of ubiquitin conjugation. Proteolysis is maximal at pH 7.8, inhibited by hemin, N ethylmaleimide, or aurintricarboxylic acid, and proceeds with an apparent Arrhenius activation energy in the range of 27 +/- 5 kcal/mol. These properties are similar to those observed for the degradation of lysozyme conjugates in lysates indicating that the partially purified protease catalyzes the "second" ATP-utilizing reaction identified previously (Hough, R., and Rechsteiner, M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 90-94; Hershko, A., Leshinsky, E., Ganoth, D., and Heller, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1619-1623; Tanaka, K., Waxman, L., and Goldberg, A. L. (1983) J. Cell Biol. 96, 1580-1585). PMID- 3003115 TI - Nucleotide sequence and in vivo expression of the ilvY and ilvC genes in Escherichia coli K12. Transcription from divergent overlapping promoters. AB - The ilvC gene of Escherichia coli K12 encodes acetohydroxy acid isomeroreductase, the second enzyme in the parallel isoleucine-valine biosynthetic pathway. Previous data have shown that transcription of the ilvC gene is induced by the acetohydroxy acid isomeroreductase substrates, acetohydroxybutyrate or acetolactate, and that this substrate induction of ilvC expression is mediated by a positive activator encoded by the ilvY gene. We report here the isolation and complete nucleotide sequence of the ilvY and ilvC genes. The ilvY and ilvC genes encode polypeptides of Mr 33,200 and 54,000, respectively. In vitro transcription translation of these gene templates results in the synthesis of gene products of these identical molecular weights. The ilvC gene is transcribed in the same direction as the genes of the adjacent ilvGMEDA operon. The ilvY gene is transcribed in a direction opposite to the ilvC and ilvGMEDA genes. The in vivo transcriptional initiation sites of the ilvY and ilvC genes have been determined by S1 nuclease protection experiments. These transcriptional initiation sites are 45 nucleotides apart, and transcription of the ilvY and ilvC genes is initiated via divergent overlapping promoters. The nucleotide sequence of the ilvY and ilvC promoters and 5'-coding regions of Salmonella typhimurium LT2 have been determined. A comparison of these sequences with E. coli K12 suggests regions important in the promotion, regulation, and translation of the ilvY and ilvC genes. A model is presented in which the ilvY-encoded activator binds to an operator site in the overlapping promoter region and reciprocally regulates the transcription of the ilvY and ilvC genes. The carboxyl-terminal amino acid sequence of threonine deaminase encoded by the ilvA gene of the ilv-GMEDA operon of E. coli K12 has been identified by homology with the previously deduced threonine deaminase amino acid sequence encoded by the ilv1 gene of Saccharomyces cerevisiae. Based on the deduced amino acid sequences of the ilvA and ilvY genes, the translational termination codons for both genes are shown to be separated by 52 nucleotides. The proximity of the ilvA and ilvY genes suggests that the 3' ends of these transcripts overlap. PMID- 3003116 TI - Expression of the gastrin-releasing peptide gene in human small cell lung cancer. Evidence for alternative processing resulting in three distinct mRNAs. AB - cDNA clones to human prepro-gastrin-releasing peptide (prepro-GRP) mRNA detect synthesis of prepro-GRP-related transcripts in 4 of 7 small cell lung cancer (SCLC) cell lines and 1 of 2 metastatic SCLC tumors examined. A correlation is noted between prepro-GRP gene expression and the occurrence of bombesin-related immunoreactivity in SCLC cell lines. Examination of the structure of prepro-GRP transcripts found in SCLC reveals three types of prepro-GRP mRNA which differ in the structure of a putative GRP-associated peptide in the pro-GRP precursor. The subcellular distribution of prepro-GRP-related RNAs and structure of SCLC-derived prepro-GRP cDNA clones suggest that all three types of transcript could function as mRNAs, although there are differences in the prevalence of the different RNA types in different cellular compartments. Comparison of the sequence of cDNA clones with the sequence of a genomic prepro-GRP clone reveals that the three forms of prepro-GRP mRNA arise from a single primary transcript which undergoes alternative processing from two splice donor sites to two splice acceptor sites. The predicted amino acid sequence of the translated products of the three mRNAs are quite distinct, leading to predicted pro-GRP molecules of differing structure. PMID- 3003117 TI - Bovine adrenocortical cytochrome P-450(17 alpha). Regulation of gene expression by ACTH and elucidation of primary sequence. AB - In the steroidogenic pathway of the adrenal cortex, 17 alpha-hydroxylase cytochrome P-450 (P-450(17 alpha)) is a major regulatory enzyme. Previous studies have shown that stimulation of 17 alpha-hydroxylase activity occurs upon treatment of bovine adrenocortical cells in culture with adrenocorticotropic hormone (ACTH), via cAMP, due to an increase in the enzyme concentration. Alterations in the levels of this enzyme result in pronounced changes in the pattern of steroids produced, with the potent glucocorticoid cortisol, as well as the sex steroids, being products of 17 alpha-hydroxylated steroid precursors. In the present study, the identification and sequencing of two cloned DNA molecules, pB17 alpha-1 and pcD17 alpha-2, which are complementary to mRNA sequences encoding bovine adrenal cortex P-450(17 alpha), are reported. Clone pcD17 alpha-2 contains an open reading frame coding for the complete amino acid sequence of P 450(17 alpha) which consists of 509 amino acids and has a molecular weight of 57,251. By comparison to other forms of cytochrome P-450, we conclude that P 450(17 alpha) is a member of a previously unidentified family within the P-450 multigene superfamily. Single bovine and human mRNA species of approximately equal to 1850 bases in length hybridize to these clones. Regulation of the concentration of this RNA has been studied using bovine adrenocortical cells in culture. Treatment with ACTH or dibutyryl cAMP causes an increase in the content of P-450(17 alpha) RNA in as little as 2 h, and its concentration increases greater than 20-fold by 8 h. In contrast, other steroidogenic enzymes that have been studied respond more slowly to ACTH. Actinomycin D and cycloheximide block induction of P-450(17 alpha) RNA, indicating that regulation of 17 alpha hydroxylase activity is controlled at the level of transcription and requires ongoing protein synthesis. PMID- 3003119 TI - Characterization of erythropoietin-like activity from mouse spleen cells. AB - Supernatants from mouse spleen cell cultures contain a factor which acts in a similar manner to erythropoietin (Ep) to stimulate the formation of 2-day erythroid (CFU-E) colonies in vitro from bone marrow or fetal liver cells. Analysis of conditioned media by high performance liquid chromatography (HPLC) on anion exchange, reverse phase, molecular size exclusion, and hydroxyapatite columns demonstrated that the erythropoietin-like activity (EpLA) has different biochemical characteristics to mouse Ep from anemic mouse serum. In addition, EpLA has a molecular weight (Mr), of 20,000 daltons determined by SDS polyacrylamide gel electrophoresis (SDS-PAGE), compared to 42,000 for mouse Ep. Partially purified EpLA was found to be active in vivo as well as in vitro. Highly purified preparations of gamma-interferon, Multilineage hemopoietic growth factor (Multi HGF), Interleukin-2 (IL-2), IL-1, and colony stimulating factor 1 (CSF-1) did not support CFU-E colony formation. Thus, it was established that EpLA could not be attributed to other known components of spleen cell conditioned medium. Titration of mouse Ep and EpLA suggests that only a portion of the Ep responsive CFU-E population in fetal liver is sensitive to EpLA. PMID- 3003118 TI - Secretory cell actin-binding proteins: identification of a gelsolin-like protein in chromaffin cells. AB - Chromaffin cells, secretory cells of the adrenal medulla, have been shown to contain actin and other contractile proteins, which might be involved in the secretory process. Actin and Ca++-sensitive actin-binding proteins were purified from bovine adrenal medulla on affinity columns using DNase-I as a ligand. Buffers that contained decreasing Ca++ concentrations were used to elute three major proteins of 93, 91, and 85 kD. The bulk of the actin was eluted with guanidine-HCl buffer plus some 93- and 91-kD proteins. These Ca++-sensitive regulatory proteins were shown to inhibit the gelation of actin using the low shear falling ball viscometer and by electron microscopy. Actin filaments were found to be shortened by fragmentation. Using antibody raised against rabbit lung macrophage gelsolin, proteolytic digestion with Staphylococcus V8 protease and two-dimensional gel electrophoresis, the 91-kD actin-binding protein was shown to be a gelsolin-like protein. The 93-kD actin-binding protein also showed cross reactivity with anti-gelsolin antibody, similar peptide maps, and a basic-shift in pHi indicating that this 93-kD protein is a brevin-like protein, derived from blood present abundantly in adrenal medulla. Purification from isolated chromaffin cells demonstrated the presence of 91- and 85-kD proteins, whereas the 93-kD protein was hardly detectable. The 85-kD protein is not a breakdown product of brevin-like or gelsolin-like proteins. It did not cross-react with anti gelsolin antibody and showed a very different peptide map after mild digestion with V8 protease. Antibodies were raised against the 93- and 91-kD actin-binding proteins and the 85-kD actin-binding protein. Antibody against the 85-kD protein did not cross-react with 93- and 91-kD proteins and vice versa. In vivo, the cytoskeleton organization of chromaffin secretory cells is not known, but appears to be under the control of the intracellular concentration of free calcium. The ability of calcium to activate the gelsolin-like protein, and as shown elsewhere to alter fodrin localization, provides a mechanism for gel-sol transition that might be essential for granule movement and membrane-membrane interactions involved in the secretory process. PMID- 3003120 TI - Hormonal responses to 1,25-dihydroxyvitamin D3 in cultured mouse osteoblast-like cells--modulation by changes in receptor level. AB - The level of 1,25(OH)2D3 receptors in cultured mouse osteoblast-like (OB) cells is modulated by the rate of cell proliferation. We have studied two 1,25(OH)2D3 induced bioresponses to ascertain whether the changes in receptor levels during growth in culture alter cell responsiveness. Nuclear receptor levels were high (127 fmol/100 micrograms DNA) in rapidly dividing (log) cells and low (25 fmol/100 micrograms DNA) in quiescent (confluent) cells. The bioresponses we studied were induction of 25(OH)D3-24-hydroxylase activity (24-hydroxylase) and inhibition of collagen synthesis. The basal levels of 24-hydroxylase were low and similar in cells at log growth phase and confluence. At a maximal induction dose of 13 nM, 1,25(OH)2D3 induced a three-fold rise in enzyme activity at long growth phase, but only caused less than two-fold rise at confluence. The half-maximal dose (ED50) was slightly shifted from 0.6 nM to 0.8 nM. Daily measurement of 1,25(OH)2D3 receptor levels and maximal induction of 24-hydroxylase activity throughout the culture cycle showed a strong correlation between receptor abundance and enzyme induction. The basal level of collagen synthesized by cells in log growth phase was approximately 5% and increased to approximately 8% at confluence. Maximal inhibition of collagen synthesis by 1,25(OH)2D3 reached 80% of control levels in log cells, but was only 40% of control in confluent cells. The ED50 was approximately 0.1 nM in the log cells and increased to approximately 1 nM at confluence. Daily assay of 1,25(OH)2D3 receptor levels and 1,25(OH)2D3 responses during the culture cycle indicated a correlation between changes in receptor level and the extent of inhibition of collagen synthesis. These changes in bioresponse at various growth phases did not occur in rat OB cells where the 1,25(OH)2D3 receptor levels were independent of cell proliferation. The results indicate that cell proliferation rate, via change in receptor levels, determines the magnitude and sensitivity of the cellular responses to 1,25(OH)2D3. PMID- 3003121 TI - Recovery of hormonal regulation in protein kinase defective adrenal cells through DNA-mediated gene transfer. AB - A cAMP-resistant mutant (Kin-8) isolated from Y1 mouse adrenocortical tumor cells harbors a specific lesion in the regulatory subunit of the type 1 cAMP-dependent protein kinase. This mutant also is resistant to the effects of corticotropin and cAMP on steroidogenesis, growth and morphology, suggesting an obligatory role for the protein kinase in regulation of adrenocortical functions. In this study, the cAMP-resistant phenotype of the Kin-8 mutant was reverted by transformation with DNA from cAMP-responsive Y1 cells, and the biochemical basis of the transformation was explored. Initially, Y1 mouse adrenocortical tumor cells were evaluated for their competence as recipients in DNA-mediated transformation experiments, by measuring their ability to incorporate and express a bacterial gene (neo) encoding resistance to neomycin. Y1 cells were transfected with the plasmid pSV2-neo (an SV40-neo hybrid vector designed for expression in animal cells) and screened for resistance to the neomycin analog, G418. Neomycin resistant transformants were recovered from Y1 cells at a frequency of approximately one per 10(3) cells per 10 micrograms of DNA, and had specific neo sequences integrated into their high molecular weight (mw) DNA. The Y1 mutant, Kin-8, then was transformed with pSV2-neo DNA plus high mw DNA prepared from cAMP responsive Y1 cells. Cells competent for transformation were recovered by selective growth in the neomycin analog G418, and these transformants were screened for recovery of morphological responses to cAMP. Several colonies capable of rounding up in the presence of cAMP were recovered after transformation with DNA from Y1 cells. These transformants also recovered the ability to round up in the presence of corticotropin, and were able to respond to both corticotropin and cAMP with increased steroidogenesis. Transformants generated from either Y1 or Kin-8 cells were unstable. Y1 cells lost resistance to neomycin when grown in the absence of G418 at a frequency of 4% per generation. Similarly, Kin-8 transformants lost their sensitivity to cAMP in subsequent culture passages. In some of the cAMP-responsive transformants, cAMP dependent protein kinase activity was recovered and approached the activity seen in cAMP-responsive Y1 cells. The recovery of a normal protein kinase by transformation appeared to have been sufficient to reverse the cAMP-resistant phenotype of Kin-8 cells. In other cAMP-responsive transformants, protein kinase activity was not appreciably affected by cAMP.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3003122 TI - Catecholamine modulation of embryonic palate mesenchymal cell DNA synthesis. AB - Development of the mammalian embryonic palate depends on the precise temporal and spatial regulation of growth. The factors and mechanisms underlying differential growth patterns in the palate remain elusive. Utilizing quiescent populations of murine embryonic palate mesenchymal (MEPM) cells in vitro, we have begun to investigate hormonal regulation of palatal cell proliferation. MEPM cells in culture were rendered quiescent by 48 hr serum deprivation and were subsequently released from growth arrest by readdition of medium containing 10% (v/v) serum. The progression of cells into S-phase of the cell cycle was monitored by autoradiographic analysis of tritiated thymidine incorporation. Palate mesenchymal cell entry into S-phase was preceded by a 6- to 8-hr prereplicative lag period, after which time DNA synthesis increased and cells reached a maximum labeling index by 22 hr. Addition of 10 microM isoproterenol to cell cultures at the time of release from growth arrest lengthened the prereplicative lag period and delayed cellular entry into S-phase by an additional 2 to 4 hr. The rate of cellular progression through S-phase remained unaltered. The inhibitory effect of isoproterenol on the initiation of MEPM cell DNA synthesis was abolished by pretreatment of cells with propranolol at a concentration (100 microM) that prevented isoproterenol-induced elevations of cAMP. Addition of PGE2 to cell cultures, at a concentration that markedly stimulates cAMP formation, mimicked the inhibitory effect of isoproterenol on cellular progression into S-phase. These findings demonstrate the ability of the beta-adrenergic catecholamine isoproterenol to modulate MEPM cell proliferation in vitro via a receptor mediated mechanism and raise the possibility that the delayed initiation of DNA synthesis in these cells is a cAMP-dependent phenomenon. PMID- 3003123 TI - Highly adherent mutants of SV40-transformed mouse fibroblasts: genetic and biochemical characterization. AB - Mutants of SV40-transformed mouse fibroblasts have been isolated that have greatly increased cell-substratum adherence. The adherent phenotype (COL-) is recessive, and all mutants analyzed belong to one complementation group. No consistent qualitative differences between wild-type and mutant cells were found with respect to the protein content of the substrate-attached material (SAM), a cell surface fraction left after removal of cells from the substrate with a gentle Ca2+-chelating agent. However, the mutants yielded 2.5-10-fold more SAM than the parental cell line, and the SAM deposited by mutants was able to mediate attachment of transformed cells to a much greater degree than was the SAM from the parental cell line. The mutation, which appears to control the generation of footpads, was shown to cosegregate with resistance to the drug 6-thioguanine, which suggests X-linkage. PMID- 3003124 TI - A comparison of the platelet-derived growth factor-dependent tyrosine kinase activity in sparse and confluent fibroblasts. AB - Confluent (density-inhibited) human foreskin fibroblasts require a higher concentration of platelet-derived growth factor (PDGF) to elicit a mitogenic response than do sparse (nondensity-inhibited) fibroblasts. The PDGF receptor number and apparent affinity were similar in the two preparations of cells. The intrinsic kinase activity of the PDGF receptor from sparse and confluent fibroblasts was therefore examined in an attempt to explain the differential mitogenic response to PDGF. When membranes from sparse and confluent cells containing equal PDGF binding capacity were incubated with increasing concentrations of PDGF, the putative PDGF receptor (a 180-kD component), was phosphorylated on its tyrosyl residues to a similar extent. The time course of tyrosine phosphorylation of the PDGF receptor from sparse and confluent cell membranes was also found to be similar. To determine whether the phosphorylation of the PDGF receptor from isolated membranes differed from the analogous phosphorylation in intact cells, sparse and confluent fibroblasts were metabolically labeled with [32P]H3PO4, stimulated with PDGF, solubilized, and the cell proteins were immunoprecipitated with a phosphotyrosine-specific antibody. The extent of PDGF-dependent tyrosine phosphorylation of the PDGF receptor from sparse vs. confluent fibroblasts was quite similar. The time course of the tyrosine dephosphorylation of the PDGF receptor was also similar in the two populations. Because comparable extents of PDGF-induced tyrosine phosphorylation of the PDGF receptor were observed despite the differential PDGF-induced mitogenic response of sparse and confluent fibroblasts, we tentatively conclude that 1) PDGF-dependent tyrosine phosphorylation of the PDGF receptor is not tightly coupled to the propagation of the mitogenic signal and 2) density dependent inhibition of growth does not reflect any measurable change in the quantity of kinase activity of the PDGF receptor. PMID- 3003125 TI - Electrophysiological differences between bone cell clones: membrane potential responses to parathyroid hormone and correlation with the cAMP response. AB - Electrophysiological measurements on three clonally derived bone cell populations showed a positive correlation between longer-term hyperpolarizing membrane potential responses to parathyroid hormone (PTH) and an intracellular cAMP response to PTH. One clone (RCJ 1.20) had no sustained electrophysiological response and no cAMP response to PTH. Another clone (ROS 17/2.8) had both a sustained hyperpolarizing response and a cAMP response to PTH. The third clone (RCB 2.2) initially had both an electrophysiological response and a cAMP response to PTH, but both responses were lost after prolonged growth in culture. Application of dibutyryl cAMP to RCJ 1.20 and ROS 17/2.8 cells produced both transient and sustained hyperpolarizing responses. Application of isobutylmethylxanthine produced a sustained hyperpolarization. These results suggest that the hyperpolarizing response to PTH is related to a cAMP-mediated increase in Ca2+ conductance, which leads to an increase in Ca2+-activated K+ conductance. The pronounced membrane potential spikes and fluctuations that occur in some of the clonal lines were shown to be unrelated to the hyperpolarizing response to PTH. This was demonstrated by the lack of correlation between the occurrence of the spikes or fluctuations and the occurrence of the hyperpolarizing response to PTH in the various cell lines, by the lack of effect of PTH on the spikes and fluctuations, and by the lack of effect on the hyperpolarizing response to PTH of verapamil and quinine, both of which significantly reduce the spikes and fluctuations. PMID- 3003126 TI - Tumor promoter enhances mitogenesis by PDGF with little effect on PDGF binding. AB - Preincubation of Swiss 3T3 cells with the tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA) at 37 degrees C is observed to cause only a small (approximately 10%) decrease in maximal binding of 125I-platelet-derived growth factor (125I-PDGF), and does not affect the affinity of 125I-PDGF binding to these cells. Under the same conditions, the affinity of the epidermal growth factor receptor is greatly reduced, possibly resulting from phosphorylation by protein kinase C. TPA is also shown to have no effect on the kinetics of internalization or degradation of bound 125I-PDGF. Although TPA has little or no effect on these properties of the PDGF receptor, it was found to act in a synergistic fashion with low, but not high, concentrations of PDGF to increase DNA synthesis by 3T3 cells. Since TPA has previously been shown to activate protein kinase C, these findings suggest that protein kinase C does not regulate the ligand-binding properties of the PDGF receptor, and that the observed synergism between TPA and PDGF in stimulating mitogenesis reflects effects of TPA on other processes in the mitogenic pathway. PMID- 3003127 TI - 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced gene sequences in human primary diploid fibroblasts and their expression in SV40-transformed fibroblasts. AB - We have isolated cDNA sequences from TPA-treated primary human fibroblasts, which indicate RNA species that are coordinately regulated after treatment of these cells with either ultraviolet light, mitomycin C, the UV-induced factor EPIF, or TPA. The levels of RNA are elevated in Bloom syndrome (cells of two out of three patients). After transformation with SV40 one of the sequences is overexpressed while another one is reduced. Both genes maintain their inducibility by the agents mentioned. PMID- 3003128 TI - The relationship between adenosine diphosphate-ribosylation and mammary gland differentiation. AB - Poly(adenosine diphosphate [ADP]-ribosyl)ation, although associated with differentiation in many systems, exhibited a reciprocal relationship with mammary gland differentiation, and both the synthetic and degradatory pathways complemented each other in this regard. Poly(ADP-ribosyl)synthetase activity declined during pregnancy and lactation, while poly(ADP-ribose) degradatory activity rose late in pregnancy and peaked during lactation. In explant cultures, similar changes occurred and appeared to be under separate hormonal control; prolactin suppressed the synthetase activity, whereas insulin stimulated the poly(ADP-ribosyl)glycohydrolase activity. This latter effect may be mediated by a decline in cAMP levels for the following reasons: the glycohydrolase is known to be inhibited by cAMp, insulin decreased cAMP concentrations in mammary explants by 70%, and cholera toxin blocked the effects of insulin on poly(ADP-ribose) degradation. This reciprocal relationship between poly(ADP-ribosyl)ation and mammary gland differentiation is further supported by pharmacological studies: in the presence of insulin, cortisol, and prolactin, an inhibitor of the synthetase stimulated alpha-lactalbumin three-fold over hormone stimulation alone. However, this inhibitor was unable to induce differentiation in the absence of prolactin. Therefore, although there is a close association between a decline in enzyme activity and mammary differentiation, the data are insufficient to support a causal relationship. PMID- 3003129 TI - Regulation of ciliary reversal in triton-extracted Paramecium by calcium and cyclic adenosine monophosphate. AB - A Triton-extracted model of Paramecium swims forwards when the Ca2+ concentration in the reactivation medium containing ATP is below 10(-6) M and swims backwards when Ca2+ concentration is above 10(-6) M. We found that cAMP (adenosine 3':5' cyclic monophosphoric acid) inhibited Ca-induced backward swimming of the model and caused forward swimming even when the [Ca2+] was above 10(-6) M. This effect of cAMP was abolished by an inhibitor of cAMP-dependent protein kinase. In order to study the possible role of phosphorylation in the regulation of ciliary orientation, ATP in the reactivation medium was replaced by an ATP analogue, ARP gamma S (adenosine 5'-O-3-thiotriphosphate), which irreversibly thiophosphorylates proteins. In ATP gamma S medium, the model ceased both swimming and ciliary beating, but the orientation of cilia was dependent on [Ca2+]. At low [Ca2+], cilia were perpendicular to the cell surface and, with increase in [Ca2+], their orientation gradually changed towards the cell anterior. Such a change in ciliary orientation corresponds roughly to the change in the swimming direction observed in ATP medium. The ciliary orientation towards the anterior of the cell in ATP gamma S medium at high [Ca2+] was maintained when [Ca2+] was decreased. In contrast, in ATP medium, the swimming direction was reversibly changed with changes in [Ca2+]. These results suggest that the ciliary orientation is regulated not only by Ca2+ but also by cAMP, probably via protein phosphorylation. PMID- 3003130 TI - Plasma membranes from adrenal cells: purification and properties. AB - A method is reported for preparing surface (plasma) membranes from adrenal tumour (Y-1) cells. The procedure is based upon homogenization in hypotonic ZnCl2 followed by sedimentation through two sucrose density gradients. The purified membranes consist of large sheets of membrane. The identity and purity of the membranes was demonstrated by: (1) microscopy (phase contrast and electron); (2) enzyme markers; and (3) functional activities associated with plasma membranes (binding of ACTH and LDL and adenylate cyclase). Phase-contrast microscopy revealed the release of membrane ghosts free from cytoplasm and nuclei. Electron microscopy showed membranes with small fragments of cytoplasm attached to the inside. Binding of ACTH was found to be specific with KD 0.12 nM and the equivalent of 2500 sites per cell. Binding of LDL was also specific with KD 0.5 nM and the equivalent of 4800 sites per cell. Specific activities of binding for ACTH and LDL were increased by 21-fold and 15-fold, respectively, relative to whole homogenate. Membranes were also prepared from beef fasciculata cells by the same method. PMID- 3003132 TI - [Value of scintigraphy in the localization of digestive hemorrhage]. AB - Scintigram imaging is a new means of localization of digestive tract hemorrhage, the radiopharmaceutic agent (technetium-labelled colloidal sulfur or technetium labelled erythrocytes) accumulating at the bleeding site. The technique used consists of early recording over 80 minutes and then later serial imaging over 24 hours after radioisotope injection. The limitations and advantages of radioisotope investigations are discussed in relation to results in 6 patients with digestive hemorrhage explored by scintigraphy. When technetium-labelled erythrocytes are used the method is a non-invasive, simple and sensitive one for locating site of bleeding during the period of active hemorrhage. It appears to be complementary to arteriography, a more aggressive and less sensitive procedure, and to endoscopy, which explores small intestine and cecum with difficulty and cannot always determine the origin of the bleeding when other lesions exist. Experimental studies in the dog have shown that scintigraphy can detect digestive bleeding of about 0.1 to 0.2 ml/min, and this method of exploration should be used routinely when endoscopy and/or arteriography have not been performed or have given inconclusive results. PMID- 3003131 TI - Distribution of cytochrome oxidase in rat brain: studies with diaminobenzidine histochemistry in vitro and [14C]cyanide tissue labeling in vivo. AB - Studies in experimental animals and post-mortem studies in humans have indicated that the level of the mitochondrial enzyme cytochrome oxidase within brain anatomical pathways is regulated by the long-term functional use of those pathways. To study this relationship, we have measured cytochrome oxidase spectrophotometrically in punch biopsies from different brain regions of rat. We compared these assays against results from the diaminobenzidine histochemical technique. We found a high degree of correlation (r = 0.90) between the density of diaminobenzidine reaction product and enzyme activity. This validates the usefulness of the diaminobenzidine technique for anatomical localization and measurement of this enzyme. To study the feasibility of using radioactive cyanide as an in vivo ligand of cytochrome oxidase, we performed quantitative autoradiographic analysis of rat brains of animals given an intravenous bolus injection of [14C]cyanide. Analysis of the arterial blood curve indicated a complex redistribution of cyanide between red blood cells, plasma, and tissues. Brain labeling reached peak levels at 1 min and then fell despite rising concentrations of free plasma cyanide. Analysis of autoradiographic images revealed good anatomical resolution. The density of labeling in individual structures over time failed to show a strong correlation with cytochrome oxidase activity or diaminobenzidine reaction product. PMID- 3003133 TI - Cushingoid changes in a 'healthy' woman. PMID- 3003134 TI - Recurrent breast cancer: an integrative CPC. PMID- 3003135 TI - Advances in diagnosis of hematologic malignancies. PMID- 3003137 TI - High-performance liquid chromatography of plasma catecholamines using 1,2 diphenylethylenediamine as precolumn fluorescence derivatization reagent. AB - A simple, rapid and highly sensitive method for the determination of catecholamines (norepinephrine, epinephrine and dopamine) in human plasma is described which employs high-performance liquid chromatography with fluorescence detection. After cation-exchange chromatography on a Toyopak SP cartridge, the catecholamines and isoproterenol (internal standard) in 500 microliters of plasma are converted into the corresponding fluorescent compounds by reaction with 1,2 diphenylethylenediamine in aqueous acetonitrile. These compounds are separated within 8 min on a reversed-phase column, TSK-gel ODS-120T, with isocratic elution using a mixture of water, methanol and acetonitrile containing a Tris- hydrochloric acid buffer (pH 7.0). The detection limit for each catecholamine is ca. 2 fmol in a 100-microliters injection volume. N-Methyldopamine can also be used as internal standard. PMID- 3003136 TI - The kidney in heart failure. PMID- 3003138 TI - Determination of pentopril, an angiotensin converting enzyme inhibitor, and its active metabolite in urine. PMID- 3003139 TI - Rapid and convenient separation of pentachlorophenol from human fat using silica Sep-Pak cartridges. PMID- 3003141 TI - Silica capillary gas chromatography of prostaglandins with electron-capture detection and its application to the forensic investigation of sexual offences. AB - Low picogram levels of the E series prostaglandins, PGE1, PGE2, 19-hydroxy PGE1 and 19-hydroxy PGE2, in human semen were analysed by silica capillary column gas chromatography with electron-capture detection after conversion to the methyl ester O-trimethylsilyl derivatives of the corresponding B series prostaglandins. The method was used to detect traces of semen on post-coital vaginal swabs, and on rectal, oral and skin swabs after simulated sexual acts. Semen was detectable on a vaginal swab taken 58 h after intercourse, and was readily detectable for at least 6 h on rectal and skin swabs. Preliminary results suggest that the ratios of prostaglandins on vaginal swabs may indicate how recently intercourse occurred. PMID- 3003140 TI - Liquid chromatographic retention behavior of organometallic compounds and ligands with amine-, octadecylsilica- and beta-cyclodextrin-bonded phase columns. AB - Effects of solvent composition and ligand variation on the retention of organometallic compounds have been studied using an amino, an octadecylsilica (ODS) and a beta-cyclodextrin (beta-CD) bonded phase column in either a normal phase or a reversed-phase mode. The retention behavior for the organometallic compounds with the amino column can be rationalized using the displacement model. The "apparent" molecular areas are greater for compounds capable of strong hydrogen bonding. The retention in the ODS column roughly follows an argument based on the expected solubility behavior while mixed retention mechanisms are involved for the solubility behavior while mixed retention mechanisms are involved for the beta-CD column, i.e. both inclusion process and solubility or solvophobic interactions are possibly operative. PMID- 3003142 TI - Activity of the stimulatory guanine nucleotide-binding protein is reduced in erythrocytes from patients with pseudohypoparathyroidism and pseudopseudohypoparathyroidism: biochemical, endocrine, and genetic analysis of Albright's hereditary osteodystrophy in six kindreds. AB - Multiple hormone resistance in many patients with pseudohypoparathyroidism (PHP) type Ia and Albright's hereditary osteodystrophy (AHO) is associated with deficient activity of the stimulatory guanine nucleotide-binding protein (Gs) of adenylate cyclase. To study further the relationship of deficient Gs activity to hormone resistance, we evaluated endocrine function and measured Gs activity of erythrocyte membranes from AHO patients with clinical hormone resistance (PHP type Ia) and from family members with AHO alone (pseudopseudohypoparathyroidism). The results of erythrocyte membrane Gs determinations were compared to those of unaffected relatives and normal subjects. Patients with pseudopseudohypoparathyroidism (pseudoPHP) had reductions in erythrocyte membrane Gs activity comparable to those in patients with PHP type Ia [43.4 +/- 11.9% (+/- SD) for PHP type Ia vs. 47.8 +/- 9.5% for pseudoPHP]. However, in contradistinction to patients with PHP type Ia, individuals with pseudoPHP did not have obvious endocrine dysfunction. Although deficient Gs activity appears to play an important role in the pathogenesis of these disorders, it is possible that Gs deficiency must be combined with other factors that limit cAMP production to cause clinically overt endocrine disease. PMID- 3003143 TI - The human fetal membranes: a target tissue for relaxin. AB - The purpose of this study was to adduce further evidence for a paracrine role for human decidual relaxin (Rlx) in the remodelling of collagen in the fetal membranes in the peripartal period. The binding of [125I]porcine Rlx to membrane enriched fractions from fetal membranes as well as from dispersed cells from the fetal membranes was used to demonstrate the presence of specific Rlx receptors. Rlx added in vitro to cultured amnion/chorion cells increased the release of plasminogen activator and collagenase into the medium. Rlx had no effect on the release of beta-glucuronidase. An in vivo correlate of these in vitro results was obtained, the detection of plasminogen activator and collagenase in amniotic fluids. The active fraction of collagenase was increased in amniotic fluids collected after spontaneous rupture of the membranes. PRL, hCG, estrogen, and progesterone added in equimolar amounts to cultured amnion/chorion cells from elective cesarean sections and normal term deliveries also effected the release of plasminogen activator and collagenase. The greatest effects were found in cells from cesarean section tissue, in terms of the stimulation of plasminogen activator release by Rlx and PRL and of collagenase release by prostaglandin F2 alpha and, to a lesser extent, by Rlx, PRL, and hCG. We conclude that human fetal membranes are targets for a number of hormones, including the decidual paracrine hormones Rlx, PRL, and prostaglandin F2 alpha as well as estrogen, progesterone, and hCG. These hormones act to release or inhibit the enzymes involved in collagen breakdown before rupture of the fetal membranes. PMID- 3003144 TI - Opioid modulation of normal and pathological human chromaffin tissue. AB - To evaluate whether opioid receptor blockade might modulate sympathetic-adrenal activity, we studied the effects of placebo or naloxone administration on plasma catecholamine (CA) levels in a group of 13 normal subjects and 15 hypertensive patients suspected to have a pheochromocytoma. Diagnostic evaluation confirmed the presence of pheochromocytoma in 9 patients. Among these, 4 had a unilateral epinephrine (E)-secreting tumor, 3 had bilateral E-secreting tumors due to multiple endocrine adenomatosis type IIa, and 2 had a unilateral norepinephrine (NE)-secreting tumor. In each subject studied, CA secretion was evaluated by calculating the area (0-30 min) under the plasma hormone curves after placebo or naloxone administration. In normal subjects naloxone caused a significant increase (P less than 0.005) of E secretion, whereas NE did not change. Similarly, in the group of hypertensive patients, E secretion increased after naloxone (P less than 0.01). In pheochromocytoma patients naloxone caused a significant increase in E (P less than 0.05) and NE (P less than 0.01) secretion from E-producing tumors but no increase in the patients with NE-secreting pheochromocytomas. The study suggests that CA secretion from normal and pathological chromaffin tissue is modulated by endogenous opioids; this modulation seems particularly evident in patients with E-secreting pheochromocytoma. PMID- 3003145 TI - Response to parathyroid hormone and 1,25-dihydroxyvitamin D3 of bone-derived cells isolated from normal children and children with abnormalities in skeletal development. AB - In order to evaluate the role of intrinsic defects in osteoblast function in the pathogenesis of diseases of skeletal development, we developed techniques which permit the evaluation of the metabolic properties of bone-derived cells in vitro. Cells from control children demonstrated a variety of properties classically attributed to osteoblasts (presence of alkaline phosphatase positive cells and synthesis of bone gla protein) and responded to PTH (cAMP production) and to 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) ([3H]25-hydroxyvitamin D3 conversion into [3H]24,25-dihydroxyvitamin D3 and bone gla protein secretion). Using these techniques we evaluated the function of cultured bone cells from patients with three rare diseases of skeletal development. Cells from a patient with rickets resistant to 1,25(OH)2D3 were resistant to 1,25(OH)2D3 but responded normally to PTH. Cells from a patient with acroosteolysis with osteoporosis responded normally to PTH and 1,25(OH)2D3. Cells from a patient with hyperphosphatasia with osteoectasia responded normally to 1,25(OH)2D3 but did not respond to PTH. The results demonstrate that bone cell cultures can provide information about the role of osteoblast dysfunction in such diseases. PMID- 3003146 TI - Plasma free cortisol during secretory episodes. AB - Plasma free cortisol fractions were determined in normal men during spontaneous cortisol secretory episodes and ACTH- or exercise-provoked cortisol peaks in the physiological range. The percentage of free cortisol was measured using ultrafiltration by centrifugation at 37 C, and total cortisol concentrations were measured by RIA. The percent free cortisol varied with the time of day, as did total cortisol. It increased parallel to total cortisol increases, whether they were spontaneous, as occurring in the early morning or postprandially, or provoked by external stimulation. A highly significant positive correlation between relative increases in free and total cortisol was found throughout the experiment. As reported previously, total cortisol increases, according to their origin, did or did not reduce the effects of subsequent stimuli. The present results indicate that the variations in the percent free cortisol are not responsible for the differences in the cortisol responses to these stimuli. PMID- 3003147 TI - The idiotype-antiidiotype network in human autoimmunity. PMID- 3003148 TI - Trafficking of lysosomal enzymes in normal and disease states. PMID- 3003149 TI - Oxalate transport by anion exchange across rabbit ileal brush border. AB - This study demonstrates the presence of oxalate transporters on the brush border membrane of rabbit ileum. We found that an inside alkaline (pH = 8.5 inside, 6.5 outside) pH gradient stimulated [14C]oxalate uptake 10-fold at 1 min with a fourfold accumulation above equilibrated uptake at 5 min. 1 mM 4,4' diisothiocyanostilbene-2,2'-disulfonate (disodium salt; DIDS) profoundly inhibited the pH-gradient stimulated oxalate uptake. Using an inwardly directed K+ gradient and valinomycin, we found no evidence for potential sensitive oxalate uptake. In contrast to Cl:HCO3 exchange, HCO3 did not stimulate oxalate uptake more than was seen with a pH gradient in the absence of HCO3. An outwardly directed Cl gradient (50 mM inside, 5 mM outside) stimulated oxalate uptake 10 fold at 1 min with a fivefold accumulation above equilibrated uptake. Cl stimulated oxalate uptake was largely inhibited by DIDS. Addition of K+ and nigericin only slightly decreased the Cl gradient-stimulated oxalate uptake, which indicates that this stimulation was not primarily due to the Cl gradient generating an inside alkaline pH gradient via Cl:OH exchange. Further, an outwardly directed oxalate gradient stimulated 36Cl uptake. These results suggested that both oxalate:OH and oxalate:Cl exchange occur on the brush border membrane. To determine if one or both of these exchanges were on contaminating basolateral membrane, the vesicle preparation was further fractionated into a brush border and basolateral component using sucrose density gradient centrifugation. Both exchangers localized to the brush border component. A number of organic anions were examined (outwardly directed gradient) to determine if they could stimulate oxalate and Cl uptake. Only formate and oxaloacetate were found to stimulate oxalate and Cl uptake. An inwardly directed Na gradient only slightly stimulated oxalate uptake, which was inhibited by DIDS. PMID- 3003150 TI - Chronic caffeine ingestion sensitizes the A1 adenosine receptor-adenylate cyclase system in rat cerebral cortex. AB - Caffeine consumption causes significant physiologic effects due to its antagonism of adenosine receptors. The A1 adenosine receptor is coupled in an inhibitory manner to adenylate cyclase. To study the effects of chronic caffeine ingestion, rats were provided with 0.1% caffeine drinking solution for 28 d. The A1 adenosine receptor agonist radioligand [3H]phenylisopropyladenosine identifies two affinity states in control rat cerebral cortex membranes with a high affinity dissociation constant (KH) of 0.40 +/- 0.08 nM and low affinity dissociation constant (KL) of 13.7 +/- 3.9 nM, with 33% of the receptors in the high affinity state. In membranes from caffeine-treated animals, all of the A1 receptors are shifted to the high affinity state with a dissociation constant (KD) of 0.59 +/- 0.06 nM. Guanylyl-imidodiphosphate (10(-4) M) decreases binding by 43% in control membrane, with no change in KH or KL, while membrane binding in caffeine-treated animals decreases by 45% with a threefold shift in KD to 1.5 +/- 0.3 nM. Concomitant with the enhanced high affinity A1 receptor state and increased sensitivity to guanine nucleotides, membranes from treated animals show a 35% enhancement in (-)-N6-(R-phenylisopropyl)adenosine-mediated inhibition of adenylate cyclase compared with controls (P less than 0.03). Photoaffinity crosslinking the receptors with [125I]N6-2-(3-iodo-4-aminophenyl)ethyladenosine reveals that A1 receptors from both groups migrate as Mr 38,000 proteins. beta adrenergic receptor binding with [125I]iodocyanopindolol shows a decrease in the number of beta-receptors from 233 +/- 7 fmol/mg protein in control membranes to 190 +/- 10 fmol/mg protein in treated membranes (P = 0.01). These data indicate that the adenosine receptor antagonist, caffeine, induces a compensatory sensitization of the A1 receptor-adenylate cyclase system and downregulation of beta-adrenergic receptors, and provides a molecular mechanism for the caffeine withdrawal syndrome. PMID- 3003152 TI - No 1,25-dihydroxyvitamin D3 receptors on osteoclasts of calcium-deficient chicken despite demonstrable receptors on circulating monocytes. AB - 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is known to stimulate osteoclastic bone resorption in vivo and whole organ bone culture systems in vitro. It has not been established whether 1,25(OH)2D3 acts directly on osteoclasts or whether its action on osteoclasts is mediated via other bone cells (e.g., osteoblasts) or recruitment of osteoclast precursor cells. Circulating monocytes have been characterized as osteoclast precursors. In the present study, vitamin D3-replete chicken on a calcium-deficient diet were studied. Circulating monocytes, whole bone cell preparations, and isolated osteoclasts (differential sedimentation) were examined for presence of 1,25(OH)2D3 receptors. Reversible, specific, and saturable binding of [3H]-1,25(OH)2D3 to a 3.5 S macromolecule was demonstrated in nuclear fractions of monocytes (maximal binding capacity, 48 fmol/mg protein; dissociation constant, 1.3 X 10(-10) M) and of whole bone cell preparations. 1,25(OH)2D3 receptors were not demonstrable in osteoclast preparations (70% pure; detection threshold, 2 fmol/mg protein). Data are consistent with indirect action of 1,25(OH)2D3 on osteoclastic bone resorption. PMID- 3003151 TI - Diabetes-induced functional and structural changes in insulin receptors from rat skeletal muscle. AB - The effect of diabetes on the structure and function of insulin receptors was studied in rats 7 d after streptozotocin injection, using solubilized, partially purified receptors from rat hindlimb muscles. Diabetes increased the number of insulin receptors per gram of muscle 60-70% without apparent change in insulin binding affinity. Incubation of receptors at 4 degrees C with [gamma-32P]ATP and insulin resulted in dose-dependent autophosphorylation of the beta-subunit on tyrosine residues; receptors from diabetic rats showed decreased base-line phosphorylation, as well as a decrease in autophosphorylation at maximally stimulating insulin concentrations. These receptors also showed diminished exogenous substrate kinase activity using histone H2b and angiotensin II as phosphoacceptors. The electrophoretic mobility (sodium dodecyl sulfate polyacrylamide gel electrophoresis) of a subpopulation of beta-subunits derived from diabetics was slightly decreased; differences in electrophoretic mobility between control- and diabetic-derived beta-subunits were enhanced by generating fragments by partial Staphylococcus aureus V8 protease digestion. Endoglycosidase H or neuraminidase treatment increased the electrophoretic mobility of beta subunits in both groups, but only neuraminidase appeared to decrease or abolish differences in electrophoretic mobility between controls and diabetics, suggesting that excess sialilation may account, in part, for the altered mobility of diabetic derived beta-subunits. All structural and functional alterations in insulin receptors were prevented by treating diabetic rats with insulin for 60 h. Peripheral insulin resistance associated with insulinopenic diabetes may be related to modifications in insulin receptor structure, resulting in impaired signal transmission. PMID- 3003153 TI - Angiotensin II binding sites in individual segments of the rat nephron. AB - The sites of action of angiotensin II along the nephron are not well defined and both proximal and distal effects are suggested. Using a microassay that permits measurement of hormone binding in discrete tubule segments, we determined the binding sites of 125I-angiotensin II along the nephron of Sprague-Dawley rats. Specific binding in proximal convoluted tubule (PCT) (at 25 degrees C, pH 7.4) was linearly related to tubule length and saturable, with an apparent maximal binding capacity of approximately 300 amol X cm-1. Binding specificity was verified in competition experiments that revealed significant (P less than 0.001) and comparable competition for radioligand binding by angiotensin II and angiotensin precursor, metabolite, and analogues, whereas unrelated peptides of similar size (bradykinin, ACTH [1-10]) were without effect. The profile of specific angiotensin II binding along the nephron was: PCT, 216 +/- 13; pars recta, 86 +/- 14; medullary thick ascending limb of Henle's loop, 46 +/- 8; cortical thick ascending limb of Henle's loop, 77 +/- 8; distal convoluted tubule, 49 +/- 10; cortical collecting tubule, 15 +/- 1; medullary collecting tubule, 32 +/- 7 amol X cm-1. These results indicate the presence of specific angiotensin II binding sites in all tubule segments studied, but binding capacity was highest in the proximal convoluted tubule, in agreement with transport studies that localize the effects of the hormone in this segment. PMID- 3003154 TI - Decreased leukotriene B4 synthesis in smokers' alveolar macrophages in vitro. AB - Recent studies have shown that alveolar macrophages (AM) are able to release leukotrienes (LTs). Since cigarette smoking inhibits the cyclooxygenase pathway of arachidonic acid metabolism in the AM, we evaluated the LT production by AM from smokers and nonsmokers. AM were obtained from 35 volunteers, 16 nonsmokers, and 19 smokers. The cells were incubated under various conditions including stimulation with 30 microM arachidonic acid, 2 microM ionophore A23187, or both. Each experiment was performed in parallel using cells from a smoker and a nonsmoker. Lipoxygenase products were analyzed by reverse-phase high performance liquid chromatography. After stimulation, nonsmokers' AM produced LTB4 and 5 hydroxy-eicosatetraenoic acid (5-HETE). In incubations of AM with arachidonic acid and ionophore, the amounts of products formed were: LTB4, 317 +/- 56 pmol/10(6) cells and 5-HETE, 1,079 +/- 254, mean +/- SEM. No metabolites were generated under control conditions (no stimulation). In all incubations performed, the peptido-LTs (LTC4, LTD4, and LTE4) were undetectable. In comparison with AM from nonsmokers, those from smokers showed a 80-90% reduction of 5-HETE and LTB4 synthesis (P less than 0.05 to P less than 0.001 according to stimulatory conditions). This defective lipoxygenase metabolite production in AM from smokers was observed over a wide range of stimuli concentrations and incubation times; AM from smokers also had lower levels of intracellular (esterified) 5-HETE than nonsmokers' AM. We also studied blood polymorphonuclear leukocytes (PMNL) and no difference in the synthesis of 5-lipoxygenase products in these cells was noticed between smokers and nonsmokers. These data show that cigarette smoking causes a profound inhibition of the 5-lipoxygenase pathway in AM but not in blood PMNL. PMID- 3003156 TI - Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line. AB - Serosal application of carbachol to T84 cell monolayers mounted in an Ussing chamber caused an immediate increase in short circuit current (Isc) that peaked within 5 min and declined rapidly thereafter, although a small increase in Isc persisted for approximately 30 min. The increase in Isc was detectable with 1 microM carbachol; half-maximal with 10 microM carbachol; and maximal with 100 microM carbachol. Unidirectional Na+ and Cl- flux measurements indicated that the increase in Isc was due to net Cl- secretion. Carbachol did not alter cellular cAMP, but caused a transient increase in free cytosolic Ca2+ ([Ca2+]i) from 117 +/- 7 nM to 160 +/- 15 nM. The carbachol-induced increase in Isc was potentiated by either prostaglandin E1 (PGE1) or vasoactive intestinal polypeptide (VIP), agents that act by increasing cAMP. Measurements of cAMP and [Ca2+]i indicated that the potentiated response was not due to changes in these second messengers. Studies of the effects of these agents on ion transport pathways indicated that carbachol, PGE1, or VIP each increased basolateral K+ efflux by activating two different K+ transport pathways on the basolateral membrane. The pathway activated by carbachol was not sensitive to barium, while that activated by PGE1 or VIP was; furthermore, their action on K+ efflux are additive. Our study indicates that carbachol causes Cl- secretion, and that this action may result from its ability to increase [Ca2+]i and basolateral K+ efflux. Carbachol's effect on Cl- secretion is greatly augmented in the presence of VIP or PGE1, which open a cAMP-sensitive Cl- channel on the apical membrane, accounting for a potentiated response. PMID- 3003155 TI - Role of protein kinases in stimulation of human polymorphonuclear leukocyte oxidative metabolism by various agonists. Differential effects of a novel protein kinase inhibitor. AB - Isoquinoline sulfonamides have recently been shown to exert novel inhibitory effects on mammalian protein kinases by competitively binding to the ATP substrate site (Hidaka, H., M. Inagaki, S. Kawamoto, and Y. Sasaki, 1984, Biochemistry, 23: 5036-5041). We synthesized a unique analog of the previously reported compounds, 1-(5-isoquinolinesulfonyl) piperazine (C-I), in order to assess the role of protein kinases in modulating the agonist-stimulated oxidative burst of human polymorphonuclear leukocytes (PMN). Compound C-I, at micromolar concentration, markedly inhibited the release of superoxide anion from human PMN stimulated with phorbol myristate acetate or the synthetic diacylglycerol, 1 oleoyl-2-acetyl glycerol. These data are consonant with previously reported data which indicate that the calcium and phospholipid-dependent protein kinase, protein kinase C, serves as the intracellular receptor for these agonists. In contrast, superoxide anion production stimulated by the complement anaphylatoxin peptide C5a or the synthetic chemotaxin formyl-methionyl-leucyl-phenylalanine were not inhibited by C-I. These data suggest that parallel pathways exist for the agonist-stimulated respiratory burst of human neutrophils, only one of which utilizes the calcium and phospholipid-dependent protein kinase. PMID- 3003157 TI - Identification of the thrombin receptor on human platelets by chemical crosslinking. AB - To identify the molecular site of thrombin binding to the platelet membrane, we covalently linked 125I-thrombin to platelets by using the bifunctional chemical cross-linking agents disuccinimidyl suberate and dithiobis(succinimidyl propionate). The proteins cross-linked to 125I-thrombin by this method were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and followed by autoradiography. Two radiolabeled thrombin complexes were identified, a major species of Mr approximately 200,000 and a minor one of Mr approximately 400,000. Hirudin prevented the formation of both complexes. The radioactivity of the approximately 200,000-Mr complex was always 7-10-fold greater than the radioactivity of the approximately 400,000-Mr complex regardless of the thrombin concentration to which the platelets were exposed (0.1-29 nM). Although 125I thrombin complexes generated with thrombasthenic platelets (lacking glycoprotein IIb/IIIa) were indistinguishable from normal, no complexes appeared when Bernard Soulier platelets (lacking glycoprotein Ib [GPIb]) were used. Complex formation was blocked by rabbit antiglycocalicin antiserum, but not by the monoclonal antibody 6D1, which is directed against the site on GPIb where von Willebrand factor (vWf) binds in the presence of ristocetin. Although cross-linking studies suggested that vWf might partially inhibit thrombin binding to platelets, this was not confirmed by equilibrium binding studies in the presence of vWf and ristocetin. The data suggest, therefore, that at all thrombin concentrations binding occurs at the same membrane site, despite evidence from equilibrium studies for high and low affinity classes of receptors, and that the approximately 400,000-Mr complex is simply a dimer of the approximately 200,000 Mr species. We conclude that the membrane site to which thrombin binds is the glycocalicin portion of platelet GPIb at a site remote from the point of ristocetin-dependent vWf binding. PMID- 3003158 TI - Adenine nucleotides and 5-hydroxytryptamine released by aggregating platelets inhibit adrenergic neurotransmission in canine coronary artery. AB - The purpose of this study was to determine the effect of vasoactive substances released by aggregating platelets on adrenergic neurotransmission in canine coronary arteries. Isometric tension was recorded in isolated ring segments of coronary artery denuded of endothelium and the release of [3H]norepinephrine was measured from strips of coronary artery preincubated with the radiolabeled transmitter. Transmural electrical field stimulation and exogenously added norepinephrine caused beta adrenergic relaxations of coronary rings contracted by prostaglandin F2 alpha. In coronary rings further contracted by the addition of aggregating platelets in numbers less than that present in blood, the response to electrical stimulation was inhibited and the sensitivity to norepinephrine reduced. Micromolar concentrations of adenosine diphosphate, adenosine triphosphate, and 5-hydroxytryptamine were released by platelets under these experimental conditions. The reduced response to electrical stimulation was in part due to inhibition of the stimulated release of [3H]-norepinephrine. The combination of the serotonergic antagonist, methiothepin, and the purinergic antagonist, theophylline, attenuated the inhibition of the responses of coronary rings; either antagonist alone failed to do so, but did significantly block the reductions caused by 5-hydroxytryptamine and adenosine diphosphate, respectively. In addition, only the combination of the two antagonists significantly attenuated the inhibition of norepinephrine release caused by platelets. These data suggest that both adenine nucleotides and 5-hydroxytryptamine are important mediators of the prejunctional and postjunctional inhibition of coronary beta adrenergic neurotransmission caused by platelets. PMID- 3003159 TI - Humoral hypercalcemia of malignancy. Release of a prostaglandin-stimulating bone resorbing factor in vitro by human transitional-cell carcinoma cells. AB - Secretion by tumor cells of circulating bone-resorbing factors may frequently underlie the hypercalcemia that occurs in patients with malignancy. Efforts to identify the responsible mediators have been hampered by a lack of available human tumor cell systems suitable for study of the pathogenesis of the humoral hypercalcemia syndrome. We have established a transitional-cell carcinoma (TCC) line in vitro from a patient with humoral hypercalcemia. These cells are tumorigenic and cause hypercalcemia in athymic nude mice. Culture medium conditioned by TCC cells contains potent bone-resorbing activity in vitro, the physical and biological properties of which are similar to those of bone resorbing activity present in the original patient's urine. The bone-resorbing activity of the TCC factor is accompanied by increased prostaglandin release from bone and is blocked by indomethacin and calcitonin. The TCC-derived bone resorbing activity coelutes with prostaglandin-stimulating activity during gel filtration with an approximate molecular weight of 15,000. This activity is nondialyzable, stable to concentrated urea and reducing agents, and destroyed by boiling. The TCC factor does not increase cyclic AMP production in bone or kidney bioassays and does not exhibit transforming growth factor activity. We conclude that a unique macromolecular factor released by TCC cells causes bone resorption by a mechanism dependent upon stimulation of bone cell cyclooxygenase, and that this factor is the probable cause of the hypercalcemia in vivo. The TCC cell line provides a new model for study of the human humoral hypercalcemia syndrome. PMID- 3003160 TI - Axo-glial dysjunction. A novel structural lesion that accounts for poorly reversible slowing of nerve conduction in the spontaneously diabetic bio-breeding rat. AB - Biochemical abnormalities in peripheral nerve are thought to precede and condition the development of diabetic neuropathy, but metabolic intervention in chronic diabetic neuropathy produces only limited acute clinical response. The residual, metabolically unresponsive neurological deficits have never been rigorously defined in terms of either persistent metabolic derangements or irreversible structural defects because human nerve tissue is rarely accessible for anatomical and biochemical study and experimentally diabetic animals do not develop the structural hallmarks of human diabetic neuropathy. Detailed neuroanatomical-functional-biochemical correlation was therefore undertaken in long-term spontaneously diabetic BB-Wistar rats that functionally and structurally model human diabetic neuropathy. Vigorous insulin replacement in chronically diabetic BB rats essentially normalized both the sural nerve fiber caliber spectrum and the decreased sciatic nerve myo-inositol and (Na,K)-ATPase levels generally associated with conduction slowing in diabetic animals; yet, nerve conduction was only partially restored toward normal. Morphometric analysis revealed a striking disappearance of paranodal axo-glial junctional complexes that was not corrected by insulin replacement. Loss of these strategic junctional complexes, which are thought to limit lateral migration of axolemmal Na channels away from nodes of Ranvier, correlates with and can account for the diminished nodal Na permeability and resultant nodal conduction delay characteristic of chronic diabetic neuropathy in this animal model. PMID- 3003161 TI - Coronary vascular occlusion mediated via thromboxane A2-prostaglandin endoperoxide receptor activation in vivo. AB - The use of enzyme inhibitors to clarify the role of thromboxane A2 in vasoocclusive disease has been complicated by their non-specific action. To address this problem we have examined the effects of thromboxane A2/prostaglandin endoperoxide receptor antagonism in a canine model of platelet-dependent coronary occlusion. Two structurally distinct thromboxane A2/prostaglandin endoperoxide receptor antagonists, 3-carboxyl-dibenzo (b, f) thiepin-5,5-dioxide (L636,499) and (IS-(1 alpha,2 beta(5Z),3 beta,4 alpha))-7-(3-((2-((phenylamino) carbonyl)hydrazino)methyl)-7- oxabicy-clo(2.2.1)-hept-2-yl)-5-heptenoic acid (SQ 29,548), were studied to ensure that the effects seen in vivo were mediated by receptor antagonism and did not reflect a nonspecific drug effect. Both compounds specifically inhibited platelet aggregation induced by arachidonic acid and by the prostaglandin endoperoxide analogue, U46619, in vitro and ex vivo, and increased the time to thrombotic vascular occlusion in vivo. When an antagonist (L636,499) was administered at the time of occlusion in vehicle-treated dogs, coronary blood flow was restored. In vitro L636,499 and a third antagonist, 13 azaprostanoic acid, specifically reversed endoperoxide-induced platelet aggregation and vascular smooth muscle contraction. Neither compound altered cyclic AMP in platelet-rich plasma before or during disaggregation. Therefore, reversal of coronary occlusion may reflect disaggregation of platelets and/or relaxation of vascular smooth muscle at the site of thrombus formation through specific antagonism of the thromboxane A2/prostaglandin endoperoxide receptor. Thromboxane A2/prostaglandin endoperoxide receptor antagonists are compounds with therapeutic potential which represent a novel approach to defining the importance of thromboxane A2 and/or endoperoxide formation in vivo. PMID- 3003163 TI - Human recombinant interleukin 1 stimulates collagenase and prostaglandin E2 production by human synovial cells. AB - The pathogenesis and progression of rheumatoid arthritis involves the production of biologically active lymphokines and monokines. Of these, interleukin 1 (IL-1) has been somewhat of a controversial molecule because it seems to evoke various biological responses in several different tissues. In these studies we demonstrate that three biological properties of human monocyte-derived IL-1 (T lymphocyte activation and human synovial cell prostaglandin E2 and collagenase production) co-purify. The complementary DNA for the prominent pI 7 form of human IL-1 was expressed, purified, and tested. Any controversy now appears resolved since homogeneous recombinant human IL-1 stimulates prostaglandin E2 and collagenase from human synovial cells as well as activates T cells in vitro. PMID- 3003165 TI - Early diagnosis of hepatocellular carcinoma: usefulness of ultrasonically guided fine-needle aspiration biopsy. AB - The results of various diagnostic procedures performed on six cases with solitary and minimal (less than 2 cm) hepatocellular carcinoma (HCC) that was later surgically resected were comparatively reviewed. The tumor was detected in 6/6 patients by ultrasonography, 1/6 by scintigraphy, 2/6 by CT, and 4/6 by angiography, and hepatocellular carcinoma was diagnosed in 3/6 by angiography and 6/6 by ultrasonically guided fine-needle aspiration biopsy. Since there are not a few HCCs that can be detected only by ultrasonography, fine-needle aspiration biopsy with ultrasound guidance is strongly recommended to confirm cases with a minimal lesion in the liver visualized only on a sonogram. PMID- 3003162 TI - Basal phosphatidylinositol turnover controls aortic Na+/K+ ATPase activity. AB - To determine whether basal phosphoinositide turnover plays a role in metabolic regulation in resting rabbit aortic intima-media incubated under steady state conditions, we used deprivation of extracellular myo-inositol as a potential means of inhibiting basal phosphatidylinositol (PI) synthesis at restricted sites and of depleting small phosphoinositide pools with a rapid basal turnover. Medium myo-inositol in a normal plasma level was required to prevent inhibition of a specific component of basal de novo PI synthesis that is necessary to demonstrate a discrete rapidly turning-over [1,3-14C]glycerol-labeled PI pool. Medium myo inositol was also required to label the discrete PI pool with [1-14C]arachidonic acid (AA). The rapid basal turnover of this PI pool, when labeled with glycerol or AA, was not attributable to its utilization for polyphosphoinositide formation, and it seems to reflect basal PI hydrolysis. Depleting endogenous free AA with medium defatted albumin selectively inhibits the component of basal de novo PI synthesis that replenishes the rapidly turning-over PI pool. A component of normal resting energy utilization in aortic intima-media also specifically requires medium myo-inositol in a normal plasma level and a free AA pool; its magnitude is unaltered by indomethacin, nordihydroguaiaretic acid, or Ca2+-free medium. This energy utilization results primarily from Na+/K+ ATPase activity (ouabain-inhibitable O2 consumption), and in Ca2+-free medium deprivation of medium myo-inositol or of free AA inhibits resting Na+/K+ ATPase activity to a similar degree (60%, 52%). In aortic intima-media basal PI turnover controls a major fraction of resting Na+/K+ ATPase activity. PMID- 3003166 TI - Depth of penetration in periodontal pockets with oral irrigation. AB - The purpose of this study was to determine the effectiveness of the Water Pik oral irrigator as a vehicle for delivering an aqueous solution into periodontal pockets. Plaque-disclosing dye diluted with sterile saline solution was applied with the irrigator toward the gingival margins of teeth at 90 degrees and at 45 degrees prior to their extraction. The mean % penetration measured between a reference notch at the gingival crest and the periodontal ligament at the bottom of the pocket showed no statistical difference between the two angles of application. Penetration ranged from 44% to 71%, the lowest being into pockets 4 7 mm; higher mean penetration was noted in both subgroups 0-3 and greater than 7 mm. No statistical difference was found between proximal and facial or lingual surfaces, maxilla and mandible, existence of tooth contact, and proximal tissue contour or consistency. The mean % penetration was independent of pocket depth (chi 2 analysis). Correlation between pocket depth and mean penetration was low for all but one subgroup (90 degrees application and pockets greater than 7 mm). The results suggest that the oral irrigator will deliver an aqueous solution into periodontal pockets and will penetrate on average to approximately half the depth of the pockets. PMID- 3003164 TI - Mapping of the X-linked agammaglobulinemia locus by use of restriction fragment length polymorphism. AB - A molecular linkage analysis in 11 families with X-linked agammaglobulinemia (XLA) localized the XLA gene to the proximal part of the long arm of the human X chromosome. Significant linkage was detected between XLA and loci defined by two polymorphic DNA probes called 19-2 for the DXS3 locus and S21 for the DXS17 locus. Both localize to the region Xq21.3-Xq22. Most likely recombination distances (theta) and associated logarithm of the odds (lod) scores for the XLA DXS3 and XLA-DXS17 pairs were theta = 0.04 morgans (lod, 3.65) and theta = 0 (lod, 2.17), respectively. Tight linkage between XLA and the locus DXS43 defined by the X short arm probe D2 (localized to Xp22-Xp21) was strongly excluded and we obtained no evidence for significant linkage between XLA and any other X short arm probe. The probe pair 19-2 and S21 should be informative for molecular linkage-based analysis of XLA segregation in the majority of families afflicted with this disorder. PMID- 3003167 TI - The localization of cytochrome oxidase in the LGN and striate cortex of postnatal kittens. AB - The distribution of cytochrome oxidase (C.O.) was examined in the lateral geniculate nucleus of the kitten during the first postnatal month and compared with the adult pattern. During the first week, most of the C.O. was localized within the perikarya of geniculate neurons. Perigeniculate neurons had darkly reactive dendrites as well as perikaya. A population of relatively large, darkly reactive neurons became distinguishable around the end of the first week, as the level of reactivity diminished to moderate-to-light within most medium and small neurons. On the basis of their relative size and pattern of distribution, most of the darkly reactive neurons are likely to represent ones that will later have class 1 morphology and develop Y receptive field properties. These cells normally undergo rapid growth earlier, and their growth is more adversely affected by early short-term monocular suture than other classes of less reactive geniculate neurons. Thus, in the LGN of developing kitten, C.O. histochemistry may be used as a functional marker for future class 1 Y-cells. The reactivity of the neuropil gradually increases as synapses with dendrites mature. At the electronmicroscopic level the increased reactivity of the neuropil is due mainly to an increase in the number of reactive mitochondria localized within the growing dendrites. In the developing striate cortex of postnatal kittens dark reactivity is localized in the outer part of layer II for the first 2 weeks and then disappears. Dark reactivity gradually increases in layer IV after the third week. The changes in C.O. reactivity accompany pathway-specific physiological and anatomical changes that occur during early postnatal development. PMID- 3003168 TI - Histopathological classification of nephroblastomas in slaughtered swine. AB - A histological classification of 74 nephroblastomas found in slaughtered swine is reported. The tumours were classified into 4 main types; nephroblastic (29 cases); epithelial (38 cases); mesenchymal (one case), and miscellaneous (6 cases), on the basis of their histological characteristics and especially on their relative contents of epithelial and mesenchymal elements. The nephroblastic type was further divided into 3 subtypes: nephroblastic only (one case), nephroblastic predominance 13 cases) and nephroblastic equal to epithelial (15 cases). Similarly, the epithelial type was classified into two subtypes: epithelial only (one case) and epithelial predominance (37 cases). The histological features of these tumours were compared with those of human nephroblastoma. The classification of swine nephroblastomas made in this study closely resembles that of human tumours. Two examples of the nephroblastic type were metastasized but none of the other types. PMID- 3003169 TI - Pathogenesis of infectious bronchitis nephritis. 1. Morphometric analysis of kidney proximal tubular epithelium in chickens. AB - The ultrastructure of the proximal tubular epithelial cells in chicken kidneys was examined throughout the course of an experimental infection with infectious bronchitis virus. A quantitative assessment of the structural changes in the cells was related to these in normal cells. Significant alterations were detected in the membrane structures and in the mitochondria. There was a reduction in surface area of the microvillus membrane, the basolateral membrane and the apical tubular membrane. There were alterations in the shape of mitochondrial profiles and a decrease in the volume density of mitochondria. Vesicular structures, which are a possible site of viral release, were observed in the lateral surface of cells. These changes in the functional components of the cells indicate impaired transport of ions and water. The results demonstrate the value of stereological methods for the study of viral-host cell interactions in the pathogenesis of viral disease. PMID- 3003170 TI - Relationship between inhibition of cardiac muscle phosphodiesterases, changes in cyclic nucleotide levels, and contractile response for CI-914 and other novel cardiotonics. AB - In the present study, the inhibitory effects of CI-914, a novel cardiotonic agent, as well as other recently identified positive inotropic agents and reference phosphodiesterase (PDE) inhibitors on the different molecular forms of cardiac PDE were examined, and correlated with changes in tissue levels of cyclic AMP and cyclic GMP, and the contractility of isolated guinea pig left atria produced by these agents. CI-914 was found to be a potent, selective inhibitor of peak III PDE, which is a low Km, cyclic AMP-specific form of the enzyme, with little effect on peak I or peak II PDE, both of which hydrolyze cyclic AMP as well as cyclic GMP. CI-914 also exerts a selective effect on cyclic nucleotides; increasing cyclic AMP levels in a concentration-dependent manner, while having no significant effect on cyclic GMP levels. Increases in contractility produced by CI-914 correlated with changes in the tissue level of cyclic AMP. Non-selective phosphodiesterase inhibitors such as theophylline, and less selective inhibitors such as papaverine, carbazeran, and milrinone increased tissue levels of both cyclic AMP and cyclic GMP. Increases in cyclic GMP, or the cyclic AMP/cyclic GMP ratio, did not appear to have an effect on contractility. These results provide support for the hypothesis that CI-914 increases contractility by selectively inhibiting the activity of the peak III phosphodiesterase. These results also indicate that cyclic AMP and cyclic GMP do not act in an opposing "yin-and-yang" fashion to regulate atrial contractility. PMID- 3003172 TI - Prophylactic dental treatment for a patient with vitamin D-resistant rickets: report of case. AB - Spontaneous oral dental abscesses in caries-free teeth has been a common sequela in patients with vitamin D-resistant rickets (VDRR). A successful attempt has been made to prevent such abscesses in a 41/2-year-old boy with VDRR by covering susceptible teeth with chrome crowns. PMID- 3003171 TI - Purification of protein kinase C from bovine rod outer segments. AB - Rod outer segments (ROS) from bovine retinae were found to have high levels of calcium/phospholipid dependent protein kinase (protein kinase C). Protein kinase C behaves as an extrinsic membrane protein and phosphorylates rhodopsin in a calcium-dependent manner. The abundance of protein kinase C in ROS is similar to that of rhodopsin kinase. Its ability to phosphorylate rhodopsin in ROS membranes suggests protein kinase C may play an important role in the regulation of signal transduction in the ROS. The limited set of extrinsic membrane proteins and abundance of protein kinase C makes this tissue an extremely useful source to purify protein kinase C. The extrinsic membrane protein fraction has 6-7 U protein kinase C activity per mg protein, and the enzyme is quite stable apparently due to the lack of proteases in the preparation. A procedure was developed using phosphatidylserine- and calcium-dependent binding of protein kinase C to phenyl-Sepharose in low ionic strength buffer to resolve protein kinase C and other calcium-binding proteins from the majority of extrinsic membrane proteins. Protein kinase C was eluted using EGTA, and peak fractions directly loaded onto a DEAE-cellulose column. The protein kinase C peak eluted from the ion-exchange column was pooled and had a specific activity greater than 1,000 nmol phosphate transferred to histone per min per mg protein with a recovery of 25 percent of the starting activity. The procedure to purify protein kinase C from ROS is simple and can be completed in one day. PMID- 3003173 TI - A comparison of the OHI-S and the PHP in an oral hygiene program. AB - This study examined two critical measurement properties of the Simplified Oral Hygiene Index (OHI-S) and the Patient Hygiene Performance method (PHP). Both scales proved to be sufficiently sensitive to decreases in plaque. PMID- 3003174 TI - Inorganic pyrophosphatase activity of purified bovine pulp alkaline phosphatase at physiological pH. AB - At physiological pH, the hydrolytic activity of purified bovine pulp alkaline phosphatase toward phosphorus compounds was observed to be in the order of inorganic pyrophosphate greater than beta-glycerophosphate greater than phosphorylcholine greater than p-nitrophenylphosphate greater than glucose-6 phosphate. Optimum pH of the enzyme toward inorganic pyrophosphate was shown to be 8.5, with around 60% of the activity at pH 7.5. The activity was increased by the addition of Mg2+, but a different pattern of activation was observed between pH 7.5 and 8.5. PMID- 3003175 TI - Seasonal prevalence of erythema infectiosum. PMID- 3003177 TI - In vivo and in vitro assessment of the role of leukotriene B4 as a mediator of rat cutaneous late-phase reactions. AB - Mast cell-dependent late-phase reactions (LPR) occur in rat skin and are characterized histologically by an early (1 to 8 hours) neutrophil-rich infiltrate, which is essential to a later (24 hours) infiltration by mononuclear cells. Although the ability of preformed mast cell-granule constituents alone to elicit LPR is clearly established, the relative pathogenetic contributions of newly generated lipid mediators to rat LPR are unknown. Leukotriene B4 (LTB4) may be generated by stimulated mast cells in a number of species and might potentially contribute to the neutrophil ingress. In order to examine this possibility in a well-characterized animal model of LPR, the capacity of LTB4 to influence rat cutaneous inflammation was studied. LTB4 (0.1 to 100 ng) alone did not induce vasopermeability in rat skin nor potentiate the blueing response to histamine. Intracutaneous LTB4 (0.1 to 100 ng) did not cause significant infiltration of neutrophils 3 to 4, 6 to 8, or 24 hours after injection; increased numbers of mononuclear leukocytes were not appreciated through 24 hours. In the same animals intracutaneous anti-IgE and intact mast cell granules both produced intense biphasic infiltration characteristic of rat LPR. In order to examine if rat polymorphonuclear leukocytes were capable of responding to LTB4, several in vitro studies were performed. Rat peritoneal and peripheral blood neutrophils migrated toward formyl-methionyl-leucyl-phenyl-alanine in vitro but not to purified human or synthetic LTB4. Rat peripheral blood and elicited peritoneal neutrophils bound only 32% and 27%, respectively, of the quantity of [3H]LTB4 bound by human neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003176 TI - [Effect of changes in the catecholamine content in the brain on the chemoreactive properties of the sensorimotor cortex neurons of rats]. PMID- 3003178 TI - Nutrition, cancer, and aging: an annotated review. I. Diet, carcinogenesis, and aging. AB - The interrelationships of diet and carcinogenesis are discussed with the focus on aging. To establish whether the elderly are more susceptible to dietary carcinogens and whether dietary prevention of cancer is a reasonable goal for this population, the mechanisms of chemical carcinogenesis, the age-related metabolic and physiologic changes, and the current cancer preventive dietary strategies are reviewed. Vulnerability to dietary carcinogens results from a combination of factors that may increase or decrease the occurrence of cancer in the elderly, and it is, therefore, a very individualized feature, unpredictable when based solely on a subject's age. Dietary prevention of cancer may be effective in advanced age, and the dietary guidelines of the National Academy of Sciences should be implemented in this population. PMID- 3003179 TI - Synaptic transmission in the rabbit inferior mesenteric ganglion. AB - Electrical properties, cholinergic neurotransmission and non-cholinergic neurotransmission in the rabbit inferior mesenteric ganglion (IMG) in vitro were examined with intracellular recording techniques. A single ganglionic neuron received an average of 42 nicotinic cholinergic synaptic inputs. An atropine sensitive slow excitatory postsynaptic potential not followed by a non cholinergic late slow excitatory postsynaptic potential (LS-EPSP) was observed in 7% of the cells. In 63% of the cells a LS-EPSP insensitive to antagonism of nicotinic and muscarinic receptors was observed following repetitive nerve stimulation. The involvement of substance P (SP) in the genesis of the LS-EPSP was tested by applications of SP, applications of SP antagonists and applications of capsaicin. Neither SP, SP antagonists nor capsaicin affected the LS-EPSP. These findings distinguish the LS-EPSP in the rabbit IMG from its counterpart in the guinea pig IMG where SP has been proposed as the mediator of the LS-EPSP. A late slow inhibitory postsynaptic potential was observed in 13% of the cells. This hyperpolarization followed repetitive nerve stimulation and was insensitive to blockade of cholinergic receptors. There is a marked convergence of subthreshold fast excitatory postsynaptic potentials (F-EPSPs) of both central and peripheral origin onto these cells. The LS-EPSP could provide a mechanism for increasing the likelihood of temporal and/or spatial summation of these fast synaptic inputs, thereby increasing the probability of action potential generation in the ganglion cells. PMID- 3003180 TI - [Conversion of alpha- and beta-receptor functions in rat submandibular glands]. PMID- 3003182 TI - Immunocytochemical localization of Na+,K+-ATPase catalytic polypeptide in mouse choroid plexus. AB - Na+,K+-ATPase plays a central role in the mechanism of cerebrospinal fluid secretion by the choroid plexus. We have used an antiserum to the 100 KD catalytic polypeptide of the enzyme purified from mouse brain (30) to localize the catalytic unit in mouse choroid plexus at the light and electron microscopic levels. Pre-embedding immunostaining with the peroxidase-conjugated second antibody technique showed that microvillar borders facing the ventricle were intensely reactive. In contrast, basal and lateral plasma membrane surfaces were devoid of activity. Identical localization was obtained with a post-embedding procedure in which protein A-gold was used to stain immunoreactive sites on thin sections of Lowicryl-embedded tissue. For comparison, immunogold staining was shown to be restricted to basolateral membranes of kidney medullary ascending thick limbs. The apical localization of Na+,K+-ATPase in choroid plexus is in striking contrast to the almost exclusive basolateral localization seen in other ion-transporting tissues. The immunocytochemical data are completely consistent with physiological data on choroidal epithelial transport and with light microscopic autoradiographic localization of [3H]-ouabain binding sites. PMID- 3003181 TI - Quantitative histochemistry of benzaldehyde dehydrogenase in hepatocellular carcinomas of vinyl chloride-treated rats. AB - Hepatocarcinogenesis in rats treated with several chemicals is associated with changes in aldehyde dehydrogenase (AlDH) activity, particularly heterogeneous expression of a "tumor specific" phenotype that is very active with aromatic aldehydes, e.g., benzaldehyde (Bz). Objectives of this study were first, to determine if liver cancers in vinyl chloride-treated rats also expressed this AlDH phenotype, and second, to quantitate the NAD- and NADP-dependent AlDH activity for the substrates Bz and acetaldehyde (Ac) in the cancers and surrounding tissue. Small cubes of tissue containing well-differentiated hepatocellular carcinoma were obtained from five Sprague-Dawley rats exposed to 2500 ppm vinyl chloride for 55 weeks. An optimized procedure was developed for AlDH histochemistry. Frozen sections were preincubated in nitroblue tetrazolium/acetone and then incubated at 20 degrees C in viscous polyvinyl alcohol media containing buffer, phenazine methosulfate, sodium azide, substrate, coenzyme, and nitroblue tetrazolium. Background activity was evaluated by omission of substrate. Activity was quantitated by computer-assisted microscopic photometry. All five carcinomas had heterogeneous staining of NADP- and NAD dependent BzDH and AcDH activity, with clusters of very high-activity cells. The magnitude of staining in the high-activity neoplastic cells was at least tenfold greater for BzDH-NADP and about twofold greater for BzDH-NAD, AcDH-NADP, and AcDH NAD than the staining in other liver cells. More neoplastic cells had high BzDH than high AcDH activity. Only BzDH-NADP was localized predominantly to the carcinoma. PMID- 3003183 TI - A new sample isolation procedure for microchemical analysis of functional liver cell heterogeneity. AB - In conjunction with the investigation of intercellular compartmentation of liver carbohydrate metabolism, a new procedure for isolation of tissue samples from freeze-dried cryosections was developed. It was designed to permit assessment of functional differences between sinusoids of portal and septal origin, and to extend investigation of liver cell heterogeneity along sinusoids to the level of the structural-functional unit. Application of this procedure, together with microchemical assays of high analytical sensitivity, enabled measurement of 50 individual glucose and glucose-6-P values in a single cross-sectional area of about 0.75 mm2 of a liver unit. Preliminary results on the distribution of glucose and glucose-6-P indicated that, in a state of overall glucose release glucose levels were significantly higher in the center than in the periphery of the unit. Overall glucose release by the liver resulted from both release and uptake of glucose along sinusoids. Glucose-6-P was highest in the periphery and decreased toward the center. Microchemical data, furthermore, indicated possible functional heterogeneity of sinusoids, insofar as both glucose and glucose-6-P gradients were steeper in "portal-central" than in "septal-central" sinusoids. PMID- 3003184 TI - Varicella-zoster viral glycoprotein envelopment: ultrastructural cytochemical localization. AB - The periodate-thiocarbohydrazide silver proteinate (PA-TCH-SP) method was used to study the envelopment process in varicella-zoster virus-infected human melanoma cells. Viral envelopment could be seen at two sites, the nuclear membrane and at virus-induced intracytoplasmic vacuoles. Virus-associated glycoconjugates were detected by the PA-TCH-SP method at the plasmalemma and on the inner membrane of the intracytoplasmic vacuoles. Virion envelopes acquired at the nuclear membrane were PA-TCH-SP negative, whereas those acquired at intracytoplasmic vacuoles were PA-TCH-SP positive. All virions found inside these vacuoles contained periodate reactive envelopes. Release of virions into the extracellular space, where virtually all virions were PA-TCH-SP positive, appeared to be via exocytosis. Thus, the PA-TCH-SP method identifies glycoprotein incorporation at specific cytoplasmic vacuoles distinct from nuclear envelope, endoplasmic reticulum, and Golgi lamellae. These results suggest that envelopment within the cytoplasm is a stage in the assembly of the varicella-zoster virion. PMID- 3003185 TI - Similarity between cytoplasmic inclusions in the paraventricular hypothalamic nucleus neurons of the turtle Mauremys caspica and the cytoplasmic structures as reaction to viral infections (hepatitis) and SIDA ultrastructural tracers. AB - Two types of "inclusions" observed in the neurons of the hypothalamic paraventricular nucleus of the turtle Mauremys caspica, "tubular systems" and "fingerprint-like" structures, may be added to the inventory of those described in apparently normal neurons. Despite their enigmatic significance, we are inclined to think that they could be a morphological expression of viral diseases in the neuron cytoplasm. These structures have a striking similarity with those observed on pathological cells (hepatitis and SIDA). PMID- 3003187 TI - Properties of poliovirus strains isolated from waste waters of nurseries in Prague in 1969-1982. AB - The rct marker and antigenic marker were studied in poliovirus strains of all the three types isolated from waste waters of selected communities of children in Prague in 1969-1982. Only three isolated strains (two of type 2 and one of type 3) i.e. 1.38% of strains from nursery waste waters and 0.99% of strains from the total Prague municipal sewerage system, differed in both markers from prototype vaccine strains. The rct marker was most frequently altered in strains of type 1; antigenic marker varied most often in strains of type 3, less conspicuously in strains of type 2 and minimum changes were found in type 1 strains. The dependence of changes in both studied markers of isolated strains on the interval between vaccination and isolation from waste waters was reflected in the dynamics of changes. The highest dynamics of changes in rct marker and antigenic marker was recorded in type 3 and type 2 strains, minimal dynamics being found in type 1 strains. Study of type 2 strains isolated for a long time in some years showed that the longer the interval between vaccination and isolation, the higher the similarity between the isolated strain and the prototype vaccine strain. PMID- 3003186 TI - [Stimulation-induced changes in the ultrastructure of the synapses in the hippocampus by post-tetanic potentiation]. AB - Within the frame of experimental brain research at present the phenomenon of potentiation (posttetanic potentiation, long-term potentiation) is considered to provide a possible mechanism of learning processes and memory formation on the synaptic level. Tetanic stimulation of the Nucleus septalis fimbrialis produces the posttetanic potentiation in synapses of the hippocampal CA 3-region, whereafter a long-lasting increase in the amplitude of evoked potentials can be recorded. By quantitative electron microscopical morphometry we investigated the morphological changes in synapses of the Stratum radiatum of the hippocampal CA 3 region of rabbits (1 hour following posttetanic stimulation). Tetanic stimulation of the Nucleus septalis fimbrialis induces an enlargement of the postsynaptic spine area significantly by 28% and a decrease of the mean area of presynaptic terminals by 24%. No changed number of synapses were found in comparison to the controls. Furthermore stimulation of the Nucleus septalis fimbrialis induces a transient decrease of synaptic vesicles in various zones of the presynaptic area, especially near the presynaptic membrane. These results demonstrate morphologically detectable changes as a consequence of synaptic activation. Therefore posttetanic potentiation is characterized by various morphologic changes reflecting probably an early phase of the learning process with transient effects on the ultrastructure of the synapses. PMID- 3003188 TI - Adrenergic receptors in hypertension: radioligand binding studies. PMID- 3003189 TI - Decreased myocardial Na+K+ATPase activity in rats with reduced renal mass-saline hypertension. AB - Inhibition of cardiovascular Na,K-pump activity has been shown to promote an increase in the contractile activity of myocardial and vascular smooth muscle and a consequent rise in blood pressure (BP). It has also been shown that vascular Na,K-pump activity and myocardial Na+K+ATPase activity [the energy source for active sodium (Na) and potassium (K) transport] are decreased in rats with various forms of low renin hypertension including rats with reduced renal mass saline (RRM-saline) hypertension. In the present study, left ventricular Na+K+ATPase activity from rats with RRM-saline hypertension was found to be decreased in membranes prepared by two independent methods: deoxycholate, sodium iodide (Nal)-treated microsomal fractions (method 1) and membranes prepared by the hypotonic, lithium bromide (LiBr) method (method 2). Relative to RRM normotensive control rats which drank distilled water, myocardial Na+K+ATPase activity from RRM-saline drinking rats was decreased by 18.2% in membranes prepared by method 1 and 33.6% in membranes prepared by method 2. The apparent affinities of Na+K+ATPase for K and for ouabain were unaltered relative to controls in membranes prepared from these hypertensive rats by method 1, and the sialic acid content and 5'-nucleotidase activity (two putative sarcolemmal markers) were unaltered in membranes from the hypertensive rats, prepared by methods 1 and 2 respectively. The Mg2+ATPase activity of membranes prepared by method 1 was increased in the RRM-saline hypertensive rats but because it was not increased in membranes prepared by method 2 the former observation does not appear to be of any pathophysiological importance. In other experiments, hypertension was reversed in RRM-saline hypertensive rats by restricting their salt intake (substitution of distilled water for drinking).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003190 TI - Effect of acute and chronic captopril infusion on blood pressure in the two kidney, one clip hypertensive rat. AB - The effect on blood pressure (BP) of acute and chronic suppression of angiotensin (ANG) II was studied in the two-kidney, one clip hypertensive rat. Conscious, chronically catheterized rats were given a bolus injection of captopril (2.5 mg/kg) followed by a chronic infusion of either dextrose or captopril (1 mg/kg per h) lasting 5 days. Blood pressure was measured continuously by a computer technique. Following the acute injection of captopril, arterial BP fell from 165.1 +/- 19.4 mmHg (mean +/- s.d.) to a minimum of 137.6 +/- 23.3 mmHg after 15 min. Twelve hours after starting the chronic infusion of captopril, BP fell to a minimum of 112.5 +/- 19.4 mmHg. This was significantly lower than that after the acute injection of captopril. Blood pressure remained lower throughout the 5-day infusion ranging, on the 5th day, from 122.1 +/- 23.4 to 136.0 +/- 30.2 mmHg. In contrast, BP continued to rise in rats given dextrose chronically ranging, on the 5th day, from 163.6 +/- 23.8 to 180.4 +/- 22.5 mmHg. Both the fall in pressure after acute captopril and that after chronic captopril were related to pre treatment levels of plasma renin concentration. These results suggest that in the two-kidney, one clip hypertensive rat ANG II, in addition to its acute vasoconstrictor property, contribute to the hypertension through a secondary effect, the mechanism of which is as yet uncertain. PMID- 3003192 TI - Association of protein kinase C activation with IL 2 receptor expression. AB - Tac antigen (as a measure of the IL 2 receptor) acquisition and regulation by IL 2, an antigen-receptor agonist (anti-T3), phorbol esters, and phytohemagglutinin (PHA) were studied. Phorbol esters stimulated de novo acquisition of Tac antigen, which was associated with the subcellular redistribution of protein kinase C (PK C) from cytosol to particulate membranes of human T lymphocytes. PHA and anti-T3 (alpha-T3) antibody also stimulated a transient redistribution and activation of PK-C that reached a maximum within 20 min after stimulation. Both phorbol esters and alpha-T3 could increase Tac expression and stimulate PK-C translocation on 5 and 12 day activated T cells that were at the G0/G1 stage of the cell cycle due to IL 2 deprivation. Tac antigen-specific mRNA was seen in the nucleus within 2 hr after stimulation. In contrast, IL 2 alone could only increase Tac expression and stimulate PK-C translocation on day 5 but not day 12 activated T cells. IL 2 synergizes with alpha-T3 and phorbol ester for the regulation of Tac expression. Although IL 2 increased expression of Tac, the majority if not all of these receptors possessed low affinity for IL 2. These data suggest that the activation of PK-C is a common transmembrane signal shared by IL 2 and antigen stimulation. The results also imply that PK-C activation is necessary for the regulation of Tac antigen expression. PMID- 3003191 TI - Activation of the Na+/H+ antiport is not required for lectin-induced proliferation of human T lymphocytes. AB - Interaction of some mitogenic lectins and growth factors with the cell surface leads to activation of the Na+/H+ antiport and a resultant cytoplasmic alkalinization. Because amiloride inhibits both Na+/H+ exchange and cell proliferation, it has been hypothesized that activation of the antiport is an obligatory requirement and may, perhaps, be the "trigger" for proliferation. However, concentrations of amiloride which inhibit the antiport also inhibit several other intracellular processes, including protein synthesis and phosphorylation. To determine whether activation of the Na+/H+ antiport is necessary for lectin-induced proliferation, we examined the inhibitory activity of a series of potent amiloride analogs by measuring [3H]thymidine incorporation, cell cycle progression, and induction of the interleukin 2 (IL 2) receptor on human lymphocytes. In medium containing bicarbonate, and at concentrations at least 10 times higher than required to inhibit the antiport, these drugs did not inhibit the proliferative response of human peripheral blood T cells to the mitogen phytohemagglutinin. The amiloride analogs also failed to inhibit induction of the IL 2 receptor. Similarly, with human thymocytes, the amiloride analogs did not inhibit the co-mitogenic effects of the lectins phytohemagglutinin and concanavalin A together with IL 2 or the phorbol ester 12 O-tetradecanoylphorbol-13-acetate. This finding suggests that Na+/H+ exchange through the antiport is not an obligatory requirement for activation or proliferation of human lymphocytes or thymocytes. PMID- 3003193 TI - Identification and characterization of a human T cell line-derived lymphokine with MAF-like activity distinct from interferon-gamma. AB - Culture supernatants of several human T cell leukemia cell lines were screened for macrophage-activating activity (MAF) as defined by induction of tumoricidal activity against human melanoma cells in a 72-hr assay. Two cell lines, MT-2 and C10/MJ2, were found to produce high levels of MAF activity constitutively, but the MT-2 cell line, unlike C10/MJ2, produced little IFN-gamma. This observation was confirmed by Northern blot analysis performed with specific IFN-gamma cDNA probe. The MT-2 cell line thus provides a useful system to evaluate the existence of lymphokines with MAF activity that are distinct from IFN-gamma. The MAF activity produced by MT-2 cells was distinguished from IFN-gamma by the following criteria. MAF activity was not removed by immunoaffinity chromatography with the use of immobilized specific polyclonal antibodies to IFN-gamma and was not neutralized by a monoclonal antibody to IFN-gamma. Heat or acid treatments of IFN gamma resulted in loss of its antiviral activity, but these treatments had no effect on MAF activity. MAF activity was not abolished by polymyxin B sulfate, suggesting that this activity is not mediated by or dependent on LPS. Initial characterization studies performed by using membrane filtration, gel filtration chromatography, and isoelectric focusing indicate that the non-IFN-gamma MAF activity produced by MT-2 cells has an apparent m.w. of 55,000 and an isoelectric point of 5.5. Collectively, these data suggest that the MT-2 human T cell line constitutively produces high levels of MAF and low levels of IFN-gamma and offers a useful source for the further purification of a unique human lymphokine with macrophage-activating activity that is distinct from IFN-gamma. PMID- 3003194 TI - Expression of the transferrin receptor gene during the process of mononuclear phagocyte maturation. AB - The expression of transferrin receptors by blood monocytes, human alveolar macrophages, and in vitro matured macrophages was evaluated by immunofluorescence, radioligand binding, and Northern analysis, using the monoclonal anti-human transferrin receptor antibody OKT9, [125I]-labeled human transferrin and a [32P]-labeled human transferrin receptor cDNA probe, respectively. By immunofluorescence, the majority of alveolar macrophages expressed transferrin receptors (86 +/- 3%). The radioligand binding assay demonstrated the affinity constant (Ka) of the alveolar macrophage transferrin receptor was 4.4 +/- 0.7 X 10(8) M-1, and the number of receptors per cell was 4.4 +/- 1.2 X 10(4). In marked contrast, transferrin receptors were not present on the surface or in the cytoplasm of blood monocytes, the precursors of the alveolar macrophages. However, when monocytes were cultured in vitro and allowed to mature, greater than 80% expressed transferrin receptors by day 6, and the receptors could be detected by day 3. Consistent with these observations, a transferrin receptor mRNA with a molecular size of 4.9 kb was demonstrated in alveolar macrophages and in vitro matured macrophages but not in blood monocytes. Thus, although blood monocytes do not express the transferrin receptor gene, it is expressed by mature macrophages, an event that probably occurs relatively early in the process of monocyte differentiation to macrophages. PMID- 3003195 TI - Regulation of the synthesis of the third component of complement and factor B in cord blood monocytes by lipopolysaccharide. AB - We compared the regulation of C3 and factor B synthesis in cord blood and adult monocytes by using techniques for identification and quantification of newly synthesized proteins, lipopolysaccharide (LPS) from several Gram-negative organisms, and precursors of LPS. Synthesis of C3 and factor B in cord blood monocytes was unaffected by lipid A (the active moiety of LPS extracted by the Westphal procedure). In contrast, adult monocytes increased C3 synthesis by 11.5 fold and factor B synthesis by 3.1-fold in response to LPS. This difference in cord blood monocyte response to LPS was specific in that other LPS-induced monocyte functions (superoxide production and phagocytosis) were stimulated comparably in both cord blood and adult monocytes by LPS. To characterize further this regulatory difference, the roles of LPS precursors, arachidonic acid metabolites, and of factor(s) released by adult monocytes were examined. Precursors of the lipid portion of LPS (lipid X and lipid Y), LPS isolated by trichloroacetic acid extraction, and endotoxin-associated protein (EAP) increased C3 and factor B synthesis in cord blood monocytes. Inhibitors of the lipoxygenase pathway (dexamethasone, ETYA) but not of the cyclooxygenase pathway (indomethacin) abrogated the response of adult monocytes to lipid A and EAP and of cord blood monocytes to EAP. Finally, co-incubation of adult monocytes and cord blood monocytes in LPS-containing medium resulted in enhancement of C3 and factor B synthesis in cord blood monocytes. These data suggest that the difference in LPS response between cord blood and adult monocytes may result from differences in lipid processing or protein recognition of LPS, differences in the production of lipoxygenase pathway products, and/or one or more regulatory factors. The availability of human mononuclear phagocytes which exhibit distinct differences in biosynthetic responsiveness to LPS should permit investigation of the molecular mechanism(s) by which LPS affects C3 and factor B gene expression. PMID- 3003196 TI - Molecular basis of macrophage activation. Expression of the low potential cytochrome b and its reduction upon cell stimulation in activated macrophages. AB - The expression of the novel b-type cytochrome, which is part of the superoxide anion (O2-)-generating system in phagocytes, has been investigated in population of mouse peritoneal macrophages heterogeneous in their capability to produce O2 ). Reduced minus oxidized difference spectra of intact cells showed the appearance of a b-type cytochrome with major peaks in the alpha region at 558 to 559 nm and in the gamma region at 426 to 428 nm. Resident peritoneal macrophages, as well as thioglycollate broth-elicited and Corynebacterium Parvum-activated macrophages and neutrophils expressed about 50 pmol cytochrome b/10(7) cells. In intact macrophages and neutrophils, Na-dithionite reduced greater than 75% of the cytochrome b measurable in disrupted cells. No correlation was found between capability to produce O2-) by different population of macrophages and their content of cytochrome b. When stimulated in strictly anaerobic conditions with phorbol myristic acetate, macrophages activated in vivo by i.p. injection of Corynebacterium Parvum reduced approximately 40% of their total cytochrome b. In resident peritoneal macrophages that produced five times lower amounts of O2-, cytochrome b reduction was instead undetectable. Potentiometric properties of cytochrome b was investigated in macrophage subcellular particles. Both resident and Corynebacterium Parvum-activated macrophages revealed the presence of b chromophores with very low potentials of -255 and -244 mV, respectively, whose content was not different in the two populations. These results show that resident and activated macrophages express the same amount of cytochrome b, but upon stimulation with PMA, activated macrophages recruit a higher number of cytochrome b molecules in parallel with an enhanced production of O2-. PMID- 3003197 TI - A monoclonal antibody against LAV gag precursor: use for viral protein analysis and antigenic expression in infected cells. AB - A murine monoclonal antibody (MAb), named C.V.K., was produced after immunization with highly purified and sonicated lymphadenopathy-associated virus (LAV). No monoclonal antibody was observed with intact virus used as immunogen. C.V.K. MAb recognizes an epitope present on the precursor gag protein of 55 kilodaltons. Western blot analysis and pulse-chase experiments support the interpretation that after p55 cleavage into p25, p18, and p13, only p18 expresses this epitope. C.V.K. MAb selectively stained only LAV-infected lymphocytes. This intracytoplasmic staining appears 3 days after the infection and is correlated with reverse transcriptase activity. Neither membrane immunofluorescence of infected lymphocytes nor neutralizing activity was observed with C.V.K. MAb. These facts suggest that p55 and p18 are not expressed at the cell membrane or on the viral envelope. C.V.K. MAb should prove useful not only in the purification of core proteins but also for detecting infected cells producing the virus in suspension or on histologic sections. PMID- 3003198 TI - Natural cytotoxicity against mouse hepatitis virus-infected target cells. I. Correlation of cytotoxicity with virus binding to leukocytes. AB - Spleen cells from uninfected control mice selectively lysed BALB/c 3T3 fibroblasts infected with mouse hepatitis virus (MHV), a murine coronavirus. Lysis of infected cells occurred within 3 hr, and histocompatibility between effector and target cells was not required. This natural, cell-mediated, virus associated cytotoxicity differed from NK cell- and T cell-mediated lysis. Spleen cells from animals infected with MHV were enriched in NK activity and were more cytotoxic to YAC-1 target cells, but did not show enhanced cytotoxicity for MHV infected target cells. Spleen cells from beige mice, which are deficient in NK cell activity, were able to lyse MHV-infected target cells, as were spleen cells from nude mice, which are deficient in T cell activity. Lysis of MHV-infected target cells could be mediated by cells from the spleen and, to a lesser extent, by cells from the bone marrow, but not by resident peritoneal cells or thymocytes. We suggest the term "virus killer (VK) activity" for this phenomenon. VK activity of splenocytes from different mouse strains correlated with the ability of the splenocytes to bind purified radiolabeled MHV virions. MHV virions caused agglutination of spleen leukocytes from susceptible mouse strains, indicating that leukocyte agglutination or adsorption may provide a useful assay for coronaviruses such as MHV which lack hemagglutinating activity. SJL mouse splenocytes did not bind MHV and did not lyse infected targets. MHV bound relatively well to splenocytes of other mouse strains, but poorly to thymocytes and erythrocytes. Binding of MHV to leukocytes was not influenced by 6 mM EDTA or EGTA, indicating a lack of requirement for Mg++ or Ca++. VK activity was also resistant to EDTA and EGTA, in contrast to NK activity, which was sensitive to those chelating agents. VK activity was also unaffected by actinomycin D, cycloheximide, or puromycin, indicating that new protein synthesis was not required for lysis. Antibody to interferon-alpha/beta did not block lysis, nor was there substantially enhanced lysis mediated by leukocytes from mice infected with virus and thus exposed to high levels of interferon. VK activity was blocked by antibody directed against the peplomeric glycoprotein E2 of MHV. VK activity required infected target cells, because cells with adsorbed MHV virions were not lysed by splenocytes.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3003200 TI - Utility of formaldehyde fixation for flow cytometry and inactivation of the AIDS associated retrovirus. AB - To maximize safety in the setting of an increasing number of requests for flow cytometric analysis of specimens potentially contaminated with the AIDS retrovirus, we evaluated some commonly used fixatives for their ability to inactivate the infectious potential of the virus. We found that both formaldehyde (0.37% v/v) and paraformaldehyde (0.5% w/v) completely inactivated the infectious activity of both free and cell-associated lymphadenopathy associated virus (LAV), the etiologic agent for the acquired immunodeficiency syndrome (AIDS). Based on encouraging preliminary results we formally evaluated the effect of formaldehyde fixation on flow cytometric parameters. In addition to inactivating LAV, 0.37% formaldehyde in phosphate buffered saline preserved light scatter and fluorescence properties of cells stained with fluorescein isothiocyanate (FITC) and beta-phycoerythrin (PE) conjugated monoclonal antibodies. These findings suggest that formalin fixation may be useful for laboratories performing flow cytometric analysis of specimens potentially contaminated with the AIDS virus. PMID- 3003199 TI - Natural cytotoxicity against mouse hepatitis virus-infected cells. II. A cytotoxic effector cell with a B lymphocyte phenotype. AB - The effector cell in mouse spleen which mediates natural cytotoxicity against mouse hepatitis virus (MHV)-infected target cells was characterized. The target cells were MHV-infected BALB/c 3T3, and the assay time was 3 hr. The effector cell, designated virus killer (VK) cell for the purpose of discussion, had the following phenotype: lymphocyte morphology, plastic-nonadherent, nylon wool adherent, nonphagocytic, cyclophosphamide-sensitive; by antibody plus complement (C) depletion studies, it was asialo GM1-, NK 1.2 alloantigen-negative, Thy-1.2-, Lyt-5-, and macrophage antigen-negative; by rosetting techniques, it was Fc receptor-positive and surface Fab+; by flow cytometry (FACS) analysis, it was Lyt 2-, MAC-1-, Ia+, IgG (gamma)+, IgM (mu)+, IgD (delta)+, and B cell lineage antibody B-220+. NK cells, measured for cytotoxicity on YAC-1 cells, were similarly tested and were found to differ from the VK cell in the following properties: nylon wool-nonadherent, asialo GM1+, NK alloantigen-positive, Lyt-5+, surface Fab-, MAC-1+, Ia-, IgG-, IgM-, IgD-, and B-220-. The VK effector cell had a phenotype highly distinguishable from NK cells, effectors most commonly associated with antiviral natural cytotoxicity. The VK cell had a phenotype identical to that of a B lymphocyte and was identified as such. Although the effector cells displayed cell surface antibody, the antibody did not appear to be involved in lysis, because lysis could not be blocked by F(ab)'2 directed against Fab, mu, or delta. Cytotoxicity was more likely associated with recognition of the B lymphocyte surface by the MHV glycoprotein E2, as shown in the accompanying companion paper. This is the first demonstration that natural cytotoxicity can be mediated by B lymphocytes. PMID- 3003201 TI - A novel immunoperoxidase procedure of using human monoclonal antibodies for the detection of cellular antigens in tissue sections. AB - A sensitive immunoperoxidase method was utilized to detect binding of human monoclonal antibodies to cellular antigens in formalin-fixed and paraffin embedded tissue sections. The method was a 2-layer system utilizing biotin labeled human monoclonal antibody and an avidin-biotin-horseradish peroxidase complex (ABC). A comparative study of this and a 4-layer peroxidase antiperoxidase (PAP) staining method was made. In the PAP system, the monoclonal was followed by rabbit anti-human IgG, swine anti-rabbit IgG as the bridge antibody and rabbit PAP complex. In both cases, the reaction was visualized by the use of aminoethylcarbazole (AEC) as chromogens. The former procedure was almost as sensitive and, most importantly eliminated an inherent problem associated with the latter namely the difficulty in distinguishing binding of the primary human monoclonal antibody from endogenous human Igs in tissue section. In addition, the 2-stage ABC procedure by comparison requires almost half the time. PMID- 3003202 TI - An improved method to quantitate phagocytosis with mineral oil particles which avoid cell flotation. AB - We have shown that actively phagocytosing human polymorphonuclear leukocytes (PMN) float on top of the incubation medium when Oil Red O containing paraffin oil particles (density 0.8740) are used as the phagocytosable material. This implies that the quantitation of phagocytosis based on the recovery of Oil Red O in phagocytosing cells pelleted after centrifugation would be underestimated. We therefore prepared particles of progressively increasing density by mixing paraffin oil (density 0.8740) with silicon oil (density 1.0802). Cell flotation also occurred with paraffin oil-silicon oil particles and could be avoided only when the density of the particles used had reached 0.9952 g/cm3. Paraffin oil silicon oil particles of a density sufficient to dissolve the dye Oil Red O were therefore used to quantitate both the initial rate of phagocytosis and the phagocytic capacity of human PMN. With this assay the initial rate of phagocytosis was found to be 400 micrograms paraffin oil-silicon oil/min/5 X 10(6) PMN, which is about 20 times higher than that reported for the same cell type using paraffin oil particles. The calculated maximum phagocytic capacity was 2.5 mg paraffin oil-silicon oil/5 X 10(6) PMN. The uptake of paraffin oil-silicon oil particles was sensitive to inhibitors of phagocytosis, such as N ethylmaleimide, papaverine and cytochalasin B, in a dose dependent manner. The assay also permitted the detection of increased phagocytosis such as occurs in myeloperoxidase deficient PMN. PMID- 3003203 TI - Visualization of the receptor for transferrin on K562 cells by a rosette-forming assay. AB - A method is described to visualize the receptor for transferrin by a rosette forming assay. K562 cells, a human pre-erythroid leukemia cell line, were used as a source of receptor-positive cells. Bovine erythrocytes coated with human transferrin by the CrCl3 method were used as indicator cells. Rosette formation occurred at 37 degrees C but not at 4 degrees C, and was inhibited by soluble transferrin in a dose-dependent manner. Human lactoferrin, which is known to have considerable amino acid sequence homology with transferrin, did not inhibit rosette formation with transferrin-coated bovine erythrocytes. A variety of blocking and non-blocking monoclonal antibodies against the human transferrin receptor (42/6, B3/25 and OKT9) inhibited rosette formation. Rosettes are able to withstand low-speed centrifugation (130 X g, 2 min) making this method potentially useful for cytochemical identification of receptor-positive or receptor-negative cells. Also, it was possible to enrich receptor-positive cells by separation of rosette-forming from non-rosette-forming cells using a density gradient centrifugation technique. PMID- 3003204 TI - The effect of delta-9-tetrahydrocannabinol and 11-hydroxy-delta-9 tetrahydrocannabinol on T-lymphocyte and B-lymphocyte mitogen responses. AB - Previous studies have shown that delta-9-tetrahydrocannabinol (THC) suppresses T lymphocyte proliferation when added to human cell cultures. We report that THC when added to mouse splenocyte cultures suppressed T-lymphocyte (Con A, PHA) and B-lymphocyte (LPS) mitogen-induced proliferation. Although the ED50 concentrations (5 micrograms/ml; 1.6 X 10(-5)M) of THC were similar for suppressing all three mitogen responses, higher threshold concentrations of drug were required to effect suppression of the T-lymphocyte mitogen responses. Complete suppression of T- and B-lymphocyte responses was achieved with THC concentrations (8 micrograms/ml or 2.6 X 10(-5)M) which were not directly toxic as judged by vital dye exclusion. The hydroxylated metabolite of THC, 11-hydroxy THC, was observed to be much less potent in the inhibition of lymphocyte proliferation. However, as with the parent compound, B-lymphocyte responses appeared to be the most affected by the drug. Additional studies demonstrated that both T- and B-lymphocyte proliferation is rapidly suppressed following THC treatment, not affected by a 24 hr. pretreatment with THC, and not as readily suppressed by THC in cultures containing 20% serum. Thus, THC appears to inhibit both T- and B-lymphocyte proliferation with B-lymphocyte responses displaying greater inhibition at lower drug concentration. The 11-hydroxy metabolite is much less suppressive in this system than the parent compound. PMID- 3003205 TI - Pathogenesis of shigella diarrhea: evidence for an N-linked glycoprotein shigella toxin receptor and receptor modulation by beta-galactosidase. AB - Pathogenic mechanisms in infectious diseases often involve specific receptor ligand interactions of cells and soluble molecules. To further elucidate structure-function relations for shigella toxin receptors, we studied binding of purified 125I-labeled toxin and biologic response under various conditions in an experimental model using HeLa cells. Response to toxin was reversibly inhibited by treatment of cells with trypsin or tunicamycin, an inhibitor of glycoprotein synthesis that also significantly inhibited toxin binding, a result indicating that the receptor is an N-linked glycoprotein. Removal of terminal beta-linked galactose from the HeLa cell surface with beta-galactosidase increased toxin binding and activity, and it also potentiated the effects of lysozyme and wheat germ agglutinin, which recognize oligomeric beta 1----4-linked N-acetyl-D glucosamine and inhibit toxin activity as well. Incubation of cells with beta-N acetylglucosaminidase, which cleaves terminal beta-linked N-acetyl-D-glucosamine, inhibited toxin activity. Effects of beta-galactosidase were reversed by readdition of galactose to cell-surface oligosaccharide acceptors. The data demonstrate that alterations of a single sugar on cell-surface glycoproteins may have a dramatic effect on receptor activity and indicate that shigella toxin is a sugar-binding protein with specificity for beta 1----4-linked N-acetyl-D glucosamine. PMID- 3003207 TI - Temporal and geographic patterns of isolates of nonpolio enterovirus in the United States, 1970-1983. AB - Surveillance data on nonpolio enterovirus (NPEV) from the Centers for Disease Control (Atlanta, Georgia) for the United States from 1970 to 1983 were analyzed for the temporal and geographic patterns of the most common types of NPEV isolated. The number of isolates varied from year to year, partly because of variation in the number of reporting laboratories and partly because of true variation in the rate of isolation. The most common types isolated also varied from year to year, yet the 15 most common types over the entire 14-year period accounted for 65%-89% of isolates for a given year. The 15 most common types of NPEV had two basic patterns of isolation, one in which a type had periodic epidemic years and the other in which it did not. In 11 of the 14 years there was one or more epidemic types, each accounting for greater than or equal to 20% of all isolates of NPEV that year. The six most common isolates in March, April, and May predicted an average of 59% of the total isolates detected in July-December of that year. PMID- 3003206 TI - Effect of acyclovir on infectious mononucleosis: a double-blind, placebo controlled study. AB - Thirty-one patients with clinical and laboratory diagnoses of infectious mononucleosis who had had symptoms for seven or fewer days were randomized for intravenous treatment with acyclovir (10 mg/kg) or placebo at 8-hr intervals for seven days in a double-blind trial. Clinical signs and symptoms were registered, and excretion of virus in the saliva as well as antibody responses in sera and saliva were assessed before, during, and at regular intervals in the six months after treatment. Acyclovir significantly (P less than .001), but reversibly, inhibited oropharyngeal shedding of Epstein-Barr virus. The humoral and cellular immune responses, however, did not differ between the two groups; nor did the development of viral latency. There were no significant (P greater than .05) differences in individual clinical symptoms or in laboratory parameters between the two groups; however, when data concerning duration of fever, weight loss, tonsillar swelling, pharyngitis, and self-assessment by the patient were combined, a significant (P less than or equal to .01) effect of treatment with acyclovir was evident. PMID- 3003208 TI - Interruption of transmission of rhinovirus colds among human volunteers using virucidal paper handkerchiefs. PMID- 3003209 TI - Monoclonal antibody to coxsackievirus B4 reacts with myocardium. PMID- 3003210 TI - Increased nuclease sensitivity of the human interferon-alpha 1-related genes and the interferon-beta 1 gene during induction by virus. AB - The chromatin structure of the human interferon (IFN) genes was evaluated during induction of human lymphoblastoid (Namalwa) cells by Sendai virus. Namalwa cells were treated with bromodeoxyuridine (BrdUrd) for 36-48 h and induced with Sendai virus for 7 h; the nuclear fraction was isolated and treated with low levels of either micrococcal nuclease or DNAse I. DNA was extracted from the nuclease treated chromatin, restricted with Eco RI and analyzed by Southern blotting using IFN-alpha 1 and -beta 1 cDNA probes. An increase in the digestibility of the IFN alpha 1-related genes and the IFN-beta 1 gene was observed in chromatin prepared from BrdUrd-treated, Sendai virus-induced Namalwa cells as compared with chromatin from uninduced Namalwa cells. Our results indicate that, during IFN induction in Namalwa cells by Sendai virus, the IFN genes assume a more open conformation consistent with increased transcriptional activity across these genes. PMID- 3003211 TI - [A case of bronchial carcinoid which developed at the right intermediate bronchus and metastasized multiply to the lower lobe of the lung]. PMID- 3003212 TI - [Lectin immunoradiometric assay versus improved radioimmunoassay: a comparison of methods for determination of desialylated forms of human chorionic gonadotropin]. AB - Two different assay systems have been developed for the measurement of asialo hCG(ashCG). One of them is an immunoradiometric assay which utilizes peanut lectin (PNA) to selectively extract ashCG and then directly quantify it with purified radiolabeled antiserum which is generated against carboxy terminal peptide of hCG beta (beta-CTP). PNA is highly specific for the terminal carbohydrate linkage Gal beta-(1----3)-Gal NAc which is only exposed to hCG when the O-serine linked oligosaccharide of its unique beta-CTP region is deficient in sialic acid. The other is an improved RIA using a specific antiserum to as beta CTP, in which extraction of hCG with monoclonal antibody-Sepharose 4B conjugate is used to obtain increased sensitivity with retention of specificity. The ashCG concentration can be measured as low as 0.01 and 0.05 pmoles/ml in the presence of an excess amount of hCG in each of these assays. These assays were applied to detect ashCG in the urine of patients with trophoblastic tumors and normal pregnant women. The proportion of ashCG to total hCG in choriocarcinoma was significantly higher than that in normal pregnancy in both assays. However, the proportion was dependent upon the amount of total hCG and it decreased as total hCG increased. PMID- 3003213 TI - Ultrastructure of leprous phlebitis. AB - An ultrastructural study on vein biopsies from six lepromatous leprosy patients was carried out. The results showed that a) the lumen-lining bacillated cells were endothelial in origin due to the presence of specific Weibel-Palade endothelial cell granules; b) endothelial cells released Mycobacterium leprae into the lumen by exophagocytosis; c) M. leprae were able to grow and multiply in the endothelial and smooth muscle cells; and d) smooth muscle cells did not show any evidence of reaction due to the presence of M. leprae in their cytoplasm. PMID- 3003214 TI - Respiration in Mycobacterium leprae. AB - Fairly pure leprosy bacilli were easily collected from nude mouse foot pad lepromas by the Ficoll density gradient centrifugation and alkali treatment methods. The yield of bacilli available for biochemical study was 42.6%. The density of Mycobacterium leprae was very heterogeneous. The percent of solid bacilli in the light bacilli fraction was 23%; that in the heavy bacilli fraction was 40%. The endogenous respiration activity in the heavy bacilli was greater than that in light bacilli. The average coefficient of respiration in M. leprae was 1 microliter O2/mg X hr. In the whole cells of M. leprae, a cytochrome b1 absorption peak and its Soret peak were detected at wavelengths of 560 nm and 426 nm, respectively. However, a cytochrome a2-like peak (which was observed in M. lepraemurium), and a cyt c and cyt a were not detected. Catalase activity was not found in whole cells, the cell-free extract, or particle fractions of M. leprae. Any catalase activity associated with M. leprae suspensions is a tissue contaminant. NAD-peroxidase activity was also not detected in the cell-free extract of the leprosy bacillus. These results would indicate that leprosy bacilli cannot degrade hydrogen peroxide. PMID- 3003215 TI - [A clinical study of calcium pyrophosphate dihydrate crystal deposition disease]. AB - Clinical features of calcium pyrophosphate dihydrate crystal deposition disease (CPPD c.d.d.) were studied and the following results were obtained. The prevalence of CPPD c.d.d. was 9.7% in 300 persons aged 50 or older, who stayed or worked at an old-age home. The most common type of CPPD c.d.d. was the asymptomatic type. Comparative studies between the patients with CPPD c.d.d. and subjects without it revealed that scoliosis of the lumbar spine, osteoarthritis like changes of the knee, subchondral bone cyst and patella wrapped around the femur were statistically more frequent in the former. The clinical study of 50 patients with CPPD c.d.d. revealed calcification not only in the articular cartilage but also in periarticular tissues and ligamentum flavum. The histological study demonstrated frequent destructive changes of the tissues around the site of CPPD crystal deposition. Formation of CPPD crystals appeared to be initiated by degenerating chondrocytes and metaplastic chondrocytes. PMID- 3003217 TI - Dietary fibre content of commonly eaten Thai plants. PMID- 3003216 TI - Cyclic AMP levels and cellular kinetics during maturation of human promyelocytic leukemia cells. AB - Differentiation of human promyelocytic leukemia cells (HL-60) in response to several classes of inducing agents is characterized by the sequential appearance of granulocytic or monocytic markers. Compounds that increase intracellular cAMP in HL-60 cells induce a program of maturation in which cells demonstrate early functional phagocytic properties. Cyclic nucleotide metabolism was studied during monocytic and granulocytic differentiation of the HL-60 cell line. In synchronous and nonsynchronous cell cultures, cyclic AMP levels were raised 300-fold or 25 fold in response to N6, O2-dibutyryl adenosine 3':5'-cyclic monophosphate or the combination of prostaglandin E2 and theophylline, respectively. No reproducible changes in intracellular adenosine 3':5'-cyclic monophosphate levels occurred in response to retinoic acid or dimethyl sulfoxide, suggesting that changes in adenosine 3':5'-cyclic monophosphate levels alone do not mediate cell maturation induced by these compounds. Agents that increased intracellular cyclic AMP in HL 60 cells slowed the progress through the S and G2-M phase of the cell cycle, whereas other agents such as dimethyl sulfoxide stimulated cells through this same period. PMID- 3003218 TI - Effects of gamma-aminobutyric acid receptor agonists on the secretion of growth hormone, luteinizing hormone, adrenocorticotrophic hormone and thyroid stimulating hormone from the rat pituitary gland in vitro. AB - The effects of the gamma-aminobutyric acid receptor agonists muscimol and baclofen were investigated on the secretion of GH, LH, ACTH and TSH from the anterior pituitary in vitro using a rapid superfusion system. A bicuculline sensitive stimulatory effect of muscimol was demonstrated on the secretion of GH, LH and ACTH but not TSH. Baclofen had no effect on the basal secretion of any of the hormones, but inhibited LH-releasing hormone-stimulated release of LH and K+- and Ba2+-stimulated release of ACTH. The benzodiazepine Roll-6896 and the barbiturate secobarbital were found to potentiate the effect of muscimol on GH secretion. These results demonstrate the presence of GABAA receptors on somatotrophs, gonadotrophs and corticotrophs, and the presence of GABAB receptors on gonadotrophs and corticotrophs. Thyrotrophs appear devoid of GABA receptors. PMID- 3003220 TI - Effect of ketoconazole on adrenocorticotrophic hormone secretion in vitro and in vivo. AB - The direct effects of ketoconazole on ACTH secretion have been investigated using a rat pituitary cell culture system. Ketoconazole had no significant effects on basal ACTH secretion, corticotrophin-releasing factor- or arginine vasopressin stimulated ACTH secretion, nor did it affect dexamethasone inhibition of ACTH secretion. In-vivo studies demonstrated an increased ACTH level (168 vs 76 ng/l) accompanied by a fall in plasma corticosterone (193 vs 307 micrograms/l) in normal rats given ketoconazole (24 mg/kg, five oral doses given 8 hourly). No effects were seen in adrenalectomized rats or at lower doses (6 mg/kg) in normal or adrenalectomized rats. A high dose of ketoconazole (24 mg/kg, twice daily oral doses) also caused increased ACTH levels in normal rats (129 vs 86 ng/l) when given for 7 days. No effects were seen in adrenalectomized rats or on plasma corticosterone levels in normal rats. Our data suggest that ketoconazole at these doses has no direct effects on pituitary ACTH secretion but causes an increase in plasma ACTH due to its inhibition of adrenal steroid synthesis. PMID- 3003221 TI - Characterization of pro-opiomelanocortin-derived peptides in pituitary and ectopic adrenocorticotrophin-secreting tumours. AB - The molecular forms of pro-opiomelanocortin in plasma of normal subjects and plasma and tissue extracts from patients with disorders of the hypothalamic pituitary-adrenal axis have been characterized using gel filtration chromatography under acid dissociating conditions, and the molecular forms further investigated by high pressure liquid chromatography and affinity chromatography. A large molecular weight MSH/ACTH fragment was observed in all plasma samples chromatographed, although the proportions of this fragment were significantly greater in patients with the ectopic ACTH syndrome. A similar profile was observed in tumour extracts: a greater proportion of large molecular weight precursors was observed. Immunoreactive-gamma-MSH in extracts of ectopic tumours displayed marked and variable heterogeneity, and affinity chromatography with Concanavalin A-Sepharose indicated that this was due to differential glycosylation. High pressure liquid chromatography of a peak of amino terminal pro-opiomelanocortin (N-POC(1-76)) and 22 000 mol. wt MSH/ACTH indicated two peaks in each peptide, again possibly due to differential glycosylation. A possible neurointermediate lobe origin of atypical invasive pituitary tumours is discussed. PMID- 3003219 TI - Testosterone maintains pituitary and serum FSH and spermatogenesis in gonadotrophin-releasing hormone antagonist-suppressed rats. AB - Groups of adult male rats were treated continuously for 30 days with either vehicle or the potent gonadotrophin-releasing hormone (GnRH) antagonist. (N-Ac-D Nal(2)1,D-pCl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10 )- GnRH (RS 68439; 35 micrograms/day). In addition, groups of vehicle- and antagonist-treated rats received s.c. testosterone implants sufficient to maintain serum testosterone concentrations 3.5- to 5-fold higher than those of vehicle-treated control rats. After 30 days of antagonist treatment serum LH, FSH and testosterone concentrations were at or below the detection limits of their respective assays and pituitary FSH content and GnRH receptor binding were reduced, relative to control animals, by 77 and 98% respectively. Testis weight in antagonist-treated rats was reduced by 75% and spermatogenesis was suppressed to an extent comparable to that observed in hypophysectomized rats. Testosterone, which caused a 40% reduction in serum FSH relative to control animals, prevented the antagonist-induced fall in both serum and pituitary FSH, but not GnRH receptors, below that observed in the vehicle plus testosterone-treated group. Furthermore, spermatogenesis in the antagonist plus testosterone-treated group was indistinguishable from that observed in control animals. It is concluded that testosterone is capable of maintaining serum and pituitary FSH levels in vivo, under conditions which presumably render the pituitary insensitive to hypothalamic GnRH. PMID- 3003222 TI - Failure of glucocorticoid feedback in males of a population of small marsupials (Antechinus swainsonii) during the period of mating. AB - In an investigation of the factors leading to the increase in the concentration of plasma free glucocorticoid, which results in immunosuppression and death after mating of all males in natural populations of a small shrew-like marsupial, the dusky antechinus (Antechinus swainsonii), the integrity of the glucocorticoid feedback control of the concentration of plasma cortisol was examined by use of dexamethasone-suppression tests. Injection of 0.2 mg dexamethasone/kg i.m. caused a marked fall in the concentration of plasma cortisol 17 h later, approximately 2 months and 2 weeks before the annual mating period in mid-July. However, the same dose had no significant effect on the increased concentration of plasma cortisol characteristic of the mid- to late July mating period. Injection of 100 i.u. ACTH/kg i.m. caused a significant increase in the concentration of plasma cortisol 6-7 h later on all occasions, indicating that the responsiveness of the adrenal cortex to ACTH did not change. Pretreatment with dexamethasone had no effect on the ACTH-stimulated cortisol concentration, ruling out a possible direct effect of dexamethasone on adrenocortical secretion in this species. Dexamethasone also reduced the concentration of plasma testosterone when the level was low, before the mating period, but not when the level was high, at the beginning of the mating period. It is concluded that, in association with a rapid increase in the concentration of plasma testosterone, an increase in aggression and intense mating activity, glucocorticoid feedback control of ACTH secretion is impaired.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003223 TI - Na-K-ATPase-inhibiting and glucose-6-phosphate dehydrogenase-stimulating activity of plasma and hypothalamus of the Okamoto spontaneously hypertensive rat. AB - The plasma of normal man and the rat, and an acetone extract of hypothalamus from the rat, have an ability to inhibit Na-K-ATPase which is related directly to salt intake. The ability of the plasma to inhibit Na-K-ATPase is raised in essential hypertension. The ability of plasma and of an acetone extract of hypothalamus from six spontaneously hypertensive (SHR) rats and six normotensive control (WKY) rats to inhibit Na-K-ATPase of fresh guinea-pig kidney was studied using cytochemical bioassay techniques. With a validated assay, which measures the capacity of biological samples to stimulate glucose-6-phosphate dehydrogenase (G6PD) as an index of their capacity to inhibit Na-K-ATPase, the mean G6PD stimulating ability of the plasma from the SHR and the WKY rat was 772.3 +/- 48.1 units/ml and 12.5 +/- 2.6 units/ml respectively (P less than 0.01) and of the hypothalamic extracts it was 2.2 +/- 1.7 X 10(8) and 4.5 +/- 1.8 X 10(4) units/hypothalamus (P less than 0.01). With a semi-quantitative cytochemical assay, which measures Na-K-ATPase activity directly, plasma and an acetone extract of hypothalamus from the spontaneously hypertensive rat had much greater capacities to inhibit Na-K-ATPase than plasma and extract from the WKY rat. These raised levels of Na-K-ATPase inhibitory activity in the plasma of the SHR rat are similar to the highest values found in the plasma of patients with essential hypertension.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003224 TI - Oestrous cycle-related LH responsiveness to naloxone: effect of high oestrogen levels on the activity of opioid receptors. AB - Endogenous opioid peptides have a tonic inhibitory control on LH secretion, participating in the functional changes of the hypothalamic-pituitary-ovarian axis. To evaluate the activity of the endogenous opioid systems during the oestrous cycle, we measured plasma LH levels after naloxone administration (5 mg/kg, s.c.) at 09.00 and 16.00 h on all days of the cycle (two further measurements were taken at 14.00 and 18.00 h on the day of pro-oestrus) and after one dose or one week's treatment with oestradiol benzoate (OB; 0.2 micrograms/rat). Concentrations of LH were measured in the same experimental models after injection of LH-releasing hormone (LHRH; 1 microgram/kg, i.p.) or saline. Naloxone induced a significant rise in LH levels on the day of oestrus, dioestrus day-1 and dioestrus day-2; this response was blunted on the morning of pro-oestrus and absent in the afternoon and after acute and chronic OB treatment. Conversely LHRH was most effective in increasing LH levels on the day of pro oestrus and in OB-treated rats. These results indicate that opioid mechanisms, independently of the time of day and the pituitary responsiveness, exhibit a reduced activity when preovulatory changes occur, probably as a result of increased oestrogen levels. PMID- 3003225 TI - Prolactin stimulates Na+-K+-ATPase activity located in the outer renal medulla of the rat. AB - The effect of rat prolactin on rat renal Na+-K+-ATPase activity was investigated by a cytochemical technique. Rat prolactin caused stimulation of Na+-K+-ATPase activity only in the outer medulla of the kidney, and not in renal cortical structures. Peak enzyme activity in cultured rat renal segments occurred after tissue had been exposed to rat prolactin for 2 min, and the time of maximal stimulation did not vary with the concentration of prolactin. There was a curvilinear response in Na+-K+-ATPase activity over the rat prolactin concentration range, 0.04-40 ng/l, but higher prolactin concentrations caused inhibition of enzyme activity. Na+-K+-ATPase response was totally blocked by specific rat prolactin antiserum. Human prolactin had no consistent effect on rat medullary Na+-K+-ATPase activity. Addition of specific tri-iodothyronine and arginine vasopressin antisera to rat prolactin was without effect, confirming that the stimulatory action of rat prolactin on Na+-K+-ATPase was not due to contamination with these hormones which are known to stimulate this enzyme. PMID- 3003226 TI - Configuration and expression of the T cell receptor beta chain gene in human T lymphotrophic virus I-infected cells. AB - We studied the configuration and expression of the gene encoding the beta chain of the T cell receptor (TCR beta) in cell lines and primary tumor cells infected by the human T cell leukemia/lymphoma (lymphotrophic) virus type I (HTLV-I). Most of the cell lines and all the primary tumor cells showed rearrangement of the TCR beta gene, and in each case the rearrangement was distinct. The majority of cases examined were clonal with respect to a particular TCR beta gene rearrangement. Primary tumor cells from one case (SD) were found to have a tandem duplication of a portion of chromosome 7; this appears to have resulted in the presence of three alleles of the TCR beta gene, each of which is arranged differently. This suggests that the chromosomal abnormality, and possibly infection by HTLV-I, occurred before TCR beta gene rearrangement. Cell lines infected by HTLV-I express levels of TCR beta mRNA similar to PHA stimulated lymphocytes, suggesting that this gene is not transcriptionally activated as a result of infection by HTLV-I. Cloned T cells of known antigen specificity that are infected by HTLV-I in vitro show impairment of immune function, including loss of antigen-specific responsiveness and the acquisition of alloreactivity. Comparison of the configuration of the TCR beta gene before and after infection revealed no changes detectable by Southern blot analysis. Levels of expression of the TCR beta gene at the mRNA level and surface expression of the T3 complex were also not significantly altered, suggesting that changes in immune function cannot be attributed to quantitative changes in the TCR molecule. The configuration of the TCR beta gene in primary tumor cells infected by HTLV-I was compared with that in the derived cell lines. In all pairs examined, the configuration in the primary tumor cells was different from that in the cell lines, strongly suggesting that the cells that grow in culture are not the original neoplastic cells. PMID- 3003227 TI - Identification of three new Ig lambda-like genes in man. AB - Three new human lambda L chain-like Ig genes are identified by restriction enzyme and nucleotide sequence analysis. Two genes, 14.1 and 16.1, have intact J and C regions, and are potentially functional, with open reading frames. A third gene, 18.1, is a pseudogene. The evolutionary lineage of these genes compared to the known functional locus lambda C1-lambda C6 can be surmised from Southern blot and nucleotide homologies. This study demonstrates that the human lambda gene family is more complex than previously recognized. PMID- 3003229 TI - Unusual responses of cultured Fundulus heteroclitus melanophores to epinephrine and ions, paradox of K+ versus Na+. AB - Fundulus heteroclitus melanophores were successfully cultured and maintained in culture for up to 2 months. Compared to other teleost melanophores in the fin, scale, or split fin preparation, the cultured melanophores show unusual responses to both epinephrine and ion solutions. First, they aggregated their melanosomes in response to concentrations of epinephrine as low as 10(-12) M. Second, the melanophores in a 6-day, or older, culture aggregated their melanosomes in response to both sodium and potassium ion solutions. This is in contrast to 4-day old cultures (and reports of noncultured melanophores) where melanosomes are aggregated in response to potassium but dispersed in sodium ion solutions. PMID- 3003228 TI - Cell surface antigens of murine leukemias induced by radiation leukemia virus. Recognition of individually distinct cell surface antigens by cytotoxic T cells on leukemias expressing crossreactive transplantation antigens. AB - The specificity of transplantation immunity and T cell cytotoxicity against leukemias induced by RadLV was examined. Subcutaneous inoculation of two RadLV leukemias induced in BALB/c mice, BALBRVB and BALBRVD, resulted in initial tumor growth in CB6F1 mice, followed by complete tumor regression. Mice that had rejected leukemias BALBRVB or BALBRVD were subsequently challenged with various tumors of BALB/c origin. The growth of all five RadLV leukemias tested, and of one radiation-induced leukemia, was significantly inhibited. Another radiation induced leukemia, a methylcholanthrene-induced sarcoma, and a leukemia induced by the Moloney leukemia virus, were not inhibited. The results indicate that RadLV leukemias share cell surface antigens that induce transplantation immunity in vivo. Cytotoxic lymphocytes were generated by coculturing spleen cells from mice that had rejected leukemia BALBRVB or BALBRVD with the corresponding leukemia cells. Direct tests and inhibition tests showed that such cytotoxic cells recognized individually specific antigens on leukemias BALBRVB and BALBRVD, distinct from the shared antigens detected in transplantation experiments. The effector cells in cytotoxicity assays were Thy-1+, Lyt-1+,-, Lyt-2+, and Lyt-3+ T cells. PMID- 3003230 TI - [Effect on health of occupational silica exposure in foundries]. PMID- 3003231 TI - Monoclonal antibodies to guinea-pig cytomegalovirus: an immunoelectron microscopic study. AB - Three groups of monoclonal antibodies which reacted with cells infected by guinea pig cytomegalovirus (GPCMV) were prepared. The first group of antibodies immunoprecipitated a 50 000 mol. wt. (50K) polypeptide of GPCMV-infected cells. This polypeptide was identified as part of the nuclear inclusion by immunofluorescence. This nuclear fluorescence was markedly diminished when the infected cells were incubated in the presence of phosphonoacetic acid. By immunoelectron microscopy the antibodies reacted mainly with filamentous structures (26 to 28 nm in diameter) in nuclear inclusions and occasionally stained nucleocapsids. Neither intracytoplasmic nor extracellular virions reacted with the antibodies. Therefore, the 50K protein with which the monoclonal antibodies reacted was nuclear inclusion-specific and a non-structural protein. The second group of antibodies reacted with a 76K polypeptide of the infected cells which was a matrix protein found in both cytoplasmic inclusions and extracellular dense virions. The third group of antibodies mainly reacted with a virion core protein by immunoelectron microscopy. PMID- 3003232 TI - Definition of two new groups of atypical rotaviruses. AB - Comparative antigenic and nuclei acid analyses were carried out on two new atypical rotavirus isolates coming respectively from chickens (D/132) and pigs (E/DC-9). Indirect immunofluorescence showed that each virus carried different group antigens which were also distinct from those of previously described rotavirus groups. By genome profile analysis each virus had a pattern of genomic RNAs clearly distinct from those of the other rotavirus groups. Comparative terminal fingerprinting of corresponding genome segments from the two viruses showed large differences between them, indicating that all of their genomic RNAs had significant differences in sequence both from each other and from the three previously defined rotavirus groups. On the basis of these results, extension of the number of rotavirus groups from three to five is proposed, with isolates D/132 and E/DC-9 being the type members of groups D and E respectively. PMID- 3003233 TI - Herpesviruses provide helper functions for avian adeno-associated parvovirus. AB - The avian herpesviruses infectious laryngotracheitis virus (ILTV) and herpesvirus of turkeys (HVT), as well as the mammalian herpesvirus pseudorabies virus (PRV) were able to provide complete helper activity for the production of infectious avian adeno-associated virus (AAAV) in chicken cells. The presence of AAAV in the infected chicken cell reduced the multiplication of HVT. ILTV or PRV, however, were not affected if used as helper viruses. Infectious AAAV was determined by an indirect immunofluorescence assay and infectious herpesvirus by plaque assays. PMID- 3003234 TI - Viral DNA sequences detected in a hamster liposarcoma induced by bovine papillomavirus type 4. AB - Following intradermal inoculation of bovine papillomavirus type 4 (BPV-4) into a Syrian hamster, a liposarcoma developed at the inoculation site 20 months later. The DNA of this tumour contained multiple copies of the BPV-4 genome which existed in a free unintegrated state. Unintegrated viral DNA and viral DNA isolated from virus particles from bovine alimentary tract papillomas revealed identical cleavage patterns with CpG methylation-resistant and -sensitive restriction enzymes: apparently there was no gross methylation of CpG sites in either case. The entire BPV-4 genome appeared to be represented in the tumour DNA. PMID- 3003235 TI - Stimulation of helper/delayed-type hypersensitivity T cells by flavivirus infection: determination by macrophage procoagulant assay. AB - West Nile virus (WNV)-immune spleen cells produced an inducer of macrophage procoagulant activity (MPCA) on restimulation with WNV in vitro. This response was specific for WNV and depended on the presence of Thy 1+, L3T4+ and also Ia+ cells but not Lyt 2+ cells. It could be induced by culture with large amounts of non-infectious, u.v.-irradiated virus or with high doses of infectious virus. Inoculation of mice with low titres of infectious, but not with u.v.-irradiated, virus primed for this in vitro response. The time of maximum production of procoagulant-inducing factor was also the period of greatest [3H]thymidine uptake and release of interleukin-2 (IL-2)-like activity. In bulk cultures, MPCA inducing factor was more readily quantified than IL-2. T cells of the helper/delayed-type hypersensitivity class are thus stimulated by WNV and this aspect of cell-mediated immunity can be readily assayed in vitro. PMID- 3003236 TI - Cloning and sequencing of 5' terminal sequences from avian infectious bronchitis virus genomic RNA. AB - The subgenomic RNAs of the fowl coronavirus infectious bronchitis virus (IBV) form a 3' co-terminal or 'nested' set. The presence of non-contiguous (leader) sequences fused to the 5' termini of murine hepatitis virus mRNAs has been demonstrated using RNase T1 oligonucleotide mapping and sequencing. The presence of a leader sequence on IBV mRNA A has been demonstrated previously. In this paper the presence of a leader identical to that present on the 5' terminus of IBV mRNA A is demonstrated to be present on the 5' terminus of IBV genomic RNA. This has been achieved by sequencing of primer extension products and cDNA clones containing the genomic leader. Analysis of these clones has revealed the presence of a sequence at the leader/genome-length RNA junction which is closely related to regions of homology identified previously within the genomic RNA sequence at the leader/body junctions of subgenomic RNAs. The implications of this finding for mechanisms of coronavirus RNA synthesis are discussed. PMID- 3003237 TI - Classification of Barmah Forest virus as an alphavirus using cytotoxic T cell assays. AB - Barmah Forest virus, an arbovirus, does not cross-react convincingly with alpha-, flavi- or bunyavirus immune sera. Secondary cytotoxic T cells generated in vitro immune to a number of alphaviruses cross-lyse Barmah Forest virus-infected target cells. Flavivirus (West Nile and Kunjin)- and Bunyamwera virus-immune Tc cells lyse homologous virus-infected target cells, but not alphavirus-infected targets. Using cytotoxic T cell assays Barmah Forest virus can be classified as an alphavirus. PMID- 3003238 TI - Mapping of the gene coding for the major late structural polypeptide on the frog virus 3 genome. AB - The gene encoding the major capsid polypeptide (MCP 48) of frog virus 3 (FV 3) has been mapped on the viral DNA. Late FV 3 messenger RNA, hybrid-selected by the SalI-F fragment or a subset of these sequences, BamHI-L and -W fragments, directed the synthesis in vitro of a 48 000 mol. wt. (48K) polypeptide. This product was recognized by monospecific antibodies raised against the major capsid polypeptide. The RNA complementary to these DNA sequences was about 1350 nucleotides in size. This transcript, encoding MCP 48, was precisely located; S1 nuclease analysis indicated that its 5' end mapped at 1250 nucleotides to the right and its 3' end at 160 nucleotides to the left of the BamHI site at the junction between the BamHI-W and -L fragments. PMID- 3003239 TI - Infection of the adrenal gland as a route to the central nervous system after viraemia with herpes simplex virus in the mouse. AB - Intravenous inoculation of 4-week-old female NIH (inbred) mice with herpes simplex virus type 1 (HSV-1) strain P2C6 (defective in thymidine kinase) produced bilateral hind limb paralysis in nearly all animals by the 5th day after inoculation; very few mice died. In male mice the incidence and severity of paralysis was considerably lower than in females. The parental strain, CL(101), produced similar paralysis but all mice died by day 7. Observations on paralysis and death after intravenous inoculation are given for other strains of HSV-1 and HSV-2. By day 1 after inoculation of P2C6 significant virus replication had occurred in the adrenal glands but in none of the other organs tested. Titres of virus were similar in the adrenal glands of male and female mice. Histology of the adrenals showed most extensive replication in the cortex with some involvement of the medulla, particularly at the corticomedullary junction. By the 2nd and 3rd days, virus was detected in the lower thoracic spinal cord of both male and female animals but clearance was possibly quicker from males. Adrenalectomy proved that virus reached the cord via the adrenals. In the cord the infection was associated with bilateral demyelination in the ventral white matter as early as day 3. PMID- 3003240 TI - Viable viruses with deletions in the left inverted terminal repeat define the adenovirus origin of DNA replication. AB - A series of human adenovirus type 2 genomes with deletions in the left inverted terminal repeat (ITR) have been constructed. Viral genomes that contained a minimum of 45 base pairs (bp) from the terminus of the genome were fully infectious and gave rise to progeny virus which maintained the deletion. In contrast, genomes containing 36 bp or less from the termini of the genome were not infectious. The boundary of a cis-acting element required for viral replication is therefore between 36 and 45 bp from the adenovirus termini and corresponds to the previously identified viral origin of replication, defined using a transfection assay to measure ori activity in vivo. The growth parameters of viruses with deletions in the left ITR were examined. These deletions had no measurable effect on plaque formation or morphology, viral DNA synthesis or early viral mRNA synthesis. Thus, it appears that DNA sequences in the left ITR, outside the replication origin, are completely dispensable for lytic viral growth in tissue culture cells. PMID- 3003242 TI - Persistence, latency and reactivation of Japanese encephalitis virus infection in mice. AB - Persistent and latent Japanese encephalitis virus (JEV) infection was studied in pregnant and non-pregnant mice. Following intraperitoneal inoculation into pregnant mice JEV persisted for 16 weeks in contrast to 4 weeks in non-pregnant mice. This was followed by a higher frequency of latent infection in pregnant mice. The virus could be reactivated during pregnancy or by cyclophosphamide treatment, the latter being more effective. PMID- 3003241 TI - In vitro packaging of foreign DNA into heads of bacteriophage T1. AB - The isolation of a collection of 44 morphologically T1-like phages is described. It is shown that these phages share some similarity with T1 in terms of cross inactivation with anti-T1 serum, particle proteins and DNA packaging in vitro by the headful process. Virion DNA extracted from these phages was treated with T1 in vitro packaging extracts and the reaction mixtures were tested for the formation of infectious phage particles. The packaging efficiencies observed varied from about 1 to 100% of that of virion T1 DNA. Phage lambda virion DNA was packaged with an efficiency of between 0.01 and 2% (5 X 10(1) to 3 X 10(3) p.f.u./micrograms DNA), the shorter deleted derivative lambda L47 being packaged more efficiently than normal length lambda C1857 DNA. Virion DNA from phages T3 and T7 was also packaged at an efficiency similar to that for lambda. The in vitro packaging of T1 DNA requires the presence of the pac sequence which initiates headful packaging from a concatemeric precursor. The high efficiency of packaging DNA from some of the T1-like phages may indicate the presence of similar packaging sequences. However, in the case of lambda L47, which is known not to contain such a sequence, the in vitro DNA packaging reaction must occur by a secondary pathway unrelated to the headful mechanism. PMID- 3003243 TI - An in vitro latency system for herpes simplex virus type 2. AB - An in vitro latency system for herpes simplex virus type 2 (HSV-2) in cultured cells has been developed. Virus replication was suppressed by infection of human foetal lung cells at the supraoptimal temperature of 42 degrees C, and, following transfer of such cell cultures to the normal growth temperature of 37 degrees C, infectious virus was generally undetectable for at least 6 days. HSV-2 was reactivated by intertypic superinfection at 38.5 degrees C with temperature sensitive mutants of HSV-1, or with human cytomegalovirus, but not by superinfection with adenovirus types 2 or 5. The HSV-1 mutant tsKsyn, which produces only immediate early polypeptides at 38.5 degrees C, was as effective as the late mutant tsIsyn, but tsK which had been irradiated with u.v. light to prevent gene expression did not reactivate HSV-2. The efficiency of reactivation was very high, since 15 to 34% of the theoretical input of infectious HSV-2 particles could be retrieved by superinfection with 0.3 p.f.u. of tsKsyn per cell. Reactivation of latent virus was not induced by cell subculture or by other treatments which alter cell metabolism. The system described here may be important for studies on the molecular basis of HSV latency. PMID- 3003244 TI - Virus excretion and cell-mediated immunity during cytomegalovirus infection among healthy infants and children. AB - Specific cell-mediated immunity (CMI) and virus isolation were examined longitudinally to clarify the mechanism of the cessation of virus excretion in inapparent cytomegalovirus (CMV) infection among healthy infants and children. We measured leukocyte procoagulant activity (LPCA) responses to CMV antigen and to a purified protein derivative of tuberculin (PPD), with results expressed as a percentage reduction of recalcification (RC) time. Based on the results in seropositive and seronegative adult control subjects, reductions in RC time of more than 10% were considered indicative of a positive LPCA response. The CMV specific LPCA response was negative in all infants shedding the virus, despite the presence of circulating antibodies, but were converted from negative to positive when the virus excretion ceased. This suggests that cessation of the virus excretion in inapparent CMV infection among healthy infants and children probably results from the specific CMI. PMID- 3003245 TI - Analysis of the genome of molluscum contagiosum virus by restriction endonuclease analysis and molecular cloning. AB - Virions of molluscum contagiosum virus (MCV), a member of the poxviridae, were isolated directly from lesions of individual patients and characterized by restriction enzyme analysis. The comparative analysis of the cleavage patterns and Southern blot hybridization of 14 independently isolated virus samples revealed that MCV isolates can be classified into two different types. The majority of MCV isolated from clinically typical skin lesions (13 of 14) showed similar DNA cleavage patterns and were termed MCV type 1, whereas one isolate derived from a vaginal lesion showed a completely different DNA cleavage pattern and therefore was termed MCV type 2. For detailed investigation of the viral genome, a defined gene library of MCV DNA sequences was established. The Bam HI DNA fragments of the viral genome of MCV type 1 prototype isolate 1/80 was inserted into the bacterial plasmid vector pAT153. With the exception of terminal fragments (fragments A and B) of the viral genome, all other DNA fragments were cloned. All cloned Bam HI DNA fragments were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridization of plasmid DNA to viral DNA. PMID- 3003246 TI - Field trial of a Japanese encephalitis diagnostic kit. AB - Serum and cerebrospinal fluid (CSF) obtained from patients in rural Thailand during an encephalitis epidemic were assayed with a Japanese encephalitis rapid diagnosis kit. Japanese encephalitis was diagnosed by detection of virus-specific IgM (JEV IgM) in CSF (1:10 dilution) or serum (1:100 dilution) with an antibody capture enzyme-linked immunosorbent assay. Specimens were assayed immediately on site at the provincial hospital and scored by visual examination within 4 h. Each specimen was retested carefully later to accurately determine its activity (units) at a single screening dilution; each was tested also at serial dilutions to determine its end-point titer. On-site kit results showed close agreement with subsequent laboratory results for detection and quantitation of JEV IgM and JEV IgG in either serum or CSF. Using the kit on site, admission CSF from 35 (73%) of 48 laboratory-proven JEV-infected patients were scored as definitely positive for JEV IgM, while all 17 CSF specimens from non-JEV infected patients were read as negative (sensitivity 73%, specificity 100%). A rapid and early diagnosis of acute Japanese encephalitis can be accomplished almost anywhere. PMID- 3003248 TI - Use of a molecular probe for detecting JCV DNA directly in human brain material. AB - A hybridot assay using a radiolabelled JC virus probe has been used to detect the presence of JCV DNA in brain biopsy and postmortem brain samples from patients with neurological disease and possible progressive multifocal leucoencephalopathy. Sixty-nine brain samples from 45 patients were examined. Eleven samples from eight patients had detectable JCV DNA sequences. In seven of the eight patients this result was confirmed by electron microscopy and/or virus isolation or immunofluorescence. PMID- 3003247 TI - Age-specific prevalence of antibodies to pyrimidine kinase enzymes of herpes simplex virus types 1 and 2, and of varicella zoster virus. AB - The age-specific prevalence of antibodies to pyrimidine kinase enzymes of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) and of varicella zoster virus (VZV) was measured in serum specimens from 360 persons. Specific inhibition of viral enzyme activity by the serum was used as an indication of the presence of antibodies to the enzyme. For HSV-1 and HSV-2 together the overall prevalence of positive sera increased with age and reached about 50% in the older age groups, the major part of the positive sera being HSV-1 positive. Sera inhibitory to the VZV pyrimidine kinase enzyme were detected only sporadically. Six percent of the tested sera repeatedly inhibited the cytosolar corresponding enzyme of uninfected host cells by an unidentified mechanism. The results of the pyrimidine kinase antibody-assay test were compared to those obtained by complement fixation (CF) and enzyme immunoassay (EIA) in a separate set of 50 sera. The correlation between the CF and the EIA tests was good in this selected serum set with CF titres from less than 8 to 64. Twenty-four percent of the CF positive sera were negative by the pyrimidine kinase antibody assay, while all pyrimidine-kinase antibody-positive sera also had antibodies detectable by the two other methods. These results confirm and extend earlier observations on the occurrence of antibodies in human sera to HSV and VZV pyrimidine kinase enzymes. PMID- 3003249 TI - GABA-noradrenergic interaction: evidence for differential sites of action for GABA-A and GABA-B receptors. AB - Treatment of mice with DSP4 (a neurotoxin that abolishes the presynaptic noradrenergic neuron; Dooley et al., 1983) resulted in: (A) a decrease in the Bmax for the low affinity GABA-B receptor site in the cerebral cortex and hippocampus, whereas the Bmax for the high affinity GABA-B receptor site was unaffected; (B) a greater potentiation of norepinephrine stimulated adenylate cyclase by baclofen in cerebral cortex slices; and (C) a decrease in the Bmax for both the high and low affinity GABA-A receptor sites in the cerebral cortex and hippocampus. These data, coupled with previous work from our laboratory, suggest that the GABA-B receptor may be associated with both the noradrenergic nerve terminal and the post-synaptic neuron receiving noradrenergic input, whereas the GABA-A receptor may be associated with the noradrenergic nerve terminal. These data further suggest a functional coupling between the noradrenergic and GABA ergic systems. PMID- 3003251 TI - Chronic treatment with the potential antidepressant drug rolipram: the effect on the behavioural responses to adrenergic and dopaminergic receptor agonists with some biochemical correlates. AB - We studied the effect of acute and chronic treatment with rolipram, a potential antidepressant drug, on the behavioural responses induced by adrenergic and dopaminergic receptor agonists in mice and rats, and on (3H)prazosin and (3H)dihydroalprenolol binding to cortical membranes and whole brain noradrenaline and dopamine utilization in rats. Chronic, but not acute, administration of rolipram potentiated a behavioural response mediated through central alpha 1 adrenoceptors, attenuated an alpha 2-adrenoceptor-mediated response and inhibited a beta-adrenoceptor-mediated response. Neither treatment affected the behavioural responses to dopaminergic stimulants. Repeated treatment with rolipram decreased the density of cortical (3H)dihydroalprenolol, but not (3H)prazosin bindings sites, and reduced brain noradrenaline, but not dopamine utilization. These results suggest that chronic administration of rolipram induces the down regulation of the central beta- and alpha 2-adrenoceptors and enhances the responsiveness of the central alpha 1-adrenoceptors with no apparent changes in the alpha 1-adrenoceptor density. PMID- 3003250 TI - Evaluation of hypothalamic dopaminergic function by neuropharmacologic means in aged women. AB - The function of the hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons involved in the control of prolactin secretion was investigated in aged subjects with the use of nomifensine, an indirect-acting dopamine (DA) agonist, domperidone, a DA receptor antagonist, and DA, which acts directly on the pituitary lactotropes. In all 33 women, aged 69-92 yr, were studied. Baseline prolactin values were slightly but significantly higher in aged women (13 +/- 1.2 ng/ml, M +/- S.E.M.) than in a group of control fertile women (8 +/- 0.7 ng/ml). Oral administration of nomifensine (200 mg), in 14 aged women suppressed plasma prolactin (greater than or equal to 30% of baseline) in 8 subjects, a proportion not different from that present in fertile women (7/15) also receiving a single oral dose of nomifensine. Intravenous infusion of DA (0.04 microgram/kg min, 120 min) induced a similar inhibition in plasma prolactin in the aged and the fertile women, while administration of domperidone (4 mg i.v.) evoked a higher plasma prolactin rise, 15 min post-administration, in fertile than aged women. In all, presence of baseline prolactin levels only slightly elevated and prolactin responsiveness to nomifensine and DA not different from that of fertile women denote preservation of TIDA neuronal function in old women. The blunted response to domperidone of the old women is likely attibutable to a reduced pituitary pool of prolactin. PMID- 3003252 TI - Chronic effects of fluoxetine, a selective inhibitor of serotonin uptake, on neurotransmitter receptors. AB - Fluoxetine administration to rats at a dose of 10 mg/kg i.p. daily up to 12 or 24 days failed to change the concentration-dependent binding of [3H]WB4101, [3H]clonidine and [3H]dihydroalprenolol to alpha 1-, alpha 2- and beta-adrenergic receptors, respectively; [3H]quinuclidinyl benzilate to muscarinic receptors; [3H]pyrilamine to histamine H1 receptors and [3H]naloxone to opiate receptors. Persistent and significant decreases in receptor number (Bmax value) without changes in the dissociation constant (KD value) of [3H]5-HT binding in cortical membranes were observed upon chronic treatment with fluoxetine administered either by intraperitoneal injection or incorporation in the diet. A detectable reduction of 5-HT1 receptor number occurred after once-daily injections of fluoxetine at 10 mg/kg i.p. within 49 hours. After pretreatment for 3 days with p chlorophenylalanine, an inhibitor of 5-HT synthesis, followed by repeated administration of fluoxetine, 5-HT1 receptor numbers were higher than those of normal rats, suggesting a dependence on synaptic concentration of 5-HT for fluoxetine to affect a receptor down-regulation. These studies provide further evidence for the selectivity of fluoxetine as an inhibitor of 5-HT reuptake, resulting in a selective down-regulation of 5-HT1 receptors in the cerebral cortex of rat brain. PMID- 3003253 TI - Potentiation of pancuronium induced neuromuscular blockade by calcium channel blockers in vitro. AB - The calcium channel blockers verapamil, methoxyverapamil, diltiazem, and nisoldipine have been tested for interactions with the nondepolarizing muscle relaxant pancuronium bromide by using the indirectly stimulated in vitro preparation of the rat hemidiaphragm. Dose response curves of each calcium channel blocker, pancuronium bromide, and combinations of both were determined. Each of the four calcium channel blocking drugs showed agonistic interaction in potentiating pancuronium induced neuromuscular blockade. PMID- 3003255 TI - Vesicular demyelination induced by raised intracellular calcium. AB - Incubation of nerve with high concentrations of the divalent cation ionophore A23187 produces myelin vesiculation (Schlaepfer 1977). This observation has now been extended using segments of rat ventral or dorsal root incubated with high (19 microM, 10 micrograms/ml) or low (1-1.5 microM) concentrations of A23187, or another divalent ionophore, ionomycin. Low concentrations of A23187 induced no vesiculation within a 2-h period. However, subsequent incubation of these roots in fresh, ionophore-free medium for 20 h, resulted in a prominent vesicular demyelination at the Schmidt-Lanterman incisures and paranodes of many fibres. At this time (22 h) the Schwann cells associated with some demyelinating internodes appeared vital upon ultrastructural examination: the cells also excluded the nuclear dye nigrosin. High concentrations of A23187 induced a similar vesicular demyelination in affected fibres within only 15-20 min. While the Schwann cells continued to exclude nigrosin for a further 4 h, their ultrastructural appearance indicated that they were probably in the early stages of necrosis. Incubation of moribund root with the ionophore produced no myelin vesiculation. At all ionophore concentrations, the myelin vesiculation was dependent upon the presence of extracellular Ca2+, and could be modulated in severity by varying this concentration. Other divalent cations (Ba2+, Co2+, Mg2+, Mn2+, Ni2+, Sr2+) could not substitute for Ca2+. The vesiculation induced by A23187 could be entirely prevented by the addition of Zn2+ (greater than or equal to 1 microM), Ni2+ (greater than or equal to 1-10 microM), Co2+ (greater than or equal to 100 microM) or Mn2+ (greater than or equal to 100 microM) to the bathing medium. A23187 applied to only part of an isolated internode resulted in a localization of the myelin disruption to that region. Ionomycin (greater than or equal to 1 microM), an ionophore with a greater selectivity for Ca2+ than A23187, also induced a prompt Ca2+-dependent myelin vesiculation. We conclude that vesicular demyelination can be initiated in vital Schwann cells by a raised intracellular Ca2+ concentration. Such demyelination does not necessarily lead to Schwann cell death. The possible relevance of the findings to vesicular demyelinating neuropathies is discussed, and a hypothesis regarding the mechanism of demyelination is advanced. PMID- 3003256 TI - Cerebrospinal fluid virus antibodies. A diagnostic indicator for multiple sclerosis? AB - Specific antibody activities (antibody per weight unit IgG) of serum and CSF against a broad variety of viruses were compared in multiple sclerosis and certain inflammatory diseases of the central nervous system, e.g. neurosyphilis, as well as herpes simplex and zoster encephalitis. No "unspecific" antiviral activities within the CSF compartment were found in the non-MS diseases. The most frequent antibodies locally produced were directed against measles, rubella and zoster antigens. A diagnostic test with these three viruses would give positive results in about 80% of patients with MS. This finding is not as frequent as the oligoclonal pattern of the CSF gamma-globulins but would have a considerably greater diagnostic significance. PMID- 3003257 TI - Herpes simplex virus encephalitis. Neuroanatomical and neurochemical selectivity. AB - Intracerebral infection of mice with HSV-1 was found to produce a 2-3-fold increase in dopamine and serotonin metabolism in cortex, striatum, diencephalon and brain stem. Neurochemical markers of GABA and acetylcholine neurones, and neurotransmitter receptor binding sites were unchanged. The immunohistochemical distribution of virus antigen revealed high levels of immunoreactivity in s. nigra, ventral tegmental area, locus coeruleus and dorsal raphe nucleus, whilst other areas of brain stem were clear of virus antigen. The changes in monoamine metabolism observed in experimental HSV encephalitis may be related to the concentration of virus in monoamine neurones. PMID- 3003258 TI - Effect of insulin, proinsulin and pancreatic extract on myelination and remyelination in organotypic nerve tissue in culture. AB - The effect of insulin, proinsulin and crude pancreatic extract was studied in organotypic nerve tissue cultures, principally in relation to the development of myelin. Cultures were exposed to media supplemented with these substances beginning on the first day of explantation. By 4 days in vitro, there was a good neuritic outgrowth from all the fragments. That from the insulin and pancreatic extract-fed were more profuse and extended further than from the control group. By 8-12 days in vitro it was also possible to observe more myelinated axons in these treated groups. The pattern of changes in the myelin associated enzyme activity, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) paralleled the differential increase in myelination. Insulin-fed cultures showed a more rapid increase in CNPase activity, which, after 21 days in vitro reached a plateau about 30-50% over that of the controls. Cultures treated with pancreatic extract showed a similar pattern of increased activity, while in proinsulin-treated explants the activity was only significantly higher after 21 days in vitro. To study the effect of these substances on remyelination, well myelinated cultures were completely demyelinated by exposure to anti-white matter antiserum and were subsequently exposed to the same normal control or supplemented media. The amount of myelin and concomitantly the CNPase activity increased rapidly and in the same proportion between the various groups as was observed previously during primary myelination. Insulin as well as crude pancreatic extract and, to some extent, proinsulin demonstrated a marked effect on the time of onset and principally on the total amount of myelin developed by treated cultures as compared to those maintained in normal nutrient medium. PMID- 3003260 TI - Cataracts induced by intermittent Decadron used as an antiemetic. AB - Recent reports have shown that Decadron (dexamethasone; Merck Sharp & Dohme, West Point, Pa) has a significant antiemetic effect on cisplatin-induced vomiting. Although the development of posterior subcapsular cataracts is a known complication of systemic steroid therapy, it usually occurs after several years of chronic administration. A young diamond dealer developed visual impairment attributed to bilateral posterior subcapsular cataracts following only four courses of intermittent Decadron used as part of a five-drug antiemetic regimen for cisplatin-associated nausea. This report is intended to alert others to the possible development of this serious complication following short-term Decadron therapy. PMID- 3003259 TI - Pulmonary toxicity with combined modality therapy for limited stage small-cell lung cancer. AB - To assess the pulmonary toxicity of radiation therapy combined with chemotherapy v chemotherapy alone, we reviewed the clinical course of 80 patients with limited stage small-cell lung cancer treated in a randomized prospective trial. Life threatening pulmonary toxicity, defined as bilateral pulmonary infiltrates extending beyond radiation ports with symptoms requiring hospital admission, developed in 11 patients (28%) receiving combined modality therapy and in two (5%) receiving chemotherapy alone. Eight of these 13 patients died from pulmonary complications with no clinical evidence of tumor in five. Pulmonary toxicity initially presented at a median of 63 days (range, 21 to 150 days) after the start of combined modality therapy and at a median of 217 days after chemotherapy alone. Biopsies obtained in 11 patients with severe toxicity revealed only interstitial fibrosis with no evidence of an infectious agent. Review of pretreatment parameters such as age, performance status, and radiation portal area failed to reveal any significant differences between patients with or without pulmonary complications. However, initial pulmonary function tests (PFTs) revealed a significantly lower vital capacity (P = .03) and forced expiratory volume (FEV/1.0 second) (P = .04) in patients with subsequent pulmonary complications. Pulmonary toxicity was significantly more common with combined modality therapy than with chemotherapy alone (P = .017) and worse than expected with radiotherapy alone. Six- or 12-month PFTs in completely responding patients revealed improvement within the chemotherapy alone group and no clear trend within the combined modality group. For the group treated with radiation therapy and chemotherapy, there was significantly less improvement after 6 or 12 months in the forced vital capacity (P less than .005) and FEV/1.0 second (P less than .005) than observed for the group treated with chemotherapy alone. Despite the increased incidence of pulmonary toxicity, overall survival favored the combined modality arm (P = .07). Enhanced local control and disease-free survival appeared to compensate for the initial increased pulmonary morbidity and mortality in the group with combined modality therapy. PMID- 3003254 TI - Treatment of peripheral neuropathies. AB - There are three general approaches to treatment of peripheral neuropathy. First, an attempt should be made to reverse the pathophysiological process if its nature can be elucidated. Second, nerve metabolism can be stimulated and regeneration encouraged. Third, even if the neuropathy itself cannot be improved, symptomatic therapy can be employed. This review outlines the options available for each approach. PMID- 3003261 TI - Potentiation of transmission at Ia-motoneuron connections induced by repeated short bursts of afferent activity. AB - Single medial gastrocnemius Ia-afferent fibers and motoneurons to which they projected were simultaneously impaled in anesthetized cats. Each Ia-afferent fiber was electrically stimulated once every 2 s with short high-frequency bursts (32 shocks at 167 Hz) followed by 1-11 test shocks. The resulting motoneuron excitatory postsynaptic potentials (EPSPs) were recorded and averaged in register. The interval between the end of one burst and the beginning of the next was 2 s; therefore, the amplitude of the first EPSP in the burst was considered to be a measure of efficacy of transmission 2 s after the burst. At most connections (23/29) the mean amplitude of the first EPSP in the burst was equal to or larger than the mean amplitude of control EPSPs produced by low-frequency (18-Hz) stimulation. Enhancement of transmission was maximum 50-100 ms after the burst, and the amplitude of the test EPSP delivered at this time was always greater than that of the control. The period of enhanced transmission appeared to decay more rapidly at connections with small EPSPs. The greatest amount of EPSP amplitude enhancement at 50 or 100 ms after the burst was observed at connections at which EPSP amplitude increased during the burst. The shape (rise time, half width) of potentiated EPSPs was the same as control EPSPs averaged during low frequency (18-Hz) stimulation. Multiple shocks delivered at low frequency between bursts revealed that enhanced transmission following the high-frequency burst is very sensitive to the effects of low-frequency test stimulation. Furthermore, increasing the number of shocks during the interval between bursts reduced the enhancement of the first EPSP in the burst. We suggest that modulation of synaptic transmission after high-frequency bursts differs across Ia-motoneuron connections. These time-dependent changes associated with short bursts of firing (which are similar in frequency to those observed in Ia-fibers supplying hind limb muscles during stepping) emphasize the necessity to consider the history of the discharge pattern of the group Ia fiber in assessing efficacy at individual Ia-motoneuron connections. PMID- 3003262 TI - Dopamine action in the nucleus accumbens. AB - The action of dopamine was studied in the nucleus accumbens of acutely prepared rabbits. Dopamine was applied iontophoretically to those cells and cell populations that responded in a monosynaptic excitatory manner to ipsilateral fimbrial stimulation. This strategy was adopted to isolate the effects of dopamine on postsynaptic receptors thus avoiding the bias resulting from activation of presynaptic dopamine receptors on dopaminergic afferents. Dopamine was found to have a suppressive effect on the excitatory (N) component of the field response and on driven extracellular unitary discharges. The specificity of dopamine's effect with receptors was indicated by the facts that fluphenazine effectively antagonized dopamine's effect, whereas bicuculline did not. The effect of dopamine was dependent on the rate of fimbrial stimulation. Dopamine has a marked suppressive effect on the fimbria-induced response at 0.5 Hz of stimulation but not at 6.0 Hz. This frequency specificity could not be linked directly to a cyclic adenosine 3',5'-cyclic monophosphate (cyclic AMP) mechanism because the iontophoresis cyclic AMP and dibutyryl cyclic AMP had suppressive effects at both 0.5 and 6.0 Hz rates of stimulation. It is suggested that dopamine acts in the nucleus accumbens to increase the "signal-to-noise" ratio. This might be a form of "contrast enhancement" of an incoming hippocampal message. PMID- 3003263 TI - Neurological deterioration after lumbar puncture below complete spinal subarachnoid block. AB - The risk of neurological deterioration after removal of cerebrospinal fluid below the level of a complete spinal subarachnoid block is generally accepted. The actual incidence of deterioration after lumbar puncture in the presence of a complete block remains unknown. The present retrospective case analysis includes a review of 100 patients found to have complete block on myelography: 50 cases with a lumbar puncture and 50 cases with a C1-2 puncture. Each group consisted of a similar age range, neurological status prior to myelography, level of block, and nature of disease. Seven patients (14%) had significant neurological deterioration after lumbar puncture, while no deterioration was seen after a C1-2 puncture. A summary of those cases in which deterioration followed lumbar puncture is presented and the possible pathophysiology is discussed. From this analysis, the estimated risk of downward spinal coning after lumbar puncture below a complete spinal subarachnoid block caused by a mass lesion is at least 14%. PMID- 3003264 TI - Antifibrinolytic therapy of experimentally grown malignant brain tumors. AB - This study was designed to evaluate the effect of an inhibitor of plasminogen activation on the growth of a human glioblastoma line grown in nude mice up to the seventh passage. The tumors produced plasminogen activators and showed histological characteristics similar to those of the original tumor. Three groups of mice were studied. Group A received 5% epsilon aminocaproic acid (EACA); Group B received 2.5% EACA; and Group C served as a control. There was no statistical difference among the three groups with regard to: 1) age at time of tumor transplantation; 2) the interval between implant and treatment; or 3) tumor volume at time of treatment. Blood measurements of EACA, performed in a limited number of animals, have shown that the drug at 5% concentration had reached toxic levels. Statistically significant differences between the three groups were noted in the following categories: 1) rate of tumor growth; 2) tumor volume at time of death, where Group A had smaller tumors than Group C; and 3) mean survival times of Groups A and B as compared to Group C. A statistically significant negative correlation was found between the rate of tumor growth and the length of survival of animals in Group C, while no correlation could be found for either Group A or B, indicating that the antifibrinolytic therapy modified this important biological variable. This study supports the hypothesis that the fibrinolytic system plays a role in the growth and development of malignant gliomas and that interference with the fibrinolytic system may retard the growth of these tumors grown in nude mice. PMID- 3003266 TI - Re-entry women in baccalaureate nursing programs: the achievement of selected developmental tasks. AB - This study sought to determine if there is a difference in the level of development, as assessed by the Student Developmental Task Inventory (SDTI-2), between the traditional age woman and the re-entry woman who has entered a baccalaureate nursing program. It builds upon the theoretical formulations of accepted principles of human growth and development and recent life cycle research, which suggests that further achievement of developmental tasks is possible throughout adulthood. The subjects consist of 156 traditional age and 50 re-entry women nursing students. Data were analyzed with the use of the subprograms "Frequencies" and "T-Test" of the Statistical Package for the Social Sciences (SPSS). Examination of the null hypotheses related to the achievement of developmental tasks and subtasks revealed that the traditional age woman and the re-entry woman did not differ on the majority of the developmental tasks and subtasks. Implications for nurse educators are discussed. PMID- 3003265 TI - A study of the cost-effectiveness of providing psychomotor practice in teaching intravenous infusion techniques. AB - Does psychomotor practice affect the student's ability to perform venipuncture? This was the primary question in a study conducted to compare two versions of an instructional program--a no-practice version and a costly version that involved practice on the simulated arm. The initial study was composed of 40 (20 each group) Junior nursing students enrolled in a baccalaureate program. The instructional program for both groups included a handout of the task-by-task description of the procedure; a performance check list, a 10-minute color videotape demonstrating the entire process, and a live demonstration on a simulated arm. Each group took a post test which covered the cognitive aspects of the task. The practice group was able to practice on the simulated arm prior to evaluation on a live subject. The cognitive scores for both groups were near 90% and the performance scores above 80%. Since there was no significant difference between the groups the study was repeated with another group of students from the same school. This group differed slightly from the original group in that they had no experience with venipuncture. The second study contained 34 students and the cognitive scores were 91% for both groups and above 80% for the performance scores. The results indicated that the least costly version was equally effective and presents nursing education with an opportunity to evaluate skills teaching to determine the most effective, efficient and/or economical method to teach skills. PMID- 3003267 TI - Faculty workload: myths and realities. AB - This survey was done to determine the current workload policies and practices in collegiate schools of nursing. A 40-item questionnaire was mailed to all schools holding membership in the American Association of Colleges of Nursing. Seventy two percent of the schools responded. Analysis of the data shows that quantifiable factors which relate directly to teaching are of considerable importance to deans in determining faculty workload while less quantifiable factors such as student advisement, research, publication and involvement in direct client care are of little or no importance in determining workload. PMID- 3003269 TI - Change in basic nursing students' attitudes toward the elderly after a nursing home experience. PMID- 3003268 TI - Reliability and validity of the Women's Health Care Skills Audit. AB - The Women's Health Care Skills Audit (WHCSA) is a behaviorally oriented instrument which is hypothesized to measure important, clinically relevant components of the basic women's health care skills of Family Nurse Practitioners. The purpose of this research was to examine the reliability and validity of the WHCSA. Two women's health care nurse practitioners independently evaluated the performance of 23 Family Nurse Practitioner students during client visits. Overall internal consistency and test-retest reliabilities of the instrument were high--.96 and .84 respectively. In addition, support for the convergent validity of the WHCSA was also obtained in the study. Results therefore indicate that the WHCSA is both reliable and valid and holds promise for use in documenting and evaluating basic Family Nurse Practitioner women's health care competencies. PMID- 3003270 TI - Clinical progress notes: a challenge for the nurse educator. PMID- 3003271 TI - Death education in baccalaureate nursing programs. AB - These findings reveal that an emphasis on death and dying in baccalaureate nursing schools has definitely increased over the past 20 years. Ninety-five percent of the schools reported here have some emphasis on death and dying. The majority of nursing students take the death and dying offerings. Overwhelmingly, the professional background of the instructor is nursing. While only 5 percent of the schools do not offer anything in death and dying, nearly half of these plan to offer something "within the next five years." If death and dying offerings influence students' attitudes, as is suggested in research cited above, baccalaureate nursing programs in the United States apparently have taken positive steps toward helping nurses cope with dying over the past 20 years. If nurses themselves cannot deal with death, caring for a dying patient may be difficult. With continued increased emphasis on death and dying in nursing curricula, both nurses, patients, and patients' families will hopefully benefit. PMID- 3003272 TI - Comprehensive curriculum evaluation. AB - The comprehensive scope of this evaluation plan, with both summative and formative components, will provide the data necessary to guide our decision making. Some aspects, such as sampling students' work, the use of simulation, and the testing of actual learning, strengthens this process. However, satisfaction with our initial attempts at evaluation has not clouded an attitude of scrutiny. Undoubtedly, revision of the evaluation methods will be necessary in the future as inadequacies are pointed out to us through experience. The faculty have become acutely aware that curriculum evaluation is a time-consuming, tedious process. However, the faculty, committed to bearing responsibility for student performance, have found their involvement educational as well as satisfying. A well-constructed curriculum evaluation system can give the teacher a sense of security; a belief that what works will be preserved while ineffective components will be deleted. As Dressel points out, "Unless continuing constructive evaluation is evident in some form, both teaching and learning degenerate to rote patterns that hardly justify the designation of education" (Dressel, 1980, p. 197). PMID- 3003273 TI - Multiculturalism in nursing: implications for faculty development. PMID- 3003274 TI - A demystification of the clinical nurse specialist role: perceptions of clinical nurse specialists and nurse administrators. AB - Differences of nurse administrators and clinical nurse specialists regarding components of the clinical specialist role were investigated. To be effective in facilitating the maximum use of the abilities of the clinical specialist, it is essential that nurse educators establish a data base about the clinical nurse specialist role from the perspective of employers and the clinical nurse specialists themselves. Subjects were 54 nurse administrators and 35 clinical specialists from a large metropolitan area. The results indicated that, generally, both groups were in agreement except for the research component. Administrators placed a higher value on research than did the clinical specialists. Both groups valued the clinical practice component with the clinical specialists tending to see themselves more heavily committed to direct specialized patient care than did the administrators. The educational component was also valued with the consultant function receiving the highest rating of all the functions surveyed. The administration component was valued least highly of all the components. It is recommended that nurse educators evaluate their educational endeavors in preparation of clinical specialists especially for research functions, and, also, consultation and those functions supportive of nursing staff. PMID- 3003275 TI - Use of nonparametric correlation analysis in graduate students research projects. PMID- 3003276 TI - The relationship of nursing deans' leadership behaviors with institutional characteristics. AB - The purpose of this study was to investigate the relationships between the leadership behaviors of nursing deans and selected organizational variables in baccalaureate and higher degree nursing programs in the United States. The sample consisted of 170 deans who provided data on their self-perceived leadership behaviors as measured by the Leadership Behavior Description Questionnaire and on institutional characteristics of both their nursing programs and parent institutions. Using correlation and analysis of variance, a significant relationship was found between the leadership dimension of Consideration and faculty expertise. Significant relationships were also found between the dimension of Initiating Structure and the nursing program variables of faculty expertise and educational task and the parent institution variables of control, educational task, and size. This study proposes that organizational variables should be included in leadership theory for nursing academic administrators. PMID- 3003277 TI - Meeting the challenge of multiple academic roles through a nursing center practice model. AB - Filling the multiple requirements of academic life--to teach, to conduct research, and to give service to the community--can be difficult for any academician. When the educator is also a nurse, meshing academic life with that of a practicing professional adds to the difficulties. Maintaining control and managing resources for practice can be time consuming and wearisome. The authors use quotes from the sociologist Paul Starr to describe and examine this professional dilemma. The process they followed in implementing a practice model in a community nursing center is described. Using this approach teaching, research, and service requirements were blended with practice for greater professional control and more productive scholarly endeavors. PMID- 3003278 TI - The dean's job satisfaction: its relationship to organizational structure. AB - The purpose of this study was to explore the relationship between formal organizational structure and the dean's job satisfaction. A two-part questionnaire was sent to 345 deans of Baccalaureate and Higher Degree nursing programs. Seventy-three percent responded. The findings showed that decentralization and some degree of complexity and formalization were related to job satisfaction. In spite of these findings, deans should consider all personnel, academic, and budgetary implications before proceeding with organizational restructuring. PMID- 3003279 TI - Critical thinking through writing: using personification to teach pharmacodynamics. PMID- 3003280 TI - Introduction of a conceptual nursing model into a fundamental baccalaureate course. PMID- 3003281 TI - Peer review--a process of socialization. PMID- 3003282 TI - Health Fairs: a non-traditional introduction to community health nursing. PMID- 3003283 TI - Faculty practice: a means to advance the discipline of nursing. PMID- 3003284 TI - A developmental approach to doctoral education. PMID- 3003285 TI - A nurse-managed Family Health Center at the University of Florida. PMID- 3003286 TI - A nursing faculty practice in a Veterans Administration Medical Center: an asset to service and education. PMID- 3003287 TI - Using literature to teach nursing. PMID- 3003288 TI - Effects of dietary zinc deprivation on the activity of angiotensin-converting enzyme in serum of rats and guinea pigs. AB - Angiotensin-converting enzyme (ACE) is a zinc-dependent peptidyldipeptide hydrolase found in blood and in the endothelium of many tissues. This study was designed to determine the effects of dietary zinc deprivation in rats and guinea pigs on ACE activity in serum. Rats were fed zinc-deficient (less than 1 mg/kg) or zinc-adequate (25 mg/kg) diets for 3 d. Guinea pigs were fed zinc-deficient (less than 1 mg/kg) or zinc-adequate (100 mg/kg) diets for 3 wk. The plasma zinc concentration in the zinc-deprived rats was 26% and that of the deprived guinea pigs 35% of the control values. When serum was diluted by one-half in the assay medium, the respective ACE activities were 42 and 59% of controls. In spite of decreased ACE activity there were no differences in plasma angiotensin II levels in either species or in bradykinin levels in the rat. To determine the effect of zinc on the kinetics of serum ACE, rat serum was treated to remove low-molecular weight components and diluted 30-fold in an assay medium that contained a final zinc concentration of 0.68 microM. Supplemental zinc (total, 24 microM) increased both Vmax and Km. EDTA inhibited the enzyme activity, and the inhibition was totally reversible by zinc. Copper at 30 microM had no effect on ACE activity. The results of this study show that ACE activity in serum of rats and guinea pigs is highly sensitive to the dietary intake of zinc and suggest that metabolism of the vasoactive hormones, angiotensin II and bradykinin, might be affected in vivo. PMID- 3003289 TI - Using density rather than mass to express the concentration of gastrointestinal tract constituents. AB - This experiment had two objectives: to develop an analytical technique to measure the density of digesta and fecal samples; to determine whether this measurement is physiologically relevant. To address the latter question, rats were provided with one of five high-fiber diets or a fiber-free control diet, and density determinations were made on samples from the cecum, proximal and distal colon, and from a passed fecal pellet. The density of colonic contents varied with the fiber component of the diet. The fiber-free control diet produced the densest stool at all sites studied (P less than 0.01), whereas wheat bran produced the least dense stool at every site (P less than 0.01). There were also density differences among bowel positions within diet treatments: the stool from those animals consuming the fiber-free, guar and pectin diets became denser as it moved distally; wheat bran, on the other hand, produced the opposite effect--the more distal the stool traveled, the less dense it became. Since the density of colonic contents varied with respect to both diet and anatomical site, these data suggest that the traditional technique of expressing the concentration of a constituent of interest (e.g., bile acid, volatile fatty acid) per gram of digesta or fecal matter may not always be appropriate. This is particularly true if exposure of the intestinal mucosal cells to a substance in the digesta or fecal stream is of interest, since density, rather than mass, reflects the diluting potential of the diet. PMID- 3003290 TI - Warning: feeding animals hydrophilic fiber sources in dry diets. AB - Feeding animals large quantities of dry hydrophilic fiber sources, such as psyllium husk or guar gum, may lead to intestinal obstruction or to other mechanical effects unrelated to the normal function of these materials in human diets. Such fiber sources should be hydrated prior to feeding, rather than being incorporated into dry diets as is. The water-holding capacity of psyllium hydrophilic mucilloid, for example, is greater than or equal to 40 g/g, compared to 2-3 g/g of wheat bran. Consumption of the psyllium product dry would be much more likely to produce intestinal dehydration than would consumption of dry bran. Because of possible untoward effects of high levels of these materials, it may also be more appropriate to feed such fiber sources in quantities approximating that of their potential human dietary consumption, rather than very high quantities that would be unlikely to be attained in human diets. PMID- 3003291 TI - Effects of absorption of large amounts of volatile fatty acids on rat liver metabolism. AB - The effects of large amounts of volatile fatty acids (VFA) on hepatic metabolism have been investigated in vivo, with rats adapted to a high fiber (HF) diet, or in vitro with isolated hepatocytes. Net absorption of glucose was negligible and lactate production was lower in rats fed the HF diet than in those fed a fiber free basal diet. VFA (particularly acetate and propionate) were absorbed in very large amounts in rats fed the HF diet. Propionate and butyrate were completely removed by the liver in both groups of rats; the efficiency of acetate uptake was higher in rats fed the HF diet than in those fed the basal diet. Gluconeogenesis was active in rats fed the HF diet, but lactate uptake was very limited in spite of high portal concentrations. Hepatocytes from rats fed the HF diet utilized VFA at different rates: acetate was poorly utilized, propionate utilization plateaued at about 1 mM external propionate, whereas butyrate utilization was utilized about twice as efficiently. Propionate and butyrate displayed opposite effects on lactate utilization. The stimulating effect of butyrate prevailed over the inhibitory effects of propionate, at high concentrations (2 mM). However, the results at lower concentrations (less than or equal to 0.5 mM) suggest that, owing to its higher portal concentrations, the effects of propionate on lactate uptake might prevail in vivo. The effects of acetate in vivo might be greater than on isolated hepatocytes. PMID- 3003292 TI - The effect of methanethiol and methionine toxicity on the activities of cytochrome c oxidase and enzymes involved in protection from peroxidative damage. AB - The tissue changes characteristic of methionine toxicity may be caused by methanethiol (CH3SH) inhibition of enzymes involved in protection from peroxidative damage. Methanethiol is an intermediate of the transaminative pathway of methionine metabolism. Glutathione peroxidase, glutathione reductase, catalase and superoxide dismutase activities were therefore tested for susceptibility of CH3SH. Cytochrome c oxidase activity was also measured because of its known inhibition by mercaptans. A 10-min exposure to CH3SH depressed hepatic cytochrome c oxidase activity to 28% of the incubated control value, while hepatic, splenic and erythrocyte catalase activities were depressed, respectively, to 53, 52 and 71% of the incubated control. Similar reductions in catalase and cytochrome c oxidase activities were observed in rats fed a diet containing 3% L-methionine as compared to rats pair-fed a control diet containing 0.3% methionine. There was no difference in the amount of lipid peroxidation as monitored by the presence of malondialdehyde in the livers of these rats. In rats injected i.p. with 50 or 75 mumol of 3-methylthiopropionate, an intermediate of methionine catabolism, the maximum levels of exhaled methanethiol coincided with depressions in liver catalase and cytochrome c oxidase activity to 40-50% of control values. The activities of these enzymes returned to control values within 2 to 4 h. The inhibition of catalase activity does not appear to be the cause of the membrane damage observed in methionine toxicity. PMID- 3003293 TI - Effects of certain dietary fibers on apparent permeability of the rat intestine. AB - Apparent intestinal permeability was determined indirectly by orally administering a poorly absorbed dye, phenol red, to rats and measuring its recovery in feces and in urine. Increased apparent permeability was recognized by increased dye recovery in urine and by an increased ratio of urinary to fecal dye recovery. Guar gum, pectin, carrageenan type I (80% kappa, 20% lambda), carrageenan type II (iota) and cellulose were each fed at levels of 5 and 15% (wt/wt) of the diet for 31 d to male Fischer 344 rats. The average initial weight of rats was 230 g. Rats fed 15% guar gum gained significantly less weight than most of the other rats (P less than 0.05). Phenol red recovery was measured at 2 and 4 wk after the beginning of the experiment. At 2 wk urinary recoveries of phenol red were high in rats fed fiber-free and carrageenan type II diets, indicating increased apparent permeability. By 4 wk, adaptation had apparently taken place. Urinary dye recoveries were lower in every diet group, and most fiber-containing diet groups gave significantly lower recoveries than did the fiber-free group. Fecal recovery of phenol red was high in the cellulose, carrageenan I, and 5% carrageenan II groups, intermediate in the 5% pectin and 15% carrageenan II groups, and low in the fiber-free, guar gum and 15% pectin groups at both 2 and 4 wk. The ratio of phenol red recovery from urine to that from feces, another index of apparent intestinal permeability, was higher in the fiber-free diet group than in all the other groups. Rats fed 15% dietary fiber had higher average ratios than those fed the same fiber at 5%. These data are consistent with the hypothesis that intestinal permeability to foreign substances may be altered considerably by diet. PMID- 3003294 TI - Comparison of the effect of cell wall and hull fiber from canola and soybean on the bioavailability for rats of minerals, protein and lipid. AB - Hull or cell wall material, isolated from canola (Brassica napus cv. Regent) or soybean (Glycine max) or cellulose was added to a basal, semipurified diet at a level of 12% and fed to growing male Wistar rats. The apparent availability of Cu, Fe, Ca, P and protein were lower when the fiber-containing diets were fed compared to the control diet. The availability of Mg was lower when the canola hull (CH) and cellulose (CE) diets were fed, whereas Zn availability was lower when the canola cell wall (CCW) and CE diets were fed, compared to the control diet. Determination of the feed transit time, by using the unabsorbable marker Cr2O3, demonstrated that the CE and soybean cell wall (SCW) diets had the fastest transit rates, followed by the CH, soybean hull (SH), CCW and control diets. Determination of the in vitro binding of the various fibers to metal ions demonstrated that CH was the strongest chelator, whereas CE had the lowest binding affinity for any of the minerals tested. At the termination of the trial, intestinal segments were removed and the in vitro mucosal transport of Cu, Fe, Mn and Zn were compared. The CCW diet consistently demonstrated lower transport for the four minerals, especially in the ileal segment. PMID- 3003295 TI - The influence of wheat bran and sugar-beet pulp on the digestibility of dietary components in a cereal-based pig diet. AB - The influence of added wheat bran or dried sugar-beet pulp on the apparent digestion of dietary components was examined in pigs fitted with duodenal and terminal ileal cannulas. The pigs were fed a basal cereal-based diet alone or substituted at a level of 33% by wheat bran or beet pulp. Neither fibrous component influenced the digestibility of starch, but inclusion of beet pulp decreased dry matter content and digestibility of ash, protein and fat in the ileum and fecal digestibility of fat. Between 11 and 37% of the nonstarch polysaccharides were degraded anterior to the ileum, and the percentage of beet pulp diet nonstarch polysaccharides that was soluble increased from 11% in the feed to 35% at the ileum. Xylose and glucose were the least degraded nonstarch polysaccharide residues in all three diets, and the non-starch polysaccharide residues varied in susceptibility to breakdown within and between diets, depending on the composition of the fiber. Almost 50% of the beet pulp, but less than 20% of the wheat bran, was degraded in the large intestine. The inclusion of wheat bran and beet pulp increased fecal output by 127 and 56%, respectively. PMID- 3003296 TI - Absence of respiratory effects in subjects exposed to low concentrations of TDI and MDI: a reevaluation. AB - Because of criticisms of the authors' previously reported study showing absence of respiratory effects in subjects exposed to low concentrations of toluene diisocyanate (TDI) and diphenylmethane diisocyanate (MDI), the data have been reanalyzed. This reanalysis confirms the accuracy of the results and the conclusions based on them. Subjects exposed to 0.0010 to 0.0015 ppm of TDI and around 0.0003 to 0.0006 ppm of MDI demonstrated a normal age- and smoking-related rate of decline in forced expiratory volume in 1 s and no evidence that it was related to isocyanate exposures. Control of isocyanate exposure sufficient to protect industrial workers against measurable deterioration in lung function appears feasible. PMID- 3003297 TI - Gestational trophoblastic neoplasms. AB - Gestational trophoblastic neoplasms have evolved from one of the most rapidly fatal malignancies to potentially one of the most curable, but these diseases have devastating emotional effects on the victims. Etiology, epidemiology, pathophysiology, diagnosis, and medical treatment are reviewed. Numerous nursing implications are discussed using crisis theory. A nursing care plan, based on nursing diagnosis, is outlined with specific nursing actions defined. PMID- 3003298 TI - Naloxone does not modify the antihypertensive effect of captopril in essential hypertensive patients. AB - It has been reported that endogenous opioid antagonism through naloxone (NAL) blunts the hypotensive effect of captopril (CAP) in normal man. For this reason a study was carried out to determine whether NAL interacts with the antihypertensive effect of CAP in patients with mild-to-moderate essential hypertension (n = 6; age 22-43 years). Patients received, according to a randomized code, placebo (PL) during saline infusion, CAP (25 mg orally) during saline infusion, PL + NAL (0.4 mg as a bolus followed by a continuous infusion of 4.0 mg/h for 2 h) and CAP + NAL at the same doses. Three-day intervals elapsed between each phase of the study. Blood pressure (BP) and heart rate (HR) were recorded before (-20 to 0 min) and every 15 min for the next 2 h after drug administration. Blood samples for plasma renin activity (PRA), noradrenaline (NA) and aldosterone (ALD) were collected before (time 0) and 120 min after drug administration. Neither PL nor NAL changed any of the parameters examined, while CAP alone reduced mean BP to a highly significant extent. Naloxone did not change the hypotensive effect of CAP to any significant extent. Captropril, either alone or associated with NAL, tended to reduce plasma ALD and increase PRA (P less than 0.05) without changing HR or plasma NA. These data indicate that, at least under the experimental conditions used, endogenous opioid antagonism does not interfere with the haemodynamic and humoral effect of CAP in essential hypertensive man. This suggests that endogenous opioids do not mediate the pharmacological actions of CAP. PMID- 3003300 TI - Blunted adrenocorticotrophic hormone release during captopril treatment. AB - High tissue levels of angiotensin II have been reported in the median eminence suggesting a possible role in the regulation of adrenocorticotrophic hormone (ACTH) secretion. To verify this hypothesis in man, the pituitary-adrenal axis response to hypoglycaemia was studied before and during captopril treatment in eight male essential hypertensive patients (stage I WHO; aged 35-52 years). Plasma levels of ACTH, cortisol and glucose were measured before and 60, 90 and 120 min after an intravenous bolus of normal saline as placebo an, 3 days later, after an intravenous bolus of rapidly acting insulin (0.1 IU/kg body weight). Captopril treatment was then started and both placebo and hypoglycaemic tests were repeated 15 days thereafter. No changes in ACTH, cortisol or glucose plasma levels were observed after acute normal saline, either before or during captopril administration. On the contrary, hypoglycaemia induced a sharp increase of ACTH plasma before captopril (from 27.7 +/- 11 to 131.30 +/- 26 pg/ml, P less than 0.01, 60 min after insulin) but not during angiotensin converting enzyme (ACE) inhibition (from 28.9 +/- 9 to 42.9 +/- 11 pg/ml, NS, at min 60 of the study). Our present data, showing a blunted ACTH response to hypoglycaemia during ACE inhibition, suggest that circulating angiotensin II may participate in the regulation of the release of the ACTH, possibly by a stimulation of angiotensin II receptors localized in the brain but outside the blood-brain barrier. PMID- 3003299 TI - The responses of adrenocorticotrophic hormone and cortisol to insulin-induced hypoglycaemic stress in man are unimpaired during chronic converting enzyme inhibition. AB - In vitro and animal studies indicate that circulating angiotensin II (ANG II) can stimulate adrenocorticotrophic hormone (ACTH) and cortisol secretion, but it is far from established that ANG II has a physiologically relevant influence on steroidogenesis. We studied the effects of hypoglycaemia induced with insulin injection (0.15 IU/kg) in patients with essential hypertension to answer this question. Hypoglycaemia was induced before and after a short term course of treatment with the converting enzyme inhibitor captopril to obtain a sustained blockade of ANG II formation. Alterations in serum glucose, plasma potassium, plasma ACTH, cortisol, renin activity and aldosterone were examined. In control studies there was a profound fall in serum glucose and plasma potassium after insulin, associated with increments in plasma renin activity, which correlated with those of aldosterone but not with those of ACTH and cortisol. Chronic captopril increased baseline plasma renin activity and lowered baseline aldosterone while leaving ACTH and cortisol unchanged. During converting enzyme inhibition the insulin-induced decrements in glucose and potassium, as well as the increments in ACTH, cortisol and aldosterone, were similar to those observed in control studies, whereas the increments in plasma renin activity were much greater. From these results it does not appear that ANG II has a relevant influence on ACTH and cortisol production, or on their responses to hypoglycaemic stress. Rather, these findings indicate that even under the present experimental conditions ANG II is the primary regulator of aldosterone secretion. However, this function can be taken over by ACTH when the generation of ANG II is blocked. PMID- 3003301 TI - Plasma vasopressin as influenced by acute and chronic blockade of the renin angiotensin system. AB - The activation of the renin-angiotensin system that occurs during the development of congestive heart failure (CHF) may be accompanied by continued secretion of vasopressin (AVP) in response to non-osmotic stimuli. Increased supine plasma AVP levels (by radio-immunoassay) were found in 31 patients with moderate to severe CHF (11.49 +/- 1.00 pg/ml s.e.m.) 24 h after the last diuretic dose, which correlated with plasma renin activity (PRA) (r = 0.37, P less than 0.05). However, acute inhibition of converting enzyme with captopril did not decrease plasma AVP levels (14.9 +/- 3.9 versus 14.0 +/- 2.8 pg/ml, n = 8). Indeed, in six out of eight patients, plasma AVP actually increased following captopril - presumably secondary to haemodynamic changes. Readministration of captopril after 4 months of captopril treatment, 12 h after the last dose, again did not change AVP levels (9.58 +/- 1.2 versus 13.1 +/- 1.9 pg/ml), whereas changes in haemodynamics, PRA and angiotensin II were as expected and similar to the first test. These results suggest that the acute haemodynamic action of captopril in CHF is not mediated via suppression of vasopressin, although in some patients with non-osmotic vasopressin, excess activation of the renin-angiotensin aldosterone system might constitute a factor. PMID- 3003302 TI - Relationship between vasopressin and the renin-angiotensin-aldosterone system in essential hypertension: effect of converting enzyme inhibitor on plasma vasopressin. AB - The effect of the chronic administration of captopril on plasma levels of vasopressin (PVP) were studied in 14 patients with moderate essential hypertension and 10 normal volunteers. All patients were studied after 10 days without drugs and under a constant diet (120 mmol sodium and 80 mmol potassium/day). Plasma levels of renin activity (PRA), aldosterone (PA) and PVP were assayed before and after captopril treatment (50-100 mg/day for 1 month). In addition to the well-known effect of captopril treatment on PRA and PA, a statistically significant reduction of PVP was observed. This finding suggests that the renin-angiotensin-aldosterone system influences vasopressin release, and its inhibitors may contribute to the absence of water retention during captopril treatment compared with the effect of other vasodilatory drugs. PMID- 3003303 TI - Will renin inhibitors influence decision-making in antihypertensive therapy? AB - Although renin was identified as playing a part in cardiovascular homeostasis by the experiments of Goldblatt in the 1930s, neither its physiological role in organs other than the kidney nor its contribution to the genesis of essential hypertension have been defined. It is difficult to interpret studies with converting enzyme inhibitors because of their multiple pharmacological effects. Specific inhibitors of renin appropriate for clinical investigation would help to resolve many questions. Four classes of compounds have been shown to be renin inhibitors of high potency: specific antibody, general peptide inhibitors of acid proteases, analogues of angiotensinogens and peptides that are related to the amino-terminal sequence of prorenin. Of these, it is likely that angiotensinogen analogues will be the first applied in human studies. The minimal substrate for renin has the sequence: His-Pro-Phe-His-Leu-Val-Tyr. Variants of this sequence have yielded competitive inhibitors. Remarkably active compounds have recently been synthesized by reducing the peptide bond that is cleaved by renin, or by incorporating the amino acid statine, found in pepstatin. These compounds have been shown to be effective in dogs, rats and monkeys and, most recently, preliminary studies have reported their efficacy in man. Recent studies with one of these inhibitors, RIP, raise questions concerning both its specificity and site of action. PMID- 3003304 TI - Non-modulating essential hypertension: a subset particularly responsive to converting enzyme inhibitors. AB - The goals of antihypertensive therapy are to normalize arterial pressure and to minimize pharmacological adverse effects. Recent data indicate that in general the first goal has been achieved but the second has not, resulting in a substantial non-compliance rate. There is increasing evidence, therefore, that the next major step forward in treating hypertensive patients is to link specific forms of therapy to subsets of the hypertensive population. The present article illustrates this point by describing the characteristics and therapeutic implications of a subset of essential hypertensives termed non-modulators and the therapeutic implications which arise. These individuals have a defect in the way that sodium intake modifies adrenal and renal vascular responses to angiotensin II, resulting in an abnormality in sodium handling. Angiotensin converting enzyme (ACE) inhibitors partially, if not completely, correct this underlying defect and thereby reduce blood pressure by restoring the renal vascular responsiveness to normal. Because of the specificity of ACE inhibitors in treating non-modulators, it is to be expected that they would produce fewer long term side effects, resulting in a better quality of life for these patients than if their blood pressures were treated non-specifically. PMID- 3003305 TI - Angiotensin converting enzyme inhibitors and quality of life: the European trial. AB - Two prospective multi-centre randomized trials were initiated to compare the relative efficacy and influence on quality of life of captopril, alone or in combination with hydrochlorothiazide, against either methyldopa, alone or in combination with hydrochlorothiazide, or oxprenolol in combination with chlorothalidone. The complaint rate, activity index and psychiatric morbidity were evaluated as indices of quality of life. Captopril was associated with a significantly (P less than 0.05) greater reduction in complaint rate compared with methyldopa and a tendency for less symptoms of depression compared with oxprenolol (P = 0.06), the latter drug being associated with an increase in depression scores. The trends in quality of life indices in the captopril-treated patients would suggest the need for double-blind placebo-controlled trials to investigate these apparent benefits. PMID- 3003306 TI - Prospectives for angiotensin converting enzyme inhibition in heart diseases. AB - Angiotensin converting enzyme (ACE) inhibitors are not known to have a direct effect on the myocardium. However, there is some evidence to suggest that they can play an important role in protecting the heart during the evolution of hypertensive and coronary arterial disease, both acutely and on a long term basis. Reduction of afterload by balanced arterial and venular dilatation has led to a sustained improvement of cardiac performance both in hypertension and heart failure. Reversal of cardiac hypertrophy has been shown to restore inotropic responsiveness to stimulators of the adenylate cyclase system. Following myocardial infarction, captopril has prevented undue ventricular dilatation and normalized left ventricular chamber stiffness; this prevented deterioration of cardiac function and improved long term survival after infarction. Control of secondary aldosteronism and prevention of hypokalaemia can play an important role in the prevention of cardiac arrhythmias. The lack of reflex sympathetic stimulation during long term captopril therapy can also play a favourable role in that respect. Although highly speculative, evidence is accumulating that ACE inhibition could have a cardioprotective effect in acute myocardial ischaemia. It is based on the demonstration that renin can be produced by myocardial cells, that angiotensin is liberated by the ischemic myocardium and that angiotensin in high renin conditions plays an active constrictor role in regulating the coronary circulation. PMID- 3003307 TI - A comparison of particulate and solid root forms of hydroxylapatite in dog extraction sites. AB - A comparison of solid root forms and particulate hydroxylapatite implanted into dog tooth extraction sites showed both types of implants to be well tolerated. Neither prevented postextraction bone resorption, but the particles were better able physically to maintain ridge height and width than the root forms. PMID- 3003308 TI - Gastrin releasing peptide in human neuroendocrine tumours. AB - Neuroendocrine tumours of the lung and gut are known to possess bombesin-like immunoreactivity. The recent observation that gastrin releasing peptide (GRP), a 27 amino acid peptide isolated from the porcine intestine, may be the mammalian analogue of bombesin led us to look for this peptide in a variety of human neoplasms. Formalin-fixed tissues from 85 tumours were examined by the immunoperoxidase technique, using specific antisera to the GRP molecule (1-27) and the GRP fragment (1-16). Intense cytoplasmic GRP immunoreactivity was seen in thyroid medullary carcinomas (3/3), carcinoids of lung, pancreas, and intestine (22/36), and paragangliomas (2/3). Less frequent staining was present in pulmonary small cell (oat cell) carcinomas (1/8) and pituitary adenomas (1/6). Complete absence of immunoreactivity was observed in three phaeochromocytomas, five Merkel cell tumours, six neuroblastomas and 15 non-neuroendocrine tumours. Normal neuroendocrine cells of the thyroid (C-cells) and bronchial mucosa (Kulchitsky cells) exhibited GRP immunoreactivity; nerve fibres from all sites failed to demonstrate staining for GRP. In each positive case, the pattern of staining for GRP (1-27) and GRP (1-16) was identical, although the GRP (1-16) immunostaining was weaker. These findings indicate that bombesin immunoreactivity in human neuroendocrine cells and tumours is attributable to GRP-like molecules and that GRP is a useful marker of neuroendocrine differentiation in many tumours. PMID- 3003309 TI - Bronchiolar and alveolar lesions in the pathogenesis of crocidolite-induced pulmonary fibrosis in mice. AB - Asbestosis is generally considered to result in restrictive pulmonary disease associated with interstitial fibrosis. Recently, however, attention has focused upon bronchiolar lesions and concomitant obstruction to air flow. The responses of conducting airways and alveoli were studied over a 20 week period following instillation of crocidolite to mice. The location of the lesions and the sequential inflammatory changes were studied by bronchoalveolar lavage, light and electron microscopy; regenerative activity was monitored by autoradiographs. Within 48 h there was multifocal necrosis of bronchiolar epithelium, maximal at bifurcations where longer fibres tend to adhere. Subsequently, intralumenal exudates were overgrown by epithelium and incorporated into the bronchiolar connective tissue where active peribronchiolar granulomas persisted until 20 weeks. Alveolar lesions were located predominantly in peribronchiolar air sacs and at the junctions of bronchioles and alveolar ducts. Focal acute injury of type 1 cells and transepithelial passage facilitated transport of short asbestos fibres to the interstitium where they were phagocytozed by macrophages. Regenerative activity was prompt with active division of type 2 epithelial cells. Biochemically, collagen increased after 4 weeks, when fibrosis was predominantly located in the bronchiolar lumens and in peribronchiolar connective tissue with lesser amounts in the centrilobular alveolar interstitium. The results suggest that the longer fibres induce bronchiolar injury and a more severe fibrotic pattern similar to the recently described changes seen in human asbestosis. PMID- 3003310 TI - Immunocytochemical evidence for neuroendocrine differentiation in human breast carcinomas. AB - Normal and neoplastic human breast tissues have been stained with antibodies recognizing neuroendrocrine differentiation. Fifteen out of 44 (34 per cent), breast carcinomas stained positively with monoclonal antibody LICRLON-E36, and 11 out of 44 (25 per cent) of these tumours stained with an antibody raised against neuron-specific enolase (NSE). Eight tumours stained positively with both antibodies. No correlation was observed between staining with these antibodies and the tumour histology, nor with the degree of cellular differentiation as indicated by staining with several cell surface directed monoclonal antibodies. Ultrastructural analysis of a series of breast tumours showed the presence of membrane-bound dense-core vesicles in almost all tumours, including E36 and NSE positive and negative tumours. The presence of these structures appears to be of little value in predicting neuroendocrine differentiation. PMID- 3003311 TI - Epithelial antigens in malignant mixed Mullerian tumours of endometrium. AB - Immunohistochemical staining of formalin fixed paraffin sections with the mouse monoclonal antibodies E29 and CAM 5.2 effectively distinguish epithelium (antigen positive) from stroma (antigen negative) in normal endometrium. These antibodies were used as epithelial markers in the study of eight malignant mixed Mullerian tumours (MMT) of endometrium and gave normal reactions with well differentiated neoplastic glands; in contrast, negative or very weak staining was observed in poorly differentiated epithelial cells, present in large numbers in three cases. Abnormal antigen-containing cells were observed in the stroma of seven MMT. In some cases these are probably due to stromal invasion by malignant epithelium but in others they may represent an early stage of epithelial differentiation in mesenchymal cells of the malignant stroma. PMID- 3003312 TI - Neurone specific enolase immunostaining in the diagnosis of breast carcinomas with neuroendocrine differentiation. Its usefulness and limitations. AB - Thirty-eight infiltrating ductal carcinomas, nine infiltrating lobular carcinomas, two tubular carcinomas and one papillary carcinoma were studied by light microscopy, immunocytochemistry and electron microscopy. Seventeen cases showed immunoreactivity for NSE. Immunostaining for different peptide-hormones was observed in 12 of these 17 cases and in none of the 10 NSE-negative cases used for controls. Scattered cells were positive for gastrin in five cases, pancreatic polypeptide in five, leu-enkephalin in three, sub-P in two, ACTH in one, bombesin in one and beta-endorphin in one case. Four cases revealed immunoreactivity for more than one peptide-hormone. Typical neuroendocrine granules were seen in five cases (all positively stained for NSE). Small, electron dense granules of possible neuroendocrine nature were not found in any of the 33 NSE-negative tumours. Our results confirm that immunoreactivity for NSE is present in a high proportion of breast carcinomas, but that neuroendocrine differentiation cannot be proved to be present in all these cases. PMID- 3003313 TI - Interhemispheric fusion. PMID- 3003314 TI - Focal glomerulosclerosis as a late sequela of Wilms tumor. PMID- 3003315 TI - Molecular epidemiology of cytomegalovirus in a nursery: lack of evidence for nosocomial transmission. AB - During a 3-year period, cytomegalovirus (CMV) was recovered from the urine of 35 hospitalized newborn infants (15 with congenital and 20 with acquired infections). Two of the infants with acquired infections lacked maternal antibody to CMV (seronegative) and received transfusions from multiple seropositive blood donors. After seronegative blood products were used exclusively for seronegative low birth weight (less than 1300 gm) infants, none of 154 seronegative infants acquired CMV. CMV was recovered from one seronegative nurse who became infected during the study period. EcoRl digestion of the DNA of the nurse's isolate and of 34 of the 35 infant isolates revealed that no two were identical. LBW seropositive infants were randomized to receive either seronegative blood products or blood products from random donors; there was no significant difference in the number of acquired CMV infections. There were no deaths among 18 infants with acquired CMV infection. Hepatosplenomegaly and worsening bronchopulmonary dysplasia developed in one LBW infant. These results prove that nosocomial transmission of CMV did not occur frequently during the 3-year period. PMID- 3003316 TI - Isolation of enteroviruses from capillary blood specimens. PMID- 3003317 TI - Bicarbonate versus citrate in oral rehydration therapy in infants with watery diarrhea: a controlled clinical trial. AB - In a double-blind, randomized trial, we compared the efficacy of bicarbonate containing oral rehydration solution vs citrate-containing solution in the treatment of infantile diarrheal dehydration and acidosis. Ninety-seven infants 3 to 24 months of age were entered in the study; 49 received bicarbonate-containing solution and 48 citrate-containing solution. The two groups were similar in all respects at the beginning of the study. Oral rehydration was successful (i.e., no intravenously administered fluids were required) in 85% of study patients; the success rate was similar in both treatment groups. Serum total CO2 concentration increased in a similar fashion in both groups, reaching near normal values at 48 hours after admission. We conclude that sodium citrate can be substituted for sodium bicarbonate in the formulation of the orally administered rehydration solution recommended by the World Health Organization for treatment of diarrheal dehydration in infants. PMID- 3003318 TI - Duchenne muscular dystrophy, glycerol kinase deficiency, and adrenal insufficiency associated with Xp21 interstitial deletion. AB - We report an interstitial deletion in the short arm of the X chromosome in a 6 year-old boy with Duchenne muscular dystrophy, glycerol kinase deficiency, adrenal insufficiency, intermittent hypoglycemia, spasticity, psychomotor retardation, and growth delay. His mother also has this deletion in an X chromosome. From our findings, we propose that the human glycerol kinase locus and the human X-linked adrenal hypoplasia locus are in the Xp21 band. PMID- 3003319 TI - Neonatal exocrine pancreatic secretory immaturity: potential mechanisms and investigative approaches. PMID- 3003320 TI - Severe hemorrhage from intestinal hemangiomatosis in Klippel-Trenaunay syndrome: pitfalls in diagnosis and management. AB - A child with Klippel-Trenaunay syndrome (KTS) and severe anemia caused by bleeding from diffuse intestinal hemangiomatosis is presented. Hemangiomas of the bowel should be considered in any child with unexplained anemia and coexisting cutaneous hemangiomas. The diagnostic workup of patients with KTS and symptomatic hemangiomatosis is outlined with respect to indications for surgical management. PMID- 3003321 TI - Treatment and prognosis of symptomatic gallbladder disease in patients with cystic fibrosis. AB - Twenty-four (3.6%) of 670 patients with cystic fibrosis seen over a 25-year period developed symptomatic gallbladder disease. Only four patients were less than 16 years old. Four patients presented with unusual problems, including one with acute cholangitis and two with atonic gallbladder, one of whom required cholecystectomy. Another patient was found to have cholangiocarcinoma of the gallbladder when an exploratory laparotomy was performed to investigate biliary obstruction. Twenty patients had cholelithiasis, 15 of whom underwent cholecystectomy. Only one patient had substantial pulmonary difficulties postoperatively. Patients who presented with classic biliary colic had no further symptoms after cholecystectomy. One patient developed intrahepatic stones 6 years later and required a choledochoduodenostomy. As the pulmonary status of most cystic fibrosis patients will eventually deteriorate, we recommend that serious consideration be given to performing a cholecystectomy as soon as practical after the diagnosis of symptomatic cholelithiasis. Our experience indicates that surgery can be performed safely unless pulmonary status is already extremely compromised and the patient is in overt respiratory failure. PMID- 3003322 TI - Testosterone-producing hepatoblastoma in a 3-year-old boy with precocious puberty. AB - The syndrome of isosexual precocious puberty (PP) associated with a primary malignant hepatic tumor is rare and previously reported in only 17 cases with poor prognosis. Twelve cases are well-documented gonadotropin-producing tumors. We here describe a new case of virilizing hepatoblastoma in a 2-year-10-month-old boy with evidence of testosterone (T) production by the tumor itself, and survival with a 3 1/2-year follow-up after an extended right hepatic lobectomy. Preoperative laboratory findings showed high levels of serum alpha-fetoprotein (AFP) and T:350,000 ng/mL and 4.92 ng/mL, respectively, which normalized after surgery. There was no circulating gonadotropin nor stimulation of the hypothalamic-pituitary axis. Testicular biopsy showed neither interstitial-cell maturation nor Leydig-cell hyperplasia. Moreover, demonstration of T secretion by tumor cells and T synthesis in presence of C14 progesterone was performed in an in vitro culture system. These data seem to provide supportive evidence of a T producing hepatoblastoma. PMID- 3003324 TI - The occult insulinoma operative localization by quick insulin radioimmunoassay. AB - Hypoglycemia secondary to organic hyperinsulinism in children can be caused by diffuse or localized pancreatic lesions. Differentiation between these two types of lesions is of utmost importance since the surgical approach will be different. Some tumors escape detection by all preoperative investigations including ultrasound, scintiscan, arteriography, and computerized tomography. We are reporting on a 12-year-old boy with organic hyperinsulinism in whom we were unable to localize a tumor preoperatively. Peroperative determination of insulin levels from a number of sites on the pancreas enabled us to localize an insulin producing pancreatic adenoma. This technique can be done easily by catheterizing the splenic and portal vein through a branch of the splenic vein and by serial sampling at 2 cm intervals along the portal and splenic veins. Insulin levels at these sites were determined by quick double-antibody radioimmunoassay, which allows the determination of the insulin levels within 50 minutes after sampling. There was a perfect biochemic and anatomic correlation allowing us to perform a precise distal pancreatectomy. The technique can be used to localize pancreatic adenomas and to decide how much pancreas to remove in diffuse lesions avoiding "blind" pancreatectomies. PMID- 3003323 TI - Conversion to resectability by intra-arterial infusion chemotherapy after failure of systemic chemotherapy. AB - A 17 cm hepatoblastoma was unresectable at initial exploration. Multiagent chemotherapy (ADR, CTX, VCR, 5FU, BLEO) incurred minimal response at 5 months. Infuse-A-Ports were placed in each hepatic artery and FUDR infused intermittently but there was no response at 6 weeks. Adriamycin and vincristine were then begun via the Infuse-A-Port, with prompt regression of the tumor mass. The intra arterial course was continued 3 months. Repeat arteriography showed vascular distortion but exploration revealed no gross tumor. A persistent alpha-feto protein elevation prompted repeat arteriography 4 months afterward. A 3 cm calcific tumor was removed via an extended left lobectomy. Selective hepatic arterial infusion appeared to potentiate chemotherapy after systemic chemotherapy had failed to produce sufficient cytoreduction to allow safe surgical excision. The intra-arterial chemotherapy for unresectable hepatoblastoma warrants further investigation. PMID- 3003326 TI - Abdominal muscular deficiency syndrome in a female child. AB - Abdominal muscular deficiency is a rare syndrome, which affects girls in less than 5% of cases. In addition, urinary tract involvement in females is unusual and these factors may suggest a sexual variation in the pathogenesis of the syndrome. PMID- 3003325 TI - Evaluation of absorbable polyglycolic acid mesh as a wound support. AB - The ideal wound-support material would reinforce a wound early in the healing process when intrinsic wound strength is the weakest, yet disappear over time, preventing many of the untoward late effects seen with currently utilized nonabsorbable materials. This study was designed to evaluate the effectiveness of a newly designed absorbable material, polyglycolic acid mesh (Dexon), as a buttress for abdominal wounds closed under moderate tension. Young male rats (n = 211) were divided into three experimental groups. Animals in groups 1 (n = 96) and 2 (n = 95) had a 1.2 cm2 midline abdominal wall defect created and closely primarily. Animals in group 2 had a 2 X 5 cm piece of polyglycolic acid mesh sutured to the anterior abdominal wall overlying the closed abdominal defect. Animals in group 3 (n = 20) were unoperated controls. The animals in groups 1 and 2 were killed 1, 2, 3, 4, and 5 weeks after surgery. The entire anterior abdominal wall was removed and placed upon a bursting strength testing device. Bursting strength determinations of the supported and unsupported abdominal closures revealed that the strength of the wounds reinforced with polyglycolic acid mesh was significantly greater than unsupported wounds at 1, 2, and 3 weeks after surgery. Wounds supported with mesh had bursting strengths similar to unoperated abdomens by the first postoperative week. This study demonstrates that abdominal wall defects in rats closed primarily develop increased wound strength when the closure is supported by absorbable polyglycolic acid mesh. The use of an absorbable material may alleviate potential late complications associated with implantation of nonabsorbable materials. The clinical application of such a material remains to be determined. PMID- 3003327 TI - Positive 99mTc-pertechnetate scan in a child with intestinal arteriovenous malformation. AB - A teenager with massive rectal bleeding had a positive 99mTc-pertechnetate abdominal scan. At laparotomy, an arteriovenous malformation of the jejunum was found. There was no evidence of a Meckel's diverticulum. The persistent problem of the positive technetium scan warrants increased utilization of preoperative visceral angiography. PMID- 3003328 TI - Cytochemical identification of lysosomal system of the rat junctional epithelium. PMID- 3003329 TI - The use of Periograf in periodontal defects. Histologic findings. AB - Hydroxylapatite (Periograf) was placed into periodontal defects around five teeth scheduled for extraction in two young adult females with excellent plaque control. On the facial surface for one tooth the material was placed in a supracrestal position. Twelve months later the teeth were extracted in block section and were examined microscopically. Hydroxylapatite crystals were seen in the histologic sections with evidence of new bone formation in juxtaposition. The hydroxylapatite was tolerated relatively well by the surrounding tissue. A "cap' of bone was present coronal and facial to those crystals placed in the supracrestal position. In some areas bone was seen attached to the root via a periodontal ligament coronal to the Durapatite crystals. The question of accidental implantation of the material into the adjacent bone versus the actual regeneration of a true new attachment was discussed. PMID- 3003330 TI - Possible involvement of beta 2-adrenoceptors in hyperthermic effect of l ephedrine in rats. AB - The experiments were conducted in order to examine the mechanism of hyperthermia induced by l-ephedrine in rats. beta-Adrenoceptor agonists have been known to enhance normal body temperature. Therefore, the effect of various beta adrenoceptor agonists on body temperature in rats was examined to clarify the mechanism of action of l-ephedrine. The results showed that drugs with beta 2 adrenoceptor agonist activity and l-ephedrine caused hyperthermia in rats and this effect was selectively inhibited by pretreatment of animals with propranolol (a mixed beta-adrenoceptor antagonist) or butoxamine (a selective beta 2 adrenoceptor antagonist). These results suggest that hyperthermic action of l ephedrine may largely be due to its effect on beta-adrenoceptors. PMID- 3003331 TI - Pharmacokinetic studies on pharmacologic response of captopril in rats. AB - After administration of captopril, a decrease in mean arterial blood pressure was observed in low sodium rats which were used as animal models of hyper-renin active pathema. In normal rats, however, this marked decrease in blood pressure was not observed, even with high dose levels. In spite of the fact that captopril does not lower the blood pressure in normal rats, it was demonstrated that converting-enzyme activity was inhibited by captopril. No differences were observed between low sodium rats and normal rats in characteristics of angiotensin-converting enzyme. It was concluded that disposition of captopril might be different in the respective rats. PMID- 3003332 TI - The effects of marijuana on human physical aggression. AB - Thirty male undergraduates received intense provocation following their ingestion of one of three doses of delta-9-tetrahydrocannabinol (THC). The subjects in the low-dose condition tended to respond in a more aggressive manner than the subjects in the moderate-and high-dose conditions. The subjects in the high-dose condition behaved in a relatively nonaggressive manner throughout the experimental session. PMID- 3003333 TI - [Analgesic activity of 1,6-trans-N,N-dimethyl-(6-benzyl-4,4-dimethyl-2 cyclohexenyl)met hylamine hydrochloride (TAI-998)]. PMID- 3003334 TI - Biochemical and genetic studies of a histidine regulatory mutant of Streptomyces coelicolor A3 (2). AB - The specific activities of three enzymes in a histidine regulatory mutant RF59 of Streptomyces coelicolor A3(2) resistant to the histidine analogue 1,2,4 triazolealanine (TRA) were measured and compared to the activity of the wild type strain. The first enzyme of the histidine pathway, phosphorybosyl-ATP pyrophosphorylase (PR-ATP-pyrophosphorylase), of mutant RF59 and the wild type was sensitive to allosteric inhibition by L-histidine and hence feed-back inhibition was not affected by mutation, although the specific activity in the mutant was 2.9 fold higher than in the wild type. The other two enzymes coded by genes from the histidine operon were significantly derepressed. The enzyme D erythroimidazoleglycerol phosphate dehydrase (IGP-dehydrase) in mutant RF59 had a 4.9 fold higher specific activity than in the wild type strain. The specific activity of the last enzyme of the pathway, histidinol-dehydrogenase (Hol dehydrogenase), in the mutant was 4.7 fold derepressed compared to the wild type strain. The results of genetic crosses revealed the mapping of RF59 regulatory mutation between argA1 and cysD18 on S. coelicolor chromosome, suggesting that the mutant RF59 is a regulatory mutant unable to fully repress genes of the histidine pathway. PMID- 3003335 TI - Cholesterol monohydrate dissolution rate studies in aqueous micellar solutions of alpha-(nonylphenyl)-omega-hydroxydeca(oxyethylene), n-alkylamines, and fatty acids. AB - The influences of electrical and nonelectrical factors in the interfacially controlled dissolution of cholesterol monohydrate by mixed micelles of alpha (nonylphenyl)-omega-hydroxydeca(oxyethylene) (polyoxyethylene [10]-nonylphenol ether; POEPE; 1) in combination with long-chain n-alkylamines or fatty acids were investigated and quantified under pH conditions where the amines and fatty acids exist in their charged and uncharged forms. The experimental findings were generally consistent with a mechanism involving micelle collision with the cholesterol surface. A significant interfacial barrier was found with neutral micelles; however, the magnitude of the barrier was smaller with uncharged mixed micelles than with the simple micelles of 1. With charged micelles, the interfacial mass transfer resistance decreased with the addition of sodium chloride to the extent that the interfacial barrier was essentially abolished and the dissolution kinetics became convective/diffusion controlled. The results are consistent with the phenomena of diffusion of charged micelles toward a charged surface, following the classical collision kinetic theory of colloids. The ease in the transfer of cholesterol monohydrate molecules from the crystal surface and into the micelles during the collision step was examined by considering factors affecting the crystal surface as well as those associated with the hydrophobic hydrophilic structure of the micelle of 1 and the distribution of charged and uncharged amphipathic additives within the basic micelle structure of 1. PMID- 3003336 TI - Nonbactericidal approach to reduce colonization of plaque microflora on teeth in vitro and in vivo. AB - A non-antibacterial, surface-modifying, perfluorosulfonamidoalkyl ester of phosphorous acid (PSAEP, 1) was adsorbed to saliva-coated hydroxyapatite disks in vitro. Pretreatment of saliva-coated hydroxyapatite beads with different concentrations of 1 markedly reduced the adherence of radiolabeled Streptococcus mutans or Actinomyces viscosus when compared with the buffer-treated controls. Pretreatment of the cells with the compound also significantly impaired their subsequent attachment to saliva-coated hydroxyapatite. In a low surface-to-volume ratio adsorption model, i.e., saliva-coated hydroxapatite disks, pretreatment with 1 for 1 min markedly reduced the adsorption of A. viscosus to the disks. A 1% solution applied topically twice a day significantly (alpha = 0.05) reduced S. mutans-induced smooth and fissure caries in rats. The effect of 0.05% 1 in a rinse was also evaluated on experimental gingivitis in beagle dogs for 6 weeks. A topical application twice a day significantly (alpha = 0.05) reduced plaque induced gingivitis when compared to that achieved with the placebo. Microbial analyses of the plaque adjacent to gingiva indicated reduction in filamentous medium and large spirochetes as compared with that seen in the pretreatment phase. Collectively, the results show that it is possible to reduce dental caries and gingivitis via an interference with the specific adsorption of organisms to teeth. PMID- 3003337 TI - Interactions in the solid state. I: Interactions of sodium bicarbonate and tartaric acid under compressed conditions. AB - The interaction of NaHCO3 and tartaric acid in powder mixtures and compressed tablets has been studied. It has been found that in an open system the reaction is simply a decarboxylation of NaHCO3 and that the effect of compression on the reaction rate can be attributed to the brittle fracture (and subsequent surface area increase) that occurs on compaction. In a closed system the decomposition of the mixture is an interaction between the acid and the base, and it is mediated by the amount of moisture in the system. This latter is a product of reaction, and a suitable kinetic scheme is described for this. It is shown that "curing" the sodium bicarbonate by heating it to, e.g., 90 degrees C stabilizes the system by virtue of the formation of surface Na2CO3, which acts as a moisture scavenger. PMID- 3003338 TI - Maprotiline: an antidepressant with an unusual pharmacological profile. AB - Maprotiline, which differs from other typical tricyclic antidepressants for its tetracyclic structure, is a highly selective inhibitor of norepinephrine reuptake. Despite its in vitro and in vivo inhibitory activity on NE uptake, 21 repeated daily injections of maprotiline (20 mg/kg i.p.) neither attenuated the norepinephrine-stimulated cAMP accumulation nor reduced the number of beta adrenergic recognition sites in rat frontal cortex. Also, the number of brain serotonin2 recognition sites labeled by [3H]ketanserin remained virtually unchanged in rats receiving 21 daily injections of maprotiline. [3H]Desmethylimipramine and [3H]mianserin specific binding sites were also unmodified by repeated maprotiline injections. However, after 3 weeks of daily administrations, maprotiline elicited a significant decrease in the number of [3H]flunitrazepam binding sites and a decrease in the apparent affinity of [3H]beta-carboline ethylester binding to crude synaptic membranes prepared from hippocampal and hypothalamic homogenates. These changes appear to be unrelated to modifications in the concentration of endogenous gamma-aminobutyric acid present in the membrane preparation, since the addition of 100 microM bicuculline to the incubation mixture decreases [3H]flunitrazepam binding to the same extent in saline- and maprotiline-treated rats. It is suggested that repeated maprotiline injections may elicit an increase in the hypothalamic and hippocampal tissue levels of an endogenous substance(s) which binds to the benzodiazepine/beta carboline recognition sites. PMID- 3003339 TI - Antagonists of central and peripheral behavioral actions of cholecystokinin octapeptide. AB - Pharmacological studies on the behavioral functions of sulfated cholecystokinin (CCK) in the gut and in the brain require potent, specific antagonists to CCK. Compounds identified as competitive antagonists at the peripheral receptors for CCK were tested for their ability to block the behavioral effects of CCK administered centrally and peripherally. Behavioral effects of CCK (8.8 X 10-10 mmol) administered centrally into the nucleus accumbens, i.e., potentiation of dopamine-induced hyperlocomotion in rats, were effectively blocked by pretreatment with proglumide (6 X 10(-5) mmol of nucleus accumbens), by benzotript (3 X 10(-5) mmol of nucleus accumbens) and by rabbit antiserum raised against CCK (0.2 microliter/nucleus accumbens), but not by CCK26-33 (1.7 X 10(-7) mmol) or unsulfated CCK26-33 (1.9 X 10(-6) mmol). The behavioral effects of peripherally administered CCK, i.e. reduced food consumption and reduced exploratory behaviors in mice, were blocked effectively by pretreatment with proglumide (0.3-0.9 mmol/kg), and by benzotript (0.03 mmol/kg), but not by CCK30 33 (0.003 mmol/kg). None of the compounds administered peripherally significantly affected food consumption or exploratory behaviors when given alone. Furthermore, none of the compounds significantly affected locomotion when administered alone into the nucleus accumbens, or significantly affected dopamine-induced hyperlocomotion when given into the nucleus accumbens before dopamine. Benzotript, proglumide and a CCK antibody appear to act as specific antagonists of the behavioral effects of CCK at both the peripheral gastrointestinal site and at the central nucleus accumbens site. Neither unsulfated CCK26-33 or CCK30-33 were effective as antagonists of peripheral or central behavioral effects of CCK. However, whereas benzotript and proglumide may be useful as pharmacologically specific antagonists, the high doses required suggest that more potent CCK antagonists are required for investigating the behavioral functions of endogenous CCK. PMID- 3003340 TI - Adenosine potentiates sympathomimetic effects of nicotinic agonists in vivo. AB - We showed previously that circulating adenosine potentiates pressor responses to nicotine in rats, apparently by enhancing the effects of nicotine in sympathetic ganglia. In the present studies, we examined the effects of adenosine (or synthetic adenosine receptor agonists) on a variety of sympathomimetic responses to nicotine (or other nicotinic cholinergic agonists). Indices of sympathetic activity examined were: blood pressure; heart rate; eyelid tension; and vas deferens perfusion pressure. Elevation of arterial plasma adenosine from its basal level (approximately 1.5 microM) to 2 to 3 microM (by i.v. adenosine infusion) had no effect on the basal value of any of these indices, but increased by 2.5 to 4-fold their peak responses to nicotine (40 microgram/kg i.v.). Adenosine also strongly enhanced sympathomimetic responses to inhaled cigarette smoke. The adenosine receptor antagonist caffeine (10 mg/kg) suppressed the ability of adenosine to potentiate these responses to nicotine. Pressor responses to the nicotinic cholinergic receptor agonists dimethylphenylpiperazinium iodide, tetramethylammonium, lobeline and cytisin were also potentiated by adenosine. Low doses (0.25-5 mu/kg) of synthetic adenosine receptor agonists potentiated all four of the tested sympathomimetic responses to nicotine, exhibiting the rank order of potency: N-Cyclopropylcarboxamidoadenosine greater than 2 Chloroadenosine greater than N6-R-Phenylisopropyl adenosine greater than N6-S Phenylisopropyladenosine. The nonpurinergic vasodilator sodium nitroprusside failed to potentiate responses to nicotine, suggesting that the influence of adenosine on responses to nicotine is not secondary to its direct circulatory effects.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003341 TI - Adenosine affects sympathetic neurotransmission at multiple sites in vivo. AB - We examined the effects of adenosine and its analogs on sympathomimetic responses of pithed rats to electrical stimulation of preganglionic sympathetic nerves (ES) or to injections of nicotine, phenylephrine (PE) or isoproterenol (ISO). Four physiological indices of sympathetic neurotransmission were measured: blood pressure, heart rate and contractions of smooth muscle in vas deferens and eyelid. Elevation of arterial adenosine levels from 1.5 to 2 to 3 microM caused a 2- to 3-fold potentiation of nicotine-induced increases in blood pressure, heart rate and smooth muscle tension. Higher adenosine concentrations (3-4 microM) produced a smaller potentiation of the effects of nicotine. At 2 to 3 microM, adenosine had no effect on sympathomimetic responses to ES or PE. Higher concentrations (3-4 microM) attenuated pressor responses to ES and PE and the contractile responses of the vas deferens to ES; these levels also potentiated positive chronotropic responses to ISO. The adenosine analogs N cyclopropylcarboxamido adenosine (N-CPCA), 2-chloroadenosine (2-CLA) and R- and S phenylisopropyl adenosine (R-PIA and S-PIA) also reduced pressor responses to both ES and PE, with the potency order: N-CPCA greater than R-PIA greater than 2 CLA greater than S-PIA. These analogs exhibited this same potency series in attenuating contractile responses to ES in the vas deferens. However, all four analogs potentiated, at the lower doses tested, the contractile response of the vas deferens to PE; at higher concentrations, inhibition predominated. N-CPCA enhanced the chronotropic effects of ISO and ES.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003342 TI - Analysis of vascular responses in rat hindquarters arterial resistance vessels and veins in situ. AB - The venous compartment plays a critical role in circulatory control. The present series of experiments was conducted to assess simultaneously effects of vasoactive drugs on arterial resistance and venous capacitance vessels of the rat hindquarters perfused with physiological salt solutions (SS). Retrograde infusion of SS into rat hindquarters via the vena cava resulted in an increase in hindquarters venous pressure (Pv). The range of mean volumes required to increase Pv to 20 and 30 mm Hg was 1.7 +/- 0.1 to 2.9 +/- 0.4 and 3.8 +/- 0.3 to 6.9 +/- 0.3 ml, respectively, in various groups of rats during a control period. Perfusion of the hindquarters with an SS containing 80 mM K+ reduced the volume required to increase Pv to 20 and 30 mm Hg to 47 +/- 2 and 42 +/- 2% of control, respectively, indicating venoconstriction. K+ (80 mM) SS also increased aterial perfusion pressure (Pa; measured from a sidearm off of the inflow catheter) to 141 +/- 9 mm Hg, indicating arterial vasoconstriction. Arterial and venous responses to 80 mM K+ were attenuated markedly by perfusion with SS containing zero Ca and 2 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, indicating dependence on extracellular Ca. Phentolamine (10(-5) M) attenuated the arterial and venous response to 80 mM K+, indicating an alpha adrenergic contribution. Arterial responses to 80 mM K+ were attenuated markedly by the Ca entry blockers nifedipine (10(-6) and 10(-5) M) and verapamil (10(-6) and 10(-5) M). In contrast, venous responses were not affected by nifedipine and were reduced slightly only at the high concentration of verapamil.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003343 TI - Identification and characterization of functional angiotensin II receptors on cultured heart myocytes. AB - Cultured neonatal rat myocytes have been investigated as a potential system to study the molecular mechanism of angiotensin II (All) stimulation of heart contractile behavior. Intact cultured cells and the membranes from these cells bind [125I] All in a biphasic manner. The data were analyzed as two classes of binding sites on membrane preparations: Kd1 = 0.65 nM; maximum binding (Bmax1) = 245 fmol/mg of protein and Kd2 = 5.57 nM, Bmax2 = 720 fmol/mg of protein. The receptor on intact cells is specific as All peptide analogs were potent at inhibiting the binding of [125I]All. An All antagonist, [125I]Sar1,Leu8-All, recognized a single class of high affinity receptors on intact cells: Kd = 0.63 nM, Bmax = 318 fmol/mg of protein, or 45,300 sites per cell. Guanine nucleotides regulated the binding of All to membranes by shifting the receptor from a high affinity to a low affinity form without changing Kd2. Conversely, these nucleotides altered the high affinity binding of [125I]Sar1,Leu8-All by increasing Kd without changing Bmax. All was found to stimulate the contractile frequency of spontaneously beating myocytes with maximal effects (a 50% stimulation) observed at 5 nM hormone. The responses were reversible with half maximal effects observed at doses around 1 nM. The antagonist, Sar1,Ala8-All, could inhibit the chronotropic stimulatory responses. It is concluded that these cultured myocytes express an All receptor system with properties consistent with a true hormone receptor system. Furthermore, studies on the pharmacology of the chronotropic responses of these cells to All demonstrate that these receptors are functional. PMID- 3003344 TI - Mechanisms of decreased cerebrospinal fluid production by radiographic contrast media: role of adenylate cyclase activation. AB - The i.v. administration of radiographic contrast media decreases cerebrospinal fluid (CSF) production as measured by negative pressure collection from the lateral ventricles of anesthetized dogs. Evidence suggests that adrenergic mediated adenylate cyclase (AC) activity controls CSF production. AC activity was measured in membrane fractions of bovine and canine choroid plexus and rat heart and lung in the presence of various concentrations of the contrast agent sodium diatrizoate. A concentration-dependent inhibition of isoproterenol-stimulated AC activity by the contrast agent was observed in vitro. Specific binding of [3H]dihydroalprenolol to membrane fractions of bovine choroid plexus was also inhibited by sodium diatrizoate. An indication that the inhibition of choroidal AC activity by contrast media is significant in vivo was obtained by measuring CSF production in dogs. The inhibition of CSF production by sodium diatrizoate was reduced 50% when the contrast agent was administered during an i.v. infusion of isoproterenol (100 ng/kg/min). These results indicate that the mechanism of decreased CSF production by contrast media may involve inhibition of beta adrenergic-stimulated AC activity. PMID- 3003345 TI - Diethyldithiocarbamate provides partial protection against pulmonary and lymphoid oxygen toxicity. AB - Prolonged exposure of C57B16 mice to pure O2 at 1 ATA induced pulmonary edema associated with involution of lymphoid system and depressed immunity. The consequences of these toxic events were evaluated by 1) mortality rate, 2) determination of pulmonary water, 3) thymic and splenic cellularity, and 4) humoral (primary antibodies) and cellular (mitogenic) immune responses. Pretreatment of mice with 125 mg kg-1 of diethyldithiocarbamate (DDC) several days before exposure to O2 resulted in 1) an increase in animal survival (92-100% vs. 59% O2 controls), 2) a reduction in pulmonary edema, 3) partial stabilization of thymus and spleen lymphocyte populations, and 4) restoration of the humoral response (specific antibodies appeared earlier than in O2 control animals) and improvement of the mitogenic proliferative response of the spleen cells after hyperoxia. None of these effects were observed when DDC treatment coincided with the beginning of exposure. Our results indicated that DDC protects mice from both pulmonary and lymphoid hyperoxic injury, but only in a partial manner. It is suggested that the mechanism of this antioxidative property is indirect. PMID- 3003346 TI - Role of 5'-nucleotidase in adenosine-mediated renal vasoconstriction during hypoxia. AB - Regulation of renal function by endogenous adenosine production was examined in isolated perfused rat kidneys. Reducing perfusate pO2 from 400 +/- 15 to 130 +/- 5 mm Hg for 20 min created an energy deficit and increased adenosine in venous perfusate (0.06 +/- 0.02 to 0.79 +/- 0.15 microM) and snap-frozen renal cortex (5.6 +/- 1.4 to 16.7 +/- 2.7 nmol/g wet wt.). A competitive inhibitor of 5' nucleotidase, alpha,beta-methyleneadenosine diphosphate (120 microM), inhibited the production of adenosine during hypoxia (perfusate, 0.26 +/- 0.05 microM and renal cortex, 3.1 nmol/g) but did not prevent the decline in cortical tissue ATP and ADP. The inhibitor was concentrated 3-fold in renal cortex compared to perfusate and could therefore inhibit both ecto and endo 5' nucleotidases. Vascular resistance increased 11.1 +/- 0.5% during hypoxia. Inhibition of 5' nucleotidase reduced the vasoconstrictive response by 40% (P less than .01). An A1 antagonist, 1,3-diprophyl-8-(2-amino-4-chlorophenyl)xanthine (10(-5) M), reduced the effect of hypoxia on vascular resistance by 60% (P less than .005). Adenosine deaminase (7-14 U/ml) added during hypoxia reduced venous adenosine from 1.0 to 0.3 microM and reduced vascular resistance by 3 +/- 1%. Neither the inhibitors nor adenosine deaminase significantly altered the response of glomerular filtration rate or sodium reabsorption to hypoxia. These results indicate that either ecto or endo 5'-nucleotidase controls the renal production of adenosine during an energy deficit and that endogenous adenosine constricts the renal vasculature. PMID- 3003347 TI - Effect of indapamide on the electromechanical properties of rat myometrium and rat portal vein. AB - The effects of indapamide were studied on membrane potentials, ionic currents and isometric contractions in pregnant rat uterine smooth muscle. Indapamide (5 X 10( 6) to 10(-3) M) depressed K contractures and twitch contractions within 3 to 6 min. At a concentration of 3 X 10(-4) M, indapamide decreased the rate of rise, amplitude and rate of repolarization of the action potential and prolonged the potential duration. The inward current, carried either by Ca or Na ions, was depressed without modification of the respective reversal potentials. The decrease in K outward current was dependent on the reduction of the Ca inward current. In both myometrium and rat portal vein, indapamide depressed the transient contractions induced in Ca-free, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid-containing solutions by either acetylcholine (10(-4) M) or norepinephrine (10(-6) M). In myometrium, the maintained and repetitive contractions induced by acetylcholine as well as the contractions produced by prolonged membrane depolarizations were not affected by indapamide. The results indicate that indapamide acts primarily on the plasma membrane of spontaneously active smooth muscles by reducing both inward (Ca and Na) currents and outward (K) current; it may also exert an effect to depress contractions supported by a release of Ca from the sarcoplasmic reticulum. PMID- 3003348 TI - Alpha-2 adrenergic receptor localization in the rat heart and kidney using autoradiography and tritiated rauwolscine. AB - To study the distribution and characterization of alpha-2 adrenergic receptors in the rat heart and kidney, we used light microscopic autoradiography and a computer-based image analyzer to quantify the localization of [3H]rauwolscine (RAUW) binding. Scintillation spectrometry of frozen sections of rat kidney demonstrated rapid binding, saturability, stereospecificity and agonist and antagonist binding characteristic of an alpha-2 adrenergic receptor. For autoradiography, sections of rat kidney and heart were incubated in several concentrations of [3H]RAUW in the absence of (total binding) and in the presence of (nonspecific binding) 10(-5) M yohimbine. The sections were processed and grain density quantified using a computer-based image analyzer. The tubules in the renal cortex had significantly more specific [3H]RAUW labeling than either the renal glomeruli or the tubules in the renal medulla at all concentrations of [3H]RAUW used (P less than .0001). Nonspecific binding was significantly higher over the cortical tubules than either the glomeruli or the tubules in the renal medulla (P less than .0001). Scatchard analysis of specific grain densities determined that the tubules in the renal cortex had the highest density of any structure studied [maximum binding (Bmax) = 1182 grains/10(-2) mm2]. The glomeruli had a Bmax of 485 grains/10(-2) mm2, whereas the tubules in the renal medulla had a Bmax of 273 grains/10(-2) mm2. There were no significant differences among these three regions in the dissociation constant of the [3H]RAUW. When analyzing the heart, we found no specific [3H]RAUW labeling over either the cardiac myocytes or the myocardial arterioles.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003349 TI - [3H]tryptamine autoradiography in rat brain and choroid plexus reveals two distinct sites. AB - In vitro autoradiographic techniques were used to examine the distribution of [3H]tryptamine binding sites in rat brain. The gross distribution and pharmacological characteristics of binding to brain sections resembled those seen in homogenate studies. Binding sites were found throughout the brain, with a preponderance of sites in the forebrain and limbic structures; highest levels were seen in the choroid plexus and the interpeduncular nucleus. Other regions exhibiting high levels of [3H]tryptamine binding include the cortex (especially lamina I), caudate putamen, hippocampus, anterior olfactory nucleus, olfactory tubercle, nucleus accumbens, amygdala, superior colliculus (superficial gray layer), locus ceruleus, the nucleus of the solitary tract and the pineal body. Although there were similarities in this distribution to that for binding sites of [3H]5-hydroxytryptamine ([3H]serotonin), the overall patterns were distinct. The binding site for [3H] tryptamine in the choroid plexus (termed T-2) was pharmacologically distinct from that in the rest of the brain (termed T-1); several compounds, including kynuramine, were potent inhibitors of [3H]tryptamine binding at the brain site, but not at the choroid plexus site. [3H]Serotonin also labels a site in the rat choroid plexus; this site was different from both [3H]tryptamine sites. Knowledge of the distribution of tryptamine binding sites in the brain will aid in efforts to ascertain the function of these sites. PMID- 3003351 TI - The immunology of cytomegalovirus infection. PMID- 3003350 TI - Effects of electrical stimulation and choline availability on the release and contents of acetylcholine and choline in superfused slices from rat striatum. AB - The presence of 5 or 20 microM choline in the eserinized medium superfusing striatal slices enhanced the spontaneous release of acetylcholine (ACh) at both concentrations and, at 20 microM, the release of transmitter evoked by electrical field stimulation. Neither the electrical stimulation nor the addition of choline altered choline acetyltransferase activity. These results show that ACh release is dependent on the availability of extracellular choline. The rate of choline efflux was 7 times higher than the rate of ACh release, was not affected by stimulation, and was increased by 40% when hemicholinium-3 (HC-3), an inhibition of choline uptake, was present. The muscarinic antagonist atropine (1 microM) increased the evoked release of ACh into both the choline-free medium and that containing 20 microM choline. An adenosine receptor antagonist, 1,3-diethyl-8 phenyl xanthine (10 microM), failed to affect ACh release or the enhancement of release produced by atropine. In medium containing HC-3, stimulation of the slices elicited ACh release for the first 20 min of the 30 min stimulation period (15 Hz); thereafter, although stimulation was continued, the rate of release decreased to that associated with spontaneous release. Tissue ACh contents were not modified by the addition of choline or atropine to the medium, but were depressed by HC-3. Neither atropine nor HC-3 altered tissue choline content. The total amount of ACh + choline released during an experiment was 5-15 times higher than the decrease in tissue levels of these two compounds during the same period of time.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003352 TI - Characterization of (3H)-spiperone binding to alpha 1-adrenergic receptors in a smooth muscle cell line. AB - Although (3H)-spiperone has been demonstrated to interact at both dopamine (D2) and serotonin (S2) receptors, it remains a popular choice for characterization of the D2-receptor using both in vitro and in vivo and in vivo assay techniques. Using a clonal smooth muscle cell culture line (DDT1 MF-2), which has previously viously been characterized as possessing alpha 1- and beta 2-adrenergic receptors, we have found that (3H)-spiperone also has a significant affinity for alpha 1-adrenergic receptors. Our results are consistent with other literature reports which have suggested that spiperone may interact at alpha 1-receptors and we have characterized this interaction. We have also found an additional, high affinity binding site for spiperone on these cells which may represent a D2 - receptor. Characterization of this high-affinity site has been difficult since it is present in very low density. We conclude that (3H)-spiperone binds with high affinity to at least three known neurotransmitter receptor sites: D2-dopamine, S2 serotoninn and alpha 1-adrenergic. The binding to the alpha 1-adrenergic receptor exhibits stereospecificity and a considerable degree of similarity in pharmacological profile to the D2-dopamine receptor. PMID- 3003353 TI - Effect of Triton X-100 on insulin and epidermal growth factor receptor binding and autophosphorylation in Golgi fractions and partially purified receptors from rat liver. AB - Triton X-100 strongly affects the receptor binding and autophosphorylation of insulin and epidermal growth factor (EGF) in rat liver Golgi fractions and partially purified microsomal receptors. At concentration 0.05% Triton X-100 decreased the insulin receptor binding by 15% and the EGF receptor binding by 70% as compared to controls. In contrast, 0.05% Triton X-100 increased insulin stimulated receptor autophosphorylation by more than 370% as compared to 87% in the control. Similarly, the same concentration of Triton X-100 increased the EGF stimulated receptor phosphorylation by 65% as compared to 14% in the control. EGF receptor binding was more sensitive to the treatment of Triton. At Triton concentrations 0.2% or more, the EGF receptor binding was totally abolished while the insulin receptor binding was decreased by 50%. On the other hand, the activity of ligand-stimulated receptor phosphorylation of both insulin and EGF receptors was only slightly decreased in the presence of 0.2% Triton. PMID- 3003354 TI - The effect of platelet protein and DNA on the measurement of human lymphocyte beta adrenergic receptor number. AB - Considerable variation exists in human lymphocyte beta adrenergic receptor measurements when the data are reported as fmole/mg protein. To investigate potential sources of measurement errors, human lymphocytes were prepared as follows: With the standard method (M1), lymphocytes were removed from heparinized whole blood by centrifugation on a ficoll gradient. The modified method (M2) was identical, except for an initial centrifugation to remove platelet-rich plasma. Beta adrenergic receptors were measured by the binding of [125I] pindolol with the results analyzed by the method of Scatchard. The M1 receptor number was 11.9 +/- 1.8 fmole/mg protein compared to 26.7 +/- 4.1 fmole/mg for M2 (p1/40.01). Since platelet-rich M1 had 71.9 micrograms protein/100 microL of sample and platelet-poor M2 and 23.8 micrograms/100 microL the difference in receptor number is a calculation artifact due to platelet protein. Similar results were obtained when data were expressed as fmole/mg DNA, because platelets also contain nucleic acids. However, when expressed as the number of binding sites/cell, neither platelet protein nor platelet-DNA affected receptor quantitation. We conclude that results are most accurately reported as sites/cell. PMID- 3003355 TI - Study using in-vivo binding of 125I-labelled hCG, light and electron microscopy of the repopulation of rat Leydig cells after destruction due to administration of ethylene-1,2-dimethanesulphonate. AB - Gonadotrophin binding to rat Leydig cells after a single administration of ethylene dimethanesulphonate (EDS) (75 mg/kg i.p.) was followed by using intratesticular microdoses of 125I-labelled hCG, whilst corresponding morphological changes in the testicular interstitium were studied with light and electron microscopy. No discernible effect on 125I-labelled hCG binding compared with controls was observed until 24 h after treatment. Between 24 and 32 h a sharp decline in binding occurred which was correlated with extensive Leydig cell destruction. By 48 h the 125I-labelled hCG binding was negligible and no morphologically recognizable Leydig cells were found at this time. The specific binding remained low until 21 days after treatment and then a marked increase occurred to give nearly normal levels by 49 days. This was consistent with a generalized repopulation of the interstitium with Leydig cells, seemingly the result of differentiation of fibroblast-like precursor cells. PMID- 3003356 TI - Percoll-gradient separation of Leydig cells from postnatal rat testes. AB - The characteristics of the fetal and adult populations of Leydig cells from postnatal rat testes were compared by Percoll gradient centrifugation. A single peak of hCG binding, due to the presence of fetal Leydig cells, was obtained after purification of intertubular cells from 8-day-old animals. Two peaks of specific hCG binding were obtained after purification of intertubular cells from 15-day-old rats: it was confirmed by autoradiographic techniques that the hCG was bound by adult-like Leydig cells in one peak and fetal Leydig cells in the other. Similarly, intertubular cell preparations from 21- and 25-day-old rats resolved into two peaks of hCG binding; adult-like Leydig cells were observed in the first peak, but fetal Leydig cells were rarely observed in the second of these peaks. These results demonstrate the separation of two Leydig cell populations from intertubular cells obtained from animals aged up to 15 days. Thereafter the pattern of the hCG binding profile is similar but is not due to the presence of the same cell types. Therefore these results emphasize the necessity for morphological identification of cell types to permit the correct interpretation of the corresponding biochemical data. PMID- 3003357 TI - Adenosine cyclic 3',5'-monophosphate and steroid production by small ovarian follicles from Booroola ewes with and without a fecundity gene. AB - The tissue contents of adenosine cyclic 3',5'-monophosphate (cAMP) in freshly dissected follicles (0.13-1.00 mm diam.) were significantly higher in Booroola ewes containing a major fecundity gene (FF and F+ ewes) compared to those values in Booroolas with no copy of the gene (++ animals; P less than 0.025). After a 1 h incubation with LH + FSH, the respective proportions of follicles with a diameter of 0.13-0.52 mm (n = 288) and 0.53-1.00 mm (n = 271) that had synthesized greater than or equal to 0.6 pmol cAMP and greater than or equal to 1.0 pmol cAMP were significantly influenced by genotype (Booroola ewes homozygous for the F-gene, FF greater than heterozygous, F+ greater than ++; P less than 0.01 for both follicle size ranges). The contents of progesterone, androstenedione, testosterone and oestradiol-17 beta in minced ethanolic extracts of freshly dissected follicles (n = 188) were undetectable regardless of Booroola genotype. However, when follicles of 0.53-1.00 mm but not 0.13-0.52 mm diameter were cultured for 48 h with LH + FSH under 70 kPa of a 50% O2, 45% N2 and 5% CO2 gas mixture, the proportions that synthesized high levels of progesterone (greater than or equal to 4.0 ng), androstenedione (greater than or equal to 3 ng), and oestradiol (greater than or equal to 0.8 ng) were significantly influenced by genotype (FF greater than F+ greater than or equal to ++; P less than 0.05 for each steroid). No significant genotypic differences were noted for testosterone synthesis. Collectively, these results show that the Booroola F-gene has an influence on the maturation of ovarian follicles from an early stage of growth. PMID- 3003358 TI - Relationship between pituitary GnRH-binding sites and pituitary release of gonadotrophins in post-partum beef cows. AB - Thirty primiparous suckling beef cows were slaughtered on Day 7, 14, 28, 42 or 56 after parturition. Some had resumed oestrous cyclicity by the time they were slaughtered on Days 42 and 56. Amongst acyclic cows between Days 7 and 42, pituitary LH concentrations and basal and GnRH-induced release of LH from pituitary explants doubled. Pituitary FSH concentration and basal release in FSH increased only by 15-20%, while GnRH-induced release of FSH in vitro was unchanged. During postpartum anoestrus, overall mean concentrations of serum FSH did not change, whereas overall mean concentrations and pulse amplitudes of serum LH increased. Numbers and affinity constants of GnRH-binding sites in pituitary glands remained constant during the post-partum period studied. We conclude that, under these experimental conditions, numbers and affinity constants of GnRH binding sites in the pituitary gland of post-partum beef cows do not limit the ability of the anterior pituitary gland to release gonadotrophins. PMID- 3003359 TI - Basic physiology of follicular maturation in the pig. AB - The pig is an excellent animal in which to study the control of folliculogenesis in a polytocous species, and particularly to examine the inter-relationships between follicles from the same animal. Follicle recruitment occurs from the proliferating pool, and various studies suggest that this recruitment occurs between Days 14 and 16 of the oestrous cycle. The growth of follicles selected for ovulation is associated with rapid atresia of smaller follicles and a block to their replacement in the proliferating pool. However, there is a considerable range in the morphological and biochemical development of the dominant follicles in the early follicular phase, suggesting that follicles are recruited at markedly different stages of development, or that recruitment continues into the follicular phase. A significant and predictable relationship has been established between follicular diameter and follicular fluid volume, and a comparison of these two characteristics demonstrates a gradual increase in follicular tissue volume as a proportion of total volume. Growth of follicles from 2 to 4 mm is associated with a proportional increase in granulosa cell numbers, but above 4 mm the relationship is very variable even in selected follicles that are steroidogenically active. Therefore, the number of granulosa cells cannot be used as an indicator of atresia in pig follicles. LH receptors are present in thecal tissue throughout development, reaching maximal levels on Day 20 of the oestrous cycle and declining on Day 21. Granulosa cells possess receptors for LH only in the later stages of maturation, and again these are maximal on Day 20. The pattern of steroidogenesis in pig follicles is consistent with the two-cell theory of steroidogenesis in that androgen produced by the theca is aromatized to oestrogen by the granulosa cells, However, in contrast to that of many other species, the theca of the pig also produces oestradiol in quantities comparable to those secreted by the granulosa. As with morphological development, the selected population of preovulatory follicles shows a considerable range of biochemical development and follicles of identical size may show great dissimilarity in follicular fluid steroid concentrations and LH binding. Androgen availability rather than aromatase activity appears to be the limiting factor for steroidogenesis. There are also several nonsteroidal factors which have been isolated from porcine tissue and play some role in follicular maturation. Although exogenous gonadotrophins are effective in promoting follicular development, other factors of extra- or intra-ovarian origin may limit follicular responsiveness to gonadotrophins.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3003360 TI - Stress, loneliness, and changes in herpesvirus latency. AB - This study used a prospective design to examine the influence of examination stress and loneliness on herpesvirus latency as measured by changes in antibody levels to three herpesviruses, Epstein-Barr virus (EBV), Herpes simplex type I (HSV-1), and cytomegalovirus (CMV). Three blood samples were obtained from 49 first-year medical students, with the first sample drawn 1 month before final examinations, the second on the first day of final examinations, and the third during the first week after their return from summer vacation. A median split on the UCLA Loneliness Scale divided subjects into high- and low-scoring loneliness groups. There were significant changes in the antibody titers to all three herpesviruses across the sample points, with the lowest levels found in the third (low stress) sample. High-loneliness subjects had significantly higher EBV antibody titers than low-loneliness subjects. These data suggest that stress related immunosuppression can significantly modulate herpesvirus latency. PMID- 3003361 TI - Molecular organization of ribosomal RNA genes clustered at variable chromosomal sites in Triturus vulgaris meridionalis (Amphibia, Urodela). AB - The ribosomal RNA genes of Triturus vulgaris meridionalis (Amphibia, Urodela) show the peculiar feature of being clustered not only at the nucleolar organizer, present in the species at a definite chromosome location, but also at "additional ribosomal sites" which are highly variable in number and chromosomal distribution among individuals. The additional ribosomal sites are most often found at specific chromosome regions, such as telomeres, C-bands and centromeres, in virtually all the chromosomes. With increasing numbers of additional clusters, the genomic dosages of ribosomal RNA genes are found to increase over a tenfold range, though not linearly. At a molecular level, the ribosomal DNA repeats differ in size because of discrete variations in the length of the non transcribed spacers. However, the resulting length heterogeneity of the gene family is rather limited within a single genome as well as within the species. Many of the ribosomal loci appear to be internally homogeneous with respect to the repeat length. Moreover, separate clusters from distant genomic regions can share the same size class of ribosomal repeats even in the same specimen. The nucleolar organizer is mostly endowed with "shorter" ribosomal repeating units, ranging in size from 13.7 X 10(3) to 15.2 X 10(3) base-pairs. The additional ribosomal sites are characterized by the occurrence of "longer" repeats, ranging in size from 16.2 X 10(3) to 19.7 X 10(3) base-pairs. The "shorter" class of ribosomal repeats is always detected in the amplified ribosomal DNA, suggesting that the nucleolar organizer locus is involved in the amplification process in most oocytes. "Longer" ribosomal repeats are also detectable in the amplified ribosomal DNA of a few females. PMID- 3003362 TI - The mitochondrial URF1 gene in Neurospora crassa has an intron that contains a novel type of URF. AB - In Neurospora crassa, a 2670 base-pair segment of the mitochondrial DNA was sequenced including a gene homologous to the mammalian URF1 that was recently shown to encode a subunit of the respiratory chain NADH dehydrogenase complex. URF1 of N. crassa is interrupted by an intron of 1118 base-pairs that divides the protein-coding sequence into two exons of 636 and 480 base-pairs length, respectively. The deduced URF1 polypeptide of 371 residues was aligned with that of other eukaryotes, revealing a degree of conservation similar to that of ubiquitous mitochondrial genes. The two highly conserved stretches coincide with the most polar regions of the otherwise hydrophobic URF1 polypeptides and may constitute functional domains of the complex I subunit. In the exon sequences of URF1, 17 codons occur that are infrequently utilized in other mitochondrial genes of N. crassa, indicating a low translational efficiency or a foreign origin of URF1. The URF1 intron is inserted in the most conserved region. It belongs to group I and contains an open reading frame of 305 codons not continuous with the upstream exon. Sequences convincingly homologous to conserved group I decapeptide motifs were not found in the URF1 intronic unassigned reading frame (URF). However, significant homology was detected to intronic URFs of the respective gene from Podospora anserina, suggesting that these reading frames constitute a novel type of group I intronic URFs. Three species of URF1 transcripts were identified. They arise most probably by subsequent removal of the intron and leader sequences from an URF1 precursor transcript. PMID- 3003363 TI - Mitochondrial DNA-like sequences in the human nuclear genome. Characterization and implications in the evolution of mitochondrial DNA. AB - Thirty-three phage clones carrying DNAs homologous to human mitochondrial DNA (mtDNA) were isolated from two independently constructed human gene libraries, and the region and extent of homology of mtDNA-like sequences carried by these clones were examined in hybridization experiments. Each phage clone contained DNA sequences homologous to various parts of the mtDNA and the extent of homology differed from clone to clone. From the efficiency of the library screening, it was estimated that human nuclear DNA contains at least several hundred copies of mtDNA-like fragments. Four clones carrying nuclear DNA sequences homologous to the mitochondrial Unidentified Reading Frame (URF) 4 and URF5 regions were chosen for further studies, and their structures were analyzed by DNA sequencing. Comparison of these mtDNA-like sequences with that of mtDNAs of several mammalian species revealed conservation of a part of the structures present in direct ancestral mtDNAs. The mtDNA fragments seem to have been continuously integrated into mammalian nuclear DNA during evolution. PMID- 3003364 TI - Fructose bisphosphatase of Saccharomyces cerevisiae. Cloning, disruption and regulation of the FBP1 structural gene. AB - Fructose bisphosphatase catalyzes a key reaction of gluconeogenesis. We have cloned the fructose bisphosphatase (FBP1) structural gene from Saccharomyces cerevisiae by screening a genomic library for complementation of an Escherichia coli fbp deletion mutation. The cloned DNA expresses in E. coli a fructose bisphosphatase activity which is precipitable with antibodies specific for the yeast enzyme and is sensitive to inhibition by fructose 2,6-bisphosphate. Evidence is presented demonstrating that the entire gene, including all cis acting regulatory sequences, has been cloned. A substitution mutation that disrupts FBP1 was incorporated into the yeast genome by transplacement to construct a fructose bisphosphatase null mutation. The fbp1 mutant strain is a hexose auxotroph, otherwise growing normally. Southern blot hybridization analysis confirmed the structure of the transplacement and demonstrated that FBP1 is present in single copy in the haploid genome. Northern blot hybridization analysis revealed an mRNA of about 1350 nucleotides, whose presence was repressible by glucose in the medium. Fructose bisphosphatase activity was not greatly overproduced when the FBP1 gene was present on a multicopy vector in yeast. PMID- 3003365 TI - Organization of the actin multigene family of Dictyostelium discoideum and analysis of variability in the protein coding regions. AB - There are 17 to 20 actin genes in the genome of the cellular slime mold Dictyostelium discoideum. Genomic clones of 15 of the genes have been isolated. Extensive nucleotide sequence within the protein-coding regions has been determined, including the complete nucleotide sequence of four genes representing the three distinct evolutionary groups of Dictyostelium actin genes. All are similar to mammalian cytoplasmic actins at diagnostic amino acid positions, and there is generally less variability among Dictyostelium actin genes than among Drosophila actin genes. Two genes, Actins 3-sub 1 and 3-sub 2 differ substantially from all the rest in terms of replacement amino acid substitutions and probably encode actin-related proteins rather than bona fide actins. Each contains several amino acid substitutions that should alter the secondary structure of the resulting proteins, and Actin 3-sub 2 encodes four additional amino acids at the C terminus. This gene is as divergent from other Dictyostelium actin genes as is the yeast or a soybean actin gene. At present, evidence suggests that all 15 genes examined are expressed, except the previously identified Actin 2-sub 2. We suggest that Dictyostelium might maintain a high number of functional actin genes for the purpose of regulating the level of actin synthesis within narrow limits, rather than because most genes perform different functions. PMID- 3003366 TI - Comparison of histidine proton magnetic resonances of human carbonmonoxyhaemoglobin in different buffers. AB - We have recorded the C-2 proton resonances of the histidines of carbonmonoxyhaemoglobin A and of four abnormal human HbCOs in different buffers and at different concentrations of haemoglobin. Resonance H assigned by Perutz et al. (1985) to His HC3(146) beta, is present at both pH 7.30 and pH 6.90, but somewhat broadened when recorded in 5 to 10% HbCO A in 0.1 M-bis-Tris. The broadening disappears on tenfold dilution of the Hb with bis-Tris and the resonance then stands out sharply. Resonance H is absent at both Hb concentrations in HbCO Cowtown (His HC3(146) beta----Leu). HbCO Fort de France (His CD3(45) alpha----Arg) in 0.1 M-bis-Tris of pH 6.90 has a spectrum similar to that of HbCO A. In the same buffer a resonance marked L by Russu et al. (1982) is absent from the spectrum of Hb Abbruzzo (His H21(143) beta----Arg), whereas resonance H is present. Hb Barcelona contains an additional histidine in position FG1(94) beta; in 0.1 M-bis-Tris buffer of pH 6.90 its resonance is not resolved and resonance H is either shifted or broadened. The resonances of both histidines are resolved in phosphate buffer. At pH 6.90, spectra in 0.1 M-bis-Tris buffer are similar to those previously recorded in 0.2 M-HEPES. Addition of 0.1 M-KCl produces marked changes. Replacement of bis-Tris by 0.2 M-KCl + 0.2 M-phosphate gives rise to a different and much better resolved spectrum. PMID- 3003367 TI - The effect of ischemia and reperfusion on sarcolemmal function in perfused canine hearts. AB - The report deals with the effect of ischemia and reperfusion on purified sarcolemma obtained from canine myocardium of perfused supported heart preparations. Perfusion was carried out with a perfluorochemical (FC-43). Ischemia was produced by intermittent total clamping of inflow and outflow followed by release until the decrease in dP/dtmax had become stabile. Purity of sarcolemmal vesicles was ascertained with marker enzymes: succinate cytochrome c reductase (for mitochondria), K+-stimulated p-nitrophenylphosphate (K+-pNPPase), (Na+/K+)ATPase and adenylate cyclase (for SL). In addition Na+/Ca2+-exchange characteristics for SL were determined. Sidedness of vesicles was ascertained by means of adenylate cyclase activity using sarcolemmal preparations treated and untreated with alamethicin. Emphasis was placed on ATP-dependent Ca2+ uptake, phosphorylation of sarcolemmal vesicles and yield of SL proteins. Ischemia and reperfusion resulted in a significant reduction in adenylate cyclase activity. This decline was significant following ischemia and reperfusion. The yield of protein recovered from SL vesicles from ischemic-reperfused heart preparations was also significantly decreased. Both initial rate of ATP-dependent Ca2+ uptake and maximal Ca2+ uptake fell significantly following ischemia and reperfusion. The initial rate of phosphorylation also dropped significantly. These disturbances in SL Ca2+ transport following ischemia and reperfusion are probably a part of the general deficit in Ca2+ translocation. PMID- 3003368 TI - Phylogenetic analysis of polymorphic DNA sequences at the Adh locus in Drosophila melanogaster and its sibling species. AB - Recent sequencing of over 2300 nucleotides containing the alcohol dehydrogenase (Adh) locus in each of 11 Drosophila melanogaster lines makes it possible to estimate the approximate age of the electrophoretic "fast-slow" polymorphism. Our estimates, based on various possible patterns of evolution, range from 610,000 to 3,500,000 years, with 1,000,000 years as a reasonable point estimate. Furthermore, comparison of these sequences with those of the homologous region of D. simulans and D. mauritiana allows us to infer the pattern of evolutionary change of the D. melanogaster sequences. The integrity of the Adh-f electrophoretic alleles as a single lineage is supported by both unweighted pair group method (UPGMA) and parsimony analyses. However, considerable divergence among the Adh-s lines seems to have preceded the origin of the Adh-f allele. Comparisons of the sequences of D. melanogaster genes with those of D. simulans and D. mauritiana genes suggest that the split between the latter two species occurred more recently than the divergence of some of the present-day Adh-s genes in D. melanogaster. The phylogenetic analyses of the D. melanogaster sequences show that the fast-slow distinction is not perfect, and suggest that intragenic recombination or gene conversion occurred in the evolution of this locus. We extended conventional phylogenetic analyses by using a statistical technique for detecting and characterizing recombination events. We show that the pattern of differentiation of DNA sequences in D. melanogaster is roughly compatible with the neutral theory of molecular evolution. PMID- 3003370 TI - Evolution of Alu family repeats since the divergence of human and chimpanzee. AB - The DNA sequences of three members of the Alu family of repeated sequences located 5' to the chimpanzee alpha 2 gene have been determined. The base sequences of the three corresponding human Alu family repeats have been previously determined, permitting the comparison of identical Alu family members in human and chimpanzee. Here we compare the sequences of seven pairs of chimpanzee and human Alu repeats. In each case, with the exception of minor sequence differences, the identical Alu repeat is located at identical sites in the human and chimpanzee genomes. The Alu repeats diverge at the rate expected for nonselected sequences. Sequence conversion has not replaced any of these 14 Alu family members since the divergence between chimpanzee and human. PMID- 3003369 TI - Comparison of human and chimpanzee zeta 1 globin genes. AB - The DNA base sequences of the entire chimpanzee zeta 1 globin gene and an additional 1 kb of DNA flanking both the human and chimpanzee genes have been determined. Whereas the human zeta 1 gene contains a termination codon in the sixth position, the chimpanzee gene appears to be functional. This finding confirms Proudfoot et al.'s suggestion that the human zeta 1 gene was recently inactivated. Like the corresponding human zeta 1 and zeta 2 genes, the first and second introns of the chimpanzee zeta 1 gene are occupied largely by tandem repeats of short oligonucleotides. These tandem repeats have undergone several rearrangements since the divergence of the human and chimpanzee zeta 1 genes. PMID- 3003371 TI - The pattern of restriction enzyme-induced banding in the chromosomes of chimpanzee, gorilla, and orangutan and its evolutionary significance. AB - The pattern of banding induced by five restriction enzymes in the chromosome complement of chimpanzee, gorilla, and orangutan is described and compared with that of humans. The G banding pattern induced by Hae III was the only feature common to the four species. Although hominid species show almost complete chromosomal homology, the restriction enzyme C banding pattern differed among the species studied. Hinf I did not induce banding in chimpanzee chromosomes, and Rsa I did not elicit banding in chimpanzee and orangutan chromosomes. Equivalent amounts of similar satellite DNA fractions located in homologous chromosomes from different species or in nonhomologous chromosomes from the same species showed different banding patterns with identical restriction enzymes. The great variability in frequency of restriction sites observed between homologous chromosome regions may have resulted from the divergence of primordial sequences changing the frequency of restriction sites for each species and for each chromosomal pair. A total of 30 patterns of banding were found informative for analysis of the hominid genealogical tree. Using the principle of maximum parsimony, our data support a branching order in which the chimpanzee is more closely related to the gorilla than to the human. PMID- 3003373 TI - In vitro biologic toxicity of native and surface-modified silica and kaolin. AB - An in vitro study of the biologic responses of surface-modified and native silica and kaolin was made to provide comparative information on the suppression of cytotoxicity by pulmonary surfactant. The release of alveolar macrophage cytoplasmic enzyme, lactate dehydrogenase (LDH), and lysosomal enzymes beta-N acetylglucosaminidase (beta-NAG) and beta-glucuronidase (beta-GLUC) and sheep blood-cell hemolysis were monitored as indicators of cell membrane damage and cytotoxicity. Surface modification of silica and kaolin with dipalmitoyl lecithin (DPL) resulted in complete abrogation of cytotoxicity of both minerals. These findings indicate that surface modification of minerals with different adsorption properties by pulmonary surfactant generally lessens their prompt adverse effects. PMID- 3003372 TI - The mouse muscle creatine kinase cDNA and deduced amino acid sequences: comparison to evolutionarily related enzymes. AB - The nucleotide sequence of cloned DNA corresponding to full-length mouse muscle creatine kinase mRNA has been determined. This 1415 base pair DNA sequence and the deduced 381 amino acid sequence of the protein have been compared to creatine kinase sequences from other vertebrate species and to invertebrate guanidino kinase sequences. These comparisons show that the vertebrate muscle creatine kinases constitute a remarkably conserved protein family with a unit evolutionary period of 30. The creatine kinases also retain marked sequence similarity with the more distantly related invertebrate guanidino kinases. A portion of the sequence, presumably part of the ATP binding site, shows similarity to other nucleotide binding proteins with diverse functions. Comparisons of the untranslated regions of the creatine kinase cDNA sequences show that the 5' untranslated regions are more highly conserved than are the 3' untranslated regions; this may point to some regulatory function in the 5' region. PMID- 3003374 TI - Bioavailability of soil-borne polybrominated biphenyls ingested by farm animals. AB - Concentrations of polybrominated biphenyl (PBB) were measured in the fat of livestock on several farms on which soil-borne PBB in confinement areas was the only source of residue. Ratios of concentrations in fat to concentrations in soil were 0.37 for dairy heifers, 0.27 for primaparous dairy cows, 0.10 for multiparous dairy cows, 0.27 for beef cows, 0.39 for beef calves, 0.37 for ewes, and 1.86 for swine. Multiparous dairy cows had lower ratios because of the excretion of PBB in milk during long-term lactation, and swine had higher ratios because they ingest greater amounts of soil than other species. Diets containing 5% PBB-contaminated soil, or 5% contaminated soil amended with activated carbon, were fed to lambs for 56 d. Accumulation of soil-borne PBB in fat, when adjusted for intake, did not differ significantly from accumulation of PBB from a diet in which PBB was added to cornmeal. Amending soil with activated carbon had no effect on residue accumulation. About 70% of PBB in a control diet with PBB added to cornmeal was absorbed, as measured by using titanium as an unabsorbed marker. Absorption of soil-borne PBB was 65% from unamended soil, 57% from soil amended with 0.3% activated carbon, and 56% from soil amended with 0.6% activated carbon. The differences were not great enough to be of practical importance. These results with PBB may be useful in assessing and managing risks of other soil borne contaminants that have chemical characteristics similar to those of PBB. PMID- 3003375 TI - Type I topoisomerase activity after infection of enucleated, synchronized mouse L cells by vaccinia virus. AB - The time course of appearance of type I topoisomerase activity after the infection of mouse L cytoplasts by vaccinia virus was determined. When the enucleation procedure was carried out with unsynchronized cell cultures, a high level of host-cell-specific type I topoisomerase activity was found associated with the resulting cytoplasts. If cells were first synchronized by the two-cycle thymidine block method and then enucleated after release, the level of host type I topoisomerase activity was also high for S-phase-enucleated cells but was very low for cytoplasts prepared from cells previously synchronized and enucleated during either the G1 or the G2 phase. After the infection of G1-phase-enucleated cytoplasts with vaccinia virus, newly synthesized type I topoisomerase activity first appeared at about 3 h postinfection. Virosomes were isolated from the infected, synchronized cytoplasts and assayed for the presence of type I topoisomerase activity. The activity remained at the top of a sucrose gradient, well resolved from the virosome fraction, at the low salt levels (0.01 M KCl, 0.01 M Tris hydrochloride, pH 8.0) normally used in the course of virosome purification. If the sedimentation was at the higher salt concentration (0.15 M KCl) at which the enzyme shows optimal activity, type I topoisomerase cosedimented with the virosome fraction onto the sucrose gradient cushion. These results show that the type I topoisomerase activity dependent upon vaccinia virus infection may be detected with high sensitivity in G1-phase-enucleated cytoplasts. The association with virosomes is consistent with an involvement of topoisomerase activity either in DNA replication or in late transcription. PMID- 3003377 TI - Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans. AB - Herpes simplex virus-specific DNA sequences have been detected by Southern hybridization analysis in both central and peripheral nervous system tissues of latently infected mice. We have detected virus-specific sequences corresponding to the junction fragment but not the genomic termini, an observation first made by Rock and Fraser (Nature [London] 302:523-525, 1983). This "endless" herpes simplex virus DNA is both qualitatively and quantitatively stable in mouse neural tissue analyzed over a 4-month period. In addition, examination of DNA extracted from human trigeminal ganglia has shown herpes simplex virus DNA to be present in an "endless" form similar to that found in the mouse model system. Further restriction enzyme analysis of latently infected mouse brainstem and human trigeminal DNA has shown that this "endless" herpes simplex virus DNA is present in all four isomeric configurations. PMID- 3003376 TI - Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade. AB - BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: protection against CB3 on HeLa, protection against CB3 RD on rhabdomyosarcoma (RD) cells, and protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of [35S]methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1 RD, CB3-RD, and CB5-RD virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and 125I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses. PMID- 3003378 TI - Mouse peritoneal cells confer an antiviral state on mouse cell monolayers: role of interferon. AB - Vesicular stomatitis virus and encephalomyocarditis virus do not multiply in the majority of peritoneal macrophages freshly explanted from 4- to 8-week-old male or female mice. However, when peritoneal macrophages were cultivated in vitro for 3 to 5 days, these cells became permissive for both viruses. The loss of antiviral state in "aged" macrophages paralleled a significant decrease in the intracellular levels of (2'-5')oligo-adenylate synthetase activity. Although biologically active interferon was not detected in the nutrient medium of macrophage cultures, freshly harvested peritoneal cells could confer an antiviral state on monolayer cultures of mouse cells (aged macrophages, embryonic fibroblasts, and L cells) but not on heterologous chicken embryo, rabbit kidney, or human cells infected with vesicular stomatitis virus or encephalomyocarditis virus. The conferred antiviral state required at least 7 h to develop in target cells and was totally inhibited by the presence of antibody to mouse interferon alpha/beta but not to interferon gamma in the cocultures. Heterologous guinea pig and rabbit peritoneal cells could not transfer an antiviral state to target mouse cells. Donor peritoneal cells from mice preinjected with antibody to interferon alpha/beta could not transfer an antiviral state to target mouse cells. This ensemble of results indicating that freshly harvested peritoneal cells transfer interferon (which is responsible for inducing an antiviral state in susceptible mouse target cells) adds further experimental evidence that interferon is spontaneously expressed in normal mice and plays an important role in maintaining some host cells in an antiviral state. PMID- 3003379 TI - Synthesis, processing, and secretion of the Marek's disease herpesvirus A antigen glycoprotein. AB - The 57,000- to 65,000-dalton (Da) Marek's disease herpesvirus A (MDHV-A) antigen glycoprotein (gp57-65) has a 47,000-Da unglycosylated precursor polypeptide (pr47), as determined by immunological detection after cell-free translation of infected-cell mRNA. Cleavage of its signal peptide yielded a 44,000-Da precursor polypeptide molecule (pr44), detected both in vivo after tunicamycin inhibition of glycosylation and in vitro after dog pancreas microsome processing of pr47. High-resolution pulse-chase studies showed that pr44 was quickly glycosylated (within 1 min) to nearly full size, a rapid processing time consistent with a cotranslational mode of glycosylation. This major glycosylation intermediate was further modified 6 to 30 min postsynthesis (including the addition of sialic acid), and mature MDHV-A was secreted 30 to 120 min postsynthesis. Limited apparent secretion of pr44 occurred only in the first minute postsynthesis, in contrast to the later secretion of most of the MDHV-A polypeptide as the fully glycosylated form described above. In addition, in the presence of tunicamycin a small fraction of the newly synthesized MDHV-A protein appeared as a secreted, partially glycosylated, heterogeneously sized precursor larger than pr44. pr44 constituted the major fraction of the new MDHV-A made in the presence of the inhibitor but the precursor was smaller than mature MDHV-A. These data indicate that there is a minor glycosylation pathway not sensitive to tunicamycin and that "normal" glycosylation is not necessary for secretion. Collectively, the data demonstrate that the rapid release of most of the fully glycosylated form of MHDV A from the cell shortly after synthesis is true secretion in a well-regulated and precisely programmed way and not the result of cell death and disruption. PMID- 3003380 TI - Nonsense mutation in open reading frame E2 of bovine papillomavirus DNA. AB - Oligonucleotide-directed mutagenesis was used to construct a nonsense mutation in open reading frame (ORF) E2 of bovine papillomavirus DNA. A single base substitution mutation was constructed which converted a TAC codon into a TAG amber stop codon at a position in the ORF that did not overlap with any other viral ORFs. Full-length viral DNA containing the mutation induced only approximately 2% of the transformed foci of mouse C127 cells that were induced by wild-type DNA. In a different transformation assay, approximately one-half of the C127 cells which had acquired the mutant DNA gave rise to colonies containing at least some cells with transformed morphology. The constructed mutation was maintained in cell lines derived from cells which had acquired the mutant viral DNA, but the viral DNA appeared to be integrated into the host cell genome. Genetic mapping experiments proved that the constructed amber mutation caused the decrease in focus-forming activity and the integration of the mutant viral DNA. These results suggest that ORF E2 encodes a protein which is involved either directly or indirectly in some aspects of oncogenic transformation by bovine papillomavirus and in maintaining the viral DNA as a plasmid in transformed cells. PMID- 3003381 TI - Human parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein: nucleotide sequence of mRNA and limited amino acid sequence of the purified protein. AB - The nucleotide sequence of mRNA for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 3 virus obtained from the corresponding cDNA clone had a single long open reading frame encoding a putative protein of 64,254 daltons consisting of 572 amino acids. The deduced protein sequence was confirmed by limited N-terminal amino acid microsequencing of CNBr cleavage fragments of native HN that was purified by immunoprecipitation. The HN protein is moderately hydrophobic and has four potential sites (Asn-X-Ser/Thr) of N-glycosylation in the C-terminal half of the molecule. It is devoid of both the N-terminal signal sequence and the C-terminal membrane anchorage domain characteristic of the hemagglutinin of influenza virus and the fusion (F0) protein of the paramyxoviruses. Instead, it has a single prominent hydrophobic region capable of membrane insertion beginning at 32 residues from the N terminus. This N-terminal membrane insertion is similar to that of influenza virus neuraminidase and the recently reported structures of HN proteins of Sendai virus and simian virus 5. PMID- 3003382 TI - Pathogenesis of acute murine cytomegalovirus infection in resistant and susceptible strains of mice. AB - We have characterized the progress of acute murine cytomegalovirus (MCMV) infection in the spleen, liver, and salivary gland of susceptible (BALB/c) and resistant (C3H) strains of mice after intraperitoneal inoculation. Viral replication was analyzed by virus titration, infectious-center assays, and in situ cytohybridization with cloned subgenomic fragments of the MCMV genome. The most striking differences between strains were observed in the spleen. At 24 h postinfection (p.i.), both strains had a similar number of infected spleen cells. At 48 h p.i., BALB/c mice showed marked dissemination of the splenic infection which continued until 96 h p.i. In contrast, the number of infected C3H spleen cells did not increase from the 24-h level but declined later on. This early block in dissemination of MCMV infection in C3H mouse spleens was not a result of the H-2k haplotype, as BALB.K (H-2k) mice, which show an intermediate level of resistance to MCMV infection, exhibited dissemination of the infection between 24 and 48 h p.i., albeit at a reduced level. However, between 72 and 96 h p.i., we observed a decline in the number of infected spleen cells in BALB.K mice similar to that observed in C3H mice. We also demonstrated by Southern blot analysis of DNA from the infected spleen cells that the termini of the MCMV genome fuse after in vivo infection. PMID- 3003384 TI - Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing. AB - Intracerebral inoculation of mice with poliovirus type 2 Lansing induces a fatal paralysis, while most other poliovirus strains are unable to cause disease in the mouse. To determine the molecular basis for Lansing virus neurovirulence, we determined the complete nucleotide sequence of the Lansing viral genome from cloned cDNA. The deduced amino acid sequence was compared with that of two mouse avirulent strains. There are 83 amino acid differences between the Lansing and Sabin type 2 strain and 179 differences between the Lansing and Mahoney type 1 strain scattered throughout the genome. To further localize Lansing sequences important for mouse neurovirulence, four intertypic recombinants were isolated by exchanging DNA restriction fragments between the Lansing 2 and Mahoney 1 infectious poliovirus cDNA clones. Plasmids were transfected into HeLa cells, and infectious recombinant viruses were recovered. All four recombinant viruses, which contained the Lansing capsid region and different amounts of the Mahoney genome, were neurovirulent for 18- to 21-day-old Swiss-Webster mice by the intracerebral route. The genome of neurovirulent recombinant PRV5.1 contained only nucleotides 631 to 3413 from Lansing, encoding primarily the viral capsid proteins. Therefore, the ability of Lansing virus to cause paralysis in mice is due to the viral capsid. The Lansing capsid sequence differs from that of the mouse avirulent Sabin 2 strain at 32 of 879 amino acid positions: 1 in VP4, 5 in VP2, 4 in VP3, and 22 in VP1. PMID- 3003383 TI - Guanine nucleotide contacts within viral DNA sequences bound by polyomavirus large T antigen. AB - Essential nucleotide contacts between the polyomavirus large T antigen and its multiple specific binding regions within the regulatory sequences of the polyomavirus genome were determined in vitro by methylation interference. Methylation of any of the guanine residues of the 5'-G(A/G)GGC-3' pentanucleotide repeats in large-T-antigen-binding regions A, B, C, and 3 (A. Cowie and R. Kamen, J. Virol. 52:750-760, 1984) interfered with T antigen binding. Within regions A, B, and C these pentanucleotides are spaced 5 or 6 base pairs apart. Therefore, the clusters of contacted nucleotides within each of these binding regions are localized along one face of the DNA helix. Methylation of guanines within the sequences between the pentanucleotide repeats did not interfere with binding. The ORI binding region contains four additional pentanucleotide sequences within a region of dyad symmetry. Methylation of only particular guanines of these pentanucleotides interfered with T antigen binding. The spatial arrangement of the pentanucleotides in the ORI is such that the clusters of contacted guanines are situated around the DNA helix, thereby forming a very different arrangement from that found in the other binding regions. A model is discussed in which cooperative interactions between T antigen protomers, recognizing individual pentanucleotides, determines the strength and the function of different T antigen DNA interactions. PMID- 3003385 TI - Role of a membrane glycoprotein in Friend virus erythroleukemia: nucleotide sequences of nonleukemogenic mutant and spontaneous revertant viruses. AB - We previously isolated spontaneous env gene mutants of Friend spleen focus forming virus that are nonleukemogenic in adult mice but form leukemogenic revertants in newborns; we found that the revertants contain secondary env mutations. To identify sites in the encoded membrane glycoprotein that are important for its pathogenic function, we molecularly cloned and partially sequenced the env genes of two mutant viruses (clone 63 and clone 4) and one revertant (clone 4REV). Clone 63 contained three noncontiguous point mutations that caused nonconservative amino acid substitutions of Gly-119----Arg-119, Cys 180----Tyr-180, and Gly-203----Arg-203 in the xenotropic-related domain of the env glycoprotein. These substitutions were presumably responsible for the altered electrophoretic and pathogenic properties of the mutant glycoprotein. The presence of these and several other G-A nucleotide substitutions at different sites in one spontaneous mutant provided striking evidence that error-rich proviruses can form during retroviral replication. Clone 4 contained a point mutation that generated a premature termination condon at amino acid residue 304 (Gln-304----Ochre-304). This termination codon was located immediately after the proposed xenotropic-ecotropic recombination site and eliminated the ecotropic related domain, including the putative membrane anchor of the glycoprotein. Clone 4REV was a true revertant derived from clone 4 in which the premature termination codon had back-mutated to re-form the wild-type sequence. These results confirm an essential role for the env gene in Friend spleen focus-forming virus pathogenesis and suggest that the encoded membrane glycoprotein contains different domains that contribute to its pathogenic function. PMID- 3003386 TI - Properties of the simian virus 40 (SV40) large T antigens encoded by SV40 mutants with deletions in gene A. AB - The biochemical properties of the large T antigens encoded by simian virus 40 (SV40) mutants with deletions at DdeI sites in the SV40 A gene were determined. Mutant large T antigens containing only the first 138 to 140 amino acids were unable to bind to the SV40 origin of DNA replication as were large T antigens containing at their COOH termini 96 or 97 amino acids encoded by the long open reading frame located between 0.22 and 0.165 map units (m.u.). All other mutant large T antigens were able to bind to the SV40 origin of replication. Mutants with in-phase deletions at 0.288 and 0.243 m.u. lacked ATPase activity, but ATPase activity was normal in mutants lacking origin-binding activity. The 627 amino acid large T antigen encoded by dlA2465, with a deletion at 0.219 m.u., was the smallest large T antigen displaying ATPase activity. Mutant large T antigens with the alternate 96- or 97-amino acid COOH terminus also lacked ATPase activity. All mutant large T antigens were found in the nuclei of infected cells; a small amount of large T with the alternate COOH terminus was also located in the cytoplasm. Mutant dlA2465 belonged to the same class of mutants as dlA2459. It was unable to form plaques on CV-1p cells at 37 or 32 degrees C but could form plaques on BSC-1 monolayers at 37 degrees C but not at 32 degrees C. It was positive for viral DNA replication and showed intracistronic complementation with any group A mutant whose large T antigen contained a normal carboxyl terminus. These findings and those of others suggest that both DNA binding and ATPase activity are required for the viral DNA replication function of large T antigen, that these two activities must be located on the same T antigen monomer, and that these two activities are performed by distinct domains of the polypeptide. These domains are distinct and separable from the domain affected by the mutation of dlA2465 and indicate that SV40 large T antigen is made up of at least three separate functional domains. PMID- 3003387 TI - Cleavage of a viral polyprotein by a cellular proteolytic activity. AB - The 200,000-dalton polyprotein encoded by the bottom component RNA of cowpea mosaic virus was synthesized in rabbit reticulocyte lysates, and this in vitro synthesized protein was isolated from the lysate reaction mixture by sucrose density gradient centrifugation. Incubation of the isolated polyprotein with buffer caused no change in the protein, but incubation with reticulocyte lysates or with fractionated lysate proteins resulted in cleavage of the protein into the expected cleavage products (32,000- and 170,000-dalton proteins). This finding indicated that reticulocytes contain a proteolytic activity that is needed for the primary cleavage reaction. A cleavage assay in which we used partially purified preparations showed that cleavage was an ATP-dependent reaction. PMID- 3003388 TI - Human papillomavirus type 16 DNA-induced malignant transformation of NIH 3T3 cells. AB - A biological function for human papillomavirus 16 (HPV 16) DNA was demonstrated by transformation of NIH 3T3 cells. HPV 16 DNA has been found frequently in genital cancer and has been classified as a papillomavirus on the basis of DNA homology. A recombinant HPV 16 DNA (pSHPV16d), which contains a head-to-tail dimer of the full-length HPV 16 genome, induced morphologic transformation; the transformed cells were tumorigenic in nude mice. Expression of transforming activity was unique because of the long latency period (more than 4 weeks) required for induction of morphologic transformation and because the transfected DNA existed primarily in a multimeric form with some rearrangements. Furthermore, virus-specific RNAs were expressed in the transformants. The transformation of NIH 3T3 cells provides a model for analyzing the functions of HPV 16, which is associated with cervical carcinomas. PMID- 3003389 TI - Hexavalent capsomers of herpes simplex virus type 2: symmetry, shape, dimensions, and oligomeric status. AB - The structures of the hexavalent capsomers of herpes simplex virus type 2 were analyzed by negative staining electron microscopy of capsomer patches derived from partially disrupted nucleocapsids. Optimally computer-averaged images were formed for each of the three classes of capsomer distinguished by their respective positions on the surface of the icosahedral capsid with a triangulation number of 16; in projection, each capsomer exhibited unequivocal sixfold symmetry. According to correspondence analysis of our set of capsomer images, no significant structural differences were detected among the three classes of capsomers, as visualized under these conditions. Taking into account information from images of freeze-dried, platinum-shadowed nucleocapsid fragments, it was established that each hexavalent capsomer is a hexamer of the 155-kilodalton major capsid protein. The capsomer has the form of a sixfold hollow cone approximately 12 nm in diameter and approximately 15 nm in depth, whose axial channel tapers in width from the outside towards the inner capsid surface. PMID- 3003390 TI - Cultivation and characterization of three strains of murine rotavirus. AB - Three distinct strains of murine rotavirus were adapted to growth in cell culture. These strains are genetically related but not identical; they are serotypically heterogeneous. The cultivatable strains were substantially more infectious (approximately 10(6)-fold) for suckling mice than heterologous simian rotaviruses were. Homologous murine rotavirus strains spread from inoculated to uninoculated litter mates and caused diarrhea, while heterologous rotaviruses did not spread and cause illness. PMID- 3003391 TI - 2.2-kilobase class of early transcripts encoded by cell-related sequences in human cytomegalovirus strain AD169. AB - In this report we describe the kinetics of appearance and fine mapping of a 2.2 kilobase (kb) class of transcripts arising from a region of the human cytomegalovirus genome which contains cell-related sequences. These transcripts are encoded by adjacent EcoRI fragments R and d (map units 0.682 to 0.713), located within the long unique segment of the genome. The 2.2-kb RNAs were first detected at 8h postinfection and appeared at comparable or slightly lower levels at 28 and 72 h postinfection. At late times (72 h) additional transcripts were detected with probes from this region. RNase, S1 nuclease, and exonuclease VII protection analyses of 8- and 28-h RNA indicated that the 2.2-kb RNAs had a complex spliced structure consisting of invariable 5' and internal exons and a heterogeneous 3' exon. The position of the 5' end of the RNA was determined with respect to the nucleotide sequence. Analysis of this sequence showed that the cell-related sequences were contained within a long open reading frame in the 5' exon. PMID- 3003392 TI - Variant influenza virus hemagglutinin that induces fusion at elevated pH. AB - The hemagglutinin (HA) glycoprotein of influenza virus performs two critical roles during infection: it binds virus to cell surface sialic acids, and under mildly acidic conditions it induces fusion of the virion with intracellular membranes, liberating the genome into the cytoplasm. The pH dependence of fusion varies for different influenza virus strains. Here we report the isolation and characterization of a naturally occurring variant of the X31 strain that fuses at a pH 0.2 units higher than the parent strain does and that is less sensitive to the effects of ammonium chloride, a compound known to elevate endosomal pH. The bromelain-solubilized ectodomain of the variant HA displayed a corresponding shift in the pH at which it changed conformation and bound to liposomes. Cloning and sequencing of the variant HA gene revealed amino acid substitutions at three positions in the polypeptide. Two substitutions were in antigenic determinants in the globular region of HA1, and the third occurred in HA2 near the base of the molecule. By using chimeric HA molecules expressed in CV-1 cells from simian virus 40-based vectors, we demonstrated that the change in HA2 was solely responsible for the altered fusion phenotype. This substitution, asparagine for aspartic acid at position 132, disrupted a highly conserved interchain salt bridge between adjacent HA2 subunits. The apparent role of this residue in stabilizing the HA trimer is consistent with the idea that the trimer dissociates at low pH. Furthermore, the results demonstrate that influenza virus populations contain fusion variants, raising the possibility that such variants may play a role in the evolution of the virus. PMID- 3003393 TI - Interaction of mRNA with proteins in vesicular stomatitis virus-infected cells. AB - The interaction of mRNA with proteins in vesicular stomatitis virus (VSV) infected cells was studied by photochemical cross-linking in intact cells. The major [35S]methionine-labeled proteins which became cross-linked by UV light to mRNA in uninfected and in VSV-infected HeLa cells were similar and had apparent mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to 135, 93, 72, 68, 53, 50, 43, and 36 kilodaltons. The proteins which were cross-linked in vivo specifically to the five mRNAs of VSV were labeled through radioactive nucleotides incorporated only into VSV mRNAs under conditions (5 micrograms of actinomycin D per ml) in which only VSV mRNAs are labeled. The same major mRNP proteins that became cross-linked to host mRNAs also became cross-linked to VSV mRNAs, although several quantitative differences were detected. Photochemical cross-linking and immunoblotting of cross-linked mRNPs with VSV antiserum demonstrated that in addition to host proteins VSV mRNAs also became cross-linked to the VSV-encoded N protein. The poly(A) segment of both host and VSV mRNAs was associated in vivo selectively with the 72-kilodalton polypeptide. The major proteins of mRNA-ribonucleoprotein complexes are therefore ubiquitous and common to different mRNAs. Furthermore, since the major messenger ribonucleoproteins interact also with VSV mRNAs even though these mRNAs are transcribed in the cytoplasm, it appears that nuclear transcription and nucleocytoplasmic transport are not necessary for mRNA to interact with these proteins. PMID- 3003394 TI - The terminal a sequence of the herpes simplex virus genome contains the promoter of a gene located in the repeat sequences of the L component. AB - The herpes simplex virus DNA genome consists of two covalently linked components, L and S. The unique sequences of the L component are flanked by 9-kilobase-pair inverted repeat sequences ab and b'a', whereas those of the S component are flanked by 6.5-kilobase-pair inverted repeat sequences c'a' and ca. We report that the 500-base-pair a sequence contains the promoter-regulatory domain and the transcription initiation site of a diploid gene, the coding sequences of which are located in the b sequences of the inverted repeats of the L component. The chimeric gene constructed by fusion of the a sequence to the coding sequences of the thymidine kinase gene and recombined into the viral genome was regulated as a gamma 1 gene. The size of the protein predicted from its sequence is 358 amino acids; it was designated as infected cell protein (ICP) 34.5. Thus, the inverted repeats flanking the unique sequences of the L component contain two genes specifying ICP0 and ICP34.5, respectively. Moreover, in addition to the cis acting sites for the inversion of L and S components relative to each other, for cleavage of unit length DNA molecules from head-to-tail concatemers, and for packaging of the DNA into capsids, the a sequence also contains the promoter regulatory domain and transcription initiation sites of a gene. PMID- 3003395 TI - Guanidine-selected mutants of poliovirus: mapping of point mutations to polypeptide 2C. AB - Sequence analysis of the genomic RNA of interstrain guanidine-resistant and antibody-resistant variant recombinants of poliovirus type 1 mapped the resistance of mutants capable of growth in 2.0 mM guanidine hydrochloride to a region located 3' of nucleotide 4444. This region of the viral genome specifies the nonstructural protein 2C. The sequence of genomic RNA encoding 2C from six independently isolated mutants resistant to 2.0 mM guanidine was determined. All six isolates contained a mutation in 2C at the same position in all cases, resulting in two types of amino acid changes. Dependent mutants were examined and found to contain two amino acid changes each within 2C. Mutants resistant to 0.53 mM guanidine were isolated and found to lack the mutations seen in variants resistant to 2.0 mM guanidine. A comparison of the amino acid sequences of the 2C proteins of poliovirus, foot-and-mouth disease virus, rhinovirus types 2 and 14, and encephalomyocarditis virus revealed a strong homology over regions totaling 115 residues. All of the mutations observed in guanidine-selected mutants were contained within this region. The amino acid region containing the mutations observed in poliovirus mutants resistant to 2.0 mM guanidine was compared with the homologous region in the other picornaviruses; a strong correlation was found between the amino acid present at this position and the sensitivity of the virus to 2.0 mM guanidine. PMID- 3003396 TI - Identification of a new glycoprotein of herpes simplex virus type 1 and genetic mapping of the gene that codes for it. AB - A type-specific monoclonal antibody, LP10, precipitated a glycoprotein with a molecular weight of approximately 59,000 from purified herpes simplex virus type 1. Although this glycoprotein was similar in size to glycoprotein D (gD), it was shown to be less abundant in both virions and infected cells, to migrate more rapidly in its precursor form, to incorporate glucosamine but not mannose, and to have a more stable precursor in tunicamycin-treated cells than the gD precursor (pgD). Immunoassays of cells infected with insertion recombinants and intertypic recombinants localized the gene coding for the target antigen of LP10 to the unique short (Us) region at map units 0.892 to 0.924 excluding gD. The target antigen of LP10 was then definitively mapped to the Us4 open reading frame by immunoprecipitation of a polypeptide synthesized by in vitro translation of a Us4 specific transcript prepared by using an SP6 cloning This newly identified glycoprotein product of the Us4 gene of herpes simplex virus type 1 is distinct from the previously identified gB1, gC1, gE1, and gH1. PMID- 3003397 TI - Effect of complement depletion by cobra venom factor on fowlpox virus infection in chickens and chicken embryos. AB - The course of infection with an attenuated strain of fowlpox virus (FPV), which is known to induce antibody-independent activation of complement via the alternative pathway, was investigated in 1- to 3-day-old chickens and 14-day-old chicken embryos by treatment with cobra venom factor (CVF). CVF was found to inhibit complement activity transiently via the alternative pathway but not via the classical pathway. In chickens treated with CVF, virus growth in the skin was enhanced, and pock lesions tended to disseminate, leading to fatal infection in some birds. Histologically, an acute inflammation at an early stage of infection (within 3 days) was inhibited, and virus content in the pock lesion was increased. In chicken embryos with immature immune capacities, CVF treatment caused changes in pock morphology from clear pocks to diffuse ones, an increase in virus content in the pock, and inhibition of cell infiltration. Thus, FPV infection was aggravated in both CVF-treated chickens and chicken embryos. These results are discussed in relation to roles of complement in the elimination of virus at an early stage of FPV infection. PMID- 3003398 TI - Dissociation of newly synthesized Sendai viral proteins from the cytoplasmic surface of isolated plasma membranes of infected cells. AB - The interaction of Sendai viral proteins with the membranes of infected cells during budding of progeny virions was studied. BHK cells infected with Sendai virus were labeled with [35S]methionine, and the plasma membranes were purified on polycationic polyacrylamide beads. The isolated membranes were incubated with various agents which perturb protein structure to dissociate viral proteins from the membranes. Incubation of membranes with thiocyanate and guanidine removed both the M and nucleocapsid proteins. Urea (6 M) removed the nucleocapsid proteins but removed M protein only in the presence of 0.1 or 1.0 M KCl. In contrast, high salt concentrations alone eluted only the M protein, leaving the nucleocapsid proteins completely membrane bound. About 65% of the M protein was eluted in the presence of 4 M KCl. The remaining membrane-associated M protein was resistant to further extraction by 4 M KCl. Thus, M protein forms two types of interaction with the membrane, one of them being a more extensive association with the membrane than the other. PMID- 3003399 TI - Ribosomal initiation at alternate AUGs on the Sendai virus P/C mRNA. AB - Peptide sera specific for the C, C/C', and P proteins of Sendai virus have been used to confirm that the viral nonstructural proteins originate from internal AUG codons and are translated in a different reading frame from that of the P protein. The C protein undergoes aberrant migration on sodium dodecyl sulfate polyacrylamide gel electrophoresis and is expressed at higher levels in infected cells than are the P and C' proteins. PMID- 3003400 TI - Characterization of a new type of human papillomavirus found in a lesion of Bowen's disease of the skin. AB - The genome of a human papillomavirus (HPV) found in a patient with Bowen's disease of the skin was molecularly cloned. Blot hybridization experiments, performed under stringent conditions, revealed no cross-hybridization between this HPV DNA and the other known HPV DNAs, showing that this HPV represents a new type, tentatively named HPV34. In relaxed hybridization conditions, the highest cross-hybridization was observed with HPV16 DNA. The physical map of HPV34 DNA was aligned with the genetic map of HPV16 DNA by heteroduplex mapping. HPV34 was not detected in 12 additional patients with Bowen's disease of the skin, but a closely related HPV DNA was found in one patient with penile Bowenoid papulosis. PMID- 3003401 TI - Nononcogenic deletion mutants of herpesvirus saimiri are defective for in vitro immortalization. AB - Herpesvirus saimiri L-DNA sequences between 0.0 and 4.0 map units (4.5 kilobase pairs) are required for oncogenicity; these sequences are not required for replication of the virus. To investigate the basis for the lack of oncogenicity of mutants with deletions in this region and to study the function of this region, we developed a reliable system for in vitro immortalization by herpesvirus saimiri. In contrast to peripheral blood lymphocytes from cotton-top tamarins (Saguinus oedipus) and owl monkeys (Aotus sp.), infection of peripheral blood lymphocytes from common marmosets (Callithrix jacchus) in vitro with herpesvirus saimiri consistently yielded continuously growing lymphoblastoid cell lines. Such cell lines were established using strains of herpesvirus saimiri from group A and group non-A, non-B; however, repeated attempts to immortalize common marmoset peripheral blood lymphocytes using strains from group B were not successful. Common marmoset cell lines immortalized by herpesvirus saimiri were T12+, T8+, T4-, and B1-, indicating that they were derived from suppressor/cytotoxic T lymphocytes. Cell lines could not be established using the nononcogenic mutants 11att and S4, both of which were derived from the group A strain 11 virus. Strain 11att has a spontaneous deletion and S4 has a constructed deletion in the 0.0 to 4.0 map unit region. Constructed strains which had these deleted sequences restored did immortalize common marmoset peripheral blood lymphocytes. Thus, the nononcogenic deletion mutants are defective for immortalization. This system should facilitate attempts to define the sequences responsible for immortalization and to determine their function. PMID- 3003402 TI - An endogenous mouse mammary tumor virus genome common in inbred mouse strains is located on chromosome 6. AB - We have examined EcoRI-restricted cellular DNA from mouse-hamster somatic cell hybrids. Results of this analysis show that the unit II mouse mammary tumor virus proviral genome is located on mouse chromosome 6. Restriction analysis of cellular DNA from (C3H/OuJ X Czech II) X Czech II backcross mice showed a strong linkage between unit II and Igk. The gene order of these markers on chromosome 6 relative to the Raf and Kirsten murine sarcoma virus ras-2 proto-oncogenes was established. PMID- 3003403 TI - Paget's disease of the urethral meatus following transitional cell carcinoma of the bladder. AB - Pagetoid extension of transitional cell carcinoma onto the urethral meatus following cystectomy is a rare complication of bladder carcinoma. We report 2 cases associated with severe dysplasia and carcinoma in situ of the periurethral glands. PMID- 3003404 TI - Solubility of calcium oxalate monohydrate and hydroxyapatite in EDTA solutions. AB - The effect of the addition of a buffer to an ethylenediaminetetracetic acid (EDTA) litholytic solution on the solubility of calcium oxalate monohydrate and hydroxyapatite is investigated. The experiments show that the addition of triethanolamine as a buffer to EDTA solutions at pH 8 and 8.5 enhances the solubility of these salts, confirming theoretical predictions. The solubility of calcium oxalate monohydrate at 25C in solutions with varying ratios of buffer to EDTA concentrations and an osmolality of 0.9 was determined. The solutions resulting in the highest solubility were finally tested for their litholytic ability at 37C. At pH 8 solubilities of 22.5 g X l-1 for calcium oxalate monohydrate and 20.2 g X l-1 for hydroxyapatite were obtained. The corresponding solubilities at pH 8.5 were respectively 24.5 g X l-1 and 19.2 g X l-1. PMID- 3003405 TI - Human prostate carcinoma causes hypercalcemia in athymic nude mice and produces a factor with parathyroid hormone-like bioactivity. AB - The mechanism of the calcium and phosphorus abnormalities associated with metastatic prostate carcinoma (CaP) is not yet understood. A tumor model was recently established in which 9479, a human CaP from a patient with prostate carcinoma-induced osteomalacia, was heterotransplanted into athymic nude mice (ANM). In the present study the effect of 9479 on ANM was evaluated. Serum calcium (Ca), phosphorus (P), parathyroid hormone (PTH), 1,25-dihydroxyvitamin D3(1,25-(OH)2D3) and urinary cAMP were measured. Ca was markedly elevated in ANM bearing 9479 vs. age-matched controls (C); the increased Ca returned to control level after tumor removal. Serum PTH was lower in 9479-bearing ANM vs. C while urinary cAMP and serum 1,25-(OH)2D3 levels were elevated. In the ANM bearing 9479, there was a decrease in serum P vs. C which returned to normal after tumor removal. Fractional P excretion was greater in 9479 animals than C. Extracts of 9479 were examined for the presence of parathyroid hormone-like bioactivity by measuring stimulation of intracellular cAMP in ROS 17/2.8 cells. Cyclic AMP stimulation which was found was shown to be inhibited by the competitive PTH antagonist [8Nle, 18Nle, 34Tyr]bPTH-(3-34) amide. These data suggest tumor induction of parathyroid hormone-like humoral modulation of calcium, phosphate and vitamin D metabolism in vivo associated with a parathyroid hormone-like prostate carcinoma product. PMID- 3003406 TI - Increased collagenase activity in early aneurysmal dilatation. AB - Increased collagenase activity has been implicated as a basic abnormality in aortic aneurysm formation. We studied a localized aneurysmal change, poststenotic dilatation, and its relation to collagenase and elastase activity of the aortic wall. Cynomolgus monkeys underwent midthoracic aortic coarctation to produce poststenotic dilatation. Serial angiography showed that poststenotic dilatation was minimal or absent at 10 days, just discernible at 3 months, and prominent at 6 months. At the 3-month time interval, collagenase activity in the region of the poststenotic dilatation increased twofold compared with the same region in aortas from animals without poststenotic dilatation (p less than 0.05). There was no change in aortic elastase activity. These data indicate that collagenolysis and aneurysmal dilatation may be induced by local modifications of pressure and/or flow. Increased collagenase activity associated with abdominal aortic aneurysms may not represent an intrinsic metabolic defect but rather a response to altered hemodynamic conditions. PMID- 3003407 TI - Task force formed to coordinate study, testing of AIDS therapies. PMID- 3003408 TI - Transmission of measles in medical settings. 1980 through 1984. AB - For the five-year period 1980 through 1984, a total of 241 persons with measles in 30 states were identified as probably having acquired their infection in a medical facility. The proportion of all measles cases acquired in medical settings increased from 0.7% for 1980 through 1982 to 2.9% for 1983 and 1984. Seventy-six percent of cases were found in patients or visitors, and 24% in personnel at the medical facility where transmission occurred. The highest proportion of cases occurred in children less than 5 years of age (54.3%), followed by persons 25 to 29 years of age (14.7%). Of spread (50.0%) and patient to-staff spread (36.7%) were most common. Medical personnel rarely transmitted disease to others. More attention needs to be given to methods of preventing spread of measles in medical facilities, such as isolation precautions, postexposure prohylaxis of potential contacts (vaccination or immune globulin), and ensuring that medical personnel are immune to measles. PMID- 3003409 TI - Leads from the MMWR. Need for malaria prophylaxis by travelers to areas with chloroquine-resistant Plasmodium falciparum. PMID- 3003410 TI - HTLV-III antibody testing: the false-negative rate. PMID- 3003411 TI - AIDS and testing for AIDS. PMID- 3003412 TI - Potential for transmission of AIDS-associated retrovirus from bisexual men in San Francisco to their female sexual contacts. PMID- 3003413 TI - Leads from the MMWR. Apparent transmission of human T-lymphotropic virus type III/lymphadenopathy-associated virus from child to mother providing health care. PMID- 3003414 TI - Reducing deaths caused by bronchial asthma. PMID- 3003415 TI - Primary therapy for Cushing's disease with metyrapone. AB - A 13-year-old boy was diagnosed as suffering from pituitary-dependent Cushing's syndrome. He was treated with 2.0 g of metyrapone daily as the sole treatment for four years. All clinical and biochemical stigmata of Cushing's disease disappeared within a few months. The patient grew 23.0 cm in four years and regained normal health. No significant side effects of metyrapone were noticed. Administering the medication at 2 PM and 8 PM allowed higher cortisol levels in the morning and noon hours than in the evening and night, approximating the normal diurnal variation in cortisol production. We conclude that metyrapone may be considered the sole treatment in patients with Cushing's disease. PMID- 3003416 TI - [Effects of dibutyryl cyclic AMP on the hemodynamics and myocardial oxygen balance in dogs with experimental myocardial infarction]. PMID- 3003417 TI - [The effect of neuromuscular blocking agents on the pre-and postjunctional receptors at the neuromuscular junction]. PMID- 3003418 TI - [Multiple primary cancers observed after surgery of lung cancer, and their countermeasures]. AB - Multiple primary cancers were found after surgery in 80 (2.5%) of 3,154 patients with lung cancer. Patients with squamous cell carcinoma most frequently suffered from multiple primary cancers. The period up to discovery of the second primary cancers was within two years in 78.8% of the patients. Second primary cancers were most frequently observed in the lung, followed by the stomach, colon and rectum. Second primary cancers were found in the lung in 26 patients, 14 of these (53.8%) having squamous cell carcinoma. Multiple primary cancers were noted in 2.8% of 1,210 lung cancer patients who underwent stage I curative operation and 2.4% of the remaining 1,944 cases. As to prognosis, 14 patients survived for more than three years after the second primary cancers were found. PMID- 3003419 TI - [Morphological examination of lymphocytes from human T-cell leukemia virus (HTLV) carriers]. PMID- 3003420 TI - [Immunoblastic lymphadenopathy complicated by Epstein-Barr virus-positive primary brain lymphoma]. PMID- 3003421 TI - [Studies on effects of eicosapentaenoic acid (EPA) on the blood coagulation fibrinolytic system and serum lipids]. PMID- 3003422 TI - [Diagnosis and therapy of adult T cell leukemia and lymphoma (ATLL)]. PMID- 3003423 TI - [Transcatheter arterial embolization treatment for rupture of hepatocellular carcinoma into the abdominal cavity]. PMID- 3003424 TI - Effect of Bordetella pertussis vaccine on bleeding time, whole blood coagulation time and platelet aggregation in animals. AB - Bordetella pertussis vaccine, a hypoglycemic agent significantly prolonged the bleeding time and whole blood coaguistion time in rabbits and dogs when administered intraperitonially. The vaccine also affects platelet aggregation in rats 4 days after its intraperitonial administration. The effect on platelet aggregation is thought to be due to its beta adrenargic receptor blocking action which may also contribute to the hypoglycemia. PMID- 3003425 TI - [Effects of dietary factors and fecal bile acids on DMH induced colon cancer in rat]. PMID- 3003426 TI - [Hemodynamics of portal blood flow in patients with hepatocellular carcinoma]. PMID- 3003427 TI - [Experimental study on liver regeneration after major hepatectomy under the portacaval shunt]. PMID- 3003429 TI - [Determination of N,N-dimethylformamide in air using silica gel tube]. PMID- 3003428 TI - [Changes in portal hemodynamics after transcatheter arterial embolization in patients with hepatocellular carcinoma]. PMID- 3003430 TI - [Synthesis of methylguanidine by active oxygen, with special reference to the significant role of hydroxyl radical]. PMID- 3003431 TI - Blood concentration and urinary excretion of enalapril in patients with chronic renal failure. PMID- 3003432 TI - [Evaluation of 3H-PK11195 as a radioligand for the in vivo study of peripheral benzodiazepine receptor]. PMID- 3003433 TI - Studies on toluene diisocyanate (TDI)-induced delayed type hypersensitivity. AB - The toluene diisocyanate (TDI)-induced delayed type hypersensitivity reaction (DTH) in the ear of male mice was investigated and compared with the picryl chloride (PC)-induced one. The results obtained were as follows: 1) When 1% TDI solution (20 microliters/ear) as a challenging concentration was used for 7 weeks old ICR mice, distinct ear swelling was observed in every group sensitized with various concentrations (1-5%, 100 microliters/animal) of TDI solution, and the swelling rate was the same or higher than that of PC-induced DTH. 2) Five, 7 and 13 weeks-old ICR mice showed a similar high response in TDI-induced DTH, whereas the reactivity of 16 weeks-old ICR mice was significantly lower than that of the above-mentioned younger mice. 3) In both TDI- and PC-induced DTH, ICR and BALB/c mice showed a similar high response, whereas the reactivity of ddY mice was significantly lower. The relationship between increase of the ear swelling and the amount of Evans' blue dye leaked being regarded as the intensity of its vasopermeability was also studied. The results obtained were as follows: 1) The dye leakage reached the maximum at 20 hr after challenge in sensitized mice. This peak preceded slightly that of the ear swelling (25 hr after challenge). 2) A positive correlation was observed between ear swelling and dye leakage (r = 0.87, P less than 0.01). The effects of dexamethasone (DX) and indomethacin (IM) were investigated with the TDI-induced DTH model. The DTH reaction was suppressed significantly by both drugs, but the suppressive effects of DX were higher than that of IM. All the above results indicate that the TDI-induced DTH model in mice taking dye leakage as an index may be useful for evaluation of drugs for disorders derived from DTH reactions. PMID- 3003434 TI - The hypotensive effect of 2-(5-chloro-2-phenoxyanilino)-2-imidazoline (FR35447). AB - The hypotensive effect of FR35447 was comparable to that of prazosin and was more potent than that of hydralazine, but its duration of action was shorter. Repeated administration of FR35447 or prazosin to hypertensive rats for 5 consecutive days induced no significant difference in the intensity or duration of the hypotensive effect. In contrast, marked tachyphylaxis to hydralazine or phentolamine was observed. FR35447 as well as prazosin induced only a transient increase in cardiac output in anesthetized dogs, whereas hydralazine induced a longlasting increase. This difference may contribute to no development of tolerance to FR35447 or prazosin. FR35447 decreased the pressor response to noradrenaline, but not that to angiotensin II or vasopressin in pithed rats, which indicates that FR35447 is an alpha-adrenoceptor antagonist. FR35447 has some selectivity for alpha 2-adrenoceptors, but the selectivity was far less than that of yohimbine. Since FR35447 induced only slight hypotension following intracerebroventricular injection in anesthetized cats, the hypotensive effect of the drug does not appear to be mediated through the central nervous system. Whereas prazosin induced a dose-dependent increase in blood glucose in rats, FR35447 showed no significant effect. PMID- 3003435 TI - [Investigation of anti-asthmatic drugs in vitro with emphasis on beta-adrenergic drugs]. PMID- 3003436 TI - [A case of Marie-Bamberger's syndrome associated with small cell carcinoma of the lung]. PMID- 3003437 TI - [Hormonal regulation of the hemostasis system in patients with ischemic heart disease during emotional stress]. AB - A total of 75 patients were examined, suffering from various forms of coronary heart disease (CHD) in conditions of emotional stress (ES). It was found that, as the blood anticoagulant activity and fibrinolysis are intensified in CHD patients in response to ES, the catecholamone effect on the blood coagulation inhibitors is decreased, the correlation between these parameters remaining high. In some patients decrease of the blood anticoagulant activity and fibrinolysis depression are observed in response to ES; decreased blood catecholamine concentrations were detected in the presence of high hydrocortisonemia, weakened hormonal effects, and emergence of negative correlation between these effects and the hemostatic system anticoagulant parameters, that evidences exhausted capacity of the anticoagulant system to defense activation. PMID- 3003438 TI - [Accumulation of pyrophosphate in the myocardium in post-infarction cardiosclerosis]. AB - A study of the relationship between the frequency of labelled pyrophosphate detection in the heart muscle and the incidence of clinical signs of heart failure or angina in 185 postmyocardial-infarction patients demonstrated that the distribution of patients with postinfarction cardiosclerosis among the positive and negative 99mTc-pyrophosphate scintigraphy groups was governed by the presence of heart failure in these patients, a finding suggestive of the scarry fields within the myocardium as the principal cause of myocardial accumulation of labelled pyrophosphate in postinfarction cardiosclerosis. Pyrophosphate accumulation in patients with postinfarction cardiosclerosis can be regarded as an indicator of unfavorable developments, as confirmed by one-year follow-up results in 114 patients. PMID- 3003439 TI - [Physical training 2 months after myocardial infarct]. AB - Two months after myocardial infarction, 30 patients were subjected to a 10-week physical training course. Following the training, their threshold capacity grew by 46%. The effect of training (increased threshold capacity, reduced double product at submaximum effort) persisted for 1 year after myocardial infarction. In 28 control-group patients, changes of threshold capacity and double product at submaximum effort were not significant over the first post-infarction year. A relatively early post-infarction physical training is not associated with complications and largely speeds up physical rehabilitation. PMID- 3003440 TI - Mineralocorticoid effect on K+ permeability of the rabbit cortical collecting tubule. AB - Mineralocorticoid hormones stimulate Na+ absorption and K+ secretion by the cortical collecting tubule. There is good evidence that this stimulation involves increasing luminal membrane Na+ permeability and the turnover rate (or number) of the Na+-K+ pumps. These experiments were designed to examine whether mineralocorticoid hormones also increase cell K+ permeability. Using 42K tracer measurements in tubules treated with amiloride to inhibit active Na+ and K+ transport, passive K+ permeation increased with increasing mineralocorticoid effect. Net Na+ absorption and the (passive) K+ efflux rate coefficient (KK) showed a linear relationship. The stimulatory effect was evident in vitro since 0.2 microM aldosterone added to the bath of tubules harvested from NaCl-loaded rabbits increased KK at 3 hrs while time controls showed no change. Since these tubules were also treated with amiloride, this increase in KK was not dependent on increasing Na+ absorption. The results indicate that in addition to the well described effects of aldosterone on Na+ permeability and cell metabolism, the mineralcorticoid effect includes an increase in cellular K+ permeability. PMID- 3003441 TI - Functional basis for the glomerular alterations in uranyl nitrate acute renal failure. AB - We have examined the acute renal failure that occurs after uranyl nitrate administration in the rat and the specific effects of pretreatment of rats with angiotensin converting enzyme inhibitor (CEI), plasma volume expansion (PVE) after uranyl nitrate, and a combination of these treatments. We utilized a combination of micropuncture measurements of glomerular hemodynamics, cage studies, and histologic examination of renal tissue to evaluate the degree of acute renal failure in all groups studied. Uranyl nitrate (UN) (25 mg/kg body wt) administration caused a reduction in the nephron filtration rate (SNGFR) (39.4 +/ 1.6 to 24.8 +/- 2.9 nl X min-1 X g kidney wt-1, P less than 0.02) as a result of a major decrease in the glomerular ultrafiltration coefficient (LpA) from control values (greater than or equal to 0.085 +/- 0.008 to 0.035 +/- 0.007 nl X sec-1 X mm Hg-1 X g kidney wt-1, P less than 0.01). Treatments with CEI, PVE, and the combination of CEI and PVE in rats receiving UN restored 0.38 +/- LpA to normal values (greater than 0.061 +/- 0.009, 0.091 +/- 0.020, and 0.138 +/- 0.020 nl X sec-1 X mm Hg-1 X g kidney wt-1, respectively). Cage studies revealed that CEI treatment prevented oliguria and resulted in major volume losses and reduction in weight. However, rats died after a similar period after UN, but probably by different mechanisms. Analysis of renal ultrastructure revealed equivalent tubular damage in all experimental groups. Alterations in LpA after UN are functional in nature and are potentially preventable and reversible by a combination of treatments with CEI and PVE. PMID- 3003442 TI - Hemodynamic effects of enalapril on neonatal chronic partial ureteral obstruction. AB - To evaluate the role of angiotensin II (ANG II) in renal vasoconstriction due to ipsilateral CPUO, guinea pigs were subjected to incomplete left ureteral stenosis within the first 48 hr of life, and were given enalapril from the 14th day of life until study at 23 +/- 3 days or 8 weeks of age. Renal blood flow (RBF) was measured using radioactive microspheres, and glomerular filtration rate (GFR) was derived from inulin extraction. The number of perfused glomeruli per kidney was determined following in vivo India ink perfusion. Enalapril treatment resulted in an 83% rise in RBF of the obstructed kidney, from 2.58 +/- 0.26 to 4.73 +/- 0.48 ml/min (P less than 0.001), which was associated with a 26% increase in number of perfused glomeruli (P less than 0.01). Mean GFR of the hydronephrotic kidney increased from 0.13 +/- 0.02 to 0.37 +/- 0.10 ml/min (P less than 0.05). Enalapril had no effect on intraureteral pressure of the obstructed left kidney after 7 to 13 days of administration, and did not affect renal mass or ureteral diameter after 6 weeks of treatment. It is concluded that hemodynamic consequences of CPUO in the neonate may be attenuated by ANG converting enzyme inhibition. The primary effect of enalapril is most likely inhibition of intrarenal ANG II formation. PMID- 3003444 TI - Reactivation of latent murine cytomegalovirus from kidney. AB - Cytomegalovirus (CMV) is presumed to cause latent infection, but the sites of infection are incompletely known. We propose that latent murine cytomegalovirus is present in kidney and may be reactivated by explanation. Immunosuppressive agents and allogeneic stimuli may enhance this process. Balb/c mice were infected 11 to 14 months previously with 10(5) pfu of Smith strain murine cytomegalovirus intraperitoneally. Kidneys from 15 infected and nine uninfected mice were washed, minced into 1 to 2 mm2 explants and placed into separate tissue culture wells containing a mouse embryo fibroblast monolayer. Explants were untreated or treated with azathioprine, cyclophosphamide, anti-thymocyte serum, or allogeneic lymphocytes. Daily observations for CPE and passage of supernatant to fresh mouse embryo fibroblast were done. Standard cultures of blood, kidney, and salivary gland were negative. However, virus was isolated from the explants of 8/15 animals, with a reactivation time of 30 to 70 days. No significant difference in reactivation time was noted between treated or untreated explants. Restriction enzyme analysis of viral DNA confirmed identity with the original strain. These data show that latent murine cytomegalovirus is present in murine kidney tissue and may be reactivated by explantation. PMID- 3003443 TI - Monoclonal antibody to Na,K-ATPase: immunocytochemical localization along nephron segments. AB - To obtain a highly specific reagent that could be utilized for ultrastructural localization of Na,K-ATPase, monoclonal antibodies were produced using microsomal preparations of outer renal medulla of dog and rat enriched for Na,K-ATPase. The monoclonal antibody (C62.4) raised against dog antigen, immunoprecipitated a 96,000 Dalton protein from membranes labeled either with 35S methionine or 3H NAB ouabain. Na,K-ATPase, Na-ATPase, and KpNPPase activity were 25, 60, and 100% maximal after reaction with C62.4. Na,K-ATPase activated with SDS was inhibited, but Na,K-ATPase in tight right-side-out membrane vesicles was not. C62.4 inhibited ouabain binding in the presence of Na,K, and Mg, but did not inhibit ouabain binding in the presence of Mg and Pi. Labeling of broken membranes was readily seen using C62.4 labeled with colloidal gold. Intact right-side-out vesicles showed no evidence of labeling, demonstrating that the antibody is directed to an epitope of the cytoplasmic domain of the enzyme. Differential localization of C62.4 along the nephron was identified. Glomeruli showed no significant antibody binding except by occasional cells in the mesangial regions. Only basal lateral membranes of cells from all tubule segments labeled with C62.4. There was no evidence of specific apical labeling. The thick ascending limb of Henle's loop demonstrated the greatest concentration of antibody binding. In the cortical and outer medullary collecting duct, only principal cells showed abundant antibody binding. Intercalated cells showed no detectable evidence of antibody binding on any surface. These studies demonstrate that Na,K-ATPase is localized exclusively to the basal lateral membrane of renal tubular epithelial cells and varies in density and distribution in different nephron segments. PMID- 3003446 TI - [Wood's disease (Barre-Masson's disease). (A review of the literature)]. PMID- 3003445 TI - Hydrochlorothiazide inhibits bone resorption in men despite experimentally elevated serum 1,25-dihydroxyvitamin D concentrations. AB - We evaluated the effects of hydrochlorothiazide administration in relation to Ca balance, the PTH and vitamin D endocrine systems, acid-base balance, and bone. We studied six healthy men fed constant diets providing only 5.1 +/- 0.7 SD mmoles Ca/day. Three of the men were also given calcitriol, 0.5 microgram 6-hrly throughout their studies. All subjects were observed during 18 control days and then during 18 days of hydrochlorothiazide (HTZ) administration, 25 mg 12-hrly. Observations during control days 11 through 16 were compared to those during days 7 through 18 of HTZ administration, inclusively. Directional changes during HTZ did not differ among subjects not given or given calcitriol. For all six subjects, control net intestinal Ca absorption, serum 1,25-(OH)2-D concentrations, serum iPTH concentrations, and daily urine cAMP excretion averaged 0.5 +/- 2.2 mmoles/day, 162 +/- 51 pM, 4.3 +/- 2.2 microliter Eq/ml and 4.2 +/- 0.9 mumoles/day, respectively; none changed during HTZ. As expected, HTZ administration was accompanied by a fall in urinary Ca excretion, averaging -1.4 +/- 0.8 mmoles/day; P less than 0.01. HTZ administration was also accompanied by less negative Ca balances, averaging +1.6 +/- 1.0 mmoles/day; P less than 0.025, and by a fall in daily urinary hydroxyproline excretion averaging -0.13 +/- 0.09 mmoles/day; P less than 0.025. We interpret these data to indicate that HTZ administration is accompanied by an inhibition of bone resorption. HTZ administration also raised serum HCO3 concentrations by +2.7 +/- 0.5 mEq/liter; P less than 0.001 and blood pH by + 0.05 +/- 0.02 units; P less than 0.005.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003447 TI - [Proteolytic enzyme activity and surgical interventions in Mooren ulcer]. AB - The authors report on two cases of Mooren's ulcer. Tear immunoglobulin levels and proteolytic enzyme activity were determined in the different stages of the disease. The latter proved to be more reliable in the assessment of the progress and prognosis of Mooren's ulcer. PMID- 3003448 TI - [A case of retinopathia oxalogenica and cytomegalic infection]. AB - A case of primary oxalosis Type I with characteristic intraocular calcium-oxalate deposition is reported. Both affected eyes show typical pathologic manifestations, which are intensified by a cytomegalic infection caused by cortisone therapy. PMID- 3003449 TI - [Active chickenpox vaccination of children with acute leukemia or other neoplastic diseases]. AB - 26 patients with acute leukemia and other malignancies susceptible to varicella were vaccinated during maintenance chemotherapy. Vaccination was done with OKA strain of live attenuated varicella vaccine developed by Takahashi 1974. All recipients showed no adverse clinical reactions. There was no spread of vaccine virus to others. Seroconversion was 94% in seronegative patients. In those having low antibody titers before vaccination in 56% booster effect was demonstrable. None of the seroconverted recipients contracted varicella in spite of documented contact exposure. Vaccine zoster was not observed. The results suggest that in immunocompromised children live varicella vaccination has a protective effect against varicella infection which has a described mortality rate up 7% in this patients. PMID- 3003452 TI - [Cardiodigin--endogenous digitalis? Unconventional comments on an old therapeutic principle]. PMID- 3003451 TI - Concomitant increase in plasma atrial natriuretic peptide and cyclic GMP during volume loading. AB - To investigate the effects of fluid expansion on endogenous atrial natriuretic peptide (ANP) and cyclic 3',5'-guanosine monophosphate (cGMP), four male volunteers were studied before, during and after intravasal volume loading. Volume expansion was performed by intravenous infusion of 2,000 ml isotonic saline solution within 30 min. Mean plasma ANP levels increased 2.5-fold from 31.2 pg/ml to 81.7 pg/ml 40 min after the start of infusion. Plasma cGMP levels paralleled the rise in ANP, showing a mean cGMP increment from 2.7 pmol/ml to a maximum of 8.2 pmol/ml. Both ANP and cGMP levels were back to basal levels 120 min after termination of the infusion. Stimulation of endogenous ANP release by volume loading suggests that ANP is involved in the regulation of fluid homeostasis in man. The parallel rise in plasma cGMP levels supports the idea that cGMP is a mediator for the effects of ANP. PMID- 3003453 TI - [Clinical picture and diagnosis of myocarditis caused by Coxsackie group B viruses]. PMID- 3003454 TI - [Role of active vitamin D3 metabolites in regulating calcium metabolism in rats during hypokinesia]. AB - The rats exposed to prolonged hypokinesia showed hypocalciemia, lower PTH and higher calcitonin concentrations in the serum, decreased calcium absorption in the small intestine, and a trend toward nephro- and arteriocalcinosis. Prophylactic administration of 24,25-hydroxy D3. 1,25-hydroxy D3 and their combinations enhanced calcium absorption and alleviated hypocalciemia. The changes in the hormonal regulation of calcium homeostasis can be viewed as a factor responsible for calcium metabolic disorders associated with hypokinesia. PMID- 3003456 TI - [Pre- and after care of the patient in endoscopic studies]. PMID- 3003455 TI - Cytoflowmetric analysis of binding pattern and a new binding kinetic constant for an enterovirus and receptor. PMID- 3003450 TI - [Significance of the endothelium of the vascular wall for maintaining hemostasis]. AB - The endothelial lining contributes in many respects to the patency of the vasculature. The production of heparan sulphate, release of prostacyclin and expression of the membrane cofactor thrombomodulin that is essentially required for the activation of protein C represent important mechanisms that warrant thromboresistance. If the integrity of the vessel wall is lost, the exposed subendothelium that has been built up by the endothelial cells serves as a highly reactive surface for platelets whose adherence is facilitated by another endothelial cell product, the von Willebrand Factor. Induction of tissue factor production after exposure to endotoxin also emphasizes an important role fur the endothelium in the pathogenesis of disseminated intravascular coagulation. Once thrombosis has occurred the release of plasminogen activator of tissue-type from the endothelium leads to dissolution of the clot and a functional restoration of the blood vessel. PMID- 3003457 TI - Distribution of the thrombomodulin antigen in the rabbit vasculature. AB - The purpose of this study was to determine the distribution of the thrombomodulin (TM) antigen in the rabbit vasculature in vivo. Acetone-fixed, frozen sections of various tissues (brain, heart, aorta, lung, liver, spleen, kidney, small intestine, large intestine, skeletal muscle, bone, and skin) were stained by indirect immunofluorescence with goat affinity-purified antibodies to TM. Both large and small vessels (including capillaries) had demonstrable TM antigen localized to the endothelium while vascular smooth muscle and connective tissue cells were negative. The parenchyma of the organs examined were also negative. Based on antibody dilutions and brightness of staining, there is an indication that lung, followed by heart and intestinal vessels, has the highest concentration of antigen; kidney glomerular capillary loops have the least. These results also indicate that TM is an excellent cell type-specific marker of endothelial cells as the endothelium of all vessels and vascular beds was positive. PMID- 3003458 TI - Localization of thrombomodulin antigen in rabbit endothelial cells in culture. An immunofluorescence and immunoelectron microscope study. AB - A total of 13 rabbit endothelial cell lines were examined for the presence, distribution, and localization of the thrombomodulin (TM) antigen. Three endothelial cell lines were derived from aorta, 5 lines from brain microvessels, and 5 lines from fat microvessels. Immunofluorescence demonstrated the presence of the TM antigen in all cell endothelial lines examined, at all passage levels, and at all stages of growth i.e., log phase, confluent, postconfluent stationary. The percentage of positive cells in a given culture and the distribution of the TM antigen within the cells, as well as on their surfaces, was a cell line characteristic. The intercellular variation in the expression of the TM antigen was insignificant in some cell lines but was striking in others. These characteristics were clonable. Immunofluorescence indicated and immunoperoxidase electron microscopy confirmed the localization of the TM antigen to the outer cell surface and in the Golgi-endoplasmic reticulum (GERL) complex. PMID- 3003460 TI - Tumor differentiation and histological type in human breast cancer. AB - Biopsies from 61 women with breast cancer were assessed for various histological features. These results were statistically analyzed for associations of gross tumor size, patient age, stage of disease, tumor hormone receptor status, with accepted systems of nuclear and histological grading for differentiation. Tumors with highly differentiated grading had smaller cell nuclei, stromal elastasis, intraduct carcinoma and tubular formations. Poorly differentiated tumors had large vesicular cell nuclei and increased mitotic indices. Gross tumor size was also linked with stromal elastosis, intraduct carcinoma, a specific pattern of stromal infiltration, and mitotic index. No relationship could be found between any of the above features and the patients clinical statuses or their tumors' hormone receptivities. The existence of two distinct histological patterns of breast carcinoma is proposed; the patterns may either represent stages in the development of the tumor or different biological types. PMID- 3003461 TI - Malignant fibrous histiocytoma of the larynx. AB - Malignant fibrous histiocytomas of the upper respiratory tract are rare, aggressive, mesenchymal neoplasms. There have been sporadic cases reported in the English language literature. This paper reports the case of a subglottic malignant fibrous histiocytoma in a 26-year-old female. A review of the important clinical-pathological features of this type of tumor is presented. The treatment of choice appears to be wide surgical resection of the tumor. Although the published experience is small, it appears that tumors of the subglottic space are characterized by a younger patient population and a better prognosis than those of the larynx. PMID- 3003459 TI - Comparison of blood and peritoneal neutrophil activity in rabbits with and without peritonitis. AB - The neutrophil (polymorphonuclear cell, or PMN) function is an essential component of the host defense against infection. However, infection itself may alter PMN activity. To investigate both the effects of infection on PMN activity and PMN activity on survival, we evaluated control and infected blood and peritoneal PMN phagocytosis, chemotaxis, and superoxide anion production in rabbits with and without peritonitis. Control blood and peritoneal PMNs were obtained from noninfected rabbits which were subjected to intraperitoneal infusion of sterile hypertonic saline. Infected blood and peritoneal PMNs were obtained from rabbits which had undergone appendiceal devascularization and ligation 18 hr earlier. Phagocytosis was similar in all groups except for a threefold increase in normal peritoneal PMNs. Chemotaxis was inhibited by infection in the blood and peritoneal PMNs. Normal peritoneal PMNs also had decreased chemotaxis. Superoxide anion production was comparable in the infected and control blood; however, both control and infected peritoneal PMNs had elevated superoxide anion production. Of the infected rabbits, four died in 5 days or less. Of the six that lived, two developed intraabdominal abscesses. Blood and peritoneal PMN activity was similar in all rabbits despite their outcome. We conclude that (1) blood and peritoneal PMNs have different basal activities and responses to infection; (2) the milieu of the peritoneal cavity appears to alter the PMNs present; and (3) PMN activity did not predict morbidity or mortality. PMID- 3003462 TI - Effect of Trichosanthes kirilowii lectin on lipolysis and lipogenesis in isolated rat and hamster adipocytes. AB - The galactose-binding lectin from the Chinese herb Trichosanthes kirilowii (Tianhuafen) stimulated the incorporation of D-[3-3H]glucose into lipids in isolated rat epididymal adipocytes. The lectin, however, did not inhibit lipolysis induced by either epinephrine or corticotropin in these adipocytes. Similarly, it did not suppress corticotropin-induced lipolysis in isolated hamster adipocytes. At a dose that did not stimulate lipolysis on its own, the lectin slightly potentiated the lipolytic effects of corticotropin and epinephrine. These findings are discussed in relation to previous observations on other galactose-binding lectins. PMID- 3003464 TI - Acupuncture treatment of sciatica and a preliminary study of the analgesic mechanism. PMID- 3003463 TI - Malignant fibrous histiocytoma in thoracic surgical practice. AB - Malignant fibrous histiocytoma is a rare, although increasingly recognized, deep seated pleomorphic sarcoma. A primitive tumor, it arises from tissue histiocytes and typically occurs in the extremities. Primary intrathoracic tumors have been reported rarely. The presentation of malignant fibrous histiocytoma in our series of seven patients has been varied. Two cases presented as solitary primary intrapulmonary tumors, two as primary chest wall tumors, one as an anterior mediastinal mass, one as a retroperitoneal tumor extending cephalad through the diaphragm, and one as a late metastasis from a primary pelvic site. Malignant fibrous histiocytoma is aggressive, with a propensity for early local and distant spread; three of the patients in our series died of progressive disease within 17 months of operation. The histologic nature of the tumor makes diagnosis on small biopsy specimens difficult and frequently misleading. We would suggest a policy of open biopsy to obtain adequate and representative specimens for histologic study and preoperative computed tomographic scanning to augment the clinical search for metastatic disease and to facilitate planning of subsequent radical, excisional operation. The preoperative use of deep x-ray therapy or the newer chemotherapeutic agents may reduce tumor bulk and thereby facilitate radical operation, which presently appears to be the most appropriate primary modality of treatment of malignant fibrous histiocytoma. PMID- 3003466 TI - [Advantages of preventing Rh isoimmunization]. AB - The authors analysed the frequency of Rh immunization from 1972 to 1983. The incidence of Rh-immunized women who after the birth of a Rh (D) positive child were not given anti-D immunoglobulin G and in subsequent pregnancies gave birth to a Rh (D) positive child was found to amount to 11.76%, while in women who were given anti-D immunoglobulin D this incidence was 0.77% (t = 5.98; p less than 0.05). Out of 29 Rh-immunized pregnant women, two developed Rh immunization in the course of the first pregnancy, three after the unsuccessful prevention of Rh immunization, and the rest after delivery or after delivery and abortion. Out of 29 Rh-immunized women, 27 (93.10%) were ABO-compatible and 2 (6.90%) ABO incompatible with their child (p less than 0.05). In the first pregnancy the incidence of Rh immunization was 1.86 per 1000 deliveries in Rh negative pregnant women and 21.19 per 1000 deliveries in subsequent pregnancies (p less than 0.05). In the period observed there were 2.24 Rh immunizations per 1000 of all deliveries. From 1972 to 1977 there were 3.19 Rh immunizations per 1000 deliveries and from 1978 to 1983 only 1.43 (t = 2.08; p less than 0.05), which is a reduction by 55.17%. The perinatal mortality rate of children affected by Rh hemolytic disease was 20%. In the last six years it has gone down by 60%, while the number of children with Rh-hemolytic diseases has been reduced by 50%. PMID- 3003465 TI - [Estrogen and progesterone receptors in ovarian carcinoma]. AB - Estrogen (ER) and progesterone (PR) receptors in 20 primary carcinomas of the ovary were studied by the customary DCC method. One of the receptors examined was found in 12 patients (60%) and both of them in 5 patients (25%). Serous types of tumour revealed a higher concentration of receptors than mucinous ones. The study of this kind is considered useful for the selection of patients in whom the adjuvant hormonal therapy could be applied. PMID- 3003467 TI - The effects of pentoxifylline on rat erythrocytes of different age. AB - Age-separated rat erythrocytes were exposed to pentoxifylline, a dimethylxanthine derivative which increases erythrocyte deformability. A comparison of drug induced effects in young and old erythrocytes yielded age-specific alterations in: (1) accumulation of intracellular Ca2+; (2) membrane protein phosphorylation; (3) ATP concentrations; and (4) membrane associated protein kinase activity. The effect of Ca2+ accumulation and membrane protein phosphorylation appears to be biphasic. Low drug concentrations (0.5-2.5 mM) reduced intracellular Ca2+ and increased membrane protein phosphorylation, whereas higher concentrations (4.0 5.0 mM) increased Ca2+ levels and reduced membrane protein phosphorylation. Young cells exhibited increased ATP levels over the whole range of pentoxifylline tested; however, older erythrocytes demonstrated higher ATP levels at 5.0 mM drug only. Membrane-associated protein kinase activity was enhanced 10% in young erythrocytes at 1.0 mM pentoxifylline and decreased to 30% of control values at 4.0 and 5.0 mM drug. Protein kinase of old erythrocytes exhibited gradual inhibition over the entire drug concentration range. In general, younger erythrocytes appear to be more responsive to pentoxifylline exposure. Based on these studies, it appears that the ageing of the erythrocyte and loss of deformability in vivo may be a consequence of increased Ca2+ entry into the cell. PMID- 3003468 TI - [Detection of anticytomegalovirus IgM in patients excreting cytomegalovirus]. PMID- 3003469 TI - [A 47-year-old male, hemophiliac, with fever, cough and dyspnea (clinicopathologic conference)]. PMID- 3003470 TI - [Familial hepatocellular carcinoma with positive surface antigen (HBsAg)]. PMID- 3003471 TI - [Increased knowledge about viruses as a cause of diarrhea. The protective effect of rotavirus vaccine is tested now]. PMID- 3003472 TI - [Taste aversions in lung cancer patients treated with cytostatics]. PMID- 3003473 TI - Beta-adrenoceptors in kidney tubules of spontaneously hypertensive and normotensive rats. AB - Beta-adrenoceptor binding characteristics were determined in different fractions of rat kidney tubules using a [125Iodo]-(-)-cyanopindolol (ICYP) binding assay. The highest amount of binding sites was found in a fraction containing predominantly distal tubular fragments. In a separate series of experiments the ICYP binding characteristics were compared in whole tubular fractions from spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rats (WKY) of different ages. The maximum number of binding sites was significantly higher both in young (3 weeks) and adult (14 weeks) SHR when compared to age-matched WKY. These studies showed the presence of beta-adrenoceptor binding sites in rat kidney tubules and support the potential importance of tubular beta-adrenoceptors in the development of spontaneous hypertension and in the mechanism of antihypertensive action of beta-blockers. PMID- 3003474 TI - Pharmacological profile of the abeorphine 201-678, a potent orally active and long lasting dopamine agonist. AB - The central dopaminergic effects of an abeorphine derivative 201-678 were compared to those of apomorphine and bromocriptine in different model systems. After oral administration, this compound induced contralateral turning in rats with 6-hydroxydopamine induced nigral lesions and exhibited strong anti-akinetic properties in rats with 6-hydroxydopamine induced hypothalamic lesions. It decreased dopamine metabolism in striatum and cortex, but did not modify noradrenaline and serotonin metabolism in the rat brain. 201-678 counteracted the in vivo increase of tyrosine hydroxylase activity induced by gamma-butyrolactone. In vitro it stimulated DA-sensitive adenylate cyclase and inhibited acetylcholine release from rat striatal slices. This compound had high affinity for 3H-dopamine and 3H-clonidine binding sites. These results indicate that 201-678 is a potent, orally active dopamine agonist with a long duration of action. Furthermore it appears more selective than other dopaminergic drugs. PMID- 3003475 TI - Ranitidine potentiates ileum contractions caused by GABA and electrical stimulation. AB - GABA-evoked contractions of the guinea pig ileum were significantly potentiated by the histamine H2-receptor antagonist ranitidine in concentrations above 10 microM. To help define the mechanism of this interaction, the present study compared the effects of ranitidine on contractile responses of the guinea pig ileum to GABA, acetylcholine (A Ch) and electrical stimulation of intrinsic cholinergic neurons. Ranitidine, at concentrations that potentiated responses to GABA, also potentiated contractions induced by transmural electrical stimulation. The ability of ranitidine to amplify these latter responses was antagonized by atropine. Contractile responses to exogenous A Ch, however, were unaffected by ranitidine at any concentration. These results suggest that prejunctional, rather than postjunctional mechanisms, are of primary importance in the interaction between ranitidine and GABA. PMID- 3003476 TI - Temperature and isoproterenol modulation of beta-adrenergic receptor characteristics. AB - Since agonists and temperature affect receptor affinity, and since these factors may influence the actual determination of receptor affinity, we assessed the in vitro effects of temperature and isoproterenol on the high and low affinity states of beta-adrenergic receptors in rat membrane preparations. There was a temperature-dependent decrease in beta-adrenergic receptor agonist affinity which was further promoted by the presence of isoproterenol. The decrease in receptor agonist affinity was reflected by a decrease in the number of receptors in the high affinity state. These data suggest that receptor desensitization may occur in membrane preparations in vitro and that the present methodology used to assess agonist affinity in vitro may itself alter the very properties being measured. PMID- 3003477 TI - ACTH increases adrenal medullary PNMT activity in neonatal rats. AB - Levels of plasma corticosterone and the activities of adrenomedullary dopamine beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) were measured in the 7-day-old rat following the administration of adrenocorticotropic hormone (ACTH) for 7 consecutive days beginning with day 1. ACTH led to significant adrenal hypertrophy and a concomitant elevation (10 to 15 fold) of plasma corticosterone concentration. Whereas DBH activity remained unchanged, adrenal PNMT activity was increased significantly following ACTH-induced elevation of plasma corticosterone levels. These results indicate that the pituitary-adreno-cortical-adrenomedullary axis is functional in the neonatal rat. Furthermore, since the transsynaptic control mechanisms are known to be non functional or immature in the 7-day-old rat, our data suggest that neonatal rat adrenal catecholamine biosynthesis may be largely controlled by the pituitary adrenocortical axis via glucocorticoids. PMID- 3003478 TI - Morphine and beta-endorphin antagonize posture and locomotor disorders induced by the injection of ACTH 1-24 in the rat locus coeruleus. AB - The unilateral microinjection of ACTH 1-24 (20 nmol) into the locus coeruleus (LC) produced a long lasting (2-3 hr) posture asymmetry and movement disorder in all rats tested. This response was readily suppressed by the subsequent local microinjection of an equimolar dose of beta-endorphin or morphine or by the intraperitoneal injection of morphine sulphate (50 mg/kg). Microinjection of naloxone (20 nmol) into the LC produced the above syndrome in a lower percentage of animals. The results support the hypothesis that ACTH peptides and opioids play opposite roles in the control of different brain functions. PMID- 3003481 TI - Brain benzodiazepine binding sites in ethanol dependent and withdrawal states. AB - The brain benzodiazepine system has been implicated to be important in both the mechanism, and treatment of ethanol related syndromes. In this report evidence is presented which indicates that "peripheral type" benzodiazepine binding sites are probably more relevant than "central type" receptors for the neurochemical consequences of ethanol dependence and withdrawal states. Utilizing radioreceptor binding techniques 20-50% increases in the binding of [3H]RO-5-4864 (a "peripheral type" ligand) to brain membranes derived from rat cerebral cortex, cerebellum and hippocampus are observed in ethanol dependent rats. These increases persist for 3 days after cessation of ethanol. The number of [3H]RO-5 4864 binding sites in cerebellum returns to normal during 4-7 days after ethanol withdrawal. In all brain areas examined no changes were observed in the "central type" benzodiazepine receptor as judged by [3H]-ethyl-Beta-carboline-3 carboxylate, BCCE binding. Scatchard analysis revealed that the number of [3H]RO 5-4864 binding sites is increased in each brain area while the affinity was unchanged. PMID- 3003479 TI - Neuropeptide Y: anatomical distribution and possible function in mammalian nervous system. AB - Neuropeptide Y (NYP) is a 36 amino acid peptide which shares considerable sequence homology with pancreatic polypeptide and peptide YY. NPY is widely distributed within neurons of the central and peripheral nervous systems, and occurs in mammalian brain in higher concentrations than all other peptides studied to date. Radioimmunoassay studies demonstrated high concentrations of NPY immunoreactivity within many regions of the hypothalamus and within the paraventricular thalamic nucleus, nucleus accumbens, the septum and medial amygdala. These findings correspond with the distribution of NPY containing terminals. Numerous cell bodies containing NPY are located within the cerebral cortex, caudate-putamen, hippocampus, hypothalamus, and nucleus tractus solitarius. Central administration of NPY causes a marked increase in ingestive behaviors, possibly related to the release of NPY from neurons in the arcuate nucleus that innervate the paraventricular nucleus of the hypothalamus. NPY projections from the arcuate nucleus to the medial preoptic area may be related to the central effects of NPY on luteinizing hormone release and sexual behavior. NPY immunoreactive terminals heavily innervated neurons within the amygdala and hypothalamus that are connected to the dorsal vagal complex, suggesting a role of NPY in central autonomic regulation. NPY terminals form a dense plexus around cerebral vessels and are probably responsible for NPY's potent vasoconstrictor effects in the cerebral cortex. Coronary vessels are also innervated heavily by NPY terminals, indicating a role for NPY in the pathogenesis of coronary vasospasm. NPY is present in pheochromocytomas and circulating levels of NPY may prove useful in the diagnosis of pheochromocytoma. Thus, anatomical and physiological studies suggest a varied, but important, function for NPY in mammalian nervous system. PMID- 3003480 TI - Steroidogenic effect of ent-kaur-16-en-15 beta-ol (kaurenol) on isolated rat adrenal cells. AB - In an in vitro bioassay system for adrenocorticotropic hormone using isolated rat adrenal cells, kaurenol, a diterpene alcohol, stimulated corticosterone production and augmented the steroidogenic effect of adrenocorticotropin or forskolin, dose-dependently. Kaurenol had no effect on cyclic AMP production by the cells. The diterpene also had no stimulatory effect on the adrenal adenylate cyclase activity in a cell free system. The results suggest that this particular diterpene exerts a steroidogenic effect through a mechanism independent of cyclic AMP generation. PMID- 3003482 TI - Diurnal rhythm of alpha 2-noradrenergic receptors in the paraventricular nucleus and other brain areas: relation to circulating corticosterone and feeding behavior. AB - The paraventricular nucleus alpha 2-noradrenergic system and the glucocorticoid hormone, corticosterone, are known to modulate feeding behavior and exhibit a circadian pattern which may be related to the natural periodicity of feeding in the rat. The results of the present study indicate that the binding of [3H]p aminoclonidine to alpha 2-noradrenergic receptors specifically in the paraventricular nucleus varies concomitantly with plasma corticosterone levels, as well as spontaneous feeding. A monophasic peak of paraventricular noradrenergic receptor binding is detected at the onset of the dark period, when corticosterone levels are highest and feeding is initiated. On the other hand, the supraoptic nucleus exhibits the reverse diurnal pattern, i.e., a significant decline of [3H]p-aminoclonidine binding at the onset of the dark period. Other hypothalamic and extra-hypothalamic areas fail to show significant changes in alpha 2-noradrenergic receptors as a function of the diurnal cycle. This study supports other evidence indicating a close interaction between circulating corticosterone and alpha 2-noradrenergic receptors in specific hypothalamic areas. It also reveals a potential importance for this interaction in control of the natural feeding rhythm. PMID- 3003483 TI - Characterization of benzodiazepine binding sites after short-wave photo-affinity labeling with flunitrazepam. AB - Membranes prepared from rat cerebral cortex were irradiated with short-wave UV light in the presence of flunitrazepam (FZ). This photo-affinity labeling (PAL) drastically reduces the potency of FZ binding to these membranes, but the binding of 3H-beta-carboline-3-carboxylate ethyl ester (3H-BCCE) was found to be essentially unchanged. 3H-BCCE binding was therefore determined in the presence of an antagonist (BCCE itself), an agonist (FZ) and a compound reported to discriminate between multiple benzodiazepine sites (CL 218,872). The results with BCCE are consistent with a single population of sites, but FZ binds to some of the sites with a reduced affinity (KI = 30 nM) and to the remaining sites with a very low affinity (KI approximately equal to 1 microM). CL 218,872 shows a reduced affinity but appears to interact with all of the sites. Taken together, these results indicate that the binding domains for BCCE and FZ are not identical, and that CL 218,872 interacts more strongly with the antagonist domain. PMID- 3003484 TI - Comparative effects of polymyxin B and compound 48/80 on histamine metabolism in rat muscle and gastric tissue. AB - Polymyxin B, administered in vivo, increased histidine decarboxylase (HDC) activity and histamine (HM) concentrations in muscle tissue homogenates and supernatants. When administered in vitro it increased HDC activity and HM concentrations in both muscle and gastric tissue. The stimulatory effect on muscle was similar to that obtained with compound 48/80, but 48/80, unlike polymyxin B, did not affect gastric tissue. In vitro additions of alpha fluoromethylhistidine inhibited both in vivo and in vitro stimulatory effects of polymyxin B. The results of these studies show that the action of compound 48/80 and of polymyxin B are similar, and that both affect HM synthesis in a manner that requires further elucidation. PMID- 3003485 TI - Intra-cerebroventricular captopril reduces plasma ACTH and vasopressin responses to hemorrhagic stress. AB - The role of the brain renin-angiotensin system in mediating the peripheral hormone response to acute hemorrhagic stress (15 ml/kg over 10 min) was studied in 6 sheep during an intracerebroventricular infusion (2.8 micrograms/min) of the angiotensin-converting enzyme inhibitor, captopril. When compared with control experiments the plasma ACTH and vasopressin (AVP) response to hemorrhage was markedly reduced and delayed during icv captopril, which did not affect the response of plasma angiotensin II (AII). These results suggest that the normal and rapid response in ACTH and AVP secretion accompanying hemorrhagic stress is dependent on increased brain production of AII. PMID- 3003486 TI - Cross-tolerance studies between caffeine and (-)-N6-(phenylisopropyl)-adenosine (PIA) in mice. AB - Chronic administration of caffeine to mice (1 mg/ml in drinking water X 14 d) led to a downward shift in the dose-response curve for the locomotor effects of caffeine. Caffeine was also less effective as an antagonist against (-)-(N6 phenylisopropyl)-adenosine (PIA)-induced analgesia in the tail flick assay in these animals. The dose-response curves of PIA for both analgesia and locomotor depression were shifted to the left in animals chronically administered caffeine. In mice chronically administered PIA (1 mg/kg/d X 14 d), the dose-response curves of PIA for both analgesia and locomotor depression were shifted to the right. The dose-response curve for the locomotor effects of caffeine was shifted to the left, and caffeine exhibited greater antagonist activity against the analgesic action of PIA in these animals. There was no change in the Kd or Bmax values of either 3H-PIA or 3H-diethylphenylxanthine (DPX, a potent adenosine receptor antagonist) in mice chronically administered PIA. The Bmax values for both 3H-PIA and 3H-DPX were significantly increased, while the Kd values were not changed in mice chronically administered caffeine. There was no detectable change in the brain levels of either PIA or caffeine in animals chronically treated with either drug. The results demonstrate that chronic administration of caffeine increases the sensitivity of mice to the actions of PIA and vice versa, providing supportive evidence for the interaction of these drugs at the same receptor, which is probably an adenosine receptor. PMID- 3003487 TI - Beta-adrenergic stimulation of fatty acid release from brown fat cells differentiated in monolayer culture. AB - The ability of brown adipocytes differentiated in monolayer culture to respond to norepinephrine was investigated. It was found that fatty acid release in confluent brown adipocytes in monolayer culture was induced by norepinephrine, thus these cells were hormone-sensitive. After confluence, the rate of fatty acid release successively declined. The norepinephrine-stimulated fatty acid release was inhibited by propranolol, but not by phentolamine, indicating a mediation via beta-adrenergic receptors. It was concluded that there exist in the brown adipose tissue of nonfetal rats preadipocytes which possess the ability to express in culture a fully developed beta-adrenergic lipolytic response. PMID- 3003489 TI - Characteristics of lipoprotein receptors of the isolated liver parenchymal cells prepared from the streptozotocin-induced diabetic rats. AB - Characteristics of lipoprotein receptors of the isolated liver parenchymal cells prepared from the streptozotocin-induced diabetic rats were investigated. Streptozotocin-induced diabetic rats fed 1.0% cholesterol showed the exaggerated hypercholesterolemia as compared to control rats fed 1.0% cholesterol. The present study was designed to elucidate the role of lipoprotein receptor mechanisms of liver parenchymal cells in the diabetic dyslipoproteinemia. 125I labeled lipoproteins (rat beta-VLDL, human LDL2 or rat HDL3) were incubated with liver parenchymal cells isolated by liver perfusion using collagenase. According to the Scatchard analysis, the apparent dissociation constant (kd) and maximum beta-VLDL binding (Bmax) for the higher affinity binding site in the diabetic rats (n = 6) were (11.9 +/- 5.1) X 10(2) ng/ml and 307.5 +/- 145.2 ng/10(6) cells, respectively. These binding characteristics of the diabetic rats were not significantly different from the control rats. Furthermore, there were no significant differences in the binding characteristics of human LDL2 and rat HDL3 between the diabetic rats and the control rats. The data presented suggest that significant role of alteration of lipoprotein receptor characteristics in liver parenchymal cells is not played in the diabetic dyslipoproteinemia. PMID- 3003488 TI - The effects of tetrachlorobiphenyls on the electron transfer reaction of isolated rat liver mitochondria. AB - A comparative study was made of the effects of several symmetrical tetrachlorobiphenyls (TCBs) on the electron transfer from succinate to oxygen of rat liver mitochondria, and some differences in effects caused by the different chlorine positions of the biphenyl ring were clarified. TCBs used in this study included 2,3,2',3'-, 2,4,2',4'-, 2,5,2',5'-, 2,6,2',6'-, and 3,4,3',4'-TCBs. The inhibitory actions of 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCBs on succinate oxidase were potent, while those caused by 2,6,2',6'- and 3,4,3',4'-TCBs were significantly weak. The inhibition sites of 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5' TCBs in succinate oxidase were succinate dehydrogenase and cytochrome b-c segment of the electron transport chain. In the cytochrome b-c segment, these TCBs acted on myxothiazol-sensitive site rather than antimycin-sensitive site. Cytochrome c oxidase was hardly affected by TCBs. These results indicate that 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCBs severely depress the electron transfer with succinate as the substrate, which secondarily reduces the synthesis of ATP. The relationship between the activity and chemical structure of TCBs is also discussed. PMID- 3003490 TI - Biochemical characterization of serotonin stimulated phosphoinositide turnover. AB - Serotonin (5HT) stimulates phosphoinositide turnover in a number of tissues, but it is not known whether this effect is due to activation of a 5HT receptor which is coupled to phosphoinositide hydrolysis or if the effect is secondary to 5HT stimulated arachidonate metabolism or to the release of another neurotransmitter. In the present study we show that neither indomethacin nor BW 755C inhibits 5HT stimulated phosphoinositide hydrolysis in rat cerebral cortex, suggesting that neither cyclooxygenase nor lipoxygenase activity is required for the response to 5HT. Proteinase inhibitors do not potentiate the response to 5HT, suggesting that 5HT's effect is not due to stimulation of release of a peptide neurotransmitter. Tetrodotoxin does not inhibit the effect of 5HT and 5HT's effect is additive with that of KCl and veratrine. These data suggest that 5HT stimulated phosphoinositide hydrolysis is not dependent upon release of another neurotransmitter. PMID- 3003491 TI - Molecular aspects of aging. PMID- 3003492 TI - Problems in nerve lesions surgery. AB - An overview of the concepts on peripheral nerve regeneration and of research on the progress in this field is carried out. Problems of regeneration and factors influencing it, both improving and worsening it, are considered, especially with respect to the clinical treatment of nerve lesions. Classical techniques of repair (sutures and grafts) as well as very new methods (e.g., direct muscular neurotization) are discussed. Postoperative treatment and result evaluation are presented. PMID- 3003493 TI - [Use of an immunoenzymatic reaction in the determination of the genetic character of poliovirus samples]. AB - Characterization of poliovirus strains by an enzyme immunoassay (EIA). An enzyme immunoassay performed with polyclonal antisera to poliovirus types 1, 2 and 3 absorbed with heterologous strains of the same types was used for the characterization of poliovirus field isolates. The assay allowed the identification of 74 (93.6%) out of 79 isolates as wild or vaccine-like strains. Applications of EIA for the study of polioviruses are discussed. PMID- 3003494 TI - Attenuated adenosine R-site effect in adipocytes in obesity. AB - Adenosine is a local hormone or a retaliatory metabolite that executes its effect via a plasma membrane receptor, the R-site. In human adipocytes it inhibits cyclic AMP accumulation and lipolysis. N6-(phenylisopropyl)adenosine is a nonmetabolizable derivative that is an R-site agonist not sharing other effects of the parent nucleoside. In subcutaneous abdominal fat cells from obese subjects (130% to 207% of ideal body weight, N = 8), the antilipolytic effect of N6 (phenylisopropyl) adenosine was markedly attenuated as compared to that in fat cells from normal weight subjects (83% to 121% of ideal body weight, N = 8). There was a negative correlation between the effectiveness of the nucleoside analog and the relative body weight of the donor. The effect of N6 (phenylisopropyl)adenosine on cyclic AMP accumulation was similarly attenuated. These findings may explain some of the metabolic alterations observed in obesity. PMID- 3003495 TI - Taurine-glutamate transaminase. PMID- 3003496 TI - D-Glutamate-D-amino acid transaminase from bacteria. PMID- 3003497 TI - Glucosamine-6-phosphate synthase. PMID- 3003498 TI - NAD synthetase. PMID- 3003499 TI - L-Glutamate decarboxylase from brain. PMID- 3003501 TI - Glutamine transaminase K from rat kidney. PMID- 3003500 TI - Glutamine transaminase L from rat liver. PMID- 3003502 TI - alpha-Keto acid omega-amidase from rat liver. PMID- 3003503 TI - Glutamyl-tRNA synthetase from Escherichia coli. PMID- 3003504 TI - Glutathione reductase. PMID- 3003505 TI - Glutathione transferases from human liver. PMID- 3003506 TI - Purification of glutamyl-tRNA synthetase from Bacillus subtilis. PMID- 3003507 TI - Cysteine conjugate beta-lyase. PMID- 3003508 TI - Cysteine S-conjugate N-acetyltransferase. PMID- 3003509 TI - Thioltransferase from human placenta. PMID- 3003510 TI - L-Aspartate alpha-decarboxylase. PMID- 3003511 TI - Aspartate kinases I, II, and III from Escherichia coli. PMID- 3003512 TI - Asparagine transaminase from rat liver. PMID- 3003513 TI - Aspartate ammonia-lyase. PMID- 3003514 TI - Kynurenine aminotransferase from kidney supernatant. PMID- 3003515 TI - Glutamate-aspartate transaminase from microorganisms. PMID- 3003516 TI - L-Kynurenine transaminase from Hansenula schneggii. PMID- 3003517 TI - Interleukin 3. PMID- 3003518 TI - The macrophage colony-stimulating factor, CSF-1. PMID- 3003519 TI - Granulocyte colony stimulating factor. PMID- 3003520 TI - Cloning of the Escherichia coli gene for the stringent starvation protein. AB - In order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein. We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences. The synthetic oligonucleotides were labelled with 32P at their 5'-termini and used as hybridization probes to detect DNA fragments containing the complementary sequences. Genomic Southern hybridization of E. coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes. The latter fragments were cloned in pBR322. By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene. PMID- 3003521 TI - An evolutionary comparison of homologous mitochondrial plasmid DNAs from three Neurospora species. AB - We have discovered a mitochondrial DNA plasmid in N. crassa 516 (Roanoke, LA) which is homologous to those previously described from N. intermedia 435 (Fiji) and N. tetrasperma 2510 (Hanalei, HA). Subsequent analysis by DNA-DNA hybridization showed that 6 of 14 other Louisiana N. crassa isolates possessed plasmids homologous to these three plasmids, but at lower copy number. Plasmids from the three named strains were studied to examine possible plasmid diversity within each isolate, the extent of the homology between the plasmids, and the possibility that these plasmids could be inherited separately from their host mitochondria. Comparison of cloned plasmids and covalently closed circular mitochondrial DNA showed that only one plasmid line was present in each of the three intensively studied isolates. DNA-DNA hybridization and restriction endonuclease site mapping showed that the mitochondrial plasmids from the three species were very similar; most of the variation was due to presumed nucleotide substitutions. Plasmids judged identical by our analysis were found in different species. The distribution of the homologous plasmids in nature and the presence of these identical plasmids in different species, suggested that these plasmids could be transmitted between isolates independently of their host mitochondria. PMID- 3003522 TI - Reversion of a truncated gene for ampicillin resistance by genetic rearrangements in Escherichia coli K12. AB - The composite transposon Tn2672 is a derivative of the Tn3-related transposon Tn902 whose bla gene providing ampicillin resistance had been inactivated by the insertion of the IS1-flanked multiple drug resistance transposon Tn2671. Most ampicillin resistant revertants of Tn2672 are due to precise excision of Tn2671. However, a rare Bla+ revertant which still retains all the previously acquired drug resistance markers was isolated. On this revertant, the 5' part of the split bla gene on Tn2672 has converted to an intact, active bla gene, and the entire Tn902 is structurally restored. In contrast, the adjacent IS1b element belonging to Tn2671 has its terminal 142 base pairs deleted. Despite of this rearrangement, the split 3' part of bla and its adjacent sequences have remained unchanged. Models are presented to explain the observed DNA rearrangements, and their similarity with gene conversion events is discussed. PMID- 3003523 TI - Structure of an amplifiable DNA sequence in Streptomyces lividans 66. AB - Spontaneous chloramphenicol-sensitive mutants of Streptomyces lividans 66 had previously been shown to be very unstable and to yield arginine auxotrophic mutants at a frequency of 25% of spores; the Arg- mutants had amplified a particular 5.7 kb DNA sequence to over one hundred tandem copies per genome. In this paper we report the cloning of the amplifiable region from amplified and wild-type strains. This showed that the amplifiable fragment is already present as a duplication in wild type cells. Hybridisation experiments also demonstrated that in the amplified strains there was a deletion of neighbouring DNA sequences to one side of the amplifiable element; sequences to the other side remain intact. PMID- 3003524 TI - Gene organization and target specificity of the prokaryotic mobile genetic element IS26. AB - The 820-bp mobile genetic element IS26 loses its ability to promote transpositional cointegration (1) by short deletions near the middle of the element causing shifts in both reading frames ORFI (left to right) and ORFII (right to left) and (2) by deletions causing substitutions of the C-terminus of ORFI but not affecting ORFII. The 702-bp ORFI is thus likely to code for the IS26 transposase. An 82-bp long sequence from the left end of IS26 contains a promoter like structure in front of the start of ORFI at coordinate 64. In appropriately constructed plasmids, this sequence promotes the expression of the galK structural gene. The observation provides additional evidence for the functional relevance of ORFI. Neither the presence nor the absence of an intact IS26 element on the same plasmid affects measurably the degree of the galK gene expression by the IS26 promoter. Sequence comparison of 14 independent integration sites of IS26 and its relatives reveals no striking rules for target selection by the element, and the distrubtion of integration sites of IS26 on small multicopy plasmids is nearly random and independent of the local AT-content. PMID- 3003525 TI - Identification of the genes and their polypeptide products responsible for aerobactin synthesis by pColV plasmids. AB - Iron acquisition via aerobactin enhances the virulence of Escherichia coli. Genes that specify functions for aerobactin synthesis and iron(III)-aerobactin transport have been identified on several ColV plasmids. Previously, we cloned the locus for aerobactin synthesis from pColV-K311 and assigned to three loci termed aerA, aerB, and aerC the functions for hydroxylation of lysine, acetylation of the 6-amino group of 6-hydroxy-lysine and coupling of N-acetyl-N hydroxy-lysine with citrate, respectively (Gross et al. 1984). In this paper we show that aerA and aerB determine polypeptides with molecular weights of 50,000 and 35,000, respectively. We identified a fourth gene designated aerD that codes for a polypeptide with a molecular weight of 60,000, and which is required for the linkage of one residue of N-acetyl-N-hydroxy-lysine to citrate. The aerC gene product completes aerobactin synthesis by coupling the second N-acetyl-N-hydroxy lysine to the monoacylated derivative citrate. The order of the genes in the operon was found to be aerD-aerB-aerC-aerA. PMID- 3003526 TI - Cloning of the tyrocidine synthetase 1 gene from Bacillus brevis and its expression in Escherichia coli. AB - The entire structural gene for tyrocidine synthetase 1 from Bacillus brevis ATCC 8185 has been cloned and expressed in Escherichia coli. Transformed E. coli cells were screened for their ability to produce tyrocidine synthetase 1 by in situ immunoassay using antibodies against gramicidin S synthetase 2 which cross-react with tyrocidine synthetase 1. The cloned gene is within a 5.2 kb fragment of B. brevis genomic DNA and requires no external promoter for its expression in E. coli. It was also observed that cloning of the 5.2 kb insert in the opposite orientation still resulted in a high level of tyrocidine synthetase 1 expression in transformed E. coli cells. In addition, protein blotting and partial purification of the gene product by gel filtration revealed a major protein of molecular weight about 100,000 with specific D-phenylalanine dependent ATP-32PPi and 2'deoxy ATP-32PPi exchange activities. These unique activities of tyrocidine synthetase 1 were not detected in protein extracts of E. coli strains carrying the vector. PMID- 3003527 TI - Role of translation in the UTP-modulated attenuation at the pyrBI operon of Escherichia coli. AB - A 273 bp DNA fragment containing the attenuator of the pyrBI operon was inserted into a synthetic cloning site early in the lacZ gene on a plasmid. By this operation the first few codons of lacZ were joined through a linker to the last 39 codons of the open reading frame for the putative pyrB leader peptide. In addition a gene fusion encoding a hybrid protein with beta-galactosidase activity was formed between the pyrB start and the rest of lacZ. This gene fusion is expressed from the lac promoter and the transcript is subject to facultative termination at the pyrBI attenuator. Different variants of the lacZ start were used that either contained a stop codon or directed the translation toward the attenuator in any of the alternative reading frames. The following results were obtained. No significant read-through of transcription over the pyrB attenuator was seen when the leader translation ended 49 nucleotide residues, or more, upstream of the attenuator symmetry, but a UTP-modulated attenuation was established if the leader translation was allowed to proceed across the attenuator as for the putative leader peptide or in a frame-shifted version. The regulation, however, was not as great as for the native pyrB gene. This is probably because the substitution of the normal start of the leader peptide by the start of lacZ alters the coupling between transcription and translation and thereby the attenuation frequency. It cannot, however, be ruled out that the pyrBI operon is regulated at the promoters in addition to the control by attenuation. PMID- 3003528 TI - Tn7-encoded proteins. AB - Proteins encoded by Tn7 have been studied in Escherichia coli maxicells harbouring either various deleted ColE1::Tn7 plasmids or Tn7 fragments cloned in pBR322. Six Tn7-encoded proteins were detected and named p18, p32, p40, p54, p85 a and p85-b according to their apparent molecular weight. Protein p18 is dihydrofolate reductase type I and p32 is probably the protein conferring resistance to streptomycin/spectinomycin. Both genes map on the left-hand part of Tn7. The genes for the four other proteins are located on the right-hand part of Tn7. We propose that they fully cover a 6.9 kb DNA fragment without any overlapping. Starting from the right-hand end towards the middle of the transposon, these four genes are in the following order: p85-a, p54, p40 and p85 b. Transposition of Tn7 onto E. coli plasmids requires the proteins p85-a, p85-b, p54 and p40. However, transposition onto the chromosome does not require the p85 b and p40 products. PMID- 3003529 TI - Cloning and characterization of the prs gene encoding phosphoribosylpyrophosphate synthetase of Escherichia coli. AB - The gene, prs, encoding phosphoribosylpyrophosphate (PRPP) synthetase of Escherichia coli was isolated from a library of E. coli genes cloned in the bacteriophage lambda D69 vector. A strain with a temperature-lethal defect in PRPP synthetase, (prs-2), was used as the host and cloning was performed by lysogenic complementation. The prs gene resided on a 5.6 kilobase-pair (kbp) DNA fragment generated by hydrolysis with restriction endonuclease BamHI. The nearby gene pth, encoding peptidyl-tRNA hydrolase, was also on this fragment. Subcloning of the fragment in the multi-copy plasmid pBR322 and subsequent deletion of parts of the insert resulted in a 1.7 kbp DNA fragment containing the entire prs gene. Bacterial strains harbouring prs-bearing plasmids showed up to 50-fold increased PRPP synthetase activity. The PRPP synthetase subunit was identified by analysis of plasmid-harbouring minicells and the subunit molecular mass established as 33,000 daltons. Analysis, by the minicell procedure, of plasmids with deletions extending into the prs gene established the direction of transcription as counterclockwise. A putative leader sequence of approximately 400 bp preceded the coding sequence. By deletion analysis and by cloning fragments of this leader sequence in a galK expression vector it was found to contain the prs promoter as well as a potential transcription termination site. PMID- 3003530 TI - Rescue of transfected genes from mammalian cells by functional selection in Escherichia coli. AB - We have established procedures for reisolating a transfected gene from mammalian cells by selection in Escherichia coli for the function of the gene product using the Herpes simplex virus thymidine kinase gene as a model. Rescue of the gene is accomplished by three different methods. The tk gene is recloned into a plasmid in which it is hooked up by either the lac promoter or a lac/tk hybrid promoter, or the original plasmid is cut out of the host cell DNA. As the lac/tk hybrid gene can be expressed and selected both in the mammalian and E. coli cells, this type of gene rescue allows investigations on mutagenesis and methylation processes. Additionally, it offers a simple way of studying the integration of the transfected gene into the mammalian genome. PMID- 3003531 TI - Genetical and functional organisation of the Escherichia coli haemolysin determinant 2001. AB - We have identified gene products corresponding to hlyC, hlyA and hlyD encoded by the Escherichia coli haemolytic determinant 2001 of human origin cloned into the recombinant plasmid pLG570. The product of hlyC is required for the "activation" of the inactive 107K polypeptide encoded by the hlyA gene. The activated 107K protein constitutes the active haemolysin secreted into the medium. hlyB and hlyD are separate regions defined by complementation studies and encode functions essential for the export of haemolysin with hlyD encoding a 53K protein. Complementation studies using subclones and Tn5 insertions into pLG570 have revealed the presence of two major promoters upstream of hlyC and hlyD which transcribe the four hly genes in the same direction. Finally, we were able to reconstitute the complete haemolysin system from three different plasmids encoding hlyC, hlyA and hlyB + hlyD, respectively. PMID- 3003532 TI - The recN locus of Escherichia coli K12: molecular analysis and identification of the gene product. AB - The recN gene which is necessary for inducible DNA repair and recombination in Escherichia coli has been cloned into the low copy plasmid vector pHSG415. Analysis of the recombinant plasmid, pSP100, revealed a 5.6 Kb HindIII insert of chromosomal DNA. Transposon inactivation of recN function and analysis of a recN::Mu(Ap lac) fusion located the coding region to a 1.4 Kb region within a 2.1 Kb BglII-AvaI DNA fragment transcribed in a clockwise direction with respect to the chromosome map. The gene product was identified in maxicells as a 60,000 dalton protein. Synthesis of this protein was increased in cells lacking LexA activity or in strains carrying recN cloned into the multicopy vector pBR322. Multiple copies of recN increase resistance to ionizing radiation in recN mutants but reduce the survival of a wild-type strain. PMID- 3003533 TI - Molecular cloning and expression of the biodegradative threonine dehydratase gene (tdc) of Escherichia coli K12. AB - The biodegradative threonine dehydratase gene (tdc) of Escherichia coli was cloned by isolating a dehydratase negative mutant after Tn5 mutagenesis, cloning the tdc::Tn5 DNA into pBR322 and then replacing the Tn5 element on the plasmid in vivo. Subcloning and nucleotide sequence data revealed two distinct procaryotic promoter-like elements each containing a potential CAP-binding site and AT-rich regions, and a Shine-Dalgarno sequence. One of these putative promoters, P2, was located immediately upstream from the tdc coding region, and a second, P1, was approximately 1 kilobase upstream from P2. Deletion of the potential CAP-binding site from P1 prevented tdc gene expression. However, removal of P2 and a large segment of the upstream DNA had no discernible effect on dehydratase synthesis. A 936-base pair open reading frame was found between P1 and the tdc coding region, which produced a polypeptide of about 32 kilodaltons. The data suggest that P1, and not P2, is necessary for tdc gene expression, and that the DNA sequences coding for the 32 KD polypeptide and threonine dehydratase are part of a single transcriptional unit. PMID- 3003534 TI - Isolation and characterization of Escherichia coli antimutators. A new strategy to study the nature and origin of spontaneous mutations. AB - To identify the nature and origin of spontaneous mutability we developed a screening procedure suitable to isolate antimutators showing a lower error rate than 10(-10) per base per replication. Among about 500,000 mutagenized colonies we found 20 mutants showing a reduced spontaneous mutability. These antimutators can be subdivided into three groups: (i) Mutants in which the level of spontaneous mutability is reduced due to an increase in efficiency of the error correcting mechanism (amu4). (ii) Mutants which are deficient in several pathways of DNA repair. This finding supports the hypothesis that much spontaneous mutability is due to error-prone repair (amu59, amu47, amu50, amu62, amu43, amu38). (iii) Mutants in which the antimutator effect seems to be the result of an auxotrophy such as Pur- (amu17), Thr- (amu1, amu28) and Ser- (amu31). This finding might support the hypothesis that metabolically induced lesions are important in spontaneous mutagenesis. Eleven of these antimutators were mapped at ten bacterial loci in the following positions: amu31 (2 min); amu4 (4 min); amu62 (82 min); amu47 (85 min); amu59 (86 min); amu17 (89 min); amu50 (95 min); amu1/amu28 (100 min); amu38 (23-27 min) and amu43 (74-81 min). PMID- 3003537 TI - Effect of mutations in the RNA polymerase gene and that of the transcription termination factor rho on expression of the Escherichia coli galactose operon with an IS2 polar insertion. AB - We analysed the effects on the expression of the gal operon of six phenotypically different mutations in the Escherichia coli RNA polymerase genes in combination with wild-type, rho, and mutant, rho15, alleles of the gene for the transcription termination factor. RNA polymerase mutations can enhance (rpoB268) or reduce (rpoB255), rpoC3, rpoB265) termination by the rho15 factor at the IS2 terminator. The rpoC1 mutation enhances the transcription of the gal operon regardless of the IS2 insertion or the rho15 mutation. Thus RNA polymerase mutations can, independently, or in combination with the rho mutation, compensate for the IS induced, specifically IS2-induced, termination, leading to a partial restoration of gene activity. PMID- 3003535 TI - Regulation of the lkyB gene expression in Escherichia coli K-12 strains carrying an lkyB-lacZ gene fusion. AB - Phage MudII301 was used to isolate new periplasmic-leaky mutants of Escherichia coli K12 carrying an lkyB-lacZ gene fusion. The properties of strain JC2299 carrying the lkyB-2299 insertion mutation were identical to those of strain JC207 carrying the previously described lkyB-207 mutation. The LkyB-beta-galactosidase hybrid protein was partially extracellular and membrane bound. It was shown that both a nonsense (envZ-22) and a polar (ompR::Tn10) mutation in the ompB operon led to an increase of beta-galactosidase activity in the lkyB-lacZ fusion strain. On the other hand, mutations in the phoB, phoR, phoS, phoT, malT or envY genes had no effect on lkyB gene expression. PMID- 3003536 TI - Multimer resolution systems of ColE1 and ColK: localisation of the crossover site. AB - We have identified and characterised a stability function encoded by the high copy plasmid ColK. The function is analogous to ColE1 cer and maximises stability by maintaining plasmids in the monomeric state. In vivo recombination between cer and ckr (which share more than 90% homology at the DNA sequence level) produced a functional hybrid. Sequence analysis of hybrids indicates that recombination involving cer and ckr is site-specific and occurs within a 35 bp region of DNA which contains palindromic symmetry. PMID- 3003538 TI - Amplification of the ArgF region in strain HfrP4X of E. coli K-12. AB - In E. coli K-12 the argF gene is flanked by ISI sequences in direct repeat. Mutants that overproduce the argF-coded enzyme ornithine transcarbamylase can be selected; we have shown that in one class of these mutants there is an approximately forty five-fold amplification of the region bounded by the ISI repeats. This class of mutants has been detected only in strains in which the F factor is integrated in cis to the region. PMID- 3003539 TI - The nature of ompA mutants of Escherichia coli K12 exhibiting temperature sensitive bacteriophage resistance. AB - A class of ompA mutants of Escherichia coli, exhibiting temperature-sensitive resistance towards phages using the OmpA protein as receptor, was analysed. The mutants produce detectable levels of the protein at 42 degrees C but not at 30 degrees C (Manning and Reeves 1976). They were found to have a deletion (one isolate) or insertions (three isolates) upstream of the coding part of the ompA gene. Several previously characterized mutants possessing insertions or a deletion in the non-translated 5' area of the gene also exhibited a similar temperature-sensitive phage resistance. This cold-sensitive phenotype is explained in terms of the recent discovery that the stability of ompA mRNA is regulated by the rate of cell growth (Nilsson et al. 1984). PMID- 3003540 TI - Molecular cloning and nucleotide sequence of the HU-1 gene of Escherichia coli. AB - The Escherichia coli HU-1 was cloned by use of mixed synthetic oligonucleotides (17-mer) predicted from a portion of its amino acid sequence. The amino acid sequence of the HU-1 protein deduced from the nucleotide sequence is in good agreement with the published sequence. The nucleotide sequence has a possible promoter and a typical ribosomal binding site upstream from the translational initiation codon (GUG) of the HU-1 gene. PMID- 3003541 TI - Isolation and organization of genes for nitrogen fixation in Rhodopseudomonas capsulata. AB - A library of Rhodopseudomonas capsulata chromosomal DNA was constructed in the broad host range cosmid vector pLAFR1. The library was used to isolate nitrogen fixation genes by complementation of R. capsulata Nif- mutants. Four complementing regions were localized on different cloned DNA fragments by Tn5 and mini-Mu mutagenesis. Additional nif genes were identified by recombination of transposons from the nif cosmids into the R. capsulata chromosome resulting in the creation of new Nif- mutations. Most of the newly cloned DNA fragments containing nif genes were found to be unlinked to any other by Southern hybridization of the cloned DNA to chromosomal DNA blots. One of the new fragments was linked to the nifHDK genes. Another cluster spanning 10-12 kilobase pairs contained a number of nif genes, possibly as many as eight. PMID- 3003542 TI - Timing of ultraviolet light induced mutagenesis in simian virus 40 relative to viral DNA replication in monkey cells. AB - Simian virus 40 (SV40) was used to probe ultraviolet light (UV)-induced mutation in mammalian cells. Viral mutations were scored as reversions of early and late temperature-sensitive (ts) mutants to the wild-type (WT) phenotype. When virus was exposed to moderate or high UV doses, WT revertants were obtained at a frequency related to the square of the dose from two early (tsA) and one late (tsBC) mutant grown at the restrictive temperature. The reversions generated in the progeny of UV-irradiated early mutants presumably arose before the onset of viral DNA replication because, at the non-permissive temperature, tsA mutants are unable to express the functions responsible for the initiation of viral DNA synthesis. Moreover, the early mutant tsA209 underwent similar levels of induced reversion at the permissive and restrictive temperatures, suggesting that the pre replicative mutational pathway might predominate for moderately and heavily irradiated virus, even under conditions where DNA synthesis can be initiated. The analysis of bursts from revertant plaques produced at the restrictive temperature was consistent with this interpretation. Although the mechanism of pre replicative mutagenesis is not known, it is likely to be mediated by cellular activities owing to the low genetic complexity of the virus. PMID- 3003544 TI - Studies of latent cytomegalovirus infection: the macrophage as a virus-harboring cell. AB - During chronic infection of mice with mouse cytomegalovirus (MCMV), the virus was isolated from various tissues by cocultivation with allogeneic mouse embryonic fibroblasts (MEF). Infectious virus was recovered from over 15% of the pancreases, salivary glands, kidneys, lacrimal glands, and spleens. When activated macrophages were obtained by intraperitoneal injection of peptone into mice infected 3 months earlier, they harbored MCMV. Macrophages or lymphocytes were infected with MCMV in vitro and injected into normal mice intravenously. The peritoneal cavities of these mice were then stimulated by peptone injection 3 months after the transfer, and peritoneal or splenic macrophages and lymphocytes were cocultured with allogeneic MEF. MCMV was recovered from the peritoneal and splenic macrophages and not from the lymphocytes. PMID- 3003545 TI - Formation of varicella-zoster virus antigens in infected Vero cells. AB - The formation of varicella-zoster (V-Z) virus-associated antigens was studied in V-Z virus-infected Vero cells by means of indirect immunofluorescence. Early antigen (EA) was first detected inside V-Z virus-infected Vero cells 4 to 6 hr after infection, whereas surface membrane antigen (SMA) was expressed on the outer surface of infected cells 2 to 3 hr later than EA, and intranuclear late antigen (LA) was detected several hours later than SMA antigen. EA expression was not inhibited by cytosine arabinoside (Ara-C) treatment, whereas LA formation was completely blocked by Ara-C. The presence of two components of SMA early SMA (ESMA) and late SMA (LSMA), was suggested by this difference in susceptibility to Ara-C. The formation of all viral antigens, EA, SMA, and LA, was blocked by inhibitors of RNA and protein synthesis. PMID- 3003543 TI - Identification of polypeptides required for the export of haemolysin 2001 from E. coli. AB - We have identified the polypeptides encoded by the haemolysin export genes from a haemolytic determinant 2001 carried by pLG570. This was previously cloned from an E. coli strain, serotype 04 isolated from a human urinary tract infection. Subclones from the recombinant plasmid pLG570 carrying hlyD analysed in vitro and in minicells showed that this gene is transcribed from an independent promoter and encodes a 53 Kd polypeptide. In contrast, detectable levels of the gene products encoded by hlyB were only observed when transcription presumably emanated from a vector promoter. This gene was found to encode at least two polypeptides apparently expressed from alternative translational start sites within a single reading frame. In minicells the major product was a 66 Kd polypeptide whilst after expression in vitro the major product was a 46 Kd polypeptide. Transposon mutagenesis leading to the synthesis of the expected truncated polypeptides was used to confirm the identity of the hlyD and the two hlyB products. Preliminary results suggest that the majority of the 53 Kd polypeptide is located in the inner membrane when cell envelopes from minicells and maxicells were fractionated using sarkosyl, although residual amounts of the 53 Kd polypeptide were also found in the outer membrane. PMID- 3003546 TI - Human papilloma virus and cervical cancer. PMID- 3003547 TI - Possible outbreak of epidemic polyarthritis. PMID- 3003548 TI - [Cancerogenic activity of silica and possible pathogenetic role of fibrogenous cellular reaction]. PMID- 3003549 TI - Serum angiotensin-converting enzyme level in silicosis. PMID- 3003550 TI - An experimental study on the epidemiology of enteroviruses: water and soap washing of poliovirus 1--contaminated hands, its effectiveness and kinetics. AB - As enteroviruses are mainly transmitted by the fecal-oral route, this study was initiated to investigate the nature of the binding of enteroviruses to human skin. Using poliovirus 1, Mahoney, we investigated the overall effectiveness of soap and water hand-washing of 1 and 5 min duration. The virus-skin interaction was studied by kinetic analysis of repeated serial washings. The following results were obtained: (1) Soap and water washing for 5 min reduced the number of infective particles on hands by 2-4 logs of ten. (2) Poliovirus binding to skin was essentially reversible. (3) Removal of virus followed a triexponential decline curve, suggesting loose, intermediate, and strong binding. (4) Washing agents more effective than soap were sand, aluminum hydroxide powder, and buffer alone, suggesting that friction was more important than emulsification. The results demonstrate the tenacity of poliovirus on skin, and offer a rationale for the epidemiology of enteroviruses on experimental grounds. From a practical point of view these results stress the need for an effective chemical hand disinfectant, particularly in hospitals. PMID- 3003551 TI - Investigation of the virulence genes of herpes simplex virus 2 by experimental infection in vivo with defined intertypic recombinants of a virulent HSV-2 X an avirulent HSV-1. AB - For determination of the gene loci for virulence and tropism of herpes simplex virus 2 (HSV-2) intertypic recombinants of HSV-1 strain HFEM and HSV-2 strain 3345 were screened for their pathogenicity in the tree shrew. HSV-1 strain HFEM is not pathogenic in the tree shrew and can be recovered as a latent virus only from the spleen of the animals; but HSV-2 strain 3345, the other parental virus of the recombinants, was found to be virulent for the tree shrew and colonized the ganglia of latently infected animals. No clinical pictures were observed when the animals were inoculated with HSV recombinants RS1(3), RB2(3), RB2(8), RB2(9), RB2(10), RB5(2), RB7(3), and RB7(7). It was found, however, that HSV recombinant RB5(0) is pathogenic for the tree show. Studies of the state of viral latency in the animals infected with these recombinants revealed that only HSV recombinant RB5(0) was able to persist as latent virus in the spleen of latently infected animals. The genome of recombinant HSV-RB5(0) contains DNA sequences of HSV-2 strain 3345 with two interruptions at 0.22-0.33 and 0.54-0.59 viral map units. In these regions of HSV-RB5(0), DNA sequences of HSV-strain HFEM at 0.26-0.33 and about 0.57-0.58 viral map units, respectively, had been recombined. The results obtained in this study indicate that the recombinant HSV-RB5(0) acquired the virulent phenotype (v+) from parental virus HSV-2 strain 3345 but lost the phenotype for colonizing the ganglia (g+). Furthermore, it acquired the phenotype of parental virus HSV-1 strain HFEM for persisting in a latent state only in the spleen (s+ and g-) of the latently infected animals. PMID- 3003552 TI - [A rare association between Klippel-Trenaunay-Parkes-Weber syndrome and Ehlers Danlos type ligamentous hyperelasticity]. PMID- 3003554 TI - [Use of calcium hydroxyapatite in oromaxillofacial surgery. III. Treatment of voluminous cysts of the maxilla. Surgical technic and presentation of cases]. PMID- 3003553 TI - [Granular cell Abrikosov's tumor. Histological and immunohistochemical study on a case with multiple oral and skin localizations]. PMID- 3003556 TI - Leading work-related diseases and injuries--United States. PMID- 3003555 TI - Apparent transmission of human T-lymphotrophic virus type III/lymphadenopathy associated virus from a child to a mother providing health care. PMID- 3003558 TI - [Treatment of patients with hepatoma and esophageal varices]. AB - Fifty-nine patients with hepatoma associated with advanced esophageal varices who received a variety of therapeutic modalities in the past 10 years at our department were reviewed. Our therapeutic modalities are hepatic resection, hepatic artery ligation (HAL) and trans-arterial embolization (TAE) for those with hepatoma and non-shunting treatment (NST; esophageal transection or Hassab's procedure) and endoscopic sclerotherapy (ST) for those with esophageal varices. A patient selection was made by our own criterial developed by a multiple regression analysis for the hepatoma and KICG value for esophageal varices. Out of 59.16 underwent hepatic resection and NST. Eight survived more than 2 years. The longest survivor has been living for 4 yr and 4 months. Two-year survival rate is 69.8%. Another 16 underwent HAL and NST. Two-year survival was 14.6%. Another 7 underwent ST following hepatic resection or HAL. Five of the 7 received an emergency ST. Hemostasis was achieved in all of them. Two-year survival was 21.8%. The remaining 20 underwent ST and TAE; 13 received an emergency ST with 85% of hemostasis rate. None of them survived more than 2 years. From these data, it is suggested that a proper selection of patients for a proper therapeutic modality improves the prognosis even in those with hepatoma associated with advanced esophageal varices. PMID- 3003557 TI - [Cyclic AMP- and ATP-mediated stimulation of DNA synthesis following partial hepatectomy by prostaglandin-E1 in D-galactosamine injured liver]. AB - D-galactosamine (D-gal) damaged rats were infused with Prostaglandin E1 (PGE1) through a peripheral vein for 40 min. before and after partial hepatectomy. DNA synthesis following 68% partial hepatectomy was severely inhibited by the pretreatment of D-galactosamine. PGE1 infusion (0.5, 1.0 microgram/kg/min) enhanced the DNA synthesis inhibited by D-gal 600 mg/kg significantly (p less than 0.01). After 20 min. of PGE1 infusion cyclic AMP levels of liver tissue was increased as compared with saline infusion in D-gal (600 mg/kg)-damaged rat (p less than 0.05). Also 20 min. and 3 hour after partial hepatectomy. ATP levels of liver tissue was enhanced in PGE1 treated group (p less than 0.05). However the doses of PGE1 infused in this investigation could not increase the hepatic tissue blood flow measured by hydrogen gas clearance method. These results suggest that PGE1 enhance DNA synthesis of injured liver after partial hepatectomy by the mechanism which PGE1 stimulate cyclic AMP production and increase ATP level in hepatic tissue. PMID- 3003559 TI - [Treatment of hepatocellular carcinoma with esophageal varices]. AB - This report describes 53 patients with hepatocellular carcinoma (HCC) complicated with esophageal varices. Esophageal varices were due to cirrhosis of the liver in all cases. Hepatic resection and blocking operations such as Sugiura procedure, transabdominal esophageal transection or Hassab's operation were performed for the treatment of HCC and esophageal varices in 6 cases with satisfactory results. Non-operative treatments such as TAE or arterial infusion chemotherapy for HCC and blocking operations for esophageal varices were performed in 17 cases. Late deaths were recognized in 10 cases. Causes of late deaths were carcinoma of the liver in 7 cases and ruptured varices in only 1 case. In 13 cases with severe hepatic failure, only endoscopic sclerotherapy was performed for the treatment of esophageal varices. However 8 cases of 13 had rebleeding from esophageal varices and died after sclerotherapy. We concluded that effective treatments for HCC complicated with esophageal varices were to perform both the hepatic resection and the blocking operation and these treatments prolong the long-term survival of patients with HCC with esophageal varices. PMID- 3003560 TI - Expression of beta-adrenergic receptors in synchronous and asynchronous S49 lymphoma cells. II. Relationship between receptor number and response. AB - We have used two experimental approaches--receptor inactivation with an irreversible antagonist and changes in receptor expression during passage of cells through the cell cycle--to explore the relationship between beta-adrenergic receptor number and response in intact S49 lymphoma cells. beta-Receptors in asynchronous cultures of S49 cells were blocked to varying degrees with the irreversible antagonist bromoacetylalprenololmenthane (BAAM). Blockade by BAAM was noncompetitive and did not alter the affinity of receptors for the agonist isoproterenol. Intracellular accumulation of cAMP in response to 1 microM isoproterenol was proportional to receptor number both at times of initial and maximal accumulation. In contrast, when intracellular accumulation of cAMP in response to isoproterenol was measured in synchronized cultures of S49 cells (obtained by centrifugal elutriation), a notably different relationship was observed. Cells were least responsive, that is, receptors appeared "uncoupled," during S phase of the cell cycle. This attenuation of response was not due to alterations of receptor number, receptor affinity for agonist, or expression of the catalytic unit of adenylate cyclase. Use of the antibiotic mycophenolic acid, a selective inhibitor of the synthesis of GTP, elicited response patterns in asynchronous cells similar to those seen in synchronized cells. These results confirm that wild-type S49 cells do not possess spare receptors. In addition to the importance of total receptor number in determining maximal response to isoproterenol, receptors may show differential efficacy in promoting cAMP accumulation as cells traverse the cell cycle. Changes in cellular levels or utilization of GTP during the cell cycle may serve to regulate the coupling of receptors to the stimulation of adenylate cyclase. PMID- 3003561 TI - Study of species specificity in growth hormone-releasing factor (GRF) interaction with vasoactive intestinal peptide (VIP) receptors using GRF and intestinal VIP receptors from rat and human: evidence that Ac-Tyr1hGRF is a competitive VIP antagonist in the rat. AB - In order to determine species specificity in growth hormone-releasing factor (GRF) interaction with vasoactive intestinal polypeptide (VIP) receptors, we have tested rat (r) GRF (with a His1 such as in VIP), human (h) GRF and position 1 substituted analogs of hGRF (Ala1, Ac-Tyr1, His1, Phe1, and Trp1 in the place of Tyr1) for their ability to inhibit 125I-VIP binding and to stimulate adenylate cyclase activity in human and rat intestinal epithelial membranes. We show that rGRF has a much higher affinity than hGRF for both human and rat VIP receptors. In humans, the Ki values for inhibiting 125I-VIP binding are 0.5 (VIP), 26 (rGRF), and 830 nM (hGRF). In rats the values are 0.6 (VIP), 46 (rGRF), and 1100 nM (hGRF). This is due in part to the presence of His1 in rGRF since the analog His1 hGRF has a higher affinity than hGRF in man and rat, i.e., Ki = 320 nM and 460 nM, respectively. Studies of adenylate cyclase stimulation reveal that rGRF and His1 hGRF are full VIP agonists in man and rat, whereas hGRF and its other analogs behave as partial agonists in both species. One of the hGRF analogs tested (Ac-Tyr1hGRF) is of great interest since it inhibits 125I-VIP binding to rat intestinal membranes with a Ki = 430 nM but has a negligible intrinsic activity in stimulating adenylate cyclase activity (about 6% of the efficacy of VIP). This analog does inhibit the VIP-stimulated adenylate cyclase activity in a dose-dependent manner, complete inhibition of the VIP (0.01-1 nM) effect being obtained with 30 microM analog. The Schild plot of the inhibitory effect further indicates competitive antagonism. In contrast, Ac-Tyr1hGRF is a partial VIP agonist in humans (about 20% of the efficacy of VIP). These results evidence the important role of His1 for peptide interaction with VIP receptors and provide the first example of a competitive VIP antagonist. PMID- 3003562 TI - Theoretical studies on the activation mechanism of the histamine H2-receptor: the proton transfer between histamine and a receptor model. AB - A proposed molecular mechanism for the activation of the H2-receptor of histamine was simulated with ab initio calculations including geometry optimization with several basis sets. The system is modeled by a proton-relay chain produced by the binding of a histamine molecule to a receptor model consisting of an anionic anchoring site, and proton donor and acceptor sites. The anchoring of histamine cation at a negative receptor site is simulated by the interaction with a hydroxyl anion or by calculations on neutral histamine; the proton donor and acceptor sites are modeled by ammonium and ammonia groups, respectively. Results of the calculations reveal that a significant decrease in the barrier for the movement of the proton from the donor site to the N1 nitrogen in the imidazole portion of histamine occurs as a consequence of the neutralization of the side chain and the simultaneous interaction of the N3 nitrogen with the proton acceptor. An increase of the driving force for the proton transfer process is produced by these interactions, as observed from the relative energies of the initial and final steps of the charge relay. The barrier and the driving force depend on the nature of the proton acceptor site. This simulation of the receptor activation mechanism provides the basis for exploration of the partial receptor activation by molecules characterized as partial agonists and the lack of activation by molecules that act as antagonists on this receptor. PMID- 3003563 TI - Isolation of plasma membranes from Drosophila embryos. AB - Cell surface components probably play an important role in early embryonic development. However, hardly any information is available on the structure or regulation of expression of the corresponding genes. As a first step in approaching this issue, we devised a procedure to obtain enriched plasma membranes from embryonic Drosophila cells. Membranes are fractionated according to two independent physical parameters: size, using velocity gradient centrifugation and density, using isopicnic gradient centrifugation. The final membrane fraction is enriched by 6 to 8 fold with respect to the plasma membrane enzyme marker Na+/K+ ATPase and substantially depleted of the mitochondrial enzyme marker cytochrome C oxidase. Two-dimensional polyacrylamide gel electrophoresis of the purified membranes reveals enrichment for specific proteins and electron microscopy reveals membrane vesicles in abundance. The enriched fraction should be suitable for the preparation of antibody probes that recognize cell surface components. PMID- 3003564 TI - Cloning and structure of a mouse interleukin-2 chromosomal gene. AB - Using non-stringent hybridization with a human interleukin-2 cDNA probe, we have isolated recombinant phages from a mouse genomic DNA library cloned in the EMBL3 phage. The sequence and organization of the mouse interleukin-2 (IL-2) gene was determined. By comparison with the human IL-2 sequence, three introns can be identified with lengths of 99, +/- 2 400, and +/- 1 900 base pairs, respectively. The mouse IL-2 gene codes for a polypeptide of 169 amino acids and contains a putative signal peptide of 20 amino acids. The homology to the human interleukin 2 is 72% at the nucleotide level in the coding part and 65% at the amino acid level. An extraordinary sequence, consisting of 12 consecutive CAG codons coding for glutamine, is found in the first exon. PMID- 3003565 TI - Genetic polymorphisms in the analysis of diabetes mellitus. PMID- 3003566 TI - [Asthma syndrome in childhood. Aspects of the definition, pathophysiology, diagnosis, differential diagnosis and therapy]. AB - The etiology and pathogenesis of childhood asthma differs from one child to another. Bronchial hyperreactivity appears to be a basic defect in asthma, as can be proven by different methods, and it is an important factor in exercise induced asthma. Exercise induced asthma leads to inactivity in some children who become isolated and psychologically depressed. To counteract this negative development it is very important firstly to establish and maintain a longterm therapeutic regime with antiasthmatic drugs, and secondly to propose an individual fitness program for such children. This fitness program must be an integral part of the total therapeutic program. PMID- 3003567 TI - [Properties of sodium- and potassium-dependent adenosine triphosphatase in the plasma membrane of albino rabbit dental pulp]. PMID- 3003568 TI - Tumor-promoting barbiturates act on DNA repair of cultured hepatocytes. AB - We have observed that two promoters of liver carcinogenesis, i.e. phenobarbital and barbital, markedly increase DNA-repair synthesis of cultured hepatocytes following treatment with the ultimate carcinogens methyl methanesulfonate, N methyl-N'-nitro-N-nitrosoguanidine, N-acetoxy-2-acetylaminofluorene, and UV light of 254 nm. Phenobarbital also increased the incorporation rates of deoxynucleoside triphosphates into nuclear DNA of permeabilized hepatocytes following carcinogen treatment. The action of these barbiturates apparently correlates with their potential to promote hepatocarcinogenesis in vivo, since the non-promoting agent barbituric acid did not modify carcinogen-induced repair synthesis. Moreover, the mechanisms of action of tumor-promoting barbiturates is different from the known enhancing action on repair synthesis of inhibitors of nuclear poly(ADP-ribose) biosynthesis. PMID- 3003569 TI - Treatment of sebopsoriasis with itraconazole. PMID- 3003570 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 9-1986. A 40-month-old girl with the acquired immunodeficiency syndrome and spinal-cord compression. PMID- 3003571 TI - Human parvovirus infection. PMID- 3003572 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 5-1986. Enlarging left hilar mass of 15 years' duration. PMID- 3003573 TI - Late-onset adrenal hyperplasia. PMID- 3003574 TI - AIDS virus at the centre. PMID- 3003575 TI - Infection mechanism? PMID- 3003576 TI - The facts on cystic fibrosis testing. PMID- 3003577 TI - Similarity of protein encoded by the human c-erb-B-2 gene to epidermal growth factor receptor. AB - A novel v-erb-B-related gene, c-erb-B-2, which has been identified in the human genome, maps to human chromosome 17 at q21 (ref. 40), and seems to encode a polypeptide with a kinase domain that is highly homologous with, but distinct from, that of the epidermal growth factor (EGF) receptor. The c-erb-B-2 gene is conserved in vertebrates and it has been suggested that the neu gene, detected in a series of rat neuro/glioblastomas, is, in fact, the rat c-erb-B-2 gene. Amplification of the c-erb-B-2 gene in a salivary adenocarcinoma and a gastric cancer cell line MKN-7 suggests that its over-expression is sometimes involved in the neoplastic process. To determine the nature of the c-erb-B-2 protein, we have now molecularly cloned complementary DNA for c-erb-B-2 messenger RNA prepared from MKN-7 cells. Its sequence shows that the c-erb-B-2 gene encodes a possible receptor protein and allows an analysis of the similarity of the protein to the EGF receptor and the neu product. As a consequence of chromosomal aberration in MKN-7 cells, a 4.6-kilobase (kb) normal transcript and a truncated 2.3-kb transcript of c-erb-B-2 are synthesized at elevated levels. The latter transcript presumably encodes only the extracellular domain of the putative receptor. PMID- 3003578 TI - fgr proto-oncogene mRNA induced in B lymphocytes by Epstein-Barr virus infection. AB - Several acute transforming retroviruses encode tyrosine-specific protein kinases which possess structural and functional relationships to cell-surface receptors for certain growth factors. One such tyrosine kinase is encoded by the onc gene, v-fgr, of Gardner-Rasheed feline sarcoma virus (GR-FeSV). Recently, we have isolated and characterized the human gene, c-fgr, corresponding to the viral onc sequence and have shown that c-fgr is a unique gene located on the short arm of chromosome 1 (ref. 7). Here we report that certain lymphomas (but not sarcomas or carcinomas) express fgr-related messenger RNA. This transcript is detected in Burkitt's lymphoma cell lines naturally infected with Epstein-Barr virus (EBV), but not in EBV-negative Burkitt's lymphoma cells. Normal umbilical cord or peripheral blood lymphocyte lines established in vitro by EBV infection also contain detectable c-fgr mRNA. Moreover, a 50-fold increase of the steady-state c fgr mRNA concentration is observed when uninfected Burkitt's lymphoma cell lines are deliberately infected with EBV. These findings demonstrate for the first time the induction of a proto-oncogene in response to infection by a DNA tumour virus. PMID- 3003579 TI - Rearrangement of two distinct T-cell gamma-chain variable-region genes in human DNA. AB - Selective cloning procedures for T-cell-specific complementary DNAs have revealed the existence of a gene designated gamma as well as the main antigen receptor alpha- and beta-chain genes. The gamma-chain genes undergo rearrangement during T cell differentiation but the patterns and complexity of such rearrangements differ markedly in mouse and human. In mouse, a panel of cytotoxic T-lymphocyte clones exhibit the same rearrangement pattern with a gamma-chain gene probe and a set of three gamma-chain variable (V) genes have been identified in the DNA. Clonal diversity in mouse seems to be confined to V-J (joining) regions. In contrast, human T-cell lines exhibit diverse rearrangements suggestive of a family of differing V gamma genes variously rearranging to the two gamma-chain constant (C) region genes. Here we report the cloning of two very different V gamma genes rearranged to J segments upstream of the two human C gamma genes. Both V gamma genes are rearranged productively but nucleotide sequence comparison shows that they possess very little homology with each other. This shows that human T-cell V gamma genes exist which differ significantly from each other at the nucleotide level and that such diverse genes can be usefully rearranged in different T cells. PMID- 3003580 TI - Evolutionary relationships of human populations from an analysis of nuclear DNA polymorphisms. AB - The genetic relationships of human populations have been studied by comparing gene frequency data for protein and blood-group loci of different populations. DNA analysis now promises to be more informative since not only do the DNA coding sequences have more variation than their corresponding proteins but, in addition, noncoding DNA sequences display more extensive polymorphism. We have now studied the frequency of a group of closely linked nuclear DNA polymorphisms (haplotypes) in the beta-globin gene cluster of normal (beta A) chromosomes of individuals from eight diverse populations. We have found that all non-African populations share a limited number of common haplotypes whereas Africans have predominantly a different haplotype not found in other populations. Genetic distance analysis based on these nuclear DNA polymorphisms indicates a major division of human populations into an African and a Eurasian group. PMID- 3003581 TI - Two roles for guanine nucleotides in the stimulus-secretion sequence of neutrophils. AB - The term 'stimulus-secretion coupling' has, since first enunciated, been held to involve the mobilization of cytosol Ca2+, which in turn is sufficient to trigger exocytotic secretory processes in metabolically competent cells. However, recent studies on a wide range of secretory cell types indicate that a role for Ca2+ can be obviated: examples are stimulation with phorbol ester (phorbol myristate acetate, PMA) which causes the activation of protein kinase C or the stimulation of platelets with collagen. Ca2+-independent exocytosis also occurs when analogues of GTP are injected through the lumen of patch pipettes directly into the cytosol of mass cells. The results presented here suggest that GTP analogues can activate secretory processes by actions at two distinct locations: one may be at the level of the receptor involving the activation of polyphosphoinositide (PPI) phosphodiesterase with consequent liberation of diacylglycerol (DG); the other involves direct activation of the exocytotic mechanism. These conclusions are based on measurements of exocytotic secretion from permeabilized neutrophils into which we have been able to introduce, individually and in combination, Ca2+ chelators (EGTA and BAPTA), Ca2+ (buffered at micromolar concentrations with EGTA), analogues of GTP and GDP and the direct activator of protein kinase C, PMA. PMID- 3003583 TI - AIDS protein made. PMID- 3003582 TI - A saturable receptor for 32P-inositol-1,4,5-triphosphate in hepatocytes and neutrophils. AB - Several receptors for neurotransmitters, hormones and growth factors cause accelerated phosphodiesteratic breakdown of polyphosphoinositides when activated. One of the soluble products of this reaction, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) is thought to act as a second messenger signalling the release of Ca2+ from intracellular stores. In support of this hypothesis, several studies have shown that Ins(1,4,5)P3 releases sequestered Ca2+ from permeable cells and microsomes. On the basis of certain structural requirements for Ca2+-releasing activity by inositol phosphates, it has been postulated that Ins(1,4,5)P3 acts by binding to a specific intracellular receptor, probably on a component of the endoplasmic reticulum. Here we report that 32P-Ins(1,4,5)P3 binds to a specific saturable site in permeabilized guinea pig hepatocytes and rabbit neutrophils, and that the properties of this binding site suggest that it is the physiological receptor for Ins(1,4,5)P3. PMID- 3003584 TI - Post-transcriptional regulation accounts for the trans-activation of the human T lymphotropic virus type III. AB - The level of synthesis of viral proteins and heterologous proteins under the control of long terminal repeat sequences of human T-lymphotropic virus type III (HTLV-III or LAV) increases dramatically in cells that constitutively express the HTLV-III trans-activator protein. Increased levels of protein synthesis occur without a comparable increase in the levels of corresponding messenger RNA. We propose that post-transcriptional events mediated by the HTLV-III trans-activator protein account for positive regulation of HTLV-III gene products in infected cells. PMID- 3003585 TI - Reciprocal changes in corticotropin-releasing factor (CRF)-like immunoreactivity and CRF receptors in cerebral cortex of Alzheimer's disease. AB - Alzheimer's disease is a progressive degenerative disease of the nervous system characterized neuropathologically by the presence of senile plaques and neurofibrillary tangles in amygdala, hippocampus and neocortex. Dysfunction and death of basal forebrain cholinergic neurones projecting to forebrain targets are associated with marked decreases in cholinergic markers, including the activity of choline acetyltransferase (ChAT). Although cortical levels of somatostatin and somatostatin receptors are reduced in Alzheimer's, no consistent changes have been reported in other neuropeptide systems. We have now examined in control and Alzheimer's brain tissues pre- and postsynaptic markers of corticotropin releasing factor (CRF), a hypothalamic peptide regulating pituitary adrenocortical secretion which also seems to act as a neurotransmitter in the central nervous system (CNS). We have found that in Alzheimer's, the concentrations of CRF-like immunoreactivity (CRF-IR) are reduced and that there are reciprocal increases in CRF receptor binding in affected cortical areas. These changes are significantly correlated with decrements in ChAT activity. Our results strongly support a neurotransmitter role for CRF in brain and demonstrate, for the first time, a modulation of CNS CRF receptors associated with altered CRF content. These observations further suggest a possible role for CRF in the pathophysiology of the dementia. Future therapies directed at increasing CRF levels in brain may prove useful for treatment. PMID- 3003586 TI - Stable expression of two variable surface glycoproteins by cloned Trypanosoma equiperdum. AB - African trypanosomes are thought to evade the host immune system by periodically changing their variable surface glycoprotein (VSG). VSG genes are activated by a complex process involving the duplicative transposition of silent basic copy genes to one of several expression sites. These expression-linked copies (ELCs) of the VSG genes are also subject to regulation within expression sites by as yet unknown mechanisms. It is generally assumed that trypanosomes can express only one VSG gene at a time. Nevertheless, the finding that they contain multiple VSG gene expression sites suggests that multiple expression is possible. We show here that Trypanosoma equiperdum can stably express two VSG genes in a simple axenic culture system and that both antigens are present on the cell surface. The two antigens do not co-cap or form heterodimers. Their corresponding genes show no cross-hybridization and are situated in different telomere-linked expression sites. Northern blot analysis reveals that both genes are active in the double expressors. PMID- 3003587 TI - Evidence for adenosine receptor-mediated isoprenaline-antagonistic effects of the adenosine analogs PIA and NECA on force of contraction in guinea-pig atrial and ventricular cardiac preparations. AB - The effects of the adenosine agonists (-)-N6-phenylisopropyladenosine (PIA) and 5'-N-ethylcarboxamideadenosine (NECA) on force of contraction, adenylate cyclase activity and normal as well as slow action potentials were studied in guinea-pig isolated atrial (left auricles) and ventricular preparations (papillary muscles). In auricles PIA and NECA exerted concentration-dependent negative inotropic effects with similar potencies (mean EC50:0.05 mumol l-1 for PIA and 0.03 mumol l 1 for NECA). Similar results were obtained in the presence of isoprenaline. In papillary muscles PIA and NECA alone had no effect on force of contraction but produced negative inotropic effects in the presence of isoprenaline (mean EC50:0.19 mumol l-1 for PIA and 0.10 mumol l-1 for NECA). In both preparations, the negative inotropic effects of PIA and NECA in the presence of isoprenaline were antagonized by the adenosine receptor antagonist 8-phenyltheophylline. In both preparations, PIA and NECA did not affect adenylate cyclase activity, both in the absence and presence of isoprenaline. In auricles the negative inotropic effects of both nucleosides were accompanied by a shortening of the action potential. This effect was also observed in the presence of isoprenaline. In papillary muscles the adenosine analogs did not detectably alter the shape of the normal action potential. Ca2+-dependent slow action potentials elicited in potassium-depolarized preparations also remained unaltered in the presence of PIA or NECA alone. However, the isoprenaline-induced enhancement of the maximal rate of depolarization of slow action potentials was attenuated by PIA or NECA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003588 TI - Increase of rat serum prolactin by adenosine analogs and their blockade by the methylxanthine aminophylline. AB - The administration of the stable adenosine analogs N6-[(R)-methyl-2 phenylethyl]adenosine (R-PIA; 0.01-1.0 mg/kg i.p.) and 1-(6-amino-9H-purin-9-yl) 1-deoxy-N-ethyl-beta-D-ribofuronamide (NECA; 0.01-1.0 mg/kg i.p.) caused a dose related (NECA) or biphasic (R-PIA) increases in rat serum prolactin. The S-isomer of PIA was inactive up to 4 mg/kg i.p. The methylxanthine aminophylline (10 and 30 mg/kg i.p.) antagonized the R-PIA- and NECA-induced elevation of prolactin suggesting an adenosine receptor-mediated effect. The dopaminergic agents L-dopa and bromocriptine antagonized the R-PIA and NECA-induced increase in serum prolactin. Haloperidol (a dopamine antagonist) and alpha-methyl-p-tyrosine (a catecholamine synthesis inhibitor) potentiated the R-PIA-induced effects. R-PIA and NECA did not displace 3H-haloperidol from rat striatal membranes nor effect in vitro prolactin release from rat anterior pituitary cells grown in culture. Based upon these findings it is postulated that R-PIA and NECA may be increasing prolactin secretion in part by inhibiting central dopamine release, although other mechanisms may also be operating. PMID- 3003589 TI - Does the carrier of chromaffin granules transport the protonated or the uncharged species of catecholamines? AB - By osmotic lysis in the presence of urea ghosts (60-100 nmol catecholamine/mg prot.) were prepared from chromaffin granules (4-6 mumol catecholamine/mg prot.) of the bovine medulla. In the presence of 1-300 mumol/l 3H-catecholamine and ATP Mg2+, ghosts show a net uptake of catecholamine. The net uptake is sensitive to reserpine or agents (uncouplers and ammonium) which diminish the electrochemical potential difference for protons at the granule membrane (delta p). The same uptake was found by 3H-counting or by fluorimetric measurements. At various pH values (pH 6.2-8.2) the Km and Vmax of the ATP-stimulated rate of uptake of 3H catecholamine into ghosts was determined (at 30 degrees C) to identify the species of catecholamine (protonated, uncharged, or anionic) which is the substrate for the granule carrier. The pH difference (delta pH = pHout - pHin) and the electrical potential difference (delta psi) were determined to calculate delta p under conditions of 3H-catecholamine uptake. When the pHout was increased (pH 6.2, 7.4, 8.2), the apparent Km of uptake decreased (50, 5, 1-2 mumol/l), showing a linear relation between pH and logarithm of Km. The Km was calculated for the uncharged catecholamine (with pK1 = 8.8 and pK2 = 10.0); it was nearly pH independent and amounted to about 0.2 mumol/l. The Vmax declined only in the extreme pH-range. Between pH 6.6 and 7.8 Vmax and delta p showed a slight increase from 16 to 20 nmoles/(mg prot. X min) and from 110 to 140 mV, resp. In the same pH-range the pHin inside ghosts increased from pH 5.2 to 5.7, whereas delta psi was constant (30 mV). At constant pHout (= 7.3) ammonium (0-30 mmol/l) caused an increase of pHin from 5.5 to 6.6. The increase of pHin was accompanied by an increase of Km from 5 to 20 mumol/l 3H-catecholamine and by a decrease of both Vmax and delta p from 20 to 5 nmoles/(mg prot. X min) and from 123 to 85 mV, respectively. From the dependence of the Km of uptake on pHout is concluded that the uncharged species of catecholamine is transported, whereas the dependence of Km on pHin suggests that the translocation of the catecholamine-carrier complex across the granule membrane is not the rate-limiting step of catecholamine uptake. PMID- 3003592 TI - [Return of adenovirus 21 to the Netherlands]. PMID- 3003591 TI - Islet-activating protein (pertussis toxin) diminishes alpha 2-adrenoceptor mediated effects on noradrenaline release. AB - The effect of islet-activating protein (IAP) on alpha 2-adrenoceptor mediated modulation of noradrenaline release in the rabbit hippocampus was studied. Slices of the hippocampus were incubated for 6 h with IAP, subsequently loaded with 3H noradrenaline and superfused continuously. IAP-pretreatment significantly enhanced the electrically evoked transmitter release and diminished the facilitatory effect of the alpha 2-adrenoceptor antagonist yohimbine. In addition, the inhibitory effect of the alpha 2-adrenoceptor agonist clonidine was reduced. These results provide circumstantial evidence that an inhibitory guanine nucleotide-binding protein, most probably Ni of a presynaptically located adenylate cyclase, is involved in the alpha 2-autoreceptor mediated modulation of noradrenaline release. PMID- 3003593 TI - [Sensitivity to GABA of the soma and dendrites of pyramidal cells in isolated sections of hippocampus in the rat]. AB - The depressing effect of GABA on excitation of nerve cells as well as the action of bicuculline, penicillin and thiopental on this process were examined on CA1 pyramidal neurons using rat hippocampal slices. It was found that GABA effectively and reversibly reduced the amplitude of antidromic population spike both in the region of somata and dendrites. The sensitivity of apical dendrites to GABA was greater by one order than that of somata, increasing along dendrites from their proximal to distal parts. The somata of pyramidal neurons showed strong desensitization to GABA. In distal parts of dendrites desensitization to GABA was absent. Bicuculline and penicillin antagonized the action of GABA at all investigated levels of CA1 pyramidal cells. Bicuculline blocked the effect of GABA on the somata and dendrites approximately equally. The antagonistic action of penicillin was 10 times larger in the pyramidal layer than in the region of dendrites. Thiopental intensified the depression produced by GABA. Potentiating effect of thiopental was stronger in dendrites. It is concluded that the membrane of CA1 pyramidal neurons has two types of bicuculline-sensitive GABA-receptors differing in their location (mainly on the soma or dendrites), in pharmacology and in ability to be desensitized by GABA. PMID- 3003590 TI - Further evidence for, and nature of, the facilitatory GABAergic influence on central noradrenergic transmission. AB - In order to explore the nature of the facilitatory GABAergic control of cerebral noradrenergic neurons, we have studied the effect of a variety of GABA mimetics (given systemically or injected locally into brain areas containing noradrenergic cell bodies or terminals) on several indices of noradrenaline turnover in the rat brain. Systemic administration of both direct and indirect acting GABA mimetics enhanced; 1) the pargyline induced accumulation of normetanephrine in the hypothalamus; 2) total DOPEG levels in a number of brain regions innervated by noradrenergic neurons; 3) both DOPAC and MOPEG levels in noradrenergic cell body areas (A1, A2 and A6). These effects are probably mediated by GABAA receptors as specific GABAA or mixed GABAA/GABAB agonists but not the GABAB agonist baclofen enhanced noradrenaline turnover. Interruption of noradrenergic impulse flow (by local injection of tetrodotoxin or by hemitransection) blocked the ability of progabide to increase DOPEG concentrations in the hypothalamus and cerebral cortex. Similarly, the co-administration of clonidine with progabide antagonized the progabide-induced increase in hypothalamic total DOPEG levels. Co administration of yohimbine with progabide provoked an additive effect on hypothalamic DOPEG levels at moderate but not at high doses of yohimbine. Thus, the acceleration of noradrenaline turnover induced by GABA mimetics appears to depend on ongoing activity in noradrenergic neurons and occurs via an increase in neuronal discharges. Local injection of muscimol into the nucleus accumbens or hypothalamus failed to affect DOPEG levels in these structures; similarly, local injection of muscimol into the locus coeruleus failed to modify DOPEG levels in corresponding noradrenergic projection areas. These data indicate that the GABAergic influence is not exerted via GABA receptors located on noradrenergic cell bodies or nerve endings. Furthermore, since systemically administered progabide still increased hypothalamic DOPEG levels after ibotenate-induced destruction of the hypothalamic neuronal cell bodies, a presynaptic modulation of noradrenergic neurons by local GABAergic interneurons is excluded. Chemical destruction of serotoninergic pathways or enhancement of 5-HT transmission by quipazine failed to alter the ability of progabide to increase cerebral DOPEG levels. Moreover, scopolamine or naloxone also failed to affect the progabide induced increase in cerebral DOPEG levels.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3003594 TI - [Effect of etimizol on the neuromuscular synapse of the frog]. AB - The effect of aethimizol, a substance activating the memory processes, was studied by intracellular recording at the frog cutaneous pectoris neuro-muscular preparation. Presynaptic action of aethimizol (0.5-2 mM) was found. This action consisted in the increase of miniature end-plate potentials (mEPP's) frequency and the modulation of quantal content of evoked end-plate potentials (EPP's) due to changes in binomial quantal release. parameters p and n. These effects depended on the initial level of quantal release. Aethimizol at the concentrations of 5-10 mM strongly decreased the amplitudes of mEPP's and EPP's while at the concentrations of 0.5-10 mM aethimizol increased the rising- and half-decay time of mEPP's and EPP's and half decay time of evoked end-plate currents presumably affecting the postsynaptic ionic channels properties. Aethimizol increased the time integral of EPP's. Effects of aethimizol are discussed in relation to its possible modes of action in the central neural system as a presumed unspecific connector. PMID- 3003595 TI - [Effect of thiamine on neuromuscular transmission in the frog]. AB - The action of thiamine on frog m. sartorius neuromuscular junction was investigated. Thiamine (1 X 10(-14)-1 X 10(-4) mol/l) reversibly increased miniature end-plate potential frequency, amplitude and quantal content of end plate potentials, probability of quantal release. The role of thiamine in the regulation of synaptic transmission and mechanism of its action are discussed. PMID- 3003596 TI - [Synaptic mechanisms of the effect of subcortical structures on motor neurons of the facial nerve in the cat]. AB - Peculiarities of synaptic processes in motoneurons of the facial nucleus evoked by stimulation of the head of caudate nucleus, globus pallidus and nucleus amygdalae centralis were studied in acute experiments on cats by means of intracellular recording technique. It is found that stimulation of the first two structures causes only polysynaptic excitation, while stimulation of the nucleus amygdalae centralis--mono- and polysynaptic excitation of motoneurons of the facial nucleus. Convergence of influences from above mentioned structures on the same motoneurons is shown. Possible ways and mechanisms of subcortical regulation of the activity of facial nucleus motoneurons are discussed. PMID- 3003597 TI - [Synaptic processes in red nucleus neurons in response to stimulation of the entopeduncular nucleus and the globus pallidus of the cat]. AB - Peculiarities of synaptic processes in red nucleus neurons evoked by stimulation of nucleus entopeduncularis and globus pallidus were studied in acute experiments on cats by means of intracellular recording technique. Stimulation of the structures mentioned was found to evoke mono- and polysynaptic excitation of the rubrospinal neurons. Analysis of time parameters of the recorded excitatory postsynaptic potentials suggested activation of axo-somatic and axo-dendritic synapses. Mechanisms of basal ganglia participation in integration of the red nucleus activity are discussed. PMID- 3003598 TI - [Features of the activation of neurons of the reticular formation of the brain stem in the cat to somatic stimuli of various modality]. AB - Neuronal responses of two reticular structures--nucleus reticularis pontis caudalis (RPC) and nucleus reticularis gigantocellularis (RGC)--to electrical nociceptive stimulation of the internal intercostal nerve and non-nociceptive, tactile stimulation of the skin in the same somatic area have been studied in chloralose anesthetized cats. It was shown that the mean number of action potentials in responses of RPC-neurons to tactile stimulation exceeded that for FGC-neurons by 20-150%. On the opposite, the average number of action potentials in RPC-neurons in response to nerve stimulation was lower by 15-54% than those of bulbar neurons. Data obtained indicate the existence of spatial rostro-caudal differentiation of somatosensory modalities in the reticular brainstem formation. PMID- 3003599 TI - [Activity of isolated interneurons of the pedal ganglia of the pteropodal mollusk]. AB - Interneurons from pedal ganglia of marine mollusc Clione limacina continued their rhythmical discharges for many hours after isolation. A discharge frequency increased with depolarization of neurons and decreased with hyperpolarization. It is concluded that the endogenous activity of interneurons underlies generation of the locomotor pattern in mollusc pedal ganglia. PMID- 3003600 TI - [Baclofen inhibition of synaptic transmission in glomeruli of the olfactory bulb of the frog]. AB - The application of (+/-) baclofen (10(-5) M) to the frog olfactory bulb greatly reduced postsynaptic components of the orthodromic potential, but the antidromic potential was not affected. The effect was not antagonized by bicuculline (10(-4) 10(-3) M). The results suggest that bicuculline-insensitive GABAB-receptors are located on the primary olfactory afferents and may realize presynaptic inhibition in the olfactory bulb. PMID- 3003601 TI - Transfection of BLV containing DNA into NIH 3T3 cells. AB - Cell DNA isolated from bovine leukosis virus (BLV) productive cell clones was transfected into the NIH3T3 cells. DNA from some cell clones was able to transform NIH3T3 cells. The transformed cells were cloned, and in 4 cell clones out of 33 bovine leukosis virus specific sequences were detected by hybridization with labeled BLV probe. According to the restriction analysis the BLV sequences were incomplete, they were rearranged, deleted, or both. The DNA from NIH3T3 transformants with BLV sequences was able to transform in the second round transfection experiments NIH3T3 cells again, but in these transformants BLV specific sequences were not detected. Cell DNA from sheep tumors induced by BLV was able to transform the NIH3T3 cells too, but BLV specific sequences were not present in the transformants. It appears that BLV specific sequences are not required for NIH3T3 cell transformation. PMID- 3003602 TI - Pulmonary tumour embolism in a case of adenoid cystic carcinoma. PMID- 3003604 TI - Renal alpha-1-adrenoreceptors in rats with obstructive jaundice. AB - Alpha 1-Adrenoreceptor affinity constants (KD) and receptor numbers (Bmax) were determined in the kidneys of 3-day-old bile-duct-ligated (BDL) jaundiced rats using 3H-prazosin. The results were compared to 3-day-old pair-fed and nonpair fed sham-operated rats as well as nonoperated rats as controls. Abdominal surgery (sham and BDL) resulted in a tendency towards a decrease in KD in all three groups of rats compared to nonoperated controls. The Bmax was also increased in the sham-operated groups compared to the nonoperated controls. In contrast, the tendency for a rise in the Bmax in the BDL group was significantly smaller than the rise seen in the two sham-operated groups. In summary, obstructive jaundice suppresses the normal renal alpha 1-adrenoreceptor response to abdominal surgery in the rat. PMID- 3003603 TI - Enhancement of 24,25-dihydroxyvitamin D levels in patients treated with continuous ambulatory peritoneal dialysis. AB - We measured 24,25-dihydroxyvitamin D [24,25(OH)2D] levels in patients treated with chronic ambulatory peritoneal dialysis (CAPD), before and after receiving vitamin D2 or 1 alpha-hydroxyvitamin D3 (1 alpha-OH-D3). Vitamin D2 administration led to an increase in 25-hydroxyvitamin D (25-OH-D) and a concomitant rise in 24,25(OH)2D. No change was observed in 1,25-dihydroxyvitamin D [1,25(OH)2D]. Administration of 1 alpha-OH-D3 resulted in an increase in 1,25(OH)2D3, and a concomitant rise in 24,25(OH)2D, but no change in 25-OH-D3. Thus, 24,25(OH)2D levels may be increased in CAPD patients by raising 25-OH-D levels, or by raising 1,25(OH)2D3 levels. Since the latter enhances specifically the renal 24-hydroxylase enzyme, we conclude that this enzyme is present in CAPD patients with kidneys in situ, and may be stimulated by adequate 1,25(OH)2D3 levels. Thus, administration of 1 alpha-OH-D3 to CAPD patients with kidneys in situ seems to be sufficient to obtain normal levels of 1,25(OH)2D3 and 24,25(OH)2D3. However, anephric patients require vitamin D2 in addition as a source of 25-OH-D, the substrate for extrarenal production of 24,25(OH)2D. PMID- 3003605 TI - [So-called glomus jugulare tumors. General report]. PMID- 3003606 TI - [Paraganglioma of the jugulo-tympanic glomus. Anatomo-pathological aspects]. AB - 15 extensive glomus jugulare tumors have been studied. Optic and ultrastructural data were very similar to those which have been reported in the literature. They possessed a local aggressiveness, demonstrated by progressive spreading in soft tissues and bone, intravascular digitations and destruction of cranial or sympathetic nerves. Dura-mater was usually preserved and prevented extension close to the brain stem. In this group, no functional activity of catecholamine secreting tumor nor metastasis proving a malignant form were observed. PMID- 3003607 TI - [Anatomical bases of the surgical approach to the posterior foramen]. PMID- 3003608 TI - [Tympano-jugular paraganglioma]. PMID- 3003609 TI - [Diagnostic and therapeutic angiography in the evaluation and treatment of glomus jugulare tumors. Apropos of 32 cases]. AB - This report is based on 32 cases of jugular paraganglioma treated by embolization, of which 7 had cervical and/or intrajugular extensions, five a giant extension involving mainly the carotid canal and one excretory activity. Aims and methods of diagnostic and therapeutic angiography are envisaged as well as results in the three angiographic types encountered: tumors vascularized by the external carotid artery only, those supplied by the internal (and external) carotid arteries and those fed by the vertebral artery or even the three arterial axes. PMID- 3003610 TI - [The infratemporal approach to glomus jugulare tumors]. PMID- 3003611 TI - [Glomus jugulare tumors. Possibilities of radiotherapy]. AB - From 1964 to 1981, twelve patients with glomus jugulare tumors involving bony lesions of base of skull were treated by high energy radiation. Ten patients presented cranial nerve pairs paralysis. Conventionally fractionated doses of 45 to 60 Gy were applied, and 11 of the 12 patients were alive and clinically stable after follow up for between 3 and 20 years. One patient died of an intercurrent infection after 16 years. Strict irradiation technique is essential to avoid principally neurologic complications. Favorable results after radiotherapy were obtained in extensive tympano-jugular forms. PMID- 3003614 TI - Regulation of multiple forms of cyclic nucleotide phosphodiesterase from bovine hypothalamus: new factors modulating enzyme activity. AB - Studies of bovine hypothalamic cyclic nucleotide phosphodiesterase (PDE) indicate the presence of several peaks of PDE activity, distinguishable by DEAE-cellulose column chromatography, displaying different substrate specificities, kinetic behavior, and regulatory properties. Evidence is presented that chromatographically separated forms of PDE activity are subject to control by Ca2+-calmodulin, cyclic nucleotides, limited proteolysis, reagents affecting sulfhydryl groups, and neurohormone "C"--one of several new cardioactive compounds isolated from hypothalamic magnocellular nuclei of animals--in a complex substrate-specific and concentration-dependent manner. Of particular interest is the finding that each of the forms of cGMP PDE, being Ca2+/calmodulin dependent, possesses sensitivity to activation by cAMP, especially under conditions favoring the oxidation of thiol groups of PDE, resulting in a loss in responsiveness of the enzyme to the activation by calmodulin. This effect appears to be relatively stable but readily reversible by sulfhydryl reducing reagents, which restore both the cGMP PDE sensitivity to competitive inhibition by cAMP and the responsiveness of the enzyme to activation by calmodulin. A reinterpretation of the regulatory properties of multiple forms of PDE is proposed. PMID- 3003613 TI - Phosphoethanolamine and phosphocholine transferases in gray matter and white matter from developing rat brain. AB - We have studied the activities of 2',3'-cyclic nucleotide 3'-phosphohydrolase, 1,2-diacylglycerol: CDPethanolamine phosphoethanolamine transferase (EC 2.7.8.1), and 1,2-diacylglycerol: CDPcholine phosphocholine transferase (EC 2.7.8.2) in developing rat brain gray matter and white matter. The specific activity of cyclic nucleotide phosphohydrolase was 5-8 fold higher in white matter than in gray matter at all ages. No significant changes were observed during development. The specific activity of phosphocholine transferase was 2 to 3 fold higher than phosphoethanolamine transferase at all ages both in gray and white matter. Both phosphocholine transferase and phosphoethanolamine transferase increased more than 2 fold in specific activity between 14 and 90 days of age. The total activity of phosphocholine transferase also showed an increase during development. The apparent Km values for nucleotides and dicaprin were similar in gray matter and white matter. Except for low Km values for nucleotides at 14 days of age, no significant changes were observed during development. Changes in rates of glycerophospholipid synthesis may be partly due to the specific activities of these enzymes but are also determined by the quantities of substrates and inhibitors and by affinities for the substrates. PMID- 3003612 TI - Conformational changes in muscarinic receptors may produce diminished cholinergic neurotransmission and memory deficits in aged rats. AB - Both clinical and laboratory studies suggest that age-related memory deficits may be due, at least in part, to disturbances in muscarinic acetylcholine (mAChR) receptors. In order to further evaluate this premise, the present studies examined the electrophysiological responses rates of hippocampal pyramidal cells to iontophoretically applied ACh in young, middle-age and aged animals. The relationship between age and muscarinic agonist and antagonist binding in the hippocampus was also examined. In addition, possible age-related changes in receptor-effector coupling were assessed by determining calmodulin levels and the activities of phospholipid methyl-transferase I and II. Analysis of electrophysiological data showed selective age-related decrements in the ability of ACh to alter burst rate but not simple spike rate. These age-related decreases in the efficacy of ACh to increase burst rate were not paralleled by decreases in mAChR density as assessed by 3H-QNB binding, but they were temporally paralleled by age-related changes in the ability of oxotremorine to inhibit 3H-QNB binding. In the young animals, the resultant Hill coefficients derived from these analyses approached 1, while in the middle and old aged animals, the Hill coefficients deviated significantly from 1, indicating the possible existence of 2 or more receptor states with differential affinity for oxotremorine in the 2 older age groups. When carbamylcholine was used to inhibit 3H-QNB, these complex binding patterns were seen even in the young, since carbamylcholine induces conformational/orientational changes in the mAChR while oxotremorine does not.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003615 TI - Cortisol effect on (Na+ + K+)-stimulated ATPase activity and on bilayer fluidity of dog brain synaptosomal plasma membranes. AB - The binding of [14C]cortisol into dog brain synaptosomal plasma membranes (SPM) follows an exponential path described by the general formula y = a.ebx. The specific activity of the SPM-bound (Na+ + K+)-stimulated ATPase was linearly increased at different concentrations of cortisol. Changes in the allosteric properties of (Na+ + K+)-stimulated ATPase by fluoride (F-) (i.e. changes of Hill coefficients) indicate that cortisol increases the membrane fluidity. The fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene-labeled SPM decreased in cortisol treated SPM compared to untreated (control) SPM, which is consistent with a general increase in membrane fluidity. This increase of fluidity by cortisol may play a role in the physiological effects of this hormone in the brain. PMID- 3003616 TI - Effects of combined administration of L-tryptophan and tricyclic antidepressants on alpha 2- and beta-adrenoceptors and monoamine levels in rat brain. AB - In order to test whether co-administration of a serotonin precursor with antidepressant drugs could potentiate the effects of the antidepressants on monoamines or adrenoceptors in rat brain, L-tryptophan (20 mg/kg) was administered to rats daily for 7 or 15 days, either alone or in combination with desipramine (10 mg/kg) or amitriptyline (10 mg/kg). After treatment with L tryptophan for 7 days, increases were observed in rat hypothalamic and frontal cortex 5-hydroxy-3-indoleacetic acid levels as well as in hypothalamic dopamine and nucleus accumbens 3,4-dihydroxyphenylacetic acid levels. After 15 days, hippocampal beta-adrenoceptor density was found to be decreased. There was no evidence of potentiation of desipramine or amitriptyline action when L-tryptophan was administered in combination with the antidepressants. On the contrary, the antidepressants appeared to interact with L-tryptophan to reduce its effects. PMID- 3003618 TI - [ACTH dependent Cushing's syndrome with an empty sella turcica: report of three cases with emphasis on diagnostic value of selective venous sampling]. AB - Three cases of ACTH dependent Cushing's syndrome are reported with emphasis on diagnostic value of selective venous sampling. Case 1. A 44-year-old female was admitted to our hospital with clinical diagnosis of Cushing's disease. Endocrinological examination revealed typical data of Cushing's disease. High resolution CT scan showed an empty sella turcica, and a chest film showed multi cystic lesion in the left lower lung field. In the first trial of selective venous sampling, central to peripheral ACTH ratio (C/P ratio) was high at the superior vena cava. So, an ectopic ACTH producing lung tumor was strongly suspected. Further examinations for lung tumor were performed, and finally showed lung cryptococcosis. Therefore, selective venous sampling was performed again, and pituitary ACTH dependency was diagnosed. An eccentric pituitary microadenoma was successfully removed by transsphenoidal surgery. Case 2. A 54-year-old female was admitted to our hospital with clinical diagnosis of Cushing's disease. In the endocrinological examinations plasma ACTH was not respond to provocation of LVP or CRF. In selective venous sampling, C/P ratios of ACTH were not greater than 2.0 at any sampling site. Further examinations showed lung tumor in the lower lobe of left lung. This tumor was surgically proved to be an ectopic ACTH producing lung carcinoid. Case 3. A 24-year-old female was admitted to our hospital for the purpose of further examination of Cushing's disease. Three years previously exploratory transsphenoidal surgery demonstrated an empty sella without adenoma. She received postoperative radiation therapy with a dose of 5000 rad. Endocrinological examination showed typical data due to Cushing's disease.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003617 TI - [The pathomechanism underlying ischemic brain edema: the role of Na+, K+-ATPase of the brain microvessels]. AB - In the present study, the anti-edema effect of AVS [1,2-bis (nicotineamide) propane] was evaluated using the cat MCA occlusion model with or without recirculation. In the prolonged ischemia (PI) group, cortical edema as assessed by the changes in specific gravity, developed in those cortical areas where the mean 1-CBF was less than 25-30 ml/100 g/min during MCA occlusion (4 hours). In the recirculation group (2 hours' ischemia followed by 2 hours' recirculation: RC group), the ischemic threshold for edema development was almost the same as in the PI group. In both groups, the drop in cortical specific gravity was significantly suppressed by AVS. Regarding the time-course of 1-CBF, there was no difference between the PI-AVS-treated and PI-saline-treated groups. In the RC group, however, the postischemic hypoperfusion was significantly ameliorated by AVS. Based on the present and previous data showing the antiedema effect of AVS, the mechanism of action of AVS was discussed in relation to the pathomechanism underlying ischemic brain edema. Our new concept of ischemic brain edema is briefly stated below. Related in vitro studies have shown the followings: (i) the influx of sodium not of proteins is the principal cause of ischemic brain edema: (ii) the eicosanoid synthetic capacity of the brain microvessel (MV) is increased simultaneous to edema development (iii) an elevation in the level of hydroperoxides enhances the activities of Na+, K+-ATPase as well as the arachidonate cascade of MV. These data suggest that free fatty acids and free radicals liberated following cerebral ischemia stimulate the activity of the MV Na+, K+-ATPase, which results in increased sodium influx across the BBB. AVS was shown to scavenge hydroxyl radicals and to inhibit the stimulatory effects of a lipid hydroperoxide (15-HPAA) on the activities of Na+, K+-ATPase and the arachidonate cascade of the MV. These actions of AVS may be linked to its antiedema effect. PMID- 3003619 TI - Brain necrosis after radiotherapy for primary intracerebral tumor. AB - Radiotherapy is a standard postoperative treatment for cerebral glioma. We have observed the onset of symptoms related to brain necrosis, as opposed to recurrent tumor, in surviving patients. This has been manifest as dementia with a computed tomographic pattern of low density in the frontal lobe uninvolved with tumor, but within the field of radiotherapy. Two patients presented with mass lesions also unrelated to recurrent tumor. We question the necessity of full brain irradiation and suggest that radiotherapy techniques be altered to target the tumor and not encompass the entire brain. PMID- 3003620 TI - Effects of valproic acid in cultured mammalian neurons. AB - Valproic acid (VPA) has been postulated to exert its anticonvulsant effects by interaction with the postsynaptic GABA receptor. To test that hypothesis, we applied VPA in clinically appropriate concentrations to cortical neurons in dissociated cell culture. VPA did not enhance the postsynaptic effect of GABA, but did decrease the generation of sodium-dependent action potentials. VPA may exert anticonvulsant effects by inhibiting spike generation, independent of the GABAergic system. PMID- 3003621 TI - Familial amyloidotic polyneuropathy diagnosed by cloned human prealbumin cDNA. AB - A diagnosis of familial amyloidotic polyneuropathy (FAP) can be made by use of restriction endonuclease NsiI, a cloned human prealbumin cDNA and Southern blot procedures. Digests of DNAs from 10 disease-free individuals showed two bands (6.6 kb and 3.2 kb) complementary to a human prealbumin cDNA, whereas digests from 11 individuals with FAP exhibited two additional bands (5.1 kb and 1.5 kb). We interpret these changes in pattern to be the result of a restriction site for NsiI located in the altered codon and associated with the mutant prealbumin gene. All these individuals with FAP were heterozygous for the prealbumin gene, carrying one normal and one mutant gene. PMID- 3003622 TI - An additional pilomotor seizure. PMID- 3003623 TI - [Acquired immunodeficiency syndrome. Review of the literature. Apropos of a pre AIDS case in a heroin addict]. AB - The Authors, after a review of the latest reports on ethiology, pathology and clinical aspects of acquired immunodeficiency syndrome (AIDS) and of linphadenopathyc syndrome (LAS or pre-AIDS), describe a case of pre-AIDS syndrome in a heroin addict characterized by typical symptoms, histological, serological and immunological patterns. PMID- 3003624 TI - Stimulation of noradrenaline synthesis by the calcium ionophore A23187 and its modulation by presynaptic receptors. AB - Noradrenaline (NA) synthesis in rat hippocampal synaptosomes is increased by calcium ionophore, A23187, in the presence of calcium. Activation of presynaptic muscarinic, gamma-aminobutyric acidB (GABAB) and alpha 2-adrenergic receptors causes inhibition of the ionophore-stimulated synthesis. The results suggest that different mechanisms mediate the depression by presynaptic receptors of NA release and NA synthesis. PMID- 3003625 TI - Long-lasting proconflict effect induced by chronic administration of the beta carboline derivative FG 7142. AB - In this study, the effect of the chronic administration of the benzodiazepine (BZD) receptor ligand FG 7142 on the rat conflict test was examined. Rats chronically treated with FG 7142 (15 mg/kg, i.p. twice a day for 10 days) had an enhanced sensitivity to punishment at 4 and 15 days after the last treatment. This 'proconflict' effect was prevented by the concurrent administration of the BZD antagonist Ro 15-1788. The hypothesis that the long-lasting proconflict effect of chronic FG 7142 administration is the consequence of a persistent down regulation of the GABAergic transmission is discussed. PMID- 3003626 TI - Pharmacological evidence for a protective role of the endogenous opioid system on electroshock-induced seizures in the mouse. AB - Acute administration of morphine produced a protective effect on electroshock (ECS)-induced seizures in mice, while naloxone and naltrexone decreased ECS seizure threshold. Chronic morphine administration in mice resulted in a decrease of ECS-induced seizure threshold evident within 24 h following the end of drug treatment. This effect disappeared 5 days after the end of chronic morphine treatment. Moreover, tolerance to the anticonvulsant effect of morphine was evident in mice chronically treated with morphine and subjected to ECS 30 min after the last injection of the drug, as well as in mice subjected to ECS 24 h after the end of chronic treatment. PMID- 3003627 TI - Enhancement of transmission at the frog neuromuscular junction by adenosine deaminase: evidence for an inhibitory role of endogenous adenosine on neuromuscular transmission. AB - Adenosine deaminase reversibly increased the amplitude and the quantum content of the end-plate potentials (EPPs) recorded from superficial muscle fibers of frog sartorius preparations in which twitches have been prevented with high-magnesium solutions. Adenosine deaminase prevented the inhibitory effect of exogenously applied adenosine but not that of 2-chloroadenosine on the amplitude of EPPs. The effect of adenosine deaminase was abolished by erythro-9(2-hydroxy-3 nonyl)adenine (EHNA). The results suggest that endogenous adenosine exerts an inhibitory 'tone' over neuromuscular transmission. PMID- 3003628 TI - Hexacarbon neuropathy: tracking a toxin. AB - This paper will present the progression of hexacarbon studies which followed the investigation of circumstances surrounding the occurrence of peripheral neuropathy in an occupational setting. The identification of methyl n-butyl ketone as the neurotoxin led to systematic studies of the biotransformation and mechanism of neurotoxicity. A consistent line of evidence showed that the neurotoxic potential of hexacarbons is directly related to the formation of the gamma-diketone metabolite, 2,5-hexanedione. The precise nature of the chemical interaction at the neurofilament is the subject of continuing investigation. PMID- 3003629 TI - The morphology of carbon disulfide neurotoxicity. AB - The morphology of carbon disulfide induced peripheral neuropathy was studied in rats exposed to three concentrations of carbon disulfide by inhalation for 90 days. Rats exposed to 800 ppm developed neurofilamentous axonal swellings in the distal portions of long fibers, including the dorsal ascending sensory and corticospinal tracts of the spinal cord. In peripheral nerve the predominant effect was seen at the level of the posterior tibial nerve. Teased fiber preparations of the muscular branch of the posterior tibial nerve showed numerous paranodal and internodal swellings as well as Wallerian degeneration. Ultrastructurally the swellings were characterized by neurofilament accumulations, decreased numbers of microtubules and thin myelin. Other features included segregation of axoplasmic organelles and cytoskeletal components, intrusion of Schwann cell processes into the axoplasm, Schwann cells with increased cytoplasmic contents, and Schwann cell proliferation around many swollen and demyelinated axons. These features draw important parallels between the morphology of carbon disulfide neuropathy and the neurofilamentous neuropathies induced by hexacarbons and beta,beta' iminodipropionitrile (IDPN). PMID- 3003630 TI - Incidence of hepatocellular carcinoma in New Zealand, 1974-78: ethnic, sex and geographical differences. AB - The incidence of hepatocellular carcinoma in New Zealand is examined for the period 1974-78. There was a significant north-south gradient for both Maori and non-Maori incidence rates, with the rates in the northern half of the North Island being approximately double those in the South Island. Within each region, Maori rates were approximately three times those of non-Maoris and incidence rates of males were approximately three times those of females. Incidence was particularly high in the Bay of Plenty, Waiapu and Otago hospital board districts. These patterns are generally consistent with available information concerning the distribution of hepatitis B surface antigen (HBsAg) carriage in New Zealand and suggest that HBsAg carriage is likely to be a major risk factor for hepatocellular carcinoma in New Zealand, as it is in other countries. PMID- 3003632 TI - Hepatocellular carcinoma. PMID- 3003631 TI - Enalapril and cough: case report. PMID- 3003633 TI - Fetal and maternal humoral immune response to cytomegalovirus infection. AB - The relationship between cytomegalovirus titers and immunoglobulin (Ig) levels in 514 paired maternal-fetal sera at term was studied. Positive cytomegalovirus titers were found in 192 (37.4%) maternal sera and 138 (26.8%) fetal sera. Fetal sera with positive cytomegalovirus titers had decreased IgM levels, and maternal sera with positive cytomegalovirus titers had increased IgG levels. There was a negative correlation between cytomegalovirus titer level and IgM concentration in fetal serum and a positive correlation between cytomegalovirus titers levels and IgG concentration in maternal serum in individual samples. After removal of IgG from samples with cytomegalovirus positive titers by column chromatography, 17 maternal and 22 fetal sera remained cytomegalovirus positive. PMID- 3003634 TI - Preterm labor associated with subclinical amniotic fluid infection and with bacterial vaginosis. AB - Maternal genital infection, particularly subclinical amniotic fluid infection, may cause preterm labor and a premature delivery. The prevalence of subclinical amniotic fluid infection was studied in 54 consecutive afebrile women in preterm labor with singleton gestations and intact fetal membranes. Microorganisms were recovered from the amniotic fluid by transabdominal amniocentesis in 13 (24%) of 54 patients. Bacteria or Candida albicans were recovered from six (11%), and genital mycoplasmas from seven (13%). Compared with women with sterile amniotic fluid, patients whose amniotic fluid contained bacteria or Candida organisms had a shorter interval from onset of preterm labor until delivery (0.6 versus 34.3 days, P less than .01), were less responsive to tocolytic therapy (0 versus 81% success rate, P less than .005), and more frequently developed subsequent intrapartum fever (83 versus 2.4%, P less than .005). In contrast, women whose amniotic fluid contained genital mycoplasmas did not differ in these parameters from those with sterile fluid. Also compared was cervical-vaginal infection among these patients in preterm labor with matched control subjects without preterm labor. In this analysis, bacterial vaginosis was identified in 43% of patients with and 14% of women without preterm labor (P = .02), yielding a relative risk of preterm labor for patients with bacterial vaginosis of 3.8. These data underscore the importance of amniotic fluid bacterial infections in preterm labor and premature delivery, and suggest that bacterial vaginosis is associated with prematurity. PMID- 3003636 TI - Estrogen replacement therapy in the patient treated for endometrial cancer. AB - Adenocarcinoma of the endometrium is considered to be an estrogen-dependent neoplasia and as such, hormone replacement therapy is said to be contraindicated. The authors are unaware of any data to substantiate that statement. Patients, who had completed their therapy for stage I carcinoma of the endometrium, were placed on estrogen hormone replacement therapy in a nonrandomized fashion. Between 1975 and 1980, 221 patients with stage I adenocarcinoma of the endometrium were managed at the Duke University Medical Center. Forty-seven patients received estrogen after their cancer therapy, whereas 174 patients did not. Risk factors for recurrence were similar between the two groups. After controlling for these known risk factors, the estimated distributions of time to recurrence for the two groups were significantly different (P less than .05), with the estrogen group experiencing longer disease-free survival. The history of endometrial cancer does not appear to be a contraindication to hormone replacement therapy in patients with stage I disease. PMID- 3003635 TI - Cervicovaginal peroxidases: markers of the fertile period. AB - The specific activity of guaiacol peroxidase was measured daily in human cervical mucus, vaginal fluids, and saliva during 45 cycles in 31 women. Also determined were basal body temperatures and serum hormones (luteinizing hormone [LH], estradiol, progesterone). The guaiacol peroxidase was extracted with 0.5 M CaCl2 and thus may be a different peroxidase from that obtained by noncalcium extraction procedures. The guaiacol peroxidase specific activity did not vary in the saliva during the cycle but fell sharply in the cervical mucus and vaginal fluid four to five days before the ovulation time, estimated by the LH peak, and rose again one to two days after ovulation. Anovulatory cycles did not show the midcycle drop in guaiacol peroxidase. Growth curve analysis gave excellent fitting of the guaiacol peroxidase data to a polynominal model. These data suggest that cervicovaginal guaiacol peroxidase may be clinically useful in detecting the fertile period for population control and for infertility treatment. PMID- 3003638 TI - Vulvar pain and dyspareunia due to glomus tumor. AB - Reported is a patient with long standing vulvar pain and severe introital dyspareunia whose symptoms were cured by removal of a glomus tumor of the vulva. PMID- 3003637 TI - Pregnancy prophylaxis: parenteral postcoital estrogen. AB - A two-dose regimen of intravenous conjugated estrogen as a postcoital interceptive was studied retrospectively. One hundred eighteen women who presented themselves at the Louisville General Hospital emergency room with a history of involuntary intercourse received 50 mg of intravenous conjugated estrogen at the conclusion of their initial medical evaluation and a second 50-mg injection approximately 24 hours later. Three pregnancies occurred among the 92 women who were seen again 30 days or more after treatment. These women reported other episodes of unprotected intercourse in the treatment cycle. Twenty-six of the women treated were lost to follow-up. Complaints of side effects were uncommon. In this series, intravenous conjugated estrogen appears to have been acceptable management for women who had been raped if no other unprotected intercourse had taken place in the treatment cycle. PMID- 3003639 TI - Glomus tumor of the vulva: a case report. AB - Glomus tumors are benign neoplasms associated with intense pain localizable to the tumor nodule. A case of a glomus tumor of the vulva is presented. Simple resection is curative. PMID- 3003640 TI - Verrucous carcinoma of the vulva associated with an unusual type 6 human papillomavirus. AB - An unusually rapid growing vulvar verrucous carcinoma found associated with human papillomavirus type 6 is described. Both human papillomavirus structural antigens and deoxyribonucleic acid (DNA) sequences were detected in the tumor biopsy. The human papillomavirus genome was isolated and compared with the genome of human papillomavirus type 6b, which was cloned from an anogenital wart. The two genomes were extremely homologous in genomic organization and DNA sequence, except for a region on the viral genome containing the virus control elements. A discussion of this observation and how it could relate to the importance of viral subtypes follows. PMID- 3003641 TI - Bilateral and synchronous squamous cell carcinoma of the cervix in a patient with uterus didelphys. AB - Reported herein is the case of a 30-year-old white woman who had a congenitally double uterus with two cervixes (uterus didelphys). Each cervix was diffusely involved by squamous cell carcinoma in situ that had progressed to a microinvasive stage of the same depth (0.8 mm) on each side. This coincidence was the more remarkable because it had taken place synchronously. A transmissible coital factor of a viral nature, ie, human papillomavirus infection, was suggested by the presence of koilocytotic atypia in both uterine cervixes. PMID- 3003642 TI - Neuroendocrine carcinoma of the uterine cervix complicated by pregnancy: case report and review of the literature. AB - A case report of neuroendocrine carcinoma of the cervix complicating pregnancy is presented. This represents the fourth reported case. Clinical staging was FIGO IB, and treatment consisted of radical surgery and aggressive chemotherapy. Electron microscopy confirmed the diagnosis of neuroendocrine carcinoma. The pregnancy-associated cases and a review of neuroendocrine cervical carcinoma are discussed. PMID- 3003643 TI - Androblastomas and thyroid disease in postmenopausal sisters. AB - Androblastomas are virilizing ovarian tumors found predominantly in premenopausal women. Reported are two postmenopausal sisters with androblastomas, the first such occurrence in the literature. An association has been proposed between the familial occurrence of these tumors and thyroid adenomas or nontoxic goiter. Of the two patients studied, one had a history of Graves disease and the other had thyroid cancer. The authors conclude that the associated thyroid pathology in the families described and in the studied patients is inconsistent and does not represent a unique multiple endocrine neoplasia syndrome. PMID- 3003644 TI - [Effect of N-acetylmuramidase on the lactate production of Streptococcus mutans]. PMID- 3003646 TI - [Malignant testicular tumors. Recommendations on diagnosis, therapy and aftercare]. AB - This manual contains recommendations for early detection, diagnostic procedures, treatment modalities and follow-up in testicular cancer as a working formulation set up by the "Testicular cancer"-group of the Tumorcenter Munich. The different histological classifications and the tumor staging are described as well as treatment strategies dependent on tumor stage and histology. For nonseminomatous testicular cancer the surgical approach as primary or secondary intervention and the indication for adjuvant, curative or palliative chemotherapy are discussed, followed by details on regimens, application and side effects of chemotherapy. For seminomas particular recommendations on radiation therapy in non-bulky disease and on chemotherapy in bulky seminoma are given. For follow-up special follow-up-programs for periodical patient examination are presented. PMID- 3003645 TI - [Diagnostic and prognostic value of determination of antibodies to herpes simplex virus in tears in ophthalmoherpes]. PMID- 3003647 TI - Bovine leukaemia virus and enzootic bovine leukosis. AB - Infection of bovines with bovine leukaemia virus (BLV) manifests itself in either of two ways: 30-70% of carriers develop persistent lymphocytosis (PL), with the viral genome integrated at a large number of different sites in the DNA of the affected B-lymphocytes, without causing any chromosomal abnormalities. Only 0,1 10% of carriers develop lymphoid tumours, which also consist of B-lymphocytes. In contrast to PL, however, they are of mono- or oligoclonal origin in terms of the integration site, which is characteristic for each tumour. All cells contain one or more copies of the viral genome, chromosomal aberrations are common and if deletions are present they are invariably found in the 5'-half of the virus DNA sequence. In both types of affected cells transcription is repressed in vivo, but transient virus production can be induced in vitro and detected by means of syncytia induction or haemagglutination. In vivo production of virus in some unknown cell is suggested by the presence of high antibody titres in infected animals, especially against the envelope glycoprotein gp51. This can be detected by various techniques such as immunodiffusion, radioimmune assay or ELISA. Monoclonal antibodies against gp51 have revealed 8 epitopes, 3 of which are recognized by neutralizing antibodies and one by a cytolytic antibody. The BLV genome, about 9 kb in size, have been cloned, and some of the information obtained on its molecular structure and function is discussed. It codes for at least 4 non-glycosylated and 2 glycoproteins. Of special interest is the recently discovered serological relationship between some of the non-glycosylated proteins and those of the human T-cell leukaemia virus. The functional role of BLV in leukaemogenesis is largely unknown. The presence of the viral genome seems to be necessary for the maintenance of the transformed state, but not its continuous expression nor an LTR-mediated promotion of transcription of cellular genes. No oncogene is carried by the virus. Although bovine leukosis is not of major economic importance, its eradication is desirable and feasible in countries with a relatively low incidence, by means of testing and elimination. For endemic situations vaccination would be preferable, and distinct possibilities exist for the development of gp51 based vaccines. PMID- 3003648 TI - Biotechnology, viral oncogenesis and jaagsiekte. AB - A brief description is given of the discovery of retroviral and cellular oncogenes and of their putative role in oncogenesis. Attempts to apply the biotechnological techniques that were so successful in the study of other retroviruses to the newly-discovered jaagsiekte retrovirus are briefly reviewed. PMID- 3003649 TI - The use of recombinant DNA technology for the development of a bluetongue virus subunit vaccine. AB - The double-stranded RNA gene coding for the surface antigen responsible for inducing neutralising antibodies has been isolated, converted to DNA, and cloned in the plasmid pBR322. So far, only plasmids containing inserts smaller than the gene have been obtained. Possible strategies for the development of a bluetongue virus subunit vaccine are discussed. PMID- 3003650 TI - Studies on bovine herpesviruses. Part 1. Isolation and characterization of viruses isolated from the genital tract of cattle. AB - Herpesviruses, previously isolated from cattle (Theodoridis, 1978), were further studied and provisionally placed in the bovid herpesvirus 4 (BHV-4) group. Major differences were found between IBR-IPV (BHV-1) and BHV-4 virus strains. In MDBK cells, all BHV-4 strains started growing at the edges of the culture, the process progressing slowly until destruction of the cells was complete by the 10th day. BHV-4 strains failed to induce neutralizing antibodies in cattle, goats and rabbits. Only the addition of mineral oil adjuvant induced neutralizing and complement fixing antibodies in goats. BHV-1 strains, in contrast, produced very potent antisera in all these systems. Cross-neutralization tests indicated the existence of 2 distinct serological groups representing BHV-1 and BHV-4. The BHV 4 strains appear to be interrelated and they could not be grouped. A BHV-1 strain showed fixation of complement with the antisera of 6 BHV-4 strains. Electron micrographs showed an accumulation of nucleocapsids in the cytoplasm and an early release of virus particles due to cell destruction. Variation in incubation temperature had a significant effect on the particle formation. At lower temperatures, the number of enveloped particles in the cytoplasm increased. On the basis of the characteristics uncovered in this study, it is possible that all the BHV-4 strains represent one and the same virus which has undergone certain biological changes, thus illustrating a phenomenon which appears to be a characteristic of the herpesviruses. PMID- 3003651 TI - Augmentation of the articular tubercle in treatment of chronic recurrent temporomandibular joint luxations. A preliminary case report. AB - Chronic recurrent luxation of the temporomandibular joint requires surgical intervention in certain cases. Following a review of the literature the authors describe their own method for augmentation of the articular tubercle by implantation of hydroxyapatite. A case report shows a good result after 9 months. PMID- 3003653 TI - [AIDS--a nightmare in our time]. PMID- 3003652 TI - The chondroid syringoma (mixed tumor of skin). Report of a case in the upper lip. AB - Sweat gland tumors are uncommon, and yet the chondroid syringoma (mixed tumor of the skin) appears most frequently on the faces of adults. Clinicians diagnosing and treating diseases of the head and neck should be aware of these tumors. A case of the chondroid syringoma in the upper lip is reported; the clinical features, histology, treatment, and follow-up are discussed. PMID- 3003654 TI - Nasopharyngeal angiofibroma in two young children. PMID- 3003655 TI - ANA urges nurses to read CDC guidelines on AIDS. PMID- 3003656 TI - Increases in meal viscosity caused by addition of guar gum decrease postprandiale acidity and rate of emptying of gastric contents in healthy subjects. PMID- 3003658 TI - [Use of permeabilized cells in the study of inhibitory products of herpes virus replication]. AB - Cells made permeable by exposure to lysolecithin following infection by HSV-1 synthesize DNA (in greater amounts than non-infected cells) in the presence of the four deoxyribonucleoside-triphosphates (dNTPs) : dATP, dCTP, dGTP, and dTTP. DNA synthesis also occurs if dTTP is replaced by dT or dTMP, indicating activity of enzymes such as thymidine kinase, thymidylate kinase, deoxyribonucleoside diphosphate kinase and ADN polymerase. Examination of DNA synthesis in permeabilized cells enables detection of antiviral activity of agents incapable of penetrating into intact cells and therefore ineffective in cell cultures. No detectable protein-tyrosine kinase activity was found in HSV-1 infected cells. PMID- 3003657 TI - [Methylglucamine antimoniate and sodium stibogluconate in the treatment of leishmaniasis. Study of 16 cases]. AB - Treatment of visceral and cutaneous leishmaniasis rests on pentavalent antimonial drugs. However, side effects are often a problem and resistance may emerge. N methyl-glucamine given for kala-azar induced hematologic or hepatic toxicity in three patients and failed in two. Four patients with cutaneous leishmaniasis recovered but two exhibited adverse side effects. Sodium stibo-gluconate failed in one of three kala-azar patients and in one of two patients with cutaneous leishmaniasis. The effects of these two drugs are discussed, as well as indications of pentamidine and splenectomy. PMID- 3003659 TI - [Augmentation by dilution of the effect of PVP iodine on poliomyelitis virus type I]. AB - The influence of dilution of povidone-iodine solutions on virucidal activity against poliovirus type 1 was investigated. After exposure to different concentrations of povidone-iodine, the reduction in virus titer was determined after separating the antiseptic by gel filtration. Diluted preparations (0.5 and 0.25%) achieved a greater reduction in virus titer (10(5) in one hour) than the 5% stock solution (10(3)). Moreover, dilute solutions exhibited a faster virucidal activity. PMID- 3003660 TI - Ultrasound of Wilms' tumor. AB - The grey scale ultrasound features of 30 cases of Wilms' tumour examined using a water delay scanner are presented. Eighty-seven percent of masses were of uniform texture, with echogenicity equal to or slightly greater than that of liver with small hypo-echoic areas. There were no totally cystic tumours in the series. In 21 cases the mass was clearly arising from the ipsilateral kidney, but in 9 cases the ipsilateral kidney could not be distinguished from the tumour. The state of the intrahepatic inferior vena cava (IVC) was correctly diagnosed in 27 cases and further evaluation was needed in two patients in whom this segment could not be demonstrated. If this segment is sonographically normal, then the IVC need not be evaluated further. Differential diagnosis is discussed, with particular reference to neuroblastoma. In the majority of patients ultrasound is the only modality needed to diagnose Wilms' tumour pre-operatively. PMID- 3003661 TI - Circulating corticosterone in the infant rat: the mechanism of age and thyroxine effects. AB - Infant rats exhibit a marked rise in serum concentrations of corticosterone between postnatal days 12 and 24. As this rise appears to be cued by L-thyroxine, the aim of the current study was to determine the physiological bases of the effects of both age and thyroid status on serum corticosterone. The in vivo response to either adrenocorticotropic hormone (60 mU/g body weight) or dibutyryl 3',5'-cyclic adenylate (0.3 mg/g body weight) increased between 10 and 16 days and was advanced and delayed by hyper- and hypothyroidism, respectively. In contrast, in vitro studies with adrenals from rats aged 10 and 16 days showed no effect of age on either basal or adrenocorticotropic hormone-stimulated production of corticosterone. Chronic administration of exogenous corticosterone to hyperthyroid animals resulted in significantly higher concentrations of serum corticosterone than did an identical administration to hypothyroid animals. As hyperthyroidism was associated with marked elevations in the concentration of corticosteroid-binding globulin and in the extent of binding of corticosterone, these results suggest that the effects of both age and thyroid status on serum concentrations of corticosterone may reflect changes in the metabolic clearance of the hormone. PMID- 3003662 TI - Phenothiazine-induced sleep apneas and the opioid system. PMID- 3003663 TI - Relationship between luminal Na+/H+ exchange and luminal K+ conductance in diluting segment of frog kidney. AB - Experiments were performed in the isolated perfused kidney of K+ adapted Rana pipiens to investigate the relationship between luminal K+ conductance and H+ transport in cells of the diluting segment. Inhibition of luminal Na+/H+ exchange by amiloride or by omission of luminal Na+ blocked luminal K+ conductance. Acidification of the kidney perfusate by elevation of pCO2 also reduced luminal K+ conductance. This effect could be prevented by furosemide. Since the steepest transcellular Na+ potential difference, directed from the lumen into the cell, is found when luminal Na+/Cl-/K+ cotransport is inhibited by furosemide, we conclude that luminal Na+/H+ exchange is most efficient at these conditions and thus could attenuate intracellular acidification. PMID- 3003664 TI - Na/H exchange at the apical membrane of guinea-pig gallbladder epithelium: properties and inhibition by cyclic AMP. AB - The properties and, in particular, the cAMP-sensitivity of apical Na/H exchange in guinea-pig gallbladder epithelium were investigated using gravimetric, pH stat, and microelectrode techniques. Proton secretion was Na-dependent, inhibited by ouabain and amiloride, and insensitive to changes in apical membrane potential. It was markedly reduced by 8-Br-cAMP and PGE1. PGE1 also attenuated the changes in intracellular Na activity produced by luminal Na removal and restoration. Our results suggest inhibition of Na/H exchange by cAMP. In conjunction with the cAMP-induced rise in apical membrane Cl permeability shown previously, this effect can account for inhibition of NaCl absorption by cAMP. PMID- 3003665 TI - The role of anions in cellular volume regulation. AB - Cellular volume can be varied substantially by replacing medium Cl- isosmotically by other univalent anions. Since K+ content changes in parallel, cellular K+ concentration is well maintained. Gluconate behaves as an impermeant anion so cells shrink. Acetate enters cells apparently by non-ionic diffusion causing marked cellular swelling. These changes in volume are fully reversed when Cl- is again restored to the medium. However, ouabain (10(-3) M) largely prevents this reversal when Cl- replaces acetate, arguing against a ouabain-insensitive volume regulating mechanism. In toad urinary bladder, serosal gluconate inhibits transepithelial Na+ transport and cells shrink. Analysis suggests that cell shrinkage results in a loss of Ba2+-sensitive highly selective basolateral membrane K+ conductance channels. PMID- 3003666 TI - How big is the electrochemical potential difference of Na+ across rat renal proximal tubular cell membranes in vivo? AB - The intracellular Na+ concentration of surface loops of proximal tubules was measured with double-barrelled Na+ -sensitive microelectrodes in micropuncture experiments on rat kidneys in situ and in vivo. With the help of a fast recording system, it was possible to select only valid measurements, by discarding all records in which the Na+ concentration rose after the impalement presumably as a result of sodium ion inflow via puncture leaks. Under free-flow conditions cell Na+ activity averaged 13.1, S.D. +/- 2.1 mmol/l which, assuming an activity coefficient of 0.75, corresponds to a Na+ concentration of 17.5 mmol/l. The simultaneously recorded cell membrane potential was -73.5 mV in agreement with best estimates derived previously. The comparison of cell sodium activity and cell pH (Yoshitomi and Fromter [17]) demonstrates that the electrochemical potential difference of Na+ is greater than that of H+, as required for the operation of Na+/H+ counter transport in the brushborder membrane but that bicarbonate efflux via the sodium bicarbonate cotransport system at the peritubular cell membrane requires a HCO3- to Na+ stoichiometry of greater than 2.0 or the involvement of other ions at least under free-flow conditions. PMID- 3003667 TI - Cellular electrophysiology of potassium transport in the mammalian cortical collecting tubule. AB - Electrophysiological studies were carried out on single perfused cortical and medullary collecting ducts to define the potassium and sodium transport properties of their apical and basolateral cell membranes. In addition, the effects of chronic mineralocorticoid hormone treatment on the mechanism of transport of potassium ions were evaluated. Studies included the measurement of transepithelial and cell potentials, and the resistance of individual cell membranes. The apical cell membrane of principal cells of the cortical collecting duct is characterized by separate potassium and sodium conductances. The basolateral cell membrane has also a potassium conductance, whereas the intercellular shunt pathway is largely permeable to chloride ions. Stimulation of potassium secretion by mineralocorticoids is associated with the following events. Increased cell potassium uptake across the basolateral cell membrane due to stimulation of Na-K ATPase and a more favorable electrical driving force for passive entry, facilitated exit of potassium from the cell to the tubule lumen by a more favorable electrochemical gradient (apical cell membrane depolarization) and enhanced potassium secretion by in increase of the potassium conductance of the apical cell membrane. Some properties of single potassium channels in the apical membrane of rabbit cortical collecting tubules are also described. PMID- 3003669 TI - [Experimental study of hepatic arterial embolization therapy by various formulations of the anticancer agent lipiodol]. PMID- 3003668 TI - Electrogenic sodium-bicarbonate symport in cultured corneal endothelial cells. AB - Intracellular potential measurements on confluent monolayers of cultured bovine corneal endothelial cells were used to define passive ion transport processes in these cells. Previous studies [11, 12] have provided the experimental basis for a cellular model, is which bicarbonate entry across the basolateral membrane in indirectly driven by a Na+/H+-exchanger, which is inhibitable by amiloride (1 mmol/l). Na+ and HCO3- leave the cell via an electrogenic bicarbonate sodium cotransport, which is inhibitable by the disulfonic stilbene derivates SITS or DIDS. This model is also compatible with transepithelial work from other groups. In this paper, we briefly review the evidence we have obtained for this model. PMID- 3003671 TI - A peptide to DNA conversion program. AB - A modification and extension of the computer program REVCUT (Blumenthal et al, Nucl. Acids Res. 10, 91-101 (1982) is described. The new program searches for restriction endonuclease recognition sites that are not coding DNA sequences of a protein of known aminoacid sequence using bit patterns. The modifications make the program more accurate and extend the range of the restriction endonucleases. PMID- 3003670 TI - Chemotherapeutic agents in the prevention and treatment of periodontal disease. PMID- 3003672 TI - MULTAN: a program to align multiple DNA sequences. AB - I describe a computer program which can align a large number of nucleic acid sequences with one another. The program uses an heuristic, iterative algorithm which has been tested extensively, and is found to produce useful alignments of a variety of sequence families. The algorithm is fast enough to be practical for the analysis of large number of sequences, and is implemented in a program which contains a variety of other functions to facilitate the analysis of the aligned result. PMID- 3003673 TI - A comprehensive package for DNA sequence analysis in FORTRAN IV for the PDP-11. AB - A computer package written in Fortran-IV for the PDP-11 minicomputer is described. The package's novel features are: software for voice-entry of sequence data; a less memory intensive algorithm for optimal sequence alignment; and programs that fit statistical models to nucleic acid and protein sequences. PMID- 3003674 TI - Cloning simulation in the cage environment. AB - The CAGE/GEM(TM) software toolkit for genetic engineering is briefly described. The system functionally uses color graphics and is menu driven. It integrates genetics and features information ("Overlays") with information based on sequence analysis ("Representations"). The system is structured around CAD (Computer Aided Design) principles. The CAGE (Computer Aided Genetic Engineering) aspects of the software are emphasized and illustrated by a simulated cloning of the hepatitis B core antigen gene into the BAMHI site of plasmid pBR322. PMID- 3003676 TI - A computer program package for restriction map analysis and manipulation. AB - Programs for the calculation, storage and analysis of restriction maps derived from the analysis of partial digestion products from end labelled DNA (1,2,3) and their correlation with digestion - and hybridisation patterns in total digestions and Southern blot experiments are described. These programs allow direct input of gel patterns from partial or complete digestion experiments using a digitizer tablet, calculation of molecular weights and restriction maps, plotting of maps and actual or predicted fragment patterns and automated identification of overlapping cosmids from partial restriction mapping results. Programs are written in PASCAL and have been implemented on a VAX/VMS system, with a HP-7221T plotter and a digitizing tablet. PMID- 3003677 TI - A program package applicable to the detection of overlaps between restriction maps. AB - A computer program package for the storage, change, and comparison of restriction maps is described. The programs are intended to detect overlaps between relatively short (about 10-40 kb; abbreviations ref.2) maps and to merge the overlapping fragments into large restriction maps. They run on a 16-bit microcomputer with limited memory and addressing capability. Due to the restricted reliability of restriction maps compared with DNA sequence data a particular storage method was used. The source code of the programs is freely available (+). PMID- 3003675 TI - A dynamic programming algorithm for finding alternative RNA secondary structures. AB - Dynamic programming algorithms that predict RNA secondary structure by minimizing the free energy have had one important limitation. They were able to predict only one optimal structure. Given the uncertainties of the thermodynamic data and the effects of proteins and other environmental factors on structure, the optimal structure predicted by these methods may not have biological significance. We present a dynamic programming algorithm that can determine optimal and suboptimal secondary structures for an RNA. The power and utility of the method is demonstrated in the folding of the intervening sequence of the rRNA of Tetrahymena. By first identifying the major secondary structures corresponding to the lowest free energy minima, a secondary structure of possible biological significance is derived. PMID- 3003678 TI - Should nucleotide sequence analyzing computer algorithms always extend homologies by extending homologies? AB - Most computer algorithms used for comparing or aligning nucleotide sequences rely on the premise that the best way to extend a homology between the two sequences is to select a match rather than a mismatch. We have tested this assumption and found that it is not always valid. PMID- 3003679 TI - Computer assisted microdensitometric analysis of footprinting autoradiographic DATA. AB - A system comprised of a linear scanning microdensitometer interfaced to a personal computer has been developed to facilitate analysis of ligand-DNA footprinting autoradiograms. The system, which can be used to record density and sequence information from autoradiographic films, enables the user to relate the area under an autoradiographic band to the concentration of radiolabeled molecules present in the electrophoresis gel. This report describes the computer program which performs the calculations and discusses the ability of the system to accurately determine oligonucleotide concentration, as a function of band separation, photographic response, and the computational algorithm used to calculate band areas. PMID- 3003680 TI - A new method of representing DNA sequences which combines ease of visual analysis with machine readability. AB - A new method of representing DNA sequences has been devised which is termed stave projection. Compared with other formats for showing the base sequences of DNA, this method greatly enhances the ease of visual analysis of the sequences of bases and it is also in a machine readable form. Using this method it is possible to identify and annotate all of the functional features found in DNA sequences. PMID- 3003681 TI - DIGICALC: a restriction fragment analysis program. AB - DIGICALC is a program designed to aid in the acquisition, storage, and analysis of nucleic acid restriction fragment data. The chief considerations during program design were (i) ease of use for people with varying degrees of computer experience, (ii) minimal hardware requirements (e.g. an IBM PC), (iii) portability and ease of modification, and (iv) improved functionality in sizing and comparing restriction fragments over manual methods. The program accepts manual or digitizer input of nucleic acid fragment mobility, calculates the fragments' sizes, and provides the means to search the fragment database and to produce charts of fragment sizes. PMID- 3003682 TI - COMAP: a comigrating analysis program for estimating the relationship of adenoviruses on the genome level. AB - The computer program COMAP is described that enables one to evaluate the relationship among adenovirus 6 strains by comigration analysis with regard to the genome. The required data were obtained by restriction analysis with several enzymes. The program is also important for identifying further adenovirus strains by DNA restriction analysis and to compare new restriction fragment patterns with stored data of known adenovirus strains. We believe that in this manner the DNA restriction analysis will become a valuable diagnostic procedure. PMID- 3003683 TI - SPLINT: a cubic spline interpolation program for the analysis of fragment sizes in one-dimensional electrophoresis gels. AB - SPLINT (SPLine INTerpolation) is a BASIC microcomputer program that uses electrophoretic data on the migration distances of DNA fragments of known size to approximate the relationship between distance moved and DNA fragment size by a piecewise cubic spline function. This function places all standards exactly on the calibration curve and then determines from it the sizes of other fragments in the gel. Special advantages of this program include the use of a digitizer to enter the fragment positions directly from a gel photograph and the optional generation of a graph of the approximating function plus the placement on it of points from any specified gel lanes. PMID- 3003684 TI - Fast analysis of DNA and protein sequence on Apple IIe: restriction sites search, alignment of short sequence and dot matrix analysis. AB - A fast restriction sites search algorithm using a quadruplet look-ahead feature has been written in 6502 assembly language code. The search time, tested on the sequence of pBR322, is 4.1 s/kilobase using a restriction site library including 112 specificities corresponding to a total site length of over 700 bases. The search for a short sequence (less than 36 bases) within a longer one (up to 9999 bases) with a given number of mismatches or gaps allowed has also been written in assembly language. Typical run time for the search of a 12 base sequence with 1, 2 or 3 gaps allowed are 6.2, 9.4 or 13.6 s/kilobase, respectively. The dot matrix analysis needs 7.5 minutes per square kilobase when using a stringency of 15 matched bases out of 25. A 7/21 matrix of two 500 amino acid proteins is obtained in 3 minutes. These three routines are included in DPSA, a general package of programs allowing manipulation and analysis of DNA and protein sequences. PMID- 3003685 TI - A DNA sequence analysis program for the Apple Macintosh. AB - This paper describes a new set of programs for analyzing DNA sequences using the Apple Macintosh computer, a computer ideally suited for this kind of analysis. Because of the Macintosh interface and the availability of high quality software only speech synthesis, these programs are truly easy to use. Instead of typing in commands, the user directs the program by making selections with the mouse, thereby eliminating most typographical and syntax errors. Output options are selected by "pressing buttons" and then clicking "OK" with the mouse. DNA sequences are confirmed by having the program speak them. The high resolution graphics on the Macintosh not only allow for explanatory diagrams to be used to aid in deciding on input parameters, but can be used to produce slides for presentations and figures for papers. Because of the clipboard and the ability of the Macintosh to readily share data among different applications, data can be saved for use directly in word processing documents (e.g. manuscripts). PMID- 3003686 TI - Personal access to sequence databases on personal computers. AB - A comprehensive package of software has been developed to access nucleic acid and protein sequence databases on stand-alone IBM personal computers. The software combines keyword search on the annotation fields of the data with pattern matching algorithms on the biological sequences. Sequences containing complex sites like promoters or kink sites can be identified as well as sequences that are similar to a query sequence. Protein sequences with particular patterns of amino acids such as hydrophobic regions can be identified as well. Considering the relatively inexpensive hard disks now available, personal computers have become a cost-effective alternative to mainframe processing for sequence databases. PMID- 3003687 TI - The complete coding sequence of the human raf oncogene and the corresponding structure of the c-raf-1 gene. AB - The complete 648 amino acid sequence of the human raf oncogene was deduced from the 2977 nucleotide sequence of a fetal liver cDNA. The cDNA has been used to obtain clones which extend the human c-raf-1 locus by an additional 18.9 kb at the 5' end and contain all the remaining coding exons. PMID- 3003688 TI - Spinach plastid genes coding for initiation factor IF-1, ribosomal protein S11 and RNA polymerase alpha-subunit. AB - The nucleotide sequence of 2.5 kbp from the cloned SalI fragments 8 and 11 of spinach plastid DNA has been determined. This region was found to encode three open reading frames for hydrophilic polypeptides of 77, 138, and 335 amino acids. Using the computer search algorithm of Lipman and Pearson (Science 227, 1435, 1985), these genes were identified as coding for homologues of E. coli initiation factor IF-1 (inFA), 30S ribosomal protein S11 (rps11), and the alpha-subunit of DNA-dependent RNA polymerase (rpoA). The spinach plastid gene organization is inFA - 381 bp spacer - rps11 - 72 bp spacer - rpoA. The genes are transcribed in vivo and appear to encode functional proteins. These findings imply that plastid chromosomes code for components of the organelle transcription apparatus. PMID- 3003689 TI - Revised assignment for the Bacillus subtilis spo0F gene and its homology with spo0A and with two Escherichia coli genes. AB - The nucleotide sequences of spo0F mutant genes which block the early sporulation process of Bacillus subtilis were determined. The mutation sites together with the results of complementation tests suggested that an open reading frame for a polypeptide of Mr = 14,229 is the spo0F gene. The deduced amino acid sequence shows striking homology with that of the spo0A gene. In addition, the upstream region involving the rib some binding site of the spo0F coding region is also similar to those of spo0A and spo0B. These homologies suggest that all three genes have a similar function in regulating the initiation of sporulation, and that their expression is controlled by a common mechanism. Clear homology is also seen between the spo0 gene products and the transcriptional control proteins, OmpR and Dye, of Escherichia coli suggesting that the spo0 gene products also are involved in the control of transcription. PMID- 3003690 TI - A new member of the plasma protease inhibitor gene family. AB - A 2.1-kb cDNA clone representing a new member of the protease inhibitor family was isolated from a human liver cDNA library. The inhibitor, named human Leuserpin 2 (hLS2), comprises 480 amino acids and contains a leucine residue at its putative reactive center. HLS2 is about 25-28% homologous to three human members of the plasma protease inhibitor family: antithrombin III, alpha 1 antitrypsin and alpha 1-antichymotrypsin. A comparison with published partial amino acid sequences shows that hLS2 is closely related to the thrombin inhibitor heparin cofactor II. PMID- 3003691 TI - Genomic and structural organization of Drosophila melanogaster G elements. AB - The properties and the genomic organization of G elements, a moderately repeated DNA family of D. melanogaster, are reported. G elements lack terminal repeats, generate target site duplications at the point of insertion and exhibit at one end a stretch of A residues of variable length. In a large number of recombinant clones analyzed G elements occur in tandem arrays, interspersed with specific ribosomal DNA (rDNA) segments. This arrangement results from the insertion of members of the G family within the nontranscribed spacer (NTS) of rDNA units. Similarity of the site of integration of G elements to that of ribosomal DNA insertions suggests that distinct DNA sequences might have been inserted into rDNA through a partly common pathway. PMID- 3003692 TI - Construction of a retroviral cDNA version of the int-1 mammary oncogene and its expression in vitro. AB - The int-1 mammary oncogene is activated by proviruses of the Mouse Mammary Tumor Virus in many different mammary tumors. We have inserted a genomic fragment containing the protein-encoding domain of the gene into the retroviral shuttle vector pZIPneoSV(X)1. After one round of virus replication we recovered recombinant proviral DNA containing a correctly spliced copy of int-1. In vitro transcription of this cDNA version of int-1 using SP6 polymerase and translation in a reticulocyte lysate yielded a protein of approximately 37,000 daltons. High expression of int-1 in NIH-3T3 cells infected with recombinant virus did not lead to morphological transformation. PMID- 3003693 TI - A BR 1 gene in Chironomus tentans has a composite structure: a large repetitive core block is separated from a short unrelated 3'-terminal domain by a small intron. AB - The large Balbiani ring (BR) genes in the dipteran genus Chironomus have been considered to be homogeneous repetitive structures. Analysis of a genomic DNA segment now reveals that a BR 1 gene in C. tentans is a composite gene, consisting of two different types of sequences. A 15-20 kb core block of tandemly arranged repeat units extends close to the 3' end of the BR 1 gene and ends in repetitive structures partly different from the repeat units in the core block. A 55 bp long intron separates the core block, which probably constitutes a single exon, from a non-related 3'-exon, comprising the final 332 bp of the translated part of the gene. According to hydrophobicity and secondary structure predictions, the 3'-exon encoded peptide is distinctly different from the repetitive core block domain and attains a globular structure. The carboxyl terminal peptide domain is likely to be a general feature of BR encoded proteins and may have important functions in the excretion and polymerisation of the secretory proteins. PMID- 3003694 TI - Structure of gene and pseudogenes of human apoferritin H. AB - Ferritin is composed of two subunits, H and L. cDNA's coding for these proteins from human liver (1,2,3), lymphocytes (4) and from the monocyte-like cell line U937 (5) have been cloned and sequenced. Southern blot analysis on total human DNA reveals that there are many DNA segments hybridizing to the apoferritin H and L cDNA probes (1,2,4,6). In view of the tissue heterogeneity of ferritin molecules (7,8), it appeared possible that apoferritin molecules could be coded by a family of genes differentially expressed in various tissues (1,2). In this paper we describe the cloning and sequencing of the gene coding for human apoferritin H. This gene has three introns; the exon sequence is identical to that of cDNA's isolated from human liver, lymphocytes, HeLa cells and endothelial cells. In addition we show that at least 15 intronless pseudogenes exist, with features suggesting that they were originated by reverse transcription and insertion. On the basis of these results we conclude that only one gene is responsible for the synthesis of the majority of apoferritin H mRNA in various tissues examined, and that probably all the other DNA segments hybridizing with apoferritin cDNA are pseudogenes. PMID- 3003695 TI - Structure and nucleotide sequence of the 5' region of the human and feline c-sis proto-oncogenes. AB - Comparative analysis of cosmid clones containing the human and feline c-sis genetic regions revealed the similar structural organization of these areas in the two species. The areas shared seven different genetic regions in and around the c-sis locus and of these was related to v-sis. Another region, 1.9 kbp in size and located about 8 kbp upstream of the v-sis homologous region in the human genome, also hybridized to the main c-sis transcriptional product of 3.5 kb. Comparison with a recently described c-sis cDNA clone (Collins et al., Nature 316, 748-750 (1985)) revealed that the 1.9 kbp DNA region contained a large 5' c sis exon of at least 1050 bp. In this exon, the presumed initiation site of the predicted PDGF-2 containing precursor protein was located and appeared to be preceded by a large untranslated region. In the region immediately upstream of this exon, a TATA box and a consensus sequence for a potential Sp1 binding site were found at similar positions in both species. This region also exhibited promoter activity when tested in an assay in which coding sequences of bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA: chloramphenicol 3-O acetyltransferase, EC 2.3.1.28) were placed under its control. The five other DNA regions were found upstream and downstream of the human c-sis transcription unit and also in an intron. Four of them contained repetitive sequences. Hybridization analysis of human and feline c-sis containing cosmid clones with a mixed synthetic nucleotide probe, which corresponded to sequences encoding amino acid residues 2-7 of chain 1 of platelet-derived growth factor (PDGF-1), suggested that the c-sis cosmid clones did not include PDGF-1-specific genetic sequences. PMID- 3003696 TI - The full length coding sequence of rat liver androsterone UDP glucuronyltransferase cDNA and comparison with other members of this gene family. AB - Cloned cDNAs coding for hepatic UDP-glucuronyltransferase (UDPGT) have been isolated from a rat liver cDNA library in the expression vector bacteriophage lambda gt11 using anti-UDPGT antibodies. Four different mRNAs have been identified by sequencing of 15 UDPGT cDNA clones. The sequences of the four classes of cDNA were determined to be 85-95% homologous. Restriction fragments were isolated from the cDNA in each class and used as class specific probes. Hybridisation of these probes to northern blots of total RNA prepared from the livers of normal and genetically deficient Wistar rats identified the cDNA in class 4 with androsterone UDPGT. Translation of the cDNA sequence of clone rlug 23, the longest member of class 4, allowed determination of the complete amino acid sequence of androsterone UDPGT. PMID- 3003697 TI - The sequence and structure of a new serum amyloid A gene. AB - The acute phase response is characterized by changes in the serum concentrations of many proteins. A 1000-fold increase in the concentration of serum amyloid A (SAA) protein occurs within 24 hours of LPS injection in the mouse. We have isolated a cDNA clone and its corresponding genomic phage for a third, previously unreported SAA protein. The sequence of the cDNA, the gene's exons and neighboring DNA are presented along with the mapping evidence supporting the gene structure. PMID- 3003698 TI - Relaxation of recognition sequence of specific endonuclease HindIII. AB - Under the standard reaction conditions, the restriction endonuclease HindIII cleaves double-stranded DNA, within the recognition sequence--A/AGCTT--at the position indicated by the arrow. In the presence of dimethyl sulfoxide the substrate specificity of this enzyme is reduced and cleavages occur at additional sites. We have determined the secondary sites in pBR322 DNA recognized by HindIII endonuclease under relaxed conditions and found that it cleaves the hexanucleotides: G/AGCTT, A/GGCTT, A/TGCTT, A/ATCTT, A/AGCCT, A/AGCAT, A/AGCGT, A/AGCTC, at the positions indicated by the arrows, producing fragments with cohesive ends. PMID- 3003699 TI - In vitro transcription of RU, a middle repetitive element of the rat genome. AB - Repetitive DNA sequences have been found within as well as adjacent to the rat growth hormone (rGH) gene (1,2). In vitro transcription of the rGH gene yields transcripts predominantly from a tandemly duplicated repeat (RU) in the second intron of the gene. The relative alpha-amanitin sensitivity of this transcription indicates that it is carried out by RNA polymerase III. Transcription initiates within a 5' flanking 15 base-pair repeat, and terminates within a stretch of A residues at the end of each repeat, a termination site that is notably unlike most of those used by polymerase III. Approximately 75% of the transcripts stop at the end of the first repeat; the remainder "read-through" to a homologous termination region at the end of the second repeat. PMID- 3003700 TI - Analysis of DNA sequences which regulate the transcription of herpes simplex virus immediate early gene 3: DNA sequences required for enhancer-like activity and response to trans-activation by a virion polypeptide. AB - The far upstream region of herpes simplex virus (HSV) immediate early (IE) gene 3 has previously been shown to increase gene expression in an enhancer-like manner, and to contain sequences which respond to stimulation of transcription by a virion polypeptide, Vmw65. To analyse the specific DNA sequences which mediate these functions, sequential deletions from each end of the far upstream region were made. The effects of the deletions on transcription in the absence or presence of the Vmw65 were measured by use of a transient expression assay. The enhancer-like activity was due to three separable elements, whereas two additional DNA regions were involved in the response to Vmw65. One of the responding elements corresponded to an AT-rich consensus (TAATGARATTC, where R = purine) present in all IE gene far upstream regions, and the other was a GA-rich sequence also present in IE genes 2 and 4/5. The TAATGARATTC element could mediate responsiveness to Vmw65 but it was fully active only in the presence of the GA-rich element. The GA-rich element was unable to confer a strong response alone but could activate an otherwise nonfunctional homologue of TAATGARATTC. PMID- 3003702 TI - Synthesis, properties and use of beta-alanyltyrosine derivatives of 2',5' oligoadenylate 5'-triphosphate. AB - beta-Alanyltyrosine methyl ester derivatives of 2-5 A, ppp-(A2'p5') A-beta-Ala Tyr, were prepared by coupling of periodate oxidizedn2-5 A with beta alanyltyrosine methyl ester, followed by reduction with sodium cyanoborohydride. The compounds were resistant to the hydrolysis by 2',5'-phosphodiesterase in the mouse L cells extract. They bound to the 2-5 A dependent RNAse (RNAse L) in the mouse L cells extract and in the rabbit reticulocyte lysate, and displaced by addition of 2-5 A. The compound, pppA2'p5'A2'p5'A2'p5'A-beta-Ala-Tyr, after iodination with 125I, was proved to be useful as a radio-labeled probe for the radiobinding assay for 2-5 A. PMID- 3003701 TI - Identification, cloning and sequence determination of the genes specifying hexokinase A and B from yeast. AB - The hexokinase A (HKA) and hexokinase B (HKB) genes of Saccharomyces cerevisiae have been cloned from a library of yeast genomic DNA. Using an in vitro glucose phosphorylation assay, the HKB gene was located on a plasmid carrying a 13.6 kb fragment of yeast DNA. After subcloning the relevant restriction fragments, the nucleotide sequence of the HKB gene was determined. Using this information, we were able to locate the HKA gene on a plasmid carrying this gene, which we then sequenced. Approximately 43% of the amino acid sequence of HKB was determined directly from 24 tryptic peptides. The results are in complete agreement with those derived from the DNA sequence and are consistent with the results of x-ray crystallography. Comparison of the amino acid sequences of HKA and HKB show that 378 out of 485 residues are identical. The 5' flanking region of the A gene contains nucleotide sequences expected for genes that are expressed at relatively high levels in yeast. The 24 base pair hyphenated palindrome at the 3' end of the HKB gene may be a site for termination of transcription of this gene. PMID- 3003703 TI - Protected 2'-deoxyaristeromycin on polymer support: a substitute for the acid labile 2'-deoxyadenosine moiety at the 3'-terminus of oligodeoxynucleotides. AB - A modified nucleoside antibiotic on polymer support was employed in the solid phase synthesis of two oligodeoxynucleotides to substitute acid labile 2' deoxyadenosine(A) at the 3'-terminus of oligomers with acid resistant 2' deoxyaristeromycin(Ar). The duplex of the decamer having sticky ends was inserted successfully into the Eco R1 site of pBR322. E. coli DH 1 was transformed with the Ar-containing plasmid. Inspection of the cloned plasmid pBR2552 by restriction endonucleases revealed that no deletion and insertion occurred near the inserted sequence. This result indicates that Ar can behave as A in DNA replication of the host cells. Therefore, Ar can be used as a substitute for 3' terminal A in the solid-phase oligonucleotide synthesis. PMID- 3003704 TI - Enzymatic purification of chemically synthesized oligodeoxyribonucleotides prior to removal from a solid-support. AB - A novel combined solid-phase DNA synthesis and solid-phase DNA purification strategy is described. Removal of all N-, cap-, and phosphate protecting groups while maintaining a 5'-blocking group on the final product followed by a 5'- specific exonuclease digestion of failure sequences with the synthetic DNA still attached to the solid phase yields an essentially pure oligomer. PMID- 3003705 TI - Determination of the complete nucleotide sequence of the Brevibacterium lactofermentum plasmid pAM330 and the analysis of its genetic information. AB - The complete nucleotide sequence of the plasmid pAM330 derived from Brevibacterium lactofermentum ATCC 13869 was determined using the dideoxy chain termination method. Analysis of the sequence allowed us to observe seven open reading frames, one of which showed the presence of the promoter and the Shine Dalgarno (SD) sequences upstream from the initiation codon. PMID- 3003706 TI - Cloning and structural analysis of a part of the human epidermal growth factor receptor gene. AB - We screened a gene library constructed with human leukocyte chromosomal DNA fragments with an oligonucleotide probe complementary to the middle part of the cDNA for the human epidermal growth factor receptor. One of the genomic DNA clones obtained contains an insert of 36 kilobase pairs encoding 54% of the mature receptor protein. From the sequencing experiments, we were able to locate the exact site of the gene rearrangement, which had been previously suggested in some epidermoid carcinoma cell lines, in the intron preceding the exon coding for the hydrophobic transmembrane domain. Such a rearrangement must result in the removal of an authentic splicing acceptor site and the separation of the 3' segment of the receptor gene which could encode a polypeptide homologous to the v erb-B oncogene product responsible for chicken carcinogenesis. PMID- 3003707 TI - Efficient production of a human tumour necrosis factor in Escherichia coli. AB - To examine the effect of altering the nucleotide sequence of the Shine-Dalgarno (SD) region and the spacer sequence between the SD sequence and the AUG translation start signal, several plasmids were constructed which directed the synthesis of mature human tumour necrosis factor (TNF) under the control of the E. coli trp leader promoter. We found that the presence of the SD sequence, AAGGAGGT, which is complementary to the 3' end of 16S rRNA, gave the higher translational efficiency, and also, the presence of the spacer sequence which consists of only A and T residues raised the production of TNF 2-3 fold. The levels of expression of TNF were elevated over 20-fold by the alteration of the SD and spacer sequences and amounted to 20% of the total cellular proteins or approx. 5 X 10(6) molecules of TNF per cell. PMID- 3003708 TI - Selective inhibition of the proliferation of herpes simplex virus type 1 thymidine kinase gene-transformed murine mammary FM3A carcinoma cells by (E)-5-(2 bromovinyl)-2'-deoxyuridine and related compounds. AB - Mouse mammary carcinoma FM3A cells deficient in thymidine kinase were transformed by a cloned gene for herpes simplex virus type 1 thymidine kinase. Among several anti-herpetic nucleoside analogues, (E)-5-(2-bromovinyl)-2'-deoxyuridine, (E)-5 (2-iodovinyl)-2'-deoxyuridine and (E)-5-(2-bromovinyl)-2'-deoxycytidine inhibited the growth of the transformed cells at concentrations 5000- to 20000-fold lower than those required to inhibit the growth of the corresponding wild-type cells. The selective inhibitory action of these compounds was due to a specific phosphorylation by the viral thymidine kinase. From the transformed cells, thymidine-auxotrophic mutants that are deficient in thymidylate synthase were isolated. These mutant cell lines should prove useful in elucidating the mechanism of action of the antiherpetic nucleoside analogues. PMID- 3003710 TI - A silica-based reversed-phase column for single-stranded oligodeoxyribonucleotides. AB - A novel, silica-based, reversed-phase column targeted to the separation of single stranded oligodeoxyribonucleotides has been developed by grafting monochlorooctadecylsilane onto silica which is a base material of reversed-phase columns (TSK 120A and 120T), in which polychlorosilane is used as a grafting reagent. By applying a shallow gradient elution of an aqueous acetonitrile solution containing 0.1 M ammonium acetate, chromatography with this column produced well-resolved peaks for large samples such as those having bases up to 26. PMID- 3003709 TI - Affinity chromatography of nucleosides and nucleic acid base derivatives with nucleic acid bases or nitrobenzeneboronic acid substituted silicas. AB - Nucleic acid bases such as adenine and uracil, and nitrobenzeneboronic acid substituted silicas were prepared by the reaction of chloromethylbenzene substituted silica with adenine sodium salt and trimethylsilylated uracil, and nitration of benzeneboronic acid substituted silica, respectively. From the results of HPLC of nucleosides and N-ethyl derivatives of nucleic acid bases using modified silicas, hydrophobic base stacking interaction, selective hydrogen bonding interaction between purine and pyrimidine bases, and reversible cyclic boronate ester formation between diols of nucleosides with boronic acid were effective for the separation of nucleic acid related compounds. Moreover, association constants for hydrogen bonding formation of nucleic acid bases were estimated. PMID- 3003712 TI - [HTLV--human T-cell leukemia virus]. PMID- 3003711 TI - Studies on griseolic acid derivatives. I. Synthesis of substituted derivatives of griseolic acid at the N1, C6, C2' or C7' position and their biological activities. AB - Griseolic acid derivatives having a different substituent at the N1,C6,C2' or C7' position of the natural product were synthesized and their structure activity relationship to cyclic nucleotide phosphodiesterase inhibitory activity was investigated. PMID- 3003713 TI - Radiologic appearances of the routes of spread of lung cancer. Importance of early recognition; some causes of failures of local treatment. TNM and TLNM system of classification. PMID- 3003714 TI - Microbiological aspects of erythromycin. AB - Almost 35 years after its discovery, erythromycin remains highly active against most strains of a broad array of clinically important organisms. Many strains of Gram-positive and Gram-negative aerobic, facultative and anaerobic bacteria, as well as Mycoplasma, treponemes and Chlamydia, are susceptible to this agent. Erythromycin acts by binding to the ribosomes of the target organisms, thereby inhibiting protein synthesis. Although a pharmacokinetically superior erythromycin estolate has been shown to be a less potent antibiotic prior to hydrolysis, it appears to contribute significantly to the therapeutic response following administration of erythromycin estolate. Clinical laboratory susceptibility testing or erythromycin may be done using standardized methods, but the laboratorian is cautioned to remember the adverse effect of acidic test conditions on the apparent activity of this antibiotic. PMID- 3003715 TI - Safety, infectivity, transmissibility and immunogenicity of rhesus rotavirus vaccine (MMU 18006) in infants. AB - In an attempt to evaluate the immunogenicity, infectivity, transmissibility and safety of rhesus rotavirus vaccine (RRV) MMU 18006, 27 infants ages 5 to 20 months participated in two randomized, double-blind placebo controlled trials, one in a day care setting to allow for child to child contact and close surveillance and the other on an outpatient basis. Fourteen infants (mean age, 8.3 months) received 10(5) plaque-forming units of RRV and 13 (mean age, 11.1 months) received placebo. In the eight infants who participated in the vaccine trial in the day care setting, there was no evidence of transmissibility of RRV, by either stool excretion or seroconversion. The data from both trials showed RRV to be 100% infective and immunogenic in the vaccinees. There were no gastrointestinal side effects although there was an association between vaccine administration and fever occurring on Days 3 and 4. Based on these encouraging preliminary results, further work is proceeding to evaluate this vaccine at lower doses in this age group of infants. PMID- 3003716 TI - Sultamicillin (ampicillin-sulbactam) in the treatment of acute otitis media in children. AB - Sultamicillin, a dimer of ampicillin and a beta-lactamase-inhibiting agent, sulbactam, was given in oral form to 50 infants and children with acute otitis media. Tympanocentesis was performed on entry into the trial. Beta-lactamase positive Haemophilus influenzae or Branhamella catarrhalis was isolated from 14 of 73 (19.2%) middle ear effusions in 9 children. Relief of symptoms (fever/otalgia) occurred in all children who completed therapy. However, in 8 children (16%), the antimicrobial agent was discontinued due to presumed adverse side effects (primarily gastrointestinal); vomiting which began prior to entry was noted in another subject who was withdrawn. An additional 14 children completed the course of treatment despite having diarrhea. Of the 41 children who completed drug therapy, 11 (26.8%) were effusion-free after 10 days, and 22 of 33 (66.7%) evaluable children were effusion-free after 6 weeks. Sultamicillin is a novel therapeutic approach to beta-lactamase-producing bacteria. In its oral form, however, diarrhea is a troublesome side effect. PMID- 3003717 TI - Diffuse recurrent herpes simplex virus skin lesions after adequate therapy with vidarabine. PMID- 3003718 TI - Overview of viral agents in pediatric enteric infections. PMID- 3003720 TI - The emerging role of adenoviruses as inducers of gastroenteritis. PMID- 3003719 TI - The role of rotaviruses in pediatric diarrhea. PMID- 3003721 TI - Other viruses with etiologic roles in childhood gastroenteritis. AB - Rotaviruses and Norwalk-like viruses are the two groups of viruses most frequently associated with gastroenteritis, but as outlined in this review several other viral agents have also been associated with acute gastroenteritis. The gastroenteritis viruses are generally fastidious, and thus traditional cell culture isolation and detection procedures are not applicable; therefore electron microscopy and immunoelectron microscopy remain among the most powerful techniques for studying these viruses. The in vitro cultivation of these viral agents will facilitate the development of diagnostic reagents and the development and evaluation of vaccines. The main need in this area is for suitable cell culture systems for isolating and growing the candidate gastroenteritis viruses. PMID- 3003723 TI - [Malignant fibrous histiocytoma of the stomach. A case report with a review of the literature]. PMID- 3003722 TI - Hyperandrogenism. Tests that simplify evaluation. PMID- 3003724 TI - [Morphology and morphogenesis of prostatic small cell cancer]. PMID- 3003725 TI - Isolation and identification of alpha-methyloctanylcarnitines from human urine. AB - Medium-chain acylcarnitines were isolated from human urine using a combination of chloroform-methanol extraction, silicic acid column and molecular sieving chromatography and preparative HPLC. Three purified acylcarnitines were analyzed by fast atom bombardment mass spectrometry and were also saponified and the free fatty acids analyzed by gas chromatography and mass spectrometry. Combined electron impact mass spectrometry and fast atom bombardment mass spectrometry and periodate oxidation for location of double bonds, demonstrated the occurrence of delta 6-octenylcarnitine, 2-methyloctanylcarnitine and 2-methyl-delta 6 octenylcarnitine. These acylcarnitines were present in the thirteen urines obtained from normal humans, but were not detected in urines from three individuals who had been on total parenteral nutrition for more than a year. The occurrence of alpha-methyl medium-chain acylcarnitines in human urine indicates a role for carnitine in excretion (detoxification) of these acyl derivatives. PMID- 3003726 TI - [Responses of plasma cortisol and ACTH to stimulation by the synthetic ovine ACTH releasing factor in normal man]. PMID- 3003727 TI - [Clinical pharmacology of enalapril]. AB - Enalapril is an inhibitor of the converting enzyme which transforms angiotensin I into angiotensin II, an octapeptide with pharmacological properties uniformly tending to increase blood pressure. The enalapril molecule is not active until it has been hydrolyzed into enalaprilic acid in the liver. This acid induces a very strong (85-95%), very early (maximum at 2 hours) and long lasting (20% at 72 hours) blockade of the angiotensin-converting enzyme, resulting in a paralleled fall in systolic and diastolic blood pressure. The fall begins with a 2.5 mg dose of the drug, is maximum with 10 mg and is not increased by higher doses. No tachyphylaxis has been observed with prolonged administration. The fall in blood pressure is due to reduction of peripheral resistance, without changes in heart rate and cardiac output. There is no sodium retention. The two main pharmacokinetic features of the drug are its very long half-life and the 100% urinary excretion of the active metabolite. The drug can be taken as a single dose at any moment of the day. The only known interaction is with propranolol, which reduces by one-third the bioavailability of enalapril. Side-effects are uncommon and mild. PMID- 3003728 TI - [Treatment of cardiac failure with enalapril]. AB - The fall in cardiac output observed in cardiac failure induces stimulation of the renin-angiotensin-aldosterone system. This results in strong constriction of the arterioles which in turn increases the fall in cardiac output and aggravates myocardial alteration. Angiotensin-converting enzyme inhibitors therefore act on a major physiological mechanism of cardiac failure. In this double-blind study 36 cardiac failure patients treated with digitalis derivatives and diuretics were divided into two groups: one group received enalapril in addition to the other drugs, the other group received a placebo. The functional symptoms, exercise tests, left ventricular function and haemodynamic values were improved in two thirds of the patients treated with enalapril, whereas no improvement was observed in those who received the placebo. Enalapril maleate was given by injection to 10 patients with acute left ventricular failure following myocardial infarction; a useful fall in pulmonary capillary pressure and systemic arterial resistance occurred within one hour. Enalapril represents an effective physiopathological treatment of severe cardiac failure. PMID- 3003729 TI - [Treatment of arterial hypertension in the aged subject with a converting enzyme inhibitor: enalapril]. AB - The purpose of this study was to investigate the effectiveness and safety of enalapril in elderly people. A double-blind, randomized, placebo-controlled trial was carried out in 32 subjects aged from 75 to 97 years (mean: 86 years) with blood pressure values equal or superior to 160/90 mmHg. After 8 weeks of treatment with enalapril in doses of 20 to 40 mg/day, the systolic pressure was lowered from 190 +/- 16 to 151 +/- 19 mmHg (P less than 0.0001) and the diastolic pressure from 102 +/- 7 to 85 +/- 11 mmHg (P less than 0.0001). Systolic and diastolic pressures were also significantly reduced in subjects under placebo (from 183 +/- 16 to 165 +/- 21 mmHg, P less than 0.001; and from 101 +/- 9 to 91 +/- 13 mmHg, P less than 0.001, respectively), but the degree of reduction was significantly superior with enalapril (systolic: 39 +/- 25 vs 18 +/- 19 mmHg, P less than 0.005; diastolic: 17 +/- 13 vs 11 +/- 12, P less than 0.001); blood pressure was inferior to 160/90 mmHg in 67% of the subjects treated, as against 35% of those under placebo. Two patients under enalapril died: one on the 27th, the other on the 47th day of treatment. No relation could be established between these deaths and the drug, and this figure of 2 is not significantly different for the number of deaths expected over the same period in a population of that age-group. Among the patients under placebo, one had pulmonary embolism on the 34th day and another had a sudden increase in blood pressure on the 6th day, requiring discontinuation of treatment. It is concluded that enalapril administered alone is effective and well tolerated. Long-term studies are needed to find out whether this angiotensin-converting enzyme inhibitor is superior to a diuretic as initial treatment of arterial hypertension. PMID- 3003731 TI - [Coronary and renal circulation in patients with cardiac failure after converting enzyme inhibitors]. AB - The cardiac output and its peripheral distribution must fulfill the metabolic and/or functional requirements of the different organs. The various techniques used to measure blood flow rates in the coronary and renal arteries provide much information on this point, but they do not tell us all we would like to know about the distribution and "utilization" of these flows in tissues. In normal subjects the myocardial oxygen consumption is not markedly different from the renal oxygen consumption, but the mechanisms that regulate the coronary and renal circulations are not the same. The flow rate in the coronary vessels is about 10% of the cardiac output, the arteriovenous oxygen gradient is superior to 10 vol % and regulation is metabolic. In the renal vessels, which are primarily "functional", the flow rate is about 25% of the cardiac output and the arteriovenous oxygen gradient is inferior to 2 vol %. In heart failure patients, despite reduced cardiac output the blood pressure is kept normal for a long time by vasoconstriction of the arterioles, a process which involves, at least partly, the renin-angiotensin system. The vasoconstriction predominates in some circuits (the renal flow rate is less than 15% of the cardiac output) and spares the "privileged" circuits (the coronary flow rate is more than 15% of the cardiac output). Under the influence of angiotensin converting enzyme inhibitors, the cardiac output increases and its distribution is modified. The coronary flow rate remains stable or is reduced in proportion to the decrease in myocardial oxygen consumption, the metabolic regulation is preserved and there is no "coronary steal". The percent increase in renal flow rate is usually superior to that of the cardiac output. This peripheral redistribution of coronary and renal blood flow rates in heart failure patients after treatment with converting enzyme inhibitors seems to correspond to the physiological purposes of the two regional blood flows. PMID- 3003730 TI - [Worldwide experience with enalapril]. AB - Up to now, the experiments carried out throughout the world with enalapril have been most encouraging. The drug gives good, even excellent responses in 54 to 66 % of patients with essential hypertension, and it is at least as effective as diuretics and beta-blockers. Compared with those of diuretics, the effects of enalapril confirm that the best responders are those patients who are most dependent on the renin-angiotensin system. When a diuretic is administered concomitantly with enalapril, almost all patients respond and the therapeutic effect is well maintained in long term. Blocadren or alpha-methyldopa can be added to hydrochlorothiazide, thus providing additional benefits to patients with severe hypertension. Enalapril reduces the undesirable metabolic effects of hydrochlorothiazide, particularly hypokalaemia. Altogether, enalapril and captopril have similar effectiveness, but enalapril is better tolerated and does not seem to produce the side-effects encountered with captopril, notably skin rashes and ageusia. As expected, enalapril and other angiotensin-converting enzyme inhibitors may be associated with azotaemia in patients with bilateral renovascular hypertension. PMID- 3003733 TI - [Mental nerve neuropathy in myeloma]. PMID- 3003732 TI - [Conversion enzyme inhibitors in the treatment of chronic congestive cardiac failure. Physiopathologic bases of their use]. AB - The treatment of chronic congestive heart failure relies on positive inotropic, diuretic, venous and arterial vasodilating drugs. Several factors are involved in the physiopathology of chronic congestive heart failure such as venous and arterial vasoconstriction mediated by the adrenergic system and/or the renin angiotensin system which increases concomitantly the extracellular volume. These factors are initially compensatory of the low-cardiac output, but aggravate the heart failure on a long-term and are the basis for the use of vasodilating drugs and diuretics. Among vasodilating drugs the converting enzyme inhibitors are of special interest since they inhibit the renin-angiotensin system which is directly involved in the physiopathology of heart failure. On the other hand, they act both as venous and as arterial vasodilators and are generally better tolerated than pure venous or pure arterial vasodilating drugs. They are also better tolerated than the oral inotropic drugs commonly used (mainly digitalis) whose efficacy is controversial in patients being in sinus rhythm. The use of converting enzyme inhibitors, which is now restricted to the more severe cases of chronic heart failure, may be enlarged in the future to the first steps of heart failure, especially if a beneficial effect of these drugs on the long term survey may be proved. PMID- 3003734 TI - [High prevalence of anti-LAV/HTLV-III antibodies in a population of heroin addicts from the Nice area]. PMID- 3003735 TI - [Nasopharyngeal carcinoma with positive Epstein-Barr virus serology in a Hodgkin's disease patient. Viral reactivation?]. PMID- 3003736 TI - [Changes in serum levels of angiotensin converting enzyme in Basedow's disease]. PMID- 3003737 TI - [Effect of somatotropin on carbohydrate metabolism and the interaction of somatotropin with insulin]. PMID- 3003739 TI - A poliovirus temperature-sensitive RNA synthesis mutant located in a noncoding region of the genome. AB - We have constructed an 8-base-pair insertion mutation in the 3' noncoding region of an infectious poliovirus cDNA clone that gives rise to a temperature-sensitive RNA synthesis mutant upon transfection into mammalian cells. The mutated cDNA was used to establish a cell line that releases the mutant poliovirus in a temperature-dependent fashion, representing a unique persistent viral infection. A poliovirus mutant mapping in the noncapsid region of the viral genome can be complemented in this cell line, implying that the cell line expresses viral proteins at the nonpermissive temperature. PMID- 3003738 TI - Involvement of bases 787-795 of Escherichia coli 16S ribosomal RNA in ribosomal subunit association. AB - A nine-base DNA oligomer [d(GTATCTAAT)] was used to probe the accessibility and function of bases in the region 787-795 of Escherichia coli 16S rRNA. Hybridization of the cDNA [d(GTATCTAAT)] to 16S rRNA in situ was carried out by binding the probe to intact 30S ribosomal subunits. Nitrocellulose filter binding showed that cDNA hybridization saturated with increasing probe concentration, suggesting that the probe was binding to a discrete site or sites. RNase H digestion of the rRNA under the DNA . rRNA hybrid and sequencing of the resultant RNA fragments verified that the cDNA probe bound specifically to the 787-795 region. Hybridization experiments using the cDNA probe showed that bases in the 787-795 region of 16S rRNA are exposed on the surface of 30S subunits. The functional role of bases 787-795 was then tested by assaying various ribosomal activities with the cDNA in place. Results of these functional assays demonstrate that this 16S rRNA region is directly involved in the association of 30S and 50S subunits. PMID- 3003740 TI - Isolation of a thermostable enzyme variant by cloning and selection in a thermophile. AB - We developed a method for rapidly generating thermostable enzyme variants. Our strategy is to introduce the gene coding for a given enzyme from a mesophilic organism into a thermophile, Bacillus stearothermophilus. Variants that retain the enzymatic activity at the higher growth temperatures of the thermophile are then selected. This strategy was applied to kanamycin nucleotidyltransferase, which confers resistance to the antibiotic kanamycin. B. stearothermophilus carrying the wild-type enzyme is resistant to the antibiotic at 47 degrees C but not at 55 degrees C and above. Variants that were kanamycin resistant at 63 degrees C were obtained by selection of spontaneous mutants, by passage of a shuttle plasmid through the Escherichia coli mutD5 mutator strain and introduction into B. stearothermophilus by transformation, and by growing the thermophile in a chemostat. The kanamycin nucleotidyltransferases purified from these variants were all more resistant to irreversible thermal inactivation than is the wild-type enzyme, and all have the same single amino acid replacement, aspartate to tyrosine at position 80. Mutants that are even more heat stable were derived from the first variant by selecting for kanamycin resistance at 70 degrees C, and these carry the additional change of threonine to lysine at position 130. This strategy is applicable to other enzymatic activities that are selectable in thermophiles or that can be screened for by plate assays. PMID- 3003741 TI - Detection of single base-pair mismatches in DNA by chemical modification followed by electrophoresis in 15% polyacrylamide gel. AB - We have developed a method for distinguishing fragments of DNA that contain single-base mismatches from their perfectly paired homologues. Single-stranded regions within a duplex fragment are accessible to 1-cyclohexyl-3-(2-[4-(4 methyl)morpholinyl]ethyl)carbodiimide, which reacts with unpaired guanidylate and thymidylate residues in DNA. Intact linear duplex DNA molecules do not react with carbodiimide, whereas DNA molecules containing single-base mismatches react quantitatively. After carbodiimide reaction, the DNA molecules are electrophoresed in high-percentage polyacrylamide gels so that modified and unmodified fragments can be resolved. Application of this technique should make it possible to locate and purify DNA fragments that exhibit sequence differences from those that do not; these might be used to signal phenotypic variation as well as to diagnose inherited disease. PMID- 3003742 TI - Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells. AB - A system in which transfected plasmid DNA replicates in the cytoplasm of poxvirus infected cells is described. A variety of recombinant plasmids was introduced into poxvirus-infected cells by transfection, and replication of input plasmid DNA was monitored by (i) digestion with restriction enzymes that discriminate between input methylated plasmid DNA and unmethylated DNA produced by replication in mammalian cells; (ii) amplification of intracellular plasmid DNA; and (iii) density shift analysis in the presence of BrdUrd. Replication of plasmid DNA was observed in the cytoplasm of cells infected with the tumorigenic leporipoxviruses Shope fibroma virus (SFV) and myxoma, and less extensively with the orthopoxvirus vaccinia, but not in uninfected cells. Unexpectedly, all input plasmids tested, including pBR322, pUC13, polyoma, PM2 phi X174 replicative form (RF), and M13 RF, replicated with equal efficiency in SFV-infected cells, indicating that no specific replication origin sequence is required. The transfected plasmid DNA was replicated concomitantly with the infecting poxviral DNA and by 24 hr post transfection, it resided predominantly in high molecular weight Dpn I-resistant head-to-tail tandem repeats. The failure to detect unreplicated Dpn I-sensitive plasmid concatemers early in replication together with the absence of significant levels of integrated plasmid sequences in the poxviral genome suggest that replication of the transfected plasmid DNA is not the consequence of nonhomologous recombination of concatemeric plasmid DNA into the poxvirus genome, but rather of an autonomous process that is dependent on trans-acting replication factors produced during virus infection, and that does not require a specific origin sequence on the substrate plasmid DNA. PMID- 3003743 TI - Mapping of the human APOB gene to chromosome 2p and demonstration of a two-allele restriction fragment length polymorphism. AB - ApoB is a large glycoprotein with an apparent molecular mass of 550 kDa on NaDodSO4/PAGE. It is a major constituent of most lipoproteins and plays an important role in their metabolism. Recently, apoB cDNA clones have been isolated from an expression library made with mRNA from a human hepatoma cell line. These clones, which were all 1.5-1.6 kilobases (kb) long and corresponded to the 3' end of apoB mRNA, were used to demonstrate that hepatic apoB mRNA is approximately 22 kb long. In the current report, a probe derived from one of these cDNA clones, pB8, was used for in situ hybridization experiments to map the human gene for apoB, APOB, to the distal half of the short arm of chromosome 2. This probe was also used to analyze somatic cell hybrids and, in agreement with the in situ hybridization studies, concordancy was demonstrated with chromosome 2. In addition, two hybrids with chromosome 2 translocations that contain only the short arm reacted with the pB8 probe. A third hybrid with a complex rearrangement of chromosome 2, which deleted an interstitial region and the tip of the short arm of chromosome 2, did not react. These data indicate that APOB maps to either 2p21-p23 or 2p24-pter. In further studies, DNA from normal individuals, digested with the restriction endonuclease EcoRI and subjected to Southern blot analysis with the pB8 probe, revealed a two-allele restriction fragment length polymorphism (RFLP). The major allele was 11 kb, and the minor allele was 13 kb. The minor allele was present with a frequency of 20-25%. The inheritance of the two alleles was studied in an informative family, and they segregated in a typical autosomal Mendelian fashion. The mapping studies provide the means for understanding the relationship of the APOB locus to others in the human genome, whereas the demonstration of an APOB RFLP increases our ability to assess the role of this locus in determining plasma lipoprotein levels. PMID- 3003744 TI - Monoclonal antibody to the type I insulin-like growth factor (IGF-I) receptor blocks IGF-I receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-I and insulin in human skin fibroblasts. AB - Insulin and insulin-like growth factor type I (IGF-I) stimulate an overlapping spectrum of biological responses in human skin fibroblasts. Although insulin and IGF-I are known to stimulate the incorporation of [3H]thymidine into DNA in these cells, the identity of the receptor(s) that mediates this effect has not been fully clarified. The mouse anti-human IGF-I receptor antibody alpha IR-3 binds with specificity to IGF-I but not to insulin receptors in human placental membranes; it also specifically inhibits the binding of 125I-labeled IGF-I but not 125I-labeled insulin to suspensions of human skin fibroblasts in a dose dependent manner. alpha IR-3 competitively inhibits IGF-I-mediated stimulation of [3H]thymidine incorporation into DNA. This inhibition is dependent on the concentration of alpha IR-3 and in the presence of a fixed antibody concentration can be partially overcome by high concentrations of IGF-I. In contrast, at concentrations of less than 1 microgram/ml, the effect of insulin to stimulate [3H]thymidine incorporation is not inhibited by alpha IR-3. However, the incremental effects of higher concentrations (greater than 1 microgram/ml) of insulin on [3H]thymidine incorporation are inhibited by alpha IR-3. alpha IR-3 is a highly specific antagonist of IGF-I receptor-mediated mitogenesis in human skin fibroblasts. By using this antibody, it is shown directly that insulin can act through the IGF-I receptor to stimulate DNA synthesis but can also activate this effect through the insulin receptor itself. PMID- 3003745 TI - Rates of nuclear DNA evolution in pheasant-like birds: evidence from restriction maps. AB - To examine the tempo of genomic evolution in birds, we mapped 161 restriction sites in the nuclear DNA of seven species of birds belonging to the pheasant superfamily Phasianoidea. The three regions mapped lie on different chromosomes and bear eight genes, coding for lysozyme c, three "alpha-like" globins, and four "beta-like" globins. Together, the three regions span about 56 kilobases, most of which is presumably noncoding. The maps differed from one another at a minimum of 77 sites and by 9 length mutations. The extent of sequence divergence due to base substitutions was inferred to be similar for all three regions, even though the three coding regions differ by 5-fold from one another in mean rate of evolution at the amino acid level. A tree relating the maps differs in branching order from that implied by the traditional classification of phasianoid birds and is supported by published protein comparisons. Five of the nodes in the tree were associated with fossil evidence and historical biogeographic information, allowing us to estimate the mean rate of DNA divergence to be 0.34-0.40% per million years. This rate is similar to that estimated for the globin gene regions of higher primates, which validates the concept of an evolutionary clock at the DNA level. Our fossil-based calibration of DNA evolution differs by a factor of almost 2 from that proposed by others on the basis of biogeography. In consequence, published estimates of divergence times for birds and primates that are based on a biogeographically calibrated DNA clock may be too long. PMID- 3003746 TI - A complete library of point substitution mutations in the glucocorticoid response element of mouse mammary tumor virus. AB - The glucocorticoid response element (GRE) of mouse mammary tumor virus (MMTV) was chemically synthesized as two complementary DNA strands bearing cohesive termini. During automated synthesis, random mutations were introduced into the DNA by "doping" each of the four nucleoside phosphoramidites (A, G, C, and T) with a low level of the other three. These preparations were annealed and cloned into an M13 phage vector to produce a library of GRE mutants. Mutations within the synthesized region were identified by sequencing phage isolates at random. All of the chemically distinct classes of transition and transversion mutations have been observed. Statistical considerations indicate that the library contains all of the possible 90 point substitution mutations within a 30-nucleotide mutagenic target. So far 88 of these substitutions have been isolated, 74 as single mutants. At least two of the three possible single mutants at each of the 30 positions have been identified. PMID- 3003747 TI - Rapid mapping of antigenic coding regions and constructing insertion mutations in yeast genes by mini-Tn10 "transplason" mutagenesis. AB - A "transplason" mutagenesis procedure was developed for the dual purposes of low resolution mapping of antigenic coding regions (using transposons) and constructing insertion mutations in yeast genes (by transplacement). Mini-Tn10 transposon derivatives containing both Escherichia coli and yeast selectable markers have been constructed. These elements are used to mutagenize lambda gt11 clones that express foreign antigens in E. coli. The transposition events are first selected in E. coli, and the effect of these insertions on antigen expression is used to locate the antigenic coding regions on the cloned DNA. Insertion mutations located within a desired yeast sequence are then substituted for the genomic copies by one-step gene transplacement. This provides a powerful method for rapidly mapping antigenic coding sequences of cloned genes and inactivating these genes in yeast to help determine their function. Several examples using this technique are presented. PMID- 3003748 TI - Shuttle mutagenesis: a method of transposon mutagenesis for Saccharomyces cerevisiae. AB - We have extended the method of transposon mutagenesis to the eukaryote, Saccharomyces cerevisiae. A bacterial transposon containing a selectable yeast gene can be transposed into a cloned fragment of yeast DNA in Escherichia coli, and the transposon insertion can be returned to the yeast genome by homologous recombination. Initially, the cloned yeast DNA fragment to be mutagenized was transformed into an E. coli strain containing an F factor derivative carrying the transposable element. The culture was grown to allow transposition and cointegrate formation and, upon conjugation, recipients were selected that contained yeast sequences with transposon insertions. The yeast DNA was removed from the vector by restriction endonuclease digestion, and the transposon insertion was transformed into yeast. The procedure required a minimum number of manipulations, and each transconjugant colony contained an independent insertion. We describe 12 transposon Tn3 derivatives for this procedure as well as several cloning vectors to facilitate the method. PMID- 3003749 TI - Detection of lymphocytes expressing human T-lymphotropic virus type III in lymph nodes and peripheral blood from infected individuals by in situ hybridization. AB - By using in situ hybridization methodology, we have directly examined primary lymph node and peripheral blood from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex for the presence of human T-lymphotropic virus type III (HTLV-III) viral RNA. Mononuclear cell preparations were hybridized with a 35S-labeled HTLV-III-specific RNA probe and exposed to autoradiographic emulsion for 2 days. HTLV-III-infected cells expressing viral RNA were detected in approximately 86% (6/7) of lymph node and 50% (7/14) of peripheral blood samples studied. However, in all patient samples examined, labeled cells were observed at very low frequency (less than 0.01% of total mononuclear cells). The HTLV-III-infected cells exhibited morphological characteristics consistent with that of lymphocytes and expressed viral RNA at relatively low abundance (20-300 copies per cell). These results demonstrate that HTLV-III expression in lymph node and peripheral blood is very low in vivo. Furthermore, the lymph node hyperplasia observed in HTLV-III-associated lymphadenopathy is not directly due to proliferation of HTLV-III-infected lymphocytes. PMID- 3003750 TI - Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugia malayi and the animal parasite Brugia pahangi. AB - A 320-base-pair repeated sequence was observed when DNA samples from the filarial parasites Brugia malayi and Brugia pahangi were digested with the restriction endonuclease Hha I. A 640-base-pair dimer of the repeated sequence from B. malayi was inserted into the plasmid pBR322. When dot hybridization was used, the copy number of the repeat in B. malayi was found to be about 30,000. The 320-base-pair Hha I repeated sequences are arranged in direct tandem arrays and comprise about 12% of the genome. B. pahangi has a related repeated sequence that cross hybridizes with the cloned B. malayi Hha I repeat. Dot hybridization with the cloned repeat shows that the sequence is present in B. malayi and in B. pahangi but not in four other species of filarial parasites. The cloned repeated DNA sequence is an extremely sensitive probe for detection of Brugia in blood samples. Hybridization with the cloned repeat permits the detection of DNA isolated from a single parasite in an aliquot of blood from animals infected with B. malayi. There are differences in the restriction sites present in the repeated sequences that can be used to differentiate between the two Brugia species. The B. malayi repeated DNA sequence is cleaved by Alu I and Rsa I but the B. pahangi sequence is not. A comparison of repeated sequences between the two species by DNA sequence analysis indicates that some regions of individual repeats are over 95% homologous, while other short regions are only 60-65% homologous. These differences in DNA sequence will allow the construction of species-specific hybridization probes. PMID- 3003751 TI - Expression of the oncogene of avian reticuloendotheliosis virus in Escherichia coli and identification of the transforming protein in reticuloendotheliosis virus T-transformed cells. AB - The genome of reticuloendotheliosis virus T (REV-T) includes a unique oncogene v rel, which is transcribed in low amounts into a 3.0-kilobase subgenomic mRNA in REV-T-transformed lymphoid cells. To identify the v-rel protein, REV-T DNA sequences were cloned into bacterial plasmid vectors designed to achieve expression of foreign DNA sequences in Escherichia coli. Portions of the v-rel gene were joined to the 5' segment of the trpE gene. Upon induction of trpE with indoleacrylic acid, large amounts of trpE-v-rel fusion proteins were produced by the bacteria carrying these recombinant plasmids. Two trpE-v-rel fusion proteins were synthesized in E. coli, which collectively represent three-quarters of the predicted v-rel protein. Polyclonal antisera were generated to trpE-v-red fusion proteins. These antisera were used in immunoblotting experiments to identify a 57 kDa v-rel protein in REV-T-transformed lymphoid cells lines and REV-T-infected chicken embryo fibroblast cultures. The v-rel gene expressed in E. coli under lac control was found to produce a 56-kDa protein. Although REV-T-transformed and Marek disease virus-transformed lymphoid cells contain c-rel mRNA transcripts, a c-rel protein could not be detected with antisera directed against v-rel fusion proteins. PMID- 3003752 TI - The anemia of thermal injury: mechanism of inhibition of erythropoiesis. AB - The anemia of thermal injury is a multifactorial process and includes hemorrhage and hemolysis. Much evidence suggests that a reduced rate of erythropoiesis contributes to this anemia. Prior studies show that this anemia is temporally related to the appearance in burn patients sera of a substance(s) capable of inhibiting erythropoiesis in vitro. Four experiments were done to elucidate the mechanism of action of this inhibitor. In all experiments sera from burn patients previously shown to be inhibitory to erythropoiesis in vitro were studied. In the first, inhibitory sera were exposed to erythropoietin solutions without loss of erythropoietic activity. Second, mouse marrow cells were preincubated with serum without loss of their ability to form erythroid colonies. Third, the inhibitory effect could not be overcome with increasing amounts of erythropoietin. Finally, erythroid colony formation was effected only if the inhibitory serum was present during the first 8 to 12 hr of culture. The data suggest that the erythropoietic inhibitor in these sera acts directly on erythroid stem cells in vitro and not by inactivating or interference with erythropoietin. PMID- 3003753 TI - Genetic obesity and dietary sucrose decrease hepatic glucagon and insulin receptors in LA/N-corpulent rats. AB - A catabolic and hypolipemic effect of glucagon has been described in normal animals. We therefore studied the role of glucagon in genetically obese, hyperlipemic rats. Twelve genetically obese hyperlipemic LA/N-cp/cp (corpulent) rats and 12 lean littermates were fed either 54% starch or 54% sucrose for 12 weeks. Plasma glucagon and insulin levels and glucagon and insulin binding to liver membranes were measured. Comparing all corpulent and lean animals regardless of diet, a significant (P less than 0.0001) phenotypical effect (cp/cp greater than lean) was observed in plasma insulin levels (464 +/- 54 vs 70.3 +/- 7.6 muu/ml, mean +/- SEM). Insulin binding (2.68 vs 16.1%/50 micrograms protein) and glucagon binding (25.6 vs 47.3%/50 micrograms protein) were both significantly lower (P less than 0.0001) in corpulent rats as compared to their lean littermates. Sucrose feeding had marginal effect on plasma insulin or insulin binding. It, however, decreased glucagon binding in corpulent rats but not in their controls. A significant negative correlation was observed between plasma insulin and insulin binding, while a positive correlation was seen for plasma glucagon and glucagon binding. A significant negative correlation was observed between plasma glucagon and lipogenic enzymes (glucose-6-phosphate dehydrogenase and malic enzyme) in liver and between glucagon binding and these enzymes. We propose that in these genetically obese rats, in addition to hyperinsulinemia, impaired glucagon activity as manifested by decreased glucagon binding to target cells may be an important contributor to the hyperlipemia and obesity. A further decrease in glucagon binding in rats fed sucrose indicates that sucrose, per se, may be an additional contributory factor. PMID- 3003754 TI - Alterations in jejunal transport and (Na+-K+)-ATPase in an experimental model of hypoxia in rats. AB - Hypoxia was induced by exposing rats to an atmosphere of 93% N2, 7% O2 for 4-48 hr. The animals became hypoxic as indicated by a decreased blood PaO2 (mean +/- SEM: 48 +/- 10 mm Hg). Hypoxia was accompanied by metabolic acidosis (pH 7.22 +/- 0.02) and decreased serum bicarbonate levels (9.0 +/- 4.0 meq/liter). Hypoxic rats also showed evidence of tissue hypoxia; liver tryptophan oxygenase levels were increased to 21 +/- 2 nmole/min/mg protein. In the hypoxic animals there was decreased jejunal mucosal (Na+-K+)-ATPase activity and an inhibition of active intestinal transport of sodium, glucose, 3-O-methylglucose, galactose, tyrosine, phenylalanine, and glycine as determined by in vivo perfusion studies. Jejunal fructose transport, which has a large passive component, was unaffected by hypoxia. The electrolyte, carbohydrate, and amino acid transport alterations produced by hypoxia were seen in the absence of an effect on jejunal cell number, DNA synthesis, or cell turnover. There was also no evidence of histological or ultrastructural damage. Furthermore, studies with a luminal macromolecular tracer, horseradish peroxidase, indicated that the jejunal lumen-to-blood barrier to macromolecules was also unaltered in these hypoxic animals. In vitro local oxygenation of the jejunum, by bubbling of 95% O2:5% CO2, markedly improved sodium and glucose (but not 3-O-methylglucose) absorption in hypoxic rats and control rats. The (Na+-K+)-ATPase activity of the jejunal mucosa of hypoxic rats was significantly enhanced by the local bubbling of 95% O2:5% CO2. Overall, our data indicate that during relatively mild conditions of hypoxia there is an inhibition of jejunal (Na+-K+)-ATPase activity and related transport processes that is prevented by in situ oxygenation. PMID- 3003756 TI - Delta 9-tetrahydrocannabinol decreases alpha/beta interferon response to herpes simplex virus type 2 in the B6C3F1 mouse. AB - This study was undertaken to determine the effect of delta 9-tetrahydrocannabinol (delta 9-THC) on polyinosinic:polycytidylic acid [poly(I):poly(C)]-induced, and on herpes simplex virus type 2 (HSV-2)-induced, alpha/beta interferon in the B6C3F1 mouse. Animals were administered delta 9-THC, or the diluent, intraperitoneally for 4 consecutive days or at various time intervals prior to administration of the interferon inducer. Poly(I):poly(C) or HSV-2 was injected intravenously on Day 4. Animals receiving poly(I):poly(C) and treated with delta 9-THC at doses ranging from 5 to 100 mg/kg exhibited significantly lower titers of interferon than mice given poly(I):poly(C) and the diluent. Diminished interferon titers occurred in HSV-2-infected animals treated with delta 9-THC in doses exceeding 15 mg/kg when compared to virus-infected animals given the diluent. This suppression of early interferon persisted through 24 hr. PMID- 3003755 TI - Estrogens with reduced catechol-forming capacity fail to induce implantation in the rat. AB - Catechol estradiol can induce implantation of the embryo in a progesterone-primed uterus, but we do not know whether conversion of estrogen to a catechol is essential for implantation. The present study examined the ability of fluorinated estradiols that have a reduced capability of catechol formation to induce implantation. Delayed implantation in rats that were hypophysectomized on the third day postcoitum was maintained by daily injection of progesterone. On the fifth day of progesterone treatment they were injected intravenously with estradiol-17 beta (E2), or various doses of 2-fluoroestradiol-17 beta (2-F1-E2) or 4-fluoroestradiol-17 beta (4-F1-E2) and examined 24 hr later for evidence of initiation of implantation. All animals treated with 25 ng of E2 showed normal numbers of implantation sites, as did those receiving 60 ng of 4-F1-E2. In contrast, 2-F1-E2 failed to initiate implantation with doses as high as 300 ng per animal; there were only single sites in two of eight rats treated with 500 ng. Initiation of implantation was not correlated with lack of uterotropic estrogenicity. The results suggest that formation of catechol estrogen may be an important step in mediating estrogen function for implantation of the embryo. PMID- 3003757 TI - Uptake of [3H]vitamin D3 from low and high density lipoproteins by cultured human fibroblasts. AB - The plasma distribution and cellular uptake of [3H]vitamin D3 was studied in vitro using cultured human fibroblasts. Incubation of [3H]vitamin D3 (cholecalciferol) with plasma followed by sequential ultracentrifugal fractionation of the lipoproteins indicated that 2-4% of the radioactivity associated with the very low density lipoprotein (VLDL), 12% with low density lipoprotein (LDL), and approximately 60% with the high density lipoprotein (HDL). The remaining radioactivity, 25%, was associated with the sedimented plasma fractions. By comparison, an average of 86% of the radioactivity from [3H]1,25 dihydroxycholecalciferol associated with the sedimented plasma fractions. The uptake of [3H]vitamin D3 from plasma, LDL, or HDL was studied in cultured human cells; uptake by normal fibroblasts was greatest from LDL and least from plasma. The cellular association of vitamin D3 was time, concentration, and temperature dependent. At a concentration of 50 micrograms LDL/ml of medium, the uptake of [3H]vitamin D3 from LDL at 37 degrees C was rapid and reached a maximum at approximately 4 hr; it was slower from HDL but continued to increase slowly up to 24 hr. The significance of these in vitro findings is uncertain since much of the vitamin D3 absorbed from the intestine reportedly associates with chylomicrons and is rapidly taken up by the liver. PMID- 3003758 TI - Human cytomegalovirus: development and progression of nuclear inclusions by primary clinical isolates and laboratory-adapted strains. AB - The morphogenesis of cytomegalovirus (CMV) nuclear inclusions (NIs) was investigated using unadapted clinical isolates and adapted laboratory strains. Both adapted and unadapted strains of CMVs induced NIs whose morphologic appearance was similar in human fibroblastic cells. Early NIs appeared as ring like structures composed of dense granular and fibrillar material, while late NIs appeared to consist of multiple electron-lucent areas containing coarse granules and bounded by electron-dense fibrillar material (cellulae). Capsids and nucleocapsids were associated primarily with the electron-dense fibrillar material; however, developing nucleocapsids were most often observed at the interface of the electron-dense and -lucent areas. Although there was some variation in the rate of development and maturation of the NIs with the intensity of infection, all CMVs examined produced late NIs with similar organizational patterns consisting of cellulae. Substitution of human fibroblastic cells derived from various tissues as cellular substrate did not appreciably affect the results. Thus, the unique organization of the CMV late NI, consisting of multiple cellulae, appears to be an intrinsic feature of CMV replication since it seems to be independent of the extent of laboratory adaptation, the virus strain, the intensity of infection, or the cell type. PMID- 3003760 TI - Platelets from aged rats aggregate more, but are more sensitive to prostacyclin. AB - The balance between vascular eicosanoids has been explored in young (1 month of age) and aged (11 month of age) rats. PRP from mature rats was more reactive to collagen (lower threshold, greater amplitude of aggregation curve and higher TxB2 formation) than PRP from young animals. Release of 6-keto-PGF1 alpha from PRP perfused isolated aortas (pg@ul) was higher and the inhibition of PRP aggregation after perfusion correspondingly greater, in mature rats. Platelets from aged rats were more sensitive to exogenous prostacyclin (higher inhibition of aggregation and greater accumulation of cAMP). The fatty acid compositions of plasma and platelet lipids were not different in the two age groups. Compensatory mechanisms were operating in the aged rats, counteracting the greater platelet aggregability with higher vascular prostacyclin production and greater sensitivity of platelets to this eicosanoid. PMID- 3003759 TI - Forskolin-induced bone resorption in neonatal mouse calvaria in vitro. AB - The role of cyclic adenosine 3',5'-monophosphate (cAMP) in inducing bone resorption was studied in neonatal mouse calvaria in vitro. Forskolin, a stimulator of adenylate cyclase, increased the medium calcium concentration at 96 hr of incubation, indicating enhanced bone resorption. Bone resorption was observed between 1 X 10(-4) and 1 X 10(-6) M forskolin; the maximal effect was at 1 X 10(-5) M and there was no effect at 1 X 10(-7) M. Lactic acid release was increased during the 96 hr of incubation in proportion to the calcium release in the media. The bone acid phosphatase activity was increased and the alkaline phosphatase activity was decreased. Bone carbonic anhydrase activity was increased more than twofold. Forskolin-induced bone resorption was significantly but incompletely inhibited by 10(-4) M acetazolamide, a carbonic acid anhydrase inhibitor. These findings support the concept that carbonic anhydrase plays a significant role in bone resorption. PMID- 3003761 TI - Benzodiazepine binding sites in mice forebrain and kidneys: evidence for similar regulation by GABA agonists. AB - Benzodiazepine binding sites in mice forebrain and kidney homogenate were labelled either in vivo (SC injection) or in vitro by 3H-flunitrazepam. The binding in both cases was carried out in vitro in the presence or absence of 10 microM unlabelled flunitrazepam during 60 min at 0 degrees C. When benzodiazepine binding sites were labelled in vivo pretreatment of animals with muscimol (0.75 and 1.5 mg/kg, IP) and fenibut (25 and 100 mg/kg, IP) in a dose-dependent manner facilitated 3H-flunitrazepam binding both in mice forebrain and kidneys. In vitro labelling of benzodiazepine binding sites demonstrated similar to in vivo labelling in forebrain but not in kidneys. The peripheral benzodiazepine binding sites in kidneys are therefore modulated similarly to benzodiazepine binding sites in forebrain by GABA-ergic drugs. PMID- 3003762 TI - Benzodiazepine-like effects of inosine on punished behavior of pigeons. AB - Behavioral effects of the putative endogenous benzodiazepine receptor ligand, inosine, were studied alone and in combination with the benzodiazepine antagonist Ro 15-1788. Keypeck responses were maintained by food under a multiple fixed interval 3-min, fixed-interval 3-min schedule of food delivery. Under the multiple schedule, the first response after 3 min produced food in the presence of either white (no punishment) or red keylights and, in addition, each 30th response produced a brief electric shock (punishment) when the keylight was red. Inosine increased the low rates of punished responding (10-100 mg/kg IM) and the higher rates of unpunished responding (30 mg/kg). The benzodiazepine antagonist Ro 15-1788 (0.03 mg/kg, IM) antagonized the rate-increasing effects of inosine but had no effect when given alone. Combinations of inosine (30 mg/kg) with higher doses of Ro 15-1788 (0.1-1 mg/kg) decreased responding much like Ro 15 1788 alone. The marked rate-decreasing effects of 1000 mg/kg inosine were not affected by concurrent administration of Ro 15-1788 (0.01-1 mg/kg). The behavioral effects of inosine alone resembled effects of benzodiazepines but not those of benzodiazepine antagonists. The response rate-increasing effects of inosine may be due to its benzodiazepine receptor binding properties, whereas the rate decreases produced by higher doses of inosine appear to be unrelated to benzodiazepine receptors. PMID- 3003763 TI - Interstrain correlation between behavioural effects of lithium and effects on cortical cyclic AMP. AB - Six inbred mouse strains were studied to explore possible correlations between lithium effects on behaviour and on cortical cyclic AMP. The strains were fed lithium in ground food for 3 weeks before behavioural tests and ex vivo evaluation of cyclic AMP accumulation. Replicating previous reports, there was a significant inverse correlation (r = 0.73, n = 6) between spontaneous activity and noradrenaline-induced cyclic AMP, and an almost significant correlation (r = 0.67, n = 6) between spontaneous activity and adenosine-induced cyclic AMP accumulation. The effect of lithium to depress spontaneous activity correlated with its effect to inhibit adenosine-induced rises in cyclic AMP (r = 0.716, n = 6). There were significant strain differences in the behavioural responses to amphetamine. In two strains where amphetamine raised activity and lithium inhibited the amphetamine-induced rise, lithium also significantly inhibited the adenosine-induced rise in cyclic AMP. Two other strains showed amphetamine induced rises in activity that were not inhibitable by lithium, and these strains showed no significant inhibition by lithium of adenosine-induced cyclic AMP accumulation. The remaining two strains showed no behavioural activity increase with amphetamine. PMID- 3003764 TI - Effects of an extract of Ginkgo biloba on noradrenergic systems of rat cerebral cortex. AB - Oral treatment of rats with Ginkgo biloba extract (Gb) elicited a biphasic effect on normetanephrine (NMN) content of cerebral cortex; an initial decrease was evident after 45 minutes, followed by a marked increase that was evident after 14 days. Chronic treatment with Gb led to decreases in the density of 3H dihydroalprenolol binding (after 27 days or 2 months) and in isoproterenol stimulated adenylate cyclase activity (after 2 months) of the cerebral cortex. Taken together, these results indicate that the effects of Gb on the central beta adrenergic system might be involved in its therapeutic actions. PMID- 3003765 TI - Monoclonal antibodies to steroid hormone receptors. PMID- 3003766 TI - Submaximal plasma prolactin response to TRH and dopamine activity in man. AB - Submaximal plasma prolactin responses to TRH were evaluated as a measure of endogenous dopamine activity in man. In 25 male subjects, plasma prolactin responses to 0.5 mg i.m. haloperidol were significantly correlated with plasma prolactin responses to 12.5 micrograms i.v. TRH but not with plasma prolactin responses to 200 micrograms i.v. TRH or with basal plasma prolactin levels. The results suggest that submaximal plasma prolactin responses to TRH may reflect the effects of endogenous dopamine activity on prolactin release more accurately than the prolactin measures previously used. PMID- 3003767 TI - Interdependence of fluence, drug dose and oxygen on hematoporphyrin derivative induced photosensitization of tumor mitochondria. PMID- 3003768 TI - Differential effects of photoactive furanyl compounds on virus functions. PMID- 3003769 TI - Cellular mechanisms of inorganic phosphate transport in kidney. PMID- 3003771 TI - [Malignant neoplasms of the ovaries]. PMID- 3003770 TI - Role of endogenous opiates and extracellular K+ accumulation in the inhibition of frog spinal reflexes by electrical skin stimulation. AB - Electrical skin stimulation of the hind limb (10-100 Hz, 30 s-5 min) at the intensity which leads only to the excitation of low threshold afferents depressed (for 1-30 min) the flexor reflex evoked in spinal frogs by nociceptive stimuli. The inhibition, which lasted for longer than 5 min was blocked by naloxone. Short term poststimulation effects were associated with an increase of extracellular K+ concentration (delta [K]e) and were not blocked by naloxone. Enkephalins or morphine applied to the spinal cord surface increased the threshold for flexor reflexes while naloxone decrease their threshold. The stimulation was followed by short-term hyperpolarization of primary afferents (PAH; 1-5 min) and by depression of dorsal root potentials (DPRs) which had a similar time course to the delta [K]e, and were not blocked by naloxone. This period was frequently followed by longlasting PAH and enhancement of DRPs (5-30 min), which were abolished by naloxone. Superfusion of the isolated spinal cord with opioids produced PAH and enhanced DRPs evoked by nociceptive stimuli, while naloxone or increase of [K] in Ringer solution depolarized primary afferents and depressed DRPs. It is suggested that the antinociceptive effects of electrical stimulation of low threshold cutaneous afferents in spinal frogs involves at least two mechanisms. The short-term effect may result from delta [K]e, especially at high stimulus strength and is equally effective against noxious and non-noxious stimuli. The longlasting effects selectively affecting nociceptive transmission appear to be produced by endogenous opioids. PMID- 3003772 TI - [Vaccine against viral hepatitis]. PMID- 3003773 TI - [Electromyography analysis of the denervation and reinnervation processes]. AB - An analysis of motor unit action potentials duration in 1065 patients suffering from various disorders of peripheral motor nerve and the results of fiber density measure in 88 patients are presented. The authors show the benefits of using of action potential duration histograms for diagnostic aims. The measurement of average potential duration is not recommended. PMID- 3003774 TI - Pro-opiomelanocortin-related peptides in cerebrospinal fluid: a study of manic depressive disorder. AB - Five peptide fragments of pro-opiomelanocortin (alpha-melanocyte-stimulating hormone, beta-lipoprotin, adrenocorticotropic hormone, beta-endorphin, and the N terminal fragment of pro-opiomelanocortin) were measured by radioimmunoassay in cerebrospinal fluid (CSF) and plasma from 31 normal volunteers and 26 euthymic lithium-treated bipolar patients (14 of whom provided a second CSF sample in the unmedicated state). With the exception of alpha-melanocyte-stimulating hormone, in the normal volunteers' CSF, levels of these peptides were highly correlated with one another, suggesting that: (1) some common regulatory factor may control the levels of these four peptides in CSF; and (2) CSF alpha-melanocyte stimulating hormone is independently regulated from the other pro opiomelanocortin products. Some of these correlations were absent in the patient groups, suggesting subtle alterations in pro-opiomelanocortin processing in manic depressive illness. No effect of lithium on the CSF levels of these peptides was observed. No group differences were found. PMID- 3003775 TI - Effects of ACTH1-24 on male rat behavior in an exploratory, copulatory and socio sexual approach test. AB - The effect of intraventricular application of ACTH1-24 on exploratory behavior, excessive grooming, and socio-sexual behavior in male rats was studied. It appeared that ACTH1-24 (1 microgram/animal) affects both excessive grooming and exploration. Sexual performance was delayed, as expressed in latency time to ejaculation, probably because of prolonged grooming behavior. The behavioral effects of ACTH are explained in terms of enhanced attention to external stimuli. PMID- 3003776 TI - Development and regeneration of motor systems under the influence of ACTH peptides. AB - ACTH peptides exert quantitative and qualitative influences on the formation and maturation of motor units in developing and regenerating neuromuscular systems. ACTH 4-10, administered daily (10 micrograms/kg. s.c.) from the day of birth, accelerated the rate at which muscle strength developed in the immature rat, the effect of this peptide being most marked in animals 11-15 days old. A similar increase in grasping time occurred in ACTH 4-10 treated animals, indicating that the peptide affects neuronal maturation at a time in development when organization and maturation of the neuromuscular system is most active. The synthetic analogue of ACTH 4-9 (Org 2766), administered in the same dosage, had little effect on these parameters, indicating a differential sensitivity to these similar peptides. Elevated circulating titers of ACTH, whether exogenous (0.2 U ACTH 1-39 IP daily), or endogenous (adrenalectomy), stimulated the formation of more functional motor units, as indicated by increased amplitude of muscle action potentials and tetanic tension following nerve stimulation. ACTH appears to favor the recovery of high threshold, small-size motor units. Fine control of muscle function in peptide-treated animals is partially restored, as indicated by the return of stepwise recruitment to an extent not seen in the reinnervated, saline treated controls. PMID- 3003777 TI - Failure of the GABAergic drug, sodium valproate, to reduce basal plasma prolactin secretion in chronic schizophrenia. AB - The psychoneuroendocrinology of schizophrenia derives from the presumption that neurotransmitter or receptor abnormalities in the limbic regions might extend to or influence the hypothalamus, which plays a role in the regulation of prolactin (PRL) secretion from the anterior pituitary gland. Since a GABA disturbance has been recently proposed in the pathogenesis of certain schizophrenic symptoms, and since a tuberoinfundibular-GABA (TI-GABA) system has been shown to modulate PRL secretion in humans, we tested the activity of this system both in controls and in chronic schizophrenic women. For this purpose the GABAergic drug sodium valproate (800 mg) was administered orally to 20 healthy women and 18 chronic schizophrenic women. Plasma PRL levels were measured before and after the drug administration. Sodium valproate decreased PRL concentrations only in the healthy women. Although the hypothesis of a GABA disturbance in schizophrenia at present is only speculative, these results might suggest a defect of the TI-GABA system in chronic schizophrenia. PMID- 3003780 TI - Evaluation of beta-adrenergic influences on cardiovascular and metabolic adjustments to physical and psychological stress. PMID- 3003779 TI - Evidence for multiple opiate receptor involvement in different phencyclidine induced unconditioned behaviors in rats. AB - Rats were used for comparing the behavioral response profiles of phencyclidine (PCP) and d,1-N-allylnormetazocine (NANM), two drugs that are proposed to exert their effects through the "PCP/sigma" receptor. Phencyclidine (1.0-5.0 mg/kg) and NANM (2.5-10.0 mg/kg) induced dose-related increases in locomotion, sniffing, repetitive head movements, non-object directed mouth movements, and ataxia. Both drugs also increased food and water consumption during the latter portion of the drug response. Ingestive behaviors induced by PCP (2.5 mg/kg), as with eating and drinking stimulated by the mu-opiate morphine (2.0 mg/kg), were blocked by a relatively low dose of the opiate antagonist naloxone (0.5 mg/kg). Multiple injections of PCP (2.5 mg/kg for 4 days) or NANM (10.0 mg/kg for 4 days) augmented several measures of behavioral activation, including horizontal locomotion, rearing, and nonfocused sniffing, but did not significantly change stereotyped behaviors or ataxia. Reciprocal cross-sensitization of locomotor activation is indicated by the finding that the response to a challenge injection of PCP (2.5 mg/kg) or to NANM (10.0 mg/kg) after 4 days of treatment with the other drug closely resembled the enhanced locomotor response observed after the chronic treatment. Phencyclidine and NANM thus appear to exert many of their effects on unconditioned behavior through common mechanisms, including interaction with sigma receptors. In addition, these findings are consistent with previous suggestions that a mu-opiate receptor system may modulate some effects of PCP. PMID- 3003778 TI - Intrinsic actions of the benzodiazepine receptor antagonist Ro 15-1788. AB - The imidazodiazepine Ro 15-1788 is a benzodiazepine receptor antagonist that was initially reported to be lacking in intrinsic activity in a variety of test situations in which benzodiazepine-like effects can be identified. However, many recent studies have shown that this compound does indeed have intrinsic activity in a variety of behavioural, neurological, electrophysiological and biochemical preparations in both animals and man. The purpose of the present review is firstly to describe these intrinsic actions, and secondly to consider to what extent these intrinsic actions of Ro 15-1788 have implications for current concepts of the functioning of the benzodiazepine receptor. PMID- 3003781 TI - Dexamethasone suppression test and familial subtypes of unipolar depression. AB - We have studied the suppression of plasma cortisol after dexamethasone (1 mg) and the peak post-dexamethasone cortisol values in 84 hospitalized patients with a diagnosis of primary unipolar depression. The non-suppressor responses in the three familial subgroups (Winokur) were: 11/22 in familial pure depressive disorder (FPDD), 21/49 in sporadic depressive disease (SDD) and 1/13 in depression spectrum disease (DSD) (FPDD vs. SDD, p less than 0.05; SDD vs. DSD, p less than 0.05). When considering the peak post-dexamethasone cortisol value, or the 8.30-9.00-hour values, the results in the DSD group were lower than in the other two groups (p less than 0.05). These results suggest a different behaviour of DSD as compared with FPDD and SDD. PMID- 3003782 TI - Charged particle cytogenetics: effects of LET, fluence, and particle separation on chromosome aberrations. AB - Induced rearrangements of chromosomes, disrupting the orderly sequence and/or separation of the genetic material, are responsible for a significant proportion of cellular lethality, genetic mutation, and, as has become increasingly apparent in recent years, human cancer. The quantitative observation of chromosomal aberrations induced by ionizing radiations led early to the realization that as linear energy transfer (LET) increased, curvilinear dose responses became increasingly linear. Those few studies that examined aberrations as a function of LET found that the optimally effective LET was about 100 keV per micrometer, results consistent with those observed for other end points. The majority of chromosomal aberrations originate from molecular interaction between pairs of lesions (misrepair), with differences in sensitivity to aberration induction through the cell cycle. In Chinese hamster V-79 cells for all LET values studied, aberrations are most frequent in G2, then G1, then S phase of the cell cycle. The variation in sensitivity through the cell cycle changes from a factor of about 5 for 10 keV/micron particles to about 3 for 80 keV/micron particles. In the G2 phase a curvilinear dose response (G1 and S being linear) is found for all LETs occurring at fluences where there are substantial distances (greater than or equal to 3 micron) between particles. It is possible that for this one phase of the cell cycle a saturation of repair capabilities occurs as a function of both fluence and LET. When cells were irradiated with associated charged particles (molecular ions) it was found that even when two particles were separated by distances of less than 100 nm their effect was much less than one particle of twice the LET (the equivalent of 0 distance separation). This implies that the vast majority of molecular interactions which result in chromosomal aberrations occur as a consequence of interaction between damaged sites formed only a few nanometers from each other. It is clear that an analysis of chromosomal aberrations produced by charged particles can provide considerable insight into basic radiobiological mechanisms and into the organization of the mammalian genome. PMID- 3003783 TI - Calculation of heavy-ion tracks in liquid water. AB - Detailed Monte Carlo calculations are presented of proton and alpha-particle tracks in liquid water. The computations treat the interactions of the primary particle and all secondary electrons on a statistical, event-by-event basis to simulate the initial physical changes that accompany the passage of an ion through water. Our methods for obtaining the cross sections needed for such calculations are described. Inelastic scattering probabilities (inverse mean free paths) are derived from a complex dielectric response function constructed for liquid water, based on experimental and theoretical data. Examples of partial cross sections for ionization and excitation by protons are shown. The computation of electron transport and energy loss includes exchange, elastic scattering, and a scheme for the delocalization of energy shared collectively by a large number of electrons in the condensed medium. Several examples of calculated proton and alpha-particle tracks are presented and discussed. The meaning and significance of the concept of a track core are briefly addressed in the light of this work. The present paper treats only the initial, physical changes produced by radiation in water (in approximately 10(-15) s in local regions of a track). The work described here is used in calculations that we have reported in other publications on the later chemical development of charged particle tracks. PMID- 3003784 TI - Progress in low-LET heavy particle therapy: intracranial and paracranial tumors and uveal melanomas. AB - The Harvard Cyclotron Laboratory in collaboration with the Department of Radiation Medicine of the Massachusetts General Hospital and the Retina Service of the Massachusetts Eye and Ear Infirmary provides low-LET heavy particle therapy with 160 MeV protons. The improved dose distribution of protons results from their physical characteristics. A total of 965 patients have been treated as of December 31, 1984. Dose is expressed in units of cobalt gray equivalent (CGE) which is the dose in Gy multiplied by the RBE (1.1) for modulated protons relative to 60Co radiation. Sixty-seven patients with chordomas or low-grade chondrosarcomas of the base of skull or cervical spine have received proton treatment. Forty-three of these patients have been followed for at least 8 months with a median follow-up of 27 months. The median dose is 69 CGE. The 3-year actuarial local control rate is 89%. Seven patients with gliomas, eight with craniopharyngiomas, and six with meningiomas have also received proton radiation treatments. A total of 615 patients with uveal melanomas have received a median dose of 70 CGE in five fractions. Tumor regression has been seen in 94% with 66% having vision of 20/100 or better. PMID- 3003785 TI - Proton therapy in Japan. AB - There are two facilities for clinical trials with protons in Japan: the National Institute of Radiological Sciences (NIRS), Chiba, and the Particle Radiation Medical Science Center (PARMS), University of Tsukuba. At the National Institute of Radiological Sciences, patient treatment with the 70 MeV proton beam began in November 1979, and 29 patients were treated through December 1984. Of 11 patients who received protons only, 9 have had local control of the tumor. Two of the 9 patients, suffering from recurrent tumor after radical photon beam irradiation, developed complications after proton treatment. In the patients treated with photons or neutrons followed by proton boost, tumors were controlled in 12 of 18 patients (66.6%), and no complications were observed in this series. Malignant melanoma could not be controlled with the proton beam. A spot-beam-scanning system for protons has been effectively used in the clinical trials to minimize the dose to the normal tissues and to concentrate the dose in the target volume. At the Particle Radiation Medical Science Center, University of Tsukuba, treatment with a vertical 250 MeV proton beam was begun in April 1983, and 22 patients were treated through February 1984. Local control of the tumor was observed in 14 of 22 patients (63.6%), whereas there was no local control in the treatment of glioblastoma multiforme. There have been no severe complications in patients treated at PARMS. The results suggest that local control of tumors will be better with proton beams than with photon beams, whereas additional modalities are required to manage radioresistant tumors. PMID- 3003786 TI - Preparatory clinical studies of Pi-mesons at TRIUMF. AB - Eighty patients have been treated with Pi-mesons (pions) at TRIUMF between 1979 1984. The patients had tumors rarely curable by standard methods and had no prior radiotherapy. The distribution by site included skin, metastatic nodules (13), brain, glioblastoma multiforme (32), pelvis, rectosigmoid (15), prostate (12), bladder (7), and ovary (1). The studies involve serial escalations of pion dose until maximum tissue tolerance is reached, monitoring the response at each dose increment. Sites were chosen for study where lack of local control is a significant cause of treatment failure with conventional radiation therapy. The low dose rate and the available beam access at TRIUMF limit the number of patients treated and the volume treatable. A 3-D treatment planning program is in use, and a 3-D display of the dose distribution delivered in brain tumor treatments has been developed using the PET scanner. In practice, new methods introduced for measurement of tissue response include tumor growth delay curves, fine-needle biopsy mapping, and PET scanning of brain tumors. The use of endoscopic assessment of the rectosigmoid region is emphasized. Treatment results of glioblastoma multiforme show that the median survival for patients treated to 125 pion cGy/fx is in the range of 187-198 days; for patients receiving 170 cGy per dose/fraction (fx) the range is 290-315 days, and for those receiving 200-220 cGy/fx the median survival is in excess of 290 days. For pelvic malignancies the local control obtained with doses of 2500 cGy or less was 50% in 12 assessable patients; it was 75% in 20 patients who had 3000 cGy or more.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003787 TI - Biomedical program for the converted 200-MeV synchrocyclotron at the Gustaf Werner Institute. AB - Between 1956 and 1977, the former synchrocyclotron in Uppsala was used for biological experiments and clinical tests with 185-MeV protons. Therapeutic irradiations have been performed since 1957 by cross-firing with pencil beams through small intracranial structures for the treatment of Parkinsonism and intractable pain and with the spread-out Bragg peak for the treatment of large malignant tumors. Radiological and radiophysical aspects of the use of charged particle beams were studied in detail. The former accelerator is now being converted to a sector-focusing, frequency-modulated cyclotron, SFSC-200, to permit acceleration of protons up to 200 MeV and other light ions to corresponding energies. Production of spallation neutrons and radionuclides for biomedical uses is expected to start this year. Experiments with charged-particle beams will begin in 1986. This paper presents a discussion of accelerator developments for planned experimental and clinical programs. PMID- 3003788 TI - Local changes after intramuscular administration of gammaphos (WR-2721) in rats. PMID- 3003789 TI - Tissue adenyl cyclase and phosphodiesterase enzymes in whole body gamma irradiated rats. PMID- 3003790 TI - Time course of 99mTc-pyrophosphate and 99mTc-gluconate uptake in experimental ischaemic myocardial necrosis. PMID- 3003791 TI - [Dynamic computed tomography and sequential scintigraphy--a comparison in cases of cerebral masses]. PMID- 3003793 TI - Cholangiocarcinoma complicating primary sclerosing cholangitis: cholangiographic appearances. PMID- 3003792 TI - Gastrointestinal complications of AIDS: radiologic features. AB - The radiologic features were examined in a retrospective review of 25 patients with gastrointestinal complications of acquired immunodeficiency syndrome (AIDS). Factors of risk for AIDS present in these patients included homosexuality (n = 10), intravenous drug abuse (n = 7), multiple blood transfusions (n = 1), and unconfirmed or unknown factors (n = 7). Gastrointestinal abnormalities identified on radiologic studies (including upper gastrointestinal, small bowel, and barium enema studies) were correlated with histopathologic specimens and the results of bacteriologic, viral, fungal, and parasitologic studies. The most common disorders (88%) were candidal esophagitis and cytomegaloviral colitis; neoplastic involvement of the gastrointestinal tract was far less common (12%), with only two patients (8%) having Kaposi sarcoma. Gastrointestinal studies, which can provide useful if not always definitive diagnostic information, are recommended to precede more invasive diagnostic studies in evaluating patients with suspected AIDS. PMID- 3003794 TI - Dietary factors in essential hypertension. AB - Dietary alteration or intervention is an ideal method of preventing or treating hypertension. Medication may be eliminated or reduced in many cases. Correction of obesity and alcohol abuse are confirmed methods of treating hypertension. Reduction of sodium intake is effective in that portion of the population which is salt-sensitive. Probably, the ratio of sodium to potassium is of importance and increasing potassium intake while reducing sodium intake is effective in many situations. Evidence is being reported which indicates that adequate intake of calcium, and perhaps magnesium, is effective in preventing hypertension. Limited information indicates that a sufficiency of dietary essential fatty acids and fibre are effective in hypertension prevention. The role of dietary protein, carbohydrates, fat, cholesterol, vitamins, and essential elements (other than those mentioned above) in the pathogenesis has not been fully elucidated at this time, but there are indications that adequate intakes are beneficial in hypertension. Water hardness may have some effect in reducing hypertension incidence, and any effectiveness would probably result from calcium and magnesium in the drinking water. Animal studies and limited human studies indicate some detrimental effects of heavy metals, such as lead and cadmium, upon the pathogenesis of hypertension. Information regarding caffeine intake is inconclusive. PMID- 3003795 TI - Transfusion-transmitted diseases: current problems and challenges. PMID- 3003796 TI - Molecular correlates of cytogenetic abnormalities in human cancer cells: implications for oncogene activation. PMID- 3003797 TI - DNA rearrangements of cell lineage specific genes in lymphoproliferative disorders. PMID- 3003798 TI - Chemical changes induced in DNA by ionizing radiation. PMID- 3003799 TI - SV40 promoters and their regulation. PMID- 3003800 TI - Molecular mechanism of inverse regulation of hepatic alpha-1 and beta-2 adrenergic receptors. AB - The adrenergic activation of glycogenolysis in the rat liver is converted from an alpha-1 to a beta-2-receptor mediated event in various conditions associated with cellular dedifferentiation. Short-term incubation of isolated hepatocytes in a serum-free medium results in a similar conversion of the adrenoceptor response, without concomitant changes in the density or affinity of alpha-1 or beta receptor binding sites. This time-dependent conversion can be prevented or reversed by inhibitors of protein synthesis, by an endogenous inhibitor of phospholipase A2 (lipomodulin), or by removal of fatty acids from the medium through a lipid-trap. Conversely, activation of phospholipase A2 or addition of exogenous arachidonic acid to freshly isolated rat liver cells induces an acute conversion from alpha-1 to beta-type response, and the effect of the latter is prevented by the cyclooxygenase inhibitor, ibuprofen. It is proposed that reciprocal changes in alpha-1 and beta-2 receptor activity in rat liver cells are triggered by inverse changes in the coupling of the two receptors to their respective post-receptor pathways. These changes are mediated by a cyclooxygenase product generated through increased phospholipase A2 activity. PMID- 3003801 TI - Course of clinical response to antidepressants. AB - The postulated pharmacological mechanisms of antidepressant effect are reviewed. The clinical profile of response to antidepressants are linked to the pharmacological mechanisms. The limitations of presently available instruments for diagnosis and measurement of change in depression are discussed. PMID- 3003802 TI - Molecular structure of benzodiazepine receptors. AB - Benzodiazepines bind to high affinity binding sites in brain and other tissues. One of the high affinity sites in brain possesses properties which make it a likely site of action for these drugs. Binding to the benzodiazepine receptor is saturable and stereospecific; a high degree of correlation can be made between binding to this site and the pharmacological potency of the benzodiazepines. Binding to the site is also enchanced in the presence of GABA, the brain's major inhibitory transmitter. Because of this allosteric activation of benzodiazepine binding and the known electrophysiological interactions of the benzodiazepines with GABA, it appears likely that the site of action of the benzodiazepines in the brain is a GABA receptor complex. Recent evidence indicates that this complex also contains an anion channel which is the site of action of some of the barbiturates and known cage convulsants. The purification of the proteins composing this complex has been carried out and the major proteins appear to be several proteins with molecular weights between 50-60 k daltons; some higher molecular weight subunits may also be present. The benzodiazepine receptor protein can be labeled with 3H-flunitrazepam which labels a protein of molecular weight about 50 k daltons. Proteolytic degradation of the photolabeled benzodiazepine receptor results in the formation of several peptides and one limiting peptide which has recently been purified. Knowledge of its structure should yield interesting information about the origins of the benzodiazepine receptor and provide a useful tool for further molecular studies. PMID- 3003803 TI - 3H-clonidine binding to membranes of platelets prepared under physiological conditions. AB - A reproducible binding assay has been established to measure alpha 2 adrenoreceptors on membranes of human platelets prepared under physiological conditions. Washed platelet suspensions were obtained from fresh blood by a modified method (Mustard et al., 1972) and membranes were prepared by mechanical homogenization. 3H-clonidine, a selective alpha 2-adrenoreceptor agonist was selected as the ligand in a concentration range of 2-64 nM. Specific binding of 3H-clonidine was defined as the difference between total bound radioactivity and that not displaced by excess cold clonidine (6.4 X 10(-5) M). Platelets from 14 healthy young male volunteers were analysed using this technique, and one high affinity binding site, with KD and Bmax values in the reported range was found (KD: 10.14 +/- 1.95 nM; Bmax: 428 +/- 53 fmol/mg protein (mean +/- S.E.M.)). This technique will be used in future studies that will examine alpha 2-adrenoreceptor changes in specific subgroups of depressed patients. PMID- 3003804 TI - Platelet 3H imipramine binding: a possible predictor of response to antidepressant treatment. AB - No significant difference in the density (Bmax) of platelet 3H imipramine recognition sites were found between the group of 20 unmedicated depressed patients and 10 healthy volunteers. The mean KD value was significantly higher in the population of depressives than in controls. Non-responders (after 2 weeks of treatment with antidepressants) had significantly lower initial Bmax values than responders or control subjects. Density of platelet 3H imipramine site may thus be a predictor of early response to antidepressant therapy. No significant sex differences were found in KD or Bmax values in the depressed group or in control subjects. There was, however, a seasonal variation in Bmax but not in KD values of platelet 3H imipramine binding. PMID- 3003805 TI - Comparative autoradiographic distribution of calcium channel antagonist binding sites for 1,4-dihydropyridine and phenylalkylamine in rat, guinea pig and human brain. AB - The in vitro autoradiographic distribution of calcium channel antagonist binding sites for 1,4-dihydropyridine and phenylalkylamine has been investigated in rat, guinea pig and human brain. 1,4-dihydropyridine ([3H] (+) PN200-110) and phenylalkylamine ([3H] (-) D-888) binding sites are identically distributed in the brain of the three mammalian species studied. High densities of calcium antagonist binding sites are present in brain areas enriched in synaptic contacts such as the hippocampus, cortex and striatum. Low to moderate densities of sites are found in other regions such as the thalamus, hypothalamus and brain stem. These data demonstrate the existence of specific calcium antagonist binding sites in mammalian brain including man. These sites are discretely distributed with highest concentrations present in the hippocampus and cortex. Moreover, the similar distribution of binding sites for [3H] (+) PN200-110 and [3H] (-) D-188 suggests that 1,4-dihydropyridine and phenylalkylamine bind to the same receptor site complex in mammalian brain. PMID- 3003806 TI - Implication of the serotoninergic system in the decreased ACTH response to stress after lesion of the amygdaloid central nucleus. AB - The present study was designed to ascertain the possible implication of the serotoninergic system and the central amygdaloid nucleus in the control of ACTH secretion in response to immobilization stress. The response to immobilization stress of intact and lesioned animals was studied by monitoring the plasma and pituitary ACTH concentration and the activity of the serotoninergic system within specific hypothalamic and amygdaloid nuclei. Bilateral lesions of the central nucleus of the amygdala significantly decreased the secretion of ACTH in response to immobilization. Moreover, the serotoninergic activity in most of the hypothalamic and in all the amygdaloid nuclei studied was greatly increased. A 60 min immobilization stress prevented this increase in the hypothalamic nuclei but not in the amygdala. These results indicate that the central nucleus of the amygdala participates in the regulation of ACTH secretion in response to immobilization stress. Furthermore, they substantiate the hypothesis of a participation of the serotoninergic system in limbic areas, particularly in nuclei which contain neurons possessing glucocorticoid receptors such as the medial, basomedial and cortical amygdaloid nuclei. PMID- 3003807 TI - Interactions between ACTH, morphine, and naloxone and their effects on locomotor behavior. AB - A series of behavioral experiments were performed to evaluate possible interactions between ACTH, morphine and naloxone with regard to locomotor activity in an open field. Manipulations included the administration of one of the drugs followed 30 min later by one of the other drugs. This allowed the examination of the effects of a drug on the ongoing behavior induced by the other drug. In two other experiments which differed in the time of day they were run, locomotor patterns as the result of the administration of combinations of the three drugs were examined for three hours. The results of the present experiments suggest a possible common mode of action of ACTH, opiate agonists and antagonists that was dependent on time of day and stress intensity. PMID- 3003809 TI - [AIDS virus]. PMID- 3003808 TI - Variability of chronic antidepressant treatments on beta-adrenergic receptor sites. AB - It has been proposed that down-regulation of beta-adrenergic receptors may be related to the therapeutic action of antidepressants. To further characterize the role of beta-adrenergic receptor regulation in the actions of antidepressants, the effects of chronic treatment with desipramine, zimelidine and bupropion on beta-adrenergic receptors in rat brain membrane preparations were studied. Chronic treatment with desipramine, but not zimelidine and bupropion decreased significantly the density of beta-adrenergic receptors. These data suggest that clinically effective antidepressants may vary considerably in their ability to down-regulate beta-adrenergic receptors after chronic treatment. PMID- 3003810 TI - [Developmental and evolutional aspects of insect cytochromes c]. PMID- 3003811 TI - [Growth factor receptors and oncogene products; EGF receptor]. PMID- 3003812 TI - [Quantitation of and neutralizing antibodies to AIDS-retroviruses; a novel system using HTLV-I carrying MT-4 cells]. PMID- 3003813 TI - [Restriction endonucleases]. PMID- 3003814 TI - Leukotriene A4-induced bronchoconstriction in the guinea pig in vivo. AB - Administration of leukotriene A4 (0.03-0.3 microgram kg-1 i.v.) to anesthetized spontaneously breathing guinea pigs produced pronounced changes in pulmonary resistance, dynamic compliance and blood pressure. The pulmonary responses were unaffected either by pretreatment with indomethacin or following desensitization to leukotriene B4 but were significantly attenuated by the leukotriene D4 receptor antagonist, FPL-55712. Following administration of leukotriene A4 increased levels of leukotriene C4-immunoreactive material were determined in the plasma and neutrophil accumulation was observed in the lung. It was concluded that leukotriene A4 induced bronchoconstriction in the guinea pig either by acting directly on the leukotriene D4 receptor site or more probably through efficient metabolism in the lung to peptido-lipid leukotrienes which in turn exerted direct bronchoconstrictor actions. PMID- 3003815 TI - Characterization of biological properties of synthetic and biological leukotriene B3. AB - Leukotriene B3 was chemically synthesized and its ability to aggregate rat polymorphonuclear leukocytes (PMN) and to enhance chemokinesis of human leukocytes demonstrated. In both these assays the potency of synthetic leukotriene B3 was marginally less than that of leukotriene B4. Rat PMN incubated with leukotriene A3 were very inefficient in the enzymatic conversion of this epoxide to leukotriene B3. However, the leukotriene B3 produced was able to aggregate rat PMN. These results suggest that unlike leukotriene B5, the proinflammatory properties of leukotriene B3 are similar to those of leukotriene B4. However, since the enzymatic conversion of leukotriene A3 to leukotriene B3 is extremely poor it seems unlikely that leukotriene B3 itself has any major role in vivo. PMID- 3003816 TI - [Enkephalins]. PMID- 3003817 TI - [Round-table conference "Biochemical diagnosis of arterial hypertension"]. PMID- 3003818 TI - Quantitative autoradiographic determination of angiotensin-converting enzyme binding in rat pituitary and adrenal glands with 125I-351A, a specific inhibitor. AB - We describe a quantitative autoradiographic technique which allows measurement of angiotensin-I-converting enzyme [ACE] (kininase II, peptidyldipeptide hydrolase, EC 3.4.15.1) levels in discrete areas of pituitary and adrenal glands in individual animals. Tissue sections were incubated with 125I-351A, a specific ACE inhibitor, and results were obtained with computerized densitometry and comparison to 125I standards. There were high levels of ACE in both the anterior and posterior lobes of the pituitary, with no detectable binding in the intermediate lobe. The maximum binding capacity (Bmax) was 920 +/- 62 fmol/mg protein for the anterior pituitary and 1162 +/- 67 fmol/mg protein for posterior pituitary. The binding affinity constant (Ka) was 0.95 +/- 0.11 X 10(9) M-1 and 1.20 +/- 0.19 X 10(9) M-1 for the anterior and posterior lobes, respectively. In the adrenal gland, there were two distinct areas of specific binding, the adrenal medulla and the adrenal capsule-zona glomerulosa area. The Bmax for the adrenal medulla was 652 +/- 80 fmol/mg protein and 294 +/- 53 fmol/mg protein for the adrenal capsule-zona glomerulosa. The Ka for 351A was 1.04 +/- 0.19 X 10(9) M-1 and 1.74 +/- 0.40 X 10(9) M-1 for medulla and adrenal capsule-zona glomerulosa respectively. The results support the existence of local ANG systems active in both the pituitary and adrenal glands. PMID- 3003820 TI - Properties of histidyl-proline diketopiperazine [cyclo(His-Pro)] binding sites in rat liver. AB - Characteristics of cyclo(His-Pro) binding sites in the rat liver were studied using 3H-labeled cyclo(His-Pro). Scatchard analysis suggested that the rat liver membrane had a single binding site with an apparent dissociation constant (Kd) of 7 X 10(-8) M. Pretreatment of membrane preparations with soybean trypsin inhibitor increased cyclo(His-Pro) binding, and the binding activity was sensitive to trypsin and phospholipase A digestion, suggesting that protein and phospholipid moieties are essential for cyclo(His-Pro) binding. Thiol reagents reduced binding activity, suggesting that the thiol group might be an important constituent of the cyclo(His-Pro) binding site. Cross-reactivities of TRH, TRH analogues, L-His and L-Pro were very low (0.2-9%). These findings indicate that specific binding sites for cyclo(His-Pro) in the rat liver have similar properties to the receptors for other polypeptides. PMID- 3003819 TI - Immunoneutralization of oxytocin attenuates stress-induced corticotropin secretion in the rat. AB - Recent studies have demonstrated that oxytocin (OT) is released during certain stresses and that OT can potentiate the activity of CRF in vitro. To better define the role of OT during stress, the effect of injections of anti-OT antiserum on stress-induced corticotropin (ACTH) secretion was studied in vivo. A dose of antiserum which completely neutralized the increase in plasma OT levels during tail-hang stress caused a 59% decrease in plasma ACTH concentrations (P less than 0.005). The data support a physiologic role for OT in the regulation of ACTH secretion. PMID- 3003821 TI - [Clinical application of radioreceptor assay]. PMID- 3003823 TI - [Effective prefilter and patient dosage]. AB - The dose of incidence at a distance of 1 m from the focus is stated as pre filtration characteristics of modern x-ray tube assemblies for estimating the patient dose. The examinations cover a constant d.c. voltage range from 40 to 150 kV and the exposure range that is of practical interest. PMID- 3003822 TI - [Applicability of nuclear spin tomography to heart diseases]. AB - Thirty-one patients with abnormalities of the heart and ten controls were examined by ECG-triggered nuclear resonance tomography. All patients had previously been completely investigated. Chamber sizes and mural thickness could be reliably demonstrated and, in addition, it was possible to show areas which could not be easily investigated by echocardiography. Visualisation of the valves was limited, but intra- and para-cardial lesions could be differentiated by a double echo technique. Absence and defects of septa could be shown exactly, as well as accurate demonstration of the relationship of the large vessels to the cardiac chambers. PMID- 3003824 TI - [Retropharyngeal air mass following injury to the mouth by a ski stick]. PMID- 3003825 TI - [Candida esophagitis with the roentgenologic appearance of a carcinoma]. PMID- 3003827 TI - Rounded atelectasis. A case report. PMID- 3003828 TI - [Scintigraphic detection of a metastasizing hepatocellular carcinoma]. PMID- 3003826 TI - [Scintigraphic visualization of a distant metastasis from a follicular thyroid carcinoma using 99mTc pertechnetate prior to thyroidectomy. Case report]. PMID- 3003829 TI - [Imaging procedures in the diagnosis of the so-called gallbladder regeneration]. PMID- 3003830 TI - [Benign pneumoperitoneum in progressive systemic sclerosis]. PMID- 3003831 TI - [Sagittal MR findings in a case of sacral lipomyelomeningocele]. PMID- 3003832 TI - [Growth of a non-ossifying fibroma in an 11-year-old boy]. PMID- 3003833 TI - [Progressive cerebrovascular occlusion]. PMID- 3003834 TI - [Nuclear spin tomographic imaging of aortic aneurysms. Initial results]. AB - Thirteen patients with aortic aneurysm (abdominal 7, thoracic 4, thoraco abdominal 2) were evaluated by magnetic resonance tomography using a super conducting magnet of a field strength of 0.35 tesla in transversal, coronal and sagittal planes. ECG gating was used in patients with thoracic aneurysm. The results of magnetic resonance imaging (MRI) were compared to angiography, computed tomography (CT) and intraoperative findings. MRI demonstrated in all 13 patients the extent and the localisation of the aneurysm. The presence of mural thrombus was better determined by MRI (10/10) than by angiography (6/10). The relationship of the brachiocephalic arteries in thoracic aneurysm was better evaluated by MRI compared to angiography and CT. All 3 aortic dissections could be demonstrated by MRI. These first results may suggest an important role of MRI in the diagnosis of aortic aneurysms. PMID- 3003835 TI - [Measurement of absolute intravascular flow velocity using digital subtraction angiography]. AB - Experiments on phantoms showed very good correlation between flow velocity, as calculated from time-density curves, and theoretical values. After further development using an inter-active computer programme, DSA measurements of flow velocity are now undergoing further experimental investigation, but are also available for routine clinical examination. PMID- 3003836 TI - [Digital subtraction angiography in traumatology]. AB - The methods, indications and results of digital subtraction angiography in traumatology are presented, based on 56 examinations. The different use of intravenous or intraarterial DSA will be discussed with respect to expanding and localisation of traumatic vascular injury. DSA is recommended as the method of choice for follow-up after vascular reconstructive procedure. PMID- 3003838 TI - [Expansion, deformation and bursting characteristics of commonly used balloon dilatation catheters. In vitro studies (1)]. AB - We evaluated the characteristics of dilatation and deformation, as well as pressure requirements, bursting point, and type of bursting of the five most common balloon dilatation catheters. Polyvinyl chloride, polyethylene, and polyurethane-nylon balloons from 4 different manufacturers were examined in water baths and an arrangement of tightly fitting teflon tubes. Defined balloon inflations were monitored and documented until balloon rupture occurred. Significant differences were found according to the different constructions and materials of the balloon catheters. Modern PVC showed a quality equal to PE material. Marked and clinically relevant differences of balloon compliance and behaviour in the water baths and within teflon tubes were demonstrated. PMID- 3003837 TI - [Initial experiences with PTA of hemodialysis shunts]. AB - Our experience of using PTA for stenoses of haemodialysis fistulae in 11 cases is described. PTA is the procedure of choice for re-establishing adequate shunt function. Intraluminal pressure measurements on the venous limb of well functioning Cimino shunts produced values of 7.5 to 44.5 mmHg, average 24.5 mmHg. These values are the basis for judging the success of a dilatation. Shunt angiography with DSA with a highly diluted non-ionic contrast medium injected via a remaining dialysis needle is an effective procedure with minimal discomfort to the patient. PMID- 3003840 TI - [Computed tomographic study of portal venous contrast medium perfusion of the liver]. AB - Contrast medium was injected in the portal vein at a low flow rate via a transumbilical catheter in twelve patients. The distribution of the contrast medium in the liver was investigated by computed tomography. The results of this study demonstrate an incomplete mixture of splenic and mesenteric venous blood in the portal vein. In addition, there is layering of the contrast medium and blood in the portal vein, causing inhomogeneous contrast medium perfusion of the liver. PMID- 3003839 TI - [Value of rapid sequential computed tomography for the diagnosis of liver cirrhosis]. AB - Thirty-eight patients with cirrhosis of the liver and thirty-seven controls were examined by dynamic computer tomography in a prospective study. Time-density difference curves for the liver, spleen, portal vein, aorta and vena cava were treated mathematically ('gamma fit'). Comparison of the values of the difference curves of liver, spleen and portal vein showed significantly lower and delayed peaks in patients with cirrhosis than in normal people. The time-density difference curves of the liver showed a highly significant shallow decline in the presence of cirrhosis. Discriminant analysis of the curve parameters using the spleen showed differentiation between normals and cirrhosis patients of 90.2%, and using the liver curve a separation of 83%. Combining these parameters of liver and spleen curves improved the separation between cirrhotic patients and normals to 94%. PMID- 3003841 TI - [Sonography and computed tomography before and after liver resection and transplantation]. AB - The diagnostic value of sonography and CT was studied in 259 patients. In the diagnosis of solid tumours, CT is superior to sonography, whereas ultrasound is more useful in the investigation of cystic lesions. In estimating the extent of intrahepatic tumours, the falciform ligament is more useful than a line from the cava to the gall bladder, both for CT and ultrasound, although in this case CT shows the extent of tumours more accurately. 3.7% of the ultrasound and 2.3% of the CT examinations were of limited value because of unavoidable technical problems. PMID- 3003842 TI - [Imaging of ventricular wall motion using venous DSA. A new method in radiologic cardiac diagnosis]. AB - Until now, angiographical and nuclear medicine examination techniques for imaging left ventricular wall motion have been presenting with difficulties endemic to the methods themselves. For the first time in cardiological diagnostics, digital subtraction angiography (DSA) makes it possible to perform a fairly non-invasive examination with good spatial and temporal resolution. Functional analytic evaluation, however, still demands time-consuming, complicated post-processing. In this article we introduce a method that uses an additive window technique for the immediate generation of wall motion images. PMID- 3003844 TI - [Studies on the angioarchitecture of polypoid colonic processes]. AB - Portions of the colon removed at operation were studied angiographically after arterial or venous injection with a barium-gelatin mixture. The aims of the investigation were: To study the differences between tumour and inflammatory angiographic appearances and the clinical value of in vivo angiography of the mesenteric arteries. A comparison of the micro-angiographic appearances of benign and malignant polypoid lesions of the colon. Adenomas and carcinomas showed patterns which are easy to distinguish and permit classification in uncertain lesions. Pseudopolyps in Crohn's disease resemble adenomas in the early stages, but later assume features characteristic of tumours. PMID- 3003843 TI - [Gas in the pancreas: value of computed tomography]. AB - Intrapancreatic gas in acute pancreatitis is generally due to infection with gas forming organisms. CT provides evidence of pancreatitis, its extent and complications. The demonstration of gas in an inflamed pancreas indicates abscess formation and is an indication for surgical intervention. A review of our own patients has shown abscess formation in six out of seven patients with gas in the pancreas. The differential diagnosis of gas collections in the region of the pancreas is discussed and illustrated by examples. PMID- 3003845 TI - [CT studies following ethanol embolization of malignant kidney tumors]. AB - CT studies were carried out in 25 patients after alcohol embolisation. It was shown that the embolisation reaches the very periphery of the renal capsule. Nevertheless, contrast enhancement in renal tumours persists, due to capsular and collateral vessels. These subsequently permit further tumour growth. Intratumoral gas could be demonstrated in all patients. In the absence of symptoms, this must be regarded as a normal post-embolic event. PMID- 3003846 TI - [Experiences with a modified nephrostomy instrument kit in ultrasound-guided kidney fistulization]. AB - During a period of 24 months, ultrasound-guided percutaneous nephrostomies were performed in 51 patients in the Hospital at Koln-Merheim. The procedure was carried out with a modified nephrostomy instrument manufactured by Angiomed, Ettlingen, according to our own design. The procedure was successful in 49 patients. In one case it had to be abandoned because of lack of patient co operation. In one patient a pyonephrosis was found and the patient was treated surgically. PMID- 3003848 TI - [MR-tomography and -spectroscopy of skeletal muscles using high magnetic field strengths]. AB - Intact as well as neuromuscular affected skeletal muscles can be precisely analysed by MR tomography with high magnetic field strengths. The substitution of muscle by adipose tissue under atrophic conditions is seen most clearly in fat images, while the morphology of small structures is predominantly shown by water images. The aim of in-vivo spectroscopy is an identification and quantification of metabolites. A relative increase in the amount of adipose tissue within atrophic muscles was confirmed by the 1-H spectrum. As concluded from 13-C and 31 P spectra there was neither a change in adipose tissue composition nor a modification of energy metabolism. PMID- 3003847 TI - [Computed tomography of soft tissue tumors of the extremities]. AB - The results of 80 CT examinations in 57 patients with malignant (32) and benign (25) soft tissue tumours of the extremities are reported. Thirty-five patients underwent surgery and the CT features (size, structure, density, necrosis, calcification, bone involvement) were compared with the pathological and histological findings. Three quarters of the tumours which were non-homogeneous on CT were malignant, one quarter benign. Only half the cases of malignant tumours showed a definite margin, but 70% of benign tumours did so. Indefinite margins were found in 50% malignant and 30% benign tumours. Soft tissue tumours of the hands and feet were difficult to differentiate. Difficulties were also found during follow-up (37 patients) because of anatomical changes following surgery. Although some histological types tend to be associated with particular CT findings, a definite histological diagnosis can only be made in the case of lipomas. PMID- 3003849 TI - Frequency, sensitivity and specificity of roentgenographic features of slight and moderate asbestos-related respiratory diseases. AB - The possibility of early detection of asbestos-related respiratory diseases was examined on the basis of four x-ray abnormalities, namely, pulmonary fibrosis, pleural plaque, diffuse pleural thickening and diaphragmatic calcification, in a group of workers exposed to chrysotile asbestos. The frequency of these phenomena was compared to the unexposed control group of similar distribution of number, sex and age. Besides the pleural plaques, which had a high specificity, the combination of minor x-ray abnormalities proved to be most characteristic of exposure to asbestos. The more frequent one of the abnormalities, the less specificity it had to asbestos exposure. PMID- 3003850 TI - [Posterior defects of the patella on CT arthrograms. Detection and differential diagnosis]. AB - Changes of the posterior bony and cartilaginous surfaces of the patella as seen on CT arthrograms are described in the case of bipartite patellae, fractures and ossification defects. Contrary to present opinion, cartilaginous lesions are frequently seen on CT arthrograms. This is also true for discreet and partial ossification defects which are not visible on conventional x-rays and are described here for the first time. The aetiology, morphogenesis and clinical examples are discussed. PMID- 3003851 TI - [Massive intraoperative pulmonary tumor thromboembolism in a case of Wilms' tumor]. PMID- 3003852 TI - Early decrease in suture line breaking strength. The effect of proposed collagenase inhibition. AB - Rats were subjected to end-to-end intestinal anastomosis. Breaking strength with the sutures in place, i.e., suture-holding capacity, was measured in different groups immediately after suture and after 24 h. The synthetic kallikrein-plasmin inhibitor S-2441, the inhibitor of plasminogen activation tranexamic acid (Cyklokapron), and the metalloprotease inhibitor tiopronin (Thiola), were studied regarding their effect on breaking strength of the intestinal anastomoses. There was a marked decrease in breaking strength at 24 h in the controls. This decrease was diminished by all of the substances tested. Their effect was probably due to an inhibition of collagenase. PMID- 3003853 TI - Inositol affects the intracellular turnover of pulmonary surfactant phospholipids in the rat. AB - Rats, given a diet supplemented with 20% inositol, had threefold increased plasma inositol concentrations. The pool size of their alveolar surfactant fraction and their lamellar body fraction were the same as in the control rats and differences in phospholipid composition of the surfactant fractions were mainly restricted to changes in the percentages phosphatidylinositol (PI) and phosphatidylglycerol (PG). The change in phospholipid composition did not affect the pressure-volume relationship of the lungs. The labeling of phosphatidylcholine (PC), saturated phosphatidylcholine (SPC) or PI in the alveolar lavage fraction was the same for both groups, whereas labeling of alveolar PG was delayed in the inositol-fed rats. The specific activity-time relationships of the lamellar body phospholipids differed significantly between the control and inositol-fed rats and the differences in disappearance rate of the label from these fractions suggest that approximately 25-30% of the lamellar body material in inositol-fed rats is directed to a third, intracellular pool. We conclude that an increase in PI and a concomitant decrease in PG content of surfactant do not affect the clearance of alveolar surfactant, but enlarge the turnover of the lamellar body fraction because of intracellular degradation. PMID- 3003854 TI - [Lipid profile and the platelet and cardiovascular activity of rats of both sexes under conditions of hyperprogesteronemia and stress]. PMID- 3003855 TI - [Experimental ethylene oxide neuropathy. Chronic exposure to 250 ppm in rats]. AB - In Wistar rats subjected to a six-hour exposure to ethylene oxide (ETO) at the concentration of 250 parts per million once a day, five times a week for 9 months, histopathologic studies of myelinated fibers of the proximal sural, distal sural and peroneal nerves and of the fasciculus gracilis at the fifth thoracic and third cervical segments of the spinal cord were performed to reveal whether ETO of such concentration produces the degeneration of the primary sensory neuron. Throughout the study, no definite abnormality of the gait or posture was observed in both control and test rats. Qualitative histologic studies disclosed preferential distal axonal degeneration of myelinated fibers in both sural nerves and gracile fascicles in test rats, although the extent of the distribution and the severity of the degenerative findings were variable among test rats. Therefore, it was concluded that exposure to 250 ppm ethylene oxide produces central-peripheral distal axonal degeneration of the primary sensory neuron in rats. PMID- 3003856 TI - Echographic characteristics and ultrasonic tissue characterization in breast tumor. AB - Early detection and correct diagnosis of breast cancer is necessary and an important determinant of prognosis. Clinical echography is increasingly being used as it offers a high degree of diagnostic accuracy and no physical hazards such as x-ray mammography. Ultrasonic tissue characterization study is an important factor in determining and refining diagnostic criteria and for developing new ultrasonic diagnostic equipment in clinical breast echography. Therefore, various echographic characteristics and their ultrasonic tissue characterization were analysed in various types of breast tumors such as medullary carcinoma, papillary carcinoma, scirrhous carcinoma, malignant lymphoma, lymphatic infiltration, benign cyst, fibroadenoma, cystosarcoma phylloides, gynecomastia and nodular fibrosis with fat necrosis, in routine clinical echograms. PMID- 3003857 TI - What are the molecular characteristics of the neutrophil receptor for chemotactic formylated peptides? PMID- 3003858 TI - Physiological studies with human leukocyte inhibitory factor. PMID- 3003859 TI - [Mediators of immediate anaphylactic reactions]. PMID- 3003860 TI - [Late anaphylactic lesions]. PMID- 3003861 TI - [Leukocytes and derivatives of arachidonic acid, prostaglandins and leukotrienes]. PMID- 3003862 TI - [Direct analysis of DNA in the prenatal and postnatal diagnosis of genetic diseases]. PMID- 3003863 TI - [LHRH agonists combined with antiandrogens in the treatment of metastasizing prostatic carcinoma]. PMID- 3003864 TI - [Acute renal insufficiency caused by rhabdomyolysis. Etiopathogenetic and therapeutic aspects]. PMID- 3003865 TI - [Linitis plastica of the colon. Report of 3 cases with special reference to its differential diagnosis from Crohn's colitis]. PMID- 3003866 TI - [Sero-epidemiology of HTLV-III/LAV antibodies in French-speaking Switzerland]. PMID- 3003867 TI - Localization of neuroendocrine tumors of the pancreas. AB - The diagnosis of neuroendocrine tumors of the pancreas is based on clinical, biochemical, and ultrastructural data. The different radiological modalities are employed only for localization, except in the case of nonfunctioning neoplasms, for which a differential diagnosis from adenocarcinoma can be suggested based on the radiographic images. Generally, the initial evaluation should consist of the noninvasive studies and should be complemented by angiography and, in cases of functioning neoplasms, with pancreatic venous sampling if the preliminary findings are inconclusive. However, the localizing work-up should be terminated when the information obtained is sufficient to determine the therapy that will follow. If the ultrasonic or computed tomographic findings preclude surgical resection, further work-up is obviously unnecessary. PMID- 3003868 TI - [Neuropathy caused by cisplatin. Clinical, electrophysiological and morphological study]. AB - Seven patients with cancer presented a sensory peripheral neuropathy induced by cisplatinum. This drug was used alone in 1 case and, in 6 other cases it was associated with drugs without any toxicity for the peripheral nervous system. Every patient had an electromyogram and motor and sensory nerve conduction studies. A sural nerve biopsy was performed in 5 cases for light and electron microscopic studies as well as for teasing and quantitative studies. Electromyograms and motor nerve conductions were normal. Sensory nerve conductions were slowed with very low amplitude sensory action potentials. Such results suggested axonal changes. Nerve biopsies showed typical axonopathic changes with secondary demyelination. Morphometric studies confirmed a loss of myelinated fibers affecting the large fibers in all cases, according to the slowed sensory nerve conductions. This study confirmed that the cisplatinum induced neuropathy is a new form of toxic distal axonal neuropathy. The hypothesis of a primary demyelination of peripheral nerves, which has been proposed, could not be retained. PMID- 3003869 TI - [Effects of a morphine-clomipramine combination on a test of experimental analgesia]. AB - Morphine (2.5 mg/kg sc) i.e. at a non-antinociceptive dose, augmented clomipramine-induced antinociceptive effect but failed to alter the values of clomipramine (10, 20, 40 mg/kg orally) levels in rats. These results suggest that the potentiation of clomipramine-induced analgesia by morphine does not result from a peripheral pharmacokinetic mechanism but probably involves a central interaction between clomipramine and opiates pathways. PMID- 3003870 TI - [Conference at the Salpetriere. June 1984. Peripheral neuropathy and vitreous opacities in a 50-year-old man]. PMID- 3003871 TI - [Neuropathies caused by thalidomide]. AB - Symptoms and signs in four patients with thalidomide-induced neuropathy developing during treatment of discoid lupus were limited for long period to distal paresthesiae with altered sensory conduction velocities. Semi-thin biopsy specimens of the distal sural nerve showed depopulation of myelinized fibers, mainly affecting those of large caliber, and signs of axonal degeneration. Study of dissociated fibers showed a high proportion of E fibers. Morphometry confirmed the axonal lesion. Ultrastructural examination demonstrated anomalies of axons of amyelinic fibers (vacuoles, lamellar figures) and of Schwann cells (stacked cytoplasmic prolongations), together with numerous collagen pockets, all non specific lesions. The disease course was slow, with disappearance of sensory symptoms in a few weeks in 3 of the 4 cases and normal clinical findings in one of the four patients one year after cessation of treatment. Definite correlations between the dose administered and the severity of the neuropathy could not be established. The still poorly understood mechanism of action is discussed. PMID- 3003872 TI - [Hypermyelination in a case of peripheral neuropathy with benign IgM monoclonal gammopathy]. AB - A 65 year-old man presented with a 8 months history of ataxia and weakness of the lower limbs. Acroparesthesias had been present for ten years in all four limbs. Serum protein electrophoresis showed a monoclonal IgM kappa spike. Bone marrow showed no increase in plasma cells. Motor conduction velocities were reduced. A biopsy from the right superficial peroneal nerve revealed several enlarged myelin sheaths in each fascicle on semi-thin sections. Under electron microscopy these large myelin sheaths were either regularly arrayed around the axon or formed several large additional loops. The outer lamellae of these abnormal myelin sheaths and of the additional loops were frequently widened. Other fibers showed a widening of some myelin lamellae (W.M.L.) with a myelin sheath of varying thickness, and these widened lamellae were frequently joined together. A direct immunopathological study could not be made. The widening of some myelin lamellae is present in most cases of peripheral neuropathy with IgM monoclonal gammopathy and a fixation of anti IgM serum on some myelin sheaths may be demonstrated by direct immunopathology. Hypermyelination has been reported in a few cases. In this patient hypermyelination was as developed as in typical cases of tomaculous neuropathy. An association in some myelin sheaths of hypermyelination and W.M.L. has been noted in serum induced myelin aberrations in peripheral nervous system cultures. PMID- 3003873 TI - [Lymphocyte subpopulations of the blood and cerebrospinal fluid in acute polyradiculoneuritis. Study of 14 cases using monoclonal antibodies]. AB - The T lymphocytes in blood and CSF of 14 patients with acute polyradiculoneuritis were studied using the OKT series of monoclonal antibodies. Results were compared with findings in two control groups. Three patients with cytomegalovirus infection showed elevation of circulating OKT 8. A significant increase in the mean ratio OKT 4/OKT 8 was noted in all other cases, corresponding in 4 of the 11 patients to a reduction in number of OKT 8 lymphocytes. The CSF was normal in 3 of the 14 cases. PMID- 3003874 TI - [Side effects of antiepileptic therapy. Study of 197 cases]. AB - A study of 197 epileptic outpatients consecutively observed by one Neurologist allowed a prospective study of the side effects of antiepileptic drugs. This study shows essentially that a clinical improvement comparable to that generally reported can be obtained with side effects limited in their number and intensity. The main principle is the routine search of these effects which are not always spontaneously mentioned by the patients, especially intellectual slowness and loss of sexual activity. The detection of the side effects through history and physical examination is far more fruitful than are plasma concentrations of drugs which can be doubly misleading: some plasma concentrations lower than recognized therapeutical levels are efficient however while others are higher and nevertheless necessary and well tolerated. Among the side effects specific to certain antiepileptic drugs, shoulder-hand syndrome due to phenobarbital was noted in 10 per cent of the cases. Furthermore, the frequency of scapulo-humeral periarthritis was significantly higher in the epileptic group than in the controls. Dupuytren's disease was less frequent (8 per cent) and the difference with the controls was not significant. Among the non-specific side effects, nystagmus is a somewhat useful sign in treatments by phenytoin but as with drugs dosages, it must be weighed within the whole clinical picture. PMID- 3003875 TI - Congenital cytomegalovirus infection. Occurrence in two socioeconomically distinct populations of a developing country. PMID- 3003876 TI - [Hepatitis associated with transfusions: epidemiology and clinical course]. PMID- 3003877 TI - [Research on anti-HTLV-III antibodies: validity of methods]. PMID- 3003878 TI - [Significance of anti-HTLV-III antibodies]. PMID- 3003879 TI - [The advantages of using monoclonal antibodies in the transfusion service]. PMID- 3003880 TI - Studies on the protein and antigen composition of individual female Onchocerca volvulus after collagenase digestion. AB - The composition of proteins and antigens of female Onchocerca volvulus from one focus of transmission was studied by SDS-PAGE and immunoblotting. Worms that had been exposed to collagenase digestion of onchocercomata for different periods of time and parasites of different age and status of reproduction were tested. Some O. volvulus antigens were found to be sensible to prolonged digestion (molecular weights: 18 KD, 21 KD, 24 KD) but the majority of the antigens was stable up to 64 hours of incubation at 34 degrees C. The composition of proteins and antigens varied with the age and the status of reproduction of the worms. Slight differences between individual filariae were found, even when worms of comparable age and status of reproduction were tested that had been exposed to nodule digestion for comparable time. PMID- 3003881 TI - [Virus diseases with different manifestations]. PMID- 3003882 TI - The "Propins" test, a method for the estimation of the participation of the beta adrenergic mechanism in ulcerogenesis. AB - In 12 ulcer patients in attack and in 6 healthy subjects the secretory test to insulin (Hollender test) was used and repeated after 48 hrs in association with propranolol (Propins test). While in controls the stimulated acid secretion was not influenced in ulcer patients a significant reduction of the secretory parameters was obtained in 60% of the cases thus confirming the existence of a beta-adrenergic component of the hydrochloric secretion. In 40% of the ulcer patients this effect was not observed. It therefore results that by using our "Propins" test it is possible to estimate the prevalence of the vagal-cholinergic or the beta-adrenergic (gastrinic) mechanism in the pathogenesis of duodenal ulcer. PMID- 3003883 TI - Fiber diet and antacids in the short-term treatment of duodenal ulcer. AB - Eighty patients with active duodenal ulcer were randomized to a diet poor or rich in fiber for a treatment period of 4 weeks. In addition, all patients received one antacid tablet (Link, 1.1 g) four times a day (total neutralizing capacity, 120 mmol HCl/day). The ulcer healed in 27 (67.5%) of the 40 patients in the high fiber group, compared with in 24 (60%) of the 40 patients in the low-fiber group (p less than 0.5). Ulcer symptoms did not differ significantly between groups during the 4-week treatment period. No serious side effects were recorded. Constipation, the most frequently registered side effect, was seen in 11 (27.5%) of the patients in the low-fiber group, compared within 4 (10%) in the high-fiber group (chi-square = 4.0; p less than 0.05). Patients with unhealed ulcer after 4 weeks' treatment received ranitidine instead of antacids. While they were receiving ranitidine treatment, no significant differences in healing rates were seen between the two dietary groups. PMID- 3003884 TI - Effect of serotonin on pentagastrin-stimulated gastric acid secretion and gastric antral motility in dogs with gastric fistula. AB - The effects of exogenous serotonin on pentagastrin-stimulated gastric acid secretion and antral motility were evaluated with regard to inhibition kinetics and receptor mediation. Conscious gastric fistula dogs were used. Serotonin inhibited the acid secretion dose-dependently, whereas the antral motility was initially stimulated and thereafter inhibited. The inhibition of secretion was counteracted by different beta-adrenergic blocking drugs and methysergide, whereas the inhibition of antral motility was blocked by methysergide and indomethacin. Dose-response analysis showed inhibition of non-competitive types. This study supports the concept of differences in the regulation of gastric acid secretion and motility, but further experiments with simultaneous registration are required. PMID- 3003885 TI - AIDS and the gut. AB - The epidemiological, immunological and early virological observations on the acquired immune deficiency syndrome (AIDS) suggested that an agent was involved which was sexually, parenterally and perinatally transmitted and perhaps tropic for T helper lymphocytes. A new subgroup of human T lymphotropic retroviruses have been identified ans seroepidemiological studies suggest that they are aetiologically related to AIDS. The syndrome is characterised by the development of tumors: such as Kaposi's sarcoma and non-Hodgkins lymphoma, with an aggressive clinical course and infection by a wide spectrum of opportunistic organisms. Both the tumours and the infections commonly involve the gut. PMID- 3003886 TI - Combined study of lymphocyte phosphoglucomutase (PGM) and adenylate kinase (AK) isoenzymes in the early characterization of bone marrow engraftment. AB - A starch-gel electrophoretic study of 2 peripheral blood lymphocyte enzymes, phosphoglucomutase (PGM) and adenylate kinase (AK), was performed in 25 donor recipient allogenic bone marrow transplantation (BMT) pairs. A different isoenzymatic pattern between donor and recipient for PGM, AK or both, was observed in 11 of these 25 pairs. From these 11 cases, an identical donor isoenzyme pattern was observed in 10 cases between 24 and 85 days (mean 41.8, SD 22.08) after BM infusion confirming engraftment, whereas in the remaining 1 the demonstration could not be substantiated due to early death. The present study demonstrates that, in addition to other cell markers, the simultaneous study of PGM and AK isoenzymes allows confirmation of BM engraftment in about 45% of cases, much earlier than is possible by means of red cell antigen analysis. Moreover, it is a simple, relatively inexpensive and rapid laboratory tool for a better characterization of the established chimera. PMID- 3003887 TI - Xenohybridization of Epstein-Barr virus-transformed cells for the production of human monoclonal antibodies. AB - Transformation of human B lymphocytes, obtained from hyperimmune donors with Epstein-Barr virus, yields polyclonal cell populations in which a minority of cells produce IgG antibodies of predetermined specificity, whereas the majority of cells produce 'non-specific' immunoglobulin (mainly of the IgM class). Such lymphoblastoid cell lines can be easily propagated in high-density cultures. Because cloning at 1 cell per well is not possible, stabilization of lymphoblastoid cell lines by limiting dilution is not feasible and most newly established lines cease to produce specific antibody within a few weeks. Xenohybrids, resulting from fusion of Epstein-Barr virus-transformed cells with NS1 mouse plasmacytoma cells, can be cloned at 1 cell per well. Stable xenohybridoma subclones, producing antibody of the desired specificity, can be isolated after a series of limiting dilutions. In a model system, we have studied the efficiency of xenohybridization of human lymphoblastoid cells. Using this system, we have constructed IgG anti-tetanus-toxoid- and IgG anti-HBsAg-producing cell lines. Next, we investigated whether transformation with Epstein-Barr virus is essential in such a two-step procedure or whether a polyclonal stimulator, such as pokeweed mitogen, could also be used. It was found that antibody producing xenohybrids can be obtained after stimulation with pokeweed mitogen. However, this latter system is subject to more variations and lacks the advantage of pre-selection of antibody-producing cells as compared to xenohybridization after transformation. PMID- 3003888 TI - Effect of zoster immunoglobulin for varicella prophylaxis in the newborn. AB - Zoster immunoglobulin (ZIG) was given for prophylaxis to 95 neonates born to mothers with perinatal varicella. The treatment had no influence on the clinical attack rate; 48 (50%) of the children developed varicella. However, the ZIG treatment clearly influenced the course of the disease for newborns at particular risk, i.e. when maternal varicella developed within 4 days before and 2 days after delivery. Of 41 such neonates, 21 (51%) contracted varicella with an incubation mean time of 11 days. 13 of the 21 developed a very mild chickenpox (no fever, less than or equal to 20 pocks), 6 had a mild to normal disease, and 2 (10%) had more severe infections; none died or got sequelae after the disease. These results should be compared with the expected rate of complications in non treated neonates in the defined risk group, where the mortality among those contracting varicella has been reported to be as high as about 30%. PMID- 3003889 TI - Rotavirus infections in newborns: an epidemiological and clinical study. AB - An outbreak of rotavirus infections among newborns at Karolinska Hospital, Stockholm, which has been going on for greater than 2 years has been followed with clinical and epidemiological investigations. About one third of the babies born in the hospital were infected at the age of 3 days. The clinical symptoms were mild, 8.8% of the rotavirus positive babies had loose stools compared to 1.9% of those who did not excrete the virus. An epidemiological survey in the neonatal intensive care unit suggested that rotavirus was introduced into the unit by babies admitted from the obstetric wards. The main reservoir of rotavirus was the babies and rotavirus was not found among staff or mothers. In the beginning hygienic measures seemed to be effective but after some weeks the colonization rate again increased. Electropherotyping of samples collected during different periods showed that one single rotavirus electropherotype belonging to the subgroup 1 of human rotavirus was found throughout the outbreak. PMID- 3003890 TI - Adult Wilms tumor. A case report. AB - We report a case of Wilms tumor in an adult. Pictorial and descriptive pathology information is included. The tumor was stage IV with pulmonary metastasis at diagnosis. The patient was treated with surgery and combined chemotherapy according to protocols used in childhood Wilms tumor. The patient is clinically free of disease at 21 months postoperatively. PMID- 3003891 TI - [HTLV-III antibodies and risk factors for healthy homosexuals in Zurich]. AB - Sera of 100 healthy homosexual men were tested for antibodies to AIDS virus HTLV III. Significant differences were found between 45 seropositive and 55 seronegative men, such as a higher number of sexual partners per year, more contacts with AIDS patients, positive serological markers for syphilis, and reduced skin reaction after intradermal application of 7 recall antigens. PMID- 3003892 TI - [Diuretic-resistant hypertension. Effect of an additional daily single dose of a ACE inhibitor]. AB - The effect of additional administration of a low dose of an ACE inhibitor when blood pressure is inadequately controlled by a diuretic alone was investigated in 12 essential hypertensive patients. 24 h blood pressure monitorings were performed with portable automatic recording devices before and after treatment with 50 mg hydrochlorothiazide (HCT) and after additional administration of 25 mg captopril. The frequency of blood pressure readings exceeding 160/95 mm Hg within 24 hours was reduced in 11 of the 12 patients after administration of the ACE inhibitor. Mean blood pressure during daytime was 158 +/- 13 / 100 +/- 8 mm Hg without treatment, 151 +/- 11 / 93 +/- 8 mm Hg during HCT alone and 140 +/- 13 / 88 +/- 9 mm Hg during HCT + captopril. The results show that in a large proportion of hypertensives inadequately controlled by a diuretic alone, the addition of a low dose of an ACE inhibitor (captopril, 25 mg once daily) may normalize the blood pressure. PMID- 3003893 TI - [Mucinous adenocarcinoma of the nose and paranasal sinuses, an occupational disease?]. AB - In Great Britain and other countries there have been reports of an increased frequency of adenocarcinoma of the nose and paranasal sinuses, mimicking histologically mucinous colonic carcinoma, among workers exposed to wood dust and workers in the leather industry. No such cases have been reported so far in Switzerland. 31 patients with this type of adenocarcinoma of the nose and paranasal sinuses observed between 1953 and 1984 have been reviewed. In 13 of these patients there was occupational exposure to wood dust, and 4 were shoemakers. The study suggests an association of this type of nasal carcinoma with occupational exposure to wood dust and leatherwork. The nature of the postulated carcinogen is unknown. The authors propose further investigations to define the magnitude of the cancer risk associated with exposure to wood dust and leather. PMID- 3003894 TI - [Etiology of atypical pneumonias. A serological study on 1494 patients]. AB - In a retrospective study the serological results from 1494 patients with community-acquired pneumonia were evaluated. An infectious etiology was found in about 40% of the cases. The majority of pneumonias was caused by Mycoplasma pneumoniae and by influenza virus type A, whereas Legionella pneumophila was the fifth most frequent pathogen. In the second part of the study, 13 hospitalized patients with community-acquired pneumonia were investigated by the whole panel of routinely used microbial methods. The etiological agent was found serologically in 3 cases and in one case by cultivation. These results suggest that the determination of serum antibodies against pathogens is frequently more useful than is generally assumed, although the yield of positive results is dependent on the epidemiological situation. The detection of elevated complement fixing titers or specific IgM antibodies often leads to diagnosis from the first serum examined. PMID- 3003895 TI - [Excretion of IBR virus, especially in milk, in experimentally infected cows]. PMID- 3003897 TI - [Some advances in angiotensin converting enzyme inhibitors]. PMID- 3003896 TI - [Superoxide free radical and superoxide dismutase]. PMID- 3003898 TI - [Recycling of cell membrane receptors]. PMID- 3003899 TI - [Advance in the research of platelet-derived growth factor]. PMID- 3003900 TI - [Physiology of locus coeruleus]. PMID- 3003901 TI - [Evaluation of tumorous parotid gland diseases by sonography]. AB - Ultrasonic evaluation was done on 62 patients with a palpable tumor in the area of the parotid gland. Morphological characteristics of the individual conditions are demonstrated. Chronic inflammations, abcesses, cysts and Warthin's tumor can be diagnosed correctly. These alterations can be distinguished from the pleomorphic adenoma. A differential diagnosis of this tumor cannot always be done, however, as it cannot be distinguished morphologically from a benign enlargement of the intraglandular lymph nodes. Malignant tumors in our patients were diagnosed correctly. Because of the small number of cases (7), however, we are not able to state whether it is possible to distinguish between benign and malignant tumors in all cases. Ultrasonic criteria of malignancy are described. PMID- 3003902 TI - Evaluation of different treatment processes with respect to mutagenic activity in drinking water. AB - Treatment processes which are applied in The Netherlands during the preparation of drinking water have been evaluated with regard to introduction and removal of organic mutagens as well as halogenated organics. It appeared that the most efficient processes in reducing mutagenic activity were activated carbon filtration and artificial dune recharge. In general these processes were also the most efficient in removing halogenated organics. Using low doses of chlorine dioxide (less than 1 mg C1O2/l) for safety disinfection of drinking water, no change or substantial less mutagenic activity than by chlorination (1 mg Cl/l) was found. This counts too for the formation of halogenated organics. Transport chlorination of stored river Meuse water was able to introduce or activate mutagenic nitro organics which have not been found previously. Ozone treatment under field conditions showed mostly a tendency to decrease the activity of organic mutagens. It was also shown that dependent on the water quality and treatment conditions a slight increase of mutagenic activity occurred, but this activity would be reduced by increasing the ozone dose. It seems possible to optimize the ozone treatment conditions regarding the level of ozone dose and the contact time to avoid an increase of mutagenic activity. Furthermore it was shown that when a mutagenic raw water source was used a proper combination of treatment processes is able to produce drinking water in which no mutagenic activity could be detected under the test conditions. Finally it is stated that before far reaching decisions with respect to use mutagenicity data for a selection of water sources or treatment processes will be made, more information on the relation mutagenic activity from drinking water and effects on human health should become available. PMID- 3003903 TI - Diet advice, with a grain of salt and a large helping of pepper. PMID- 3003904 TI - Translocation of protein kinase C activity may mediate hippocampal long-term potentiation. AB - Protein kinase C activity in rat hippocampal membranes and cytosol was determined 1 minute and 1 hour after induction of the synaptic plasticity of long-term potentiation. At 1 hour after long-term potentiation, but not at 1 minute, protein kinase C activity was increased twofold in membranes and decreased proportionately in cytosol, suggesting translocation of the activity. This time dependent redistribution of enzyme activity was directly related to the persistence of synaptic plasticity, suggesting a novel mechanism regulating the strength of synaptic transmission. PMID- 3003905 TI - Equine infectious anemia virus gag and pol genes: relatedness to visna and AIDS virus. AB - Comparison of HTLV-III, the putative AIDS virus, with other related viruses, may help to reveal more about the origin of AIDS in humans. In this study, the nucleotide sequence of the gag and pol genes of an equine infectious anemia virus (EIAV) proviral DNA clone was determined. The sequence was compared with that of HTLV-III and of visna, a pathogenic lentivirus of sheep. The results show that these viruses constitute a family clearly distinct from that of the type C viruses or the BLV-HTLV-I and -II group. Within the family, EIAV, HTLV-III, and visna appear to be equally divergent from a common evolutionary ancestor. PMID- 3003906 TI - Induction of HTLV-III/LAV from a nonvirus-producing T-cell line: implications for latency. AB - When the human T-cell line A3.01 is infected with HTLV-III/LAV, the virus associated with the acquired immune deficiency syndrome (AIDS), most of the cells are killed. However, a small number of cells that lack the Leu-3 surface marker survive. Under normal conditions these surviving cells do not produce virus, nor can they be infected by the virus, but they can be induced to produce virus by treatment with 5-iodo-2'-deoxyuridine. This response can be induced for as long as 3 months after the initial infection, suggesting that the cells may harbor a latent form of HTLV-III/LAV. PMID- 3003907 TI - Stress-induced inhibition of reproductive functions: role of endogenous corticotropin-releasing factor. AB - In the adult castrated male rat, exposure to inescapable, intermittent electroshocks inhibited the pulsatile pattern of luteinizing hormone release and markedly lowered its plasma concentrations. The central administration of the corticotropin-releasing factor (CRF) antagonist alpha-helical ovine CRF residues 9 to 41 reversed the inhibitory action of stress. Neither its peripheral injection, nor the intraventricular injection of the inactive CRF analog des-Glu to Arg ovine CRF was effective. These results suggest that endogenous CRF may mediate some deleterious effects of noxious stimuli on reproduction. PMID- 3003908 TI - Detection of papillomavirus DNA in human semen. AB - Human papillomavirus DNA has been detected in the semen of three patients, two of whom have severe chronic wart disease. These data support the contention that sexual transmission of human papillomavirus DNA could occur via semen, a possibility suggested by epidemiological data on the sexual transmission of human papillomavirus. PMID- 3003909 TI - The NOD mouse: recessive diabetogenic gene in the major histocompatibility complex. AB - Examination of the histocompatibility region of the nonobese diabetic (NOD) mouse with antibodies against class II glycoproteins (products of immune response genes of the major histocompatibility complex I-A and I-E), hybrid T-cell clones, and mixed-lymphocyte cultures and analysis of restriction fragment length polymorphisms indicate that the NOD mouse has a unique class II major histocompatibility complex with no expression of surface I-E, no messenger RNA for I-E alpha, and an I-A not recognized by any monoclonal antibodies or hybrid T cell clones studied. In crosses of NOD mice with control C3H mice, the development of diabetes was dependent on homozygosity for the NOD mouse's unique major histocompatibility region. PMID- 3003910 TI - High titers of autoantibodies to topoisomerase I (Scl-70) in sera from scleroderma patients. AB - Patients with rheumatic diseases often have circulating autoantibodies to nuclear components. The clinical significance of the antibodies is controversial, although in some cases they are valuable in the diagnosis of the disease. This report presents results of a study of Scl-70, an autoantigen recognized by sera of many patients with the most severe form of progressive systemic sclerosis. It was possible to show, by three independent criteria, that Scl-70 is the abundant nuclear enzyme DNA topoisomerase I. Therefore, antibody probes of high titer and high affinity are now available for the study of this important nuclear enzyme. PMID- 3003911 TI - Ubiquitin moves to the cell surface. PMID- 3003912 TI - A surprising action for the AIDS virus. PMID- 3003913 TI - Cell surface molecule associated with lymphocyte homing is a ubiquitinated branched-chain glycoprotein. AB - Partial amino acid sequence analysis of a purified lymphocyte homing receptor demonstrates the presence of two amino termini, one of which corresponds precisely to the amino terminus of ubiquitin. This observation extends the province of this conserved polypeptide to the cell surface and leads to a proposed model of the receptor complex as a core polypeptide modified by glycosylation and ubiquitination. Independent antibodies to ubiquitin serve to identify additional cell surface species, an indication that ubiquitination of cell surface proteins may be more general. It is proposed that functional binding of lymphocytes to lymph node high endothelial venules might involve the ubiquitinated region of the receptor; if true, cell surface ubiquitin could play a more general role in cell-cell interaction and adhesion. PMID- 3003915 TI - Progress on a vaccine for Epstein-Barr virus. PMID- 3003914 TI - Expression cloning of a lymphocyte homing receptor cDNA: ubiquitin is the reactive species. AB - The lymphocyte cell surface receptor for the high endothelial venules (HEV's) of peripheral lymph nodes is specifically recognized by the monoclonal antibody MEL 14. Three independent complementary DNA (cDNA) clones, each of which encodes the protein ubiquitin, were detected by virtue of the expression of the MEL-14 antigenic determinant on cDNA-beta-galactosidase bacterial fusion proteins. The antigenic determinant defined by MEL-14 resides in the carboxyl terminal 13-amino acid proteolytic peptide of ubiquitin, but is undetected in intact undenatured ubiquitin and other cellular ubiquitinated proteins. Antisera and monoclonal antibodies to ubiquitin determinants bind to the surface of both HEV-receptor positive and negative cell lines. The MEL-14-identified cDNA clones hydridize to RNA transcripts that encode tandemly repeated ubiquitins. Sequence analysis of these polyubiquitin cDNA's does not identify a leader sequence for export to the cell surface. The expression of the MEL-14 epitope of ubiquitin depends upon its local environment. The steady-state levels of expression of the ubiquitin messenger RNA's do not correlate with either the tissue derivation of the RNA or the expression of the lymphocyte HEV receptor. Regulation of the expression of the HEV receptor is not likely to reflect the transcriptional control of ubiquitin genes, but rather to reflect control of the expression of the HEV core polypeptide or its level or form of ubiquitination. PMID- 3003916 TI - Human papilloma virus and cervical cancer. PMID- 3003918 TI - FDA approves Pasteur's AIDS test kit. PMID- 3003917 TI - Three-year incidence of AIDS in five cohorts of HTLV-III-infected risk group members. AB - The incidence of the acquired immune deficiency syndrome (AIDS) among persons infected with human T-lymphotropic virus type III (HTLV-III) was evaluated prospectively among 725 persons who were at high risk of AIDS and had enrolled before October 1982 in cohort studies of homosexual men, parenteral drug users, and hemophiliacs. A total of 276 (38.1 percent) of the subjects were either HTLV III seropositive at enrollment or developed HTLV-III antibodies subsequently. AIDS had developed in 28 (10.1 percent) of the seropositive subjects before August 1985. By actuarial survival calculations, the 3-year incidence of AIDS among all HTLV-III seropositive subjects was 34.2 percent in the cohort of homosexual men in Manhattan, New York, and 14.9 percent (range 8.0 to 17.2 percent) in the four other cohorts. Out of 117 subjects followed for a mean of 31 months after documented seroconversion, five (all hemophiliacs) developed AIDS 28 to 62 months after the estimated date of seroconversion, supporting the hypothesis that there is a long latency between acquisition of viral infection and the development of clinical AIDS. This long latency could account for the significantly higher AIDS incidence in the New York cohort compared with other cohorts if the virus entered the New York homosexual population before it entered the populations from which the other cohorts were drawn. However, risk of AIDS development in different populations may also depend on the presence of as yet unidentified cofactors. PMID- 3003919 TI - Endonucleolytic activity that cleaves immunoglobulin recombination sequences. AB - An endonucleolytic activity has been identified in nuclear extracts of chick embryo bursa and mouse fetal liver cells. The activity introduces a double-strand cut in the vicinity of the recombination site of immunoglobulin joining gene segments. The cleavage occurs at the dinucleotide pair TG-AC. This activity is a good candidate for the putative endonuclease involved in recombination of the immunoglobulin variable, diversity, and joining regions. It is distinct from the endonuclease activities previously reported by others. PMID- 3003920 TI - Quantitative imaging of neuroreceptors in the living human brain. AB - Positron emission tomography (PET) makes it possible for the first time to examine in living humans the chemistry of the brain, which relates the structures of the brain to the functions of the mind. PET scans make it possible to assess the state of neurotransmitter receptors, such as the dopamine, serotonin, muscarinic cholinergic, opiate, and benzodiazepine receptors, in different regions in normal persons and patients with neuropsychiatric diseases. One of the most striking findings to date is that dopamine receptors fall significantly between the ages of 19 and 73 years, to a greater degree in men than in women. The effects of neurotropic drugs, such as haloperidol and methadone, can be assessed in an individual patient, providing for the first time an objective indicator of the specific desired effects of such drugs in the treatment of nervous or mental disease. Studies of patients with diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease, bipolar disease, and schizophrenia are in progress. At present, the patients are being examined as part of research protocols, but it is likely that clinical trials will not be far off. PMID- 3003922 TI - [Zovirax]. PMID- 3003921 TI - Expression of platelet proteins during the in vitro and in vivo differentiation of megakaryocytes and morphological aspects of their maturation. PMID- 3003923 TI - Norwalk virus outbreak at a college campus. AB - An epidemic of nonbacterial gastroenteritis affected nearly 100 students at a college campus in Jefferson County, Alabama. The outbreak closely resembled food poisoning, since there was a rapid occurrence of multiple cases within a short period. Vomiting occurred in 79% and diarrhea in 64%; fever was uncommon. We found a significant association between the illness and the eating of lettuce at a meal one day before the outbreak began. Paired serologic specimens showed evidence of Norwalk virus infection. Twenty acutely ill students had leukocytosis (mean WBC 12,780/cu mm) and lymphopenia--a pattern that may be characteristic of Norwalk virus gastroenteritis. Outbreaks of Norwalk virus infection as well as other nonbacterial gastroenteritis may closely mimic epidemics caused by more familiar foodborne pathogens such as staphylococci. PMID- 3003924 TI - Breast cancer: presentation and prognosis in the community hospital. AB - A retrospective analysis of all breast cancer primarily treated at this institution during the ten-year period from 1967 to 1977 revealed 336 cases; in 308 of these cases, complete follow-up of five or more years was available. Clinical staging using the TNM system (tumor, node, metastasis) showed stage I disease in 60 patients, stage II in 94, stage III in 105, and stage IV in 49. Pathologic classification of these 308 cases showed a high incidence of infiltrating ductal carcinoma (88%). Early in the study operative treatment consisted of radical mastectomy, with modified radical mastectomy being more common in the later years. Simple mastectomy was occasionally done palliatively. Adjuvant treatment using cyclophosphamide (Cytoxan), methotrexate, 5 fluorouracil, radiation, and/or hormonal manipulation was then given according to a set protocol. In general, five- and ten-year survival improved with this combined treatment. The mortality among patients with stage I infiltrating ductal carcinoma not given adjuvant therapy suggests the need for proper selection of patients in this group who should receive adjuvant chemotherapy because they are at increased risk. PMID- 3003925 TI - Lung cancer: is the increasing incidence due to radioactive polonium in cigarettes? AB - This paper presents clinical, experimental, and epidemiologic evidence to help explain the rapidly increasing incidence of primary lung cancer, with recently observed reversal in leading cell type from squamous cell to adenocarcinoma. It postulates that this may be due to changes in modern cigarettes, with or without filters, which allow inhalation of increased amounts of radioactive lead and polonium and decreased amounts of benzopyrene. This hypothesis is based upon measurements of increased concentrations of radioactive polonium in the lungs of cigarette smokers, in modern tobaccos grown since 1950, and in high-phosphate fertilizers used for tobacco farming in industrialized countries. Critical support for this thesis is based upon experimental animal studies in which lung cancers that resemble adenocarcinomas are induced with as little as 15 rads of radioactive polonium, equal to one fifth the dosage inhaled by cigarette smokers who average two packs a day during a 25-year period. PMID- 3003926 TI - Genital condylomas in immunosuppressed women: a therapeutic challenge. AB - Of 30 immunosuppressed women with genital condylomas, 20 (67%) had associated neoplasia of the lower genital tract. We retrospectively studied 11 patients with flat condylomas confined to the cervix; all lesions were eradicated by conventional therapy. Three patients studied retrospectively and 16 studied prospectively had condylomatous lesions involving several sites within the lower genital tract. These lesions were unusually resistant to various treatment methods. Maintenance therapy with 5-fluorouracil (5-FU) cream resulted in a significantly higher control rate (6/7 patients, 86%) than could be achieved otherwise (2/9 patients, 22%; P less than .025). We conclude that ablative therapy by surgery or topical chemical agents followed by maintenance therapy with 5-FU cream may be the treatment of choice for condylomas and other human papillomavirus-associated lesions of the lower genital tract in immunosuppressed women. PMID- 3003927 TI - Laetrile intoxication and hepatic necrosis: a possible association. AB - A 65-year-old woman with cirrhosis and hepatoma lapsed into deep coma, hypotension, and acidosis after ingestion of 3 gm of Laetrile, a cyanogenetic glucoside. After initial treatment, the patient regained consciousness, but massive hepatic damage led to her death. We suggested a possible relationship between Laetrile poisoning and massive hepatic necrosis. PMID- 3003928 TI - Immortalization of xeroderma pigmentosum cells by simian virus 40 DNA having a defective origin of DNA replication. AB - A simian virus 40 (SV40) DNA fragment, encompassing the whole early region and having a defective origin of DNA replication, has been used to transform human fibroblast cells derived from two xeroderma pigmentosum (XP) patients. Two of the SV40-transformed XP cell lines, belonging to complementation group C, had acquired the characteristic of indefinite life-span in culture. These XP cell lines synthesize T antigen as shown by immunofluorescence and retain the high sensitivity to UV irradiation. Detailed karyotype analysis shows very few chromosomal changes, while the transfecting SV40 DNA is integrated into cellular DNA sequences. These are the first immortalized XP cell lines derived from complementation group C. In view of the extreme difficulty in obtaining immortalized human fibroblasts, we suggest a possible advantage of replication defective SV40 DNA molecules for immortalizing human fibroblast cells of any source. PMID- 3003929 TI - Novel mutants of CHO cells resistant to adenosine analogs and containing biochemically altered form of adenosine kinase in cell extracts. AB - Stable mutants which are approximately five- and eightfold resistant to an inosine analog, formycin B (Fomr) have been selected in a single-step from Chinese hamster ovary cells at a frequency of approximately 10(-6). Cross resistance studies with these mutants show that the Fomr mutants exhibit increased resistance to all adenosine analogs (N-and C-nucleosides) examined and, in accordance with their cross-resistance pattern, the mutants exhibited decreased cellular uptake and phosphorylation of formycin B and various adenosine analogs. In cell hybrids formed with sensitive cells, the drug-resistant phenotype of these mutants behaved recessively. However, unlike mutants resistant to adenosine analogs that have been obtained previously, which contain no measurable activity of adenosine kinase (AK) in cell extracts, the two Fomr mutants studied contained about 60 and 110% of the enzyme activity (compared to the parental cells) in their cell extracts. Biochemical studies with AK from the mutant cells show that in comparison to the wild-type enzyme, the mutant enzymes required much higher concentrations of the adenosine analog N7-(delta 2 isopentenyl) formycin A for similar inhibition of [3H]adenosine phosphorylation. These results indicate that AK from the Fomr mutants has lower affinity for phosphorylation of adenosine analogs in comparison to the enzyme from the parental cells. The genetic lesion in the Fomr mutants may thus be directly affecting the structural gene for AK. PMID- 3003930 TI - Mitotic segregation of mitochondrial DNAs in human cell hybrids and expression of chloramphenicol resistance. AB - The relationship between the chloramphenicol (CAP)-resistant phenotype and the mtDNA genotype was investigated in segregating human, HeLa X HT1080, somatic cell hybrids. The parental mtDNAs were quantitated in heteroplasmic cells by using restriction fragment length polymorphisms (RFLPs) detected in Southern blots. CAP resistant (R) X CAP-sensitive (S) hybrids selected and grown in CAP for brief periods had as little as 25% CAP-R mtDNA. With prolonged selection, the CAP-R mtDNA increased to 90-95%. Hybrids selected and passaged without CAP either retained both mtDNAs or progressively lost one mtDNA (mitotic segregation). The CAP-resistance phenotype of these hybrids changed abruptly when the proportion of CAP-R mtDNAs fluctuated around approximately 10% (threshold effect). Hybrids with greater than 25% HT1080 mtDNA had an additional characteristic. They cloned better with CAP than without. The cloning efficiency in CAP of hybrids having 90% HT1080 mtDNA was more than fivefold greater than the control. PMID- 3003931 TI - Analysis of homologous recombination in cultured mammalian cells in transient expression and stable transformation assays. AB - Recombination between plasmid molecules, each containing a nonoverlapping deletion mutation in the hamster adenine phosphoribosyltransferase gene, was measured after coinjection into rat cells. Using these two plasmids, as linear or circular molecules, the recombination efficiency was measured soon after injection in a transient expression assay or after selection for stable transformants. The transient assay revealed that linear molecules were a better substrate for recombination, with double strand breaks within the region of homology stimulating recombination more than breaks outside the region of homology. A 20 to 70-fold increase in the efficiency of recombination was observed when two linear molecules were coinjected as compared to two circular molecules. Linear molecules were found to not only stimulate recombination but also to facilitate stable integration of the recombinant molecule into the host genome. PMID- 3003932 TI - Genetic mapping of Pim-1 putative oncogene to mouse chromosome 17. AB - Pim-1 is a putative oncogene activated in T-cell lymphomas induced by Moloney and AKR mink cell focus forming (MCF) viruses. We have determined the chromosomal localization of the Pim-1 gene in mice by Southern blot analysis of DNAs obtained from a panel of mouse-Chinese hamster somatic cell hybrids. The Pim-1 gene was localized on chromosome 17, a chromosome frequently aberrant in T-cell lymphomas. Two chromosomal regions, containing sequences homologous to regions within the Pim-1 locus, were localized on chromosome 6 and 16. PMID- 3003933 TI - [Neurophysiologic evaluation of the proximal part of peripheral nerves in uremic neuropathies]. PMID- 3003934 TI - [Parathormone and peripheral neuropathy in patients on chronic hemodialysis]. PMID- 3003935 TI - [Serum angiotensin-converting enzyme activity in disorders of thyroid gland function--clinical and experimental findings]. PMID- 3003938 TI - [Anticoagulation protein C: physiological aspects]. PMID- 3003936 TI - Causes of hepatomegaly at King Edward VIII Hospital, Durban. A prospective study of 240 black patients. AB - In this prospective study of 240 black patients with liver enlargement admitted to the medical wards of King Edward VIII Hospital, Durban, a cause for the hepatomegaly was found in 92.5% of cases (63.8% without recourse to biopsy, 28.7% after liver biopsy). The commonest cause was congestive heart failure (36.7%), followed by amoebic liver abscess (7.1%), hepatocellular carcinoma (5.8%) and cirrhosis (5.4%). Liver biopsy provided the diagnosis in 90.8% of patients with initial unexplained hepatomegaly. The diagnostic yield of liver biopsy was increased by submitting 3 biopsy specimens for histological examination. The 3 specimens are obtained using a single intercostal entry site and redirecting the biopsy needle, without increasing the risk of complications. Hepatic tuberculosis was present in 9.2% of patients who underwent biopsy. There were no consistent clinical findings in these patients. Therefore, in communities in which tuberculosis is endemic, all patients with unexplained hepatomegaly require liver biopsy since it provides the only means of making this diagnosis. PMID- 3003937 TI - Obesity in perspective. AB - Attitudes to obesity are changing. It is currently regarded as a common, multifactorial disorder with serious medical and psychological consequences. It is also resistant to treatment. Recent research with experimental animals has given new insights into the molecular pathology of this condition and gives some hope of novel therapeutic intervention. PMID- 3003939 TI - [Anti-HTLV-III in hemophilic patients in Barcelona]. PMID- 3003940 TI - Salivary glands. AB - A review of the more common inflammatory and neoplastic conditions affecting salivary glands has been presented. The use of hydration, massage, antibiotics, and steroids is effective initial treatment for suppurative sialadenitis and usually negates the need for surgical drainage. Total excision of the salivary gland and its duct is necessary in procedures for recurrent infection. Our technique for closure of the floor of the mouth after excision of the submandibular gland and Wharton's duct is described. Salivary neoplasms involving the parotid gland, the submandibular gland, and the minor salivary glands are treated on the basis of their histologic and local findings. Stepwise illustrations of our technique of parotidectomy and surgical considerations, including the counseling of a patient with a parotid mass, are presented to assist surgeons who care for patients with salivary disorders. PMID- 3003941 TI - Ethiodized oil emulsion enhanced computerized tomography in the preoperative assessment of metastases to the liver from the colon and rectum. AB - Selective reticuloendothelial (RE) cell uptake of ethiodized oil emulsion 13 (EOE 13), an emulsion of Ethiodol (ethiodized oil) roentgenographic contrast material in a phosphate buffer, permits detection of small metastatic lesions of the liver and spleen through enhancement of roentgenographic density differences on computerized tomography (CT) between tissues containing and not containing RE cells. To determine the efficacy of this contrast material in the assessment of patients with metastatic disease of the liver, routine CT and emulsion enhanced tomography (EOE) were performed in a series of 15 patients prior to surgical exploration for treatment of carcinoma of the colon and rectum. All patients were suspected of harboring hepatic metastasis on the basis of clinical examination, liver function tests or radionuclide scans. EOE consistently demonstrated the nature and location of hepatic defects. Surgical exploration failed to locate one metastasis that was judged to be real because of progressive enlargement on EOE and CT over a period of two years. CT scans detected metastases in three patients subsequently shown to have normal livers and failed to detect disease in one patient subsequently shown to have metastases. EOE contrast material provides a more sensitive and accurate picture of metastatic liver involvement from carcinoma of the colon and rectum than is available on routine CT. The information provided by the results of this test can be useful in preoperative planning when treatment of disease of the liver is considerable feasible. PMID- 3003942 TI - Treatment of fibrolamellar hepatoma with partial or total hepatectomy and transplantation of the liver. AB - Fourteen patients with fibrolamellar hepatoma were treated with radical excision. In eight, a subtotal hepatic resection was performed from 16 months to more than 16 years ago. None of the patients have died and recurrences have been seen in only one patient. Six other patients had total hepatectomy and hepatic replacement. Two of these six patients have died of metastases and a third is living with recurrent tumor. This experience has justified the continuing use of quite aggressive extirpative procedures for the treatment of fibrolamellar hepatoma. PMID- 3003943 TI - [Results of radiotherapy and chemotherapy in small-cell bronchial carcinoma]. AB - At the Radiotherapeutic Department of the Faculty of Medicine in Istanbul, 35 masculine patients with microcellular bronchial carcinoma, limited disease, were treated for two years, i.e. between 1980 and 1981, with a combination of radiotherapy and chemotherapy. Nine out of these patients are tumor-free after at least 46 months, i.e. about four years. This corresponds to a tumor-free survival rate of 25.7%. PMID- 3003944 TI - Inhibition of reduction in the testicular weight by WR-2721 in relation to the body weight after whole-body gamma irradiation. AB - Male Swiss albino mice were irradiated with 60Co gamma rays at 3, 6 and 8 Gy dose levels in separate groups in the presence and absence of the radioprotective drug, WR-2721. The drug treated animals received intraperiotoneal injection of 400 mg/kg body weight aqueous solution (pH 6.2) of WR-2721. Animals were weighted and sacrificed at various post-irradiation intervals between day 1/4 to day 28. Testes were weighed for determining the testis/body weight ratios. The results indicated that testis/body weight ratio reduced after radiation exposure and the extent of this reduction depended on the dose of exposure. Injection of WR-2721 inhibited the body weight loss including the testicular mass loss after irradiation, as depicted by the significantly higher testis/body weight ratio in the drug pretreated animals. PMID- 3003945 TI - Selective impairment of nutrient absorption from intestines with chronic venous hypertension. AB - Malnutrition is frequently associated with advanced cirrhosis. To investigate the role of portal hypertension in nutritional impairment, we developed an animal model to isolate and characterize the effects of chronic intestinal venous hypertension on intestinal nutrient absorption. We performed mesenteric arteriovenous anastomosis combined with portal vein banding in rats. Hepatic architecture and excretory function (bile flow and bile salt output) were unaltered, while severe and persistent intestinal venous hypertension was produced. We then measured in vivo absorption rates of three test nutrients (vitamin D3, valine, and tryptophan) and water. Vitamin D3 absorption was significantly impaired by intestinal congestion, while amino acid absorption was unaffected. Splanchnic hypertensive rats absorbed less water than controls. We conclude that chronic intestinal venous hypertension alone selectively impairs nutrient absorption. PMID- 3003946 TI - [Criteria for the selection of drugs for the pathogenetic treatment of Itsenko Cushing disease]. AB - The authors presented the results of a study of the level of corticotropin and cortisol in the blood at different time after a single intake of peritol or parlodel in 48 patients with Icenko-Cushing disease at the active stage. Corticotropin and cortisol concentrations were determined by radioimmunoassay with the help of standard kits. The study showed a different sensitivity of patients with Icenko-Cushing disease to peritol and parlodel. According to the expression of the reaction of the hypophyseal-adrenal system all the patients were divided into the following groups: with a noticeable reaction to one or both drugs, with a slightly noticeable decrease in the level of hormones or a paradoxical reaction to the drugs. A single test with inhibitors of hypophyseal corticotropic function provided an opportunity for an individual choice of an effective drug for pathogenetic treatment of patients with Icenko-Cushing disease. The optimum time for blood examination to determine the concentration of corticotropin was 1 and 2 hrs, the level of cortisol 2-4 hrs after the intake of peritol and parlodel. PMID- 3003948 TI - Brain and meningeal biopsy in patients with acquired immunodeficiency syndrome. PMID- 3003947 TI - [Relation between blood levels of thyroid hormones and insulin and lipid metabolism and the clinical course of chronic forms of ischemic heart disease]. AB - Ninety patients aged 41 to 68 years with the chronic patterns of coronary heart disease (CHD) were examined for the content of triiodothyronine (T3), total T4 and free thyroxine (FT4), thyrotropic hormone (TTH), adrenocorticotropic hormone (ACTH), and insulin. At the same time the patients were examined with the aid of the glucose tolerance test, determination of blood concentration of cholesterol, triglyceride, high density lipoprotein cholesterol, fibrinogen and soluble fibrin. The patients with CHD showed a decrease in the basal level of T3, T4, FT4 and elevation of TTH, ACTH and insulin in blood. A correlation was found between the basal of insulin and thyroid hormones and lipid metabolism in CHD patients. It was shown that thyroid hypofunction, unmarked clinically but detectable by the lowering of the content of thyroid hormones and rise of thyrotropic hormone in blood of CHD patients, might promote the development of hyperinsulinemia. PMID- 3003949 TI - [Immunologic (serologic) identification of sexually transmitted infections]. PMID- 3003951 TI - [Survey of drug prescriptions in a French-speaking black African city]. PMID- 3003950 TI - [Bilateral stenosis of the renal artery under enalapril without deterioration of renal function]. PMID- 3003953 TI - Urokinase antigen in plasma of patients with liver cirrhosis and hepatoma. AB - Plasma urokinase antigen levels were studied in 78 patients suffering from liver diseases. Blood was drawn before any specific medication was initiated. Impairment of liver function was comparable in all patients. In both groups of cirrhotic liver disease (alcoholic and non-alcoholic), normal levels of plasma urokinase antigen were found as compared to age-matched control groups. In both groups of patients with hepatomas (with or without a history of liver cirrhosis), however, significantly increased plasma urokinase antigen levels could be determined. These data indicate that an increase in plasma urokinase antigen might rather relate to malignant growth in liver disease than to impaired liver function. PMID- 3003952 TI - Studies on platelets of patients with inherited platelet disorders suggest that collagen-induced fibrinogen binding to membrane receptors requires secreted ADP but not released alpha-granule proteins. AB - Collagen induces a saturable 125I-fibrinogen binding to normal human platelets. A role for secreted ADP in this process is supported by studies on 2 patients with the Chediak-Higashi syndrome. Both collagen-induced nucleotide release and 125I fibrinogen binding were strongly reduced while ADP-induced fibrinogen binding was normal. Platelets from 2 patients with the gray platelet syndrome bound normal amounts of 125I-fibrinogen in the presence of ADP or collagen despite the severe reduction of secretable alpha-granule proteins. Binding did not occur to collagen stimulated type I thrombasthenic platelets which lacked GPIIb-IIIa complexes but was detected in amounts which correlated with the residual concentrations of GPIIb-IIIa in the platelets of a patient with type II disease. Our results allow us to propose that collagen-induced fibrinogen binding to normal platelets requires the presence of GPIIb-IIIa complexes and secreted ADP but proceeds independently of alpha-granule release. PMID- 3003954 TI - One year follow-up study of T-cell subsets and incidence of seropositivity for HTLV-I and HTLV-III antibodies in patients treated "on demand" or sporadically with clotting concentrates. AB - A 1-year follow-up study of the T-cell subset abnormalities was carried out in 16 severe haemophilia A patients, treated "on demand" with an average amount of 500 U/kg/yr of factor VIII concentrate (group A) and in 15 mild haemophiliacs or von Willebrand patients treated only sporadically with less than 3000 U of factor VIII and no longer exposed to any other blood component in the 2 years preceding the beginning of the study (group B). In group A, 50% and 70% of patients showed a reduced or inverted T 4/T 8 ratio, respectively, at the beginning and at the end of follow-up. These values were of 30% and 20% in patients of group B, suggesting a long-lasting effect of concentrate therapy on T-cell subsets. The low T 4/T 8 ratio was mainly due to an increase of suppressor cells. None of the patients was found positive for anti HTLV-I, whereas 3 patients, all belonging to the group A, showed antibodies against HTLV-III. Thus, in these patients, HTLV III seems not to be the only cause of low T 4/T 8 ratio. PMID- 3003955 TI - Anticoagulant activity in cell-free peritoneal fluid of an experimental pancreatic ascites tumor. AB - The ascitic form of a chemically-induced pancreatic ductal adenocarcinoma in the Syrian golden hamster was very bloody and indistinguishable from blood macroscopically. Unlike blood, the bloody fluid remained unclotted at room temperature. To explore the possibility of presence of anticoagulants, we mixed 40% cell-free fluid with 60% normal human plasma and tested the clottability of the mixture with standard techniques. Plasma containing the fluid showed markedly prolonged activated partial thromboplastin time (APTT), thrombin time (TT) and recalcification time (RCT), and normal prothrombin time (PT) and reptilase time (RT). Comparing the prolongation of APTT of samples containing the fluid to those containing a commercial heparin, the fluid contained an anticoagulant activity equivalent to 0.436 +/- 0.03 unit heparin per ml (mean +/- SEM, n = 14). In addition to prolonging the APTT, TT and RCT, the fluid also inhibited the clotting and amidolytic activities of thrombin. "Heparsorb" had nearly completely neutralized the anticoagulant activity in fluid samples, while protamine sulfate was only partially effective. Incubation of fluid with pronase or phospholipase did not affect its anticoagulant activity; incubation with heparinase had only a minimal effect. Electrophoresis of an alkali digested fluid on cellulose acetate revealed the presence of heparan sulfate. The native ascitic fluid also contained other hemostatic components including platelets, fibrinogen and antithrombin III, but their concentrations were much lower than in blood. Apparently, heparan sulfate in the neoplastic effusion is largely responsible for the bloody ascites tumor remaining unclotted. PMID- 3003956 TI - Influence of beta 2-glycoprotein-I upon the content of cAMP and cGMP in human blood platelets. AB - The influence of beta 2-glycoprotein-I on the cyclic nucleotide content in human gel filtered platelets (GFP) was studied: beta 2-G-I up to a concentration of 5 mg/ml does not alter the basal cAMP level in resting GFP and also does not influence the action of PGE1 which strongly inhibits platelet aggregation by elevation of cAMP. Collagen induced aggregation of GFP leads to an increase of the platelet cGMP content. Preincubation of GFP with 0.2 mg/ml of beta 2-G-I enhances significantly the cGMP increase by collagen. In a similar manner beta 2 G-I also amplifies the increase in cGMP concentration induced by ADP. In this case, a concomitant alteration of the aggregation pattern is observed. Thrombin produces a much higher cGMP elevation in GFP than ADP or collagen. The admixture of 0.2 mg/ml of beta 2-G-I is not able to enhance further the thrombin effect on cGMP. The results are discussed in view of the possible significance of these observations for platelet aggregation in vivo. PMID- 3003957 TI - The prevalence of HTLV-III and HTLV-I antibodies in serum of hemophiliacs. PMID- 3003958 TI - Interaction of stable prostaglandin endoperoxide analogs U46619 and U44069 with human platelet membranes: coupling of receptors with high-affinity GTPase and adenylate cyclase. AB - It has been demonstrated using a membrane preparation of human platelets that stable analogs of PGH2, U46619 and U44069, control the activity of adenylate cyclase and a high-affinity hormone-sensitive GTPase. At 10(-8)-10(-6) M, the analogs inhibit the basal activity of adenylate cyclase by 20-25%. With a further rise in U46619 and U44069 concentrations up to 10(-5)-10(-4) M, the inhibition is abolished and adenylate cyclase activity is stimulated in a dose-dependent fashion. In the presence of PGE1, only inhibitory action of U46619 was observed at all the concentrations tested. The inhibitory action of the analogs on adenylate cyclase correlates with the activation of the high-affinity hormone sensitive GTPase. It is concluded that U46619 and U44069 inhibit human platelet adenylate cyclase via specific receptors coupled to the GTP-binding inhibitory protein. PMID- 3003959 TI - Plasma thrombospondin in patients with chronic renal failure, liver disease and splenectomy. AB - Thrombospondin (TSP), is a major constituent of human blood platelet alpha granules. Stimulation of platelets causes the release of TSP in parallel with other alpha-granule constituents such as beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4) but the thrombospondin plasma in vivo half life is significantly greater than beta-TG and PF4. The aim of this study was to assay TSP levels in plasma of patients with chronic renal failure (CRF), liver disease (LD) and following splenectomy. The TSP values were then compared to the patients plasma levels of two traditional markers of platelet activation, beta-TG and PF4, and to fibronectin (FN) and von Willebrand factor (VIII:vWF). Plasma TSP levels (67.6 +/- 16.9 ng/ml) assayed in 14 CRF patients were significantly higher (p less than 0.05) than those measured in 28 donors (55.5 +/- 11.7 ng/ml). No correlation was observed, in CRF patients, between the TSP level and PF4 (2.5 +/- 1.5 ng/ml), beta-TG (131.1 +/- 21 ng/ml), FVIII:vWF (252 +/- 85%), or FN (102 +/- 33%) plasma levels. The TSP plasma level in CRF patients was significantly correlated (p less than 0.02) with that of fibrinopeptide A (4.1 +/- 1.9 ng/ml). Although the beta-TG (23.5 +/- 6.9 ng/ml) and PF4 (2.9 +/- 2 ng/ml) plasma levels in six LD patients were normal, the TSP levels (82.5 +/- 39.1 ng/ml) were significantly increased (p less than 0.01). Thrombospondin plasma levels (77.1 +/ 20.1 ng/ml) in 14 patients having undergone splenectomy were significantly increased (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3003960 TI - Differential labeling of platelet alpha 2 adrenoceptors by 3H dihydroergocryptine and 3H yohimbine in patients with myeloproliferative disorders. AB - Platelet alpha adrenoceptor status was examined using the radioligands 3H yohimbine (3H-YOH) and 3H-dihydroergocryptine (3H-DHE) in 14 patients with myeloproliferative disorder (MPD) and 10 normal controls. Platelets from normal controls and MPD patients sensitive to adrenaline induced aggregation exhibited approximately 50% more binding sites identified by 3H-DHE than 3H-YOH, whereas MPD platelets insensitive to adrenaline showed selective loss of these 'extra' 3H DHE sites. In functional studies after 30 minutes preincubation with the unlabelled antagonists, DHE was more potent than YOH at inhibiting adrenaline induced aggregation in normal platelets. In addition, the affinity constant for DHE was virtually identical in binding and functional experiments, whereas for YOH the affinity constant for binding was approximately 10 fold more potent than that for aggregation. These results suggest that the alpha adrenoceptor binding site on human platelets labelled by 3H-DHE may be of more functional relevance than that labelled by 3H-YOH alone. PMID- 3003962 TI - Effects of aspirin, indomethacin, and sodium salicylate on human erythrocyte membranes as detected with electron spin resonance spectroscopy. AB - Electron spin resonance spectroscopy of probed samples was used to determine the structural changes in human erythrocyte membranes prior to and at intervals following ingestion of either 10 grains acetylsalicylic acid, 10 grains sodium salicylate, or 50 mg indomethacin by both male and female subjects. Analysis of erythrocytes from female subjects indicated a time-dependent disordering of the membrane over the eight hour period following aspirin ingestion while the cells of male subjects showed a slight membrane ordering over the same time period. Erythrocytes drawn from females at the beginning of the menstrual cycle showed the greatest amount of membrane disordering at one hour following aspirin ingestion, but by eight hours, the membrane structure had returned to that of control. The time dependent disordering in membrane structure of cells from females in the middle of the menstrual cycle was biphasic. Ingestion of indomethacin induced only slight membrane changes in both male and female subjects over the times examined. Ingestion of sodium salicylate by either men or women did not induce significant changes in erythrocyte membrane order. Washed erythrocytes when mixed with salicylate, aspirin, or indomethacin were either identical to control cells or slightly more ordered. This study suggests that aspirin-induced alterations in membrane structure may depend upon steroid hormone levels. PMID- 3003961 TI - Characterization of human platelet beta-adrenoceptors. AB - The widespread use of beta-adrenoceptor antagonists against hypertension, angina pectoris and migraine or as a preventive treatment after myocardial infarction has encouraged us to investigate the effects of these drugs on platelet function. The aim of this study was to examine whether beta-blocking drugs interfere with platelet beta- adrenoceptors and whether this dependency is related to their selectivity for beta-adrenoceptor subtypes. Beta-adrenoceptor stimulation of human platelets with isoprenaline increased cyclic AMP (cAMP), which is known to inhibit platelet aggregation. Furthermore, our studies showed that cAMP formation in vitro was stimulated by non-selective and beta 2-selective agonists, but not by the predominant beta 1-agonist prenalterol. Isoprenaline- stimulated cAMP formation was blocked by the non- selective beta-adrenoceptor antagonists propranolol, timolol, and alprenolol, while the beta 1-selective antagonists atenolol and metoprolol had no influence on an isoprenaline-induced cAMP formation. Receptor binding studies using (3H)-dihydroalprenolol revealed an IC50 value for propranolol of 85 nM, while metoprolol only displaced the bound (3H) dihydroalprenolol at far higher concentrations (IC50, 20 microM). We conclude that the human platelet beta-adrenoceptors are mainly of the beta 2- subtype and that beta-adrenoceptor antagonists, especially the non-selective antagonists interfere with platelet function assessed as platelet cAMP formation. PMID- 3003963 TI - Diagnosis of submucosal tumors by injecting a water soluble contrast medium: diagnosis of extra-gastric tumors and gastric varices. AB - Extra-gastric compression caused tumescent lesions that are difficult to differentiate from gastric submucosal tumors, and gastric varices similar in appearance to circumscribed tumors were sometimes experienced clinically. Up to now, the differential diagnosis of these lesions has been done by the palpation through x-ray examination or the tactile test under endoscopic examination. Even by the recent use of the CT scan, the differential diagnosis still remains unsatisfactory except in a few specific cases. Under these present circumstances, submucosography is recommended for routine screening test for outpatients. Our method is simple, safe and time-saving. Recently, it has become easy to diagnose hemangiomas as well as varices in the stomach by application of submucosography. Accordingly, in the cases of vascular tumors or tumescent lesions caused by extra gastric compression, the risk of incidental major bleeding or perforation can be prevented by using the submucosography. PMID- 3003964 TI - Acute and subchronic toxicity of a nonsulfhydryl angiotensin-converting enzyme inhibitor. AB - Acute and 1-month toxicity studies with SCH 31846, a nonsulfhydryl anti hypertensive agent which acts by inhibiting angiotensin-converting enzyme, were initiated to evaluate its toxicity. The oral LD50s in mice and rats were approximately 1.8 and 2.5 g/kg, respectively, while the iv LD50 was approximately 450 mg/kg in mice and 150 mg/kg in rats. Signs of acute toxicity in rats and mice included salivation, hypoactivity, ataxia, prostration, and convulsions. In a 1 month dog study at oral doses of 25, 75, or 150 mg/kg, there was a dose-related increase in emesis between 1 and 2 hr after dosing. Absorption studies showed peak blood concentrations occurring in dogs between 0.3 and 1 hr after dosing. No other noteworthy antemortem changes were observed. In a 1-month rat study at oral doses of 30, 180, or 600 mg/kg, the hematocrit and hemoglobin values of the 600 mg/kg-dosed female rats were slightly but significantly (p less than 0.05) decreased and the blood urea nitrogen was slightly but significantly (p less than 0.05) increased in all SCH 31846-dosed male rats and the 600 mg/kg-dosed female rats. Absorption studies in male rats at doses of 30, 180, and 600 mg/kg indicate that SCH 31846 is well absorbed in rats. The 150 mg/kg-dosed dogs and the 180- and 600 mg/kg-dosed rats had a slight increase in the number of renin-containing granules in the renal juxtaglomerular cells. No other compound-related microscopic changes were observed. These data are similar to data reported for Captopril and suggest that in the dog and rat the toxicity of ACE inhibitors is not dependent upon the presence or absence of a sulfhydryl group. PMID- 3003965 TI - Alteration of in vivo and in vitro effects of heroin by esterase inhibition. AB - Selective inhibition of peripheral esterases by tri-ortho-tolyl phosphate in the mouse resulted in an increase in the analgetic activity of heroin, without affecting the activity of morphine. In vitro inhibition of esterases by paraoxon reduced the affinity of heroin for the opiate receptor, while that of morphine was unaffected. These results suggest that both central and peripheral esterases are involved in the metabolism of heroin and that interference with critical esterases can alter its pharmacologic and toxicologic effects. PMID- 3003966 TI - The effect of intragastric administration of delta 9-tetrahydrocannabinol on the growth and development of fetal mice of the A/J strain. AB - Pregnant A/J mice were intubated with vehicle (sesame oil:Tween 80:water) or 60, 120, or 240 mg/kg of delta 9-tetrahydrocannabinol on Days 11 and 12, 12 and 13, or 13 and 14 (vehicle and 240 mg doses only) of gestation. Mice were killed on Day 20 of gestation, and examined for number of corpora lutea and live and resorbed fetuses. Fetuses were weighed and examined for gross external and internal malformations. Each treatment group consisted of a minimum of 10 litters with about 10 pups per litter. In a few groups the effects of feed deprivation on Day 12 or of glucocorticoid administration on Days 12 and 13 (positive control) were assessed. Intubation with vehicle or delta 9-tetrahydrocannabinol, or feed deprivation did not affect number of live fetuses, incidence of resorption, fetal weights, or gross malformations other than cleft palate. Intubation of delta 9 tetrahydrocannabinol on gestational Days 12 and 13 or 13 and 14 increased the mean frequency of cleft palate formation. The increase was 2- to 2.5-fold at the 240-mg dose, being significant (p = 0.05) in the Days 12 and 13 group. Cortisone acetate and corticosterone injection induced both resorption and cleft palate formation. Other developmental or reproductive parameters were not influenced by delta 9-tetrahydrocannabinol treatment. We conclude that delta 9 tetrahydrocannabinol administered by gavage during Days 12 and 13 of gestation retards normal palatal development. PMID- 3003967 TI - Induction of renal and hepatic mixed function oxidases in the hamster and guinea pig. AB - A marked species difference exists in the induction of renal and hepatic mixed function oxidase (MFO) activity between rats and rabbits. However, little is known about MFO induction in these organs from other laboratory animals. Male Golden Syrian hamsters and male Hartley guinea pigs were administered phenobarbital (PB) or beta-napthoflavone (BNF) at 70 and 40 mg/kg, respectively, as daily i.p. injections for 4 days. Polybrominated biphenyl (PBB) (Firemaster BP 6) was given as a single i.p. injection (50 mg/kg). Hamster hepatic microsomal ethoxyresorufin-O-deethylase (EROD) and benzphetamine-N-demethylase (BPND) were selectively induced by BNF and PB, respectively. PBB administration induced both hamster hepatic EROD and BPND. In contrast, hepatic microsomal MFO activity from the guinea pig was inducible by PB, PBB and BNF. Renal microsomal MFO activity in both species was inducible by BNF and PBB as arylhydrocarbon hydroxylase and EROD were induced approximately 10-fold. On the other hand, hamster BPND was induced by PB whereas guinea pig MFO activity was unaffected. Total renal cytochrome P 450 content was not affected by any of these inducers in either species. These data demonstrate selective patterns of induction in both hamster and guinea pig liver and kidney suggesting the involvement of multiple forms of cytochrome P 450. PMID- 3003968 TI - Effect of mixtures of dietary fibres on the enzyme activity of the rat caecal microflora. AB - The enzyme activity of the caecal microflora from weanling rats was determined after feeding 1 of 3 basal diets (purified fibre-free; purified plus cellulose; and stock), with or without additional dietary fibre (pectin, i-carrageenan or carboxymethylcellulose 5% w/w). The wet weight of caecal contents and total bacterial numbers were similar for the purified fibre-free and purified plus cellulose diets, yet were significantly higher in animals fed the stock diet. Pectin supplementation of the basal diets had no effect of caecal bacterial numbers, but significantly increased total nitrate reductase activity per caecum except when added to stock diet. Carrageenan decreased caecal bacterial numbers and most enzyme activities with both purified diets, and to a lesser extent with the stock diet. Carboxymethylcellulose increased bacterial numbers and enzyme activities, particularly beta-glucosidase and nitrate reductase when added to the purified diet but not when added to either the purified diet plus cellulose or the stock diet. The results demonstrate that the effects of dietary fibre components on the rat caecal microflora are dependent upon the initial fibre content of the diet base. PMID- 3003969 TI - Venom properties of the rattlesnakes (Crotalus) inhabiting the Baja California region of Mexico. AB - Seven species of rattlesnakes (genus Crotalus) from Baja, Mexico, were investigated for venom yield, protein content, lethal toxicity, 'Mojave toxin' content and hemorrhagic, esterase (BAEE), phosphodiesterase and protease activities. Venom yield was lowest in C. catalinensis and C. enyo enyo and highest in C. ruber ruber. All venoms exhibited esterase and phosphodiesterase activities, except that three (of 14) specimens of C. e. enyo lacked esterase activity. Protease activity was extremely low or absent in adult C. e. enyo, adult C. mitchellii mitchellii, adult C. viridis caliginis and juvenile C. r. ruber venoms. The venom of C. m. mitchellii was the only venom lacking hemorrhagic activity. This venom also had the highest lethal toxicity (i.p. LD50 0.13 - 0.24 mg/kg) in mice, and was the only venom that exhibited a toxin antigenically related to 'Mojave toxin'. PMID- 3003971 TI - Mestranol: the assignment of its 1H and 13C NMR spectra by means of two dimensional NMR spectroscopy, and its photochemical decomposition. AB - The 1H- and 13C-nmr spectra of mestranol were assigned with the help of a 2 D-J resolved, a 2D spin echo J-correlated (SECSY) and a 2D 1H-13C hetero-shift correlation experiment. The analysis of the spectra facilitated the identification of some of the photodecomposition products of mestranol. It was shown that, upon irradiation with UV-B light in water-ethanol (1:1, v/v), products are formed by oxidation of rings B and C of the steroid. PMID- 3003970 TI - An enzyme-linked immunosorbent assay for fetal steroid binding protein of human serum. AB - The binding characteristics and quantitation of the recently reported fetal steroid binding protein (FSBP) cannot be determined on unpurified samples; an immunoassay was therefore desirable. The protein was purified to homogeneity in order to raise a highly specific polyclonal antibody. An enzyme-linked immunosorbent assay applicable to unpurified samples was developed. Intra- and inter-assay coefficients of variation are 8.0% and 9.2% respectively; there is a sensitivity of 30 fmol FSBP per well, and there is no cross-reactivity with other binding proteins. Results obtained with the assay correlate with the more complex ligand binding assay (r = 0.85; p less than 0.02). Measurement of sera showed that FSBP levels are higher in women than in men (51.2 +/- 10.62 nM; 41.2 +/- 13.65 respectively; p less than 0.05) and are elevated in cirrhotic women (66.4 +/- 18.67; p less than 0.05) and in males with hepatocellular carcinoma (62.2 +/- 13.05; p less than 0.002). Use of the enzyme-linked immunosorbent assay confirmed the identity of FSBP separate from sex hormone binding globulin. PMID- 3003972 TI - [Vitamin D deficiency and hip fracture]. AB - Vitamin D deficiency is common in the elderly, especially in patients with hip fracture. Elderly people infrequently stay outside in the sunshine, and nutrition is deficient in vitamin D. In addition, the hydroxylation of vitamin D into active metabolites decreases with age. Vitamin D deficiency ultimately leads to osteomalacia, but in an earlier stage it causes secondary hyperparathyroidism, which is accompanied by increased bone turnover and cortical bone loss. Along these pathways vitamin D deficiency may contribute to the pathogenesis of hip fractures. In a survey in Amsterdam vitamin D deficiency was observed in more than 60% of the patients with hip fracture. Transilial bone biopsy showed signs of high turnover and cortical bone loss in more than 20% of patients. The elderly which are institutionalized carry an increased risk. Prevention or vitamin D deficiency is possible by adequate exposure to ultraviolet light. Primarily, the elderly should be encouraged to go out into the sunshine regularly. Advice on nutrition may be given additionally. When sunshine exposure is negligible, as in many disabled and institutionalized elderly, a daily supplement of vitamin D3 400 IU should be given. Preventive measures have to be evaluated prospectively. Vitamin D deficiency is not the most important risk factors for hip fractures, but the easiest to correct. PMID- 3003974 TI - A Wj-negative patient with anti-Wj. PMID- 3003973 TI - HLA antibodies as a cause of false-positive reactions in screening enzyme immunoassays for antibodies to human T-lymphotropic virus type III. AB - 15,680 volunteer blood donors were screened by enzyme-linked immunosorbent assay (EIA) for antibodies to human T-lymphotropic virus Type III (HTLV-III). On at least two of three determinations, 0.37 percent were found to be reactive. When the first 38 of these samples were sent for repeat EIA and Western blot, the EIA was positive in 14. In only three of these 14 samples was the EIA result confirmed by Western blot. Eight of these eleven false-positive samples were from women who were found to have HLA antibodies. In seven of these women, the HLA antibodies were directed to Class II antigens expressed on the cell used to grow HTLV-III in the preparation of screening EIA kits. We conclude that HLA antibodies are an important cause of false-positive reactions in the screening test for HTLV-III antibody. PMID- 3003975 TI - Protein kinase activation and the immunosuppressant cyclosporine. AB - Cyclosporine (CsA), a potent immunosuppressant for the prevention of transplant rejection, modulates T lymphocyte activation by blocking antigen stimulation and the production of interleukin-2. The mode of action by which CsA generates this immunosuppressive effect is unknown. We have studied two early intracellular enzymes associated with mitogen activation. They include calcium/phospholipid dependent protein kinase (C-kinase) and cAMP-dependent protein kinase (cAMPd PK). Changes in protein kinase activation were correlated with the immunosuppression of polyamine and DNA synthesis measured by ornithine decarboxylase (ODC) induction and 3H-thymidine incorporation, respectively. These studies utilized murine T cell tumor lines sensitive to the effects of CsA. Similar to the mitogen activation of human peripheral blood lymphocytes, CsA was capable of inhibiting the induction of ODC and 3H-thymidine uptake of T cell tumor lines cultured with either fresh serum or mitogen. In contrast, C-kinase and cAMPd PK activation stimulated by the addition of fresh serum was not affected by CsA. Further, CsA did not inhibit the direct activation of C-kinase with phorbol esters or the activation of cAMPd PK with exogenous cAMP. We conclude that CsA does not affect the activation of either C-kinase or cAMPd PK in T cell tumor lines when activated by either fresh serum or when stimulated with chemical agents. The suppression of ODC induction and 3H-thymidine incorporation associated with CsA treatment cannot be accounted for with changes in C-kinase and cAMPd PK activation. PMID- 3003976 TI - Difference in sensitivity to cyclosporine in vitro of human alloreactive lines and clones. AB - The effectiveness of cyclosporine (CsA) as immunosuppressive agent in human kidney graft rejection is well established. However, in spite of efforts to maintain optimal plasma levels, a fraction of transplanted patients undergo rejection episodes and/or irreversible chronic rejection. This suggests that immunosuppression by CsA cannot control the alloreactive response if there is a high degree of histoincompatibility for HLA or non-HLA antigens, or it has little effect on the "high responder" patient. Both possibilities are difficult to test in the human system. A third hypothesis, the existence of individual CsA resistance, was tested by evaluating the in vitro inhibitory activity of CsA on alloreactive T cell lines from several individuals. A different degree of in vitro sensitivity to the drug was observed among alloreactive lines generated from different individuals and among clones obtained from the same bulk line. The variability at the individual level and at the clonal level may account for the onset of CsA-resistant rejection assuming that in vivo a positive selection in the presence of the drug occurs and allows for the resistant clones, if present, to dominate the sensitive ones. PMID- 3003977 TI - Severe immunosuppression in a renal transplant recipient with HTLV-III antibodies. PMID- 3003978 TI - Increased Leu-7-positive T lymphocytes during cytomegalovirus infection following allogeneic bone marrow transplantation for hematologic malignancies. PMID- 3003979 TI - Comparison of splenic and renal subcapsular islet autografting in dogs. PMID- 3003980 TI - [Effect of hypoxia on the structure of the presynaptic grid of the interneuronal contacts of the rat neocortex]. AB - Hypoxic effect (a 6 minute asphyxia) on the presynaptic grid structure and on the amount of synapses in the neocortex has been studied in white rats using the method of selective staining of synapses with phosphoric tungsten acid. Neurofilamentous formations of the presynaptic grid appeared to be most labile structures. Dense projections of the presynaptic grid are most sensitive to hypoxia: their height, distinctness of the frame, and the intensity degree of phosphoric tungsten acid staining decrease. The content of intermediate form contacts with low indistict dense projections increases with the increase in the number of light type changed synapses. The principle organization of the presynaptic grid does not change in the posthypoxic period: hexagonal division of dense projections and places of vesicular attachment are kept. The hypertrophy of the presynaptic grid is also kept during that process. PMID- 3003981 TI - [Ultracytochemical detection of nucleoside phosphatase activity in human peripheral blood cells]. AB - The authors elaborated and described the optimum conditions for fixation, incubation and preparation of human blood cell samples in minimum quantities for ultrastructural and ultracytochemical investigations of 5'-nucleotidase and ATPase activities. The best preservation of the blood cell ultrastructure was obtained after fixation with buffered 1% glutaraldehyde solution followed by postfixation in buffered 1% OsO4 solution. The best ultracytochemical demonstration of 5'-nucleotidase and ATPase activities was achieved after fixation in buffered 2% formaldehyde prior to cytochemical incubation. DMSO added to either fixation or incubation media was shown to damage the plasmalemma and glycocalyx structure in cell suspensions. ATPase in 5'-nucleotidase activities were revealed in plasmalemma, cytoplasmic reticulum, Golgi complex, mitochondria and in the nuclei, in particular, in the perinuclear space, nucleolus and chromatin. With respect to the localization and activity of nucleosidephosphatases, lymphocytes proved to be most heterogenic, with the enzyme activity level directly depending on the rate of ultrastructural differentiation in lymphocytes. PMID- 3003982 TI - [Heavy water inhibition of alkali cation transport across the muscle membrane. II. A comparison of the action of D20 and ouabain on the sodium efflux and rubidium influx in magnesium media]. AB - The action of heavy water and ouabain on sodium effluxes and rubidium influxes has been measured and compared in frog muscles (m. sartorius, R. temporaria). Approximately half of muscle sodium was substituted by lithium by preliminary incubation in mixed sodium-lithium media. The ratio of the ouabain-sensitive parts of rubidium influx and sodium efflux is 7.3:10.5, and that of D2O-sensitive parts of corresponding parameters is 7.5:11.3. A conclusion is made that D2O effect on the Na, K-ATPase system of muscles under investigation resembles ouabain-effect on sodium effluxes as well as on rubidium influxes. PMID- 3003983 TI - [Transformation of mice by a prokaryotic gene for dihydrofolate reductase]. AB - In mice obtained after microinjection into the male pronucleus of fertilized eggs of the plasmid, containing the bacterial gene of dihydrofolate reductase (DHFR), under the control of the early promotor of the simian virus 40 (SV40), an integration of the foreign DNA into the mouse genome is found. About 30% of the treated animals contain the integrated plasmid DNA sequences, i.e. are transgenic. In 2 of 7 mice, containing the introduced plasmid in their genome, the methotrexate-resistant DHFR activity is found in the kidney and spleen, which may be due to the expression of gene DHFR. The plasmid DNA sequences and the ability to synthesise the methotrexate-resistant enzyme DHFR are transmitted to the next generation of mice. PMID- 3003984 TI - [Effect of ouabain on the Na+, K+-ATPase function and multiplication of transformed cells in culture]. AB - A study was made of the ouabain effect (10(-3] on cell proliferation and the dependence of ATP hydrolysis on Na/K-concentration in homogenates of mouse hepatoma (XXIIa) and of L-cells, both sensitive and resistant to etidium bromide. Na+, K+-ATPase activity was found in homogenates of cells from sparse cultures in the presence of ouabain, the activity being stimulated by the Na/K-ratio pecular for the maximum enzymatic activity in cells from the dense cultures. The effect of ouabain on the cell proliferation is similar to the effect of transition of sparse cultures to dense ones. PMID- 3003985 TI - Counter immunoelectroosmophoresis in the diagnosis of infectious bursal disease of poultry. AB - Infectious bursal disease virus antigen was detected in the bursa of Fabricius of infected birds by the use of the counter immunoelectroosmophoresis technique. Eight suspected outbreaks were confirmed in this way and 82 out of 89 bursal samples submitted for laboratory confirmation were positive. The test was also suitable for the detection of antibody to infectious bursal disease virus in sera of infected birds. Precipitin lines were visible within 30 min as compared with 18 to 24 h with the Ouchterlony agar gel precipitation test. Counter immunoelectroosmophoresis is recommended for rapid confirmation of infectious bursal disease of poultry. PMID- 3003986 TI - [Nicotinamide coenzyme levels and activity of enzymes of their biosynthesis and degradation in animal tissues after exposure to extreme conditions and pathogenic factors]. AB - Studies of enzymic systems of biosynthesis and decay of nicotinamide coenzymes under the effect of extremal and pathogenic factors have shown that the activation of enzymes of the nucleosidase cleavage of coenzymes is the most essential link in the mechanism of concentration disturbance of metabolically active vitamin PP forms under cooling and development of inflammatory processes in the myocardium and liver. Inhibition of the enzymic activity in the biosynthesis of coenzymes under allergic myocarditis and hepatitis may play a definite role in the mechanism of disturbance of the coenzymic function of vitamin PP. PMID- 3003987 TI - [Na+,K+-ATPase activity of the subcellular fractions of the rat brain in aging]. AB - The Na+, K+-ATPase activity in the homogenate and in subcellular fractions of different parts of the brain of adult and old rats was studied in comparison. The content of cholesterol in the above fractions was also determined. In old age the Na+, K+-ATPase activity in the homogenate and microsomal fraction of the cerebral hemispheres' cortex decreases, while the Mg2+-ATPase activity in the cortex microsomal fraction increases. The age-related Na+, K+- and Mg2+-ATPase activity in the myelin of the stem in the synaptic plasma membranes of hemispheres and the brain stem remains unchanged whereas in the myelin fraction of hemispheres it grows. The content of cholesterol in the brain of old rats as compared with adult ones increases in the microsomal fraction and remains unchanged in synaptic membranes. The possible role of age-related modification of lipid component of plasma membranes in the above changes of Na+, K+-ATPase activity is discussed. PMID- 3003988 TI - [The role of membrane phosphoinositides in mediating hormonal effects]. AB - The present notions on the pathways of phosphoinositide metabolism in a cell and on the functional significance of the given process are reported. The enzymic systems providing catabolism of phosphoinositides are analyzed. The data indicating the dose interrelation between the stimulation of the phosphatidyl inosite and polyphosphoinositides metabolism by hormones and the Ca2+ transport into a cell are generalized. Universality, biochemical mechanisms and functional significance of the "phosphatidyl inosite" response are discussed. The phosphoinositide metabolism is considered from the standpoint of its significance for other cell processes: synthesis of eicosanoids, provision of Ca2+-dependent processes in the synapse, cell proliferation, activation and the "anchoring" of enzymes on membranes. PMID- 3003989 TI - Electron microscopy what izzits. PMID- 3003990 TI - Application of electron microscopy to diagnosis in gynecologic neoplasms and tumorlike conditions. PMID- 3003992 TI - Malignant myoepithelioma of parotid salivary gland. PMID- 3003991 TI - Tubular Krukenberg tumor of the ovary: an ultrastructural study. PMID- 3003993 TI - Primitive neuroectodermal tumor (peripheral neuroblastoma). PMID- 3003994 TI - Ultrastructural and immunohistochemical features of common lung tumors: an overview. PMID- 3003995 TI - Small cell undifferentiated carcinomas of the lung with nonneuroendocrine features. PMID- 3003996 TI - [The Na,K pump, the cellular receptor for digitalis glycosides]. PMID- 3003997 TI - Three-dimensional reconstruction of the shape of human wart virus using spatial correlations. AB - Correlation averaging was used to enhance the three-dimensional spherical harmonic expansion of human wart virus particles from randomly-oriented negatively-stained electron microscopic images in holey grids. The reconstruction reveals the coat protein arrangement and variations in stain distribution inside the virus core. Selection rules imposed by the spherical symmetry on the harmonic expansion component are obtained indeed for angular momenta, l, lower than 11, but cannot be shown for l greater than or equal to 11, due to non-spherical distortions of about 1/11th of the radius of the virus. This prevents us from resolving fine details of packing of the protomers on the surface lattice. Distribution of stain inside the virus core, and its spherical symmetry, is reconstructed and may be related to nucleic acids and core protein structure. PMID- 3003998 TI - [Diagnosis of sphenoiditis by contrast roentgenolgic examination]. PMID- 3003999 TI - [Giant laryngopharyngeal angiofibroma and an organ-sparing method for its removal]. PMID- 3004001 TI - Neoplasia and hyperplasia of pancreatic endocrine tissue in the rat: an immunocytochemical study. AB - Spontaneously occurring neoplasms and non-neoplastic proliferative changes of the pancreatic cells in aging Sprague-Dawley and Long-Evans rats were examined for the presence and distribution of pancreatic hormones using immunocytochemical techniques. Islet cell tumors were indistinguishable in the two rat strains. They were composed principally of insulin-containing beta cells, but had additional and variable small proportions of cells that stained for somatostatin, glucagon, or rarely, pancreatic polypeptide. The heterogeneity in these spontaneous islet cell neoplasms was similar to that reported in humans as well as those induced in rats by streptozotocin. Hyperplasia of the islet cells also mainly affected the beta cells, but the overall pattern of immunocytochemical staining usually remained similar to that of normal islets, a point of distinction from islet cell neoplasms. In addition, rats with exocrine atrophy and fibrosis were found to have considerable disruption and focal proliferation of the islets. PMID- 3004000 TI - [The kallikrein-kinin system in acute peritonitis and methods of correcting its disorders]. AB - In experimental fecal peritonitis in 140 albino rats the kallikrein-kinin system was shown to undergo the following changes: its inhibition during the first day was followed by an activation on the second day and inanition on the third day. Under conditions of hyperbaric oxygenation the normalizing effect on the kinin system was observed during two days. The application of salicylates against the background of HBO was shown to enhance the normalizing effect which lasted for three days. PMID- 3004002 TI - Feline leukemia virus-induced thrombocytopenia and macrothrombocytosis in cats. AB - The Kawakami-Theilen strain of feline leukemia virus (FeLV-KT), an exogenous anemia-inducing retrovirus, induced significant macrothrombocytosis and thrombocytopenia during acute infections of cats. The geometric mean platelet volumes of both freshly isolated fixed platelets and ethylenediamine tetraacetic acid-sphered platelets from infected cats were increased, but the ratio of fresh fixed to ethylenediamine tetraacetic acid-sphered platelet volumes, an estimate of ethylenediamine tetraacetic acid-induced isovolumetric shape change, was normal. Total plasma membrane increased as the platelet volumes increased in FeLV KT-infected cats, but estimated surface connected canalicular system surface area did not. Platelet concentrations were decreased 2 to 6 weeks after inoculation, but marked macrothrombocytosis resulted in a trend toward increased platelet mass on weeks 5 and 6 after inoculation. Thus, FeLV-KT-induced macrothrombocytosis may serve as a model of impaired platelet volume regulation. The platelet volume and platelet membrane surface area abnormalities found suggest that this model would allow studies of the megakaryocyte/platelet axis, particularly in the area of membrane formation and territorial demarcation. PMID- 3004003 TI - Central nervous system lesions in suckling mice and rats inoculated intranasally with sialodacryoadenitis virus. AB - Suckling CD-1 and CFW mice and WI Wistar rats were inoculated intranasally with sialodacryoadenitis virus. In animals inoculated during the first week of life, there was an acute necrotizing encephalitis with malacia and minimal inflammatory cell response. In mice and rats which survived for up to 12 days post inoculation, loss of brain substance, gitter cells and occasionally mineralized debris were seen. Viral antigen was readily demonstrated in neurons and nasal epithelium in trypsin-treated, paraffin-embedded sections, and corresponded with the presence of lesions. There was no compelling evidence that sialodacryoadenitis virus invaded the central nervous system directly via the cribriform plate, and hematogenous spread was considered to be a likely route of spread to the brain. An age-related resistance to the encephalitic form was evident beginning at 10 days of age in both species. Thus it is conceivable that encephalitis could occur in young suckling rats exposed during naturally occurring epizootics of sialodacryoadenitis. PMID- 3004004 TI - Multihormonal islet cell carcinoma in a greater bushbaby (Galago crassicaudatus argentatus). PMID- 3004005 TI - Cultivation of bovine herpesvirus 2 by incubation at reduced temperature. PMID- 3004006 TI - Parvovirus vaccination. PMID- 3004007 TI - Transmission of foot-and-mouth disease from African buffalo virus carriers to bovines. PMID- 3004008 TI - Parvovirus vaccination. PMID- 3004009 TI - Presence of bluetongue virus vectors on Rhodes. PMID- 3004010 TI - An enzyme-linked immunosorbent assay method for the simultaneous measurement of antibody titer to multiple viral, bacterial or protein antigens. AB - A model enzyme-linked immunosorbent assay (ELISA) system was developed which permits the user to evaluate replicate single sera dilutions of 46 test and control serum samples for the presence of specific antibody against up to eight different antigens in one assay. The system makes use of a transplanting device for the simultaneous transfer of aliquots of independently diluted replicate test samples along with conjugate and substrate from a serum reservoir plate or from a reagent reservoir onto different antigen coated target plates. Kinetically read, raw absorbance data from tests are transmitted to a microcomputer, where absorbance values from all tests are quickly reduced to predicted titer levels by computer analysis. All titer computations involve the use of a single prescribed standard curve for predicting antibody titer against the different antigens. Between assay repeatability of this procedure is high and its potential for replacing a number of different conventional assays for epidemiological studies has been evaluated. PMID- 3004011 TI - Propagation of infectious bovine rhinotracheitis (bovine herpes-1) virus in murine primary cell cultures. AB - Virus synthesis in BALB/C mouse lung and kidney primary cultures infected with infectious bovine rhinotracheitis (IBR) virus started between 6 and 8 hours after virus inoculation and reached a maximum titer of 5.5 log10 plaque forming units (PFU) at 48 hours postinfection (PI). Cytopathic effect (CPE) in cell cultures occurred at 8-10 hours and over 90% of the cells had CPE by 48 to 72 hours PI. The bulk of the newly replicated virus (60-80%) was cell-associated as determined by plaque assay of extracellular and intracellular virus. Pulse-chase experiments demonstrated incorporation of radioactive precursors into viral DNA and protein macromolecules. Viral DNA synthesis was initiated between 2 and 4 hours PI and was maximum at 4 to 6 hours. Viral proteins were detected at 4 hours and peaked between 6 and 8 hours PI. Enzyme-linked immunosorbant assay (ELISA) confirmed synthesis of specific viral proteins, which gradually increased during the virus growth cycle. Abstract in French is given at the end of the article. PMID- 3004012 TI - [Experiments to purify and concentrate the foot-and-mouth disease virus]. AB - Experiments were carried out for the purification and concentration of the foot and-mouth disease virus. For the purpose the virus suspensions were treated with chloroform and 8 per cent polyethylene glycol. The precipitated virus was eluated with phosphate buffer (of pH 7.6) up to 1:100 of the initial volume. The concentrated virus was subjected to gradient ultracentrifugation on gradient of CsCl. The fractions obtained were investigated through radial immunodiffusion reaction with early sera, containing IgM antibodies. The fractions that formed circles of precipitation were studied under electron microscope at negative contrasting. It was found that the fractions taken from between the CsCl layer of 1.42 g/cm3 density and the underlying CsCl layer of 1.50 g/cm3 density, which produced precipitation circles in the radial immunodiffusion test, contained purified virions of intact structure in concentration of 10(9). PMID- 3004013 TI - Monoclonal antibodies against CEA-related components discriminate between pancreatic duct type carcinomas and nonneoplastic duct lesions as well as nonduct type neoplasias. AB - The expression of CEA and related antigens in formalin-fixed paraffin-embedded tissues of normal pancreas and different pancreatic neoplasms was studied immunocytochemically using three monoclonal antibodies (MAbs) recognizing different epitopes on CEA and related antigens. Additionally, a number of extrapancreatic malignancies were tested. The epitope recognized by MAb 250 (present on CEA and NCA 95) was expressed in all but one pancreatic ductal adenocarcinoma and ampullary carcinoma (42/43). The MAb 431 defined epitope (present only on CEA) was less frequently found (27/43). MAb 374, defining an epitope on CEA, NCA 95 and NCA 55 proved to be nearly as sensitive as MAb 250, but also reacted with normal duct epithelium. In contrast, MAb 250 and MAb 431 discriminated clearly between reactive duct lesions and malignant duct changes. Moreover, these MAbs differentiated between pancreatic duct carcinomas and nonduct type carcinomas as well as benign pancreatic tumours. In duct type carcinomas, the strongest staining was observed in well differentiated tumours. No discrimination was possible between pancreatic carcinomas and other adenocarcinomas of the gastrointestinal tract nor between most of the lung carcinomas and some other malignancies, specified below. MAb 250 and MAb 431 failed to react with hepatocellular carcinomas, renal cell carcinomas, carcinoids, sarcomas and melanomas. The findings suggest that paraffin-embedded tissues of pancreatic duct type carcinomas, in contrast to nonduct type tumours and normal ducts, are distinguished by the presence of a CEA and NCA 95 related epitope. PMID- 3004015 TI - Epithelioid hemangioendothelioma. Report of a case with immuno lectinhistochemical and ultrastructural demonstration of its vascular nature. AB - The epithelioid hemangioendothelioma (EHE) is a rare vascular tumour of borderline malignancy, first described as a separate entity in 1982 by Weiss and Enzinger. The abundant cytoplasm of endothelial cells mimiking epithelioid appearances, prominent cytoplasmic vacuolization and barely perceptible lumina even in reticulin stains may result in EHE being mistaken for a signet ring cell carcinoma. In our case, difficulties in differential diagnosis were enhanced by the location of the tumour within an inguinal lymph node. The usefulness of FVIIIR: Ag- and UEA I- histochemistry in ascertaining the endothelial nature of this tumour is demonstrated, in correlation with electron microscopic data. The different reaction sites of these markers are striking and typical: FVIIIR: Ag displays a granular or diffuse cytoplasmic reaction, whereas UEA I provides a linear staining of vacuoles and luminal surfaces. PMID- 3004016 TI - Isolation of poly(A)-rich mRNA from the chorioallantoic membrane of Sendai virus infected chick embryos. AB - Total RNA was extracted from the chorioallantoic membrane (CAM) of Sendai virus infected chick embryos by the original Kecskemethy-Schafer method and by a modification of this technique, including phenol treatment, in order to ensure complete removal of proteins. The purity of the total RNA extracted by the modified technique was ascertained by spectral analysis. Chromatography on oligo(dT)-cellulose of the total RNA led to the isolation of poly(A)-rich mRNA- the material retained by oligo(dT)-cellulose and eluted with the buffer of the lowest ionic strength. The ability of the poly(A)-rich mRNA thus obtained to stimulate incorporation of 14C-leucine into protein was demonstrated in a cell free protein-synthesizing assay system. PMID- 3004017 TI - Effect of the oral administration of NIVGRIP inactivated influenza vaccine on virus carriage in the nasopharynx of children aged 0-5 years. AB - The NIVGRIP inactivated influenza vaccine was administered by oral route to 11 children of a semi-closed pre-school community, found to carry viruses in their nasopharynx. Periodic virological investigations demonstrated that after vaccination virus carriage disappeared completely in 7 children; in the remaining 4 cases there was a considerable decrease (from 14 to 6) in the number of virus strains isolated. The virus isolates obtained after vaccination were represented exclusively by enteroviruses (Coxsackie B1 and B3, poliovirus type 1), in contrast with the wider range of viral agents (adeno-, parainfluenza, herpes, Coxsackie, and polioviruses) detected prior to vaccine administration. PMID- 3004014 TI - Immunohistochemical study of granular cell tumours. Demonstration of neurone specific enolase, S 100 protein, laminin and alpha-1-antichymotrypsin. AB - Nine granular cell tumours were investigated with poly- or monoclonal antisera to neurone specific enolase (NSE), glial enolase (GE), S 100 protein, alpha-1 antichymotrypsin, lysozyme, laminin, neurofilament (NF), glial fibrillary acidic protein (GFAP), brain creatine kinase (CK), different cytokeratins (Keratin Dako, PKK1), tissue polypeptide antigen (TPA), carcinoembryonic antigen (CEA), desmin, myoglobin and leukocyte common antigen (LCA), using immunoperoxidase-methods on formalin fixed paraffin embedded sections. While five tumours from adults show specific cytoplasmic staining for NSE and S 100, three congenital tumours, two from the gingiva and one from palatine, show only a weak reaction for NSE, reflecting a possible origin from mature and immature Schwann cells, respectively. However, one subcutaneous tumour from near the clavicule of a ten year old girl differs from the other eight tumours by its specific cytoplasmic staining for alpha-1-antichymotrypsin only, supporting the view that there are granular cell tumours of histiocytic origin. In addition, the five adult NSE-S100 tumours show strong laminin-immunostaining around the single small or syncytial granular cells, whereas pericellular laminin is not detectable in the histiocytic nor in the three congenital tumours. None of the tumours shows any staining for lysozyme, epithelial, muscular, leukocyte, neurofilament or glial antigens. PMID- 3004018 TI - [Intracellular changes induced by the canine infectious laryngotracheitis virus (CAV-2)]. AB - Canine infectious laryngotracheitis virus, an adenovirus designated CAV-2, induces numerous changes in the nucleus and cytoplasm of canine kidney cells (MDCK). The following features could be observed on ultrathin sections examined in the electron microscope: a shift of the nucleolus towards the nuclear membrane and a compression of the latter by the virus particles; the release of virions into the cytoplasm following the tearing or evagination of the nuclear membrane; the attachment of virus particles onto the numerous microtubules of the infected cell. Microfilaments appear to be involved into the vectorial movement of virus particles towards the cytoplasmic membrane in the final phase of infection. PMID- 3004019 TI - Preliminary data on the encapsulation of biologically active materials in liposomes. AB - Multilamellar and unilamellar phospholipid liposomes were prepared and investigated as regards their properties and the capacity of encapsulating biologically active materials, such as CrO4-(2-)ions, basic dyes, proteins, DNA, as well as Sendai virus particles. The efficiency of encapsulation ranged from 10% for CrO4-(2-)ions to about 80% for toluidine blue; it was found to depend not only on the type of encapsulated material, but also on the method used for liposome preparation and on liposome composition. PMID- 3004020 TI - Detection by immunofluorescence of possible viral implications in "idiopathic" peripheral facial paralysis. AB - The presence of viral antigens was detected by the indirect immunofluorescence technique in exfoliated cells occurring in the pharyngeal exudate of 18 out of 29 patients with peripheral facial paralysis. The most frequently encountered antigens were: Coxsackie A and B virus (33.3%), adenovirus (16.7%), and the association Coxsackie B virus + adenovirus (16.7%). The possibility that some of the so-called "idiopathic" peripheral facial paralyses may have a viral etiology is discussed. PMID- 3004021 TI - Immunofluorescence study on the prevalence of some respiratory viruses in subjects vaccinated against influenza and in unvaccinated controls. AB - The circulation of respiratory viruses was investigated comparatively in a group of 24 subjects vaccinated intranasally with the NIVGRIP inactivated influenza vaccine and in 20 unvaccinated controls. The study relied on the detection by immunofluorescence reactions of viral antigens in exfoliated cells present in the nasopharyngeal secretions collected in January, February and March 1984. In January-February the prevalence of influenza, parainfluenza and adenovirus type 5 antigens was considerably lower in vaccinees than in controls. Simultaneous detection of more than two viral antigens in the same subject was more frequent in controls. The data demonstrate that the influenza vaccine administered not only conferred specific protection, but also stimulated the mechanisms of local nonspecific antiviral defence. PMID- 3004022 TI - Mapping of the varicella zoster virus deoxypyrimidine kinase gene and preliminary identification of its transcript. AB - The varicella-zoster virus (VZV) deoxypyrimidine kinase (dPK) gene was mapped by transfection of cloned viral DNA fragments into thymidine kinase-deficient mouse L (LTK-) cells and subsequent biochemical transformation of these cells to the LTK+ phenotype. Such transforming activity was limited to the BamHI-H and EcoRI-D fragments of the VZV genome, which overlap by 2.2 kb between map units 0.50 and 0.52. Biochemically transformed cells were shown to contain a high copy number of viral DNA sequences that had integrated into the cellular DNA. Extracts of these cells showed a higher level of dPK activity than did extracts of parental LTK- cells. With the use of Northern hybridization analysis of transformed and VZV infected cell RNAs, it was possible to tentatively assign a 1.8-kb transcript to the VZV dPK. PMID- 3004023 TI - A detailed kinetic analysis of the in vitro synthesis and processing of encephalomyocarditis virus products. AB - Translation of encephalomyocarditis virus RNA in rabbit reticulocyte lysates has been used to analyse the pathway of proteolytic processing of the primary translation products. A minimum of two distinct proteases is required to account for the results: one for the excision of the capsid precursor protein, A1, from the nascent polyprotein, and the other for all other cleavages including cleavage at the F/C junction. The excision of A1 is an extremely rapid reaction which occurs as soon as the cleavage site has been synthesised and is resistant to all the proteolytic inhibitors tested and to high temperature, characteristics which are more consistent with an intramolecular cleavage catalysed by a virus-coded protease than cleavage by an endogenous reticulocyte protease. Once excised, A1 remains stable until translation has reached the middle of the region of the genome coding for C, at which time a number of events occur in rapid succession: F is excised in its mature form probably via an intramolecular cleavage; a proteolytic activity capable of secondary processing of A1 to A, B, D1, alpha, gamma and epsilon appears; and a polypeptide of molecular weight about 32,000 appears. This protein (p32) originates from the N-terminal portion of C, and maps in the same position on the genome as p22, the protein previously identified as the virus-coded protease. Polypeptide p32 is derived from C by a single step cleavage generating E as the other product, a processing pathway at least as important, if not more important than the step-wise route via D as an intermediate. Since p32 first appeared at the same time as the start of secondary processing, whilst p22 was first detected much later, it is argued that at least the early stages of processing of the capsid precursor must have been carried out by p32 rather than p22. PMID- 3004024 TI - Regulation of herpes simplex virus 1 genes: alpha gene sequence requirements for transient induction of indicator genes regulated by beta or late (gamma 2) promoters. AB - This laboratory reported earlier that chimeric genes consisting of the structural sequences of the thymidine kinase (TK) gene fused to the promoter-regulatory domains of late (gamma 2) genes were regulated as bonafide gamma 2 genes when resident in the herpes simplex virus 1 genome but could not be differentiated from beta genes when introduced by transfection and stably integrated into the environment of the host genome (S. Silver and B. Roizman, Mol. Cell. Biol. 5, 518 528, 1985). We report here that beta-TK and the chimeric gamma 2-TK gene transfected into TK- baby hamster kidney (BHKtk-) were induced by alpha 4 and alpha 0 but not by the other alpha genes. Specifically: Both TK genes were induced by cotransfection with DNA fragments carrying an intact alpha 4 or an intact alpha 0 gene, but not by fragments carrying only the promoter-regulatory domain or the structural sequences of the alpha 4 gene or intact alpha 22, alpha 27, and alpha 47 genes. An alpha 4 gene carrying a 2700-bp deletion in its 3' coding sequence also induced both genes, although less efficiently. RNA homologous to the alpha 4 gene recovered from the cytoplasm of cells transfected with either the intact or truncated alpha 4 gene mapped to the bonafide site of transcription initiation of the alpha 4 gene. RNA homologous to the chimeric TK gene extracted from the cytoplasm of cells transfected with both gamma 2-TK and the alpha 4 gene was transcribed from the bonafide gamma 2 gene capping site fused to the TK gene. These results indicate that the alpha 4 gene and the alpha 0 gene are each capable of inducing the expression of both beta and gamma 2 genes resident in the environment of the cellular genome, that the active site responsible for induction is located near the N terminus of the alpha 4 protein, and reinforce the conclusion that gamma 2 genes resident in the environment of the host cell cannot be used to identify the authentic determinants of gamma 2 gene regulation by currently available tests. PMID- 3004025 TI - Expression in Escherichia coli of cDNA fragments encoding the gene for the host protective antigen of infectious bursal disease virus. AB - The larger segment of the IBDV genome codes for a 32-kDa host-protective antigen. Inserts from a cDNA library in pBR 322, containing overlapping cDNA fragments of varying sizes and covering the entire large segment of the IBDV genome, were subcloned into a mixture of expression vectors pUR 290, 291, and 292. Clones expressing the host-protective antigen, or parts of it, were identified by an immunoblot assay and the fusion proteins were further characterized by Western blot analysis using a monoclonal antibody specific for the 32-kDa polypeptide. Hybridization of inserts from expressing clones to the original cDNA library led to the identification of the region of the IBDV genome that codes for the 32-kDa host-protective antigen. Clone D1 which encodes approximately 50% and clone D6 which encodes the entire 32-kDa protein were selected for further studies. The fusion proteins from clones D1 and D6 were affinity purified and tested for their immunogenicity in chickens. Both fusion proteins induced the synthesis of antibodies in both primed and unprimed chickens that reacted specifically with denatured 32-kDa viral protein, but less well with intact virus. It was concluded that the response to the fusion proteins was to linear rather than conformational epitopes on the 32-kDa viral protein. PMID- 3004026 TI - Primary structural relationships may reflect similar DNA replication strategies. AB - The primary structures of several proteins of bacterial and viral origin involved in the initiation of DNA synthesis and its subsequent elongation were compared. It was found that the known sequences of DNA polymerases and the single-stranded DNA binding proteins from phage T7 and Escherichia coli aligned well. Furthermore, segmental homologies were found in the phage phi 29 and adenovirus polymerases as well as in their DNA binding proteins. These results suggest similar mechanisms of DNA synthesis for E. coli and T7 on the one hand and for phi 29 and adenovirus on the other. PMID- 3004027 TI - Detection and characterization of the protein encoded by the v-rel oncogene. AB - To identify the protein encoded by v-rel, the oncogene of reticuloendotheliosis virus (REV-T), antisera have been raised to three synthetic peptides derived from the translation of our previously published v-rel DNA sequence [R.M. Stephens, N.R. Rice, R.R. Hiebsch, H.R. Bose, Jr., and R.V. Gilden, Proc. Natl. Acad. Sci. USA 80, 6229-6233 (1983)]. Sera to all three peptides precipitate a 59,000 Da protein from REV-T-transformed chicken lymphoid cells. This protein is not detectable in uninfected chick embryo fibroblasts, and its observed size is in good agreement with the 56,000 Da predicted by the DNA sequence. We conclude that this protein is the v-rel product and designate it p59rel. To search for evidence of post-translational processing of this protein, cells were grown in the presence of glycosylation inhibitors. These resulted in no detectable difference in the size of p59rel. Nor was its size detectably altered during the course of a pulse-chase experiment. Growth of cells in the presence of [32P] orthophosphate, however, revealed that p59rel is a phosphoprotein. It is also closely associated with a protein kinase activity, for precipitation with one of the peptide antisera (but not the other two) resulted in strong kinase activity in the immune complex pellet. During this reaction, p59rel itself becomes phosphorylated. Kinase activity was retained in the immune complex following detergent and high salt washes, leaving open the possibility that p59rel is itself a kinase. PMID- 3004028 TI - MPLV: a retrovirus complex inducing an acute myeloproliferative leukemic disorder in adult mice. AB - A novel murine retrovirus complex was derived from the in vivo passage of a molecularly cloned Friend ecotropic helper virus. The virus isolate, myeloproliferative leukemia virus (MPLV), causes an acute (2-3 weeks) and generalized myeloproliferative disorder in adult mice. All strains of mice examined, including the C57BL/6J strain, developed the acute syndrome. This syndrome is characterized by a rapid hepatosplenomegaly, no thymus or lymph node involvement, granulocytosis, thrombocytosis, and erythroblastosis leading to polycythemia. The most prominent feature at the terminal phase of the disease is a granulocytic hyperplasia. The MPLV isolate replicates in vitro on NIH 3T3 fibroblasts but does not induce foci of transformed cells. Thus, MPLV exhibits unique biological properties that distinguish it either from the Friend virus complexes or from acutely transforming sarcomatogenic murine retrovirus which also induced a rapid splenomegaly. PMID- 3004030 TI - A sequence in HpaI-P fragment of herpes simplex virus-1 DNA determines intraperitoneal virulence in mice. AB - The virulence of herpes simplex virus-1 (HSV-1) strains by the intraperitoneal (ip) route of injection in mice depends on the presence of an intact sequence in the HpaI DNA fragment P within coordinates 0.762 to 0.787. Deletion of the HpaI-P region (e.g., strain HFEM) abrogates the ability of the virus to infect mice by the ip route without affecting pathogenicity by the intracerebral (ic) route. A recombinant virus (M1C1) derived from DNA of the HSV-1 HFEM strain and the MLUIDNA fragment (coordinates 0.761 to 0.796) spanning the HpaI-P sequence of the pathogenic strain F regained pathogenicity for mice by the ip route. PMID- 3004029 TI - Molecular cloning and characterization of human papillomavirus type 7 DNA. AB - Human papillomavirus type 7 (HPV-7) was first described in 1981 but so far could not be molecularly cloned. It has been found almost exclusively in hand warts of butchers. We have cloned the complete genome in pBR 322, established its physical map, demonstrated the colinear genome organization with HPV-18 and analyzed the degree of homology with other HPV types and bovine papillomavirus (BPV) types. In order to investigate whether HPV-7 might be a so far unidentified bovine virus, we screened 37 bovine tumor DNAs using Southern blot analysis for its presence, with exclusively negative results. From our data we conclude that the HPV-7 genome shows all characteristics of a papillomavirus genome and that its origin is most likely human. PMID- 3004031 TI - [Transformation of cholecalciferol (vitamin D3) into a steroid hormone. Effective metabolites of cholecalciferol]. PMID- 3004033 TI - [Effect of diet on transport ATPase activity in myocardial and liver membranes]. AB - Distinct impairments were found in membranes of rat myocardium and liver tissue in animals kept on food, which was deficient in retinol, tocopherol, ascorbic acid and essential amino acids lysine, methionine and threonine. Deficiency in these dietary components led to a decrease in content of phosphatidyl ethanolamine and phosphatidyl serine and in activity of total ATPase and Ca2+ ATPase in membranes of myocardial sarcoplasmic reticulum as well as to decrease Na+, K+-ATPase activity in liver plasmatic membrane. PMID- 3004032 TI - [Aminoacyl-tRNA synthetases and their high molecular weight complexes in autolysis of the swine heart]. AB - In pig myocardial extracts autolyzed within 15 min alanyl-, glycyl-, glutamyl-, leucyl- and seryl-tRNA synthetase activities were increased as compared with controls. The enzymatic activities were decreased after autolysis for 30 min. The 15 min autolysis was shown to decrease the molecular mass of the glycyl-tRNA synthetase complex. Within both 15 min and 30 min autolysis inorganic pyrophosphatase was markedly activated either in myocardium extracts or in high molecular complexes; this phenomenon may be responsible for activation of a number of aminoacyl-tRNA synthetases. PMID- 3004034 TI - [Phosphoprotein phosphatase of the myocardium. Isolation of individual molecular forms and their regulation]. AB - Individual molecular forms of phosphoprotein phosphatase (PPase) and their inhibitors of the protein nature were isolated from rat heart muscle by means of column chromatography on DEAE Sephadex A-50. All the three fractions of PPase were capable to dephosphorylate casein but did not affect pyrophosphate. PPase I dephosphorylated trimethaphosphate or casein. The enzyme molecular forms were different from each other by the value of specific activity and sensitivity to heat treatment. Two protein inhibitors eluted by buffers of different ionic strength - 0.2 M and 1.6 M - were identified. One of them was inactivated during heat treatment (95 degrees 5 min), while the second inhibitor maintained completely its activity in these conditions. PMID- 3004035 TI - [Effect of adrenocorticotropic hormone on the biosynthesis of glycerolipid components in the rat liver]. AB - Single administration of ACTH led to stimulation of synthesis of saturated and monoenic fatty acids in liver tissue as well as to their esterification with formation of triacyl glycerols. The hormone inhibited elongation and desaturation of fatty acids and decreased the formation of polyenic fatty acids in liver tissue. Cycloheximide alone or simultaneously with ACTH activated synthesis of unsaturated and monoenic fatty acids and stimulated the glycerophosphate shunt of triacyl glycerols synthesis. The tropic hormone activated glyceroneogenesis contributing to an increase in concentration of both alpha-glycerophosphate and long-chain fatty acids in hepatocytes; either ACTH or cycloheximide stimulated the triacyl glycerols synthesis via induction of the key enzymes by excess of the substrates. Consumption of acetyl-CoA, derived from labeled leucine, for biosynthesis of individual lipids was regulated by other mechanisms distinct from those involved in consumption of acetyl-CoA pool, derived from labelled acetate. PMID- 3004036 TI - [Restriction endonuclease Sau 6782]. AB - Data are described on identification, isolation and purification of restricting endonuclease Sau 6782 as well as on estimation of the enzyme recognition site. Conditions were developed for growing of Staphylococcus aureus 6782 strain, which enabled to produce a maximal yield of the restricting activity containing minimal level of nucleases. The procedure for isolation and purification of restrictase Sau 6782 involved affinity chromatography on Blue Sepharose and cation exchange chromatography on phosphocellulose PII. The enzyme preparation obtained was free from impurities of unspecific nucleases. The yield of the Sau 6782 restrictase constituted 1,000 un from 1 g of the culture cells. Restrictase Sau 6782 recognized the nucleotide sequence 5'...GATC...3' and was the isoshizomere of the Sau 3A enzyme. PMID- 3004037 TI - [Peroxisomes in human neutrophils]. AB - Complex electronocytochemical and stereological methods showed that human neutrophil contained usually 442 +/- 14 peroxidasosomes, which occupied 7.8 +/- 0.2% of the cell cytoplasm volume (nuclear volume excluded). The mean diameter of the peroxidasosome was 406 +/- 14 nm and the volume--3.5 +/- 0.09 X 10(7) nm3. The EPR method exhibited that approximately 2.2 X 10(7) molecules of myeloperoxidase contained in each neutrophil, i.e. one peroxidasosome included about 5 X 10(4) molecules of myeloperoxidase. PMID- 3004038 TI - [Age-dependent characteristics of the effect of insulin on 5'-nucleotidase activity in liver plasma membranes]. AB - Vmax of 5'-nucleotidase was found to be similar in liver plasmatic membranes of both 4-5 months old male rats and 24-26 months old animals. At the same time, Km value was higher in the membranes of old rats than in young animals. Insulin (administered at a dose of 2 un per 100 g of body mass) increased the enzyme Vmax without some age-dependent differences and decreased Km value in old animals as compared with controls. PMID- 3004040 TI - [Resection of the liver in tumors]. AB - Seventeen cases of liver resection for tumors of various origin are discussed. The results of 245 liver resections suggest a wider application of the procedure in tumor patients. A case of extended liver resection is described. The surgical procedure of liver resection for tumor is discussed. Radical surgery should be performed in all patients irrespective of tumor size unless portal fissure is obstructed. As few as 2 out of 17 operated cases were fatal. PMID- 3004039 TI - [Increased levels of transferrin in the rabbit heart muscle in experimental myocardial infarction]. AB - Concentration of transferrin was distinctly increased in the infarction zone of heart muscle of rabbits with experimental myocardium infarction within 24 hrs and 168 hrs after coronary occlusion. Under conditions of cell lysis apotransferrin appears to bind free iron in rabbit heart muscle. The data obtained suggest that apotransferrin may serve as a screening agent of free iron, while caeruloplasmin is able to exhibit the superoxide dismutase activity. PMID- 3004041 TI - [Pathogenesis of virus-induced diabetes (a review of the literature)]. PMID- 3004042 TI - [Acquired immunodeficiency syndrome (a review of the literature)]. PMID- 3004045 TI - Navigating the sea of eicosanoids. PMID- 3004044 TI - Primary hepatocellular carcinoma--recent advances and future prospects. PMID- 3004047 TI - [Thyroid function in persons exposed to hydrocyanic acid]. PMID- 3004046 TI - [Klinefelter's syndrome and neoplasms]. PMID- 3004048 TI - Principles of nucleic acid hybridization and comparison with monoclonal antibody technology for the diagnosis of infectious diseases. AB - Until the 1980s the diagnosis of specific etiologic agents of infectious diseases rested with their isolation in vitro and identification by analysis of their phenotypic characteristics. In the 1970s the concept of a microbial species evolved from phenotypic analysis to nucleic acid homology. Currently, nucleic acid sequences specific for a given species are being isolated and amplified and utilized not only to identify the pathogen after it has been grown in vitro but also elucidate it directly in biological material. The procedures for making nucleic acid hybridization probes are analogous to the generation of monoclonal antibody tests. Currently, research and development are centered in choosing the particular nucleic acid to analyze, establishing the most efficient vector system for amplifying the nucleic acid, generating an efficient means of selecting the particular nucleic acid fragment specific for the microorganism, and in measuring the hybridization reaction. While immunological techniques have been utilized in the clinical laboratory for over thirty years, the means of detecting nucleic acid hybridization reactions are just beginning to be usable in the clinical diagnostic laboratory. Much of nucleic acid hybridization research is proprietary, and a particular challenge is to develop a means whereby information can be used for the progress of science as a whole when generated by private ownership. PMID- 3004049 TI - [Diagnosis of lymphatic metastases in intraoperative thoracic cytology]. AB - The diagnosis of metastases to lymph nodes of the thoracic or neck region reveals great importance for the thoracic surgery. In the last two years we investigated materials taken from 730 patients undergoing thoracotomies, mediastinoscopies and by exstirpations of lymph nodes. Altogether there were imprints from 1400 lymph nodes cytologically screened. Different tumor types exhibited different numbers of metastases. Usually we took smears from 2 till 12 lymph nodes from one patient. We got the imprints from several planes of the lymph node. All imprints were stained by Giemsa fast staining. The results of this cytological method are not inferior to the histological section technique. PMID- 3004050 TI - [Binding activity of circulating HBsAg particles for polymerized human albumin in acute hepatitis B]. AB - The binding activity of circulating HBsAg particles to polymerized human serum albumin (pHSA) was determined by radioimmunoassay in 18 patients with acute hepatitis B at follow-up and compared with other hepatitis B virus (HBV) markers. All patients had serum HBsAg particles with binding sites for pHSA during the acute phase of disease. Elimination of HBsAg particles with pHSA receptors occurred within 1 to 8 weeks in uncomplicated courses and in one case with a protracted course of 16 weeks before total elimination of HBsAg. The pHSA-test became negative after elimination of HBsAg in most instances. Three patients developed chronic hepatitis with persistence of pHSA-binding HBsAg particles. Circulating HBcAg dissociated by chaotropic ions was detectable in the first week of observation in 4 of 15 patients who recovered and persisted in 3 cases with transition to chronic disease. The results show that acute hepatitis B is associated with circulating pHSA-binding HBsAg particles which can serve as a prognostic marker of the early phase of the disease. PMID- 3004052 TI - [Virus infections of the skin and mucous membranes and malignant tumors]. AB - The relationship between virus infections of the skin and mucosae and the development of malignant tumors is discussed. Guided by seroepidemiological data, virus infections with herpes simplex have been associated with the development of cervical carcinoma, genomes specific for HSV, however, have only occasionally been detected in malignant tumor tissue from the cervix. In contrast, DNA specific for papilloma virus could be identified to a high percentage in cervical cancerous and precancerous tissue as well as in bowenoid papulosis in both sexes. Aside from oncogenic papilloma virus, malignant degeneration seems to be dependent on both endogenic cofactors such as immunodeficiency and exogenic cofactors like sunlight or additional virus infections. PMID- 3004051 TI - The potential use of fiber polysaccharides in enteral feeding. PMID- 3004053 TI - [Age-dependent regulation of cardiac glycoside receptors]. AB - Specific binding of cardiac glycosides to their receptors precedes their actions on the myocardium. Thus changes in the number and affinity of these membrane bound receptors will vary the response to cardiac glycoside therapy and the incidence of side-effects. Several studies have reported an age-dependent decrease in the number of cardiac glycoside receptors in animals as well as in human erythrocytes. These results may explain the increase in cardiac glycoside sensitivity with age which is not accounted for by the reduction in kidney function. Furthermore, changes in both binding affinity and capacity are known to occur in several diseases which are more common in older patients. Therefore, we have measured the number and affinity of cardiac glycoside receptors in atrial samples from 209 patients and in papillary muscle samples from 59 patients taken during coronary bypass graft surgery or mitral valve replacement. Further, the maximal increase in force of contraction was measured using papillary muscle strips from some of these patients. Our results show no significant age-dependent alteration in the characteristics of the cardiac glycoside receptors but a reduced myocardial receptor density in males (3.47 +/- 0.14 X 10(14)/g protein) compared with females (4.44 +/- 0.21 X 10(14)/g protein) (p less than 0.001) which is more pronounced in older patients. About 30% fewer cardiac receptors either per g protein or per g wet weight are present in patients with coronary heart disease or dilative cardiomyopathy. The maximal inotropic effect of ouabain in human papillary muscle strips is correlated with the number of cardiac glycoside receptors present.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004043 TI - Prostaglandins, thromboxanes and leukotrienes in clinical medicine. AB - Although prostaglandin research began about 50 years ago, many of the most important advances in understanding the biochemistry, physiology and pharmacology have taken place within the past five to ten years. There is great potential for the extension of this research to the clinical practice of medicine. At this time, the most common interaction that clinicians have with the prostaglandin field is in administering nonsteroidal anti-inflammatory drugs, which function by inhibiting prostaglandins. The uses of these drugs include treating not only inflammation, but also dysmenorrhea, some renal disease, thrombotic diseases and some metabolic disorders. Prostaglandin analogs, with their potent effects on uterine contraction, are in common use in obstetrics. Other analogs, with gastric and duodenal cytoprotective effects are useful in treating peptic ulcer disease. Future benefits from prostaglandin and leukotriene research may include new therapy for inflammatory and hypersensitivity diseases such as asthma, inflammatory bowel diseases and dermatitis. PMID- 3004055 TI - [Some recent biochemical and physiologic aspects of the vascular endothelium]. PMID- 3004054 TI - [Functional morphology of the endothelium with special reference to the coronary vessels]. AB - Contrary to our former belief, endothelial cells can no longer be classified as a homogeneous cell population which, as a living border layer between the blood and the extravascular space, serves exclusively as a selective filter. The technique of routinely culturing endothelial cells from various sources has provided new insights into an unexpected multitude of synthetic and metabolic capacities of these cells, e. g. the degradation of arachidonic acid, the enzymatic activation of angiotensin I, and many others. However, many of these results are still contradictory, and therefore make any critical review of the data almost impossible. Some examples of this are briefly outlined in this contribution. Irrespective of this, these new results have to be taken into account, although the possibly high specificity of the endothelium will render the interpretation of future data, particularly those obtained from animal models, much more difficult. However, to simply neglect these complexities would be disastrous for all future endothelial research. PMID- 3004056 TI - [Recent knowledge about the metabolic regulation of coronary circulation, with a contribution on adenine nucleotide metabolism of coronary endothelial cells]. AB - The adenosine hypothesis in its original form neglects other myocardial tissues besides the predominant cardiomyocyte compartment. We could, however, demonstrate that the coronary endothelium comprises a metabolically very active adenosine and adenine nucleotide compartment of the heart, and functions as an impermeable metabolic barrier for interstitially or intravascularly accumulating adenosine if the vasoactive nucleoside is present at concentrations less than 10(-6) M. As a consequence, the vasodilatory action of intracoronarily applied adenosine cannot result from a direct action on the smooth muscle cells of the arterioles, but must be mediated by the endothelium. Since high molecular weight derivatives of adenosine, which are clearly confined to the coronary system, can also induce a very prompt coronary flow increase when applied intravascularly, smooth muscle relaxation must be triggered by an extracellular adenosine receptor at the luminal surface of the endothelium. According to preliminary pharmacological studies, this receptor belongs to the A2-type and thus stimulates the endothelial adenylate cyclase system. On the basis of our findings it is evident that with respect to the vasodilating effect of adenosine one has to distinguish between its action from the interstitial space directly via the putative receptor at the surface of the arteriolar smooth muscle cells, and its action from the intravascular space via the newly detected endothelial A2-receptor. It is a matter of further experimentation to determine to what extent both receptor populations actually participate in the metabolic regulation of coronary flow under physiological and pathophysiological conditions. PMID- 3004057 TI - [Regulation of vascular tone by the endothelium]. AB - In the last few years, experimental evidence has accumulated which suggests a substantial role for the endothelium in the control of vascular tone. Endothelium dependent dilations have been demonstrated in various arteries of numerous mammalian species including man. Among the stimuli which elicit endothelium dependent dilatation are such different stimuli as increases in blood flow and hypoxia as well as endogenous (acetylcholine, ATP, ADP, bradykinin, substance P) and pharmacological agents (calcium ionophore A 23 187, ergometrine, hydralazine, melittin). The functional importance of endothelium-dependent dilatation is emphasized by the fact that the direct vasoconstrictor effects of some of these substances (acetylcholine, histamine, norepinephrine, serotonin) on vascular smooth muscle is attenuated or even reversed by their simultaneous stimulatory effect on endothelial cells resulting in the release of a vasodilator signal. Bioassay experiments have shown that a humoral vasodilator agent with a biological half-life in the range of seconds is released from the endothelium (native or cultured) during stimulation with acetylcholine, ATP and calcium ionophore. Experimental data are presented which suggest that EDRF may act by direct stimulation of guanylate cyclase, resulting in smooth muscle relaxation due to increased smooth muscle cyclic GMP levels. The chemical nature of this nonprostaglandin endothelium-derived relaxant factor (EDRF) is still not known. The possible physiological and pathophysiological significance of endothelium dependent dilatation in situ is discussed. Special attention is paid in this context to the potential role of EDRF activity in coronary vasomotor control. PMID- 3004058 TI - [Changes in nutrition habits in obesity and hyperlipoproteinemia]. AB - Lowering of cardiovascular risk factors has a favorable effect on the manifestation of arteriosclerotic vascular diseases. The dietetic measures constitute the therapeutic basis of this; atherogenic low-density lipoproteins (LDL) are lowered and at the same time the high-density lipoproteins (HDL) are raised. The lowering of alimentary cholesterol intake and selection of suitable fats with a high percentage of polyunsaturated fatty acids is at the forefront. The diet should be supplemented by roughage and soya proteins. In particular, obese patients with disorders of lipid metabolism should pay attention to the guidelines indicated. PMID- 3004059 TI - [Tasks within the scope of psychosocial treatment of suicidal patients: crisis intervention and social help, psychiatric evaluation and after care]. PMID- 3004060 TI - [Compliance problems in suicidal patients]. PMID- 3004061 TI - [Experimental basis for the use of collagenase preparations to prevent aging of the skin]. PMID- 3004062 TI - [Cytochemical studies of myeloperoxidase and non-enzyme cationic proteins in various dermatoses]. PMID- 3004063 TI - [Neuronal activity of the caudate nucleus and centrum medianum of the thalamus in the cat during formation of an instrumental defensive conditioned reflex]. AB - Implanted tungsten microelectrodes were used for studying the neuronal activity of CN and MC at initial stage of formation of instrumental defensive behaviour. The linear correlation coefficient was not above +/- 0.15 during recording of the background impulse activity before presentation of the positive stimulus to the animal or during its presentation not more than 100 times. In the course of formation of the instrumental reflex the correlation coefficient increased up to +/- 0.3 for 19 cells and up to +/- 0.31--+/- 0.56 for 9 cells. Inhibitory conditioned reactions were characteristic of 34 CN neurones (out of 56) and responses of 42 MC neurones (out of 51) had an activating form. Reactions of both structures were of tonic character with most pronounced changes at the start of the conditioned signal. In dynamics of instrumental behaviour formation, the intensity correlation coefficient of CN and MC neurones reactions recorded simultaneously, increased. PMID- 3004064 TI - [Effect of acute and chronic deprivation of catecholaminergic system activity induced by 6-hydroxydopamine on the behavior of the rat]. AB - On 202 male rats of Wistar line, a study was carried out of the effect of chronic and acute deprivations of the brain CA-systems activity resulting from administration of 6-OHDA on investigating behaviour and learning. Chronic deprivation of CA-systems activity by neonatal administration of 6-OHDA (100 mg/kg subcutaneously) and their acute deprivation by intracerebral administration of 6-OHDA to adult rats (150 mkg in each lateral ventriculus) was accompanied by similar deep changes of behaviour. Both forms of deprivation reduced the investigating activity of the animals in the open field. In both cases, the above 6-OHDA dozes sharply impeded the learning of animals with emotionally negative reinforcement, with no significant influence on learning with emotionally positive reinforcement. Both forms of deprivation of CA-systems activity weakened the reaction of frustration elicited by a sharp reduction of food reinforcement. PMID- 3004065 TI - [Smoking behavior and cytology of the cells of smokers]. AB - Cytological examinations of lung impression preparations from 131 smoker lungs revealed that the content of smoker cells within lung tissue increases up to a daily consumption of 40 cigarettes. Additional cigarette consumption does not raise the number of smoker cells further. Determination of the nuclei content in the smoker cells of groups with different consumption rates showed that the number of macrophages with more than two nuclei increases in proportion to the number of cigarettes smoked. If more than 50 cigarettes a day were smoked, many multinucleated giant cells were observed. Anamnestic inquiries proved that these cytological changes in the lungs were caused exclusively by the smoking habits of the deceased. Not only the number of cigarettes used per day, but also the manner of inhalation and peculiarities of cigarette smoking are reflected in the morphological changes of lung tissue. For the forensic pathologist, examination of lung impression preparations from smoker lungs makes it possible to note the quantity of daily cigarette consumption of a dead person to help to identify the deceased. PMID- 3004066 TI - [Course and regulation of the menstrual cycle from Robert Schroder to the present]. AB - The fundamental histological findings about endometrial and ovarian changes during the menstrual cycle by Robert Schroder (1884-1959) are passed in review and some actual conceptions of the regulation of the ovarian cycle are cited. In addition, general findings of molecular processes during gonadotropin actions in follicular cells are described, e.g. activation and desensitization of adenylyl cyclase, the role of 3',5'-adenosinmonophosphate and down-regulation of gonadotropin receptors. In general, the progress in cycle research since the work of R. Schroder was done consists in the transition from the histological description of endometrial changes to recognition of intracellular events which have to be considered by gynaecologists. PMID- 3004067 TI - [Herpes simplex virus type 2 infection. Etiology, clinical aspects, diagnosis, therapy, HSV-2 and cervix cancer]. AB - The genital herpes infection is mostly caused by HSV-2. The analysis of epidemic data supports the common impression that herpes genitalis markedly increased. The HSV-2 is with few exceptions a sexually transmitted virus, the infectionsity is about 80%. The primary infection with HSV-2 occurs in the youth. HSV-2 survives in a latent stage in the human body for life and causes as a rule to recurrent diseases. The clinical picture is variable. The most severe complication is the sepsis of newborn. The virological diagnosis is made by isolation of virus. The therapy of herpes genitalis is problematic. It is proposed that the HSV-2 plays a key role as an initiating factor in the development of human genital cancer. PMID- 3004068 TI - [Cytostatic treatment of pseudomyxoma peritonei]. AB - Clinic, pathomorphology and therapy of pseudomyxoma peritonei are explained and discussed by means of a women of 54 years. The prognosis in former times very bad could be improved by a combination of radical surgery and polychemotherapy. PMID- 3004069 TI - Polyradiculoneuritis in children groups during simultaneous circulation of enteroviruses and adenoviruses. AB - Two cases of polyradiculoneuritis in children were noted during January, 1984. Each girl was a member of a different group of children: the first group was newly constituted of children from various remote regions of Czechoslovakia in a Medical Institution, the second one included children attending the same nursery school for a long time. In both groups, all or most of the children went through one or two respiratory infections which preceded the development of a paralytic disease. From the first girl, coxsackievirus A9 was recovered in nasopharyngeal swabs and in a stool sample. Among contacts in the Children's Medical Institution, a concurrent circulation of this enterovirus and of an adenovirus type 3 was demonstrated by isolation attempts and confirmed by serological examinations. From the second girl, coxsackie A9 and an adenovirus type 29 were demonstrated in the same stool sample and a simultaneous circulation of both virus species among the nursery school and family contacts was proved by isolation attempts and by serological investigations. The concurrent, overlapping or sequential circulation of adenoviruses and enteroviruses may perhaps contribute to a compromised immunity resulting in a manifestation of paralysis. PMID- 3004070 TI - [Typing of Streptococcus group A isolated from patients and healthy persons]. AB - The typing of 80% of 381 streptococcal strains, group A, under study was accomplished with a set of diagnostic anti-T sera obtained from the Sevak Institute (Czechoslovakia). None of the T-types could be related with certainty to the localization of the infective agent in the human body (the pharynx, the skin). Different T-types were shown to circulate in definite regions of the USSR. To enhance the differentiating capacity of T-typing, the enzymatic (lipoproteinase and NADase) activity of the strains was determined, thus permitting the subdivision of the T-types into still smaller groups. The typing of OF+ strains of unknown M-specificity could be carried out by means of the blood sera of healthy persons, containing antibodies to streptococcal lipoproteinase. The conclusion on the expediency of using the determination of lipoproteinase and NADase as an additional marker in the typing of group A streptococci was made. PMID- 3004071 TI - [Comparative study of the action of Vibrio cholerae enterotoxin, thermolabile Escherichia coli toxin and a Salmonella typhimurium filtrate on the small intestine of laboratory animals]. AB - The results of the comparative study of the effects produced by V. cholerae enterotoxin E. coli thermolabile toxin (TL-toxin) and S. typhimurum 415 filtrate on the loops of the small intestine of rabbits and C57BL/6 mice are presented. The results obtained in mice were considerably more uniform than those obtained in rabbits. For the first time the dose-effect curve for V. cholerae enterotoxin and TL-toxin, obtained on both animal species, was found to be bell-shaped. The concentration relationship for dibutyryl-cAMP proved to be similar in character. The authors suggest that these effects are based on the specific relationship between the cellular processes, controlling the transport of electrolytes and water, and the level of cAMP. PMID- 3004072 TI - [Preparation of diagnostic sera to enteroviruses 69, 70 and 71 having medical importance]. AB - The use of enteroviruses, types 69, 70 and 71, purified with freon and concentrated with polyethylene glycol, for immunizing rabbits, following 4 immunization schedules, has made it possible to obtain active virus-neutralizing diagnostic sera with antibody titers of up to 1:2560, 1:806 and 1:9123, respectively. PMID- 3004073 TI - [Detection of Epstein-Barr viral antibodies in patients with acute viral hepatitis]. AB - Serum samples obtained from 44 patients with virus A hepatitis, 23 patients with virus B hepatitis, 65 patients with virus non A, non B hepatitis and 100 healthy adults were studied for the presence of Epstein-Barr (EB) virus in the indirect immunofluorescence test. In this work lymphoblastoid cell lines PH3-J-1 and CN37 were used. Among patients with different forms of hepatitis, the statistically significant elevation of the titers of antibodies to EB virus was detected only in the group of patients with virus non A, non B hepatitis, and in 6 cases the etiological role of EB virus was confirmed by serological and hematological methods. PMID- 3004074 TI - The interaction of metal ions with nucleic acids. NMR, EPR, CD and spectrophotometric study of the copper (II) interaction with 6-ketopurine ribosides. AB - The copper (II)-inosine system in water-DMSO solutions was investigated as a function of pH and the molar ratio between the ligand and copper(II) ion by the EPR, NMR, CD and visible absorption spectrometric methods. It was concluded that a simple M.[-N]L copper(II)-inosine 1:1 complex is formed over the pH range 1.4 5.0, while M.[-N]L2 complexes are present in the solutions of pH 5.0-6.2. From pH 6.2 to 7.8 a diamagnetic, hydroxybridged complex M2.(OH)2.[-N]L4 dominates. At pH values of 7.8-9.2 an insoluble, oxybridged species (M.O.[-N]L)n is formed in addition to the soluble paramagnetic M.[-N-1)L4 complex. Above pH 9.1 the nitrogenbridged polymeric complex (M.[-N-1].M[-N-7] )n is formed which is stable up to pH 12.5, and above pH 12.5 the only species found is the M.[-OH]L2 chelate complex in which inosine is coordinated to copper through the two ionized hydroxyl groups. PMID- 3004075 TI - Characterization of the 5'-half of Leishmania brasiliensis (Y) 20S ribosomal DNA and its adjacent spacer. PMID- 3004076 TI - Acquired immune deficiency syndrome. A viral disease. PMID- 3004077 TI - Drugs recently released in Belgium: epirubicin and enalapril maleate. PMID- 3004078 TI - Cytologic diagnosis of male breast cancer with nipple discharge. A case report. AB - The cytologic findings in a nipple discharge from a male patient with breast cancer are described. Malignant epithelial cells and cell clusters believed to be derived from ductal carcinoma were observed. The subsequent mastectomy specimen contained a ductal carcinoma with minute foci of stromal invasion. PMID- 3004079 TI - Carcinomatous meningitis as the initial manifestation of breast cancer. AB - Report is made of an elderly woman in whom carcinomatous meningitis was the initial manifestation of breast cancer. The patient presented with nonfocal neurologic symptoms. Cytologic examination of cerebrospinal fluid (CSF) revealed malignant cells arranged either in loose clusters or as isolated single cells. The morphology of the cells, some of which had a signet-ring configuration with a crescent-shaped nucleus as well as prominent round nucleoli, suggested a breast primary, which was proven by subsequent histologic study. PMID- 3004080 TI - Endoscopic "salvage" cytology in neoplasms metastatic to the upper gastrointestional tract. AB - Endoscopic "salvage" cytology was the only successful nonoperative diagnostic method in two patients with malignancy metastatic to the upper gastrointestinal tract. Smears and cell blocks of centrifuged material aspirated from the endoscope channel provided diagnoses of malignancies when other diagnostic techniques had been nonproductive. The results in these cases support the general utility of this technique and indicate its successful application in this specific clinical setting. PMID- 3004081 TI - Intracytoplasmic eosinophilic inclusion bodies in benign breast cyst fluids. AB - Intracytoplasmic eosinophilic inclusion bodies (EIBs) observed in cytologic preparations of benign breast cyst fluid aspirates were morphologically similar to the well-known EIBs in urine specimens. EIBs have not been previously described in breast cyst fluids. A chi-square test proved a statistically significant correlation between the presence of intracellular EIBs and cellular degeneration. The possible significance of this finding with regard to the origin of EIBs is discussed. PMID- 3004082 TI - Postradiation pleomorphic malignant fibrous histiocytoma of the breast. AB - A case of primary malignant fibrous histiocytoma (MFH) of the breast occurring two years after surgical excision and radiation therapy for a carcinoma of the left breast is reported. Fine needle aspiration was positive for malignant cells, consistent with a pleomorphic sarcoma. Cytologic examination revealed giant cells with marked pleomorphism. Some cells showed single large nuclei with cytoplasmic vacuoles while others revealed multinucleation with foamy cytoplasm, phagocytosed erythrocytes and cellular debris. These findings are considered useful in the cytologic diagnosis of the pleomorphic variant of MFH. PMID- 3004084 TI - Type 1 bronchioloalveolar carcinoma. PMID- 3004083 TI - Fine needle aspiration cytology of sclerosing hemangioma of the lung, a mimicker of bronchioloalveolar carcinoma. AB - A rare "sclerosing hemangioma" of the lung in a 24-year-old man was initially interpreted as a bronchioloalveolar carcinoma by fine needle aspiration (FNA) cytology. The similarity of these two tumors in fine needle aspirates is discussed. Benign sclerosing hemangioma should be considered in the differential diagnosis when numerous atypical proliferating bronchiolar or alveolar cells are obtained by FNA. PMID- 3004085 TI - Bile cytology in the diagnosis of sclerosing cholangitis and cholangiocarcinoma. PMID- 3004086 TI - Effect of angiotensin II on plasma ACTH in patients with Addison's disease. AB - Short-term angiotensin II (AII) infusions (3 ng/kg/min) were performed in 5 patients with Addison's disease in order to assess the effect of AII on ACTH secretion. Base line ACTH levels were elevated due to a 9-h time lag between hydrocortisone administration and onset of the study. In 2 separate infusion periods of 30-min duration, AII had no unidirectional effect on plasma ACTH. Mean ACTH increased slightly but insignificantly. Mean blood pressure rose by about 10 mmHg. The degree of angiotensinaemia induced is probably similar to the state of moderate to severe sodium deficiency. Short-term changes of AII in this order of magnitude have obviously no major effect on ACTH secretion. PMID- 3004087 TI - Dissociation of the triiodothyronine receptor from the nucleus induced by cations: evidence for divalent cation sensitive and insensitive nuclear sites. AB - Aliquots of purified rat liver nuclei were diluted at 0 degrees C in isotonic buffers containing monovalent (Na+) or divalent (Ca2, Mg2+) cations. At different times following dilution the nuclear suspensions were centrifuged and the T3 receptor was measured in KCl extracts of the nuclear pellets. The rate of receptor loss after dilution in EDTA was 0.0025 min-1. Dilution in the presence of cations caused a fast release of receptor during the first 10 min. This phase, which was not observed when the nuclei were diluted in EDTA without salt, was followed by a second phase where the receptor was released at the same rate as in EDTA. Receptor release was only dependent on the presence of cations in the dilution buffer during the first 10 min after dilution. The amounts of receptor remaining in the nuclei after the first 10 min of dilution were 51.8 +/- 9.2%, in the presence of Ca2+ and Mg2+, 38.6 +/- 8.9% in 0.15 M NaCl, and 18.0 +/- 4.8% in 0.15 M NaCl in the presence of Ca2+ and Mg2+. The release of receptor was not influenced by the integrity of the nuclear membrane. These results suggest the presence of divalent cation sensitive and insensitive nuclear sites for the T3 receptor, in amounts which could be estimated to be about 48 and 52%, respectively. Other interpretations are also possible, such as the presence of a high proportion of free receptors in the nucleosol, which could be released during the first phase of dilution if the negative charges in chromatin are blocked by cations to avoid redistribution of receptors immediately after dilution. PMID- 3004088 TI - Effect of etomidate on cortisol biosynthesis: site of action after induction of anaesthesia. AB - To determine the site of inhibition of etomidate on cortisol biosynthesis, plasma cortisol, aldosterone, 17 alpha-hydroxyprogesterone, 11-deoxycortisol and ACTH levels were measured in healthy women before and after the administration of a single dose of either 0.20 mg kg-1 etomidate (mean value, n = 10) or 3.15 mg kg-1 thiopental (n = 9) for induction of anaesthesia in a randomized trial. Etomidate produced a smaller increase in plasma cortisol and had a later onset of action than thiopental. Plasma ACTH levels, however, rose higher in the etomidate induced patients to reach peak levels 6 h after drug administration. In the same group, plasma aldosterone remained below the control levels but still within the normal range, whereas it rose about 2-fold in the thiopental group. Plasma levels of 17 alpha-hydroxyprogesterone and 11 beta-deoxycortisol were hardly modified after thiopental but increased significantly and remained high for 6 h after etomidate injection. This marked rise in precursors together with a blunted and delayed cortisol response to high ACTH levels, and slightly lowered plasma aldosterone concentration indicates a blockage of 11 beta-hydroxylation in adrenal cortisol synthesis after induction of anaesthesia with etomidate. PMID- 3004089 TI - Regulation of 11 beta/18-steroid hydroxylation in newborn rat adrenal cells in primary culture. AB - In newborn rat adrenal cells in primary culture, the level of activity of the 11 beta/18-steroid hydroxylase system involved in the last step of the corticosteroid biosynthesis is increased by ACTH. A parallel study of 11 beta- and 18-hydroxylation showed the same apparent Km values (64 microM) for both hydroxylations. The Vmax values differed: 11.5 micrograms/10(6) cells/h for corticosterone and 6.9 micrograms/10(6) cells/h for 18-hydroxyDOC. A dose response study of the ACTH effect, measured by the bioconversion of deoxycorticosterone to corticosterone and 18-hydroxyDOC, showed maximum hydroxylation with a dose of 2.2 mU of ACTH/ml. Addition of ACTH after several weeks in culture produced a smaller increase in 11 beta/18-hydroxylation. Removal of ACTH after several weeks of treatment produced an immediate decrease in corticosteroid production; readdition of ACTH produced an increase to the previous level in the case of the 22 mU/ml dose, but not in the case of the 2.2 mU/ml dose. The use of actinomycin D demonstrated that ACTH affects mainly the biosynthesis of protein which must be renewed approximately every 24 h. Finally, the effect of pretreatment or co-treatment with various concentrations of the end products of the reaction showed no inhibition or destruction of the 11 beta/18 hydroxylating enzyme system. Therefore, the regulation of the 11 beta/18-steroid hydroxylase system in these cell cultures seems to be accomplished through the induction by ACTH of the transcription involved in the biosynthesis of cytochrome P450(11) beta and the amount of available precursor furnished by endogenous steroidogenesis. PMID- 3004090 TI - Antler cycle and thermolabile and thermostable alkaline phosphatase in white tailed deer; circannual and circadian rhythms and variation after thyroxine, dexamethasone and ACTH administration. AB - The plasma levels of thermolabile (TLAP) and thermostable (TSAP) alkaline phosphatase were investigated in adult male white-tailed deer. Distinct seasonal variation of TLAP (with highly elevated levels in July) were observed, whereas TSAP exhibits low concentration with a small increase in October. No circadian rhythm was found for TLAP or TSAP. A close correlation (r = 0.98) between TLAP activity and antler weight was found. Administration of ACTH or dexamethasone were ineffective in influencing AP activity. On the other hand, variations of triiodothyronine (T3) levels in plasma induced by thyroxine (T4) injections correlated well with concentration of TLAP. PMID- 3004091 TI - Concentrations of immunoreactive tonin in submandibular glands and saliva of the rat. AB - The submandibular gland (SMG) of the rat contains tonin, an enzyme of serine proteases, which specifically cleaves angiotensinogen and angiotensin I to yield angiotensin II directly. Using a specific RIA for rat tonin, the present study was performed to examine the concentrations of immunoreactive (IR) tonin in SMG of normal Wistar male rats with various ages, those in SMG and saliva of normal adult males and females, and also its SMG levels of castrated males and testosterone-treated females. The concentrations of IR-tonin in SMG of immature 4 week old male rats were very low but rose exponentially with increasing age to reach adult levels (3-6 micrograms/mg wet tissue) in male rats after 6 week old or more. SMG of the adult male rat contained 10-fold more IR-tonin than that of the female rat and male saliva contained 5-fold more IR-tonin than that of females. The concentrations of SMG IR-tonin in the castrated males fell to about one-tenth the levels of normal males, whereas SMG IR-tonin levels in the testosterone-treated females increased about ten times more than those of normal females. These data confirm the sexual dimorphism of rat SMG with respect to the concentrations of tonin and indicate that androgens play an important role in regulating the synthesis and/or storage of tonin in rat SMG. It is also suggested that tonin is secreted into saliva in concentrations reflecting its SMG levels. However, biological significance of tonin in SMG and saliva remains obscure. PMID- 3004092 TI - Evaluation of sodium and potassium pump activity and number in diabetic erythrocytes. AB - The Na+,K+-ATPase (= Na pump), which produces the concentration gradient of Na+ and K+ across the cell membrane, was studied in diabetic erythrocytes. The activity and number of Na pumps, which are functional and quantitative expression of the Na+,K+-ATPase in erythrocytes, were measured by ouabain-sensitive 42K or 43K influx and by [3H]ouabain binding, respectively. The turnover rate of the pumps was calculated from the two measurements to evaluate in situ activity of the pump. The Na pump activity was found to be higher in the diabetic (193 +/- 12 nmol K+/h . 10(9) cells) than in the normal group (164 +/- 6) (P less than 0.05), though the affinities of the pump for extracellular K+ were not different. The number of Na pumps and the Kd of the pumps for ouabain in the diabetic group were not significantly different from those in the normal group (354 +/- 12 site/cell and 4.33 +/- 0.20 nM). The turnover rate of the diabetic group tended to be higher than that of the normal group. The rate was about one third of the molecular activity reported for Na+,K+-ATPase under optimum conditions. These results indicate that the enzymatic properties as well as the number of Na pumps were not altered in diabetic erythrocytes despite the increased Na pump activity. Therefore the increased activity of the erythrocyte Na pump in diabetes mellitus suggests an increase of cation permeability associated with a possible disorder in the diabetic membrane. PMID- 3004093 TI - Alpha- and beta-adrenergic receptor activity in circulating blood cells of patients with phaeochromocytoma: effects of adrenalectomy. AB - In many tissues adrenergic responsiveness may be impaired by chronic exposure to adrenergic agonists. We examined the effects of high levels of circulating norepinephrine and epinephrine to alpha 2- and beta 2-adrenoceptors on platelets and mononuclear leucocytes (MNL) of patients with phaeochromocytoma. When compared with controls the density of beta 2-receptors prior to removal of the tumour was diminished by 67% (500 +/- 160 sites/cell, n = 7 vs 1500 +/- 350 sites/cell, n = 29; P less than 0.001). Binding affinities for [3H]dihydroalprenolol (apparent KD = 0.45 +/- 0.16 nmol/l in phaeochromocytoma, 0.52 +/- 0.19 nmol/l in controls) were not significantly different in MNL preparations from the two groups. MNL adenylate cyclase activities in response to 10 mumol/l (-) isoproterenol was lower in the patients (7.5 vs 22 pmol cAMP/10(6) MNL/10 min; P less than 0.001). Conversely, the number of alpha 2-adrenoceptors on platelets and the affinity of these receptors for [3H]yohimbine did not differ between patients and controls. Ensuing adrenalectomy the regeneration of the beta adrenergic cAMP-system results from a two-phase process. A prompt but moderate restoration was followed by a gradual rise to the normal range within about 1 to 2 months indicating the dynamic character of the beta 2-adrenoceptor cAMP-system in MNL. We conclude that the downregulation observed in MNL, which appears not to be operative in human platelets, represents only one mechanism of feedback control in the sympatho-adrenal-system protecting tissues from prolonged stimulation by excessive catecholamines. PMID- 3004094 TI - Incidentally discovered ACTH-dependent adrenal adenoma presenting as 'pre Cushing's syndrome'. AB - An adrenal tumour was incidentally discovered with no clinical signs of Cushing's syndrome. The endocrine evaluation revealed the unique hormonal constellation of an increased urinary cortisol excretion rate, unequivocal suppressibility of plasma and urinary cortisol by dexamethasone, but only to a residual level in the low normal range which probably reflected ACTH-independent 'autonomous' cortisol secretion. After removal of the adrenal mass, urinary cortisol secretion and dexamethasone suppressibility were normalized. In vitro, the tumour cells were as sensitive towards ACTH as 'normal' human adrenal cells, but showed a reduced cortisol production rate per cell. We suppose that the adrenal mass participated in the diurnal rhythm of ACTH-mediated cortisol secretion in vivo, which resulted in an increased cortisol secretion. During the night, when ACTH levels were low, the cortisol production decreased and the hormone levels were probably too low to suppress ACTH. We regard the hormonal findings in our patients as 'Pre-Cushing's syndrome', although the absence of clinical features of Cushing's syndrome remains unclear. We suggest that every patient with an incidentally discovered adrenal mass should have an endocrinological evaluation because the results may help to decide whether or not the adrenal tumour should be removed. PMID- 3004096 TI - Neutrophil function in leukemia patients--the correlation between bactericidal capacity and generation of oxygen radicals, especially hydroxyl radical. PMID- 3004095 TI - Plasma adrenocorticotrophin, cortisol and aldosterone responses to ovine corticotrophin-releasing factor and vasopressin in sheep. AB - Ovine corticotrophin-releasing factor (oCRF) (1 microgram/kg) and arginine vasopressin (AVP) (1 microgram/kg) were injected iv in sheep, both separately and in combination. Plasma levels of immunoreactive ACTH (IR-ACTH), cortisol, and aldosterone were measured for 3 h after the injections. Mean levels before injections were 8 +/- 4 pmol/l for ACTH, 7 +/- 3 nmol/l for cortisol, and 28 +/- 9 pmol/l for aldosterone. CRF caused a rapid rise in IR-ACTH and a peak level of 125 +/- 52 pmol/l was obtained 15 min after injection. Highest values for cortisol and aldosterone levels were 40 +/- 9 nmol/l and 64 +/- 13 pmol/l, respectively, 30 min after injection. AVP also increased IR-ACTH (maximum level: 202 +/- 77 pmol/l at 5 min) and aldosterone (128 +/- 36 pmol/l at 15 min), whereas the cortisol increase was lower than after CRF. Simultaneous injection of CRF and AVP produced an addition of the IR-ACTH response (295 +/- 82 pmol/l at 15 min), but the changes in cortisol levels were similar to those obtained after CRF alone and those in aldosterone levels resembled those induced by AVP alone. Plasma Na and K, osmolality, and plasma renin activity (PRA) were not modified by either CRF or AVP. It is suggested that the increase in aldosterone levels after CRF could be mediated by ACTH and that after AVP by an IR-ACTH peptide with less effect on cortisol secretion. PMID- 3004097 TI - Epstein-Barr virus-positive primary brain lymphoma complicating immunoblastic lymphadenopathy. AB - A 54-year-old man developed primary brain lymphoma after immunosuppressive treatment for immunoblastic lymphadenopathy. The lymphoma cells were positive for the Epstein-Barr virus genome and had the surface phenotype of early B cells and a normal karyotype. PMID- 3004098 TI - Muscle blood flow after amputation. Increased flow with medullary plugging. AB - At below-knee amputation for arterial insufficiency in 31 patients, the muscle blood flow of quadriceps and triceps surae was measured by clearance of 99mTc pertechnetate pre- and postoperatively. In 15 patients, myoplastic amputation was performed and in 16 patients the medullary cavity of the tibial stump was plugged with cortex of the removed bone as well. Plugging caused a two-third increase in muscle blood flow. PMID- 3004099 TI - [Use of a physical model in the study of the dispersion of an alternative electrical field and its application in the per- and postoperative control of an extracochlear implant]. AB - A study of the dispersion of an alternative electrical field by a physical model, and its application in the control of unicanal extra-cochlear implant is discussed. The authors think that this method is indispensable for an objective control of the correct function of the extra-cochlear implant. PMID- 3004101 TI - Morphological studies on the mechanism of hepatocellular injury during acute phase of infection in marmosets inoculated with hepatitis A virus. AB - The histological changes during the acute phase of infection in the livers of marmosets inoculated with hepatitis A virus were examined. The acute phase was divided into four stages according to the liver enzyme changes and serological markers for the viral infection (Stage I, II, III, IV). Round cell infiltration in the portal tracts was first recognized in Stage I. Localization of parenchymal changes was predominantly periportal in Stage I, II, and IV, whereas the lesion was diffuse in Stage III. Hepatitis A virus antigen (HAVA) was widely distributed but spotty and the largest amount of HAVA was found in Stage II by immunofluorescent and immunoperoxidase study. By electron microscopy the endoplasmic reticulum was altered in the liver cells and in some area there was interaction between the hepatocytes and lymphocytes. These findings suggest that hepatocellular damages seen in this model are the result of immune response rather than cytotoxic effect. PMID- 3004100 TI - Fluctuation of specific IgA antibodies in human milk. AB - The concentration of secretory IgA and the levels of IgA specific antibodies against Escherichia coli labile-toxin, Shigella flexneri 6, and rotaviruses were determined in milk samples obtained serially from women during the first 16 weeks postpartum. The mean concentration of secretory IgA followed the expected pattern; the levels of specific antibodies fluctuated in an unpredictable manner and independently of milk secretory IgA content, becoming undetectable in many instances. Under some circumstances, continued breast-feeding may not guarantee continued intake of antibodies against intestinal pathogens by the breast-fed infant. PMID- 3004103 TI - Glucocorticoid-suppressible hyperaldosteronism. Ultrastructural observation of a case. AB - A 56-year-old woman presented with intracranial hemorrhage. Laboratory examinations revealed severe hypertension, hypokalemia, elevated aldosterone excretion, and suppressed plasma renin activity. Left adrenocortical tumor was suspected and adrenalectomy was performed. The laboratory data after operation, however, showed no significant difference from the preoperative data. On the basis of further examinations, dexamethasone was administered and returned blood pressure to normal, and also normalized serum potassium, plasma aldosterone, and renin activity. The patient's illness was diagnosed as glucocorticoid suppressible hyperaldosteronism. Light microscopically, the zona glomerulosa was hypertrophic and the outer zona fasciculata decreased in lipid droplets and was centrifugally arranged in small alveoli. Electronmicroscopically, the cells of the outer zona fasciculata had several lipid droplets and well-developed sER. Mitochondria were round to oval with lamellar or lamellovesicular cristae. These findings were evidence of hyperfunction. The cytoplasm of the cells also contained spironolactone bodies. Therefore, it is assumed that the aldosterone, which induced the disorder, was produced mainly in the outer zona fasciculata. PMID- 3004102 TI - Lung cancer in a Thorotrast administered patient. AB - A 79-year-old man developed small cell carcinoma in the lung 43 years after Thorotrast-injection. The tumor with interstitial fibrosis arose in the periphery of the lung. Thorotrast particles were observed in the liver, spleen, bone marrow, and lymph nodes but not in the lungs. Four other Japanese cases of lung cancer after Thorotrast injection were reviewed. PMID- 3004104 TI - Malignant fibrous histiocytoma with granular cells, mimicking a granular cell tumor. Light, electron microscopic and immunohistochemical observation of a case. AB - The tumor that occurred subcutaneously in the left hypochondriac region of an 87 year-old female is reported. Light microscopically, this tumor was composed of two kinds of cells, spindle cells and granular cells. The spindle cells showed various pictures such as fascicular arrangement, pleomorphic giant cells in the myxoid stroma and areas with abundant blood vessels. The granular cells contained PAS positive and diastase-resistant granules, and these granules, being electron microscopically phagocytic lysosomes, showed the same findings as the granules seen in the granular cell tumor. The immunohistochemical studies for S-100 protein, neuron specific enolase, CEA, myoglobin, and lysosome were negative, but alpha-antichymotrypsin was positive. From the microscopic and immunohistochemical findings, this tumor was diagnosed as a malignant fibrous histiocytoma with granular cell changes of tumor cells. PMID- 3004105 TI - Dissociation between renal prostaglandin E2 and renin release. Effects of glucagon, dopamine and cyclic AMP in dogs. AB - To examine the relationship between prostaglandin E2 (PGE2) and renin release, glucagon, dopamine and dibutyryl cyclic AMP (DB-cAMP) were infused into dog kidneys during autoregulatory dilation of preglomerular vessels. Autoregulatory vasodilation, which enhances PGE2 and renin release, was induced by renal arterial constriction or ureteral occlusion. Glucagon infusion increased both PGE2 and renin release during autoregulatory vasodilation, and renin release was almost abolished after inhibiting PGE2 release by indomethacin. In contrast, dopamine and DB-cAMP infused during autoregulatory vasodilation increased renin release without significantly changing PGE2 release. Stimulation of renin release was not dependent on vasodilatory effects, which for all drugs were greatly diminished during autoregulatory vasodilation. Hence, glucagon stimulates both PGE2 and renin release. Most of the increase in renin release during glucagon infusion is prostaglandin-dependent since indomethacin greatly reduced the stimulatory effect. In contrast, dopamine and DB-cAMP stimulate renin release without increasing PGE2 release as previously found for beta-adrenergic stimulation. PMID- 3004107 TI - Cyclic AMP potentiates glucose-induced insulin release from mouse pancreatic islets without increasing cytosolic free Ca2+. AB - The effects of various stimulants of insulin release on cytosolic free Ca2+, [Ca2+]i, in dispersed and cultured pancreatic beta-cells from ob/ob-mice were studied using the indicator quin-2, which in itself has only slight effects on the glucose-induced insulin release and the metabolism of the sugar. The resting [Ca2+]i was 158 +/- 7 nM. After increasing glucose to 20 mM there was a lag period of 1-2 min before [Ca2+]i gradually rose, reaching a new plateau 60% higher after 5-6 min. Increasing intracellular cyclic AMP by adding forskolin did not further increase [Ca2+]i; on the contrary there was a slight temporary reduction despite a doubling of insulin secretion. The maintenance of the beta cell function was evident from a marked increase of cytosolic [Ca2+]i after depolarization evoked by high extracellular K+. Also dibutyryl cyclic AMP and theophylline lacked the ability to raise [Ca2+]i beyond that obtained by glucose. The results suggest that cyclic AMP potentiates glucose-induced insulin release by sensitizing the secretory machinery to changes of [Ca2+]i rather than by increasing the cytosolic concentration of the ion. PMID- 3004106 TI - Influence of thyroid hormones on transport function and Na+-K+-ATPase activity in the rat choroid plexus. AB - The sympathomimetic stimulation of choroid plexus transport and secretion in rat seems to be mediated by beta-adrenergic receptors. In the present report the effect of induced changes in thyroid function on transport mechanisms in the rat choroid plexus was studied. Following induction of hyperthyroidism (treatment with T3 for 10 days) the tissue/medium ratio (T/M) for choline uptake in choroid plexus in vitro decreased significantly by 68%. The Na+-K+-ATPase activity showed a statistically significant increase of about 16%. Also following cervical sympathectomy, T3 caused a reduction of the T/M for choline, to the same level as in the non-sympathectomized animals, while the effect of T3 on the Na+-K+-ATPase activity was changed into a 22% decrease. Hypothyroidism (administration of PTU in the drinking water) had little or no effect on the uptake and accumulation of choline in the choroid plexus. The Na+-K+-ATPase activity was reduced by 40%, in contrast to the stimulating effect of T3. There is, hence, reason to believe that the transport of choline in the choroid plexus is only partly regulated by adrenergic mechanisms acting via Na+-K+-ATPase. The major effect of T3 on the choline uptake may be exerted by a mechanism different from the ATPase activity and not involving adrenergic receptors. PMID- 3004108 TI - Pre- and postjunctional modulation of cholinergic neuroeffector transmission by adenine nucleotides. Experiments with agonist and antagonist. AB - Adenosine, 2-chloroadenosine, AMP, AMPNH2, ADP, ATP, AMPPNP and beta, gamma meATP dose-dependently and reversibly inhibited contractile responses to nerve stimulation in longitudinal plexus-containing muscle preparations of guinea pig ileum. All the purines, except for 2-chloroadenosine, were virtually equipotent and elicited maximal inhibition within 2 min after application. Adenosine deaminase abolished inhibition by adenosine but did not block the effect of 2 chloroadenosine or the adenine nucleotides. During transmural nerve stimulation, the nucleotidase-resistant analogues alpha, beta meADP and AMPPNP significantly depressed acetylcholine release measured by gas chromatography-mass spectrometry. The inhibitory effect of all the purines was competitively, reversibly and at comparable pA2 values antagonized by 8-p-sulphophenyltheophylline. The results indicate that nucleotides per se can inhibit neurotransmission via a prejunctional receptor common to nucleosides and nucleotides. An excitatory effect by all the di- and triphosphate nucleotides was also observed. The excitation was unaltered by atropine, tetrodotoxin and 8-p sulphophenyltheophylline, thus suggesting an action at postjunctional ADP- and ATP-like receptive sites. PMID- 3004109 TI - Distribution of ouabain-binding sites along the dog nephron. AB - To quantify the relative amount of ouabain bound to different segments of the nephron after in vivo injection of the drug, an autoradiographic (ARG) study was carried out. After intrarenal injection of [3H]ouabain (120 nmol kg-1 body wt, 0.9-1.2 Ci mol-1) to intact kidneys of three anaesthetized dogs, 69-89% of renal Na,K-ATPase activity was inhibited. Sodium reabsorption decreased by 21-54%. Sections for ARG were obtained from tissue slices frozen in liquid Freon, freeze dried and embedded in resin. Almost no loss of activity occurred during processing and background activity was negligible after 23-36 days' exposure. The density of [3H]ouabain grains per mu 2 of tubular walls was 3.8 times higher over medullary ascending limbs of Henle's loop (MAL) and distal cortical tubules (DT) as compared to proximal tubules (PT). In terms of tubular length, the grain density of MAL exceeded that of PT by merely 35% since the cross-sectional area of the MAL was only 25% of that of PT. In DT, grain density in terms of tubular length was lower than in PT by 10%. Based on previous estimate of the absolute ouabain-binding capacity in MAL of 60 fmol mm-1 tubule, the ouabain-binding capacity in PT and DT would equal 45 and 40 fmol mm-1, respectively. From composite microphotographs, the relative volume of PT was estimated to be 42% of the total renal volume. This means that 47% of the total renal ouabain-binding sites are localized to PT, whereas MAL and DT together contain 51%. PMID- 3004110 TI - ACTH, cortisol and growth hormone 24-hour profiles in major depressive illness. AB - Circadian rhythms of ACTH, cortisol and growth hormone have been studied in eighteen major depressives (eight unipolar and ten bipolar) as well as in eight normal controls. Both unipolar and bipolar depressed patients secreted more growth hormone than normal men. This hypersecretion occurred during waking hours rather than during sleep. An early sleep GH increase was found in all but one of the normal men, but was absent in seven of the eight unipolar depressed patients, who had instead a presleep increase. No consistent disturbance of the temporal association between sleep onset and GH secretion was found in bipolar depressed patients. Both unipolar and bipolar depressed patients had higher 24 h mean cortisol levels than normal men, but no significant difference was found for 24 h ACTH levels. An early timing of the nadir of ACTH-cortisol secretion which was observed in our depressed patients also suggest that disorders of circadian time keeping may characterize major endogenous depression. PMID- 3004111 TI - Effects of morphine on hypothalamic corticotropin-releasing factor (CRF), norepinephrine and dopamine in non-stressed and stressed rats. AB - The effects of morphine on the hypothalamic corticotropin-releasing factor (CRF), norepinephrine (NE) and dopamine (DA) concentrations were investigated in non stressed and stressed rats. Acutely administered morphine stimulated both the synthesis and release of CRF in the hypothalamus, thereby activating the pituitary-adrenocortical system in non-stressed rats, but inhibited the stress induced CRF synthesis and ACTH-corticosterone secretion. Either a morphine or ether-laparotomy stress reduced NE and DA concentrations in the hypothalamus. A pretreatment with morphine inhibited the stress-induced reduction in the hypothalamic NE and DA concentrations, and induced a significant increase in the DA concentration. These observations suggest that hypothalamic NE and DA are involved in morphine-induced changes in hypothalamo-pituitary-adrenocortical (HPA) activity and that endogenous opiates have a role in regulating CRF secretion by interacting with hypothalamic biogenic amines. PMID- 3004112 TI - Regional difference in depolarization-elicited accumulation of cyclic AMP in cobalt-induced epileptic cortex of the rat. AB - The appearance of epileptiform discharges in electrocorticograms was induced by a unilateral injection of CoCl2 solution into the sensorimotor cortex of rats. Accumulation of cyclic AMP elicited by ouabain or a high concentration of potassium ions was determined in slices from different cortical areas of rats 9 or 10 days after the injection. In the anterior cortex, the depolarization elicited accumulation of cyclic AMP was significantly higher in the cortical area ipsilateral to the injection site than in the contralateral cortical area. In the posterior cortex, a similar but not significant difference in the accumulation of cyclic AMP was noted. PMID- 3004114 TI - Alcohol abstinence in alcoholic liver disease. PMID- 3004113 TI - Converting enzyme inhibition in mild and moderate essential hypertension. I. Acute effects on blood pressure, the renin-angiotensin system and blood bradykinin after a single dose of captopril. AB - The acute effects of 25 mg captopril on blood pressure, heart rate, components of the renin-angiotensin system and blood concentration of bradykinin were followed in a single-blind placebo study of untreated (group A, n = 15) and thiazide treated (group B, n = 13) patients with mild or moderate essential hypertension. A drug-related fall in blood pressure was seen in both groups. The blood pressure reduction was more marked in group B than in group A. Heart rate remained unchanged. Plasma concentrations of angiotensin II decreased significantly with concurrent increases in plasma concentrations of renin and angiotensin I, indicating the in vivo inhibition of converting enzyme. Blood concentrations of bradykinin showed no systemic changes. The magnitude of blood pressure reduction was correlated both with the pretreatment levels and the concurrent decreases in plasma angiotensin II. Inhibition of angiotensin II formation can explain a large part of the acute hypotensive pharmacological action of captopril. Other vasoactive systems may be involved. The kallikrein-kinin system does not appear to participate as indicated by the unchanged concentrations of kinin in blood. PMID- 3004116 TI - Neurosurgical treatment of Cushing's disease in children and adolescents. AB - The authors report on 15 unselected consecutive children and adolescents treated for hypothalamo-pituitary Cushing's disease by transsphenoidal sella exploration and microadenomectomy. The diagnosis was established by dynamic endocrine testing. Peri- and post-operative measurements of ACTH levels were used to monitor the effectivity of the surgical procedure. In one patient no microadenoma was found. Hypercortisolism was corrected in 13 of the 15 patients. Although transient secondary adrenocortical insufficiency occurred in all of the successfully operated patients, no permanent damage of the anterior or posterior pituitary was found by postoperative endocrinological follow-up testing. One patient died of pneumonia as a direct consequence of surgery. PMID- 3004115 TI - Intradiscal collagenase for treatment of lumbar disc herniations. A comparison of clinical results and computed tomography follow-up. AB - In a series of 34 patients with herniated lumbar discs, treated by intradiscal injection of highly purified collagenase, the post-treatment course has been followed-up clinically and by repeated computed tomographies (CT). Good or excellent results have been achieved in 17 patients. An only slight improvement of pain was noted in 2 patients. Fifteen patients had to be operated on due to not improved or worsened clinical symptoms. The most striking result of our CT follow-up was a tendency of the disc herniation to increase initially after collagenase injection. About two thirds of the patients had such an increase at the one week after injection control. After 6 weeks this rate had decreased to only about one quarter, but in the meantime 13 patients had to be operated. Only after 6 months most hernias of the up till then not operated patients were smaller and none were larger than before treatment. There was also a transient density decrease of the treated disc, most pronounced one week after collagenase injection. At controls 6 months later density had reached again pre-treatment levels. It is likely that the volume increase tendency of the disc material after collagenase injection is responsible for a worsening of the clinical symptoms, which not seldomly occurs during the initial post-treatment period, and in some patients makes an operation necessary. PMID- 3004117 TI - Pathology of midline brain tumors. Immunocytochemical tumor markers and classificatory aspects. AB - Our understanding of the pathology of human brain tumors has considerably increased in recent years thanks to investigation of nervous tissue markers which can be demonstrated by immunocytochemical techniques in sections of neurosurgical tissue specimens. This development is presented here in a review of the most important entities among midline brain tumors. Detection of neural differentiation markers such as glial fibrillary acidic protein (GFAP), S 100 protein (S 100 p), neurofibrillary proteins and neuron-specific enolase (NSE) greatly contributed to clarifying the histogenesis and differentiation potential of undifferentiated small-cell tumors grouped as primitive neuroectodermal tumors (PNETs) which include cerebellar medulloblastomas and pinealoblastomas as well as neuroblastomas and ependymoblastomas. Among medulloblastomas, the desmoplastic variety (formerly called by some "arachnoidal sarcoma of the cerebellum") shows frequent glioneuronal differentiation. Monoclonal antibodies recognizing antigenic determinants specific for tumor types and grades of malignancy/differentiation will gain significance in brain tumor diagnosis, as demonstrated in some examples. Despite the development of more objective ("scientific") criteria for typing human brain tumors, the "art" of classical histopathologic evaluation is not replaced but supplemented by the wealth of data supplied by such modern accomplishments. PMID- 3004118 TI - Assay of succinate-cytochrome c reductase in human brain. PMID- 3004119 TI - The Blym oncogenes. PMID- 3004120 TI - Retrovirus-induced acquired immunodeficiencies. PMID- 3004121 TI - The molecular action of platelet-derived growth factor. PMID- 3004122 TI - Cooperation between multiple oncogenes in rodent embryo fibroblasts: an experimental model of tumor progression? PMID- 3004123 TI - Advances in nonisotopic immunoassay. PMID- 3004124 TI - Oncogenes. AB - Many of the genes that are likely participants in the pathogenesis of human neoplasia have been identified. The major classes of events that activate these genes in tumors have also been described. We expect that continuing research on the function of oncogenes will greatly inform our understanding of fundamental growth control processes and, eventually, influence our approaches to treating cancer patients. PMID- 3004125 TI - Myoclonic encephalopathy of infants. PMID- 3004126 TI - Urea myoclonus: possible involvement of glycine. PMID- 3004127 TI - Leukotrienes and related compounds. PMID- 3004128 TI - Leukotriene B4 stimulates phospholipase-C-mediated formation of inositol trisphosphate in neutrophils: implications for convergence of the phospholipase A2 and C pathways. PMID- 3004129 TI - Pathways of phosphoinositide metabolism in human platelets. PMID- 3004130 TI - Regulation of inositol phospholipid metabolism and arachidonic acid release: inhibitory mechanism of arachidonic acid release by prostaglandin E2 and cAMP. PMID- 3004131 TI - Ca2+ requirement in hydrolysis of phosphatidylinositol-4,5-bisphosphate in human platelets. AB - The activity of partially purified phospholipase C from human platelets was totally dependent on Ca2+, and approximately 800 microM Ca2+ was required for half-maximal activity. The enzyme hydrolyzed endogenous substrates in the order DPI greater than TPI greater than PI in a Ca2+-dependent manner. Hydrolysis of TPI in thrombin-stimulated platelets was dependent on the amount of the agonist, and it was not affected by the presence or absence of extracellular Ca2+. Hydrolysis was inhibited by preincubation with Quin-2AM in the absence of extracellular Ca2+. The intracellular Ca2+ concentration was significantly lowered below the basal level by such treatment. These observations suggested that TPI breakdown in thrombin-stimulated platelets is mediated by agonist receptor coupling and requires at least the basal level of intracellular Ca2+. PMID- 3004132 TI - Biosynthesis of leukotriene A4 from 5-hydroperoxyeicosatetraenoic acid: possible involvement of 8-lipoxygenase reaction. PMID- 3004134 TI - Modulation of arterial cholesteryl ester metabolism by prostacyclin and prostaglandin E2. PMID- 3004135 TI - Measurement of leukotriene B4 in vitro and in vivo by radioimmunoassay. AB - The concentration of LTB4 in in vitro incubations of leukocytes and in experimentally-induced inflammatory exudate can be determined reliably by direct RIA; i.e., extraction and chromatography of samples prior to RIA are not mandatory. It is probable that the RIA for LTB4 is also suitable for monitoring the levels of LTB4 in clinical samples (e.g., synovial fluid, blister fluid from involved psoriatic skin). PMID- 3004133 TI - Hydroperoxide availability in the regulation of the arachidonate cascade. PMID- 3004136 TI - Antiinflammatory effects of eicosapentaenoic acid: relevance to icosanoid formation. PMID- 3004137 TI - Possible mechanisms of antithrombotic effect of eicosapentaenoic acid: species specificity. PMID- 3004138 TI - Biological and chemical implications of a diet high in eicosapentaenoic acid: its influence on hemostasis and prostaglandin metabolism. PMID- 3004139 TI - Effects of eicosapentaenoic acid on hemostatic function and serum lipids in humans. PMID- 3004140 TI - Aza-substituted omega-side-chain modifications of 7-oxabicyclo[2.2.1]heptane thromboxane A2 receptor antagonists: structure-activity relationships. PMID- 3004141 TI - Chemical and enzymatic syntheses of lipoxin A: stereochemical assignment of natural lipoxin A and its possible biosynthesis. PMID- 3004142 TI - Subclass-specific receptors for sulfidopeptide leukotrienes. AB - Separate receptors for LTC4 and LTD4 have been identified and characterized by physiologic and radioligand binding criteria. LTD4 receptors appear to be plasma membrane-associated and to display characteristics previously described for many hormone receptors, such as inhibition of agonist binding by guanine nucleotides and Na+, suggesting a postreceptor linkage to adenylate cyclase of an inhibitory nature. LTC4 receptors have a substantial subcellular distribution; agonist binding to the receptor is not influenced by monovalent ions or guanine nucleotides but is enhanced by divalent cations. Within these two major subclass receptors with specificity for LTC4 and LTD4, respectively, there is an additional heterogeneity in terms of affinities. It is considered likely that a third receptor mediates the distinct actions of LTE4 on a guinea pig tracheal spirals, where this agonist exhibits greater contractile potency than LTD4 and LTC4 and elicits hyperreactivity to histamine that is not a nonspecific response to a prior contraction. PMID- 3004143 TI - Infectious-inflammatory environment alters prostaglandin regulation of cyclic AMP levels in human peritoneal macrophages. PMID- 3004144 TI - Inhibition of DNA synthesis and cell cycle by prostaglandins independent of cyclic AMP. PMID- 3004145 TI - Metabolism of leukotriene A4 in the rat kidney and isolated glomeruli. PMID- 3004146 TI - Peptide stimulation of cytochrome P-450-related arachidonate metabolism in renomedullary cells: formation of Na+ -K+ ATPase inhibitor. PMID- 3004147 TI - Localization of prostaglandin bindings in the central nervous system. PMID- 3004148 TI - Interaction between prostaglandin and purine systems on neurotransmission in the kidney. PMID- 3004149 TI - High-performance anion-exchange chromatography of leukotrienes. PMID- 3004150 TI - Quantitative analysis of leukotriene B4 in human serum. PMID- 3004151 TI - Evidence for leukotriene B4 receptors in human neutrophils. AB - [3H]LTB4 binds concentration-dependently to intact human PMNs. The binding is saturable, reaches equilibrium in 10 min at 4 degrees C, and is readily reversible. Mathematical modeling analysis reveals biphasic binding of [3H]LTB4, indicating two discrete populations of binding sites. The high-affinity binding sites have a dissociation constant of 0.46 X 10(-9) M and a Bmax of 1.96 X 10(4) sites per neutrophil; the low-affinity binding sites have a dissociation constant of 541 X 10(-9) M and a Bmax of 45.16 X 10(4) sites per neutrophil. Competitive binding experiments with structural analogues of LTB4 demonstrate that the interaction between LTB4 and the binding site is stereospecific and correlates with the relative biological activity of the analogues. At 25 degrees C, [3H]LTB4 is rapidly dissociated from the binding site and metabolized to 20-OH- and 29 COOH-LTB4. Purification of neutrophils in the presence of 5-lipoxygenase inhibitors significantly increases specific [3H]LTB4 binding, suggesting that LTB4 is biosynthesized during the purification procedure. These data suggest that stereospecific binding and metabolism of LTB4 in neutrophils are tightly coupled processes. PMID- 3004152 TI - Up-regulation of prostaglandin D2 receptors by agonist binding to beta adrenoceptors in rabbit platelets. PMID- 3004153 TI - The renal collecting tubule as a model for examining the mechanism of action of prostaglandins: interactions between prostaglandin E2 and vasopressin. PMID- 3004154 TI - Regulations of transmitter release by prostaglandins on vascular and tracheal smooth muscle tissues. PMID- 3004155 TI - Platelet-activating factor-binding protein in human serum. AB - The occurrence of a kind of protein (PBP) with stronger PAF-binding ability than albumin was shown in human serum. The molecular weight of PBP was approximately 160K to 180K. PBP seemed to be a pentamer composed of five 32K subunits. PAF bound to PBP revealed platelet aggregation and was resistant to acetyl-hydrolase. PAF synthesized in neutrophils stimulated with the ionophore A23187 was released by PBP from cells. PMID- 3004156 TI - Beneficial effects of (RS)-2-methoxy-3-(octadecylcarbamoyloxy)propyl 2-(3 thiazolio)ethyl phosphate, a specific PAF antagonist, in endotoxin and anaphylactic shock. PMID- 3004157 TI - Selected animal herpesviruses: new concepts and technologies. PMID- 3004158 TI - Pathobiology of animal platelets. PMID- 3004159 TI - The pathology of prosimians, especially lemurs. PMID- 3004160 TI - Reactive oxygen species and drug therapy for inflammatory diseases. AB - To understand drug action on R.O.S., it helps to distinguish effects on the one hand that are anti-radical/anti-peroxide etc. (where a drug such as phenylbutazone may be fairly potent) from other effects that are anti lipoperoxide. The same drug may show variable activity, depending on conditions pertaining e.g. 'peroxide tone', what activates cellular R.O.S. production etc. We still lack useful/acceptable clinical criteria to effectively monitor the performance of antioxidants in vivo. Without these, we cannot formulate guidelines for adding antioxidants to (or withdrawing them from) the overall therapy for sustained inflammatory disorders. PMID- 3004162 TI - [Calpain and alpha-crystallin in the lens of cac mouse]. PMID- 3004161 TI - [The biosynthesis of leukotriene B4 in human cataractous lens and in bovine lens]. PMID- 3004163 TI - [Studies on cytomegalovirus endophthalmitis. Virus isolation and immunohistological studies after intraperitoneal and intraocular inoculation]. PMID- 3004164 TI - [Studies on cytomegalovirus endophthalmitis. Histopathological studies after intraocular inoculation of guinea pig cytomegalovirus]. PMID- 3004165 TI - [Extrarenal nephroblastoma: report of a case and review of the literature]. AB - A 3-year-old girl with an extrarenal nephroblastoma arising from the right retroperitoneal space is described. She was admitted to our hospital with the chief complaint of abdominal pain. On physical examination, she was found to have a large (12 X 10 cm in size), firm and nontender mass in the right upper quadrant of the abdomen. The mass did not extend beyond the midsagital line. The physical examination did not reveal any particular findings or any congenital anomalies. Urinalysis and hematological data were within the normal limit. Radiological examinations including CT scan showed that the solid tumor was related to the right kidney. Under the diagnosis of right nephroblastoma, 15 micrograms/kg/day of actinomycin D was given intravenously for 5 days from October 18, 1982. The regression rate of the tumor was 78 percent on CT scan after chemotherapy. On November 22, 1982, transperitoneal nephrectomy was performed through a right paramedian incision. The tumor was found to adhere tightly to the upper pole of the kidney. The surgical specimen was 76 g in weight. A section of the surgical specimen showed an extrarenal tumor located completely outside the kidney and separated from the renal cortex by a thickened renal capsule. Histological diagnosis was extrarenal nephroblastoma showing renal capsular invasion by epithelial tumor cells. No teratomatous components were encountered in the tumor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004166 TI - [A case of malignant paraganglioma within the renal sinus]. AB - A case of malignant paraganglioma within the renal sinus is presented. A 51-year old woman underwent left transabdominal nephrectomy for renal tumor. Pathologically, the tumor was malignant nonchromaffin paraganglioma. This tumor seemed to originate from parasympathetic paraganglions around the left renal artery. Paraganglioma is rare in the literature. Especially, this may be the first report of such a lesion in the renal sinus. PMID- 3004167 TI - [A case of signet ring cell carcinoma of the urinary bladder]. AB - A 60-year-old female complained of gross hematuria and urinary frequency on November 27th, 1982. Cystoscopic examination revealed papillary invasive tumor around the bladder neck and a transurethral biopsy showed signet ring cell carcinoma. Since there was no adenocarcinoma in any other organs, we diagnosed it as primary signet ring cell carcinoma of the urinary bladder. Total cystectomy with ileal conduit and post-operative irradiation were performed, but she died on May 13th, 1983. We summarize 16 cases of primary signet ring cell carcinoma of the urinary bladder including this case and discuss this rare condition. PMID- 3004169 TI - [Facilitation of the onset of tetanic responses induced by 4-methylesculetol in amphibian isolated neuromuscular preparations]. AB - Previous observations have shown that the 4-Methylesculetin (4ME) and the calcium provoke an increased response when the motor somatic and autonomic nerve-endings are stimolated. In the present research, we have studied the influence of the bioflavonoid and of an increased medium [Ca++] on the tetanic responses obtained from the isolated frog "sciatic nerve-gastrocnemius muscle" preparation. The 4ME has always facilitated the beginning of a complete tetanic response and has also frequently increased the developed mechanical tension. These effects appeared more evident when the bioflavonoid was employed together with the ascorbic acid. Moreover, an increased medium [Ca++] has induced similar effects. The nifedipine and the flunarizine, well-known Ca++ -antagonists, have constantly reduced or even abolished the facilitating effect induced by the bioflavonoid. On the basis of these results, we could hypothize that these observed facilitating effects are dependent on both an increased Ach-release and an increased muscle mechanical efficiency. Our previous results and also some literature data suggest that these effects could be due to an increased Ca++-passage through the cell membrane of the motor nervendings and of the muscle fibres too. PMID- 3004168 TI - Comparative study of mechanical responses of hepatic arteries strips to adrenalin in presence of pyridoxine and pyridoxal-5'-phosphate. AB - Since it is known that PLP inhibits "in vitro" the COMT much more than pyridoxine; the influence of pyridoxine to the response of hepatic arteries isolated to AD has been compared to the influence of PLP to the same arteries. As for as the increase in percent is concerned, the result is that PLP gives rise to a greater answer to AD than pyridoxine. Such effects lacked when pyrogallol, a powerful COMT inhibitor, was present. Taking such results as a basis, it has been concluded that the mechanism of action of pyridoxine and of PLP was metabolic and that it was based on the COMT inhibition, even for the hepatic arteries. It was been also deduced that the higher efficiency of PLP compared to the pyridoxine's was due to its greater capacity to inhibit the COMT. PMID- 3004170 TI - Plasma vitamin C (ascorbic acid) levels in asthmatic children. AB - Plasma concentration of ascorbic acid was determined in fifty-one asthmatic children and a group of matched controls. The mean ascorbic acid level of 0.54 mg/100 ml among the asthmatics was significantly lower than a mean of 0.84 mg/100 ml for controls (P less than 0.001). Ascorbic acid level was directly related to the socio-economic class (SC) since asthmatic children from SC I, II and II had significantly higher ascorbic acid levels than those from SC IV and V. There was however, no relationship between the plasma ascorbic acid level and atopy, frequency of asthmatic attacks over the previous 12 months and the duration of asthma. It is postulated that if plasma ascorbic acid level was related to the susceptibility to viral respiratory tract infections, the observed low level of the vitamin in the asthmatics would make them more liable to such infections which are capable of precipitating acute asthmatic attacks. Confirmation of our results would indicate the need for regular ascorbic acid supplement in some children with bronchial asthma. PMID- 3004171 TI - Hypersensitivity to paw-paw (Cerica papaya): report of a case. AB - A 23-year-old female presented with classical features of immediate hypersensitivity reaction after contact with paw-paw (Cerica papaya). She had a total serum IgE of 2500 i.u./ml. Prick tests with paw-paw extracts were positive. Her serum gave a positive P-K test on her siblings. PMID- 3004172 TI - Management of the lost IUD. AB - Records of twenty-six patients who presented at the University College Hospital, Ibadan with 'lost' IUDs between 1 July 1980 and 30 April 1982 were analysed. During the 22-month study period there were 3476 IUD insertions. Routine evaluation showed that eighteen IUDs (69.2%) were intrauterine whilst eight IUDs (30.8%) were extrauterine. Seventeen (65.4%) intrauterine devices were recovered by routine D and C. Of the eight extrauterine devices, six (23.1%) were removed by laparoscopy, one (3.8%) was removed by laparotomy and one (3.8%) was removed by colpotomy. It is concluded that it is preferable for all extrauterine devices to be removed in order to discourage psychosomatic symptomatology commonly associated with forgotten devices. PMID- 3004173 TI - Social, cultural and economic factors in the management of diabetes mellitus in Nigeria. AB - The problems of caring for diabetic patients in the developing world have been getting increasing attention recently. While the focus has often been on peculiar clinical characteristics such as pancreatic diabetes, malnutrition diabetes, J type and other similar forms, there is a need to explore further the impact of the prevailing social, cultural and economic milieu on the patients and the management of their disease. The socio-economic status of 147 Nigerian diabetic patients was assessed by the use of an interview format and also their knowledge, attitudes, beliefs, expectations and concerns about diabetes was determined. The study confirmed that most of the patients are illiterate, poor and with very little understanding of the nature of their disease. Their greatest concern was the impact of diabetes on their ability to work and the extreme difficulty in getting their medications regularly and at affordable prices. A survey of twelve pharmacies revealed that many of the oral hypoglycemic agents, insulin, syringes, needles and materials for urine testing are not stocked by most of them. Some suggestions have been made as to possible solutions which will involve efforts on the parts of health care providers, patients, governments, pharmacies and private organizations in these countries and the international community as well, including the international drug companies. It is hoped that the problem will remain high on the agenda of all those concerned about diabetes and the care of diabetic patients throughout the world. PMID- 3004174 TI - Reproductive performance following active management of diabetic pregnancies at the University College Hospital, Ibadan, Nigeria. AB - This article presents a report of the first 5 years of an active approach to the management of diabetic pregnancies at the University College Hospital, Ibadan. During this period the incidence rate of diabetic pregnancy was 0.64 per 1000 deliveries per year. The mean birthweight (3.40 +/- 1.68 kg) of babies whose mothers had good diabetic control was lower than the mean birthweight (4.19 +/- 1.05 kg) of babies born to mothers with poorly-controlled diabetes. The difference was however not statistically significant (P less than 0.5). Whilst the overall perinatal mortality rate was 10.8%, there was a statistically significant difference in the perinatal mortality associated with poor control of diabetes (42.9%) when compared with good control (0.0%): P less than 0.02. The authors conclude that earlier booking in pregnancy, stricter control of diabetes by multiple insulin injections and improved cooperation of the patients will in future help to lower the perinatal mortality rate associated with diabetic pregnancy. PMID- 3004175 TI - Serum alpha 1-antitrypsin levels in asthmatic children. AB - Serum levels of alpha 1-antitrypsin (AAT) were determined by an enzymatic assay method in fifty-five asthmatic children and in the same number of controls. The mean AAT level was significantly lower in asthmatics (1.65 mumol/min/ml) than in controls (2.0 mumol/min/ml) (P less than 0.02). A significantly higher proportion of asthmatics than controls (P less than 0.05) had levels below 2.1 mumol/min/ml which is the lower limit of normal, thus suggesting a higher prevalence of partial (heterozygous) AAT deficiency in the asthmatics. There was no relationship between the mean AAT levels and age, duration of asthma or frequency of asthmatic attacks. Although there is some controversy about the relationship between heterozygous AAT deficiency and pulmonary disease, severe (homozygous) AAT deficiency has been linked with emphysema which is also a complication of asthma. There was however, no evidence of emphysema in either the asthmatic child or the control who had no detectable serum AAT. There were three asthmatics whose chest radiographs showed hyperinflation, but had a mean AAT level that was not significantly different from that in those without such changes. Further studies, including phenotype determination in a larger group of asthmatic children, are required in order to determine the prevalence of both homozygous and heterozygous AAT deficiencies which may be risk factors in the development of emphysema and other pulmonary complications of asthma. PMID- 3004176 TI - Single dose treatment of gonococcal urethritis with augmentin in Ibadan. AB - Augmentin, a new orally absorbed broadspectrum antibacterial agent comprising of amoxycillin trihydrate and potassium clavulanate, was investigated in the treatment of gonococcal urethritis in Ibadan, Nigeria, where penicillinase producing Neisseria gonorrhoeae (PPNG) constitute about 80% of the circulating strains of gonococci. Two different formulations of the agent were employed in the study. The first formulation consisting of 3.0 g amoxycillin and 125 mg clavulanic acid, achieved a cure rate of 75% (i.e. eighteen out of twenty-four patients) among PPNG infections, but 100% cure rate among nine patients with non PPNG infections. The second formulation consisting of 3.0 g amoxycillin and 250 mg clavulanic acid, had a cure rate of 86% (i.e. fifty-seven out of sixty-six patients) among PPNG infections, and 91% (i.e. ten out of eleven patients) among non-PPNG infections. Clavulanic acid appears to potentiate and enhance the activity of amoxycillin against the beta-lactamase produced by the gonococci. Augmentin seems to be a good and acceptable agent for the treatment of gonococcal infections, in this environment and further studies on its efficacy are therefore justified, such as the simultaneous administration of probenecid. PMID- 3004177 TI - Study of incidence of post-operative pain among Nigerian patients. AB - The incidence of post-operative pain was determined among 200 adult Nigerian patients who presented for a variety of surgical procedures. The overall incidence of moderate to severe post-operative pain was 68% while the remaining 32% complained of only mild pain. When the results were categorized according to the site and type of surgery, the highest incidence of pain were found following surgery of anorectal region (90%), thoracotomy (88%), major joints (88%) and upper abdomen (80%). The use of opiates pre-operatively for premedication and anaesthetic techniques using intra-operative opiates or ketamine delay the onset and reduce the severity of post-operative pain. No sex difference was observed but elderly patients (especially above 50 years of age) tended to have lower pain ratings. PMID- 3004178 TI - Burkitt's lymphoma in South Ethiopia. AB - In a retrospective study eleven cases with Burkitt's lymphoma have been diagnosed at three rural hospitals in the Sidamo and Gamu Gofa Regions of South Ethiopia. The clinical picture is heterogeneous with an abdominal mode of presentation being most frequently observed. It is concluded that South Ethiopia has an incidence of Burkitt's lymphoma between that of endemic countries (e.g. Uganda) and non-endemic countries such as the USA. PMID- 3004180 TI - Plasma renin activity and plasma aldosterone concentrations in normotensive pregnant Nigerians. AB - Plasma renin activity (PRA) and plasma aldosterone concentrations were determined in thirty-four normotensive pregnant subjects, sixteen subjects who were examined 6 weeks post-partum and sixteen non-pregnant controls. Plasma renin activity and plasma aldosterone concentrations increased sequentially in pregnancy i.e. there were progressive significant increases when the values of the first, second and third trimesters were compared. The values for the post-partum subjects and the non-pregnant controls were not significantly different. There was no significant correlation between any pair of the following indices: mean arterial pressure (MAP), PRA and plasma aldosterone. The results are discussed in the light of the present state of knowledge of the pathogenesis of pregnancy-induced hypertension (PIH). PMID- 3004179 TI - Changes in blood chemistry and liver histopathology of rabbits during experimental tetanus toxicity. AB - Changes in blood chemistry, especially blood glucose, were studied in rabbits developing tetanus after injection of tetanus toxin. Blood glucose levels increased significantly above control values after the animals developed tetanus. The increase in glucose level paralleled the depletion of liver glycogen, detected by means of PAS staining. The observed changes were not affected by adrenergic receptor block or catecholamine depletion by reserpine, suggesting non mediation of the sympathetic nervous system in the response. A direct action of the toxin on the liver, initiating glycogen depletion, is being postulated. PMID- 3004181 TI - Preliminary report on the in vitro antibacterial activity of Bryophyllum pinnatum leaf juice. AB - The juice from the leaves of Bryophyllum pinnatum S. Kurtz (Crassulaceae) was tested for antibacterial activity. The extract at 5% v/v was bactericidal to a wide spectrum of Gram-positive and Gram-negative bacteria such as Bacillus subtilis, Staphyllococcus aureus, Streptococcus pyogenes; Streptococcus faecalis; Escherichia coli; Proteus spp; Klebsiella spp; Shigella spp; Salmonella spp; Serratia marcescens; and Pseudomonas aeruginosa including the clinical isolates of these organisms possessing multiple antibiotic resistance. PMID- 3004183 TI - Hepatoblastoma. PMID- 3004182 TI - Uptake of 111In-labeled leukocytes by tumor. AB - The 111In-labeled leukocyte scan is a sensitive and specific technique for the detection and localization of abscesses. However, in reviewing an unselected series of 249 scans, six (2.3%) were false-positive cases in which leukocyte uptake by tumor mimicked an abscess. This represented 12% (6/51) with known tumor at the time of imaging. Five of the cases were primary or metastatic tumors to the soft tissues; the sixth was a skeletal metastasis. The intensity of tumor activity has been characterized as mild in the few cases of leukocyte uptake reported in the literature, suggesting that the degree of uptake is helpful in distinguishing tumor from abscess. In this study, however, variable degrees of tumor-associated activity were seen ranging from mild to very intense. These findings indicate that tumor accumulates labeled leukocytes more frequently than has been previously appreciated, that primary and metastatic neoplasms involving both the soft tissues and skeleton are involved, and that the intensity of uptake is not a reliable criterion to distinguish tumor from abscess. PMID- 3004184 TI - Relative contribution of inotropic and vasodilator effects to amrinone-induced hemodynamic improvement in congestive heart failure. AB - The relative contribution of inotropic and vasodilator effect to amrinone-induced hemodynamic improvement in congestive heart failure (CHF) is unknown. In 9 patients with CHF, the effects of amrinone and nitroprusside on hemodynamic and radionuclide measurements were compared to determine whether reduced afterload accounts for the amrinone-induced decrease in left ventricular end-systolic volume. In each patient, the end-systolic pressure-volume relation was derived using nitroprusside. After terminating nitroprusside treatment, intravenous amrinone (3 mg/kg) caused end-systolic volume to decrease from 148 +/- 32 ml/m2 (mean +/- standard deviation) to 133 +/- 32 ml/m2 (p less than 0.05), causing an increase in cardiac index from 1.9 +/- 0.8 to 2.7 +/- 0.8 liters/min/m2 (p less than 0.001). Arterial end-systolic pressure decreased in all patients during amrinone administration, from 96 +/- 22 to 84 +/- 19 mm Hg (p less than 0.005), as did systemic vascular resistance. Nitroprusside doses needed to match the decrease in LV end-systolic volume induced by amrinone caused significantly greater decreases in arterial end-systolic pressure than did amrinone (p less than 0.01). The amrinone-induced decrease in end-systolic volume exceeded that predicted for a pure vasodilator based on arterial end-systolic pressure and the nitroprusside-derived pressure-volume relation in 6 patients. In 3 patients, the decrease in end-systolic volume did not exceed that expected for a pure vasodilator. In conclusion, after amrinone treatment, afterload reduction occurs in all patients with severe CHF and is the sole effect in some.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004185 TI - Provocation of hyper- and hypokalemic sudden death during treatment with and withdrawal of converting-enzyme inhibition in severe chronic congestive heart failure. PMID- 3004186 TI - Alterations in cardiovascular structure and function with advancing age. AB - The greatest difficulty in studying the effects of aging on cardiovascular structure and function lies in separating the effects of aging itself from those of the inextricably entwined disease processes and life-style changes that accompany aging. Age-related stiffening of the arterial tree results in an increased systolic blood pressure, which appears to impose a greater load on the heart. Probably as an adaptive mechanism to maintain normal wall stress, a modest age-associated concentric left ventricular hypertrophy develops, which approximates 30% between ages 25 and 80. The decrease in mitral valve E-F slope and increase in the left atrial dimension, which are seen with advancing age, may be construed as the consequences of a thicker-walled, less compliant left ventricle. Systolic left ventricular function, measured at rest either by echocardiography or radionuclide techniques, is unaffected by aging. Aerobic exercise capacity, whether measured as total work performance or maximal oxygen consumption declines with age, although in subjects who maintain a high level of physical activity, the decline appears to be approximately half of the 10% per decade decrease seen in sedentary persons. An age-related decline in maximal exercise heart rate has been a universal finding. Although several studies over the past 4 decades have found cardiac output to decrease with age, both at rest and during exercise, a recent study of subjects carefully screened to exclude latent coronary artery disease found no such decline in cardiac output. In these subjects, the age-related decline in maximal heart rate and systolic emptying at peak exercise was offset by an increased utilization of the Frank-Starling mechanism. An attractive hypothesis for explaining the hemodynamic profile of a decreased maximum heart rate, increased preload and decrease in ejection fraction at maximal effort, despite elevated norepinephrine levels, is an age-associated, diminished end-organ responsiveness similar to beta-adrenergic blockade. PMID- 3004188 TI - Increasing starch intake in the human diet increases fecal bulking. AB - Fecal bulking occurs through water holding by dietary residue as well as by enhanced bacterial mass as a result of bacterial utilization of dietary fiber. In this study, increasing the energy intake of human subjects by supplementing the carbohydrate content of the diet with corn starch increased fecal weights and fecal nitrogen content. This indicates that the carbohydrate content, more specifically the starch content, of the diet influences fecal bulking by possibly increasing available substrates for colonic bacterial proliferation which are dependent largely on undigested fiber in the colon. This may explain why high fecal weights occur in the tropics on comparable intakes of dietary fiber but on high starch intakes. PMID- 3004187 TI - Effectiveness of converting enzyme inhibition (enalapril) for mild congestive heart failure. AB - The present study investigates the effectiveness of converting enzyme inhibition (CEI) on cardiac performance of patients with congestive heart failure (New York Heart Association functional class II). Outpatients (n = 12) were treated with enalapril, 5 to 10 mg twice daily, in addition to stable doses of digitalis and diuretic drugs. Before and after 4 and 12 weeks of treatment a treadmill exercise test and echocardiography were performed. Maximal oxygen uptake and exercise tolerance increased significantly and mean arterial pressure at rest and on exertion decreased significantly. Heart rate did not change. Left ventricular end diastolic diameter decreased significantly. Serum angiotensin converting enzyme activity was reduced to nearly 0; plasma renin concentration, which was already elevated, increased further. Plasma norepinephrine levels did not change significantly. Treatment was tolerated well by all patients. CEI decreased preload and afterload, suggesting that they might have had an inappropriately elevated arteriolar and venous tone owing to a moderately stimulated renin angiotensin system and sympathetic nervous system. These conditions may lead to further deterioration of cardiac performance. By means of CEI one may be able to interrupt these pathogenetic mechanisms, relieving the already damaged heart from inappropriate elevations of preload and afterload and delaying or even preventing further deterioration of cardiac performance. PMID- 3004190 TI - Comparison of two commercial enzyme-linked immunosorbent assays for detection of herpes simplex virus antigen. AB - Two commercial enzyme-linked immunosorbent assays (ELISA) for detection of herpes simplex virus (HSV) antigen in direct clinical specimens were compared with cell culture in primary rabbit kidney and MRC-5. With a total of 238 specimens, the sensitivity and specificity of the Dako ELISA method were determined to be 70.1% and 82.4%, respectively, and 60.4% and 88.2%, respectively, for the Ortho Antigen ELISA. PMID- 3004192 TI - Receptor repartee redux. PMID- 3004191 TI - Acid-fast bacilli with cytomegalovirus and herpesvirus inclusions in the skin of an AIDS patient. AB - A 66-year old acquired immunodeficiency syndrome (AIDS) patient presented with cutaneous lesions suspicious for Kaposi's sarcoma. Biopsies disclosed granulomatous infiltrates with acid-fast bacilli and cytomegalovirus inclusions within macrophages and endothelial cells in one biopsy. Herpesvirus vesicle, necrotizing folliculitis, and vasculitis were observed in a second biopsy taken concurrently. These findings emphasize the polymorphous presentation of infectious disorders in AIDS and the need for multiple biopsies and for work-up with special stains in these patients. PMID- 3004189 TI - The effect of diet on bile acid kinetics and biliary lipid secretion in gallstone patients treated with ursodeoxycholic acid. AB - Effects of specific dietary alterations in patients with radiolucent gallstones treated with ursodeoxycholic acid (UDCA, 750 mg at bedtime) were investigated. Patients were allocated randomly to one of four diets: standard (500 mg cholesterol/day), low-cholesterol (250 mg/day), added-bran (30 g/day), or substituted medium-chain triglycerides (MCT) oil (20% of fat). Dietary intake and good compliance were verified by computerized analysis of dietary diaries. Bile acid kinetics (26 patients) or secretion of biliary lipids (23 other patients) were determined at enrollment and at 6 and 9 mo, respectively, during treatment. Although MCT further decreased the UDCA-induced decrease in the synthesis of chenodeoxycholic acid, it did not lessen desaturation of bile. Otherwise, compared to the standard diet, no experimental diet significantly altered the UDCA-induced increase of the pools of total bile acids and UDCA or the UDCA induced decrease in synthesis of bile acids and in biliary secretion or saturation of cholesterol. If these dietary manipulations facilitate dissolution of gallstones by UDCA, they do so by other mechanisms. PMID- 3004193 TI - Congenital cytomegalovirus infection. Information for educational personnel. PMID- 3004194 TI - Wilms' tumor detection in patients with sporadic aniridia. Successful use of ultrasound. AB - Eleven patients with sporadic aniridia have been followed up for periods of time ranging from eight months to ten years. The initial renal evaluation included an intravenous pyelogram and ultrasound. Ultrasound evaluation was then performed every four to six months until 8 years of age. After 8 years of age, evaluation was performed every six to 12 months. Two of 11 patients were found to have Wilms' tumors. Ultrasound was sensitive enough to find small lesions prior to extension from the kidney. Ultrasound evaluation permits early detection without repeated radiation, sedation, or contrast and offers an easy technique for follow up of patients with sporadic aniridia who have a high likelihood of developing a Wilms' tumor. PMID- 3004195 TI - Ileal loss of available carbohydrate in man: comparison of a breath hydrogen method with direct measurement using a human ileostomy model. AB - The breath hydrogen technique has suggested that a considerable amount of available plus unavailable carbohydrate enters the large intestine after the consumption of starchy foods (white bread 11%, wholemeal bread 8%, and red lentils 18%). Direct measurement of the available carbohydrate in ileal effluent after the consumption of test meals by three individuals with ileostomies gave values similar to those determined by the breath hydrogen technique (white bread 10%, wholemeal bread 8%, and red lentils 22%). These studies confirm that considerable amounts of "available carbohydrate" may be lost to the small intestine and physiologically must be considered as dietary fiber. The implications of this with respect to metabolism and colonic disease remain to be investigated. PMID- 3004196 TI - The effect of dietary guar on serum cholesterol, intestinal transit, and fecal output in man. AB - In six healthy subjects serum cholesterol was unchanged during a 2-wk period of controlled diet (2750 kcal) followed by a 2-wk period of free diet but showed a 16% decrease (p less than 0.05) during a further 2-wk period of controlled diet to which guar (5.7 g bid) was added. Stool weight, frequency and whole gut transit time did not differ in the diet periods with and without guar. Of guar ingested 82-95% was metabolized in the gut. Mouth to cecum transit of a meal measured by the hydrogen breath test in the same subjects was unaffected by the addition of 5.7 g of guar. PMID- 3004198 TI - Demonstration of antibodies to human T-lymphotropic retrovirus type III in lymphoadenopathy syndrome patients and in individuals at risk for acquired immune deficiency syndrome (AIDS) in Italy. AB - An epidemiologic survey of the distribution of lymphoadenopathy syndrome in six Italian cities and its correlation with human T-lymphotropic retrovirus III (HTLV III) is reported. Serum samples of nine patients with acquired immune deficiency syndrome (AIDS) were tested, 180 from patients with lymphoadenopathy and 349 from individuals belonging to groups such as homosexuals, drug addicts, hemophiliacs, and polytransfused considered at increased risk for AIDS. Prevalence of HTLV-III antibodies was 78% in AIDS patients and 61% in 180 patients with lymphoadenopathy syndrome (variation among drug abusers by city from 51% in Cagliari to 87% in Rome). The percentage of positive sera in individuals at risk for AIDS or lymphoadenopathy ranged from 0% in polytransfused to 8.5% in homosexuals, 14% in drug addicts, and 19.5% in hemophiliacs. No positive sera were found among 660 healthy individuals including relatives of patients with AIDS or lymphoadenopathy or in 342 patients suffering from immunologic or infectious diseases. These results strongly suggest that HTLV-III is the causative agent of AIDS and lymphoadenopathy. Since none of the healthy subjects were positive while a substantial percentage of people at risk for AIDS showed antibodies to HTLV-III, it may be presumed that this infection is also prevalent in the Italian population group in which AIDS and lymphoadenopathy are most likely to develop. PMID- 3004197 TI - A strategy for management of the irritable bowel. AB - The irritable bowel is one of the most common complaints facing clinicians. Its management is difficult but a strategy is proposed that is useful in most instances. It is well to remember that most people with these symptoms do not complain to a doctor. The reason for the visit to the physician may, therefore, hold a clue as to the best course of management. The physician must first establish his credibility through a careful history, physical examination, and sigmoidoscopy. He needs to identify the precipitating factors such as dysentery, drugs, diet, emotion, or an important life event. In accordance with the patient's needs, the physician should comfort and reassure. Bran appears to be a safe method of treatment and many are satisfied with it. A follow-up visit is important to ensure compliance and comprehension. The biggest risk of the IBS lies in uncertainty, which generates needless anxiety, costly investigation, fruitless drug side effects, and even the hazards of surgery. If the patient is unimproved at the second visit, one must consider diagnostic alternatives while resisting the temptation to order irrevelant investigative procedures. Continued empathy with the patient is important and certain drugs might be considered either for their placebo effect or because of their effect on a specific complaint such as diarrhea. In the patient who continues to be dissatisfied, the physician must offer continued support and reassurance. Certain referral options may be considered but if possible, the physician should retain the initiative. PMID- 3004199 TI - A comprehensive investigation of immunity to poliomyelitis in a developing country. AB - A comprehensive nationwide surveillance program of serologic immunity of two-year old black children, combined with evaluation of vaccine quality and distribution, was carried out in South Africa during 1983-1984. Sera were randomly collected from urban and rural groups and cluster samples collected from the semi-urban group. The sample represented 0.23% of the total target population. Satisfactory levels of immunity were found in the urban (80%) and semi-urban (71%) groups but a disquietingly low level was found for the rural group (59%). Individual districts in the rural group could be singled out for directed cluster sampling at a later stage. History and documentation of immunization corresponded well to serologic findings and revealed also a fairly substantial level of natural immunization among individuals who, on history, had received no vaccine. Some 95% of random samples of vaccine recalled from the field showed satisfactory levels of potency. An immunity surveillance program such as this is ideally suited and highly cost-effective for developing countries with incomplete immunization to prevent large-scale buildup of immunity deficit. The technique, however, is too insensitive to detect localized community immunity defects. PMID- 3004200 TI - The virology of variola minor. Correlation of laboratory tests with the geographic distribution and human virulence of variola isolates. AB - Several groups of variola isolates were compared in DNA structure, and by four independent biologic markers. Isolates of variola minor from Europe and South America (alastrim virus) could be distinguished from African isolates of variola minor by DNA structure and by two of the four biologic markers. Taken as a group, the properties of African isolates, in general, differed from those of variola major, but this difference was confined to properties which depended (in the laboratory) on the recent history of the virus concerned. The suggestion made previously that there was an "intermediate" or "African" variety of variola virus is discounted. Laboratory tests did not distinguish any individual African isolate from variola major virus. It is concluded that a virus which may be called "alastrim" represents a "fixed" variant of variola virus, whose distribution is consistent with the dramatic spread of variola minor through the Americas and Europe in the early part of this century, and that variola minor in Africa in recent years was due to variola virus which was not alastrim and which laboratory evidence fails to identify as an entity distinguishable from variola major virus. PMID- 3004201 TI - Intracellular survival of Candida albicans in peritoneal macrophages from chronic peritoneal dialysis patients. AB - Candidal peritonitis is a tenacious infection in patients undergoing chronic peritoneal dialysis. Since little is known about host defenses of the human peritoneal cavity against fungi, we investigated the interaction of peritoneal macrophages (PM phi) from uninfected dialysis patients with Candida albicans blastospores. Chemiluminescence (CL) techniques were used to assess the respiratory burst activity of these cells, and candidacidal activity was evaluated with a fluorochrome microassay. In sharp contrast to peripheral blood polymorphonuclear leukocytes (PMNs) from healthy donors, which gave a brisk luminol-enhanced CL response to opsonized blastospores and killed 35% of cell associated organisms, PM phi produced barely detectable luminol-enhanced CL and killed only 13% of intracellular Candida. These findings appeared to be associated with a decreased level of myeloperoxidase in PM phi. The mechanism of intracellular survival of C albicans also appeared to be related to relatively poor triggering of superoxide production during phagocytosis of viable blastospores. The CL response of PMNs to C albicans was opsonin-dependent, and peritoneal dialysis effluent was devoid of opsonic activity. These studies suggest that local cellular and humoral mechanisms of defense are inadequate for protection of peritoneal dialysis patients against candidal peritonitis. PMID- 3004202 TI - Further evidence for the dispersion of the human fibrillar collagen genes. AB - Recombinant DNA probes specific for the human pro alpha 1(II) and pro alpha 1(III) collagen chains have been used for the chromosomal localization of the two genes. Restriction endonuclease analysis of DNA from human-rodent hybrid cell lines in conjunction with in situ hybridization of human metaphasic chromosomes have shown that the gene coding for the pro alpha 1 chain of type II collagen (COL2A1) is located on chromosome 12 in the segment 12q131----12q132. Likewise, the gene coding for the pro alpha 1 chain of type III collagen (COL3A1) was assigned to the segment 2q31----2q323 of chromosome 2. PMID- 3004204 TI - Duplication of the human immunoglobulin heavy chain gamma 2 gene. AB - The five C gamma genes in the human immunoglobulin heavy chain region show nonrandom association and segregation as haplotypes. From the study of genetic variation in C gamma genes of 58 healthy Caucasian volunteers, we have identified a haplotype that involves a duplication of C gamma 2. This haplotype contains both the 13.5-kilobase (kb) and 25-kb BamHI fragment alleles of C gamma 2. In addition, the patterns and relative intensity of BamHI fragments containing C gamma genes were those expected for genomic DNA containing three copies of C gamma 2 for every two copies of the four other C gamma genes. A new EcoRI polymorphism in C gamma 4 was useful in defining the haplotype containing the duplication. Alleles of the C gamma genes in the duplication haplotype, including Gm markers of C gamma 1 and C gamma 3 and DNA polymorphisms of C psi gamma, C gamma 2, and C gamma 4, were consistent with its origin from an unequal crossover between the two common C gamma haplotypes, H1 and H2. This recombinant haplotype, which has been designated H2;1(gamma 2 dup) to reflect its origin, occurred with a frequency of .043 in a random sample of 116 chromosomes. PMID- 3004203 TI - Chromosome 13 homozygosity in osteosarcoma without retinoblastoma. AB - We provide evidence that some human osteosarcomas arise subsequent to the development of homozygosity at loci on the long arm of chromosome 13. The resulting chromosome 13q homozygosity allows the phenotypic expression of any recessive allele on that chromosome. Clinical evidence suggests that it is the retinoblastoma locus within 13q14 that is involved in the formation of these bone tumors. PMID- 3004205 TI - The analysis of multiple polymorphic loci on a single human chromosome to exclude linkage to inherited disease: cystic fibrosis and chromosome 4. AB - Classical linkage programs analyze the segregation of two markers in informative families. When several markers are available for one human chromosome, pairwise analysis can exclude linkage between each marker and an inherited disease. The identification of restriction fragment length polymorphisms has made many new informative markers, assigned to chromosomes, available. We have adapted the multipoint linkage program MLINK developed by Lathrop et al. in order to exclude linkage between cystic fibrosis and several markers known to be on human chromosome 4. The exclusion obtained is greater than that for a pairwise analysis. PMID- 3004206 TI - A deletion map of the human Y chromosome based on DNA hybridization. AB - The genomes of 27 individuals (19 XX males, two XX hermaphrodites, and six persons with microscopically detectable anomalies of the Y chromosome) were analyzed by hybridization for the presence or absence of 23 Y-specific DNA restriction fragments. Y-specific DNA was detected in 12 of the XX males and in all six individuals with microscopic anomalies. The results are consistent with each of these individuals carrying a single contiguous portion of the Y chromosome; that is, the results suggest a deletion map of the Y chromosome, in which each of the 23 Y-specific restriction fragments tested can be assigned to one of seven intervals. We have established the polarity of this map with respect to the long and short arms of the Y chromosome. On the short arm, there is a large cluster of sequences homologous to the X chromosome. The testis determinant(s) map to one of the intervals on the short arm. PMID- 3004208 TI - Implications of hypercalcemia with respect to diagnosis and treatment of lung cancer. AB - This study had two objectives. The first was to determine if hypercalcemia presents as an isolated finding in patients with lung cancer prior to the development of abnormalities on chest radiography. The second was to correlate the presence of hypercalcemia with survival after surgical therapy. A review of clinical material over a seven-year period yielded 67 patients with diagnoses of hypercalcemia and lung cancer. No patient presented with surgically curable lung cancer associated with hypercalcemia. A review of the literature disclosed only four cases in which a putative cure occurred in the presence of hypercalcemia. Review of the clinical material and the literature revealed no instance in which hypercalcemia per se led to the diagnosis of clinically occult lung cancer. Hypercalcemia was almost always associated with large tumor masses. Median survival after the discovery of hypercalcemia complicating carcinoma of the lung was one month. PMID- 3004207 TI - New mutation and prenatal diagnosis in ornithine transcarbamylase deficiency. AB - Ornithine transcarbamylase (OTC) (E.C.2.1.3.3) is an X-linked hepatic enzyme in the urea cycle necessary for ammonia detoxification. Deficiency of OTC results in neonatal hyperammonemia, coma, and death in childhood. Because fibroblasts do not express OTC, prenatal diagnosis in the past has required fetal liver biopsy. Using a complementary DNA (cDNA) for OTC for Southern blot analysis of genomic DNA, we have found probands with complete OTC deficiency from two unrelated families in whom the same TaqI restriction endonuclease site has been altered because of independent, but not necessarily identical, mutations in the OTC gene, suggesting that this site may be a relative hotspot for mutation at a location that is critical for normal gene function. This TaqI alteration has allowed the identification of the individual in each family in whom the mutation originated as well as the exclusion of a recurrence of OTC deficiency in a male fetus at risk for the disease. OTC deficiency joins the growing list of genetic disorders for which Southern blot analysis allows accurate heterozygote detection and prenatal diagnosis in conditions for which they were not previously available. PMID- 3004209 TI - Dermatomyositis without creatine kinase elevation. A poor prognostic sign. AB - Serum muscle enzyme levels are usually elevated in patients with untreated polymyositis and dermatomyositis. Creatine kinase is the muscle enzyme most often used to diagnose inflammatory myopathies. Seven patients with dermatomyositis and normal creatine kinase levels are described. Five of the seven patients had either an associated malignancy or severe interstitial lung disease. The one-year survival of the six patients followed for that length of time was 33 percent. A lack of creatine kinase elevation in patients with dermatomyositis is a poor prognostic sign. PMID- 3004210 TI - Non-glycoside, non-catecholamine inotropic agents in the treatment of congestive heart failure. PMID- 3004211 TI - Digitalis and other positive catecholamine-like inotropic agents in the management of congestive heart failure. AB - Positive inotropic agents are used to improve the impaired cardiac contractility that characterizes chronic heart failure. Digitalis is the traditional drug given for this purpose. However, there is controversy about the effectiveness of digitalis in chronic heart failure. Analysis of the available data indicates the efficacy of digoxin in mild heart failure (i.e., New York Heart Association functional classes I and II) and the relative lack of efficacy in advanced heart failure (i.e., NYHA functional class IV). Further, digoxin can be stopped in a substantial number of patients without recurrence of congestive heart failure. In selected patients whose condition no longer responds to digoxin, the long-term administration of dobutamine may be an effective alternative approach. PMID- 3004213 TI - Protease production by microorganisms associated with reproductive tract infection. AB - Factors influencing pathogenicity of various microbes found in the female lower genital tract remain incompletely understood. Protease production by cervico/vaginal microorganisms may alter or inactivate a variety of proteins important in host defense and structural-functional integrity including collagen containing chorioamniotic membranes and uterine cervix. Host tissues may be made more susceptible to other organisms' virulence factors by protease-producing members of genital tract local flora. Microorganisms themselves may also be influenced by the presence of other microbial protease. Nonspecific protease, gelatinase, collagenase, and elastase production was examined for in vitro with use of aerobic (30) and anaerobic (25) strains of microorganisms typical of those isolated from the lower genital tract of women with premature rupture of membranes, chorioamnionitis, and puerperal infection. Microorganisms including Bacteroides bivius, Bacteroides melaninogenicus, Bacteroides fragilis, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Proteus species, and Propionibacterium acnes produce various proteases. Protease production by both acknowledged pathogenic and commensal bacteria may contribute to the occurrence of reproductive tract morbidity including premature rupture of membranes and preterm labor. PMID- 3004212 TI - Short-term changes in blood ketone body ratios in the phase immediately after hepatic artery embolization: their clinical significance. AB - Changes in arterial and hepatic venous blood ketone bodies were investigated following transcatheter hepatic artery embolization (THAE) in patients with hepatocellular carcinoma. Acetoacetate/ beta-hydroxybutyrate ratio (ketone body ratio) in arterial blood was positively correlated with those of hepatic venous blood (r = 0.960, p less than 0.001), which reflects the mitochondrial redox potential in the embolized lobe. Nine cirrhotic patients were classified into three groups according to the changes in arterial blood ketone body ratio following THAE: Type A without decrease to below 0.7; Type B with a transient decrease to below 0.7, followed by its restoration within 5 hours; and Type C with decrease to below 0.7 without recovery within 5 hours. There were no serious complications in Type A and B patients. By contrast, severe sepsis and hepatic failure developed in Type C patients, possibly due to the extended embolization of both lobes. It is suggested that THAE can be successfully performed even in severely cirrhotic patients, as long as the embolized area is restricted to one lobe. In addition, changes in arterial blood ketone body ratios can give early information about the likely consequences of the THAE procedure just performed. PMID- 3004214 TI - Human papillomavirus deoxyribonucleic acid in cervical carcinoma from primary and metastatic sites. AB - Tissue from 13 cervical cancers and pelvic or para-aortic lymph nodes from the same patient were evaluated by deoxyribonucleic acid hybridization with a human papillomavirus type 16 deoxyribonucleic acid probe for the presence of human papillomavirus-related deoxyribonucleic acid sequences. Twelve of the primary malignancies were squamous cancers and one was an adenocarcinoma. Eight of the primary tumors contained human papillomavirus type 16 deoxyribonucleic acid sequences, and five contained viral sequences closely related to human papillomavirus type 16. Histopathologic diagnosis confirmed malignant cells in six of 13 lymph nodes; three of these specimens contained human papillomavirus type 16 sequences while three had human papillomavirus type 16-related sequences. One lymph node that failed to show malignant cells also contained human papillomavirus type 16 deoxyribonucleic acid. The remaining lymph nodes did not contain malignant cells by either histologic examination or deoxyribonucleic acid hybridization. The human papillomavirus deoxyribonucleic acid sequences in the lymph nodes were similar to those in the matched primary cancer in all cases. These data provide further evidence implicating human papillomavirus in the etiology of cervical cancer. PMID- 3004215 TI - Epithelial regulation of prolactin effect on amnionic permeability. AB - The permeability of human amnion to tritiated water is reduced in the presence of both human and ovine prolactin. The cellular composition of amnion is such that the action of prolactin on this membrane probably occurs by way of the epithelium lining the fetal surface. The present study sought to confirm an epithelial site of action of prolactin on the permeability of amnionic membrane to tritiated water. In addition, radioautography and competition experiments were conducted to determine a possible receptor-mediated mechanism for prolactin action. Membrane permeability to tritiated water was found to be equivalent for both intact membranes and membranes enzymatically stripped of the lining epithelial cells. However, when ovine prolactin was presented to the fetal surface of amnion, only intact membrane displayed decreased permeability to tritiated water. Although localization of iodine 125-labeled prolactin to the light cell population of amniotic epithelium was observed, positive evidence of a receptor-mediated mechanism could not be established. The results indicate that the permeability of human amnion to water is influenced principally by cells of the epithelium in response to prolactin. PMID- 3004216 TI - Amniotic fluid collagenase inhibitor: correlation with gestational age and fetal lung maturity. AB - Concentrations of collagenase inhibitor in amniotic fluid correlated with gestational age and with indices of fetal lung maturity such as the lecithin/sphingomyelin ratio and the presence of phosphatidylglycerol. Possible sources of amniotic fluid collagenase inhibitor were sought, and a number of tissues and fluids, both maternal and fetal in origin, were found to contain or synthesize this glycoprotein in significant quantities. The highest concentrations were achieved in cultures of lung fibroblasts implicating the fetal lung as a major source. Although collagenase inhibitor is largely present in a free or available state, its specific role, other than that of a general antiproteinase, has not been determined in amniotic fluid. However, the quantitation of amniotic fluid collagenase inhibitor provides an index of the maturation of the connective tissue of the fetal lung and may reflect the ability of the extracellular matrix to meet neonatal demands. PMID- 3004217 TI - Male pseudohermaphroditism due to 17-ketoreductase deficiency: report of a case without gynecomastia and without vaginal pouch. AB - A black adult subject, exhibiting complete virilization at puberty with psychological female orientation, was investigated. An older sister had similar physical habitus. Hormonal data showed an androstenedione/testosterone ratio greater than 1. This led us to the diagnosis of 17-ketoreductase deficiency, with possible genetic transmission of the defect. PMID- 3004218 TI - Herpes simplex virus testing of an obstetric population with an antigen enzyme linked immunosorbent assay. AB - Commercial herpes simplex virus antigen enzyme-linked immunosorbent assay kits were compared to conventional culture for herpes simplex virus with 3237 genital specimens from obstetric patients. These rapid enzyme-linked immunosorbent assay tests had a sensitivity of 34.3% and specificity of 98.1% with primarily cervical specimens from asymptomatic patients. Specimens from vulvar swabs had higher positive rates than those from cervical swabs from the same patient, whether symptomatic or asymptomatic with both culture and enzyme-linked immunosorbent assay. Fifty-six women had enzyme-linked immunosorbent assay false positive tests; use of enzyme-linked immunosorbent assay testing alone could have resulted in 1.7% unnecessary cesarean deliveries. Six cases of neonatal herpes simplex virus infection were identified; two of the mothers had negative cervical cultures the week of delivery preceded by positive vulvar cultures. PMID- 3004219 TI - Ovarian carcinoma of low malignant potential: staging and treatment. AB - A review of 65 cases of ovarian carcinoma of low malignant potential registered at the Manitoba Cancer Treatment and Research Foundation over a 7 1/2-year period was undertaken. Eighty-four percent of patients presented with Stage I disease, which was confirmed by primary staging laparotomy in 25%. The average age at diagnosis was younger than that commonly found in patients with invasive carcinoma. Fourteen patients received postoperative chemotherapy, of whom 10 were evaluated with second-look laparotomy. No patient with macroscopic residual disease after initial surgery was cured by chemotherapy. This report emphasizes the need for a prospective controlled study to evaluate adjunctive chemotherapy in the treatment of these tumors. PMID- 3004220 TI - Inhibition of gonadotropin-releasing hormone receptors in rat anterior pituitary monolayer cell cultures by danazol. AB - To study possible cellular antigonadotropic effects of danazol, monolayer cultures of anterior pituitary cells from immature female rats were treated with danazol. Measurements of luteinizing hormone release in response to 10(-8) mol/L gonadotropin-releasing hormone challenge and iodine 125-labeled gonadotropin releasing hormone binding activity were done after exposure to increasing concentrations of danazol and for increasing lengths of time. It was found that luteinizing hormone secreted by pituitary cells in response to gonadotropin releasing hormone was inhibited after danazol treatment in a dose- and time dependent manner when compared to controls. Also, a 45% decrease in gonadotropin releasing hormone receptor binding capacity was observed in pituitary cells cultured in the presence of increasing concentrations of danazol in the range of 10(-8) to 10(-4) mol/L when compared to controls. Furthermore, exposure to danazol for 25 to 96 hours caused a marked decrease in gonadotropin-releasing hormone binding activity (p less than 0.005). Under these experimental conditions danazol treatment decreased the pituitary receptors for gonadotropin-releasing hormone in a dose- and time-dependent manner. Scatchard analysis of saturation curves for the binding of gonadotropin-releasing hormone to cellular gonadotropin releasing hormone receptors indicated that the observed decrease in gonadotropin releasing hormone binding in the danazol-treated group was due to a change in the number of gonadotropin-releasing hormone binding sites rather than a change in the affinity. It is therefore concluded that the antigonadotropic activity of danazol appears to be related to a decrease in gonadotropin-releasing hormone receptors in the pituitary. PMID- 3004221 TI - Plasma growth hormone concentration in the chronically catheterized ovine fetus during spontaneous term delivery and premature delivery induced by continuous intravascular infusion of low doses of adrenocorticotropin or cortisol to the fetus. AB - Fetal plasma growth hormone concentrations were measured in 15 pregnant ewes over the last 5 days before delivery. In five chronically catheterized pregnant ewes that underwent spontaneous vaginal delivery 146 +/- 2 days (mean +/- SD) of gestation, fetal plasma growth hormone concentrations fell from 124.6 +/- 44.1 ng X ml-1 5 days before delivery to 35.2 +/- 31.3 ng X ml-1 at delivery. In three fetuses in which premature delivery was induced by the infusion of cortisol to the fetus at 128 days of gestation, fetal plasma growth hormone levels fell from 195 +/- 19.1 ng X ml-1 to 70.7 +/- 25.5 ng X ml-1 over the last 5 days of intrauterine life. In seven fetuses in which premature delivery was induced with infusion of synthetic adrenocorticotropin to the fetus beginning at 120 or 130 days of gestation, the fetal plasma growth hormone level did not fall (175.6 +/- 75.7 to 158.9 +/- 60.1 ng X ml-1). Fetal plasma cortisol concentrations at delivery were significantly higher in the cortisol-infused fetuses (214 +/- 38.4 ng X ml-1) than in both control (94.4 +/- 33.7 ng X ml-1) and adrenocorticotropic hormone-infused fetuses (94.5 +/- 31.9 ng X ml-1). The fall in the fetal plasma growth hormone level in cortisol-induced fetuses may be due to the higher fetal plasma cortisol concentrations achieved in the cortisol-infused compared with the adrenocorticotropic hormone-infused fetuses in which no comparable decrease in growth hormone was observed. These findings suggest that there are significant differences in the fetal response to various experimental regimens used for the induction of premature labor in the sheep. PMID- 3004222 TI - Biochemical and histologic effects of sequential estrogen/progestin therapy on the endometrium of postmenopausal women. AB - Medroxyprogesterone acetate in doses of 10, 5, and 2.5 mg was administered sequentially to three groups of postmenopausal women receiving 0.3 mg, 0.625 mg, and 1.25 mg of conjugated equine estrogens, respectively. Serial endometrial biopsies were performed on these women before therapy, during estrogen therapy alone, and during sequential estrogen-progestin therapy. Endometrial histology and estrogen receptor concentrations were assessed. A linear increase of cytosolic estrogen receptor concentration occurred over the dosage range of conjugated equine estrogen. When medroxyprogesterone acetate was added to the estrogen therapy, the concentrations of estrogen receptors fell. Within the groups of women receiving 0.3 mg and 0.625 mg of conjugated equine estrogen, all doses of medroxyprogesterone acetate were equally effective in reducing the levels of cytosolic receptor to pretreatment levels. However, at the conjugated equine estrogen dose of 1.25 mg, only 5 mg and 10 mg doses were effective in reducing the cytosolic receptor concentration to pretreatment levels. Histologically, little effect was observed from the lowest doses of either drug. However, even though 5 and 10 mg of medroxyprogesterone acetate were identical biochemically, the 10 mg dose was the only one producing a homogeneous, secretory pattern within the endometrium. PMID- 3004223 TI - Heat flux and oxygen consumption of the pregnant uterus. AB - Heat flux (conductive and convective heat) and oxygen consumption of the pregnant uterus and its content were measured simultaneously in the same group of pregnant ewes during the acute postoperative period, during a chronic resting period, and during alpha- and beta-adrenergic-receptor stimulation with norepinephrine and ritodrine. Results indicated four conclusions. First, an excellent correlation existed between heat flux and oxygen consumption in the acute and chronic resting condition as well as during increasing uteroplacental vascular resistance and decreasing blood flow produced by norepinephrine infusion; the correlation was not as good during ritodrine infusion. Second, during rest, about 85% of heat generated by the pregnant uterus is eliminated through the uteroplacental circulation while the remaining heat diffuses through the myometrium. Third, during decreasing uteroplacental blood flow and elevated resistance, the pregnant uterus is able to maintain a normal thermostasis by widening the temperature difference in the blood entering and leaving the uterus and by increasing the myometrial heat exchange; oxygen consumption also is maintained at normal level through increase in oxygen extraction. Fourth, with the exception of uteroplacental circulation, the circulatory, metabolic, and thermal conditions of the pregnant ewe are not different after 5 hours from 5 to 7 days after the surgical procedure. PMID- 3004224 TI - Isolation of the human T-cell leukemia/lymphotropic virus type III from the cornea. AB - Corneoscleral donor tissue from a donor with a positive serum antibody to HTLV III but without the overt clinical signs of the acquired immune deficiency syndrome (AIDS) was cultured for the presence of the human T-cell leukemia/lymphotropic virus type III (HTLV-III). The virus was isolated from the two corneal specimens in this patient after the tissue had been stored for four days in McCarey-Kaufman medium. The presence of HTLV-III was confirmed by the detection of viral core proteins (approximately 24,000 protein, termed P24 gag), by immunofluorescence of a touch preparation of the corneal epithelium as well as in cells cultured in vitro. The percentage of immunofluorescent cells detected by HTLV-III anti-P24 antibody ranged between 2% and 3%. These findings emphasize the possibility of transmission of this virus via corneal transplantation surgery. Although no documented cases of AIDS have occurred in corneal transplant recipients, serologic screening of donors before the use of the tissue for transplantation is advisable. PMID- 3004225 TI - The risk posed by HTLV-III-infected corneal donor tissue. PMID- 3004226 TI - Crocidolite-induced pulmonary fibrosis in mice. Cytokinetic and biochemical studies. AB - The responses of pulmonary alveolar and bronchial cells to asbestos exposure were studied by relating the cytokinetic changes of injury and repair to the inflammatory process and subsequent fibroblastic activity. The lesions were induced by intratracheal instillation of 1 mg crocidolite asbestos in mice, which were killed up to 20 weeks thereafter; 3H-thymidine was injected 1 hour before death. A rapid inflammatory response with elevated polymorphonuclear leukocytes and lysosomal enzyme release was largely over by 2 weeks, but the increase in alveolar macrophages was maintained. Focal necrosis of bronchial epithelial cells was repaired by cell regeneration, whereby new epithelial cells overgrew luminal exudates to incorporate long asbestos fibers into the peribronchial interstitium, where macrophagic granulomas formed. Increased collagen levels were largely due to stimulation of peribronchial fibroblasts. A lesser reaction of epithelial damage, Type 2 cell proliferation, and fibroblast stimulation also occurred in the alveolar walls. The results suggest that macrophage-fibroblast interactions associated with enhanced fibrosis occur readily in the peribronchial interstitium following injury and repair of epithelial cells by long asbestos fibers. PMID- 3004228 TI - Skull base surgery. PMID- 3004227 TI - Coxsackievirus B-3 myocarditis. Identification of different pathogenic mechanisms in DBA/2 and Balb/c mice. AB - DBA/2 and Balb/c mice, both H-2d, develop myocardial inflammation and necrosis when infected with a heart-adapted strain of coxsackievirus Group B, Type 3. Similar inoculation of C57Bl/6 (H-2b) animals results in minimal myocarditis despite equivalent heart virus titers in the three stains. Thus, the host's genetic constitution influences the pathogenesis of the infection. Anti-mouse thymocyte serum and monoclonal Iad antibody effectively prevent myocarditis induction in DBA/2 and Balb/c mice, which demonstrates the importance of the immune system in this disease. Cytolytic T lymphocytes lysing virus-infected and uninfected myocytes and heart-reactive autoantibodies occur in both myocarditis susceptible strains. Cellular immunity causes the myocardial injury in Balb/c mice. Complement depletion of Balb/c mice using cobra venom factor fails to alter the disease. Similar treatment of DBA/2 animals abrogates inflammation and necrosis, which suggests that heart-reactive antibodies in this strain are primarily responsible for initiating myocardial damage. PMID- 3004229 TI - Localization of binding sites for alpha-rat atrial natriuretic polypeptide in rat kidney. AB - To determine the intrarenal localization of receptors for atrial natriuretic polypeptide (ANP) in the rat, we performed autoradiography and binding assay by using 125I-labeled alpha-rat ANP (alpha-rANP). Autoradiography at the slice and microscopic level in the kidney and binding assay in isolated glomeruli demonstrated that receptors in the renal cortex were distributed mainly in glomeruli. Although dense silver grains were distributed diffusely both in inner medulla and outer stripe of outer medulla, a marked displacement of the grains was observed only in the inner medulla. Autoradiography at the microscopic level also showed that silver grains were distributed in the renal artery, renal pelvis, and inner medullary collecting tubule (IMCT) prepared by the microdissection method, but not in the arcuate artery, interlobular artery, and afferent or efferent arterioles. Specific binding was demonstrated in the isolated glomeruli and the preparation was rich in fragments of IMCT. Apparent binding affinity (Kd) and receptor density (R) for 125I-labeled alpha-rANP in isolated glomeruli and IMCT were Kd = 3.2 and 21 X 10(-9) M, and R = 320 and 420 fmol/mg protein, respectively. These observations suggest that alpha-rANP has a physiological action on glomeruli and possibly on the inner medullary collecting tubules in addition to the renal artery. PMID- 3004230 TI - Sodium absorption and potassium secretion in rabbit colon during sodium deficiency. AB - Reducing the daily Na intake of rabbits from approximately 4.4 to 0.1 meq/kg body wt increases plasma aldosterone levels and the rate of amiloride-sensitive Na transport in the descending colon two- to threefold. The stimulation of Na transport is a result of an increase in the maximum transport capacity of the epithelium, whereas the affinity of Na to its transport system is not altered. Simultaneous with enhanced Na absorption, there is statistically significant K secretion of 0.25 mu eq . cm-2 . h-1 under short-circuit conditions. Transepithelial current-voltage relations in the absence and presence of amiloride were used to determine the Na permeability of the apical membrane and the intracellular Na activity of the Na-transporting cells. The Na content of the amiloride-sensitive cells was estimated from the kinetics of absorptive Na tracer fluxes. The stimulation of active Na transport under conditions of dietary Na restriction is associated with parallel increases in apical membrane Na permeability and the Na content of the amiloride-sensitive cells, but the intracellular Na activity and the activity of the epithelial Na-K-ATPase are not significantly altered. Taken together, these results suggest that endogenous aldosterone increases the number of conducting Na entry sites in the apical membrane of colonic epithelium and that there is activation of additional Na pump units in the basolateral membrane, brought about by cell swelling and possibly by an increase in the fraction of epithelial cells that participate in active Na transport. PMID- 3004231 TI - Dissociation of CO2 hydration and renal acid secretion in the dogfish, Squalus acanthias. AB - Maximal rates of renal hydrogen ion secretion and bicarbonate reabsorption in the dogfish were stimulated by intravascular infusion of acidic and basic buffers: bicarbonate, phosphate, phenol red, dimethadione (DMO), imidazole, and piperazine N,N'-bis(2 ethanesulfonic acid) (PIPES). There was no difference in titratable acid secretion or urinary pH after bicarbonate infusion despite a sevenfold increase in plasma bicarbonate. Bicarbonate reabsorption was increased 12-fold and showed no evidence of reaching a maximum. This was not altered by methazolamide, as expected, since there is no renal carbonic anhydrase in seagoing fish. Imidazole resulted in the greatest augmentation of renal titratable acid secretion (33----390 mueq . h-1 . kg-1) and did not alter urinary pH. Inhibition of organic base secretion by Darstine had no effect on the imidazole-induced maximal rate of acid secretion. This rate was compared with that of hydrogen ion generation calculated from the uncatalyzed reactions of CO2 and H2O or OH-, maximizing PCO2 and OH- gradients and reaction volumes in vivo. These calculated chemical rates could only account for 9-14% of the measured maximal acidification rate. Thus the powerful process that maintains constant acid urine pH is not only independent of carbonic anhydrase but can function well in a low CO2 environment in which the reactions CO2 + H2O or CO2 + OH- do not furnish enough protons for H+ secretion or HCO3- reabsorption. We conclude that following the cellular protolysis of water, processes other than those involving CO2 buffering of OH- permit H+ to engage in the formation of urine. PMID- 3004232 TI - Supply-to-demand ratio for oxygen determines formation of adenosine by the heart. AB - To investigate the basic mechanism for formation of adenosine, cardiac work was increased using an isolated guinea pig working heart preparation. Cardiac metabolism was stimulated by intracoronary infusion of isoproterenol, norepinephrine (NE), ouabain, and the cardiotonic agent 1H-imidazo[4,5 b]pyridine, 2-[2-methoxy-4-(methylsulfinyl)-phenyl] (AR-L 115), and the release of adenosine into the effluent was determined. Besides their inotropic effects on myocardial tissue, these substances affect differently the tone of coronary arteries. Thus they influence the supply-to-demand ratio for O2 and the tissue cyclic AMP content differently. When myocardial O2 consumption was increased to the same extent, the changes in coronary flow induced followed the order of AR-L 115 greater than isoproterenol greater than NE greater than ouabain. Conversely, the rank order of potency causing adenosine and inosine release was NE greater than ouabain greater than isoproterenol greater than AR-L 115. When NE-induced vasoconstriction was abolished by prazosin, the release of adenosine and inosine was significantly diminished. Enhancement of O2 supply by overperfusion of the coronary arteries in isoproterenol-stimulated hearts was associated with a significant reduction of nucleoside release into the effluent. Our findings suggest that the major stimulus for myocardial adenosine formation is an imbalance between O2 delivery and O2 demand and not the metabolic rate as such. In the isolated heart, adenosine is formed as a feedback signal and can compete with NE- and ouabain-induced vasoconstriction. PMID- 3004233 TI - Na+-H+ exchange is present in sarcolemmal vesicles from dog superior mesenteric artery. AB - The Na+ concentration inside vascular smooth muscle cells is an important regulator of vascular smooth muscle function, but the mechanisms that mediate Na+ influx are not known. We studied Na+ transport in a newly described vesicle preparation preferentially enriched in sarcolemma and obtained by Mg2+ aggregation and differential centrifugation of homogenized dog superior mesenteric artery. In the presence of an outwardly directed proton gradient (pHout = 7.5, pHin = 5.0), 1 mM 22Na+ uptake was stimulated over twofold relative to the absence of a pH gradient (pHin = 7.5 or 5.0). pH gradient-stimulated Na+ uptake was inhibited by 1 mM amiloride. 22Na efflux was stimulated by an inwardly directed proton gradient (pHin = 7.5, pHout = 5.7 vs. 7.5). The rate of proton efflux from acid-loaded vesicles was measured by acridine orange fluorescence and was stimulated by 100 mM Naout but not by Nain = Naout = 100 mM. H+ gradient stimulated Na+ transport and Na+ gradient-stimulated H+ transport were not due to electrical coupling between the two cations. The pH gradient-stimulated component of Na+ transport in the final vesicles, an intermediate fraction, and microsomes were proportional to the respective enzyme marker activities for sarcolemma but not for sarcoplasmic reticulum or mitochondrial membranes. We conclude that Mg2+ aggregation and differential centrifugation of homogenized vascular smooth muscle yield a vesicle preparation preferentially enriched in sarcolemma. Furthermore, the sarcolemma of vascular smooth muscle contains an amiloride-sensitive Na+-H+ proton countertransport system. PMID- 3004234 TI - Mechanisms of adrenergic control of blood pressure in developing rats. AB - The rat is a species in which the sympathetic nervous system (SNS) is highly immature at birth. Blood pressure was measured directly in anesthetized preparations starting on the first postnatal day (days 1, 5, 9, 20, 40, 55, and 85), and pharmacological tests were used to evaluate the functional development of the vasomotor nerves (maximum pressor response to tyramine), the sensitivity of the vasculature to direct stimulation of alpha 1-adrenoceptors (maximum and 50% effective dose of methoxamine pressor response), and relative magnitude of the SNS contribution to resting blood pressure (hypotensive response to ganglionic blockade divided by resting blood pressure). The SNS contribution was not significant on postnatal day 1, but the relative magnitude was comparable to the adult by the end of the first postnatal week. During week 1 the vasomotor nerves were functionally immature (tyramine response was 48% of mature value on day 5), and the vasculature was supersensitive to alpha 1-adrenoceptor stimulation (154% of mature value). Conversely, in postnatal weeks 2 and 3, when the developing SNS is known to be hyperactive, the vasculature was subsensitive to noradrenergic stimulation (60-70% of adult). The net effect was to attenuate the SNS contribution to resting blood pressure during this period (55% of adult value). We conclude that there is an inverse relation between the level of tonic SNS activity and vascular sensitivity to noradrenergic stimulation in the developing rat. Supersensitivity may be critical for cardiovascular adjustments to asphyxia perinatally when the vasomotor nerves are functionally immature; subsensitivity may act homeostatically to prevent hypertension during the developmental period when SNS is hyperactive. PMID- 3004235 TI - Can cultured teleost hepatocytes show temperature acclimation? AB - Hepatocytes were prepared from 15 degrees C acclimated catfish (Ictalurus punctatus) and maintained in primary culture for 20 days on biomatrix at 7, 15, and 25 degrees C without hormones or serum to determine if cells can directly adapt to temperature. Specific activities of cytochrome-c oxidase, NADH cytochrome c reductase, citrate synthase, and glucose-6-phosphate dehydrogenase showed acclimatory rate compensation (7 greater than 15 greater than 25 degrees C cultured); 6-phosphogluconate dehydrogenase had activity changes of 15 greater than 7 greater than 25 degrees C cultured; activity of lactate dehydrogenase occurred in the series 7 greater than 15 = 25 degrees C. Protein synthesis of freshly isolated hepatocytes from catfish acclimated to the three temperatures exhibited acclimatory rate compensation. In contrast, protein synthesis of cultured hepatocytes occurred in the series 15 greater than 25 greater than 7 degrees C cultured. Protein degradation was highest at 25 degrees C followed by cells at 15 and 7 degrees C. Cultured hepatocytes showed incomplete temperature acclimation in vitro by way of enzyme activity changes and of protein synthesis. This suggests that some factor(s), such as hormones, is probably necessary to mediate the full temperature-acclimation process. PMID- 3004236 TI - Renin, ACTH, and adrenocortical function during hypoxia and hemorrhage in conscious rats. AB - We studied the effect of chronic hypoxia on the renin, adrenocorticotropin (ACTH), aldosterone, and corticosterone responses to acute hemorrhage in conscious male rats with chronic femoral arterial catheters. Rats were exposed to 21, 12.5, or 10% O2 (n = 7 per group). At 42 h of exposure, animals underwent a rapid 6 ml/kg hemorrhage. O2 at 12.5 and 10% led to significant hypoxemia (arterial PO2 = 52 +/- 1 and 43 +/- 1 Torr, respectively) and respiratory alkalosis. Significant increases in plasma sodium to 145 +/- 2 meq/l and decreases in plasma potassium to 3.53 +/- 0.12 meq/l were also observed during hypoxia. Hypoxia per se had no significant effect on blood pressure, plasma renin activity, ACTH, and corticosterone. O2 at 12.5% led to a significant reduction in aldosterone levels (0.9 +/- 0.8 ng/dl) compared with normoxia (4.2 +/- 0.9 ng/dl). The mean arterial pressure, plasma renin activity, and aldosterone responses to hemorrhage were unaltered by hypoxia. ACTH and corticosterone responses to hemorrhage were potentiated by exposure to 10% O2. We conclude that chronic exposure to severe hypoxia augments the pituitary-adrenal but not the renin-aldosterone response to hemorrhage. PMID- 3004237 TI - Evidence for an opioid neurotransmission mechanism in adult rumination. PMID- 3004238 TI - The effects of infant feeding on rotavirus-induced gastroenteritis: a prospective study. AB - The relationship between feeding method and risk of rotavirus infection was studied by following a cohort of 197 infants from low income households through the winter diarrhea season of 1983-84. Fecal specimens were systematically collected and tested for the presence of rotavirus particles by electron microscopy, confirmed by ELISA. The attack rates of rotavirus gastroenteritis were similar for breast-fed and bottle-fed infants (20 per cent, 17 per cent, respectively); however, the clinical course of rotavirus gastroenteritis was quite different. Infants who were breast-fed had illnesses which were characterized by milder symptoms of shorter duration. Of the 10 breast-fed infants who acquired rotavirus gastroenteritis, nine (90 per cent) were classified as mild illnesses while of the 25 bottle-fed infants who acquired rotavirus gastroenteritis, only nine (36 per cent) were classified as having mild illnesses. These data suggest that factors associated with breast-feeding, although not affecting rotavirus infection rates, may moderate the clinical course of rotavirus gastroenteritis. PMID- 3004239 TI - Prevalence of hepatitis A virus antibody among Navajo school children. AB - Previous studies of the prevalence of immunity to hepatitis A (anti-HAV) in the United States have used urban settings or institutions for the mentally handicapped. In a rural setting among normal children, a serologic investigation of prevalence of anti-HAV was conducted in a boarding school adjacent to the Navajo reservation. The results show rates of anti-HAV that are the highest reported at the ages tested in any subpopulation in the United States, comparable only with those in developing countries. PMID- 3004240 TI - Identification of the physical and chemical characteristics of volcanic hazards. PMID- 3004241 TI - Evaluation of physical health effects due to volcanic hazards: crystalline silica in Mount St. Helens volcanic ash. AB - This investigation has shown that crystalline silica has been identified as being present in the Mount St. Helens volcanic ash at levels of 3 to 7 per cent by weight. This identification has been established using X-ray powder diffraction, infrared spectrophotometry, visible spectrophotometry, electron microscopy, and Laser Raman spectrophotometry. Quantitative analysis by IR, XRD, and visible spectrophotometry requires a preliminary phosphoric acid digestion of the ash sample to remove the plagioclase silicate material which interferes with the determination by these methods. Electron microscopic analysis as well as Laser Raman spectrophotometric analysis of the untreated ash confirms the presence of silica and at levels found by the XRD and IR analysis of the treated samples. An interlaboratory study of volcanic ash samples by 15 laboratories confirms the presence and levels of crystalline silica. Although several problems with applying the digestion procedure were observed in this hastily organized supply, all laboratories employing the digestion procedure reported the presence of crystalline silica. These results unequivocally put to rest the question of the presence of silica in the volcanic ash from eruptions of Mount St. Helens in 1980. PMID- 3004242 TI - Use of cytological preparations in the frozen section diagnosis of central nervous system neoplasia. AB - Because cytologic preparations capture the fine cellular detail that frozen sections often obscure, touch and smear preparations are valuable adjuncts to intraoperative histologic diagnoses. PMID- 3004243 TI - Pulmonary scar cancer. A pathologic reappraisal. AB - A total of 49 consecutive specimens of lung cancer were collected prospectively at surgical resection or autopsy from 40 men and nine women, aged 40-74 years. Of the 49 tumors, the gross appearance of 22 fitted the description of a scar cancer, i.e., a tumor with pleural puckering and central pigmentation. Nineteen of the "scar cancers" were peripheral (17 adenocarcinomas and two squamous cell carcinomas); three were central (one squamous cell carcinoma and two adenocarcinomas). In the 19 peripheral "scar cancers," elastic stains demonstrated the presence of collapsed, unfibrosed lung tissue at the center with traction of the overlying pleura toward it. Elsewhere in the tumor, the elastic framework was either destroyed or expanded by tumor filling the alveolar spaces. None of the "scar cancers" had a significant desmoplastic reaction that might otherwise explain the scarred appearance. It appeared that local atelectasis was solely responsible for the pleural puckering and central pigmentation. On the other hand, atelectatic lung tissue was not seen in the 27 cancers that did not have the appearance of a scar cancer. Tuberculosis was found in 10 of the 49 lung specimens. In only one specimen was the tuberculous lesion anatomically associated with the tumor. There was no evidence of pulmonary infarct in any of the specimens. The term "scar cancer" was considered inappropriate as there was no preformed fibrous tissue. The scarred appearance was thought to be the result of localized pulmonary atelectasis owing to small airways obstruction by tumor. Association with tuberculosis was considered incidental. PMID- 3004244 TI - Functioning oncocytic islet-cell carcinoma. Report of a case with electron microscopic and immunohistochemical confirmation. AB - A case of a 58-year-old woman with an unusual variant of malignant islet-cell tumor showing oncocytic features is described. Using the light microscopy technique, the tumor appeared comprised of solid nests of uniform cells with abundant, eosinophilic cytoplasm and round nuclei with granular chromatin. Ultrastructurally, the cells contained numerous abnormal mitochondria, dilated rough endoplasmic reticulum, and scattered dense-core neurosecretory granules, often associated with cytoplasmic filaments. Tumor cells were focally immunoreactive for insulin, glucagon, and somatostatin and diffusely immunoreactive for alpha 1-antitrypsin as assayed by the avidin--biotin technique. The tumor was immunonegative for human chorionic gonadotropin, gastrin, adrenocorticotropic hormone, and serotonin. The patient exhibited some of the clinical features associated with glucagonoma syndrome, including diabetes mellitus and chronic diarrhea. The tumor behaved in a malignant fashion, with widespread lymphatic involvement and bony metastases at the time of presentation. This report of an oncocytic islet-cell carcinoma supports the concept of oncocytic differentiation in islet-cell tumors in a fashion analagous to oncocytic carcinoids. PMID- 3004246 TI - Mesonephric adenosarcoma of the renal pelvis with heterologous elements. AB - A polypoid tumor located in the renal pelvis with both epithelial and sarcomatous elements is described. The epithelial component was histologically benign and resembled urothelium. The sarcomatous component showed undifferentiated cells and rhabdomyoblastic elements. The term mesonephric adenosarcoma is proposed to describe this biphasic tumor. Although this tumor might be histogenetically related to Wilms' tumor, it is morphologically distinct, and might behave more aggressively. PMID- 3004245 TI - Rapid frozen section in pediatric pathology. AB - Frozen section examination in pediatrics differs from that in adult practice in two ways. First, there is a high proportion of undifferentiated small-cell cancers in which it it difficult to make a definitive diagnosis without additional information. Second, there are special categories of congenital disorders where one is seeking not neoplasia but the presence, absence, or size of normal structures. In 520 pediatric frozen sections, there was a comparatively high incidence of deferred (5.6%) or inaccurate diagnoses (3.5%). However, as there were 99 small-cell cancers, it is perhaps surprising that the number was not greater. In the nervous system (208 cases), it was sometimes difficult to distinguish between malignant ependymoma and medulloblastoma. In other neoplasms, e.g., soft tissues and bone, the most important requirement was adequate clinical and radiological information. In Hirschsprung's disease (132 cases), ganglion cell detection was 100% accurate. In measuring the diameter of bile ducts in the porta hepatis during surgery for biliary atresia (18 cases), it was sometimes difficult to recognize ducts that had lost their epithelial lining. PMID- 3004248 TI - Hyaline cells in chondroid syringoma. PMID- 3004247 TI - Primary mucinous carcinoma of the skin with metastases to the lymph nodes. AB - Primary mucinous carcinoma is a rare sweat-gland neoplasm of the skin with a tendency to grow slowly. Although the neoplasm persists locally in nearly half of the cases after attempts at removal, metastases to regional lymph nodes and widespread metastases are uncommon. We present a case of primary mucinous carcinoma in an axilla with metastases to the axillary lymph nodes and propose a hypothesis explaining the slow rate of growth, based on our findings of a paucity of both blood vessels and macrophages in the neoplasm. Electron microscopy revealed mucin production by the dark cell and its extracellular secretion, which supports the theory of eccrine differentiation of the neoplasm. PMID- 3004249 TI - Humoral autoimmune manifestation in subacute thyroiditis. AB - To study the autoimmune manifestations in subacute thyroiditis (SAT), the patterns of thyroid antibodies, thyroglobulin and circulating immune complexes were investigated in 10 patients during the course of the disease. Eight patients were thyrotoxic at diagnosis, and became euthyroid during recovery with a median observation of 8 months (4-30 months). Thyroid stimulating immunoglobulins were measured as TSH binding inhibiting immunoglobulins (TBII) and as thyroid stimulating antibodies (TSAb). TBII were present in all patients at least once during the observation period and remained detectable in six patients after recovery. TSAb were detected in three patients without relation to the hyperthyroid state. Thyroglobulin antibodies (TgAb) were present in four patients and persisted in three, while microsomal antibodies (MAb) were negative. Thyroglobulin (Tg) in the TgAb negative patients (n = 6) was high at diagnosis (median 229 micrograms/l, range 55-375) and fell rapidly during the course of SAT. Circulating immune complexes (CIC), which were found in all patients, reached maximal levels shortly after the onset of the disease and persisted after recovery. No correlation could be demonstrated between the different thyroid antibodies, and there was no clear relation between the levels of CIC and presence of the autoantibodies. However, the changes in CIC paralleled the changes in TBII, and it is suggested that immune complex formation is a major feature of the regulatory mechanisms controlling the immune responses in SAT. PMID- 3004251 TI - Separation of [1-3H]cellooligosaccharides by thin-layer chromatography: assay for cellulolytic enzymes. AB - A thin-layer chromatographic method for the separation with high resolution of [1 3H]cellooligosaccharides on silica gel plates has been developed. Reducing end labeled glucose through cellohexaose were separated on silica gel plates with three ascents of ethyl acetate:water: methanol (40:15:20; v:v) and each was extracted with an efficiency of 88 +/- 3%. Separations of cellooligosaccharides using other adsorbents, solvents, and impregnants are also described. This thin layer chromatographic method facilitated analysis of the activity of cellulolytic enzymes. PMID- 3004250 TI - [Bupivacaine 0.5% CO2 in spinal anesthesia]. AB - The clinical usage of 0.5% bupivacaine-CO2 for spinal anaesthesia was tested in 45 patients. The use of bupivacaine CO2 is safe, segmental dificits were not observed. Compared to bupivacaine-HCl the latency period is shorter (7.7 min), whereas the duration of maximal analgesic spread (105 +/- 31 min) is identical. 93% of the patients had a complete motor blockade. The solubility of bupivacaine CO2 in CSF (1.75 mg ml-1) is more than double the solubility of bupivacaine-HCl (0.8 mg ml-1), thus lowering the risk of precipitation. PMID- 3004252 TI - Use of high-performance liquid chromatography to detect hydroxyl and superoxide radicals generated from mitomycin C. AB - Distinguishing between short-lived reactive oxygen species like hydroxyl and superoxide radicals is difficult; the most successful approaches employ electron spin resonance (ESR) spin-trapping techniques. Using the spin trap 5,5-dimethyl-l pyrroline N-oxide (DMPO) to selectively trap various radicals in the presence and absence of ethanol, an HPLC system which is capable of separating the hydroxyl- and superoxide-generated DMPO adduct species has been developed. The radical generated DMPO adducts were measured with an electrochemical detector attached to the HPLC system and confirmed by spin-trapping techniques. The HPLC separation was carried out on an ODS reverse-phase column with a pH 5.1 buffered 8.5% acetonitrile mobile phase. The advantage of the HPLC system described is that it permits the separation and detection of hydroxyl and superoxide radicals without requiring ESR instrumentation. The antineoplastic bioreductive alkylating agent mitomycin C, when activated by NADPH-cytochrome c reductase, was shown to generate both hydroxyl and superoxide radicals. PMID- 3004253 TI - An enzymatically coupled assay for rat brain polyphosphoinositide phosphodiesterase in an optimized reaction mixture. AB - Bovine intestinal alkaline phosphatase was found to hydrolyze inositol phosphates many times faster than the monoester phosphate groups of the polyphosphoinositides. A convenient and sensitive in vitro assay for the Ca2+ dependent polyphosphoinositide phosphodiesterase was devised in which inositol trisphosphate released from exogenous phosphatidylinositol 4,5-bisphosphate was hydrolyzed by alkaline phosphatase. The resulting inorganic phosphate was measured by an automated method after solubilization of the reaction mixture with sodium dodecyl sulfate. The phosphodiesterase was maximally stimulated by combining the known positive effects of cetyltrimethylammonium bromide (at the optimum detergent-to-substrate ratio of 2.3), monovalent cations (0.1 M KCl), and Ca2+ (0.5 mM) with the additional enhancement by Triton X-100 (0.2% w/v). Activities obtained for rat brain homogenates and microsomal and cytosol fractions were 126 +/- 3.8 (17), 110 +/- 5.7 (10), and 252 +/- 15.5 (8) nmol X min-1 X mg protein-1 (mean +/- SE for n determinations), respectively. PMID- 3004255 TI - Preparation of chromosomal protein A24 (uH2A) by denaturing gel filtration and preparation of its free nonhistone component ubiquitin by ion-exchange chromatography. AB - Chromosomal protein A24 (uH2A) is unique in that it comprises the nucleosomal core histone H2A in isopeptide linkage with the highly conserved, globular, and stable nonhistone protein ubiquitin. Some 10% of the chromatin complement of H2A is modified in this way and studies to elucidate a role for this modification have concentrated on observations requiring no purification of A24 due to the difficulty in isolating the protein in large and pure quantities. We describe a method for isolating A24 by chromatography on Pharmacia G-100 gel filtration medium under urea denaturing conditions. A24 prepared by this method is structurally intact and is available in the quantities required for studies of the behavior and influence of the protein on histone-histone, histone-DNA, and enzymatic interactions. In conjunction with this method we describe a procedure for the isolation of large quantities of free ubiquitin of far greater purity than previously reported. PMID- 3004254 TI - Fluorometric assay using high-pressure liquid chromatography for the microsomal metabolism of certain substituted aliphatics to 1,N6-ethenoadenine-forming metabolites. AB - Monohaloacetaldehydes and monohalooxiranes are early oxidative metabolites of several carcinogenic haloaliphatics. Since monohaloacetaldehydes and supposedly monohalooxiranes react with adenines to form fluorescent 1,N6-ethenoadenines, it was hypothesized that in vitro metabolic systems that produce an ethenoadenine forming metabolite could be assayed quantitatively by trapping the metabolite in situ with an adenine and identifying it by its characteristic retention and fluorescence during HPLC. Bromoacetaldehyde was chosen as a model haloacetaldehyde to develop an assay based on this concept for measurements in a microsomal system. The optimal trapping reaction requires a postmetabolic step involving acidification and heating. Cyclic AMP was found to be a suitable adenine for the trapping reaction under these conditions. The chromatographic analysis utilizes tetrabutylammonium phosphate and a nonsilica reversed-phase stationary phase (Hamilton PRP-1). The chromatography is isocratic and allows an analysis time of less than 5 min per sample. The titration of bromoacetaldehyde in a microsomal system is affected by typically studied metabolic conditions: incubation time, pH, and protein concentration. Using this assay, the following were found to be metabolized by rat liver microsomes to etheno-adenine-forming products: 1,2-dibromoethane, 1,2-dichloroethane, cyclophosphamide, vinyl chloride, and acrylonitrile. Chloroacetone and 1,3-dichloroacetone also are fluorochromogenic without metabolism but the latter apparently forms a positively charged, nonetheno adduct. The proposed assay should be useful for in vitro metabolic studies of 1,2-dihaloethanes and mustards and has potential application for similar studies of monohalogenated ethanes, ethanols, and ethenes. The positive results with acrylonitrile suggest also that many types of substituted aliphatics may be studied with this proposed assay. PMID- 3004256 TI - Redox changes in coenzyme Q in the millisecond time range: an approach using rapid quenching and high-performance liquid chromatography. AB - We have combined a rapid-quenching protocol with HPLC analysis to measure the kinetics of reduction of coenzyme Q in a mitochondrial enzyme complex. The method has a time resolution of several milliseconds and will readily measure 1-20 nmol of the Q derivatives under investigation. By changing the HPLC solvent, either Q6 or Q10 can be studied. PMID- 3004257 TI - A method for purifying the platelet membrane glycoprotein IIb-IIIa complex. AB - A method has been developed for the rapid isolation of platelet membrane glycoproteins (GP) IIb and IIIa. This method produces an excellent yield and does not require the prior isolation of platelet membranes. Outdated platelets were washed and solubilized in Triton X-100. Concanavalin A affinity chromatography was used to purify a platelet glycoprotein fraction. The concanavalin A-retained glycoproteins were eluted and adsorbed with a heparin-Sepharose column to remove a major contaminant, thrombospondin. Sephacryl S-300 gel filtration was used as the final purification step to remove most fibrinogen and low-molecular-weight contaminants. Wheat germ agglutinin affinity chromatography was used to completely remove trace amounts of fibrinogen. The purified GP IIb and GP IIIa were analyzed by sucrose gradient sedimentation and found to consist of heterodimer complexes. PMID- 3004258 TI - A pH-metrical method of determining glucose-6-phosphatase activity. AB - A simple, rapid, and reproducible method of determining glucose-6-phosphatase activity is described. The glucose 6-phosphate hydrolysis is accompanied by the disappearance of the protons from the medium owing to a phosphate species pK change from 6.1 (in glucose 6-phosphate) to 6.9 (in inorganic phosphate). Alkalization is registered by a pH meter with a recorder. The method described in this paper may be used in routine determinations of glucose-6-phosphatase activity. PMID- 3004259 TI - Spinal sufentanil effects on spinal pain-transmission neurons in cats. AB - The ability of sufentanil to suppress noxiously evoked activity of wide dynamic range (WDR) neurons was studied in decerebrate, spinal-cord-transected cats. Sufentanil, 2.5 micrograms (n = 7) or 5.0 micrograms (n = 7), when administered spinally, produced a significant, dose-dependent suppression of noxiously evoked (51 degrees C radiant heat stimulus) activity of WDR neurons in the dorsal horn of the spinal cord. Spontaneous recovery from sufentanil suppression was not seen for up to 2 h. Reversal following intravenous naloxone, 0.12 mg, although present, was not as complete as that seen following other spinal opiates. Intravenous sufentanil, 5.0 micrograms/kg (n = 4), produced significant but short lasting depression of noxiously evoked WDR neuron activity. A comparison of the results of this study with data from a previous fentanyl study suggests that sufentanil may be more appropriate than fentanyl for spinal or epidural administration because of a possible longer duration of action. However, the lesser degree of naloxone reversal seen in this study may suggest that, clinically, reversal of sufentanil effects may be more difficult. PMID- 3004260 TI - Mechanism of antagonism by physostigmine of acute flunitrazepam intoxication. AB - The effect of physostigmine on the loss of consciousness and respiratory depression induced in rabbits by flunitrazepam, 1 mg/kg, was studied to demonstrate whether the restoration of consciousness and respiration rate results from an increase in central cholinergic activity or from an interference by physostigmine with specific binding of flunitrazepam to its receptors. Physostigmine, 0.1-0.4 mg/kg iv, caused a dose-related reversal of consciousness and respiration rate within 15 min of its injection, which lasted 15-30 min depending on the dose. This was associated with peak inhibition of acetylcholinesterase (AChE) in the frontal cortex and medulla, at 15 min, ranging from 35-51%. The analeptic effect of physostigmine in flunitrazepam-treated rabbits was prevented by pretreatment with scopolamine, 1 mg/kg. The effective dose range for physostigmine, 3-12 mumol/kg, is close to concentrations of this agent that inhibit activity in solubilized preparations of AChE from rabbit cortex, 1-3 X 10(-8) M. However, physostigmine, 10(-9) -10(-4) M, failed to displace 3H flunitrazepam from specific binding sites on membranes prepared from rabbit cerebral cortex. It is concluded that physostigmine antagonizes the somnolence and respiratory depression induced by benzodiazepines by restoring cholinergic transmission to normal levels. The effective dose range of physostigmine is small, and serious side effects from overdose can occur as a result of excess cholinergic activity at neuromuscular synapses. PMID- 3004261 TI - The influence of hypothermic cardiopulmonary bypass on neuromuscular transmission in the absence of muscle relaxants. PMID- 3004262 TI - Serial changes of plasma cortisol levels during various types of asthmatic responses due to allergen inhalation. AB - To observe possible changes in adrenal function during asthmatic attacks due to allergen inhalation, plasma cortisol levels were measured serially in asthmatic patients who showed immediate asthmatic responses, late asthmatic responses, and dual asthmatic responses were compared with their diurnal levels. There were slightly different patterns of cortisol responses among these groups. In the five patients who showed late asthmatic responses alone, cortisol levels fell significantly during late-type attacks although the decrease was quantitatively small. In the six patients who showed immediate asthmatic responses alone, cortisol levels tended to increase slightly during immediate-type attacks. Cortisol levels in the group with dual asthmatic responses increased slightly during immediate attacks and significantly decreased during late-type attacks, although the decrease was also small. PMID- 3004263 TI - [Application of hepatitis B virus DNA cloning in the diagnosis of infections related to hepatitis B virus]. PMID- 3004264 TI - [Comparison of 2 reagents for the measurement of serum 5'-nucleotidase activity (kinetic methods)]. PMID- 3004265 TI - [Analgesia with an implanted device for repetitive intrathecal injections of morphine]. AB - The use of intraspinal narcotics has been widely accepted as pain relief treatment for intractable cancer pain. Intraspinal low doses of morphine induce a potent selective long lasting analgesia. To avoid repetitive lumbar puncture, a drug delivery device was surgically implanted in 41 patients. The surgical procedure is described. The mean amount of morphine needed was 1.48 +/- 0.25 mg per day at time of surgery, rising to 6.86 +/- 1.47 mg per day after a mean survival time of 65 days. Tolerance became a major problem in 18 patients, which nearly all were selected at a late disease stage and previously received narcotics for pain relief. However, no clear-cut prognostic factor had a predictive value for the appearance of tolerance. In some cases, it could be successfully treated by intraspinal injection of local anaesthetics or clonidine. CSF leakage was noted in 11 patients; this was a challenge for us, as no other authors reported such a high rate for this complication. Aseptic meningitis was noted three times. In all cases but one, the symptoms resolved with appropriate treatment. PMID- 3004266 TI - Sequence relationships of United States prototype and wild-type bluetongue virus RNA genomes investigated by northern blot hybridization analysis. AB - The 10 double-stranded RNA (dsRNA) genome segments of various isolates of bluetongue virus (BTV) were separated on a polyacrylamide gel, denatured in NaOH, and blotted onto 2-aminophenylthioether paper. Blotted dsRNA segments were detected, using radioactive probes, a cloned copy of DNA 70% fragment of genome segment 7 of BTV-17, whole genome BTV-17 copy DNA, or whole genome BTV-17 dsRNA. These probes detected sequence diversities in different isolates of BTV and these diversities are discussed in relation to the serotype and the electrophoretic migration patterns of the isolates. PMID- 3004267 TI - Reclassification of North American leptospiral isolates belonging to serogroups Mini and Sejroe by restriction endonuclease analysis. AB - The genomes of North American strains of leptospires belonging to serogroups Mini and Sejroe were analyzed and compared with those of reference strains by cleavage with restriction endonucleases. The isolates selected for this study, when typed by the serologic method, were identified as serovars szwajizak, hardjo, and balcanica. However, the results of restriction endonuclease analysis (REA) indicated that a different classification existed. The 2 isolates typed as serovar szwajizak seem to be georgia by REA. Isolates belonging to serovars balcanica and hardjo had REA patterns that differed from both reference strains. Differences were not observed in the REA patterns between balcanica and hardjo isolates. All hardjo and balcanica isolates examined are suggested to be classified into a previously described hardjo, REA subtype hardjobovis. Using the enzyme Hha1, these isolates were subdivided into 3 subgroups. When examining the REA pattern of the 17 reference strains in serogroup Sejroe, 3 identical pairs were observed: wolffi and roumanica; sejroe and polonica; and istrica and nyanza. The REA again indicated that it will be a valuable method for the classification of leptospires. PMID- 3004268 TI - Virus isolation from semen of bulls serologically positive for bluetongue virus. AB - Isolation of bluetongue virus was attempted from 85 semen samples taken from 3 long-term seropositive bulls and 9 short-term seropositive bulls in an artificial breeding service unit. Two types of cell cultures susceptible to bluetongue virus were used for virus isolation. Extended sonication, centrifugation of specimens, and treatment of cell cultures with dimethyl sulfoxide and diethylaminoethyl dextran were used to enhance virus attachment and infection of cell cultures. Virus isolation results were negative on all specimens. These results indicate that at the limits of the methods used, bluetongue virus-seropositive bulls do not have long-term latent bluetongue virus in their semen. PMID- 3004269 TI - Acetylcholine-induced pulmonary vasodilation in lung vascular injury. AB - Recent work with isolated blood vessels has emphasized the importance of intact endothelium when the relaxation of vascular smooth muscle is induced by acetylcholine (ACh). However, the physiologic significance of this endothelial dependent ACh response in a complete organ circulation is unclear. We questioned whether diminished ACh vasodilation would result from damage of lung vascular endothelium and whether this response could be used as an indication of endothelial injury. We therefore induced pulmonary endothelial cell injury in one rat model by repeated injections of alpha-naphthyl thiourea (ANTU) and in a second rat model by exposing rats for 52 h to 100% oxygen at a barometric pressure of 760 torr (hyperoxia). Rats injected with Tween 80, the solvent for ANTU, or exposed to ambient Denver air served as the respective control animals. The isolated lungs of these rats were perfused with a recirculating cell- and plasma-free, physiological salt solution to study the effect of ACh or NaCl infusion on pulmonary perfusion pressure and vascular responsiveness. ANTU treated rats demonstrated an intact vasodilatory response after ACh infusion when compared with the solvent control animals. The immediate pulmonary vasodilation after ACh infusion was slightly enhanced in the hyperoxic rat lung when compared with the rats exposed to ambient air, but there was no difference between these groups in the prolonged depression of vascular responsiveness to hypoxia or angiotensin II. Thus, in both models of lung endothelial cell injury, the pulmonary vascular responses to ACh were intact.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004270 TI - Lung neutrophils in the adult respiratory distress syndrome. Clinical and pathophysiologic significance. AB - Although neutrophils are of pathogenetic importance in various animal models of acute lung injury, their role in the adult respiratory distress syndrome (ARDS) is unclear. To study the significance of lung neutrophils in this disorder, patients with ARDS (n = 11) were evaluated by bronchoalveolar lavage within 24 h of admission to the intensive care unit. Patients with non-ARDS respiratory failure requiring mechanical ventilation (n = 4) and normal volunteers (n = 12) were also studied. Neutrophils constituted 67.6 +/- 9.8% of recovered lavage cells in patients with ARDS compared with only 4.0 +/- 2.4% of cells in mechanically ventilated control patients and 0.8 +/- 0.2% in normal volunteers (p less than 0.005, both comparisons). Furthermore, in patients with ARDS (n = 6) evaluated serially by bronchoalveolar lavage at 72-h intervals, neutrophil percentages decreased from 91 +/- 3.2% (initial lavage) to 42.8 +/- 12% (final lavage) (p less than 0.005). Lung neutrophils also predicted the severity of abnormalities in gas exchange and lung protein permeability. That is, the percentage of neutrophils correlated directly with the alveolar-arterial Po2 difference (r = 0.69, p less than 0.01) and lavage fluid total protein concentrations (r = 0.62, p less than 0.01). Because large numbers of lung neutrophils were present in these patients, ARDS lavage fluid was assayed for neutrophil mediators relevant to the pathogenesis of acute lung injury. Neutrophil elastase activity was not detected in any ARDS lavages, although elastase was antigenically present in most samples and appeared to be complexed to alpha-1-antitrypsin. In contrast to elastase, neutrophil collagenase was readily detectable in ARDS fluid.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004271 TI - Colloidal gold immunoultrastructural localization of rat surfactant. AB - Using a polyclonal antiserum against the nonserum proteins in purified rat surfactant, we have localized protein antigen within the lamellar bodies of rat alveolar Type II cells perfusion-fixed with 2% cacodylate-buffered paraformaldehyde and postfixed with 0.5% osmium. A postembedment indirect immunogold ultrastructural localization was used and 20 nm gold particles were localized over the lamellae in Type II cell lamellar bodies, in tubular myelin, and in some of the secondary lysosomes of alveolar macrophages. Occasional labeling was seen in the rough endoplasmic reticulum and multivesicular bodies in some Type II cells, but the amount of this staining was not different from nonspecific background. There was, however, an invariant lack of labeling over all other lung cell types. These results demonstrate the presence of surfactant proteins within the lamellar body secretory product and support the idea that the surfactant lipoprotein complex is formed within intracellular sites prior to its secretion into the alveolar space. PMID- 3004272 TI - Opsonic receptor function is reduced on the surface of newborn alveolar macrophages. AB - Neonates have an increased susceptibility to infections. Because optimal phagocytosis of offending organisms by alveolar macrophages (AM) requires recognition and attachment of opsonized organisms to the AM cell membrane, the expression of opsonic receptors on the surface of newborn and adult rat AM was investigated using immunologic techniques, cell culture, and flow cytometry. We investigated the expression of Fc, C3b, fibronectin, and lectin receptors on newborn (1 to 5 wk) AM and compared them with those of adult AM. The expression of Fc receptors (FcR) was significantly less on the surface of newborn AM, particularly during the first week of life, as determined by their binding of aggregated IgG, E(IgG), and opsonized 125I-Listeria monocytogenes. A similar depressed receptor function was observed for C3b, fibronectin, and some lectins. The possible effect of cell size on receptor expression was examined by morphometry and flow cytometry. The results indicated that, while mean AM size was approximately 12% smaller during the first week of life, it attained adult levels by the second week. Thus, a difference in size did not account for either the magnitude of decreased receptor expression or the diminished intensity of staining for surface-aggregated IgG that was detectable for up to 5 wk. Binding of a variety of lectins to the AM surface was decreased during the first week after birth, but approximated adult levels by 4 wk. By contrast, soybean and peanut agglutinin, lectins that bind to D-GaINAC moieties, showed a transient increase in binding to AM during the second and third weeks of life.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004274 TI - [Respiratory infections associated with a cytomegalovirus]. AB - We report 27 children with respiratory tract disease in whom cytomegalovirus was isolated. These group excludes transplant patients and those on hemodialysis and mononucleosis. At the time of virus studies 7 had pneumonia, 3 chronic bronchopneumopathy, 2 bronchopneumonia, 8 pertussoid syndrome, 6 bronchitis or bronchiolitis and 1 laryngitis with glottic oedema. Virus studies consisted in cell cultures of biological products (pharyngeal exudates and urine). They were positive in 18 pharyngeal exudates, 24 urines, 2 bronchial brushings and 1 bronchoaspiration. In only 5 patients a complete serologic study was performed, with 3 seroconversions and one case of persistent high titers. Three patients had severe immune disease (2 hipogammaglobulinemias) and 1 dysgammaglobulinemia. These findings are discussed. PMID- 3004273 TI - The effect of furosemide during normoxemia and hypoxemia. AB - The effect of furosemide infusion was studied in 6 normal subjects and 6 patients with severe COPD and right ventricular failure during normoxemia and hypoxemia. In normal subjects, hypoxemia alone caused an insignificant (p less than 0.15) fall in plasma aldosterone concentrations (PAC). Intravenously administered furosemide resulted in significant (p less than 0.05) increases in PAC during both normoxemia and hypoxemia. After furosemide treatment mean arterial pressure (MAP) was significantly lower and arginine vasopressin (AVP) was significantly higher (p less than 0.05) with hypoxemia than with normoxemia. These changes in MAP and AVP were strongly correlated (r = -0.84). Urinary losses of water and sodium were similar after furosemide treatment with hypoxemia and normoxemia. In patients with right ventricular failure, neither changes in oxygenation nor furosemide infusion affected the markedly elevated baseline PRA and PAC levels. Arginine vasopressin levels were significantly higher with hypoxemia than with normoxemia, but urinary losses of water and salt did not differ between the 2 study days. We conclude that hypoxemia does not affect the PAC increase or urinary volume and sodium response to furosemide. PMID- 3004275 TI - An overnight high-dose dexamethasone suppression test for rapid differential diagnosis of Cushing's syndrome. AB - We have developed a high-dose dexamethasone suppression test that can be administered overnight with a single 8-mg dose and used the new procedure in the differential diagnosis of 83 patients with Cushing's syndrome. In 76 patients with surgically or pathologically proven cause--60 with Cushing's disease, 7 with the ectopic adrenocorticotrophic hormone syndrome, and 9 with adrenal tumors- suppression of plasma cortisol levels to less than 50% of baseline indicated a diagnosis of Cushing's disease. The test had a sensitivity of 92%, a specificity of 100%, and a diagnostic accuracy of 93%. These values equal or exceed those of the standard 2-day test whether based on suppression of urinary 17 hydroxycorticosteroids or plasma cortisol. We conclude that this overnight, high dose dexamethasone suppression test is practical and reliable in the differential diagnosis of Cushing's syndrome. PMID- 3004277 TI - Human T-lymphotropic virus type III in high-risk, antibody-negative homosexual men. AB - A cohort of 215 asymptomatic homosexually active men from a Boston community health center are being prospectively followed to assess the natural history of the human T-lymphotropic virus type III (HTLV-III) infection. To determine if certain asymptomatic persons who are HTLV-III antibody negative may be viremic, an algorithm was developed that defined high-risk characteristics (a sexual partner with the acquired immunodeficiency syndrome [AIDS]; more than 100 homosexual partners; or leukopenia, lymphopenia, neutropenia, or thrombocytopenia). Of 33 asymptomatic homosexual men who did not have antibody to HTLV-III and whose cases have not been previously reported, 2 had HTLV-III recovered from their lymphocytes. Clinical, behavioral, and hematologic data from seronegative persons did not distinguish between those with negative or positive viral cultures. Asymptomatic carriage of HTLV-III in high-risk seronegative persons underscores the need to base preventive educational strategies and behavioral modification on the assessment of risk factors and not solely on the results of HTLV-III antibody screening. PMID- 3004278 TI - Organophosphorus esters and polyneuropathy. PMID- 3004276 TI - Microbial causes of proven pelvic inflammatory disease and efficacy of clindamycin and tobramycin. AB - Thirty-six women with suspected pelvic inflammatory disease were examined by laparoscopy and endometrial biopsy. Acute salpingitis was diagnosed by laparoscopy in 22. Among women with evaluable biopsy samples, plasma cell endometritis was present in 14 of 20 with acute salpingitis and in 1 of 13 without acute salpingitis (p less than 0.001). Chlamydia trachomatis, Neisseria gonorrhoeae, or both were identified in the endometrium or fallopian tubes in 11 of 14 women with both salpingitis and endometritis, in 2 of 9 with salpingitis or endometritis alone, and in 0 of 13 without salpingitis or endometritis (p less than 0.0001). Anaerobic or facultative bacteria or mycoplasmas were isolated from tubes or peritoneum from 9 of 14 women with both salpingitis and endometritis, 2 of 9 with salpingitis or endometritis alone, and 3 of 13 without salpingitis or endometritis. Therapy with clindamycin plus tobramycin produced an adequate short term clinical response in 16 of 19 patients, although patients with severe salpingitis at laparoscopy responded slowly. PMID- 3004280 TI - Neurologic dysfunction in the idiopathic hypereosinophilic syndrome. PMID- 3004279 TI - Enzyme-linked immunosorbent assay of antibodies to Epstein-Barr virus nuclear and early antigens in patients with infectious mononucleosis and nasopharyngeal carcinoma. AB - A sensitive enzyme-linked immunosorbent assay was used to measure titers of IgG antibodies against bacterially synthesized Epstein-Barr virus nuclear antigen and early antigen in sera from 100 healthy North Americans, 40 North American patients with infectious mononucleosis, and 48 Asian patients with nasopharyngeal carcinoma. All healthy persons previously infected with Epstein-Barr virus had antibodies to nuclear antigen, and 70% had very low but detectable antibody titers to early antigen. In contrast, patients with mononucleosis had nondetectable or very low levels of antibodies to nuclear antigen and high antibody levels to early antigen. High levels of antibody to early antigen also were seen in patients with nasopharyngeal carcinoma, and a decrease in this response during the first 12 months after diagnosis and treatment was a significant prognostic indicator of survival. The probability of survival was 75% for patients whose antibody concentration to early antigen remained constant or decreased, and near 0% for patients with increasing levels of antibody. PMID- 3004282 TI - [A unusual manifestation of multiple sclerosis: sciatica]. AB - The authors recall the possibility of a typical sciatica in multiple sclerosis and describe seven new cases. Their main clinical features are: early onset before any other signs of the disease, hyperalgic, paroxysmal, short, repetitive episodes, absence of mechanical or effort triggers, absence of improvement with rest or anti-inflammatory drugs, well defined course. The intra-medullar origin of these attacks is suggested by the clinical criteria and the aspect of evoked potentials of the lower limbs recorded in 2 of the 7 patients. PMID- 3004281 TI - [Hepatic adenoma and oral contraceptives: personal case]. PMID- 3004283 TI - [Objective and subjective needs of mental patients 1 year after their discharge from a sectorized service]. AB - The present paper is based on a study of 50 non-randomized psychiatric patients who had left the hospital one year before. A semi-structured interview was used to acquire the data. Two types of data have been collected: objective and subjective. Objective data concern the diagnosis, marital status, living conditions, economic situation, educational and professional levels and medical care after hospitalization. As a whole living conditions were found to be very poor. Subjective data concern patients complaints, financial difficulties, their needs and their desire to get in contact with the community health center during hospitalisation. Most patients were in favour of starting after care just prior to discharge. All data are discussed. The most interesting finding is, that despite the many socio-economic hardships, the primary need expressed by the patients is a valuable personal relationship. PMID- 3004284 TI - Small proteins affect Na+ gradient-dependent D-glucose transport in isolated renal brush-border membrane vesicles. PMID- 3004285 TI - Na+-dependent D-glucose transport in brush-border membrane vesicles of chick small intestine: relation to Na+/H+ exchange and H+ permeability. PMID- 3004286 TI - Sodium-dependent conformational changes in the intestinal glucose carrier. PMID- 3004288 TI - Regulation of Na+/H+ exchange in lymphocytes. PMID- 3004287 TI - Cotransport systems for inorganic sulfate and phosphate in small intestine and renal proximal tubule. PMID- 3004289 TI - Properties of the renal Na+-H+ exchanger. PMID- 3004290 TI - Regulation of the activity of the Na+-H+ antiporter in brush-border membrane vesicles from the proximal tubule. PMID- 3004291 TI - Relative rates of Na+-H+ and Cl(-)-OH- exchange reactions in isolated intestinal cells. PMID- 3004292 TI - Principles of carrier catalysis elucidated by comparing two similar membrane translocators from mitochondria, the ADP/ATP carrier and the uncoupling protein. PMID- 3004293 TI - Structural and functional asymmetry of the ADP/ATP carrier from mitochondria. PMID- 3004294 TI - Proton electrochemical gradients and active transport: the saga of lac permease. PMID- 3004295 TI - Lactose: H+ carrier of Escherichia coli: kinetic mechanism, purification, and structure. PMID- 3004296 TI - Melibiose-cation cotransport system of Escherichia coli. PMID- 3004297 TI - Myo-inositol transport in bacteria: H+ symport and periplasmic binding protein dependence. PMID- 3004298 TI - Isolation and culture of murine renal proximal tubule cells: a system to study solute transport in mutants. PMID- 3004299 TI - Development of Na+-dependent hexose transport in cultured renal epithelial cells (LLC-PK1). PMID- 3004300 TI - Hormonal effects on sodium cotransport systems. PMID- 3004301 TI - Further studies of the effect of thyroid hormones on Na+-H+ exchange in renal brush-border membrane vesicles (BBMV). PMID- 3004302 TI - Glucose and sodium transport in brush-border membrane vesicles from fetal rabbit kidney. PMID- 3004303 TI - Regulation of cytochrome P-450(17)alpha activity and synthesis in bovine adrenocortical cells. PMID- 3004304 TI - Adenoid cystic carcinoma presenting as an orbital apex syndrome. AB - Adenoid cystic carcinomas (cylindromas) are distinctive neoplasms characterized by slow growth, perineural spread, extensive local invasion, frequent recurrence, and high mortality. A case of paranasal sinus adenoid cystic carcinoma presenting as an orbital apex syndrome is described. PMID- 3004305 TI - [Placental microvillus: study of the chemico-physical characteristics with electron paramagnetic resonance spectroscopy. I: The normal placenta]. PMID- 3004306 TI - [Approaches to the infra-spleno-temporal fossa and the lateral wall of the nasopharynx]. AB - The authors suggest replacement of the term "infra-temporal fossa" (indicating in the international anatomical nomenclature the pterygo-maxillary fossa) by the term "infra-spheno-temporal fossa" which would seem anatomically more appropriat. In the context of an operation performed in this region for a recurrence of a nasopharyngeal angiofibroma, a review and anatomical study of the different approaches has been undertaken and a classification established. The antero inferior approach via the cervico-trans-oral route would seem to be of particular interest, offering good exposure of the anatomical features of the region and avoiding the parotid bed ans dissection of the facial nerve. PMID- 3004307 TI - [Granular-cell tumor (Abrikosov). Immunohistological study of 4 cases with review of the literature]. AB - Granular-cell tumour (GCT) is a benign neoplastic proliferation first described by Abrikossoff (1), who considered it to be of muscular origin. Since then, however, the histogenesis of GCT has been a matter of serious controversy, and various cell types have been considered as being the origin of GCT. In this work we investigated the immunohistochemical profile of four cases of GCT using antibodies to several neural differentiation markers: S-100 protein, neurofilaments (NF), glial fibrillary acidic protein (GFAP). The results were compared to those obtained on three cases of schwannomas (S) and to those already reported in literature. Four cases of GCT were retrieved from the files of the Laboratory of Histopathology of the Clinic of Dermatology, Hop. E.-Herriot. The tumours had been observed during a two-year-period (1983-1984), fixed in Bouin's fixative and embedded in paraffin. In parallel, three cases of S that were treated in the same way were also studied. This was performed on 3 mu-thick paraffin sections using the avidin-biotin-peroxidase complex method (kit Vectastain, Vector Lab., Burlingame, USA). The following antibodies were used: a) antiserum to protein S-100 (Dakopatts, Denmark) (working dilution 1: 50); b) monoclonal antibody to 200 Kd NF (Labsystem, Helsinki) (working dilution 1: 40): C) monoclonal antibody to GFAP (Biosoft, Paris) (working dilution 1: 10).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004308 TI - [Histiocytofibroma and Dupuytren's palmar fibromatosis. Comparative autohistoradiographic study of their proliferative and biosynthetic activities]. AB - The proliferative rate and synthesis of proteins was studied in histiocytofibromas and in nodules of Dupuytren's palmar fibromatosis. Incorporations of tritiated thymidine and tritiated proline were used and revealed by autohistoradiography. In the two diseases, proliferation and protein synthesis occurred predominantly in cells of the vascular walls while the other cells constitutive of the lesions appeared less active. PMID- 3004309 TI - [Glucagonoma syndrome]. PMID- 3004310 TI - [Vegetating iododerma following lymphography]. PMID- 3004311 TI - [Verruciform epidermodysplasia and papillomavirus: 2 case reports. Presence of HPV-5 in a lymph node metastasis]. PMID- 3004312 TI - [Buschke-Lowenstein tumor. 3 vulvar localizations. Association with HPV-6 in one case]. PMID- 3004313 TI - [Gingival pigmentation caused by electrogalvanism: an entity studied with new technics: biochemical and ultrastructural analysis using a Castaing probe]. PMID- 3004314 TI - [Use of an electrode selective for perchlorate ions in the potentiometric analysis of quaternary ammonium by tetraphenylborate ions]. PMID- 3004316 TI - Oncogenes and human cancer--a surgeon's perspective. PMID- 3004315 TI - Paget's disease of the nipple after subcutaneous mastectomy for cancer with primary reconstruction. AB - Three cases of Paget's disease of the nipple are presented occurring in women treated for carcinoma of the breast with subcutaneous mastectomy and primary silicone reconstruction. These cases represent a 0.5% incidence of this problem in 584 women similarly treated over fourteen years. A suggested method of management is outlined. PMID- 3004317 TI - [The renin-angiotensin system and large arteries in the hypertensive patient]. AB - The large arteries are impaired in uncomplicated, permanent, essential arterial hypertension. The peripheral arteries such as the humeral artery or the common carotid artery have a normal or increased diameter, reduced blood flow and, especially, reduced compliance. Reduction of the arterial compliance reflects an impairment peculiar to the large blood vessels, independent of the pressure. Antihypertensive medicines, for a given drop in pressure, may increase, diminish or not change arterial compliance. This is an important point to be taken into account in relation to cardiovascular morbidity and mortality of treated hypertensives. It has been particularly well studied in the context of inhibitors of the renin-angiotensin system. PMID- 3004318 TI - Alpha-1 adrenoceptors are decreased in human epileptic foci. AB - Cortical alpha-1 adrenoceptors were measured in tissues obtained from 10 patients immediately following temporal lobectomy for intractable partial epilepsy. At operation each patient exhibited spontaneous spiking restricted to either the anterior (n = 5) or posterior (n = 5) portion of the first two temporal gyri. Control samples were obtained from the nonspiking half of the same gyrus. Receptor-binding assays were performed on isolated cortical membranes using [3H]prazosin. There was a reduction (p less than 0.01) in the receptor density (beta max) of the sites in the epileptic foci without any change in affinity (mean +/- SEM): spiking--beta max, 160.5 +/- 11.3 fmol/mg protein; affinity, 0.17 +/- 0.04 nM; nonspiking--beta max, 218.8 +/- 15.6 fmol/mg protein; affinity 0.17 +/- 0.04 nM. This relative decrease in alpha-1 adrenoceptor density may be the substratum of a noradrenergic hyposensitivity that could contribute to a localized diminution in inhibitory mechanisms in epileptic foci. PMID- 3004319 TI - Thrombotic cerebral vasculopathy associated with herpes zoster. AB - We describe the clinical, radiographic, and pathological findings in 3 patients with large-vessel cerebral vasculopathy following herpes zoster. Two of the patients were studied at postmortem examination, and a brain biopsy was performed in the third. Each of the 3 patients suffered thrombotic occlusions of large vessels without notable inflammatory or granulomatous changes following trigeminal or segmental herpes zoster infection. In the 2 autopsied patients, varicella-zoster virus (VZV) antigens were detected by immunoperoxidase staining within the media of the affected cerebral arteries. Little or no inflammation was associated with the foci of the VZV antigens. These studies provide evidence that the vasculopathy following herpes zoster may result from direct VZV infection of the artery and the in situ thrombosis can develop within the infected vessels in the absence of clear inflammatory vasculitis. PMID- 3004320 TI - Anticerebellar antibodies in serum and cerebrospinal fluid of a patient with oat cell carcinoma of the lung and paraneoplastic cerebellar degeneration. AB - A 56-year-old man was seen with subacute cerebellar degeneration and was found to have oat cell carcinoma of the lung. Antibodies to cerebellar Purkinje cells and granule cells were detected in both serum and cerebrospinal fluid (CSF), and intrathecal antibody synthesis was suggested by serum CSF antibody ratios, CSF IgG index, and CSF IgG synthesis rate. The patient's condition improved slightly with plasmapheresis, corticosteroids, and therapy of his underlying tumor, but he continued to exhibit a severe cerebellar deficit until his death more than one year later. Paraneoplastic cerebellar degeneration may be accompanied by synthesis of anticerebellar antibodies, both systemically and within the central nervous system. PMID- 3004321 TI - Ecto-5'nucleotidase activity in B and T lymphocytes and monocytes of patients with multiple sclerosis. AB - Ecto-5'nucleotidase activity in the plasma membrane of B and T lymphocytes and cultured and noncultured monocytes from patients with multiple sclerosis and from age- and sex-matched control subjects was determined by radioisotopic assay. The activity on lymphocytes and noncultured monocytes was normal, but the activity on cultured monocytes was found to be higher than normal in 70% of patients with active disease and lower than normal in 85% of patients with chronic inactive disease. Immunofluorescence staining with monoclonal antibodies, carried out in parallel with each assay, did not show any phenotypic differences between each cell population and its control. PMID- 3004322 TI - [Characteristics of the derepressed mutant of the F-like plasmid pAP18-1 coding for tetracycline resistance in Escherichia coli]. AB - A transfer function derepressed mutant of the F-like plasmid pAP18-1 (Tc, ColV) was induced with the help of N-methyl-N'-nitro-N-nitrozoguanidine. The mutant plasmid pAP18-1drd belongs to the FVII incompatibility group of the F-like plasmids. The plasmid pAP18-1drd is characterized by the loss of the capacity for inhibiting the tra-genes functions of the Flac plasmid and is sensitive to the Tra-function inhibitors of the reference plasmids of the FinV and FinW groups. PMID- 3004323 TI - Pharmacokinetic evaluation of UK-49,858, a metabolically stable triazole antifungal drug, in animals and humans. AB - The pharmacokinetic profile of UK-49,858 (fluconazole), a novel triazole antifungal agent which is being developed for oral and intravenous use, was determined in mice, rats, dogs, and humans. Comparative data following oral and intravenous administration showed that bioavailability was essentially complete in all four species. Peak concentrations in plasma of drug normalized to a 1 mg/kg dose level following oral administration, were relatively high: 0.7, 0.6, 1.1, and 1.4 micrograms/ml in mice, rats, dogs, and humans, respectively. The volumes of distribution ranged between 1.1 liter/kg in mice and 0.7 liter/kg in humans, which are approximate to the values for total body water. Whole body autoradiography studies in mice following intravenous administration of [14C]UK 49,858 demonstrated that the drug was evenly distributed throughout the tissues, including the central nervous system and the gastrointestinal tract. Plasma protein binding was low (11 to 12%) in all species. Marked species differences were observed in elimination half-lives, with mean values of 4.8, 4.0, 14, and 22 h in mice, rats, dogs, and humans, respectively. The major route of elimination of the drug was renal clearance, with about 70% of the dose being excreted unchanged in the urine in each species. Studies with [14C]UK-49,858 on metabolism and excretion (intravenous and oral) in mice and dogs showed that about 90% of the dose was recovered as unchanged drug in urine and feces, confirming the metabolic stability of the drug. This pharmacokinetic profile is markedly different from that of imidazole antifungal drugs and undoubtedly contributes to the excellent efficacy of UK-49,858 in vivo. PMID- 3004324 TI - Uptake, intracellular activity, and influence of rifampin on normal function of polymorphonuclear leukocytes. AB - Quinone and hydroquinone forms of rifampin accumulated in normal polymorphonuclear leukocytes (PMN) (maximal cellular to extracellular concentration ratio [C/Emax] +/- standard error of the mean, 9.36 +/- 0.54 and 8.82 +/- 0.65, respectively, after 5 to 10 min) and chronic granulomatous disease PMN (C/Emax, 13.76 +/- 0.77 and 14.29, respectively). Uptake of rifampin was influenced by incubation temperature and extracellular pH but not by phorbol myristate acetate stimulation or metabolic inhibitors. At extracellular concentrations between 0.06 and 5.0 mg/liter, rifampin significantly reduced the number of staphylococci surviving inside chronic granulomatous disease PMN, thus compensating for the bactericidal defect inherent with this disease. Spontaneous migration and chemotaxis of normal PMN were unaffected by rifampin. However, phagocytosis of yeast particles and oxygen consumption of stimulated PMN were moderately depressed, and O2- production and chemiluminescence were significantly depressed in a dose-dependent manner. The bactericidal activity of normal PMN was not impaired. Inhibition of chemiluminescence and O2- release were also observed in a cell-free system. We conclude that rifampin possesses favorable characteristics for the effective elimination of intracellular microorganisms. Further studies are needed to evaluate the in vivo significance of ion scavenging by rifampin, which could be hazardous to immunocompromised patients. PMID- 3004325 TI - Susceptibility of Campylobacter species to nalidixic acid, enoxacin, and other DNA gyrase inhibitors. AB - Nalidixic acid-resistant mutants of Campylobacter jejuni and C. coli as well as "C. laridis" strains showed cross-resistance to another DNA gyrase subunit A inhibitor, enoxacin (MIC, 32 micrograms/ml), whereas C. fetus subsp. fetus, C. fetus subsp. venerealis, and "C. hyointestinalis" strains were all susceptible to enoxacin (MIC, less than or equal to 2 micrograms/ml). All Campylobacter species were resistant to novobiocin (MIC, 32 to 512 micrograms/ml), but most strains were susceptible to the other DNA gyrase subunit B inhibitors coumermycin A1 and clorobiocin. PMID- 3004326 TI - The need for new antiviral agents. AB - Population density and immune status, vectors and virulence of infection, nutritional status, sanitation, genetic susceptibility and medical management of cases, are important factors influencing the incidence and/or severity of virus infections. Thus, the prevalence and clinical importance of virus infections and the need for antiviral drugs differ from place to place and from time to time. National and World Health Statistics of notifications of disease give some index of the incidence of infections but not all virus infections are notifiable. Such statistics can be misleading also through failures to notify from sloth on the part of the physician or, in the absence of pathognomonic symptoms or signs, from errors in diagnosis. Any assessment of the need for new antiviral drugs should consider the availability, safety, effectiveness and cost of alternative measures, including prevention of spread of infection by control of vectors, immunization by use of viral vaccines, or treatment with existing antiviral drugs. Early start of treatment of acute virus infections with existing drugs gives the best results and, where the clinical diagnosis is uncertain, accurate rapid virus diagnosis is of paramount importance. Many virus infections are asymptomatic or of trivial importance and without sequelae. However, new or improved antiviral drugs are needed for the prevention and/or treatment of a number of significant conditions caused by viruses which are not at present adequately controlled. These include upper and lower respiratory tract infections, influenza, chronic hepatitis, gastroenteritis, infectious mononucleosis, measles, rabies, haemorrhagic fevers and warts. Furthermore, such drugs might prove of therapeutic value in the prevention or treatment of virus associated tumours, such as hepatoma, nasopharyngeal carcinoma, Burkitt's lymphoma, Kaposi's sarcoma and possibly carcinoma of the cervix. PMID- 3004328 TI - Development of methods to measure virus inactivation in fresh waters. AB - This study concerns the identification and correction of deficiencies in methods used to measure inactivation rates of enteric viruses seeded into environmental waters. It was found that viable microorganisms in an environmental water sample increased greatly after addition of small amounts of nutrients normally present in the unpurified seed virus preparation. This burst of microbial growth was not observed after seeding the water with purified virus. The use of radioactively labeled poliovirus revealed that high percentages of virus particles, sometimes greater than 99%, were lost through adherence to containers, especially in less turbid waters. This effect was partially overcome by the use of polypropylene containers and by the absence of movement during incubation. Adherence to containers clearly demonstrated the need for labeled viruses to monitor losses in this type of study. Loss of viral infectivity in samples found to occur during freezing was avoided by addition of broth. Finally, microbial contamination of the cell cultures during infectivity assays was overcome by the use of gentamicin and increased concentrations of penicillin, streptomycin, and amphotericin B. PMID- 3004327 TI - The inactivation of herpes simplex virus by some Solanaceae glycoalkaloids. AB - The infectivity of herpes simplex virus Type I in tissue culture was inhibited by prior incubation with aqueous suspensions of glycoalkaloids in order of activity alpha-chaconine greater than alpha-tomatine greater than alpha-solasonine but not by the corresponding aglycones, solanidine, tomatidine and solasodine. However, inhibition was not only dependent on the presence of a sugar moiety since the glycone alpha-solanine was inactive under the conditions used. The glycones, but not the aglycones, showed cytopathic effects on cellular membranes of Vero cells and erythrocytes; therefore, it is suggested that inactivation of virus results from insertion of the glycones into the viral envelope. PMID- 3004329 TI - Method for detecting viruses in aerosols. AB - A simple method with poliovirus as the model was developed for recovering human enteric viruses from aerosols. Filterite filters (pore size, 0.45 micron; Filterite Corp., Timonium, Md.) moistened with glycine buffer (pH 3.5) were used for adsorbing the aerosolized virus. No virus passed the filter, even with air flow rates of 100 liters/min. Virus recovery from the filter was achieved by rapid elution with 800 ml of glycine buffer, pH 10. The virus in the primary eluate was reconcentrated by adjusting the pH to 3.5, adding AlCl3 to 0.0005 M, collecting the virus on a 0.25-micron-pore Filerite disk (diameter, 25 mm) and and eluting with 6 ml of buffer, pH 10. With this method, virus could be detected regularly in aerosols produced by flushing when 3 X 10(8) PFU of poliovirus were present in the toilet bowl. Poliovirus-containing fecal material from two of four infants who had recently received oral polio vaccine also yielded virus in the aerosols when feces containing 2.4 X 10(7) to 4.5 X 10(7) PFU of virus had been added to the toilet bowl. Persons infected with a variety of natural enteric viruses are known to excrete this amount of virus in their daily stools. PMID- 3004330 TI - Immunoperoxidase method with human immune serum globulin for broad-spectrum detection of cultivable human enteric viruses: application to enumeration of cultivable viruses in environmental samples. AB - The detection and enumeration of most cultivable human enteric viruses from water is possible if samples are first inoculated onto a suitable cell line such as MA 104 or BGM. Virus growth is then detected by an indirect immunoperoxidase method with human immune serum globulin as the source of antibody to most enteric viruses. The number of positive cell cultures in the immunoperoxidase assay is used to calculate the virus titer (as a most probable number) in the sample assayed. PMID- 3004331 TI - Evaluation of methods for concentrating hepatitis A virus from drinking water. AB - By using recently developed cultivation and assay systems, currently available methods for concentrating enteric viruses from drinking water by adsorption to and subsequent elution from microporous filters followed by organic flocculation were evaluated for their ability to recover hepatitis A virus (HAV). Cell culture adapted HAV (strain HM-175) in seeded tapwater was efficiently adsorbed by both electronegative (Filterite) and electropositive (Virosorb 1MDS) filters at pH and ionic conditions previously used for other enteric viruses. Adsorbed HAV was efficiently eluted from these filters by beef extract eluents at pH 9.5. Eluted HAV was further concentrated efficiently by acid precipitation (organic flocculation) of eluents containing beef extract made from powdered, but not paste, sources. By using optimum adsorption conditions for each type of filter, HAV was concentrated greater than 100-fold from samples of seeded tapwater, with about 50% recovery of the initial infectious virus added to the samples. The ability to recover and quantify HAV in contaminated drinking water with currently available methods should prove useful in further studies to determine the role of drinking water in HAV transmission. PMID- 3004332 TI - Concentration of viruses from water by using cellulose filters modified by in situ precipitation of ferric and aluminum hydroxides. AB - Untreated cellulose filters adsorbed only small amounts of poliovirus 1, echovirus 5, coxsackievirus B5, or bacteriophage MS2 that were added to tap water or to solutions of imidazole-glycine buffer at pH 5 to 7. Modification of filters by in situ flocculation of ferric and aluminum hydroxides greatly increased the ability of the filters to adsorb viruses. Viruses adsorbed to the modified filters could be recovered by treating the filters with 3% beef extract (pH 9.5). Greater than 60% of the enteroviruses and greater than 55% of the MS2 added to tap water or buffer could be recovered in the beef extract eluate. PMID- 3004333 TI - Enzyme regulation in C4 photosynthesis: purification, properties, and activities of thioredoxins from C4 and C3 plants. AB - Procedures are described for the purification to homogeneity of chloroplast thioredoxins f and m from leaves of corn (Zea mays, a C4 plant) and spinach (Spinacea oleracea, a C3 plant). The C3 and C4f thioredoxins were similar immunologically and biochemically, but differed in certain of their physiochemical properties. The f thioredoxins from the two species were capable of activating both NADP-malate dehydrogenase (EC 1.1.1.37) and fructose-1,6 bisphosphatase (EC 3.1.3.11) when tested in standard thioredoxin assays. Relative to its spinach counterpart, corn thioredoxin f showed a greater molecular mass (15.0-16.0 kDa vs 10.5 kDa), lower isoelectric point (ca. 5.2 vs 6.0), and lower ability to form a stable noncovalent complex with its target fructose bisphosphatase enzyme. The C3 and C4 m thioredoxins were similar in their specificity (ability to activate NADP-malate dehydrogenase, and not fructose-1,6 bisphosphatase) and isoelectric points (ca. 4.8), but differed slightly in molecular mass (13.0 kDa for spinach vs 13.5 kDa for corn) and substantially in their immunological properties. Results obtained in conjunction with these studies demonstrated that the thioredoxin m-linked activation of NADP-malate dehydrogenase in selectively enhanced by the presence of halide ions (e.g., chloride) and by an organic solvent (e.g., 2-propanol). The results suggest that in vivo NADP-malate dehydrogenase interacts with thylakoid membranes and is regulated to a greater extent by thioredoxin m than thioredoxin f. PMID- 3004334 TI - Kinetic properties of the insulin receptor tyrosine protein kinase: activation through an insulin-stimulated tyrosine-specific, intramolecular autophosphorylation. AB - The insulin receptor is an insulin-activated, tyrosine-specific protein kinase. Previous studies have shown that autophosphorylation of tyrosine residues on the Mr 95,000 is associated with an activation of the protein kinase activity toward exogenous protein substrates. We have employed the highly purified insulin receptor, immobilized on insulin-Sepharose or eluted in an active form, to define the metal/ATP requirements for kinase activation, the relationship of receptor autophosphorylation to activation, and the kinetic properties of the autophosphorylated, activated receptor kinase. Prior incubation of the immobilized receptor with 2 mM ATP, 10 mM Mg (or 10 mM Mn), followed by removal of these reactants, served to abolish the upward curvilinearity in the rate of histone 2b (tyrosine) phosphorylation measured subsequently. This treatment also markedly increased the rate of histone 2b phosphorylation as compared to that observed with the unmodified, immobilized receptor, as estimated under conditions that per se minimized further activation. The extents of maximal activation of receptor histone 2b (tyrosine) kinase obtained on preincubation with MgATP or MnATP are identical; however, the affinity of the receptor for MnATP is approximately 10-fold higher than that for MgATP. The higher affinity of the receptor for MnATP is observed for both autophosphorylation/autoactivation and histone 2b tyrosine kinase activity (Km MnATP approximately 0.01 mM; Km MgATP approximately 0.1 mM). Autophosphorylation/autoactivation per se does not significantly alter the apparent affinity for MeATP (or protein substrate, as previously reported) but increases Vmax. Activation of receptor histone 2b (tyrosine) kinase is due to tyrosine-specific autophosphorylation of the Mr 95,000 (beta) subunit; thus the extent of total 32P incorporation into the beta subunit correlates precisely with the extent of kinase activation, both over time and at a wide variety of Me2+ ATP concentrations. Sequential treatment of the autophosphorylated receptor with elastase and trypsin yields a single, basically charged 32P-peptide, Mr less than 2000. The functional properties of the unphosphorylated and fully phosphorylated receptor were compared after elution from insulin-Sepharose. The insulin binding characteristics of the two forms of the receptor were indistinguishable; the kinase properties differed greatly; whereas the histone 2b activity of the unphosphorylated receptor was low in the basal state, and activated 10-fold by insulin, the fully autophosphorylated receptor exhibits maximal histone 2b kinase in the basal state and is unaffected by insulin addition.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3004336 TI - Hydroxyl radical formation as a result of the interaction between primaquine and reduced pyridine nucleotides. Catalysis by hemoglobin and microsomes. AB - Kinetic, circular dichroism, and NADH and NADPH fluorescence quenching studies indicate that these compounds interact with the antimalarial drug primaquine (PQ). The affinity of both pyridine nucleotides for PQ is similar. The data are in contrast with a previous report (Thornalley et al. (1983) Biochem. Pharmacol. 32, 3571-3575) suggesting specificity for the interaction with NADPH. The complex was seen to facilitate electron transfer from NAD(P)H to oxygen, generating oxygen-free radicals which were detected by the spin-trapping technique and to flavin nucleotides, giving rise to flavin semiquinone radicals which were demonstrated by direct ESR spectroscopy under anaerobic conditions. A twofold increase in oxygen uptake and hydroxyl radical generation by the NAD(P)H-PQ complex was observed in the presence of hemoglobin. This effect was independent of heme concentration (in the range 1 X 10(-5)-1 X 10(-4) M) and oxidation state of the iron. Under anaerobic conditions, the NAD(P)H-PQ complex reduces Fe-III to Fe-II hemoglobin, and under aerobic conditions about 65% of the heme chromophore is irreversibly destroyed. Superoxide dismutase inhibits hydroxyl radical generation by the NAD(P)H-PQ pair; this effect is not observed in the presence of hemoglobin. In the presence of microsomes there is a 10-fold increase in both oxygen consumption and hydroxyl radical generation by the NAD(P)H-PQ pair. The fact that both pyridine nucleotides are active, and the inability of SKF 525A in decreasing hydroxyl radical generation, suggests that microsomal reductases are involved in the catalysis. PMID- 3004335 TI - Functional characterization of the uncoupler-insensitive Na+ pump of the halotolerant bacterium, Ba1. AB - Respiration initiates Na+ efflux from Na+-preloaded cells of the halotolerant bacterium, Ba1. This efflux can take place against the concentration and electrochemical gradients. Since it is not inhibited by carbonylcyanide-p trifluoromethoxyphenyl-hydrazone or N'N'-dicyclohexylcarbodiimide, it seems unlikely that either delta p (electrochemical potential difference of H+ across the membrane) generated by the primary proton pump or ATP play a role in the transduction of the energy supplied by electron transport. The electrogenic extrusion of Na+ causes passive counterflow of protons and/or simultaneous flux of permeant anions. In the absence of permeant anions the charge compensation attained by influx of protons is not complete. The membrane potential which persists in this case is inside negative and insensitive to uncoupler. The influx of protons builds up a delta pH of reversed sign (more acid inside), which is insensitive to uncoupler. The simultaneous efflux of Na+ and permeant anions diminishes the intracellular salt content and, as a corollary, causes volume contraction. Thus, the respiration-linked, uncoupler-insensitive Na+ pump may play a role in the regulation of the intracellular salt content. PMID- 3004337 TI - In vivo spin trapping of free radicals generated in brain, spleen, and liver during gamma radiation of mice. AB - Spin trapping techniques combined with electron spin resonance spectroscopy were used to capture and detect free radicals generated in vivo during exposure to ionizing radiation. Tissue extracts of mice given an intraperitoneal or intragastric dose of the spin trap, alpha-phenyl-t-butyl nitrone prior to exposure to gamma radiation (2 to 5 Gy), contained a radical adduct with hyperfine splitting constants characteristic of spin adducts of carbon-centered lipid radicals. Considerably more radicals were trapped in tissues when the trap was given 3 h before radiation as compared to 30 min before exposure. The radicals observed may either be secondary species resulting from an attack on cellular components by products of water radiolysis, or primary radicals resulting from direct interaction of the radiation with biological molecules. The results indicate that the spin trapping agent is able to penetrate well into animal tissues, and to capture radical species under conditions where the latter would be expected to occur. PMID- 3004338 TI - Iron and xanthine oxidase catalyze formation of an oxidant species distinguishable from OH.: comparison with the Haber-Weiss reaction. AB - O2- was produced by gamma irradiation of formate solutions, by the action of xanthine oxidase on hypoxanthine and O2, and by the action of ferredoxin reductase on NADPH and paraquat in the presence of O2. Its reaction with H2O2 and various iron chelates was studied. Oxidation of deoxyribose to thiobarbituric acid-reactive products that was appropriately inhibited by OH. scavengers, or formate oxidation to CO2, was used to detect OH(.). With each source of O2-, and by these criteria, Fe(EDTA) efficiently catalyzed this (Haber-Weiss) reaction, but little catalysis was detectable with iron bound to DTPA, citrate, ADP, ATP, or pyrophosphate, or without chelator in phosphate buffer. O2- produced from xanthine oxidase, but not from the other sources, underwent another iron dependent reaction with H2O2, to produce an oxidant that did not behave as free OH(.). It was formed in phosphate or bicarbonate buffer, and caused deoxyribose oxidation that was readily inhibited by mannitol or Tris, but not by benzoate, formate, or dimethyl sulfoxide. It did not oxidize formate to CO2. Addition of EDTA changed the pattern of inhibition to that expected for a reaction of OH(.). The other chelators all inhibited deoxyribose oxidation, provided their concentrations were high enough. The results are compatible with iron bound to xanthine oxidase catalyzing production of a strong oxidant (which is not free OH.) from H2O2 and O2- produced by the enzyme. PMID- 3004339 TI - Cyanide- and carbon monoxide-resistant mutants of Vitreoscilla: altered cytochromes and respiratory properties. AB - Two respiratory mutants of the aerobic bacterium, Vitreoscilla, have been studied: a CO-resistant mutant that can grow in 50% CO-50% oxygen, and a cyanide resistant mutant that can grow in 1 mM KCN. Wild-type cells are unable to grow under either condition. This report presents evidence that the resistance of the CO mutant is due to an altered membrane-bound cytochrome o [cytochrome o(m)], and that of the cyanide mutant is due to the presence of an increased amount of cytochrome d, which has a lower affinity for cyanide than cytochrome o(m). The evidence was obtained from spectral studies on the three types of intact cells as well as enzymatic and ligand-binding techniques on the cytoplasmic cytochromes o[cytochrome o(s)] and the respiring membrane vesicles isolated from these cells. Carbon monoxide difference spectra of intact cells revealed a 5-nm shift in an absorption maximum of a CO-binding pigment in the CO mutant relative to that of the wild type. The formation of oxygenated cytochrome o(s) and its conversion to the reduced form when the cells became anaerobic due to cellular respiration were inhibited when 1 mM KCN was added to a cell suspension of wild-type cells; the cyanide mutant cells showed resistance to cyanide in this experiment. Cytochrome o(s) purified from all three cell types had identical physical, electron transferring, and ligand binding properties within experimental error. Respiring membrane vesicles isolated from the two mutants showed more resistance to inhibition by cyanide and carbon monoxide than those from the wild type. Carbon monoxide difference spectra of these membrane vesicles revealed that there was a fivefold increase in the amount of cytochrome d in the cyanide mutant relative to the wild type. A CO absorption band of the membrane-bound cytochrome o in the CO mutant membrane vesicles showed a 5-nm shift relative to that of the wild type. PMID- 3004341 TI - Heparin modulates conformational states of plasma fibronectin: an electron spin resonance spin label approach. AB - We have examined the interaction between heparin and human plasma fibronectin using electron spin resonance (ESR) spin label methods. The titratable sulfhydryl groups of plasma fibronectin were modified with a maleimide spin label [Lai and Tooney (1984) Arch. Biochem. Biophys. 228, 465-473]. Addition of heparin resulted in a decrease in the maximum splitting value of the ESR spectrum of spin-labeled fibronectin from 66.8 to 64.3 G, suggesting that heparin induces a conformational alteration of plasma fibronectin. This heparin effect was noticeable at a heparin to-fibronectin ratio of 20 to 1 and reached a plateau at about 100 to 1. Other sulfated carbohydrates were tested; dextran sulfate was found to be as effective as heparin but chondroitin sulfates were ineffective. The results presented suggest that the binding of heparin changes the molecular conformation of plasma fibronectin to a more relaxed or flexible state. PMID- 3004340 TI - Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts by reversible phosphorylation: modulation of enzymatic activity by low density lipoprotein, sterols, and mevalonolactone. AB - 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase exists in interconvertible active and inactive forms in cultured fibroblasts from normal and familial hypercholesterolemic subjects. The inactive form can be activated by endogenous or added phosphoprotein phosphatase. Active or partially active HMG CoA reductase in cell extracts was inactivated by a ATP-Mg-dependent reductase kinase. Incubation of phosphorylated (inactive) HMG-CoA reductase with purified phosphoprotein phosphatase was associated with dephosphorylation (reactivation) and complete restoration of HMG-CoA reductase activity. Low density lipoprotein, 25-hydroxycholesterol, 7-ketocholesterol, and mevalonolactone suppressed HMG-CoA reductase activity by a short-term mechanism involving reversible phosphorylation. 25-Hydroxycholesterol, which enters cells without the requirement of low density lipoprotein-receptor binding, inhibited the HMG-CoA reductase activity in familial hypercholesterolemic cells by reversible phosphorylation. Measurement of the short-term effects of inhibitors on the rate of cholesterol synthesis from radiolabeled acetate revealed that HMG-CoA reductase phosphorylation was responsible for rapid suppression of sterol synthesis. Reductase kinase activity of cultured fibroblasts was also affected by reversible phosphorylation. The active (phosphorylated) reductase kinase can be inactivated by dephosphorylation with phosphatase. Inactive reductase kinase can be reactivated by phosphorylation with ATP-Mg and a second protein kinase from rat liver, designated reductase kinase kinase. Reductase kinase kinase activity has been shown to be present in the extracts of cultured fibroblasts. The combined results represent the initial demonstration of a short-term regulation of HMG-CoA reductase activity and cholesterol synthesis in normal and receptor negative cultured fibroblasts involving reversible phosphorylation of both HMG CoA reductase and reductase kinase. PMID- 3004343 TI - Phosphoinositide phosphorylation and hydrolysis in pancreatic islet cell membrane. AB - Membranes were isolated from dispersed rat pancreatic islet cells by attachment to Sephadex beads. When these membranes were exposed to [gamma-32P]ATP, formation of 32P-labeled phosphatidate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate was observed. Carbamylcholine, added 10 s prior to lipid extraction, caused a dose-related fall in 32P-labeled phospholipids. The effect of the cholinergic agent was suppressed by atropine, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, and verapamil, and simulated, in part, by an increase in Ca2+ concentration. When the membranes were derived from islet cells prelabeled with [U-14C]arachidonate, carbamylcholine stimulation, in addition to decreasing labeled polyphosphoinositides, was accompanied by an increased production of labeled diacylglycerol, without a concomitant increase in labeled phosphatidylinositol. These results indicate that activation of a plasma membrane-associated phospholipase C directed against polyphosphoinositides represents a primary event in the functional response of the pancreatic beta cell to cholinergic agents. PMID- 3004342 TI - Interaction of the extrinsic potential-sensitive molecular probe diS-C3-(5) with pigeon heart mitochondria under equilibrium and time-resolved conditions. AB - Some aspects of the interaction of the extrinsic, potential-sensitive, molecular probe diS-C3-(5) with pigeon heart mitochondria are reported in this paper. Binding studies based on fluorimetry indicate that the ratio of the dissociation constant to the maximum number of binding sites, KD/n, is larger for succinate containing mitochondria than that for cyanide-inhibited preparations. These observations suggest that the basis of the energy-dependent diS-C3-(5) optical signals is the ejection of the probe from the mitochondrial membrane. A more detailed analysis indicated that the major change in the binding parameters is a reduction in the maximum number of binding sites, n, when a charge gradient is formed at the expense of substrate. Using rapid mixing techniques, the time course of the passive association of diS-C3-(5) with mitochondria, that of the glutamate- and ATP-dependent optical signals, and the effect of this probe on the rate at which the energy-dependent cytochrome c oxidase Soret band shift signal develops have been monitored. Retardation the ATP-dependent cytochrome c oxidase Soret band shift signal suggests that the probe readily permeates the mitochondrial membrane. The first-order rate law that the glutamate-dependent signal obeys suggests that the rate-limiting step in the development of this signal is the dissociation of the dye from the mitochondrial membrane or the permeation of this membrane by the probe. The faster phase of the ATP-induced signal likely reflects the initial transfer of dye from the bulk aqueous phase followed by a slower probe permeation process that obeys a first-order rate law. This probe appears to distribute across the mitochondrial membrane in accordance with the transmembrane potential as judged by its effect on the ATP-dependent cytochrome c oxidase Soret band shift signal. DiS-C3-(5) also appears to inhibit the NADH dehydrogenase. PMID- 3004344 TI - Metabolism of leukotriene E4 by rat tissues: formation of N-acetyl leukotriene E4. AB - Leukotriene E4 was incubated with subcellular fractions from rat liver homogenates. A product identified as 5-hydroxy-6-S-(2-acetamido-3-thiopropionyl) 7,9-trans-11,14- cis-eicosatetraenoic acid (N-acetyl leukotriene E4) was formed. Enzymes catalyzing the reaction were associated with particulate fractions sedimenting between 600 and 8500 g and 20,000 and 105,000 g. Acetyl coenzyme A served as the donor of the acetyl group. N-Acetyl leukotriene E4 was also formed by the 105,000g sediment fractions from kidney, spleen, skin, and lung. The myotropic activity of N-acetyl leukotriene E4 on isolated guinea pig ileum was reduced over 100-fold compared to that of leukotriene D4. PMID- 3004345 TI - Covalent modification of Escherichia coli ADPglucose synthetase with 8-azido substrate analogs. AB - Two photoaffinity labeling agents, 8-azido-ATP and 8-azido-ADPglucose, are substrate site specific probes of the Escherichia coli ADPglucose synthetase. In the presence of light (254 nm), the analogs specifically and covalently modify the enzyme with concomitant loss of catalytic activity. The substrate ADPglucose completely protects the enzyme from covalent modification by these 8-azido analogs. ATP, another substrate, also provides nearly 100% protection from 8 azido-ATP inactivation but is less efficient in protection of inactivation by 8 azido-ADPglucose. In the absence of light, however, ADPglucose synthetase can utilize either 8-azido-ATP or 8-azido-ADPglucose as substrates. PMID- 3004346 TI - Effects of ATP and monovalent cations on Mg2+ inhibition of (Na,K)-ATPase. AB - The hydrolysis of ATP catalyzed by purified (Na,K)-ATPase from pig kidney was more sensitive to Mg2+ inhibition when measured in the presence of saturating Na+ and K+ concentrations [(Na,K)-ATPase] than in the presence of Na+ alone, either at saturating [(Na,Na)-ATPase] or limiting [(Na,0)-ATPase] Na+ concentrations. This was observed at two extreme concentrations of ATP (3 mM where the low affinity site is involved and 3 microM where only the catalytic site is relevant), although Mg2+ inhibition was higher at low ATP concentration. In the case of (Na,Na)-ATPase activity, inhibition was barely observed even at 10 mM free Mg2+ when ATP was 3 mM. When (Na,K)-ATPase activity was measured at different fixed K+ concentrations the apparent Ki for Mg2+ inhibition was lower at higher monovalent cation concentration. When K+ was replaced by its congeners (Rb+, NH+4, Li+), Mg2+ inhibition was more pronounced in those cases in which the dephosphorylating cation forms a tighter enzyme-cation complex after dephosphorylation. This effect was independent of the ATP concentration, although inhibition was more marked at lower ATP for all the dephosphorylating cations. The K0.5 for ATP activation at its low-affinity site, when measured in the presence of different dephosphorylating cations, increased following the sequence Rb+ greater than K+ greater than NH+4 greater than Li+ greater than none. The K0.5 values were lower with 0.05 mM than with 10 mM free Mg2+ but the order was not modified. The trypsin inactivation pattern of (Na,K)-ATPase indicated that Mg2+ kept the enzyme in an E1 state. Addition of K+ changed the inactivation into that observed with the E2 enzyme form. On the other hand, K+ kept the enzyme in an E2 state and addition of Mg2+ changed it to an E1 form. The K0.5 for KCl induced E1-to-E2 transformation (observed by trypsin inactivation profile) in the presence of 3 mM MgCl2 was about 0.9 mM. These results concur with two mechanisms for free Mg2+ inhibition of (Na,K)-ATPase: "product" and dead-end. The first would result from Mg2+ interaction with the enzyme in the E2(K) occluded state whereas the second would be brought about by a Mg2+-enzyme complex with the enzyme in an E1 state. PMID- 3004347 TI - The role of respiration during adaptation of the freshwater cyanobacterium Synechococcus 6311 to salinity. AB - Growth of the freshwater cyanobacterium Synechococcus 6311 under saline conditions stimulated respiration tenfold during the first 24 h, while growth and photosynthesis were inhibited. The elevated respiration rate was seen under both light and dark conditions, was uncoupler and cyanide sensitive, and did not decrease upon salt removal. Membrane preparations from salt-grown cells exhibited a tenfold increase in cytochrome oxidase activity, while electron transfer rates from NADPH to cytochrome c only increased threefold. Cytochrome oxidase activities were correlated with levels of EPR detectable Cu2+ in the salt and control membranes. Sodium-driven proton (antiproter) gradients in salt-grown cells were sensitive to cyanide but not dicyclohexylcarbodiimide, indicating the direct role of respiratory electron transport in maintaining low intracellular sodium levels. PMID- 3004349 TI - Respiration-linked proton flux in Wolinella succinogenes during reduction of N oxides. AB - Formate uncoupled proton translocation in formate-grown Wolinella succinogenes cells supplied with N-oxides as terminal electron acceptors. In suspensions containing KSCN (but not valinomycin), H2 supported proton translocation when NO3 , NO2-, and NO were provided as oxidants. H+/N-oxide ratios were 4.77 for NO3-, 2.49 for NO2-, and 1.75 for NO. KSCN inhibits N2O reduction thus precluding use of N2O as oxidant. Repeated exposure of cells to NO inhibited their ability to translocated protons with NO as oxidant but only slightly diminished and did not eliminate their capacity for NO3(-)- or NO2(-)-dependent proton flux. Substituting reduced benzyl viologen for H2 and measuring proton uptake provided results consistent with an extramembranal location for the N- oxide reductases. The uncoupler, carbonyl cyanide m-chlorophenylhydrazone, collapsed proton gradients, permitted uptake of 2 mol H+/mol NO3- or NO2-, but unaccountably inhibited NO3- reduction by 50% while leaving H+ uptake stoichiometry of the cells unaffected. PMID- 3004348 TI - Mechanism of gastric antisecretory effect of thiocyanate: further evidence for the thiocyanate-induced impediment in gastric H+,K+-ATPase function. AB - Two hypotheses have recently been proposed for the thiocyanate inhibition of gastric acid secretion--a protonophore mechanism and an uncoupling model. The mechanistic aspects for the latter scheme have been examined on the following basis: capability of generating verifiable predictions, supporting evidence that is unambiguous, and compatibility with experimental realities. Gastric microsomes bind 5 nmol of SCN-/mg, and a "pure" and highly active fraction of H+,K+-ATPase prepared from gastric microsomes binds about 15 nmol of SCN-/mg. The affinity of SCN- binding to gastric microsomes changes from 10 to 25 mM in the presence of 20 mM K+ suggesting competition between K+ and SCN-. Potassium also displaces the bound SCN- from "pure" H+,K+-ATPase with a Ki of about 25 mM. Of the cations tested--Tl+, K+, Rb+, Cs+, NH4+, Na+, and Li+--Tl+ was the most effective in displacing bound SCN- while Na+ and Li+ were without effect. The effects of anions such as Cl-, NO3-, and gluconate were found to be nonspecific and absolutely dependent on K+ as cocation. Sulfate and OCN-, on the other hand, showed an ability to displace SCN- as both K+ and Na+ salts. For SO4(-2) the K+ form was much more effective than the Na+ salt. Besides these antagonistic effects of K+ and congeners with the H+,K+-ATPase-bound SCN-, a competition between K+ and SCN- was also observed at the level of gastric K+-stimulated pNPPase reaction. The effects of SCN- and two other unrelated anions, F- and NO2 , on artificial delta pH across the microsomal vesicles exhibited a lack of appreciable change up to 5 mM and a small (about 13%) reduction between 10 and 20 mM. However, a combination of CCCP and nigericin or valinomycin completely abolished the delta pH under identical conditions. The present data in conjunction with other reports suggest that the proton impediment model best explains the gastric antisecretory effects of SCN-. PMID- 3004350 TI - 1H-NMR spectroscopic manifestations of ligand binding to the kringle 4 domain of human plasminogen. AB - Structural aspects of the binding of the linear ligands N alpha-acetyl-L-lysine (AcLys) and epsilon-aminocaproic acid (epsilon ACA) and of the cyclic analogs trans-(aminomethyl)-cyclohexanecarboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) to the intact plasminogen kringle 4 domain have been investigated by 1H-NMR spectroscopy at 300 and 600 MHz. Ligand binding results in consistent shifts of the His-II (His31), Trp-I (Trp25?), Trp-II (Trp62?), Trp-III (Trp72), Tyr-II (Tyr50), and Phe64 ring signals. BASA tends to induce larger shifts than elicited by the aliphatic ligands, most noticeably on Trp-II and on Trp72, suggesting that the ligand aromatic ring interacts with the two indole groups. Trp-II and, to lesser extent, Trp-I interact with an acidic side chain group, in a manner that is blocked by BASA. BASA binding also perturbs Tyr-II (Tyr50), Tyr III (Tyr41), and Tyr-IV (Tyr74) over a wide pH range and lowers the pKa* of His31 from approximately 4.8 to approximately 4.6. His-III (His33) responds to BASA and AMCHA but is relatively insensitive to the linear ligands. His33 carries a sterically shielded side chain which, in conjunction with Leu46, Trp-I, Tyr50, and Tyr74, participates in structuring the kringle hydrophobic core, contiguous to the binding site. Pronounced shifts are observed for aliphatic resonances stemming from the kringle-bound molecules of AMCHA, AcLys, and epsilon ACA. It is proposed that the lysine-binding site is mostly supported by the loop that extends from Cys51 through Cys71 and that aromatic residues, which include Trp II, Trp72, and Phe64, play a major role in interacting with the nonpolar segment of the ligand molecule. The binding site also encompasses Tyr50, Tyr74, His31, and His33 although it is not clear the extent to which these residues interact directly with the ligand. PMID- 3004351 TI - Purification and properties of two DNA ligases from human placenta. AB - Two DNA ligase activities have been separated, purified, and characterized. The resolution of the two enzymes from crude extracts was initially achieved through Polymin P precipitation. The ligation activity precipitating with the nucleic acids on treatment with Polymin P is designated as DNA ligase I, and an activity remaining in the supernatant fraction, as DNA ligase II. DNA ligase I and II are ATP and Mg2+-dependent enzymes with pH optima of 7.8 and 8.0 and isoelectric points of 6.9 and 7.6, respectively. The purified I and II DNA ligase activities have molecular weights of 83,000 and 89,000, respectively. Both activities are inhibited by dATP and inorganic pyrophosphate. However, in the presence of optimum rATP levels, dATP stimulates DNA ligase II activity, whereas DNA ligase I is inhibited under the same conditions. Both activities are DNA specific and ligation follows reaction steps similar to those described for the Escherichia coli DNA ligase. PMID- 3004353 TI - Separation of dihydropyridine binding sites from cardiac junctional sarcoplasmic reticulum. AB - When microsomes from feline ventricular muscle are centrifuged on continuous linear sucrose gradients, the major peak for the distribution pattern of the dihydropyridine binding sites corresponds in position and shape with the distribution of the Mr 300K polypeptide marker for junctional sarcoplasmic reticulum (SR). Plasma membrane vesicles are also present in those gradient fractions and appear to be joined to the junctional SR as native dyads. We now report that when such putative dyads are passed through the French press, both the dihydropyridine binding sites and the plasma membrane marker band together at a new isopycnic point distinct from the junctional SR. We conclude that as has been found in the skeletal muscle system the dihydropyridine binding sites are a marker for the junctional domain of the plasma membrane and that separation of the dyad components of the mammalian myocardium can be attained. PMID- 3004352 TI - Utilization of membranous lipid substrates by membranous enzymes: activation of the latent sphingomyelinase of hen erythrocyte membrane. AB - Previous studies demonstrated that hen erythrocytes have an inoperative, latent sphingomyelinase which is activated when the cells are hemolyzed in a hypotonic medium. Within minutes after hemolysis about 60-80% of the sphingomyelin (SPM) of the RBC "ghost" membrane was hydrolyzed. In this paper, expression of sphingomyelinase activity was further investigated. The percentage of total SPM hydrolyzed depended on the volume of the hypotonic hemolyzing buffer. Thus, suspending the erythrocytes in 4 vol of the buffer resulted in clumping of the hemolyzed "ghosts" and no hydrolysis of SPM. In comparison, suspension in 19 vol of the hypotonic buffer showed no clumping and sphingomyelinase activity was fully expressed. But centrifugation of the latter or, alternatively, addition of concanavalin A induced clumping and elimination of sphingomyelinase activity. Hen RBC could also be hemolyzed in an isotonic medium in the presence of Triton X 100, mellitin, halothane, and phospholipase C. Activation of the latent sphingomyelinase occurred at concentrations of these reagents which caused cell lysis. Hen RBC were dispersed in an isotonic medium containing glutaraldehyde (0.1%) or formaldehyde (10%). This rendered the cells resistant to hemolysis, even when subsequently dispersed in a hypotonic medium or water. But incubation of the "fixed" cells in a hypotonic or isotonic medium activated the enzyme, resulting in hydrolysis of 60% of the cellular SPM. In contrast, when glutaraldehyde was included in the hypotonic buffer, hemolysis occurred but sphingomyelinase activity was eliminated. PMID- 3004354 TI - Purification and characterization of ribulose-5-phosphate kinase from spinach. AB - An efficient purification procedure utilizing affinity chromatography is described for spinach ribulose-5-phosphate kinase, a light-regulated chloroplastic enzyme. Gel filtration and polyacrylamide gel electrophoresis of the purified enzyme reveal a dimeric structure of 44,000 Mr subunits. Chemical crosslinking with dimethyl suberimidate confirms the presence of two subunits per molecule of native kinase, which are shown to be identical by partial NH2 terminal sequencing. Based on sulfhydryl titrations and on amino acid analyses, each subunit contains four to five cysteinyl residues. The observed slow loss of activity during spontaneous oxidation in air-saturated buffer correlates with the intramolecular oxidation of two sulfhydryl groups, presumably those involved in thioredoxin-mediated regulation. PMID- 3004355 TI - 1H-NMR assignments of GM1-oligosaccharide in deuterated water at 500 MHz by two dimensional spin-echo J-correlated spectroscopy. AB - The 1H-NMR spectra of the oligosaccharide derived from monosialoganglioside GM1 (GM1 = beta-D-galactosyl-(1-3)-beta-D-N-acetylgalactosaminyl-(1-4)- [alpha-N acetylneuraminyl-(2-3)-]-beta-D-galactosyl-(1-4)-b eta-D-glucosylceramide) (GM1OS) and its reduced form (GM1OS-R) have been obtained at 500 MHz in D2O. Through the combined use of one-dimensional and homonuclear two-dimensional spin echo J-correlated (2D SECSY) spectra of GM1OS-R, the assignments for the ring protons of GM1OS are made. Data on chemical shifts and coupling constants of GM1OS including the alpha-linked neuraminic acid protons, in aqueous solution, are tabulated. Due to the very small coupling constants (less than 2 Hz) and the closeness in chemical shifts (less than 0.04 ppm) for the pair of correlated peaks in the two-dimensional spectrum, the information on the connectivities of the H5 ring protons of the neutral sugar residues is missing. Second-order coupling also blurs this information. Data are compared with those obtained for ganglioside GM1 in dimethyl sulfoxide (DMSO; the actual composition therein was 97% DMSO-d6 and 3% D2O) by T. A. W. Koerner, J. H. Prestegard, P. C. Demou, and R. K. Yu (1983, Biochemistry 22, 2676). While the heterogeneity of chemical shifts for the H5, H6a, and H6b protons diminishes in D2O, that for A-9a and A-9b remains. The latter suggests an intraneuraminic acid conformation involving the glycerol side chain unaffected by the solvent. Moreover, the chemical shifts of the III-1, III-2, and A-4 protons (and perhaps the II-4, IV-2, and A-8 protons) in D2O exhibit unusual upfield shifts compared with those in DMSO. This indicates that the intramolecular interactions between GalNAc residue III and neuraminic acid present in DMSO are weakened in D2O. The effect of temperature on the conformation is also examined and appears to be minimal (less than 0.02 ppm) in the range 22-50 degrees C. PMID- 3004356 TI - Similarity of kinetics of three types of myeloperoxidase from human leukocytes and four types from HL-60 cells. AB - Km values for H2O2 and Vmax values for three types of myeloperoxidase (MPO) from human leukocytes (MPO-I, -II, and -III) and four types from human myeloid leukemia HL-60 cells (MPO-IA, -IB, -II, and -III) were determined. Km values of human leukocyte MPOs decreased with increasing pH from 4.4 to 6.2 and increased with increasing NaCl concentration from 0.025 to 0.14 M. There was no significant difference among Km values of leukocyte MPO-I, -II, and -III. NaBr also showed a tendency similar to that of NaCl with regard to the effects of pH and halide concentration on Km values. However, Km values in the presence of NaBr were lower than those in the presence of NaCl. Effects of pH and NaCl concentration on Vmax values of MPO-I, -II, and -III were also examined. Vmax values of MPO-I, -II, and -III were higher at pH 4.9 and 5.4 and increased with increasing NaCl concentration. In addition, no difference was observed between Km values of leukocyte and those of HL-60 cells. MPO-IB, the half-molecular-weight enzyme of HL-60 cells, also had the same Km values as the others. Furthermore, inhibition of the activities of seven MPOs of leukocytes and HL-60 cells by H2O2 was similarly observed at concentrations above 1 mM at pH 5.4 but not at pH 4.4. These results indicate that there is no difference in the affinity to H2O2 among leukocyte MPO-I, -II, and -III and HL-60 cell MPO-IA, -IB, -II, and -III. PMID- 3004357 TI - The reactions of the phosphorylated pathway of L-serine biosynthesis: thermodynamic relationships in rat liver in vivo. AB - The effect of the induction of the enzymes of the phosphorylated pathway of L serine biosynthesis on the thermodynamic relationships among the reactions has been determined in rat liver in vivo. The mass action ratios of the reactions involved were calculated from the concentrations of appropriate metabolites in freeze-clamped liver from animals fed normal and low-protein diets for 2 weeks. These ratios were compared with the equilibrium constants of the same reactions previously determined under physiological conditions and the results previously obtained in the rabbit. The thermodynamic relationships in the pathway were different between the normal rat and rabbit as might have been expected, because of the significantly lower activities of the L-serine biosynthetic enzymes in the former animal. Although the delta G for the overall pathway is nearly identical in the rat and rabbit (-5.8 versus -5.5 kcal/mol, respectively), the distribution of delta G among the reactions is different. The disequilibrium in the pathway in rat liver is nearly equally divided between the L-phosphoserine phosphatase (EC 3.1.3.3) step and the other two reactions [D-3-phosphoglycerate dehydrogenase (EC 1.1.1.95) and L-phosphoserine aminotransferase (EC 2.6.1.52)], whereas in rabbit the phosphatase reaction accounts for nearly the entire delta G. Feeding the rat a low protein diet, however, induced the activity of D-3-phosphoglycerate dehydrogenase 12-fold, that of L-phosphoserine aminotransferase 20-fold, and that of L-phosphoserine phosphatase 2-fold. With the induction of the pathway, L phosphoserine appeared in the tissue, there was a more than 3-fold rise in L serine in the liver, and the pattern of delta G in the rat liver approached that in the rabbit. PMID- 3004359 TI - Inhibition of fructose bisphosphatase and stimulation of phosphofructokinase by a stable isosteric phosphonate analog of fructose 2,6-bisphosphate. AB - We describe the synthesis of a mixture of D-manno- and D-gluco-2,5-anhydro-1 deoxy-1-phosphonohexitol 6-phosphate via a Horner-Emmons reaction of 2,3,5-tri-O benzyl-beta-D-arabinofuranose followed by phosphorylation of the equivalent 6 position and subsequent deprotection. This mixture inhibits fructose-1,6 bisphosphatase; the concentration required for half-maximal effect in the presence of 25 microM AMP is approximately 6 microM. The mixture of analogs also stimulates 6-phosphofructo-1-kinase from rabbit liver; the concentration required to reach one-half Vmax was found to be ca. 25 microM at 0.25 mM fructose 6 phosphate and 50 microM AMP. These analogs have replaced the labile anomeric phosphate of fructose 2,6-bisphosphate with a stable methylenephosphonate, and could be of great interest due to their appropriate physiological effects and their chemical stability. PMID- 3004360 TI - Extracellular ATP activates polyphosphoinositide breakdown and Ca2+ mobilization in Ehrlich ascites tumor cells. AB - The effects of extracellular ATP on phosphoinositide metabolism and intracellular Ca2+ homeostasis were studied in Ehrlich ascites tumor cells. Cytosolic [Ca2+] was measured using either quin 2 or the recently described indicator fura 2. Addition of 0.5-25 microM extracellular ATP to intact cells results in a rapid mobilization of Ca2+ from a nonmitochondrial, intracellular Ca2+ store. Likewise, direct addition of 0.2-2 microM myo-1,4,5-inositol trisphosphate (IP3) to digitonin-permeabilized Ehrlich cells induces a rapid and reversible release of Ca2+ from a nonmitochondrial pool. Under the same conditions which facilitate intracellular Ca2+ mobilization, extracellular ATP also triggers a rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and accumulation of IP3. A maximal 18% decrease of the polyphosphoinositide is observed 40-60 s after the addition of 25 microM ATP; within 5 min PtdIns(4,5)P2 returns to or exceeds the original, prestimulus level. These conditions also trigger a rapid accumulation of phosphatidic acid (1.7-fold increase within 5 min). Paralleling these ATP-induced changes in phospholipid levels is a substantial accumulation of the mono-, bis-, and trisphosphate derivatives of inositol; most significantly, a 2-fold increase in the IP3 level is observed within 30 s after ATP addition. These results suggest that in these tumor cells, extracellular ATP elicits changes in phosphoinositide metabolism similar to those produced by a wide variety of Ca2+-mobilizing hormones and growth factors. PMID- 3004358 TI - Oscillatory response of photosynthesis in leaves to environmental perturbations: a mathematical model. AB - Oscillations in the yield of chlorophyll fluorescence, in oxygen evolution, and in CO2 uptake observed with leaves upon perturbation of steady-state conditions are suggested to be due to the interdependence of turnover of adenylates and Calvin cycle intermediates. This suggestion is quantified in a mathematical model; the behavior of the model system in the neighborhood of the singular point of the system is analyzed. The linearized system is solved analytically, a condition for the occurrence of oscillations is given, and explicit expressions for the oscillation period and the damping constant are derived. The model is shown to be capable of exhibiting oscillations with the period observed with algae or leaves, whereas calculated values of the damping constant are higher than those measured for leaves or algae. PMID- 3004361 TI - Isozymes of phosphoglyceromutase from the developing endosperm of Ricinus communis: isolation and kinetic properties. AB - The isozymes of phosphoglyceromutase from the developing endosperm of Ricinus communis have been partially purified. The purified cytosolic and plastid isozymes have specific activities of 622.8 and 83.8 mumol min-1 mg protein-1, respectively. They both have relative molecular masses of approximately 64,000. The cytosolic enzyme has lower Km values for both 2-phosphoglycerate and 3 phosphoglycerate than the plastid enzyme. The Km values for 3-phosphoglycerate are 330 +/- 25 and 430 +/- 48 microM for the cytosolic and plastid isozymes, respectively. The corresponding Km values for 2-phosphoglycerate are 60 +/- 10 and 112 +/- 22 microM. The two isozymes also have different pH optima and heat labilities. Neither isozyme requires 2,3-bisphosphoglycerate or a divalent cation and neither is regulated by metabolites. PMID- 3004362 TI - [Pathogenesis and selection of treatment modalities for hepatocellular carcinoma]. AB - Factors determining the prognosis of patients with hepatocellular carcinoma (HCC) were analyzed. Small HCC was compared to large HCC. Small HCC is less encapsulated, but has less frequent vascular invasion with favorable prognosis if resectable. Liver transplantation would be indicated in small HCC, unresectable because of far-advanced liver cirrhosis. PMID- 3004363 TI - [A cooperative phase II study of cisplatin in patients with head and neck cancer]. AB - A cooperative phase II study of cisplatin in head and neck cancer was conducted in 23 institutions. Eighty-nine patients were entered into this trial, of which 73 were evaluable. Two different regimens were employed in this study. Regimen A: cisplatin 10 mg/m2 intravenous (i.v.) infusion daily, days 1-5, q 3 wk. Regimen B: cisplatin 50 mg/m2 i.v. infusion, day 1, q 3 wk. Two patients achieved complete response and 17 achieved partial response with an overall response rate of 26.0%. By histological types, the response rate was 26.3% in the case of squamous cell carcinoma. Partial response were observed in 2 cases of adenocarcinoma and in one case each of adenoid cystic carcinoma and transitional cell carcinoma. The response rate was 19.4% for previously treated patients, as compared to 63.6% for the previously untreated group. Toxic effects were observed in 94.7% of 76 evaluable cases. From 50 to 68% of patients experienced nausea, vomiting and anorexia. No patient exhibited a serum creatinine level exceeding 2 mg/dl. Anemia and leukopenia were observed in 58.9% and 32.9% respectively. It is therefore concluded that cisplatin is markedly useful for the treatment of head and neck cancer. PMID- 3004364 TI - [Basic study of anti-cancer agents suspended in lipiodol]. AB - Anti-cancer agents suspended in Lipiodol have been proved to be effective in the targeting of chemotherapy for cancer of the digestive organs. 5-FU, ADM and MMC were suspended both separately and collectively in Lipiodol. These preparations were terned FULIP, ADRLIP, MMCLIP and FAMLIP, respectively. Forty percent 5-FU was released from FULIP within 24 hours. ADM and MMC were released more slowly from ADRLIP and MMCLIP when compared with FULIP. ADM W/O type emulsion (ADM was dissolved in 60% Urographin/Lipiodol) released ADM so rapidly that 37% of the drug was detected in the water phase within 10 hours. However, the ADM W/O emulsion with Arlacel-A was much slower. From these results, it was suggested that the release speed of anti-cancer agents can be controlled by modifying the drug dosage and form. FAMLIP released 5-FU, ADM and MMC independently and the release speeds were equal to FULIP, ADRLIP and MMCLIP. It was also suggested that the biological activities of 5-FU, ADM and MMC in FAMLIP were stable in FULIP, ADRLIP and MMCLIP. It should therefore be possible to use a large amount of anti cancer agent suspended in Lipiodol, as a combined drug cancer therapy. PMID- 3004366 TI - Distinctive eruption characterized by linear supravenous papules and erythroderma following broxuridine (bromodeoxyuridine) therapy and radiotherapy. AB - A distinctive cutaneous eruption occurred in two patients with central nervous system tumors who were treated with broxuridine (bromodeoxyuridine) and radiotherapy. Findings included the initial appearance of papular lesions within skin directly overlying acral superficial veins and the later development of a more generalized eruption eventuating into exfoliative erythroderma. PMID- 3004365 TI - [Cyclic alternating combination chemotherapy of small cell carcinoma of the lung]. AB - Twenty-six consecutive previously untreated patients with small cell carcinoma of the lung were treated with cyclic alternating combination chemotherapy. COAM consists of cyclophosphamide 140 mg/m2 i.v. day 1 through 5, vincristine 1.3 mg/m2 i.v. day 1 and 15, ACNU 50 mg/m2 i.v. day 1, methotrexate 7 mg/m2 i.v. day 1 through 5 repeated at 4 week intervals. VAP consists of VP-16 100 mg/m2 p.o. day 1 through 5, adriamycin 30 mg/m2 i.v. day 1, procarbazine 100 mg/m2 p.o. day 1 through 14 repeated at 4 week intervals. The order of alternating course was determined by the time of entry into the study. There were 12 complete and partial responses (86%) among 14 patients treated with COAM-VAP including 6 complete responses (43%), whereas there were 12 responses (100%) including 4 complete responses (33%) among 12 patients treated with VAP-COAM. There was no significant difference between patients receiving COAM-VAP and VAP-COAM with respect to response, duration of response, and survival. Overall median survival was 9.8 months. There was no significant difference between patients with and without distant metastasis with respect to response, duration of response and survival. Two patients were alive at 24 months. The addition of second combination chemotherapy did not increase the response rate. Most patients had mild or moderate leukopenia. Four patients developed interstitial pneumonia. PMID- 3004367 TI - [Trial of automated recognition between 2 cytologic types of bronchial carcinoma]. PMID- 3004368 TI - [2 articles on glomus tumors of the hand]. PMID- 3004370 TI - [Interaction between calcium inhibitors and vascular alpha-adrenergic receptors]. AB - A survey shall be given of the pharmacology of calcium antagonists or calcium entry blockers (CEB) in various types of cardiovascular disease. It emphasises their vasodilator potency, the common characteristic of all CEB and probably the property underlying most of their therapeutic effects apart from the antiarrhythmic activity of verapamil. All CEB relax vascular smooth muscle, particularly in arteriolar beds, thus reducing peripheral vascular resistance. Arteriolar relaxation appears to be associated with a specific inhibition of transmembrane calcium influx. Calmodulin is probably not generally involved, and neither are intracellular processes. Vasoconstriction induced by stimulation of vascular postsynaptic alpha2-adrenoceptors, using selective agonists, is reduced by CEB by a non-competitive mechanism. This interaction has been observed for different CEB like nifedipine and other dihydropyridines, verapamil and diltiazem. The effect on alpha2-adrenoceptor mediated vasoconstriction is directly associated with the calcium antagonistic potency of the drugs. On the other hand, vasoconstriction evoked by selective excitation of vascular alpha1 adrenoceptors remains virtually uninfluenced by CEB. The selective interference of CEB with alpha2-adrenoceptor stimulation. Furthermore this mechanism may contribute to the vasodilator effect of CEB, by inhibiting that part of vascular tone evoked by the stimulation of alpha2-adrenoceptors by endogenous catecholamines. PMID- 3004369 TI - Corticosteroid therapy in Epstein-Barr virus infection. Effect on lymphocyte class, subset, and response to early antigen. AB - Corticosteroid treatment of impending upper airway obstruction due to Epstein Barr virus (EBV) infectious mononucleosis did not alter the pattern of lymphocyte changes induced by this viral infection during the first two weeks following administration of prednisone. By 12 weeks, 11 treated students had significantly fewer lymphocytes, including B, total T, helper, and T-suppressor cell numbers, than 11 untreated EBV-infected students, and values were closer to those noted in uninfected controls. Corticosteroid therapy did not alter the serologic response to early antigens of EBV. Fever and lymphadenopathy resolved somewhat more quickly in treated students. PMID- 3004371 TI - Atypical mesoblastic nephroma. Pathologic characterization of a potentially aggressive variant of conventional congenital mesoblastic nephroma. AB - A case of an aggressive variant of conventional congenital mesoblastic nephroma (CMN) occurred in a 10-month-old male infant. The tumor proved to be fatal, with two abdominal recurrences, but no metastases were found. Of the various terms used to designate the tumor, atypical mesoblastic nephroma (AMN) is preferred. We reviewed 17 previously reported cases to attempt pathologic characterization of AMN. The features that distinguish AMN from CMN are as follows: (1) atypical gross features consisting of one or more of the following: fleshy areas, foci of hemorrhage, necrosis, involvement of adjacent structures other than connective tissue; and (2) high cellularity and mitotic index. Aggressive behavior was noted at surgery or on follow-up in seven of the 18 cases. It was characterized by one or more of the following: (1) invasion of adjacent structures, such as adrenal gland, spleen, colon, and diaphragm (three cases); (2) one or more recurrences (four cases); and (3) metastasis (two cases). Atypical mesoblastic nephroma should be recognized as a potentially aggressive lesion separate from CMN. PMID- 3004372 TI - Bilateral cystic nephroblastomas and botryoid sarcoma in a child with Dandy Walker syndrome. AB - We report an autopsy case of an 8-month-old male infant with bilateral cystic nephroblastomas, a botryoid sarcoma involving the urinary bladder, microencephaly, the Dandy-Walker syndrome, bilateral cataracts, hypospadias, and male pseudohermaphroditism. An unusual combination of congenital malformation and tumors may reflect the presence of a mutant gene. PMID- 3004374 TI - Congenital mesoblastic nephroma. When should we worry? PMID- 3004373 TI - Hypertriglyceridemia and lipid inclusions in a case of Philadelphia chromosome positive, acute nonlymphocytic leukemia. AB - We report a case of a 71-year-old female patient with an unusual morphological variant of Philadelphia chromosome-positive, acute nonlymphocytic leukemia. The myeloblasts exhibited an extreme degree of lipid vacuolization and the serum exhibited hyperlipidemia. The initial serum triglyceride level was 756 mg/dL. There were 26,000 white blood cells per cubic millimeter with 19% myeloblasts. The bone marrow contained greater than 80% myeloblasts that were myeloperoxidase- and chloracetate esterase-positive and typed positive for OKM1 and Leu 1 myeloid cellular markers. At remission, the lipid inclusions disappeared and the serum triglyceride levels returned to normal. Both abnormalities recurred at relapse. The cause of the hyperlipidemia and lipid inclusions was most likely an acquired hyperlipoproteinemia and secondary absorption of lipids into the malignant cells. PMID- 3004375 TI - Minute hepatoma with excessive copper accumulation. Report of two cases with resection. AB - Minute hepatomas with prominent copper accumulations were resected in two women, aged 60 and 62 years, who had never suffered from jaundice. Mild elevation of serum alpha-fetoprotein level was found in both patients. One tumor was diagnosed by celiac angiography, and the other was determined by an ultrasonic echogram. Microscopically, these two tumors were relatively well-differentiated hepatocellular carcinoma, though having less-differentiated foci. Many cancer cells contained numerous copper granules stained by orcein, Victoria blue, and p dimethylaminobenzylidene rhodanine. Ultrastructurally, cancer cells contained many secondary lysosomes with an electron-dense material. We concluded that the excessive copper in the cancer cells was aggregated lysosomal copper metallothionein, and that it might not be carcinogenic but stored by an altered metabolism of copper and copper-binding proteins with the neoplastic transformation. PMID- 3004377 TI - Pleomorphic adenoma of the larynx. AB - A 40-year-old woman was hoarse for five months due to a pleomorphic adenoma in the false vocal cord. The peripheral, ulcerated portion of the tumor had undergone squamous metaplasia, and this was initially misdiagnosed as squamous cell carcinoma. For that reason, the patient received radiation treatment, but the tumor remained unchanged. It was then locally excised, and the patient was still free of disease 14 years later. This case illustrates the hazard of misinterpreting squamous metaplasia in pleomorphic adenoma, the resistance of this tumor to irradiation, and the satisfactory long-range response to local excision. PMID- 3004376 TI - Malignant fibrous histiocytoma arising in the liver. AB - A unique type of hepatic sarcoma in an adult was composed of pleomorphic fibrohistiocytic cells with benign cystic structures. The malignant mesenchymal component was morphologically similar to malignant fibrous histiocytoma of the soft tissue. The cystic epithelial structures appeared to be formed by the cystic transformation of the entrapped bile ducts and ductules. Malignant fibrous histiocytoma can arise in the liver, but the original architecture of the liver may affect or modify the histologic characteristics of the sarcoma. PMID- 3004378 TI - Trauma serum suppresses superoxide production by normal neutrophils. AB - The effect of trauma serum on superoxide production by normal neutrophils was studied in 47 serum samples from 18 patients with multiple trauma. Ten patients became septic and eight patients remained nonseptic. Incubation in trauma serum significantly suppressed superoxide production by normal neutrophils compared with incubation in normal serum: 3.6 +/- 1.44 vs 4.04 +/- 1.64 nmole of superoxide produced by 10(6) neutrophils (mean +/- SD). There was no difference in the suppressive effect between septic and nonseptic trauma serum samples. The chemiluminescence response of normal neutrophils was likewise suppressed following incubation in trauma serum compared with incubation in normal serum. The chemiluminescence response correlated with superoxide reduction of cytochrome C. In addition, the chemiluminescence response was significantly less in septic trauma serum than in nonseptic-trauma serum. Suppressive serum was found to inhibit the neutrophil-membrane depolarization response to latex particles, as measured by flow cytometry. We conclude that trauma serum suppresses superoxide production by normal neutrophils, and that such suppression can be detected reliably using the clinically applicable technique of chemiluminescence. A normal chemiluminescence response excludes serum-mediated suppression of neutrophil superoxide production. In addition, chemiluminescence may be of value in detecting altered resistance to sepsis following injury, while superoxide determinations do not seem to be helpful in this regard. The mechanism of action of the suppressor may involve reversible inhibition of membrane depolarization necessary for the production of bactericidal oxygen species. PMID- 3004379 TI - Forskolin (cyclic adenosine monophosphate)-dependent protein phosphorylation in isolated gastric glands. AB - Improved management of peptic ulcer disease requires elucidation of cellular processes underlying gastric secretion. The intracellular execution of regulatory commands to secretory cells involves protein phosphorylation. We studied cyclic adenosine monophosphate (cAMP)-dependent phosphorylation in isolated gastric glands (IGGs) using forskolin, which directly stimulates adenylate cyclase. Forskolin stimulated secretion by both parietal and chief cells. In a separate set of studies, IGGs were incubated for 45, 90, and 105 minutes in modified Ham's F-10 medium containing orthophosphate labeled with phosphorus 32. The forskolin (10(-4) M) was added to some IGG preparations at 90 minutes. The reaction was terminated with sodium dodecyl sulfate and boiling. The proteins were resolved on sodium dodecyl sulfate-polyacrylamide gels, stained with Coomassie blue, and autoradiographed. Incorporation of phosphorus 32 increased progressively at 45, 90, and 105 minutes. Forskolin enhanced phosphorylated bands around 92 kilodaltons. These results are consistent with the major role of cAMP in the regulation of gastric cellular function. The study of cAMP-stimulated phosphorylation may be an important tool in the elucidation of intracellular regulatory mechanisms of gastric secretion. Modulation of these mechanisms may be the ideal therapeutic modality for treatment of acid-secretory disorders. PMID- 3004381 TI - The interaction of aluminum and other metal ions with calcium-calmodulin dependent phosphodiesterase. AB - In the search for the mode of action of aluminum ions in dialysis encephalopathy, their interaction with calmodulin has been assessed, and compared with those of Ca2+, Pb2+, Mn2+, Hg2+ and Cd2+. The basal and calmodulin-dependent activity of phosphodiesterase was measured in the presence of the ions, and their binding to calmodulin was assessed by competition with 45Ca2+ in flow dialysis. Al3+, Mn2+, Hg2+, and Cd2+ cannot be regarded as exclusive calmodulin antagonists or agonists, but rather interact with the phosphodiesterase itself. Pb2+ however, mimicks Ca2+ in every system tested, and is active in the same concentration range as Ca2+. Our results indicate that the assumed role of Al3+ ions in dialysis encephalopathy or other neurological disturbances is not linked with calmodulin. PMID- 3004382 TI - 3H-dopamine uptake and 3H-haloperidol binding in striatum after administration of methyl mercury to rats. AB - Methyl mercury inhibits dopamine (DA) and serotonin (5-HT) uptake by brain synaptosomes and decreases antagonist binding to striatal dopaminergic D2 receptors in vitro. To assess the effects in vivo, adult rats were given methyl mercury, either as a single dose (10 mg/kg by gavage) or a cumulative total dose of 50 mg/kg in 2 weeks. The repeated dosing decreased body weight and caused neuromuscular dysfunction. In spite of this overt toxicity, neither 3H-DA uptake nor 3H-haloperidol binding changed in striatal synaptosomes. There were no significant alterations in 3H-5-HT uptake by hypothalamic synaptosomes or 3H flunitrazepam binding in cerebellar synaptosomes. The results suggest that monoaminergic synapses and the benzodiazepine binding sites, associated with cerebellar GABA receptors, remain functionally normal at doses of methyl mercury that are otherwise toxic. The results also emphasize the importance of due care when extrapolating cellular or biochemical data to the level of the whole organism. PMID- 3004380 TI - [Determination of the average duration of food in the digestive tract of sheep]. AB - The average time the test feed remains in sheep was determined with ewes during three stages of the reproduction cycle. The feed was labelled with 51Cr2O3. Experiment conditions are described under which the experiments can be carried out within the permissible limits. PMID- 3004383 TI - Effect of acrylamide and related compounds on glycolytic enzymes in rat sciatic nerve in vivo. AB - The effect of acrylamide and six analogues on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase in sciatic nerve was examined in rats after their prolonged administration in drinking water. After 15 days' treatment with acrylamide and N-isopropylacrylamide, slight signs of peripheral neuropathy were produced with no changes in the activity of either enzyme. N,N dimethylacrylamide, a non-neurotoxic analogue, produced only body weight loss at this stage. After 30 days' treatment, acrylamide and N-isopropylacrylamide produced moderate signs of neuropathy, but no changes in enzyme activity. N,N dimethylacrylamide produced a reduction in GAPDH activity as well as body weight loss. After 45 days' treatment, acrylamide, N-isopropylacrylamide, N hydroxymethylacrylamide and N-methylacrylamide produced severe signs of neuropathy as well as body weight loss. All these analogues also produced a reduction in the two enzyme activities, except for enolase after N isopropylacrylamide. N,N-dimethylacrylamide produced inhibition of GAPDH as well as body weight loss without neuropathy. N-tert-butylacrylamide and N,N'-methylene bis-acrylamide induced neither neuropathy nor inhibition of either enzyme. PMID- 3004384 TI - Replication of alphaviruses in cultures of donkey monocytes. AB - Representative strains of Venezuelan equine encephalitis virus (VEEV) and Eastern equine encephalitis virus (EEEV) were compared for their ability to grow in cultures of unstimulated leucocytes and monocytes derived from donkey peripheral blood. Replication of epizootic and vaccine strains of VEEV, but not of enzootic strains was observed in this system. Only a minority of monocytes supported virus replication as detected by immunofluorescence, electron microscopy and infectious center assays. EEEV did not appear to replicate in this cell system although virus attached to and was internalized by monocytes. PMID- 3004385 TI - Replication of infectious bursal disease virus in lymphoid cells. AB - Replication of infectious bursal disease virus in lymphoid cells from the bursa of Fabricius occurs preferentially in a population of proliferating cells characterized by increased thymidine incorporation and by blast cell formation. This was demonstrated by enrichment of virus producing cells in gradients of bovine serum albumin and by mitogenic stimulation of cells with fetal calf serum or an extract prepared from bursae of susceptible chickens. Susceptibility of lymphoid bursa cells is not correlated with the expression of immunoglobulins on their surface. This conclusion was reached after the enrichment of Ig-bearing cells by rosetting techniques and by separation through a cell sorter. PMID- 3004386 TI - Bovine herpesvirus 1: molecular and antigenic characteristics of variant viruses isolated from calves with neurological disease. AB - This report presents data showing that several virus isolates recovered in Argentina, mainly from calves with non-purulent meningo-encephalitis, represent a hitherto unrecognized antigenic variant of BHV-1. The following experimental approaches have been adopted to demonstrate both the unique features among and the relatedness with BHV-1 of these isolates: i) crossed serum neutralization test with rabbit immune sera, ii) analysis by SDS-polyacrylamide gel electrophoresis of radio-labeled virus induced polypeptides and glycoproteins, iii) discriminating reactivity of a panel of monoclonal antibodies which recognize known virus types, and iv) restriction endonuclease analysis of viral DNA. Another strain of BHV-1, which exhibits a specific neuropathogenic potential [Hall et al., Austral. Vet. J. 42, 229-237 (1966)] shares all major features with the viral strains originating from Argentina. Our results imply that antigenic variants of BHV-1 exist and that they can be accurately and easily identified and differentiated by the available methods. PMID- 3004387 TI - Interactions between herpes simplex virus and murine bone marrow macrophages. AB - Macrophages from murine bone marrow (strain C3Hf Bu/Kam) were cultured in vitro in L-cell conditioned medium. After 0, 2, 4, 6, 8 and 10 days, they were infected with a clinical strain of herpes simplex virus type 1 and the outcome followed morphologically, by phagocytic index, infectious virus yields, immunofluorescence, expression of Fc receptors and major histocompatibility complex (MHC) Class II antigens. At a multiplicity of infection of 1-5, little morphological difference was apparent between infected and uninfected cultures at early stages in vitro but marked changes occurred later with reduction in cell numbers in the infected cultures. Indirect immunofluorescence failed to detect cells expressing early viral antigens, and yields of infectious virus indicated that permissive infection was not taking place. While phagocytic index and Fc receptor expression did not change 24 hours post-infection, MHC Class II antigen expression was increased. Thus, although the bone marrow macrophages seem predominantly resistant to infection with HSV-1, they may be modified by the presence of the virus. Since macrophages may act as antigen presenting cells for the immune system, this type of mechanism may be important in the generation of local immune responses. PMID- 3004388 TI - Cytomegalovirus infection of rat endothelial cells in vitro. AB - Rat endothelial cells were productively infected with rat cytomegalovirus in vitro. A typical CMV-like cytopathic effect resulting in a lytic infection was observed. Intracytoplasmic and intranuclear virus and viral antigens were detected by fluorescence and electron microscopy. Receptors for the Fc region of IgG in the cytoplasm and on the cell membrane were observed. Infectious virus was released in the supernatant of the infected cell cultures. PMID- 3004389 TI - Genetic relatedness among animal rotaviruses. AB - The genomic relatedness among representative rotavirus strains was examined by employing cross-hybridization techniques. Single stranded (ss) RNA prepared by in vitro transcription of purified rotavirus particles and labeled with either 32P or 125I was hybridized to denatured genomic, double stranded (ds) RNAs. The hybrids formed were analyzed by polyacrylamide gel electrophoresis (PAGE) or by testing their sensitivity to digestion with single strand specific nuclease (S-1 nuclease). A relatively high degree of genomic homology was found to exist among several bovine rotavirus strains obtained from different geographical areas. Similarly, a high degree of homology was found between two different simian rotavirus strains, and also between two porcine strains. The human Wa strain exhibited a low degree of genomic homology with simian, bovine and canine strains whereas a higher level of homology was detected between the human Wa strain and the porcine strains. The observed RNA sequence divergences of rotaviruses isolated from different animal species are in agreement with the restricted host range of these viruses and their known antigenic differences and suggest a divergent evolution of their genomes. PMID- 3004391 TI - Genome variation of 5 clinical adenovirus type 2 isolates determined by restriction enzyme digestion analysis. Brief report. AB - The restriction enzymes Eco RI, Hind III and Pst I have been used to show that 5 adenovirus type 2 isolates can be divided into 2 genome types and within each type there are different genome variations. PMID- 3004390 TI - Human exposure to bovine polyomavirus: a zoonosis? AB - A competitive-type solid phase radioimmunoassay (RIA) was developed for the detection of antibody to bovine polyomavirus. Comparison of RIA and counter immunoelectrophoresis (CIE) results on 273 cattle sera indicated that both techniques were detecting antibody of like specificity. Human sera from 256 blood donors, 219 people recently vaccinated against polio, rubella or rabies, 50 immunosuppressed patients and 472 people with various occupational exposure to cattle were tested for antibody to bovine polyomavirus, the foetal rhesus monkey kidney strain, (anti-FRKV) by RIA. Apart from one blood donor and one of 108 rabies vaccinees only those in close contact with cattle possessed anti-FRKV. Compared with 62 per cent seropositive in the natural hosts, cattle, 71 per cent of veterinary surgeons, 50 per cent of cattle farmers, 40 per cent of abattoir workers, 16 per cent of veterinary institute technical staff and 10 per cent of veterinary students were anti-FRKV positive. Our findings indicate that the theoretical hazard of FRKV infection from undetected contamination of current tissue culture derived vaccines may, in practice, be remote. Proposed wider use of primate kidney cells as substrates for new vaccines may increase this risk. PMID- 3004392 TI - Atypical rotavirus from South African neonates. Brief report. AB - Rotaviruses displaying an RNA profile different from other human rotaviruses were detected in stools from six healthy neonates. These viruses shared the common group A antigen, unlike most other atypical human rotavirus electropherotypes described to date. PMID- 3004394 TI - [Cytological diagnosis of epithelial dysplasias]. AB - The attempt is undertaken to determine the possibilities of the dysplasia cytological diagnosis taking as an example the gynecological smears, smears prints of the gastrobiopsies and mammary gland punctures. The difficulties are noted with which the cytologist is faced. The correctness of the cytological diagnosis "epithelial proliferation with atypia" and the possibilities of using the term "severe dysplasia" in the pronounced epithelial cells alterations, signs of their disordered differentiation is pointed out. PMID- 3004393 TI - [Studies on leukocyte procoagulant activity antigenically stimulated by purified protein derivative and varicella virus antigen]. PMID- 3004395 TI - [Morphology of dysplasia and small cancer of the breast]. AB - The results of histological study of material from sectoral resection of 285 mammary glands (MG) from 265 patients with fibrocystic disease (FCD) are presented. 112 cases of a nonproliferative FCD and 173 cases with different variants and degree of proliferation and atypia of the duct and lobules epithelium are recorded. Slight proliferation is observed in 66 cases, mild in 43 cases, pronounced proliferation corresponding to the atypical hyperplasia according to the WHO classification of MG tumours (1981) is found in 30 cases; ductal and lobular carcinoma in situ is observed in 16 cases. A long-term follow up showed the invasive MG carcinoma in the preserved part of MG to develop in 3 of 172 FCD cases (1.7%). A frequent combination of the atypical hyperplasia foci with an intraductal and lobular carcinoma in situ, revealed in 60 cases of the MG small carcinoma (from 3 to 10 mm in size) with a focal invasion, is in favour of the morphogenetic link between dysplasia and MG carcinoma. Electron-microscopic examination of a proliferative FCD (12 observations) and invasive carcinoma (15 observations) established an enhancement of the specific ultrastructural differentiation in parallel with an increase of the epithelial proliferation but did not reveal the ultrastructural signs of malignant transformation. PMID- 3004397 TI - [CSF eosinophils in malignant tumors: a case report]. AB - It is presented a case of a patient with a cerebral malignant astrocytoma in which the spinal fluid cytomorphology showed numerous eosinophilic granulocytes. PMID- 3004396 TI - [Changes in the gastroduodenal system in chronic nonspecific lung diseases]. AB - Chronic non-specific pulmonary diseases are frequently followed by the development of inflammatory-degenerative and erosive-ulcerative processes in the mucous membrane of the stomach and duodenum. The literature date are presented on the frequency of combination of these conditions, the attempt is made to assess their link from the viewpoint of pathogenesis. Disturbances of respiratory lung function and hypoxia as well as non-respiratory metabolic functions of the pulmonary tissue are regarded as etiological factors of pathological processes in the gastro-duodenal area. PMID- 3004398 TI - Surgery for lesions of the brachial plexus. AB - Current diagnostic workup and surgical management of stretch injuries, gunshot wounds, lacerations, iatrogenic injuries, tumors, and thoracic outlet syndromes involving the brachial plexus are reviewed. Use of appropriate radiologic and electrodiagnostic studies to work up such patients is summarized as is selected literature concerning the more controversial aspects of their management. Some of the arguments both for and against operation on stretch injuries are presented and it is concluded that surgery can be of value for well-selected patients. Although a number of patients with gunshot wounds involving the plexus recover spontaneously, many still require an operation. Experience with tumors arising from the plexus suggests the need for early and relatively aggressive removal. Use of magnification and intraoperative recording permits removal of some but not all neurofibromas without further deficit. Timing for repair of lacerating injuries to the plexus, as well as iatrogenic injuries, selection of the few patients with thoracic outlet syndrome who require operation, and a brief review of plexus neuropathy are also presented. Importance of evaluating individual plexus injuries in terms of how complete or incomplete loss is in the distribution of each individual element is stressed. Development of intraoperative stimulation and recording methods to help sort out lesions, use of magnification for repair, and improved grafting techniques where gaps result from resection have helped to restimulate interest in managing these patients. PMID- 3004399 TI - Unusual acute neurologic presentations with Epstein-Barr virus infection. AB - We describe two patients with serologic evidence of active Epstein-Barr virus infection who presented with unusual neurologic manifestations and minimal systemic findings of infectious mononucleosis. One girl developed an acute hemiplegic migraine syndrome followed by acute psychosis, and the other patient had severe, generalized chorea. The wide spectrum of presenting central nervous system findings associated with Epstein-Barr virus infections underscores the need to suspect this agent in a variety of acute neurologic syndromes. PMID- 3004400 TI - Lafora's disease. Comparison of inclusion bodies in skin and in brain. AB - A patient had the clinical and neuropathologic signs of Lafora's disease. Skin biopsy specimens from the midcalf area confirmed earlier findings by showing numerous periodic acid-Schiff-positive inclusion bodies in eccrine sweat gland duct cells. In our patient, however, inclusion bodies were more abundantly present in the apocrine sweat gland duct cells of the axilla skin. In brain biopsy specimens and autopsy material the same periodic acid-Schiff-positive inclusion bodies were found. From these data it can be stated that skin biopsy of the axilla is the method of first choice in confirming the diagnosis. PMID- 3004401 TI - Isolation of Ross River virus from epidemic polyarthritis patients in Australia. AB - Ross River virus was isolated from two out of four seronegative serum samples obtained from epidemic polyarthritis patients in Australia. Virus isolated from each patient was found to be phenotypically diverse. These isolations suggest that previous failure to isolate virus from epidemic polyarthritis patients in this country may have been due to failure to obtain material (serum) from patients early enough in the course of the disease and to the use of relatively insensitive isolation techniques. PMID- 3004402 TI - Experimental infection with Murray Valley encephalitis virus: galahs, sulphur crested cockatoos, corellas, black ducks and wild mice. AB - Orally infected Culex annulirostris or intravenous injections were used to infect 10 Galahs, 15 Sulphur-crested cockatoos, 12 Corellas, 4 Black ducks and 10 wild mice with Murray Valley encephalitis (MVE) virus. The birds produced moderate viraemias of titres of log 10(2.0)-10(6.0) suckling mouse intracerebral (SMIC) LD50/ml for durations of 1-9 d. However, the wild mice developed low grade viraemia for 1-4 d. Recipient Cx annulirostris feeding on viraemic birds sustained infection rates of 0-10% but, by extrapolation from vector competence data, some titres were capable of infecting greater than or equal to 50% of recipient Cx annulirostris. HI antibody responses were moderately high for most birds (reciprocal titres of 1 in 640-2560) by 14 d and usually persisted at detectable levels for 140 d and, in some individuals, for 6 months. However, 6 of 12 Corellas and 1 of 4 Black ducks became seronegative after 42 and 56 d, respectively. As with previously tested animals, any IgM response was absent 4 wks post-infection. The viraemias attained indicate that these bird species may be considered as potential hosts of MVE, but the limited viraemic response of wild mice suggests that they might play only a minor role. PMID- 3004403 TI - Tomaculous neuropathy: hereditary predisposition to pressure palsies. AB - A family is described in which six members in three generations have been affected by a remittent and pressure-sensitive mononeuritis or mononeuritis multiplex. In addition, nerve conduction studies have demonstrated the presence of peripheral neuropathy in clinically unaffected as well as affected family members, thus providing evidence of an autosomal dominant inheritance pattern. A sural nerve biopsy from one of of an autosomal dominant inheritance pattern. A sural nerve biopsy from one of the clinically affected members of this family showed 'sausage-shaped' swellings of myelin sheaths characteristic of tomaculous neuropathy. This rare condition, which is briefly reviewed, appears to be a distinctive clinicopathological entity and usually follows a benign course. PMID- 3004404 TI - Characterization of Neurospora crassa cyclic AMP phosphodiesterase activated by calmodulin. AB - Activation of cyclic AMP phosphodiesterase I by brain or Neurospora calmodulin was studied. The stimulation required micromolar concentrations of Ca2+, and it was observed at cyclic AMP concentrations between 0.1 and 500 microM. Activation was blocked by EDTA and some neuroleptic drugs such as chlorpromazine and fluphenazine. These drugs inhibit the elongation of N. crassa wild-type aerial hyphae. These results reinforce the evidence towards the recognition of Ca2+ calmodulin as one of the systems controlling cyclic nucleotide concentrations in Neurospora. PMID- 3004405 TI - Rat fat-cells have three types of adenosine receptors (Ra, Ri and P). Differential effects of pertussis toxin. AB - Activation of rat adipocyte R1 adenosine receptors by phenylisopropyladenosine (PIA) decreased cyclic AMP and lipolysis; this effect was blocked in cells from pertussis-toxin-treated rats. In contrast, the ability of 2',5'-dideoxyadenosine to decrease cyclic AMP was not affected by pertussis-toxin treatment. Addition of adenosine deaminase to the medium in which adipocytes from control animals were incubated resulted in activation of lipolysis. Interestingly, adipocytes from toxin-treated rats (which had an already increased basal lipolysis) responded in an opposite fashion to the addition of adenosine deaminase, i.e. the enzyme decreased lipolysis, which suggested that adenosine might be increasing lipolysis in these cells. Studies with the selective agonists N-ethylcarboxamidoadenosine (NECA) and PIA indicated that adenosine increases lipolysis and cyclic AMP accumulation in these cells and that these actions are mediated through Ra adenosine receptors. Adenosine-mediated accumulation of cyclic AMP was also observed in cells preincubated with pertussis toxin (2 micrograms/ml) for 3 h. In these studies NECA was also more effective than PIA. Our results indicate that there are three types of adenosine receptors in fat-cells, whose actions are affected differently by pertussis toxin, i.e. Ri-mediated actions are abolished, Ra-mediated actions are revealed and P-mediated actions are not affected. PMID- 3004406 TI - Regulation of the translocation of phosphatidate phosphohydrolase between the cytosol and the endoplasmic reticulum of rat liver. Effects of unsaturated fatty acids, spermine, nucleotides, albumin and chlorpromazine. AB - The translocation of phosphatidate phosphohydrolase between the cytosol and the microsomal membranes was investigated by using a cell-free system from rat liver. Linoleate, alpha-linolenate, arachidonate and eicosapentenoate promoted the translocation to membranes with a similar potency to that of oleate. The phosphohydrolase that associated with the membranes in the presence of [14C]oleate or 1mM-spermine coincided on Percoll gradients with the peak of rotenone-insensitive NADH-cytochrome c reductase, and in the former case with a peak of 14C. Microsomal membranes were enriched with the phosphohydrolase activity by incubation with [14C]oleate or spermine and then incubated with albumin. The phosphohydrolase activity was displaced from the membranes by albumin, and this paralleled the removal of [14C]oleate from the membranes when this acid was present. Chlorpromazine also displaced phosphatidate phosphohydrolase from the membranes, but it did not displace [14C]oleate. The effects of spermine in promoting the association of the phosphohydrolase with the membranes was inhibited by ATP, GTP, CTP, AMP and phosphate. ATP at the same concentration did not antagonize the translocating effect of oleate. From these results and previous work, it was concluded that the binding of long-chain fatty acids and their CoA esters to the endoplasmic reticulum acts as a signal for more phosphatidate phosphohydrolase to associate with these membranes and thereby to enhance the synthesis of glycerolipids, especially triacylglycerol. The translocation of the phosphohydrolase probably depends on the increased negative charge on the membranes, which could also be donated by the accumulation of phosphatidate. Chlorpromazine could oppose the translocation by donating a positive charge to the membranes. PMID- 3004408 TI - Isolation of pig thyroid lysosomes. Biochemical and morphological characterization. AB - Open thyroid follicles were prepared by mechanical disruption of pig thyroid fragments through a metal sieve. This procedure allowed preparation of thyroid cell material depleted of colloid thyroglobulin. Open thyroid follicles were used to prepared a crude particulate fraction, which contained lysosomes, mitochondria and endoplasmic reticulum. These organelles were subfractionated by isopycnic centrifugation on iso-osmotic Percoll gradients. A lysosomal peak was identified by its content of acid hydrolases: acid phosphatase, cathepsin D, beta galactosidase and beta-glucuronidase. The lysosomal peak was well separated from mitochondria and endoplasmic reticulum. The lysosomal peak, from which Percoll was removed by centrifugation, was taken as the purified lysosome fraction (L). Lysosomes of fraction L were purified 45-55-fold (as compared with the homogenate) and contained about 5% of the total thyroid acid hydrolase activities. Electron microscopy showed that fraction L was composed of an approx. 90% pure population of lysosomes, with an average diameter of 220 nm. Acid hydrolase activities were almost completely (80-90%) released by an osmotic pressure-dependent lysis. Thyroglobulin was identified by polyacrylamide-gel electrophoresis as a soluble component of the lysosome fraction. In conclusion, a 50-fold purification of pig thyroid lysosomes was achieved by using a new tissue disruption procedure and isopycnic centrifugation on Percoll gradient. The presence of thyroglobulin indicates that the lysosome population is probably composed of primary and secondary lysosomes. Isolated thyroid lysosomes should serve as an interesting model to study the reactions whereby thyroid hormones are generated from thyroglobulin and released into the thyroid cells. PMID- 3004407 TI - Adenosine-receptor-mediated stimulation of low-Km GTPase in guinea-pig cerebral cortex. AB - Inhibition of receptor-coupled adenylate cyclase by hormones is proposed to be associated with GTP hydrolysis. Since adenosine inhibits cerebral-cortical adenylate cyclase via A1 adenosine receptors, the present study attempts to verify this mechanism for A1-selective adenosine derivatives. In guinea-pig cortical membranes N6-(phenylisopropyl)adenosine (PIA) increased the Vmax. of the low-Km GTPase, with an EC50 (concentration causing 50% of maximal stimulation) of about 0.1 microM, and the stimulatory effect was competitively antagonized by 5 microM-8-phenyltheophylline. The rank order of potency of the stereoisomers of PIA and of 5-(N-ethylcarboxamido)adenosine (NECA) to stimulate GTPase correlated with their ability to inhibit adenylate cyclase activity (R-PIA greater than NECA greater than S-PIA). Competition binding studies with (-)-N6- ([125I]iodo-4 hydroxyphenylisopropyl)adenosine suggest that adenylyl imidodiphosphate (p[NH]ppA), an essential component of the GTPase assay system, is a more potent A1-receptor agonist than ATP, with an IC50 (concentration giving half-maximal displacement of radioligand binding) of 7.9 microM. On the basis of the p[NH]ppA concentration used in the GTPase assay (1.25 mM), enzyme stimulation by adenosine seems to be highly underestimated. Nevertheless, adenosine-induced GTP hydrolysis reflects an increased turnover of guanine nucleotides at the Ni regulatory site and appears to be a crucial step in the sequence of events processing the inhibitory signal to adenylate cyclase. PMID- 3004409 TI - Cytidine monophosphate-dependent synthesis of phosphatidylglycerol in permeabilized type II pneumonocytes. AB - Results of previous investigations support the proposition that, in type II pneumonocytes, CMP is involved in integration of the synthesis of phosphatidylcholine and phosphatidylglycerol for lung surfactant. In the present investigation, the amount of CMP in rat type II pneumonocytes was altered directly and resultant changes in the synthesis of phosphatidylglycerol were examined. Type II pneumonocytes were made permeable to CMP by treatment with Ca2+ free medium, and phosphatidylglycerol synthesis was then assessed by measurement of the incorporation of a radiolabelled precursor, [14C]glycerol 3-phosphate, that was not effectively utilized by cells that resisted permeabilization. Incorporation of [14C]glycerol 3-phosphate into phosphatidylglycerol (but not into other lipids) was stimulated greatly by CMP (half-maximal stimulation at approx. 0.1 mM). CMP stimulated the incorporation of [14C]glycerol 3-phosphate into both the phosphatidyl moiety and the head group of phosphatidylglycerol. Incorporation of [14C]palmitate into phosphatidylglycerol was also stimulated by CMP. myo-Inositol, at concentrations found in foetal-rat serum (0.2-2.0 mM), inhibited CMP-dependent incorporation of [14C]glycerol 3-phosphate into phosphatidylglycerol and promoted, instead, CMP-dependent incorporation into phosphatidylinositol. These data, when extrapolated to foetal type II pneumonocytes, are consistent with the view that the developmental increase in the synthesis of phosphatidylglycerol for surfactant by foetal lungs is promoted by the increase in intracellular CMP and the declining availability of myo inositol that were found previously to be associated with this period of development. PMID- 3004410 TI - Novel retinoid-binding proteins from filarial parasites. AB - The present study deals with the discovery and partial characterization of specific binding proteins for retinol and retinoic acid from filarial parasites (worms of the superfamily Filarioidea), including those from two species of Onchocerca. These binding proteins, which are distinct in their physicochemical properties and in the mode of ligand interactions from the host-tissue retinoid binding proteins, may be involved in the mediation of the putative biological roles of retinoids in the control of parasitic growth, differentiation and reproduction. Parasite retinol-binding protein and retinoic acid-binding protein exhibited specificity for binding retinol and retinoic acid respectively. Both the binding proteins showed an s20,w value of 2.0 S. On gel filtration, both proteins were retarded to a position corresponding to the same molecular size (19.0 kDa). On preparative columns, the parasite binding proteins exhibited isoelectric points at pH 5.7 and 5.75. Unlike the retinoid-binding proteins of mammalian and avian origin, the parasite retinoid-binding proteins showed a lack of mercurial sensitivity in ligand binding. The comparative amounts of retinoic acid-binding protein in five parasites, Onchocerca volvulus, Onchocerca gibsoni, Dipetalonema viteae, Brugia pahangi and Dirofilaria immitis, were between 2.7 and 3.1 pmol of retinoic acid bound/mg of extractable protein. However, the levels of parasite retinol-binding protein were between 4.8 and 5.8 pmol/mg, which is considerably higher than the corresponding levels of cellular retinol-binding protein of mammalian and avian origin. Both retinol- and retinoic acid-binding protein levels in O. volvulus-infected human nodules and O. gibsoni-infected bovine nodules were similar to their levels in mammalian tissues. Also, these nodular binding proteins, like the host-binding proteins, exhibited mercurial sensitivity to ligand interactions. PMID- 3004411 TI - Covalent labelling of ligand binding sites of human placental S adenosylhomocysteine hydrolase with 8-azido derivatives of adenosine and cyclic AMP. AB - S-Adenosylhomocysteine hydrolase (AdoHcyase) has previously been identified as a cytoplasmic adenosine and cyclic AMP binding protein. In order to examine the relationship between the adenosine and cyclic AMP binding sites on this enzyme we have explored the use of 8-azido analogues of adenosine and cyclic AMP as photoaffinity reagents for covalently labelling AdoHcyase purified from human placenta. 8-Azidoadenosine (8-N3-Ado), like adenosine, inactivated AdoHcyase, and the rate of inactivation was greatly increased by periodate oxidation. In addition, 8-N3-Ado was found to participate in the first step in the catalytic mechanism for AdoHcyase, resulting in conversion of enzyme-bound NAD+ to NADH, although it was not a substrate for the full enzyme-catalysed reaction. Radioactively labelled 8-N3-Ado, its periodate-oxidized derivative and 8 azidoadenosine 3', 5'-phosphate (8-N3-cAMP) bound specifically to adenosine binding sites on AdoHcyase and, after irradiation, became covalently linked to the enzyme. Photoaffinity-labelled enzyme could be precipitated by monoclonal antibody to human AdoHcyase. Two observations suggested that cyclic AMP and adenosine bind to the same sites on AdoHcyase. First cyclic AMP and adenosine each blocked binding of both radioactively labelled 8-N3-Ado and 8-N3-cAMP, and second, digestion with V8 proteinase generated identical patterns of peptides from AdoHcyase that had been photolabelled with [32P]8-N3-cAMP and [3H]8-N3-Ado. Binding sites for cyclic AMP on AdoHcyase were found to differ functionally and structurally from cyclic AMP binding sites on the R1 regulatory subunit of cyclic AMP-dependent protein kinase. PMID- 3004412 TI - Microtubules and nucleoside diphosphate kinase. Nucleoside diphosphate kinase binds to co-purifying contaminants rather than to microtubule proteins. AB - Nucleoside diphosphate (NDP) kinase has been postulated to generate GTP from the GDP bound to tubulin. The purified chick brain enzyme was studied with respect to its kinetic parameters, and the protein-protein interactions between the NDP kinase and tubulin were examined. No specific interaction is observed between the enzyme and assembled microtubules, tubulin dimers, or tubulin-microtubule associated protein (MAP) oligomers under a variety of nucleotide conditions. The apparent association is demonstrated to result from NDP kinase binding to a co purifying contaminant. The absence of detectable NDP kinase-tubulin interactions indicates that NDP kinase does not directly charge up tubulin-GDP. PMID- 3004413 TI - Effects of pH and ionic strength on the binding of L-tri-iodothyronine to the solubilized nuclear receptor. AB - K'A (apparent association constant) and Bmax. (total receptor concentration) describing the interaction of tri-iodothyronine (T3) and its solubilized rat liver nuclear receptor (R) are found to be moderately consistent in successive preparations, but both quantities diminished after a few days. To achieve comparability in the effects of ionic strength (I) and of pH on K'A and Bmax, appropriate measurements have been made simultaneously on single preparations. K'A and Bmax. were found to be effectively unchanged over the range I0.05-0.60. Both parameters have been measured over the range pH 6.4-9.0 and the values of K'A analysed in terms of the 4'-OH ionization of T3 and that of a cationic acidic group, shown to require pK' = 7.6. This group could be identified either with the terminal alpha-NH3+ of T3 or with a group (RH+) in the receptor site. On the balance of evidence the first possibility is the more likely, in which case the variation of Bmax. with pH is ascribed to conformational changes in the receptor protein. PMID- 3004414 TI - Nucleoside transport in Walker 256 rat carcinosarcoma and S49 mouse lymphoma cells. Differences in sensitivity to nitrobenzylthioinosine and thiol reagents. AB - The characteristics of nucleoside transport were examined in Walker 256 rat carcinosarcoma and S49 mouse lymphoma cells. In Walker 256 cells the initial rates of uridine, thymidine and adenosine uptake were insensitive to the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR) (1 microM), but were partially inhibited by dipyridamole (10 microM), another inhibitor of nucleoside transport. In contrast, the transport of these nucleosides in S49 cells was completely blocked by both inhibitors. Nucleoside transport in Walker 256 and S49 cells also differed in its sensitivity to the thiol reagent p chloromercuribenzenesulphonate (pCMBS). Uridine transport in Walker 256 cells was inhibited by pCMBS with an IC50 (concentration producing 50% inhibition) of less than 25 microM, and inhibition was readily reversed by beta-mercaptoethanol. In S49 cells uridine transport was only inhibited at much higher concentrations of pCMBS (IC50 approximately equal to 300 microM). In other respects nucleoside transport in Walker 256 and S49 cells were quite similar. The Km and Vmax. values for uridine transport were nearly identical, and the transporters of both cell lines appeared to accept a broad range of nucleosides as substrates. Uridine transport in Walker 256 cells was non-concentrative and did not require an energy source. These studies demonstrate that nucleoside uptake in Walker 256 cells is mediated by a facilitated-diffusion mechanism which differs markedly from that of S49 cells in its sensitivity to the transport inhibitor NBMPR and the thiol reagent pCMBS. PMID- 3004415 TI - Calcium ions and glycogen act synergistically as inhibitors of hepatic glycogen synthase phosphatase. AB - We investigated the inhibitory effect of Ca2+ in the micromolar range on the activation of glycogen synthase in crude gel-filtered liver extracts [van de Werve (1981) Biochem. Biophys. Res. Commun. 102, 1323-1329]. The magnitude of the inhibition was highly dependent on the glycogen concentration in the final liver extract. Ca2+ inhibited the activation of purified hepatic synthase b by the G component of synthase phosphatase, as present in the isolated glycogen-protein complex. The cytosolic S-component was not inhibited. Maximal inhibition of the crude G-component occurred at 0.3 microM-Ca2+. The inhibition was not influenced by the addition of either calmodulin or calmodulin antagonists, or by various proteinase inhibitors. The use of purified G-component revealed that the inhibition by 0.3 microM-Ca2+ increased from 45% to 85% when the concentration of glycogen was raised from 1.5 to 20 mg/ml. Muscle glycogen synthase, extensively phosphorylated in vitro, was also used as substrate for purified G-component. Activation and dephosphorylation were similarly inhibited by 0.3 microM-Ca2+, but the magnitude of the inhibition was much greater with the hepatic substrate. No effect of 0.3 microM-Ca2+ was found on the activity of phosphorylase phosphatase in various liver preparations. We conclude that the inhibition of synthase activation by Ca2+ is one of the mechanisms by which cyclic AMP-independent glycogenolytic hormones promote the inactivation of glycogen synthase in the liver, especially in the fed state. PMID- 3004416 TI - Regulation of CTP: phosphocholine cytidylyltransferase activity in type II pneumonocytes. AB - Phosphatidylcholine synthesis by rat type II pneumonocytes was altered either by depleting the cells of choline or by exposing the cells to extracellular lung surfactant. Effects of these experimental treatments on the activity of a regulatory enzyme, CTP:phosphocholine cytidylyltransferase, were investigated. Although choline depletion of type II pneumonocytes resulted in inhibition of phosphatidylcholine synthesis, cytidylyltransferase activity (measured in cell homogenates in either the absence or presence of added lipids) was greatly increased. Activation of cytidylyltransferase in choline-depleted cells was rapid and specific, and was quickly and completely reversed when choline-depleted cells were exposed to choline (but not ethanolamine). Choline-dependent changes in enzymic activity were apparently not a result of direct actions of choline on cytidylyltransferase and they were largely unaffected by cyclic AMP analogues, oleic acid, linoleic acid or cycloheximide. The Km value of cytidylyltransferase for CTP (but not phosphocholine) was lower in choline-depleted cells than in choline-repleted cells. Subcellular redistribution of cytidylyltransferase also was associated with activation of the enzyme in choline-depleted cells. When measured in the presence of added lipids, 66.5 +/- 5.0% of recovered cytidylyltransferase activity was particulate in choline-depleted cells but only 34.1 +/- 4.5% was particulate in choline-repleted cells. An increase in particulate cytidylyltransferase also occurred in type II pneumonocytes that were exposed to extracellular surfactant. This latter subcellular redistribution, however, was not accompanied by a change in cytidylyltransferase activity even though incorporation of [3H]choline into phosphatidylcholine was inhibited by approx. 50%. Subcellular redistribution of cytidylyltransferase, therefore, is associated with changes in enzymic activity under some conditions, but can also occur without a resultant alteration in enzymic activity. PMID- 3004417 TI - Identification and characterization of the chicken transferrin receptor. AB - Among a panel of monoclonal antibodies that recognize chicken haematopoietic differentiation antigens, one, JS 8, was found to immunoprecipitate a 95000-Mr cell-surface protein from chicken erythroblasts transformed with avian erythroblastosis virus. This protein was shown, by affinity chromatography on immobilized chicken transferrin (conalbumin), to be the chicken transferrin receptor. Although immunologically unrelated to the human transferrin receptor, biochemical comparison of the chicken transferrin receptor to the human receptor showed similarities with respect to the pattern of biosynthesis, degree of glycosylation, dimerization in the absence of reducing agents and subcellular localization. The present report contrasts with recent ones describing the chicken transferrin receptor isolated from embryonic tissues as a 58000-Mr protein. PMID- 3004418 TI - Guanine nucleotide regulation of agonist binding to muscarinic cholinergic receptors. Relation to efficacy of agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization. AB - The efficacies of a series of six muscarinic cholinergic receptor agonists for stimulation of phosphoinositide breakdown and unidirectional efflux of 45Ca2+ in 1321N1 human astrocytoma cells were compared with the relative capacity of these agonists for formation of a GTP-sensitive high-affinity binding state in washed membranes. Carbachol and methacholine were 'full' agonists as regards phosphoinositide breakdown and Ca2+ mobilization, whereas bethanechol, arecoline and oxotremorine were 'partial' agonists for these two responses. Pilocarpine was the least efficacious of the six drugs tested. Except for pilocarpine, competition curves generated with the agonists and [3H]quinuclidinyl benzilate did not follow the Law of Mass Action for ligand interaction at a single site. Non-linear regression analyses of these data indicated that the data significantly better fit a two-, rather than a single-, site model with a high- and a low-affinity binding component. Competition curves generated in the presence of GTP were shifted to the right, and the extent of receptors in the high-affinity agonist-binding state was decreased. The relative efficacies of the six agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization were significantly correlated with the difference in affinities (KL/KH) between the two affinity states for each agonist. The relative efficacy of the agonists for stimulation of Ca2+ mobilization also was significantly correlated with the extent of receptors in the high-affinity state (%H) for each agonist. The results suggest that interaction with an as-yet unidentified guanine nucleotide regulatory protein is important in the mechanism whereby muscarinic receptors stimulate phosphoinositide breakdown in 1321N1 astrocytoma cells. PMID- 3004420 TI - Guanine nucleotides stimulate production of inositol trisphosphate in rat cortical membranes. AB - The guanine nucleotides guanosine 5'[beta, gamma-imido]triphosphate (Gpp[NH]p), guanosine 5'-[gamma-thio]-triphosphate (GTP gamma S), GMP, GDP and GTP stimulated the hydrolysis of inositol phospholipids by a phosphodiesterase in rat cerebral cortical membranes. Addition of 100 microM-Gpp[NH]p to prelabelled membranes caused a rapid accumulation of [3H )inositol phosphates (less than 30 s) for up to 2 min. GTP gamma S and Gpp [NH]p caused a concentration-dependent stimulation of phosphoinositide phosphodiesterase with a maximal stimulation of 2.5-3-fold over control at concentrations of 100 microM. GMP was as effective as the nonhydrolysable analogues, but much less potent (EC50 380 microM). GTP and GDP caused a 50% stimulation of the phospholipase C at 100 microM and at higher concentrations were inhibitory. The adenine nucleotides App[NH]p and ATP also caused small stimulatory effects (64% and 29%). The guanine nucleotide stimulation of inositide hydrolysis in cortical membranes was selective for inositol phospholipids over choline-containing phospholipids. Gpp[NH]p stimulated the production of inositol trisphosphate and inositol bisphosphate as well as inositol monophosphate, indicating that phosphoinositides are substrates for the phosphodiesterase. EGTA (33 microM) did not prevent the guanine nucleotide stimulation of inositide hydrolysis. Calcium addition by itself caused inositide phosphodiesterase activation from 3 to 100 microM which was additive with the Gpp[NH]p stimulation. These data suggest that guanine nucleotides may play a regulatory role in the modulation of the activity of phosphoinositide phosphodiesterase in rat cortical membranes. PMID- 3004419 TI - Castanospermine inhibits glucosidase I and glycoprotein secretion in human hepatoma cells. AB - We studied the effect of the plant alkaloid castanospermine on the biosynthesis and secretion of human hepatoma glycoproteins. The HepG-2 cells, grown in the presence or absence of the alkaloid, were labelled with [2-3H]mannose and then the labelled glycopeptides were prepared by Pronase digestion. This material was analysed by gel filtration on Bio-Gel P-4 before and after treatment with endo beta-N-acetylglucosaminidase H. Castanospermine caused an accumulation of high mannose oligosaccharides, by 70-75% over control. The major accumulated product, which could also be labelled with [3H]galactose and was only partially susceptible to alpha-mannosidase digestion, was identified by h.p.l.c. as a Glc3Man9GlcNAc. Thus the alkaloid inhibits glucosidase I in the human hepatoma cells. Analysis of total glycoproteins secreted by the cells into the medium revealed the presence of only complex oligosaccharides in both control and treated cultures, and the amount of the oligosaccharides labelled with radioactive mannose, galactose or N-acetylmannosamine, secreted by treated cells, was decreased by about 60%. The rate of secretion of total protein labelled with [35S]methionine and precipitated from the medium with trichloroacetic acid was inhibited by up to 40% in the presence of castanospermine. Pulse-chase studies utilizing [35S]methionine labelling were performed to study the effect of the alkaloid on secretion of individual plasma proteins. Immunoprecipitation at different chase times with monospecific antisera showed that castanospermine markedly decreased the secretion rates of alpha 1-antitrypsin, caeruloplasmin and, to a lesser extent, that of antithrombin-III. Secretions of apolipoprotein E, a glycoprotein containing only O-linked oligosaccharide(s), and albumin, a non glycosylated protein, were not affected by the drug. It is suggested that castanospermine inhibits secretion of at least some glycoproteins containing N linked oligosaccharides, owing to the inhibition of oligosaccharide processing. PMID- 3004421 TI - The origin of human cartilage proteoglycan link-protein heterogeneity and fragmentation during aging. AB - Human articular-cartilage link proteins are resolved into three components by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicative of three different structures. The action of the proteinase clostripain yields a single link-protein component with electrophoretic properties analogous to those of the smallest (most mobile) native link protein, suggesting that this link protein may be derived naturally from one or both of the larger molecules by proteolytic cleavage in situ. Upon chemical deglycosylation of native link protein two components are resolved, suggesting that two of the link proteins differ only in their degree and/or type of oligosaccharide substitution. This pattern is compatible with a proteolytic origin for the smallest link protein. During aging further proteolytic fragmentation occurs, though it is only apparent on reduction of disulphide bonds. This fragmentation occurs at identical sites in all three native link proteins, indicating the existence of a large region common to all the link proteins, which appears to consist predominantly of the C-terminal half of the molecules. These observations are compatible with the variation in oligosaccharide and proteolytic heterogeneity occurring at the N-terminus of the link proteins. PMID- 3004422 TI - Inhibition of foetal pulmonary choline-phosphate cytidylyltransferase under conditions favouring protein phosphorylation. AB - Choline-phosphate cytidylyltransferase (EC 2.7.7.15) activity from 25- and 29-day foetal rabbit lungs was inhibited in both the cytosolic and the microsomal fractions by preincubation with MgATP. The inhibition of the cytosolic enzyme was greater when measured with added phosphatidylglycerol (PG) than without (78-89% versus 50-55%), whereas the inhibition of the microsomal enzyme did not exhibit this distinction (66-72% versus 60-70%). When preincubated with the buffer alone, the cytosolic enzyme was activated to a greater extent by added PG than was the microsomal enzyme (13-14-fold versus 2-3-fold). However, after preincubation with MgATP, the cytosolic enzyme was activated to a smaller extent by added PG (3-6 fold). The inhibition of the enzyme by MgATP required a preincubation and was absent when ADP or AMP was substituted for ATP. Moreover, ATP analogues such as adenosine 5'-[beta, gamma-methylene]triphosphate and adenosine 5'-[gamma thio]triphosphate also failed to inhibit the enzyme when substituted for ATP in the preincubation. The inhibition by MgATP was not affected by including cyclic AMP in the preincubation, but Ca2+ ions alone or plus diacylglycerol in the preincubation increased the inhibition slightly. The inhibition was abolished by including an inhibitor of cyclic-AMP-dependent protein kinase in the preincubation. These observations, taken collectively, point to the inhibition of foetal pulmonary cytidylyltransferase through the phosphorylation of a protein and suggest that this key enzyme in lung surfactant production may be regulated through this mechanism. PMID- 3004423 TI - Chemiluminescence of luminol caused by interaction of cytochrome P-450 and cytochrome C with cumene hydroperoxide: comparative studies. AB - A comparative study of the interaction of cumene hydroperoxide with cytochrome P 450LM2 and with cytochrome C has been undertaken using the chemiluminescence method in the presence of luminol. Considerable differences in the mechanisms of action of both hemoproteins have been revealed with various inhibitors of radical stages, i.e. superoxide dismutase, mannitol, sodium azide, and alpha-tocopherol. It is shown that molecular oxygen participates in the process of hemoprotein catalyzed hydroperoxide oxidation of luminol. PMID- 3004424 TI - A simplified quick method for determination of sialic acid in serum. AB - A modification of the method reported by R. J. Shamberger (J. Clin. Chem. Clin. Biochem. 22, 647 (1984) for detection of serum sialic acid is described. The whole procedure takes less than 1 h. It is based on the release of bound sialic acid by heating with 5% perchloric acid. After cooling and brief centrifugation, the supernatant is heated for 15 min with Ehrlich's reagent at 100 degrees C. Thereafter the absorbance of the color developed in the sample is measured at 525 nm. PMID- 3004425 TI - Madin-Darby canine kidney cells display type I and type II insulin-like growth factor (IGF) receptors. AB - Using affinity cross-linking techniques, we report the presence of type I IGF and type II IGF receptors in Madin-Darby canine kidney cells, a line of cells lacking insulin receptors. The IGF receptors were further characterized by competition binding studies and found to be similar to IGF receptors in other tissue types. In Madin-Darby canine kidney cells, the type I IGF receptor binds IGF-I greater than IGF-II greater than insulin and the type II IGF receptor binds IGF-II and IGF-I with approximately the same affinity, but does not bind insulin. PMID- 3004426 TI - The receptor binding properties of the 20K variant of human growth hormone explain its discrepant insulin-like and growth promoting activities. AB - The 20K variant of native (22K) hGH is a full agonist for the growth promoting and lactogenic properties of the hormone in vivo but has been reported to have weak or absent insulin-like properties. To explore if these differences may be explained at the receptor level, we compared the ability of 22K and 20K hGH to inhibit the binding of 125I-22K hGH to receptors in isolated rat adipocytes, a target for the insulin-like effects of the hormone and in IM-9 cultured human lymphocytes, more specific for growth effects. Our data show that while 20K hGH is a potent agonist of native 22K hGH in the IM-9 lymphocyte assay, its potency in the rat adipocyte binding assay is only 3%, even when both cells are incubated together in identical conditions. Thus, the receptors for hGH appear to be different on various target cells, explaining why the 20K variant has different relative biological potencies at different sites of action. PMID- 3004427 TI - An endogenous brain substance, CDS (clonidine-displacing-substance), inhibits the twitch response of rat vas deferens. AB - The effect of CDS, an endogenous brain substance that specifically displaces bound [3H]clonidine and [3H]rauwolscine in rat brain membranes and human platelets, has been tested in isolated, field-stimulated rat vas deferens. CDS, obtained after an extensive purification procedure as a single peak from an HPLC sizing column, inhibited the electrically stimulated rat vas deferens similarly to the inhibitory action of clonidine, an alpha 2-agonist. The effective dose of CDS as an inhibitor of the vas deferens is equivalent to its effective dose in displacing specifically bound [3H]-clonidine in rat brain membranes. Furthermore, the CDS inhibition of the twitch response is reversed by two alpha 2-adrenergic antagonists, yohimbine and phentolamine. From these results, it is suggested that CDS extracted from brain, with affinity for clonidine sites, may be involved in the nonadrenergic fast response of the sympathetic transmission of the vas deferens. PMID- 3004428 TI - A DNA glycosylase for oxidized thymine residues in Drosophila melanogaster. AB - A DNA glycosylase activity which excises fragmented thymine residues has been identified in cell extracts from Drosophila melanogaster embryos. The enzyme has an apparent Mr = 40,000, acts preferentially on double-stranded polydeoxyribonucleotide substrate and requires no co-factors. The DNA glycosylase presumably acts in excision-repair of pyrimidines damaged by hydroxyl radicals and other oxidizing species. This is the first identification of a DNA glycosylase in Drosophila cells. PMID- 3004429 TI - Calmodulin-like protein from Bacillus subtilis. AB - The first example of a calmodulin-like activity in a Gram-positive bacterium, Bacillus subtilis, is reported. A calcium ion-dependent, 3', 5' cyclic-AMP phosphodiesterase-stimulating activity was found in the soluble fraction of cell free extracts of cells sporulating in a chemically-defined medium; activation was reversed by trifluoperazine. The activity was heat stable, bound to phenothiazine agarose in a calcium ion-dependent manner and was eluted therefrom with buffer containing EGTA, and displaced authentic beef brain calmodulin from its antibody in a radioimmunoassay. PMID- 3004430 TI - Evidence for an antagonist specific receptor that does not bind mineralocorticoid agonists. AB - Kinetics of association--dissociation, competition and chromatography on two different resins, all revealed the presence of a new binding site which: specifically accepts 7-alpha-propyl spirolactone (3H-RU-26752), has little affinity for aldosterone, is present only in the target tissue (rat kidney), and is wanting in a non-target organ (liver). The presence of such sites could explain syndromes of mineralocorticoid excess where even trace amounts of an unusual aldosterone analogue, with little affinity for the classical mineralocorticoid receptor, can nevertheless produce hypertension through the intervention of an entirely new and abundant receptor system. This new molecule thus forms a novel tool to understand the nature and function of the soluble mineralocorticoid receptor in target organs. PMID- 3004431 TI - 6'-O-dansyl-gamma-aminobutyryl atractyloside, a fluorescent probe of the ADP/ATP carrier: exploration of conformational changes of the membrane-bound ADP/ATP carrier elicited by substrates and inhibitors. AB - A fluorescent atractyloside analogue, the 6'-O-dansyl-gamma-aminobutyryl atractyloside (DGA), has been used to probe the binding of the inhibitors carboxyatractyloside (CATR) and bongkrekic acid (BA) and nucleotide substrates to the membrane-bound ADP/ATP carrier protein in beef heart mitochondria. Binding and release of DGA were followed by fluorescence responses. Specifically bound DGA was fully released by CATR alone, or by BA in the presence of micromolar amounts of ADP. In the absence of the inhibitors, ADP increased the rate of the specific binding of DGA. The effect of ADP was shared by transportable nucleotides. Non transportable nucleotides were ineffective. These data are consistent with the previously described CATR and BA conformations of the ADP/ATP carrier that are able to bind CATR and BA respectively, the transition between the two conformations being accelerated by micromolar concentrations of transportable nucleotides. PMID- 3004433 TI - ANF-like peptide(s) in the peripheral autonomic nervous system. AB - The recent demonstration of the atrial natriuretic factor (ANF) within the brain has been extended in the present study by the additional localization of ANF-like activity in the peripheral nervous structures. Using a sensitive radioimmunoassay, it was possible to detect ANF-like immunoreactive peptide(s) in crude and chromatographically separated extracts of parasympathetic rat ganglia. The partially purified ANF-like peptide exhibited a biological action similar to cardiac ANF. This finding supports a possible involvement of ANF in the regulation of both, central and peripheral neuronal activities. PMID- 3004432 TI - Effects of trypsin and calcium chloride on signal IIs in oxygen-evolving PS II preparations. AB - Photosystem II oxygen-evolving preparations exhibited a reversible loss of signal IIs hyperfine structure when treated with 1.0 M CaCl2. A progressive irreversible loss of hyperfine structure was observed upon trypsin treatment of these preparations. These treatments appear to alter the environment of the radical responsible for signal IIs. Gel electrophoresis of trypsin-treated photosystem II preparations indicates that three polypeptides (45, 32-34, and 26 kDa) are altered with the same kinetics as observed for the trypsin-induced loss of signal IIs. Two of these polypeptides (45 and 32-34 kDa) are core components of photosystem II. PMID- 3004434 TI - Stimulation of pyruvate kinase phosphatase activity by insulin in isolated rat hepatocytes. AB - Addition of insulin (10(-8)M) to hepatocytes, incubated either in the absence or in the presence of a suboptimal concentration of glucagon, caused the reactivation of pyruvate kinase and simultaneously provoked a transient stimulation of pyruvate kinase phosphatase activity (40-70% over control values). The stimulatory effect of insulin on pyruvate kinase phosphatase activity was dose-dependent (ED50 = 1 to 2 X 10(-11)M) and persisted after Sephadex G-25 filtration or ammonium sulfate precipitation of hepatocytes extracts. Our results demonstrate that insulin exerts a short-term regulation on hepatic pyruvate kinase phosphatase activity. PMID- 3004435 TI - Co-activation of protein kinase C and NADPH oxidase in the plasma membrane of neutrophil cytoplasts. AB - Enucleated, granule-free neutrophil cytoplasts, which in hypotonic media fully release cytosolic components and generate ghosts, have been used to study the cell localization of protein kinase C (PK-C). Treatment of cytoplasts with phorbol myristate acetate, a potent activator of neutrophil functions, triggers translocation of PK-C from the cytosol to the plasma membrane, with an activity recovery of 83 +/- 16%. In the ghost fraction, PK-C catalyzes the phosphorylation of polypeptides with an apparent mol. wt. of 115K, 89K, 79K, 62K, 47K and 19K. From the plasma membrane PK-C can be extracted in an active form by Triton X-100 but not by EGTA. Translocation of PK-C is already evident at 5 sec and plateaus at about 50 sec. Activation of plasmalemmal, O-2 generating NADPH oxidase by the phorbol ester is delayed by about 20 sec with respect to the activation of PK-C. Dose/response experiments show that the pattern of activation of O-2 generation by cytoplasts strictly superimposes with the pattern of PK-C translocation. PMID- 3004436 TI - Transcription but not translation is required for EDTA-induced autolysis in Escherichia coli. AB - Rifampicin, but not chloramphenicol or other inhibitors of translation, inhibited EDTA-induced autolysis in Escherichia coli. Inhibition of EDTA-induced autolysis in E. coli was also observed with nalidixic acid and novobiocin, inhibitors of topoisomerase II. Rifampicin or nalidixic acid-resistant mutants of E. coli were resistant to the inhibitory effect of the respective antibiotic on EDTA-induced autolysis. The implications of these studies in regard to our understanding of the regulation of autolysis in E. coli are discussed. PMID- 3004437 TI - Characterization of D-myo-inositol 1,4,5-trisphosphate phosphatase in rat brain. AB - Rat brain homogenates contain significant amounts of inositol 1,4,5-trisphosphate phosphatase in both 180,000xg (60 min) particulate and supernatant fractions. As other membrane-bound enzymes (e.g. guanylate cyclase), particulate inositol 1,4,5 trisphosphate phosphatase activity is highly sensitive to low concentrations of Triton X-100 (0.03%). Higher concentrations of detergent (1%) partially solubilized the enzyme. Thiol blocking agents (e.g. p-hydroxymercuribenzoate) inactivate inositol 1,4,5-trisphosphate phosphatase activity (an effect reversed with 2-mercaptoethanol). It is thus suggested that enzymatic activity requires the presence of -SH groups. PMID- 3004438 TI - Affinity modification of creatine kinase and ATP-ADP translocase in heart mitochondria: determination of their molar stoichiometry. AB - Oxidized dialdehyde analogs of ADP or ATP (oADP and oATP) were shown to inhibit irreversibly adenine nucleotide translocator (T) and creatine kinase (CK) in heart mitochondria. Inactivation of T and CK was parallel with carboxyatractyloside - sensitive and (ADP + phosphocreatine) - sensitive incorporation of o[3H]ADP into mitochondria, respectively. o[3H]ADP incorporation sensitive to CAT or ADP+phosphocreatine was used to determine T and CK contents in mitochondria. T content in cardiac mitochondria from rat, rabbit, dog, and chicken was calculated to be 2.6 - 2.9 moles/mole cyt.aa3. The same value of T/cyt.aa3 ratio was found in liver mitochondria with lower cytochrome aa3 content. In all types of cardiac mitochondria CK content was found to be 2.4 - 2.6 moles/mole cyt.aa3. The data show that T and CK are present in molar ratio 1:1 in all types of cardiac mitochondria. PMID- 3004439 TI - Intracellular Ca2+ requirements for zymosan-stimulated phosphoinositide hydrolysis in mouse peritoneal macrophages. AB - The phosphatidylinositol cycle has been demonstrated to be involved in the control of Ca2+ cytosolic levels in several cellular types. The Ca2+ requirements of phospholipase C activity and the described stimulation of phosphoinositide hydrolysis by Ca2+ ionophores make unclear the relationship between phosphatidylinositol cycle and Ca2+ mobilization. The results reported here suggest that intracellular Ca2+ is necessary for zymosan-stimulated phospholipase C activation in macrophages. PMID- 3004440 TI - Cationic amphiphilic drugs perturb the metabolism of inosititides and phosphatidic acid in photoreceptor membranes. AB - Incubation of purified bovine photoreceptor rod outer segments with [gamma 32P]ATP resulted in the labeling of phosphatidylinositol 4-phosphate (PIP) and phosphatidic acid (PA) with little labeling of phosphatidylinositol 4,5 bisphosphate (PIP2). Propranolol inhibited in a dose-dependent manner the labeling of PA and enhanced that of PIP. Various cationic amphiphilic drugs also were tested for these effects. Propranolol had the same effects on high-speed rat brain particulate material. While this particular preparation displayed more labeling of PIP2, propranolol was ineffective, as it was on retinal PIP-kinase. Ca2+-activated polyphosphoinositide phosphodiesterase activity in nerve-ending membranes also was inhibited by propranolol. It is concluded that cationic amphiphilic drugs can inhibit diacylglycerol kinase and the polyphosphoinositide phosphodiesterase and stimulate the phosphatidylinositol-kinase (but not PIP kinase). PMID- 3004441 TI - Fluorescence probing of the function-specific cysteines of rat microsomal NADPH cytochrome P-450 reductase. AB - Titration of NADPH-cytochrome P-450 reductase with a fluorigenic maleimide suggests that approximately four cysteines are initially accessible and in close proximity to four tryptophans. Perturbation of the cysteines and/or tryptophans results in concomitant decreases in enzymic activity. These cysteines were correlated with functional components by binding studies and subsequent tryptic peptide mapping on the acid mobile phase-reverse phase HPLC. Adenine nucleotides and cytochrome c block labelling of the more hydrophilic peptides, while detergents facilitate labelling of the more hydrophobic peptides. The more hydrophobic peptides contain the microsomal binding site of cytochrome P-450. Removal of the prosthetic flavins exposes more cysteines in the more hydrophilic and hydrophobic regions of the peptide map, associating the former with FAD and the latter with FMN binding sites. PMID- 3004443 TI - Cyclic AMP suppresses expression of v-rasH oncogene linked to the mouse mammary tumor virus promoter. AB - Clone 433.3 of NIH 3T3 cells is a stable carrier of the MMTV LTR:v-rasH chimeric DNA. Only in the presence of dexamethasone (a synthetic glucocorticoid), 433.3 cells exhibit an induced level of p21 transforming protein and phenotypic transformation. N6,O2'-dibutyryl cAMP (DBcAMP) antagonized the effect of dexamethasone in a time - and concentration - dependent manner. DBcAMP (5 X 10( 4)M) added 18 hr prior to the addition of dexamethasone (10(-7)M) almost completely blocked the hormone effect: cells contained levels of p21 20% of that in the cells treated with dexamethasone alone, and formed flat, contact inhibited monolayers. On the basis of these results together with our previous data on mammary carcinomas in vivo, we postulate that cAMP may be an intracellular suppressor acting at a regulatory locus of both cellular and viral ras genes. PMID- 3004442 TI - "In vitro" method of assembling a synthetic gene. AB - Without prior in vitro enzymatic ligation a DNA duplex was assembled successfully by directly transforming competent cells with a mixture containing six synthetic complimentary oligodeoxyribonucleotides and a linearized plasmid. One out of 100 transformants was positive in colony hybridization with one of the synthetic fragment probe. The sequence of the DNA duplex inserted into the plasmid was confirmed by dideoxy sequencing method. PMID- 3004444 TI - Nucleotide sequence of chick 14K beta-galactoside-binding lectin mRNA. AB - cDNA for chick 14K beta-galactoside-binding lectin mRNA was cloned and the nucleotide sequence determined. The deduced amino acid sequence and the results of in vitro translation of its mRNA suggest that this lectin does not include any cleavable signal sequence while it exists in extracellular matrix. Comparison of the primary structures has shown that chick 14K lectin includes some regions homologous to those in discoidin I, which is also known to be located in extracellular matrix and lack signal peptide. The results imply some relation between these two lectins in spite of their great phylogenetic separation. PMID- 3004445 TI - Osmotic activation of the Na+/H+ antiport in protein kinase C-depleted lymphocytes. AB - The Na+/H+ antiport of rat thymic lymphocytes is activated when protein kinase C is stimulated by phorbol esters. A similar activation of the antiport is obtained when the cells are treated with hypertonic solutions. We tested the possibility that protein kinase C also mediates the osmotic activation of Na+/H+ exchange. Protein kinase C was depleted by preincubation of thymocytes for 24 hr in the presence of high concentrations of phorbol ester. Disappearance of the enzyme was assessed by direct measurement of phosphotransferase activity, and by the loss of biological responses to phorbol esters. The Na+/H+ antiport in protein kinase C depleted cells was not stimulated by addition of phorbol ester, but responded normally to hypertonic treatment. The results indicate that the osmotic activation of countertransport does not require stimulation of protein kinase C. PMID- 3004446 TI - Conversion of leukotrienes A4 to C4 in cell-free systems. AB - A procedure for assaying leukotriene C4 synthase activity in cell-free extracts has been presented. Leukotriene A4 methyl ester was as active a substrate as leukotriene A4 (Na salt) for the synthesis. The methyl ester is the substrate of choice, because (1) it is more stable than the sodium salt, (2) it is not a substrate of epoxide hydrolase for leukotriene B4 synthesis, and (3) it gives a lower blank than an equimolar concentration of leukotriene A4. The enzyme activity in rat liver, guinea pig and human lungs, and human nasal polyp was chiefly membrane-bound, although the cytosol contained some activity. PMID- 3004447 TI - Inhibition of the catalytic subunit of phosphorylase phosphatase by oxalyl thioesters and its possible relevance to the mechanism of insulin action. AB - Oxalyl thioesters, especially S-oxalylglutathione, are shown to be effective inhibitors of the catalytic subunit of phosphorylase phosphatase. The amount of inhibition was found to be time dependent and partially reversed by thiols, thus suggesting that at least part of the inhibition is due to oxalylation of an enzymic thiol group. The possibility that the inhibition of the phosphatase by oxalyl thioesters may be important in vivo and that oxalyl thioesters may be functioning as negative intracellular messengers for insulin is discussed. PMID- 3004448 TI - Phenylmethylsulfonyl fluoride (PMSF) inhibits nerve growth factor binding to the high affinity (type I) nerve growth factor receptor. AB - Dorsal root ganglia were extirpated from 9-day old embryonic chickens and solubilized in phosphate buffered saline containing 0.5% Noniodet P 40 detergent. When nerve growth factor binding studies are performed on these samples, the expected curvilinear Rosenthal (Scatchard) plot is obtained. However, when the solubilized cell sample is made 1-2 mM in phenylmethylsulfonyl fluoride and nerve growth factor binding is determined, a linear Rosenthal (Scatchard) plot is obtained. The equilibrium dissociation constant obtained from the slope of the line is 1.9 X 10(-9) M, identical to the equilibrium dissociation constant of the low affinity receptor. A similar phenomenon is observed when rat pheochromocytoma cells are solubilized in the non-ionic detergent and nerve growth factor binding is determined. No high affinity binding can be detected for either cell type when detergent solubilized cells are incubated with phenylmethylsulfonyl fluoride. PMID- 3004449 TI - Identification of gonadotropin inducible, high density lipoprotein receptors in the solubilized membranes from rat ovary. AB - Specific receptors for high density lipoproteins (HDL3) were solubilized from membranes of rat corpus luteum using different detergents. Among the detergents tested, octyl-beta-D-glucoside (40 mM) was most effective with respect to recovery of binding activity. The receptor activity released into 105,000 X g supernatant, can be assayed directly or with the precipitate obtained after dilution of the soluble supernatant. The 125I-HDL3 binding activity in the precipitated extract was linear with time, proportional to the amount of protein in the incubation mixture and saturable with increasing concentrations of 125I HDL3. The solubilized receptor has an equilibrium dissociation constant (Kd) of 21.2 micrograms/ml and the binding activity was insensitive to Ca+2, EDTA and NaCl. These properties are similar to the membrane associated receptor. Administration of gonadotropin induced the HDL3 receptor in the solubilized membranes, suggesting that this receptor represents the physiologic receptor in the ovary. PMID- 3004450 TI - Isolation and characterization of a novel cytochrome P-450-like pseudogene. AB - A rabbit liver P-450-like pseudogene has been isolated from a lambda phage genomic library. Sequence analysis revealed structural homology with respect to the rat P-450b and P-450e genes as well as a similar intron-exon organization. A 5'-proximal TATA box-like sequence and two 3'-distal putative polyadenylation signals were identified, and all putative intron-exon boundaries except at the 3' splice site of intron 2 were found to follow the GT/AG rule. With allowance for apparent deletions and insertions, the structural homology of the amino acid sequence deduced from the pseudogene with respect to rabbit P-450 isozyme 2 is lower for exons 1 through 4 (18-28%) than for exons 5 through 9 (42-65%). S1 nuclease mapping showed that mRNAs complementary to the DNA sequence of exon 9 are expressed. However, due to the alterations in the pseudogene, it appears that functional P-450 would not be produced from such mRNAs. PMID- 3004451 TI - A simultaneous quantitation of leukotriene B4 and its omega-oxidized products by gas chromatography-mass spectrometry. AB - We developed a highly sensitive and specific method for the simultaneous quantitation of leukotriene B4 (LTB4) and its omega-oxidized metabolites (20 hydroxy-LTB4 and 20-carboxy-LTB4) by mass fragmentography using deuterated compounds as internal standards. The ions produced by the cleavage of the C12-13 bond of the methyl ester dimethylisopropylsilyl ether derivatives of LTB4 and its metabolites were measured by selective ion monitorings. The detection limit of LTB4 was less than 10 pg and about 100-fold lower than that by high performance liquid chromatography. By using this method, the synthesis and further metabolism of LTB4 in human polymorphonuclear leukocytes were investigated. PMID- 3004452 TI - Intracellular localization of ATP:AMP phosphotransferase in Escherichia coli. AB - ATP and AMP were immediately converted into ADP by intact cells of Escherichia coli in the presence of Mg2+, while ADP was also rapidly converted into ATP and AMP under the same conditions. Adenylate kinase was released when E. coli cells were converted to spheroplasts by treatment with lysozyme-EDTA or osmotic shock. Adenylate kinase activities detected in the cytoplasm, periplasm and membrane fractions were approximately 58%, 36% and 6% of the total cellular activity, respectively. These results indicate that adenylate kinase in E. coli occurs in the periplasm as well as the cytoplasm. PMID- 3004453 TI - Tyrosine kinase activity of brain insulin and IGF-1 receptors. AB - Lectin-purified rat brain preparations demonstrate specific [125I]insulin and [125I]-IGF-1 binding. Insulin-stimulable tyrosine kinase activity as measured by exogenous substrate phosphorylation was present in brain and liver lectin purified preparations with the delta kinase activity/B/F of brain approximately 2.5 fold greater than that of liver. Insulin-stimulable tyrosine kinase activity was abolished in liver but decreased by only approximately 50 percent in brain after immuno-depletion with antiserum which recognizes insulin but not IGF-1 receptors. Insulin and IGF-1 dose responses for phosphorylation of the immunodepleted brain preparations suggested that the remaining tyrosine kinase activity was IGF-1 receptor mediated. Thus, functional IGF-1 receptors are present in rat brain, and the doses of insulin typically used to evaluate insulin receptor tyrosine kinase activity will stimulate IGF-1 receptor tyrosine kinase activity as well. PMID- 3004454 TI - Human lung tumor sensitivity to difluoromethylornithine as related to ornithine decarboxylase messenger RNA levels. AB - The differential response to polyamine depletion has been studied in two types of human lung tumor cells. Small cell lung carcinoma cells die following polyamine depletion by difluoromethylornithine treatment while non-small cell lines demonstrate a typical cytostatic response. We now report that a small cell line, NCI H82, has a lower apparent capacity for polyamine biosynthesis than does a representative non-small cell, NCI H157. In subconfluent cultures, the ornithine decarboxylase activity is 25 times lower in the small cell than the non-small cell and by comparison, the polyamines in the small cell line are markedly reduced. Most significantly, levels of mRNA coding for ornithine decarboxylase are approximately 100-fold lower in the small cell than the non-small cell line, and this difference does not appear to be a result of gene rearrangement. These results suggest that differential sensitivity to polyamine depletion is related to different steady-state levels of ornithine decarboxylase mRNA. PMID- 3004456 TI - Identification of receptors for insulin-like growth factor II in two insulin-like growth factor II producing cell lines. AB - Specific high affinity membrane receptor(s) for insulin-like growth factor II have been characterized in two cell lines which produce this hormone and have the ability to proliferate in serum-free media. These receptor(s) have no affinity for either insulin or biosynthetic insulin-like growth factor I. Affinity cross linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent Mr of 250K which does not change with disulfide bond reduction. Our findings are consistent with an autocrine function for insulin-like growth factor II and indicate that these continuous cell lines may provide unique systems for further investigations of this hormone and its receptor. PMID- 3004457 TI - Stimulation of Na,K-ATPase activity of frog skeletal muscle by insulin. AB - Na,K-ATPase activity of a plasma membrane fraction obtained from frog skeletal muscles was increased approximately two-fold by exposing muscles to insulin, whereas the addition of insulin to a membrane preparation suspension has no effect on Na,K-ATPase activity. The effect of insulin on Na,K-ATPase activity of whole muscles was specific to insulin and insulin derivatives that had the ability of receptor-binding and was not inhibited by actinomycin D. Insulin also induced a development of Na,K-ATPase activity in muscles whose Na,K-ATPase activity had been blocked by ouabain-pretreating. Such a insulin action was inhibited by monensin. These observations suggest that insulin stimulates the monensin-sensitive intracellular transport of membrane proteins which should be responsible for the increase in Na/K pumping activity. PMID- 3004455 TI - Stimulus-dependent mobilization of protein kinase C. AB - Protein kinase C and its associated phorbol myristate acetate receptor moved from cytosol to membranes in human neutrophils stimulated with direct activators of the kinase, calcium ionophores, or chemotactic peptides. However, the peptides acted only in the presence of cytochalasin B and neither platelet-activating factor nor leukotriene B4 (+/- cyclochalasin B) induced this movement. Thus, protein kinase C appears variably involved in neutrophil responses: physiological agents may bypass or depend minimally upon the phosphorylating enzyme to elicit function. PMID- 3004458 TI - The production of topoisomerase II-mediated DNA cleavage in human leukemia cells predicts their susceptibility to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA). AB - Protein-associated DNA cleavage is produced in mammalian cells treated with active antileukemic DNA intercalating agents such as 4'(9 acridinylamino)methanesulfon-m-anisidide (m-AMSA). We have examined the ability of m-AMSA to produce DNA cleavage in 3 human myeloid leukemic cell lines with different sensitivities to the cytotoxic actions of m-AMSA to see if the magnitude of DNA cleavage correlated with the degree of m-AMSA sensitivity. DNA alkaline elution was used to quantify DNA cleavage. The amount of m-AMSA-induced DNA cleavage in the two lines sensitive to m-AMSA was 1-2 orders of magnitude greater than that in an m-AMSA-resistant leukemic line. The m-AMSA resistant line had been developed by prolonged exposure of one of the sensitive lines to m-AMSA. This finding was not secondary to a decreased uptake of m-AMSA in the resistant cell line. m-AMSA treatment of the nuclei isolated from the three lines produced DNA cleavage frequencies comparable to the cleavage frequencies produced by m AMSA treatment of the whole cells from which the nuclei were isolated. The DNA cleaving ability stimulated by m-AMSA is thought to be mediated by drug-induced effects on topoisomerase II, a nuclear enzyme that mediates alterations in DNA conformation. Alterations in the manner in which this enzyme interacts with antineoplastic agents may explain the emergence of resistant cells following initially successful chemotherapy. PMID- 3004459 TI - A rapid method for the preparation of 125I-labelled human growth hormone for receptor studies, using reverse-phase high performance liquid chromatography. AB - Human growth hormone was labelled with 125 Iodine by the stoichiometric modification of the chloramine-T method to a specific activity of 50-80 microCi/microgram, and the iodinated mixture was purified by reverse-phase high performance liquid chromatography using a C18 column (SynChropak RP-P) and a linear gradient. Compared with the usual Sephadex G-100 chromatography, HPLC gave a much better separation, with a higher yield and a considerably reduced analysis time (30 min vs 5 h). The [125I]-labelled preparation had normal binding to IM-9 lymphocyte receptors. The maximum bindability of the HPLC-purified preparation approximated 90%, which is the best value so far reported for human growth hormone. It is concluded that HPLC is a fast, convenient and reproducible method for obtaining an improved [125I]-labelled human growth hormone for receptor studies. PMID- 3004460 TI - Partial dependency of 1-oleoyl-2-acetyl-glycerol-induced superoxide production by human neutrophils on calcium ions and cytochalasin B. AB - Superoxide production by neutrophils induced by 1-oleoyl-2-acetyl-sn-glycerol at concentrations below 100 microM was enhanced by extracellular calcium ions, while that of phorbol myristate acetate was unaffected. Verapamil, a calcium-channel blocker, more effectively inhibited the superoxide production induced by 1-oleoyl 2-acetyl-sn-glycerol than that of phorbol myristate acetate. Cytochalasin B at 5 micrograms/ml significantly potentiated superoxide production by 1-oleoyl-2 acetyl-sn-glycerol at concentrations below 100 microM, but not that of phorbol myristate acetate. It is suggested that neutrophil activation induced by the former have different features from that of the latter. PMID- 3004461 TI - Recovery of transferrin receptors on hepatocytes membrane after collagenase perfusion. AB - Hepatocyte cell suspensions obtained by collagenase perfusion method did not have transferrin (TF) receptors. However, after incubation at 37 degrees, they appeared to gain TF receptors, the number of which was the function of incubation time at 37 degrees. It is suggested that hepatocyte TF receptors are collagenase sensitive. This study can explain previous observations that hepatocyte isolated with collagenase treatment of the tissue do not bind TF at 4 degrees but take up TF at 37 degrees. PMID- 3004462 TI - Catalysis of singlet oxygen production in the reaction of hydrogen peroxide and hypochlorous acid by 1,4-diazabicyclo[2.2.2]octane (DABCO). AB - The kinetics of the singlet oxygen production in the hydrogen peroxide plus hypochlorous acid reaction were studied by measuring the time course of the singlet oxygen emission at 1268 nm. The addition of 1,4-diazabicyclo[2.2.2]octane (DABCO) increased the peak intensity of the chemiluminescence, but decreased its duration. The increased rate of singlet oxygen production likely accounts for the enhancement of singlet oxygen dimol emission reported in 1976 by Deneke and Krinsky (J. Am. Chem. Soc. 98, 3041-3042). This phenomenon was not seen when singlet oxygen was generated with the reaction of hypobromous acid and hydrogen peroxide. Thus, the enhancement of red chemiluminescence by DABCO should not be regarded as a general test for the production of singlet oxygen in complex biochemical systems. PMID- 3004463 TI - A signal sequence is required for the functions of the signal recognition particle. AB - It is shown that removal of the signal sequence of carp preproinsulin by gene technology abolishes the interaction between the polypeptide chain and the signal recognition particle with respect to both its functions: the inhibition of translation by the signal recognition particle in the absence of microsomal membranes, and the reconstitution of the protein translocation competence of high salt inactivated microsomes. It is concluded that a signal sequence is absolutely required for the functions of the signal recognition particle. PMID- 3004464 TI - Molecular cloning of rat mitochondrial glutamic oxaloacetic transaminase mRNA and regulation of its expression in regenerating liver. AB - cDNA clones for rat mitochondrial glutamic oxaloacetic transaminase (mGOT) have been isolated from a rat liver cDNA library. One of the clones, designated p501, contained a cDNA insert of 1.4 kilobase pairs in length and hybridized to a mRNA of 2.4 kilobases from rat liver. We measured mGOT mRNA content in a regenerating rat liver. In a regenerating rat liver, mGOT activity was increased and reached maximum (170% of control activity) at about 48 h following the operation. Using the cDNA of mGOT, it was revealed that the increase of mGOT in the regenerating rat liver depended on its mRNA content. PMID- 3004466 TI - Inhibitory effects of theophylline and dibutyryl cAMP on murine erythroleukemia cell differentiation. AB - Murine erythroleukemia cells can be induced to differentiate by a variety of compounds. We have previously shown that 5'-methylthioadenosine, an inhibitor of cAMP phosphodiesterase, blocks induction of these cells. The present study demonstrates that theophylline, another cAMP phosphodiesterase inhibitor, also blocks murine erythroleukemia cell differentiation in a concentration-dependent manner. Northern blot analysis indicates that this agent inhibits accumulation of alpha- and beta-globin transcripts. These findings are extended by demonstrating that dibutyryl cAMP exerts similar effects. Furthermore, theophylline and dibutyryl cAMP are synergistic in inhibiting appearance of the mature erythroid phenotype. The results thus suggest that cAMP regulates induction of murine erythroleukemia cell differentiation. PMID- 3004465 TI - Na+/H+ exchange is present in basolateral membranes from rabbit small intestine. AB - Fluorescence quenching of the pH gradient sensitive dye acridine orange and that of the membrane potential sensitive dye Di-S-C3(5) have been studied in purified basolateral membrane vesicles obtained from rabbit small intestine. Basolateral membranes contain an electroneutral, carrier mediated, Na+/H+ exchange activity. They also appear to contain an electrogenic pathway for H+ movement. Based on the comparison of acridine orange fluorescence quenching in the presence of an outwardly directed Na+ gradient and in the presence of known K+ diffusion gradients it can be estimated that at least 50% of the observed proton fluxes are due to the activity of the exchanger. Acridine orange fluorescence recovery measurements have been used to assess the kinetic properties of the exchanger. PMID- 3004467 TI - Atrial natriuretic factor and cGMP inhibit amiloride-sensitive Na+ transport in the cultured renal epithelial cell line, LLC-PK1. AB - The renal cell culture model, LLC-PK1, which contains an amiloride-sensitive conductive Na+ transport pathway and a Na+/H+ exchanger, was utilized to examine the direct effects of atriopeptin II and cGMP on Na+ transport in epithelial cells. Exposure of cells to atriopeptin II (10(-7) M) increased cGMP production within 2 min of addition to cells in monolayer. Atriopeptin II (10(-7) M) or exogenous 8-bromo-cGMP (10(-3) M) maximally inhibited the uptake of 22Na+ through the conductive pathway which accounted for up to 60% of total 22Na+ uptake. The apparent Ki for this inhibition by atriopeptin II was 2 X 10(-11) M. Amiloride inhibited 22Na+ uptake to a similar extent as atriopeptin II, and the effects of the presence of both agents was not additive. In contrast, neither atriopeptin II nor cGMP blunted the increment in 22Na+ uptake induced by a pH gradient. Thus atriopeptin II can directly inhibit Na+ transport in renal epithelial cells, probably through its stimulation of cGMP. PMID- 3004468 TI - Gonadotropin releasing hormone enhances polyphosphoinositide hydrolysis in rat pituitary cells. AB - Addition of gonadotropin releasing hormone to myo-[2-3H]inositol-prelabeled rat pituitary cells in primary culture evoked a dose-dependent increase of the accumulation of [3H]inositol phosphates with a rise of inositol triphosphate within 30 sec of stimulation, followed by a rise in inositol diphosphate and inositol monophosphate. Inositol phosphate accumulation was enhanced up to 5-to-8 fold and was time-dependent between up to 15 min incubation without further increase beyond this time period. Without preincubation with LiCl2, there was no measurable increase of GnRH-induced inositol phosphate accumulation compared to controls. The presence of calcium in the incubation medium did not affect the increase of inositol phosphates. These data give evidence, that polyphosphoinositide breakdown may be an early step in the action of gonadotropin releasing hormone on gonadotropin secretion. PMID- 3004469 TI - Reversal by protein kinase C inhibitor of suppressive actions of phorbol-12 myristate-13-acetate on polyphosphoinositide metabolism and cytosolic Ca2+ mobilization in thrombin-stimulated human platelets. AB - In the presence of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C in vitro, phorbol-12-myristate-13-acetate (PMA) did not suppress the thrombin-induced increase of cytosolic Ca2+ concentration in human platelets. The H-7 reversal of the inhibitory action of PMA was also observed in thrombin-induced polyphosphoinositide breakdown by phospholipase C. These results provide additional support to the developing theory that the inhibition of PMA on Ca2+ mobilization and phosphoinositide turnover may be mediated by protein kinase C activation. PMID- 3004470 TI - Phosphoinositide phosphorylation precedes growth in rat mammary tumors. AB - DMBA-induced rat mammary tumors were used to study the possible association of phosphoinositide phosphorylation to tumor growth. These membranous enzymatic activities were measured during various stages of tumor growth induced by pharmacological manipulation of plasma prolactin level. An increase in phosphorylation of both phosphatidyl inositol and phosphatidyl inositol 4 phosphate preceded the growth induced by prolactin concomitantly with an increase in tyrosine phosphorylation. Good correlation (r = 0.87) existed between the tyrosine kinase activity and phosphatidyl inositol kinase activity of 21 individual tumors taken from animals at different stages of hormonal manipulation. Phosphoinositide phosphorylation was inhibited by quercetin and was not affected by cAMP, similar to tyrosine kinase. Phosphorylation of angiotensin II by tyrosine kinase was inhibited by 0.2 mg/ml phosphatidyl inositol 4 phosphate or phosphatidyl inositol 4,5-bisphosphate. PMID- 3004471 TI - Characterization of [3H]pentagastrin binding in guinea pig gastric glands--an alternative convenient ligand for receptor binding assay. AB - The binding of [3H]pentagastrin to guinea pig gastric glands was specific, saturable and of high affinity (Kd = 5 nM). The relative order of potencies for gastrin and CCK analogs in displacing [3H]pentagastrin binding correlated well with those obtained using [125I]gastrin and their reported biological potencies for stimulating acid secretion. Nonselective CCK/gastrin antagonists including carbobenzoxy-CCK (26-32), proglumide and benzotript, but not the selective peripheral CCK antagonist, asperlicin, inhibited specific [3H]pentagastrin binding. The results indicate that [3H]pentagastrin labels physiologically relevant gastrin receptors in guinea pig gastric glands. PMID- 3004472 TI - Purification of SV40 T-antigen by SV40 DNA-sepharose affinity chromatography. AB - T-antigen from SV40-infected BSC-1 cells was purified approximately 30,000 fold using a rapid purification procedure consisting of ammonium sulfate fractionation followed by chromatography on hydroxylapatite, blue-sepharose, and SV40 DNA sepharose. The SV40 DNA-sepharose was optimized for the binding of T-antigen by the covalent attachment of the SV40 DNA at its BamHI site to cyanogen bromide activated sepharose. The most highly purified T-antigen appeared as a single polypeptide of 94 K daltons by polyacrylamide gel electrophoresis. PMID- 3004473 TI - Synthesis of carboxy-nifedipine and its use in the preparation of an affinity resin for the 1,4-dihydropyridine receptor. AB - Affinity chromatography represents a potentially valuable approach to study the calcium antagonist receptor in many tissues. Methods have been developed to synthesize carboxy-analogues of the 1,4-dihydropyridine calcium antagonists. Carboxy-nifedipine ([+/-]1,4-dihydro-2,6 dimethyl-4-[3-nitrophenyl] pyridine-3 carboxylic acid-5-carboxylic methyl ester) was prepared with a yield of 50% and its structure has been thoroughly characterized. Carboxy-nifedipine has been coupled to a hexamine-agarose resin through an acid chloride intermediate producing an affinity resin (1.6 mumol of drug/ml). Experiments have shown that this affinity resin is capable of binding the [3H]nitrendipine receptor solubilized from transverse tubule membranes. PMID- 3004474 TI - GTP-activated GTP binding protein(Gs) in membranes achieved by hormone plus GDP does not serve as a substrate for ADP-ribosylation by cholera toxin. AB - Adenylate cyclase in the presence of GTP became active by the addition of cholera toxin irrespective of the presence of glucagon, and under the same condition the Gs of these activated enzymes were good acceptor of an ADP-ribose moiety. On the other hand, the cyclase in the presence of GDP remained inactive with cholera toxin but became active by the further addition of glucagon. However, neither of these Gs served as a cholera toxin substrate. Glucagon reduced an inhibitory action of added GDP for cholera toxin plus GTP-stimulated adenylate cyclase activity but did not for toxin plus GTP-enhanced ADP-ribosylation of Gs. These results demonstrate that Gs-GTP complex formation alone is not sufficient for Gs to serve as a cholera toxin substrate, and suggest an additional GTP binding site responsible for ADP-ribosylation by the toxin. Hormone dependent preferential interaction between the GTP binding site on Gs coupled with adenylate cyclase regulation and membrane-associated nucleoside diphosphate kinase is discussed. PMID- 3004475 TI - ApoE deficiency: markedly decreased levels of cellular ApoE mRNA. AB - Apolipoprotein (apo) E deficiency is a rare genetic disease characterized by palmar and tuberoeruptive xanthomas, type III hyperlipoproteinemia, and premature atherosclerotic vascular disease. The plasma level of apoE in apoE deficiency is less than 0.05 mg/dl by radioimmunoassay, and no structural variants of apoE were detected by immunoblot of plasma or VLDL separated by two-dimensional gel electrophoresis. The apoE gene is present in the apoE deficient patient, and there are no major insertions or deletions in the gene by Southern blot analysis. Blood monocyte-macrophages isolated from a patient with apoE deficiency contain 1 3% of the level of apoE mRNA present in monocyte-macrophages isolated from normal subjects. The apoE mRNA in the monocyte-macrophages of the apoE deficient patient is similar in size to normal apoE mRNA. The deficiency of plasma apoE in the patient with apoE deficiency is due to a markedly decreased level of apoE mRNA and decreased production of the E apolipoprotein. The decreased apoE mRNA may be due to a defect in transcription or processing of the primary transcript of the apoE gene or to instability of the apoE mRNA. The decreased plasma level of apoE results in delayed clearance of remnants of triglyceride rich lipoproteins, hyperlipidemia, and a type III phenotype. PMID- 3004476 TI - Influence of dietary zinc deficiency and parenteral zinc on rat liver fructose 1,6-bisphosphatase activity. AB - Fructose 1,6-bisphosphatase activity in liver of rats fed a zinc deficient diet was decreased to 60% of that in zinc adequate controls. Activity in the zinc deprived rats was not restored to control values by in vitro addition of EDTA. When a physiological dose of zinc was tube fed to the depleted rats, activity increased approximately 150% within 0.5 hr of the dose, and by 1 hr plateaued to a level seen in zinc adequate controls. A significant transient decrease in activity occurred following an intraperitoneal zinc load. This is reversible by in vitro addition of EDTA. These results suggest that rat liver fructose 1,6 bisphosphatase activity is highly sensitive to zinc in vivo as has been demonstrated in vitro. PMID- 3004477 TI - In vitro interactions between Sertoli cells and steroidogenic cells. AB - Both the cell and the species specificities of the steroidogenic potentiating activity (SPA) of Sertoli cells on Leydig cells were studied using a coculture system. Coculture of purified pig Leydig cells with rat or pig Sertoli cells in the presence of FSH led in both cases, to a significant increase in hCG receptor number and in hCG-stimulated testosterone production. Similarly, coculture of bovine adrenal cells with rat or pig Sertoli cells enhanced the steroidogenic response of adrenal cells to ACTH and angiotensin II. Such effects were not observed when pig Leydig cells or bovine adrenal cells were cocultured with bovine aortic endothelial cells. Coculture of Sertoli and Leydig cells in the presence of hCG, resulted in a significant increase in FSH receptor number and in FSH-induced plasminogen activator activity. Such effects did not occur when Sertoli cells were cocultured with either adrenal or aortic endothelial cells. PMID- 3004478 TI - Phorbol esters inhibit alpha 1-adrenergic receptor-stimulated phosphoinositide hydrolysis and contraction in rat aorta: evidence for a link between vascular contraction and phosphoinositide turnover. AB - We investigated the actions of two biologically active phorbol esters, phorbol dibutyrate (PDB) and phorbol myristate acetate (PMA), on receptor-stimulated phosphoinositide hydrolysis in rat aorta. We found both PDB and PMA potently inhibited norepinephrine (NE) stimulated PI hydrolysis in rat aortic rings. The biologically inactive phorbol, 4-alpha-phorbol was ineffective. In the presence of the calcium channel antagonist nitrendipine, PDB potently inhibited both the phasic and tonic components of NE-induced contraction. These results suggest a functional coupling between receptor-stimulated PI turnover and vascular contraction. They also suggest a mode of feed-back regulation in vascular tissue involving phorbol esters in receptor-stimulated PI hydrolysis. PMID- 3004479 TI - Intracellular potassium promotes antibody binding to an antigen associated with the Na/K pump of sheep erythrocytes. AB - Anti-Lp antibody is known to bind to sheep red cells and to stimulate the Na/K pump. The antibody acts by reducing the affinity of the pumps for intracellular K as a noncompetitive inhibitor. We now show that intracellular K enhances the extent of anti-Lp binding to the pump-associated antigens. Cells made with approximately 60 mmol/1 K bound approximately 60% more anti-Lp than cells with O K; binding was assayed by measuring the extent of stimulation of the pump mediated K influx. PMID- 3004480 TI - Evidence that mammalian lignans show endogenous digitalis-like activities. AB - Enterolactone, a lignan that has been identified in biological samples from man and several mammals, shares with ascorbic acid and cardiac glycosides a gamma butyrolactone. It displaces 3H-ouabain from its binding sites on cardiac digitalis receptor and inhibits, dose dependently, the Na+, K+-ATPase activity of human and guinea-pig heart. The time dependence of this inhibition resembles that of dihydroouabain, a cardiac glycoside in which the lactone ring does not contain conjugated double bonds. The active concentrations of enterolactone as inhibitor of Na+,K+-ATPase are in the 10(-4) M range and, at those concentrations, the cross-reactivity with antidigoxin antibodies is low. Lignans may contribute to the putative digitalis-like activity found in tissues, blood and urine of several mammals including man. PMID- 3004481 TI - 5S RNA gene specific transcription factor (TFIIIA) changes the linking number of the DNA. AB - The purified 5S gene specific transcription factor A (TFIIIA), when incubated with the relaxed 5S gene in the presence of topoisomerase I alters the conformation of the DNA, resulting in a change in the linking number. This interaction introduces a change of one bp per TFIIIA binding site at a low concentration of DNA to protein (1:2) which increases to an extent of 0.9 turns (9 bp) per TFIIIA binding site at a higher protein concentration (1:12). These analyses support the notion that the binding of TFIIIA to the 5S DNA introduces a minimal change in the topology of circular DNA molecules. PMID- 3004482 TI - Expression of aldolase A messenger RNAs in human adult and foetal tissues and in hepatoma. AB - 3 specific cDNA clones for human aldolase A were isolated from a human muscle library. One of them was subcloned in M 13 phage, then used as a probe to investigate the patterns and the levels of aldolase A mRNA in various human tissues. Two mRNA species differing in length were observed. The lighter one 1550 bases- was found specific to skeletal muscle; its amount increased during muscle development. The heavier aldolase A mRNA -1650 bases- accounted for foetal and ubiquitous presence of aldolase A isozyme. The resurgence of aldolase A in hepatomas occurred through this latter mRNA species. PMID- 3004483 TI - Methylation and expression of the human thyroglobulin gene. AB - The DNA methylation pattern at the 5'end of the human thyroglobulin gene has been determined in different tissues. Out of the four HpaII/MspI sites (5'-CCGG-3') present in this region, three were found to be non-methylated in thyroid DNA, while full methylation was observed in liver, salivary gland and sperm DNA. This demethylation therefore correlates with expression of the thyroglobulin gene. However, all four sites were found to be non-methylated in placental DNA, regardless of the activity of the gene. PMID- 3004484 TI - Modulation of prolactin binding sites in vitro by membrane fluidizers. IV. Differential effects on plasma membrane and Golgi fractions of male prostate and female liver in the rat. AB - In vitro treatment of crude particulate fractions of male rat ventral prostate and female rat liver with membrane fluidizers (aliphatic alcohols) has been previously reported by us to increase prolactin (PRL) receptor levels, presumably by unmasking cryptic prolactin receptors. The objective of this study was to determine if similar in vitro treatment of purified plasma membrane- and Golgi rich fractions of male rat prostate and female rat liver with ethanol produced differential effects on prolactin binding in these two subcellular fractions. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. 125I-PRL specific binding to Golgi-rich fractions of male ventral prostate and female liver was approximately 4-fold higher than that observed in plasma membrane-rich fractions. The microviscosity parameter, inversely related to lipid fluidity, was consistently lower in Golgi-rich fractions than that in plasma membrane-rich fractions in both prostate and liver. In vitro ethanol treatment of prostatic and hepatic plasma membrane fractions produced a dose-related increase and then decline in prolactin binding and a maximal (60-75%) increase in prolactin binding was observed at 4.8% and 2.0% ethanol in prostatic and hepatic membranes, respectively. This in vitro treatment also produced a significant increase in apparent lipid fluidity of plasma membrane-rich fractions of prostate gland and liver. However, similar in vitro ethanol treatment of Golgi fractions of both prostate gland and liver exhibited little increase in prolactin binding without changing microviscosity. Our observations are consistent with the direct relationship between membrane fluidity and prolactin receptor levels. The changes in prostatic and hepatic plasma membrane fractions following in vitro ethanol treatment suggest that prolactin receptors located on the plasma membranes may be modulated (via membrane lipid microviscosity changes) in vivo to a greater extent by various physiological agents than those located within the Golgi fraction. PMID- 3004485 TI - Kinetic properties of glycogen synthase from skeletal muscle after phosphorylation by glycogen synthase kinase 4. AB - Glycogen synthase I (EC 2.4.1.11) from rat and from rabbit skeletal muscle was phosphorylated in vitro by glycogen synthase kinase 4 (EC 2.7.1.37) to the extent of 0.8 phosphates/subunit. For both phosphorylated enzymes, the activity ratio (activity without glucose 6-P divided by activity with 8 mM glucose 6-P) was 0.8 when determined with low concentrations of glycogen synthase and/or short incubation times. However, the activity ratio was 0.5 with high enzyme concentrations and longer incubation times. It was found that the lower activity ratios result largely from UDP inhibition of activity measured in the absence of glucose 6-P. Inhibition by UDP was much less pronounced for glycogen synthase I, indicating that a major consequence of phosphorylation by glycogen synthase kinase 4 is an increased sensitivity to UDP inhibition. PMID- 3004486 TI - Monoclonal antibody detection of transformation associated protein (TAP) in ts110 Moloney murine sarcoma virus transformed 6M2 cells that are different from the mos gene product. AB - By the use of a highly specific monoclonal antibody (designated MC), we were able to detect three radiolabeled bands with molecular weights of 60,000, 63,000, and 66,000 daltons in the ts-110 Moloney murine sarcoma virus mutant-transformed rat kidney cells known as 6M2. Expression of transformation properties as well as these three bands in 6M2 cells was found to be temperature sensitive. Therefore, MC detected factors that are apparently associated with the transformation of 6M2 cells. These factors are tentatively referred to as transformation associated proteins. These transformation proteins were found in two other Moloney murine sarcoma virus-transformed rat cell lines. These proteins were found to differ from known gene products of the ts-110 Moloney murine sarcoma virus mutant and do not have kinase activity. The transformation associated proteins may represent rat cellular factors activated during the transformation of rat cells by Moloney murine sarcoma virus. PMID- 3004487 TI - Transfer of the Epstein-Barr virus (EBV) DNA fragment coding for EBNA-1, the putative transforming antigen of EBV, into normal human lymphocytes: gene expression without cell transformation. AB - Fresh human lymphocytes were transfected with cloned EBV DNA fragments containing the coding exon for EBNA-1. DNA-loaded reconstituted Sendai Virus envelopes (RSVE) were used for efficient gene transfer. EBNA was detected by immunofluorescence in 1-5% of cells transfected with either the cloned BamHl K fragment of EBV DNA (5.1 kb) or the recombinant plasmid pSV3neoEBNA1, containing only the 2.0kb EBNA-1 coding exon. EBV-specific mRNA was detected by hybridization up to 14 days after DNA transfer. Quantitation of mRNA by laser densitometry revealed that the transcription level was similar to that obtained after infection with an intact virus. However, no effect on cellular proliferation was observed by (3H)-thymidine incorporation assays, and transformation was not achieved. We conclude that though EBNA-1 may be necessary for cellular immortalization by EBV, it alone is not sufficient. PMID- 3004488 TI - Kinetic evidence for activating and non-activating components of autophosphorylation of the insulin receptor protein kinase. AB - Reduced and carboxamidomethylated-lysozyme (RCAM-lysozyme) is an excellent substrate (Km = 13 microM) and a potent inhibitor of receptor autophosphorylation (Ki = 0.6 microM). By using these properties of RCAM-lysozyme autophosphorylation was resolved into two kinetically and functionally distinct components involving formation of phosphotyrosine on the receptor's beta-subunits: 1. Insulin stimulated autophosphorylation is independent of autophosphorylation at other sites; activation of insulin receptor-catalyzed substrate phosphorylation is dependent upon this component of autophosphorylation, which is inhibited by RCAM lysozyme. 2. Autophosphorylation at saturating RCAM-lysozyme concentration is insensitive to insulin and has little effect on substrate phosphorylation. Thus, only insulin-dependent receptor autophosphorylation is responsible for activation of kinase-catalyzed substrate phosphorylation. PMID- 3004489 TI - Polyamine-activated protein phosphatase activity in HeLa cell nuclei. AB - Protein phosphatase activity towards endogenous nuclear substrates in sonicates of isolated nuclei was activated 2-4-fold by spermine. Exogenous casein was dephosphorylated by these preparations only in the presence of spermine. Activation by spermine was half maximal at about 0.1 mM. Spermidine also activated, with half maximal stimulation at 1mM; putrescine activated poorly. Mg++ and Ca++ appeared to activate the same phosphatase activity but were only 50% as effective as spermine. Spermine activation was inhibited by 200 mM NaCl, 50 mM NaF, or 40 mM beta-glycerol phosphate. Nuclear phosphatase activity, with or without spermine, was inhibited 50% by inhibitor 2 of protein phosphatase 1. These observations suggest that protein phosphatase 1 is a major nuclear protein phosphatase and that its activity against endogenous nuclear substrates is activated by physiological concentrations of spermine. PMID- 3004491 TI - An interferon analogue, [Ala 30,32,33]HuIFN-alpha 2, acting as a HuIFN-alpha 2 antagonist on bovine cells. AB - We have studied the biological and receptor binding properties of a human alpha 2 interferon (HuIFN-alpha 2) analogue, [Ala30,32,33] HuIFN-alpha 2, which is shown in the accompanying paper (1) to be biologically inactive on homologous cells. Here we demonstrate that this analogue is also devoid of biological activity on bovine MDBK cells. However, whereas the analogue did not inhibit the binding of radiolabeled HuIFN-alpha 2 to WISH cells, it did compete for binding to receptors on the bovine cells. This behavior suggested that [Ala30,32,33] HuIFN-alpha 2 could act as an antagonist of HuIFN-alpha 2 on bovine cells and indeed coaddition of the analogue and native HuIFN-alpha 2 to MDBK cells competitively inhibited both the antiviral and antiproliferative activity of HuIFN-alpha 2. PMID- 3004490 TI - Differential regulation of multiple neuroreceptors in a somatic cell hybrid by inhibitors of glycoprotein processing. AB - Specific binding of [3H][D-Ala2,D-Leu5]enkephalin, [3H]ethylketocyclazocine, 5 [3H]hydroxytryptamine, and [3H]spiperone was examined in neuroblastoma-brain hybrid cell line NCB-20 following exposure to inhibitors of N-linked protein glycosylation (tunicamycin, TM) and oligosaccharide processing (swainsonine, SW). TM treatment reduced ligand binding at delta- and sigma-opiate receptors and neuroleptic binding sites (20 to 50% of control), with no discernible effect on the binding properties of 5HT1-serotonin receptors. In contrast, exposure to SW resulted in a three-fold increase in binding capacity of sigma-receptors, while decreasing receptor affinity for ligand. SW treatment did not alter ligand interactions with either sigma-receptors or neuroleptic binding sites, but did reduce specific binding of serotonin to 5HT1-receptors. The effects of TM and SW on distinct receptor subpopulations were further demonstrated by attenuation of opiate and serotonin-mediated regulation of intracellular cyclic AMP. PMID- 3004492 TI - Enzymatic synthesis of mevalonate-5-(2-thiodiphosphate). AB - Mevalonate-5-(2-thiodiphosphate), a substrate analog for diphosphomevalonate decarboxylase, has been enzymatically prepared from mevalonate-5-phosphate and adenosine-5'-0-(3-thiotriphosphate) using phosphomevalonate kinase as a catalyst, in a 37% yield. The substrate properties of the synthesized compound are compared to those of the normal substrate of the enzyme. PMID- 3004494 TI - Effect of zinc on the binding and action of growth hormone in isolated rat adipocytes. AB - Ionic zinc has been shown to exert a dose-dependent (250-500 microM) stimulation of 125I-human growth hormone specific binding to isolated rat adipocytes. The effect was rapid, being observed in less than 15 min exposure to zinc, and was due to an increased number of available binding sites as determined by Scatchard analysis. The well-known insulin-like effects of both zinc and growth hormone on lipogenesis in this tissue were additive at submaximal doses of each agonist, but zinc did not appear to potentiate growth hormone action. A similar, but smaller effect of zinc on growth hormone specific binding was observed in rabbit liver membranes at lower zinc concentrations (5-100 microM). At higher zinc doses a marked increase in non-specific binding caused a reduction of specific binding in liver membranes. These studies raise the possibility that zinc may be a modulator of growth hormone binding in vivo. Although our initial experiments suggest that this may be independent of an effect on growth hormone action, further studies are required to assess the possibility that zinc's effect at the receptor level may be to increase the sensitivity of tissues to growth hormone, thereby promoting a full manifestation of effects at lower growth hormone concentrations. PMID- 3004493 TI - Characterization of nucleosidediphosphate (NDP)-kinase-associated GTP binding proteins from human recombinant interleukin 2 (rIL-2)-treated mouse NK cells. AB - Nucleosidediphosphate (NDP)-kinase-associated proteins from rIL-2-treated mouse NK cells have been biochemically characterized. The associated proteins could be separated from partially purified NDP-kinases by the 5-25% glycerol density gradient centrifugation method after treatment with 6 M urea in the presence of 1 mM EDTA. The associated proteins (approx. Mr 20,000) were defined as GTP binding proteins, since only [alpha-32P]GTP was bound to these proteins in the presence of 5 mM Mg2+ at 37 degrees C. We also found that these GTP binding proteins hydrolyzed only GTP in the presence of 5 mM Mg2+. The data presented here for: GTP specific binding activity; GTPase activity; and molecular size (approx. Mr 20,000) of the NDP-kinase-associated GTP binding proteins are similar to those reported for ras oncogene products (p21 proteins). PMID- 3004495 TI - The inhibitory effect of enfenamic acid (Tromaril) on hepatic gluconeogenesis in Swiss albino mice. AB - Enfenamic acid, a new non-steroidal anti-inflammatory drug was studied for its effect on hepatic gluconeogenesis and some of the enzymes involved in this process in mice. Incubation of liver cells in the presence of 1.0 mM enfenamic acid inhibited the output of glucose. And also the in vitro addition of various concentrations of enfenamic acid (0.25 to 3.0 mM) to the tissue extracts of liver inhibited the activities of important gluconeogenic enzymes such as pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK) and fructose 1,6 diphosphatase (FDPase). The oral and intraperitoneal administrations of the drug for 15 and 3 days respectively, exhibited significant decrease in the hepatic PC, PEPCK and FDPase. These findings indicated that the impairment of gluconeogenesis might be due to the inactivation of the enzymes by the drug. PMID- 3004497 TI - Determination of the size of polyphosphates with polyphosphate glucokinase. AB - A procedure for determining the size of inorganic polyphosphates of chain lengths up to about 750 is described. It involves reducing the size with polyphosphate glucokinase to a chain length that can be accurately determined by polyacrylamide gel electrophoresis. This measurement along with determination of the glucose-6-P formed and the total phosphate of the original polyphosphate permits calculation of the chain length. The accuracy of this method has been demonstrated by comparison with other reliable procedures. Thus far, it is the only method available for sizing long chain polyphosphates with nmol quantities. PMID- 3004496 TI - Hexaammineruthenium as an electron donor to mitochondrial cytochrome oxidase: membrane potential generation in the absence of cytochrome c. AB - Cytochrome c oxidase can generate membrane potential in the absence of cytochrome c (e.g., in cytochrome c-deficient mitochondria or in proteoliposomes) with hexaammineruthenium as an artificial electron donor. Of several other redox mediators tested, phenazine methosulfate was found to be an efficient artificial substrate for membrane energization by cytochrome oxidase, whereas TMPD, DAD, DCPIP or ferrocyanide are virtually ineffective. The ability of Ru(NH3)6(2+) and phenazine methosulfate to support the generation of delta psi by cytochrome c oxidase correlates with their effectiveness as electron donors to cytochrome a in the cyanide-inhibited membrane-bound enzyme. PMID- 3004498 TI - N-terminal amino acid sequence of cytochrome c-552 from Nitrosomonas europaea. AB - Nitrosomonas europaea is an ammonia-oxidizing bacterium which contains multiple c type cytochromes. Few of these components have been assigned physiological roles, but on the basis of molecular weight and redox potential cytochrome c-552 has been considered to be an analogue of the mitochondrial cytochrome-c family of proteins. We present the N-terminal amino acid sequence (47 residues) of cytochrome c-552 and show that this protein is most closely related to the group of small cytochrome-c components from pseudomonads (cytochromes c-551) and is probably evolutionarily distant from the analagous protein (cytochrome c-550) from the nitrite-oxidizing bacterium Nitrobacter agilis. PMID- 3004499 TI - Multiple mu opiate receptors: biochemical and pharmacological evidence for multiplicity. PMID- 3004500 TI - Novel kainic acid analogues. Effects on cyclic GMP content of adult rat cerebellar slices. AB - On the basis of previous electrophysiological studies, it has been proposed that there are three main classes of excitatory amino acid receptor in the mammalian central nervous system, which are activated preferentially by kainic acid, quisqualic acid and N-methyl-D-aspartate respectively. Although the pharmacology of the N-methyl-D-aspartate receptor has been investigated extensively, potent and selective ligands which act at the kainate or quisqualate sites are lacking. In this study, we report that a number of novel kainate analogues possess either agonist or antagonist activity in a system which permits investigation of receptor-mediated coupled responses, viz. the ability of excitatory amino acids to elevate cyclic GMP concentrations in incubated cerebellar slices prepared from the adult rat. The data reported here provide some clues as to the likely structural requirements for developing effective kainate antagonists. PMID- 3004502 TI - The characteristics of low and high affinity [3H]-prazosin binding to membranes from rat renal cortex. AB - The characteristics of [3H]-prazosin binding in renal cortical membranes of the rat have been assessed under a variety of buffer conditions. At 37 degrees, in Krebs' phosphate and Tris buffer, [3H]-prazosin bound to two sites, a small population of high affinity sites with properties of alpha1-adrenoceptors and a much larger population of low affinity sites with different characteristics. High affinity [3H]-prazosin binding was insensitive to Na+, K+, Ca2+ and Mg2+ ions, but low affinity [3H]-prazosin binding was markedly increased in Krebs' phosphate or sodium phosphate buffer and further enhanced in membranes pretreated with EGTA. Binding was decreased in the presence of Ca2+, the decrease in binding mainly being due to a decrease in the number of low affinity sites labelled by the ligand. Low affinity [3H]-prazosin binding was increased at 37 degrees and relatively insensitive to alpha-adrenoceptor antagonists which were weak competitors while catecholamines failed to compete for low affinity binding. Scatchard plots of [3H]-prazosin binding performed using (-)-noradrenaline (1 mM) to define non-specific binding defined binding only to alpha 1-adrenoceptors. This provides a means of differentiating high and low affinity [3H]-prazosin binding. PMID- 3004501 TI - Inhibition of the leukotriene synthetase of rat basophil leukemia cells by diethylcarbamazine, and synergism between diethylcarbamazine and piriprost, a 5 lipoxygenase inhibitor. AB - Diethylcarbamazine inhibited the formation of sulfidopeptide leukotrienes in rat basophil leukemia (RBL) cells (50% inhibitory concentration, EC50, 3 mM). Similar concentrations also inhibited the formation of leukotriene C4 (LTC4) by LTC synthetase, a detergent-solubilized cell free particulate enzyme from RBL cells which is capable of coupling LTA4 to glutathione. By contrast, the conversion of LTA4 to LTC4 using enzymes from rat liver was at least ten times less sensitive to this inhibitor. The EC50 for inhibition of the leukotriene C synthetase of RBL cells was directly proportional to the LTA4 concentration in the incubations, ranging from 1.5 mM at 10 microM LTA4 to over 40 mM at 500 microM LTA4. Kinetic analysis revealed that the inhibition of the leukotriene C synthetase reaction by diethylcarbamazine was competitive with respect to LTA4. In contrast to diethylcarbamazine, piriprost (U-60,257; 6,9-deepoxy-6,9-(phenylimino)-delta 6,8 prostaglandin I1), which inhibits the formation of sulfidopeptide leuktrienes in RBL cells at the 5-lipoxygenase step (EC50 5 microM), did not inhibit the leukotriene synthetase of these cells. On the other hand, low concentrations of piriprost, which had no demonstrable inhibitory activity on leukotriene formation by themselves, markedly synergized the inhibitory activity of diethylcarbamazine. These results are consistent with the interpretation that both piriprost and diethylcarbamazine inhibit leukotriene formation but that they act on sequential steps in the biosynthetic pathway in such a manner as to synergistically interfere with the availability or utilization of LTA4 in the leukotriene C synthetase reaction. PMID- 3004503 TI - Stimulatory effect of Silibinin on the DNA synthesis in partially hepatectomized rat livers: non-response in hepatoma and other malign cell lines. PMID- 3004505 TI - Xenobiotic-metabolizing enzyme activity in human non-small-cell derived lung cancer cell lines. AB - Human lung cancer cell lines in culture were investigated for the expression of monooxygenase and other xenobiotic-metabolizing enzyme activities. Two bronchiolo alveolar carcinoma derived cell lines (NCI-H322 and NCI-H358) and two small-cell carcinoma derived cell lines (NCI-H128 and NCI-H69) were used. Previous work has shown that NCI-H322 has ultrastructural features of Clara cells while NCI-H358 shows characteristics of alveolar type II cells [Schuller et al., Proc. Am. Ass. Cancer Res. 26, 27 (1985)]. NCI-H128 and NCI-H69 show very poor differentiation of cytoplasmic organelles. Cytochrome P-450 levels were spectroscopically detectable only in NCI-H322. Both NCI-H322 and NCI-H358, but not NCI-H69 and NCI H128, exhibited aryl hydrocarbon hydroxylase (using benzo[a] pyrene as substrate) and ethoxycoumarin O-deethylase activities. These activities were highly inducible following pretreatment with the polycyclic aromatic hydrocarbons (PAH) beta-naphthoflavone or benzo[a] anthracene. The PAH produced a 2-fold increase in spectroscopically detectable cytochrome P-450 levels in NCI-H322. Following induction, cytochrome P-450 was also spectroscopically detectable in NCI-H358. No aldrin epoxidase activity was present in either untreated or pretreated cell lines. Pretreatment with phenobarbitone or dexamethasone did not induce the aryl hydrocarbon hydroxylase activity in either NCI-H322 or NCI-H358. The ethoxycoumarin O-deethylase activity in beta-naphthoflavone-pretreated NCI-H322 and NCI-H358 was inhibited in a concentration-dependent manner by ellipticine, alpha-naphthoflavone, cimetidine or metyrapone. Untreated NCI-H322 and NCI-H358 also contained cytochrome b5, NADPH cytochrome c reductase and epoxide hydrolase activities. None of these enzyme activities measured was detectable in the untreated or pretreated small-cell derived cancer cell lines (NCI-H128 and NCI H69). These data show that the two bronchiolo-alveolar carcinoma derived cell lines (NCI-H322 and NCI-H358) exhibit cytochrome P-448-dependent monooxygenase activity and may thus prove useful to study the processes of xenobiotic activation in human lung. PMID- 3004504 TI - Hydroxyl radicals and the toxicity of oral iron. PMID- 3004506 TI - Reductive metabolism of nitrofurantoin by rat lung and liver in vitro. AB - In the present study, the metabolism of NF has been examined in detail in both rat lung and liver 9000 g supernatants using a specific radiometric HPLC assay. Over 92% of the total radioactivity chromatographed with authentic NF after incubations from either organ were carried out under oxygen for 60 min. Under anaerobic conditions, only 19% and 5% of the total unbound radioactivity corresponded to unchanged NF in lung and liver respectively. At least 4 metabolites were evident from the HPLC trace (M1, M2, M3, M4 according to increasing retention times). In the absence of oxygen, liver 9000 g supernatants generated 65% more M1 and 260% more M3 than did lung 9000 g supernatants, but the lung produced significantly more M4. Covalent binding to tissue macromolecules was similar in both tissues under oxygen but was 7 times greater in lung than in liver in the absence of oxygen (compared per unit protein). Neither piperonyl butoxide nor indomethacin affected NF metabolism. However, allopurinol almost completely inhibited the anaerobic and aerobic (superoxide generation measured by the rate of acetylated cytochrome c reduction) metabolism in the lung with little or no effect in the liver. The data indicate a quantitative difference in NF metabolism between the two tissues that may be related to the organ-selective toxicity of the drug. PMID- 3004507 TI - Human melanoma cells sensitive to deoxyadenosine and deoxyinosine. AB - In an in vitro study conducted without the use of adenosine/deoxyadenosine deaminase inhibitors, two human melanoma cell lines, MM96L and MM127, were found to be highly sensitive to killing by continuous treatment with deoxyadenosine (dAdo) (D37 47 microM and 68 microM respectively) compared with fibroblasts (D37 440 microM), Hela cells (D37 1.1 mM) and other melanoma cell lines (D37 0.8 to 2.5 mM). Cross-sensitivity was found to deoxyinosine (dIno) and in part to adenosine but not to related metabolites such as inosine or hypoxanthine. Hypersensitivity to dAdo was associated with deficiency in cell membrane 5' deoxynucleotidase but not in deaminase activity. dAdo toxicity could be prevented in MM96L by addition of the other three deoxynucleosides together but not by removing dAdo after a brief (2 hr) treatment. Resistant melanoma cells, however, required more than 24 hr dAdo treatment to produce toxicity. DNA synthesis in MM96L cells was reversibly inhibited, and cells tended to accumulate in G1/S. No DNA strand breaks were detected. These results showed that in contrast to the resistant cell line, asynchronous MM96L cells are highly sensitivity to brief treatment, toxicity resulting from an effect associated with inhibition of DNA synthesis. dAdo and dIno, either combined with a deaminase inhibitor or as deaminase-resistant derivatives, may have a favourable therapeutic index for some melanomas in vivo. PMID- 3004508 TI - [Synthesis and biological activity of a lysine-containing cyclic analog of [Leu5]enkephalin]. AB - The cyclic analogue of [Leu5]enkephalin--cyclo (Lys-Tyr-Gly-Gly-Phe-Leu) and two corresponding linear hexapeptides with lysine residue attached to the N- or C terminus of the molecule have been synthesized by classical methods of peptide chemistry. The addition of lysine residue to the N-terminus of cyclization of the molecule reduce the interaction of these analogues with both central and peripheral opiate receptors. The addition of lysine residue to the C-terminus of the molecule through the epsilon-amino group does not affect the interaction of the analogue with mu-receptors but reduces approximately tenfold its affinity for delta-receptors. All three analogues have analgesic potency similar to that of [Leu5]enkephalin as assayed after intracisternal administration to mice. PMID- 3004509 TI - [General approach to the engineering of synthetic DNA]. AB - A useful and efficient approach to the synthesis of DNA duplexes of practically unlimited length has been developed. The proposed methodology is based on the use of temporary restriction sites for subcloning and assembling the segments of the desired DNA. It allows the utilization of chemically synthesized oligonucleotides of various length (from 10- to 100-mers) for the duplex construction. The application of this approach to the synthesis of a gene for the functionally active bacteriorhodopsin fragment is described. PMID- 3004510 TI - [Substrate specificity of restriction endonuclease Eco781]. AB - The recognition sequence and cleavage point of restriction endonuclease Eco781 have been determined as 5'-GGCGCC-. There are several known enzymes recognizing the same sequence, although the prototype NarI and isoschizomers NdaI and NunII cleave the substrate to produce 5'-protruding ends, whereas cleavage with isoschizomer BbeI results in 3'-protruding ends. Therefore, restrictase Eco78I, generating flush ends, may be regarded as an enzyme with new specificity among the restriction endonucleases recognizing the 5'-GGCGCC-sequence. PMID- 3004511 TI - [Isolation and properties of recombinant DNA from a plasmid and filamentous phage]. AB - In the course of studying extrachromosomal DNA with composite replicons, a hybrid has been constructed by the in vitro recombination of the filamentous phage M13mp2 DNA (RF) and plasmid pUR222 (ApR). Both parental DNAs contain a fragment of lac-operon (ca. 800 bp), which includes the distal end of lacI gene, lacPO segments, and the lacZ gene proximal region coding for 145 N-terminal amino acid residues of beta-galactosidase and thus providing for alpha-complementation, the effect being cancelled with a polynucleotide insertion at the unique EcoRI site in the lacZ gene segment. E. coli BMH71-18 cells were transformed with the ligated mixture of EcoRI restricts of both DNAs. A phage-like nucleoprotein was isolated from colourless plaques (on the Xgal- and IPTG-supplemented medium); its deproteinization yielded a DNA which contains the ApR-determinant and, according to PAGE, structurally specific staining, restriction analysis, sequencing by the Sanger procedure, and electron microscopy data, is a linear double-stranded molecule comprising the phage and plasmid genomes in an equimolar ratio. Since the hybrid DNA does not display the alpha-complementation effect, both bacterial inserts are in the opposite orientation. Transformation of both phage (F+) and plasmid (F-) hosts with the hybrid DNA led to cultures which, after precipitation of the nucleoprotein from the extracellular medium and deproteinization, afforded the same composite DNA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004512 TI - [Synthesis and analysis of conformationally restricted analogs of peptide inhibitors of the angiotensin-converting enzyme]. AB - To investigate conformations of peptide inhibitors of the angiotensin-converting enzyme in the enzyme-inhibitor complex, the synthesis, studies of inhibitory activity, and conformational calculations of analogues of bradykinin-potentiating peptides with N-methylalanine or D-alanine in place of L-proline or L-alanine residues have been carried out. All the analogues showed a sharp decrease of inhibitory activity in comparison with the natural peptides, that might be considered as an indirect confirmation of the earlier proposed "conformation of inhibition" of the above-mentioned peptides. PMID- 3004513 TI - [Neurotoxin of the black widow spider and its interaction with receptors from the rat brain]. AB - A presynaptic neurotoxin isolated from the venom of the Central Asia spider karakurt (Black Widow Spider, Latrodectus mactans tredecimguttatus) is shown to consist of two identical subunits of mol. weight about 118 kDa. The iodinated neurotoxin binds to the rat brain synaptosomal plasma membranes with Kd 0.1 nM (Bmax 0.1 pmol/mg of protein) at 37 degrees C, and with Kd 0.35 nM (Bmax 0.2 pmol/mg of protein) at 5 degrees C. At intermediate temperatures both types of receptors are detectable. It is supposed that the dimeric form of the toxin interacts with a single class of receptors possessing lateral mobility in the membrane. By the use of different bifunctional reagents it is revealed that the neurotoxin interacts with a presynaptic membrane protein of mol. weight 95 kDa. A protein of the same size accompanied by a 71 kDa protein was isolated by the affinity chromatography of solubilized synaptosomal membranes on the absorbent, containing immobilized neurotoxin. PMID- 3004515 TI - Alterations in macrophage collagenase secretion induced by gold sodium thiomalate. AB - The effects of gold sodium thiomalate (GST) on the production of specific collagenase by thioglycolate-elicited macrophages was investigated. Our studies demonstrated that GST administration can significantly decrease collagenase production in a dose-dependent manner. These effects were observed with levels of GST attainable in serum or synovial tissue during routine chrysotherapy. In addition, GST altered lysozyme secretion by activated macrophages in a pattern distinct from that of collagenase alteration. These effects of enzyme secretion were not secondary effects of GST on viability, general protein secretion, or the specific assay procedures utilized, and were not attributable to the thiomalate moiety. Thus, GST may exert its therapeutic effect in rheumatoid arthritis through interference with the production of degradative proteolytic enzymes, which are important effector molecules mediating tissue destruction. PMID- 3004514 TI - Hereditary joint disorder in progressive ankylosis (ank/ank) mice. II. Effect of high-dose hydrocortisone treatment on inflammation and intraarticular calcium hydroxyapatite deposits. AB - Mice homozygous for the progressive ankylosis trait develop an inflammatory joint disorder associated with the intraarticular deposition of calcium hydroxyapatite. When affected (ank/ank) mice were treated with high doses of hydrocortisone, synovitis receded, development of cartilaginous and bony osteophytes halted, and calcium hydroxyapatite accumulated in and distended the synovial spaces. No changes, however, occurred in the joint morphology of hydrocortisone-treated normal (ank/+) mice. Since inhibition of inflammation by hydrocortisone treatment did not block apatite accumulation, intraarticular deposition of hydroxyapatite occurs independent of inflammation in progressive ankylosis. PMID- 3004516 TI - Fibrin glue safety: inactivation of potential viral contaminants by pasteurization of the human plasma components. AB - Fibrin glue has become an indispensable tool in salvaging operations of the parenchymatous organs of the abdomen, in vascular and ophthalmic plastic surgery as well as neurosurgery. Currently available glues contain clotting factors from human plasma and thus carry the potential risk of transmitting viral infections like hepatitis or acquired immunodeficiency syndrome (AIDS). Combined efforts in selection of plasma donations as well as pasteurization of the human plasma products allow the manufacturing of a product (Beriplast) with virtually no risk of transmission of viral infections as demonstrated by in vivo and in vitro experiments with a variety of human pathogenic viruses. No changes in activity or antigenicity of the clotting factors by the pasteurization procedure have been encountered. PMID- 3004517 TI - Increase in the cardiotonic action and in the therapeutic margin of digoxin by the adrenergic beta-stimulant doxaminol in cats. AB - The interaction between digoxin and the beta-sympathomimetic drug doxaminol was investigated in cats with acute heart failure induced by pentobarbital sodium. Doxaminol, 50 micrograms/kg X min, infused for 60 min caused a dose-dependent rise in dp/dtmax with little increase in heart rate. The maximum increase of 4.3 mHg/s was obtained after about 37 min. Digoxin, 10 micrograms/kg X min, and the combination of both drugs were infused until cardiac arrest. The maximum increase of dp/dtmax was observed after 29 min in both experiments; it was 5.7 mHg/s with digoxin alone and 7.3 mHg/s with the combination (p = 0.025). The combined infusion of epinephrine (0.3 micrograms/kg X min) plus digoxin (10 micrograms/kg X min) caused a maximal increase of dp/dtmax by 7.9 mHg/s. The cardiotoxic dose of digoxin was markedly reduced by epinephrine, not by doxaminol. The relevance of this difference to man cannot be assessed definitely because the ECG changes produced by digoxin in cats are different from those seen in man. PMID- 3004518 TI - The calcium antagonist nifedipine inhibits arterial smooth muscle cell proliferation. AB - Migration of smooth muscle cells from the media to the intima of the arterial wall and proliferation of intimal smooth muscle are major early events in the formation of an atherosclerotic lesion. The start of proliferation requires that the cells have passed through a modulation from contractile to synthetic phenotype and that they are stimulated with growth factors. Here, we have examined the effects of the calcium antagonist nifedipine on phenotypic modulation and growth of isolated rat arterial smooth muscle cells cultivated in vitro. The results indicate that micromolar concentrations of nifedipine slow down the rate of transformation of the cells from a contractile to a synthetic phenotype and inhibit initiation of DNA synthesis as well as cellular proliferation. The inhibitory effect on DNA synthesis was seen both in cells stimulated with whole blood serum and with purified platelet-derived growth factor. The results raise the possibility that nifedipine may be used to prevent atherogenesis and to inhibit progression of fibromuscular lesions by interfering with the proliferation of arterial smooth muscle cells. PMID- 3004519 TI - Effect on human platelets of catecholamines at levels achieved in the circulation. AB - Epinephrine at concentrations varying between 3.3 and 12.5 nM had no effect on blood platelets when added alone, but augmented the in vitro platelet response to collagen and thrombin. Both aggregation and secretion responses were enhanced. Norepinephrine produced similar effects but was 50-60% less active than epinephrine. The minimum concentration of epinephrine or norepinephrine to achieve potentiation of platelet responses was even lower in the presence of 5 hydroxy- tryptamine. In contrast to the effects observed with higher concentrations of catecholamines, the synergistic interaction of these low concentrations of catecholamines with other agonists was not transient. The augmented response to catecholamines was mediated by platelet alpha 2 adrenoceptors. The response was inhibited by aspirin indicating that metabolism of arachidonic acid contributes to the synergy between low concentrations of catecholamines and other agonists. These studies show that the levels of the hormones epinephrine and norepinephrine obtained in circulating blood in humans, can be sufficient to enhance platelet responses. The action of catecholamines on platelets may be important in hemostasis and could provide an explanation for the association between certain risk factors and cardiovascular disease. PMID- 3004520 TI - Primary culture of endothelial cells from atherosclerotic human aorta. Part 1. Identification, morphological and ultrastructural characteristics of two endothelial cell subpopulations. AB - Endothelial cells (EC) were harvested by 0.1% collagenase treatment for adult human thoracic aortas obtained 1-3 h after sudden death. At least 35-70% of EC were removed from the intimal surface of aorta, 90-95% of them being viable. Plating efficiency was 70-80%. Monolayer formation was achieved at a seeding density of 5-8 X 10(2) cells/mm2. The cells were identified as endothelium by the presence of Factor VIII antigen, Weigel-Palade bodies and typically endothelial morphology at confluence. Unlike endothelial cultures derived from human umbilical veins and infant aortas, primary cultures obtained from human adult aortas contain multinuclear EC with Factor VIII antigen and Weibel-Palade bodies. The number of multinuclear EC in cultures isolated from aortas affected by atherosclerosis was 2-fold higher (P less than 0.05) than in cultures obtained from grossly normal aortas taken from donors of the same age. EC with numerous lipid inclusions revealed by oil-red-O staining were present in all the EC primary cultures derived from aortas affected by atherosclerosis. No oil-red-O positive cells were detected among the EC cultured from infant aorta, aorta of young donors, and umbilical vein. An electron microscopic examination of EC from atherosclerotic aorta in culture and in situ failed to reveal any ultrastructural peculiarities distinguishing multinuclear EC from the mononuclear EC. PMID- 3004521 TI - Opioid receptors. PMID- 3004523 TI - Ten years of opioid peptides--retrospectives and perspectives. PMID- 3004522 TI - Opioid physiology. PMID- 3004524 TI - Comparison between delta-9-tetrahydrocannabinol and cannabis extract on resorption rate in mice. AB - Delta-9-tetrahydrocannabinol, the principal psychoactive ingredient in cannabis extract, and cannabis extract containing an equivalent amount of delta-9 tetrahydrocannabinol were administered to different groups of pregnant mice on gestation days 7-15 inclusive. There was a significant dose-related increase in resorptions but no significant difference between compounds in resorption rate, nor was there any significant interaction between drug and dosage. The results suggest that compounds in cannabis extract do not modify the actions of delta-9 tetrahydrocannabinol on resorption rate. PMID- 3004525 TI - Differences in drug delivery with peripheral and central venous injections: normal perfusion. AB - The differences between central venous (CV) and peripheral venous (PV) injections with respect to drug delivery times and peak concentrations in the left ventricle are examined. In this drug-delivery model, anesthetized dogs (n = 7) received both PV and CV injections of a radionuclide tracer. Measurements of radioactivity levels were made over each ventricle during normal perfusion. There was no statistically significant difference in left ventricular delivery times or peak radioactivity levels between the two different routes. When delivery time was corrected for variations in flow rate, a small but statistically significant difference was seen, the CV route being faster than the PV route (P less than 0.05). The magnitude of the observed difference for normal perfusion (2 seconds) is unlikely to be of clinical significance. PMID- 3004527 TI - Emergency intraosseous infusions in children. AB - Vascular access during advanced life support is essential. Vascular access in the critically ill child can be particularly difficult and often causes unacceptable delay. Intraosseous infusion provides safe, rapid, reliable access to the venous circulation. A case is presented illustrating the value of familiarity with this procedure. Use of the bone marrow for emergency administration of fluids and medications should be considered early in resuscitation until vascular access is obtained. PMID- 3004526 TI - Pediatric resuscitation without an intravenous line. AB - The case of a 3-month-old male infant who was found unresponsive and cyanotic in a crib at home is presented. On arrival in the emergency department the child was receiving basic cardiopulmonary resuscitation (CPR) by a rescue squad and was without vital signs in asystole. The patient achieved a stable rhythm and blood pressure before intravenous access was obtained. Epinephrine and atropine were given via the endotracheal route and sodium bicarbonate through intraosseous infusion. PMID- 3004528 TI - Amitriptyline plasma protein binding: effect of plasma pH and relevance to clinical overdose. AB - Reversing ventricular ectopy with plasma alkalinization following acute tricyclic antidepressant overdose is a recognized mode of therapy. The mechanism responsible for this effect is unclear. Changes in plasma protein binding of free drug, effects of the sodium ion on the myocardium, and alterations of plasma concentrations of alpha-1-acid glycoprotein may all interact to alter toxicity of tricyclics in overdose. An in vitro investigation using equilibrium dialysis was designed to examine the effect of altering plasma pH on percentage of free amitriptyline at clinical overdose plasma concentrations. A 1973 report on this effect lacked adequate controls and was faulty in experimental protocol. The current investigation used plasma concentrations typically present in amitriptyline overdose, a sensitive gas liquid chromatographic assay to detect total and free drug, and adequate control of plasma pH. The results of two separate experiments demonstrated a significant decrease in percentage of free amitriptyline of 20% over a pH range of 7.0-7.4 (P less than 0.05) and 42% over a pH range of 7.4-7.8 (P less than 0.05). The rate of change in slope in both experiments was not significantly different (P less than 0.01) indicating similar effects of pH change on plasma protein binding of amitriptyline within the two groups. PMID- 3004529 TI - Calcium pyrophosphate and pseudogout. AB - Calcium pyrophosphate deposition disease (CPDD) is a condition in which calcium pyrophosphate dihydrate crystals are deposited in joint articular cartilage, menisci, and synovium. The main clinical presentations of CPDD are chondrocalcinosis--calcification of cartilage, pseudogout--acute joint inflammation due to crystal-induced synovitis, and pyrophosphate arthropathy- degenerative joint disease similar to osteoarthritis associated with calcium pyrophosphate crystal deposition. The clinical importance of CPDD for the arthroscopist is the ability to recognize the condition so that appropriate treatment can be instituted. Arthroscopy is valuable for diagnosis as well as lavage and intraarticular debridement or meniscectomy. Tissue removed for microscopic examination should be sent to the laboratory in saline, since formalin dissolves the crystals. Postarthroscopy treatment of CPDD should include oral antiinflammatory medication. Asymptomatic chondrocalcinosis does not require treatment. PMID- 3004530 TI - Hepatocellular carcinoma in the University Hospital. PMID- 3004531 TI - Burkitt's lymphoma. Distinction of subgroups by morphometric analysis of the characteristics of 55 cell lines. AB - In an attempt to clarify the controversy about the distinction between Burkitt's and non-Burkitt's small noncleaved lymphomas, 55 cell lines derived from 48 Burkitt's lymphoma patients were characterized by morphometry on plastic-embedded sections. The results of the measurements permitted the identification of five main cytologic types, with regard to nuclear size, nuclear area dispersion and irregularity of nuclear profiles. The presence of the Epstein-Barr virus (EBV) and the geographic origin of the tumors seemed to play essential roles in the determination of nuclear size, with a significantly larger size seen in EBV positive cell lines, and especially in the African lines among these. Immunoglobulin profile and monoclonal antibody expression also correlated with the nuclear size. Two conclusions may be drawn from this analysis. An in vivo transformation of the cells of Burkitt's lymphoma can be postulated to explain the wide morphologic spectrum of lymphomas presenting a rearrangement of chromosome 8. The fact that typical and atypical Burkitt's lymphomas cannot be differentiated by study of their derived cell lines raises the question as to the validity of the distinction between the two subtypes of small noncleaved lymphomas. PMID- 3004532 TI - Peripheral nerve morphometry for daily practice. AB - Quantitative methods for the routine diagnostic analysis of sural nerve biopsies were evaluated. Semiautomatic image analysis was used to compare three sampling methods and to test the accuracy and reproducibility of measurements of various elements in biopsy specimens from seven cases of paraproteinemic neuropathy. Systematic nonrandom sampling gave better results than did completely random sampling, as compared with systematic covering sampling. The accuracy of measurements was optimal at a final magnification of 3,200X. Comparison of single data of double measurements revealed a mean relative frequency of class change of 7.4%. Computation of regression showed good conformity, with r = 1.0. In conclusion, systematic nonrandom sampling at a final magnification of 3,200 X provided good analysis of the total population of structural elements with sufficient accuracy and reproducibility. PMID- 3004533 TI - Sympathetic ganglia in diabetes. An ultrastructural and stereologic study. AB - An ultrastructural and stereologic study was performed on sympathetic ganglia collected at surgery from eight diabetic patients, seven age-matched non diabetics and six subjects with glucose intolerance. All patients underwent surgery because of arteriosclerosis obliterans. Volume density (Vv) and mean thickness (tau) of the satellite cell layer were found to be significantly higher in diabetics (P less than .001 and P less than .002, respectively) than in the other two groups, between which no difference was found. Vv and tau of the endothelial basal lamina were found to be increased in the diabetic and glucose intolerance groups at a low level of significance (P less than .05). Satellite cells are thought to exhibit a close similarity to Schwann cells. Ultrastructurally, cytoplasmic edema, increase of subcellular organelles and lipid inclusions were consistently observed in satellite cells from diabetic patients. The results suggest that there may be a metabolic impairment of these cells in diabetes. PMID- 3004534 TI - Stereologic correlates of steroid receptor concentration in invasive ductal breast cancer. AB - The hypothesis was tested that morphometric parameters of tumor cell nuclei correlate with the steroid receptor concentration in mammary carcinoma. In 50 consecutive mastectomy specimens with a diagnosis of invasive ductal cancer in which estrogen receptor (ER) and progesterone receptor (PR) concentrations had been assayed quantitatively, morphometric measurements were performed on four visual fields of two sections per case. The fields were sampled from the most cellular regions of the tumor. The number of tumor cell nuclear profiles per tissue area, the nuclear profile area and the long and short nuclear profile axes and their ratios were measured with a semiautomatic image analysis system. Estimates of the number of tumor cell nuclei per tissue volume (Nv) and of the mean tumor cell nuclear volume (V) were obtained by standard stereologic techniques. Association between the morphometric and biochemical parameters was tested by Spearman's rank correlation coefficient. Nv correlated positively with the steroid receptor concentration whereas V correlated negatively with both ER and PR concentrations. A correlation of the receptor concentrations to the standard deviation of the nuclear area or the mean ratio of the nuclear axes could not be demonstrated. These results suggest that receptor-rich tumors have a large number of small tumor cell nuclei whereas receptor-poor tumors have a small number of large tumor cell nuclei per tissue volume in the actively proliferating, highly cellular regions. These differences are not accompanied by significant changes in nuclear size variability or nuclear shape. PMID- 3004535 TI - The molecular genetics of human monogenic diseases. PMID- 3004537 TI - Dentists found at small risk to AIDS: LA task force. Virus may be killed easily; autoclaving, sterilization recommended. PMID- 3004536 TI - Myotonic dystrophy and gene mapping on human chromosome 19. PMID- 3004538 TI - Hepatitis B outbreak. Two deaths spur CDC investigation. Nine cases occur in 1984; tenth reported in February. PMID- 3004540 TI - Content and accessibility of sialic acid on the surface of rat hepatocytes during primary culture. AB - The content and accessibility of terminal sialic acid and galactose residues of rat hepatocytes in primary culture were determined by in situ labeling using either periodate or sialidase/galactose oxidase treatment followed by sodium borotritiide reduction. Rat erythrocytes which were used for comparison showed a strongly enhanced tritium incorporation into galactose after sialidase treatment. In contrast, with freshly prepared rat hepatocytes only a small amount of galactose labeling was achieved after sialidase treatment. The amount of galactose labeled following sialidase treatment increased with time in culture up to day 6 and roughly paralleled the increase of the total sialic acid content. Major changes of sialic acid-containing glycoconjugates were restricted to the gangliosides. There was a transient drop in surface labeling of ganglioside associated sialic acid on the first day in culture. The specific radioactivity of the in situ-tritiated ganglioside-sialic acid also fell by 50% in this period. Between day 2 and 4, there was an increase in gangliosidesialic acid labeling but the specific radioactivity of the sialic acid remained constant. This indicates that newly synthesized gangliosides but not the preexisting ones were accessible to periodate oxidation. The data allow conclusions about turnover and topology of the sialic acid-containing glycolipids. PMID- 3004539 TI - Thalamus as a relay station for catalepsy and rigidity. AB - The aim of the study was to determine to what extent catalepsy and tonic rigidity of muscles induced by muscimol administration into the ventral thalamic nuclei disturb the motor activity of rats. This study also aimed to test whether the ventromedial thalamic nucleus (Vm) was involved in transmitting effects evoked by the systemic injection of neuroleptics or opioids. For this purpose muscimol and/or picrotoxin was injected into the ventral thalamic nuclei and the behaviour of the animals was assessed in a series of test situations. It was found that muscimol administration to the Vm disturbs not only the initiation and performance of voluntary movements but also the occurrence of avoidance when the animal's life is endangered. Postural reflexes remained, however, undisturbed. Those effects seemed to be GABA- and site-specific to Vm. The haloperidol catalepsy was strongly inhibited by administration of picrotoxin to the Vm while the morphine catalepsy remained unchanged after picrotoxin. The Vm plays a crucial role in the motor behaviour and transmission of cataleptogenic effects of haloperidol, whereas similar effects produced by morphine appear to by-pass the investigated thalamic region. PMID- 3004541 TI - Kaposi's sarcoma and HTLV-III infection. Virus-like particles in skin lesions: experimental observations. AB - Skin biopsies from a subject affected by KS and AIDS were examined by means of EM. Samples were obtained both in correspondence of a typical nodule, and from an apparently normal area. The disease process had started at least 8 mo. before, and the patient showed a reduced number of OKT4 cells, with an inversion of the normal T4/T8 ratio. Besides that, high titre antibodies versus HTLV-III were present, whereas antibodies versus CMV and EBV were not demonstrable. Light and electron microscopy demonstrated the typical picture of KS, with marked proliferation of undifferentiated endothelial and spindle-like cells within a network of collagen fibers. Neoformed capillaries very rarely showed an organization comparable to small vessel of normal dermis. Retro-virus-like particles recalling the ones described in lymph-nodes of subjects affected by AIDS by Armstrong et al. (1984) were observed in the skin tissue obtained in correspondence of the Kaposi lesion; the same were either isolated or gathered in small groups within cytoplasmic vescicles. Similar particles were not evidenced in the tissue obtained from normal skin. The interpretation of morphological aspects requests of course a great caution. However the observation seems to be noteworthy, specially if one considers the demonstrated association between retro virus of HTLV-III group and AIDS, and that its unusual frequency as well as the malignancy in an immunodepressed host are as yet poorly understood. PMID- 3004542 TI - Screening for antibody to HTLV-III in blood donors of Liguria. Ligurian Committee for the Study of Infections Transmitted through Blood and Blood Products. AB - Anti-HTLV III had been detected in 12,689 serum samples collected within August 1985 from 12 Transfusion Centres of Liguria. In the course of the first test, carried out by ELISA using disrupted virions as antigen, 12,666 samples (99.81%) resulted non reactive; 14 (0.1%) belonged to the grey-zone and 9 (0.08%) were clearly reactive. The samples of the last two classes were tested again in the same way and, whenever the cases were suspect as positive, by Western-blot technique. All positive cases, but three, were linked with risk factors such as intravenous drug use or drug addicts partners. Even if the prevalence of blood donors anti-HTLV III positive resulted, on the whole, very low (0.063%), the investigations carried out prove the importance of regular screening of blood units for the prevention of HTLV III infection. PMID- 3004543 TI - Prevalence of infections caused by AIDS and hepatitis B viruses in jailed people. AB - Prevalence of positive subjects to anti-HTLV III and HBV markers (HBsAg; anti HBc; anti-HBs) has been studied both among jailed people and wardens of Sanremo Jail. Out of 92 subjects in custody, 11 were anti-HTLV III positive and 44 had acquired HBV infection markers (antigen and/or antibodies). One of the wardens resulted anti-HTLV III positive whilst 14 appeared to have been infected by HBV. All anti-HTLV III positive subjects, but the warden, were intravenous drug users. The study of prevalence was the first step of a perspective monitoring program in Ligurian Jails. PMID- 3004544 TI - Immunohistochemical reactivity of anti-LAV p18 monoclonal antibody in lymph nodes from PGL and AIDS patients. AB - This study deals with the immunohistochemistry of a monoclonal antibody (Mab) raised against the p18 protein of the LAV virus in lymphnodes from 20 cases of persistent generalized lymphoadenopathy (PGL) (2) and 6 of acquired immunodeficiency syndrome (AIDS) (3). In all the PGL cases that have been studied, we observed a very important disruption of the follicular dendritic cell's (FDC) framework in the germinal centers which is associated with the presence of an increased number of Leu2a+ lymphocytes inside the germinal centers. This observation is consistent with the main morphological feature of the lymphnodes in the PGL syndrome which is mainly characterized by lesions of FD cells. PMID- 3004545 TI - No effect of ethanol ingestion on beta-adrenoceptor-mediated circulatory responses to isoprenaline in man. AB - The acute effects of ethanol on circulatory responses to isoprenaline and atropine were investigated in 21 and 15 normal male subjects respectively. Each subject acted as his own control by participating twice, once after consumption of ethanol (1.0 ml kg-1, 20% v/v in orange juice) and once after orange juice. Ethanol increased baseline heart rate and forearm blood flow, but had no effect on heart rate and forearm blood flow responses to isoprenaline, or on heart rate responses to atropine. Baseline blood pressure and blood pressure responses to isoprenaline were also unaffected by ethanol. It is concluded that acute ethanol ingestion has no physiologically significant effect on beta-adrenoceptor responsiveness as assessed by the cardiovascular responses to bolus doses of isoprenaline. PMID- 3004546 TI - Pharmacokinetics of enalapril in normal subjects and patients with renal impairment. AB - The pharmacokinetics of enalaprilat were studied after administration of single and multiple doses of enalapril maleate to people with normal and impaired renal function. Renal impairment was associated with higher serum concentrations of enalaprilat, longer times to peak concentrations, slower decline of serum concentrations and with reduced urinary elimination. Urinary elimination of enalaprilat was closely related to renal function. In patients with severe renal impairment (GFR values below 30 ml min-1 1.73 m-2) significantly smaller doses of enalapril maleate will be required than in patients with normal or less severely impaired renal function. PMID- 3004547 TI - Relationship between ploidy and steroid hormone receptors in primary invasive breast cancer. AB - The relationship between ploidy, as measured by flow cytometry, and the presence of oestrogen and progesterone receptors was investigated in 145 primary invasive breast cancers. The tumours were considered as an integral group, and as subgroups of lobular and ductal carcinomas. An association was found between the presence of aneuploid stemlines and an absence of oestrogen receptors (ER), for the total tumour population (P less than 0.02), and for the ductal carcinoma group (P less than 0.05). An association between aneuploidy and an absence of progesterone receptors (PR) was observed for the total tumour group (P less than 0.05). Evaluation of a combined oestrogen and progesterone receptor status indicated that the association between aneuploidy and an absence of both receptors was highly significant. The probability of such an association was P less than 0.001 for the total tumour population, and P less than 0.01 for the ductal tumour group. Assessment of progesterone receptor expression by breast cancers containing oestrogen receptors indicated that aneuploid tumours were as likely to express PR as were diploid tumours. Hence, the biological activity of oestrogen receptors appears unmodified by the presence of aneuploid nuclei. PMID- 3004548 TI - Immunological analysis of the specificity of the autologous humoral response in breast cancer patients. AB - The autologous and allogeneic immunological humoral response of breast cancer patients to breast tumours was investigated by ELISA assay of both the serum and the supernatants of transformed lymphocytes from the patients and controls. No specificity or increased titre relative to the controls was observed in serum antibody. However, when the response was dissected by the use of clones of transformed lymphocytes from the patients, considerable specificity could be demonstrated in certain clones while other clones showed a generalised specificity which contributed to the masking of the specific response in the serum. Some of these clones may have clinical potential as diagnostic and prognostic tools. PMID- 3004549 TI - The adrenal medulla and the airway. PMID- 3004551 TI - Mixed tumours of the lung: a report of three cases. AB - Three patients with mixed tumours of the lung are presented. The difficulties making a histological diagnosis are stressed and the value of extensive examination of large amounts of tissue, and electron microscopy is emphasized. PMID- 3004550 TI - Adrenal function in tuberculosis. AB - In a study of 41 African Zulus with acute pulmonary tuberculosis, 55% were found to have a suboptimal cortisol response to Synacthen and all had very low plasma dehydroepiandrosterone levels. Following a 2-week course of antituberculous chemotherapy there was an improvement in adrenal corticosteroid function with a reduction to 30% of those showing an impaired cortisol response to Synacthen, but adrenal androgen function did not improve in any. Patients who received rifampicin as part of their treatment appeared to show less improvement in adrenal corticosteroid function when compared to a group who received antituberculous treatment which did not include rifampicin. PMID- 3004552 TI - Inhalation and injection studies in rats using dust samples from chrysotile asbestos prepared by a wet dispersion process. AB - Long term inhalation studies and intraperitoneal injection studies in rats were undertaken with a series of chrysotile asbestos dusts. Three dust samples were generated from chrysotile modified by the wet dispersion process (WDC) and one was from unmodified chrysotile. Following a 1 year inhalation period, all the chrysotile samples proved extremely fibrogenic and carcinogenic and there were no significant differences between the WDC dusts and normal chrysotile. In all experimental groups approximately 25% of animals developed pulmonary carcinomas and in the oldest rats advanced interstitial fibrosis occupied on average 10% of all lung tissue. In the injection studies all the dust samples produced mesotheliomas in over 90% of animals. Very little chrysotile remained in the lungs of the animals that survived longest following dust inhalation and what there was was present as individual chrysotile fibrils. It is suggested that chrysotile is potentially the most harmful variety of asbestos as shown in these and other animal studies but that it is removed from lung tissue quite rapidly. In the long lived human species this may mean that except where exposure levels are very high and of long duration, chrysotile should be less hazardous than other asbestos types. PMID- 3004553 TI - Red cell volume distribution curves and intracellular globin chain precipitation in the alpha-thalassaemic mouse, Hbath-J. AB - Red cell volume distribution curves were studied in alpha-thalassaemic mice (Hbath-J/+ mice) and normal mice (+/+ mice) of various ages. Individual Hbath-J/+ mice could not be reliably distinguished from their +/+ littermates on the basis of modal cell volume either at birth or during the first 3 weeks of life. However, between the ages of 4 and 30 weeks Hbath-J/+ mice displayed a degree of microcytosis that enabled them to be readily distinguished from their normal littermates using the criterion of modal red cell volume. Preliminary studies of alpha:beta globin chain synthesis ratios given by blood reticulocytes of Hbath J/+ and +/+ mice after incubation with 3H-leucine for 5 min and 2 h suggest that there is little or no proteolysis of excess beta-chains in the alpha-thalassaemic mouse. Electron microscope studies revealed that the erythroblasts, marrow reticulocytes and circulating red cells of Hbath-J/+ but not +/+ mice contain stellate and branching intracytoplasmic inclusions, presumed to consist of precipitated beta-chains. These inclusions were ultrastructurally similar to the inclusions which have been previously reported in the erythroblasts and marrow reticulocytes of people with various alpha-thalassaemia syndromes. The proportion of erythropoietic cell profiles with inclusions was higher in Hbath-J/+ mice (in which two of the four alpha-globin genes are deleted) than in Thai patients with HbH disease (in whom there is usually a deletion of three of the four alpha globin genes); this finding is probably related to a relatively low proteolytic capacity in the more mature mouse erythroid cells when compared with human cells. The presence of inclusion-containing red cells (mainly reticulocytes) in the peripheral blood of unsplenectomized Hbath-J/+ animals contrasts with the absence of such cells in unsplenectomized patients with alpha-thalassaemia I trait and HbH disease; this difference seems to be at least partly due to a poorly developed pitting function in the mouse spleen. PMID- 3004554 TI - Growth fraction of tumour cells and infiltration density with natural killer-like (HNK1+) cells in non-Hodgkin lymphomas. AB - Two markers for cells in the growth fraction, the T9 antigen (i.e. the transferrin receptor) and the T10 antigen were investigated in frozen tissue sections of 105 non-Hodgkin lymphomas (NHL). The results were correlated with the histological subtype and the pattern of tumour infiltration by reactive cells. Special attention was directed to the density of natural killer (NK)-like cells using the anti-HNK1 (Leu7) antibody since the transferrin receptor (tfr) or other growth-associated membrane structures may serve as target for NK cells. Our study confirms a relationship between number of tumour cells with the T9 marker and tissue infiltration by HNK1+ cells in NHL of low (chronic lymphocytic leukaemia, hairy-cell leukaemia, immunocytic lymphoma, centroblastic-centrocytic lymphoma) and intermediate (centrocytic and centroblastic lymphoma) but not in NHL of high malignant grade (immunoblastic and lymphoblastic lymphoma). Comparable results were obtained with the T10 antigen although the correlation was less close. The percentage of cells in the growth fraction, defined by the expression of the T9 and T10 marker, corresponded with prognostically unfavourable subgroups with the remarkable exception of follicular NHL of centroblastic-centrocytic type. This lymphoma showed high numbers of cells with the T9 and the T10 marker in a microenvironment resembling normal germinal centres in many aspects. PMID- 3004555 TI - Health effects among refrigeration repair workers exposed to fluorocarbons. AB - Refrigeration repair workers may be intermittently exposed to fluorocarbons and their thermal decomposition products. A case of peripheral neuropathy (distal axonopathy) in a commercial refrigeration repairman prompted an epidemiological investigation of the health of refrigeration repair workers. No additional cases of peripheral neuropathy were identified among the 27 refrigeration repair workers studied. A reference group of 14 non-refrigeration repair workers was also studied. No differences were noted between groups for the ulnar (motor and sensory), median (motor and sensory), peroneal, sural, or tibial nerve conduction velocities. Refrigeration repair workers reported palpitations and lightheadedness significantly more often than workers in the reference group. No clinical neurological or electroneurophysiological abnormalities were detected in eight refrigeration repair workers followed up for three years during continuous employment. PMID- 3004556 TI - Choroidal metastasis of adenocystic carcinoma of the salivary gland. AB - A case of adenocystic carcinoma of the salivary gland arising in the hard palate metastatic to the choroid is presented. The histopathology of the tumour is discussed. PMID- 3004557 TI - Antibodies to herpes simplex virus type I in intraocular fluids of patients with acute retinal necrosis. AB - The intraocular fluids and sera from three patients with acute retinal necrosis (ARN) and from 120 age-matched controls were tested for the presence of antibodies to herpes simplex virus type I (HSV-I). Antibodies to HSV-I were detected in both intraocular fluids and sera of two cases, and in intraocular fluids alone in one case of ARN. Among 120 control patients undergoing routine intraocular surgery antibodies to HSV-I were detected in the sera of 85 (79.2%) but in the intraocular fluids of only two (1.7%). The presence of antibody within the eye may indicate local antibody production, antibody sequestration within the eye, or damage to the blood ocular barrier. PMID- 3004558 TI - In vivo effects of cis- and trans-diamminedichloroplatinum(II) on SV40 chromosomes: differential repair, DNA-protein cross-linking, and inhibition of replication. AB - The mechanism of action of the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, was investigated by using the approximately 5200 base pair (bp) chromosome of simian virus 40 (SV40) as an in vivo chromatin model. Comparative studies were also carried out with the clinically ineffective isomer trans-DDP. Although 14 times more trans- than cis-DDP in the culture medium is required to inhibit SV40 DNA replication in SV40-infected green monkey CV-1 cells, the two isomers are equally effective at inhibiting replication when equimolar amounts are bound to SV40 DNA in vivo. Since both isomers are transported into CV-1 cells at similar rates, differential uptake cannot account for the greater ability of cis-DDP to inhibit SV40 DNA replication. Rather, this result is explained by the finding that cis-DDP-DNA adducts accumulate continuously over the incubation period, whereas trans-DDP binding to DNA reaches a maximum at 6 h and thereafter decreases dramatically. We suggest that the different accumulation behavior of cis-DDP and trans-DDP on DNA is due to their differential repair in CV-1 cells. A variety of non-histone proteins, including SV40 capsid proteins but virtually no histones, are cross-linked to SV40 DNA in vivo by either cis- or trans-DDP. More DNA-protein cross-links are formed by trans-DDP than by cis-DDP at equivalent amounts of DNA-bound platinum.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004559 TI - Effect of the vesicular stomatitis virus matrix protein on the lateral organization of lipid bilayers containing phosphatidylglycerol: use of fluorescent phospholipid analogues. AB - In order to investigate the mode of interaction of peripheral membrane proteins with the lipid bilayer, the basic (pI approximately 9.1) matrix (M) protein of vesicular stomatitis virus was reconstituted with small unilamellar vesicles (SUV) containing phospholipids with acidic head groups. The lateral organization of lipids in such reconstituted membranes was probed by fluorescent phospholipid analogues labeled with pyrene fatty acids. The excimer/monomer (E/M) fluorescence intensity ratios of the intrinsic pyrene phospholipid probes were measured at various temperatures in M protein reconstituted SUV composed of 50 mol % each of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). The M protein showed relatively small effects on the E/M ratio either in the gel or in the liquid-crystalline phase. However, during the gel to liquid-crystalline phase transition, the M protein induced a large increase in the E/M ratio due to phase separation of lipids into a neutral DPPC-rich phase and DPPG domains presumably bound to M protein. Similar phase separation of bilayer lipids was also observed in the M protein reconstituted with mixed lipid vesicles containing one low-melting lipid component (1-palmitoyl-2-oleoylphosphatidylcholine or 1 palmitoyl-2-oleoylphosphatidylglycerol) or a low mole percent of cholesterol. The self-quenching of 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence, as a measure of lipid clustering in the bilayer, was also studied in M protein reconstituted DPPC-DPPG vesicles containing 5 mol % NBD-phosphatidylethanolamine (NBD-PE). The quenching of NBD-PE was enhanced at least 2-fold in M protein reconstituted vesicles at temperatures within or below the phase transition.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004560 TI - Hydrogen exchange of individual amide protons in the F helix of cyanometmyoglobin. AB - Hydrogen exchange of the individual amide protons of alanine-90 (F5), glutamine 91 (F6), serine-92 (F7), and histidine-93 (F8) residues in cyanometmyoglobin of sperm whale has been studied by 1H nuclear magnetic resonance spectroscopy at 360 MHz. The amide proton resonance of F5, F6, and F7 have been assigned by use of the selective nuclear Overhauser effect between the consecutive amide protons. At pH 6.8, and in the temperature range of 5-20 degrees C, these protons show a 10(4)-fold retardation compared to the rates in free peptides. Apparent activation enthalpies for hydrogen exchange of F5, F6, and F8 protons are 18.5 +/ 0.4, 9.5 +/- 0.3, and 18.5 +/- 0.3 kcal/mol, respectively. Some implications of these results on the nature of the opening processes involved in hydrogen exchange are considered. PMID- 3004561 TI - Resonance assignment of the 500-MHz proton NMR spectrum of self-complementary dodecanucleotide d-GGATCCGGATCC: altered conformations at BamHI cleavage sites. AB - Resonance assignments of nonexchangeable base and sugar protons of the self complementary dodecanucleotide d-GGATCCGGATCC have been obtained by two dimensional NMR methods and strategies derived from interproton distance calculations on different secondary structures of nucleic acids. Conformational details about the glycosidic dihedral angle and sugar pucker have been derived from the relative intensities of cross peaks in the two-dimensional J-correlated and nuclear Overhauser enhancement correlated spectra in D2O solution. It is observed that d-GGATCCGGATCC assumes a predominantly B-type conformation with sequence-dependent changes along the chain. The recognition site of BamHI shows a distinctly different geometrical environment. The sugar rings of G1 and G7 assume a C3'-endo geometry while the rest of the sugars possess C2'-endo geometry. PMID- 3004562 TI - Mapping of gonadotropin-releasing hormone receptor binding site. AB - On the basis of the spatial conformation of gonadotropin-releasing hormone (GnRH), we have predicted that aromatic amino acids and at least one carboxyl group are involved in the recognition site of the receptor. Therefore, various specific reagents were examined for their ability to interfere with the binding of GnRH to its receptor. Pretreatment of pituitary membrane preparations with sodium periodate decreased the specific binding in a dose-dependent manner (IC50 = 0.5 mM) due to a decrease in receptor affinity. This indicated the presence of a sugar moiety in the binding site. Tryptophan is another constituent that participates in the GnRH binding site, as pretreatment of pituitary membranes with 2-methoxy-5-nitrobenzyl bromide inhibited the binding (IC50 = 0.22 mM) by decreasing receptor affinity. In addition, the native hormone conferred on the binding site a protective effect against inactivation by 2-methoxy-5-nitrobenzyl bromide. Pretreatment of membranes with p-diazobenzenesulfonic acid also inhibited the binding of 125I-Buserelin (IC50 = 0.1 mM), indicating the presence of tyrosine within or near the binding site. Pretreatment of pituitary membrane preparations with dithiothreitol also inhibited the binding due to a decrease in the binding affinity, which was accompanied by an increase in receptor number. These data suggest that there are disulfide bonds within or near the binding region. Treatment with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and glycine ethyl ester also prevented binding in a dose-dependent manner and implies that free carboxylic groups are involved in the binding site.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004563 TI - Effects of tyrosine-26 and tyrosine-64 nitration on the photoreactions of bacteriorhodopsin. AB - In the dark, all titratable tyrosine residues of bacteriorhodopsin have pK's of greater than 11.0, which may be caused by the hydrophobic environment for buried residues and by high negative charge density for surface residues [Scherrer, P., & Stoeckenius, W. (1984) Biochemistry 23, 6195-6202]. Under illumination, deprotonation of only one tyrosine is observed in the micro- and millisecond time ranges of the photocycle; this is Tyr-64. Nitration of Tyr-64 decreases the chromophore absorbance, shifts the absorption maximum to 535 nm, and affects photocycle kinetics. However, restoring its native pK by reduction after nitration has no effect on the changes in photocycle kinetics or absorbance of the chromophore. Nitration of Tyr-64 apparently causes a conformational change in bR, which is independent of the pK of its phenolic group. These observations contradict earlier conclusions that in the photocycle a tyrosine residue directly interacts with the Schiff base during its deprotonation or reprotonation. The protonation state of Tyr-26 and the alkaline chromophore transition are correlated, as shown earlier (Scherrer & Stoeckenius, 1984). Lowering the pK of Tyr-26 by nitration decreases the M-decay rate, and this effect is partially reversed by reduction of the nitro group. We conclude that Tyr-26 may be located close to the chromophore and interact with it; but its protonation state does not change at physiological pH and in the microsecond time range of the photocycle. Tyr-64 is apparently located at or close to the external surface; its modification strongly affects the chromophore but apparently indirectly and not through its protonation changes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004565 TI - Matrix free Ca2+ in isolated chromaffin vesicles. AB - Isolated secretory vesicles from bovine adrenal medulla contain 80 nmol of Ca2+ and 25 nmol of Mg2+ per milligram of protein. As determined with a Ca2+-selective electrode, a further accumulation of about 160 nmol of Ca2+/mg of protein can be attained upon addition of the Ca2+ ionophore A23187. During this process protons are released from the vesicles, in exchange for Ca2+ ions, as indicated by the decrease of the pH in the incubation medium or the release of 9-aminoacridine previously taken up by the vesicles. Intravesicular Mg2+ is not released from the vesicles by A23187, as determined by atomic emission spectroscopy. In the presence of NH4Cl, which causes the collapse of the secretory vesicle transmembrane proton gradient (delta pH), Ca2+ uptake decreases. Under these conditions A23187-mediated influx of Ca2+ and efflux of H+ cease at Ca2+ concentrations of about 4 microM. Below this concentration Ca2+ is even released from the vesicles. At the Ca2+ concentration at which no net flux of ions occurs the intravesicular matrix free Ca2+ equals the extravesicular free Ca2+. In the absence of NH4Cl we determined an intravesicular pH of 6.2. Under these conditions the Ca2+ influx ceases around 0.15 microM. From this value and the known pH across the vesicular membrane an intravesicular matrix free Ca2+ concentration of about 24 microM was calculated. This is within the same order of magnitude as the concentration of free Ca2+ in the vesicles determined in the presence of NH4Cl.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004564 TI - Resonance Raman study on photoreduction of cytochrome c oxidase: distinction of cytochromes a and a3 in the intermediate oxidation states. AB - Occurrence of photoreduction of bovine cytochrome c oxidase was confirmed with the difference absorption spectra and oxygen consumption measurements for the enzyme irradiated with laser light at 406.7, 441.6, and 590 nm. The resonance Raman spectra were obtained under the same experimental conditions as those adopted for the measurements of oxygen consumption and difference absorption spectra. The photoreduction was more effective upon irradiation at shorter wavelengths and was irreversible under anaerobic conditions. However, upon aeration into the cell, the original oxidized form was restored. It was found that aerobic laser irradiation produces a photo steady state of the catalytic dioxygen reduction and that the Raman scattering from this photo steady state probes cytochrome a2+ and cytochrome a3(3)+ separately upon excitations at 441.6 and 406.7 nm, respectively. The enzyme was apparently protected from the photoreduction in the spinning cell with the spinning speed between 1 and 1500 rpm. These results were explained satisfactorily with the reported rate constant for the electron transfer from cytochrome a to cytochrome a3 (0.58 s-1) and a comparable photoreduction rate of cytochrome a. The anaerobic photoreduction did give Raman lines at 1666 and 214 cm-1, which are characteristic of the ferrous high-spin cytochrome a3(2)+, but they were absent under aerobic photoreduction. The formyl CH = O stretching mode of the a3 heme was observed at 1671 cm-1 for a2+a3(2)+CO but at 1664 cm-1 for a2+a3(2)+CN-, indicating that the CH = O stretching frequency reflects the pi back-donation to the axial ligand similar to the oxidation state marker line (v4). PMID- 3004566 TI - Contribution of water to free energy of hydrolysis of pyrophosphate. AB - The energy of hydrolysis of phosphate compounds varies depending on whether they are in solution or bound to the catalytic site of enzymes. With the purpose of simulating the conditions at the catalytic site, the observed equilibrium constant for pyrophosphate hydrolysis (Kobsd) was measured in aqueous mixtures of dimethyl sulfoxide, ethylene glycol, or polymers of ethylene glycol. The reaction was catalyzed by yeast inorganic pyrophosphatase at 30 degrees C. All the cosolvents used promoted a decrease of Kobsd. Polymers of ethylene glycol were more effective than dimethyl sulfoxide or ethylene glycol in decreasing Kobsd. The higher the molecular weight of the polymer, the lower the value of Kobsd. A decrease in Kobsd from 346 M (delta G degree obsd = -3.5 kcal mol-1) to 0.1 M (delta G degree obsd = 1.3 kcal mol-1) was observed after the addition of 50% (w/v) poly(ethylene glycol) 8000 to a solution containing 0.9 mM MgCl2 and 1 mM Pi at pH 8.0. The association constants of Pi and pyrophosphate for H+ and Mg2+ were measured in presence of different ethylene glycol concentrations in order to calculate the Keq for hydrolysis of different ionic species of pyrophosphate. A decrease in all the Keq was observed. The results are interpreted according to the concept that the energy of hydrolysis of phosphate compounds depends on the different solvation energies of reactants and products. PMID- 3004567 TI - Protein 4.1 is involved in a structural thermotropic transition of the red blood cell membrane detected by a spin-labeled stearic acid. AB - Proteins involved in a structural transition in red blood cell membranes detected at 8 +/- 1.5 degrees C by a stearic acid spin-label have been investigated. Calcium loading of red blood cells with ionophore A23187 caused the disappearance of the 8 degrees C transition. Protein 4.1 appears to be the most susceptible protein to Ca2+ treatment. Antibodies specific for spectrin, band 3 (43K cytoplasmic domain), and protein 4.1 have been utilized as specific probes to modify membrane thermotropic properties. The 8 degrees C transition was eliminated by anti-4.1 protein antibodies but was not modified by the other antibodies. To further characterize the protein(s) involved in the transition, ghosts were subjected to sequential extraction of skeletal proteins. The extraction of band 6, spectrin, and actin did not modify the 8 degrees C transition. In contrast, high-salt extraction (1 M KCl) of spectrin-actin depleted vesicles, a procedure that extracts proteins 2.1 and 4.1, was able to eliminate the 8 degrees C transition. Rebinding of purified protein 4.1 to the high salt extracted vesicles restored the 8 degrees C transition. These results indicate the involvement of protein 4.1 in the transition and suggest a functional membrane association of this protein. The binding of protein 4.1 to the membrane seems to contribute significantly to the thermotropic properties of red blood cells. PMID- 3004568 TI - Deuterium and phosphorus nuclear magnetic resonance studies on the binding of polymyxin B to lipid bilayer-water interfaces. AB - Deuterium and phosphorus NMR methods have been used to study the binding of polymyxin B to the surface of bilayers containing lipids that were deuterated at specific positions in the polar head-group region. The binding of polymyxin B to acidic dimyristoylphosphatidylglycerol (DMPG) membranes induces only small structural distortions of the glycerol head group. The deuterium spin-lattice relaxation times for the different carbon-deuterium bonds in the head group of the same phospholipid are greatly reduced on binding of polymyxin B, indicating a restriction of the motional rate of the glycerol head group. Only very weak interactions were detected between polymyxin B and bilayers of zwitterionic dimyristoylphosphatidylcholine (DMPC). In mixed bilayers of the two phospholipid types, in which either of the two phospholipids was deuterated, the presence of polymyxin B caused a lateral phase separation into DMPG-enriched phospholipid peptide clusters and a DMPG-depleted phase. Complete phase separation did not occur: peptide-containing complexes with charged phosphatidylglycerol contained substantial amounts of zwitterionic phosphatidylcholine. Exchange of both phospholipid types between complexes and the bulk lipid matrix was shown to be fast on the NMR time scale, with a lifetime for phospholipid-peptide association of less than 1 ms. PMID- 3004569 TI - Site-selected fluorescence spectra of porphyrin derivatives of heme proteins. AB - The emission spectra of the porphyrin in metal-free and Zn cytochrome c and in metal-free mesoporphyrin derivatives of horseradish peroxidases A and C, leghemoglobin, and myoglobin were examined as a function of temperature and excitation wavelength. At room temperature, the emission spectra were unresolved and were independent of excitation wavelength. At low temperature (4.2 K), the spectra depended upon excitation wavelength: using narrow-band excitation into the high-energy side of the 0-1 and 0-0 bands gave unresolved emission spectra whereas excitation into the low-energy side produced quasi-line spectra. The resolved spectra were different for the five proteins and further varied with pH, indicating chromophore-protein interactions. The spectra are interpreted in terms of site selection and phonon interactions. PMID- 3004570 TI - Slow refolding kinetics in yeast iso-2 cytochrome c. AB - In refolding of iso-2 cytochrome c from Saccharomyces cerevisiae, there are two slow folding reactions, tau 1a and tau 1b. The slower of the slow reactions, tau 1a = 100-200 s, is observed only by absorbance changes, while tau 1b (10-20-fold faster) is detected by fluorescence changes. The temperature dependence of the rates of these reactions has been measured: for kinetic experiments ending below the folding-unfolding transition zone (pH 7.2, 0.3 M guanidine hydrochloride, 5 30 degrees C), the activation enthalpies are delta H++ = 27 kcal/mol for tau 1a and 21 kcal/mol for tau 1b. Double-jump (unfolding, then refolding) experiments demonstrate that the two sets of species responsible for the slow folding reactions are generated slowly but at different rates under unfolding conditions (3 M guanidine hydrochloride, pH 7.2, 20 degrees C). Finally, as a test for changes in the population of the slow refolding species under different unfolding conditions, the amplitudes for slow refolding have been measured as a function of the initial unfolding conditions with the final refolding conditions held constant. Over the range accessible to measurement in the absence of interference from other reactions, the amplitudes for fluorescence-detected (alpha 1b) and absorbance-detected (alpha 1a) slow folding are independent of guanidine hydrochloride concentration and pH in the initial conditions. Although a full description requires a more complex explanation, many of the properties of the slow folding species are those expected for proline imide bond isomerization. PMID- 3004571 TI - Nucleotide sequence of cloned cDNA for human pancreatic kallikrein. AB - Cloned cDNA sequences for human pancreatic kallikrein have been isolated and determined by molecular cloning and sequence analysis. The identity between human pancreatic and urinary kallikreins is indicated by the complete coincidence between the amino acid sequence deduced from the cloned cDNA sequence and that reported partially for urinary kallikrein. The active enzyme form of the human pancreatic kallikrein consists of 238 amino acids and is preceded by a signal peptide and a profragment of 24 amino acids. A sequence comparison of this with other mammalian kallikreins indicates that key amino acid residues required for both serine protease activity and kallikrein-like cleavage specificity are retained in the human sequence, and residues corresponding to some external loops of the kallikrein diverge from other kallikreins. Analyses by RNA blot hybridization, primer extension, and S1 nuclease mapping indicate that the pancreatic kallikrein mRNA is also expressed in the kidney and sublingual gland, suggesting the active synthesis of urinary kallikrein in these tissues. Furthermore, the tissue-specific regulation of the expression of the members of the human kallikrein gene family has been discussed. PMID- 3004572 TI - DNA filter retention assay for exonuclease activities. Application to the analysis of processivity of phage T5 induced 5'-exonuclease. AB - The 5'-exonuclease of phage T5 has been purified nearly to homogeneity by using a simple and fast procedure. The kinetic properties of the purified enzyme have been studied by using a new sensitive assay based upon retention by nitrocellulose filters of DNA with short protruding single-stranded ends. The enzyme is specifically stimulated by KCl. Its Km is 2.2 X 10(-7) M at 30 degrees C, and its turnover number is 0.33 DNA molecule transformed per minute. The filter retention assay shows that the T5 exonuclease acts by a semiprocessive mechanism, removing from DNA ends about 30 nucleotides on the average per cycle. The degree of enzyme processivity increases with increasing magnesium concentrations. PMID- 3004573 TI - Calcium binding to complexes of calmodulin and calmodulin binding proteins. AB - The free energy of coupling for binding of Ca2+ and the calmodulin-sensitive phosphodiesterase to calmodulin was determined and compared to coupling energies for two other calmodulin binding proteins, troponin I and myosin light chain kinase. Free energies of coupling were determined by quantitating binding of Ca2+ to calmodulin complexed to calmodulin binding proteins with Quin 2 to monitor free Ca2+ concentrations. The geometric means of the dissociation constants (-Kd) for Ca2+ binding to calmodulin in the presence of equimolar rabbit skeletal muscle troponin I, rabbit skeletal muscle myosin light chain kinase, and bovine heart calmodulin sensitive phosphodiesterase were 2.1, 1.1, and 0.55 microM. The free-energy couplings for the binding of four Ca2+ and these proteins to calmodulin were -4.48, -6.00, and -7.64 kcal, respectively. The Ca2+-independent Kd for binding of the phosphodiesterase to calmodulin was estimated at 80 mM, indicating that complexes between calmodulin and this enzyme would not exist within the cell under low Ca2+ conditions. The large free-energy coupling values reflect the increase in Ca2+ affinity of calmodulin when it is complexed to calmodulin binding proteins and define the apparent positive cooperativity for Ca2+ binding expected for each system. These data suggest that in vitro differences in free-energy coupling for various calmodulin-regulated enzymes may lead to differing Ca2+ sensitivities of the enzymes. PMID- 3004574 TI - Stereochemistry of the guanyl nucleotide binding site of transducin probed by phosphorothioate analogues of GTP and GDP. AB - The stereochemistry of the guanyl nucleotide binding site of transducin from bovine retinal rod outer segments was probed with phosphorothioate analogues of GTP and GDP. Transducin has markedly different affinities for the five thio analogues of GTP, as measured by their effectiveness in inhibiting GTPase activity, competing with GTP for entry into transducin, and displacing GDP bound to transducin. The order of binding affinities is GTP gamma S = (Sp)-GTP alpha S greater than (Rp)-GTP alpha S greater than (Sp)-GTP beta S much greater than (Rp) GTP beta S. The affinity of transducin for GTP gamma S is greater than 10(4) higher than that for (Rp)-GTP beta S. These five analogues have the same relative potencies in eliciting the release of transducin from the membrane and in activating the phosphodiesterase. Transducin hydrolyzes (Sp)-GTP alpha S with a l/e time of 55 s, compared with 28 s for GTP. In contrast, (Rp)-GTP alpha S, like GTP gamma S, is not hydrolyzed on the time scale of several hours. The order of effectiveness of thio analogues of GDP in displacing bound GDP is (Sp)-GDP alpha S greater than GDP greater than (Rp)-GDP alpha S greater than GDP beta S. The affinity of transducin for (Sp)-GDP alpha S is about 10-fold higher than that for GDP beta S. Mg2+ is required for the binding of GTP and GDP to transducin. Cd2+ does not lead to a reversal of stereospecificity at either the alpha- or beta phosphorus atom of GTP. These results lead to the following conclusions: The pro R oxygen atom at the alpha-phosphorus of GTP does not bind Mg2+ but instead interacts with the protein. The pro-S oxygen at the alpha-phosphorus does not appear to be involved in a critical interaction with transducin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004575 TI - Electron transfer in photosystem II at cryogenic temperatures. AB - The photochemistry in photosystem II of spinach has been characterized by electron paramagnetic resonance (EPR) spectroscopy in the temperature range of 77 235 K, and the yields of the photooxidized species have been determined by integration of their EPR signals. In samples treated with 3-(3,4-dichlorophenyl) 1,1-dimethylurea (DCMU), a single stable charge separation occurred throughout the temperature range studied as reflected by the constant yield of the Fe(II)-QA EPR signal. Three distinct electron donation pathways were observed, however. Below 100 K, one molecule of cytochrome b559 was photooxidized per reaction center. Between 100 and 200 K, cytochrome b559 and the S1 state competed for electron donation to P680+. Photooxidation of the S1 state occurred via two intermediates: the g = 4.1 EPR signal species first reported by Casey and Sauer [Casey, J. L., & Sauer, K. (1984) Biochim. Biophys. Acta 767, 21-28] was photooxidized between 100 and 160 K, and upon being warmed to 200 K in the dark, this EPR signal yielded the multiline EPR signal associated with the S2-state. Only the S1 state donated electrons to P680+ at 200 K or above, giving rise to the light-induced S2-state multiline EPR signal. These results demonstrate that the maximum S2-state multiline EPR signal accounts for 100% of the reaction center concentration. In samples where electron donation from cytochrome b559 was prevented by chemical oxidation, illumination at 77 K produced a radical, probably a chlorophyll cation, which accounted for 95% of the reaction center concentration. This electron donor competed with the S1 state for electron donation to P680+ below 100 K.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004576 TI - Studies on the effect of copper deficiency on rat liver mitochondria. III. Effects on adenine nucleotide translocase. AB - Liver mitochondria from Cu-deficient rats exhibit impaired State 3 respiration (oxygen consumption in the presence of exogenous ADP) compared with Cu-adequate controls, whereas State 4 respiration (oxygen consumption after depletion of exogenous ADP) and ADP/O are unaffected. In view of previous observations (Davies, N.T., Lawrence, C.B., Mills, C.F. and Nicol, F. (1985) Biochim. Biophys. Acta 809, 351-361) it seemed that a decline in cytochrome c oxidase activity (EC 1.9.3.1) could not fully account for these findings. Cu deficiency resulted in a significant decline (40%, P less than 0.01) in [14C]ADP uptake by liver mitochondria which suggests there is a reduced activity of the adenine nucleotide translocase. The reduced translocase activity was not associated with any marked change in fatty-acid composition of either intact mitochondria or inner mitochondrial membranes. Inhibitor titrations with the irreversible inhibitor carboxyatractyloside showed that 'Cu-deficient' mitochondria required the same concentration of inhibitor to produce 100% inhibition of State 3 respiration as control mitochondria, suggesting that the amount of functional translocase enzyme present is unaffected. When the translocase assay was allowed to proceed until equilibrium was established between external and internal nucleotides, it was apparent that the exchangeable adenine nucleotide pool of Cu-deficient mitochondria was 36% lower than in controls. Analysis of mitochondria for their ATP, ADP and AMP contents showed that, whereas the AMP content was unaffected, ATP and ADP contents were 39 and 40% lower, respectively, which resulted in a significantly reduced pool of total adenine nucleotides (ATP + ADP + AMP) and a reduced 'energy charge' [(ATP + 0.5 ADP)/(ATP + ADP + AMP)]. These results are discussed in relation to current concepts of the regulation and control of mitochondrial respiration. PMID- 3004577 TI - Identification of ubiquinone-binding proteins in yeast mitochondrial ubiquinol cytochrome c reductase using an azido-ubiquinone derivative. AB - An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyloctyl) 1,4-benzoquinone, was used to study the ubiquinone-protein interaction and to identify the ubiquinone-binding proteins in yeast mitochondrial ubiquinone cytochrome c reductase. The phospholipids and Q6 in purified reductase were removed by repeated ammonium sulfate precipitation in the presence of 0.5% sodium cholate. The resulting phospholipid- and ubiquinone-depleted reductase shows no enzymatic activity; activity can be completely restored by the addition of phospholipids and Q6 or Q2. The ubiquinone- and phospholipid-replenished ubiquinonol-cytochrome c reductase is also fully active upon reconstituting with bovine succinate-ubiquinone reductase to form succinate-cytochrome c reductase. When an azido-ubiquinone derivative was added to the ubiquinone and phospholipid depleted reductase in the dark, followed by the addition of phospholipids, partial reconstitutive activity was restored, while full ubiquinol-cytochrome c reductase activity was observed when Q2H2 was used as substrate in the assay mixture. Apparently, the large amount of Q2H2 present in the assay mixture displaces the azido-ubiquinone in the system. Photolysis of the azido-Q-treated reductase with long-wavelength ultraviolet light abolishes about 70% of both the restored reconstitutive activity and Q2H2-cytochrome c reductase activity. The activity loss is directly proportional to the covalent binding of [3H]azido ubiquinone to the reductase protein. When the photolyzed, [3H]azido-ubiquinone treated sample was subjected to SDS-polyacrylamide gel electrophoresis followed by analysis of the distribution of radioactivity among the subunits, the cytochrome b protein and a protein with an apparent molecular weight of 14 000 were heavily labeled. The amount of radioactive labeling in both these proteins was affected by the presence of phospholipids. PMID- 3004579 TI - Activation of the ADP/ATP carrier from mitochondria by cationic effectors. AB - The ADP/ATP carrier from the mitochondrial inner membrane was found to be influenced by cationic substances from the hydrophilic surroundings. Under low ionic-strength conditions, addition of these cationic effectors fully activated the reconstituted adenine nucleotide translocator. The list of activators included divalent cations, polyamines, peptides and cationic proteins. The minimum requirement for an activator to be effective was the presence of at least two positive net charges, regardless of the size of the molecule. Cationic molecules were not activating when an intramolecular charge compensation was possible or when the two charges were too far apart from one another. The affinity of these activators varied from several hundred microM (diaminoalkanes, divalent cations) to 1 microM (cytochrome c, spermine) and even down to a few nM (polylysine). The activation by cations was fully reversible and was not due to fusion processes. It was not mediated by an interaction with the anionic substrates ADP and ATP, nor by interaction with the liposomes. The stimulation could directly and functionally be correlated to the reconstituted carrier protein. Activation was not observed in intact mitochondria, but could be demonstrated when the outer mitochondrial membrane had been removed by treatment with digitonin. These mitoplasts were stimulated by polycations similar to the ADP/ATP carrier in the reconstituted system. PMID- 3004578 TI - The effect of Cl- depletion and X- reconstitution on the oxygen-evolution rate, the yield of the multiline manganese EPR signal and EPR signal II in the isolated Photosystem-II complex. AB - The role of Cl- in photosynthetic O2 evolution has been investigated by measurement of the steady-state O2 rate and EPR of the electron donors responsible for the S2 multiline signal and Signal IIs upon Cl- depletion and substitution in Photosystem II membranes. Cl- removal has three effects upon the donor side of Photosystem II. (1) It abolishes O2 evolution reversibly, while decreasing the yield of the S2 multiline signal indicative of the manganese site of the O2-evolving complex in the S2 oxidation state. This decrease is brought about by (2) the reversible disconnection of the manganese complex from the reaction center; and by (3) deactivation of S1 centers having reduced primary acceptor QA to form SO centers having a reduced Signal IIs species. Reactivation of O2 evolution by anions confirms earlier work showing a requirement for a univalent anion of optimum charge density. The observed order of reactivation is Cl- greater than Br- approximately NO3- much greater than OH- approximately F-. Reactivation of the S2 multiline signal follows Cl- approximately Br- greater than NO3- approximately OH- greater than F-, in near correspondence with reactivation of O2-evolution rates. Cl- titrations of F- -inhibited samples reveal two binding sites for Cl- which differ in binding affinity by 11-fold. The higher-affinity site reactivates the S1----S2 light reaction, while the lower affinity site reactivates the S3----S0 light reaction. The high affinity site is located within the O2-evolving complex at an undetermined site, while the lower affinity site functions in coupling the reaction center photochemistry to the O2 evolving complex. The results are compared with Cl-/F- exchange equilibria for Mn3+ in solution. A model for the lower S-state transitions is presented in which specific oxidation state assignments are made for some of the donors and acceptors of Photosystem II. PMID- 3004580 TI - Effect of fatty acyl chain length of phosphatidylcholine on their transfer from liposomes to erythrocytes and transverse diffusion in the membranes inferred by TEMPO-phosphatidylcholine spin probes. AB - TEMPO-phosphatidylcholine (PC) spin probes which have homologous saturated acyl chains of 10, 12, 14 and 16 carbon atoms, were synthesized as analogues of PC. Transfer of TEMPO-PCs from liposomal membrane to the ghost membrane of human erythrocyte and transverse diffusion of TEMPO-PCs within the membrane of intact erythrocytes were determined by measurement of spontaneous increase and decrease in signal amplitude of an anisotropic triplet spectrum, due to dilution of the label by natural phospholipid of the membrane and reduction of the label by the cytoplasmic content of the erythrocyte, respectively. TEMPO-PC molecules in TEMPO PC liposomes, except dipalmitoyl TEMPO-PC, were rapidly incorporated into the ghost membrane by incubation at 37 degrees C; the PC having shorter acyl chains was transferred faster. The cytoplasmic content of the erythrocyte rapidly reduced the nitroxide radical of the spin probe. The central peak height of ESR signal was once increased by incorporation of TEMPO-PC into the erythrocyte membrane and then was spontaneously decreased during further incubation at 37 degrees C. This decrease indicates that PC molecules traverse from the outer to the inner layer of the membrane lipid bilayer. The decrease of signal amplitude was faster with PC of shorter acyl chain. These findings suggest that both transfer between membranes and transverse diffusion in the membrane may be favored to the PC species with shorter acyl chains. PMID- 3004582 TI - Immunological identification of rat tissue kallikrein cDNA and characterization of the kallikrein gene family. AB - A tissue kallikrein cDNA was identified by direct immunological screening with affinity-purified anti-rat tissue kallikrein antibody from a rat submandibular cDNA library constructed with the expression vector pUC8. Sequence analysis of the kallikrein cDNA revealed an encoded protein 97% homologous to the partial amino acid sequence of rat submandibular kallikrein. This cDNA was used to hybrid select kallikrein-specific RNA from submandibular gland. Translation of the hybrid-selected RNA in a cell-free assay system resulted in the production of a 37 kDa peptide representing the preproenzyme. In addition, hybrid-selection of RNA under less stringent conditions showed cross-hybridization with other submandibular gland mRNA species. In correlation with these results, analysis of rat genomic DNA showed extensive hybridization, suggesting a family of closely related kallikrein-like genes. Consequently, a Charon 4A rat genomic library was screened for kallikrein genes by hybridization with rat tissue kallikrein cDNA. Thirty-four clones were isolated and found to be highly homologous by hybridization and restriction enzymes analyses. Fourteen unique clones were identified by restriction enzyme site polymorphisms within DNA segments which hybridized to the kallikrein cDNA probe and it was estimated that at least 17 different kallikrein-like genes are present in the rat. Sequence and structural analysis of one of the genomic clones revealed a gene structure similar to that of other serine proteinases. Comparison of the partially sequenced exon regions of the gene with the sequence of rat tissue kallikrein cDNA reveals 89% identity when aligned for the greatest homology. However, the genomic sequence predicts termination codons in all three translational reading frames, implying that this gene is nonfunctional, i.e., a pseudogene. Comparison of the rat genomic sequence to a kallikrein-like gene from the mouse reveals extensive preservation of exons, less identity within introns and no significant homology between extragenic regions. PMID- 3004581 TI - Sodium and buffer cations inhibit dephosphorylation of (Na+ + K+)-ATPase. AB - Effects of various cations on the dephosphorylation of (Na+ + K+)-ATPase, phosphorylated by ATP in 50 mM imidazole buffer (pH 7.0) at 22 degrees C without added Na+, have been studied. The dephosphorylation in imidazole buffer without added K+ is extremely sensitive to K+-activation (Km K+ = 1 microM), less sensitive to Mg2+-activation (Km Mg2+ = 0.1 mM) and Na+-activation (Km Na+ = 63 mM). Imidazole and Na+ effectively inhibit K+-activated dephosphorylation in linear competitive fashion (Ki imidazole 7.5 mM, Ki Na+ 4.6 mM). The Ki for Na+ is independent of the imidazole concentration, indicating different and non interacting inhibitory sites for Na+ and imidazole. Imidazole inhibits Mg2+ activated dephosphorylation just as effective as K+-activated dephosphorylation, as judged from the Ki values for imidazole in the two processes. Tris buffer and choline chloride, like imidazole, inhibit dephosphorylation in the presence of residual K+ (less than 1 microM), but less effectively in terms of I50 values and extent of inhibition. Tris inhibits to the same extent as choline. This indicates different inhibitory sites for Tris or choline and for imidazole. These findings indicate that high steady-state phosphorylation levels in Na+-free imidazole buffer are due to the induction of a phosphorylating enzyme conformation and to the inhibition of (K+ + Mg2+)-stimulated dephosphorylation. PMID- 3004583 TI - Regulation of acetohydroxy acid synthase activities in Escherichia coli K-12 by small metabolites. AB - The effects of several metabolites (indole acetic acid, imidazole acetic acid and indole) on acetohydroxy acid synthase activities have been examined in both cya+ and cya- strains. Specifically, indole acetic acid caused an increase in the rate of acetohydroxy acid synthase synthesis under both in vivo and in vitro conditions. Taken together, these data suggest that small metabolites, other than cAMP, can alter acetohydroxy acid synthase gene expression. PMID- 3004584 TI - Conserved restriction sites within the ribosomal RNA genes of vertebrates. AB - We have mapped the cleavage sites of four restriction enzymes which recognize six base sequences within the nuclear ribosomal (rRNA) genes of twelve vertebrates, including several placental mammals (Homo sapiens, man; Bos taurus, cow; Equus caballus, horse; Sus scofra, pig; Ovis aries, sheep; Rattus rattus, rat), a marsupial (Didelphis marsupialis, opossum), a bird (Gallus domesticus, chicken), an amphibian (Xenopus laevis), a reptile (Alligator mississipiensis), a bony fish (Cynoscion nebulosus, sea trout), and a cartilagenous fish (Carcharhinus species, requiem shark). These animals represent a span of approx. 400 million years of evolutionary divergence. Our data identify restriction sites in the rRNA genes which are highly conserved among higher vertebrates and therefore are likely to be in functionally important regions. Additionally, the restriction enzyme sites identified will be useful in cloning and sequencing the rRNA genes in any vertebrate. Finally, the consistent size and conserved sequence homology suggests that these rRNA gene segments will be useful as internal controls in hybridization experiments involving other genomic regions in vertebrates. PMID- 3004585 TI - Purification and characterization of ATP:AMP phosphotransferase from Mycobacterium marinum. AB - ATP:AMP phosphotransferase (EC 2.7.4.3) (adenylate kinase) has been purified 1746 fold from Mycobacterium marinum (ATCC 927) by successive column chromatography on DEAE-cellulose (DE-53), Reactive Blue agarose, Sephadex G-75, hydroxyapatite and, finally, DEAE-Sephadex A-50. The final enzyme preparation had a specific activity of 576 mumol/min per mg protein with an overall yield of 51%. The preparation was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was estimated to have an Mr of 29500 and an isoelectric point of 6.7, properties which generally resemble those of the mitochondrial enzyme. Indeed, the two enzymes failed to separate when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The extinction coefficient (at 276 nm) was calculated to be 3.114 X 10(4) M-1 X cm-1 and E1%1cm = 10.556. Adenylate kinase was present at a concentration of 0.06 mg/g (wet weight) bacteria. Enzyme was stable for months in 60% glycerol in the freezer; at 4 degrees C, less than 5% of the activity was lost over a 7 day period. PMID- 3004586 TI - Microenvironment around the essential cysteine residues in chicken liver fructose 1,6-bisphosphatase as analyzed by ESR spin labelling. AB - Chemical modification and electron spin resonance spectroscopy (ESR) spin labelling techniques have been employed to investigate the local environment of the essential sulfhydryl groups of chicken liver fructose-1,6-bisphosphatase. The results demonstrate the presence of two distinct classes of sulfhydryl groups in this enzyme. The first class react preferentially with iodoacetate and its spin labelled derivative, and this results in an increase in catalytic activity, while the second class react preferentially with N-ethylmaleimide and its spin-labelled derivative, and this leads to a decrease in catalytic activity. The ESR spectral data strongly suggest that the first class of sulfhydryl groups are located in a deep cleft of the enzyme molecule, while the second class of sulfhydryl groups are located in a shallow crevice. The environment of the second class of the sulfhydryl groups appears to undergo a significant change after the modification of the first class of sulfhydryl groups by iodoacetate. PMID- 3004587 TI - Spin-density distribution in the [FeO2]-complex. Electron spin resonance of myoglobin single crystals. AB - X-irradiated oxymyoglobin (MbO2) exhibits electron spin resonance (ESR) spectra at 77 K due to two distinct [FeO2]-centers formed by electron addition to the dioxygen. In single crystals with 17O and 57Fe isotope enrichment in the heme ligand complex, the full set of spectral parameters is analyzed for one of the centers (gmax = 2.23, gint = 2.13, gmin = 1.97; HOmax = 2.66 mT, HOint = 1.61 mT, HOmin = 0.57 mT; HFe max = 1.62 mT, HFe int = 0.57 mT, HFe min = 0.49 mT) and the iron-dioxygen spin-density distribution and bonding geometry is derived. The g tensor is evaluated for the second species at 77 K (gmax = 2.25, gint = 2.11, gmin = 1.95). Both centers transform into secondary species at 180 K for which the g-tensor elements are analyzed (gmax = 2.31, gint = 2.18, gmin = 1.93; gmax = 2.35, gint = 2.21, gmin = 1.91). PMID- 3004588 TI - Role of apolipoproteins in cellular cholesterol efflux. AB - The effects of serum apolipoproteins, particle size and concentration on the effectiveness of phosphatidylcholine (PC)-containing acceptor particles in causing release of cholesterol from cells growing in culture have been investigated. The acceptor particles were prepared by detergent-dialysis procedures and were either egg PC small unilamellar vesicles (SUV) or discoidal complexes of egg PC with apoproteins from human high-density lipoprotein (HDL). Gel filtration chromatography was employed to isolate particles of defined composition and size. The half-times (t 1/2) for the unidirectional efflux of cholesterol from cells prelabeled with [3H]cholesterol were measured as a function of acceptor PC concentration in the extracellular medium. HDL apolipoprotein-egg PC discoidal complexes at 100 micrograms PC/ml gave the following t 1/2 values when incubated with rat Fu5AH hepatoma, human HepG2 hepatoma, human GM3468 skin fibroblast, L-cell and mouse J774 macrophage-tumor cells: 11 +/- 2, 22 +/- 5, 84 +/- 18, 17 +/- 2 and 32 +/- 6 h, respectively. Equivalent experiments using purified apolipoprotein A-I or the total apolipoprotein C fraction to form the egg PC complexes showed that the t 1/2 values for the hepatoma cells were unaltered. However, with the fibroblasts, L cells and J774 macrophages, the apolipoprotein C complexes gave significantly longer t 1/2 than complexes of egg PC with either apolipoprotein A-I or HDL apolipoprotein which gave the same t 1/2. An analysis based on the theory of fast coagulation of colloid particles to describe collisions between desorbed cholesterol molecules and acceptor particles predicts that the dependence of t 1/2 for cholesterol efflux from a given cell to different acceptors should be normalized when the extracellular level of acceptors is expressed in terms of the product of the radius of the particle times the number concentration of acceptor particles. The decrease in t 1/2 for cholesterol efflux from fibroblasts when the egg PC acceptor was changed from an SUV to an apolipoprotein HDL discoidal complex is consistent with the above concepts. The primary effect of the apolipoproteins in promoting cellular cholesterol efflux seems to be the solubilization of PC so that the PC is present in the extracellular medium as many small particles. PMID- 3004589 TI - Studies on the binding and degradation of human very-low-density lipoproteins by human hepatoma cell line HepG2. AB - The regulation of the hepatic catabolism of normal human very-low-density lipoproteins (VLDL) was studied in human-derived hepatoma cell line HepG2. Concentration-dependent binding, uptake and degradation of 125I-labeled VLDL demonstrated that the hepatic removal of these particles proceeds through both the saturable and non-saturable processes. In the presence of excess unlabeled VLDL, the specific binding of 125-labeled VLDL accounted for 72% of the total binding. The preincubation of cells with unlabeled VLDL had little effect on the expression of receptors, but reductive methylation of VLDL particles reduced their binding capacity. Chloroquine and colchicine inhibited the degradation of 125I-labeled VLDL and increased their accumulation in the cell, indicating the involvement of lysosomes and microtubuli in this process. Receptor-mediated degradation was associated with a slight (13%) reduction in de novo sterol synthesis and had no significant effect on the cellular cholesterol esterification. Competition studies demonstrated the ability of unlabeled VLDL, low-density lipoproteins (LDL) and high-density lipoproteins (HDL) to effectively compete with 125I-labeled VLDL for binding to cells. No correlation was observed between the concentrations of apolipoproteins A-I, A-II, C-I, C-II and C-III of unlabeled lipoproteins and their inhibitory effect on 125I-labeled VLDL binding. When unlabeled VLDL, LDL and HDL were added at equal contents of either apolipoprotein B or apolipoprotein E, their inhibitory effect on the binding and uptake of 125I-labeled VLDL only correlated with apolipoprotein E. Under similar conditions, the ability of unlabeled VLDL, LDL and HDL to compete with 125I labeled LDL for binding was a direct function of only their apolipoprotein B. These results demonstrate that in HepG2 cells, apolipoprotein E is the main recognition signal for receptor-mediated binding and degradation of VLDL particles, while apolipoprotein B functions as the sole recognition signal for the catabolism of LDL. Furthermore, the lack of any substantial regulation of beta-hydroxy-beta-methylglutaryl-CoA reductase and acyl-CoA:cholesterol acyltransferase activities subsequent to VLDL degradation, in contrast to that observed for LDL catabolism, suggests that, in HepG2 cells, the receptor-mediated removal of VLDL proceeds through processes independent of those involved in LDL catabolism. PMID- 3004591 TI - Specific incorporation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid into phosphatidylcholine in human endothelial cells. AB - The incorporation of hydroxyeicosatetraenoic acids (HETEs) into cellular lipids was studied in cultures of human umbilical vein endothelial cells. 5-[3H]HETE was incorporated into the phospholipids (8%) and neutral lipids (15.5%). The uptake was at half maximum after 15 min and reached a plateau after 1 h. The incorporation occurred mainly into phosphatidylcholine (6.3%) with minimal uptake into phosphatidylserine and phosphatidylinositol (0.6%) or phosphatidylethanolamine (1.2%). There was no uptake of 12-[3H]HETE, 15-[3H]HETE or [3H]leukotriene B4 into phospholipids. Treatment of the phosphatidylcholine fraction with phospholipase A2 released 64% of the 5-[3H]HETE with 26% remaining in the lysophosphatidylcholine fraction. This indicates that the majority of the 5-HETE was in the sn-2 position. Unlabeled 5-HETE and arachidonic acid inhibited the uptake of 5-[3H]HETE into phosphatidylcholine with an ID50 of 2.5 and 1.25 microM, respectively. Stearic acid and 15-HETE were not effective inhibitors. Histamine, which activates phospholipases, increased the uptake of 5-[3H]HETE into phosphatidylcholine by 3-fold. Both 5-[3H]HETE and 12-[3H]HETE were incorporated into the neutral lipids of the cells. Analysis of the neutral lipid fraction revealed that 5-[3H]HETE was incorporated into mono-, di- and triacylglycerols but not cholesterol esters. Incorporation of 5-HETE into cellular lipids reduced histamine- and arachidonic acid-stimulated synthesis of 6 ketoprostaglandin F1 alpha and prostaglandin E2 in a concentration-related manner. Angiotensin I converting enzyme activity was not changed. Thus, 5-HETE is incorporated specifically into phosphatidylcholine and glycerol esters of human endothelial cells and this incorporation inhibits prostaglandin synthesis in these cells. PMID- 3004590 TI - CTP:phosphocholine cytidylyltransferase in intestinal mucosa. AB - CTP:phosphocholine cytidylyltransferase is thought to be a rate-limiting enzyme in phosphatidylcholine synthesis. This enzyme has not been well studied in intestine. We found that activity was greater in the non-lipid stimulated state (cytosolic form of the enzyme) than any previous tissue investigated (2.7 nM/min per mg protein). On addition of lysophosphatidylethanolamine, the enzyme only increased in activity 2.4-fold which is less than any previously reported tissue on lipid stimulation. As compared to liver, the enzyme was resistant to inhibition by chlorpromazine (gut, 100% activity remaining at 80 microM; 14% in liver). Tetracaine and propranolol were found to be impotent as inhibitors of the intestinal enzyme. Octanol-water partitioning showed that both chlorpromazine and tetracaine were hydrophobic, propranolol was not. pKa studies demonstrated that at the reaction pH, chlorpromazine would be uncharged. Physiologic experiments in which de novo phosphatidylcholine synthesis was either stimulated by bile duct fistulization and triacylglycerol infusion or suppressed by including phosphatidylcholine in a lipid infusion demonstrated that the enzyme (cytosolic enzyme) responded by decreasing Vmax but that the Km remained the same. In sum, these studies suggest that CTP:phosphocholine cytidylyltransferase in intestine is unique as compared to other tissues and that its response to a physiological stimulus is counter to that which would be adaptive. PMID- 3004592 TI - Effects of different nutritional conditions on chick liver mevalonate-activating enzymes. AB - The response of mevalonate kinase, mevalonate-5-phosphate kinase and mevalonate-5 pyrophosphate decarboxylase of chick liver to different dietary situations has been investigated. Fasting inhibited mevalonate kinase and mevalonate-5 pyrophosphate decarboxylase activities, while mevalonate-5-phosphate kinase remained practically unaltered. Refeeding after 72 h of starvation restored mevalonate kinase activity to normal levels after 120 h of refeeding. Likewise, decarboxylase activity reached normal levels at 72 h of refeeding the standard diet and slightly supranormal levels after 120 h. In addition, the sequential response of the three enzymes to a high cholesterol diet was followed throughout a 120 h period. Feeding a 5% cholesterol diet to 13-day-old chicks previously fed with a standard diet from hatching reduced considerably the activity of mevalonate-5-pyrophosphate decarboxylase, while the kinases were less affected. The present results support the idea of a coordinate regulation of the enzymes implied in cholesterol biosynthesis and suggest that mevalonate-5-pyrophosphate decarboxylase may play a significant role in this regulation. PMID- 3004593 TI - An improved preparation of phosphatidylglycerophosphate. AB - Phosphatidylglycerophosphate is not normally accumulated in the reaction mixture. It was found that octyl-beta-D-glucopyranoside greatly stimulated the synthesis and inhibited the degradation of phosphatidylglycerophosphate. The phenomenon could be used for the preparation of phosphatidylglycerophosphate from CDPdiacylglycerol and the sn-glycero-3-phosphate with the crude membrane enzyme preparation. The optimal concentration of octyl-beta-D-glucopyranoside to be used for such a purpose was 24.5 mM. PMID- 3004594 TI - Leukotrienes stimulate phosphatidylcholine secretion in cultured type II pneumocytes. AB - We previously reported that arachidonic acid stimulates secretion of phosphatidylcholine in cultures of type II pneumocytes and, based on studies with cyclooxygenase and lipoxygenase inhibitors, suggested that this effect was mediated by lipoxygenase products of arachidonic acid metabolism (Gilfillan, A.M. and Rooney, S.A. (1985) Biochim. Biophys. Acta 833, 336-341). We have now examined the effect of leukotrienes on phosphatidylcholine secretion in type II cells as well as the effect of a leukotriene antagonist, FPL55712, on the stimulatory effect of arachidonic acid. Leukotrienes C4, D4 and E4 stimulated phosphatidylcholine secretion and this effect was dependent on concentration in the range 10(-12)-10(-6) M. Leukotriene E4 was the most stimulatory, followed by D4 and C4. Leukotriene B4 had no effect. Incubation of the cells with 10(-7) M leukotriene E4 for 90 min resulted in a 107% increase in the rate of phosphatidylcholine secretion. Incubation with 10(-6) M leukotrienes D4 and C4 for the same period resulted in 81% and 63% stimulation, respectively. The leukotrienes had no effect on cellular phosphatidylcholine synthesis or on lactate dehydrogenase release. The stimulatory effects of leukotrienes E4 and D4 were abolished by FPL55712. Similarly, the stimulatory effect of 6 X 10(-6) M arachidonic acid on phosphatidylcholine secretion was reduced from 74% to 25% by 10(-5) M FPL55712. Thus, the stimulatory effect of arachidonic acid on surfactant phospholipid secretion in type II cells is mediated at least in part by leukotrienes. PMID- 3004595 TI - NADP phosphatase as a marker in free-flow electrophoretic separations for cisternae of the Golgi apparatus midregion. AB - Based on cytochemical analysis, the enzyme NADP phosphatase is most concentrated in the so-called intercalary cisternae from the mid-region of the Golgi apparatus stack. Using free-flow electrophoresis to separate different Golgi regions of rat liver Golgi apparatus, the NADP phosphatase activity, based on estimation of the rate of release of inorganic phosphate from NADP under standard conditions, was similarly localized to membrane fractions from the center of electrophoretic separations. Peak specific activities for both a putative cis marker (NADH cytochrome c reductase) and an established trans marker (galactosyltransferase) coincided with minima in NADP phosphatase activity, in agreement with the cytochemical observations. The pattern of distribution of enzyme activity for NADP phosphatase differed from that of both acid phosphatase and glucose-6 phosphatase. The pH optimum was 5.0, the Km for NADP was 0.6 mM and a corresponding production of NAD and inorganic phosphorus was shown. Taken together with other markers for free-flow electrophoresis separation, the NADP phosphatase will provide considerable utility as a specific marker to help identify intercalary cisternae of the mammalian Golgi apparatus and to monitor electrophoretic separations. PMID- 3004596 TI - Contamination of commercial preparations of calmodulin by phospholipase A2. AB - In the course of studies of the possible regulation of cellular phospholipase A2 activities by calcium and calmodulin, it was observed that some of the commercial preparations of calmodulin contained significant phospholipase A2 activity. Six commercially available calmodulin sources were compared for the presence of contaminating phospholipase A2 activity, relative purity by SDS-gel electrophoresis, and relative biological activity in stimulating calmodulin deficient phosphodiesterase. One of the commercial calmodulin sources contained a relatively high specific phospholipase A2 activity (1.30 +/- 0.11 nmol [1 14C]arachidonic acid released/mg protein per h) and yielded two major bands in SDS-gel electrophoresis. Two of the calmodulin sources tested were relatively free of phospholipase A2 activity, were quite pure (one band on SDS-gel) and had high biological activity in stimulating calmodulin-deficient phosphodiesterase. Thus, investigators using commercially available preparations of calmodulin should be aware of the contamination of some of these sources by phospholipase A2 activity. These findings may be of importance to investigators considering the role of calmodulin in activating a variety of calcium-dependent enzymes, including phospholipase A2. PMID- 3004597 TI - Tubulin polymerization with ATP is mediated through the exchangeable GTP site. AB - Glycerol-induced tubulin polymerization supported by non-guanine nucleotides was examined. The electrophoretically homogeneous tubulin was devoid of nucleoside diphosphate kinase activity and 95% saturated with exchangeable GDP and nonexchangeable GTP. All purine ribonucleoside 5'-triphosphates were active but no polymerization occurred with CTP or UTP. All polymerization reactions, as a function of nucleotide concentration, were similar: above a minimum (threshold) concentration, as the amount of nucleotide increased the reaction became progressively more rapid and extensive with a progressively shorter nucleation period. Threshold concentrations of ATP, XTP, ITP and GTP were 0.6 mM, 0.3 mM, 30 microM and 7 microM, respectively. Most ribose- and polyphosphate-modified ATP analogs also supported polymerization at high concentrations, but the activity of these analogs relative to ATP was very similar to the activity of cognate GTP analogs relative to GTP. Polymerization with ATP was associated with an ATPase reaction. ATP hydrolysis was potently inhibited by GDP and GTP and altered by antimitotic drugs in parallel with the effects of these agents on GTP hydrolysis. Substantial amounts of [8-14C]GDP bound in the exchangeable site of tubulin were displaced during polymerization with GTP or ATP, but much higher concentrations of ATP were required for equivalent displacement of the tubulin-bound GDP. Polymerization with GTP or ATP was inhibited in a qualitatively similar manner by GDP, with increasing concentrations of GDP causing a progressive prolongation of the nucleation period and reduction in reaction rate and extent. However, complete inhibition of polymerization required that GDP:GTP much greater than 1, but that GDP:ATP much less than 1. Inhibition appeared to be primarily competitive, since with higher triphosphate concentrations higher GDP concentrations were required for comparable inhibition. We conclude that ATP effects on tubulin polymerization are mediated through a feeble interaction at the exchangeable GTP site. PMID- 3004598 TI - The action of pulsed magnetic fields on cyclic AMP levels in cultured fibroblasts. AB - Pulsed magnetic fields, similar to those used clinically to promote bone repair, have been applied to cultured fibroblasts obtained from chick embryo tendons and rabbit bone marrow. Cyclic adenosine monophosphate (cAMP) levels were found to be lower in field-treated cultures in response to hormones such as prostaglandin E2 and isoproterenol, and the fibroblasts appear less sensitive to environmental perturbation prior to hormone incubation. We propose that the adenylate cyclase complex is temporarily inactivated by prolonged exposure to pulsed magnetic fields, and that this effect might be analogous to desensitisation phenomena. PMID- 3004600 TI - A 200 kDa surface glycoprotein of human fibroblasts encoded by a gene on chromosome 11 is regulated by cyclic AMP. AB - A gene mapped to the long arm of human chromosome 11 was previously characterized to code for a cell-surface glycoprotein of an apparent molecular mass of 200 kDa in fibroblasts. We now report that this surface protein is expressed in an increased amount when human fibroblasts or human X Chinese-hamster hybrid cells containing human chromosome 11 are treated with 1 mM dibutyryl cyclic AMP. A detailed analysis of this phenomenon of induction was performed using a long established, stable cell line of human X Chinese-hamster hybrid cells, J1 clone, which contained human chromosome 11 and expressed the 200 kDa glycoprotein of human origin. It was shown that the 200 kDa protein of J1 cells was inducible with 1 mM dibutyryl cyclic AMP, 10 mM unmodified cyclic AMP, 0.2 mM 8-para chlorophenylthio cyclic AMP or 1 nM cholera toxin. Induction was potentiated by the addition of a phosphodiesterase inhibitor, Ro 20-1724. These results are consistent with the finding in human fibroblast, confirming that human fibroblasts express a 200 kDa glycoprotein on their surface which is regulated by intracellular concentrations of cyclic AMP. The regulation may be at the level of transcription since the addition of actinomycin D prevented the induction. PMID- 3004601 TI - Effect of hypothyroidism on the cytosolic free Ca2+ concentration in rat hepatocytes during rest and following stimulation by noradrenaline or vasopressin. AB - The mean resting concentration of cytosolic free Ca2+ [( Ca2+]i) in parenchymal liver cells, as determined with the intracellular Ca2+ indicator quin2, was lowered by about 30% in hypothyroidism (0.17 microM vs. 0.27 microM in normal cells). The [Ca2+]i level in hypothyroid cells at 10 s following stimulation by noradrenaline (1 microM) was about 64% lower than in normal cells (0.33 microM vs. 1.0 microM). The response to noradrenaline in hypothyroid cells was slower in onset (significant at 5 s vs. 3 s in euthyroid cells), and the maximum of the initial [Ca2+]i increase was reached later (14 s vs. 8 s in normal cells). In hypothyroid hepatocytes the initial increase was followed by a slow but prolonged secondary increase in [Ca2+]i. With vasopressin similar results were found. Chelation of extracellular Ca2+ with EGTA immediately prior to stimulation had no effect on the initial [Ca2+]i increase. Treatment with T3 in vivo (0.5 micrograms/100 g body weight daily during 3 days) completely restored the basal and stimulated [Ca2+]i in hypothyroid cells. The half-maximally effective dose of noradrenaline was the same in euthyroid and hypothyroid liver cells (1.8 X 10(-7) M). Hypothyroidism had no significant effect on the number of alpha 1-receptors determined by [3H]prazosin labeling in crude homogenate fractions, while the Kd for [3H]prazosin was 21% lower than in the euthyroid group. These results show that thyroid hormone has a general stimulating effect on intracellular Ca2+ mobilization by Ca2+-mobilizing hormones, probably at a site distal to the binding of the agonist to its receptor. The results also support our idea that thyroid hormone may control metabolism during rest and activation, at least partially, by altering Ca2+ homeostasis. PMID- 3004599 TI - Localization and role of pyruvate kinase isoenzymes in the regulation of carbohydrate metabolism and pyruvate recycling in rat kidney cortex. AB - This work was performed to gain more information on the role of pyruvate kinase isoenzymes in the regulation of renal carbohydrate metabolism. Immunohistochemically, pyruvate kinase type L is shown to be localized in the proximal tubule of the nephron and pyruvate kinase type M2 in the distal tubule and the collecting duct. a tight relationship between gluconeogenesis and pyruvate recycling was found. The rate of gluconeogenesis (8 mumol/g wet wt. per 30 min) was of the same order of magnitude as the rate of pyruvate recycling (10.92 mumol/g wet wt. per 30 min). Stimulation of gluconeogenesis from 20 mM lactate in kidney cortex slices of 24-h-starved rats by dibutyryl-cAMP, alanine and parathyroid hormone was connected with a decrease in pyruvate recycling; inhibition of gluconeogenesis due to a lack of Ca2+ in the incubation medium was linked with an increase in pyruvate recycling. The degradation of [6-14C]glucose to lactate, pyruvate, ketone bodies and CO2 and of [2-14C]lactate was unaffected by dibutyryl-cAMP, alanine, epinephrine, vasopressin or the omission of Ca2+ from the incubation medium. 1 mM dibutyryl-cAMP or 5 mM alanine did not alter the activities of oxaloacetate decarboxylase, 'malic' enzyme and malate dehydrogenase from rat kidney cortex. Since aerobic glycolysis in the distal tubules and the collecting ducts is not influenced by hormones, dibutyryl-cAMP and Ca2+, pyruvate kinase type M2 residing in this tissue is unlikely to be a control point of glycolysis. Since this tissue degrades only one-seventh of the glucose formed via gluconeogenesis, it does not contribute significantly to pyruvate recycling. Therefore, the decrease of pyruvate recycling in the presence of dibutyryl-cAMP and alanine in rat kidney cortex slices, leading to increased renal gluconeogenesis, has to be ascribed to the regulation of pyruvate kinase type L. PMID- 3004602 TI - Effects of the polyamine spermine on binding of follicle-stimulating hormone to membrane-bound immature bovine testis receptors. AB - The effect of the polyamine, spermine, on the interaction of human 125I-labeled FSH with membrane-bound receptors derived from bovine calf testes has been examined. Concentrations of spermine less than 0.01 M resulted in a slight but insignificant (P greater than 0.10) enhancement of FSH concentrations of 0.01 M and above caused a progressive reduction of FSH binding. Membrane receptors incubated in the presence of spermine at concentrations inhibitory to human 125I FSH binding (0.01-0.04 M) resulted in an 8-50% decrease in the apparent FSH receptor concentration and a 10-65% decrease in the affinity constant as determined by computerized analysis of the isothermic ligand-binding data. Within the temperature range 4-20 degrees C, simultaneous addition of spermine (0.025 M) increased the reversibility of human 125I-FSH binding approx. 10% (P less than 0.005). Delayed addition of spermine (0.01-0.04 M) resulted in a dose-related dissociation of human 125I-FSH already bound to its receptor (P less than 0.05). However, preincubation of membrane receptors with spermine (0.002-0.04 M) at 4 degrees C or 34 degrees C followed by washing and addition of human 125I-FSH, resulted in an increase in hormone binding (P less than 0.05) over that of controls. If membrane receptor was incubated at 34 degrees C with spermine in the absence of radioligand, the usual loss of hormone binding was reduced (P less than 0.05), while membrane receptor incubated with spermine at 4 degrees C exhibited hormone binding greater (P less than 0.05) than that observed before treatment. Thus, the mechanism of inhibition of human 125I-FSH binding to membrane receptors appears to be correlated with an increase in reversibility of the membrane receptor-human 125I-FSH complex and is expressed as a decrease in the calculated receptor concentration and affinity constant of that interaction. Second, spermine appears to stabilize the membrane receptor in the absence of ligand, presumably through a membrane effect. These data suggest that spermine, and possibly other polyamines, which are endogenous to eukaryotic cells and undergo increases in concentration following stimulation by trophic hormone may play a role in the modulation of the ligand-membrane receptor interaction, in part, through direct effects on the membrane and/or the receptor. PMID- 3004603 TI - Characterization of cyclic-nucleotide phosphodiesterase activities in resting and N-formylmethionylleucylphenylalanine-stimulated human neutrophils. AB - Cyclic nucleotide phosphodiesterase activities in human neutrophils were characterized. Neutrophil sonicates exhibited high-affinity and low-affinity cAMP phosphodiesterase activities, with apparent Km values of 1.9 microM and 112 microM, respectively. No cGMP phosphodiesterase activity was detected. Approx. 70% of cAMP phosphodiesterase activity measured at 1 microM cAMP was present in the soluble subcellular fraction, and the remainder was localized in the particulate fraction. Chromatography of the soluble subcellular fraction on DE-52 ion-exchange resin yielded a low-affinity cAMP phosphodiesterase activity and a high-affinity cAMP phosphodiesterase activity. The soluble high-affinity cAMP phosphodiesterase activity exhibited moderate calmodulin sensitivity. After incubation of intact neutrophils with N-formylmethionylleucylphenylalanine (fMet Leu-Phe), a 25-30% increase in the activity of the high-affinity cAMP phosphodiesterase activity was observed in the sonicate and in the soluble fraction. Maximal increases were achieved after 2 min of incubation and the increases persisted for at least 10 min. The increase in activity was independent of calmodulin and guanine nucleotide regulatory proteins. These results indicate that a soluble high-affinity cAMP phosphodiesterase comprises the majority of phosphodiesterase activity in neutrophils and that increases in this activity may contribute to the regulation of cAMP levels in neutrophils during activation. PMID- 3004604 TI - The Mr 17500 region of the A chain of urokinase is required for interaction with a specific receptor in A431 cells. AB - We have found the existence of specific receptors for the plasminogen activator, urokinase, in A431 human epidermoid carcinoma cells, cultures in plasminogen-free conditions. Two subsets of receptors have been recognized on the basis of 125I labelled urokinase binding analysis: about 1 X 10(3) high-affinity (Kd = 5.0 X 10(-11) M) and 1 X 10(5) low-affinity (Kd = 9 X 10(-9) M) receptors per cell. The electron microscopic observation of a urokinase: ferritin conjugate has shown single and clustered receptors at the cell surface. Down-regulation of the receptors (T1/2 = 3.77 h) follows the binding of urokinase. The binding does not involve an intact catalytic site and is inhibited by a monoclonal antibody against the Mr 17500 proteolytic fragment of the A chain of urokinase. PMID- 3004605 TI - Effect of angiotensin II infusion on glomerular angiotensin II receptor in rats. AB - Previous studies by other investigators have shown that sodium depletion causes down-regulation of angiotensin II receptors in renal glomeruli, which is ascribed to elevation of circulating angiotensin II levels. The present study was designed to determine whether elevation of circulating angiotensin II levels alone can down-regulate its own receptors without changes in the sodium balance. Rats were infused with angiotensin II at 50 ng/min intraperitoneally for 24 h or 7 d, then glomeruli were isolated by a mechanical sieving technique and used in a radioreceptor assay for angiotensin II. Angiotensin II infusion for 24 h and for 7 d both significantly suppressed plasma renin activity, and elevated plasma angiotensin II level (3-fold) but did not affect the plasma sodium and potassium or differences between dietary intake and urinary excretion of these electrolytes. By Scatchard analyses significant down-regulation of angiotensin II receptor number was demonstrated in renal glomeruli derived from rats infused with angiotensin II for either 24 h (32% decrease from the control value) or 7 d (37% decrease). No significant changes in receptor affinity were observed after 24 h of angiotensin II infusion, although the infusion for 7 d slightly but significantly decreased Kd (to 2.57 +/- 0.08 nM from 3.17 +/- 0.19, P less than 0.01). From these results, we conclude that circulating angiotensin II level itself can regulate the number of its own receptors in renal glomeruli even in the absence of changes in the sodium balance. PMID- 3004606 TI - Direct demonstration of intrinsic follicle-stimulating hormone receptor-binding activity in acid-treated equine luteinizing hormone. AB - After dissociating equine gonadotropins as a function of time at pH 3, we examined them by radioligand assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis under nondissociating conditions (low, 0.1% SDS). Equine follicle stimulating hormone (FSH) rapidly lost its receptor-binding activity, and low SDS polyacrylamide gels demonstrated dissociation into subunits. Maximum dissociation occurred after 20-30 min of pH 3 incubation. Equine luteinizing hormone (LH), however, retained most biologic activity and was largely intact after 72 h of pH 3 incubation. Dose-response curves of acid-treated equine LH and FSH and intact equine LH and FSH were compared in five types of radioligand receptor assays. LH and FSH receptor-binding activities of equine LH were unaffected by pH 3. Equine LH showed 19- and 32-times more activity in the rat testis FSH assay than it did in chicken or horse FSH assays, respectively, directly demonstrating the intrinsic FSH receptor-binding activity of equine LH and the relative lack of specificity for these hormone preparations of the rat FSH receptor. Acid-treated 95% of its biologic activity in FSH assays. In LH assays, the slight (0.2%) activity of equine FSH was relatively unaffected by acid treatment, suggesting that contamination by equine LH accounts for this activity. PMID- 3004607 TI - [Efficiency of associative memory inherent in post-tetanic potentiation]. AB - An associative memory is modeled in networks of cells that are assumed to have the short-term plasticity of the neuromuscular junction of the frog. The data relating synaptic transmission efficiency and stimulation frequency for post tetanic potentiation of the neuromuscular junction are represented by polynomial expansions. Simulation of storage and retrieval demonstrates that functional associative memory is feasible based on this particular synaptic plasticity. Retrieval reaches a maximum efficiency at a delay of three minutes after storage and is lost after about 9 min. The signal to noise ratio of the retrieved pattern drops steadily as additional associations are stored in memory but retrieval appears to be possible with up to four stored associations. Although the data are derived from synapses not normally proposed as a basis for memory functions, the results here will generalize to other synaptic junctions located more centrally that have similar characteristics. This simulation technique allows the efficiency of associative memory based on various types of synaptic plasticity to be evaluated. PMID- 3004608 TI - [Effect of a highly dispersed zinc powder on the cAMP phosphodiesterase activity in mouse thymocytes]. AB - The inhibition of cAMP phosphodiesterase activity by high dispersed zinc powder and zinc ions has been found in vitro experiments. The enzyme activity dependence on concentration of inhibitors is characterized by sigmoidal curve with Hill coefficients 1,8-2,1 and 1,2-1,3 correspondingly that may indicate the presence of positive cooperativity on inhibitor in enzyme. Magnesium ions influence the inhibited effect of zinc ions and the preparation of high dispersed zinc powder. PMID- 3004609 TI - ACTH in plasma and CSF in the rhesus monkey. AB - Adrenocorticotrophic hormone (ACTH) and other biosynthetically related peptides are found both in the brain and peripherally, but the function and regulation of these substances differ in the brain and in the periphery. It has been suggested that measurement of peptide hormones in the cerebrospinal fluid (CSF) might provide information relevant to the diagnosis and pathophysiology of neurological and psychiatric illnesses. We report experiments using a rhesus monkey model to evaluate parameters affecting CSF ACTH concentrations. We found that CSF ACTH concentrations follow a diurnal rhythm that is markedly different from that in plasma, concentrations of ACTH in monkey CSF, but not in plasma, increased significantly after 4 days of social separation, and CSF ACTH concentrations did not change after hypophysectomy. These results suggest that CSF ACTH concentrations reflect the activity of brain and not peripheral ACTH systems. PMID- 3004610 TI - Heterogeneity in anorexia nervosa. PMID- 3004611 TI - ECT, opioid system, and motor response in Parkinson's disease . PMID- 3004612 TI - Seasonal variation of platelet serotonin uptake and 3H-imipramine binding in normal and depressed subjects. AB - Density of 3H-imipramine binding sites and serotonin (5-HT) uptake in blood platelets were repeatedly recorded in normal controls (n = 9) and depressed patients (n = 7 for the imipramine binding assay and n = 4 for the serotonin uptake) over a 1-year period. The study demonstrated a striking seasonal variation of both parameters in both groups, with lower values in winter and spring than in summer and fall. No difference in the density of 3H-imipramine binding sites was found between the two populations throughout the year, but serotonin uptake was significantly decreased in depressed patients in May and September. These results underscore the importance of studying controls and patients at the same time of the year. PMID- 3004613 TI - Deuterium oxide (D2O) enhances the photosensitivity of Stentor coeruleus. AB - Stentor coeruleus exhibits negative phototaxis and step-up photophobic response (avoiding reaction) to visible light (maximum at 610-620 nm in both responses). In the presence of deuterium oxide (D2O) the step-up photophobic response was markedly enhanced, whereas the phototactic orientation response was inhibited. The induction time for the step-up photophobic response was longer in D2O than in H2O, and the duration of ciliary reversal for the response was also longer in D2O than in H2O, indicating that certain steps of the sensory transduction chain are subject to solvent deuterium isotope effects. The enhancement of the step-up photophobic response in D2O was canceled by LaCl3, while the inhibition of the phototactic orientation response in D2O was partially removed by LaCl3, even though LaCl3 did not affect the phototactic orientation response. These results suggest that the sensory transduction mechanisms for the two photoresponses are different, although the photoreceptors (stentorin) are the same. PMID- 3004615 TI - Preparation of oxygen-18-labeled lipoxygenase metabolites of arachidonic acid. AB - Plasma pseudocholinesterase and porcine liver esterase were used to catalyse the incorporation of the stable isotope oxygen-18 into the carboxyl moiety of lipoxygenase metabolites of arachidonic acid. This simple method produces eicosanoid products containing two oxygen-18 atoms; but the enzymes studied were found to display large substrate specificity in the efficiencies at which oxygen 18 could be incorporated into the lipoxygenase metabolites. Furthermore, [18O2]LTB4 was found not to back exchange during in vitro incubation with human neutrophils. The methods involved for stable isotope incorporation are simple, efficient and produce highly enriched species in a short time. By varying the type of esterase, the amount of esterase or the length of incubation highly enriched species of all eicosanoids tested could be prepared. PMID- 3004614 TI - Oriented secondary structure in integral membrane proteins. I. Circular dichroism and infrared spectroscopy of cytochrome oxidase in multilamellar films. AB - The circular dichroism (CD) of cytochrome oxidase in solution indicates the presence of both alpha-helix (approximately 37%) and B-sheet (approximately 18%). In oriented films generated by the isopotential spin-dry method, the CD measured normal to the film shows a marked decrease in the negative bands at 222 and 208 nm, and a decrease and red shift in the positive band near 195 nm, relative to solution spectra. These features are characteristic of alpha-helices oriented with their helix axes along the direction of light propagation. A quantitative estimate of the orientation, based on the ratio of the rotational strengths of the 208-nm band in the film and in solution, leads to an average angle between the helix axis and the normal to the film, phi alpha of approximately 39 degrees. A method for analyzing infrared (IR) linear dichroism is developed that can be applied to proteins with comparable amounts of alpha-helix and beta-sheet. From analysis of the amide I band, phi alpha is found to lie between 20 and 36 degrees, depending on the angle that the amide I transition moment forms with the helix axis. A survey of the literature on the amide I transition moment direction indicates that a value of approximately 27 degrees is appropriate for standard alpha-helical systems, such as those in cytochrome oxidase. A larger value, near 40 degrees, is reasonable for systems that have distorted alpha-helices, as evidenced by amide I frequencies above 1,660 cm-1, as is the case of bacteriorhodopsin. This conclusion supports phi alpha approximately 36 degrees from IR linear dichroism, in agreement with the CD results. Linear dichroism in the amide I and amide II region indicates that the beta-sheet in cytochrome oxidase is oriented with the carbonyl groups nearly parallel to the plane of the membrane and the chain direction inclined at approximately 40 degrees to the normal. Comparison of these results with tentative identification of transmembrane helices from sequence data suggests that either some of the transmembrane helices are inclined at an unexpectedly large angle to the normal, or the number of such helices has been overestimated. Some putative transmembrane helices may be beta-strands spanning the membrane. PMID- 3004616 TI - Temperature-dependent effects of EDTA on the membrane glycoprotein IIb-IIIa complex and platelet aggregability. AB - In agreement with previous studies, we observed that incubation of washed human platelets with EDTA at 37 degrees C for short periods caused an irreversible loss of their aggregation response to adenosine diphosphate and markedly diminished their capacity to bind fibrinogen. AP-2 is a monoclonal antibody that reacts with a determinant specific to the glycoprotein (GP) IIb-IIIa complex. We now report that in a direct binding assay, the number of sites for AP-2 on platelets incubated with EDTA at 37 degrees C fell to approximately 30% of those present on control platelets. This effect of EDTA was not observed at room temperature. Analysis of the treated platelets by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed normal amounts of GP IIb and GP IIIa. However, studies using crossed immunoelectrophoresis with 125I-AP-2, 125I-Tab (anti-GP IIb), or 125I-AP-3 (anti-GP IIIa) in intermediate gels showed that at 37 degrees C, EDTA was inducing an irreversible change in GP IIb-IIIa complexes. A reduction in size and probable dissociation of the GP IIb-IIIa precipitate was accompanied by the appearance of precipitates having the characteristics of those given by free GP IIb and free GP IIIa and the location of a major new cathodal precipitate, which bound Tab and AP-3 but not AP-2. Membrane modifications associated with the loss of antigenic determinants on GP IIb-IIIa may explain EDTA-induced loss of platelet aggregability at 37 degrees C. PMID- 3004617 TI - Pathogenesis of B cell lymphoma in a patient with AIDS. AB - Lymphoma occurs at increased frequency in patients with the acquired immunodeficiency syndrome (AIDS). We studied, using serologic and molecular techniques, one such lymphoma for (a) evidence of infection with human T lymphotropic virus, type III (HTLV-III), and Epstein-Barr virus (EBV), (b) monoclonal rearrangement of immunoglobulin and T cell receptor genes, and (c) rearrangement of the c-myc oncogene. Immunoglobulin and T cell receptor gene studies demonstrated that the tumor was of monoclonal B cell origin. Similar to cases of Burkitt's lymphoma unrelated to AIDS, there were DNA sequences in the lymphoma that hybridized to EBV-specific probes and demonstrated evidence of c myc rearrangement. HTLV-III sequences were not detected in the malignant B cells. The pathogenesis of some B cell neoplasms in patients with the syndrome may involve transformation by EBV and deregulation of oncogene expression without direct infection of the malignant B cells by HTLV-III. PMID- 3004618 TI - Cytogenetic and immunophenotypic analysis of cell lines established from patients with T cell leukemia/lymphoma. AB - Cell lines were established from five patients with T cell malignancies. Two patients had T cell lymphoblastic lymphoma (T-LL), whereas three patients had T cell acute lymphoblastic leukemia (T-ALL). Both T-LL cell lines expressed cell surface antigens characteristic of midthymocytes (Leu 2, 3, 6+). One T-ALL cell line also expressed this immunophenotype, one expressed suppressor/cytotoxic antigens (Leu 2+; Leu 3, 6-), and one expressed antigens of a mature but uncommitted T cell (Leu 4+; Leu 2, 3, 6-). Cytogenetic analysis showed that each cell line had 46 chromosomes with pseudodiploidy. The three T-ALL cell lines had only a few chromosome changes; one cell line had one deletion, another had two deletions, and the third had a translocation and two deletions (including loss of part of 9p). In comparison, both T-LL cell lines had complex chromosome changes, including most notably a rearrangement of band 14q11.2. The immunophenotypes and chromosome breakpoints showed patterns of interlock between the T-LL and T-ALL cell lines because common abnormalities occurred at six distinct chromosome sites. Cell lines with limited and specific chromosomal abnormalities are important because they can provide the basic material for molecular genetic studies that could elucidate the genetic mechanisms involved in neoplasia. PMID- 3004619 TI - Spontaneous tumor cytolysis mediated by inflammatory neutrophils: dependence upon divalent cations and reduced oxygen intermediates. AB - The role of divalent cations and reactive products of the respiratory burst were investigated in spontaneous tumor lysis mediated by inflammatory neutrophils (PMNs). Murine peritoneal PMNs, obtained five hours after intraperitoneal injection of bacteria, conjugated and lysed teratocarcinoma cells in chromium release and single-cell cytotoxicity assays. The presence of extracellular magnesium was required and was sufficient for tumor cell binding to PMNs. Postbinding lytic events depended upon the simultaneous presence of extracellular calcium and magnesium. Catalase and superoxide dismutase inhibited postbinding lytic events, indicating that production of reduced oxygen moieties was important. Scavengers of hydroxyl radicals could inhibit tumor cell binding, but none could affect postbinding lytic events. Neither could inhibitors of myeloperoxidase decrease tumor lysis. The ability of conjugating PMNs to lyse their bound targets correlated with their reduction of nitro blue tetrazolium (NBT). Optimal concentrations of phorbol myristate acetate (PMA) markedly increased the NBT positivity of PMNs and the killing of bound tumor cells. Even with optimal stimulation of the respiratory burst, however, there was still a significant number (19%) of bound targets that escaped lysis, suggesting active resistance to oxygen-mediated tumor cell injury. PMID- 3004620 TI - Hepatocellular carcinoma and focal nodular hyperplasia associated with norethandrolone-therapy: a case report. AB - A 33-year-old woman was treated for severe aplastic anemia with norethandrolone over a period of four years, with a cumulative dose of 25 g. In the fifth year of therapy two intrahepatic tumors were detected and were classified as hepatocellular carcinoma and as focal nodular hyperplasia, respectively. PMID- 3004621 TI - Assessment of malignancy potential in so-called interval mammary carcinomas. AB - Forty-two so-called interval cancers of the breast were studied. The mammograms were reviewed and the tumors were classified as 14 'unrecognized', 9 'observer error', 1 'technical error' and 18 'true' interval cancers. By using a scoring system based upon histologic differentiation, axillary nodal status, estrogen receptor level, and nuclear DNA content, eight ductal carcinomas with high malignancy potential were identified. All of these tumors belonged to the group 'true' interval cancer. The data indicate that the mammographic subgroup 'true' interval cancer identifies the highly malignant tumors. However, even this strictly selected subgroup is heterogeneous since it also includes some tumors with low malignancy potential. PMID- 3004623 TI - Dependence of tetanic hyperpolarization on pump conductance in the cyanide treated Xenopus node. PMID- 3004622 TI - Cyclic nucleotide levels in human breast cancer and in rat mammary tissues during tumor development. AB - The levels of cyclic adenosine 3':5'-monophosphate (cAMP) and cyclic guanosine 3':5'-monophosphate (cGMP) were studied in dimethylbenz(a)anthracene (DMBA) induced mammary tumors of Sprague-Dawley rats and in human breast cancer. In the rat carcinomas, these levels were significantly lower than in non-malignant tissues when calculated on the basis of DNA content, but higher (cAMP) or equal (cGMP) when calculated on the basis of weight. In human breast cancer the cyclic nucleotide levels were higher than in non-malignant tissues according to both methods of calculation. No correlation was found in human carcinomas between the cyclic nucleotide levels and mitotic index, nuclear grade, tumor size, or lymph node involvement. The rat tumors were subclassified according to state of differentiation, mitotic index, and state of development. Not all the sub-groups had cAMP levels different from normal values. Differences in cAMP levels between the sub-groups could not be correlated with tumor growth rates and/or mitotic index. Thus, cyclic nucleotides may not be useful in prognosis of breast cancer. PMID- 3004625 TI - Adenoid cystic carcinoma of the prostate. PMID- 3004624 TI - T-lymphotropic retroviruses of non-human primates. PMID- 3004626 TI - Paget's disease of the nipple occurring after conservation management of early infiltrating breast cancer. PMID- 3004627 TI - Influence of local surgery and radiotherapy on the natural history of pleomorphic adenomas. AB - We have analysed the interval between first treatment and tumour recurrence in 65 patients with parotid pleomorphic adenomas which had recurred following local excision. Our results indicate that 5 years is an inadequate period of follow-up and that 10-20 years may be more realistic. Radiotherapy given to 17 patients after local excision of their tumours was found to have had no significant advantageous effect in terms of recurrence-free interval either before or following formal parotidectomy or in limiting the ultimate surgery required. Major complications directly attributable to radiotherapy developed in at least 3 of these 17 patients. Malignant transformation of the pleomorphic adenoma has occurred in three patients, two of whom had been subjected to radiotherapy. We advise that caution is exercised in the interpretation of results of local excision and radiotherapy in this disease. In view of the fact that an alternative and apparently superior treatment is available in the form of formal parotidectomy, we urge that this should be universally adopted for the management of both primary and recurrent pleomorphic adenomas. PMID- 3004629 TI - AIDS and lymphadenopathy. PMID- 3004628 TI - The surgeon and HTLV-III infection. PMID- 3004630 TI - Clinical investigations of lymphadenopathy, including lymph node biopsies, in 24 homosexual men with antibodies to the human T-cell lymphotropic virus type III (HTLV-III). AB - The findings of 27 lymph node biopsies performed on 24 homosexual patients with lymphadenopathy are presented. Six had acquired immune deficiency syndrome (AIDS) and 18 lymphadenopathy only, of whom one subsequently developed AIDS. All these patients had antibodies to the human T-cell lymphotropic virus type III (HTLV III) suggesting that HTLV-III is currently the commonest cause of lymphadenopathy in homosexual men. The histopathological findings of six of seven nodes from AIDS patients showed either follicular depletion alone or follicular and paracortical lymphocyte depletion. Nodes from four patients showed Kaposi's sarcoma, three of which also showed follicular hyperplasia. In two of these patients there were no cutaneous manifestations of this condition. One lymph node from a patient with persistent generalized lymphadenopathy (PGL) showed Mycobacterium tuberculosis. Six nodes from six other patients have had features of toxoplasmosis although there was no serological or clinical evidence of recent toxoplasma infection. The remaining 11 lymph nodes from patients with PGL and one node from a patient with transient lymphadenopathy, showed reactive follicular hyperplasia only. We conclude that homosexuals with lymphadenopathy who are HTLV-III antibody positive do not need a routine node biopsy unless an alternative diagnosis is strongly suspected. PMID- 3004631 TI - Surgical biopsy for persistent generalized lymphadenopathy. AB - Lymph node biopsy was performed in 39 homosexual men with unexplained persistent generalized lymphadenopathy (PGL). Thirty-seven (95 per cent) of these patients had antibodies to human T-lymphotropic virus type III (HTLV-III), at the time of biopsy. Histology in all but one showed only follicular hyperplasia, the exception showed caseating granulomata typical of tuberculosis. Clinical differentiation between lymphadenopathy associated with HTLV-III and other causes of generalized lymphadenopathy is difficult; however, the presence of antibodies to HTLV-III probably identifies patients in whom surgical biopsy will only occasionally reveal a specific histological diagnosis. It is suggested that the presence of antibodies to HTLV-III in patients with PGL justifies a more selective approach to lymph node biopsy. PMID- 3004632 TI - Strategy for lymph node biopsy in homosexual men suspected of having LAV/HTLV-III related disease. AB - Since January 1985 we have changed our policy regarding lymph node biopsy in male homosexuals presenting with lymphadenopathy. Before that date all such patients underwent biopsy if there was no apparent cause. We no longer perform biopsy in male homosexuals presenting with uncomplicated persistent generalized lymphadenopathy (enlargement to more than 1 cm of lymph nodes in more than one extra-inguinal site for more than 3 months with no apparent cause) provided that the patient is positive for the antibody to lymphadenopathy associated virus/human T-lymphotropic virus type III. Such a policy should reduce the need for open biopsy procedures in this group of patients. PMID- 3004633 TI - Experimental colorectal cancer: the relationship of diet and faecal bile acid concentration to tumour induction. AB - Epidemiological studies have consistently suggested an aetiological relationship between certain dietary constituents, faecal bile acid (FBA) concentration and colorectal cancer. This study was designed to examine the effect of the dietary manipulation of fat and fibre on tumour induction and on various faecal characteristics in Albino Swiss rats. A total of 232 animals were maintained on one of four different diets for 4, 20 and 28 weeks respectively. The diets were classified as high fat high fibre, low fat high fibre, high fat low fibre and low fat low fibre. The groups were further sub-divided according to the administration of systemic azoxymethane (10 mg/kg per week) or saline over 12 consecutive weeks. The high fat low fibre diet was associated with the highest risk for tumour production and the low fat high fibre diet with the lowest risk. Statistically significant differences between all the diets were noted with the exception of a comparison between the high fat high fibre and low fat high fibre diets. The highest total concentration of free FBA was found in the faeces from animals fed low fibre containing diets. The results show a clear influence of both fat and fibre on tumour induction while, in this model, fibre was the principle determinant of faecal bile acid concentration. PMID- 3004635 TI - Glucomannan and risk of oesophageal obstruction. PMID- 3004634 TI - Distinct IgG recognition patterns during progression of subclinical and clinical infection with lymphadenopathy associated virus/human T lymphotropic virus. AB - Longitudinal IgG recognition patterns of viral proteins were studied in 15 men who had seroconverted for lymphadenopathy associated virus/human T lymphotropic virus (LAV/HTLV-III). Antibodies to the major viral core protein p24, which is a cleavage product of the gag gene encoded precursor protein pr55, appeared first. These were soon followed by antibodies to pr55 and more gradually by antibodies to the other gag gene encoded cleavage product p18, the env gene encoded transmembrane glycoprotein gp41, the env gene encoded glycoproteins gp65 and gp110, and the putative pol gene product p33. In 13 subjects who remained healthy the reactivity to the different proteins increased or stabilised with time, while in two men who developed acquired immune deficiency syndrome (AIDS) the reactivity, most noticeably to gag encoded proteins, diminished before or at the onset of symptoms. PMID- 3004636 TI - Rare cancers and specialist centres. PMID- 3004637 TI - Action potential characteristics of demyelinated rat sciatic nerve following application of 4-aminopyridine. AB - The sciatic nerves of rats were demyelinated by microinjection of lysophosphatidylcholine. A variety of abnormalities such as conduction slowing and block were present. Application of the potassium channel blocker 4 aminopyridine (4-AP) to the lesion site, led to an increase in area of the compound action potential recorded across the site of demyelination. Single axon recordings revealed three types of changes that may account for the 4-AP-induced increase in the compound response. One group showed broadening of the action potential. Other axons showed hyperexcitability following 4-AP, as manifest by spontaneous firing and multiple spike discharge following a single stimulus. In some of the axons studied, 4-AP led to overcoming of conduction block. Although many axons showed increased excitability properties in the presence of 4-AP, the frequency-following ability of the axons was reduced, and the absolute refractory period of the axons was increased. These results indicate that pharmacological blockade of potassium channels with 4-AP not only leads to action potential broadening in demyelinated axons, but to a variety of excitability changes. These heterogeneous effects of 4-AP should be considered in the rationale for its clinical use. PMID- 3004638 TI - Examination of spinal monoamine receptors through which brainstem opiate sensitive systems act in the rat. AB - The microinjection through stereotaxically implanted guide cannulae of morphine (5 micrograms/0.5 microliter) into the periaqueductal gray, the n. raphe magnus or the n. reticulogigantocellularis results in a significant elevation in the latency of a thermally evoked, spinally mediated reflex (tail-flick) and a supraspinally organized response (hot-plate). Spinal serotonin, noradrenalin, opiate and dopamine receptors were antagonized by the injection through chronically implanted intrathecal catheters of methysergide, phentolamine, naloxone and cis-flupenthixol, respectively. After a significant elevation of the tail-flick response latencies with intracerebral injections of morphine into the periaqueductal gray, the magnitude of the reversal produced by the intrathecally administered antagonists was phentolamine = methysergide much greater than naloxone = cis-flupenthixol = 0. After a significant elevation of the tail-flick response latency with intracerebral injections of morphine into the n. raphe magnus, the magnitude of the reversal produced by the intrathecally administered antagonist was methysergide greater than phentolamine greater than naloxone much greater than cis-flupenthixol = 0. After a significant elevation of the tail flick response latency was produced by the microinjection of morphine into the n. reticulogigantocellularis, the magnitude of the reversal produced by intrathecal antagonists was phentolamine greater than naloxone much greater than methysergide = cis-flupenthixol = 0. None of the intrathecal antagonists reversed the elevation of the hot-plate response latencies produced by morphine injections into the n. raphe magnus and n. reticulogigantocellularis injections. A significant, but clearly subtotal reversal of the elevated hot-plate response latencies produced by periaqueductal gray morphine was produced by intrathecal phentolamine and methysergide. It is concluded that discrete populations of brain opiate receptors in the periaqueductal gray, n. raphe magnus and n. reticulogigantocellularis differentially activate spinal monoamine and opioid receptors to modulate thermally evoked spinally mediated reflexes. The general failure of treatments which reverse the segmental reflex inhibition to reverse the hot-plate effects suggests that: other spinopetal pathways are operative; that descending pathways activated by these manipulations do not contribute to the analgesic effects of brainstem morphine; and/or that in addition to spinopetal modulation, brainstem opiate receptors modulate nociceptive transmission at the brainstem level. PMID- 3004639 TI - Frontal decortication and adaptive changes in striatal cholinergic neurons in the rat. AB - Interruption of the corticostriatal pathway by undercutting the cortex resulted in a reduction of glutamate uptake by 55% and in a depression of acetylcholine (ACh) synthesis by 30% in striatum after two postlesion weeks without affecting the content of ACh and choline, the specific binding of [3H]dexetimide to muscarinic receptors, the activity of choline acetyltransferase and the levels of noradrenaline, serotonin, dopamine and 3,4-dihydroxyphenylacetic acid. The influence of this excitatory pathway on striatal cholinergic neuropharmacology was investigated. It was found that the effect of a number of agonists (R apomorphine, bromocriptine, lisuride, quinpirole, JL-14389, 2-chloroadenosine, oxotremorine and methadone), capable of depressing cholinergic activity in the striatum through receptor-mediated responses--reflected as an increase in ACh content--is operative only when the corticostriatal pathway is intact. By contrast, antagonists capable of decreasing ACh content, i.e. the typical neuroleptics pimozide, haloperidol and the atypical ones clozapine, L-sulpiride, as well as the anti-muscarinic agent scopolamine, were not influenced by the lesion. The possibility that the lesion non-specifically damaged striatal cells on which the agonists, but not the antagonists acted was excluded by results showing, firstly, that the increase in striatal ACh elicited by the ACh precursor, choline, was not blocked by decortication, and secondly, that the degeneration of the corticostriatal neurons did not prevent the ACh-increasing effect of bromocriptine, a long-acting ergot alkaloid, when sufficient time was allowed for the drug to act. It was furthermore possible to restore the inhibitory action of apomorphine on cholinergic neurons either by short-term chemical lesion of the nigrostriatal dopaminergic input or by the administration of choline.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004640 TI - Dopaminergic facilitation of recovery from amygdaloid lesions which affect hypothalamic defensive attack in cats. AB - Influence of dopamine agonists, methamphetamine (MAT, 1 mg/kg, i.p.) and apomorphine (APO, 1 mg/kg, i.p.), on the effects of amygdaloid lesions on thresholds for the defensive attack behavior elicited by electrical stimulation of the ventromedial hypothalamic nucleus was studied in cats. The thresholds were measured in two situations, one with provocation by a human and the other without provocation. Electrolytic lesions of the basolateral part of the amygdala attenuated markedly facilitatory influences of the visual provocation on the thresholds, but subsequent administration of MAT or APO rapidly abolished the effects of the lesions. The effects of MAT lasted for at least 3.5 h while the effects of APO were of shorter duration. The results were discussed from the viewpoint of possible rapid compensation by the remaining intact tissue of the amygdala which was produced by excessive dopaminergic inputs to it. PMID- 3004641 TI - Zinc and paired-pulse potentiation in the hippocampus. AB - Zinc was added to the perfusate of rat hippocampal slices to determine the effect on the potentiation of evoked responses in subfield CA3 following paired-pulse stimulation of mossy fibres. Paired-pulse potentiation across a range of interpulse intervals and currents was significantly depressed by 50 and 500 microM zinc chloride. Additionally but only at the higher concentration, the amplitude of the first evoked potential was significantly increased. These results provide further evidence of a role for zinc in hippocampal neurotransmission. PMID- 3004642 TI - The effect of vanadate on Na+,K+-ATPase activity of mouse cerebral cortex during bicuculline-induced seizures. AB - The effect of bicuculline-induced seizures on Na+,K+-ATPase activity of mouse cerebral cortex homogenates, using two different procedures of sample preparation (freezing in situ or decapitation of animals without freezing) is described. Regardless of tissue treatment Na+,K+-ATPase activities during bicuculline induced seizures did not differ significantly from the appropriate controls when vanadate-free ATP was used as substrate. The response of Na+,K+-ATPase to K+ activation was also similar; the increase in potassium concentration from 2 to 20 mM caused a 33.0 and 32.3% increase of enzyme activity in cortical homogenates from control and convulsing mice, respectively. Vanadate added to the assay medium inhibited Na+,K+-ATPase activity in a dose-dependent manner; with both types of tissue treatment there was, however, a tendency towards lesser inhibition of the enzyme from convulsing mice and at 1 X 10(-7) M vanadate this difference, though slight, was statistically significant: -22.59 vs -27.55% (freezing) and -28.73 vs -38.42% (decapitation) for seizures vs controls, respectively. The reduced sensitivity of Na+,K+-ATPase towards vanadate inhibition in cortical homogenates prepared from mice with convulsions suggests that vanadate might play a role in the modulation of enzyme activity during seizures in vivo. PMID- 3004643 TI - A decrease in the number of GABAergic somata is associated with the preferential loss of GABAergic terminals at epileptic foci. AB - Previous studies have indicated that a loss of GABAergic terminals occurs at epileptic foci. The present study was undertaken to investigate if this loss is associated with a loss of GABAergic neuronal somata. Seven juvenile monkeys (M. mulatta) received alumina gel injections to the pre-central gyrus of the left cerebral hemisphere to produce epileptic foci. Four of these monkeys were chosen for further quantitative study. One was sacrificed prior to seizure onset ('pre seizure'), one had seizures for 3 days ('acute'), and two had a seizure record of one month ('chronic'). Sections of tissue from the epileptic cortex and from the contralateral, non-epileptic cortex were processed for glutamate decarboxylase (GAD) immunocytochemistry at the light microscopic level. Quantitative analysis revealed that a loss of GAD-positive neuronal somata ranging from 24 to 52% occurred at epileptic foci for all monkeys. This decrease was significant (P less than 0.01) for the two chronic monkeys. There was also a slight decrease in GAD positive neurons 1 cm distal to the focus ('parafocus') in the chronic monkeys, but not in the acute or pre-seizure animals. In addition, small GAD-positive somata (50-150 micron2) were more severely decreased in number at epileptic foci than larger ones (200-250 micron2). As an experimental control, an additional monkey was given a surgical lesion in area 4 of one cerebral hemisphere. It did not display seizure activity prior to sacrifice and did not show a loss of GAD positive neurons proximal to the control lesions. The results of this study indicate that a loss of GABAergic neuronal somata is associated with a loss of GABAergic terminals at epileptic foci, and that this loss may be more specific for the small GABAergic neurons. PMID- 3004645 TI - Astroglial contribution in human temporal lobe epilepsy: K+ activation of Na+,K+ ATPase in bulk isolated glial cells and synaptosomes. AB - Potassium activation of Na+,K+-ATPase within glial cells and synaptosomes, bulk isolated from temporal neocortices of 15 patients with hippocampal-amygdalar epilepsy were compared to two patients with extratemporal complex partial epilepsies and 8 post-mortem temporal neocortices from patients with no known neurological ailments. Temporal neocortices were obtained from 17 patients who had undergone 'en bloc' anterior temporal lobectomy. In 15 patients with hippocampal amygdalar epilepsy, enzymatic activities of glial fractions were lower than those shown in 2 control lobectomy specimens and post-mortem brains. Glial enzymes had no or poor activation when K+ concentrations increased from 3 to 18 mM. Two patients served as control lobectomy specimens since they had normal neuropathological studies, and electroclinical correlations indicated an extratemporal lobe origin for complex partial seizures. In these two control specimens, glial Na+,K+-ATPase activities were similar to normal animals or post mortem specimens. Na+,K+-ATPase activities were also slightly decreased in the synaptosomal fractions of patients with hippocampal-amygdalar epilepsy. K+ response of enzyme activities, however, corresponded to what had been described in controls, i.e. hyperbolic curves saturated at 3-6 mM K+. These results indicate that a defect in glial Na+,K+-ATPase exists in temporal neocortices of patients with hippocampal-amygdalar epilepsy. PMID- 3004644 TI - Comparison of antinociceptive action of morphine in the periaqueductal gray, medial and paramedial medulla in rat. AB - Microinjection of morphine (5 micrograms) through stereotaxically implanted microinjection cannulas into the periaqueductal gray (104 sites), medial (n. raphe magnus; 26 sites) and paramedial (n. reticulogigantocellularis; 49 sites) medulla resulted in an increase in the latency of supraspinally (hot-plate) and spinally (tail-flick)-mediated responses evoked by thermal stimuli. This effect of intracerebral morphine on both hot-plate and tail-flick was dose-dependent, and reversed by systemically administered naloxone as well as by naloxone administered by microinjection into the same site. On the basis of frequency of occurrence, time of onset and magnitude of effect of the minimum effective dose, we could demonstrate no difference between the efficacy of morphine acting at sites in the periaqueductal gray, n. raphe magnus or n. reticulogigantocellularis on the supraspinally mediated response. In all areas examined, morphine was able to produce the maximum elevation in response latency. The microinjection of morphine into the periaqueductal gray frequently produced a total block of the thermally evoked spinally mediated tail-flick reflex. Unlike the periaqueductal gray, the systems through which opiates act in the n. raphe magnus or the n. reticulogigantocellularis to suppress spinal reflex activity displayed a clear plateau in their physiological effects. Microinjections of morphine into the n. raphe magnus or n. reticulogigantocellularis never produced a complete block of the spinal reflex. Further increases in inhibition could not be achieved by either a 3-fold increase in dose or bilateral injections into the paramedial medulla. The failure to block spinal reflex activity often occurred at sites where morphine would completely block the hot-plate response. These observations indicate that opiate receptor-linked systems in the mesencephalon and medulla can significantly attenuate the coordinated escape behavior otherwise evoked by a high-intensity thermal stimuli. We find there is no difference in the physiological efficacy of morphine acting in those regions on supraspinally mediated measures of pain responding. The differential effect on spinally mediated reflex function suggests that these several opiate linked systems produce their effect by discriminable mechanisms. PMID- 3004646 TI - Histaminergic axons in the neostriatum and cerebral cortex of the rat: a correlated light and electron microscopic immunocytochemical study using histidine decarboxylase as a marker. AB - Histaminergic nerve fibers and their axonal varicosities in the neostriatum and cerebral cortex were light and electronmicroscopically examined by means of peroxidase-antiperoxidase immunocytochemistry with histidine decarboxylase (HDC) as a marker. A majority of HDC-like immunoreactive axonal varicosities observed in serial thin sections for electron microscopy exhibited no synaptic contacts in either the neostriatum or cerebral cortex. The remaining small proportion of immunoreactive axonal varicosities formed synaptic contacts with non immunoreactive dendritic shafts and spines. PMID- 3004647 TI - The action of oxygen and oxygen at high pressure on inhibitory transmission. AB - The effect of 100% oxygen at ambient pressure, 100% oxygen at 1.7 Atmospheres Absolute (ATA), 100% oxygen at 5.1 ATA, helium at 1.7 ATA and helium at 5.1 ATA on inhibitory synaptic transmission was studied using the lobster walking leg neuromuscular preparation. Exposure to 100% oxygen at ambient pressure, at 1.7 ATA or at 5.1 ATA produced a decrease in inhibitory transmission manifest as a fall in inhibitory synaptic conductance (Ginh). The largest decrease in Ginh was seen in 100% oxygen at ambient pressure, while a progressively smaller decrease was seen in 100% oxygen at 1.7 ATA and 5.1 ATA, respectively. Also associated with 100% oxygen at ambient pressure was the disappearance of inhibitory junction potentials. Pressurization with helium produced a fall in Ginh at 5.1 ATA but no change or a slight increase at 1.7 ATA. The action of either 100% oxygen at ambient and at 1.7 or 5.1 ATA or helium at 1.7 or 5.1 ATA was shown to be on presynaptic parameters since the percent decrease in Ro induced by exogenous application of gamma-aminobutyric acid (GABA), the inhibitory transmitter, was the same in either 100% oxygen at ambient pressure, 100% oxygen or helium at 1.7 ATA and 5.1 ATA. The similarity in action of oxygen to the action of isoniazid, a known glutamic acid decarboxylase (the enzyme that catalyzes the production of GABA) inhibitor in the same preparation suggests that one possible site of oxygen action is on GABA production. PMID- 3004648 TI - Astrocytes have both M1 and M2 muscarinic receptor subtypes. AB - In astrocytes, carbachol evoked the turnover of membrane inositol phospholipids prelabelled with [3H]inositol, as revealed by [3H]inositol phosphate accumulation in the presence of 5 mM lithium. This effect was blocked by atropine and by pirenzepine (IC50 2.2 nM and 56 nM, respectively). Carbachol partially attenuated the isoproterenol-stimulated cyclic adenosine 3',5'-monophosphate production in astrocytes by a direct effect on adenylate cyclase, an effect blocked by atropine and pirenzepine. These results suggest that astrocytes express muscarinic receptor subtypes. PMID- 3004649 TI - Differential effects of striatal injections of dopaminergic, cholinergic and GABAergic drugs upon swimming behavior of rats. AB - The present study provides a detailed report about similarities and dissimilarities between the effects of neostriatally applied dopaminergic (apomorphine, 250-300 ng; haloperidol, 250-500 ng), cholinergic (carbachol, 50 100 ng; scopolamine, 200-500 ng), and GABAergic (muscimol, 1-2 ng; bicuculline, 5 35 ng) drugs upon swimming of rats. The used swimming test consisted of 4 parts: (a) open-field test for analyzing drug-induced changes in normal behavior; (b) 'swimming without escape' test for analyzing drug-induced changes in the ability to switch from one type of behavior to another; (c) 'swimming with escape' test for analyzing drug-induced changes in the ability to switch from ongoing swimming behavior to climbing behavior by allowing the rats to escape via a rope; and (d) 'rope' test for analyzing drug-induced changes in the kind of contact behaviors needed to switch to the latter climbing behavior. In the open-field test the drugs produced neither abnormal behavior nor motor disturbances, which prevented the display of normal behavior in the remaining tests. Both apomorphine and carbachol produced identical effects in all tests. Muscimol produced overall effects which were not only opposite to those of apomorphine and carbachol, but also comparable to those of scopolamine. All effects elicited by apomorphine, carbachol and muscimol were antagonized by their corresponding antagonists: haloperidol, scopolamine and bicuculline respectively, whereas the effects of the latter were suppressed by their corresponding agonists. These data globally show that dopamine and acetylcholine act in the same direction but opposite to that of GABA as far as it concerns the regions investigated. The finding that haloperidol injected into the GABA target area produced effects which were not only similar to those of haloperidol injected into the dopamine target area, but also dissimilar to those of muscimol and bicuculline injected into the GABA target area, shows that the effects were drug-specific rather than region-specific. Though 3 distinct cholinergic regions were investigated, cholinergic-specific effects could only be elicited from one region, suggesting that the neostriatum is heterogeneous in this respect. Finally, well-delineated dissimilarities between haloperidol-, scopolamine-, and muscimol-treated rats were found in the rope test. These data show that behavior-relevant information transmitted by GABAergic drugs surmounted that transmitted by cholinergic drugs which, in turn, surmounted behavior-relevant information transmitted by dopaminergic drugs. PMID- 3004650 TI - Discordant changes between immunoreactive ACTH and beta-endorphin in rat brain and pituitary during early development. AB - Discordant changes in brain concentrations of immunoreactive (IR-) adrenocorticotropic hormone (ACTH) or beta-endorphin, peptides derived from pro opiomelanocortin (POMC), have been reported during the latter part of development and with subsequent aging. These changes were believed to be due to age-related alterations in the regulation of metabolism of POMC-related peptides. However, concentrations of IR-ACTH and IR-beta-endorphin have not been simultaneously studied during early development when many changes in brain development and behavior are occurring. To determine whether concentrations of IR-ACTH and IR beta-endorphin change during early development and whether changes are discordant, concentrations of IR-ACTH and IR-beta-endorphin were measured in several brain regions and pituitary in rats at 10, 29 and 80 days after birth. Whereas IR-ACTH in extrahypothalamic brain increased at day 29, and decreased at day 80, it did not change in hypothalamus and pituitary. Between day 10 and 29, IR-beta-endorphin rose in all brain regions, but subsequent changes in different tissues were variable at day 80. Because concentration changes can be mediated by alterations in one or more regulatory mechanisms, chromatographic profiles of hypothalamus and amygdala and molar ratios of all tissues were subsequently studied to give further insight into the mechanisms of the discordant changes. Molecular profiles of hypothalamic IR-ACTH and amygdalar IR-beta-endorphin exhibited lesser proportions of large molecular forms and greater proportions of ACTH and beta-endorphin during development. Molar ratios of IR-ACTH/IR-beta endorphin in all tissues and ratios of ACTH1-39/beta-endorphin in hypothalamus and amygdala changed during development. CONCLUSIONS: (1) changes in IR-ACTH and IR-beta-endorphin occur in rat pituitary and several brain regions at different ages during early development and are frequently discordant; (2) molecular profiles suggested that the activity of processing enzymes for POMC and its derivatives vary in hypothalamus and amygdala with respect to type of derivative, brain region, and developmental age; and (3) changes in some molecular profiles and changes in molar ratios of IR-ACTH/IR-beta-endorphin and ratios of ACTH1 39/beta-endorphin suggest that changes in processing and possibly changes in neurosecretion and degradation contribute to concentration changes independent of possible alterations in biosynthesis of POMC. PMID- 3004651 TI - Dorsal column postsynaptic neurons in the cat are excited by myelinated nociceptors. AB - Some (25-50%) dorsal column postsynaptic (DCPS) neurons respond only to innocuous mechanical stimuli; the remainder (50-75%) responds to both innocuous and noxious mechanical stimuli. Those that respond to noxious mechanical stimuli (pinch) are assumed to be excited by input from nociceptive primary afferents, but it is conceivable that their pinch-evoked responses are produced by the inadvertent activation of those low-threshold mechanoreceptive primary afferents that respond to stretching the skin. Because nociceptive primary afferents respond reliably to noxious heat and low-threshold mechanoreceptors do not, we tested DCPS neurons in the cat lumbar spinal cord with a series of noxious heat stimuli (48 degrees C or 50 degrees C-56 degrees C; 30 s duration). Seven of eight pinch-responsive neurons responded to noxious heat, but only after their receptive fields had been sensitized by prolonged or repeated heating. The results show that (1) many DCPS neurons in the cat are excited by nociceptive primary afferents and (2) these nociceptive afferents are probably myelinated high-threshold mechanoreceptors. PMID- 3004653 TI - Synaptic transmission of excitation from the retina to cells in the pigeon's optic tectum. AB - Intracellular recordings were used to study the synaptic excitation of optic tectum neurons in the pigeon. Electrical stimulation of both contralateral optic nerve and ipsilateral optic tract evoked in the tectal neurons EPSPs which in most cases were followed by an IPSP. An extrapolation procedure based on response latency was used to reveal that the EPSPs were mediated by way of mono-, di- and polysynaptic connections with the retinal endings. The laminar location of the recorded cells was estimated according to the field potential and the recording depth with the exception of one cell which was intracellularly stained with HRP. Monosynaptic EPSPs were recorded from cells in the retinorecipient region (sublayers IIa-f) as well as in the non-retinorecipient region (sublayers IIg-j and layer III) of the tectum, while di- and polysynaptic EPSPs were never recorded from the input layers. Tectofugal projections arise largely from layer III neurons. Thus, these results indicate that retinal excitation is transmitted to the output tectal cells by way of mono-, di- or polysynaptic pathways. The conduction velocities of most retinal fibers mediating the EPSP ranged from 4 to 22 m/s (average 12 m/s). However, in a number of retinal fibers the conduction velocities were in a faster range, up to 36 m/s. PMID- 3004652 TI - Somata-selective lesions induced by cobaltous chloride: a parametric study. AB - Repeated injections of cobaltous chloride destroy neuronal somata while sparing fibers-of-passage. This effect is dose-dependent and excess cobalt destroys fibers-of-passage as well as somata. By manipulating the concentration and volume of cobaltous chloride injected into rat lateral geniculate nuclei, we defined a range of parameters producing highly selective destruction of somata for regions 1 mm in diameter or smaller. PMID- 3004654 TI - Spinal noradrenergic neurotransmission and the analgesia induced by brief footshock. AB - Antinociception induced by brief footshock as well as by 5-methoxy-N,N dimethyltryptamine was antagonized by lesions of the descending bulbospinal noradrenergic (NA) pathways by intrathecal injections of 6-hydroxydopamine. The alpha 2-adrenoceptor antagonist, yohimbine, injected intrathecally also blocked both types of nociceptive effects in the tail-flick and hot-plate tests. 5 Methoxy-N,N-dimethyltryptamine (1 mg/kg) potentiated shock-induced antinociception and this potentiation was also antagonized by decreased NA neurotransmission. These findings suggest an important role for spinal NA innervation, and possibly alpha 2-adrenoceptors in antinociception induced by brief footshock and serotonergic receptor stimulation induced analgesia in rats. PMID- 3004655 TI - Noradrenergic stimulation of supraoptic neuronal activity and vasopressin release in vitro: mediation by an alpha 1-receptor. AB - Using the isolated rat hypothalamoneurohypophysial system, the effects of noradrenaline (NA) and some adrenoceptor agonists on vasopressin (VP) release and supraoptic neuronal activity were assessed in vitro. Extracellular action potentials driven antidromically by neural stalk stimulation were recorded while fractions of the perifusate were collected every minute for radioimmunoassay of VP. NA and the alpha 1-agonist phenylephrine stimulated VP release and elicited bursts in tuberal supraoptic neurons, whereas isoprenaline (a beta-agonist) and clonidine (an alpha 2-agonist) did not. The peak rate of VP release was reached shortly after the onset of burst discharge. when intraburst firing rate was highest, and both effects were dose-dependent when tested to different concentrations of phenylephrine. Thus VP secreting neurons are excited to release VP by alpha 1-adrenoceptor activation. PMID- 3004656 TI - Functional relations of the rodent claustral-entorhinal-hippocampal system. AB - Electrophysiological studies of the claustrum of rat demonstrate functional connections with the limbic system (dentate gyrus, hippocampus) via the entorhinal cortex. The pathways show reciprocal connectivity, a degree of topological organization and the capacity for a long-term response potentiation in the claustral projection to entorhinal cortex. The hypothesis that claustrum acts to functionally link neocortical regions with components of the limbic system is considered. PMID- 3004657 TI - Interaction of neuropeptides and biogenic amines on cyclic adenosine monophosphate accumulation in hypothalamic nuclei. AB - Neuropeptides and biogenic amines known to be present in neurons or afferent terminals in the paraventricular nucleus (PVH), supraoptic nucleus (SON) and/or lateral hypothalamus (LH) were added to small areas of these structures obtained by micropuncture and cyclic adenosine monophosphate (cAMP) levels were measured. cAMP accumulation occurred in PVH, SON and LH in response to neuropeptides of the secretin family, such as vasoactive intestinal peptide (VIP) and in response to catecholamines. Bradykinin, alpha-melanocyte-stimulating (alpha-MSH), luteinizing hormone-releasing hormone (LH-RH), oxytocin and carbamylcholine stimulated cAMP accumulation selectively in one or two of the above structures. Glucagon, cholecystokinin (CCK), somatostatin (SRIF), corticotropin-releasing factor (CRF), thyrotropin-releasing hormone (TRH), adrenocorticotropin (ACTH), melanocyte stimulating hormone (MSH), methionine enkephalin (Met-Enk), beta-endorphin, neurotensin, bombesin and angiotensin II did not effect cAMP levels while leucine enkephalin (Leu-Enk), arginine vasopressin and gamma-aminobutyric acid (GABA) elicited regionally selective decreases in basal levels of cAMP. When interactions between some of these compounds were measured, VIP and norepinephrine exerted a more than additive effect on cAMP elevation in the PVH, while the effect on cAMP of the SON and LH was additive. PMID- 3004658 TI - Effect in cat of locus coeruleus lesions on the response of cerebral blood flow and cardiac output to altered paCO2. AB - In pentobarbital-anesthetized cats, over arterial paCO2 values of 20-60 mm Hg, cerebral blood flow (CBF, Xenon) and cardiac output (CO, thermal dilution) show positively inflicted curves with slopes significantly greater than zero. To examine the role of the locus coeruleus (LC) in these responses, bilateral stereotactic thermo-coagulation lesions of the LC were made. The effect of lesions confirmed to involve the LC bilaterally (n = 10), were compared with the effects of misdirected lesions placed in the cerebellum and lateral to the LC (n = 10) and sham lesions (n = 10). Ten days after the lesioning procedure, the animals were re-anesthetized with pentobarbital and paCO2 response curves were determined for CBF and CO prior to and following intravenous administration of propranolol (1 mg/kg, i.v.). The results obtained with the sham-operated animals and the animals with lesions outside of the LC were indistinguishable. Bilateral LC lesions had no significant effect on normocapnic CBF as compared to control animals. They did, however, significantly reduce the slope of the CBF paCO2 response curve. Propranolol produced a significant reduction in CBF in lesioned and non-lesioned animals measured at all levels of pCO2 and did not alter the slope of the pCO2 response curve for any group as compared to predrug values. Bilateral lesions of the LC did not significantly alter either normocapnic CO or the slope of the CO-paCO2 relationship, but did reduce the elevation in mean arterial blood pressure otherwise observed during hypercarbia. Measurement of norepinephrine levels in cortex indicate a close correlation between the ability of the lesion to reduce norepinephrine content and produce the observed physiological effects. The results of these experiments suggest that the hypercapnic response of CBF, but not CO to arterial paCO2 is modulated by systems which traverse the dorsal brainstem. The role of the locus coeruleus catecholamine cell bodies in this effect, however, must be considered speculative until further transmitter-selective interventions are carried out. PMID- 3004659 TI - The effects of nerve section on the non-quantal release of ACh from the motor nerve terminal. AB - The spontaneous release of acetylcholine (ACh) from motor nerve terminals is now thought to occur by two mechanisms: (a) quantal release, giving rise to miniature endplate potentials; and (b) non-quantal release. In this study we have examined the effect of nerve section on spontaneous non-quantal ACh release, and have compared the time-course of cessation of non-quantal and quantal ACh release. Non quantal ACh release, measured by an electrophysiological technique, declined 4 h after nerve section to approximately 50% of the control value. At 8-10 h it briefly rose again, then gradually declined to undetectable levels. Spontaneous quantal release (frequency of miniature endplate potentials) in the same muscle fibers remained close to control levels for 8 h after nerve section, and also increased prior to failure. Decline of non-quantal ACh release appears to be the earliest change in the nerve terminal following nerve transection; it may therefore be relevant in understanding the effects of denervation on the consequent changes in muscle properties. PMID- 3004660 TI - The responses of simultaneously recorded Purkinje cells to the perturbations of the step cycle in the walking ferret: a study using a new analytical method--the real-time postsynaptic response (RTPR). AB - Experiments were performed in ambulating decerebrate ferrets to examine the activity of up to 6 simultaneously recorded Purkinje cells oriented sagittally during unperturbed and perturbed locomotion using a new analytical technique, the real-time postsynaptic response (RTPR), which permits a trial-by-trial assessment of the action of the recorded neurons on a simulated cerebellar nuclear cell. The data illustrate that the responsiveness of the Purkinje cells located in a specific region of lobules V and VI is most dramatically modulated by the perturbations of the locomotor cycle and that this responsiveness in the perturbed trials is related to the degree of synchrony of the activated climbing fiber inputs to the cells of the set. The data were interpreted as supporting the gain change hypothesis of climbing fiber function. PMID- 3004661 TI - Cyclic AMP-dependent protein phosphorylation is reduced in rat striatum after chronic ethanol treatment. AB - Endogenous protein phosphorylation by cyclic AMP-dependent protein kinase was found reduced in striatal membranes obtained from chronic ethanol-treated rats. Experiments using an exogenous substrate show that the decreased response is due to a deficiency in the phosphorylating activity of the cyclic AMP-dependent protein kinase and not to a lack of endogenous substrate for phosphorylation. PMID- 3004662 TI - Evidence for a catecholamine-mediated slow hyperpolarizing synaptic response in parasympathetic ganglia. AB - Stimulation of preganglionic nerve trunks in the presence of muscarinic and nicotinic cholinoceptor and purinoceptor antagonists produced a slow hyperpolarizing synaptic potential that was mimicked by exogenously applied norepinephrine. Both responses were blocked by yohimbine, an alpha-adrenoceptor antagonist, and enhanced by imipramine and cocaine, inhibitors of norepinephrine reuptake. These findings fulfill pharmacological criteria suggesting that norepinephrine is a neurotransmitter in cat bladder parasympathetic ganglia. PMID- 3004663 TI - Synaptic organization of inhibitory circuits in the pigeon's optic tectum. AB - The synaptic organization of inhibitory systems in the pigeon's optic tectum was studied with intracellular recording techniques. An extrapolation procedure based on response latency was used to determine the synaptic delay of the postsynaptic potentials (PSPs) and the velocity of conduction of the associated retinal axons. Tectal cells receive mostly disynaptic, trisynaptic or polysynaptic inhibition from retinal ganglion cells. However, evidence was found which together with previous studies raised the possibility of the existence of a direct inhibitory retino-tectal path. Our present results also suggest that inhibition is transmitted from the retina to the tectal cells by way of both, feedforward and feedback pathways. PMID- 3004664 TI - Neostriatal projections from individual cortical fields conform to histochemically distinct striatal compartments in the rat. AB - A number of recent studies have demonstrated two chemically distinct compartments in the neostriatum: opiate receptor-rich patches and a surrounding matrix. Using axonal transport and receptor localization techniques in the rat brain, we have found that striatal projections from architectonically distinct cortical fields conform to this compartmentalized plan. The prelimbic cortex has bilateral projections that concentrate within the striatal patches. In contrast, the agranular motor cortex and cingulate cortex have bilateral projections to the matrix, while the somatic sensory cortex and visual cortex have ipsilateral matrix projections. Each matrix input occupies a characteristic striatal district. The projection to the patches distributes prelimbic input throughout the striatum, which may allow for prelimbic interactions with input from all other cortical areas. PMID- 3004666 TI - Dark-rearing affects the development of benzodiazepine receptors in the central visual structures of rat brain. AB - The postnatal development of [3H]flunitrazepam binding to benzodiazepine receptors has been studied in the retina, lateral geniculate nucleus, superior colliculus, frontal cortex and visual cortex of the rat. In the frontal and visual cortices, binding reached the highest level at postnatal day 25 and then decreased slightly until adulthood. In the superior colliculus, the adult value of [3H]flunitrazepam binding is already reached at postnatal day 25, whereas in the retina the highest binding was found at postnatal day 50. The ontogeny of benzodiazepine binding sites in the visual regions does not essentially differ from that in other brain regions, suggesting that the appearance of [3H]flunitrazepam binding sites in the visual system is not correlated with the development of retinal function and the functional maturation of the visual system with regard to processing of light stimuli. Raising rats in complete darkness from birth until the age of 25 days resulted in significantly decreased binding levels in the lateral geniculate nucleus and in the superior colliculus by 29% and 17%, respectively, as compared to controls. [3H]Flunitrazepam binding in the other regions studied was not affected by dark-rearing. Presumably, the complete lack of visual experience interferes with the functional development of GABAergic mechanisms involved in the gating function of the subcortical visual centres for visual information transfer. PMID- 3004665 TI - The ontogeny of specific prolactin binding sites in the rat choroid plexus. AB - The development of prolactin receptors in the choroid plexus of the rat was examined using the in vivo autoradiographic approach employing the principle of competitive binding. Experimental animals were injected with [125I]prolactin alone (total binding) while control animals received [125I]prolactin and a 500 fold excess of unlabelled prolactin (non-specific binding). Newborns as well as animals 10, 14 and 18 days postnatal were studied. Three minutes following hormone injection animals received an intracardiac perfusion with fixative and tissues were prepared for quantitative light microscopic autoradiography. The choroid plexus first demonstrated specific binding of prolactin, i.e. a statistically significant difference in the autoradiographic reactions between experimental and control animals, at 14 days postnatal. The lactogen specificity of these binding sites was further defined by the ability of [125I]prolactin to be displaced by unlabelled human growth hormone, which is lactogenic in rats, and not by unlabelled insulin, which is structurally dissimilar to prolactin. Morphometric analyses were performed on electron micrographs of choroid plexus from 10- and 14-day postnatal rats. The volume densities of constituents known to be enriched in polypeptide hormone receptors were measured and compared. Small cytoplasmic vesicles and tubules were statistically significantly more abundant in 10-day-old rats than in 14-day-old animals. It is conjectured that these vesicles and tubules contain an intracellular pool of prolactin receptors whose decrease at 14 days parallels the expression of specific binding sites at the cell surface. PMID- 3004667 TI - Autoradiographic study of beta 1-adrenergic receptor development in the mouse forebrain. AB - The development of beta 1-adrenergic receptors has been studied in the mouse forebrain from embryonic day 14 (E14) to adulthood, using autoradiographic visualization of [125I]iodocyanopindolol (ICYP) binding sites. From E14, ICYP binding sites are detected in moderate amounts in the striatum and basal forebrain and in very low concentration in the cortical plate. At E17, binding sites have increased in number in the deep layers of the embryonic cortex and extend over the whole thickness of the cortical ribbon at birth. On postnatal day 4 (P4), ICYP binding sites are more abundant in the superficial than in the inner cortex. By P10 the adult pattern of ICYP binding site distribution is achieved, namely: a high concentration in ventral pallidum, striatum and cortical layers I, II and III, a moderate concentration in layers V and VI and a lower density in septal areas and in cortical layer IV. It is well established that norepinephrine fibers arrive in the embryonic cortex early in development. The present results show that the development of norepinephrine fiber and beta 1 receptor systems are coincident in the mouse. PMID- 3004668 TI - Characterization of opioid receptor binding in adult and fetal sheep brain regions. AB - Opioid receptor binding was studied in 3 brain regions from maternal and fetal sheep at various gestational ages. [3H]dihydromorphine [( 3H]DHM) and [3H]D Ala2,D-Leu5-enkephalin ([3H]DADLE) were employed as radioligands to characterize mu- and delta-opioid receptor binding sites, respectively. [3H]DHM binding was found to be highest in maternal cerebellum, intermediate in frontal cortex, and lowest in hippocampus. [3H]DADLE binding was highest in frontal cortex, intermediate in hippocampus and lowest in cerebellum. Cerebellum was the only tissue studied which contained more [3H]DHM than [3H]DADLE binding sites. Dissociation constants for [3H]DHM binding were similar in all 3 brain regions from both maternal and fetal sheep, while the dissociation constant for [3H]DADLE binding was significantly higher in cerebellum than in frontal cortex or hippocampus. Binding of both mu- and delta-receptor-selective ligands was 70% of maternal values in fetal cerebellum at 97-101 days of gestation and gradually increased over the remainder of the gestational period studied. Levels of [3H]DHM binding in frontal cortex and hippocampus were also similar to maternal levels at all timepoints studied. In contrast, [3H]DADLE binding was only 40-45% of maternal levels in fetal frontal cortex and hippocampus prior to 110 days of gestation, followed by a rapid increase in binding in both brain regions. PMID- 3004669 TI - Ontogeny of receptors for thyrotropin-releasing hormone in the rat brain. AB - Developmental changes in the thyrotropin-releasing hormone (TRH) receptor concentrations of hypothalamic and certain extrahypothalamic structures in the rat were evaluated from 2 days after birth until sexual maturity. The maturational pattern of TRH receptors in hypothalamus, striatum and amygdala follow that of the pituitary and all show the same kinetic properties. The developmental pattern also coincides with that of other components of the hypothalamic-pituitary-thyroid axis. In all tissues, the concentration of receptors increased in early life to reach a maximum between 10 and 30 days of age and then decreased gradually to adult values. Development of TRH receptors reflected changes in the number of receptor sites but not in binding affinity, which remained constant and suggests that the TRH receptor is identical in all 4 tissues studied. These results indicate the parallel development of pituitary and central nervous system TRH receptors. PMID- 3004670 TI - Microtubule organization and synaptogenesis in the vestibular sensory cells. AB - Microtubule organization in type I hair cells has been investigated during the synaptogenesis of vestibular receptors in mammals. The different steps in the maturation of the synapse between the hair cell and the nerve chalice were: a slight symmetrical membrane densification; the appearance of synaptic bodies alongside microtubules closely associated with densified presynaptic membranes; the disappearance of synaptic bodies and the persistence of microtubules. During this development, microvesicules were never seen to be associated with microtubules. PMID- 3004671 TI - Further behavioral analysis of GABA-mediated inhibition in the early chick embryo. AB - Five mumol of gamma-amino-N-butyric acid (GABA) completely inhibited the spontaneous motility of 5-day-old chicken embryos. A molar equivalent dose of baclofen, a specific GABAB receptor agonist, produced a much smaller (but still significant) inhibition of motility. The (-)-isomer of baclofen was significantly more potent than the (+)-isomer. GABAB receptors controlling overt behavior appear in the chick embryo at least as early as day 5. Neither 5 mumol of histamine nor 5 mumol of bradykinin, both potent cerebral vasodilators, inhibited spontaneous motility in 5-day-old chicken embryos. One mumol of serotonin, a potent cerebral vasoconstrictor, did not antagonize the significant (but submaximal) inhibition of motility caused by 1 mumol of GABA, GABA-mediated inhibition of overt behavior in the early chick embryo cannot be the result of GABA-mediated cerebral vasodilation. PMID- 3004672 TI - Morphological modulation of cultured rat brain astroglial cells: antagonism by ganglioside GM1. AB - Secondary cultures of neonatal rat astroglial cells, maintained in a serum-free, chemically defined medium were treated with several agents thought to activate cyclic AMP-synthesizing systems. Dibutyryl cyclic AMP (dBcAMP), forskolin and cholera toxin promoted, within 2 h, the near-complete conversion of 1-day-old (D1) astroglial cells from a flat, epithelioid morphology to a stellate (star shaped) morphology. With all 3 agents, cell susceptibility to morphological change declined with culture age, 5-day-old cultures failing to respond altogether. D1 cultures, after 48 h of treatment, had reverted to the flat morphology. Gangliosides reported to stimulate adenylate cyclase were also tested, using purified GM1 X GM1 failed to stimulate the conversion to stellate morphologies. GM1, however, did affect these astroglial cells by causing a block or reversal of their morphological response to dBcAMP, forskolin or cholera toxin. The GM1 response was specific for the intact ganglioside molecule, asialo GM1 and sialic acid having no effect. Gangliosides GD1a, GD1b and GT1b were also active, being effective at ca. 4-fold lower concentrations. The response to GM1 appeared to involve a direct interaction with the astroglial cell, rather than influencing either substratum or medium components. PMID- 3004673 TI - Ontogeny of the GABA receptor complex in chick brain: studies in vivo and in vitro. AB - The ontogeny of the gamma-aminobutyric acid-A (GABAA) receptor complex in the chick brain was studied by specific binding of [3H]muscimol, [3H]flunitrazepam (Flu) and [35S]t-butylbicyclophosphorothionate (TBPS) to isolated membranes. During development in ovo, the specific binding of muscimol and flunitrazepam increased from day 8 and reached 50% of adult levels of day 20, while a comparable level of TBPS binding was achieved by day 17. The increases in TBPS and Flu binding were reflected in Bmax rather than Kd changes. In embryonic brain, only a low-affinity site for muscimol (Kd = 23 nM) was observed while an additional high-affinity site (Kd = 0.4 nM), as well as the low-affinity site, was found in adult tissue. Similar studies were carried out with cultures of cerebral neurons prepared from 8-day embryos. The level of specific binding of muscimol, Flu and TBPS increased in culture, achieved one half of the maximum level by days 4-5, maximal levels by day 10 and decreased slowly thereafter. The maximal levels in culture corresponded, respectively, to 27%, 67% and 57% of these found in the 18-day embryo. The binding of Flu to membranes from neurons, embryos and adults was enhanced by addition of GABA while TBPS binding was inhibited. The EC50 and IC50 values for these effects corresponded to those for gating of chloride channels. These findings indicate a coordinated expression of receptors for GABA, benzodiazepines and convulsant/TBPS during neuronal maturation both in vivo and in vitro. The schedule for this postsynaptic ontogeny is very similar to that for presynaptic markers of GABAergic neurons (see companion paper). PMID- 3004674 TI - Effects of thermal trauma on dog erythrocyte ATPase and shape. AB - Studies have been carried out on dog erythrocytes to determine the effects of heating to 57 degrees C on membrane ATPase and erythrocyte shape. The results of these studies showed that after heating the blood there was approximately a 30 per cent decrease in membrane ATPase and an alteration of erythrocyte shape from biconcave to spherical. Additional studies of the effects of addition of arachidonic acid to erythrocyte-membrane homogenates demonstrated that this substance produced a dose-related inhibition of ATPase. If the additions were made to membranes from blood which had previously been heated, the effects of arachidonic acid and heat were additive. Addition of the hydroxyl-radical scavenger catalase to the blood before heating prevented the observed decrease in ATPase and prevented the change in shape to spherical. These results confirm the previous findings of Bessis that heat will cause a change in the erythrocyte shape from biconcave to spherical and suggest that the mechanism responsible for this change is an alteration in membrane ATPase. The observation that a similar response can be produced by arachidonic acid suggests that metabolism of this substance may generate agents which are capable of inhibiting erythrocyte membrane ATPase. The observation that catalase will prevent the ATPase inhibition and the change in erythrocyte shape suggests that hydroxyl-free radicals play an important part in this thermal trauma-induced response. PMID- 3004675 TI - [Stimulation of adenyl cyclase and fixation of iodine in human cancerous thyroid cells in culture]. AB - In primary cultures of cells from human thyroid cancer, functional activity was investigated by adenylate cyclase responsiveness and radioiodine uptake. In cells from well-differentiated follicular carcinomas (2 cases), TSH stimulation of cAMP accumulation is similar to that of normal thyroid cells. In contrast, in papillary carcinomas (11 cases), the mean dose-response curve is much lower than normal. In thyroid cancers, radioiodine uptake by the cells has been detected in some cases after 3 days of 125I incorporation. As previously observed, our results suggest the existence of a relationship between adenylcyclase responsiveness and 125I uptake by the cells. PMID- 3004676 TI - [Hypnogenic effects of des-acetyl-alpha-MSH and CLIP (ACTH 18-39) in the rat]. AB - In the rat, the intraventricular administration of ACTH (adrenocorticotropic hormone) has no effect upon the sleep-waking cycle. However, administration of two ACTH-derived peptides is followed by a selective and significant increase of each sleep state: Des-alpha-MSH (Des-acetyl-alpha-melanocyte stimulating hormone) administration induces an increase of slow wave sleep, while CLIP (corticotropin like intermediate lobe peptide) is followed by an increase of paradoxical sleep. PMID- 3004677 TI - [Human monoclonal antibodies against surface antigens of Langerhans cells from lymphocytes of diabetics transformed by Epstein-Barr virus]. AB - Human monoclonal antibody against islet cell surface antigens was generated from a pre-diabetic patient's peripheral blood lymphocytes transformed with Epstein Barr virus. Reactivity of these transformed lymphocytes was evaluated using indirect immunofluorescence on rat islet cell suspensions and frozen sections of human pancreas. Several lymphoblastoid cell lines that react with islet cell surface were obtained. Preliminary immunoblots with enriched rat islet cell membrane antigens suggest a reactivity toward a 64 kdalton antigen. PMID- 3004678 TI - Examination of the porcine fetus. AB - The primary purpose of necropsy of the porcine fetus is the collection of tissues to be submitted to the laboratory to assist in identification of the causative agent(s). Proper collection and handling of tissues are critical for optimum identification of infectious agents. Gross examination is an aid in differentiating stillbirth and neonatal death, determining relative time of fetal death, and identifying congenital defects. PMID- 3004680 TI - Intraoral pleomorphic adenoma. Report of a case presenting in an unusual location. PMID- 3004679 TI - The effect of aluminum on the rate of dissolution of calcium hydroxyapatite--a contribution to the understanding of aluminum-induced bone diseases. AB - Aluminum ions, Al3+, as these ions exist in aqueous solutions at pH approximately equal to 7, adsorb onto calcium hydroxyapatite (HAP) crystals and severely inhibit the dissolution process of these crystals at 0.1 microM concentrations of aluminum. Human bone crystals have also been shown to adsorb these ions or molecules. A mechanism explaining why aluminum, in connection with bone resorption, causes less demineralization in the osteoid/calcified bone region than deeper in the calcified bone, is suggested. PMID- 3004681 TI - Oxygen defense systems in obligately thermophilic bacteria. AB - Ten strains of Gram-negative, aerobic, obligately thermophilic bacteria were examined for their response to oxygen toxicity by comparing static with shaken cultures. All of the organisms tested had measurable levels of superoxide dismutase, catalase, and peroxidase. Aeration generally did not result in an increased level of superoxide dismutase in any of the thermophiles. Aeration of organisms obligate for n-alkane substrate caused an increase in cellular peroxidase levels and a corresponding decrease in catalase. The thermophiles that grew on either n-alkanes or complex media did not grow on the hydrocarbon in aerated culture but on a complex medium, aeration effected an increased level of catalase. With the exception of a pink-pigmented thermophile which, when aerated, did not have an increased level of the three oxygen defense enzymes, most of the thermophiles surveyed had an increased level of catalase or peroxidase when exposed to increased oxygen tension. The activity of the enzymes was determined at temperatures from 25 to 65 degrees C and the former temperature was satisfactory for these experiments. PMID- 3004682 TI - Effect of relative humidity on the airborne survival of rhinovirus-14. AB - Rhinovirus-14, suspended in tryptose phosphate broth supplemented with uranine (physical tracer) and an antifoam, was aerosolized by use of a Collison nebulizer. The aerosols were held in a rotating drum with the relative humidity at either the low (30 +/- 5%), medium (50 +/- 5%), or high (80 +/- 5%) level at 20 +/- 1 degrees C. An all-glass impinger was used to recover the virus from the air in the drum, with the first air sample being collected after a 15-min period of aerosol stabilization. Subsequent air samples were withdrawn at 2, 4, 8, and 14 h after stabilization of the aerosol. At the low and medium relative humidity levels, the infectivity of the airborne virus was rapidly lost and less than 0.25% could be detected in the first air sample. At the high RH level, however, the airborne virus had a half-life of 13.7 +/- 1.91 h and nearly 30% of the input infectious virus could be detected in the drum air even after 24 h of aerosolization. These findings suggest that under certain environmental conditions, notably high relative humidity, air may act as a vehicle for the spread of rhinovirus infections. PMID- 3004683 TI - Influence of inoculum size, incubation temperature, and cell culture density on virus detection in environmental samples. AB - The influence of inoculum size and cell culture density on virus titer by cytopathic effect or plaque assay was studied using poliovirus type 1 and BGM (Buffalo green monkey) cells as a model for this evaluation. With a plaque assay system, a linear relationship was observed for an inoculum size of up 1 mL/25 cm2; a marked decrease in the number of plaques was observed when over 1 mL of sample was inoculated on this surface area. Cell culture density also affected virus titer; maximal titers were observed when cells were seeded at 25 000 to 75 000 cells/mL and incubated for 6 days before infection with the virus. Viral density, evaluated as most-probable-number and measured by cytopathic effect under liquid overlay, revealed that the viral titer was similar up to 1 mL inoculum and increased only when over 1 mL was inoculated. Cell density had no significant effect on the viral titer measured by the most-probable-number method and cytopathic effect. Inactivation of inoculum due to an incubation temperature of 37 degrees C for a short period was shown to be minimal for poliovirus type 1, reovirus type 2, coxsackievirus B-5, and the simian rotavirus SA-11. Longer inactivation time led to a 2 logs reduction of the infectious titer of coxsackievirus B-5 (in 48 h) while the other viruses showed a significant reduction in titer only after 96 h. PMID- 3004684 TI - Isolation and characterization of pyrimidine mutants of Salmonella typhimurium altered in expression of pyrC, pyrD, and pyrE. AB - Parental strains of Salmonella typhimurium having a specific pyr gene (pyrC, pyrD, or pyrE) fused to the structural genes of the lac operon through the specialized transducing phage Mu dl (ApRlac) were used to construct thermostable derivatives for purposes of conducting a genetic and biochemical characterization of the individual pyr genes. The direction of transcription of each pyr gene in relation to the current linkage map was defined with both pyrC and pyrE being transcribed counterclockwise and pyrD exhibiting clockwise transcription. Mutants displaying increased pyr gene expression were isolated employing a genetic strategy which is of general applicability. Among the mutants, only one isolate was found to possess a mutation which was unlinked to the specific pyr gene under study; the other isolates harbored linked mutations which were inferred to be cis acting. Additional studies demonstrated that in conditions of severe pyrimidine limitation, further derepression could still occur in the mutant strains. PMID- 3004685 TI - Catalase, superoxide dismutase, and the production of O2-sensitive mutants of Bacillus coagulans. AB - A number of facultatively anaerobic members of the genus Bacillus were screened for their catalase, diaminobenzidine peroxidase, and superoxide dismutase activities. A strain of Bacillus coagulans (7050) lacking peroxidatic activity and containing single catalatic and superoxide dismutase activities was selected. Responses of the superoxide dismutase activity and catalase level to the partial pressure of oxygen, and Fe and Mn levels, as well as to aerobic and fermentative metabolism, were determined. There appeared to be a relationship between high endogenous catalase levels and the high H2O2 evolution and KCN insensitivity of B. coagulans respiration. Bacillus coagulans 7050 was mutagenized with N-methyl N'-nitro-N-nitrosoguanidine and screened for the expression of oxygen intolerance. All of the 38 stable oxygen sensitive mutants obtained had very low or completely absent catalatic activity and catalase protein. No mutant lacked superoxide dismutase, although five showed significantly lowered levels of the enzyme. Exogenous bovine liver catalase restored aerotolerance and reduced cell pleomorphism in the mutants. PMID- 3004686 TI - AIDS and taurolin. PMID- 3004687 TI - Human T-lymphotropic virus: exposure and prognosis. PMID- 3004688 TI - Conservative management of intraductal breast carcinoma with tumorectomy and radiation therapy. AB - Between 1967 and 1983, 54 patients with strictly noninvasive intraductal breast carcinoma were treated with tumorectomy and radiation therapy. Median follow-up was 55 months. Three patients had a recurrence in the treated breast; two were noninvasive, and one was invasive. One patient died of disease. Actuarial 5-year disease-free survival rate was 95.2%. No axillary node recurrences occurred in patients treated with irradiation to the breast and regional nodes (34 patients), or in patients treated with breast irradiation alone (20 patients). These preliminary results suggest that combined tumorectomy and radiation therapy could be a valuable conservative alternative to mastectomy in the treatment of noninvasive intraductal breast carcinoma. PMID- 3004689 TI - Serial plasma carcinoembryonic antigen measurement for monitoring patients with advanced lung cancer during chemotherapy. AB - The plasma carcinoembryonic antigen (CEA) levels in 243 patients with untreated advanced lung cancer were studied to assess their value for prognosis and for indicating the effectiveness of chemotherapy. Of patients with adenocarcinoma, small cell carcinoma, squamous cell carcinoma, and large cell carcinoma, 43%, 24%, 7%, and 13%, respectively, had elevated CEA levels of 20 ng/ml or greater before treatment. Pretreatment CEA levels were elevated to above 20 ng/ml for 38% of 163 patients with extensive disease and for 22% of 80 patients with limited disease (P less than 0.02). In patients with adenocarcinoma of the lung, the pretreatment CEA levels were not correlated with response to chemotherapy and patients' survival. Serial measurement of plasma CEA was a useful noninvasive technique for monitoring the response to chemotherapy in patients whose pretreatment levels were 20 ng/ml or higher. All of 18 patients with complete or partial responses and 5 of 6 patients with minor responses showed greater than 36% decrease in the CEA level compared with the pretreatment level. In all of nine patients with progressive disease, the CEA levels increased after chemotherapy. Therefore, an increase of greater than 36% beyond the baseline level was a useful guideline criterion for a significant change for determination of tumor response to chemotherapy, although 41% of 22 patients with stable disease exceeded the pretreatment level by 36% or more in either direction (mean percent change +/- standard deviation, -4.1% +/- 52.2%), and 4 of 9 patients with progressive disease did not have levels greater than 36% above the baseline levels. PMID- 3004690 TI - Are prognostic factors for local control of breast cancer treated by primary radiotherapy significant for patients treated by mastectomy? AB - Recent follow-up studies of patients with mammary carcinoma treated with breast conserving primary radiotherapy identified a triad of pathologic features significantly associated with local treatment failure. These unfavorable characteristics of the primary tumor were: poor or undifferentiated nuclear grade; intraductal carcinoma within the tumor mass; and intraductal carcinoma in breast tissue outside the perimeter of the primary lesion. The current study was undertaken to assess the impact of these same factors on the prognosis of 573 consecutively treated women, with invasive duct carcinomas 5 cm or less in diameter, and who underwent mastectomy. Histologic sections of all primary tumors were reviewed, and the lesions were classified according to the distribution of intraductal carcinoma present: only within the tumor (IN, 247 cases, 43%), only outside the tumor (OUT, 25 cases, 4%), within the outside (IN-OUT, 158 cases, 28%), or not seen (IFDC, 143 cases, 25%). The median follow-up period for the entire series was 56 months. Ninety-five (17%) patients were dead of disease (median time to death, 36 months). Variables that proved to be statistically significant for overall survival were nodal status (P less than 0.001), nuclear grade (P less than 0.03), and histologic grade (P less than 0.007). Nodal status (P less than 0.001), histologic grade (P less than 0.001), and tumor size (P = 0.01) were significant predictors of disease-free survival. The pattern of intraductal carcinoma, when present, was not predictive of the risk for recurrence or survival in women treated by mastectomy. These findings provide a rationale for additional surgical treatment for women whose tumors have features more likely to be associated with local failure following primary radiotherapy. To permit more detailed pathologic examination of the primary lesion, the initial excision should be carried out separately from the treatment when limited resection and radiation are to be considered as a treatment option. PMID- 3004691 TI - Local recurrences in patients with breast cancer at the North Carolina Memorial Hospital (1970-1982). AB - A study of predictive factors for locoregional recurrences after curative surgery for breast cancer was undertaken. Specifically, the authors wished to determine whether such recurrences correlated with either hormonal receptor status or a delay between the initial biopsy and the definitive surgery. A retrospective chart review was done on all women with breast cancer who had surgery for cure between 1970 and 1982. Factors analyzed included, among others, size of the tumor, clinical and pathologic status of the axilla, estrogen and progesterone receptors status, and delay between biopsy and definitive surgery. There were 404 patients studied. Pathologic axillary nodal status was the most important predictor of locoregional recurrence, with failures in 36 of 188 (19%) node positive but only 9 of 216 (4%) node-negative patients (P = 0.0001). In node positive patients, tumor size was a predictor of local recurrence, with failure in only 4 of 51 (8%) of tumors less than 2 cm, but in 14 of 44 (32%) of tumors greater than 6 cm (P = 0.004). Progesterone receptor (PR) status correlated with locoregional recurrence, but estrogen receptor status did not. In node-positive women, there were 4 of 14 PR-negative but 0 of 15 PR-positive local failures (P = 0.017); this result has not been previously reported. The presence of palpable axillary disease was also found to be a predictor of local recurrence. Finally, no increase in locoregional recurrence could be attributed to the delay between biopsy and definitive surgery. Two new predictors for locoregional recurrence in breast cancer, not previously emphasized, are PR and clinical axillary status. Should these findings be substantiated, patients at high risk for locoregional recurrence could then be more readily identified. PMID- 3004693 TI - Liver cell dysplasia in normal, cirrhotic, and hepatocellular carcinoma patients. AB - An assessment was made of the frequency of liver cell dysplasia and the mean age of each group in 56 normal, 13 cirrhotic, and 50 hepatocellular carcinoma (HCC) patients, 40 with cirrhosis, from southern Africa. Dysplasia increased from 7.1% in normal subjects to 38.5% in cirrhotic, 40% in noncirrhotic HCC, and 52.5% in cirrhotic HCC patients, three statistically similar frequencies. Average patient ages were as follows: patients with normal livers, 37.3 years; with cirrhosis, 42.4 years; with noncirrhotic HCC, 36.5 years; and with cirrhotic HCC, 34 years, the mean age with dysplasia being lower than that of the whole group. With no increase in frequency of dysplasia from cirrhosis to HCC with cirrhosis, with a similar high frequency in HCC without cirrhosis, and with a mean age of all HCC patients 8 years less than that of cirrhotics and 3 years less than normals, chronologic evolutionary progression from cirrhosis to dysplasia to HCC in southern Africa cannot be demonstrated. PMID- 3004692 TI - Adenoid cystic carcinoma of the esophagus. Complete response to combination chemotherapy. AB - A 55-year-old man had adenoid cystic carcinoma of the esophagus metastatic to the lungs and right supraclavicular fossa. He was treated with local radiation therapy to the esophagus and supraclavicular fossa, followed by combination chemotherapy with doxorubicin, mitomycin C, and 5-fluorouracil (5-FU). After a modest initial response, disease progression was noted in the pulmonary nodules. He was then treated with cisplatin, cyclophosphamide, vincristine, and doxorubicin. After two cycles of this regimen, there was complete regression of his pulmonary nodules, which was sustained for 5 months. A review of 44 literature cases of esophageal adenoid cystic carcinoma contrasted with adenoid cystic carcinoma of salivary gland origin indicated that the esophageal adenoid cystic carcinomas have a high tendency to metastasize (76% of cases) and a much poorer prognosis, with only 23% 1-year survival rate. It was concluded that esophageal adenoid carcinoma is clinopathologically distinct from the salivary gland variant, and that combination chemotherapy may be an effective treatment modality for this cancer. PMID- 3004694 TI - Bronchioloalveolar carcinomas. Cell types, patterns of growth, and prognostic correlates. AB - Forty-five bronchioloalveolar carcinomas were studied, including 27 cases by electron microscopy. Bronchioloalveolar carcinomas can be classified by routine sections or by diastase-digested periodic acid-Schiff (PAS) stains, but electron microscopy is useful in confirming Clara cell or type II pneumocyte (nonmucinous) differentiation and excluding metastases. Mucinous bronchioloalveolar carcinoma can mimic metastatic adenocarcinoma histologically and ultrastructurally. Of the nine tumors with mucinous differentiation, eight had aerogenous dissemination (multifocal or with pneumonic spread), and seven of those eight were fatal. Twenty-four of 36 nonmucinous bronchioloalveolar tumors had aerogenous spread; all of the 24 patients died or were living with distant metastases. The 12 nonmucinous tumors without aerogenous dissemination had a 5-year survival rate of 61%. Among these, the smaller tumors had a better prognosis. The presence of alveolar spread, rather than cell type, was the most important feature predicting survival. PMID- 3004695 TI - Cytologic diagnosis of bronchioloalveolar carcinoma by fine-needle aspiration biopsy. AB - From 1970 to June 1984, 275 patients with bronchioloalveolar carcinoma were admitted to the Toronto General Hospital. Of these, 181 (190 aspiration biopsies, including nine repeat samples) had this diagnosis made following the use of transthoracic fine-needle aspiration biopsy. Based on the cytomorphologic features observed in the aspiration preparations, the tumor was subclassified into three types: nonsecretory, secretory, and poorly differentiated. The cytologic features of these three types of bronchioloalveolar carcinoma are presented and illustrated. Cytomorphologically, the three types of this tumor are distinctly different and their features are sufficiently distinctive from those of bronchogenic adenocarcinoma and metastatic adenocarcinomas to be of diagnostic value. Transthoracic fine-needle aspiration biopsy appears to be a definitive minimally invasive means of establishing the diagnosis of bronchioloalveolar carcinoma preoperatively and especially to be of value for those small peripheral cancers which are relatively inaccessible to direct method of study and are potentially surgically curable. PMID- 3004696 TI - In vitro hyperdiploidy in a family with nasopharyngeal cancer. AB - In this in vitro study of 28 members of a family with nasopharyngeal cancer (NPC) and nasopharyngeal angiofibromas (NPA) in consecutive generations, in vitro hyperdiploidy, (IVH) in dermal fibroblast cultures was associated with NPC in 2 affected members, NPA in 4 affected members, and 4 of 16 members considered to be at increased genetic risk for these tumors. None of the six controls, all spouses, had IVH. As IVH has been shown to be associated with high specificity (approximately 99%) with other heritable single tumors, IVH appeared to be a potential biomarker also for genetic predisposition for NPC. As NPC and NPA did not occur in a single affected family member, any association other than with IVH has not been established. IVH may be the in vitro expression of a gene/genes conveying tumor proneness per se, or it may determine tissue specificity of tumor pathology. As cellular hyperdiploidy has been considered to convey genomic instability, chromosome instability in numerically normal (diploid) and altered (tetraploidy and hyperdiploid) dermal fibroblasts from two NPC-affected members and two control spouses were evaluated by a parameter of chromosome stability- the frequency of sister chromatid exchanges (SCE) per cell. Irrespective of donor source, the diploid and tetraploid fibroblasts exhibited little evidence of chromosome instability, whereas there was a statistically significant difference in the mean frequency of SCE/cell between hyperdiploid and diploid cells in cultures derived from NPC-affected members (p less than 0.01). PMID- 3004697 TI - The Philadelphia chromosome: a model of cancer and molecular cytogenetics. AB - Recent developments in molecular biology related to the Ph chromosome lead us to an evaluation of knowledge regarding this chromosome. The molecular advances are related to two cellular oncogenes, c-abl and c-sis, and also to the identification and molecular cloning of specific areas of DNA (e.g., band 22q11), permitting the isolation of a probe specific for the translocation breakpoint domain. In the preponderant number of cases examined, it was found that the breakpoints at 22q11 occur within a limited region of up to 5-6 kb, for which the term "breakpoint cluster region" (bcr) has been suggested. In contrast, breaks at 9q34 seem to occur within a much larger region at the molecular level. Yet to be established is the exact genetic composition of the bcr and a determination as to whether or not the breaks leading to the disease occur preferentially within specific areas. In spite of this level of knowledge, we do not understand how the Ph chromosome participates in CML. If Ph-positive CML is ultimately associated with a cascade of gene activations, the unraveling of their nature and chronology will undoubtedly tell us much of their contribution to the biology of CML, in particular, and to neoplasia, in general. In this respect, the rather clear description of CML in cytogenetic, clinical, and laboratory terms, the relatively long chronic phase of the disease, and the association of the blastic phase with nonrandom chromosome changes (at least in the initial phases of the disease) make Ph-positive CML an excellent candidate for a model for the study of molecular events in human neoplasia. PMID- 3004698 TI - Cytogenetics of a renal adenocarcinoma in a 2-year-old child. AB - Chromosomal abnormalities in a 2.4-year-old boy with renal adenocarcinoma (Grawitz tumor) are described. Renal adenocarcinoma is extremely rare in childhood, compared with nephroblastoma (Wilms' tumor). In all tumor cells the same 46,XY,t(X;1) (p11.2;q21.2) karyotype was found. This karyotype is compared with the cytogenetic descriptions of renal adenocarcinoma in adults. PMID- 3004699 TI - Cytogenetic characterization of selected small round cell tumors of childhood. AB - Small, round, blue-cell tumors (SRCT), including rhabdomyosarcoma, Ewing's sarcoma of bone and soft tissue, mesenchymal chondrosarcoma, small cell osteosarcoma, hemangiopericytoma, neuroblastoma, peripheral neurectodermal tumor (peripheral neuroepithelioma of bone and soft tissue), and the malignant small cell tumor of the thoracopulmonary region described by Askin (Askin's tumor), are often difficult to distinguish by light microscopy. We have evaluated the cytogenetics of these tumors by studying 24 tumor explants in short-term culture and 22 tumor cell lines. In Ewing's sarcoma (a tumor of unknown histogenesis), and in peripheral neuroepithelioma and Askin's tumor (tumors with evidence of neural origin), we have observed an indistinguishable t(11;22) translocation. PMID- 3004701 TI - Growth-inhibitory effects of epidermal growth factor and overexpression of its receptors on human squamous cell carcinomas in culture. AB - The role of epidermal growth factor (EGF) and its receptors in human cancers was studied using 24 human cell cultures including 15 of squamous cell carcinoma (SCC) of the skin, oral cavity, and esophagus. EGF was found to inhibit the growth and colony formation of all the SCC cells at doses that are mitogenic in many other cells, including epidermal keratinocytes and dermal fibroblasts. This inhibitory effect of EGF on SCCs was specific, because EGF did not inhibit and in some cases slightly stimulated the growth of other tumor cells, such as adenocarcinomas of the stomach, cervix, and breast and sarcomas. The amounts of EGF receptors on these SCC cells were measured by immunoprecipitation of labeled proteins with anti-EGF receptor polyclonal antibody and binding assay of membrane preparations using 125I-EGF. Of 13 SCC cell cultures tested, all except 3 of esophageal SCC showed higher levels of EGF receptor than normal epidermal keratinocytes, which contain 1.5 X 10(5) binding sites/cell. In general, SCCs of the skin and oral cavity had large amounts of EGF receptor on the order of 10(6)/cell, whereas the receptor of esophageal SCCs was on the order of 10(5)/cell. Some SCC cells had about twice as many EGF receptors as A431 cells. The values for the equilibrium dissociation constant (Kd) of these cells were on the order of nM. The sensitivity to the inhibitory effect of EGF correlated well with the elevated level of EGF receptors in 12 SCC cell lines, and higher significance was obtained when data on esophageal SCCs were excluded. The present observations suggest that EGF and EGF receptors play a role in the development of SCCs. PMID- 3004700 TI - Role of gastrin and gastrin receptors on the growth of a transplantable mouse colon carcinoma (MC-26) in BALB/c mice. AB - We recently reported trophic response of transplantable mouse colon cancer cells (MC-26) to pentagastrin, in vivo, and demonstrated gastrin receptors on MC-26 cells, in vitro. In the present study, growth of MC-26 cells in mice, in response to pentagastrin, was studied in relation to binding kinetics and capacity of gastrin receptor. Gastrin receptor levels on mouse fundic and colonic membranes and on MC-26 cellular membranes were determined before MC-26 cell inoculation and designated as Day 0 levels. Four groups of mice were next inoculated with MC-26 cells and given injections of either pentagastrin (treated) or normal saline (control) for 10 or 15 days. At the end of the treatment periods, body, tumor, fundic, and colon weights were noted and gastrin receptor measured. tumor and fundic weights increased significantly within 15 days of pentagastrin treatment, compared to control values. In control (non-pentagastrin treated) mice, the binding affinity of gastrin receptor on tumor membranes was significantly decreased and associated with the complete loss of high-affinity gastrin receptor (Kd = less than 0.5 nM) by Day 15 of tumor growth. On the other hand, both the binding affinity and gastrin receptor levels of tumor membranes were maintained at Day 0 values by pentagastrin treatment. Endogenous gastrin was therefore ineffective in maintaining high-affinity gastrin receptor on control tumors. A significant number of low-affinity gastrin-binding sites (Kd = less than 2 nM) appeared in control tumors by Day 15, which could reflect rapid dedifferentiation or conformational changes of gastrin receptor in the absence of high levels of normal regulatory hormones. These studies demonstrate that the trophic effects of gastrin on MC-26 cells are probably mediated by its regulation and maintenance of the binding affinity and capacity of gastrin receptor on the cancer cells, in vivo. PMID- 3004702 TI - Repair of ionizing radiation DNA base damage in ataxia-telangiectasia cells. AB - Micrococcus luteus endonuclease sensitive sites were measured by alkaline elution in normal human and ataxia-telangiectasia (AT) fibroblasts after ionizing radiation. Due to the sensitivity of this assay, repair of base damage after 3 to 6 kilorads has been measured after oxic or hypoxic radiation. With 5.5 kilorads of oxic radiation, more than 50% of the base damage was removed after 1.5 h of repair incubation in all cells, including exr+ and exr- AT cells, and approximately 75% was removed by 4 h. After 3 or 4.5 kilorads of hypoxic X irradiation, repair was equivalent in normal and exr- AT cells. This study included three exr- AT strains which have been reported to be deficient in the removal of gamma-ray base damage at higher doses. Since these strains repaired ionizing radiation base damage normally at lower doses, which are more relevant to survival, it is concluded that the X-ray hypersensitivity of AT cells is probably not related to the repair of base damage. PMID- 3004703 TI - Relationship between colonic luminal pH, cell proliferation, and colon carcinogenesis in 1,2-dimethylhydrazine treated rats fed high fiber diets. AB - The comparative effects of different fibers on colonic luminal pH, crypt cell proliferation, and colon carcinogenesis were studied in 120 male Sprague-Dawley rats. The animals were divided into five equal groups and fed either a basal fiber free diet or the basal diet supplemented with 10% pectin, cellulose or guar, or 20% oat bran for up to 30 weeks. 1,2-Dimethylhydrazine was given at 20 mg/kg body weight as a weekly s.c. injection for 12 weeks. Food intake and weight gain were similar in all diet groups. At sacrifice, in vivo pH measurements showed that compared to fiber free rats, all fibers significantly acidified large bowel luminal contents (P less than 0.05). In the guar group 62.5% of rats developed colonic tumors compared to 33.4% of the fiber free rats (P less than 0.05). The yield of proximal colonic adenocarcinomas in the oat bran, pectin, and guar groups was increased by 4.5 to 5 times over the fiber free level (P less than 0.05-0.025). Pectin and guar provided the greatest stimulus to cell proliferation. A lower luminal pH was associated with a higher tumor yield and increased epithelial cell proliferation. Thus, acidification of colonic contents by high fiber diets failed to inhibit rat colon carcinogenesis, while the consumption of soluble fibers, such as oat bran, pectin, and guar, was associated with enhancement of proximal colon carcinogenesis. PMID- 3004704 TI - Mechanisms of growth inhibition by anti-transferrin receptor monoclonal antibodies. AB - In previous studies, an immunoglobulin A, anti-transferrin receptor antibody (42/6) inhibited growth of a variety of normal and malignant human hemopoietic cells. To determine its mechanism of growth inhibition, we compared effects of 42/6 and B3/25, an immunoglobulin G anti-transferrin receptor antibody which does not inhibit lymphocyte growth, on transferrin (TF) binding and uptake. As in previous studies, affinity constants of TF and anti-TF receptor antibodies for human TF receptors at 4 degrees C were similar, but the number of calculated binding sites was higher for the antibodies. Antibody B3/25 did not inhibit TF binding at either 4 degrees C or 37 degrees C. At 4 degrees C, antibody 42/6 inhibited TF binding to normal, mitogen-stimulated mononuclear cells. However, TF did not inhibit 42/6 binding, suggesting 42/6 inhibited TF binding by noncompetitive, possibly steric, mechanisms. When cells were simultaneously exposed to labeled TF and unlabeled anti-TF receptor antibodies at 37 degrees C, 42/6 inhibited TF binding only slightly. Initial uptake of antibodies and TF at 37 degrees C was rapid, but when mononuclear cells or HL60 cells were cultured with either 42/6 or B3/25 for 2 days, TF binding and immunoreactive TF receptor sites decreased. However, TF bound to cells cultured with B3/25 continued to enter the cell, whereas cells cultured with 42/6 would no longer take up bound TF. Studies using HL60 cells grown with soluble iron in lieu of TF showed that changes in TF binding sites and TF uptake were not secondary to growth inhibition. We conclude that incubation with both inhibitory (42/6) and noninhibitory (B3/25) anti-TF receptor antibodies results in decreased TF binding sites. However, exposure to 42/6 also inhibits TF uptake and causes growth inhibition by iron deprivation. Monoclonal antibodies to receptors transporting critical nutrient molecules, such as iron, may inhibit cell growth by blocking ligand access to the cell's interior. PMID- 3004705 TI - Effect of phorbol esters on the activity of purified transforming growth factors. AB - Application of either phorbol esters or transforming growth factors to normal cells in culture may induce the appearance of a malignant phenotype. Since the mechanism of transformation by both chemical tumor promoters and growth factors is unknown, it was of interest to investigate whether phorbol esters might potentiate the activity of transforming growth factors. The combination of beta transforming growth factor (beta-TGF) and epidermal growth factor (EGF) causes the normally anchorage-dependent cells (NRK-49F) to form colonies in soft agar. In the presence of maximally stimulating concentrations of EGF and suboptimally stimulating concentrations of beta-TGF, phorbol 12-myristate-13-acetate (PMA) enhances the soft agar colony growth of NRK-49F cells in a concentration dependent manner. However, PMA alone or in combination with either EGF or beta TGF does not stimulate soft agar growth. In contrast to the stimulation of PMA, 4 alpha-phorbol 12,13-didecanoate, an inactive phorbol ester, does not potentiate the effects of EGF plus beta-TGF on soft agar growth. PMA does not stimulate the growth of NRK-49F cells in monolayer, nor does it further potentiate the monolayer growth induced by EGF with or without beta-TGF. Because the addition to NRK-49F cells of compounds which potentiate beta-TGF activity increases EGF receptor number, the effects of PMA on the EGF receptor were studied. A short exposure to PMA (30 min) induces a 50% decrease in EGF receptors of NRK cells whether or not they have been exposed to beta-TGF. Scatchard analysis shows that this decrease involves primarily high affinity EGF receptors. However, cells treated with PMA for longer periods of time (4, 6, and 24 h) show no change in EGF or beta-TGF receptor binding. PMA therefore must potentiate the activity of purified TGFs without causing an increase in EGF or beta-TGF receptor binding. PMID- 3004706 TI - Effects of amiloride on thermosensitivity of Chinese hamster cells under neutral and acidic pH. AB - The modifying effects of amiloride on the thermosensitivity of Chinese hamster V 79 cells were examined under both neutral (pH 7.3) and acidic (pH 6.6) conditions. Amiloride, a diuretic drug, is known to inhibit the Na+/H+ exchange activity. Under the extracellular pH of 7.3, amiloride (0.1-0.5 mM) enhanced the thermal cell killing powers of 42 degrees C hyperthermia with increasing concentration and exposure time of the drug. The age response of cells to 42 degrees C hyperthermia in the presence or absence of amiloride (0.5 mM) showed that amiloride sensitized cells to heat, especially those at G1-S boundary through middle S phases. On the other hand, the lowering of extracellular pH to 6.6 enhanced cell killing by 42 degrees C hyperthermia. When cells were exposed to 42 degrees C hyperthermia in the presence of amiloride at pH 6.6, cell survival decreased still more. The thermosensitizing effects of the lowered pH at 6.6 and amiloride appeared to be additive. From these results, it is suggested that the thermosensitization by amiloride is probably due, in part, to the inhibition of cellular Na+/H+ exchange activity. The present study proposes the possibility that amiloride may be useful as a hyperthermic sensitizer in a clinical treatment of cancer. PMID- 3004707 TI - Anchorage dependency effects on difluoromethylornithine cytotoxicity in human lung carcinoma cells. AB - Difluoromethylornithine (DFMO), a specific, irreversible, enzyme-activated inhibitor of ornithine decarboxylase activity, the first and rate-limiting step in polyamine biosynthesis, has been shown to inhibit neoplastic cell proliferation in culture. In most cases, such inhibition is not accompanied by cell loss, with the exception of multiple cell lines of human small cell lung carcinoma (SCC), a human leukemia cell line (HL-60), and possibly the B16 melanoma cell line. The first two cell types grow as anchorage-independent suspension cultures, the HL-60 as single cells and the SCC as multicellular spheroid aggregates. Moreover, in the spectrum of human lung carcinoma cells in culture, the SCC cells respond in a cytotoxic manner to DFMO, whereas the non small cell lung carcinoma (non-SCC) cells, which are anchorage dependent, show only growth inhibition, without actual cell loss. In the present study, we have investigated relationships between anchorage-dependent and -independent growth patterns of cells in culture and their response to DFMO treatment. Two non-SCC lung cancer cell lines, which normally grow as anchorage-dependent monolayers, show growth inhibition but no cell loss with the addition of DFMO. When these anchorage-dependent cells were forced to grow as multicellular aggregates, by coating the culture flask with Teflon, the cells developed an increased sensitivity to DFMO. They showed not only inhibition of cell proliferation but also cell death. Two SCC cell lines, which normally grow as anchorage-independent spheroids, developed adherence to the culture dishes coated with fibronectin. These cells, which show a cytotoxic response to DFMO during normal anchorage independent growth, developed a decreased sensitivity to DFMO, showing only cell growth inhibition, but no cell death when treated during anchorage-dependent growth. Our data thus suggest that the state of anchorage dependence of lung cancer cells in culture is a critical factor in determining their response to polyamine depletion during treatment with DFMO. PMID- 3004708 TI - Long-term maintenance therapy of established human small cell variant lung carcinoma implants in athymic mice with a cyclic regimen of difluoromethylornithine. AB - We report that cyclic p.o. administration of alpha-difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, is an effective long-term (1 year) maintenance therapy for established implants of cultured human small cell lung carcinoma in athymic (nude) mice. Human small cell lung carcinoma cells, from a line which exhibited cell death in culture in the presence of DFMO, were inoculated into athymic mice and permitted to grow to palpable tumors (3-5-mm nodules with mean volume of 0.04 cm3). The animals were then randomized into untreated, continuous treatment and cyclic (3 weeks of 4 beginning 1 week after 8 weeks continuous) treatment groups. Treatment consisted of 3% DFMO in the drinking water (5.1 g/kg/day). The tumors in the untreated group grew to 27 cm3 by 8 weeks and the animals had a median survival of 7.6 weeks. Tumor growth was inhibited by 99% (0.3 cm3) in the continuous treatment group in comparison to untreated controls. Survival was prolonged with 93% survival at 10 weeks and a 101% increase in median survival to 15.3 weeks (P less than 0.05). The cyclic DFMO group had a 98.3% inhibition in tumor growth for longer than 1 year (0.56 cm3; P less than 0.05). Survival was also markedly prolonged compared to the untreated group with 100% survival up to 24 weeks and a median survival of 54.3 weeks (P less than 0.05). No significant toxicities were observed in the first 10 weeks of DFMO treatment even though antitumor effects were observed. With continuous DFMO treatment, the animals eventually became debilitated and developed marked weight loss and thrombocytopenia; by 20 weeks, mortality was 79%. With cyclic therapy, the animals resumed weight gain, recovered from thrombocytopenia and, at 20 weeks, had 0% mortality. By 55 weeks, mortality was 50% which, however, was not significantly different (P approximately 0.50) from mortality of a control group of nontumorous, athymic mice that had weekly body weight and skin fold measurements concurrently with the experimental, tumor bearing animals. Thus, the observed mortality is ascribable to continuous encroachment on the normally sterile environment. These data suggest a role for DFMO in long-term therapy of sensitive human tumors such as small cell lung carcinoma, especially in patients with a low tumor burden. Furthermore, a cyclic regimen might be an important tool in maintaining clinical remissions induced by conventional combination chemotherapy. PMID- 3004709 TI - Expression of retroviral sequences and oncogenes in murine hepatocellular tumors. AB - The expression of endogenous retroviral sequences and of three cellular oncogenes was examined in three hepatocellular adenomas and in four carcinomas induced in male C57BL/6JDp X C3Hf/Dp F1 (hereafter called B6C3F1) mice by a single dose of nitrosodiethylamine, in five carcinomas that arose spontaneously in male C3Hf mice, and in the livers of normal age-matched control mice. In all of these adenomas and carcinomas, there was increased expression of Moloney murine leukemia virus- and intracisternal A particle-related sequences. The retrovirus like VL30 sequence was expressed at significant levels in the normal liver of these mice and increased expression of this sequence was found in only 4 of the 12 tumors examined. Expression of endogenous mouse mammary tumor virus-related sequences was not detected in the normal livers or in any of the liver tumors. With respect to cellular oncogenes, increased expression of c-myc was seen in all of the B6C3F1 tumors. Two of five normal liver samples and all of the tumors of the C3Hf mice displayed significant expression of c-myc. There was a slight increase in expression of c-Ha-ras in some of the tumors. Increased expression of c-fos was found in only 1 of the 12 tumors. Taken together, these studies indicate that both carcinogen-induced and spontaneous liver tumor formation in mice is associated with abnormalities in the expression of endogenous retrovirus related DNA sequences and also specific cellular oncogenes. PMID- 3004710 TI - Heterogeneity in the hormonal responsiveness of clones derived from the 13762NF rat mammary tumor. AB - The transplantable hormone-responsive rat mammary adenocarcinoma 13762NF was dissociated with collagenase and hyaluronidase. Cells were cloned directly or lines were established from mass cultures and cells from these lines were cloned. Clones differed in cellular morphology, colony morphology on plastic or in collagen gel, growth rate, growth response to hormones, and hormone receptor levels. Growth response to prolactin, estradiol, progesterone, cortisol, and epidermal growth factor (EGF) was determined by culturing the cells within collagen gel and using a serum-free medium base of DME/F12 (1:1) with insulin, linoleic acid, and BSA. The clones varied in their hormone responses, with all 20 of the clones tested responding to cortisol in combination with EGF. Some clones would respond to EGF, cortisol, or progesterone when used alone. None of the clones tested could be stimulated by prolactin or estradiol. Receptor levels for estradiol, progesterone, glucocorticoids, and EGF were assessed in 3 selected clones differing in their hormone responsiveness. Receptor levels appeared to correlate with hormonal sensitivity. Selected clones transplanted into female F344 rats produced carcinomas with histopathologies similar to the original tumor. PMID- 3004711 TI - Characterization of acquired epipodophyllotoxin resistance in a Chinese hamster ovary cell line: loss of drug-stimulated DNA cleavage activity. AB - Recent evidence indicates that type II DNA topoisomerases mediate epipodophyllotoxin-induced DNA damage and may be intrinsic to the drug's antitumor effects. Using an epipodophyllotoxin-resistant cell line, we have now further defined the relationship between DNA damage and cell death and delineated the significance of certain drug-enzyme interactions. When compared to wild-type cells, the mutant Chinese hamster ovary cell line, VpmR-5, exhibits marked resistance to both the cytotoxic and DNA cleavage activities of etoposide (VP 16). Steady-state concentrations of radiolabeled VP-16 are identical in both cell lines. Catalytic activity in crude nuclear extracts from wild-type and VpmR-5 cells is equal and is equally sensitive to inhibition by VP-16. However, using an assay that specifically measures generation of 5' protein-linked breaks in 32P labeled 3' DNA, we have found that DNA cleavage activity in nuclear extract from the VpmR-5 line is profoundly resistant to stimulation by VP-16. Further, a somatic cell hybrid line of VpmR-5 cells and drug-sensitive EOT-3 cells exhibits recovery of VP-16 sensitivity in concert with reconstitution of DNA cleavage activity. These data indicate that stimulation of enzyme-mediated DNA cleavage, rather than loss of normal topoisomerase function, is responsible for epipodophyllotoxin-induced cytotoxicity. PMID- 3004712 TI - Cross-resistance to intercalating agents in an epipodophyllotoxin-resistant Chinese hamster ovary cell line: evidence for a common intracellular target. AB - Several intercalating agents, as well as the epipodophyllotoxins, appear to effect DNA damage through their interaction with type II DNA topoisomerases. However, the relationship of this phenomenon to anti-tumor activity remains unproven. Our studies with an epipodophyllotoxin-resistant cell line not only provide additional evidence that the enzyme is a multidrug target but also serve to implicate it as a mediator of cytotoxic effect. When compared to wild-type cells, the epipodophyllotoxin-resistant Chinese hamster ovary cell line, VpmR-5, exhibits cross-resistance to both the cytotoxic and DNA cleavage activities of 4',9-acridinylaminomethanesulfon-m-anisidide, mitoxantrone, and Adriamycin. Steady-state concentrations of radiolabeled-4',9-acridinylaminomethanesulfon-m anisidide and daunomycin are identical in both cell lines. Sharp plateaus in the VpmR-5 dose-response curves for Adriamycin-induced DNA strand breaks and cytotoxicity appear to be related to interference with type II topoisomerase mediated cleavage of DNA at high concentrations of the intercalator. These data support a direct role for DNA strand scission in cell death and also suggest that multidrug resistance may be acquired by a qualitative change in type II topoisomerase that alters interaction of drug with the enzyme or enzyme-DNA complex. PMID- 3004713 TI - DNA sequence selectivity of guanine-N7 alkylation by three antitumor chloroethylating agents. AB - The DNA sequence selectivities of guanine-N7 alkylation produced by three chloroethylating antitumor agents, 1-(2-chloroethyl)-3-(cis-2-hydroxy) cyclohexyl 1-nitrosourea (cis-2-OH CCNU), 2-chloroethyl (methylsulfonyl)methanesulfonate, and 8-carbamoyl-3-(2-chloroethyl)imidazo-[5,1-d]-1,2,3,5-tetrazin-4(3H )-one (mitozolomide), were examined using a modification of the Maxam and Gilbert sequencing technique. In a region of pBR322 DNA, 2-chloroethyl (methylsulfonyl)methanesulfonate produced approximately the same degree of alkylation at all guanines. cis-2-OH CCNU, however, preferentially alkylated the middle guanines in runs of three or more guanines; the intensity of the reaction increased with the number of adjacent guanines in the DNA sequence. Mitozolomide produced the same pattern of preferential alkylation but not as intensely as cis 2-OH CCNU. Three other nitrosoureas, 1-(2-chloroethyl)-3-cyclohexyl-1 nitrosourea, 1-(2-fluorethyl)-3-cyclohexyl-1-nitrosourea, and 1-(2-chloroethyl)-1 nitrosourea gave similar patterns of alkylation to that of cis-2-OH CCNU at pH 7.2. The ratio of 7-hydroxyethylguanine to 7-chloroethylguanine was approximately the same following treatment of the synthetic polymers dGn X dCn and (dG X dC)n with cis-2-OH CCNU, indicating that 7-chloroethylation and 7-hydroxyethylation were enhanced similarly by the presence of adjacent guanines. Ethylnitrosourea produced relatively little alkylation preference. The results suggest that the alkylating intermediates, 2-chloroethyldiazohydroxide and 2 hydroxyethyldiazohydroxide, tend to react preferentially with those guanine-N7 positions the electronegativity of which is enhanced by the presence of neighboring guanines. This is consistent with the presence of cationic character in the alkylating centers of these intermediates. 2-Chloroethyl (methylsulfonyl)methanesulfonate and ethyldiazohydroxide would not be expected to have strong cationic character, in agreement with their lack of sequence selectivity. PMID- 3004714 TI - Insulin and epidermal growth factor receptors in rat liver after administration of the hepatocarcinogen 2-acetylaminofluorene: ligand binding and autophosphorylation. AB - The livers of male F344 rats which were fed 0.02% 2-acetylaminofluorene (2-AAF) for two days or more had decreased binding of insulin and epidermal growth factor (EGF) to their hepatic receptors in microsomal and Golgi fractions. Hepatic receptors which were partially purified from carcinogen-fed rats by Triton X-100 solubilization and wheat germ agglutinin affinity column chromatography also had decreased binding activity compared to receptors from normal rats. Scatchard analysis indicated that the decrease in insulin receptor binding was due to decreased receptor number whereas the change in EGF receptor binding was attributed to decreased receptor affinity. Insulin receptor phosphokinase activity was also decreased in 2-AAF-fed rats and correlated with the decrease in receptor binding. EGF receptor phosphokinase activity was unchanged in 2-AAF-fed rats when stimulated with a high concentration (1 microM) of EGF but was decreased when stimulated with low concentrations (0.01-0.1 microM) of EGF. No EGF or insulin competing activity for receptor binding was found using acid ethanol extracts of 2-AAF-altered liver. These results suggest that 2-AAF causes different alterations in the insulin and EGF receptors of the rat liver. PMID- 3004715 TI - Connective tissue degradation by invasive rat bladder carcinomas: action of nonspecific proteinases on collagenous matrices. AB - The invasive RBTCC-8 rat bladder carcinoma cell line (passage number, greater than 100) and its derivates, the RBTCC-8 tumor isografts and the 1-RBTCC-8 daughter cell line (fourth passage), express proteolytic activities of broad substrate specificity, which allow them to efficiently degrade extracellular (collagenous) matrices. Cell-associated, collagenolytic activity is evidenced by the release of hydroxyproline from collagen substrates of types I and IV, by visualizing the low-molecular-weight collagen breakdown products on sodium dodecyl sulfate-polyacrylamide gels, and by the depth of invasion into extracellular matrices in our bone invasion assays. Fractionated by diethylaminoethyl column chromatography, the major collagenolytic activities against collagens of types I and IV coelute in a relatively narrow peak within a NaCl gradient. The pooled collagenolytic diethylaminoethyl fractions contain: (a) two chymotrypsin-like, catheptic activities; (b) activity against a synthetic elastase substrate; (c) gelatinase activity; and (d) caseinolytic activity. Despite efficient collagenolysis, a vertebrate-type collagenase cannot be detected in any of our tumor samples, even after trypsin activation of the tumor cell extracts. The mechanism of action of these nonspecific proteinases is thought to be that of collagen "crosslinkases." The neutral proteinase activities are highest in RBTCC-8 tumor isografts, intermediate in the fourth passage 1 RBTCC-8 carcinoma cell line, and lowest in the RBTCC-8 carcinoma cell line of high passage number. The levels of these nonspecific enzyme activities are well correlated with the depth of invasion into bony matrices, as shown by our invasion assays. PMID- 3004717 TI - Comparison of amplified and unamplified c-myc gene structure and expression in human small cell lung carcinoma cell lines. AB - A human small cell lung cancer cell line (H82) demonstrates 40- to 50-fold amplification of the c-myc gene but expresses at least 250-fold more steady-state c-myc messenger RNA than an unamplified small cell lung cancer cell line (H378) with no detectable expression of c-myc. We compared the chromatin structure of c myc in H82 to that in H378 using DNase I sensitivity and DNA methylation patterns. DNase I hypersensitivity sites were identical in H82 and H378 and were similar to the pattern seen in a B-lymphoblastoid cell line, despite extensive amplification of c-myc in H82. Methylation patterns were also very similar in H82 and H378, with hypomethylation or partial methylation at the c-myc coding regions and the flanking 5' sequences, despite the absence of detectable c-myc expression in H378. Therefore, the predominant chromatin structural patterns do not appear to correlate with observed differences in gene expression. In addition, these studies demonstrate that the patterns of DNase I hypersensitivity and of methylation can remain intact during a 40- 50-fold gene amplification, as observed for the c-myc gene in H82. PMID- 3004716 TI - DNA damage by antitumor acridines mediated by mammalian DNA topoisomerase II. AB - Antitumor drugs from many chemical classes have been shown to induce protein linked DNA breaks in cultured mammalian cells and in vitro in the presence of purified mammalian DNA topoisomerase II. The possibility that mammalian DNA topoisomerase II is an intracellular target which mediates drug-induced DNA breaks is supported by the following studies using 4'-(9-acridinylamino)methane sulfon-m-anisidide (m-AMSA): (a) a single m-AMSA-dependent DNA cleavage activity copurified with calf thymus DNA topoisomerase II activity at all chromatographic steps of the enzyme purification; (b) m-AMSA-induced DNA cleavage by this purified activity resulted in the covalent attachment of protein to the 5'-ends of the DNA via a tyrosyl phosphate bond. This covalently linked protein has the same reduced molecular weight as purified calf thymus DNA topoisomerase II. The possibility that topoisomerase II-mediated DNA breaks may be responsible for cytotoxicity has also been investigated using a number of m-AMSA-related acridines. The level of topoisomerase II-mediated DNA breaks in vitro strongly correlates with the level of protein-linked DNA breaks in cultured cells and drug induced cytotoxicity. These results suggest that mammalian DNA topoisomerase II may be a cytotoxic target of antitumor acridines. PMID- 3004718 TI - Effector mechanism in antitumor activity of monoclonal antibodies produced against an ascitic mouse mammary tumor. AB - Therapeutic effects of monoclonal antibodies with different immunoglobulin classes, detecting the same antigenic determinant of tumor specific antigen expressed on ascitic mouse mammary tumor MM46, were examined. With i.v. administration of gamma 2a, gamma 2b, or gamma 1 antibody we were able to keep a significant proportion of mice tumor free against i.p. inoculation of 5 X 10(4) MM46 cells with doses as small as 0.5 micrograms, but with administration of mu, gamma 3, or alpha antibody we were not able to keep mice tumor free with doses up to 5 micrograms. With 200 micrograms of antibody, however, an antitumor effect was observed even with mu or gamma 3 antibody, although alpha antibody still showed no effect at all. The therapeutic effect of gamma 2a was further examined in mice challenged with an increasing dose of tumor cells, and a significant effect was demonstrated against 1 X 10(6) cells with 200 micrograms of antibody but not against 5 X 10(6) cells. To assess the effector cells in antitumor activity of antibody in vivo, a histological examination of the tumor cells treated with each class of antibody was carried out. The tumor treated by gamma 2a antibody revealed a remarkable cell infiltration consisting predominantly of mononuclear cells, whereas those treated by mu, gamma 3, or alpha antibody did not. The tumor cells treated by gamma 1 or gamma 2b antibody showed a moderate cellular reaction. Next, a Winn assay was carried out in athymic C3H/HeN-nu/nu mice using gamma 2a antibody. A significant antitumor effect was observed even in those mice, indicating that T-cells are not predominant effector cells. The role of macrophages was studied in mice treated with two macrophage toxic agents, carrageenan and silica particles. These agents were shown to reduce the antitumor effect of gamma 2a antibody in both Winn assays and therapeutic experiments. Thus, histological examination and the blocking effect of macrophage toxic agents suggested the participation of host macrophages as effector cells in antibody mediated tumor cell suppression in vivo. PMID- 3004719 TI - Cytotoxic murine monoclonal antibody LAM8 with specificity for human small cell carcinoma of the lung. AB - The reactivity of the murine immunoglobulin monoclonal antibody LAM8 directed against a membrane antigen of human small cell carcinoma (SCC) of the lung was investigated on human cell lines and tissues. Indirect immunofluorescence staining, radioimmunoassays, and cytotoxicity assays showed LAM8 antibody to selectively react with SCC but not with non-SCC lung cancer cell lines and extrapulmonary tumor cell lines. Unlike other SCC antibodies, including those we have previously described, highly preferential reactivity with SCC tissues was also demonstrated by immunoperoxidase staining of deparaffinized formalin-fixed tissue sections. Membrane and cytoplasmic staining was seen in of 9 of 12 SCC tissues. No significant staining was seen in non-SCC lung cancer and a wide range of other tumors, including mesothelioma and bronchial carcinoids. Significant LAM8 reactivity was also absent in normal tissues of all major organs. Few tumors and epithelial tissues, including bronchial epithelium had rare LAM8 positive cells which were always less than 2% of the entire cell population. In vitro treatment with antibody and human complement was highly cytotoxic to SCC cells, but had not effect on bone marrow progenitor cells. Immunoblotting of membrane extracts separated on sodium dodecyl sulfate-polyacrylamide gels showed the LAM8 antigen to have a band of an approximate molecular weight of 135,000 and a cluster of bands with approximate molecular weights of 90,000. This reactivity was lost after incubation of the extracts with periodate. LAM8 antibody shows a highly preferential reactivity with SCC cell lines and formalin-fixed paraffin embedded SCC tissues and is selectively cytotoxic to cells expressing LAM8 antigen. PMID- 3004720 TI - Rescue of a biologically active Epstein-Barr virus from nonproducer cells. AB - We recently established an epithelial/hybrid cell line, A2L/AH, by fusion of 8 azaguanine-resistant epithelial cells, Ad-AH, with a nonproducer lymphoblastoid cell line, A2L, using lymphocytes derived from the human nasopharynx transformed with the B95-8 strain of Epstein-Barr virus. The treatment of the parental A2L lymphoid cells with iododeoxyuridine led to the expression of Epstein-Barr virus early antigen, but neither virus capsid antigen or induction of Epstein-Barr virus DNA synthesis was observed. However, the treatment of the A2L/AH hybrid cells with iododeoxyuridine led to early antigen, virus capsid antigen and virus DNA synthesis, and the formation of virus particles. The virus rescued from the A2L/AH hybrid cells transforms human cord blood lymphocytes but is not able to induce early antigen in superinfected Raji cells. PMID- 3004721 TI - Anomalous adenosine cyclic 3':5'-monophosphate responses to glucagon in patients with hepatocellular carcinoma. AB - Glucagon resistance has been reported in rat hepatoma models. We studied the responses to glucagon challenge in 35 patients with hepato-cellular carcinoma. They have increased cyclic AMP and decreased glucose responses to glucagon (2 mg) challenge when compared with normal controls. Possible explanations for increased cyclic AMP responses include special membrane properties of hepatoma cells and increased adrenergic stimulation of adenylate cyclase during hypoglycemia. Decreased glucose responses are most apparent in patients with overt hypoglycemia. This may be related to a number of postulates, including depleted glycogen store of liver, impaired glycogenolysis, fatty metamorphosis, or insulin like activities secreted by hepatoma. In this study, the increased cyclic AMP responses do not support the postulation that glucagon receptors are damaged in hepatocellular carcinoma. PMID- 3004722 TI - Establishment in long term culture of megakaryocytic leukemia cells (EST-IU) from the marrow of a patient with leukemia and a mediastinal germ cell neoplasm. AB - Megakaryocytes are the bone marrow cells that generate platelets. They are relatively rare cells, comprising between 0.03 and 0.06% of all nucleated marrow cells (R. L. Berkow et al., J. Lab. Clin. Med., 103: 811-818, 1984). The study of human megakaryocyte differentiation and function has been hampered by the small number of these cells available for study. Recently we have established a human cell line (EST-IU) from the marrow of a patient with an acute nonlymphocytic leukemia and a mediastinal germ cell tumor. While this cell line seems to express many of the phenotypic characteristics of human megakaryocytes, it does not appear to express any phenotypic properties associated with cells of the erythroid, lymphoid, granulocytic, or monocytic lineages. Transmission electron microscopy demonstrates frequent multinucleated cells. Staining for platelet peroxidase reactivity revealed darkening of the perinuclear envelope and the endoplasmic reticulum, a characteristic of cells of the megakaryocytic lineage. Indirect immunofluorescence assays reveal that EST-IU expresses reactivity with anti-platelet glycoprotein antisera, anti-Factor VIII-related antigen antisera, anti-Factor V antisera, anti-thrombocyte antisera, Tab (monoclonal anti-platelet glycoprotein IIb-IIIa), and anti-fibronectin antisera. Flow cytometry-derived DNA histograms demonstrate populations with multiple ploidies, ranging from approximately 4N to 32N. Based upon morphological and histochemical characteristics, antigenic expression, and nuclear characteristics, EST-IU cells appear to have a phenotype that closely resembles human megakaryocytes. This cell line should be useful in the further cell study of the molecular and cell biology of human megakaryocytopoiesis. PMID- 3004723 TI - Presence of human papillomavirus type 16 related sequences in verrucous carcinoma of the larynx. AB - Verrucous carcinoma of the larynx clinically resembles laryngeal papilloma in that both are wart-like masses on the vocal cords and may be characterized by multifocality and recurrence. Human papillomavirus (HPV) infection is an etiological factor in laryngeal papilloma, and recent evidence implicates HPV in squamous neoplasias. To determine whether HPV is also associated with verrucous carcinoma of the larynx, we analyzed tissue specimens from six patients with verrucous carcinoma of the larynx by Southern and DNA dot blot hybridization for HPV DNA. From three patients, specimens of normal laryngeal epithelium were also studied. All tissues showed evidence of HPV sequences related but not identical to HPV-16. They were negative for HPV-11. In contrast, four squamous cell carcinomas of the larynx and three normal laryngeal tissues were negative for HPV DNA. Histological sections of the six verrucous lesions were found to contain koilocytosis. Immunoperoxidase staining for HPV capsid antigens was negative in all these cases. The consistent and specific association of HPV with the verrucous carcinomas in this report suggests the possibility of a pathogenic involvement. PMID- 3004725 TI - Pharmacokinetic evaluation of increasing dosages of etoposide in a chronic hemodialysis patient. AB - A chronic hemodialysis patient was treated for small cell lung cancer with a combination therapy consisting of doxorubicin, cyclophosphamide, and etoposide. The dose of etoposide was increased to normal in five steps because of the possible accumulation and higher plasma peak levels due to terminal renal failure. In order to study this phenomenon, plasma, urine, and dialysate levels of etoposide were determined during five treatment courses with a high performance liquid chromatographic method combined with electrochemical detection; this proved that the pharmacokinetic curves for etoposide fit a two compartment model. Peak plasma levels were 7.96, 10.84, 18.24, 21.0, and 24.0 micrograms/ml for dosages of etoposide of 75, 100, 150, 200, and 250 mg, respectively. No accumulation occurred, half-life times of the elimination phase were between 8.7 and 23.0 hours, apparent distribution volumes of the central compartment were between 9.2 and 14.5 L, and the total-body clearances were between 2.1 and 2.8 L/hour. Thus, pharmacokinetic parameters were comparable with those obtained in patients with normal renal function. Hemodialysis did not disturb the exponential decay of plasma etoposide concentration during the elimination phase. No etoposide was found in the dialysate and only minimal etoposide was recovered from the urine. The data presented here indicate that renal clearance is not indispensable for eliminating etoposide. PMID- 3004724 TI - Cyclic alternating chemotherapy for small cell carcinoma of the lung. AB - Eighty-two previously untreated patients with small cell cancer of the lung were treated with six cycles of two alternating drug regimens: a new combination of mitomycin, methotrexate, and etoposide; and cyclophosphamide, doxorubicin, and vincristine. No maintenance chemotherapy was used. Consolidative thoracic irradiation and prophylactic cranial irradiation were employed. The median survival time for 32 limited-disease patients was 59 weeks, and for 50 extensive disease patients was 35 weeks. Four-year survival was 12% for limited-disease patients and 2% for extensive-disease patients. These results were not superior to conventional combination chemotherapy regimens. PMID- 3004726 TI - Phase II trial of 5-FU, vincristine, and mitomycin (FOMi) in metastatic bronchioloalveolar cell lung cancer: a Southwest Oncology Group Study. AB - Twenty-three previously untreated patients with bronchioloalveolar cell lung cancer who had measurable disease and distant metastases (stage IIIM1, extensive) were treated with combination chemotherapy including 5-FU, vincristine, and mitomycin. Two of 23 patients (9%) achieved partial response lasting 5 and 6 months. Two patients (9%) died of sepsis while neutropenic. The current study does not justify the use of 5-FU, vincristine, and mitomycin combination chemotherapy in patients with metastatic bronchioloalveolar cell lung cancer. PMID- 3004727 TI - Vinblastine plus cisplatin in advanced non-small cell lung cancer: lack of advantage for vinblastine infusion schedule. AB - We have evaluated 31 patients with advanced non-small cell lung cancer treated by short-term 5-day vinblastine infusion combined with bolus cisplatin. Nine of 31 patients (29%) had partial and complete responses. Although five of nine (55%) of the responders were alive at greater than or equal to 1 year, three of the 31 patients experienced drug-related mortality. Our experience, as well as a review of previously reported trials in the literature, suggests that the infusion schedule of vinblastine offers no advantage over the bolus schedule. PMID- 3004728 TI - Cytarabine: an inactive drug for extensive-stage small cell lung cancer. PMID- 3004729 TI - Phase II evaluation of cisplatin in advanced hepatocellular carcinoma and cholangiocarcinoma: a Southeastern Cancer Study Group Trial. PMID- 3004730 TI - Synthesis of methyl 6-(ammonium 2-acetamido-2-deoxy-alpha-D-glucopyranosyl phosphate)-alpha-D-mannopyranoside and use of this compound for the determination of N-acetylglucosamine-1-phosphotransferase. AB - Methyl 6-(ammonium 2-acetamido-2-deoxy-alpha-D-glucopyranosyl phosphate)-alpha-D mannopyranoside was synthesized and identified by 1H-n.m.r. and 13C-n.m.r. data, acid hydrolysis, and elemental analysis. It was utilized for the determination of UDP-N-acetylglucosamine-1-phosphotransferase in an assay procedure that employed methyl alpha-D-mannopyranoside as an acceptor. The assay product was identified and characterized by thin-layer chromatography with the title reference compound. The present technique does not require [32P]UDP-N-acetylglucosamine, but effectively uses commercially available UDP-[14C]GlcNAc. PMID- 3004731 TI - Differential subcellular localization of ACTH and alpha-MSH in corticotropes of the rat anterior pituitary. AB - Specific antisera to alpha-melanotropin (alpha-MSH) and corticotropin (ACTH 1-39) were used to obtain immunocytochemical evidence for the differential localization of alpha-MSH and ACTH in the secretory granules of corticotropes of rat anterior pituitary. The specificity of the antisera was established by binding 131I labeled alpha-MSH and ACTH 1-39 to their respective antisera. Double-labeling immunocytochemistry (for alpha-MSH, ferritin; for ACTH, colloidal gold) was performed. Some secretory granules were labeled with ferritin particles (alpha MSH), whereas others contained gold particles (ACTH). Only a few granules showed both ACTH and alpha-MSH. In typical corticotropes (stellate in form with a small number of secretory granules aligned along the cell periphery) only some of the secretory granules that were labeled with anti-ACTH serum were also immunoreactive to anti-alpha-MSH. In atypical corticotropes (polygonal in shape and containing a large number of secretory granules) almost all of the immunoreactive ACTH secretory granules were also positive to anti-alpha-MSH serum. An intermediate type of corticotrope was observed containing a small number of secretory granules, almost all of which were labeled with anti-alpha MSH. Thus, rat anterior pituitary corticotropes may be classified into three types according to the distribution and content of alpha-MSH. The light microscopic immunocytochemistry provided similar results. PMID- 3004732 TI - The adrenal: a new target organ of the calciotropic hormone 1,25-dihydroxyvitamin D3. AB - Target cells for 1,25-dihydroxyvitamin D3 were demonstrated in the adrenal medulla by frozen-section autoradiography. The appearance of these target cells was age-dependent in neonatal mice. Immunocytochemical staining for phenylethanolamine-N-methyltransferase revealed that both epinephrine and non epinephrine cells concentrate 1,25-dihydroxyvitamin D3 in their nuclei. In contrast, immunocytochemical staining for "vitamin D-dependent calcium-binding protein" (D-CaBP) demonstrated that D-CaBP immunoreactivity is localized in only a small percentage of adrenomedullary cells, in mice and rats. Comparison of PNMT and D-CaBP immunoreactivities in sequential sections showed that epinephrine producing cells do not contain D-CaBP. These results indicate that adrenal medullary cells have receptors for 1,25-dihydroxyvitamin D3 and that 1,25 dihydroxyvitamin D3 may directly affect certain functions of these endocrine cells. PMID- 3004735 TI - [Relation between the mortality of primary hepatic carcinoma and viral hepatitis in the surveillance areas of Jiangmen City]. PMID- 3004734 TI - Acute response of testicular interstitial tissue in rats to the cytotoxic drug ethane dimethanesulphonate. An ultrastructural and hormonal assay study. AB - The cytotoxic effects of ethane dimethanesulphonate upon rat Leydig cells were examined ultrastructurally up to 3 days after treatment and related to changes in serum levels of gonadotrophins and testosterone. Six hours after administration of ethane dimethanesulphonate the usual tubulo-vesicular morphology of Leydig cell smooth endoplasmic reticulum was converted to small vesicles and the Golgi apparatus showed focal hypertrophy into anastomosing tubules. These changes became more marked by 12 h with many Leydig cells exhibiting karyopyknosis and hyperchromatism. Necrotic Leydig cells were often engulfed by macrophages, the latter containing pyknotic fragments of Leydig cells within their cytoplasm. One day after administration, advanced necrosis of Leydig cells occurred, many of which were phagocytosed by macrophages, and on day 3, destruction of Leydig cells was complete resulting in their elimination from the interstitial tissue, which contained only loose connective tissue and macrophages. Structural alterations to the Leydig cells from 6-24 h was reflected by a significant reduction in serum testosterone levels which further declined to the limits of detection accompanying the abolition of Leydig cells on day 3. These changes were paralleled by a significant elevation of serum LH and FSH levels suggesting diminished feedback regulation of pituitary gonadotrophin secretion. The results indicate that ethane dimethanesulphonate is a rapidly acting Leydig cell toxin which may be a useful experimental tool in further studies of spermatogenic function mediated via Sertoli cell-Leydig cell interaction. PMID- 3004733 TI - Short- and long-term effects of ACTH on the adrenal zona glomerulosa of the rat. A coupled stereological and enzymological study. AB - Short-term ACTH treatment provoked a decrease in volume of the lipid-droplet compartment in rat zona glomerulosa cells, and a rise in plasma and intracellular concentrations of corticosterone and aldosterone. It enhanced activities of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), 11 beta-hydroxylase (11 beta OH) and 18-hydroxylase (18OH). Long-term ACTH administration produced a hypertrophy of the zona glomerulosa and its parenchymal cells, a result of the increase in volume of the smooth endoplasmic reticulum and the mitochondrial compartment. The surface area per cell of mitochondrial inner membranes increased; the tubular cristae were transformed into a homogeneous population of vesicles. The plasma and intracellular concentrations of corticosterone further increased, whereas those of aldosterone fell below basal levels (the "aldosterone-escape" phenomenon). The activities of 3 beta HSD and 11 beta OH were enhanced, that of 180H decreased. Therefore, ACTH stimulates zona glomerulosa growth and transforms parenchymal elements into zona fasciculata cell-types. Cyanoketone nullified acute ACTH effects on plasma and intracellular concentrations of corticosterone and aldosterone, but did not affect the activities of 11 beta OH and 18OH. Chronic ACTH treatment produced similar results, although 18OH activity was not suppressed. The mechanism underlying the "aldosterone-escape" phenomenon may thus involve a rise in the intracellular concentration of corticosterone, caused by the enhanced synthesis and activation of 3 beta HSD and 11 beta OH. PMID- 3004736 TI - [Inactivation of polioviruses by the methane-producing fermentation method]. PMID- 3004737 TI - [Epidemiology of rota-virus gastro-enteritis]. PMID- 3004738 TI - Universal code equivalent of a yeast mitochondrial intron reading frame is expressed into E. coli as a specific double strand endonuclease. AB - The intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae (r1 intron) possesses a 235 codon long internal open reading frame (r1 ORF) whose translation product determines the duplicative transposition of that intron during crosses between intron-plus strains (omega+) and intron-minus ones (omega ). Using site-directed mutagenesis, we have constructed a universal code equivalent of the r1 ORF that, under appropriate promoter control, allows the overexpression in E. coli of a protein identical to the mitochondrial intron encoded "transposase". This protein exhibits a double strand endonuclease activity specific for the omega- site. This finding demonstrates, for the first time, the enzymatic activity of an intron encoded protein whose function is to promote the spreading of that intron by generating double strand breaks at a specific sequence within a gene. PMID- 3004739 TI - Homologous recognition of a promoter domain common to the MSV LTR and the HSV tk gene. AB - We have partially purified, from rat liver, a nuclear protein fraction with sequence-specific affinity to a promoter domain shared by the herpesvirus tk gene and the Moloney murine sarcoma virus LTR. For both promoters, the protein-binding domain occurs roughly 80 bp upstream of the mRNA cap site and harbors the pentanucleotide sequence 5'-CCAAT-3'. The MSV LTR pentanucleotide occurs on the coding strand in an orientation pointing toward the retroviral transcription unit. The HSV tk pentanucleotide occurs on the noncoding strand in an orientation pointing away from the gene. Assays in microinjected frog oocytes and transfected mouse L cells indicate that equivalent point mutations, introduced at each residue of the CAT pentanucleotide of each promoter, lead to similar changes in promoter activity. Furthermore, they similarly alter binding of the nuclear protein fraction. Surprisingly, a C to G transversion at the first residue of the CAT pentanucleotide, which severely impairs the activity of both promoters, appears to increase affinity of the CAT binding protein. PMID- 3004741 TI - Ras p21 proteins with high or low GTPase activity can efficiently transform NIH/3T3 cells. AB - We sought to determine whether decreased in vitro GTPase activity is uniformly associated with ras p21 mutants possessing efficient transforming properties. Normal H-ras p21-[Gly12-Ala59] as well as an H-ras p21-[Gly12-Thr59] mutant exhibited in vitro GTPase activities at least fivefold higher than either H-ras p21-[Lys12-Ala59] or H-ras p21-[Arg12-Thr59] mutants. Microinjection of as much as 6 X 10(6) molecules/cell of bacterially expressed normal H-ras p21 induced no detectable alterations of NIH/3T3 cells. In contrast, inoculation of 4-5 X 10(5) molecules/cell of each p21 mutant induced morphologic alterations and stimulated DNA synthesis. Moreover, the transforming activity of each mutant expressed in a eukaryotic vector was similar and at least 100-fold greater than that of the normal H-ras gene. These findings establish that activation of efficient transforming properties by ras p21 proteins can occur by mechanisms not involving reduced in vitro GTPase activity. PMID- 3004740 TI - Detection of a subunit of cellular Pol II within highly purified preparations of RNA polymerase isolated from rabbit poxvirus virions. AB - A large (approximately 200 kd) subunit of cellular RNA polymerase II (Pol II) and a virus-encoded subunit of rabbit poxvirus (RPV) RNA polymerase (137 kd) react with common monoclonal antibodies. Hybridization studies with viral and cellular DNA clones confirm that the viral and cellular proteins are related. Following RPV infection, the Pol II subunit is translocated from the nucleus to the cytoplasmic virosomes, then packaged into mature virus, and is found associated with the viral RNA polymerase purified from virions. The results suggest that the cellular Pol II subunit may be directly involved in the transcription of viral genes. PMID- 3004742 TI - The human glucose transporter can insert posttranslationally into microsomes. AB - RNA transcripts encoding the complete human glucose transporter (GT) or fragments of GT corresponding to the NH2-terminal 340 amino acids (GT-N) or the COOH terminal 148 amino acids (GT-C) were synthesized in vitro from SP6 plasmids. The corresponding polypeptides were synthesized in reticulocyte or wheat germ cell free systems and their insertion into pancreatic microsomes was assessed by endoglycosidase H and trypsin digestion or alkaline extraction of membranes. The following observations were made: both GT and GT-N can insert posttranslationally into microsomes; GT contains at least two distinct signal sequences; both co- and posttranslational insertion of GT-N are mediated by a signal recognition particle (SRP)-SRP receptor-dependent mechanism; and SRP is required both for targeting the polypeptide to the membrane and for initiating the actual insertion process. We propose a model for the biosynthesis of GT and proteins of similar membrane topology which does not require a tight ribosome-membrane interaction. PMID- 3004743 TI - Lymphocyte homing receptors. PMID- 3004744 TI - An altered DNA conformation in origin region I is a determinant for the binding of SV40 large T antigen. AB - Seventeen base pairs of DNA from SV40 origin region I encode a tripartite binding site for a dimeric mass of SV40 large T antigen. Two binding components are the directly repeated pentanucleotide sequences 5'-GAGGC-3'/5'-GCCTC-3'. The third component is the asymmetric sequence 5'-TTTTTTG-3'/5'-CAAAAAA-3' that separates the pentanucleotides. Nucleotide-specific features of this spacer element stabilize binding to the adjacent pentanucleotides. We report here that the spacer sequence determines a DNA conformation that correlates with high affinity binding of T antigen. The nature of the spacer sequence suggests that the DNA is bent. We propose that binding of T antigen to region I proceeds through monomer pentanucleotide interactions and either protein-protein or protein-spacer interactions directed by the spacer-encoded structure. PMID- 3004745 TI - Different localization of the product of the v-rel oncogene in chicken fibroblasts and spleen cells correlates with transformation by REV-T. AB - Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus that transforms early lymphoid cells in vivo and in vitro, but REV-T does not transform chicken embryo fibroblasts (CEF). Using antisera to p59v-rel, the v-rel oncogene product of REV-T, we show that p59v-rel is expressed at equal levels and is a phosphoprotein in REV-T infected spleen cells and CEF. Biochemical fractionation and immunofluorescence of REV-T infected nontransformed CEF show that p59v-rel is loosely associated with the nucleus. However, in REV-T transformed spleen cells p59v-rel is primarily a cytoplasmic protein. MSB-1 cells, a Marek's disease virus transformed T cell leukemic line, and E26 virus transformed myeloid cells show nuclear staining of p59v-rel when they are infected by REV-T. Our results indicate that there is a correlation between a cytoplasmic localization of p59v-rel and transformation by REV-T, and they suggest that p59v-rel cannot transform cells in which it assumes solely a nuclear location. PMID- 3004746 TI - The presequences of two imported mitochondrial proteins contain information for intracellular and intramitochondrial sorting. AB - Gene fusion experiments were used to identify signals that direct imported precursor proteins to specific intramitochondrial locations in yeast. The amino terminus of alcohol dehydrogenase III (ADHIII, a mitochondrial matrix enzyme) transported attached mouse dihydrofolate reductase (DHFR, a cytosolic enzyme) into the mitochondrial matrix. The presequence of cytochrome c1 (a mitochondrial inner membrane protein protruding into the intermembrane space) transported attached DHFR into the intermembrane space. The first half of the cytochrome c1 presequence, which resembles the ADHIII presequence, is a matrix-targeting sequence: it transported attached DHFR into the matrix. The second half of the cytochrome c1 presequence contains a stretch of 19 uncharged amino acids and may thus be a stop-transfer sequence. We conclude that intramitochondrial sorting involves matrix-targeting and stop-transfer sequences within the cleavable presequence. PMID- 3004747 TI - New pharmacologic approaches to spinal cord injury: opiate antagonists and thyrotropin-releasing hormone. PMID- 3004748 TI - [Advances in the field of receptors]. PMID- 3004749 TI - Reduction of the metallochromic indicators murexide and tetramethylmurexide to their free radical metabolites by cytoplasmic enzymes and reducing agents. AB - Murexide underwent reduction by rat liver cytosolic fraction or a hypoxanthine xanthine oxidase system to produce a free radical metabolite. Reduction of murexide by the freshly prepared cytosolic fraction depended upon the presence of ascorbic acid. N1-Methylnicotinamide, xanthine or hypoxanthine, in that order, could also serve as a source of reducing equivalents for the production of that free radical by the cytosolic fraction. Several thiol compounds (GSH, cysteine, and cysteamine), pyridine nucleotides (NADH, NADPH) and ascorbic acid were also effective in generating the murexide-derived free radical. Tetramethyl murexide was also reduced to its free radical derivative by a hypoxanthine-xanthine oxidase system. PMID- 3004751 TI - Lethal and adjuvant activities of cord factor (trehalose-6,6'-dimycolate) and synthetic analogs in mice. PMID- 3004750 TI - Regulatory effect of calcium-binding protein isolated from rat liver cytosol on activation of fructose 1,6-diphosphatase by Ca2+-calmodulin. PMID- 3004752 TI - Preparation of an affinity gel for opiate receptors. PMID- 3004753 TI - Binding characteristics of [3H]dihydroalprenolol to beta-adrenergic receptors of rat brain: comparison with those of rat heart treated with neuraminidase. PMID- 3004754 TI - Measurements of the adhesive force between particles of powdered organic substances and a glass substrate by means of the impact separation method. II. Effect of addition of light anhydrous silicic acid on the adhesive force of potato starch. PMID- 3004755 TI - [Morphological characteristics of aldosteronoma]. PMID- 3004756 TI - [Ultrastructural changes of neonatal thymuses of mice infected with virus]. PMID- 3004757 TI - [Pathological appraisal of human gastric carcinoma cell line culture in vitro]. PMID- 3004758 TI - [Ultrastructural study on bronchioloalveolar carcinoma]. PMID- 3004759 TI - [Ultrastructural study of carcinomas of pancreatic head and ampullary region]. PMID- 3004761 TI - [Study on ultrastructural features of osteogenic sarcoma]. PMID- 3004760 TI - [Malignant fibrous histiocytoma: an ultrastructural study of 12 cases]. PMID- 3004762 TI - [Pituitary adenomas: pathologic analysis of 150 cases with light and electron microscopy]. PMID- 3004763 TI - [Pathological and immunohistochemical studies of medullary carcinoma of the thyroid]. PMID- 3004764 TI - [Pathological analysis of 208 cases of lung carcinoma diagnosed by fibroptic bronchoscope]. PMID- 3004765 TI - [Studies on the effect of lipoproteins and apoproteins on lipoprotein receptors. I. Isolation of lipoproteins and LDS by one-step ultracentrifugation]. PMID- 3004766 TI - [Tumor transforming growth factor II. Growth inhibition of human pulmonary adenocarcinoma cell lines with antiepidermal growth factor receptor antibody]. PMID- 3004767 TI - [Analysis of the genome of rotavirus from the feces of adult infectious diarrhea]. PMID- 3004768 TI - [Bronchogenic carcinoma (personal observations)]. AB - The authors report the results achieved in the treatment of 62 patients with pulmonary cancer "non-microcytoma". The survival rate was 20.9% at 5 years (actuarial). The best prognosis is for patients with epidermoid cancer: 35.5%. The authors emphasize the importance of the exact stage of the neoplasm and the evaluation of the postoperative cardiopulmonary functional adaptation in the prognostic evaluation of resection for pulmonary carcinoma. PMID- 3004769 TI - [A case of vascular leiomyosarcoma of the superficial soft tissues]. AB - The authors report a case of vascular leiomyosarcoma of the superficial soft tissues, and point out its epidemiologic (rareness of the report), biologic (high degree of malignancy, trend to local relapse and remote conversion into metastases), histopathologic aspects. They consider significant as to prognostic value the appraisal of the "mitotic index" and the opportunity of a radical intervention, followed by radiotherapy. They, moreover, think it useful, in consideration of the frequency of the relapses, to perform a periodic checking of the patient. PMID- 3004770 TI - [Partial nephrectomy in nephroblastoma]. AB - The authors analyse their 18 partial nephrectomies performed on 12 children with either a bilateral or a Wilms' tumor of a single remaining kidney: 13 affected kidneys were otherwise normal, the 5 others were sites of a diffuse nephroblastomatosis. The short term results are good (one technical failure only). The long term results were excellent for Wilms' tumors on otherwise healthy kidneys (no recurrence), as opposed to kidneys with diffuse nephroblastomatosis, where invariable tumor recurrence was observed. Because of the low risk of tumor development in the controlateral healthy kidney, and the established value of classical total nephrectomy, the authors, however, consider partial nephrectomy only in highly selected cases of unilateral nephroblastoma. PMID- 3004771 TI - Calmodulin and alpha tocopherol as additional binding sites for doxorubicin. AB - The hydrophobic probes anthroylcholine (9AC), 8-anilino-1-napthalene sulfonate (ANSE), and 2-P-toluidinyl naphthalene 6-sulfonate (TNS) increased calcium calmodulin (Ca2+-aM) fluorescence. This fluorescence was decreased by doxorubicin (DXR) in a dose-dependent fashion. The Ca2+ ion was an absolute requirement for the observed effects of DXR. DXR bound to the Ca2+-CaM complex (Kd = 4.2 X 10(-5) M, Bmax = 1.8) and to alpha tocopherol. The binding of untransformed (native) DXR to CaM was a reversible process. These data support a previous finding that DXR inhibits stimulation of calmodulin-deficient PDE (a CaM target enzyme) using either the Ca2+ CaM complex or alpha tocopherol by interacting with these agents, and suggest that other target enzymes for CaM may be similarly affected. PMID- 3004773 TI - Ascorbate-copper induced DNA lesions and repair in Escherichia coli K12 cells. AB - Multiple lines of evidence show that oxidation products of ascorbic acid (vitamin C) are capable of inducing a variety of genetic alterations in microbial and mammalian cells. We have studied the inactivation kinetics in repair proficient and deficient Escherichia coli K12 cells treated with oxidized solutions of ascorbic acid, in the presence of catalytic amounts of copper. Our results suggest that the repair pathways controlled by the recA and uvrA gene products (the latter in a recA strain) contribute to cell survival. However, the lack of beta-galactosidase induction, in the SOS chromotest, implies a role for the RecA protein other than SOS induction. Catalase and thiourea suppress the toxic effects of oxidized ascorbate solutions, confirming that H2O2 and hydroxyl radicals are intermediate agents in the damaging action. Single-strand breaks were detected in DNA from treated cells. PMID- 3004772 TI - The effect of dose on the bioavailability of oral etoposide. AB - The bioavailability of orally administered etoposide varies considerably. The effect of dose on bioavailability has not previously been investigated. In this study six patients were each treated with oral etoposide at doses of 200, 400, and 600 mg, and the pharmacokinetics determined. Each patient acted as his own control. The area under the plasma concentration-time curve (AUC) was proportionately greatest at the lowest dose. Doubling the dose from 200 mg to 400 mg increased AUC by only 50%, and a further increase of only 2.2% occurred at a dose of 600 mg. These data show nonlinear bioavailability of etoposide within the range in clinical use and may explain the variable results of reported studies. The data may have important implications for chemotherapy regimens with oral etoposide. PMID- 3004774 TI - Radiation-induced anchorage-independent growth and collagenase production in diploid human fibroblasts. AB - Early-passage human foreskin fibroblasts were exposed to X-ray doses ranging from 100 to 600 rad. The X-ray treatments resulted in cell killing in a dose-dependent manner as judged by the colony-forming efficiency of the cells. When cultures exposed to radiation were serially passaged and checked at various times for growth in semi-solid medium they showed the presence of cells with the ability to grow under anchorage-independent conditions (in agarose) at 24 population doublings. The frequencies of colonies with the ability to grow in agarose increased with increasing doses of X-rays above the levels observed for control cultures under similar conditions. When assayed for plasminogen activator levels, the X-ray-treated cultures at various passages showed insignificant differences from levels observed in control cultures. However, the amounts of collagenase (type IV collagen-specific) increased significantly by 32 population doublings in the X-ray-treated cultures compared with control cultures. Our results suggest that the production of type IV collagen-specific collagenase could be useful as an in vitro marker for the transformation of human diploid fibroblasts by X irradiation. PMID- 3004775 TI - Induction of microchromosomes by chemical carcinogens correlates with SV40-DNA amplification in SV40-transformed Chinese hamster cells. AB - Radiations and chemical carcinogens induce the amplification of viral DNA inserts and of their cellular flanking sequences in SV40-transformed Chinese hamster embryo cells. In the cell line CO60, the phenomenon is easily measured by in situ hybridization using SV40-DNA as a probe. We found that the appearance of microchromosomes (MC) in CO60 metaphases correlated well with the induction of SV40-DNA amplification (SDA) mediated in the same cells by chemical carcinogens. SDA and MC formation had the same inducers and were essentially transient phenomena whose occurrence and disappearance were simultaneous. The banding properties of MC and the respective time courses of induction of chromosome aberrations and MC formation/disappearance indicated that MC were not chromosome or chromatid breaks but rather acentric double minute-like chromosomes. Double minute chromosomes (DM) have been shown to contain amplified genes. They specifically occur in established cell lines and in vivo tumor cells. Therefore, the reported correlations between SDA and MC formation and the homologies between MC and DM in CO60 cells further support the existence of specific relationships between mutagenesis, gene amplification, in vivo selective pressures and tumor cytogenetics. PMID- 3004776 TI - Molecular cloning of sequences activated during multi-stage carcinogenesis in mouse skin. AB - Recombinant DNA techniques were employed to isolate sequences that were activated during multi-stage carcinogenesis in the skin of NMRI mice. Differential screening of 5000 cDNA clones made from poly(A)+ RNA of squamous cell carcinomas induced by 7,12-dimethylbenz[a]anthracene (DMBA) and the tumor promoter 12-O tetradecanoyl-phorbol-13-acetate (TPA) resulted in the identification of 35 cDNA clones displaying a different hybridization signal. Eight cDNA clones, three of which proved to be identical, were used as probes in transfer hybridization experiments. These cDNA clones, designated pmal-1 to pmal-6, showed a strong signal with carcinoma RNA but either a weak or no signal with RNA from normal epidermis. Clone pmal-2 contained repetitive sequences as shown by Southern analysis resulting in a smeared RNA hybridization signal in RNA blots whereas the other five clones corresponded to discrete size classes of mRNA. Studies on the expression pattern of mal-1,-2 and -3 related sequences revealed transcriptional activation already in the benign papilloma stage of multi-step carcinogenesis. PMID- 3004777 TI - Pharmacologic manipulation of the peripheral vasculature in shock: clinical and experimental approaches. AB - An improved understanding of the patho-physiological and biochemical changes that occur in shock states has led to new and innovative pharmacologic approaches to shock reversal. In this article, we review the actions of several pharmaceutical agents on the peripheral vasculature in shock states. Agents with known efficacy, probable utility, and possible usefulness are each discussed. A model of factors modulating alpha-1 adrenergic receptor action is presented. PMID- 3004778 TI - Regulation of adenosine formation by the heart. AB - We think that the available data on adenosine formation suggest the two signals are responsible for adenosine release from cardiac myocytes: (1) the ratio of oxygen supply to demand and (2) agonist-triggered release of extracellular adenine nucleotides. We do not believe that the available data support the oxygen consumption hypothesis. The few studies which allow us to judge the relative importance of these two signals suggest that both hypoxia and sympathetic nerve stimulation release adenosine primarily by decreasing O2 supply:demand. Agonist triggered nucleotide release may be quantitatively important in situations in which decreased O2 supply/demand cannot explain increased release, i.e., isoproterenol and acetylcholine administration. PMID- 3004780 TI - Adenosine 3',5'-cyclic-monophosphate-dependent regulation of alpha 1-adrenergic receptor number in rabbit aortic smooth muscle cells. AB - The purpose of this study was to determine whether a cyclic adenosine 3',5' monophosphate-dependent process can be involved in the regulation of vascular smooth muscle alpha 1-adrenergic receptor responsiveness. Experiments were performed in cultured rabbit aortic smooth muscle cells which were characterized previously according to alpha-adrenergic receptor-binding characteristics and receptor-coupled norepinephrine-stimulated 45Ca++ efflux. The addition of dibutyryl-cyclic adenosine monophosphate to the cell culture medium for 24 hours resulted in a concentration-related decrease in maximal [3H]prazosin-binding capacity (41 +/- 4% decrease with 1 mM dibutyryl-cyclic adenosine monophosphate) without an effect on [3H]prazosin-binding affinity. Prostaglandin E1 (10 microM) and forskolin (10 microM) caused similar decreases in maximal [3H]prazosin binding capacity, whereas butyrate (1 mM) and dibutyryl-guanosine-3',5' cyclic monophosphate (1 mM) had no effect. Dibutyryl-cyclic adenosine monophosphate (1 mM) caused significant potentiation of the decrease in [3H]prazosin-binding caused by a submaximal (10 nM) but not a maximal (10 microM) concentration of norepinephrine, suggesting that cyclic adenosine monophosphate may act at a distal step in common with norepinephrine to reduce alpha-adrenergic receptor number. Despite the approximately 41% reduction in alpha-adrenergic receptor number following 24-hour incubation of cells with dibutyryl-cyclic adenosine monophosphate, maximal norepinephrine-stimulated 45Ca++ efflux was not reduced, consistent with the markedly nonlinear relationship between alpha-adrenergic receptor occupancy and maximal norepinephrine-stimulated 45Ca++ efflux in this cell system. These data provide evidence for a novel mechanism by which hormones or drugs which increase cyclic adenosine monophosphate levels can modulate alpha adrenergic responsiveness in vascular smooth muscle. PMID- 3004779 TI - Hypothalamic beta 2-adrenoceptor control of renal sympathetic nerve activity and urinary sodium excretion in conscious, spontaneously hypertensive rats. AB - The contributions of beta 1-, B2-, and alpha 2-adrenoceptors in the posterior hypothalamus to the increased renal sympathetic nerve activity and decreased urinary sodium excretion resulting from environmental stress (air jet) in conscious spontaneously hypertensive rats were examined. Air stress increased mean arterial pressure and renal sympathetic nerve activity (54% from 7.0 +/- 0.7 integrator resets/min), and decreased urinary sodium excretion (44% from 2.7 +/- 0.4 microEq/min per 100 g body weight). After bilateral injection of ICI 118,551 (beta 2-adrenoceptor antagonist) into the posterior hypothalamus of the same spontaneously hypertensive rats, air stress had no effect on renal sympathetic nerve activity (8% from 4.8 +/- 0.7 integrator resets/min) or urinary sodium excretion (2% from 5.2 +/- 0.8 microEq/min per 100 g body weight), but still increased mean arterial pressure. Bilateral injection of isoproterenolol (beta adrenoceptor agonist) into the posterior hypothalamus enhanced the renal sympathetic nerve activity and urinary sodium excretion (but not mean arterial pressure) responses to air stress. Air stress had no effect on renal sympathetic nerve activity or urinary sodium excretion when ICI 118,551 was given into the posterior hypothalamus before isoproterenol. Atenolol (beta 1-adrenoceptor antagonist) had no effect on the renal responses to air stress when given alone or before isoproterenol. Similarly, ICI 118,551 administered into the lateral hypothalamus or lateral cerebral ventricle, or guanabenz (alpha 2-adrenoceptor agonist) given into the posterior hypothalamus, had no effects on the renal or mean arterial pressure responses to air stress. Thus, beta 2-adrenoceptors in the posterior hypothalamus mediate the increased renal sympathetic nerve activity and antinatriuresis resulting from environmental stress in conscious spontaneous hypertensive rats. PMID- 3004781 TI - Peripheral-type benzodiazepine receptors in the living heart characterized by positron emission tomography. AB - The presence of specific benzodiazepine binding sites in the hearts of dogs and human beings was demonstrated in vivo by a noninvasive method, positron emission tomography (PET). An antagonist of the peripheral-type benzodiazepine binding site, PK 11195, was labeled with carbon-11, a short-lived positron emitter. When injected at high specific activity, 11C-PK 11195 was concentrated in the myocardium. As increasing amounts of unlabeled PK 11195 were added to the radioactive ligand, the myocardial ligand concentration was proportional to myocardial regional perfusion up to quantities of 40 nmol/kg body weight. Above 40 nmol/kg the ligand concentration reached a maximum value (6000 pmol/cm3), which could be considered as the total number of binding sites per unit heart volume. The specificity of 11C-PK 11195 binding to canine heart was demonstrated from a study on the inhibition of binding for radioligand by an excess of several agonists or antagonists of benzodiazepine receptor. The distribution and specificity of 11C-PK 11195 was similar in dogs and in human beings. PET thus opens the way to the investigation of the peripheral-type benzodiazepine receptor in a clinical situation, since it has recently been shown that this receptor could be coupled to the calcium channel in the heart. PMID- 3004782 TI - Reduction of the size of infarction by allopurinol in the ischemic-reperfused canine heart. AB - This study was performed to assess the effect of allopurinol in a canine preparation of myocardial infarction. Dogs underwent occlusion of the left circumflex coronary artery for 90 min, followed by reperfusion for 6 hr. Three groups were studied: (1) control, (2) dogs receiving 25 mg/kg allopurinol 18 hr before occlusion and 50 mg/kg 5 min before occlusion, and (3) dogs receiving allopurinol as above plus 5 mg/kg superoxide dismutase over 1 hr beginning 15 min before reperfusion. Infarct size expressed as a percentage of the area at risk was 40 +/- 4 in the control group, 22 +/- 5 in the allopurinol group (p less than .05 vs control), and 17 +/- 4 in the allopurinol plus superoxide dismutase group (p less than .05 vs control). The differences in infarct size were not due to differences in myocardial oxygen supply or demand. Neutrophil superoxide anion production was not altered by allopurinol treatment. The results suggest that myocardial xanthine oxidase may generate oxygen radicals that play a role in myocardial injury due to ischemia and reperfusion. PMID- 3004783 TI - Partial reversal of asymmetry in microvessel neurochemical changes after ischemia by corpus callosum section. AB - Common carotid occlusion in the rat significantly decreases the density of beta adrenergic receptors in preparations of microvessels obtained from ipsilateral and contralateral cerebral cortices. The disruption of nerve pathways connecting the hemispheres (callosal transection) partially reverses the effect of common carotid occlusion on beta-adrenergic receptor density in capillaries of the contralateral cortex. In addition, the destruction of the central noradrenergic system by intraventricular injection of 6-hydroxydopamine abolishes the effect of ischemia on capillary beta-adrenergic receptor function in both hemispheres. The results suggest that beta-adrenergic receptors located on microvessels are partially regulated by neuronal pathways and that focal ischemia induces neurochemical and functional changes in remote areas of the brain. PMID- 3004784 TI - Disparate effects of the calcium-channel blockers, nifedipine and verapamil, on alpha 2-adrenergic receptors and thromboxane A2-induced aggregation of human platelets. AB - Calcium-channel blockers inhibit human platelet aggregation in vitro and ex vivo. To further evaluate the mechanism(s) responsible for the inhibition induced by this structurally heterogeneous group of compounds, we studied the effect of nifedipine and verapamil on human platelet aggregation in vitro. Neither 10 microM nifedipine nor 10 microM verapamil consistently inhibited the aggregation response of platelet-rich plasma to threshold concentrations of ADP, sodium arachidonate, epinephrine, or collagen. However, both 10 microM nifedipine and 10 microM verapamil epinephrine-potentiated, thromboxane A2 (TXA2)-induced aggregation of aspirin-incubated, gel-filtered platelets. Aggregation of similarly prepared platelets induced by TXA2 alone was abolished by 10 microM nifedipine but not by 10 microM verapamil. Even 100 microM verapamil gave only partial and inconsistent inhibition of aggregation. Both drugs had essentially the same effects on platelet aggregation induced by the stable endoperoxide and TXA2 mimic, U46619, with or without epinephrine. Neither 10 microM nifedipine nor 10 microM verapamil elevated platelet cyclic AMP. Verapamil (10 microM) inhibited binding of [3H]-yohimbine (an alpha 2-adrenergic receptor antagonist) to intact human platelets (KD 10.5 nM vs 2.4 nM for control platelets) without altering the number of binding sites. In contrast, 10 microM nifedipine had no effect on KD or number of binding sites. These results indicate that nifedipine and verapamil inhibit epinephrine-potentiated, TXA2-induced human platelet aggregation by different mechanisms. Verapamil inhibits the epinephrine contribution to the aggregation response by blocking alpha 2-adrenergic receptor binding. Nifedipine blocks the platelet response to TXA2 without affecting alpha-adrenergic receptor binding. These observations have potential clinical implications with regard to the mechanisms by which calcium-channel blockers inhibit vascular spasm and myocardial ischemia. PMID- 3004785 TI - [Applications of competitive radioassay in studies of acupuncture mechanism]. PMID- 3004786 TI - [Role of locus coeruleus in the effects of electroacupuncture on hippocampal pyramidal cells spontaneous firing in the rat]. PMID- 3004787 TI - [The afferent pathway of the hippocampus on participation in acupuncture analgesia. A study of retrograde transport method]. PMID- 3004789 TI - Evaluation of an immunoinhibition kit that does not require a sample blank to eliminate interference from adenylate kinase. PMID- 3004788 TI - A peroxidase-coupled kinetic enzymatic procedure evaluated for measuring serum and urinary creatinine. AB - We evaluated an enzymatic procedure for the automated determination of serum and urinary creatinine. The procedure was sensitive and precise for serum creatinine concentrations as low as 3 mg/L, and the standard curve was linear to 200 mg/L. Measurements of creatinine in clinical serum and urine specimens agreed with those obtained by two other enzymatic methods and four picric acid methods. There was no interference from picrate-reactive substances tested. Among other substances, bilirubin (50-100 mg/L) interfered negatively with the assay, decreasing apparent creatinine concentrations by as much as 6 mg/L. The advantages of specificity, speed, and convenience make this enzymatic procedure an attractive primary or secondary method for creatinine determinations in clinical laboratories. PMID- 3004790 TI - Inherited Xp21 deletion in a boy with complex glycerol kinase deficiency syndrome. PMID- 3004791 TI - The interaction of ephedrine with beta-adrenoceptors in tracheal, cardiac and skeletal muscles. AB - Three beta-adrenoceptor mediated effects were measured in vitro on tissues from guinea-pig: (a) relaxation of the trachea (mainly beta 2), (b) increase in the force of contraction of the papillary muscle (beta 1), and (c) depression of the subtetanic contractions of the soleus muscle (beta 2). The relaxant effect of ephedrine on the trachea was weak but was resistant to reserpine pretreatment. There was no appreciable agonistic effect of ephedrine on the soleus muscle. Ephedrine per se had a marked positive inotropic effect on the papillary muscle with a pD2 of about 6.0. This effect disappeared completely after pretreatment with reserpine. Ephedrine inhibited the response to isoprenaline. The apparent pA2 was about 4.8 in all tissues studied. PMID- 3004792 TI - The protective effects of hyperimmune anti-murine cytomegalovirus antiserum against lethal viral challenge: the case for passive-active immunization. AB - The administration of 0.2 ml of hyperimmune anti-mouse cytomegalovirus (CMV) antiserum intraperitoneally (ip) or intravenously provided complete protection against lethal challenge (10(5.8) PFU ip) with murine CMV. Antiserum protection was complete when the antiserum was administered as long as 24 hr after viral challenge. The administration of antiserum had little effect on the titers of virus in the organs of these animals. Ammonium sulfate-treated antiserum provided similar complete protection. Animals rechallenged with 10(6)-10(6.5) PFU of murine CMV 1 month after initial challenge, at a time when the administrated antiserum was no longer detectable, all survived. We conclude that hyperimmune antiserum can provide significant protection against otherwise lethal murine CMV infection, that the protecting material lies within the immunoglobulin fraction, and that long-term immunity results from the combined exposure to virus and antiserum. Such passive-active protection could be useful in protecting against human CMV infection. PMID- 3004793 TI - [A case of Kearns-Sayre-Shy syndrome with neuropathy and high lactate content in CSF]. PMID- 3004794 TI - [A case of chronic sensory neuropathy accompanied with orthostatic hypotension, hypohidrosis and loss of toes]. PMID- 3004795 TI - Epstein-Barr virus infection associated with rhabdomyolysis and acute renal failure. PMID- 3004796 TI - The anatomy and physiology of reverse cholesterol transport. PMID- 3004797 TI - Evaluation of an isotope washout technique to measure skin vascular resistance and skin perfusion pressure: influence of age, site and arterial surgery. AB - A simplified isotope washout technique has been devised to calculate the skin perfusion pressure (SPP) and skin vascular resistance (SVR). This test is simple, requires inexpensive equipment and is well tolerated by patients. SPP and SVR were calculated in 20 patients less than 30 years of age, 13 patients greater than 30 years of age and in 15 patients with peripheral vascular disease (PVD). With increasing age the SPP and SVR were increased. The SPP was similar to the mean arterial pressure in normal individuals but was decreased in patients with PVD. The SPP is a useful indicator of the severity of the PVD. The SPP and SVR were higher in the calf than in the foot. This is probably related to the decrease in pressure in the distal arterial tree. SPP was increased by 110% and skin blood flow by 190% by arterial reconstructive surgery. This test may be of use in assessing the effectiveness of arterial surgery. PMID- 3004798 TI - The effect of cyclical hormonal changes on erythrocyte electrolyte transport mechanisms. AB - Electrolyte transport characteristics were examined in erythrocytes from 13 normal men and from two groups of women: taking combined oral contraceptive preparations (O/C, n = 10), and ovulatory women (non-O/C, n = 10) pre- and post ovulation, at the same time intervals (days 7-10 and days 15-18) during a menstrual cycle. With rubidium (86Rb+) used as a potassium analogue, co-transport (ouabain-resistant, frusemide-sensitive 86Rb+ influx) values were found to be lowest in non-O/C women (28 +/- SE 2.5 nmol h-1 10(-9) cells) and highest in men (56 +/- 5.7, P less than 0.001), with results between the two in women taking O/C (42 +/- 4.2, P less than 0.05 vs men, P less than 0.01 vs non-O/C). Passive 86Rb leak (frusemide-and ouabain-resistant) was significantly lower in men (13 +/- 1.6 nmol h-1 10(-9) cells) than in both groups of women (non-O/C 29 +/- 1.8, P less than 0.001; O/C 25 +/- 1.2, P less than 0.001). There was no cyclical variation within either group of women. Maximum ouabain binding (number of NA+,K+-ATPase units) was the same in all groups. Na+,K+-ATPase activity, as determined by ouabain-sensitive 86Rb influx, was the same in men and non-O/C groups, but was significantly suppressed in O/C compared with both men (P less than 0.01) and non O/C women (P less than 0.05). The differences found were not due to alterations in either progesterone or aldosterone, but could represent an androgenic effect in vivo of the 19-nortestosterone derivatives in combined oral contraceptive preparations. PMID- 3004799 TI - Chemical specificity of coughing in man. AB - The purpose of this study was to test whether cough response to inhaled ultrasonically nebulized fluid is dependent on the ionic content of the fluid. Coughing was recorded in human volunteers during inhalation of aqueous solutions in a series of double blind randomized experiments. The occurrence of cough was found to be dependent on the concentration of chloride ions in the inhaled fluid, cough frequency progressively increasing as chloride ion concentration was reduced. It is proposed that the ion composition of the surface lining fluid of the airway may moderate the cough response by means of a chemo-receptor, although tonicity may also be important. PMID- 3004800 TI - Alopecia associated with long-term metyrapone use. PMID- 3004801 TI - Diagnosis and pathophysiology of Cushing's syndrome. AB - Cushing's syndrome is the consequence of a sustained overproduction of cortisol (hydrocortisone) by the adrenal cortex. This may be due to excessive secretion of cortisol by functioning adrenocortical tumors or to "nontumorous" adrenocortical hyperfunction. The latter may be a result of stimulation of the adrenal cortex by increased release of corticotropin (ACTH) from a small pituitary tumor or from nonpituitary nonadrenal tumor. Carcinoids or carcinomas of the lung or pancreas, and even pheochromocytomas have caused the syndrome of ectopic ACTH production. The problems involved in the diagnosis of Cushing's syndrome are establishing its presence and determining the underlying cause. Treatment is then dependent upon the underlying pathogenetic lesion. PMID- 3004802 TI - [The persistence of activity of H1N1 (swine) influenza virus in pig breeding units during non-epidemic phases]. AB - A study is carried on in a group of 16 commercial breeding-finishing units in Brittany (France). The aim of this study is to try to reveal the persistence of activity of the Influenza Virus within these intensive units after an acute epizootic. In each farm two batches of pigs are selected, individually identified and followed from suckling period to slaughter. Serological controls are conducted every month on the same pigs. Both Influenza and Aujeszky's disease antibodies are apprehended. Furthermore nasal swabs and lungs are submitted to analysis for virus isolation purposes and data are collected from each herd for epidemiological studies. Influenza passive H.I. Antibodies rapidly decrease in the piglets. Later a seroconversion is observed in 10% of the investigated piglets. These pigs belong to 4 herds. The conversion happens usually after 3 months of age. All attempts to isolate the virus failed. Special herd conditions may be associated with a persistence of the viral activity inside the units between epizootics: bad demography (unbalance in the breeding stock), inadequate hygiene policy in the fattening house (with permanent arrivals of pigs coming from subsequent batches of sows), a high prevalence of respiratory and specially pulmonary enzootic diseases. A seroconversion against Aujeszky's disease is pointed out in 3 out of the 4 herds concerned with Influenza virus activity suggesting that closely the same conditions may be related both to influenza and Aujeszky's disease virus activity. PMID- 3004803 TI - The duration of the foot-and-mouth disease virus carrier state in African buffalo (i) in the individual animal and (ii) in a free-living herd. AB - The maintenance of a virus depends on a number of factors, including the duration of infectivity and the size of the available host population. In this work, foot and-mouth disease virus was shown to persist in individual African buffalo (Syncerus caffer) for up to at least five years; thus, the duration of infectivity is more than adequate to cover the normal periods between calving peaks. In a small isolated free-living population which varied from 30 to 100 buffalo, two immunological types of foot-and-mouth disease virus were maintained for at least 24 years and through several generations. PMID- 3004805 TI - The separation of sheep and human serum "A"-esterase activity into the lipoprotein fraction by ultracentrifugation. AB - Using sheep and human serum the relationship between centrifugation time and yield of total lipoprotein, HDL-cholesterol and "A"-esterase in lipoprotein was studied employing different centrifuge rotors. More rapid separation of these components was obtained with a vertical rotor than with an angled rotor. The procedures commonly employed for lipoprotein separation gave low yields of lipoprotein "A"-esterase and HDL-cholesterol. The separation of sheep serum "A" esterase into the lipoprotein fraction was not in phase with that of HDL cholesterol and the pattern of separation was different from that in human serum. These results provide further evidence that serum "A"-esterase activity is associated with different species of HDL-particle. PMID- 3004804 TI - The diagnosis of enzootic bovine leukosis. AB - This paper reviews the clinical and virological diagnostic procedures for enzootic bovine leukosis (EBL). The clinical diagnosis must be always confirmed by a specific laboratory test for Bovine Leukaemia Virus (BLV). Many virological tests were proposed. The sensitivity of all the diagnostic methods is sufficient to do an early detection of a BLV infection on an individual base. Advantages of the highly sensitive methods like RIA and ELISA appear when the samples to be tested have naturally very low antibody titers (individual milk, bulk milk, pooled sera). PMID- 3004806 TI - Inositol lipid turnover and adenosine 3,5 cyclic monophosphate in the salt secreting rectal gland of the dogfish (Scyliorhinus canicula). AB - The rectal gland of the dogfish is rich in inositol lipids. Total phospholipids from the gland contained 9.1 mol% of phosphatidylinositol (PtdIns), 1.0 mol% of phosphatidylinositol 4-phosphate (PtdIns4P) and 0.9 mol% of phosphatidylinositol 4,5-biphosphate (PtdIns4,5P2). [32P]Orthophosphate was readily incorporated into PtdIns, phosphatidic acid (PtdA) and especially into PtdIns4P and PtdIns4,5P2 in salt gland slices incubated in elasmobranch Ringer with glucose and no other additions over a 2 hr period. The calcium ionophore A23187 stimulated incorporation into PtdIns and PtdA, but not into PtdIns4P or PtdIns4,5P2. Oxygen uptake by rectal gland slices was maximally stimulated by 0.08mM forskolin, 2.5mM 8-chlorophenylthio cyclic AMP, 2.0mM dibutyryl cyclic AMP and 0.25mM theophylline. Stimulated oxygen uptake was inhibited by 0.1mM ouabain in all cases. Incorporation of [32P]orthophosphate into PtdIns, PtdA, PtdIns4P and PtdIns4,5P2 was inhibited by 0.08mM forskolin and 2.0mM dibutyryl cyclic AMP over a 2 hr period. The results are discussed in relation to the control of salt secretion by the rectal gland. PMID- 3004807 TI - A comparison of glucose metabolism and related hormonal parameters in two strains of mice having differing hepatic glucokinase activities. AB - Parameters of glucose metabolism in the livers of two inbred strains of mice, C3H/He and C58, which have high- and low-glucokinase activities respectively, have been determined. Unlike insulin concentrations, the plasma glucagon concentrations are similar in the two strains. Certain of the numbers of insulin receptors per hepatocyte cell surface area were higher in starved than fed animals of the same strain but affinities were the same, while only small differences in receptor numbers were found between the strains in starved animals. The difference in glucokinase activity, determined spectrophotometrically and confirmed by measurements of detritiation of [2 3H]glucose by hepatocytes incubated in vitro, does not apparently influence the minimal rate of glucose recycling as measured by the relative loss of 3H and 14C from [2-3H, U-14C]glucose. The development profiles for the two strains show a marked developmental difference arising around 20 days after birth. PMID- 3004809 TI - Use of meglumine iotroxate in the detection of liver tumors by computed tomography. AB - Experience with contrast enhanced computed tomographic (CT) examinations of 13 patients with hepatic tumors using a new cholangiographic contrast material, meglumine iotroxate, is described. After infusion, small density differences between the tumor and liver were accentuated. In 11 cases postcontrast CT using meglumine iotroxate improved visualization of lesions compared with conventional pre-and postcontrast CT using urographic contrast material. Our results indicate that contrast enhanced CT using meglumine iotroxate is a promising alternative to conventional CT in the detection of hepatic tumors. PMID- 3004808 TI - Rat intestinal brush border membrane trehalase: some properties of the purified enzyme. AB - Rat intestinal brush border trehalase (EC 3.2.1.28) solubilized by Triton X-100 or Emulphogen BC 720 has been purified almost to homogeneity in a five steps procedure including DEAE cellulose, Sephadex G-200, preparative flat bed electrofocusing and hydroxylapatite. The apparent molecular weight was estimated to be about 65,500 daltons by mannitol density gradient ultracentrifugation. The optimum pH of the enzyme was between 5.5 and 5.7 in phosphate, maleate or citrate buffers. The apparent Km for trehalose was found to be 10 mM in maleate buffer pH 6.0. The isoelectric point was 4.9. Tris, P-aminophenylglucoside, sucrose and maltose are fully competitive inhibitors with Kis of 2.2, 1.8, 7.7 and 170 mM, respectively. The inhibition by Phloridzin appeared to be of the mixed type with a Ki of 1.7 mM. Trehalase is heat stable up to 50 degrees C and the activation energy is 10.96 kcal/mol. Schiff's staining on polyacrylamide gel and interaction with Con-A-Sepharose indicate that rat trehalase is a glycoprotein. PMID- 3004810 TI - Neonatal cytomegalovirus infections due to blood. AB - Infants with very low birthweights (less than 1250g) are immunocompromised and have immature hematopoietic systems. They require frequent blood transfusions and have an increased susceptibility to infection. These very low birthweight infants who lack passively acquired antibody against CMV, acquire transfusion-associated CMV infections with a frequency of approximately 30%. These infections are associated with significant morbidity and mortality. The source of these postnatally acquired CMV infections are seropositive blood donors. These infections can be prevented by appropriate donor selection and/or blood processing. Recent but limited data suggests that all infants (regardless of birthweight or the presence of antibody against CMV) should receive CMV seronegative blood products if they are likely to receive multiple transfusions from multiple donors. PMID- 3004811 TI - Tumors and tumor-like lesions of the kidney. PMID- 3004813 TI - Ultraviolet erythema: causes and consequences. PMID- 3004812 TI - Furocoumarin photochemistry and its main biological implications. PMID- 3004814 TI - Regulation of cAMP-dependent protein kinase in cultured cells. PMID- 3004815 TI - Sensitivity of metabolic fluxes to covalent control. PMID- 3004816 TI - NMR studies of the mechanism of action and regulation of protein kinase. PMID- 3004817 TI - The role of protein kinase in the lens. PMID- 3004818 TI - Regulation of yeast fructose-1,6-bisphosphatase by phosphorylation dephosphorylation. PMID- 3004819 TI - Subunit structure and regulation of phosphorylase phosphatase. PMID- 3004820 TI - The coordinated control of metabolic pathways by broad-specificity protein kinases and phosphatases. PMID- 3004821 TI - cAMP-triggered proteolysis of cAMP-dependent protein kinase in brush border membranes. PMID- 3004822 TI - Regulation of mammalian cytosolic Ca2+-requiring neutral proteinases. PMID- 3004823 TI - Modification of yeast fructose-1,6-bisphosphatase. PMID- 3004824 TI - Production and utilization of reactive oxidants by neutrophils. PMID- 3004825 TI - Fructose 2,6-bisphosphate versus cyclic AMP in the liver and in lower eukaryotic cells. PMID- 3004826 TI - Regulation of mammalian pyruvate and branched-chain alpha-keto acid dehydrogenase complexes by phosphorylation-dephosphorylation. PMID- 3004828 TI - The calmodulin regulatory system. PMID- 3004827 TI - Fructose 2,6-bisphosphate and enzymatic activities for its metabolism in ascites tumor. PMID- 3004829 TI - Mode of calcium activation of calmodulin-regulated enzymes. PMID- 3004830 TI - Mechanism of enzyme regulation by calmodulin and Ca2+. PMID- 3004831 TI - Homology among oncogenes. PMID- 3004832 TI - Hepatitis B virus (HBV) DNA in leucocytes in acquired immune deficiency syndrome (AIDS). AB - Earlier reported findings of hepatitis B virus (HBV) DNA in white blood cells of patients with hepatoma, and in a patient with autoimmune haemolytic anaemia, led to the examination of HBV DNA in a series of twenty three patients with acquired immune deficiency syndrome (AIDS), including nine with opportunistic infections and fourteen with Kaposi's sarcoma, by Southern blot hybridization method, using 32P labelled HBV DNA specific probe obtained by nick translation of HBV DNA cloned into plasmid pBR325. Four of the patients were found to be positive for HBV DNA or HBV related DNA in their leucocytes. The HBV DNA was found free or integrated in the leucocytes of the patients. PMID- 3004833 TI - Glucocorticoid receptors of lymphoid cells. Cell biological and clinical aspects. PMID- 3004834 TI - Pulmonary cell interactions with asbestos fibers in vivo and in vitro. PMID- 3004835 TI - Cellular and molecular mechanisms of asbestosis. PMID- 3004836 TI - Immunologic investigations in asbestos-exposed workers. PMID- 3004837 TI - The pathogenesis of silicosis. State of the art. PMID- 3004838 TI - Modulation by T cells of pulmonary inflammation and fibrosis in an experimental model of silicosis. PMID- 3004839 TI - Ultrastructural study of 62 cases of human primary hepatic carcinoma. PMID- 3004840 TI - [Antigenic analysis of 100 strains of poliomyelitis type I virus by monoclonal antibody]. PMID- 3004841 TI - [Comparison of two schedules for administering trivalent oral poliovaccine]. PMID- 3004842 TI - [A clinical pathologic study of lymphatic metastasis of rectal cancer]. PMID- 3004843 TI - [Physiologic hepatic dearterialization and 5-FU intra-arterial perfusion in primary hepatic carcinoma]. PMID- 3004844 TI - [Dissolution of uric acid calculi]. PMID- 3004845 TI - [Antiviral action and therapeutic effect of acycloguanosine in herpes simplex infection]. PMID- 3004846 TI - Flow cytometric and radioisotopic determinations of platelet survival time in normal cats and feline leukemia virus-infected cats. AB - This study demonstrates the potential usefulness of a flow cytometric technique to measure platelet survival time in cats utilizing autologous platelets labeled in vitro with fluorescein isothiocyanate (FITC). When compared with a 51Cr method, no significant differences in estimated survival times were found. Both the 51Cr and FITC-labeling procedures induced similar changes in platelet shape and collagen-induced aggregation. Platelets labeled with FITC had significantly greater volumes compared with those of glutaraldehyde-fixed platelets. These changes were primarily related to the platelet centrifugation and washing procedures rather than the labels themselves. This novel technique potentially has wide applicability to cell circulation time studies as flow cytometry equipment becomes more readily available. Problems with the technique are discussed. In a preliminary study of the platelet survival time in feline leukemia virus (FeLV)-infected cats, two of three cats had significantly reduced survival times using both flow cytometric and radioisotopic methods. These data suggest increased platelet turnover in FeLV-infected cats. PMID- 3004847 TI - Pancreatic carcinoma is associated with delayed gastric emptying. AB - Fifteen patients with histologically confirmed pancreatic carcinoma, without evidence of gastroduodenal invasion or obstruction, were prospectively studied to determine the frequency of gastric emptying disorders as determined by a solid phase gastric emptying study. Nine of these (60%) had gastric emptying curves more than two standard deviations below normal mean values. The majority of patients did not have symptoms of gastric stasis. Nausea and/or vomiting was present in 33% of patients with abnormal gastric emptying and in none of those with normal emptying. Abdominal and/or back pain was present in 8/9 with delayed gastric emptying and in 3/6 with normal emptying. Disordered gastric emptying did not correlate with tumor stage, histology, location, or hyperbilirubinemia. Delayed solid-food gastric emptying may be responsible for the nonspecific abdominal complaints that occur during the course of pancreatic carcinoma, although more frequently, gastroparesis exists on a subclinical level. PMID- 3004849 TI - Effect of thyroid status on lung and heart beta-adrenergic receptors in fetal and newborn sheep. AB - The effect of altered thyroid status on the development of beta-adrenergic receptor (BAR) density and affinity was investigated in ovine fetal and newborn heart and lung. Fetal (119-121 days gestation) and newborn (2-3 days of age) sheep underwent either thyroidectomy alone, thyroidectomy plus infusion of a large dose of T3 or sham operation. Eight days later BAR were measured in heart and lung using the tritiated radioligand dihydroalprenolol. There was no apparent effect of altered thyroid status on fetal heart or lung BAR density or affinity. In contrast, the newborns thyroidectomized and infused with T3 had a 96% increase in heart BAR density and an 83% increase in lung BAR compared to the thyroidectomized only or sham-operated newborns which were similar. These results suggest that in the near term ovine fetus heart and lung tissue BAR are neither dependent on nor responsive to thyroid hormones, whereas in the newborn period heart and lung BAR are highly responsive to thyroid hormones. PMID- 3004848 TI - Severe osteomalacia associated with renal tubular acidosis in Crohn's disease. AB - Severe renal tubular acidosis associated with massive osteomalacia is described in a patient with Crohn's disease. To our knowledge this association has not been previously recognized. The possible role of renal tubular acidosis in this patient's osteomalacia is discussed and the factors that could be involved in renal tubular acidosis in the context of Crohn's disease are analyzed. PMID- 3004850 TI - Effects of ouabain on monovalent cation transport in mammalian tissue. AB - We measured the effects of ouabain on sodium-potassium ATPase (Na-K ATPase) specific activity (SA) and rubidium (Rb) transport in newborn (NB) and adult (Ad) sheep myocardium (myo), guinea pig myo and red blood cells (RBC), and human RBC. 50% inhibition of Na-K ATPase SA and Rb transport occurred at ouabain concentrations between 10(-4.8) and 10(-7.6) M in different species; within a species, 50% inhibition of Rb transport occurred at lower ouabain concentrations than 50% inhibition of Na-K ATPase SA. No differences between mature and immature tissues were found. Ouabain binding stability to Na-K ATPase extracted from myo and RBC did not vary with age. Binding studies revealed a KD X 10(-8) M of 0.87 +/- 0.09 for NB human RBC and 0.88 +/- 0.14 for Ad human RBC. Calculated RBC receptor density however, was higher in the Ad (1.25 +/- 0.08 sites/micron2) than in the NB (1.03 +/- 0.04 sites/micron2, p less than 0.01). Binding studies in extracted myocardial specimens showed a KD X 10(-8) M of 1.23 +/- 0.27 for NB and 1.09 +/- 0.46 for Ad; mM ouabain bound X 10(-12)/Na-K ATPase unit were 355 +/- 120 (SEM) for NB and 316 +/- 51 for Ad. These studies of cardiac glycosides reveal few age-related differences in drug binding or inhibition of monovalent cation transport. PMID- 3004851 TI - Binding of the human glucocorticoid receptor to defined regions in the human growth hormone and placental lactogen genes. AB - An in vitro competition assay was used to investigate whether binding sites for the human glucocorticoid receptor occur in the human genes for growth hormone (hGH) and placental lactogen (chorionic somatomammotropin, hCS). These genes display 95% sequence homology. Two receptor-binding regions were found in the hGH gene, one of which is located within 290 bp upstream, and one within 251 bp downstream from the transcription initiation site. Two binding regions homologous to those in the hGH gene were found in the hCS gene. The receptor-binding DNA fragment from the structural part of the genes, but not that from their promoter area, contained a sequence homologous to a 15-bp consensus sequence proposed earlier for the glucocorticoid receptor binding site. It is unlikely that the putative difference in glucocorticoid sensitivity between the hGH and hCS genes is accounted for by major differences in glucocorticoid receptor binding pattern. PMID- 3004852 TI - A highly modular cloning vector for the analysis of eukaryotic genes and gene regulatory elements. AB - We have developed a highly modular vector, pDSP1, which contains two independent mammalian transcription cassettes. Each cassette contains SV40 early gene regulatory elements controlling the expression of an easily assayable, selectable Escherichia coli marker gene, either galK or xgprt. The regulatory elements of the galK cassette are bounded by multiple unique and nearly unique restriction sites allowing for the easy removal and replacement of either the regulatory sequences or of the galK gene itself. Expression of the marker genes is monitored by transient transfection into mammalian cells followed by filter enzyme assays. Expression of xgprt serves as an internal control and the relative expression of galK/xgprt is used to quantitate modifications made to the vector. We have used this system to analyze many eukaryotic polyadenylation regions as well as several other eukaryotic gene regulatory elements. We have also removed the galK gene and replaced it with other mammalian genes. The entire galK cassette is contained on a Sal I restriction fragment that can be readily removed and placed into a unique Sal I site in one of our Epstein-Barr virus (EBV), bovine papilloma virus (BPV), or BK defective viral stable expression vectors. We believe that pDSP1 is a powerful vector system for studying eukaryotic gene regulation, and in conjunction with our stable expression vectors, it represents a unified system for exploring expression in mammalian cells both transiently and stably. PMID- 3004853 TI - [Establishment of highly metastatic human tumor cell line in nude mice]. AB - A total of 22 adult nude mice (7 approximately 17 weeks old) of BALB/cA origin were inoculated subcutaneously (SC) with human tumor cell lines derived from rhabdomyosarcoma and lung carcinomas, including squamous cell carcinoma, bronchiolo-alveolar carcinoma, small cell carcinoma, giant cell carcinoma and 2 types of adenocarcinoma. 12 neoplasms (54.5%) was confirmed at the initial transplantation and 7 serially transplantable tumors were established in 201 nude mice. Metastatic behavior of 7 human tumor cell lines grown in nude mice was judged histologically after sacrifice and SC transplanted tumors could either be highly metastatic (giant cell carcinoma), moderately metastatic (adenocarcinoma PAb), poorly metastatic (squamous cell carcinoma and adenocarcinoma PAa) or non metastatic (small cell carcinoma and bronchiolo-alveolar carcinoma). The tumor from SC inoculated giant cell carcinoma (PG) could produce regional and/or distant lymph node metastases (12/12) and lung metastases (9/12) in BALB/cA nude mice maintained under semi-barrier system conditions. Six NIH nude mice developed metastatic tumor nodules both in the lymph node and lung (6/6) under the same condition. The highly metastasizing property of PG transplanted tumors remained after passage for as many as 18 times. The limitations of nude mice as an in vivo model for the study of tumor biology are discussed, eg. malignant neoplasms rarely metastasize when transplanted into nude mice. The use of such model (PG) could lead to a better understanding of the biology of metastasis, thus contributing to the development of new approaches to the therapy of metastasis. PMID- 3004855 TI - [Erythrophagocytic cancer cells]. AB - The phagocytosis of senescent red cells by the monocyte-macrophage system and neutrophils is a normal physiological phenomenon. However, the erythrophagocytosis by malignant tumor cells was also reported, such as plasma cells of multiple myeloma, acute lymphoblastic leukemia cells and oat cells of bronchogenic carcinoma, etc. This paper describes the erythrophagocytosis by signet-ring cells of adenocarcinoma of the lung observed by electronmicroscope in 1982 and supported by light microscopy and cytochemical tests. PMID- 3004854 TI - [Forestomach carcinoma and hepatoma induced in mice and rats by N-3-methylbutyl-N 1-methylacetonylnitrosamine (MAMBNA)]. AB - MAMBNA is a new N-nitroso compound isolated from Fusarium moniliforme-inoculated corn meal. In the present studies the carcinogenicity of MAMBNA is shown by the induction of forestomach carcinomas and liver tumors in mice and rats following the gastric intubation of this compound. Among 42 mice of Kunming (KM) stock treated with MAMBNA (10-20 mg/week), 22 showed epithelial hyperplasia of the esophagus, 29 papillomas and 4 squamous carcinomas in the forestomach, and there were 6 liver adenomas and 3 hepatomas. One mouse had forestomach carcinoma and carcinoma in the liver. Most tumors developed in mice receiving the treatment for 136-317 days, with a total dose of 210-670 mg (Table 1). No tumor was found in 12 controls observed for 239-357 days. In the experiment with 29 Wistar strain rats, administration of MAMBNA (20-120 mg/week) resulted in 11 epithelial hyperplasias in the lower esophagus, 14 atypical hyperplasias and papillomas, and 11 squamous carcinomas in the forestomach. The earliest forestomach carcinoma appeared in a rat treated for 454 days, receiving 4480 mg of MAMBNA, and the other 10 carcinomas occurred in animals treated for 518-640 days. Hyperplasia of liver cells was noted in 4 rats and liver adenoma in 7 and hepatocellular carcinoma in 8. Most hepatomas developed in rats treated for 480-640 days, and 5 rats had both forestomach carcinoma and hepatoma (Table 2). In 11 untreated rats observed for 411-644 days only one forestomach papilloma was noted. PMID- 3004856 TI - [Malignancy of the uterine body after radiotherapy for carcinoma of uterine cervix--report of 12 cases]. AB - Twelve cases of malignancy of the uterine body occurred 5-19 years after radiotherapy for 8,704 cases of carcinoma of cervix-an incidence of 0.14%. 10 cases were malignant mixed mesodermal tumor and 2 endometrial carcinoma. The radiation dose varied from 5,500 to 12,000 rad to the area of uterine body. Eight patients had vaginal discharge and 4 were asymptomatic in the follow-up examination in which enlarged uterus was found in all except one. The tumor had extented out of the uterus in 6 patients. 6 of the 12 patients were diagnosed by endometrial biopsy before treatment. Eight patients were treated by operation, 3 by radiation and 1 was untreated. Eleven patients died within 3 years, one recurred in 4.5 years after treatment. It is believed that the malignant tumor of the uterine body is closely related to radiotherapy and the prognosis is poor. Therefore, in the follow-up examination, if vaginal discharge and enlarged uterus are found, endometrial biopsy should be done to ensure correct diagnosis and early operation in order to obtain a better result. PMID- 3004858 TI - [Fibrobronchoscopy in lung cancer--an analysis of 560 cases]. AB - 560 cases of lung cancer were detected from 1100 cases examined by fibrobronchoscopy. 91.2% of these cases were diagnosed by histology or/and cytology. 71.9% were squamous cell carcinoma. The fibrobronchoscopy gave a positive detection rate of 89.8%, and in 51.1% of cases, it was able to see the tumor or the tumor with necrosis. The positive rate of the cellular brushings was 48%, and that of biopsy, 73.7%. The fibrobronchoscopy is most efficient in localizing the occult lung cancer (positive cytology but negative chest films). The false negative causes of the biopsy or cellular brushings are discussed. PMID- 3004857 TI - [Ligation of hepatic artery combined with radiotherapy in the treatment of unresectable primary liver carcinoma]. AB - The ligation of hepatic artery combined with radiotherapy is effective in the treatment of unresectable liver carcinoma. Our data show that some of the lesions, upon ligation of hepatic artery, underwent shrinkage and became darkened in color, were designated as "hypervascular type". The others which did not give these responses were designated as "oligovascular type". Yet both belonged to the hepatocellular carcinoma pathologically. In this series the total response rate was 71.4% (10/14). There were 3 patients who had complete regression. The 6-month survival rate was 85.7% (12/14), the one-year, 57.1% (8/14), and the 2-year, 21.4% (3/14). The median survival interval was 14 months (ranging from 2-36 months). The "mass type" which was localized and "hypervascular" responded well to treatment. The application of radiotherapy according to the location and the size of the tumor after the ligation of hepatic artery may be able to improve the result and reduce the side effects of irradiation. PMID- 3004859 TI - [Pulmonary oncocytic carcinoid--a case report]. AB - A pulmonary oncocytic carcinoid arising from the medial basal segment of the right lung is presented. The abundant oncocytic granules in the cytoplasm were observed by light-microscope. The ultrastructure by electronmicroscope showed that these granules originated from the hyperplasia of mitochondria, and there were membrane-bordered electric dense core granules (neuro-secretive granule) in the cytoplasm of this tumor. According to the morphological features observed by light and electronmicroscope, this tumor is considered as a subtype of bronchial carcinoid and belongs to the APUD system which should be differentiated from the metastatic tumor, the granular cell myoblastoma and the chemodectoma of lung. PMID- 3004860 TI - [Presynaptic effects of anticholinesterase substances in the sympathetic ganglia]. PMID- 3004861 TI - [Characteristics of the electroencephalogram reaction to voluntary hyperventilation in cancer patients]. PMID- 3004862 TI - [Spontaneous and temperature-induced recombination in Drosophila strains with altered cAMP metabolism]. PMID- 3004863 TI - [Molecular mechanism of transposition memory in the mdg4-locus cut system of Drosophila melanogaster]. PMID- 3004864 TI - [Molecular cloning of human genes coding tumor necrosis factors: tandem arrangement of alpha- and beta-genes in a short segment (6 thousand nucleotide pairs) of human genome]. PMID- 3004865 TI - [Nucleotide sequence of cDNA and primary structure of alpha-subunit of Na+,K+ ATPase from the swine kidney]. PMID- 3004866 TI - [Transactivation of the long terminal repeat promoter of the bovine leukemia virus in infected cells]. PMID- 3004867 TI - [Interaction of the N- and O-derivatives of aminodicarboxylic acids with CNS glutamate receptors]. PMID- 3004868 TI - [Transposition and reversion of mutations induced in Drosophila melanogaster by viral DNA]. PMID- 3004869 TI - [Activity of cyclic nucleotide phosphodiesterase in rod outer segments of the gopher retina]. PMID- 3004870 TI - [Antimetastatic effect of a liposome-entrapped synthetic analog of muramyl dipeptide]. PMID- 3004871 TI - [Potentiation by phorbol ester, a protein kinase activator, of the effect of prostaglandin E1 on human platelets]. PMID- 3004872 TI - [Construction of expression vectors with strong promoters on the basis of plasmid pBR327]. PMID- 3004874 TI - [Thyroliberin receptors in the brain of ground squirrels (Citellus undulatus) during winter hibernation]. PMID- 3004873 TI - [Modifying effect of antioxidants on the biotransformation of dialkyl-N nitrosamines in microsomes. A spin trap method]. PMID- 3004876 TI - [Changes of the cyclic AMP content in experimental atherosclerotic lesions]. PMID- 3004875 TI - [A preliminary observation on changes of the platelet cyclic nucleotides and platelet function in coronary heart disease]. PMID- 3004877 TI - [Acquired receptor deficiencies. Pathophysiologic basis and clinical significance]. PMID- 3004878 TI - [HTLV-III in persons with intravenous drug abuse. Correlation of antibodies against HTLV-III with neopterin and TH/TS]. AB - Antibodies against HTLV-III, neopterin levels in blood and urine, TH/TS ratio and hepatitis marker were determined in 34 clinically symptom-free persons known to be intravenous drug abusers. 15 persons were positive in the ELISA and Western blot tests. There was a strong reaction to protein p24 compared with that to protein p41. In 12 of 14 persons who were antibody-positive the neopterin level in morning urine was elevated; an abnormal TH/TS ratio was present in nine of 13 persons. In future, determination of neopterin and of antibodies against certain proteins of HTLV-III may make it possible to provide a simple way of prognosticating on the course of an HTLV-III infection. PMID- 3004879 TI - [Hereditary receptor defects as a cause of disease]. PMID- 3004880 TI - [HTLV-III/LAV and resuscitation]. PMID- 3004881 TI - [Bone marrow transplantation in chronic myeloid leukemia. Influence of gnotobiotic measures, especially antiviral prophylaxis]. AB - Bone marrow from HLA-identical siblings was transplanted in 14 patients with chronic myeloid leukaemia (CML), including one patient in acceleration phase and one in chronic phase following 2 blast crises. Restoration of the bone-marrow occurred in all cases and Philadelphia chromosome could not be detected in any of the patients after the transplantation. Two patients died due to lung complications. Twelve patients who received antiviral prophylaxis (aciclovir per os, anti-CMV-hyperimmunoglobulin) after the transplantation are in very good condition with 140 to 790 days complete clinical and cytogenetic remission. Bone marrow transplantation, as a curative measure for patients with CML up to the age of 45 (50), should be included in therapy schemes when an HLA-identical sibling is available. PMID- 3004882 TI - [Bronchoalveolar lavage. The humoral parameter spectrum in bronchial carcinoma and chronic bronchitis]. AB - The concentrations of tissue-polypeptide antigen (TPA), ferritin, alpha 1-acid glycoprotein (alpha 1-aGP), transthyretin (TBPA), alpha 1-antitrypsin (alpha 1 Pi), alpha 2-macroglobulin (alpha 2-MG), C-reactive protein and IgA were determined in broncho-alveolar lavage fluid of 13 patients with chronic bronchitis and 11 with bronchial carcinoma and accompanying bronchitis. Measurement of TPA, alpha 1-Pi, ferritin and transthyretin provides useful additional information in the diagnosis of bronchial carcinoma. The ratios of TPA/TBPA, alpha 1-Pi/TBPA and alpha 1-aGP/TBPA differentiate highly sensitively between bronchial carcinoma and chronic bronchitis. PMID- 3004883 TI - [New viewpoints on the role of vitamin D. Current knowledge and outlook]. PMID- 3004884 TI - [Epidermodysplasia verruciformis--an example of viral carcinogenesis]. PMID- 3004885 TI - Pathomorphological changes in gills of fish fingerlings (Cirrhina mrigala) by linear alkyl benzene sulfonate. AB - Fish fingerlings (Cirrhina mrigala) exposed to 0.005 ppm (25% of LC50) concentration to detergents (linear alkyl benzene sulfonate) showed marked behavioral changes and distorted appearance of primary and secondary lamellae along with damage to gill epithelium under scanning electron microscopy at various magnifications. Mucosal cells of gills were found to secrete mucus showing primary reactions for membrane damage leading to dysfunction in respiration and osmoregulation. PMID- 3004886 TI - Effect of different saline, alkaline salts, fertilizers, and surfactants on the movement of some phosphorus-containing pesticides in soils. AB - The influence of organic matter, calcium carbonate, flyash, saline and alkaline salts, inorganic fertilizers, surfactants, and exchangeable ions on the mobility of different phosphorus pesticides has been studied using soil thin-layer chromatographic techniques. The variations in Rf, RB, and RM values of DDVP, diazinon, Ekatin, Folithion, malathion, metasystox, parathion methyl, and Rogor under different treatments is reported and explained on the basis of adsorption and leachability. PMID- 3004887 TI - [Changes in the biochemical parameters of lymphocyte functional activity in simian adenovirus SA7(C8)-induced carcinogenesis]. AB - A functional defect of lymphocytes in hamsters with tumours during carcinogenesis induced by monkey adenovirus SA7(C8) was found and estimated by means of the blast transformation reaction. An increased activity of transport ATPases, the disturbance of cyclic nucleotide levels (cAMP, cGMP) and a decrease in both basal and Con A-activated calcium transportation were observed simultaneously with changes in the activation of immunocompetent cells under Con A influence. PMID- 3004888 TI - Proliferation of mouse uterine epithelial cells in vitro. AB - Although estrogens can stimulate the growth of uterine epithelial cells in vivo, there is no clear effect of estrogens on the in vitro growth of epithelial cells from reproductive tract tissues; thus, we have established a defined culture system for mouse uterine epithelial cells. Pieces of uteri from immature CD-1 mice (21-23 days of age) were treated with trypsin, and the epithelial fragments were separated, enriched by Percoll gradient centrifugation, and seeded on collagen gels prepared from rat tail tendon. Initially, the cells were cultured in a 1:1 mixture of Ham's F-12 and Dulbecco's Modified Eagle's Medium supplemented with epidermal growth factor (EGF; 10 ng/ml), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), hydrocortisone (0.1 micrograms/ml), and vitamin A (10 ng/ml). The cells formed a monolayer on the collagen gel within 1-2 days, but with time, cells began to detach from the gel. Further studies revealed that the attachment and growth of these cells on collagen were markedly influenced by the calcium concentration. It was found that lowering the calcium concentration from 1.05 to 0.05-0.1 mM dramatically suppressed cell detachment; the number of cells doubled after 7 days of culture. Proliferation of uterine epithelial cells was enhanced by EGF, but not by fibroblast growth factor, platelet-derived growth factor, nerve growth factor, multiplication-stimulating activity, or somatomedin-C. The uterine epithelial cells exhibited a single class of high affinity binding sites for [125I]iodo-EGF (Kd, approximately 1.8 nM), with approximately 5 X 10(4) receptors/cell; binding was inhibited by EGF but not by the other polypeptides. This cell culture system will aid in our investigations on hormonal effects on the growth and differentiation of estrogen target cells. PMID- 3004889 TI - Location of epidermal growth factor receptors on porcine thyroid follicle cells and receptor regulation by thyrotropin. AB - By using a direct electron microscopic autoradiographical technique, we were able to demonstrate that the epidermal growth factor (EGF) receptor is asymmetrically located on polarized porcine thyroid follicle cells cultured on a collagen gel matrix. More than 80% of autoradiographical grains from [125I]EGF were associated with the basolateral cell surface compared to only about 2.5% at the apical cell margin. EGF stimulated growth only when the collagen gels were floating, indicating a basolateral location of functional EGF receptors. Preincubation with TSH or another stimulator of cAMP synthesis, forskolin, increased binding of [125I]EGF to the cells. Moreover, the subsequent mitogenic response to EGF was potentiated by such pretreatment. The results are in accordance with the hypothesis that TSH potentiates the response to EGF in the stimulation of thyroid growth. PMID- 3004890 TI - Effect of the Hyp mutation and diet-induced hyperparathyroidism on renal parathyroid hormone- and forskolin-stimulated adenosine 3',5'-monophosphate production and brush border membrane phosphate transport. AB - The present study was undertaken to investigate the effect of the Hyp mutation and diet-induced hyperparathyroidism on renal responsiveness to PTH and forskolin and to determine whether the renal brush border membrane phosphate transport defect is expressed in the vitamin D- and calcium-deficient Hyp mouse. Our results indicate that PTH is a potent activator of cAMP synthesis in renal slices obtained from vitamin D-replete normal mice and Hyp littermates. However, in the mutants, the amount of cAMP produced in response to similar concentrations of PTH (0.005-5 U/ml) is 55% of normal (P = 0.0076). PTH-dependent cAMP synthesis is significantly reduced in both normal and Hyp mice fed the vitamin D-deficient low calcium diet, and under these conditions, genotype differences are no longer apparent. In contrast, forskolin-stimulated cAMP production is identical in vitamin D-replete normal and mutant mice. Furthermore, cAMP accumulation in response to forskolin is not decreased by diet-induced hyperparathyroidism in either genotype. Vitamin D and calcium deprivation results in a significant decrease in renal brush border membrane Na+-dependent phosphate transport in both genotypes, and under these conditions, the phosphate transport defect persists in Hyp mice. Finally, vitamin D and calcium deprivation has no effect on renal brush border membrane alkaline phosphatase activity. The present results suggest that the catalytic subunit of adenylate cyclase is intact in the mutant strain and that the blunted renal response to PTH in vitamin D-replete Hyp mice as well as in vitamin D- and calcium-deficient mice may be due to uncoupling of the PTH receptor-adenylate cyclase system. The data also suggest that the expression of the renal phosphate transport defect in Hyp mice is independent of PTH status and alkaline phosphatase activity. PMID- 3004891 TI - Hormonal control of polyamine levels in bovine adrenocortical cells. AB - The implication of polyamines in cellular growth and differentiation processes and the existence of a polyamine-mediated protein phosphorylation system in adrenocortical cells suggest that polyamines may be examined as potential intracellular messengers in the pleiotypic action of ACTH. Bovine adrenocortical cells in culture exhibit a specific, energy-dependent, partly sodium-supported, inward polyamine transport system, independent of the A, L, and N aminoacid uptake systems. Steroidogenic concentrations of ACTH (10(-12) to 10(-9)M) induced a rapid activation of the polyamine uptake, resulting in a 2- to 3-fold increase in intracellular polyamine content, over 1 h. The ACTH dose-response curves for steroidogenic activity and for polyamine uptake were similar. Other adrenocortical effectors such as angiotensin II, acetylcholine, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) activated polyamine uptake with a pattern parallel to their steroidogenic potency (i.e. ACTH greater than angiotensin II greater than acetylcholine, TPA). Steroidogenic concentrations of 8-bromo-cAMP displayed no effect on the adrenocortical polyamine uptake, suggesting that cAMP does not mediate this action of ACTH. On the other hand, ACTH induced a large increase in ornithine decarboxylase (ODC) activity in bovine adrenocortical cells, after a 6- to 8-h lag period, resulting in an average 2 fold increase in cell putrescine, spermidine, and spermine content. However, when the cells were previously polyamine loaded, ACTH-dependent ODC induction was suppressed. Adrenocortical cell polyamine content thus appears to be under hormonal control. ACTH may act through two possible pathways: 1) the rapid activation of the cell polyamine accumulation from an extracellular source and 2) the delayed increase in polyamine biosynthesis secondary to induction of ODC activity, when the cells are relatively depleted of the polyamines. These observations suggest that polyamines may function as intracellular messengers for some of the ACTH effects in bovine adrenocortical cells. PMID- 3004892 TI - Processing of insulin-like growth factors I and II by capillary and large vessel endothelial cells. AB - Cultured endothelial cells from bovine capillaries and pulmonary arteries were incubated with highly purified [125I]insulin-like growth factor I ([125I]IGF-I), [125I]IGF-II, or [125I]insulin for periods up to 120 min, the cells were washed, and the cell-bound radioactivity was allowed to dissociate from the cells into fresh incubation medium. For insulin, 85-95% of the 125I dissociated from cells in 15 min, with 75% dissociating by 5 min. The 125I material released into the medium during the first 5 min of dissociation was entirely intact insulin, while the material released in the next 10 min was 80% intact insulin. For the [125I]IGFs, several differences were observed. First, after 5 min of dissociation, only 30-45% of the cell-bound 125I was released into the medium. For IGF-I, this rapidly dissociating material was entirely intact peptide, whereas for IGF-II, up to 55% of the dissociated radioactivity was degraded peptide. Second, during the next 10 min of dissociation, an additional 20% of the 125I was released from the cells. For IGF-I, this was 85% intact peptide; however, for IGF-II, this dissociated fraction contained as little as 25% intact peptide. Third, for both IGFs, after 15 min of dissociation, 40-65% of the initial cell-associated 125I remained within the endothelial cells; after the 15 min dissociation period, 95% of the remaining internalized 125I was intact IGF-I, whereas for IGF-II, the internalized 125I was 90% intact IGF-II after short periods of association (less than 15 min) and progressively decreased to 65% intact peptide after 60 min of association. We conclude that in addition to having separate surface receptors for insulin, IGF-I, and IGF-II, endothelial cells also process each hormone by distinct pathways. PMID- 3004893 TI - Genetic regulation of androgen-induced accumulation of mouse renal beta glucuronidase messenger ribonucleic acid. AB - Mouse kidney beta-glucuronidase is induced by androgens in a receptor-dependent fashion. Genetic regulatory mutations have been described which govern this response. In mice carrying the Gus-ra allele (A/J), the induction of enzyme activity is 3-5 times greater than in animals with the Gus-rb allele (C57BL/6J). To study this hormonal and genetic control at the molecular level, we measured changes in beta-glucuronidase mRNA concentrations in these two mouse strains using cloned cDNA. The specificity of the regulation was assessed by following changes in the concentration of kidney androgen-regulated protein (KAP) mRNA, which is also under androgen control in mouse kidney. Female mice were treated with Silastic implants that released 120 micrograms testosterone/day over a 20 day time course. Induction of specific mRNA was analyzed by either Northern blot hybridization or a more quantitative assay in which renal poly(A) RNA was covalently bound to aminophenylthioether paper discs. The induction of beta glucuronidase mRNA was about 10-fold higher in the strain carrying the Gus-ra regulatory allele, indicating that the Gus-r locus controls the beta glucuronidase structural gene (Gus-s) at the level of mRNA accumulation. Regulation by Gus-r was specific for beta-glucuronidase mRNA as kidney androgen regulated protein mRNA accumulation did not differ between these two strains of mice after androgen treatment. PMID- 3004895 TI - Thyroxine and 3,5,3'-triiodothyronine bind to the same putative receptor in hepatic nuclei of Rana catesbeiana tadpoles. AB - Previous studies have provided conflicting evidence as to whether tadpole liver nuclei contain the same number of high affinity binding sites for T4 as they do for T3. Inability to prepare stable tadpole liver nuclei may have contributed to these inconsistencies. Thus, a study was carried out in which the in vivo binding of T4 and T3 by tadpole liver nuclei was reexamined with a greatly improved method for isolating liver nuclei. It was found that the maximum binding capacities of the nuclei for T3 and T4 were not significantly different [0.161 +/ 0.015 (+/- SEM) vs. 0.185 +/- 0.046 pmol/mg DNA]. However, the affinity of the binding sites for T3 was 2-3 times their affinity for T4 (Kd, 1.65 +/- 0.31 vs. 4.32 +/- 0.92 X 10(-12) M). Furthermore, the nuclear binding of [125I]T4 or [125I]T3 was decreased to comparable levels by administration of saturating amounts of T3 and T4 given either before or with the 125I-labeled hormone, indicating that both hormones were competing for the same set of sites. It is suggested that at least some of the previous conflicting findings related to the number of putative thyroid hormone receptors in tadpole liver nuclei are attributable to inadequate methodology which resulted in an overestimation of the maximum binding capacity for T4. PMID- 3004894 TI - Reduction of lactogenic receptors in female hamster liver due to the human growth hormone analog produced by plerocercoids of the tapeworm, Spirometra mansonoides. AB - The inductive effect of GH on hepatic lactogenic receptors is suspected of being due to a direct somatogenic action. Plerocercoid larvae of the tapeworm, Spriometra mansonoides, produce a factor that stimulates body growth, suppresses endogenous GH, and specifically displaces [125I]human (h) GH from hepatic receptors. Plerocercoid growth factor (PGF) mimics the growth-promoting actions of GH, but it has not been shown to duplicate all of the activities reported for GH. An important function of GH is its role in the maintenance of liver receptors for lactogenic hormones. This study was undertaken to determine if treatment of female hamsters with PGF would increase, decrease, or have no effect on liver receptors that bind hGH. Since hGH binds to somatogenic as well as lactogenic receptors, it was necessary to demonstrate the specificity of PGF's effects on [125I]hGH binding. PGF-treated (15 pleocercoids sc) hamsters had accelerated body growth, suppressed serum GH, and a marked reduction in [125I]hGH and [125I]ovine PRL binding to hepatic microsomes. Specific binding of [125I] bGH was unaltered by PGF treatment. The difference in [125I] hGH binding was due to a reduction in receptor number and not to receptor occupancy or reduced affinity. Serum GH was normalized after 10 days of estradiol benzoate (25 micrograms/day) injections, but the binding capacity for [125I]hGH of the PGF-treated group was less than half that of the control group. The fact that estrogen injections normalized serum GH, but not hGH binding, indicates that down-regulation of these receptors by PGF cannot be entirely explained on the basis of reduced levels of serum GH. The lack of any effect of PGF treatment on [125I]bGH binding suggests that the hepatic somatogenic receptors were not involved and that the reduction in receptors for [125I]hGH was associated with the lactogenic component of hGH. PMID- 3004896 TI - Dexamethasone increases 1,25-dihydroxyvitamin D3 receptor levels and augments bioresponses in rat osteoblast-like cells. AB - Glucocorticoids increase the level of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors in primary cultures of rat calvarial osteoblast-like (OB) cells. The present study investigated how this dexamethasone (DEX) up-regulation of 1,25 (OH)2D3 receptors modulates three 1,25-(OH)2D3 bioresponses: inhibition of collagen synthesis, stimulation of osteocalcin synthesis, and induction of 25 hydroxyvitamin D3-24-hydroxylase activity. Pretreatment of OB cells with 13 nM DEX for 24 h doubled the 1,25-(OH)2D3 receptor level without changing receptor affinity for 1,25-(OH)2D3 to study bioresponses. After DEX treatment to increase the 1,25-(OH)2D3 receptor level, the magnitude and sensitivity of all three 1,25 (OH)2D3 bioresponses were enhanced. The maximal 1,25-(OH)2D3 inhibition of collagen synthesis was increased by DEX pretreatment compound to control values: 30 to 50% (1 day treatment) and 50 to 70% (2 day treatment). The sensitivity to 1,25-(OH)2D3, as measured by reduction of the half-maximal inhibitory dose (ED50), was increased 50%. This potentiation of 1,25-(OH)2D3 inhibitory action on collagen synthesis was still evident after correction for the inhibitory effect on collagen synthesis by DEX alone. The maximal stimulation of osteocalcin by 1,25-(OH)2D3 was also enhanced from 2- to 3-fold in controls to over 4- to 5-fold by DEX pretreatment. Similarly, the ED50 of the response was reduced 50%. For the induction of 25-hydroxyvitamin D3-24-hydroxylase activity, DEX doubled the enzyme activity over that seen with 1,25-(OH)2D3 alone, but only slightly affected the sensitivity of the enzyme induction. In conclusion, after DEX up-regulation of 1,25-(OH)2D3 receptor levels, there was a general potentiation of 1,25-(OH)2D3 bioresponses in rat OB cells. However, the detailed patterns of the augmented responses were different for each of the three biological functions we studied. PMID- 3004897 TI - Gonadotropin-releasing hormone stimulates mass changes in phosphoinositides and diacylglycerol accumulation in purified gonadotrope cell cultures. AB - GnRH stimulates rapid and specific hydrolysis of the phosphoinositides and their conversion to diacylglycerols (DAGs) in enriched gonadotrope cultures. In short term labeling studies, we observed stimulation of 32P incorporation into inositol phospholipids; no corresponding change was seen in other major phospholipids. The observation that this response continues in the presence of the Ca2+ channel antagonist D600 suggests that, unlike LH release, stimulation of phosphatidylinositol (PI) production is independent of mobilization of extracellular Ca2+. Agents that substitute for endogenous DAGs (phorbol myristate acetate) or mobilize Ca2+ directly (ionophore A23187) did not stimulate 32P incorporation into PI. Relative mass changes in PIs in response to GnRH were measured in a 32P/33P protocol. These showed a fall (40%) in the level of the inositol phospholipids within 45 sec, with a new steady state achieved at a lower level by 5 min. Phosphatidic acid formation was stimulated by GnRH within 2 min, with a 150% increase above control values at the highest dose of GnRH tested (10( 7) M). In cells prelabeled with [3H]arachidonic acid, GnRH caused a transient increase in DAG formation (twice the control value) within 1 min (with or without D600) which declined thereafter. This effect of GnRH was not seen in cells stimulated to release LH with ionophore A23187 or phorbol myristate acetate, suggesting that these may act distally to inositol phospholipid turnover. The data suggest that GnRH stimulates the hydrolysis of newly synthesized PIs, leading to the formation of DAGs. This pathway appears to be regulated independently of mobilization of extracellular Ca2+. PMID- 3004898 TI - Corticotropin-releasing factor binding to the anterior pituitary receptor is modulated by divalent cations and guanyl nucleotides. AB - The binding of ovine CRF to bovine anterior pituitary membranes was characterized with the radioligand [Nle21, m-125I Tyr32]ovine CRF. The specific binding of CRF was increased by millimolar concentrations of the divalent cations Mg2+, Ca2+, and Mn2+, but was unaffected by the monovalent cations Na+, Ki+, and Li+. The increase in specific binding was not caused by a change in the affinity, but resulted from an apparent increased number of high affinity binding sites. In the presence of 10 mM Mg2+, CRF binding was saturable, specific, and of high affinity. At room temperature, under optimum conditions, binding reached equilibrium within 70 min, remained stable for at least 4 h, was reversible by excess unlabeled peptide, and was characterized by a Kd of 1.3 nM (0.74-2.4) and a total number of sites, R degree, of 90 fmol/mg protein (55-145). The affinity for the bovine membranes was the same as that for rat anterior pituitary membrane homogenates, and the concentrations of sites were similar. The relative binding affinities for the CRF receptor of selected agonists and antagonists in the bovine and rat systems were similar, and both showed good correlation with the relative in vitro potencies at stimulating or antagonizing, respectively, ACTH release from cultured rat anterior pituitary cells. The nonhydrolyzable GTP analog 5'-guanylylimidodiphosphate [Gpp(NH)p], caused a dose-dependent inhibition of bound radioligand, with an EC50 of about 0.5 microM. In the presence of 1 microM Gpp(NH)p, the number of high affinity CRF-binding sites was reduced, but the affinity was unchanged. In the presence of 5 microM Gpp(NH)p, there was no detectable high affinity binding, and the rate of dissociation of previously bound radioligand was increased. The other guanyl nucleotides, GTP and GDP, inhibited binding but to a lesser extent, whereas GMP and (Bu)2cGMP were ineffective. The inhibition was specific for guanyl nucleotides. In view of the established effects of guanyl nucleotides and divalent cations on adenylate cyclase-linked receptors, these data support the involvement of the adenylate cyclase system in the mechanism of action of CRF on the anterior pituitary. PMID- 3004899 TI - Adenosine 3',5'-monophosphate-mediated induction of 17 alpha-hydroxylase and C 17 20 lyase activities in cultured mouse Leydig cells is enhanced by inhibition of steroid biosynthesis. AB - We recently reported that treatment of mouse Leydig cell cultures for 5 days with LH or cAMP caused an induction of the microsomal cytochrome P-450 activities 17 alpha-hydroxylase and C17-20 lyase. We also have shown that the microsomal P-450s are very sensitive to oxygen-mediated loss of these activities and that this decrease is enhanced by steroids produced during acute cAMP stimulation of Leydig cells. In the present study, we investigated whether steroids produced during chronic cAMP treatment of Leydig cells limit the extent of induction of 17 alpha hydroxylase and C17-20 lyase. Treatment of Leydig cell cultures with 8-bromo-cAMP in the presence of aminoglutethimide, an inhibitor of cholesterol side-chain cleavage, resulted in a 4- to 7-fold enhancement of cAMP-mediated induction of the 17 alpha-hydroxylase and C17-20 lyase activities and, after 11 days of treatment, completely restored the activities to those found in freshly isolated Leydig cells. Treatment with aminoglutethimide in the absence of cAMP had no effect on these enzyme activities. Addition of the steroid products androstenedione and/or testosterone (5 microM) to cAMP-plus amino-glutethimide treated cultures caused a significant reduction in cAMP-mediated induction of microsomal P-450, while addition of estradiol (50 nM) had no significant effect. 3 beta-Hydroxysteroid dehydrogenase-isomerase, another microsomal enzyme that is not a P-450 enzyme, was not induced by cAMP in either the presence or absence of aminoglutethimide. The data suggest that Leydig cell microsomal P-450 activities are maintained in vivo by a balance between two processes: cAMP-mediated induction and steroid product-induced degradation. PMID- 3004900 TI - Solubilization of a guanine nucleotide-sensitive parathyroid hormone-receptor complex from canine renal cortex. AB - Canine renal cortical PTH receptors were solubilized after occupancy of membrane associated receptors with the agonist ligand [125I]bovine (b) PTH-(1-34). Stabilization of binding during solubilization required the use of high concentrations of BSA (optimally 5%) and appropriate detergents (0.5% 3-[(3 cholamidopropyl)dimethylammonio]2-hydroxy-1-propanesulfonate, 0.5% 3-[(3 cholamidopropanyl)dimethylammonio]1-propanesulfonate, or 0.5-1.0% digitonin). The soluble fraction (240,000 X gav supernatant) contained [125I]bPTH-(1-34) associated with macromolecular components as well as unbound [125I]bPTH-(1-34) that dissociated during solubilization. The soluble macromolecular complex had functional properties expected of a ternary complex consisting of [125I]bPTH-(1 34) receptor stimulatory guanine nucleotide-binding protein (Ns). Thus, the dissociation of labeled PTH at 30 C was slow (t1/2 = 75 min); in the presence of GTP (10(-4) M), 75% of the sites displayed rapid dissociation kinetics (t1/2 = 2.3 min). This effect was nucleotide specific, with GTP approximately equal to GTP gamma S approximately equal to GDP greater than GDP beta S greater than ITP approximately equal to guanylylimidodiphosphate much greater than GMP approximately equal to App(NH)p. ATP was ineffective. GTP produced a half-maximal response at a concentration of 200 nM. These results are consistent with the reported nucleotide specificity and affinity of purified Ns. Treatment of membranes with N-ethylmaleimide during the binding reaction rendered the solubilized complex refractory to GTP. Gel filtration chromatography (Sepharose 6B) revealed a GTP-sensitive complex that eluted in the position expected of a detergent-free spherical protein of 180,000 daltons. This complex may consist of the 60,000 to 70,000-dalton PTH-binding subunit (previously identified by photoaffinity labeling) together with Ns. PMID- 3004901 TI - Effects of 1,25-dihydroxyvitamin D3 on osteoblastic MC3T3-E1 cells. AB - 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] was examined for a possible stimulative effect on osteoblastic MC3T3-E1 cells. During the early period of culture, 1,25 (OH)2D3 had a stimulative effect. During the growth phase, however, the steroid had little effect on either the protein or DNA content of the cultures. 1,25 (OH)2D3 increased bone-liver-kidney-type alkaline phosphatase activity in a dose related manner up to a concentration of 5 pg/ml; the increase was 2.2-fold over the control value. Studies on the effect of actinomycin D or cycloheximide treatment indicated that the vitamin may enhance de novo synthesis of ALP. The steroid also stimulated type I collagen production dose dependently via an increase in collagen synthesis rather than by inhibition of collagen degradation. MC3T3-E1 cells have a specific receptor for 1,25-(OH)2D3 which has a dissociation constant of 4.17 X 10(-11) M and a sedimentation coefficient of 3.67S. The receptor concentration varied with the period of culture, being higher during the growth phase and lower at confluence, but its affinity did not change. The results indicate that 1,25-(OH)2D3 has a direct specific anabolic effect on osteoblastic cells in vitro during the growth phase and that this effect is related to receptor concentration. PMID- 3004902 TI - Growth factors suppress and phorbol esters potentiate the action of dibutyryl adenosine 3',5'-monophosphate to stimulate aromatase activity of human adipose stromal cells. AB - In previous studies, we observed that the stimulatory effect of (Bu)2cAMP on aromatase activity of human adipose stromal cells was markedly attenuated when fetal calf serum was present in the culture medium. To determine whether growth factors may be the inhibitors of (Bu)2cAMP-stimulated aromatase activity in serum, the effects of growth factors and phorbol esters on aromatase activity of human adipose stromal cells in monolayer culture were investigated. Epidermal growth factor (EGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) were all without effect on aromatase activity when added by themselves, but markedly inhibited aromatase activity stimulated by (Bu)2cAMP. On the other hand, nerve growth factor, multiplication-stimulating activity, relaxin, and insulin had no effect on aromatase activity, either by themselves or in the presence of (Bu)2cAMP. Thus, EGF, PDGF, and FGF can mimic the inhibitory action of fetal calf serum on (Bu)2cAMP-stimulated aromatase activity of these cells. By contrast, none of these substances was capable of mimicking the effect of serum to facilitate the stimulatory action of dexamethasone on aromatase activity of these cells. The phorbol esters phorbol-12-myristate-13-acetate, phorbol-12,13-didecanoate, and phorbol-12,13-diacetate were also capable of facilitating the action of (Bu)2cAMP to stimulate aromatase activity, but had little or no action on dexamethasone-stimulated aromatase activity or when added by themselves. It is concluded that aromatase is under multifactorial regulation in human adipose stromal cells. The activity is induced by glucocorticoids and by agents that stimulate cAMP-dependent protein kinase; the latter effect is potentiated by factors that stimulate protein kinase C, but is suppressed by growth factors such as EGF, FGF, and PDGF, whose actions are believed to be mediated by receptor-linked tyrosine kinase activity. PMID- 3004903 TI - Studies on the glycoprotein nature of the thyrotropin receptor: interaction with lectins and purification of the bovine protein with the use of Bandeiraea (Griffonia) simplicifolia I affinity chromatography. AB - The TSH receptor from Triton-solubilized bovine microsomal membranes was found to bind to a substantial extent to columns of the immobilized lectins Bandeiraea (Griffonia) simplicifolia I, Ricinus communis I, wheat germ, and Concanavalin A, whereas it was not retained by Dolichos biflorus. Elution of TSH receptor activity from these lectins could be achieved with the appropriate saccharides in all cases except Concanavalin A. The most extensive adsorption of the receptor occurred on B. simplicifolia I-agarose (84%), and the terminal alpha-D-galactosyl specificity of this interaction was substantiated by its susceptibility to alpha galactosidase treatment. Whereas TSH itself was not bound to this immobilized lectin, a complex of this hormone with its receptor did interact and could be eluted with methyl-alpha-D-galactoside. Purification (800-fold) of the bovine TSH receptor was achieved by a combination of TSH and B. simplicifolia I affinity chromatographies. Polyacrylamide gel electrophoresis of the purified TSH receptor after radioiodination revealed three major components with apparent mol wt of 316,000, 115,000, and 54,000. PMID- 3004904 TI - Measurement of cytosolic free Ca2+ concentrations in human and rat osteosarcoma cells: actions of bone resorption-stimulating hormones. AB - Influx of extracellular Ca++ into bone cells has been postulated as an early action of PTH and other bone resorption-stimulating factors. To test this hypothesis directly, we measured the cytosolic free Ca2+ concentration ([Ca2+]i) in two hormone-responsive human (SaOS-2 and G-292) and two rat osteosarcoma cell lines (Ros 25/1 and Ros 17/2.8) and in primary cultures of bone cells from neonatal mouse calvaria using the fluorescent Ca2+ indicator Quin 2. Actions of bovine PTH-(1-34), vasoactive intestinal peptide, epidermal growth factor, prostaglandin E2, and ionomycin were studied. Medium cAMP (20 min; 37 C; 25 microM 3-isobutyl-1-methylxanthine) was quantitated by RIA. Basal [Ca2+]i was: SaOS-2, 126 +/- 8 nM; G-292, 61 +/- 6 nM; Ros 25/1, 109 +/- 15 nM; Ros 17/2.8, 363 +/- 42 nM; and primary cultures, 266 +/- 39 nM (mean +/- SE; n = 3-14). In each cell type, no acute (1 sec to 20 min) spike in [Ca2+]i was observed in response to PTH (24-120 nM), vasoactive intestinal peptide (100 nM), epidermal growth factor (17 nM), or prostaglandin E2 (2.8 microM). However, in SaOS-2 cells only, PTH reproducibly increased [Ca2+]i 10-15% above basal values beginning about 3 min after hormone addition, and this small increase returned to baseline at 15-20 min. Ionomycin (100 nM) elicited an immediate spike in [Ca2+]i to levels 2- to 4-fold above basal in all cells; the peak [Ca2+]i decayed rapidly (within 4 5 min) to baseline in G-292, Ros 25/1, and Ros 17/2.8 cells. The decay of peak [Ca2+]i in SaOS-2 was prolonged. To test for intact hormone responses in Quin 2 loaded cells, cAMP accumulation was measured. In SaOS-2 and Ros 17/2.8, both control and Quin 2-loaded cells showed similar increases in cAMP in response to PTH. Considering the limitations of the Quin 2 technique, we conclude that in the four hormone-responsive bone cell lines and primary cultures of bone cells tested, acute elevation of [Ca2+]i is not an inevitable consequence of receptor occupancy and/or adenylate cyclase activation by bone resorption-stimulating hormones. PMID- 3004905 TI - Characterization of a specific, high affinity [3H]arginine8 vasopressin-binding site on liver microsomes from different strains of rat and the role of magnesium. AB - A single class of high affinity, low capacity, specific binding sites for [3H]arginine8 vasopressin (AVP) has been characterized in a plasma membrane enriched microsomal fraction of the rat liver. Specific binding was saturable, linear with protein concentration, reversible, and 40-65% of the total binding. Binding at 25 C achieved a plateau after 30 min of incubation, whereas at 4 C, equilibrium was reached more slowly, and the level of binding was reduced. The presence of magnesium (Mg2+) in the assay medium enhanced the affinity of specific binding, while calcium and higher levels of sodium and potassium decreased binding. Scatchard analysis of binding in the presence of Mg2+ (5 mM) revealed an apparent mean +/- SE equilibrium dissociation constant (Kd) of 0.29 +/- 0.08 nM, with a maximal site density (Bmax) of 150.4 +/- 25.0 fmol/mg; in contrast, the Kd was 1.93 +/- 0.33 nM and the Bmax was 113.4 +/- 40.0 fmol/mg in the absence of Mg2+. No significant differences in Kd and Bmax were observed among membrane fractions derived from spontaneously hypertensive rats, Wistar Kyoto rats, Long-Evans rats, and Brattleboro rats. Plasma levels of AVP were similar in spontaneously hypertensive, Wistar-Kyoto, and Long-Evans rats, but AVP was not detectable in the plasma from DI rats. Competitive inhibition of specific [3H]AVP binding by unlabeled AVP and related peptides showed the following Ki values: AVP, 0.19 nM; LVP, 1.7 nM; oxytocin, 41.4 nM; desamino AVP, 0.38 nM; [1 (beta-mercapto-beta, beta-cyclopentamethylene propionic acid) 4-Val,8-D-Arg] VP2 (O-methyl)tyrosine]AVP, 1.8 nM; desglycinamide AVP, 2.2 microM. The neuropeptide metabolite of AVP [[pGlu4,Cyt6] AVP-(4-9)], angiotensin II, and other unrelated peptides did not displace [3H]AVP, demonstrating the specificity of AVP and its related biologically active peptides for this binding site. Moreover, the rank order of potency for displacement of [3H]AVP binding by these various peptides parallels their reported glycogenolytic activity in liver and/or their agonistic or antagonistic potency in vascular smooth muscle. Finally, the Mg2+-induced increase in the affinity of [3H]AVP for this liver binding site is similar to the reported effect of Mg2+ on the contractile responses of vascular smooth muscle to AVP (i.e. increased affinity). The results are consistent with the interpretation that the high affinity receptor site characterized in rat liver microsomes is of the V1 type. PMID- 3004906 TI - Dopamine inhibits prolactin release when cyclic adenosine 3',5'-monophosphate levels are elevated. AB - We purified lactotrophs from pituitary tumors induced by estrogen in ovariectomized female Fischer 344 rats from 80% of the population before to more than 90% after purification through a continuous Percoll density gradient. The percentage of lactotrophs was evaluated by immunofluorescence. The patterns of PRL release stimulated by 100 nM TRH, 20 microM A23187 (a Ca++ ionophore), 50 nM 12-O-tetradecanoyl-phorbol-13-acetate (a C-kinase activator), or combinations of these agents, or inhibited by 10 microM dopamine were similar in perifused primary cultures of tumor lactotrophs to patterns in cultures of anterior pituitary cells from female retired breeders used previously. In particular, dopamine completely inhibited the release stimulated by forskolin. Intracellular cAMP concentrations and PRL accumulation in the medium were measured in monolayer cultures of purified tumor lactotrophs. In 9 separate experiments, forskolin (10 microM) increased intracellular cAMP concentrations more than 60-fold above control after 30 min of incubation. Preincubation (30 min) with dopamine (10 microM) reduced the cAMP accumulation caused by forskolin, but levels were still at least 20-fold above basal levels in most experiments. PRL release was stimulated 2-fold with forskolin alone, but there was no stimulation of PRL release by forskolin in the presence of dopamine even though cAMP levels were elevated above basal. Therefore, a decrease in cAMP levels is not necessary to inhibit PRL release, and dopamine must have a mechanism for inhibiting PRL release in addition to inhibiting adenylate cyclase. PMID- 3004907 TI - Covalent cross-linking of growth hormone-releasing factor to pituitary receptors. AB - We have used a bifunctional cross-linker, disuccinimidyl suberate, to covalently attach [125I]human pancreatic GH-releasing factor (GHRF) (-1-40)OH to bovine pituitary membranes and rat anterior pituitary cells. Covalently radiolabeled membrane and cell preparations were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing and nonreducing conditions. In the former case, we observed the specific labeling of a polypeptide with an apparent mol wt of 75,000 +/- 3,000. The labeling of this species was specific for GHRF, as evidenced by the fact that it was inhibited in a dose-dependent fashion with increasing concentration of unlabeled GHRF. Furthermore, the radiolabeling was inhibited in the presence of excess unlabeled GHRF analogs but not unrelated peptides such as insulin and rat GH. The size of the radiolabeled band was the same in both bovine pituitary membranes and rat anterior pituitary cells. The extent of radiolabeling was dependent on the amount of membrane or the number of cells present during the binding reaction. These observations indicate that the mol wt 75,000 species is a ligand-binding subunit of the GHRF receptor in the pituitary. Under nonreducing conditions, a species much larger than mol wt 200,000 was specifically radiolabeled, again in both bovine pituitary membranes and rat cells. This result suggests the possibility that the ligand-binding subunit might be disulfide-linked to other subunit(s) forming homo- and heterooligomers. PMID- 3004908 TI - Long term culture of primary rat pituitary adrenocorticotropin/endorphin producing cells in serum-free medium. AB - Insulin, transferrin, and serum albumin were found to be essential additives for maintenance of primary rat pituitary ACTH/endorphin cells in complete serum-free defined medium (CSFM). Primary anterior pituitary cultures maintained in CSFM exhibited a 3- to 5-fold increase in cell content of ACTH/endorphin-related peptide during the first 2 weeks in culture, and this level remained stable for at least the next week. Immunocytochemical and morphometric studies indicated that the number of corticotropes increased only slightly, so that hormone content per corticotrope increased. Anterior pituitary cultures maintained in CSFM exhibited a basal secretory rate of 0.3-0.4% of cell hormone content/h throughout the 3-week period in culture, and total hormone production increased 6-fold. Primary anterior pituitary cultures maintained chronically in CSFM containing 10 nM CRF demonstrated a 15- to 20-fold increase in total ACTH/endorphin production over 3 weeks in culture. Chronic treatment with CRF brought about a sustained 6 fold increase in secretory rate (2.5% of cell hormone content/h), and corticotrope content of hormone was diminished 3-fold. Corticotropes maintained chronically in CSFM containing CRF did not increase in number and exhibited a rim of immunocytochemically identifiable hormone around the cell periphery. Anterior pituitary cultures maintained chronically in CSFM containing 100 nM dexamethasone (DEX) exhibited decreased cell hormone content and an unaltered secretory rate. In the DEX-treated cultures the number of immunocytochemically identifiable corticotropes declined, as did the staining intensity per corticotrope. Primary cultures of rat intermediate pituitary cells maintained in CSFM exhibited a 1.5- to 2-fold increase in hormone content after 1-2 weeks in culture, maintained a constant basal secretory rate of 0.4-0.5% cell content/h, and were not responsive to chronic treatment with CRF or DEX. PMID- 3004909 TI - Peroxide formation and glucose oxidation in calf thyroid slices: regulation by protein kinase-C and cytosolic free calcium. AB - We have determined the effects of tetradecanoyl phorbol acetate (TPA) and of the calcium ionophore A23187 on two thyroid responses to TSH previously reported to be cAMP-independent. We observed that TPA and A23187, at doses of 1.0 microM, stimulated both hydrogen peroxide generation and glucose oxidation in calf thyroid slices. A subthreshold dose of A23187 (0.1 microM) added to a submaximal dose of TPA (0.5 microM) acted synergistically, stimulating H2O2 production to the same degree as a maximally effective dose of TSH (50 mU/ml). Forskolin (25 microM), a direct stimulator of adenylate cyclase, actually inhibited both glucose oxidation and hydrogen peroxide generation. Lithium chloride (25 mM) had no effect on either response, either in the basal state or with TSH stimulation. The calcium channel antagonist verapamil (50 microM) decreased the basal activity of glucose oxidation and peroxide generation but did not substantially inhibit the effect of TSH on H2O2 generation under the conditions studied. These data support the concept that TSH induces changes in the thyroid phosphatidylinositol metabolism which activates protein kinase-C (c-kinase) and raises cytosolic free calcium. These events appear to act in concert to mediate certain metabolic responses in differentiated thyroid tissue. PMID- 3004910 TI - Thyrotropin regulates thyrotroph responsiveness to dopamine in vitro. AB - The effect of conditioned vs. fresh culture medium on the dopaminergic inhibition of TSH and PRL secretion by primary cultures of male rat anterior pituitary cells has been studied. In the presence of conditioned medium (that had been in contact with the cells over the 3-day culture period) 10(-6) M dopamine (DA) inhibited PRL secretion by 50% and TSH secretion by 30%. After 4 h of incubation with fresh medium 10(-6) M DA still inhibited PRL secretion by 50% but increased TSH release by 20%. TSH release was rapid and could be prevented by 10(-6) M prazosin, an alpha 1 adrenoreceptor antagonist. Fresh medium did not alter TRH induced TSH release. In parallel cultures and under identical conditions fresh medium reduced [3H]dihydroergocryptine (DHE) binding to DA receptors from 2.5 +/- 0.4 fmol/10(5) cells to 0.95 +/- 0.3 fmol/10(5) cells (means +/- SEM, n = 5, P less than 0.001). The effect of fresh medium was dose dependent against the dopaminergic inhibition of TSH secretion and against DA receptor binding. If 1 mU TSH was included, in fresh medium, the dopaminergic inhibition of TSH secretion remained unchanged and [3H]DHE binding to DA receptors did not fall. The rank order of potency of thyroid stimulators was bovine TSH (21 U/mg) greater than semipurified bovine TSH (Thytropar, 1.4 U/mg) greater than endogenous rat TSH (0.03 U/mg expressed as NIADDK-rat TSH-RP2) greater than Graves' immunoglobulin G (0.01 U/mg) when either DA or bromocriptine was used as the dopaminergic agonist. When anterior pituitary cells from hypothyroid rats were examined, the effects of culture medium on the dopaminergic inhibition of TSH and on DA receptor binding were approximately twice those observed in normal cells, but the inclusion of 1 mU TSH in the fresh medium completely prevented the loss of DA function and binding. PRL, human CG, ACTH, insulin, glucagon, and heat-inactivated TSH were unable to prevent the effect of medium replacement on dopaminergic inhibition of TSH and DA receptor binding. The data suggest a mechanism whereby TSH may control its own secretion via DA. PMID- 3004911 TI - Cytochromes P-450scc, P-450(17)alpha, adrenodoxin, and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase in bovine follicles and corpora lutea. Changes in specific contents during the ovarian cycle. AB - To investigate the basis for the pattern of ovarian steroid production during the bovine estrous cycle, the tissue concentrations of major steroidogenic enzymes, 17 alpha-hydroxylase cytochrome P-450 and cholesterol side-chain cleavage cytochrome P-450 (cytochrome P-450scc), and their respective electron donors, NADPH-cytochrome P-450 reductase and adrenodoxin, were estimated and compared with those of the nonsteroidogenic enzymes, cytochrome c oxidase and F1-ATPase. The levels of these enzymes were estimated in medium sized (9-11 mm) and large (14-18 mm) follicles after removal of follicular fluid by centrifugation, and corpora lutea from the early, early-mid late-mid, and late stages of the luteal phase (n = 5 per group). The specific contents of all enzymes and electron donors were determined by immunoblot analysis, except for cytochrome c oxidase, which was quantified by determination of specific activity. The specific (per microgram of tissue homogenate protein) and total (per follicle or corpus luteum) tissue contents of 17 alpha-hydroxylase cytochrome P-450 increased 0.5- and 5-fold respectively from medium sized to large follicles, but then decreased to undetectable levels in corpora lutea in the early luteal phase, and remained undetectable throughout the luteal phase. In contrast, the specific content of NADPH-cytochrome P-450 reductase was similar between follicles and corpora lutea. The specific contents of cytochrome P-450scc, adrenodoxin and cytochrome c oxidase in follicles were similar to those of corpora lutea of the early luteal phase. However, by the early-mid luteal phase the specific contents of luteal cytochrome P-450scc (490 +/- 46 vs. 5709 +/- 982 cpm/micrograms protein) and adrenodoxin (44 +/- 15 vs. 705 +/- 229 cpm/micrograms protein) were increased, by 12- and 15-fold, respectively (P less than 0.05). In contrast, cytochrome c oxidase activity (29.1 +/- 10.1 vs. 108.6 +/- 20.7 nmol/mg tissue protein X min) and the specific content of F1-ATPase increased only 3- to 4-fold reflective of an increase in numbers of mitochondria. The levels of these enzymes remained elevated until the late luteal phase when they declined markedly. It is concluded that the induction of synthesis of P-450scc and adrenodoxin after ovulation is specific and does not merely reflect biogenesis of mitochondria during luteinization. Moreover the changes in the types of steroids produced by the ovarian compartments throughout the estrous cycle are a reflection of changes in the tissue content of steroidogenic enzymes. PMID- 3004912 TI - In vitro stimulation and inhibition of adrenocorticotropin release by pituitary cells from ovine fetuses and lambs. AB - Short term cultured pituitary cells from fetal (63-144 days old) and young lambs (30-120 days old) were tested for their in vitro ability to release immunoreactive ACTH and cAMP in response to stimulation by ovine (o) oCRF1-41, arginine vasopressin (AVP), epinephrine, and forskolin, both in the absence and presence of corticosteroids. For each culture, the percentage of corticotrophs was determined by immunocytochemistry using ACTH antibodies, and the net responses (stimulated-baseline) were expressed per 10(5) corticotrophs. During gestation, basal ACTH release did not change significantly except at 125 days where it was 2-fold higher than at other fetal stages. Basal ACTH release was 2 fold higher in lambs than in fetuses. In the presence of oCRF1-41, a significant increase in ACTH secretion over basal value was observed at all stages studied. The maximal response decreased from 7.89 +/- 1.19 ng ACTH 10(5) corticotrophs-1 3 h-1 at 63 days of gestation to 3.49 +/- 0.88 ng at 115 days, then remained fairly constant in prepartum animals and lambs. No significant change in the ED50 was observed. The cAMP output induced by oCRF1-41 decreased progressively between 63 and 133 days of gestation from 10.72 +/- 1.84 to 2.21 +/- 0.62 pmol, then increased in lambs to values similar to that of 63-day-old fetuses. The ACTH response to AVP was higher than that to oCRF1-41 at 115 days, decreased dramatically in late gestation without modification of the ED50, and remained low in lambs. The ACTH response to epinephrine was always very low. Forskolin-induced ACTH release was lower between 115 and 144 days of gestation than at other stages. The synergistic effect of AVP and epinephrine on both cAMP and ACTH productions stimulated by oCRF1-41 decreased at the end of gestation. The ACTH response to each stimulus was inhibited by dexamethasone, cortisol, and corticosterone (10(-9) and 10(-7) M) throughout the period studied. This effect was maximum at 63 days of gestation. cAMP release was not altered by glucocorticoids. These results indicate that 1) maximal ACTH response in vitro of corticotrophs to AVP is achieved during fetal life; 2) the capacity of fetal corticotrophs to release ACTH in response to oCRF1-41 is similar to that of lambs throughout the last month of gestation; and 3) corticotrophs are sensitive to the glucocorticoid-negative feedback at least during the second half of fetal life. PMID- 3004914 TI - Spontaneous and stimulated adrenocorticotropin and vasopressin pulsatile secretion in the pituitary venous effluent of the horse. AB - Plasma ACTH, arginine vasopressin (AVP), and catecholamines were measured at 5 min intervals in the pituitary venous effluent of the unanesthetized horse. Pulses of ACTH and AVP were found to be surprisingly brief (usually of less than 10-min duration) and frequent (averaging between 15-25 min). A highly significant relationship in the changes in concentration of these two hormones was demonstrated (P less than 0.0002) both at rest and after a mild hypoglycemic stimulus. Although there was also a significant correlation (P less than 0.005) between simultaneous plasma ACTH and AVP values the pulse amplitude ratio of AVP to ACTH showed a considerable variation. A rise in cortisol appeared to have a greater suppressive effect on the amplitude of ACTH than AVP pulses. The gradient in hormonal concentration between pituitary effluent and jugular plasma was at times over 50-fold for ACTH, and 500-fold for AVP. A gradient was also found for epinephrine, norepinephrine, and dopamine. A highly significant correlation (P less than 0.005) was demonstrated between changes in norepinephrine, ACTH, and AVP concentrations, but no such relationship could be shown for epinephrine and dopamine. It is concluded that there is a close temporal relationship between changes in ACTH, AVP, and norepinephrine concentrations. Pulses of these hormones are greater in amplitude and more frequent than would have been suspected from sampling peripheral plasma. The variability in the pulse amplitude ratio of ACTH and AVP may suggest that other factors are affecting ACTH secretion. The ability to sample frequently for several hormones and to obtain a marked gradient in hormonal secretion between the pituitary venous effluent and jugular plasma suggest that the horse should provide an excellent animal model in which to study the regulation of hypothalamic and pituitary hormone secretion. PMID- 3004913 TI - Growth hormone enhances follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells. AB - Suppression of serum GH levels in immature rats is associated with delayed onset of puberty and decreased ovarian steroidogenic responsiveness to FSH. To investigate possible direct effects of GH on the differentiation of ovarian cells, granulosa cells from hypophysectomized estrogen-treated rats were cultured with FSH in the presence or absence of GH for 3 days. FSH stimulated granulosa cell LH receptor formation and steroid production in a dose-dependent manner. Concomitant treatment with GH increased LH receptor content by enhancing the action of low doses of FSH without substantial increases in the maximal response. This increase was due to an elevation in the receptor number rather than changes in their affinity for hCG. At 3 ng/ml FSH, concomitant treatment with ovine or bovine GH increased LH/hCG binding in a dose-dependent manner, with 300 ng/ml GH increasing the FSH action by about 3-fold. LH receptors in the GH-treated cells were functional, as indicated by the enhanced cAMP production of these cells in response to LH treatment. The cellular protein content in the FSH-treated cultures was slightly increased by GH (18%), but cell number and viability were unaffected. The change in cell protein content could not account for the increases in the amount of LH receptors. In addition to its effects on LH/hCG receptor content, GH also augmented FSH-stimulated progesterone and 20 alpha hydroxy-4-pregnen-3-one production in a dose-dependent manner, with 100 ng/ml GH causing significant increases in FSH-induced progesterone production. In contrast, GH treatment did not significantly affect FSH-stimulated estrogen production. The augmentating effects of GH on LH receptor formation and progestin biosynthesis were associated with an enhancement of FSH-stimulated cAMP production. In addition, GH increased forskolin- and 8-bromo-cAMP-induced LH receptor formation and progestin production. Thus, GH-augmented LH receptor induction and progestin biosynthesis may be due to both increased cAMP production and enhanced action of cAMP. The present data have demonstrated that GH augments gonadotropin-stimulated differentiation of ovarian granulosa cells, suggesting an important regulatory role of GH in follicular growth and pubertal development. PMID- 3004915 TI - Ontogeny of the stress response in the rat: role of the pituitary and the hypothalamus. AB - The neonatal rat shows a period of decreased responsiveness to noxious stimuli during the first 3 weeks of life, but the nature of this impairment is still controversial. To test the functionality of the hypothalamus-pituitary-adrenal axis during this period, we studied pituitary and adrenal responsiveness to exogenous ovine CRF and the ability of various stressors (ether vapors, electroshocks, and hypoxia) to elicit ACTH and corticosterone secretion. We also measured hypothalamic CRF content and pituitary ACTH content as well as CRF binding sites in the anterior pituitary. From days 3-10, small elevations in plasma ACTH and corticosterone levels were observed after a 3-min exposure to ether vapors or electroshocks. In contrast, during this period, a 20-min exposure to hypoxia (5% O2 in N2) was unable to trigger measurable ACTH secretion, while corticosterone was significantly elevated. From days 14-21, plasma ACTH and corticosterone levels increased significantly after exposure to ether stress, hypoxia, and, to a lesser extent, electroshocks. By contrast, administration of urethane (1.2 g/kg BW) caused a significant increase in ACTH secretion on days 3, 5, and 10, an effect that was partially suppressed by pretreatment with an anti CRF serum. This suggests that endogenous CRF can be released by at least some stimuli as early as day 3. Direct stimulation of the pituitary with synthetic oCRF (10 micrograms/kg BW) caused significant elevations in plasma ACTH levels at all ages tested (days 3 through 21), though these increases were significantly (P less than or equal to 0.01) smaller on day 3 (2.7-fold) than on day 21 (4.3 fold). Hypothalamic CRF content as well as ACTH content increased gradually with age, but the values reached by the third week of life were still low compared to the values on day 45. Finally, anterior pituitary CRF-binding sites averaged 317 +/- 48 fmol/mg protein on day 5 and 158 +/- 22 fmol/mg protein on day 17. The affinity (Kd) of the receptor for CRF was not significantly different on day 5, 17, or 45. These results show that although pituitary corticotrophs appear to be functional at birth, exposure to stress does not elicit marked increases in plasma ACTH until day 14 of age. PMID- 3004917 TI - The characterization of the ascorbic acid-mediated alpha-amidation of alpha melanotropin in cultured intermediate pituitary lobe cells. AB - Previous studies have shown that cultured rat intermediate pituitary lobe cells lose the ability to form ACTH-(1-13)NH2-related molecules (alpha MSH) and instead produce ACTH-(1-14)-related peptides. In vitro studies have shown that peptidylglycine alpha-amidating monooxygenase the enzyme responsible for the conversion of ACTH-(1-14) to ACTH-(1-13)NH2, requires ascorbate, CuSO4, and molecular oxygen as cofactors. In the present study we have demonstrated that cultured intermediate pituitary lobe cells require long term supplementation of the medium with ascorbate for continued production of immunoactive alpha-amidated alpha MSH. When the relative quantities of alpha-amidated and COOH-terminally glycine-extended forms of alpha MSH were assessed in biosynthetic labeling experiments, it was shown that either L-ascorbate or an epimer of the vitamin, D isoascorbate, was capable of supporting cellular alpha-amidation. However, the potency of isoascorbate was approximately 4- to 5-fold lower than that of ascorbate. The ascorbate-mediated reestablishment of alpha-amidation ability was shown to be dependent on the presence of sodium in the medium; at physiological levels of ascorbate (35 microM), the EC50 for sodium was about 40 mM. Time-course experiments indicated that the time of exposure of the cultured cells to ascorbate could be decreased to as little as 30 min. However, a total incubation time of 6 h was required after such an exposure to convert biosynthetically labeled ACTH-(1-14)-related peptides to labeled ACTH-(1-13)NH2-related peptides. The time course of the effects of ascorbic acid on the reestablishment of alpha amidation, as well as the relative stereospecificity for L-ascorbic acid and the sodium requirement, are all consistent with the hypothesis that ascorbic acid must be transported into the intermediate pituitary lobe cells to participate in the peptidyl alpha-amidation reaction. Moreover, it is apparent that the length of time required to reestablish alpha-amidation ability (6 h) is a function of transport of the cofactor into the granules, the rate of the peptidylglycine alpha-amidating monooxygenase-catalyzed reaction, or both. PMID- 3004916 TI - Ascorbate transport by AtT20 mouse pituitary corticotropic tumor cells: uptake and secretion studies. AB - Ascorbate is an important cofactor in the biosynthesis of alpha-amidated endocrine and neural peptides. Peptidylglycine alpha-amidating monooxygenase (PAM) is the enzyme responsible for the generation of mature COOH-terminal alpha amidated peptides from COOH-terminal glycine-extended peptides, and this enzyme requires ascorbate for full activity in vitro. Also, cultured intermediate pituitary lobe cells contain PAM and require ascorbate for the COOH-terminal alpha-amidation of alpha MSH. Since pituitary cells are not capable of synthesizing ascorbate, the ability of the cells to accumulate the cofactor must play an important role in the biosynthesis of alpha-amidated peptides. The AtT20 corticotropic pituitary tumor cell line also contains PAM and a potential site for COOH-terminal alpha-amidation of the pro-ACTH/endorphin-derived hinge peptide and was, thus, used for the study of cellular ascorbate transport. Radiolabeled L [1-14C]ascorbate ([1-14C]ascorbate) was incubated with the cells under various conditions, and the accumulation of radioactivity by the cells was followed. Reverse phase HPLC was used to identify the integrity of the labeled ascorbate, both intra- and extracellular, during the course of the experiments. The uptake of [1-14C]ascorbate was saturable (Km = 31.5 microM), sodium and temperature dependent, and stereoselective. The products of ascorbate autooxidation, dehydroascorbate and 2,3-diketogulonic acid, did not inhibit [1-14C]ascorbate uptake. To study the presence of ascorbate in the secretory granules, cells were incubated with [1-14C]ascorbate and then induced to secrete with isoproterenol or 8-bromo-cAMP. A 2- to 6-fold stimulation of ACTH secretion over the basal secretion rate was observed; however, the secretion of intracellular [1 14C]ascorbate did not change significantly with stimulation, suggesting that very little of the cellular ascorbate was contained within secretory granules. PMID- 3004919 TI - Rapid glucose-dependent increases in phosphatidic acid and phosphoinositides in rat pancreatic islets. AB - Glucose effects on islet phospholipids were examined during direct incubation or after 3 days of 32P prelabeling in primary culture. In both cases, glucose increased the 32P content of phosphatidic acid (PA), phosphatidylinositol (PI), and polyphosphoinositides (PPI). Glucose-induced increases in PA, PI, and PPI in the culture-prelabeling experiments were evident within 1 min, dose related, and reflective of increases in phospholipid mass, which was confirmed in direct incubations by measurement of PI phosphorus. Thus, in addition to increasing PI PPI hydrolysis, glucose increases de novo phospholipid synthesis in pancreatic islets. The latter may result from enhanced glycolysis and substrate availability for PA-PI-PPI synthesis, since glyceraldehyde and pyruvic acid also increased PI levels. Our findings raise the possibility that increases in PA, PI, and PPI synthesis could serve as a mechanism to enhance the generation of intracellular mediators, which are purported to regulate insulin secretion. PMID- 3004918 TI - Regulation of thyroxine 5'-deiodinase by thyroid hormones and activators of protein kinase C in GH4C1 cells. AB - The regulation of T4 5'-deiodinase activity was studied in cultured GH4C1 cells. Enzyme activity was measured in cell sonicates as the release of radioiodide from [125I]T4. Enzyme activity was stimulated 2- to 3-fold by hypothyroid serum and activators of protein kinase C, such as TRH and phorbol esters. The hypothyroid serum effect was maximal by 3 h, whereas TRH and phorbol esters required 6 h to achieve a maximal effect. The hypothyroid serum effect was gone within 4 h of returning the cells to control medium. In contrast, the TRH and phorbol ester effects persisted 24-48 h after removal of those agents. Both T4 and rT3 were at least as potent as T3 in blocking the effect of hypothyroid serum. The stimulation of 5'-deiodinase induced by hypothyroid serum was additive with that induced by kinase C activators. Trifluoperazine blocked the effect of TRH and phorbol esters, but not that of hypothyroid serum. It is concluded that stimulation of 5'-deiodinase activity can occur by at least two independent mechanisms: one involving hypothyroidism and another involving activation of protein kinase C. The relative potencies of various iodothyronines for abolishing the hypothyroid effect differ markedly from the relative binding affinities of these agents for the nuclear T3 receptor, suggesting that this thyroid hormone effect may not be mediated by the classical nuclear thyroid hormone receptor. PMID- 3004921 TI - Thyrotropic activity of acidic isoelectric variants of human chorionic gonadotropin from trophoblastic tumors. AB - Previous studies have indicated that hCG has a weak intrinsic thyroid-stimulating activity. Differences in the molecular composition and biological activity of hCG in patients with trophoblastic diseases and pregnant women occur, but are not well defined. Therefore, we have studied the effect of serum samples and purified hCG preparations from patients with trophoblastic diseases on T3 release from human and porcine thyroid slices in vitro. We examined 30 serum samples from 13 patients with nonseminomatous testicular germ cell tumors, 3 from women with choriocarcinoma, and 5 from patients with hydatidiform moles. In all but 1 serum sample from the tumor patients, but in none of 11 serum samples of pregnant women, T3-releasing activity was found. Two patients with testicular cancer and 1 patient with molar pregnancy experienced episodes of frank hyperthyroidism. Isoelectric focusing on polyacrylamide gels of tumor sera (n = 15) revealed substantial amounts of acidic isoelectric variants, pI 3.3-3.9, which were only barely detectable in pregnancy sera. The percentage of acidic hCG variants with pI 3.3-4.0 to total hCG with pI 3.3-5.2, as determined by hCG (+hCG beta) RIA of the eluted fractions of polyacrylamide gel isoelectric focussing, varied from 12 45% in sera of tumor patients and from 0-4% in pregnant sera. We purified the acidic variants of hCG with pI 3.6-3.8 (hCGav) from the urine of our patients. The beta-subunit of purified hCGav had a slightly higher mol wt (35,750) than that of hCG CR 119 (34,190) on polyacrylamide gel electrophoresis. The hCGav showed a dose-dependant stimulation of T3 release and cAMP generation from human thyroid slices, whereas the other hCG fractions on isoelectric focussing had no thyrotropic effect in similar dose levels. The TSH-like activity of hCGav could be roughly estimated as 10 mIU TSH/IU hCGav. Anti-hCG (+hCG beta) antiserum, but not anti-hTSH antiserum, neutralized the biological activity of hCGav. These findings strongly suggest that acidic hCG variants act as functional stimulators of the human thyroid in vitro. Since these molecular variants of hCG can exist in patients with trophoblastic diseases in significant amounts, they could be responsible for some cases of hyperthyroidism in trophoblastic diseases. PMID- 3004920 TI - Increased water excretion in hyperprolactinemic rats. AB - The role of PRL in mammalian salt and water balance remains controversial. To avoid the methodological problems of exogenous PRL administration, endogenous hyperprolactinemia was established in normal rats by implantation of extra anterior pituitary glands under the kidney capsule. To eliminate excess glucocorticoid secretion in these rats, they were adrenalectomized, implanted with corticosterone replacement pellets, and given 0.9% saline drinking fluid. A highly significant increase in urine flow was found in pituitary-grafted male Fischer rats compared to control rats (38.9 +/- 3.8 vs. 20.7 +/- 1.6 ml/24 h X 100 g BW; P less than 0.0005). Urine osmolality was lower in pituitary-grafted rats, but sodium and potassium excretion were not abnormal. Although fluid intake was also greater in hyperprolactinemic rats, the urine flow remained elevated when adjusted for fluid intake. Renal binding sites for radioactive PRL were not decreased in pituitary-grafted rats. Thus, the hyperprolactinemic rat has increased water excretion that may be attributed to a direct renal effect rather than to glucocorticoid excess or a primary change in thirst. Since it is possible that water absorption in the gut is also increased by PRL, multiple effects of PRL may be responsible for the diuresis observed in hyperprolactinemic rats. PMID- 3004922 TI - Human parathyroid hormone inhibits renal 24-hydroxylase activity of 25 hydroxyvitamin D3 by a mechanism involving adenosine 3',5'-monophosphate in rats. AB - The in vivo effect of PTH on renal 24-hydroxylase activity of 25-hydroxyvitamin D3 (25OHD3) was examined in vitamin D-deficient thyroparathyroidectomized rats by a recently developed sensitive in vitro assay of 25OHD3-hydroxylases using rat kidney homogenates and by an in vivo assay measuring the accumulation of tritiated metabolites in plasma 5 h after injection of 25OH[3H]D3. Infusion for 20 h of either human PTH-(1-34) (except at 800 pmol/h) or cAMP did not significantly change the plasma levels of calcium and phosphorus compared with those in thyroparathyroidectomized rats given 125 ng 1,25-dihydroxyvitamin D3 to induce renal 24-hydroxylase activity. On the other hand, human PTH-(1-34) markedly inhibited renal 24-hydroxylase activity and stimulated 1-hydroxylase activity. The effective concentration of human PTH-(1-34) was much lower for inhibiting 24-hydroxylase than for stimulating 1-hydroxylase activity. Infusion of less than 100 nmol/h cAMP similarly inhibited 24-hydroxylase activity without enhancing 1-hydroxylase activity. Either theophylline (1.0 mumol/h) or a submaximal dose (25 pmol/h) of human PTH-(1-34) alone inhibited 24-hydroxylase activity only slightly, but the concomitant infusion of both chemicals markedly inhibited 24-hydroxylase activity without stimulating 1-hydroxylase activity. These effects of human PTH-(1-34) and cAMP occurred similarly in both the in vitro and the in vivo assays. The present study clearly indicates that besides its well known action in stimulating 1-hydroxylase activity, PTH inhibits renal 25OHD3-24-hydroxylase activity by a mechanism involving cAMP. PMID- 3004924 TI - Differentiation of rat ovarian thecal cells: evidence for functional luteinization. AB - To determine if thecal cells of rat preovulatory (PO) follicles become functionally luteinized, theca from small antral (SA) and PO follicles were isolated before and 8 h after iv injection of an ovulatory dose (10 IU) of hCG. Thecal explants were cultured for 30 days in Dulbecco's Modified Eagle's Medium Ham's F-12 medium containing 1% fetal calf serum (FCS) with or without 5 ng/ml ovine LH or 10 microM forskolin. Whereas theca from SA, hCG-treated SA, and PO follicles were dependent on LH or forskolin to maintain progesterone (greater than 10 ng/ml) and androstenedione (greater than 10 ng/ml) accumulation, luteinizing theca (hCG-treated PO) accumulated more than 10 ng/ml progesterone and more than 2 ng/ml androstenedione with or without LH or forskolin for 30 days. Granulosa cells were isolated from these same follicles and cultured under similar conditions, including 10 ng/ml testosterone and 25 ng/ml ovine FSH. Only granulosa cells isolated from luteinizing follicles (hCG-treated PO) maintained progesterone (greater than 20 ng/ml) and estradiol (10 ng/ml) accumulation with or without FSH or forskolin for 30 days. Basal concentrations of cAMP were 5 to 10-fold higher in thecal and granulosa cells from luteinizing follicles than in these tissues isolated from SA or PO follicles. We conclude that thecal cells as well as granulosa cells of rat PO follicles respond to the LH/hCG surge by becoming functionally luteinized, less dependent on LH, and capable of maintaining an increased accumulation of basal cAMP. Furthermore, the data suggest that one luteinizing thecal explant produces a similar amount of progesterone as one follicle equivalent of luteinizing granulosa cells. Thus, luteinized theca have the potential of contributing significantly to progesterone secretion by the mature rat corpus luteum. PMID- 3004923 TI - Interaction of insulin-like growth factor I/somatomedin-C with cultured rat chondrocytes: receptor binding and internalization. AB - We have characterized the interaction of insulin-like growth factor I/somatomedin C (IGF-I/Sm-C) with its plasma membrane receptors on cultured rat chondrocytes. Our studies have demonstrated that [125I]IGF-I/Sm-C binding to these receptors is a relatively specific, reversible, and time-, temperature-, pH-, and concentration-dependent process. Insulin displaces [125I]IGF-I/Sm-C from its receptors on rat chondrocytes, but with a potency only 10(-4) that of IGF (I and II). Using the known lysosomotropic agents chloroquine and ammonium chloride as well as the substituted diamine monodansylcadaverine, we have shown that this 125I-labeled Sm is internalized and partially degraded via the lysosomal pathway. These conclusions have been further supported by photoaffinity labeling studies which, surprisingly, demonstrate that the predominant IGF receptor on chondrocytes is the type II receptor, and that [125I]IGF-I/Sm-C is bound primarily in this ligand-receptor complex which is internalized and degraded, in part, by lysosomes. PMID- 3004925 TI - Angiotensin-converting enzyme localized in the rat pituitary and adrenal glands by [3H]captopril autoradiography. AB - We have localized angiotensin-converting enzyme (EC 3.14.5.1) in the rat pituitary and adrenal glands by [3H]captopril autoradiography. The maximal numbers of [3H] captopril-binding sites are: posterior pituitary, 4500 fmol/mg protein; anterior pituitary, 1950 fmol/mg; intermediate pituitary, less than 100 fmol/mg; adrenal medulla, 480 fmol/mg; and adrenal cortex, less than 25 fmol/mg. The distribution within the posterior pituitary and adrenal medulla is homogeneous, whereas that in the anterior pituitary is patchy. Subcellular fractionation of the bovine adrenal medulla reveals enrichment of angiotension converting enzyme in plasma membrane fractions, but not in chromaffin granules. [3H]Captopril autoradiography in the rat pituitary gland is unaltered by dehydration, adrenalectomy, or reserpine treatment and in Brattleboro rats. [3H]Captopril binding in the adrenal medulla is increased by 75% 3 weeks after hypophysectomy and is elevated by 80% after reserpine treatment. The change after hypophysectomy is not reversed by dexamethasone treatment. PMID- 3004926 TI - Identical transcription initiation sites for proinsulin messenger ribonucleic acid in three insulin-expressing tissues. AB - Proinsulin mRNA was analyzed by RNA blot hybridization from four insulin expressing tissues of the rat, including adult and fetal pancreas, yolk sac, and an insulinoma cell line (RIN 5F). The proinsulin mRNA transcripts from the insulinoma cell line and fetal pancreatic tissue were estimated to be, respectively, 100 and 50 bases larger than the transcript from adult pancreas. Yolk sac proinsulin mRNA comigrated with the fetal transcript. While glucose is an important regulator of proinsulin mRNA in the adult, there is a marked increase in the concentrations of proinsulin mRNA and insulin in the developing rat, although plasma glucose levels are quite low. Expression of proinsulin mRNA independent of glucose levels is also found in insulinoma tissue. In addition, there is a second TATA sequence upstream of the putative start site in rat insulin II gene, and the transcription initiation site(s) has not been mapped in all of these tissues. These observations suggested that alternative transcription initiation sites may place the genes under different promoter control during development. To map the 5' end of the gene, primer extension was performed using a synthetic oligonucleotide primer complementary to the first 20 bases of the coding portion of the rat insulin I gene. The extended products of the proinsulin mRNAs from adult insulinoma cell line and fetal pancreas were identical and consistent in size with those predicted if transcription occurs at the putative start site. The 3' ends of the proinsulin mRNA transcripts were evaluated by ribonuclease H digestion, and it was shown that the noted size differences could be accounted for by different lengths of 3'-polyadenylation. Northern blot analysis of proinsulin mRNA from animals treated under conditions where mRNA varied from low to high levels failed to show any modulation of polyadenylation. The role of polyadenylation of proinsulin mRNA in the physiological regulation of insulin biosynthesis, if any, is currently unknown. PMID- 3004927 TI - Effect of o,p'-DDD on cortisol metabolism in Cushing's syndrome of various etiology. AB - Effects of o,p'-DDD on parameters of cortisol metabolism were studied in 3 patients with Cushing's syndrome (ectopic ACTH-syndrome, Cushing's disease, and adrenal cancer). Before o,p'-DDD treatment, plasma cortisol, urinary 17OHCS, and urinary free cortisol were elevated in all patients. These parameters correlated well with each other in ectopic ACTH-syndrome and Cushing's disease. However, in adrenal cancer, urinary 17OHCS did not correlate with either plasma cortisol or urinary free cortisol, while the latter two parameters did. During o,p'-DDD, urinary 17OHCS rapidly declined in a patient with ectopic ACTH syndrome and a patient with Cushing's disease before plasma cortisol or urinary free cortisol decreases. Consequently the positive correlations of urinary 17OHCS with the other parameters were lost. In a case of adrenal cancer, urinary 17OHCS again did not correlate with plasma cortisol or urinary free cortisol. In these conditions, plasma cortisol and urinary free cortisol still significantly correlated. The present results demonstrated the limit of urinary 17OHCS as the index of the cortisol secretion rate both in some cases of adrenal cancer and in patients taking o,p'-DDD. It is suggested that urinary free cortisol should be utilized as a more accurate index for the cortisol secretion rate in such circumstances. PMID- 3004928 TI - Brain corticotropin-releasing factor (CRF) and catecholamine responses in acutely stressed rats. AB - Ether-laparotomy stress produced a rapid increase in rat hypothalamic CRF concentration, followed by a rapid reduction and subsequent increase. Cold restraint stress significantly reduced hypothalamic CRF concentration at 15 min after stress onset. Serum ACTH and corticosterone levels were significantly elevated at 15 min after the onset of both stresses. The CRF responses in the medulla oblongata were not similar to the hypothalamic CRF responses. Norepinephrine concentration in the hypothalamus was reduced, whereas dopamine concentration in the hypothalamus and medulla oblongata was significantly increased. Epinephrine concentrations in these tissues did not show any significant change throughout the stress period. The observations lead to the following conclusions: hypothalamic CRF plays a major role in stimulating ACTH secretion under acute stress; the reduction in hypothalamic CRF is due to an excess release in the early phase of acute stress; hypothalamic CRF and medulla oblongata CRF are controlled by different mechanisms; norepinephrine in the hypothalamus may not be involved in stimulating hypothalamic CRF secretion in the early phase of acute stress; and catecholamines are regulated differently in the hypothalamus and medulla oblongata. PMID- 3004929 TI - Responses of plasma adrenocorticotropin and cortisol to intravenous injection of synthetic ovine corticotropin releasing factor in the morning and early evening in normal human subjects. AB - This study was designed to compare the responsiveness of adrenocorticotropin (ACTH) and cortisol secretion to corticotropin-releasing factor (CRF) in the morning and early evening in normal human subjects. Synthetic ovine CRF (1.0 micrograms/kg) or normal saline, was administered as an i.v. bolus injection to six normal males at 900 h and 1700 h. Blood samples were obtained before and 15, 30, 60, 90 and 120 min after CRF or saline injection. Significant increases in plasma ACTH and cortisol levels were observed in all subjects at the both time of testing after CRF injection. The net increments in the areas under the concentration curve (areas in the CRF experiment minus those in the saline control experiment) were not statistically different for both ACTH (mean +/- SEM: 41.0 +/- 10.6 pg/ml h in the morning: 51.1 +/- 8.9 pg/ml h in the evening) and cortisol (mean +/- SEM: 28.5 +/- 5.0 micrograms/dl h in the morning; 36.2 +/- 4.0 micrograms/dl h in the evening). Also no significant difference was observed in net increment, peak level and the ratio of peak level to the basal level of ACTH and cortisol after CRF injection. There were no appreciable changes in plasma concentrations of growth hormone, thyroid-stimulating hormone or prolactin, although slight but statistically significant rises in plasma levels of luteinizing hormone and follicle-stimulating hormone were observed. These results suggest that there is no significant difference in responsiveness of the pituitary-adrenal axis to CRF in the morning (900 h) and early evening (1700 h), and thus the time of day will not necessarily have to be considered when CRF is used between these times in a clinical test to evaluate pituitary ACTH reserve. PMID- 3004930 TI - Comparative anticonvulsant activity and neurotoxicity of felbamate and four prototype antiepileptic drugs in mice and rats. AB - Felbamate (2-phenyl-1,3-propanediol dicarbamate), phenytoin, phenobarbital, ethosuximide, and valproate were evaluated in mice and rats with a battery of well-standardized anticonvulsant test procedures. The results obtained indicate that felbamate exhibits a wider range of experimental anticonvulsant activity than either phenytoin or ethosuximide and a somewhat more restricted range than either phenobarbital or valproate. Felbamate is effective in nontoxic intraperitoneal doses in mice by the maximal electroshock seizure (MES), pentylenetetrazol (s.c. PTZ), and picrotoxin (s.c. Pic) tests but ineffective against bicuculline- and strychnine-induced seizures; it is effective after nontoxic oral doses in both mice and rats by the MES and s.c. PTZ tests. When compared on the basis of protective indices (PI = TD50/ED50) calculated from the intraperitoneal data in mice, the PIs for felbamate were from 1.05 to 2.37 times higher than those of the prototype antiepileptics. Overall, except for the s.c. PTZ test in mice and rats after oral administration, the PIs were equal to or higher than those of the prototype agents. The PIs for the s.c. PTZ test in mice and rats after oral administration were within the range of the prototype agents. These data indicate that felbamate is a relatively nontoxic agent with a unique profile of anticonvulsant action. PMID- 3004931 TI - Effects of training on enzyme activities involved in purine nucleotide metabolism in Thoroughbred horses. PMID- 3004933 TI - Expression of the human proenkephalin gene in mouse pituitary cells: accurate and efficient mRNA production and proteolytic processing. AB - A recombinant plasmid containing the human proenkephalin gene ligated to pBR322 was introduced into a mouse pituitary cell line (AtT-20D16v) that normally expresses pro-opiomelanocortin but not proenkephalin. The plasmid was introduced by co-transformation with the G418-selectable plasmid, pRSVneo. Stable transformants were isolated and analyzed for the presence of the human proenkephalin gene. AtT-20 transformants which had one or more copies of the human proenkephalin gene integrated stably into the mouse chromosomal DNA expressed a 1.45 kb mRNA identical in size to human proenkephalin mRNA. Primer extension analysis indicated that the human proenkephalin gene was accurately and efficiently transcribed from its own promoter. AtT-20 transformants that expressed the 1.45 kb human proenkephalin mRNA also expressed proenkephalin protein and cleaved the protein to form free Met-enkephalin. This is of particular interest because these cells do not cleave all of the available pairs of basic amino acids in the endogenous protein, pro-opiomelanocortin, the precursor to ACTH, beta-endorphin and melanocyte stimulating hormones. The release of both ACTH and Met-enkephalin from these cells is stimulated by corticotropin releasing factor, a natural secretagogue for ACTH, indicating that the two classes of peptide share a related secretory pathway. PMID- 3004934 TI - Molecular cloning of S-protein, a link between complement, coagulation and cell substrate adhesion. AB - cDNA clones coding for human S-protein have been isolated using monoclonal antibodies to screen a cDNA library in pEX. These clones are shown to be authentic S-protein clones on the basis of sequence, composition and immunological criteria. The complete open reading frame sequence for S-protein has been determined and shows it to be a single polypeptide chain of 459 amino acids preceded by a cleaved leader peptide of 19 residues. No evidence was found for polymorphism of S-protein suggesting that different molecular weight forms arise by proteolytic degradation. Of the first 44 amino-terminal residues 42 are identical with the so-called somatomedin B peptide suggesting that S-protein is the somatomedin B precursor. Striking homology is found in the rest of the sequence with the serum spreading factor, vitronectin, which has also been shown to contain somatomedin B sequences at its amino terminus. We conclude that S protein and vitronectin are identical and discuss the relevance of this finding to the coagulation and complement pathways. PMID- 3004932 TI - Pump currents generated by the purified Na+K+-ATPase from kidney on black lipid membranes. AB - The transport activity of purified Na+K+-ATPase was investigated by measuring the electrical pump current induced on black lipid membranes. Discs containing purified Na+K+-ATPase from pig kidney were attached to planar lipid bilayers in a sandwich-like structure. After the addition of only microM concentrations of an inactive photolabile ATP derivative [P3-1-(2-nitro)phenylethyladenosine 5' triphosphate, caged ATP] ATP was released after illumination with u.v.-light, which led to a transient current in the system. The transient photoresponse indicates that the discs and the underlying membrane are capacitatively coupled. Stationary pump currents were obtained after the addition of the H+, Na+ exchanging agent monensin together with valinomycin to the membrane system, which increased the permeability of the black lipid membrane for the pumped ions. In the absence of ADP and Pi the half saturation for the maximal photoeffect was obtained at 6.5 microM released ATP. The addition of ADP decreased the pump activity. Pump activity was obtained only in the presence of Mg2+ together with Na+ and Na+ and K+. No pump current was obtained in the presence of Mg2+ together with K+. The electrical response was blocked completely by the Na+K+-ATPase specific inhibitors vanadate and ouabain. No pump currents were observed with a chemically modified protein, which was labelled on the ATP binding site with fluoresceine isothiocyanate. The method described offers the possibility of investigating by direct electrical measurements ion transport of Na+K+-ATPase with a large variety of different parameters. PMID- 3004935 TI - Sequence structures of a mouse major urinary protein gene and pseudogene compared. AB - Laboratory mouse strains carry approximately 35 major urinary protein (MUP) genes per haploid genome, tightly clustered together on chromosome 4. Most belong to two main groups (Groups 1 and 2). The available evidence strongly suggests that the Group 1 genes are active while the Group 2 genes are pseudogenes. Here we present the complete sequence of a Group 1 gene and a Group 2 gene and 700 bp of flanking sequence. The sequence of the Group 1 gene is consistent with its being active. The Group 2 gene contains two stop codons and a frame-shift mutation in the reading frame defined by the Group 1 gene, and would code for a signal peptide 25 rather than 19 amino acids long. The Group 2 gene differs from the Group 1 gene in other ways: a deletion upstream of the TATA box and another in intron 3, a base change in the TATA box itself, a 2 bp duplication at the splice acceptor boundary of intron 6, an altered poly(A) addition signal and a 1-base deletion 5' to the initiation codon. Some of these differences may explain the 10 to 20-fold higher level of Group 1 mRNA in mouse liver, and the fact that Group 1 and Group 2 transcripts are mainly spliced differently. The presence of the stop codon means that the Group 2 gene is a pseudogene in the context of the Group 1 gene. However, there is some evidence that the mature hexapeptide that it would code for may have biological activity. The 12 acceptor splice sites of the two genes all contain the identical sequence ACAG at the exon boundary.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004936 TI - Analysis of mouse major urinary protein genes: variation between the exonic sequences of group 1 genes and a comparison with an active gene out with group 1 both suggest that gene conversion has occurred between MUP genes. AB - Here we compare the exonic sequences of four Group 1 mouse major urinary protein (MUP) genes and four Group 1 cDNA sequences. These define seven different nucleotide sequences which differ from each other by 0.35% of bases on average, and which would code for seven different MUP proteins that could probably be resolved physically into at least five classes. The sequences differ at 13 nucleotide positions and at six codons, and although they are closely related their descent cannot be described by a simple series of duplications. We also describe the sequence of another liver cDNA (pMUP15) which has diverged from the Group 1 consensus sequence in 14.6% of bases. The divergence is much greater over exons 1-3 than over exons 4-6, suggesting that an ancestral gene conversion event has occurred. pMUP15 also differs from the Group 1 genes in having a longer signal peptide sequence and a different splice configuration between exons 6 and 7. Unlike the Group 1 sequences, pMUP15 contains a potential N-linked glycosylation site. Other published work has shown that a shorter cDNA clone which is identical over their common sequence to pMUP15 codes for MUP proteins that are unusually large in size and acidic in pI. We show here that mouse urine does indeed contain a glycosylated MUP protein with those properties, presumably the product of the gene that corresponds to pMUP15. PMID- 3004937 TI - Phosphatidylinositol turnover and transformation of cells by Abelson murine leukaemia virus. AB - The transforming protein of the Abelson murine leukaemia virus encodes a protein tyrosine kinase. Previously, we have shown that in Abelson-transformed cells, the Abelson kinase regulates the phosphoserine content of ribosomal protein S6. Phorbol 12-myristate 13-acetate (TPA), which activates protein kinase C, induces the phosphorylation of S6 at the same five phosphopeptides as found in S6 isolated from Abelson-transformed cells. We have investigated three models whereby the Abelson kinase might regulate S6 phosphorylation via the activation of protein kinase C. First, the Abelson kinase could phosphorylate protein kinase C on tyrosine. However, we do not detect significant amounts of phosphotyrosine in protein kinase C in vivo. Second, it has been suggested that protein-tyrosine kinases might phosphorylate phosphatidylinositol. This could increase the intracellular levels of diacylglycerol and thereby activate protein kinase C. Our data strongly suggest that direct phosphorylation of phosphatidylinositol by the Abelson protein-tyrosine kinase has no physiological role. Third, an indirect activation of protein kinase C may occur via an increase in the rate of phosphoinositide breakdown. We have found that phosphoinositide breakdown appears to be constitutively activated in Abelson-transformed cells. The implications of these observations are discussed with regard to S6 phosphorylation and the mechanism of Abelson-induced transformation. PMID- 3004938 TI - Activation of c-erbB in avian leukosis virus-induced erythroblastosis leads to the expression of a truncated EGF receptor kinase. AB - Chicken erythroblastosis caused by avian leukosis virus (ALV) is thought to be mediated by activation of the c-erbB/EGF receptor oncogene by a promoter insertion mechanism. Here we study the proteins expressed by two ALV-induced leukemias and compare them with the avian EGF receptor and with the oncogene product of avian erythroblastosis virus (v-erbB) which was shown to be a truncated EGF receptor. It appears that the two leukemias express truncated EGF receptors of slightly different sizes with intrinsic tyrosine kinase activity. Hence, acute and chronic retroviruses utilize a common pathway for transformation. Moreover, the proteins expressed in the leukemias are similar to the avian EGF receptor with respect to their phosphopeptide maps, suggesting that they do not carry the C-terminal deletion characteristic of v-erbB. PMID- 3004939 TI - Chromatin structure of the murine c-myc locus: implications for the regulation of normal and chromosomally translocated genes. AB - To assess possible alterations of c-myc transcriptional control in murine B-cell tumors, we have investigated the pattern of DNaseI hypersensitive sites in the gene's putative regulatory region and within the gene in a variety of genomic contexts. A number of such sites were found in several cell types, but none of these was detectable in a gene which was shown to be transcriptionally silent by the criterion of elongation of nascent transcripts in isolated nuclei. These results differ from those of a previous study, in which a DNaseI-hypersensitive site approximately 2 kb upstream of the gene was proposed to be associated with negative regulation of c-myc transcription in human cells. An analysis of DNA sequences presented here reveals that this region is highly homologous between mouse and human, suggesting that these upstream hypersensitive sites do not reflect species-specific regulatory elements. We also present data indicating that this hypersensitive site distinguishes the c-myc alleles in translocation positive plasma cell tumors which lack c-myc rearrangement. Furthermore, we report the existence of hypersensitive sites within the gene. One of these appears to be associated with cryptic promoters that are employed only when the normal promoters are lost as a consequence of chromosome translocation. These results are discussed in the context of c-myc translocation and gene breakage and with respect to possible stage-specific regulation of the gene's transcriptional competence. PMID- 3004941 TI - The 5' region of the p53 gene: evolutionary conservation and evidence for a negative regulatory element. AB - The 5' regions of the mouse, rat and human functional p53 genes were isolated and analysed. All three genes possess a non-coding exon, comprising exclusively 5' untranslated sequences. This exon contains extensive diad symmetry near the 5' end of p53 mRNA, possibly allowing for the formation of a stable hairpin structure in this mRNA. The nucleotide sequence within this hairpin element is highly conserved among the species. A DNA stretch of 225 bp preceding the p53 mRNA cap site possesses distinct promoter activity when assayed in the CAT system. However, this activity is practically abolished when further upstream p53 sequences (approximately 120 bp) are included in front of the CAT gene. This suggests that the control of p53 gene expression is complex and involves a negative regulatory element. PMID- 3004940 TI - Duplicated gene pairs and alleles of class I genes in the Qa2 region of the murine major histocompatibility complex: a comparison. AB - DNA restriction maps of the major histocompatibility complex and hybridization with low copy probes have previously revealed strong homology between the Q6-Q7 and the Q8-Q9 class I gene pairs in the Qa2 region of the C57BL/10 mouse. After DNA sequence analysis of the Q7, Q8 and Q9 genes, we have compared the Q7 gene with its apparent allele, 27.1, from the BALB/c mouse; the 99% homology between Q7 and 27.1 indicates that this is a non-polymorphic gene. Comparison of Q7 with Q9, its homologue in the Q8-Q9 gene pair, revealed greater than 99% homology, thus supporting our proposal that the Qa2 region has evolved by the duplication of gene pairs. Q7 was also found to be homologous (93%) to Q8, the second member of the Q8-Q9 pair. However, the first exon (encoding the leader sequence) as well as the first intron of Q7 and Q8, which are presumably not subject to strong selective pressure, are essentially identical in nucleotide sequence (having only one mismatch), which suggests that greater than 200 bp of DNA may have been exchanged by gene conversion. Furthermore, transcripts of both Q7 and Q8 would have termination codons derived from the exon that normally encodes the transmembrane domain, thus these genes could encode either membrane-bound class I proteins that lack a cytoplasmic protein domain or class I proteins that are secreted. PMID- 3004942 TI - Selection of mouse neuroblastoma cell-specific polyoma virus mutants with stage differentiative advantages of replication. AB - Two mouse neuroblastoma cell lines were analyzed for their permissivity for polyoma virus growth. One (N18) is fully permissive for polyoma replication, the other (41A3) shows limited permissivity and the viral genome persists, without noticeable cell death. Virus persistence does not seem to alter the cells' ability to differentiate in vitro and leads to selection of viral mutants altered in the untranscribed regulatory region of the genome. The mutant types obtained appear to be related to the degree of host cell differentiation. Nucleotide sequence analysis of the restriction fragment covering the regulatory region shows that duplications are present in all mutants, while deletions in the non duplicated segment are only present in mutants selected from less differentiated cells. These alterations involve both domains of the regulatory region that are considered to be essential for DNA replication and for enhancer activity. Mixed infections with polyoma wild type show that the selected mutants have cis advantage in replication in neuroblastoma cells and not in 3T6 cells. Mutants carrying the deletion in the non-duplicated segment of the enhancer show a selective advantage in replication over the undeleted one in mixed infection. This advantage is much stronger in neuroblastoma cells in suspension (less differentiated stage) than in monolayer cells (more-differentiated stage). An interpretation of the overall structure of the regulatory enhancer region, based on the observed differences between the mutants selected at different stages of differentiation in neuroblastoma and previously described mutants selected in undifferentiated cells, is discussed. PMID- 3004943 TI - Cellular proteins expressed in herpes simplex virus transformed cells also accumulate on herpes simplex virus infection. AB - The cell proteins expressed in rat embryo cells transformed by herpes simplex virus (HSV) have been analysed by immunoprecipitation assays to determine those polypeptides which can be identified by immunoprecipitation with the sera of tumour-bearing animals and also with antisera to herpes simplex infected cells. Cell polypeptides commonly recognised by both these sera have been further characterised using a monoclonal antibody directed against a cellular polypeptide which accumulates on HSV-2 lytic infection. This monoclonal antibody recognises in HSV-transformed cells polypeptides of mol. wts. 90 000, 40 000 and 32 000. Further studies show that the accumulation of these polypeptides in HSV transformed cells is not HSV specific but is a common feature of transformation or of cells which have been immortalised. We suggest that cellular polypeptides accumulating as a result of HSV infection may be of importance in the initiation of transformation by HSV, i.e., at the level of immortalisation of cells. PMID- 3004944 TI - Recombinant vaccinia virus induces neutralising antibodies in rabbits against Epstein-Barr virus membrane antigen gp340. AB - The Epstein-Barr virus membrane antigen gene gp340 was isolated, inserted into several strains of vaccinia virus and expressed under the control of a vaccinia virus promoter. The EBV-derived protein which was produced by the recombinant vaccinia viruses was heavily glycosylated, readily labelled with threonine, could be detected at the surface of infected cells and had a mol. wt. of approximately 340 kd, all of which are properties of the authentic gp340. Polyclonal rabbit antisera against gp340 and an EBV-neutralising anti-gp340 monoclonal antibody both recognised cells infected with the recombinant vaccinia viruses. Moreover, rabbits vaccinated with one of the recombinants produced antibodies that recognised EBV-containing lymphoblastoid cells and neutralised EBV. PMID- 3004945 TI - The abnormal location of cytoplasmic SV40 large T is not caused by failure to bind to DNA or to p53. AB - We have examined the large T encoded by an SV40 mutant, d10, which fails to localize to the nucleus. The DNA sequence of the mutant predicts the alteration of Lys 128----Thr within the sequence 127 Lys Lys Lys Arg Lys 131 of large T. The results show that d10 large T is capable of binding to SV40 DNA, to cellular DNA and to the cellular phosphoprotein p53 as well as wild-type large T. These data suggest that the cytoplasmic location of d10 large T is not due to an inability of the protein to be retained within the nucleus, but argues instead that the protein fails to reach the nucleus because it contains a defective nuclear location signal. PMID- 3004946 TI - Inhibition of DNA synthesis by aphidicolin induces supercoiling in simian virus 40 replicative intermediates. AB - Highly torsionally stressed replicative intermediate SV40 DNA molecules are produced when ongoing replicative DNA synthesis is inhibited by aphidicolin, a specific inhibitor of DNA polymerase alpha. The high negative superhelical density of these molecules can be partially released by intercalating drugs such as chloroquine or ethidium bromide. The torsionally stressed replicative intermediates bind to monoclonal anti-Z-DNA antibodies. Electron microscopy of anti-Z-DNA cross-linked to torsionally stressed replicative intermediates shows that the antibody specifically binds close to the replication forks. The superhelical structures are not formed when SV40 DNA replication is inhibited by both aphidicolin and novobiocin, suggesting that a topoisomerase type II-like enzyme is somehow involved in the introduction of torsional strain in replicative intermediate DNA. One interpretation of our data is that fork movement continues to some rather limited extent when SV40 DNA synthesis in replicative chromatin is blocked by aphidicolin. After deproteinization, the exposed single-stranded DNA branches reassociate to form paranemic DNA structures with left-handed helical stretches, while the reduced linking number of the parental strands induces a high negative superhelical density. PMID- 3004947 TI - The SV40 enhancer influences viral late transcription in vitro and in vivo but not on replicating templates. AB - We have examined transcription from the SV40 late promoter in vitro and in vivo. In HeLa whole cell extracts, late transcription is efficient in the absence of T antigen but is impaired by enhancer specific point mutations. In vivo, when replication is prevented, transcription from the late promoter requires T antigen as well as a functional enhancer. However, enhancer sequences fail to potentiate late transcription from replicating templates although, under such conditions, enhancer binding factors do not become limiting. It appears that the SV40 late transcription unit is refractory to enhancer-mediated activation when it is located on a replicating template. PMID- 3004949 TI - Role of the 5' hairpin structure in the splicing accuracy of the fourth intron of the yeast cob-box gene. AB - The splicing mechanism of the maturase-coding introns is poorly understood. We have systematically examined the phenotypes of a large number of revertants from the mitochondrial mutation G2457. This mutation results from a single base change near the 5' splicing site. We show here that this base change does not completely block the splicing of the intron but rather affects the specificity of the splicing process. We examine four classes of revertants which allow us to characterize the crucial role of a stem and loop structure in the accuracy of the intron excision process. An unexpected class of revertant suggests that other elements are involved in this mechanism. Reversion of G2457 can also occur via the excision in the mitochondrial genome of the intron coding sequence. These results are discussed in relation to the possible role fulfilled by the maturase in the control of intron splicing. PMID- 3004948 TI - Structure, expression and regulation of a nuclear gene encoding a mitochondrial protein: the yeast L(+)-lactate cytochrome c oxidoreductase (cytochrome b2). AB - The yeast L(+)-lactate cytochrome c oxidoreductase or cytochrome b2 is a component of the mitochondrial intermembrane space. The protein is encoded by the nuclear genome, synthesized as a larger precursor in the cytoplasmic compartment, and then proteolytically processed to its mature form during its import into the mitochondria. The structural gene for yeast cytochrome b2 has been cloned. The complete nucleotide sequence of the gene with its 5' and 3' flanking regions was determined. The deduced primary structure of the cytochrome b2 precursor reveals an unusually long amino terminal extension of 80 amino acids. A variety of potentially significant sequences were identified in the region flanking the structural portion of the gene. Transcript mapping with both S1 nuclease and primer extension methods reveals that the site of RNA synthesis is 56-66 bp downstream from a putative TATA box. By Northern blot analysis and gene disruption, it is shown that there is only a single copy of the cytochrome b2 gene per haploid yeast nucleus. The cloned cytochrome b2 gene was used to probe specific mRNA levels and demonstrate that cytochrome b2 expression is transcriptionally repressed by glucose and induced by lactate. The inactivation of the chromosomal cytochrome b2 gene by integrative transformation led to a deficiency in L(+)-lactate dehydrogenase activity and consequently to the inability to use L(+)-lactate as a sole source of carbon. This is the first reported mutation affecting the structural gene of cytochrome b2. PMID- 3004950 TI - Transcription of a transposed trypanosome surface antigen gene starts upstream of the transposed segment. AB - The non-telomeric variant surface glycoprotein (VSG) genes in Trypanosoma brucei are activated by a duplicative transposition to a telomeric expression site. We have determined the 5' end of the transposed segment of the gene for VSG 117 and infer from comparison with similar data obtained by others that the crossover can occur at variable positions within short repeats present upstream of this gene and in the expression site. We have analysed nascent and steady state transcripts of the transposed gene and its neighbouring expression site DNA. The results indicate that transcription starts upstream of the transposed gene segment in the expression site and that transcripts are rapidly processed at specific points identified by protection of DNA-RNA hybrids against digestion by nuclease S1 or Exo VII. Hence, this gene appears to be activated by a process akin to promoter addition. PMID- 3004951 TI - Mutations that alter the allosteric nature of cAMP receptor protein of Escherichia coli. AB - Mutations which permit cAMP binding protein (CRP) to act in the absence of cAMP have been isolated by in vitro mutagenesis of a plasmid containing the cloned crp gene. Adenylate cyclase deficient cells harbouring the mutant (crp*) plasmids exhibited a variety of fermentation profiles on MacConkey indicator plates containing various sugars. beta-galactosidase synthesis in cells carrying the crp* plasmids was activated most by the addition of cGMP as well as cAMP. The sites of mutations which are responsible for the cAMP independent phenotype were determined by in vitro recombination and DNA sequencing. The amino acid substitutions in the mutant proteins were found in two specific regions of the crp gene encoding residues 53-62 and 141-148 of CRP polypeptide. The first region may participate in cAMP binding, while the second appears to be the inter-domain region of the N-terminal cAMP-binding and C-terminal DNA-binding domains. PMID- 3004952 TI - Two operator sites separated by 599 base pairs are required for deoR repression of the deo operon of Escherichia coli. AB - DeoP1 and deoP2 promoter fragments from the deo operon of Escherichia coli have been transcriptionally fused to the galactokinase gene. From single-copy expression of these fusions it is shown that the deoR binding site of both deoP1 and deoP2 are necessary to achieve full repression of the deo operon by the deoR repressor. Repression of the promoters can be achieved either by supplying extra deoR repressor in trans or by introduction of an extra deoR binding site at a position between 224 and 997 bp upstream of the promoter. Furthermore, the deoP2 promoter is shown to be regulated in a cumulative way by both the deoR and the cytR repressors, while deoP1 is only regulated by the deoR repressor. DeoP2 is a strong promoter being 20 times stronger than araPBAD and four times stronger than deoP1. PMID- 3004953 TI - Priming immunization against cholera toxin and E. coli heat-labile toxin by a cholera toxin short peptide-beta-galactosidase hybrid synthesized in E. coli. AB - A synthetic oligodeoxynucleotide encoding for a small peptide was employed for the expression of this peptide in a form suitable for immunization. The encoded peptide, namely, the region 50-64 of the B subunit of cholera toxin (CTP3), had previously been identified as a relevant epitope of cholera toxin. Thus, multiple immunizations with its conjugate to a protein carrier led to an efficient neutralizing response against native cholera toxin. Immunization with the resulting fusion protein of CTP3 and beta-galactosidase, followed by a booster injection of a sub-immunizing amount (1 microgram) of cholera toxin, led to a substantial level of neutralizing antibodies against both cholera toxin and the heat-labile toxin of Escherichia coli. PMID- 3004954 TI - Conservation of genes and their organization in the chromosomal replication origin region of Bacillus subtilis and Escherichia coli. AB - The organization of six open reading frames which were deduced from the nucleotide sequence of some 10 kb from the replication origin region of Bacillus subtilis resembles the organization of the genes in the rnpA-dnaA-gyrB region of the Escherichia coli chromosome. Based on the detection of homology with the E. coli genes the open reading frames were found to represent the Bacillus 'rnpA', 'rpmH', 'dnaA', 'dnaN', recF and gyrB genes. Only the latter two have also been defined by genetic analysis. Two regulatory regions containing nine and four copies of a repeating sequence, DnaA-box, which is identical with the DnaA protein-binding sequence repeated four times in the E. coli origin of replication, flank the 'dnaA' gene of B. subtilis. One or both of them are proposed to function as origins in the initiation of chromosomal replication. Transcription of the 'dnaA' gene of Bacillus starts in one of these regions and appears to be coupled to initiation of chromosomal replication. We propose that the conserved gene organization in the 'dnaA'-'gyrB' region of B. subtilis is representative of the replication origin region of a primordial replicon. The oriC sequence of E. coli has either been translocated to its present location 44 kb away from the primordial origin or has independently evolved there. PMID- 3004955 TI - The SecY membrane component of the bacterial protein export machinery: analysis by new electrophoretic methods for integral membrane proteins. AB - The product of the secY (prlA) gene (the SecY protein) involved in protein export in Escherichia coli was overproduced and localized in the cytoplasmic (inner) membrane. Because of its strong interaction with a non-ionic detergent (NP40), it partitioned into the detergent layer during electroblotting through a NP40 containing gel (detergent blotting), and it formed a horizontal streak in the O'Farrell two-dimensional gel electrophoretic system. Consequently, we developed an alternative two-dimensional gel procedure, which proved useful for analysis of integral membrane proteins, especially in combination with detergent blotting. SDS-gel electrophoresis was carried out successively through gels of lower (first dimension) and higher (second dimension) sieving effects. Many membrane proteins, unlike soluble proteins, formed spots off and above the diagonal line, and all of these spots partitioned exclusively into the detergent layer. A characteristic pattern of integral membrane proteins of E. coli was thus obtained and the spot of the SecY protein in the cytoplasmic membrane was identified even when it was not overproduced. These results show that the gene secY specifies an integral membrane component of the protein export machinery. PMID- 3004956 TI - Transposon Tn554: complete nucleotide sequence and isolation of transposition defective and antibiotic-sensitive mutants. AB - The complete nucleotide sequence of the Staphylococcus aureus transposon Tn554, which encodes resistance to erythromycin and spectinomycin, was determined by the dideoxy chain termination method. The transposon was found to be 6691 bp in length and to contain six open reading frames of greater than 125 amino acids. Small insertion and deletion mutations were obtained in each of these by in vitro mutagenesis at restriction endonuclease cleavage sites and the mutants characterized with respect to transposition functions and antibiotic resistance markers. Three of the reading frames, designated tnpA, tnpB and tnpC, encode functions that are required for transposition of Tn554; genetic analysis indicated that these three genes define distinct complementation groups of transposition-defective mutants. Two of the open reading frames correspond to the resistance determinants spc and ermA, the sixth, designated ORF, has no known function. Tn554-specific peptides corresponding to tnpA, and spc were identified in a coupled transcription-translation system in vitro. PMID- 3004957 TI - The B chain of PDGF alone is sufficient for mitogenesis. AB - The platelet-derived growth factor (PDGF) is a mitogen derived from human platelets consisting of two related polypeptides termed A and B chains. The entire B chain of PDGF is highly (96%) homologous to a portion of p28sis, the transforming protein of simian sarcoma virus. It has been suggested that p28sis exerts its transforming potential by mimicking the growth promoting activity of PDGF and stimulating the cell in an autocrine manner. We have directly examined the mitogenic potential of p28sis and the B chain homologous region by expressing these heterologous sequences in the yeast Saccharomyces cerevisiae. In our constructions, these proteins are encoded by portions of the v-sis gene. Expression and secretion from the yeast cell is achieved by using a yeast promoter and the alpha-factor pheromone secretory leader. The sis proteins thus expressed and secreted are immunoreactive with anti-PDGF antisera and are mitogenic for cultured fibroblasts. Furthermore, they mediate this mitogenic activity by specific binding to the PDGF cell surface receptor. Gel electrophoresis and cell binding analysis indicates that the mitogenic species is primarily a disulphide-bonded dimer. We are able to conclude that p28sis is a mitogen and that a polypeptide corresponding to the B chain alone is sufficient to account for the mitogenic activity attributed to PDGF. PMID- 3004958 TI - Two types of receptor for insulin-like growth factors in mammalian brain. AB - Two types of receptor for insulin-like growth factors (IGFs) have been identified on adult rat and human brain plasma membranes by competitive binding assay, affinity labelling, receptor phosphorylation and interaction with antibodies to insulin receptors. The type I IGF receptor consists of two species of subunits: alpha-subunits (mol. wt. approximately 115 000), which bind IGF I and IGF II with almost equal affinity and beta-subunits (mol. wt. approximately 94 000), the phosphorylation of which is stimulated by IGFs. The alpha-subunits of type I IGF receptors in brain and other tissues differ significantly (mol. wt. approximately 115 000 versus 130 000), whereas the beta-subunits are identical (mol. wt. approximately 94 000). The type II IGF receptor in brain is a monomer (mol. wt. approximately 250 000) like that in other tissues. Two antibodies to insulin receptors, B2 and B9, interact with type I but not with type II IGF receptors. B2 is more potent than B9 in inhibiting IGF binding and in immunoprecipitating type I IGF receptors, in contrast to their almost equal effects on insulin receptors. This pattern is characteristic for IGF receptors in other cells. The presence of two types of IGF receptor in mammalian brain suggests a physiological role of IGFs in regulation of nerve cell function and growth. Since IGF II, but not IGF I, is present in human brain, we propose that IGF II interacts with both types of IGF receptor to induce its biological actions. PMID- 3004959 TI - Permanent expression of p53 in FR 3T3 rat cells but cell cycle-dependent association with large-T antigen in simian virus 40 transformants. AB - The p53 oncogene is thought to play a role in the proliferation of normal and transformed cells and its expression was postulated to be cell cycle dependent. Using flow cytofluorimetry sorting of populations of exponentially growing cells, coupled to a radioimmune assay, we have investigated the accumulation of p53 along the cell cycle in normal FR 3T3 rat cells as well as in two types of SV40 transformed derivatives, one of which only expresses the large-T protein during the G2 phase of the cell cycle. p53 was accumulated at a constant level throughout the cell cycle in FR 3T3 cells. Its level and stability increased to different extents in the two types of transformants. However, the formation of complexes between p53 and large-T was modulated by the G2-restricted accumulation of large-T, thus leading to a differential increase in the levels of p53 both in exponentially growing cells and along the cell cycle. This increase in the levels of p53 appeared to be regulated at a post-transcriptional level. PMID- 3004960 TI - Retention of herpes simplex virus type II sequences in bglII n-transformed cells after co-transfection with a selectable marker. AB - Transformation of mammalian cells by total u.v.-inactivated herpes simplex virus II (HSVII) or cloned fragments thereof (BglII n, BglII C) has been complicated both by a low efficiency of oncogenic transformation and the disappearance of viral DNA and/or viral products initially detected in the transformed cell lines. In an attempt to effect a stable integration of BglII n and to elucidate the role of HSVII in oncogenic transformation, we have co-transfected NIH 3T3 cells with pAG60, a plasmid which confers resistance to the G418 antibiotic, and plasmids containing either BglII n in its entirety (pNB2) or one of five subfragments of BglII n. Several isolated clones exhibit a transformed phenotype as expressed by rapid growth in low serum concentrations and colony formation in soft agar. We have obtained a markedly reduced frequency of biochemical transformants when co transfecting pNB2 in comparison with the numbers obtained when cotransfecting the five subfragments. Furthermore, a greater proportion of subfragment-transfected colonies contain viral DNA, and in higher copy number, than observed in the pAG60/pNB2 clones. We have also found viral DNA to be more stably integrated in the subfragment-transfected clones than in the pNB2-transfected clones. PMID- 3004961 TI - Hormonal regulation of cell surface expression of the major histocompatibility antigen H-2Ld in transfected cells. AB - The murine major histocompatibility antigens are cell surface glycoproteins which play an important role in the recognition of foreign antigens by cytotoxic T lymphocytes. Modulation of the level of expression of histocompatibility antigens could therefore be useful for the study of the interaction between the antigen presenting cells and T lymphocytes. The glucocorticoid hormone-inducible promoter, located in the long terminal repeat of mouse mammary tumor virus, was used to replace the promoter region of a cloned H-2Ld class I gene. The chimeric gene was introduced into cultured cells. Glucocorticoid induction of MMTV LTR H 2Ld mRNA could be shown by blot analysis. An S1 nuclease protection assay indicated that the transfected cells accurately initiate the chimeric mRNA. Immunoprecipitation of H-2Ld protein with a specific monoclonal antibody showed inducibility also at the cellular protein level. Fluorescence-activated cell sorter analysis monitored a 3-fold increase of H-2Ld on the cell surface when the transfected cells were grown in the presence of dexamethasone. This increase of H 2Ld expression was accompanied by a corresponding decrease on the cell surface of the endogenous H-2Kk. PMID- 3004962 TI - Separable cis-acting control elements for expression of the white gene of Drosophila. AB - The white gene of Drosophila is required for the pigmentation of the eyes, ocelli, Malpighian tubules and testis sheath. We have examined the regulation of white expression in germ line transformants that carry the mRNA-coding sequences of white flanked by varying amounts of the sequences normally located adjacent to them. A segment with as little as 1.9 kb of 5'- and 2.1 kb of 3'-flanking DNA was expressed to give essentially wild-type pigmentation in all of these tissues and interacted in trans with the zeste-1 allele. In contrast, transformants having segments with 1.1 kb of 5'-flanking DNA had reduced levels of pigment in their eyes, no detectable pigment in their testes and did not interact with zeste-1. The eyes, Malpighian tubules and ocelli of transformants with as little as 0.4 kb of 5'-flanking DNA were pigmented and the eye pigmentation exhibited dosage compensation. We conclude that there are sequence elements present in the region from 1.1 to 1.9 kb upstream from the 5' end of the white transcript which are required for expression in the testes and interaction of white and zeste-1. The same or other elements in this region appear to augment the pigmentation of the eye. PMID- 3004963 TI - Multiple upstream regulatory elements control the expression of the Drosophila white gene. AB - Constructions containing the Drosophila white gene and different amounts and arrangements of its regulatory region were introduced into the germ line of white mutant flies by P-mediated transformation. The results obtained with the different transposon constructions show that different parts of the 1.8-kb region preceding the transcription start are required for the expression of the gene in different tissues and at different developmental stages. Different sequences independently control the expression of the gene in the adult testes, in the larval and adult Malpighian tubules and in the eye. Another sequence located greater than 1080 bp upstream of the transcription start is the target of zeste interaction. The results also suggest that sequences required for dosage compensation are contained between -216 and the transcription start site. We show that at least some of these regulatory elements are equally functional if their distance from the promoter is varied or if their orientation is inverted. Their properties suggest that they act as enhancer-like elements to regulate the activity of the white promoter and, at least in the case of the zeste regulatory site, that they can act also in 'trans' on a white promoter locked in close physical proximity by homologous chromosome pairing. PMID- 3004964 TI - The first twelve amino acids of a yeast mitochondrial outer membrane protein can direct a nuclear-coded cytochrome oxidase subunit to the mitochondrial inner membrane. AB - We have used an in vivo complementation assay to test whether a given polypeptide sequence can direct an attached protein to the mitochondrial inner membrane. The host is a previously described yeast deletion mutant that lacks cytochrome oxidase subunit IV (an imported protein) and, thus neither assembles cytochrome oxidase in its mitochondrial inner membrane nor grows on the non-fermentable carbon source, glycerol. Growth on glycerol and cytochrome oxidase assembly are restored to the mutant if it is transformed with the gene encoding authentic subunit IV precursor, a protein carrying a 25-residue transient pre-sequence. No restoration is seen with a plasmid encoding a subunit IV precursor whose pre sequence has been shortened to seven residues. Partial, but significant restoration is achieved by an artificial subunit IV precursor in which the authentic pre-sequence has been replaced by the first 12 amino acids of a 70-kd protein of the mitochondrial outer membrane. If this dodecapeptide is fused to the amino terminus of mouse dihydrofolate reductase (a cytosolic protein), the resulting fusion protein is imported into the matrix of yeast mitochondria in vitro and in vivo. Import in vitro requires an energized inner membrane. We conclude that the extreme amino terminus of the 70-kd outer membrane protein can direct an attached protein across the mitochondrial inner membrane. PMID- 3004965 TI - Mutational analysis of the maize chloroplast ATPase-beta subunit gene promoter: the isolation of promoter mutants in E. coli and their characterization in a chloroplast in vitro transcription system. AB - This paper describes the use of Escherichia coli to isolate Bal31 deletion mutants and single-base substitution mutants that functionally define the promoter of the maize chloroplast beta-ATPase gene (atpB). Promoter function in E. coli and in a chloroplast in vitro transcription system was determined by S1 nuclease protection experiments using RNA products from each mutant. The results show that in vitro the chloroplast RNA polymerase responds to the promoter point mutations in a quantitatively similar fashion to the E. coli RNA polymerase. Deletion analysis demonstrates that sequences 5' of the -35 region are not necessary for chloroplast promoter function in vitro and that the presence of an adjacent promoter drastically decreases the transcriptional activity of the atpB promoter in E. coli. PMID- 3004966 TI - Mapping of the mouse X chromosome using random genomic probes and an interspecific mouse cross. AB - Two libraries enriched in murine X chromosome material have been constructed in the lambda vector NM 1149 from flow-sorted chromosomes. Inserts of unique genomic sequence DNA were purified and their X chromosome specificity characterised by hybridisation to a panel of somatic cell hybrid lines. Of the first five such X chromosome-specific probes characterised, all detect restriction fragment length polymorphisms (RFLPs) between inbred mouse laboratory strains such as C57BL/6 and BALB/c and the SPE/Pas mouse strain established from a wild Mus spretus mouse, when their DNAs are digested with the restriction enzyme TaqI. Taking advantage of these RFLPs, all five probes have been localised on the X chromosome using an interspecific backcross between the B6CBARI and SPE/Pas mouse strains segregating the X chromosome markers hypoxanthine phosphoribosyl transferase (Hprt) and Tabby (Ta). Three of the probes map to the region between the centromere and Hprt, and two distal to Ta. Since such X-specific sequence probes detect RFLPs between M. spretus and M. musculus domesticus DNAs with high frequency, a large panel of well localised probes should soon be available for studies of biological problems associated with the X chromosome which can best be approached using the murine species. PMID- 3004967 TI - Regulation of c-fos transcription in mouse fibroblasts: identification of DNase I hypersensitive sites and regulatory upstream sequences. AB - In quiescent mouse fibroblasts, the c-fos gene is expressed at very low levels, but is rapidly and transiently inducible by peptide growth factors. In this study, we have identified in quiescent cells five DNase I-hypersensitive sites located -1700, -290, +10, +240 and +700 bp relative to the 5' cap site. After serum stimulation, the distinct nuclease hypersensitive site at position +10 rapidly disappeared, and instead a broad region of DNase I accessibility between positions 0 and +250 occurred. Nucleotide sequence analysis of the 5'-flanking region of the mouse c-fos gene showed that the hypersensitive site around position -290 is located in a region that is highly conserved between mouse and human, and that contains an enhancer-like structure. When the mouse c-fos promoter and 351 bp of 5'-flanking sequences were linked to the bacterial chloramphenicol acetyl transferase (CAT) gene and transfected into NIH3T3 cells efficient, constitutive expression of CAT activity was observed, even in unstimulated, quiescent cells. However, removal of a 256-bp stretch upstream from position -95 completely abolished CAT expression, indicating that sequences within a region of approximately 350 bp upstream from the cap site are indispensable for c-fos transcription. In addition, our findings point to the existence of other sequence elements that exert negative regulation in the absence of growth factor stimulation. Such sites may be found around the growth factor-responsive nuclease hypersensitive sites in the vicinity of the cap site. PMID- 3004968 TI - Aberrant c-myc RNAs of Burkitt's lymphoma cells have longer half-lives. AB - BL67 and BL18 are Burkitt's lymphoma cell lines with t(8;14) translocations (the breakpoint is in the first exon and first intron, respectively) in which the mu heavy chain switch region is fused to the c-myc gene in head to head orientation. In both cell lines only aberrant c-myc RNAs are found. BL67 cells contain two c myc RNA species of 2.4 and 3.5 kb. The 2.4-kb RNA is initiated at several cryptic promoters in the first intron. The 3.5-kb RNA is transcribed from the immunoglobulin heavy chain anti-sense strand across the breakpoint of the translocation into the first exon of the c-myc gene and is then normally spliced using the physiological splice donor and acceptor sites of the c-myc gene. BL18 contains c-myc RNA of 2.4 kb initiated at cryptic promoters in the first intron and additional RNAs of 0.90 kb and 0.74 kb transcribed from the dual c-myc promoters on the reciprocal fragment of the translocation. The cytoplasmic turnover of these RNAs differs significantly from that of the normal c-myc message. The 3.5-kb RNA of BL67 cells and the 0.90-kb and 0.74-kb RNAs of BL18 cells, which are both hybrid molecules consisting of c-myc and immunoglobulin sequences, have a half-life of several hours in contrast to the normal c-myc message with a half-life of 15 min. The aberrant 2.4-kb c-myc RNAs of BL67 and BL18 cells are also more stable than the normal c-myc message and disappear with a half-life of 50 min.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004969 TI - Functional domains of the Xenopus laevis 5S gene promoter. AB - To study the fine structure of the Xenopus laevis somatic 5S gene internal control region, we have created 15 different transversions using mutagenic oligonucleotide primers. The effects of these mutations on 5S DNA transcription in vitro as well as on stable complex formation with transcription factor TF III A and TF III C in crude nuclear extracts were analyzed. Mutations in the common class III 5' promoter element (nucleotides 50-61 in the 5S gene) interfere with transcription activity and stable complex formation whenever they contradict the tDNA box A consensus sequence. The second promoter element is defined by a major sequence block (nucleotides 80-89, box C) and two additional internal residues (70 and 71) at a distance of roughly one helical turn from both the major 3' and 5' control sequences; these two 3' elements contain the primary TF III A binding domain. The remaining nucleotides (62-69 and 71-79) when mutated do not interfere with transcription activity or factor binding and thus they constitute two spacer elements within a symmetrically structured 5S gene promoter. An increase in the relative spacing of box A and box C by insertion of 3 bp between nucleotides 66 and 67 leads to a drastic reduction in transcription activity and the ability to form a stable complex with TF III A and/or TF III C. Thus, accurate spacing is essential for the proper orientation of TF III A on 5S DNA and/or TF III C binding. PMID- 3004971 TI - Human T lymphocyte clones specific for malaria (Plasmodium falciparum) antigens. AB - Peripheral blood mononuclear cells (PBM) from a patient who had lived in a malarial-endemic area were cultured in the presence of malarial antigens (a lysate of Plasmodium-infected erythrocytes). Responding cells were grown in IL-2 containing medium and then cloned, and subsequently subcloned, in the presence of phytohemagglutinin and allogeneic irradiated PBM. Ten clones were specific for malarial antigens. They proliferated in response to P. falciparum extract, but not to a lysate of uninfected erythrocytes. The response was HLA-restricted. All the clones tested responded to lysates of cells infected with parasites of either African or Asian origin. Six clones had the T4+/T8- phenotype and four the T4 /T8+ phenotype. Two of the T4+ clones recognised a parasite antigen of apparent mol. wt. approximately 50 000. All of the clones tested produced gamma-interferon following antigen stimulation. PMID- 3004970 TI - Topogenesis of microbody enzymes: a sequence comparison of the genes for the glycosomal (microbody) and cytosolic phosphoglycerate kinases of Trypanosoma brucei. AB - To determine how microbody enzymes enter microbodies, we are studying the genes for cytosolic and glycosomal (microbody) isoenzymes in Trypanosoma brucei. We have found three genes (A, B and C) coding for phosphoglycerate kinase (PGK) in a tandem array in T. brucei. Gene B codes for the cytosolic and gene C for the glycosomal isoenzyme. Genes B and C are 95% homologous, and the predicted protein sequences share approximately 45% amino acid homology with other eukaryote PGKs. The microbody isoenzyme differs from the cytosolic form and other PGKs in two respects: a high positive charge and a carboxy-terminal extension of 20 amino acids. Our results show that few alterations are required to redirect a protein from cytosol to microbody. From a comparison of our results with the unpublished data for three other glycosomal glycolytic enzymes we infer that the high positive charge represents the major topogenic signal for uptake of proteins into glycosomes. PMID- 3004972 TI - Polymorphism of the precursor for the major surface antigens of Plasmodium falciparum merozoites: studies at the genetic level. AB - The gene for the precursor of Plasmodium falciparum merozoite surface antigens has been cloned. The entire sequence of the gene from a Thai isolate of the parasite is reported. It provides evidence for a signal peptide, a region containing short repeating peptides and an anchor sequence. In addition, the 5' end of a Papua New Guinea isolate has been sequenced. Comparison of these and other sequences defines, at the genetic level, a polymorphic region in the protein, and suggests that other parts of the protein are less susceptible to variation. Furthermore it appears that several signal peptides of P. falciparum exhibit extensive sequence homologies. PMID- 3004973 TI - SV40 enhancer activation during retinoic acid-induced differentiation of F9 embryonal carcinoma cells. AB - The transient expression vector pSV2CAT, which carries the bacterial chloramphenicol acetyl transferase (CAT) gene under the control of the SV40 early promoter, was used to transfect the murine embryonal carcinoma cell line F9 at various times during the retinoic acid-induced differentiation of these cells. Expression of the CAT gene under SV40 promoter control was found to increase markedly on F9 cell differentiation, measured relative to expression from the thymidine kinase promoter in the same cells. A series of constructs was prepared to identify the features of the SV40 early promoter required for transcription in differentiated and undifferentiated cells, as well as the factors limiting transcription in each case. The increased transcription seen on F9 cell differentiation was not observed when cells were transfected with molecules lacking a functional enhancer. It appears that as embryonal carcinoma cells differentiate, increased SV40 transcription results from enhancer sequence activation. In both differentiated and undifferentiated cell types the level of transcription was found to be limited by the availability and/or activity of cellular factors necessary for enhancer function. PMID- 3004974 TI - All six GC-motifs of the SV40 early upstream element contribute to promoter activity in vivo and in vitro. AB - Recombinants in which the six GC-motifs (I-VI) present in the upstream element of the SV40 early promoter region have been point mutated either individually or in pairs were used to determine the possible contribution of each GC-motif to the function of the overlapping early-early and late-early SV40 promoters. GC-motif I, and to a lesser extent, GC-motifs II and III, are critical for initiation at the early-early start sites. GC-motifs IV-VI play a subsidiary role. Mutations in GC-motifs I and II do not decrease the activity of the late-early promoter, whereas mutations in the GC-motifs III-VI have a moderate effect on it. The in vivo phenotype of the GC-motif mutants can be almost fully reproduced in vitro using a nuclear extract. DNase I protection footprinting experiments using wild type or mutated templates and nuclear extracts indicate that each GC-motif behaves principally as an independent protein-binding site, presumably for transcription factor Sp1. The effect of changing the position of the 21-bp repeat region on initiation from the early-early and late-early start sites indicates that there is little flexibility in the position in which this upstream element can efficiently activate initiation of transcription from these start sites. PMID- 3004975 TI - In vitro and in vivo synthesis of the hepatitis B virus surface antigen and of the receptor for polymerized human serum albumin from recombinant human adenoviruses. AB - We have developed an adenovirus vector to express foreign proteins under the control of the adenovirus E1a promoter. Two recombinant plasmids, harbouring either the S gene or the pre-S2 region and the S gene of hepatitis B virus under the control of the E1a promoter, were used to construct two recombinant adenoviruses. These two viruses direct the synthesis of hepatitis B virus surface antigen (HBsAg) particles during the time course of an infectious cycle. When the pre-S2 region is present in the constructed virus, the synthesis of particles carrying the receptor for polymerized human serum albumin (pHSA) is observed. Moreover, the inoculation of rabbits with this latter purified recombinant adenovirus elicits the production of antibodies that react with both HBsAg and pHSA receptor. PMID- 3004976 TI - Deletion of a yeast small nuclear RNA gene impairs growth. AB - We have cloned and sequenced the single copy gene SNR10 which encodes the yeast small nuclear RNA, snR10. This species does not show obvious primary sequence homology to any previously identified small nuclear RNA. As an inital step towards determining the function of snR10, we have introduced insertions and deletions into the chromosomal copy of the gene. Strains lacking an intact copy of SNR10 are viable but considerably imparied in growth, particularly at elevated osmotic strengths or low temperatures; at 25 degrees C the doubling time of snr10 strains is 47% greater than that of otherwise isogenic SNR10 strains. As judged by the incorporation of radioactive precursors, snr10- strains are impaired in net RNA synthesis at low temperatures. The identification of a leaky, conditional phenotype associated with the deletion of this small nuclear RNA gene was entirely unexpected since the defect in snR10 synthesis is complete and non conditional. PMID- 3004977 TI - Coupled transcription and processing of mouse ribosomal RNA in a cell-free system. AB - An in vitro processing system of mouse rRNA was achieved using an RNA polymerase I-specific transcription system, (S100) and recombinant plasmids consisting of mouse rRNA gene (rDNA) segments containing the transcription initiation and 5' terminal region of 18S (or 41S) rRNA. Pulse-chase experiments showed that a specific processing occurred with transcripts of the plasmid DNAs when the direction of transcription was the correct orientation relative to the 18S rRNA coding sequence, but not with transcripts of the DNA templates in which this coding sequence was in the opposite orientation. From the S1 nuclease protection analyses, we concluded that there are several steps of endonucleolytic cleavage including one 105 nucleotides upstream from the 5' end of 18S rRNA. Intermediates cleaved at this site were identified in in vivo processing of rRNA. This result indicates that endonucleolytic cleavage takes place 105 nucleotides upstream from the 5' terminus of 18S rRNA prior to the formation of mature 18S rRNA. Trimming or cleavage of the 105 nucleotides may be involved in the formation of the 5' terminus of mature 18S rRNA. PMID- 3004980 TI - Detection of anti-HTLV-III/LAV antibody by enzyme-linked immunosorbent assay in high-risk individuals in Switzerland 1974-1985. AB - Anti-HTLV-III antibody was tested in frozen sera from high-risk individuals, collected between 1974 and 1985 in Switzerland. All but one of the 262 sera of a control group were negative. Among drug addicts, none of the 78 samples collected from 1974 to 1979 was positive. Antibody was first detected in 1980 and thereafter with progressively increasing frequency. Three of 63 samples from 24 dialyzed patients who had received multiple transfusions showed borderline positive values. None of the seropositive subjects has developed AIDS or AIDS related complex. PMID- 3004978 TI - Nucleotide sequence and structural organization of an insertion sequence element (IS231) from Bacillus thuringiensis strain berliner 1715. AB - This paper describes the structural organization of a repetitive DNA sequence isolated from plasmids of Bacillus thuringiensis strain berliner 1715. DNA sequence analysis of this repetitive sequence (RS) revealed all the characteristic features of an insertion sequence (IS). This 1656-bp element is delineated by two 20-bp inverted repeats which are flanked by two 11-bp direct repeats. A long open reading frame spans almost the entire sequence and is preceded by potential transcriptional and translational signals. A structural homology was observed between this RS and the Escherichia coli IS4 element in the length of their direct repeats, the sequence of their inverted repeats and the sequence of their putative transposases. These data strongly suggest that this sequence is an authentic insertion sequence. Therefore the name IS231 is proposed for this first Bacillus IS element. Further relationships with other known IS sequences were also found and are discussed. PMID- 3004979 TI - Influence of vagal cooling on cardiac output in normal and beta-blocked exercising dogs. AB - To study the relative influence of parasympathetic and sympathetic innervation on the early adaptation of cardiac output (CO) to exercise, we determined the time constant and amplitude of the CO change in dogs following a stepwise increase in treadmill velocity. The animals were studied during control conditions, beta blockade, vagal blockade and combined beta-blockade and vagal blockade. To measure CO, an electromagnetic flow probe was implanted around the ascending aorta. Vagal activity was blocked with coolers, implanted around the cervical vagosympathetic trunks. The time constant during beta-blockade (12.1 s) was not different from the control situation (11.4 s), but during vagal cooling it increased significantly (16.2 s), and with combined vagal cooling and beta blockade it rose to 20.7 s. Thus the increase in cardiac output with exercise is accelerated most by the loss of vagal tone and to a lesser degree by sympathetic activation. The amplitude of the change in CO during control was 112%. Heart rate (HR) rose by 74% and stroke volume (SV) by 22%. Beta-blockade lowered the initial CO but did not alter the percentage increase. Vagal cooling, with or without beta blockade, caused an increased initial HR but did not influence basal CO because of a concomitant reduction in SV. Exercise now increased HR less (21% and 30%, respectively) and SV more (52% and 52%) but the increase in CO did not change significantly (87% and 97%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3004981 TI - Assignment of conserved amino acid residues to the ATP site in the protein kinase domain of the receptor for epidermal growth factor. AB - The ATP substrate site in the epidermal growth factor (EGF) receptor was mapped by using a series of 26 ATP derivatives with modifications at the base, ribose or triphosphate moiety. Ki values for these derivatives were determined by competition with [gamma-32P]ATP. The enzyme seems to interact specifically with the beta-phosphate in an ion-pair bond with the N-6 amino group at the adenine in a hydrogen bond. With ribosyl-2-aminopurine triphosphate and GTP, the enzyme most likely recognizes the 2-amino group in a hydrogen bond. This high specificity for ATP and GTP is unique for the ATP site in the EGF receptor among all investigated protein kinases. The available data on the interaction between ATP derivatives and protein kinases were used to assign conserved amino acid residues found in diverse protein kinases to the ATP site in this type of enzyme. PMID- 3004982 TI - Nucleotide sequence and transcription of the fbc operon from Rhodopseudomonas sphaeroides. Evaluation of the deduced amino acid sequences of the FeS protein, cytochrome b and cytochrome c1. AB - The fbc operon from Rhodopseudomonas sphaeroides encodes the three redox carriers of the ubiquinol-cytochrome-c reductase (b/c1 complex): FeS protein, cytochrome b and cytochrome c1 [Gabellini, N. et al. (1985) EMBO J.2, 549-553]. The nucleotide sequence of 3874 bp of cloned R. sphaeroides chromosomal DNA, including the three structural genes fbcF, fbcB and fbcC has been determined. The reading frames of the fbc genes could be identified readily since the encoded amino acid sequences are highly homologous with the sequences of the corresponding mitochondrial polypeptides. Initiation and termination points for transcription have been investigated by S1 nuclease protection analysis. The transcription of the fbc operon starts approximately 240 base pairs upstream from the start codon of the fbcF gene and terminates 120 base pairs downstream from the stop codon of the fbcC gene. Nucleotide sequences resembling recognition signals for the binding and release of the RNA polymerase were identified. The N-terminal amino acid sequence of the mature cytochrome c1 was obtained by automated Edman degradation of the isolated subunit, confirming the fbcC reading frame and indicating that the bacterial preapocytochrome c1 has a transient leader sequence including 21 residues. The N-terminal sequence of one hydrophilic peptide of the FeS protein has been also obtained confirming the fbcF reading frame. The deduced amino acid sequences are discussed in relation to the known primary structures of the homologous proteins from mitochondria and chloroplasts. The primary structures of the polypeptides are evaluated with respect to their topology in the membrane, their biogenesis, the structure of the catalytic sites and subunit interactions. PMID- 3004983 TI - Expression of the polyoma middle-size T antigen in Escherichia coli. AB - We constructed a plasmid encoding a hybrid protein, consisting of the N-terminal signal sequence of the major outer membrane lipoprotein (lpp) of Serratia marcescens joined to the polyoma middle-size T antigen (mT antigen). The hybrid protein expressed under the control of a lpp-lac hybrid promoter was synthesized at levels up to 5% of newly synthesized protein and could be accumulated in Escherichia coli strains carrying the Cap R mutation. The mT antigen produced in E. coli was precipitated by polyoma antitumor serum, and by serum directed against a synthetic peptide corresponding to the C terminus of the authentic mT antigen. The protein was secreted into the periplasmic space, from which it could be released by osmotic shock. The bacterial mT antigen had no detectable associated protein kinase activity. PMID- 3004984 TI - Functional incorporation of beef-heart cytochrome c oxidase into membranes of Streptococcus cremoris. AB - Beef heart mitochondrial cytochrome c oxidase has been incorporated into membrane vesicles derived from the homofermentative lactic acid bacterium Streptococcus cremoris. Proteoliposomes containing cytochrome c oxidase were fused with the bacterial membrane vesicles by means of a freeze/thaw sonication technique. Evidence that membrane fusion has taken place is presented by the demonstration that nonexchangeable fluorescent phospholipid probes, originally present only in the bacterial membrane or only in the liposomal membrane, are diluted in the membrane after fusion and, by sucrose gradient centrifugation, indicating a buoyant density of the membranes after fusion in between those of the starting membrane preparations. The fused membranes are endowed with a relatively low ion permeability which makes it possible to generate a high proton motive force (100 mV, inside negative and alkaline) by cytochrome-c-oxidase-mediated oxidation of the electron donor system ascorbate/N,N,N',N'-tetramethyl-p phenylenediamine/cytochrome c. In the fused membranes this proton motive force can drive the uptake of several amino acids via secondary transport systems. The incorporation procedure described for primary proton pumps in biological membranes opens attractive possibilities for studies of proton-motive-force dependent processes in isolated membrane vesicles from bacterial or eukaryotic origin which lack a suitable proton-motive-force-generating system. PMID- 3004985 TI - The complete amino acid sequence of adenylate kinase from baker's yeast. AB - The complete amino acid sequence of cytosolic adenylate kinase (MgATP + AMP--- MgADP + ADP) from baker's yeast has been determined. Tryptic and clostripaic cleavage of the protein yielded 27 and 10 fragments, respectively. They were sequenced with either a solid-phase sequencer or a gas-phase sequencer. Alignment of the clostripaic fragments was deduced from the sequence of peptides obtained by endoproteinase Lys-C and cyanogen bromide cleavages. The N-terminus is blocked by an acetyl group as shown by proton magnetic resonance. Carboxypeptidase A digestion of the whole protein showed that the C-terminal sequence is -Lys-Asn, in agreement with the sequence of peptides from tryptic, clostripaic and 2 iodosobenzoic acid cleavages. The enzyme is a monomer of 220 amino acids with Mr 24077. Comparison of the sequence of the cytosolic adenylate kinases from yeast and pig shows 25% identity with highly conserved segments in the putative active site region of the enzyme. After position 111, however, there is an insertion of 32 residues in the yeast species, similar to the adenylate kinase and the GTP:AMP phosphotransferase from beef heart mitochondria. PMID- 3004986 TI - Formation of an aspartyl phosphate intermediate in the reactions of nucleoside phosphotransferase from carrots. AB - The nucleoside phosphotransferase from carrots forms N-phosphorylhydroxylamine when substrates are hydrolysed in the presence of hydroxylamine. Denaturation of the enzyme after short incubation with the substrates leads to a protein, in which, after reduction with [3H]NaCNBH3 and complete hydrolysis with 6 M HCl, labelled homoserine can be detected. The first experiment provides evidence for an activated phosphorylenzyme, the second experiment shows that the intermediate is an acyl phosphate formed by a nucleophilic attack of an aspartate beta carboxylate group. PMID- 3004987 TI - Purification and characterization of calcitonin receptors in rat kidney membranes by covalent cross-linking techniques. AB - We have characterized the binding parameters of renal receptors (Scatchard analysis revealed the presence of two binding sites: site I, Ka1 = 1.29 X 10(9) M 1, number of binding sites = 9.9 X 10(6)/micrograms protein; site II, Ka2 = 0.93 X 10(8) M-1, number of binding sites = 4.27 X 10(8)/micrograms protein) and studied the effect of solubilization. The high-affinity sites are preserved during affinity chromatography and the process results in a 6080-fold purification of those sites. The lower-affinity sites are also preserved but the overall purification factor is about 40% lower than that obtained using molecular sieving. The purification of the renal calcitonin receptor by molecular sieving (Sephacryl S-200) is accompanied by total loss of the high-affinity site; however, the low-affinity site is enriched over 1642-fold. Binding parameters were obtained for the purified fractions. Synthetic salmon calcitonin was also bound to renal membranes using the bifunctional reagent disuccinimidyl suberate and photo-affinity cross-linking using hydroxysuccinimidyl azidobenzonate reagent. Cross-linked receptor eluted in the same volume as solubilized membranes specifically binding salmon calcitonin (S-200 chromatography). Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of purified fractions showed several protein bands with apparent molecular masses ranging from 18 000 Da to 100 000 Da in the presence or absence of a reducing agent (2-mercaptoethanol). Autoradiography of polyacrylamide gels of cross-linked calcitonin receptor showed only three protein bands specifically binding salmon calcitonin. Their molecular masses were 70 000 Da, 40 000 Da and 33 000 Da respectively. The 40 000-Da molecule represents a major band (47% total binding species). This suggests that these three proteins are the principal components of the calcitonin receptor and that S-S bonds are not involved in the assembly of the receptor subunits. PMID- 3004988 TI - Production of anti-(ADP-ribose) antibodies with the aid of a dinucleotide pyrophosphatase-resistant hapten and their application for the detection of mono(ADP-ribosyl)ated polypeptides. AB - Previous attempts to produce anti-(ADP-ribose) antibodies by immunization of rabbits with ADP-ribose conjugated to serum albumin had resulted in the production of 5'AMP-specific antibodies [Bredehorst et al. (1978) Eur. J. Biochem. 82, 105-113]. To obtain true anti-(ADP-ribose) antibodies an antigen was constructed that was resistant to enzymic degradation at the pyrophosphate group. The enzymically active beta-methylene derivative of NAD (NAD[CH2]) was synthesized from ADP containing a methylene bridge (CH2) instead of an oxygen in the diphosphate group. NAD[CH2] was converted to its N6-[(2 carboxyethyl)thiomethyl] derivative and hydrolyzed to the corresponding ADP[CH2] ribose derivative which was then coupled to bovine serum albumin. The antibodies obtained with this antigen were specific for free or protein-bound ADP-ribose groups, except for a cross-reaction with FAD, AMP, ADP, ATP or poly(ADP-ribose) interfered with [3H]ADP-ribose tracer binding only at higher concentrations. No interference was observed with poly(A), RNA and DNA at 6000-fold excess. The antibodies were purified on a novel type of affinity matrix. This was formed from NAD and guanidinobutyrate by a cholera-toxin-catalyzed reaction and the product, ADP-ribosyl guanidinobutyrate, was bound to Affi Gel by carbodiimide-aided condensation. The purified antibodies allowed the detection of ADP-ribose conjugated to polypeptides in amounts lower than 1 pmol as demonstrated by immunoblotting of [14C]ADP-ribosylated elongation factor 2. They also could be used to observe in situ, by indirect immunofluorescence, the increased mono(ADP ribosyl)ation of nuclear proteins in dimethyl-sulfate-treated cells, and to show that histone H2B was the principal histone acceptor of single ADP-ribose groups in alkylated 3T3 cells. PMID- 3004989 TI - Fractionation of DNA restriction fragments with ion-exchangers for high performance liquid chromatography. AB - The fractionation abilities of several ion-exchangers of the high-performance liquid chromatography type for two sets of DNA restriction fragments, ranging from 7 base pairs (bp) to about 650 bp and differing in their mean base composition, have been studied. The ion-exchangers tested comprise the RPC-5, the 5-PW DEAE and the Mono Q as polymer-based resins, and the Nucleogens 500 and 4000, both prepared from silica beads. The results indicate that all the ion exchangers except the 5-PW DEAE perfectly separate fragment sizes up to about 90 bp, the 5-PW DEAE separating to 45 bp only. Above 200 bp only the Mono Q resin works in a satisfactory way provided that about 100 micrograms DNA mixture, containing less than 25 fragments within the given size range, is loaded per milliliter of packed resin. Appreciable base-pair specificities were detected for most of the resins which cause substantial retardations of the d(A + T)-rich fragments with respect to the eluting salt concentration. If the latter dominate in the DNA sample, acceptable results were only obtained with the Mono Q resin when the column was operated at elevated temperature. PMID- 3004990 TI - High antigen concentration inhibits T cell proliferation but not interleukin 2 production: examination of limiting dilution microcultures and T cell clones. AB - The effect of high antigen dose on the activation of cytochrome c peptide-primed lymph node cells was determined in several strains of mice by a limiting dilution analysis. It was found that proliferation of cytochrome c peptide-specific T cells was completely inhibited at high antigen concentration in C57BL/6 but only partially in DBA mice and had no effect in SJL mice. Clones derived from DBA mice showed a differential capacity to be inhibited by high antigen dose. On the other hand, interleukin 2 production by these clones was not impaired regardless of the antigen concentrations used. PMID- 3004991 TI - Mechanism of action of B-HT 933 (azepexole) in rat vas deferens and guinea-pig ileum. AB - The mechanism of action of B-HT 933 (azepexole) was studied on the rat vas deferens and myenteric plexus-longitudinal muscle (MP-LM) of the guinea-pig ileum. The drug caused a concentration-dependent inhibition of the twitch response in both preparations. The maximal inhibition in both preparations was 80 90%. B-HT 933 did not affect the cumulative dose-response curves of the vas deferens and of MP-LM to noradrenaline (NA) and acetylcholine (Ach) respectively. Yohimbine antagonized in a competitive way the twitch inhibitory effect of B-HT 933 on vas deferens and on MP-LM; the pA2 values were 8.62 and 8.5, respectively. The twitch inhibitory effects of B-HT 933 were not antagonized by propranolol. The results suggest that the action of B-HT 933 is mediated by stimulation of presynaptic alpha 2-receptors. PMID- 3004992 TI - GABAB receptors in the rabbit uterus may mediate contractile responses. AB - The effects of gamma-aminobutyric acid (GABA) and related compounds on the contractility of isolated rabbit uterus were examined. GABA and baclofen (10(-6) 10(-4) M) stimulated the spontaneous motility in a dose-dependent manner and showed cross-desensitization. The effect of baclofen was stereoselective for the (-)-enantiomer. Muscimol was ineffective, while bicuculline evoked marked contractions. Contractions elicited by submaximal electrical stimulation could be further increased by GABA and baclofen. The effects of GABA and baclofen on spontaneous contractility could not be antagonized by atropine, phentolamine, or tetrodotoxin. These findings indicate: (1) the presence of GABAB receptors in the rabbit uterus; (2) their involvement in the modulation of uterine contractility; (3) the extraneuronal location of uterine GABAB receptors which are thus most probably on smooth muscle cells; and (4) a possible role of inhibitory GABAA receptors in the modulation of spontaneous movements of the uterus. PMID- 3004994 TI - gamma-Aminobutyric acid, benzodiazepine binding sites and gamma-aminobutyric acid concentrations in epileptic E1 mouse brain. AB - All E1 mice provoked by postural stimulation since the age of 4 weeks had convulsions between 22 and 24 weeks of age, the refractory period ranging from 20 to 30 min. As compared to ddY mice, the maximal number of high-affinity [3H]muscimol binding sites was larger and the affinity was lower in the brains of the E1 mice, which had or had not experienced repeated seizures caused by postural stimuli. The basal and gamma-aminobutyric acid (GABA)-stimulated [3H]flunitrazepam binding sites, and GABA concentration in the brains of the E1 mice did not differ from those of the ddY mice. In E1 mice following provoked convulsions, there were no temporary changes in [3H]muscimol binding, or [3H]flunitrazepam binding with or without exogenous GABA stimulation. The GABA concentration in the brains of the E1 mice increased immediately after seizures, and returned to the control values within 60 min. PMID- 3004993 TI - Studies aimed at elucidating the mechanism of action of CI-914, a new cardiotonic agent. AB - CI-914 is a novel positive inotropic agent whose cardiotonic activity is not due to inhibition of Na+, K+-ATPase or to stimulation of cardiac beta-receptors. CI 914 also has no direct effect on sarcoplasmic reticulum, mitochondria or adenylate cyclase activity. CI-914 does, however, exert a potent inhibitory effect on cardiac phosphodiesterase activity. In evaluating the effect of this agent on the different molecular forms of phosphodiesterase present in cardiac muscle, CI-914 was found to selectively inhibit PDE III, which is a low Km, cAMP specific form of the enzyme (IC50 = 6.1 microM). This inhibitory effect was found to be competitive with respect to the substrate. Papaverine and theophylline on the other hand were found to inhibit all three forms of phosphodiesterase present in cardiac muscle. The role of phosphodiesterase inhibition in mediating the positive inotropic response to CI-914 is supported by the finding that this agent: (i) significantly elevates cyclic AMP levels in ventricular tissue; (ii) shifts the normal concentration-response to the beta-receptor stimulant isoproterenol to the left: and (iii) restores contractility to K+-depolarized papillary muscles. PMID- 3004995 TI - Effect of ACTH-(4-10) on equilibrium compensation after unilateral labyrinthectomy in the squirrel monkey. AB - Daily injection of ACTH-(4-10) was given to squirrel monkeys for a 28 day period to modify the characteristics of the post-unilateral labyrinthectomy symptoms along the course of the equilibrium compensation. When compared to the results of the control group, the ACTH-(4-10) injection groups (daily dose 250 micrograms/kg or 500 micrograms/kg) showed a significant improvement of acquisition and maintenance of compensation both in the spinal locomotor balance function and the oculomotor balance function. PMID- 3004996 TI - [3H]beta-funaltrexamine binds covalently to brain opioid receptors. PMID- 3004997 TI - Evidence that antagonism by delta-aminovaleric acid of GABAB receptors in the guinea-pig ileum may be due to an interaction between GABAA and GABAB receptors. AB - The antagonism by delta-aminovaleric acid has been investigated using electrically stimulated segments of isolated guinea-pig ileum. The depression of the twitch response by baclofen was antagonized by delta-aminovaleric acid and this antagonism is also mimicked by the selective GABAA agonists, 3 aminopropanesulphonic acid and isoguvacine. The GABAA antagonist bicuculline reversed the antagonism of the baclofen response. These results indicate that this antagonism of GABAB receptors may be due to GABAA receptor modulation of GABAB receptors. PMID- 3004998 TI - Effects of atriopeptins on relaxation and cyclic GMP levels in rat and rabbit aortas. AB - The effects of atriopeptins I and II on relaxation and cyclic GMP levels were studied on rat and rabbit aortas. Atriopeptin I was 2- and 100-fold less potent than atriopeptin II in causing relaxation of rat and rabbit aortas, respectively. The atriopeptin-elevated cyclic GMP levels generally correlated with the amount of relaxation. These results demonstrate that the vasodilator profile and, presumably, the receptor for atrial natriuretic factor, varies among different blood vessels and species. PMID- 3004999 TI - Antidepressant-like effects of diphenylhydantoin in mice: involvement of alpha adrenoceptors. AB - Several studies have indicated that alpha-adrenergic systems are implicated in the anticonvulsant activity of diphenylhydantoin. We now report that in mice prazosin (0.125 mg/kg), a blocker of alpha 1-adrenoceptors, reverses the effects exerted by diphenylhydantoin (256 mg/kg) in tests which are predictive of antidepressant activity: reserpine-induced ptosis and immobility which are caused by inescapable, aversive situations. These date indicate that alpha-adrenoceptors are not only involved in the anticonvulsant action of diphenylhydantoin, but also in its antidepressant-like effects. PMID- 3005000 TI - The effect of chronic administration of caffeine on morphine-induced analgesia, tolerance and dependence in mice. AB - Morphine-induced analgesia, and the development of morphine-induced tolerance and dependence was determined in mice which had drunk caffeinated water (1 mg/ml) for 14 days or in mice which had received (-)-N6-(phenylisopropyl)-adenosine (PIA) 1 mg/kg i.p. for 14 days. Analgesia was assessed by the tail flick assay. The development of dependence was assessed by determining the ED50 of naloxone to precipitate withdrawal jumping (3 h after 100 mg/kg morphine pretreatment or 72 h after s.c. implantation of a morphine 75 mg pellet) and by determining the extent of naloxone-precipitated hypothermia in morphine-implanted animals. In mice chronically administered caffeine, the ED50 for morphine-induced analgesia was significantly decreased while the naloxone ED50 for withdrawal jumping increased by 2-fold after both types of morphine pretreatment. In control animals (tap water for 14 days), doses of 1 and 10 mg/kg of naloxone caused significant hypothermia in morphine-implanted animals. Doses of naloxone up to 100 mg/kg did not cause significant hypothermia in morphine-implanted animals which had received chronic caffeine. The development of tolerance was determined by computing the morphine potency ratio for the tail flick assay (tolerant ED50/control ED50). In mice chronically administered caffeine, the potency ratio was decreased significantly in morphine-implanted animals when compared to control. Morphine-induced analgesia, tolerance and dependence was not changed significantly in animals chronically administered PIA. Neither the distribution of morphine to the brain nor the opioid receptor binding parameters for [3H]etorphine and [3H]naltrexone were altered in mice chronically administered caffeine or PIA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3005001 TI - Baclofen, a GABA derivative, inhibits stress-induced prolactin release in the rat. AB - The effect of baclofen, beta-(4-chlorophenyl)GABA, on prolactin secretion was investigated in rats under several experimental conditions. In adult male rats subjected either to immuobilization, ether, swimming or cold stress there was a rapid increase of serum prolactin levels; acute pretreatment with baclofen, 10 mg/kg i.p. inhibited the hormone response to all these stresses. The same blocking effect of the drug was observed in prepubertal male and female rats and in adult gonadectomized animals. In basal conditions, i.e. in undisturbed male rats, baclofen did not change the hormone titers significantly. Taken together our results indicate that baclofen blocks prolactin release when release of the hormone is dynamically stimulated by stress and this effect is relatively independent of the endocrine status of the rat. PMID- 3005002 TI - Serotonin 5-HT1 receptors mediate inhibition of cyclic AMP production in neurons. AB - In purified striatal and cortical neurons in primary culture, serotonin (5-HT) stimulated basal cyclic AMP production (EC50, 0.5 microM) 2.5- and 1.5-fold, respectively. The 5-HT1 selective agonists, RU 24969 and 8-hydroxy-2-(di-n propylamino)tetralin (PAT), did not stimulate cyclic AMP production. However, 5 HT, RU 24969 and PAT inhibited VIP-stimulated cyclic AMP formation in a dose dependent manner. The actions of selective agonists and antagonists at 5-HT receptors mediating attenuation of cyclic AMP production suggest that they may be of the 5-HT1 subtype. PMID- 3005003 TI - Dopaminergic modulation of ACTH-induced grooming. AB - ACTH-(1-24)-induced grooming was studied after administration of the peptide into the substantia nigra or intracerebroventricularly (i.c.v.). The modulation of dopamine receptors in neostriatum (with haloperidol and apomorphine) and nucleus accumbens (with 3,4-dihydroxyphenylamino-2-imidazoline hydrochloride; DPI and ergometrine) was investigated. In the nucleus accumbens, the modulatory effects of ergometrine and DPI on ACTH-(1-24)-induced grooming were based on their affinity for dopamine receptors and not on their affinity for adrenoceptors. Intrastriatal application of dopaminergic agents inhibited i.c.v. ACTH-(1-24) induced excessive grooming, whereas the grooming score was enhanced if ACTH-(1 24) was administered into the substantia nigra. The finding of differential effects of dopaminergic agents on ACTH-induced excessive grooming depending on the route of administration indicate that i.c.v. ACTH-induced excessive grooming is not mediated solely through the substantia nigra. The increase in grooming behavior seen after the intrastriatal administration of dopaminergic agents - when ACTH was injected into the substantia nigra - suggests the involvement of the striato-nigral GABAergic pathway. Local injections of ACTH-(1-24) into the periaqueductal gray also induced excessive grooming. Since a second injection of ACTH-(1-24) into the periaqueductal gray did not lead to a grooming response, irrespective of where the first injection of ACTH-(1-24) was given (i.c.v. into the nigra or via the periaqueductal gray) it is suggested that this structure seems to play a primary role in the induction of excessive grooming. Therefore the modulatory effects of the dopaminergic influence on ACTH-(1-24)-induced grooming may be exerted via the striato-nigro-collicular pathway. PMID- 3005004 TI - Heterogeneity of mammalian alpha 2-adrenoceptors delineated by [3H]yohimbine binding. AB - [3H]Yohimbine binding to membrane preparations of human colon, cerebral cortex, kidney, spleen and platelets was compared with binding to preparations of animal tissues (rabbit spleen, kidney and cerebral cortex; rat cerebral cortex; guinea pig and cat spleen). Specific binding to all preparations was saturable and indicative of binding to a uniform population of sites. The equilibrium dissociation constants (KD) of [3H]yohimbine ranged from 1.6 to 2.6 nM for human tissue and from 5.1 to 9.4 nM for the animal tissues. Binding to all tissues was displaced by drugs with an order of potency yohimbine greater than phentolamine greater than prazosin, indicating an alpha 2-adrenoceptor classification of the labelled sites. Whilst certain drugs (phentolamine, corynanthine) possessed similar affinities for all alpha 2-adrenoceptors, others (prazosin, idazoxan, WY 26392) exhibited differential potencies for alpha 2-adrenoceptors in certain species. The pharmacological characteristics of human alpha 2-adrenoceptors were conserved within the tissues examined. These results suggest that human alpha 2 adrenoceptors differ in a number of ways from those present in tissues from the other mammalian species examined. The possible existence of a spectrum of alpha 2 adrenoceptors is discussed in the light of these findings. PMID- 3005005 TI - Effects of verapamil on activation of arachidonic acid metabolism in guinea-pig lungs. AB - The effects of (+/-)-verapamil and its optical isomers on the activation of arachidonic acid (AA) in guinea-pig isolated perfused lungs were investigated. The calcium ionophore A23187 (6-15 nmol), histamine (20-50 nmol) and leukotriene E4 (1-2 nmol) induced the release of thromboxane A2 (TxA2), which was detected by bioassay and by radioimmunoassay of the stable metabolite TxB2. Racemic (+/-) verapamil (0.4-40 microM) caused concentration-dependent inhibition of A23187 induced release of TxA2 without affecting the conversion of exogenous AA to TxA2. Both (+)-verapamil and (-)-verapamil (1-10 microM) caused concentration-dependent inhibition of histamine-induced release of TxA2. In contrast, racemic (+/-) verapamil did not inhibit leukotriene E4 (LTE4)-induced release of TxB2. Calcium depletion (with 2 mM ethylenediamine tetra acetate) significantly reduced both histamine-induced release of TxB2 from 8.6 +/- 2.6 to 1.8 +/- 0.8 ng/min (P less than 0.05) and LTE4-induced release of TxB2 from 4.9 +/- 0.9 to 0.5 +/- 0.2 ng/min (mean +/- S.E.M.) (P less than 0.05). Since histamine stimulates phospholipase A2 in guinea-pig lungs, these results suggest that (+/-)-verapamil inhibits phospholipase A2 and that A23187 activates AA metabolism by stimulating phospholipase A2. Although all three agents activate AA metabolism by calcium dependent processes, LTE4 may stimulate calcium entry via separate mechanisms because it is not inhibited by (+/-)-verapamil. PMID- 3005006 TI - Dopaminergic control of respiration as shown by effects of 4-aminopyridine. AB - The dopaminergic control of respiration in conscious and urethane-anaesthetized rabbits, was studied by comparing the respiratory effects of 4-aminopyridine alone (4-AP; 1 mg/kg i.v.) and those after the administration of dopamine antagonists (domperidone and haloperidol; 1 mg/kg each). The respiratory rate in conscious rabbits was increased by 4-AP. After domperidone this increase was reduced and preceded by a transient decrease. In spontaneously breathing, anesthetized rabbits there was a transient reduction after which the respiratory rate was increased by 4-AP; tidal volume was affected in an inverse manner. After domperidone, the excitatory effect of 4-AP on respiratory rate and the inhibitory effect on tidal volume were blocked. The effects of 4-AP on respiratory rate were prevented by vagotomy. In anesthetized, vagotomized, paralyzed and artificially ventilated rabbits (VPV animals) the peak amplitude of the integrated phrenic nerve activity ("phrenic activity') was increased by 4-AP. After pretreatment with haloperidol this effect of 4-AP on phrenic activity was reduced while the respiratory rate was now increased. In VPV animals with denervated carotid bodies the excitatory effect of 4-AP on phrenic activity was strongly enhanced and respiratory rate was increased. These effects were slightly reduced but not blocked by haloperidol. It is concluded that endogenous dopamine is involved in the control of respiration through effects on peripheral mechanisms (inhibition of inspiratory activity and enhancement of respiratory rate) as well as on central mechanisms (stimulation of inspiratory activity and reduction of respiratory rate). PMID- 3005008 TI - Interaction between theophylline and ouabain in the rabbit heart: effect on (Na+ + K+)-ATPase. AB - In electrically stimulated rabbit ventricular strips, theophylline (0.3-30 mM) antagonized the increased contractility produced by ouabain (0.8 microM). Initial velocity of specific [3H]ouabain binding to homogenates prepared from the muscle strips was used to determine the fraction of binding sites occupied by ouabain. Theophylline decreased the binding of ouabain to (Na+ + K+)-ATPase. It is concluded that the effect of theophylline on ouabain-induced positive inotropism may be mediated by decreased ouabain binding to (Na+ + K+)-ATPase. PMID- 3005007 TI - Centrally administered beta-endorphin produces prolonged changes in delta-opioid ligand activity in vivo. AB - Spontaneous reflex bladder contractions were recorded isometrically in urethane anesthetized rats. Bladder contractions were depressed by intracerebroventricular injections of the mu-opioid receptor agonist [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO) and the delta-agonist [2D-penicillamine,5D-penicillamine]enkephalin (DPDPE) respectively. The effect of DPDPE was selectively antagonized by ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH; Aib = alpha-aminoisobutyric acid). However following the administration of beta-endorphin the antagonistic action of ICI 174,864 could no longer be observed. In addition ICI 174,864 exhibited agonistic activity following beta-endorphin and the effects of DPDPE were prolonged in a dose related manner by beta-endorphin. These observations suggest that beta-endorphin may produce complex changes in central delta-opioid receptor activity. PMID- 3005009 TI - Specific receptors for thyroxine in nuclear non-histone proteins of rat thymus during early, medium and late period of life. AB - Specific binding of 125I-thyroxine to thymus nuclear non-histone proteins of rats aged 7, 14, 45, 80 and 365 days was studied. In 0.4 mol l-1 NaCl nuclear extract of thymocytes practically the same values of Ka were found in rats aged 7, 14, 45 and 80 days (5.72 +/- 0.11 X 10(9) l mol-1, 3.44 +/- 1.65 X 10(9) l mol-1, 5.00 +/- 1.09 X 10(9) l mol-1 and 5.55 +/- 0.95 X 10(9) l mol-1, respectively). As compared to groups of animals of 7, 45 and 80 days of age a marked diminution of Ka (= 1.30 +/- 0.25 X 10(9) l mol-1) with P less than 0.001, less than 0.02 and less than 0.01, respectively, was found in rats aged 365 days. The highest value representing maximum binding capacity (MBC) of 125I-thyroxine was noted in rats aged 45 days (MBC = 6.16 +/- 0.58 X 10(10) molecules per mg of protein) and in adult rats (80 days old) (MBC = 5.82 +/- 1.28 X 10(10) molecules mg-1). In rats aged 7 days a lower value for MBC (= 1.92 +/- 0.77 X 10(10) molecules mg-1; P less than 0.01) was observed as compared to the animals aged 45 days (see above). Similar decrease was found also in old rats aged 365 days (MBC = 3.46 +/- 0.69 X 10(10) molecules mg-1; P less than 0.02). The data quantify the affinity and the specific 125I-thyroxine binding capacity in nuclear non-histone proteins of rat thymus during early, adult and late period of life. PMID- 3005010 TI - The possible potassium involvement in the modulation of the long-term trophic action of ACTH on the rat adrenal zona fasciculata. AB - The effects of chronic potassium loading on the zona fasciculata of the rat adrenal cortex were investigated. It was found that potassium stimulates the growth and the steroidogenic capacity of the zona fasciculata presumably by enhancing the trophic effects of ACTH. PMID- 3005011 TI - Mature T cells are part of adherent cells in human long-term bone marrow cultures. AB - We determined the proportion of lymphocytes in nonadherent and adherent fractions of human bone marrow cells cultured by a Dexter-type continuous marrow culture method. T-lymphocytes, B-lymphocytes, and common alloantigen (CALLA)-positive cells (cells with receptors for CALLA) were sequentially enumerated using commercially available appropriate monoclonal antibodies. Nonadherent cells from weeks 2-5 of culture contained relatively fixed proportions of OKT3-positive (4% 10.4%) and CALLA-positive (5%-6.6%) cells. The adherent cells during the culture period between weeks 6 and 18 contained an average of 34% OKT3-positive cells with a range from 6% to 68.5%, despite a high hydrocortisone concentration of 5 X 10(-5) M added to the growth medium. The T cells retrieved from the adherent layers and resuspended in steroid-free medium responded to PHA stimulation and to mixed leukocyte culture in the same manner as did freshly drawn peripheral blood leukocytes. These results indicate that adherent cell populations include mature T-lymphocytes in human long-term bone marrow cultures. In view of well-documented interactions of nonlymphoid hematopoietic progenitors with T-lymphocytes in the clonogenic culture system, it can be speculated that the adherent T cells also may play a role in proliferation and differentiation of granulocyte-macrophage and erythroid progenitors in this long-term culture system. PMID- 3005012 TI - Recent advances in sarcoidosis. PMID- 3005013 TI - Lung abscess in small cell carcinoma of the lung during chemotherapy and corticosteroids: an analysis of 276 consecutive patients. AB - Two hundred and seventy-six consecutive patients with small cell carcinoma of the lung (SCCL) treated with combination chemotherapy and in 79 cases with "high dose" steroids (greater than 40 mg of prednisone per day) were reviewed for the presence of lung abscess. This was diagnosed in 17 patients, in 4 (1.5%) at the time of their malignant diagnosis and 13 (4.9%) during chemotherapy. Five of 79 patients receiving "high-dose" glucocorticoid therapy and 8 of 184 patients not receiving steroids developed lung abscess (no statistical difference, P greater than 0.05). "High-dose" steroids do not facilitate the development of lung abscess. Eleven patients presented with a lung abscess within a month of initiation of chemotherapy. Median survival of these patients was 182 days and not significantly different from a median survival of 224 days (P greater than 0.05) observed in 31 compatible patients without lung abscess. Lung abscess per se in patients with SCCL should not prevent the use of intensive combination chemotherapy and "high-dose" steroid therapy. PMID- 3005014 TI - Cortically and lingually induced postsynaptic potentials in trigeminal motoneurons after axotomy. AB - The membrane properties and the efficacy of excitatory and inhibitory synapses were studied in cat masseteric motoneurons (Mass Mns) after axotomy. In axotomized Mass Mns the slope of the primary range in the frequency-current relationship showed a higher gain than that of normal Mass Mns. The safety of antidromic invasion was increased and the initial segment component of antidromic action potentials could not be separated from the soma-dendritic component. In normal Mass Mns a single shock delivered to the orbital gyrus or the lingual nerve induced long-lasting inhibitory postsynaptic potentials (IPSPs). In two thirds of Mass Mns explored 30 days after axotomy, a single shock delivered to the orbital gyrus or the lingual nerve evoked a mixture of inhibitory and excitatory synaptic potentials. In Mass Mns 50 days after axotomy, we have demonstrated that the major fraction of the total sample of explored Mass Mns showed long-lasting excitatory postsynaptic potentials followed by IPSPs. The results suggest that in Mass Mns, axotomy is followed by the decline of synaptic efficacy of inhibitory rather than of excitatory synapses. PMID- 3005016 TI - Quantitative distribution of GABA-immunoreactive neurons in the visual cortex (area 17) of the cat. AB - Cortical neurons using the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) are known to contribute to the formation of neuronal receptive field properties in the primary visual cortex (area 17) of the cat. In order to determine the cortical location of GABA containing neurons and what proportion of cortical neurons might use GABA as their transmitter, we analysed their distribution quantitatively using a post-embedding GABA immunohistochemical method on semithin sections in conjunction with stereological procedures. The mean total numerical density of neurons in the medial bank of the lateral gyrus (area 17) of five adult cats was 54,210 +/- 634 per mm3 (mean +/- SD). An average of 20.60 +/- 0.48% (mean +/- SEM) of the neurons were immunoreactive for GABA. The density of GABA-immunoreactive neurons was somewhat higher in layers II, III and upper VI, compared with layers I, IV, V and lower VI, with the lowest density being in layer V. The proportion of GABA-immunopositive cells relative to immunonegative neurons gradually decreased from the pia to the white matter. Layer I was different from other layers in that approximately 95% of its neurons were GABA-immunoreactive. The results allowed the calculation of the absolute numbers of GABAergic neurons in each layer under a given cortical surface area and could provide the basis for the quantitative treatment of cortical circuits. PMID- 3005015 TI - Short latency inputs to phrenic motoneurones from the sensorimotor cortex in the cat. AB - Short latency responses were recorded from C5 phrenic roots and intracellularly from phrenic motoneurones following stimulation of the pericruciate cortex or medullary pyramids in cats anaesthetized with Nembutal or chloralose-urethane. Focal stimulation of the cortical surface (single pulses, 0.5-2 ms, 0.3-8 mA) during inspiration evoked EPSPs (latency 4.7 +/- 1.7 ms, rise time 1.9 +/- 1.1 ms, amplitude 0.22 to 3.94 mV) in 42% of motoneurones studied (n = 107). The EPSPs were absent, or on average 60% smaller, following stimulation during expiration. In all but two motoneurones, during both inspiration and expiration, hyperpolarizing potentials were observed either following the initial depolarization or alone. They could be reversed by hyperpolarizing current or chloride injection. Stimulation of the pyramidal tract at mid medullary level (1 to 3 pulses, 0.2 ms) evoked short latency excitation in phrenic motoneurones only with currents of more than 200 microA. Smaller stimuli applied to the medial reticular formation above the pyramidal tract evoked excitation (onset latency 1.5-3.2 ms) in which the earliest part was probably monosynaptic. These results show that the corticospinal responses in phrenic motoneurones are both excitatory and inhibitory. They are not transmitted through the pyramidal tract and are at least disynaptic. Excitation evoked from the medullary pyramidal tract can be explained by current spread beyond the pyramidal tract fibres. PMID- 3005017 TI - The heteronymous monosynaptic actions of triceps surae group Ia afferents on hip and knee extensor motoneurones in the cat. AB - Monosynaptic connections from the group Ia afferents of triceps surae onto quadriceps, anterior biceps and semimembranosus motoneurones have been demonstrated in the cat. They appear to be equivalent to those found between triceps surae and thigh muscles in man. PMID- 3005018 TI - Long-term potentiation and depression in hippocampal slices. AB - Antidromic stimulation of hippocampal CA 1 cells, in the presence of Mg2+ to eliminate synaptic transmission and with a pattern of impulses which when applied via a synaptic pathway produced long-term potentiation, was found to decrease the response of the CA 1 cells to subsequent synaptic activation. It was found that stimulation via synapses with the same pattern of stimuli caused long-term potentiation in normal conditions but not in the presence of 25 mM Mg2+. PMID- 3005019 TI - Alpha 2 adrenoceptors modulate the nociceptive jaw-opening reflex in rats. AB - The jaw-opening reflex in lightly anesthetized rats induced by intrapulpal (left maxillary) electrical tooth pulp stimulation and quantified by the electromyograms (threshold values) recorded from the ipsilateral digastric muscle was used as the experimental pain index. The threshold for the jaw-opening reflex was significantly elevated by clonidine (12.5 to 50 micrograms/kg, i.v.) and was inversely correlated with the frequency of stimulation. The analgesia elicited by clonidine was antagonized by pretreatment with the alpha 2-adrenoceptor antagonist yohimbine (1 mg/kg, i.v.). All doses of clonidine produced an initial transient pressor response followed by a sustained hypotension and bradycardia. However, there was no direct correlation between the antinociceptive and cardiovascular effects of clonidine. It is proposed that clonidine modulates jaw opening reflex analgesia by specifically activating alpha 2-adrenoceptors. PMID- 3005020 TI - Responses of morphologically identified cortical neurons to intracellularly injected cyclic GMP. AB - Cyclic nucleotides are thought to act as second messengers of neurotransmission inside central neurons, and cyclic guanosine monophosphate (cGMP) has been postulated to act as a messenger for muscarinic, cholinergic transmission. Nonetheless, the action of cGMP has not yet been established in identified cortical neurons. We injected cGMP and horseradish peroxidase (HRP) intracellularly in neurons of the motor cortex of awake cats. Fifty-four percent of injected cells responded to cGMP and HRP with an increase in input resistance within 30 s after injection. None of a control group of cells injected with HRP without cGMP so responded. In cells receiving intracellular depolarizing current sufficient to produce repeated spike discharge at the time of injection, the increase in input resistance after cGMP persisted for as long as the cells could be held. There was no significant increase in firing rate after injection of cGMP. Cells responding to cGMP with an increased input resistance were identified as pyramidal cells of layer V. One inverted pyramidal cell of layer VI also showed an increase in input resistance in response to cGMP. Previous studies have suggested that muscarinic cholinergic agents produce an increased input resistance (thought to reflect a decreased potassium conductance) underlying an increased rate of discharge in neocortical neurons. Our results favor a dual action of muscarinic cholinergic transmission in mammalian cortical neurons--the increase in input resistance in layer V pyramidal cells mediated by cGMP, and the increase in rate of discharge mediated by other means. PMID- 3005021 TI - Responses of morphologically identified cortical neurons to intracellularly injected cyclic AMP. AB - Intracellular injections of cyclic adenosine monophosphate (cAMP) and horseradish peroxidase (HRP) were made in neurons of the motor cortex of awake cats. Eighty six percent of injected cells responded to cAMP and HRP with a rapid decrease in input resistance. The decreases in input resistance occurred immediately after injection and began to return toward baseline 2 to 3 min later. The decreases were significantly greater than the small decreases in input resistance normally seen in uninjected cells held for 2 min or more after penetration and exceeded comparably small decreases in input resistance seen after control injections of 5'-AMP plus HRP. Pyramidal cells of layer V were identified as responding to cAMP with a decreased input resistance. A spiny stellate cell of layer III and a pyramidal cell of layer VI were also identified that showed similar responses. Increased rates of discharge were also observed after penetration with electrodes containing cAMP, but significant changes in input resistance were not found in association with the increased rates of discharge. After pressure injection of cAMP, the rates of discharge decreased toward more normative values. Our findings indicate that cAMP has an effect on cortical neurons similar to that found in some types of invertebrate (molluscan) neurons and dissimilar to the effect of cyclic guanosine monophosphate. PMID- 3005022 TI - Depletion of brain mitochondria cytochrome oxidase in the mottled mouse mutant. AB - The mouse mutant Mobr is an animal model of Menkes kinky hair syndrome with a similar defect in copper utilization. The copper-dependent enzyme, cytochrome oxidase from the brain, liver, and heart mitochondria was examined. The brain and heart from Mobr/y had significantly less cytochrome alpha + alpha 3 than normal animals' when the cytochrome absorption spectra of tissue samples from animals 11 to 13 days of age were analyzed. Liver cytochrome was not significantly different. When brain mitochondrial cytochrome oxidase spectrograms from animals of different ages were examined, a major change was found to occur during the 2nd week of life. When cytochrome oxidase activity from brain mitochondria was measured, assaying the rate of oxidation of cytochrome c, the results were similar to those from spectrogram analysis. PMID- 3005024 TI - Proton and potassium fluxes in rat red blood cells incubated with sugar phosphates. AB - Fructose-1,6-diphosphate counteracts potassium ejection and proton uptake induced in rat red blood cells by valinomycin and an uncoupler. The effect on potassium ejection is reduced in the presence of ouabain and divalent cations. PMID- 3005023 TI - Benzodiazepine receptors resolved. AB - To date, attempts to map the distribution and density of benzodiazepine receptors in the CNS have been dominated by radiohistochemical techniques with conventional receptor binding. Their limited resolution, however, prompted us to try an immunohistochemical approach. Purified GABA/benzodiazepine receptors, prepared from bovine cerebral cortex, have been used to raise monoclonal antibodies for this purpose. Immunoreactive sites in rat brain, spinal cord and retina as well as in bovine and post-mortem human brain were found to be concentrated on neuronal cell bodies and processes in those regions known to be innervated by GABAergic neurons. Electron microscopic analysis revealed a selective staining of axosomatic and axodendritic pre- and postsynaptic contacts. PMID- 3005026 TI - Early membrane damage during ischemia in rat heart. AB - Effects of ischemia on cell membrane of rat heart were investigated. The endothelial surface revealed the existence of ruthenium red-positive glycocalyx at the anionic site. Membrane bound enzyme as Na-K ATPase was mostly located in the inner side and pinocytotic vesicles of endothelial cell. The clumping and dispersion in glycocalyx of endothelial cells was observed in an ischemic heart and it may prove the functional disturbance of plasma membrane. A potential and functional defect with reduced activity of Na-K ATPase occurred within 1 hr of vascular ligation. The membrane dysfunction due to these molecular changes has been proved by the membrane permeability alteration as well as the intracytoplasmic localization of horseradish peroxidase as tracer. PMID- 3005025 TI - Influences of the chemical structure on the activity of new inhibitory compounds of the angiotensin-converting enzyme. AB - The ACE inhibitory activity of some perimidines, chinazolinones and amidinohydrazones is described. Relations were found between the chemical structure and the inhibitory activity on the ACE. PMID- 3005027 TI - Peroxidase activity in rat tracheal epithelium and gland. AB - Endogenous peroxidase activity in the upper tracheal epithelium and submucosal gland of specific pathogen-free rats was examined cytochemically using the DAB method in animals bred in a conventional room without special equipment for air filtration (conventional system), and those bred under a semibarriered system in which fresh air was filtered and controlled to flow in one way (semibarriered system). Peroxidase activity was consistently positive in the serous cells of the gland of all the rats in both groups. In 22 (71.0%) of the 31 rats in the conventional system, peroxidase activity was demonstrated also in ciliated cells, mucous cells, and goblet cells of the tracheal epithelium, and in mucous cells and duct cells of the tracheal submucosal gland. However, the activity was detected in these cells only in 2 (6.7%) of the 30 rats in the semibarriered system. Goblet cells were not observed in the latter group of rats. The fine localization of peroxidase activity was similar among the peroxidase positive cells, and was demonstrated in the cisternae of the nuclear envelope and rough surfaced endoplasmic reticulum, some parts of Golgi apparatus, secretory granules, and in some cases in the intraductal spaces of the gland. The present study indicated that environmental conditions markedly influence peroxidase activity in the upper tracheal epithelium and gland of rats. PMID- 3005028 TI - Preliminary study on Hip-Phe-Arg substrate for human urinary kininase assay. PMID- 3005030 TI - [Adrenoreceptors of the heart]. PMID- 3005029 TI - [Problems of drug recognition. Imipramine receptors]. PMID- 3005031 TI - [Effect of Nakom on the activity of the hypothalamo-hypophyseo-adrenal system]. AB - The effect of nakom, containing the dopamine precursor levodopa, on the activity of the hypothalamo-hypophysial-adrenal system (HHAS), the content of dopamine in the hypothalamus and the cerebral hemisphere cortex and cAMP in tissues, was studied in experiments on rats. It was revealed, that the preparation in the 2 mg per 100 g dose has a marked inhibiting action on the activity of the HHAS, that is expressed in 2 h by the drop of the level of ACTH, corticosterone, aldosterone in blood that is maintained for 10 hours and disappears 24 hours after the administration. One of the possible mechanisms of the inhibiting action of the preparation on HHAS activity is the increase of dopamine content in the hypothalamus and cerebral hemisphere cortex. The inhibiting action of the preparation on HHAS activity is dose-dependent. The inhibiting influence of nakom on the HHAS activity is realized through the adenylate cyclase system. PMID- 3005032 TI - [Ceruloplasmin activity and content of the blood of persons with acute and chronic alcoholic intoxication]. AB - The concentrations of the enzymatically active and immunoreactive ceruloplasmin (CP) were determined in the blood serum of healthy men by means of the spectrophotometric and immuno-electro-photometric methods. Part of the total (immunoreactive) CP (15%), circulated in the blood channel in an enzymatically inactive state. The increase of the time of suffering from chronic alcoholism from 1 to 29 years leads to the growth of the concentrations of CP possessing enzymatic activity and of immunoreactive CP. The share of the enzymatically inactive protein remains practically constant. During acute alcohol intoxication there is a temporal drop of the C1 share in enzymatically active state with a constant concentration of the total protein in the blood serum. PMID- 3005033 TI - Comparison of the internalization efficiency of LDL and transferrin receptors on L2C guinea pig lymphocytes. AB - We demonstrate that L2C lymphocytes have about 10-times more receptors for transferrin (Tf) than healthy lymphocytes, as has been shown in the case of LDL receptors. The dissociation constant is the same in the two cell types (about 4 X 10(-7) M). In contrast to LDL, Tf enters L2C lymphocytes with very rapid kinetics. It is shown by cross-reaction that each receptor is internalized independently of the other. PMID- 3005034 TI - Bradykinin stimulates GTP hydrolysis in NG108-15 membranes by a high-affinity, pertussis toxin-insensitive GTPase. AB - In membranes of neuroblastoma x glioma hybrid (NG108-15) cells, bradykinin (EC50 approximately equal to 5 nM) stimulates GTP hydrolysis by a high-affinity GTPase (Km approximately equal to 0.2 microM). The octapeptide, des-Arg9-bradykinin, was inactive. Stimulation of GTP hydrolysis by bradykinin and an opioid agonist was partially additive. Treatment of NG108-15 cells with pertussis toxin, which inactivates Ni, eliminated GTPase stimulation by the opioid agonist but not by bradykinin. The data suggest that bradykinin activates in NG108-15 membranes a guanine nucleotide-binding protein which is not sensitive to pertussis toxin and which may be involved in bradykinin-induced stimulation of phosphoinositide metabolism in these cells. PMID- 3005035 TI - Identification of a c-myc oncogene lacking the exon 1 in the normal cells of a patient carrying a thyroid carcinoma. AB - In this paper we describe an alteration of the c-myc oncogene present in the white blood cells and normal as well as neoplastic thyroid cells of a subject carrying a thyroid carcinoma. Restriction enzyme mapping and hybridization to human c-myc probes specific for different regions of this gene demonstrate that this subject carries, in addition to the normal one, a c-myc oncogene lacking the first exon and part of the first intron. The levels of the c-myc mRNA in thyroid cells of this subject do not show differences with respect to thyroid cells from other subjects. Taken together, these findings indicate that the deletion of the first exon of the c-myc oncogene, in itself, does not produce overtranscription of this oncogene nor hematopoietic malignancies. PMID- 3005036 TI - Thyrotropin and cyclic AMP regulation of ras proto-oncogene expression in cultured thyroid cells. AB - We examined the effect of thyrotropin (TSH) on intracellular levels of c-ras mRNA in a line of differentiated rat thyroid cells obtained from normal Fischer rat thyroids. These cells are totally dependent on TSH for growth. TSH stimulation of quiescent cells increased c-ras mRNA content, with a maximal response (730% of basal) after 6 h, and a decline towards basal levels after 24 h. Dibutyryl cAMP and forskolin mimicked this stimulatory effect of TSH on c-ras, but did not enhance beta-actin mRNA content. This study demonstrates hormonal and cyclic nucleotide control of c-ras expression in a well-differentiated, non-tumorogenic mammalian cell. PMID- 3005037 TI - Primary structure of the beta-subunit of Torpedo californica (Na+ + K+)-ATPase deduced from the cDNA sequence. AB - DNA complementary to the Torpedo californica electroplax mRNA coding for the beta subunit of (Na+ + K+)-ATPase has been cloned by screening a cDNA library with an oligodeoxyribonucleotide probe. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 305 amino acid residues (including the initiating methionine). The transmembrane topology and the potential N glycosylation sites of this polypeptide are discussed. PMID- 3005038 TI - Regulatory subunit of type II cAMP-dependent protein kinase as substrate and inhibitor of protein phosphatase-1 and -2A. AB - The dissociated regulatory subunit (RII) of autophosphorylated cAMP-dependent protein kinase II was dephosphorylated by the catalytic subunits of protein phosphatase-1 and -2A (phosphatase-1c and -2Ac) and by a high-Mr polycation dependent form of phosphatase-2A (2Ao) with Km values of 5, 0.3 and 1 microM, respectively. Dissociation of protein kinase by cAMP preferentially increased the dephosphorylation of RII by phosphatase-1c, whereas polycations (histone Hl or polybrene) markedly stimulated phosphatase-2Ac and -2Ao even in the absence of cAMP. Thiophosphorylated RII inhibited the dephosphorylation of phosphorylase a by these phosphatases with half-maximum inhibitory concentrations of 0.1-0.36 microM. PMID- 3005039 TI - Quantitative changes in the catalytic and regulatory subunits of nuclear cAMP dependent protein kinases type I and type II during isoproterenol-induced growth of the rat parotid gland. AB - To quantify the cAMP-dependent protein kinases I and II in parotid gland nuclei independent of the enzyme activity, monospecific antisera against their subunits were applied in a sensitive enzyme immunoassay. About 3% of total catalytic subunit in the homogenate was found in the isolated nuclei. During beta-agonist induced proliferation of the parotid gland the nuclear concentration of catalytic and regulatory subunits changed. Related to the number of nuclei, the catalytic subunit and the regulatory subunit RI increased about 3-fold whereas the regulatory subunit RII remained unchanged. PMID- 3005040 TI - Gastric (H+ + K+)-ATPase: modulation of the inhibitory properties of the novel potent antisecretagogue Ro 18-5364 by sulfhydryl reagents and nucleotides. AB - The sulfoxide Ro 18-5364, a potential metabolite of the IND Ro 18-5362, is a powerful inhibitor of gastric mucosal (H+ + K+)-ATPase, decreasing enzymatic activity with an apparent Ki of 0.1 microM. Exposure of Ro 18-5364-treated gastric membranes to dithiothreitol fully restored (H+ + K+)-ATPase activity. ATP protected the enzyme against Ro 18-5364-induced inactivation of enzymatic activity. In addition, Ro 18-5364 inhibited vesicular proton uptake. In proton translocation experiments reduced lipoic acid methyl ester partially restored transport properties. Dithiothreitol and mercaptoethanol were without effect. The results are discussed with respect to the possible location of essential sulfhydryl groups for enzyme activity and proton transport. PMID- 3005041 TI - Biochemical properties of macrophage fractions and their relation to the mechanism of superoxide production. AB - Guinea pig alveolar macrophages are separable by density gradient centrifugation into three subpopulations whose capacity for biological activity (e.g. O2- production and chemotaxis) varies directly with buoyant density [(1983) J. Reticuloendothel. Soc. 33, 157-164]. This study demonstrates that the activity per cell of various other enzymes remains constant among the subpopulations. When normalized for cell volume, enzyme activity diminishes with decreasing buoyant density. Intracellular calcium mobilization, linked to formyl peptide and concanavalin A-stimulated O2- production, similarly diminishes. Formyl peptide receptor distribution and affinity remain constant. Decreased responsiveness of lower density cells is probably due to lower concentration of enzyme(s) involved in the transduction of signal distal to ligand recognition (or binding). PMID- 3005042 TI - Resonance Raman evidence for an exchangeable protein hydrogen associated with the heme a group of cytochrome oxidase. AB - When cytochrome-c oxidase is soaked in D2O, downshifts of the cytochrome a formyl C = O stretching mode are seen in the resonance Raman (RR) spectra (413.1 nm excitation) of both the resting and reduced forms. Other changes observed in the reduced protein RR spectra are consistent with involvement of the cytochrome a formyl group in the deuterium effect. The D2O-induced RR changes are fully developed during 3-5 days incubation, but are incomplete after 1 h. Extraction of the heme a chromophore in deuterated solvents eliminates these changes, implying that the exchangeable proton is on a protein group in the cytochrome a pocket which H-bonds to the heme formyl. The rate of the D2O exchange process is unaffected by enzyme turnover, thus reducing the likelihood that the cytochrome a formyl H-bond is directly involved in the redox-linked mechanism of proton pumping. PMID- 3005043 TI - Tris(picolinato)manganese(II): a chemical model for the mechanism and function of mitochondrial superoxide dismutase. AB - The reaction of HO2. with the allylic groups of lipids initiates their peroxidation and auto-oxidation, and probably represents the most serious biological hazard of O2.- -derived species. The presence of tris(picolinato)manganese(II) [MnII(PA)2(PAH)(H2O)], a model complex for mitochondrial superoxide dismutase, (i) efficiently catalyzes the disproportionation of O2.-, (ii) precludes the formation HO2., and thereby (iii) prevents hydrogen abstraction from allylic and thiol groups. Such protection demonstrates that a primary function of superoxide dismutase is to block the formation of HO2., which is the obligatory intermediate for the nonenzymatic proton-induced disproportionation process. This requires that the primary step for the enzyme-O2.- reaction be kinetically favored and dominant relative to the protonation reaction (HA + O2.-). PMID- 3005044 TI - A chemiluminescent probe with a Cypridina luciferin analog, 2-methyl-6-phenyl-3,7 dihydroimidazo[1,2-a]pyrazin-3-one, specific and sensitive for O2- production in phagocytizing macrophages. AB - When a Cypridina luciferin analog (the title compound) was added to a macrophage suspension in Hank's balanced salt solution (control), the system emitted a weak, but detectable light, which was not altered in the presence of superoxide dismutase. The same system, however, emitted a much stronger light, just after the addition of a trigger, opsonized zymosan. The luminescence was suppressed to the control level in the presence of superoxide dismutase, while it was only slightly influenced, if at all, by NaN3, a scavenger of singlet oxygen and an inhibitor of myeloperoxidase. Some other results obtained also indicate the participation of O2- in the luciferin analog-dependent luminescence in macrophages during phagocytosis. PMID- 3005045 TI - Microsomal membrane subfractionation by a lectin affinity method. AB - Concanavalin A-agarose treatment of rat liver post-mitochondrial supernatant removes a fraction rich in cholesterol and 5'-nucleotidase activity but low in glucose-6-phosphatase. At the same time, radiolabel associated with the cell surface is removed. We interpret these findings as evidence that concanavalin A binds to, and under these circumstances will remove, fragments of plasma membrane present in the microsomal fraction and believe that this may be of use in the gentle, and rapid subfractionation of microsomal membranes. PMID- 3005046 TI - EGF and insulin action in fibroblasts. Evidence that phosphoinositide hydrolysis is not an essential mitogenic signalling pathway. AB - Chinese hamster lung fibroblasts (CHL) arrested in G0 by serum starvation reinitiate DNA synthesis in response to either EGF, thrombin or serum. Arrested cells, prelabelled to equilibrium with [3H]inositol and receiving 20 mM LiCl prior stimulation, released rapidly large amounts of inositol phosphates when stimulated with thrombin or serum. In sharp contrast, EGF alone, or in association with insulin, failed to induce phosphoinositide breakdown at either early or late stages of EGF stimulation or in growing cells in EGF-supplemented serum-free medium. Phospholipase C remained, however, highly activatable by thrombin at all stages of EGF stimulation. Since EGF and thrombin are equally potent mitogens for CHL, we conclude that hydrolysis of polyphosphoinositides is not an exclusive signalling pathway for commitment to DNA replication and cell division. PMID- 3005047 TI - Stable transformation of Drosophila Kc cells to antibiotic resistance with the bacterial neomycin resistance gene. AB - By transfection with a plasmid containing the APH(3') gene under control of the HSV I thymidine kinase promoter, independent series of stably transformed Drosophila cells were established and grown for more than one and a half years under highly selective pressure (2 mg G 418/ml). Analysis of transformed Drosophila cell DNAs shows that the APH(3') gene was integrated into the genome. Neomycin phosphotransferase is constitutively expressed in transformed cells. This efficient selective system by a dominant marker makes it possible to introduce, by cotransfection, any DNA sequence of interest into the genome of cultured Drosophila cells. PMID- 3005049 TI - Acquired immune deficiency syndrome and the fertility clinic. PMID- 3005048 TI - Sequence rearrangements may alter the in vivo superhelicity of recombinant plasmids. AB - Electrophoretic resolution of topoisomers was used to compare the in vivo superhelicity of recombinant plasmids containing a fragment of cDNA for an immunoglobulin light chain, cloned in the two possible orientations into the BamHI site of pBR313 or pBR322. Previously, frequent transpositions of IS1 or IS5 were observed into the sequence upstream to the cloned fragment in recombinants in one orientation [(+) plasmids] but not in recombinants in the opposite, (-) orientation [(1982) Nucleic Acids Res. 10, 4525-4542]. The results of the present analyses show that, on average, (-) plasmids are less negatively supercoiled than (+) plasmids, or pBR322. These results suggest that primary sequence rearrangements in plasmids could affect their in vivo topological state, and consequently, perhaps, their effectiveness as recipients of transposable elements. PMID- 3005050 TI - Comparison of the action of nonoxynol-9 and chlorhexidine on sperm. AB - The effect on sperm motility of nonoxynol-9 chlorhexidine diacetate was compared in semen and cervical mucus. Both compounds had similar spermicidal potency in semen, abolishing sperm motility within 3 minutes at 0.5 mg/ml. When these compounds were allowed to diffuse into mucus, the subsequent survival of sperm in the mucus was different. Restricted penetration and loss of motility occurred rapidly after treatment with 0.1 mg/ml chlorhexidine, whereas sperm survived normally in mucus after prolonged contact with 200 mg/ml chlorhexidine. When the compounds were mixed directly with the mucus before sperm penetration was attempted, chlorhexidine still immobilized sperm, but concentrations of nonoxynol 9 that would be spermicidal in semen had no effect in mucus. PMID- 3005052 TI - Is Provera the ideal progestogen for addition to postmenopausal estrogen therapy? AB - In a dose-ranging study, medroxyprogesterone acetate, 2.5, 5, or 10 mg daily, was given for 12 days of each calendar month to postmenopausal women also receiving conjugated estrogens, 0.625 mg daily, continuously. Endometrial biopsy specimens were taken on the sixth day of combined therapy for histologic, ultrastructural and biochemical evaluation. Medroxyprogesterone acetate induced secretory and ultrastructural changes within the endometrium, but the responses were variable and inconsistent. Suppression of epithelial deoxyribonucleic acid synthesis appeared dose-dependent. The levels of nuclear estradiol receptor, although reduced to within the secretory phase range, were not significantly lower than the values observed during the estrogen-only phase of treatment. Induction of both estradiol and isocitrate dehydrogenase activities was to within the secretory phase ranges, but the magnitude of these responses appeared less than those observed previously with other progestogens. Both morphologically and biochemically, medroxyprogesterone acetate, even at high dosage, produced suboptimal responses. Further studies are required to establish whether this is a dose-related effect. PMID- 3005053 TI - Treatment of persistent trophoblastic tissue after salpingostomy with methotrexate. AB - Salpingostomy has become a common treatment of unruptured ectopic pregnancies. A complication of salpingostomy is persistent trophoblastic tissue. All reported cases to date of salpingostomy with persistent trophoblastic tissue have required salpingectomy. We reported a case of persistent trophoblastic tissue after salpingostomy treated successfully with methotrexate. PMID- 3005051 TI - Lowered levels of bicarbonate in seminal plasma cause the poor sperm motility in human infertile patients. AB - Both the adenylate cyclase activity and the motility of human sperm were stimulated by bicarbonate with the same concentration dependency. The correlation between bicarbonate levels in semen and the motility of sperm from the patients with male infertility was investigated. Bicarbonate in semen was found to originate mainly from the seminal vesicles, and a significant positive correlation was observed between bicarbonate levels and volume of semen. The motility of infertile sperm was also found to correlate positively to the seminal levels of bicarbonate. These results suggest that the lowered levels of bicarbonate in semen are at least in part responsible for the poor sperm motility in infertile patients, as a result of the failure in the activation of sperm adenylate cyclase. PMID- 3005055 TI - [Various ways of assessing the structure of trace reactions of neurons of the medial geniculate body]. AB - Afterdischarges of the medial geniculate body units were recorded in anesthetized cats following different sound stimuli. With traditional PSTH-technique, a periodicity of afterdischarges was found in some neurons whereas others showed diffuse and prolonged changes of their activity. To estimate the diffuse afterdischarges a special method was used. The procedure of eliminating time shift in neuronal responses was employed followed by averaging the individual PSTHs. A consequent diminution of time dispersion of neuronal responses revealed a distinct time structure in the diffuse afterdischarges. PMID- 3005054 TI - [Calmodulin--second messenger. History of research, physiologic role]. PMID- 3005056 TI - [Participation of angiotensin II and acetylcholine in central mechanisms of alcoholic motivation in the rat]. AB - Chemical sensitivity to acetylcholine (Ach) and angiotensin II (A-II) of the neurons from the perifornical area of the posterio-lateral hypothalamus was studied in rats after 30 days of alcoholization with 20% ethanol solution instead of water. The artificially formed alcohol motivation was accompanied by profound changes of the neuronal sensitivity to Ach and A-II. The latter changes were manifested by specific reorganization of time--interval patterns of neuronal activity. Changes of sensitivity to Ach were less obvious and involved prevalence of 30--40 msec intervals in the unit activity. These changes of chemical sensitivity to peptides and classical neurotransmitters may reflect profound changes in the system of intramembranous and intracellular cyclic nucleotides during alcoholization. PMID- 3005057 TI - [Study of the beta-adrenoreceptive function of the erythrocyte membrane of the guinea pig using spectral luminescence analysis]. AB - The binding of DSM fluorescent probe with erythrocyte membranes in conditions of the organism high and low adrenoreactivity was studied in guinea pig. The dependence of DSM binding on the level of adrenoreactivity was shown by probe luminescence in the range of 615 nm. Specific and unspecific types of the DSM bindings with erythrocyte membranes were revealed. In high adrenoreactivity, an increase of specific binding places (Nsp) and a decrease of specific binding constant (Ksp) were found whereas in low adrenoreactivity the Nsp decreased and the Ksp increased. PMID- 3005058 TI - [Activity of various renal enzymes during maximal secretion of xenobiotics]. PMID- 3005059 TI - [Mechanism of inhibition of cardiac activity by the stellate ganglion in the cat and guinea pig]. AB - In acute and chronic experiments on guinea pigs (43) and cats (49), the stellate ganglion inhibition of the heart activity was revealed to be a consequence of excitation of the vagus cholinergic fibers converging with branches of the stellate ganglion on their route to the heart. Alpha-adrenorecptors were shown to exert no noticeable effect on the heart work. Neither the hypothesis of switching of the sympathetic fibers over to intracardiac cholinergic neurons, nor the hypothesis of the cholinergic link in the mechanism of catecholamines release by the sympathetic nerve terminals, were confirmed. The Dayle principle stating that one neuron exerts its efferent affect by means of one transmitter, seems to be justified. PMID- 3005060 TI - [Comparative analysis of changes in intercentral relations of the brain as a result of administration of delta-sleep peptide and ACTH]. AB - Elucidation of the mechanisms of the DSIP effect on the CNS functional state is one of the leading problems of neuroendocrinology. The investigation of DSIP physiological properties as antistress factor in systemic mechanisms of emotional stress is of great interest. The purpose of our studies was to investigate the character of changes in intercentral interrelationships among hypothalamus, limbic system and reticular formation in augmentation of corticosteroids level up to the stressful one. We also studied the character of changes in intercentral interrelationship under the DSIP effect. The latter depends on the initial level of corticosteroids in the blood and is more obvious at the stress level. DSIP, ACTH and steroid hormones facilitate the process of the self-organization of the brain as well as the formation of the current behavioral responses. PMID- 3005061 TI - [Studies on the thyrotropic activity of hCG and its derivatives]. AB - The placenta may be involved in the regulation of the maternal thyroid function during pregnancy. As human chorionic gonadotropin (hCG) is a primary protein from the placenta, we examined the thyrotropic activity of hCG and its derivatives using tissue culture and monolayer cell culture of human thyroid glands. The TSH receptor preparation was made from thyroid tissues, and its binding affinity with hCG and Asialo hCG (As-hCG) was examined. hCG did not bind to TSH receptor, but As-hCG did. In the experiment with tissue culture, TSH, hCG, hCG alpha, beta subunit and As-hCG were added to the culture medium. Secretions of L-thyroxine (T4) and L-triiodothyronine (T3) in the culture medium were measured at regular time intervals. While TSH showed increases of T4 and T3 secretion, other hCG derivatives did not show any differences from the control values. When TSH and hCG were added together, the secretion of thyroid hormone was the same as that obtained by TSH single administration. On the other hand, the secretion of T4 and T3 was inhibited with co-administration of TSH and As-hCG. Only TSH and none of the other hCG derivatives showed the dose-dependent stimulation of T4 and T3 secretion. A similar experiment was carried out in a monolayer cell culture obtained by trypsin treatment of human thyroid tissue. The secretions of cyclic AMP (c-AMP), T4 and T3 were measured. As in the previous tissue experiment, the thyrotropic activity of TSH was not modified by hCG, but the secretions of T4, T3 and especially c-AMP were inhibited by co-administration of As-hCG and TSH. These findings suggest that hCG and its subunits do not show thyrotropic activity in human thyroid glands, but As-hCG acts as an antagonist of TSH. PMID- 3005062 TI - [Perfusion of monolayer-cultured B cells of the neonatal rat pancreas--effect of 2-deoxy-2-fluoroglucose]. AB - The effects of 2-deoxy-2-fluoroglucose in promoting the function of neonatal rat pancreatic B cells in vitro are presented. Functional studies of insulin secretion were carried out by perifusion procedures. At day 0 of culture, B cells showed a minimal monophasic insulin secretion in response to a 16.7 mM single dose of glucose, whereas in the presence of 10 microM forskolin or 1 mM 3 isobutyl-1-methylxanthine, the same dose of glucose stimulated insulin secretion in a biphasic fashion. In contrast, after 7 days of culture in medium supplemented with 1 mM 2-deoxy-2-fluoroglucose, B cells showed an adult-like biphasic pattern regardless of the presence of forskolin or 3-isobutyl-1 methylxanthine. In addition, the stimulatory effect of either leucine or 2 ketoisocaproate was also significantly increased, when compared to that of B cells at day 0. Furthermore, when exposed to a linear gradient stimulation by glucose, these competent B cells secreted insulin in a dose-dependent fashion. Moreover, in cultures supplemented with 2-deoxy-2-fluoroglucose the basal or stimulating levels of cAMP were about 20-fold higher than at day 0. In conclusion, the data presented here demonstrate that the function of neonatal B cells matures during culture, and suggest that this effect of 2-deoxy-2 fluoroglucose may be mediated by activation of either adenylate cyclase or catabolic enzymes of amino acids in B cells. PMID- 3005063 TI - [Clinical significance of serum angiotensin I-converting enzyme in essential hypertension]. AB - The present study was designed to clarify the role of serum angiotensin I converting enzyme (ACE) in the occurrence and maintenance of hypertension in essential hypertension (EH). For this purpose, following experiments were carried out: 1) Correlations between serum ACE activity and renin activity (PRA), aldosterone concentration (PAC) and bradykinin concentration (PBC) in plasma, and blood pressure (BP) as well as serum creatinine levels. 2) Circadian rhythm of serum ACE activity. and 3) Effect of furosemide, upright posture, both furosemide and upright posture, propranolol, indomethacin, 9 alpha-fluorocortisol or angiotensin II (A-II) on the serum ACE activity, PRA, PAC and circulating plasma volume (CPV). The following results were obtained: The serum ACE activity was 30.2 +/- 5.0 U/ml (means +/- SD) in EH as a group, which was significantly higher than that (27.3 +/- 3.9 U/ml) in age matched normotensive subjects (NT) (p less than 0.001). While there was no significant difference in the enzyme activity between low-renin EH (LREH) and NT, a significant difference was found between normal- (NREH) or high-renin EH (NREH) and NT (p less than 0.05 for NREH, p less than 0.01 for HREH). A negative correlation was observed between enzyme activity and age in EH (r = -0.221, 0.05 less than p less than 0.10) as well as in NT (r = -0.306, p less than 0.05). No significant relationships were observed between enzyme activity and BP in either EH or NT. There was a significant positive correlation between enzyme activity and PRA in NT. (r = 0.501, p less than 0.001), NREH (r = 0.658, p less than 0.001) and HREH (r = 0.695, p less than 0.001). However, no significant relationship was found between them in LREH. The enzyme activity was significantly correlated to PAC in NT (r = 0.368, p less than 0.01), NREH (r = 0.567, p less than 0.001) and HREH (r = 0.529, p less than 0.01), but not in LREH. Although no significant correlation was observed between enzyme activity and PBC in NT, NREH and HREH, a significant relationship was found in LREH (r = -0.460, 0.05 less than p less than 0.10). The enzyme activity was not related to serum creatinine levels in EH as well as in NT. In NT, the serum levels of ACE activity reached a maximum values at 6:00 a.m. or 9:00 a.m., and gradually decreased between 6:00 p.m. and 3:00 a.m. An almost similar circadian rhythm of enzyme activity was found in EH.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3005064 TI - Epidermodysplasia verruciformis associated with Bowen's carcinoma, B lymphocytopenia and decreased immune functions. AB - A case of epidermodysplasia verruciformis associated with Bowen's carcinoma, persistent B lymphocytopenia and decreased immune functions is reported. Human papilloma virus 5 (HPV-5) DNA was shown to be associated with the DNA from the tumor tissue by Southern blot hybridization using P-labeled HPV DNA sequences cloned on plasmid vectors. The associated Bowen's carcinoma in our case may be caused by multiple-factor relationships which include (a) an oncogenic potential of infected virus; (b) an inherited abnormality in immune function, and (c) the decreased immune function resulting from the viral infection itself. A marked B lymphocytopenia appears to be associated with persistent viral infection. PMID- 3005065 TI - Magnesium trisilicate induced fixed drug eruptions. PMID- 3005066 TI - Identification and measurement of D-glycero D-ido octulose 1,8-bisphosphate: D altro-heptulose 7-phosphotransferase enzyme in tissues with L-type pentose phosphate pathway activity. AB - The enzyme D-glycero D-ido octulose 1,8-bisphosphate:D-altro-heptulose 7 phosphotransferase (abbreviated to phosphotransferase, PT) catalyses the transfer of the phosphate ester group at C-1 between altro-heptulose (sedoheptulose) and octulose phosphate intermediates of the L-type pentose pathway. Using synthetically prepared and 14C-labelled octulose mono- and bisphosphates, two methods are described for the measurement of the catalytic capacity of the PT reaction operating in both the "forward" and "reverse" modes of L-type pentose pathway operation. PT activity was found in normal, regenerating and foetal rat liver, rat heart, rat epididymal fat pad, rat kidney, brain and skeletal muscle, extracts of C. fusca, pea leaf and a variety of tumour tissues. The highest activity of the enzyme was found in the neoplasms. The Michaelian kinetic constants, temperature and pH optima for the reaction of the enzyme from rat liver together with an assortment of its substrate specificities have been determined. Vanadate anion was found to inhibit the enzyme and the pattern of inhibition suggests that the PT may act by a sequential mechanism. Neither arabinose 5-phosphate nor inorganic phosphate showed any effect on the catalytic activity of the PT enzyme in liver. PMID- 3005067 TI - Polypeptide components of bovine heart cytochrome c oxidase. Cross identifications in two high-resolution polyacrylamide-gel electrophoresis systems. AB - Two-dimensional polyacrylamide-gel electrophoresis has been used to correlate polypeptide components of bovine heart cytochrome c oxidase that are resolved by two high resolution systems. The systems utilise chloral hydrate (2,2,2 trichloroethane-1,1-diol), which resolves fifteen components, and sodium dodecyl sulphate and urea, which resolves thirteen components. Seven components have been isolated and identified from their amino acid compositions in terms of polypeptides for which the amino acid sequence is known. Full resolution of all components present in this enzyme cannot be accomplished using any single dimension system currently available. PMID- 3005068 TI - Interactions between ATP and cholesterol side-chain cleavage in mitochondria isolated from superovulated rat ovaries. AB - ATP stimulated the rate of [4-14C]cholesterol side-chain cleavage in mitochondria isolated from superovulated rat ovaries. The effect of ATP was apparently similar to the stimulatory effect of choriogonadotropin on mitochondrial [4 14C]cholesterol utilization. Enhancement of the rate of steroidogenesis by ATP and choriogonadotropin were not additive. ATP seemed to promote both cholesterol uptake into the inner mitochondrial membrane and the supply of electrons for [4 14C]cholesterol utilization from both endogenous substrate and succinate. PMID- 3005069 TI - Activated murine alpha-globin gene is not preferentially associated with the nuclear matrix. AB - The association of the murine alpha-globin gene with the nuclear matrix was studied in three different states of the gene: inactive (EAT cells), potentially active (MEL cells) and active (induced MEL cells). When "native" nuclei were digested with DNase I it was found that the nuclear matrix was not enriched in alpha-globin DNA sequences in all three different types of cells. A nuclease hypersensitive site in the 5'-flanking region of the alpha-globin gene was detected in the induced MEL-cells. PMID- 3005070 TI - Measurement and activity of cytosolic deoxyribonucleoside-activated nucleotidase in various cell types from rat liver and spleen. AB - The enzyme activity was measured in hepatocytes, Kupffer cells, endothelial cells and spleen cells. Hepatocytes showed proportionality between enzyme activity and cytosol concentration, but with Kupffer cells, endothelial cells and spleen cells the specific activity decreased with decreasing cytosol concentration when the amount of cytosol protein in 250 microliters incubation mixture was below 80, 60 and 20 micrograms, respectively. The specific activities in hepatocytes, Kupffer cells, endothelial cells and spleen cells were 2, 16, 18 and 115 nmol/min per mg of cytosol protein, respectively. PMID- 3005071 TI - An ADPase from sarcoplasmic reticulum of rabbit muscle cleaves ADP bound to F actin. AB - We have found that sarcoplasmic reticulum from rabbit muscle contains an ADPase which cleaves ADP bound to F-actin. The interaction is not of the simple Michaelis-Menten type, the order of the reaction being larger than the first. A possible explanation of this behaviour could be that ADPase binds to two adjacent actin monomers with a preferred orientation thus cleaving preferentially the nucleotide of one of the two monomers. PMID- 3005072 TI - The proteolytic system involving calpains. PMID- 3005073 TI - Recent advances in high-performance liquid affinity chromatography columns. PMID- 3005074 TI - Recent advances in automated sample preparation of biological materials for high performance liquid chromatographic analysis. PMID- 3005075 TI - Chemical characterization of lung surfactant apoproteins: amino acid composition, N-terminal sequence and enzymic digestion. PMID- 3005076 TI - Isolation of the gamma-aminobutyric acid/benzodiazepine receptor. PMID- 3005077 TI - Monoclonal antibodies as tools for the study of membrane receptors. PMID- 3005078 TI - Receptor-dependent generation of intracellular signals from inositol phospholipids in parotid gland and brain. PMID- 3005079 TI - Functional control of the delta-opiate receptor by the inhibitory guanine nucleotide-binding protein. PMID- 3005080 TI - Molecular characterization of a corticotropin (ACTH) receptor. AB - We have used a new methodology to generate a monospecific antiserum to the corticotropin (ACTH) receptor on mouse Y-1 adrenal cells. Using immunoaffinity chromatography the ACTH receptor was purified, and the molecular structure and 125I-ACTH binding characteristics were determined. A molecular weight (Mr) of 225 000 was determined for the complete ACTH receptor as analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The receptor was composed of 4 subunits with Mr 83 000, 64 000, 52 000 and 22 000. The 83 and 52 kDa subunits were disulfide linked and non-covalently associated with the 64 and 22 kDa subunits. The ability to specifically bind 125I-ACTH was localized to the 83 kDa subunit. The purified receptor possessed binding affinities of 3.4 X 10(10) M-1 and 1.0 X 10(9) M-1 as determined by Scatchard analysis. PMID- 3005082 TI - Stereospecific transport of triiodothyronine to cytoplasm and nucleus in GH1 cells. AB - We have recently demonstrated substantial stereospecific nuclear/cytosolic free triiodothyronine (T3) gradients within T3 responsive rat tissues in situ. These studies have now been extended to examine T3 transport in a rat pituitary tumor cell line, GH1. L-T3 had a 7.6-fold higher affinity for the nuclear receptor when assayed in whole cell incubations in comparison to isolated nuclei, though D-T3 affinity was not altered under these conditions. An apparently higher number of receptors for D-T3 was explained by racemic contamination of the isotopes used. Measurement of free hormone concentration ratios for both enantiomers revealed a small step up from medium to cytosol for L-T3 (1.65) but a reverse ratio for D-T3 (0.46). The nuclei were able to concentrate both enantiomers, though stereospecificity was maintained (nucleus/cytosol, L-T3, 4.5, D-T3 1.7). Transport of L-T3 at both boundaries could be inhibited by monodansylcadaverine. Thus, stereospecific transport functions are found within GH1 cells, though the magnitude of the free nucleus/cytosol gradient is reduced from those seen in rat tissues in situ. PMID- 3005081 TI - Vanadium inhibits ACTH-mediated but not cyclic AMP-dependent adrenal steroidogenesis. AB - We have studied the effect of vanadium on steroidogenesis using dispersed cells from rat adrenals. It was found that when the cells were stimulated by ACTH both vanadate and vanadyl sulfate inhibited adrenal steroidogenesis. In contrast, when steroidogenesis was supported by cAMP analogues, no inhibition was observed. The inhibitory action of vanadium compounds was also seen when cholera toxin or forskolin was used as a stimulant instead of ACTH, indicating that vanadate does not act on the ACTH-receptor protein. When adrenal cells were stimulated by ACTH, cholera toxin- or forskolin-supported cAMP levels were diminished equally by vanadate. In addition, the mitochondrial steroid hydroxylation per se was not inhibited by vanadium compounds using either endogenous or exogenous steroid substrates. Based on these results, it is concluded that the site of inhibition by vanadium is located in the vicinity of the guanine nucleotide-binding protein and the catalytic unit of adenylate cyclase. PMID- 3005083 TI - Modulation of rat testicular LH/hCG receptors by membrane lipid fluidity. AB - The specific binding of [125I]hCG to rat testicular membrane preparations was investigated as a function of membrane fluidity changed by lipids. Membrane fluidity was measured either by fluorescence depolarization of diphenylhexatriene or ESR spectra of I(1,14), I(12,3) and CAT 16 incorporated into the membrane. Incubation of membrane with cholesteryl-hemisuccinate increased both the rigidity of membrane lipids and the specific binding of [125I]hCG. A similar rigidifying action of saturated fatty acids was, however, not accompanied by increased accessibility of LH/hCG receptors. Treatment of testicular membranes with unsaturated fatty acids enhanced membrane fluidity but specific binding of the gonadotropin disappeared. In spite of the increase of LH/hCG receptors in cell membranes treated with cholesteryl-hemisuccinate, Leydig cells showed decreased sensitivity to cAMP response to LH stimulation. The results indicate that newly exposed LH/hCG receptors are not coupled with the adenylate cyclase system. PMID- 3005084 TI - Occupied and unoccupied FSH receptors in follicles of cyclic, hypophysectomized or hypophysectomized/gonadotropin-treated hamsters. AB - Occupied and unoccupied FSH receptors were measured in follicles from intact cyclic hamsters or in animals hypophysectomized on day 1 (= estrus) which were untreated or given ovine FSH on day 1 plus LH on days 1-4 or FHS and LH plus 1 mg progesterone on days 1-4. Intact hamsters, from days 2-4, the number of occupied and unoccupied FSH receptors and receptor affinity was unchanged in antral follicles. For the untreated hypophysectomized hamsters, serum FSH was undetectable beyond day 3 but receptor affinity increased on day 2 and the number of occupied FSH receptors was still very high on day 4. Thus, in the hypophysectomized untreated hamster the ovary is not hormonally deprived - at least up until day 4. Treatment of the hypophysectomized hamsters with FSH-LH restored all follicular values of levels of intact hamsters on days 2 and 3. On day 4, although serum FSH was undetectable since day 3 the number of occupied FSH receptors was 70% of control values. The FSH-LH regimen developed by day 4 large antral follicles in the hypophysectomized hamsters. Collectively, the experiments demonstrate that in intact hamsters as a result of the prolonged peak of serum FSH on day 1 FSH binds avidly to receptors in granulosa cells and persists until day 4. Thus, the follicles in the hypophysectomized groups develop in response to exogenous LH but with hamster FSH still present, bound to granulosa cells. PMID- 3005085 TI - Characterization of luteinizing hormone-releasing hormone receptor binding in rat pituitary cell monolayer cultures; influence of intercellular communication. AB - Receptors for the luteinizing hormone-releasing hormone (LHRH) were characterized in rat pituitary cells cultured for 3 days as monolayers on coverslips using 125I [D-Ala6-Pro9-LHRH-NEt] as the labeled ligand. The monolayers were left intact during the binding assay. Specific binding displayed the various characteristics of binding to the physiological LHRH receptor. Various kinetic data corresponded to those reported previously. However, in these cultured cells, in which binding was tested in a physiological medium, the dose response of competition for binding by LHRH agonists ranged over a smaller concentration range (less than 2 orders of magnitude) than that by LHRH antagonists. In a cation-free buffer competition curves of agonists and antagonists were parallel but the apparent dissociation constant was lower than in the physiological medium. In cultures of pituitary cell populations separated by unit gravity sedimentation, the specific binding increased with the proportional number of gonadotrophs in the various populations. However, when the gonadotroph-richest population (approximately equal to 70% gonadotrophs) was cultured after recombination with gonadotroph-poor populations, binding capacity significantly increased. Microscopic examinations suggested that this phenomenon was the consequence of disrupting cellular contacts among gonadotrophs. It is concluded that certain characteristics of LHRH receptors tested on cells in a tissue-like organization and in a physiological environment are different from those reported previously in disrupted cells or monodispersed cell suspensions and that intercellular communication is an important factor controlling LHRH receptors. PMID- 3005086 TI - Studies on dissociation of mouse prolactin from mouse hepatic receptors. AB - The influence of pH, temperature, ethylene glycol, urea, chaotropic anions and excess unlabelled secreted mouse prolactin (smPRL) on the dissociation kinetics of 125I-iodosmPRL from mouse hepatic receptors was investigated. The destabilization of smPRL-receptor complexes by chaotropic anions followed the typical trend of the Hofmeister series: I- greater than Br- greater than Cl- greater than F-. Increasing the temperature of the dissociation reaction from 8 degrees C to 23 degrees C and 30 degrees C caused partial dissociation of 125I iodosmPRL-receptor complexes. Dissociation of 125I-iodosmPRL from mouse hepatic receptors was pH dependent, with the slowest rate of dissociation occurring at pH 8 and the fastest rate of dissociation occurring at pH 5 and 6. Both ethylene glycol and urea accelerated the rate of dissociation of 125I-iodosmPRL from mouse hepatic receptors in a concentration-dependent manner. Dissociation of 125I iodosmPRL from mouse hepatic receptors was 6-fold faster in the presence of excess unlabelled smPRL than in its absence. The results of these investigations suggest that both protonation/de-protonation reactions and hydrophobic interactions play important roles in stabilizing the smPRL-receptor complex. In addition, they suggest that cooperative interactions may be involved in the binding of smPRL to mouse hepatic receptors. PMID- 3005088 TI - Distribution of epidermal growth factor receptors in the rat ovary. AB - The distribution of epidermal growth factor (EGF) was studied in the ovary using light microscope radioautography which was performed at different time intervals (2-60 min) after intravenous (i.v.) injection of [125I]EGF into adult rat at random stages of the estrous cycle and also after topical localization of iodinated EGF on slide-mounted frozen ovarian sections. After i.v. injection of label, the labeling was mostly observed in the theca interna cells of secondary, preovulatory and atretic follicles and luteal cells of corpus luteum. Primordial and primary follicles did not show any significant labeling. The time-course study performed on luteal cells showed that, 2 min after injection, most silver grains were found at the periphery of the cells. At the 10 min time interval, silver grains were found both at the periphery and over the cytoplasm of these cells. The number of grains was very reduced over the cytoplasm at the 60 min time interval. In the in vitro study, a positive radioautographic reaction was seen in the same cellular elements as found in vivo, with the additional labeling of the granulosa cells of growing and preovulatory follicles. Control experiments indicated that the radioautographic labeling was due to specific interaction of [125I]EGF with its receptor. These results clearly indicate that EGF binding sites are present in luteal, thecal and granulosa cells, and provide support for the inhibitory and stimulatory actions of EGF on different parameters of ovarian cells. PMID- 3005087 TI - Formation of parathormone 8-34 by cathepsin-D digestion of parathormone and its efficacy as a hormone antagonist. AB - It previously has been shown that digestion of bovine parathormone (bPTH) with cathepsin-D results in rapid cleavage of the hormone between Phe34 and Val35 yielding PTH(1-34) and PTH(35-84). Since bPTH also contains a Phe at residue 7 we have conducted additional studies to determine whether cleavage at this position could occur. We have found that following longer incubation periods of hormone and enzyme, 2 additional peptides are generated; PTH(8-34) and PTH(1-7). Time course studies demonstrated that these 2 fragments are formed from the (1-34) peptide generated through the initial cleavage at Phe34-Val35 of PTH. The identification of the bPTH(8-34) was accomplished through amino acid analysis and N-terminal sequencing. bPTH(8-34) behaved as a PTH antagonist in an in vitro mouse calvarial bone resorption assay. Although bPTH(8-34) did not affect the PTH stimulated cAMP response when added simultaneously with PTH, preincubation of bone cells with this peptide caused desensitization of the PTH-stimulated cAMP response. PMID- 3005089 TI - Morphological identification of sperm receptors above egg microvilli in the polychaete, Neanthes japonica. AB - A fine-structural study of sperm-egg interactions in the polychaete Neanthes japonica was carried out. Unfertilized eggs are surrounded by a chorion 0.6-0.7 micrometers thick. Oocyte microvilli are inserted into the inner layer of the chorion. The outer layers of the chorion are opened just above the tips of the microvilli, where a membrane vesicle (microvillus tip vesicle, about 0.2 micrometers in diameter) plugs the chorion's opening. During fertilization, the acrosomal process of the sperm fuses with an egg microvillus within 1 min of insemination. All the microvillus tip vesicles disappear from the chorion surface within 5 min of insemination. When eggs, which are prefixed with glutaraldehyde, are inseminated, numerous sperm undergoing the acrosome reactions attach to the eggs. In the majority of these sperm, the tip of acrosomal process which is coated with the acrosomal content, adhere to a microvillus tip vesicle. These findings suggest that the microvillus tip vesicle serves as a sperm receptor, which induces the acrosome reactions and adhere to the sperm acrosomal process. The adhesion of the acrosomal process to the microvillus tip vesicle seems to be a prerequisite event for its fusion with the microvillus. PMID- 3005090 TI - Spontaneous release of transmitter from the growth cones of Xenopus neurons in vitro: the influence of Ca2+ and Mg2+ ions. AB - To determine whether spontaneous release of transmitter from the growth cones of neurons exhibits properties similar to the spontaneous release which occurs from the neurons at the neuromuscular junction, release of transmitter from the growth cones of Xenopus neurons in culture was monitored in salines containing varying calcium and magnesium concentrations. Release was monitored by use of an outside out piece of muscle membrane attached to a patch clamp electrode. Spontaneous release of transmitter from the growth cones in standard saline (2 mM CaCl2, 1 mM MgCl2) produces clusters of single-channel openings in the muscle membrane. Clusters are seen to consist of two types: a series of high-frequency channel openings, called "bursts," and clusters of low-frequency channel openings called "singles." The bursts were identified and examined for their possible relationship to MEPP-producing release, and the singles were identified and examined for their possible relationship to "leak" release of the neuromuscular junction. When the external saline contains high calcium (10 mM CaCl2, 1 mM MgCl2) or high magnesium (2 mM CaCl2, 9 mM MgCl2), the frequencies of both "bursts" and "singles" was greatly reduced. This reduction in release persists if the neurons are grown in the high-calcium or high-magnesium solutions. When the saline is a low-calcium solution (0 mM CaCl2, 3 mM MgCl2) the growth cones release transmitter at rates similar to those from standard saline. These results indicate that although the spontaneous release from the growth cone shares one characteristic with the leak release, neither the burst nor the singles release from the growth cones share exact relationship with either the MEPP producing release or the leak release. This suggests that further development of the mechanisms for spontaneous release of neurotransmitter occurs after nerve-muscle contact. PMID- 3005091 TI - Progesterone and cAMP-dependent protein kinase regulate in vivo the level of phosphorylation of two proteins (Mr 20,000 and Mr 32,000) in Xenopus oocytes. AB - The [32P]phosphoproteins and [35S]thiophosphoproteins were analyzed by electrophoresis and autoradiography after microinjection of [gamma-32P]ATP or of [35S]ATP-gamma-S into living Xenopus oocytes. The level of 32P incorporation into a 20-kDA protein was decreased following progesterone treatment (between 1 and 2 hr). This 20-kDa protein was partially thiophosphorylated in vivo by [35S]ATP gamma-S. Furthermore it was found that this phosphoprotein was partially purified by TCA (1%) extraction and heat treatment. Microinjection of the C-subunit of cAMP-dependent protein kinase (0.6 to 1.2 pmole) inhibited maturation and provoked an increase in the level of phosphorylation of the 20-kDa protein and of a 32-kDa protein, indicating that both proteins were in vivo substrates (directly or indirectly) for cAMP-dependent protein kinase. When inhibitor-1 of protein phosphatase-1 was microinjected (5 to 10 pmole per oocyte) meiotic maturation was inhibited and the level of phosphorylation of the 32-kDa protein was increased; the same result was obtained following ATP-gamma-S (1 mM) microinjection. Altogether these results suggest that a 20-kDa phosphoprotein, whose level of phosphorylation is decreased by progesterone, could be involved in the regulation of maturation by lowering the level phosphorylation of a 32-kDa phosphoprotein. An attractive hypothesis would be that the 20-kDa phosphoprotein is an inhibitor of protein phosphatase-1. PMID- 3005092 TI - Cell-cell contact and cAMP regulate the expression of a UDP glucose pyrophosphorylase gene of Dictyostelium discoideum. AB - UDP glucose pyrophosphorylase (UDPGP) (EC.2.7.7.9) is a developmentally regulated enzyme of Dictyostelium discoideum. Two polypeptides of UDPGP are translated from Dictyostelium mRNA. Recently we isolated a cDNA clone which encodes one of the UDPGP polypeptides (B. R. Fishel, J. A. Ragheb, A. Rajkovic, B. Haribabu, C. W. Schweinfest, and R. P. Dottin (1985). Dev. Biol. 110, 369-381). By hybridization with the cDNA and by in vitro translation and immunoprecipitation, we examined the effect of cell-cell contact and cAMP on the regulation of UDPGP expression. Disaggregation of slugs resulted in a rapid loss of UDPGP mRNA. Addition of cAMP to these cells resulted in increased levels of UDPGP mRNA, though not to the same extent as seen during normal development. The two UDPGP polypeptides observed in vitro are coordinately regulated. Unaggregated cells, starved and shaken rapidly in suspension, did not show UDPGP mRNA accumulation. However, addition of cAMP to these cells caused UDPGP induction, suggesting that the requirement for cell-cell contact could be bypassed in part by cAMP addition. PMID- 3005094 TI - Cold exposure reverses the diabetogenic effects of high-fat feeding. AB - Long-term cafeteria feeding, cold exposure, and the combination of treatments increased energy intake in female Wistar rats by 25%, 113%, and 150%, respectively, in comparison with controls (P less than 0.01). Although cafeteria feeding at room temperature markedly increased the insulin response to an intravenous glucose tolerance test (IVGTT), glucose tolerance was deteriorated (P less than 0.01). In contrast, cold exposure significantly improved glucose tolerance in the presence of a reduced insulin response in Purina- and cafeteria fed animals. Moreover, cold exposure also decreased body weight gain and increased brown adipose tissue mass, total cytochrome-oxidase activity, and cellularity by approximately 600-800%. The results suggest that cold exposure enhances insulin sensitivity of peripheral tissues, whereas hyperphagia on a high fat, low-protein diet leads to insulin resistance. In addition, the results demonstrate that prolonged stimulation of energy expenditure by cold exposure not only reverses the diabetogenic effects of cafeteria feeding but also improves glucose tolerance. This phenomenon could result from a combination of two factors: (1) a cold-induced prevention of obesity; and (2) an enhanced disposal of circulating glucose into peripheral tissues, including brown adipose tissue. PMID- 3005093 TI - Putative hypothalamic glucoreceptors play no essential role in the response to moderate hypoglycemia. AB - The response to insulin-induced hypoglycemia includes increased plasma levels of glucagon, epinephrine, norepinephrine, cortisol, and growth hormone. This integrated response is thought to be mediated via sympathetic afferent pathways emanating from the ventromedial hypothalamus. However, the precise loci of the receptors that sense glucopenia are not known. In this study, we investigated the importance of putative forebrain glucoreceptors to the systemic response to hypoglycemia. Three protocols were performed. Protocol 1: the systemic response was observed in conscious dogs to hypoglycemia induced by infusion of insulin at a high rate (150 mU/min). Protocol 2: the effect of concomitant bilateral, intracarotid glucose infusion on the response to intravenous insulin was examined. Intracarotid glucose infusion rates were chosen to yield central euglycemia in the face of systemic hypoglycemia. Protocol 3: as a control for protocol 2, glucose was infused at low rates into the systemic circulation, yielding hypoglycemia in both central and systemic blood. When insulin was infused at 150 mU/min, without glucose replacement (protocol 1; N = 6), plasma insulin increased from 14 +/- 3 to 335 +/- 35 microU/ml at 60 min (P less than 0.001). Glucose fell from basal (104 +/- 3 mg/dl) to 38 +/- 3 mg/dl (P less than 0.001). Significant increases were observed in epinephrine (basal, B: 63 +/- 8; steady state, SS: 1762 +/- 582 pg/ml; P less than 0.007), norepinephrine (B: 209 +/- 33, SS: 650 +/- 133 pg/ml; P less than 0.01), and glucagon (B: 256 +/- 35, SS: 467 +/- 35 pg/ml; P less than 0.03). In addition, endogenous glucose production (Ra) increased from 72 +/- 4 to 108 +/- 9 mg/min (P less than 0.02) despite frank hyperinsulinemia. Infusion glucose into the carotid arteries at 204 +/- 10 mg/min (protocol 2; N = 7) during a 4-h systemic insulin infusion was sufficient to prevent jugular hypoglycemia [jugular glucose, Gj, B: 100 +/- 3, SS (90-160 min): 101 +/- 3 mg/dl; P greater than 0.70], but not peripheral hypoglycemia (Gp, B: 102 +/- 2, SS: 55 +/- 3 mg/dl; P less than 0.001). Despite carotid glucose replacement, counterregulatory responses were still observed in epinephrine (B: 98 +/- 14, SS: 466 +/- 127 pg/ml; P less than 0.04) and norepinephrine (B: 213 +/- 19, SS: 474 +/- 133 pg/ml; P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3005095 TI - Mitogenic activity and receptor reactivity of hybrid molecules containing portions of the insulin-like growth factor I (IGF-I), IGF-II, and insulin molecules. AB - Insulin and the insulin-like growth factors IGF-I and IGF-II are thought to exert their mitogenic effects in cultured chick embryo fibroblasts and human skin fibroblasts via IGF receptors rather than via insulin receptors. These effects appear to be mediated by the type I subtype of IGF receptor, which is structurally similar to the insulin receptor and exhibits significant cross reactivity with insulin. As a first step in our long-range goal of defining those features of the IGF-I and IGF-II molecules that confer enhanced mitogenic activity and reactivity with these mitogenic type I IGF receptors, we have prepared two hybrid insulin-IGF molecules and examined their mitogenic and binding activities: (1) A27-insulin, containing an elongated 27-residue A-chain (in which the 6-residue D-domain of IGF-II was added to the carboxy-terminus of the 21-residue A-chain of insulin) combined with the B-chain of insulin; and (2) A insulin-B IGF-1, containing the A-chain of insulin and the synthetic 30-residue B-domain of IGF-I. Both hybrid molecules stimulated DNA synthesis and inhibited 125I-IGF-I binding to type I IGF receptors in both chick embryo and human fibroblast cultures. A27-insulin had considerably greater mitogenic potency and binding potency than A insulin-B IGF-I. Neither hybrid molecule was more potent in these assays than insulin, indicating that the presence of D IGF-II or B IGF-I by itself was not sufficient to increase the mitogenic potency of insulin in fibroblasts. By contrast, A insulin-B IGF-I showed enhanced reactivity with an antiserum to IGF-I. A27-insulin retained significant insulin-like metabolic activity despite the presence of the D-domain of IGF-II. PMID- 3005096 TI - Effect of clonidine on platelet alpha 2-adrenoreceptors and plasma norepinephrine of children with Tourette syndrome. AB - Clonidine, an alpha 2-adrenoreceptor agonist, is useful for treating some patients with Tourette syndrome, and it has been suggested that their noradrenergic receptors are 'subsensitive'. The authors measured plasma norepinephrine and specific binding of 3H-clonidine and 3H-yohimbine, an alpha 2 adrenoreceptor antagonist, to receptors on platelet membranes from children with Tourette syndrome. Before clonidine treatment, plasma norepinephrine, the maximum number of binding sites and the dissociation constants for both ligands were the same as for the controls. After two weeks of treatment there was little clinical improvement, but the number of binding sites for 3H-yohimbine decreased and plasma norepinephrine also decreased in four of the five patients. Over the next six months all five patients continued to improve clinically, but both indices of noradrenergic activity returned towards baseline values. The data suggest that clonidine's action may be independent of its prominent effects on alpha 2 adrenergic receptors and norepinephrine release. PMID- 3005097 TI - Restriction fragment length polymorphism of the insulin gene region in Japanese diabetic and non-diabetic subjects. AB - A polymorphic locus flanking the 5' end of the insulin gene was studied in 154 unrelated Japanese diabetic and nondiabetic subjects. A predominance of the small allele was found with the following frequency: of 64 nondiabetic subjects, only 3 of 128 alleles were of the large class (2%); none of 78 alleles were of the large class in 39 Type 1 (insulin-dependent) diabetic subjects, and 4 of 102 alleles (4%) were of the large class in 51 Type 2 (non-insulin-dependent) diabetic subjects. The very low frequency of large allele may relate to the lower prevalence of atherosclerosis in Japanese. However, this possibility requires further examination. PMID- 3005098 TI - Glucagon-like peptide-1 does not have a role in hepatic carbohydrate metabolism. AB - Glucagon-like peptide-1 does not have specific, high-affinity receptors on rat liver membranes, does not displace glucagon from glucagon receptors on these membranes and does not stimulate the production of cyclic AMP by isolated rat hepatocytes. In the presence of glucagon, high concentrations of glucagon-like peptide-1 do not significantly alter the production of cyclic AMP. Thus, glucagon like peptide-1 appears unlikely to have a direct action on hepatic carbohydrate metabolism. PMID- 3005099 TI - Toxicity and carcinogenicity of chlorodibromomethane in Fischer 344/N rats and B6C3F1 mice. AB - Chlorodibromomethane, a trihalomethane found in water supplies after chlorination, was administered by gavage in corn oil to male and female Fischer 344/N rats and B6C3F1 mice in toxicity studies of 13 weeks and 2 years duration. Doses used in the 13-week study were 0, 15, 30, 60, 125, and 250 mg/kg in rats and mice. At 250 mg/kg, hepatic and renal toxicity was produced in male and female rats and male mice, and mortality was increased in male and female rats. In the chronic study, male and female rats were administered the chemical at 0, 40, and 80 mg/kg. No adverse effects on survival in treated rats were observed. Male rats receiving 80 mg/kg had reduced body weight gains relative to controls. Fatty change and cytoplasmic changes were seen in the liver of treated male and female rats. The male and female mice in the chronic study received doses of 0, 50, and 100 mg/kg of chemical. A dosing accident rendered the number of low-dose male mice inadequate for statistical analysis. High-dose male mice had reduced survival relative to controls. Survival was similar in dosed and control female mice. Dosed male and high-dose female mice had reduced body weight relative to controls. Non-neoplastic hepatic lesions were seen in treated male mice (necrosis and hepatocytomegaly) and in treated female mice (calcification and fatty change); nephrosis was seen in treated male mice. The incidence of hepatocellular adenoma and carcinoma (combined) was increased in treated female mice, but only marginally so in treated male mice. Chlorodibromomethane, like chloroform, is toxic to liver and kidneys, and like chloroform induces hepatocellular tumors in mice. PMID- 3005101 TI - Hormonal consequences of organophosphate poisoning. AB - The hormonal changes were estimated after poisoning with an extremely toxic organophosphate, soman (pinacolyl methylphosphonofluoridate). Soman produced a significant (p less than or equal to 0.05) increase in serum corticosterone, thyroxine, and triidothyronine concentrations at 3, 6, and 9 hr after poisoning. However, by 22 hr the levels were not significantly different from control. Plasma ACTH levels were decreased at 3 hr after soman at 100 micrograms/kg but not after 287 micrograms/kg. Serum testosterone levels were decreased significantly (p less than or equal to 0.01) 6 and 9 hr after soman poisoning but had returned to control levels by 22 hr. Soman (100 micrograms/kg) produced an immunosuppressive response when administered at 24 hr after an antigen (sheep red blood cells). However, a similar response was obtained in adrenalectomized mice suggesting that some other mechanism other than elevated corticosterone was responsible for the immunosuppressive effect. Soman poisoning produced an intense hypothermia. It is possible that the hormonal changes noted after soman poisoning are due to a reduction in metabolism and excretion, a result of the hypothermia and not a direct action of soman. PMID- 3005100 TI - Distribution and elimination of the stereoisomers of soman and their effect on brain acetylcholine. AB - The four stereoisomers of soman (O-(1,2,2-trimethylpropyl)-methyl fluorophosphonate) have been analyzed in vivo in mouse blood and tissues after administration of doses corresponding to 0.75 X LD50 of the two diastereoisomeric pairs of soman (Sc- and Rc-soman). The disappearance of the four isomers has been studied in vitro in the presence of enzymes involved in the toxicity and detoxification of soman, e.g., acetyl- and pseudocholinesterase, aliesterase, and phosphorylphosphatase. The effect of Sc- and Rc-soman on brain acetylcholine was studied in the mouse. The analytical methods used are based on gas chromatography mass spectrometry with deuterated internal standards. Rc-Rp- and ScRp-soman, the two isomers that preferentially react with acetylcholinesterase, were found in blood and liver. In liver the concentration of ScRp was higher than that of RcRp and could be followed for 18 hr. In blood only ScRp could be found. Its presence there could be followed during 18 hr. The levels were, however, lower than in liver. The results indicate that the liver might be a depot for soman and that ScRp might be responsible for the delayed intoxication noted after treatment with antidotes. Rc-soman was found to have a more pronounced effect on the acetylcholine synthesizing system than has Sc-soman, which might explain its higher in vivo toxicity. PMID- 3005102 TI - Denture plaque and denture cleansers: review of the literature. PMID- 3005103 TI - Noncirrhotic portal fibrosis after Wilms' tumor therapy. AB - A 9-yr-old girl developed massive hemorrhage from esophageal varices 2 yr after combined modality therapy for Wilms' tumor. Evaluation showed a patent extrahepatic portal venous system and an elevated splenic pulp pressure. In contrast to previous reports of hepatopathy after irradiation injury, histologic sections of the liver did not demonstrate occlusion of the central veins, but rather a diffuse obliteration of intrahepatic portal venous radicles. This pattern of noncirrhotic portal fibrosis has not been described following antitumor therapy. PMID- 3005104 TI - Stimulation of interstitial collagenase in co-cultures of rat hepatocytes and sinusoidal cells. AB - Although the fibrosis observed during chronic liver injury is the result of a complex process, the striking accumulation of collagen in end stage liver disease has provoked interest in the mechanisms that regulate both collagen production and degradation in the diseased liver. The present studies have examined the cell interactions that may be important in the regulation of collagen degradation. Although minimal amounts of interstitial collagenase activity were noted in cultures of normal hepatocytes and sinusoidal cells, the co-cultures of these cells in the presence of lipopolysaccharide showed a substantial increase in collagenase activity. When the hepatocytes were obtained from rats that had been treated with carbon tetrachloride in vivo, the enhanced activity seen in the co cultures did not require the addition of lipopolysaccharide. Further characterization of this interaction suggested that the increase in collagenolytic activity was partially due to the elaboration of soluble factors by the hepatocyte, which stimulated collagenase production by the sinusoidal cell population. Elaboration of collagenase activity by the sinusoidal cells was inhibited by cycloheximide, suggesting that protein synthesis was required. The proteolytic activity was abrogated by inhibitors of metalloproteinases but not by serine or thiol proteinase inhibitors. The degradation products of type I collagen were typical of the expected products seen with vertebrate collagenases. Thus, it appears that the increased collagenolytic activity detected in this co culture system is attributable to the production of interstitial collagenase by the sinusoidal cell population. Such cell-cell interactions may play an important role in the maintenance of normal connective tissue structure of the liver during disease processes. PMID- 3005105 TI - Endoscopic removal of a granular cell tumor of the stomach. PMID- 3005106 TI - Nonsurgical management of rapidly progressive periodontitis. PMID- 3005107 TI - [New method for the synthesis and cloning of cDNA]. AB - A simple method for cloning cDNA has been suggested. The plasmid pUC18 was digested with Pst1. A plasmid primer for cDNA synthesis was prepared by dT tailing with terminal transferase. After synthesis of cDNA, dG tails were added and then 3' ends blocked with rG. The plasmid was digested with Kpn1 and dC tails were added, after which annealing took place and RNA:DNA hybrids were used for Escherichia coli transformation. The efficiency of approx. 10(4) transformants per microgram of starting mRNA has been obtained. PMID- 3005108 TI - Transcriptional antitermination activity of the synthetic nut elements of coliphage lambda. I. Assembly of the nutR recognition site from boxA and nut core elements. AB - An active nutR antiterminator was reconstructed from two synthetic modules, one containing the 8-bp boxA (5'-CGCTCTTA) and the other the 17-bp nutR core (5' AGCCCTGAAAAAGGGCA) sequence. The modules were synthesized with HindIII cohesive ends, which upon annealing and ligation created an 8-bp spacer (5'-CAAAGCTT) between the boxA and nutR core. The 8-bp length was the same as in the native nutR (5'-CACATTCC), but the sequence showed less than 38% homology. The antitermination mediated by the synthetic nutR, was 68-80% efficient when tested in the pp-nutR-N-tL1-galK expression plasmid, analogous to that used by Drahos and Szybalski [Gene, 16 (1981) 261-274]. The cloned boxA by itself has no activity, while the nutR core alone shows only marginal (5-10%) antiterminator function. Increasing the distance between boxA and the nutR core from 8 bp to 20 28 bp, i.e., by one to two turns of the DNA helix (about 10 bp per turn), has little effect on the antiterminator function, whereas use of spacers with length about halfway between 8 and 20 bp results in reduced antitermination. It appears that both the sequences and spacial arrangement of the boxA and nut elements are important for efficient antiterminator function. PMID- 3005111 TI - Expression of tetracycline resistance in pBR322 derivatives reduces the reproductive fitness of plasmid-containing Escherichia coli. AB - Plasmid pBR322 and its numerous derivatives are used extensively for research and in biotechnology. The tetracycline-resistance (TcR) genes in these plasmids are expressed constitutively and cells carrying these plasmids are resistant to tetracycline. We have shown that expression of the TcR gene has an adverse effect on the reproductive fitness of plasmid-containing bacteria in both glucose limited batch and chemostat cultures. If the TcR genes are inactivated at any one of three different restriction sites, mixed cultures of plasmid-free and plasmid containing bacteria grow at the same rate. PMID- 3005109 TI - Cloning and characterization of the multifunctional his-3 gene of Neurospora crassa. AB - We have cloned the his-3 gene of Neurospora crassa and determined its nucleotide sequence. The gene specifies a protein of 863 amino acids (aa) and contains a 59 bp intron which interrupts aa 800, a proline residue. The 5' end of the his-3 transcript is heterogeneous with major starts 122 and 124 bp upstream from the start codon. There are three possible polyadenylation sites, 119, 120 and 121 bp after the UAA stop codon. The protein shows two regions of homology to the yeast HIS4 gene which correspond to the hisIE and hisD genes of Escherichia coli and Salmonella typhimurium. Northern analysis shows that the level of his-3 mRNA increases severalfold in cultures subjected to histidine starvation. PMID- 3005110 TI - The RNA polymerase I initiation site and the external transcribed spacer of the fission yeast Schizosaccharomyces pombe ribosomal RNA genes. AB - A 5.45-kb fragment containing the 5' end of the ribosomal RNA transcriptional unit from the fission yeast Schizosaccharomyces pombe was cloned in the yeast-E. coli shuttle vector YEp13. The transcription start point was mapped by R looping and S1 nuclease protection. The sequence of the entire external transcribed spacer (ETS) and its flanking regions was determined. Comparison of the sequence around the transcription start point with those of four budding yeasts (Saccharomycetoideae) reveals a consensus sequence from position -9 to -4 from the start. This sequence is likely to be an important element of the promoter for yeast RNA polymerase I (Pol.I). Comparison of all known Pol.I promoter sequences reveals a strong bias for nucleotides (nt) at several positions between -16 and +10. These nt may have a critical role in the transcription initiation process. The S. pombe ETS, which comprises 1355 bp, is significantly longer than those of the budding yeasts and lacks any significant sequence homology with the Saccharomyces cerevisiae ETS. R-loop analysis reveals a putative processing site within the ETS of S. pombe. PMID- 3005112 TI - Nucleotide sequence of the central non-transcribed spacer region of Physarum polycephalum rDNA. AB - In Physarum polycephalum the rRNA genes are present on linear extrachromosomal molecules, each containing two transcription units arranged as a giant palindrome. In the center of the molecule, between the two transcription units, is the 23-kb central spacer, previously shown to contain the replication origins and several regions of direct and inverted repeats. Segments of all the repeats in the spacer have been sequenced and their overall organization determined. The entire spacer consists of reiterations of only 1200 bp of different DNA sequences. The sequence surrounding the transcription start point is not repeated in the spacer, as it is in Xenopus and Drosophila. Sequencing of purified, uncloned rDNA localized some of the methylcytosine residues in the spacer. The repetitious sequences appear to be undergoing concerted evolution. PMID- 3005113 TI - Expression of a rat brain creatine kinase-beta-galactosidase fusion protein in Escherichia coli and derivation of the complete amino acid sequence of rat brain creatine kinase. AB - Expression of a rat brain creatine kinase (CKB)/beta-galactosidase fusion protein in Escherichia coli has allowed isolation of a rat CKB cDNA clone by direct antibody screening of a rat brain gamma gt11 expression library. This clone is 1416 bp long and includes 202 bp of 3'-untranslated region and 29 bp of 5' untranslated region. The coding sequence of this clone has enabled us to deduce the complete amino acid (aa) sequence of rat CKB protein. PMID- 3005115 TI - A rapid procedure for DNA sequencing using transposon-promoted deletions in Escherichia coli. AB - A simple procedure has been developed for sequencing long fragments of DNA. The fragment (which can be several kb in length) is cloned in pAA3.7X, and subdivided into many overlapping segments by Tn9-promoted deletions. The deletions are isolated by positive selection for galactose resistance. A rapid plasmid preparation from several hundred galactose-resistant colonies is fractionated by agarose gel electrophoresis to pick a series of deletions terminating at approx. 200-bp intervals across the entire length of the fragment. Selected plasmids are purified by rapid alkaline extraction, and used directly for supercoil sequencing with a primer derived from IS1. Sequences of adjacent deletions contain overlaps which are used to connect individual sequences to give the complete sequence. PMID- 3005114 TI - Vectors with restriction site banks. IV. pJRD184, a 3793-bp plasmid vector with 49 unique restriction sites. AB - An improved restriction site bank vector has been constructed from plasmid pJRD158. The new version is smaller and contains 43 unique restriction sites. It should greatly facilitate cloning versatility by providing unique sites for most commercially available restriction enzymes. PMID- 3005116 TI - EcoK restriction during in vitro packaging of coliphage lambda DNA. AB - The K restriction system of Escherichia coli works in vitro [Meselson and Yuan, Nature 217 (1968) 1110-1114]. E. coli C lacks the K restriction system. I show that in vitro packaging in standard E. coli K-12-derived systems effects a loss of plaque-former output from K-unmodified lambda DNA relative to K-modified lambda DNA when compared with packaging in the E. coli C-derived system of Rosenberg et al. [Gene 38 (1985) 165-175]. I conclude that the EcoK restriction system is active in standard in vitro packaging systems. EcoK restriction during in vitro packaging could specifically depress recovery of some lambda and cosmid clones of eukaryotic DNA or any other DNA not modified for EcoK restriction. PMID- 3005117 TI - Structure and expression of kappa-chain genes in two IgE-producing rat immunocytomas. AB - The light chain expression in two IgE-producing rat immunocytomas, IR2 and IR162, was studied. Both immunocytomas produce light chains of the kappa type. The kappa chains were characterized at the protein level by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and amino acid (aa) sequencing. cDNA clones corresponding to the kappa-chain mRNA were also prepared and sequenced. The results showed that rat kappa chains have the same structure as their mouse counterparts with respect to signal sequence cleavage, somatic mutations in the V J region and invariance of all the aa positions which are strongly conserved in the frame work regions of mouse V kappa chains (greater than 95% conservation). Results from studies on kappa-chain transcription lend support to the allelic exclusion model with only one functionally expressed light chain in each immunocytoma. PMID- 3005119 TI - Mapping a mammalian mRNA cap site by restriction digestion of hybridized cDNA. AB - A method using restriction enzymes to locate the cap site in a cloned and sequenced 5' region of a gene is described. With a cDNA fragment (or synthetic oligonucleotide sequence), complementary to any portion of an mRNA, the position of the cap site nucleotide in a genomic clone can be precisely located. The steps include: (1) hybridization of a cDNA fragment to an mRNA sample, and reverse transcription from this primer to produce labeled, fully extended cDNA molecules, (2) hybridization of the extended cDNA to a genomic clone containing the region in which initiation occurs (previously sequenced), (3) restriction endonuclease digestion of the hybrid with two or more enzymes within the putative first exon, and (4) size analysis of the short labeled cDNA fragments produced. Multicut restriction enzymes are most useful in this technique. The sizes of the hybridized fragments correspond to the unique distances from the 5' end of the message to each of the different cleavage sites allowing the cap site to be positioned in the genomic sequence. PMID- 3005118 TI - Nonfunctional immunoglobulin light chain transcripts in two IgE-producing rat immunocytomas; implications for the allelic exclusion and transcription activation processes. AB - The rearrangement and expression of immunoglobulin light-chain genes have been studied in two IgE-producing immunocytomas, IR2 and IR162. In the IR2 tumor only one of the kappa-chain alleles is rearranged, expressing a full-length kappa chain polypeptide. In IR162 one of the kappa-chain alleles is functionally rearranged, expressing a 1200-nucleotide (nt) long mRNA, which encodes a functional 23-kDal kappa-chain polypeptide. The second kappa-chain allele is aberrantly rearranged; i.e., a different V region is connected to a position that is located between the J cluster and the C kappa exon. Two mRNAs which are 750 and 850 nt are transcribed from the aberrantly rearranged allele, both of which appear to encode a 12-kDal polypeptide consisting of a signal sequence that is connected directly to the C region. The levels of expression from the two kappa chain alleles are approximately the same, suggesting that no specific mechanism exists to suppress expression of a nonfunctional allele. The rat genome contains a single lambda-chain locus which includes two C-region exons. Although this locus remains in the germ-line configuration in the IR2 and the IR162 tumors, transcripts from the C lambda I and C lambda II regions were detected at a low level in both tumors. These transcripts were detected in RNA from the immunocytomas but not in rat liver RNA indicating that expression is tissue specific. They lacked V-region sequences and resemble so-called sterile transcripts which are expressed at a low level from unrearranged mu- and kappa chain genes. PMID- 3005120 TI - Cloning and sequence analysis of porcine myoglobin cDNA. AB - Porcine myoglobin cDNA clones have been isolated from a cDNA library prepared from enriched heart-myoglobin mRNA. Sequence analysis revealed 59 nucleotides (nt) in the 5'-untranslated, 462 nt in the amino acid (aa)-coding, and 590 nt in the 3'-untranslated regions. The myoglobin cDNA showed a high G + C content (60%). When the nt sequence of the porcine myoglobin cDNA is compared with those of seal and human myoglobin cDNAs deduced from the corresponding genomic myoglobin genes [Blanchetot et al., Nature 301 (1983) 732-734; Weller et al., EMBO J. 3 (1984) 439-446; Akaboshi, Gene 33 (1985) 241-249], a high degree of homology is observed in the 5'-untranslated region and in parts of the 3' untranslated region, as well as in the coding region. PMID- 3005121 TI - Cloning of the Escherichia coli K-12 guaC gene following its transposition into the RP4::Mu cointegrate. AB - The guanosine 5'-monophosphate reductase gene, guaC, has been cloned into the multicopy vector pBR325 from the RP4::Mu cointegrate, pKGM62-1, and the gene product identified by in vitro transcription/translation as a protein of Mr 36 000. Strains harbouring the recombinant plasmid had increased levels of GMP reductase activity. PMID- 3005122 TI - Promoter activity and transcript mapping in the regulatory region for genes encoding ribosomal protein S15 and polynucleotide phosphorylase of Escherichia coli. AB - The genes encoding ribosomal protein S15 (rpsO) and polynucleotide phosphorylase (pnp) occupy adjacent positions and are oriented in the same direction on the Escherichia coli chromosomes. The nucleotide sequence of the region controlling the expression of these two genes has been determined. Two in-phase gene fusions between pnp and lacZ were constructed. The fusions define the translational reading frame of the pnp gene and indicate that the expression of pnp is independent of the upstream rpsO gene. Transcript mapping with nuclease S1 demonstrated that the two genes are transcribed from separate promoters and that the rpsO-pnp intergenic space contains a strong transcriptional terminator. The transcriptional start points have been localized. PMID- 3005123 TI - Plasmids supplying the Q-qut-controlled gam function permit growth of lambda red- gam- (Fec-) bacteriophages on recA- hosts. AB - A plasmid, pgam, has been constructed which expresses the phage lambda gene, gam, under the control of the lambda late promoter, p'R, contained in a form of a p'R qut-t'R1 module. Lambda red- gam-, which normally do not grow on recA- hosts, are able to grow on recA- hosts containing pgam, because their Q function can turn on the gam gene expression. This facilitates cloning with lambda red- gam- vectors in recA- hosts. PMID- 3005124 TI - Control of cloned gene expression by promoter inversion in vivo: construction of the heat-pulse-activated att-nutL-p-att-N module. AB - We have constructed a prototype of gene-expression plasmids with three novel properties: its "OFF phase" is absolute in all common hosts because the expression promoter is facing away from the studied gene and is blocked by a strong terminator; the "ON phase" is attained by the rapid and efficient inversion of the promoter; only a short heat pulse or exposure to other inducing agents is required to initiate this two-stage process. In the first stage, synthesis of the phage lambda Int protein is induced by the transient derepression of the properly engineered lambda xis- cIts857 prophage. In the immediately following second stage, Int causes inversion of a promoter cloned between the inverted ----P'OP phage att site and the normally oriented ----delta PO delta P' pseudo-bacterial att site. The inverted promoter can now control the expression of the studied gene and also of the lambda N gene cloned in tandem. The N product, in conjunction with the nutL site placed downstream of the promoter, permits efficient antitermination of any terminators present in the att sites, in the plasmid or in the cloned DNA, making this system efficient and of practical value. Employing the promoter-inverting plasmid, it was possible to obtain rapid onset and a high level of galactokinase synthesis from the cloned galK gene. Only a transient, 10-min induction at 42 degrees C was employed, permitting protein synthesis at 30 degrees C, which might be of importance for thermosensitive products. Furthermore, the entire promoter-inversion module can be transferred to any plasmid as a 1.3-kb AvaI-ClaI fragment (see Fig. 1).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3005125 TI - Expression of hepatitis B virus surface antigen P31 gene in Escherichia coli. AB - A hepatitis B virus surface antigen (HBsAg) P31-coding DNA was constructed from a DNA fragment of the plasmid pHBr330 containing the entire hepatitis B virus (HBV) adr DNA and a chemically synthesized adaptor. The P31 gene was inserted into an expression vector, pTRP771, having an Escherichia coli tryptophan operon (trp) promoter to give a recombinant plasmid pTRP P31-R. The distance between the Shine Dalgarno (SD) sequence and the initiation codon of P31 gene was adjusted to 9 bp. The expression level of HBsAg by E. coli 294[pTRP P31-R] was significantly elevated, in contrast to that of HBsAg by E. coli 294[pTRP SS-6]. Western blotting analysis has shown that E. coli[pTRP P31-R] synthesizes a specific polypeptide P31 of about 31 kDal, which reacts with anti-HBsAg antibody. The binding studies with polyalbumins from various species have also suggested that HBsAg P31 specifically binds to polymerized human serum albumin. PMID- 3005126 TI - Nonrandom insertion of Tn5 into cloned human adenovirus DNA. AB - The bacterial transposable element Tn5 displays regional selectivity in target sites for transposition. To examine this integration specificity of Tn5, we have mapped 57 insertion events in a plasmid pXC1 containing a eukaryotic viral DNA fragment as a target for Tn5 insertional mutagenesis. We found a nonrandom distribution of integration sites in pXC1, suggesting preferred targets for transposition. However, DNA sequence analysis of seven mutants revealed no target site sequence specificity for Tn5 insertion. We demonstrated that the majority of these insertions mapped downstream from a fortuitous promoter sequence which was present and active in this cloned insert in pXC1. Furthermore, when this promoter region was removed, Tn5 was able to transpose into previously unused upstream target sequences. Our data suggest that transcriptional activity may influence Tn5 transposition. PMID- 3005127 TI - Stable gene amplification in the chromosome of Bacillus subtilis. AB - We constructed five different structures, consisting of a genetic marker flanked by directly repeated sequences 2-4 kb long, in the Bacillus subtilis chromosome. When a selective pressure was applied amplification of the marker and one of the repeats was observed in all cases. Amplification was not detected with two markers which were not flanked by the repeated sequences. The maximum amplification level observed with the different structures varied between 5 and 50. The size of the most amplified structure corresponded to 7.5% of the chromosome. Amplification was stable upon growth of cells under non-selective conditions. Each copy of an amplified gene was expressed with equal efficiency. These results indicate that chromosomal gene amplification may be useful for constructing genetically engineered B. subtilis strains. PMID- 3005128 TI - Expression of chicken egg white lysozyme by Saccharomyces cerevisiae. AB - An efficient yeast promoter was isolated using a beta-galactosidase (beta Gal) promoter probe vector. This promoter was then used to express chicken egg white lysozyme in yeast using a complete intron-free lysozyme-coding sequence constructed by in vitro recombination between a cDNA clone lacking the 5' end and the corresponding 5' end from a nuclear DNA clone. The resulting lysozyme is efficiently exported into the growth medium suggesting that the chicken signal sequence is recognized by the yeast secretion process. PMID- 3005129 TI - Nucleotide sequence of the rpsU-dnaG-rpoD operon from Salmonella typhimurium and a comparison of this sequence with the homologous operon of Escherichia coli. AB - In Escherichia coli the genes encoding ribosomal protein S21 (rpsU), DNA primase (dnaG), and the 70-kDal sigma subunit of RNA polymerase (rpoD) are contained in a single operon. These gene products are involved in the initiation of translation, DNA replication, and transcription, respectively. We have examined the homologous region in the closely related bacterium Salmonella typhimurium and have found that the same three genes are similarly organized. We have sequenced the DNA for this operon in S. typhimurium and have compared the (nt) nucleotide and amino acid (aa) sequences with E. coli. In the coding regions, the sequence conservation varies from extremely high for rpsU to moderate for dnaG with respect to both nt and aa sequence. In the noncoding regions, sequences thought to be important for the regulation of transcription are conserved, while other sequences are not conserved. aa differences in DNA primase and sigma are not randomly distributed and suggest regions that may be important for protein structure or function. PMID- 3005131 TI - A common precursor for the two subunits of the penicillin acylase from Escherichia coli ATCC11105. AB - Penicillin acylase (PA) is an industrial enzyme that is used to convert penicillin G into a precursor for semisynthetic penicillins. We have cloned a segment of DNA that codes for the two subunits required for PA activity. We also report the nucleotide sequence of a DNA fragment that codes for (i) the small subunit, (ii) the N-terminal region of the large subunit and (iii) a putative connecting peptide. These results confirm the existence of a common precursor for both peptides. PMID- 3005130 TI - Characterization of Tn3926, a new mercury-resistance transposon from Yersinia enterocolitica. AB - A new transposon coding for mercury resistance (HgR), Tn3926, has been found in a strain of Yersinia enterocolitica, YE138A14. The element has a size of 7.8 kb and transposes to conjugative plasmids belonging to different incompatibility groups. A restriction map has been established. DNA-DNA hybridization indicates that Tn3926 displays homology with both Tn501 and Tn21; the greatest homology is shown with the regions of these transposons that encode HgR. Weaker homology is observed between Tn3926 sequences and those regions of Tn501 and Tn21 that encode transposition functions. Complementation experiments indicate that the Tn3926 transposase mediates transposition of Tn21, albeit somewhat inefficiently, but not of Tn501, while the resolvase mediates resolution of transposition cointegrates formed via Tn21, Tn501, or Tn1721. PMID- 3005132 TI - Transient assay, by [3H]guanine incorporation of Escherichia coli xanthine guanine phosphoribosyl transferase (GPT) in transfected human fibroblasts. AB - We have used [3H]guanine incorporation as a rapid and sensitive assay of xanthine guanine phosphoribosyl transferase (GPT) activity in SV40 transformed human fibroblasts. The SV40 early promoter is more efficient than the Rous sarcoma virus long terminal repeat for transient expression of the gpt gene. The assay works well in a derivative of AT5BIVA which lacks hypoxanthine-guanine phosphoribosyl transferase (hprt-) and we show here how the assay has been adapted to work in the hprt+ AT5BIVA parent. PMID- 3005133 TI - Expression of an Escherichia coli beta-galactosidase fusion gene in Aspergillus nidulans. AB - We inserted in frame the Escherichia coli lacZ gene into the protein-coding region of the Aspergillus nidulans trpC gene and introduced the resultant fused gene into the A. nidulans genome. A functional beta Gal fusion protein was produced. Removal of the trpC transcription and translation initiation sequences from the fusion gene abolished production of the fusion protein, showing that expression is dependent on these sequences. Thus, lacZ fusions should be of use for estimating gene activity in a. nidulans. PMID- 3005135 TI - Colorectal cancer in the elderly. PMID- 3005134 TI - Adrenocortical responsiveness to graded ACTH infusions in normal young and elderly human subjects. AB - The responses of plasma cortisol, aldosterone, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S) to graded ACTH infusions (from 50 mU/h to 1,000 mU/h) in elderly subjects were compared with those in young subjects. There were no significant differences between young and elderly subjects in terms of the levels of plasma cortisol during ACTH infusion. The increment in median serum cortisol increase observed in elderly subjects was also equal to that found in young subjects. Plasma aldosterone concentration showed a gradual increase in response to ACTH infusion in both young and elderly individuals. There was no significant difference between the response of young and aged subjects. Significant increases in serum DHEA in response to ACTH infusion were observed in both young and aged individuals, however, the median increase of serum DHEA (delta DHEA) in the elderly subjects was markedly lower than that in the young ones. Serum DHEA-S concentrations prior to ACTH infusion were significantly lower in the elderly subjects. With graded infusions of ACTH, plasma DHEA-S concentrations in young subjects tended to increase gradually, whereas there was no significant increase in plasma DHEA-S concentrations in the elderly. These results are indicative that the responses of adrenal androgens in elderly subjects to small, graded doses of ACTH infusion are preferentially impaired; however, the responses of cortisol and aldosterone are well maintained. PMID- 3005136 TI - [Sources and levels of the irradiation of the inhabitants of the Far North]. PMID- 3005137 TI - [Course of lead poisoning after vitamin D administration]. PMID- 3005138 TI - Effects of the fibre components pectin, cellulose, and lignin on bile salt metabolism and biliary lipid composition in man. AB - Randomised crossover studies in three separate groups of 10 healthy volunteers were undertaken to determine the effects of biliary lipid composition and bile salt metabolism of daily dietary supplementation for four weeks with the purified fibre components pectin (12 g/day), cellulose (15 g/day) and lignin (12 g/day). The subjects' biles were initially unsaturated with cholesterol and no significant changes in the lithogenic indices or mean percentages of cholesterol, phospholipid, or total bile acids after any of the supplements were observed. After pectin, the mean (+/- SD) percentage of cholic acid decreased significantly from 42.8 (+/- 10.8) to 39.0 (+/- 11.2), the mean (+/- SD) percentage of deoxycholic acid increased significantly from 18.2 (+/- 13.7) to 25.4 (+/- 13.5) and C14-deoxycholate metabolites were raised significantly by 65%. After cellulose, the mean (+/- SD) percentage of chenodeoxycholic acid was increased significantly from 33.6 (+/- 6.3) to 35.4 (+/- 7.0), the mean (+/- SD) percentage of deoxycholic acid decreased significantly from 18.6 (+/- 9.6) to 14.2 (+/- 8.3) and C14-deoxycholate metabolites halved. Lignin did not exert any significant effects. Though these results show that individual fibre components are associated with quite different effects on bile acid metabolism, in the short term no significant effect on biliary cholesterol saturation was observed in bile initially unsaturated with cholesterol. The bile acid changes most likely result from the different effects on colonic metabolism induced by the individual fibre components. PMID- 3005139 TI - Effects of antimicrobial therapy on faecal bulking. AB - It has recently been postulated that dietary fibre acts as a substrate for colonic flora, and that the resultant microbial growth bulks the faeces. Antimicrobial therapy was used in this study to assess the effect of reduction in colonic microbial proliferation on faecal output in human subjects on a constant dietary fibre intake. Six healthy young male subjects were maintained on constant daily diets and metronidazole (1 g/day) and ampicillin (1 g/day) were administered in divided doses for one week after an initial baseline study period of two weeks. After antimicrobial therapy, mean faecal weights rose from 176.0 +/ 27.0 g to 348.1 +/- 37.7 g/day. Faecal solids increased from 32.9 +/- 4.2 g to 46.1 +/- 5.8 g/day. Faecal neutral detergent fibre increased from 1.92 +/- 0.42 g to 15.19 +/- 2.58 g/day. The mean transit times and mean daily faecal nitrogen remained the same, both before and after treatment. Substantial breakdown of dietary fibre occurs in the human colon which may decrease faecal bulk, suggesting that water holding by dietary fibre is probably of greater importance for faecal bulking. PMID- 3005140 TI - Bowel function measurements of individuals with different eating patterns. AB - Bowel function was assessed in 51 subjects: 10 women and seven men who habitually consumed an omnivorous, vegetarian, or vegan diet. The subjects on these diets had a mean intake of fibre of 23 g, 37 g, and 47 g respectively. Mean transit times were variable and not significantly different between the groups. Vegans, however, had a greater frequency of defecation and passed softer stools. All measurements of bowel function were significantly correlated with total dietary fibre. As dietary fibre increased mean transit time decreased, stool frequency increased and the stools became softer. Men produced a greater quantity of softer, less formed faeces than women. During the luteal phase of the menstrual cycle women excreted harder stools and had a significantly longer mean transit time. The finding that mean transit time was more highly correlated with faecal form than any of the other bowel function measurements could be of practical importance. PMID- 3005143 TI - Sudan black B and peroxidase positivity in cultured lymphoid cells from patients with B-type chronic lymphocytic leukemia. PMID- 3005141 TI - Serum CA 125 in ovarian pathology and its variation in ovarian carcinoma after integrated therapy. AB - Serum CA 125 was detected by RIA in a total of 66 patients with various ovarian pathologies (16 malignant and 50 benign). Six patients with ovarian carcinoma were monitored during the 1st week after surgery (2 patients underwent only explorative laparatomy) and during chemotherapy for a total of 150 days of treatment. We observed that CA 125 serum level is consistently above the normal range (greater than 35 U/ml) in all malignant diseases. In benign pathology, levels above the normal were found to be represented almost exclusively by ovarian endometriosis. Furthermore, the results demonstrate that the chemotherapeutic regime alone is capable of lowering CA 125 serum levels. PMID- 3005144 TI - [Peripheral and central neurological syndrome in chronic idiopathic intestinal pseudo-obstruction]. PMID- 3005142 TI - Usefulness of naphthol-AS-D-chloroacetate esterase (NASDCAE) in the diagnosis of AML: report of a case. PMID- 3005145 TI - [The murine mammary tumor virus (MuMTV) and the association with breast cancer]. PMID- 3005146 TI - [Retroviruses, AIDS and systemic lupus erythematosus]. PMID- 3005148 TI - The characteristics of hGH binding to the liver macrophages. AB - Macrophages isolated from female rat liver as well as hepatocytes bind 125I-hGH. This study compares the effect of sex of the rat, hypophysectomy (hypox) and preincubation of the cells with oPrl on the binding of 125I-hGH to the cells. The percent of 125I-hGH to the hepatocytes was decreased in cells from hypox female and male rats, and hepatocytes preincubated with oPrl to 0.43, 0.21 and 0.39, respectively, of that observed in hepatocytes from normal female rats. In the hepatocytes from normal female, hypox female, and male rats, hGH was the most effective competitor for 125I-hGH binding with an ID50 of 0.73-0.99 nM. The concentration of oPrl, bGH and rGH that produced half-maximal inhibition (ID50) of 125I-hGH binding to hepatocytes from female rat liver was 6.3, 100, and 420 nM respectively. In hepatocytes from male and hypox female rats, and hepatocytes preincubated with oPrl, the ID50 for bGH and rGH varied from 2.1 to 15.9 nM. The percent of 125I-hGH bound by the macrophages from hypox female and male rats, and macrophages preincubated with oPrl was 0.06, 0.15 and 0.18, respectively, of that bound by macrophages from normal female rat liver. In contrast to hGH binding to the hepatocytes, the ID50 for hGH was 6 to 180-fold greater in macrophages from hypox female and male rats, and macrophages preincubated with oPrl compared to that observed in macrophages from normal female rats, Rat GH was the most effective competitor for 125I-hGH binding in the macrophages from the hypox female and male rat liver with ID50 of 5.5 and 85 respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3005147 TI - Involvement of cyclic AMP-dependent protein kinase on the phosphorylase kinase inhibition by glucose-6-phosphate in adipose tissue extracts. AB - In order to achieve further clarification of the regulation of glycogenolysis in adipose tissue, we studied the effect of glucose-6-phosphate on phosphorylase activation in Sephadex G-25 filtrate of adipose tissue. The activity of phosphorylase kinase was decreased by 50% and by 75% in the presence of 0.5 mM and 2 mM of glucose-6-phosphate, respectively. This inhibition could be partially prevented by 0.5 mM AMP. Furthermore, we investigated the influence of glucose-6 phosphate on the effect of cyclic-AMP-dependent protein kinase on the activation of phosphorylase. The addition of cyclic-AMP and cyclic-AMP-dependent protein kinase caused a decrease in the inhibition of the phosphorylase activation by glucose-6-phosphate. Also, the glucose-6-phosphate at physiological concentration, decreased adipose tissue cyclic-AMP-dependent protein kinase activity. PMID- 3005149 TI - Purification and comparison of the receptors for insulin and insulin like growth factor I. AB - The insulin-like growth factors are structurally and biologically similar to insulin. The receptor sites for insulin-like growth factor-I have recently been shown to have a sub-unit structure very similar if not identical to insulin. We have compared the behavior of insulin and insulin-like growth factor-I receptors during purification using gel filtration, lectin affinity chromatography, insulin affinity chromatography, and gel electrophoresis. We demonstrate the remarkably similar physicochemical characteristics of these two receptors, but have achieved complete separation by the use of insulin affinity chromatography. PMID- 3005150 TI - Dopaminergic control of aldosterone secretion in hypertensive patients chronically treated with an angiotensin converting enzyme inhibitor. AB - To investigate whether dopamine plays a role in the regulation of aldosterone secretion during long-term blockade of the renin-angiotensin system, we studied the effect of metoclopramide, a competitive antagonist of dopamine, in 6 patients with essential hypertension chronically treated with the angiotensin converting enzyme inhibitor enalapril. All but one of these patients received a diuretic in addition to enalapril. Six hours after the daily morning dose of enalapril (10-40 mg p.o.) a 10 mg bolus dose of metoclopramide was injected intravenously. In one patient a hypotensive episode developed following metoclopramide administration. In the 5 other patients plasma aldosterone significantly rose within 30 min after metoclopramide from 51 +/- 8.7 to 128.2 +/- 29.2 pg/ml. This metoclopramide induced release of aldosterone occurred in the absence of concomitant changes in circulating angiotensin 11, potassium and ACTH levels. Metoclopramide given during chronic blockade of the renin-angiotensin system caused anxiety and agitation in 2 patients. The increase in plasma aldosterone following competitive dopamine blockade in the face of chronic angiotensin converting enzyme inhibition, unchanged plasma potassium and ACTH levels strongly suggests that in hypertensive patients, dopamine exerts a direct inhibitory effect on aldosterone secretion. PMID- 3005151 TI - Effects of tetrahydrocannabinol on melatonin secretion in man. PMID- 3005152 TI - Clonidine and the hormonal responses to graded exercise in healthy subjects. AB - The aim of this study was to assess the acute effects of clonidine, an alpha 2 adrenergic agonist, on hormonal responses to graded exercise in 8 healthy young men. After fasting overnight, each subject was tested on 2 mornings, 1 week apart. On one occasion he was given 200 micrograms clonidine orally and on the other identical placebo tablets, the order being randomized in a double-blind fashion over the 2 days. Thereafter each subject performed 2 successive treadmill runs, equivalent to 60 and 100%, respectively, of maximal aerobic power. Clonidine pretreatment blunted the maximal increase in plasma catecholamines by more than 60% of the control response (p less than 0.01), without significantly altering the rise of plasma cortisol or ACTH. Furthermore, clonidine significantly reduced the exercise-induced maximal rise in plasma glucose, without modifying the slight decline in mean plasma insulin or increase in pancreatic glucagon levels. The drug did not affect the maximal increments in plasma growth hormone or prolactin occurring after exercise. It was concluded that a single dose of clonidine suppressed peripheral sympathetic responses, without altering central (pituitary) alpha-adrenergic-mediated hormonal responses, to short-term exercise in healthy men. PMID- 3005153 TI - Secretory response and immunochemical heterogeneity of glucagon in plasma and tumor extracts of a patient with glucagonoma. AB - The secretory response and immunoreactive heterogeneity of glucagon was investigated in a patient with glucagonoma syndrome. After glucose administration, abnormal insulin release accompanied by glucose intolerance were observed, whereas the high glucagon circulating levels were only partially blocked after glucose or somatostatin infusion. Chromatographic fractionation of plasma samples, before and after arginine administration showed that most of the immunoreactivity eluted as true glucagon. Furthermore, when aliquots of the tumor extracts were fractionated by column chromatography or by polyacrylamide gel electrophoresis, most of the immunoreactivity eluted in the 3,500 molecular weight peak. In contrast with previous reports, our results indicate that neoplasia A cells can also manufacture and release into the bloodstream great amounts of genuine glucagon rather than larger glucagon immunoreactive forms. In spite of such findings, in this patient neither diabetes nor hyperglycemia were present. PMID- 3005154 TI - Follow-up care for chronic mentally ill: whose job? PMID- 3005155 TI - Generalized venocentric lesions in the virus-associated hemophagocytic syndrome. AB - Clinical and autopsy findings in a 41/2-year-old boy who had been healthy until the onset of virus-associated hemophagocytic syndrome are presented. In addition to findings previously reported in association with the syndrome, widely disseminated perivenular expansile nodules of bland necrosis were seen. These nodules were suggestive of vessel injury, with extravasation of vascular contents and focal destruction of normal elements. The morphologic similarities of these nodules to early lesions of pulmonary veno-occlusive disease are discussed. PMID- 3005156 TI - Primary hepatocellular carcinoma with hepatitis B virus-DNA integration in a 4 year-old boy. AB - The autopsy findings in a case of hepatocellular carcinoma with hepatitis B virus (HBV)-DNA integration in a Japanese boy, 4 years and 10 month of age, are reported. The boy was an HBV carrier born in a typical familial cluster of HBV infection in Japan. He had been asymptomatic until the sudden manifestation of the liver tumor. Histopathologic examination revealed a well-differentiated, adult-type hepatocellular carcinoma without hepatic cirrhosis. HBV-DNA sequences were detected in the tumor cell DNA by the Southern blot hybridization method. This is the youngest patient with hepatocellular carcinoma with HBV-DNA integration reported to date, suggesting that HBV may manifest its oncogenic properties after a shorter incubation period than generally believed. PMID- 3005157 TI - Cell-mediated immunity in neurologic disease. PMID- 3005158 TI - Pathologic features of AIDS encephalopathy in children: evidence for LAV/HTLV-III infection of brain. AB - The neuropathologic findings in 11 children with a new CNS disorder that occurs in children with the acquired immunodeficiency syndrome (AIDS) and is postulated to be due to LAV/HTLV-III, the virus that causes AIDS, are reported. The children, who ranged in age from 4 months to 11 years, died of AIDS complicated by progressive encephalopathy. Ten of the children either had positive serum antibody for LAV/HTLV-III or had received blood products from donors later found to be antibody-positive. Examination of the brains of these children at autopsy revealed a unique constellation of findings, including varying degrees of diminished brain weight in all cases, inflammatory cell infiltrates in nine brains, multinucleated cells in eight, three of which also contained multinucleated giant cells, vascular calcification in ten, vascular and perivascular inflammation in five, and white matter changes in nine. Inflammatory and vascular lesions were most prominent in basal ganglia and pons. LAV/HTLV-III retroviral particles, associated with multinucleated giant cells, were observed in two brains on electron microscopic examination. These two and one additional brain had evidence of the LAV/HTLV-III genome by hybridization studies. Only one brain had a recognizable opportunistic infection. PMID- 3005160 TI - A short term evaluation of ultrasonically delivered medications in the treatment of moderate periodontal disease. PMID- 3005159 TI - Introduction of lymphadenopathy associated virus or human T lymphotropic virus (LAV/HTLV-III) into the male homosexual community in Amsterdam. AB - To establish when lymphadenopathy associated virus or human T lymphotropic virus (LAV/HTLV-III) was introduced into the Netherlands, we studied a cohort of homosexual men who participated in a hepatitis B vaccine efficacy study between 1980 and 1982. On entry into the study (November 1980 to December 1981) five (0.7%) out of 685 participants were found to have antibodies to LAV/HTLV-III, and during follow up 15 seroconversions were detected among the 680 who had been seronegative initially (end point attack rate 3%). LAV/HTLV-III was not transmitted by the heat inactivated hepatitis B virus (HBV) vaccine used. Anal sexual contact and antibodies to cytomegalovirus (CMV) were found to correlate with seropositivity or seroconversion for LAV/HTLV-III. Six out of 15 men who seroconverted reported a mononucleosis like illness, but three of them had other concurrent virus infections. To date, only one of the 20 seropositive men has developed the acquired immune deficiency syndrome (AIDS), three years after his seroconversion. This study shows that the introduction of LAV/HTLV-III into the Dutch male homosexual community took place at the end of the 1970s, a few years before the first case of AIDS in a native Dutchman. PMID- 3005161 TI - Herpes simplex virus-specific lymphoproliferation: an analysis of the involvement of lymphocyte subsets. AB - Herpes simplex virus (HSV)-immune murine splenocytes incorporated significant levels of tritiated thymidine when incubated with UV-inactivated, heat inactivated, and active preparations of HSV. Normal splenocytes incubated with the HSV preparations did not exhibit such proliferation. Maximum incorporation by the immune splenocytes occurred on the fifth day of culture and was mediated by Thy-1+, Lyt-1+, and Lyt-2+ cells. Attempts to correlate lymphoproliferation with other HSV-specific cellular immune responses demonstrated the complexity of this response. While T cells mediating delayed type hypersensitivity responses and cytotoxic T lymphocytes were involved in the lymphoproliferative response, neither could be considered as being exclusively associated with lymphoproliferation. Instead, lymphoproliferation appeared to be indicative of HSV-specific Lyt-1+ helper cells. Evidence was also presented that suppressor cells appeared to be involved in the regulation of the lymphoproliferative response. PMID- 3005162 TI - Alterations in epidermal handling of HSV-1 antigens in vitro induced by in vivo exposure to UV-B light. AB - In order to investigate the cellular interactions involved in the immune response to herpes simplex virus type 1 (HSV-1) in a murine model, an in vitro antibody induction system was developed. This comprised HSV-1-primed T cells from infected mice, trinitrophenol (TNP)-primed B cells from mice primed with TNP-coupled calf erythrocytes, and TNP-HSV-1 as antigen. When antigen-presenting cells (APC) were removed from the assay system, the induced antibody response disappeared but could be reconstituted by the addition of APC derived from the peritoneal cavity or skin of normal mice. Since HSV-1 is an epidermal pathogen, it was decided to investigate the role of skin APC in HSV-1 immunity. Skin APC from mice irradiated 3 days previously with a suberythemal dose of ultraviolet (UV)-B were found to have a decreased capacity to present HSV-1 antigen in vitro. However, the APC capacity of their peritoneal cells was unaffected. The reduction in APC capacity is not only a local effect at the irradiated site, as APC from mice exposed to UV B with one ear protected by black electrical tape were equally affected in both ears. PMID- 3005163 TI - Immunological alterations in anti-HTLV-III negative haemophiliacs and homosexual men in Hungary. AB - Hungary can be considered as a low risk area for AIDS since no patient with full blown AIDS or AIDS-related complex has been found in the country. A complex clinical and immunological (T cell subsets, DNCB sensitization test, circulating immune complexes, acid-labile alpha interferon) investigation was performed between November, 1983 and June, 1984 in order to study whether alterations found in symptom-free homosexuals and haemophiliacs in the risk areas for AIDS can be observed in Hungary as well. 38 patients with mild haemophilia, 35 patients with severe haemophilia and 40 homosexual men were investigated in parallel to 37 heterosexual blood donors as controls. Anti-HTLV-III antibodies were measured later in the stored serum aliquots from the same subjects by the indirect membrane immunofluorescence assay. Although specific anti-HTLV-III antibodies were not detected in the haemophiliacs or homosexuals, immunological alterations characteristic for the members of AIDS risk groups in the high risk areas (decrease in the percentage of OKT4 cells and/or decrease of the OKT4/8 ratio) were found in one-third of the homosexuals and haemophiliacs tested. In addition, a significant part of these subjects did not develop delayed type hypersensitivity skin reaction on DNCB rechallenge. These findings indicate that an immunodeficiency independent of HTLV-III infection can be present in two major AIDS risk groups, in homosexual men and haemophiliacs. PMID- 3005164 TI - Adrenal cortical reserve in leprosy. PMID- 3005165 TI - Diagnosis of intrauterine cytomegalovirus infection by IgM antibody test. PMID- 3005166 TI - Human parvovirus infection in children. PMID- 3005167 TI - Coxsackie A-9 in the etiology of poliomyelitis-like diseases. PMID- 3005168 TI - Dopaminergic binding and inhibitory effect in the bovine adrenal zona glomerulosa. AB - Dopaminergic mechanisms may be involved in the regulation of aldosterone secretion in humans and in the rat. Whether these effects are indirect or are exerted directly at the adrenal level has not yet been resolved. We now report the identification of dopaminergic binding sites in the bovine adrenal zone glomerulosa using [3H]spiperone, a butyrophenone with high affinity for D2 dopamine receptors. Specific [3H]spiperone binding (defined as binding displaceable by 10 microns (+)-butaclamol) reached equilibrium within 20 minutes at 22 degrees C, was reversible, and was heat labile (60 degrees C). Binding was of high affinity and saturable with a Kd of 1.8 +/- 0.2 nM and maximal specific binding of 38 +/- 8 fmol/mg (means +/- SEM; n = 18). [3H]Spiperone binding was unaffected by coincubation with angiotensin II, adrenocorticotropic hormone, or KCl. Binding characteristics, including a dissociation constant at the nanomolar range, greater potency of the D2-agonist LY 171555 relative to the D1-agonist SKF 38393 in inhibiting [3H]spiperone binding, and lack of stimulation of cyclic adenosine 3',5'-monophosphate by dopamine (10(-4) M), were consistent with a predominantly D2-receptor. In vitro studies with collagenase-dispersed adrenal zona glomerulosa cells showed that dopamine (10(-4) M) attenuated angiotensin II stimulated aldosterone secretion. These observations are consistent with a direct inhibitory effect of dopamine on aldosterone secretion in the adrenal zona glomerulosa. PMID- 3005169 TI - Renal and endocrine response to saline infusion in essential hypertension. AB - To assess the contribution of the renin-angiotensin-aldosterone system and renal hemodynamics to acute renal sodium handling in essential hypertension we studied 21 subjects who had essential hypertension (16 with normal renin, 5 with low renin) and 9 normal subjects. All were in balance on a 10 mEq sodium intake before receiving a small sodium load, 60 mEq intravenously over 1 hour. Hypertensive subjects with low renin showed the anticipated exaggerated natriuresis, which was transient and occurred without a rise in blood pressure. Natriuresis in hypertensive subjects with normal renin was either normal or blunted; delayed sodium excretion occurred in a subset, along with delayed suppression of the renin-angiotensin-aldosterone system by the saline load. Neither renal plasma flow nor glomerular filtration rate changed during the saline load. After 72 hours of converting enzyme inhibition with enalapril, renal plasma flow increased substantially more in the subjects with a blunted renin response and their natriuretic response to the sodium load returned to normal. These results indicate that when prior sodium intake is controlled, large sodium loads are avoided, and low renin hypertension is removed as a confounding variable, blunted rather than exaggerated natriuresis is the common feature of essential hypertension. This abnormality is reversed by angiotensin converting enzyme inhibition, perhaps because of converting enzyme inhibition-induced renal vasodilatation. PMID- 3005170 TI - Effect of sieved buckwheat (Fagopyrum esculentum) flour supplementation on lipid profile and glucose tolerance. AB - Buckwheat (Fagopyrum esculentum) was picked up for a study of its effects on lipid profile and glucose tolerance in view of its relatively high fibre content. In earlier studies, we demonstrated that supplementing the daily diet with 100 g whole buckwheat flour raised the high density lipoprotein cholesterol (HDL C)/cholesterol ratio and improved glucose tolerance. In the present study, 12 human volunteers replaced part of their cereal intake at lunch by a preparation made from 100 g sieved buckwheat flour for a period of 4 weeks. At the end of 4 wk, there was a significant rise in HDL-C from 42.8 +/- 11.4 mg/100 ml to 55.2 +/ 15.3 mg/100 ml (P less than 0.05), and in HDL-C/cholesterol ratio from 26.7 +/- 7.0% to 33.8 +/- 10.2% (P less than 0.02): The other changes in lipid profile were not significant. There was also no significant change in fasting blood glucose or oral glucose tolerance in the 5 subjects on whom the test was done. Comparing the results with the observations made earlier on whole buckwheat, it is still not possible to say to what extent the effects of buckwheat on lipids may be attributed to its fibre content. The effects on glucose tolerance, on the other hand, seem to be more directly related to the fibre content. PMID- 3005171 TI - Inhibition of neutrophil killing of Candida albicans pseudohyphae by substances which quench hypochlorous acid and chloramines. AB - Using a microtiter plate killing assay, we investigated the in vitro killing of Candida albicans by human neutrophils and by hypochlorous acid/hypochlorite ion (HOCl/OCl-) or chloramine solutions to evaluate the inhibition of this process by quenchers of these oxidants. Methionine, tryptophan, and alanine were able to effectively inhibit neutrophil killing of candida pseudohyphae. These substances were capable of quenching the oxidant activity of NaOCl, monochloramine (NH2Cl), and to a lesser extent, taurine chloramine. NaOCl and NH2Cl were able to kill C. albicans in the absence of inhibitors in concentrations of less than 5 microns M, whereas greater than 100 microns M taurine chloramine was required for killing. Methionine and tryptophan were capable of markedly inhibiting killing by all three oxidants, whereas alanine affected only killing by NaOCl. The oxidant activity of NaOCl was more readily quenched by opsonized or unopsonized Candida yeast than was the oxidant activity of either NH2Cl or taurine chloramine. These results suggest that some substances which quench the oxidizing activity of the products of the neutrophil myeloperoxidase system can inhibit the killing of C. albicans by these cells. PMID- 3005172 TI - Contributions of C1q, bacterial lipopolysaccharide, and porins during attachment and ingestion phases of phagocytosis by murine macrophages. AB - In contrast to the S-form of Salmonella minnesota, its Re mutant binds to mouse peritoneal macrophages. The binding reaction triggers an oxidative burst, measured by a chemiluminescent reaction. The oxidative burst was abolished in the presence of either purified lipopolysaccharide or porins (outer membrane proteins) extracted from the Re mutant, suggesting that both components are involved in binding of the Re mutant to macrophages. In addition, Fc-recognizing membrane structures on the macrophage surface bind the Re mutant. Preincubation of macrophages with the Re mutant abolishes immunoglobulin G-sensitized erythrocyte-induced chemiluminescence. Macrophages preincubated with immunoglobulin G-sensitized erythrocytes had a low chemiluminescent signal, and after treatment of the cells with the Re mutant, there was an additional, higher signal. Binding of purified C1q to the Re mutant decreased the adherence of the Re mutant to macrophages, resulting in a diminished chemiluminescent signal. Blocking of endogenous macrophage membrane-associated C1q with a monoclonal antibody [F(ab')2 fragment] directed against mouse macrophages (recognizes the A and B chains of C1q) diminished the oxidative burst. Therefore, the endogenous C1q of macrophages also appears to be involved in attachment of the S. minnesota Re mutant. PMID- 3005173 TI - Protection against Brucella abortus in mice with O-polysaccharide-specific monoclonal antibodies. AB - Mice injected with either of two monoclonal antibodies specific for the O polysaccharide of Brucella abortus prior to challenge infection had viable counts in spleens and livers significantly below those in control groups 1 and 4 weeks later. Two monoclonal antibodies specific for the porin of B. abortus failed to confer protection. PMID- 3005174 TI - Induction of a protective immune IgE response in rats by injection of defined antigens of schistosomulum-released products: immunochemical properties of the target antigens. AB - Brown Norway rats were injected without adjuvant with the soluble products liberated in a 16-hour culture by schistosomula (schistosomula-released products, SRP-A). A strong cytotoxic and protective IgE response was elicited, mainly directed against 22- and 26-kilodalton (kDa) SRP-A molecules. In the present study, we have attempted to characterize further those molecules. Metaperiodate denaturing treatment of the SRP-A glycans before injection into rats did not modify the immunogenicity of the SRP-A antigens. Results obtained by lectin affinity suggested that the 22- and 26-kDa molecules were glycoconjugates binding to ConA. Preparative sodium dodecylsulfate electrophoresis has allowed the separation of enriched fractions of 22- and 26-kDa molecules which have been injected separately into rats. The corresponding sera were tested in antibody dependent cell cytotoxicity and displayed a significant cytotoxic IgE response (65 and 53%, respectively) towards the larvae. These results lend further support to the view that the 22- and 26-kDa antigens are the major targets of the protective IgE response and thus appear as potentially protective antigens. PMID- 3005175 TI - Clinical analysis of 33 patients with adult T-cell leukemia (ATL)-diagnostic criteria and significance of high- and low-risk ATL. AB - The clinical characteristics of 33 patients with adult T-cell leukemia (ATL) are described. All patients were born and have lived in Miyazaki Prefecture (southwest of Japan). Because of a wide range of clinical presentations and courses, they were subdivided into 2 groups. In the high-risk group, patients presented with high white-cell counts (WBC greater than or equal to 20,000/microliter) and over 30% of abnormal lymphoid cells (18 patients) and hypercalcemia with a low percentage of leukemic cells (5 patients). In this group the median survival time was only 3 months despite various modes of treatment. In contrast, patients of the second group exhibited a low percentage of abnormal lymphoid cells (WBC less than 20,000/microliter and/or leukemic cells less than 30%) and had no hypercalcemia (8 patients). Their clinical course was chronic with a median survival of 8 months, regardless of modalities of treatment. Two patients went through a period when the number of circulating leukemic cells was low (less than or equal to 5%) before overt leukemia appeared. Other clinical features, signs, symptoms, routine laboratory data, serum anti-ATL-associated antibody, cell membrane markers and cytogenetic studies were similar to those observed in other districts of Kyushu island. PMID- 3005176 TI - Epstein-Barr virus status and tumour cell phenotype in sporadic Burkitt's lymphoma. AB - Burkitt's lymphoma (BL) biopsy cells and derived cell lines can be grouped according to their patterns of reactivity with 6 selected monoclonal antibodies (MAbs) against B cell-associated surface antigens. Group I cells react only with MAbs J5 and 38.13, recognising the common acute lymphoblastic leukaemia antigen and a BL-associated antigen respectively; group II cells react with J5 and 38.13 and with one or more of a set of MAbs (Ki-24, MHM6, AC2, Ki-1) against "lymphoblastoid" antigens; group III cells react only with these anti "lymphoblastoid" MAbs. Tumour biopsy cells from 17 cases of sporadic BL, 9 positive for the Epstein-Barr (EB) virus genome and 8 negative, have been analysed during the process of cell line establishment in vitro. In early passage the EB virus-negative BL cells showed either a group I phenotype or gave an additional reactivity with MAb Ki-24 which placed them in group II; these phenotypes remained essentially stable with continued growth of the cell lines for up to 50 passages. By contrast the EB virus-positive BL cells were much more susceptible to phenotypic change in vitro. Although such cells displayed a group I or group II phenotype in early passage, many of the lines soon moved into group III whilst retaining the karyotypic markers indicative of their malignant origin. These observations suggest that a resident EB virus genome can drive the in vitro progression of BL cells towards a more "lymphoblastoid" phenotype. This was confirmed in subsequent experiments where virus-negative BL cell lines were converted to EB virus positivity by in vitro infection. Clearly, therefore, phenotypic analysis of long-established lines can lead to false distinctions being drawn between the EB virus-positive and -negative forms of sporadic BL; both may derive from the same sub-population of target B cells in vivo. PMID- 3005177 TI - Epstein-Barr viral serology in nasopharyngeal carcinoma patients in the USSR and Cuba, and its value for differential diagnosis of the disease. AB - Serological responses to Epstein-Barr virus (EBV)-associated antigens were studied in nasopharyngeal carcinoma (NPC) patients in 2 countries non-endemic for the disease: the USSR (77 cases) and Cuba (55 cases). Two age- and sex-matched control groups were available, one consisting of patients with other head-and neck tumours (OHNT) (171 from the USSR and 56 from Cuba), and the other of normal individuals (blood donors) (83 from the USSR and 80 from Cuba). Unlike the control groups, NPC patients from both countries had high levels of IgG and IgA antibodies, similar to those seen in patients from endemic areas. The only difference between NPC patients in the USSR and those in Cuba was lower (2-2.5 fold) anti-VCA IgG and IgA antibody titres. Using one-factor and multi-factor statistical methods the diagnostic value of different titres of EBV-specific IgG and IgA antibodies and their combinations for NPC patients (in the USSR) was evaluated. It was found that with simple mathematical analysis of EBV-specific antibody titres a differential diagnosis of NPC could be made to a significance level of 90%. The data obtained demonstrated the importance and reliability of EBV serology in the diagnosis of NPC in areas of low incidence of the disease. PMID- 3005179 TI - Characteristics of in vitro transformed cells essential for their in vivo survival, selection and metastasizing activity. AB - Two questions were investigated: (1) the dynamics of in vivo selection of tumor cells possessing metastasizing activity (MA) at the time preceding the appearance of visible lung metastases, and (2) the possible connection of in vivo selection of metastatic tumor cells with their susceptibility to H2O2 produced by the cytotoxic oxidative burst of activated neutrophils, monocytes and macrophages. Spontaneously in vitro transformed Syrian hamster embryo cells (strain STHE), never selected in vivo and possessing low MA, were used as parental cells. STHE cells were injected i.v. and, after various time intervals (1 to 21 days), isolated from lungs of injected hamsters. A total of 43 STHE cell variants were isolated from individual animals. Their MA, natural-resistance-depressing (NRD) activity and susceptibility to H2O2 were determined. Selection of metastatic tumor cell variants capable of forming experimental metastasis (EM) begins a few hours after injection. The vast majority of injected cells die and the few cells which survive do not proliferate for a long period. The majority of variants isolated at days 5 to 6 after injection and later possess increased MA, depending on the duration of the in vivo selection, and varying among individual hosts. The ability to form spontaneous metastasis (SM) in the lungs and in other organs appears among cell variants isolated at days 10 to 21 after inoculation. This qualitatively new characteristic of cell variants correlates with the restoration of their proliferative activity and in most cases with their NRD activity. With respect to susceptibility to H2O2, parental STHE cells and their 5 in vivo isolated cell variants, all possessing low MA, were highly susceptible to H2O2 injury; 15 highly-metastatic variants were 10 to 200 times more resistant; and 4 variants obtained from s.c. transplants of parental cells were highly resistant to H2O2. Hamster embryo cells in vitro transformed with SV40 or bovine adenovirus type 3, were susceptible to high doses of H2O2 (14-28 mM) but resistant to lower doses. In contrast, the same cells, once passaged in vivo and the cells of tumors induced in vivo by the same viruses were resistant to high doses of H2O2. In vitro transformed cells and their in vivo selected variants thus appear to differ widely in terms of their susceptibility to H2O2. Extremely rare cell variants which are resistant to H2O2 and apparently present in the original populations of in vitro-transformed cells possess an essential selective advantage in vivo, that is resistance to H2O2 appears necessary for in vivo survival of tumor cells and their growth in s.c. nodules and metastases. PMID- 3005178 TI - Transmission of human T-cell leukemia virus (HTLV-I) by blood transfusion: demonstration of proviral DNA in recipients' blood lymphocytes. AB - Human T-cell clones bearing antigens encoded by human T-cell leukemia/lymphoma virus (HTLV-I) were isolated from 6 patients who produced antibodies against HTLV I after having received anti-HTLV-I-positive blood units containing cell components. On the other hand, it was not possible to isolate clonal cells carrying viral antigens from the recipients who did not produce antibodies. The clonal cell lines had the same surface markers as neoplastic cells of adult T cell leukemia and had the HLA phenotype of the recipients themselves. Proviral DNA of HTLV-I was demonstrated in each of the clonal cell lines. The site of integration was different in each case even if the clones were derived from the same recipient. These results indicate that blood transfusion can cause persistent HTLV-I infection. PMID- 3005180 TI - Use of artificial liver support. AB - Artificial liver support is used to remove toxic substances accumulating in the circulation of patients with fulminant hepatic failure. Charcoal haemoperfusion with infusion of prostacyclin (PGI2) to prevent platelet damage has become a routine treatment and has led to improved survival. In 76 patients treated 29 (38%) survived to leave hospital and of these 31 patients were treated when in Grade III coma and 20 (65%) survived. Charcoal haemoperfusion also reduced the incidence of cerebral oedema which is a major cause of death in fulminant hepatic failure. Most episodes (congruent to 80%) of cerebral oedema can be managed using the osmotic agent mannitol. In a complete liver support system both protein-bound and compounds of a middle relative molecular mass as well as water soluble compounds should be removed. An albumin-coated resin has been developed to remove these compounds and in preliminary clinical studies it has been shown to have good blood compatibility and satisfactory adsorption properties. The importance of combined systems has been confirmed in studies on the removal of inhibitors of brain Na+K+-ATPase, where both resin and charcoal columns removed significant amounts of the inhibitory activity. PMID- 3005181 TI - Pharmacodynamics and population pharmacokinetics of enalapril and lisinopril. AB - The di-acid metabolite of enalapril, enalaprilat, and its lysine analogue lisinopril are potent inhibitors of angiotensin converting enzyme (ACE); they do not contain sulphydryl groups. Both drugs can be assayed by high pressure liquid chromatography and by radioimmunoassay and plasma ACE inhibition remains stable under normal storage conditions. It is therefore possible to study their pharmacokinetics as well as their pharmacodynamic effects in man. Enalaprilat and lisinopril as well as ACE activity have been measured in blood taken during the course of two studies of the effects of these drugs on blood pressure and autonomic responsiveness. A population pharmacokinetic analysis approach applied to a few concentration-time data points in each of a relatively large number of subjects provided average population parameter estimates of the absorption rate constant, volume of distribution and clearance which correspond closely with the limited published data based on conventional pharmacokinetic approaches. It also provided estimates of pharmacodynamic parameters and the concentration of the drug required to produce a 50% ACE inhibition. Population drug concentration data obtained in the course of early clinical evaluations of new drugs may provide a rational basis for dosage regimens with improved efficacy and, in particular, reduced concentration-related toxic effects. PMID- 3005182 TI - Angiotensin-converting enzyme inhibitors in hypertension: a review. AB - Angiotensin-converting enzyme (ACE) inhibitors are a new class of drugs, whose main indications are the treatment of hypertension and of heart failure. Data obtained with captopril, the first orally active ACE inhibitor, affords an understanding of the rationale of their therapeutic use based on the knowledge of their mechanisms of action, efficacy, contraindications and precautions, dosage and frequency of administration, side-effects, interactions and advantages. ACE inhibitors appear to exert their haemodynamic effect mainly by inhibiting the renin-angiotensin-aldosterone system, but also by modulating sympathetic nervous system activity and by increasing prostaglandin synthesis. Therefore they act both on vasoconstrictor and volume factors, since they cause vasodilation (the main effect) and mild natriuresis without affecting the heart rate and contractility and, probably, favourably influencing renal, coronary and cerebral circulation. So far it appears that ACE inhibitors can be usefully employed in the treatment of heart failure, in which they reduce both pre- and after-load, and mainly of hypertension. In the past captopril has been used to treat only severe and or resistant hypertension and some secondary forms, like renal parenchymal and renovascular hypertension, but now it seems that captopril is useful also to treat mild to moderate essential hypertension. Their efficacy in reducing blood pressure is similar to that of thiazide diuretics and of beta blockers, the two drugs now considered of first choice and they exert their hypotensive action without the development of pseudotolerance or tolerance. ACE inhibitors seem, at the moment, contraindicated in pregnancy and in hyperkalaemic syndromes and must be used with caution in patients with collagen disease (mainly associated with renal failure), with severe bilateral renal artery stenosis (and with severe artery stenosis of a solitary kidney) and with severe sodium depletion. It is now established that captopril has a flat dose response curve and that it must be given (twice daily) at a dose not exceeding 150 mg/day. The same pharmacological approach must be used with future ACE inhibitors in order to establish the right posology and the frequency of administration. In this respect enalapril seems to be a promising ACE inhibitor with a prolonged action (at least 24 hours). The exact posology of ACE inhibitors might be crucial, since it has been shown that the side-effects of captopril (skin rashes, fever, taste disturbances, proteinuria and neutropenia) are dose dependent.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3005183 TI - Herpetiform protothecosis. PMID- 3005184 TI - Enzymes of adenosine metabolism in Hymenolepis diminuta (Cestoda). PMID- 3005185 TI - Synthesis and binding properties of specific photoaffinity ligands for mu and delta opioid receptor subtypes. AB - We report the synthesis and binding properties of specific photoaffinity ligands for mu and delta opioid receptor subtypes. These ligands are derived from DAGO: Tyr-D-Ala-Gly-NMePhe-Gly-ol, a mu selective probe and DTLET: Tyr-D-Thr-Gly-Phe Leu-Thr, a delta selective probe by modifying the Phe 4 residue. These modifications are: i) a nitro group on the para position of Phe ring as Phe(4 NO2) or Nip, ii) an azido group as Phe(4 N3) or AZ. Pharmacological responses on mouse vas deferens (delta sites) and guinea pig ileum (mu sites), as well as competition experiments with [3H] DAGO and [3H] DTLET on crude rat brain membranes have been performed. The nitro group on the phenyl ring of the Phe residue preserves the affinity and selectivity of each probe: NipDAGO for the mu sites, NipDTLET for the delta ones. However the nitro probes do not appear to be photoactivable by u.v. irradiation. Likewise, azidation of the phenyl ring of the Phe residue does not change the receptor selectivity of each probe, but AZDAGO has less affinity than its parent molecule DAGO, while AZDTLET has more affinity than DTLET. These compounds are photoactivable and provide an efficient tool to characterize and isolate the different receptor subtypes, especially the delta site. PMID- 3005186 TI - Phagocytic activity and bactericidal capacity of polymorphonuclear leukocytes in children with recurrent otitis media. AB - In 19 children with recurrent otitis media the phagocytic activity of blood granulocytes was studied and indirectly also their bactericidal capacity by means of the NBT test and myeloperoxidase activity. The mean value of granulocytes containing latex particles was 44.32% in the patients group, which differed significantly from the mean value established in controls: 66.54%. The mean phagocytic number in the patients group, 1.82 was also significantly diminished in comparison to controls: 2.41. The average number of nitroblue tetrazolium (NBT)-positive cells in patients, 86.74% as well the mean myeloperoxidase index, 2.52 did not differ from the mean control values, 92% and 2.42, respectively. In two patients a lower percentage of NBT-positive cells was determined. They are probably heterozygous carriers of an enzyme deficiency related to a depressed bactericidal activity of phagocytes. Patients with recurrent otitis media should be tested on possible phagocytic dysfunctions which may be helpful in understanding the pathogenesis of infections and also in identifying infants at particular risk of otitis media. PMID- 3005187 TI - Differential effects of dPTyr(Me)AVP, a vasopressin antagonist, upon foot shock analgesia. AB - Acute exposure to either prolonged intermittent foot shock (PIFS) or brief continuous foot shock, (BCFS) decreases the sensitivity of rats to noxious stimuli, but differ in their mechanisms of actions. Since the peptide vasopressin (VP) has been implicated in analgesic and stress-related processes, the present study examined whether antagonism of central VP receptors with dPTyr(Me)AVP would alter the analgesic responses following PIFS or BCFS. While intracerebroventricular administration of dPTyr(Me)AVP, a V1 receptor antagonist, significantly attenuated the analgesic response to PIFS, it potentiated the analgesic response to BCFS. It should be noted that the form of PIFS employed in the present study was not blocked by naloxone. These results are discussed in terms of multiple forms of pain-inhibitory systems that may utilize collateral inhibition as a means of providing selective activation. PMID- 3005188 TI - Biochemical background of the development of gastric mucosal damage in pylorus ligated plus aspirin-treated rats. AB - Gastric mucosal damage was produced in rats after pyloric ligation by intragastric administration of 200 mg/kg aspirin diluted in 2 ml 150 mmol/l HCl. The animals in the control group received 2 ml saline solution, or submitted to pyloric ligation only. The animals were killed 4 h after the pyloric ligation, when the number and severity of gastric lesions (ulcers), and the gastric fundic mucosal level of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), cyclic adenosine monophosphate (cAMP) and lactate, were noted and measured. The adenylate pool (ATP + ADP + AMP) and the energy charge (ATP + 0.5ADP). (ATP + ADP + AMP)-1 were calculated. It was found that: the gastric H+ output decreased significantly in the pylorus-ligated plus aspirin treated animals; the number and severity of gastric lesions increased significantly in the pylorus-ligated aspirin-treated animals; the extent of ATP transformation into the ADP decreased significantly in the pylorus-ligated aspirin-treated animals; the extent of ATP transformation into the cAMP decreased significantly during the aspirin treatment; the values of adenylate pool and of "energy charge" remained unchanged in the different groups of animals. It is concluded that: the decreased H+ output in the pylorus-ligated plus aspirin treated group can be obtained by the decreased extent of ATP transformation into the ADP by membrane ATPase, and the biochemical changes in the gastric mucosa indicate a decreased energy turnover. PMID- 3005189 TI - Immunity of hospital personnel against polio virus. AB - Sera from 275 employees of three hospitals were tested for the presence of antibodies to polio virus. Immunity was found to increase with age, and was higher in personnel directly involved in patient care than in the others. Previous exposure to poliomyelitis as well as a history of previous immunization did not correlate with the immunological status of the employee. When antibodies against one of the three polio types or more could not be demonstrated, the employee was vaccinated by an inactivated polio virus vaccine. PMID- 3005190 TI - Dopamine influences the light peak in the perfused mammalian eye. AB - Dopamine, cis-flupenthixol, and dibutyryl cAMP affected the standing potential and light-evoked responses of the perfused cat eye in vitro. At micromolar concentrations, dopamine, a retinal neurotransmitter, increased the standing potential of the eye. At slightly higher concentrations, dopamine abolished the "light peak," a slow voltage response to light generated by the retinal pigment epithelium. The light peak was depressed by cis-flupenthixol, a dopamine antagonist, at micromolar concentrations. Dibutyryl cAMP produced effects similar to those produced by dopamine. A possible interpretation is that the generation of the light peak involves a catecholamine-stimulated adenylate cyclase in retinal pigment epithelium that is influenced by dopamine released from retinal neurons. PMID- 3005191 TI - Epstein-Barr virus specific antibodies in patients with coeliac disease. PMID- 3005192 TI - Human growth hormone gene deletion without antibody formation or growth arrest during treatment--a new disease entity? AB - Using restriction endonuclease analysis of genomic DNA hybridized to a human chorionic somatomammotropin (hCS) complementary (c)DNA probe, we studied four young Jewish patients with isolated growth hormone deficiency (IGHD), and 15 family members. One family originated in Iraq, two in Yemen and one in Iran. Each patient was homozygous for a deletion of approximately 7.5 kilobases, which included the hGH-N gene. Three of the deletions were associated with the same restriction fragment length polymorphism haplotype, while the deletion in the child of Iranian descent was associated with a different haplotype. All the patients were treated with three injections per week of pituitary human growth hormone (hGH) for periods of 2 1/2 to 14 1/2 years. All had a good growth response. Three reached normal and one almost normal height. Repeated serum analyses revealed absence of anti-hGH antibodies. Thus, the presently described patients differ from those previously reported from Switzerland, Argentina and Japan, all of whom developed anti-hGH antibodies during treatment, with resultant slowing or arresting of growth. Expression of heterozygosity in family members was variable with regard to stature, hGH reserve and insulin-like growth factor I (IGF-I) levels. It is hypothesized that hGH-N gene deletion is not the sole determinant of immune response during hGH treatment, and that the difference between the current series and other cases needs further investigation. PMID- 3005193 TI - Mineral intake and blood levels in vegetarians. AB - Concern has been raised that a long-term high-fiber diet may lead to mineral deficiencies. In this study, mineral intake and blood levels were investigated in 92 ovolacto vegetarians and 113 omnivores. The intake of iron, zinc, calcium and magnesium was adequate in both groups. The intake of iron and magnesium was significantly higher in the vegetarians. Mean blood levels of iron, iron binding capacity, calcium, phosphorus, alkaline phosphatase, zinc and magnesium were within normal limits in both groups. Serum magnesium levels were significantly higher in male vegetarians. Iron binding capacity was significantly lower in vegetarians of both sexes. It is concluded that a long-term ovolacto vegetarian diet does not lead to mineral deficiencies. PMID- 3005194 TI - Case report of two primary tumors: mullerian adenosarcoma and endometrial adenocarcinoma. AB - A rare case of two concomitant primary uterine neoplasmas in a 64-year-old patient who had received conjugated estrogens for the 3 preceding years is presented. Mullerian adenosarcoma, heterologous type, with stromal components suggestive of rhabdomyosarcoma, and adenocarcinoma of endometrium, were localized in different sites of the endometrium with no intermingling between them (combination tumor). The diagnostic and therapeutic problems are discussed. PMID- 3005195 TI - [Retroviruses in dermatology]. AB - Retroviruses have a genomic RNA and can induce malignant tumors. Study of the retroviruses led to the discovery of retroviral oncogenes, which were found to be responsible for the development of malignancies. Later on, cellular genes were detected that are closely related to the viral oncogenes but not oncogenic per se. There is strong evidence that retroviruses are associated with cutaneous lymphomas (HTLV I) and AIDS (LAV/HTLV III). PMID- 3005197 TI - Electron spin resonance dosimetric properties of bone. AB - The characteristics of electron spin resonance (ESR) dosimetry using bovine bone samples are described. The number of paramagnetic centers created by gamma radiation in the inorganic bone matrix was measured as a function of absorbed dose. The minimum detectable dose was 0.5 Gy for 60Co gamma rays. The response was linear up to the maximum dose studied (30 Gy) and independent of dose rate up to the maximum dose rate used (1.67 Gy min-1). For different bone samples the reproducibility was 5%. This method may be valuable for nuclear accident dosimetry. PMID- 3005196 TI - Influence of oxidizing or reducing agents on gastrointestinal absorption of U, Pu, Am, Cm and Pm by rats. AB - Absorption of 233U, 238Pu, 241Am, and 244Cm from the gastrointestinal (GI) tract was measured in rats, fed ad libitum or fasted, that were gavaged with solutions containing ferric iron, ferrous iron, iron powder, quinhydrone or ascorbic acid. Absorption and retention of all of these actinides was increased substantially by fasting and by the addition of mild oxidizing agents, ferric iron and quinhydrone. In contrast, absorption and retention were decreased to below the fasted level by all the reducing agents except ascorbic acid, which caused diarrhea and an increase in absorption. Absorption of the lanthanide element 147Pm from the intestine of fasted rats was also increased by ferric iron. Some of these actinide elements are polyvalent and are, in some cases, known to be absorbed from the GI tract more readily in their higher oxidation states. This suggested an oxidation-reduction mechanism for the effect of fasting and the action of the chemical agents used. However, the improbability that either 241Am(III) 244Cm(III) or 147Pm is converted to a different oxidation state under these conditions makes that mechanism unlikely. Other explanations are suggested. PMID- 3005198 TI - Nutritional goals from COMA and NACNE: how can they be achieved? AB - A simple way of altering the average British diet to make it comply with the short-term NACNE nutritional goals was discovered by comparing the diet of a cross-section of Cambridge adults with that of a subgroup whose diet approached the desired objectives. Between 1977 and 1979, 105 men and 112 women aged 18-57 completed 7-day semi-weighed records of food consumption. Although no subject achieved all of the NACNE or COMA goals, seven men and three women had diets in which fat provided less than 35 per cent of energy, alcohol less than 10 percent, with fibre more than 20 g/day; total sugars provided 25 per cent of energy. When these diets were compared with those of the rest of the sample (excluding diets where alcohol provided more than 10 per cent of energy), it was seen that men and women in the low-fat, high-fibre group ate more white bread, high-fibre breakfast cereals, low-fat meat dishes, and vegetables, and less brown bread, biscuits, eggs, poultry and fried fish. Only small changes were needed to make the average Cambridge diet consistent with the short-term NACNE goals for fat and fibre. Achieving the COMA or long-term NACNE goals will require more radical change, based on strategies to provide the necessary foodstuffs and nutrition education to allow informed individual choice. PMID- 3005200 TI - The response of atelectasis from lung cancer to radiation therapy. AB - Between January 1981 and June 1983, 33 newly diagnosed patients with lung cancer presented with radiological findings of atelectasis. These patients were treated by primary radiation therapy, with doses ranging from 1200 to 6000 cGy. The response of atelectasis to radiation therapy was established on the basis of follow-up chest roentgenograms. Of the 28 patients with non-small cell carcinoma of lung, there were 17 (61%) who had improvement of the atelectasis. Among these, 13 patients were treated with doses ranging from 5000 to 6000 cGy in 5 to 8 weeks; 9 of these (70%) responded. By histological subtype, the numbers, though small, show that three of eight patients with adenocarcinoma responded, as compared to 2 out of 4 with large cell undifferentiated carcinoma and 12 of 16 patients with squamous cell carcinoma. In patients treated by more than 5000 cGy, four of eight (50%) patients with squamous cell carcinoma had a complete response and three (37.5%) had a partial relief of atelectasis, for a total response of 87.5%. The study indicates the importance of radiation therapy in the management of atelectasis caused by primary lung cancer. PMID- 3005199 TI - A monoclonal antibody that recognizes the alpha chain of HLA-DR antigens. AB - A monoclonal antibody, HC2.1, has been generated that specifically reacts with both the denatured and the in vitro translated alpha chain of the DR antigen. Although HC2.1 antibody reacted with the alpha chain of protein immunoprecipitated by two DR-specific monoclonal antibodies, L227 and LB3.1, it did not react with the alpha chain of the DQ1 antigen immunoprecipitated by the monoclonal antibody, Genox 3.53. The isoelectric focusing pattern of the alpha chain precipitated by HC2.1 antibody was invariant across a range of DR specificities within a panel of lymphoblastoid cells. The alpha chain of DR antigen from a B cell line was purified by HC2.1-Sepharose immunoaffinity chromatography and limited amino acid sequence analysis was carried out with Staphylococcus aureus SV8 protease fragments purified by high-pressure liquid chromatography. The sequence analysis confirmed that the antigen reactive with HD2.1 antibody is encoded by the DR alpha chain gene. PMID- 3005201 TI - 90Yttrium antiferritin--a new therapeutic radiolabeled antibody. AB - A new radiolabel 90Yttrium has been chelated to antiferritin antibodies for the treatment of hepatocellular cancer. The isotope 90Yttrium has the advantage of no significant external radiation to other individuals, that is, outpatient therapy and potentially more therapeutic power with an increase from 0.3 Mev 131I beta radiation to 0.9 Mev 90Yttrium pure beta radiation. Six patients treated in the Phase I study have had modest hematologic toxicity and two have had partial remissions of their primary tumors. One of these patients has had complete remission of a pulmonary metastasis. The use of external radiation (900 rad) to the primary tumor in advance of radiolabeled antibody administration has increased antibody uptake and increased tumor dose rate and total dose. An extensive study of 90Yttrium antiferritin is planned. PMID- 3005202 TI - Evaluating the reliability of diagnostic test results. PMID- 3005203 TI - The p40x of human T-cell leukemia virus type I is a trans-acting activator of viral gene transcription. AB - Human T-cell leukemia virus type I has a unique sequence pX and the product p40x was proposed to be a specific trans-acting transcriptional activator of expression of the viral gene. Recently, a second pX protein p27x-III in addition to p40x was identified; these two proteins are encoded by overlapping frames III and IV (x-lor). For determination of which product is the trans-acting activator, site-directed mutations were introduced into the pX sequence which was placed under the metallothionein promoter. On cotransfection with pLTR-CAT (a plasmid containing the LTR of HTLV-I and chloramphenicol acetyltransferase gene), only the mutations that affected p40x expression inactivated the transcriptional activation from the LTR. PMID- 3005204 TI - Myristylation of gag protein in human T-cell leukemia virus type-I and type-II. AB - We found that p19gag of HTLV-I and p23gag of HTLV-II are myristylated. The p28, which is immunologically cross-reactive with monoclonal antibody against p19gag of HTLV-I was also shown to be myristylated in the HTLV-I-infected cell lines MT 2 and HUT102. However, no myristylated p28 was found in HTLV-II-infected cell lines, Mo and Ton1. PMID- 3005206 TI - Oral infection of a common marmoset with human T-cell leukemia virus type-I (HTLV I) by inoculating fresh human milk of HTLV-I carrier mothers. AB - To obtain definitive evidence that milk-borne infection plays a critical role in the endemy or mother-to-child transmission of human T-cell leukemia virus type-I (HTLV-I), we inoculated concentrated fresh human milk cells obtained from HTLV-I carrier mothers into the oral cavity of a common marmoset (Callithrix jacchus). Twenty-eight milk samples were collected (5-10 ml each) from 17 carrier mothers in the first week after delivery. Cells in the milk were centrifuged down and resuspended in 1/10 vol of the milk fluid. The concentrated cell suspensions were successively inoculated into the oral cavity of a common marmoset. The marmoset was found to be seroconverted by indirect immunofluorescence assay at 2.5 months after the first inoculation of the milk (3.5 X 10(8) cells in total), and was later confirmed to be infected with HTLV-I by the detection of viral antigen expression in short-term cultures of its peripheral blood T-lymphocytes. The results strongly support the working hypothesis that milk-borne infection plays a significant role in the mother-to-child transmission of HTLV-I. PMID- 3005207 TI - Casein and histone kinases of a rat ascites hepatoma as compared with those of rat liver. AB - Seryl/threonyl-protein kinases in cytosolic and particulate fractions from rat liver and AH-13, a rat ascites hepatoma, have been studied by chromatographing these fractions on DEAE-cellulose and assaying the eluates with casein, phosvitin, histone and protamine as substrates. Liver cytosolic fraction contains a group of well-characterized seryl/threonyl-protein kinases, namely, casein kinases I and II and histone kinases I and II. Liver particulate fraction, on the other hand, is almost totally devoid of casein kinase I and histone kinase I but contains an additional peak of casein kinase tentatively designated casein kinase III. In AH-13, cytosolic casein kinase I is markedly increased and particulate associated casein kinases II and III are moderately increased as compared with liver. Moreover, it was found that in AH-13, the histone kinase I level is high in the particulate fraction but markedly decreased in the cytosolic fraction. It is suggested that particulate-associated histone kinase I may be of cytosolic origin. PMID- 3005205 TI - Hypomethylation of c-myc and epidermal growth factor receptor genes in human hepatocellular carcinoma and fetal liver. AB - The methylation state of cellular oncogenes (c-oncs) and epidermal growth factor (EGF) receptor gene from human liver tissues was examined by means of restriction endonuclease analysis. c-myc and EGF receptor gene from hepatocellular carcinoma and fetal liver were substantially hypomethylated in comparison with those genes from normal liver, while the extents of methylation of c-mos and c-Ki-ras genes were the same among these tissues. It can be speculated that the specific hypomethylation of c-myc and EGF receptor genes may be associated with the development of hepatocellular carcinoma. PMID- 3005208 TI - Creatine kinase B subunit as a biomarker for small cell carcinoma of the lung: comparison with gamma-enolase. AB - Concentrations of creatine kinase (CK) B subunit (CK-B) in tumor tissues and in sera of patients with various lung carcinomas were determined, together with the concentrations of neuron-specific gamma-enolase (gamma subunit of a gamma and gamma gamma enolases), by the use of a sensitive enzyme immunoassay method. The CK-B and gamma-enolase levels were enhanced in tissues of small cell carcinoma of the lung. The average tissue contents of CK-B in small cell carcinoma (SCCL), adenocarcinoma (ADCL) and squamous cell carcinoma (ECCL) of the lung, and normal lung were 2320, 308, 163, and 372 ng/mg protein, respectively. The contents of gamma-enolase in those tissues were 1460, 276, 225, and 42.7 ng/mg protein, respectively. Serum CK-B concentrations in healthy adults (n = 100) were 0.53 +/- 0.22 ng/ml and ranged from 0.25 to 1.44 ng/ml, but they were significantly increased (greater than 1.5 ng/ml) in some patients with SCCL (26/42 cases, 62%), ADCL (7/36, 19%), ECCL (7/37, 19%), and large cell carcinoma of the lung (LCCL, 4/13, 31%). Serum CK-B was also enhanced in some patients with breast carcinoma and in a few cases in carcinomas of the stomach, colon and pancreas. Serum concentrations of CK-B were well correlated with those of gamma-enolase in patients with SCCL (r = 0.667, n = 83, P less than 0.01) and LCCL (r = 0.689, n = 20, P less than 0.01), but poorly in patients with ADCL and ECCL. Since serum CK B concentrations in patients with SCCL changed in parallel with the clinical course during treatment, serum CK-B may also be a useful biomarker, as well as neuron-specific gamma-enolase, for monitoring the clinical course of patients with SCCL. PMID- 3005209 TI - A close association of adriamycin resistance with expression of cell surface antigens in adriamycin-resistant cell lines of herpes simplex virus type 2 transformed Syrian hamster cells. AB - We determined the properties of three adriamycin (ADM)-resistant cell lines isolated from a clone of herpes simplex virus (HSV) type 2-transformed Syrian hamster cells. The ADM-resistant lines (ADMR-6, -7 and -9) established by continuous exposure of the cells to ADM were 20 to 30 times more resistant to ADM than was the parent line. These resistant lines exhibited cross-resistance to daunomycin, actinomycin D, chromomycin A3 and vincristine, but not to mitomycin C, bleomycin or methotrexate. Uptake and efflux studies with [3H]ADM indicated that one line (ADMR-9) incorporated and retained lesser amounts of [3H]ADM than did the parent line but the other two lines accumulated and retained as much of the drug as did the parent line. Expression of cell surface antigens, detected by immunofluorescence tests with antiserum to HSV type 1, was at a low level (about 8% positive) in the parent line after replating of the cells, although it was enhanced by treatment of the cells with ADM (0.25 micrograms/ml). However, the antigens were strongly expressed (greater than or equal to 35% positive) on the surface of cells of the three resistant lines in the absence of ADM. Both antigen expression and ADM resistance of the cells were relatively stable in in vitro cultivation but were unstable in in vivo cultivation, suggesting that the constitutive expression of the antigens is closely associated with the phenotype of ADM resistance. PMID- 3005210 TI - Mechanism of the cytotoxic effect of tumor necrosis factor. AB - The mechanism of murine tumor necrosis factor (TNF) cytotoxicity against tumor cell lines (L929, HeLa, K562) was investigated. Electron microscopic observation revealed that most of the organellas of L929 cells incubated with partially purified murine TNF underwent almost complete lysis with no drastic disruption of the cytoplasmic membrane, while injection of the TNF into the cytoplasm or nuclei of L929 cells caused no apparent morphological change or growth inhibition. Preincubation of the TNF with tumor cells (L929, HeLa, K562) resulted in a decrease in cytotoxic activity which was proportional to their susceptibility to TNF, thus indicating their absorption of TNF. The susceptibility of L929 tumor cells to TNF was apparently suppressed by treatment with proteases, suggesting the existence of protease-sensitive recognition sites for TNF on the tumor cell. PMID- 3005211 TI - Treatment of primary radiogenic C57BL mouse cell leukemia/lymphoma by 1,3-bis(2 chloroethyl)-1-nitrosourea chemotherapy and adjuvant cellular therapy. AB - Primary radiation-induced or radiation leukemia virus (RadLV)-induced T leukemias/lymphomas were treated in vivo in an early to advanced state by using 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU was given at various times after the tumor induction procedure. Death from RadLV lymphomas which had been initiated in 33 +/- 3 day old C57BL mice by intrathymic injection of RadLV was scored in untreated, BCNU-treated or BCNU and cellular adjuvant treated mice. Intrathymic RadLV injection in 33 +/- 3 day old mice produced tumors in 98% of injected mice. Median survival time (MST) was increased by BCNU and by BCNU plus bone marrow cell therapy whether done 33 or 47 days after RadLV. There was increased in MST from 108 days to 171 days by BCNU and bone marrow cell therapy given 33 days after tumor initiation and to 195 days when therapy was given 47 days after initiation. In radiation-induced lymphomas produced by 190 rad every week X 4 of 33 +/- 3 day old mice, spleen cell (X 1) therapy or BCNU treatment increased the MST of treated mice from 142 days to 177 days after iv spleen cells or to 195 days after iv-ip spleen cells, and this protocol produced 31% long-term cures. Cellular adjuvant therapy combined with BCNU chemotherapy was effective for curing the lymphomas but cellular adjuvant therapy alone was also highly effective for therapy. PMID- 3005212 TI - Asperlicin, a novel non-peptidal cholecystokinin antagonist from Aspergillus alliaceus. Fermentation, isolation and biological properties. AB - The fermentation and isolation of a new, non-peptide cholecystokinin antagonist, asperlicin, produced by Aspergillus alliaceus is described. The potent and specific interaction of asperlicin with cholecystokinin receptors was shown using in vitro biochemical assays. PMID- 3005213 TI - Studies on antiviral agents. III. Synthesis and in vitro antiviral activity of 1 N-higher-acyl-3"-N-functionalized acylkanamycin A derivatives. AB - The synthesis and antiviral activity of 1-N-palmitoyl- or 1-N-(3 hydroxytetradecanoyl)-kanamycin A derivatives (7,8) having various type of acyl substituents at the N-3" position were investigated. The structure-activity relationships between the antiviral activity and the substituent at the N-3" position is described. In this series, 3"-N-acetyl-1-N-palmitoyl-kanamycin A (7c) showed the excellent antiviral activity against HSV-I and influenza virus. Further, we examined the synthesis and the antiviral activity of 3"-N glycylkanamycin A derivatives (9) having a higher-acyl group at the N-1 position. The 3"-N-glycyl-1-N-pentadecanoylkanamycin A (9a) also exhibited excellent antiviral activity. PMID- 3005214 TI - Immunoactive peptides, FK 156 and FK 565. IV. Activation of mouse macrophages. AB - We investigated the effects of the immunoactive peptides, FK 156 and FK 565, on functions of mouse macrophages. FK 156 and FK 565 given parenterally or orally to mice enhanced spreading of peritoneal macrophages, phagocytosis of latex particles and intracellular killing of bacteria by peritoneal macrophages. FK 156 and FK 565 also enhanced the production of superoxide anion and lysosomal enzyme activities of macrophages. The peptides also activated mouse spleen macrophages, and the kinetics of this activation differed from that of the peritoneal macrophages. In addition, both drugs directly enhanced the production of superoxide anion by mouse peritoneal macrophages treated in vitro and enhanced the functions of peritoneal macrophages of athymic nude mice. Both these phenomena suggest that direct activation might be one of the mechanisms of macrophage activation by the peptides. PMID- 3005215 TI - Identification of urinary metabolites of rifamycin LM 427 in man. PMID- 3005216 TI - Foroxymithine, a new inhibitor of angiotensin-converting enzyme, produced by actinomycetes. PMID- 3005218 TI - K-26, a novel inhibitor of angiotensin I converting enzyme produced by an actinomycete K-26. AB - A novel inhibitor of angiotensin I converting enzyme (ACE), designated K-26, was isolated from the broth filtrate of an actiomycete K-26. K-26 is a water soluble, acidic peptide composed of an equal mol of L-isoleucine, L-tyrosine and 1(R)-1 amino-2-(4-hydroxyphenyl)-ethylphosphonic acid. The IC50 of K-26 for ACE inhibition was 6.7 ng/ml when hippuryl-L-histidyl-L-leucine was used as a substrate of ACE. K-26 possesses hypotensive activity in vivo. PMID- 3005219 TI - Recent advances in the evolution of drug resistance. PMID- 3005217 TI - Studies on a new epidermal growth factor-receptor kinase inhibitor, erbstatin, produced by MH435-hF3. PMID- 3005220 TI - An investigation of the hydrophobicity of the quinolones. PMID- 3005221 TI - In-vitro susceptibility of beta-lactamase-producing Haemophilus influenzae to the combinations ampicillin with mecillinam and ampicillin and VD2085. PMID- 3005222 TI - In-vitro susceptibility of the Bacteroides fragilis group to cefoperazone, ampicillin, ticarcillin and amoxycillin combined with beta-lactamase inhibitors. PMID- 3005223 TI - Phosphomannosyl receptor in bovine and human tissues determined by a sensitive radioimmunoassay method. AB - A highly sensitive radioimmunoassay method has been established for the phosphomannosyl receptor using an antibody to the bovine receptor. The amount of the receptor in extracts from total membrane fractions varied remarkably in different tissues and species. The amount in liver was the highest and that in brain was the lowest among bovine tissues. Human tissues contained significant amounts of material cross-reacting to the antibody, the highest in spleen and the lowest in kidney. The physiological significance of the receptor is discussed in terms of the intracellular transport of lysosomal enzymes in human tissues. PMID- 3005224 TI - Monovalent cation-insensitive hydrophobic region on calmodulin facilitates the rapid isolation and quantitation of calmodulin free from other Ca2+-dependent hydrophobic proteins. AB - Calmodulin binds quantitatively to phenyl-Sepharose through its Ca2+-induced hydrophobic binding region. Troponin C and S-100 protein, as well as several other proteins present in rat tissues, also bind to phenyl-Sepharose in a Ca2+ dependent manner. While the Ca2+-dependent binding of calmodulin to phenyl Sepharose is not altered appreciably by monovalent cations, they do appear to compete for Ca2+ binding to most of the other proteins, including S-100 protein, which exhibit Ca2+-induced interaction with phenyl-Sepharose. The selective elution of these proteins from the phenyl-Sepharose column can be achieved with a 0.5 M concentration of monovalent cations such as K+, Na+, and NH4+ in the presence of a low (100 microM) Ca2+ concentration. Calmodulin-binding proteins associated with calmodulin in crude cell extracts can prevent the interaction of calmodulin with the phenyl-Sepharose, resulting in low recoveries of calmodulin from these tissues. The majority of these interfering proteins are heat labile so that heat treatment (boiling) of the cell extract for a limited time (5 min) negates any binding of these proteins to calmodulin and allows the quantitative recovery of calmodulin by hydrophobic interaction chromatography. This procedure allows the rapid and quantitative recovery of highly purified calmodulin from both cytosolic and Triton X-100-solubilized particulate fractions prepared from various rat tissues. Calmodulin isolated in this manner can be accurately and reliably quantitated by direct protein determination with Coomassie brilliant blue dye or fluorescamine or by the cyclic nucleotide phosphodiesterase stimulation assay. PMID- 3005225 TI - The alveolar macrophage. AB - The alveolar macrophage is one of the few tissue macrophage populations readily accessible to study both in the human and in animals. Since harvesting of these cells by bronchoalveolar lavage was first described in 1961, alveolar macrophages have been extensively investigated. This population is the predominant cell type within the alveolus, and undoubtedly serves as the first line of host defense against inhaled organisms and soluble and particulate molecules. Early studies focussed on this endocytic role and delineated the cells' phagocytic and microbicidal capacities. More recent investigations demonstrated an extensive synthetic and secretory repertoire including lysozyme, neutral proteases, acid hydrolases and O2 metabolites. In addition, the complex immunoregulatory role of the macrophage has also been appreciated. These cells have been shown to produce a wide variety of pro- and anti-inflammatory agents including arachidonic acid metabolites of the cyclooxygenase and lipoxygenase pathways, cytokines which modulate lymphocyte function and factors which promote fibroblast migration and replication. PMID- 3005228 TI - Serum angiotensin converting enzyme in respiratory diseases. PMID- 3005227 TI - Hepatic infusional chemotherapy in metastatic colorectal carcinoma and hepatoma. PMID- 3005229 TI - [Principles in the follow-up of breast fibroadenomas]. PMID- 3005226 TI - Induction of tumorigenesis and chromosomal abnormalities in human amniocytes infected with simian virus 40 and Kirsten sarcoma virus. AB - Cell cultures of epithelial-like human amniocytes were infected with simian virus 40 (SV40) and Kirsten sarcoma virus (KSV) in various sequential orders, and tested for agar growth, chromosome abnormalities, and tumorigenesis in the nude mouse assay. We observed that regardless of the order in which the viruses were introduced, the doubly infected cells always exhibited the typical SV40 premalignantly transformed phenotype before changing to the malignant phenotype. All doubly transformed cells from different cell donors produced tumors in adult and suckling nude athymic mice, classified as poorly differentiated sarcomas. Infection with SV40 alone conferred extended life span and accelerated growth without the malignant capability of tumor production. Kirsten sarcoma virus alone produced only focal cell alterations with no change in cell longevity or tumorigenesis. Chromosome studies of the premalignant and malignant cells from one cell donor did not reveal any significant clonal development for marker chromosomes in either cell line. Chromosome 12, which carries the homologous cellular oncogene to KSV, had no increase in aberrations in the malignant cells. Chromosome 8 was most often involved in aberrations, and the most frequent aberration for both series was dicentric chromosomes due to telomere fusion. For other translocations the breakpoints were almost exclusively in the centromere regions. The vulnerability of telomere and centromere regions to the free virus present in these precrisis cells is discussed, and similarities in regard to types of aberrations in transfection experiments are noted. PMID- 3005230 TI - [Radioisotope imaging in cardiology]. PMID- 3005231 TI - Isolation and sequence analysis of the gene (cpdB) encoding periplasmic 2',3' cyclic phosphodiesterase. AB - The cpdB gene encodes a periplasmic 2',3'-cyclic phosphodiesterase (3' nucleotidase). This enzyme has been purified previously and the gene is located at 96 min on the Escherichia coli chromosome. In this study the cpdB gene was cloned from ClaI-cleaved DNA, and the gene product was identified. DNA blotting experiments showed that the recombinant plasmid contains a deletion with respect to the expected genomic fragment of approximately 4 kilobases, which extends into the vector. Furthermore, the gene was absent from three other recombinant libraries. Together, these findings suggest the presence in the genome of an adjacent gene whose product is lethal when it is present on a multicopy plasmid. The nucleotide sequence of the cpdB gene was also determined. The 5' and 3' untranslated sequences contain characteristic sequences that are involved in the initiation and termination of transcription, including two possible promoters, one of which may contain two overlapping -10 sequences. A strong Shine-Dalgarno sequence is followed by an open reading frame which corresponds to a protein having a molecular weight of 70,954. The first 19 amino acid residues have the characteristics of a signal peptide. The 3' untranslated sequence contains two putative rho-independent transcription terminators having low thermodynamic stability. PMID- 3005232 TI - Gene sequence encoding early enzymes of arginine synthesis within a cluster in Bacillus subtilis, as revealed by cloning in Escherichia coli. AB - From a partial Sau3A gene library of Bacillus subtilis chromosomal DNA in the expression plasmid pRK9, four hybrid plasmids were isolated carrying overlapping segments of the argA-argF-cpa cluster. The complementation patterns within Escherichia coli arginine auxotrophs of these hybrids and deletion derivatives provided the gene order argC-argA-argE-argB-argD-cpa-argF. PMID- 3005233 TI - Plasmid-mediated tetracycline resistance in Campylobacter jejuni: expression in Escherichia coli and identification of homology with streptococcal class M determinant. AB - Plasmid pUA466, a 45-kilobase transmissible tetracycline resistance plasmid from Campylobacter jejuni was mapped with AvaI, AvaII, BclI, HincII, PstI, XhoI, and XbaI. The resistance determinant was cloned and expressed in Escherichia coli and was homologous with a class M determinant from Streptococcus spp. PMID- 3005234 TI - Molecular cloning of genetic determinants for inhibition of fungal growth by a fluorescent pseudomonad. AB - Pseudomonas fluorescens HV37a inhibits growth of the fungus Pythium ultimum in vitro. Optimal inhibition is observed on potato dextrose agar, a rich medium. Mutations eliminating fungal inhibition were obtained after mutagenesis with N methyl-N'-nitro-N-nitrosoguanidine. Mutants were classified by cosynthesis and three groups were distinguished, indicating that a minimum of three genes are required for fungal inhibition. Cosmids that contain wild-type alleles of the genes were identified in an HV37a genomic library by complementation of the respective mutants. This analysis indicated that three distinct genomic regions were required for fungal inhibition. The cosmids containing these loci were mapped by transposon insertion mutagenesis. Two of the cosmids were found to contain at least two genes each. Therefore, at least five genes in HV37a function as determinants of fungal inhibition. PMID- 3005235 TI - Isolation of a mutation resulting in constitutive synthesis of L-fucose catabolic enzymes. AB - A ribitol-positive transductant of Escherichia coli K-12, JM2112, was used to facilitate the isolation and identification of mutations affecting the L-fucose catabolic pathway. Analysis of L-fucose-negative mutants of JM2112 enabled us to confirm that L-fucose-1-phosphate is the apparent inducer of the fucose catabolic enzymes. Plating of an L-fuculokinase-negative mutant of JM2112 on D-arabinose yielded an isolate containing a second fucose mutation which resulted in the constitutive synthesis of L-fucose permease, isomerase, and kinase. This constitutive mutation differs from the constitutive mutation described by Chen et al. (J. Bacteriol. 159:725-729, 1984) in that it is tightly linked to the fucose genes and appears to be located in the gene believed to code for the positive activator of the L-fucose genes. PMID- 3005236 TI - Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant. AB - A recombinant plasmid carrying the recA gene of Vibrio cholerae was isolated from a V. cholerae genomic library, using complementation in Escherichia coli. The plasmid complements a recA mutation in E. coli for both resistance to the DNA damaging agent methyl methanesulfonate and recombinational activity in bacteriophage P1 transductions. After determining the approximate location of the recA gene on the cloned DNA fragment, we constructed a defined recA mutation by filling in an XbaI site located within the gene. The 4-base pair insertion resulted in a truncated RecA protein as determined by minicell analysis. The mutation was spontaneously recombined onto the chromosome of a derivative of V. cholerae strain P27459 by screening for methyl methanesulfonate-sensitive variants. Southern blot analysis confirmed the presence of the inactivated XbaI site in the chromosome of DNA isolated from one of these methyl methanesulfonate sensitive colonies. The recA V. cholerae strain was considerably more sensitive to UV light than its parent, was impaired in homologous recombination, and was deficient in induction of a temperate vibriophage upon exposure to UV light. We conclude that the V. cholerae RecA protein has activities which are analogous to those described for the RecA protein of E. coli. PMID- 3005237 TI - Lateral diffusion of proteins in the periplasm of Escherichia coli. AB - We have introduced biologically active, fluorescently labeled maltose-binding protein into the periplasmic space of Escherichia coli and measured its lateral diffusion coefficient by the fluorescence photobleaching recovery method. Diffusion of this protein in the periplasm was found to be surprisingly low (lateral diffusion coefficient, 0.9 X 10(-10) cm2 s-1), about 1,000-fold lower than would be expected for diffusion in aqueous medium and almost 100-fold lower than for an equivalent-size protein in the cytoplasm. Galactose-binding protein, myoglobin, and cytochrome c were also introduced into the periplasm and had diffusion coefficients identical to that determined for the maltose-binding protein. For all proteins nearly 100% recovery of fluorescence was obtained after photobleaching, indicating that the periplasm is a single contiguous compartment surrounding the cell. These data have considerable implications for periplasmic structure and for the role of periplasmic proteins in transport and chemotaxis. PMID- 3005238 TI - Substrate-induced membrane association of phosphatidylserine synthase from Escherichia coli. AB - To better establish the intracellular location of the phosphatidylserine synthase of Escherichia coli and hence better understand how it is regulated in the cell, we compared the size, function, and binding properties of the enzyme made in vitro with the enzyme found in cell lysates and with the purified enzyme. The enzyme made either in vivo or in an active form in vitro was found primarily associated with the ribosomal fraction of the cell and had the same apparent molecular mass as the purified enzyme. These results were unaffected by the presence of protease inhibitors. Addition of unsupplemented E. coli membranes or membranes supplemented with phosphatidylethanolamine did not affect the subcellular distribution of the enzyme in these experiments. However, addition of membranes supplemented with either the lipid substrate, CDP-diacylglycerol, or the lipid product, phosphatidylserine, resulted in membrane association by the enzyme rather than ribosomal association. Addition of membranes supplemented with acidic lipids also brought about membrane association, but this association was primarily ionic since it was disrupted by high salt concentrations. These results strongly suggest that the ribosomal location of this enzyme is not the result of some modification event occurring after cell lysis and that the normal functioning of the enzyme involves membrane association which is primarily induced by the presence of a membrane-associated substrate. PMID- 3005239 TI - Classification of Histoplasma capsulatum isolates by restriction fragment polymorphisms. AB - Twenty isolates of the dimorphic, pathogenic fungus Histoplasma capsulatum were divided into three classes based on comparisons of restriction enzyme digests of their mitochondrial DNA and rDNA. The majority of isolates, including most North American strains and the African H. capsulatum var. duboisii variants, belong to class 2. Isolates from Central America and South America make up class 3. The attenuated Downs strain is the only member of class 1. PMID- 3005241 TI - Alkalophilic Bacillus firmus RAB generates variants which can grow at lower Na+ concentrations than the parental strain. AB - Obligately alkalophilic Bacillus firmus RAB cannot grow well on media containing less than 5 mM Na+. However, variant strains can be isolated on plates containing 2 to 3 mM Na+. These variants are observed only rarely in cultures that are plated before being subjected to repeated transfers in liquid medium. Cultures which have been transferred several times produce variants at an apparent frequency of 2 X 10(-4). Most of these variants are unstable, generating parental types at the high frequency of 10%; however, stable variants can be isolated. These strains grow better than the parental strain at very high pH values in the presence of 5 mM Na+ and have enhanced activity of the Na+ -H+ antiporter that has been implicated in pH homeostasis. By contrast, Na+ -coupled solute uptake is indistinguishable from that of the parental strain, and no obvious changes in the respiratory chain components are apparent in reduced versus oxidized difference spectra. The membranes of the variants show a marked enhancement, on sodium dodecyl sulfate-polyacrylamide gradient electrophoresis, in one polypeptide band with a molecular weight in the range of 90,000. The findings are discussed from the point of view of genetic mechanisms that might confer adaptability to even more extreme environments than usual and in view of earlier models relating the Na+ -translocating activities of the alkalophiles. PMID- 3005240 TI - Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector. AB - A highly efficient protoplast transformation system for Streptococcus faecalis has been developed by systematically optimizing different parameters. Up to 10(6) transformants per micrograms of DNA were consistently obtained within 3 days, and cell wall regeneration of protoplasts was virtually 100%. A systematic search for useful vectors showed that the broad-host-range plasmid pIP501 could transform S. faecalis at a high frequency (6.3 X 10(4) transformants per microgram). By combining a high-copy-number derivative of pIP501, designated pGB354, with the Escherichia coli vector pACYC184, we constructed a new E. coli-S. faecalis shuttle vector (pAM401) having nine unique restriction sites. In a shotgun cloning experiment, we ligated a tetracycline resistance determinant from Streptococcus sanguis chromosomal DNA into pAM401 by direct transformation of S. faecalis, establishing the utility of the protoplast transformation system and of the new shuttle vector. PMID- 3005243 TI - Tn4351 transposes in Bacteroides spp. and mediates the integration of plasmid R751 into the Bacteroides chromosome. AB - The gene for resistance to erythromycin and clindamycin, which is carried on the conjugative Bacteroides plasmid, pBF4, has been shown previously to be part of an element (Tn4351) that transposes in Escherichia coli. We have now introduced Tn4351 into Bacteroides uniformis 0061 on the following two suicide vectors: (i) the broad-host-range IncP plasmid R751 (R751::Tn4351) and (ii) pSS-2, a chimeric plasmid which contains 33 kilobases of pBF4 (including Tn4351) cloned into the IncQ plasmid RSF1010 and which is mobilized by R751. When E. coli HB101, carrying either R751::Tn4351 or R751 and pSS-2, was mated with B. uniformis under aerobic conditions, Emr transconjugants were detected at a frequency of 10(-6) to 10(-5) (R751::Tn4351) or 10(-8) to 10(-6) (R751 and pSS-2). In matings involving pSS-2, all Emr transconjugants contained simple insertions of Tn4351 in the chromosome, whereas in matings involving R751::Tn4351, about half of the Emr transconjugants had R751 cointegrated with Tn4351 in the chromosome. Of the Emr transconjugants, 13% were auxotrophs. Bacteroides spp. which had R751 cointegrated with Tn4351 in the chromosome did not transfer R751 or Tn4351 to E. coli HB101 or to isogenic B. uniformis, nor did the intergrated R751 mobilize pE5-2, an E. coli-Bacteroides shuttle vector that contains a transfer origin that is recognized by R751. PMID- 3005245 TI - Depression correlated with cellular immunity in systemic immunodeficient Epstein Barr virus syndrome (SIDES). AB - We conducted studies on the peripheral blood of 12 depressed patients with previous diagnoses of mood and/or personality disorders. These patients, and other depressives we observed informally, were resistant to infectious mononucleosis during an epidemic of that illness. All 12 had serologic evidence of a chronic or recrudescent viremia caused by the Epstein-Barr virus (EBV), the infectious agent in infectious mononucleosis. Additional evidence that EBV viremia may be causally related to depression was provided by a strong correlation between the intensity of depressive symptoms and the cellular immune response to the EBV infection. PMID- 3005242 TI - Subcellular and submitochondrial localization of phospholipid-synthesizing enzymes in Saccharomyces cerevisiae. AB - Using highly enriched membrane preparations from lactate-grown Saccharomyces cerevisiae cells, the subcellular and submitochondrial location of eight enzymes involved in the biosynthesis of phospholipids was determined. Phosphatidylserine decarboxylase and phosphatidylglycerolphosphate synthase were localized exclusively in the inner mitochondrial membrane, while phosphatidylethanolamine methyltransferase activity was confined to microsomal fractions. The other five enzymes tested in this study were common both to the outer mitochondrial membrane and to microsomes. The transmembrane orientation of the mitochondrial enzymes was investigated by protease digestion of intact mitochondria and of outside-out sealed vesicles of the outer mitochondrial membrane. Glycerolphosphate acyltransferase, phosphatidylinositol synthase, and phosphatidylserine synthase were exposed at the cytosolic surface of the outer mitochondrial membrane. Cholinephosphotransferase was apparently located at the inner aspect or within the outer mitochondrial membrane. Phosphatidate cytidylyltransferase was localized in the endoplasmic reticulum, on the cytoplasmic side of the outer mitochondrial membrane, and in the inner mitochondrial membrane. Inner membrane activity of this enzyme constituted 80% of total mitochondrial activity; inactivation by trypsin digestion was observed only after preincubation of membranes with detergent (0.1% Triton X-100). Total activity of those enzymes that are common to mitochondria and the endoplasmic reticulum was about equally distributed between the two organelles. Data concerning susceptibility to various inhibitors, heat sensitivity, and the pH optima indicate that there is a close similarity of the mitochondrial and microsomal enzymes that catalyze the same reaction. PMID- 3005244 TI - Apparently unidirectional polyamine transport by proton motive force in polyamine deficient Escherichia coli. AB - A transport system for polyamines was studied with both intact cells and membrane vesicles of an Escherichia coli polyamine-deficient mutant. Polyamine uptake by intact cells and membrane vesicles was inhibited by various protonophores, and polyamines accumulated in membrane vesicles when D-lactate was added as an energy source or when a membrane potential was imposed artificially by the addition of valinomycin to K+-loaded vesicles. These results show that the uptake was dependent on proton motive force. Transported [14C]putrescine and [14C]spermidine were not excreted by intact cells upon the addition either of carbonyl cyanide m chlorophenylhydrazone, A23187, and Ca2+ or of an excess amount of nonlabeled polyamine. However, they were excreted by membrane vesicles, although the degree of spermidine efflux was much lower than that of putrescine efflux. These results suggest that the apparent unidirectionality in intact cells has arisen from polyamine binding to nucleic acids, thus giving rise to a negligible free intracellular concentration of polyamines. Polyamine uptake, especially putrescine uptake, was inhibited strongly by monovalent cations. The Mg2+ ion inhibited spermidine and spermine uptake but not putrescine uptake. PMID- 3005246 TI - Establishment of an efficient purification method and further characterization of 32K calmodulin-binding protein in testis. AB - A heat-stable 32K calmodulin-binding protein has been purified approximately 3,670-fold from porcine testis to apparent homogeneity as judged by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and polyacrylamide gel electrophoresis under native conditions. The purification employed calmodulin Sepharose 4B affinity chromatography; elution was performed with a free Ca2+ gradient. This provided a simple and efficient procedure, and approximately 1.62 mg of pure heat-stable calmodulin-binding protein was obtained from 390 g of porcine testis with a yield of 47% in activity. The purified protein was asymmetric (f/fo = 1.89) and consisted of a single polypeptide of Mr = 32,000. It is a highly acidic protein (pI = 3.9) with a diffusion coefficient of 5.4 X 10( 7) cm2/s, a sedimentation coefficient of 1.43 S, and a Stokes radius of 39.5 A in its free form and 41.3 A in its complex form with calmodulin. The extent of inhibition of phosphodiesterase by the calmodulin-binding protein was affected by the order of addition of the agents to the reaction mixture. The extent of inhibition was maximal when phosphodiesterase was added last, while it was minimal when the calmodulin-binding protein was added last. This protein was indistinguishable from a heat-stable calmodulin-binding protein in rat testis (Ono, T., Koide, Y., Arai, Y., & Yamashita, K. (1984) J. Biol. Chem. 259, 9011 9016). PMID- 3005247 TI - Dynamic behavior of the imino protons of the gamma OR3 17mer in H2O solution studied by high-resolution NMR. AB - The imino proton resonances of gamma OR3 17mer in water were observed at 500 MHz with the time-shared Redfield pulse train. All of the 17 imino proton resonances could be assigned specifically to individual base pairs by utilizing the trace of NOE connectivities between the imino and adenine C2H protons and between imino protons themselves. AT1 and 17 showed abnormally high chemical shifts in comparison with the other AT pairs. On raising the temperature, broadening of the signal occurred in a sequential manner from the terminals except for AT10 and AT11, which were broadened at a lower temperature than GC12. The relaxation rates of the imino protons were measured by the inversion recovery method. The rates at higher temperatures represent the exchange rates of the imino protons. From the temperature dependences, activation energies of about 15 kcal/mol for the AT imino protons and 23-26 kcal/mol for the GC imino protons were obtained. PMID- 3005248 TI - Expression and phosphorylation of transferrin receptors in mitogen-activated peripheral blood lymphocytes. AB - Expression of transferrin receptors of cultured human lymphocytes has been investigated by using monoclonal antibody (5E9) specific for human transferrin receptors. When isolated lymphocytes were cultured in a medium containing fetal calf serum, the biosynthesis of transferrin receptor was barely detectable. The addition of concanavalin A or human serum to the medium caused a slight stimulation of the biosynthesis. The addition of concanavalin A and human serum in combination caused the highest biosynthetic activity. Appearance of the receptor on the cell surface increased in parallel with the degree of the synthesis. Treatment of concanavalin A- and human serum-treated cells with 12-O tetradecanoylphorbol-13-acetate (TPA) resulted in a marked stimulation of the phosphorylation of the receptor. Enhancement of phosphorylation occurred within 20 min after the addition of TPA. The density of the receptor on the cell surface slightly increased upon TPA treatment of cells, and the treatment was without effect on iron incorporation from transferrin into the cells. The density of newly synthesized receptor in TPA-treated cells was similar to that in non treated cells. These results indicated that TPA treatment of mitogen-activated human lymphocytes stimulated the phosphorylation of transferrin receptors, but TPA had no effect on the expression of the receptors thereafter. PMID- 3005249 TI - Occurrence of O-glycosidically peptide-linked oligosaccharides of poly-N acetyllactosamine type (erythroglycan II) in the I-antigenically active Sendai virus receptor sialoglycoprotein GP-2. AB - Unique high molecular weight (M.W. 4,000-9,000) sugar chains termed erythroglycan II have been obtained from alkali/sodium borohydride digests of I-active asialoglycoprotein derived from sialoglycoprotein GP-2, which was isolated recently from bovine erythrocyte membranes as Sendai virus receptor (Suzuki, Y. et al. (1983) J. Biochem. 93, 1621-1633; (1984) ibid, 95, 1193-1200). It was found that these sugar chains comprise about 40% of total alkali-labile oligosaccharides of asialo GP-2 and contain endo-beta-galactosidase (Flavobacterium keratolyticus)-resistant highly branched and heterogeneous oligosaccharides of poly-N-acetyllactosamine type which are linked O glycosidically to the peptide backbone through N-acetylgalactosamine. Erythroglycan II also contains endo-beta-galactosidase-susceptible straight terminal polylactosaminyl side chains. A major oligosaccharide released by the enzyme cochromatographed with Gal beta 1-4GlcNAc beta 1-3Gal. Inhibitory activity of Sendai virus-mediated hemagglutination and the receptor activity for the virus were reduced significantly but not completely by the endo-beta-galactosidase. These results indicate that both linear and branched sialosylpolylactosamine sequences in erythroglycan II are important for the reception of the virus into the target cells. PMID- 3005251 TI - Structural comparisons between mouse and human prealbumin. AB - In an attempt to construct model systems for familial amyloidotic polyneuropathy, prealbumin cDNA was cloned from a mouse liver cDNA library, using previously cloned human prealbumin cDNA as a hybridization probe. The primary structure of mouse prealbumin deduced from the cDNA sequence shows that it consists of 147 amino acids, including a whole prealbumin sequence (127 amino acids) and a putative signal sequence (20 amino acids). These numbers are in complete agreement with those determined for the human prealbumin. Among the 127 amino acid residues of the mature human prealbumin, 25 are replaced by different amino acids in the mouse prealbumin. Interestingly, 24 out of the 25 substituted amino acids are located at the outer surface of the protein, and the regions corresponding to the core and central channel of the protein are almost completely conserved. The cloned cDNA provided essential information for manipulating amyloidosis in mice. PMID- 3005250 TI - The role of Ca2+ and Ca2+-activated phospholipid-dependent protein kinase in degranulation of human neutrophils. AB - The degranulation reactions of human neutrophils induced by 1-oleoyl-2 acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and calcium ionophore A23187 or their combinations, were studied. OAG in the absence of the Ca2+-ionophore A23187 stimulated the releases of both lysozyme and lactoferrin, constituents of the specific granules, but did not stimulate the release of beta glucuronidase, an enzyme of the azurophil granules. Electron microscopy revealed a selective decrease in the numbers of the specific granules in this case. The combined effects of A23187 at a concentration higher than 0.1 microM and OAG were essentially additive. W-7, known to be an inhibitor of both Ca2+-activated phospholipid-dependent protein kinase (C-kinase) and calmodulin, inhibited the degranulation induced by OAG or PMA, while it inhibited the reaction induced by A23187 less markedly. The release of lysozyme reached a plateau at about 0.1 microM A23187 and increased again at higher concentrations of A23187. The observations suggest that degranulation can be induced by the activation of the C kinase, and the degranulation by A23187 at low concentrations may be due to the activation of the C-kinase; the effects of A23187 at high concentrations, however, could not be explained only in terms of the activation of the C-kinase. PMID- 3005253 TI - Histopathological studies on experimental murine salivary gland tumours treated with adriamycin: evidence for an anti-myoepithelial cell action. AB - Mice bearing pleomorphic salivary tumours induced by polyoma virus underwent chemotherapy with Adriamycin. Animals were killed at 2 day intervals and the tumours examined histologically. Two days after a single dose of Adriamycin, gross vacuolation degeneration was seen around ductal cells at sites corresponding to myoepithelial cells. At subsequent time periods there was progressive degeneration of other tumour elements. It is proposed that myoepithelial cell death in salivary gland tumours induced by Adriamycin may be an important factor in the early response of this tumour to chemotherapy. PMID- 3005252 TI - One of two copper atoms is not necessary for the cytochrome c oxidase activity of Pseudomonas AM 1 cytochrome aa3. AB - From Pseudomonas AM 1 grown in a medium deficient in Cu, aa3-type cytochrome c oxidase was purified which contained 2 molecules of haem a and one atom of Cu per molecule. The enzyme showed absorption peaks at 428 and 595 nm in the oxidized form and at 442 and 604 nm in the reduced form, and its CO complex showed peaks at 432 and 602 nm. The enzyme in the oxidized state showed an obscure absorption peak around 800 nm instead of a peak at 820 nm. One mol of the enzyme oxidized maximally 76, 75, and 98 mol of the ferrocytochromes c of Candida krusei, horse and Pseudomonas AM 1 per sec, respectively. These reactions were 50% inhibited by 7 microM KCN. The product of reduction of O2 catalyzed by the enzyme was concluded to be H2O on the basis of the ratio of ferrocytochrome c oxidized to O2 consumed. PMID- 3005255 TI - Metal ion binding to tetrameric lima bean lectin. AB - The binding of Mn2+ and Ca2+ to tetrameric lima bean lectin has been examined by equilibrium dialysis and magnetic resonance techniques. Demetalized lectin prepared by acid treatment binds either 1 Mn2+ or 2 Ca2+/monomer. When demetalized lectin is presaturated with Ca2+, only 1 Mn2+ binds per dimer. Water proton relaxation rate enhancements and Mn2+ electron spin resonance spectra were used to monitor metal ion association processes. Following Mn2+ binding to demetalized lectin, a conformational change with activation energy of 16 kcal/mol was detected; this is similar in magnitude to that observed for a conformational change with the lectin concanavalin A. The pH dependence suggests that a histidine residue is involved. ESR spectroscopy shows clearly that 1 Mn2+ binds to each demetalized subunit, but that Ca2+ induces dissociation of half the Mn2+; this result is in agreement with the equilibrium dialysis studies. PMID- 3005254 TI - Apatite crystallite alignment in sound human tooth enamel studied by E.S.R. AB - The degree of microcrystal alignment in enamel of clinically sound upper and lower central incisors, canines and premolars was determined by means of electron paramagnetic resonance. The results show that the degree of alignment depends on the individual properties of the enamel as determined by biological variations, tooth morphology and tooth position. Upper incisors and canines have a much higher degree of crystallite alignment as compared to all other teeth investigated. These results indicate that the caries susceptibility of tooth enamel is not defined only by the degree of microcrystal alignment. PMID- 3005256 TI - The rho-115 mutation in transcription termination factor rho affects its primary polynucleotide binding site. AB - We have investigated the effect of the rho-115 mutation on the catalytic properties of the Escherichia coli termination protein, rho. Comparison of the primary and secondary polynucleotide binding sites activities reveals dramatic differences between the mutant and wild-type molecules. Wild-type rho must bind single-stranded polynucleotides to activate its nucleotide triphosphatase (NTPase) activity, and either poly(C), or poly(dC) plus oligo(C), will suffice. In contrast, attempted activation of the rho-115 NTPase with poly(C) in the presence of poly(dC) showed the latter to be a potent inhibitor. Inclusion of small oligonucleotides such as oligo(C) in the activation assay does not inhibit the poly(C)-induced NTPase reaction of either wild-type rho or rho-115. This would indicate, in the two polynucleotide binding site model for rho proposed by Richardson (Richardson, J.P. (1982) J. Biol. Chem. 251, 5760-5766), that the mutation in rho-115 affects the primary polynucleotide binding site. Transcription termination in vitro at the rho-dependent site trp t' showed dramatically reduced termination with rho-115 protein compared to wild-type rho. In the presence of rho-115, the transcript is longer and termination occurs over a narrower range of nucleotides than with wild-type rho. This suggests that the primary polynucleotide binding site is important not only for efficient termination of transcription but may also be involved in determining the terminal end point of the transcript itself. PMID- 3005257 TI - Disulfide cross-linking of H,K-ATPase opens Cl- conductance, triggering proton uptake in gastric vesicles. Studies with specific inhibitors. AB - An S-S cross-linking reagent, Cu2+-o-phenanthroline, increased the 36Cl-/Cl- exchange rate across the hog gastric vesicle membrane, which contains H,K-ATPase, but did not affect the 86Rb+/Rb+ exchange rate. The results show that closed Cl- conductance can be opened by S-S cross-linking. Gastric vesicles with opened Cl- conductance could take up H+ upon addition of MgATP without prolonged preincubation in a solution containing K+. Preincubation of gastric vesicles with picoprazole, which is a specific inhibitor of H,K-ATPase and binds to 100-kDa polypeptides of the enzyme, dose dependently inhibited opening of the Cl- conductance by Cu2+-o-phenanthroline, indicating that the Cl- conductance is part of the function of the H,K-ATPase. The effect of picoprazole was greater at alkaline pH than at acidic pH. Another H,K-ATPase inhibitor, 2-[2-(3,5-dimethyl-4 methoxy)-pyridylmethylsulfinyl] (5-methoxycarbonyl-6-methyl)-benzimidazole (H compound), had a similar but stronger effect on the Cl- conductance than that of picoprazole. A pungent ingredient of curry, allylisothiocyanate, caused similar pH-dependent inhibition to that of picoprazole. However, another substituted benzimidazole, omeprazole, did not inhibit Cl- conductance. Substituted benzimidazoles, such as picoprazole, H compound, and omeprazole, inhibited the H,K-ATPase activity progressively with a decrease in pH of the medium. This pH dependence was the reverse of that in inhibition of Cl- conductance, suggesting that the inhibitory site of Cl- conductance is different from that of the H,K ATPase activity and that the conformational states of the two sites change in different ways with change in pH of the medium. PMID- 3005258 TI - Generation of Na+ electrochemical potential by the Na+-motive NADH oxidase and Na+/H+ antiport system of a moderately halophilic Vibrio costicola. AB - Cells of Vibrio costicola at pH 8.5 generate both membrane potential (inside negative) and delta pH (inside acidic) in the presence of a proton conductor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). The generation of CCCP-resistant membrane potential was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide that is known to inhibit the Na+-motive NADH oxidase of Vibrio alginolyticus. NADH oxidase, but not lactate oxidase, of inverted membrane vesicles prepared from V. costicola required Na+ for a maximum activity and was inhibited by 2-heptyl-4 hydroxyquinoline-N-oxide. By the oxidation of NADH, inverted membrane vesicles generated concentration gradients of Na+ across the membrane, whose magnitude was always larger than that of delta pH by about 50 mV. In contrast, magnitudes of delta pH and Na+ concentration gradients generated by the oxidation of lactate were similar. Na+ translocation in the presence of lactate was inhibited by CCCP but little affected by valinomycin. On the other hand, Na+ translocation in the presence of NADH was resistant to CCCP and stimulated by valinomycin. Amiloride, an inhibitor for a eucaryotic Na+/H+ antiport system, inhibited the lactate dependent Na+ translocation but had little effect on the NADH-dependent Na+ translocation. These results indicate that a primary event of lactate oxidation is the translocation of H+, which then causes the generation of Na+ concentration gradients via the secondary Na+/H+ antiport system. We conclude that the NADH oxidase of V. costicola translocates Na+ as an immediate result of respiration, leading to the generation of Na+ electrochemical potential. PMID- 3005259 TI - Regulation of free cytosolic Ca2+ in the peptic and parietal cells of the rabbit gastric gland. AB - Quin 2-loaded isolated rabbit gastric glands and purified peptic cells were used to measure free cytosolic Ca2+ ([Ca2+]i) during hormone stimulation. Rabbit gastric glands are composed of peptic and parietal cells with less than 1% endocrine cells. Although both cell types responded to the same hormones, they may be distinguished in terms of the source of Ca2+ bringing about the change in [Ca2+]i. Experiments were designed to assign changes in [Ca2+]i to either the peptic or parietal cells and to attempt to maintain these distinctions in the mixed cell population of gastric glands. It was shown that the peptide cholecystokinin octapeptide induced a rapid and transient increase in [Ca2+]i of isolated peptic cells. This signal was independent of medium Ca2+ and insensitive to the Ca2+ channel blockers La3+ and nifedipine. In gastric glands, the Ca2+ outdependent increase in (Ca2+)i (the secondary transient) was slower and dose dependently blocked by La3+ and nifedipine. This allowed [Ca2+]i levels in the physiologically more intact rabbit gastric glands to be dissected and correlated with fluorescence changes of quin 2 in either cell type. The transient increase in [Ca2+]i coincided with a burst of pepsin but not acid secretion. A subsequent slower phase of pepsin secretion took place while the cells restored near resting [Ca2+]i. Using a combination of the Ca2+ ionophore A23187 and the protein kinase C activating phorbol ester 12-O-tetra-decanoylphorbol 13-acetate, the hormone response pattern of pepsin secretion could be mimicked. The intracellular Ca2+ stores of the peptic cells in the gastric gland remained depleted of Ca2+ until specific antagonists were added. The reloading of intracellular stores required medium Ca2+ although [Ca2+]i was maintained at resting level during the entire reloading period. Hence, a specialized pathway of Ca2+ reloading is postulated. PMID- 3005260 TI - A study on the role of evolutionarily invariant leucine 32 of cytochrome c. AB - To investigate the role of evolutionarily invariant leucine 32 of horse cytochrome c, analogs of residues 28-38, (28-38), each containing a substituted amino acid at positions 32 or 35 were synthesized using Merrifield's method. Position 35 is leucine in horse cytochrome c but replaced by nonpolar amino acids in some species. The ability of the analogs to bind to the two-fragment complex of ferri- or ferro heme fragment (1-25)H and apofragment (39-104) was measured using gel filtration and equilibrium dialysis. Replacement of leucine 32 with isoleucine, for example, increased the dissociation constant by more than 400 fold for the ferrous complex. In contrast, replacement of leucine 35 with isoleucine seems to increase it only by a small degree. Since both leucine 32 and leucine 35 are completely buried within the structure, hydrophobic interaction would not explain this striking difference. However, thermodynamic analyses and absorption spectra of the ferric complex have indicated that replacement with norvaline of leucine 32 increases both delta H and delta S (more positive) associated with formation of an intermediate three-fragment complex and decreases both delta H and delta S (more negative) associated with transformation from the intermediate to the ground state, resulting in weakening the methionine 80--S- heme-Fe bond formed in the latter step. Taking the results together with the fragment exchange studies on the ferrous complex and available evidence, we suggest that the interaction involving leucine 32 would be coupled not only with the methionine 80--S--heme-Fe bond but also with the energy state of other distant residues such as tryptophan 59, generating extra energy for modulating the binding of the complex, i.e. the force of folding. In contrast, leucine 35 would be less important even if it were involved in such coupling. PMID- 3005261 TI - Thyrotropin-releasing hormone and GTP activate inositol trisphosphate formation in membranes isolated from rat pituitary cells. AB - Stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by a phospholipase C to produce inositol trisphosphate (InsP3) and 1,2-diacylglycerol appears to be the initial step in signal transduction for a number of cell-surface interacting stimuli, including thyrotropin-releasing hormone (TRH). In suspensions of membranes isolated from rat pituitary (GH3) cells that were prelabeled to isotopic steady state with [3H]inositol and incubated with ATP, [3H] PtdIns(4,5)P2, and [3H]phosphatidylinositol 4-phosphate, the polyphosphoinositides, and [3H]InsP3 and [3H]inositol bisphosphate, the inositol polyphosphates, accumulated. TRH and GTP stimulated the accumulation of [3H]inositol polyphosphates in time- and concentration-dependent manners; half maximal effects occurred with 10-30 nM TRH and with 3 microM GTP. A nonhydrolyzable analog of GTP also stimulated [3H] inositol polyphosphate accumulation. Moreover, when TRH and GTP were added together their effects were more than additive. Fixing the free Ca2+ concentration in the incubation buffer at 20 nM, a value below that present in the cytoplasm in vivo did not inhibit stimulation by TRH and GTP of [3H]inositol polyphosphate accumulation. ATP was necessary for basal and stimulated accumulation of [3H]inositol polyphosphates, and a nonhydrolyzable analog of ATP could not substitute for ATP. These data demonstrate that TRH and GTP act synergistically to stimulate the accumulation of InsP3 in suspensions of pituitary membranes and that ATP, most likely acting as substrate for polyphosphoinositide synthesis, was necessary for this effect. These findings suggest that a guanine nucleotide-binding regulatory protein is involved in coupling the TRH receptor to a phospholipase C that hydrolyzes PtdIns(4,5)P2. PMID- 3005262 TI - Voltage-sensitive nitrendipine binding in an isolated cardiac sarcolemma preparation. AB - Nitrendipine binding has been evaluated in a highly enriched sarcolemma preparation isolated from canine ventricle. The binding was found to be specific, saturable, rapid, and reversible. The dissociation constant (Kd) determined by equilibrium binding studies at 20 degrees C was 0.0880 nM. The Kd increased to 0.670 nM at 37 degrees C. The maximal binding capacity of this preparation ranged from 437 to 1775 fmol/mg protein and was not significantly affected by changes in temperature between 20 and 37 degrees C. The Kd, determined kinetically from the ratio of the dissociation and association rate constants (k-1/k1), was 0.112 and 0.285 nM at 20 and 37 degrees C, respectively. In order to test the hypothesis that nitrendipine binding changes with membrane potential potassium, Nernst potentials were developed, in the presence of valinomycin, by the establishment of potassium gradients across the vesicular membrane. Evaluation of the rates of dissociation of [3H]nitrendipine from the sarcolemma preparation identified a component of binding that was rapidly lost when the transmembrane potential was polarized to inside-negative values. The magnitude of the loss of nitrendipine binding was 25-27% at the most negative potentials examined. Evaluation of the rate of association of nitrendipine revealed that the component of binding that was rapidly lost upon hyperpolarization of the membrane returned over a time course similar to the rate of dissipation of the membrane potential, suggesting that the effects of potential on nitrendipine binding are reversible. These findings are consistent with the hypothesis that nitrendipine binding affinity changes with membrane potential. PMID- 3005264 TI - Synergistic inhibition of hepatic glycogenolysis in the presence of insulin and a cAMP antagonist. AB - The effects of insulin on the ability of the specific intracellular cAMP dependent protein kinase antagonist, the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate, to inhibit glycogenolysis induced by the Sp diastereomer was studied in hepatocytes isolated from fed rats. Addition of the cAMP agonist, (Sp)-cAMPS, to hepatocytes resulted in a concentration-dependent increase in glycogenolytic glucose production concomitant with the cAMP-dependent activation of phosphorylase and inhibition of glycogen synthase. Activity curves were shifted to the right in the presence of the cAMP antagonist, (Rp)-cAMPS. Preincubation of the hepatocytes with a maximally effective concentration of insulin did not affect the concentration of (Sp)-cAMPS required for half-maximal activation of phosphorylase but did result in a 10-fold shift in the concentration of (Sp)-cAMPS required for half-maximal inactivation of glycogen synthase. Preincubation of hepatocytes with a combination of the cAMP antagonist, (Rp)-cAMPS, and insulin resulted in synergistic inhibition of (Sp)-cAMPS-induced phosphorylase activation, glycogen synthase inactivation, and glycogenolytic glucose production. Since neither phosphorothioate diastereomer was hydrolyzed significantly during the course of the experiments, the synergistic effects of insulin are postulated to be working through a mechanism subsequent to the phosphodiesterase activation step. PMID- 3005263 TI - Li+ as substrate of the synaptosomal Na+/H+ antiporter. AB - The influence of replacing external Na+ by choline+ on Li+ uptake into rat cortical synaptosomes was studied. Tetraphenylphosphonium+ and methylamine distribution techniques were used to estimate the plasma membrane potential and the transmembrane H+ gradients, respectively. H+ efflux was monitored by automatic titration in the pH-stat mode. In the Na+- and K+-free medium, synaptosomes concentrated Li+ about 10-fold at 1 mM Li+o in the presence of ouabain. Varying external free Ca2+ between 13 and 300 microM, or addition of MgCl2 had no effect on Li+ uptake. Ouabain-insensitive Li+ transport was separated into two components: 1) non-saturable Li+ influx with a rate constant of 0.6/min; 2) saturable uptake, which obeyed Michaelis-Menten kinetics (Km, 2.0 mM Li+; Vmax, 7.3 mmol of Li+/liter and min). Li+ uptake was competitively inhibited by amiloride (Ki, 3.2 microM; Hill coefficient, 1.0) and external Na+ (Ki, 5.8 mM). External Li+ scarcely accelerated Na+ efflux and phloretin failed to inhibit Li+ uptake, indicating that Li+ uptake was not directly coupled to Na+ gradient. Because of a reversal of the H+ transport by the pHi-regulating system, synaptosomes accumulated acid in the Na+-free medium. Li+ influx was electroneutral, but impaired H+ gradients and was coupled to the simultaneous release of stoichiometric amounts of H+ at less than 3 mM Li+o. Uptake of Li+ was linearly related to H+ gradients imposed onto the plasma membrane by varying external pH. In the steady state, internal Li+ was close to the value predicted for passive distribution. It is concluded that in Na+-free media Li+ uptake at low external Li+ is predominantly driven by transmembrane H+ gradients. The stoichiometric exchange of Li+ for H+ is mediated by the Na+/H+ antiporter. The Li+ distribution ratio is close to the electrochemical activity coefficient since protons are passively distributed across the synaptosomal plasma membrane in the absence of external Na+. PMID- 3005265 TI - Secretion of metalloproteinases by stimulated capillary endothelial cells. I. Production of procollagenase and prostromelysin exceeds expression of proteolytic activity. AB - We have characterized the biosynthesis of two metalloproteinases, procollagenase and prostromelysin, by rabbit brain capillary endothelial cells (RBCE) by means of immunochemical, biosynthetic, and functional assays. Unstimulated RBCE secreted no detectable metalloproteinases. Secretion of both procollagenase and prostromelysin was induced within 6 h by treating the cells with 50 ng/ml 12-O tetradecanoylphorbol-13-acetate. In treated cells, the two proenzymes accounted for up to 20% of the [35S]methionine-labeled secreted proteins; about 15 micrograms of each protein was secreted in 48 h by 10(6) RBCE. Although RBCE secreted approximately as much procollagenase and prostromelysin as did rabbit fibroblasts, virtually no enzyme activity could be measured in RBCE-conditioned medium, even after activation of the proenzymes by trypsin or an organomercurial agent. PMID- 3005266 TI - Secretion of metalloproteinases by stimulated capillary endothelial cells. II. Expression of collagenase and stromelysin activities is regulated by endogenous inhibitors. AB - Rabbit brain capillary endothelial cells treated with 12-O-tetradecanoylphorbol 13-acetate produce the metalloproteinases, procollagenase and prostromelysin, as up to 20% of their total secreted protein. However, little or no catalytic activity of these enzymes can be found after treatment with either trypsin or an organomercurial agent, which are able to activate the proenzymes in the medium from stimulated rabbit fibroblasts. We now have shown that enzyme activities of procollagenase and prostromelysin are revealed after conditioned medium is analyzed by gel filtration chromatography or by electrophoresis on sodium dodecyl sulfate-substrate gels. In both systems, the metalloproteinases were separated from metalloproteinase inhibitors. The major inhibitor of Mr = 30,000 from capillary endothelial cells was immunologically identical with the rabbit tissue inhibitor of metalloproteinases. Two additional inhibitors of metalloproteinases at Mr = 22,000 and 19,000 were also observed. Inhibitors were present in the conditioned medium from rabbit fibroblasts in much lower quantities and were also qualitatively different. When gel filtration chromatography was used to remove the tissue inhibitor of metalloproteinases from medium conditioned by stimulated capillary endothelial cells, both activatable procollagenase and prostromelysin were readily demonstrable. These data suggest that endogenous inhibitors regulate the expression of metalloproteinases secreted by endothelial cells. PMID- 3005268 TI - Association of the 3,5,3'-triiodo-L-thyronine nuclear receptor with the nuclear matrix of cultured growth hormone-producing rat pituitary tumor cells (GC cells). AB - The iodothyronine nuclear receptor, a nonhistone chromatin protein, mediates growth hormone gene transcription in cultured GC cells (Yaffe, B.M., and Samuels, H. H. (1984) J. Biol. Chem. 259, 6284-6291). To determine whether the 3,5,3' triiodo-L-thyronine (T3) receptor was localized to the nuclear matrix, we studied the subnuclear distribution of [125I]T3-receptor complexes after treatment of nuclei with DNase I and 2 M NaCl to facilitate removal of histones. After incubation with 5 nM [125I]T3 to exchange with 80-90% of nuclear T3 receptors, the nuclear matrix fraction contained less than 1% of nuclear DNA, 16.5% of nuclear protein, and 30-50% (mean, 40.0 +/- 2.3%) of specifically bound [125I]T3. Control experiments showed that nuclear matrix [125I]T3 did not appear exchangeable with added 5 nM T3 and did not result from release and nonspecific precipitation of [125I]T3 or [125I]T3-receptor complexes during nuclear matrix preparation. Studies with the T3 photoaffinity probe, N-2-diazo-3,3,3 trifluoropropionyl-[125I]T3 resulted in the finding of limited capacity receptor forms, 58,000 and 46,000 kDa, in the nuclear matrix. These receptor forms were identical to those observed when N-2-diazo-3,3,3-trifluoropropionyl-[125I]T3 labeled receptors were solubilized directly from nuclei. Lastly, limited capacity [125I]T3 binding was demonstrated in 0.4 M KCl buffer extracts of nuclear matrix. Binding displacement studies suggested that 46% of the binding activity solubilized from nuclear matrix exchanged with [125I]T3 and that the apparent equilibrium association constant of this binding activity was similar to that of 0.4 M KCl extracts of isolated nuclei. These results suggest that an appreciable fraction of nuclear T3 receptors is localized to the nuclear matrix and may influence the expression of thyroid hormone action. PMID- 3005267 TI - Deletion of clustered O-linked carbohydrates does not impair function of low density lipoprotein receptor in transfected fibroblasts. AB - A single exon in the gene for the receptor for plasma low density lipoprotein (LDL) encodes a region of clustered serine and threonine residues that is immediately external to the membrane-spanning sequence. This region has been proposed as the site of clustered O-linked carbohydrate chains. In the current studies we have deleted the 144 base pairs (48 amino acids) that encode this serine- and threonine-rich region from the cDNA for the human LDL receptor. Upon transfection into receptor-deficient hamster fibroblasts, this mutated cDNA encoded a shortened receptor that no longer showed an anomalously high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling with [3H]glucosamine confirmed the lack of clustered O-linked sugars and further revealed that the shortened receptor and the normal receptor both contained isolated O-linked carbohydrate chains attached to the NH2-terminal portion of the protein. The ratio of clustered to isolated O-linked sugar chains in the normal receptor was estimated to be approximately 4-6 to 1. Despite the loss of clustered O-linked carbohydrate, the LDL receptor encoded by the deletion-bearing cDNA bound and internalized LDL normally. It also recycled normally and exhibited a normal half-life. We conclude that: 1) the serine- and threonine-rich region of the LDL receptor is the site for addition of clustered O-linked carbohydrates; 2) the receptor contains a small number of isolated chains of O-linked carbohydrates in addition to the clustered chains; and 3) the clustered O-linked carbohydrates are not essential for LDL receptor function in cultured hamster fibroblasts. PMID- 3005269 TI - Newly synthesized Na,K-ATPase alpha-subunit has no cytosolic intermediate in MDCK cells. AB - Recently published data indicate that the alpha-subunit of Na,K-ATPase, a transmembrane protein of animal cell plasma membranes, is synthesized as a soluble precursor. In the present experiments we demonstrate that an apparent "soluble" form can indeed be detected in crude cytosolic fractions prepared by centrifugation from MDCK cells disrupted by sonication. We find, however, that this form has no precursor-product relationship with membrane-associated alpha subunit. The quantity of unsedimentable alpha-subunit can be greatly diminished by increasing the centrifugal field employed to remove membranous vesicles from the cytosol fraction. Sonication of membrane pellets generates alpha-subunit which, like the "soluble" form, resists pelleting. Finally, cytosol fractions prepared from cells disrupted by sonication contain membrane-bound vesicles which can be immunoadsorbed on Staphylococcus aureus cells coated with a monoclonal antibody directed against alpha-subunit. We find, therefore, that the previously observed soluble precursor of alpha-subunit is actually a component of small unpelleted membrane vesicles generated by harsh disruption conditions. When cells are disrupted by less violent techniques no newly synthesized alpha-subunit can be detected in the cytosol fraction. We calculate that to escape detection under our experimental conditions a bona fide soluble precursor of alpha-subunit must have a cytosolic t1/2 less than 20 s. We conclude that the alpha-subunit is most probably inserted into the bilayer cotranslationally. PMID- 3005270 TI - Heart myosin light chain 2 gene. Nucleotide sequence of full length cDNA and expression in normal and hypertensive rat. AB - We have isolated and characterized a cDNA recombinant plasmid (pRLC429) specific for the rat heart myosin light chain 2 (MLC2). The cDNA insert consists of 446 base pairs, including a 72-base pair segment of the 3'-untranslated region. Additional 5'-sequence, not present in plasmid pRLC429, was obtained by primer extension of the cDNA. The extended cDNA sequence combined with the plasmid pRLC429 sequence provided the codon information for the entire MLC2 polypeptide and partial sequences for the 3'- and 5'-noncoding regions of MLC2 mRNA. The predicted amino acid sequence for rat heart MLC2 showed a high homology with the sequences available for the chicken (83%) and human heart (80%) MLC2s. However, the homology between rat heart MLC2 and its counterpart in rat skeletal muscle is relatively low (67%). On the basis of the nuclease S1 protection assay with uniformly labeled single-stranded pRLC429 DNA, subcloned into M13mp18 phage vector, we conclude that the rat atrial muscle also contains MLC2 of the ventricular type. In an attempt to ascertain whether structural variants of MLC2 are expressed in hypertrophic heart muscle, we examined the RNAs from spontaneously hypertensive rat where there is a natural progression of hypertrophy associated with an increase in blood pressure. The RNA isolated from 7-, 13-, and 18-week-old spontaneously hypertensive rat hearts protected the same length DNA against S1 nuclease as was observed with RNAs from the age-matched normal rat hearts, suggesting that there is a single MLC2 gene transcript expressed in both the normal and hypertrophic heart muscle cells. PMID- 3005271 TI - Thyrotropin-releasing hormone activates a Ca2+-dependent polyphosphoinositide phosphodiesterase in permeable GH3 cells. GTP gamma S potentiation by a cholera and pertussis toxin-insensitive mechanism. AB - Numerous hormones are known to rapidly activate polyphosphoinositide turnover in target cells by promoting phosphodiesteratic cleavage of the phospholipids; however, little is known about the enzymology of receptor-mediated phosphoinositide breakdown. In the present study, thyrotropin-releasing hormone (TRH) stimulation of polyphosphoinositide turnover has been characterized in electrically permeabilized, [3H]myoinositol-labeled GH3 cells. The permeable cells allow the influence of small molecular weight (Mr less than or equal to 1000) cofactors to be determined. We present evidence for the following: 1) TRH stimulates inositol phosphate generation in permeable cells; 2) optimal hormone stimulated inositol phosphate generation requires Mg2+, ATP, and Ca2+; 3) Mg2+ and ATP requirements reflect polyphosphoinositide kinase reactions; 4) in the absence of MgATP, TRH stimulates the phosphodiesteratic breakdown of pre-existing polyphosphoinositides in a reaction which requires only low Ca2+ (10(-7) M); 5) hormone activation is potentiated in the presence of the stable guanine nucleotide, GTP gamma S; neither TRH-stimulated nor GTP gamma S-potentiated hydrolysis is inhibited by cholera or pertussis toxin treatment. These results demonstrate that hormone-induced phospholipid hydrolysis involves activation of a phosphoinositide phosphodiesterase; activation results in lowering the Ca2+ requirement of the phosphodiesterase such that maximal activity is observed at Ca2+ levels characteristic of a resting cell (10(-7) M). Furthermore, TRH regulation of polyphosphoinositide hydrolysis is modulated by guanine nucleotides; however, nucleotide regulation appears to involve a GTP-binding factor (Np) other than Ns or Ni. PMID- 3005272 TI - Isolation and characterization of the structural gene for secreted acid phosphatase from Schizosaccharomyces pombe. AB - The Schizosaccharomyces pombe acid phosphatase structural gene (PHO 1) was isolated by complementation of an S. pombe acid phosphatase mutant with a wild type S. pombe DNA recombinant plasmid library. Northern analysis indicates that acid phosphatase is encoded by a 1.4-kilobase mRNA of which approximately 100 bases are 3'-poly(A). The gene contains no introns and the 3' and 5' untranslated regions are short. According to DNA and amino acid sequence data, the S. pombe acid phosphatase has a molecular weight of 50,600. An 18-amino acid sequence at the N terminus was found that is similar to previously identified signal peptides in other eukaryotic secretory proteins. This signal peptide is apparently removed during secretion, since it is absent in the mature secreted acid phosphatase. The gene can be induced 2--3-fold by starvation for phosphate. The signals required for this induction are contained on the isolated DNA clone. Although the gene can be expressed in Saccharomyces cerevisiae, secretion is abnormal. PMID- 3005273 TI - Alpha-amylase of Escherichia coli, mapping and cloning of the structural gene, malS, and identification of its product as a periplasmic protein. AB - Mal+ lacZ operon fusions, inducible by maltose, were isolated in Escherichia coli, strain MC4100. One fusion strain, SF1707, was analyzed in detail. This fusion did not map in any of the known genes of the malA or malB region, but its expression was under control of malT, the positive regulator gene of the maltose regulon. The gene in which the fusion occurred mapped between xyl and mtl at 80 min on the linkage map and was transcribed clockwise. We define this gene as malS. The malS-lacZ fusion was transferred onto a phage lambda vector and the 5' portion of malS was subcloned into pBR322. The resulting plasmid was used as a probe to identify the intact malS gene in a lambda library of E. coli chromosomal HindIII fragments. The phage that hybridized with the probe contained a 12 kilobase insert. The malS containing portion was subcloned into pBR322 as a 4 kilobase ClaI-HindIII fragment. This plasmid directed the malT and maltose dependent synthesis of a periplasmic protein of 66,000 apparent molecular weight. The purified enzyme hydrolyzed maltodextrins longer than maltose including cyclic dextrins. The primary products of hydrolysis were glucose, maltose, and maltotriose, even when maltotetraose was used as a substrate. These properties differentiate this periplasmic enzyme from the cytoplasmic amylomaltase and define it as an alpha-amylase. PMID- 3005274 TI - Characterization of a cyclic nucleotide- and calcium-independent neurofilament protein kinase activity in axoplasm from the squid giant axon. AB - The phosphorylation activity associated with a neurofilament-enriched cytoskeletal preparation isolated from the squid giant axon has been studied and compared to the phosphorylation activities in intact squid axoplasm. The high molecular weight (greater than 300 kDa) and 220-kDa neurofilament proteins are the major endogenous substrates for the kinases in the axoplasm and the neurofilament preparation, whereas 95- and less than 60-kDa proteins are the major phosphoproteins in the ganglion cell preparation. The squid axon neurofilament (SANF) protein kinase activity appeared to be both cAMP and Ca2+ independent and could phosphorylate both casein (Km = 40 microM) and histone (Km = 180 microM). The SANF protein kinase could utilize either ATP or GTP in the phosphotransferase reaction, with a Km for ATP of 58 microM and 129.4 microM for GTP when casein was used as the exogenous substrate; and 25 and 98.1 microM for ATP and GTP, respectively, when the endogenous neurofilament proteins were used as substrates. The SANF protein kinase activity was only slightly inhibited by 2,3-diphosphoglycerate and various polyamines at high concentrations and was poorly inhibited by heparin (34% inhibition at 100 micrograms/ml). The failures of heparin to significantly inhibit and the polyamines to stimulate the SANF protein kinase indicate that it is not a casein type II kinase. The relative efficacy of GTP as a phosphate donor indicates that SANF protein kinase differs from known casein type I kinases. Phosphorylated (32P-labeled) neurofilament proteins were only slightly dephosphorylated in the presence of axoplasm or stellate ganglion cell supernatants, and the neurofilament-enriched preparation did not dephosphorylate 32P-labeled neurofilament proteins. The axoplasm and neurofilament preparations had no detectable protein kinase inhibitor activity, but a strong inhibitor activity, which was not dialyzable but was heat inactivatable, was found in ganglion cells. This inhibitor activity may account for the low phosphorylation activity found in the stellate ganglion cells and may indicate inhibitory regulation of SANF protein kinase activity in the ganglion cell bodies. PMID- 3005275 TI - Synthetic peptides corresponding to the site phosphorylated in 6-phosphofructo-2 kinase/fructose-2,6-bisphosphatase as substrates of cyclic nucleotide-dependent protein kinases. AB - The specificities of cAMP-dependent and cGMP-dependent protein kinases were studied using synthetic peptides corresponding to the phosphorylation site in 6 phosphofructo-2-kinase/Fru-2,6-P2ase (Murray, K.J., El-Maghrabi, M.R., Kountz, P.D., Lukas, T.J., Soderling, T.R., and Pilkis, S.J. (1984) J. Biol. Chem. 259, 7673-7681) as substrates. The peptide Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro Gln was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase on predominantly the first of its 2 seryl residues. The Km (4 microM) and Vmax (14 mumol/min/mg) values were comparable to those for the phosphorylation of this site within native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. An analog peptide containing only two arginines was phosphorylated with poorer kinetic constants than was the parent peptide. These results suggest that the amino acid sequence at its site of phosphorylation is a major determinant that makes 6-phosphofructo 2-kinase/Fru-2,6-P2ase an excellent substrate for cAMP-dependent protein kinase. Although 6-phosphofructo-2-kinase/Fru-2,6-P2ase was not phosphorylated by cGMP dependent protein kinase, the synthetic peptide corresponding to the cAMP dependent phosphorylation site was a relatively good substrate (Km = 33 microM, Vmax = 1 mumol/min/mg). Thus, structures other than the primary sequence at the phosphorylation site must be responsible for the inability of cGMP-dependent protein kinase to phosphorylate native 6-phosphofructo-2-kinase/Fru-2,6-P2ase. Peptides containing either a -Ser-Ser- or -Thr-Ser- moiety were all phosphorylated by cGMP-dependent kinase to 1.0 mol of phosphate/mol of peptide, but the phosphate was distributed between the two hydroxyamino acids. Substitution of a proline in place of the glycine between the three arginines and these phosphorylatable amino acids caused the protein kinase selectively to phosphorylate the threonyl or first seryl residue and also enhanced the Vmax values by 4-6-fold. These results are consistent with a role for proline in allowing an adjacent threonyl residue to be readily phosphorylated by cGMP dependent protein kinase. PMID- 3005276 TI - Identification of a putative thromboxane A2/prostaglandin H2 receptor in human platelet membranes. AB - The binding of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist (9,11-dimethylmethano-11, 12-methano-16-(3-aza-15 alpha beta-omega tetranor-TXA2) ([125I]PTA-OH) to membranes prepared from human platelets was characterized. [125I]PTA-OH binding to membranes from human platelets was saturable, displaceable, and dependent on protein concentration. Scatchard analysis of equilibrium binding carried out at 30 degrees C revealed one class of binding sites with a Kd of 30 +/- 4 nM and a Bmax of 1.8 +/- 0.3 pmol/mg of protein (n = 5). Kinetic analysis of the binding of [125I]PTA-OH at 0 degrees C yielded a k1 of 1.35 X 10(6) M-1 min-1 and a k-1 of 0.032 min-1, Kd = k-1/k1 = 24 nM. The potencies of a series of TXA2/PGH2 antagonists as inhibitors of [125I]PTA OH binding was correlated with their potencies as inhibitors of platelet aggregation induced by the TXA2/PGH2 mimetic, U46619 (1 microM) (r = 0.93, p less than 0.01). A series of TXA2/PGH2 mimetics also displaced [125I]PTA-OH from its binding site, and their potencies as inhibitors of [125I]PTA-OH binding were correlated with their potencies as stimulators of platelet aggregation (r = 0.91, p less than 0.05). The IC50 values for displacement of [125I]PTA-OH by PGF2 alpha, PGD2, and the stable PGI2 analog Iloprost were greater than 25 microM, suggesting that [125I]PTA-OH does not bind to other known platelet prostaglandin receptors. These data are consistent with the notion that this binding site may represent the platelet TXA2/PGH2 receptor. PMID- 3005278 TI - Interleukin 2 modulation of adenylate cyclase. Potential role of protein kinase C. AB - Interleukin 2 (IL 2) stimulated DNA synthesis of murine T lymphocytes (CT6) in a concentration-dependent manner, over a range of 1-1000 units/ml. This proliferative effect of IL 2 was attenuated by simultaneous exposure to prostaglandin E2 (PGE)2. In intact cells, IL 2 inhibited both basal and PGE2 stimulated cAMP production; the amount of cAMP generated was dependent upon the relative concentrations of IL 2 and PGE2. The effect of IL 2 on CT6 cell proliferation and cAMP production was mimicked by the phorbol ester 12-O tetradecanoylphorbol-13-acetate (TPA), which, like IL 2, causes a translocation and activation of protein kinase C. While PGE2 stimulated adenylate cyclase activity in membrane preparations, neither IL 2 nor TPA inhibited either basal or stimulated membrane adenylate cyclase activity. However, when CT6 cells were pretreated with IL 2 or TPA and membranes incubated with calcium and ATP, both basal and PGE2-and NaF-stimulated membrane adenylate cyclase activity was inhibited. This inhibition of adenylate cyclase activity was also observed if membranes from untreated cells were incubated with protein kinase C purified from CT6 lymphocytes in the presence of calcium and ATP. The data suggest that the decreased cAMP production which accompanies CT6 cell proliferation results from an inhibition of adenylate cyclase activity mediated by protein kinase C and that these two distinct protein phosphorylating systems interact to modulate the physiological response to IL 2. PMID- 3005277 TI - Expression of transferrin receptors in phytohemagglutinin-stimulated human T lymphocytes. Evidence for a three-step model. AB - Resting human T-lymphocytes show an elevated intracellular concentration of ferritin, whereas transferrin receptors are not detectable. Stimulation by phytohemagglutinin markedly lowers their ferritin content, while inducing the synthesis of transferrin receptors. Addition of iron salts (ferric ammonium citrate) in activated T-lymphocyte cultures causes a marked enhancement of both [3H]uridine and [3H]thymidine incorporation. Nevertheless, it also induces a concentration-dependent decrease in transferrin receptor synthesis, associated with a marked rise of ferritin production. Hemin treatment exerts the same effects. Addition of picolinic acid in phytohemagglutinin-stimulated cultures causes a decrease of [3H]thymidine incorporation, whereas transferrin expression is markedly enhanced. The action of iron salts and chelators is specific for transferrin receptors, since the expression of other membrane markers of activated human T-lymphocytes (interleukin-2 receptor, insulin receptor, and HLA DR antigen) is not modified by treatment with iron or picolinic acid. These observations suggest that expression of transferrin receptors in activated T lymphocytes is specifically modulated by their intracellular iron level, rather than their proliferative rate. Addition of picolinic acid to resting T lymphocytes in the absence of mitogen induces a marked decrease of their ferritin content, but not the appearance of transferrin receptors. On the basis of these results, we suggest a three-step model: (a) in resting T-lymphocytes, the gene for transferrin receptor is apparently "closed," in that it is not expressed under both normal conditions and following iron deprivation. (b) After mitogen stimulus, T-lymphocytes are reprogrammed into cell cycle progression, which necessarily entails synthesis of transferrin receptors (c) Expression of these receptors is modulated by the intracellular iron level, rather than the rate of proliferation per se. PMID- 3005279 TI - Redox cycling of anthracyclines by cardiac mitochondria. II. Formation of superoxide anion, hydrogen peroxide, and hydroxyl radical. AB - In the accompanying paper (Davies, K. J. A., and Doroshow, J. A. (1986) J. Biol. Chem. 261, 3060-3067), we have demonstrated that anthracycline antibiotics are reduced to the semiquinone form at Complex I of the mitochondrial electron transport chain. In the experiments presented in this study we examined the effects of doxorubicin (Adriamycin), daunorubicin, and related quinonoid anticancer agents on superoxide, hydrogen peroxide, and hydroxyl radical production by preparations of beef heart submitochondrial particles. Superoxide anion formation was stimulated from (mean +/- S.E.) 1.6 +/- 0.2 to 69.6 +/- 2.7 or 32.1 +/- 1.5 nmol X min-1 X mg-1 by the addition of 90 microM doxorubicin or daunorubicin, respectively. However, the anthracycline 5-iminodaunorubicin, in which an imine group has been substituted in the C ring quinone moiety, did not increase superoxide production over control levels. In the presence of rotenone, initial rates of oxygen consumption and superoxide formation were identical under comparable experimental conditions. Furthermore, H2O2 production increased from undetectable control levels to 2.2 +/- 0.3 nmol X min-1 X mg-1 after treatment of submitochondrial particles with doxorubicin (200 microM). The hydroxyl radical, or a related chemical oxidant, was also detected after the addition of an anthracycline to this system by both ESR spectroscopy using the spin trap 5,5 dimethylpyrroline-N-oxide and by gas chromatographic quantitation of CH4 produced from dimethyl sulfoxide. Hydroxyl radical production, which was iron-dependent in this system, occurred in a nonlinear fashion with an initial lag phase due to a requirement for H2O2 accumulation. We also found that two quinonoid anti-cancer agents which produce less cardiotoxicity than the anthracyclines, mitomycin C, and mitoxantrone, stimulated significantly less or no hydroxyl radical production by submitochondrial particles. These experiments suggest that injury to cardiac mitochondria which is produced by anthracycline antibiotics may result from the generation of the hydroxyl radical during anthracycline metabolism by NADH dehydrogenase. PMID- 3005280 TI - Direct anesthetic-like effects of forskolin on the nicotinic acetylcholine receptors of PC12 cells. AB - Forskolin is thought to be a highly specific activator of adenyl cyclase. However, when applied to rat pheochromocytoma (PC12) cells at concentrations of 1 microM or higher it caused an immediate, concentration-dependent inhibition of carbachol-stimulated uptake of 86Rb+ through the nicotinic receptors, which did not appear to be related to activation of adenyl cyclase. The inhibition of receptor activation occurred instantaneously whereas cellular cAMP content did not increase for a measureable period of time. Normal receptor function was recovered rapidly upon removal of forskolin. Additional evidence that this effect of forskolin was not related to cAMP was obtained when 1,9-dideoxyforskolin (an analog of forskolin which does not activate adenyl cyclase) also caused a rapid, concentration-dependent, rapidly reversible inhibition of receptor-mediated influx of 86Rb+ into the cells. An examination of the effect of forskolin on 86Rb+ uptake at various concentrations of carbachol showed that forskolin was not acting by competing with carbachol for the receptor activation site. Given the lipophilic nature of forskolin, it probably acts like a general anesthetic to perturb the plasma membrane lipid structure and alter the function of the nicotinic acetylcholine receptors, possibly by increasing the rate of closure of open channels. PMID- 3005281 TI - Transfer RNA is required for conjugation of ubiquitin to selective substrates of the ubiquitin- and ATP-dependent proteolytic system. AB - Degradation of intracellular proteins via the ubiquitin- and ATP-dependent proteolytic pathway involves several steps. In the initial event, ubiquitin, an abundant 76-residue polypeptide is covalently linked to the protein substrate in an ATP-requiring reaction. Proteins marked by ubiquitin are selectively proteolyzed in a reaction that also requires ATP. Ubiquitin conjugation to proteins appears also to be involved in regulation of cell cycle and cell division, and probably in the regulation of gene expression at the level of chromatin structure. We have previously shown (Ciechanover, A., Wolin, S. L., Steitz, J. A., and Lodish, H. F. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1341 1345) that transfer RNA is an essential component of the ubiquitin pathway. Ribonucleases strongly and specifically inhibited the degradation of 125I-labeled bovine serum albumin, while tRNA purified from reticulocyte extract could restore the proteolytic activity. Specifically, pure tRNAHis isolated by immunoprecipitation with human autoimmune serum could restore the proteolytic activity. Here we demonstrate that tRNA is required for conjugation of ubiquitin to some but not all proteolytic substrates of the ubiquitin mediated pathway. Conjugation of 125I-labeled ubiquitin to reduced carboxymethylated bovine serum albumin, alpha-lactalbumin, and soybean trypsin inhibitor was strongly and specifically inhibited by ribonucleases. Consequently, the ATP-dependent degradation of these substrates in the cell-free ubiquitin-dependent reticulocyte system was inhibited as well. Addition of tRNA to the ribonuclease inhibited system (following inhibition of the ribonuclease) restored both the conjugation activity and the ubiquitin- and ATP-dependent degradation of these substrates. Conjugation of ubiquitin to some endogenous reticulocyte proteins was also inhibited by ribonucleases and could be restored by the addition of tRNA. In striking contrast, the conjugation of radiolabeled ubiquitin to lysozyme, oxidized RNase A, alpha-casein, and beta-lactoglobulin was not affected by the ribonuclease treatment, and the degradation of these substrates was significantly accelerated by the ribonucleases. These findings indicate that there are at least two distinct ubiquitin conjugation systems. One requires tRNA, and the other is tRNA independent. These pathways, however, must share some common component(s) of the system, since the inhibition of one system accelerates the other. The possible function of tRNA in the selective conjugation reaction and the possible role of the two distinct ubiquitin marking mechanisms are discussed. PMID- 3005282 TI - Ethanol increases the expression of functional delta-opioid receptors in neuroblastoma x glioma NG108-15 hybrid cells. AB - Ethanol inhibits opioid peptide binding to the delta-opioid receptor. When neuroblastoma x glioma NG108-15 hybrid cells are grown with 25-200 mM ethanol, opioid receptor density increases up to 2-fold without a change in receptor affinity. Since changes in neurotransmitter receptor density may be important in neuronal adaptations to ethanol, we investigated the underlying mechanisms and functional consequences of this phenomenon. The opiate antagonist, naloxone, also increased opioid receptor number, but produced a smaller effect than ethanol with greater fractional inhibition of binding; long term enhancement of binding by ethanol is therefore not a simple function of acute receptor inhibition. Ethanol did not inhibit receptor down-regulation by etorphine, an opiate agonist, and therefore is not likely to increase receptor expression through interference with tonic down-regulation by endogenous opioid peptides. Ethanol increased opioid receptor expression in NG108-15 cells treated with actinomycin D, but not cycloheximide; hence, normal protein synthesis, but not DNA transcription, may be required for this response. The opioid receptors induced in ethanol-treated cells were subject to normal up-regulation by naloxone, down-regulation by etorphine, and acute inhibition of agonist binding by Na+. Etorphine maximally inhibits cyclic AMP accumulation in NG108-15 cells with only fractional occupancy of opioid receptors. Chronic ethanol exposure increased the receptor reserve for this response, resulting in a 3.5-fold increase in the potency of etorphine for inhibiting phenylisopropyladenosine-stimulated cyclic AMP accumulation. Neuronal adaptation to ethanol may involve changes in the density of receptors that regulate cellular levels of cyclic AMP. PMID- 3005283 TI - Relationship of major phosphorylation reactions and MgATPase activities to ATP dependent shape change of human erythrocyte membranes. AB - Human erythrocyte ghosts prepared by hemolysis and washing in hypotonic Tris are crenated by salt and divalent cations, but undergo shape change to smooth biconcave discs and stomatocytic forms when incubated with MgATP at 37 degrees C. This is normally accompanied by protein and lipid phosphorylations in which the major phosphate acceptors are the spectrin beta-chain and inositol phospholipids, respectively. The system was manipulated in several ways to demonstrate the independence of ATP-dependent shape change from the major phosphorylation reactions. Salt-extracted membranes incubated with adenosine, an inhibitor of spectrin and phosphatidylinositol kinases, underwent normal shape change despite reductions of greater than 90% in spectrin and phospholipid labeling by [gamma 32P]ATP. ATP-dependent shape change was blocked by vanadate at micromolar concentrations (half-maximal inhibition at less than 1 microM), but vanadate did not inhibit membrane autophosphorylation reactions or turnover of spectrin- or lipid-bound phosphate. Vanadate inhibited part of the ATP hydrolysis that accompanies shape change and is expressed in the presence of ouabain and EGTA. The vanadate-sensitive MgATPase activity was approximately 3 nmol Pi X min-1 X mg of protein-1. The results implicate it in ATP-dependent shape change. PMID- 3005284 TI - Phosphatidylinositol synthase from Saccharomyces cerevisiae. Reconstitution, characterization, and regulation of activity. AB - Purified membrane-associated phosphatidylinositol synthase (CDP diacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) from Saccharomyces cerevisiae was reconstituted into unilamellar phospholipid vesicles. Reconstitution of the enzyme was performed by removing detergent from an octylglucoside/phospholipid/Triton X-100/enzyme mixed micelle mixture by Sephadex G-50 superfine column chromatography. The average diameter of the vesicles was 40 nm and chymotrypsin treatment of intact vesicles indicated that over 90% of the reconstituted enzyme had its active site facing outward. The enzymological properties and reaction mechanism of reconstituted phosphatidylinositol synthase were determined in the absence of detergent. The reconstituted enzyme was used as a model system to study the regulation of activity. Phosphatidylinositol synthase was constitutive in wild type cells grown in the presence of water-soluble phospholipid precursors as determined by enzyme activity and immunoblotting. Reconstituted enzyme was not effected by water soluble phospholipid precursors or nucleotides. Maximum activity was found when the enzyme was reconstituted into phosphatidylcholine: phosphatidylethanolamine: phosphatidylinositol: phosphatidylserine vesicles. Phosphatidylserine stimulated reconstituted activity, suggesting that the local phospholipid environment may regulate phosphatidylinositol synthase activity. PMID- 3005285 TI - Biochemical changes during sucrose deprivation in higher plant cells. AB - The mobilization of stored carbohydrates (sucrose and starch) during sucrose starvation was studied with sycamore (Acer pseudoplatanus) cells. When sucrose was omitted from the nutrient medium, vacuolar sucrose was first consumed. When a threshold of intracellular sucrose concentration was attained the cytoplasmic phosphorylated compounds decreased whereas cytoplasmic Pi increased symmetrically. Such a situation triggered starch breakdown. When almost all the intracellular sucrose pool had disappeared, the cell respiration rates (normal and uncoupled) declined progressively. The decrease in the rate of respiration triggered by sucrose starvation was attributable neither to the availability of substrate for mitochondrial respiration nor to a decrease in the maximal rate of O2 consumption by mitochondria expressed in terms of nanomole of O2 consumed per min/mg of mitochondrial protein. In fact, the uncoupled respiration rates decreased in parallel with the decrease in total intracellular cardiolipin or cytochrome aa3. These results demonstrate therefore that after a long period of sucrose starvation the progressive decrease in the uncoupled rate of O2 consumption by sycamore cells was attributable to a progressive diminution of the number of mitochondria/cell. PMID- 3005286 TI - Polarized distribution of the Na+/H+ exchange system in a renal cell line (LLC PK1) with characteristics of proximal tubular cells. AB - Studies of Na+ and H+ transport by confluent monolayers of the epithelial cell line LLC-PK1 were performed to verify the presence of a Na+/H+ exchange system. The presence of an outwardly directed H+ gradient produced a large stimulation of Na+ influx measured under net flux conditions. Amiloride (10(-3) M) completely inhibited Na+ influx stimulated by the H+ gradient and part of the Na+ influx measured in the absence of a pH gradient. Half-maximal inhibition of the Na+ influx stimulated by a pH gradient at 143 mM Na was observed at 5 microM amiloride. The presence of an inwardly oriented proton gradient also stimulated Na+ efflux from Na+-loaded cells. The stimulation was completely inhibited by the presence of 10(-3) M amiloride in the washout medium. These results indicate that this system could operate in the opposite direction depending on the orientation of the Na+ and H+ gradient. Incubation in Na+-free medium or in the presence of 10(-3) M ouabain resulted in a dramatic decrease of H+ release from LLC-PK1 cells. This H+ release was largely, although not completely, inhibited by 10(-4) M amiloride. Neither chloride substitution by the impermeable anion isethionate nor incubation in the presence of the ionophore valinomycin in high K+ medium affected Na+ influx by stimulated by a pH gradient. Inhibition of the Na+ influx by amiloride occurred only from the apical side of the monolayer. These results indicate that the Na+/H+ exchange system in LLC-PK1 monolayers is specifically localized in the apical membrane of the epithelial cells. PMID- 3005288 TI - Reversible change in light scattering following formation of ADP-sensitive phosphoenzyme in Na+,K+-ATPase modified with N-[p-(2 benzimidazolyl)phenyl]maleimide. AB - An increase in light scattering (3.5 +/- 0.2%) was observed when pig kidney Na+,K+-ATPase preparations modified with N-[p-(2-benzimidazolyl)phenyl] maleimide were phosphorylated by ATP in the presence of 2 M Na+ with Mg2+ to form ADP sensitive phosphoenzyme (E1P), which had a negative fluorescence intensity (-1.5 +/- 0.3%). Addition of K+ or ouabain to E1P reduced the light scattering to the original level observed in the absence of ATP. Stopped flow measurements showed that the fluorescence change accompanying the E1P formation (t1/2 = 0.1 s) occurred preceding the light-scattering change (t1/2 = 1 s). Oligomycin affected the rate of the scattering increase little, but it diminished the effect of K+ on E1P to reduce the light scattering and increase the fluorescence. The addition of 2 M Na+ to K+-sensitive phosphoenzyme (E2P) immediately decreased the fluorescence (t1/2 = 0.02 s) to form E1P which was followed by a slow increase in the light scattering (t1/2 = 0.25 s). Oligomycin reduced both rates of the above changes accompanying the transition of E2P to E1P. The data suggest the sequential appearance of species of E1P that precede E2P formation during the hydrolysis of ATP. PMID- 3005287 TI - Amino acid replacements in yeast iso-1-cytochrome c. Comparison with the phylogenetic series and the tertiary structure of related cytochromes c. AB - The structural and folding requirements of eukaryotic cytochromes c have been investigated by determining the appropriate DNA sequences of a collection of 46 independent cyc 1 missense mutations obtained in the yeast Saccharomyces cerevisiae and by deducing the corresponding amino acid replacements that abolish function of iso-1-cytochrome c. A total of 33 different replacements at 19 amino acid positions were uncovered in this and previous studies. Because all of these nonfunctional iso-1-cytochromes c are produced at far below the normal level and because a representative number are labile in vitro, most of the replacements appear to be affecting stability of the protein or heme attachment. By considering the tertiary structure of related cytochromes c, the loss of function of most of the mutant iso-1-cytochromes c could be attributed to either replacements of critical residues that directly interact with the heme group or to replacements that disrupt the proper folding of the protein. The replacements of residues interacting with the heme group include those required for covalent attachment (Cys-19 and Cys-22), ligand formation (His-23 and Met-85), and formation of the immediate heme environment (Leu-37, Tyr-53, Trp-64, and Leu-73). Proper folding of the protein is prevented by replacements of glycine residues at sites that cannot accommodate side chains (Gly-11 and Gly-34); by replacements of residues with proline, which limit the torsion angle (Leu-14 and His-38); and by replacements apparently unable to direct the local folding of the backbone into the proper conformation (Pro-35, Tyr-72, Asn-75, Pro-76, Lys-84, Leu-99, and Leu 103). Even though most of the missense mutations occurred at sites corresponding to evolutionarily invariant or conserved residues, a consideration of the replacements in functional revertants indicates that the requirement for residues evolutionarily preserved is less stringent than commonly assumed. PMID- 3005289 TI - Reconstruction of adenovirus replication origins with a human nuclear factor I binding site. AB - Nuclear factor I is a host-coded DNA-binding protein that stimulates initiation of adenovirus DNA replication. To understand the mechanism of action of nuclear factor I, we have constructed, by recombinant DNA techniques, origins of replication in which the adenovirus type 5 nuclear factor I binding site (FIB site) has been replaced by a FIB site isolated from human genomic DNA (Gronostajski, R. M., Nagata, K., and Hurwitz, J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4013-4017). Assays of such recombinants for initiation and elongation in vitro showed that nuclear factor I was active only when the FIB site was relatively close to the DNA terminus, i.e. the FIB site was centered at nucleotides 30-36 from the end of the DNA. Nuclear factor I was active in either orientation within this distance range. The presence of one or two additional FIB sites in the downstream region had no effect. The implications of these results for the mechanism of nuclear factor I action are discussed. PMID- 3005290 TI - Alterations in components of adenylate cyclase associated with transformation of chicken embryo fibroblasts by Rous sarcoma virus. AB - Regulation of adenylate cyclase coincident with transformation of chicken embryo fibroblasts by Rous sarcoma virus is manifest as a 10-50% decrease in basal, Mg2+ , and forskolin-stimulated activities; activities elicited by fluoride and guanosine 5'-O-(3-thiotriphosphate) are unaltered. The level of the catalytic component of adenylate cyclase, assessed with activated stimulatory guanine nucleotide-binding protein (Gs), increases approximately 1.5-fold. The level of the beta subunit common to Gs and the inhibitory regulatory protein assessed by enzyme-linked immunotransfer blotting, increases 2.7-fold. The isoelectric behavior of the beta subunit is unaltered. The amount of radiolabel incorporated into the alpha subunit of Gs (Mr = 45,000) upon incubation of membranes with 32P labeled NAD and cholera toxin increases 3-fold upon transformation. Detergent extracts prepared from membranes of untransformed and transformed fibroblasts nevertheless exhibit equivalent abilities to reconstitute fluoride-stimulated activities to membranes of the cyc-variant of mouse S49 lymphoma cells. Islet activating protein catalyzes incorporation of radiolabel from 32P-labeled NAD into 39,000- and 41,000-dalton proteins; the extent of radiolabel incorporation does not change upon transformation. Modest alterations in the isoelectric behaviors of substrates for cholera toxin and islet-activating protein occur. PMID- 3005291 TI - Structure of rodent helix-destabilizing protein revealed by cDNA cloning. AB - A cDNA library of newborn rat brain poly(A+) RNA in lambda gt 11 was screened with a synthetic oligonucleotide probe corresponding to a five amino acid sequence in the N-terminal region of the calf helix-destabilizing protein, UP1. Six positive phage were isolated after testing 2 X 10(5) recombinants, and each phage was plaque purified. Four of these phage clones were positive with a second oligonucleotide probe corresponding to a 5 amino acid sequence in the C-terminal region of calf UP1; one of the clones positive with both probes was selected for detailed study. This phage, designated lambda HDP-182, contained a 1706-base pair cDNA insert corresponding to an mRNA with a poly(A) sequence at the 3' terminus and a single open reading frame starting 63 bases from the 5' terminus and extending 988 bases. The 3' untranslated region of the mRNA contained 718 bases, including an AAUAAA signal 21 bases from the poly(A) sequence and a 16-residue poly(U) sequence flanked on each side by oligonucleotide repeats. Primer extension analysis of newborn rat brain poly(A+) RNA suggested that the cDNA insert in lambda HDP-182 was full length except for about 35 nucleotide residues missing from the 5' end untranslated region, and Northern blot analysis revealed one relatively abundant mRNA species of approximately the same size as the cDNA insert. The 988-residue open reading frame in the cDNA predicted a 34,215-dalton protein of 320 amino acids. Residues 2 through 196 of this rat protein are identical to the 195-residue sequence of the calf helix-destabilizing protein, UP1. The 124-amino acid sequence in the C-terminal portion of the 34,215-dalton protein is not present in purified calf UP1. This 124-residue sequence has unusual amino acid content in that it is 11% asparagine, 15% serine, and 40% glycine and consists of 16 consecutive oligopeptide repeats. Computer-derived secondary structure predictions for the 34,215-dalton protein revealed two distinct domains consisting of residues 1 through approximately 196 and residues approximately 197 to 320, respectively. PMID- 3005292 TI - Solubilization and hydrodynamic characterization of the dihydropyridine receptor from rat ventricular muscle. AB - The dihydropyridine receptor-calcium channel complex, prelabeled with (+) [3H]PN200-110, was solubilized from rat heart membranes with a detergent mixture of digitonin and Triton X-100. The dissociation of (+)-[3H]PN200-110 was slow enough to permit the hydrodynamic characterization of the complex by means of sucrose gradient sedimentation and gel filtration. The hydrodynamic properties of the complex were determined in several detergents, including Tween 80, 3-[(3 cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), and digitonin. S20,w values of 12.5, 15.4, and 21.0 S were obtained in sucrose gradients prepared in Tween 80, CHAPS, and digitonin, respectively. A Stokes radius of 86 87 A was obtained in each of the three detergents. Determination of the partial specific volume of the protein-detergent complex in each case revealed that the differences in S20,w values could be explained by the differences in the properties of the bound detergent species. Partial specific volumes of 0.796, 0.730, and 0.730 ml/g, corresponding to molecular weights of 595,000, 540,000, and 740,000 were obtained for the complex in Tween 80, CHAPS, and digitonin, respectively. This indicated that Tween 80 readily exchanged for the solubilizing mixture of digitonin and Triton X-100, whereas CHAPS did not. Detergent exchange with Tween 80 made it possible to determine the fractional contribution of the receptor protein to the molecular weight of the protein-detergent complex. The molecular weight of the dihydropyridine receptor-calcium channel complex was estimated to be 370,000. The protein-detergent complex was found to have a frictional coefficient of 1.39, consistent with a large transmembrane protein. PMID- 3005294 TI - Methylamine dehydrogenase and cytochrome c552 from the bacterium W3A1. AB - We describe a two-step purification of the methoxatin-containing enzyme methylamine dehydrogenase from crude extracts of the bacterium W3A1, and a longer purification of cytochrome c552 from the same organism. Some of the kinetic properties of the dehydrogenase are presented, together with the demonstration that c552 is an electron acceptor for this enzyme. Cytochrome c552 is the only hemeprotein we observed in the visible spectrum of intact W3A1 cells that were grown under the same culture conditions used for the protein purifications. Addition of methylamine to whole cells causes an increase in the rate of O2 uptake together with an abrupt reduction of c552. We propose that, in vivo, the electrons from the amine reach the hemeprotein through the dehydrogenase. PMID- 3005293 TI - Mechanism of ribonucleotide reductase from herpes simplex virus type 1. Evidence for 3' carbon-hydrogen bond cleavage and inactivation by nucleotide analogs. AB - Isotope effects of 2.5, 2.1, and 1.0 were measured on the conversion of [3' 3H]ADP, [3'-H]UDP, and [5-3H] UDP to the corresponding 2'-deoxynucleotides by herpes simplex virus type 1 ribonucleotide reductase. These results indicate that the reduction of either purine or pyrimidine nucleotides requires cleavage of the 3' carbon-hydrogen bond of the substrate. The substrate analogs 2'-chloro-2' deoxyuridine 5'-diphosphate (ClUDP), 2'-deoxy-2'-fluorouridine 5'-diphosphate, and 2'-azido-2'-deoxyuridine 5'-diphosphate were time-dependent inactivators of the herpes simplex virus type 1 ribonucleotide reductase. Incubation of [3' 3H]ClUDP with the enzyme was accompanied by time-dependent release of 3H to the solvent. Reaction of [beta-32P]ClUDP with the reductase resulted in the production of inorganic pyrophosphate. These results are consistent with the enzyme-mediated cleavage of the 3' carbon-hydrogen bond of ClUDP and the subsequent conversion of the nucleotide to 2-methylene-3(2H)furanone, as previously reported with the Escherichia coli ribonucleotide reductase (Harris, G., Ator, M., and Stubbe, J. A. (1984) Biochemistry 23, 5214-5225; Ator, M., and Stubbe, J. A. (1985) Biochemistry 24, 7214-7221). PMID- 3005295 TI - Interaction of plasma gelsolin with ADP-actin. AB - In the presence of Ca2+, gelsolin forms a very tight, stoichiometric complex with 2 molecules of ADP-G-actin. Removal of free Ca2+ causes the 1:2 complex to dissociate to a 1:1 complex. Gelsolin accelerates the very slow polymerization of ADP-actin, apparently by accelerating the rate of nucleation, but the number concentration of filaments formed is probably less than the gelsolin concentration, indicating that the GA2 complex is not a true nucleus. These results are similar to those obtained for the interaction of gelsolin with ATP-G actin. Both kinetic and equilibrium measurements demonstrate that the critical concentration of gelsolin-capped ADP-actin filaments (8 microM in 1 mM MgCl2 and 0.2 mM ADP) is the same as for the uncapped filaments, proving that the critical concentration is the same at both ends of the equilibrium polymer in ADP as predicted by theory. The association and dissociation rate constants for the addition of ADP-G-actin at the pointed end of an ADP-F-actin filament are estimated to be 4.6 X 10(4) M-1 s-1 and 0.4 s-1, respectively, about 15-fold lower than the rate constants at the barbed end. PMID- 3005296 TI - Carbodiimide inactivation of Na,K-ATPase. A consequence of internal cross-linking and not carboxyl group modification. AB - Irreversible inhibition of Na,K-ATPase and K+-dependent p-nitrophenylphosphatase activities was produced by incubation of purified Na,K-ATPase enzyme with 1-ethyl 3(3-dimethylaminopropyl)carbodiimide (EPC). Inhibition was time and [EPC] dependent and displayed first order kinetics with respect to time. The [EPC] to reduce the enzyme velocity by 50% for Na,K-ATPase and phosphatase activities was 1.6 and 2.2 mM, respectively. Analysis of the kinetics of inhibition by EPC indicated that reaction at one site was sufficient to produce inhibition. Inhibition was greatly reduced by the presence of Mg2+, Na+, K+, choline, or Tris (decreasing order of effectiveness); ATP was without effect. This suggests that cation-bound enzyme forms were less reactive with the carbodiimide than free enzyme; ATP-bound enzyme was as reactive. Apparently the cations Na+, Mg2+, Tris, and choline stabilize E1 forms of the enzyme which are different from the E1 form stabilized by ATP. Addition of [14C]glycine ethyl ester (Gly-OEt) resulted in incorporation of radioactivity into both alpha and beta subunits that was dependent upon the presence of EPC, and the incorporation was reduced by the cations which reduced the inhibition due to EPC. Simultaneous addition of Gly-OEt with EPC prevented inhibition, although 14C incorporation still took place. If Gly-OEt addition was delayed the initial inactivation was not affected, but little subsequent inactivation occurred. The protection against inactivation by EPC occurs on the addition of other exogenous nucleophiles, such as aminoethane or ethylenediamine. Dicyclohexylcarbodiimide, a more potent hydrophobic carbodiimide inhibitor, shows similar effects; the inhibition due to dicyclohexylcarbodiimide is also prevented by the simultaneous presence of a nucleophile. After treatment with a carbodiimide and exogenous nucleophile the Na,K-ATPase has modified carboxyl residues but is not inhibited. Thus, modification of the cation-protectable carboxyl groups does not by itself cause inhibition. It seems likely that the inhibition of activity due to carbodiimide alone is not due to the modification of a carboxyl group per se but to the formation of an intramolecular bond between the carbodiimide-activated carboxylic acid and an endogenous nucleophile. The formation of such bonds suggests the close juxtaposition of amine and carboxyl groups in the secondary structure of the enzyme. PMID- 3005297 TI - Internalization and recycling of transferrin and its receptor. Effect of trifluoperazine on recycling in human erythroleukemic cells. AB - When human erythroleukemic (K562) cells were incubated with 25 microM trifluoperazine (TFP), a drug that inhibits both calmodulin-dependent and calcium activated phospholipid-dependent kinases, the number of transferrin receptors detected on the cell surface was reduced to approximately half with no change in the affinity of the remaining surface receptors. Removal of the TFP from the incubation medium reversed the loss of surface receptors and they returned to the cell surface in an apparently synchronous manner. As a result, the number of receptors detected on the cell surface exceeded the original level but later returned to normal. Measurements of the total number of receptors available to transferrin in TFP-treated cells suggested that the lost receptors were not participating in the internalization and recycling pathway but instead were probably trapped at an intracellular location. However, those receptors that remained on the cell surface continued to internalize transferrin and to recycle apotransferrin to the cell surface albeit more slowly than in cells that had not been treated with TFP. Using transferrin that had been labeled with iron-59, it was found that although iron uptake was reduced in line with the diminished number of surface receptors, iron still accumulated within TFP-treated cells, suggesting that in the presence of the drug, transferrin-transferrin receptor complexes continued to migrate through an intracellular compartment that contained a low pH. PMID- 3005298 TI - Purification and characterization of the human brain insulin receptor. AB - The insulin receptor from human brain cortex was purified by a combination monoclonal antibody affinity column and a wheat germ agglutinin column. This purified receptor preparation exhibited major protein bands of apparent Mr = 135,000 and 95,000, molecular weights comparable to those for the alpha and beta subunits of the purified human placental and rat liver receptors. A minor protein band of apparent Mr = 120,000 was also observed in the brain receptor preparation. Crosslinking of 125I-insulin to all three receptor preparations was found to preferentially label a protein of apparent Mr = 135,000. In contrast, cross-linking of 125I-labeled insulin-like growth factor I to the brain preparation preferentially labeled the protein of apparent Mr = 120,000. The purified brain insulin receptor was found to be identical with the placental insulin receptor in the amount of neuraminidase-sensitive sialic acid and reaction with three monoclonal antibodies to the beta subunit of the placental receptor. In contrast, a monoclonal antibody to the insulin binding site recognized the placental receptor approximately 300 times better than the brain receptor. These results indicate that the brain insulin receptor differs from the receptor in other tissues and suggests that this difference is not simply due to the amount of sialic acid on the receptor. PMID- 3005299 TI - Limited proteolytic digestion and dissociation of smooth muscle phosphatase-I modifies its substrate specificity. Preparation and properties of different forms of smooth muscle phosphatase-I. AB - Smooth muscle phosphatase-I (SMP-I), a protein phosphatase purified from turkey gizzard smooth muscle, is composed of 2 regulatory subunits (Mr = 60,000 and 55,000) and a catalytic subunit (Mr = 38,000). Two other forms of this enzyme have been prepared and characterized. The free catalytic subunit, termed SMP-Ic, was prepared by ethanol treatment of SMP-I, and a form devoid of the 55,000-Da subunit, termed SMP-I2, was prepared by limited tryptic digestion. Exposure of SMP-I to proteases like trypsin and chymotrypsin results in a rapid degradation of the 55,000-Da polypeptide. Degradation of the catalytic subunit is observed only upon prolonged digestion. The 60,000-Da polypeptide appears to be resistant to the action of trypsin and chymotrypsin. SMP-I dephosphorylates myosin light chains but is not active toward intact myosin or heavy meromyosin. However, when the catalytic subunit is dissociated from both regulatory subunits or from the 55,000-Da polypeptide, the enzyme becomes active toward myosin suggesting that the 55,000-Da polypeptide inhibits the activity of the catalytic subunit toward myosin. In addition to alteration of the substrate specificity, the regulatory subunits also modulate the effect of divalent cations, like Mn2+, on the activity of the enzyme. PMID- 3005300 TI - Characterization of affinity-purified insulin receptor/kinase. Effects of dithiothreitol on receptor/kinase function. AB - The insulin receptor/kinase was purified to near homogeneity from human placenta. The purified kinase exhibited a specific activity of 300 nmol/min/mg of protein at 30 degrees C using the synthetic peptide, Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr Ala-Ala-Arg-Gly, as substrate in the presence of insulin. Treatment of the receptor/kinase with dithiothreitol (DTT) reduced insulin binding by 40-50% and also inhibited tyrosine kinase activity. Phosphorylation and activation of the receptor/kinase did not prevent the DTT-induced loss of binding but completely protected it from the deleterious effects of reducing agent on enzymic activity. Analyses of the structure of the receptor/kinase following phosphorylation and treatment with DTT indicated that the class I disulfide bonds were reduced under the conditions employed, but the tetrameric structure of the receptor/kinase was essentially unaltered. These findings indicate that intact class I disulfides are required for insulin binding but are not necessary for maintenance of the preactivated kinase. DTT was also found to enhance the autoactivation of the insulin receptor/kinase and to promote the reversal of the autophosphorylation reaction. Thus disulfide bonds appear to have multiple roles in the function of the insulin receptor/kinase. PMID- 3005301 TI - Effects of collagenase on the structure of the lutropin/choriogonadotropin receptor. AB - The structure of the lutropin/choriogonadotropin (LH/CG) receptor of a clonal strain of cultured Leydig tumor cells (designated MA-10) and primary cultures of porcine granulosa cells was studied by cross-linking 125I-labeled derivatives of human CG and ovine LH with bifunctional succinimidyl esters. We show that in both cell types, both subunits of the receptor-bound hormone become cross-linked to a single cellular component of Mr = 106,000, when analyzed in the absence of reducing agents, and of Mr = 83,000 when analyzed in the presence of reducing agents. We also present a detailed investigation on the effects of several collagenase preparations on the structure and some functions of the LH/CG receptor. Our results show that the LH/CG receptor is exquisitively sensitive to degradation by these preparations of collagenase; degradation products can be detected only in the presence of reducing agents; the enzyme(s) responsible for degradation is not collagenase itself, but rather a contaminating enzyme(s), presumably a protease(s); and receptor degradation has little effect on the ability of the cells to bind hormone or to respond with increased steroid biosynthesis. Since normal gonadal cells are usually isolated following dispersion of the tissue with collagenase, our results suggest that these cells are likely to bear a degraded (albeit functional) form of the LH/CG receptor, and thus should not be used in studies dealing with the structure of this receptor. PMID- 3005302 TI - Studies on the lipid dependency and mechanism of the translocation of the mitochondrial precursor protein apocytochrome c across model membranes. AB - The lipid dependency of apocytochrome c binding to model membranes and of the translocation of the precursor protein across these membranes was studied by using large unilamellar, trypsin-containing vesicles. These vesicles were improved with respect to those used in a previous article (Rietveld, A., and de Kruijff, B. (1984) J. Biol. Chem. 259, 6704-6706), in the sense that a lower amount of trypsin was enclosed. In mixed egg phosphatidylcholine/bovine brain phosphatidylserine vesicles, both the Kd of apocytochrome c binding (about 20 microM) and the number of phosphatidylserine molecules interacting with the protein was found to be constant. When the phosphatidylserine fraction in the vesicles is more than 15-30% apocytochrome c addition results in the exposure of (a part of) the protein to the internal, trypsin-containing vesicle medium, which process we conceive as a translocation event. Also the interaction of apocytochrome c with vesicles composed of phosphatidylcholine and another acidic phospholipid in a 1:1 ratio, leads to the translocation of the protein across the model membrane. The affinity of this binding was found to be in the order cardiolipin greater than phosphatidylglycerol greater than phosphatidylinositol greater than phosphatidylserine. By varying the lipid composition of the vesicles, it could be demonstrated that the translocation requires a fluid bilayer. In addition, protein specificity was shown for the translocation process. Although apocytochrome c-lipid interaction causes vesicle aggregation, fusion by lipid mixing could not be detected. Due to the apocytochrome c-lipid interaction also, protein aggregates and oligomers have been formed. These results will be discussed in the light of a model for translocation of a precursor protein across a model membrane. The relevance for the mitochondrial system will also be discussed. PMID- 3005303 TI - Tumor-promoting phorbol esters increase the Km of the ATP-binding site of the insulin receptor kinase from rat adipocytes. AB - Phorbol ester treatment of isolated rat adipocytes inhibits insulin stimulation of the glucose transport system. We studied whether this effect is related to a modification of the insulin receptor kinase. Insulin receptor of tetradecanoyl beta-phorbol acetate (TPA)-treated adipocytes was solubilized and partially purified, and its kinase activity was studied in vitro. We found that insulin (10(-7) M) increased the tyrosine autophosphorylation of the insulin receptor kinase from TPA-treated cells only 3-fold in contrast to a 12-fold stimulation in control cells. The rate of insulin-stimulated 32P incorporation into the receptor of TPA-treated cells at insulin concentrations between 10(-10) M and 10(-7) M and at nonsaturating [32P]ATP levels (5 microM) was reduced to only 5-8% of the values found with receptor from control cells. 125I-insulin binding to the solubilized receptor of TPA-treated cells was reduced as well, apparently due to a decreased affinity of the binding site. Decreased binding was however, not sufficient to explain the difference of kinase activity. The inhibition of kinase activity of the receptor from TPA-treated cells decreased when the concentration of [gamma-32P]ATP in the phosphorylation assay was increased. A Lineweaver-Burk analysis of receptor phosphorylation revealed that the Km for ATP of the receptor kinase from TPA-treated cells was increased to greater than 100 microM compared to 25 microM for the receptor of control cells. An analogous change of the Km for ATP was found when we studied the phosphorylation of a synthetic polymer of tyrosine and glutamic acid as a substrate of the receptor kinase. We conclude from the data that phorbol treatment of rat adipocytes modulates the kinase activity of the insulin receptor by increasing its Km for ATP and that this is part of a mechanism leading to insulin resistance in these cells. PMID- 3005304 TI - The effect of bovine thrombomodulin on the specificity of bovine thrombin. AB - Bovine lung thrombomodulin is purified and used to investigate the basis of the change in substrate specificity of bovine thrombin when bound to thrombomodulin. Bovine thrombomodulin is a single polypeptide having an apparent molecular weight of 84,000 and associates with thrombin with high affinity and rapid equilibrium, to act as a potent cofactor for protein C activation and antagonist of reactions of thrombin with fibrinogen, heparin cofactor 2, and hirudin. Bovine thrombomodulin inhibits the clotting activity of thrombin with Kd less than 2.5 nM. Kinetic analysis of the effect of bovine thrombomodulin on fibrinopeptide A hydrolysis by thrombin indicates competitive inhibition with Kis = 0.5 nM. The active site of thrombin is little perturbed by thrombomodulin, as tosyl-Gly-Pro Arg-p-nitroanilide hydrolysis and inhibition by antithrombin III are unaffected. Insensitivity of the reaction with antithrombin III is likewise observed with thrombin bound to thrombomodulin on intact endothelium. Antithrombin III-heparin, human heparin cofactor 2, and hirudin inhibit thrombin-thrombomodulin more slowly than thrombin. These effects may arise from a decrease in Ki of the inhibitors for thrombin-thrombomodulin or from changes in the active site not detected by tosyl-Gly-Pro-Arg-p-nitroanilide or antithrombin III. Bovine prothrombin fragment 2 inhibits thrombin clotting activity (Kd less than 7.5 microM) and acts as a competitive inhibitor of protein C activation (Kis = 2.1 microM). The data are consistent with a mechanism whereby thrombomodulin alters thrombin specificity by either binding to or allosterically altering a site on thrombin distinct from the catalytic center required for binding or steric accommodation of fibrinogen, prothrombin fragment 2, heparin cofactor 2, and hirudin. PMID- 3005305 TI - The degradation of guanidinated lysozyme in reticulocyte lysate. AB - Egg white lysozyme, treated with O-methylisourea to convert lysine to homoarginine residues, was used as a substrate for the ATP-dependent proteolytic pathway in rabbit reticulocyte lysates. Although guanidinated lysozyme was degraded by an ATP-dependent, hemin-sensitive process, ubiquitin conjugates of this protein were present at less than 5% the level of conjugates between ubiquitin and nonguanidinated lysozyme. When lysates were chromatographed on DEAE cellulose to produce Fractions I and II of (Hershko et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3107), ubiquitin-depleted Fraction II was capable of degrading nonguanidinated lysozyme, but the degradation of guanidinated lysozyme was markedly reduced or abolished. Glycerol-stabilized Fraction II, on the other hand, supported the degradation of both proteins in an ATP-dependent process stimulated by ubiquitin. The degradation of the two proteins differed, however, in that guanidinated lysozyme was more sensitive to competitive substrates, and higher concentrations of ubiquitin were required for its maximal proteolysis. Despite ubiquitin stimulation of guanidinated lysozyme degradation, only trace amounts of higher molecular weight species of guanidinated lysozyme attributable to ubiquitin conjugation were observed in ubiquitin-supplemented, glycerol stabilized Fraction II even when special precautions were employed to preserve labile covalent bonds. These results indicate that covalent attachment of ubiquitin to the epsilon-amino group of substrate lysines is not mandatory for ATP-dependent proteolysis in rabbit reticulocyte lysates. The observation that ubiquitin stimulates proteolysis of guanidinated lysozyme, without extensive conjugation to it, suggests that ubiquitin may have essential functions for proteolysis other than direct marking of the protein substrate. PMID- 3005306 TI - Purification and characterization of the human platelet alpha 2-adrenergic receptor. AB - Human platelet alpha 2-adrenergic receptors have been purified approximately 80,000-fold to apparent homogeneity by a five-step chromatographic procedure. The overall yield starting from the membranes is approximately 2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated protein from purified receptor preparations shows a single major band of Mr 64,000. The specific binding activity of the alpha 2-adrenergic receptor after four chromatographic steps is 14.5 nmol/mg protein. This value is consistent with the expected theoretical specific activity (15.6 nmol/mg) for a protein with a molecular mass of 64,000 daltons if it is assumed that there is one ligand binding site/receptor molecule. The purified protein can be covalently labeled with the alkylating alpha-adrenergic ligand, [3H]phenoxybenzamine. This labeling is specific, and it shows that the Mr 64,000 protein contains the ligand binding site of the alpha 2-adrenergic receptor. In addition, the competitive binding of ligands to the purified receptor protein shows the proper alpha 2-adrenergic specificity. The alpha 2-adrenergic receptor contains an essential sulfhydryl residue. Thus, exposure of the purified receptor to the sulfhydryl-specific reagent, phenylmercuric chloride, resulted in an 80% loss of binding activity. This loss of binding activity was prevented when exposure to phenylmercuric chloride was done in the presence of alpha 2-adrenergic ligands, and it was reversed by subsequent exposure to dithiothreitol. Partial proteolysis of purified alpha 2-adrenergic receptors was obtained with Staphylococcus aureus V-8 protease, alpha-chymotrypsin, and papain. In a comparison with purified beta 2 adrenergic receptors, no common partial proteolytic products were found. PMID- 3005308 TI - Human apolipoprotein A-I. Post-translational modification by fatty acid acylation. AB - The human apolipoproteins are secretory proteins some of which have been shown to undergo proteolytic processing and post-translational addition of carbohydrate. Apolipoprotein A-I (apo-A-I), the predominant protein associated with high density lipoproteins, undergoes co-translational proteolytic processing as well as post-translational conversion of proapo-A-I to mature apo-A-I following cellular secretion. Utilizing the human hepatoma cell line HEP-G2, we have established that, in addition to proteolytic processing, secreted nascent apo-A-I is acylated with palmitate. Uniformly labeled [14C]palmitate and [1-14C]palmitate were each incorporated into apo-A-I when analyzed by sodium dodecyl sulfate gel electrophoresis and autoradiography. The acylation of apo-A-I with palmitate was confirmed by immunoprecipitation and gas chromatography/mass spectrometry. Hydroxylamine treatment resulted in the deacylation of apo-A-I. Although three of the apo-A-I isoforms analyzed by two-dimensional gel electrophoresis were shown to contain radio-labeled palmitate, 80% of acylated apo-A-I was in the proapolipoprotein A-I isoform. [14C]Oleate was not incorporated in secreted apo-A I, indicating the specificity of the acylation of apo-A-I. Incubation of [14C] palmitate-acylated apo-A-I in serum and plasma under conditions in which proapo-A I is proteolytically cleaved to mature apo-A-I did not result in deacylation. These data establish that fatty acid acylation occurs in human secretory proteins in addition to the previously reported acylation of cellular membrane proteins. These results suggest that the covalent linkage of lipids to apolipoproteins may play a critical role in apolipoprotein and lipoprotein metabolism. PMID- 3005307 TI - Functional reconstitution of the alpha 2-adrenergic receptor with guanine nucleotide regulatory proteins in phospholipid vesicles. AB - We describe the successful reconstitution of functional interactions between an inhibitory adenylate cyclase-coupled receptor and various nucleotide-binding regulatory proteins in phospholipid vesicles. The receptor is the alpha 2 adrenergic receptor (alpha 2AR) which has been partially purified (approximately 500-5000-fold) from human platelet membranes. The nucleotide-binding regulatory proteins include purified preparations of human erythrocyte Ni and Ns, bovine retinal transducin and the recently discovered bovine brain No. Addition of the physiologic ligand, epinephrine, to vesicles containing the alpha 2AR and Ni results in stimulation of the GTPase activity in Ni. This stimulation of GTPase activity by epinephrine is prevented in the presence of the alpha-adrenergic antagonist, phentolamine, which indicates that a functional reconstitution of the alpha 2AR and Ni has been established. The maximum turnover number for the alpha 2AR-mediated epinephrine-stimulated GTPase activity in Ni is similar to the maximal turnover numbers obtained for the beta-adrenergic receptor-mediated isoproterenol-stimulated GTPase activity in Ns and the rhodopsin-mediated light stimulated GTPase activity in transducin (0.5-1.5 mol of Pi released per min per mol of nucleotide regulatory protein). Functional similarities between the alpha 2AR and rhodopsin are observed in their interactions with the various nucleotide binding regulatory proteins. Thus, both of these receptor proteins are capable of promoting the maximal activation of Ni and No while being much less effective in promoting the activation of Ns. However, there are differences between the alpha 2AR and rhodopsin in their interactions with transducin. Specifically, while rhodopsin will maximally activate transducin, the alpha 2AR is much less effective in promoting this activation (i.e. approximately 20% as effective as rhodopsin). Overall, these results suggest the following specificities of interaction: for rhodopsin, transducin approximately equal to Ni approximately equal to No much greater than Ns; while for alpha 2AR, Ni approximately equal to No greater than transducin greater than or equal to Ns. PMID- 3005309 TI - Proton translocation by cytochrome oxidase vesicles catalyzing the peroxidatic oxidation of ferrocytochrome c. AB - Coupled with the peroxidatic oxidation of ferrocytochrome c under anaerobic conditions, proteoliposomes reconstituted with a purified preparation of bovine heart cytochrome oxidase ejected protons into the external medium with an apparent H+/e- ratio of 0.9. At the same time, protons in the intravesicular space were consumed. Dicyclohexylcarbodiimide significantly inhibited the proton translocation. Cyanide (0.14 mM) completely inhibited both the peroxidase and proton translocating activities. On the contrary, in the presence of 1 mM CO the proton ejection was abolished almost completely, but 50% of the peroxidase activity persisted. This result suggests the operation of multiple mechanisms in the peroxidase reaction and that the CO-sensitive one is coupled to the proton translocation. PMID- 3005310 TI - Thyrotropin and dibutyryl cyclic AMP increase levels of c-myc and c-fos mRNAs in cultured rat thyroid cells. AB - We have studied the effects of thyrotropin (TSH) on the growth and on the levels of the mRNAs of the cellular proto-oncogenes, c-myc, and c-fos, in the specific target of TSH action, the thyroid follicular cell. FRTL5 cells, a cloned line from normal rat thyroid gland that depends upon TSH for its replication, were maintained in a quiescent state for 5 days by keeping them in a medium devoid of serum or TSH. The addition of bovine TSH (bTSH, 1 nM) increased DNA synthesis and stimulated cell proliferation after a lag period of 24 h. This growth response was anteceded by prompt, but transient, increases in the levels of c-myc and c fos mRNAs, with peak responses at 60 and 30 min, respectively. The minimally and maximally effective concentrations of bTSH were 0.01 mM and 1.0 nM, respectively. Dibutyryl cAMP (Bt2cAMP) stimulated cell growth and increased the level of c-myc mRNA in a concentration-dependent manner, with maximum effects at a Bt2cAMP concentration of 1 mM. At the single concentration tested (1 mM), Bt2cAMP also increased the level of c-fos mRNA. Hence, bTSH-stimulated mitogenesis in quiescent FRTL5 cells is associated with rapid, but short-lived, increases in the levels of the mRNAs of the proto-oncogenes, c-myc and c-fos. Since bTSH is known to stimulate adenylate cyclase in these cells, and since the effect of TSH on c myc and c-fos mRNAs is mimicked by Bt2cAMP, it is possible that these responses to bTSH are mediated, at least in part, by cAMP. PMID- 3005311 TI - Follicle-stimulating hormone enhances somatomedin C binding to cultured rat granulosa cells. Evidence for cAMP dependence. AB - The ovarian granulosa cell has recently been shown to be the site of Somatomedin C (Sm-C) production, reception, and action. To further elucidate the relevance of Sm-C to granulosa cell physiology, we have undertaken to study the regulation of its receptor under in vitro conditions using a primary culture of rat granulosa cells. Granulosa cells cultured without treatment for 72 h displayed limited, albeit measurable, specific Sm-C binding. However, continuous treatment with increasing concentrations of follicle-stimulating hormone (FSH) for the duration of the 72-h incubation period resulted in dose-dependent increments in Sm-C binding (1.7-, 2.9-, 3.9-, and 3.6-fold increases over untreated controls for 50, 100, 180, and 330 ng/ml of FSH, respectively). This apparent up regulatory action of FSH proved time-dependent, with a minimal time requirement of 24-48 h. Granulosa cell Sm-C binding was similarly enhanced following elevation of the intracellular cAMP content by a series of cAMP-generating agonists, inhibition of cAMP-phosphodiesterase activity, or the provision of nondegradable cAMP analogs. Our findings further indicate that high dose forskolin, like FSH, is capable of augmenting Sm-C binding by itself, that a relatively inactive low dose of forskolin synergizes with FSH in this regard, but that combined treatment with maximal stimulatory doses of both agonists does not prove additive. Taken together, these observations indicate that FSH is capable of exerting a stimulatory effect on granulosa cell Sm-C binding and that cAMP, its purported intracellular second messenger, may play an intermediary role in this regard. PMID- 3005312 TI - A critical base in the yeast mitochondrial nonanucleotide promoter. Abolition of promoter activity by mutation at the -2 position. AB - The yeast mitochondrial promoter comprises a highly conserved nonanucleotide sequence, 5'-ATATAAGTA-3'. Transcription is initiated both in vivo and in vitro at the 3'-terminal A (+1). The G at the -2 position is strictly conserved. The requirement for this nucleotide is established by comparing the in vitro promoter activity of nonanucleotides containing any of the four bases at the -2 position, obtained as synthetic oligonucleotides. Only the synthetic promoter having G at the -2 position exhibits any promoter activity. PMID- 3005313 TI - Amino acid sequence of the phosphorylation site of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase. AB - Fructose-1,6-bisphosphatase from the yeast Saccharomyces cerevisiae has properties similar to other gluconeogenic fructose-1,6-bisphosphatases, but an unusual characteristic of the yeast enzyme is that it can be phosphorylated in vitro by cAMP-dependent protein kinase. Phosphorylation also occurs in vivo, presumably as part of a signalling mechanism for the enzyme's degradation. To probe the structural basis for the phosphorylation of yeast fructose-1,6 bisphosphatase, we have developed an improved procedure for the purification of the enzyme and then performed sequence studies with the in vitro-phosphorylated protein as well as with tryptic and chymotryptic peptides containing the phosphorylation site. As a result of these studies, we have determined that yeast fructose-1,6-bisphosphatase has the following 24-residue NH2-terminal amino acid sequence: Pro-Thr-Leu-Val-Asn-Gly-Pro-Arg-Arg-Asp-Ser-Thr-Glu-Gly- Phe-Asp-Thr Asp-Ile-Ile-Thr-Leu-Pro-Arg. The site of phosphorylation is located at Ser-11 in the above sequence. The amino acid sequence around the site of phosphorylation contains the sequence - Arg-Arg-X-Ser- associated with many of the better substrates of cAMP-dependent protein kinase. The sequence of residues 15-24 above is highly homologous with the sequence of residues 6-15 of pig kidney fructose 1,6-bisphosphatase, showing 7 out of 10 residues in identical positions. The yeast enzyme, however, has a dissimilar NH2-terminal region which extends beyond the NH2 terminus of mammalian fructose-1,6-bisphosphatases and contains a unique phosphorylation site. PMID- 3005314 TI - Structure and function of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p dioxin. Species difference in molecular properties of the receptors from mouse and rat hepatic cytosols. AB - Molecular properties of cytosolic Ah receptors from livers of Sprague-Dawley rats and C57BL/6N mice were assessed by velocity sedimentation on sucrose gradients and by gel permeation chromatography on Sephacryl S-300. Analyses were done under conditions of both moderate ionic strength (presence of 0.1 M KCl) and high ionic strength (0.4 M KCl). [3H] 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was used as the radioligand. In conditions of moderate ionic strength the receptor from Sprague-Dawley rat liver sedimented at 8.8 +/- 0.05 S, had a Stokes radius of 7.0 +/- 0.21 nm, and an apparent relative molecular mass (Mr) of 257,000 +/- 7,700. In conditions of high ionic strength the Ah receptor from rat hepatic cytosol dissociated to a [3H]TCDD-binding subunit which sedimented at 5.6 +/- 0.58 S, had a Stokes radius of 5.2 +/- 0.24 nm, and an apparent Mr of 121,000 +/- 5,600. The Ah receptor from liver of C57BL/6N mice, in moderate ionic strength conditions, sedimented at 9.4 +/- 0.54 S, had a Stokes radius of 7.1 +/- 0.12 nm, and an apparent Mr of 277,000 +/- 4,800. Whereas the Ah receptor from rat liver readily dissociated into a [3H]TCDD-binding subunit during brief exposure to 0.4 M KCl, the mouse Ah receptor resisted dissociation. When exposed to 0.4 M KCl for 2 h, the mouse Ah receptor remained at the same molecular size that it had exhibited in moderate ionic strength conditions. Prolonged exposure (16 h) to 0.4 M KCl prior to analysis partially converted the mouse Ah receptor into a smaller [3H]TCDD-binding subunit which sedimented at 4.9 +/- 0.07 S, had a Stokes radius of 5.2 +/- 0.19 nm, and an apparent Mr of 105,000 +/- 3,800. The potency of seven different Ah receptor agonists in competing with [3H]TCDD for specific receptor sites was slightly different in mouse cytosol than in rat cytosol. By criteria of size, response to high ionic strength environments, and ligand binding preferences the mouse and rat Ah receptors appear to be similar but not identical molecular species. PMID- 3005315 TI - Altered retinal metabolism in diabetes. II. Measurement of sodium-potassium ATPase and total sodium and potassium in individual retinal layers. AB - Pathological changes in retinas of diabetics include specific morphological, biochemical, and functional abnormalities. As biochemical manifestations of the disease, increased sorbitol and decreased myo-inositol were found in retinas of experimentally diabetic animals. Similar alterations in polyol metabolism have been associated in nerves of diabetics with a reduction of Na+-K+-ATPase activity. To determine whether this association extends to the retinas of diabetic animals, we applied quantitative histochemical techniques to measure ATPase activities and the amounts of sodium and potassium in samples from nine individual layers of cryostat sections of rabbit retina. ATPase activities were determined fluorimetrically, and the ions were measured by atomic absorption with a carbon rod atomizer. The activity of Na+-K+-ATPase was reduced in the retinal pigmented epithelium (retinal pigment epithelium) and in selected layers of the neural retina, and total sodium in the retinal pigment epithelium layer was elevated in diabetes. The retinal pigment epithelium forms the outer component of the blood-retinal barrier and partly determines the composition of the retinal interstitial fluid. Changes in retinal pigment epithelium biochemistry and function might alter the intraretinal environment, predisposing neural retina or retinal blood vessels to disease. The morphologically and functionally well defined retinal pigment epithelium may provide a useful model for studying the pathogenesis of diabetic complications. PMID- 3005316 TI - Angiotensin II and dopamine modulate both cAMP and inositol phosphate productions in anterior pituitary cells. Involvement in prolactin secretion. AB - Despite their opposite effects on prolactin secretion, both dopamine and angiotensin II inhibit adenylate cyclase activity in homogenates of anterior pituitary cells in primary culture. Dopamine and angiotensin II inhibition of adenylate cyclase was not additive, suggesting that both neurohormones inhibit the adenylate cyclase of the lactotroph cells. Pretreatment with Bordetella pertussis toxin (islet activator protein) completely suppressed the dopamine induced inhibition of both adenylate cyclase and prolactin secretion. The islet activator protein also reversed the angiotensin II-induced inhibition of the adenylate cyclase activity. In contrast, angiotensin II stimulation of prolactin release was not affected by the toxin. Angiotensin II also induced a dose dependent stimulation of inositol phosphates (250%) with an EC50 of 0.1 nM, close to that observed for prolactin secretion. Islet activator protein pretreatment did not block the stimulation of inositol phosphate production. Dopamine inhibited the angiotensin II-stimulated prolactin release and the production of inositol phosphates induced by angiotensin II. It is concluded that angiotensin II and dopamine receptors of lactotroph cells are able to modulate both cAMP and inositol phosphate production. The dopamine receptor of lactotrophs appears to be the first example of a receptor which is negatively coupled to the production of inositol phosphates. PMID- 3005318 TI - The involvement of calpain in the activation of protein kinase C in neutrophils stimulated by phorbol myristic acid. AB - The Ca2+/phospholipid-dependent protein kinase (protein kinase C) of human neutrophils is converted to a proteolytically modified Ca2+/phospholipid independent form (Inoue, M., Kishimoto, A., Takai, Y.U., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616) on incubation with neutrophil membranes in the presence of micromolar concentrations of Ca2+ and an endogenous Ca2+-requiring proteinase (Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., and Horecker, B. L. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6435 6439). We have now demonstrated the appearance of a similar Ca2+/phospholipid independent kinase in intact human neutrophils stimulated by phorbol 12-myristate 13-acetate (PMA). The following evidence supports the conclusion that the Ca2+/phospholipid-independent protein kinase recovered from the PMA-treated cells is a proteolytically modified form of the "native" protein kinase C. 1) In cells exposed to PMA, the rate of disappearance of Ca2+/phospholipid-dependent protein kinase C activity is correlated with the rate of appearance of the Ca2+/phospholipid-independent kinase. 2) The chromatographic behavior of the new protein kinase and its molecular size (approximately 65 kDa) are identical to those previously reported for the proteolytically modified form of protein kinase C. 3) The modified protein kinase no longer binds to the cell membrane and is recovered almost entirely in the cytosol fraction. 4) In neutrophils preloaded with inhibitors of the Ca2+-requiring proteinase, stimulation with PMA results in translocation of protein kinase C from the cytosol fraction to the particulate fraction, but the appearance of the soluble, Ca2+/phospholipid-dependent form is prevented. We conclude that binding of protein kinase C to the plasma membrane and its proteolytic conversion are related, but independent, processes both elicited by exposure of neutrophils to the phorbol ester. Proteolytic cleavage of the membrane-bound protein kinase C provides an alternative mechanism for its activation and may account for certain of the cellular responses observed in PMA stimulated neutrophils. PMID- 3005317 TI - Rat Sertoli cells secrete a growth factor that blocks epidermal growth factor (EGF) binding to its receptor. AB - The conditioned medium from Sertoli cells contains a potent mitogen(s) that can markedly stimulate the proliferation of 4 different cell lines of endoderm or mesoderm origin in the presence or absence of serum. With A431 cells, conditioned medium produced in a dose-dependent manner up to a 5.2-fold increase in cell number after 5 days in culture. Addition of follicle-stimulating hormone (FSH), testosterone, retinol, and insulin to the Sertoli cells increased the secretion of the mitogenic activity. The ability of Sertoli cell conditioned medium (SCCM) to displace 125I-labeled epidermal growth factor (125I-EGF) from formalin-fixed A431 cells was also examined. The SCCM from Sertoli cells incubated with insulin contained 1.42 ng eq of EGF/ml; testosterone, retinol, and FSH (in the presence of insulin) further increased the secretion of this EGF competing activity to 2.09, 2.56, and 3.22 ng eq/ml, respectively. The amount of EGF competing activity was positively correlated with mitogenic activity. Separation of SCCM by gel filtration on Bio-Gel P-10 produced three major peaks of EGF-competing activity at apparent Mr = 1800-2100, 3800-4200, and 8000-9500. Chromatographing SCCM (in the presence of protease inhibitors) on size exclusion high performance liquid chromatography revealed two peaks of EGF competing activity at Mr about 8000 and 2000 coincident with and proportional to peaks of mitogenic activity. This activity was heat-sensitive and resistant to reducing agents, and addition of an equivalent amount of EGF as that present in SCCM produced an inhibition in growth of the A431 cells compared to a 3-fold stimulation with SCCM. Thus, the Sertoli cells secrete a potent mitogen that is distinct from EGF and alpha TGF. This factor that we have termed Sertoli cell-secreted growth factor is hormonally regulated by FSH, testosterone, and retinol and may play an important role in controlling spermatogenesis. PMID- 3005319 TI - Structural analysis of cloned cDNA for mRNA of microsomal cytochrome P-450(C21) which catalyzes steroid 21-hydroxylation in bovine adrenal cortex. AB - We have isolated cDNA clones of the mRNA for cytochrome P-450 that catalyzes the steroid C-21 hydroxylation (P-450(C21)), which specifically catalyzes 21 hydroxylation of steroids in the microsomes of bovine adrenal cortex by using synthetic oligonucleotides as probes. Sequence determination of the cloned cDNA showed that it contains 2157 nucleotides and a poly(A) chain and that a single open reading frame of 1488 nucleotides codes for a polypeptide of 496 amino acids with a molecular weight of 56,113. The deduced amino acid composition is in agreement with that determined by direct amino acid analysis of purified P 450(C21) and the predicted primary structure contained amino acid sequences of N terminal region and two internal tryptic fragments of the protein so far analyzed. Comparing the amino acid sequence with those of other forms of P-450 reveals that a conserved amino acid sequence containing a putative heme-binding cysteine is present in the equivalent position, proximate to the COOH terminus of the molecules and that P-450(C21) is phylogenically situated in an intermediate position between steroidogenic mitochondrial cytochrome P-450 which catalyzes the side-chain cleavage of cholesterol (P-450(SCC)) and drug-metabolizing microsomal P-450s. However, the amino acid sequence of P-450(C21) is much closer to that of drug-metabolizing P-450s than to that of P-450(SCC). PMID- 3005320 TI - Epidermal growth factor stimulates the synthesis of its own receptor in a human breast cancer cell line. AB - The MDA 468 human breast carcinoma cell line was examined for changes in epidermal growth factor (EGF) receptor synthesis and degradation under the influence of EGF. This cell line was used because it overexpresses the EGF receptor such that each cell has 10(6) receptors, but unlike the well-studied A431 cell, its receptor gene is amplified but is not rearranged. On exposure to EGF, total cellular receptor protein, measured by immunoprecipitation with monoclonal antibody B1D8, is reduced. The half-life of receptor metabolically labeled with L-[35S]methionine is 24 h in the absence of EGF and is reduced to 12 h in the presence of 10(-9) M EGF. To measure the effect of EGF on synthesis of the receptor, pulse labeling conditions were selected in which the rate of synthesis of the receptor precursor were followed. EGF had no significant effect on the rate of general protein synthesis in these cells, yet stimulated the synthesis of the EGF receptor 1.8-fold over the unstimulated rate. This increase in receptor precursor synthesis showed time and dose dependence. Stimulation could be detected after 3 h exposure to EGF with a maximum at 6-8 h. A concentration of 10(-11) M EGF gave detectable stimulation with maximal stimulation occurring at 10(-9) M. Longer times and higher concentrations gave submaximal stimulation. A similar dose-response relationship was observed when the rate of mature 170-kDa receptor protein synthesis was measured. These studies demonstrate that EGF stimulates the synthesis of it own receptor. Downregulation of the receptor by EGF results from an increased rate of receptor degradation and not decreased synthesis. PMID- 3005321 TI - Purification and partial amino acid sequence of a bovine cartilage-derived collagenase inhibitor. AB - An inhibitor of mammalian collagenase from bovine scapular cartilage has been purified to homogeneity. The inhibitor, extracted from cartilage using 2 M NaCl, was applied to an A-1.5m gel filtration column. Inhibitor eluted at an apparent Mr of 28,000. Further purification was achieved by ion exchange chromatography, gel filtration, and reverse-phase high performance liquid chromatography. A purification of greater than 1,000-fold was achieved. The inhibitor was judged homogeneous by the appearance of a single band on a silver-stained 15% sodium dodecyl sulfate-polyacrylamide gel. Reduced inhibitor had an Mr of 27,400, unreduced inhibitor had an Mr of 23,900. NH2-terminal sequence data were obtained for the first 45 residues. The bovine cartilage-derived inhibitor exhibits greater than 65% homology over the first 23 residues with a collagenase inhibitor purified from human skin fibroblasts maintained in cell culture. This is the first demonstration that collagenase inhibitors extracted directly from tissue may be similar to those obtained from culture medium. PMID- 3005322 TI - Characterization of the cell surface receptor for granulocyte-macrophage colony stimulating factor. AB - 125I-labeled recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to characterize receptors specific for this lymphokine on the surface of cells of both myelomonocytic and T-cell origin. GM-CSF binding to these cells was specific and saturable. Equilibrium binding studies revealed that on all cell types examined, GM-CSF bound to a single class of high affinity receptor (1000-5000 receptors/cell) with a Ka of 10(8)-10(9) M-1. More extensive characterization with P388D1 cells showed that binding of GM-CSF was rapid at 37 degrees C with a slow subsequent dissociation rate. Among a panel of lymphokines and growth hormones, only unlabeled natural or recombinant GM-CSF were able to compete for the binding of 125I-GM-CSF to these cells. Affinity cross-linking experiments with the homobifunctional cross-linking reagents disuccinimidyl suberate, disuccinimidyl tartrate, and dithiobis(succinimidyl propionate) resulted in the identification of a receptor protein with a Mr of 130,000 on five out of the seven cell types examined. This protein was extremely sensitive to proteolysis and in the absence of protease inhibitors was degraded to a form with an approximate Mr of 70,000. A receptor protein of Mr 180,000, in addition to the Mr 70,000 protein, was found on bone marrow cells and on P815 cells. The potential tissue-specific molecular heterogeneity associated with the GM-CSF receptor may help to explain some of the diverse biological effects associated with this growth and differentiation factor. PMID- 3005323 TI - Type X collagen contains two cleavage sites for a vertebrate collagenase. AB - Type X collagen was cleaved at two sites by a purified human skin collagenase. Two experimental approaches were used to identify the location of the cleavage sites. First, native type X collagen was digested with the enzyme, and the rotary shadowed products were visualized in the electron microscope. The major collagenase fragment of type X contained the epitope recognized by a monoclonal antibody (X-AC9). The antibody was used as a point of reference to locate the position of the cleavage fragment within the native molecule. Second, the digestion of radiolabeled type X collagen substrates was analyzed by gel electrophoresis. The complete cleavage of type X generated three products with 32 , 18-, and 9-kDa chains. The 32-kDa peptides were present in a triple-helical conformation and demonstrated a midpoint denaturation temperature of 43 degrees C in CD experiments. The 18-kDa peptide contained the tyrosine-rich globular domain of the molecule. The 9-kDa peptide was derived from the triple-helical end of the native molecule. Type X collagen was cleaved more rapidly by the vertebrate collagenase than was type II collagen in in vitro solution studies. PMID- 3005324 TI - The fatty acid synthase gene in avian liver. Two mRNAs are expressed and regulated in parallel by feeding, primarily at the level of transcription. AB - The rates of synthesis of fatty acid synthase and the levels of its mRNA are high in livers of chicks, ducklings, or goslings fed high-carbohydrate mash diets and low in livers of starved birds, indicating pretranslational regulation of fatty acid synthase activity. Determination of the step(s) at which the nutritional state regulates the fatty acid synthase mRNA level was the objective of this study. Total RNA extracted from gosling or duckling liver contains two discrete fatty acid synthase transcripts, one of about 12,200 nucleotides and the other about 10,800 nucleotides. Both mRNAs are transcribed from the same gene because there is only one fatty acid synthase gene/haploid genome. A combination of 1) comparison of restriction fragment lengths in genomic DNA and cloned fatty acid synthase cDNAs, 2) differential hybridization of cloned cDNAs to the two mRNAs, and 3) sequence analysis indicates that the longer mRNA is a 3'-extension of the shorter one. The half-lives for fatty acid synthase mRNAs in fed ducklings and in starved ducklings were estimated from the rate at which mRNA level approached steady state during starvation or refeeding. The amount of fatty acid synthase mRNA in total liver RNA increased rapidly when starved ducklings were fed a high carbohydrate mash diet, reaching an apparent steady state of 10 times the initial level after 9 h. The kinetics of accumulation suggested a half-life of 4-6 h for fatty acid synthase mRNA in fed ducklings. When fed ducklings were starved, fatty acid synthase mRNA decayed with a half-life of about 3 h. Therefore, the half life for fatty acid synthase mRNA appeared to be little affected by feeding or starvation. The levels of both mRNAs changed in parallel indicating that half lives of the two mRNAs were not regulated differentially. Transcription of the fatty acid synthase gene, as measured in isolated nuclei, increased about 10-fold when starved ducklings were refed for 24-30 h. Most of the increase in transcription occurred within 45 min after feeding was initiated. However, when fed ducklings were starved, the initial decrease in fatty acid synthase mRNA level occurred more rapidly than the decrease in transcription of the fatty acid synthase gene, indicating some degree of post-transcriptional regulation. Nevertheless, after 48 h of starvation, both mRNA level and transcription were decreased to the same extent. Nutritional state, therefore, regulates the transcription of two fatty acid synthase mRNAs from a unique gene. In addition, transient regulation occurs at an as yet undefined post-transcriptional step. PMID- 3005325 TI - Regulation of pigment organelle translocation. I. Phosphorylation of the organelle-associated protein p57. AB - Treatment of goldfish xanthophores with adrenocorticotropin (ACTH) or cyclic AMP (cAMP) induces the centrifugal movement of their pigment organelles from the center of the cells. Using purified xanthophores pulse labeled with 32Pi, we have shown that the dispersion of the organelles is accompanied by the phosphorylation of a pair of polypeptides, termed p57. After fractionation on sucrose gradients, nearly all of the p57 is found associated with the pigment organelles. The phosphorylation induced by ACTH or cAMP apparently occurs at multiple sites on p57. The minimal effective doses of ACTH or cAMP required to induce full pigment dispersion also fully stimulate the phosphorylation of p57. Increased phosphorylation of p57 is detectable within a minute after stimulating the cells and appears to be near completion during the early phases of pigment dispersion. Upon withdrawal of ACTH, these events are reversed; the pigment organelles reaggregate toward the center of the cells and p57 is dephosphorylated. Again, dephosphorylation commences soon after ACTH is withdrawn and is complete before the organelles have completely reaggregated. These results suggest a novel mechanism for governing the movement of these organelles which acts on the organelles themselves through the phosphorylation and dephosphorylation of p57. PMID- 3005326 TI - Regulation of pigment organelle translocation. II. Participation of a cAMP dependent protein kinase. AB - In intact goldfish xanthophores, the phosphorylation of a pigment organelle (carotenoid droplet) protein, p57, appears to play an important role in adrenocorticotropin (ACTH)- or cAMP-induced pigment organelle dispersion while the dephosphorylation of this protein upon withdrawal of ACTH or cAMP is implicated in pigment aggregation. In this paper, we report the cAMP-dependent phosphorylation of this protein in cell-free extracts of xanthophores as determined by the incorporation of 32P from [gamma-32P]ATP. As is the case in intact cells, p57 is the predominant protein phosphorylated in the presence of cAMP. The cAMP-dependent protein kinase which phosphorylates p57 is not bound to the isolated organelles but is found in the soluble portion of the cell extracts. Hence, the phosphorylation of p57 requires the carotenoid droplets bearing the substrate, soluble extract containing the kinase, cAMP (half-maximal activation at 0.5 microM), and Mg2+ (optimal at 5 mM or higher). The presence of protein phosphatase(s) in these extracts was shown indirectly by the stimulation of phosphorylation by fluoride. The phosphorylation of p57 does not appear to require a cell-specific kinase as soluble extracts of goldfish dermal nonpigment cells also phosphorylate p57 associated with isolated carotenoid droplets. Furthermore, using a constant amount of carotenoid droplets, a linear relationship was demonstrated between the rate of p57 phosphorylation and the amount of extract present in the assays. These results suggest that p57 is phosphorylated directly by a cAMP-dependent protein kinase and that the activity of this enzyme is important in regulating the intracellular movement of the pigment organelles of the xanthophore. PMID- 3005327 TI - Comparison between calcium transport and adenosine triphosphatase activity in membrane vesicles derived from rabbit kidney proximal tubules. AB - Characteristics of Ca2+ uptake were studied in a vesicular preparation of proximal tubule plasma membranes from rabbit kidney and compared with the properties of both membrane-bound and solubilized Ca2+-ATPase activities. Calcium uptake required both ATP and MgCl2 and revealed two kinetic components with respect to Ca2+ concentration requirements, one with a high affinity for Ca2+ (1.8 microM), operative in the range of cytosolic Ca2+ activity, and one with a low affinity for Ca2+ (250 microM) which may become active only at abnormally high cytosolic Ca2+ concentrations. The high- and low-affinity components were stimulated to similar extents by phosphate, and required similar concentrations of ATP (0.6 mM) for half-maximal activity. The amount of membrane-bound phosphoenzyme formed from ATP in the presence of Ca2+ was the same regardless of whether only one or both sites were saturated, suggesting that occupancy of the second Ca2+ binding site accelerates the enzyme turnover. Inhibition of Ca2+ transport by Na+ was reversed by the addition of ouabain or an ATP-regenerating system, indicating that this inhibitory effect of Na+ on Ca2+ uptake may be due to the accumulation of ADP in the medium as a result of Na+ pump activity. Low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and valinomycin (2.5 and 1 microM, respectively) were without effect on Ca2+ uptake in the presence of phosphate, whereas higher concentrations of the ionophores (200 and 100 microM, respectively) reduced uptake by 60% or more. The calmodulin antagonist 48/80 also reduced Ca2+ uptake with half-maximal effectiveness at 100 micrograms/ml. None of these drugs affected either ATPase activity or the EGTA induced Ca2+ efflux from preloaded vesicles. The Ca2+ dependence of ATP hydrolysis by the membrane-bound enzyme preparation was similar to that observed for Ca2+ uptake by the vesicles. However, with solubilized enzyme, concentrations of Ca2+ similar to that found in the plasma reduced Ca2+-stimulated ATP hydrolysis to one-half of its maximal rate. This indicates that peritubular Ca2+ may play a role in the regulation of Ca2+ transport across the tubular epithelium. ATP could not be replaced by ITP as a substrate for Ca2+ uptake, and the (Ca2+ + Mg2+)ITPase activity of soluble enzyme was 25-fold lower than in the presence of ATP. This is an indication that the active Ca2+ pumping mechanism in proximal tubules is critically dependent on the nucleoside moiety of the substrate. PMID- 3005329 TI - Adenine nucleotides promote dissociation of pertussis toxin subunits. AB - Pertussis toxin is composed of an enzymatically active A subunit and a binding component (B oligomer). Both the holotoxin and the isolated A subunit have previously been shown to exhibit NAD glycohydrolase activity although the A subunit is more active on a molar basis than the holotoxin. We have investigated the mechanism by which ATP stimulates the activity of this toxin. Since dissociation of pertussis toxin subunits would result in increased NAD glycohydrolase activity, the ability of ATP to promote release of the A subunit from the B oligomer was examined. In the presence of the zwitterionic detergent 3 (3-cholamidopropyldimethyl)-1-ammonio)-propanesulfonate, concentrations of ATP as low as 1 microM promoted subunit dissociation. The concentration of ATP required for release of the A subunit was similar to that required for stimulation of NAD glycohydrolase activity. Both ATP and ADP promoted subunit dissociation and stimulated NAD glycohydrolase activity. In contrast, AMP and adenosine did not alter NAD glycohydrolase activity or affect subunit structure. The ability of ATP to decrease the affinity of the A subunit for the B oligomer may play a role in nucleotide stimulation of pertussis toxin activity. PMID- 3005328 TI - Isolation and characterization of the apolipoprotein E receptor from canine and human liver. AB - Previous results have demonstrated that liver membranes possess two distinct lipoprotein receptors: a low density lipoprotein (LDL) receptor that binds lipoproteins containing either apolipoprotein (apo-) B or apo-E, and an apo-E specific receptor that binds apo-E-containing lipoproteins, but not the apo-B containing LDL. This study reports the isolation and purification of apo-B,E(LDL) and apo-E receptors from canine and human liver membranes. The receptors were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate and were partially purified by DEAE-cellulose chromatography. The apo-B,E(LDL) receptor was isolated by affinity chromatography on LDL-Sepharose. The apo-E receptor, which did not bind to the LDL-Sepharose column, was then purified by using an HDLc (cholesterol-induced high density lipoprotein)-Sepharose affinity column and an immunoaffinity column. Characterization of the receptors revealed that the hepatic apo-B,E(LDL) receptor is similar to the extrahepatic LDL receptor with an apparent Mr = 130,000 on non reducing sodium dodecyl sulfate-polyacrylamide gels. The apo-E receptor was found to be distinct from the apo-B,E(LDL) receptor, with an apparent Mr = 56,000. The purified apo-E receptor displayed Ca2+-dependent binding to apo-E-containing lipoproteins and did not bind to LDL or chemically modified apo-E HDLc. Antibodies raised against the apo-B,E(LDL) receptor cross-reacted with the apo-E receptor. However, an antibody prepared against the apo-E receptor did not react with the apo-B,E(LDL) receptor. The apo-E receptor also differed from the apo B,E(LDL) receptor in amino acid composition, indicating that the apo-E receptor and the apo-B,E(LDL) receptor are two distinct proteins. Immunoblot characterization with anti-apo-E receptor immunoglobulin G indicated that the apo E receptor is present in the hepatic membranes of man, dogs, rats, and mice and is localized to the rat liver parenchymal cells. PMID- 3005330 TI - Influence of urapidil on alpha- and beta-adrenoreceptors in pithed rats. AB - The interaction of urapidil with pre- and postsynaptic alpha-adrenoreceptors and with postsynaptic beta-adrenoreceptors was studied in pithed normotensive rats and compared to the effects of clonidine, prazosin, and atenolol. I.v. injection of urapidil did not substantially change blood pressure, while clonidine raised blood pressure. Urapidil dose-dependently antagonized the pressor effects of the alpha 1-agonist L-phenylephrine (pDR2 6.8) and of the alpha 2-agonist azepexole (pDR2 5.2). Compared to urapidil, prazosin was a more potent antagonist of phenylephrine at postsynaptic vascular alpha 1-adrenoreceptors (pDR2 8.7) and of azepexole at alpha 2-adrenoreceptors (pDR2 5.6). Urapidil inhibited the tachycardia produced by discontinuous or continuous electrical stimulation of the thoracic spinal outflows (ID50 4.8 and 27.2 mumol/kg, respectively). In contrast to the inhibitory action of clonidine (ID50 0.039 and 0.023 mumol/kg, respectively), the inhibition by urapidil was not reversed by the selective alpha 2-antagonist rauwolscine (10 mumol/kg). Prazosin did not change stimulation evoked tachycardia but atenolol caused pronounced inhibition (ID50 0.158 mumol/kg, discontinuous stimulation). Urapidil dose-dependently antagonized the tachycardic effect of isoprenaline at beta 1-adrenoreceptors (pDR2 5.1) but also exhibited intrinsic activity by increasing basal heart rate (maximum effect of urapidil was 30% of that of isoprenaline). Urapidil did not change the vasodilatory beta 2-adrenoreceptor-mediated effect of isoprenaline. The results suggest that urapidil is an antagonist at postsynaptic vascular alpha 1- and alpha 2-adrenoreceptors, with a greater potency against alpha 1-adrenoreceptors. An agonistic interaction of urapidil with presynaptic alpha 2-adrenoreceptors could not be demonstrated in pithed rats. Instead, the inhibition by urapidil of stimulation-evoked tachycardia could be accounted for by its beta 1 adrenoreceptor antagonistic effect. PMID- 3005331 TI - Cell multiplication of an ascites tumour in the presence of superoxide dismutase and catalase. AB - Superoxide dismutase and catalase were demonstrated to favour the multiplication of ascites tumour cells in vitro. It is proposed that these enzymes neutralize the O2-. and H2O2 that may accumulate in the neoplastic cell and that cell damage occurs because the cellular levels of both enzymes are low. PMID- 3005332 TI - Direct observation of steady-state microtubule dynamics. AB - Different types of unusual dynamic behavior have been reported for steady-state microtubules. While almost all earlier reports relied on kinetic measurements of bulk polymerization, we have directly visualized the steady-state addition of subunits to individual microtubules through the use of tubulin derivitized with biotin. Biotinylated tubulin was used both as an internal "seed" for polymerization and as a marker for assembly onto the ends of microtubules composed of purified tubulin. Biotinylated segments were distinguished from unmodified tubulin by double-label immunofluorescence. Microtubule lengths, number concentrations, and segment lengths have been monitored with time at steady state under two buffer conditions. The results indicate that the microtubule steady state under these conditions is a balance between a majority of slowly growing microtubules and a minority of rapidly depolymerizing ones as described by the "dynamic instability" model (Mitchison T., and M. Kirschner, 1984, Nature (Lond.)., 312:232-242). Microtubules show no evidence of treadmilling; instead most show progressive growth off both ends at steady state. Although solvent conditions markedly influence the growth rates, qualitatively the behavior is unchanged. PMID- 3005334 TI - A re-examination of the interaction of vinculin with actin. AB - Vinculin prepared by published procedures (i.e., Feramisco, J. R., and K. Burridge, 1980, J. Biol. Chem., 255:1194-1199) contains contaminants that have been shown by Evans et al. (Evans, R. R., R. M. Robson, and M. H. Stromer, 1984, J. Biol. Chem., 259:3916-3924) to reduce the low-shear viscosity of F-actin solutions. In this study we separated contaminants from conventional vinculin preparations by hydroxylapatite chromatography. We found that although the contaminants represented a small fraction (less than or equal to 5%) of the total protein in the conventional vinculin preparations, they were responsible for practically all of the filament capping and bundling activities previously attributed to vinculin. In addition, we examined the size of the molecule(s) responsible for the observed capping activity and found that its apparent molecular weight under denaturing conditions in sodium dodecyl sulfate (SDS) polyacrylamide gels fell within a broad range of 23,000-33,000. These results contrast with the observation that under nondenaturing conditions, the activity migrated in gel filtration columns at a position that corresponded to the Stoke's radius of a much bigger molecule. Since the migration of the activity in these chromatographic experiments is independent of the presence of vinculin, it is unlikely that the active protein associates with vinculin with high affinity under the conditions examined. PMID- 3005333 TI - Regulation of reactivated elongation in lysed cell models of teleost retinal cones by cAMP and calcium. AB - Teleost retinal cones elongate in the dark and contract in the light. In isolated retinas of the green sunfish Lepomis cyanellus, cone myoids undergo microtubule dependent elongation from 5 to 45 micron. We have previously shown that cone contraction can be reactivated in motile models of cones lysed with Brij-58. Reactivated contraction is both actin and ATP dependent, activated by calcium, and inhibited by cAMP. We report here that we have obtained reactivated cone elongation in lysed models prepared by the same procedures. Reactivated elongation is ATP dependent, activated by cAMP, and inhibited by calcium. The rate of reactivated elongation is proportional to the cAMP concentration between 10 microM and 0.5 mM, but is constant between 10 microM and 1.0 mM Mg-ATP. No elongation occurs if cAMP or Mg-ATP concentration is less than or equal to 5 microM. Mg-ATP is required for both cAMP-dependent and cAMP-independent processes, suggesting that Mg-ATP is required both for a regulatory process entailing cAMP-dependent phosphorylation and for a force-producing process. Free calcium concentrations greater than or equal to 10(-7) reduce the elongation rate by 78% or more, completely inhibiting elongation at 10(-5) M. This inhibition is not due to competition from calcium-activated contraction. Cytochalasin D blocks reactivated contraction, but does not abolish calcium inhibition of reactivated elongation. Thus calcium directly affects the elongation mechanism. Calcium inhibition is calmodulin dependent. The calmodulin inhibitor trifluoperazine abolishes calcium inhibition of elongation. Furthermore, calcium blocks elongation only if present during the lysis step; subsequent calcium addition has no effect. However, if calcium plus exogenous calmodulin are subsequently added, elongation is again inhibited. Thus calcium inhibition appears to require a soluble calmodulin which is lost shortly after lysis. PMID- 3005336 TI - Commitment to expression of the metalloendopeptidases, collagenase and stromelysin: relationship of inducing events to changes in cytoskeletal architecture. AB - Agents that alter the morphology of rabbit synovial fibroblasts induce synthesis of the metalloendopeptidases, collagenase and stromelysin. We studied the relationship of cytoskeletal changes to the commitment to expression of these metalloendopeptidases. Cells treated with cytochalasin B (CB) or 12-O tetradecanoylphorbol-13-acetate rounded, and only cells that had lost their stress fibers expressed collagenase and stromelysin, as determined by immunofluorescence. We concentrated on the effects of CB because of its rapid reversibility. When CB was added for 1-24 h, then removed, the cells respread within 30-60 min. The minimum period of CB treatment that committed cells to the subsequent synthesis of collagenase and stromelysin was 3 h. After initial treatment with 2 micrograms/ml CB for 3-24 h, or with various concentrations of CB (0-2 micrograms/ml) for 24 h, both enzyme activity and biosynthesis of the proenzymes showed a graded increase when measured at 24 h. Even after treatment with 2 micrograms/ml CB for only 3 h, greater than 85% of all cells were positive for both collagenase and stromelysin when cells were monitored by immunofluorescence. In contrast, when the dependence of collagenase and stromelysin expression on the inducing concentration of CB was examined, there was a dose-dependent increase in the number of cells positive for collagenase and stromelysin, as determined by immunofluorescence. Thus, at low concentrations of CB (less than 0.5 micrograms/ml), a heterogeneous population response was observed. These results suggest that the commitment of fibroblasts to induction of the metalloproteinases is a stochastic process in which a second signal that correlates with the disruption of the actin cytoskeleton may be rate-limiting for collagenase and stromelysin gene expression. PMID- 3005335 TI - Disialogangliosides GD2 and GD3 are involved in the attachment of human melanoma and neuroblastoma cells to extracellular matrix proteins. AB - Human melanoma cells express relatively large amounts of the disialogangliosides GD3 and GD2 on their surface whereas neuroblastoma cells express GD2 as a major ganglioside. Monoclonal antibodies (Mabs) directed specifically to the carbohydrate moiety of GD3 and GD2 inhibit melanoma and neuroblastoma cell attachment to various substrate adhesive proteins, e.g. collagen, vitronectin, laminin, fibronectin, and a heptapeptide, glycyl-L-arginyl-glycyl-L-aspartyl-L seryl-L-prolyl-L-cysteine, which constitutes the cell attachment site of fibronectin. Cells that are preattached to a fibronectin substrate can also be induced to detach and round up in the presence of purified anti-ganglioside Mab. Moreover, when melanoma cells that contain both GD2 and GD3 are incubated with Mabs directed to both of these molecules an additive inhibition is observed. The specificity of this inhibition is demonstrated since Mabs of various isotypes directed to either protein or carbohydrate epitopes on a number of other major melanoma or neuroblastoma cell surface antigens have no effect on cell attachment. A study of the kinetics involved in this inhibition indicates that significant effects occur during the first 5 min of cell attachment, suggesting an important role for GD2 and GD3 in the initial events of cell-substrate interactions. The role of gangliosides in cell attachment apparently does not directly involve a strong interaction with fibronectin since we could not observe any binding of radiolabeled fibronectin or fragments of the molecule known to contain the cell attachment site to melanoma gangliosides separated on thin-layer chromatograms. An alternative explanation would be that gangliosides may play a role in the electrostatic requirements for cell-substrate interactions. In this regard, controlled periodate oxidation of terminal, unsubstituted sialic acid residues on the cell surface not only specifically destroys the antigenic epitopes on GD2 and GD3 recognized by specific Mabs but also inhibits melanoma cell and neuroblastoma cell attachment. In fact, the periodate-induced ganglioside oxidation and the inhibition of cell attachment are equally dose dependent. These data suggest that cell-substratum interactions may depend in part on the electrostatic environment provided by terminal sialic acid residues of cell surface gangliosides and possibly other anionic glycoconjugates. PMID- 3005337 TI - The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells. AB - Nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells has been described to be synergistically potentiated by the simultaneous addition of dibutyryl cAMP. To elucidate further the role of cAMP in NGF-induced neurite outgrowth we have used the adenylate cyclase activator forskolin, cAMP, and a set of chemically modified cAMP analogues, including the adenosine cyclic 3',5'-phosphorothioates (cAMPS) (Rp)-cAMPS and (Sp)-cAMPS. These diastereomers have differential effects on the activation of cAMP-dependent protein kinases, i.e., (Sp)-cAMPS behaves as a cAMP agonist and (Rp)-cAMPS behaves as a cAMP antagonist. Our data show that the establishment of a neuritic network, as observed from PC12 cells treated with NGF alone, could not be induced by either forskolin, cAMP, or cAMP analogues alone. The presence of NGF in combination with forskolin or cAMP or its agonistic analogues potentiated the initiation of neurite outgrowth from PC12 cells. The (Sp)-cAMPS-induced stimulation of NGF mediated process formation was successfully blocked by the (Rp)-cAMPS diastereomer. On the other hand, NGF-stimulated neurite outgrowth was not inhibited by the presence of the cAMP antagonist (Rp)-cAMPS. We conclude that the morphological differentiation of PC12 cells stimulated by NGF does not require cAMP as a second messenger. The constant increase of intracellular cAMP, caused by either forskolin or cAMP and the analogues, in combination with NGF, not only rapidly stimulated early neurite outgrowth but also exerted a maintaining effect on the neuronal network established by NGF. PMID- 3005338 TI - PC12 cell mutants that possess low- but not high-affinity nerve growth factor receptors neither respond to nor internalize nerve growth factor. AB - Four mutant PC12 pheochromocytoma cell lines that are nerve growth factor (NGF) nonresponsive (PC12nnr) have been selected from chemically mutagenized cultures by a double selection procedure: failure both to grow neurites in the presence of NGF and to survive in NGF-supplemented serum-free medium. The PC12nnr cells were deficient in all additional NGF responses surveyed: abatement of cell proliferation, changes in glycoprotein composition, induction of ornithine decarboxylase, rapid changes in protein phosphorylation, and cell surface ruffling. However, PC12nnr cells closely resembled non-NGF-treated PC12 cells in most properties tested: cell size and shape; division rate; protein, phosphoprotein, and glycoprotein composition; and cell surface morphology. All four PC12nnr lines differed from PC12 cells in three ways in addition to failure of NGF response: PC12nnr cells failed to internalize bound NGF by the normal, saturable, high-affinity mechanism present in PC12 cells. The PC12nnr cells bound NGF but entirely, or nearly entirely, at low-affinity sites only, whereas PC12 cells possess both high- and low-affinity NGF binding sites. The responses to dibutyryl cyclic AMP that were tested appeared to be enhanced or altered in the PC12nnr cells compared to PC12 cells. Internalization of, and responses to, epidermal growth factor were normal in the PC12nnr cells ruling out a generalized defect in hormonal binding, uptake, or response mechanisms. These findings are consistent with a causal association between the presence of high-affinity NGF receptors and of NGF responsiveness and internalization. A possible relationship is also suggested between regulation of cAMP responses and regulation of NGF responses or NGF receptor affinity. PMID- 3005340 TI - Lysosomal enzyme trafficking in mannose 6-phosphate receptor-positive mouse L cells: demonstration of a steady state accumulation of phosphorylated acid hydrolases. AB - During their transport from the endoplasmic reticulum to lysosomes, newly synthesized acid hydrolases are phosphorylated in the Golgi apparatus to generate a common recognition marker, mannose 6-phosphate (Man 6-P). The phosphorylated acid hydrolases then bind to the Man 6-P receptor and are transported by an unknown route to lysosomes. To learn more about the delivery pathway, we examined the fate of the phosphorylated oligosaccharides synthesized by a Man 6-P receptor positive line of mouse L-cells. In contrast to the rapid degradation of the recognition marker previously observed in mouse lymphoma cells (Gabel, C. A., D. E. Goldberg, and S. Kornfield. 1982. J. Cell Biol., 95:536-542), the number of high mannose oligosaccharides phosphorylated by the L-cells after a 30-min pulse labeling with [2-3H]mannose increased continuously during a subsequent 4-h chase period to a maximum of 9.3% of the total cell-associated structures. After 19 h of chase the absolute number of phosphorylated oligosaccharides declined, but the loss was accompanied by a general loss of cellular oligosaccharides such that 7.4% of the cell-associated high mannose oligosaccharides remained phosphorylated. The longevity of the Man 6-P recognition marker in the L-cells was verified by analyzing the ability of an individual acid hydrolase, beta glucuronidase, to serve as a ligand for the Man 6-P receptor. At least 60% of the steady state beta-glucuronidase molecules isolated from the L-cells could undergo receptor-mediated endocytosis into enzyme-deficient human fibroblasts. Dense lysosomal granules isolated by metrizamide gradient centrifugation from [3H]mannose-labeled L-cells were found to be highly enriched in their content of phosphomonoester-containing oligosaccharides. The data indicate that acid hydrolases may retain their Man 6-P recognition markers within lysosomes, and suggest the possibility that dephosphorylation occurs at a nonlysosomal location through which the newly synthesized enzymes pass en route to lysosomes. PMID- 3005341 TI - Exposure of K562 cells to anti-receptor monoclonal antibody OKT9 results in rapid redistribution and enhanced degradation of the transferrin receptor. AB - When the human erythroleukemia cell line K562 is treated with OKT9, a monoclonal antibody against the transferrin receptor, effects on receptor dynamics and degradation ensue. The apparent half-life of the receptor is decreased by greater than 50% as a result of OKT9 treatment. The transferrin receptor is also rapidly redistributed in response to OKT9 such that a lower percentage of the cellular receptors are displayed on the cell surface. OKT9 treatment also leads to a decrease in the total number of receptors participating in the transferrin cycle for cellular iron uptake. The reduction in iron uptake that results from the loss of receptors from the cycle leads to enhanced biosynthesis of the receptor. Receptors with bound OKT9 continue to participate in multiple cycles of iron uptake. However, OKT9 treatment appears to result in a relatively small increase per cycle in the departure of receptors from participation in iron uptake to a pathway leading to receptor degradation. Radiolabeled OKT9 is itself degraded by K562 cells and this degradation is inhibitable by leupeptin or chloroquine. In the presence of leupeptin, OKT9 treatment results in the enhanced intracellular accumulation of transferrin. Because the time involved in the transferrin cycle is shorter (12.5 min) than the normal half-life of the receptor (8 h), a small change in recycling efficiency caused by OKT9 treatment could account for the marked decrease in receptor half-life. In this paper the implications of these findings are discussed as they relate to systems in which receptor number is regulated by ligand. PMID- 3005339 TI - Nerve-induced remodeling of muscle basal lamina during synaptogenesis. AB - To identify mechanisms that regulate the deposition of the junctional basal lamina during synaptogenesis, immunocytochemical experiments were carried out on cultured nerve and muscle cells derived from Xenopus laevis embryos. In some experiments successive observations were made on individual muscle cells after pulse-labeling with a fluorescent monoclonal antibody specific for a basal lamina proteoglycan. In others, old and new proteoglycan molecules were differentially labeled with antibody conjugated to contrasting fluorochromes. These observations revealed that surface deposits of antibody-labeled proteoglycan remain morphologically stable for several days on developing muscle cells. Over the same period, however, new sites of proteoglycan accumulation formed that contained primarily those antigenic sites recently exposed at the cell surface. When muscle cells became innervated by cholinergic neurites, new proteoglycan accumulations were induced at the developing neuromuscular junctions, and these too were composed almost exclusively of recently deposited antigen. In older muscle cultures, where many cells possessed relatively high background concentrations of antigen over their surfaces, developing neuromuscular junctions initially showed a markedly reduced proteoglycan site-density compared with the adjacent, extrajunctional muscle surface. Much of this perineural region eventually became filled with dense, nerve induced proteoglycan plaques at later stages of synapse development. Motoneurons thus appear to have two, superficially paradoxical effects on muscle basal lamina organization. They first cause the removal of any existing, extrajunctional proteoglycan from the path of cell contact, and then induce the deposition of dense plaques of recently synthesized proteoglycan within the developing junctional basal lamina. This observation suggests that the proteolytic enzyme systems that have already been implicated in tissue remodeling may also contribute to the inductive interaction between nerve and muscle cells during synaptogenesis. PMID- 3005342 TI - Class specificity of transferrin as a muscle trophic factor. AB - The specificity of transferrin (Tf) in its exertion of a growth-promoting effect on myogenic cells was examined using serum Tfs from chick, dove, goose, turkey, bovine, horse, rabbit, rat, and swine and primary myogenic cells from chick, duck, quail, rabbit, and rat, and rat L6 cells. Avian Tfs were effective on avian cells but not on mammalian cells, while mammalian Tfs were effective on mammalian cells but not on avian cells. Dove and bovine Tfs were exceptional in that they were effective on some class-heterologous cells at higher concentrations and less so or completely ineffective on some class-homologous cells. Despite these exceptions, however, the relationship between Tfs and cells can be summarized as a class specificity. To exert the growth-promoting effect, it is prerequisite for Tf to bind its specific receptor on the cell surface. Using quail and L6 cells, we found that the binding of 125I-labeled chick and rat Tfs to the respective receptors of quail and L6 myoblasts was competitively inhibited by other kinds of effective Tfs, but not by ineffective ones. We conclude that the class specificity in myotrophic activity of Tf is due to the affinity between Tf and Tf receptor. PMID- 3005343 TI - Binding and metabolism of leukotriene B4 by neutrophils and their subcellular organelles. AB - The subcellular distribution of leukotriene (LT)B4 binding and metabolizing sites was investigated in human neutrophils. Cells were disrupted by nitrogen cavitation and fractionated by Percoll density gradient centrifugation to yield cytoplasm, membranes, azurophilic granules, and specific granules. Only membrane fractions contained high affinity [3H]LTB4 binding sites. Binding of radiolabeled ligand to membranes was rapid, reversible, and saturable; it was blocked by a series of LTB4 analogues at concentrations corresponding to their respective potencies in 1) blocking [3H]LTB4 binding to whole cells and 2) stimulating neutrophil degranulation responses. In contrast, [3H]LTB4 was metabolized by fractions enriched with markers for cytoplasm plus endoplasmic reticulum. The metabolic activity was sedimented by ultracentrifugation, enhanced by NADPH, and inhibited at 4 degrees C. The cell-free system, like intact cells, metabolized [3H]LTB4 to omega-oxidized product rapidly and quantitatively at 37 degrees C but was inactive at 4 degrees C. Whole cells converted radiolabel to 20-hydroxy (approximately 30% of product) and 20-carboxy (approximately 70% of product) derivatives; the cell-free system formed principally 20-hydroxy-[3H]LTB4. These products were less bioactive than LTB4. Nevertheless, metabolism of LTB4 played little role in limiting the cells' response to the ligand: neutrophils completed degranulation and became desensitized to LTB4 within 3-5 min of exposure. Within this time frame, they oxidized less than 30% of the stimulus, and the extracellular fluid of these neutrophil suspensions was fully capable of activating fresh cells. We conclude that neutrophils transmit bioactions of LTB4 via a specific receptor integrally associated with their plasmalemma and/or endoplasmic reticulum. They inactivate the stimulus via a particulate omega oxidase. At the level of the individual cell, receptor down-regulation, rather than ligand metabolism, appears to limit functional responses such as degranulation. PMID- 3005344 TI - The modulation of transcobalamin II (TC-II) production by cyclic adenosine 3',5' monophosphate in the murine macrophage cell line J774: relationship to growth behavior. AB - We undertook a study to define the role of cyclic AMP [cAMP] in modulating the secretion of transcobalamin II (TC-II) in the mouse macrophage like cell line J774. J774 was observed to secrete large amounts of TC-II, particularly in the presence of 8-bromo cAMP or cholera toxin or when grown in medium supplemented with low concentrations of horse serum (1% or 5%) or in serum-free medium. Variant cell lines derived from J774 and deficient either in adenylate cyclase (ac -) or cAMP-dependent protein kinase (pk -) activity showed very low and intermediate levels of basal secretory activity of TC-II, respectively, compared to J774. Maximum secretory activity of TC-II was observed in J774 under conditions in which growth was poorest (in the presence of 8-bromo-cAMP or 1% or 5% horse serum-supplemented medium or in serum-free medium). Cells grown in serum free medium were found to have elevated basal adenylate cyclase activity and cAMP levels compared to those grown in medium supplemented with 20% horse serum. The data from this study demonstrate a negative correlation between growth activity and TC-II secretion in the J774 cell line. The stimulatory effect of exogenous cAMP on TC-II secretion by J774, the reduced secretory activity of the variant lines ac- and pk- and the observed increase in cell cAMP levels under conditions of serum starvation in which TC-II secretion is considerably enhanced, suggest that cell cAMP is an important modulator of TC-II secretion and growth behavior in the J774 cell line. PMID- 3005345 TI - Molecular basis of cell recognition during fertilization in higher plants. AB - The molecular basis of recognition between plant cells is incompletely understood. Some principles established for recognition between animal cells may well apply to plant cell recognition, although, in contrast to animal cells, plant cells are encased by cell walls that play an active role in plant cell-cell recognition. The interaction that controls fertilization in flowering plants involves recognition between pollen or pollen tubes and the female sexual tissues. In many flowering plant families, self-incompatibility (S) genes operate to prevent inbreeding. In plants that have gametophytically controlled self incompatibility, recognition of common S alleles in pollen tube and style results in arrest of pollen tube growth within the style. Self-incompatibility therefore provides a model cell-cell recognition system that is genetically defined. We have taken two approaches to defining cell recognition involved in gametophytic self-incompatibility in Nicotianas alata. Firstly, we have established the major features of the pollen tube wall and the matrix of the style transmitting tissue that are in contact with the growing pollen tube. Secondly, we have established the nature of style glycoproteins that are associated with the S genotype and have initiated a program to clone the genes coding for the protein component of these glycoproteins. Analyses of the pollen tube are consistent with the major polymers being a (1----3)-beta-D-glucan (callose) and a (1----5)-alpha-L arabinan. The pollen tube has two distinct layers: gold immunocytochemistry using a monoclonal antibody directed to terminal alpha-L-arabinosyl residues shows the binding is confined to the outer layers. The major component of the extracellular matrix of the style transmitting tissue is a family of proteoglycans, the arabinogalactan-proteins. A major glycoprotein that segregates with the S2 allele is present in extracts of mature styles. This component has a high pI (greater than 9.5) and an apparent molecular weight of 32 X 10(3). It is not present in extracts of immature styles of N. alata genotypes bearing the S2 allele, or in extracts from other organs of N. alata or styles of other members of the Solanaceae. The isolated glycoprotein is an effective inhibitor of in vitro pollen tube growth. This evidence suggests that the S2-associated glycoprotein is either the product of the S2 allele, or a gene closely associated with the S gene. We have prepared a cDNA library from styles of one genotype and are screening this library with mRNA from mature and immature styles.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3005346 TI - Correlation of Na+,K+-ATPase content and plasma membrane surface area in adapted and de-adapted salt glands of ducklings. AB - During salt-water adaptation, an increase occurs in Na+,K+-ATPase content and surface area of the basolateral plasma membrane of the principal cell of the duck salt gland. To determine the degree to which these changes are correlated, accepted morphometric methods were used to determine numerical cell densities and plasma membrane surface densities of peripheral and principal cells. After adaptation, the plasma membrane surface area per principal cell was five times greater than in controls. Following de-adaptation, the plasma membrane content in principal cells returned to 1.9 times control levels. Two other cell constituents, mitochondria and lipid droplets, displayed similar quantitative changes. Na+,K+-ATPase content increased about fourfold with adaptation and decreased to near control levels with de-adaptation. Thus, changes in Na+,K+ ATPase content and basolateral plasma membrane surface area in adapting and de adapting secretory epithelia of the salt gland occur nearly in parallel. These quantitative data enable Na+,K+-ATPase synthesis and degradation to be investigated in relation to membrane biogenesis. PMID- 3005348 TI - The oncogene of BAV-3 as a mutagen. AB - We studied the mutagenic and carcinogenic effects on mammalian cells of two EcoRI DNA fragments of bovine adenovirus type3 (BAV-3) integrated into the pBR325 plasmid. Fragment D located between 3.6 and 19.7 map units, contains the viral oncogene, fragment C, located between 44.3 and 63.7 map units, has no oncogenic activity. The BAV-3 oncogene was shown to increase significantly the frequency of 6-mercaptopurine (6MP)-resistant mutants in Chinese hamster calls. Fragment C, pBR325 without viral sequences and DNA from normal Syrian hamster cells did not have any mutagenic effect. We also looked at the combined action of the viral DNA fragments and the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which enhances the transforming effect of carcinogens. TPA was shown to increase the mutant yield on exposure to the viral oncogene but not to induce mutagenic activity in those types of DNA that are unable to transform cells. Probably TPA does not affect the initiation of the mutation process, but acts on later stages just as it affects carcinogenic activity. Thus the results obtained confirm the existence of parallelism between the mutagenic and transforming effects of viral DNA and show that both activities are mapped in the same region of viral DNA - its oncogene. PMID- 3005347 TI - Acinar structure and membrane regionalization as a prerequisite for exocrine secretion in the rat submandibular gland. AB - The significance of glandular organization in exocrine secretion was examined by analysing the functional and morphological features of the dissociated rat submandibular gland with special reference to the acinar structure and luminal specialization. The digestion of the gland with collagenase (C preparation) produced relatively large cellular masses having well-preserved acinar structures. When EGTA and the proteolytic enzyme Dispase were added to the C preparation (CED preparation), the gland was dissociated into small cellular aggregates in which the acinar structure disintegrated. Upon stimulation with either isoproterenol or dibutyryl cyclic AMP, a large amount of peroxidase, one of the secretory products of the rat submandibular gland, was released from C treated cells, while discharged peroxidase was greatly reduced after the CED preparation was used. Measurements of dye exclusion, oxygen consumption, protein synthetic activity and receptor binding, as well as ultrastructural features and the absence of inhibitory effects of EGTA and Dispase, suggested that the reduced secretory response of CED-treated cells was not attributable to cellular death, denaturation of receptors or the inhibitory effects of EGTA and Dispase. When the localization of dipeptidyl aminopeptidase IV was surveyed by both enzyme histochemistry and immuno-histochemistry, the luminal plasma membrane was the exclusive site for the reaction in C-treated cells as well as intact acini, whereas the entire cell surface was reaction-positive in CED-treated cells. In addition, the luminal microfilament system and tight junctions, as revealed by nitrobenz-oxadiazole-phallacidin staining and freeze-replica studies, respectively, were well-preserved in the C-treated cells, but considerably disorganized in the CED-treated cells. All these results strongly suggest that: (1) luminal specialization plays an important role in exocrine secretion; and (2) normal acinar arrangement provides the luminal specialization. PMID- 3005349 TI - Antibodies in cardiovascular diagnosis and therapy. PMID- 3005350 TI - Management of pediatric cancer. PMID- 3005351 TI - Myasthenia gravis and arrows of fortune. PMID- 3005352 TI - Human chorionic gonadotropin stimulation of estradiol production and androgen antagonism of gonadotropin-stimulated responses in cultured human granulosa luteal cells. AB - Baseline and gonadotropin-stimulated estradiol production were examined in long term cultures of human granulosa-luteal cells isolated from women undergoing in vitro fertilization. Estradiol production declined by 70% during the first 48 h in culture and was minimally stimulated by the addition of hCG to the culture medium. During subsequent culture from 48-120 h estradiol production was significantly increased over control levels by hCG concentrations greater than 0.1 IU/ml. Incubation with testosterone stimulated estradiol production 100-fold in the presence and absence of gonadotropin. hCG (0.01-10 IU/ml) stimulated a 3- to 13-fold increase in progesterone production. However, at hCG concentrations greater than 1 IU/ml, coincubation with testosterone (10(-7) M) significantly inhibited progesterone production. Dihydrotestosterone also inhibited progesterone production, but to a lesser extent than testosterone. Freshly isolated granulosa-luteal cells specifically bound small amounts of [125I]hCG (less than 1,000 cpm/10(5) cells). Glycine buffer wash was shown to reversibly remove more than 88% of bound hCG and, in freshly isolated cells, increased [125I]hCG binding by 100%. In 5-day cultures, specific [125I] hCG binding nearly doubled from 52,000 cpm/10(5) cells in control cultures to 87,000 cpm/10(5) cells in cultures treated with hCG (0-5 IU/ml). At the highest concentration of hCG (5 IU/ml), testosterone (10(-7) M) significantly inhibited the amount of [125I]hCG specifically bound. In summary, estradiol production in long term cultures of granulosa-luteal cells appears to be gonadotropin dependent. In addition, the presence of testosterone (10(-7) M) antagonizes hCG-stimulated progesterone and LH receptor production by these cells. PMID- 3005353 TI - Responsivity of adrenocorticotropin to corticotropin-releasing hormone and lack of suppressibility by dexamethasone are related phenomena in Cushing's disease. AB - The ACTH and cortisol responses to 100 micrograms ovine corticotropin-releasing hormone (CRH) in 19 consecutive patients with Cushing's disease were compared with those in 13 normal subjects. In 2 patients with Cushing's disease, plasma ACTH and cortisol did not increase after CRH administration. Compared to the normal subjects, maximal ACTH increments [135 +/- 25 (+/- SEM) vs. 42 +/- 6 pg/ml; P less than 0.001, by Wilcoxon's two-sample test] and maximal cortisol increments (17.7 +/- 1.8 vs. 9.4 +/- 1.1 micrograms/100 ml; P less than 0.01 by Wilcoxon's test) after CRH were significantly higher in the 17 CRH-responsive patients with Cushing's disease. In the normal subjects, there was a significant negative correlation between the basal cortisol level and the maximal ACTH (r = 0.65; P less than 0.05, by Spearman's rank correlation test) and cortisol (r = 0.95; P less than 0.001, by Spearman's test) responses to CRH. In contrast, in the CRH-responsive Cushing patients, there was no significant correlation between the basal cortisol level and the maximal ACTH (r = 0.10; P greater than 0.10, by Spearman's test) and cortisol (r = 0.14; P greater than 0.10, by Spearman's test) increments after CRH treatment. In the normal subjects, there was no significant correlation between the basal ACTH level and the maximal ACTH increments after CRH administration (r = -0.24; P greater than 0.10, by Spearman's test). Again in contrast, in the CRH-responsive Cushing patients, maximal ACTH increments after CRH treatment correlated positively with basal ACTH levels (r = 0.69; P less than 0.005, by Spearman's test). Moreover, in these patients, the maximal ACTH increments after CRH were positively correlated with the ACTH levels after suppression with higher and lower doses of dexamethasone. We conclude that in Cushing's disease, unlike in normal subjects, circulating cortisol is not the major modulator of ACTH and cortisol responses to CRH, and that in these patients, responsivity of ACTH to CRH and lack of suppressibility by dexamethasone are related phenomena. PMID- 3005354 TI - Selective resistance to parathyroid hormone in cultured skin fibroblasts from patients with pseudohypoparathyroidism type Ib. AB - We measured cAMP production in response to agonists in cultured skin fibroblasts from subjects with pseudohypoparathyroidism type Ib (PHP Ib; normal phenotype, resistance to PTH only, normal guanine nucleotide stimulatory coupling protein activity) and skin fibroblasts from normal subjects. There were no significant differences in basal or prostaglandin E1- and forskolin-stimulated cAMP production in PHP Ib vs. normal fibroblasts. Fibroblasts from 7 of 10 subjects with PHP Ib had significantly reduced peak cAMP responses to PTH [3.95 +/- 0.88 vs. 15.9 +/- 4.2 pmol/100 micrograms protein (mean +/- SD); n = 7 for both groups; P less than 0.001]. PTH-stimulated cAMP production was significantly reduced in the 7 subjects with PHP Ib at all concentrations of PTH tested [3-1000 ng/ml human PTH-(1-34)]. In the other 3 subjects with PHP Ib, the cAMP response to PTH was either normal (2 subjects) or above the normal range (1 subject). Thus, skin fibroblasts from many, but not all, subjects with PHP Ib have selective resistance to PTH in terms of cAMP response. Since the defect is hormone specific and persists in culture, we suggest that an intrinsic defect in the PTH receptor may cause PTH resistance in certain subjects with PHP Ib. The cause of PTH resistance in the subjects with a normal cAMP response to PTH is not known, but the data suggest heterogeneity even within the PHP Ib subgroup. PMID- 3005355 TI - The human erythrocyte insulin-like growth factor I receptor: characterization and demonstration of ligand-stimulated autophosphorylation. AB - To characterize the insulin-like growth factor I (IGF-I) receptor on human erythrocytes, cells were purified from peripheral blood by Ficoll-Hypaque gradient centrifugation and incubated with [125I]IGF-I. Specific binding was maximal at pH 8.0 after 24 h at 4 C and increased linearly with cell number to 3.9 +/- 0.2% (+/- SEM) for 3.0 X 10(9) cells/ml. The Scatchard plot of the binding data was linear, with 7 fmol [125I]IGF-I bound/10(9) cells and an affinity constant (K) of 1.8 X 10(9) M-1. Unlabeled IGF-I inhibited tracer binding half-maximally at 6 ng/ml. Multiplication-stimulating activity (or rat IGF-II) was 40% as potent (ED50, 15 ng/ml), whereas insulin and proinsulin were 30- to 500-fold less potent. A monoclonal antibody to the IGF-I receptor (alpha IR-3) inhibited IGF-I binding by 50% at a 1:1000 dilution and by 80% at a 1:250 dilution. Insulin binding was unaffected by the same dilutions. IGF-I receptor phosphorylation was studied in erythrocyte ghosts prepared by hypotonic lysis and solubilized in 1% Triton. The extract was preincubated with and without 100 ng/ml IGF-I or porcine insulin and incubated with [gamma-32P]ATP in the presence of Mn2+, and the receptor was identified by immunoprecipitation with alpha IR-3 antibody and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IGF-I stimulated 4-fold the incorporation of 32P into a protein of 95,000 mol wt, which was immunoprecipitated by alpha IR-3; insulin produced a 2-fold stimulation of this protein. This protein corresponds to the beta-subunit of the IGF-I receptor. These data demonstrate that human erythrocytes have specific receptors for IGF-I, and that this IGF-I receptor, like the insulin receptor, undergoes ligand stimulated autophosphorylation. Thus, analysis of erythrocyte IGF-I binding and receptor phosphorylation may be useful tools for the study of patients with a variety of growth disorders. PMID- 3005356 TI - Isolated growth hormone (GH) deficiency type 1A associated with a double deletion in the human GH gene cluster. AB - The gene deletions responsible for isolated GH deficiency type 1A were characterized by direct analysis of genomic DNA prepared from the leukocytes of two affected children. The probands had typical symptoms of severe isolated GH deficiency complicated by antibody development and growth arrest after human (h) GH treatment. DNA analysis using the restriction endonucleases Eco RI, Bam HI, and Hind III revealed that the restriction fragment containing the hGH-N gene was absent along with those bearing the human chorionic somatomammotropin (hCS)-A and -B and hGH-V sequences. A total of about 40 kilobases DNA were absent due to two separate deletions flanking the hCS-L gene. The two affected siblings are homozygous for this rearrangement of the hGH/hCS gene cluster, which could have been generated by homologous crossing over between two different chromosomes, one bearing one of the previously described deletions of the hGH-N gene, and one bearing a deletion of DNA containing the hCS-A, hCS-B, and hGH-V sequences. Alternatively, this abnormality could have been generated by a complex intrachromosomal rearrangement. The parents, who are consanguinous, have DNA restriction patterns consistent with heterozygosity for this double deletion. This type of deletional mutation is the first involving multiple deletion of the hGH and hCS gene cluster. PMID- 3005357 TI - Responses of plasma adrenocorticotropin, cortisol, and dehydroepiandrosterone to ovine corticotropin-releasing hormone in healthy aging men. AB - To assess the effects of age on both the pituitary ACTH response to corticotropin releasing hormone (CRH) and the secretory responses of cortisol (F) and dehydroepiandrosterone (DHEA) to endogenous rises in ACTH, we measured evening basal and ovine CRH (oCRH; 1 mu/kg)-stimulated plasma concentrations of ACTH,F, and DHEA in 49 healthy men, aged 21-86 yr. By analysis of variance, we found no change with age in either the basal concentration of ACTH or the magnitude of the peak ACTH response to oCRH. Older men had higher basal F levels (P less than 0.05), while basal plasma levels of CBG and ratios of F to CBG did not vary significantly with age (P greater than 0.1). We also found no significant increase with age in the magnitude of the peak F response to oCRH (P greater than 0.2), although peak F responses occurred significantly earlier (P less than 0.03) in the older men. Basal plasma levels of DHEA decreased significantly with age (P less than 0.001), as did the magnitude of peak DHEA responses to endogenous ACTH rises (P less than 0.01). There was no alteration in the timing of the peak DHEA response with age (P greater than 0.7). We conclude that while ACTH and F responses to evening injections of oCRH are well maintained in healthy aging men, that of DHEA is discordantly decreased. The present findings are compatible with the hypotheses that there is a diminished sensitivity of ACTH secretion to negative feedback regulation by glucocorticoids in older men, and there is an ACTH-independent age-related diminution in adrenal androgen secretion. PMID- 3005359 TI - Brain inflammatory exudate in Junin virus-infected rats: its characterization by the immunoperoxidase (PAP) technique. AB - Morphologic changes in cyclophosphamide (CY)-suppressed vs. control non suppressed new-born rats infected i.c. with XJC13 strain of Junin virus were compared and the cells involved in CNS lesions were identified by the PAP technique. Fifty per cent of the control rats exhibited widespread cerebral necrosis vs. only 15% of the immunosuppressed animals. The first cells to reach Junin virus-infected CNS in controls were T lymphocytes, which destroyed viral antigen-laden target neurons and astrocytes. B lymphocytes and macrophages, presumably attracted by viral antigen and/or by lymphokines, made their appearance a day or two later. Activated macrophages phagocytosed necrotic cells and perhaps exerted a cytotoxic effect upon target neural cells, whereas the actual role of B lymphocytes requires further explanation. In CY-treated rats, cerebral lesions were smaller and the cellular exudate, though similar, proved much scantier than in controls. A similar extent of cerebellar necrosis was observed in both groups. PMID- 3005358 TI - Pituitary and recombinant deoxyribonucleic acid-derived human growth hormones alter glucose metabolism in 3T3 adipocytes. AB - 3T3-F442A adipocytes were used to compare the effects on glucose metabolism of pituitary human (h) GH and methionyl-hGH produced by recombinant DNA techniques (met-hGH 22K). Pituitary hGH and met-hGH 22K were similar in their ability to inhibit glucose oxidation and lipid accumulation after 48 h. After 4 h of incubation, both forms of hGH stimulated glucose oxidation transiently in 3T3 adipocytes. A bacterially produced form of the 20,000-dalton variant of hGH (met hGH 20K) also stimulated glucose oxidation at 4 h and inhibited glucose oxidation and lipid synthesis after 48 h. All three forms of hGH had a similar ability to inhibit [125I]iodo-met-hGH 22K binding to 3T3-adipocytes. Thus, met-hGH 22K and 20K directly produce in 3T3 adipocytes the transient stimulation and delayed inhibition of glucose metabolism attributed to pituitary hGH, indicating that these metabolic effects are intrinsic to the hGH molecule. PMID- 3005360 TI - Specificity of human IgM monoclonal antibodies from patients with peripheral neuropathy. AB - Some patients with peripheral neuropathy and gammopathy have IgM monoclonal antibodies that react with the myelin-associated glycoprotein (MAG), some 20-26 kDa glycoproteins present only in the peripheral nervous system (PNS), and some acidic glycolipids that are also PNS-specific. This communication describes an investigation of 18 patients with IgM paraproteinemia and neuropathy to test for the presence of antibodies that react with each of these components. Eleven patients had IgM that reacted with MAG, and in all cases the IgM also reacted with the lower Mr glycoproteins and the acidic glycolipids that are specific for the PNS. With respect to the other 7 patients that did not react with MAG, in no instance did immune-staining of electroblots reveal the presence of reactivity with the 20-26 kDa glycoproteins of the PNS or with any other protein antigen in the PNS or central nervous system (CNS). However, these 7 patients fell into 3 categories with regard to reactivity with acidic glycolipids: three reacted with the acidic glycolipid fraction of both PNS and CNS tissue; two reacted with the acidic glycolipid fraction of the PNS but not the CNS; and two showed no reactivity with the acidic glycolipids from either PNS or CNS. PMID- 3005361 TI - Effects of fibrinogen derivatives upon the inflammatory response. Studies with human fibrinopeptide B. AB - Fibrin formation and turnover are intimately associated with inflammation and wound healing. To explore whether fibrin(ogen)-derived peptides exert direct effects upon cells involved in inflammation and tissue repair we examined the capacity of human fibrinopeptide B (hFpB), a thrombin-derived proteolytic cleavage product of the fibrinogen B beta-chain, to stimulate neutrophils (PMN), monocytes, and fibroblasts. hFpB caused directed cell migration of PMN and fibroblasts that was optimal at approximately 10(-8) M. This chemotactic activity was blocked by preincubating hFpB with antiserum to hFpB. hFpB was not chemotactic for monocytes. The chemotactic potency of hFpB for PMN was equivalent to that of anaphylatoxin from the fifth component of human complement (C5a), leukotriene B4 (LTB4), and formyl-methionyl-leucyl-phenylalanine (fMLP), and for fibroblasts its chemotactic activity was comparable to that of platelet-derived growth factor. hFpB did not interact with PMN receptors for C5a, LTB4, or fMLP as (a) desensitization with 10(-7) M hFpB abolished chemotaxis to hFpB but had no effect upon chemotaxis to C5a, LTB4, or fMLP and (b) induction of chemotactic responses to fMLP and LTB4 in neutrophilic leukemic cells (HL-60 cells) by incubation with dimethylsulfoxide did not extend to hFpB. Like fMLP, hFpB caused a rapid, dose-dependent increase in PMN cytoskeletal associated actin, but unlike fMLP, hFpB did not cause PMN aggregation, release of lysosomal enzymes (lysozyme and beta-glucuronidase), or the production of superoxide anion. These results suggest that hFpB may have a role in recruiting PMN and fibroblasts at sites of fibrin deposition and turnover. The capacity of hFpB to cause PMN chemotaxis without causing concurrent release of lysosomal enzymes or the production of superoxide anion is further evidence for the complexity of PMN responses to chemotactic agents. PMID- 3005362 TI - Sodium-hydrogen exchange and glucose transport in renal microvillus membrane vesicles from rats with diabetes mellitus. AB - Diabetes mellitus is associated with important changes in renal hemodynamics and transport function. Disturbances in solute transport have also been characterized in nonrenal tissues during hyperglycemia and insulinopenia. The purpose of this study was to determine if diabetes is associated with adaptive changes in function of the brush-border membrane of the proximal tubule. We studied Na+ and glucose transport in rat microvillus membrane vesicles isolated from the renal cortex of streptozotocin-induced and BB/W autoimmune diabetic rats. Untreated diabetes was associated with an increase in pH-stimulated total and amiloride sensitive 22Na+ uptake into vesicles. Insulin treatment returned vesicle 22Na+ uptake to control levels. The increased Na+/H+ exchange was shown to be a result of increased net renal acid production rather than a specific response to insulinopenia because treatment with NaHCO3 also returned 22Na+ uptake to control levels. On the other hand, Na+-glucose cotransport, which was depressed in vesicles from untreated diabetics, returned to control levels with insulin but not NaHCO3 administration. This decreased Na+-glucose cotransport was not secondary to reduction in transport sites in untreated diabetics. These results show that in diabetes mellitus, increased Na+/H+ exchange activity is not the direct result of insulinopenia. However, the diabetic state appears to alter the functioning of the luminal Na+-glucose cotransporter. PMID- 3005364 TI - Superoxide-mediated modification of low density lipoprotein by arterial smooth muscle cells. AB - Extracellular superoxide was detected in cultures of monkey and human arterial smooth muscle cells as indicated by superoxide dismutase inhibitable reduction of cytochrome c. Superoxide production by these cells in the presence of Fe or Cu resulted in modification of low density lipoprotein (LDL). The degree of LDL modification was directly proportional to the rate of superoxide production by cells. Superoxide dismutase (100 micrograms/ml), and the general free radical scavengers butylated hydroxytoluene and butylated hydroxyanisole (50 microM), inhibited Fe- and Cu-mediated modification of LDL by monkey smooth muscle cells, while catalase (100 micrograms/ml) and mannitol (25 mM) had no effect. The chelators desferrioxamine and diethylenetriamine pentaacetic acid completely inhibited Fe- and Cu-promoted modification of LDL, while EGTA had no inhibitory effect. EDTA stimulated Fe-promoted modification in the 1-100 microM range while inhibiting Cu-mediated modification of LDL. LDL modified by smooth muscle cells in the presence of 10 microM Fe or Cu stimulated [14C]oleate incorporation into cholesteryl ester by human macrophages and murine J774 cells to a degree comparable to that produced by acetylated LDL. LDL incubated with smooth muscle cells and metal ions in the presence of superoxide dismutase failed to enhance macrophage cholesteryl ester accumulation. Thus, arterial smooth muscle cells in culture generate superoxide and modify LDL by a superoxide-dependent, Fe or Cu catalyzed free radical process, resulting in enhanced uptake of the modified LDL by macrophages. Neither hydroxyl radicals nor H2O2 are likely to be involved. Superoxide-dependent lipid peroxidation may contribute to biological modification of LDL, resulting in foam cell formation and atherogenesis. PMID- 3005363 TI - Exposure of fibrinogen receptors in human platelets by surface proteolysis with elastase. AB - Human platelets that were preincubated with porcine elastase aggregated spontaneously upon the addition of fibrinogen. Maximal aggregation to fibrinogen was observed with platelets pretreated with an elastase concentration of 111 micrograms/ml, and half-maximal aggregation occurred after treatment with 11 micrograms/ml elastase. Binding of radiolabeled fibrinogen to elastase-treated platelets was specific, saturable, and showed a single class of 48,400 +/- 9,697 fibrinogen-binding sites per platelet with a dissociation constant of 6.30 +/- 1.48 X 10(-7) M. ATP, apyrase, and the stimulators of platelet adenylate cyclase forskolin, prostaglandin E1, prostacyclin, and N6, 2'-O-dibutyryl cyclic AMP did not inhibit the fibrinogen-induced aggregation of elastase-treated platelets. EDTA completely blocked the initiation of aggregation and reversed the fibrinogen induced aggregation of elastase-treated platelets. Monoclonal and polyclonal antibodies directed against glycoproteins (GP) IIb and IIIa completely blocked the fibrinogen-induced aggregation of elastase-treated platelets. Immunoprecipitates with these antibodies obtained from detergent extracts of surface-radiolabeled, intact, and elastase-treated platelets contained the glycoproteins IIb and IIIa. We conclude that surface proteolysis by low concentrations of elastase can expose fibrinogen-binding sites associated with GPIIb and GPIIIa on the platelet surface, resulting in spontaneous aggregation upon the addition of fibrinogen. These findings may be relevant to hemostatic changes observed in patients with increased levels of circulating elastase. PMID- 3005365 TI - Complement membrane attack complex stimulates production of reactive oxygen metabolites by cultured rat mesangial cells. AB - To explore possible mechanisms by which complement membrane attack complexes (MAC) that are deposited in the glomerular mesangium might be pathogenic, we stimulated rat glomerular mesangial cells grown in vitro with nascent MACs formed from the purified human complement components C5b6 and normal human serum and measured production of superoxide ion (O2-) and hydrogen peroxide (H2O2). Mesangial cells incubated with C5b6 + serum, which results in cell membrane interaction with the MAC, produce 0.9 +/- 0.15 nmol O2-/10(5) cells per 30 min, which was significantly greater than the amount produced by cells incubated with C5b6 alone, serum alone, or decayed MACs that can no longer interact with the cell membrane (0.3 +/- 0.2, 0.4 +/- 0.1, 0.3 +/- 0.2 nmol O2-/10(5) cells per 30 min, respectively; P less than 0.02). Production of O2- after stimulation with MACs increased during the first 20 min of incubation but then plateaued. Cells exposed to decayed MACs produced small amounts of O2-, which did not increase from 20 to 60 min. Production of H2O2 was also observed after stimulation with MACs, and continued to increase during 60 min of incubation (1.22 +/- 0.16 nmol H2O2/10(5) cells per 60 min), whereas H2O2 production could not be detected after exposure to decayed MACs. Cell viability was not adversely affected by exposure to nascent MACs as determined by trypan blue exclusion or chromium-51 release. These results demonstrate that glomerular mesangial cell membrane interaction with the MAC stimulates the production of the toxic oxygen metabolites O- and H2O2. Activation of the terminal complement pathway by mesangial immune deposits in vivo might lead to tissue injury by stimulation of local production of toxic oxygen-free radicals. PMID- 3005366 TI - Proton secretion by the sodium/hydrogen ion antiporter in the human neutrophil. AB - The reducing equivalents used by the human neutrophil respiratory burst oxidase are derived from NADPH generated by the hexose monophosphate shunt. The CO2 generated by the HMP shunt is spontaneously hydrated and the protons (H+) are secreted upon the dissociation of carbonic acid. The mechanism and significance of H+ secretion by the resting and stimulated neutrophil was investigated. A basal rate of H+ secretion by resting neutrophils observed in a choline buffer was augmented with the addition of sodium (Na+) (Km for Na+ was 3.22 +/- 0.32 mM). Amiloride, a Na+/H+ antiporter inhibitor, reduced H+ secretion in Na+ containing buffers with a Ki = 1.02 microM. This Na+/H+ exchange mechanism was also operative in cells stimulated with a variety of agonists, and an increased H+ flux, relative to resting cells, was observed at higher Na+ concentrations. Cytoplasts incorporating acridine orange were also used to assess Na+-H+ flux. Cytoplasts were used to avoid alteration of the fluorescent pH probe by HOCl formed in intact neutrophils. Alkalinization of the cytoplasm was dependent on extracellular Na+ in concentrations similar to that found to augment H+ secretion in intact cells. Also, amiloride competitively inhibited H+ secretion by the cytoplasts. Both superoxide (O2-) production and lysozyme release in cells stimulated with opsonized zymosan or concanavalin A was significantly inhibited in the absence of Na+, restored to normal with the addition of Na+ in low concentrations, and inhibited again in the presence of amiloride. A Na+/H+ antiporter similar to that found in other cell types is present in the human neutrophil and appears linked to activation of the respiratory burst and degranulation. PMID- 3005367 TI - Transferrin synthesis by inducer T lymphocytes. AB - Transferrin (Tf) is a growth factor that transports iron in plasma. It is essential for proliferation of activated T lymphocytes. Previous studies have suggested that peripheral blood cells are capable of synthesizing Tf. Using in situ hybridization techniques and human Tf complementary DNAs as probes, peripheral blood cells have been examined for sites of Tf messenger RNA (mRNA) transcription. The studies described here demonstrate that Tf is synthesized by a specific subset of T lymphocytes, the T4+ inducer subset. T lymphocyte proliferation is dependent upon the presence of both interleukin 2 (IL-2) and Tf, even though resting cells do not possess receptors for either. The present studies indicate that during T cell activation, induction of IL-2 mRNA transcription and IL-2 receptor expression precede the transcription of Tf mRNA and expression of Tf receptors, respectively. These events in turn precede the initiation of DNA synthesis. Transferrin and its receptor appear to be involved in an autocrine pathway which is functionally linked to the IL-2/IL-2 receptor autocrine loop. PMID- 3005368 TI - Role of reactive oxygen in bile salt stimulation of colonic epithelial proliferation. AB - Our previous studies had suggested a link between bile salt stimulation of colonic epithelial proliferation and the release and oxygenation of arachidonate via the lipoxygenase pathway. In the present study, we examined the role of reactive oxygen versus end products of arachidonate metabolism via the cyclooxygenase and lipoxygenase pathways in bile salt stimulation of rat colonic epithelial proliferation. Intracolonic instillation of 5 mM deoxycholate increased mucosal ornithine decarboxylase activity and [3H]thymidine incorporation into DNA. Responses to deoxycholate were abolished by the superoxide dismutase mimetic CuII (3,5 diisopropylsalicylic acid)2 (CuDIPS), and by phenidone or esculetin, which inhibit both lipoxygenase and cyclooxygenase activities. By contrast, indomethacin potentiated the response. Phenidone and esculetin suppressed deoxycholate-induced increases in prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and 5, 12, and 15-hydroxyeicosatetraenoic acid (HETE), whereas CuDIPS had no effect. Indomethacin suppressed only PGE2. Deoxycholate (0.5-5 mM) increased superoxide dismutase sensitive chemiluminescence 2-10-fold and stimulated superoxide production as measured by cytochrome c reduction in colonic mucosal scrapings or crypt epithelium. Bile salt-induced increases in chemiluminescence were abolished by CuDIPS, phenidone, and esculetin, but not by indomethacin. Intracolonic generation of reactive oxygen by xanthine-xanthine oxidase increased colonic mucosal ornithine decarboxylase activity and [3H]thymidine incorporation into DNA approximately twofold. These effects were abolished by superoxide dismutase. The findings support a key role for reactive oxygen, rather than more distal products of either the lipoxygenase or cyclooxygenase pathways, in the stimulation of colonic mucosal proliferation by bile salts. PMID- 3005370 TI - Insulin receptor of human cerebral gliomas. Structure and function. AB - The insulin receptor from human brain tumors of glial origin was examined for the first time using intact cells (from an established cultured human glioblastoma cell line) and partially purified solubilized membranes (from cultured cells and freshly isolated human brain tumors). The structure of the glial insulin receptor subunits was assessed by affinity cross-linking of 125I-insulin with the alpha subunit of the receptor, neuraminidase treatment of the cross-linked receptor, behavior of the receptor on lectin columns, and electrophoretic mobility of the phosphorylated beta-subunit. The functions of the insulin receptor were examined by measuring specific 125I-insulin binding (receptor concentration, affinity, specificity, pH-, time-, and temperature dependence), insulin-induced down regulation of the receptor, insulin-stimulated autophosphorylation of the beta subunit, and phosphorylation of exogenous substrates as well as insulin stimulated glucose uptake in glioblastoma cells. All of these properties were typical for the insulin receptor from target tissues for insulin action. The insulin receptor of the normal human brain showed the altered electrophoretic mobility and lack of neuraminidase sensitivity of its alpha-subunit previously reported for the rat brain receptor. There was no difference, however, in the functions of the receptor subunits (binding, phosphorylation) from the normal brain tissue and the eight human gliomal tumors. Since the glial elements compose a majority of the brain cells, the "normal" structure and function of their insulin receptor might provide a key to understanding the role of insulin in the carbohydrate metabolism of the human central nervous system. PMID- 3005369 TI - Exudation primes human and guinea pig neutrophils for subsequent responsiveness to the chemotactic peptide N-formylmethionylleucylphenylalanine and increases complement component C3bi receptor expression. AB - After circulating in the vascular system a short time, polymorphonuclear leukocytes (PMN) migrate to extravascular sites in response to chemotactic stimuli. Prestimulation of PMN in vitro by secretagogues has been shown to increase their number of N-formylmethionylleucylphenylalanine (fmet-leu-phe) and complement component C3bi (CR3) receptors. We investigated whether the same phenomenon occurred in vivo, comparing characteristics of human skin chamber and guinea pig peritoneal exudate and blood PMN. Exudate PMN of both species contained approximately 28% less of the specific granule marker vitamin B12 binding protein (P less than 0.01) but a similar amount of the azurophil granule marker beta-glucuronidase. The total number of fmet-leu-phe receptors was 5.9 times higher in guinea pig exudate than in blood PMN (P less than 0.01) and 2.9 times higher in human exudate than in blood PMN (P less than 0.02). All exudate PMN and most blood PMN preparations showed a high affinity receptor (Kd approximately 2.3 X 10(-8) M) and a low affinity receptor (approximately 1.5 X 10(-7) M). The upregulation of fmet-leu-phe receptors in exudate PMN correlated with an improved responsiveness to fmet-leu-phe induced membrane depolarization, oxidative metabolism, and chemotaxis. In addition, the concentration of fmet-leu phe that produced a half-maximal response of chemotaxis, superoxide production, and membrane potential depolarization was 10-fold lower in exudate PMN than in blood PMN. Human exudate PMN had a twofold increased C3bi receptor expression compared with blood PMN. Thus, a preferential loss of specific granules is associated with increased number of high and low affinity fmet-leu-phe receptors and increased C3bi receptor expression not only in vitro, but also in vivo. The data indicate that exudation primes PMN for their subsequent responsiveness to fmet-leu-phe, a modification that may be crucial for efficient antimicrobial host defense. PMID- 3005371 TI - Study of childhood renal tumours using antisera to fibronectin, laminin, and epithelial membrane antigen. AB - Using a peroxidase-antiperoxidase staining procedure, formalin fixed paraffin embedded sections of fetal and normal kidney; benign (mesoblastic nephroma); and malignant tumours (Wilms' tumour, clear cell renal carcinoma, rhabdoid renal tumour, and bone metastasising renal tumour of childhood (BMRTC] were examined for their reactivity with antisera to fibronectin, laminin, and epithelial membrane antigen. Mesoblastic nephroma contained fibronectin but no laminin. Most Wilms' tumours lacked both fibronectin and laminin; 50% of rhabdoid renal tumours were positive for fibronectin and laminin--rhabdoid tumours as recognised morphologically may, in fact, be two separate entities. BMRTC and clear cell renal carcinoma lacked both fibronectin and laminin. Epithelial membrane antigen was present in most of the tubular Wilms' tumour but absent in blastemal Wilms' tumours. The presence of epithelial membrane antigen in rhabdoid tumours was surprising, as histologically, this type of tumour shows no sign of epithelial differentiation. Epithelial membrane antigen antiserum stained clear cell renal carcinomas: epithelial membrane antigen is found in the distal and not the proximal tubules of fetal and normal kidneys. Thus an obvious interpretation is that clear cell renal carcinomas originate from distal rather than from proximal tubules, as has always been thought. On the basis of these results and data from other published findings some possible histogenetic origins of childhood renal tumours were proposed. PMID- 3005372 TI - Aplastic and hypoplastic episodes in sickle cell disease and thalassaemia intermedia. AB - Aplastic and hypoplastic crises are well recognised complications of sickle cell disease. Recent evidence has shown that most of these crises are caused by parvovirus infection. Five cases of aplastic or hypoplastic crises in patients born and living in this country were studied. Three patients had clear evidence of parvovirus infection, while in two evidence of parvovirus infection was lacking. One patient had evidence of concurrent parvovirus and Mycoplasma pneumoniae infection. Recurrent crises may occur, and reticulocyte monitoring during infection in patients with chronic haemolytic states is therefore important. PMID- 3005373 TI - Laminin and fibronectin in adenoid cystic carcinoma. AB - The distribution of fibronectin and laminin was examined by immunohistochemistry in 11 adenoid cystic breast carcinomas, six adenoid cystic carcinomas of mouth and salivary gland, and six cribriform ductal breast carcinomas. Both proteins were present lining cystic lumina and around tumour islands in all the adenoid cystic breast carcinomas and in five of six salivary gland tumours. Abundant laminin and fibronectin were dispersed among adenoid cystic tumour cells arranged in sheets. One adenoid cystic carcinoma from buccal mucosa showed a transition from a cribriform tumour positive for both fibronectin and laminin to a cribriform tumour negative for fibronectin and laminin to undifferentiated carcinoma. Fibronectin and laminin seemed to disappear simultaneously from tumour cell surfaces. Another adenoid cystic carcinoma from buccal mucosa was negative for fibronectin and laminin from the time of initial biopsy. This was the only tumour that gave rise to disseminated metastases, resulting in the death of the patient within two years of surgery. In cribriform invasive ductal breast carcinomas the linings of cystic lumina were always negative for fibronectin and laminin. Varying quantities were present at the tumour boundaries. We suggest that staining for fibronectin and laminin may be a valuable aid to the diagnosis of adenoid cystic carcinomas and that the absence of these proteins may have important prognostic implications. PMID- 3005374 TI - Distinguishing lymphoma and small cell anaplastic carcinoma of the thyroid by immunocytochemistry. PMID- 3005375 TI - Clinical pharmacokinetics of suramin in patients with HTLV-III/LAV infection. AB - Suramin has been reported to inhibit the reverse transcriptase activity of a number of retroviruses and to reduce the in vitro infectivity and cytopathic effect of HTLV-III/LAV, the etiologic agent of acquired immune deficiency syndrome (AIDS). The clinical pharmacokinetics of suramin were investigated as part of a pilot study to evaluate the safety and efficacy of this drug for the treatment of patients with diseases caused by HTLV-III/LAV. A dose of suramin 6.2 g was given intravenously over a five-week period to four patients. After the last dose, the plasma half-life of suramin was 44 to 54 days. This is among the longest half-lives reported for any therapeutic substance given to humans. Total plasma levels of suramin were greater than 100 micrograms/mL for several weeks. In vitro activity of suramin was found at concentrations as low as 50 micrograms/mL. Metabolites were not found in plasma, and urinary excretion accounts for elimination of most of the drug. Suramin is approximately 99.7% bound to plasma proteins. The results from these initial clinical pharmacokinetic studies might assist the design of further therapeutic trials of suramin, especially the selection of frequency of dosing and adjustments for renal impairment. PMID- 3005376 TI - Enalaprilat in hypertensive emergencies. AB - Enalaprilat (MK-422), an intravenously administered angiotensin-converting enzyme inhibitor, which is the parent compound of the oral angiotensin-converting enzyme inhibitor enalapril (MK-421), was studied in 11 patients with asymptomatic accelerated hypertension. Each patient received an initial intravenous dose of 1 mg, followed at one-hour intervals by enalaprilat 10 mg, furosemide 40 mg, and enalaprilat 40 mg. Six of 11 patients responded with a drop in mean arterial pressure greater than 15 mm Hg to diastolic levels below 110 mm Hg; there were four partial responders and one nonresponder. Pretreatment renins were not predictive of blood pressure response. No patient had any adverse reaction to the drug; there were no significant changes in posttreatment laboratory values. We conclude that enalaprilat is an effective, well-tolerated agent for the treatment of uncomplicated accelerated hypertension and its use does not imperil nonresponding uncomplicated patients. PMID- 3005378 TI - Naloxone withdrawal exacerbates Tourette syndrome. PMID- 3005377 TI - Tolerance and pharmacology of ciprostene, a stable epoprostenol (prostacyclin) analogue in humans. AB - Safety, tolerance, and pharmacology of 9-beta-methylcarbacyclin calcium (ciprostene calcium) was investigated in healthy male volunteers. This stable prostacyclin analogue was infused intravenously into groups of 12, 11, and three volunteers for three, six, and eight hours, respectively, in doses up to 480 ng/kg/min. Based on the tolerance data obtained, a single-blind, placebo controlled study was conducted. Seven subjects were infused for 8 hr/d for three days with ciprostene at a maximum dose of 160 ng/kg/min and seven subjects received placebo. One subject from each group did not complete the infusion schedule, and they were not included in the final analysis. During infusion of ciprostene, consistent changes in blood pressure and heart rate did not occur. Most frequent adverse drug reactions consisted of headache, restlessness, nausea, perspiration, flushing, and jaw pain. As compared with placebo, ADP-induced platelet aggregation was inhibited during the infusion period (P = .048). Significant (P = .04) elevations of platelet cyclic AMP were observed in subjects during infusion of ciprostene. Pre- versus postinfusion routine laboratory evaluations, fibrinogen concentration, antiplasmin activity, and plasminogen and template bleeding times remained unchanged. Placebo- and drug-treated subjects had a daily postinfusion shortening of euglobulin clot lysis time (ECLT). The preinfusion minus postinfusion ECLT for ciprostene subjects on days 2 and 3 (133 and 118 min, respectively) compared with placebo (239 and 217 min) suggest a trend to increased fibrinolytic activity. Based on the outcome of this trial, it is estimated that ciprostene is about 15 times less potent than prostacyclin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3005379 TI - Laminar distribution and patchiness of cytochrome oxidase in mouse superior colliculus. AB - The cytochrome oxidase (CO), acetylcholinesterase (AChE), myelin, and Nissl stains were studied and compared to develop an anatomical system identifying the laminar architecture of the mouse superior colliculus. The CO and myelin stains are shown to define collicular laminae more distinctly than does the Nissl stain. The layer of large rostrocaudally coursing fiber bundles that has formerly been referred to in the rodent literature as stratum album intermediale (SAI; layer V) is renamed as a sublayer of the stratum griseum intermediale (SGI; layer IV) to conform with the nomenclature for the cat superior colliculus of Kaneseki and Sprague ('74, J. Comp. Neurol. 158:319-338). Patches of CO activity in layer IV (SGI) are shown that contain intensely stained, large, multipolar cell bodies. The CO patches do not correspond to those previously reported for AChE. The CO, myelin, and AChE stains all indicate the presence of a large lateral extension termed the flank of layer IV (SGI). In contrast to the classical lamination pattern of the superior colliculus, the flank has no overlying layer II (stratum griseum superficiale, SGS) or layer III (stratum opticum, SO). PMID- 3005380 TI - Lobar intensity differences of the liver on MR imaging. AB - Differences in signal intensity involving lobes of the liver were noted in seven cases of liver tumor. The clinical significance and possible cause of these differences in lobar intensity are discussed. PMID- 3005381 TI - MR imaging of rhabdomyolysis. AB - The use of magnetic resonance (MR) imaging in two cases of rhabdomyolysis, one resulting from prolonged muscle compression and one from electrical burns, is described. The involved muscles were clearly demonstrated with MR. Recognition and assessment of the extent of rhabdomyolysis are important since life threatening sequelae including severe metabolic disorders are possible. In one case, spin-echo and inversion-recovery MR imaging provided greater detail of muscle abnormalities than did 99mTc-pyrophosphate radionuclide scanning. Both cases illustrate the usefulness of MR in evaluation of skeletal muscle disorders. PMID- 3005383 TI - The effect of the Keyes technique on the structure and function of oral mucosa. PMID- 3005382 TI - CT findings of lobar bronchioloalveolar carcinoma. AB - The CT findings in a case of lobar bronchioloalveolar carcinoma included stretching, spreading, and uniform narrowing of the involved bronchi without obstruction. PMID- 3005385 TI - Solubilization of striatal D-2 dopamine receptors: evidence that apparent loss during aging is not due to membrane sequestration. AB - D-2 dopamine receptors from mature (3 to 6 months) and senescent (24 to 25 months) rat striatal membranes were solubilized with the detergent 3-(3 cholamidopropyl) dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO). Essentially all of the receptors were recovered following CHAPSO treatment. Approximately 80 to 90% of the receptors were detected in the supernatant fraction under these conditions, with a small percentage remaining membrane bound in both age groups. A 10-fold reduction in binding affinity for [3H]spiperone was observed for solubilized receptors of both age groups. Ultra high speed centrifugation of untreated preparations (200,000 X g) revealed essentially no hidden D-2 dopamine receptors in the light membrane fraction from either mature or senescent rats. These results suggest that a decreased biosynthetic rate (Henry & Roth, 1984; Leff et al., 1984) rather than membrane sequestration accounts for loss of D-2 dopamine receptors with age. PMID- 3005384 TI - Double-blind comparison of captopril and enalapril in mild to moderate hypertension. AB - To compare the antihypertensive and humoral effects of the angiotensin-converting enzyme inhibitors captopril and enalapril, 20 patients with essential hypertension, not receiving treatment for 2 weeks and consuming a prescribed sodium ion intake, were randomly assigned to two parallel, double-blind treatment groups with stratification based on race and untreated seated diastolic blood pressure. These groups received a placebo (day -1) followed by either captopril, 200 mg every 12 hours (n = 9), or enalapril maleate, 20 mg every 12 hours (n = 11), alone (days 1 to 14) and then with hydrochlorothiazide, 25 mg every 12 hours (days 16 to 28). Captopril and enalapril were coadministered alone (day 15) and with hydrochlorothiazide (day 29) to assess whether further decreases in blood pressure would occur. Captopril and enalapril alone caused comparable decreases (p less than 0.05) in the mean 12 hour time-averaged seated diastolic blood pressure from values on day -1 (placebo), on day 1 (11 and 9 mm Hg, respectively) and day 14 (8 and 7 mm Hg, respectively). The addition of hydrochlorothiazide further decreased (p less than 0.05) blood pressure in each group (7 and 8 mm Hg, respectively) from values on day 14. Combined use of captopril and enalapril did not result in further reduction. Coupled with the comparable changes observed in each treatment group in serum angiotensin-converting enzyme activity, plasma renin activity and plasma aldosterone concentration, these data support the view that captopril and enalapril have similar antihypertensive effects and mechanisms. PMID- 3005387 TI - Acquired immunodeficiency syndrome manifested as disseminated cryptococcosis. AB - A 32-year-old male homosexual presented to the emergency department (ED) with the clinical picture of a nonspecific illness. While in the ED, he experienced a first-time seizure. Computed tomography (CT) showed an enhancing mass lesion. Antibacterial therapy was started and continued until a second lumbar puncture (LP), 36 hours after admission, showed distinct yeast forms. Subsequent institution of appropriate therapy did not prevent the patient's death. The cause of death was disseminated cryptococcosis secondary to acquired immunodeficiency syndrome (AIDS). PMID- 3005386 TI - Bicarbonate therapy for the cardiovascular toxicity of amitriptyline in an animal model. AB - The beneficial hemodynamic effects of sodium bicarbonate as treatment for tricyclic antidepressant poisoning were investigated in an animal model. Seven adult dogs (17.5 to 20 kg) were poisoned by an intravenous infusion of amitriptyline. Toxicity was defined as a doubling of the initial QRS width. A continuous infusion was used to maintain toxicity for 30 minutes after which 44.5 mEq of sodium bicarbonate was administered intravenously. Five of the animals survived to completion of the experiment. Three of the surviving animals developed dysrhythmias. All dysrhythmias ceased within one minute of administration of sodium bicarbonate. An increase in mean blood pressure (P less than .05) and serum pH (P less than .05) and a decrease in mean QRS width (P less than .05) occurred following administration of sodium bicarbonate. The maintenance of toxicity for 30 minutes suggests that this model can be used for future studies of tricyclic antidepressant poisoning. PMID- 3005388 TI - Primary hepatocellular carcinoma localised by a radiolabelled monoclonal antibody. AB - A rat monoclonal antibody, YPC2/38.8, was selected from a panel of antibodies derived by immunising rats with fresh human colorectal carcinoma. It was found to bind to a 30 000 dalton protein present on the cell surface of normal colon and liver. This protein was increased 10-fold on primary hepatocellular carcinoma (PHC) cells. After labelling with 131I, YPC2/38.8 was shown to localise human PHCs grown as xenografts in immunosuppressed mice. Sixteen patients with PHC were given 1 mg of purified antibody labelled with 1 mCi of 131I by slow intravenous injection. In 8 out of 9 patients with PHC arising in non-cirrhotic livers, good tumour images were obtained on gamma camera or rectilinear scans, but in 7 patients who had developed PHC on the background of established hepatic cirrhosis, no tumour images were seen. Subsequent studies revealed that the 30K antigen recognised by this antibody was present in increased quantity on PHC cells and the regenerating liver cells in cirrhosis. We conclude that YPC2/38.8 may have potential for diagnostic localisation and possibly thence for the selective targeting of drugs or toxins in patients with PHC arising in a liver unaffected by significant parenchymal disease. PMID- 3005389 TI - Granular cell myoblastoma of the esophagus. PMID- 3005390 TI - Immunocytochemical localization of a tartrate-resistant and vanadate-sensitive acid nucleotide tri- and diphosphatase. AB - Purified rabbit antiserum to a tartrate-resistant and vanadate-sensitive acid phosphatase (nucleotide tri- and diphosphatase) prepared from rat bone was used in immunocytochemical studies. The antigen was localized in sections of fixed, decalcified tissue (head from rat) using the peroxidase-antiperoxidase bridge (PAP) or the avidin-biotin-peroxidase complex (ABC) technique. Both techniques resulted in similar and specific immunostaining in the following cells and tissues: osteoclasts situated in resorption lacunae, epithelium overlying enamel free areas of tips of cusps of unerupted molars, cilia of respiratory epithelium, and tissue macrophages. This distribution corresponds to the cellular sites of tartrate-resistant acid phosphatase activity, as revealed by enzyme histochemistry. With the ABC method, staining in osteoclasts was obtained with antiserum dilutions of up to 1:10,000. Biochemical studies revealed that vanadate sensitive acid ATPase activity in liver subcellular fractions was almost exclusively confined to lysosomes. Thus, the immunostaining has revealed the presence of the tartrate-resistant and vanadate-sensitive nucleotide phosphatase in many cells associated with tissue resorption and phagocytosis. PMID- 3005391 TI - Thiamine monophosphatase: a genuine marker for transganglionic regulation of primary sensory neurons. AB - Thiamine monophosphatase (TMPase) has been selectively localized in small dorsal root ganglion cells and in their central and peripheral terminals. Light microscopic localization of TMPase, and its alterations due to transganglionic effects, are identical with those of fluoride-resistant acid phosphatase (FRAP), but are not contaminated by the ubiquitous lysosomal reaction inevitable in trivial acid phosphatase-stained sections. TMPase is inhibited by 0.1 mM NaF, which is slightly less than the concentration needed to inhibit FRAP (0.2-0.4 mM). It is assumed that TMPase and FRAP are identical enzymes. In the perikaryon of small dorsal root ganglion cells, TMPase is located in the cisterns of the endoplasmic reticulum and in the Golgi apparatus. The central terminals of these cells are scalloped (sinusoid) axon terminals, surrounded by membrane-bound TMPase activity. Central terminals outline substantia gelatinosa Rolandi throughout the spinal cord, as well as the analogous nucleus spinalis trigemini in the medulla. TMPase-active central terminals outline "faisceau de la corne posterieure" in the sacral cord, as well as Lissauer's tract in the thoracic, upper lumbar, and sacral segments, and the paratrigeminal nucleus and the terminal (sensory) nucleus of the ala cinerea in the brainstem. Peripheral terminals displaying TMPase activity are fine nerve plexuses of C fibers. The TMPase activity of the central terminals disappears after dorsal rhizotomy in the course of Wallerian degeneration, and is depleted in the course of transganglionic degenerative atrophy (after transection of the related peripheral sensory nerve). TMPase is an outstanding genuine marker for the study of transganglionic regulation in Muridae. PMID- 3005392 TI - [Morphologic correlates of the electrical interactions between neurons in the limbic system]. AB - Electron microscopical results on "special structural relations" between neighbouring neuronal elements (pericarya, dendrites) are demonstrated in different regions of the rat limbic system: field CA3 and field CA1 of the hippocamp, dentate gyrus, area entorhinalis (L V) and area retrosplenialis granularis (L III). The "special structural relations" are to be characterized as follows: direct apposition of membranes of the neighbouring neurons including an extracellular space of relatively constant width over a larger distance without neuroglia intervening this space. Areas of membrane appositions have been observed between neuronal somata ("soma-somatic appositions"), between dendrites ("dendro-dendritic appositions") and between both, somata and dendrites ("dendro somatic appositions"). In the elmigraphs their length varied between 1 and 6 microns. In the entorhinal cortex and in the retrosplenial cortex soma-somatic appositions were in the majority, whereas in the hippocampal regions dendrodendritic appositions, in the dentate gyrus all types seem to be predominant. In the literature such direct appositions of neuronal membrane often have been described in the CNS of different species. They are considered as a possible morphological correlate of the ephaptic interaction, which is caused by field effects between neighbouring neurons. Following the literature the results of the ephaptic interactions vary between a weak facilitation and synchronous discharge of an unstimulated neuron by a stimulated one. Although such direct appositions are present in different groups of neurons in the limbic system, especially in the hippocamp, up to now their functional meaning is unclear. No correlation seem to exist between neuronal membrane appositions/ephaptic interactions and such a phenomenon like the hippocampal longterm potentiation; mechanisms of cooperativity included in the LTP like coactivation effects of pre- or postsynaptic elements still remain to be clarified. PMID- 3005393 TI - Inhibitory action of morphine on adenosine triphosphatase content in the whole and individual nuclei of mouse brain during the tolerance-dependence development and its reversal by naloxone. AB - Fluctuations in the content and distribution of adenosine triphosphatase in the brain of mice during the period of morphine tolerance-dependence development as well as normal and and naloxone induced withdrawal have been studied. The histochemical investigation revealed the enzyme activity in the neurons, neuroglia and blood vessels. In the control animals the nucleus caudatus putamen, globus pallidus, hippocampus, nuclei of amygdala, nuclei hypothalami, substantia grisea centralis, griseum pontis, nucleus trapezoideus, nucleus prepositus hypoglossi, nucleus parabrachialis, nucleus vestibularis, nucleus nervi hypoglossi, nucleus dorsalis nervi vagi, nucleus olivaris and nucleus centralis superior are found to be very rich in ATPase. However, morphine treatment inhibited the enzyme in all the above nuclei and it was linear with the increase in dose and the duration of the treatment. Cytophotometric studies reveal that the differences in the enzymatic activity varies from nuclei to nuclei. Surprisingly enough, normal withdrawal as well as naloxone induced withdrawal significantly elevated the enzyme levels. All above findings have been confirmed biochemically. The study gives a firm support of the earlier finding that morphine inhibits ATPase. In addition to this, the present work also reveals a direct antagonistic effect of naloxone on the content of the enzyme in the morphine treated animals. This suggests that the observed inhibition of the enzyme is narcotic specific. The role of ATPase in the Na+, K+ transport is discussed with respect to morphine action. In the light of the present investigation, the effect of morphine on the neurotransmitter release, and the cause and effect there upon has been analyzed. It has been suggested that ATPase might be the enzyme responsible for the observed pharmacological responses of the neurons to the application of the drug by affecting the Na+, K+ flux and neurotransmitter release. PMID- 3005394 TI - Escherichia coli as a genetic tool. AB - The study of Escherichia coli and its plasmids and bacteriophages has provided a vast body of genetical information, much of it relevant to the whole of biology. This was true even before the development of the new techniques, for cloning and analysing DNA, that have revolutionized biological research during the past decade. Thousands of millions of dollars are now invested in industrial uses of these techniques, which all depend on discoveries made in the course of academic research on E. coli. Much of the background of knowledge necessary for the cloning and expression of genetically engineered information, as well as the techniques themselves, came from work with this organism. PMID- 3005395 TI - Intraepithelial leukocytes contain a unique subpopulation of NK-like cytotoxic cells active in the defense of gut epithelium to enteric murine coronavirus. AB - Initially the intraepithelial leukocytes (IEL) of specific pathogen free (SPF) mice were compared with those of mice held without isolation and were found to differ markedly in total number and distribution of cell surface antigens. The IEL from SPF mice expressed significantly less Thy-1, Lyt-1, and Lyt-2 antigens than their conventional counterparts. The local cell-mediated immune response of mucosal lymphocytes to an enteric murine coronavirus (MHV-Y) was studied in inbred strains of naive SPF mice. A potent in vitro cytotoxic activity was demonstrated by mucosal leukocytes, especially IEL, and spleen cells for MHV-Y infected syngeneic and allogeneic target cells. The cytotoxicity was not restricted by the major histocompatibility complex. Targets infected with Pichinde virus, an enveloped nonenterotropic virus, were not lysed by these cells. The phenotype of the IEL effector cell was asialo GM1+, Thy-1-, Lyt-1-, Lyt-2-. This cell represents a small subpopulation of the total IEL. After the in vivo administration of anti-asialo GM1 sera, the virus-specific cytotoxic function of the IEL was markedly diminished in in vitro assays, and there was enhanced persistence of virus in gut tissues in vivo. The IEL effector population is defined as a natural killer-like cell that appears to be active in the defense of the gut epithelium to a murine enteric coronavirus. PMID- 3005396 TI - IL 2 alone is mitogenic only for Tac-positive lymphocytes in human peripheral blood. AB - To investigate the ability of interleukin 2 (IL 2) alone to induce proliferation of resting human lymphocytes, we stimulated human peripheral blood mononuclear cells (PBMC) with an immunopurified preparation of IL 2 or with phytohemagglutinin (PHA). Proliferation and the percent of cells expressing IL 2 receptors were assessed over 6 days of culture. Regardless of the stimulus, the percent of cells bearing an IL 2 receptor paralleled the amount of proliferation, and proliferation was inhibited by an anti-IL 2 receptor monoclonal antibody (anti-Tac). When stimulated by IL 2 alone, less than 8% of PBMC expressed an IL 2 receptor after 24 hr of culture. Stimulation by IL 2 caused both proliferation and IL 2 receptor expression to increase over the entire culture period (routinely to 75,000 cpm and 50% respectively). When colchicine was added (to inhibit cell division), the percent of cells bearing an IL 2 receptor did not increase. IL 2 alone also induced proliferation of PBMC depleted of accessory cells, with the same kinetics but reduced peak response. Both accessory cells and supernatants that showed IL 1 but not IL 2 activity augmented this proliferation 50 to 100%. In contrast to the effect of IL 2, 25 to 50% of PBMC stimulated by PHA expressed an IL 2 receptor after 24 hr of culture. PHA-induced proliferation and IL 2 receptor expression peaked early in the culture period (routinely to 100,000 cpm and 50% respectively within 3 days), and colchicine did not inhibit the early induction of IL 2 receptors on PBMC. Our findings indicate that unlike PHA, IL 2 induces proliferation of PBMC (or PBMC depleted of accessory cells) by expanding the small percentage of cells in a resting population that already express IL 2 receptors. IL 2 does not appear to induce IL 2 receptors on cells previously lacking this molecule. We also find that IL 1 can enhance the response to IL 2 alone. PMID- 3005398 TI - Characterization of tubular basement membrane antigens in human kidney. AB - Tubular basement membrane (TBM) was prepared from normal human kidneys and solubilized with various enzymes. Collagenase digestion released antigenic moieties from the TBM. All four anti-TBM antibodies we studied, three from patients with idiopathic tubulo-interstitial nephritis (TIN) and one from a renal allograft recipient, distinctively reacted with collagenase-digested (CD) TBM during enzyme-linked immunoassay and could discriminate among sera of normal controls or of other nephritis patients, including anti-glomerular basement membrane (anti-GBM) nephritis. When digested with pronase, trypsin, or pepsin, antigenicity of the TBM decreased. We studied the TBM antigens with immunoprecipitation and immunoblotting. After incubation of radio-iodinated CDTBM with anti-TBM sera, immunoprecipitates were identified by single-dimension SDS polyacrylamide gel electrophoresis or two-dimension gel electrophoresis, followed by autoradiography. All four antibodies had identical results on immunoprecipitation; under nonreducing conditions, they gave two protein bands with m.w. of 54,000 and 48,000 and with pI 7.0 to 8.0 and 6.5 to 7.0. Electrophoresis performed under reducing conditions disclosed only one band at the m.w. of 48,000 and pI of 6.5, suggesting that the 54-kDa component is composed of peptides linked by interchain disulfide bonds. Immunoblot analysis showed that the anti-TBM antibodies were heterogeneous; three antibodies from the idiopathic TIN patients reacted with the 54-kDa band, but the one from the renal allograft recipient reacted with neither band. This finding suggests that there are two antigenic determinants on the 54-kDa component. One such determinant that was resistant to denaturation with SDS was detected by the first three antibodies, and the other that was sensitive to such denaturation bound to the last antibody. The 48-kDa component seemed not to be immunoreactive after incubation with SDS. We studied TBM antigens reactive with anti-GBM antibodies. By immunoblotting, all four sera from patients with anti-GBM nephritis stained TBM proteins of 45 to 50 kDa and 25 to 27 kDa at pH 8.0 to 9.0; this was similar to the staining pattern of CDGBM with the same sera, but the highly cationic (pH greater than 9.0) components were specifically detected in the CDGBM. By inhibition ELISA, the binding of the anti-GBM sera to denatured CDTBM decreased with preincubation of the sera with CDGBM, suggesting that the anti-GBM antibodies recognize the same epitope(s) on the GBM and the TBM. PMID- 3005397 TI - Decreased natural killer activity in thalassemia major: a possible consequence of iron overload. AB - We have investigated the natural killer (NK) activity of both fractionated (Percoll density gradient) and unfractionated mononuclear cells from patients with beta-thalassemia major who are iron overloaded as a consequence of chronic transfusion therapy. These patients were found to have significantly decreased NK activity against K562 targets at all effector:target ratios tested (p less than 0.001). Both splenectomized and nonsplenectomized patients had normal proportions of Leu-11b-staining (NK) cells. Since they had normal to elevated absolute white cell and lymphocyte counts, a change in the absolute number of NK cells could not account for the decreased killing. We also found that the decrease in NK activity was transfusion related (r = -0.603, p less than 0.005). To determine whether or not this decrease in NK activity could be related to iron overload, we preincubated patient effector cells with desferrioxamine (DFO) or 2,3 dihydroxybenzoic acid (DHB) for 6 hr before addition of K562 targets. Both of these iron-chelating agents consistently increased the NK activity of cells from thalassemia patients. DHB had the greater effect, being able to increase patient NK activity to virtually normal levels. On the other hand, preincubation of cells from normal controls with DHB caused only a slight increase in NK activity, while similar treatment with DFO had little or no effect. When target cells were preincubated with the chelating agents before addition of either normal or patient effector cells, no change in cytotoxicity was seen, demonstrating that the chelating agents act at the effector cell level. Furthermore, if the chelating agents were saturated with iron prior to preincubation with the effectors, no increase in the cytotoxicity of thalassemic NK cells was observed. These results indicate that thalassemia patients have a reversible, transfusion related decrease in NK function which may arise as a consequence of iron overload. PMID- 3005399 TI - Interferon-beta and recombinant IL 2 can both enhance, but by different pathways, the nonspecific cytolytic potential of T3- natural killer cell-derived clones rather than that of T3+ clones. AB - We studied the enhancement of cytolytic activity of T3- natural killer cell derived clones, of T3+ T cell activated killer (AK) clones, and of fresh peripheral blood lymphocytes (PBL) by various crude and recombinant interferon (r IFN) as well as IL 2 preparations. It was found that IFN-beta had the highest cytotoxicity inducing potency as compared to crude or r-IFN-alpha or -gamma preparations. This enhancement was blocked by anti-IFN-beta antibodies but not by anti-IFN-gamma antibodies. IL 2 also strongly enhances cytolytic activity in cloned T3- killer cells that express the IL 2 receptors as determined with the anti-Tac monoclonal antibody (MAb) at concentrations of IL 2 (25 U/ml) which induced one-half of the maximal proliferation capacity in human T cells and murine CTLL cells. For enhancement of cytolytic activity in fresh NK cells, a much higher concentration of IL 2 is required. In addition, the enhancement of cytolytic activity by r-IL 2 but not that by IFN-beta can be reduced by anti-Tac MAb, suggesting that the IL 2 receptor is involved in the enhancement by IL 2, but not by IFN. Both IFN-beta and IL 2 were able to enhance (over threefold) the cytolytic activity of T3- cloned killer cells against a variety of tumor target cell types. Another remarkable observation was that K562 cells, the most commonly used target cell for determining NK cell cytolytic activity, are not the most suitable targets to assess enhancement of nonspecific lytic activity as compared to Daudi or lung tumor-derived cell lines. No enhancement of anti-body-dependent cellular cytotoxicity was observed. Finally, the effects of these biological response modifiers were much more pronounced on "fresh" and cloned T3- natural killer cell-derived than on T3+-activated killer mature T cell-derived clones. PMID- 3005401 TI - Bacterially expressed antigenic peptide from foot-and-mouth disease virus capsid elicits variable immunologic responses in animals. AB - A fusion protein consisting of beta-galactosidase (GZ) to which was attached at its N-terminus the amino acid sequence corresponding to residues 142-160 of the immunogenic protein VP1 of foot-and-mouth disease virus (FMDV) has been expressed in E. coli. A chemically synthesized section of DNA corresponding to the amino acid sequence 142-160 was inserted into a vector (pXY410) designed to express fusion proteins with the carboxy terminal 1015 amino acids of GZ. The hybrid protein immunopurified by a GZ-specific monoclonal antibody was soluble, retained full GZ activity, and induced virus-neutralizing antibody in guinea pigs and mice. There were significant differences between the responses of individual mice to the FMDV peptide sequence, although the titers against GZ were uniformly high. This variable pattern did not change after hyperimmunization and was demonstrable in a range of mouse strains of different haplotype. The same results were obtained whether the response was measured by virus neutralization or by RIA against the FMDV peptide sequence. The possible reasons for the variable recognition of the FMDV epitopes by individual mice are discussed. PMID- 3005400 TI - Chronic infection due to Mycobacterium intracellulare in mice: association with macrophage release of prostaglandin E2 and reversal by injection of indomethacin, muramyl dipeptide, or interferon-gamma. AB - As a model for the study of human atypical mycobacterial disease, we explored the basis for the prolonged mycobacteriosis in mice infected with Mycobacterium intracellulare. Two weeks after i.v. injection of mycobacteria, peritoneal macrophages were found to be activated, as indicated by their capacity to produce large amounts of superoxide anion (O2-) in response to phorbol myristate acetate (PMA) or viable M. intracellulare. However, 4 wk after infection, despite the continued presence of large numbers of mycobacteria in the spleen, macrophages from infected animals produced low amounts of O2-. Unfractionated spleen cells from mice infected 4 wk earlier produced increased amounts of interleukin 2 and interferon (IFN) when stimulated with the mitogen concanavalin A, but less of these lymphokines than unstimulated cells when exposed to antigens derived from M. intracellulare, suggesting production of an inhibitory factor. Spleen cells from infected mice were not stimulated to incorporate [3H]thymidine by exposure to mycobacterial antigens; but this unresponsiveness could be reversed by addition of indomethacin to the cultures. Additional investigation showed that macrophages from infected animals produced large amounts of prostaglandin E2 (PGE2) when stimulated by mycobacterial antigens. In vitro, such concentrations of PGE2 inhibited uptake of [3H]thymidine by stimulated spleen lymphocytes from infected animals. Thus, it seemed likely that in infected animals, macrophage derived PG suppressed production of IFN-gamma by lymphocytes, which in turn prevented activation of the macrophages to full microbicidal activity. To test this hypothesis, we administered either indomethacin, IFN-gamma, or muramyl dipeptide to infected mice. Mice treated with each of these agents showed decreased spleen and lung weights, and decreased numbers of viable mycobacteria in these organs. These results support the concept that interaction between the host and M. intracellulare is modulated profoundly by PG and suggest that administration of agents that directly promote macrophage activation can enhance resistance to infection by this organism. PMID- 3005403 TI - Rabbit major histocompatibility complex. I. Isolation and characterization of three subregions of class II genes. AB - Approximately 300 kb of DNA from the rabbit major histocompatibility complex class II region has been cloned from two genomic libraries. Four alpha chain genes and ten beta chain genes were identified in the recombinant phage and cosmid clones by hybridization with human class II cDNA probes. These genes were classified into three subregions (R-DP, R-DQ, and R-DR) based on hybridization analyses with human DP, DQ, and DR subregion genes under stringent conditions. Two alpha genes and one beta gene were assigned to the R-DP subregion, one alpha gene and one beta gene to the R-DQ subregion, and one alpha gene and eight beta genes to the R-DR subregion. In each subregion, the alpha gene and at least one beta gene were closely linked and were oriented in a manner similar to those in the homologous subregions of human and mouse. A combination of cloning data and genomic blot analyses indicated that the rabbit genome contains a minimum of four alpha-chain genes. The results suggested that there has been evolutionary conservation of the subunit organization of the alpha- and beta-chain genes as well as the coding sequences of these genes and that the mammalian ancestor possessed three distinct MHC class II subregions before diversification of human, mouse, and rabbit. PMID- 3005402 TI - Variations in the susceptibility to Coxsackievirus B3-induced myocarditis among different strains of mice. AB - This study was undertaken to examine the inherent predisposition of different inbred strains of mice to develop Coxsackievirus B3-induced myocarditis. A time course study established the pertinent, differential parameters of the disease and their corresponding genetic control. The A.BY/SnJ (H-2b), A.SW/SnJ (H-2s), A.CA/SnJ (H-2f), B10.S/SgSf (H-2s), B10.PL/SgSf (H-2u), and C3H.NB/SnJ (H-2p) strains were found to vary widely in the extent and duration of viremia, in the temporal appearance and titer of neutralizing antibody, and in the prevalence, severity, and duration of myocardial disease. The A.BY/SnJ (H-2b), A.SW/SnJ (H 2s), A.CA/SnJ (H-2f), and C3H.NB/SnJ (H-2p) mice developed continuing, chronic myocardial disease, whereas B10.S/SgSf (H-2s) and B10.PL/SgSf (H-2u) did not. The four strains that displayed prolonged myocarditis also produced heart-specific myocardial autoantibodies. Heart-specific autoantibodies were not found in the B10.S/SgSf and B10.PL/SgSf animals. Differences in prevalence and titer of these heart-specific autoantibodies were noted among the three A strain H-2 congenic lines. The influence of the major histocompatibility complex (MHC) on disease production was demonstrated by comparison of the three A strain and two B10 strain H-2 congenics. Differences between A.SW/SnJ (H-2s) and B10.S/SgSf (H-2s) suggested non-MHC control of disease. These studies additionally indicate that the genetic regulation of susceptibility to CB3 infection and the direct virus induced inflammation differ from the later immunopathic myocarditis. PMID- 3005405 TI - The role of the immune response in TMEV infection and the development of late onset demyelination. AB - The nature of the role of the immune response to TMEV in the development of LODD after TMEV infection was examined by different means. Immunosuppression by both cyclophosphamide (postinfection) or adult thymectomy and ATS treatment (preinfection) did not inhibit clinical LODD. Adoptive transfer of spleen cells from infected donors with LODD facilitates LODD in the recipients compared with i.v. transfer of the freeze-thaw supernatants from such suspensions (although virus is present in splenocyte suspensions). The immune response to TMEV, both lymphoproliferative and antibody, reaches a peak coincidental with the development of LODD. The role of the immune response is equivocal depending on the stage of the viral infection, the timing of immunosuppressive treatment, and assessment. PMID- 3005404 TI - High efficiency gene transfer into murine T cell clones using a retroviral vector. AB - To establish a gene transfer and expression system for murine T cell clones, we have introduced the neomycin phosphotransferase gene encoding resistance to the neomycin analogue, G418, into non-neoplastic inducer T cell clones by using a replication-defective retroviral vector. This method allowed highly efficient gene transfer (20 to 40%) into two inducer T cell clones. The level of viral RNA expression in G418r T cells was 0.1% of poly(A)+ RNA. The infected G418r cells retained physiologic responsiveness to specific antigen as judged by antigen specific proliferation and production of IL 3. PMID- 3005406 TI - Interferon-gamma-induced IA expression in WEHI-3 cells is enhanced by the presence of 1,25-dihydroxyvitamin D3. AB - It has been suggested recently that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is involved in the regulation of the immune functions of lymphocytes and in the differentiation of monocytic cells. This report examined the possibility that 1,25(OH)2D3 influences immune functions mediated by monocytic cells by studying its effect on the murine myelomonocytic line WEHI-3. We found that WEHI-3 cells possess 3.3S receptor proteins with high affinity (Kd = 3.3 X 10(-10) M) for 1,25(OH)2D3 that are capable of binding to DNA. Also we found that 1,25(OH)2D3 enhances the interferon-gamma (IFN-gamma)-induced expression of the class II major histocompatibility complex antigens (Ia molecules), and such enhancement leads to increased capacity of the WEHI-3 cells to stimulate antigen-specific Ia restricted T cell activation. Finally, 1,25(OH)2D3 inhibits the proliferation of WEHI-3 cells, and this inhibition is enhanced in the presence of IFN-gamma. The 1,25(OH)2D3 modulation of IFN-gamma induction of Ia antigens suggests that the hormone might promote monocytes to function more efficiently as antigen presenting cells. PMID- 3005407 TI - The specificity of H-2-restricted cytotoxic T lymphocytes directed to AKR/Gross leukemia virus-induced tumors. III. Coordinate alterations in viral gp70 antigen expression and restoration of CTL-susceptibility to insusceptible variant tumors. AB - Two variant subclones, called cl.18-5 and cl.18-12, were derived from the AKR.H 2bSL1 tumor cell line that were, in contrast to the parental cells, selectively insusceptible to H-2-restricted anti-AKR/Gross virus cytotoxic T lymphocytes (CTL). Cell surface expression of viral envelope (env) and group-specific antigens (gag) on these CTL-resistant variants were analyzed and compared with the expression of these antigens on AKR.H-2bSL1 and two other CTL-susceptible clones, cl.1 and cl.5, also derived from AKR.H-2bSL1. Although normal levels of gag-encoded and H-2 antigens were displayed on the CTL-resistant variants, the expression of five distinct determinants of viral gp70 antigen as defined by monoclonal antibodies was significantly decreased on these CTL-resistant variants relative to their expression on the CTL-susceptible cell lines. However, similar dramatic changes in cell surface gp70 antigen expression were undetectable as defined by anti-gp70-specific antiserum. Immunoprecipitation and gel electrophoretic analysis revealed that gp70 molecules from cl.18-5 cells had a lower m.w. than those of AKR.H-2bSL1, but there were no differences in the m.w. of gp70 antigens from AKR.H-2bSL1, cl.5, and cl.18-12 cells. Expression of the five gp70 antigenic determinants mentioned above was completely restored by exposure of cl.18-5 and cl.18-12 cells to the halogenated pyrimidine, iododeoxyuridine (IudR). Treatment of cl.18-5 and cl.18-12 cells with IudR simultaneously restored CTL susceptibility of these cells to anti-AKR/Gross virus CTL without affecting gag and H-2 antigen expression. Viral gp70 antigen immunoprecipitated from IudR-treated cl.18-5 cells had a mobility slightly lower, but different from that of untreated cl.18-5 cells. Pulse-labeling with [35S] methionine showed that IudR treatment of cl.18-5 cells caused the expression of an additional high m.w. gp70 precursor protein originally absent in untreated cl.18-5 cells but present on parental AKR.H-2bSL1 cells. Collectively, these results pointed to the involvement of viral gp70 antigenic determinants in the recognition of AKR/Gross virus-induced tumor targets by anti-AKR/Gross virus CTL. PMID- 3005408 TI - Translocation of protein kinase C during membrane immunoglobulin-mediated transmembrane signaling in B lymphocytes. AB - Previous studies have implicated a role for protein kinase C (PKC) in transmembrane signal transduction by B cell surface immunoglobulin (Ig). Specifically, the pharmacologic PKC activator phorbol myristate acetate mimics the biologic effects of mIg cross-linking ligands, and cross-linking of membrane Ig (mIg) induces polyphosphoinositide hydrolysis generating diacylglycerol, a potent activator of PKC. Studies described here additionally implicate PKC in mIg mediated signaling by demonstrating rapid translocation of activatable PKC (PKCa) from cytosol to Triton-soluble membrane fractions after cross-linking of cell surface IgM or IgD. This response, which is also induced by phorbol myristate acetate and lipolysaccharide, is detectable within 1 min of mIg cross-linking and is followed within 4 min by additional translocation of PKCa to a Triton insoluble particulate compartment. The ability of dbcAMP plus theophylline to inhibit polyphosphoinositide hydrolysis, PKCa translocation, and the B cell's subsequent biological response suggests that these events may be causally related. PMID- 3005409 TI - Desferoxamine blocks IL 2 receptor expression on human T lymphocytes. AB - Thymidine uptake by PHA-stimulated human lymphocytes is reduced in the presence of 100 microM or greater concentrations of the iron-chelating agent desferoxamine (DF). We assessed expression of IL 2 receptor, 4F2 and Ia antigens, IL 2 production, and cell cycle progression by blood mononuclear cells (MNC) stimulated by PHA in the presence or absence of DF to determine whether the lack of T cell proliferation was a manifestation of inhibition of an earlier activation event. Tac antigen expression on PHA-stimulated MNC was inhibited by DF throughout 8 days of culture, and those cells which were positive had a low density of Tac antigen as compared with controls without DF. Expression of other activation antigens, 4F2 and Ia, was not impaired by DF. The supernatants of the DF-containing and control cultures contained equivalent IL 2 activity, as measured on the HT-2 cell line. Cell cycle analysis of these cultures shows that the addition of DF at the beginning of culture blocks most cells from undergoing G0 to G1 transition, whereas later addition of DF arrests the progression of the T cell blasts through the cell cycle. Separation of cells cultured with PHA and DF into Tac+ and Tac- subsets showed that progression from G0 to G1 was restricted to the former subset. These results suggest that interference with IL 2 receptor expression might contribute to the block in mitogen-induced proliferation caused by DF. PMID- 3005410 TI - Interferon-gamma enhances expression of cellular receptors for tumor necrosis factor. AB - Incubation of several human tumor cell lines with human interferon-gamma (IFN gamma) increased the specific binding of subsequently added 125I-labeled recombinant human tumor necrosis factor (TNF). A similar increase in TNF binding was seen in murine L929 cells after incubation with murine IFN-gamma, but not after incubation with human IFN-gamma. Increased TNF binding to cells incubated with IFN-gamma was due to an increase in the number of TNF receptors, with no demonstrable change in binding affinity. In one out of two human cell lines tested, IFN-alpha and IFN-beta also produced increased TNF binding, albeit with a lower efficacy than IFN-gamma. A maximal increase in TNF binding was seen after about 6 to 12 hr of incubation with IFN. Increased TNF binding due to enhanced TNF receptor expression may contribute to the enhancement of TNF cytotoxicity seen in some tumor cell lines after INF treatment. Modulation of TNF receptor expression by IFN may also influence other biological activities of TNF. PMID- 3005411 TI - Induction of the synthesis of tumor necrosis factor receptors by interferon gamma. AB - Human HT-29 colon carcinoma and HeLa D98/AH2 and SK-MEL-109 melanoma cells were sensitive to synergistic growth inhibition by concentrations of recombinant human tumor necrosis factor (rTNF) and interferon-gamma (rIFN-gamma) which individually were only slightly inhibitory. We investigated whether this synergism could be explained by the presence of an increased number of TNF receptors in cells treated with rIFN-gamma. These receptors were measured by incubating cells resuspended from monolayers with 125I-rTNF. HT-29 cells treated for a few hours with rIFN-gamma could bind more 125I-rTNF than control untreated cells, but this binding returned to the level of control cells after 24 hr. The treatment with rIFN-gamma did not change the binding affinity of TNF receptors, but increased their number to 1800 per cell from a basal level of about 800 per cell. Inhibitors of RNA synthesis prevented this increase. HT-29 cells were significantly more growth-inhibited when treated first for 6 to 12 hr with rIFN gamma and then with rTNF, than when treated first with rTNF and then with rIFN gamma. Untreated HeLa D98/AH2 and SK-MEL-109 cells had 2400 and 9000 receptors per cell, with a KD similar to that of HT-29 cells (approximately 2 X 10(-10)M). A significant increase in TNF receptors after treatment with rIFN-gamma was observed in HeLa D98/AH2, but not in SK-MEL-109 cells. No increase in TNF receptors was detected in cells treated with rIFN-alpha 2. These results indicate that the synergism between rTNF and rIFN-gamma may be due, at least in part, to a transient induction of the synthesis of TNF receptors by rIFN-gamma in cells with a relatively low number of these receptors. PMID- 3005412 TI - Interleukin 2 (IL 2) up-regulates its own receptor on a subset of human unprimed peripheral blood lymphocytes and triggers their proliferation. AB - Several reports indicate that human peripheral blood lymphocytes (PBL) seeded in culture with purified or recombinant interleukin 2 (IL 2) immediately after separation from the blood display a substantial level of proliferation at day 5 or 6, even in the absence of any activating signal. The spontaneously IL 2 proliferating cells are large lymphocytes, and they co-purify on a Percoll gradient in the large granular lymphocytes (third (LGL) fraction) together with the natural killer (NK) activity. When LGL were separated into NKH1 (an NK specific surface marker)-positive and NKH1-negative cells by fluorescence activated cell sorting (FACS), proliferating cells were mainly found in the NKH1 negative fraction. On the contrary, when cells from Percoll fraction 3 were separated into OKT3-negative and positive cells, the majority of the proliferating cell was found in the OKT3-positive cells. These results indicate that spontaneously IL 2 proliferating (SIP) cells most probably belong to the T cell lineage, but are distinct from NK cells. Surprisingly, cells from this Percoll fraction examined immediately after separation from the blood do not express detectable amounts of IL 2 receptors as assessed by three different techniques: binding of [3H]IL 2, binding of [125I]anti-Tac antibodies, and FACS analysis with the use of anti-Tac antibodies. However, after 18 hr of culture in IL 2-supplemented medium, 5 to 7% of these cells became Tac-positive by FACS analysis. Additional analysis of IL 2 receptor induced in culture with IL 2 was performed by [125I]anti-TAC binding and by [3H]IL 2 binding. Scatchard analysis of [3H]IL 2 binding, in the range of concentrations leading to the detection of high-affinity binding sites, showed an affinity constant similar to that of conventional phytohemagglutinin blasts. The role of IL 2/IL 2 receptor interaction in the proliferation process was confirmed by the fact that proliferation, in contrast with NK activation, was clearly inhibited by anti-Tac antibodies. When LGL activated with IL 2 for 60 hr were sorted into Tac+ and Tac- cells, equal levels of NK activity was found in the two fractions. Proliferation, however, was only observed in the Tac+ population. We interpret these results to indicate that SIP cells are preactivated cells circulating in the blood. They are large cells and represent a very small proportion of circulating lymphocytes (0.3%). They express a subliminal amount of IL 2 receptor. Cultivated in the presence of IL 2, IL 2 receptor expression is enhanced to a detectable level, and the SIP cells begin to proliferate. These SIP cells could be activated T cells present in every normal individual. PMID- 3005413 TI - Interleukin 2-dependent phosphorylation of interleukin 2 receptors and other T cell membrane proteins. AB - The addition of IL 2 to Con A-activated splenic T cells induced the rapid and time-dependent phosphorylation of membrane proteins with m.w. of 115,000 to 105,000, 90,000, and 66,000, and to a lesser extent 55,000 to 58,000, 40,000, and 34,000. Immunoprecipitations conducted with an anti-IL 2 receptor antibody indicated that the murine IL 2 receptor (55,000 to 58,000) was included in the set of IL 2-dependent phosphoproteins. Phosphorylation of these same proteins was also seen after IL 2 treatment of PHA-activated T cells and of the IL 2-dependent line CTLL-2. Membrane phosphorylation was dependent on physiologically relevant IL 2 concentrations (0.2 to 1 ng/ml), and was detected as early as 1 min after IL 2 addition, with maximal levels of phosphorylation achieved by 15 min. In contrast to these observations, the pattern of cytoplasmic protein phosphorylation remained unchanged after IL 2 addition, although IL 2 did augment the level of preexisting cytoplasmic phosphorylation induced by lectin. The pattern of membrane protein phosphorylation induced by IL 2 also overlapped in part with that induced after stimulation of Con A-activated T cells with the phorbol ester PMA. IL 2-stimulated phosphorylation was inhibited by the addition of agents that both stimulate cyclic AMP-dependent protein kinases and block lymphocyte mitogenesis. No effect was seen upon addition of agents that enhance cyclic GMP-dependent protein kinases. These observations support a role for specific membrane as opposed to cytoplasmic protein phosphorylation in the regulation of lymphocyte growth by IL 2, and also suggest that protein kinase A, and perhaps protein kinase C, participate as regulators of the IL 2 signaling mechanism. PMID- 3005415 TI - Deactivation of the respiratory burst in activated macrophages: evidence for alteration of signal transduction. AB - Enhanced function of the respiratory burst, measured as stimulated release of superoxide anion (O2-) or hydrogen peroxide, characterizes activated macrophages. Activated macrophages undergo a decline in their capacity to release O2- (a deactivation) when placed in culture for 3 days. To better understand the molecular basis for the enhanced respiratory burst of activated macrophages, we explored the mechanisms underlying deactivation of activated mouse peritoneal macrophages. Deactivation was observed when the assay was performed in a physiologic Na+ buffer, and by day 3 of culture, release of O2- from activated macrophages stimulated with phorbol myristate acetate (PMA) was almost identical to that in resident (nonactivated) macrophages. In contrast, when the assay was performed in a buffer in which Na+ was replaced by K+, release of O2- from activated macrophages on day 3 was equal to or greater than that on day 0, suggesting that the enzyme responsible for the respiratory burst was not altered during culture. The number and affinity of PMA receptors were not changed during culture and were not affected by high external K+. Continuous assay of O2- release by coverslip-adherent macrophages in a cuvette indicated that the lag time between addition of stimulus and release of O2- was reduced, and the initial rate of O2- release was enhanced in K+ buffer. The potency of monovalent cations to support O2- release was K+ greater than Rb+ greater than choline+ greater than Cs+ = Na+ greater than Li+, suggesting that characteristics such as ionic radius or molecular size influence this effect, and the effect is not due simply to absence of Na+. Extracellular Ca2+ or Mg2+ was required for the maximal effect of high external K+, and enhancement by high K+ and divalent cations increased progressively during culture. These findings suggest that deactivation is caused primarily by changes in signal transduction from PMA receptors to the respiratory burst enzyme, rather than by changes in these receptors or the enzyme itself, and that signal transduction can differ in different macrophage populations. PMID- 3005416 TI - Reversal of virus-induced immune suppression. AB - We studied the immunosuppressive capacity of splenic lymphocytes from rabbits at different stages of progressive myxosarcoma induced by malignant rabbit fibroma virus (MV). Spleen cells taken from rabbits 7 days after virus inoculation proliferate poorly in response to Con A, and suppress normal responses to the mitogen. Those from animals 11 days after virus injection have recovered partially from MV-induced suppression. Further, their Con A responses are no longer suppressed by day 7 spleen cells. Supernatants from cultures of spleen cells from rabbits given MV 7 days previously suppress both antibody-producing and proliferative responses to unrelated antigens. Comparable supernatants from rabbits receiving MV 11 days before sacrifice neither suppress nor augment such responses. Mixing cells from 7 or 11 day MV rabbits with normal spleen cells gives similar results. When supernatants from spleen cell of rabbits with tumors induced 7 and 11 days previously are mixed, the supernatants from rabbits with 11 day-old tumors inhibit the suppressive capacity of those from animals with 7-day old tumors. Similarly, mixing spleen cells from rabbits given MV 7 and 11 days previously results in culture supernatants that do not suppress normal antibody and proliferative responses. The ability of cells from rabbits given MV 11 days before to inhibit the effects of cells from rabbits given MV 7 days previously does not involve the production of interferon. Thus, despite progressive tumor burden, immunologic recovery is observed in rabbits 11 days after tumor virus inoculation. One factor in this recovery may be the generation of active inhibitors of virus-induced immunosuppression. Similar mechanisms may apply to recovery of immunologic function in other virus infections as well. PMID- 3005414 TI - Enhancement of plasma levels of biologically active leukotriene B compounds during anaphylaxis in guinea pigs pretreated by indomethacin or by a fish oil enriched diet. AB - The changes in arterial plasma concentrations of immunoreactive leukotriene B (LTB) were compared after antigen challenge of two groups of sensitized, mepyramine-treated, and mechanically ventilated guinea pigs, one fed a diet enriched with fish oil and the other a control diet enriched with beef tallow. The lung tissue of animals fed a fish oil-enriched diet (FFD) for 9 to 10 wk incorporated eicosapentaenoic acid (EPA) and docosahexaenoic acid to constitute 8 to 9% of total fatty acid content, whereas these alternative fatty acids constituted less than 1% of the total fatty acid content of the lung tissue of animals on a beef tallow-supplemented diet (BFD). The maximum increase after antigen challenge in immunoreactive LTB4 from 0.16 +/- 0.04 ng/ml to 0.84 +/- 0.25 ng/ml in BFD animals and from 0.47 +/- 0.11 to 5.1 +/- 1.4 ng/ml immunoreactive LTB (LTB4 and LTB5) in FFD animals was significant (p less than 0.02) for each. Furthermore, the increase in total immunoreactive LTB in mepyramine-treated FFD animals was significantly greater than the increase in LTB4 in mepyramine-treated BFD guinea pigs at 2 to 8 min after antigen challenge (p less than 0.05). Resolution of arterial plasma immunoreactive LTB from pooled samples by reverse-phase high-performance liquid chromatography demonstrated that the sum of LTB4 and LTB5 in FFD animals exceeded that of LTB4 in BFD animals and that the quantity of LTB4 in the FFD animals was at least as great as that in the BFD animals during anaphylaxis. The products eluting at the retention times of LTB4 and LTB5 exhibited the chemotactic activity of their respective synthetic standards. The combination of indomethacin and mepyramine markedly augmented the antigen-induced increase in arterial plasma immunoreactive LTB4 concentrations in BFD animals, but had no effect on immunoreactive LTB levels in FFD animals. Limited in vivo measurements showing a lesser increase of plasma immunoreactive thromboxane B2 in the FFD relative to the BFD animals during anaphylaxis and ex vivo measurements showing a decreased LTB4-stimulated (cyclooxygenase product dependent) contractile response of pulmonary parenchymal strips from the FFD relative to the BFD animals provide evidence for blockade in the cyclooxygenase pathway in the FFD animals. The measurements of arterial plasma LTB indicate that indomethacin treatment alone, which inhibits cyclooxygenase activity, and FFD treatment each augment the metabolism of arachidonic acid by the 5-lipoxygenase pathway in animals pretreated with mepyramine.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3005418 TI - ELISA detection of human IgG subclass antibodies to Streptococcus mutans. AB - A sensitive enzyme-linked immunosorbent assay (ELISA) has been developed to measure IgG subclass antibodies against whole cells of Streptococcus mutans and to a purified streptococcal antigen (SA I/II). Bacterial cells were bound to the solid phase using methyl glyoxal and mouse monoclonal antisera against IgG and each IgG subclass were used to detect antibodies. Natural antibodies to S. mutans were predominantly of the IgG1 and IgG2 subclasses, though IgG3 and IgG4 antibodies were detectable in most subjects, and were the majority response in a few subjects. Antibodies to SA I/II were predominantly of the IgG1 subclass with virtually no activity detectable in the IgG3 and IgG4 subclasses. Inhibition studies suggested some restriction of IgG subclass responses to bacterial antigens since SA I/II and c polysaccharide could inhibit binding of all subclasses to whole cells of S. mutans equally, whereas glucosyltransferase, lipoteichoic acid and dextran showed greatest inhibition of the IgG3 and IgG4 subclasses. PMID- 3005417 TI - Helper T cells against tumor-associated antigens (TAA): preferential induction of helper T cell activities involved in anti-TAA cytotoxic T lymphocyte and antibody responses. AB - This study establishes assay systems for helper T cell activities assisting cytotoxic T lymphocyte (CTL) and antibody responses to tumor-associated antigens (TAA) and demonstrates the existence of TAA that induce preferentially anti-TAA CTL helper and B cell helper T cell activities in two syngeneic tumor models. C3H/HeN mice were immunized to the syngeneic X5563 plasmacytoma or MH134 hepatoma. Spleen cells from these mice were tested for anti-TAA helper T cell activity capable of augmenting anti-trinitrophenyl(TNP) CTL and anti-TNP antibody responses from anti-TNP CTL and B cell precursors (responding cells) by stimulation with TNP-modified X5563 or MH134 tumor cells. The results demonstrate that cultures of responding cells plus 85OR X-irradiated tumor-immunized spleen cells (helper cells) failed to enhance anti-TNP CTL or antibody responses when in vitro stimulation was provided by either unmodified tumor cells or TNP-modified syngeneic spleen cells (TNP-self). In contrast, these cultures resulted in appreciable augmentation of anti-TNP CTL or antibody response when stimulated by TNP-modified tumor cells. Such anti-TAA helper activities were revealed to be Lyt 1+2- T cell mediated and TAA specific. Most interestingly, immunization with X5563 tumor cells resulted in anti-TAA helper T cell activity involved in CTL, but not in antibody responses. Conversely, TAA of MH134 tumor cells induced selective generation of anti-TAA helper T cell activity responsible for antibody response. These results indicate that there exists the qualitative TAA heterogeneity as evidenced by the preferential induction of anti-TAA CTL- and B cell-helper T cell activities. The results are discussed in the light of cellular mechanisms underlying the preferential anti-TAA immune responses, and the interrelationship between various types of cell functions including CTL- and B cell-help. PMID- 3005419 TI - A radioimmunoassay that sandwiches human interleukin-2 between radiolabeled monoclonal antibody and the receptor on a hematopoietic cell line. AB - Two monoclonal antibodies were raised against human interleukin-2 (IL-2) produced by E. coli harboring recombinant complemental DNA. Both antibodies did not neutralize its activity, nor did they inhibit the binding of IL-2 to the receptor on target cells. Taking advantage of the ability of monoclonal antibodies to detect IL-2 that had bound to the receptor, a radioimmunoassay was developed that sandwiched IL-2 between the radiolabeled monoclonal antibody and the receptor on a hematopoietic cell line infected with human T cell leukemia virus Type I. The assay had the advantage of detecting only IL-2 with the ability to bind to the receptor, and displayed a linear dose-response relationship over concentrations ranging from 5 to 100 ng/ml. PMID- 3005420 TI - A simple method for the production of specific antiserum to the second nuclear antigen (EBNA-2) of Epstein-Barr virus (EBV). AB - A simple technique for raising specific antiserum to the native molecule of Epstein-Barr virus (EBV)-encoded nuclear antigen-2 (EBNA-2) from Raji cells is described. The procedure involves the use of immunoblotting to identify the EBNA 2 polypeptide followed by subsequent excision from an SDS-gel and immunization of experimental animals. The anti-EBNA-2 antiserum recognized a single polypeptide of 86-87 kDa on immunoblots prepared from extracts of EBV-positive Raji or B95-8 cells, while it did not react with any proteins form P3HR-1 or Daudi cells, which carry EBNA-2-defective virus. The method might be applicable to other systems where isolation of purified proteins for immunization is either difficult or unfeasible. PMID- 3005421 TI - Field, ward, and laboratory: where the infectious disease physician worked. PMID- 3005422 TI - Applied molecular genetics: new tools for microbiologists and clinicians. PMID- 3005423 TI - Hepatitis B virus DNA in mononuclear cells and analysis of cell subsets for the presence of replicative intermediates of viral DNA. AB - To determine whether peripheral blood mononuclear cells (PBMCs) contain replicating forms of hepatitis B virus (HBV) DNA and to define which cell subset may be permissive for viral replication, we analyzed the PBMC DNA from 14 carriers positive for hepatitis B surface antigen (HBsAg) by Southern blot hybridization. HBV-related DNA, which was present exclusively in an extrachromosomal state, was found in the PBMCs of all five hepatitis B e antigen (HBeAg)-positive and three of nine HBeAg-negative carriers. Serum-associated HBV DNA was detected only in those HBsAg carriers whose PBMCs contained HBV DNA forms resembling replicative intermediates (1.0-3.2 kilobase pairs in the EcoRI digests). Analysis of PBMC subsets revealed that replicating forms of the HBV genome were present primarily in monocytes. Low levels of hybridization also were detected in B cells, whereas the T cell fraction (which contained natural killer cells) appeared to be devoid of these replicating forms. PMID- 3005424 TI - Risk factors for cytomegalovirus infection after human marrow transplantation. AB - The records of 545 patients were reviewed for risk factors associated with cytomegalovirus (CMV) infection after marrow transplant. CMV infection occurred among 36% of seronegative patients and 69% of seropositive patients. Among seronegative patients, significant risk factors for CMV infection included positive serology of the marrow donor (relative rate, 2.3) and the use of granulocyte transfusions from seropositive donors (relative rate, 2.5). Among both seronegative and seropositive patients, the occurrence of acute graft-versus host disease (GVHD) significantly increased the risk of CMV infection (average relative rate, 1.8) and of subsequent CMV pneumonia (average relative rate, 2.6). CMV excretion and viremia were each associated with subsequent pneumonia, but the positive predictive values were low. One-third of long-term survivors excreted CMV at greater than 250 days after transplantation. The only risk factor for late excretion was CMV infection that occurred in the first 150 days after transplantation. In contrast to the effect of acute GVHD on CMV infection, CMV infection did not increase the risk of either acute or chronic GVHD. PMID- 3005425 TI - Protective effect of interferon on infections with hand, foot, and mouth disease virus in newborn mice. AB - The protective effect of interferon on infection with coxsackievirus type A 16 (CA-16) or enterovirus type 71 (EV-71) in newborn mice was examined. Subcutaneous administration of murine interferon (MuIFN-alpha/beta) into the infected mice produced a protective effect against infection with CA-16 or EV-71. It was found that the time of administration of MuIFN was important in relation to the cycle of infection. Protection was observed when MuIFN was given once daily for several days, from one day before or after infection with the lethal dose of CA-16 or EV 71. These results suggest that interferon may directly suppress infection with CA 16 and not indirectly suppress it by the medium of macrophages, natural killer cells, and T cells. PMID- 3005426 TI - Effects of cyclosporine and cortisone on the pathogenesis of primary infection with cytomegalovirus in the guinea pig. AB - Effects of the immunosuppressive agents cyclosporine (CsA) and cortisone on the pathogenesis of primary infections with cytomegalovirus (CMV) were investigated in the guinea pig model. All animals received 10(4) 50% tissue culture infectious doses of virulent salivary gland-passaged guinea pig CMV (GPCMV) subcutaneously on day 0. Oral CsA (20 mg/kg per day) and/or subcutaneous cortisone (10 mg/kg per day) were administered until the animals were killed on day 14. Untreated controls developed lymphocytosis, and GPCMV was isolated from 19.4% of cocultivated tissues. Animals treated with CsA alone developed lymphopenia, and GPCMV was isolated from 53% of their tissues, including 16 of 16 lungs. Histopathology showed widespread viral inclusions and minimal inflammatory response to GPCMV in CsA-treated animals. Guinea pigs treated with either cortisone or CsA/cortisone did not develop lymphopenia, and their rates of isolation of GPCMV were significantly lower than those of CsA-treated animals. PMID- 3005427 TI - Infection with two genotypes of Epstein-Barr virus in an infant with AIDS and lymphoma of the central nervous system. PMID- 3005428 TI - Varicella-Zoster virus does not become more resistant to acyclovir during therapy. PMID- 3005429 TI - Effect of intravenous recombinant gamma-interferon on the respiratory burst of blood monocytes from patients with AIDS. PMID- 3005431 TI - Transformation of a plasmid encoding an adhesin of Staphylococcus aureus into a nonadherent staphylococcal strain. AB - Plasmid DNA was isolated and purified from a strain of Staphylococcus aureus demonstrating adherence to human epithelial cells, as determined by an assay quantitating adherence of radiolabeled S. aureus to HeLa cells in tissue culture. Other phenotypic characteristics of the donor strain are resistance to ampicillin and absence of hemolysis. A 23.5-kb (kilobase) plasmid was transformed into a nonadherent, ampicillin-sensitive, beta-hemolytic recipient of S. aureus, rendering it both ampicillin-resistant and adherent to a degree approaching that of the donor strain. Plasmid analysis of the donor and transformant strains revealed three identical EcoRI fragments of 7.6 kb, 6.5 kb, and 2.2 kb, together with a 3.6-kb EcoRI fragment in the transformant that demonstrated homology with the last 7.2-kb fragment in the donor. We conclude that an adhesin of S. aureus is encoded by this 23.5-kb penicillinase-encoding plasmid and that techniques of molecular genetics may be utilized to clarify the mechanisms of adherence of S. aureus. PMID- 3005430 TI - A comparative analysis by restriction endonucleases of herpes simplex virus type 1 isolated in Japan and Kenya. PMID- 3005432 TI - Production of encephalitis restricted to the temporal lobes by experimental reactivation of herpes simplex virus. AB - A model was developed in which reactivation of latent infections with herpes simplex virus type 1 was induced in trigeminal ganglia and central nervous system olfactory centers of rabbits by administration of cyclophosphamide and dexamethasone. Latent infections were reactivated at a significantly higher frequency in rabbits infected with a highly virulent strain of virus than in those infected with a strain of lower neurovirulence. Electroencephalographic abnormalities that were largely confined to the posterior lateral cerebral hemispheres and corresponding inflammatory lesions were seen in rabbits acutely infected with the highly virulent strain, whereas no brain electrical abnormalities and only mild inflammatory lesions were seen in rabbits acutely infected with the strain of low neurovirulence. Reactivation of the highly neurovirulent strain produced focal brain necrosis restricted to the temporal lobes, which was similar to the disease produced in humans and which correlated with progressive worsening of brain electrical abnormalities. PMID- 3005434 TI - Serological characterization of HTLV-III infection in AIDS and related disorders. AB - Current efforts to test blood donors and other persons for exposure to the human T cell lymphotropic virus type III (HTLV-III), the etiologic agent of the acquired immunodeficiency syndrome (AIDS), are based on the measurement of serum antibodies to viral antigens. We studied presence of serum antibodies to HTLV-III related antigens from 767 individuals with AIDS or AIDS-related complex (ARC) or asymptomatic persons at risk for AIDS by using ELISA and immunoblot techniques. Of the 280 specimens from AIDS and ARC subjects that were tested, 99% were ELISA reactive and 96% were immunoblot reactive. Greater than 96% of the seropositive subjects manifested antibodies to the p24 core antigen, whereas only 88% had antibodies to the gp41 envelope-related glycoprotein. Contrary to previous reports, a short incubation time in the immunoblot assay failed to detect low titer or low-affinity antibodies that were detected by overnight incubation. There was no apparent difference in pattern of antibodies to HTLV-III-related antigens in symptomatic vs. asymptomatic seropositive individuals. PMID- 3005433 TI - A rabbit model of focal herpes simplex encephalitis. AB - Results from studies designed to create a model of focal encephalitis caused by herpes simplex virus (HSV) are reported. Anesthetized rabbits underwent exposure and inoculation of the olfactory bulb with three different doses of a wild-type HSV. Lethal infection resulted in 69% of the animals, without evidence for a dose response relationship. Necropsy specimens obtained on or before day 10 after inoculation routinely yielded HSV in culture. In 76% of the animals with positive cultures for virus, these cultures originated exclusively or primarily from the pyriform (or temporal) cortex and frontal lobes. Virus could not be cultured from animals killed more than two weeks after inoculation. Histological examination of brains obtained three or more days after inoculation demonstrated evidence of viral infection, with more severe involvement of temporal cortex than of the surrounding brain in 80%. Immunohistochemical demonstration of viral antigens persisted for up to three weeks after inoculation. PMID- 3005435 TI - Preparation of a prototype inactivated hepatitis A virus vaccine from infected cell cultures. AB - Studies were conducted on the preparation, inactivation, safety, and immunogenicity of a prototype hepatitis A virus vaccine prepared from infected cell cultures. BS-C-1 cells maintained in medium 199 without serum were infected with the HM175 strain of hepatitis A virus and harvested after 21-28 days. The harvested virus preparation contained 6.8-7.4 (log 10) cell culture infectious doses/ml. After exposure to 1:4,000 formalin at 35 C, the infectivity titer decreased 10(6)-fold in 30 hr at an exponential rate, although virus was detected in 5.0-ml vaccine samples for up to three days. Three separate vaccine lots elicited antibody in all the guinea pigs given three doses. Owl monkeys given three doses of vaccine did not have any evidence of HAV infection but developed antibodies identifiable by radioimmunoassay and serum neutralization tests. After either oral or intravenous challenge with at least 10(6) monkey infectious doses of a virulent field strain of hepatitis A virus, none of the vaccinated monkeys shed virus in their feces or had elevated serum levels of alanine aminotransferase. The findings suggest that an effective inactivated whole virus hepatitis A vaccine can be prepared from cell culture. PMID- 3005436 TI - Typing of herpes simplex virus with synthetic DNA probes. AB - Three single-stranded oligonucleotide probes, 22 bases long, homologous to unique regions of herpes simplex virus (HSV) types 1 (HSV-1) and 2 (HSV-2) and a region common to both were chemically synthesized with use of a modified phosphochloridite protocol. For hybridization experiments each probe was labeled with use of polynucleotide kinase and [gamma-32P] ATP to a specific activity of approximately 2 X 10(9) cpm/micrograms. Two hundred one clinical isolates of HSV (96 HSV-1 and 105 HSV-2) collected from vesicles in the mucocutaneous junction of the mouth or from the genital area were analyzed. There was a 99% (199 of 201) agreement between hybridization and monoclonal antibody typing; the two discrepant isolates of HSV-2 that were negative by monoclonal antibody typing were confirmed as HSV-2 by restriction endonuclease analysis. The probes detected between 10(4) and 10(5) HSV infectious units and from 150 to 600 HSV-infected Vero cells. No binding was detected between any of the three probes and isolates of cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus. PMID- 3005437 TI - Polypeptide-specific antibody response to human cytomegalovirus after infection in bone marrow transplant recipients. AB - Human cytomegalovirus (HCMV) infection continues to be the most important infection occurring after allogeneic bone marrow transplantation (BMT). Although T cell-specific antiviral immunity appears to be necessary for control of the infection, the humoral immune reaction also contributes to a complete immune response. In this paper we report our findings concerning the antibody response to the individual polypeptides of HCMV in patients who had undergone BMT and subsequently had displayed evidence of HCMV infection. Sera obtained from the subjects during and after HCMV viremia were studied by using both a radioimmunoprecipitation assay and an immunoblot assay, and the results were compared with the pattern of antibody response in normal individuals. The results show that the BMT patient can make antibodies to individual proteins of HCMV in a pattern similar to that displayed by persons with natural infection. The polypeptide-specific antibody response present most frequently was directed to proteins of 64 kDa, 50 kDa, and 36 kDa. Some BMT recipients also produced antibody to a wide range of HCMV proteins: 183 kDa, 155 kDa, 130 kDa, 110 kDa, 92 kDa, 86 kDa, 74 kDa, 71 kDa, 69 kDa, 39-44 kDa, 33 kDa, and 29 kDa. PMID- 3005438 TI - Viral envelope protein of HTLV-III is the major target antigen for antibodies in hemophiliac patients. PMID- 3005439 TI - Enhancement of human neonatal natural killer cytotoxicity to herpes simplex virus with use of recombinant human interferons: lack of neonatal response to gamma interferon. PMID- 3005441 TI - The gate control theory re-revisited. PMID- 3005440 TI - Frozen deglycerolized blood and transmission of Epstein-Barr virus. PMID- 3005443 TI - Plethysmography: history and recent advances. AB - A short historical development of pneumoplethysmography and its clinical usefulness has been given and the older instruments have been compared with a modern computerized pneumoplethysmograph. The computer aided plethysmogram, with proper programming, makes possible numerous computations which give new and useful information about the circulation. New applications of the CAP will quickly develop and the data collected will be only limited by the imagination. PMID- 3005442 TI - Spiromustine and intracarotid artery cisplatin in the treatment of glioblastoma multiforme. AB - Glioblastoma multiforme is a highly malignant and rapid-growing primary brain tumor. It constitutes one-fourth of all intracranial tumors and about half of all gliomas. Survival rate following conventional treatment is only 12 to 18 months. At the National Institutes of Health, two promising therapies are currently undergoing clinical trials. Spirohydantoin mustard (spiromustine) is a combination of a nitrogen mustard and a derivative of phenytoin, an anticonvulsant drug that rapidly penetrates the blood-brain barrier and localizes in brain tumors. Intracarotid administration of cis-diamminedichloroplatinum (cisplatin) increases drug delivery to the tumor and, through hemodialysis, systemic exposure is reduced. Nursing management of patients receiving these two agents requires precise planning and implementation of an individualized plan of care to ensure a successful chemotherapeutic regimen. PMID- 3005444 TI - Studies of catecholestrogen metabolism during normal pregnancy: changes of plasma catecholestrogen levels after DHA-S or E1-S injection. AB - So as to evaluate metabolism of catecholestrogens during pregnancy, changes in plasma unconjugated estradiol (E2), estrone (E1), 2-methoxyestradiol (2-mE2) and 2-methoxyestrone (2-mE1) concentrations were measured after a bolus injection of 40mg of estrone-sulfate (E1-S) or 100mg of dehydroepiandrosterone-sulfate (DHA-S) to normal pregnant women near term. The injection of E1-S resulted in a marked rise in plasma 2-mE1 levels followed by a rapid marked increase in the plasma E1 concentration. The injection of DHA-S produced a small increase in the plasma 2 mE2 levels in spite of a marked increase in the plasma E2 concentration. No significant correlation between E2 and 2-mE2 was found, however, significant correlations between E1 and 2-mE1, between E1 and 2-mE2 and between 2-mE2 in in both groups were demonstrated. The marked increase in 2-mE1 compared to a small increase in 2-mE2 after DHA-S injection indicates that E2 may participate in the formation of catecholestrogen via its conversion to E1 during pregnancy. These results suggest that the main route for the formation of catecholestrogen is via E2----E1----(2OHE1)----2-mE1 in normal pregnancy near term. PMID- 3005445 TI - [Clinical experiences with RF thermotherapy for locally advanced hepatocellular carcinoma]. PMID- 3005446 TI - [Blood level of 5-fluorouracil (5-FU) by oral administration of 1-hexylcarbamoyl 5-fluorouracil (HCFU) and tegafur in patients with hepatocellular carcinoma]. PMID- 3005447 TI - [Studies on treatment of peritonitis carcinomatosa in gastric cancer by intraperitoneal administration of 198Au colloid]. PMID- 3005448 TI - [Epidemic myalgia associated with frequent electrocardiographic abnormalities in a local prison]. PMID- 3005449 TI - [Adrenocorticotropic hormone (ACTH) and growth hormone (GH) deficiency with empty sella of normal size due probably to post-partum hemorrhage]. PMID- 3005450 TI - [A case of isolated ACTH deficiency with a myopathy of rare type and hyponatremia]. PMID- 3005451 TI - Changes in oxidative phosphorylation, adenylate energy charge, and respiratory components in chloramphenicol-treated regenerating rat liver. AB - To clarify the functional adaptability of mitochondria in the regenerating liver, the concentrations of respiratory components, hepatic energy charge levels, cytochrome oxidase activity, and phosphorylative activity were studied in mitochondria obtained from regenerating liver of rats treated with chloramphenicol (CAP). In the hepatectomized groups with CAP treatment, a dose dependent decrease occurred in the concentrations of cytochrome a(+ a3), cytochrome b, cytochrome c + c1, flavoprotein, and pyridine nucleotide. Cytochrome oxidase activity and phosphorylative activity per milligram of mitochondrial protein also decreased dose dependently in CAP-treated hepatectomized groups, with a significant increase in these values per unit of cytochrome a(+ a3). Hepatic energy charge levels significantly decreased in the hepatectomized groups. However, no significant differences were seen among the hepatectomized groups. However, no significant differences were seen among the hepatectomized groups with or without CAP treatment. Our results suggest that hepatic energy charge is maintained at the same relative level by a compensatory enhancement in phosphorylative capacity associated with cytochrome oxidase activity per unit of cytochrome a(+ a3), in spite of a remarkable decrease in the concentrations of respiratory components. PMID- 3005452 TI - Spontaneous hydrogen peroxide release from alveolar macrophages of some cigarette smokers. AB - Alveolar macrophages were retrieved by bronchoalveolar lavage (BAL) from 30 patients, 24 smokers and six nonsmokers. The macrophages were separated from other cells in the BAL fluid by glass adherence. The amount of hydrogen peroxide released into the media by these macrophages was then measured by a new method of determining hydrogen peroxide concentration. Two groups were found. Group 1, who did not spontaneously release hydrogen peroxide, were mostly nonsmokers (six of nine), and group 2, who spontaneously secreted hydrogen peroxide (87.5 +/- 17.08 nmol/10(6) macrophages [mean +/- SEM]), were all smokers (21 of 21). When the alveolar macrophages in group 1 were stimulated with phorbol myristate acetate, they secreted as much hydrogen peroxide as the stimulated macrophages of group 2 (group 1: 125.0 +/- 92.08 nmol/10(6) macrophages, group 2: 116.7 +/- 14.82 nmol/10(6) macrophages). We conclude that there is a subset of smokers whose alveolar macrophages spontaneously release hydrogen peroxide. PMID- 3005454 TI - Juvenile nasopharyngeal angiofibroma treated by radiotherapy. AB - A case of juvenile nasopharyngeal angiofibroma is reported and its response to radiotherapy demonstrated. The literature is reviewed and the use of radiotherapy for advanced tumours is advised. PMID- 3005453 TI - Von Willebrand factor-dependent agglutination of washed fixed human platelets by insoluble collagen isolated from bovine aorta. AB - Adult bovine aortic tissue was homogenized in a neutral phosphate buffer containing proteinase inhibitors. The insoluble residue was rehomogenized in Tris buffered 6 mol/L guanidinium chloride (pH 7.4). An insoluble fibrillar protein, floating above the main pellet after recentrifugation, was harvested. This material agglutinated washed fixed human platelets in the presence of either normal human plasma or purified von Willebrand factor (vWF). No such reaction was seen when either buffer or plasma from patients with severe von Willebrand's disease was added instead. The extent of platelet agglutination was measured photometrically, similarly to the ristocetin cofactor assay. The agglutination reaction was strongest at neutral pH and was impaired after either addition of EDTA or previous digestion of the fibrillar material by collagenase or pepsin. By light microscopy platelets were seen to adhere onto isolated fibers. Amino acid composition, subunit polypeptides, substrate properties, and interaction with fibronectin of this fibrillar protein were comparable to those of collagen. Therefore, we tentatively denote the induction of platelet agglutination by vWF protein in the described test system as "vWF-collagen cofactor" activity. Comparison of this activity in 65 plasma samples, containing various concentrations of vWF, with ristocetin cofactor activity showed good correlation between results obtained in both tests (r = 0.91). PMID- 3005455 TI - Fractionation of rat alveolar macrophages by isopycnic centrifugation: morphological, cytochemical, biochemical, and functional properties. AB - Studies on alveolar macrophages have usually been performed on a single cell suspension obtained by lung lavage. However, recent evidence on the diversity of functions of the alveolar macrophage suggests that the macrophage is not a single population, but one composed of several subpopulations of macrophages. One approach toward determining if alveolar macrophages are heterogeneous would be to separate subpopulations based on density. To accomplish this, we developed a continuous gradient of iso-osmotic colloidal silica (Percoll) that separated alveolar macrophages from Fischer 344 rats into 18 density-defined subpopulations. The density-defined alveolar macrophage subpopulations were then characterized and were shown to be significantly different based on morphological, cytochemical, biochemical, and functional analysis. The results of this study suggest that alveolar macrophages are heterogeneous and that a continuous iso-osmotic gradient of colloidal silica is an efficient and reproducible method for separating subpopulations. PMID- 3005456 TI - In vitro effects of platelet factor 4 on normal human neutrophil functions. AB - Platelet factor (PF4) prepared from human outdated platelets by heparinagarose affinity chromatography was confirmed to be chemotactic for human neutrophils and in a concentration-dependent fashion caused significant release of lysosomal enzymes (myeloperoxidase, lysozyme, beta-glucuronidase) from human neutrophils treated with cytochalasin B. Lysosomal enzyme release from PF4-stimulated neutrophils was rapid and reached a plateau by 1-3 min. PF4 did not cause release of the cytoplasmic enzyme lactate dehydrogenase which indicates that exocytosis of granule-containing lysosomal enzymes did not result from cytolysis. In contrast, superoxide anion generation from human neutrophils stimulated with PF4 was undetectable even at the highest PF4 concentration tested (2 X 10(-5) M). Pretreatment of neutrophils with PF4 caused significant increased adherence of neutrophils to plastic surfaces and cultured pulmonary artery endothelial cells. The concentration of PF4 that elicited neutrophil chemotaxis, lysosomal enzyme release and increased adherence is slightly higher than those concentrations found in normal human sera. However, the results suggest that PF4 may be an important mediator in neutrophil-platelet interactions and the induction of acute inflammation especially at sites of platelet microthrombi where the concentration of PF4 would be elevated. PMID- 3005458 TI - [Value of arteriography in the exploration of subungual glomus tumors. Apropos of a case]. AB - Difficulties in the diagnosis of subungual glomus tumors are increased when clinical examination and standard radiographic images are normal. In these cases arteriography is a useful procedure, opacification in the arterial phase of a small vascular lake confirming the diagnosis and localizing the lesion. A case is described that illustrates the interest of this examination, although it is indispensable only in patients with glomus tumors of atypical clinical expression or when symptoms recur after excision. PMID- 3005457 TI - Adenoid cystic carcinoma of the major and minor salivary glands. A clinicopathological study of 17 cases. AB - Local recurrence and distant metastasis were studied in relation to treatment modality and histological findings in 17 patients with adenoid cystic carcinoma of the major and minor salivary glands. Fifteen patients underwent surgical resection with or without postoperative irradiation and the others were treated by irradiation alone. Local recurrence developed in nine patients (52.9%), mostly within two years of the treatment. Incomplete removal was the major reason for the failure to control the primary lesion. Postoperative irradiation was of value in preventing recurrence in patients who demonstrated microscopic residual disease at the surgical margins, whereas it was not effective if gross residual tumour was recognized. Direct surgical intervention on the tumour tissue could have been the possible cause for the distant metastasis that developed in 10 (58.8%) of the 17 patients. Surgical excision with a wider safety margin than the visibly affected area, followed by postoperative irradiation and chemotherapy was essential to obtain a better prognosis. PMID- 3005459 TI - Modulation of thyrotrophin release from an intracellular pool by pre-exposure to thyrotrophin-releasing hormone and dibutyryl cyclic AMP. AB - Amplification of desensitization of TSH response to thyrotrophin-releasing hormone (TRH) may be important mechanisms in the regulation of its secretion. We have investigated this possibility in vitro, using monolayer culture of rat anterior pituitary cells. Cells (1-1.5 X 10(5)/250 microliters per well) were cultured for 72 h, exposed to TRH or dibutyryl cyclic AMP (dbcAMP) for 6 or 8 h, washed, and then treated for 4 h with various doses of TRH, or with K+ (55 mmol/l) as a non-specific secretagogue. Pretreatment with TRH (20 nmol/l) for 8 h reduced subsequent TSH release: basal release fell to 64% of the control value (1.01 +/- 0.10 micrograms/l pretreated, 1.58 +/- 0.16 control) and release in response to TRH (100 nmol/l) to 69% of the control (2.7 +/- 0.19 micrograms/l vs 3.98 +/- 0.22); K+ response was reduced to 86% of the control (3.77 +/- 0.21 micrograms/l vs 4.39 +/- 0.20), significantly less than the other reductions. The extent of the parallel downward shift of the TRH dose-response curve was proportional to dose and duration of prior TRH exposure. There was no significant change in the dose of TRH required to cause half-maximal TSH release (ED50: pretreated 4.8, control 2.8 nmol TRH/l) suggesting depletion of an intracellular pool of TSH rather than 'desensitization'. After 6-h pretreatment with dbcAMP, subsequent TSH responses were augmented: basal release was 130% of the control, response to TRH (100 nmol/l) was 137% and to K+ it was 132% of the control, with a parallel upward shift of the TRH dose-response curve but no change in cellular TSH content.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3005460 TI - Efficacy and specificity of human parathyroid hormone analogues as antagonists in intact clonal osteogenic sarcoma cells. AB - Five synthetic analogues of human parathyroid hormone (hPTH), (Tyr34)hPTH(3-34) amide, (5-34) amide, (7-34) amide, (8-34) amide and (9-34) amide, were tested for their ability to antagonize hPTH action specifically in intact cultured cells. Clonal rat osteogenic sarcoma cells were used (UMR 106-06 line) which respond to PTH with an increase in cyclic AMP (cAMP) formation. The most potent antagonists were (Tyr34)hPTH(3-34) amide and (5-34) amide, which inhibited the effect of hPTH(2.4 nmol/l) with half maximally effective concentrations of 0.1 mumol/l. When conditioned medium was used from a human lung cancer cell line producing osteoblast adenylate cyclase-stimulating activity, these two analogues were capable of inhibiting the increase in cAMP production. The specificity of the antagonism was indicated by the inability of the analogues to influence the effects of prostaglandin E2 or of calcitonin, which are alternative stimulators of cAMP production in the osteogenic sarcoma cells. Only (Tyr34)hPTH(3-34) amide showed some PTH-like agonist activity at high concentrations. These analogues should prove valuable in the investigation of PTH actions on target cells and of tumour products which appear to act through the PTH receptor. PMID- 3005461 TI - Influence of acute intracerebroventricular (i.c.v.) administration of adrenocorticotrophin (ACTH) on LH secretion in male rats: effect of pretreatment (i.c.v.) with ACTH antiserum on the serum LH response to an acute ether stress. AB - Adult male rats with chronic indwelling intracerebroventricular (i.c.v.) and jugular catheters were given an i.c.v. injection (over 1 min) of 1, 10, 100 ng or 1 microgram ACTH(1-24), or 300 ng ACTH(4-10) or saline, and blood samples were taken 0, 5, 15, 30 and 60 min later. Increasing dosages of ACTH(1-24) caused a dose-related rise in serum LH levels. Peak levels of serum LH (ranging from 157 to 473% of pretreatment levels) were reached 5-15 min after treatment, and then serum LH values returned to pretreatment levels by 60 min. The serum LH response to 1 microgram ACTH(1-24) did not differ from the response to 100 ng ACTH(1-24). Administration (i.c.v.) of 300 ng ACTH(4-10) was also effective in increasing serum LH values. Repeated withdrawal of blood during the experiment increased serum corticosterone values in all groups (including saline-treated), but i.c.v. administration of ACTH(1-24) or ACTH(4-10) did not further increase serum corticosterone levels. Two additional groups of rats were injected i.p. with either saline or pentobarbital (30 mg/kg body weight) 1 h before i.c.v. administration of 10 ng ACTH(1-24) and blood samples were taken as in the other groups. The animals in these groups did not show a rise in serum LH concentrations in response to ACTH(1-24). In a third experiment, rats were pretreated (i.c.v.) with either ACTH antiserum (ACTH-Ab) or normal rabbit serum 15 min before a 2-min ether stress. The ether stress evoked a significant rise in serum LH concentrations within 15 min.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3005462 TI - The physiological significance of arginine vasopressin in potentiating the response to corticotrophin-releasing factor in sheep. AB - In order to assess the physiological importance of endogenous arginine vasopressin (AVP) in augmenting the ACTH response to corticotrophin-releasing factor (CRF), the response to CRF during hypertonic saline infusion in six Coopworth sheep was examined. A 4-h infusion of 5% (w/v) NaCl (3.8 ml/min) resulted in significantly (P less than 0.01) greater rises in ACTH and cortisol, but not aldosterone, than were observed after CRF alone. Infusion of hypertonic saline without CRF resulted in a highly significant (P less than 0.001) rise in plasma osmolality and AVP but no significant change in plasma ACTH, cortisol or aldosterone. It is concluded that a marked but physiological increase in peripheral (and presumably central) levels of AVP does not result in any demonstrable change in plasma ACTH concentration. However, under these conditions, the ACTH and cortisol responses to CRF are considerably augmented. PMID- 3005463 TI - Endocrine control of smoltification in anadromous salmonids. AB - The parr-smolt transformation (smoltification) of juvenile anadromous salmonids involves a morphological, physiological and behavioural metamorphosis of the fish from a freshwater-adapted form to a salt-water-adapted form. Several endocrine glands are activated during the period of smoltification, including pituitary, thyroid, and interrenal tissues. The pituitary-thyroid axis appears to be the endocrine system most directly involved in controlling smoltification. A plasma thyroid hormone surge occurs during smoltification which appears to influence various tissues and other endocrine systems, and to induce the well-documented developmental changes associated with smoltification. The pituitary-interrenal axis has been implicated in several smoltification-related events, including development of hypo-osmotic regulatory ability. A plasma cortisol surge closely follows the thyroid hormone surge during smoltification, but in contrast to anuran metamorphosis, the peaks do not coincide. Despite recent attention, the role of the corticosteroids in development of hypo-osmotic regulatory ability remains unclear. The other endocrine tissues of the salmonids appear to be acting trophically with the thyroid hormones, or to have little involvement in the control of smoltification. PMID- 3005464 TI - High-affinity receptor-mediated internalization and degradation of interleukin 2 in human T cells. AB - Receptor-mediated internalization and degradation of IL-2 were investigated in cell lines carrying human T cell leukemia/lymphoma (lymphotrophic) virus type I (HTLV-I) and PHA-treated normal PBL. The HTLV-I-carrying cell lines ILT-Yan and TL-Mor, and the PBL expressed both high- and low-affinity IL-2-R. However, another HTLV-I-carrying T cell line, MT-1, expressed mainly low-affinity receptors. Greater than 50% of the IL-2 bound to high-affinity receptors was internalized within 10 min when these cells were incubated at 37 degrees C. The internalized IL-2 was rapidly degraded and the products were excreted into the culture fluid. The t1/2 of IL-2 degradation in these cells was estimated as 60-80 min at 37 degrees C. The internalization and degradation of IL-2 were both temperature dependent. Light-microscopic autoradiography with 3H-labeled IL-2 confirmed the internalization of IL-2, and suggested that some IL-2 might be carried to the nucleus. PMID- 3005465 TI - Genetic basis for expression of the idiotypic network. One unique Ig VH germline gene accounts for the major family of Ab1 and Ab3 (Ab1') antibodies of the GAT system. AB - Ig germline genes have been isolated from recombinant clones prepared in separate libraries constructed from adult BALB/c liver DNA either in pBR328 plasmid or in EMBL 3 phage. Three clones that gave a very strong positive hybridization signal with a VH anti-GAT-specific probe were completely characterized and sequenced. All three were greater than 95% homologous, with the exception of the 5' noncoding region, which was only 85% homologous but contained characteristic regulatory signals. One of these genes, H10, had a sequence that was completely identical to that of a cDNA derived from a GAT-specific BALB/c hybridoma. Southern blot analysis using Eco RI-digested DNA from rearranged GAT-specific hybridomas revealed that the same gene was used for other GAT-specific VH regions, including one differing from the H10 sequence by 12 nucleotides, which must have been generated by a somatic mechanism. The same H10 germline gene was also used, in most cases without any nucleotide substitution, in hybridomas of the Ab1' set of the GAT idiotypic cascade, suggesting that immunization with Ab2 (antiidiotypic) antibodies preferentially stimulates the direct expression of VH germline genes. Finally, the previous hypothesis that NPa and GAT VH genes were derived from the same germline gene was definitively confirmed, both from sequence data and Southern blot analysis. PMID- 3005466 TI - Susceptibility to Theiler's virus-induced demyelination. Mapping of the gene within the H-2D region. AB - Demyelination induced by Theiler's virus was examined in mouse strains with congeneic recombinant haplotypes. Light and electron microscopy of spinal cord sections from mice with s, q, v, p, and f H-2D alleles showed perivascular inflammation and primary demyelination. The presence of susceptible haplotypes in the K or I region did not correlate with pathologic abnormalities. The Qa, Tla, PgK, and UpG genes did not appear to be critical in determining susceptibility to disease. However, mutation in the H-2D genes altered the susceptibility to virus induced demyelination. B10.D2dm1 mice, which have deletions in the 3' end of Dd and the 5' end of Ld, showed prominent demyelination and clinical deficits. In contrast, BALB/c-dm2, which have a deletion of the entire L gene, showed no pathologic changes. Central nervous system virus titers correlated with susceptibility to demyelination; both resistant and susceptible strains had a strong humoral immune response to the virus. The findings in the congeneic recombinant mice and in mice mutant in the H-2D region strongly suggest that at least one of the genes critical for determining virus-induced demyelination maps to the 3' end of the H-2D gene. PMID- 3005469 TI - Diazepam in blood of Danish drivers: occurrence as shown by gas-liquid chromatographic assay following radioreceptor screening. PMID- 3005468 TI - Identification of a second class of IgG Fc receptors on human neutrophils. A 40 kilodalton molecule also found on eosinophils. AB - We describe a newly recognized 40 kD FcR for IgG on human neutrophilic granulocytes. An mAb (IV3) developed against the IgG FcR of K562 cells, and specific as well for a 40 kD FcR on human monocytes and platelets, was found to purify by affinity adsorption a 40 kD protein from detergent lysates of surface radioiodinated neutrophils. This protein, proteolytically degraded to 33 kD when purified in the absence of diisopropylfluorophosphate, is distinct from the 51-73 kD protein precipitated by the anti-neutrophil FcR mAb, 3G8, previously described by others. Complete inhibition of binding of rabbit IgG-coated erythrocytes to neutrophils was achieved only when both antibodies, IV3 and 3G8, were used. Fab fragments of IV3 were as effective inhibitors as the intact molecule. IV3 IgG or Fab fragments completely and selectively inhibited immune complex-mediated generation of superoxide by human neutrophils; superoxide generation by other stimulants was not abrogated by IV3. This antibody (IV3) bound also to human eosinophils and completely inhibited the binding of IgG-coated erythrocytes to eosinophils. IV3 appears to define the human homolog of the murine macrophage FcRII identified initially by mAb 2.4G2 and present in the mouse on both neutrophils and eosinophils. PMID- 3005467 TI - Delineation of a defect in T cell receptor beta genes of NZW mice predisposed to autoimmunity. AB - In an attempt to determine whether genes involved in T cell antigen recognition are structurally abnormal and thereby promote murine systemic lupus, we analyzed the structural integrity of the D, J, and C region elements of the T cell receptor alpha and beta chain genes in all major lupus strains and several normal strains. Within the limits of restriction fragment length polymorphism analysis, all strains had an identical genomic organization, except the NZW mice, in which a deletion of the C beta 1-D beta 2-J beta 2 elements was found. Sequence analysis of NZW genomic elements containing this deletion placed its probable origin within the first exon of C beta 1, and extending to a complementary region within the first exon of C beta 2. The significance of this abnormality in the pathogenesis of systemic autoimmune disease remains to be determined. PMID- 3005470 TI - Mode of inhibitory action of Zn2+, Hg2+ and UO2(2+) on 5'-nucleotidase of mouse hepatic microsomes. PMID- 3005471 TI - Prevalence of anti-oncogenic virus antibodies in patients with malignancies, hemophilia and systemic lupus erythematosus. PMID- 3005472 TI - Interactions of lithium and protons with the sodium-proton exchanger of dog red blood cells. AB - Passive movements of Li in dog red blood cells (RBC) ar like those of Na and protons in being stimulated by osmotic cell shrinkage and inhibited by amiloride. Li and protons have similar asymmetrical effects on Na-H exchange. When the intracellular fluid is made rich in Li or protons, Na-H exchange is stimulated. When the extracellular fluid is enriched in Li or protons, Na-H exchange is inhibited. In the case of protons, these effects can override alterations in driving force that are created by the experimental conditions. For example, acidification of the cytoplasm stimulates outward Na movements, while acidification of the medium inhibits Na efflux. Thus, protons (and, by analogy, Li) can interact with the Na-H exchanger not only as substrates but also as modulators. In previous experiments, the only way to activate the Na-H exchanger in dog RBC was to shrink the cells in hypertonic media. The influences of Li or protons, however, are so strong as to preempt the volume effects, so that the pathway can be activated even in swollen cells and deactivated in shrunken ones. PMID- 3005473 TI - Energy-dispersive X-ray microanalysis of membrane-associated inclusions in hydrogenosomes isolated from Trichomonas vaginalis. AB - Energy-dispersive X-ray microprobe analysis of electron-dense inclusions in hydrogenosomes isolated from the aerotolerant anaerobic protozoal human parasite Trichomonas vaginalis, Bushby strain, indicated the presence of high levels of Mg, P and Ca. This suggested that divalent cation (e.g. Ca2+ or Mg2+) accumulation by hydrogenosomes may be important in the regulation of intracellular ion concentrations. PMID- 3005474 TI - Evidence for the presence of cAMP, cAMP receptor and transcription termination factor rho in different gram-negative bacteria. AB - Cyclic AMP has been shown to be present in 12 different Gram-negative bacteria and the regulation of its concentration, as a function of growth conditions, is similar to that described for Escherichia coli K12. Antibodies raised against catabolite activator protein (CAP) and Rho protein of E. coli K12 were used to check for the occurrence of cross-reactive antigens. Using radioimmunological assays, immunoblotting techniques and biochemical criteria we showed a wide distribution of CAP and Rho, structurally and functionally closely related to the corresponding E. coli K12 proteins. These results suggest that transcription is similarly regulated by these factors in Gram-negative bacteria. PMID- 3005476 TI - Cloning of genes determining the production of vero cytotoxin by Escherichia coli. AB - Sequences encoding the production of a cytotoxin (VT) active on Vero cells were cloned in Escherichia coli K12 from a VT-determining phage that originated in E. coli strain H19 of serotype O26.H11. Subcloning resulted in the identification of a 2.5 kb fragment that still coded for VT production. Mutagenesis with transposon Tn1000 was used to map VT sequences and a 0.75 kb probe was developed. In colony hybridization tests with strains isolated from patients with haemolytic uraemic syndrome or diarrhoea, this probe derived from the H19 VT genes detected only some of the VT+ strains belonging to serogroup 0157. A VT+ strain, E32511, serotype 0157.H-, which was negative in colony hybridization was the source of another VT-determining phage from which VT sequences were cloned. Southern hybridization of the VT genes from E32511 with the H19 probe was negative under stringent conditions but there was weak homology under conditions of low stringency. These results indicate that there are differences in the VT genes of pathogenic E. coli. PMID- 3005475 TI - The fumarase genes of Escherichia coli: location of the fumB gene and discovery of a new gene (fumC). AB - The fumB gene of Escherichia coli, which complements the fumarase deficiency of a fumA mutant when present in multiple copies, has been located at 93.5 min in the E. coli linkage map and its product has been identified as a polypeptide of 61 kDal. Four overlapping ColE1-fumB+ plasmids representing a continuous segment of 23.3 kb of bacterial DNA have been isolated from the Clarke-Carbon E. coli gene bank and the location of the fumB gene relative to the restriction map and the adjacent mel operon has been defined. Hybridization studies have shown that the fumB gene is homologous to the fumA gene, which complements the fumA1 mutation in single and multi-copy situations, and encodes an analogous 61 kDal product formerly regarded as the E. coli fumarase. The hybridization studies also showed that the Bacillus subtilis fumarase gene (citG) is homologous to an independent gene, fumC (formerly g48), which lies adjacent to the fumA gene at 35.5 min in the E. coli linkage map. The N-terminal sequences of the citG and fumC products exhibit a 51% identity over 88 residues. It is possible that the fumC and citG genes are fumarase structural genes of E. coli and B. subtilis, and that the fumA gene may encode a differentially-regulated fumarase or be a positive regulator gene which is essential for the expression of fumC (but not citG). If so, the fumB gene may encode a related enzyme or activator that can replace the fumA function when amplified. PMID- 3005477 TI - Multivariate analysis of Neisseria DNA restriction endonuclease patterns. AB - Chromosomal DNA was extracted from eleven Neisseria meningitidis and seven Neisseria gonorrhoeae isolates and cleaved with the restriction enzyme HindIII. The DNA fragments were separated according to their size, using a 4% polyacrylamide gel. The band patterns obtained were digitized and statistically analysed by the SIMCA method. To develop the models for N. meningitidis (class 1) and N. gonorrhoeae (class 2), all eleven meningococci and seven gonococci, were used. All strains were classified correctly and showed an extremely good class separation. PMID- 3005479 TI - Plasma membrane proteins and glycoproteins induced by human cytomegalovirus infection of human embryonic fibroblasts. AB - An analysis of the plasma membrane proteins of human embryonic fibroblasts (HEF) infected with human cytomegalovirus strain AD169 (HCMV) was performed using in vitro radioactive labelling techniques followed by PAGE. Of the 12 virus-induced proteins detected in infected cells, glycoproteins of mol. wt. 34000 (34K), 53K to 55K, 60K to 63K, 70K to 72K, 98K to 103K and 145K to 150K and proteins of 130K to 133K and 260K to 270K were considered significant. The 60K to 63K, 70K to 72K and 130K to 133K components were detectable at early stages of infection (8 h), although only the latter two were labelled by surface iodination. The others only appeared in the membrane from 48 h to 80 h after infection. Serological studies indicated that the 34K, 70K to 72K, 98K to 103K and 145K to 150K components may be HCMV-specified virion constituents, as these glycoproteins reacted with antibodies raised against virions and extracted envelope glycoproteins. Of immunological importance was the exposure on the cell surface of the protein moieties of 70K to 72K and 130K to 133K proteins at 8 h and 53K to 55K, 60K to 63K, 70K to 72K and 145K to 150K components at 80 to 90 h after infection. Pooled human immune sera contained antibodies which reacted with these exposed proteins, as well as with three other virus-induced membrane components of 230K to 240K, 98K to 103K and 78K to 80K. PMID- 3005478 TI - Evolution of vesicular stomatitis virus in athymic nude mice: mutations associated with natural killer cell selection. AB - BHK-21 cells readily produce tumours in athymic nude mice, but BHK-21 cells persistently infected with wild-type vesicular stomatitis virus (VSV) do not. However, rare persistently infected virus-shedding tumours (VSV-P tumour cells) were independently derived by in vivo selection on three different occasions. Cloned viruses isolated from each of these (VSV-P virus mutants) carried mutations determining the VSV-P phenotype because they all allowed growth of virus-shedding tumours in nude mice when they were used to persistently infect normal (unselected) BHK-21 cells. Treatment of nude mice with anti-asialo-GM1 allowed BHK cells persistently infected with wild-type VSV to form tumours, and BHK cells persistently infected with VSV-P were resistant to natural killer (NK) cells in vitro; this implicates NK cells in the in vivo rejection of persistently infected tumours and in the selection of the VSV-P variant. In this paper, we have sequenced the glycoprotein (G protein), matrix (M) and non-structural (NS) proteins of three independently derived VSV-P type mutants to find mutations associated with in vivo passage of persistently infected nude mouse tumours and with resistance to NK cells. We found extensive mutation in the G protein of VSV P but relatively few mutations in the M and NS proteins. This suggests but does not prove a role for the G protein in NK cell killing of infected cells. PMID- 3005480 TI - Evidence for the presence of duck hepatitis B virus in wild migrating ducks. AB - A virus closely related to duck hepatitis B virus (DHBV) was isolated from serum and liver samples of wild migratory ducks (mallards) caught in two separate wildlife reserve parks in France. In the first one (Dombes region) 12% of wild mallards were positive for DHBV, and in the second (River Somme) 3% of mallards were found positive. The DHBV isolated from the serum of wild mallards was also associated with an endogenous DNA polymerase activity capable in vitro of completing a partially double-stranded viral DNA into a fully double-stranded DNA of 3 kb. The various replicative DNA forms reported for DHBV were also detected in the liver of wild viraemic mallards. The DNA restriction enzyme pattern of the wild mallard strain differed from that of American and French strains of DHBV. The wild mallard strain DHBV was experimentally transmitted to mallard and Pekin ducklings and induced a chronic viraemia in both varieties of infected birds. This strain might be the common ancestor of all DHBV strains isolated from domestic ducks world-wide. The discovery of a DHBV-related virus in the natural wild population might be an important clue in the study of the different roles of environmental, host and viral factors in the pathogenesis of DHBV infection, and their possible oncogenic action in ducks. PMID- 3005481 TI - Replication of atypical ovine rotavirus in small intestine and cell culture. AB - Colostrum-deprived lambs experimentally infected with an atypical ovine rotavirus isolated from naturally scouring animals, and a naturally infected colostrum deprived lamb, were examined by immunofluorescence and immunoperoxidase labelling, and electron microscopy. A hyperimmune serum to the virus was produced in a gnotobiotic lamb and used to demonstrate antigen in the villous epithelial cells of the small intestine from infected animals. Scanning and transmission electron microscopy of tissues from infected animals revealed giant multinucleate syncytia composed of fused epithelial cells. Infected cells contained intracytoplasmic virus particles which resembled group A rotaviruses in morphology and morphogenesis. Small numbers of infected cells in MA104 cultures inoculated with ovine atypical rotavirus could be detected by immunofluorescence but virus growth could not be maintained by passage. Virus particles were seen by thin-section electron microscopy but their morphology and morphogenesis were abnormal; they were each composed of a single electron-dense shell, with no core, and were associated with envelopes of smooth membrane rather than rough endoplasmic reticulum. PMID- 3005482 TI - Diarrhoea in mice infected with a human rotavirus. AB - Oral inoculation of newborn mice with the MET strain of human rotavirus produced transient diarrhoeal disease. Light and scanning electron microscopy showed typical rotavirus-induced morphological lesions in the villous epithelium of the small intestine consisting of extensive cytoplasmic vacuolation, villous necrosis and atrophy. Virus recovered from intestinal suspensions of infected mice showed the typical electrophoretic profile of the genome of the inoculated strain. Rotavirus antibody appeared in infected mice 10 to 20 days after inoculation but not in controls or nursing dams. The availability of a small animal model for experimental infection with human rotaviruses should prove useful for virulence and protection studies. PMID- 3005483 TI - Human papillomavirus type 16 DNA in genital tumours: a pathological and molecular analysis. AB - The presence of human papillomavirus type 16 (HPV16) DNA in 34 genital tract tumours of Italian female patients was investigated by Southern blot hybridization in high stringency conditions. HPV16 DNA was detected in 16 neoplasias, including cervical invasive and intraepithelial lesions as well as vulvar intraepithelial neoplasias and, to a lesser extent, vulvar invasive carcinomas. Appropriate control tissues included in the study were negative. The data suggest that integration of viral DNA had occurred in most tumours, both in invasive and in intraepithelial lesions. HPV16 variants or defective genomes, lacking the BamHI restriction site, were detected in three tumours. PMID- 3005484 TI - Molecular epidemiology of rotavirus infections in Uppsala, Sweden, 1981: disappearance of a predominant electropherotype. AB - The molecular epidemiology of rotavirus infections was studied in children with acute gastroenteritis in Uppsala, Sweden, during 1981. Altogether 118 virus strains were investigated by analysis of the RNA migration pattern in silver stained polyacrylamide gels. Six different electropherotypes were seen: two with "short" and four with "long" RNA migration patterns. Forty-two strains (36%) exhibited "short" patterns. The seasonal distribution showed that strains with "long" and "short" RNA patterns cocirculated in equal frequency during the first seven months of the year, until the predominant "short" RNA electropherotype suddenly disappeared. More than 11 RNA segments were seen in two stool specimens. A complete correlation was found between the electrophoretic migration of segments 10 and 11 and the serological defined subgroup specificity. PMID- 3005485 TI - Development of a competitive enzyme-linked immunosorbent assay for detecting cytomegalovirus antibody. AB - A competitive enzyme-linked immunosorbent assay (ELISA) for detecting cytomegalovirus (CMV) antibody was developed. The competitive ELISA was five times more sensitive than the complement fixation test (CFT) and twice as sensitive as indirect ELISA. Testing of paired sera from cardiac transplant patients taken before and after transplantation showed good correlation between results of competitive and indirect ELISA and CFT. The competitive ELISA was more successful than CFT or indirect ELISA in detecting passively acquired antibody, but detection of CMV antibody by competitive ELISA immediately after primary CMV infection was unreliable, possibly because of the high affinity of the monoclonal antibody chosen for the horseradish peroxidase conjugate. However, competitive ELISA may well prove to be more suitable than indirect ELISA for detecting CMV antibody in blood donations. PMID- 3005486 TI - Protection of weanling hamsters from experimental infection with wild-type parainfluenza virus type 3 (para 3) by cold-adapted mutants of para 3. AB - Parainfluenza virus type 3 (para 3) was adapted to replicate at 20 degrees C, a nonpermissive temperature for wild-type (wt) para 3. Serial passage at 20 degrees C resulted in the generation of cold-adapted (ca) and temperature-sensitive (ts) mutants. These mutant viruses have been characterized both in vitro and in vivo [Belshe and Hissom (1982): Journal of Medical Virology 10:235-242; Crookshanks and Belshe (1984): Journal of Medical Virology 13:243-249]. We now report the evaluation of three mutants (clone 1150, passaged 12 times in the cold [cp12], clone 1146, passaged 18 times in the cold [cp18], and clone 1328, passaged 45 times in the cold [cp45]) for their ability to protect hamsters from infection by wild-type para 3. Ether-anesthetized male syrian hamsters were intranasally vaccinated with either wt para 3 (clone 127) or one of the ca para 3 mutants and on day 28 post-vaccination; each animal was intranasally challenged with 10(5.0) pfu of wt para 3. On days 1, 2, 3, and 4 post-challenge, 4 to 13 hamsters from each group were sacrificed, and the quantity of para 3 in the nasal turbinates and lungs was determined. Wt virus induced protection from challenge. cp12, cp18, and cp45 reduced the peak titer of wt replication in the lungs by greater than 100-fold, tenfold, and tenfold, respectively. The duration of virus replication was shortened also by intranasal vaccination with the mutants. These data give evidence of an inverse relationship between the degree of protection induced by vaccination with cold-adapted mutants and the number of passages of the virus in the cold. PMID- 3005487 TI - Detection of cytomegalovirus antigen and antibodies in the urine of small infants and children. AB - The diagnostic efficacy of an enzyme immune assay to detect cytomegalovirus (CMV) antigen in the urine of infected infants and children was determined, and the effect that CMV-specific antibodies present in the urine have on the sensitivity of the test was ascertained. The antigen was detected in 84.4% of the CMV-culture positive samples, while CMV-specific IgA was detected in 24% of CMV-culture positive specimens. The absence of CMV-specific IgA correlated with detection of CMV antigen in the CMV-culture-positive urine specimens (p less than 0.05). PMID- 3005488 TI - Characterization of rotaviruses and subgroup F adenoviruses from acute summer gastroenteritis in South Africa. AB - Six hundred and sixteen specimens were collected from black children hospitalised with acute gastroenteritis during the summer and autumn of 1982-1983 (October to May). Eighty-five children (13.8%) shed rotavirus and at least 40 (6.5%) shed adenovirus (Ad) type 40 or 41 belonging to subgroup F. The highest monthly prevalence of shedding subgroup F adenoviruses (10.1%) coincided with a peak in admissions in midsummer, whereas the highest monthly prevalence of shedding rotaviruses (41.9%) coincided with a peak in admissions in autumn. There were at least five genome types of rotavirus, at least three genome types of Ad40, and at least five genome types of Ad41 circulating in the Johannesburg-Soweto area during the study period. The high rate of rotavirus shedding in autumn could not be attributed to an upsurge in infections by any particular rotavirus strain. PMID- 3005489 TI - Immunological reactivity of human sera with individual herpes simplex proteins: a comparative study of sera from patients with preinvasive or invasive cervical cancer and from controls. AB - Forty-three human sera collected from patients with preinvasive or invasive cervical carcinoma were analyzed for their repertoire of herpes simplex virus (HSV) specific antibodies reactive with individual viral HSV-1 and HSV-2 proteins. The reactivity was compared to that of sera from 27 control persons. The patients and controls were clinically and histologically characterized in a previous study we carried out, where the analysis of the HLA-antigen pattern was compared among the groups [Vass-Sorensen, 1984]. Immunoprecipitation analysis showed that only a subset of the infected cell proteins was precipitable by the human sera. The major proteins identified in the polyacrylamide gels were the glycoproteins B and D, the ICP-5 and ICP-8. There was no difference between the results obtained with patients and control sera. Immunoblot analysis showed that a different subset of HSV proteins reacted with the human sera, but the variability among individuals was significant. Rank data showed that sera from both patients and controls reacted most frequently with proteins belonging to the "35-family" [Braun et al, 1984] and with the glycoproteins B and D of HSV-1 and HSV-2. PMID- 3005490 TI - Coxsackie B virus-specific IgM responses in coronary care unit patients. AB - An enzyme-linked immunosorbent assay (ELISA) test using polyvalent antigens and antisera was used to detect Coxsackie B virus-specific IgM responses in 329 patients admitted to the Coronary Care Unit, Wellington Hospital, New Zealand over a 12-month period. The sera of 30 of 153 (19.6%) patients with acute myocardial infarction (AMI), 16 of 98 (18.4%) with chest pain, and 7 of 46 (15.2%) patients with arrhythmia were positive for Coxsackie B virus-specific IgM. Four of 12 (25%) patients with heart failure were also positive. Over the same period, 178 sex- and age-matched normal blood donors were also studied. Eleven of 178 (6.2%) matched blood donors were positive for Coxsackie B virus specific IgM. The rates of occurrence of Coxsackie B virus-specific IgM in patients with AMI and in a group of matched controls showed a significant difference (chi 2 = 5.64, p = 0.02). PMID- 3005491 TI - Adenoid cystic carcinoma of the base of the tongue. PMID- 3005492 TI - Kinetics and physical parameters of rat brain opioid receptors solubilized by digitonin and CHAPS. AB - Rat brain opioid receptors were solubilized with digitonin and a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). The yield of solubilization was 70-75% with digitonin and 30-35% with CHAPS. Kinetic and equilibrium studies performed from digitonin extracts resulted in KD values comparable with those of the membrane fractions. Two [3H]naloxone binding sites were obtained in the extracts similarly to membrane fractions. The rank order potency of drugs used in the competition experiments did not change during solubilization. The distributions of mu, delta, and kappa opioid receptor binding sites were similar in membrane and digitonin-solubilized fractions (48-50% mu, 35 37% kappa, and 13-17% delta subtypes). The hydrodynamic properties of digitonin- and CHAPS-solubilized preparations were studied by sucrose density gradient centrifugation and Sepharose-6B chromatography. In all cases, two receptor populations were identified with the following parameters: sedimentation coefficients for the digitonin extracts were 9.2S and 13.2S and for CHAPS extract 8S and 15.6S; the Stokes radii were 45 A and 65A for the digitonin extract and 31A and 76A for the CHAPS-solubilized preparation. PMID- 3005493 TI - Differential sensitivity of "central" and "peripheral" type benzodiazepine receptors to phospholipase A2. AB - The effects of preincubating cerebral cortical membranes with phospholipase A2 (PLA2) were examined on radioligand binding to benzodiazepine receptors of the "central" and "peripheral" types. PLA2 (0.005-0.1 U/ml) increased [3H]flunitrazepam and [3H]3-carboethoxy-beta-carboline binding by increasing the apparent affinities of these ligands with no concomitant change in the maximum number of binding sites. In contrast, neither gamma-aminobutyric acid (GABA) enhanced [3H]flunitrazepam binding nor [3H]Ro 15-1788 binding was altered by preincubation with PLA2 at concentrations as high as 2 U/ml. Both pyrazolopyridine (SQ 65,396)- and barbiturate (pentobarbital)-enhanced [3H]flunitrazepam binding and [35S]t-butylbicyclophosphorothionate (TBPS) binding were markedly reduced by as little as 0.0025-0.005 U/ml of PLA2. These findings suggest that PLA2 inactivates the TBPS binding site on the benzodiazepine-GABA receptor chloride ionophore complex, which results in a selective loss of allosteric "regulation" between the components of this complex. PLA2 also reduced the apparent affinity of [3H]Ro 5-4864 to peripheral-type benzodiazepine receptors in cerebral cortical, heart, and kidney membranes, but increased the number of [3H]PK 11195 binding sites with no change in apparent affinity. These data demonstrate that PLA2 can differentially affect the lipid microenvironment of "central" and "peripheral" types of benzodiazepine receptors. PMID- 3005494 TI - Effects of cell division, cell density, and cyclic nucleotides on choline acetyltransferase activity in a cholinergic neuroblastoma cell line (S-20Y). AB - We investigated the effects of a number of experimental perturbations on choline acetyltransferase (ChAT) in a cholinergic mouse neuroblastoma cell line (S-20Y). ChAT specific activity increased by 4.5-fold during growth, suggesting that enzyme activity is dependent on increased cell density. This was confirmed by assessing enzyme activity at differential initial seeding densities. ChAT activity was also markedly enhanced by 1 mM dibutyryl cyclic-3',5'-AMP (dBcAMP), an effect that was blocked by cycloheximide. Confirmation of the dBcAMP effect was achieved with forskolin, a compound known to enhance intracellular cyclic AMP; forskolin (100 microM) caused a significant increase in ChAT activity. After a 20-h latent interval ChAT activity was also enhanced significantly by cytosine arabinoside. The common element in these diverse effects on ChAT activity may be cessation of cell division, although cell-cell interactions at the level of the cell membrane may also be important in the control of ChAT in S-20Y. PMID- 3005495 TI - Acetylcholine synthesis and release by a sympathetic ganglion in the presence of 2-(4-phenylpiperidino) cyclohexanol (AH5183). AB - These experiments measured the release and the synthesis of acetylcholine (ACh) by cat sympathetic ganglia in the presence of 2-(4-phenylpiperidino) cyclohexanol (AH5183), an agent that blocks the uptake of ACh into synaptic vesicles. Evoked transmitter release during short periods of preganglionic nerve stimulation was not affected by AH5183, but release during prolonged stimulation was not maintained in the drug's presence, whereas it was in the drug's absence. The amount of ACh releasable by nerve impulses in the presence of AH5183 was 194 +/- 10 pmol, which represented 14 +/- 1% of the tissue ACh store. The effect of AH5183 on ACh release was not well antagonized by 4-aminopyridine (4-AP), and not associated with inhibition of stimulation-induced calcium accumulation by nerve terminals. It is concluded that AH5183 blocks ACh release indirectly, and that the proportion of stored ACh releasable in the compound's presence represents transmitter in synaptic vesicles available to the release mechanism. The synthesis of ACh during 30 min preganglionic stimulation in the presence of AH5183 was 2,448 +/- 51 pmol and in its absence it was 2,547 +/- 273 pmol. Thus, as the drug decreased ACh release it increased tissue content. The increase in tissue content of ACh in the presence of AH5183 was not evident in resting ganglia; it was evident in stimulated ganglia whether or not tissue cholinesterase was inhibited; it was increased by 4-AP and reduced by divalent cation changes expected to decrease calcium influx during nerve terminal depolarization.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3005496 TI - Autoradiographic localization of benzodiazepine receptor binding in dissociated cultures of fetal mouse cerebral cortex. AB - Autoradiography utilizing photoaffinity labelling with [3H]flunitrazepam was used in living cultures of fetal mouse cerebral cortex in situ to localize benzodiazepine receptor binding sites. There was a predominant localization of silver grains over neurons; however, substantial labelling also occurred over nonneuronal background cells. Clonazepam (0.1 microM) and Ro 5-4864 (0.1 microM) displaced substantial numbers of silver grains over neurons and background cells, respectively. In addition, clonazepam displaced 58-68% of specific grains over background cells and Ro 5-4864 displaced 30% of grains over neurons, suggesting that multiple cell types in the CNS may participate in the neuropharmacologic actions of the benzodiazepines. PMID- 3005497 TI - Invalidity of criticisms of the deoxyglucose method based on alleged glucose-6 phosphatase activity in brain. AB - The observations made by Sacks et al. [Neurochem. Res. 8, 661-685 (1983)] on which they based their criticisms of the deoxyglucose method have been examined and found to have no relationship to the conclusions drawn by them. (1) The observations of Sacks et al. (1983) of constant concentrations of [14C]deoxyglucose and [14C]deoxyglucose-6-phosphate, predominantly in the form of product, reflects only the postmortem phosphorylation of the precursor during the dissection of the brain in their experiments. When the brains are removed by freeze-blowing, the time courses of the [14C]deoxyglucose and [14C]deoxyglucose-6 phosphate concentrations in brain during the 45 min after the intravenous pulse are close to those predicted by the model of the deoxyglucose method. (2) Their observation of a reversal of the cerebral arteriovenous difference from positive to negative for [14C]deoxyglucose and not for [14C]glucose after an intravenous infusion of either tracer is, contrary to their conclusions, not a reflection of glucose-6-phosphatase activity in brain but the consequence of the different proportions of the rate constants for efflux and phosphorylation for these two hexoses in brain and is fully predicted by the model of the deoxyglucose method. (3) It is experimentally demonstrated that there is no significant arteriovenous difference for glucose-6-phosphate in brain, that infusion of [32P]glucose-6 phosphate results in no labeling of brain, and that the blood-brain barrier is impermeable to glucose-6-phosphate. Glucose-6-phosphate cannot, therefore, cross the blood-brain barrier, and the observation by Sacks and co-workers [J. Appl. Physiol. 24, 817-827 (1968); Neurochem. Res. 8, 661-685 (1983)] of a positive cerebral arteriovenous difference for [14C]glucose-6-phosphate and a negative arteriovenous difference for [14C]glucose cannot possibly reflect glucose-6 phosphatase activity in brain as concluded by them. Each of the criticisms raised by Sacks et al. has been demonstrated to be devoid of validity. PMID- 3005498 TI - Extracellular calcium-induced neuroblastoma cell differentiation: involvement of phosphatidylinositol turnover. AB - The rat CNS neuroblastoma B50 cell line is known to differentiate on addition of 1 mM dibutyryl cyclic AMP or on withdrawal of serum. In this report it is shown that high levels of extracellular calcium (10-25 mM) cause neurite extension, an important component of morphological differentiation. Stimulation of calcium influx with the ionophore A 23187 or blockade of calcium efflux with lanthanum are less efficient than extracellular calcium in stimulating neurite extension. These data suggest that intracellular calcium is not sufficient to cause full expression of a calcium-dependent differentiated state. Furthermore, phosphatidylinositol turnover is sharply altered as early as 1 h after addition of calcium to the medium while cyclic nucleotide levels remain unaffected. This suggests that activation of the phosphatidylinositol second-messenger system by calcium at the level of the cell membrane is the initial step in the cascade of events leading to neurite extension. Later events include a decrease in DNA synthesis (6-10 h after addition of calcium), and increase in intracellular calcium levels (12-24 h after calcium addition) concurrent with neurite extension. The intracellular increase in calcium levels is facilitated by synergistic action of 1 mM dibutyryl cyclic AMP with high external calcium (10-25 mM). This combined treatment results in a more complex pattern of neurite formation characterized by many synaptic-like junctions; this pattern is not obtained when either dibutyryl cyclic AMP or calcium is used as sole inducer. PMID- 3005499 TI - Enzymatic reconstitution of brain membrane and membrane opiate receptors. AB - A new method using lysophosphatide and acyl-CoA as detergents has been used to solubilize the rat brain opiate receptor. After solubilization, lysophosphatide and acyl-CoA can be almost completely removed by an enzymatic reaction that uses an acyltransferase from rat liver microsomes and reconstitutes the solubilized receptor in membranous vesicles. Morphological studies performed with negative staining and freeze-fracture electron microscopy revealed that the general appearance and intramembrane particle distribution of fracture faces in the reconstituted membrane are similar to those of the native membrane; this indicates that hydrophobic protein components of the original membrane were incorporated during reconstitution. Reconstituted membrane, however, contained higher levels of phosphatidylcholine and lower levels of cholesterol. The activities of the membrane-bound enzymes Na+, K+-ATPase and Ca2+, Mg2+-ATPase in the reconstituted system were 24 and 3%, respectively, those of the native membrane. Although binding of opiate ligands to the reconstituted membrane was stereospecific and saturable, higher concentrations of some of the unlabeled ligands were required to inhibit binding of the radiolabeled ligands. These changes in receptor characteristics are likely due to changes in lipid composition, physical state, and/or distribution of the lipids in the reconstituted membrane bilayer. This conclusion is supported by an increase in the affinity of opiate ligands for reconstituted membrane after adjustment of the latter's lipid composition to match more closely that of the original membrane. This was accomplished by treatment with phospholipid exchange protein to remove the excess phosphatidylcholine and by incorporation of cholesterol into the reconstituted membrane. PMID- 3005500 TI - Stimulation of the serotonin autoreceptor prevents the calcium-calmodulin dependent increase of serotonin biosynthesis in rat raphe slices. AB - The role of the serotonin (5-hydroxytryptamine) autoreceptor in the regulation of the activity of tryptophan hydroxylase was investigated in rat raphe slices. The activity of tryptophan hydroxylase was estimated by measuring the accumulation of 5-hydroxytryptophan in the presence of inhibition of aromatic L-amino acid decarboxylase using 3-hydroxy-4-bromobenzyloxy-amine by HPLC with fluorescence detection. Serotonin and its agonists N,N-dimethyl-5-methoxytryptamine and 1-(m chlorophenyl)-piperazine reduced the formation of 5-hydroxytryptophan to 50-60% at 10(-5) M. The effect of serotonin was reversed by 10(-5) M methiothepin, an antagonist of the serotonin autoreceptor. The calmodulin antagonists N-(6 aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and N-(6-aminohexyl)-1 naphthalenesulfonamide (W-5), dose-dependently reduced the basal formation of 5 hydroxytryptophan to 40-50% at 10(-6) and 10(-4) M, respectively. W-7 also reduced the activated formation by A-23187 or dibutyryl cyclic AMP in a dose dependent manner. W-7 had no effect on 5-hydroxytryptophan formation reduced by serotonin at 10(-5) M. These results suggest that the role of the serotonin autoreceptor was related to the prevention of the calcium-calmodulin-dependent activation of tryptophan hydroxylase. PMID- 3005501 TI - Interaction of [3H](-)-SKF-10,047 with brain sigma receptors: characterization and autoradiographic visualization. AB - The sigma opiates differ from other opiates in their stimulatory and psychotomimetic actions. The sigma opiate [3H](-)-SKF-10,047 has been used to characterize sigma receptors in rat nervous tissue. Binding of [3H](-)-SKF-10,047 to rat brain membranes was of high affinity, saturable, and reversible. Scatchard analysis revealed the apparent interaction of this drug with two distinct binding sites characterized by affinities of 0.03 and 75 nM (5 mM Tris-HCl buffer, pH 7.4, at 4 degrees C). Competition analyses involving rank order determinations for a series of opiates and other drugs indicate that the high-affinity binding site is the mu opiate receptor. The lower-affinity site (revealed after suppression of mu and delta receptor binding) has been identified as the sigma opiate/phencyclidine receptor. In vitro autoradiography has been used to visualize neuroanatomical patterns of receptors labeled using [3H](-)-SKF-10,047 in the presence of normorphine and [D-Ala2,D-Leu5]enkephalin to block mu and delta interactions, respectively. Labeling patterns differ markedly from those for mu, delta, or kappa receptors. The highest densities (determined by quantitative autoradiography) are found in the medial portion of the nucleus accumbens, amygdaloid nucleus, hippocampal formation, central gray, locus coeruleus, and the parabrachial nuclei. Receptors in these structures could account for the stimulatory, mood-altering, and analgesic properties of the sigma opiates. Although not the most selective sigma opiate ligand, [3H](-)-SKF-10,047 binds to sigma opiate receptors in brain, and this interaction can be readily distinguished from its interactions with other classes of brain opiate receptors. PMID- 3005502 TI - Phosphatidylinositol:myo-inositol exchange activity in intact nerve endings: substrate and cofactor dependence, nucleotide specificity, and effect on synaptosomal handling of myo-inositol. AB - Micromolar concentrations of CMP produced a large increase in Mn2+-dependent phosphatidylinositol:myo-inositol exchange activity in isolated nerve endings or synaptosomes. The apparent Km for CMP was 2 microM, and that for myo-inositol was 38 microM. Only cytidine nucleotides were capable of enhancing activity, and this effect is probably specific for CMP, because the synaptosomal preparation rapidly converted CTP or CDP to CMP. Manganese did not affect the uptake of myo-inositol into the synaptosomal cytosolic fraction or myo-inositol levels. Determinations of myo-inositol specific activity showed that the Mn2+-enhanced labeling of phosphatidylinositol was not accompanied by a decrease of label content in free myo-inositol. This lack of an effect on intrasynaptosomal myo-inositol and the dependence of exchange on cytidine nucleotides whereas cytidine itself was previously found to be without effect show that for the bulk of Mn2+-dependent exchange activity, it is the myo-inositol in the incubation medium that is being directly incorporated into membrane-bound phosphatidyl-inositol. Because CMP dependence is the hallmark of exchange catalyzed by CDP-diacylglycerol:inositol phosphatidyl transferase, this enzyme is likely to be responsible for most of the exchange activity in synaptosomes. The strong affinity of this exchange system for CMP suggests that endogenous levels of this nucleotide might support Mn2+ dependent exchange in the absence of added nucleotide. PMID- 3005503 TI - A study of the cerebral cortex cholecystokinin receptor using two radiolabelled probes: evidence for a common CCK 8 and CCK 4 cholecystokinin receptor binding site. AB - This study was directed at the issue of whether or not subpopulations of cholecystokinin (CCK) receptors exist within the CNS. This was achieved through the use of two radiolabelled probes, namely [125I] Bolton-Hunter (BH) CCK 8 and [3H]pentagastrin (Boc-beta-Ala CCK 4), in comparative studies under identical conditions. Both probes bound with high affinity to the mouse cerebral cortical CCK receptor binding site with apparent equilibrium dissociation constants (KD) of 1.9 nM and 1.4 nM for [3H]pentagastrin and [125I]BH CCK 8, respectively. The maximal binding capacity was 1.05 and 1.15 pmol/g weight for the tritium and iodinated probes, respectively. Hill analysis yielded Hill numbers close to unity, suggesting the absence of more than one binding site and the lack of cooperativity of CCK receptor binding. Kinetic studies revealed binding site homogeneity in that no evidence of multiphasic dissociation curves was seen. Computerised analysis of displacement binding data using LIGAND established that both radiolabelled probes bound to a single site, with the one-site model providing the best fit of the data. Similar rank orders of potency were obtained for various fragments of CCK 8 in competing for the CCK receptor, labelled with either probe. Both CCK 8 and CCK 4 bound with roughly equinanomolar affinity. These studies demonstrate that both CCK 8 and its shorter C-terminal fragment CCK 4 bind to a single class of high-affinity binding site, with as yet no evidence of CNS CCK receptor multiplicity. PMID- 3005505 TI - Subcellular compartmentation of opioid receptors: modulation by enkephalin and alkaloids. AB - A subclone of NG108-15 neuroblastoma-glioma hybrid cells was used to study the intracellular distribution of opioid receptors. Subcellular organelles were separated on self-generating Percoll-sucrose gradients and the enzymes beta glucuronidase, galactosyltransferase, 5'-nucleotidase, and glucose-6-phosphatase were used as markers to localize the various structures. Analysis of the receptor distribution from untreated cells shows that the plasma membranes contained the highest receptor density, but a significant portion of the opioid binding sites was unevenly distributed between the lysosomes, microsomes, and Golgi elements. The enzyme markers indicated that appearance of opioid receptors in these intracellular structures does not result merely from contamination with plasma membranes. About 11% of the receptors appeared in a fraction lighter than plasma membranes. The antilysosomal agent chloroquine altered the intracellular compartmentation of the receptors, possibly by blocking their translocation in the cells. Leu-enkephalin induced time-dependent loss of receptors from all four intracellular compartments examined, but a kinetic analysis showed that the rate of receptor loss in these fractions was not identical. Thus, the percent of receptors appearing in the lysosomal fraction that could still bind [3H]D-Ala2-D Leu5-enkephalin in vitro was increased on treatment with Leu-enkephalin. As an additional approach to follow the intracellular fate of the receptors, cells were labeled with [3H]diprenorphine, chased with various unlabeled opiates, and the distribution of 3H-ligand-receptors in the cells was monitored. Leu-enkephalin and etorphine altered the distribution of receptor-bound [3H]diprenorphine between the plasma membranes, lysosomes, and Golgi elements, whereas morphine had no such effect. The study sheds light on the role of intracellular structures in the metabolism of opioid receptors in untreated and opioid-treated cells. PMID- 3005504 TI - Distinct subtypes of the opioid receptor with allosteric interactions in brain membranes. AB - The binding isotherms of opioid receptors in rat brain membranes with [3H]D-Ala2 D-Leu5-enkephalin ([3H]DADLE), [3H]dihydromorphine ([3H]DHM), and [3H]etorphine were analysed to show the effects of Mg2+, Na+, and guanine nucleotides. Four opioid receptor subtypes of delta, kappa, mu 1, and mu 2 specificities were differentiated, where necessary with the aid of specific displacing ligands. Both a guanine nucleotide [guanosine-5'-(beta, gamma-imido)triphosphate] and the cations (Na+, Mg2+) affect the affinity state of all four subtypes of the receptor. The opioid binding behaviour is found on detailed inspection to be complex, with cases of "half-of-the-sites" reactivity and of cooperativity. By their behaviour under the various ionic conditions noted, it was concluded that these subtypes are distinct, without the need to assume interconvertibility by such agents. The evidence suggests that the formation of heterologous kappa-delta or mu 1-mu 2 receptor complexes is required for stabilization of the high affinity conformational state of the receptor. Important effects of cations in increasing the binding and regulating the equilibria of receptor association dissociation were observed when these studies were conducted, not in the Tris-HCl buffer commonly used in opioid binding assays, but in N-tris[hydroxymethyl] methyl-2-aminoethanesulphonate (K+) buffer (TES-KOH; 10 mM, pH 7.5): it was found that ionic species of Tris can substitute for divalent cations. Dithiothreitol effects on agonist binding in the presence and absence of the cations suggested that those cation effects involve the exchange of -SH/-SS- bonds between receptor subunits. All of the behaviour is interpreted in terms of a model involving association-dissociation equilibria of homologous and/or heterologous receptor subunits of an oligomeric opioid receptor structure. PMID- 3005506 TI - Identification of the subunit structure of rat pineal adrenergic receptors by photoaffinity labeling. AB - The adrenergic receptors of rat pineal gland were investigated using radiolabeled ligand binding and photoaffinity labeling techniques. 125I-2-[beta-(4 hydroxyphenyl)ethylaminomethyl]tetralone (125I-HEAT) and 125I-cyanopindolol (125I CYP) labeled specific sites on rat pineal gland membranes with equilibrium dissociation constants (KD) of 48 (+/- 5) pM and 30 (+/- 5) pM, respectively. Binding site maxima were 481 (+/- 63) and 1,020 (+/- 85) fmol/mg protein. The sites labeled by 125I-HEAT had the pharmacological characteristics of alpha 1 adrenergic receptors. 125I-CYP-labeled beta-adrenergic receptors were characterized as a homogeneous population of beta 1-adrenergic receptors. The alpha 1- and beta 1-adrenergic receptors were covalently labeled with the specific photoaffinity probes 4-amino-6,7-dimethoxy-2-(4-[5-(4-azido-3 [125I]iodophenyl) pentanoyl]-1-piperazinyl) quinazoline (125I-APDQ) and 125I-p azidobenzylcarazolol (125I-pABC). 125I-APDQ labeled an alpha 1-adrenergic receptor peptide of Mr = 74,000 (+/- 4,000), which was similar to peptides labeled in rat cerebral cortex, liver, and spleen. 125I-pABC labeled a single beta 1-adrenergic receptor peptide with a Mr = 42,000 (+/- 1,500), which differed from the 60-65,000 peptide commonly seen in mammalian tissues. Possible reasons for these differences are discussed. PMID- 3005507 TI - Dihydropyridine [methyl-3H]PN 200-110 binding and myogenesis in intact muscle cells in vitro. AB - The radioligand dihydropyridine [methyl-3H]PN 200-110 binds to contracting myotubes in culture derived from chick embryo pectoralis muscle. [methyl-3H]PN 200-110 binds specifically to high-affinity sites, with nonspecific binding only between 15 and 30% of the total binding. A Scatchard plot of the specific binding revealed a single high-affinity binding site with a KD (dissociation constant) of 0.5 nM +/- 0.2 nM and Bmax (number of binding sites) of 100 fmol/10(6) nuclei. We employed this sensitive assay to probe the appearance of high-affinity [methyl 3H]PN 200-110 binding sites during myogenesis. The time course of appearance of high-affinity binding sites lags behind that of fusion. Low-calcium media prevented the differentiation of myoblasts and blocked the appearance of high affinity sites. Chelation of intracellular calcium before or after fusion of myoblasts with the calcium indicator Quin 2 prevented the appearance of dihydropyridine binding sites. These findings are consistent with the view that the expression of dihydropyridine receptors is modulated by the intracellular calcium. PMID- 3005508 TI - Forskolin potentiates the stimulation of rat striatal adenylate cyclase mediated by D-1 dopamine receptors, guanine nucleotides, and sodium fluoride. AB - We report here that forskolin acts in a synergistic manner with dopaminergic agonists, guanine nucleotides, or sodium fluoride to potentiate the stimulation of rat striatal adenylate cyclase mediated by these reagents. In the presence of 100 microM GTP, 100 microM guanyl-5'-yl imidodiphosphate [Gpp(NH)p], or 10 mM NaF, there is a greater than additive increase in forskolin-stimulated enzyme activity as well as a concomitant decrease (two- to fourfold) in the EC50 value for forskolin stimulation of striatal enzyme activity. In the presence of various concentrations of forskolin (10 nM-100 microM), the stimulation of adenylate cyclase elicited by GTP, Gpp(NH)p, and NaF is potentiated 194-1,825%, 122-1,141%, and 208-938%, respectively, compared with the stimulation by these agents above basal activity in the absence of forskolin. With respect to 3,4 dihydroxyphenylethylamine (dopamine) receptor-mediated stimulation of striatal enzyme activity, the stimulation of enzyme activity by dopaminergic agonists, in the absence or presence of forskolin, was GTP-dependent and could be antagonized by the selective D-1 antagonist SCH23390 (100 nM), indicating that these effects are mediated by D-1 dopamine receptors. In the presence of 100 microM GTP, forskolin at various concentrations markedly potentiates the stimulation elicited by submaximal as well as a maximally effective concentrations of dopamine (100 microM) and SKF38393 (1 microM). At higher concentrations of forskolin (10-100 microM) the stimulation elicited by the partial agonist SKF38393 is comparable to that of the full agonist dopamine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3005509 TI - [3H]5-hydroxytryptamine binding to brain astroglial cells: differences between intact and homogenized preparations and mature and immature cultures. AB - [3H]5-hydroxytryptamine ([3H]serotonin) binds with high affinity (KD 2-12 nM) to a finite number of sites on brain astroglial cells. The number of binding sites in the C6 glioma line is decreased significantly (Bmax = 315 versus 30 fmol/mg) by homogenization. In intact primary cultures, derived from newborn rat brain, the number of binding sites is far greater in cultures of immature astrocytes than in cultures treated with dibutyryl cyclic AMP (Bmax = 1,520 versus 580 fmol/mg). A role for these receptors in development is suggested. PMID- 3005510 TI - Genetic expression of cyclic GMP phosphodiesterase activity defines abnormal photoreceptor differentiation in neurological mutants of inherited retinal degeneration. AB - We have examined cyclic GMP concentrations, guanylate cyclase activities, and cyclic GMP phosphodiesterase (PDE) activities in developing retinas of congenic mice with different allelic combinations at the retinal degeneration (rd) and retinal degeneration slow (rds) loci. Although guanylate cyclase activities were found to be uniformly low in the mutant retinas, striking differences in PDE activity and cyclic GMP levels were observed in retinas of the various genotypes. Homozygous rds mice, which lack receptor outer segments, showed reduced retinal PDE activity and cyclic GMP concentration in comparison to normal animals. In heterozygous rds/+ mice with abnormal outer segments, the levels were intermediate. In retinas of homozygous rd mice, PDE activity was lower than in rds retinas and cyclic GMP levels were much higher. In mice homozygous for both rd and rds genes, retinal PDE activities were even lower than in single homozygous rd mice; the cyclic GMP level reached the same high value as in the rd animals, persisted for a longer time at this high level, and did not correlate with the rate of photoreceptor cell loss. Thus, a marked variation in PDE activity appears to be the major manifestation of abnormal outer segment differentiation and eventual degeneration of photoreceptor cells in these neurological mutants. An increased cyclic GMP level seems to be an essential corollary in the expression of the rd gene even in the absence of outer segments, but it appears unlikely that an abnormally high nucleotide level in itself causes photoreceptor cell death. PMID- 3005511 TI - Activation of beta-adrenergic receptors stimulates release of an inhibitory transmitter from astrocytes. AB - Activation of beta-adrenergic receptors on astrocytes in primary cell culture results in the release of taurine, an inhibitory transmitter. Taurine release occurs via a cyclic AMP-mediated intracellular pathway, because (a) taurine release and intracellular cyclic AMP accumulation have similar pharmacologies and time courses of activation and (b) N6,O2'-dibutyryl cyclic AMP stimulates release with a time course similar to that observed with the beta-adrenergic agonist isoproterenol. These results describe a previously unrecognized physiological function of astrocytes in the CNS-receptor-mediated release of the neuroactive amino acid taurine. This observation indicates that astrocytes may function as local regulators of neuronal activity. PMID- 3005512 TI - Is GABA-stimulated [3H]flunitrazepam binding modulated by benzodiazepine receptor ligands? PMID- 3005513 TI - Herpes simplex virus: a role in the aetiology of Alzheimer's disease? PMID- 3005514 TI - Legionella brain stem encephalopathy and peripheral neuropathy without preceding pneumonia. PMID- 3005515 TI - Collagenase activity in skin fibroblasts of patients with amyotrophic lateral sclerosis. AB - In the current study we have measured collagenase activity released from skin explants and fibroblasts of patients with both Guam-type and sporadic amyotrophic lateral sclerosis and controls. The rationale for such a study derives from work reported more than 20 years ago demonstrating abnormalities in skin collagen metabolism in patients with the disease. We were not able to find significant differences in collagenase activity when fibroblasts were compared relative to the total protein secreted. This is explained, in part, by our finding of an increase in total protein released from fibroblasts of the amyotrophic lateral sclerosis patient group. Increased collagenase release did occur when activity was expressed per number of cells plated but was not statistically significant. In addition, increased release followed a 3-day lag period in skin organ culture. These results suggest that collagenase and other enzymes known to activate collagenase, such as plasminogen activator, capable of degrading extracellular matrix components might be responsible for the increased collagenolytic activity previously observed in amyotrophic lateral sclerosis patients' skin. Further evaluation of extracellular-acting degradative enzymes from skin, muscle, nerve and central nervous system may be important to follow-up such leads in understanding the pathogenesis of this enigmatic and fatal disorder. PMID- 3005516 TI - Localization of cytomegalovirus proteins and genome during fulminant central nervous system infection in an AIDS patient. AB - Approximately one-half of autopsied acquired immune deficiency syndrome (AIDS) patients demonstrate probable human cytomegalovirus (CMV) infection of the central nervous system (CNS). Because CMV in brain tissue or cerebrospinal fluid is difficult to culture, we used antisera, and radioactive probes to diagnose CMV infection in the brain of an autopsied AIDS patient, who died of a fulminant CNS and systemic infection with CMV, suggesting a complete seeding of the ependymal regions possibly followed by a uniform ventriculofugal spread of the virus deep into the parenchyma. Cytomegalic cells were observed in optic nerve, retina, ependymal and subependymal regions of the brain and in the motor (but not sensory) root-CNS junctions. Immunocytochemistry demonstrated viral antigen predominantly in cytomegalic cells, which also stained positively for glial fibrillary acidic protein, S-100, or neuron-specific enolase, but not a common leukocyte antigen. Virions were visible in these cells examined by electron microscopy. No viral replication was observed in pineocytes, pituicytes or the choroid plexus. Morphologically normal cells that were CMV antigen-negative proved to be infected after in situ hybridization with well-defined human CMV DNA fragments. Hence, morphologically normal glia and neurons show restricted replication of CMV, indicating that such cells may be latently infected. PMID- 3005517 TI - Microscopic globular bodies in the human brain. AB - Microscopic globular bodies (MGB) in the neuropil of gray matter are different from other previously reported structures such as corpora amylacea, axonal spheroids and Hirano bodies. They are brilliantly eosinophilic with the hematoxylin and eosin (H&E) stain, measure 1-10 microns in diameter, and are seen in the neuropil of the gray matter, chiefly in the cerebral cortex. Ultrastructurally, MGB are osmiophilic and globular, and located within a cytoplasmic process. They are homogeneous, finely granular, and surrounded by a ruffled membrane. The processes in which MGB are found, contain a few organelles or may be empty. Synaptic junctions are often present on the cytoplasmic process containing MGB. Quantitative studies of MGB reveal a striking correlation with age. Compared with controls, MGB are fewer in number in neurological disorders with mental retardation or dementia. Microscopic globular bodies (MGB) are similar to the dense microspheres described by Averback and have been previously reported by the author as eosinophilic globular bodies. The present study discloses some features of MGB to be similar to the bodies described by Averback, although there are some discrepancies in the reported staining reactions and in the interpretation that these bodies have some relationship to senile plaques. In the few animals studied (five rats, one pig and one monkey) MGB were found only in the pig. PMID- 3005518 TI - Presidential address: the histopathology of meningiomas. A reflection of origins and expected behavior? AB - Meningiomas and their principal cells of origin, the arachnoidal "cap" cells are unique in their morphology with multiple and sometimes seemingly contradictory features related to their origins and basic character. Some meningiomas express mesenchymal features either in histologic pattern (fibroblastic, lipo-myxochondro osteoblastic differentiation), participation in other disease processes, e.g. taking part in the formation of rheumatoid nodules, or in storage phenomena shared with other mesenchymal cells of the body. At the same time they may display epithelial features, such as well-formed desmosomes ultrastructurally, papillary formations and intracellular lumina in cells that stain positively for various cell markers usually considered to characterize epithelial cells. Histologic similarities of meningiomas to various gliomas, schwannomas, neuroblastomas, fibrous histiocytomas, myxomas, chordomas, metastatic carcinomas, and in the cases of meningiomas with marked inflammatory infiltrates, to benign or malignant lymphoproliferative disorders involving the meninges may pose serious diagnostic problems. The localization and resectability of meningiomas are important factors related to long-term prognosis. Of the histologic features hemangiopericytomatous pattern, papillary formations, high cellularity (focal or diffuse) and invasion of the brain appear to correlate with potentially aggressive behavior, whereas cytologically aneuploidia, large number of mitoses, prominent nucleoli and cell necrosis suggest a guarded prognosis. However, some meningiomas with no detectable histologic features of malignancy may nevertheless metastasize to distant sites. PMID- 3005519 TI - Ultrastructure of the rectifying electrotonic synapses between giant fibres and pectoral fin adductor motor neurons in the hatchetfish. AB - Synapses formed by giant fibres on pectoral fin adductor motor neurons were identified by horseradish peroxidase (HRP) injection. The synapses were distributed in clusters on the somata and proximal dendrites of the motor neurons. All of the labelled synapses contained synaptic vesicles and often had clearly defined active zones characteristic of chemical synapses. Some synapses also showed gap junctions with the motor neuron soma, often directly adjacent to an active zone. The gap junctions were asymmetrical, with a thick layer of electron dense material on the postsynaptic side. Previous electrophysiological data indicate that giant fibre inputs to motor neurons are purely electrotonic and that these electrical synapses rectify. PMID- 3005520 TI - Cerebral haemorrhage in arteriovenous malformation associated with Klippel Trenaunay syndrome. AB - The computed tomography, magnetic resonance imaging and angiographic findings are described in a patient with Klippel-Trenaunay syndrome, who also had a cerebral haemorrhage from an arteriovenous malformation. The resulting aphasia disappeared completely after resorption of the haemorrhage. In this syndrome, the occurrence of a cerebral angioma has not previously been mentioned in the literature. PMID- 3005521 TI - Epirubicin: a review of the pharmacology, clinical activity, and adverse effects of an adriamycin analogue. AB - Epirubicin (4'-epidoxorubicin) is an antineoplastic agent derived from doxorubicin. The compounds differ in the configuration of the hydroxyl group at the 4' position. Epirubicin, like doxorubicin, exerts its antitumor effects by interference with the synthesis and function of DNA and is most active during the S phase of the cell cycle. Epirubicin is administered by intravenous (IV) injection. It is metabolized by the liver and primarily eliminated in the bile. About 10% of the drug is eliminated in the urine. Dosage adjustments are recommended for patients with liver metastases or elevated liver function tests. The elimination half-life of epirubicin is 30 to 40 hours. Clinical studies indicate activity in breast cancer, non-Hodgkin's lymphomas, ovarian cancer, soft tissue sarcomas, and pancreatic cancer. There is also evidence of activity against gastric cancer, small-cell lung cancer, and acute leukemia. Epirubicin has limited activity as a single agent against head and neck tumors or non-small cell lung cancer, but may be beneficial in combination with other agents. The overall activity of epirubicin appears to be comparable with that of doxorubicin. However, more studies are needed to define its role in combination chemotherapeutic regimens. The acute dose-limiting toxicity of epirubicin is myelosuppression. Nausea, vomiting, and alopecia are also common. Epirubicin may cause transient cardiac arrhythmias and alterations of the electrocardiogram. Chronic therapy is limited, but available data indicate that epirubicin can be administered in higher cumulative doses than doxorubicin before cardiotoxicity limits further therapy. PMID- 3005522 TI - Generator potentials and spike initiation in auditory fibers of goldfish. AB - Responses were recorded extra- or intracellularly from single, small afferent fibers of the goldfish saccule, that is, S2 fibers, to clarify how the spontaneous and sound-evoked firings in these fibers are initiated from generator potentials. Spontaneously active units randomly selected from the saccular nerve of five goldfish (total 78 units) were classified by the coefficient of variation (CV) of interspike intervals into irregular (59 units; CV greater than 0.3), intermediate (10 units), and regular types (9 units; CV less than 0.23). The irregular type showed a burst (38 units) or random (21 units) pattern of firing in spontaneous activity. In cases where a clear generator potential could be recorded in response to each sound wave, spontaneous generator potentials could also be observed in the absence of stimulus sound. These spontaneous potentials were irregular in amplitude and time course, and often contained components much slower than the sound-evoked generator potentials. The sound-evoked generator potentials in S2 fibers were produced with a delay of 0.4-0.85 ms following each sound wave, and had a time course comparable to the sound-evoked excitatory postsynaptic potentials (EPSPs) of large S1 fibers. They also shared other properties such as adaptive decline in amplitude, incremental and decremental responses, and off-suppression. The mean amplitude of the sound-evoked generator potentials in S2 fibers was linearly related to the rate of afferent firing. There was no apparent difference in the action potential threshold amplitude for spontaneous and sound-evoked generator potentials. It may be concluded that the generator potentials that underlie the spontaneous and sound-evoked firing of S2 fibers are produced by the release of transmitter by hair cells. PMID- 3005523 TI - Probabilistic determination of synaptic strength. AB - This work was carried on to analyze the presynaptic components of synaptic efficacy, which is designated by the term of synaptic strength. To assess the relations between synaptic strength and innervation density, the properties of unitary Cl(-)-dependent inhibitory postsynaptic potentials (IPSPs) evoked in the potentials (IPSPs) evoked in the goldfish Mauthner (M-) cell by single impulses in individual presynaptic cells, including quantal release parameters, were compared with the histological features of the same neurons, determined from their reconstructions after intracellular injection with horseradish peroxidase (HRP). Because the M-cell is a stereotyped target neuron, comparison of the synaptic strength from different experiments was accomplished by using as a quantitative measure of this parameter the mean unitary IPSP amplitude normalized with respect to the reversal potential for Cl- (i.e., the driving force). In 108 experiments at low stimulus frequency, the majority of the normalized responses (63%) were grouped in a rather narrow range, varying about fourfold, or from 1.5 to 6% of the driving force, with results from stained (n = 46) and unstained (n = 62) cells being the same. In contrast, for the same restricted set of responses, the number of presynaptic terminals (histological n) encompassed a larger range, varying from 3 to 52. Impulses in neurons with quite different complements of terminal boutons could evoke similarly sized, normalized IPSPs, and these two parameters were poorly correlated, with there being, at most, a tendency for the responses to increase with histological n for small values of the latter. Quantal fluctuations in IPSP amplitudes were analyzed according to a binomial model having three parameters, p, which is the probability of release, n, which is the number of releasing units and was previously shown to equal the number of presynaptic boutons or active sites, and q, or the quantal size. The normalized quantal size varied randomly, with a mean value of 0.51% (SD = 0.20) and was relatively independent of n. In contrast, the distribution of p, which ranged from 0.17 to 0.74 (mean = 0.40, SD = 0.155), was skewed to the right; this parameter tended to decrease as a function of increasing n. The normalized unitary inhibitory conductance (g'IPSP) underlying an IPSP is equal to the product of npg'q, where g'q is the normalized quantal conductance.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3005524 TI - Changes in excitability induced by herpes simplex viruses in rat dorsal root ganglion neurons. AB - The physiological properties of rat sensory neurons infected with herpes simplex type 1 viruses and maintained in cell culture were studied using intracellular recording techniques. Two syncytial (cell fusing) and two nonsyncytial strains of virus were examined; individual strains of virus had different effects on neuronal excitability. The nonsyncytial viruses caused a loss of tetrodotoxin sensitive low-threshold action potentials and blocked hyperpolarization-activated inward rectification, but did not alter the resting membrane potential, depolarization-activated outward rectification, or render the cells leaky. These effects develop progressively over the period 5-15 hr postinfection. One syncytial strain of virus induced spontaneous electrical activity that appeared to be the result of discrete electrical coupling between sensory neuron processes; as a result, action potential discharge is synchronized in coupled neurons. A second syncytial strain of virus rendered neurons inexcitable; however, in these experiments the input resistance fell to low values, possibly as a result of extensive coupling between sensory neurons. Viral replication in sensory neurons was demonstrable with indirect immunofluorescence using an antibody to herpes simplex viruses and correlated with the onset of virally induced changes in excitability. Virally triggered changes in excitability were blocked by the specific herpes virus antimetabolite acyclovir, suggesting that viral adsorption and penetration are by themselves insufficient to evoke changes in excitability. These results suggest that herpes viruses have selective effects on the excitable mechanisms in sensory neurons that are not simply the result of a general loss of membrane conductances or the disruption of transmembrane ion gradients. PMID- 3005525 TI - Long-term enhancement of hippocampal synaptic transmission and the acquisition of spatial information. AB - The hypothesis that memories are stored as a specific distribution of strengths in a population of modifiable synapses was examined by the bilateral induction of long-term enhancement in synapses of the main afferent fiber system to the hippocampal formation in rats. Brief, high-frequency activation of the perforant pathway in chronically prepared animals resulted in a persistent increase in the field EPSP and population spike, measured extracellularly in fascia dentata. This treatment resulted in a profound and persistent deficit in the acquisition of new spatial information in a task requiring spatial "reference" memory, and disruption of recently acquired spatial information. Well-established spatial memory was completely unaffected, however, as was the acquisition of spatial information into short-term "working" memory. These results support the hypothesis that, during the formation of "cognitive maps," spatial information must be temporarily stored at modifiable synapses at the input stage to the hippocampal formation, but that this information is not needed once the representation of the environment is well established. Spatial working memory, in a familiar environment, appears not to depend on the distribution of synaptic strengths in this system at all. PMID- 3005526 TI - Primary lymphoma of the brain: transient disappearance of the CT image under corticosteroid therapy. One case. PMID- 3005527 TI - Phase II trial of interferon-beta for treatment of recurrent glioblastoma multiforme. AB - Twelve patients were admitted to a Phase II study on the treatment of recurrent glioblastoma multiforme with interferon-beta (IFN-beta). All patients had previously undergone craniotomy and received a standard course of radiation therapy. Recurrence was inferred from enlargement of the lesion on computerized tomography (CT) scanning and in each case was confirmed by CT-guided stereotaxic biopsy. Treatment consisted of combined intravenous (10 X 10(6) IU/day) and intratumoral (1 X 10(6) IU every other day) administration of IFN-beta over three 10-day cycles. This regimen was well tolerated, with toxicity requiring temporary dose modifications in five patients. As judged from data from historical cases, however, the patients admitted to this study demonstrated no clear improvement in mean survival time. The findings of this study also emphasize the importance of distinguishing between radiation necrosis and tumor recurrence. PMID- 3005528 TI - Computer-assisted stereotaxic laser resection of intra-axial brain neoplasms. AB - Computer interpolation of stereotaxic computerized tomography (CT) scanning data allows the transposition of a tumor volume in stereotaxic space. A stereotaxically directed and computer-monitored CO2 laser is then utilized to vaporize that volume as the surgeon monitors the position of a cursor representing the laser beam against planar contours of the tumor displayed on an operating room computer monitor. Computer-assisted stereotaxic laser microsurgery provides precise three-dimensional control for aggressive resection of deep seated tumors from neurologically important areas with acceptable postoperative results. Thus, a significant cytoreduction can be achieved in addition to providing a tissue diagnosis and internal decompression. The authors report 83 computer-assisted stereotaxic laser procedures for tumor excision in 78 patients. The tumors were located in the thalamus/basal ganglia in 15 patients, ventricular system in five, corpus callosum in four, brain stem in three, and deep and centrally in the hemispheres in 51. Histologically, there were 26 glioblastomas, seven grade III astrocytomas, 14 grade II astrocytomas, 14 metastatic tumors, nine vascular lesions, and eight miscellaneous lesions. Resection of these subcortical lesions was confirmed by postoperative contrast-enhanced CT scanning. Neurological examinations performed 1 week after the 83 procedures revealed that 48 patients had improved from their preoperative level and 23 were unchanged (12 were neurologically normal preoperatively). Twelve patients had an increase in a preoperative neurological deficit, three of whom died in the postoperative period: one from infection, one from pulmonary emboli, and one from brain-stem edema. The average survival period (37.6 weeks) of patients having glioblastomas treated by this technique and irradiation was no different from that of patients having glioblastomas in more favorable locations treated by conventional surgery and irradiation. Patients with circumscribed lower-grade astrocytomas did better in terms of morbidity and completeness of resection than those with infiltrative neoplasms. Other circumscribed lesions, such as metastatic tumors, vascular lesions, and intraventricular tumors, were easily resected by the technique described. PMID- 3005529 TI - Effects of collagenase and chymopapain on spinal nerves and intervertebral discs of cynomolgus monkeys. AB - In order to test the safety and efficacy of Nucleolysin, a collagenase for intradiscal chemotherapy, laminectomies were performed on the L2-3 intervertebral discs of four groups of three young adult Cynomolgus monkeys. One primate from each group was injected with half the recommended human dose of Nucleolysin, chymopapain, or the same volume of sterile water. The remaining half of the human dose of each drug or equal volume of sterile water was equally divided and placed upon the right L-3 and L-4 nerve roots at their vertebral foramina. The right L-4 nerve root was first compressed for 10 seconds with an aneurysm clip. These procedures were done to simulate inadvertent contact of enzyme with spinal nerves in patients undergoing chemonucleolysis. After 4 weeks of observation, the 12 primates were humanely killed and examined post mortem. The effects of both enzymes were limited to those tissues with which they came in direct contact. Complete digestion of the nucleus pulposus of all enzyme-injected intervertebral discs was observed. Variable portions of the anulus fibrosus (from 2.3% to 57.4%) were also dissolved. Direct contact of Nucleolysin with lumbar nerve roots caused minor perineural reaction and no more intraneural changes than seen in sterile water controls. Chymopapain induced mild to severe perineural skeletal muscle necrosis and fibrosis with perineural arterial lesions as well as a degenerative neuropathy which was more marked in the traumatized nerve. The results of this study suggest that Nucleolysin and chymopapain are approximately equally effective on intervertebral discs, and that Nucleolysin is less injurious to spinal nerve roots and perineural tissue at the doses used. PMID- 3005530 TI - Surgical experience following intervertebral discolysis with collagenase. AB - Of 410 patients with refractory herniated lumbar disc disease treated with intradiscal collagenase, 82 (20%) did not respond to enzyme treatment and subsequently underwent surgery. Failure to improve in 6 to 8 weeks was the predominant cause for surgical intervention (53 patients). Increased pain (18 patients), progressing neurological deficit (10 patients), and disc-space infection (one patient) were the other indications for surgery. At surgery, extrusions and/or sequestrations were found in 46 patients, undigested protrusions in 16 patients, and other causes of treatment failure in 14 patients. Six patients had normal findings. There was no evidence of adverse enzyme activity on the surrounding structures. Surgical results showed an overall success rate of 87%, and did not appear to be compromised by the previous enzyme therapy. PMID- 3005531 TI - Peripheral nerve entrapment due to steroid-induced lipomatosis of the popliteal fossa. Case report. AB - A case of peroneal nerve entrapment is reported in a patient with scleroderma. Compression was due to a lipoma in the popliteal fossa and resulted in increasingly severe foot-drop. Complete recovery occurred after the lipoma was resected. A brief review of peroneal nerve palsies and lipomatosis is presented. PMID- 3005532 TI - Extracranial internal carotid artery occlusion associated with ipsilateral cerebral glioblastoma. AB - A case of left extracranial internal carotid artery occlusion, associated with ipsilateral parietal glioblastoma, is reported. The clinical and radiological aspects are discussed. The Authors stress the usefulness of a complete investigation in patients with symptoms of cerebrovascular insufficiency. PMID- 3005534 TI - Excision of sclerosing osteomyelitis and reconstruction with particulate hydroxylapatite. AB - A new technique for treating diffuse sclerosing osteomyelitis is described. The entire lesion is eliminated and the ridge bulk and form are immediately reconstructed by implantation of hydroxylapatite. Three cases are described. PMID- 3005533 TI - Studies on the early changes in rat hepatic fructose 2,6-bisphosphate and enzymes in response to a high protein diet. AB - Pertinent hepatic metabolites and enzymes were examined in rats fed a high carbohydrate (HC) diet and during the first 24 h of either starvation or feeding a high protein (HP) diet. Consumption of the HC diet induced slight but definite 24-h oscillations in hepatic concentrations of cyclic AMP, glycogen, glucose 6 phosphate, fructose 2,6-bisphosphate, fructose 1,6-bisphosphate and phosphoenolpyruvate, as well as the activities of 6-phosphofructo-2 kinase/fructose 2,6-bisphosphatase and phosphoenolpyruvate carboxykinase. The transition to starvation or the HP diet induced, within 12 h, concurrent increases in cyclic AMP and phosphoenolpyruvate and decreases in glycogen, glucose 6-phosphate, fructose 6-phosphate, fructose 2,6-bisphosphate and fructose 1,6-bisphosphate. These changes were associated with a decrease in the ratio of 6 phosphofructo-2-kinase/fructose-2,6-bisphosphatase and an increase in phosphoenolpyruvate carboxykinase. These results suggest that the activity of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle is similar during the first 24 h of starvation or HP consumption. PMID- 3005535 TI - Biomechanical optimization of a model particulate composite for orthopaedic applications. AB - Particulate composites are a potential solution to the need for an injectable, biocompatible, resorbable material that could be used to reinforce fractures and defects in bone and temporarily to stabilize porous ingrowth prostheses. We have developed a model system for producing and testing particulate composites to determine if mechanical properties suitable for orthopaedic applications can be achieved. The experiments used bovine cortical bone and various forms of hydroxyapatite for the particulate phase and a collagen and particulate reinforce gelatin-resorcinol-formaldehyde (G-R-F) adhesive for the matrix phase. Using unconfined compression testing, we measured the effects of variation in particulate type, size, shape, and volume fraction on the material properties of the particulate composites. We found that compressive strengths greater than 10 MPa and compressive moduli greater than 100 MPa could be achieved in this model system. Rough and irregular particulates exhibited higher compressive strengths and moduli than smooth and spherical particulates. Mechanical properties were largely independent of particulate size in the range of 125-850 microns diameter. This model system suggests that, with the development of new biocompatible matrix materials, particulate composites with mechanical properties suitable for orthopaedic applications can be achieved. PMID- 3005536 TI - Increased incidence of herpes zoster in normal children infected with varicella zoster virus during infancy: community-based follow-up study. AB - We surveyed outbreaks of varicella zoster virus (VZV) and herpes zoster virus, involving 31 outbreaks of chicken pox, in a semiclosed institution in Osaka Japan during the 34 years between 1949 and 1984. Eight hundred forty-nine infants and children who had had clinical varicella during the first 4 years of life and those who had resided in the institution at least 12 to 144 months after the onset of varicella were included in the study. Nine cases of zoster were observed among children who had acquired varicella during the first year of life, but there was no case of zoster in those who had acquired varicella after 1 year of age. In 61,800 person-months of observation, the overall incidence rate of zoster was calculated as 0.15 per 1000 person-months for the population at risk. The incidence rate in children infected with VZV when younger than 2 months was 1.0 per 1000 person-months during the first decade of life. This rate was significantly (P less than 0.005) greater than that (0.19 per 1000 person-months) in children who had varicella when they were 2 to 11 months of age. These observations suggest that zoster occurs at a significantly shorter interval if VZV infection is acquired during infancy. More than 85% of subjects with prior infection were intimately reexposed to epidemic varicella during their residency in the institution, before having zoster. Epidemic reexposure to varicella during follow-up resulted in enhancement of preexisting immunologic reactivity, but did not prevent subsequent zoster in the population studied. PMID- 3005537 TI - Role of respiratory viruses in exacerbations of primary nephrotic syndrome. AB - To determine whether respiratory virus infections (URI) are associated with exacerbation of nephrotic syndrome (NS) in childhood, a prospective two-winter study of 32 children with NS was done. We obtained pre- and post-season viral serologic studies, biweekly nose and throat viral cultures, daily urinalysis, biweekly telephone follow-up for URI and renal complaints, and clinical assessments as indicated. When a URI occurred, viral cultures were done weekly if the child was at home and twice weekly if hospitalized. Sixty-one URIs occurred; the agent was identified in 33 (51.6%) (respiratory syncytial virus 14, influenza virus five, parainfluenza virus five, varicella zoster virus four, adenovirus three, Mycoplasma pneumoniae one, and Chlamydia trachomatis one). Forty-one exacerbations occurred, 71% with URI; 29% had no URI during the preceding 10 days (P less than 0.01). Total relapse occurred in 29 of 41 exacerbations, 69% with URI and 31% without URI (P less than 0.01). Patients with unstable NS had more exacerbations than those with stable NS (15 of 19 (79%) vs four of 13 (31%), P less than 0.001) and more URI (2.32 vs 1.46 per child, P less than 0.05). Exacerbations in patients with minimal change, mesangioproliferative, and focal glomerulosclerosis occurred in 40%, 60%, and 64%, respectively. We conclude that exacerbations and relapses of childhood NS are temporally related to URI. Inasmuch as multiple viral agents were associated with exacerbations, nonspecific host response to infection, not viral antigen or antibody response, may be the link to NS. PMID- 3005538 TI - Further studies of genetic variation in Schistosoma mansoni. AB - Genetic studies on the human blood fluke Schistosoma mansoni were undertaken using starch gel electrophoresis to detect new gene loci and allelic variation. The number of enzyme staining systems useful with S. mansoni was increased from 14 to 34. It was found that unmated female worms stained as well as male worms. Three new polymorphic loci, fructose biphosphatase (FBP), gly-leu dipeptide peptidase (PEP-4), and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were detected. This brings the known number of polymorphic loci to 10 for this species. One locus (FBP) was found to be polymorphic in the PR-1 strain of S. mansoni. This strain was previously reported to be invariant. PMID- 3005539 TI - Hirschsprung's disease: catecholamine content, alpha-adrenoceptors, and the effect of electrical stimulation in aganglionic colon. AB - In order to assess abnormalities in the adrenergic mechanism in the intestine of Hirschsprung's disease, catecholamine concentrations, alpha-adrenoceptors, and the effect of electrical field stimulation were examined in aganglionic segments of colon or rectum. The aganglionic segment had a higher concentration of norepinephrine, assayed with high performance liquid chromatography with an electrochemical detector, whereas concentrations of epinephrine or dopamine were similar in normal and pathological segments. In four patients with extensive aganglionosis, the norepinephrine concentration in aganglionic colon segments decreased progressively in descending, transverse, and ascending colon. The tissue content of alpha-adrenoceptors and their affinity assayed from the specific binding of [3H]dihydro-alpha-ergocryptine appeared similar in normal and aganglionic segments of the rectosigmoidal colon. Electrical field stimulation of normal rectosigmoidal colon segments caused relaxation at low frequencies and contraction at a very high frequency. Relaxation was not abolished by blocking concentrations of propranolol or phentolamine. In aganglionic segments, the predominant response to electrical field stimulation was contraction, which was inhibited by either atropine or tetrodotoxin. These results indicate that an alpha-adrenergic system and cholinergic innervation apparently exist in aganglionic colon segments and that dysfunction of the colon appears to result from lack of a nonadrenergic inhibitory system. PMID- 3005540 TI - Hepatocellular carcinoma associated with congenital macronodular cirrhosis in a neonate. AB - Hepatocellular carcinoma is rare in infancy. This report describes the first documented case of congenital hepatocellular carcinoma associated with macronodular cirrhosis. PMID- 3005541 TI - Effects of diethyldithiocarbamate, a metabolite of disulfiram, on the pharmacokinetics of alcohol and acetaldehyde in the rat. AB - The effects of diethyldithiocarbamate (DDC), a metabolite of disulfiram which is known as Antabuse, on the blood concentrations of alcohol and acetaldehyde were determined simultaneously by head space gas chromatography in rats. After an intravenous injection of alcohol, the blood concentration of acetaldehyde was much lower than that of the alcohol. A pharmacokinetic model featuring the liver compartment for acetaldehyde was used to estimate pharmacokinetic parameters on the assumption that the distribution volumes of the central compartments were same for alcohol and acetaldehyde, and that the elimination rate of acetaldehyde from liver was large enough to isolate the liver compartment from the central compartment. The results showed that the clearance of alcohol was 0.0226 l/min/kg and the elimination rate constant of acetaldehyde from the liver compartment was very large and 35 min-1. The administration of DDC decreased the above significantly to 0.0132 l/min/kg and 20 min-1, respectively. After intravenous infusion of acetaldehyde, the time course of the blood concentration of acetaldehyde was analyzed by the one compartment model. The estimated elimination rate constants from blood and the distribution volume were in good agreement with those calculated from alcohol injection, indicating the appropriateness of the method used in this study. DDC had no effect on the elimination of infused acetaldehyde from blood indicating that the elimination may be due to the loss from lungs into breath, from skin surfaces and/or from the kidney but not by metabolism in the liver. PMID- 3005543 TI - Involvement of different receptor subtypes for the production of in vitro and in vivo effects in a series of synthetic enkephalin analogues. AB - Enkephalin analogues of tyrosyl group on the N-terminal and Phe-ol or phenylethylamine (PHA) group on the C-terminal which are connected with different chain length were tested for their activities in vitro and in vivo. The inhibitory effect of the synthetic peptides on the electrically evoked contractions of isolated longitudinal muscle strips of guinea pig ileum were weaker than that of morphine or Leu-enkephalin and tended to decrease by increasing the number of methylene group,-(CH2)n-, n = 1-5, between N- and C terminal. Compounds with PHA group on the C-terminal, n = 4 and 5, showed the least activity. The effect of peptides with short chains of methylene groups, n = 1 or 2, and Phe-ol on the C-terminal were antagonized by naloxone but others were insensitive to naloxone. Differing from in vitro activity, compounds with PHA on the C-terminal with a chain of 4 methylene groups produced short lasting analgesia after i.c.v. injection as well as the compounds with Phe-ol on the C terminal and 1 or 2 methylene groups. The analgesic effect of these compounds were completely antagonized by naloxone. At a high i.c.v. dose, all the synthetic peptides, except the one with PHA on the C-terminal with a chain of 4 methylene group, produced convulsions and/or ipsilateral rotation to the injection side. These behavioral effects were not antagonized by naloxone. Thus, minor alterations in the chemical structure of enkephalin analogues resulted in the changes of their receptor selectivity and potencies in vitro and in vivo. PMID- 3005542 TI - Suppression of phenacetin-induced methemoglobinemia by diethyldithiocarbamate and carbon disulfide and its relation to phenacetin metabolism in mice. AB - Oral pretreatment with diethyldithiocarbamate (DTC) and carbon disulfide (CS2) prevented mice from methemoglobinemia induced by phenacetin. This treatment resulted in marked elevation of plasma p-phenetidine concentrations, prolongation of phenacetin levels, and lowering of N-acetyl-p-aminophenol and p-aminophenol levels. Both DTC and CS2 also suppressed p-phenetidine-induced methemoglobinemia with a delay in plasma p-phenetidine disappearance. In vitro, methemoglobin formation by p-phenetidine was decreased in liver microsomes isolated from DTC- or CS2-treated mice. The liver microsomal phenacetin and p-phenetidine O deethylation activities and p-phenetidine N-hydroxylation activity decreased 1 h after administration of DTC or CS2, whereas deacetylation of phenacetin and N acetyl-p-aminophenol by microsomes and acetylation of p-phenetidine by a soluble fraction from a liver homogenate were scarcely affected. The suppression of methemoglobinemia by DTC and CS2 may result from an inhibition of metabolic conversion of p-phenetidine to methemoglobin-forming substances such as N-hydroxy p-phenetidine which is of most importance, p-aminophenol and 2-hydroxy-p phenetidine by the microsomal cytochrome P-450-containing monooxygenase system in the liver. PMID- 3005544 TI - [Studies on Persicae semen. III. Oxygen radical scavenging activity of PR-B, an anti-inflammatory protein of Persicae semen]. PMID- 3005545 TI - Uptake of nourseothricin by the producing microorganism, Streptomyces noursei. AB - The uptake of 14C-(U)-nourseothricin by stationary phase mycelium of Streptomyces noursei JA 3890 b-NG 13/14 was demonstrated. An energy-dependent transport system appears to be involved in the transport of the antibiotic. Relatively large quantities of the antibiotic were adsorbed to the surface of mycelium. Degradation of nourseothricin by the producing microorganism was not detectable. PMID- 3005546 TI - Airway hyperresponsiveness induced by platelet-activating factor: role of thromboxane generation. AB - The effect of platelet-activating factor (acetyl glyceryl ether phosphorylcholine; PAF), a potent inflammatory mediator, on airway responsiveness was studied. In six dogs airway responsiveness was determined by measuring the provocative concentration of acetylcholine aerosol that increased total pulmonary resistance (RL) by 5 cm H2O X L-1 X s, before and after inhalation of PAF (1 mg). PAF caused a 2.3-fold increase in RL (P less than .001) that lasted approximately 30 min. Airway hyperresponsiveness was maximal at 3 hr (mean 3.7-fold increase, P less than .001), persisted at 6 hr (P less than .005) and disappeared by 24 hr. Inhalation of 0.9% NaCl had no effect on RL or on responsiveness. PAF caused an 8 fold increase in neutrophil recovery in bronchoalveolar lavage fluid at 3 hr. OKY 046, a thromboxane synthetase inhibitor, inhibited PAF-induced bronchoconstriction and hyperresponsiveness but did not alter the increase in neutrophil recovery. We tested the specificity of the effect of OKY-046 on the release of cyclooxygenase products from canine neutrophils in vitro; OKY-046 suppressed PAF-induced generation of thromboxane and caused a small increase in prostaglandin F2 alpha release. The studies suggest that the airway hyperresponsiveness induced by PAF may depend on thromboxane generation. PMID- 3005548 TI - Changes in adenosine receptor sensitivity in morphine-tolerant and -dependent mice. AB - Treatment of mice with either a 75-mg morphine pellet (72 h) s.c. or 100 mg/kg of morphine s.c. (3.5 h) did not alter the ED50 of (-)-N6-(phenylisopropyl)adenosine (PIA) in the tail-flick assay. Under the same treatment conditions, caffeine became a more potent antagonist of PIA-induced analgesia, and the dose-response curve for the locomotor effects of caffeine was shifted to the left. There was no change from control in the distribution of caffeine to the brain in mice pretreated with morphine. Brain levels of PIA were decreased significantly at the two highest doses used to elicit analgesia in morphine-implanted mice when compared to control. Adenosine receptor binding assays, utilizing [3H]PIA and [3H]diethylphenylxanthine as agonist and antagonist ligands, respectively, revealed significant increases in the Bmax values for both ligands without changes in Kd in morphine-implanted mice when compared to control. These data suggest that there is an increase in sensitivity to drugs which interact with adenosine receptors in morphine-tolerant and -dependent mice. PMID- 3005547 TI - Glucose transport in human platelets and its inhibition by forskolin. AB - Hexose transport in washed human platelets and its inhibition by forskolin was examined by using 3-O-methylglucose (MG). MG influx in platelets exhibited saturable kinetics with the Km of 1.74 mM and the Vmax of 0.242 mumol/ml of cells X sec at 20 degrees C. Forskolin was found to reduce the Vmax without appreciably affecting the Km, suggesting a noncompetitive mode of inhibition. Forskolin inhibited MG influx with an IC50 value of approximately 2 microM. Forskolin also inhibited the influx of other carbohydrates, including galactose, fructose and ribose. However, forskolin had no effect on adenosine transport. MG transport was abolished instantaneously by forskolin before a significant increase in cellular cyclic AMP content. While both forskolin and prostaglandin l2 stimulated cyclic AMP formation severalfold, only forskolin inhibited MG influx. These findings suggest that the inhibitory action of forskolin on MG transport is unrelated to its well established ability to activate adenylate cyclase and that human platelets possess a hexose transport which is unresponsive to cyclic AMP. PMID- 3005549 TI - Effect of continuous intraventricular estrogen or catechol estrogen treatment on catecholamine turnover in various brain regions. AB - The effect of 7-day i.v.t. administration of catechol estrogens (CE) or estrogens (5 micrograms/day) on the catecholamine turnover rate of various brain areas was examined in ovariectomized rats. Norepinephrine turnover was increased significantly in the hypothalamus and cerebral cortex by estradiol treatment but not by any CEs tested when compared to control values. However, the turnover rate of dopamine in the cerebral cortex was increased compared to control values only by the 2-hydroxyestrogens (2-hydroxyestradiol and 2-hydroxyestrone) and estradiol was without effect. Only estrogens and CEs with physiologically significant estrogen receptor binding affinities (17 beta-estradiol, moxestrol, 2 hydroxyestradiol and 4-hydroxyestradiol) decreased the turnover rate of dopamine in the corpus striatum compared to control values. Estrogens (17 alpha-estradiol and 2-hydroxyestrone) which are weak ligands for the estrogen receptor did not affect striatal dopamine turnover. In addition, body weight gain measured during estrogen treatment was reduced by CEs and estrogens which have significant estrogen receptor affinities. These results suggest that the CEs may play a role in central modulation of catecholaminergic function by estrogens either through direct actions of the catechol moiety or activation of estrogen receptors. PMID- 3005550 TI - Muscarinic cholinergic ligand binding to intact mouse pituitary tumor cells (AtT 20/D16-16) coupling with two biochemical effectors: adenylate cyclase and phosphatidylinositol turnover. AB - (-)-[3H]Quinuclidinyl benzilate (QNB) binding to muscarinic receptors on intact mouse pituitary tumor cells (AtT-20/D16-16) was characterized in an attempt to correlate radioligand binding properties with receptor-coupled biochemical responses. Performing rinse time studies for 2 hr produced a remarkably improved ratio of specific/total (+)-[3H]QNB binding (85%). Kinetic experiments yielded association (k+1) and dissociation (k-1) rate constants of 2.2 X 10(8) M-1 min-1 and 6.8 X 10(-3) min-1, respectively. Receptor occupancy curves demonstrated a uniform population of specific, saturable (-)-[3H]QNB binding sites with a Hill coefficient equal to 1.0 and an apparent dissociation constant (Kd) equal to 34 pM under our conditions. Stereoselectivity was observed with the enantiomers (dexetimide and levetimide) of benzetimide (a factor of 4300). Concentrations of carbachol that produced a half-maximal inhibition of cyclic AMP formation and a concentration of carbachol for producing half-maximal stimulation of phosphatidylinositol turnover in the intact cells were 0.45 and 170 microM, respectively. Schild analysis revealed that pirenzepine, a nonclassical muscarinic antagonist, had a 40-fold greater affinity for reversing carbachol stimulated phosphatidylinositol turnover (inhibition constant or Ki = 7 nM), compared to its antagonism of the carbachol-mediated inhibition of isoproterenol stimulated cyclic AMP formation (Ki = 280 nM). Interestingly, pirenzepine inhibited (-)-[3H]QNB binding with a Ki value of 72 nM. In contrast, atropine was nearly equipotent (Ki = 0.3-0.5 nM) in binding studies and in both effector systems. PMID- 3005551 TI - Beta-1 receptor is the predominant beta-adrenoreceptor on rat brown adipose tissue. AB - A new in vitro radioligand binding assay is described for brown adipose tissue using the beta adrenergic antagonist [3H]CGP 12177 (4-(3-t-butylamino-2 hydroxypropoxy)-[5,7-3H]benzimidazol-2-one). Binding was saturable and stereoselectively inhibited by propranolol. There was 60 to 80% specific binding using either 30 microM l-isoproterenol or 10 microM l-propranolol to define nonsaturable binding. [3H]CGP 12177 was bound to partially purified membranes from collagenase-separated brown adipocytes with a Kd of 0.84 nM, as determined from kinetic studies, and 1.24 +/- 0.13 nM as found by equilibrium binding studies; maximum binding was 14.2 +/- 0.9 fmol/mg of protein. Membranes from whole-pad homogenates had a similar Kd of 1.17 +/- 0.14 nM but twice the maximum number of binding sites (28.5 +/- 4.4 fmol/mg of protein). Intact brown adipocytes had a Kd of 0.55 nM and a maximum binding of 29.4 +/- 1.5 fmol X 10( 6)/cell or 17,700 sites per cell. Competitive binding studies showed about 80% of the binding sites to be of the beta-1 and 20% of the beta-2 subtype. The pA2 values derived from inhibition of isoproterenol-stimulated in vitro oxygen consumption in intact brown adipocytes by the beta-1 selective antagonist metoprolol and beta-2 selective lCl 118551 were in close agreement with their respective K1 values at the beta-1 receptor as derived from competitive binding studies. These data strongly suggest that the beta-1 adrenoreceptor on brown adipose tissue is primarily responsible for the initiation of thermogenesis in this tissue. PMID- 3005552 TI - D-1 and D-2 dopamine receptor blockade: interactive effects in vitro and in vivo. AB - Haloperidol, at low concentrations that block D-2 dopamine (DA) receptors but not D-1 DA receptors (less than 10 microM), potentiated the enhancement of adenylate cyclase activity produced by the D-1 agonist SKF 38393. Low concentrations of haloperidol (less than or equal to 5 microM) also potentiated the K+-evoked release of [3H]acetylcholine from superfused striatal tissue slices. Both of these effects of haloperidol were blocked by nanomolar concentrations of SCH 23390, a D-1 receptor antagonist. In addition, SCH 23390 reduced the ability of haloperidol to antagonize the inhibition of [3H]acetylcholine release produced by the DA agonist apomorphine. By itself, SCH 23390 did not alter either basal adenylate cyclase activity or the K+-evoked release of [3H]acetylcholine. These findings suggest that SCH 23390 can attenuate in vitro responses to D-2 receptor blockade. Likewise, in vivo, very low doses (less than 1 microgram/kg) of SCH 23390 reduced the ability of haloperidol to elevate striatal DA metabolite concentrations and plasma prolactin concentrations. Thus, D-1 receptor blockade may attenuate the effects of D-2 DA receptor blockade both in vitro and in vivo. PMID- 3005553 TI - Alpha-1 adrenoceptors mediate splanchnic nerve inhibition of pentagastrin-induced gastric acid secretion and mucosal blood flow in rats. AB - Effects of stimulation of the sympathetic nervous system on pentagastrin-induced increases in gastric acid secretion and mucosal blood flow (MBF) were studied in anesthetized, gastric fistula rats. Gastric acid secretion and MBF were increased by i.v. infusion of a submaximal dose of pentagastrin (1.0 microgram/kg/min). Stimulation of the splanchnic nerve was carried out under the steady, increased state of pentagastrin-stimulated acid secretion and MBF. Stimulation of the splanchnic nerve significantly reduced gastric acid secretion, while MBF was little affected. Thus, the delta MBF/delta H+ ratio was significantly increased during this stimulation. Phentolamine, but not propranolol, abolished the inhibitory effects of splanchnic nerve stimulation on the pentagastrin-stimulated gastric acid secretion. In addition, prazosin, but not yohimbine, abolished the inhibitory effects of splanchnic nerve stimulation on the pentagastrin-induced gastric acid secretion. These results suggest that splanchnic nerves directly inhibit pentagastrin-induced gastric acid secretion through alpha-1 adrenoceptors, independently of changes in the MBF, in rats. PMID- 3005555 TI - Antagonism by nifedipine of alpha-1 and alpha-2 adrenoceptor-mediated responses of human digital arteries. AB - This study examines the effects of calcium-entry blockade by nifedipine on alpha 1 and alpha-2 adrenoceptor-mediated contractile responses of the human isolated digital artery to exogenous agonists and sympathetic nerve stimulation. Spiral strips of digital artery were mounted for isometric tension recording in physiological salt solution. Contractile responses to TL-99 and to norepinephrine in the presence of prazosin were effectively antagonized by rauwolscine and are therefore presumably mediated by alpha-2 adrenoceptors. Responses to norepinephrine in the presence of rauwolscine were antagonized by prazosin and are therefore mediated predominantly by alpha-1 adrenoceptors. Responses to methoxamine were antagonized by prazosin and, to a lesser extent, by rauwolscine, suggesting that this agonist is also activating alpha-2 adrenoceptors. Nifedipine antagonism of contractile responses was assessed as the percentage of inhibition of the area under the agonist concentration-effect curve or nerve stimulation frequency-effect curve. Nifedipine (10(-9) to 10(-7) M) inhibited responses to TL 99 and methoxamine to similar extents. Nifedipine (10(-8) and 10(-7) M) also had similar effects on the alpha-1 and alpha-2 adrenoceptor-mediated responses (in the presence of rauwolscine and prazosin, respectively) to either exogenous norepinephrine or sympathetic nerve stimulation. At concentrations up to 10(-6) M, nifedipine failed to reduce the efflux of [3H]norepinephrine produced by sympathetic nerve stimulation at either 2 or 8 Hz. We conclude that the alpha-1 and alpha-2 adrenoceptor-mediated contractile responses of the human digital artery are dependent to similar extents on calcium entry through the slow calcium channels of the cell membrane. PMID- 3005554 TI - Determination of molecular size of alpha-1 and alpha-2 adrenoceptors in rat mesenteric artery by radiation inactivation. AB - Radiation inactivation of alpha-1 and alpha-2 adrenoceptors in the purified plasma membranes of rat mesenteric artery has been performed with high energy electrons at -45 to -55 degrees C. Alpha-1 and alpha-2 adrenoceptor inactivation was monitored with [3H] prazosin and [3H]yohimbine binding, respectively. Internal endogenous and external standards of known molecular weight were used in these studies to determine the molecular size. The average value of D37 for the [3H]prazosin binding site was 6.75 +/- 0.62 Mrad (n = 4) with an estimated molecular size of 122,921 +/- 11,329 Daltons. However, the average value of D37 for the [3H] yohimbine binding site was higher (D37 = 10.05 +/- 0.91 Mrad) and accordingly the molecular size of this binding site was less than the [3H]prazosin binding sites (molecular weight = 82,540 +/- 7478 Daltons; n = 4). Irradiation did not change the dissociation constant of either radioligand, suggesting that the loss of the radioligand binding sites after radiation is due to receptor protein inactivation. These results confirm our earlier finding that [3H]prazosin and [3H]yohimbine bind to two distinct sites in the plasma membranes of rat mesenteric artery. Whether both of these sites are the subunits of a common macromolecule of alpha adrenoceptor on vascular smooth muscle in rat mesenteric artery cannot be concluded from these results. This report is the first one in the literature on the molecular size of alpha-1 and alpha-2 binding sites in vascular smooth muscle. PMID- 3005556 TI - Binding of [3H]prazosin to porcine aortic membranes: interaction of calcium antagonists with vascular alpha-1 adrenoceptors. AB - The characteristics of [3H]prazosin binding and the interaction of Ca antagonists with alpha-1 adrenoceptors in the porcine aortic membranes were investigated. The binding characteristics of [3H]prazosin, namely, the kinetics and affinity of binding, saturability, competition by adrenergic agonists and antagonists, stereoselectivity and the localization of binding sites, indicated that [3H]prazosin binds specifically to the alpha-1 adrenoceptors in the sarcolemma of porcine aortic smooth muscle cells. In the inhibition study by several Ca antagonists, the specific binding of [3H]prazosin to aortic membranes was inhibited by verapamil (Ki = 0.66 microM), D600 (Ki = 0.86 microM), nicardipine (Ki = 2.3 microM) and d-cis diltiazem (Ki = 9.8 microM). Nifedipine and nitrendipine, potent dihydropyridine Ca antagonists, only partially inhibited the [3H]prazosin binding, up to 10(-4) M. l-Cis and dl-trans diltiazem, the less potent stereoisomers as Ca channel blockers compared with the d-cis form, showed a similar and greater potency as a competitor to alpha-1 adrenoceptors, respectively. These observations indicate that verapamil, D600, nicardipine and diltiazem interact with vascular alpha-1 adrenoceptors and that the potency of these compounds as a competitor to alpha-1 adrenoceptors does not parallel their potency as Ca channel blockers. PMID- 3005557 TI - Central alpha-2 adrenoceptor-mediated hypertensive response to clonidine in conscious, normotensive rats. AB - Possible involvement of central alpha-2 adrenoceptors in the hypertensive response to i.c.v. injected clonidine was investigated in free-moving, normotensive rats. Clonidine (2-50 micrograms) injected i.c.v. produced a dose dependent and long-lasting pressor response associated with bradycardia in conscious rats, but a long-lasting depressor response in anesthetized rats. The pressor response to clonidine (20 micrograms i.c.v.) was antagonized in a dose dependent manner by central (i.c.v.) pretreatment with yohimbine (20-100 micrograms) and was abolished by a high dose (100 micrograms), whereas the same dose of yohimbine injected i.v. had less effect on the response. Central pretreatment with prazosin (10 and 20 micrograms) inhibited, but did not abolish, the pressor response to clonidine. However, systemic (i.v.) pretreatment with the same dose of prazosin (10 and 20 mu) was more effective in reducing the clonidine induced pressor response than central pretreatment with the drug. The pressor response to clonidine (20 micrograms i.c.v.) was not significantly modified by central pretreatment with pyrilamine (50 and 100 micrograms), cimetidine (50 and 100 micrograms), ketanserin (50 and 100 micrograms) or procaine (100 micrograms). The selective alpha-2 adrenoceptor agonist, BHT-920, injected i.c.v. (5-50 micrograms) also produced a dose-dependent pressor response which was abolished by either anesthesia or central pretreatment with yohimbine, but not with prazosin, whereas the selective alpha-1 adrenoceptor agonist, methoxamine (10-100 micrograms i.c.v.), caused a slight increase in mean blood pressure only at higher doses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3005558 TI - Interaction of the renin-angiotensin system and the renal nerves in the regulation of rat kidney function. AB - Stimulation of the renal sympathetic nerves in pentobarbitone anaesthetized rats achieved a 13% reduction in renal blood flow, did not change glomerular filtration rate, but reduced urine flow by 37%, absolute sodium excretion by 37%, and fractional sodium excretion by 34%. Following inhibition of converting enzyme with captopril (0.38 mmol kg-1 h-1), similar nerve stimulation reduced both renal blood flow and glomerular filtration rate by 16%, and although urine flow and absolute sodium excretion fell by 32 and 31%, respectively, the 18% fall in fractional sodium excretion was significantly less than that observed in the absence of captopril. Renal nerve stimulation at low levels, which did not change either renal blood flow or glomerular filtration rate, reduced urine flow, and absolute and fractional sodium excretions by 25, 26 and 23%, respectively. In animals receiving captopril at 0.38 mmol kg-1 h-1, low-level nerve stimulation caused small increases in glomerular filtration rate of 7% and urine flow of 12%, but did not change either absolute or fractional sodium excretions. At one-fifth the dose of captopril (0.076 mmol kg-1 h-1), low-level nerve stimulation did not change renal haemodynamics but decreased urine flow, and absolute and fractional sodium excretions by 10, 10 and 8%, respectively. These results showed that angiotensin II production was necessary for regulation of glomerular filtration rate in the face of modest neurally induced reductions in renal blood flow and was compatible with an intra-renal site of action of angiotensin II preferentially at the efferent arteriole. They also demonstrated that in the rat the action of the renal nerves to decrease sodium excretion was dependent on angiotensin II. PMID- 3005559 TI - Adenosine increases synaptic facilitation in the in vitro rat hippocampus: evidence for a presynaptic site of action. AB - The effect of adenosine on paired synaptic responses was characterized in the CA1 region of the rat hippocampus in vitro. Adenosine increased the degree of synaptic facilitation at a 40 ms conditioning-testing interval under all conditions tested. Even when the stimulation intensity was increased so as to counteract the direct depressant effect of adenosine on synaptic transmission, its effect on facilitation was maintained. The ability of adenosine to increase synaptic facilitation was a complex function of several variables. The effect was enhanced by increasing the calcium concentration of the medium, and was most pronounced at short conditioning-testing intervals and at low response amplitudes. Adenosine was particularly efficacious in blocking the depression of synaptic responses observed in high-calcium medium at short conditioning-testing intervals. Because this depression most probably reflects depletion of the available store of releasable transmitter, one mechanism by which adenosine could reverse this effect would be by blocking the depletion of transmitter. These results suggest that adenosine diminishes transmitter release via an action at the presynaptic terminal. The reduction in the release of neurotransmitter, particularly at excitatory synapses, may be responsible for the depressant effects of adenosine upon the central nervous system. PMID- 3005560 TI - Systemic sclerosis and malignancy--are they related? PMID- 3005561 TI - Purine enzyme levels in rheumatoid arthritis. AB - We studied purine metabolism in rheumatoid arthritis (RA), adenosine deaminase (ADA), 5'-nucleotidase (5'NU) and purine nucleoside phosphorylase (PNP) activities by measuring the circulating mononuclear cells of patients with RA and healthy controls. Patients had significantly lower levels of ADA and 5'NU but not of PNP than controls. The decreases could not be related to age, antiinflammatory therapy, decreased percentages of T cells or imbalance between major T cells subsets. Differences in cell maturation or traffic could account for our observation. Alternatively, abnormalities of purine metabolism are not definitely excluded in RA if the lower enzyme activity is not sufficient to perform the metabolic steps. PMID- 3005562 TI - IgMk monoclonal antibody directed against peripheral nerve myelin: clinical peripheral neuropathy and longterm rheumatic disease. PMID- 3005563 TI - Malignant histiocytosis. PMID- 3005564 TI - Cutaneous metastasis (cancer en cuirasse and carcinoma erysipelatoides): a non invasive search for the primary cancer using microscopical techniques on urine and skin. PMID- 3005565 TI - Synthesis, structure, and antitumor activity of N-salicyloyl-N'-(2 furylthiocarbonyl)hydrazine and its copper(II) complex. AB - N-Salicyloyl-N'-(2-furylthiocarbonyl)hydrazine (H2sfth) and its Cu(II) complex [Cu(sfth)] were prepared and characterized by physicochemical studies. The IR and ESR spectral studies imply dibasic tetradentate behavior of the ligand bonding through "thiolo" sulfur, enolic oxygen, and hydrazinic nitrogens in a polymeric structure. The electronic spectrum of the complex indicates a square-planar geometry around Cu(II). Maximum antitumor activity was observed when 25 mg/kg dose levels of H2sfth and Cu(sfth) were injected intraperitoneally in mice bearing either solid fibrosarcoma or ascites Dalton's lymphoma. However, H2sfth appeared to possess better antitumor activity as demonstrated by higher T/C (percent) values than those observed for Cu(sfth). The appearance of lymphocytes, leukocytes, and macrophages within the tumor mass 2-6 days after treatment are indicative of involvement of the host's immune system. PMID- 3005566 TI - Synthesis and antiviral activity of (E)-5-(2-bromovinyl)uracil and (E)-5-(2 bromovinyl)uridine. AB - (E)-5-(2-Bromovinyl)uracil (BVU) and (E)-5-(2-bromovinyl)uridine (BVRU) were synthesized starting from 5-formyluracil via (E)-5-(2-carboxyvinyl)uracil or starting from 5-iodouridine via (E)-5-(2-carbomethoxyvinyl)uridine and (E)-5-(2 carboxyvinyl)uridine, respectively. Depending on the choice of the cell system, BVU and BVRU exhibited a marked activity against herpes simplex virus type 1 (HSV 1) in vitro. Although BVU and BVRU were less potent than the reference compound (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), their antiviral activity spectrum was remarkably similar to that of BVDU. The latter findings suggest that BVU and BVRU are metabolically converted to BVDU or a phosphorylated product thereof. In vivo, BVU protected mice against a lethal disseminated HSV-1 infection. PMID- 3005567 TI - Glycine antagonists. Synthesis, structure, and biological effects of some bicyclic 5-isoxazolol zwitterions. AB - The bicyclic 5-isoxazolol zwitterions 4,5,6,7-tetrahydroisoxazolo[4,3-c] pyridin 3-ol (3, iso-THPO), 5,6,7,8-tetrahydro-4H-isoxazolo [4,3-c]azepin-3-ol (12, iso THAO), and 5,6,7,8-tetrahydro-4H-isoxazolo [3,4-c]azepin-3-ol (13, iso-THIA), which are structurally related to the glycine antagonist 5,6,7,8-tetrahydro-4H isoxazolo[3,4-d]azepin-3-ol (iso-THAZ), have been synthesized and tested biologically. All of these compounds were glycine antagonists approximately equipotent with iso-THAZ during microelectrophoretic ejection near cat spinal neurons. In contrast to iso-THAZ, which also interacts with 4-aminobutyric acid (GABA) receptors in rat brains, neither 12 nor 13 show any significant affinities for GABA binding or uptake mechanisms in vitro. The glycine antagonist 3 was, however, shown also to be a moderately potent inhibitor of GABA uptake. The structure of 12 was established by an X-ray analysis. The bond lengths of the 5 isoxazolol anionic moiety of 12 are in agreement with a pronounced delocalization of the negative charge of this compound. PMID- 3005568 TI - Antiarrhythmic activity of 17 beta-aminoestratrienes. Comparison of 3-ols and 3 acetates with the corresponding 3-(3-amino-2-hydroxypropyl) ethers. AB - The antiarrhythmic efficacy of 17 beta-amino- and 17 beta-amino-16 alpha hydroxyestratrien-3-ols and 3-acetates (group 1) was compared with the efficacy of corresponding 3-[2-hydroxy-3-(isopropylamino)propyl] and 3-[2-hydroxy-3-(tert butylamino)propyl] ethers (group II), substituents which are usually associated with beta-adrenoceptor blocking activity. Group I compounds exerted potent antiarrhythmic activity against both aconitine-induced arrhythmias in mice and ischemia-induced arrhythmias in rats and reduced the maximum following frequency of isolated guinea pig atria. Electrophysiological studies indicated that their mechanism of action is due to an ability to reduce the fast inward sodium current in cardiac cells (class I antiarrhythmic action). Group II compounds were inactive in the aconitine and atrial tests and electrophysiological studies confirmed that they were devoid of class I activity. However, these compounds, like both class I antiarrhythmic and beta-adrenoceptor blocking drugs, were active against ischemia-induced arrhythmias. Group II compounds, unlike group I compounds, exerted nonspecific beta-adrenoceptor blocking actions, which may account for their activity in the rat test. It was concluded that introduction of the 3-substituted ether group did not confer any advantage over the parent 3-ol or 3-acetate compounds. PMID- 3005569 TI - Conformationally restricted inhibitors of angiotensin converting enzyme: synthesis and computations. AB - A series of inhibitors of angiotensin converting enzyme (ACE, dipeptidyl carboxypeptidase, EC 3.4.15.1) is described which addresses certain conformational aspects of the enzyme-inhibitor interaction. In this study the alanylproline portion of the potent ACE inhibitor enalaprilat (2) is replaced by a series of monocyclic lactams containing the required recognition and binding elements. In order to more fully assess the lactam ring conformations and the key backbone angle psi as defined in 3 with respect to possible enzyme-bound conformations, a series of model lactams was investigated with use of molecular mechanics. The results point to a correlation between inhibitor potency (IC50) and the computed psi angle for the lowest energy conformation of the model compounds. Thus the psi angle as defined in 3 is an important determinant in the binding of inhibitors to ACE. The inhibition data in conjunction with the computational data have served to define a window of psi angles from 130 degrees to 170 degrees which seems to be acceptable to the ACE active site. PMID- 3005570 TI - Mapping the turkey erythrocyte beta receptor: a distance geometry approach. AB - Extensions and refinements of the receptor mapping method as originally developed by Crippen are presented. In a set of newly developed algorithms measures are taken to reduce the number of required energy parameters to a statistically acceptable degree. The most important measure is the incorporation of lipophilicity as a hydrophobic bonding parameter to describe the binding of parts of the ligands to lipophilic areas on the receptor. In order to test the applicability of our set of programs, we mapped the turkey erythrocyte beta receptor using a data set of Bilezikian. It was found that the experimentally determined free energies of binding can be reasonably described using a nine point geometrical representation of the receptor site and only six energy parameters. The deduced model predicts that the phenyl rings of phenylethanolamines and phenoxypropanolamines occupy different parts of the receptor site. PMID- 3005571 TI - Pyrazolo[4,5-c]quinolines. 2. Synthesis and specific inhibition of benzodiazepine receptor binding. AB - A series of 1-aryl-3,5-dimethyl-4,5-dihydro-1H-pyrazolo[4,5-c]quinolin-4-ones (2a e) and 1-aryl-3-methyl-1H-pyrazolo[4,5-c]quinolines (3-7a-e) bearing different substituents at position 4 were prepared and tested for their ability to displace specific [3H]flunitrazepam binding from bovine brain membranes. The 5-N-methyl derivatives 2a-c,e were the compounds that bound with the highest affinity within this class. The replacement of the carbonyl group with other substituents and the resulting aromatization of the pyridine moiety greatly decreased the binding affinity. From a Lineweaver-Burk analysis on the most active compound 2b, it appears that the inhibition is a competitive one. PMID- 3005572 TI - Crystal structure of the dihydropyridine Ca2+ antagonist felodipine. Dihydropyridine binding prerequisites assessed from crystallographic data. AB - The molecular structure of the dihydropyridine Ca2+ antagonist felodipine (ethyl methyl 1,4-dihydro-2,6-dimethyl-4-(2,3-dichlorophenyl)-3,5-pyridinedicarboxy late) has been determined by X-ray crystallographic methods. The dihydropyridine ring in this potent smooth muscle relaxant is among the flattest found in such structures. This is in qualitative agreement with previous investigations of dihydropyridine Ca2+ antagonists; deviations from planarity in the dihydropyridine ring are generally smallest in the most active compounds. Hydrogen-bonding patterns observed in the crystal lattices of several dihydropyridine Ca2+ antagonists are compared. Antiperiplanar carbonyl groups are partly shielded from forming hydrogen bonds in compounds with relatively bulky ortho phenyl substituents. Conformational prerequisites for a favorable hydrogen bonding geometry toward a receptor site may thus involve synperiplanar carbonyl groups. PMID- 3005574 TI - Ribose-modified adenosine analogues as adenosine receptor agonists. AB - Analogues of the potent adenosine receptor agonist (R)-N-(1-methyl-2 phenylethyl)adenosine (R-PIA), modified at N9, were prepared and evaluated for adenosine A1 and A2 receptor binding and in vivo central nervous system and cardiovascular effects. The modifications at N9 include deoxy sugars, 5' substituted-5'-deoxyriboses, non-ribose sugars, sugar ring homologues, and acyclic sugar analogues. Most of the derivatives have poor affinity for adenosine receptors. Only minor modifications at C5' and C3' maintain potent binding. In general, those derivatives exhibiting in vivo behavioral or cardiovascular effects also have the highest affinity for adenosine receptors. PMID- 3005573 TI - Influence of alkyl-chain fluorination on the action of mammary tumor inhibiting 2,3-bis(hydroxyphenyl)butanes and 2,3-bis(hydroxyphenyl)but-2-enes. AB - trans-1,2-Bis(trifluoromethyl)-1,2-bis(4- and 3-hydroxyphenyl)ethenes 2 and 4 were prepared by reductive coupling (TiCl4/Zn/pyridine) of the methoxy substituted alpha, alpha, alpha-trifluoroacetophenones, separation of the resulting cis- and trans-stilbene derivatives, and ether cleavage with BBr3. The cis-stilbenes were catalytically hydrogenated to give meso-1,1,1,4,4,4-hexafluoro 2,3-bis(4- and 3-hydroxyphenyl)butanes 6 and 8. Compounds 2, 4, 6, and 8 showed 2 to 10-fold increased binding affinities for the estradiol receptor (E2R) and enhanced estrogenicity in the uterine weight test of the immature mouse compared to their unfluorinated analogues. Compound 8 exhibited a 46% inhibition of the estrone-stimulated uterine growth. Antitumor activity was evaluated with use of the transplantable, hormone-dependent MXT mammary tumor of the BD2F1 mouse. All compounds showed tumor growth inhibitory activity corresponding to their RBA values. The most interesting compound 8 led to a significant inhibition of the tumor growth on the DMBA-induced hormone-dependent mammary carcinoma of the Sprague-Dawley rat. PMID- 3005575 TI - New structure-activity relationships of the quinolone antibacterials using the target enzyme. The development and application of a DNA gyrase assay. AB - A series of 60 newly synthesized and known quinolone antibacterials, including quinoline- and 1,8-naphthyridine-3-carboxylic acids, pyrido[2,3-d]pyrimidine-6 carboxylic acids, and some monocyclic 4-pyridone-3-carboxylic acids, were tested and compared in a newly established, easy to perform, DNA gyrase assay. The results were correlated with minimum inhibitory concentrations (MICs) against a variety of organisms. Among the known quinolones were 14 clinically significant drugs (oxolinic acid, norfloxacin, ciprofloxacin, enoxacin, etc.) which were used as standards and compared side-by-side. The study focused on the changes in DNA gyrase inhibition brought about by certain features of the molecules, namely, the C6-fluorine or the nature of the C7 substituent. The intrinsic gyrase inhibition of the fused parent rings, quinoline vs. naphthyridine vs. pyrido[2,3 d]pyrimidine, was also explored. In all cases, loss of enzyme inhibition produced poor MICs, but some compounds with good DNA gyrase inhibition did not correspondingly inhibit bacterial growth. Possible explanations for this phenomena and the benefits of a DNA gyrase-MIC strategy for developing future structure-activity relationships are discussed. PMID- 3005577 TI - Synthesis and antiviral activity of sulfonamidobenzophenone oximes and sulfonamidobenzamides. AB - To find antiviral agents, various sulfonamidobenzophenone oximes (II) were synthesized from the appropriate m-sulfonamidobenzophenones by hydroxylamine reaction. The reaction products were generally obtained as syn/anti mixtures which were separable by fractional crystallization. The anti isomer had more potent antipoliovirus activity than the syn isomer. Various sulfonamidobenzamides (III) which were structurally related to II were synthesized by the reactions of amino-substituted benzamides with sulfuryl chloride or amines with (aminosulfonyl)benzoyl chloride. Antiviral activity was examined by the plaque inhibition test. Compounds 5, 36, and 69 exhibited strong antipicornavirus activity. The structure-activity relationships are discussed. PMID- 3005576 TI - Angiotensin converting enzyme inhibitors as antihypertensive agents: 1-[(2 mercaptocycloalkyl)carbonyl]-L-prolines. AB - The synthesis of 1-[(2-mercaptocyclopentyl)carbonyl]-L-prolines, 1-[(2 mercaptocyclobutyl)carbonyl]-L-prolines and related benzoyl derivatives as pure isomers is described. The abilities of all the compounds to inhibit angiotensin converting enzyme (ACE) in vitro and in vivo and to lower the systolic blood pressure in renal hypertensive dogs were determined. Three of them, namely 1-[[2 (benzoylthio)cyclopentyl]carbonyl]-L-proline (10f(R,S], 1-[(2 mercaptocyclopentyl)carbonyl]-L-proline (10g(R,S], and 1-[[2 (benzoylthio)cyclobutyl]carbonyl]-L-proline (16f(R,S], were found to be as potent as captopril in reducing blood pressure. The influence of chirality and ring size on the ACE inhibition is described. PMID- 3005578 TI - Antipicornavirus activity of substituted phenoxybenzenes and phenoxypyridines. AB - Phenoxybenzenes and phenoxypyridines were prepared and tested for the effect of substituents on antipicornavirus activity. The most active compound, 2-(3,4 dichlorophenoxy)-5-nitrobenzonitrile (8), demonstrated broad-spectrum antipicornavirus activity. Compound 8 and several analogues each given orally prior to and during infection protected mice against an otherwise lethal challenge with coxsackievirus A21. PMID- 3005579 TI - Meiotic recombination between two polymorphic restriction sites within the beta globin gene cluster. AB - Analysis of beta globin gene haplotypes for prenatal diagnosis of beta thalassaemia has revealed a recombination event within the beta globin gene cluster. Both a change in the AvaII polymorphic site within the beta globin gene and a change in the phenotype of the beta globin gene were observed. Paternity was established by the pedigree analysis of hypervariable 'minisatellite' DNA polymorphisms and the most probable explanation of the recombination event is a crossover between the psi beta globin gene and the beta globin gene. The data provide direct evidence in support of a DNA region 3' to the beta globin gene with a recombination frequency much higher than expected, and have important implications for the prenatal diagnosis of beta thalassaemia by linked restriction fragment length polymorphisms. PMID- 3005580 TI - Evidence against the structural gene encoding type II collagen (COL2A1) as the mutant locus in achondroplasia. AB - The structure of the locus encoding the major cartilage collagen gene (COL2A1) was studied in a total of 19 cases of achondroplasia. No gross rearrangements were seen. The segregation of COL2A1 was examined in three affected kindreds using restriction site and length variants as genetic markers. In two kindreds discordant segregation between the achondroplasia and COL2A1 loci was demonstrated. Paternity/maternity was confirmed using a 'minisatellite' core sequence probe which reveals cross hybridising polymorphic loci. PMID- 3005581 TI - Haemagglutination by the Bacteroides fragilis group. AB - Adhesive properties of five species of Bacteroides were compared by direct haemagglutination with erythrocytes of different origin. Only strains of Bacteroides fragilis agglutinated erythrocytes and different patterns of haemagglutination were observed. None of eight carbohydrates tested inhibited haemagglutination. The activity was destroyed by heat and by periodate treatment, but not by three proteases tested. PMID- 3005583 TI - Intracellular calcium activity in split frog skin epithelium: effect of cAMP. AB - Measurement of intracellular calcium activity (acCa) by ion-selective microelectrodes has previously been technically limited to relatively large cells (greater than or equal to 20 micron). We now report results obtained with this technique in the small epithelial cells (less than or equal to 10 micron) of split frog skin using microelectrodes having an outer tip diameter of less than 0.2 micron. The basolateral membrane potential was measured with Ca2+-selective microelectrodes (EscCa) and with reference micropipettes (psi sc) either sequentially or simultaneously in 15 successful experiments. Under baseline conditions, acCa was measured to be 215 +/- 39 nM (mean +/- SE), in close agreement with the mean values estimated from published data obtained with Necturus proximal tubule. Stimulation of Na+ transport across six skins with 1 mM serosal 8 p-chlorophenylthio-3,5' cyclic AMP (CPTcAMP) increased acCa by a factor of 2.6 +/- 0.6. The increase in acCa preceded the CPTcAMP-induced increase in Isc. The results of the present study indicate that electrometric determination of intracellular calcium activity is now feasible in a much wider range of cell systems than heretofore possible. CPT cAMP elevates intracellular Ca2+ activity; this phenomenon is an early event, preceding the natriferic effect of CPTcAMP. PMID- 3005585 TI - The effects of dietary fibre in a liquid diet on bowel function of mentally retarded individuals. AB - A soy polysaccharide at a level of 20-22.3 g/day was added to a liquid formula diet provided to constipated, tube-fed, nonambulant, severely or profoundly mentally retarded individuals. This amount of polysaccharide contained 15.6-17.4 g of dietary fibre or 4.9-5.5 g of neutral detergent fibre. The fibre formula was well tolerated by the subjects and increased stool size and improved stool consistency. At these levels of fibre and short duration of the study, defecation rate and need for elimination aids were unaffected. Transit times of 5 to 6 days were unchanged but mean daily stool weights increased to a level equivalent to low normal values for healthy adults on a low fibre intake. PMID- 3005584 TI - Substrate and inhibitor specificity of anion exchangers on the brush border membrane of rabbit ileum. AB - In previous studies we have found that several anions can be transported by an exchange process in rabbit ileal brush border membranes. We demonstrated exchanges of C1 for OH or HCO3, SO4 for OH, oxalate for OH, and oxalate for C1. The purpose of these studies was to determine the number of distinct carriers mediating these exchanges. We utilized substrate and inhibitor specificity studies to distinguish between different anion exchange transporters. We conclude that SO4:OH and oxalate:OH exchange occur on the same carrier because: (i) pH gradient stimulated transport of both 14C-oxalate and 35SO4 were equally sensitive to cis-inhibition by unlabeled SO4 or oxalate; and (ii) both were inhibited equally by K. We conclude that oxalate:OH and oxalate:C1 exchanges occur on different carriers because: (i) C1 or SO4 caused unequal cis-inhibition of these two exchanges; and (ii) as compared to oxalate:C1 exchange, oxalate:OH exchange was more sensitive to inhibition by probenecid and K and less sensitive to inhibition by bumetanide. Finally, we conclude that oxalate:C1 exchange and C1:HCO3 exchange occur on different carriers because: (i) C1:HCO3 exchange was almost completely insensitive to cis-inhibition by oxalate; and (ii) oxalate:C1 exchange was more sensitive to inhibition by DIDS and bumetanide than C1:HCO3 exchange. Thus we have found that there are at least three separate anion exchangers on rabbit ileal brush border: a Cl:HCO3 exchanger; a SO4:OH exchanger, which also transports oxalate; and an oxalate:C1 exchanger. PMID- 3005582 TI - Evaluation of ion gradient-dependent H+ transport systems in isolated enterocytes from the chick. AB - The experiments reported here evaluate the capability of isolated intestinal epithelial cells to accomplish net H+ transport in response to imposed ion gradients. In most cases, the membrane potential was kept constant by means of a K+ plus valinomycin "voltage clamp" in order to prevent electrical coupling of ion fluxes. Net H+ flux across the cellular membrane was examined at pH 6.0 (the physiological lumenal pH) and at pH 7.4 using methylamine distribution or recordings of changes in media pH. Results from both techniques suggest that the cells have an Na+/H+ exchange system in the plasma membrane that is capable of rapid and sustained changes in intracellular pH in response to an imposed Na+ gradient. The kinetics of the Na+/H+ exchange reaction at pH 6.0 [Kt for Na+ = 57 mM, Vmax = 42 mmol H+/liter 30MG (3-O-methylglucose) space/min] are dramatically different from those at pH 7.4 (Kt for Na+ = 15 mM, Vmax = 1.7 mmol H+/liter 30MG space/min). Experiments involving imposed K+ gradients suggest that these cells have negligible K+/H+ exchange capability. They exhibit limited but measurable H+ conductance. Anion exchange for base equivalents was not detected in experiments performed in media nominally free of bicarbonate. PMID- 3005586 TI - Immunocytochemical demonstrations of cytoplasmic and cell-surface EGF receptors in A431 cells using cryo-ultramicrotomy, surface replication, freeze-etching and label fracture. AB - In this paper we describe the use of a number of complimentary methods to visualize cytoplasmic and cell-surface located epidermal growth factor (EGF) receptors in cultured A431 cells. Cryo-ultramicrotomy in combination with immuno gold labelling will be shown to provide an excellent method in visualizing cytoplasmic located EGF receptors in addition to cell-surface located EGF receptors. An important aspect in this method involves the possible effects of the fixatives on antigenicity. Using radioactive labelled anti EGF receptor antibodies, it was shown that formaldehyde as a fixative had no significant effect on label-efficiency. The density and lateral distribution of EGF receptors at the cell surface has been studied by three methods, i.e. surface replication, freeze etching and label fracture, all methods in conjunction with immuno-gold labelling. These methods allow in principle a quantitation of the surface distribution of the EGF receptors. The surface-replication method involves, however, dehydration and critical-point drying steps, and using radioactive labelled anti EGF receptor antibodies it was shown that in particular OsO4 fixation and dehydration caused a significant loss of cell-associated antibodies. This disadvantage is overcome by freeze etching and the label-fracture method, and as such these techniques provide the best methods for quantitative analysis of the planar distribution of cell-surface located EGF-receptors. PMID- 3005588 TI - Acromegaly: a case report. PMID- 3005587 TI - Modulation of mammalian immunity by electromagnetic radiation. AB - There have been reports that electromagnetic radiation (EMR) alters the function of the immune system; however, these reports are often contradictory. This review reexamines the literature and attempts to evaluate the data on potential mechanisms of interaction of EMR on mammalian immune function. This report concludes that there is no convincing evidence that EMR effects on the human immune system are a health hazard. It was suggested by some authors that long term EMR exposure may impair immune surveillance, and hypothetically thus facilitate tumor growth. Additional research is needed to prove or disprove this hypothesis. Available data indicate that EMR exposure does not affect the ability of cells of the immune system to respond to a subsequent challenge. However, the time-course and magnitude of the response may be affected by exposure following stimulation. Research to date provided evidence that at least at some frequencies and/or amplitude and pulse modulations, the site of primary interaction of EMR is at the cell membrane. However, it was shown that one specific response, the increase in B complement-receptor positive lymphocytes (Cr+) in the mouse is under genetic control by a single gene localized on chromosome 5. It is suggested that cells of the immune system are a convenient model for further studies on mechanisms of EMR interaction with living systems. Future research should be directed at exploring beneficial medical applications of EMR modulation of immune responses. PMID- 3005589 TI - The Vi element. A transposon-like repeated DNA sequence interspersed in the vitellogenin locus of Xenopus laevis. AB - A repeated DNA element in Xenopus laevis is described that is present in about 7500 copies dispersed throughout the genome. It was first identified in the 5' flanking region of one vitellogenin gene and was therefore named the Vi element. Seven copies are present within the vitellogenin gene region, three of them within introns of the genes A1, A2 and B2, and the other four copies in the gene flanking regions. Four of these copies have been sequenced. The Vi element is bounded by a well-conserved 13 base-pair inverted repeat; in addition, it is flanked by a three base-pair direct repeat that appears to be site-specific. The length of these four copies varies from 112 to 469 base-pairs; however, sequence homology between the different copies is very high. Their structural characteristics suggest that length heterogeneity may have arisen by either unequal recombinations, deletions or tandem duplications. Altogether, the characteristics and properties of the Vi element indicate that it might represent a mobile genetic element. One of the four copies sequenced is inserted close (position -535) to the transcription initiation site of the vitellogenin gene B2 in a region otherwise showing considerable homology with the closely related gene B1. Nevertheless, the presence of the Vi element does not seem to influence significantly the estrogen-controlled expression of gene B2. In addition, three alleles of this gene created by length polymorphism in intron 3 and in the Vi element inserted near the transcription initiation site are described. PMID- 3005591 TI - Cloning and expression of Clostridium pasteurianum galactokinase gene in Escherichia coli K-12 and nucleotide sequence analysis of a region affecting the amount of the enzyme. AB - The Clostridium pasteurianum galactokinase gene was cloned by complementation, of the galK locus, into Escherichia coli. Restriction enzyme analysis subcloning and Tn5 mutagenesis indicated that the gene was located on a 1.8 X 10(3) base-pair ClaI-Sau3A fragment that encoded a polypeptide of approximately 40 Mr. Although the C. pasteurianum and the E. coli galactokinases have similar subunit molecular weights, Southern hybridization analysis indicated no strong homology between their genes. Even though this clone showed a low level of galactokinase expression, the Gal+ phenotype, provided by the clostridial galactokinase, was unstable in E. coli, and the gene was frequently inactivated by the spontaneous acquisition of insertion sequences. A second clone containing this gene on a large restriction fragment was isolated by hybridization. This clone was unable to grow on galactose-containing media due to the overproduction of galactokinase. Comparison of the plasmids from these two clones revealed that the second contained an additional 300 base-pairs located at one end of the galactokinase gene. Appropriate operon fusions with a promoter-less E. coli galactokinase gene indicated that these additional 300 base-pairs had promoter activity in E. coli. The DNA sequence of this region which lies upstream of the C. pasteurianum galactokinase gene was determined and compared with that from several clones producing high, low or undetectable amounts of galactokinase. The reasons for the high and low level expression and for the instability of the C. pasteurianum galactokinase in E. coli are discussed. The presence of the galactokinase suggests that galactose is used in C. pasteurianum through the Leloir pathway via galactose 1-phosphate. PMID- 3005590 TI - Intramolecular recombination of linear DNA catalyzed by the Escherichia coli RecE recombination system. AB - Transformation of different Escherichia coli strains by linear dimers of pBR322 containing different tet alleles was investigated. Linear dimers transformed wild type strains 0.1 to 1% as efficiently as circular dimers. In contrast, linear dimers transformed recBrecCsbcA strains, where the RecE recombination system is functional, as efficiently as circular dimers. The transformants contained plasmids that had a single recombinant monomer genotype, indicating that transformation was mediated by a recombination-dependent cyclization reaction. Altering the position of the double-strand break changed the frequency of recovering different recombination products, but had no effect on the frequency of transformation. Both the frequency of transformation and the production of Tcr recombinants were decreased by recE mutations, while recA and recF mutations were slightly stimulatory (twofold). Several recombination models consistent with these results are presented. PMID- 3005592 TI - Structure of ferricytochrome c' from Rhodospirillum molischianum at 1.67 A resolution. AB - The structure of ferricytochrome c' from Rhodospirillum molischianum has been crystallographically refined to 1.67 A resolution using a combination of reciprocal space and restrained least-squares refinement methods. The final crystallographic R-factor for 30,533 reflections measured with I greater than sigma (I) between infinity and 1.67 A is 0.188. The final model incorporates 1944 unique protein atoms (of a total of 1972) together with 194 bound solvent molecules. The structure has been analysed with respect to its detailed conformational properties, secondary structural features, temperature factor behavior, bound solvent sites, and heme geometry. The asymmetric unit of the cytochrome c' crystal contains a dimer composed of chemically identical 128 residue polypeptide chains. Although the refined structure shows the monomers to be very similar, examination of the differences that do occur allows an evaluation of how different lattice contacts affect protein conformation and solvent binding. In particular, comparison of solvent binding sites in the two subunits allows identification of a common set that are not altered by lattice interactions. The preservation of these solvent interactions in different lattice environments suggests that they play a structural role in protein stabilization in solution. The refined structure additionally reveals some new features that relate to the ligand binding properties and unusual mixed-spin state character of cytochrome c'. Finally, comparison of the heme binding geometry in cytochrome c' and other structurally unrelated c-type cytochromes shows that two alternative, but sterically favorable, conformational variants occur among the seven examples examined. PMID- 3005593 TI - Molecular analysis of the Adh region of the genome of Drosophila melanogaster. AB - A small region of the genome of Drosophila melanogaster has been cloned in a series of overlapping phage. A length of 165 X 10(3) base-pairs of contiguous DNA that spans polytene chromosome region 35A4 to 35B1 and includes the structural gene for alcohol dehydrogenase (Adh) as well as at least two other genes, outspread (osp) and no-ocelli (noc), has been characterized by mapping chromosome aberrations to the DNA. The relationship between osp and Adh is surprising: of nine osp alleles associated with chromosome breakpoints, five map distal (i.e. 5') to Adh and four map proximal (i.e. 3') to this gene. None affects the expression of Adh. As defined by these and other breakpoints, the osp gene spans at least 52 X 10(3) base-pairs and overlaps the Adh gene. The noc gene, as defined by the mapping of nearly 30 breakpoints, is at least 50 X 10(3) base pairs in size. Alleles of noc and noc- deletions show either of two kinds of interaction with the recessive lethality of l(2)br29ScoR+1, a lethal that maps immediately distal to noc. One class of noc allele is viable when heterozygous with ScoR+1, while the other class is lethal or semi-lethal. Both classes, however, are homozygous or hemizygous viable. The locations of these two classes of noc allele on the DNA fall into two clusters, with those that are viable with ScoR+1 located proximal to those that are not. The physical boundary between these classes lies at a site just distal to that of the breakpoint of the inversion associated with ScoR+1 itself. PMID- 3005594 TI - Processive action of terminase during sequential packaging of bacteriophage lambda chromosomes. AB - Bacteriophage lambda chromosomes are packaged in a polarized, sequential fashion from a multimeric DNA substrate. Mature chromosomes are generated when terminase introduces staggered nicks in the cohesive end sites (cos sites) bounding a chromosome. Packaging is polarized, to the initial and terminal cos sites for packaging a chromosome can be defined. To initiate packaging, terminase binds to cos at cosB, and subsequently cuts at cosN. To terminate packaging of a chromosome, a functional cosB is not required at the terminal cos. To explain this finding, it was proposed earlier that terminase scans for the terminal cosN, rather than any subsequent cosB, during packaging. In the work described here we performed helper packaging experiments to see whether processive action of terminase occurs during sequential packaging of lambda chromosomes. The helper packaging experiments involve trilysogens; strains carrying three prophages in tandem. Infection by a hetero-immune helper phage results in packaging of the repressed prophage chromosomes, since the prophage structure is analogous to the normal DNA substrate. Two chromosomes can be packaged from between the three cos sites of the prophages of a trilysogen. Both chromosomes are packaged even when the central cos is cosB-. Our interpretation of these data is that terminase is brought to the central cos by packaging; following cleavage of the central cos, the terminase remains bound to the distal chromosome; and terminase acts to begin packaging of the distal chromosome. The frequency at which terminase reads across the central cos to initiate packaging of the distal chromosome is in the range from 0.3 to 0.5 in our experiments. Reading across cos was found not to be greatly dependent on the state of cosB, indicating that cosB binding is only needed for packaging the first chromosome in a packaging series. A multilysogen was constructed in which the initial cos was cos+ and the distal cos sites were all cosB-. The initial and downstream chromosomes were found to be packaged. This result indicates that terminase that is brought to the central cos by packaging is not only able to initiate packaging of a downstream chromosome, but can also scan and terminate packaging of the downstream chromosome. A model is presented in which processive action of terminase is the basis for sequential packaging of lambda chromosomes. PMID- 3005595 TI - Adenosine metabolism in microvessels from heart and brain. AB - Activities of several adenosine metabolizing enzymes were examined in capillary preparations isolated from rabbit ventricle. Vmax and Km values for 5' nucleotidase were 2.3 nmol/min/mg and 10 microM, respectively. For adenosine deaminase the corresponding values were 7.8 nmol/min/mg and 32 microM. S-adenosyl homocysteine hydrolase, which forms adenosine by the hydrolysis of S-adenosylhomo cysteine, was also present (Vmax, 0.07 nmol/min/mg; Km, 0.81 microM), as were adenosine kinase (Vmax, 0.2 nmol/min/mg; Km, 0.52 microM) and purine nucleoside phosphorylase (Vmax, 13.8 nmol/min/mg; Km, 96 microM). These enzymes were also present in microvessels (capillaries and arterioles) purified from rabbit brain. Activities of several enzymes, especially 5'-nucleotidase and adenosine deaminase, were much lower in myocytes isolated from rabbit ventricle. The study provides evidence that endothelial cells of the microvasculature from heart and brain are capable of activity forming and degrading adenosine. It is possible that adenosine formed by these cells may contribute to the local regulation of blood flow. PMID- 3005596 TI - Polyamine metabolism and function in the heart. PMID- 3005598 TI - XII world congress of the International Society for Heart Research. 9-13th February 1986, Melbourne, Australia. Abstracts. PMID- 3005597 TI - 1,25-Dihydroxyvitamin D3 receptors identified in the rat heart. AB - Specific receptors for 1,25-dihydroxyvitamin D3, the active hormonal form of vitamin D3, were demonstrated in low salt chromatin preparations from normal rat hearts. Sucrose gradient analysis of KCl-extracted chromatin yielded a significant (P less than 0.005) peak of specific [3H]1,25-dihydroxyvitamin D3 binding in the 3.6S region. The peak of [3H]1,25-dihydroxyvitamin D3 binding was abolished by excess 1,25-dihydroxyvitamin D3, but not by 50 nM 25-hydroxyvitamin D3 nor by 1.0 microM levels of estradiol-17B, cortisol, or promegestone, demonstrating steroid specificity characteristic for such receptors. Upon Scatchard analysis this putative cardiac 1,25-dihydroxyvitamin D3 receptor yielded a single binding component with high affinity (KD = 0.36 nM) and low capacity (Nmax = 33 fmol/g tissue). Coupled with evidence for the presence of calcium binding proteins in this tissue, these observations suggest functional roles for 1,25-dihydroxyvitamin D3 and its receptors in cardiac muscle, possibly in regulating intracellular effects of calcium. PMID- 3005599 TI - Hepatitis B immune globulin as treatment of CMV infections in patients with AIDS. AB - This report presents three case histories of acquired immunodeficiency syndrome (AIDS) patients with cytomegalovirus (CMV) who showed considerable improvement after receiving hepatitis B immune globulin. PMID- 3005600 TI - AIDS: no time for apathy. PMID- 3005601 TI - Benzo[a]pyrene dione-benzo[a]pyrene diol oxidation-reduction couples; involvement in DNA damage, cellular toxicity, and carcinogenesis. AB - Three isomeric quinone metabolites of the environmental carcinogen benzo[a]pyrene undergo reversible, univalent oxidation-reduction cycles involving the corresponding benzo[a]pyrene diols and intermediate semiquinone radicals. Under anaerobic conditions, benzo[a]pyrene 1,6-dione, benzo[a]pyrene 3,6-dione, and benzo[a]pyrene 6,12-dione are readily reduced by mild biological agents such as NADH and glutathione. The benzo[a]pyrene diols, in turn, are very rapidly autooxidized to diones when exposed to air. Substantial amounts of hydrogen peroxide are produced during these autooxidations. The benzo[a]pyrene diol/benzo[a]pyrene dione interconversions proceed by one-electron steps; the corresponding semiquinone radicals were detected as intermediates when the reactions were carried out at high pH. Benzo[a]pyrene diones are electron acceptor substrates for NADH dehydrogenase. Catalytic amounts of these metabolites, together with this respiratory enzyme, function as cyclic oxidation reduction couples to link NADH and molecular oxygen in the continuous production of hydrogen peroxide. Benzo[a]pyrene diones induce strand scissions when incubated with T7 DNA. The damage is modified by conditions that indicate that reduced oxygen species propagate the reactions responsible for strand scission. Benzo[a]pyrene diones are cytotoxic at low concentrations to cultured hamster cells. The cytotoxic effect can be substantially reduced by depletion of oxygen from the growth medium and the atmosphere in which the cells are incubated. The results support the hypothesis that the biological activity of benzo[a]pyrene diones is due to the regenerative oxidation-reduction cycles involving quinone and hydroquinone forms; activated oxygen species and semiquinone radicals formed during these cycles are most likely responsible for the observed cytotoxic action. The role of activated oxygen species in carcinogenesis is discussed. PMID- 3005603 TI - Effects of monoalkyl dithiocarabamates on mobilization of cadmium in the mouse. AB - Monoalkyl dithiocarbamates are capable of mobilizing cadmium from aged intracellular deposits in which the cadmium is largely present in metallothionein. The sodium salts of monomethyl, ethyl, n-propyl, and n-butyl dithiocarbamates have been prepared, characterized, and examined for their relative ability to mobilize cadmium from such aged deposits in the kidneys, liver, spleen, testes, brain, and pancreas as well as from the whole body, using sodium diethyl dithiocarbamate as the positive control. Alterations in the structure of the dithiocarbamates can be correlated with alterations in organ cadmium levels. The acute toxicity of these compounds is sufficiently greater than disubstituted dithiocarbamates that their use would appear to possess few advantages, although they do seem to be more effective in reducing testicular cadmium levels. PMID- 3005604 TI - Response of Japanese quail fed seed meal from sunflowers grown on a municipal sludge-amended soil: elevation of cadmium in tissues. AB - Sunflowers were grown on soil amended with 224 metric tons/ha of municipal sewage sludge from Syracuse, N.Y. The yield of sunflower seeds was reduced by 47.2% by the sludge addition. The harvested seeds contained 1.71 ppm dry weight of cadmium. Deoiled seed meal was incorporated as 25 and 50% of semipurified diet and fed to male and female Japanese quail. The concentrations of cadmium were higher in kidney, liver, muscle, and eggs of birds fed the sludge-grown seed meal as compared to control quail. Tissue concentrations of cadmium increased with increasing dietary levels of sludge-grown seed meal. No significant differences (p greater than 0.05) were observed between dietary treatments in the activity of hepatic microsomal p-nitroanisole O-demethylase or aminopyrine N-demethylase in the male birds. Additionally, no mutagenic activity, either direct or with metabolic activation, was found in quail eggs. No observable changes in tissue ultrastructure were observed under electron microscopy in any of the treatment groups. There were no significant (p greater than 0.05) differences among the dietary treatment groups in feed intake, growth rate, egg production, or egg hatchability. PMID- 3005605 TI - Woven polyglycolic acid mesh has increased the opportunity for safe splenic repair. PMID- 3005602 TI - Serum angiotensin-converting enzyme levels in patients with silicosis. AB - In a group of control subjects, the mean serum angiotensin-converting enzyme (ACE) level was 38.1 +/- 10.6 nmol/min X ml (n = 30), and in a group of silicosis patients the mean serum ACE level was 45.2 +/- 16.0 (n = 26). Thirteen of these patients were classified as having nodular silicosis, and their mean serum ACE level was 44.2; 10 of these patients were classified as having progressive massive fibrosis, and their mean serum ACE level was 39.4. Three of these patients had confirmed silicotuberculosis, and their serum ACE levels were 63, 67, and 77 (mean = 69); these serum ACE levels are somewhat higher than those having been reported for patients with acute sarcoidosis. Thus, when serum ACE levels are being used to assist in distinguishing between silicosis and sarcoidosis, the possibility of silicotuberculosis must be also considered when high serum ACE levels are encountered. PMID- 3005606 TI - An improved radioimmunoassay for the anabolic agent hexoestrol, using a monoclonal antibody. AB - A monoclonal antibody was raised against hexoestrol coupled to bovine serum albumin. The antibody cross-reacted with the stilbenes, diethylstilboestrol (10%) and dienoestrol (4%), but had no cross-reaction (less than 0.01%) with other anabolic agents. A radioimmunoassay method using the monoclonal antibody has been validated and used to measure residues of hexoestrol in the urine of treated cattle. The limit of detection was 0.6 pg/ml urine at the 95% confidence limit. The results were compared with those obtained using polyclonal antibodies. Although there was a good correlation between the results, the use of monoclonal antibody gave more reliable results than those obtained with available polyclonal antibodies. The monoclonal antibody, because of its quality and theoretically limitless supply, is very suitable for use in large scale screening or monitoring programmes for regulating the use of hexoestrol. PMID- 3005607 TI - Characterization and translation of transmissible gastroenteritis virus mRNAs. AB - Three protein species were identified in purified transmissible gastroenteritis virus particles (strain Purdue). They are thought to represent constituents of the peplomer (E2; molecular weights of 280,000 and 240,000), the envelope (E1; molecular weights of 28,000, 31,500, and 33,000), and the nucleocapsid (N; molecular weight of 48,000). In infected cells, proteins with molecular weights of 195,000 (E2), 48,000 (N), and 28,000 (E1) were detected. Tunicamycin, an inhibitor of N glycosylation, prevented the appearance of polypeptides with molecular weights of 195,000 and 28,000 in infected cells; instead, proteins with molecular weights of 160,000 and 25,000 were observed. One minor and five major mRNA species were detected in porcine cells after infection. Their size was determined to be 23.6 kilobases (kb) (RNA1), 8.4 kb (RNA3), 3.8 kb (RNA4), 3.0 kb (RNA5), 2.6 kb (RNA6), and 1.9 kb (RNA7). The RNAs were translated in vitro. RNA7 was shown to code for the N protein. Although complete separation of RNA6 could not be achieved, it was shown to encode an unglycosylated (molecular weight of 25,000) precursor of E1 (molecular weight of 28,000). RNA4 was translated into a nonstructural protein with a molecular weight of 24,000. Translation of RNA3 resulted in proteins with molecular weights of 250,000 and 130,000 and smaller molecules which could be precipitated with a monoclonal antibody directed against E2. PMID- 3005608 TI - trans activation of the latent Epstein-Barr virus (EBV) genome after transfection of the EBV DNA fragment. AB - Transfection of Epstein-Barr virus (EBV)-nonproducer Raji cells with the BamHI Z fragment of EBV DNA induced antigens that were detected with human antiserum against EBV-specific early antigens. Northern blot analysis of transfected cells revealed that one intense RNA band hybridized with the BamHI H and F fragments but not with the BamHI Z fragment. Cooperation between the BamHI H, F, and BamHI Z regions was also confirmed in baby hamster kidney cells that were cotransfected with both fragments. These results indicate that the transfected BamHI Z fragment of EBV DNA induces a trans-acting factor which activates the gene expression of the BamHI H and F region and that the BamHI Z region possibly plays an important role in the latency of EBV. PMID- 3005609 TI - Characterization of the genes encoding herpes simplex virus type 1 and type 2 alkaline exonucleases and overlapping proteins. AB - A detailed sequence analysis of the herpes simplex virus type 1 (HSV-1) and HSV-2 DNA encoding the alkaline exonuclease mRNA clusters has been completed. Three partially colinear mRNAs (2.3, 1.9, and 0.9 kilobases) are completely encoded within the DNA sequence presented. The putative promoter regions of the transcripts were inserted upstream of a plasmid-borne chloramphenicol acetyl transferase (CAT) gene and assayed for their ability to induce transcription of the CAT gene upon low multiplicity of infection with HSV in transient expression assays. We conclude that the expression of all three transcripts appear to be controlled by individual promoters. The 2.3-kilobase mRNA contains an open translational reading frame sufficient to encode 626 amino acids for the HSV-1 alkaline exonuclease enzyme; this value is 620 amino acids for HSV-2. A comparison of the predicted amino acid sequences of the HSV-1 and HSV-2 alkaline exonuclease enzymes revealed significant amino acid differences in the N-terminal portions of the two proteins; however, computer analyses suggest that the three dimensional structures of the HSV-1 and HSV-2 nuclease enzymes are very similar. The 0.9-kilobase mRNA contains an open reading frame which shares a small amount of out-of-phase overlap with the C-terminal portion of the alkaline nuclease open reading frame. This open reading frame has the capacity to encode a 96-amino-acid polypeptide (10,500 daltons). PMID- 3005610 TI - Ecotropic and mink cell focus-forming murine leukemia viruses integrate in mouse T, B, and non-T/non-B cell lymphoma DNA. AB - Structures of somatically acquired murine leukemia virus (MuLV) genomes present in the DNA of a large panel of MuLV-induced C57BL and BALB/c B and non-T/non-B cell lymphomas were compared with those present in MuLV-induced T-cell lymphomas induced in the same low-"spontaneous"-lymphoma-incidence mice. Analyses were performed with probes specific for the gp70, p15E, and U3-long terminal repeat (LTR) regions of ecotropic AKV MuLV and a mink cell focus-forming virus (MCF)-LTR probe annealing with U3-LTR sequences of a unique endogenous xenotropic MuLV, which also hybridizes with U3-LTR sequences of a substantial portion of somatically acquired MCF genomes in spontaneous AKR thymomas. The DNAs of both T- and B-cell tumors induced by neonatal inoculation with the highly oncogenic C57BL derived MCF 1233 virus predominantly contain integrated MCF proviruses. In contrast, the DNAs of more slowly developing B and non-T/non-B cell lymphomas induced by poorly oncogenic ecotropic or MCF C57BL MuLV isolates mostly contain somatically acquired ecotropic MuLV genomes. Approximately 50% of the spontaneous C57BL lymphoma DNAs contain somatically acquired MuLV genomes. None of the integrated MuLV proviruses annealed with the MCF-LTR probe, which indicates a clear difference in LTR structure with a substantial portion of the somatically acquired MuLV genomes present in the DNA of spontaneous AKR thymomas. This study stresses a dominant role of MuLV with ecotropic gp70 and LTR sequences in the development of slowly arising MuLV-induced B and non-T/non-B cell lymphomas. PMID- 3005611 TI - Ecotropic murine leukemia virus-induced fusion of murine cells. AB - Extensive fusion occurs upon cocultivation of murine fibroblasts producing ecotropic murine leukemia viruses (MuLVs) with a large variety of murine cell lines in the presence of the polyene antibiotic amphotericin B, the active component of the antifungal agent Fungizone. The resulting polykaryocytes contain nuclei from both infected and uninfected cells, as evidenced by autoradiographic labeling experiments in which one or the other parent cell type was separately labeled with [3H]thymidine and fused with an unlabeled parent. This cell fusion specifically requires the presence of an ecotropic MuLV-producing parent and is not observed for cells producing xenotropic, amphotropic, or dualtropic viruses. Mouse cells infected with nonecotropic viruses retain their sensitivity toward fusion, whereas infection with ecotropic viruses abrogates the fusion of these cells upon cocultivation with other ecotropic MuLV-producing cells. Nonmurine cells lacking the ecotropic gp70 receptor are not fused under similar conditions. Fusion is effectively inhibited by monospecific antisera to gp70, but not by antisera to p15(E), and studies with monoclonal antibodies identify distinct amino- and carboxy-terminal gp70 regions which play a role in the fusion reaction. The enhanced fusion which occurs in the presence of amphotericin B provides a rapid and sensitive assay for the expression of ecotropic MuLVs and should facilitate further mechanistic studies of MuLV-induced fusion of murine cells. PMID- 3005612 TI - Transforming Sloan-Kettering viruses generated from the cloned v-ski oncogene by in vitro and in vivo recombinations. AB - The Sloan-Kettering viruses (SKVs) are replication-defective retroviruses that transform avian cells in vitro. Each of the three SKV isolates is a mixture of viruses with genomes ranging in size from 4.1 to 8.9 kilobases (kb) with a predominant genome of 5.7 kb. Using a cDNA representing a sequence, v-ski, that is SKV specific and held in common by the multiple SKV genomes, we generated a restriction map of the 5.7-kb SKV genome and molecularly cloned a ski-containing fragment from SKV proviral DNA. Southern hybridization and sequence analysis showed that the cloned DNA fragment consisted of the 1.3-kb ski sequence embedded in the p19gag sequence and followed by the remaining 5' half of the gag gene and small portions of both the pol and env genes. A large deletion encompassing the 3' half of gag and the 5' 80% of pol was mapped to a position about 1 kb downstream from the 3' ski-gag junction. To determine whether the cloned ski sequence had transforming activity, the ski-containing fragment and a cloned Rous associated virus 1 (RAV-1) genome were used to construct an analog of the 5.7-kb SKV genome, RAV-SKV. Cotransfection of chicken embryo cells with RAV-SKV and RAV 1 yielded foci of transformed cells whose morphology was identical to that induced by the natural SKVs. The transformed transfected cells produced transforming virus with a 5.7-kb ski-containing genome and synthesized a gag containing polyprotein of 110 kilodaltons (kDa). Several nonproducer clones of RAV-SKV-transformed cells were analyzed, and most were found to synthesize a 5.7 kb SKV RNA and a 110-kDa polyprotein. One clone was found to contain an 8.9-kb SKV RNA, and this clone synthesized a 125-kDa polyprotein. Since both the 5.7- and 8.9-kb genomes and the 110- and 125-kDa polyproteins had been identified in studies on the natural SKVs, the present results not only demonstrate the transforming activity of these individual SKVs but also suggest mechanisms for their generation. PMID- 3005613 TI - Characterization of an Epstein-Barr virus-induced thymidine kinase. AB - Previous work from our laboratory suggested that the selective inhibition of Epstein-Barr virus (EBV) replication by 1-beta-D-arabinofuranosylthymine in human lymphoid cell lines involved the induction of a new thymidine kinase (TK) able to phosphorylate the thymidine analog. We further characterized this enzyme induced in various EBV-positive cell lines after viral genome activation with a combination of sodium butyrate and 12-O-tetradecanoylphorbol-13-acetate. The following results confirmed the existence of an EBV-specific deoxypyrimidine kinase: induction of EBV-related TK was connected with the appearance of viral early antigens in EBV-carrying cells; unexpected behaviors of the enzyme activity upon different fractionating treatments led to the conclusion that EBV-induced TK was extracted as a complex molecular form, larger than other known cellular or viral isozymes; enzymatic properties distinguished EBV-induced TK from host lymphoid cell isozymes but made it resemble other herpesvirus-specific deoxypyrimidine kinases, i.e., by partial inhibition by dTTP or ammonium sulfate, insensitiveness to dCTP, and nonstringent specificity for normal TK substrates. Genetic evidence is required to definitively ensure that EBV-specific TK actually is virus coded in EBV-transformed human lymphoid cells. PMID- 3005614 TI - Rous-associated virus 1-induced erythroleukemic cells exhibit a weakly transformed phenotype in vitro and release c-erbB-containing retroviruses unable to transform fibroblasts. AB - Avian leukosis viruses induce erythroblastosis in chicks by integrating into the c-erbB gene and thus activating c-erbB transcription. We characterized Rous associated virus 1-induced leukemic erythroblasts in vitro and showed that they mostly resemble erythropoietin-independent but otherwise normal erythroid progenitors. Some leukemic cells, however, were able to both differentiate and proliferate extensively in vitro. All 14 leukemias studied expressed high levels of erbB-related proteins that were 5 to 10 kilodaltons larger but otherwise very similar to the gp74erbB protein of avian erythroblastosis virus ES4 with respect to biosynthesis, glycosylation, and cell surface expression. Two leukemias contained and released retroviruses that transduced erbB. Chicken embryo fibroblasts fully infected with these viruses expressed high levels of erbB RNA and protein but retained a normal phenotype. Our results suggest that certain forms of c-erbB, activated by long terminal repeat insertion or viral transduction, are capable of inducing erythroleukemia but unable to transform fibroblasts. PMID- 3005617 TI - Papillomavirus DNA associated with pulmonary fibromatosis in European elks. AB - Multiple beadlike fibromas have been observed in the lungs of European elks bearing cutaneous fibromas and fibropapillomas. DNA extracted from the lung fibromas was found to contain multiple copies of unintegrated European elk papillomavirus DNA, indicating an association between pulmonary fibromatosis and papillomavirus. No virus particles were observed in the tumor tissue by electron microscopy. Histological examination revealed that the lung fibromas had a morphology similar to that of cutaneous fibromas. PMID- 3005615 TI - Effects of cyclosporin A on humoral immune response and resistance against vesicular stomatitis virus in mice. AB - The effect of cyclosporin A (CS-A) on the antiviral humoral response was studied by using vesicular stomatitis virus (VSV); VSV provided the opportunity to simultaneously assess both T-independent and T-dependent antibody responses. The T-independent anti-VSV immunoglobulin M (IgM) response was virtually unaffected, whereas the T-dependent primary anti-VSV IgG response was suppressed by CS-A; in contrast, the secondary IgG response was highly resistant to CS-A. Moreover, once the switch from IgM to IgG had occurred, the primary response also became refractory to suppression by CS-A. We concluded that the effect of CS-A on the primary anti-VSV antibody response was mediated via impairment of a T-dependent mechanism; in contrast, memory T cells or memory B cells or both were quite resistant to the suppressive effects of CS-A. CS-A treatment rendered mice highly susceptible to VSV infection; under CS-A treatment, mortality was 100% after infection via footpads, whereas immunocompetent mice survived. Since CS-A does not impair induction of early T-independent anti-VSV IgM neutralizing antibodies, this high mortality in CS-A treated mice illustrates the crucial role of CS-A sensitive cells in resistance against VSV. PMID- 3005616 TI - Murine cytomegalovirus infection of mouse testes. AB - With the aim of illustrating a mechanism of cytomegalovirus (CMV) venereal transmission, we induced murine CMV infection in the mouse testes of immunologically competent mice. Using in situ cytohybridization, we were able to show that murine CMV-specific DNA was associated with spermatocytes and mature sperm. Electron microscopy studies also supported sperm infection. The virus could be reisolated from infected epididymal sperm by cocultivation with mouse embryo fibroblasts. We found no difference in either the sexual performance or the fertilization efficiency of the sperm between infected and uninfected males. PMID- 3005618 TI - Identification and separation of the two subunits of the herpes simplex virus ribonucleotide reductase. AB - The herpes simplex virus ribonucleotide reductase is associated with two viral proteins which are both immunoprecipitated by monoclonal antibodies specific for the enzyme. We separated the two proteins and showed that individual antibodies react solely with one or the other. In addition, antibodies to either protein can neutralize enzymatic activity. Our data demonstrate that the proteins are associated in a complex and constitute the subunits of the enzyme. PMID- 3005619 TI - Monoclonal antibodies specific for v-abl- and c-abl-encoded molecules. AB - Monoclonal antibodies specific for regions of the transforming protein of Abelson murine leukemia virus were prepared. Antibodies directed against the kinase domain inhibited the autophosphorylation of v-abl proteins, and all of the antibodies reacted with the products of the murine and human c-abl loci. PMID- 3005620 TI - Construction of poliovirus intertypic recombinants by use of cDNA. AB - We investigated the use of infectious cDNA for the production of poliovirus type 1-type 3 recombinants. One such recombinant virus was produced, but a second construct involving the transfer of part of the capsid protein region was not infectious. Our results suggest that the approach may prove valuable but that not all cDNA constructs will give rise to viable viruses. PMID- 3005621 TI - New common nomenclature for glycoprotein genes of varicella-zoster virus and their glycosylated products. AB - The accumulation of recent data concerning the reactivity of monoclonal antibodies with particular varicella-zoster virus (VZV) glycoproteins and the mapping of several of their respective genes on the VZV genome has led to a unified nomenclature for the glycoprotein genes of VZV and their mature glycosylated products. Homologs to herpes simplex virus glycoprotein genes are noted. PMID- 3005622 TI - Heterogeneity in base sequence among different DNA clones containing equivalent sequences of rotavirus double-stranded RNA. AB - The nucleotide sequences for several complementary DNA clones of the rotavirus genome were determined. When the sequences obtained from different clones for the same regions (16,000 bases) were compared, differences in eight base positions were observed. These discrepancies, approximately 1 in 2,000 bases, may be due to differences in individual RNA genomes resulting from multiple passages; infidelity of DNA synthesis in the cloning procedure; or both factors. Whatever the cause, this frequency of base substitution found in sequences of complementary DNA obtained from the same isolate should be considered when comparing DNA sequences obtained from independent isolates. On the other hand, the frequency of base changes observed suggests that the rotavirus genome is very conserved since the virus used for cDNA synthesis has been continuously passaged for 6 years without plaque purification. PMID- 3005623 TI - High-frequency RNA recombination of murine coronaviruses. AB - The RNA genome of coronaviruses consists of a single species of nonsegmented RNA. In this communication, we demonstrate that the RNA genomes of different strains of murine coronaviruses recombine during mixed infection at a very high frequency. Susceptible cells were coinfected with a temperature-sensitive mutant of one strain of mouse hepatitis virus (MHV) and a wild-type virus of a different strain. Of 21 randomly isolated viruses released from the coinfected cells at the nonpermissive temperature, 2 were recombinants which differed in the site of recombination. After three serial passages of the original virus pool derived from the mixed infection, the majority of the progeny viruses were recombinants. These recombinant viruses represented at least five different recombination sites between the two parental MHV strains. Such a high-frequency recombination between nonsegmented RNA genomes of MHV suggests that segmented RNA intermediates might be generated during MHV replication. We propose that the RNA replication of MHV proceeds in a discontinuous and nonprocessive manner, thus generating free segmented RNA intermediates, which could be used in RNA recombination via a copy choice mechanism. PMID- 3005624 TI - Activation of enhancer sequences in type II human T-cell leukemia virus and bovine leukemia virus long terminal repeats by virus-associated trans-acting regulatory factors. AB - The ability of the sequences present in the long terminal repeats (LTRs) of human T-cell leukemia viruses type I and II (HTLV-I and HTLV-II) and of bovine leukemia virus to function as enhancer elements was investigated. Recombinant plasmids that contained the HTLV-I, HTLV-II, and bovine leukemia virus LTRs at a distance from a simian virus 40 promoter element located 5' to the bacterial gene encoding chloramphenicol acetyltransferase (EC 2.3.1.28) were constructed. We report that all three LTR sequences contain enhancer elements capable of increasing the level of gene expression directed from a distal heterologous promoter. The enhancer present in the HTLV-I LTR was active in uninfected cells of lymphoid and nonlymphoid origin. In contrast, the enhancer activity of the HTLV-II and bovine leukemia virus LTR sequences was evident only in virus-infected cells. This activity is likely due to virus-associated trans-acting transcriptional factors previously shown to be present in HTLV- and bovine leukemia virus-infected cells. The implication of these observations for virus replication and transforming activity are discussed. PMID- 3005625 TI - Characterization of envelope proteins of infectious bovine rhinotracheitis virus (bovine herpesvirus 1) by biochemical and immunological methods. AB - Ten glycoproteins of molecular weights of 180,000, 150,000, 130,000, 115,000, 97,000, 77,000, 74,000, 64,000, 55,000, and 45,000 (designated as 180K, 150K, etc.) and a single nonglycosylated 107,000-molecular-weight (107K) protein were quantitatively removed from purified bovine herpesvirus 1 (BHV-1) virions by detergent treatment. Immunoprecipitations with monospecific and monoclonal antibodies showed that three sets of coprecipitating glycoproteins, 180K/97K, 150K/77K, and 130K/74K/55K, were the major components of the BHV-1 envelope. These glycoproteins were present in the envelope of the virion and on the surface of BHV-1-infected cells and reacted with neutralizing monoclonal and monospecific antibodies. Antibodies to 150K/77K protein had the largest proportion of virus neutralizing antibodies, followed by antibodies to 180K/97K protein. Monoclonal antibodies to 130K/74K/55K protein were neutralizing but only in the presence of complement; however, monospecific antisera produced with 55K protein did not have neutralizing activity. Analysis under nonreducing conditions showed that the 74K and 55K proteins interact through disulfide bonds to form the 130K molecule. Partial proteolysis studies showed that the 180K protein was a dimeric form of the 97K protein and that the 150K protein was a dimer of the 77K protein, but these dimers were not linked by disulfide bonds. The 107K protein was not glycosylated and induced antibodies that did not neutralize BHV-1. The 64K protein was not precipitated by anti-BHV-1 convalescent antisera, and monospecific antisera to this protein precipitated several polypeptides from uninfected cell lysates, suggesting that 64K is a protein of cellular origin associated with the BHV-1 virion envelope. PMID- 3005626 TI - DNA-binding region of the simian virus 40 tumor antigen. AB - The simian virus 40 (SV40) tumor (T) antigen was purified by immunoaffinity chromatography and cleaved with small amounts of trypsin, and the resulting fragments were subjected to SV40 DNA cellulose chromatography. A 44,000-molecular weight fragment (44K fragment) from the left end of the molecule and a 30K fragment mapping from approximately Lys 131 to Lys 371 bound to the column and were eluted with 1 M NaCl. In a second series of experiments, T antigen was immunoprecipitated with hamster anti-T serum or various monoclonal antibodies and partially digested with trypsin. Fragments that were solubilized by this treatment were tested for DNA-binding activity by using an SV40 DNA fragment binding assay. A 17K fragment which originated from the amino-terminal region of the polypeptide had no apparent binding activity in this assay. On the other hand, larger fragments (76K, 46K, and 30K) whose amino termini were mapped around Lys 131 did display DNA-binding activity. Finally, complexes consisting of SV40 DNA and T-antigen fragments were precipitated in the DNA-binding assay with monoclonal antibodies that recognize the central region of the protein; however, antibodies with specificities to the amino- or carboxy-terminal regions were inactive. These results strongly suggest that the DNA-binding region of T antigen lies approximately between Lys 131 and Lys 371, corresponding to 0.51 and 0.37 map units on the DNA. PMID- 3005627 TI - Differentiation of strains of varicella-zoster virus by changes in neutral lipid metabolism in infected cells. AB - Eleven isolates of varicella-zoster virus were tested for their effects on the incorporation of [14C]acetate into lipids in infected human embryonic lung cells. By relative percent, all virus isolates demonstrated a shift from polar lipid synthesis to neutral lipid, especially triglyceride, synthesis. By data expressed as counts per minute per microgram of protein, the VZV strains could be separated into two groups: those strains which depressed lipid synthesis and those strains which did not depress, and may even have stimulated, lipid, especially triglyceride, synthesis. These results may be useful in understanding the development of lipid changes seen in children affected with Reye's syndrome following chickenpox. PMID- 3005628 TI - Genetic relatedness and colinearity of genomes of equine herpesvirus types 1 and 3. AB - The arrangement and location of homologous DNA sequences within the genomes of equine herpesvirus type 1 (EHV-1) and EHV-3 were investigated by using Southern blot hybridization analyses conducted under stringent conditions. Recombinant plasmid libraries comprising 95 and 84% of the EHV-1 and EHV-3 genomes, respectively, were labeled with 32P-deoxynucleotides by nick translation and were used as probes in filter hybridization studies. The DNA homology between the EHV 1 and EHV-3 genomes was dispersed throughout the genomes in a colinear arrangement. Significant hybridization was detected between the EHV-1 short region inverted repeat sequences, which are known to encode immediate early transcripts, and the corresponding EHV-3 inverted repeat sequences. Interestingly, probes derived from the EHV-1 heterogeneous region, which is adjacent to the EHV-1 short region, hybridized strongly to EHV-3 DNA sequences within a similar genomic location, but did not reveal any corresponding heterogeneity within the EHV-3 genome. Our results demonstrated that there is a highly conserved evolutionary relationship between EHV-1 and EHV-3 and provided the foundation for further investigations to determine whether similarities in protein function underpin the genetic relatedness between these two herpesviruses. PMID- 3005630 TI - Biosynthesis and properties of the adenovirus 2 L1-encoded 52,000- and 55,000-Mr proteins. AB - The adenovirus type 2 L1 region, which is located at 30.7 to 39.2 map units on the viral genome, is transcribed from the major late promoter during both early and late stages of virus replication, and a 52,000-Mr (52K) protein-55K protein doublet has been translated in vitro on L1-specific RNA. To investigate the biosynthesis and properties of the L1 52K and 55K proteins, we prepared antibody against a synthetic peptide encoded near the predicted N terminus. As determined by immunoprecipitation and immunoblot analysis, the antipeptide antibody recognized major 52K and 55K proteins synthesized in adenovirus type 2-infected cells that appeared to be identical to the 52K-55K doublet translated in vitro. The immunoprecipitated 52K and 55K proteins were very closely related, as shown by a peptide map analysis. Both L1 proteins were phosphorylated, and they were phosphorylated at similar sites. No precursor-product relationship was detected between the 52K and 55K proteins by a pulse-chase analysis. Biosynthesis of the L1 52K and 55K proteins began about 6 to 7 h postinfection, after biosynthesis of the early region 1A and early region 1B 19K (175R) T antigens, and reached a maximum rate at about 15 h; the maximum rate was maintained until at least 25 h postinfection. At all times, the 55K protein appeared to be synthesized at a severalfold-higher level than the 52K protein. Both proteins were quite stable and accumulated until late times after infection. Viral DNA replication was not essential for formation of the L1 proteins. Thus, the L1 52K-55K gene appears to be regulated in a manner different from the classical early and late viral genes but similar to the protein encoded by the i-leader (Symington et al., J. Virol. 57:849-856, 1986). The L1 proteins were detected in the cell nucleus by immunofluorescence microscopy with antipeptide antibody and were found to be primarily associated with the nuclear membrane by an immunoblot analysis of subcellular fractions. PMID- 3005629 TI - Bovine leukemia virus protease: purification, chemical analysis, and in vitro processing of gag precursor polyproteins. AB - Bovine leukemia virus protease was purified to homogeneity and assayed by using murine leukemia virus Pr65gag, a polyprotein precursor of the viral core structural proteins, as the substrate. A chemical analysis of the protease, including an amino acid composition and NH2- and COOH-terminal amino acid sequence analysis, revealed that it has an Mr of 14,000 and is encoded by a segment of the viral RNA located between the gag gene and the putative reverse transcriptase gene. As expected from the nucleotide sequence data (Rice et al., Virology 142:357-377, 1985), the reading frame for the protease is different from both the gag and reverse transcriptase reading frames. The 5' end of the protease open reading frame extends 38 codons upstream from the codon for the NH2-terminal residue of the mature viral protease and overlaps the gag open reading frame by 7 codons. The 3' end of the protease open reading frame extends 26 codons beyond the codon for the COOH-terminal residue of the mature protease and overlaps 8 codons of the reverse transcriptase open reading frame. Several lines of evidence, such as protein mapping of the gag polyprotein precursor, the characteristic structure of the mRNA, and promotion of the synthesis of a gag polyprotein precursor by lysine tRNA in vitro, suggest that the protease could be translated by frameshift suppression of the gag termination codon. In vitro synthesized bovine leukemia virus gag-related polyproteins were cleaved by the protease into fragments which were the same size as the known components of bovine leukemia virus, suggesting that the specificity of cleavage catalyzed in vitro by the purified protease is the same as the specificity of cleavage found in the virus. PMID- 3005631 TI - Biosynthesis of adenovirus type 2 i-leader protein. AB - The i-leader is a 440-base-pair sequence located between 21.8 and 23.0 map units on the adenovirus type 2 genome and is spliced between the second and third segments of the major tripartite leader in certain viral mRNA molecules. The i leader contains an open translational reading frame for a hypothetical protein of Mr about 16,600, and a 16,000-Mr polypeptide (16K protein) has been translated in vitro on mRNA selected with DNA containing the i-leader (A. Virtanen, P. Alestrom, H. Persson, M. G. Katze, and U. Pettersson, Nucleic Acids Res. 10:2539 2548, 1982). To determine whether the i-leader protein is synthesized during productive infection and to provide an immunological reagent to study the properties and functions of the i-leader protein, we prepared antipeptide antibodies directed to a 16-amino acid synthetic peptide which is encoded near the N terminus of the hypothetical i-leader protein and contains a high acidic amino acid and proline content. Antipeptide antibodies immunoprecipitated from extracts of adenovirus type 2-infected cells a major 16K protein that comigrated with a 16K protein translated in vitro. Partial N-terminal amino acid sequence analysis by Edman degradation of radiolabeled 16K antigen showed that methionine is present at residue 1 and leucine is present at residues 8 and 10, as predicted from the DNA sequence, establishing that the 16K protein precipitated by this antibody is indeed the i-leader protein. Thus, the i-leader protein is a prominent species that is synthesized during productive infection. The i-leader protein is often seen as a doublet on polyacrylamide gels, suggesting that either two related forms of i-leader protein are synthesized in infected cells or that a posttranslational modification occurs. Time course studies using immunoprecipitation analysis with antipeptide antibodies revealed that the E1A 289R T antigen and the E1B-19K (175R) T antigen are synthesized beginning at 2 to 3 and 4 to 5 h postinfection, respectively, whereas the i-leader protein is synthesized starting at about 8 h postinfection and continues unabated until at least 25 h postinfection. The i-leader protein is very stable, as determined by pulse-chase labeling experiments, and accumulates continuously from 8 to 25 h postinfection, as shown by immunoblot analysis. The synthesis of i-leader protein does not depend upon viral DNA replication. Thus, the i-leader protein is a viral gene product of unknown function and high stability that is made in large quantities at intermediate times of productive infection.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3005632 TI - Expression of 100,000-Mr simian virus 40 (SV40) tumor antigen in mouse fibroblasts transfected with replication-defective SV40 genomes. AB - Simian virus 40 early region mutants which are partially or completely replication defective were tested for their ability to transform postcrisis mouse fibroblasts. All mutants tested were capable of generating anchorage-independent transformants. We have previously reported the presence of a variant tumor antigen of 100,000 Mr (100K protein) generated upon transformation by wild-type simian virus 40 virions which correlates with anchorage-independent growth (Chen et al., Mol. Cell. Biol. 1:994-1006, 1981). In this study, none of the mutants tested produced the 100K variant protein at early (before the fifth) passage. Long-term passage (greater than 20 weeks) permitted the expression of this 100K variant in half of the transformants. Thus the phenotype of these mutants is different from both wild-type simian virus 40 (frequently production of 100K by the third passage, and always by the tenth passage) and the origin-minus class of mutants (no production of 100K at any passage). PMID- 3005633 TI - Regulation of cytomegalovirus late gene expression: gamma genes are controlled by posttranscriptional events. AB - We investigated the control of human cytomegalovirus (CMV) late (gamma)-gene expression in human fibroblast cells. Transcriptional activity of two gamma genes, encoding ICP27, a structural component (matrix or tegument) of virions, and ICP36, a major DNA-binding protein family, was followed by analysis of steady state RNA levels during viral infection. Synthesis of the protein products of these genes was analyzed with specific monoclonal antibodies in conjunction with sensitive immunoblot or immunoprecipitation analysis. Although accumulation of ICP27 and ICP36 was not abundant until late times, both late genes were as transcriptionally active at early times (4 h postinfection) as at late times (48 h postinfection). Reduced amounts (less than 5% of late levels) of the protein products were detected at early times, demonstrating that a small proportion of the ICP27 and ICP36 RNA made at this time was translated. These observations establish that expression of at least two CMV gamma genes is regulated through posttranscriptional events. The very early transcriptional activation of late genes and the relative importance of posttranscriptional regulation to late-gene expression distinguishes CMV from other well-studied herpesviruses and does not appear analogous to late-gene regulation in any other DNA animal virus. PMID- 3005635 TI - RNA virus genomes hybridize to cellular rRNAs and to each other. AB - In this communication we show that the RNA genomes of vesicular stomatitis, Sindbis, and reoviruses can specifically hybridize under stringent conditions to the large rRNAs present in HeLa cell cytoplasmic extracts. In addition, we show that some virus genome RNAs can also hybridize to each other. On the basis of our previous detailed studies identifying specific regions of hybridization between the poliovirus genome and 28S rRNA, we suggest that a similar phenomenon of "patchy complementary" may be responsible for the interactions described here (M. A. McClure and J. Perrault, Nucleic Acids Res. 13:6797-6816, 1985). The possible biological implications of these cross-reacting hybridizations and practical considerations in the use of viral probes for diagnosis are discussed. PMID- 3005634 TI - Control of expression of an integrated Rous sarcoma provirus in rat cells: role of 5' genomic duplications reveals unexpected patterns of gene transcription and its regulation. AB - Rat cells transformed by Rous sarcoma virus frequently contain duplications of viral (and sometimes cellular) DNA 5' to the integrated provirus, suggesting that such rearrangements favor provirus expression. In one cell line, A11, the duplication includes the viral src gene and proviral sequences that flank it. We examined three possible roles for this structure. Since the proviral v-src gene transformed recipient cells upon DNA transfer and was the major template for v src transcription in A11 cells, the presence of v-src in the duplication is presumably not necessary for transformation. Since the size and structure of transcripts from the proviral v-src gene in A11 cells were conventional, the duplication does not facilitate transformation by providing a novel transcriptional strategy. Thus, we favor the concept that the duplication either attenuates a negative effect of flanking elements at the host chromosome integration site or augments the positive regulation of conventional provirus expression or both. Gene transfer and transcription analyses with both genomic and cloned DNA showed that the mechanisms of such regulatory phenomena are complex. Identical sequences in the provirus and the 5' duplication displayed different patterns of expression in A11 cells that could be disrupted in segments of cloned DNA. Among the elements that influenced such expression were sequences from the gag-pol region of the provirus. PMID- 3005636 TI - Stereo images of vesicular stomatitis virus assembly. AB - Viral assembly was studied by viewing platinum replicas of cytoplasmic and outer plasma membrane surfaces of baby hamster kidney cells infected with vesicular stomatitis virus. Replicas of the cytoplasmic surface of the basilar plasma membrane revealed nucleocapsids forming bullet-shaped tight helical coils. The apex of each viral nose cone was anchored to the membrane and was free of uncoiled nucleocapsid, whereas tortuous nucleocapsid was attached to the base of tightly coiled structures. Using immunoelectron microscopy, we identified the nucleocapsid (N) viral protein as a component of both the tight-coil and tortuous nucleocapsids, whereas the matrix (M) protein was found only on tortuous nucleocapsids. The M protein was not found on the membrane. Using immunoreagents specific for the viral glycoprotein (G protein), we found that the amount of G protein per virion varied. The G protein was consistently localized at the apex of viral buds, whereas the density of G protein on the shaft was equivalent to that in the surrounding membrane. These observations suggest that G-protein interaction with the nucleocapsid via its cytoplasmic domain may be necessary for the initiation of viral assembly. Once contact is established, nucleocapsid coiling proceeds with nose cone formation followed by formation of the helical cylinder. M protein may function to induce a nucleocapsid conformation favorable for coiling or may cross-link adjacent turns in the tight coil or both. PMID- 3005637 TI - Herpes simplex virus amplicon: cleavage of concatemeric DNA is linked to packaging and involves amplification of the terminally reiterated a sequence. AB - Herpes simplex virus-infected cells contain large concatemeric DNA molecules arising from replication of the viral genome. The large concatemers are cleaved to generate unit-length molecules terminating at both ends with the a sequence. We have used constructed defective virus vectors (amplicons) derived from herpes simplex virus to study the mechanism of cleavage of viral DNA concatemers and the packaging of viral DNA into nucleocapsids. These studies revealed that (i) a 248 base-pair a sequence contained the signal(s) required for cleavage-packaging, (ii) the cleavage of viral DNA concatemers was coupled to packaging, (iii) the a sequence contained the information required for its own amplification, and (iv) cleavage-packaging occurred by a novel process involving the amplification of the a sequence. PMID- 3005638 TI - Herpes simplex virus type 1-induced hydrocephalus in mice. AB - Adult ICR/Slc or BALB/c mice developed hydrocephalus when attenuated herpes simplex virus type 1 (HSV-1) (strain Ska) was injected intracerebrally 2 to 4 weeks earlier and then after mice were challenged with the same virus or virulent HSV-1. Initial inoculation of the Ska strain elicited acute meningitis and ependymitis with transient mild hydrocephalus. Viral antigen was seen in the meninges and subependymal areas, and the virus was titrated during the acute phase of infection. After the second virus inoculation, more prominent inflammation was evoked in the same area, and the animals developed hydrocephalus, although viral antigen and infectious virus were hardly detected. When the mice were immunosuppressed with cyclophosphamide, they ceased to develop hydrocephalus. BALB/c nude mice did not show the same pathology, even though they were treated in the same way. When irradiated mice, which had been infected with the Ska strain intracerebrally 2 weeks earlier, received syngeneic immune spleen cells, they developed hydrocephalus. The T-cell nature of the effector cells was confirmed by the elimination of the pathology after treatment of the donor cells with anti-Thy-1.2 plus complement. No hydrocephalic mice were observed after treatment of the donor cells with anti-Lyt-1.2 plus complement, which gave further evidence of the T-cell nature of the effector cells as the Lyt-1+.2+ antigen-bearing subsets. Intervals between priming and challenge virus inoculation could be more than 18 months. The presence of purified HSV-1 envelope protein was feasible for the development of the hydrocephalic animals. PMID- 3005639 TI - The soluble glycoprotein of vesicular stomatitis virus is formed during or shortly after the translation process. AB - Gs protein is a shorter, soluble form of the viral G protein of vesicular stomatitis virus (VSV) lacking the membrane-anchoring domain. Production of Gs protein appears to be a general property of VSV because infection of BHK-21 cells by five different isolates of the VSV serotype Indiana led in all cases to the synthesis of Gs protein. Moreover, it is formed in a variety of eucaryotic cell lines after VSV infection. In pulse-chase experiments, we observed a time dependent change in the ratio of G to Gs protein released into the growth medium, suggesting that Gs is formed intracellularly rather than on the cell surface. Further experiments revealed that Gs protein can be synthesized in vitro in the reticulocyte lysate system after addition of a viral mRNA fraction and in a coupled transcription-translation system with VSV core particles. In the presence of microsomal membranes both G and Gs protein were glycosylated in the reticulocyte lysate, confirming that the authentic Gs protein is synthesized in vitro. The addition of various protease inhibitors to the cell-free system and variation of the incubation conditions did not alter the ratio of G to Gs formation. Taken together, these experiments suggest strongly that Gs protein is not a product of a membrane-associated proteolytic activity but is formed during or shortly after the translation process. Our attempts to detect a specific, shorter mRNA coding for the Gs protein by molecular hybridization procedures did not reveal the existence of such a mRNA species. PMID- 3005640 TI - Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins. AB - Defined segments of the cloned foot-and-mouth disease virus genome corresponding to all parts of the coding region were expressed in Escherichia coli as fusions to the N-terminal part of the MS2-polymerase gene under the control of the inducible lambda PL promoter. All constructs yielded large amounts of proteins, which were purified and used to raise sequence-specific antisera in rabbits. These antisera were used to identify the corresponding viral gene products in 35S labeled extracts from foot-and-mouth disease virus gene products in the nucleotide sequence, to identify precursor-product relationships, and to detect several foot-and-mouth disease virus gene products not previously identified in vivo or in vitro. PMID- 3005641 TI - Persistent infection of chimpanzees with human T-lymphotropic virus type III/lymphadenopathy-associated virus: a potential model for acquired immunodeficiency syndrome. AB - The lymphadenopathy-associated virus (LAV) prototype strain of human T lymphotropic virus type III/LAV was transmitted to juvenile chimpanzees with no prior immunostimulation by (i) intravenous injection of autologous cells infected in vitro, (ii) intravenous injection of cell-free virus, and (iii) transfusion from a previously infected chimpanzee. All five animals that received more than one 50% tissue culture infective dose were persistently infected with LAV or chimpanzee-passaged LAV for up to 18 months. During this time they developed no illnesses, but they exhibited various degrees of inguinal and axillary lymphadenopathy and significant reductions in rates of weight gain. Detailed blood chemistry and hematologic evaluations revealed no consistent abnormalities, with the exception of immunoglobulin G (IgG) hypergammaglobulinemia, which became apparent in one animal 6 months postinfection and continued at more than 1 year postinfection. Transient depressions followed by increases in the numbers of T4 cells to levels greater than normal were observed in all animals after virus inoculation. However, the number of LAV-infected peripheral blood cells decreased with time after infection. Results of enzyme immunoassays showed that all infected animals seroconverted to IgG anti-LAV within 1 month postinfection and that antibody titers remained high throughout the period of observation. In contrast, only three of the five LAV-infected chimpanzees had detectable IgM antibody responses, and these preceded IgG-specific serum antibodies by 1 to 2 weeks. Virus morphologically and serologically identical to LAV was isolated from peripheral blood mononuclear cells of all infected animals at all times tested and from bone marrow cells taken from one animal 8 months after infection. One chimpanzee that was exposed to LAV only by sharing a cage with an infected chimpanzee developed lymphadenopathy and an IgM response to LAV, both of which were transient; however, no persistent IgG antibody response to LAV developed, and no virus was recovered from peripheral blood cells during a year of follow up. Thus, LAV readily infected chimpanzees following intravenous inoculation and persisted for extended periods despite the presence of high titers of antiviral antibodies. However, the virus was not easily transmitted from infected to uninfected chimpanzees during daily cage contact. PMID- 3005642 TI - Isolation of an MH2 retrovirus mutant temperature sensitive for macrophage but not fibroblast transformation. AB - A conditional mutant of the MH2 avian retrovirus, termed ts41MH2, was isolated. Unlike wtMH2, ts41MH2 permitted transformed macrophages to differentiate during a 5- to 7-day temperature shift from 37 to 42 degrees C. Mutant-infected cells incubated at 42 degrees C exhibited a flattened morphology and then fused to form giant multinucleated cells that closely resembled normal macrophage maturation in vitro. These differentiated cells reacted strongly with a myeloid-macrophage specific monoclonal antibody. The process of differentiation was inhibited when ts41MH2-transformed nonproducer clones were superinfected before the temperature shift with the myc gene-containing MC29 or OK10 viruses. By contrast, no inhibition was observed in clones superinfected with the MH2-PA200 virus that contains only the mil gene. The mutant also demonstrated a reduced oncogenic potential relative to that of wtMH2 when it was inoculated intravenously into young birds. However, in contrast to the results obtained with hematopoietic cells, none of the five fibroblast transformation parameters tested for ts41MH2 were altered from those induced by wtMH2. These results suggest that the mutation in ts41MH2 is located in a region of myc required for macrophage transformation, but not required for fibroblast transformation. PMID- 3005643 TI - Genetically determined resistance to murine cytomegalovirus and herpes simplex virus in newborn mice. AB - Mice which were infected with the herpesvirus murine cytomegalovirus or herpes simplex virus type 1 on the day of birth exhibited mouse strain-dependent differences in the development of lethal disease. The pattern of resistance among the strains was distinct for each virus and closely resembled that reported in adult mice. However, much lower doses of the viruses were required in newborn mice to reveal these resistance patterns. For murine cytomegalovirus, both H-2 associated and non-H-2 genes conferred resistance, and, as has been shown for adults, there was a 25-fold difference in the dose required to kill 50% of the animals belonging to the most resistant and susceptible strains. The resistance of newborn mice to herpes simplex virus type 1 was conferred by non-H-2 genes in C57BL/6 mice, as has been reported for adults, and newborn C57BL/6 mice were considerably more resistant than mice of susceptible strains. Resistance was also reflected in the titer of these viruses in the spleen or liver early in infection and, with murine cytomegalovirus, in the survival time of infected mice. The resistance of newborn mice to lethal disease was not conferred postnatally by the mother. This appears to be the first report of genetically determined resistance to herpesviruses in newborn mice. Such autonomous virus-specific resistance may provide a significant barrier to naturally acquired infection in genetically resistant strains. Similar genetically regulated mechanisms may protect the newborns of many species, including humans, against infection with herpesviruses. PMID- 3005644 TI - A subclass of polyomavirus middle tumor antigen binds to DNA cellulose. AB - We examined the binding of polyomavirus large (L-T)-, middle (M-T)-, and small tumor antigens to DNA cellulose. At pH 6.0, the majority of L-T bound to calf thymus DNA cellulose, while little or no small tumor antigen was retained under these conditions. Unexpectedly, a small but reproducible proportion of M-T bound to both native and denatured DNA cellulose. M-T encoded by polyomavirus mutant dl 8, which expressed shortened L-T and M-T, bound to DNA, indicating that the deleted sequences are not required for DNA binding. Also, M-T from transformed BMT-1 rat cells, which synthesize exclusively this polyomavirus tumor antigen, bound to DNA, indicating that its binding is not due to association with other polyomavirus-encoded proteins. Using the DNA fragment immunoassay, we found that, under conditions in which L-T bound specifically to DNA fragments containing viral regulatory sequences, no viral DNA fragments were bound by M-T. The existence of distinct subpopulations of M-T that differ in their DNA-binding properties was indicated by rebinding experiments in which M-T that had bound to DNA cellulose rebound very efficiently, while that which had not been originally retained by DNA cellulose rebound poorly. Furthermore, the M-T-pp60 c-src complex did not bind to DNA cellulose. These data suggest that polyomavirus M-T is heterogeneous, consisting of populations of molecules that differ in their interactions with DNA cellulose. PMID- 3005645 TI - Spontaneous deletion mutants resulting from a frameshift insertion in the simian virus 40 agnogene. AB - The 61-amino-acid agnoprotein is a nonessential polypeptide encoded by the late leader region of simian virus 40 which appears to play a role in viral assembly. A 2-base-pair (bp) insertion mutant (in2379) was created by altering the coding region of this protein. This mutation prevents the synthesis of the agnoprotein and, in contrast to the more extensive deletion mutations previously described in this region, might be expected to have a lesser effect on the template for late viral transcription. In fact, the 2-bp insertion mutant grew significantly less well than most mutants containing larger deletions in the agnoprotein region and frequently gave rise to stock containing second-site alterations in the same region. These observations suggested that the defect in mutant in2379 extends beyond the loss of the agnoprotein. Characterization of a number of second-site mutants indicated that all of them grew more efficiently than the original 2-bp insertion mutant. Based on the nucleotide sequence of these mutants, we suggest possibilities for the deleterious effect induced by the insertion in mutant in2379. PMID- 3005646 TI - Characterization and mapping of a nonessential pseudorabies virus glycoprotein. AB - Antigenic variants of pseudorabies virus (PRV) containing mutations in a viral glycoprotein with a molecular weight of 82,000 (gIII) were isolated by selecting for resistance to a complement-dependent neutralizing monoclonal antibody (MCA82 2) directed against gIII. These mutants were completely resistant to neutralization with MCA82-2 in the presence of complement. Two mutants selected for further studies either did not express gIII or expressed an improperly processed form of the glycoprotein. The mutations were also associated with an altered plaque morphology (syncytium formation). The gIII gene was mapped by marker rescue of a gIII- mutant with cloned restriction enzyme fragments to the long unique region of the PRV genome between 0.376 and 0.383 map units. This corresponds to the map location of a glycoprotein described by Robbins et al. (J. Mol. Appl. Gen. 2:485-496, 1984). Since gIII is nonessential for viral replication in cell culture and has several other characteristics in common with the herpes simplex virus glycoprotein gC, gIII may represent the PRV equivalent to herpes simplex virus gC. PMID- 3005647 TI - Specific lysis of varicella zoster virus-infected B lymphoblasts by human T cells. AB - Epstein-Barr virus-transformed human B cells expressed cell surface varicella zoster virus (VZV) antigens after superinfection with VZV although they did not form infectious centers in a plaque assay. The VZV-superinfected cells were lysed by autologous VZV-stimulated T-cell lines and their derivative clones. The effector cells were specific for VZV and an HLA DR antigen and were T4+. The specificity of lysis of Epstein-Barr virus-transformed, VZV-superinfected targets by prestimulated mononuclear cells in this system contrasted with the unrestricted lysis seen when the targets were VZV-infected fibroblasts. PMID- 3005648 TI - Synthesis and processing of the envelope gp55-116 complex of human cytomegalovirus. AB - The envelope of human cytomegalovirus has been reported to contain between three and eight glycoproteins. Major constituents of the envelope include two abundant glycoproteins with estimated molecular weights of 55,000 (gp55) and 116,000 (gp116). These two glycoproteins have been shown to exist as a disulfide-linked complex (gp55-116) within the envelope of mature virions. Utilizing a panel of monoclonal antibodies reactive with the gp55-116 complex, we characterized the synthesis and processing of these two virion proteins. Infected cells were shown to contain two glycosylated proteins of 160,000 and 150,000 daltons as well as the mature gp55 and gp116. Pulse-chase analysis indicated that gp150 was a precursor protein of gp160. The mature gp55 and gp116 were generated, in turn, by cleavage of gp160. Antigenic and structural analysis revealed that gp55 and gp116 shared little structural homology and no detectable antigenic cross-reactivity. The results of this study are discussed in relation to the synthesis of envelope proteins of other herpesviruses. PMID- 3005649 TI - Functional and molecular analyses of the avirulent wild-type herpes simplex virus type 1 strain KOS. AB - It has been documented that KOS, a laboratory strain of herpes simplex virus type 1, is several orders of magnitude less neurovirulent than most other wild-type strains. Studies initiated to determine the functional nature of the block to neuroinvasiveness and to establish the genes involved have determined that, after footpad inoculation of mice, strain 17 syn+ induced neuropathologic signs (paralysis) at titers of 10(2) and yielded a PFU/50% lethal dose ratio of 10(4). In contrast, KOS was not lethal and did not induce paralysis at inoculation doses of 10(8) PFU. This reduced neurovirulence of KOS could not be explained by the lack of thymidine kinase activity, its inability to replicate in mouse cells maintained in culture at 38.5 degrees C, or its inefficient replication in nonneural tissues in vivo. Kinetic experiments tracing the virus through the nervous system after footpad inoculation showed that KOS was blocked at the level of the spinal ganglia. A cosmid library of strain 17 syn+ was utilized in recombination and in vivo selection experiments with strain KOS to establish the genomic region involved in 17 syn+ neuroinvasiveness. A cosmid clone containing the HindIII A fragment (0.25 to 0.53 map units) of strain 17 syn+ in mixed transfections with full-length KOS DNA yielded recombinants with enhanced neuroinvasiveness. PMID- 3005650 TI - Lack of evidence for VPg priming of poliovirus RNA synthesis in the host factor dependent in vitro replicase reaction. AB - Anti-VPg immunoprecipitable RNA labeled in vitro during a poliovirus RNA polymerase reaction was formed by the elongation of VPg-containing template fragments rather than by initiation with VPg. The reaction was dependent on a host factor (terminal uridylyl transferase). The incorporation of labeled UTP could be detected with only the host factor present. PMID- 3005651 TI - Mapping 5' termini of JC virus late RNA. AB - The 5' termini of late mRNAs were mapped 17 to 19 days after primary human fetal glial cells were infected with JC virus. The major 5' start sites spanned a region of approximately 250 nucleotides, starting at nucleotide 5114, which was on the early side of the replication origin, and extending to nucleotide 242, which was on the late side of the 98-base-pair (bp) repeats. The sequence TATATAT was contained within each of the 98-bp repeats but does not specify 5' start sites in vivo. However, the sequence TACCTA, which occurred 25 to 30 bp upstream of the simian virus 40 nucleotide position 325 start site (J. Brady, M. Radonovich, M. Vodkin, V. Natarajan, M. Thoren, G. Das, J. Janik, and N. P. Salzman, Cell 31:625-633, 1982) and functions as a surrogate TATA box, was present 30 bp upstream of two JC virus start sites. PMID- 3005652 TI - Cloning and characterization of the DNA of a new human papillomavirus from a woman with dysplasia of the uterine cervix. AB - A previous analysis of 121 female genital tract lesions from the United States and South America had revealed that a large number contained DNA sequences that were weakly homologous to a panel of human papillomavirus (HPV) probes. The DNA sequences of one of these viruses have been molecularly cloned and shown to be a new type of HPV which is called HPV 31. Among the cloned HPV genomes, HPV 31 is most closely related to HPV 16. Although absent from all genital condylomas studied, HPV 31 was present in approximately 20% of the mild and moderate dysplasias and in 6% of the invasive cervical cancers PMID- 3005653 TI - Leukocyte migration inhibition demonstrates a human T-cell response to a membrane protein expressed in latent Epstein-Barr virus infection. AB - Leukocyte migration inhibition tests show that lymphocytes of Epstein-Barr virus seropositive individuals recognize a Raji cell membrane antigen and a membrane protein encoded by Epstein-Barr virus in latently infected cells. Antiserum against the latter blocks the leukocyte migration inhibition triggered by both preparations, suggesting that the two antigens are associated with the same protein complex. PMID- 3005654 TI - Orientation and patching of the latent infection membrane protein encoded by Epstein-Barr virus. AB - Epstein-Barr virus is known to encode three nuclear proteins and one membrane protein (LMP) in latently infected growth-transformed cells. Studies of the plasma membrane localization and orientation of LMP by protease digestion of live cells and by immunofluorescence indicated the following. (i) At least 30% of LMP is in the plasma membrane, as opposed to other cytoplasmic membranes. (ii) A small LMP domain which corresponds to a previously proposed outer reverse turn between the first two transmembrane domains is exposed on the outer cell surface (and two other proposed outer-reverse-turn domains may be exposed), whereas all or almost all of the rest of the protein is not exposed on the outer cell surface. (iii) LMP is present in patches in the cell plasma membrane. PMID- 3005655 TI - Spontaneous curing of a minute virus of mice carrier state by selection of cells with an intracellular block of viral replication. AB - We previously described a persistent infection established by the lymphotropic minute virus of mice in mouse L cells at the level of the cell population (D. Ron, P. Tattersall, and J. Tal, J. Virol. 52:63-69, 1984). This carrier state is maintained by a series of consecutive phenotypic changes which take place in both the cells and the virus and is cured spontaneously after 150 to 200 cell generations (D. Ron and J. Tal, J. Virol. 55:424-430, 1985). We show here that the cure was caused by the selection of virus-resistant cells in the culture. The resistance of these survivor cells to virus replication was due to an intracellular block. Infection of a spontaneously cured culture with the fibrotropic parental minute virus of mice resulted in a restrictive infection in which the viral replicative-form DNA was formed and amplified, but the synthesis of single-stranded progeny DNA was markedly reduced. The lymphotropic strain was blocked in these cells at an earlier stage, with little or no amplification of viral replicative-form DNA observed. These data indicate that the replication of minute virus of mice requires host-coded helper functions in at least two stages of its growth cycle. PMID- 3005657 TI - Short repeats cause heterogeneity at genomic terminus of bovine herpesvirus 1. AB - Analysis of the genomes of different bovine herpesvirus 1 strains revealed a UL terminal HindIII fragment differing in size (from 2.4 to 2.8 kilobases). This fragment polymorphism occurred in the DNA of a wild-type isolate, in highly passaged, apathogenic tissue culture derivatives, and in plaque-purified substrains. This heterogeneity was due to variations in the copy number of a 14 base-pair tandem repeat comprising the base sequence 5'-GCTCCTCCTCCCTC-3', which also exists, with some differences, in other short reiteration sequences of herpes simplex virus type 1, Epstein-Barr virus, and related human cellular DNA. Furthermore, the tandem repeat array was located in close proximity to the left end of the viral genome and may functionally be involved in viral replication. PMID- 3005656 TI - Pathogenicity in mice of herpes simplex virus type 2 mutants unable to express glycoprotein C. AB - Herpes simplex virus type 2 (HSV-2) mutants that were unable to express glycoprotein C (gC-2) were isolated. Deletions were made in a cloned copy of the gC-2 gene, and recombinant viruses containing these deletions were screened by using an immunoreactive plaque selection protocol. The viruses did not display a syncytial phenotype. Intravaginal inoculation of BALB/cJ mice with one of the HSV 2 gC-2- viruses produced local inflammation followed by a lethal spread of the viral infection into the nervous system in a manner identical to that produced by parental HSV-2 strain 333. Similarly, intracerebral inoculation of DBA-2 mice with the gC-2- virus produced a lethal neurological disease paralleling that caused by HSV-2 strain 333. These results indicate that gC-2 is not required for the spread of HSV-2 infections in mice. PMID- 3005659 TI - The 89,000-Mr murine cytomegalovirus immediate-early protein activates gene transcription. AB - To study trans-activation of gene expression by murine cytomegalovirus (MCMV) immediate-early (IE) proteins, the IE coding region 1 (ie1), which encodes the 89,000-Mr IE phosphoprotein (pp89), was stably introduced into L cells. A cell line was selected and characterized that efficiently expressed the authentic viral protein. The pp89 that was constitutively expressed in L cells stimulated the expression of transfected recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of viral promoters. The regulatory function of the ie1 product was confirmed by transient expression assays in which MCMV IE genes were cotransfected into L cells together with recombinant constructs of the CAT gene. For CAT activation by the ie1 product, a promoter region was required, but there was no preferential activation of a herpes simplex virus type 1 delayed-early promoter. All plasmid constructs that contained the intact coding sequences for pp89 induced gene expression in trans. The MCMV enhancer region was not essential for the expression of a functional IE gene product, and testing of the cis-regulatory activity of the MCMV enhancer revealed a low activity in L cells. Another region transcribed at IE times of infection, IE coding region 2, was unable to induce CAT expression and also did not augment the functional activity of ie1 after cotransfection. PMID- 3005658 TI - Structure and processing of the p2 region of avian sarcoma and leukemia virus gag precursor polyproteins. AB - We have purified two low-molecular-weight polypeptides from the Prague C strain of Rous sarcoma virus and have identified these as products of the gag precursor Pr76 by protein sequencing and by amino acid analysis. Both polypeptides are derived from a stretch of 22 amino acids within Pr76 that separates p19 and p10. We refer to this region as p2. Together the two cleavage products form the entire p2 region. The junctions of p19 with the amino-terminal fragment of p2 and of p10 with the carboxy-terminal fragment of p2 define two new processing sites within the gag precursor, Tyr-155-His-156 and Gly-177-Ser-178. Both polypeptides are major cleavage products of Pr76 that occur in Prague C Rous sarcoma virus at an estimated 1,000 copies per virion. They also are prominent components of avian myeloblastosis virus. The combination of gel filtration and reverse-phase high pressure liquid chromatography, which was used for the isolation of the two fragments of p2, resolved over a dozen other low-molecular-weight polypeptides from avian sarcoma and leukemia viruses that previously were undetected. This technique thus should serve as a useful procedure for further characterization of viral components. PMID- 3005660 TI - Tropism of sheep lentiviruses for monocytes: susceptibility to infection and virus gene expression increase during maturation of monocytes to macrophages. AB - Visna lentiviruses have a natural tropism for cells of the macrophage lineage of sheep and goats, but virus replication in these cells in vivo is restricted so that only small quantities of virus are produced. One restricting factor suggested in previous studies is that virus replication is dependent on the maturity of the cells: the more mature the cell, the less restrictive the replication of the virus. Since monocytes in peripheral blood are precursors of macrophages, we investigated the effect of cell maturation on virus replication under limited control conditions in vitro by inoculating blood leukocytes with virus and retarding the maturation of monocytes to macrophages during cultivation in serum-free medium. Using enzyme markers that identified the cells in their resting monocytic stage (peroxidase) and mature macrophage stage (acid phosphatase) along with quantitative in situ hybridization and immunocytochemistry with viral reagents to trace the efficiency of virus replication, we correlated virus replication with cell maturation. Only a few monocytes were susceptible to infection, and virus replication did not extend beyond a low level of transcription of viral RNA. In the acid phosphatase positive, maturing macrophage, susceptibility of the cells to infection was increased and virus replication was greatly amplified to the level of translation of viral polypeptides. However, virus maturation was delayed by 3 days until further cell maturation had occurred. Thus, the entire life cycle of the virus, from its attachment to the target cell to its maturation in the cell, was dependent on the level of maturation/differentiation of the monocytic cell. PMID- 3005661 TI - Insertion of several different DNAs in reticuloendotheliosis virus strain T suppresses transformation by reducing the amount of subgenomic mRNA. AB - The highly oncogenic retrovirus reticuloendotheliosis virus (Rev) strain T (Rev T) has, relative to its helper virus Rev strain A, a substitution of the oncogene v-rel for most of the env gene and a large deletion of gag and pol sequences. When the helper virus sequences that are deleted in Rev-T are replaced, the recombinant virus is nontransforming (I. S. Y. Chen and H. M. Temin, Cell 31: 111 120, 1982). We show that suppression of transformation occurs when several different DNA sequences are inserted in Rev-T and that suppression is correlated with a reduction in the amount of v-rel mRNA and v-rel protein in infected cells. The reduced amount of v-rel protein is insufficient for transformation. PMID- 3005662 TI - Sendai virus-erythrocyte membrane interaction: quantitative and kinetic analysis of viral binding, dissociation, and fusion. AB - A kinetic and quantitative analysis of the binding and fusion of Sendai virus with erythrocyte membranes was performed by using a membrane fusion assay based on the relief of fluorescence self-quenching. At 37 degrees C, the process of virus association displayed a half time of 2.5 min; at 4 degrees C, the half time was 3.0 min. The fraction of the viral dose which became cell associated was independent of the incubation temperature and increased with increasing target membrane concentration. On the average, one erythrocyte ghost can accommodate ca. 1,200 Sendai virus particles. The stability of viral attachment was sensitive to a shift in temperature: a fraction of the virions (ca. 30%), attached at 4 degrees C, rapidly (half time, ca. 2.5 min) eluted from the cell surface at 37 degrees C, irrespective of the presence of free virus in the medium. The elution can be attributed to a spontaneous, temperature-induced release, rather than to viral neuraminidase activity. Competition experiments with nonlabeled virus revealed that viruses destined to fuse do not exchange with free particles in the medium but rather bind in a rapid and irreversible manner. The fusion rate of Sendai virus was affected by the density of the virus particles on the cell surface and became restrained when more than 170 virus particles were attached per ghost. In principle, all virus particles added displayed fusion activity. However, at high virus-to-ghost ratios, only a fraction actually fused, indicating that a limited number of fusion sites exist on the erythrocyte membrane. We estimate that ca. 180 virus particles maximally can fuse with one erythrocyte ghost. PMID- 3005664 TI - An outbreak of a herpesvirus infection in harbor seals (Phoca vitulina). AB - During an outbreak of a herpesvirus infection in juvenile harbor seals, 11 out of 23 seals died. The duration of the disease in these 11 animals varied from 1-6 days. Nasal discharge, inflammation of the oral mucosa, vomiting, diarrhea and fever up to 40 degrees C were observed in the first days of the disease. In later stages coughing, anorexia and lethargy occurred. Severe necrosis of the liver and interstitial pneumonia were the most striking histopathological findings. PMID- 3005665 TI - Canine parvovirus infection in wolves (Canis lupus) from Minnesota. PMID- 3005663 TI - Studies on emerging radiation leukemia virus variants in C57BL/Ka mice. AB - To analyze the emergence of radiation leukemia virus (RadLV) variants in primary X-ray-induced C57BL/Ka thymoma and to identify the virus responsible for the very high leukemogenic potential of passaged Kaplan strain BL/VL3 preparation, we cloned several primary and passaged ecotropic RadLV infectious genomes. By restriction analysis, we found that BL/VL3 cells harbor three related but different ecotropic RadLVs. Their restriction map differs significantly from those of primary RadLVs. Hybridization analysis also indicated that BL/VL3 and primary RadLVs differ in their p15E and long terminal repeat (LTR) regions. As compared with the LTR sequence of the putative parental endogenous ecotropic provirus, the LTR sequence of primary weakly leukemogenic RadLV has only one change, a C-rich sequence, generating a 6-base-pair direct repeat just in front of the promotor. The LTR of the primary nonleukemogenic RadLV only showed few base changes, mainly clustered in R and U5. The LTR from a moderately leukemogenic passaged BL/VL3 RadLV had conserved the C-rich sequence and acquired a 43-base-pair direct repeat in U3 and several other point mutations, small insertions, and deletions scattered in U3, R, and U5. All cloned primary RadLVs were fibrotropic, and some were weakly leukemogenic. All cloned BL/VL3 RadLVs were thymotropic and nonfibrotropic. The block of their replication was found to be after the synthesis of unintegrated linear and supercoiled viral DNA. Most of the BL/VL3 RadLVs were moderately leukemogenic, and one (V-13) was highly leukemogenic, being as virulent as the Moloney strain. We propose a model for the emergence of the RadLV variants and show that the virus responsible for the high leukemogenic potential of BL/VL3 preparation is a nondefective, ecotropic, lymphotropic, nonfibrotropic, unique retrovirus which most likely arose from a parental primary RadLV similar to those studied here. PMID- 3005666 TI - Occurrence of antibodies to the etiologic agents of infectious bovine rhinotracheitis, parainfluenza 3, leptospirosis, and brucellosis in white-tailed deer in Minnesota. AB - A serologic survey was conducted on 628 white-tailed deer (Odocoileus virginianus) in 1976 and 1979-1980. Tests for antibodies to the etiologic agents of infectious bovine rhinotrancheitis (IBR), parainfluenza 3 (PI3), leptospirosis, and brucellosis produced positive results of 15%, 20%, 3% and 0%, respectively. Adult deer had significantly higher prevalence of antibodies to IBR virus and PI3 virus than fawns. These data provide a basis for monitoring these disease agents in Minnesota's white-tailed deer. PMID- 3005667 TI - Serosurvey for selected pathogens in hunter-killed pronghorns in western Nebraska. AB - Exposure of pronghorns (Antilocapra americana) in western Nebraska in 1983 to selected livestock pathogens was examined by serology and attempted virus isolation. Antibodies were present to the agents of bluetongue, epizootic hemorrhagic disease, and bovine respiratory syncytial virus. There were no serologic reactors to Brucella, and attempts to isolate the viruses of bluetongue and epizootic hemorrhagic disease were negative. PMID- 3005668 TI - Antagonism of xylazine hydrochloride with yohimbine hydrochloride and 4 aminopyridine in captive wapiti. AB - Eight captive wapiti (Cervus elaphus nelsoni) were injected with xylazine hydrochloride on two occasions during March and April 1984. Animals were grouped into a modified Latin square design and were given either successive injections of yohimbine hydrochloride and 4-aminopyridine (4-AP) to antagonize the sedative effects of xylazine hydrochloride or permitted an unantagonized recovery. Induction times ranged from 3 to 26 min with excited and wild animals requiring a supplementary dose. Time until walking was significantly (P less than 0.005) shorter in the group given successive injections (given i.v.) of the reversal drugs yohimbine hydrochloride (0.15 mg/kg) and 4-AP (0.30 mg/kg) than those animals during unantagonized recoveries. Marked increase in heart rate and respiratory rate were observed in animals within 3 min after successive injections of yohimbine hydrochloride and 4-AP. There was no occurrence of convulsions and animals did not relapse to profound sedation. Slight muscle tremors were observed in one animal which received a dose of 0.35 mg/kg of 4-AP. This drug combination can reduce markedly the duration of recovery from xylazine hydrochloride-induced sedation in wapiti. PMID- 3005670 TI - Leads from the MMWR. Leading work-related diseases and injuries--United States. PMID- 3005669 TI - The effect of antacids on the absorption of simultaneously ingested iron. AB - Most discussions of iron therapy include a statement about the ineffectiveness of iron ingested simultaneously with antacids. This study was designed to determine whether or not antacids inhibit iron absorption. A small-dose iron tolerance test was used to compare absorption of iron with and without various antacids. Liquid antacid containing aluminum hydroxide and magnesium hydroxide did not significantly decrease iron absorption. Sodium bicarbonate and calcium carbonate caused the plasma iron increase to be 50% and 67% less than the control values, respectively. However, when calcium carbonate was present in a multivitamin-plus minerals tablet, the plasma iron change was not significantly different from control trials. Presumably the competitive binding of iron by ascorbic acid in the vitamin pill allowed uninhibited absorption of the iron. Our results suggest that certain antacids may be combined with iron therapy without reducing the efficacy of the iron. PMID- 3005671 TI - Predicting the predisposition to osteoporosis. Gonadotropin-releasing hormone antagonist for acute estrogen deficiency test. AB - A reliable predictive test to identify perimenopausal women who are vulnerable to the adverse effects of severe estrogen deficiency may be useful in deciding for whom and when to begin estrogen replacement therapy. Primate models were employed to determine whether short-term treatment with gonadotropin-releasing hormone antagonist allows prospective identification of individuals at greatest risk for a negative calcium balance during overt estrogen deficiency; whether high-dose clomiphene citrate is sufficiently estrogenic to abate urinary calcium loss and to sustain vaginal and perineal tissues after ovariectomy; and whether clomiphene citrate will provide these beneficial estrogenic effects without inducing endometrial proliferation and menstruation after progestin withdrawal. The data indicate the capability of short-term gonadotropin-releasing hormone-antagonist test to identify individuals likely to lose calcium rapidly after ovariectomy. This result has potential usefulness in the prediction of susceptibility to osteoporosis after medical castration or spontaneous menopause. At high doses, clomiphene citrate therapy was nearly as effective as high-dose conjugated equine estrogens for conservation of urinary calcium, yet clomiphene citrate did not cause endometrial proliferation or withdrawal bleeding after progesterone therapy. PMID- 3005672 TI - Hypertensive emergencies and urgencies. PMID- 3005673 TI - HTLV-III/LAV-seronegative, virus-negative sexual partners and household contacts of hemophiliacs. PMID- 3005674 TI - The prevalence of HTLV-III/LAV antibodies in heterosexuals. PMID- 3005675 TI - Minimal risk of transmission of AIDS-associated retrovirus infection by oral genital contact. PMID- 3005676 TI - Female-to-male transmission of HTLV-III. PMID- 3005677 TI - Condoms prevent transmission of AIDS-associated retrovirus. PMID- 3005678 TI - Preventing the spread of AIDS. PMID- 3005679 TI - Treatment of uncomplicated infections due to Neisseria gonorrhoeae. A review of clinical efficacy and in vitro susceptibility studies from 1982 through 1985. PMID- 3005680 TI - Leads from the MMWR. Additional recommendations to reduce sexual and drug abuse related transmission of human T-lymphotropic virus type III/lymphadenopathy associated virus. PMID- 3005681 TI - Pharmacological comparison of captopril and MK-422 by a new method for measuring activity of angiotensin converting enzyme (ACE). AB - The authors recently reported the development of a new method for measuring angiotensin converting enzyme (ACE) by means of a highly sensitive angiotensin II RIA technique. We have carried out a comparative study of the pharmacological properties of captopril and MK-422, two ACE inhibitors recently developed as new antihypertensive agents. In this study, in vivo and in vitro animal experiments were performed using the Gottingen Mini-pig (Mini-pig G) animal model of the human disease. In the in vivo experimental system, each drug was administered by intravenous injection at a dose of 1 mg/kg, and a slight difference was found in the time-course of the per cent inhibition of ACE in the blood. In the in vitro system (cultured aortic endothelial cells), the ACE inhibitory activities of the two drugs were compared in terms of the 50%-inhibition point on the dose response curve, and it was found that MK-422 was about 100 times more potent than captopril. These results indicate that our newly-developed experimental system can be useful in the establishment of the clinical dose of vasoactive drugs that act on the renin-angiotensin system. PMID- 3005682 TI - The effects of phospholipase A2 on beta adrenoceptor function in isolated cardiac cells. AB - The role of phospholipids in the maintenance of beta-adrenoceptor function was investigated in isolated canine myocytes prepared from eight adult mongrel dogs by using collagenase. The characteristics of beta-adrenoceptors were assessed by determining the number and the affinity of receptors by a radioactive ligand binding assay using 125I-iodocyanopindolol. The increase in cyclic AMP content induced by isoproterenol or forskolin was also determined by radioimmunoassay with or without pretreatment with phospholipase (PLase) A2. The amount of free fatty acids released from isolated myocytes by PLase A2 was measured by high performance liquid chromatography. PLase induced a significant decrease in the number of beta-adrenoceptors but did not affect their affinity. Although the isoproterenol-stimulated increase in cyclic AMP was significantly inhibited by the pretreatment with PLase A2, the forskolin-stimulated increase was not affected. Responsive accumulation of cyclic AMP to isoproterenol was much more impaired than the decrease in beta-adrenoceptor number. These results indicate that PLase A2 deteriorates the function of the adenylate cyclase system linked beta-adrenoceptor, and suggest that PLase A2 affects both beta-adrenoceptors and the coupling of beta-adrenoceptors with adenylate cyclase. PMID- 3005683 TI - [Antagonistic effect of dibutyryl cyclic AMP on the action of d-tubocurarine]. PMID- 3005685 TI - [The role of lymphadenectomy in the treatment of renal cell carcinoma]. AB - The usefulness of radical nephrectomy combined with lymphadenectomy for improving the prognosis of renal cell carcinoma was evaluated in 146 patients with this disease during the 21-year period from 1963 to 1983. Eighty-six out of the 146 underwent radical nephrectomy alone, while the remaining 60 patients received radical nephrectomy combined with lymphadenectomy. In cases of stage I and stage IV B, lymphadenectomy produced no improvement in the survival rate. On the other hand, of the 58 patients who had stage II and stage III disease, in the 23 who underwent lymphadenectomy, the survival rate improved significantly as compared to the 35 who did not. PMID- 3005684 TI - [Effect of anxiolytic music on endocrine function in surgical patients during operations under epidural anesthesia]. PMID- 3005686 TI - [Sequential measurement of serum CA 125 levels in Krukenberg's tumor]. AB - CA 125 is a tumor marker for ovarian cancer developed by hybridoma technology. In the present study, the levels of CA 125 were sequentially measured in the serum of five patients with Krukenberg's tumor originating in the gastrointestinal tract. Four out of the five (80%) had elevated serum CA 125 levels, i.e., greater than 35 U/ml and, in most cases, these levels decreased after resection of the ovarian tumor. Serum CA 19-9 and CEA levels were also increased in two (40%) and three (60%) patients, respectively, and, in these positive cases, reflected the clinical course. The results indicated the clinical usefulness of these cancer markers in Krukenberg's tumor. PMID- 3005687 TI - [Prognostic factors influencing the improvement of allogeneic bone marrow transplantation in acute leukemia based on a national survey in Japan]. AB - The records of 115 patients with acute leukemia who underwent allogeneic bone marrow transplantation from 1975 through August 1984 were collected by a national survey in Japan. One-year survival significantly improved from 9% in group I (before 1980) to 52% in group II (after 1981). Multivariate analysis indicated that selection of the patients in remission without infection at the time of transplant, as well as progress in technology of total body irradiation and selection of a platelet donor with negative cytomegalovirus titer resulted in the significant improvement in survival after 1981. PMID- 3005688 TI - [Metastatic intracerebral malignant fibrous histiocytoma from the lung]. AB - Malignant fibrous histiocytoma usually develops in the extremities, peritoneal or retroperitoneal space and the thigh. The occurrence in the lung and brain is quite rare. We report two cases of metastatic intracerebral malignant fibrous histiocytoma from the lung. The first patient developed cerebral metastasis six years after treatment of the primary site, and the second one died two months after the diagnosis of generalized metastases. Both were quite resistant to radiation therapy. PMID- 3005689 TI - [A case of triple neoplasm with acute myelogenous leukemia, squamous cell carcinoma of the lung and adenocarcinoma of the stomach]. AB - A 70-year-old man had undergone middle and lower lobectomy of the right lung for pulmonary squamous cell carcinoma in January 1979. He was treated with anticancer drugs including alkylating agents. In February 1983, he was diagnosed as AML (FAB, M2). Although cytoreduction effect was observed, he died of prolonged pneumonia on the 30th admission day. Autopsy revealed a well-differentiated tubular adenocarcinoma of the stomach (Borrman II type) and a poorly differentiated epidermoid carcinoma of the right hilar lymph node. PMID- 3005690 TI - [An evaluation of carcinoembryonic antigen (CEA), beta 2-microglobulin (BMG), and immunosuppressive acidic protein (IAP) in the serum of primary lung cancer patients]. AB - The correlation between histological type and serum CEA, BMG, and IAP was studied in 98 patients with primary lung cancer. In particular, the results showed that the positive ratio in squamous cell carcinoma was increased by measuring serum BMG or IAP simultaneously with serum CEA more than by measuring serum CEA alone. Also, when these values were compared with histological type, adenocarcinoma showed an elevation of the serum CEA level, whereas squamous cell carcinoma showed an elevation of the serum BMG or IAP level. It was suggested that serum CEA, BMG and IAP levels were closely correlated with the histological type in primary lung cancer. PMID- 3005692 TI - [A case of carcinoma of the esophagogastric junction with marked eosinophil infiltration]. AB - A case of carcinoma of the esophagogastric junction with marked eosinophil infiltration is reported. The patient was a 56-year-old man. He had complained of abdominal discomfort for three months. After examination, a total gastrectomy was performed. Carcinoma of Borrmann III type was found at the esophagogastric junction. Pathohistological findings showed that mucinous adenocarcinoma and marked eosinophil infiltration were present in the primary lesion and metastatic lymph nodes, but there was no evidence of eosinophilia. These results suggested that the tumor might produce eosinophil chemotactic factor. However, the factor could not be detected. PMID- 3005691 TI - [Whole abdominal irradiation of ovarian cancer in combined treatment with OK-432 picibanil]. AB - One hundred seventeen patients with postoperative ovarian cancer who were treated with whole abdominal irradiation by the open-field technique were analyzed as to the effectiveness of combined therapy with or without OK-432. OK-432, 0.2 to 2.0 (KE) (kev) daily, has been used to prevent bone marrow suppression since 1978 at NIRS. Cumulative five-year survival rates were 63.6% in the OK-432 group (37 patients) and 54.5% in the NON-OK-432 group (34 patients). The complete rates of previously arranged treatment schedules were 81% and 66% in the two groups, respectively, as we originally intended. PMID- 3005693 TI - [Three cases of lung cancer with bulla]. AB - We present three cases of lung cancer with bulla, which were preoperatively detected by TBLB or brushing cytology. Case 1:55 year-old man. Large-cell carcinoma of the right upper lobe. A giant bulla had been observed eight years earlier. The carcinoma protruded into the bulla just like a pedunculated polyp. Case 2: 67-year-old woman. Adenocarcinoma with severe fibrosis adjoined to the bulla of the right upper lobe. Case 3: 63-year-old man. Adenocarcinoma surrounding the central margin of the subpleural giant bula of the right lower lobe. We also discussed the clinical and pathological problems of lung cancer with bullous disease. PMID- 3005694 TI - [Wegener's granulomatosis]. PMID- 3005695 TI - [Renal tubular acidosis]. PMID- 3005696 TI - [Transport of H+ and HCO3- in the kidney]. PMID- 3005697 TI - [Transport of hydrogen and carbonic acid ions in distal nephrons]. PMID- 3005698 TI - [Hydrogen ion pump in the bladder cell membrane of turtles and toads]. PMID- 3005699 TI - [Acid accumulation of urine and influencing factors]. PMID- 3005700 TI - [Ammonium chloride and the calcium chloride loading test for the diagnosis of renal tubular acidosis]. PMID- 3005701 TI - [Sodium bicarbonate loading test and urine-blood carbon dioxide partial pressure for the diagnosis of renal tubular acidosis]. PMID- 3005702 TI - [Sodium sulfate and furosemide loading tests for the diagnosis of renal tubular acidosis]. PMID- 3005703 TI - [Definition, classification and diagnosis of renal tubular acidosis]. PMID- 3005705 TI - [Distal renal tubular acidosis. a. Classic type]. PMID- 3005704 TI - [Classification and diagnosis of proximal renal tubular acidosis]. PMID- 3005706 TI - [Distal renal tubular acidosis. C. Rate-dependent type]. PMID- 3005707 TI - [Type III renal tubular acidosis]. PMID- 3005708 TI - [Type IV renal tubular acidosis]. PMID- 3005709 TI - [Metabolic disturbances in renal tubular acidosis: water-electrolyte balance]. PMID- 3005710 TI - [Metabolic disturbances in renal tubular acidosis: calcium and phosphorus metabolism]. PMID- 3005711 TI - [Thyroid and parathyroid diseases associated with renal tubular acidosis]. PMID- 3005712 TI - [Liver disease and renal tubular acidosis]. PMID- 3005713 TI - [Urologic kidney disease and acid excretion disorders--with special reference to medullary sponge kidney]. PMID- 3005714 TI - [Primary renal tubular acidosis]. PMID- 3005715 TI - [Inborn errors of metabolism and renal tubular acidosis]. PMID- 3005716 TI - [Genetic diagnosis of familial amyloidotic polyneuropathy]. PMID- 3005717 TI - [Acquired immune deficiency syndrome]. PMID- 3005718 TI - [Diagnosis of leukemia by genetic engineering]. PMID- 3005719 TI - [Human oncogenes]. PMID- 3005720 TI - [Leukemogenic viruses]. PMID- 3005721 TI - [Carcinoembryonic antigen in genital Paget's disease]. PMID- 3005722 TI - [Eosinophilic granules of myrmecia]. PMID- 3005723 TI - [Effects of hyperthermia on the proliferation and viability of cultured human hepatobiliary carcinoma cells]. PMID- 3005724 TI - [Dietary fiber intake and diverticular disease of the colon--a case control study]. PMID- 3005725 TI - [The dual energy myocardial SPECT of 99mTc-PYP and 201TlCl for diagnosis of acute myocardial infarction]. PMID- 3005726 TI - [Basic and clinical evaluation of neuron-specific enolase (NSE) RIA kit]. PMID- 3005727 TI - [Attempts at the development of vaccines against adult T-cell leukemia]. PMID- 3005728 TI - Effect of captopril on converting enzyme activity in chemically sympathectomized, spontaneously hypertensive rats. AB - Effect of subacute angiotensin converting enzyme (ACE) blockade on the converting enzyme activity (ACE activity) in plasma, aorta, lung, kidney and whole brain was evaluated in chemically-sympathectomized (with 6-hydroxydopamine) normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) using captopril given peripherally via the intraperitoneal (i.p) route and centrally through intracerebroventricular (i.c.v.) administration. Daily i.p. injection of 25 mg/kg for 8 days reduced the blood pressure of both WKY rats and SHR, and the ACE activity in the aorta, lung and plasma of both WKY rats and SHR were correspondingly depressed. The brain ACE activity remained unaltered in both strain of rats. The ACE activity in the kidney of WKY was depressed, while that of SHR remained unchanged. These observations are independent of peripheral sympathectomy with 6-hydroxydopamine (6-OHDA). Daily central captopril administration at a dose of 2 mg/kg, i.c.v., for 8 days significantly reduced the blood pressure of SHR but not WKY rats, whereas the ACE activity of the whole brain of both WKY and SHR were depressed. Central sympathectomy with 6-OHDA did not alter these responses. It is concluded that captopril exerts its antihypertensive effect not only via reduction of the ACE activity in the plasma and lungs as reported earlier, but also that of other organs, principally the aorta, and that these effects are independent of the sympathetic nervous system. PMID- 3005729 TI - Inhibition of phospholipase A2 by tiaramide in rabbit platelets. AB - The mechanism by which tiaramide inhibited platelet aggregation was investigated using phospholipid labelling techniques by 14C-arachidonic acid (AA) and thin layer chromatography. Tiaramide did not affect cyclo-oxygenase nor thromboxane synthetase, because TXB2 was detected in tiaramide-treated platelets, unlike aspirin-treated ones, and PGE2 and PGD2 did not increase, unlike in platelets treated with OKY-1581 (an inhibitor of thromboxane synthetase). Total phospholipid radioactivity was 82.5% of radioactivity recovered before aggregation, and this decreased to 49.0% (n = 5, P less than 0.05) after aggregation by collagen (30 micrograms/ml). AA radioactivity was 9.6% before aggregation and 40.0% after. Tiaramide (100 microM) restored total phospholipid and AA levels to those before aggregation. Tiaramide decreased the amount of AA liberated from 2-(3H-arachidonyl)phosphatidylcholine by whole platelet phospholipase A2 (PLA2). Tiaramide at 10 microM inhibited collagen-induced aggregation, but not that by AA. Tiaramide did not affect 45Ca-uptake by itself nor collagen-induced 45Ca-uptake from the external medium. Tiaramide did not inhibit intracellular Ca mobilization, and it did not affect the calmodulin dependent cyclic nucleotide phosphodiesterase of rabbit brain. These facts suggest that tiaramide inhibits platelet PLA2 through mechanisms other than the blockade of Ca-influx and intracellular Ca mobilization or antagonism to calmodulin. PMID- 3005730 TI - Eugenol-mediated superoxide generation and cytotoxicity in guinea pig neutrophils. AB - Eugenol is the medicament used routinely as an anodyne and antiseptic in dentistry and a food flavour and fragrance agent. The drug stimulates the superoxide (O2-.) release of guinea pig neutrophils without a lag time. The production rate increases with the drug concentration and reaches a plateau at 5 mM. However, the induction accompanies the cytotoxicity. The stimulation system of O2-. production is sensitive to mild heating (45 degrees C, 15 min). The system proceeds without artifacts which may be mediated by a radical chain reaction with H2O2 and hydroxy radical, since neither catalase, mannitol nor azide changes the rate. Ca2+ and Ni2+ in the medium enhance the activity, but Mg2+ and Zn2+ have no effect. EDTA inhibits completely, suggesting that intracellular metal ions are involved in this system. Phenolic compounds used as dental medicaments other than eugenol act as potent stimulators of the O2-. production, with the following order of potency: thymol greater than eugenol greater than o-cresol. Resorcinol, guaiacol and hexachlorophene show little activity. This order of potency agrees with the order of hydrophobicity of these chemicals and that of the cytotoxicity to neutrophils. The data suggest that phenolic antiseptic drugs bind to the cell surface hydrophobically, trigger the oxygen burst and make the plasma membrane fragile at a high dose of drugs. PMID- 3005731 TI - Effects of enalapril and captopril on urinary excretion of kinins and electrolytes in stroke-prone spontaneously hypertensive rats. AB - Urinary excretion of kinins in stroke-prone spontaneously hypertensive (SHRSP) rats was unchanged during oral enalapril (10 mg/kg) or captopril (30 mg/kg) treatment once a day for 8 days compared to vehicle treatment. However, a significant decrease in urinary kinin excretion was observed on the 5th and 7th day compared to the pretreatment value in rats treated with enalapril. Both enalapril and captopril produced a significant reduction in blood pressure when compared to the vehicle. These findings provide no positive evidence to support the hypothesis of possible involvement of renal kinins in the antihypertensive effect of converting enzyme inhibitors in SHRSP rats. PMID- 3005733 TI - [Clinicopathological study of sclerosing hemangioma of the lung--a case report and review]. PMID- 3005732 TI - Effect of GTP on the affinity of denopamine, a new cardiotonic agent, for beta adrenergic receptors of turkey erythrocytes and rat reticulocyte membranes. AB - Affinities of denopamine, a new cardiotonic agent, and several beta-adrenergic drugs for turkey erythrocyte membranes (TEM) and rat reticulocyte membranes (RRM) which contain homogeneous beta 1- and beta 2-receptors, respectively, were studied by receptor binding. The order of potencies of denopamine and several beta-adrenergic agonists in displacing 3H-dihydroalprenolol binding (Ki, nM) in TEM was isoproterenol (Iso, 27) greater than norepinephrine (Nor, 360) greater than epinephrine (Epi, 860) greater than dobutamine (DB, 1380) greater than denopamine (1540) greater than dopamine (DA, 49500). The order in RRM was Iso (7.3) greater than Epi (58) greater than DB (750) greater than Nor (1090) greater than denopamine (2300) greater than DA (26800). In the presence of GTP, competition curves for full agonists like Iso, Epi and Nor shifted to the low affinity side (Ki values increased by 300-500% in TEM and 200-460% in RRM), and the slopes were steepened in both membrane preparations. The Ki value for denopamine increased only in TEM (70%) and that in RRM was not influenced by GTP. This suggests that denopamine has an agonist property at the beta 1-receptor but not at the beta 2-receptor and that the intrinsic activity at the beta 1-receptor of the drug is lower than full agonists. Affinities of DB and DA for TEM were influenced by GTP as well as those for RRM, although the extent of the rightward shift was less than full agonists. PMID- 3005735 TI - [A case of interstitial pneumonia with autoimmune hemolytic anemia complicated with cytomegalovirus pneumonia]. PMID- 3005734 TI - [Localization of tissue angiotensin-converting enzyme activity in rabbit lung- comparison between normal lung and the lung with granulomas induced by Freund's complete adjuvant]. PMID- 3005736 TI - [Effect of dobutamine and terbutaline on adenylate cyclase activity of the kidney pelvis and ureter in the adult rat]. PMID- 3005737 TI - [Effect of isoprenaline and the barium ion on the relaxation of the smooth muscle of the guinea pig trachea]. PMID- 3005738 TI - [1H nuclear magnetic resonance relaxation times through VX2 carcinoma growth in rabbit bladder]. PMID- 3005739 TI - Lymphosarcoma in a bullock inoculated with lymphocytes from bovine leukemia virus infected cattle. PMID- 3005740 TI - Status of Japanese encephalitis in cattle: survey of antibodies in various geographical locations in Japan. PMID- 3005741 TI - Pathology of active hepatitis in athymic nude mice caused by a mutant strain of mouse hepatitis virus, MHV-2-CC. PMID- 3005742 TI - Perspectives on the role of protein kinase C in stimulus-response coupling. PMID- 3005743 TI - Interference of mouse mammary tumor virus replication by PTT.119 [p-fluoro-L-phe m-bis-(2-chloroethyl)amino-L-phe-met ethoxy HCl]--an antitumor agent. AB - PTT.119 [p-fluoro-L-Phe-m-bis-(2-chloroethyl)-amino-L-Phe-Met ethoxy HCl] is a new bifunctional alkylating compound that possesses both cytolytic and antiviral activities. Continuous treatment of mouse mammary tumor cells with 15 microM PTT.119 reduced production of the B-type retrovirus murine mammary tumor virus (MuMTV). MuMTV levels in PTT.119-treated cultures were reduced by 31% in the first 24 hours; an additional 24 hours of treatment resulted in a further reduction of 70%. These reductions were significantly greater than could be accounted for by the effects of PTT.119 tumor cell metabolism and viability. Under identical treatment conditions, equimolar concentrations of L-phenylalanine mustard reduced MuMTV production by only 3%. PTT.119 inhibition of MuMTV replication was also apparent when mammary tumor cells were exposed for periods as short as 0.5-4 hours; persistent decreases in virus production were detected even 7 days following tripeptide treatment. Analyses of the polypeptide composition of MuMTV by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that virions from these PTT.119-treated mammary tumor cultures contained significant reductions in the relative level of the nonglycosylated 24,000-dalton (p24) viral polypeptide associated with the nucleoprotein core. Decreases in p24 were observed in MuMTV produced in the presence of the tripeptide and 1-7 days after removal of PTT.119. Examination of the steady-state levels and rates of intracellular MuMTV protein synthesis suggested that PTT.119 interferes with late steps in MuMTV processing and maturation. PMID- 3005744 TI - Acute leukemia after chemotherapy. PMID- 3005745 TI - [Elevated serum angiotensin-converting enzyme in miliary tuberculosis]. PMID- 3005746 TI - [Tissue angiotensin-converting enzyme activity in various tuberculous lesions]. PMID- 3005747 TI - [Determination of the size of a myocardial infarct by the technic of single photon emission computer tomography]. AB - A study of 38 patients with acute myocardial infarction verified by clinical, laboratory and electrocardiographic data made use of single-photon emission computered tomography, a new radiodiagnostic method for the assessment of the extent of myocardial lesion. Its findings were compared with cardiospecific enzyme activity data and showed good correlation. The new technique was found to be more informative as compared to plane scintigraphy. Possibilities of single photon emission computered tomography are discussed with reference to the diagnosis of acute infarction at different sites and showing different radionuclide inclusion patterns. PMID- 3005748 TI - [Interaction of adrenocorticotropic hormone, cortisol and insulin during emotional tension among ischemic heart disease patients]. AB - Blood levels of the adenocorticotrophic hormone (ACTH), cortisol and insulin were measured in normal subjects and patients with different forms of coronary heart disease (CHD), aged 35 to 60 years and showing no signs of diabetes mellitus, obesity and arterial hypertension, under simulated emotional stress. The majority of the patients, particularly those with unfavorable course of the CHD, showed stress-induced disturbances of hormonal control due to depressed insulin levels in the presence of an insignificant ACTH increment and high cortisolemia. Hormonal rations were shown to be of predictive value in stress-exposed normal subjects and coronary patients alike. PMID- 3005749 TI - [Alpha 2-adrenoreceptor regulation in the human body]. AB - The affinity of platelet alpha-2-adrenoreceptors for adrenaline is decreased in vivo and in vitro exposure of platelets to catecholamines at physiological concentrations in normal subjects. The direction of these alterations is consistent with the direction of the desensitization phenomena observed in the intact platelet. Hypertensive subjects display an abnormality characterized by an inability to reduce the affinity of their platelet alpha-2-adrenoreceptors in response to increased circulating levels of catecholamines. The potential importance of this defect in the etiology and clinical manifestations of essential hypertension remains to be ascertained. PMID- 3005751 TI - Outcome of children with acute renal failure. PMID- 3005750 TI - [Role of the monoaminergic system and steroid-producing glands in the development of emotional stress and the pathogenesis of arterial hypertension in monkeys]. AB - Radioimmune assays of blood cortisol and testosterone levels were carried out in mature and prepubertal male baboons exposed to repeated immobilization stress. Sympathoadrenal activity was assessed on the basis of total daily urinary catecholamine excretion. Repeated immobilization stress resulted in stable arterial hypertension persisting for 6 months after stress exposure in prepubertal baboons, whereas mature males showed transitory arterial BP elevation. In both samples, repeated stress was associated with sympathoadrenal activation and elevated blood cortisol, with higher cortisol concentrations and extremely low testosterone in prepubertal animals, while mature males only showed episodic blood testosterone drops. It is suggested that rapid development of stable hypertension in prepubertal animals may be related to specific hormonal support of their response to stress. PMID- 3005752 TI - [Possibilities of nuclear magnetic resonance in the diagnosis of breast diseases]. PMID- 3005754 TI - [Malignant fibrous histiocytoma of soft tissues simulating hematoma]. PMID- 3005753 TI - [Surgical treatment of cancer of the sigmoid]. PMID- 3005755 TI - [Mediators of inflammation]. PMID- 3005756 TI - Adrenocortical responsiveness after discontinuous corticosteroid therapy. AB - Plasma cortisol levels and the response of the adrenal gland to 0.25 mg ACTH administration were measured in 12 patients receiving 15 mg or more of a short acting steroid (fluocortolone or prednisone) on three out of four days, in 10 patients on daily steroid treatment (15 mg prednisone or more per day), and in 9 normal subjects. The basal plasma cortisol level of the first group was between that of patients on daily prednisone treatment and that of normal subjects. The adrenal function in patients on the three out of four day treatment schedule appeared to be slightly diminished, yet still within the accepted limits for adrenals capable of responding adequately to stress. The adrenal function proved insufficient in all patients on daily steroids. The three out of four day steroid regimen thus offers a solution with many advantages over continuous steroid treatment for patients refractory to the alternate-day schedule. PMID- 3005757 TI - Missing effect of etomidate on testosterone secretion in man. AB - We studied the effect of low dosage (0.26 mg/kg as a single induction dose) and high dosage (30 mg/h for long term sedation) etomidate on serum testosterone and serum luteinizing hormone (LH) concentrations in males. During high dose etomidate we found inhibition of both 11 beta-hydroxylase and cholesterol-side chain cleavage enzyme with unresponsiveness of progesterone, 17 alpha OH progesterone and 11-deoxycortisol to stimulation with ACTH. However, neither high dosage nor low dosage etomidate had any influence on serum testosterone or LH concentrations. We conclude that, in contrast to other substituted imidazole derivatives, etomidate does not interfere with testicular testosterone synthesis. It therefore may be possible to find clinically useful imidazole derivatives with endocrine actions confined to either the adrenals or the testes. PMID- 3005760 TI - [Adenomatosis of the lungs]. PMID- 3005759 TI - HTLV III antibodies and immunological alterations in hemophilia patients. AB - The clinical, immunological, and serological status of 28 patients with hemophilia A and of 13 patients with hemophilia B was investigated. Thirty-four patients were treated regularly by clotting factor concentrates and 7 patients had been substituted only 1 to 4 times. Almost all patients with severe hemophilia suffered from hepatopathy. No patient had clinical evidence of the acquired immunodeficiency syndrome (AIDS). Asymptomatic hemophiliacs showed a decreased number of T-helper (OKT 4) cells and an increased number of T suppressor (OKT 8) cells, which resulted in an inversed OKT 4/OKT 8 cell ratio. Natural killer cell activity of all patients was decreased compared to controls. After culture there was no significant difference of NK cell activity between hemophiliacs and controls. This phenomena was interpreted as a possible maturation defect of NK-cells in vivo. No relationship between immunological alterations and hepatopathy, hepatitis markers, CMV antibodies, amount and source of required factor concentrates, and the kind of hemophilia was observed. IgG immunoglobulins were higher and the OKT 4/OKT 8 ratio lower in the eight patients with lymphadenopathy than in patients without lymphadenopathy. The prevalence of antibodies to human T-lymphotropic virus (HTLVIII) was measured in 35 hemophiliacs and in 25 polytransfused patients, most of whom were suffering from acute leukemia. In 8 of 35 hemophiliacs antibodies to HTLVIII virus were detected by an enzyme linked immunosorbent assay (ELISA) and confirmatory tests. All seropositive patients were treated by blood products from the United States. Eight hemophiliacs treated by factor concentrates from German donors only were seronegative. In comparison 2 of 25 examined non-hemophilia patients receiving multiple blood products from local donors were seropositive for HTLVIII. The results show that hemophilia patients treated by imported clotting factor concentrates have a high risk of HTLVIII positivity. Hemophiliacs substituted by blood products obtained by local donor pools have only a small risk of infection. Because non-hemophiliac polytransfused patients had HTLVIII antibodies, there must be asymptomatic virus carriers in the local donor pool. The HTLVIII antibody screening of all donors and the heat treating of factor concentrates will give better therapeutic safety. PMID- 3005758 TI - Dementia of Alzheimer type (DAT)--a review of its morbid anatomy. AB - The most important morphological findings in dementia of Alzheimer type (DAT) are Alzheimer neurofibrillary tangles, senile plaques, amyloid angiopathy, granulovacuolar degeneration and Hirano bodies. The morphological and immunocytochemical findings in these changes are described, in particular those related to the pathological cytoskeleton. Their possible relationship to the disturbed synthesis of neurotransmitters recently demonstrated is considered, and their relevance for the clinical syndrome of dementia is discussed. Hypothetical etiologies (ageing per se, genetics, infection and chronic intoxication) are briefly mentioned. PMID- 3005761 TI - [Radiobiologic basis of the quality factor of protons and helium ions]. AB - Reported data and experimental results of measuring the relative biological effectiveness of protons of different energies and helium ions of 4 GeV/nuclon were analyzed to determine quality factors of the major components of cosmic radiations. It is recommended to use quality factors equal to 1.30-1.45 for 100 730 MeV protons and equal to 1.75 for 9 GeV protons and 4 GeV/nuclon helium ions. It is also suggested to employ them as standards for solving practical problems of radiation safety in space flights. PMID- 3005762 TI - [Radioprotective and therapeutic efficacy of carrageenan during exposure to proton radiation]. PMID- 3005763 TI - Ectrodactyly and syndactyly in a common marmoset (Callithrix jacchus). AB - This paper describes previously unreported malformations of both fore and hind feet in a liveborn common marmoset. Ectrodactyly of both fore feet and left hind foot and syndactyly of the right hind foot were observed. PMID- 3005764 TI - Enterotropic mouse hepatitis virus infection in nude mice. AB - The cause of emaciation and diarrhea in athymic nude mice was found to be hyperplastic typhlocolitis resulting from infection with enterotropic mouse hepatitis virus (MHV). The disease was reproduced in experimentally-inoculated nude mice using intestinal homogenates from affected mice and cell culture derived virus. Material derived from an experimental mouse was passed into neonatal Swiss mice and caused acute typhlocolitis. Virus failed to grow in NCTC 1469 cells and 17Cl-1 cells, which are normally permissive for MHV, but grew to low titer in a mouse rectal carcinoma cell line, CMT 93. These results show that an enterotropic strain of MHV can cause chronic enteric disease in athymic nude mice. The pattern of infection differs markedly from the more common MHV wasting syndrome in nude mice caused by non-enteric strains of MHV. PMID- 3005765 TI - Diagnostic exercise: tumors in a baboon. PMID- 3005766 TI - Shaping the too fluid bilayer. PMID- 3005767 TI - Alterations in hepatocyte plasma membrane in carbon tetrachloride poisoning. Freeze-fracture analysis of gap junction and electron spin resonance analysis of lipid fluidity. AB - The plasmalemma of the livers of rats treated with carbon tetrachloride (CCl4) were examined by freeze-fracture and electron spin resonance probe techniques. The rodents received by mouth either mineral oil alone (0 to 4.5 hours before sacrifice) or CCl4 in mineral oil (1:1) (2.5 ml of CCl4/kg, 0 to 3 hours before sacrifice). Rats were anesthetized with ether and livers were perfused in situ with saline either at ambient temperature or at 4 degrees C. After perfusion, livers were fixed in situ and processed for freeze-fracture and electron microscopy. Hepatocytes were isolated and incubated with 12-doxylstearic acid and subjected to electron spin resonance analysis. Electron microscopy revealed greater than a 2.5-fold increase in the individual mean gap junction size when rats were treated with mineral oil alone for 4.5 hours and the livers were processed at room temperature. The mean gap junction size in rats dosed with CCl4 for 0.5 hours before sacrifice equalled those of the group treated with mineral oil for 4.5 hours. Increases in gap junction size with CCl4 were progressive with time; by 3 hours, a 3.5-fold increase over controls was observed (p less than 0.05). When livers were perfused with iced saline, rats treated with mineral oil for 1.5 hours had a slight decrease (not significant) in mean gap junction size as compared to controls, while the size in rats treated for the same amount of time with CCl4 increased almost 5-fold over controls (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3005768 TI - Quantification of beta-adrenergic receptors in canine cardiac myocytes using autoradiography and an internal standard. AB - The type and amount of adrenergic receptors in different tissues are important determinants of their response to adrenergic stimulation in normal and pathologic states. Autoradiographic analysis of receptor binding has the advantage that receptor sites can be localized over different tissue compartments. However, quantitative assessment of receptor sites through autoradiography can only be made by using radioactive standards. The purpose of this study was to investigate the use of an internal standard method for quantitative autoradiographic receptor studies by analyzing the binding of the beta-receptor antagonist, [3H]dihydroalprenolol, to canine cardiac tissue. Sections from dog heart were incubated in [3H]dihydroalprenolol to label the beta-adrenergic receptors in the absence of (total binding) and in the presence of (nonspecific binding), 10(-5) M (+/-) propranolol. Specific binding was calculated as total minus nonspecific binding. Half of the sections were scraped off of the slides and assayed by scintillation spectrometry, and half of the sections were set up for light microscopic autoradiography. The autoradiograms were exposed, developed and stained, and grain density was quantified by using an automated image analyzer. By plotting grain density per tissue section against cpm per tissue section for total, nonspecific and specific binding, curves for efficiency of the autoradiographs were obtained. There were no significant differences among the slopes of the three lines. Also, there were no significant differences among the efficiencies of the total, nonspecific and specific binding at different concentrations of [3H]dihydroalprenolol using a two way analysis of variance. Thus, grain counts from the autoradiographs can be used to estimate the concentration of beta-adrenergic receptors in cardiac myocytes. By using these methods it was calculated that canine cardiac myocytes have 5.12 X 10(9) receptor sites/mm3. PMID- 3005769 TI - Enhanced androgen production by rabbit adrenocortical cells stimulated chronically with corticotropin: evidence for increased 17 alpha-hydroxylase activity. AB - The effects of prolonged treatment with corticotropin (ACTH1-24, 200 micrograms s.c. daily during 12 days) on the production of androgens and glucocorticoids were studied on rabbit dispersed adrenocortical cells. The steroidogenic capacity of adrenocortical cells, expressed in terms of the maximal response to ACTH of glucocorticoid (i.e. corticosterone and cortisol) production, was significantly increased after treatment with ACTH. This was associated with a loss of sensitivity to this peptide: indeed, the concentration of ACTH required to induce a half maximal secretory response was one order of magnitude higher with cells from ACTH-treated animals. Among the C21 steroids measured the changes observed involved the 17 alpha-hydroxylated compounds (cortisol, cortisone, 11 deoxycortisol) while corticosterone production was significantly depressed. This effect of prolonged ACTH treatment on steroidogenic pathways involving 17 alpha hydroxylation, was further evidenced by a clear-cut enhancement in androgen secretion (dehydroepiandrosterone, androstenedione and testosterone) by adrenocortical cells from ACTH-treated animals. The changes observed after treatment of the animal with ACTH were equally obvious, whether the adrenocortical cells were incubated with ACTH or with dibutyryl-c-AMP. PMID- 3005770 TI - Influence of triamcinolone, estradiol-17 beta and testosterone on 1,25 dihydroxyvitamin D3 binding performances to its chick intestinal receptor. AB - We have investigated the effects of large molar excesses (20,000) of triamcinolone, estradiol-17 beta and testosterone on the binding performances of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] to its receptor. The source of receptor was a duodenal chromatin fraction of vitamin D replete chickens which exhibited a high level of positive cooperativity (Hill coefficient = 1.50 +/- 0.12; n = 4) in the binding of 1,25(OH)2D3 to the two sites of its intestinal receptor. Triamcinolone did not modify the affinity, cooperativity level and maximum binding capacity of the 1,25(OH)2D3 receptor. Estradiol-17 beta induced a slight but significant increase of 13 +/- 1% (P less than 0.01) of the receptor capacity and testosterone a 29 +/- 6% (P less than 0.02) increase of the receptor affinity. A combination of estradiol-17 beta and testosterone did not modify the 1,25(OH)2D3 receptor's binding performances. In conclusion, the effects of corticoids, estradiol-17 beta and testosterone under in vitro conditions on the 1,25(OH)2D3 receptor's binding performances were found to be marginal in our system. Other studies under in vivo conditions, possibly at the pre transcriptional level, of these steroids effects on the 1,25(OH)2D3 receptor gene regulation expression would be of great interest. PMID- 3005772 TI - Conversion of androgen to estrogen by the rat fetal and neonatal female gonad: effects of dcAMP and FSH. AB - Female gonads of fetal (on days 14.5, 16.5, 18.5 and 20.5 postcoitum) and neonatal rats (on days 4.5 and 8.5 postpartum) were cultured in Medium 199 in the presence of [3H]testosterone and the conversion into [3H]estrone and [3H]estradiol was estimated. Formation of both estrogens was found in all fetal and neonatal ovaries explanted in control medium. Dibutyryl cAMP (1 mM) had a clear-cut stimulatory effect as early as 16.5 days postcoitum, but had little or no effect at 8.5 days postpartum. In contrast, ovine or rat FSH (0.3 or 1 microgram/ml, respectively) increased the aromatase activity only from 20.5 days postcoitum. The effects of FSH and dibutyryl cAMP were more obvious after preculture for 48 h in control medium. These results indicate that: a biochemical sex differentiation, revealed by the difference in aromatase activity levels between ovaries and testes or other tissues occurs in female gonads as early as 14.5 days postcoitum; aromatase activity in the ovaries increases markedly after birth; functional FSH receptors are absent before 20.5 days postcoitum in the ovaries. PMID- 3005771 TI - Adrenal cholesterol esters as substrate source for steroidogenesis. AB - Adrenocortical cells obtained from tissues of unstimulated rats and which contained a high concentration of endogenous esterified cholesterol, were labeled in vitro with unesterified [4-14C]cholesterol, or incubated in the presence of [2 14C]acetate or lipoprotein [4-14C]cholesterol oleate (LP-CE). Incubations were conducted in the absence and presence of ACTH, and changes in the specific radioactivity (SA) of the secreted corticosterone were used to assess the primary sources of cholesterol substrate used for steroidogenesis. Incubations of cells containing [4-14C]cholesterol with ACTH resulted in a marked increase in the output of corticosterone mass, but not of labeled corticosterone. Thus, the SA of corticosterone when cells were incubated with ACTH was only 6.5% of that obtained from cells incubated in the absence of ACTH. During incubations with [2 14C]acetate, the ACTH-induced increase in the output of corticosterone mass was not associated with increased isotope output, and the SA of corticosterone was only 15% of that in control incubations. This dilution was not altered in cells isolated from adrenals of rats treated with 4-aminopyrazalopyrimidine (4-APP), in which increased cholesterogenesis was demonstrable. The uptake, and hydrolysis of LP-CE, and formation of labeled corticosterone was lipoprotein concentration dependent, and was not influenced by ACTH. However, in the presence of ACTH, the SA of the secreted corticosterone was only 4-8% of that in unstimulated cells. The consistent dilution of the SA of corticosterone in ACTH-treated cells in all studies suggest that the large stores of cytoplasmic cholesterol esters in these cells may normally serve as a primary source of the immediate precursor sterol used for steroidogenesis. PMID- 3005773 TI - Modulation of oestrogen excretion profiles by adjuvant chemotherapy in pre- and postmenopausal breast cancer. AB - Modulation of steroid status by conventional chemotherapy was studied in 31 breast cancer patients receiving CMF and in 31 age-matched breast cancer patients without any therapy, taken as controls. This was achieved through the study of oestrogen excretion profiles using previously identified parameters and referring not only to classical but also to the "other", namely catechol and unusual, oestrogen metabolites. After CMF treatment the premenopausal patients exhibit a modified excretion pattern, mainly concerning a marked and significant reduction of classical oestrogens, as shown by pattern indices. Because there is evidence that oestriol metabolism is not markedly affected by CMF treatment, such a significant decrease in classical oestrogens must be attributed to the secretory function, presumably ovarian ab origine. To the contrary, after treatment, pattern indices show significantly higher median values in postmenopausal patients. Mean oestriol ratio values also display a significant increase, thus supporting the hypothesis that conventional cytotoxic drugs may act by enhancing oestrogen metabolic rates. In fact, the postmenopausal treated subgroup proved to have significantly higher excretion levels of most of the oestrogens considered to date. Surprisingly, E1 + E1-S fractions were strongly reduced in this subgroup and this leads to the suggestion of an increased steroid metabolic rate by CMF treatment. However, comparing 9 breast cancer patients, when having had both short-term and non-short-term CMF treatment, the effects on steroid excretion patterns appear to arise at an early stage. PMID- 3005775 TI - Ergot alkaloids and central monoaminergic receptors. AB - The interactions of ergolines and of ergopeptines with dopamine (DA), alpha 1 and alpha 2 central adrenoreceptors were studied. Ergolines and ergopeptines exert agonist activities at central DA receptors and exhibit antiparkinsonian activities in monkeys with unilateral ventromedial tegmental lesions of the brain stem. Both ergolines and ergopeptines are used in treatment of Parkinson's disease and their therapeutic efficacy, as well as their propensity to develop undesirable side effects is under investigation. The interactions of ergolines and of ergopeptines differ with DA receptor subtypes and states. The former are regulated by guanine nucleotides, but not the latter. Hydergine is used in treatment of disorders associated with senile dementia, and its interaction with DA, alpha 1 and alpha 2 adrenoreceptors may affect the monoaminergic imbalance in the aging brain. PMID- 3005774 TI - Increasing the response rate to cytotoxic chemotherapy by endocrine means. AB - Cloned cell lines of human breast cancer can be growth inhibited by tamoxifen and this inhibition can be reversed by estrogen. We wondered whether tamoxifen inhibition of breast cancer followed by estradiol reversal would increase the efficacy of chemotherapy by increasing the fraction of rapidly cycling cells. We describe a clinical trial in which 110 patients were prospectively randomized to chemotherapy consisting of cytoxan 750 mg/m2 and adriamycin 30 mg/m2 on Day 1 plus 5-FU 500 mg/m2 and methotrexate 40 mg/m2 on Day 8 vs the same chemotherapy plus tamoxifen 20 mg/m2 Days 2-6 and premarin 0.625 mg Q 12-H X 3 on Day 7. Chemotherapy was given in 21-day cycles. 108 patients were evaluable. No difference exist for any important prognostic variables. The first 55 patients were randomized to a regimen in which 5-FU preceded methotrexate by 24 h; thereafter, all patients received methotrexate followed in 1 h by 5-FU. No difference in any response parameter was seen between these two 5-FU methotrexate schedules. No differences in percent of protocol chemotherapy administered or observed toxicity was seen between the 2 regimens. Objective response rate was nearly identical--57% without and 64% with additional hormones. Prior adjuvant chemotherapy with L-PAM had no observable effect on response rate, response duration or survival. In a limited number of patients with inflammatory breast cancer we saw a significantly higher response rate (93 vs 61%; P = 0.03) than in patients with recurrent metastatic disease. Time to progression (13 vs 17 months) and survival (17 vs 23 months) of responders significantly favored the treatment arm including tamoxifen and premarin. Greater benefits of additional tamoxifen and premarin were seen in partial vs complete responders. This may have resulted from lower doses of chemotherapy given to patients achieving a complete remission. An additive effect of hormones plus chemotherapy cannot be entirely excluded as the explanation for the improved results seen with the addition of tamoxifen for 4 days plus 1 day of premarin. We believe that our results suggest that further efforts to increase the efficacy of chemotherapy by perturbing tumor growth rates may be worthwhile. PMID- 3005776 TI - The role of adjuvant therapy after resection of T1 N1 M0 and T2 N1 M0 non-small cell lung cancer. AB - Thirty-four consecutive patients with non-small cell lung cancer plus N1 nodal metastases (eight with T1 N1 M0 and 26 with T2 N1 M0) were retrospectively reviewed. Nineteen had adenocarcinoma, 11 had squamous disease, and four had large cell carcinoma. Eleven patients had surgical resection alone (32.3%), with a median survival of 13 months. Seven patients (20.6%) had resection followed by radiation therapy, with a median survival of 19.2 months. Sixteen patients (47.1%) had resection followed by radiation therapy and chemotherapy, consisting of cyclophosphamide, doxorubicin, methotrexate, and procarbazine. Median survival for the latter group was 45.5 months, significantly greater than for those treated with resection alone (p less than 0.005). We did not observe any relationship between survival and age, cell type, number or location of diseased hilar nodes, distance of tumor from the resected bronchial margin, tumor size, the presence or absence of visceral pleural involvement, or the type of resection performed. Resection in combination with adjuvant radiation therapy and chemotherapy offers improved median survival over resection alone in patients with T1 N1 M0 and T2 N1 M0 non-small cell lung cancer. PMID- 3005777 TI - The effect of renin-angiotensin system blockade on visceral blood flow during and after thoracic aortic cross-clamping. AB - Surgical procedures necessitating clamping of the thoracic aorta are associated with a high incidence of postoperative renal dysfunction. Plasma renin activity is elevated during and after thoracic aortic occlusion in animals. The pathophysiology of the renal dysfunction may involve the renin-angiotensin system. Blockade of the renin-angiotensin system was studied in a canine model during occlusion of the thoracic aorta. Saralasin, a competitive blocker of angiotensin II, and the converting enzyme inhibitor MK422 were studied. Sixteen animals were separated into three treatment groups: control (five animals), saralasin (five), and MK422 (six). All dogs underwent clamping of the thoracic aorta for 60 minutes. In control animals, plasma renin activity increased from 0.16 +/- 0.04 to 6.41 +/- 1.57 ng/ml/hr at 30 minutes after thoracic aortic occlusion (p less than 0.05). Thirty minutes after cross-clamp release, plasma renin activity remained 10 times greater than baseline, 1.47 +/- 0.20 ng/ml/hr (p less than 0.05). Renal blood flow was measured with 15 micron microspheres before, during, and after thoracic clamping. In control animals, renal cortical blood flow decreased during cross-clamping and remained below baseline after clamp release: baseline, 7.05 +/- 0.98 ml/gm/min (standard error of the mean); 30 min after clamp release, 3.77 +/- 0.43 ml/gm/min (standard error of the mean) (p less than 0.05). In the MK422 group, renal cortical blood flows returned to baseline after cross-clamp release: baseline, 6.38 +/- 0.49 ml/gm/min; 30 minutes after clamp release, 7.30 +/- 1.6 ml/gm/min. Infusion of MK422 after placement of the thoracic aortic cross-clamp resulted in normal renal blood flow after clamp release. This protective effect was not seen with saralasin. The resumption of normal renal cortical blood flow after the administration of the converting enzyme inhibitor MK422 suggests that elevated plasma renin activity may contribute to renal dysfunction after thoracic aortic occlusion. PMID- 3005778 TI - Additional c-abl/bcr rearrangements in a CML patient exhibiting two ph1 chromosomes during blast crisis. AB - Recent data suggest that two human genes, c-abl on chromosome 9 and bcr on chromosome 22, are involved in the generation of Ph1-positive CML. To examine a possible role of these sequences in transition from chronic towards blastic phase, rearrangements within bcr were analysed in 4 patients with Ph1-positive CML during chronic and acute phase. In 3 patients bcr rearrangements were identical in both phases, while in a fourth patient with duplicated Ph1 an amplified additional bcr fragment was detected in acute phase. Northern blot analysis of blast cells of the latter patient showed a novel 10.3 kb RNA species that replaced the altered 8 kb RNA transcript usually found in Ph1-positive CML. PMID- 3005779 TI - Improved culture of individual muscle fibres with and without spinal cord explants in a collagen gel. AB - Suspension culture of single adult rat flexor digitorum brevis (FDB) muscle fibres in Vitrogen, a purified collagen, on tissue culture plastic or glass with mesh ring supports is superior to culture upon other substrates including collagen-, laminin-, or Vitrogen-coated tissue culture plastic. The Vitrogen gel fibre mixture which attaches to glass or plastic provides at least 10 times more fibres per dish than does plating fibres on other substrates. Use of Vitrogen gel permits variable plating densities and the production of adequate numbers of cultures for long-term experimental comparisons of acetylcholinesterase (AChE) and rhodamine-alpha-bungarotoxin (RBTX) distribution on muscle fibres. Use of 40 micrograms/ml ovotransferrin (OT) instead of chick embryo extract in the culture medium significantly improves long-term survival. Cultured fibres, with or without the addition of ventral spinal cord explants. may also be examined with electrophysiological techniques. PMID- 3005780 TI - Progress in pediatric solid tumors. AB - Since 1980, impressive progress in the treatment of solid tumors has altered the practice of pediatric surgical oncology. Because these advances have been balanced by both liberal and conservative viewpoints, an accurate reassessment of traditional approaches has been possible. Noteworthy alterations in chemotherapy, radiation therapy, and surgical attitudes have improved survival rates and lessened the morbidity associated with malignant disease. Close cooperation among surgeons, pediatricians, and oncologists remains the common denominator in the successful treatment of pediatric solid tumors. This article presents examples of progress in the treatment of Hodgkin's disease, metastatic pulmonary disease, Wilms' tumor, and rhabdomyosarcoma and discusses the role of surgical intervention in conjunction with chemotherapy and radiation therapy. PMID- 3005781 TI - Forskolin- and dibutyryl cyclic AMP-mediated inhibition of chondrogenesis. AB - The regulatory role of cyclic AMP in various cellular activities is well known. It has been documented that both the notochord and extracellular matrix materials (ECM) induce somite chrondrogenesis. We believe that the ECM modulates the intracellular cAMP level during chondrogenic differentiation. The studies indicated that notochordal induction, which resulted in somite chondrogenesis (reflected by increased sulfated glycosaminoglycan synthesis) reduced the intracellular cAMP level in somites. Addition of forskolin and dibutyryl cAMP resulted in increased intracellular cAMP levels and decreased synthesis of sulfated glycosaminoglycans (decreased chondrogenesis). In the case of dibutyryl cAMP, the inhibition of sulfated glycosaminoglycan synthesis was related to the length of exposure time. Thus, the inverse relationship between cAMP content and enhanced chondrogenesis supports the theory that, in somites, a decrease in the intracellular cAMP level may be necessary to trigger chondrogenic differentiation. PMID- 3005782 TI - [Adrenomyeloneuropathy. Clinical and ultrastructural study of a case]. PMID- 3005784 TI - Arthritis associated with crystals containing calcium. AB - Varying combinations of acute inflammatory and/or chronic degenerative arthritis have been found to be associated with crystals of calcium pyrophosphate dihydrate (CPPD) and/or basic calcium phosphates (BCPs). Since the arthropathies associated with CPPDs and/or BCPs occur in older individuals, while diagnosis and treatment for monosodium urate monohydrate crystal deposition disease (gout) have become extremely precise and effective, joint problems associated with calcium crystals have become more common than those associated with monosodium urate monohydrate crystals. The classification, pathogenesis, clinical manifestations, and treatment of CPPD and BCP crystal deposition are discussed. PMID- 3005783 TI - Mechanisms of inflammation and leukocyte chemotaxis in the rheumatic diseases. AB - Understanding the mechanisms of inflammatory cell accumulation, as well as how such cells mediate tissue destruction, provides better insights into the pathogenesis and therapeutics of the rheumatic diseases. This article discusses the role of the inflammatory process in normal immune function, the mechanisms of inflammatory cell accumulation, and how the local accumulation of inflammatory cells can lead to the clinical signs and symptoms associated with rheumatic disorders. PMID- 3005786 TI - [Case for diagnosis. Mixed tumor of the skin--chondroid syringoma, Lever's mucinous hidradenoma]. PMID- 3005785 TI - [Immune staining of Paget's cells]. AB - The authors studied with the technic of PAP a case of the Paget extramammary. The presence of the precursors of the human milk in these cells in a date for to thing than Paget extramammary is the origin eccrine or apocrine. PMID- 3005788 TI - [From opiate receptors to opioid receptors]. PMID- 3005787 TI - The disposition and metabolism of captopril. PMID- 3005789 TI - Pertussis toxin enhances the beta-adrenergic and blocks the alpha 2-adrenergic regulation of renin secretion in renal cortical slices. AB - The adrenergic regulation of renin secretion was studied in renal cortical slices from control and pertussis toxin-treated rats. Pertussis toxin was used to study the role of adenylate cyclase in the control of renin release. It was observed that isoproterenol and epinephrine stimulated renin secretion and that clonidine decreased both basal and isoproterenol-stimulated renin secretion in the control group. Pertussis toxin: a) increased significantly basal renin secretion, b) displaced to the left the concentration-response curve for isoproterenol and epinephrine and magnified the response to epinephrine and c) abolished the inhibitory effect of clonidine on renin secretion. This work confirms our previous results obtained in vivo and suggests a direct effect of pertussis toxin on the cells that secrete renin. PMID- 3005790 TI - Discriminative and aversive properties of beta-carboline-3-carboxylic acid ethyl ester, a benzodiazepine receptor inverse agonist, in rhesus monkeys. AB - Rhesus monkeys were trained to discriminate injections of saline from those of beta-carboline-3-carboxylic acid ethyl ester (beta-CCE), a compound that binds to the benzodiazepine receptor, but often has actions opposite to those of the benzodiazepines. A benzodiazepine agonist midazolam and low doses of a specific benzodiazepine antagonist, Ro 15-1788, reversed the discriminative effects of beta-CCE. Higher doses of Ro 15-1788 produced stimulus effects similar to beta CCE. In a separate experiment, monkeys responded to terminate intravenous infusions of beta-CCE, but not midazolam. This aversive effect of beta-CCE was reversed by Ro 15-1788. The behavioral effects of beta-CCE in these non-human primates are consistent with other data that have shown it to act on benzodiazepine receptors, and support the hypothesis that beta-CCE can be considered an inverse agonist at this receptor. PMID- 3005791 TI - Behavioral and receptor binding studies of phencyclidine (PCP) and lithium interaction in the rat. AB - Three groups of female Sprague-Dawley rats (n = 4) were conditioned to drink water during a daily 2 hr session. The water was then changed to a solution of 1.0 mg/ml lithium chloride producing average doses between 62.9 and 72.1 mg/kg/day for Groups I and II. These rats were challenged with 4 mg/kg PCP i.p. before and during lithium treatment. Group I was tested for spontaneous locomotor activity in the open field apparatus. Lithium alone did not affect activity. After 1, 2, and 3 weeks of chronic lithium, PCP-induced activity increased 2.1, 1.7, and 2.8 fold, respectively, relative to PCP-induced activity during limited access to water only. Whole brain homogenates from Group II, after one week of chronic lithium, were used for receptor binding experiments using [3H] PCP; Group III served as water controls. The Kd (nM +/- S.E.M.) was not different in untreated (146.39 +/- 18.95) and lithium-treated (181.22 +/- 14.35) rats. The Bmax (pmole/mg protein +/- S.E.M.), however, was increased 48% (p less than 0.01) from 1.50 +/- 0.08 to 2.22 +/- 0.10 after lithium. These preliminary results suggest that chronic administration of lithium modifies the behavioral effects of PCP possibly via alterations at the receptor level. PMID- 3005792 TI - The influence of Bordetella pertussis and its constituents on the beta-adrenergic receptor in the guinea pig respiratory system. AB - In the present study, the effect of vaccination of guinea pigs with Bordetella pertussis was investigated, 4 days after treatment, on the cholinergic and beta adrenergic receptor function in isolated tracheal spirals and the number of beta adrenoceptor binding sites in guinea pig lung. It was found that B. pertussis caused an impairment in the beta-adrenoceptor function and a decrease in its number. Similar results were obtained with endotoxin. Leucocytosis promoting factor, however, was ineffective. These results indicate that endotoxin is the constituent responsible for the beta-adrenoceptor blocking effects of the bacterium. Also the combined whole cell diphtheria, B. pertussis and tetanus toxoid vaccine induced a beta-adrenoceptor blockade; the acellular vaccine was less effective. The results obtained with the B. pertussis vaccines are discussed in relation to the possible side-effects that sometimes occur after immunization of infants. PMID- 3005793 TI - Human chorionic gonadotropin binding to rat testis receptors is inhibited by a thymus factor. AB - An interrelationship between immune and reproductive systems has been postulated, and involves, among others, bidirectional effects between gonads and thymus. To this effect a rat thymus fraction of about 28000 mol wt has been reported to inhibit the effect of hCG on in vitro suspension of Leydig cells. We have investigated the antigonadotropin activity of thymus extracts on rat testis receptors. Acetonic powder obtained from thymus of 14 day-old rats was separated by molecular sieve chromatography. The effect of the collected fractions on the 125I-hCG binding to receptor sites in rat testes was evaluated. A fraction corresponding to 27000-28000 mol wt named thymus factor (TF), was found to inhibit the binding activity of 125I-hCG to its testicular receptor. The inhibitory effect of TF on hCG binding is dose related. By Scatchard analysis a competitive interaction at the receptor level between TF and hCG was demonstrated. The Ka values of hCG binding were diminished in the presence of TF while no significative changes were detected in the number of receptor sites. Present results strongly suggest a modulation function of TF at the testis receptor level. PMID- 3005794 TI - Functionalized congeners of 1,3-dipropyl-8-phenylxanthine: potent antagonists for adenosine receptors that modulate membrane adenylate cyclase in pheochromocytoma cells, platelets and fat cells. AB - Six amine, amino acid and peptide derivatives derived from 1,3-dipropyl-8-(p carboxymethylphenyl)xanthine, a functionalized congener of 1,3-dipropyl-8 phenylxanthine, have been investigated as antagonists at A2 adenosine receptors stimulatory to adenylate cyclase in membranes from rat pheochromocytoma PC 12 cells and human platelets and at A1 adenosine receptors inhibitory to adenylate cyclase from rat fat cells. The functionalized congeners and conjugates have affinity constants ranging from 80 to 310 nM at A2 receptors of PC 12 cells and from 25 to 135 nM at those of platelets. The affinity of the xanthine derivatives at A1 receptors of fat cells are in the 15 to 30 nM range. Thus, the amino acid and peptide conjugates have high potencies at both receptor subclasses and show some selectivity toward A1 adenosine receptors. Derivatives of the congeners should be useful as receptor probes and as radioiodinated ligands. PMID- 3005795 TI - Receptors for atrial natriuretic factor in cultured vascular smooth muscle cells. AB - The presence of receptors for atrial natriuretic factor (ANF) was previously demonstrated in the mesenteric vascular bed in rats. Cultured vascular smooth muscle cells obtained from mesenteric arteries of rats were examined for binding of ANF. Saturation and competition experiments demonstrated the presence of a single class of receptors for ANF with high affinity (16 pM) and low capacity. Binding was specific. Kinetic studies showed a dissociation constant which agreed with that obtained at equilibrium in saturation and competition experiments. The exposure of the cells to unlabeled ANF for at least 24 hours showed that ANF may regulate its own receptors in smooth muscle under certain physiological conditions. PMID- 3005797 TI - Activation of adenylate cyclase in Acanthamoeba palestinensis. AB - Preincubation of Acanthamoeba palestinensis homogenates in 0.25M sucrose-TM (2mM MgSO4 and 5mM Tris-HCl, pH 7.4) at 0 degree C for increasing periods of time up to 3 h, leads to a progressive increase in the activity of adenylated cyclase. In contrast, preincubation of isolated membrane fractions enriched in enzyme activity in the same medium results in no activation. However, preincubation of membrane fractions in medium containing a high density of sugars (sucrose, glucose or fructose) mimics the activation obtained with homogenates. The high density sugar activation is time and temperature dependent, and reversible upon return to a low density medium. The high osmotic pressure of the sugars utilized may be a factor, since high concentrations of the sucrose polymer, Ficoll, which has low osmotic activity, causes not activation. Soluble activators, protein synthesis and changes in cyclic nucleotide phosphodiesterase activity were all eliminated as possible effectors of the apparent activation of adenylate cyclase. In contrast to mammalian adenylate cyclase, the endoplasmic reticulum localized enzyme of Acanthamoeba is inhibited by NaF and is unaffected by GTP, adenosine, epinephrine, prostaglandin E1, propranolol, and meclofenamic acid. These data indicate that the adenylate cyclase of Acanthamoeba is structurally different from that of most mammalian cells. PMID- 3005796 TI - ACTH-(1-24) and alpha-MSH antagonize dopamine receptor-mediated inhibition of striatal dopamine and acetylcholine release. AB - The effects of ACTH-(1-24), alpha-MSH and ACTH-(4-10) were studied on the electrically evoked release of 3H-dopamine and 14C-acetylcholine from striatal slices in the absence and presence of the dopamine receptor agonist TL-99. None of the peptides affected transmitter release when TL-99 was not present. ACTH-(1 24) and alpha-MSH concentration-dependently antagonized the inhibition of striatal transmitter release induced by dopamine receptor stimulation due to the presence of TL-99. ACTH-(1-24), 10(-7)M, reduced the TL-99-induced inhibition of the release of both dopamine and acetylcholine by approximately 50%, and 5 X 10( 6) M ACTH-(1-24) restored the release fully to control values. alpha-MSH was less effective by a factor 20-30 in counteracting the release-inhibiting effect of TL 99. ACTH-(4-10) had no effect at any of the concentrations tested. These results show that ACTH/MSH-like neuropeptides may act by modulating dopamine receptor functions in rat striatum. PMID- 3005798 TI - Suppression by dexamethasone of isoproterenol-mediated changes in fatty acyl-CoA desaturase activity of Tetrahymena microsomes. AB - Preincubation of Tetrahymena pyriformis cells with dexamethasone inhibited the microsomal fatty acyl-CoA desaturase activities of isoproterenol-induced modulation; that is, an increase in delta 9-desaturase activity accompanied by a decrease in delta 12-desaturase activity. Although isoproterenol caused an increase in delta 9-terminal component activity with decreased delta 12-terminal component activity, dexamethasone reduced these isoproterenol-mediated activity changes. In cells treated with dexamethasone prior to isoproterenol administration, stimulation of cyclic AMP accumulation by isoproterenol was inhibited. These results suggest that dexamethasone may repress isoproterenol modulation of the activity of terminal components (cyanide-sensitive factor) in the fatty acyl-CoA desaturase system by reducing the cyclic AMP level. PMID- 3005800 TI - Familial isolated hypoparathyroidism: a molecular genetic analysis of 8 families with 23 affected persons. AB - Abnormalities in the parathyroid hormone (PTH) gene as a cause of hypoparathyroidism were evaluated by linkage analysis with DNA polymorphisms adjacent to the PTH gene in 8 families in which members were affected with familial isolated hypoparathyroidism (FIH). We found that in none of the 23 affected individuals was there absence of the parathyroid hormone gene or abnormal restriction patterns to suggest recognizable deletions, insertions, or rearrangements. To determine if subtle mutations within the PTH gene were associated with hypoparathyroidism in these families, we used the Pst I and Taq I restriction-site polymorphisms in linkage analysis as markers to differentiate between PTH alleles. In 4 families, affected sibs inherited different PTH gene alleles, implying that hypoparathyroidism was not due to an abnormality in the PTH gene. In 2 other families, linkage analysis was uninformative because of inability to differentiate between PTH alleles. In 2 families, concordance was found between the inheritance of hypoparathyroidism and specific PTH alleles in affected members, suggesting that in these families, hypoparathyroidism may be due to an alteration in or near the PTH structural gene. We conclude that FIH is a diverse group of disorders and is characterized by genetic and molecular heterogeneity. In some forms of FIH the mutation that leads to PTH deficiency does not lie within the region of the structural gene for PTH. Linkage analysis using DNA polymorphisms within the PTH gene is of benefit in identifying individuals with disorders of PTH secretion or synthesis in whom DNA sequencing and expression studies of the PTH gene might succeed in establishing the molecular basis of the disease. PMID- 3005799 TI - Clinical and laboratory evaluation of cytomegalovirus-induced mononucleosis in previously healthy individuals. Report of 82 cases. AB - The present report describes the clinical and laboratory profile of 82 previously healthy individuals who developed cytomegalovirus (CMV)-induced mononucleosis. Many of these patients posed initial diagnostic problems and were hospitalized with diagnoses such as fever of undetermined origin, active viral hepatitis, acute leukemia, probable systemic lupus erythematosus, autoimmune hemolytic anemia, and severe pancytopenia. These patients underwent a variety of diagnostic biopsies, including liver biopsies (6) and bone marrow aspirations (9). Four patients had exploratory laparotomies, 1 for a ruptured spleen, and another had a splenectomy following an erroneous initial diagnosis of agnogenic myeloid metaplasia. There was no apparent clinical response to a short course of steroid therapy in 3 of 5 cases and acyclovir in another. The vast majority of these patients demonstrated infectious mononucleosis-type reactive blood smears, negative heterophil antibody studies, mildly or moderately elevated aspartate aminotransferase activity, and evidence for subclinical hemolysis on serial specimens. The peak serum bilirubin levels were above 2.0 mg/dl in only 2 of 71 cases tested, both of the latter patients having significant hemolysis (hemoglobin values 8.6-9.3 g/dl). The CMV-IgM test had a high sensitivity for detection of CMV macroglobulins (positive in 81 of 82 cases). In contrast, complement-fixing antibodies to CMV showed diagnostic four-fold titer changes in only 39/82 cases (47.6%). Despite its great sensitivity, the CMV-IgM test is limited by a one-way crossreaction of acute Epstein-Barr virus (EBV)-IM sera and spurious positive reactions in some sera due to the presence of rheumatoid factors. Based on EBV-specific serologic studies, the 82 patients with CMV-IM could be divided into 4 groups: 3 patients without antibodies to EBV; 2) 69 patients with uncomplicated serologic data indicative of long-past EBV infections; (3) 6 patients with unusual antibody profiles, e.g., anti-D responses; and (4) 5 patients, including 1 originally susceptible to EBV, with apparent dual CMV/EBV infections. At the conclusion of our study, final diagnoses and initial hematologic data were correlated in 750 cases in which CMV macroglobulins were searched for. The vast majority of patients with active CMV infections initially demonstrated either markedly or moderately reactive peripheral blood smears. These data support our impression that diagnostic tests for CMV, as well as for EBV, are seldom indicated in symptomatic previously healthy patients whose blood smears during the acute phase (first several weeks) of their illnesses are either nonreactive or minimally reactive. PMID- 3005801 TI - Evidence for a delayed, integral, and proportional phase of glucocorticoid feedback on ACTH secretion in normal human volunteers. AB - To investigate the mechanisms responsible for glucocorticoid feedback on nonstress-induced ACTH secretion in normal subjects, 24 volunteer subjects (14 males and 10 females, 21 to 43 years) were divided into six groups in a randomized fashion and studied. Each subject received a single midnight dose of 30 mg/kg per body weight of metyrapone and then cortisol was administered according to different protocols in the next morning to provide extreme variations of the input signal. It was found that no obvious inhibition in plasma ACTH levels was shown during the first 15 min despite the fact that cortisol was given at rather larger doses and short time intervals. However, a significant suppression in plasma ACTH levels began to manifest approximately 30 min after cortisol administration in each study group and it became apparent that the degree of inhibition of ACTH level at 75 min correlated with the plasma cortisol concentrations at the same moment (r = 0.97, P less than 0.01) as well as with the dosage of cortisol during this time, whatever administered (r = 0.99, P less than 0.01). In summary, our data provided evidence for a delayed, proportional, and integral phase of glucocorticoid feedback on nonstress-induced ACTH secretion in normal human volunteers. PMID- 3005803 TI - Plasmid vectors for the genetic analysis and manipulation of rhizobia and other gram-negative bacteria. PMID- 3005802 TI - Improved preparative methods for isolation and purification of tobacco chloroplast ribosomes, ribosomal proteins, and rRNA. PMID- 3005804 TI - Coronavirus inhibitor in human sera: age distribution and prevalence. AB - We studied the distribution, in human sera, of non antibody serum inhibitor active towards human OC43 and bovine NCDCV coronaviruses. Antibodies to coronaviruses, present with high prevalence in human sera, were filtered by Protein A Sepharose CL 4B column. In the present work we tested sera of newborns, infants, young children and adults. Inhibitory activity was revealed in all sera except these from newborns. This non antibody inhibitor was tested by a plaque reduction assay. PMID- 3005805 TI - Serum inhibitor of coronaviruses OC43 and NCDCV: a study in vivo. AB - It is well known that some strains of coronaviruses, such as human OC43 and bovine NCDCV, are highly inhibited by non antibody factors present in sera of different mammalian species and sensitive to phospholipase-C treatment. So far this inhibitor has only been revealed in vitro experimental procedures with coronavirus strains adapted to growth in human lung fibroblast cell cultures. The purpose of this work was to ascertain whether this inhibiting activity was also effective "in vivo". In order to detect the "in vivo" activity, we used suckling mice which have been shown to be the animals most sensitive to infection with these viruses. Our results demonstrated that the same non antibody inhibitor is able to neutralize viral infection both "in vivo" and "in vitro". PMID- 3005806 TI - Plasmid mediated gentamicin resistance in strains of Klebsiella from hospital acquired infections. AB - Two gentamicin resistant strains of Klebsiella were isolated at different times from patients with hospital acquired urinary tract infections. Both strains contained a conjugative 81-megadalton plasmid that encoded resistance to gentamicin, ampicillin, cephalothin and mercuric chloride. The plasmid conferred to Escherichia coli CSH26 the same pattern of resistance to eight aminoglycoside antibiotics and gave similar DNA fragments after restriction endonuclease digestion. PMID- 3005807 TI - Effect of near-UV (366 nm) on the activity of certain nucleic acid enzymes in Verticillium agaricinum. AB - Verticillium agaricinum when grown for 60 min under near-UV irradiation (366 nm) followed by 24 h in darkness produced maximal activity of a number of nucleic acid enzymes (DNase I, endonuclease, nuclease, RNase A, and RNase T1). Total protein and nucleic acid on the other hand showed a decrease under the same conditions. The nucleic acid enzymes which are involved in reversible reactions seem to favour nucleic acid degradation in this study. PMID- 3005808 TI - Aftercare following miscarriage. PMID- 3005809 TI - [Effect of cyclic 3',5'-adenosine monophosphate on the growth rate of Escherichia coli]. AB - Cyclic 3',5'-adenosine monophosphate (cAMP) inhibits the rate of Escherichia coli growth in media with glucose. When the exogenous nucleotide is added, the generation time and the lag phase become longer. These parameters decrease if cAMP is entirely absent from the cya- mutant as compared to the parent cya+ strain. The nucleotide exerts a low activity in media with glycerol. The action of cAMP is highly specific. PMID- 3005811 TI - [Mechanism of action of intravenous anesthetics]. PMID- 3005810 TI - Gastric mucus--physical properties in cytoprotection. PMID- 3005813 TI - Infection of the oral mucosa with defined types of human papillomaviruses. AB - Biopsies from 9 different oral papillomatous proliferations were analysed for human papilloma viral (HPV) sequences of types 1 to 19 and 21 to 26 by Southern blot analysis with 32p-labelled cellular DNA. HPV sequences were detected in 7 out of 9 biopsies obtained from individual patients. Of three cases with the clinical diagnosis focal hyperplasia Heck, two contained HPV-6 related sequences and one HPV 13. In addition, one tongue base papilloma contained HPV 11. A papilloma of the palate revealed HPV 11 sequences. HPV 6 could be demonstrated twice in the remaining 4 oral papillomatous proliferations. Two biopsies remained negative. The data shows that HPV DNA can be regularly demonstrated in oral papillomas. PMID- 3005812 TI - Analysis of benign and malignant urogenital tumors for human papillomavirus infection by labelling cellular DNA. AB - A total of 268 biopsies from the genital region was screened for the presence of human papillomavirus DNA. The specimens included carcinoma of the vulva, vagina, cervix, corpus uteri, ovaries and penis, and Bowen's carcinomas, Bowenoid papuloses, Bowen's disease, cervical intraepithelial neoplasias (CIN I to III), Buschke-Lowenstein tumors, a cervical polyp, decidua, endometrium and histologically normal biopsies. Of 45 carcinomas, 18 contained either HPV 16 and/or 18 and 3 HPV 6-related sequences. In a few individual biopsies double or even triple infections were noted. Unusual was the presence of HPV 2-related DNA in one biopsy from Bowen's disease, whereas 2 condylomata acuminata contained HPV 3-related DNA and one contained HPV DNA related to a group of epidermal HPV's found in epidermodysplasia verruciformis lesions. PMID- 3005815 TI - Breast cancer after treatment for osteosarcoma. AB - Two cases of patients with primary osteosarcoma who developed subsequent new primary infiltrating ductal carcinoma of breast are presented. The relationship of irradiation from diagnostic radiology, chemotherapy given, and possible genetic factors are discussed. A recommendation for the lifetime follow-up program of a patient with osteosarcoma should include careful attention to breast self-examination and regular breast examination by the attending physician. PMID- 3005814 TI - Neurovirulence and latency in inbred mice of two HSV-1 intrastrain variants of divergent pathogenicity. AB - The pathogenicity pattern of the HSV-1 strain ANG which is nonencephalitogenic in mice is compared with that of a selected neurovirulent variant of this strain in DBA-2 mice. After i.p. inoculation both variants replicate to high titers in the mouse peritoneum and build up a virus reservoir in the spleen. Both viruses have no effect on visceral mouse organs other than the spleen; both viruses lead to an inefficient and masked viraemia and both replicate efficiently in CNS tissue after direct intracranial injection. Only the pathogenic variant, however, spreads to the CNS and leads to lethal encephalitis upon intraperitoneal infection. The assumption that infection of the CNS would be mediated by hematogenous transport is not supported by the data obtained from transfer and cocultivation experiments with lymphocytes or experiments involving artificial viraemia. In a model to analyse the capacity of the viruses to invade nerve axons and to induce a latent infection both viruses were found to be latency positive in dorsal root ganglia. It is clear that non-neurovirulent HSV-1 strains are subjected to a postganglionic block of virus spread from the periphery to the CNS. The experiments led to the hypothesis that axonal transport even beyond the dorsal root ganglia to the CNS proceeds unrestricted, whereas lethal CNS invasion is prevented by a restriction of viral replication of HSV-1 ANG in the CNS by a virus-induced host defence mechanism. PMID- 3005816 TI - Simultaneous leptomeningeal and intramedullary spinal metastases in small cell lung carcinoma. AB - A patient with small cell lung carcinoma and simultaneous intramedullary spinal and leptomeningeal metastases is described. A review of the literature revealed that 28 cases of intramedullary spinal metastases have been reported in small cell lung carcinoma patients since 1978. Fifteen of these were in patients who had coexisting carcinomatous meningitis. The pathogenesis of intramedullary spinal metastases is discussed and a potential mechanism is proposed to explain the simultaneous occurrence of intramedullary and leptomeningeal metastases. Therapy for leptomeningeal metastases should be considered in patients with small cell lung carcinoma and intramedullary spinal metastases. PMID- 3005817 TI - Congestive heart failure, hypertension, and hyperreninemia in bilateral Wilms' tumor: successful medical management. AB - Congestive heart failure is an unusual complication of the hyperreninemia of Wilms' tumors. Cases with bilateral tumors present a difficult management problem. This is a report of the successful medical management of a child with congestive heart failure secondary to hyperreninemia from bilateral Wilms' tumor. Hypertension and hyperreninemia were extensively documented. Their etiologic relation to the congestive heart failure was supported by the patient's improved cardiac function following specific renin-angiotensin blockade. With unilateral tumors, surgical excision corrects the hypertension; however, with large bilateral tumors, excision is out of the question. A unique feature of this case is the ability to control the blood pressure with saralasin. With subsequent antitumor therapy, renin concentrations decreased proportional to tumor size, and renin angiotensin blocking therapy could be discontinued. PMID- 3005819 TI - [Demonstration of rotaviruses in fecal samples of children with several rapid methods of agglutination]. PMID- 3005820 TI - [Gigantism caused by a double-secreting (GH-PRL) acidophile adenoma in a 6-year old girl]. PMID- 3005821 TI - Acquired immunodeficiency syndrome. A contemporary overview. PMID- 3005822 TI - Additional recommendations to reduce sexual and drug abuse-related transmission of human T-lymphotropic virus type III/lymphadenopathy-associated virus. PMID- 3005818 TI - Uses of lac fusions for the study of biological problems. PMID- 3005823 TI - Acquired immunodeficiency syndrome in correctional facilities: a report of the National Institute of Justice and the American Correctional Association. PMID- 3005824 TI - Dopamine, acting through D-2 receptors, inhibits rat striatal adenylate cyclase by a GTP-dependent process. AB - This report demonstrates that the D-2 dopamine receptors that are present in rat striatum can directly inhibit the activity of adenylate cyclase in a GTP dependent manner. N-n-propylnorapomorphine evoked a more pronounced inhibition than did dopamine. However, in the presence of the D-1-selective antagonist, SCH 23390, dopamine was also observed to inhibit the enzyme. Forskolin facilitated the detection of D-2 receptor-mediated inhibition by markedly stimulating striatal adenylate cyclase activity. The inhibition was antagonized in a dose dependent manner by the D-2 receptor antagonist spiperone (Ki value = 70 pM) and was absolutely dependent on the presence of both GTP and sodium ions. Inhibition produced via D-2 receptors was additive with that produced via opiate or adenosine A1 receptors. The nonhydrolyzable GTP analogue, 5' guanylylimidodiphosphate [Gpp(NH)p], did not substitute for GTP in promoting the D-2 receptor-mediated inhibition. It thus appears that D-2 receptors mediate adenylate cyclase inhibition by processes that have been observed for other neurotransmitters in the striatum and elsewhere. In addition, Gpp(NH)p displayed a Ca2+/calmodulin dependency for its inhibitory effects that was not shared by receptor-mediated, GTP-dependent inhibition. PMID- 3005826 TI - Characterization of angiotensin converting enzyme by [3H]captopril binding. AB - We demonstrate that [3H]captopril selectively labels angiotensin converting enzyme (EC 3.14.15.1) (ACE) and employ this technique to probe enzyme-inhibitor interactions. [3H]Captopril binding sites copurify with ACE activity from rat lung or rat brain. At each stage of the purification the Vmax/Bmax ratio, or kcat is 17,000 min-1 with hippuryl-L-histidyl-L-leucine as substrate. The specificity of [3H]captopril binding is apparent in the similar pharmacologic profile of inhibition in crude and pure enzyme preparations. Furthermore, binding sites and enzyme activity comigrate in gel filtration and sucrose gradient sedimentation experiments. Equilibrium analysis of [3H]captopril binding to purified ACE reveals a Bmax of 6 nmol/mg of protein (KD = 2 nM), demonstrating the presence of one inhibitor binding site per polypeptide chain. The kinetics of [3H]captopril binding are characterized by monophasic association and dissociation rate constants of 0.026 nM-1 min-1 and 0.034 min-1, respectively. The affinity of ACE for both [3H] captopril and enalaprilat is greater at 37 degrees than at 0 degree, demonstrating that these interactions are entropically driven, perhaps by an isomerization of the enzyme molecule. The ionic requirements for [3H]captopril binding and substrate catalysis differ. Chloride and bromide ion, but not fluoride, are about 100-fold more potent stimulators of binding than catalysis. When the active site Zn2+ ion is replaced by Co2+, catalysis was stimulated 2 fold, whereas binding activity was decreased by 70%. PMID- 3005825 TI - A functionalized congener approach to adenosine receptor antagonists: amino acid conjugates of 1,3-dipropylxanthine. AB - 1,3-Dipropyl-8-phenylxanthine, a synthetic analog of theophylline and a potent antagonist of adenosine at A1 and A2-adenosine receptors, has been attached covalently through a functionalized chain to amino acids and oligopeptides. The xanthine conjugates have been studied as competitive inhibitors of the specific binding of [3H]N6-cyclohexyladenosine to A1-receptors of rat cerebral cortical membranes and for inhibition of cyclic AMP accumulation elicited by 2 chloroadenosine in guinea pig brain slices through A2-receptors. A free amino group on the extended chain generally resulted in high potency at A1-receptors. The potency (in some cases extending into the subnanomolar range) and selectivity for A1-receptors (up to 200-fold) suggest that this approach can yield a versatile class of "functionalized congeners" of adenosine receptor antagonists in which distal modifications of the attached moiety ("carrier") can serve also to improve pharmacodynamic and pharmacokinetic parameters. The water solubility in many of the more potent analogs has been enhanced by two orders of magnitude over that of simple, uncharged 8-phenyl xanthine derivatives. Analogs in which the carrier contains D-tyrosine have potential for development of iodinated radioligands for adenosine receptors. The functionalized congener approach is potentially applicable to other drugs and for development of prodrugs. PMID- 3005827 TI - Biochemistry of misonidazole reduction by NADPH-cytochrome c (P-450) reductase. AB - The biochemical mechanism for the reduction of misonidazole [1-(2-nitro-1 imidazolyl)-3-methoxy-2-propanol] by purified rabbit liver NADPH-cytochrome c (P 450) reductase, the primary nitroreductase of liver, has been studied. Neither the anaerobic nor the futile aerobic reduction velocities exhibited signs of Michaelis-Menten saturation at concentrations less than 5 and 10 mM, respectively. The anaerobic reduction of misonidazole resulted in the formation of glyoxal from fragmentation of the imidazole ring in 25% yield. The rate of glyoxal formation was linear with time and paralleled the reduction of misonidazole, suggesting that it was derived from the partitioning of a reactive intermediate between at least two alternative pathways. Negligible amounts of the 2-amino derivative of misonidazole were formed, however, indicating the existence of alternative reduction/fragmentation pathways. PMID- 3005830 TI - Effects of temperature and membrane phase transitions on ligand binding to alpha 2-receptors of human platelets. AB - The binding of agonists and antagonists to alpha 2-adrenergic receptors of human platelets was studied. The receptors showed homogeneous affinities for antagonists but two affinity states for the agonist (-)-epinephrine, which were modulated by guanine nucleotides. Van't Hoff plots of antagonist binding had a break point at about 18 degrees and considerable diversity between 18 degrees and 0 degree. Agonist binding to both affinity states showed a similar break point; agonist binding to the high affinity state was characterized by a large entropy component compared to the low affinity state. This entropy component was reduced at higher concentrations of sodium, indicating that it may be due to liberation of sodium ions. Measurements of the fluorescence of 1-anilin-8 naphthalenesulfonate showed thermotropic phase transitions of the platelet membranes at about 17 degrees. The transition temperature was decreased to about 12 degrees by addition of 10 mM octanoic acid. Octanoic acid also shifted the break points of the van't Hoff plot of antagonist and low affinity agonist binding from 18 degrees to 12 degrees. High affinity agonist binding, however, remained unchanged. It is concluded that agonist-specific thermodynamic characteristics of ligand binding to alpha 2-receptors of human platelets can only be investigated by regarding differences between high and low affinity agonist binding. These differences include an entropy increase upon ligand binding, which is in part due to enhanced liberation of sodium ions, and a loss of sensitivity to fluidity changes in the outer layer of the plasma membrane. PMID- 3005828 TI - Effect of islet-activating pertussis toxin on the binding characteristics of Ca2+ mobilizing hormones and on agonist activation of phosphorylase in hepatocytes. AB - Islet-activating protein (IAP, a Bordetella pertussis toxin) was employed to test the hypothesis that the inhibitory GTP-binding regulatory protein of adenylate cyclase (Ni) mediates GTP effects on the binding of Ca2+-mobilizing hormones to liver plasma membranes and is involved in calcium mobilization stimulated by these agonists. IAP added to normal liver plasma membranes catalyzed the incorporation of radioactivity from [32P]NAD into a 41,000-Da peptide (presumably the alpha-subunit of Ni). However, no such incorporation was observed in liver membranes prepared from rats 24 hr after intraperitoneal injection of IAP. Angiotensin II attenuated glucagon-stimulated increases in cAMP in hepatocytes prepared from control but not IAP-treated rats. In contrast, following IAP treatment, no changes were observed in the ability of glucagon, vasopressin, angiotensin II, or epinephrine to activate phosphorylase; nor did this treatment alter [3H]vasopressin binding or epinephrine displacement of [3H]prazosin binding. However, IAP treatment decreased [3H]angiotensin II binding affinity when studies were performed in the absence but not the presence of 5' guanylylimidodiphosphate (GppNHp). This shift was small and represented only 5-8% of the shift in apparent Kd elicited by GppNHp in untreated membranes. In vitro studies with IAP confirmed the results of the radioligand binding studies using in vivo IAP treatment. The effects of NaCl on [3H]angiotensin II binding were also tested but were not typical of other receptors which couple to Ni. The data suggest that, although a small population of hepatic angiotensin II receptors couple to Ni and attenuate glucagon-stimulated increases in cAMP, vasopressin, alpha 1-adrenergic, and the majority of angiotensin II receptors do not interact significantly with Ni. Thus, although there is evidence that agonist-induced Ca2+ mobilization requires a GTP-binding regulatory protein, this protein does not appear to be Ni in rat liver. PMID- 3005829 TI - Synthesis and characterization of a high affinity radioiodinated probe for the alpha 2-adrenergic receptor. AB - The availability of radioiodinated probes has facilitated the localization and molecular characterization of cell membrane receptors for hormones and neurotransmitters. However, such probes are not available for the study of the alpha 2-adrenergic receptor. This report describes the synthesis and characterization of functionalized derivatives of the selective alpha 2 adrenergic antagonists, rauwolscine and yohimbine, which can be radiolabeled to high specific activity with 125I. Following demethylation of rauwolscine or yohimbine, the resultant carboxylic acid derivatives were reacted with 4 aminophenethylamine to yield the respective 4-aminophenethyl carboxamides, 17 alpha-hydroxy-20 alpha-yohimban-16 beta-[N-4-amino-phenethyl]carboxamide (rau pAPC) and 17 alpha-hydroxy-20 beta-yohimban-16 alpha-[N-4 aminophenethyl]carboxamide. In competitive inhibition studies using rat renal membranes and the radioligand [3H]rauwolscine, rau-pAPC (Ki = 11 +/- 1 nM) exhibited a 14-fold greater affinity than the corresponding yohimbine derivative (Ki = 136 +/- 45 nM). The higher affinity compound, rau-pAPC, was radioiodinated by the chloramine T method, and the product, 125I-rau-pAPC [17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-3 -[125I]iodophenethyl)carboxamide], was purified by reverse phase HPLC to high specific activity (2175 Ci/mmol) and its binding characteristics were investigated in rat kidney membranes. Specific binding of 125I-rau-pAPC was saturable and of high affinity as determined by Scatchard analysis (KD = 1.8 +/- 0.3 nM) or from kinetic studies (KD = k2/k1 = 0.056 +/- 0.013 min-1)/4.3 +/- 0.2 X 10(7) M-1 min-1 = 1.3 +/- 0.3 nM). In competition studies, alpha-adrenergic antagonists and agonists inhibited the binding of 125I-rau-pAPC with a potency order consistent with an interaction at alpha 2-adrenergic receptors (rauwolscine greater than phentolamine greater than prazosin; clonidine greater than (-)-epinephrine greater than (-)-norepinephrine greater than dopamine greater than (+)-epinephrine). In rat liver and human platelet membranes, high affinity binding of 125I-rau-pAPC was also observed (liver, KD = 1.2 +/- 0.4 nM; platelet, KD = 3.2 +/- 1.5 nM). In addition, the density of alpha 2-adrenergic receptors identified from binding studies with 125I rau-pAPC in kidney, liver, and platelet membranes was similar to that observed in parallel studies with [3H]rauwolscine. These findings indicate that 125I-rau-pAPC is a high affinity probe that selectively identifies alpha 2-adrenergic binding sites. Availability of this radioligand should facilitate the localization and biochemical characterization of this alpha-adrenergic receptor subtype. PMID- 3005831 TI - Solubilization of [3H]leukotriene D4 receptor complex from guinea pig lung membranes. AB - Guinea pig lung membrane leukotriene D4 (LTD4) receptors were prelabeled with [3H]LTD4 and solubilized using digitonin, 3-[(3-cholamidopropyl)- dimethylammonio]-1-propane sulfonate, and other non-ionic, zwitterionic, and ionic detergents. [3H]LTD4 remains tightly associated with the receptor complex in the digitonin solubilized state. The dissociation rate of [3]LTD4 from the soluble receptor complex was increased in the presence of guanine nucleotides and sodium ions in a manner similar to that observed for the receptors in the membrane-bound state. The soluble [3H]LTD4 receptor complex was retained on wheat germ lectin affinity columns and destabilized by heat (40 +/- 4 degrees), trypsin, and chymotrypsin treatment, suggesting that the receptor is a glycoprotein. Size exclusion high pressure liquid chromatography of the soluble receptor complex showed that an apparent molecular weight of the soluble receptor complex, in the presence of digitonin, is in the range of 240,000-500,000. An approximately 20-fold enrichment of receptor-radioligand complex was achieved by passing the solubilized LTD4 receptor preparation successively through size exclusion and wheat germ lectin chromatography columns. These data provide the first step toward the purification and chemical characterization of LTD4 receptors. PMID- 3005832 TI - Uncoupling of gamma-aminobutyric acid B receptors from GTP-binding proteins by N ethylmaleimide: effect of N-ethylmaleimide on purified GTP-binding proteins. AB - Treatment of membranes from bovine cerebral cortex with N-ethylmaleimide (NEM) resulted in inhibition of gamma-aminobutyric acid (GABA) binding to GABAB receptors. The binding curve for increasing concentrations of agonist was shifted to the right by NEM treatment. Guanine nucleotide had little effect on the binding of GABA to NEM-treated membranes. The addition of purified GTP-binding proteins, which were the substrates of islet-activating protein (IAP), pertussis toxin, to the NEM-treated membranes caused a shift of the binding curve to the left, suggesting modification of GTP-binding proteins rather than receptors by NEM. Therefore, the effect of NEM on two purified GTP-binding proteins, Gi (composed of three subunits with molecular weight of alpha, 41,000; beta, 35,000; gamma, 10,000) and Go (alpha, 39,000; beta, 35,000; gamma, 10,000) was studied. NEM did not significantly change guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding and GTPase activity of these two proteins. In contrast, NEM-treated Gi and Go were not ADP-ribosylated by IAP and did not increase GABA binding to NEM treated membranes. When alpha and beta gamma subunits were treated with NEM and then mixed with nontreated alpha and beta gamma to form Gi or Go, respectively, both oligomers with NEM-treated alpha-subunits lost their abilities to be IAP substrates and to couple to receptors. These results indicate that NEM uncoupled GTP-binding proteins from receptors by modifying alpha-subunits of GTP-binding proteins, and the site seemed to be on or near the site of ADP-ribosylation by IAP. When alpha and beta gamma subunits were treated with NEM and then mixed to form Gi or Go, GTP gamma S binding in the absence of Mg2+ and GTPase activity were changed, although they were not affected when oligomers were treated with NEM. The results suggest the existence of another sulfhydryl group which is protected from NEM by the association of subunits. The modification of this sulfhydryl group by NEM appeared to interfere with the interaction between alpha and beta gamma. PMID- 3005834 TI - Modulation of calmodulin function and of Ca2+-induced smooth muscle contraction by the calmodulin antagonist, HT-74. AB - The relationship between the functions of calmodulin (CaM) and Ca2+-induced smooth muscle contraction was investigated using a newly synthesized CaM antagonist, 3-(2-benzothiazolyl)-4,5-dimethoxy-N-[3-(4- phenylpiperidinyl)propyl]benzenesulfonamide (HT-74). We noted a selectivity of HT 74 for CaM, compared to other calcium-binding proteins and target enzymes of CaM. As HT-74 had no significant effect on the intensity of 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence in the presence of the Ca2+-CaM complex, the HT 74-binding sites may differ from those of naphthalenesulfonamides and phenothiazines which decrease ANS fluorescence. The Ca2+ binding to CaM was inhibited significantly by 1.0 microM HT-74, in sharp contrast to phenothiazines and naphthalenesulfonamides which increase the extent of the Ca2+ binding to CaM. Increasing CaM concentrations reversed the HT-74-induced inhibition of CaM dependent enzymes such as myosin light chain kinase and Ca2+-dependent cyclic nucleotide phosphodiesterase, with Ki values of 0.5 microM and 0.4 microM, respectively. In the presence of 0.3 microM HT-74, potassium-depolarized rabbit aortic strips pre-contracted with 0.3 mM CaCl2 relaxed, and this relaxation was completely reversed by the addition of an excess amount of CaCl2 (10 mM). This compound shifted the dose-response curve for CaCl2 to the right, in a competitive manner. However, HT-74 inhibited the phenylephrine-induced contraction elicited in Ca2+-free solution and the calcium ionophore A23187-induced contraction in the presence of calcium ion. Therefore, this agent affects intracellular actions of Ca2+ rather than membrane receptors or the influx of Ca2+. HT-74 is a CaM antagonist which binds to CaM in a manner different from that heretofore reported. It inhibits Ca2+ binding to CaM and produces a competitive inhibition of Ca2+-induced contractions of depolarized vascular smooth muscle. PMID- 3005833 TI - Inhibition of Ns-stimulated human platelet adenylate cyclase by forskolin. AB - The diterpene, forskolin, increases basal adenylate cyclase activity in membranes of human platelets to more than 20-fold with an EC50 of about 5 microM. However, when the platelet adenylate cyclase was activated via the stimulatory coupling component, Ns, e.g., by the hormone, prostaglandin E1, or the stable GTP analog, guanosine 5'-[gamma-thio]triphosphate, added in combination with a protease, forskolin was able to inhibit the enzyme. The inhibition was half-maximal and maximal (40-50% inhibition) at 0.01 and 0.1 microM forskolin, respectively, and occurred without apparent lag phase. At a maximally inhibitory concentration, forskolin largely reduced the apparent affinity of the Ns-stimulated platelet adenylate cyclase for its substrate MgATP in a noncompetitive manner, which resulted in a pronounced inhibition by forskolin at low substrate concentrations and a further increase in activity at high MgATP concentrations. Treatment of intact platelets or platelet membranes with agents known to interfere with Ni mediated adenylate cyclase inhibition did not diminish but even increased the forskolin-induced inhibition of the adenylate cyclase. However, inhibition of the prostaglandin E1-stimulated adenylate cyclase by forskolin and the inhibitory hormonal agents, thrombin and epinephrine, were not additive at maximally inhibitory concentrations. Furthermore, increasing concentrations of Mg2+ and Mn2+ reduced (Mg2+) or even reversed (Mn2+) the forskolin-induced inhibition. The data indicate that forskolin apparently has two distinct effects on the platelet adenylate cyclase, namely inhibition and stimulation. The data furthermore suggest that the adenylate cyclase inhibition by forskolin is not mediated by the inhibitory guanine nucleotide-binding protein Ni, but may be due to an action of the diterpene at the adenylate cyclase catalytic moiety, particularly when activated by Ns, or a closely related membrane component. PMID- 3005835 TI - The effect of 16 beta-substitution on the structure and activity of digitoxigenin: is there an additional binding interaction with Na+,K+-ATPase? AB - We have studied the basis of the effect of 16 beta-substitution on the structure and activity of digitoxigenin derivatives by examining the crystal structures of these compounds and their inhibitory activity toward the receptor for these drugs, Na+,K+-ATPase. To understand the increase in inhibitory activity of the 16 beta-ester compounds and the decrease in activity of gitoxigenin (16 beta hydroxydigitoxigenin), both with respect to digitoxigenin, we have compared the observed conformations of gitoxigenin, gitoxigenin 16 beta-formate, and other 16 beta-esters to that of digitoxigenin. Our data do not support the possibility of hydrogen bonding between the 16 beta-hydroxyl of gitoxigenin and the lactone ring, previously suggested to account for the decreased activity of gitoxigenin vis a vis digitoxigenin, but, rather, suggest that the decreased activity may be due to an intramolecular hydrogen bond between the hydroxyls on C-14 and C-16 and an unusual D-ring conformation which combine to alter the carbonyl oxygen of the lactone ring away from the putative active position. In contrast, the 16 beta ester moiety has a preferred conformation which may serve to fix the lactone ring in the active conformation. Thus, the increased activity of the 16 beta-esters cannot be explained by altered carbonyl oxygen position and may be related to an additional receptor binding site for the ester moiety. PMID- 3005836 TI - Effects of mercury (II) compounds on the activity of dUTPases from various sources. AB - The deoxyuridine triphosphate nucleotidohydrolases (dUTPases, EC 3.6.1.23) from Escherichia coli K-12-,Acholeplasma laidlawii B-PG9-, human KB cell-, and the herpes simplex virus (HSV) type 1- and 2-induced dUTPases were purified and used to determine the effect of various mercury (II) compounds on their activities. Mercuric acetate, 5-mercuri-dUTP (HgdUTP), and 5-mercuri-dCTP (HgdCTP) acted as irreversible active site-directed inhibitors of the dUTPases purified from eukaryotic organisms but not those from prokaryotic organisms. The inhibition constants (Ki) were estimated for the KB, HSV-1, and HSV-2 dUTPases to be 8 +/- 2, 12 +/- 3, and 9 +/- 2 microM for mercuric acetate, 204 +/- 25, 121 +/- 15, and 111 +/- 10 microM for HgdUTP, and 775 +/- 25 and 651 +/- 23 microM for HgdCTP, respectively. The conversion of HgdUTP to its mercurithio-derivative resulted in a decrease in the affinity of the derivative for the eukaryotic dUTPases. The 5 mercurithioethylene glycol derivative of dUTP did not act as a substrate for the KB dUTPase but it did act as a substrate for the HSV-1- and HSV-2-induced dUTPases with Ki values of 526 +/- 47 and 483 +/- 32 microM, respectively. These results demonstrate that the eukaryotic dUTPases can be distinguished based upon differences in their affinities for the mercurithio-derivatives of dUTP and suggest that there are differences in the steric binding properties of the nucleotide-binding site of these enzymes. PMID- 3005837 TI - Regional difference in brain benzodiazepine receptor carbohydrates. AB - The ability to photolabel benzodiazepine receptors from various regions of the rat brain with 3H-flunitrazepam has allowed for the structural examination of these receptors by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. Results for all regions studied revealed the labeled receptor to consist of a single major band of radioactivity with the apparent molecular weight of approximately 50,000. Under our conditions of labeling we do not significantly label any higher molecular weight forms of the receptor. Exposure of the benzo-diazepine receptors to either of the glycosidases neuraminidase (N) and endoglycosidase-H (E) results in the specific removal of sialic acids and complete asparagine-linked carbohydrate moieties, respectively. This type of structural modification of the receptor resulted in an apparent decrease in the molecular weight, as determined by increased mobility on sodium dodecyl sulfate polyacrylamide gel electrophoresis, for all regions examined (cortex + N + E, 8,000-10,000; hippocampus + N, 7,000, + E, 12,000; cerebellum + N, none, + E, 4,000). These results point to a heterogeneity in the posttranslational glycosylation of the benzodiazepine receptor that may be due to brain region-specific differences in glycosylation. The removal of these carbohydrate moieties alters the binding of agonists and antagonists to the benzodiazepine receptor. Cortical agonist binding following either glycosidase treatment resulted in no apparent shift in the Kd but a significant decrease in the Bmax. The Bmax change may be the result of a large decrease in affinity or denaturation of a subpopulation of benzodiazepine receptors. Antagonist binding also showed no apparent Kd shift but a significant increase in the Bmax. The increase may have resulted from the activation of "hidden" benzodiazepine receptors or a shift of low affinity sites to sites of higher affinity. Cerebellar agonist or antagonist binding was not altered, in terms of either Kd or Bmax, by either enzyme treatment, correlating well with the small amount of carbohydrate removal seen following such treatments. The ability of these enzymes to modify the apparent molecular weight of the benzodiazepine receptors and the strong correlation to altered ligand binding, in a regional specific manner, generally parallel the description given of type 1 and type 2 benzodiazepine receptors. PMID- 3005838 TI - Proposals for the mu-active conformation of the enkephalin analog Tyr-cyclol(-N gamma-D-A2-bu-Gly-Phe-Leu-). AB - The conformational behavior of the sterically restricted cyclic peptide Tyr cyclo(-N gamma-D-A2-bu-Gly-Phe-Leu-), proposed recently as an enkephalin analog with high opiate activity, is examined by theoretical investigations. The method used allows the search of conformational energy minima associated with cyclic structures fitting a hypothetical opiate pharmacophore. The results obtained show that, despite the fact that many cyclic structures of low conformational energy can be found for this compound, only one of them can be retained as a conformer presenting the characteristic features of the imposed pharmacophore. This conformation is stabilized by an intramolecular H-bond between the D-A2bu carbonyl and the Leu NH group so that a beta-turn is formed. This structure also presents a high mobility of the Tyr1 side-chain which can fit the tyramine moiety of rigid opiates with minor loss of conformational energy. A two-step binding mechanism is proposed for the interactions of this cyclic peptide with its receptor which could be an intermediate between the "zipper" model proposed for flexible linear peptides and the "lock-and-key" model adapted to rigid molecules. The selectivity of enkephalin analogs for mu and delta opioid receptors is discussed in light of the present theoretical investigations. PMID- 3005839 TI - [Amplification of the adenylate cyclase gene in Escherichia coli cells]. AB - Amplification of the cya gene of E. coli on the plasmid pBR325 leads to an increase of adenylate cyclase activity proportional to the gene dosage. In strains harboring hybrid plasmids with cya gene the intracellular level of cAMP and the rate of nucleotide secretion are also elevated. The adenylate cyclase activity in cells with truncated cya gene cloned on pBR322 remains sensitive to glucose inhibition. Amplification of the cya gene leads to considerable resistance of beta-galactosidase synthesis to transient repression by alpha methylglucoside, but does not influence the permanent repression caused by glucose. PMID- 3005841 TI - [The role of ATP and membrane potential in the penetration of phage T17 DNA into the cell during infection]. AB - The role of ATP and membrane potential in phage T7 DNA injection into E. coli during infection has been studied. Entrance of phage T7 genes of class II and III was shown to be prevented by arsenate, indicating the requirement for phosphorylated macroergs in the phage DNA injection. The injection process was also inhibited by exposition of the cells to the uncoupler of oxidative phosphorylation. Dependence of the injection efficiency on the membrane-potential value has been shown to be sigmoidal, which suggests a regulatory role of the membrane potential in phage T7 DNA injection from the virion into the host cell. PMID- 3005840 TI - [Restriction analysis of DNA from phage SM of Pseudomonas aeruginosa]. AB - The DNA of temperate phage SM P. aeruginosa has one PvuII site, two BamHI sites, three HindIII sites and five EcoRI sites. Using these restrictases the physical map of the phage genome has been constructed. The DNA of phage SM has in their structure cohesive ends similar to cos-sites of phage lambda DNA. EcoRI-fragments with cohesive ends have molecular masses 2.9 and 4.9 MDa. PMID- 3005843 TI - [Cloning of the DNA fragment of a transgenic mouse containing an integrated recombinant plasmid]. AB - The "plasmid rescue" method has been used to isolate the recombinant plasmid pMA3 fragment and flanking sequences from the transgenic mouse genome containing the fragment in integrated form. The "rescued" plasmid, pMAR1, lacks all virus sequences and retains only those regions of pBR322 that are responsible for the plasmid replication and Escherichia coli ampicillin resistance. The plasmid pMA3 deletion has occurred at its integration into the mouse genome after microinjection into the zygote. The integrated fragment of the plasmid is adjacent to the genome repeated sequence that is highly conservative in evolution. PMID- 3005842 TI - [Replication in yeasts of plasmid pE194 from Staphylococcus aureus]. AB - The ability of the plasmid pE194 from S. aureus to serve as an autonomously replicating sequence (ARS) in yeast was shown. The hybrid plasmid pLD744 that contains pE194 and the yeast LEU2 gene sequences is unstable in yeast like other YRp-vectors: the mitotic stability of the pLD744 was as much as 1%. The plasmid pLD712 that differs from pLD744 by the existence of a centromeric sequence from the chromosome III of yeast Saccharomyces cerevisiae reveals about one order greater stability. The observation that there are some sequences in the primary structure of the pE194 which strongly conform to the ARS consensus in yeast inclines us to infer that the existence of ARS consensus on pE194 DNA is not sufficient for its effective replication in yeast. PMID- 3005844 TI - [Interaction of EcoRII restriction and modification enzyme with synthetic DNA fragments. IV. DNA duplexes with phosphoamide and pyrophosphate internucleotide bonds--the substrates for the study of single-strand breaks]. AB - A set of DNA duplexes with repeated EcoRII, EcoRI and AluI restriction endonuclease recognition sites in which EcoRII scissile phosphodiester bonds were replaced by phosphoramide or uncleavable pyrophosphate bonds have been synthesized. Endonuclease EcoRII was found not to cleave the substrate at the phosphoramide bond. The substrates containing non-nydrolysable pyrophosphate or phosphoramide bonds in one of the chains of EcoRII recognition sites were used to show that this enzyme is able to catalyze single-strand scissions. These scissions occur both in dA- and dT-containing chains of the recognition site. Endonuclease EcoRII interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of the other. Synthesized DNA duplexes are cleaved specifically by EcoRI and AluI endonucleases, this cleavage being retarded if the modified bonds are in the recognition site (EcoRI) or flank it (AluI). For EcoRII and AluI this effect is more pronounced in the case of substrates with pyrophosphate bonds than with the phosphoramide ones. PMID- 3005845 TI - [Similar organization of beta,beta'-subunit RNA-polymerase genes and adjacent ribosomal protein genes in Enterobacteriaceae and Pseudomonas putida]. AB - The genes coding for the RNA-polymerase beta,beta'-subunits and adjacent ribosomal protein genes in Escherichia coli, Salmonella typhimurium, Shigella flexneri, Serratia marcescens, Proteus mirabilis and Pseudomonas putida are compared by the Southern hybridization procedure. In all the species studied close clustering of the genes rplKAJL and rpoBC is demonstrated. Preliminary physical maps for these genes in S. typhimurium, S. flexneri, S. marcescens and P. mirabilis are proposed. Rifampicin is shown to stimulate the beta,beta' subunit synthesis in all the species studied, suggesting the existence of attenuators localized in front of the rpoBC genes. The similar arrangement of the genes rplKAJLrpoBC in a number of bacterial species is proposed to be due to common mechanisms of their coordinate expression. PMID- 3005846 TI - [Electron microscopic study of jugulo-tympanic paraganglioma. Rhomboid crystals in the tumor cells]. PMID- 3005847 TI - [Fatal central nervous system metastases from clinically cured primary small cell carcinoma of the lung. Case report and general chemotherapeutic conclusions]. PMID- 3005848 TI - TSH receptor antibodies. PMID- 3005849 TI - The effect of gamma-irradiation on mu DNA transposition and gene expression. AB - Mobile genetic elements are a ubiquitous presence in the genomes of all well studied organisms. The effect of genomic stress on the status and transposition of these elements has not, as yet, been extensively characterized. We have been using temperate, transposable bacteriophage Mu as a model system to examine the behavior of mobile genetic elements and have previously shown that many DNA damaging agents did not induce a Mu prophage to enter the lytic cycle of multiple rounds of DNA transposition. To extend these results and to examine the possibility that they were a reflection of damage to the DNA substrate for Mu transposition, we have constructed a mini-Mu plasmid, pMD12, which contains the early region of Mu, flanked by both extremities required for transposition in cis, and the beginning of the transposase gene A fused in frame to the lacZ gene. This A'-lacZ fusion protein maintains beta-galactosidase enzymatic activity under the control of the expression of the Mu transposase A gene and thus, the capacity for Mu transposition can be easily monitored by assaying for beta-galactosidase. By measuring the amount of beta-galactosidase after various doses of gamma irradiation, we found that doses of up to 75 krad had no effect on the expression of the Mu transposase gene A. This was confirmed by the lack of induction of a Mu prophage in strains containing a chromosomally inserted Mu genome. Although the plaque-forming units per colony-forming unit of strain CSH67, containing a chromosomally inserted lambda prophage, increased approximately 100-fold from 0 to 75 krad, no stimulation of induction of prophage Mu lytic growth was observed. We also found that plasmid pMD12 did not transpose and chromosomally associate upon gamma-irradiation. This supports the assertion that DNA-damaging agents, including gamma-rays, do not induce the transposition of prokaryotic mobile genetic elements. PMID- 3005850 TI - Analyses of mutation in pSV2gpt-transformed CHO cells. AB - We have developed a system to study mutations which affect expression of the E. coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) in hypoxanthine-guanine phosphoribosyl transferase-deficient (HPRT-) Chinese hamster ovary (CHO) cells that have been transformed by the plasmid pSV2gpt. Several gpt transformed cell lines have been isolated and characterized with respect to integrated pSV2gpt sequences, expression of the gpt gene, and cytotoxic and mutagenic responses to UV light. While the gpt-transformed CHO and wild-type CHO K1-BH4 cell lines have similar cytotoxic responses to UV light, the gpt transformed cell lines respond differently from the parental CHO-K1-BH4 cell line in terms of mutation induction. As with CHO-K1-BH4 HPRT mutants, spontaneous or induced XPRT mutants derived from the gpt+ cell lines can be selected for 6 thioguanine resistance (TGr). Analysis of cell-free extracts from a number of these TGr clones indicates that the mutant phenotype is due to the absence of XPRT activity. One transformant, designated AS52, has previously been described in limited detail. Here we describe additional characteristics of this cell line, as well as several related transformants. PMID- 3005851 TI - Transposable genetic elements, spontaneous mutations and the doubling-dose method of radiation genetic risk evaluation in man. AB - The principal aspects of the 'doubling-dose method' currently used by the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) and the Committee on the Biological Effects of Ionizing Radiation (BEIR) of the U.S. National Academy of Sciences, for the evaluation of genetic radiation hazards in man are briefly reviewed. With this method, which is primarily applicable to autosomal dominant and X-linked disorders, the expected increase in risk from radiation is expressed as a fraction of the current prevalence of these disorders, and thus in relation to an understandable frame of reference. Since the doubling dose is estimated as a ratio of spontaneous to induction rates of mutations, its magnitude is susceptible to changes in either the numerator (spontaneous rate) or the denominator (induction rate). Studies during the past 20 years or so with a number of experimental systems have demonstrated the existence of mobile DNA sequences in the genome and their causal role in the origin of spontaneous mutations, although the proportion of the latter among all spontaneous mutations is not known for any species. If a major proportion of spontaneous mutations in man is mediated by these mobile DNA sequences, and if their mobility is unaltered by radiation exposures, the calculation of the doubling dose in the manner mentioned above, and its use in risk evaluations becomes questionable. However, considerations based on the organization of the human genome would suggest that it is unlikely that a major fraction of spontaneous mutations that lead to disease states in man is due to mobile genetic elements. Consequently, the use of the doubling-dose method for the evaluation of genetic radiation hazards in man would appear to be valid at the present time. PMID- 3005852 TI - Enhanced induction of SV40 replication from transformed rat cells by fusion with UV-irradiated normal and repair-deficient human fibroblasts. AB - Fusion of SV40-transformed rat (BRKSV) cells which do not spontaneously produce infectious virus, with permissive monkey cells resulted in a low level of production of infectious virus in the heterokaryons. UV-Irradiation of the BRKSV cells prior to fusion did not result in increased virus production, but irradiation of the monkey cells prior to fusion did result in enhanced induction (EI) of SV40, as compared to control experiments in which neither cell type was irradiated. This indicated that rat cells lack the ability to initiate replication of integrated SV40 upon UV-irradiation and do not contain "permissiveness" factors that are required to support SV40 replication. In contrast, monkey cells do contain such permissiveness factors which seem to be temporally enhanced by UV-irradiation, and thus may be responsible for the EI phenomenon. Expression of EI was dose-dependent and reached maximum values approximately 24 h after UV-irradiation. The kinetics of EI resembled that of EI previously established for SV40 induction in semi-permissive cells, and of enhanced reactivation (ER) and enhanced mutagenesis (EM) of SV40 in monkey cells. Similar kinetics of EI were obtained when human diploid fibroblasts were used for fusion with BRKSV cells. Similar levels of EI were found with normal human cells and repair-deficient xeroderma pigmentosum (XP) cells of complementation groups A and C, and XP variant cells. This suggests that expression of EI is not related to excision repair. Since EI is also normally expressed in XP cells which display an abnormal ER of HSV and in XP variant cells which show a delayed EM of HSV, we conclude that EI may occur independently of ER and EM. Finally it was shown that treatment of human cells with N-ethyl-N-nitrosourea results in similar induction of EI as irradiation with UV-light, and that addition of TPA in fusion experiments has no effect on EI. PMID- 3005853 TI - Chromosomal changes induced by chrysotile fibres or benzo-3,4-pyrene in rat pleural mesothelial cells. AB - The induction of chromosomal aberrations in rat pleural mesothelial cells (RPMC) following in vitro treatment with chrysotile fibres has been demonstrated. The production of chromosomal aberrations was also observed after treatment of the cells with benzo-3,4-pyrene (BP). The yield of abnormal metaphases was dose dependent and reached 58% at a BP dose of 2 micrograms/ml. Chrysotile fibres at 7 micrograms/ml induced 21% abnormal metaphases and the frequency decreased with further increases in fibre concentration. Their decline is possibly related to a lethal effect. Chrysotile-induced chromosomal aberrations were primarily of the chromatid type and included breaks and fragments. BP induced chromosome exchanges which were not seen following chrysotile treatment. Minutes and double minutes were detected in BP-treated RPMC and occasionally found after chrysotile application. These results confirm that chrysotile fibres are clastogenic for some cultured cells and demonstrate that the fibres induce chromosome damage in target RPMC. PMID- 3005854 TI - Role of the uvr gene in the SOS function inducing activity of two tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, in Escherichia coli K12. AB - Two tryptophan pyrolysis products, Trp-P-1 and Trp-P-2 were assayed in the SOS chromotest using PQ 37 (uvr A) and PQ 35 (uvr+) E. coli K12 strains, in the presence of S9 fraction from Aroclor-induced rats. Both compounds were able to induce the expression of SOS functions in uvr A bacteria, in the following order: Trp-P-1 less than Trp-P-2 less than aflatoxin B1, at low concentrations (less than 125 ng/assay). In this range, the induction of SOS functions was significantly decreased in the uvr+ strain. This implies that the uvr gene product plays an important role in the repair of genotoxic damage induced by Trp P-1 and Trp-P-2. At higher concentrations (125-500 ng/assay), Trp-P-1 became more efficient in inducing SOS functions than Trp-P-2 and excision repair was less efficient than at low concentration. PMID- 3005855 TI - Evaluation of putative inhibitors of DNA excision repair in cultured human cells by the rapid nick translation assay. AB - A human fibroblast nick translation assay has been applied to an examination of 48 diverse chemical agents to assess their ability to specifically interfere with the DNA excision-repair process following ultraviolet irradiation. Certain inhibitors of DNA polymerase, ribonucleotide reductase and purine and pyrimidine biosynthesis are shown to inhibit the resynthesis step of repair while DNA intercalators and inhibitors of DNA topoisomerases appear to inhibit the incision step. A variety of other agents previously implicated as inhibitors of DNA repair was also examined and found to have no such effect. This type of analysis should prove useful in the rapid identification of new classes of compounds that antagonize normal cellular repair functions and that might, therefore, act as comutagens or cocarcinogens. PMID- 3005856 TI - Motor neuropathy associated with a facilitating myasthenic syndrome. AB - We report a patient with progressive muscle weakness, areflexia, and no sensory loss. Electromyography revealed normal sensory nerve conductions, mild slowing of motor nerve conduction velocities, low amplitude compound muscle action potentials, a neuromuscular transmission defect characterized by prominent facilitation, and diffuse fibrillation potentials. Muscle biopsies showed acute denervation atrophy, and at autopsy, there was anterior horn cell loss and gliosis in the spinal cord. The findings suggest the coexistence of a motor neuropathy and a facilitating myasthenic syndrome. PMID- 3005857 TI - Widespread outbreaks of clam- and oyster-associated gastroenteritis. Role of Norwalk virus. AB - Consumption of raw shellfish has long been known to be associated with individual cases and sporadic outbreaks of enteric illness. However, during 1982, outbreaks of gastroenteritis associated with eating raw shellfish reached epidemic proportions in New York State. Between May 1 and December 31, there were 103 well documented outbreaks in which 1017 persons became ill: 813 cases were related to eating clams, and 204 to eating oysters. The most common symptoms were diarrhea, nausea, abdominal cramps, and vomiting. Incubation periods were generally 24 to 48 hours long, and the duration of illness was 24 to 48 hours. Bacteriologic analyses of stool and shellfish specimens did not reveal a causative agent. Norwalk virus was implicated as the predominant etiologic agent by clinical features of the illness and by seroconversion and the formation of IgM antibody to Norwalk virus in paired serum samples from persons in five (71 percent) of seven outbreaks in which testing was done. In addition, Norwalk virus was identified by radioimmunoassay in clam and oyster specimens from two of the outbreaks. Determining the source of the shellfish was not always possible, but northeastern coastal waters were implicated. The magnitude, persistence, and widespread nature of these outbreaks raise further questions about the safety of consuming raw shellfish. PMID- 3005858 TI - Infections with herpes simplex viruses (1). PMID- 3005859 TI - Infections with herpes simplex viruses (2). PMID- 3005860 TI - The AIDS epidemic: multidisciplinary trouble. PMID- 3005861 TI - Treatment of serious cytomegalovirus infections with 9-(1,3-dihydroxy-2 propoxymethyl)guanine in patients with AIDS and other immunodeficiencies. AB - The drug 9-(1,3-dihydroxy-2-propoxymethyl)-guanine (DHPG) was used to treat serious cytomegalovirus infections in 26 patients with underlying immunodeficiency (including 22 with the acquired immunodeficiency syndrome). In 17 of 22 patients in whom cytomegalovirus was virologically confirmed, clinical status improved or stabilized, although in 4 of them the status of some affected organs deteriorated or did not improve. Fourteen of 18 patients with adequate viral-culture data had clearing of cytomegalovirus from all cultured sites. Patients with cytomegalovirus pneumonia often responded poorly; four of seven died before completing 14 days of DHPG therapy. The condition of 11 of 13 patients with cytomegalovirus retinitis and 5 of 8 with gastrointestinal disease stabilized or improved. However, clinical and virologic relapses occurred in 11 of 14 patients (79 percent) when DHPG was discontinued. Neutropenia was the most frequent adverse reaction. We conclude that DHPG offers promise for the therapy of severe cytomegalovirus infections in some immunodeficient patients, but further study will be necessary to establish its efficacy and safety. PMID- 3005862 TI - Defective regulation of Epstein-Barr virus infection in patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related disorders. AB - Patients with the acquired immunodeficiency syndrome (AIDS) acquire undifferentiated B-cell lymphomas that are similar to African Burkitt's lymphoma and contain Epstein-Barr virus (EBV). Using an in vitro assay system that measures a complex of cellular responses to EBV-infected lymphocytes, we found that B cells from 7 patients with AIDS and from 10 patients with AIDS-related disorders produced abnormally low numbers of immunoglobulin-secreting cells (P less than 0.001 as compared with normal controls) and that T-cell suppression, which was greater than 80 percent in EBV-seropositive normal controls, was absent. Instead, the patients' T cells markedly increased immunoglobulin production induced by EBV. In further studies, we determined that the mean frequency of circulating EBV-infected B cells capable of spontaneous outgrowth in vitro was 13 per 10(6) B cells in 7 patients with AIDS and 21 per 10(6) B cells in 10 patients with AIDS-related disorders--figures that were significantly higher than the mean in normal controls (P less than 0.001). Thus, patients with AIDS or AIDS-related disorders may be predisposed to the development of EBV containing lymphomas, because they have a profound defect of T-cell immunity to EBV and abnormally high numbers of EBV-infected B cells in the circulation. PMID- 3005863 TI - Reduced sensitivity of lymphocyte beta-adrenergic receptors in patients with endogenous depression and psychomotor agitation. PMID- 3005864 TI - Animal model for Epstein-Barr lymphoma. PMID- 3005865 TI - Cloning of cDNA encoding the murine IgG1 induction factor by a novel strategy using SP6 promoter. AB - Complementary DNA encoding the IgG1 induction factor, the first lymphokine directed to B lymphocytes, from a murine T-cell line has been cloned using a new strategy. The putative primary amino-acid sequence was deduced from the nucleotide sequence determined. The lymphokine synthesized by the direction of this cloned cDNA has many other functions, such as production of B-cell growth factor-1 and induction of Ia on B cells. PMID- 3005866 TI - Inhibition of growth factor-induced differentiation of PC12 cells by microinjection of antibody to ras p21. AB - The protein products (p21) of the ras cellular proto-oncogenes are thought to transduce membrane signals necessary for the induction of cell division. However, there is uncertainty as to the precise role of ras p21 in mediating ligand membrane receptor signals leading to cell differentiation. Treatment of rat phaeochromocytoma cells (PC12) with nerve growth factor (NGF) results in the induction of a number of phenotypic characteristics of sympathetic neurones, including cessation of cell division and outgrowth of neuronal processes (neurites). Here we report that microinjection of antibody to ras p21 into PC12 cells inhibited neurite formation and resulted in temporary regression of partially extended neurites, an effect which was observed up to 36 h after initiation of NGF treatment. Neurite formation induced by cyclic AMP was unaffected by injection of anti-p21 antibody. These results indicate that p21 is involved in the initiation phase of NGF-induced neurite formation in PC12 cells and has a role in hormone-mediated cellular responses distinct from cell proliferation. PMID- 3005868 TI - AIDS. PMID- 3005867 TI - Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K+), K+- and Ca2+-ATPases. AB - The plasma membrane ATPase of plants and fungi is a hydrogen ion pump. The proton gradient generated by the enzyme drives the active transport of nutrients by H+ symport. In addition, the external acidification in plants and the internal alkalinization in fungi, both resulting from activation of the H+ pump, have been proposed to mediate growth responses. This ATPase has a relative molecular mass (Mr) similar to those of the Na+-, K+- and Ca2+-ATPases of animal cells and, like these proteins, forms an aspartylphosphate intermediate. We have cloned, mapped and sequenced the gene encoding the yeast plasma membrane ATPase (PMA1) and report here that it maps to chromosome VII adjacent to LEU1. The strong homology between the amino-acid sequence encoded by PMA1 and those of (Na+ + K+), Na+-, K+ and Ca2+- ATPases is consistent with the notion that the family of cation pumps which form a phosphorylated intermediate evolved from a common ancestral ATPase. The function of the PMA1 gene is essential because a null mutation is lethal in haploid cells. PMID- 3005869 TI - AIDS virus and HTLV-I differ in codon choices. PMID- 3005870 TI - Origin of the human AIDS virus. PMID- 3005871 TI - Human p53 gene localized to short arm of chromosome 17. AB - The p53 gene codes for a nuclear protein that has an important role in normal cellular replication. The concentration of p53 protein is frequently elevated in transformed cells. Transfection studies show that the p53 gene, in collaboration with the activated ras oncogene, can transform cells. Chromosomal localization may provide a better understanding of the relationship of p53 to other human cellular genes and of its possible role in malignancies associated with specific chromosomal rearrangements. A recent study mapped the human p53 gene to the long arm of chromosome 17 (17q21-q22) using in situ chromosomal hybridization. Here, by Southern filter hybridization of DNAs from human-rodent hybrids, we have localized the p53 gene to the short arm of human chromosome 17. PMID- 3005873 TI - Lack of antibodies to adult T-cell leukaemia virus and to AIDS virus in Israeli Falashas. PMID- 3005872 TI - Stable transformation of maize after gene transfer by electroporation. AB - The graminaceous monocots, including the economically important cereals, seem to be refractory to infection by Agrobacterium tumefaciens, a natural gene transfer system that has been successfully exploited for transferring foreign genes into higher plants. Therefore, direct transfer techniques that are potentially applicable to all plant species have been developed using a few dicot and monocot species as model systems. One of these techniques, electroporation, uses electrical pulses of high field strength to permeabilize cell membranes reversibly so as to facilitate the transfer of DNA into cells. Electroporation mediated gene transfer has resulted in stably transformed animal cells and transient gene expression in monocot and dicot plant cells. Here we report that electroporation-mediated DNA transfer of a chimaeric gene encoding neomycin phosphotransferase results in stably transformed maize cells that are resistant to kanamycin. PMID- 3005874 TI - Lack of HTLV-I antibodies in Africans. PMID- 3005876 TI - Liposomal subunit vaccine against Epstein-Barr virus-induced malignant lymphoma. PMID- 3005875 TI - Ecto-protein kinase activity on the external surface of neural cells. AB - ATP is secreted in association with neurotransmitters at certain synapses and neuromuscular junctions. Extracellular ATP is known to exert potent effects on the activity of cells in the nervous system, where it can act as a neurotransmitter or as a modulator regulating the activity of other neurohormones. We have suggested that such modulation may involve the activity of extracellular protein phosphorylation systems. It is well known that intracellular protein kinases are important in the regulation of various neuronal functions, but protein kinases which use extracellular ATP to phosphorylate proteins localized at the external surface of the plasma membrane (ecto-protein kinases) have not been demonstrated in neuronal cells. Here we present direct evidence for the existence of an ecto-protein kinase and demonstrate endogenous substrates for its activity at the surface of intact neural cells. The phosphorylation of one of these surface proteins is selectively stimulated during cell depolarization. In addition, neuronal cell adhesion molecules (N-CAMs) appear to be among the substrates of ecto-protein kinase activity. These results suggest a role for surface protein phosphorylation in regulating specific functions of developing and mature neurones. PMID- 3005877 TI - Swiss prize shared. PMID- 3005878 TI - AIDS therapy by blocking CD4 + cells. PMID- 3005879 TI - Modulation of visual cortical plasticity by acetylcholine and noradrenaline. AB - During a critical period of postnatal development, the temporary closure of one eye in kittens will permanently shift the ocular dominance (OD) of neurones in the striate cortex to the eye that remains open. The OD plasticity can be substantially reduced if the cortex is infused continuously with the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) during the period of monocular deprivation, an effect that has been attributed to selective depletion of cortical noradrenaline. However, several other methods causing noradrenaline (NA) depletion leave the plasticity intact. Here we present a possible explanation for the conflicting results. Combined destruction of the cortical noradrenergic and cholinergic innervations reduces the physiological response to monocular deprivation although lesions of either system alone are ineffective. We also find that 6-OHDA can interfere directly with the action of acetylcholine (ACh) on cortical neurones. Taken together, our results suggest that intracortical 6-OHDA disrupts plasticity by interfering with both cholinergic and noradrenergic transmission and raise the possibility that ACh and NA facilitate synaptic modifications in the striate cortex by a common molecular mechanism. PMID- 3005880 TI - T-cell recognition of antigen and the Ia molecule as a ternary complex. AB - T-lymphocyte co-recognition of antigen and major histocompatibility complex (MHC) encoded molecules (such as murine Ia molecules) is thought to be mediated by a single cell-surface receptor, although the molecular mechanism by which this occurs is controversial (reviewed in ref. 1). One possibility is that the antigen molecule and the Ia molecule interact physically, either before or after encountering the T-cell antigen-specific receptor. Alternatively, both molecules could bind to the receptor independently of one another, accounting for the dual specificity of the receptor without postulating a physical interaction between a limited number of Ia molecules present in any given animal and the myriad antigens to which T cells can respond. Here, we used a recently described approach for analysing the relative avidity of the T-cell receptor for different ligands to address these two possibilities. We describe a T-cell clone whose response to a single antigen, presented in the context of two different Ia molecules, strongly suggests that the antigen and the Ia molecule interact physically. PMID- 3005881 TI - AIDS. Pasteur plans to pursue patent suit on virus. PMID- 3005882 TI - Opioid activities of human beta-casomorphins. AB - Opioid activities of human beta-casomorphin-4, -5, -7 and -8 and, for comparison, of the corresponding bovine beta-casomorphins were studied in the guinea-pig ileum preparation. Binding parameters, i.e. KD-values and binding site concentrations, for the interaction of human and bovine beta-casomorphins with opioid receptors in rat brain homogenates were determined in inhibition experiments, using [3H]-(D-Ala2, MePhe4, Gly-ol5)enkephalin, [3H]-(D-Ala2, D Leu5)enkephalin and [3H]ethylketazocin as mu-, delta- and kappa-opioid receptor ligands. Analysis of binding data was performed using a non-linear curve fitting program. All beta-casomorphins examined displayed opioid activity. The affinity was highest for mu-receptors, less so for delta-receptors and lowest for kappa receptors. It is suggested that human beta-casomorphins might play a role as "food hormones". PMID- 3005883 TI - Interaction of Pseudomonas aeruginosa cytotoxin with plasma membranes from Ehrlich ascites tumor cells. AB - Biologically active 125I-cytotoxin from Pseudomonas aeruginosa binds to plasma membranes from Ehrlich ascites tumor cells in a saturable manner. The Scatchard plot indicated a single binding site with a capacity of 260 pmoles/mg of membrane protein and a KD of 2 X 10(-8) M. Specific binding was dependent on temperature, pH and ionic strength. Thus constant levels of bound 125I-cytotoxin were attained either within 30 min at 30 degrees C or within 3 h at 4 degrees C. Binding was 30 fold higher at 4 degrees C vs 30 degrees C and 2-6-fold higher at pH 5.3 vs pH 8.3. Binding was not effected by 50 mM sugar or sialic acid. 300 mM sucrose, however, instead of phosphate buffer, reduced binding by 50%. Pretreatment of plasma membranes with trypsin or papain led to a significant decrease in 125I cytotoxin binding. A pretreatment with phospholipase C or D had no effect, whereas phospholipase A2 induced a decrease by 34%. The collected data suggest that the binding site for 125I-cytotoxin within the plasma membrane from Ehrlich ascites tumor cells is a membrane protein. Correlation of 125I-cytotoxin binding and membrane action of the unlabelled cytotoxin can be observed through (a) increased lowering of the cellular K+ and Na+ gradient by decrease of medium pH, (b) decreased toxicity after substitution of ions by sugar, and (c) increased breakdown of cellular cationic gradient after temperature shift from 4 degrees C to 37 degrees C. PMID- 3005884 TI - Influence of neuronal uptake on the contribution of smooth muscle alpha 2 adrenoceptors to vasoconstrictor responses to noradrenaline in SHR and WKY isolated tail arteries. AB - The effects of the selective alpha-adrenoceptor antagonists idazoxan (alpha 2) and prazosin (alpha 1) were examined on responses to exogenous noradrenaline and to sympathetic nerve stimulation in SHR and WKY rat isolated perfused proximal tail artery segments. The influence of inhibition of neuronal uptake with cocaine on the effects of these antagonists was also determined. The following results were obtained: Prazosin (10 nmol/l) was equieffective in antagonising responses to exogenous noradrenaline and sympathetic nerve stimulation in both SHR and WKY arteries and the degree of antagonism was similar in either the presence or the absence of neuronal uptake inhibition. In contrast to prazosin, the effects of idazoxan (100 nmol/l), on both exogenous noradrenaline and sympathetic nerve stimulation were dependent on the degree of inhibition of neuronal uptake. In SHR arteries, the degree of antagonism of responses to exogenous noradrenaline by idazoxan (100 nmol/l) decreased progressively as the concentration of cocaine was increased to 4 and 40 mumol/l; in WKY arteries, even in the absence of cocaine, idazoxan (100 nmol/l) did not antagonise responses to exogenous noradrenaline. In SHR arteries, the responses to sympathetic nerve stimulation were reduced to a lesser extent by idazoxan (100 nmol/l) when the concentration of cocaine was increased to 4 mumol/l than in the absence of cocaine. In WKY arteries, idazoxan (100 nmol/l) reduced the responses to sympathetic nerve stimulation in the absence of cocaine. However, this concentration of idazoxan increased the responses to nerve stimulation in the presence of cocaine. Our results indicate that smooth muscle alpha 2-adrenoceptors are present in SHR tail arteries, both intra- and extrajunctionally.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3005886 TI - Apamin distinguishes two types of relaxation mediated by enteric nerves in the guinea-pig gastrointestinal tract. AB - Eight smooth muscle preparations from the stomach, small intestine and large intestine of the guinea-pig were used to compare apamin's actions in reducing the effectiveness of transmission from enteric inhibitory nerves and in reducing responses to inhibitory agonists alpha, beta-methylene ATP, VIP and isoprenaline. The effects of apamin on inhibitory reflexes in the ileum and colon were also evaluated. Apamin had little or no effect on responses to VIP and isoprenaline in any region, but consistently and substantially reduced responses to alpha, beta methylene ATP. Responses to stimulation of enteric inhibitory neurons were substantially reduced by apamin in the antrum circular muscle, ileum longitudinal and circular muscle, taenia coli and distal colon longitudinal muscle, but it was ineffective in the fundus circular muscle, proximal colon longitudinal muscle and distal colon circular muscle. It caused a small reduction of the relaxation of the ileal circular muscle caused reflexly by distension, but did not modify the similar descending inhibitory reflex in the circular muscle of the colon. It is concluded that apamin can be used to distinguish two types of non-noradrenergic transmission from enteric inhibitory nerves to gastrointestinal muscle. Furthermore, neither VIP nor ATP can be the sole transmitter chemical released from enteric inhibitory neurons throughout the gastrointestinal tract. PMID- 3005885 TI - Opioid peptides decrease noradrenaline release and blood pressure in the rabbit at peripheral receptors. AB - Effects of dynorphin-(1-13), Leu5-enkephalin, D-Ala2,D-Leu5-enkephalin (DADLE), and for comparison bremazocine, on plasma noradrenaline concentration and mean arterial pressure (MAP) were studied in pithed rabbits. In the first series of experiments, the sympathetic outflow was stimulated electrically via the pithing rod at 2 Hz twice for 3 min each (S1, S2). Drugs were administered before S2. Bremazocine 10 micrograms/kg + 2 micrograms/kg/h and 100 micrograms/kg + 20 micrograms/kg/h, dynorphin 1 and 3 micrograms/kg/min, Leu5-enkephalin 100 micrograms/kg/min and DADLE 10 and 30 micrograms/kg/min all diminished the electrically-evoked increase in plasma noradrenaline and MAP. The effects were antagonized by naloxone. In the second series, an infusion of noradrenaline (2 micrograms/kg/min) was given twice for 3 min each (N1, N2). Drugs were administered before N2. Bremazocine 100 micrograms/kg + 20 micrograms/kg/h slightly enhanced the pressor effect of exogenous noradrenaline, whereas dynorphin 3 micrograms/kg/min, Leu5-enkephalin 100 micrograms/kg/min and DADLE 30 micrograms/kg/min caused no significant change. In the third series, the sympathetic outflow was stimulated continuously at 2 Hz, and the interaction of dynorphin and DADLE was studied. Dynorphin 1 microgram/kg/min and DADLE 10 micrograms/kg/min initially decreased MAP to a similar extent. The effect of DADLE faded with time. When, during continuous infusion of DADLE 10 micrograms/kg/min, and after return of MAP to the pre-DADLE level, dynorphin 1 microgram/kg/min or DADLE 10 micrograms/kg/min was infused additionally, the effect of dynorphin was unchanged, whereas that of DADLE was almost abolished. We conclude that the opioid peptides as well as bremazocine decrease action potential-evoked release of noradrenaline and, secondarily, blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3005887 TI - [Epstein-Barr virus infection: immunology and immunopathology]. PMID- 3005888 TI - [Is there sufficient awareness of thalassemia syndromes? A screening method for these hereditary disorders]. PMID- 3005889 TI - [Tentative position determination of the new virostatic agent acyclovir]. PMID- 3005890 TI - [In-situ ductal carcinoma of the breast; a retrospective clinico-pathological study]. PMID- 3005891 TI - [Value of physical therapy in the therapy of Parkinson patients]. PMID- 3005892 TI - Preoperative gland localization by 201thallium and 99mtechnetium subtraction scintigraphy in persistent secondary hyperparathyroidism. PMID- 3005893 TI - [Immunological aspects of amyotrophic lateral sclerosis]. PMID- 3005894 TI - [Multifocal glioma of the brain--an autopsy case]. AB - This is an autopsy case report of a 76 year old woman with multifocal glioblastoma multiforme. CT scan revealed three separate mass lesions in the left temporal lobe, left parietal lobe and right fronto-parietal lobes. CT-guided needle biopsy of the right fronto-parietal mass revealed glioblastoma multiforme. At autopsy, serial horizontal sections of the brain confirmed the CT scan findings and demonstrated extension of the tumor in the left parietal lobe to the subarachnoid space. The lesion in the right fronto-parietal region infiltrated a large area and was not demarcated. Microscopic examination of all three lesions revealed glioblastoma multiforme. The left temporal lobe tumor consisted of gemistocytic astrocytes predominantly. Reaction of the tumor to glial fibrillary acid protein (GFAP) antibody ranged from markedly positive in the gemistocytic astrocytes to negative in the anaplastic cells. The reported incidence of multifocal glioma is about 2-10%. The main pathophysiologic mechanisms invoked for this phenomenon are simultaneous neoplastic transformation and seeding from one tumor. The pathophysiology of this particular case is discussed in reference to the differences in the histopathology of the 3 separate foci of glioblastoma multiforme. PMID- 3005895 TI - Regulation of thyrotropin secretion by the central epinephrine system. Studies in the chronically cannulated rat. AB - In the present investigation CNS epinephrine (EPI) biosynthesis was selectively interrupted with specific norepinephrine N-methyltransferase (NMT) inhibitors, SK&F 64139 (Smith, Kline & French Laboratories) and LY 78335 (Eli Lilly & Co. Research Laboratories), to determine the effects of central EPI depletion on basal and cold, thyrotropin-releasing hormone, and hypothalamic somatostatin antiserum induced thyrotropin (TSH) secretion in chronically cannulated rats. Because these NMT inhibitors also are alpha 2-adrenergic receptor blockers, the effects of alpha 2- and alpha 1-adrenergic blockade and alpha 2-activation on plasma TSH were assessed with rauwolscine and corynanthine and B-HT 933, respectively. Serum T4 and plasma corticosterone were also measured. Blockade of CNS EPI synthesis resulted in inhibition of basal and cold and thyrotropin releasing hormone induced TSH release, suppression of serum T4, and increased corticosterone release. The stimulatory effect of SRIF antiserum on plasma TSH was not altered by SK&F 64139. alpha 2-adrenergic blockade suppressed plasma TSH levels, but not to the same degree as the NMT inhibitors; activation of alpha 2 receptors enhanced TSH secretion. Thus, it is possible that part of the effect of the NMT inhibitors on TSH was due to alpha 2-blockade. alpha 1-adrenergic blockade also lowered plasma TSH. These results indicate that central EPI systems have a stimulatory role in TSH regulation, possibly mediated by alpha 2 adrenergic receptors, cold-induced TSH release is mediated, in part, by EPI, and central EPI systems exert an inhibitory effect on the hypothalamic-pituitary adrenal axis. PMID- 3005896 TI - Effects of castration and testosterone on the pituitary and adrenal responses of the newborn rat to ether inhalation. AB - In 8-day-old rat newborns, the pituitary response to 2 min of ether inhalation was noted to vary according to sex. Plasma ACTH levels were similarly increased in males and females at the end of ether exposure; however, during the following 30 min, ACTH levels were always higher in females than in males. In order to verify that the putative masculinization of some neuroendocrine pathways involved in the pituitary response to ether stress was the result of the transitory surge of testosterone at birth, fetuses at term were delivered by cesarean section and thereafter immediately castrated or sham-castrated under cold anesthesia (males), injected with testosterone heptylate (1 mg s.c.) or olive oil used as solvent (females) before being put in the care of a nurse. The rise in plasma testosterone levels during the 1st h after birth was prevented or stopped in males put at 2 degrees C. At the 8th postnatal day, the newborns were subjected to 2 min of ether inhalation; they were sacrificed either just, before or after the end (0 and 30 min) of the stress procedure. Plasma immunoreactive ACTH level and adrenal corticosterone content were measured. The pituitary response, shown by the ACTH increase, in castrated or sham-castrated males and testosterone injected females was similar to that of intact males but very different from that observed in olive-oil-injected or intact females. The rise in adrenal corticosterone content 30 min after ether inhalation was greater in intact and olive-oil-injected females than in testosterone-injected ones or in males; adrenal response was well correlated with the maintenance of ACTH release in the former and the decrease following transitory surge in the latter.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3005897 TI - GnRH release from the mediobasal hypothalamus. In vitro regulation by oxytocin. AB - The effects of oxytocin (OT) on gonadotropin-releasing hormone (GnRH) release from the adult male rat mediobasal hypothalamus and median eminence were studied in an in vitro incubation system. OT induced a dose-related (10(-13) to 10(-9) M) inhibition of both basal and KCl-stimulated GnRH release from the mediobasal hypothalamus. OT, but not arginine vasopressin, also inhibited GnRH release from the isolated median eminence, and this inhibition was blocked by treatment with an OT receptor antagonist. Moreover, antagonist alone stimulated GnRH release from the isolated median eminence. These results demonstrate that OT can inhibit in vitro release of GnRH by an OT receptor mediated mechanism at the level of neurosecretory terminals in the median eminence and suggest that the hypothalamic OT neuronal system may play an inhibitory role in the modulation of GnRH secretion. PMID- 3005898 TI - Characteristics of the receptors which mediate the stimulation of ACTH secretion by vasopressin in conscious dogs. AB - In order to characterize the receptors which mediate the adrenocorticotropic hormone (ACTH) response to vasopressin in conscious animals, plasma 11 hydroxycorticosteroid concentration was measured in conscious dogs during the infusion of vasopressin or structural analogs of vasopressin which exhibit selective antidiuretic or vasoconstrictor activity. Vasopressin (1.0 ng/kg/min for 60 min) increased mean arterial pressure, decreased heart rate and increased plasma corticosteroid concentration from 1.0 +/- 0.2 to 2.2 +/- 0.2 micrograms/dl (p less than 0.001). A specific antagonist of the vasoconstrictor activity of vasopressin, d(CH2)5MeTyrAVP (10 micrograms/kg), completely blocked the cardiovascular and corticosteroid responses to vasopressin. A selective vasoconstrictor (V1) agonist, PheOrnOT (1.0 ng/kg/min), which produced the same cardiovascular responses as vasopressin, increased plasma corticosteroid concentration from 1.1 +/- 0.1 to 2.9 +/- 0.9 micrograms/dl (p less than 0.005). In marked contrast, a selective antidiuretic (V2) agonist, dDAVP (1.0 ng/kg/min) had no effect on blood pressure, heart rate or plasma corticosteroid concentration. These results indicate that the stimulation of ACTH release by vasopressin in conscious dogs is mediated by receptors which resemble vasoconstrictor-type (V1) receptors rather than antidiuretic-type (V2) receptors. PMID- 3005899 TI - Centrally administered inhibitors of the generation and action of angiotensin II do not attenuate the increase in ACTH secretion produced by ether stress in rats. AB - The role of the brain renin-angiotensin system in the ACTH response to ether stress in rats was investigated by injecting the angiotensin II receptor blocking drug saralasin, the angiotensin II converting enzyme inhibitors enalaprilat and captopril, and the renin inhibitor L 363714 intraventricularly and measuring the ACTH and corticosterone concentration in plasma 10 min after ether stress. ACTH and corticosterone were elevated to at least the same level in rats treated with the inhibitors as they were in rats treated with the corresponding vehicles; indeed, ACTH values were somewhat greater in stressed rats treated with the converting enzyme inhibitors and the renin inhibitor. ACTH values in the absence of ether were not affected by saralasin, enalaprilat, and captopril and were increased by L 363714. The data do not support the hypothesis that the brain renin-angiotensin system is involved in the maintenance of ACTH secretion or that it mediates the increase produced by ether stress. PMID- 3005900 TI - Serotonin and dopamine independently regulate pituitary beta-endorphin release in vivo. AB - Serotonin and dopamine neurons have been shown to exert a stimulatory and inhibitory control, respectively, over pituitary release of beta-endorphin-like immunoreactivity (beta-END-LI). In the present study we sought to determine whether an interaction exists between these two reciprocal mechanisms regulating beta-END-LI in the rat. The intraperitoneal (i.p.) administration of 5 mg/kg quipazine, a serotonin receptor agonist, or 2.5 mg/kg haloperidol, a dopamine receptor antagonist, each elevated circulating levels by beta-END-LI 5-fold over control levels by 30 min post-injection. Pretreatment (1 h) with 5 mg/kg, i.p., cinanserin, a serotonin receptor antagonist, completely blocked the quipazine induced rise in beta-END-LI without affecting the elevated levels of beta-END-LI in haloperidol-treated animals. Conversely, pretreatment (2 h) with 1 mg/kg, i.p., bromocriptine, a dopamine receptor agonist, had no effect on quipazine induced release of beta-END-LI but did completely prevent the rise in plasma beta END-LI due to haloperidol treatment. Gel filtration chromatography revealed that quipazine and haloperidol treatments elevated plasma levels of both beta-END-size immunoreactivity and beta-lipotropin (beta-LPH)-sized immunoreactivity though to different relative degrees. However, since circulating levels of beta-LPH serve as a marker for anterior lobe (AL) beta-END-LI secretion, serotonin and dopamine appear to exert stimulatory and inhibitory control, respectively, over AL beta END-LI release. Further, the quipazine-induced rise in total plasma beta-END-LI primarily resembled beta-LPH in size and was blocked by cinanserin but not bromocriptine pretreatment. And conversely, bromocriptine but not cinanserin prevented the haloperidol-induced rise in circulating beta-END-LI.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3005901 TI - Biochemical profile of midalcipran (F 2207), 1-phenyl-1-diethyl-aminocarbonyl-2 aminomethyl-cyclopropane (Z) hydrochloride, a potential fourth generation antidepressant drug. AB - The present study of midalcipran (F 2207), 1-phenyl-1-diethyl-aminocarbonyl-2 aminomethyl-cyclopropane(Z) hydrochloride, was undertaken to determine its biochemical profile. The properties of midalcipran, in inhibiting the uptake of monoamines were tested and compared with that of imipramine. In vitro, midalcipran was found to inhibit the uptake of radiolabelled serotonin and noradrenaline (IC50 = 203 and 100 nM, respectively), but not that of dopamine, into brain slices. Hyperthermia induced by the centrally-acting displacers of monoamines, H77/77 and H75/12, were almost equipotently antagonized by midalcipran, confirming the inhibition of the uptake of serotonin and noradrenaline by midalcipran in vivo (ID50 = 11 and 4.8 mg/kg, respectively). Midalcipran showed no inhibition of the activity of monoamine oxidase in vitro or in vivo. The interaction between midalcipran and neurotransmitter receptors and binding sites in the CNS was studied in the rat in comparison with imipramine and desipramine. In contrast to these two antidepressant drugs, midalcipran showed no affinity for alpha- or beta-adrenoceptors, muscarinic, histaminergic H1, dopaminergic D2 or serotonergic 5-HT2 receptors, suggesting a general absence of anticholinergic, sedative and other side-effects. Midalcipran was equipotent with imipramine at inhibiting the binding of [3H]imipramine. Chronic administration of midalcipran to rats did not alter the number of beta-adrenergic receptors in the cortex, in contrast to imipramine and desipramine which decreased the binding of beta-adrenoceptors. Thus midalcipran appears to act exclusively presynaptically, inhibiting the uptake of serotonin and noradrenaline. This activity, coupled to the total absence of interaction at postsynaptic sites, suggests that midalcipran may be a useful and novel antidepressant drug. PMID- 3005903 TI - Selective inhibition of monoamine oxidase by p-aminosubstituted phenylalkylamines in catecholaminergic neurones. AB - The in vivo inhibition of monoamine oxidase (MAO) inside and outside noradrenergic and dopaminergic nerve terminals in the hypothalamus and striatum, respectively, was examined in the rat after oral administration of a series of substituted p-aminophenethylamines and some related compounds. This was achieved by measuring their ability to protect MAO from irreversible inhibition by phenelzine, determined by the deaminating activity of synaptosomal preparations in the absence and presence of maprotiline, a selective inhibitor of the uptake of noradrenaline, or of amfonelic acid, a potent inhibitor of the uptake of dopamine, with small (0.25 microM) concentrations of [14C]noradrenaline or [14C]dopamine as substrate. It was found that several of these compounds were much more potent in protecting MAO within the noradrenergic neurones than MAO in other cells. Since the inhibitors of the uptake of noradrenaline, desipramine and CPP 199 antagonized this preference for noradrenergic MAO it is concluded that these MAO inhibitors are accumulated in the noradrenergic neurones by the membranal uptake carrier. Hence the selectivity for MAO within noradrenergic neurones seems to reflect the ability of the compounds to be transported by this carrier. The structure-activity relationship obtained showed the greatest selectivity for the unsubstituted p-dimethylamino-(FLA 289), p-methylamino-(FLA 727) and p-amino-(FLA 334)-amphetamines, whereas the 2-fluoro compound (FLA 558) had the greatest potency. N,N-didesmethylamiflamine [FLA 668(+)] had an almost specific effect in the noradrenergic nerve terminals. The primary p-amino derivatives, FLA 334 and FLA 668, produced a marked selective protection of MAO in dopaminergic nerve terminals, whereas the tertiary and secondary derivatives had much less preference for dopaminergic MAO.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3005902 TI - Amine-mediated toxicity. The effects of dopamine, norepinephrine, 5 hydroxytryptamine, 6-hydroxydopamine, ascorbate, glutathione and peroxide on the in vitro activities of creatine and adenylate kinases in the brain of the rat. AB - The effects of several concentrations of amines and reducing agents on the activity of creatine (CK) and adenylate (AK) kinases were determined in homogenates of the brain of the rat at 0 and 37 degrees C. The order of decreasing irreversible inhibition of the enzymes was peroxide, 6 hydroxydopamine, dopamine, norepinephrine, 5-hydroxytryptamine. At 37 degrees C, approx. 50% of the activity of creatine kinase was lost in 30 min in the presence of 20 microM dopamine. 5-Hydroxytryptamine was several orders of magnitude less toxic. The action of dopamine was not prevented by inhibition of monoamine oxidase, chelation of metals or the addition of a catalase, indicating that formation of peroxide by monoamine oxidase was not the primary cause of the loss of enzyme. Although auto-oxidation of dopamine to a toxic quinone was considered, the degree of inhibition of creatine kinase was not affected when auto-oxidation was prevented under anaerobic conditions. Glutathione (GSH), present during the incubation, protected the enzymes but could not restore activity after exposure to amine. Concentrations of glutathione above 5 mM and of oxidized glutathione as low as 10 microM inhibited creatine kinase. Ascorbate protected the enzymes even when present at a concentration much less than that of the amine, but ascorbate was itself toxic. The findings indicate that dopamine, at concentrations attained after drug-induced release or ischemia, can be toxic to a metabolic enzyme present in the synaptosomal membrane. PMID- 3005904 TI - Effect of clonidine on motoneuron excitability in spinalized rats. AB - In a dose of 0.1 mg/kg clonidine, an alpha-2 receptor agonist, depressed the spontaneous EMG activity of the biceps and quadriceps femoris in chronically spinalized rats. It also antagonized in a dose-dependent manner the stimulating effect of 5-hydroxytryptophan (5-HTP, 100 mg/kg). Doses of more than 0.1 mg/kg were less potent in antagonizing the effect of 5-HTP. Clonidine reduced the tonic activity of the hindlimb muscles but allowed walking movements. The depressant effect of clonidine in animals pretreated with 5-HTP was prevented by yohimbine (1.25 mg/kg), while the depressant action of the serotonin antagonist, cyproheptadine was not. In chronically-spinalized rats, clonidine (0.1 mg/kg) increased the threshold of electrically-induced flexor and extensor reflexes and decreased their amplitude. No significant modification of reflexes was seen with this dose 24 hr after spinalization. Thus, clonidine in doses of 0.1 mg/kg or less reduced directly or indirectly the excitability of motoneurons. Clonidine may prove to be a useful therapeutic adjunct in the treatment of spasticity. PMID- 3005905 TI - Striato-nigral denervation increases type II benzodiazepine receptors in the substantia nigra of the rat. AB - The degeneration of the striato-nigral projection induced by the injection of kainic acid into the striatum produced a 30% increase in the density of type II benzodiazepine binding sites (measured as the proportion of [3H]flunitrazepam which remained after the addition of 2 X 10(-7) M CL 218872). The lesion did not change the number of type I benzodiazepine binding sites (measured using [3H]ethyl-beta-carboline-3-carboxylate). The increase of type II benzodiazepine binding sites persisted and was markedly enhanced in the substantia nigra, previously lesioned with kainic acid. In fact, the injection of kainic acid into the nigra caused, 3 weeks after the treatment, a 80% decrease in the total number of type I benzodiazepine binding sites, and no change in the number of type II benzodiazepine binding sites. The density of type II sites increased by 70% following a subsequent injection of kainic acid into the striatum, homolateral to the lesioned substantia nigra. The results suggest that type I benzodiazepine binding sites in the nigra are located on kainic acid-sensitive elements (probably intrinsic neurones), while type II benzodiazepine binding sites, the number of which increased after degeneration of the striato-nigral pathway, are localized on kainic acid-resistant structures (probably axons or terminals) that receive an input from striatal afferents and from interneurones in the nigra. PMID- 3005906 TI - Antagonistic action of uranyl nitrate on presynaptic neurotoxins from snake venoms. AB - Uranyl ion (UO2+2) antagonized the neuromuscular blocking action and phospholipase A2 activity of neurotoxins which act presynaptically [beta bungarotoxin (beta-BuTX) and crotoxin] but did not affect the action of alpha bungarotoxin and tetrodotoxin. On the basis of the kinetic analysis of the UO2+2 and strontium ion (Sr2+) antagonism of muscle paralysis induced by beta bungarotoxin, it was found that they inhibited both the binding of the toxin and the steps following binding that brought about the neuromuscular blocking action of beta-bungarotoxin. Uranyl ion was about 50 times more potent than Sr2+ in antagonizing beta-bungarotoxin. High Ca2+ (10 mM) abolished but low Ca2+ (0.25 1.25 mM) medium enhanced the antagonizing action of UO2+2 and Sr2+. In low Ca2+ medium, UO2+2 markedly potentiated the amplitude of the twitch, subsequent addition of beta-bungarotoxin produced three phases of effects on the twitches, e.g. an initial depression, followed by the second facilitation and finally a rapid depression of twitches; however, approx. 70 min after beta-bungarotoxin the small twitches reached a steady state which persisted for more than 350 min. Therefore, it is evident that UO2+2 is the most potent antagonist of beta bungarotoxin so far tested. PMID- 3005907 TI - Hydrodynamic parameters of opioid receptors from frog brain. AB - Active opioid receptors were solubilized from frog (Rana esculenta) brain membranes using 1% digitonin. The solubilized preparation was sedimented in sucrose density gradient and applied to Sepharose-6B column. In the ultracentrifugation experiments, two distinct molecular forms of the opioid receptors were observed with apparent s20, w values of 15.7 and 10.8 S. The estimated molecular weights were 470 and 180 kD. The Stokes radii of the two separate forms were determined by gel filtration and found to be 71 and 42 A. The corresponding molecular weights were 500 and 140 kD indicating a good correlation with data obtained from the sedimentation experiments. PMID- 3005908 TI - A model of neuronal network learning based on variations in the efficiency of excitatory and inhibitory synapses. PMID- 3005909 TI - Glutamate decarboxylase activity in the pituitary in the presence of a change in the corticotropin level in the rat organism. PMID- 3005910 TI - Association of short-latent processes in the rabbit visual system with pre excitation inhibition in the retina. AB - An electrographic correlate of short-latent processes in the visual system corresponding to the ERG a-wave was detected in chronic experiments on awake rabbits. The formation of an early negative potential (ENP), small in amplitude, that precedes the positive component of the primary responses (PR) to light and is thus the earliest signal concerning the arrival of information from the retina was detected during a special recording regimen in the optic tract, superior colliculi, outer geniculate body, and visual cortex. The shift in the latent periods with respect to the onset of the development of the ERG a-wave comprised 2-3 msec in all the above structures. The suppression of all ERG components except the a-wave by glycine injected into the vitreous body was accompanied in all structures by the suppression of all PR components except the very initial ENR. Since the pre-excitatory inhibition of ganglionic elements corresponds to the ERG a-wave at the exit from the retina, the role of this inhibition in the transmission of information concerning the very first instant of the visual signal both to subcortical centers and the visual region of the cerebral cortex should be recognized. PMID- 3005911 TI - Selective inhibition of responses of feline dorsal horn neurones to noxious cutaneous stimuli by tizanidine (DS103-282) and noradrenaline: involvement of alpha 2-adrenoceptors. AB - The effects of tizanidine (DS103-282) were compared with those of noradrenaline and other adrenoceptor agonists on responses of laminae IV and V neurones in the lumbar dorsal horn to noxious and innocuous cutaneous stimuli in the anaesthetized cat. Tizanidine, noradrenaline and the alpha 2-receptor agonist, clonidine, depressed spontaneous activity and responses to noxious, but not those to innocuous, stimuli when administered iontophoretically, either near the recording site in laminae IV-V, or into laminae II-III, i.e. 300-900 microns dorsal to the recording site. Iontophoretic ejection of dopamine, the beta agonist isoprenaline and the alpha 1-agonists phenylephrine and amidephrine had no effect at either site, or only relatively weak and sometimes non-selective depressant actions on neuronal responses to cutaneous stimuli. The preferential depressant actions of tizanidine, noradrenaline and clonidine were antagonized by the selective alpha 2-antagonist RX781094 administered iontophoretically at the same site as the agonists, and by intravenously administered yohimbine. In contrast, the alpha 1-antagonists, prazosin and WB4101, the beta-antagonist, sotalol and opiate antagonist, naloxone did not alter the depressant actions of these agonists on laminae IV and V neurones. These findings indicate that the selective inhibitory effect of tizanidine and noradrenaline on responses of laminae IV and V neurones to noxious peripheral stimuli are mediated at alpha 2 adrenoceptors situated in either laminae IV and V or laminae II-III. The possible physiological relevance of these receptors is discussed. PMID- 3005912 TI - Dye-coupled magnocellular peptidergic neurons of the rat paraventricular nucleus show homotypic immunoreactivity. AB - Magnocellular neurons in rat hypothalamic slices are known to exhibit dye coupling: the transfer of the fluorescent dye, Lucifer Yellow, from an intracellularly-injected neuron to one or more nearby neurons. The question of the hormonal identity of coupled cells and the possibility of dye coupling as an artefact led us to determine the immunoreactivity of dye-coupled magnocellular neurons in the paraventricular nucleus of the rat hypothalamus using antisera to oxytocin- and vasopressin-associated neurophysins. In 23 pairs, one triplet, and one quadruplet, immunoreactivity to one or the other antiserum was always exclusive, and dye coupling was always homotypic, that is, coupled neurons in each instance were reactive to the same antiserum. The quadruplet, triplet and 17 pairs were immunoreactive to vasopressin-associated neurophysin, and oxytoxin associated neurophysin immunoreactivity was observed in the remaining pairs. Immunoreactivity to each antiserum was found for somasomatic and non somasomatic modes of coupling and for coupled neurons in the three magnocellular areas of the nucleus. A relationship between mode of coupling and hormone content was not detected. The data support the hypothesis that coupling is a real, functionally significant mechanism for coordinating neuronal activity in this nucleus, particularly under conditions of high hormone demand. They do not support the idea that coupling is artefact. The possibility of a relationship between hormone content and mode of coupling, and the projection pathway(s) of the coupled neurons of each type require further study. PMID- 3005913 TI - Release and turnover of noradrenaline in isolated median eminence: lack of negative feedback modulation. AB - A low volume (tissue holder, 100 microliter; dead space, 300 microliter) perfusion system has been developed for measuring [3H]noradrenaline release from isolated median eminence, where supramaximal electrical field stimulation can be applied. In tissue preloaded with [3H]noradrenaline, the resting release (0.4-2% of the content) was enhanced by electrical stimulation (2-10-fold increase). That the released radioactivity in response to electrical stimulation is mainly due to release of [3H]noradrenaline was confirmed by high pressure liquid chromatography combined with radiochemical detection. Evidence has been obtained that of the stimulation-evoked release of radioactivity 70-80 percent originates from noradrenergic neurons, however, the release observed at rest was not affected by 6-hydroxydopamine pretreatment. 6-Hydroxydopamine pretreatment selectively reduced the concentration of noradrenaline of the median eminence without affecting its dopamine content. The release evoked by electrical stimulation was [Ca2+]- and tetrodotoxin-sensitive. 4-Aminopyridine enhanced both the resting and stimulation-evoked release. The ratio between the amount of [3H]noradrenaline released by two consecutive stimulation periods at 2 Hz (120 shocks) was constant, 0.94 +/- 0.08. In contrast with other noradrenergic axon terminals, the release of [3H]noradrenaline in the median eminence was not subject to negative feedback modulation, yohimbine and xylazine had no effect. This conclusion was substantiated by in vivo study showing that yohimbine, an alpha2-adrenoceptor antagonist enhanced the turnover rate of noradrenaline in the cortex but not in the median eminence. Since noradrenergic axon terminals in the median eminence do not make synaptic contact and the released noradrenaline does not modulate its own release via alpha2-adrenoceptors, it is an interesting anatomical arrangement: the modulatory alpha2-adrenoceptors are located exclusively on the terminals of the hormone-containing neurons. PMID- 3005914 TI - Serotoninergic but not noradrenergic neurons in rat central nervous system adapt to long-term treatment with monoamine oxidase inhibitors. AB - Repeated administration of monoamine oxidase inhibitors induces a transient decrease in the firing rate of serotoninergic neurons followed by complete recovery, whereas it results in a persistent reduction of the firing rate of noradrenergic neurons. Under these conditions, serotoninergic, but not noradrenergic, neurons undergo a desensitization of their somatic autoreceptors. Serotoninergic neurons therefore show the capacity to free themselves from their autoregulatory control, a property which noradrenergic neurons appear to be lacking. The time course of the recovery in the firing rate of the serotoninergic neurons is consistent with the delayed antidepressant effect of monoamine oxidase inhibitors. PMID- 3005915 TI - Neuronal mechanisms of the interaction of Deiters' nucleus with some brainstem structures. AB - The effects of stimulation of the interstitial nucleus of Ramon y Cajal, as well as the nucleus of Darkschewitsch, inferior olive and nucleus reticularis tegmenti pontis on the neuronal activity of the lateral vestibular nucleus of Deiters were studied by means of an intracellular recording technique. Stimulation of these structures is shown to lead to antidromic and orthodromic activation of Deiters' neurons. Collaterals of vestibulospinal neurons entering these structures are revealed electrophysiologically. It was shown that stimulation of the rostral part of the inferior olive evoked in Deiters' neurons predominantly antidromic responses, whereas stimulation of the caudal part of the inferior olive leads mostly to synaptic activation. Stimulation of Ramon y Cajal's and Darkschewitsch's nuclei evokes mono- and/or polysynaptic excitatory and inhibitory postsynaptic potentials in Deiters' neurons. Mono-, oligo- and/or polysynaptic inhibitory postsynaptic potentials were also evoked by stimulation of nucleus reticularis tegmenti pontis, as well as the rostral and particularly, caudal parts of the inferior olive. Stimulation of the caudal part of the inferior olive evoked mono-, oligo- and/or polysynaptic excitatory postsynaptic potentials in Deiters' neurons. Convergence of influences from the stimulated structures on the vestibular neurons under study was shown. A topical correlation between Deiters' nucleus and the brainstem nuclei mentioned above was found. The presence of inhibitory and excitatory interaction of these structures with Deiters' nucleus was established. PMID- 3005916 TI - Effects of valproate on biogenesis and function of liver mitochondria. AB - The mitochondrial protein content of liver increased, and mitochondria enlarged after prolonged administration of valproic acid (VPA: N-dipropylacetic acid) to rats. The mitochondria showed low specific activities of membrane-bound enzymes, decreased content of cytochrome aa3, and decreased respiration in states 3 and 4. In vitro studies of amino acid incorporation suggested a decrease in protein synthesis in liver mitochondria from VPA-treated rats. Prolonged administration of VPA seems to impair mitochondrial structure and function. PMID- 3005917 TI - [Plasma beta endorphin in man during partial immersion in water using the therapeutic method]. AB - The following study was designed to evaluate plasma beta-endorphin (beta-EP) variations in healthy volunteers during thermoneutral head-out water immersion while prevalently in the standing position. The type of immersion was similar to that currently adopted for therapeutic rehabilitation. Plasma beta-EP was evaluated by RIA previous beta-lipotropin stripping and Sep-Pack cartridge methanol extraction. Plasma beta-EP levels significantly decreased during water immersion from a value of 12.71 +/- 2.04 pmol/l to 7.46 +/- 1.09 pmol/l at 15 min (P less than 0.05) and to 6.08 +/- 1.87 pmol/l at 30 min (P less than 0.01). Thirty min after the end of immersion, plasma beta-EP levels showed a slight increase to 6.98 +/- 1.88 pmol/l but were still significantly below the basal level (P less than 0.05). These results are consistent with the previously demonstrated decrease of ACTH and prolactin and the increase of plasma dopamine and decrease of norepinephrine, suggesting that thermoneutral head-out water immersion is not a stressful condition in healthy subjects. Further studies are necessary in order to clarify the mechanism involved in beta-EP decrease in normal subjects during thermoneutral head-out water immersion. PMID- 3005919 TI - [Role of surgery in the prognosis of AIDS and related syndromes. Case reports ]. AB - The authors review the epidemiologic and etiopathogenetic aspects of AIDS, in agreement with data published in december by the Center for Disease Control and by the WHO for the States, Europe and Italy. In their experience twenty-one patients, with clinical clues of AIDS, undergoing lymph node biopsy, have been analysed (18 LAS-ARC and 3 AIDS) and the role of prophylaxis for the surgical approach to the patient with aids or related syndrome has been underlined. PMID- 3005918 TI - [Value of serology and endoscopy and clinical aspects in idiopathic rectocolitis]. AB - The clinical course of ulcerative colitis is characterized by recurring acute attacks and interspersed with periods of remission. The present study correlates the clinical, endoscopic and laboratory results in the follow-up of 22 rectocolitis patients in order to identify any data that might serve as a warning of recurrences. It is claimed that laboratory examinations like titering of alpha 1-antitrypsin, antithrombin III collagenase, C3 and C4 offer useful support for endoscopy which is generally considered the only reliable examination in the diagnosis of idiopathic rectocolitis. PMID- 3005920 TI - [Primary hepatocarcinoma. Epidemiologic case-control study in the province of Bergamo]. AB - This is the first epidemiological study of Hepatocellular Carcinoma (HCC) in the province of Bergamo, an area well-known to have a high incidence of HBsAg (9.1%) and chronic alcoholic liver disease. 72 cases of HCC (60 male, 12 female) al from the province of Bergamo and encountered in 1980-84 were subjected to an epidemiological case-control study. Analysis of the results confirmed the role of certain known Risk Factors (RF) with a prevalence of male sex (83.3%), age (mean age 63), association with live cirrhosis (79.2%) and HBsAg+ (31.9%). Such findings are in line with the Italian average found in previous studies. No difference between the sexes was found in these RF except for alcoholic abuse which was significantly higher in the males (53.3%, p less than 0.05). The case control correlation analysis revealed no difference in the prevalence of alcohol addiction and previous HBV infection (HBV-Ab+) between the HCC (with or without cirrhosis) and the various control groups (Group A: patients with no liver pathology. Group B: patients with cirrhosis of the liver). HBsAg+ was significantly higher among HCC patients without cirrhosis (46.6%, p less than 0.001), but there was no significant difference between HCC + cirrhosis and cirrhosis alone (88%). The difference between "expected" and "observed" HCC HBsAg+ was highly significant (p less than 0.001). The overall Relative Risk (RR) of HCC for the RF-HBsAg+ was 4.6. When divided into subgroups this gave: RR = 1 for patients with cirrhosis of the liver. RR = 11.5 for patients with no liver pathology. These data confirm the importance of current HBV infection (HBsAg+), in the province, though the presence or absence of cirrhosis probably influences its significance. The approximate incidence of HCC is 9.7%/100,000/year. Considering the limitations of data on a small monocentric study in a limited area (USSL no. 30) the figure is probably underestimated and the real incidence probably higher. PMID- 3005921 TI - [Pregnancy and malignant tumors. Considerations on 5 cases]. PMID- 3005922 TI - Prolonged changes in dopaminergic terminal excitability and short changes in dopaminergic neuron discharge rate after short peripheral stimulation in monkey. AB - Time-courses of responses to peripheral somatosensory stimulation were studied in the nigrostriatal dopamine (DA) system by comparing rates of neuronal discharges with changes in nerve terminal excitability, an indicator of DA release. The excitability of DA nerve terminals in the putamen was assessed as probability for evoking an antidromic response in substantia nigra DA cells with electrical stimulation in an anesthetized monkey. At about 30-60% decrease of excitability was seen during and about 15 min beyond pain pinch stimulation (PPS) in 12 of 17 tested DA neurons, while 4 neurons showed a 40% increase. Discharge rates were decreased in 7 and increased in 5 of the 17 DA neurons during, but not after PPS. It is concluded that the release of DA in the striatum may be controlled in two ways: rapid reactions would be mediated by changes in discharge rate, while slower, prolonged responses could be due to presynaptic interactions with other striatal afferents. PMID- 3005923 TI - Influence of proopiomelanocortin-derived peptides on the sleep-waking cycle of the rat. AB - The effects of i.c.v. injections of different proopiomelanocortin-derived peptides were tested on the rat sleep-waking cycle: adrenocorticotropic hormone (ACTH) had no effect. In contrast, administration of two ACTH-derived peptides was followed by a selective and significant increase of each sleep state: desacetyl-alpha-melanocyte-stimulating hormone induced an increase of slow-wave sleep, while corticotropin-like intermediate lobe peptide was followed by an increase of paradoxical sleep. Both alpha-melanocyte-stimulating hormone in its acetylated form and alpha-endorphin were inactive. PMID- 3005924 TI - The roles of alpha 2-adrenoceptors in the nucleus reticularis gigantocellularis and vagal mechanism in the cardiovascular suppressive effects of guanabenz in the rat. AB - In pentobarbital-anesthetized rats, pretreatment with yohimbine (10 micrograms), which was microinjected into the bilateral nucleus reticularis gigantocellularis (NRGC), significantly antagonized the reduction in arterial pressure, and the force and rate of heart contraction normally promoted by systemic administration of guanabenz (10 micrograms/kg, i.v.). At the same time, the vasodepressive as well as negative inotropic and chronotropic effects of direct application of guanabenz (500 ng) into the NRGC were attenuated by bilateral cervical vagotomy or atropine sulfate (1 mg/kg, i.v.). We conclude that guanabenz may promote antihypertension by activating the alpha 2-adrenoceptors in the NRGC, which in turn elicits cardiovascular suppression by at least facilitating the vagal outflows to the heart. PMID- 3005925 TI - Inhibition of inferior olivary transmission by mesencephalic stimulation in the cat. AB - Cerebellar climbing fiber responses (CFRs) evoked in anesthetized cats by stimulation of peripheral nerves, contralateral inferior olive and cerebellar white matter were investigated by recording unit activity and surface field responses in anterior lobe of cerebellar cortex. When nerve and olive stimulation was preceded at long intervals (greater than 35 ms) by weak electrical stimulation of an ipsilateral mesencephalic area close to the locus coeruleus and brachium conjunctivum, CFRs could be virtually abolished in the pars intermedia but not in the vermis. White-matter evoked CFRs were not affected; thus the site of the inhibition was the inferior olive. PMID- 3005926 TI - Frequency- and reserpine-dependent chemical coding of sympathetic transmission: differential release of noradrenaline and neuropeptide Y from pig spleen. AB - The importance of impulse pattern and stimulation frequency for the release of noradrenaline (NA) and the coexisting peptide neuropeptide Y (NPY) in relation to vasoconstriction (perfusion-pressure increase) was studied in the blood-perfused pig spleen in vivo. Splenic nerve stimulation with intermittent bursts at high frequency (20 Hz) caused a several-fold larger release of NPY-like immunoreactivity (-LI) in relation to NA than a continuous stimulation at a low frequency (2 Hz), giving the same total number of impulses. alpha-Adrenoceptor blockade by phentolamine enhanced markedly both NA and NPY release, especially at low stimulation frequency, suggesting prejunctional adrenergic inhibition of release. Addition of propranolol unmasked a large remaining perfusion-pressure response to nerve stimulation. Reserpine treatment reduced the NA content of the spleen as well as the stimulation-evoked NA release by greater than 90%. However, the perfusion-pressure increase in response to nerve stimulation was well maintained. A marked increase in the stimulation-evoked release of NPY-LI occurred after reserpine. Adrenoceptor blockade after reserpine treatment reduced only slightly the perfusion-pressure response in parallel with a decline in NPY output. NPY caused an adrenoceptor-resistant perfusion-pressure increase at plasma concentrations that were in the same range as the maximal increase during nerve stimulations. In conclusion, the present data suggest a frequency dependent, chemical coding of sympathetic transmission with preferential release of the classical transmitter NA at low, continuous frequencies and release of NPY, mainly at high frequencies. Reserpine treatment enhances markedly NPY release, which may explain why the functional response is largely intact in spite of adrenoceptor blockade and marked NA depletion. PMID- 3005927 TI - Supraspinal depressant action of diazepam on the crossed extensor reflex. AB - The effects of diazepam on the crossed extensor reflex (CER) were compared between intact and spinally transected rats and also between IVth and lateral ventricular administration. The CER of the chronic spinal rat was not influenced by 0.05-2 mg/kg doses of diazepam, which was 5-200 times larger than the ED50 of the intact preparation. The IVth ventricular injection required only half as much diazepam compared to the lateral ventricular injection and the elapsed time between drug administration to the peak effect was significantly shorter. According to these findings, benzodiazepines seem to act primarily on the supraspinal structure near the IVth ventricle and thus depress the CER. PMID- 3005928 TI - Bilateral projections of neurons in the lateral geniculate nucleus and nucleus lateralis posterior to the visual cortex in the neonatal rat. AB - The projection from the lateral geniculate nucleus (LGN) and nucleus lateralis posterior (LP) to the visual cortex was examined in rat pups 3-7 days of age using the fluorescent tracers True Blue, Fast Blue and Nuclear Yellow. Our data provide the first evidence that (1) these projections are bilateral, (2) the ipsilateral projection from these nuclei to the visual cortex in the neonatal rat is well localized and is similar in distribution and organization to that reported by others in the adult and (3) bilaterally projecting geniculocortical cells are morphologically heterogeneous; bilaterally projecting cells in LP are morphologically homogeneous. PMID- 3005929 TI - Metabolic myopathies. PMID- 3005930 TI - Radionuclide scans to define patterns of occult myonecrosis. PMID- 3005931 TI - Multiform atrial tachycardia. PMID- 3005932 TI - Plants as a source of vitamin D3 metabolites. PMID- 3005933 TI - The management of small cell lung cancer. PMID- 3005934 TI - Neoplasia arising in dysgenetic gonads. AB - We analyzed the clinicopathologic characteristics of 140 cases of neoplasia arising in dysgenetic gonads. These 140 cases were found on a review of the medical literature published between 1953 and 1983. The age of the patients at the time of diagnosis was recorded in 133 patients and ranged from 6 months to 45 years. The mean age at diagnosis was 18 years 8 months. Thirteen (9.8 per cent) patients were below the age of 10 at the time of diagnosis. A menstrual history was recorded in 109 cases. Amenorrhea was present in 103 (94.5 per cent). A Y chromosome or Y-chromosome fragment was present in 90.7 per cent of the 119 patients who had karyotype analysis. Bilateral tumors were found in 54 instances (38.6 per cent). Thus, a total of 194 neoplasms were found. Of these 103 (53.1 per cent) were gonadoblastomas, 38 (19.6 per cent) dysgerminomas, 34 (17.5 per cent), gonadoblastoma with areas of dysgerminoma, and 19 (9.8 per cent) were of other histologic types. Patients with dysgenetic gonads and Y chromosome material are at risk for development of ovarian neoplasm. A dysgerminoma of dysgerminomatous component was present in 37 per cent of the reviewed tumors. These neoplasms have been discovered as early as 6 months of age and 9.8 per cent of the cases occurred in patients below the age of 10. Early exploration and bilateral gonadectomy should be performed in patients with gonadal dysgenesis and Y-chromosome material in their karyotype. PMID- 3005935 TI - [Chemotherapy with mitomycin C, vindesine and ifosfamide in the treatment of inoperable non-small cell bronchial carcinoma]. AB - 27 patients (24 male und 3 female) with inoperable non-small-cell lung cancer were treated with combined mitomycin C, vindesine, and ifosfamide. The performance status of all patients was over 70%. 20 patients had extensive disease, 7 limited disease. The most frequent histology was squamous cell carcinoma found in 20 patients. Only 3 patients had large cell carcinoma and 4 patients adenocarcinoma. 2 complete (7.7%) and 9 partial remissions (34.6%) were achieved. 3/26 (11.5%) patients showed a minor response. In 7/26 patients (26.9%) there was no change, 5/26 (19.2%) showed progressive disease. Because of the short observation time, no deductions regarding the duration of remission and survival time of the patients can be made. The toxicity of the regimen was relatively mild. In particular, the subjective gastro-intestinal symptoms occurred only in 50% of our patients. The hematological toxicity was acceptable. Also the renal toxicity did not pose problems. Considering an exact lung function analysis with diffusing capacity and spirometric data, we found an unexpectedly high proportion of pulmonary toxicity in about 30%, but clinically obvious in only 10% of patients. According to the remission rates, this regimen is comparable to combinations with cisplatin and appears to have much less toxicity. PMID- 3005936 TI - [Lymphoid cells in the venous blood of 100 female patients with breast carcinoma. Follow-up studies]. AB - The following blood cells were examined in 100 patients with invasive carcinoma of the breast and 100 control patients without malignant disease: leukocytes, lymphocytes, monocytes and different sub-populations of lymphoid cells. Lymphoid cells were characterized by E-, EA- and EAC-rosetting technique, alphanaphtyl acetate esterase activity and by the ability of latex phagocytosis. Both groups showed no significant differences. In 81 patients undergoing postoperative radiation therapy lymphocytes and lymphoid cells were reduced considerably. These changes were still evident after 12 months (6 months after completion of radiotherapy). Mainly the T lymphocytes were effected by this decrease. A negative effect of the radiation induced reduction of immune competent cells must be discussed and examined further by clinical trials. PMID- 3005937 TI - [Total abdominal irradiation following combination chemotherapy and second-look laparotomy in the treatment of advanced ovarian cancer]. AB - From 1980 to 1984 fifty-four patients with advanced ovarian carcinoma after operation and concluding chemotherapy with alkeran (n = 7) or cis-platin/alkeran +/- hexamethylmelamine (n = 47) as well as second-look laparotomy received follow up radiotherapy either with the moving-strip technique (n = 35) or later the open field technique (n = 19). 32 patients in CR received radiation therapy. 15 patients in CR are without relapse after undergoing open-field radiation therapy and a mean observation period of 25 months. At this point of time 5 of 17 patients had relapses under the moving-strip radiation treatment. The frequency of the relapses is apparently due to the very long periods of radiation and numerous interruptions in treatment. If residual tumors were present at the begin of ray therapy, a CR could only be achieved in cases where the previous monotherapy was with alkeran. PMID- 3005938 TI - Modulation of lens-induced uveitis by superoxide dismutase. AB - Recent studies on reactive oxygen metabolites have suggested that these products may be important mediators in the early tissue damage that develops from immunopathologic inflammations. The superoxide anion appears to be the principal product of the respiratory burst. In order to determine the effects of removing superoxide with superoxide dismutase on modulating the inflammation in experimental phacoanaphylactic endophthalmitis, the animals with this experimental disorder were treated with superoxide dismutase. The treatment resulted in significant reduction of choroidal inflammation, retinal edema and vasculitis, suggesting phlogogenic role of superoxide in the ocular immune complex disease. PMID- 3005939 TI - Effects of female hormones on the muscarinic and alpha 1-adrenergic receptors of the nasal mucosa. An experimental study in guinea pigs. AB - During pregnancy aggravation of nasal allergic symptoms is occasionally observed in subjects with nasal allergy. To evaluate the effect of female hormones on the muscarinic receptor and the alpha 1-adrenergic receptor of the nasal mucosa, receptor binding assay was performed using the nasal mucosa of pregnant guinea pigs and of male guinea pigs; the latter were treated either with estrogen or with progesterone. Pregnancy induced a significant decrease of the density of the alpha 1-adrenergic receptor of the nasal mucosa (p less than 0.05). Estrogen induced a significant increase of the density of the muscarinic receptor (p less than 0.05), while progesterone induced a significant decrease of the density of the alpha 1-adrenergic receptor of the nasal mucosa of guinea pigs. When similar changes of the receptor can be induced by female hormones in the nasal mucosa of the human subjects, this may facilitate secondary development of hyperreactive nasal symptoms in subjects who have been sensitized before without apparent nasal symptoms. PMID- 3005940 TI - [The effect of vitamin-D-3 loading and thyroxine therapy on low 25-hydroxy vitamin-D-3 levels in patients with hypothyroidism]. PMID- 3005942 TI - [Pancreatitis and cytomegalic infection in a child with Burkitt-type lymphoma]. PMID- 3005941 TI - [Hepatitis A virus infection, an etiologic factor in chronic active diseases of the liver? IgM antibody of the hepatitis A virus]. PMID- 3005943 TI - [Results of the treatment of neoplasms of the external auditory meatus and middle ear]. PMID- 3005944 TI - Management of facial angiofibromas in tuberous sclerosis: use of the carbon dioxide laser. AB - Facial angiofibromas are estimated to occur in 90% of patients with tuberous sclerosis and can cause considerable cosmetic disfigurement, emotional distress, obstruction of vision, and hemorrhage when abraded. Postoperative wound management associated with skin grafting and dermabrasion is often difficult because patients are mentally retarded and noncooperative. Three patients with extensive facial angiofibromas were treated successfully with the carbon dioxide (CO2) laser, with follow-up period ranging from 8 to 48 months. Uncomplicated wound healing occurred in each patient with minimal recurrence of lesions. Ablation with the CO2 laser is our treatment of choice for angiofibromas associated with tuberous sclerosis. PMID- 3005945 TI - AIDS update: HTLV-III/LAV infection. PMID- 3005946 TI - Autotomy in rats after peripheral nerve section: lack of effect of topical nerve or neonatal capsaicin treatment. AB - The influence of capsaicin on autotomy was studied in adult rats in which the sciatic and saphenous nerves were sectioned. Capsaicin was administered subcutaneously to neonatal rats or applied topically to the sciatic and saphenous nerves in adult animals. Quantification of neurogenic plasma extravasation in skin areas subserved by these nerves and of the number of small type B neurones in lumbar sensory ganglia were used to confirm the effectiveness of capsaicin induced lesions of unmyelinated sensory nerves. Neonatal capsaicin treatment significantly reduced neuronal numbers in ganglia and, compared to control responses, plasma extravasation was nearly abolished after both neonatal and peripheral nerve treatment with capsaicin. Despite these deficits in sensory neurones function, no differences in any parameters of autotomy were observed between animals receiving both capsaicin treatment and nerve section and those given nerve section alone. Animals in both control and experimental groups exhibited high autotomy scores. These results suggest that capsaicin-sensitive primary sensory neurones do not have a significant role in precipitating autotomy characterized by high incidence and severity. PMID- 3005947 TI - Effects of prior anaesthesia on autotomy following sciatic transection in rats. AB - The animal model for chronic pain following sciatic nerve section in the rat has been studied varying the sensory afferents prior to nerve section, using the anaesthetic blocking of the sciatic nerve. The experimental parameters used were the day of onset of autotomy and the time course of autotomy. The results show that the anaesthetic blocking prior to nerve section significantly reduces the degree of autotomy. PMID- 3005948 TI - Effects of anethole dithiolthione and 2(3)-tert-butyl-4-hydroxyanisole on schistosome granuloma formation. AB - Administration of the antioxidants 2(3)-tert-butyl-4-hydroxyanisole (BHA) or 5-(P methoxyphenyl)-3H-1,2-dithiol-3-thione (ADT) to female CD-1 mice starting 4 weeks after infection with 70 cercariae of Schistosoma mansoni resulted in a decrease in the size of the inner fibrotic region of the hepatic granuloma. The cellular composition of the granuloma was not altered by treatment with these two compounds. The administration of the specific superoxide scavenger copper diisopropylsalicylate (CuDIPS) resulted in a similar decrease in granuloma size, suggesting a role of superoxide radicals in the granulomatous response. PMID- 3005949 TI - [Immunological status of hemophiliacs: study of blood T-lymphocyte populations, serum immunoglobulins, and the prevalence of anti-LAV antibodies]. AB - Blood T-lymphocyte subsets and serum immunoglobulin levels were studied in a group of 52 haemophiliacs (44 patients with haemophilia A and 8 patients with haemophilia B). None of the patients had AIDS or belonged to any AIDS high-risk group. Patients were exclusively treated with clotting fractions obtained from healthy volunteers in metropolitan France. As compared to a group of 52 normal donors, haemophiliacs had increased numbers of suppressor lymphocytes, which resulted in depressed helper/suppressor (H/S) ratios, and increased levels of serum IgG and IgA. 21 haemophiliacs (40,3%) had a H/S ratio less than 1.4. Among patients with haemophilia A a higher mean IgG level was found in patients presenting lymphadenopathy. Decreased mean H/S ratio and increased mean serum IgG level were found in patients receiving more than 50 000 U of factor VIII during the 2 years preceding the study. No striking difference in mean serum IgG, IgA and IgM levels was found in patients with haemophilia A when H/S ratios were higher and lower than 1.4 respectively. As AIDS and immunological abnormalities among haemophiliacs probably share a common viral origin, this study emphasize the need to discourage blood donation from donors who belong to any AIDS high risk group, and to screen sera from the blood donor population for antibodies to LAV/HTLV III. PMID- 3005950 TI - Common epithelial tumours of the ovary--a new look. PMID- 3005951 TI - Antenatal diagnosis of severe beta thalassemia during the first trimester of pregnancy. AB - Prenatal diagnosis of hemoglobinopathies may be obtained during the first trimester of pregnancy by a combination of chorion biopsy and DNA mapping. This study illustrates 2 DNA approaches which are available for identification of abnormal globin genes. These include restriction fragment length polymorphisms (RFLPs) and direct gene mapping. PMID- 3005953 TI - Cirrhosis and hepatocellular carcinoma in a patient with heterozygous (MZ) alpha 1-antitrypsin deficiency. AB - A patient is described with micronodular cirrhosis, partial (heterozygous, MZ) deficiency of alpha-1-antitrypsin (AAT) and hepatocellular carcinoma. The patient did not drink alcohol and all serological markers of infection with hepatitis B virus were absent. Death was due to intra-peritoneal bleeding from a multifocal liver tumour. Histology revealed multiple intracytoplasmic AAT globules in hepatocytes at the periphery of the cirrhotic nodules. Copper granules, present in the same non-neoplastic liver cells may have resulted from minor cholestasis. Within the neoplastic hepatocytes AAT globules were sparse and copper deposits co existed with the globular variant of Mallory bodies. The case is presented in support of the postulated association of partial deficiency of AAT, chronic liver disease and hepatic neoplasia. PMID- 3005952 TI - Rapid diagnosis of viral infections with fluorescent antisera. AB - Viral immunofluorescence tests were performed with commercial antisera on 1046 specimens of respiratory, conjunctival corneal and dermal origin to compare the diagnostic effectiveness of direct examination by immunofluorescence with culture. The fluorescence assays were found to be highly sensitive for respiratory syncytial and measles virus in nasopharyngeal aspirates from children, but less satisfactory for other respiratory viruses. The test detected 38% of the adenovirus positives from conjunctival specimens and 67% of the combined herpes simplex virus positives from the latter 3 groups. Despite the reduced sensitivity of fluorescence for some viruses, a case is presented for more widespread use of fluorescence in routine microbiology laboratories because of its simplicity, speed and cost-effectiveness compared with culture which is labour-intensive and time consuming. PMID- 3005955 TI - Cardiopulmonary resuscitation. PMID- 3005954 TI - [Role of the opioid system in the functional regulation of the hypothalamo hypophyseal-adrenal system in traumatic shock]. PMID- 3005956 TI - Nutrition and infant lung functions: report of a National Heart, Lung and Blood Institute symposium. PMID- 3005957 TI - Supersensitivity of the renal tubule to catecholamines in the chronically denervated canine kidney. AB - Experiments were performed on anesthetized dogs to study whether or not renal tubules of the chronically denervated kidney show supersensitivity toward circulating catecholamines. In one kidney the influence of plasma catecholamines was inhibited by intrarenal administration of the alpha adrenergic receptor blocker phenoxybenzamine (POB, 2 micrograms/min), and renal parameters of the infused kidney were compared to those of the contralateral noninfused organ. Before POB infusion urine flow (V), urinary sodium and potassium excretion (UNa V, UKV) as well as clearance of inulin and PAH (GFR, CPAH) were similar in infused and contralateral kidneys in all groups studied. In dogs (n = 8) with two innervated kidneys POB infusion elevated V and UNa V by 53 +/- 13% and 102 +/- 34% (p less than 0.05). In dogs (n = 8) with acute bilateral renal denervation POB administration failed to alter any of the measured parameters. In contrast, V and UNaV from chronically denervated kidneys (n = 7) were increased after POB infusion by 40 +/- 9% and 103 +/- 34% (p less than 0.05). Glomerular filtration rate, CPAH and UKV were not changed by alpha adrenoceptor blockade in any of the groups. In an additional group of animals (n = 8) acute unilateral renal denervation increased V and UNaV to a significantly higher extent (by 282 +/- 85% and 330 +/- 106%) than POB administration did in the innervated kidney and elevated UKV (44 +/- 10%), too.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3005958 TI - 2-(4-phenylpiperidino) cyclohexanol (AH5183) decreases quantal size at the frog neuromuscular junction. AB - AH5183 was studied because it inhibits acetylcholine transport into synaptic vesicles. The drug apparently is a slow-acting anti-cholinesterase, so further experiments were performed with this enzyme inhibited. Soaking for hours in AH5183 in Ringer does not decrease quantal size. However, a few minutes of tetanic nerve stimulation results in a marked decrease in quantal size. Quantal size also decreased after hours in a hypertonic Ringer containing the drug. PMID- 3005959 TI - [Transarterial chemoembolization therapy of hepatocellular carcinoma using anticancer agents (mitomycin C and/or adriamycin) suspended in lipiodol]. PMID- 3005960 TI - [Effect of combined radiotherapy, hyperthermia, radioprotective agent and hypoxic cell sensitizer on mice testis--testicular weight as indicator of the combined effect]. PMID- 3005961 TI - [A new evaluation method of the topical drug concentration by means of X-ray CT and water soluble contrast media]. PMID- 3005962 TI - Transcatheter internal radiotherapy of hepatoma using radioactive iodized oil (I 131 Lipiodol). PMID- 3005963 TI - [The evaluation of the effect of transcatheter hepatic arterial embolization (T.A.E.) for hepatocellular carcinoma (H.C.C.). The assessment of efficacies of T.A.E. for the resected specimens in comparison with changes of medical imagings]. PMID- 3005964 TI - [Intraarterial infusion therapy with polysaccharide solution as a carrier of anticancer drugs]. PMID- 3005965 TI - [GABA receptor function and manic depressive disease]. PMID- 3005966 TI - Structure- and sequence-specificity of ozone degradation of supercoiled plasmid DNA. AB - Ozone-reactive sites on the nucleobase moieties in supercoiled pBR322 DNA were investigated by using sequencing procedures. Ozonolysis in the absence of salt resulted in degradation of thymine residues in the A + T rich region located at 3100-3400bp. In the presence of salt, such as NaCl or MgCl2, a conformational change of plasmid DNA was induced. Subsequently the thymine and guanine residues in the loop of the cruciform located at 3120bp and 3220bp were degraded. In addition, central thymine residues present in sequences GTA, GTT and ATA were also degraded. PMID- 3005967 TI - RNA-protein cross-linking in Escherichia coli ribosomal subunits: localization of sites on 16S RNA which are cross-linked to proteins S17 and S21 by treatment with 2-iminothiolane. AB - Treatment of E. coli ribosomal subunits with 2-iminothiolane coupled with mild ultraviolet irradiation leads to the formation of a large number of RNA-protein cross-links. In the case of the 30S subunit, a number of sites on 16S RNA that are cross-linked to proteins S7 and S8 by this procedure have already been identified (see ref. 6). Here, by using new or modified techniques for the partial digestion of the RNA and the subsequent isolation of the cross-linked RNA protein complexes, three new iminothiolane cross-links have been localized: Protein S17 is cross-linked to the 16S RNA within an oligonucleotide encompassing positions 629-633, and protein S21 is cross-linked to two sites within oligonucleotides encompassing positions 723-724 and positions 1531-1542 (the 3' end of the 16S RNA). PMID- 3005968 TI - In vitro splicing of simian virus 40 early pre mRNA. AB - The products of splicing of simian virus 40 early pre mRNA in HeLa cell nuclear extracts have been characterized. Of the two alternative splicing patterns exhibited by this precursor in vivo, which involve the use of alternative large T and small t 5' splice sites and a single shared 3' splice site, only one, producing large T mRNA, was found to occur in vitro. A number of possible intermediates and byproducts of splicing of large T mRNA were observed, including free large T 5' exon, lariat form intron joined to 3' exon and free lariat and linear forms of large T intron. The formation of these products argues strongly for a basic similarity in the mechanism underlying large T and other, non alternative splices. A collection of RNAs resulting from protection of early pre mRNA at specific points from an endogenous 5' to 3' exonuclease activity in vitro have also been observed. The regions of the precursor RNA protected map to positions immediately upstream of the 5' splice sites of large T and small t and the lariat branchpoint, and may represent interaction of these regions with components of the splicing machinery. PMID- 3005969 TI - Expression of ribosomal insertion in Drosophila: sensitivity to intercalating drugs. AB - Ribosomal insertions in Drosophila are transcribed at very low levels. The abundance of the most prominent 0.8 kb type 1 insertion transcript increased up to 60-fold when cultured cells were exposed to the DNA intercalating drug chloroquine. After injection of insertion-containing rDNA in circular form into Xenopus laevis oocytes an apparently identical 0.8 kb insertion transcript was synthesized, and its accumulation was stimulated several fold by coinjection of chloroquine or ethidium bromide. We suggest that ribosomal insertions are assembled in a chromatin conformation that lacks unconstrained torsional stress, accounting for the inactivity of these DNA regions; introduction of stress by intercalation results in activation of transcription from the insertion sequences. PMID- 3005970 TI - Identification and characterisation of PmaCI an endonuclease of novel specificity from Pseudomonas maltophila. AB - We report the use of MonoQ FPLC (Fast Protein Liquid Chromatography) for the rapid purification of a novel Type II restriction endonuclease PmaCI, from Pseudomonas maltophila, which recognises the sequence 5'-CAC decreases GTG-3'. The resulting enzyme is free of other nucleases to a level suitable for its characterisation by multiple-substrate digestion and DNA sequencing techniques. This method appears to be widely applicable and we have used it for the isolation of restriction endonucleases of comparable purity from a range of other organisms. Also described is a rapid method for screening a library of small inserted regions in recombinant M13 molecules for the presence and subsequent screening of restriction sites of interest. PMID- 3005971 TI - Tetrahymena conjugation-induced genes: structure and organization in macro- and micronuclei. AB - The physical organization of eight Tetrahymena genes active during conjugation (meiosis) was examined in the somatic (macro-) and germinal (micro-) nuclei of this organism. Three of these genes make transcripts that are only detected during meiotic prophase. Southern blot analyses indicated that all genes examined were present in both nuclei. Except for one gene family (pC6), all appeared to be non-repetitive and there were no detectable sequence rearrangements in or near the genes. The exceptional gene was repeated approximately five to seven times and DNA rearrangement occurred in or near each of these copies. A comparison of cDNA and macro- and micronuclear DNA restriction maps indicated that one of the genes (cnj B) contains introns. This is the first report of evidence for introns in a non-ribosomal gene in Tetrahymena. A site specific modification probably due to adenine methylation was seen in the macronuclear copy of another gene (cnj C). PMID- 3005972 TI - Secondary structure of Tetrahymena thermophilia 5S ribosomal RNA as revealed by enzymatic digestion and microdensitometric analysis. AB - The secondary structure of [32P] end-labeled 5S rRNA from Tetrahymena thermophilia (strain B) has been investigated using the enzymes S1 nuclease, cobra venom ribonuclease and T2 ribonuclease. The results, analyzed by scanning microdensitometry and illustrated by three-dimensional computer graphics, support the secondary structure model of Curtiss and Vournakis for 5S rRNA. Aberrent mobility of certain RNA fragments on sequencing gels was observed as regions of band compression. These regions are postulated to be caused by stable internal base-pairing. The molecule was probed with T2 RNase in neutral (pH 7.5) and acidic (pH 4.5) buffers and only minor structural differences were revealed. One of the helices was found to be susceptible to enzymatic attack by both the single strand and double-strand specific enzymes. These observations are evidence for the existence of dynamic structural equilibria in 5S rRNA. PMID- 3005974 TI - CD spectra and some properties of deoxyoligonucleotide duplexes having a C:G terminus (nucleosides and nucleotides. Part 69). AB - In the course of an investigation of the mode of recognition of nucleotide sequences with restriction endonucleases, several deoxyoligonucleotide duplexes having G:C terminal base pairs were synthesized. The oligonucleotides having a 5' C (3'-G) terminus showed unusual CD spectra with a negative band at the longer wavelength region, when compared to those of the same internal sequences but a 5' G (3'-C) terminus, which showed a positive band like the B- or A-DNA type. The nature of these CD spectra was compared with those of the Z-DNAs on the effect of salt concentrations, intercalation with ethidium bromide, or 31P-NMR spectra. These unusual spectra may be attributed to the terminal effect of the 5'-C:3'-G pairs. PMID- 3005973 TI - Nucleotide sequence of the chicken 5-aminolevulinate synthase gene. AB - 5-Aminolevulinate synthase, the first and rate-controlling enzyme of heme biosynthesis, is regulated in the liver by the end-product heme. To study this negative control mechanism, we have isolated the chicken gene for ALA-synthase and determined the nucleotide sequence. The structural gene is 6.9 kb long and contains 10 exons. The transcriptional start site for ALA-synthase was determined by primer extension analysis. A fragment of 291 bp from the 5' flanking region including 34 bp of the first exon shows promoter activity when introduced upstream of a chicken histone H2B gene and injected into the nuclei of Xenopus laevis oocytes. PMID- 3005975 TI - Determination of the complete nucleotide sequence of the Sendai virus genome RNA and the predicted amino acid sequences of the F, HN and L proteins. AB - We previously determined the 3' proximal 5,824 nucleotides of the Sendai virus genome RNA (Nucleic Acids Res. 11, 7317-7330, 1983; Nucleic Acids Res. 12, 7965 7973, 1984), and present here the sequence of the remaining 5' proximal 9,559 nucleotides. Thus, this is the first paramyxovirus to have its genome organization elucidated. The set of complementary DNA clones used was prepared by the method of Okayama and Berg from polyadenylylated viral genome RNA. We sequenced the region containing the 5' proximal half of the F gene, and the subsequent HN and L genes, and predicted the complete amino acid sequence of the products of these genes. Sequence analyses confirmed that all the genes are flanked by consensus sequences and suggest that the viral mRNAs are capable of forming stem-and-loop structures. Comparison of the F and HN glycoproteins of Sendai virus with those of simian virus 5 strongly suggests that the cysteine residues are highly important for maintenance of the molecular structures of these glycoproteins. PMID- 3005976 TI - Nucleotide sequence of the hygromycin B phosphotransferase gene from Streptomyces hygroscopicus. AB - The nucleotide sequence of a 1467 bp fragment of Streptomyces hygroscopicus DNA containing the gene (hyg) encoding a hygromycin B phosphotransferase (HPH) has been determined. The N-terminal amino acid sequence of HPH determined by automated Edman degradation has allowed the coding sequence of the hyg gene to be identified. The translation initiation triplet is GTG and 5 bp preceding it there is a sequence complementary to the 3'-end of 16S rRNA from S. lividans. The transcriptional start and termination sites have been determined; the presumptive promoter region has only partial homology to that of the Streptomyces vinaceus vph gene and is different to the promoter sequences of other Streptomyces genes. PMID- 3005977 TI - A human DNA-binding protein is methylation-specific and sequence-specific. AB - A nuclear protein isolated from human placenta, methylated DNA-binding protein (MDBP), binds selectively to DNA enriched in 5-methylcytosine. We now demonstrate that MDBP is a sequence-specific, as well as methylation-specific, DNA-binding protein. From ten restriction fragments of pBR322 DNA methylated with human DNA methyltransferase, one was bound to MDBP very much more strongly than any of the others. For this preferential binding to MDBP, the DNA had to be methylated. By a DNase I protection experiment (DNase I footprinting), a 22-base sequence within this methylated restriction fragment was shown to be specifically protected by MDBP. The sequence-specificity of MDBP coupled with its dependence on DNA methylation suggests that this is one of the proteins which modulates important functions of human DNA methylation in vivo. PMID- 3005978 TI - Cloning and sequence analysis of cDNA encoding active phosphoenolpyruvate carboxylase of the C4-pathway from maize. AB - A recombinant clone, pM52, containing cDNA for maize phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) was isolated from a maize leaf cDNA library constructed using an expression vector in Escherichia coli. The screening of the clone was conveniently performed through its ability to complement the phenotype (glutamate requirement) of PEPCase-negative mutant of E. coli. The enzyme encoded by this clone was identical with the major PEPCase in maize, a key enzyme in the C4-pathway, as judged from its allosteric properties and immunological reactivity. The cloned cDNA (3093 nucleotides in length) contained an open reading frame of 2805 nucleotides, the 3'-untranslated region of 222 nucleotides and the poly(dA) tract of 64 nucleotides. The deduced amino acid sequence (935 residues) of the enzyme showed higher homology with that of an enterobacterium, E. coli (43%) than that of a cyanobacterium (blue-green alga), Anacystis nidulans (33%). PMID- 3005979 TI - Tissue restricted and stage specific transcription is maintained within 411 nucleotides flanking the 5' end of the chicken alpha-skeletal actin gene. AB - alpha-skeletal actin message levels have been shown to be tightly regulated in chicken primary myoblast cultures. To test for gene elements required for muscle cell specific expression, DNA sequences containing the 5'-flanking regions of the chicken alpha-skeletal actin, beta-cytoplasmic actin, and the histone H2b genes were linked to the coding sequences of the chloramphenicol acetyltransferase gene and transfected into myogenic and non-myogenic cells. In contrast to beta-actin CAT hybrids, the alpha-skeletal actin CAT constructions displayed restricted CAT expression in transfected non-myogenic cells. We showed that a 411 nucleotide fragment flanking the 5' end of of the alpha-skeletal actin gene was responsible for a 9-15 fold increase in CAT enzymatic activity during myoblast fusion, versus only a transient 2 fold rise for the beta-actin and histone flanking sequences. These results indicate that DNA sequences within 411 bp of the 5' terminus of the alpha-skeletal actin gene influenced its cell type and stage specific expression. PMID- 3005980 TI - Complete DNA sequence of the short repeat region in the genome of herpes simplex virus type 1. AB - We report the complete DNA sequence of the short repeat region in the genome of herpes simplex virus type 1, as 6633 base pairs of composition 79.5% G+C. This contains immediate early gene 3, encoding the IE175 protein, an important transcriptional activator of later virus genes. The IE175 coding region was identified as a 3894 base sequence of 81.5% G+C DNA. The base composition of this gene is thus the most extreme yet determined, and the IE175 predicted amino acid composition is correspondingly biased, most notably with an alanine content of 20.9%. Functionally important regions of the IE175 polypeptide were tentatively identified by comparison with the sequence of the homologous protein from varicella-zoster virus and from locations of ts mutations, and were correlated with properties of the amino acid sequence. Aspects of the evolution of such an extreme composition DNA sequence were discussed. PMID- 3005981 TI - Alphaherpesviruses possess a gene homologous to the protein kinase gene family of eukaryotes and retroviruses. AB - The US3 genes of herpes simplex virus serotypes 1 and 2, and the corresponding gene of varicella-zoster virus, encode proteins whose sequences are clearly homologous to members of the protein kinase family of eukaryotes and retroviruses. Similarity is most characteristic, and strongest, in an 80 residue region comprising part of the catalytic structure of the kinases. In this region the herpesvirus proteins are most like a yeast cell division control protein, and least like the retrovirus protein-tyrosine kinases. We consider that the herpesvirus proteins are probably involved in modulation of cellular processes during lytic infection, although other roles are also possible, for example in latent infection. PMID- 3005983 TI - Rapid synthesis of cDNA for cloning into lambda vectors. PMID- 3005982 TI - Stabilization of T4 polynucleotide kinase by macromolecular crowding. AB - T4 polynucleotide kinase rapidly loses activity during its reaction on duplex DNA termini. Addition of high concentrations of nonspecific polymers reverses or prevents this inactivation. In contrast, additions of related materials of lower molecular weight are relatively ineffective in stabilizing the kinase. Such a pattern suggests that the stabilizing effects of polymers on kinase activity are due to macromolecular crowding. An effect of crowding on the known tendency of the kinase to undergo oligomerization reactions is consistent with our observations. PMID- 3005984 TI - RFLP for HP30, D11S28, an anonymous genomic clone localised to 11p12. PMID- 3005985 TI - An anonymous human single copy genomic clone, D11S29 (L7) at 11q23, identifies a moderately frequent RFLP. PMID- 3005986 TI - An anonymous single copy X-chromosome clone, pTAK8, identifies a frequent RFLP at Xp11-q12(HGM8 no. DXS146). PMID- 3005987 TI - ApaI RFLP 5.4 kb 5' to the human apolipoprotein AI (APO A1) gene. PMID- 3005988 TI - An anonymous human single copy genomic clone, D5S6 (M4) on chromosome 5 identifies a three allele RFLP. PMID- 3005989 TI - DNA polymorphic sites in the human ApoAI-CIII-AIV cluster: Taq I and Ava I. PMID- 3005990 TI - RFLP for the human erbA2 gene. PMID- 3005991 TI - An anonymous single copy chromosome 18 probe associated with a frequent RFLP (D18S5). PMID- 3005992 TI - Isolation of a polymorphic DNA sequence (lambda 82B, D8S2) from chromosome eight. PMID- 3005993 TI - EcoRI RFLP linked to the human myb gene. PMID- 3005995 TI - [A case of paralysis of the right diaphragm with paralysis of the contralateral brachial plexus]. AB - We describe a case of unilateral diaphragmatic paralysis following a distocic delivery. We think that the rarity of this syndrome in only apparent while, if investigated with fluoroscopy, much more cases can be discovered. Our case is interesting too since the Erb's palsy was contralateral to the diaphragmatic paralysis; that occurs in very few cases. PMID- 3005994 TI - [Controlled study on the prevention of respiratory distress syndrome with betamethasone in pregnancy and ACTH and cortisol levels in the premature newborn infant]. AB - The influence of maternal corticosteroid administration was studied on the ACTH and cortisol concentrations in neonatal blood of 24 premature infants whose mothers received betamethasone for prevention of RDS, compared with 11 untreated subjects. Cord blood was taken at birth and from venous sample in 5th day. All samples were analyzed for ACTH and Cortisol by radioimmunoassay. No statistically significant differences between these groups were noted. Additional analysis of ACTH and Cortisol levels in 9 RDS premature infants versus 26 control ones failed to demonstrate any deficiency of corticosteroids in newborn infants with RDS. The findings provide a justification for the prepartum treatment of respiratory distress syndrome with glucocorticoids because this dose of betamethasone does not expose the newborn to potentially harmful effects. PMID- 3005996 TI - Cytomegalovirus in hospitals. PMID- 3005997 TI - Restriction endonuclease analysis of cytomegalovirus DNA from strains isolated in day care centers. AB - Cytomegalovirus (CMV) strains isolated in a previous study from children in group day care have been analyzed by restriction endonuclease cleavage of DNA. CMV was isolated from 16 of 60 children (27%) in several centers in Stockholm. In one center (Center A) 7 of 20 children excreted CMV and all were in the same group. In another center (Center B) 5 of 16 children were CMV-positive. In Center A three children excreted CMV strains with identical DNA cleavage patterns. The other four strains had different unique patterns as had those isolated in Center B. This finding provides evidence that CMV can be transmitted between children in group day care. PMID- 3005999 TI - Aerosol transmission of experimental rotavirus infection. AB - Several aspects of the epidemiology of rotavirus suggest the possibility that transmission may occur by nonenteral routes. We utilized the mouse model of rotavirus infection to investigate the experimental transmission of rotavirus infection by respiratory droplets. Following exposure to a defined dose of aerosolized rotavirus, the kinetics of viral replication within the lung and gastrointestinal tract was studied using a double antibody enzyme immunoassay and indirect immunofluorescence. These studies documented the efficient transmission of rotavirus infection by means of aerosol in all exposed animals. Rotavirus antigen was detected as early as 12 hours after infection in the pulmonary and gastrointestinal tracts of the infected animals and antigen remained detectable in both sites for at least 8 days following infection. Gastrointestinal illness was clearly demonstrable in the animals while pulmonary pathology was not evident. These studies document that rotavirus infection can be transmitted by aerosol droplets under experimental conditions. PMID- 3005998 TI - Frozen deglycerolyzed blood prevents transfusion-acquired cytomegalovirus infections in neonates. AB - Frozen deglycerolyzed blood (FDB) was used for routine transfusions to 63 low birth weight newborns (less than or equal to 1500 g) over a 12-month period in an effort to decrease transfusion-acquired cytomegalovirus (CMV) infections. Nine of the 63 infants also received nonfrozen blood products (platelets, liquid blood). Seventy-two percent of the donor blood units were CMV-seropositive. Urine cultures and serum titers for anti-CMV antibody (immunoglobulins G and M) were obtained prior to the initial transfusion and sequentially throughout the study. No infant (0 of 54) who received only FDB acquired CMV, whereas 3 of 9 infants (33%) who received non-frozen blood and FDB acquired CMV, as evidenced by CMV viruria and/or a 4-fold rise in immunoglobulin G anti-CMV antibody titers. These results suggest that transfusions with frozen deglycerolyzed blood decrease the risk in low birth weight infants of acquiring CMV, regardless of the CMV serologic status of the donor. PMID- 3006000 TI - Nosocomial transmission of cytomegalovirus. PMID- 3006001 TI - Role of the polymorphonuclear neutrophils in the phototoxic reaction in porphyria cutanea tarda. AB - We have compared the superoxide production of polymorphonuclear cells (PMNLs) from healthy donors, when incubated with either control or porphyria cutanea tarda (PCT) sera at 4, 24 and 48 hours after exposure in vivo to UVA light. Serum from UVA-irradiated (1-5 J/cm2) PCT patients generated significantly greater amounts of superoxides from PMNLs than serum from UVA-irradiated (8-12 J/cm2) normal controls. This indicates that serum factors activated by porphyrin plus UVA stimulate neutrophilic granulocytes to liberate superoxides, triggering a series of events that cause tissue damage. The vascular lesions in patients with PCT could be, at least in part, due to this cytotoxic effect. PMID- 3006002 TI - Evaluation of prostatic cancer histology and grade distribution: experience with the Colorado Central Cancer Registry. AB - Although well-defined grading schemes for prostatic adenocarcinoma have been developed, they are not yet universally accepted by practicing surgical pathologists. The complexity of these schemes often leads surgical pathologists to develop their own modified Broders scheme. This study compared the grade distribution as obtained by a community of surgical pathologists with that obtained using the National Prostatic Cancer Project (NPCP) and Gleason grading schemes. In 1978, 308 cases of prostate cancer were reported to the Colorado Central Cancer Registry (CCCR) from the Denver Standard Metropolitan Statistical Area. Two hundred eighteen of these cases were regraded. The grade distribution as reported by the CCCR revealed a predominance of low-grade tumors (grade I-41%, grade II-28%, grade III-17%, grade IV-3%). Regrading of these same cases revealed a shift to higher grades (NPCP: grade I-14%, grade II-11%, grade III-37%, grade IV-29%; Gleason: pattern scores less than 6-30%, score 6-24%, score 7-12%, score 8-11%, score 9-10%, score 10-4%). Twenty-one cases of histologic variants which were not originally diagnosed were also noted (six mucinous, 12 ductal, two endometroioid, one squamous). There were 18 cases in which no evidence of carcinoma was confirmed. These results suggest that there is a tendency to underestimate grade and potentially malignant behavior when well-defined prostatic cancer grading schemes are not applied. PMID- 3006003 TI - Metastatic tumor of the hand from malignant cystosarcoma phylloides of the breast. A case report. AB - In this case report, a malignant cystosarcoma phylloides of the breast metastasized to the pulp of the little finger in a 47-year-old woman. It initially masqueraded as a whitlow. The diagnosis was helped by radionuclide bone scanning, which showed multiple areas of increased focal uptake including the terminal phalanx of the fifth finger, and the diagnosis was established by a frozen section biopsy of the tumor. The patient underwent a palliative fifth ray resection. She died within six weeks of surgery from extensive pulmonary and osseous metastases. Review of literature revealed only one other case of malignant cystosarcoma phylloides of the breast that metastasized to the hand and was initially misdiagnosed as a whitlow. PMID- 3006004 TI - Surgical technique and results of limb sparing surgery for high grade bone sarcomas of the knee and shoulder. AB - Thirty-three patients with high grade bone sarcomas of the knee and shoulder treated by limb sparing surgery were evaluated. The histological diagnoses were osteosarcoma (25), chondrosarcoma (3), malignant fibro-histiocytoma (3), fibrosarcoma (1) and unclassified (1). The Surgical Stages were: Stage IIA (3), Stage IIB (28) and Stage III (3). The operative procedure consisted of three phases: tumor resection, skeletal reconstruction and soft tissue reconstruction. All resections obtained negative margins and were classified as, marginal excision (3), intracompartmental resections (28) and radical resections (2). Overall survival was 77%. Four of 33 patients (12.4%) required a secondary amputation. Local recurrence was 6% (2/33) with an average follow-up of 37.2 months. The most common complications were flap necrosis (33%) and transient nerve palsies (33%). There were 2 infections and one prosthetic dislocation. We believe that limb salvage surgery for high grade bone sarcomas need not be reserved for only those without extraosseous extension. Careful preoperative selection and attention to the three stages of a limb sparing procedure are important for a successful outcome. Presently, we consider the following as contraindications to resection: vascular involvement, pathologic fracture, inappropriate biopsy and infection. PMID- 3006005 TI - Acute and latent effect of poliomyelitis on the motor unit as revealed by electromyography. AB - When polio virus attacks the motor neuron it may be completely destroyed, damaged, or unaffected. Muscle fibers of a destroyed motor neuron are orphaned or reinnervated. Nearby functioning motor units will then send terminal axon sprouts to reinnervate the orphaned muscle fibers. If there are too many orphaned muscle fibers and not enough surviving motor units to reinnervate them, the orphaned muscle fibers will continue to fibrillate until they atrophy and die. The resultant effect of poliomyelitis upon the affected muscle is an overall loss of motor units with the remaining units innervating many more muscle fibers than they originally did. There appears to be a late effect of polio upon these larger reinnervated motor units. After approximately 20 to 30 years, impulse transmission to the muscle fibers of the large reinnervated motor unit begins to fail. These transmission difficulties increase with age and time from recovery. These late onset transmission abnormalities may be factors in patient complaints of fatigue and progressive weakness. PMID- 3006006 TI - Resorbable materials and composites. New concepts in orthopedic biomaterials. AB - For the last several decades, research in orthopedic biomaterials has remained focused on homogeneous plastics and metals. Initial research resulted in rapid improvements and a number of standardized materials emerged. With time, development of these materials has become a slower, more deliberate process. Recently, however, investigators have sought to broaden the scope of orthopedic biomaterials research. New materials systems are now under consideration. These systems include natural and synthetic resorbable polymerics and resorbable ceramics. A variety of composite materials are under primary investigation or in clinical trials. Our emerging understanding of these new materials is rapidly leading to new surgical applications not possible with conventional metals and plastics. PMID- 3006007 TI - Melanotic neuroectodermal tumor of infancy. AB - Melanotic neuroectodermal tumor of infancy is a specific but unusual tumor of infancy for which only sporadic cases have been reported in the literature. This paper presents a case in an infrequent site, the epididymis, and summarizes the literature on the subject. PMID- 3006008 TI - Meningeal tumors of infancy and childhood. AB - Seventeen meningeal tumors were examined for pathology with electron microscopy and immunohistochemistry including glial fibrillary acidic protein (GFAP), S-100 protein, muramidase, and factor VIII. These tumors included seven meningiomas, one hemangiopericytoma, three meningeal sarcomas (1 pleomorphic-cell type and 2 myxofibrosarcomas), two fibrous histiocytomas, and four malignant melanomas. Two of seven children with meningioma had a poor outcome despite the benign histological features of the tumor. S-100 protein was present in the two tumors. All three children with meningeal sarcoma had a rapid downhill clinical course, although the myxofibrosarcoma initially had a relatively benign histological appearance. The two children with fibrous histiocytoma did well despite the aggressive histological features. Muramidase was a good marker of such tumors. Because of the morphological difficulties associated with childhood meningeal tumors, both electron microscopy and immunohistochemistry can be of diagnostic benefit. PMID- 3006009 TI - Case 2. Cystic cellular mesoblastic nephroma. AB - Mesoblastic nephroma is a benign mesenchymal tumor of the kidney generally diagnosed during the first 3 months of life. A case of the cellular variant of mesoblastic nephroma associated with prominent cystic change and renal vein invasion arising in an 8-month-old female is described. This unusual case expands the morphologic spectrum of mesoblastic nephroma. PMID- 3006010 TI - Case 3. Fetal hepatoblastoma with placental metastases. PMID- 3006011 TI - Cytomegalovirus and inflammatory bowel disease. AB - An 80 year old man was admitted to hospital with a 4 month history of diarrhoea with blood and mucus. The diarrhoea could not be controlled by a variety of drugs and he died 7 weeks later. Rectal biopsy showed both intranuclear and intracytoplasmic inclusional bodies consistent with cytomegalovirus infection. PMID- 3006012 TI - [Carcinoembryonic antigen (CEA) and tissue polypeptide antigen (TPA) in the diagnosis of small cell and non-small cell bronchial cancer]. PMID- 3006013 TI - [Autopsy findings in acquired immunodeficiency syndrome (AIDS)]. PMID- 3006014 TI - [The secretin test in a case of pancreatic somatostatinoma]. PMID- 3006015 TI - [Identification of C or D virus-type particles in the germinal centers of lymph nodes sampled at the lymphadenopathy stage of the acquired immunodeficiency syndrome]. PMID- 3006016 TI - [Value of the use of monoclonal antibodies in the diagnosis of cytomegalovirus infections]. PMID- 3006017 TI - [Normal pregnancy in a female acromegaly patient after x-ray therapy]. PMID- 3006018 TI - [So-called transitional or atypical carcinoids]. PMID- 3006020 TI - [Effect of adrenocorticotropic hormone on the secretory activity of the gonads and adrenals of male hamadryas baboons]. AB - The time course of the secretion of steroid hormones to the adrenal and seminal veins following administration of synthetic ACTH was studied in experiments on mature male hamadryas baboons. Testosterone, 5 alpha-dihydrotestosterone, androstendion, 17-oxyprogesterone and 17-oxypregnenolone levels in the venous blood were determined by means of radioimmunoassays with preliminary chromatographic isolation of steroids on cellite. The rate of hormone secretion by the adrenal and seminal glands was calculated by the gradient of the hormone concentration in the peripheral blood and in the blood outflowing from the steroid secreting glands. Sharp activation of the seminal secretion of testosterone, 5 alpha-dihydrotestosterone, androstendion and 17-oxyprogesterone was recorded 10-30 min after ACTH administration. The time course of testosterone secretion by the testes in ACTH administration showed correlation with changes in the hormone concentration in the peripheral blood. The data obtained were indicative of the seminal origin of a short-term rise of testosterone concentration in male hamadryas baboons in the initial period of ACTH action. A possible mechanism of the ACTH effect on the biosynthesis of steroids in the testes was discussed. PMID- 3006019 TI - [Psychotropic properties of corticotropin and its analogs]. AB - The authors presented evidence for the effect of 6 analogues of ACTN4-10 on teaching of albino rats in a labyrinth with negative electrocutaneous reinforcement. Sixteen series of experiments on 192 rats demonstrated a positive effect of corticotropin fragments on memory processes. The activating ACTH fragment effect was shown in recording the background and bioelectrically induced rabbit brain activity in response to a photo-flash in 36 experiments on 18 animals. Three groups of 30 patients with alcohol withdrawal syndrome, posttraumatic craniocerebral effect and schizophrenia received injections of corticotropin as a therapeutic agent. A high efficacy of corticotropin in the treatment of the alcohol withdrawal syndrome and after effect of craniocerebral injury and a low efficacy and sometimes aggravation of symptoms in schizophrenia patients were shown. PMID- 3006021 TI - [Significance of endogenous factors and pharmacological agents in regulating prolactin synthesis and secretion]. PMID- 3006022 TI - Linkage analysis for psychiatric disorders. II. Methodological considerations. AB - Recent advances in molecular biology make genetic linkage analysis an increasingly attractive tool for the identification and characterization of genes involved in the etiology of psychiatric illnesses. However, the complex nature of psychiatric illnesses engenders a host of methodological difficulties not encountered in linkage analyses of simple, Mendelian genetic traits. A previous paper reviewed the basic concepts of genetic linkage analysis. This paper focuses on the methodological difficulties associated with the application of genetic linkage methods to psychiatric illnesses. PMID- 3006023 TI - Genetic analysis of the fragile-X mental retardation syndrome with two flanking polymorphic DNA markers. AB - The fragile-X mental retardation syndrome, one of the most prevalent chromosome X linked diseases (approximately equal to 1 of 2000 newborn males), is characterized by the presence in affected males and in a portion of carrier females of a fragile site at chromosomes band Xq27. We have performed a linkage analysis in 16 families between the locus for the fragile-X syndrome, FRAXQ27, and two polymorphic DNA markers that correspond to the anonymous probe St14 and to the coagulation factor IX gene F9. Our results indicate that the order of loci is centromere-F9-FRAXQ27-St14-Xqter. The estimate of the recombination fraction for the linkage F9-FRAXQ27 is 0.12 (90% confidence limits: 0.044-0.225) and 0.10 for FRAXQ27-St14 (90% confidence limits: 0.040-0.185). Recombination between St14 and F9 does not appear to be significantly different in normal and fragile-X families. The two flanking probes were used for diagnosis of the carrier state and for detection of transmission of the disease through phenotypically normal males. They should also allow first-trimester diagnosis with a reliability of about 98% in 40% of the families. Used in conjunction with the cytogenetic analysis, the segregation studies with both probes should improve the genetic counseling for the fragile-X syndrome and should be useful for the formal genetic analysis of this unique disease. PMID- 3006024 TI - Amplification units containing human N-myc and c-myc genes. AB - The amplification units in human tumors containing amplified myc genes were examined. The amplification unit in all cases consisted of a large genomic region coamplified with the coding region of the myc genes themselves. In eight independent neuroblastomas containing N-myc amplifications, the amplification unit was estimated to be 290 to 430 kilobases. This amplification unit was highly conserved among the different neuroblastomas, with some neuroblastomas containing almost identical units. In contrast, five tumor cell lines containing c-myc amplifications exhibited amplification units that were more variable in size (90 to 300 kilobases) and sequence content; at least three different patterns of c myc amplification units could be discerned. PMID- 3006025 TI - Genetic instability in Drosophila melanogaster: the genetics of an MR element that makes complete P insertion mutations. AB - An MR element that maps to a specific locus in chromosome 3 of Drosophila melanogaster has the unusual feature of producing complete P (a mobile element) sequence mutations at several X chromosome loci. This MR also increases the frequency of mitotic recombination. Evidence is given for the transposition of MR. The complete P insertion mutations are autonomously unstable and are capable of causing otherwise stable incomplete (defective) P insertion mutations to revert. These results complement the analysis of P element functions with a synthetic complete P derivative. The genetic basis for the mutational-mitotic recombinational components of "hybrid dysgenesis" is conveniently explicable in terms of MR elements present in the genomes of flies present in the wild. PMID- 3006026 TI - Evidence for increased recombination near the human insulin gene: implication for disease association studies. AB - Haplotypes for four new restriction site polymorphisms (detected by Rsa I, Taq I, HincII, and Sac I) and a previously identified DNA length polymorphism (5' FP), all at the insulin locus, have been studied in U.S. Blacks, African Blacks, Caucasians, and Pima Indians. Black populations are polymorphic for all five markers, whereas the other groups are polymorphic for Rsa I, Taq I, and 5' FP only. The data suggest that approximately equal to 1 in 550 base pairs is variant in this region. The polymorphisms, even though located within 20 kilobases, display low levels of nonrandom association. Population genetic analysis suggests that recombination within this 20-kilobase segment occurs 24 times more frequently than expected if crossing-over occurred uniformly throughout the human genome. These findings suggest that population associations between DNA polymorphisms and disease susceptibility genes near the insulin gene or structural mutations in the insulin gene will be weak. Thus, population studies would probably require large sample sizes to detect associations. However, the low levels of nonrandom association increase the information content of the locus for linkage studies, which is the best alternative for discovering disease susceptibility genes. PMID- 3006027 TI - Elevated erythrocyte adenosine deaminase activity in patients with acquired immunodeficiency syndrome. AB - Acquired immunodeficiency syndrome (AIDS) is an often fatal disease caused by a retrovirus frequently resulting in malignancy and/or opportunistic infection. Because the immune deficiency in AIDS is similar to that in some purine enzyme deficiencies, we measured erythrocyte adenosine deaminase (ADA) and purine nucleoside phosphorylase activities in patients with AIDS, heterosexual controls, and a high-risk asymptomatic population. We found that erythrocyte ADA activity was significantly elevated in patients with AIDS (40 +/- 11 nmol/mg of hemoglobin per hr, mean +/- SD) relative to heterosexual controls (25 +/- 10, P less than 0.001). We also measured ADA activity in a group of individuals at high risk for AIDS and found that approximately half had significantly elevated ADA activities (45 +/- 4, P less than 0.002) that correlated with the presence of antibody to the lymphadenopathy retrovirus. Purine nucleoside phosphorylase activity was relatively normal in patients with AIDS as well as in individuals at risk for AIDS. Increased ADA appears to be a diagnostic marker of AIDS and may be useful in conjunction with antibody to the AIDS-related retrovirus in detecting the presence of infection in asymptomatic high-risk individuals. These data also suggest that, in addition to the lymphocyte, the erythroid cell line may also be infected by the AIDS-related retrovirus. PMID- 3006028 TI - Genes involved in Haemophilus influenzae type b capsule expression are part of an 18-kilobase tandem duplication. AB - Encapsulated Haemophilus influenzae type b produce nonencapsulated variants at high frequency (0.1-0.3%). Cosmid cloning was used to investigate the genetic mechanism responsible for this instability. Analysis of three independently derived cosmid clones showed that the b+ parental strain contains an 18-kilobase tandem duplication of genes involved in type b capsule expression. Loss of one complete copy of the 18-kilobase tandem duplication occurred following transformation of the cosmid clones into Rec+, but not Rec-, Escherichia coli, and in H. influenzae strains that had spontaneously lost capsule expression. These results suggest that high-frequency loss of type b capsule expression is due to rec-dependent recombination between the two copies of the 18-kilobase tandem repeat. This is further supported by our finding that introduction of the H. influenzae rec-1 mutation stabilized type b capsule expression. PMID- 3006029 TI - Control of the light-regulated current in rod photoreceptors by cyclic GMP, calcium, and l-cis-diltiazem. AB - The effect of calcium ions on the cGMP-activated current of outer segment membrane was examined by the excised-patch technique. Changes in the extracellular calcium concentration had marked effects on the cGMP-activated current, while changes in intracellular calcium concentration were ineffective. Changes in calcium concentration in the absence of cGMP had little, if any, effect on membrane conductance. These results suggest that both intracellular cGMP and extracellular calcium can directly affect the conductance underlying the light response in rod cells. The pharmacological agent l-cis-diltiazem reversibly inhibited the cGMP-activated current when applied to the intracellular side of an excised patch. When superfused over intact rod cells, l-cis-diltiazem reversibly blocked much of the normal light response. The isomer, d-cis-diltiazem, did not significantly affect either patches or intact rod cells. Thus, the light regulated conductance has binding sites for both calcium and cGMP that may interact during the normal light response in rod cells and a site specific for l cis-diltiazem that can be used to identify and further study the conductance mechanism. PMID- 3006030 TI - Single protein from human leukocytes possesses 5-lipoxygenase and leukotriene A4 synthase activities. AB - The activity of leukotriene A4 (LTA4) synthase in crude human leukocyte homogenates was found to have a similar requirement for Ca2+ and ATP as had been noted previously for 5-lipoxygenase activity. Purification of the 5-lipoxygenase using ammonium sulfate fractionation, AcA 44 gel-filtration chromatography, and HPLC on anion-exchange and hydroxyapatite columns demonstrated that LTA4 synthase activity copurified with the 5-lipoxygenase with similar recoveries and increases in specific activity. Furthermore, the two enzymatic activities coeluted exactly on three different HPLC systems. Maximal activity of purified LTA4 synthase required the addition of three nondialyzable stimulatory factors, two of which were cytosolic and one of which was membrane-bound. These findings were identical for 5-lipoxygenase activity. When incubated with arachidonic acid, the purified 5 lipoxygenase converted approximately equal to 15% of its endogenously generated 5 hydroperoxyicosatetraenoic acid (5-HPETE) to LTA4. LTA4 production was more efficient when the enzyme utilized 5-HPETE generated from arachidonic acid than when 5-HPETE was exogenously supplied as substrate. These findings suggest that a single protein from human leukocytes possesses 5-lipoxygenase and LTA4 synthase activities and that the synthesis of LTA4 from 5-HPETE is controlled by the same complex multicomponent system that regulates the 5-lipoxygenase reaction. PMID- 3006031 TI - Mapping of phosphomonoester and apparent phosphodiester bonds of the oncogene product p53 from simian virus 40-transformed 3T3 cells. AB - The oncogene product p53, isolated from SV3T3 cells where it forms a complex with simian virus 40 large tumor antigen (T antigen) in the nucleus, has been found to be phosphorylated at at least four distinct sites on the 390 amino acid protein. Separation of tryptic phosphopeptides has permitted identification of two sites as Ser-312 and Ser-389, and permitted analysis of the types of phosphate bonds. The peptide containing Ser-312 separates electrophoretically into three charged forms; two are resistant to dephosphorylation by both alkaline phosphatase and alkaline hydrolysis, suggesting a phosphodiester. The carboxyl-terminal phosphopeptide containing Ser-389 was alkaline phosphatase-resistant and liberated four ribonucleoside monophosphates upon base or RNase hydrolysis, suggesting that Ser-389 may be covalently linked to RNA. Phosphorylation of Ser 389 decreased markedly at the nonpermissive temperature in simian virus 40 tsA58 transformed cells, indicating a dependence on native T antigen function and a possible role in transformation by T antigen. Two additional phosphorylation sites, one involving serine and one involving threonine, probably reside in the amino-terminal segment of p53 and appear to be peptide-phosphate monoesters. PMID- 3006032 TI - Structural features of the 5' noncoding region of the rabbit globin messenger RNAs engaged in translation. AB - Accessible sites in the 5' noncoding region of the rabbit alpha- and beta-globin mRNAs were identified and compared in deproteinized RNA and in the mRNAs engaged in translation in the reticulocyte lysate. Preparations of RNA and lysate were subjected to limited nuclease digestion by RNase T1 and Neurospora endonuclease, and the cleavage sites were analyzed by a nuclease S1 mapping procedure. The free alpha-globin mRNA contained few nuclease-sensitive sites and its initiation codon AUG was masked. The free beta-globin mRNA contained a larger number of accessible sites and its AUG was highly exposed. The distribution of sensitive sites differed considerably in the lysate. In both mRNA species, a site near the 5' terminus became the one most accessible to Neurospora endonuclease. Also the accessibility of the AUG in beta-globin mRNA decreased considerably. The distribution of accessible sites in the lysate was the same when the mRNAs were undergoing rapid initiation and when initiation became limited after prolonged incubation. Inhibition of initiation by the cap analogue 7-methylguanosine 5' triphosphate was accompanied by increased sensitivity of some of the sites in both mRNA species. One of the accessible sites in each mRNA species had a sequence complementary to the 3'-terminal portion of the 18S ribosomal RNA. PMID- 3006033 TI - Bacteriophage T4 DNA topoisomerase mediates illegitimate recombination in vitro. AB - We have found that purified T4 DNA topoisomerase promotes recombination between two phage lambda DNA molecules in an in vitro system. In this cross, T4 DNA topoisomerase alone is able to catalyze the recombination and produce a linear monomer recombinant DNA that can be packaged in vitro. ATP is not required for this recombination, while oxolinic acid stimulates it. The recombinant DNA molecules contain duplications or deletions, and the crossovers take place between nonhomologous and nonspecific sequences of lambda DNA. Therefore, the recombination mediated by the T4 DNA topoisomerase is an illegitimate recombination that is similar to that mediated by Escherichia coli DNA gyrase. A model was proposed previously in which DNA gyrase molecules that are bound to DNA associate with each other and lead to the exchange of DNA strands through the exchange of DNA gyrase subunits. This model is also applicable to the recombination mediated by T4 DNA topoisomerase. PMID- 3006034 TI - Purification and characterization of a membrane-associated 3,3',5-triiodo-L thyronine binding protein from a human carcinoma cell line. AB - A membrane-associated binding protein for 3,3',5-triiodo-L-thyronine (T3) was purified to apparent homogeneity from A431 human epidermoid carcinoma cells. A431 cells were specifically labeled with the N-bromoacetyl derivative of T3 labeled with 125I at the 3' position (BrAc[125I]T3) and were extracted with 3-[3 (cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic detergent. The solubilized BrAc[125I]T3-labeled protein was successively purified by chromatography on Sephadex G-200 and QAE-Sephadex followed by NaDod-SO4/PAGE. Approximately 0.2 mg of purified protein was obtained from 2.5 X 10(9) cells, which represents a 3000-fold purification. The membrane-associated T3 binding protein is an acidic protein with a pI of 5.1 and an apparent molecular mass of 55,000 daltons determined by NaDodSO4/PAGE. Polyclonal antibodies against the 55 kDa protein were prepared and used in indirect immunofluorescence to show that the 55-kDa protein was mainly found in the nuclear envelope and endoplasmic reticulum. PMID- 3006035 TI - Are C14-C15 single bond isomerizations of the retinal chromophore involved in the proton-pumping mechanism of bacteriorhodopsin? AB - Resonance Raman spectroscopy is used to examine the possibility that C14-C15 single bond isomerizations of the retinal prosthetic group are involved in the photochemical reactions of bacteriorhodopsin. Normal mode calculations show that the vibration that contains predominantly C14-C15 stretch character is approximately equal to 70 cm-1 lower in frequency in the 14-s-cis conformer than in the s-trans case. This geometric effect is insensitive to out-of-plane twists and should be observed in the sterically hindered 13-cis, 14-s-cis retinal protonated Schiff base, which has been proposed as the chromophore in the K and L intermediates of bacteriorhodopsin. Resonance Raman spectra were obtained of K625 by using the low temperature (77 K) spinning-cell technique. Isotopic substitutions with 13C and 2H show that significant C14-C15 stretch character is observed in normal modes at approximately equal to 1185-1195 cm-1. The relatively high frequency of the C14-C15 stretch argues that K625 contains a 13-cis, 14-s trans chromophore. Similarly, isotopic derivatives show that L550 has a localized C14-C15 stretch at 1172 cm-1, consistent with a 14-s-trans chromophore. These results argue that the primary step in bacteriorhodopsin is a C13=C14 trans--- cis photoisomerization that does not involve C14-C15 s-cis structures. PMID- 3006036 TI - Correlation between hormone dependency and the regulation of epidermal growth factor receptor by tumor promoters in human mammary carcinoma cells. AB - The effects of the tumor promoter phorbol 12-tetradecanoate 13-acetate (TPA) on the epidermal growth factor (EGF) receptor levels were investigated in hormone dependent (MCF-7, T-47-D, and ZR-75-1) and hormone-independent (MDA-MB-231, HBL 100, and BT-20) human mammary carcinoma cell lines. In the absence of TPA, hormone-independent cell lines contained high concentrations of low-affinity EGF receptors (apparent Kd = 8 X 10(-10) M), whereas hormone-dependent cell lines exhibited low concentrations of high-affinity receptors (apparent Kd = 1 X 10( 10) M). TPA causes a change of the receptor from a high- to the low-affinity state in hormone-dependent cell lines (MCF-7, T-47-D, and ZR-75-1), as well as in the hormone-independent HBL-100, whereas the affinity remained unchanged in MDA MB-231 and BT-20 cells. In addition, progesterone receptor levels are decreased after TPA treatment in the hormone-dependent cell lines MCF-7, T-47-D, and ZR-75 1, whereas the estrogen receptor levels remained unchanged. Tumor promoters such as TPA or teleocidin inhibited the proliferation of these cell lines at concentrations above 10 microM with the exception of the T-47-D cells. The most sensitive cell line towards growth inhibition by tumor promoter was the hormone dependent MCF-7 cell line. Evaluation of different TPA analogs indicated a positive correlation between the growth-inhibitory effects and their ability to stimulate the subcellular redistribution of protein kinase C activity in MCF-7 cells. These data suggest a protein kinase C-mediated down-regulation of the progesterone receptor concentration and of the EGF receptor affinity, which is supposed to mediate the mitogenic response. Furthermore, these results support the hypothesis that the tumor-derived growth factors induced by estradiol act via the EGF receptor in hormone-dependent mammary carcinoma cells. PMID- 3006037 TI - Lateral diffusion of specific antibodies bound to lipid monolayers on alkylated substrates. AB - We have measured the lateral mobility of fluoresceinated monoclonal IgG antibodies bound specifically to a spin label lipid hapten in phospholipid monolayers supported on alkylated silicon oxide surfaces. Dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine monolayers containing 5 mol% of the lipid hapten were transferred by conventional Langmuir-Blodgett techniques onto substrates alkylated with hydrocarbon chains containing 10, 16, and 18 carbon atoms. We show that the diffusion of the bound antibodies depends on their lateral density, the composition of the lipid monolayer, and the nature of lipid coupling to hydrocarbon chains on the alkylated substrate. Antibody diffusion coefficients at low antibody densities are within a factor of 2 of those displayed by the lipid hapten in the absence of the bound antibody. High antibody densities result in reduced antibody mobility, but the lateral diffusion of unbound lipids is unaffected. PMID- 3006038 TI - Phosphodiesterase activation by photoexcited rhodopsin is quenched when rhodopsin is phosphorylated and binds the intrinsic 48-kDa protein of rod outer segments. AB - Each photoexcited rhodopsin (R*) molecule catalyzes binding of GTP to many copies of the guanine nucleotide-binding protein transducin, which, in its GTP-binding form, then activates cGMP phosphodiesterase (PDEase). Subsequent deactivation of this light-activated enzyme cascade involves hydrolysis of the GTP bound to transducin, as well as decay of the activating capacity of R*. We report here that deactivation of PDEase in rod outer segment suspensions is highly enhanced by addition of ATP and purified 48-kDa protein, which is an intrinsic rod outer segment protein that is soluble in the dark but binds to photolyzed rhodopsin that has been phosphorylated by rhodopsin kinase and ATP [Kuhn, H., Hall, S.W. & Wilden, U. (1984) FEBS Lett. 176, 473-478]. To analyze the mechanism by which ATP and 48-kDa protein deactivate PDEase, we used an ATP-free system consisting of thoroughly washed disk membranes, whose rhodopsin had been previously phosphorylated and chromophore-regenerated, and to which purified PDEase and transducin were reassociated. Such phosphorylated membranes exhibited a significantly lower (by a factor less than or equal to 5) light-induced PDEase activating capacity than unphosphorylated controls. Addition of purified 48-kDa protein to phosphorylated membranes further suppressed their PDEase-activating capacity; suppression could be as high as 98% (as compared to unphosphorylated membranes), depending on the amount of 48-kDa protein and the flash intensity. Addition of ATP had little further effect. In contrast, PDEase activation or deactivation with unphosphorylated control membranes was not influenced by 48-kDa protein, even in the presence of ATP, provided rhodopsin kinase was absent. Our data suggest that 48-kDa protein binds to phosphorylated R* and thereby quenches its capacity to activate transducin and PDEase. PMID- 3006039 TI - Construction of heterodimer tyrosyl-tRNA synthetase shows tRNATyr interacts with both subunits. AB - The tyrosyl-tRNA synthetase (EC 6.1.1.1) from Bacillus stearothermophilus is a dimer of two identical subunits. The dimer shows "half-of-the-sites" reactivity in that only one molecule of tyrosyladenylate is formed and one molecule of tRNATyr binds per dimer. To identify whether the tRNATyr binds to a single subunit in the dimer, or to both subunits, heterodimers were constructed by mixing two variant dimers together in 8 M urea. As the unfolded protein is electrophoresed into a native polyacrylamide gel, it refolds and reassociates, and heterodimers can be purified from the parental dimers. Kinetic analysis of heterodimers formed between variant enzymes with defective tyrosine activation or tRNA aminoacylation shows that a molecule of tRNATyr interacts with the N terminal region of one subunit and the C-terminal region of the other subunit in the dimer. PMID- 3006040 TI - Direct comparison of Ca2+ requirements for calmodulin interaction with and activation of protein phosphatase. AB - The mechanism of Ca2+-dependent protein-protein interaction and enzyme activation by calmodulin was investigated with the phosphoprotein phosphatase, calcineurin. Dimethylaminonaphthalene (dansyl)-calmodulin, a fluorescent derivative used to monitor complex formation, produced similar maximal activation (10- to 12-fold) with a Ca2+ dependence (Ka = 17 microM) identical to that of native calmodulin. The Ca2+-dependent increase in fluorescence intensity of dansyl-calmodulin was enhanced 100-150% by calcineurin, indicating complex formation; the concentration of Ca2+ required for a half-maximal increase in fluorescence was the same (K1/2 approximately equal to 7 microM) with and without calcineurin. Since the Ca2+ concentration required for activation appeared to differ from that necessary for protein-protein interaction, a method was devised to measure both the formation of complexes between dansyl-calmodulin and calcineurin and enzyme activity in the same samples. Direct comparison of interaction (measured by polarization of fluorescence) and enzyme activity demonstrated different Ca2+ requirements for the two events. Whereas dansyl-calmodulin-calcineurin interaction, measured in the presence of phosphoprotein substrate, exhibited very little cooperativity (Hill coefficient = 1.2, Ca2+ concentration required for the half-maximal increase in fluorescence, K1/2, approximately equal to 6 microM), phosphatase activation was highly cooperative (Hill coefficient = 3.5) and required 3 times higher Ca2+ concentration for half-maximal stimulation. Equivalent results were obtained with p-nitrophenyl phosphate as substrate. These data are consistent with a sequential mechanism for interaction and activation wherein filling of perhaps two Ca2+ sites permits calmodulin interaction with the phosphatase; this complex is inactive, requiring further binding of Ca2+ for activation. Such a scheme would provide a sensitive switch for control of enzyme activity within a narrow range of free Ca2+ concentration. PMID- 3006041 TI - Cloning of the Escherichia coli gene for primosomal protein i: the relationship to dnaT, essential for chromosomal DNA replication. AB - The Escherichia coli gene encoding one of the primosomal proteins, protein i, was cloned by the use of synthetic oligonucleotide probes. Nucleotide sequence analysis revealed a coding region for protein i of 537 base pairs preceded by a possible promoter sequence. The gene is located adjacent to the dnaC locus, probably both being in a single operon. The protein i gene was shown to be closely related to the dnaT locus based on the following observations. (i) A multicopy plasmid carrying only the protein i gene suppresses the temperature sensitive phenotype of a dnaT strain and restores the ability of the strain to carry out stable DNA replication in the absence of protein synthesis. (ii) An extract from a dnaT strain does not support replication of the plasmid pBR322 in vitro; addition of purified protein i restores its activity. These results indicate that protein i is encoded by dnaT and that it is essential for chromosomal DNA replication and is involved in the induction of stable DNA replication during the SOS response. PMID- 3006042 TI - Intracellular activation of protein kinase C and regulation of the surface transferrin receptor by diacylglycerol is a spontaneously reversible process that is associated with rapid formation of phosphatidic acid. AB - The effect of the synthetic diacylglycerol, sn-1,2-dioctanoylglycerol (diC8), on the expression of the surface transferrin receptor reveals that exogenous diC8 can act as an intracellular activator of protein kinase C and stimulate both down regulation and increased receptor phosphorylation in a manner similar to that induced by the active tumor promotor, 4 beta-phorbol 12,13-dibutyrate. Unlike the spontaneously irreversible effect noted when 4 beta-phorbol 12, 13-dibutyrate is added, this same effect mediated by diC8 is brief, lasting only minutes, and is spontaneously reversible. The rate of reversibility is dependent on the concentration of diC8 added, and it is associated with rapid formation of a newly detected intracellular phospholipid that corresponds to sn-1,2-dioctanoyl phosphatidic acid. These data, in conjunction with findings that demonstrate that exogenous diacylglycerols (including diC8) when added to cells do not stimulate cellular phospholipase A2 or C, argue that protein kinase C is activated only briefly in this system since exogenous diC8 is subject to rapid intracellular metabolism to phosphatidic acid. PMID- 3006043 TI - Studies of the radical species in compound ES of cytochrome c peroxidase altered by site-directed mutagenesis. AB - Yeast cytochrome c peroxidase reacts with hydrogen peroxide to form an intermediate, compound ES, in which the heme iron atom is converted to a ferryl function (Fe4+ = O) and a radical center is generated on a reversibly oxidizable amino acid residue of uncertain identity. As methionine-172 is a possible site of this radical, we have constructed specific variants of cytochrome c peroxidase in which methionine-172 is replaced by serine or cysteine. These mutants and the wild-type enzyme have been expressed in Saccharomyces cerevisiae, purified, and crystallized. Both mutant enzymes are fully active. A stable, reversible, peroxide-induced intermediate with optical properties characteristic of compound ES is observed for the three forms of the enzyme. The electron paramagnetic resonance spectrum of this intermediate at 93 K for the serine mutant exhibits the narrow free-radical signal and hyperfine structure observed for the wild-type enzyme. However, a broader component of the signal that is observed for the wild type enzyme at this temperature is absent from the spectrum observed with the serine mutant. These results demonstrate that the narrow component of the free radical signal observed at 93 K cannot reside at methionine-172. The absence of the broader component of the signal for the serine mutant may reflect the loss of spin density on methionine or, alternatively, could arise from conformationally induced changes in the properties of the radical. The results are consistent with a heterogeneity of radical species in the ES complex. PMID- 3006045 TI - Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution. AB - Exotoxin A of Pseudomonas aeruginosa is a secreted bacterial toxin capable of translocating a catalytic domain into mammalian cells and inhibiting protein synthesis by the ADP-ribosylation of cellular elongation factor 2. The protein is a single polypeptide chain of 613 amino acids. The x-ray crystallographic structure of exotoxin A, determined to 3.0-A resolution, shows the following: an amino-terminal domain, composed primarily of antiparallel beta-structure and comprising approximately half of the molecule; a middle domain composed of alpha helices; and a carboxyl-terminal domain comprising approximately one-third of the molecule. The carboxyl-terminal domain is the ADP-ribosyltransferase of the toxin. The other two domains are presumably involved in cell receptor binding and membrane translocation. PMID- 3006044 TI - Gyration is required for 5S RNA transcription from a chromatin template. AB - We have assembled transcriptionally active chromatin on 5S DNA plasmids by using a Xenopus oocyte supernatant and the 5S-specific transcription factor IIIA (TFIIIA). In this system, the 5S RNA gene is accurately transcribed at a rapid rate of 50 transcripts per gene per hr. By following the time course of RNA synthesis during chromatin assembly, the dose response to TFIIIA addition, and the effect of novobiocin on the assembled nucleoprotein, we show that there is a strict correlation between transcriptional activity and the generation of torsionally strained DNA supercoils in "dynamic chromatin." Transcription cannot be the cause of the dynamic structure, because the assembly of this chromatin is unaffected by alpha-amanitin levels that completely block RNA polymerase III. Surprisingly, the dynamic chromatin remains transcriptionally active after relaxation with DNA topoisomerase I, which implies that the essential parameter for chromatin transcription is gyration per se, and not its effect on DNA topology. PMID- 3006046 TI - Isotope-detected 1H NMR studies of proteins: a general strategy for editing interproton nuclear Overhauser effects by heteronuclear decoupling, with application to phage lambda repressor. AB - A strategy for editing interproton nuclear Overhauser effects (NOEs) in proteins is proposed and illustrated. Selective incorporation of 13C- (or 15N)-labeled amino acids into a protein permits NOEs involving the labeled residues to be identified by heteronuclear difference decoupling. Such heteronuclear editing simplifies the NOE difference spectrum and avoids ambiguities due to spin diffusion. Isotope-detected 1H NMR thus opens to study proteins too large for conventional one- and two-dimensional NMR methods (20-75 kDa). We have applied this strategy to the N-terminal domain of phage lambda repressor, a protein of dimer molecular mass 23 kDa. A tertiary NOE from an internal aromatic ring (Phe 51) to a beta-13C-labeled alanine residue (Ala-62) is demonstrated. PMID- 3006047 TI - Direct measurements of intramolecular electron transfer rates between cytochrome c and cytochrome c peroxidase: effects of exothermicity and primary sequence on rate. AB - Rapid mixing of ferrocytochrome c peroxidase [cyt c peroxidase(II)] and ferricytochrome c [cyt c(III)] results in the reduction of cyt c(III) by cyt c peroxidase(II). In 10 mM phosphate, pH 7.0, the rate of decay of cyt c peroxidase(II) and the rate of accumulation of cyt c(II) give equal first-order rate constants: k = 0.23 +/- 0.02 s-1. Equivalent results are obtained by pulse radiolysis using isopropanol radical as the reducing agent. This rate is independent of the initial cyt c(III):cyt c peroxidase(II) ratios. These results are consistent with unimolecular electron transfer occurring within a cyt c(III) cyt c peroxidase(II) complex. When cyt c is replaced by porphyrin cyt c (iron free cyt c), a complex still forms with cyt c peroxidase. On radiolysis, using e aq as the reducing agent, intracomplex electron transfer occurs from the porphyrin cyt c anion radical to cyt c peroxidase(III) with k = 150 s-1. This large rate increase with increasing delta G degrees suggests that the barrier for intracomplex electron transfer is large. Finally, we have briefly investigated how the cyt c peroxidase(II)----cyt c(III) rate depends on the primary structure of cyt c(III). We find the reactivity order to be as follows: yeast (k = 3.4 s-1) greater than horse (k = 0.3 s-1) greater than tuna (k = 0.2 s-1). These results mirror a report [Ho, P. S., Sutoris, C., Liang, N., Margoliash, E. & Hoffman, B. M. (1985) J. Am. Chem. Soc. 107, 1070-1071] on excited state reactions of the cyt c/cyt c peroxidase couple. PMID- 3006048 TI - Erythrocyte-neutrophil interactions: formation of leukotriene B4 by transcellular biosynthesis. AB - Studies on the mechanism of leukotriene B4 biosynthesis in suspensions composed of neutrophils plus erythrocytes indicate that human erythrocytes convert neutrophil-derived leukotriene A4 into leukotriene B4. Leukotriene B4 formation by neutrophils in the presence of erythrocytes exceeded that from corresponding suspensions of neutrophils alone. The increase was proportional to the erythrocyte content of the suspension. The erythrocyte-dependent increase in leukotriene B4 biosynthesis did not equal the arithmetic sum of calcium ionophore dependent biosynthesis by neutrophils plus calcium ionophore-dependent biosynthesis by erythrocytes, since erythrocytes produced no leukotriene B4 upon incubation with ionophore A23187. Erythrocytes did not stimulate 5-lipoxygenase activity within neutrophils, since the erythrocyte effect was confined to enzymatic hydration: leukotriene B4 increased coincident with decreased formation of 5,12-dihydroxyicosatetraenoic acids derived from nonenzymatic hydration. Biosynthesis of leukotriene B4 within the erythrocyte, from neutrophil-derived leukotriene A4, was established by comparing the effect of normal erythrocytes with erythrocytes containing a leukotriene A4 hydrolase that was inactivated by the substrate. In the latter case, leukotriene B4 formation increased by only 30 40%; in the former case, it increased by 100-200%. Transcellular biosynthesis of leukotriene B4 from erythrocyte-neutrophil interactions explains the paradoxical presence of leukotriene A4 hydrolase within erythrocytes, a cell incapable of synthesizing leukotriene A4; affords a mechanism to overcome rate limitations or "suicide inactivation" of leukotriene A4 hydrolase in neutrophils; exploits a cryptic capacity within erythrocytes, provisionally dormant cells in terms of icosanoid biosynthesis; indicates that the biosynthetic capacity of cell combinations is not necessarily equivalent to the sum of their separate capacities. PMID- 3006049 TI - Mutations of the Drosophila myosin heavy-chain gene: effects on transcription, myosin accumulation, and muscle function. AB - Mutations of the myosin heavy-chain (MHC) gene of Drosophila melanogaster were identified among a group of dominant flightless and recessive lethal mutants (map position 2-52, 36A8-B1,2). One mutation is a 0.1-kilobase deletion in the 5' region of the MHC gene and reduces MHC protein in the leg and thoracic muscles of heterozygotes to levels found in 36AC haploids. Three mutations are insertions of 8-to 10-kilobase DNA elements within the MHC gene and produce truncated MHC transcripts. Heterozygotes of these insertional mutations possess levels of MHC intermediate between those of haploids and diploids. An additional mutation has no gross alteration of the MHC gene or its RNA transcripts. Although leg and larval muscles function normally in each mutant heterozygote, indirect flight muscles are defective and possess disorganized myofibrils. Homozygous mutants die during embryonic or larval development and display abnormal muscle function prior to death. These findings provide direct genetic evidence that the MHC gene at 36B (2L) is essential for both larval and adult muscle development and function. The results are consistent with the previous molecular evidence that Drosophila, unlike other organisms, has only a single muscle MHC gene per haploid genome. Quantitative expression of both copies of the MHC gene is required for function of indirect flight muscle, whereas expression of a single MHC gene is sufficient for function of larval muscles and adult tubular muscles. PMID- 3006050 TI - The nerve growth factor receptor gene is at human chromosome region 17q12-17q22, distal to the chromosome 17 breakpoint in acute leukemias. AB - Genomic and cDNA clones for the human nerve growth factor receptor have been used in conjunction with somatic cell hybrid analysis and in situ hybridization to localize the nerve growth factor receptor locus to human chromosome region 17q12 q22. Additionally, part, if not all, of the nerve growth factor receptor locus is present on the translocated portion of 17q (17q21-qter) from a poorly differentiated acute leukemia in which the chromosome 17 breakpoint was indistinguishable cytogenetically from the 17 breakpoint observed in the t(15;17)(q22;q21) translocation associated with acute promyelocytic leukemia. Thus the nerve growth factor receptor locus may be closely distal to the acute promyelocytic leukemia-associated chromosome 17 breakpoint at 17q21. PMID- 3006052 TI - Monoclonal antibodies specific for H-2K and H-2D antigens on cytotoxic T cells can inhibit their function. AB - Antibodies specific for murine major histocompatibility gene complex (MHC) class I H-2K and H-2D molecules present on cytotoxic T (Tc) cells have been shown to inhibit their function of target cell lysis. This could only be demonstrated by using a more sensitive assay for T-cell-mediated lysis, and many monoclonal antibodies of different Ig class, origin, and specificity can be shown to inhibit alloreactive as well as MHC-restricted Tc cells. These antibodies inhibit different activated T-cell populations to varying extents, and anti-H-2K but not anti-H-2D antibodies show a synergistic effect with anti-Lyt-2 antibodies. Data here suggest that MHC molecules may be located in or near the T-cell receptor complex on these cells. PMID- 3006051 TI - Evidence the yeast STE3 gene encodes a receptor for the peptide pheromone a factor: gene sequence and implications for the structure of the presumed receptor. AB - Haploid yeast cells of the a mating type secrete a peptide pheromone, a factor, which acts on cells of the alpha mating type to prepare them for conjugation. We show that the STE3 gene, which is required for mating only by alpha cells and is transcribed only in alpha cells, likely encodes a cell-surface receptor for a factor. This view is based on three findings. First, wild-type Ste3 product is required for response to the pheromone: mutants with any one of five different ste3 mutations are unresponsive to a factor. Second, a hybrid Ste3-beta galactosidase protein encoded by a STE3-lacZ gene fusion fractionates to the particulate fraction of yeast cell extracts, suggesting that Ste3 is a membrane protein. Finally, the DNA sequence of STE3, which we report here, encodes a protein of 470 amino acid residues that contains seven distinct hydrophobic segments of sufficient length to span a lipid bilayer. PMID- 3006054 TI - Autoradiographic comparison of the distribution of the neutral endopeptidase "enkephalinase" and of mu and delta opioid receptors in rat brain. AB - The neutral endopeptidase EC 3.4.24.11, also designated enkephalinase, has been visualized by in vitro autoradiography using the tritiated inhibitor [3H]-N [(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl] glycine, ([3H]HACBO-Gly). Specific binding of [3H]HACBO-Gly (Kd = 0.4 +/- 0.05 nM) corresponding to 85% of the total binding to brain slices was inhibited by 1 microM thiorphan, a selective inhibitor of enkephalinase, but remained unchanged in the presence of captopril, a selective inhibitor of angiotensin-converting enzyme. Very high levels of [3H]HACBO-Gly binding were found in the choroid plexus and the substantia nigra. High levels were present in the caudate putamen, globus pallidus, nucleus accumbens, olfactory tubercle, and in the substantia gelatinosa of the spinal cord. Moderate densities were found in parts of the amygdala, the periaqueductal gray matter, the interpeduncular nucleus, and the molecular layer of the cerebellum. The distribution of enkephalinase was compared to that of mu and delta opioid receptors, selectively labeled with [3H]Tyr-D-Ala-Gly-MePhe glycinol and [3H]Tyr-D-Thr-Gly-Phe-Leu-Thr, respectively. In the caudate putamen, [3H]HACBO-Gly binding overlapped the clustered mu sites but appeared more closely related to the diffusely distributed delta sites. High levels of enkephalinase and mu opioid binding sites were present at the level of the periaqueductal gray matter and in the substantia gelatinosa of the spinal cord, regions where only sparse delta opioid receptors could be detected. The association of enkephalinase with delta and mu opioid receptors in these areas is consistent with the observed role of the enzyme in regulating the effects of opioid peptides in striatal dopamine release and analgesia, respectively. Except for the choroid plexus and the cerebellum, the close similarity observed in numerous rat brain areas between the distribution of enkephalinase and that of mu and/or delta opioid binding sites could account for most of the pharmacological effects elicited by enkephalinase inhibitors. PMID- 3006053 TI - Biogenesis of the platelet receptor for fibrinogen: evidence for separate precursors for glycoproteins IIb and IIIa. AB - Congenital absence of platelet glycoproteins IIb and IIIa (GPIIb and GPIIIa) results in a severe bleeding disorder characterized by defective platelet aggregation and failure of fibrinogen to bind to platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIb and GPIIIa are present as a heterodimeric, noncovalent complex in the platelet plasma membrane and function as the fibrinogen receptor. To characterize synthesis of these two proteins, RNA isolated from a human leukemia cell line that contains GPIIb and GPIIIa was translated in a wheat germ cell-free system. Polyclonal antibodies specific for each protein immunoprecipitated distinct [35S]methionine-labeled precursors, indicating that GPIIb and GPIIIa are translated from separate mRNAs. Moreover, using specific antibodies against either intact unreduced GPIIb or the beta subunit, we obtained evidence for synthesis of a common polypeptide precursor for GPIIb alpha and GPIIb beta. Based on experiments using microsomal membranes, it appears that GPIIb is integrated into the platelet membrane with little or no cytoplasmic component. These results suggest that precursors of GPIIb and GPIIIa may be encoded by separate genes and that each precursor is processed before delivery to the plasma membrane. PMID- 3006055 TI - Immunoreactivity for a calmodulin-dependent protein kinase is selectively increased in macaque striate cortex after monocular deprivation. AB - Immunocytochemical methods were used to localize type II Ca2+/calmodulin dependent protein kinase in the macaque primary visual cortex. Neurons that stain for the kinase include both pyramidal and nonpyramidal cells and they appear to form a subset of cortical neurons. They are densely packed in layers II and IVB, somewhat more sparse in layers III, IVC beta, and VI, and nearly absent in layer V. In normal animals the distribution of kinase-positive cells within each layer is relatively uniform. However, in animals in which one eye is removed 7-14 days before sacrifice or sutured shut for 9 or 11 weeks, the cells in layer IVC beta are divided into alternating lightly and darkly stained bands. Comparison of immunocytochemically stained sections with adjacent sections stained for the mitochondrial enzyme, cytochrome oxidase, reveals that the kinase staining increases in ocular dominance columns originally driven by the removed or closed eye. These findings suggest that either the concentration of type II Ca2+/calmodulin-dependent protein kinase or its accessibility to the antibody probe increases dramatically and selectively in neurons of macaque primary visual cortex that have been deprived of their normal visual input. This may indicate that changing levels of activity in cortical neurons can alter their regulatory machinery. PMID- 3006056 TI - Large introns in the 3' end of the gene for the pro alpha 1 (IV) chain of human basement membrane collagen. AB - Using a recently characterized cDNA clone (HT-21) coding for the pro alpha 1 (IV) chain of human type IV procollagen, we have isolated three clones from a bacterio phage lambda Charon 4A library of human genomic DNA. The intron/exon structure of the pro alpha 1 (IV) genomic clones was analyzed by heteroduplex electron microscopy and nucleotide sequencing. The analysis showed that the introns separating exons 2-9 are large and have a total length of over 12,000 base pairs (bp). Six of seven exons at the 3' end of the gene coded for -Gly-Xaa-Yaa-repeats of the collagenous part of the chain. Five of the -Gly-Xaa-Yaa- coding exons (numbers 5-9) varied in size between 72 bp and 134 bp, and none of them were 54 bp or multiples thereof. A sixth exon (exon 4) was a junction exon containing 71 bp coding for -Gly-Xaa-Yaa- sequences and 142 bp coding for the carboxyl-terminal noncollagenous domain (NC-1). The seventh exon (exon 3, 178 bp) coded for sequences of the NC-1 domain. Five of the six -Gly-Xaa-Yaa- coding exons began with the second base coding for glycine, and only one exon began with a complete glycine codon at the 5' end. The results (i) suggest that the gene for the pro alpha 1(IV) chain of human basement membrane collagen is significantly larger than the genes for fibrillar collagens and (ii) show that it lacks the 54-bp exon repeats characteristic of fibrillar collagen genes. PMID- 3006057 TI - Hepatitis B virus gene function: the precore region targets the core antigen to cellular membranes and causes the secretion of the e antigen. AB - The core gene of the hepatitis B virus genome contains two conserved in-phase initiation codons separated by about 90 nucleotides. This region ("the precore region") encodes largely hydrophobic amino acids. We have expressed the coding sequence of the core gene with or without the precore region by using a simian virus 40-derived vector in heterologous mammalian cells. The results show that the precore region is not required for the expression either of core antigen (cAg) or of a related hepatitis B virus antigen, the e antigen (eAg). However, the precore region causes the cAg to become associated with cytoplasmic membranes, probably the endoplasmic reticulum. Further, the presence of the precore sequence results in the secretion of eAg. Our results suggest that the precore region plays a role in targeting core proteins to the membrane; this may be the direct cause of eAg secretion and also may aid in the interaction of the core and surface antigens in the formation of the viral particle. PMID- 3006058 TI - Protein kinase C phosphorylates topoisomerase II: topoisomerase activation and its possible role in phorbol ester-induced differentiation of HL-60 cells. AB - DNA topoisomerase II from Drosophila was phosphorylated effectively by protein kinase C. With a Km of about 100 nM, the reaction was rapid, occurring at 4 degrees C as well as at 30 degrees C and requiring as little as 0.6 ng of the protein kinase per 170 ng of topoisomerase. About 0.85 mol of phosphate could be incorporated per mol of topoisomerase II, with phosphoserine as the only phospho amino acid produced. The reaction was dependent on Ca2+ and phosphatidylserine and was stimulated by phorbol esters. Calmodulin-dependent protein kinase II, but not cyclic AMP-dependent protein kinase, was also able to phosphorylate the topoisomerase. Phosphorylation of topoisomerase II by protein kinase C resulted in appreciable activation of the topoisomerase, suggesting that it may represent a possible target for the regulation of nuclear events by protein kinase C. This possibility is supported by the finding that the phorbol ester-induced differentiation of HL-60 cells was blocked by the topoisomerase II inhibitors novobiocin and 4'-(9-acridinylamino)methanesulfon-m-anisidide(m-AMSA), but not by the inactive analog o-AMSA. PMID- 3006059 TI - Hepatitis B virus DNA contains a glucocorticoid-responsive element. AB - It has recently been shown that hepatitis B virus (HBV) contains a transcriptional enhancer element. In order to determine whether this enhancer responds to glucocorticoids, a series of derivatives of plasmid pA10CAT2 was constructed containing the HBV enhancer and variable lengths of further upstream sequences. Transient expression of chloramphenicol acetyltransferase (CAT) was determined after introduction of these plasmids into PLC/PRF/5, Hep 3B, Hep G2, HeLa, and mouse L cells. Highest CAT activity was noted in the human hepatocellular carcinoma line PLC/PRF/5, which contains integrated HBV DNA sequences. Dexamethasone augmented CAT expression in all cell lines tested with 40% of maximal induction at 10 nM and maximum stimulation (3- to 8-fold) at 1 microM dexamethasone. Dexamethasone augmentation of CAT expression was observed only when constructs contained HBV DNA sequences residing upstream to map position 735 from the EcoRI site. This indicates that the glucocorticoid responsive region is distinct from the previously defined HBV enhancer sequence located at map position 1080-1234. These studies suggest that HBV DNA contains a glucocorticoid-responsive element, which may mediate expression of HBV genes in infected mammalian cells. PMID- 3006060 TI - In vitro synthesis of the iron-molybdenum cofactor of nitrogenase. AB - Molybdate- and ATP-dependent in vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of nitrogenase requires the protein products of at least the nifB, nifN, and nifE genes. Extracts of FeMo-co-negative mutants of Klebsiella pneumoniae and Azotobacter vinelandii with lesions in different genes can be complemented for FeMo-co synthesis. Both K. pneumoniae and A. vinelandii dinitrogenase (component I) deficient in FeMo-co can be activated by FeMo-co synthesized in vitro. Properties of the partially purified dinitrogenase activated by FeMo-co synthesized in vitro were comparable to those of dinitrogenase from the wild-type organism; e.g., ratios of acetylene- to nitrogen reduction activities, as well as those of acetylene reduction activities to EPR spectrum peak height at g = 3.65, were very similar. A. vinelandii mutants UW45 and CA30 have mutations in a gene functionally equivalent to nifB of K. pneumoniae. PMID- 3006062 TI - DNA binding site for a factor(s) required to initiate simian virus 40 DNA replication. AB - Efficient initiation of DNA replication in the absence of nonspecific DNA repair synthesis was obtained by using a modification of the system developed by J.J. Li and T.J. Kelly [(1984) Proc. Natl. Acad. Sci. USA 81, 6973-6977]. Circular double stranded DNA plasmids replicated in extracts of CV-1 cells only when the plasmids contained the cis-acting origin sequence for simian virus 40 DNA replication (ori) and the extract contained simian virus 40 large tumor antigen. Competition between plasmids containing ori and plasmids carrying deletions in and about ori served to identify a sequence that binds the rate-limiting factor(s) required to initiate DNA replication. The minimum binding site (nucleotides 72-5243) encompassed one-half of the simian virus 40 ori sequence that is required for initiation of replication (ori-core) plus the contiguous sequence on the late gene side of ori-core containing G + C-rich repeats that facilitates initiation (ori-auxiliary). This initiation factor binding site was specific for the simian virus 40 ori region, even though it excluded the high-affinity large tumor antigen DNA binding sites. PMID- 3006061 TI - DNA supercoiling of recombinant plasmids in mammalian cells. AB - We have used chloroquine/agarose gel electrophoresis and a blot-hybridization technique to study the modulation of superhelicity of extrachromosomal DNA in mammalian cells. The high sensitivity of the procedure has allowed us to measure the change in the specific linking difference or superhelical density (sigma) of a plasmid, psvo alpha 1p3d, after its introduction into COS-7 cells by DNA transfection. Because the molecular weight of psvo alpha 1p3d is approximately the same as that of simian virus 40 (SV40) DNA, the latter can be used as a standard for estimating the average linking difference or number of superhelical turns (tau) of psvo alpha 1p3d after separation of the different supercoiled species on chloroquine/agarose gels. It was found that transfection of monkey cells with either fully supercoiled psvo alpha 1p3d isolated from bacteria (tau = -27 +/- 1, sigma congruent to -0.051) or its relaxed form after treatment with DNA topoisomerase I yields psvo alpha 1p3d samples of the same tau and sigma values of -20 +/- 1 and -0.038, respectively. The difference between the tau values of psvo alpha 1p3d and SV40 in COS-7 cells, in which both plasmids undergo rounds of replication, corresponds to an average difference of 5 +/- 1 superhelical turns. Plasmid psvo alpha 1p3d remains at this lower level of superhelicity for at least 72 hr. The distribution in linking numbers of the topoisomers of psvo alpha 1p3d isolated from transfected COS cells is also more heterogeneous than that of SV40 DNA. These results suggest that the regulation of DNA supercoiling and chromatin assembly may be closely associated with specific DNA sequences. The approach presented here should have a wide application in the study of the regulation and functional role(s) of DNA supercoiling of plasmids in mammalian cells. PMID- 3006063 TI - AMP-insensitive fructose bisphosphatase in Escherichia coli and its consequences. AB - Inhibition of fructose bisphosphatase (EC 3.1.3.11) by AMP is thought to be an important control mechanism, and, for the Escherichia coli enzyme, is the only control known. We here report on a mutant E. coli fructose bisphosphatase almost insensitive to this inhibition. The purified enzyme is normal in other respects. A strain with this enzyme instead of the wild-type enzyme grows normally in a variety of media. A strain with a very high level of the wild-type enzyme also grows normally. A strain with the very high level of mutant enzyme, however, does show growth abnormalities, but they are not clearly associated with the AMP insensitivity. PMID- 3006064 TI - ATP inhibits nuclear and mitochondrial type I topoisomerases from human leukemia cells. AB - Type I topoisomerases have been purified from nuclei and mitochondria of human acute lymphoblastic leukemia cells. Both of these ATP-independent enzymes are actually found to be inhibited by ATP at physiologically significant concentrations. Other adenine nucleotides showed varying effects: ADP inhibited only at high concentrations; AMP had no effect on either topoisomerase. Both enzymes were also inhibited by dATP. The importance of the adenine ring structure was confirmed by the lack of an inhibitory effect observed with equivalent levels of GTP, UTP, CTP, or their deoxy counterparts. Assays performed in the presence of nonhydrolyzable analogs of ATP suggest that hydrolysis of ATP does not accompany this enzyme inhibition. This was supported by direct determination of the ATPase activity of the purified enzymes. Type I topoisomerase from calf thymus and HeLa cells were also found to be sensitive to ATP. These results suggest that mammalian type I topoisomerases in general may possess a nucleotide binding site that may be involved in regulation of enzyme activity. PMID- 3006065 TI - Bovine parathyroid cells: cultures maintained for more than 140 population doublings. AB - Primary cultures of bovine parathyroid cells were developed using Coon's modified Ham's F-12 medium containing low (0.3 mM) concentrations of calcium and supplements of bovine hypothalamic extract, bovine pituitary extract, epidermal growth factor, insulin, transferrin, selenous acid, hydrocortisone, triiodothyronine, retinoic acid, and galactose. These cells were cultured serially on serum-coated dishes for 140 population doublings before signs of senescence were detected. The cells were epithelioid and maintained a high degree of differentiation as evidenced by calcium regulation of both growth and secretion and by prostaglandin E1 stimulation of cAMP formation and hormone release. PMID- 3006066 TI - Identification and preferential expression in thymic and bursal lymphocytes of a c-ets oncogene-encoded Mr 54,000 cytoplasmic protein. AB - The avian retrovirus E26 is unique among acute leukemia viruses in its ability to induce transformation of cells belonging to either the myeloid or erythroid lineage. The genome of E26 carries two oncogenes, v-myb and v-ets, that are derived from distinct cellular loci, c-myb and c-ets. We have constructed a plasmid vector that allows expression of part of the coding region of v-ets in a bacterial host. Antisera to the bacterially synthesized ets protein specifically precipitated the E26-encoded P135gag-myb-ets transforming protein. These antisera permitted us to identify a chicken c-ets-encoded protein of Mr 54,000 (P54c-ets) that shares 7 out of 10 of its major [35S]methionine-containing tryptic peptides with the v-ets-encoded domain of P135gag-myb-ets. Unlike P135gag-myb-ets and the Mr 75,000 translation product of c-myb (P75c-myb), which are nuclear proteins, P54c-ets was found to be predominantly cytoplasmic. P54c-ets is expressed at low levels in most cell lines and tissues tested, including bone marrow cells and circulating lymphocytes. P54c-ets, together with a minor but closely related Mr 56,000 protein, was found to be expressed at high levels in chicken thymocytes and bursal lymphocytes. PMID- 3006067 TI - DNA amplification and neoplastic transformation mediated by a herpes simplex DNA fragment containing cell-related sequences. AB - The transforming potential of the herpes simplex virus type 2 (HSV-2) BamHI fragment E (map position 0.533-0.583) encoding the 140-kDa ribonucleotide reductase was assayed by transfection in established Rat-2 cells. Foci of refractile, morphologically distinguishable cells were induced at lower efficiency and after a longer incubation period as compared to the human tumor oncogene EJ-Ha-ras. Focus-derived BamHI fragment E-transformed cell lines formed medium-to-large (0.1-0.25 mm) colonies in soft agar and were tumorigenic in immunocompetent syngeneic rats. Southern blot analysis of normal rat DNA after EcoRI digestion revealed specific DNA segments homologous to HSV-2 BamHI fragment E DNA. In BamHI fragment E-transformed and tumor-derived lines, about 8- to 30 fold amplification was detected in a subset of the specific HSV-related DNA segments. In addition, extrachromosomal DNA was isolated from transformed cells by plasmid rescue and contained the left-hand 70% of HSV-2 Bam HI fragment E fused to rat DNA. These results indicate the presence in normal cells of nonrepetitive DNA segments, related to the transforming HSV-2 fragment, that can be targeted for genetic alterations associated with neoplastic transformation. PMID- 3006068 TI - Norepinephrine-induced alteration in the coupling of alpha 1-adrenergic receptor occupancy to calcium efflux in rabbit aortic smooth muscle cells. AB - To determine whether alpha-adrenergic desensitization of vascular smooth muscle is due to an alteration in alpha 1-adrenergic receptor coupling, we determined the relationship between receptor occupancy and maximal receptor-coupled Ca2+ efflux in cultured rabbit aortic smooth muscle cells (i) under basal conditions as defined by receptor inactivation with phenoxybenzamine and (ii) after 48 hr of exposure to several concentrations of 1-norepinephrine (NE). Neither phenoxybenzamine nor NE exposure caused a change in binding affinity for [3H]prazosin or NE. Maximal [3H]prazosin binding capacity and maximal NE stimulated 45Ca2+ efflux decreased progressively with exposure of incubated cells to increasing concentrations of phenoxybenzamine or NE. An approximately 80% decrease in maximal [3H]prazosin binding capacity caused by either phenoxybenzamine or NE resulted in complete loss of NE-stimulated 45Ca2+ efflux, indicating that under these conditions approximately 20% of alpha 1-adrenergic receptors are not coupled to the Ca2+ efflux. Under basal conditions, the relationship between maximal [3H]prazosin binding capacity and maximal NE stimulated 45Ca2+ efflux was markedly nonlinear, so that a near maximal response could be elicited by occupancy of only approximately 40% of the receptors. In contrast, after a 48-hr incubation of cells with NE, occupancy-response coupling was considerably less efficient, so that even full occupancy of the 35% of receptors that remained after NE exposure resulted in only approximately 20% of maximal NE-stimulated 45Ca2+ efflux. Thus, an alteration in occupancy-response coupling at a step proximal to Ca2+ mobilization and/or influx, rather than a reduction in receptor number, is of primary importance in the process of agonist induced alpha-adrenergic receptor desensitization of vascular smooth muscle cells. PMID- 3006069 TI - Double-strand gap repair results in homologous recombination in mouse L cells. AB - Previous studies have demonstrated that the presence of double-strand breaks or double-strand gaps increases the frequency of homologous recombination between two cotransferred DNAs when they are introduced into cultured mammalian cells. Here we demonstrate that the repair of these double-strand gaps is a major mechanism for homologous recombination between exogenous DNAs. In particular, when a plasmid DNA containing a 104-base-pair (bp) gap in its tk gene (herpes simplex virus gene for thymidine kinase) undergoes recombination in mouse L cells to generate an intact gene, the majority of events result from direct repair of the double-strand gap using a cotransferred DNA as the template. We analyzed the recombination events by comparing the frequency of tk+ colonies, Southern blotting of cloned tk+ cell lines, and cloning recombined functional tk genes by plasmid rescue. In addition, by creating double-strand breaks within or adjacent to heterologous insertions in a mutant tk gene, we estimate that the L cell can generate a double-strand gap of between 152 and 248 bp and then can repair the gap to create a functional tk gene. We conclude that double-strand breaks and double-strand gaps are recombinogenic in transferred plasmid DNAs because they serve as intermediates in homologous recombination by double-strand gap repair, a nonreciprocal exchange of DNA or gene conversion event. PMID- 3006070 TI - DNA modification of a maize transposable element correlates with loss of activity. AB - An unstable allele of the bronze 2 (bz2) locus was isolated from a Robertson's Mutator Zea mays line containing a family of active transposable Mu elements. This mutation is somatically unstable, resulting in numerous revertant purple sectors on a bronze kernel. By following the variegated kernel phenotype through two generations, several lineages have been identified that have a distorted transmission of the mutant phenotype (fewer variegated kernels are produced than expected). Southern blot analysis of Mu elements in these plants demonstrates a correlation between an inhibition of digestion of Mu elements by certain restriction enzymes and the loss of somatic reversion at the mutant allele. The DNA modification can occur in all the Mu elements in a plant within one generation; however, plants have been identified that contain both modified and unmodified elements, suggesting that the modification can occur in a progressive manner. We hypothesize that the DNA modification results in nonfunctional elements. PMID- 3006072 TI - Quantitation of insertion sequence IS10 transposase gene expression by a method generally applicable to any rarely expressed gene. AB - We have found that IS10 transposase is synthesized in tiny amounts, about 0.15 polypeptide chain per cell per generation on average, as judged from the beta galactosidase activity of a single chromosomal copy of a suitable transposase lacZ gene fusion. Enzymatic activity from the fusion gene is a factor of 10 lower in a permeabilized whole cell assay than in cell extracts. Probably, most cells contain fewer than four polypeptide chains, and these chains can assemble into active tetramers only after cell disruption. This interpretation permits formulation of two equations relating enzyme activities to transcription and translation rates, solution of which reveals that the fusion gene is expressed at the average rate of only 0.25 transcript per cell per generation, with an average of only 0.58 translation product per transcript. This methodology is generally applicable to analysis of any gene from which fewer than four polypeptide chains are synthesized per cell per generation. PMID- 3006071 TI - A chimeric plasmid from cDNA clones of poliovirus and coxsackievirus produces a recombinant virus that is temperature-sensitive. AB - We have inserted a 405-nucleotide fragment from the 5' noncoding region of the coxsackievirus B3 genome into an infectious cDNA copy of the poliovirus RNA genome. Transfection of plasmid DNA containing this hybrid genome construct into cultured monkey cells produced infectious virus. Recombinant virus stocks displayed a temperature-sensitive phenotype for growth at 37 degrees C. We found that there is a dramatic reduction in the level of viral proteins and viral RNAs in HeLa cells infected with the recombinant at 37 degrees C compared to that obtained at 33.5 degrees C. Thus, insertion of a portion of the coxsackievirus genome into the poliovirus genome produces a temperature-sensitive recombinant virus. That this substitution occurs in a region of the poliovirus genome that, to date, has not been shown to have any coding function suggests that RNA sequences involved in replicase recognition or ribosome binding may contribute to the temperature-sensitive phenotype of the recombinant virus. PMID- 3006073 TI - Translation of open reading frame E5 of bovine papillomavirus is required for its transforming activity. AB - A series of mutations in open reading frame (ORF) E5 of bovine papillomavirus type 1 has been constructed to determine whether this putative gene is required for in vitro oncogenic transformation by viral DNA. Frameshift mutations at either of two different positions located exclusively in ORF E5 cause a substantial reduction in the ability of the cloned viral DNA to induce the appearance of transformed foci of mouse C127 cells. A genetic mapping experiment with one of the mutants indicates that this characteristic transformation defect is actually due to the constructed mutation in ORF E5. Analysis of 10 different mutants with sequence changes at a single position in the ORF showed that there is an exact correspondence between transformation-competence and the ability of the 3' half of ORF E5 to be correctly translated. The transformation defect of an ORF E5 frameshift mutant can be suppressed by a second mutation that restores the correct reading frame to most of the ORF, but not by one that restores the reading frame near the 3' end of the ORF. These results constitute strong genetic evidence that translation of ORF E5 is required for efficient transformation of mouse C127 cells by bovine papillomavirus DNA. Wild-type ORF E5 has the potential to encode a short hydrophobic protein or polypeptide domain. PMID- 3006074 TI - Information transfer between duplicated chromosomal sequences in mammalian cells involves contiguous regions of DNA. AB - We have investigated the nature of information transfer that appears to occur nonreciprocally between duplicated chromosomal sequences in cultured mouse L cells. We have studied gene conversion between two different defective thymidine kinase genes derived from two closely related strains of type 1 herpes simplex virus and that share a silent restriction site polymorphism. Our results demonstrate that this silent site can be coconverted along with the selected mutant sites. The findings are consistent with a mechanism of gene conversion that involves contiguous blocks of DNA differing in length, position, or both. An additional finding is that the products of coconversion events involving the silent site are unequally recovered although the rates of conversion observed at four different selected sites are similar. PMID- 3006075 TI - Assignment of the gene for the beta subunit of thyroid-stimulating hormone to the short arm of human chromosome 1. AB - The chromosomal locations of the genes for the beta subunit of human thyroid stimulating hormone (TSH) and the glycoprotein hormone alpha subunit have been determined by restriction enzyme analysis of DNA extracted from rodent-human somatic cell hybrids. Human chorionic gonadotropin (CG) alpha-subunit cDNA and a cloned 0.9-kilobase (kb) fragment of the human TSH beta-subunit gene were used as hybridization probes in the analysis of Southern blots of DNA extracted from rodent-human hybrid clones. Analysis of the segregation of 5- and 10-kb EcoRI fragments hybridizing to CG alpha-subunit cDNA confirmed the previous assignment of this gene to chromosome 6. Analysis of the patterns of segregation of a 2.3-kb EcoRI fragment containing human TSH beta-subunit sequences permitted the assignment of the TSH beta-subunit gene to human chromosome 1. The subregional assignment of TSH beta subunit to chromosome 1p22 was made possible by the additional analysis of a set of hybrids containing partially overlapping segments of this chromosome. Human TSH beta subunit is not syntenic with genes encoding the beta subunits of CG, luteinizing hormone, or follicle-stimulating hormone and is assigned to a conserved linkage group that also contains the structural genes for the beta subunit of nerve growth factor (NGFB) and the proto-oncogene N-ras (NRAS). PMID- 3006076 TI - Sodium-23 magnetic resonance imaging of the eye and lens. AB - In order to develop a better understanding of cataract and to evaluate the effectiveness of potential drugs, noninvasive techniques must be devised to detect early metabolic changes. As a prelude to these goals, sodium-23 imaging experiments operating at 29.8 MHz (2.7 teslas) were performed on the bovine eye and lens. A spatially localized transverse relaxation time (T2)-weighted spin density map of the sodium-23 within the lens is presented, with a resolution better than 250 micron. Due to the presence of short-T2 (3 msec) components within the lens, only the use of the planar-integral projection reconstruction (PPR) imaging scheme allowed sufficiently short echo-times (1 msec) to permit sodium-23 signal detection. These noninvasive imaging results show differences in the apparent sodium concentration within the lens that are consistent with separate, invasive measurements of sodium concentration. Separate analysis (with no spatial localization) at 79.4 MHz (7.2 teslas), using a shift reagent (dysprosium) to distinguish extracellular from intracellular sodium, indicates that approximately 62% of the detected sodium-23 signal is intracellular. These results are consistent with observations based on invasive measurements and further support the existence of the pump-leak system and a sodium gradient within the lens. PMID- 3006078 TI - In vivo inhibition of enterocyte metabolism by delta 9-tetrahydrocannabinol. AB - Mice were given 10 to 100 mg/kg by stomach tube of delta 9-tetrahydrocannabinol (THC) in a single dose or for 4 consecutive days. [3H]Thymidine or [3H]glucosamine was given 3 or 24 hr before sacrifice. Enterocytes were isolated, and the incorporation of radioactivity into the acid insoluble fraction was measured. THC significantly inhibits in a dose-related fashion (from 10 to 90%) in vivo enterocyte metabolism. This inhibition is found in all enterocytes whatever their position in the intestinal tract; it is also independent of the state of differentiation of enterocytes. After a single ingestion of THC, crypt cells which synthesize DNA incorporate 37 to 45% less thymidine, and villus cells, which synthesize important amounts of glycoproteins, incorporate 15 to 39% less glucosamine. After 4 days of THC administration, the inhibition of thymidine incorporation is even more significant (up to 88%). PMID- 3006077 TI - Inhibition of the in vitro infectivity and cytopathic effect of human T lymphotrophic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) by 2',3'-dideoxynucleosides. AB - Human T-lymphotropic virus type III (HTLV-III)/lymphadenopathy-associated virus (LAV) is a a newly discovered lymphotropic retrovirus that is cytopathic for helper/inducer T cells in vitro. This virus is the etiologic agent of the acquired immunodeficiency syndrome and related diseases. In the current study, we tested the capacity of purine and pyrimidine nucleoside derivatives to inhibit the infectivity and cytopathic effect of human T-lymphotropic virus type III in vitro. With the ribose moiety of the molecule in a 2',3'-dideoxy configuration, every purine (adenosine, guanosine, and inosine) and pyrimidine (cytidine and thymidine) nucleoside tested suppressed the virus, although the thymidine derivative seemed to have substantially less activity in our system than the others. In general, we observed essentially complete suppression of the virus at doses that were lower by a factor of 10 to 20 than those needed to inhibit the proliferation of the target T cells and the immune reactivity of normal T cells in vitro. An analysis of five adenosine congeners, which differed only in the sugar moiety, revealed that reduction (an absence of hydroxyl determinants) at both the 2' and 3' carbons of the ribose was necessary for an anti-viral effect, and an additional reduction at the 5' carbon nullified the anti-viral activity. These observations may be of value in developing a new class of experimental drugs for the therapy of human T-lymphotropic virus type III infections. PMID- 3006079 TI - Effects of propranolol and indomethacin on muscarinic airway reactivity in unanesthetized guinea pigs. AB - To investigate whether endogenous beta-adrenergic stimulation or cyclooxygenase products normally affect muscarinic reactivity in conscious, spontaneously breathing guinea pigs, we measured specific airway resistance (SRaw) during acetylcholine (ACh) infusion before and after treatment with propranolol (10 mg/kg ip) or indomethacin (30 mg/kg ip). Airway reactivity was assessed by measuring changes in SRaw upon increasing ACh infusion. We found that propranolol treatment increased reactivity to parenteral ACh, but did not change baseline SRaw. Furthermore, propranolol reduced the range in muscarinic reactivity for the group, and it enhanced thr reproducibility of measurements in individual animals. In contrast, indomethacin had no effect on either baseline SRaw or muscarinic reactivity. Our results suggest that beta-blockade of endogenous adrenergic stimulation increases the muscarinic reactivity of guinea pig airways, but does not influence resting airway tone. It appears that propranolol treatment allows a more reproducible assessment of muscarinic reactivity in the guinea pig. In contrast, cyclooxygenase products do not seem to significantly affect baseline airway resistance, reactivity, or reproducibility in the guinea pig. PMID- 3006081 TI - Radiotherapy of small cell lung cancer. An analysis with special reference to autopsy findings. PMID- 3006080 TI - Cell-mediated immune responses to Chlamydia trachomatis in mothers and infants. AB - Cell-mediated immunity to Chlamydia trachomatis was studied in pregnant women with chlamydial infection of the cervix, in infants born vaginally to these women, and in infants presenting with chlamydial conjunctivitis. Uninfected pregnant women and their infants were studied as controls. McCoy cell cultures were used to isolate C. trachomatis from clinical specimens. Cell-mediated immunity was measured by lymphocyte proliferative responses in vitro to stimulation by chlamydial antigens. Chlamydial IgG antibody in serum specimens was detected by a microenzyme-linked immunosorbent assay technique. The mean lymphocyte proliferative responses to chlamydial antigens were greater in infected women than in uninfected women both during pregnancy and in the postpartum period. Lymphocyte responsiveness in infected pregnant women, however, was less than in postpartum women. Despite failure to detect chlamydial infection in exposed infants, lymphocyte proliferative responses were greater in umbilical cord blood and later in peripheral blood samples from neonates born to infected mothers than in infants born to uninfected mothers. These responses were also greater in infants with chlamydial conjunctivitis than in infants of uninfected mothers. These data suggest that cellular immune responses to chlamydial antigens are increased in infected mothers and infants and that infants may acquire chlamydial cell-mediated immunity transplacentally. PMID- 3006082 TI - Structure-activity studies on gonadotropin-releasing hormone in teleosts, amphibians, reptiles and mammals. PMID- 3006083 TI - Fecal mutagens as a function of diet. PMID- 3006084 TI - Urinary excretion of TXB2 after angiotensin converting enzyme inhibition in hypertensive patients. AB - Urinary excretion of thromboxane B2 (TXB2), a stable metabolite of thromboxane A2 (TXA2), was measured by radioimmunoassay in 7 essential hypertensive patients before and after a converting enzyme inhibitor, SQ 14225, administration. When a single oral dose of SQ 14225 (50mg) was given to 7 patients with essential hypertension, urinary excretion of TXB2 was increased significantly (from 58.9 +/ 18.1 to 116.1 +/- 20.7 pg/min, mean +/- SE, P less than 0.02) with simultaneous increase in plasma renin activity, urine volume, urinary sodium, urinary potassium and urinary excretion of PGE (from 58.8 +/- 12.8 to 135.1 +/- 30.0 pg/min, mean +/- SE, P less than 0.05). These results indicate that SQ 14225 stimulates vasoconstricting TXA2 production as well as vasodilating PGE production. PMID- 3006086 TI - Locomotor depression in mice by norcocaine does not involve central alpha 2 adrenergic or presynaptic dopamine receptors. AB - The inhibition of spontaneous locomotor behavior of mice by norcocaine was antagonized neither by the adrenoceptor antagonists yohimbine and phentolamine, nor by the neuroleptics haloperidol and spiperone, at low doses aimed at presynaptic dopamine receptors. In contrast, the antagonists were effective in reducing the hypomotility induced by clonidine and apomorphine, respectively. These results make it unlikely that central alpha 2-adrenergic or presynaptic dopamine receptors are involved in the hypomotive effect of norcocaine. PMID- 3006085 TI - Anticonflict effects of low doses of the dopamine agonist apomorphine in the rat. AB - The effects of low, "autoreceptor" doses (3.13-100 micrograms/kg, SC) of the dopamine (DA) agonist apomorphine were investigated in a modified Vogel's conflict paradigm. The compound was found to exert a marked, dose-dependent increase in the number of shocks taken in the conflict situation (maximum: approximately 230% of control responding, obtained at 12.5 micrograms/kg), thus indicating an anxiolytic action. However, the dose-response curve was biphasic, inversely U-shaped, with the highest dose tried actually suppressing the punished response rate to below control levels. Neither low- nor high-dose apomorphine modified the rats' drinking "motivation" (glucose intake after 48 hr of water deprivation). On the other hand, while unaltered by 12.5 micrograms/kg, the pain threshold tended to be lowered by 100 micrograms/kg. It is suggested that the anxiolytic-like action of apomorphine might be due to central DA autoreceptor stimulation, possibly in limbic/cortical forebrain regions. The conflict promoting effect seen at 100 micrograms/kg is likely related to the concomitantly elicited hyperalgesia. The possibility of developing novel DA-modulating agents for the treatment of anxiety is raised. PMID- 3006088 TI - Relationship of CNS tryptaminergic processes and the action of LSD-like hallucinogens. AB - Tryptamine produces pharmacologic effects in man and the chronic spinal dog which are similar to those produced by LSD, mescaline, psilocin, DMT, DOM and DOB. These effects include tachycardia, tachypnea, mydriasis, hyperreflexia, behavioral changes and in man, hallucinations. Chronic spinal dogs treated chronically with LSD became tolerant to its ability to produce mydriasis, tachycardia, tachypnea and hyperreflexia, and were cross tolerant to the ability of tryptamine, psilocin, mescaline, DMT, DOM and DOB to produce these same effects. Further, it was found that the brain and spinal cord contained tryptamine and could release it. Further tryptamine levels were higher in the brainstem and spinal cord above the level of transection in the chronic spinal dog that in intact dogs, and the same in the spinal cord below the level of transection. These observations suggested that there were both ascending and descending tryptaminergic pathways. Supporting this hypothesis were the observations that L-tryptophan also produced hyperreflexia in the acute, but not the chronic, spinal dog and cat, and that L-tryptophan hyperreflexia was antagonized by alpha-methyldopa but not pCPA. These observations and others argue that the spinal cord and brain have tryptaminergic mechanisms which are distinct from serotoninergic mechanisms, and that LSD-like hallucinogens act in part through a tryptaminergic mechanism. PMID- 3006087 TI - Use of naltrexone as a provocative test for hypothalamic-pituitary hormone function. AB - Naltrexone (50 mg) administration to normal adult women during the early follicular phase of the menstrual cycle (day 1 to day 4 following onset of menstruation) induced a significant elevation in plasma LH, prolactin, ACTH and cortisol levels. Orally administered naltrexone appears to be a safe and effective compound for assessing function of the hypothalamic-anterior pituitary axis in women. PMID- 3006089 TI - Opioid-hallucinogen interactions. AB - Before the advent of neuroleptics, opioids such as morphine were used occasionally in the treatment of schizophrenia and other mental disorders. Recent interest in the possible therapeutic role of endogenous opioid peptides in various mental states has prompted a new look at the opioids. The present paper summarizes the research to date in the author's laboratory on opioid-hallucinogen interactions. A model behavioral state was induced in rats with N,N dimethyltryptamine (DMT) or lysergic acid diethylamide-25 (LSD). Several mu opioid agonists, antagonists, and synthetic enkephalin analogs interacted with DMT and LSD. Adult male Holtzman rats trained on a positive reinforcement fixed ratio four (FR4) behavioral schedule (i.e., a reward of 0.01 ml sugar-sweetened milk was earned on every fourth bar press) were used in these studies. DMT (3.2 and 10.0 mg/kg) given with a 0.9% NaCl pretreatment IP, disrupted established food rewarded FR4 bar pressing behavior in a dose related fashion. Pre-determined behaviorally ineffective doses of mu opioid agonists showed selective biphasic effects against DMT and LSD. Low doses antagonized the effects of both hallucinogens, whereas larger doses enhanced their effects. In contrast to the antagonistic effects of low doses of mu opioid agonists, the mu-kappa opioid antagonist (-)-naloxone enhanced the effects of DMT and LS. (-)-Naloxone enhanced the effects of DMT and LSD. Potentiation of DMT-induced behavioral disruption was attributed to a stereospecific opioid antagonist effect of (-)-naloxone in that the (+)-naloxone enantiomer failed to potentiate the effects of DMT. Further studies are indicated to determine hallucinogen-opioid interactions in various species, including man.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006090 TI - Hallucinogenic drug research--if so, so what? Symposium summary and commentary. AB - The conference brought a critical focus both on older findings and new strategies in hallucinogenic drug research as well as on behavioral and electrophysiologic tools that correlate with neurobehavioral and neurochemical events. A number of unsolved problems to be investigated were noted. The sensitizing effects of both mescaline and LSD on other inputs at motor nuclei or at limbic sites, and effects on afferent processes were examples of important new directions as were drug discrimination tests that can determine the relative roles of aminergic systems in drug effect. Fixed ratio operant schedules can differentiate serotonin blocking agents, detect active indole psychotomimetics and demonstrate temporal and tolerance parameters relevant to drug induced neurochemical changes. Data and critique as well as pharmacokinetics no longer support the presynaptic disinhibition model of LSD effects, nor is LSD induced "slowed turnover" of serotonin seen as an accurate description of neurochemical changes. The distinct nerve ending biochemical changes after LSD are reviewed. Subcellular compartmental analyses require a radical revision of the picture of the ratio in the nerve ending of cytoplasmic and vesicular amine as well as factors normally regulating accessibility of amine to intraneuronal MAO. PMID- 3006091 TI - Calcium antagonists and low density lipoproteins metabolism by human fibroblasts and by human hepatoma cell line HEP G2. AB - The effect of Ca2+ antagonists (CA) on the receptor-mediated low density lipoprotein pathway has been investigated "in vitro" in human skin fibroblasts (HSF) and in human hepatoma cell line Hep G2. The specific binding and internalization of human 125I-labeled LDL are dose-dependently increased in HSF by CA of the verapamil series (verapamil, anipamil, gallopamil, ronipamil, and diltiazem), but neither by CA of the dihydropyridine series (nifedipine, nitrendipine) nor by flunarizine. BAY K 8644, a Ca2+ agonist, elicited an opposite effect. In the presence of the tested CA, LDL degradation is either unaffected (lower concentrations) or inhibited (higher concentrations). 125I-LDL uptake is stimulated also in fibroblasts from type IIa hypercholesterolemic patients, heterozygous for defective expression of LDL receptor. The enhanced cellular uptake of 125I-LDL was prevented by cycloheximide and by alpha-amanitin. CA of the verapamil series including diltiazem retained their effect in human hepatoma cell line Hep G2, a model proposed for hepatic metabolism of LDL. Our studies show that a) CA stimulate the high affinity binding and internalization of LDL in HSF and in human hepatoma cell line Hep G2; b) this stimulation involves DNA transcription and new protein synthesis; c) this effect is specific to one subgroup of Ca2+ antagonists (the verapamil class only). PMID- 3006093 TI - Heterogeneous effect of flavonoids on K+ loss and lipid peroxidation induced by oxygen-free radicals in human red cells. AB - Treatment of fresh erythrocytes with phenazine methosulfate, an intracellular generator of oxygen-free radicals, and diethyldithiocarbamate an inhibitor of superoxide dismutase results in membrane damage consisting in lipid peroxidation and increase in passive K+ permeability. Various flavonoids which have previously been reported to act as oxygen-free radical scavengers were tested on this erythrocyte model. Surprisingly, flavonoids did not exhibit the same effect on the oxygen free radical-stimulated K+ permeability. It was possible to classify these agents into four groups: protective (those decreasing the oxygen-free radical-stimulated K+ permeability): kaempferol, naringenin, apigenin, naringin; toxic (those increasing the deleterious effect of oxygen-free radicals): myricetin, delphinidin, quercetin; biphasic effective (characterized by opposite effects depending on the concentration): phloretin, cyanin, catechin, morin and inactive: rutin, phloridzin. In addition, a similar classification was observed when membrane lipid peroxidation was examined, i.e. kaempferol decreased lipid peroxide formation whereas myricetin enhanced it, morin exhibited a biphasic effect and rutin has no effect. The previously reported metal chelating effect of flavonoids could not totally explain the protective effect of kaempferol as was demonstrated by the partial protective effect exhibited by desferrioxamine. Moreover, this study suggests that a generation of oxygen-free radicals in red cells induced a K+ loss which probably results from membrane lipid peroxidation. PMID- 3006092 TI - Converting enzyme activity in sow oviducts at different stages of the sex cycle. Influence of inhibitors of prostaglandin synthesis and its possible role in the inotropic effects of bradykinin. AB - The existence of angiotensin I converting enzyme (CE) activity in isolated sow Fallopian tubes and the "in vitro" influences of exogenous bradykinin (BK) on the spontaneous motility of sow oviducts, were explored. Independently of the stage of the sex cycle, the ampullary region of sow Fallopian tubes presented a higher basal CE activity than the isthmic one. On the other hand a greater CE activity was found in the isthmus at estrus and metestrus than at proestrus. Acetylsalicylic acid and mefenamic acid, two inhibitors of prostaglandin synthesis, reduced the CE activity only in ampullary segments obtained at metestrus. BK (0.06 microgram.ml-1) stimulates the spontaneous contractions of isthmic and ampullary tubal segments, triggering tonic responses. The positive inotropism had a longer duration in the isthmus than in the ampulla; presented at estrus a higher magnitude than at proestrus and was enhanced by captopril (50 microgram.ml-1). The data suggest that the low CE activity found in the isthmic tubal region could be subserving the positive inotropic action of BK in the isthmus, long lasting in comparison to that evoked in the ampulla. Moreover, at metestrus some tissue eicosanoid might be modulating the CE activity of ampullary tubal segments. Alternatively the foregoing results could be explained assuming the presence of a carboxy-peptidase different than CE in one region of the oviducts and not in the other, i.e., either one with different sensitivity towards sex hormones and prostaglandin synthesis blockers. PMID- 3006095 TI - Antiviral effect of harmine, a photoactive beta-carboline alkaloid. PMID- 3006094 TI - Norepinephrine and the antibody response. PMID- 3006096 TI - Investigation of the antiviral action of the photoactive compound phenylheptatriyne. PMID- 3006097 TI - The effects of ultraviolet light on host cell reactivation and plaque size of herpes simplex virus type I in C3H/10T1/2 mouse cells. PMID- 3006099 TI - The nucleotide sequences of the replication origins of plasmids ColA and ColD. AB - The nucleotide sequences of the replication origin regions of plasmids ColA-CA31 and ColD-CA23 were obtained. Analysis of the nucleotide sequences showed a high degree of homology of these regions with the plasmid ColE1 region responsible for its autonomous replication. In the ColA and ColD regions involved in the regulation of replication, sites have been revealed identical to those participating in the transcription initiation of the ColE1 plasmid RNA I and primer RNA. The presumed RNA polymerase binding sites and the RNA polymerase recognition sequences are identical in ColA, ColD, and ColE1 plasmids. In spite of the differences in the nucleotide sequences, RNA I and the preprimer RNA of ColA and ColD may form structures analogous to the respective structures of ColE1. PMID- 3006098 TI - Deficits in the control of food intake after hypothalamic paraventricular nucleus lesions. AB - Noradrenergic mechanisms of the hypothalamic paraventricular nucleus (PVN) have been shown to play an important role in the stimulation of feeding To determine the influence of this nucleus in monitoring and controlling responses to physiological and pharmacological challenges, PVN electrolytic lesion rats were tested for their behavioral responsiveness to agents known to affect the alpha-2 noradrenergic system as well as release of corticosterone, and to short- and long term periods of food deprivation. Discrete lesions of the PVN produced enhanced feeding, particularly of carbohydrate, in freely-feeding rats maintained on a macronutrient self-selection paradigm. Lesion rats demonstrated a behavioral deficit in food intake regulation (a decrease in carbohydrate ingestion) in response to 5-hr and 24-hr fasts, showed a disturbance in circadian feeding, and exhibited a dramatic decrease in circulating corticosterone. However, feeding in response to 2-deoxy-D-glucose and insulin remained intact, suggesting that noradrenergic receptors within the PVN are not involved in the mediation of glucoprivic-induced feeding. PMID- 3006100 TI - Self-cloning in the cyanobacterium Anacystis nidulans R2: fate of a cloned gene after reintroduction. AB - Functional analysis of cloned genes often makes use of complementation after introducing these genes into cells of a mutant strain. Problems with this self cloning step in the cyanobacterium Anacystis nidulans R2 have been encountered, which were mainly due to recombinational instability of gene and vector after transformation. Therefore, conditions determining the exchange of material between chromosome, insert and plasmids were studied to achieve the necessary stability. The fate of plasmid pME1, containing a wild-type methionine gene from A. nidulans R2, was investigated after its introduction into a Tn901-induced methionine mutant strain as recipient, so that the mutant chromosomal gene could be distinguished from the plasmid-borne wild-type copy. Two different recipients were constructed, one containing and one lacking the resident plasmid pCH1, which is a derivative of the indigenous small plasmid pUH24. When using the pCH1-free strain and with combined selection for both wild-type gene and vector, the original configuration of the genes in chromosome and vector was retained in the majority of the transformed cells, while the remaining transformants were reciprocal recombinants; under conditions of single selection mainly nonreciprocal recombination or loss of the vector was observed. When the recipient strain contained pCH1 additional recombinational events took place. The results show that under appropriate conditions a chromosomal gene cloned on a plasmid vector can be stably maintained in a majority of the transformants, thus making self-cloning experiments feasible in A. nidulans R2. On the other hand, the introduction of foreign DNA into the chromosome can be achieved by deliberately exploiting recombination between chromosome and plasmid. PMID- 3006101 TI - Polypeptides encoded by cryptic plasmids from Neisseria gonorrhoeae. AB - Almost all clinical isolates of Neisseria gonorrhoeae harbor a small, phenotypically cryptic plasmid of approximately 4.1 kb. In this study several polypeptides encoded by two variants of such plasmids, one (pSB01C) having a deletion of approximately 50 bp as compared to the other (p31788C), have been identified, and the position of the genes for two of the proteins determined. The cryptic plasmids were cloned into the HindIII site of the vectors pBR322 and pACYC184. The resulting recombinant plasmids were transformed into the Escherichia coli minicell producing strain DS410 (minA, minB) and the plasmid encoded proteins analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pSB01C derivatives express two distinct proteins of 22 and 16 kDa and p31788C two other proteins of 24 and 18.5 kDa. Additionally, both plasmids express common proteins of 32.5, 9, and 7.5 kDa. The genes coding for the 24- and the 7.5 kDa proteins have been mapped by restriction enzyme analysis of Tn5 insertions suppressing the expression. The additional 50 bp in p31788C are localized to the coding region of the 24-kDa protein, and the 22-kDa protein of pSB01C is possibly a shortened form of the former due to the lacking 50 bp. PMID- 3006102 TI - The EcoR124 and EcoR124/3 restriction and modification systems: cloning the genes. AB - The Escherichia coli plasmid R124 codes for a type I restriction and modification system EcoR124 and carries genetic information, most probably in the form of a "silent copy," for the expression of a different R-M specificity R124/3. Characteristic DNA rearrangements have been shown to accompany the switch in specificity from R124 to R124/3 and vice versa. We have cloned a 14.2-kb HindIII fragment from R124 and shown that it contains the hsdR, hsdM, and hsdS genes which code for the EcoR124 R-M system. An equivalent fragment from the plasmid R124/3 following the switch in R-M specificity has also been cloned and shown to contain the genes coding for the EcoR124/3 R-M system. These fragments, however, lack a component present on the wild-type plasmid essential for the switch in specificity. Restriction fragment maps and preliminary heteroduplex analysis indicate the near identity of the genes that encode the two different DNA recognition specificities. Transposon mutagenesis was used to locate the positions of the hsdR, hsdM, and hsdS genes on the cloned fragments in conjunction with complementation tests for gene function. Indirect evidence indicates that hsdR is expressed from its own promoter and that hsdM and hsdS are expressed from a single promoter, unidirectionally. PMID- 3006103 TI - Segregational instability of pUB110-derived recombinant plasmids in Bacillus subtilis. AB - To study plasmid instability in Bacillus subtilis the pUB110-derived hybrid plasmid pLB2 (3.6 kb) and the bifunctional replicon pLB5 (5.9 kb), able to replicate in B. subtilis and Escherichia coli, were constructed. In both vectors homologous B. subtilis, or heterologous E. coli DNA fragments of various lengths were inserted. Irrespective of the source of the cloned DNA, the segregational stability of the recombinant plasmids in B. subtilis was severely affected by the DNA inserts. In contrast, no instability was observed in E. coli. In B. subtilis a steep inverse relationship existed between the size of the inserts and the level of stability. Increased size of the pLB plasmids resulted in strongly reduced copy numbers. This seems to be the primary cause of the size-dependent segregational instability. PMID- 3006104 TI - DNA segments of the IncX plasmid R485 determining replication and incompatibility with plasmid R6K. AB - The expression of incompatibility properties between the IncX plasmids R6K and R485 of Escherichia coli was examined. For small autonomously replicating derivatives of both plasmid elements, the requirements for incompatibility expression include a functional R485 replicon and an active R6K beta-origin region. Functional R6K alpha and gamma origins are not directly involved in incompatibility expression between R6K and R485. A trans-acting replication system was constructed for plasmid R485. It consists of a 3.2-(kb) DNA fragment of R485 that specifies a product(s) in trans which supports replication from an R485 origin plasmid. A minimal R485 origin region of 591 bp was derived utilizing this trans-acting replication system and the nucleotide sequence of this origin region determined. The most striking feature of the sequence is the presence of six tandem 22-bp nucleotide sequence direct repeats. PMID- 3006106 TI - A restriction map of IncFIV plasmid R124. AB - A physical and genetic map of the 125.7-kb IncFIV plasmid R124 was constructed using the restriction enzymes Sal1 and EcoR1. Two discrete regions involved in plasmid replication were identified on the plasmid genome. One region was located on a 4.66-kb segment of an EcoR1 fragment at map coordinates 73.87 to 78.53 kb. Another was located within an 8.05-kb segment of an EcoR1 fragment at map coordinates 113.40 to 121.45. This region was very unstable but, when ligated to the 3.21-kb EcoR1 fragment E13 located at map coordinates 18.83 to 22.06 kb, replication was stable. Thus, at least three regions of R124 widely separated around the genome are associated with plasmid replication and stable maintenance. Each of these three regions expressed incompatibility with R124. The Tc resistance gene of R124 was located on the contiguous EcoR1 fragments E8 and E12 located at map coordinates 100.49 to 113.40. PMID- 3006105 TI - Saccharomyces cerevisiae ARS on a plasmid from Staphylococcus aureus. AB - Staphylococcus aureus plasmid pC194 carries three sequences closely related to a consensus sequence defined previously by analysis of different genetic elements which replicate autonomously in yeast Saccharomyces cerevisiae. Two of these enable the plasmid to replicate in yeast, the third does not. A new consensus sequence A/T T T T A T R T T T, 1 bp shorter than the previous one, can be deduced from our results. Replacement of the T with G at the position 9 of the sequence abolishes its activity. The presence of the two active sequences on pC194 genome can be explained by the A + T-rich base composition of the plasmid. PMID- 3006107 TI - Genetic analysis of promoters on the insertion sequence IS21 of plasmid R68.45. AB - Tandem duplication of a 2.1-kb DNA sequence on R68 leads to the active insertion element IS21 on the enhanced chromosome mobilizing plasmid R68.45. The HindIII/SalI fragment which carries the single copy or the tandem duplication of IS21 was cloned from R68 and R68.45, respectively, into the multicopy plasmid pED815. Promoters on the two HindIII/SalI fragments were subsequently identified by cloning Sau3A fragments into the BglII site of the promoter cloning vector pGA46. Three promoters were identified on the HindIII/SalI fragment derived from R68 or R68.45, two of them mapped on Sau3A fragments of 214 bp and 82 bp, respectively, on IS21. The promoter on the 82 bp Sau3A fragment which maps at the SmaI site close to the left end of IS21 reads inward. The Sau3A fragment of 214 bp contains the left end of IS21 and transcription from its promoter proceeds outward. In R68.45, readthrough from this preexisting promoter located near the junction of the tandem copies of IS21 proceeds from the right-hand copy into the left, opposing the reading direction of the promoter mapped at the SmaI site of IS21. The expression of genes on one copy of IS21 by readthrough from a promoter on the other one is a possible explanation for the transpositional activity of the tandem configuration of IS21. The similarity of IS21 to other insertion sequences and especially to "mobile promoters" is discussed. PMID- 3006108 TI - Forskolin: from an ayurvedic remedy to a modern agent. PMID- 3006109 TI - Monoclonal antibodies to pseudorabies virus produced by mouse hybridomas. AB - Monoclonal antibodies to pseudorabies viral capsid protein were prepared by fusing Sp2/0-Ag14 myeloma cells with pseudorabies virus (PrV)-primed Balb/c splenocytes. These monoclonal antibodies were specific for PrV. No crossreaction with Herpes Simplex Virus (HSV, type I) or infectious bovine rhinotrachitis virus (IBR) was found. The reaction between these monoclonal antibodies and PrV was demonstrated by immunofluorescent staining technique and enzyme-linked immunoblotting assay. These antibodies could be a useful diagnostic reagent for PrV as well as a good tool for the study of the capsid protein of PrV. PMID- 3006110 TI - Effects of chlordiazepoxide training dose on the mixed agonist-antagonist properties of benzodiazepine receptor antagonist Ro 15-1788, in a drug discrimination procedure. AB - In experiment 1, rats (n = 12) were trained to discriminate the benzodiazepine (BDZ) compound chlordiazepoxide (CDP, 20 mg/kg, IP) from saline in a two-lever food-reinforced procedure, and subsequently were tested for stimulus control with different doses of CDP, Ro 15-1788 (a proposed BDZ receptor antagonist) and Ro 15 1788 plus 20 mg/kg CDP. Ro 15-1788 (0.63-40 mg/kg) dose-dependently antagonized CDP, and induced predominantly saline appropriate responding when administered alone. Thereafter, the same rats were retrained by progressively decreasing the training dose, to discriminate 2.5 mg/kg CDP from saline, and were tested again with the same compounds. Ro 15-1788 (0.16-40 mg/kg) now failed to antagonize CDP (2.5 mg/kg) and increased the percentage of drug-appropriate responding in a dose related manner when administered alone. In experiment 2, separate groups of rats (n = 10) were similarly trained to discriminate either 15 or 3 mg/kg CDP from saline. Tests with CDP, Ro 15-1788 and Ro 15-1788 plus CDP (either 15 or 3 mg/kg) yielded similar results to experiment 1, suggesting that the training dose effects on generalization and antagonism of Ro 15-1788 were not affected by the manner in which the lower CDP dose acquired drug stimulus control. It is concluded that mixed agonist-antagonist properties are apparent after variations of the BDZ training dose in a drug discrimination procedure. PMID- 3006111 TI - Effects of central dopamine depletion on the d-amphetamine discriminative stimulus in rats. AB - The discriminative stimulus properties of d-amphetamine, cocaine, and apomorphine were assessed in rats trained in a two-lever, food-reinforced drug discrimination paradigm to discriminate 1.0 mg/kg d-amphetamine from saline. After determination of these dose-response relationships, the rats were divided into two groups. One group (6-OHDA) was given injections of desmethylimipramine (DMI, 30 mg/kg, IP) and 6-hydroxydopamine (6-OHDA, 100 micrograms/10 microliters each ventricle), while the other group (sham) was given the same dose of DMI and 6-OHDA vehicle (intraventricularly). Beginning approximately 45 days after intraventricular injections, dose-response relationships were redetermined for all three drugs. Larger doses of d-amphetamine were required to elicit the same response in the 6 OHDA group (i.e. the dose-response relationship was shifted to the right), while no change was observed in the sham group. Any changes in the dose-response relationships for cocaine and apomorphine were comparable in the 6-OHDA and sham group. The rate-decreasing effects were not altered in either group for any of the drugs. Upon sacrifice, dopamine (DA) was found to be significantly depleted in the accumbens, caudate and rest of brain of the 6-OHDA group. Levels of norepinephrine and serotonin were unaltered. These data suggest that central DA containing neurons play a role in the discriminative stimulus properties of psychomotor stimulants in rats. PMID- 3006112 TI - Effects of lithium and rubidium on shock-induced changes in open-field activity. AB - Lithium chloride and rubidium chloride were tested under conditions in which the effects of their chronic administration on aversively-controlled behavior could be assessed. Lithium attenuated shock-induced suppression of open-field activity when that suppression was under the control of mild or moderate stimulus parameters, but had no effect on the suppression produced by the presence of shock itself. Rubidium, on the other hand, increased shock-induced suppression under all conditions. When shock was removed and extinction of the activity suppression was investigated, lithium subjects failed to return to their original baseline activity levels, while subjects receiving rubidium recovered baselines in a manner indistinguishable from that observed in control animals. PMID- 3006113 TI - Effects of antidepressant drugs, selective noradrenaline-or 5-hydroxytryptamine uptake inhibitors, on apomorphine-induced hypothermia in mice. AB - Antidepressant drugs which are selective noradrenaline (NA) uptake inhibitors (desipramine, maprotiline, oxaprotiline, talsupram: 0.625-10 mg/kg) antagonized dose-dependently hypothermia induced by 16 mg/kg apomorphine (APO) in mice. Of the two stereoisomers of oxaprotiline, only that inhibiting NA uptake was active. Antidepressants which are selective 5-hydroxytryptamine (5-HT) uptake inhibitors (citalopram, fluvoxamine: 2.5-40 mg/kg) did not affect APO (16 mg/kg)-induced hypothermia. Neither NA nor 5-HT uptake inhibitors counteracted hypothermia induced by 1 mg/kg APO, a dose which is easily antagonized by low doses of dopamine receptor blockers. The antagonistic action of desipramine towards APO (16 mg/kg)-induced hypothermia was prevented by phenoxybenzamine, prazosin and ( )-propranolol, while (+)-propranolol and cyproheptadine were inactive. St. 587 (an alpha 1-adrenoceptor agonist) or salbutamol (an agonist of beta adrenoceptors) attenuated APO (16 mg/kg)-induced hypothermia: given jointly, the drugs completely reversed it. m-CPP, a 5-HT receptor agonist, did not affect APO (16 mg/kg)-induced hypothermia. In conclusion, the antagonistic action of antidepressant drugs towards APO (16 mg/kg)-induced hypothermia in mice did not reflect their "antidepressant properties", dopamine antagonism or their action on 5-HT receptors, only their effects on the NA uptake and/or NA transmission. Both alpha 1 and beta-adrenoceptors are involved in this antagonistic action. PMID- 3006115 TI - Natural history of acromegalic peripheral neuropathy. AB - Eleven sural nerve biopsies from nine acromegalic patients, with and without peripheral neuropathy, were examined utilizing routine sections, single teased fibres and electron microscopy. The initial basic pathological lesion in both groups consisted of demyelination combined with hypertrophic formations affecting the Schwann cell system of the small diameter fibres. Second biopsies from two patients with neuropathy demonstrate the natural history of the lesion. The progression to eventual end-stage neuropathy is documented and is due to the marked onion bulb formation. Recovery is unlikely. Our findings indicate that the development of peripheral neuropathy in acromegaly is a serious complication. Deterioration, both clinically and pathologically, can be expected if the growth hormone remains persistently elevated. PMID- 3006114 TI - The kinetics of D3-3H metabolism in tropical sprue. AB - The metabolism of vitamin D3-3H was studied in a small group of controls and subjects with tropical sprue after the oral or intravenous administration of 8 to 10 microCi of D3-3H. The biological half life of D3-3H upon the administration of the isotope by the intravenous route was normal in 2 controls, very low in a subject with tropical sprue who had steatorrhea, and decreased in a subject with tropical sprue who did not present steatorrhea. After the administration of the isotope by the oral route, the biological half life was 35 hours in the control and no radioactivity could be detected in the plasma of the subject with tropical sprue who had steatorrhea. Twenty four hours after the intravenous dose the percentage of radioactivity in the plasma as HCC-3H was two times higher in the tropical sprue subjects than in the controls. When the dose was given orally the net absorption was 50.5% in the subject with tropical sprue and steatorrhea and 86.8% in the subject with tropical sprue who was partially treated. These results showed rapid clearance of the D3-3H in the subject with tropical sprue and steatorrhea indicating depletion of vitamin D stores in the tissues and decrease in the net absorption of the dose when given orally. The presence of a higher percentage of the dose in the plasma as HCC-3H after the intravenous and oral administrations in the tropical sprue subjects when compared to controls indicates that the diseased state does not alter vitamin D3 metabolism. PMID- 3006116 TI - Amino acid sequence, haem-iron co-ordination geometry and functional properties of mitochondrial and bacterial c-type cytochromes. PMID- 3006117 TI - [Radiation-induced free radicals in DNA, DNA level in tissues and tissue radiosensitivity]. AB - Radiochemical yields (G-values) of H-adducts of thymine bases of DNA (TH) in frozen (77 K) gamma-irradiated mouse and rat tissues were measured. The content of DNA and the number of TH radicals formed in DNA mass of 10(12)D at a dose of 1 Gy (beta parameter) were determined for each of the studied tissue. It was shown that beta parameter, which indicated DNA in situ radiosensitivity, was different for different tissues: it was higher for radiosensitive tissues. The possible causes of the effect observed are discussed. PMID- 3006118 TI - [Paramagnetic centers formed in mouse blood gamma-irradiated at 77K]. AB - The method of low-temperature ESR-spectroscopy was used to study the primary products of radiation injury of mouse blood. It was shown that when blood samples were exposed to gamma-radiation at 77 K radicals of water, lipids and proteins and also oxyhemoglobin adducts (Fe3+--O2(2-)) were formed. It was shown that oxyhemoglobin electron adducts were protonated during stepwise annealing to form complexes (Fe3+--HO2-). High spin methemoglobin is probably the final product of transformations of the oxyhemoglobin complexes. PMID- 3006119 TI - [Diagnostic problems of alveolar cell carcinoma]. AB - The clinical and radiological symptoms and differential diagnosis of four out of seven cases of alveolar cell carcinomas of the lung, which were observed during the last years, have been compared. Some characteristics are shown, which make the differentiation possible between tuberculosis, sarcoidosis, chronic carcinogenic pneumonia and interstitial lung diseases. Early diagnosis by histological investigation of cells from sputum or from bronchial secretion and by needle biopsy seems possible. During the earliest stage alveolar cell carcinoma can be cured by surgery. PMID- 3006120 TI - [The solitary alveolar cell carcinoma]. AB - Alveolar-cell carcinoma was first described by Malassez in 1876. It contributes 5 per cent to all malignant tumors of the lung. In a retrospective study 75 patients with alveolar-cell carcinoma were analyzed. Clinical symptoms are non specific and the tumor will often be discovered on the occasion of a routine chest X-ray as a small peripheral and infiltrative lesion. The most frequent form is a nodule which generally shows a regular and well defined outline, but may sometimes be irregular and have spiky margins too. Very often an air bronchogram and the tail sign can be seen so the combination seems typical of solitary alveolar-cell carcinoma. In the case material investigated the authors found it in 36 per cent. The early stage of localized alveolar-cell carcinoma has a good prognosis when treated by surgery. Multilocular and advanced cases, however, have a very poor prognosis. Therefore, early radiological diagnosis is most important. The value of needle aspiration biopsy in the diagnosis of alveolar-cell carcinoma is emphasized. PMID- 3006121 TI - Temporal bone region: high-resolution MR imaging using surface coils. AB - Specially designed surface coils for the region of the temporal bone enable high resolution magnetic resonance (MR) imaging of the structures of the inner ear. Eight healthy volunteers and 21 patients (six with cholesteatomas, five with acoustic neuromas, five with glomus tumors, and five with mastoiditis) were examined using a 0.5-T MR imager. The demarcation of tumor extent with MR imaging was better than with computed tomography because of improved soft-tissue contrast and because the surrounding bony tissue did not generate any signal. High resolution MR imaging is particularly useful for small acoustic neuromas because of its higher specificity compared with gas cisternography. PMID- 3006122 TI - Cholangiocarcinoma: imaging by MR. AB - Magnetic resonance (MR) images and computed tomographic (CT) scans of nine patients with histologically proved cholangiocarcinoma were compared retrospectively to assess the potential of MR imaging in the detection and staging of the disease. Cholangiocarcinomas were demonstrated as soft-tissue masses by both techniques in seven of the nine patients. In three patients, the masses were more apparent with MR because of a greater degree of contrast between the tumor and the surrounding tissues. In all four patients with the scirrhous subtype of cholangiocarcinoma, the soft-tissue masses showed decreased signal intensity on the second spin-echo image (echo time = 56 msec). Displacement or encasement of the adjacent vessels was well demonstrated by MR. Distal extension of the tumor (hepatic metastases, regional lymphadenopathy) appeared on both MR images and CT scans but was more apparent with MR. Both MR and CT demonstrated intrahepatic bile duct dilatation, but CT demonstrated it more readily. MR appears to be an effective modality for the detection and staging of cholangiocarcinoma. PMID- 3006123 TI - [Is prevention without qualified practitioners workable?]. PMID- 3006124 TI - [Jacket crown technics. 2]. PMID- 3006125 TI - [The Maryland bridge]. PMID- 3006126 TI - [Inner temporomandibular joint diseases and their significance in correct diagnosis. 1]. PMID- 3006127 TI - [The significance of intra-articular soft tissue changes in the diagnosis of temporomandibular joint diseases. 2]. PMID- 3006128 TI - [Materials for gold foil fillings. Structure and indications]. PMID- 3006129 TI - [Restoration of small defects]. PMID- 3006130 TI - [Matrix technic. 1]. PMID- 3006131 TI - [Newest results of preventive dentistry in Sweden]. PMID- 3006132 TI - [New uses for resin-bonded anchors of etched metal]. PMID- 3006133 TI - [Matrix technic. 2]. PMID- 3006134 TI - [Jacket crown technics. 3]. PMID- 3006135 TI - [Care of the patient after periodontitis therapy]. PMID- 3006136 TI - [A new mini-microfilled composite resin]. PMID- 3006137 TI - [Inner temporomandibular joint diseases and their significance in correct diagnosis. 2]. PMID- 3006138 TI - [Occlusal adjustment: a critical evaluation]. PMID- 3006139 TI - [Diagnosis, differential diagnosis and therapy of acute temporomandibular joint inflammation]. PMID- 3006140 TI - [Amalgam filling: transfer and polishing]. PMID- 3006141 TI - [Through the eye of the scanning electron microscope]. PMID- 3006142 TI - [Diagnostic microbiology and periodontal diseases: theory and practice]. PMID- 3006143 TI - [Jacket crown technic. 4]. PMID- 3006144 TI - [Inner temporomandibular joint diseases. 3. Diagnosis of orofacial pain syndromes]. PMID- 3006145 TI - [Condensation, carving and polishing amalgam fillings]. PMID- 3006147 TI - [Instruments and technics for oral hygiene]. PMID- 3006146 TI - [Inner temporomandibular joint diseases. 4. Therapy of orofacial pain syndromes]. PMID- 3006148 TI - [Mastication and treatment objectives--how many teeth does a person need?]. PMID- 3006149 TI - [Composites in restorative dentistry. Status of technics 1985]. PMID- 3006150 TI - [5-year clinical study of UV-cured composites in the posterior region]. PMID- 3006151 TI - [Inner temporomandibular joint diseases. 5. Therapy of orofacial pain syndromes]. PMID- 3006152 TI - [Precision casting for split bridges and combined dentures. The Krupp pouring technic]. PMID- 3006153 TI - [The Kuwata technic. 1]. PMID- 3006154 TI - Leydig cell function in the pubertal rat following local testicular irradiation. AB - Dose- and time-response relationships were measured after irradiation of the pubertal rat testis with between 1 and 20 Gy of 300 kVp X-rays. The threshold dose for Leydig cell dysfunction was about 5 Gy. Dysfunction after higher doses was observed by 2 weeks post-irradiation as a dose-dependent decrease in serum testosterone (T) concentrations, and the levels were undetectable after 15 or 20 Gy. Despite the recovery of serum T by 24 and 36 weeks, dysfunction of the Leydig cell population was still observed as an increase in luteinizing hormone (LH) secretion (1.5 to 2-fold increase after 15 or 20 Gy at 24 weeks; 2 to 3-fold increase after 10 or 20 Gy at 36 weeks). The endocrine changes were probably due to the observed loss of Leydig cells following irradiation. These results indicate that the Leydig cells of pubertal rats are more radioresponsive than those of the adult. PMID- 3006155 TI - Interaction of [125I]-Tyr4-bombesin with specific receptors on normal human pancreatic membranes. AB - The binding of bombesin to its receptors on normal human pancreatic membranes was investigated using high specific activity, radioiodinated bombesin ([125I]-Tyr4 bombesin), prepared by an oxidative method with chloramine-T. Binding was specific, temperature-dependent, saturable, reversible and linearly related to membranes protein concentration. After a 30 min period of incubation with membranes the degradation of the tracer has never been found superior to 20%. Scatchard analysis of binding data was compatible with a single class of binding sites with a high affinity (0.96 nM) and a Bmax of 753 fmol/mg protein. [125I] Tyr4-bombesin binding to human pancreatic membranes was competitively inhibited by (1-Tyr4-)bombesin, GRP, the nonapeptide of bombesin and litorin but not by unrelated hormones such as somatostatin, CCK, human gastrin, etc. These results describe for the first time the presence of specific receptors for bombesin on human pancreatic membranes. The binding characteristics obtained are comparable with those found in other species. PMID- 3006156 TI - Differential insulin responses to oral and intravenous glucose in the transplantable rat insulinoma--a role for GIP. AB - We have previously demonstrated an impaired insulin response to intraperitoneal glucose and arginine by the transplantable NEDH rat insulinoma. The nature of this tumour B-cell defect has been further studied by investigating the response of insulinoma-bearing rats to intravenous and intragastric glucose. Intravenous glucose failed to stimulate plasma immunoreactive insulin (IRI) above high basal levels (14.5 +/- 1.1 micrograms/L). However, significant elevation of the plasma IRI concentration was observed following an intragastric glucose load (17.1 +/- 1.5 micrograms/L; P less than 0.02). In view of the different effects of oral and intravenous glucose on insulin secretion in the RIN, implicating an involvement of incretin factors from the gut, the response of the tumour to GIP was investigated. Plasma IRI concentrations rose significantly in these animals (20.6 +/- 2.5 micrograms/L at 5 min, P less than 0.02). We conclude that (a) the transplantable rat insulinoma is responsive to GIP, and (b) that whilst the tumour B-cell has lost its insulin responsiveness to hyperglycaemia produced by intraperitoneal or intravenous glucose, it retains its ability to respond to intragastric glucose. This could be due to incretin factors from the gut of which GIP is currently the strongest candidate. PMID- 3006157 TI - Measurement and partial characterization of the multiple forms of neurokinin A like immunoreactivity in carcinoid tumours. AB - An antiserum raised against neurokinin A has been used to demonstrate storage and release of neurokinin A-like immunoreactivity by carcinoid tumours. The antiserum showed reactivity towards members of the tachykinin family of polypeptides in the order: neurokinin A greater than eledoisin greater than neurokinin B greater than kassinin greater than substance P greater than physalaemin but the magnitude of the cross-reactivity with substance P and physalaemin was less than 1% of that of neurokinin A. A sensitive (IC50 238 fmol/ml; minimum detectable concentration, 9 fmol/ml) radioimmunoassay was set up using this antiserum. Extracts of metastatic tumour tissue from four patients with a primary carcinoid tumour in the midgut contained both neurokinin A-like immunoreactivity (NKA-LI) and substance P-like immunoreactivity (SP-LI). The concentrations (pmol/g wet weight) of NKA-LI and SP LI in the tumours were: patient A 210, 201; patient B 2276, 6849; patient C 1198, 834 and patient D 424, 379. Analysis of the tumour extracts by reverse phase HPLC indicated that the NKA-LI was heterogeneous. Under two different conditions of chromatography, one component was eluted with the same retention time as neurokinin A. Two further components were more hydrophobic than neurokinin A but were not eluted with the retention time of neurokinin B. Analysis of these components by gel filtration indicated a molecular weight in the 3000-4000 range suggesting that they may be related to neuropeptide K, an N-terminally extended form of neurokinin A. NKA-LI and SP-LI were undetectable in the plasma of patients A and D but were elevated in patient B (NKA-LI 1005 +/- 114; SP-LI 345 +/- 85 fmol/ml) and patient C (NKA-LI 80 +/- 31; SP-LI 21 +/- 13 fmol/ml). PMID- 3006158 TI - [Uranium, thorium and potassium contents and radioactive equilibrium states of the uranium and thorium series nuclides in phosphate rocks and phosphate fertilizers]. AB - Uranium, thorium and potassium contents and radioactive equilibrium states of the uranium and thorium series nuclides have been studied for 2 phosphate rocks and 7 phosphate fertilizers. Uranium contents were found to be rather high (39-117 ppm) except for phosphate rock from Kola. The uranium series nuclides were found to be in various equilibration states, which can be grouped into following three categories. Almost in the equilibrium state, 238U approximately 230Th greater than 210Pb greater than 226Ra and 238U greater than 230Th greater than 210Pb greater than 226Ra. Thorium contents were found to be, in general, low and appreciable disequilibrium of the thorium series nuclides was not observed except one sample. Potassium contents were also very low (less than 0.3% K2O) except for complex fertilizers. Based on the present data, discussions were made for the radiation exposure due to phosphate fertilizers. PMID- 3006159 TI - [Production of 52Fe by the 55Mn(p,4n)52Fe reaction and milking of 52mMn from 52Fe]. AB - The excitation functions were measured for the nuclear reactions of 55Mn(p,4n)52Fe and 55Mn(p,n)55Fe by using thin manganese disks, specially prepared under a pressure of 200-250 kg/cm2 at 400-500 degrees C for 0.5-1 hour. The maximum cross section in the excitation function for the 55Mn(p,4n)52Fe reaction was found to be 1.4mb at Ep = 54 MeV. From the yield curve, 24.8 MBq/microA h (670 muCi/microA h) of 52Fe was estimated to be produced with 0.45% 55Fe contamination in the energy region between 73 and 39 MeV. Iron-52 was produced in the yield of 85-93% to the expected value from the yield curve in the energy region of 44-60 MeV. For the separation of 52Fe, the radiochemical yield of 52FeCl3 was 70-92% and its radionuclidic purity was higher than 99%. Manganese 52m was obtained repeatedly by eluting the anion exchange column adsorbing 52Fe with 6N-HC1 in 99.9% radionuclidic purity and 86% yield to the expected value. The amount of 55Fe in a large quantity of 52Fe could be determined in 1-2 days from end of bambardment (EOB) by analyzing decay curve of Mn-Ka-X ray from 52Fe and 55Fe. PMID- 3006160 TI - Distribution of 201Tl in the blood. AB - Thallium-201 distribution in the blood was investigated both in vivo and in vitro. Thallium-201 was distributed into the erythrocytes and plasma with the ratio of 1.4 +/- 0.3 to 1.0, immediately after its administration. The uptake of 201Tl into the erythrocytes in vitro were affected by the incubation temperature and the presence of ouabain and KCl; indicating that the 201Tl was uptaken into cells partly through their membranes Na, K-ATPase. Erythrocytes could retain 201Tl in it, whereas 201Tl was present as free ion in the plasma. Thallium-201 was flew out of erythrocytes into the plasma, keeping the ratio of 201Tl in erythrocytes/plasma to be 1.9 +/- 0.2/1.0. PMID- 3006161 TI - [Sialography, echography and computerized tomography in the study of the parotid region]. AB - The diagnostic accuracy of sialography and ultrasonography (US) in the evaluation of parotid masses is evaluated. Furthermore the role of computed tomography (CT) in this pathology is discussed. In the personal experience US proved to be the best method in the recognition of a parotid tumor while sialography was superior in defining the intra or extraparotid site. The two investigations showed the same accuracy in the definition of benign or malignant nature of the mass. Therefore we consider US the only investigation in most instances; sialography could be performed when the site of the lesion is uncertain or an inflammatory lesion is suspected. CT is never the first investigation; its use is limited to a low number of cases, mainly for the evaluation of large masses and when the association US-sialography does not allow a sure diagnosis. PMID- 3006162 TI - [38 year-old male with bilateral pulmonary infiltrates and the septic picture of fatal development]. PMID- 3006163 TI - [Program system for quantitative evaluation of computed tomograms using a digital masking technic for isolation of organs of interest and organ regions from the value matrix]. AB - A program system for the quantitative analysis of CT images is described and its versatile applicability is demonstrated by practical examples. Using a semi automatic digital masking technique, the system allows to isolate single organs or regions of organs with good reproducibility and to process their CT values separately. For processing of the complete image matrix or of isolated areas of the matrix, evaluation programs have been developed, which--in addition to the standard image evaluation functions available in modern CT systems--provide the possibility for various CT value transformations and special statistical analyses of original and transformed CT values. The demonstrated applications of the program system include determination of the venous capacity of the lower extremities by CT, separation of different components of the brain from a cranial CT, ventilation analyses of the respiratory lung parenchyma, calculation of the fat content in a fatty liver and approximate measurement of the iodine concentration in a contrast-enhanced kidney. PMID- 3006164 TI - [Dynamic CT of lymphoma manifestations of the liver and spleen]. AB - Fourteen patients with lymphoma involving the liver and/or spleen were examined by dynamic CT. Lymphoma lesions show little enhancement with respect to the spleen and usually also when compared with liver tissue. On two occasions there was a definite increase in density during the arterial phase and dynamic CT could not distinguish the lesions from tumours of hepatic origin. One circumscribed lymphomatous lesion of the liver and six splenic infiltrates were only visible during the first few seconds after a bolus of contrast material had been injected. Dynamic CT does not usually differentiate diffuse involvement of the liver and spleen. In this situation, lack of a trabecular structure of the spleen during the arterial phase and a low peak of the time-density curve may suggest diffuse infiltration. PMID- 3006165 TI - [Results of the diagnosis of adrenal masses using percutaneous CT-guided fine needle biopsy]. AB - This paper reports the results of 42 CT-aided fine needle biopsies of adrenal masses in 40 patients (26 were smaller than 3 cm., six smaller than 1 cm.). There were 25 adenomas, seven metastases, one primary carcinoma, one case of tuberculosis, two phaeochromocytomas and four post-inflammatory lesions. Diagnostic material was obtained in 88%. There was one incorrect diagnosis and on five occasions the result was indefinite, leading to an accuracy of 73%. Exclusion or demonstration of malignancy was, however, correct in 85%. There were no complications except for one unimportant pneumothorax. PMID- 3006167 TI - [High-resolution realtime sonography of varicoceles]. AB - An attempt has been made to classify varicoceles by means of real time sonography in accordance with the findings on palpation. The maximal diameter of the pampiniform plexus and of individual veins was determined sonographically and compared with the results of palpation in 74 patients with varicoceles. The sonographic findings are more accurate than is palpation. Fifteen subclinical varicoceles were discovered by sonography which could not be detected by palpation. It has been found convenient to classify the lesions in four sonographic stages. Maximal transverse diameter of the pampiniform plexus up to one centimetre is regarded as normal. A diameter of one to two centimetres corresponds to grade I, two to three centimetres corresponds to grade II and greater than three centimetres to grade III. PMID- 3006166 TI - [MR tomography of gynecologic diseases of the minor pelvis]. AB - The results of MR examinations of 31 women with gynaecological diseases are described; all had CT and histological confirmation at operation. The extent and relationship of the lesions were well demonstrated by the three-dimensional sections of MR, in some cases better than on CT. Variations in the signals allowed better differentiation of myomas and ovarian carcinomas from normal tissue. Like CT, MR tomography was not reliable in showing the extent of infiltration into neighbouring structures. PMID- 3006168 TI - [Realtime sonography in the diagnosis and follow-up of malignant tongue tumors. II. Detection and staging]. AB - 92 sonographic examinations performed in 62 patients were evaluated to determine the possible application of sonography in the diagnosis and follow-up of malignant tumours of the tongue. Squamous cell carcinomas were by far dominant and proved to be hypoechoic, mainly inhomogeneous and ill-defined masses. In most cases, they ranged between 2 and 4 cm. in diameter. In 50% of the lesions an infiltration of the pharynx wall was confirmed, and exulcerations were correctly detected by sonography in 75%. In preoperative determination of the size of the tumour, sonography correctly detected the size in 93% of the cases and was thus markedly superior to the clinical palpatory examination, which determined a mere 43% correctly. Sonography should be included in the pretherapeutic staging of tumours of the tongue to objectify the clinical findings. It is also adequate to document tumour behaviour during radiation. PMID- 3006169 TI - [Videodensitometric determination of relative blood regurgitation volume in aortic valve insufficiency]. AB - A simple video densitometric method based on the indicator distribution principle in the region of interest permits the determination of the relative amount of regurgitation between the ascending aorta and the left ventricle in cases of aortic valve insufficiency. The experimental basis and early clinical measurements are described and are compared with the more conventional methods of cine angiography and radionuclide ventriculography. PMID- 3006171 TI - [Expansion, deformation and bursting characteristics of frequently used balloon dilatation catheters. In vivo studies on canine vessels (2)]. AB - In an experimental angioplasty study in dogs, we evaluated the characteristics of dilatation and deformation, as well as the pressure requirements, bursting points, and types of bursting of four different balloon dilatation catheters. The balloons, which were selected 0%, 30%, and 80-100% larger than the arterial lumen, measured 4, 6, and 8 mm in diameter and were inflated with an automated pump which simultaneously recorded balloon pressure and volume. Changes to the vessel wall were assessed radiographically and by histology. In vivo, balloon dilatation catheters demonstrated a markedly different behaviour than in vitro. Significantly higher pressures were tolerated due to the support of the arterial sleeve. Non-compliant oversized balloons caused more damage to the arterial wall than compliant ones. Oversized balloons attained their required diameters only with high inflation pressures of 10 to 12 atm or after rupture of the artery. PMID- 3006170 TI - [Percutaneous transluminal angioplasty in Brescia-Cimino shunt insufficiency]. AB - Forty-eight patients with failing arteriovenous dialysis fistulas were evaluated in an 18-month period. Forty-six of dialysis access fistulas were of the Brescia Cimino type; two were fistulas in the upper forearm. PTA was technically feasible in twenty-three patients (44%). 54% dilatations with polyethylene balloons were initially successful and patent at 3 months; 43% were patent at 6 months; and 24% after more than 9 months. Two complications were encountered. PTA is recommended in Brescia-Cimino type dialysis fistulas, as the procedure prevents damage to the arterial and venous vessels suitable for a new fistula at the same forearm. The procedure may be performed on an outpatient basis. PMID- 3006172 TI - [Diagnosis of deep venous thrombosis of the leg. Phlebofluorography versus Doppler sonography]. AB - In this retrospective study, the results of Doppler sonography and phlebography are compared. In about 30% cases the diagnosis of thrombosis made by Doppler sonography could not be confirmed. Agreement was better in patients with acute thrombosis or following pulmonary emboli than in those with a longer history of swelling of the lower extremities. These results agree with the findings of other authors using impedance plethysmography; this also shows lack of agreement in about 30% of the cases. PMID- 3006173 TI - [Initial experiences with nuclear spin tomography of bone diseases]. AB - The results of MRT in 41 patients with bone diseases are reported. MRT proved to be a sensitive method in evaluating localised bone lesions. MR provided definite advantages in comparison with computed tomography and plain films in the determination of the intramedullary and extraosseous extent of circumscribed bone lesions. A better characterisation of the nature and histology of bone changes was not possible with MRT. Diffuse bone marrow disease are well demonstrable via MRT. PMID- 3006174 TI - [High-resolution nuclear spin tomography of the hip joint using a Helmholtz surface coil]. AB - A specially constructed Helmholtz surface coil enables one to obtain high resolution nuclear tomograms of the hip. Anatomical structures which are clinically important and which can regularly be shown are described. A pilot study has been carried out on 16 patients and typical findings are described in necrosis of the femoral head, osteoarthritis and changes resulting from tumours in the region. Possible further applications are discussed. PMID- 3006176 TI - [Incidence of pineal calcification in the first 18 years of life]. AB - There are sparse or contradictory data on frequency and pathological significance of pineal calcifications in childhood. This is particularly so for children younger than 6 years of age. We therefore looked for pineal gland calcifications in 1044 consecutive a.p. and lateral skull films. Pineal calcification was diagnosed, if accepted by both authors, and if the calcified spot fitted into at least 2 of 4 localisation methods. 80 patients with pineal calcifications were detected using this method. In 40 of these patients CCT confirmed the calcification. The frequency of pineal calcification was 3% in the first 12 months of life rising gradually to 7.1% in children of 10 years of age. From 10 years onwards, there is a marked increase of frequency of calcifications of the pineal gland up to 33% in the group of children of 18 years of age. In contrast to some statements in literature, pineal calcifications seem to be physiological in a limited percentage even below the age of 6 years. PMID- 3006175 TI - [Indications for magnetic resonance tomography in the diagnosis of cerebral diseases. Report of experiences with 1200 cases]. AB - The current paper is a review of our experience of 1200 examinations of the brain, using magnetic resonance tomography (MRT), derived from a mixed neurological and neuro-surgical clinic. MRT has advantages in localisation and tissue characterisation. Localisation is improved by the ability to obtain coronal and sagittal images and by the absence of artifacts due to bone in lesions in the middle and posterior fossa. Some tissues and structures (blood, fat, vessels) produce relatively characteristic signals on MRT, which may be important in clinical diagnosis (eg. chronic subdural haematoma). A significant advantage of MRT is its excellent contrast resolution of soft tissues; this results in better demonstration of normal brain anatomy (eg. corticomedullary contrast), areas of demyelinisation (eg. multiple sclerosis) and certain cerebral tumours (eg. astrocytomas). On the basis of our experience, certain indications are defined, depending on the physical characteristics of the method; these are illustrated by clinical examples. PMID- 3006177 TI - [Amphetamine analog of ruthenocene as a radiodiagnostic agent: brain affinity of ruthenocenoyl-isopropylamine]. AB - N-alkyl derivatives of 1-ruthenocenoyl-2-aminopropane (Rc-amphetamine) were labelled with 103Ru and their organ distributions were measured in mice and rats. All Rc-amphetamines showed a high affinity for brain and lung. A concentration ratio brain/blood up to 17:1 and lung/blood up to 80:1 was measured. The advantages of 97Ru labelled Rc-amphetamine compared to 123I labelled amphetamine for measurement of regional cerebral blood flow are discussed. PMID- 3006178 TI - [Cerebral toxoplasmosis in AIDS]. PMID- 3006179 TI - [Topographic diagnosis of chordomas using magnetic resonance tomography (MRT)]. PMID- 3006180 TI - Periosteal ganglion. PMID- 3006181 TI - [Desmoplastic fibroma]. PMID- 3006182 TI - Computerised tomographic demonstration of the "crescent sign" confirming gangrenous sloughing of the lung associated with a primary lung carcinoma. PMID- 3006184 TI - [Castleman's lymphoma]. PMID- 3006183 TI - [Malignant lymphoma of the kidney and hydronephrosis]. PMID- 3006185 TI - [Immunologic markers of the risk of cytomegalovirus infection among immunosuppressed patients]. AB - ELISA and complement fixation test (CF) were compared for the determination of the Cytomegalovirus (CMV) humoral immune status of donors and recipients before and after graft. ELISA-IgG was more sensitive than the CF test and permitted a better identification of the high risk donors and recipients. The presence of IgM antibodies in the recipient at the day of transplantation is an index of greater susceptibility to chronic or recurrent infection at a later date. In primary renal allograft recipient the detection of specific IgM antibodies by ELISA provides more rapid diagnosis than that by CF. In bone marrow transplants, ELISA IgM showed a better correlation with the virus isolation than the CF test. In the reinfected renal allograft patients the presence of IgM was related to clinical manifestations. Using ELISA, specific decreases of CMV antibody titers were detected before virus isolation. PMID- 3006186 TI - [Markers of exposure and early lesions in cancer epidemiology]. AB - The biological markers of risk can be separated into two categories: markers of exposure to a specific substance, based on several biochemical, physical and immunological methods (usually able to measure the absorbed dose); markers of early lesions which usually are not specific for a particular substance and do not estimate the dose of exposure. In epidemiological research on cancer etiology, biological markers have a different role according to whether cases are subjects with early lesions or with invasive cancers. The first approach has the advantage of shortening the observation time between exposure and the appearance of a biological effect. On the other hand, the biological relevance of the study could be limited due to the non univocal relation between early lesions and cancer. With the second approach, invasive cancer can be related to specific markers of exposure (this would permit to draw direct inference on disease etiology) or to markers of early lesions (etiological interpretation in this case would be less straightforward). The two approaches should be considered in the frame of two basic problems: What is the advantage of measuring exposure at the individual level? What is the advantage of a biological marker as compared to traditional epidemiological methods? With studies at the group level the correlation between exposure variables and disease frequency can be estimated. Nevertheless only studies at the individual level can exclude the fact that the observed correlation is due to an artefact. In this case we are left with the second question and many answers are possible.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006187 TI - [Peripheral neuropathies associated with monoclonal gammapathies. Value of electrophysiologic studies]. AB - An electrophysiological study was performed in 19 patients with polyneuropathy associated with a monoclonal gammopathy. This study has shown some difference according to the type of the gammopathy and the benign or malignant nature of the disease. Patients with benign IgM gammopathy are in an homogenous group. In every case the neuropathy is of the demyelinating type. In the other groups, the neuropathy is less homogenous. Most of the time it was predominantly of axonal type. Within the myeloma group, the neuropathy is more severe and the denervation pattern is marked. PMID- 3006188 TI - [Tomaculous neuropathy. Electrophysiologic study]. AB - In two cases with recurrent palsies, the results of electrophysiological studies led to nerve biopsy showing typical tomacula. The first case was an inherited neuropathy with liability to pressure palsies. The second case was an apparently sporadic painless recurrent brachial neuropathy. Electrophysiological alterations were diffuse and sensory fibres of the median nerve between index and wrist were the most involved. Conduction blocks were observed without palsy in narrow anatomical passageways where nerve compressions are frequent (ulnar nerve at the elbow, peroneal nerve at the fibula). A compression by a neighbouring anatomical structure could make the prognosis worse and justify nerve decompression. The nerves with slowest conduction have the most important risk of palsy and the patients should be given advice to avoid their compression. PMID- 3006189 TI - [Muscle-nerve ephapse in chronic denervation? Axonal localization]. AB - Muscle-to-nerve ephaptic responses, taking place between a reinnervated motor unit and a motor axon, have been reported in the case of chronic denervation (Roth and Magistris, 1985). One of these responses is described here. Its main features are that it manifests itself as a motor unit potential, which, when present, is evoked either as a direct tardive response (RE), or as an indirect F response, the two potentials never being observed together on any recording. The characteristics of these potentials show that two distinct competitive pathways lead to the same muscular fiber group which suggests the existence of an ephaptic connection. Furthermore, a slight difference in the shape of the RE and F potentials suggests an axon terminal branch localisation of this ephapse. PMID- 3006190 TI - [Peritoneal metastasis of hepatocarcinoma. Report of 11 cases with laparoscopic diagnosis]. PMID- 3006191 TI - [The endocochlear potential in experimental renal insufficiency]. AB - Endocochlear Potential (EP) was measured in male Wistar rats under control conditions and renal failure produced by means of bilateral nephrectomy. Results show a statistically significant (p less than 0.01) decrease in EP measured in animals with renal failure. This finding is in accordance with the decrease in Na K ATPase activity found in the cochlea and other structures in human and experimental kidney insufficiency. PMID- 3006192 TI - [Radioimmunologic determination of ACTH in the rabbit: stress-ACTH-corticosteroid relationship and diarrhea]. AB - Plasma ACTH was measured in young and adult rabbits with a CEA-SORIN kit. The reliability of the analysis was controlled by liquid chromatography, several biochemical tests such as parallelism and reproducibility and several physiological tests such as plasma ACTH response to ethylic ether stress, to adrenalectomy, to dexamethasone and metopyrone. In young diarrhoeic rabbit plasma, ACTH (293 +/- 45 pg/ml) and corticosteroid (111 +/- 13 ng/ml) levels were significantly much higher than in young healthy rabbits (ACTH: 130 +/- 35 pg/ml; corticosteroids: 66 +/- 7 ng/ml). Furthermore, transporting young rabbits by car caused such violent stress that plasma ACTH levels increased from 52 +/- 11 to 130 +/- 35 pg/ml. A causal relationship between stress - ACTH and diarrhoea is discussed. PMID- 3006193 TI - Effect of bilirubin on the spectrophotometric and radionuclide assay for serum angiotensin-converting enzyme. AB - The effect of bilirubin on serum angiotensin-converting enzyme (ACE) activity was studied with spectrophotometric and radionuclide assays. In the spectrophotometric assay addition of bilirubin to normal serum from dog, mouse, and human produced a dose-related inhibition of ACE activity. A 50% decrease in human ACE activity was produced by the addition of approximately 250 mg/L in vitro. Serum from icteric patients with elevated bilirubin was also associated with a reduction in ACE activity in the spectrophotometric assay. A 50% decrease in ACE activity in these samples was associated with a serum bilirubin of approximately 220 mg/L. In the radionuclide assay, however, addition of bilirubin to normal human serum failed to reduce measured ACE activity. The use of a radionuclide assay for serum ACE in clinical samples offers the advantage of less interference from serum bilirubin. PMID- 3006194 TI - Alterations in vascular alpha and beta adrenergic responsiveness in the Dahl rat: a preliminary report. AB - The effect of a high salt diet on in vitro vascular adrenergic responsiveness of Dahl salt-sensitive (DS) and salt-resistant (DR) rats was investigated. Female DS and DR rats were fed either a normal (low salt) or high salt (8%) diet for 7 weeks. DS rats on the high salt diet showed a progressive increase in systolic blood pressure (SBP) and were considered hypertensive (SBP greater than 150 mmHg) at the time of experimentation. SBP did not change appreciably in either the DS rats on a low salt diet or in the DR rats on either diet. Isoproterenol induced relaxation of rings of aortic smooth muscle from DS rats on the high salt diet was significantly attenuated when compared to the other three groups. However, relaxation in response to sodium nitrite was similar in the 4 groups. An enhanced aortic smooth muscle response to norepinephrine stimulation was observed in both the DS and DR rats maintained on the high salt diet, whereas no difference was seen in the contractile response to KC1 depolarization. In contrast to previous reports, the present study shows that alterations do occur in in vitro vascular adrenergic responsiveness of the Dahl rat. PMID- 3006195 TI - Superoxide generation by 1-nitropyrene in rat lung microsomes. AB - 1-Nitropyrene (1-NP) is a potent mutagen which has been found in diesel exhaust particulates and photocopy toners. 1-NP is known to undergo reduction and oxidation by mammalian enzymes. Research into the biological activity of 1-NP has concentrated on its possible mutagenic and carcinogenic properties. Because other nitro compounds, such as nitrofurantion, are reduced to a nitro anion radical which can react with molecular oxygen to produce superoxide, it was of interest to determine if 1-NP produced superoxide. This study used acetylated cytochrome c/superoxide dismutase as a detection system in a lung microsomal system. Results show that 1-NP catalyzes the formation of superoxide, that the reaction obeys Michaelis-Menten kinetics and that this reaction is saturable. The significance of this study is that 1-NP may have toxic effects which are related to oxidant stress. PMID- 3006197 TI - Assessment of activity in chronic sarcoidosis: usefulness of serum angiotensin converting enzyme and gallium scan. AB - Gallium scan and serum angiotensin converting enzyme (SACE) activity were performed in 39 patients, who were not receiving corticosteroids and had histologically proven chronic sarcoidosis of at least 2 years duration. Results of these investigations were compared with clinical assessment of disease activity. In patients with radiographic evidence of pulmonary parenchymal disease, the positive and negative predictive values for gallium scan and/or SACE were 96 and 100% respectively; however, in patients with no radiographic evidence of parenchymal disease the positive and negative predictive values were 50 and 100%, respectively. These findings suggest that SACE and gallium scan are useful in assessing the activity and hence the need for therapy in patients with chronic sarcoidosis with radiographic evidence of parenchymal disease. PMID- 3006196 TI - Role of prostaglandins in marihuana-induced bronchodilation. AB - In vitro evidence suggests that physiological effects of marihuana may be mediated by prostaglandins via the stimulation of phospholipase A2. To verify if marihuana could act by this route in vivo, we tested the effects of acetylsalicylic and mefenamic acids, inhibitors of cyclooxygenase, on marihuana induced bronchodilation and tachycardia. In 11 healthy volunteers, marihuana smoking (7 mg/kg, 1.7% delta 9-tetrahydrocannabinol) produced a significant increase in specific airway conductance (from 0.262 +/- 0.033 to 0.360 +/- 0.050 s-1 X cm H2O-1, mean +/- SE, p less than 0.01), forced expiratory volume in 1 s (4.02 +/- 0.22-4.27 +/- 0.25 liter, p less than 0.05) and heart rate (73.2 +/- 2.0-108.5 +/- 5.2 beats/min, p less than 0.001). In a second session, acetylsalicylic or mefenamic acid was taken for 30 h before marihuana smoking. No inhibition of marihuana-induced increase of specific airway conductance, forced expiratory volume in 1 s and heart rate was found. These findings suggest that the bronchodilation and the tachycardia induced by marihuana smoking in humans are not mediated by prostaglandins. PMID- 3006198 TI - Frequency of distribution according to histological types of lung cancer in the tracheobronchial tree. AB - The incidence of the location within the bronchi related to the cell types was investigated with the flexible fiberoptic bronchoscope in 355 cases of lung carcinoma. In 5 patients carcinoma was situated only in the trachea. In the other 350 cases the cell types other than adenocarcinoma were found to show different locations following their cell type. Epidermoid carcinoma was found more frequently in the two upper lobes (p less than 0.001), while small cell carcinomas showed predilection for the main bronchus on the right side, and the upper lobe in the left (p less than 0.001). No difference could be found between the upper, lower lobes and main bronchi for adenocarcinoma. It was also observed that large cell carcinomas were situated more often in the right upper lobe. The most important finding in this investigation was that, apart from adenocarcinoma, the other types were located mainly in the upper lobes, and much less frequently in the lower lobes. The predilection of localization of epidermoid and small cell carcinomas in the upper lobes suggests a possible relationship to tobacco smoke inhalation as these regions have been shown to be more affected by the smoke. PMID- 3006199 TI - [Relationship among myocardial norepinephrine, cyclic AMP and ventricular fibrillation in experimental infarction]. PMID- 3006200 TI - [Immunohistochemical study of condyloma and and carcinoembryonic antigens in the uterine cervix in human papilloma virus infection and neoplasia]. PMID- 3006202 TI - Diet in the etiology of cancer. PMID- 3006203 TI - [Parvovirus infections in humans]. PMID- 3006201 TI - [Use of serological markers in the diagnosis and prognosis of chronic viral hepatitis]. PMID- 3006204 TI - [Adrenal hyperplasia as a result of 21-hydroxylase deficiency: prenatal diagnosis and treatment. Neonatal diagnosis]. PMID- 3006205 TI - [Local radiation therapy of rheumatoid arthritis patients]. PMID- 3006208 TI - [Hepatocellular adenoma]. PMID- 3006206 TI - Opportunistic infections in patients with AIDS: clues to the epidemiology of AIDS and the relative virulence of pathogens. AB - The frequency of nine reactivating or opportunistic infections and Kaposi's sarcoma among patients with the acquired immunodeficiency syndrome (AIDS) was reviewed. The diagnoses of 87 patients reported from the Colorado AIDS registry and 359 others from literature reports were abstracted, and data were placed in one of 11 categories on the basis of the risk group of the patient. Pneumocystis carinii infection was significantly commoner among blood or blood-product recipients than among natives of the tropics (P less than .001). Tuberculosis and toxoplasmosis each were significantly commoner among natives of the tropics than natives of developed countries (P less than .001), whereas disseminated Mycobacterium avium-Mycobacterium intracellulare infections were present more often in the latter group. Among natives of the tropics treated in developed countries, cytomegaloviral infection was diagnosed significantly less often (22%) than among persons from developed countries in whom sexual transmission was presumed (47%; P = .0005). These data suggest that the pattern of infections manifested in AIDS could provide clues about transmission and that there may be a hierarchy of reactivation of latent infections in which populations with exposure to multiple agents manifest these preferentially to Pneumocystis carinii. PMID- 3006207 TI - [Diagnostic value of the assay of carbohydrate antigen 19-9 in patients with primary bronchial cancer]. AB - We studied the levels of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9) in 90 patients with primary lung cancer. One or both markers were increased in 45 patients (50%): CEA only was increased in 13, CA 19-9 only was increased in 19, and both CEA and CA 19-9 were increased in 13. Increase of markers did not differ according to histologic subtype of cancer. Increase or decrease of the markers (mainly CA 19-9) usually parallelled evolution of the disease in our patients. Thus, measurement of both CEA and CA 19-9 levels are of diagnostic value in half the patients with primary lung cancer. PMID- 3006209 TI - [Effects and cardiovascular dangers of betamimetics]. PMID- 3006211 TI - [ELISA-IC for the detection of the rotavirus group antigen, in the stools of children with acute gastroenteritis]. PMID- 3006210 TI - [Diagnostic and therapeutic use of human anti-D (Rho) monoclonal antibodies. Evaluation and perspectives]. AB - Human monoclonal antibodies will be essential in medicine. They are valuable tools for biological diagnosis and therapeutics. Our model, human monoclonal antibodies directed against the Rhesus D antigen can be used for the determination of the Rhesus D phenotype and for the suppression of Rh(D) immunisation in women. These new products require new procedures of preparation, new regulations for the quality controls, which will be discussed in this paper. PMID- 3006212 TI - Hepatitis A antibodies in two socioeconomically distinct populations of Sao Paulo, Brazil. PMID- 3006213 TI - Fanconi syndrome and osteomalacia without hyperparathyroidism. PMID- 3006214 TI - [The role of neuroendocrine-immunological interrelations in the development of tumors]. AB - There is at present an experimental evidence of neuroendocrine-immunological interactions in tumor development both in experimental models and man. In fact, different hormone receptors have been found in both experimental and spontaneous tumors in animals and man. Such tumors are therefore under the influence of the neuroendocrine system. In addition, some hormone-metabolic alterations such as those observed in advancing age are often associated with a high incidence of the neoplastic growth. Interestingly, tumor incidence increases with advancing age in both man and animals and experimental tumors are more easily transplanted in old mice than in young adult mice. On the ground of these observations it can be suggested that tumor growth results from the interactive action of both the immunological and neuroendocrine systems. PMID- 3006215 TI - [Are endocrine receptors determining factors for the prognosis of carcinoma of the breast?]. AB - The capacity of hormone receptors to predict sensitivity to hormone therapy in breast cancer has been demonstrated since many years; on the other hand, its importance in the prognosis of these patients, expressed as disease-free survival (DFS) or overall survival (OS), is still discussed. The authors have reviewed the literature data on this problem, paying particular attention to studies homogeneous for disease extent and therapeutic approaches (surgical or post surgical). At the moment of mastectomy, the presence of estrogen or progesterone receptors in primary tumors was not clearly related to the patients' DFS. The higher probability of DFS was observed in the group of patients who received adjuvant hormone therapy after mastectomy, with increasing amounts of each receptor in primary tumors and when both hormone receptors were contemporaneously present. In patients with breast carcinoma, the data on the relationship between hormone receptors and overall survival were more homogeneous: the presence of the cellular hormone receptor improves the probability to have a longer OS in patients treated by radical mastectomy. The opportunity to analyze these relationships in some reports which processed a statistical multivariate analysis permits us to confirm the hormone receptor values also as a prognostic parameter which is not dependent from other (axillary lymph nodes involvement, tumor size and therapy) till now considered parameters. PMID- 3006216 TI - The central auditory conduction at term date and three months after birth. I. Composite group averages of brainstem (ABR), middle latency (MLR) and auditory cortical responses (ACR). AB - We are investigating the maturation of auditory evoked responses, as a potential diagnostic tool for the neurological examination of premature infants at the Intensive Care Unit for Prematures. In this communication we report the group composite averages of the BMC-ARs in a group of 25 mature and healthy newborns with a follow-up at 3 months of age. The ABRs showed a remarkable variability in wave I latency, with a relatively stable I-V central conduction time (CCT) of the brainstem. The ipsilateral II-V CCT does not differ appreciably from the contralateral II-V CCT. The MLR components NoPoNa and Nb, Nc, Nd are easy to identify in the group averages. Voltage asymmetry between stimulated versus non stimulated side is slight but persistent in the newborn period as well as at 3 months of age. The group averaged ACRs, show an early complex within the latency reach of 100 ms and a slow W-shaped late complex, most distinct at 3 months. The results indicate that the protocol as applied constitutes a tool for the testing of conduction function of the auditory afference in newborns, even beyond the level of the brainstem. Group averaging offers a method to determine the intragroup stability of evoked potentials under investigation. PMID- 3006217 TI - Kidney Na,K-ATPase activity in streptozotocin-diabetic rats. AB - Measurements of the ouabain-sensitive Na,K-ATPase activity in the kidney was performed on female Wistar rats after 50 days' duration of diabetes, and compared to an age-matched control group. The measurements showed a 23% increase in the Na,K-ATPase activity per mg protein, and a 66% increase in the total Na,K-ATPase activity in the whole kidney. The increase Na,K-ATPase activity can be seen as a response to the increase in tubular sodium load which is a consequence of the increased glomerular filtration rate in diabetes. PMID- 3006219 TI - Dietary fiber and peptic ulcer. PMID- 3006218 TI - Haemodynamic and humoral effects of lower body negative pressure in normal, sodium-replete man during angiotensin-converting enzyme inhibition with captopril. AB - The significance of the renin-angiotensin system (RAS) for circulatory homeostasis during gravitational stress (10 min of lower body negative pressure, LBNP, at -40 mmHg) was investigated in eight men on liberal sodium intake. The function of RAS was inhibited by a single oral dose of 100 mg captopril, an angiotensin-converting enzyme inhibitor. Plasma concentrations of renin and angiotensin I were normal before and increased after captopril and during LBNP. Plasma concentration of angiotensin II was normal before captopril, increased during LBNP, and fell to low values after captopril. Systolic blood pressure decreased more during LBNP after captopril than in the control situation. In three cases, the LBNP experiment after captopril had to be interrupted due to marked hypotension. Heart rate and plasma concentration of adrenaline increased above pre-captopril levels. In six subjects, plasma concentration of noradrenaline increased more during LBNP after captopril, less in two subjects, whereas the arginine vasopressin concentration increased more after captopril in all five subjects where measurements were available. The results demonstrate that RAS participates in blood pressure homeostasis also in sodium-replete, normal man. The enhanced increases in heart rate and plasma catecholamines after captopril do not suggest that sympathetic reflex activity during gravitational stress is blunted after captopril, in contrast to the evidence from animal experiments. PMID- 3006220 TI - Placental calcification: ultrastructural and X-ray microanalytic studies. AB - Calcification is common in human placentas and is widely recognized as a normal part of maturation and aging of this organ. Eleven human placentas were studied by light and electron microscopy to elucidate the mechanism of placental calcification. Earliest mineral deposits were seen along the trophoblastic basement membrane of the chorionic villi undergoing fibrinoid degeneration. Transmission electron microscopic examination revealed crystalline deposits within small membrane-bound vesicles; the latter appear to be derived from degenerating cells and were particularly numerous within the basement membrane. X ray microanalysis of these deposits revealed calcium and phosphorus peaks and the pattern of calcium hydroxyapatite was noted by electron diffraction. This pattern of calcification, i.e., precipitation of calcium hydroxyapatite in association with extracellular membrane bound vesicles, is similar to that seen in physiologic and pathologic calcifications of other tissues. PMID- 3006222 TI - Myeloperoxidase-deficient polymorphonuclear leucocytes (VI): Relation to cytogenetic abnormalities in primary myelodysplastic syndromes. AB - Relations between cytogenetic status, FAB-classification and an abnormal subpopulation of myeloperoxidase (MPO)-deficient polymorphonuclears (PMN) in 45 patients with myelodysplastic syndrome (MDS) are reported. Clonal abnormalities were demonstrated in 85% of the patients, with a lower incidence in the RA+ group (refractory anaemia with ring sideroblasts) compared to the others (p = 0.004). In 12 patients a spontaneous progression in cytogenetic aberrations occurred and in 7 of these (60%) a simultaneous progression in FAB-subtype was seen. The appearance of MPO-deficient PMNs was observed in 6 of these patients (55%). A progression in FAB-subtype was noted in further 4 patients and 2 additional patients developed MPO-deficient PMNs. Only one sufficient cytogenetic investigation was available in these patients. Thus 100% of the fully studied patients showed progression in cytogenetic abnormalities when a progression in FAB-subtype or a development of MPO-deficient PMNs was seen. 3 (49%) of the 8 patients developing MPO-deficient PMNs too showed a progression in FAB-subtype. Although no significant correlation to specific categories of structural aberrations or abnormalities in specific chromosome pairs could be demonstrated, clonal cytogenetic aberrations seem to be involved when the disease progresses and when MPO-deficient PMN develop. PMID- 3006221 TI - Enzyme markers in acute myeloid leukaemia. AB - The levels of TdT, AdA, 5'-N, 20 alpha-SDH and TK1 were analyzed in different FAB subgroups of AML. AdA, 5'-N, 20 alpha-SDH and TK1 showed large variations both within and between the different subgroups. It therefore seems unlikely that measurements of these enzymes will be able to aid morphological subclassification of AML. Neither could analysis of these enzymes give prognostic information. TdT was detectable in 46% of AML but the levels were only 1/10-1/100 of those found in ALL. The enzyme was detected in leukaemic granulocytopoietic cells while leukaemic cells of monocytic origin were negative in all but 1 case. In addition, increased TdT activity was positively correlated to the length of survival. PMID- 3006223 TI - Clinical severity of non-deletion form of HbH disease (--Med/alpha alpha thal). AB - We carried out alpha-globin gene analysis by restriction endonuclease mapping in a family with 2 cases of HbH disease. These data show that HbH disease in this family results from the interaction between a common deletional defect and a less common non-deletion alpha-thal lesion (--Med/alpha alpha thal). Furthermore, the presence of a beta-thal determinant in this family was investigated by beta gene polymorphism study. We showed that a patient with HbH disease also inherited a beta-thal determinant from the mother and although this was a beta O-thal gene, it was not sufficient to mask the severe alpha chain deficiency. The --Med/alpha alpha thal genotype is more severe than other types of alpha thalassaemia interactions causing HbH disease, probably because the expression of alpha alpha thal determinant may be lower than that of an alpha-thal determinant containing just a single alpha gene (-alpha) and the output so poor that the presence of one beta-thal gene does not significantly change the clinical picture. PMID- 3006225 TI - Scandinavian symposium on herpes virus infections and acyclovir (Zovirax). Copenhagen, Denmark, March 13-15, 1985. PMID- 3006224 TI - Myeloperoxidase-deficient polymorphonuclear leucocytes (VII): Incidence in untreated myeloproliferative disorders. AB - In 98 patients with chronic myeloproliferative disorders (45 chr. myeloid leukaemia (CML), 19 myelofibrosis primaria (MP), 28 polycythaemia vera (PV) and 6 idiopathic thrombocythaemia (IT)) the incidences of increased numbers of MPO deficient polymorphonuclear (PMN) were 60% in CML, 32% in MP, 7% in PV and 0% in IT patients. The CML figure differed significantly from the others (p less than 0.001). This study confirms the finding of low NAP scores in CML compared to normal or high NAP scores in the other groups of the myeloproliferative syndrome. The incidences of increased numbers of MPO-deficient PMN in this study are comparable to those found in the primary myelodysplastic syndromes and in acute myeloid leukaemia. The finding supports the view that some of the CML cases and may be other cases of the chronic myeloproliferative disorders may be fundamentally the same disease as in primary myelodysplastic syndromes and in acute myeloid leukaemias. PMID- 3006226 TI - Acyclovir treatment in primary Epstein-Barr virus infection. A double-blind placebo-controlled study. AB - Acyclovir (ACV), which effectively inhibits in vitro Epstein-Barr virus (EBV) production, was tested in 31 patients with clinical and laboratory diagnosis of infectious mononucleosis (IM) in a double-blind trial. Patients with symptoms not exceeding 7 days were randomised to intravenous ACV 10 mg/kg or placebo (PLO) treatment every 8 h for 7 days. Clinical, virological and immunological parameters were followed in each patient before, during and after treatment. There were no significant differences (p greater than 0.05) between the treatment groups for any single clinical symptom between the treatment groups. If data concerning duration of fever, weight loss, tonsillar swelling, sore throat and patient self-assessment of general health were combined, a significant effect (p less than or equal to 0.01) favouring ACV treatment was determined. ACV significantly inhibited oropharyngeal EBV shedding (p less than or equal to 0.001), but the salivary EBV titer returned within 3 weeks after cessation of treatment. Specific cellular and humoral immunity was not affected, nor was the development of virus latency. PMID- 3006227 TI - Herpesvirus infections in the immunocompromised patient. AB - The herpes group viral infections in the T cell immunocompromised host may be seen as a disturbance of a delicate balance between the host and well adapted commensals. The clinical aspect of this disturbance is highly variable but may cause the destruction of the host due to widespread organ damage or prolonged debilitating illnesses. An important aspect is the immunosuppressive effect of the infections themselves leading to a number of superinfections, often with unusual pathogens, difficult to treat. Further problems arise from the potential onchogenic effect of some of these viruses such as EBV induced lymphomas. On the threshold to the era of effective antiviral chemotherapy, two major problems are still present: identification of the precise immunological mechanisms controlling the latency/reactivation of the different viruses and the establishment of reliable methods for rapid diagnosis, crucial for the treatment of patients with atypical clinical presentation and atypical serological responses. PMID- 3006228 TI - Acyclovir prophylaxis in bone marrow transplant recipients. AB - Forty-two patients undergoing bone marrow transplantation were included in a randomised, double-blind and placebo controlled trial of prolonged acyclovir prophylaxis against infections with viruses of the herpes group. Twenty patients were allocated to receive acyclovir and 22 to receive placebo. Acyclovir or placebo was administered i.v. at a dose of 250 mg/m2 twice daily, starting 5 days before transplantation. At 5 weeks after transplantation, administration was changed to tablets, 400 mg three times daily (children less than 6 years, 200 mg three times daily) and continued until 6 months after transplantation. In the placebo group, 10 acute herpes simplex virus (HSV) infections occurred in 7 patients (5 HSV-1 and 2 HSV-2), and another patient repeatedly shed HSV in throat washings. Five patients developed herpes zoster. Among patients receiving acyclovir only one episode of HSV infection occurred and no herpes zoster. The difference in the number of infection episodes and the number of infected patients was strongly significant (p = 0.0002 and 0.0017, respectively). The only acyclovir patient who reactivated HSV was terminally ill, and it is highly likely that she did not absorb a sufficient amount of the orally administered drug to control infection. All HSV and varicella zoster virus (VZV) infections were reactivations, and 9 of 10 patients who developed HSV infections or shed virus had a pre-transplantation HSV IgG titer of greater than 10 000 (ELISA). Acyclovir had no effect on cytomegalovirus (CMV), time of engraftment, or graft versus host disease (GVHD). Apart from a possible allergic reaction (skin rash) to acyclovir tablets, no adverse reactions were seen during this long prophylaxis with acyclovir. PMID- 3006229 TI - Sensitivity of HSV strains isolated before and after treatment with acyclovir. AB - Sequential isolates of herpes simplex virus (HSV) from 5 patients, treated for six episodes with orally administered acyclovir, were tested for in vitro sensitivity to the drug. In addition, isolates from an acyclovir treated immunocompromised patient with oral and genital herpes infections were examined for sensitivity to acyclovir. All isolates were tested in a production (24 h yield) inhibition assay and defined as sensitive or resistant by the parallel examination of thymidine kinase (TK) positive and negative reference strains of HSV. No evidence for decreased or altered sensitivity to acyclovir was recorded of the genital HSV strains, isolated during seven episodes of recurrences. However, in agreement with the clinical response to therapy, genital isolates obtained from the immunocompromised patient pre- and post-treatment with acyclovir displayed in vitro sensitivity values indicating a rapid and complete development of resistance. PMID- 3006230 TI - Viral resistance, clinical experience. AB - Over 1500 herpes simplex virus isolates from over 600 patients have been examined in the Wellcome Research Laboratories during the past 5 years using the dye uptake method in Vero cells to determine acyclovir sensitivity. No significant change in sensitivity of those isolates to acyclovir has been noted during that period, and the few isolates whose sensitivity significantly diminished during therapy were generally not associated with a clinical lack of response to therapy. In patients who were severely immunocompromised and who had received prolonged or repeated courses of therapy, however, less sensitive viruses were occasionally associated with poorly healing ulcers. The significance of these findings are discussed, as are problems in the interpretation of results of in vitro antiviral sensitivity testing and their extrapolation to clinical practice. PMID- 3006231 TI - Present and future of acyclovir. AB - Acyclovir is now established as an effective and well tolerated therapeutic agent for the management of at least the more common infections of the herpes virus group. Evaluation of the drug nevertheless continues, primarily to verify its value in those infections caused by cytomegalovirus (CMV) and Epstein-Barr virus (EBV). Furthermore with the development of analogues of acyclovir with better absorption profiles or enhanced anti-viral activity the future for this area of anti-viral therapy looks optimistic. PMID- 3006232 TI - Laboratory diagnosis of herpesviruses. AB - Laboratory diagnosis is made either by demonstration of the presence of virus, viral antigens or virus-specific nucleic acid sequences in suitable specimens or by measurement of antibodies--rise in titer or presence of viral-specific IgM--in blood samples from the patients. A number of biological features characteristic for the herpes viruses can be utilised for detection: the infectivity (isolation in tissue culture), the morphology (visualisation by electronmicroscopy), the antigenicity of viral specified proteins produced by infected cells (immunofluorescence, ELISA and RIA), and unique sequences in viral DNA (hybridisation). The different methods are described and evaluated in regard to viral detectability and speed of performance. Concerning diagnostic serology only the ELISA systems for measurement of viral-specific IgG, total antibodies and IgM have been described because these methods now have been introduced in most viral diagnostic laboratories, and many commercial available kits also operate according to these techniques. PMID- 3006233 TI - Herpesvirus infection in man. AB - Herpesviruses which affect man are herpes simplex virus type 1 and type 2, varicella-zoster virus, cytomegalovirus and Epstein-Barr virus. The review deals with the more common clinical manifestations of human herpesvirus infections, which occur ubiquitously in all populations throughout the world. Primary infections most commonly occur in childhood. It is a characteristic feature of herpesvirus that they generally remain in a latent form after clearance of the primary infection. The overall majority of clinical problems are related to activated latent infections, viz. recurrent lesions of herpes simplex, herpes zoster with remaining neuralgia in older patients, and in particular herpesvirus infections and reactivation in immunodeficient individuals. Herpes infection during pregnancy may result in severe generalised infection of the newborn. A possible relationship between herpesvirus and cancer, especially between herpesvirus type 2 and cervical cancer and between Epstein-Barr virus and Burkitt's lymphoma are of major interest today. PMID- 3006234 TI - Genital herpes. AB - Due to increasing frequency genital herpes has received much attention in medical as well as non-medical literature throughout the Western world. This article concentrates on reviewing the various clinical manifestations of the disease. Different diagnostic methods, prophylactic and therapeutic measures (with the exception of acyclovir) are discussed, as well as transmission and spread of the virus. PMID- 3006235 TI - Virological aspects on herpesvirus infections. AB - The family herpesviridae include five important human pathogens, herpes simplex virus types 1 and 2 (HSV-1, HSV-2), varicella-zoster virus (VZV) and cytomegalovirus (CMV). The five viruses are represented in all three herpesvirus subfamilies, a subdivision based on differences in phenotypic properties such as length of replication cycle, cytotoxic properties and host cell range. The herpesviruses have DNA genomes sharing characteristic anatomical features. They replicate in the nuclei of eukaryotic cells and produce virions consisting of enveloped icosahedral nucleocapsids. Although all herpesviruses can establish latent infections in selected target cells, conditions for establishment and maintenance of latency vary between the members of the herpesvirus family. PMID- 3006236 TI - [Diagnostic tests in parathyroid diseases]. AB - The diagnosis of primary hyperparathyroidism (PHP) depends increasingly on laboratory tests, since the majority of patients are elderly people without typical symptoms. A mean plasma calcium level close to the upper normal limit serves to diagnose hypercalcemia. To rule out malignant disease, the most common cause of hypercalcemia, measurement of plasma PTH is the most appropriate test. Determination of blood phosphorus, chloride, and alkaline phosphatase, and of urinary calcium and phosphorus, contribute to the investigation of the metabolic effects of the given disease but are not very useful for causal diagnosis. Urinary and nephrogenous cyclic AMP reflect PTH secretion but can be elevated in paraneoplastic hypercalcemia. Diagnosis of subtle forms of PHP by dynamic tests is largely of scientific interest, since they do not necessarily need treatment. The diagnosis of hypoparathyroidism is primarily clinical. PTH measurements rarely distinguish normal from low values. In severe hypocalcemia of non parathyroid origin, plasma PTH is elevated (except in hypomagnesemia). In borderline cases, measurement of urinary cyclic AMP or of plasma PTH after attempted stimulation by EDTA infusion is helpful, especially in distinguishing between subtle hypoparathyroidism and tetany induced by hyperventilation. PMID- 3006237 TI - [Kala-azar imported from Yugoslavia]. AB - Two months after a vacation in Bosnia and at the Dalmatian coast, a 52-year-old Jugoslav male resident in Switzerland developed slowly progressive fever with arthralgia. Two months later his temperature became septic and his general condition deteriorated. After many wrong diagnostic tracks, six months after onset of the illness kala-azar was finally diagnosed. Treatment with Pentostam cured the disease completely. PMID- 3006238 TI - [Hypothalamic factors: current findings]. AB - The last few years have seen the isolation, characterization and synthesis of two new hypothalamic peptides, the growth-hormone releasing factor (GRF) and the corticotrophin-releasing factor (CRF). GRF selectively stimulates pituitary growth hormone. It is interesting to note that GRF was isolated from two pancreatic tumors which were responsible for an acromegalic condition in the two patients. Hypothalamic GRF (1-44) has been found to be identical to this pancreatic GRF. CRF was isolated first from ovine hypothalamus and later characterized in several species and in the human. CRF specifically stimulates the secretion of ACTH and of beta-endorphins, and other fragments of the common precursor named pro-opiomelanocortin. Chemical synthesis of analogues of hypothalamic peptides, and in particular of GnRH (gonadotrophin-releasing hormone), has made available new molecules which form agonists as well as antagonists. After a short period of gonadotrophin stimulation GnRH agonists induce desensitization of the pituitary and a decrease in secretion of the gonadotrophins and the sex steroids by the gonads. Their usefulness is presently being tested in several conditions such as prostate cancer, endometriosis, breast cancer and idiopathic precocious puberty, and as a contraceptive method. On the other hand, pulsatile administration of GnRH restores deficient reproductive functions in certain conditions such as anovulation or azoospermia. PMID- 3006239 TI - [Carbohydrates and dietary fiber in the diabetic diet]. AB - Dietary fibre has a blood-glucose reducing effect, as is manifested by a diminished glycemic index. In 1980 the consumption of dietary fibre in Switzerland is 22 g/day in comparison to 27 g in 1956. Thirty-five patients with diabetes (15 with insulin and 20 without) were treated with a high carbohydrate high fibre diet (55% of total calories) for a period of 12 months. There were no changes in postprandial blood glucose values, HbA1, cholesterol or bodyweight (in insulin-treated patients the weight decreased p less than 0.05) despite a high carbohydrate diet. PMID- 3006240 TI - The molecular genetics of hemophilia. PMID- 3006241 TI - Two elements in the bovine leukemia virus long terminal repeat that regulate gene expression. AB - The bovine leukemia virus, like the human T-cell leukemia viruses (HTLV-I and HTLV-II), are unusual biologically in that viral transcripts are not detected in tumors or infected tissues. The bovine leukemia virus long terminal repeat (BLV LTR) functions as a transcriptional promoter only in cell lines productively infected with BLV. Deletion mapping indicated that at least two regions of the LTR, on the 5' and 3' sides of the RNA start site, influenced gene expression. An analysis has now been made of the effects of coupling sequences from these LTR regions to a heterologous core promoter derived from the SV40 early promoter unit. Through the use of the transient expression of the bacterial chloramphenicol acetyltransferase (CAT) gene to monitor transcriptional activity in vivo, two independent, regulatory elements were identified in the BLV LTR. One was present in a fragment of 75 base pairs derived from the U3 region of the LTR and behaved much like other enhancer elements. It may be a major determinant of BLV expression in productively infected cell lines, since it enhanced transcription controlled by the heterologous core promoter only in these cells. The second element was contained in a 250-bp fragment derived from LTR sequences in the R region, located downstream from the RNA start site. Its activation of CAT expression was not dependent on BLV infection and was evident only when the fragment was located immediately downstream from the RNA start site. BLV expression thus appears to be regulated in part by a cell-specific enhancer element upstream from the core promoter and a novel sequence downstream from the RNA initiation site in the viral LTR. PMID- 3006242 TI - AIDS drug shows promise in preliminary clinical trial. PMID- 3006243 TI - A new HTLV-III/LAV protein encoded by a gene found in cytopathic retroviruses. AB - The DNA of the HTLV-III/LAV group of retroviruses contains certain additional open reading frames that are not found in typical avian or mammalian retroviruses. The role of these sequences in encoding for gene products that may be related to pathogenesis remains to be resolved. An open reading frame whose 5' end overlaps with the pol gene, but is unrelated to the env gene, has been observed in HTLV-III/LAV and visna virus, both cytopathic mammalian retroviruses. Evidence presented here shows that this open reading frame is a bona fide coding sequence of HTLV-III/LAV and that its product, a protein with a molecular weight of 23,000, induces antibody production in the natural course of infection. PMID- 3006244 TI - Replicative and cytopathic potential of HTLV-III/LAV with sor gene deletions. AB - The genome of the human T-lymphotropic virus type III (HTLV-III/LAV) has the potential to encode at least three polypeptides in addition to those encoded by the gag, pol, and env genes. In this study, the product of the sor (short open reading frame) region, which overlaps the 3' end of the pol gene, was found to be a protein with a molecular weight of 23,000. An assay was developed for testing the ability of cloned HTLV-III proviruses to produce viruses cytopathic for T4+ lymphocytes. In the cell line used, C8166, neither the HTLV-III sor gene product nor the complete 3'-orf gene product were necessary for the replication or cytopathic effects of the HTLV-III. PMID- 3006245 TI - Identification of HTLV-III/LAV sor gene product and detection of antibodies in human sera. AB - The nucleotide sequence of the genome of HTLV-III, the infectious agent etiologically associated with the acquired immune deficiency syndrome, predicts a small open reading frame, termed sor, located between the pol and env genes. A DNA segment containing 82 percent of the sor region was inserted into a prokaryotic expression vector, pJL6, to determine whether sor encodes a viral protein and to gain some insight into its possible function. The bacterially synthesized sor protein reacted with sera from individuals infected with HTLV III, indicating that sor is expressed as a protein product or products that are immunogenic in vivo. Antibodies to the purified, bacterially synthesized sor protein were found to react specifically with the same protein and also with a protein of molecular weight 23,000 (23K) in HTLV-III-infected H9 cell extracts. The 23K protein comigrated with a protein immunoprecipitated by the serum of a hemophiliac patient with antibodies to HTLV-III, suggesting that this protein is probably the sor gene product. PMID- 3006246 TI - Antiserum to a synthetic peptide recognizes the HTLV-III envelope glycoprotein. AB - In a study performed to determine which regions of the human T-cell lymphotrophic virus type III (HTLV-III) may represent vaccine candidates to prevent the acquired immune deficiency syndrome (AIDS), a synthetic peptide corresponding to amino acid sequence 735 to 752 of the precursor envelope glycoprotein of HTLV-III was used to immunize rabbits. The resulting rabbit antiserum to the synthetic peptide specifically recognized the precursor envelope glycoprotein (gp160) of HTLV-III. Human sera positive for antibody to HTLV-III reacted with this peptide. These findings indicate that synthetic peptides can be used to induce an immune response directed against a native envelope glycoprotein epitope of HTLV-III. The data are discussed in terms of using synthetic peptides to identify antigenic determinants involved in the induction of protective immunity and possibly as vaccine candidates against the etiologic agent of AIDS. PMID- 3006248 TI - Inhibition of vasopressin action by atrial natriuretic factor. AB - Atrial natriuretic factor results in diuresis in animals and humans, perhaps because atrial natriuretic factor increases renal blood flow. The possibility that this diuresis is due to direct inhibition of renal tubular epithelial water transport was examined in rabbit collecting tubules perfused in vitro. Atriopeptin III inhibition of the hydraulic conductivity response to the hormone arginine vasopressin but not to either 3'5'-cyclic adenosine monophosphate or forskolin was found. These results suggest that atriopeptin III acts proximal to cyclic adenosine monophosphate formation to directly affect vasopressin stimulated water transport in the mammalian nephron. They also suggest a potential role for inhibition by atrial natriuretic factor of the renal response to arginine vasopressin as a contributor to a diuretic state. PMID- 3006247 TI - Nucleotide sequence of SRV-1, a type D simian acquired immune deficiency syndrome retrovirus. AB - Simian acquired immune deficiency syndrome (SAIDS) in the macaque genus of monkeys at the California Primate Research Center is apparently caused by infection by a type D retrovirus. The complete nucleotide sequence (8173 base pairs) of a molecular clone of the prototype SAIDS virus isolate, SRV-1, reveals a typical retrovirus structure with long terminal repeats (346 base pairs) and open reading frames for the gag (663 codons), pol (867 codons), and env (605 codons) genes. SRV-1 also has a separate open reading frame of 314 codons between the gag and pol genes that defines the viral protease gene (prt) and a short open reading frame of unknown significance downstream from the env gene. The SRV-1 protease region shows a high degree of homology to its counterpart in the hamster intracisternal A-type particle genome; both these protease genes are about twice as long as the analogous region of other retroviruses. SRV-1 has no notable similarity in either genetic organization or sequence to the human AIDS retroviruses. PMID- 3006249 TI - Tissue-specific expression in transgenic mice of a fused gene containing RSV terminal sequences. AB - Transgenic mice were generated with pRSV-CAT, a chimeric gene construct containing the long terminal repeat of Rous sarcoma virus (RSV) linked to the bacterial gene encoding chloramphenicol acetyltransferase (CAT). CAT expression, detected in adult animals of five independent strains, was preferentially directed to organs rich in tendon, bone, and muscle. This pattern reflects the disease specificity of the intact virus and suggests that the tissue tropism of RSV is determined at least in part by the presence of endogenous tissue-specific factors that can promote expression of genetic information linked to the long terminal repeat. In two of the mouse strains, insertion of the pRSV-CAT DNA resulted in developmental abnormalities. One of these strains was characterized by a dominant trait of embryonic lethality, the other by a recessive trait of fused toes in all four feet. PMID- 3006250 TI - Human beta-adrenoceptors: relation of myocardial and lymphocyte beta-adrenoceptor density. AB - In human right atria obtained from 21 patients during open-heart surgery, beta adrenoceptor density [assessed by iodine-125-labeled (-)-cyanopindolol binding] and responsiveness (positive inotropic responses to isoprenaline) were linearly related to the beta-adrenoceptor density in the corresponding circulating lymphocytes. This direct relation of human myocardial and lymphocyte beta adrenoceptor alterations, therefore, makes it possible to monitor drug- or disease-induced beta-adrenoceptor changes in tissues not easily accessible in humans. PMID- 3006252 TI - Vegetables, fruits, and oncologists. PMID- 3006251 TI - Perturbation of red cell membrane structure during intracellular maturation of Plasmodium falciparum. AB - An experimental approach, which in this study was applied to the malarial system, can be used to analyze the molecular structure and organization of individual phospholipids in a wide variety of biological membranes. Electron spin resonance spectroscopy was used to investigate the structural modifications of the major red cell phospholipids that occur in erythrocyte membranes infected with the human malarial parasite, Plasmodium falciparum. These modifications were correlated with the intracellular developmental stage of the parasite. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine were increasingly disordered (fluidized) as infection progressed. This disordering occurred at different rates and to varying extents. PMID- 3006253 TI - In vivo competition between a metallothionein regulatory element and the SV40 enhancer. AB - The human metallothionein-IIA (hMT-IIA) gene contains an enhancer element within its 5' regulatory region. This enhancer element can compete with the SV40 enhancer for one or more cellular factors in vivo. The competition between the two elements is modulated by cadmium, an inducer of hMT-IIA transcription. The data presented are consistent with a model in which heavy metal ions control the ability of the hMT-IIA enhancer to bind a positive factor, leading to increased transcription. The same factor is required for maximal activity of the SV40 enhancer, which suggests that viruses utilize factors that have a normal role in cellular gene expression to control their own genes. PMID- 3006254 TI - Receptor-coupled activation of phosphoinositide-specific phospholipase C by an N protein. AB - Cleavage of phosphatidylinositol 4,5-bisphosphate by phospholipase C results in the production of two important second messengers: inositol-1,4,5-trisphosphate and 1,2-diacylglycerol. Although several receptors promote this cleavage, the molecular details of phospholipase C activation have remained unresolved. In this study, occupancy of a Ca2+-mobilizing receptor, the oligopeptide chemoattractant receptor on human polymorphonuclear leukocyte plasma membranes, was found to lead to the activation of a guanine nucleotide regulatory (N) protein by guanosine 5' triphosphate. The activated N protein then stimulated a polyphosphoinositide specific phospholipase C by reducing the Ca2+ requirement for expression of this activity from superphysiological to normal intracellular concentrations. Therefore, the N protein-mediated activation of phospholipase C may be a key step in the pathway of cellular activation by chemoattractants and certain other hormones. PMID- 3006255 TI - New relatives of AIDS virus found. PMID- 3006256 TI - New human T-lymphotropic retrovirus related to simian T-lymphotropic virus type III (STLV-IIIAGM). AB - This report describes serologic evidence for a virus similar to that known as simian T-lymphotropic virus type III of African Green monkeys (STLV-IIIAGM) infecting apparently healthy people in Senegal, West Africa, and the isolation of virus from these individuals. Serum samples from selected healthy West African people showed unusual serologic profiles when tested with antigens of HTLV III/LAV, the etiologic agent of AIDS, and of STLV-IIIAGM. The samples reacted strongly with all of the major viral antigens of STLV-IIIAGM but showed variable or no reactivity with the major viral antigens of HTLV-III/LAV by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A new human T-lymphotropic virus (HTLV-IV) isolated from these people was grown in vitro and shown to have retroviral type particles, growth characteristics, and major viral proteins similar to those of the STLV-III and HTLV-III/LAV group of retroviruses. The gp120/160, gp32, p64, p55, p53, p24, and p15 proteins precipitated were the same size as and reactive with STLV-IIIAGM proteins. The serologic data suggest that this virus shares more common epitopes with STLV-IIIAGM than with the prototype HTLV-III/LAV that infects people in the United States and Europe. Further study of this virus and of the origin of the HTLV-III/LAV group of viruses may expand our understanding of the human AIDS virus. PMID- 3006257 TI - Malignant astrocytic neoplasms: classification, pathologic anatomy, and response to treatment. PMID- 3006258 TI - Biology of gliomas: heterogeneity, oncogenes, growth factors. PMID- 3006259 TI - The role of positron emission tomography in the study of cerebral tumors. PMID- 3006260 TI - Social networks and the spread of infectious diseases: the AIDS example. AB - Conceptualizing a population as a set of individuals linked together to form a large social network provides a fruitful perspective for better understanding the spread of some infectious diseases. Data related to AIDS (the acquired immune deficiency syndrome) were used to illustrate the potential usefulness of a network approach in evaluating the infectious agent hypothesis when studying a disease or disease outbreak of unknown etiology and in developing strategies to limit the spread of an infectious agent transmitted through personal relationships. PMID- 3006262 TI - [Peripheral neuropathies. General facts]. PMID- 3006261 TI - [Clinical signs and nursing care in peripheral neuropathies]. PMID- 3006263 TI - Herpes without vesicles: limited, recurrent genital lesions in an immunodebilitated host. AB - We have reported a case of herpes genitalis in a man with acquired immunodeficiency syndrome (AIDS), who was receiving chemotherapy for Hodgkin's disease. The herpes infection was recurrent but limited, without vesicles or the progressive lesions usually seen in AIDS. Studies excluded known causes, and the unusual character of the infection long obscured and delayed diagnosis. Even atypical, culture-negative penile ulcers in immunodebilitated patients may be herpetic. Retrospective study of our patient's penile biopsy specimen using immunoperoxidase reaction was positive for herpesvirus. The existence of effective treatment mandates vigorous pursuit of the diagnosis. PMID- 3006264 TI - Sonographic diagnosis of a liver cell adenoma. AB - As shown by the case we have reported, ultrasonography should be the first imaging method used in a young woman with pain in the right upper quadrant and a history of contraceptive use. Possible biliary system abnormalities, a more common cause of pain in the right upper quadrant, can be simultaneously evaluated. PMID- 3006265 TI - Malignant fibrous histiocytoma manifesting as a cavitary lung metastasis. AB - Exploration prompted by acute abdominal symptoms in a 29-year-old woman discovered to have an asymptomatic right upper lobe cavitary lesion disclosed a malignant fibrous histiocytoma, the primary lesion of which was in the duodenum. At autopsy, the lung lesion was found to be metastatic. We believe this is the first recorded instance of cavitary lung metastasis from this type of tumor. PMID- 3006266 TI - Susceptibility to varicella-zoster virus in Thai children and young adults. AB - During 1982-1983, susceptibility to varicella-zoster virus (VZV) in 224 Thai subjects at high risk for varicella infection was studied. The immune adherence hemagglutination (IAHA) and VZ skin test were carried out to determine VZV immunity in immunocompromised children and young adults. The history of varicella and herpes zoster from each subject was recorded. The mean +/- SD age in children and young adults were 7.3 +/- 2.8 and 19.6 +/- 1.2. Negative IAHA test was found in 74.2% of 62 children and 35.2% of 162 young adults. The increase in immune individuals was demonstrated with advancing age. Response to VZ skin test showed positive results in 79 of 162 (48.8%) young adults. The seronegativity was related to the negative VZ skin test (p less than 0.001, X2 test). Regardless of antibody detection or VZ skin test, 47 of 162 (29%) young adults were susceptible. According to the positive history of varicella and of herpes zoster obtained from 95 young adults, 80% had developed varicella during 1 to 10 years of age and 8.8% had positive history of herpes zoster. The findings suggest that the IAHA and VZ skin test should be used together for assessing VZ immunity. Varicella vaccination is highly recommended for susceptible persons who may develop severe illness. PMID- 3006268 TI - The influence of the solvent on the toxicity and radioprotective effect of thiophosphate WR2721 in mice. AB - The influence of various solvents on the toxicity as well as the protective efficiency of thiophosphate WR2721 in X-irradiated male mice is described. The use of Sorensen buffer as solvent with a pH-value of 7.4 leads to a statistically significant lower lethality in animals than aqua bidest. However, the radiation efficacy of thiophosphate with respect to the acute lethality within 30 days after irradiation is not influenced by the choice of the solvent used. A dose reduction factor of 2.49 +/- 0.03 is gained for the LD50/30 d at 500mg WR2721 per kg animal weight. PMID- 3006267 TI - [Use of a sodium bicarbonate jet sprayer and oro-tracheo-bronchial lesions]. PMID- 3006269 TI - [Combined therapy of tumors in adults]. AB - A significant amelioration of treatment results is achieved by sequential chemotherapy and radiotherapy in patients with lymphogranulomatosis of stage IIb to IVb and in patients with non-Hodgkin's lymphomas in corresponding stages. Similar results will probably be obtained in patients with small cell bronchial carcinomas in a limited stage. Patients suffering from an initially inoperable ovarian cancer often reach an operable condition by sequential chemotherapy and radiotherapy. In the stages Dukes B2 and C of the rectum carcinoma, preoperative and/or postoperative irradiation significantly reduces the recurrence rates and increases the survival times. A considerable reduction of recurrence rates is obtained by postoperative radiotherapy in soft tissue sarcomas of the stages T1 to T3. Another improvement is anticipated by a neutron or neutron boost irradiation for stage T3 and by adjuvant chemotherapy for G3 tumors. In the osteosarcoma of adult persons, the results of the limb-sparing sequential therapy will not be worse than the results achieved by amputation. Retrospective analyses of the long-term results of radical mastectomy and conservative operation with postoperative irradiation in case of mammary carcinoma did not show any difference for the stages T1 to T3, N0 to N1. PMID- 3006270 TI - Pediatric ocular sarcoidosis. AB - Sarcoidosis is an uncommon cause of childhood uveitis. However, the ophthalmologist familiar with the clinical features of childhood sarcoidosis can play a key role in the diagnosis and treatment of this disorder. Two subsets of pediatric sarcoidosis are identified. The 8-15 year age group has almost universal lung involvement, with the eye, skin, liver, and spleen involved in 30 40% of cases. Children 5 years of age and under are characterized by the triad of uveitis, arthropathy, and skin rash. The epidemiology, clinical features, diagnostic evaluation, and ocular management of pediatric sarcoidosis are reviewed. The clinical and laboratory findings that distinguish sarcoidosis from other causes of childhood uveitis are discussed. PMID- 3006271 TI - Factors relating to the sensory acuity of limbs with peripheral vascular insufficiency. AB - We examined several possible causes for the high incidence of poor sensory acuity in the limbs of 176 patients with moderate to severe peripheral vascular insufficiency. We investigated the relationships of diabetes, alcoholism, and smoking, as well as the severity of peripheral vascular disease, to the integrity of basic sensory modalities such as two-point discrimination and perception of light touch. The presence or absence of diabetes exerted the strongest effect on peripheral sensation. In patients who did not have diabetes, sensation in the limbs was most strongly affected by whether the patient was an alcoholic. Smoking did not have a significant effect on limb sensation. Among nondiabetic, nonalcoholic patients, there was a weak residual effect related to the severity of the peripheral vascular insufficiency. Even among these patients, however, systemic factors predominated in determining the loss of sensation. We also examined the extent to which loss of sensation might be related to the development of ulcers. Among patients who were not diabetic, there was a highly significant relationship between loss of sensation and the presence of limb ulceration. Surprisingly, however, there was no discernable relationship between the presence of ulcers in diabetic patients and the degree of loss of peripheral sensation. This result suggests that a large percentage of ulcers seen in diabetic patients are not of neurogenic origin. PMID- 3006272 TI - Surgical treatment of 109 patients with symptomatic and asymptomatic hepatocellular carcinoma. AB - A total of 109 patients with histologically proved hepatocellular carcinoma (HCC) have undergone hepatic resection during the 56-month period from October 1978 to May 1983. There were two sources of patients: those with symptomatic HCC (n = 47) and those with asymptomatic HCC (n = 62). A family tendency of HCC was noted in 11% of the patients studied. The percent of positive hepatitis B surface antigen (HBsAg) was 87%, and the serum alpha-fetoprotein was less than 20 ng/ml in 30% in the group with symptoms. The operative mortality rate was 3% and the hospital mortality rate was also 3%. The postoperative course was complicated with pleural effusion in 10%, bile leakage in 4%, subphrenic abscess in 4%, and upper gastrointestinal bleeding caused by gastritis in 1% of the patients. The actual survival rate for the 103 cases was 84% for 350 days and 28% for 1400 days. However, in the group with asymptomatic HCC with an average tumor size of 3.35 +/ 1.49 cm in diameter, the rate was 92% for 350 days and 44% for 1400 days. In the group with symptomatic HCC with an average tumor size of 10.6 +/- 5.1 cm in diameter, the rate was 76% for 350 days and 8% for 1400 days. The survival rate of the group with asymptomatic HCC was far better than that of the group with symptoms (p less than 0.05). In analysis of factors that might affect the patient's survival, only second or third operations (p less than 0.05), typical gross findings of tumor appearance (p less than 0.05), and an adequate margin were closely related (p less than 0.001). Neither the tumor size, the status of accompanying liver cirrhosis, the tumor location, nor the patient's sex and age affected the patient's survival (all p greater than 0.05). PMID- 3006273 TI - [AIDS--this century's catastrophe? 1. Origin and cause]. PMID- 3006274 TI - [New substance for treatment of herpes infection]. PMID- 3006275 TI - Immunohistochemical evidence of papillomavirus antigen in focal epithelial hyperplasia. PMID- 3006276 TI - [Current status of AIDS]. PMID- 3006277 TI - Bronchioloalveolar carcinoma arising in longstanding lung cysts. PMID- 3006278 TI - [Preventive measures against HTLV-III/LAV infections among hemophiliacs and their relatives]. PMID- 3006279 TI - [Feline leukemia: current status regarding prevention]. AB - Particularly as a result of the increasing popularity of keeping cats as pets, feline leukaemia constituted a risk to the cat population for many years; it was uncontrollable because of its insidious course and the spread of the virus, which passed unnoticed. During the past decade, diagnostic laboratory tests resulted in progress in the control of the infection for the first time. Dissemination of the virus could then at least be checked to some extent. Prevention by vaccination did not appear to be possible for many years. Promising developments thanks to biotechnical progress and experiments with other retrovirus systems were only recently reported. A vaccine for use in practice is currently being launched in the United States and different countries in Europe. It remains to be seen whether this vaccine, the manufacture of which is quite expensive, will prove successful and whether new developments will occur in the near future, which will bring further improvements concerning protection and reduction of the cost of production. PMID- 3006280 TI - [Big spleen, big problems]. AB - The history of a Moroccan girl is described with splenomegaly, lymphadenopathy and pancytopenia after a holiday in her native country. Bone marrow smears were considered negative for Leishmaniasis in four different laboratories. All other diagnostic options could also not be confirmed. Reexamination of the bone marrow smears in a laboratory for tropical diseases revealed Leishmania donovani organisms. Treatment with sodium antimony gluconate was successful. Epidemiology, symptoms and diagnostic problems are discussed. PMID- 3006281 TI - Polymorphisms within the HLA-DR4 haplotypes. Various DQ subtypes detected with monoclonal antibodies. AB - Polymorphisms among HLA class II molecules expressed by cells with different HLA DR4 haplotypes were analysed biochemically (isoelectrofocussing and 2D gels), cellularly (HLA-Dw) and serologically (monoclonal antibodies). The results confirm the correlation which exists between HLA-D specificity and DR beta chain isoelectric point polymorphism. Furthermore, a biochemical polymorphism was observed among DQw3 molecules. No correlation was found with HLA-Dw types. On the other hand a correlation was found between DQ-polymorphism and TA10 and 2B3 specificities defined by monoclonal antibodies. The comparison of different methods defining polymorphisms of HLA class II molecules will be discussed. PMID- 3006283 TI - The effects of protease inhibitor homologues from mamba snake venoms on autonomic neurotransmission. AB - Five protease inhibitor homologues isolated from mamba venoms were tested for facilitatory actions on autonomic neurotransmission using isolated smooth muscle preparations. Dendrotoxin from the eastern green mamba (Dendroaspis angusticeps) was the most consistent in augmenting the responses to sympathetic stimulation in vas deferens preparations and to parasympathetic stimulation in chick oesophagus preparations. Toxin I from the black mamba (D. polylepis) venom augmented the neurally evoked responses in vas deferens preparations, and toxin K from the same venom augmented neurally evoked responses in chick oesophagus preparations. Proteins B and E from D. polylepis venom, as well as bovine pancreas trypsin inhibitor, had no significant facilitatory action on either smooth muscle preparation. The mechanism of the augmentation of neurally evoked responses produced by toxin I on vas deferens preparations, and dendrotoxin on chick oesophagus preparations, was investigated using a variety of drugs which interfere with cholinergic and adrenergic transmission. It is concluded that dendrotoxin and toxin I increase evoked transmitter release in the autonomic nervous system by a direct action on nerves. PMID- 3006282 TI - Functional hepatocellular heterogeneity determined by the hepatotoxins allyl alcohol and bromobenzene in immature and adult Fischer 344 rats. AB - Adult (male, 75-90 days old) and immature rats (both sexes, 11-12 days old) were treated with allyl alcohol or bromobenzene to induce periportal or centrilobular hepatic injury, respectively. Histologically confirmed liver lesions were produced in adult rats with both treatments. In adult rats, allyl alcohol decreased hepatic cytochrome P-450, benzphetamine N-demethylation, and ethoxyresorufin O-deethylation activities all by about 30%, whereas bromobenzene influenced these parameters differently: cytochrome P-450 was lowered by 55%, benzphetamine N-demethylation by 80%, and ethoxyresorufin O-deethylation by 90%. Cytochrome c reductase, 5'-nucleotidase, glucose-6-phosphatase, and glutamate pyruvate transaminase activities were not significantly influenced. In immature rats, allyl alcohol did not produce histopathological alterations in liver, but did lower both cytochrome P-450 concentration (30%) and ethoxyresorufin O deethylation (75%). Benzphetamine N-demethylation was not significantly affected. Bromobenzene produced typical centrilobular liver damage and a decrease of both cytochrome P-450 (20%) and ethoxyresorufin O-deethylation (50%). Benzphetamine N demethylation was increased slightly, but not significantly. The differences in effects of the two hepatotoxins in adult vs immature rats seem to indicate that the hepatocellular heterogeneity of xenobiotic metabolism which is seen in adult liver (perivenous vs periportal areas) is not well developed in the immature animal. PMID- 3006284 TI - Effects of crotoxin on autonomic neuromuscular transmission in the guinea-pig myenteric plexus and vas deferens. AB - The effects of crotoxin, the neurotoxic complex from the venom of the South American rattlesnake Crotalus durissus terrificus on mammalian autonomic neuromuscular transmission, have been investigated. In the longitudinal muscle of the guinea-pig ileum, crotoxin induced a dose-dependent contraction which was followed by relaxation, in spite of the continued presence of the toxin. The contractile response was inhibited by indomethacin, tetrodotoxin, verapamil or nifedipine, but was unaffected by atropine, propranolol, mepyramine or methysergide. In addition, crotoxin caused a presynaptic inhibition of the electrically-evoked twitch of the longitudinal muscle of the guinea-pig ileum. In the guinea-pig vas deferens crotoxin also caused an inhibition of the response to field stimulation. The inhibition was reversible after washing and the preparation remained insensitive to further doses of the toxin. The inhibitory effects of crotoxin were not mediated by noradrenaline and were not due to a non specific smooth muscle depression, because it was not associated with any reduction in motor responses to acetylcholine, ATP, bradykinin or substance P. Pre-incubation of the guinea-pig vas deferens with indomethacin blocked the inhibitory effects of the toxin. This suggests that the presynaptic activity of crotoxin in the vas deferens might be mediated by prostaglandins. PMID- 3006285 TI - Muscle paralyzing effect of the juice from the trunk of the banana tree. AB - The effect of an extract from the trunk of the banana tree (Musa sapientum) was investigated in isolated skeletal muscle preparations from the chick, mouse and frog using twitch tension and intracellular recording techniques. The extract produced, in the same concentration range and after an initial period of twitch augmentation, paralysis of skeletal muscle in both directly and indirectly stimulated preparations. It also had a dose-dependent stimulant effect on the muscle causing a contracture. The neuromuscular blockade was reversed by calcium, but only when added before complete paralysis of the muscle. On the other hand, neostigmine usually hastened the blockade and aggravated the contracture. The frequency of the miniature endplate potential in the mouse phrenic nerve diaphragm preparation greatly increased initially, declining to an elevated plateau. Effects on quantal content of endplate potentials (e.p.p.s) were studied in the transected mouse phrenic nerve-hemidiaphragm using trains of e.p.p.s. In the presence of the extract, only a few e.p.p. trains could normally be evoked, probably due to nerve terminal block. When quantal content could be measured at low concentrations of the extract, an increase was usually obtained. Muscle action potentials in the frog sartorius muscle were decreased in amplitude until no further potentials could be generated. The results suggest that the nature of the block produced by the extract resembles that of a potent local anaesthetic with an initial atypical labilizing effect on cell calcium rather than a conventional curariform block. PMID- 3006286 TI - Lack of effect of tetrodotoxin and of an extract from the posterior salivary gland of the blue-ringed octopus following injection into the octopus and following application to its brachial nerve. AB - Lack of effect of tetrodotoxin and of an extract from the posterior salivary gland of the blue-ringed octopus following injection into the octopus and following application to its brachial nerve. Toxicon 23, 997-999, 1985. Injections of the blue-ringed octopus salivary gland extract and tetrodotoxin into the blue-ringed octopus have no ill-effect on the animals. Similarly, in vitro nerve preparations from the animal were not affected by these materials although they are both extremely potent on bioelectrically excitable preparations from other species. PMID- 3006287 TI - Pathomorphological study on thorotrast-induced hepatic malignancies. PMID- 3006288 TI - Dose distribution and lung cancer incidence in thorotrast patients. PMID- 3006290 TI - Skeletal dosimetry of injected 226Ra in young adult beagles. PMID- 3006289 TI - Activity concentration of exhaled 220Rn and burden of 228Th in workers working at the Bai Yuan iron mine in Innermongolia and in inhabitants living in the high background radiation area in China. PMID- 3006291 TI - Argonne-Utah studies of 224Ra endosteal surface dosimetry. PMID- 3006292 TI - Receptors and recognition mechanisms of Leishmania species. AB - This paper examines briefly receptors and recognition mechanisms involved in binding Leishmania parasites to the lumen of the sandfly gut and to cells of the vertebrate host's mononuclear phagocyte system. In particular, work carried out in our laboratory on the relative roles of the macrophage plasma membrane receptor (CR3) for the cleaved third complement component (iC3b) and the mannosyl/fucosyl receptor (MFR) in binding, ingestion and respiratory burst (RB) response elicited by promastigotes versus amastigotes of L. donovani, is discussed. In the absence of serum, soluble inhibitors (e.g. mannan) of the MFR cause a dose-dependent reduction in promastigote binding to murine resident peritoneal macrophages and in the proportion of bound parasites eliciting a RB response. For amastigotes, no consistent reduction in binding in the presence of mannan is observed but the proportion of parasites eliciting a RB is reduced. Serum-independent binding of promastigotes, which are good activators of the alternative complement pathway, is also inhibited by the anti-CR3 monoclonal antibody M1/70, by Fab anti-C3, and by an inhibitor of C3 fixation, sodium salicyl hydroxamate. With amastigotes, which are poor activators of the alternative pathway, a lesser effect is observed. These results provide strong evidence that cleaved macrophage-derived C3 (iC3b) mediates promastigote binding to CR3. Modulation experiments in which either CR3 or MFR are rendered inaccessible demonstrate that both receptors must be present on the segment of macrophage membrane with which the promastigote makes contact to mediate binding and ingestion. These receptor interactions may be important determinants of the infectivity and survival of Leishmania parasites in the vertebrate host. PMID- 3006293 TI - The effects of silica on lymph nodes and vessels--a possible mechanism in the pathogenesis of non-filarial endemic elephantiasis. AB - Non-filarial tropical elephantiasis, which occurs in certain volcanic areas of the world, has been postulated to be an obstructive lymphopathy due to the fibrogenic effects of silica absorbed through the plantar skin of bare-footed people. Animal experiments involving the direct intralymphatic injection of fine silica particles have been carried out in order to assess the extent to which this substance can engender lymphatic obstruction and to determine its main site of action. Intralymphatic silica provoked an immediate and intense macrophage reaction with later fibrosis both within lymph vessels and to a lesser extent within lymph nodes. Lymphography indicated that the consequent obstruction resulted more from the effects of silica on vessels than on nodes. PMID- 3006294 TI - Visceral leishmaniasis unresponsive to antimonial drugs. I. Clinical and immunological studies. AB - Ten Kenyan patients with visceral leishmaniasis, unresponsive to sodium stibogluconate at a dose of 16 to 20 mg Sb/kg/day given for 30 to 98 days, have been studied clinically and immunologically and compared with 57 antimony responsive patients. Pulmonary tuberculosis and previous treatment with antimonial drugs were the only factors which were more common in unresponsive patients. The degree of immunosuppression and rate of recovery of immunoreactivity did not differ between antimony-responsive and -unresponsive patients. Only one patient had never been treated before (primary unresponsiveness). In the other nine patients secondary unresponsiveness occurred after one or more treatment courses, suggesting that the parasite developed resistance to antimony. Antimony-unresponsiveness in visceral leishmaniasis is a serious problem numerically, clinically and economically. A plea is made that the initial treatment of visceral leishmaniasis should be adequate in dose and duration. PMID- 3006295 TI - Visceral leishmaniasis unresponsive to antimonial drugs. II. Response to high dosage sodium stibogluconate or prolonged treatment with pentamidine. AB - Ten Kenyan patients with visceral leishmaniasis unresponsive to sodium stibogluconate, at a dose of 16 to 20 mg Sb/kg body-weight/day given for 30 to 98 days, were treated with 20 mg Sb/kg bw given every eight hours. This regimen was modified or abandoned in six patients because of suspected toxicity, although toxicity was difficult to assess because of intercurrent illness. Toxic effects included lethargy, anorexia, vomiting, electrocardiographic changes, fall in haemoglobin and rise in liver enzymes. One patient died, probably from a cardiac arrhythmia. Two patients were cured, four responded partially and four showed no response. Pentamidine, at a dose of 4 mg/kg body-weight given one to 3 times per week for 5 to 39 weeks, was given as initial treatment in one patient and after failure of sodium stibogluconate in seven. Toxic effects included nephritis, hepatitis, transient diabetes and subcutaneous abscesses. Two patients were cured, two responded partially, three showed no response and one, after apparent cure, relapsed and was unresponsive to additional pentamidine treatment. Low frequency, long-duration pentamidine was often useful in maintaining any improvement made during treatment with the less well tolerated high-dose, high frequency sodium stibogluconate. We observed the step-wise development of resistance to both sodium stibogluconate and pentamidine. The problems of managing patients with visceral leishmaniasis which is unresponsive to conventional doses of pentavalent antimonials are discussed and some tentative suggestions put forward. PMID- 3006297 TI - The postnatal decline in antibody to hepatitis A virus. PMID- 3006296 TI - Visceral leishmaniasis unresponsive to antimonial drugs. III. Successful treatment using a combination of sodium stibogluconate plus allopurinol. AB - Five patients with long-standing visceral leishmaniasis who were unresponsive to sodium stibogluconate, 20 mg antimony/kg body-weight once or twice daily, were treated for 14 to 54 days with a combination of sodium stibogluconate at the same dose plus allopurinol at a dose of 20 mg/kg body-weight per day in three divided doses. This combination was safe and effective. Negative splenic aspirate smears were obtained from all patients within 19 days, and none has relapsed in at least 12 months of follow-up. PMID- 3006298 TI - Calcium homeostasis during therapeutic plasma exchange. AB - Because the removal of substantial quantities of plasma calcium during plasma exchange is rarely attended by clinically significant hypocalcemia, we evaluated calcium homeostasis during this procedure. Twenty-one procedures were performed on 10 patients with various neurological disorders. The reduction by plasma exchange in the serum concentrations of total calcium, ionized calcium, magnesium, and phosphate was significantly less than predicted (p less than 0.001) based on plasma volume of the patient and size of the exchange. However, N terminal parathomone (PTH) levels increased to 242 +/- 120 percent midway into the procedure and were 207 +/- 84 percent after plasma exchange; urinary cyclic adenosine monophosphate (cAMP) levels rose by 165 +/- 35 percent. These data demonstrate a rapid compensatory response in N-terminal PTH and urinary cAMP to the reduction by plasma exchange of serum concentrations of total calcium, ionized calcium, and phosphate. The routine administration of supplemental calcium during plasma exchange may therefore be unnecessary in patients with normal parathyroid function. PMID- 3006299 TI - Automatic special case consultations in transfusion medicine. AB - In the past, consultations in immunohematology were usually delivered only when there was a blood or component shortage, or when the time required for compatibility testing was prolonged due to the presence of a difficult recipient antibody problem. More recently, transfusion medicine physicians have increased their role as clinical consultants concerned about the appropriate indications for blood transfusion. This communication presents a way to begin a sound program of clinical transfusion medicine consultation. The program can fit with community wide teaching efforts, community or hospital transfusion audits, and specific physician education programs. Automatic or unsolicited consultations appear to have been both accepted well and beneficial in large hospitals and in communities where they have been provided for more than 7 years. PMID- 3006300 TI - A comparison of four commercial test kits for detection of cytomegalovirus antibodies in blood donors. AB - Four commercial test kits for detecting cytomegalovirus (CMV) antibodies (indirect hemagglutination assay, indirect fluorescent antibody technique, enzyme immunoassay, and passive latex agglutination technique) were compared according to their technical demand, hands-on time, turnaround time, concordance with other techniques, reagent cost per test, and objectivity. The indirect hemagglutination assay, the enzyme immunoassay, and the passive latex agglutination technique produced identical results in 85 donors, detecting 63 positive and 22 negative samples. The indirect fluorescent antibody technique showed discrepant results in four samples. The passive latex agglutination technique rated best overall since it was technically the easiest and required the least hands-on and turnaround times; the short turnaround time (10 minutes) rendered the latex technique a more flexible test for blood bank use because both scheduled and emergency CMV screening of donors could be accommodated. The comparatively high reagent cost of the latex test kit could be offset by savings on technologist time. PMID- 3006301 TI - Results of nationwide screening of blood and plasma for antibodies to human T cell lymphotrophic III virus, type III. AB - From April 22 to July 28, and from October 7 to November 3, 1985 the American Red Cross, the Council of Community Blood Centers, the American Association of Blood Banks, and the American Blood Resources Association provided the Food and Drug Administration with data at 2-week intervals on human T-lymphotropic virus type III (HTLV-III) test kit results at blood and plasma collection centers. Reports were received from over 150 blood collection centers that screened 2,502,829 units of blood for antibody to HTLV-III by enzyme-linked immunosorbent assay (ELISA) during nine 2-week surveillance intervals. Of these, 25,324 or 1.01 percent were initially reactive and 8443 or 0.34 percent were repeatedly reactive. The repeatedly reactive rate for women was 0.33 percent and for men 0.30 percent. Data for source plasma was collected at 275 locations and tested at eight central laboratories showed that for 2,603,652 units screened (which may represent multiple collections from the same donor) 3978 or 0.15 percent were repeatedly reactive. Screening results from both blood and plasma collection centers varied over time and among kits. PMID- 3006302 TI - Relative specificity of enzyme-linked immunosorbent assays for antibodies to human T-cell lymphotrophic virus, type III, and their relationship to Western blotting. AB - A population of 73 donor samples was assembled on the basis of reactive results in routine screening with three different licensed human T-lymphotrophic virus type III (HTLV-III) antibody enzyme-linked immunosorbent assay (ELISA) procedures. The samples were retested by a number of licensed and developmental tests and by Western blot analysis. Our data indicate that nonspecific results are generated by ELISA tests and that many of these reactions appear to be directed against the cell substrate used to grow the virus. These findings suggest that combinations of currently licensed ELISA tests, based upon HTLV-III grown in H-9 cells, cannot be used to confirm the specificity of reactive samples. PMID- 3006303 TI - Inactivation and partition of human T-cell lymphotrophic virus, type III, during ethanol fractionation of plasma. AB - Because of concern about the safety of immune globulins prepared for injection, we studied the effects of ethanol fractionation of human plasma on human lymphotropic virus, type III, (HTLV-III) by spiking the products of various fractionation steps with HTLV-III. Tests of inactivation and removal indicated that the ratio of residual live virus in plasma fractions/live virus in starting plasma was about 1 X 10(-15) for precipitate II from which immune globulin for injection is manufactured. The results are reassuring regarding the potential safety of immune globulin. PMID- 3006304 TI - [Transposition and reversion of mutations induced in Drosophila by viral DNA]. AB - Injection of solutions of highly polymerized DNA isolated from nuclear polyhedrosis virus of Galleria mellonella into adult males induced with a considerable frequency visible mutations, two of which were studied in detail. They were detected in about 30 000 flies in the progeny of treated males. Much less than Notched-wings much greater than (Ndw, chromosome 3, location 87.9, dominant) independently arose 12 times, much less than thickened-veins much greater than (thi, chromosome 2, location 71.4, recessive) independently arose 7 times. No mutations were detected in the control of the same size. It was found that both Ndw and thi mutations gave frequent transpositions and reversions in mature, immature germ cells and in somatic cells, in latter cases leading to mosaicism. These results demonstrate for the first time that mutations induced by exogenous DNA are capable of transposition and reversion. PMID- 3006305 TI - [Manifestation of instability in the leucine-dependent Bacillus subtilis auxotroph induced by herring DNA]. AB - Unstable leucine-depending Bacillus subtilis auxotroph previously induced by the herring DNA was studied. Instability was manifested as revertability in the leucine-free medium. Average frequency of revertants amounted to 50% of the number of viable cells. A clone analysis has shown that most of such colonies are mosaics consisting of leucine-independent and leucine-sensitive cells. Unstable mutation was cotransformed with leuA marker. Heating followed by material storage in demineralized water stimulated transposition of unstable mutation under study to the tryptophan operon region. PMID- 3006306 TI - [Plasmid pBR325tk as a mutagen]. PMID- 3006307 TI - Correlation of histologic grade and lymph node status with some histopathologic discriminants in breast cancer. AB - In fifty non selected ductal carcinomas of the breast we found that a marked tumoral inflammatory infiltrate (P less than 0.025), perinodal tumoral infiltrate (P less than 0.01), sinus catarrh (P less than 0.05), follicular hyperplasia (P less than 0.025), mixed pattern in lymph nodes (P less than 0.01) and with 54 years of age or younger (P less than 0.01) correlated significantly with lymph node metastases and/or high histologic grade. On the contrary, elastosis (P less than 0.05), scanty or absent inflammatory infiltrate (P less than 0.01), sinus histiocytosis (P less than 0.001) and endothelial hyperplasia were statistically related to low histologic grade and/or lack of metastases. Elastosis is considered a defensive host response. Groups of lymphocytes in the perinodal fat is usually found in metastasized lymph nodes and may indicate metastasis should be sought in a lymph node which otherwise seems to be tumor-free. PMID- 3006308 TI - Cystic nephroma in children. Report of a case. AB - A case of cystic nephroma is described in a 20-month-old female. The diagnosis of this rare renal pathologic lesion is essentially based on its pathologic features. The treatment consisted of nephrectomy followed by a brief course of chemotherapy. The literature is reviewed with regard to the pathologic features and treatment of this lesion, which has a favorable prognosis. PMID- 3006309 TI - [Scintigraphy of the thyroid gland with technetium 99m pertechnetate]. PMID- 3006310 TI - [Brain scintigraphy for the demonstration of intracranial tumors. Illustration of its use and limitations of the method]. PMID- 3006312 TI - [Extraorganic retroperitoneal paraganglioma associated with a horseshoe kidney]. PMID- 3006311 TI - [Characteristics of prooxidant and antioxidant processes in chronic pyelonephritis in children]. PMID- 3006313 TI - A method for studying inhibitory activity in whole urine. AB - A method has been developed for inducing and quantifying calcium oxalate crystallisation in whole human urine. The propensity of a given urine to induce crystal formation was described in two ways: its ability to resist spontaneous nucleation of calcium oxalate crystals was assessed by titrating 20 mls of the urine with increasing quantities of sodium oxalate (0-150 mumol) to determine its practical metastable limit. This limit was inversely related to the endogenous calcium concentration; its capacity to inhibit crystal growth was quantified by determining the rate of growth of calcium oxalate crystals precipitated in response to a fixed oxalate load (30 mumol) above its metastable limit. The crystals produced were predominantly calcium oxalate dihydrate and were morphologically identical to those occurring naturally in urine. Citrate had no effect on the metastable limits of 3 urines examined, but markedly inhibited crystal growth. Pyrophosphate had a similar effect on crystal growth, and in addition, raised the metastable limit of one of the urine samples. PMID- 3006315 TI - [Comparative evaluation of the surgical approaches to nasopharyngeal tumors, and our suggestions]. PMID- 3006314 TI - [Characteristics of the development of diseases of the upper respiratory tract in workers exposed to chrysotile and asbestos dust]. PMID- 3006317 TI - Measurement of antibodies after parvovirus vaccination. PMID- 3006316 TI - [Various problems in the pathogenesis and surgical treatment of patients with hypercortisolism]. AB - Based on an analysis of clinical and laboratory data, results of histological examination of removed adrenals and tumors of the cortical layer as well as an assessment of the functional state of the hypothalamo-hypophyseal-adrenal system in 132 patients the authors discuss some debatable questions of pathogenesis and selection of methods of treatment of the disease known as the Icenko--Cushing disease and syndrome. PMID- 3006318 TI - A possible viral candidate for the aetiology of turkey rhinotracheitis. PMID- 3006319 TI - Outbreak of bovine herpes mammillitis in Cumbria. PMID- 3006320 TI - The sympatho-adrenal system and plasma levels of adrenocorticotropic hormone, cortisol and catecholamines in equine grass sickness. AB - Plasma levels of adrenocorticotropic hormone (ACTH), cortisol and catecholamines were used to study the role of the sympatho-adrenal system in equine grass sickness. Statistical evaluation determined differences of hormone levels between seven horses with grass sickness (one acute, five subacute and one chronic), six horses with colic (one with laminitis) and 16 control horses before and after mild stress. Plasma levels of the hormones were higher in horses with acute and subacute grass sickness than in the other groups. No differences were detected between horses with colic and stressed control horses but some hormone levels differed between control and colic horses and control horses before and after stress. It is possible that hyperactivation of the sympatho-adrenal system is caused by stress but it is uncertain whether the stress is only a result of the severity of the disease or also plays a role in its aetiology. PMID- 3006321 TI - Experimental infection of red deer (Cervus elaphus) and cattle with a herpesvirus isolated from red deer. PMID- 3006322 TI - Hemagglutinating activity associated with bovine herpesvirus type 1. AB - Using C57BL/HPB mouse erythrocytes, hemagglutination has been observed with the Los Angeles and Colorado-1 strains of bovine herpesvirus type 1 and with 12 other Canadian field isolates as well. The specificity of the hemagglutination observed with the viral strains has been confirmed by a hemagglutination-inhibition assay. PMID- 3006323 TI - Parvovirus infection in pigs with necrotic and vesicle-like lesions. AB - Porcine parvovirus was isolated from many visceral organs and also from the brain, serum and skin specimens of swine with vesicular-like conditions. Severe lesions were reported to have occurred in the mouth, on the tongue and snout, on the coronary band and in the interdigital spaces. Also, parvoviral antigens were demonstrated, by immunofluorescence, in the outer layers of hair follicles in skin adjacent to coronary band lesions. PMID- 3006324 TI - Comparison of Mycoplasma ovipneumoniae isolates using bacterial restriction endonuclease DNA analysis and SDS-PAGE. AB - Sixteen isolates of Mycoplasma ovipneumoniae recovered from the nasal tract or lungs of sheep from different flocks in New Zealand were examined by bacterial restriction endonuclease DNA analysis (BRENDA) using EcoR1 and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). All isolates gave BRENDA patterns which differed entirely from one another. Following 20 serial passages (corresponding to approximately 67 generations) of an isolate, no change was detected in the BRENDA pattern. When eight isolates were examined by SDS-PAGE most bands were common but, nevertheless, each isolate was unique in the sense that they differed from one another in one or more bands. The marked heterogeneity of patterns observed when strains of M. ovipneumoniae are compared by BRENDA, together with the stability of such patterns over many generations, will enable this approach to be used to study the epidemiology of individual strains of M. ovipneumoniae within a flock. PMID- 3006325 TI - [Malignant fibrous histiocytoma of the bone (x-ray diagnosis)]. PMID- 3006326 TI - A feline retrovirus induced T-lymphoblastoid cell-line that produces an atypical alpha type of interferon. AB - A cell-line, designated LSA-1, was derived from a thymic lymphosarcoma that occurred in a cat with experimentally induced feline leukemia virus (FeLV) infection. LSA-1 cells possessed surface receptors and antigens of normal T lymphocytes, but were unresponsive to interleukin-2 stimulation. The LSA cell line was found to constitutively produce and release an interferon into the culture supernatants. Production of this interferon was enhanced in certain clones of the original LSA-1 cell lines. The interferon produced by LSA-1 cells and some of its clones was compared to the standard alpha, beta, and gamma interferons of cats. Unlike alpha and beta interferons, which were acid, SDS, and heat stable, LSA interferon was acid labile and SDS and heat stable. In comparison, standard feline gamma interferon was acid, SDS, and heat labile. LSA interferon had a molecular weight of 20,000 daltons, compared to 17-19,000 daltons for gamma, 19-25,000 for beta, and 25-45,000 daltons for alpha interferons. Standard feline interferons were active only on cat cell lines, with the exceptions of alpha interferon, which also reacted with MDCK canine cells. LSA interferon resembled the standard feline alpha interferon because it also reacted with feline and canine cells. It was concluded that LSA interferon was an atypical acid labile alpha interferon, resembling in this respect the abnormal alpha interferon seen in humans with AIDS and SLE, and mice with retrovirus infections. LSA-1 cells produced high levels of FeLV structural proteins but very little infectious virus. This effect was due to endogenously produced interferon; LSA cell clones that were selected for low interferon production produced much higher levels of infectious FeLV than parent cells or clones selected for high interferon production. Cat cells pretreated with LSA or with standard feline alpha and beta interferons, and then infected with FeLV, produced high levels of FeLV proteins but very little infectious virus. PMID- 3006327 TI - Intestinal antibody response after vaccination and infection with rotavirus of calves fed colostrum with or without rotavirus antibody. AB - The intestinal and systemic antibody response of calves vaccinated and/or challenged with rotavirus was studied employing isotype-specific ELISAs for the detection of IgG1, IgG2, IgM and IgA antibodies to rotavirus. Monoclonal antibodies to bovine immunoglobulin isotypes of proven specificity were used as conjugated or catching antibody. Five days after oral inoculation (dpi) of a 5 day-old gnotobiotic calf with rotavirus, IgM rotavirus antibodies were excreted in faeces, followed 5 days later by IgA rotavirus antibodies. The increase in IgM rotavirus antibody titre coincided with the inability to detect further rotavirus excretion. Faeces IgM and IgA rotavirus antibody titres fell to low levels within 3 weeks post infection. IgG1 and IgG2 rotavirus antibodies were not detected in faecal samples. In serum, antibodies to rotavirus of all four isotypes were detected, starting with IgM at 5 dpi. Two SPF-calves, which were fed colostrum free of rotavirus antibodies, were vaccinated with a modified live rotavirus vaccine and challenged with virulent rotavirus 6 days later. Upon vaccination, the calves showed an antibody response similar to the response of the infected gnotobiotic calf. Intestinal IgM rotavirus antibodies were excreted before or on the day of challenge and appeared to be associated with protection against challenge infection with virulent virus and rotavirus-induced diarrhoea. In 3 control calves, which were challenged only, the antibody patterns also resembled that of the gnotobiotic calf and again the appearance of IgM rotavirus antibodies coincided with the end of the rotavirus detection period. Two other groups of 3 SPF-calves were treated similarly, but the calves were fed colostrum with rotavirus antibodies during the first 48 h of life. These calves excreted passively acquired IgG1 and IgG2 rotavirus antibodies in their faeces from 2 to 6 days after birth. After vaccination, no IgM or IgA antibody activity in serum or faeces was detectable. Upon challenge, all calves developed diarrhoea and excreted rotavirus. Seven to 10 days after challenge low levels of IgM rotavirus antibody were detected for a short period. These data indicate that the intestinal antibody response of young calves to an enteric viral infection is associated with the excretion of IgM antibodies, immediately followed by IgA antibodies. This response is absent or diminished in calves with passively acquired specific antibodies which may explain the failure to induce a protective intestinal immune response by oral vaccination with modified live rotavirus of calves fed colostrum containing rotavirus antibodies. PMID- 3006328 TI - Immunohistochemical study of carbonic anhydrase in mixed tumours from major salivary glands and skin. AB - Immunohistochemical distribution of carbonic anhydrase isoenzyme I and II was studied in mixed tumours of major salivary glands and skin. The normal salivary glands displayed strong carbonic anhydrase activity in both ductal epithelium and serous acinar cells and the serous demilune cells in the submandibular glands, including the eccrine ducts. Pleomorphic adenoma salivary gland origin exhibited positive staining in the inner-layer of epithelial cells of tubular, duct-like and glandular structures. No enzymatic staining was noted in the outer layer of tumour cells in these structures. Spindle tumour cells or the fibroblast-like cells with long cytoplasmic processes identified in the adjacent hyalin and myxomatous stroma were rarely positive, while chondroidal and osteo-chondroidal cells were highly reactive. Mixed tumours of eccrine gland origin showed the most reactive staining cells scattered throughout neoplastic epithelium in all tissues examined. Immunohistochemical stainability was usually higher for carbonic anhydrase II than I for both normal and tumour tissues. The biological roles of the distribution profiles of carbonic anhydrase are discussed. PMID- 3006329 TI - Virus-neutralizing activity, serologic heterogeneity, and retrovirus isolation from homosexual men in the Los Angeles area. AB - Human retroviruses have been causally associated with the development of the acquired immune deficiency syndrome (AIDS) in groups of individuals at high risk including intravenous drug users, hemophiliacs, and homosexual men. Aside from classic AIDS, homosexual men also develop persistent generalized lymphadenopathy (PGL) which is considered a part of the AIDS-related complex (ARC). We have isolated 70 strains of retroviruses related to human T-lymphotropic viruses type III (HTLV-III) from patients with PGL, AIDS, or ARC. analyses of sera from these patients indicated a high degree of serologic heterogeneity in the antibody titers and their reactivities toward various HTLV-III viral proteins. In addition, we have detected virus-neutralizing antibodies in approximately 50% of the serum samples tested from patients with PGL or AIDS. This is the first comprehensive virologic and serologic report on more than 100 patients studied at one institution in Los Angeles. PMID- 3006330 TI - Serological characterization of human T-cell leukemia (lymphotropic) virus, type I (HTLV-I) small envelope protein. AB - Murine monoclonal antibodies were developed against the protein products produced by murine C127 cells which had been transfected with a recombinant plasmid clone containing the human T-cell leukemia (lymphotropic) virus type I (HTLV-I) proviral DNA coding regions for part of env, px, and the 3' LTR. Four antibodies with different binding patterns were obtained. One of these antibodies, F1.6, reacted against HTLV-I infected cells but not against noninfected cell lines. This antibody also reacted with sucrose-gradient purified HTLV-I and -II particles with preferential binding against the HTLV-I preparation. The F1.6 antibody bound to two proteins of approximately 21 and 43 kDa in gradient purified HTLV-I preparations, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots, and to a 43-kDa protein in cell lysates of HTLV-I infected cell lines; the F1.6 antibody did not bind significantly to any HTLV-II proteins in western blots. The three other antibodies F1.1, F1.2, and F1.5, recognized the same size proteins, 21 and 43 kDa in the gradient purified viral preparations of HTLV-I and in the case of the F1.2 antibody, the same size proteins in purified virus preparation of HTLV-II and III. The F1.2 and F1.5 antibodies bound not only to purified HTLV particles but also to a variety of cellular proteins in HTLV-I infected and noninfected cells suggesting that they recognized epitopes which were shared between HTLV proteins and normal cells. The identity of the 21-kDa viral protein is most likely that of the small envelope glycoprotein. The identity of the larger protein is undetermined. PMID- 3006331 TI - Multiple adjacent or overlapping loci affecting the level of gC and cell fusion mapped by intratypic recombinants of HSV-1. AB - We have prepared and analyzed 40 HSV-1 intratypic recombinants with regard to plaque morphology and glycoprotein C(gC) phenotypes. Vero cells have been cotransfected with the intact genome of HSV-1(F) and cloned or uncloned DNA fragments from HSV-1(MP) and recombinants inducing the fusion of Vero cells [syncytial (Syn) recombinants] have been selected and purified. Marker transfer of the Syn phenotype has been observed with the cloned BamHI L and B fragments (0.706-0.745 and 0.745-0.810 map units, respectively) as well as with the uncloned HpaI TXO fragment (0.710-0.761) from MP DNA. No marker transfer has been observed with F DNA alone or with the cloned BamHI N fragment (0.863-0.898 map units). When viruses expressing the Syn phenotype in Vero cells were tested in HEp-2 cells, three kinds of recombinants were observed. Members of the first class expressed a wild type, cytoaggregating (Syn+), plaque morphology in these cells. Members of the second class induced the complete fusion (Syn phenotype) of the cells. Members of the third class induced an intermediate plaque morphology, characterized by the formation of groups of polykaryocytes (fused cells) but without formation of a complete syncytium. All recombinants expressing the Syn+ phenotype in HEp-2 cells were also gC+, whereas recombinants expressing the Syn phenotype in these cells were gC- with one exception, in which low levels of gC could be detected (but clearly less than with HSV-1(F]. Concerning polykaryocytic class of recombinants, some of them were gC+ while others expressed only low amounts of gC; no gC- virus was observed within this class of recombinants. The three classes of recombinants were observed with each of the cloned BamHI L and B fragments and also with the HpaI TXO fragment, suggesting the existence of multiple adjacent or overlapping loci affecting plaque morphology and the control of the accumulation or the synthesis of gC at both sides of 0.745 map units. PMID- 3006332 TI - Distribution of type D retrovirus sequences in tissues of macaques with simian acquired immune deficiency and retroperitoneal fibromatosis. AB - Horizontally acquired SAIDS retrovirus type 2 (SRV-2), a type D retrovirus related to the Mason-Pfizer monkey virus, has been associated with the simian acquired immunodeficiency syndrome (SAIDS) including retroperitoneal fibromatosis (RF) in several macaque species at two primate research centers. Virus specific gene sequences are present in lymphoid and RF tissues but not in muscle tissue of diseased macaques or in any tissues of uninfected normal monkeys. Serologic and restriction endonuclease mapping techniques have defined unique SRV-2 strains in the Celebes (SRV-2C) and rhesus (SRV-2R) macaques at the Oregon Regional Primate Center, SRV-2 is related to both MPMV and SAIDS type 1 retroviruses and it has no detectable molecular homology with the human AIDS retroviruses. PMID- 3006333 TI - Production of human monoclonal antibodies against Epstein-Barr virus-specific antigens by the virus-immortalized lymphoblastoid cell lines. AB - The possible production of human monoclonal antibodies against Epstein-Barr virus (EBV) was assessed through the EBV immortalization technique. When individual lymphocyte samples from 50 clinical patients and healthy donors were immortalized by EBV, 4 lymphoblastoid lines yielded antibodies to EBV antigens. These positive lines were cloned and each line yielded cultures that secreted monoclonal antibodies against either viral capsid antigen (VCA) or membrane antigen (MA) component. Above all, a clonal line TAKA-SP-8 produced 5 micrograms MA antibody/10(6) cells/ml for more than 12 months. The culture fluid specifically immunoprecipitated a single polypeptide with a size of 93K from both P3HR-1 and B95-8 cell extracts. FUKA-SP-3, on the other hand, secreted 5 micrograms VCA antibody/10(6) cells/ml for at least 8 months. This antibody recognized two polypeptides with sizes of 123K and 120K, from P3HR-1 and B95-8 cell extracts, respectively. When B95-8 and P3HR-1 EBV were treated with the human MA monoclonal, both nuclear antigen (EBNA) synthesis and early antigen (EA) induction were strongly inhibited. All EBV antibody-producing cultures were exclusively achieved from splenic lymphocytes of patients with autoimmune diseases, but not from other donors. PMID- 3006334 TI - Characterization of viruses infecting a eukaryotic Chlorella-like green alga. AB - Nineteen plaque-forming viruses of the unicellular, eukaryotic Chlorella-like green alga, strain NC64A, were isolated from various geographic regions in the United States and characterized. Like the previously described virus, PBCV-1, all of the new viruses were large polyhedrons, sensitive to chloroform, and contained large dsDNA genomes of ca. 300 kbp. All of the viral DNAs contained 5 methyldeoxycytidine which varied from 0.1 to 47% of the deoxycytidine. In addition, 10 of the viral DNAs contained N6-methyldeoxyadenosine which varied from 8.1 to 37% of the deoxyadenosine. These viruses, along with 11 previously described viruses which replicate in the same Chlorella host, were grouped into 11 classes based on at least one of the following properties: plaque size, reaction with PBCV-1 antiserum, or the nature and abundance of methylated bases in their genomic DNA. PMID- 3006336 TI - Genetic analysis of the 3' early region transformation and replication functions of bovine papillomavirus type 1. AB - Cell transformation by BPV-1 is dependent on sequences confined to a region comprising only 69% of the viral DNA. Analysis of this subgenomic fragment of the viral genome has revealed a composite of functions. To further study this region of the BPV-1 genome, we constructed mutants affecting the 3' half of the transforming region. These mutations have revealed a plasmid maintenance function for the E2 ORF and a requirement that the 3' half of E5 be intact for in vitro mouse cell transformation. Cotransfection experiments revealed that phenotypic complementation (wild type transformation efficiency and autonomous replication of viral DNA) resulted in recombination between input plasmids in the majority of cases. However, rare cases were encountered where the wild type phenotype was observed with no evidence of recombination between input plasmids. These data suggest trans-acting functions of the E2 and E5 ORFs in viral DNA replication and the E5 ORF in cell transformation. PMID- 3006335 TI - Identification, properties, and gene location of a novel glycoprotein specified by herpes simplex virus 1. AB - We report the identification of a novel herpes simplex virus 1 (HSV-1) glycoprotein reactive with type specific monoclonal antibody H1379. The monoclonal antibody reacted with two broad bands with apparent mol wt of 60K to 68K and 44K to 48K formed by infected cell lysates subjected to electrophoresis in denaturing polyacrylamide gels and electrically transferred to a nitrocellulose sheet. Early in infection the H1379 reactive protein was found in the faster migrating band. The rate of accumulation was highest late in infection and only the slower migrating form incorporates significant amounts of glucosamine. The epitopic site recognized by H1379 was not uniformly distributed among strains. Analyses of HSV-1 X HSV-2 recombinants with monoclonal antibodies to HSV-1 and HSV-2 glycoproteins mapping in the S component of the HSV genomes and marker transfer experiments indicated that the gene specifying the H1379 reactive protein maps within BamHI fragment J to the left of gD most probably within the open reading frame designated as US4 (D. J. McGeoch, A Dolan, S. Donald, and F. J. Rixon, 1985, J. Mol. Biol. 181, 1-13). The gene specifying a recently discovered HSV-2 glycoprotein designated as gG-2 (B. Roizman, B. Norrild, C. Chan, and L. Pereira, 1984, Virology 133, 242-247) maps in the corresponding domain of the HSV-2 genome and marker transfer experiments suggest that the H1379 reactive protein and gG-2 are collinear. We have therefore designated the novel HSV-1 glycoprotein as gG-1. PMID- 3006337 TI - Lack of correlation between the accumulation of plus-strand leader RNA and the inhibition of protein and RNA synthesis in vesicular stomatitis virus infected mouse L cells. AB - The inhibition of protein synthesis in mouse L cells infected by vesicular stomatitis virus (VSV) requires expression of two regions (one large and one small) of the viral genome, as determined by target size analysis. The inhibition of host RNA synthesis was also shown to be dependent on expression of two regions of the VSV genome, most likely the same ones. In some cases, such as in cells infected by mutants T1026R1, or tsG41 at 40 degrees, or moderately uv irradiated VSV, only one of the two regions was expressed, yet cellular protein and RNA synthesis was decreased. This suggests that the product of each region of the viral genome can act independently. In these instances the severity of the inhibition was dependent on both the length of the infection period and the multiplicity of infection. The identity of neither gene product is known, but it has been suggested that small product is plus-strand leader RNA. As shown herein, however, there was no correlation between the extent of host macromolecular synthesis inhibition and the quantity of leader RNA in infected cells. PMID- 3006338 TI - A cryptic transcription promoter in the myb oncogene of avian myeloblastosis virus. AB - The potential regulatory signals contained in the v-myb oncogene of avian myeloblastosis virus have been inserted upstream to the herpes simplex type 1 thymidine kinase gene in order to test their promoter activity. The isolation of TK+ transformants after transfection of clone 1D(TK-) mouse cells with the resulting recombinant DNAs indicated that the expression of the TK gene was made possible by the myb-derived sequences. Analysis of the TK specific RNA expressed in different TK+ transformants revealed that the regulatory signals contained in v-myb correspond to a weak functional promoter. PMID- 3006339 TI - Visualisation, by immunocytochemistry, of p53 at the plasma membrane of both nontransformed and SV40-transformed cells. AB - The cellular protein p53 normally functions in the control of cell proliferation; but, when expressed abnormally p53 also contributes towards the process of cell transformation. The functioning of p53 is thought to involve interaction with specific cellular targets and, in SV40- and other transformed cells, p53 is located in the nucleus: thus p53 may function via interacting with specific nuclear components. In addition, there is indirect evidence that in SV40 transformed cells p53 is also associated with the plasma membrane; however, this is not evident in cells stained by immunofluorescence. We have sought to obtain direct evidence for p53 at the plasma membrane by staining with a very sensitive immunocytochemical procedure. We have compared SV40-transformed BALB 3T3 cells with BALB 3T3, NIH 3T3, and NIH 3T3 transfected with v-mos provirus. The last cell line is tumorogenic. We found direct evidence for p53 at the plasma membrane for each cell line. In nontransformed and SV40-transformed cells the association of p53 with the plasma membrane was restricted to the period of mitosis. These results indicate that association of p53 with the plasma membrane is a normal, rather than a transformation-related phenomenon, and is temporally linked to the period of mitosis. PMID- 3006340 TI - Analysis of the autophosphorylation activity of transformation defective mutants of avian erythroblastosis virus. AB - The v-erb B protein of avian erythroblastosis virus (AEV) possesses an associated protein kinase activity in vitro. Analysis of temperature-sensitive mutants, and nonconditional host range mutants of AEV demonstrated that there was no simple correlation between this autophosphorylation activity and the transformation ability of the various AEV mutants. These data suggest that although this kinase activity may be central to transformation by AEV it is in itself insufficient. PMID- 3006341 TI - Differential antibody responses of individuals infected with AIDS-associated retroviruses surveyed using the viral core antigen p25gag expressed in bacteria. AB - Infection with the retrovirus that is the etiological agent of acquired immune deficiency syndrome (AIDS) is characterized by the development of antiviral antibodies. To generate reagents for studying immune responses to individual viral proteins, we have produced viral antigens in microorganisms by recombinant DNA techniques. Large amounts of the major core protein (p25gag) of an isolate of the AIDS retrovirus (AIDS-associated retrovirus; ARV-2) have been directly expressed in Escherichia coli. Recombinant p25gag (R-p25gag) has been purified and used in an enzyme-linked immunosorbent assay (ELISA) for antibodies to p25gag. Serum samples obtained from 100 individuals with AIDS, AIDS-related complex (ARC), or potential exposure to the virus through sexual contact with AIDS or ARC patients (contacts) were tested first in an ELISA with disrupted whole virus to determine which of the subjects had mounted an antibody response to the virus (virus seropositive) and then in the p25gag ELISA to determine if they had antibodies to this particular viral antigen. We observed a decrease in the proportion of virus seropositive individuals with antibodies to p25gag among patients groups in which the disease was more advanced; contacts were often positive (71%), ARC patients less frequently positive (48%), and AIDS patients only rarely positive (16%). Our results suggest that monitoring p25gag seropositivity of infected individuals may be useful for predicting either the prognosis or the stage of the disease. PMID- 3006342 TI - Cleavage of the cap binding protein complex polypeptide p220 is not effected by the second poliovirus protease 2A. AB - Poliovirus protein 2A contains a short amino acid sequence that also occurs in the putative active site of the known viral proteinase, 3C, previously shown to be responsible for glutamine/glycine cleavages in the poliovirus polyprotein precursor. Experimental evidence indicates that 2A is a second viral proteinase that mediates the cleavage of two tyrosine/glycine cleavages in the generation of virus-specific proteins. Since poliovirus inhibition of host cell protein synthesis correlates with the specific cleavage of the 220,000-Da component of the cap binding protein complex, we have tested whether viral protein 2A contains the p220 cleavage activity. The results show that 2A does not copurify with p220 cleavage activity, partially purified fractions containing high p220 cleavage activity contain no detectable 2A sequences in the form of either mature or precursor protein, and anti-2A serum or IgG does not inhibit p220 cleavage in vitro. PMID- 3006343 TI - Complete nucleotide sequence of the matrix protein mRNA of vesicular stomatitis virus (New Jersey serotype). AB - The complete nucleotide sequence of the mRNA of the matrix (M) protein of vesicular stomatitis virus [New Jersey serotype, VSV(NJ)] was derived from a cDNA clone and mRNA. The mRNA is 758 nucleotides long (excluding polyadenylic acid) and encodes a protein of 229 amino acids. The predicted amino acid sequence was compared with that of the corresponding protein of Indiana serotype [VSV(IND)] and a fish rhabdovirus, spring viremia of carp virus (SVCV). An amino acid identity of 62% was found between the M proteins of VSV(NJ) and VSV(IND) while only 24% was present between VSV(NJ) and SVCV. A highly basic NH2-terminal domain followed by a proline-proline-X-tyrosine sequence was present in all the three M polypeptides. Except for the L gene sequence, the complete nucleotide sequence of the four genes of VSV(NJ) are now known. The comparison of the amino acid sequences between the Indiana and New Jersey serotypes demonstrates a high degree of homology between these genes except for the phosphoprotein gene, NS. PMID- 3006344 TI - Host and phage-coded functions required for coliphage N4 DNA replication. AB - Escherichia coli strains containing mutations in various deoxyribonucleic acid synthesis cistrons have been tested for their ability to support bacteriophage N4 growth and, specifically, N4 DNA synthesis. N4 DNA synthesis is independent of the activity of the products of the E. coli dnaA, dnaB, dnaC, dnaE, dnaG, and rep genes. In contrast, N4 DNA replication requires the products of the dnaF, (ribonucleotide reductase) and lig (DNA ligase) genes of E. coli. N4 DNA replication, specifically processing of short DNA fragments requires the 5'-3' exonuclease activity of the polA gene product. However, its DNA polymerizing activity is not required. In addition, the sensitivity of N4 DNA synthesis to inhibitors or temperature-sensitive mutants of E. coli DNA gyrase suggests that this activity is required for N4 DNA synthesis. To date, we have found five N4 gene products required for N4 DNA replication: dbp (a single-stranded DNA binding protein), dnp (a DNA polymerase), dns (unknown function), vRNAp (the N4 virion associated, DNA-dependent RNA polymerase) and exo (a 5'-3' exonuclease). PMID- 3006345 TI - Phosphorylation of simian virus 40 large T antigen: cytoplasmic and nuclear phophorylation sites differ in their metabolic stability. AB - The turnover of phosphate residues in simian virus 40 (SV40) large T antigen (large T) was determined by pulse-chase labeling with 32Pi and subsequent two dimensional peptide mapping. Three groups of phosphorylation sites with respect to metabolic stability were distinguished with half-lives of about 8, 3 to 5, or 2 hr, respectively. Sites phosphorylated in the cytoplasm were relatively stable, whereas most of the sites phosphorylated in the nucleus exhibited high turnover rates. We suggest that sites with low turnover represent permanent modifications whereas sites with high turnover might contribute to the dynamic properties of large T, e.g., its interaction with the viral genome. When the phosphate turnover in various subclasses of large T was compared the monomeric and oligomeric forms showed no significant differences. Likewise, the DNA-binding and nonbinding fractions exhibited similar phosphate turnover. However, in the DNA-binding fraction the 3H label decreased faster than the 32P label indicating that large T in this fraction had been replaced by newly synthesized molecules which were not labeled with 3H but nevertheless with 32P. this latter result suggests that a certain degree of phosphorylation might be required for DNA binding. PMID- 3006346 TI - Oncogenic retrovirus from spontaneous murine osteomas. II. Molecular cloning and genomic characterization. AB - An N-ecotropic murine leukemia virus (OA MuLV), originally isolated from spontaneous osteomas of strain 101 mice, was molecularly cloned. The virus induces osteomas, osteopetrosis, and malignant lymphomas in NMRI mice. The cloned virus was analyzed by heteroduplex analysis, restriction enzyme mapping, and oligonucleotide mapping. The data show a very close relationship to the endogenous Akv prototype virus with some differences in the gag and the env region. The nucleotide sequence of the U3 region of OA MuLV LTR revealed a structure within the presumable enhancer region very similar to the U3 sequences of the FBJ murine sarcoma virus and its associated helper virus. The significance of these specific structures for the oncogenicity of the virus and the development of the typical disease pattern is discussed. PMID- 3006347 TI - Compartmentation of second messenger action: immunocytochemical and biochemical evidence. PMID- 3006348 TI - Vitamin K-dependent formation of bone Gla protein (osteocalcin) and its function. PMID- 3006349 TI - [Effect of cytochrome c on bioenergetic processes in the liver in severe compression trauma]. AB - After severe mechanical trauma of rat soft tissue content of ATP, total adenine nucleotides, amounts of "energy charge" of the adenine nucleotide system and cytochrome c were decreased in liver tissue, while content of AMP and Pi was increased. Parenteral administration of cytochrome c at a dose of 10 mg/kg into animals after the trauma normalized the energy processes in liver tissue and increased the content of endogenous cytochrome c. PMID- 3006351 TI - Antibodies to the human T-cell lymphoma/leukemia virus type I in Dutch haemophiliacs. AB - 95 Dutch haemophiliacs were tested for antibodies to membrane antigens on cells infected with human T-cell leukemia virus type I (HTLV-I-MA) by indirect immunofluorescence and to purified HTLV-I by enzyme-linked immunosorbent assay. Antibodies to HTLV-I-MA were present in 8 of 95 (8%) haemophiliacs, but only 3 (3%) had antibodies to purified HTLV-I. No clinical disease was related to HTLV-I seropositivity. The importance of iatrogenic HTLV-I transmission to haemophiliacs is discussed. PMID- 3006352 TI - [Response to thyroliberin of prolactin, corticotropin and cortisol among women with Itsenko-Cushing disease]. PMID- 3006353 TI - [Nonchromaffin paraganglioma of the stomach]. PMID- 3006350 TI - Transmission of lymphotropic retroviruses (HTLV-I and LAV/HTLV-III) by blood transfusion and blood products. PMID- 3006354 TI - [Disorder of the regulation of the contractile ability of the heart in myocardial lesions]. PMID- 3006355 TI - [Corticosteroid and corticotropin levels in patients with hypocorticism before and after the transplantation of cultured cells of the adrenal cortex]. PMID- 3006356 TI - [Functional diagnosis of myocardial diseases in patients exposed to various chemical substances]. PMID- 3006357 TI - [Further study of viral non-A, non-B hepatitis]. AB - The paper describes the study of non-A-non-B virus hepatitis with parenteral mechanism of the infection transmission. Immunofluorescence method was used to test 9 liver biopsies from patients with non-A-non-B hepatitis using blood sera from convalescents after this disease. In 6 liver preparations, diffuse fluorescence of hepatocyte cytoplasm was observed. No markers of hepatitis A or hepatitis B were found. In the control group of 17 patients with HB and HA, no non-A-non-B hepatitis antigen was detected. Analysis of the blood sera from the acute period by the ELISA demonstrated the presence of anti-HBs in 28.2% of the patients with non-A-non-B virus hepatitis which corresponds to the frequency of their detection in the normal population. Antibody to HBeAg were found in 59.5%, i. e. significantly more frequently than anti-HBs in non-A-non-B virus hepatitis (p less than 0.03) and anti-HBe in the normal population (15%, p less than 0.01). Experiments of DNA-DNA hybridization demonstrated the homology of HBV DNA and that isolated from the blood serum and liver of patients with non-A-non-B virus hepatitis. The above results suggest that the agent or one of the agents of parenteral non-A-non-B virus hepatitis belongs to the group of Hepadnaviruses. PMID- 3006358 TI - [Leukocyte interferon reaction in patients with primary immunodeficiencies]. AB - The capacity of leukocytes from children with primary immunodeficiency to produce alpha- and gamma-interferons in vitro was studied. Interferon response of leukocytes in most of the patients examined was found to be practically unchanged. The immunostimulating therapy in some cases exerted a regulating effect on leukocyte capacity for interferon production. It is assumed that the interferon-producing function of T lymphocytes may be preserved in patients suffering from primary immunodeficiency. PMID- 3006360 TI - [Antiviral effect of an extract of Luffa cylindrica (L 043) on Japanese B encephalitis virus infection in vivo]. PMID- 3006361 TI - [Progress in the diagnosis and treatment of insuloma]. PMID- 3006359 TI - [Shifts in the antioxidant activity in the membrane structures of virus-infected cells]. AB - It is assumed that the process of virus infection of cells causes significant changes in the conditions of lipid peroxidation in membrane structures of such cells. Experiments with virus-infected HeLa cells demonstrated noticeable decrease in the capacity of membrane lipids for peroxidation induced by ultraviolet irradiation and iron monoxide ions. Similar changes were also observed in membranes of chick embryo liver cells infected with viruses. The established reduced capacity of lipids in membranes of virus-infected cells to induced peroxidation (interpreted as the increase of their antioxidant activity) is characteristic of whole membranes only, because the lipids extracted from infected and control cells showed no differences in their capacity to peroxidation in liposomal membranes when oxidation was induced by iron monoxide. PMID- 3006362 TI - [Experiences with acyclovir in herpes virus infections]. AB - 46 patients suffering from various malignancies (17 non Hodgkin lymphomas, 12 Hodgkin's diseases, 11 acute leukaemias, 4 myelomas, 2 carcinomas), 6 patients with haematological disorders such as ITP, SAA, myeloproliferative disease, LAS and 3 patients without preexisting disease were treated with acyclovir for herpes virus infection diagnosed by clinical means. All but 7 patients had been given intensive treatment with various cytostatic agents and/or irradiation. Most patients were treated with 1500 mg acyclovir daily for 5 to 13 days. Dosage was adjusted according to renal function and clinical response in the remaining 10 cases. 11 patients received intravenous immunoglobulins in addition. Side effects were negligible (local irritation, minimal rise in serum creatinine levels in 5 patients). All patients responded to treatment; 6 patients complained of severe neuralgia lasting for more than one month; 5 patients relapsed. PMID- 3006363 TI - [Experiences with an organ-saving and adjuvant-therapy concept in stage 1 breast cancers after 6 years of use within the scope of a controlled clinical study]. AB - Between 1979 and 1983 56 women with stage I breast cancer underwent tumorectomy plus lymphadenectomy, postoperative irradiation and adjuvant antioestrogen therapy in cases of hormone receptor positive tumours. All patients with hormone receptor negative tumours received 6 cycles of combined CMF chemotherapy instead of antioestrogen therapy. It was possible to complete optimal dosage of CMF or tamoxifen in more than 85% of our patients. Leukopenia occurred in only 3 patients given chemotherapy, whilst intolerable gastrointestinal side effects occurred in 4 patients given antioestrogen therapy. No interference was detected between irradiation and chemo- or hormone therapy and no serious local complications with functional impairment were registered. 23% of the women had positive axillary nodes at the time of operation. The preoperative staging proved incorrect in 16% of cases, the tumour size exceeding 2 cm. After a mean follow-up period of 36 months the local recurrence rate was 3.6% and distant metastases appeared in 9% of patients, whereby there was a significant dependence on lymph node status. No patient died of cancer during the observation period. In view of the hypothesis that breast cancer is a systemic disease more attention should be paid to local cosmetic results and to systemic adjuvant therapy. PMID- 3006364 TI - cAMP and cGMP (EVE) changes in immune competent cells under sensibilization and anaphylactic shock of animals of different age. AB - The studies performed proved that there were no substantial differences in intracellular content of cAMP and cGMP in immune systems of both old and young animals. The influence of equine serum upon lymphocytes in accompanied by the most substantial changes in concentrations of cyclic nucleotides in spleen B cells. The amplitude of level changes in cAMP and cGMP in these cells under anaphylactic shock doubles the value of old animals. Thus together with genetically preprogrammed change in functional activity of immune competent cells, expressed in different force of immune response, the correlative changes in cyclase system of animals of different ages have been observed. PMID- 3006365 TI - [Infectious diseases in pregnancy]. PMID- 3006366 TI - [Reconstructive operations in tracheal stenoses and acquired tracheoesophageal fistulas]. PMID- 3006367 TI - [Status of detecting bronchial cancer in the Newbrandenburg district]. AB - All 489 cases of bronchogenic carcinoma reported during the years 1982 and 1983 in the county of Neubrandenburg were analysed and compared with an analogous examination of the period from 1962 to 1969. In spite of a considerable shortening of delay between first symptoms and final diagnosis there was a shift to a greater proportion of more advanced tumor stages III and IV. Consequently, the rate of patients in whom resection could be performed, decreased. In general, patients detected by mass x-ray examinations are in an earlier stage. The analysis of clinical symptoms showed that especially in cases with obstructive pneumonia a careful examination is mandatory to early diagnosis of bronchogenic carcinoma with central localization. PMID- 3006368 TI - [Changes in blood pressure and serum lipids with fish diets in patients with mild essential hypertension]. AB - 14 male patients with moderate essential hypertension were treated in the cross comparison with a mackerel and herring diet, respectively, for two weeks. The mackerel diet contained double as much eicosapentaenic (EPA) and docosahexaenic acid (DHA) as the herring diet which served as control. In the serum triglycerides particularly DHS, in the cholesterol esters above all EPA were enriched. In the phospholipids the increase of the two fatty acids was approximately the same. At the end of the mackerel period the serum triglycerides, the total and LDL-cholesterol, the activity of the lecithin cholesterol-acyl-transferase (CALT) and the serum sodium were significantly decreased. On the other hand, the HDL-cholesterol and the uric acid in the serum significantly increased. Under influence of the herring diet the parameters mentioned appeared only slightly changed. After the mackerel diet also a significantly lower systolic blood pressure was found. The systolic and diastolic blood pressure during a standardized psychophysiological stress test was more diminished at the end of the mackerel period than after the herring diet. The plasma renin activity (PRA) was increased after the mackerel diet. Its increase under the stress test could no more be proved at the end of the mackerel diet. In similar way the stress-conditioned increase of thromboxane B2 could no more be observe both after mackerel and after herring diet. When the results should confirm themselves in long-term studies, a mackerel diet in practicable dosage can be recommended as non-medicamentous treatment of moderate hypertension. PMID- 3006369 TI - [Psoriasis in acquired immunodeficiency syndrome--new aspects for an immunopathogenesis of psoriasis]. PMID- 3006370 TI - [Psoriatic osteoarthropathy from the current viewpoint]. AB - Application of scintigraphic techniques, especially bone scintigraphy, to a group of unselected psoriatic patients provided us with new knowledge about frequency, dimension, and valence of psoriatic osteoarthropathy. Thus scintigraphy was able to prove up to 90% of the incidences, whereas clinical and radiological investigation only revealed 7%. This result was achieved on account of better registration of the polytopical character as well as the evidence of potential early manifestations which could not be detected clinically or radiologically. In addition, we proved extraarticular manifestations of psoriatic osteopathy, especially in the skull, the long marrow bones, the ribs, and the distal phalanges. PMID- 3006371 TI - [Failure to form wrinkles on the finger tips in the warm water test in lepromatous nerve damage]. AB - In order to estimate the extent of nerval defects, we recommend the soaking test which may reveal affected areas indicated by the loss of wrinkling at the finger tips after soaking the hands in warm water for half an hour. Thus we found impairment in the formation of wrinkles in 6 out of 7 tested patients suffering from different types of leprosy. In order to study the significance of the soaking test with regard to acute nerval signs and symptoms, one patient was reexaminated when he showed acute painful swelling of the right n. ulnaris. There was normal wrinkling observed at the affected hand, which did not differ from the outcome of the preceding test. This finding indicates that the soaking test is only appropriate to the examination of chronic nerval lesions in leprosy. PMID- 3006372 TI - [Virus papillomas and malignant genital tumors]. AB - Morphological and epidemiological studies point to a close correlation between viral genital warts and genital cancer. Benign condylomata acuminata are commonly induced by HPV-6 and HPV-11. In precancerous bowenoid genital lesions, HPV-16 and HPV-18 could be cloned directly from cervical cancer tissue. Whereas the occurrence of HPV-6 and HPV-11 seems to be a low risk infection, HPV-16 and HPV 18 may represent a high risk for malignant conversion. PMID- 3006373 TI - [Endocrinologic reactions following exposure to fluorescent lamps]. AB - Some reports on an increase of malignant skin tumors following fluorescent lighting made us investigate the systemic effects after exposure to fluorescent rays. Fluorescent rays induce small wavy fluctuations of the plasma concentration of alpha-MSH within normal ranges. Some test persons revealed a steep increase of cortisol values shortly after beginning of fluorescent lighting, indicating that fluorescent rays may induce endocrinological reactions. The significance of these findings with regard to the development of diseases, however, cannot be assessed by now. PMID- 3006374 TI - Nuclear inclusions in inferior olivary nucleus neurons of the cat and the ground squirrel (Citellus citellus L.). PMID- 3006375 TI - [Sexually differentiated lysosomal breakdown of exogenous cytochrome C in the rat nephron]. PMID- 3006376 TI - Ultrastructural changes elicited in the Tetrahymena by primary exposure (imprinting) and reexposure to hormone. PMID- 3006377 TI - [Role of insulin in regulating carbohydrate metabolism]. PMID- 3006379 TI - [Stromal reaction in breast cancer]. PMID- 3006378 TI - [Immunohistochemical studies with retrovirus-specific antibodies in benign and malignant diseases of the breast]. PMID- 3006381 TI - [Morphologic studies of the stroma of breast cancers]. PMID- 3006380 TI - [Leukocytic cell infiltrates in breast cancer. Studies using monoclonal antibodies]. PMID- 3006383 TI - [Metastatic behavior of breast cancer correlated with TNM classification and malignancy grade. An autopsy study]. PMID- 3006384 TI - [Lymphoreticular infiltrates in axillary lymph node metastases of invasive ductal breast cancers: histological and immunohistological findings]. PMID- 3006382 TI - [Relation of cell proliferation to regression with special reference to epithelial tumors]. PMID- 3006386 TI - [Statistical analysis of lectin and hormone receptor findings in 306 breast cancers]. PMID- 3006387 TI - [Prognostic significance of tumor grading in breast cancer]. PMID- 3006385 TI - [Morphometric correlates of the steroid receptor content in invasive ductal breast cancers]. PMID- 3006388 TI - Nuclear diameters in the invasive ductal carcinoma of the breast with a predominant intraductal component. PMID- 3006389 TI - [Cytochemical and roentgen microanalytic studies of the development of microcalcinosis in the breast]. PMID- 3006390 TI - [Topography and kinetics of epithelial proliferations of the lactiferous ducts. A scanning electron microscopy and endocrinologic study]. PMID- 3006391 TI - [Cytodiagnosis of invasive ductal breast cancer]. PMID- 3006392 TI - [DNA determinations in breast cancers: cytophotometric proliferation behavior and cyto/histologic malignancy grade]. PMID- 3006393 TI - [Cytomorphology and prognosis of invasive breast cancer--recommendation for cytologic grading and personal experiences]. PMID- 3006394 TI - [Ultrastructure and electron microscopy classification of noninvasive and invasive breast cancers]. PMID- 3006396 TI - [Morphology, topography and growth pattern of benign proliferative breast lesions and pre-invasive cancers]. PMID- 3006395 TI - New aspects of the natural history of in situ and invasive carcinoma in the female breast. Results from autopsy investigations. PMID- 3006397 TI - [Hydroxylapatite. Bone substitute material in jaw]. PMID- 3006398 TI - [Hydroxylapatite in preprosthetic surgery]. PMID- 3006399 TI - [What should the dentist know about AIDS?]. PMID- 3006400 TI - [The role of endoneural edema in the pathogenesis of diabetic neuropathy]. AB - Average endoneurial area was correlated with measurements of myelinated fiber density in sural nerves from diabetics and controls. Although, in general, myelinated fiber density decreased with increasing endoneurial area, this relationship was not significant for diabetic nerves. The increase in endoneurial area was attributed to expansion of the endoneurial space as a result of endoneurial edema. A possible role for such edema in the pathogenesis of diabetic neuropathy is considered. PMID- 3006401 TI - Spindle-cell squamous carcinoma (pseudosarcoma) of the breast. AB - A spindle-cell tumour of the breast developed in a woman after mastectomy for carcinoma and subsequent radio therapy. The final diagnosis of spindle-cell squamous carcinoma was corroborated using electron microscopy and immunohistochemical staining for keratin and vimentin. PMID- 3006402 TI - [Prosthetic problems following ridge augmentation with hydroxylapatite]. PMID- 3006403 TI - [Hydroxylapatite augmentation of the maxilla for denture wearing]. PMID- 3006404 TI - Non-chromaffin paraganglioma of the orbit. Case report. AB - The non chromaffin paraganglioma of the orbit is a relatively rare tumor. To our knowledge only 25 cases have been reported in the world literature. We report on the case of a 39-year-old woman who was treated surgically for the removal of an orbital paraganglioma 10 years ago. She had complained of proptosis and her right eyeball was slightly displaced upwards and laterally. A transcranial operation was performed and the tumor, located medically and weighing 9'5 grs., was completely removed; it was encapsulated. 10 years after this total excision there was no evidence of recurrence. From a histological point of view the tumor cells closely resembled those of paragangliomas of the carotid body and glomus jugulare. PMID- 3006405 TI - Recovery of reoviruses from tap water. AB - Samples of piped water were taken from the source of the springs in ten villages in Lorraine. Usually this water has not been subjected to disinfection. The water was tested for fecal indicator bacteria, phages and animal viruses. Of the ten samples examined, seven contained no fecal indicator bacteria, phages or animal viruses, in three samples, however, Reovirus type 2 was found. Among the three positive samples only one contained fecal bacteria and phages. Consequently, the two other samples could be regarded as drinking water, despite the presence of viruses. Such an approach poses the problem of testing drinking water for viruses and suggests that viral tests should be undertaken as part of the water quality control. PMID- 3006406 TI - Study of immunity induced by the inactivated oil-adjuvant vaccine after natural infection with the virus of infectious bovine rhinotracheitis (IBR) in cattle. PMID- 3006407 TI - Appearance of haemagglutinability of infectious bronchitis virus following in vitro and in vivo tracheal passages. PMID- 3006408 TI - [Antibodies to measles, smallpox, influenza and arenaviruses in schizophrenic patients]. AB - The levels of hemagglutinating antibodies to measles, smallpox, influenza viruses and of complement-fixing antibody to lymphocytic choriomeningitis, Takaribe, Amapari viruses were studied, in the blood sera of 77 schizophrenics and 44 normal donors. The investigation revealed definite differences. The schizophrenic patients had a statistically significant elevation in the titres of anti-smallpox antibodies. A certain increase in levels of antibodies to the measles virus was observed. There were no considerable differences in titres of antibodies to arenaviruses and influenza virus. A relationship between antibody production, on the one hand, and the severity and course of the disease and also the age of those examined, on the other, was noted. PMID- 3006409 TI - Serum cyclic 3',5'-nucleotide phosphodiesterase activity in myocardial infarction. AB - The activities of cyclic 3',5'-nucleotide phosphodiesterases which hydrolyze cyclic 3',5'-nucleotides were measured in sera from patients with an acute myocardial infarction, angina pectoris and other heart diseases. Cyclic AMP and cyclic GMP phosphodiesterase activities were significantly elevated in acute myocardial infarction, but not in angina pectoris and other cardiovascular diseases. The peak activity appeared approximately within 24 hours following the acute attack of chest pain, and then gradually decreased as the patient recovered. The observed changes of cyclic 3',5'-nucleotide phosphodiesterase activity were similar to that of the other enzyme activities such as GOT, CPK and LDH in sera of acute myocardial infarction. These data reflect damage of myocardial cells during myocardial infarction. PMID- 3006410 TI - Receptors, endocytosis and the clinician. PMID- 3006411 TI - [Myocardial contusion]. PMID- 3006412 TI - Clomiphene citrate induces pituitary GnRH receptors in ovariectomized rats: its possible role in induction of ovulation. AB - Since our previous studies have shown that clomiphene citrate (clomiphene) acts directly on the pituitary gland and exerts a facilitatory role on oestradiol-17 beta (E2)-induced LH surge in chronically ovariectomized rats, the effect of clomiphene on pituitary GnRH receptors was investigated. A single ip injection of either 5 micrograms E2 or 200 micrograms clomiphene did not induce LH release in adult rats ovariectomized 1-2 weeks before the injection. However, a significant increase in serum LH was noted 24 h after a single injection of E2 in the ovariectomized rats, if clomiphene was pre-injected 48 h before the E2 injection. The content of pituitary GnRH receptors in the ovariectomized rats (62 +/- 9 fmol/pituitary) remained almost unchanged until 24 h after a single injection of clomiphene but significantly increased 48 h after the injection (105 +/- 13 fmol/pituitary) without any alterations in the affinity for GnRH. To determine steroid specificity for the increase in pituitary GnRH receptors, other classes of steroids were injected in the ovariectomized rats. A single dose of E2 increased GnRH receptors, but either progesterone or 5 alpha-dihydrotestosterone failed to show any effect on the level of GnRH receptors. These results suggest that clomiphene may augment oestrogen-induced pre-ovulatory LH surge in anovulatory women, at least in part by increasing the number of pituitary GnRH receptors. PMID- 3006413 TI - Effect of angiotensin II on aldosterone and its precursor steroid production in adrenal zona glomerulosa cells from heparin-treated rats. AB - To assess the nature of the heparin-induced aldosterone deficiency, we investigated the stimulatory effect of angiotensin II (AII) on aldosterone and its precursor steroids in adrenal zona glomerulosa cells from heparin-treated rats compared with those in the cells from vehicle-treated rats. Heparin-treated rats had low plasma aldosterone levels, high plasma renin activity and plasma AII levels, and normal plasma corticosterone level 6 weeks after the treatment (1500 IU/kg, twice daily). Basal aldosterone production, when corrected to a uniform number of cells per group, was similar in the cells from heparin- and vehicle treated rats. The cells from heparin-treated rats had a less sensitive and lower response of aldosterone production to AII; an increase by 4 orders of magnitude in the threshold dose for AII and a decrease in the maximum AII-stimulated level. The maximum AII-stimulated levels, but not the basal levels, of pregnenolone, corticosterone and 18-OHB production were low in the cells from heparin-treated rats. ACTH caused a similar stimulatory effect on aldosterone production in the cells from heparin- and vehicle-treated rats. The cells from heparin-treated rats had a less sensitive and lower response of aldosterone production to potassium; an increase by one order of magnitude in the threshold dose for potassium and a decrease in the maximum potassium-stimulated level, presumably because of the glomerulosa hyporesponsiveness to AII. These results suggest that our heparin treated rats have selective impairment of adrenal zona glomerulosa cells, involving the specific receptors and the aldosterone biosynthesis, to AII. PMID- 3006414 TI - Effect of an opiate antagonist on the responses of circulating catecholamines and the renin-aldosterone system to acute sympathetic stimulation by hand-grip in man. AB - The effect of naloxone on the neurohumoral responses to acute sympathetic stimulation by sustained hand-grip in normal man was investigated. Six normal males were studied fasting at 08.30 h, on two occasions at 7-day intervals, with each subject sustaining 30% of his maximal hand-grip on a hand dynamometer for 5 min. Naloxone (8 mg bolus) in 20 ml normal saline, or saline alone, was given 5 min before hand-grip in a randomised double-blind cross-over trial. Blood was sampled for plasma renin activity, serum aldosterone and plasma catecholamines. The study was repeated in the absence of hand-grip. Sustained hand-grip produced significant elevations in mean blood-pressure, circulating adrenaline, noradrenaline and aldosterone. Naloxone, which had no effect on basal catecholamines, plasma renin activity or aldosterone, significantly enhanced the responses in plasma adrenaline, plasma renin activity and serum aldosterone to hand-grip. The increments in blood pressure and noradrenaline were not affected. These results suggest that endogenous opioids modulate the response of the sympathoadrenal and renin-aldosterone systems to acute sympathetic stimulation by a mild stress in man. PMID- 3006415 TI - Enhancement of vitamin D3 effect on bone metabolism in weanling rats orally administered zinc sulphate. AB - The interaction of vitamin D3 and zinc on bone metabolism was investigated in the femur of weanling rats. Oral administration of vitamin D3 (1.0 micrograms/100 g body weight) did not cause any increase in the zinc accumulation in the femoral tissue following treatment with zinc sulphate (1.0 mg Zn/100 g). Administration of vitamin D3 or zinc produced significant increases the alkaline phosphatase activity and DNA content of the femoral diaphysis but not of the epiphysis. The increase in alkaline phosphatase activity was enhanced additionally by simultaneous administration of vitamin D3 and zinc. Moreover, the increase in DNA content was enhanced markedly (about 4 times) by these treatments. At a dose of 0.5 micrograms of vitamin D3 per 100 g, DNA content was at the control level. This level was increased about 2 times by simultaneous administration of zinc (1.0 mg/100 g). The increase in alkaline phosphatase activity following simultaneous administration of vitamin D3 and zinc was significantly inhibited by treatment with cycloheximide, actinomycin D, or mitomycin C. Also, the increase in DNA content was completely inhibited by mitomycin C treatment. The present data suggest that the combination of vitamin D3 and zinc has a multiple effect on the stimulation of bone growth and mineralization in weanling rats, and that this effect is based on a stimulation of the DNA synthesis in bone cells. PMID- 3006416 TI - Myeloperoxidase activity of polymorphonuclear leukocytes in iron deficiency anemia and anemia of chronic disorders. AB - Myeloperoxidase (MPO) activity was studied in adults with iron deficiency anemia (IDA) or anemia of chronic disorders (ACD). MPO activity (biochemical quantitation) was found to be decreased in IDA when compared to the control group (p less than 0.05); ACD subjects also had lower values although the difference was not significant (p less than 0.05). MPO scores (MPO staining) were significantly lower in IDA and ACD subjects than in the healthy control group (p less than 0.05). A significant positive correlation was noted between ferritin (R = 0.40) and percent transferrin saturation (R = 0.37) and MPO activity (p less than 0.001) in IDA and for the healthy controls. PMID- 3006417 TI - Mistletoe lectin I binding sites on the synaptosomes of the rat cerebral cortex. AB - By means of combined lectinological and immunological methods were demonstrated mistletoe lectin I binding sites on rat cerebral cortex synaptosomes. The mistletoe lectin I binds specifically D-galactose. Galactosyl residues were established on the junctional and nonjunctional synaptosomal membrane, on the synaptic vesicles, mitochondria and on myelin contamination. The relative number of mistletoe lectin I receptors per unit area of synaptosomal membrane was calculated. PMID- 3006418 TI - Localization of nucleoside phosphatases (ATPase and 5'-nucleotidase) and nuclear ribonucleoproteins in the human endometric glandular cells during the secretory phase. AB - Endometrial curettings from 3 healthy women between 16th and 19th d of the menstrual cycle were investigated employing ultracytochemical methods. In endometrial glandular cells ATPase and 5'-nucleotidase activities were demonstrated in the nuclei, nucleoli, and nuclear channel system (NCS), i.e., mainly in structures characterized ++as ribonucleoproteins according to the Bernhard method. The functional relationship between the NCS and the molecular biology of the endometrial glandular cell is discussed. PMID- 3006420 TI - [Light and electron microscopic studies on glycogen inclusions in the parathyroid cell nuclei of cattle]. PMID- 3006419 TI - Inhibition of corticosteroidogenesis by etomidate. AB - The effects of the intravenous anesthetic etomidate have been investigated on ACTH-induced steroidogenesis in vitro, using purified isolated rat adrenal cells. It was found that etomidate almost completely blocked corticosterone production induced by physiological concentrations of ACTH at doses of 200 ng or greater. The mean inhibitory etomidate concentration resulting in 50% inhibition approximated 1.5 X 10(-7)M which is in the range of concentrations measured after clinical doses of etomidate. PMID- 3006421 TI - [Anatomo-clinical evaluation of 426 cases of Hansen's disease under observation in the dermatology service of C.H.U. de Treichville (Abidjan)]. PMID- 3006422 TI - In vitro analysis of BCNU-sensitivity in human malignant gliomas. II. Cross resistance studies with cisplatinum and nitrosoureas. AB - With the use of the Human Brain Tumor Stem Cell Assay (HBTSCA) in a cross resistance study, four early (3-4) culture passages of human malignant gliomas (glioblastoma multiforme) were tested for in vitro chemosensitivity with three of the most effective single agents for brain tumor chemotherapy: BCNU, CCNU and cisplatinum (DDP). The shapes of the dose-response curves indicated complete cross-resistance between BCNU and CCNU, i.e. two chloroethyl-nitrosoureas sharing a common alkylating-carbamoylating activity, with no evident cross-resistance between the two nitrosoureas and the DDP, a DNA binder with a putatively different antitumor action. Probably because of differences in drug delivery kinetics or in the cytotoxic mechanism, DDP might play a role in the treatment of nitrosourea-resistant gliomas. PMID- 3006423 TI - Best's vitelliform macular dystrophy. AB - We examined and evaluated the ophthalmological findings of 47 patients with Best's Vitelliform Macular Dystrophy (BVMD) and 5 cases suffering from related conditions to this macular disorder. Our sample re-confirm that BVMD is a progressive disease which may have several appearances in the course of its evolution. The heredity of this disorder is autosomal dominant with reduced penetrance and variable expressivity. Some contradictions exist regarding the nature of the primary defect in this entity. Electrooculographic and angiographic investigations lend support to the belief that the basic pathological changes are located in the retinal pigment epithelium. However, recent histopathological findings and flicker electroretinographic results indicate the possibility that the photoreceptor cells are equally involved, even before the pigment epithelium. In view of the existing disagreements about the pathogenesis of this disorder, certain considerations were advanced which suggest that the basic pathologic process in this entity produces a disorganisation in the structural and functional interdependance of both the photoreceptor cells and pigment epithelium. PMID- 3006424 TI - [Cochleo-vestibular attacks of viral origin]. AB - The viral etiology of some cochlear or vestibular lesions is well documented. Concerning the cochlear congenital lesions, the cytomegalovirus is becoming the main etiologic factor, while the rubella vaccination has shown its efficiency in the United States. The acquired hearing or vestibular diseases were briefly reviewed as well as the pathology and their therapeutic considerations. PMID- 3006425 TI - Impairment of polymorphonuclear leucocyte function during therapy with synthetic ACTH in children affected by epileptic encephalopathies. AB - Therapy with synthetic ACTH (zinc tetracosactide) in children affected by epileptic encephalopathy is often associated with a large number of infectious complications. We studied the phagocytic activity of polymorphonuclear leucocytes (PMN) in 9 children with West or Lennox-Gastaut syndrome, measuring PMN superoxide anion production during the phagocytosis of particles of Zymosan and after phorbol myristate acetate (PMA) stimulation. The test was performed before, during and after therapy with zinc tetracosactide (0.02 mg/kg/day for 15 days). At the same time plasma immunoglobulins, C3, C4, C3 activator and cortisol were determined. During treatment PMN phagocytic function was significantly reduced but returned to normal levels after suspension of therapy. The other hematological parameters considered remained within the normal range. During the follow-up of the patients we observed 15 infectious episodes (3 mucocutaneous candidiasis, 2 enterocolitis, 4 urinary tract infections, 1 otitis media, 3 bronchiolitis, 2 pneumonia). One of the patients died of a bilateral pneumonia. Three children were treated with ACTH on alternating days. In these patients PMN phagocytic activity was less impaired and 2 infectious episodes rapidly resolved. Alternate day ACTH therapy seems to be preferable. PMID- 3006426 TI - Long-term treatment with corticosteroids/ACTH in asthmatic children. II. Hypothalamic-pituitary-adrenal function. AB - Hypothalamic-pituitary-adrenal (HPA) function was studied in 23 children with severe bronchial asthma during and after long-term treatment with prednisolone and/or ACTH1-24 depot tetracosactrin) by means of ACTH stimulation test and insulin tolerance test. In the 14 children primarily treated with depot tetracosactrin, the cortisol levels in insulin tests were within normal limits both during and after treatment. An enhanced response to ACTH stimulation was found during the treatment period. During treatment with prednisolone a marked impairment of the adrenocortical function was found, with low basal plasma cortisol levels and subnormal response to ACTH stimulation, more marked the lower the age at the start of treatment and the higher the dose per kg body weight. After substitution with depot tetracosactrin the HPA-function was restituted, with plasma cortisol levels within normal limits. Growth hormone levels after insulin induced hypoglycemia were greater than or equal to 7 ng/ml during and after treatment with depot tetracosactrin. As long-term treatment with depot tetracosactrin has little side-effects in terms of suppression of the HPA-axis it is a useful alternative to oral prednisolone in severe asthma in children. PMID- 3006427 TI - [Radioligand binding assay of a beta-adrenoceptor on duck erythrocyte membranes]. PMID- 3006428 TI - Cortical beta- and alpha 2- adrenoceptor binding, hypothalamic noradrenaline and pineal melatonin concentrations measured at different times of the day after repeated treatment of rats with imipramine, zimeldine, alaproclate and amiflamine. AB - The effect of repeated treatment of rats for 21 days with the monoamine reuptake inhibitors imipramine, zimeldine, alaproclate (in each case 10 mumol/kg b.i.d.) and the reversible monoamine oxidase-A inhibitor amiflamine (3 mumol/kg b.i.d.) on brain noradrenergic mechanisms measured at different times of the day and night was investigated. Imipramine treatment produced a down-regulation of the Bmax for 3H-dihydroalprenolol binding to cortical beta-adrenoceptors that was not dependent upon the time of day the animals were killed. Zimeldine, on the other hand, reduced both Bmax and Kd of binding for day-time, but not night-time samples. Alaproclate and amiflamine were without effect on the binding. Twenty four hour mean values for 1 nM 3H-p-aminoclonidine binding to alpha 2 adrenoceptors were lower for the zimeldine-treated rats than for the saline treated rats. Pineal melatonin concentrations, which are regulated by beta adrenoceptors, showed a pronounced diurnal rhythm, with the highest concentrations being found at 02:00. At this time point, a lower pineal melatonin content was found after amiflamine treatment, whereas imipramine, zimeldine and alaproclate were without significant effect. The importance of the use of more than one time point and the use of more than one biochemical test for the determination of the effects of repeated antidepressant treatment on central noradrenergic systems measured ex vivo is discussed. PMID- 3006429 TI - Fluorocarbons and cardiac arrhythmia: does difluorodichloromethane (FC 12) inhibit cardiac metabolism? AB - Certain fluorocarbons, such as difluorodichloromethane (FC 12), depress the cardiovascular system by diminution of all the transmembrane ionic conductances in cardiac tissues. Does FC 12 also inhibit active transport and thus enzymatic activity and cellular energy? We measured phosphocreatine (PC), adenosine triphosphate (ATP) and cyclic adenosine monophosphate (AMPc) in rat hearts. Rats were randomly divided into 4 groups; 2 control groups: one breathing a mixture of oxygen (21%) and nitrogen (79%) (group C) and the other breathing the same mixture but simultaneously perfused with 1 microgram/kg/min. epinephrine (groupe E-C); 2 trial groups T and E-T where nitrogen was replaced by FC 12. The maximal FC 12 concentration of 720 micrograms/ml in arterial blood produced no significant difference in the concentrations of these three metabolites compared with controls. PMID- 3006430 TI - Noradrenaline evokes an alpha-adrenoceptor-mediated inotropic effect in human ventricular myocardium. PMID- 3006431 TI - Evidence for a role of brain serotonergic neurotransmission in avoidance learning. AB - This thesis has analyzed the role of brain serotonergic (5-HT) neurotransmission in avoidance learning in the male rat using neurochemical, pharmacological and behavioural approaches. The acute and long-term effects of p-chloroamphetamine (PCA) on one-way and two-way active avoidance (AA) acquisition and retention and passive avoidance (PA) retention and on central monoamine concentrations were examined in the male rat. The effects of PCA were compared with the 5-HT synthesis inhibitor p-chlorophenylalanine (PCPA). To characterize the effects of PCA the following neurotoxins were used: 5,6- and 5,7-dihydroxytryptamine (5,6- and 5,7-DHT) and N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). The concentrations of biogenic monoamines and their metabolites in discrete brain regions were determined by high pressure liquid chromatography with or without electrochemical detection. The effects on 5-HT receptors in vitro and in vivo were measured by ligand binding studies (using 3H-5-HT and 3H-ketanserin as radioligands) and with behavioural techniques, respectively. Administration of the 5-HT releasing compound PCA prior to training (pre-training) produced a dose- and time-related impairment of one-way AA acquisition and retention and PA retention. A series of studies indicated that the avoidance learning deficits caused by PCA are produced by release of 5-HT, resulting in stimulation of postsynaptic 5-HT receptors. For instance, the avoidance deficit was blocked by pretreatment with the 5-HT uptake inhibitors alaproclate and zimeldine, which inhibit the 5-HT release induced by PCA. The avoidance deficits could not be related to changes (direct or indirect) in NA and DA transmission. Lesion experiments in combination with biochemical analyses provided evidence that the avoidance deficit caused by PCA involves 5-HT terminals of the forebrain, while the descending 5-HT projections seem to play a minor role. The AA acquisition deficit induced by PCA appears to be mediated via stimulation of 5-HT2 receptors, whereas the PA retention is mediated via 5-HT1 receptors. Analysis of the PCA induced AA deficit indicated that it is mediated by non-associative factors. Thus, the performance of the PCA-treated rats was susceptible to interference from extratask contextual stimuli. Pre-training administration of PCA was found to produce a time-dependent loss in memory retention (PA retention) in animals which had acquired the response. This finding indicates that serotonin also has a role in associative learning processes e.g. in the way information is processed in the rat brain following acquisition.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3006432 TI - Studies of neurotensin-dopamine receptor interactions in striatal membranes of the male rat. The influence of 6-hydroxydopamine-induced dopamine receptor supersensitivity. PMID- 3006433 TI - [3H]muscimol and [3H]quinuclidinyl benzilate binding in rat cortex in the abstinence after long-term barbital treatment. PMID- 3006434 TI - GABA antagonists increase the release of [3H]acetylcholine from the chick retina. AB - The release of [3H]acetylcholine from the chick retina was studied. A 5 mM increase in K+-concentration caused an increased release, which was Ca2+ dependent. The effect of 5 mM K+ was neither potentiated by bicuculline nor inhibited by isoguvacine or muscimol. This indicates that the K+-induced release is not controlled by GABA. However, bicuculline and picrotoxin increased the spontaneous efflux of radioactivity, whereas GABA had no significant effect. The results suggest that cholinergic neurons are tonically inhibited by a continuous release of endogenous GABA. Neither glycine or strychnine, nor dopamine or haloperidol had any effect on the spontaneous release. PMID- 3006435 TI - Effects of repeated mammographic screening on breast cancer stage distribution. Results from a randomised study of 92 934 women in a Swedish county. AB - A randomised controlled trial of mass screening for breast cancer by single-view mammography was begun in Sweden in 1977. All women aged 40 and older and resident in the counties of Kopparberg and Ostergotland were enrolled. The present report is confined to the Ostergotland study, which started in 1978 and comprised 92 934 women. After randomisation, which was done on the basis of communities rather than individuals, 47 001 women were allocated to the study group and offered repeated mammographic screening; 45 933 were allocated to the control group. As compliance among women over 74 years of age was poor these were excluded from the present report. The yearly incidence of stage II or more advanced breast cancers after the initial screening round up to and including the second was reduced by 40 per cent in the study group compared with the controls. This effect was less marked in the age group 40-49. After 5.5 years average from the date of entry the absolute number of women with stage II-IV disease in the control group exceeded that for the study group by 44, whereas there was a large excess of cancer in situ and stage I cancer in the study group. PMID- 3006436 TI - Chest irradiation as an attempt to improve the response after induction chemotherapy in small cell lung carcinoma. AB - Forty-six patients with small cell lung carcinoma received cyclic chemotherapy with cisplatin-VP 16 and vincristine, doxorubicin, and cyclophosphamide. The responding patients were given prophylactic cranial irradiation. Patients without metastases not achieving a complete response (CR) following induction chemotherapy were given chest irradiation. The response rate was 73.9 per cent. Response was improved by radiation therapy in only 9 per cent of the patients with limited disease. Median survival was 39 weeks, with 2 patients surviving for longer than 24 months. The duration of response and survival in complete and partial responders was similar; absence of radiation therapy in the patients with CR might explain this finding. PMID- 3006437 TI - The effect of surgery, radiation therapy, and combined radiation therapy and chemotherapy on immunocompetence in patients with lung carcinoma. AB - The immunologic status of 59 patients with lung carcinoma was determined by analysis of peripheral venous blood samples. The following tests were performed: total leucocyte and lymphocyte counts, the number of acid alpha-naphthyl acetate esterase (ANAE) positive cells (T-cells), and phytohaemagglutinin (PHA) and tuberculin (PPD) transformation tests. The patients were divided into three treatment groups: a surgery group (S), a radiation therapy group (R), and a combined cytostatic and radiation therapy group (C). A follow-up was carried out 4 to 6 months after treatment. The therapeutic measures, resection, irradiation, and chemotherapy, produced a decrease in the total leucocyte and lymphocyte counts, in the number of T-cells, and in the leucocyte transformation response to PPD. In the surgically treated group the decrease was transient. In the groups treated with radiation therapy and combined cytostatic and radiation therapy the values remained low throughout the follow-up. The lymphocyte response to PHA was not altered in any of the groups during initial treatment or follow-up. The results did not suggest a correlation between the immunologic parameters used and the stage and histologic type of lung cancer. The tests were of no clinical value in the determination of the individual prognosis. PMID- 3006438 TI - Detection of neoplastic lymph nodes in Hodgkin's disease and non-Hodgkin lymphoma. Comparison between tomography and lymphography. AB - During a period of 17 months, 98 consecutive patients with malignant lymphoma were examined for initial staging before therapy. Both CT and lymphography were performed in 58 patients (19 patients with Hodgkin's disease (HD) and 39 patients with non-Hodgkin lymphoma (NHL], and these were included in the investigation. The results were discrepant in 26 cases where lymph node lesions were detected by only one of the two methods. In 10 patients, 5 with HD and 5 with NHL, the positive finding by one of the methods was taken as determinant of the stage. The conclusion drawn was that CT cannot completely replace lymphography without losing important information. Owing to limited resources for lymphography and CT a reduced staging programme is proposed. Judging by the present results, this reduced programme would probably mean only a minimal loss of information. PMID- 3006439 TI - Radical irradiation of T2 and T3 bladder carcinoma. A retrospective investigation. AB - The relative 5-year survival was 32 per cent in 97 patients with T2 or T3 bladder carcinoma (1972-1978) treated with high dose radiation therapy (CRE greater than or equal to 1700 reu). A slight but significant difference was noted between the two T categories. Fifty-three patients who were tumour-free 3 to 4 months after irradiation had a 55 per cent survival. No other prognostic parameters were identified on the basis of histologic or radiologic findings. In 14 patients severe radiation complications were observed (bladder 9, small bowel 3, combined 2). In patients with radiation sensitive bladder carcinoma, radical radiation therapy seems to be a comparable alternative to total cystectomy. PMID- 3006440 TI - Quantitative evaluation of spontaneous and radiation-induced polyploidisation processes in human and murine testes. AB - Flow cytometric analysis of human and murine testicular tissue was performed in order to determine whether cells exist with a DNA content differing from the expected categories 1 c, 2 c and 4 c, and to estimate the frequencies of diploid elongated spermatids. About 1.5 per cent of the murine testicular cells had an 8 c DNA content. In human testis, no 8 c cells were detected. A slight increase of 8 c cells was observed in the murine testis, following irradiation. An average of 1.8 per cent of the elongated spermatids in 10 control mice were diploid. In 12 human testicular biopsies, an average of 4.7 per cent of diploid elongated spermatids was observed among all elongated spermatids in mice. Acute or split dose exposure, with 15 Gy roentgen rays, to spermatocytes increased the spontaneously occurring level of diploid elongated spermatids 25-fold. PMID- 3006441 TI - The use of computed tomography numbers in dose calculations for radiation therapy. AB - Although corrections for 'beam hardening' and 'scattering' have been implemented in currently available CT scanners, systematic differences exist between a real CT image and an ideal, artefact-free and monochromatic image. The appearance and magnitude of these differences are discussed. Conversion to the ideal image, i.e. conversion from CT number to X-ray attenuation coefficient at diagnostic photon energies, turns out to be possible with an accuracy of 5 per cent. In order to use the CT 'density' information from patients, in clinical photon and electron beam dose calculations, conversions must be made from the X-ray attenuation coefficient at diagnostic energies to relevant high energy radiation interaction properties. These conversions turn out to be possible within an accuracy also of 5 per cent. These limited accuracies cause errors in the photon beam dose calculation of less than 1 per cent of the dose maximum and errors in electron beam dose calculations of less than 2 per cent of the dose maximum. PMID- 3006442 TI - Human blood granulocyte macrophage progenitors (GM-CFU) during extended field radiation therapy. AB - The rate of the clonogenicity of granulocyte macrophage forming cells (GM-CFU) in agar was analysed using mononuclear cells from the peripheral blood of cancer patients undergoing total body irradiation (TBI, 5 patients, 1.5 Gy in 15 days) or hemicorporal irradiation of the lower (LHBI, 5 patients, 15 Gy in 15 days) or upper (UHBI, 4 patients, 15 Gy in 15 days) part of the body. Although the GM-CFU level in the blood is low compared with that in the bone marrow, and shows considerable variability, the preliminary results demonstrated that in most cases the GM-CFU level was increased around the 10th day after the beginning of irradiation, followed by a return to base level within a few days, for LHBI and TBI but not for UHBI. The question of whether the observed peak for the clonogenicity was due to stem cells leaving a radiation-injured compartment of the bone marrow or to vicarious stem cells migrating from non-irradiated regions, or was the result of enhanced activity of colony-stimulating factors, remains to be investigated. PMID- 3006443 TI - Intracytoplasmic immunoglobulins in the differential diagnosis of lymphocytic lymphomas of the B-CLL type and immunocytic lymphomas. AB - One hundred and thirty-three consecutive cases originally classified either as a lymphocytic lymphoma of the B-CLL type or as an immunocytic (IC) lymphoma could be reclassified morphologically and analyzed for the presence of cytoplasmic immunoglobulins (cIg) with the PAP-technique. The morphologic reclassification confirmed the initial diagnosis in most cases, whereas after staining for cIg, the diagnosis was changed in a large number of cases, i.e. from B-CLL to IC, or the reverse, or from IC of the polymorphic subtype (ICp) to 'high-grade' non Hodgkin lymphoma (NHL). Cases classified as IC were often localized (stage I+II: 22/43) with a long disease-free survival after local radiation therapy, while B CLL were usually generalized. For patients in stage IV, the prognosis of B-CLL was significantly superior to that of IC, which in turn was superior to the prognosis of cases referred to as 'high-grade' NHL. The difficulties in the morphologic distinction between B-CLL and IC on one hand and between ICp and some 'high-grade' NHL on the other hand, as well as the clinical significance of these distinctions, are discussed. PMID- 3006444 TI - Cycling S-phase cells in animal and spontaneous tumours. I. Comparison of the BrdUrd and 3H-thymidine techniques and flow cytometry for the estimation of S phase frequency. AB - Evaluation of the proliferative activities of cell populations has mainly been restricted to the use of autoradiography and flow cytometric measurements. The introduction of a new BrdUrd specific antibody makes it possible to determine exactly the DNA synthesizing cells. The BrdUrd technique is safe with respect to handling and the results are obtained within five hours. The suitability of the BrdUrd labelling procedure has been studied in different cell lines and compared with 3H-thymidine autoradiography and flow cytometry. PMID- 3006445 TI - RNA and protein synthesis of irradiated Ehrlich ascites tumour cells. II. Amount of m-RNA and in vitro investigations. AB - Poly(A)-containing RNA (m-RNA) was studied in in vivo growing Ehrlich ascites tumour cells following a roentgen irradiation dose of 5 Gy. m-RNA increased significantly during the first 12 hours after irradiation. Thus, the observed decrease in protein synthesis rate during this time seems not to be due to radiation induced changes at the transcriptional level. The protein synthesis rate of in vivo irradiated cells incubated in vitro in culture medium was unchanged. On the other hand, the protein synthesis rate of non-irradiated cells incubated in vitro in ascites fluid from irradiated animals was decreased. We concluded that factor(s) inhibiting protein synthesis or the lack of factor(s) promoting protein synthesis in the ascites fluid is(are) of significance for the reduced protein synthesis of tumour cells found in irradiated in vivo growing cells. PMID- 3006446 TI - Influence of pancreatic secretion on late radiation enteropathy in the rat. AB - In female Wistar rats a 10 cm long exteriorized mid small intestinal segment was roentgen irradiated 3 weeks after a pancreatic duct-occluding operation/sham operation. Roentgen doses were 19 and 21 Gy as single exposures. Radiation injury was assessed 2, 8 and 26 weeks after irradiation using one macroscopic and 7 histopathologic parameters. The parameters were graded according to severity, and a radiation injury score was calculated by adding the scores for the individual parameters. Two and 8 weeks following irradiation there was no difference between pancreatic duct-occluded and sham operated animals. Twenty-six weeks after irradiation all parameters of radiation injury except the extent of vascular sclerosis were less marked in pancreatic duct-occluded rats than in controls. It is concluded that exocrine pancreatic secretions may influence the development of late changes following irradiation, and that these changes seem to be mainly independent of the degree of vascular sclerosis. PMID- 3006447 TI - Cortisol insufficiency caused by electroconvulsive therapy? A case report. AB - A 30-year-old woman was treated with a series of electroconvulsive therapy (ECT) due to a personality disorder with depressive symptoms. Three days after the last ECT, anisocoria was noticed. It subsided after 2 days, and attacks of syncope, vertigo, anorexia and weight loss started. These symptoms ceased by administration of cortisone acetate and fluoro-cortisone. During an observation time of five years, repeated attempts to omit the cortico-steroids or reduce the cortisone dose to less than 20 mg/day have resulted in immediate symptoms of cortisol deficiency. Plasma adrenocorticotrophin (ACTH) and cortisol and urinary cortisol were low during cortisol withdrawal. Cortisol response to ACTH stimulation and cortisol and ACTH response to hypoglycemia were normal. The cortisol deficiency was considered to be due to a defect in the central nervous regulation of ACTH secretion. As it occurred in close connection to ECT, it seems likely that the treatment induced a defect, or aggravated a preexisting one, in neural pathways controlling corticotrophin-releasing factor and ACTH secretion. PMID- 3006449 TI - Converting enzyme inhibition in mild and moderate essential hypertension. II. AB - In 24 patients with mild/moderate essential hypertension, we studied the effects of captopril with/without hydrochlorothiazide (Htz) on blood pressure, the renin angiotensin system, blood bradykinin concentration (BBK), plasma volume, exchangeable sodium and glomerular filtration. Daily captopril doses of 75 and 150 mg were equally effective in reducing the blood pressure. Addition of Htz caused further blood pressure reductions. Nineteen patients attained a diastolic blood pressure less than or equal to 90 mmHg. Angiotensin converting enzyme inhibition with captopril led to a fall in plasma concentrations of angiotensin II (PAII) and renin substrate, and an increase in plasma concentrations of renin and angiotensin I. Patients starting with Htz had a higher PAII and subsequently a larger fall in blood pressure on captopril than untreated patients. BBK remained unchanged, indicating that the hypotensive action of captopril does not involve an accumulation of circulating kinin. Body fluid volumes and renal function were not affected by the various treatment regimens. PMID- 3006448 TI - Plasma lipoproteins and fatty acid composition during a moderate eicosapentaenoic acid diet. AB - The effect of a diet rich in marine fatty acids, especially eicosapentaenoic acid, on plasma lipids (total plasma cholesterol, HDL cholesterol, total triglycerides and apolipoproteins A and B) and fatty acid composition in plasma phosphatidylcholine (PC) was studied in 10 healthy men. They were maintained for 11 weeks on their normal diet which was partly replaced by 150-200 g of fatty fish per day. In the same individuals this diet had previously caused a delay in primary haemostasis and a decrease in platelet aggregability similar to that caused by acetylsalicylic acid, a known inhibitor of thromboxane A2 formation. Apart from its effect on haemostasis, the fish diet substantially reduced serum triglycerides (by 43%, p less than 0.01) but caused no changes in total plasma or HDL cholesterol or apolipoproteins A and B. After three weeks on the diet the proportion of plasma PC omega-3 polyunsaturated fatty acids increased (C20:5 and C22:6) and omega-6 fatty acids decreased (C18:2 and C20:3). The relative plasma PC content of arachidonic acid was unaffected throughout. These alterations in plasma PC fatty acid composition were principally in accordance with those seen in platelet membrane PC. There was a linear correlation between the content of omega-3 and of omega-6 fatty acids in plasma PC with that of platelet PC as well as in predominate individual fatty acids of the two series. Six weeks after the volunteers had resumed their usual diet, total triglycerides and the fatty acid composition of plasma PC had returned to the original state. PMID- 3006450 TI - Photosensitization of mitochondrial adenosine-triphosphatase and adenylate kinase by hematoporphyrin derivative in vitro. AB - Mitochondrial ATPase and adenylate kinase activity of hepatoma cells were inhibited by hematoporphyrin derivative (HPD) followed by photoirradiation. Inhibition of ATPase activity was a dose- and time-related event. Malonaldehyde (MDA) content of mitochondrial membranes was markedly increased by HPD plus light. The content of mouse liver microsomal cytochrome P-450 was greatly increased after intraperitoneal injection of HPD for 4 days (5 mg/kg/day). The liver weight, and levels of liver microsomal G-6-phosphatase, MDA and triglyceride (TG) showed no difference in treated vs. control animals. The data presented here demonstrate that mitochondria may be a sensitive site of action of HPD photosensitization, and inactivation of ATPase and adenylate kinase may be an important contributing factor to tumor cell damage and death. PMID- 3006451 TI - HL-A and disease. AB - Of more than 500 diseases or syndromes studied for HL-A markers, more than 40 are known to be associated with an allele of class I, II, or III. Seven are linked to the HL-A region: six are recessive (idiopathic hemochromatosis, C2, C4A, and C4B deficiencies, congenital and late-onset deficiencies) and one is dominant (spinocerebellar ataxia). In addition, insulin-dependent diabetes mellitus is also linked to HL-A with more than one single locus. HL-A typing is of practical interest for diagnosis of ankylosing spondylitis by B27 antigen determination and for prevention of idiopathic hemochromatosis by genotyping of siblings of the index case. Prenatal diagnosis of 21-OH deficiency by genotyping fetal cells permits genetic counseling. Indeed, the discovery of the relationship between HL A and disease can be considered a new approach to medical genetics. Extensive use of HL-A technology will probably allow better prediction of risk and may elucidate the mechanisms of certain diseases. For the first time the study of one single immunogenetic system may have a significant effect on public health through the possibility of wide-scale prevention. PMID- 3006452 TI - Viral infections in renal transplant recipients: an evolutionary problem. PMID- 3006453 TI - The role of ethanol in the etiology of primary liver cancer. PMID- 3006454 TI - Direct and indirect thermogenic effects of anorectic drugs. PMID- 3006455 TI - The dietary management of diabetes. PMID- 3006456 TI - Genetics of human alcohol and aldehyde dehydrogenases. PMID- 3006458 TI - Influence of a diet rich in eicosapentaenoic acid on the development of rat paw oedema and on the formation of prostaglandins I2 and E2. AB - The influence of feeding a marine oil (MaxEPA) with a high content of eicosapentaenoic acid (EPA) to rats for 10 weeks on the development of carrageenin oedema was studied. Since prostaglandins (PGs) are involved in the development of this acute experimental inflammation, the influence of EPA feeding on PG release from aorta (PGI2) and from the subplantar skin of the inflamed foot (PGI2, PGE2) was investigated also. MaxEPA was fed in two daily doses containing 50 or 100 mg EPA/kg/day. In both rat groups there was no influence of EPA on the development of the oedema. However, the capacity of aorta and skin of the plantar region of the experimentally inflamed foot to release PGI2 was strongly reduced by EPA. On the other hand, the release of PGE2 from the skin was not reduced. Indomethacin at a low dose (2 mg/kg perorally) reduced the development of the paw oedema as well as the release of PGs in control rats and rats on an EPA containing diet. It is concluded that EPA did not influence carrageenin oedema because there was an adequate production of the oedema promoting substance PGE2. PMID- 3006457 TI - Endothelium-derived relaxing factor (EDRF). AB - Recent studies on endothelium-dependent vasorelaxation have been briefly reviewed and analyzed. The following processes appear to subserve this mechanism: In the endothelial cell: receptor activation, activation of phospholipases, mobilization of intracellular Ca2+, synthesis and release of 'endothelium-derived relaxing factor(s) (EDRF); In the smooth muscle cell: activation of guanyl cyclase and protein kinase, protein phosphorylation/dephosphorylation, relaxation. Alterations of this mechanism could be involved in certain cardiovascular disorders. PMID- 3006460 TI - Dynamic CT features of arterioportal shunts in hepatocellular carcinoma. AB - Dynamic computed tomographic (CT) findings of 42 patients with hepatocellular carcinoma having angiographically proven arterioportal shunt were reviewed. CT findings related to arterioportal shunt were as follows: early enhancement of the portal vein, which showed a time-density curve similar to that of the aorta, markedly prolonged enhancement of the portal vein, dilated, abnormal intrahepatic vessels often accompanied by irregular, transiently enhanced areas, transient high attenuation of lobar or segmental distribution in the lobe contralateral to the main tumor, and transient wedge-shaped enhancement peripheral to the tumor. Dynamic CT usually detected arterioportal shunts involving larger portal veins as represented by any of the first four findings (14 of 17), whereas detection of arterioportal shunts involving smaller portal veins was lower in frequency (8 of 25), but such a shunt could be demonstrated as transient wedge-shaped enhancement peripheral to the tumor. PMID- 3006459 TI - Biochemical mechanisms in 5-hydroxytryptamine-induced human platelet aggregation. AB - The activation of human platelets by 5-hydroxytryptamine (5-HT) is not accompanied by detectable release of ATP or TXB2. The process is unaffected by cyclooxygenase, thromboxane synthetase or combined cyclooxygenase/lipoxygenase inhibition (suprofen, indomethacin, R 19091, dazoxiben, N.D.G.A, BW755C, esculetin), indicating the absence of involvement of arachidonic acid metabolites. Transmembrane Ca2+-entry blockers (flunarizine, nifedipine, nimodipine) have no effect either, indicating that the activator calcium released by 5-HT comes from intracellular stores. The 5-HT-induced platelet activation is inhibited by stimulators of adenylate cyclase (PGE1, PGE2, isoprenaline, adenosine) and inhibitors of cAMP phosphodiesterase (papaverine, anagrelide, RA233), indicating that also for this type of platelet activation cAMP behaves as a unidirectional, inhibitory regulator. PMID- 3006461 TI - CT of hepatic masses: significance of prolonged and delayed enhancement. AB - Hepatic masses showing higher density than that of liver after 3 min (prolonged enhancement) and/or having prominent enhancement after the arterial-dominant phase (delayed enhancement) of dynamic CT (bolus enhancement followed by serial scans) were reviewed. Prolonged enhancement was noted in any hepatic masses, but mainly in cavernous hemangioma and capsule of hepatocellular carcinoma, whereas delayed enhancement occurred in capsule of hepatocellular carcinoma and occasionally in metastatic tumor. The volume of arterial and portal blood supply, turnover rate of blood, extent of the interstitial space, and diffusion rate between the vascular and interstitial space, as well as dose and speed of contrast agent administered, seemed to be important factors in such contrast enhancement. Prolonged enhancement and delayed enhancement are nonspecific but still are of some value in the differentiation of hepatic masses on dynamic CT. PMID- 3006462 TI - Glioblastoma multiforme masquerading as a more benign process. PMID- 3006463 TI - CT evaluation of the greater sciatic foramen in patients with sciatica. AB - Sciatic and lower extremity neurologic symptoms may be from pathologic involvement of the sacral plexus or sciatic nerve in the region of the greater sciatic foramen. Twenty-five patients were reviewed who presented consecutively over a 4 year period with sciatic symptoms secondary to pathologic changes in the greater sciatic foramen. Malignant neoplasm alone (18 patients) and malignant neoplasm associated with infection (two patients) account for most of these cases. Neurogenic tumors (three patients), both benign and malignant, and infection alone (three patients) were less frequent. Although sciatic symptoms usually derive from spinal abnormalities, the evaluation of sciatic symptoms should not be considered complete without CT scanning of the greater sciatic foramen. PMID- 3006464 TI - Comparison of the electrocardiographic and hemodynamic responses to ionic and nonionic radiocontrast media during left ventriculography: a randomized double blind study. AB - The ECG and hemodynamic responses to a standard ionic radiographic contrast agent (diatrizoate) were measured and compared to those induced by iopamidol, a newly developed nonionic agent, during left ventriculography. Studies were performed using randomized double-blind techniques in 46 patients with suspected coronary artery disease who were scheduled for cardiac catheterization. A nuclear probe was used to measure left ventricular ejection fraction and relative ventricular volume before and immediately after left ventriculography. Bolus injections of diatrizoate and iopamidol induced similar significant decreases in left ventricular end-diastolic and end-systolic volume and similar significant increases in both left ventricular end-diastolic pressure (p less than 0.05) and systolic ejection fraction (p less than 0.01 vs baseline). Both agents induced modest increases in heart rate, but only the increase induced by diatrizoate was significant (p less than 0.01). The maximal rate of left ventricular pressure rise was not significantly altered by either agent. Iopamidol induced a slight increase in QRS duration (p less than 0.05); neither agent effected a significant change in QT duration. We conclude that the hemodynamic effects during left ventriculography using diatrizoate and iopamidol are similar. These findings do not justify the large-scale substitution of more expensive nonionic radiographic contrast agents for standard ionic agents such as diatrizoate in left ventriculography. PMID- 3006465 TI - Possible contribution of digitalis-induced coronary constriction to toxicity. PMID- 3006466 TI - Adrenergic activity and left ventricular function during treatment of essential hypertension with calcium antagonists. AB - The effects of 2 calcium antagonist drugs, verapamil and nifedipine, on blood pressure, heart rate (HR), plasma catecholamines, plasma renin activity and some echocardiographic indexes of left ventricular anatomy and function were studied in 67 patients with essential hypertension. The short- and long-term antihypertensive effect of verapamil was not associated with significant changes in HR, plasma catecholamines or plasma renin activity; the decrease in blood pressure after nifedipine was associated with a significant increase in HR and plasma catecholamines (mainly noradrenaline) (p less than or equal to 0.05). These findings were confirmed in a crossover comparison in 12 hospitalized patients treated with verapamil and nifedipine for 8 days each. The dose of isoproterenol that increased HR by 25 beats/min was significantly increased during verapamil treatment (p less than 0.05) and decreased during nifedipine treatment (p less than 0.01). Stroke volume and shortening fraction increased slightly but significantly (p less than 0.05) with 3 months of nifedipine treatment, while no change was detected with verapamil treatment. Left ventricular mass was significantly decreased after effective antihypertensive treatment for 3 months with verapamil or nifedipine (p less than or equal to 0.05). PMID- 3006468 TI - Amiodarone-induced decrease in lymphocyte beta-adrenergic receptor density. PMID- 3006467 TI - Calcium antagonism--a new concept for treating essential hypertension. AB - Research on calcium antagonists has been prompted by the observation that the powerful vasodilatory effect of verapamil, as well as other calcium antagonists, is enhanced in hypertensive patients. Increased vascular resistance, seen in most types of hypertension, is determined by the intracellular free calcium concentration. The finding of an increased vascular responsiveness to calcium channel blockade and the direct relation between the degree of antihypertensive response and the height of pretreatment blood pressure indicate abnormal intracellular calcium handling in patients with essential hypertension. This is supported by the observation that the intracellular free calcium concentration was significantly increased in patients with essential hypertension compared with normotensive subjects. The decrease in blood pressure with calcium antagonists was directly correlated with the patient's age and inversely with the pretreatment plasma renin activity. There was comparable antihypertensive efficacy among verapamil, nifedipine and nitrendipine. Increased understanding of pathophysiologic mechanisms in essential hypertension and pharmacotherapeutic studies have led to a new strategy for treatment of high blood pressure--in which calcium antagonists may be used, at least in part, as alternatives to diuretic drugs primarily in older and low renin patients with essential hypertension. PMID- 3006469 TI - Animal pharmacology of guanfacine. AB - The pharmacologic data obtained from animal experiments with guanfacine, a novel, centrally acting antihypertensive agent, are reviewed. When given orally, guanfacine lowers systemic blood pressure in conscious DOCA-NaCl-hypertensive rats, Grollman rats and spontaneously hypertensive rats in a dose-dependent manner. It is also effective in renal hypertensive cats. Guanfacine reduces blood pressure in cats, rabbits and rats after injection into the lateral cerebral ventricle and in dogs after infusion into the vertebral artery at intravenously ineffective doses. Vagally mediated reflex bradycardia in dogs is enhanced. The preganglionic splanchnic (sympathetic) nerve activity is reduced in cats. In rats, guanfacine reduces the noradrenaline turnover in the brain stem. All these findings indicate a central site of action. Peripheral alpha-adrenoceptor stimulant properties of guanfacine have been demonstrated in various studies. In addition to postsynaptic stimulant effects, presynaptic guanfacine-induced inhibition of sympathetic heart nerve stimulation is antagonized by rauwolscine but not by prazosin, indicating a highly preferential alpha 2-agonistic presynaptic action of the drug. In receptor binding studies using rat cortex membranes and human platelets, guanfacine exhibited a high selectivity for alpha 2 adrenoceptors. Guanfacine has the advantage over other centrally acting antihypertensives of being less sedative and causing no rebound hypertension after discontinuation of treatment. The latter is mainly due to its pharmaco kinetic properties. PMID- 3006470 TI - Long-term effect of wholemeal bread on stool weight, transit time, fecal bile acids, fats, and neutral sterols. AB - Stool weight, fecal constituents, bile acids, fat, neutral sterols, and intestinal transit time were recorded in 28 subjects over 18 mo. During the first 12 mo the subjects ate white bread. They were studied for an initial period of 7 days, and after 6 mo (study period 1). For the first 6 mo they ate their usual intake of bread, they then increased their white bread intake by 62 g/day for 6 mo (study period 2). The subjects ate a self-selected diet throughout the 18 mo study. During the last 6 mo (study period 3) the subjects replaced white bread by the same amount of wholemeal bread as in study period 2. No increase in stool weight occurred until study period 3 when there was an increase of 20%. There developed a linear relationship between stool weight and intestinal transit time which was not found during the initial first and second study periods. A seasonal influence on serum cholesterol was not observed during the wholemeal bread period. Fecal bile acid excretion was unchanged throughout the experiment. PMID- 3006472 TI - Apparent absorption and retention of Ca, Cu, Mg, Mn, and Zn from a diet containing bran. AB - To establish conditions for comparisons of mineral bioavailability from plant sources, seven male subjects consumed a constant diet containing bran fiber and phytate. Absorption and retention of Ca, Cu, Mg, Mn, and Zn were measured for 7 day periods through wk 2-7. Intakes of Mg, Mn, and Zn significantly exceeded the RDA; Ca and Cu intakes were only slightly in excess of RDA. All mineral retentions fluctuated from week to week but only Mg and Mn showed a consistent positive trend over time. Phytate excretions showed characteristic individual patterns, but did not appear to change with time. In contrast to previous observations fecal recovery of polyethyleneglycol (PEG) (MW = 4000) was consistently lower than recovery of simultaneously ingested Cr. Only five of the seven subjects returned close to 100% of Cr within 7 days. It was concluded that at least 4 wk were needed for adaptation in investigations involving more than one mineral when the experimental diet is adequate in the nutrients under investigation, that measurements of responses to treatment required 2-3 wk each, and that successive isotopically labeled test meals may overlap if they are spaced at 7-day intervals. PMID- 3006471 TI - Interaction of dietary sucrose and fiber on serum lipids in healthy young men fed high carbohydrate diets. AB - High sucrose diets may cause increased serum triglycerides and decreased high density lipoprotein concentration. To determine whether dietary fiber protects against these effects, four groups of six healthy young men were assigned to one of four very high carbohydrate diets providing 0, 18, 36, or 52% of calories as sucrose. Each diet was fed in both low (less than 14 g) and high (greater than 34 g) levels of dietary fiber for 10 days each. Triglycerides increased during the 36 and 52% sucrose diets compared to 0 and 18% sucrose diets, and fiber protected partially against this rise. Serum cholesterol and LDL cholesterol were lower during the 0 and 18% sucrose diets than the 36 or 52% sucrose diets but fiber had no effect. HDL cholesterol decreased during all low fat diets, with a trend toward a greater decrease during the high sucrose diets. The results suggest that fiber protects against carbohydrate-induced lipemia but has no effect on cholesterol during very high carbohydrate diets. PMID- 3006473 TI - Adriamycin chemotherapy for malignant mixed mesodermal tumor of the ovary. A Gynecologic Oncology Group Study. AB - Malignant mixed mesodermal tumor (MMMT) of the ovary is a rare, but usually fatal, cancer for which there exists no proven postsurgical therapy. The Gynecologic Oncology Group has evaluated adriamycin in this disease at a dose of 75/m2. Among 10 evaluable cases with measurable disease there was one partial response and no complete responses. Of 21 cases with nonmeasurable tumor four remain clinically free of cancer from 2 to 45 months. There were two treatment related deaths. We conclude that adriamycin alone as first-line chemotherapy in patients with MMMT of the ovary is not sufficiently active to be clinically useful. PMID- 3006474 TI - Intrahepatic doxorubicin in unresectable hepatocellular carcinoma. The unfavorable role of cirrhosis. AB - To investigate the relationship between the presence of cirrhosis and the antitumor effects of locoregional chemotherapy with doxorubicin, 16 patients with nonresectable hepatocellular carcinoma (HCC) and satisfactory baseline clinical conditions (Child class A or B, Karnofsky index greater than 70%) were studied. Eight patients had post-necrotic cirrhosis, five had serum HBsAg. The dose of doxorubicin was 0.3 mg/kg body weight/day, given by continuous intracoeliac infusion for 8 consecutive days. Eight patients (six with cirrhosis) died prematurely after the first course of chemotherapy. Six (2 with cirrhosis) responded to therapy; they survived 3-33 months (median: 10). In these patients, the type and severity of drug-related side effects were comparable to those reported for patients treated by intravenous chemotherapy. The implication that in many patients with cirrhosis intrahepatic chemotherapy with doxorubicin may hasten death, lessens our interest in its use for nonresectable HCC. In fact, in Italy these cancers frequently occur in association with cirrhosis. PMID- 3006475 TI - Comparison between histologic type, estrogen receptor, and nuclear DNA content in mammary carcinoma. AB - Histological specimens from 80 invasive breast carcinomas comprising typical cases of 16 ductal, nine papillary, 14 comedo, 13 colloid (mucous), 15 lobular, and 13 medullary carcinomas were examined with respect to nuclear DNA and estrogen receptor content. In agreement with previous studies, ductal carcinomas were found to exhibit different types of nuclear DNA distribution patterns, i.e., tumors with DNA values in the normal diploid or tetraploid regions indicative of good prognosis (euploid tumors) or those with values exceeding the normal tetraploid region indicative of poor prognosis (aneuploid tumors). The majority of the papillary and colloid tumors were euploid, while comedocarcinomas in general had aneuploid profiles. These findings are in agreement with expected survival within these patient groups. In lobular breast carcinomas, the correlation between the DNA distribution patterns and expected patient survival was less obvious; and in medullary carcinomas where the vast majority of the tumors showed aneuploid DNA profiles, the correlation to expected patient survival was low. Thus, lobular carcinoma in general seems to have a worse prognosis than is expected from nuclear DNA analysis, whereas medullary carcinomas in general seem to carry a better prognosis than indicated from DNA measurements. In agreement with earlier reports there was a good correlation between nuclear DNA content of the tumor cells and cytosol estrogen receptor values, i.e., euploid tumors in general exhibited relatively high receptor levels, whereas aneuploid tumors had low or unmeasurable estrogen receptor levels. PMID- 3006476 TI - Immune response of infants and children to low-passage bovine rotavirus (strain WC3). AB - A bovine rotavirus (strain WC3) was isolated from a calf in Pennsylvania and adapted to growth in continuous Cercopithecus cell line CV1. A pool for human vaccine trials was produced at the 12th cell culture passage level. After preliminary testing in adults and older children, a dose of 3 X 10(7) plaque forming units was given by mouth to 52 infants and children aged 5 months to 6 years. No clinical sequelae were detected, and shedding in feces was detected in only 30% of tested infants. A serum-neutralizing antibody response was induced in 95% of 21 infants aged 5 to 11 months; response rates were slightly reduced in older infants. The antibody response was primarily directed toward bovine rotavirus, but a response to human serotype 3 rotavirus was also observed in approximately 50% of vaccinees. After vaccination with WC3, infants with preexisting antibody to rotaviruses of human serotype 1 or 3 frequently exhibited a booster response to those serotypes. WC3 is a candidate rotaviral vaccine deserving larger trials in children. PMID- 3006477 TI - Thyroid lymphoma with gastrointestinal involvement: report of three cases. AB - Three elderly females are reported who presented with high-grade lymphoma of the thyroid and subsequently were found to have extensive gastrointestinal (GI) lymphoma that dominated their clinical courses. One of the patients remains free of disease 30+ months after extensive resection of involved bowel and combination chemotherapy. Two died from disseminated lymphoma. Optimal delivery of therapy in both of the latter patients was impeded by massive gastrointestinal hemorrhage. A review of previously reported cases of thyroid lymphoma, plus those described here, suggests a predilection for these tumors to involve the GI tract independent of other organ metastases. PMID- 3006478 TI - Activation of platelet function in Fabry's disease. AB - Increased platelet aggregation and high plasma concentration of beta thromboglobulin were observed in hemizygotes and heterozygotes of Fabry's disease. Carbamazepine and phenytoin administered for the treatment of pains in these patients showed no significant effect on platelet aggregation. No activation of platelets was observed after the addition of ceramide trihexoside, the storage lipid of this disease. Mitral valve prolapse was found in eight of 12 patients. Although the pathogenesis of platelet activation and mitral valve prolapse are not known, the platelet activation could be an early indicator and an accelerating factor of thromboembolic vascular change in this disease. PMID- 3006479 TI - Use of molecular haplotypes specific for the human pro alpha 2(I) collagen gene in linkage analysis of the mild autosomal dominant forms of osteogenesis imperfecta. AB - Autosomal dominant osteogenesis imperfecta (OI) is a heterogeneous group of disorders. Molecular haplotypes associated with the pro alpha 2(I) gene of human type I procollagen were used for genetic linkage studies in a group of 10 families with OI. The clinical phenotypes of the families studied were those of OI type I and OI type IV. Evidence for linkage was highly suggestive in the four families with OI type IV (Z = 3.91 for theta = 0). In contrast, little or no indication for linkage was found in the six families with OI type I (Z = .055 for theta = .415). Heterogeneity between the two groups of families was highly significant (chi 2 = 11.14, P = .0008), suggesting that at least two separate gene defects may be the cause of the autosomal dominant forms of OI. PMID- 3006480 TI - A highly polymorphic locus in human DNA revealed by probes from cosmid 1-5 maps to chromosome 2q35----37. AB - The highly polymorphic locus D2S3 is revealed by three single-copy probes from cosmid C1-5. These probes, 1-30, 1-32, and 2-96, collectively reveal seven restriction fragment length polymorphisms. Fifty-three of 56 unrelated individuals (93%) were heterozygous at one or more of the seven loci, making the compound locus a very useful marker for gene mapping. Chromosomal assignment of D2S3 was obtained using a panel of human X hamster and human X mouse somatic cell hybrids. Molecular hybridization of EcoRI-digested DNA from these cell lines with the DNA inserts from subclones 1-30, 1-32, and 2-96 showed that all three probes mapped to the long arm of chromosome 2. Additionally, in situ hybridization of [3H]-labeled probe 2-96 to metaphase chromosome preparations allowed more precise assignment of the locus to the region 2q35----37. PMID- 3006481 TI - Linkage studies of polymorphic, repeated DNA sequences in centromeric regions of human chromosomes. AB - The DNA at human centromeric regions was characterized by using a repetitive sequence, 308, which localizes in situ exclusively to centromeres of all chromosomes. We previously noted that this sequence is enriched on chromosome 6 and has chromosome-specific organization on 6, 3, 7, 14, X, and Y. In addition to this basic organization, sequences homologous to 308 are polymorphic among normal individuals. The variants are transmitted in a Mendelian manner within a family. To determine the chromosome origin of the variants, we studied their linkage to markers of various chromosomes. Linkage analysis of one pedigree segregating two polymorphisms shows that the 2.6-kilobase (kb) BamHI and 2.6-kb TaqI fragments are linked to each other and to the HLA loci on chromosome 6. Data from another family shows that 2.8-kb TaqI, 4.0-kb TaqI, and 1.3-kb BamHI polymorphic fragments are linked and are probably near the Fy locus on chromosome 1. By dot blot analysis, we determined that the relative amount of these sequences in the genome is not measurably different between unrelated individuals. Thus, the polymorphisms represent changes in homologous 308 sequences on specific chromosomes and can be used as chromosome-specific markers. Linkage studies using polymorphisms of repeated sequences will be most useful within a kindred, especially from an inbred population, because polymorphic repeats of the same restriction size may be heterogeneous in origin. PMID- 3006482 TI - The origin of 45,X males. AB - Maleness in association with the karyotype 45,X is a very rare and hitherto unexplained condition previously described in only four or five patients. This study was carried out to determine whether such males might actually possess Y chromosomal material. Of the two 45,X males studied, one was found to be a low grade mosaic with a 46,XY karyotype in less than 3% of fibroblasts; all lymphocytes karyotyped were 45,X. Fibroblast DNA from this individual was found to contain Y-specific repeated sequences in 1%-3% the amount observed in the father, consistent with mosaicism for a 46,XY cell line. No Y-specific repeated sequences were detected in the other patient, in whom all mitoses were 45,X. In neither patient were there detectable amounts of any of the single-copy Y specific DNA sequences for which we tested. Studies of Xg blood groups and of X linked restriction fragment length polymorphisms indicated that the single X chromosome was of maternal origin in both 45,X male probands. In contrast to the situation in XX males, we can exclude paternal X-Y interchange as the etiology in the cases described here. Our findings are compatible with mosaicism being the explanation of at least some "45,X" males. PMID- 3006483 TI - Human mitochondrial DNA types in two Israeli populations--a comparative study at the DNA level. AB - Variations in human mtDNA restriction endonuclease fragment patterns were investigated in a sample number of 81 Israelis--Jews and Arabs--using total blood cell DNA. Eight new morphs were observed using five enzymes: HpaI, BamHI, HaeII, MspI, and AvaII. Of the 18 different combinations of fragment patterns (mtDNA types), only three were shared by both groups, but with striking frequency differences. The Arab sample disclosed "African" characteristics and was found to be slightly more polymorphic than the Israeli sample. One of the new types filled a "missing link" originally postulated in a phylogeny of mtDNA human types. PMID- 3006484 TI - A population genetic survey of the haptoglobin polymorphism in Melanesians by DNA analysis. AB - We have determined the haptoglobin (Hp) genotypes of 831 Melanesians from Vanuatu, Papua New Guinea, and New Caledonia by Southern blot analysis of DNA extracted from umbilical cord and peripheral blood samples. There was complete agreement between these genotypes and the protein phenotype in cases where both were determined, and genotyping was possible in cases where no serum haptoglobins were measurable. Subtyping of Hp1 alleles using restriction enzymes showed that Melanesians, like Mongoloids and Australian Aboriginals, have only the Hp1S allele. Three cases of Hp Johnson were found in Vanuatu, and further restriction mapping supported a partial gene triplication model for the structure of this variant. We also report a new common BclI restriction enzyme polymorphism upstream of the Hp1 gene. The advantages of using DNA for haptoglobin typing are discussed. PMID- 3006486 TI - Association of human T lymphotropic virus type III antibodies with sexual and other behaviors in a cohort of homosexual men from Boston with and without generalized lymphadenopathy. AB - Forty asymptomatic homosexually active men seen at a Boston community health center and 39 men with generalized lymphadenopathy were interviewed and filled out detailed epidemiologic questionnaires. Twenty percent of the asymptomatic men and 92 percent of those with lymphadenopathy had antibodies to human T lymphotropic virus type III (HTLV-III). None of the men have subsequently had the acquired immune deficiency syndrome (AIDS). Seropositivity was associated with receptive anal intercourse and oral exposure to ejaculate, a history of hepatitis B, anal gonorrhea, or intestinal parasites, but no other sexually transmitted diseases, and did not correlate with the use of recreational drugs. More of the seropositive men had multiple partners from New York City. An association with seropositivity was less evident in relation to the numbers of partners from San Francisco or Los Angeles, since the whole cohort generally had fewer contacts with partners from these cities. The data suggest that educational programs among homosexual men attempting to decrease AIDS risk should focus on decreasing the number of partners, receptive anal intercourse, oral exposure to ejaculate and other intimate rectal contact, and sexual contact with men from areas of increased HTLV-III seroprevalence. PMID- 3006485 TI - Human T cell leukemia virus type III antibody, lymphadenopathy, and acquired immune deficiency syndrome in hemophiliac subjects. Results of a prospective study. AB - A cohort of 63 hemophiliac subjects was followed for clinical and immunologic abnormalities related to the acquired immune deficiency syndrome (AIDS). When evaluated in early 1984, antibody to human T cell leukemia virus type III (HTLV III) was detected in the serum of 59 percent (24 of 41) of factor VIII or IX concentrate recipients, but in none (0 of six) of the cryoprecipitate/fresh frozen plasma recipients. HTLV-III-seropositive hemophiliac subjects, on average, had been exposed to twice as much concentrate during the previous year as seronegative hemophiliac subjects. The seropositive group had a significantly lower mean helper/suppressor T cell ratio and absolute helper T cell level than the seronegative group. By early 1984, 13 hemophiliac subjects in the study population had lymphadenopathy and one had AIDS. Antibody to HTLV-III was detected in the serum of 13 of these 14 hemophiliac subjects with overt clinical disease. The prevalence of lymphadenopathy or AIDS among HTLV-III-seropositive hemophiliac subjects was 54 percent (13 of 24). It is concluded that HTLV-III antibody occurs with high frequency in hemophiliac subjects, and is related to the amount of factor VIII or IX concentrate infused. Over half of HTLV-III seropositive hemophiliac subjects in this population had overt clinical disease with either lymphadenopathy or AIDS. PMID- 3006487 TI - Hemodynamic effects of vanadate administration in rats with different levels of sodium intake. AB - The acute hemodynamic effect of an intravenous (IV) bolus of vanadate (10 mumol/kg bwt) followed by an IV infusion at 0.5 mumol/kg min was studied in Wistar rats at three different levels of Na intake: low Na+ (0.5 mEq/24 h), normal Na+ (1.5 mEq/24 h), and high Na+ (15 mEq/24 h). Hemodynamic changes were measured using the radioactive microsphere method. Vanadate decreased cardiac output by 52.0 +/- 6.4% in low Na+ rats, by 41.2 +/- 3.3% in normal Na+, and by 29.2 +/- 3.1% in high Na+ group. Total peripheral resistances increased by 47.1 +/- 26% in high Na+ rats, by 80.1 +/- 6.6% in normal Na+, and by 96.3 +/- 4.5% in low Na+ group. Renal blood flow decreased by 70 +/- 4% in low Na+, 58 +/- 3% in normal Na+, and 39 +/- 3% in high Na+. Cerebral, testicular, and splanchnic blood flow showed smaller changes. These results demonstrate that intravenous vanadate induces marked hemodynamic changes that depend on Na+ intake being more striking when the intake of sodium is reduced. PMID- 3006488 TI - Hyperthyroidism following hypothyroidism. AB - Two patients are presented who developed autonomous thyrotoxicosis following a diagnosis of primary hypothyroidism. In one of these patients, antibodies to the TSH receptor were typical of Graves' disease when measured as thyrotropin binding inhibitor immunoglobulins (TBII) and as human thyroid adenylate cyclase stimulating (HTACS) activity, while a needle biopsy of the thyroid gland was consistent with lymphocytic thyroiditis. Twenty-one other reported cases of this unusual sequence found in the literature are reviewed. This occurrence is more common than is generally appreciated. PMID- 3006489 TI - Renal sodium excretion and atrial natriuretic factor. PMID- 3006490 TI - DNA linkage studies in the fragile X syndrome suggest genetic heterogeneity. AB - Previously, we showed genetic heterogeneity for linkage between the fra(X) locus and a factor IX DNA RFLP (Brown et al, 1985). When fra(X) families were predivided into two classes, one containing those with non-penetrant (NP) males and one with apparent full penetrance (P), evidence of significant heterogeneity was present. We have now extended this analysis by adding DNA linkage information on 2 additional probes, 52A and ST14, studied in 16 fra(X) kindreds. These data were combined with information on 16 published fra(X) families. There were 7 NP families and 25 P families. We confirmed our previous findings of a higher recombination fraction between factor IX and fra(X) in P families (0 = .32 with lod of .67) compared to as NP families (0 = .06 with lod of 6.11) which was significant at p less than .01. In comparing recombination fractions for the additional probes, more recombination between 52A and the other loci was consistently seen in P compared to NP families which suggested that there may be a higher rate of recombination proximal to the fra(X) locus in P kindreds. A strikingly higher recombination fraction between 52A and factor IX was present in comparing all fra(X) families (.18) to normal families (.02) which was significant at p less than .001. These results suggest genetic heterogeneity with respect to recombination is present both among fra(X) pedigrees and between fra(X) and normal pedigrees. PMID- 3006491 TI - An assessment of the use of flanking DNA markers for fra(X) syndrome carrier detection and prenatal diagnosis. AB - One hundred and three individuals in 11 unrelated families with the fragile-X [fra(X)] syndrome were tested for polymorphisms identified by probes flanking the fra(X) site at Xq27.3. Two probes distal and 2 proximal to the fra(X) site were used. Thirteen known female carriers were analyzed retrospectively. DNA markers gave probabilities of carrying the mutation of 99% in 1 female, 89% in 8 females, and 10-55% in the other 4 females. We also estimated the probability of having inherited the mutation for 16 individuals of unknown fra(X) status using DNA markers and corrections for incomplete penetrance. The DNA marker test gave risks for females of 1-6% (7 females), 15% (1 female), and 97% (1 female). In males the risks were 1-3% (6 males) and 91% (1 male). In 3 families, DNA marker data were used to calculate probabilities of greater than or equal to 98.5% that transmission of the fra(X) mutation had occurred through normal males. In the retrospective studies, only 1 of 7 retarded males could have been diagnosed prenatally as having the fra(X) mutation with a probability of 99%. DNA marker analysis was uninformative in 5 of these males. When fra(X) carrier status cannot be established by chromosome analysis, DNA marker studies provide an alternative test that can be used to calculate individual risks more precisely. However, linkage analysis of the probe loci in these 11 families suggests that the recombination frequency between the fra(X) locus and the factor IX gene (F9) and DXS52 may be greater than previously suggested. Until the true recombination frequencies are established and the question of heterogeneity among families is fully analyzed, caution in using DNA markers as a predictive test is advised. PMID- 3006492 TI - Speculation on the role of transposable elements in human genetic disease with particular attention to achondroplasia and the fragile X syndrome. AB - We suggest that mutations for fragile X-positive Martin-Bell syndrome, and perhaps also for achondroplasia, may result from the insertion of transposable elements (TE's). Loss of genetic function could result from either the insertion of TE's within or adjacent to a normal chromosomal gene or, in the case of fragile X, from the loss of genes distal to the site of TE insertion following subsequent TE excision without ligation of the resulting discontinuity. The phenotypically and often cytogenetically normal transmitting males in fragile X pedigrees are interpreted not as "nonpenetrant" transmitters of a fully formed fragile X but rather as transmitters of some or all of the factors necessary for TE insertion at Xq27. We consider it likely that such insertion frequently first occurs, both in soma and especially in the germline, in their daughters. Our models predict that father to son transmission of causative factors would be a common occurrence in fragile X pedigrees. The absence of documented father to son transmission either points to a flaw in the models or reflects systematic bias in the collection of pedigree information. PMID- 3006493 TI - Inheritance of fragile X syndrome: an hypothesis. AB - The fragile X (fra(X), or Martin Bell-MB) syndrome is considered an X-linked recessive trait. However, clinically normal male transmitters of the condition have been observed occasionally. The occurrence of "carrier" males and the observation of other unusual genetic characteristics in the MBS suggest that this condition is not a standard X-linked recessive trait. We propose that the MBS is due to a transposable genetic element which can exist in 3 different chromosomal states and effect 2 different extrachromosomal environments. This model can account for the peculiar genetic behavior of the fragile X syndrome. PMID- 3006495 TI - Prenatal diagnosis of fetal renal mesoblastic nephroma. AB - A rare case of fetal renal mesoblastic nephroma diagnosed prenatally by ultrasonography is presented. PMID- 3006494 TI - Corticotropin-releasing factor can stimulate gonadotropin secretion by human fetal pituitaries in superfusion. AB - In previous studies, corticotropin-releasing factor was found to elicit a rise in circulating adrenocorticotropic hormone in human subjects and laboratory animals, but no stimulatory effect of corticotropin-releasing factor on other pituitary hormones was detected. Since stress may be associated with luteinizing hormone changes as well as with those of corticotropin-releasing factor and adrenocorticotropic hormone, we quantified gonadotropin responses to corticotropin-releasing factor and arginine vasopressin in 11 human fetal pituitaries with use of both superfusions and static incubations. Exposure to corticotropin-releasing factor brought about a significant increase in adrenocorticotropic hormone and gonadotropin concentrations in the effluent media by means of the superfusion system. Similar concentrations of corticotropin releasing factor significantly increased adrenocorticotropic hormone secretion into the medium by dispersed fetal pituitary cells cultured on an extracellular matrix but failed to increase luteinizing hormone and follicle-stimulating hormone secretion. Exposure to 3 mmol/l 8-bromo-cyclic adenosine monophosphate caused an increase in all three peptides, both in superfusion and static incubations. Dose-response studies showed that the effect on gonadotropin secretion occurred at concentrations of 8-bromo-cyclic adenosine monophosphate two orders of magnitude lower than those affecting adrenocorticotropic hormone secretion. The purity of corticotropin-releasing factor and arginine vasopressin used in these studies was confirmed by high-performance liquid chromatography. These in vitro results are consistent with a paracrine effect of corticotropes acting on gonadotropes. The combination of static incubation and superfusion has proved useful in elucidating the effects of different secretagogues on pituitary cells. PMID- 3006496 TI - Basal and forskolin-stimulated cyclic adenosine monophosphate in intact human platelets during the menstrual cycle. AB - In previous studies we observed modifications of cyclic adenosine monophosphate and adenylate cyclase activity in human endometrium during the menstrual cycle. In the present study our intension was to verify whether these modifications occur in isolated intact platelets. The results demonstrate that in nine normal women platelet cyclic adenosine monophosphate content varies during the menstrual cycle both in basal and in stimulated conditions (in vitro addition of forskolin). In fact, significantly higher levels of cyclic adenosine monophosphate were consistently observed during the proliferative phase. These findings provide evidence that platelet cyclic adenosine monophosphate metabolism normally varies during the menstrual cycle, which suggests a possible involvement of this system in some important clinical events. PMID- 3006498 TI - HTLV-III screening by eye banks. PMID- 3006497 TI - Immune complex containing herpesvirus antigen in a patient with acute retinal necrosis. AB - A 21-year-old man with acute retinal necrosis showed a marked increase in the convalescent titer to herpes simplex type 1 virus, especially in the aqueous humor obtained by anterior chamber paracentesis. Using an enzyme-linked immunosorbent assay, we tried to detect the herpesvirus antigen in the circulating immune complex obtained from this patient. The immune complex contained an antigen or antigens that reacted with antiherpes simplex type 1 antibody. PMID- 3006500 TI - Effect of adenosine 3',5'-cyclic monophosphate on volume and cytoskeleton of MDCK cells. AB - We examined the effect of N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP) on the volume and cytoskeleton of confluent cultures of Madin-Darby canine kidney (MDCK) cells. A 90-min exposure to 1 mM DBcAMP resulted in a 20% reduction in volume as measured by [14C]-urea water space. The volume in cells exposed to isobutylmethylxanthine (IBMX, 0.1 mM) was reduced by 24%. In control cultures F-actin, revealed by staining with nitrobenzoxadiazole-phallacidin, was found at the base of the cell as fibers, at the junctional region as a circumferential band, and on the apical cell surface as a mottled fluorescence. A dense pattern of microtubules, revealed by indirect immunofluorescence, was seen throughout the cell. Exposure to DBcAMP for 90 min resulted in a change of F actin fibers into dense bundles near the periphery of the cell. This effect was even more striking when cells were exposed to IBMX. Cytochalasin B disrupted F actin and resulted in a volume reduction similar to that in DBcAMP. Neither DBcAMP nor IBMX affected the distribution of microtubules. Moreover, colchicine, which completely disrupted the microtubules, did not change MDCK cell volume. The results suggest that DBcAMP and F-actin play a role in volume control in MDCK cells. PMID- 3006499 TI - Ultrastructural morphometric investigation of early lesions in the pulmonary alveolar region of pigs during experimental swine influenza infection. AB - Experimental infection of specific-pathogen-free pigs with swine influenza virus by the intratracheal route resulted in a severe respiratory disease that closely resembled natural swine influenza in clinical course and pathologic lesions. Alveolar epithelial necrosis with sloughing of necrotic cells occurred from 24 to 96 hours after inoculation (p.i.) and was associated with alveolar edema and diffuse interstitial pneumonitis. The latter, initially of neutrophilic character, became histiocytic 48 hours p.i. Ultrastructural analysis of alveolar parenchyma disclosed viral replication in epithelial cells beginning at 5 hours p.i. and lasting to 96 hours. Budding of pleomorphic virus particles from the surface of alveolar epithelial cells and accumulation of viral proteins within the nucleus and cytoplasm of epithelial cells were seen. The extent of parenchymal lesions as quantified by stereologic morphometry within the whole lung was characterized by a marked relative and absolute volume increase of interalveolar septa and increased air-blood tissue barrier thickness. The volume increase of interalveolar septa was due to an increase of interstitial tissue volume by 85% in pigs at 96 hours p.i., compared with control pigs with similar lung volumes. PMID- 3006501 TI - Effect of valinomycin on thyroid iodide transport and TSH-stimulated cAMP formation. AB - The K+ ionophore valinomycin, in concentrations as low as 0.1 microM, induces an inhibition of thyroid-stimulating hormone (TSH)-stimulated cAMP formation in cat and pig thyroid slices and isolated, trypsin-collagenase-dispersed beef thyroid cells. Valinomycin was also shown to inhibit histamine and prostaglandin E1 stimulation of thyroid cAMP formation. The inhibitory effect of valinomycin could be partially overcome by elevated (81 mM) K+ concentrations. In the absence of valinomycin, the ability of TSH to stimulate thyroid cAMP formation was dependent on extracellular K+. Chronic removal or addition of K+ to medium bathing thyroid sections was accompanied by inhibition of TSH-stimulated cAMP formation. Maximum TSH stimulation was observed at an extracellular K+ of 2.7 mM. Valinomycin had no significant effect on thyroid ATP content but did reduce the ATP-to-ADP ratio. However, chronic removal of K+ had no effect on either ATP or the ATP-to-ADP ratio. Varying extracellular Na+ from 26 to 144 mM or addition of tetrodotoxin did not affect TSH action. Valinomycin addition to thyroid slices was associated with a reduction in iodide transport as measured by the ratio of tissue to extracellular iodide concentrations. The effect of valinomycin on iodide transport was accompanied by an increase in iodide efflux that was not greater than that observed with perchlorate ion, suggesting a reduced recirculation of released iodide in valinomycin-treated tissue. These findings suggest that alterations in thyroid cell K+ permeability or intracellular K+ concentration may be accompanied by changes in TSH-induced stimulation of thyroid cAMP formation. PMID- 3006502 TI - Ontogeny of pepsin secretory response to secretagogues in isolated rat gastric glands. AB - By use of isolated gastric glands from rats at various ages, we demonstrated that full-term neonate and 1-day-old rats showed no response to cholecystokinin octapeptide (CCK-OP), carbachol, or Ca2+ ionophore. The same glands, however, were responsive to dibutyryl cAMP. A mature response was not found until the pups were 2 days old. Injection of hydrocortisone into newborn rats led to an increase in pepsinogen concentrations in gastric glands and also an increased responsiveness to CCK-OP, carbachol, and Ca2+ ionophore A23187 24 h after administration. Hydrocortisone thus caused precocious maturation of both pepsinogen accumulation and pepsinogen secretory responsiveness of gastric glands in rat pups. PMID- 3006503 TI - Analysis of intracellular oxygenation of isolated adult cardiac myocytes. AB - The influence of cellular shape, cellular O2 consumption rate, and intracellular diffusion coefficient for O2 on the magnitude of intracellular O2 gradients during hypoxia was analyzed with the model of Boag (Curr. Top. Radiat. Res. 5: 141-195, 1969) to determine whether these parameters could account for the experimentally measured O2 dependence curves for myoglobin (Mb) oxygenation and cytochrome a + a3 oxidation in heart cells. The analysis shows that the intracellular diffusion coefficient for O2 must be below 4 X 10(-6) cm2/s for a substantial intracellular gradient to occur. The intracellular diffusion coefficient was calculated from the difference in half-maximal oxidation (P50) values for isolated Mb and intracellular Mb and was found to be 1.76 X 10(-6) cm2/s. Use of this value and appropriate geometric models satisfactorily described the O2 dependence of Mb oxygenation and cytochrome a + a3 oxidation in cells over an eightfold range of O2 consumption rates. However, the analysis does not account for the correspondence of intracellular P50 values of Mb oxygenation and cytochrome a + a3 oxidation. This implies that there exists an intracellular heterogeneity of either Mb distribution, mitochondrial distribution, or mitochondrial respiratory characteristics. Such heterogeneity would further contribute to diffusion limitation of O2 supply during hypoxia and could be a major factor underlying the cardiac myocyte structure-function relationship. PMID- 3006504 TI - Eicosonoid metabolism and beta-adrenergic mechanisms in coronary arterial smooth muscle: potential compartmentation of cAMP. AB - Beta-Adrenergic relaxation in bovine coronary arteries is enhanced by inhibition of eicosonoid metabolism and inhibited by its stimulation. We investigated the interaction between eicosonoid metabolism and beta-adrenergic mechanisms by studying the effect of perturbations of eicosonoid metabolism on vascular adenosine 3',5'-monophosphate (cAMP) content and the cAMP-dependent relaxation of isometric force and activation of glycogen phosphorylase. KCl (35 mM) elicited a contraction, activated phosphorylase, and slightly decreased cAMP content. Isoproterenol (10(-7) M) relaxed the KCl contraction, further increased phosphorylase activity, and increased cAMP. Neither indomethacin (5 X 10(-6) M) nor arachidonic acid (3 X 10(-5) M) affected the KCl contraction, but arachidonic acid increased both cAMP and phosphorylase activity and indomethacin decreased cAMP. Indomethacin potentiated the relaxation induced by isoproterenol but inhibited the activation of phosphorylase and had no effect on the isoproterenol induced increase in cAMP. Arachidonic acid, on the other hand, inhibited the isoproterenol-induced relaxation but potentiated both the increases of phosphorylase activity and cAMP. Thus neither relaxation nor phosphorylase activity was related in a straightforward manner to the total cAMP content. A direct relation between cAMP, relaxation, and phosphorylase can be reconciled with the antiparallel effects of alterations of eicosonoid metabolism observed in this study by a proposed model in which the effects of cAMP are assumed to be functionally compartmentalized. PMID- 3006505 TI - Mapping subcellular distribution of Na+-K+-ATPase in rat parotid gland. AB - Recent subcellular fractionation studies have raised the possibility that Na+-K+ ATPase might be present in both the apical and the basal-lateral membranes of exocrine gland acinar cells. Analytical fractionation and immunofluorescence microscopy studies of rat parotid glands were performed to confirm this interpretation. The distributions of biochemical markers after analyses based on differential sedimentation, equilibrium density-gradient centrifugation, and partitioning in an aqueous polymer two-phase system defined a total of 15 physically and biochemically distinct membrane populations. Among these populations, it was possible to select one (designated population i) with the characteristics expected of acinar cell basal-lateral plasma membranes. It contained Na+-K+-ATPase enriched 33-fold, and gamma-glutamyl transpeptidase enriched 23-fold with respect to the initial homogenate. A second population (designated population c) had the characteristics expected of acinar cell apical plasma membranes; it contained Na+-K+-ATPase enriched 28-fold, and gamma-glutamyl transpeptidase enriched 53-fold with respect to the initial homogenate. Although the identification of population c remains provisional, immunofluorescence studies verified that Na+-K+-ATPase is present in both the apical and the basal lateral acinar cell plasma membranes. In view of these results, it is likely that the apical Na+-K+-ATPase would participate in series with basal-lateral sodium- and chloride-entry pathways in driving the secretory electrolyte fluxes. PMID- 3006506 TI - Downregulation of vasopressin receptors in toad bladder. AB - Binding of tritium-labeled vasopressin [( 3H]AVP) to a broken epithelial cell preparation of the toad's urinary bladder has been related to hormonal action on water and urea transport across the intact tissue. Hormone binding to receptor sites and permeability changes were initiated at the same concentration of hormone (0.4 nM). Half-maximal urea and water permeability responses were observed with 3.1 and 5.6 nM AVP, respectively, although half-maximal receptor saturation required considerably higher concentrations of hormone (less than 500 nM). Because maximal permeability responses were obtained with occupation of approximately 200 fmol/mg protein receptor sites and the total receptor density was in excess of 2,000 fmol/mg protein, there is apparently a receptor reserve in this tissue. The antidiuretic hormone employed by the toad is vasotocin (AVT). This compound was 60-fold more effective than AVP in displacing [3H]AVP from receptor sites. Preincubation of bladders with AVT resulted in downregulation of receptor sites. Although the magnitude of receptor loss was equivalent in the presence or absence of a transmembrane osmotic pressure gradient, the capacity of AVT to induce permeability changes was more markedly reduced in the presence of an osmotic gradient. This observation suggests that the negative-feedback signal initiated by water flow through the hormone target cell diminishes sensitivity to hormone by a mechanism other than by a reduction in the number of surface receptors or by a decrease in their affinity for the hormone. PMID- 3006507 TI - Effects of forskolin and cyclic nucleotides on isometric force in rat aorta. AB - The present study was undertaken to determine the extent to which cyclic nucleotide-induced relaxation in the intact rat aorta is mediated at the level of the contractile system. The relaxant effects of the cyclic nucleotide analogues [8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) and dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP)] and forskolin were examined in both the intact vessel and a Triton X-100-skinned preparation of rat thoracic aorta. Relaxation of a norepinephrine-induced contraction was essentially complete 30 min after the addition of 50 microM 8-BrcGMP [% relaxation = 87.2 +/- 4.4% (n = 4)], 100 microM DBcAMP [98.2 +/- 1.2% (n = 4)], and 1 microM forskolin [107.0 +/- 3.3% (n = 5)]. These same doses were ineffective in relaxing precontracted skinned rat aortic rings compared with the relaxation achieved in the intact vessel. The largest relaxation in the skinned aortas was achieved with the addition of 1 microM forskolin [17.4 +/- 1.5% (n = 4)]. The addition of catalytic subunit of cAMP-dependent protein kinase had no effect on isometric tension in the precontracted skinned aorta. Preincubation with the cyclic nucleotide analogues or forskolin in a low-Ca2+ solution (pCa less than 8) was also ineffective in inhibiting subsequent isometric tension development. Our results suggest that only a very small fraction of the relaxation with cyclic nucleotides and forskolin in the intact rat aorta is due to the action of these agents at the level of the contractile system. PMID- 3006508 TI - Control of gap junction formation in canine trachea by arachidonic acid metabolites. AB - This study examined whether the synthesis of the metabolites of arachidonic acid (AA) was involved in gap junction formation by 4-aminopyridine (4-AP) treatment in vitro in canine trachealis. Studies were made of the effects on gap junction formation of putative inhibitors of the cyclooxygenase and of both this and the lipoxygenase pathway of AA metabolism and the direct effects of prostaglandins (PG) E2 and I2. The number of gap junctions of similar size was increased after brief exposure to 4-AP. After indomethacin (IDM), 4-AP treatment decreased the number of gap junctions but did not affect their size. Pretreatment with 5,8,11,14-eicosatetraynoic acid or nordihydroguiaretic acid, putative inhibitors of cyclooxygenase and lipoxygenase enzymes, inhibited both the 4-AP-induced increase and decrease in the number of gap junctions. FPL 55712, a putative antagonist of leukotriene C4, did not alter either the number or the size of gap junctions when added alone or in combination with IDM. AA alone increased the number of gap junctions, but after IDM, AA decreased the number of gap junctions compared with the controls. Incubation of trachealis strips in vitro for 30 min with PGE2 increased the number of gap junctions by about threefold along with an increase in the size of the gap junctions. Similar incubation with PGI2, however, increased the number of gap junctions by approximately 60% without any change in the size. In the course of some control experiments, an interaction between carbachol and alcohol was observed such that alcohol caused an IDM-sensitive relaxation of carbachol-induced contractions, which was not observed when serotonin was the contractile agent. These results strongly suggest that PGE2 and PGI2 increase the formation of gap junctions in canine trachealis and that these prostanoids are released by 4-AP treatment. Leukotrienes may also be inhibitory in the formation of gap junctions, but FPL 55712 did not affect either the increase or the decrease in gap junctions after 4-AP. PMID- 3006509 TI - Effect of opiate-receptor blockade on normoglycemic and hypoglycemic glucoregulation. AB - By use of the opiate antagonist naloxone, we have examined the hormonal and metabolic responses to opiate-receptor blockade under basal conditions and during insulin-induced hypoglycemia in normal dogs. Naloxone treatment had no measurable effect on glucose concentration, turnover, and norepinephrine levels, but stimulated plasma epinephrine, glucagon, and cortisol and inhibited insulin release. Insulin (7 mU X kg-1 X min-1) decreased plasma glucose to 42 +/- 4 mg/dl due to an initial decrease in glucose production and an increase in glucose disappearance. Glucose production then increased, and plasma glucose plateaued. After 50 min of insulin infusion, epinephrine levels increased 26-fold (P less than 0.05), norepinephrine and glucagon 3-fold (P less than 0.02), and cortisol 4 fold (P less than 0.01). Similarly, plasma beta-endorphin and adrenocorticotropin (ACTH) were elevated (6-fold, P less than 0.01, and 16-fold, P less than 0.05, respectively). When naloxone was given during insulin-induced hypoglycemia, there was earlier release of epinephrine, glucagon, beta-endorphin, ACTH, and cortisol as well as a greater release of glucagon (P less than 0.001) and cortisol (P less than 0.0001). This resulted in a greater increase in glucose production (P less than 0.01), thus lessening the insulin-induced hypoglycemic excursion. In conclusion, in the dog, endogenous opiates may play a small role in the regulation of basal insulin and glucagon release and can inhibit the pituitary adrenal axis under basal conditions and during hypoglycemia. Thus increased glucose production in response to insulin-induced hypoglycemia is consistent with the excessive response of counterregulatory hormones during opiate-receptor blockade. PMID- 3006510 TI - Effect of hydrogen ion concentration on corticosteroid secretion. AB - The direct effects of changes in extracellular hydrogen ion (H+) concentration on corticosteroid secretion under basal and ACTH-stimulated conditions were studied in isolated, perfused canine adrenal glands. Changes in extracellular H+ concentration were produced by altering either PCO2 or [HCO-3] of the Krebs bicarbonate perfusate. Alkalosis markedly inhibited ACTH-stimulated aldosterone secretion. Moreover, within the range of pH from 7.19 to 7.85, there was a positive correlation between H+ concentration and the fractional secretion of aldosterone but a negative correlation between H+ concentration and the fractional secretion of corticosterone and 18-hydroxycorticosterone in response to ACTH. In contrast, neither acidosis nor alkalosis had a significant, direct effect on basal or ACTH-stimulated cortisol secretion. We conclude that 1) H+ concentration modulates the stimulatory effect of ACTH on aldosterone secretion by a direct action on the adrenal cortex, 2) acid-base disturbances are specific to the zona glomerulosa of the canine adrenal gland, and 3) H+ concentration may influence events occurring late in the pathway for aldosterone biosynthesis. PMID- 3006511 TI - Long-term effects of hypothalamic paraventricular lesion on CRF content and stimulated ACTH secretion. AB - The effect of short-term (1 wk) and long-term (6 wk) lesion of the paraventricular nucleus (PVN) on the hypothalamopituitary-adrenal axis was studied. Six weeks after PVN lesion there was no change in resting morning plasma ACTH and corticosterone levels. The increase of plasma ACTH levels that occurs 8 days after adrenalectomy was inhibited 6 wk after placing a lesion in the PVN. In contrast, 6 wk after PVN lesion the plasma ACTH response measured 3 min after laparatomy and intestinal traction under ether anesthesia was not significantly different from that in the controls. The responsiveness to corticotropin releasing factor (CRF)-41 of anterior pituitary segments incubated in vitro increased slightly at 6 wk after PVN lesion. The amount of CRF-41-like immunoreactive material in the stalk-median eminence decreased to approximately 14% of the control, while that in neural lobe failed to change. We suggest that the ACTH hypersecretion after adrenalectomy is driven predominantly by CRF-and/or AVP-producing neurons in and around the PVN, whereas other sources of CRF-41, increased pituitary sensitivity or other hypothalamic factors, may restore stress induced ACTH release in the absence of the region of the PVN. PMID- 3006513 TI - Ammonium as a substrate for Na+-K+-ATPase in rabbit proximal tubules. AB - The role of the ammonium ion (NH+4) as a substrate for Na+-K+-ATPase was determined in intact rabbit proximal tubules. Since ouabain-sensitive oxygen consumption and Na+-K+-ATPase transport activity are tightly coupled in the proximal tubule with a stoichiometry of 12 K+ pumped/oxygen consumed, we used the ouabain-sensitive oxygen consumption of rabbit proximal tubule suspensions as an assay of Na+-K+-ATPase pump activity. The addition of NH+4 to K+-depleted tubules in nominally K+-free media resulted in a dose-dependent increase in oxygen consumption with an apparent affinity (Km) of 0.4 mM NH+4. Oxygen consumption was increased by 39.3 +/- 3% over control (n = 7) by 5 mM NH4Cl. This stimulation was completely inhibited by the addition of 5 X 10(-4) M ouabain. Under the same conditions, the addition of 5 mM KCl stimulated oxygen consumption by 52.4 +/- 2.9% (n = 7) with a Km of 0.5 mM. This stimulation was also completely inhibited by ouabain. Ouabain was also found to decrease the initial rate of NH+4 uptake into the proximal tubule cells. K+ and NH+4 competed with each other for active uptake into tubule cells. These results demonstrate that NH+4 can substitute for K+ on the Na+-K+-ATPase of the rabbit proximal tubule cell. Based on these data we have developed a kinetic model that predicts that the competition between NH+4 and K+ for transport on the Na+-K+-ATPase is not significant in the cortical labyrinth but potentially very significant in the inner medulla. PMID- 3006512 TI - Effects of increase in plasma calcium concentration on renal handling of NaCl and NaHCO3. AB - Recollection micropuncture experiments were carried out in thyroparathyroidectomized volume-expanded rats to examine the effects of CaCl2 infusion on the renal and nephronal segmental handling of chloride and bicarbonate. In group 1A, a 0.23 mM increase in plasma calcium concentration [delta(Ca)P] reduced urinary total CO2 (tCO2) excretion from 401 +/- 90 to 166 +/ 43 nmol X min-1 X g kidney wt-1 (P less than 0.05), whereas tCO2 filtered load was slightly diminished from 34,086 +/- 3,627 to 28,904 +/- 2,496 nmol X min-1 X g kidney wt-1 (NS). In group 1B [delta(Ca)P, 0.73 mM], whole kidney filtered loads were significantly lowered, as was urinary tCO2 excretion; however, urinary excretion of sodium, chloride, and water remained constant. Calcium infusion inhibited the proximal reabsorption of chloride 25% and water 16%; however, calcium infusion caused the end-proximal tCO2 concentration to significantly decrease so that the absolute and fractional tCO2 reabsorption remained constant. In group 2 [delta(Ca)P, 0.43 mM], whole kidney filtered load was unchanged for chloride and water but decreased for bicarbonate; urinary tCO2 excretion was reduced, whereas chloride and water excretion increased. In this group, early distal micropunctures evidenced that superficial single-nephron filtered loads were significantly reduced during calcium infusion; early distal chloride delivery was enhanced from 348 +/- 32 to 441 +/- 36 pmol X min-1 X g kidney wt-1 (P less than 0.05), whereas tCO2 delivery decreased from 47 +/- 5 to 38 +/- 4 pmol X min-1 X g kidney wt-1 (P less than 0.05). In group 3 of time control animals, whole kidney and early distal data were unchanged during second period. In group 4, H+ secretion in the collecting duct, as assessed by analyzing the relationship between urine-minus-blood PCO2 and urinary bicarbonate concentration in maximally alkaline urine, was not modified during CaCl2 infusion [delta(Ca)P, 0.79 mM]. We conclude that increase in plasma calcium concentration inhibits proximal NaCl and water reabsorption, whereas it stimulates the bicarbonate transport relative to that of chloride, leading to an enhanced proximal and renal bicarbonate-to-chloride reabsorptive ratio that could generate metabolic alkalosis; and decreases urinary bicarbonate excretion by also lowering the bicarbonate filtered load. PMID- 3006514 TI - Parathyroid hormone inhibits water flow in the isolated toad bladder. AB - These experiments studied the effect of parathyroid hormone (PTH) (1-84) on water and Ca transport in isolated toad bladder sacs and toad bladder epithelial cells. Serosal addition of PTH significantly inhibited maximal water flow induced by vasopressin or exogenous cyclic AMP. This effect was seen over a wide range of concentrations, with the threshold for the effect occurring at 1 ng/ml. Pretreatment of the toad bladder sacs with prostaglandin inhibitors (indomethacin or ibuprofen, 1 X 10(-6) M) or preincubation in low-Ca medium (0.089 mM) abolished the effect of PTH on vasopressin-stimulated water flow. Pretreatment of the toad bladders with lanthanum (5 X 10(-5) M) also abolished the effect of PTH on vasopressin-stimulated water flow. Synthetic PTH (1-34) inhibited vasopressin stimulated water flow only at a high concentration (1 microgram/ml). PTH increased 45Ca uptake by toad bladder epithelial cells but had no effect on 45Ca efflux. These results demonstrate that PTH inhibits water transport beyond the generation of cyclic AMP. That the effect of PTH was abolished in a low-Ca medium or by pretreatment with lanthanum suggests that cell Ca uptake is required for the effect of PTH on water transport. That prostaglandin inhibitors also block the effect of PTH on vasopressin-stimulated water flow suggests that prostaglandin synthesis is required for the effect. These data suggest that the effect of PTH on water flow is mediated by an increased cellular uptake of Ca that stimulates prostaglandin release. Prostaglandin release, in turn, appears to mediate the inhibitory effect of PTH on vasopressin-stimulated water transport.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006515 TI - Characteristics of the Na+-H+ antiporter in the intact renal proximal tubular cell. AB - The characteristics of the proximal tubular Na+-H+ antiporter were determined in isolated proximal tubular cells to ascertain whether the features of this transport system in intact cells are comparable with those previously described for isolated brush-border membrane vesicles. A method is described for the rapid isolation of a purified preparation of cells that demonstrate morphological and functional characteristics of the renal proximal tubule. The cells maintain their polarity while in suspension, and adenylate cyclase activity is enhanced by parathyroid hormone but not by arginine vasopressin. The cells display gluconeogenic function and Na+-dependent alpha-methyl-D-glucose and organic phosphate cotransport, processes that confirm their proximal tubule origin. O2 consumption rates and cytosolic adenosine triphosphate levels indicate functional integrity. Na+-H+ antiport activity was defined in these cells by measuring amiloride-sensitive Na+ uptake. At intracellular pH = 6.4 vs. extracellular pH = 7.4, KtNa was 10.1 +/- 2.8 mM, and maximal sodium flux was 0.89 +/- 0.13 nmol X 10(6) cells-1 X K0.5 for amiloride and ethyl-isopropyl amiloride, measured at an external Na+ concentration of 1 mM, was observed at 2.5 X 10(-5) M and 2.9 X 10( 6) M, respectively. The external and internal loci of the exchanger displayed asymmetric affinity for the hydrogen ion: the apparent pK for the external site was 7.20-7.26 vs. less than 6.5 for the internal site. The internal site demonstrated features of positive cooperativity. In summary, the Na+-H+ antiporter present in the luminal membrane of the renal proximal tubule has been characterized in the intact cell and displays functional and kinetic parameters closely resembling those described in isolated brush-border membrane vesicles. PMID- 3006516 TI - Effects of verapamil and diltiazem on human platelet function. AB - In this study the antiplatelet properties of two calcium channel blockers, verapamil and diltiazem, were evaluated. In 20 random aspirin-abstaining donors, both diltiazem and verapamil (0.01-10 microM) reduced epinephrine-induced aggregation [46 +/- 6% (SE) inhibition] and demonstrated a dose-dependent inhibition of epinephrine-induced [14C]serotonin release (43 +/- 3% reduction). However, at equimolar concentrations, verapamil was twice as effective. Neither drug altered ADP, collagen, thrombin, or calcium ionophore-induced platelet aggregation or platelet granule secretion. Neither drug prevented formation of thromboxane B2 during secondary aggregation. Verapamil, but not diltiazem, increased the Kd of [3H]yohimbine binding from 2.03 to 46.99 nM without altering the calculated number of binding sites per platelet (124 sites/platelet). Supplemental calcium added to citrated platelet-rich plasma reversed both verapamil and diltiazem-induced inhibition of platelet aggregation. We conclude that, at the concentrations tested, both verapamil and diltiazem are specific inhibitors of epinephrine-induced platelet activation. Clearly, both agents may be acting by preventing epinephrine-induced increases in plasma membrane permeability to calcium. However, the greater potency of verapamil compared with diltiazem with only verapamil binding to alpha2-adrenergic receptors suggests that alpha-blockade represents a significant component of verapamil-induced platelet inhibition. PMID- 3006517 TI - Differences in affinity of cardiac beta-adrenergic receptors for [3H]dihydroalprenolol. AB - We performed quantitative light microscopic autoradiography of [3H]dihydroalprenolol (DHA) binding to frozen sections of canine myocardium to test the hypothesis that there are differences in the density or affinity of beta adrenergic receptors on various tissue compartments. In one study, with concentrations of [3H]DHA from 0.34 to 5.1 nM, specific binding to cardiac myocytes was saturable, whereas nonspecific binding was linear with ligand concentration. Arterioles had more specific grain counts than muscle cells (P less than 0.0001), and Scatchard analysis showed that the arterioles had a much higher affinity for [3H]DHA than myocytes. In a second study with lower concentrations of [3H]DHA (0.19-1.98 nM), binding to the arterioles saturated, whereas binding to the cardiac myocytes did not. Specific binding to arterioles was significantly higher (P less than 0.0001) than binding to myocytes at all concentrations of [3H]DHA. The dissociation constants for the subendocardial and subepicardial myocytes were 1.57 and 1.71 nM, respectively, while the dissociation constant for the arterioles was 0.26 nM. The maximum number of binding sites was 911 grains/0.9 X 10(-2) mm2 for subepicardial myocytes, 936 for subendocardial myocytes, and 986 for arterioles. The large nerves accompanying an epicardial artery also demonstrated specific [3H]DHA binding. Thus this study has demonstrated major differences in the distribution and affinity of beta adrenergic receptors, which may help to explain various physiological responses to beta-adrenergic stimulation. PMID- 3006518 TI - Alterations in beta-adrenergic stimulation of myocardial adenylate cyclase in endotoxic rats. AB - The effect of in vivo endotoxin administration on adenylate cyclase in rat ventricular membranes was studied. Basal, fluoride-, and 5' guanylylimidodiphosphate-stimulated adenylate cyclase activities were not affected throughout the period of endotoxicosis. Isoproterenol-stimulated adenylate cyclase activity was not different at 0.5 or 3 h after endotoxin. At the agonal stage, the isoproterenol dose-response curve was shifted significantly to the right in myocardial membranes from endotoxic rats, but there was no significant decrease in maximum stimulated activity. These data indicate that endotoxicosis does not inhibit the adenylate cyclase enzyme system but does decrease stimulation of adenylate cyclase via beta-adrenergic receptors when the animal is approaching death. PMID- 3006519 TI - Dissociation of sympathetic and thermogenic activity in brown fat of Syrian hamsters. AB - Syrian hamsters (Mesocricetus auratus) were fed a high-fat (HF) diet for up to 16 wk. Sympathetic and thermogenic activities in their brown adipose tissue (BAT) were assessed by measuring norepinephrine content and turnover and mitochondrial GDP binding and cytochrome c oxidase activity. Chronic ingestion of the HF diet resulted in significant increases in carcass lipid and interscapular BAT wet weight by the end of the second week. HF-fed hamsters were slightly hyperphagic during the first 2 wk of HF feeding only. Significant weight gains persisted beyond the period of hyperphagia. Hypertrophy of interscapular BAT after 16 wk on the HF diet was accompanied by increases in protein and DNA content, indicating growth of functional tissue. Norepinephrine turnover and content in BAT were decreased throughout the entire period of HF feeding, regardless of changes in caloric intake or body weight. Mitochondrial cytochrome c oxidase activity and GDP binding were increased after 16 wk on the HF diet, a time when the HF-fed animals were obese but not hyperphagic. These results demonstrate a dissociation of BAT thermogenesis from sympathetic activity in the tissue. It appears that sympathetic nervous system activity in BAT was suppressed by the HF diet, whereas thermogenic activity of the tissue was activated when the hamsters became obese. PMID- 3006520 TI - Interaction between CRF and angiotensin II in control of ACTH and adrenal steroids. AB - These experiments were designed to test for interactions between plasma angiotensin II (ANG II) and corticotropin-releasing factor (CRF) in the control of plasma adrenocorticotropin (ACTH), aldosterone, and corticosteroids, mean arterial pressure (MAP), and heart rate (HR) in conscious dogs. Five trained dogs with exteriorized carotid arteries were studied. Each dog was infused with saline and with CRF at three rates (2.5, 5, and 10 ng X kg-1 X min-1) and ANG II at three rates (5, 10, and 20 ng X kg-1 X min-1) for 60 min. The same animals were also coinfused with 10 ng X kg-1 X min-1 ANG II at each rate of CRF infusion and with 10 ng CRF X kg-1 X min-1 at each rate of ANG II infusion. Infusion of ANG II alone caused dose-related increases in aldosterone, corticosteroids, and MAP but did not alter ACTH or HR. Infusion of CRF alone increased ACTH, aldosterone, and corticosteroids but not MAP or HR. Coinfusion of CRF and ANG II caused ANG II dose-related ACTH responses but did not alter the sensitivity of the ACTH responses to CRF. Thus it appears that ANG II alone does not stimulate ACTH release but requires increased CRF concentrations to effect ACTH release. PMID- 3006521 TI - Absence of fast negative feedback control of ACTH and renin in fetal and adult sheep. AB - Fetal adrenocorticotropin (ACTH) and renin secretion are increased by a variety of stimuli and decreased by cortisol negative feedback inhibition. However, the time courses of these interactions are unknown. The present studies were designed to test for rapid feedback negative suppression of ACTH and renin secretion in fetal and adult sheep. In chronically catheterized fetal sheep, ACTH and renin secretion were stimulated by intravenous infusion of sodium nitroprusside, a vasodilator drug. Vehicle or cortisol, infused at rates of 1, 2, or 4 micrograms/min for 2 min before and during the infusion of nitroprusside did not significantly alter the fetal ACTH or renin responses to nitroprusside. In five nonpregnant ewes, chronically prepared with skin loops containing the carotid arteries, nitroprusside (20 micrograms X kg-1 X min-1) was infused beginning 2 min after infusion of vehicle or cortisol (3.5 or 7 micrograms X kg-1 X min-1). Cortisol infusion produced a rising plasma cortisol concentration similar to that after stress but did not alter the magnitude of the ACTH response to nitroprusside. The results indicate that cortisol-induced suppression of ACTH secretion does not occur rapidly in the fetal or adult sheep and that the cortisol-induced suppression of fetal plasma renin activity is a slow process. PMID- 3006522 TI - High-affinity imipramine binding and serotonin uptake in platelets of eight adolescent and ten adult obsessive-compulsive patients. AB - The authors evaluated high-affinity [3H]imipramine binding and [3H]serotonin uptake to platelets in eight adolescent and 10 adult patients who met DSM-III criteria for obsessive-compulsive disorder in comparison with those of normal control subjects of similar ages. The maximal binding of [3H]imipramine was significantly lower in adults and adolescents with obsessive-compulsive disorder than in the control subjects. No differences between groups in the affinity of [3H]imipramine to its binding sites or in serotonin uptake kinetic measures were detected. The lower density of [3H]imipramine binding sites in platelet membrane in patients with obsessive-compulsive disorder might implicate involvement of the serotonergic system or might represent an adaptive response to a chronic disease. PMID- 3006523 TI - Viral diagnosis by in situ hybridization. Description of a rapid simplified colorimetric method. AB - An improved method of colorimetric in situ hybridization for the diagnosis of viral infections in standard formalin-fixed, paraffin-embedded tissue sections has been developed. This method employs a 2-hour hybridization with biotin labeled DNA probes followed by direct colorimetric detection with avidin-alkaline phosphatase complexes. Visual results are obtained within 8 h of cutting the tissue section. Specific histologic localization of cytomegalovirus and adenovirus genetic information has been achieved in infected lung tissues from autopsy or biopsy. Simultaneous denaturation of tissue and probe DNA at elevated temperature (100-105 degrees C) resulted in increased signal. It is our suggestion that these denaturing conditions may be required to denature more fully formalin cross-linked tissue DNA and favor penetrance of probe into the tissues. Comparison of the results of hybridization and viral culture for the diagnosis of CMV infections suggest that in clinical situations hybridization will allow specific diagnosis of productive viral infection more rapidly than viral culture with some loss in sensitivity. Colorimetric in situ DNA hybridization offers the surgical pathologist a powerful new technique that provides an alternative to immunocytochemistry and electron microscopy in the diagnosis of viral pathogens. PMID- 3006524 TI - Postradiation malignant fibrous histiocytoma of bone. A clinicopathologic study of 20 patients. AB - Among the 130 primary or secondary malignant fibrous histiocytomas of bone diagnosed and treated at Memorial Hospital for Cancer and Allied Diseases during the previous half century, 20 (15.4%) arose as a direct consequence of irradiation. This type is the commonest secondary osseous malignant fibrous histiocytoma at this institution. It affects the ilium, the scapula, and the distal end of the femur most frequently, predominantly in patients whose age peaks in the fifth decade of life, when their sarcomas developed. Grounds for the irradiation were either nonosseous conditions (13 patients) or preexistent skeletal lesions (seven patients). Reasons for the incidental bone irradiation included Hodgkin's disease; carcinoma of cervix, breast, or lung; bilateral retinoblastoma, and others; giant cell tumors predominated among the irradiated skeletal lesions. The mean and the median radiation doses were 6,040 and 5,700 rads, respectively. The latent period between irradiation and the appearance of the bone sarcoma ranged from 4 to 47 years with a mean of 16.5 and a median of 14.5 years, respectively. The cumulative disease-free survival rate at 3 years was 58%. Although all patients who received their radiation therapy for a preexistent bone lesion survived, only 27% of patients whose bone was normal at the time of irradiation are alive and well at the 3-year mark. PMID- 3006525 TI - Papillary adenoma of type 2 pneumocytes. AB - A case of papillary adenoma of type 2 pneumocytes is reported. A 57-year-old man had an unusual coin lesion in the periphery of the right lung without any symptoms. When detected in a mass survey examination, it was approximately 1.5 cm in diameter, well circumscribed, and located in S4, involving the sixth-order bronchus of B4. Light-microscopic examination revealed cuboidal tumor cells arranged in a papillary pattern. Ultrastructurally, the cells had characteristic osmiophilic lamellar bodies. By immunoperoxidase staining, the tumor cells were shown to have intracytoplasmic surfactant apoproteins. The postoperative course was uneventful, and there is no evidence of disease 8 years later. PMID- 3006526 TI - Florid papillomatosis of the nipple. A study of 51 patients, including nine with mammary carcinoma. AB - The present study was undertaken to review the pathology of florid papillomatosis (FP) of the nipple and to examine the relationship of FP to breast carcinoma. Clinical features of 49 women studied did not differ appreciably from those noted on prior reports, except that in one instance the lesion was probably congenital. Histologically, three distinct growth patterns were found: sclerosing papillomatosis (17 cases), papillomatosis (12 cases), and adenosis (3 cases). In 17 other cases, mixtures of these proliferative patterns were seen. FP with the sclerosing papillomatosis pattern more frequently had areas of focal necrosis in hyperplastic ducts and scattered mitoses, features that might be interpreted as evidence of carcinoma. No prognostic significance can be attributed to these patterns, since all types were cured by excision with follow-up that averaged 8.3 years. Seven of the 49 women had carcinoma in the same breast as FP: Two women had invasive carcinoma that appeared to arise from FP, and four women had concurrent invasive carcinomas that were separate from the FP; the seventh woman developed diffuse intraductal carcinoma 10 years after FP was excised from the same breast. Three of the seven women were also treated for contralateral breast carcinoma. Also reviewed were lesions from two men who had carcinoma arising in FP. One had intraductal carcinoma with Paget's disease and the other had invasive carcinoma. Appreciation of the diverse histological patterns of FP may be helpful in avoiding an erroneous diagnosis of carcinoma. Features indicative of carcinoma arising in FP are Paget's disease and areas of invasion. FP of the nipple is rarely the substrate for mammary carcinoma and is adequately treated by local excision. Coexistence with carcinoma elsewhere in the same or opposite breast occurs often enough to warrant thorough examination of the breasts when FP of the nipple is diagnosed. The risk of subsequent carcinoma following excision of FP appears to be low, but clinical follow-up is prudent. PMID- 3006527 TI - Concentration of Plasmodium ovale- and Plasmodium vivax-infected erythrocytes from nonhuman primate blood using Percoll gradients. AB - Plasmodium vivax and Plasmodium ovale schizont-infected erythrocytes were separated from peripheral blood by centrifugation using discontinuous Percoll (colloidal silica) gradients. Infected Aotus monkey or chimpanzee blood was diluted and placed on a discontinuous gradient containing 30%, 40%, 45%, and 50% Percoll (v/v in media) layers before centrifugation at 1,450 X g. Parasitized erythrocytes were concentrated to greater than 95% schizont-infected cells in two bands that contained an average of one leukocyte per 500 infected cells. Mononuclear cells and trophozoites were isolated in another band and noninfected red cells, ring-infected cells, and granulocytes were pelleted to the bottom. The yield of parasitized erythrocytes ranged from 50% to close to 100% of the estimated number of infected cells in the original whole blood. Use of this Percoll procedure results in a high yield of concentrated parasitized erythrocytes and separation of these cells from host white blood cells. PMID- 3006528 TI - In vitro culture of exoerythrocytic parasites of the North Korean strain of Plasmodium vivax in hepatoma cells. PMID- 3006529 TI - In vitro and in vivo effects of itraconazole against Trypanosoma cruzi. AB - The synthetic imidazole, itraconazole, was examined for in vitro and in vivo activity against Trypanosoma cruzi. Mice treated with concentrations as low as 15 mg itraconazole/kg/day were completely protected against death due to infection with any of three different and highly virulent strains of T. cruzi. Treatment of infected mice with 120 mg itraconazole/kg/day for seven to nine weeks apparently resulted in the parasitologic cure as determined by negative hemocultures and subinoculations, negative serology for T. cruzi, and absence of parasites in histologic sections following completion of therapy. Peak serum levels of itraconazole after treatment of mice with the dose of the drug that provided protection against death were less than 1 microgram/ml. Experiments in vitro revealed that concentrations of itraconazole as little as 0.001 microgram/ml inhibited replication of intracellular amastigotes in macrophages. These results indicate that itraconazole has a remarkable activity against T. cruzi. Further investigation of intraconazole as a therapeutic agent for Chagas' disease may be warranted. PMID- 3006530 TI - Epidemiology of rotavirus in Guayaquil, Ecuador. AB - Detection of rotavirus by electron microscopy was conducted with fecal specimens from 1,722 infants and young children with acute diarrhea, during a 41-month survey from April 1978 through December 1981 in Guayaquil, Ecuador; 376 of these specimens (21.8%) were positive. The detection rate was higher during the dry season (May to November; 25.2%) than during the rainy season (December to April; 14.7%). When rotaviruses isolated from 59 patients hospitalized with diarrhea (from April 1979 to July 1981) were subjected to genome RNA analysis by polyacrylamide gel electrophoresis, a single dominant electropherotype was found with other less common electropherotypes. An atypical rotavirus with a unique property was also found. PMID- 3006531 TI - Apparent cure of a difficult treatment problem in a patient with mucosal leishmaniasis. PMID- 3006532 TI - Multicentricity in nonpalpable breast carcinoma and its implications for treatment. AB - During a 9 year period, 300 consecutive women underwent breast biopsies solely because of nonpalpable, mammographically suspicous findings. One hundred clinically occult breast carcinomas were found, 65 of which were invasive and 35 noninvasive. Eighty-three mastectomy specimens were evaluable for evidence of multifocal carcinoma in another quadrant of the breast or at a distance of 5 cm and residual cancer outside the excisional biopsy cavity. Multicentricity was present in 47 percent and residual tumor in 60 percent of the whole group. When only clinically occult invasive carcinomas were considered, other foci of invasive carcinoma were demonstrated in 26 percent of the patients and residual invasive cancer in 35 percent. The rate of bilaterality was 14 percent, occurring synchronously in 11 percent of the patients. Any therapeutic procedure for nonpalpable breast carcinoma, whether invasive or noninvasive ductal carcinoma, should be directed to the whole breast. Mammography of the contralateral side should be an integral part of the preoperative work-up of patients with palpable lesions ipsilaterally. PMID- 3006533 TI - In vitro buffering capacity of Alka Seltzer Effervescent. A comparison with magnesium trisilicate mixture B.P. and sodium citrate 0.3 M. AB - The variety of antacids used as a prophylaxis for acid aspiration syndrome reflects dissatisfaction with each agent. Alka Seltzer Effervescent is a proprietary product without aspirin. We have shown that it can easily be dissolved in a small volume, readily mixes with hydrous fluids, and has a satisfactory neutralising capacity. Its non-particulate nature, storage in individual packages and palatability, indicated by commercial acceptance overseas, show that further trials in vivo as a method of raising gastric pH may be useful. PMID- 3006534 TI - [Changes in acid-base status, potassium ions and glucose concentrations in blood during cardiopulmonary resuscitation. Study of the significance of animal experiment results in emergency medicine case examples]. AB - Results obtained during animal investigations cannot be directly applied to patients because of the anatomical and physiological differences present. Experience has shown that cases of sudden cardiac arrest can only be effectively helped if cardiac massage and artificial ventilation are carried out as soon as possible, and that no sodium bicarbonate need be given when resuscitation measures are begun immediately, or very soon after arrest. In this situation it is better to use adrenaline only, in order to improve organ perfusion. The examples presented show that giving bicarbonate can alter potassium kinetics. Very low serum potassium concentrations are often measured during and following successful resuscitation, and giving too large an amount of buffer solution can worsen this hypokalaemia and lead to a further arrest. The infusion of sodium bicarbonate and the rapid increase in blood sugar levels during resuscitation can lead to a marked rise in serum osmolality which in turn can jeopardize the return of cerebral function. Because in the presence of reduced cerebral perfusion, high blood glucose levels increase the degree of cerebral lactacidosis blood glucose levels should be measured and corrected as necessary. PMID- 3006535 TI - A continuous spectrophotometric assay for pyrimidine-5'-nucleotidase. AB - A simple method is presented for the determination of pyrimidine-5'-nucleotidase activity using a continuous spectrophotometric assay system. Activity is determined by measuring inorganic phosphate generation using a linked indicator system that produces uric acid in the presence of inosine, purine nucleoside phosphorylase, and xanthine oxidase. This method has several advantages over any of the methods currently in use. PMID- 3006537 TI - Characterization of phosphohydrolase activity in bovine spleen extracts: identification of a bis(p-nitrophenyl)phosphate-hydrolyzing activity (phosphodiesterase IV) which also acts on adenosine triphosphate. AB - Several bovine spleen enzymes with acid pH optima, some of which hydrolyze bis(p nitrophenyl)phosphate and therefore fit the definition of "phosphodiesterase IV," were partially separated by isoelectric focusing and ion-exchange techniques. The activities were characterized by zymogram analysis with the aid of p-nitrophenyl and 4-methylumbelliferyl phosphate and phosphonate substrates. A number of these enzymes meet the criteria for phosphodiesterase I or other phosphodiesterases. However, the predominant phosphodiesterase I hydrolyzes the bis(p-nitrophenyl) and 4-methylumbelliferyl phosphates, p-nitrophenyl and 4-methylumbelliferyl phenylphosphonate, and ATP at the beta-gamma bond, but not p-nitrophenyl or 4 methylumbelliferyl 5'-thymidylate (the usual PDE I substrates). These properties, as well as the pH optimum, distinguish the activity from the previously described, alkaline pH optimum PDE I. A second phosphodiesterase hydrolyzes only the phenylphosphonates. Several other activities, less well described, are apparent on zymograms. None of the phosphodiesterases IV was also a phosphodiesterase II (no hydrolysis of 4-methylumbelliferyl 3'-thymidylate). PMID- 3006536 TI - Detection of glycosaminoglycans at the one-nanogram level by 125I-cytochrome c. AB - The basic protein cytochrome c forms stable ionic complexes with all known glycosaminoglycans. When labeled with 125I, cytochrome c is capable of detecting exceptionally small quantities of glycosaminoglycans. Subsequent to electrophoresis on cellulose acetate strips using pyridine formate buffer at pH 3, followed by ethanol fixation, and treatment with 125I-cytochrome c, all the known glycosaminoglycans are detected at minimum levels of 1 ng/0.25-microliter application. The method can be used for quantification of glycosaminoglycans in other electrophoretic buffer systems also. PMID- 3006538 TI - Quantitation of zinc in nitric acid-digested plasma by atomic absorption spectrophotometry. AB - Human plasma, digested in a screw-capped Teflon vial for 1 h at 130 degrees C in concentrated nitric acid, is assayed for zinc by atomic absorption spectrophotometry using a single-slot, 10-cm burner and air/acetylene flame. The assay is linear to 10.00 mg Zn/liter, recovery averages 100.9%, and inter- and intraassay coefficients of variation are 5.9 and 1.9%. With this method, there is no burner clogging or adjustment necessary for sample viscosity. Sodium chloride does not interfere with the assay. The linear regression data of the standard curve for milligrams of Zn per liter (x) and milliabsorbance units (y) is y = 40x + 0.001. PMID- 3006539 TI - Mg2-dependent phosphatidate phosphohydrolase of rat lung: development of an assay employing a defined chemical substrate which reflects the phosphohydrolase activity measured using membrane-bound substrate. AB - An assay of pulmonary phosphatidate phosphohydrolase activity has been developed that employs a chemically defined liposome substrate of equimolar phosphatidate and phosphatidylcholine. Enzyme assays employing this substrate resolved two distinct activities based upon their requirements for Mg2+. Assays were performed in the presence and absence of 2 mM MgCl2 and the Mg2+-dependent phosphatidate phosphohydrolase activity calculated by difference. The Mg2+-independent phosphatase activity resembled that found using aqueous dispersions of phosphatidate (PAaq). Approximately 90% of the Mg2+-dependent phosphatidate phosphohydrolase activity was recovered in the cytosol and the remainder was associated with the microsomal fraction. The Mg2+-dependent phosphatidate phosphohydrolase activity has kinetic parameters of Km = 55 microM, Vmax = 1.6 nmol/min/mg protein for the microsomal fraction, and Km = 215 microM, Vmax = 6.8 nmol/min/mg protein for the cytosolic fraction. These parameters resembled those found using the microsomal membrane-bound (PAmb) substrate. In addition, the pH optima and sensitivity to detergents and thermal inactivation are equal to those for the PAmb-dependent phosphatidate phosphohydrolase activity. In the course of these studies the microsomal and cytosolic activities were qualitatively equal, indicative of a single enzyme in two subcellular locations. In conclusion, the assay of Mg2+-dependent phosphatidate phosphohydrolase activity measured using equimolar phosphatidate and phosphatidylcholine liposomes is equivalent to that activity previously described using microsomal membrane-bound substrate. However, the chemically-defined system provides a more simplified starting point for further studies on this important enzyme. PMID- 3006540 TI - Enzymatic method for continuous monitoring of inorganic pyrophosphate synthesis. AB - A sensitive method for the analysis of inorganic pyrophosphate (PPi) which utilizes the enzymes ATP sulfurylase and firefly luciferase is described. The assay is based on continuous monitoring of the ATP formed in the ATP sulfurylase reaction using purified firefly luciferase. The assay can be completed in less than 2 s and is not affected by inorganic phosphate. The method has been used for continuous monitoring of formation of PPi in Rhodospirillum rubrum chromatophores. The assay is extremely sensitive, the linear range of the assay being 1 X 10(-9) - 5 X 10(-7) M PPi. It is suitable for routine applications. It is also possible to use the method for determination of low amounts of adenosine 5'-phosphosulfate. PMID- 3006541 TI - Separation of nuclear cAMP independent protein kinases NI and NII from their chromosomal protein substrates and enzyme inhibitors by the use of a casein phosvitin-Sepharose column. AB - A casein-phosvitin-Sepharose chromatography column allows separation of nuclear protein kinases from their chromosomal phosphoprotein substrates and from at least some protein kinase inhibitors in a single step. The additional step of passing the eluted material through a partially hydrolyzed, dephosphorylated casein-Sepharose column separates the two protein kinases, NI and NII, from each other. PMID- 3006542 TI - Enzyme-linked coagulation assay. II. A sensitive assay for tissue factor and factors II, VII, and X. AB - We have developed a solid-phase clotting assay which uses peroxidase-fibrinogen in solution and fibrinogen bound to microtiter plates as a substrate for the thrombin generated from the clotting cascade. We have developed this assay for measurement of the extrinsic pathway factors thromboplastin (tissue factor, factor III), VII and VIIa, X, and II. Using long incubation times (40-90 min), thromboplastin could be measured in extracts of human brain at very low concentrations. Specificity for thromboplastin was demonstrated by showing a requirement for factors II, V, X, and VII but not for VIII, IX, XI, or XII; both substrate plasmas monodeficient in single factors and mixtures of the pure factors were used in demonstrating this specificity. The assay was modified to measure factors II, VII, VIIa, and X using appropriate deficient plasmas. The limit of detection was 2-3 orders of magnitude lower than a one-stage clotting test for all factors assayed. This assay has the advantages of convenience, specificity comparable to standard clotting tests, and high sensitivity. PMID- 3006543 TI - Half-site editing: an in vitro mutagenesis procedure for truncating a DNA fragment and introducing a new restriction site. AB - Half-site editing is an in vitro mutagenesis procedure designed for use in making precise plasmid constructions. Like many in vitro mutagenesis techniques, this procedure involves hybridization of a mutagenic oligonucleotide primer to single stranded template DNA followed by polymerization with DNA polymerase I (Klenow). Half-site editing differs from other techniques in two main ways. First, T4 DNA polymerase treatment truncates the target DNA at a point determined by the primer and repairs any mismatches to the sequence specified by the primer. Second, a blunt-end ligation step is included. This ligation exploits the symmetry inherent in most restriction sites to create a desired restriction site at the truncated end of the target DNA fragment. Half-site editing has been used to place ClaI restriction sites at the 3' end of the yeast pyruvate kinase promoter and at two positions at the 5' end of the yeast acetolactate synthase coding sequence. PMID- 3006545 TI - Light and electron microscopic study on the osteoclastic phagocytosis of cells in the rat. AB - By means of micromorphological and cytochemical investigations of the tibia metaphyses of growing rats the phagocytosis of cells by multinucleated osteoclasts could be demonstrated. On the average 1.9% of osteoclasts exhibited included cells. With very few exceptions the phagocytosed cells belonged to the monocyte/macrophage lineage, as shown by evidence from the ultrastructure. After the animals were treated with parathyroid hormone the number of osteoclasts increased significantly, but not, however, the number of osteoclasts with a cell inclusion. PMID- 3006544 TI - Detection of femtomole quantities of proteases by high-performance liquid chromatography. AB - Small amounts (femtomoles) of proteases, as might be present in cell extracts or secretions, were detected using reverse-phase high-performance liquid chromatography. Carboxymethylated lysozyme and cytochrome c were incubated with trypsin and chymotrypsin. Peptide peaks were present in the column elution profiles (as detected by absorbance, 206 nm) from incubations with as little as 0.1 fmol of chymotrypsin and 5 fmol of trypsin. In addition, the disappearance of the substrate peak or the increase in peptide peaks could be quantitated by integrating the areas under the peaks. In this way estimates of relative enzyme concentrations or duration of incubation can be determined. However, when [14C]lysozyme was used as a substrate and the radioactivity of collected peaks was measured, the assay was less sensitive than that using uv absorbance. This finding probably is related to the selective radiolabeling of the substrate, in contrast to uv detection, which should detect all the peptides. The technique reported in this paper should prove to be a sensitive indicator of proteolytic activity in cell or tissue preparations where the use of synthetic ester or amide substrates might lead to erroneous conclusions regarding the nature of the enzymatic activity present. Furthermore, by the collection of the peptides generated, one would have the ability to determine amino acid compositions or sequences and thus ascertain the specificity of enzymatic cleavage. PMID- 3006546 TI - Plasma membrane reregionalization in cultured mouse hepatocytes. AB - The cytochemical localization of alkaline phosphatase (ALPase) activity and the autoradiographic distribution of glucagon receptors were examined in the plasma membrane of cultured mouse hepatocytes. After 24 hours of culture, ALPase activity was exclusively localized on the plasma membrane in areas of cell-cell contact, and glucagon receptors were more numerous in the plasma membrane at the periphery of re-formed cell trabeculae. These results indicate that plasma membrane regionalization of hepatocytes, lost by cell isolation, reappeared during culture. The cells maintained this plasma membrane regionalization until 48 hours of culture. By 72 hours of culture, however, ALPase activity was seen on the external surface of all regions of plasma membrane, and the glucagon receptors decreased markedly in number and became scattered in all regions of plasma membrane. Thus, the re-formed plasma membrane regionalization disappeared in the cells by 72 hours of culture. PMID- 3006547 TI - Cytochemical and biochemical glucose 6-phosphatase activity in skeletal muscle cells of mice. AB - Cytochemical and biochemical glucose 6-phosphatase (G6Pase) activity and fiber type composition were studied in soleus (SOL) and gastrocnemius (GC) muscles of mice. The SOL is a red muscle which contains numerous type I fibers (60%) and relatively few type II fibers (40%). The GC is a white muscle which contains numerous type II fibers (90-100%) and very few type I fibers (0-10%). In the SOL and GC, cytochemical G6Pase activity was localized in the sarcoplasmic reticulum, lateral elements of triads, myonuclear envelope, and in the endoplasmic reticulum and nuclear envelope of endothelial cells. Differential centrifugation showed that G6Pase activity was recovered in the 105,000g pellet (microsomal fraction). Histochemical enzyme activity in type II fibers was slightly higher than that in type I fibers. Biochemical G6Pase activity in the GC was significantly higher than that in the SOL. The possible functional significance of G6Pase in skeletal muscles was discussed. PMID- 3006549 TI - Leukotrienes in pulmonary edema fluid after cardiopulmonary bypass. PMID- 3006548 TI - In vitro effects of prolactin and dexamethasone on rat Leydig cell aromatase activity. AB - In Percoll purified adult rat Leydig cells, the estradiol secretion, in presence of exogenous testosterone (200 ng/ml) is stimulated 2-fold by either LH (100 ng/ml) or dbcAMP (1 mM). The addition of prolactin (1 microgram/ml) or dexamethasone (10(-7) M) to the Leydig cell incubation medium induces a 20% increase of the basal estradiol production, whereas, under LH or dbcAMP stimulations, 46 and 41% decreases are noted; moreover, a synergistic effect between prolactin and dexamethasone was observed in presence of dbcAMP leading to a 53% diminution of the estradiol synthesis. There results suggest that either hyperprolactinemia or high doses of glucocorticoids might inhibit the Leydig cell aromatase activity in presence of LH. PMID- 3006550 TI - Naloxone does not antagonize the analgesic effects of inhalation anesthetics. AB - A previous demonstration that the ratio of analgesic to anesthetic endpoints is not constant across inhalation anesthetic agents implies that more than one mechanism of action may be operant in general anesthesia. We hypothesized that the endogenous opiate systems might account for this observed disparity in ratios. The tail flick ED50 (TFED50) in response to a heat stimulus, as an index of analgesia, and MAC as an index of anesthesia, were determined in rats treated with either saline or naloxone, 20 mg/kg, and exposed to halothane, enflurane, or isoflurane. Our findings confirmed those of Deady et al., showing a lack of uniformity of ratios of TFED50/MAC, with values of 0.90 +/- 0.03 for halothane, 0.80 +/- 0.04 for enflurane, and 0.70 +/- 0.04 for isoflurane. Naloxone had no effect on TFED50, MAC, or their ratio. If the endogenous opiate system were involved in the analgesic effect of general anesthetics, naloxone would have affected the ratios. We conclude that opiate systems are not involved in the analgesic action of general anesthetics. PMID- 3006551 TI - A microelectrode study of the effects of atracurium on neuromuscular transmission. AB - Using standard microelectrode recording techniques, we studied the effects of atracurium on neuromuscular transmission in a concentration range between 10(-13) and 10(-5) M in the rat diaphragm. Both intact and cut diaphragm preparations were used. Atracurium produced no significant alteration of miniature endplate potential (MEPP) frequency (P greater than 0.05). Increasing concentrations of atracurium (10(-6)-10(-5) M) caused a linear decrease in MEPP amplitude from 70.5 +/- 2.3% to 28.0 +/- 2.5% of baseline levels (P less than 0.05). In the cut diaphragm preparation, atracurium increased the degree of rundown and decreased quantum content of the endplate potential (EPP). The above observations suggest that atracurium interferes with neuromuscular transmission by, first of all, producing cholinergic receptor block and secondly, producing frequency-dependent inhibition of release of acetylcholine from the nerve terminal. PMID- 3006552 TI - Selective kappa opioid agonist for spinal analgesia without the risk of respiratory depression. AB - The effects of the selective kappa agonist, U-50,488H, on the motor response to noxious mechanical stimulation when administered intrathecally, as well as on respiratory rate and PaCO2 when administered by intracerebroventricular injection, were compared with those of morphine in experiments on rats. The agonist U-50,488H caused a dose-dependent increase in the threshold to noxious stimulation but did not have a potential for depression of resting ventilation. The results suggest that selective kappa opioid agonists may be suitable for clinical spinal (epidural) analgesia without the associated risk of respiratory depression. PMID- 3006554 TI - Midazolam infusion for sedation in the intensive care unit: effect on adrenal function. PMID- 3006553 TI - The effects of nitrous oxide on oxygen consumption by isolated cerebral cortex mitochondria. AB - The influence of N2O on O2 consumption by mitochondria isolated from the cerebral cortex of goats was examined in incubations preequilibrated with N2O-O2 or N2-O2. Rates of O2 consumption were measured polarographically in a closed system while adenosine triphosphate (ATP) formation was maximal (after addition of excess adenosine diphosphate (ADP), state 3 respiration) and then when it was at zero (after addition of excess oligomycin, state 4 respiration). Compared with 90% N2, 90% N2O produced no change in the rate of state 3 respiration; but an observed 9% decrease in the state 4 rate and an 11% increase in the state 3: state 4 ratio were statistically significant (P less than 0.05). These differences were not seen with N2 and N2O at 70% rather than at 90%, or when succinate rather than pyruvate-malate was used as the respiratory substrate. We conclude the following: Unlike other inhalation anesthetics, N2O at comparable anesthetic concentrations does not inhibit mitochondrial electron transport or ATP formation coupled to it (oxidative phosphorylation). N2O does inhibit one or more other processes, as yet unidentified, which are energetically coupled to electron transport. The increased cerebral O2 consumption that accompanies N2O anesthesia cannot be attributed to a direct effect of N2O on mitochondrial respiration. PMID- 3006556 TI - Bovine herpesvirus-1 and Pasteurella haemolytica aerobiology in experimentally infected calves. AB - Eight calves (2 calves in each of 4 groups) were exposed to an aerosol of bovine herpesvirus-1 (BHV-1) and 4 days later to an aerosol of Pasteurella haemolytica. Samples of tracheal and exhaled air were taken simultaneously beginning 1 day before viral exposure and once a day up to 3 to 4 days after the bacterial exposure. Samples were also taken during the period of aerosol exposure. Only 0.04% to 0.42% of P haemolytica-carrying droplets of the bacterial aerosol passed beyond the cranial part of the respiratory tract to the trachea. Nevertheless, numbers of bacteria as few as 1 bacterium/L of tracheal air were sufficient to produce fatal disease in the lungs of BHV-1-infected calves. In 1 of 4 groups, BHV-1 was isolated from most daily samples of exhaled and tracheal air. Pasteurella haemolytica was isolated 7 times more frequently from air when calves were kept at 1 C than when calves were kept at 23 C. The number of P haemolytica carrying droplets in exhaled air was low (less than 1/L of air); however, samples obtained during the time that calves were coughing contained up to 10 P haemolytica-carrying droplets/L of air. It was learned that the cranial part of the respiratory tract serves as an efficient filter on inhalation and exhalation, but this filter is deficient in the animal when coughing occurs. This process expels infective droplets of size suitable for inhalation by other cattle in close proximity. PMID- 3006555 TI - Infection control and the pregnant health care worker. AB - Health care workers may be exposed to a variety of infectious agents in the workplace. The pregnant health care worker presents additional concerns because of the potential risk of infection to the developing fetus. Health care workers often misunderstand the basic elements of infection transmission. The result of this misinformation is that personnel are most often concerned about the agents that are least transmissible. To develop an infection control program that is rational and workable, the infection control practitioner must have a thorough understanding of the mechanisms of disease transmission. With this foundation, an infection control program for the pregnant health care worker will rarely involve transfer to alternative assignments or work restriction based on pregnancy alone. The approach outlined in this article stresses a more generic approach to infection control by isolating the disease and not the employee. PMID- 3006557 TI - Effect of selenium deficiency on caprine polymorphonuclear leukocyte production of leukotriene B4 and its neutrophil chemotactic activity. AB - These studies were designed to measure leukotriene B4 (LTB4) production by polymorphonuclear leukocytes (PMN) from selenium (Se)-deficient and Se-adequate goats. Leukotriene B4 was measured by both radioimmune and chemotactic activity assays in supernatants of PMN stimulated by calcium ionophore A23187. The concentration of LTB4 produced by PMN from Se-deficient goats was significantly (P less than 0.05) lower than the concentration of LTB4 produced by PMN from Se adequate goats. Neutrophil chemotactic activity of LTB4 was found to be directly dependent (r2 = 0.85) on the LTB4 concentration. When supernatants from Se deficient and Se-adequate goats were adsorbed with caprine neutrophils, significant (P less than 0.05) reduction in LTB4 was found in supernatants from both groups. Furthermore, neutrophil adsorption of LTB4 from supernatants of both groups was indicated by significantly (P less than 0.05) decreased chemotaxis to LTB4 supernatants. Decrease in chemotactic response was, however, significantly (P less than 0.05) lower when neutrophils were treated with supernatants from Se deficient goats. These results indicate that Se deficiency decreases the production of LTB4 by caprine PMN and, hence, LTB4-mediated neutrophil chemotaxis. PMID- 3006558 TI - A genetically engineered, mutant human alpha-1-proteinase inhibitor is more resistant than the normal inhibitor to oxidative inactivation by chemicals, enzymes, cells, and cigarette smoke. AB - Alpha-1-proteinase inhibitors (alpha 1PI) containing methionine (Met-358) or valine (Met----Val-358) at the reactive center were synthesized in and purified to homogeneity from recombinant yeast. The pure proteins were exposed to 1 of 4 different oxidizing systems: N-chlorosuccinimide (chemical oxidation), myeloperoxidase plus peroxide and halide (enzymatic oxidation), activated neutrophils (cellular oxidation), or gas-phase cigarette smoke. The effect of these treatments on the leukocyte elastase inhibitory function of both proteins was then assessed. After brief exposures, substantial inactivation of the normal inhibitor occurred, whereas the mutant inhibitor remained fully active. More prolonged exposures led to complete inactivation of the normal protein and partial inactivation of the mutant inhibitor. These results suggest that the reactive center methionyl residue in alpha 1PI is more rapidly affected by oxidants than are other oxidizable residues in the inhibitor; however, Met-358 is not the only residue in alpha 1PI whose modification can lead to the inactivation of the elastase inhibitory capacity of this protein. PMID- 3006560 TI - Renin-angiotensin II-aldosterone and ACTH-cortisol control during acute hypoxemia and exercise in patients with chronic obstructive pulmonary disease. AB - Acute hypoxia has been reported to induce a decrease in aldosterone levels despite no change or increases in plasma renin activity and ACTH. Converting enzyme inhibition and/or mild hypokalemia have been suggested as possible mechanisms for this dissociation. We studied 15 patients with chronic obstructive pulmonary disease (COPD) who used continuous ambulatory O2 therapy (home O2). Group A (n = 10) had O2 discontinued for 30 min before exercise to induce hypoxemia; Group B (n = 5) had O2 continued for 30 min (time control). Discontinuation of home O2 in Group A resulted in a significant fall in Pao2 from 77 +/- 6 to 51 +/- 2 torr. Arterial CO2 tension decreased and the pHa increased slightly. Renin, angiotensin II, plasma potassium, and sodium did not change during hypoxemia, whereas ACTH increased significantly. Despite this, aldosterone decreased from 26 +/- 5 to 18 +/- 2 ng/dl. Group B (time control) did not exhibit significant changes in hormones over 30 min, indicating that the effects observed in Group A were specific to O2 discontinuation. Exercise in Group A induced significant increases in ACTH, potassium, and aldosterone. We conclude from these data that acute hypoxemia in patients with COPD results in a decrease in aldosterone not related to converting enzyme inhibition, ACTH, or plasma potassium. PMID- 3006559 TI - The role of phospholipase in reduced beta-adrenergic responsiveness in experimental asthma. AB - This investigation was designed to elucidate the role of phospholipase (PLase) in relation to reduced beta-adrenergic responsiveness in guinea pigs subjected to experimental asthma. In the in vivo experiment, guinea pigs that had developed asthma-like symptoms after exposure to an aerosol of 2% ovalbumin for 7 to 8 min for 10 successive days were used as the experimental asthma group. The control group was exposed to saline. The endogenous PLase activity was determined by high performance liquid chromatography using ditridecanoyl phosphatidylcholine as a substrate. PLase activity of lung membranes in the experimental asthma group was significantly elevated by 50% compared with that in the control group. Phospholipid content of lung membranes in the experimental asthma group was decreased by 11% compared with that in the control group. The experimental asthma group showed a 37% decrease in the number of beta-adrenoceptors in lung membranes and a 54% decrease in isoproterenol-stimulated adenylate cyclase activity in lung membranes compared with the control group. Although forskolin-stimulated adenylate cyclase activity was also reduced by 24%, decreases in forskolin stimulated activity were less than decreases in isoproterenol-stimulated activity. In the in vitro experiment phospholipids in lung membranes were degraded by pretreatment with 0.1 U of PLase A2. After pretreatment of lung membranes with PLase A2, the number of beta-adrenoceptors was reduced by 25% compared with that in the control group, and adenylate cyclase activity stimulated by isoproterenol and forskolin were also reduced by 67 and 28%, respectively. PLase A2 had a minor effect on forskolin-stimulated activity as compared with isoproterenol-stimulated activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006561 TI - Studies of membrane receptors and phagocytosis in subpopulations of rat alveolar macrophages. AB - Recent data suggest that alveolar macrophages may be a heterogeneous group of cells with several subpopulations. This study was undertaken to determine if there is heterogeneity among rat alveolar macrophages with respect to receptors for zymosan, immunoglobulin, and complement. In addition, avidity for the immunoglobulin IgG was defined by opsonizing sheep red blood cells (SRBC) with different amounts of IgG. Alveolar macrophages were harvested by bronchoalveolar lavage and separated into 18 density-defined fractions by centrifugation through a continuous isosmotic gradient of colloidal silica. All density-defined alveolar macrophages exhibited similar abilities to attach zymosan and SRBC opsonized with a high level of IgG. In contrast, macrophages of a density of 1.046 g/ml to 1.075 g/ml exhibited high receptor activity capable of attaching and phagocytizing SRBC opsonized with small amounts of IgG. Similarly, attachment indices for complement coated SRBC exhibited a peak activity among fractions exhibiting similar high avidity receptors for IgG. These results demonstrate the functional heterogeneity with respect to IgG and complement receptors among density-defined rat alveolar macrophage subpopulations. PMID- 3006562 TI - Nonpalpable breast lesions: wire localization and excisional biopsy. AB - From January, 1984, through July, 1984, 53 women with 56 nonpalpable suspicious mammographic breast lesions underwent breast biopsy directed by pre-operative needlewire localization. All of the suspicious lesions were removed at the first operation, and, although in 10 patients, the localization wire was greater than 2 cm from the lesion, this did not increase the number of attempts required to excise the lesion. Once the specimen and wire were removed, a radiograph was taken to assure removal of the suspicious area. Also, once the blocks were cut, they were x-rayed to pinpoint further which histopathologic sections would include the area in question. Six of the 56 suspicious mammographic lesions removed were intraductal breast carcinomas. All were Stage I tumors. The remainder of the lesions were benign including 24 with nonproliferative fibrocystic changes, 31 with proliferative fibrocystic changes and only one demonstrating proliferative changes with atypical hyperplasia. This procedure can be performed effectively on outpatients utilizing local anesthesia. Excellent communication among surgeon, radiologist, and surgical pathologist is imperative. PMID- 3006563 TI - Modified radical mastectomy with immediate breast reconstruction. AB - The authors reviewed 100 consecutive patients with breast cancer treated by modified radical mastectomy and immediate breast reconstruction in order to assess the safety and efficacy of this procedure in terms of cancer control. The study began in 1978 and is continuing. The procedure involves a two-team approach with both the general surgeon and the plastic surgeon interviewing the patient preoperatively. Virtually all breast reconstruction involved the use of a submuscular silicone saline type of implant. The median follow-up is 36 months. Slightly over half of these cases were Stage 0 or Stage I. There have been eight recurrences, including five local or regional and three distant. No patient has died of her disease at this point. Cosmesis was equal to or superior to that seen in delayed breast reconstruction. There have been no hidden recurrences. Postoperative depression has been significantly less. We conclude that modified radical mastectomy with immediate breast reconstruction is a safe and effective alternative to modified radical mastectomy alone. PMID- 3006564 TI - [The Roberts-SC phocomelia syndrome. Apropos of a case without cytogenetic changes and review of the literature]. PMID- 3006565 TI - Marrow transplantation for severe aplastic anemia. Long-term outcome in fifty "untransfused" patients. AB - Fifty patients with severe aplastic anemia had no transfusions of blood products until just before marrow transplantation from HLA-identical family members. Of the 50, 42 are still alive 1 to 12 years after transplantation (median, 7 years). By actuarial standards, the 10-year probability of survival is 82%. Of the 42 surviving patients, 37 have Karnofsky performance status scores of 100% and 5 with chronic graft-versus-host disease have scores ranging from 50% to 90% (median, 80%). The 8 deaths were caused by early infection in 1, graft rejection in 1, acute graft-versus-host disease in 3, and chronic graft-versus-host disease in 3. All deaths occurred within two years after transplantation. The incidence of graft failure was 10%. Acute graft-versus-host disease developed in 14 of 44 patients at risk and chronic graft-versus-host disease, in 15 of 41. Risk factors for development of chronic graft-versus-host disease included increased age (p = 0.008) and presence of acute graft-versus-host disease (p = 0.001). The only factor associated with increased risk of death was development of acute graft versus-host disease (p = 0.05). Results of this study extend our previous finding that patients with severe aplastic anemia who have transplants before the onset of transfusion-induced sensitization have an excellent probability of long-term survival and a normal life. PMID- 3006566 TI - Rapid immunodiagnosis of cytomegalovirus pneumonia by bronchoalveolar lavage using human and murine monoclonal antibodies. AB - Bronchoalveolar lavage material from 54 immunocompromised patients with interstitial pneumonia was examined by immunofluorescence with cytomegalovirus specific monoclonal antibodies. Twelve patients (22%) had cytomegalovirus detected in their lavaged cells, and 9 of these patients (17%) had proven cytomegalovirus pneumonitis. This assay detected all samples with cytomegalovirus when the virus was detected by established methods either at the time of lavage or after any other procedure in the subsequent 2 months; that is, it had a sensitivity of 100%. Cytomegalovirus could be detected within 3 hours of the lavage, and a clear correlation was seen between the number of fluorescent cells and the presence of cytomegalovirus pneumonia. All 9 patients with pneumonitis had more than 0.5% fluorescent cells, whereas the 3 patients in whom cytomegalovirus was detected without pneumonia had significantly fewer fluorescent cells. This method provides a sensitive, rapid, and quantifiable system for detection of cytomegalovirus, facilitating the early diagnosis and treatment of cytomegalovirus pneumonia. PMID- 3006567 TI - Antibody to hepatitis B core antigen as a paradoxical marker for non-A, non-B hepatitis agents in donated blood. AB - The relationship between the presence of antibody to hepatitis B core antigen (anti-HBc) in donor blood and the development of hepatitis in recipients of that blood was studied in 6293 blood donors and 481 recipients who were followed for 6 to 9 months after transfusion. Of 193 recipients of at least 1 unit of blood positive for anti-HBc, 23 (11.9%) developed non-A, non-B hepatitis compared with 12 (4.2%) of 288 recipients of only anti-HBc-negative blood (p less than 0.001). Donor anti-HBc status was not significantly associated with the development of hepatitis B in the recipient and was negatively associated with the development of cytomegalovirus hepatitis. The relationship of donor anti-HBc status and the development of non-A, non-B hepatitis in the recipient was independent of transfusion volume and elevated donor transaminase level. Although 88% of anti HBc-positive blood units were not associated with recipient non-A, non-B hepatitis, calculation of maximal corrected efficacy predicted that exclusion of anti-HBc-positive donors might have prevented 43% of the cases of non-A, non-B hepatitis with a donor loss of 4%. Because of the serious chronic consequences of non-A, non-B hepatitis, surrogate tests for non-A, non-B virus carriers must be seriously considered. PMID- 3006568 TI - Long-term seropositivity for human T-lymphotropic virus type III in homosexual men without the acquired immunodeficiency syndrome: development of immunologic and clinical abnormalities. A longitudinal study. AB - The long-term effects of seropositivity for human T-lymphotropic virus type III (HTLV-III) on T-lymphocyte subsets and health status were evaluated in longitudinal studies of 250 initially healthy homosexual men. The relative risk of having an inverted T-lymphocyte helper-to-suppressor ratio rose from 14.3-fold among short-term seropositive subjects (less than 19 months) to 46.9-fold among long-term seropositive subjects (greater than 29 months) in comparison with the risk among seronegative subjects. Overall, 91.7% of long-term seropositive men had inverted ratios, compared with 12.9% of seronegative men. None of the seropositive men who developed an inverted ratio later reestablished a normal ratio. Both decreased T-helper cell number and percentage (p = 0.003) and increased T-suppressor cell number and percentage (p = 0.03) were significantly correlated with duration of seropositivity. Among seropositive persons, lymphadenopathy was a highly significant short-term as well as long-term consequence, whereas diarrhea, oral thrush, and herpes zoster were correlated with long-term seropositivity. Overall, 50% of long-term seropositive men compared with 16% of seronegative men developed at least one of five clinical symptoms (p less than 0.003). We conclude that a high proportion of persons infected with HTLV-III will develop measurable immunologic and clinical abnormalities. PMID- 3006569 TI - Centers for Disease Control guidelines for prevention and control of Chlamydia trachomatis infections. Summary and Commentary. AB - Recent guidelines issued by the Centers for Disease Control discuss prevention and control of Chlamydia trachomatis infection. Chlamydia trachomatis is the commonest sexually transmitted infection in the United States. The rate of infection has increased in the past 10 years. The guidelines do not discuss control of trachoma and only briefly discuss lymphogranuloma venereum. Education of health care professionals and the public about C. trachomatis is recommended, along with the establishment of a nationwide surveillance system. PMID- 3006570 TI - New frontiers in genetic medicine. AB - Disorders determined wholly or in part by genetic factors constitute a substantial number of human diseases. This realization has grown during the past 2 decades with the recognition of many specific heritable conditions and the identification of familial risk factors for common disorders. New technologies, such as fetal visualization, chorionic villus sampling, molecular cloning methods, and gene transfer technology, provides a framework for dealing with genetically determined illness in unprecedented ways. Several current and potential applications of these methods are examined, as is the use of restriction fragment length polymorphisms to survey the variability within the genome and to generate markers permitting the prospective detection of genetic disorders. The promise and limitations of chorionic villus biopsy sampling are considered for early prenatal diagnosis. The future of gene therapy in hereditary diseases is examined, and some of the substantial social and ethical considerations engendered by these new developments are explored. PMID- 3006572 TI - Acquired immunodeficiency syndrome. Health and Public Policy Committee, American College of Physicians; and The Infectious Diseases Society of America. PMID- 3006571 TI - Advanced poorly differentiated carcinoma of unknown primary site: recognition of a treatable syndrome. AB - We describe the clinical characteristics and prognostic features of 71 patients with advanced poorly differentiated carcinoma of unknown primary site. These patients had at least one component of the extragonadal germ cell cancer syndrome that we have previously described. Of 68 patients who received therapy, 62 were given intensive cisplatin-based combination chemotherapy that is used for treatment of germinal neoplasms. Fifteen patients (22%) had complete responses, and 9 patients (13%) have remained free of tumor after a minimum follow-up of 36 months (range, 36 to 67 months). Tumor in the mediastinum, retroperitoneum, and lymph nodes was associated with a favorable outcome of treatment when compared with tumor in other locations (p = 0.0016, Cox regression analysis). Although the histogenesis of tumors in many of these patients remains unclear, we believe the tumors most likely originate from germ cells. Patients with advanced poorly differentiated carcinoma should be considered for treatment with cisplatin-based combination chemotherapy, particularly if tumors occur predominantly in the mediastinum, retroperitoneum, or lymph nodes. PMID- 3006573 TI - Breakthrough of cytomegalovirus infection despite 9-(1,3-dihydroxy-2 propoxymethyl)guanine therapy. PMID- 3006574 TI - Cytomegalovirus infection, ascending myelitis, and pulmonary embolus. PMID- 3006575 TI - Pneumococcal vaccine and isolates for the Centers for Disease Control. PMID- 3006576 TI - Genetically induced enzyme anomalies: insights into normal cellular processes. PMID- 3006577 TI - [Diagnosis and treatment of human papilloma virus infections in cervico-vaginal screening]. PMID- 3006578 TI - Surgical treatment of metachronous primary lung carcinomas. AB - Eleven patients were operated on for metachronous primary lung carcinomas. Most patients were heavy smokers. The incidence of primary metachronous carcinomas suitable for the operation was 0.45% of primary lung malignancies operated on during the same period. The mean interval between the first and second operations was 47.3 months. Surgical mortality was 0 after the first operation and 18% (2 out of 11 patients) after the second operation. The second primary malignant tumours were more advanced than the first ones. Two patients were alive at the follow-up 9 and 52 months after the second operation. The cause of the death was pulmonary carcinoma in five patients and respiratory and renal failure in one patient and respiratory insufficiency in one patient. Our findings suggest that reoperation for second primary lung malignant tumours should only be performed if the diagnosis is established early enough and if the primary operation was considered to be radical. PMID- 3006579 TI - The relationship of beta-endorphin and ACTH in the cerebrospinal fluid and plasma of adult surgical patients. AB - Cerebrospinal fluid and plasma beta-endorphin/beta-lipotropin immunoreactivity and adrenocorticotropin hormone were determined in simultaneously obtained samples from 25 healthy, adult surgical patients about to undergo spinal anesthesia using radioimmunoassay techniques. Cerebrospinal fluid adrenocorticotropin concentrations were significantly higher than those in plasma (25.76 +/- 2.11 fm/ml vs. 8.83 +/- 0.84 fm/ml), whereas beta-endorphin/beta lipotropin concentrations in cerebrospinal fluid were significantly lower than those in plasma (6.60 +/- 0.43 fm/ml vs. 3.35 +/- 0.30 fm/ml). In cerebrospinal fluid, a significant positive correlation was found between beta-endorphin/beta lipotropin and adrenocorticotropin (r = 0.64, p less than 0.01), whereas no such relationship could be demonstrated in plasma. This suggests that beta endorphin/beta-lipotropin and adrenocorticotropin might enter the cerebrospinal fluid via a mechanism unrelated to their entry into plasma. This may have implications for the pharmacologic manipulation of these peptides within the central nervous system. PMID- 3006580 TI - [The CT scan coupled to the injection of lipiodol into the hepatic artery. Preliminary trial]. AB - A preliminary study of CT findings for 13 malignant liver tumors evaluated after intra-arterial hepatic lipiodol injection is presented. Comparison with ultrasound, conventional CT before and after contrast material injection, and arteriography revealed the superiority of intra-arterial lipiodol injection coupled with CT examination for both the detection of hepatomas and disease extension workups, thanks to the detection of small multifocal lesions. There is no lipiodol uptake by the tumor in cases of metastases or cholangiocarcinoma. The same technique can be applied to non-surgery patients for therapeutic hepatic artery injections of an extemporaneous lipiodol emulsion, hydrosoluble contrast materials and oncostatic drugs. A review of over 100 cases in the Japanese literature emphasizes the efficacy of this therapeutic approach. PMID- 3006581 TI - Cytochrome oxidase status in protein-energy-deficient rats. AB - Effects of changes in dietary protein have been investigated on three mitochondrial enzymes, succinate dehydrogenase, isocitrate dehydrogenase and cytochrome oxidase. Weanling rats (21 days old) were fed for 30 days on (a) a commercially produced diet (CPD) containing 21.0% dietary protein and (b) a low protein-high carbohydrate diet (LPD) containing 3.47% dietary protein. Signs of protein-energy malnutrition developed in the animals having the low protein diet. The mitochondrial enzymes were assayed. Some of the experimental rats were repleted by feeding them on a protein-rich diet for 3 weeks, and the same mitochondrial enzymes were assayed. The activity of mitochondrial cytochrome oxidase, which fell to 24% of the control values during the period of deficiency, rose to 91% of the values for control rats during rehabilitation. The activities of succinate dehydrogenase and NAD+-isocitrate dehydrogenase fell to 75 and 73% of the control values, respectively, during depletion and rose to 83 and 88% during repletion in line with the general rate of recovery of the malnourished rats as reflected by the changes in the body weights during repletion. These results show that mitochondrial cytochrome oxidase is very sensitive to changes in dietary protein. Its activity drops sharply with reduction in dietary protein intake and rises rapidly, outstripping the rate of general recovery on reverting to a protein-rich diet. PMID- 3006582 TI - Monoclonal antibody detection of 2-acetyl-aminofluorene-modified DNA probes for the specific detection of nucleic acids in hybridization procedures. AB - We describe the use of acetoxy-acetyl-aminofluorene-modified DNA probes in several hybridization techniques. Hybrids were detected with the help of a monoclonal antibody raised against AAF-guanosine and a second antibody coupled to an enzyme. The sensitivity achieved with AAF-DNA probes routinely detected 0.25 pg DNA bound to a filter. AAF-DNA probes were highly stable and were prepared by simple chemical modification of DNA. Their use as a possible diagnostic tool is discussed. PMID- 3006584 TI - [Inferior vena cava thrombus in nephroblastoma in a child. Diagnostic, surgical and prognostic problems]. AB - The authors report a case of a child Wilm's tumour with inferior vena cava obstruction. The diagnostic, technical and prognostic features are discussed. Ultrasonography appears to be the best diagnostic method to confirm thrombus in the vena cava and allows the choice of an appropriate surgical technique. The presence of vena cava thrombus does not alter the prognosis. PMID- 3006583 TI - High and low density PHA- (but not ConA-) activated T cells stimulate the autologous mixed lymphocyte reaction. AB - In this study, PHA- and ConA-activated cells (PAC and CAC) were used as stimulators in mixed lymphocyte reactions (MLR) using autologous (auto) and allogeneic (allo) peripheral mononuclear cells as responders. PAC, but not CAC, were stimulatory in allo- and auto-MLR, and this stimulation was not due to residual PHA. In PAC which have been activated for 96 h, auto-MLR was due to determinants present on low density T-cell blasts, while with PAC which had been stimulated for more than 192 h, the determinants seemed to be associated with high density T cells. Anti-T3 monoclonal antibodies and certain anti-DR suppressed auto- and allo-MLR mediated by PAC when present throughout the entire MLR assays. CAC suppressed PAC-mediated auto-MLR in a dose-dependent fashion. This inhibition was not DR-restricted and was reversed by the addition of exogenous IL-2. Our results indicate that: depending upon the length of activation, both low density and high density PHA-activated T cells exhibited strong stimulatory capacity in auto-MLR; ConA-activated T cells failed to stimulate auto- or allo-MLR and suppressed MLR mediated by PAC; this suppression was due to suppressor cells, not to suppressor factors, and was readily reversed by exogenous IL-2; pretreatment of CAC with anti-TAC did not reverse the inhibition. PMID- 3006585 TI - Organizations reconsider reusing disposables issue. PMID- 3006586 TI - Rapid physical mapping by transposon Tn5 mutagenesis to localize the cloned yeast ILV2 gene. AB - A rapid method for Tn5 mutagenesis of cloned genes on multicopy plasmids was used to map a yeast ILV2 mutant allele encoding a sulfometuron methyl-resistant acetolactate synthase. Twenty-one of 40 independent Tn5 insertions were within the 5.6-kilobase-pair cloned segment. Of these, seven adjacent transposition events inactivated the sulfometuron methyl resistance determinant, localizing the ILV2 gene to a minimum 1.4-kilobase-pair region. PMID- 3006588 TI - Cloning in Streptococcus lactis of plasmid-mediated UV resistance and effect on prophage stability. AB - Plasmid pIL7 (33 kilobases) from Streptococcus lactis enhances UV resistance and prophage stability. A 5.4-kilobase pIL7 fragment carrying genes coding for both characters was cloned into S. lactis, using plasmid pHV1301 as the cloning vector. The recombinant plasmid was subsequently transferred to three other S. lactis strains by transformation or protoplast fusion. Cloned genes were expressed in all tested strains. PMID- 3006587 TI - Concentration of viruses in beef extract by flocculation with ammonium sulfate. AB - Bacteriophages and enteroviruses in water were adsorbed to positively charged filters (Virosorb 1MDS [AMF Cuno, Inc., Meriden, Conn.] or Seitz S [Republic Filters, Milldaler, Conn.]). Adsorbed viruses were eluted by treating the filters with 10% beef extract, pH 9. Organic flocculation of the beef extract at pH 3.5 permitted recovery of more than 40% of the enteroviruses tested but less than 15% of the bacteriophages present. A method was developed that uses salts at pH 7 to flocculate beef extract. Two volumes of saturated ammonium sulfate were added to beef extract, and both enteroviruses and bacteriophages were adsorbed to the flocs that formed. Greater than 70% of the enteroviruses and bacteriophages were recovered by centrifuging the sample and suspending the flocs in a small volume of distilled water. PMID- 3006589 TI - Inactivation of human and simian rotaviruses by chlorine. AB - The inactivation of simian rotavirus SA-11 and human rotavirus type 2 (Wa) by chlorine was compared at 4 degrees C by using single-particle virus stocks. Both virus types were usually more readily inactivated at pH 6.0 than at pH 8.0 when low chlorine concentrations (0.05 to 0.2 mg/liter) were used. A complete (5 log) reduction of both was obtained within 20 s at all pH levels when chlorine concentrations were increased to 0.3 mg/liter. Slight differences in the chlorine sensitivities of SA-11 and human rotavirus type 2 were noted but were not considered to be significant. PMID- 3006590 TI - Activation and inactivation of an enzyme catalyzed by a single, bifunctional protein: a new example and why. AB - Recent studies have shown that the light-dark mediated regulation of the leaf photosynthetic enzyme pyruvate, Pi dikinase results from interconversion between an active nonphosphorylated form of the enzyme and an inactive form phosphorylated on a threonine residue. These phosphorylation and dephosphorylation reactions are apparently catalyzed by a single protein termed the pyruvate, Pi dikinase regulatory protein and, notably, both reactions are mechanistically unique. We consider the evidence that this regulatory protein belongs to a group of unusual bifunctional enzymes that catalyze opposing reactions, apparently at separate catalytic sites on the same polypeptide. In three of the four known cases these bifunctional enzymes interconvert the active and inactive forms of another enzyme. The possible advantages of such opposing reactions being catalyzed by the same protein are considered. PMID- 3006591 TI - An analysis of the g values in semi-met forms of hemerythrin and in related proteins. AB - The two semi-met (FeIII, FeII) forms of hemerythrin prepared by either oxidation of the deoxy form or reduction of the met form, exhibit rather different EPR spectra. It is shown that a very weak difference in the rhombic distortion of the ferrous site is sufficient to account for this large shift of the g values. It is proposed that the important departure from g = 2.00 and the large anisotropy of the g tensor reflect directly the octahedral coordination of the ferrous ion. Such a coordination could then be present in other proteins which contain binuclear clusters characterized by similar EPR spectra. PMID- 3006592 TI - Salinity adaptation in fish: interaction of thyroxine with fish gill mitochondria. AB - When the freshwater fish Sarotherodon mossambicus is exposed to an ionoosmotic stress, extensive changes take place in the energetics of the gill mitochondria. These changes are reversed when thyroxine is administered to the fish prior to exposure to stress [K. Shivakumar and J. Jayaraman (1984) Arch. Biochem. Biophys. 233, 728]. The presence of a thyroxine binding component in the mitochondrial inner membrane, its characteristics, and its possible involvement in the salinity adaptation process are discussed. PMID- 3006593 TI - Membrane potentials in reconstituted cytochrome c oxidase proteoliposomes determined by butyltriphenyl phosphonium cation distribution. AB - Equilibration of the butyltriphenyl phosphonium (BTPP+) cation into cytochrome c oxidase reconstituted proteoliposomes was measured potentiometrically. The maximum membrane potential (delta psi) generated by oxidase activity was estimated to lie between -65 and -90 mV, vesicle interior negative, when internal BTPP+ binding is taken into account. Formation of delta psi was completely prevented by valinomycin and carbonyl-cyanide-p-trifluoromethoxyphenylhydrazone but only 10% inhibited by levels of N',N'-dicyclohexylcarbodiimide that abolish proton pumping by the oxidase. delta psi is thus maintained by at least one charge transfer process that does not involve proton movement. A nonlinear relationship was obtained between oxidase activity and steady-state delta psi. The value of delta psi estimated by BTPP+ distribution was lower than that calculated using the optical probes safranine and a carbocyanine dye. Possible reasons for this discrepancy are discussed. PMID- 3006594 TI - [Recent advances in the management of chemotherapy-induced emesis]. AB - Nausea and vomiting induced by cisplatin are very severe and intractable to standard antiemetics. During the past several years, many studies of antiemetic management in the patients receiving cisplatin have been performed. In this paper recent advances in the management of cisplatin-induced emesis were reviewed. To date, high dose (2 mg/kg every 2 hours 3-5 times) metoclopramide is considered to be the most effective drug against emesis induced by high-dose (100-120 mg/m2) cisplatin, and a combination therapy of metoclopramide, dexamethasone, diphenhydramin and lorazepam appears to be the most effective. In antiemetic management the problem of anticipatory emesis and delayed or persistent emesis must be considered as well as acute chemotherapy-induced emesis. On the other hand, we have experienced that antiemetic trials are more ineffective in women than in men, so it is more important to control chemotherapy-induced emesis in women. For obtaining more effective control of cisplatin-induced emesis, a combination of antiemetic agents affecting more than one of several neurotransmitter receptors is necessary. In Japan, antiemetic trials have only strated, so we must make an effort to work towards reducing the distressing emesis induced by cisplatin. PMID- 3006595 TI - [The simultaneous evaluation of serum NSE as a tumor marker in lung cancer compared with serum CEA--high sensitivity and specificity defined in the TP-FP curve]. AB - Serum NSE and serum CEA levels were simultaneously examined in 40 patients with primary lung cancer and in 70 other patients without cancer, including 45 young healthy subjects, 13 cases of chronic obstructive pulmonary diseases (COPD), and 12 cases of pulmonary tuberculosis. The primary lung cancers consisted of 13 small cell carcinomas, 12 adenocarcinomas, 11 epidermoid carcinomas, 3 large cell carcinomas, and 1 unclassified. The mean serum NSE level in the 40 primary lung cancer cases was 18.4 +/- 65.7 ng/ml, the levels for small cell carcinomas, adenocarcinomas, epidermoid carcinomas, and large cell carcinomas being 46.8 +/- 46.7 ng/ml, 3.8 +/- 1.0 ng/ml, 4.6 +/- 2.8 ng/ml and 3.6 +/- 0.3 ng/ml, respectively. The levels in young healthy subjects, COPD cases, and pulmonary tuberculosis cases were 3.0 +/- 0.59 ng/ml, 3.2 +/- 0.51 ng/ml and 5.0 +/- 1.5 ng/ml, respectively. In order to evaluate the efficacy of NSE as a tumor marker, we calculated the true positive ratios and false positive ratios for serum NSE, as on indicator of sensitivity and specificity. It was concluded that serum NSE is an excellent new tumor marker compared with CEA, especially for small cell carcinomas. PMID- 3006597 TI - [Chemoembolization therapy of liver cancer combined with lipiodol and OK-432 intra-arterial administration]. PMID- 3006596 TI - [PMU therapy of recurrent gastric cancer. A case report]. AB - A 56-year-old woman with recurrent gastric cancer treated with PMU therapy, combined CDDP 75 mg/m2 i.v. MMC 10 mg/body i.v. and UFT 400mg/body/2 alpha/day p.o., was reported. She was admitted because of cervical lymph node swelling and abdominal tumor (para-aorta lymph node swelling). She was treated two times with this therapy and induced into complete remission. The serum CEA level, more than 500 ng/ml before the treatment, was reduced to 6.7 ng/ml after treatment. She has currently been free of disease for more than four weeks. We conclude that this PMU therapy is extremely effective for indurable gastric cancer. PMID- 3006598 TI - [Transcatheter oily chemoemobolization of hepatocellular carcinoma using adriamycin, lipiodol and gelfoam: pharmacokinetics of adriamycin]. PMID- 3006599 TI - A comparison of the Tzanck smear and viral isolation in varicella and herpes zoster. AB - We compared the usefulness of the Tzanck smear to viral culture in patients with varicella and herpes zoster. All 11 patients with early lesions of varicella had a positive Tzanck preparation, while only seven (64%) of 11 had a positive culture. Twelve (80%) of 15 patients with zoster had a positive Tzanck preparation, while only nine (60%) of 15 had a positive culture. The predominance of the Tzanck yield over the culture is probably due to the increased difficulty in isolation of the varicella zoster virus. These results suggest that the Tzanck preparation is a valuable tool in the diagnosis of patients with varicella and zoster and offers a much more immediate answer than does viral culture, which often takes one to two weeks. These results contrast with those of our previous study of the Tzanck preparation and herpes simplex, in which the viral culture was more accurate than the Tzanck preparation. PMID- 3006601 TI - A new era in antimycotic agents. PMID- 3006600 TI - What is the basic defect in genetic disease? PMID- 3006602 TI - Itraconazole therapy in lymphangitic and cutaneous sporotrichosis. AB - Itraconazole, a new orally absorbable azole derivative, was used for the treatment of 17 patients with cutaneous and lymphangitic sporotrichosis. The drug, administered at a dose of 100 mg/day, proved to be effective in all cases. Lesions disappeared and cultures became negative after 90 to 180 days of therapy. There were no major side effects. Posttherapy evaluations, done in 14 of 17 cases for an average of 115 days, revealed no relapses. Objective evaluation of the treatment by means of a scoring system indicated complete resolution of the pretherapy abnormalities at varying periods; thus, 35.3% (six of 17) of the patients had recovered by 90 days, 45.4% (five of 11) by 120 days, and 83.3% (five of six) by 150 days of therapy. Consequently, therapy with itraconazole is an adequate alternative to iodide treatment in sporotrichosis. PMID- 3006603 TI - Response to ACTH in the newborn. AB - Adrenocortical function was studied in 52 newborn infants who had been divided into three groups: preterm well, preterm ill, and term ill. Basal plasma 17 hydroxyprogesterone concentrations were significantly increased in both groups of preterm infants. There was no significant difference in basal plasma cortisol concentrations, although they were highest in preterm ill infants. All infants responded to adrenocorticotrophic hormone (ACTH) stimulation (36 micrograms/kg intramuscularly) with a two to three fold increase in the concentration of both steroids. The peak plasma 17-hydroxyprogesterone response was significantly higher in preterm ill infants. A subgroup of five infants, who were highly stressed but had undetectable basal plasma cortisol concentrations, also showed an appropriate response to ACTH. The results provide useful reference data to assess adrenal function in the infant of a mother given glucocorticoids during pregnancy. There is also a change from the pattern of fetal adrenal steroidogenesis soon after birth, which may be affected by exogenous ACTH stimulation. Roughly 10% of stressed newborns failed to synthesise cortisol basally; temporary glucocorticoid replacement for such infants may be appropriate. PMID- 3006604 TI - Dietary survey of diabetics. AB - This study of 168 diabetic children from Tyneside and Teeside aimed to record what the children actually ate and to compare this with both their prescribed diet and current recommendations. The amounts of energy consumed were similar to those expected of non-diabetic children, but the components of the diabetic children's diets were different, consisting of more fat and fibre, but less sugars and carbohydrates. They ate more carbohydrate than prescribed but less than current recommendations as there was a shortfall between the amount prescribed and that suggested in the recommendations. Diabetic control was related to the amount of fibre consumed and to compliance with the prescribed diet, but not to the proportion of energy taken as carbohydrate. The insulin dose was slightly lower in those children eating more fibre. PMID- 3006605 TI - Norwalk like viruses: study of an outbreak. AB - An outbreak of acute non-bacterial gastroenteritis is reported, during which a ward had to be closed, and stool samples from 15 patients showed a virus structurally similar to the Norwalk agent. PMID- 3006606 TI - Leukopheresis for treatment of psoriasis: is therapeutical benefit related to reduced activities of neutral proteinases of polymorphonuclear leukocytes? AB - Ten patients were treated with repeated leukophereses performed one to three times per week for 2-5 weeks. Two of the patients was cleared completely, four exhibited regression of more than one-half of the lesions, and four showed only a slight improvement. The therapy did not markedly affect the granulocyte count in peripheral blood, and the beneficial clinical response was not related to the number of polymorphonuclear leukocytes (PMNs) removed by leukophereses. During therapy, the activities of elastase, cathepsin G, lysozyme, and myeloperoxidase in PMNs were determined by spectrophotometry. PMNs isolated using a Haemonetics 30 blood-cell separator were about 50% deficient in these activities in comparison to cells obtained directly from peripheral blood. Thus, leukopheresis induces a marked degranulation of PMNs. Repeated leukophereses were found to generate significant variations in the activities of circulating PMN granule enzymes and in the levels of acid-soluble proteins. Remission or great improvement were observed in patients who, during therapy, exhibited decreased PMN elastase and cathepsin G activities, whereas a poor clinical response was accompanied by high enzymatic activities. PMID- 3006607 TI - Treatment of bovine-papillomavirus-type-1 (BPV)-transformed mouse cells with aromatic retinoid and retinoic acid. PMID- 3006608 TI - Epidermodysplasia verruciformis. Clinical and light- and electron-microscopic observations during etretinate therapy. AB - Three patients with epidermodysplasia verruciformis (EV) were treated with etretinate for 9-13 months. The patients had lesions characteristic of EV, including flat warts, common genital warts, pityriasis-versicolor-like lesions and malignant changes such as actinic keratosis and Bowenoid cancer in situ. During etretinate treatment, some flattening of the warts was observed in all three patients, and the lesions on the chest and back became less red and scaling. However, none of the lesions disappeared completely, and when the treatment was discontinued, the lesions relapsed. No malignant changes were detected during the period of therapy. Electron microscopy revealed the presence of typical large, clear cells containing viral particles in the upper epidermis. Etretinate therapy induced the same type of fine-structural changes as those seen in keratinization disorders and genodermatoses. The clear cells and virus particles persisted throughout the treatment period. More long-term, controlled studies are necessary to make possible an estimate of the curative and cancer inhibitory effect of etretinate treatment in patients with EV. PMID- 3006609 TI - Cyclic AMP and cyclic AMP-dependent protein kinase in mouse skin. II. In vitro effects of isotretinoin and etretinate. AB - Skin from hairless mice was incubated with two synthetic retinoids, isotretinoin and etretinate, and the cAMP content as well as the activity of cAMP-dependent protein kinase were determined. A crude plasma membrane preparation was used to measure adenylyl cyclase activity. Neither isotretinoin (10(-6) and 10(-5)M) nor etretinate (10(-6)-10(-4)M) produced any significant changes in adenylyl cyclase activity. Tissue cAMP levels also remained unaltered after treatment with these retinoids. Although the protein kinase activity ratios remained constant over the concentration range of each retinoid, absolute protein kinase activity was stimulated by treatment with etretinate. These data suggest that cAMP may not mediate the action of retinoids in skin, and that the stimulation of protein kinase activity caused by etretinate probably involves an alternative mechanism. PMID- 3006610 TI - Studies of the effect of D-penicillamine and sodium aurothiomalate therapy on superoxide anion production by monocytes from patients with rheumatoid arthritis: evidence for in vivo stimulation of monocytes. AB - The capacity of monocytes from patients with rheumatoid arthritis to generate superoxide anion in vitro after stimulation with serum treated zymosan (STZ) or IgG treated zymosan (IgTZ) was studied before and during therapy with penicillamine (n = 9) or sodium aurothiomalate (AuTM) (n = 12). Significant increases in rates of STZ (p less than 0.01) and IgTZ (p less than 0.02) stimulated superoxide anion production were seen after successful therapy (14 patients), which were paralleled by a significant increase in serum thiol levels. Patients who did not respond clinically to therapy (n = 4) showed a smaller mean increase in serum thiol levels and had high mean rates of in vitro superoxide production before and after second-line therapy. Three patients were withdrawn from the study. The data suggest that successful therapy with penicillamine or AuTM may be associated with monocyte activation, and possible mechanisms are discussed. PMID- 3006611 TI - Rheumatoid arthritis, juvenile arthritis, iridocyclitis and the Epstein-Barr virus. AB - In order to examine the relation of Epstein-Barry virus (EBV) infection to chronic arthritis in children antibodies to EB virus capsid antigen (EBVCA) and rheumatoid arthritis nuclear antigen (RANA) were analysed in sera from 133 patients classified as juvenile rheumatoid arthritis (JRA) or pauciarticular, polyarticular, or systemic juvenile chronic arthritis. Except for an increased frequency in the systemic subgroup, the prevalence of antibodies to EBVCA and titres of anti-RANA antibodies was similar in patients and controls. These data do not support an aetiological role for EBV in chronic arthritis in children, including JRA, and suggest that the mechanisms which may account for the higher titres of anti-RANA antibodies in adult RA do not occur in children. PMID- 3006612 TI - Antioxidant action of antimalarials. AB - The effects of antimalarials, chloroquine and quinacrine, on the generation of reactive oxygen species were examined both in polymorphonuclear leucocytes and in the xanthine-xanthine oxidase system. Antimalarials showed inhibitory effects on the production of reactive oxygen species probably by affecting cell functions, such as membrane phospholipid methylation. It is suggested that antimalarial agents can work as antioxidants at the site of inflammation protecting against auto-oxidative tissue damage with resultant anti-inflammatory effects. PMID- 3006613 TI - Superoxide production by polymorphonuclear leucocytes in rheumatoid arthritis and osteoarthritis: in vivo inhibition by the antirheumatic drug piroxicam due to interference with the activation of the NADPH-oxidase. AB - The superoxide (O2-) production of stimulated polymorphonuclear leucocytes is increased in patients with rheumatoid arthritis and osteoarthritis compared with controls. Treatment of these different groups with pharmacological amounts of the non-steroidal anti-inflammatory drug piroxicam in vivo resulted in a decrease of about 25% in O2- secretion by isolated granulocytes. In vitro experiments showed that piroxicam inhibits O2- production of granulocytes by interference with the stimulation of the NADPH-oxidase. Piroxicam caused diminished O2- production of membrane fragments if it was present during the stimulation of the NADPH-oxidase of the intact cells. During the actual O2- production of the stimulated membrane fragments piroxicam had no effect. It is concluded that piroxicam is able to inhibit granulocyte O2- production by blocking the activation of NADPH-oxidase, which results in diminished tissue destruction by oxygen free radicals in inflammatory diseases. PMID- 3006614 TI - PMN superoxide radical production following a metabolic-endocrine simulation of trauma. AB - Serious infections following major trauma remain inexplicably high. Metabolic and endocrine changes after injury have been suggested as being responsible for many of the documented defects in the polymorphonucleocyte (PMN). The in vitro bactericidal activity of normal human PMNs has been examined in this laboratory by assaying the activity of the PMN membrane bound enzyme NADPH oxidase and hence O2- production of the PMN in a metabolic/endocrine milieu designed to simulate moderately severe trauma. This was accomplished by incubating the PMN with physiological and trauma serum concentrations of insulin, glucose, cortisol, epinephrine, and glucagon. The results indicate that the O2- production of the PMN is significantly enhanced in this environment. It would appear that exogenous glucose alone was responsible for this enhanced O2- production. PMID- 3006615 TI - Diagnosis for interstitial lung disease in patients with acquired immunodeficiency syndrome (AIDS): a prospective comparison of bronchial washing, alveolar lavage, transbronchial lung biopsy, and open-lung biopsy. AB - This study was undertaken to compare prospectively the diagnostic yield of the various bronchoscopic techniques with that of open-lung biopsy for interstitial lung disease in patients with acquired immunodeficiency syndrome (AIDS). Under general anesthesia, 15 patients sequentially underwent bronchial washing, transbronchial lung biopsy, alveolar lavage, and open-lung biopsy in the same segment of lung. Of nine patients with Pneumocystis carinii, seven were diagnosed by means of the transbronchial lung biopsy, eight by the open-lung biopsy, and all nine by alveolar lavage. Of the six patients with cytomegalovirus, five were diagnosed by the open-lung biopsy, five by the transbronchial lung biopsy, and three by alveolar lavage. The sensitivities of the procedures for identifying infection were washings (15%), transbronchial lung biopsy (50%), alveolar lavage (73%), and open-lung biopsy (88%). Combined, transbronchial lung biopsy and alveolar lavage showed a diagnostic yield (85%) for infections comparable to that of open-lung biopsy (88%), thereby obviating the need for open-lung biopsy for such diagnoses. However, open-lung biopsy was the only procedure that diagnosed Kaposi's sarcoma in lung. PMID- 3006616 TI - Pharmacological effects of SCH 30497--a novel analgesic substance. AB - SCH 30497 (2-[3-(1,2,3,6-tetrahydro-4-2(methylphenyl)-pyridin-1-yl) -propyl] 1,2,4-triazolo[4,3-a] pyridin-3-(2H)-one) was tested for analgesic effects in the rat, mouse and squirrel monkey. SCH 30497 showed dose-related analgesic effects in the rat yeast-paw test; at peak times the ED7sec (95% confidence limits) in mg/kg via the oral, subcutaneous, intramuscular and intravenous routes of administration were as follows, respectively: 4.7 (2.1-9.8), 5.7 (3.4-9.6), 6.4 (4.1-9.8) and 1.4 (0.6-3.2). SCH 30497 was also analgesic in the acetic acid induced writhing test in mice (ED50 = 1.9 mg/kg p.o.) and the squirrel monkey shock titration test (ED50 = 14.5 mg/kg p.o.). It was inactive in the mouse (100 mg/kg p.o.) and rat (40 mg/kg p.o.) tail-flick tests. Thus, SCH 30497 was efficacious versus chemical, mechanical and electrical nociceptive stimuli. Naloxone antagonism of the analgesic effects of SCH 30497 was species specific with significant inhibition observed only in the rat and not in the mouse or monkey. SCH 30497 did not produce Straub tail or hyperactivity in mice. Twice daily dosing at 30 mg/kg p.o. to rats for 5 days failed to produce tolerance; in separate experiments, daily injections for 10 days at 20 or 100 mg/kg p.o. failed to induce signs of dependence following naloxone challenge. SCH 30497-induced analgesia was not attenuated in rats previously made tolerant to narcotics by implantation of a morphine pellet. SCH 30497 showed a weak ability to displace 3H Met5-enkephalin from its binding sites on rat brain membranes (IC50 = 48 microM). SCH 30497 (100 microM) did not affect prostaglandin synthesis in vitro. In vivo, the drug did not have anti-inflammatory or ulcerogenic effects up to 80 mg/kg p.o. Acute behavioral, neurological and autonomic side effects were primarily depressant in rodents and occurred at doses greater than 15 times those that were analgesically relevant. Moderate doses in the monkey (2.5 times the ED50) and high doses in mice produced convulsions. It is hypothesized that SCH 30497-like drugs represent a new class of analgesics based on this unique pharmacological spectrum of activity. PMID- 3006617 TI - Physical performance during sustained converting enzyme inhibition with Hoe 498 in conscious dogs. AB - The effect of sustained converting enzyme (CE) inhibition on modest levels of exercise performance was investigated using German Shepherds. The dogs were trained to run on an inclined treadmill and to tolerate repeated jugular venous puncture. The effects of exercise were assessed by monitoring heart rate and respiratory rate and plasma catecholamine- and serum electrolyte concentrations on each of two control runs. Plasma CE- and renin activity were also determined. Inhibition of CE-activity was established thereafter by high dose treatment with 10 mg/kg Hoe 498 p.o. once daily for one week and the run repeated. These large doses of Hoe 498 were well tolerated. Neither running performance nor cardiorespiratory responses were altered by CE-inhibition. Plasma K+ and catecholamines were taken as measures of peripheral sympathetic activity, and CE inhibition did not alter the increase in either of these in response to exercise. It is concluded that converting enzyme inhibition with Hoe 498 does not impair exercise performance despite any direct effects the loss of angiotensin II may have on reflex sympathetic functions. PMID- 3006618 TI - [Prospective study of the value of echocardiography and myocardial scintigraphy with Tc 99m pyrophosphate during the acute stage of right ventricular infarction]. AB - The aim of this study was to compare the clinical values of 99mTc pyrophosphate scintigraphy and M mode and 2D echography in the diagnosis of right ventricular infarction and in the predicting of some of its complications. Fifty-two patients were prospectively studied by echocardiography and scintigraphy at the acute stage of inferior wall infarction. Scintigraphy was performed in the antero posterior and 45 degrees left anterior oblique incidences during the first 3 days of infarction. Right ventricular infarction was diagnosed if the right ventricular fixation was separated from the left ventricle by fixation at the base of the septum. Echocardiography was performed at an early stage by the usual 3 incidence. Dilatation of the right ventricle on the parasternal and submitral incidences; abnormal right ventricular contraction was searched for in all the incidences. The following results were obtained: scintigraphy showed a localised fixation allowing a topographic study in 40/52 patients (77%); satisfactory echocardiographic studies were obtained in 46/52 patients (88.5%). Signs of right ventricular infarction: scintigraphy showed signs of necrosis of the right ventricle in 12/40 patients (30%) who had a localised fixation; echocardiography showed dilatation (greater than 25 mm) of the superior part of the right ventricle with a right/left ventricle ratio greater than 0.5 in 16/46 patients (37%) with interpretable studies and obvious abnormalities of right ventricular wall motion in 7 patients (15.2%), less obvious in 10 other patients (22%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006619 TI - [Predictability and prevention of hypokalemia induced by hydrochlorothiazide]. AB - In a double blind study, 104 hypertensive patients were randomly allocated to two different stepped care programs for 6 months. Following a placebo run-in-phase, patients were given either Enalapril (EN) 20 mg once-a-day, or a Placebo (PL). Drugs were added as follow in a parallel stepwise sequence, until the goal blood pressure was obtained. First Hydrochlorothiazide (HCTZ) and then, if necessary, Oxprenolol (OXP) and Dihydralazine (DIH). The two goals of this study were to lower diastolic blood pressure below 90 mmHg and to maintain plasma potassium above 3.5 mmol/l. Amiloride was prescribed if plasma potassium was lower than 3.5 mmol/l. At the end of the study the EN group's blood pressure was 130 +/- 12/83 +/- 6 mmHg, the tablet's daily number was 2.6 +/- 1.8 and Amiloride was necessary in 15 patients. These parameters were significantly different from those observed in the Pl group (BP: 136 +/- 9/87 +/- 5 mmHg, tablets 4.2 +/- 2.4; amiloride necessary in 34 patients). The effects of HCTZ were evaluated in 32 patients previously treated by EN compared to 39 patients receiving PL. The converting enzyme inhibition minimized the fall in plasma potassium induced by HCTZ (3.6 t +/- 0.4 vs 3.3 +/- 0.5 mmol/l, p less than 0.05). Related to the HCTZ dose (mg/kg), the plasma potassium fall is lower in the EN group (0.46 +/- 1.1) than in the PL group (0.94 +/- 0.9, p less than 0.05). The plasma potassium reduction could not be predicted by age, plasma renin activity, plasma and urinary aldosterone or urinary kallikrein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006621 TI - [Circulating inhibitor of sodium active transport in essential hypertension and volemic expansion]. AB - Circulating inhibitors of the Na+ pump have been proposed as participating in sodium excretion, extracellular and vascular volume regulation and as hypertensiogenic agents. The presence of digitalis-like compounds in human plasma has been investigated by measuring its ability to compete with tritiated ouabain for binding to the digitalis site of red blood cells. Their activities in plasma from either hypertensive or volume expanded patients were compared. High levels were found in plasma from 37 p. cent of the untreated patients with essential hypertension, 64 p. cent of patients with end-stage renal failure and 71 p cent of acromegalic patients in the hypersecreting phase. The patients of these two last classes have been selected as being normotensives and without family history of hypertension. An increased activity of the inhibitor should more likely be linked to the positive Na+ balance and the volemic expansion which characterise these last two diseases than to high blood pressure. The observations that the activity of the inhibitor is correlated with the plasma volume in acromegalic patients, it returns to normal values after hemodialysis in renal insufficiency or successful therapy of acromegaly and the decrease in its activity is proportional to the weight lost during dialysis in uremic patients, agree with this proposal. PMID- 3006620 TI - [Phosphoinositide metabolism in essential and experimental hypertension]. AB - The metabolism of phosphoinositides, a class of membrane lipids that appears to be intimately involved in the regulation by the membrane of the intracellular Ca2+ level, has been reported to be modified in the erythrocyte of the spontaneously hypertensive rat (SHR and SHR/SP). In order to elucidate the link between the phosphoinositide alteration and hypertension, the metabolism of phosphoinositides was studied in human essential hypertension and in Sabra rats under various patho-physiological conditions. Experiments were performed in vitro on isolated ghost membranes by measuring the radioactivity incorporated into triphosphoinositides (PI-P2) and diphosphoinositides (PI-P) following the incubation of membranes with [gamma 32P]-ATP. 32P-PI-P2, in moderate untreated essential hypertensive controls (n = 31) was higher than in normotensives (n = 30) (1.18 +/- 0.06 vs 0.92 +/- 0.04, 32P nmol/15 min/mg prot, p less than 0.005); 32P-PI-P2 and 32P-PI-P in hypertensive patients treated with beta-blocking agents (n = 20) did not differ from the values observed in untreated hypertensives. In Sabra rats, 32P-PI-P2 values were 0.79 +/- 0.03 and 1.32 +/- 0.08 for SbN and SbH, respectively (8-11 animals per group); difference was significant. 32P-PI-P values varied similarly. Both 32P-PI-P2 and 32P-PI-P did not change significantly when animals were fed a high sodium diet or were injected with DOCA, though such treatments rose the blood pressure. Our data indicate that the modification of phosphoinositide metabolism that we observed both in rat and human hypertension is not a consequence of the blood pressure elevation, but may be considered as an intrinsic membrane defect. Changes in the phosphoinositide metabolism may therefore be associated with the functional and structural alterations concerning the transmembrane Na+ and Ca2+ fluxes which may be of pathogenic importance. PMID- 3006622 TI - [Chronic dietary sodium overload and release of a circulating Na+-K+ pump inhibitor]. AB - High Na+ intake has been proposed to induce a rise in the activity of a circulating inhibitor of the Na+, K+-pump. The effects on male Wistar rats of a high sodium diet (8 per cent NaCl) on the activity of such a plasma Na+, K+ ATPase inhibitor were investigated. Systolic blood pressure, body weight, urinary Na+ excretion, haematocrit, intraerythrocytic Na+ content and the activity of a Na+ dependent transport system, i.e. the uptake of 5-HT by blood platelets were measured in parallel. After one week, neither systolic blood pressure nor intraerythrocytic Na+ content were modified, but the ability of the plasma extracts to inhibit renal Na+, K+-ATPase increased (70.9 +/- 1.7 vs 76.3 +/- 2.1 mumol Pi/mg/h; p = 0.05). After two weeks, the plasma inhibitory activity, the systolic blood pressure and the intraerythrocytic Na+ content were higher than that of control animals (65.5 +/- 1.6 vs 79.1 +/- 2.8 mumol Pi/mg/h, p less than 0.001; 132 +/- 2 vs 114 +/- 4 mmHg, p. +/- 0.001 and 4.95 +/- 0.32 vs 3.81 +/- 0.36 mmol/l.cells, p less than 0.05). After three months, the ability of plasma extracts to inhibit the Na+ pump and the systolic blood pressure were still elevated (57.8 +/- 1.8 vs 72.9 +/- 1.8 mumol Pi/mg/h, p less than 0.001; 145 +/- 4 vs 118 +/- 2 mmHg, p less than 0.001) whereas intraerythrocytic Na+ content had returned to control levels and 5-HT uptake was not modified.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006623 TI - Differential sensitivities to glucose and galactose repression of gluconeogenic and respiratory enzymes from Saccharomyces cerevisiae. AB - The synthesis of isocitrate lyase was induced by the presence of ethanol in the chemostat reaching a specific activity of 200 mU X mg-1 at this induced state. In glucose-limited, derepressed cells, 20 mU X mg-1 were detected and under repressed conditions isocitrate lyase activity was not detected. The sensitivity of gluconeogenic enzymes: cytoplasmic malate dehydrogenase; fructose 1,6 bisphosphatase and isocitrate lyase as well as the mitochondrial enzymes NADH dehydrogenase and succinate cytochrome c oxidase to glucose and galactose repression were studied in chemostat cultures. Our results show that galactose was less effective as a repressor than glucose. Malate dehydrogenase was completely inactivated by glucose, whereas galactose only produced a 78% decrease of specific activity. Fructose 1,6-bisphosphatase and isocitrate lyase were completely inactivated by both sugars but at different rate. Glucose produced an 85% decrease of specific activity of the mitochondrial enzymes whereas galactose only decrease an 67%. PMID- 3006624 TI - Some properties of fructose 1:6 bisphosphatase activity of buffalo seminal plasma. PMID- 3006625 TI - Effect of cations on hydrolysis of fructose 1:6 bisphosphate in bull seminal plasma. PMID- 3006627 TI - Gastrointestinal autonomic nerve (GAN) tumor. Ultrastructural evidence for a newly recognized entity. AB - We describe three cases of spindle cell tumors arising in the gastrointestinal tract, each with ultrastructural features that recapitulate the ultrastructural morphology of the enteric autonomic nervous system. These features include the presence of small, dense core granules in the synthetic-secretory Golgi structures, sparsely distributed within the ample tumor-cell cytoplasm, near plasma-membrane surfaces and within axons. Tumor cells had elongated processes and axons contained small granular vesicles, clear vesicles, or large, dense vesicles. Specific features diagnostic for smooth-muscle cell, Schwann cell, or fibroblast cellular origin were absent. PMID- 3006626 TI - [New insights into the biological effects and the physiological role of relaxin in domestic animals]. PMID- 3006628 TI - Primary duodenal small-cell neuroendocrine carcinoma with production of vasoactive intestinal polypeptide. AB - A primary duodenal small-cell neuroendocrine carcinoma was found in an elderly man who presented with upper abdominal pain. Although metastatic small-cell carcinoma was documented by liver biopsy, the primary lesion was not identified until postmortem examination. The latter tumor, which ulcerated the duodenal mucosa, was composed of small ovoid cells with sparse cytoplasm and granular chromatin. Electron microscopy revealed cytoplasmic dense-core granules. Immunocytochemical study demonstrated the presence of neuron-specific enolase, Leu 7 antigen, chromogranin, epithelial membrane antigen, and vasoactive intestinal polypeptide within tumor cells. However, there was no evidence of a clinical endocrinopathy. This case emphasizes the need to include the duodenum as a possible primary site when metastatic small-cell neuroendocrine carcinoma is seen in the absence of apparent pulmonary disease. PMID- 3006629 TI - Mucinous carcinoid tumor of the appendix presenting as bilateral ovarian tumors. AB - Mucinous carcinoid tumor of the vermiform appendix, an uncommon variant of appendiceal carcinoid, may present clinically with ovarian metastases. We studied a tumor by immunohistochemistry and electron microscopy and reviewed eight similar cases from the literature. The primary and metastatic tumors in our case were composed of mucin-producing cells and small argyrophilic cells arranged in cords and acini. Tumor cells in both primary and metastatic sites exhibited identical patterns of immunoreactivity for epithelial antigens (epithelial membrane antigen, carcinoembryonic antigen) and neuroendocrine antigens (serotonin, vasoactive intestinal polypeptide, adrenocorticotropic hormone). Ultrastructurally, the cells contained either mucin vacuoles or dense-core neurosecretory granules; rare individual cells contained both types of inclusions. When bilateral solid mucinous ovarian tumors are discovered at laparotomy, diagnostic appendectomy is indicated if no obvious extraovarian primary tumor can be found. PMID- 3006631 TI - The choice between conservative and radical surgery in cancer in the upper abdomen. PMID- 3006630 TI - Adrenergic receptors in insensitive skin of spinal cord injured patients. AB - The factors responsible for the increased susceptibility to decubitus ulcers of the insensitive skin of spinal cord injury (SCI) patients are not known. Autonomic dysfunction leading to defective vascularity is a possibility. SCI removes cerebral control of the isolated nervous system which may mimic denervation hypersensitivity of autonomic neural synapses, where an increase in number and a scattering of the receptors on the postsynaptic membrane leads to abnormal responses. Since adrenergic receptors mediate vascular tone and regulate blood flow in the skin, it would be of great interest to determine whether the number and the concentration of receptors in the insensitive skin of SCI patients is modified as a function of time since injury. To achieve this aim, alpha and beta adrenergic receptors were measured in biopsies obtained from intact skin used to surgically repair decubitus ulcers in SCI patients admitted to The Institute for Rehabilitation and Research. Receptors were identified by competitive radioligand-binding assays in whole skin homogenates. Patients were divided into two groups: patients injured less than five years ("early") and patients injured more than five years ("late"). Alpha and beta adrenergic receptors in both cervical and thoracic SCI patients decreased in density in the "late" patients. The small sample size and the inherent large errors of the assay precluded achievement of statistically significant differences. Nevertheless, a definite trend is seen: Disconnection of the adrenergic neurons from brain integration may mimic denervation and lead to abnormal vascular responses in the insensitive skin. PMID- 3006632 TI - Thrombogenic effects of xenobiotics. AB - The mechanisms by which xenobiotics may cause or promote thrombosis include vascular damage, induction of a hypercoagulable state and disturbances of blood flow. This paper discusses the methods available to detect various types of thrombogenic substances. Pathomorphological techniques are best suited to demonstrate thrombosis caused by localized vascular damage or generalized endothelial lesions. For the assessment of disseminated microcirculatory thrombosis, the consumption of platelets and clotting factors and the appearance of specific platelet proteins and fibrinogen and fibrin split products can be determined in the blood. Hypercoagulability which is defined as a perturbation of the hemostatic equilibrium resulting in a shift in the direction of thrombosis, is of particular importance in toxicology. Many in vitro, ex vivo and in vivo methods have been proposed to detect and to measure the ability of xenobiotics to induce a prethrombotic state. Their usefulness is demonstrated with several examples. PMID- 3006633 TI - The effects of methylglyoxal on central synaptic transmission in the isolated nerve cord of the cockroach. (Periplaneta americana L). AB - Methylglyoxal (10(-5) to 1.5 X 10(-4) M) was found to have excitatory effects on synaptic transmission in the isolated 6th abdominal ganglion of the cockroach. There was a concentration-dependent depolarization of the giant interneurones which was accompanied by an increase in the amplitude and duration of electrically evoked excitatory postsynaptic potentials. The frequency of spontaneous activity was also increased. PMID- 3006635 TI - Separation of immature and adult rat hepatocytes into distinct subpopulations by centrifugal elutriation. AB - Several subpopulations of hepatocytes with increasing cell diameters were isolated, the smaller cells were attributed to the periportal area, the larger ones to the perivenous region. Profiles of total cytochrome P-450, benzphetamine N-demethylation and ethoxyresorufin O-deethylation, cytochrome c-reductase, glucose-6-phosphatase and GPT activities were determined. With adult hepatocytes an increasing cytochrome P-450 concentration with increasing cell diameter could be observed, paralleled by increasing activities of monooxygenases. Glucose-6 phosphatase and GPT also revealed increasing activities with increasing cell diameter, but cytochrome c-reductase did not show a distinct zonation. Immature hepatocytes (age 11-15 days) were smaller, more fragile, and could not be isolated with the same enzyme solution as adult hepatocytes. They did not show any zonation of cytochrome P-450 whereas the zonation of the monooxygenases was almost fully developed. For cytochrome c-reductase a zonation with higher activities in the perivenous cells could be demonstrated, in contrast to the lack of zonation in adult rats. Glucose-6-phosphatase showed a decline with increasing cell diameter in immature hepatocytes, whereas GPT did not show any zonation. In rats aged 20 days the zonation of these parameters in liver was in between younger and older animals. PMID- 3006634 TI - The role of receptors in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. AB - There is good evidence that the Ah-(TCDD-) receptor plays a role in the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its congeners. TCDD and other chlorinated aromatic hydrocarbons with chlorine atoms in lateral positions (2,3,7,8-tetrachlorodibenzofuran, 3,3',4,4'-tetrachloroazoxybenzene, 3,3',4,4'(5,5')-tetra(hexa)chlorobiphenyl), all bind to the receptor and show a similar pattern of toxicity, although there is a wide range in potency. The Ah receptor is viewed as the major product of the regulatory gene of the Ah-locus in the mouse. Several of the toxicities of TCDD and congeners (teratogenesis, thymic involution and hepatic porphyria) have been shown to segregate with the Ah-locus. In vitro studies using keratinizing cells or fetal thymus organ culture have shown a good correlation between activity as ligands of the receptor and toxicity for the compounds discussed. The great differences in toxic potency of these compounds in vivo may therefore be a result of variation in rate of metabolism and excretion rather than differences in affinity for the Ah-receptor. The physiological role of the Ah-receptor is discussed, whether it has developed as a response to exposure to toxic substances in the environment, as a means of induction of P-450-dependent polysubstrate mono-oxygenase activities in order to make those substances more liable for excretion--or is there a physiological ligand? TCDD has a long half-life in the body, and a sustained competition for binding to the receptor between TCDD and a ligand of importance for normal cell functions may result in toxicities such as the wasting syndrome. This tentative ligand could be of varying importance in different species, which might explain the great variation in sensitivity between species, the hamster being about 5000 times less sensitive than the guinea pig. PMID- 3006637 TI - Effect of dithiocarbamate fungicides and thiurams on 3H-haloperidol binding in rat brain. AB - Dithiocarbamates and disulfiram have been shown to be neurotoxic. As one biochemical indicator of the toxicity, dopamine-beta-hydroxylase is inhibited in the brain. Consequently, noradrenaline synthesis is decreased and dopamine tends to accumulate in the brain. It was decided to examine whether cerebral dopaminergic D2-receptor binding is affected by these agents after their short term administration to rats. It appeared that ferbam, maneb, nabam, zineb, disulfiram, thiram and ethylene thiourea did not change significantly D2-receptor binding at doses which were already overtly toxic. PMID- 3006636 TI - The oncogene and its potential role in carcinogenesis. AB - Cellular onc genes are a group of evolutionarily conserved sequences which are homologous to the transforming genes (v-onc) of oncogenic retroviruses. Although their functions in normal cells are largely not known, the sequence homology between viral and cellular onc genes is consistent with the idea that neoplastic transformation may, in some cases, be due to abnormal levels of cellular onc gene expression. Several models can be proposed for such a mechanism, including the insertion nearby of a viral promoter, alteration of the physiological promoter by a mutagenic agent, gene amplification, relocation in a transcriptionally active region of the genome as a consequence of chromosomal rearrangements and point mutations induced by external factors. Examples of these different mechanisms of onc gene activation can be found in animal and human tumors. Finally, the detail description of one cellular onc gene (c-sis), its relation to the viral gene and to a known cellular growth factor and its possible mode of activation in neoplastic transformation is presented. PMID- 3006638 TI - Enteric adenoviruses. Brief review. PMID- 3006639 TI - Biochemical characterization of a foot-and-mouth disease virus strain attenuated for cattle. Brief report. AB - Wild-type, virulent (A-24 Cruzeiro subtype) foot-and-mouth disease virus (FMDV), a related attenuated strain and revertants of the attenuated strain were examined by titration on primary bovine kidney (PBK) and baby hamster kidney (BHK-21) cells, as well as, by infection of unweaned mice. Wild type virus grew equally well in all three systems, whereas the attenuated strain had a titer 2-3 log lower in PBK cells than in the other 2 assays. Within 9 successive passages in BHK-21 cells the attenuated strain gave rise to revertants that had regained the growth properties of wild-type virus in PBK cells. After cloning of the attenuated strain by plaque isolations, the same revertant phenotype was obtained within 9 successive passages. Oligonucleotide mapping indicated that the attenuated strain differed from the wild-type and revertants by at least one additional oligonucleotide. Differences in poly(C) length were not found among any of the three strains of FMDV. These results correlate attenuation and virulence with point mutation(s) and not with deletions. Possible reversions in nature with this attenuated strain may be anticipated. PMID- 3006640 TI - L and S species of Machupo virion RNA contain a mixture of complementary strands. Brief report. AB - RNase resistance analyses of native, heat-denatured and self-annealed L and S species of Machupo virion RNA revealed the presence of complementary sequences. Self-annealing was concentration-dependent. The data indicate, that complementary sequences were present in separate RNA strands. PMID- 3006641 TI - A solid phase fluorescent immunoassay for the rapid detection of virus antigen or antibodies in fetuses infected with porcine parvovirus. AB - A solid phase fluorescent immunoassay using polyacrylamide beads coated with rabbit anti-porcine parvovirus antibodies has been developed and utilized in the diagnosis of porcine parvovirus infection. The antibody-coated beads (immunobeads) were used both to detect virus in mummified fetal tissues and to demonstrate specific antibodies in serum and ovarian follicular fluid. The immunobeads assay (IBA) was as sensitive as ELISA but more sensitive than virus isolation using tissue culture and haemagglutination tests. Both mummified and normal fetuses were obtained after experimental infection of SPF pregnant gilts. Using immunobeads, porcine parvovirus antigen was found in all mummified fetuses, but was found in only 1 out of 17 normal fetuses. PMID- 3006643 TI - [The role of arachidonic acid metabolism on the generation of active oxygens in human granulocytes]. PMID- 3006642 TI - Murine cytomegalovirus adrenalitis in athymic nude mice. AB - During studies of the pathogenesis of murine cytomegalovirus (MCMV) infection in athymic nude mice, we noted striking virus involvement of the adrenal glands. Because patients with the acquired immunodeficiency syndrome (AIDS) have recently been reported to have adrenal necrosis and evidence of infection of the adrenal gland with human cytomegalovirus (HCMV), we have further evaluated adrenal gland involvement during MCMV infection. Following virus inoculation, MCMV replicated to high titer in the adrenal glands of T-cell deficient, homozygous nude mice, but not heterozygous littermates with intact T-cell function. Concomitant with the high titers of virus, there appeared overt histological evidence of herpes virus virus infection accompanied by patchy necrosis of adrenal cortical and medullary tissues. Acyclovir, which inhibits growth of MCMV, reduced virus replication in the adrenal gland. Similarly, virus replication was diminished in homozygous nude mice immunologically reconstituted by infusion of normal spleen cells three weeks prior to infection. Thus, in the absence of functioning T lymphocytes, MCMV can infect and replicate in adrenal tissues causing a progressive destructive adrenalitis. PMID- 3006645 TI - [Electron-microscopic study of monophasic spindle-cell synovial sarcomas]. AB - Ultrastructural analysis of 9 monophasic spindle-cell synovial sarcomas revealed both undifferentiated tumour cells and histiocyte-like and fibroblast-like elements. Specialized contacts between tumour cells are found in 8 tumours, formation of the cavities of varying size (including vessel-like structure in 2 cases) in 7 tumours. Other findings included a discontinuous external lamina in 4 tumours; filopodium-like cell protrusions in 5, and spindle-shaped collagen bundles with long striation in 2 cases. The observation of cell contacts and cavities is of a diagnostic importance for monophasic synovial sarcoma. The differential diagnosis of these sarcomas and other spindle-cell tumours is discussed. Ultrastructural findings are interpreted as evidence of the monophasic spindle-cell synovial sarcoma origin from mesenchymal stem cells. PMID- 3006644 TI - [Ultrastructural and ultracytochemical characteristics of the terminal villi of the placenta in EPH-toxemia of pregnancy]. AB - The main types of the terminal villi in the EPH-toxicosis of pregnancy with various degrees of severity are studied with light, scanning and transmission electron microscopy and ultracytochemistry. The incipient stages of the transition from normal to pathologically changed villi could be detected by means of ultrastructural study only. Enhancement of the degenerative processes in placenta occurs as the severity of this complication increases. PMID- 3006646 TI - Herpesvirus in the hippocampus as a cause of Alzheimer's disease. PMID- 3006647 TI - Proton beam therapy of uveal melanomas. PMID- 3006648 TI - Ciliochoroidal melanomas treated with a narrow medical proton beam. AB - We treated 63 patients with intraocular melanomas by means of a narrow medical proton beam. Tumors were irradiated with 2,500 rad at each of four to five sessions, with an interval of one to two days between sessions. The melanomas ranged in diameter from 8 to 20 mm and were from 3.0 to 13.7 mm in thickness. Patients were followed up for three months to seven years. In 11 cases, the tumor was fully resorbed. Complications included radiation cataract, postradiation glaucoma, radiation retinopathy, and exudative retinal detachment. In 12 cases, enucleation was performed because tumor growth persisted. Four patients died during follow-up period because of metastasis. The eye was preserved in 47 cases. PMID- 3006649 TI - Progressive blindness caused by metastatic occult signet-ring cell gastric carcinoma. AB - We described a metastatic signet-ring cell gastric adenocarcinoma in a 60-year old woman who complained of progressive visual loss. The discovery of signet-ring cells in the cerebrospinal fluid established the diagnosis of mucus-secreting adenocarcinoma. Histologic study of an ulcerated lesion in the lesser curvature of the stomach established the diagnosis of primary gastric adenocarcinoma. When confronted by a patient with impaired function of the cerebral cortex, cranial nerves, and spinal nerve roots that results in progressive neurologic deterioration, leptomeningeal carcinomatosis should be considered and repeated cytologic examinations of spinal fluid should be obtained. PMID- 3006650 TI - Inclusion body pancreatitis in guinea fowl (Numida meleagris). PMID- 3006651 TI - Disturbances in exploratory behavior and functional recovery in the Y and radial mazes following dopamine depletion of the lateral septum. AB - The effects of 6-hydroxydopamine (6-OHDA) injected into the lateral septum in rats were investigated for spontaneous alternation behavior in a Y maze and for spatially oriented behavior in an 8-arm radial maze. The performance of the animals in these tests was assessed under two physiological states, food-satiated and food-deprived. The selective depletion of septal dopaminergic concentrations leads to behavioral disturbances in both the Y and the radial mazes. These deficits disappeared when the animals with 6-OHDA lesions were food-deprived. These results confirm other studies from our laboratory and support two conclusions. First, lesions of dopaminergic neurons lead to behavioral impairments which resemble those found after the total lesion of the structure they innervate. Second, these behavioral impairments are responsive to therapeutic treatments, manipulations of the internal or external environment, or the level of arousal, since under certain conditions a recovery of function can occur in the absence of dopaminergic neurons. These two points provide additional support for a nonspecific role for the dopaminergic neurons originating in the ventral tegmental area. These neurons could have a permissive role in the functioning of the forebrain structures they innervate. PMID- 3006652 TI - The origin of aneuploidy in humans. PMID- 3006653 TI - Neoplasia and cytogenetic abnormalities. PMID- 3006654 TI - Detection by low-temperature magnetic circular-dichroism spectroscopy of optical absorption bands due to molybdenum (V) in the form of xanthine oxidase giving the Desulpho Inhibited e.p.r. signal. AB - The magnetic circular-dichroism (m.c.d.) spectra in the temperature range 1.5-100 K and the electronic absorption spectra at 4.2 and 295 K were measured for a number of desulpho xanthine oxidase derivatives. There were no significant differences between the absorption spectra that could be attributed to molybdenum. However, the visible-region m.c.d. spectrum of the ethanediol-treated metalloprotein (which gives rise to the Desulpho Inhibited e.p.r. signal) contained features assignable to Mo(V) absorption bands. This is the first report of the detection of optical bands of Mo(V) in an enzyme in the presence of other chromophoric centres. PMID- 3006655 TI - Induction of intra- and extra-cellular phospholipids in the lungs of rats exposed to silica. AB - Intracellular and extracellular compartments of phospholipids in the lungs of rats were examined 28 days after intratracheal injection of silica (200 mg/kg). All compartments containing phospholipids were elevated, but the largest increases were seen in the intracellular and extracellular pulmonary surfactant. Intracellular pulmonary surfactant increased 123-fold from 1.18 +/- 0.65 to 144.9 +/- 53.8 and the extracellular surfactant increased 22-fold from 1.17 +/- 0.04 to 25.1 +/- 7.1 mg per pair of rat lungs respectively. The phospholipid composition of intracellular and extracellular surfactant did not change in response to silica, except for an almost 2-fold increase in the percentage of total phosphatidylinositol in both compartments. The phospholipid content of the lungs increased from 24.9 +/- 4.6 to 268.6 +/- 20.8 mg, with the intracellular and extracellular surfactant accounting for 59.1 and 24.6% of this total increase respectively. These data demonstrate that the major increases in the phospholipid content of the lungs induced by silica is associated with the pulmonary surfactant system. PMID- 3006656 TI - The kinetics of bivalent metal ion dissociation from myosin subfragments. AB - Bivalent metal ions have multiple roles in subunit association and ATPase regulation in scallop adductor-muscle myosin. To help elucidate these functions, the rates of Ca2+ and Mg2+ dissociation from the non-specific high-affinity sites on the regulatory light chains were measured and compared with those of rabbit skeletal-muscle myosin subfragments. Ca2+ dissociation had a rate constant of about 0.7 s-1 in both species, as measured by the time course of the pH change on EDTA addition. Mg2+ dissociation had a rate constant of 0.05 s-1, as monitored by its displacement with the paramagnetic Mn2+ ion. It is concluded that the exchange between Ca2+ and Mg2+ at the non-specific site, on excitation of both skeletal and adductor muscles, is too slow to contribute to the activation itself. The release of bivalent metal ions from the non-specific site is, however, the first step in release of the scallop regulatory light chain (Bennett & Bagshaw (1986) Biochem. J. 233, 179-186). In scallop myosin additional specific sites are present, which can bind Ca2+ rapidly, to effect activation of the ATPase. In the course of this work, Ca2+ dissociation from EGTA was studied as a model system. This gave rates of 1 s-1 and 0.3 s-1 at pH 7.0 and pH 8.0 respectively. PMID- 3006657 TI - Reaction of cyanide with cytochrome aa3 in isolated perfused rat head in situ. AB - The reaction of cyanide with cytochrome aa3 in intact mitochondria is known to differ significantly from the reaction with the isolated enzyme. To examine the cyanide reaction with cytochrome aa3 in situ, we studied the spectral characteristics and the reaction kinetics of cyanide with reduced brain cytochrome aa3 in an isolated perfused rat head preparation. Anaesthetized rats underwent bilateral carotid-arterial cannulation. The head (skull intact, muscle removed) was perfused with a crystalloid solution containing Na2S2O4, and the animal was then decapitated. By means of reflectance spectrophotometry the reaction of cyanide with cytochrome aa3 was continuously monitored with the use of the 590 nm-575 nm, 610 nm-575 nm and 590 nm-610 nm wavelength pairs. We found that: the kinetics of the absorbance change at 590 nm and 610 nm were similar, with almost identical apparent rate constants, suggesting that these spectral changes are the results of the formation of a single complex; the difference spectrum obtained on addition of cyanide to the fully reduced preparation showed a peak at 588 nm and a trough at 610 nm, consistent with spectral characteristics of the cyanide-ferrocytochrome aa3 complex in isolated enzyme and isolated mitochondria in vitro; this observation underscores the accuracy of monitoring the effects of inhibitors of mitochondrial function on cytochrome redox reactions in situ; the half-maximal (K0.5) effect was approx. 50 microM, significantly lower than that in vitro. The lower apparent K0.5 for cyanide in this preparation in situ may be due to a difference in the pH of the two systems. This approach provides the means to study the inhibitors of mitochondrial function in intact brain under a physiological environment. PMID- 3006659 TI - The effects of stress and injury on the activity of phosphoenolpyruvate carboxykinase in the liver of the rat. AB - The effects of stress (diethyl ether anaesthesia for 4-8 min, or intravenous injection of 0.05 ml of a dimethyl sulphoxide/water mixture) and of a scald injury given under ether anaesthesia on hepatic PEPCK (phosphoenolpyruvate carboxykinase, EC 4.1.1.32) were studied in the post-absorptive rat. Injury raised PEPCK activity by about 70% in 2 h and by over 100% in 4 h, over three times as fast as in animals that had only been handled (controls). The two stresses, both of types commonly imposed in animal experiments, had almost as much effect as injury for the first 2 h, although much less thereafter. The roles of sympathetic stimulation and corticosterone in mediating these rises were studied by using alpha beta-blockers and trilostane respectively as inhibitors. (Trilostane only decreased corticosterone concentrations to a little above control values.) The ether-induced increase was somewhat decreased by alpha beta blockade, but was only eliminated by combined alpha beta-blockade and trilostane. After injury, however, PEPCK synthesis was unaffected by either alpha beta blockade or trilostane, although it was decreased by their combined action; and it seems that either corticosterone or sympathetic stimulation was sufficient to stimulate PEPCK synthesis maximally. Stimulation by corticosterone was much greater than reported previously by others, for reasons that are discussed. Sympathetic stimulation may have been mediated by glucagon and cyclic AMP, since injury raised portal glucagon concentrations, and stress and injury raised those of hepatic cyclic AMP. PEPCK synthesis was, however, stimulated despite increases in portal insulin concentration, and was not related to the [insulin]/[glucagon] ratio. Thus stress and injury over-rode normal control mechanisms. PMID- 3006658 TI - K+-stimulated p-nitrophenyl phosphatase is not a partial reaction of the gastric (H+ + K+)-transporting ATPase. Evidence supporting a new model for the univalent cation-transporting ATPase systems. AB - Studies with intact and lysed gastric microsomal vesicles demonstrate that there are two pNPP (p-nitrophenyl phosphate)-and one ATP-hydrolytic sites within the gastric H+, K+-ATPase [(H+ + K+)-transporting ATPase] complex. Whereas the ATPase site is located exclusively on the vesicle exterior, the pNPPase sites are distributed equally on both sides of the bilayer. Competition by ATP for the pNPPase reaction on the vesicle exterior suggests that both ATP and pNPP are hydrolysed at the same catalytic site present at the outside surface of the intact vesicles. However, a biphasic inhibition of the K+-pNPPase (K+-stimulated pNPPase) by ATP in the lysed vesicles suggest the pNPPase site of the vesicle interior to have very low affinity (Ki approximately equal to 1.2 mM) for ATP compared with the vesicle exterior (Ki approximately equal to 0.2 mM). Studies with spermine, which competes with K+ for the K+-pNPPase reaction without inhibiting the H+, K+-ATPase, suggest there are two separate K+ sites for the pNPPase reaction and another distinct K+ site for the ATPase reaction. In contrast with the K+ site for the ATPase, which is located opposite to the catalytic site across the bilayer, both the K+ and the catalytic site for the pNPPase are located on the same side. The data clearly demonstrate that the pNPPase is not a manifestation of the phosphatase step of the total H+, K+-ATPase reaction. The K+-pNPPase associated with the Na+, K+-ATPase also has properties strikingly similar to the gastric K+-pNPPase system, suggesting a resemblance in the basic operating principle of the two ion-transporting enzymes. A unified model has been proposed to explain the present data and many other observations reported in the literature for the ATPase-mediated transport of univalent cations. PMID- 3006660 TI - The gamma-aminobutyrate/benzodiazepine receptor from pig brain. Enhancement of gamma-aminobutyrate-receptor binding by the anaesthetic propanidid. AB - The binding of [3H]muscimol, a gamma-aminobutyrate (GABA) receptor agonist, to a membrane preparation from pig cerebral cortex was enhanced by the anaesthetic propanidid in a concentration-dependent manner. At 0 degrees C, binding was stimulated to 220% of control values, with 50% stimulation at 60 microM propanidid. At 37 degrees C, propanidid caused a more powerful stimulation of [3H]muscimol binding (340% of control values). Propanidid (1 mM) exerted little effect on the affinity of muscimol binding (KD approx. 10 nM), but increased the apparent number of high-affinity binding sites in the membrane by 2-fold. Enhancement of [3H]muscimol binding was observed only in the presence of Cl- ions, half-maximal activation being achieved at approx. 40 mM-Cl-. Picrotoxinin inhibited the stimulation of [3H]muscimol binding by propanidid with an IC50 (concentration causing 50% inhibition) value of approx. 25 microM. The enhancement of [3H]muscimol binding by propanidid was not additive with the enhancement produced by secobarbital. Phenobarbital inhibited the effect of propanidid and secobarbital. The GABA receptor was solubilized with Triton X-100 or with Chaps [3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate]. Propanidid and secobarbital did not stimulate the binding of [3H]muscimol after solubilization with Triton X-100. However, the receptor could be solubilized by 5 mM-Chaps with retention of the stimulatory effects of propanidid and secobarbital. Unlike barbiturates, propanidid did not stimulate the binding of [3H]flunitrazepam to membranes. It is suggested that the ability to modulate the [3H]muscimol site of the GABA-receptor complex may be a common and perhaps functional characteristic of general anaesthetics. PMID- 3006662 TI - Presence of fucosamine in teichuronic acid of the alkalophilic Bacillus strain C 125. AB - Cell walls of the alkalophilic Bacillus strain C-125 are composed of gamma peptidoglycan, teichuronic acid and a polymer of glucuronate and glutamate. An amino sugar that was a main component of the teichuronic acid did not correspond to any of the commercially available hexosamines. The amino sugar was purified into crystalline form from the hydrolysate of the teichuronic acid by ion exchange chromatography and then partition chromatography on a cellulose column. The amino sugar was identified as D-fucosamine (2-amino-2,6-dideoxy-D-galactose) by 400 MHz n.m.r. spectrometric analysis, measurement of optical rotation and elemental analysis. PMID- 3006661 TI - The gamma-aminobutyrate/benzodiazepine receptor from pig brain. Purification and characterization of the receptor complex from cerebral cortex and cerebellum. AB - The gamma-aminobutyrate/benzodiazepine-receptor complex has been purified from a Triton X-100 extract of crude synaptic membranes from pig cerebral cortex and cerebellum by a combination of affinity and ion-exchange chromatography. [3H]Flunitrazepam binding activity was purified 2200-fold from cortex with an overall yield of 2%. The dissociation constants for the binding of [3H]muscimol and [3H]flunitrazepam to the receptor complex were 14 +/- 3 nM and 14 +/- 2 nM respectively. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was in the range 2.2-2.8. There appeared to be no selective inactivation of either binding site during the purification procedure. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed two major polypeptides of Mr 49 000 and 55 000 from both cortex and cerebellum. When the receptor from cortex was photoaffinity labelled with [3H]flunitrazepam, radioactivity was incorporated predominantly into the Mr-49 000 polypeptide, although some radioactivity was detectable in the Mr-55 000 band. The cerebellar receptor was photoaffinity labelled on the 49 000-Mr polypeptide but not on the polypeptide of Mr 55 000. In addition, some radioactivity was detected in a minor polypeptide of Mr 43 000. When purified in the presence of 3-[(3 cholamidopropyl)dimethylammonio]propanesulphonate the same major polypeptide components (Mr 49 000 and 55 000) were isolated, but the receptor now retained its ability to be modulated by secobarbital and by the anaesthetic propanidid. PMID- 3006663 TI - Specific proteolytic modification of creatine kinase isoenzymes. Implication of C terminal involvement in enzymic activity but not in subunit-subunit recognition. AB - We are using the isoenzymes of creatine kinase (CK) to investigate the effect of specific proteolytic modification on the abilities of enzyme subunits to establish precise subunit-subunit recognition in vitro. Previous work by others has shown that treatment of the MM isoenzyme of rabbit CK with Proteinase K results in a specific proteolytic modification and inactivation of the enzyme. In the present work, we show that both the MM and BB isoenzymes of chicken CK are also specifically modified by Proteinase K, resulting in over 98% loss of catalytic activity and approx. 10% decreases in subunit molecular masses of the enzymes. Similar reactions appear to occur when the isoenzymes are treated with Pronase E. Limited amino acid sequence analysis of intact and Proteinase K modified MM-CK suggests that the proteolytic modification results from a single peptide-bond cleavage occurring between alanine residues 328 and 329, about 50 amino acid residues from the C-terminal end; the active-site cysteine residue was recovered in the large protein fragment of modified M-CK subunits. Proteolytically modified M-CK and B-CK subunits were able to refold and reassociate into dimeric structures after treatment with high concentrations of LiCl and at low pH. Thus the proteolytically modified CK subunits retain their ability to refold and to establish precise subunit-subunit recognition in vitro. PMID- 3006664 TI - Activation of V1-receptors by vasopressin stimulates inositol phospholipid hydrolysis and arachidonate metabolism in human platelets. AB - The activation of platelet V1-receptors by vasopressin (0.01-1 microM) induces the rapid formation of inositol phosphates, 1,2-diacylglycerol and phosphatidic acid, indicating inositol phospholipid hydrolysis by phospholipase C. Vasopressin immediately induces the formation of inositol bisphosphate and inositol trisphosphate. Accumulation of inositol 1-monophosphate and inositol 4 monophosphate occurs later after a time lag of 15 s. Low concentrations (10-100 nM) of vasopressin only activate phospholipase C, whereas high concentrations (1 microM) induce activation of phospholipase C and subsequently the production of arachidonate metabolites. Cyclo-oxygenase metabolites are associated with further activation of phospholipase C, release reaction and irreversible platelet aggregation. Vasopressin requires for its action extracellular Mg2+, but not Ca2+. The described platelet changes are not induced by 1-desamino-[8-D arginine]vasopressin, a V2-receptor agonist, and are blocked by a specific V1 receptor antagonist. The results indicate that platelets possess a V1-receptor that is coupled to polyphosphoinositide hydrolysis by phospholipase C, leading to the formation of 1,2-diacylglycerol and inositol trisphosphate. Those compounds may act as second messengers for platelet responses induced by vasopressin, whereas endoperoxides and thromboxane A2 stimulated by vasopressin may serve as amplifiers for platelet activation. PMID- 3006666 TI - The amino acid sequence of cytochrome c-555 from the methane-oxidizing bacterium Methylococcus capsulatus. AB - The amino acid sequence of the cytochrome c-555 from the obligate methanotroph Methylococcus capsulatus strain Bath (N.C.I.B. 11132) was determined. It is a single polypeptide chain of 96 residues, binding a haem group through the cysteine residues at positions 19 and 22, and the only methionine residue is a position 59. The sequence does not closely resemble that of any other cytochrome c that has yet been characterized. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50131 (12 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment. PMID- 3006667 TI - Evidence for the rapid internalization and recycling of lutropin receptors in rat testis Leydig cells. AB - A study into the binding of 125I-human chorionic gonadotropin (hCG) to the lutropin (LH) receptor in rat testis Leydig cells, and subsequent internalization of the hormone-receptor complex, has been carried out. The results show that there is rapid internalization of the hormone-receptor complex; 240 receptors/cell (from a total of approx. 4000 receptors/cell) were internalized each minute in the first hour after exposure to hCG. Radioactivity was released from the cell 1 h after internalization and was found to be associated with highly degraded hCG. The endocytic process was found to have two temperature sensitive steps. At 4 degrees C, movement of the hormone-receptor complex inside the cell did not occur, and at 21 degrees C hormone accumulated within the cytoplasm but was not degraded or released from the cell. At 34 degrees C, internalization, degradation and loss of the degraded hormone from the cell occurred. These processes appeared to reach a steady state after 2 h. Even though there is rapid internalization of the hormone-receptor complex following exposure to hCG, the binding sites on the cell surface were maintained for at least 4 h. The number of binding sites on the cell surface was not decreased by a protein synthesis inhibitor but was reduced to undetectable levels by monensin. This compound inhibits acidification of endocytic vesicles, which is known to be an important prerequisite to receptor cycling. It is concluded that, in the rat testis Leydig cells, following binding of hCG to the LH receptor there is rapid internalization of the complex and that recycling of the receptor occurs to the cell surface. This process may be essential in maintaining the capacity of the Leydig cell to bind fresh hormone. PMID- 3006668 TI - Sequence and interspecies transfer of an aminoglycoside phosphotransferase gene (APH) of Bacillus circulans. Self-defence mechanism in antibiotic-producing organisms. AB - The APH gene of a butirosin-producing Bacillus circulans was cloned and shown to be expressed in Escherichia coli and Streptomyces lividans. The gene was sequenced and a possible developmentally regulated promoter identified. When the deduced protein sequence was compared with those from transposon Tn5, transposon Tn903, Streptomyces fradiae, Staphylococcus aureus and Streptococcus faecalis, significant homology was found, indicating that the genes may have a common origin. PMID- 3006665 TI - Extracellular ATP: effects, sources and fate. PMID- 3006669 TI - Inhibition of the apparent affinity of the epidermal growth factor receptor caused by phorbol diesters correlates with phosphorylation of threonine-654 but not other sites on the receptor. AB - Addition of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) to A431 human epidermoid carcinoma cells causes a marked increase in the phosphorylation state of the epidermal growth factor (EGF) receptor with a concomitant inhibition of both the high-affinity binding of 125I-EGF and the receptor tyrosine kinase activity. It was found in the present studies that the diuretic drug amiloride has no effect on the action of PMA to inhibit the binding of 125I-EGF. However, amiloride was observed to inhibit markedly the effect of PMA to cause a 3-fold increase in the phosphorylation state of the EGF receptors. In the presence of PMA and amiloride, the increase in the phosphorylation state of the EGF receptors was found to be only 1.2-fold over controls. Analysis of the EGF receptor phosphorylation sites by phosphopeptide mapping by reverse-phase h.p.l.c. demonstrated that PMA increases the phosphorylation state of the EGF receptor at many sites. One of these sites has been identified as a C-kinase substrate, threonine-654. In the presence of amiloride, PMA causes phosphorylation of threonine-654 to the same stoichiometry as that observed in the absence of amiloride. However, the marked increase in the phosphorylation state of the EGF receptor at other sites caused by PMA is abolished in the presence of amiloride. We conclude that the extensive phosphorylation of the EGF receptor at several sites caused by the addition of PMA to A431 cells is not required for the action of PMA to inhibit the high-affinity binding of 125I-EGF. The results indicate that the phosphorylation state of threonine-654 may play a role in this process. PMID- 3006670 TI - Inactivation of 3 alpha-hydroxysteroid dehydrogenase by superoxide radicals. Modification of histidine and cysteine residues causes the conformational change. AB - 3 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1.50) from Pseudomonas testosterone was inactivated by superoxide radicals generated by the aerobic xanthine oxidase reaction. Superoxide dismutase, NAD+, bovine serum albumin and histidine and cysteine as free amino acids partially protected the enzyme from inactivation. NADH-binding properties were determined by fluorescence spectroscopy, and no variation was found between native enzyme and the unmodified fraction of the partly inactivated one. The fluorescence emission maximum for the completely inactivated enzyme was shifted 10 nm to a longer wavelength when compared with the native one, and it seems possible that the modification of histidine and cysteine residues by superoxide radicals causes the conformational change of the enzyme and the consequent loss of catalytic activity. PMID- 3006672 TI - Characteristics of complement subcomponents C1r and C1s synthesized by Hep G2 cells. AB - The association and activation states of complement subcomponents C1r and C1s biosynthesized by Hep G2 cells were studied. C1r and C1s are secreted in stoichiometric amounts; in the presence of Ca2+ they are associated in a complex that sediments similarly to plasma C1r2-C1s2. Both compounds are synthesized as monomer proteins of apparent Mr 86 000. C1r is secreted as a dimer. Secreted C1r is not autoactivatable but undergoes proteolysis by exogenous C1r; secreted C1s is also proteolysed by exogenous C1r. In the presence of immune-complex-bound C1q, secreted C1r and C1s are able to reconstitute C1, but normal activation requires extrinsic C1r2-C1s2. PMID- 3006671 TI - Interrelationships in rats of tissue pools of cholecalciferol and 25 hydroxycholecalciferol formed in u.v. light. AB - Vitamin D-deficient rats were irradiated with u.v. light three times weekly for 30 min for several weeks. D3 (cholecalciferol) and 25(OH)D3 (25 hydroxycholecalciferol) concentrations in skin, plasma, muscle and adipose tissue were measured. In other experiments, isolated skin or the whole animal was irradiated once and the cholecalciferol response monitored. Only a small fraction of the 7-dehydrocholesterol in skin is converted into D3 (less than 2%), and the presence of fur decreases the proportion converted into 20% of that occurring in shaved rat skin. D3 formed in the skin disappears relatively slowly, so that about 90% has gone after 7 days. In normal rats 10 micrograms of D3 formed over 2 h irradiation only caused a small rise in plasma D3 concentration over the following week, indicative of a high rate of clearance from this tissue. Irradiation of vitamin D-deficient rats for a prolonged period raised plasma D3 and 25(OH)D3 concentrations to a constant value. D3, but not 25(OH)D3, could be found in adipose tissue and muscle. Prolonged irradiation of normal rats showed these tissues and plasma could hold very large amounts of D3. Pharmacokinetic analysis of the changes in D3 concentration in rats showed that the disposition kinetics of D3 was explained by a two-compartment model with half-lives of 13.8 and 7.7 days. The volume of distribution of the more-slowly-turning-over compartment was 500 ml, which presumably reflects the large amounts of D3 that can accumulate in adipose tissue. Rat skin can synthesize about 0.85 ng of D3/mJ of u.v. light energy, but it seems that not all this is available to the rat. Adipose-tissue D3 is available for use by the rat, the t1/2 being 12.0 days. PMID- 3006674 TI - Accumulation of hepatic collagen following long-term administration of sake to rats. AB - Sake, a rice wine, induced hepatic collagen accumulation in rats. This was noted after 16 weeks of a liquid diet containing 35% ethanol by caloric content. However, under similar experimental conditions, whiskey and ethanol did not produce any changes. Hepatic collagenase activities in sake-fed rats were slightly, but significantly, higher than in the whiskey group. The central and pericellular liver fibrosis in sake-fed rats was caused possibly by either accelerated collagen synthesis or maturation, exceeding the increased collagen degradation. Mechanisms of enhanced fibrogenesis in sake-fed rats were discussed. PMID- 3006673 TI - Biochemical effects of mild iron deficiency and cold acclimatization on rat skeletal muscle. AB - Most of the previous studies on the effects of iron deficiency on skeletal muscle respiratory capacity and work performance have been investigated in severe or moderate iron-deficiency anemia. We report here that even in mild iron deficiency where the hemoglobin concentration was 10 g/dl and the iron stores in livers and spleen were not completely depleted, a marked reduction in succinate dehydrogenase was observed in skeletal muscles but not in heart. Similarly, cytochrome oxidase activities were reduced. Although no significant change in glycerophosphate dehydrogenase was detected in the iron-deficient rats, exposure to cold in this group greatly reduced this enzyme activity. As cold acclimatization accelerates marrow erythropoiesis (20) which in turn, demands more iron, it seems that in the iron-insufficient state, this iron demand for marrow activity may persist at the expense of the tissue iron pool, resulting in a marked reduction in glycerophosphate dehydrogenase activities. Since succinate dehydrogenase plays a significant role in the impairment of mitochondrial function and early fatigue of iron-deficient muscle (11), the present study shows that even in mild iron deficiency, some loss of muscle functions could result as succinate dehydrogenase activities were greatly reduced. PMID- 3006676 TI - Anticarcinogenic effect of retinoids on 7,12-dimethylbenz(a)anthracene-induced mammary tumor induction, and its relationship to cyclic AMP-dependent protein kinase. AB - Administration of 13-cis retinoic acid and N-(4-hydroxyphenyl) retinamide daily in the diet to female Sprague-Dawley rats beginning one day after intubation with 7,12-dimethylbenz(a)anthracene (DMBA) prolonged the latency periods and inhibited the percentage incidence of mammary tumors. A significant reduction in the total number of tumors was also evident. The inhibition of mammary tumor growth by retinoids was associated with a significant increase (3-fold) in cytosolic cAMP binding and histone kinase activities. The increase of histone kinase activity was almost totally in the cAMP-dependent protein kinase Type II. Retinoic acid increased the amount of the regulatory subunit (R11) rather than altering its cAMP binding affinity. These results suggest that cAMP-dependent protein kinase Type II may be involved in mediating the retinoid action in the inhibition of mammary tumor growth in vivo. PMID- 3006675 TI - Endogenous "ouabain-like" activity in bovine plasma. AB - An endogenous inhibitor of the sodium pump has been detected and concentrated 1000-fold from bovine plasma. The steps of purification included deproteinization and extraction with methanol, removal of lipids by coextractions with a lipophilic solvent, desalting and further concentration by adsorption on C18 SepPack cartridges and HPLC fractionation on a weak anionic exchange column. The material isolated displaces 3H-ouabain from brain synaptosomes, inhibits red cell membrane Na,K-ATPase without inhibiting Mg-ATPase or Ca,Mg-ATPase. Deproteinization of plasma by boiling may lead to appearance of non-specific inhibitors. The procedures developed should now permit isolation of sufficient amount of material for further purification and structural characterization. PMID- 3006677 TI - Specific, covalent binding of an azidoretinoid to cellular retinoic acid-binding protein. AB - Two C(5)-azido substituted aromatic retinoids were evaluated as photoaffinity probes for studying the mechanism of retinoid action. The secondary azide 1 and the tertiary azide 2 were equipotent with the parent C(5)-geminal-dimethyl substituted aromatic retinoid 3 in stimulating F9-cell differentiation. Both azides bound covalently to cellular retinoic acid-binding protein upon photolysis, but the secondary azide was twice as efficient, likely because of lesser steric hindrance. The covalent binding of azide 1 was specific, since it was inhibited by retinoic acid. Thus substitution of a photolabile group onto aromatic retinoids does not abolish biological activity and affinity for cellular retinoic acid-binding protein. PMID- 3006678 TI - Redistribution of protein kinase C during mitogenesis of human B lymphocytes. AB - G0 human tonsillar B-lymphocytes were stimulated to divide by the polyclonal mitogen Staphylococcus Aureus Cowan strain 1 (SAC) and by the combined use of 12 O-tetradecanoyl phorbol-13-acetate (TPA) and the calcium ionophore ionomycin. The activities of protein kinase C, which requires Ca++ and phospholipid as co factors, and a proteolytically cleaved form of this enzyme (protein kinase M), which is independent of calcium and phospholipid control, were determined in soluble and particulate fractions obtained from activated B cells. Treatment of G0 B cells with SAC or TPA together with ionomycin caused redistribution of protein kinase C from the soluble to the particulate fraction where the 80,000 Dalton protein kinase C was cleaved to give rise to a 50,000-Dalton form of the kinase which was also found in the cytoplasm. These data suggest that redistribution and proteolytic cleavage of protein kinase C are key signal transduction events in B cell mitogenesis. PMID- 3006679 TI - Characterization of the guinea pig adipocyte thyrotropin receptor. AB - 125I-TSH binding to porcine thyroid and guinea pig fat resulted in curvilinear Scatchard plots with similar dissociation constants for the high and low affinity binding components. Antibodies from the sera of patients with Graves' disease inhibited binding to the high and low affinity binding components of both tissues. Covalent cross-linking of 125I-TSH to membranes from each tissue resulted in the specific labeling of two protein bands. The guinea pig fat receptor subunits have Mr values of 52,000 and 38,000, whereas the porcine thyroid receptor subunits have values of 46,000 & 35,000. The labeling of the receptor subunits was inhibited by preincubation with Graves' autoantibodies. Despite possessing a different subunit composition, the receptors from these tissues exhibit similar affinity for TSH and share similar antigenic determinants for Graves' autoantibodies. PMID- 3006680 TI - Peroxidative free radical formation and O-demethylation of etoposide(VP-16) and teniposide(VM-26). AB - The peroxidative activation of the antitumor drugs, etoposide (VP-16) and teniposide (VM-26), has been studied in vitro. Both of these drugs, in the presence of horseradish peroxidase or prostaglandin synthetase, formed phenoxy radical intermediates. Furthermore, this activation also resulted in the formation of two metabolites from each of the drugs. Using HPLC and mass spectrometry, one of the metabolites was shown to be the reactive o-quinone derivative of the parent drug which resulted from the peroxidative O demethylation. It appears that O-demethylation catalyzed by peroxidases may be an important mechanism for the formation of reactive intermediates and may play a role in the mechanism of action of VP-16 and VM-26. PMID- 3006681 TI - The allergenicity of complex cations. AB - A homologous series of eight quaternary ammonium salts (quats) were used as complex cations in a survey of contact hypersensitivity in guinea pigs. Two of the quats tested were found to be strong allergens which was due to stable association with membrane lipids at the surface of epidermal cells. This surface complexation reaction was studied in detail by using a spin-labelled quat of intermediate allergenicity. Electron spin resonance was used to show that stable "ion pairs" are formed between membrane receptor sites and the two strong allergens. Information was obtained on the specificity and kinetics of immunogenic complex formation as well as on the position and orientation of these haptens on epidermal receptor sites in vivo. PMID- 3006682 TI - Receptors for insulin-like growth factors and insulin on murine fetal cortical brain cells. AB - Fetal murine neuronal cells bear somatomedin receptors which can be classified according to their affinities for IGF-I, IGF-II and insulin. Binding of 125I-IGF I is half-maximally displaced by 7 ng/ml IGF-I while 15- and 700-fold higher concentrations are required for, respectively, IGF-II and insulin. Linear Scatchard plots of competitive-binding data with IGF-I suggest one single class of type I IGF receptors (Ka = 2.6 X 10(9) M-1; Ro = 4500 sites per cell). The occurrence of IGF-II receptors appears from the specific binding of 125I-IGF-II and competition by unlabeled IGF-II; the IGF-II binding sites display a low affinity for IGF-II and no affinity for insulin. IGF-II also interacts with insulin receptors although 50- to 100-fold less potent than insulin in competing for 125I-insulin binding. The presence of distinct receptors for IGF-I, IGF-II and insulin on fetal neuronal cells is consistent with a role of these peptides in neuronal development, although our data also indicate that IGF-I receptors could mediate the growth promoting effects of insulin. PMID- 3006683 TI - Oncogene expression in human hepatoma cells PLC/PRF/5. AB - The expression of 7 cellular oncogenes in a human hepatoma cell line PLC/PRF/5 was studied using Northern blot analyses. Among the oncogenes tested, c-abl, c fes, c-fms, c-myc, c-Ha-ras and c-sis were expressed. The oncogene c-Ki-ras was not expressed. The length of the mRNAs expressed was almost consistent with published data. Compared to the oncogene expression in Daudi lymphoma cells, the same kind of oncogenes were expressed in PLC/PRF/5 cells, but the intensity of the signal in each oncogene expression was stronger in Daudi cells than in PLC/PRF/5 cells. Considering the cellular localization and the function of each oncogene, the oncogene survey in hepatoma cells broadens the knowledge of hepatocarcinogenesis and the character of human hepatoma cells. PMID- 3006684 TI - Phorbol esters enhance basal and stimulated adenylate cyclase activity in a pituitary cell line. AB - We reported in anterior pituitary cells that hormone stimulation of cyclic AMP levels is amplified by agents that activate protein kinase C (e.g., phorbol esters). We utilized the 235-1 pituitary cell line to explore the mechanism of this response. PGE1- and forskolin-stimulated cyclic AMP accumulation and adenylate cyclase activity are enhanced by exposing viable cells to phorbol esters. Adenylate cyclase activity in the presence of PGE1 demonstrated a biphasic stimulatory, then inhibitory response to increasing GTP concentrations; phorbol esters attenuated this inhibition. These data support the hypothesis that protein kinase C can covalently change the functional state of the adenylate cyclase holoenzyme, amplifying its response to certain hormones. PMID- 3006685 TI - Circulating factor with ouabain-like immunoreactivity in patients with primary aldosteronism. AB - Circulating factor with ouabain-like immunoreactivity was studied in patients with primary aldosteronism. Anti-ouabain antibody was prepared from specific pathogen-free rabbits. In the plasma of patients with primary aldosteronism, the level of a factor with ouabain-like immunoreactivity was 2.59 +/- 1.39 pmol ouabain equivalent/ml plasma. This value was significantly (p less than 0.05) higher than that of age-matched normotensive subjects, 1.06 +/- 0.86 pmol ouabain equivalent/ml plasma. The plasma level of ouabain-like immunoreactivity correlated significantly (p less than 0.05) with blood pressure. These results indicate that the factor with ouabain-like immunoreactivity may play a pathophysiological role in the maintenance of the high blood pressure observed in patients with primary aldosteronism. PMID- 3006686 TI - PDGF modifies phosphoinositide metabolism and inhibits aggregation and release in human platelets. AB - While platelet derived growth factor (PDGF) did not induce any platelet aggregation nor secretion, it modified the polyphosphoinositide metabolism of human platelets prelabeled with 32P-orthophosphate. We found a decrease of 32P associated with phosphatidylinositol 4,5 bisphosphate after 3 min, with parallel increase of 32P-phosphatidylinositol 4 phosphate and 32P-phosphatidylinositol using 100 ng/ml of PDGF. This modification was PDGF concentration dependent. PDGF inhibited thrombin and collagen induced platelet aggregation and 14C-serotonin release in a dose dependent manner, but was without effect when arachidonic acid was used. These results suggest that PDGF (i) stimulated the hydrolysis of polyphosphoinositides (ii) and could exert a negative feedback control on platelet activation induced by thrombin or collagen. PMID- 3006687 TI - Interaction of spermine with polyphosphoinositides containing liposomes and myo inositol 1,4,5 triphosphate. AB - The interaction of spermine with liposomes containing 2% phosphatidylinositol, phosphatidylinositol 4 phosphate and phosphatidylinositol 4,5 biphosphate was inferred from the ability of these liposomes to interfere with spermine binding to the resin heparin-Sepharose. The inositol phospholipids tested showed different affinities for spermine: the order of binding strength appear to be phosphatidylinositol phosphatidylinositol 4 phosphate phosphatidylinositol 4,5 biphosphate. The ability of vesicles containing 2% polyphosphoinositides to interact with spermine is comparable to that of either single stranded RNAs or highly negatively charged liposomes. Myo-inositol 1,4,5 triphosphate has a much lower ability to bind spermine. PMID- 3006688 TI - Intracellular free calcium transients induced by norepinephrine in rat aortic smooth muscle cells in primary culture. AB - In cultured rat arterial smooth muscle cells treated with quin 2, cytosolic Ca2+ transients induced by norepinephrine were recorded microfluorometrically. In the presence or absence of extracellular Ca2+, norepinephrine induced transient and dose-dependent elevations in cytosolic Ca2+, with a similar time course, the peak levels being observed at 2 min. These transient elevations in cytosolic Ca2+ were dose-dependently inhibited by alpha-adrenergic antagonists, the order of potency being prazosin greater than phentolamine greater than yohimbine, irrespective of the presence of extracellular Ca2+. We propose that with or without extracellular Ca2+, norepinephrine activates mainly alpha-1 adrenoceptors leading to a release of Ca2+ from intracellular stores. This would explain the transient elevation in cytosolic Ca2+ in rat aortic vascular smooth muscle cells in primary culture. PMID- 3006689 TI - Role of calcium ion in hormone-stimulated lipolysis. AB - Using the flask-incubated fat cell system, the effects of Ca2+ removal from the incubation medium on the lipolytic system were studied. The removal of Ca2+ resulted in a total abolition of the lipolytic response and the increased cyclic AMP accumulation produced by ACTH. The lipolytic response to isoproterenol and forskolin were reduced approximately 40% by Ca2+ removal, but cyclic AMP accumulation was not altered in the presence of either of these agents using a Ca2+-free medium. The lipolytic response to the dibutyryl analog of cyclic AMP was also reduced by omission of Ca2+ from the incubation medium. It is concluded the Ca2+ is required for the interaction of ACTH with its receptor and the resultant activation of adenylate cyclase. Ca2+ also is required at some step in the lipolytic process distal to cyclic AMP. PMID- 3006690 TI - Eicosapentaenoic acid as a modulator of inflammation. Effect on prostaglandin and leukotriene synthesis. AB - Products derived from arachidonic acid (AA) via both the cyclo-oxygenase and lipoxygenase pathways play a role in inflammation: prostaglandins (PGs), particularly PGE2, contribute to the formation of oedema, erythema and hyperalgesia whereas leukotriene B4 (LTB4), a product of the 5' lipoxygenase, may modulate the recruitment of leukocytes. We have previously reported that supplementation of a standard rat diet with eicosapentaenoic acid (EPA) caused a significant increase in the formation of LTB5, which is less active biologically than LTB4, and a decrease in the synthesis of LTB4 by stimulated leukocytes. Now we have assessed the effects of administration of highly purified EPA ethyl ester (79% pure), in two models of acute inflammation. Supplementation of a standard rat diet with 240 mg/kg/day EPA for 4 weeks significantly decreased the concentration of PGE2 and TXB2 in inflammatory exudate derived from implantation of carrageenin impregnated sponges: neither the concentration of LTB4 nor the cell number were reduced significantly. Triene prostaglandins were not detected in the exudate, however, significant levels of LTB5 were present. In the second model, oedema induced by injection of carrageenin into rat paws was significantly reduced in animals fed an EPA-rich diet. Supplementation of the diet with EPA could, by mainly reducing the synthesis of prostaglandins, offer a novel and non toxic approach to the modulation of an inflammatory response. PMID- 3006691 TI - Multiple molecular forms of cyclic nucleotide phosphodiesterase in cardiac and smooth muscle and in platelets. Isolation, characterization, and effects of various reference phosphodiesterase inhibitors and cardiotonic agents. AB - Multiple molecular forms of cyclic nucleotide phosphodiesterase have been identified previously in several tissues and cell types using a variety of different isolation methods. In the present study, the different molecular forms of phosphodiesterase (PDE) were isolated from cardiac muscle (guinea pig left ventricle), vascular smooth muscle (bovine coronary arteries) and human platelets using the same isolation procedure in each instance. These enzymes were then characterized kinetically, and the effects of various reference PDE inhibitors and cardiotonic agents on each form were examined. A low Km, low Vmax form of phosphodiesterase (PDE I) was found in all three tissue/cell types. PDE I activity was stimulated by calmodulin in cardiac and smooth muscle, but not in platelets. In smooth muscle and platelets, PDE I preferentially hydrolyzed cyclic GMP, whereas cardiac muscle PDE I hydrolyzed cyclic AMP and cyclic GMP equally. A high Km, high Vmax form of phosphodiesterase (PDE II) was found in cardiac muscle and platelets, but not in smooth muscle. PDE II activity was not stimulated by calmodulin and there was no substrate specificity. A low Km, low Vmax cyclic AMP specific form of phosphodiesterase (PDE III) was found in all three tissue/cell types. The activity of PDE III was not stimulated by calmodulin. The reference inhibitors theophylline and papaverine exerted non-specific inhibitory effects on all forms of phosphodiesterase. Other reference inhibitors (M & B 22,948 and dipyridamole) and several cardiotonic agents (AR-L 57, CI-914, CI-930, amrinone, and MDL 17,043) exerted selective inhibitory effects on only one molecular form of phosphodiesterase. The degree of selectivity was often dependent upon the tissue or cell from which the molecular form of phosphodiesterase was isolated. These studies demonstrate that there is heterogeneity regarding the number of phosphodiesterases present in various tissue/cell types, as well as their substrate specificity and their ability to be stimulated by calmodulin, and these different molecular forms of phosphodiesterase can be selectively inhibited by different pharmacological agents. The possibility exists that such selective inhibitors may produce discrete changes in cyclic AMP or cyclic GMP levels, and that these changes may be produced in specific tissues and/or cells. PMID- 3006692 TI - Potentiation of prostaglandin E1-stimulated cAMP formation by 12-O tetradecanoylphorbol-13-acetate in BALB/c mouse 3T3 cells. AB - Prostaglandin E1 (PGE1: 0.1-100 microM), forskolin (0.1-100 microM), and cholera toxin (20 ng/ml) stimulated cAMP formation of BALB/c 3T3 cells. The pretreatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced PGE1 (10 microM)-stimulated cAMP formation in a concentration- and a time-dependent manner. If the cells were pretreated with TPA (0.1 microM) for only 1 hr, the above augmentation was not observed. Maximal enhancement was observed by pretreatment of the cells for 5 hr with 0.1 microM TPA. Basal cAMP formation was not affected by TPA pretreatment. Other tumor promoters, such as teleocidin and mezerein, showed a potentiating effect similar to that of TPA on the PGE1 stimulated cAMP formation. However, phorbol which is not a tumor promoter, failed to potentiate PGE1 action significantly. These results suggest that the above TPA action may share some common mechanisms with the tumor-promoting action of this agent. On the other hand, the forskolin- and cholera toxin-stimulated cAMP formations were not changed by pretreatment of the cells with TPA. Therefore, our results indicate that the potentiating action of TPA on PGE1-stimulated cAMP formation in 3T3 cells is not due to the activation of the catalytic unit or the stimulatory guanine nucleotide binding protein (Ns) of adenylate cyclase (AC) system by this agent. It is highly likely that TPA induces some alterations on PGE1 receptors or on PGE1 receptor-Ns coupling systems and consequently induces an augmentation of PGE1-stimulated cellular cAMP response. PMID- 3006693 TI - A new class of adenosine receptors in brain. Characterization by 2 chloro[3H]adenosine binding. AB - Micromolar concentrations of adenosine and its analogs have profound depressant effects on neuronal firing and synaptic transmission in many brain areas. Using the adenosine agonist 2-chloro[3H]adenosine (Cl[3H]Ado), we have identified a distinct class of micromolar-affinity adenosine binding sites in rat forebrain membranes. Specific Cl[3H]Ado binding was reversible and saturable with an apparent KD of 9.1 microM and a Bmax of 61 pmoles/mg protein. The present studies were conducted using washed brain membrane fractions not treated with adenosine deaminase. Specific Cl[3H]Ado binding under these conditions was insensitive to ( )-N6-(R-phenylisopropyl)adenosine ((-)PIA) and treatment with 3 mM N ethylmaleimide, unlike high-affinity A1 adenosine receptor binding. Treatment of membranes with adenosine deaminase revealed an additional population of binding sites sensitive to (-)PIA. Inhibition of Cl[3H]Ado binding by adenosine analogs exhibited an order of potency ClAdo greater than 5'-N-ethylcarboxamide adenosine (NECA) greater than (-)PIA which differs from that of both A1 and A2 adenosine receptors. The potent A1 and A2 receptor antagonist 8-phenyltheophylline had no significant effect on binding up to 10 microM. Specific binding, however, was inhibited by the adenosine antagonists 8(p-sulfophenyl)theophylline, isobutylmethylxanthine, theophylline, and caffeine. Micromolar Cl[3H]Ado binding was highly selective for adenosine agonists and antagonists. These results suggest that the micromolar-affinity Cl[3H]Ado binding sites may represent a novel central purinergic receptor, distinct from the A1 and A2 adenosine receptors involved in the regulation of adenylate cyclase. PMID- 3006694 TI - The inhibition of rat adipocyte ecto-5'-nucleotidase by xanthines is not related to lipolysis. PMID- 3006696 TI - Inhibition of rat neutrophil functional responses by azapropazone, an anti-gout drug. AB - Azapropazone at concentrations of 0.1 to 1 mM inhibited by 30-70% rat neutrophil migration, aggregation, and degranulation in response to the synthetic chemotactic peptide fMet-Leu-Phe. Binding studies using fNle-Leu-[3H]Phe, a radiolabeled analog of fMet-Leu-Phe, showed that azapropazone did not inhibit these responses by interfering with fMet-Leu-Phe binding. Azapropazone also decreased both the apparent rate of production and maximal levels of superoxide anion (O2-) generated by cells stimulated with 100 ng/ml phorbol-12-myristate-13 acetate (PMA). The concentrations of azapropazone that inhibit these neutrophil responses in vitro approximate those previously found in vivo after administration of therapeutic doses of drug to rats or humans. Taken together, the data suggest that the efficacy of azapropazone in gouty arthritis may be partly due to its ability to inhibit key neutrophil functional responses in vivo. PMID- 3006695 TI - Ketoconazole inhibits the biosynthesis of leukotrienes in vitro and in vivo. AB - Ketoconazole inhibits in vitro (IC50:2.6 X 10(-5) M) the formation of 5-HETE and LTB4 by isolated, carrageenin-elicited rat peritoneal PMN leukocytes, challenged with the Ca2+-ionophore A23187 in the presence of [14C]-arachidonic acid ([14C] AA). The relative potency of various compounds tested in this respect is NDGA greater than nafazatrom greater than phenidone greater than ketoconazole greater than BW 755C. In contrast to the other compounds studies, ketoconazole in vitro, up to 1 X 10(-4) M, has no effect on the fatty acid cyclo-oxygenase or the 12 lipoxygenase-mediated metabolism of [14C]-AA by isolated human platelets; however, it stimulates the 15-lipoxygenase activity in phenylhydrazine-induced rabbit reticulocytes. After oral administration (10-40 mg/kg, -2 hr), ketoconazole inhibits in a dose-dependent way, the leukotriene-mediated anaphylactic bronchoconstriction in guinea pigs. This study demonstrates that ketoconazole is a comparatively specific and orally active inhibitor of the 5 lipoxygenase activity bearing on the production of leukotrienes derived from arachidonic acid. PMID- 3006697 TI - Renal mitochondrial integrity during continuous gentamicin treatment. AB - Rats were given gentamicin over a period of 21 days. At 5, 10, 14 and 21 days renal cortical mitochondria were isolated, and respiratory and Ca2+ transport functions and cytochrome concentrations were determined. The mitochondrial data were correlated with indicators of deteriorating renal function and tissue gentamicin accumulation. During the first 10 days of chronic gentamicin treatment, mitochondrial cytochrome oxidase and cytochrome c concentrations declined significantly. This decline was followed by a partial spontaneous recovery by days 14 and 21. Cytochrome b concentration was not significantly different from normal. Parallel with the cytochrome concentration changes, State 3 respiratory activities with all substrates studied and the rates of Ca2+ accumulation declined during the first 10 days and recovered spontaneously thereafter. It is concluded that chronic gentamicin treatment leading to renal failure inhibits mitochondrial energy-linked functions, which inhibition is induced by rate-limiting synthesis of those mitochondrial respiratory chain enzymes coded outside the mitochondrion. PMID- 3006698 TI - Purine nucleotides inhibit the binding of DL-[3H] 2-amino-4-phosphonobutyrate (DL [3H] APB) to L-glutamate-sensitive sites on rat brain membranes. AB - The effects of purine nucleotides on the binding of DL-[3H] 2-amino-4 phosphonobutyrate (DL-[3H] APB) to rat brain membranes were investigated. Certain guanine nucleotides, especially cyclic GMP and GTP, were found to be potent inhibitors of binding. Kinetic studies revealed that both cyclic GMP and GTP acted to decrease receptor affinity without affecting significantly binding site density. These endogenous substances may therefore play an important role in the regulation of excitatory amino acid receptor function. PMID- 3006699 TI - [Phosphorylation as a method of regulating inorganic pyrophosphatase activity in E. coli. I. Phosphorylation and enzyme activation induced by ATP]. AB - ATP phosphorylates the regulatory center of E. coli inorganic pyrophosphatase with the resultant 1,5-fold increase in the activity of the enzyme. The maximal incorporation of the ATP gamma-group into pyrophosphatase is 3 moles per mole of the protein. Pi likewise phosphorylates the enzyme regulatory center and lowers the pyrophosphatase activity by 10-15%. The ATP- and Pi-mediated phosphorylation processes are interrelated; ATP prevents phosphorylation by Pi and brings about rapid dephosphorylation of Pi-modified protein. PMID- 3006700 TI - [Phosphorylation as a method of regulating inorganic pyrophosphatase activity in E. coli. II. Identification of the types of chemical bonds between phosphate and the enzyme]. AB - A bond formed by phosphate with Pi-phosphorylated pyrophosphatase from E. coli was found to be labile in acidic and alkaline media and to be rapidly cleaved by hydroxylamine at neutral pH. N-Methylhydroxylamine modifies also activated carboxyl groups of the enzyme. Interaction of inorganic pyrophosphatase with ATP produces an alkali-resistant phosphoamide bond. A phosphorylated amino acid, identified as phosphohistidine, was isolated from the alkaline hydrolyzate of the ATP-phosphorylated pyrophosphatase. PMID- 3006701 TI - Treatment of rheumatoid synovitis of the knee with intraarticular injection of dysprosium 165-ferric hydroxide macroaggregates. AB - One hundred eight knees of 93 patients with seropositive rheumatoid arthritis and persistent synovitis of the knee were treated with an intraarticular injection of 270 mCi of dysprosium 165 bound to ferric hydroxide macroaggregate. Leakage of radioactivity from the injected joint was minimal. Mean leakage to the venous blood 3 hours after injection was 0.11% of the injected dose; this corresponds to a mean whole body dose of 0.2 rads. Mean leakage to the liver 24 hours after injection was 0.64% of the injected dose; this corresponds to a mean liver dose of 3.2 rads. In 7 additional patients examined, there was negligible or near negligible activity found in the draining inguinal lymph nodes. One-year followup was possible for 74 knees (63 patients). Sixty-one percent of the knees had good results, 23% had fair results, and 16% had poor results. There was a direct correlation between the radiographic stage and response to treatment. In knees with stage I radiographic changes, 72% showed good results; 93% showed improvement. In knees with stage II changes, 59% showed good results; 81% showed improvement. These preliminary results indicate that dysprosium 165-ferric hydroxide macroaggregate is an effective agent for radiation synovectomy. The low leakage rates observed offer a definite advantage over agents previously used. PMID- 3006703 TI - Light and electron microscopic findings in POEMS, or Japanese multisystem syndrome. PMID- 3006702 TI - Type C retroviruses of the human T cell leukemia family are not evident in patients with systemic lupus erythematosus. AB - Type C retroviruses of the human T cell leukemia virus (HTLV) family have been implicated in immune aberrations observed in patients with leukemias, lymphomas, and the acquired immune deficiency syndrome. We have investigated whether retroviruses of the HTLV family are involved in the etiopathogenesis of systemic lupus erythematosus (SLE) by using 1) an immunoenzymatic assay to measure antibodies to HTLV-I and HTLV-III proteins in the sera of 30 patients with SLE, and 2) nucleic acid hybridization techniques (Southern transfer) to detect proviral sequences of HTLV-I, HTLV-II, and HTLV-III in DNA extracted from peripheral blood mononuclear cells of 10 of these patients. Sera from 20 normal individuals served as controls. None of the 30 sera from SLE patients or the 20 sera from normal controls had any detectable antibodies to HTLV-I or HTLV-III. Nucleic acid hybridization studies also failed to detect any HTLV proviral sequences. These results suggest that viruses of the HTLV family do not participate in the etiopathogenesis of SLE. PMID- 3006704 TI - [Behavior of the second messenger cAMP in status asthmaticus before and following administration of theophylline-ethylenediamine]. AB - The present investigation provides evidence that the second messenger cyclic adenosine monophosphate (cAMP) shows a different reaction before and after treatment of asthmatic attack. 18 patients suffering from an asthmatic attack admitted to hospital as internal emergency cases were given intravenously 0.24 g theophylline-ethylenediamine (Euphyllin) and 10 mg benzoctamine. Blood was taken and cAMP-concentrations were tested during status asthmaticus, i.e. before theophylline was injected, and after symptoms had decreased. After injection of theophylline a relation could be shown between the clinical effect and a significant biochemical correlate. The cAMP-concentrations increased from 15.5 to 20.2 pmol/ml. The results are significant on the 1% level. It should be discussed whether there is a malfunction or a lack of receptors in asthmatic disease. The cAMP-concentrations found during status asthmaticus are low compared to those found during paroxysmal tachycardia (40 pmol/ml) and are slightly higher than those found in healthy persons. There was no depressant effect on respiration by benzoctamine; in spite of that good sedative component could be seen. PMID- 3006705 TI - Cell membrane associated protein kinase C as receptor of diterpene ester co carcinogens of the tumor promoter type and the phenotypic expression of tumors. AB - For investigations of biochemical and molecular mechanism(s) involved in carcinogenesis the three stage initiation/promotion/progression approach in mouse skin provides one of the most developed experimental models. As initiators (and as progressors), solitary carcinogenic polycyclic aromatic hydrocarbons (PAH), such as 7,12-dimethylbenz [a]anthracene (DMBA), are used. As promoters in the last about 15 years, cocarcinogenic skin irritant polyfunctional diterpene esters of plant origin have become most powerful tools. Their low activity "cryptic forms" are activated metabolically by hydrolases yielding highly active skin irritants and cocarcinogens of the "ultimate promoter" type such as the phorbol 12,13-diester TPA and congeners of the ingenane- and daphnane type. In their target cells the ultimate promoters are bound to specific binding sites both through their ester moieties as well as through their diterpene moieties as revealed by structure/activity relationships. In such investigations most recently, also fine structural comparison of threedimensional computergraphs of prototype promoters were included. In correlation with their irritant and promoting activities, diterpene ester promoters exhibit agonist type specific (non-covalent) binding to the particulate fraction of epidermis and other organs or cells. The specifically bound portion of certain electron spin and photoaffinity labeled phorbol ester analogs indicates their distinct molecular orientation in membranes and, in their microenvironment, presence of phospholipids essential for activity of the Ca++ dependent proteinkinase C (PKC). Specific bindings to and activation of PKC was shown for TPA and congeners particularly also in mouse epidermis as one of the target organs of initiation/promotion by DMBA/TPA. Diterpene ester promoters are postulated to interact with the second messenger system operated by inositolphospholipid/diacylglycerol which is linked to (i) receptors of certain (endocrine) hormones or growth factors and (ii) to certain oncogene derived (autocrine) molecular signals. In this way, diterpene ester promoters, mostly taylor-made by partial syntheses, have lead to new dimensions the investigations of specific mechanistic problems of processes of carcinogenesis. Interlinkage of parts of these processes with receptor research opens up new and fascinating aspects of mechanisms of carcinogenesis at the cell and/or molecular level. They will provide important leads in gaining a more differentiated system of assessment of environmental risk factors of cancer for cancer prevention and in developing more purposeful and selective antineoplastic drugs for clinical use. PMID- 3006706 TI - Receptors involved in the regulation of vascular tone. AB - In vascular smooth muscle the occurrence of several rather different receptors can be demonstrated: alpha 1/ alpha 2- and beta 1/beta 2-adrenoceptors; muscarinic (mainly M2)-cholinoceptors; dopaminergic (DA1 and DA2), angiotensin II and serotonergic receptors (5HT1 and 5HT2). Apart from this variety in receptor subtypes a distinction should be made between pre- and postsynaptic (pre- and postjunctional) receptors. The characteristics and functionality of these numerous receptor subtypes are the subject of the present survey, with an emphasis on the selective agonists and antagonists which interact with the various receptor populations. Although dopaminergic, serotonergic and angiotensin II-receptors can be demonstrated to exist in vascular smooth muscle and also in the sympathetic system which innervates the circulatory tract, their relevance with respect to the regulation and maintenance of vascular tone remains obscure. It seems clear, in any way, that the levels of the circulating agonists of these receptors, that is dopamine, angiotensin II and serotonin, are too low to cause any significant degree of receptor activation. As far as can be judged at present, vascular tone is predominantly maintained by sympathetic stimuli, via the mediation of postsynaptic alpha 1- and alpha 2-adrenoceptors, vascular beta 2 receptors being of limited importance. Muscarinic receptors of the M2-subtype should be considered as well, although their quantitative role is probably rather variable and less important than that of alpha-adrenoceptors. PMID- 3006707 TI - Role of autoreceptors in the function of the peripheral and central nervous system. AB - Neurotransmitter receptors, located on the nerve terminal from which this transmitter is released, are termed presynaptic autoreceptors. Evidence for their existence and functional role has been obtained by experiments carried out in vitro and in vivo. For example, noradrenergic, dopaminergic, serotoninergic, cholinergic and GABAergic neurons are endowed with presynaptic autoreceptors mediating a negative feedback loop. These receptors play a physiological role in the fine regulation of transmitter release in the peripheral and/or central nervous system, and, thus, may modulate any function controlled by the respective neurones. The physiological role of inhibitory or facilitatory autoreceptors for peptide cotransmitters on, e.g., noradrenergic and serotoninergic neurones is less well established. Alterations of the number or responsiveness of autoreceptors may play a role in the pathogenesis of diseases which are related to a disturbed function of the respective neurones in the peripheral or central nervous system. As an example, the potential importance of autoinhibitory alpha 2 adrenoceptors and autofacilitatory beta 2-adrenoceptors (activated by the cotransmitter adrenaline; both receptors located on sympathetic nerve fibres) and of autoinhibitory presynaptic 5-HT1B receptors (located on central serotoninergic nerves) in the development of essential hypertension is discussed. Autoreceptors may play a role in the therapeutic effect of currently available drugs, and it is probable that new classes of drugs which act predominantly via this site will be developed. PMID- 3006708 TI - Thyrotropin receptor antibodies. AB - The thyrotropin (TSH) receptor is an integral membrane protein which contains 2 subunits linked by a disulphide bridge. The A subunit (mol. wt. 50,000) is water soluble and forms the binding site for TSH, whereas the B subunit (mol. wt. 30,000) penetrates the lipid bilayer and probably forms the site for interaction with adenylate cyclase. Autoantibodies to the TSH receptor are found in the sera of patients with Graves' disease. The antibodies bind to the same region of the receptor's A subunit as TSH and usually act as TSH agonists, causing hyperthyroidism. Occasionally, TSH receptor autoantibodies are found which can act as TSH antagonists. Isoelectric focusing and binding studies indicate that these antibodies also bind to the same region of the receptor A subunit as TSH. PMID- 3006710 TI - Tissue levels, tissue angiotensin converting enzyme inhibition and antihypertensive effect of the novel antihypertensive agent alacepril in renal hypertensive rats. AB - Tissue levels, tissue angiotensin I converting enzyme (ACE) inhibition and hypotension were examined 20 min, 1, 5 and 14 h after oral administration of 1 [(S)-3-acetylthio-2-methylpropanoyl]-L-prolyl-L-phenylalanine (alacepril, DU 1219) (37.5 mg (92 mumol)/kg) or 1-[(S)-3-mercapto-2-methylpropanoyl]-L-proline (captopril) (20.0 mg (92 mumol)/kg) in renal hypertensive rats, using 14C-labeled compounds. Alacepril exerted a more gradual and more sustained antihypertensive effect than captopril. The maximal hypotension was observed 1 and 5 h after administration of captopril and alacepril, respectively. After administration of [14C]captopril, serum level reached the maximum at 20 min and then decreased rapidly. After administration of [14C]alacepril, serum level reached the maximum at 1 h and decreased more slowly than after [14C]captopril. Time course patterns of tissue levels were essentially in parallel with those of serum levels. Captopril exerted the maximal reduction of ACE activity in tissues 20 min after oral administration and thereafter, the reduction was diminished with time rapidly. [14C]Alacepril showed gradual reduction (the maximum at 1 h) and recovery of ACE activity relative to captopril. After oral administration of [14C]alacepril, tissue unbound fractions contained captopril and its derived metabolites while serum unbound fraction contained the intermediate metabolite desacetyl-alacepril (DU-1227) as well. Correlations between ACE inhibition and tissue levels and between changes in tissue ACE inhibition and in blood pressure with time after oral administration of the two agents were discussed. Furthermore, the direct comparison of alacepril and captopril was attempted by the difference in blood pressures and in ACE inhibitions induced after oral administration of the agents. PMID- 3006709 TI - Possible involvement of eicosanoids in the pharmacological action of bromelain. AB - Bromelain, a proteolytic enzyme extracted from pineapple plants, was investigated for its capacity to interfere with arachidonic acid metabolism, since prostaglandins and other eicosanoids are well-known to be involved in the pathogenesis of inflammatory diseases. Bromelain was tested for its ability to interfere with eicosanoids generation in vivo in two experimentally-induced inflammatory reactions in the rat. Also antiplatelet aggregation activity of bromelain was studied in ex vivo rat platelets. The results seem to indicate an interference of bromelain with arachidonic acid cascade, which, however, deserves further investigation to be better assessed. PMID- 3006711 TI - Metabolism of protein conjugate of desacetyl-alacepril and its effect on angiotensin converting enzyme in renal hypertensive rats. AB - The fate of protein conjugate of desacetyl-alacepril (DU-1227) and its effect on angiotensin I converting enzyme (ACE) activity in renal hypertensive rats were studied. [14C]DU-1227-protein conjugate was prepared by ultrafiltration method and administered intravenously in rats. Elimination of radioactivity of [14C]DU 1227-protein from plasma after injection seemed much slower than that reported of [14C]alacepril (1-[(S)-3-acetylthio-2-methylpropanoyl]-L-prolyl-L-phenylalanine, DU-1219) given orally. In the plasma unbound fraction, captopril and captopril cysteine were detected. Most tissue levels were higher than plasma levels. Significant reduction of tissue ACE activity was seen after administration of the conjugate. Radioactivity was mostly excreted in feces. Captopril, captopril disulfide and captopril-cysteine were found as urinary metabolites. These findings indicate that protein-bound DU-1227 readily dissociated and released DU 1227 was converted to captopril in vivo and can therefore participate in prolonged hypotensive effect exerted by alacepril. PMID- 3006712 TI - Effect of alacepril on renin-angiotensin-aldosterone system and kallikrein-kinin prostaglandin system in experimental animals. AB - The effects of 1-[(S)-3-acetylthio-2-methylpropanoyl]-L-prolyl-L-phenylalanine (alacepril, DU-1219), an orally active angiotensin converting enzyme (ACE) inhibitor, on humoral factors which participated in the blood pressure control were examined with various experimental animals. In conscious renal hypertensive dogs, alacepril (3 mg/kg p.o.) showed decreases in plasma ACE activity and plasma aldosterone concentration, and increases in plasma renin activity and plasma angiotensin I concentration accompanied by a significant reduction in blood pressure. In conscious normotensive dogs, alacepril (1 and 3 mg/kg p.o.) showed an increase in urinary excretion of bradykinin accompanied by increases in urinary water and sodium excretion. In spontaneously hypertensive rats, alacepril (30 and 100 mg/kg p.o.) showed increases in urinary excretion of bradykinin and 6 keto-prostaglandin Fl alpha, and a decrease in that of aldosterone accompanied by increased in excretion of water and sodium. These results indicate that the antihypertensive activity of alacepril is due to the suppression of renin angiotensin-aldosterone system and the enhancement of kallikrein-kinin prostaglandin system through the inhibition of ACE (kininase II) activity in vivo. PMID- 3006713 TI - Differentiation of central and peripheral cholecystokinin receptors by new glutaramic acid derivatives with cholecystokinin-antagonistic activity. AB - Three glutaramic acid derivatives provided with a potent antagonistic activity on the contractions elicited by the carboxyl terminal octapeptide CCK-8 in the guinea pig gallbladder have been evaluated for their capacity to inhibit the binding of [125I]-(Bolton-Hunter)-CCK-8 to both central and peripheric cholecystokinin (CCK) receptors. The most active compound inhibits the CCK binding to rat pancreas acini at a concentration 10(-7) mol/l, but only at 10(-4) mol/l on cerebral cortex membranes, confirming the existence of at least two different populations of CCK receptors. PMID- 3006714 TI - Maternal herpes infection complicated by prolonged premature rupture of membranes. AB - Three cases of patients who developed genital herpes virus infections after prolonged, premature rupture of membranes (PROM) at 28-31 weeks gestation are reported. These patients were expectantly managed without immediate intervention at the time of diagnosis of the genital herpes virus infection. In all three cases, there was no evidence of neonatal herpes virus infection at the time of delivery or before hospital discharge. The spectrum of decisions facing the physician managing a patient with prolonged PROM and a genital herpes virus infection is discussed, and a rational approach to management presented. PMID- 3006716 TI - Dietary fat, calories and cancer. PMID- 3006715 TI - Synovial proliferative disorders: differential diagnosis. AB - The proper treatment of synovial proliferative disorders depends on an accurate diagnosis and a knowledge of the natural history of these afflictions. A simple classification of the major synovial disorders with a brief general description of each, highlighting the prominent clinical features, is helpful. A thorough history and physical examination should enable the clinician to categorize the nature of the synovial disorder. Further evaluation and consultation can then be directed within the appropriate category. Radiologic, laboratory, and microscopic studies aid in arriving at a definitive diagnosis. Once a diagnosis has been established, therapy or further evaluation can be instituted. PMID- 3006718 TI - Marihuana and driving. AB - A review was performed of the marihuana and driving literature, both epidemiological and experimental. It was noted that epidemiological studies face considerable difficulties in obtaining estimates of risks involved for drivers utilizing marihuana due to the rapid decline in blood levels of tetrahydrocannabinol. On the other hand, experimental studies examining the relationship between administered marihuana dose and performance have identified many driving-related areas as exhibiting impairment. Areas impaired include coordination, tracking, perception, vigilance and performance in both driving simulators and on the road. Other behavioral areas of lesser importance for driving also exhibited evidence of impairment by marihuana. Areas for further research are suggested. PMID- 3006719 TI - [Serological study of cytomegalovirus, herpes simplex and rubella virus, hepatitis B and Toxoplasma gondii in 2 populations of pregnant women in Santiago, Chile]. PMID- 3006717 TI - DNA image cytometry in acquired immune deficiency syndrome (AIDS). AB - In nine cases with the acquired immune deficiency syndrome (AIDS), including four stage I cases, three stage II cases and two stage III cases, DNA image cytometry was performed on Feulgen-stained lymph node imprint smears. Diploidy was found in three cases, tetraploidy in three cases and octoploidy in two cases. Aneuploid DNA distribution patterns were not seen. The lymphoid cells showed an enormously increased proliferation rate. Two cases in stage I revealed characteristic intranuclear DNA inclusions in lymphoid cells. These results indicate that DNA image cytometry may be useful as an adjunct to surgical pathology in certain cases to assist in the differential diagnosis between AIDS and benign conditions of the lymphoid system as well as between AIDS and malignant lymphomas, which usually have aneuploid DNA patterns. PMID- 3006720 TI - [Etiology of hepatitis in Mexico City]. PMID- 3006721 TI - Patterns of cytochrome oxidase activity in the frontal agranular cortex of the macaque monkey. AB - The laminar pattern of cytochrome oxidase activity was studied in the agranular frontal cortex (area 4-6 complex) of the macaque monkey. The cortex, stained with this method, showed 6 stripes of different enzymatic activity. On the basis of their characteristics and of the presence of highly active cells, the agranular frontal cortex could be parcellated in 5 areas (F1-F5). F1 very likely corresponds to area FA of von Bonin and Bailey. Rostral to F1 two large regions could be distinguished, one located medial to the spur of the arcuate sulcus and its imaginary caudal extension, the other laterally. The superior region was formed by areas F2 and F3. The first was located on the dorsomedial cortical surface, the other on the mesial surface. F3 possibly corresponds to the supplementary motor area. The inferior region was formed by areas F4 and F5. The rostral area (F5) showed transition characteristics that rendered it somehow similar to the prefrontal areas. It may correspond to the cytoarchitectonic area FCBm. The cytocrome oxidase technique is a useful means of parcellating the agranular frontal cortex and may greatly help in physiological and behavioral experiments. PMID- 3006722 TI - Deficits in spatial-memory tasks following lesions of septal dopaminergic terminals in the rat. AB - The behavioral effects of 6-hydroxydopamine, injected bilaterally into the lateral septum, were investigated in two tests of spatial memory (radial 8-arm and T-maze). Three different experiments were conducted in the radial maze. In experiment I, rats were permitted to learn the task with food reinforcement in all arms of the maze. In experiment II, retention of the spatial information (working memory) learned in experiment I was tested by interposing various time intervals between choice 4 and 5 of each trial. In experiment III, reference and working memory were simultaneously assessed by only reinforcing 4 choices in the radial maze. Performances were compared in spaced versus massed trials. In the T maze, the rats were first tested for learning a spatial discrimination between the two arms of the maze, and subsequently for reversal of the previously learned response. The results showed that the rats with lesions were impaired in all experiments. This impairment was particularly marked in some aspects of the procedures used: (1) in the search for the last 4 pellets in experiment I, (2) in the first presentations of various intervals interposed between choices 4 and 5, (3) in the search for food in the baited arms when the trials were massed in experiment III and (4) in the reversal of previously learned spatial discrimination in the T-maze. These behavioral deficits in the rats with septal dopaminergic lesions were interpreted as an increased susceptibility to interference. The lesions were shown to have selectively depleted dopamine concentrations in the septum without damaging noradrenergic terminals or cholinergic cell bodies. It was concluded that dopaminergic neurons could have a modulatory influence on memory processes. PMID- 3006724 TI - Degradation of myocardial structural proteins in myocardial infarcted dogs is reduced by Ep459, a cysteine proteinase inhibitor. AB - The purpose of this study is to clarify whether cysteine proteinases play an important role in the degradation of myocardial proteins in the infarcted tissue. We studied the effects of a cysteine proteinase inhibitor, Ep459, on degradation of cardiac structural proteins caused by ischemia due to coronary artery ligation for 24 h. Proteolytic effects of purified cysteine proteinases on isolated cardiac tissue were also examined. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, degradation of cardiac structural proteins, particularly of myosin heavy chain, alpha-actinin and troponin-I was observed in the infarcted tissue. Treatment with Ep459 significantly reduced protein degradation and total activity of cathepsins B and L in the infarcted tissue, compared with the findings in the untreated group. The electrophoretic pattern of the infarcted myocardium was similar to that of myofibrillar proteins degraded by cathepsins B and L. These results suggest that cysteine proteinases, particularly cathepsins B and L, are involved in degradation of myofibrillar proteins in myocardial infarction. PMID- 3006723 TI - Biochemical basis of alcoholism: statements and hypotheses of present research. AB - Experimental results and theoretical considerations on the biology of alcoholism are devoted to the following topics: genetically determined differences in metabolic tolerance; participation of the alternative alcohol metabolizing systems in chronic alcohol intake; genetically determined differences in functional tolerance of the CNS to the hypnotic effect of alcohol; cross tolerance between alcohol and centrally active drugs; dissociation of tolerance and cross tolerance from physical dependence; permanent effect of uncontrolled drinking behavior induced by alkaloid metabolites in the CNS; genetically determined alterations in the function of opiate receptors; and genetic predisposition to addiction due to innate endorphin deficiency. For the purpose of introducing the most important research teams and their main work, statements from selected publications of individual groups have been classified as to subject matter and summarized. Although the number for summary-quotations had to be restricted, the criterion for selection was the relevance to the etiology of alcoholism rather than consequences of alcohol drinking. PMID- 3006725 TI - Studies on cytochrome-c oxidase, XIII. Amino-acid sequence of the small membrane polypeptide VIIIc from bovine heart respiratory complex IV. AB - The isolation and complete amino-acid sequence analysis of the cytoplasmically synthesized polypeptide VIIIc from bovine heart cytochrome-c oxidase is described. The protein is a stoichiometric constituent of the mitochondrial respiratory complex IV. Its primary structure is deduced from N-terminal sequencing and peptides obtained by enzymatic cleavage with Staphylococcus aureus proteinase and chemical cleavage with cyanogen bromide. The small protein consists of 56 amino acids summing up to a total Mr of 6243. From position 34 to 51 the chain contains a hydrophobic sequence of 18 residues. This probably membrane-spanning segment also contains the 2 cysteine residues of the chain. The function of this subunit in the respiratory complex IV is still unknown. PMID- 3006726 TI - Digitalis in chronic renal insufficiency. AB - Cardiac dysfunction is common in patients with terminal renal failure. However, no consensus has been reached with respect to the indications for digitalis therapy. Depression of myocardial contractility may occur as a result of circulating toxic factors, parathyroid hormone, and altered catecholaminergic responsiveness. On the other hand, paradoxical positive inotropic effects have been observed possibly as a result of a circulating natriuretic factor (an endogenous digitalis analogue) which inhibits Na,K-ATP'ase. Pharmacokinetics and pharmacodynamics of digitalis steroids are altered in uremia. Elimination half lives of strophanthin and digoxin are prolonged, whereas the elimination half life of digitoxin is unchanged. Altered protein binding and volume of distribution have been noted. Despite its long elimination half-life, most nephrologists favor administration of digitoxin because of its insensitivity to changes in renal function. PMID- 3006728 TI - A human hepatoma cell line modulates its insulin receptors under different culture conditions. PMID- 3006727 TI - Diphasic or prolonged course of viral hepatitis A in children. AB - A study on nine cases of diphasic viral hepatitis A was carried out in 130 children admitted to pediatric hospital from January to December 1982. One hundred and eight children (83.0%) showed IgM anti-HAV (one of them was a chronic HBsAg carrier), 19 (14.6%) were HBsAg positive at the admission and 3 (2.3%) became positive for anti-HBc IgM marker during the course of the illness. Nine anti-HAV IgM positive children showed an atypical course of their disease in that after a short period of progressive enzyme level normalization, a relapse occurred without signs of subsequent HBV, CMV or EBV infection. Probable although hypothetical interpretations of these cases are discussed. PMID- 3006730 TI - Plasma cyclic AMP response to calcitonin: a potential clinical marker of bone turnover. AB - A noninvasive marker of bone turnover would be useful in predicting which patients are at risk of rapid bone loss and in monitoring response to therapy. Calcitonin (CT)-induced hypocalcemia correlates with bone turnover but has not been a clinically useful test because changes in serum calcium are small, and test duration is long. CT rapidly increases plasma cAMP levels. We conducted studies in rats and in man to characterize the origin of this rise, and to determine its suitability as a potential clinical marker of bone resorption. Administration of CT to rats resulted in a prompt and sustained rise in plasma cAMP. This effect was not blunted by nephrectomy and was greater in calcium deprived rats with increased bone resorption. Thus, it appears to reflect CT action on bone. An intramuscular injection of 100 units salmon CT elevated plasma cAMP in 18 normal men and women. This effect was observed by 20 min after injection and persisted over 2 h. Although basal levels of plasma cAMP were similar in healthy postmenopausal women and men, the response to CT was greater in women. The response of women with hyperparathyroidism was greater than that of normal women, and the response of pagetic men was greater than that of normal men. The rise in plasma cAMP following CT is rapid and easily measured and appears to correlate with the state of bone remodeling. Additional studies will be required in osteoporotic subjects with high and low remodeling activity before the clinical utility of this test can be determined. PMID- 3006729 TI - [Adaptation to defined chemical conditions of the in vitro growth of PLC/PRF/5 cells]. PMID- 3006731 TI - Effect of pyrophosphate and two diphosphonates on 45Ca and 32Pi uptake and mineralization by matrix vesicle-enriched fractions and by hydroxyapatite. AB - Inorganic pyrophosphate (PPi) and two diphosphonates, ethane-1-hydroxy-1, 1 diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl2MDP), were found to inhibit in vitro mineralization induced by matrix vesicle-enriched fractions from chicken epiphyseal cartilage. Inhibitor concentrations from 0.20 to 20 microM caused a dosage-dependent decrease in 45Ca and 32Pi uptake by the vesicle fraction. These inhibitors were also tested in a hydroxyapatite (HA)-seeded system to help distinguish between effects on the mineral vs nonmineral portions of the vesicle fraction. The order of inhibition of the HA-seeded system was EHDP greater than PPi greater than Cl2MDP, except for inhibitor concentrations of 0.20 microM where EHDP was the least inhibitory. This variation may be due to differences in the binding of the inhibitors to HA crystals. In general, inhibition of HA mineralization was greatest during later time periods, whereas vesicle ion uptake was affected more during early stages of incubation. The differential effects of the three inhibitors were most obvious at the 2.0 microM concentration. With PPi substantial inhibition of HA-seeded mineralization was observed even in late stages of the study; in contrast, with time the vesicle fraction overcame this inhibition. This suggests that alkaline phosphatase, an enzyme notably enriched in matrix vesicles, catalyzed the hydrolysis of PPi, reducing its concentration to a level where mineralization could proceed. Our findings show that matrix vesicle-induced mineralization differs significantly from apatite-induced mineralization. The data support the concept that vesicle alkaline phosphatase acts, at least in part, to remove physiological crystal growth inhibitors. PMID- 3006733 TI - The lifespan of osteoclasts: experimental studies using the giant granule cytoplasmic marker characteristic of beige mice. AB - Osteoclasts are large multinucleated skeletal cells that form by fusion of bloodborne mononuclear precursors. Fusion with mononuclear precursors occurs throughout life, and survival of osteoclasts is believed to be dependent upon continued replenishment by fusion. This study examined osteoclast lifespan, defined as maximal survival without fusion, in normal mice irradiated to eliminate host stem cells and rescued with stem cells from beige (bg) mice whose osteoclasts have a distinctive phenotype. Osteoclasts of donor phenotype appeared during the second week and progressively increased so that by the sixth week no osteoclasts of host phenotype were present. Radiation alone did not produce any change in osteoclast phenotype. These data are interpreted to indicate that the maximal survival of osteoclasts without fusion of precursors is less than 6 weeks. PMID- 3006732 TI - Effect of bisphosphonates on production of interleukin 1-like activity by macrophages and its effect on rabbit chondrocytes. AB - Bisphosphonates are potent inhibitors of bone resorption, but their mode of action is still unknown. Since interleukin 1 (IL-1)-like activity has been shown to stimulate bone resorption in vitro, we have studied whether bisphosphonates inhibit either the production of IL-1-like activity or its effect on one type of connective tissue cell, chondrocytes. The production of IL-1-like activity was examined using rabbit peritoneal macrophages and the murine macrophage cell line P388D1, and the effect of IL-1-like activity was assessed by measuring the secretion of collagenase and prostaglandin E2 (PGE2) by rabbit chondrocytes. Production of IL-1-like activity was unaffected by bisphosphonates, whereas the effect of IL-1-like activity on collagenase and prostaglandin E2 secretion by rabbit chondrocytes was increased rather than inhibited by bisphosphonates. Finally, bisphosphonates increased DNA and cell number in chondrocyte cultures, but this effect was blocked when IL-1-like activity was added to the cultures. Thus, our results provide no evidence for a direct inhibitory effect of bisphosphonates on either the production of IL-1-like activity or the action of IL-1-like activity on chondrocytes. PMID- 3006734 TI - Serum galactosyltransferase isoenzyme patterns of cancer patients with liver involvement. AB - The level of galactosyltransferase activity was measured in the serum of 220 patients with a variety of solid tumours. There was a significantly greater proportion of patients with elevated galactosyltransferase in the group with metastatic disease (43%) than for the group with localised disease (16%). Galactosyltransferase was elevated in 69% of patients with liver metastasis compared to 32% of patients with metastatic disease at sites other than liver and this difference was also significant. High resolution agarose isoelectric focusing was used to determine the 'isoenzyme' pattern of serum galactosyltransferase of 6 patients with liver metastasis and 2 patients with primary hepatoma and these were compared to those of 6 patients with similar primary tumours without liver involvement. There were no qualitative differences in the patterns from the two groups. The average peak height for each of the 19 peaks of activity identified was generally higher in the group with liver involvement, except for those peaks known to contain little or no attached sialic acid. Liver involvement appears not to contribute in any specific way to the altered pattern of serum galactosyltransferase often seen in patients with solid tumours. The tumour rather than the liver is therefore the most likely source of these alterations. PMID- 3006736 TI - c-Ki-ras amplification in human lung cancer. PMID- 3006735 TI - Changes in the response of the RIF-1 tumour to melphalan in vivo induced by inhibitors of nuclear ADP-ribosyl transferase. AB - The effect of inhibitors of nuclear ADP-ribosyl transferase (ADPRT) on the cytotoxicity of melphalan (L-PAM) in the RIF-1 tumour in vivo was investigated. A large single dose of nicotinamide (1000 mg kg-1) enhanced the tumour cell killing by L-PAM as measured by tumour cell survival. This enhancement was maximum when nicotinamide was administered within 1 h before injecting the L-PAM. When given at this time, the nicotinamide had a dose-modifying effect on all L-PAM doses tested, giving rise to a mean enhancement ratio (ER) of 2.2. Nicotinamide did not appear to inhibit the recovery from L-PAM induced potentially lethal damage. L PAM (6 mg kg-1) produced a transient drop in mouse body temperature. This effect was both increased and prolonged by nicotinamide. In addition the inhibitor also delayed the clearance of L-PAM from the plasma of C3H mice, such that the half life of the chemotherapeutic agent was extended from 41 min to 143 min. The effect of combining L-PAM with nicotinamide doses below 1000 mg kg-1 was also investigated. The results showed that as the nicotinamide dose was decreased, the enhancement of the effects on body temperature, pharmacokinetics and white blood cell counts were reduced. However, a concomitant loss in the enhancement of tumour cell killing was also observed. Similar results were obtained using 3 aminobenzamide, a more efficient inhibitor of ADPRT. PMID- 3006737 TI - Proteolytic enzymes in blister fluids from patients with dermatitis herpetiformis. AB - Proteolytic enzymes may be involved in blister formation in dermatitis herpetiformis (DH). We have examined collagenase, gelatinase and elastase-like enzyme activities in fluids collected from spontaneous blisters and from suction blisters raised on developing DH-lesions induced by application of potassium iodide. Control suction blisters were raised on unaffected DH-skin and on healthy volunteers. High enzyme activities were found in spontaneous blisters, and suction blister fluids obtained from developing lesions showed increased levels of gelatinase and elastase-like enzymes. Inhibitor studies revealed that a part of the elastase-like enzyme activity might be derived from inflammatory cells. Gel filtration chromatography disclosed two separate elastase-like enzymes which, as they had high molecular weights, could be either enzyme-inhibitor complexes or aggregates. PMID- 3006739 TI - Feasibility of prenatal diagnosis of beta thalassaemia by DNA polymorphisms in an Italian population. AB - A feasibility study of prenatal diagnosis of beta thalassaemia in a northern Italian population has been carried out. Twenty-five families have been studied, each consisting of two parents and a homozygous beta thalassaemia child, thus enabling linkage analysis of restriction fragment length polymorphisms (RFLPs) to the normal and the thalassaemic chromosomes. Using seven standard RFLPs, 19/25 families could be offered prenatal diagnosis; inclusion of the recently described Ava II psi beta polymorphism increased this figure to 23/25 (92%) of the families. PMID- 3006738 TI - Lymphocytes from patients receiving lithium do not inhibit CFU-C growth. AB - Lymphocyte subset levels and function were examined in 12 patients on lithium therapy and in 11 healthy hospital personnel. Co-culture of allogeneic human bone marrow cells with monocyte-depleted lymphocyte preparations revealed that CFU-C formation was significantly reduced (mean 43% inhibition) in the presence of normal lymphocytes but not with the patients' lymphocytes (less than 5% inhibition). This did not reflect numerical changes in lymphocyte subsets, since these were similar for control and lithium subjects. T colony formation was significantly depressed in the patient group (P less than 0.05), whereas B colony numbers were similar in both groups (P greater than 0.1). The possible role of HLA-incompatibility affecting CFU-C growth was investigated in co-culture experiments, using lymphocytes from HLA-identical twins, one of whom was receiving lithium. In four separate co-culture experiments, the inhibitory effect was shown with lymphocytes from the non-lithium twin but was not demonstrated by the lithium subject. Addition of lithium in vitro to co-cultures of normal marrow and lymphocytes was found to negate the inhibitory phenomenon in a dose-related manner. It is postulated that granulocytosis induced by the administration of lithium may be a manifestation of changes in a lymphocytic control system. PMID- 3006740 TI - Purine degradative enzymes and immunological phenotypes in chronic B-lymphocytic leukaemia: indications that leukaemic immunocytoma is a separate entity. AB - Investigations of the purine degradative enzymes adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and ecto-5'-nucleotidase (5'NT) have been shown to be of value in defining subsets of lymphoid malignancies. We have studied the activities of these enzymes in the circulating malignant cells of 35 patients with chronic B lymphocytic leukaemia and have correlated the biochemical data with immunological phenotypes. Classification of the cases into those without evidence of secretory activity ('true' CLL, 14 patients) and those with cytoplasmic immunoglobulin (CIg) ('immunocytoma'; 21 patients) revealed that immunocytomas are phenotypically and biochemically associated with more mature features. Malignant cells without CIg were characterized by low activities of ADA, PNP and 5'NT. In malignant cells with evidence of secretory activity (immunocytoma), low activity of ADA was also observed, but the activities of PNP and 5'NT were relatively high and approached the range of normal B lymphocytes. The differences in PNP (P less than 0.05) and in 5'NT (P less than 0.01) between these two groups were significant. Phenotypically the cells without CIg were predominantly associated with IgM (+k light chains) as surface membrane immunoglobulin (SmIg) whereas expression of IgG was more often observed in the leukaemic cells with CIg. No correlation between enzyme patterns and the stage of the disease was apparent. Thus both biochemical and immunological criteria show that cases of CLL vary within a range of maturity and that those with CIg might be more mature in the B cell axis. The present study emphasizes the value of purine enzyme studies in defining subsets of B cell neoplasia. PMID- 3006741 TI - Comparison of phenotypic assessment and the use of two restriction fragment length polymorphisms in the diagnosis of the carrier state in haemophilia B. AB - Carrier detection in haemophilia B has previously involved pedigree analysis and assessment of the coagulation phenotype. At best using these methods, carrier evaluation may be made with about 80% certainty (Orstavik et al, 1981). With isolation and cloning of the factor IX gene, DNA probes are now available to detect intragenic nucleotide changes. This study uses one such probe which detects restriction fragment length polymorphisms with the restriction endonucleases Taq 1 and Xmn 1. Seventy-eight individuals from eight haemophilia B kindred were initially evaluated for factor IXC and factor IXAg levels. DNA from these individuals was then digested with the two restriction enzymes and after Southern blotting, analysed for restriction fragment length polymorphisms (RFLPs) with the radiolabelled genomic probe. Four kindred proved informative with RFLP linkage assessment. In three families evaluation was possible with both Taq 1 and Xmn 1 polymorphic markers but in the fourth pedigree only Xmn 1 was informative. Using these techniques five potential carriers have been definitively assigned as either normal or carriers of the haemophilia B gene defect. Problems of phenotypic assessment are well illustrated in pedigree 4 where polymorphic linkage positively identified a carrier whose coagulation data were normal. PMID- 3006742 TI - Purification and characterization of a membrane-associated phosphatidylserine synthase from Bacillus licheniformis. AB - A CDP-diacylglycerol-dependent phosphatidylserine synthase was solubilized from Bacillus licheniformis membranes and purified to near homogeneity. The purification procedure consisted of CDP-diacylglycerol-Sepharose affinity chromatography followed by substrate elution from blue dextran-Sepharose. The purified preparation showed a single band with an apparent relative molecular mass of 53 000 daltons when subjected to sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Proteolytic digestion of the enzyme yielded a smaller (41 000 daltons) active form. The preparation was free of any phosphatidylglycerophosphate synthase, phosphatidylserine decarboxylase, CDP diacylglycerol hydrolase, and phosphatidylserine hydrolase activities. The utilization of substrates and the formation of products occurred with the expected stoichiometry. Radioisotopic exchange patterns between related substrate and product pairs suggest a sequential Bi-Bi reaction as opposed to the ping-pong mechanism exhibited by the well-studied phosphatidylserine synthase of Escherichia coli [Larson, T. J., & Dowhan, W. (1976) Biochemistry 15, 5212-5218]. The B. licheniformis enzyme was also found to be markedly dissimilar to the E. coli enzyme with regard to association with detergent micelles, affinity for ribosomes, and antigenicity. PMID- 3006744 TI - Interaction of guanosine cyclic 3',5'-phosphate dependent protein kinase with lin benzoadenine nucleotides. AB - Using the activated cGMP-dependent protein kinase in the presence of the phosphorylatable peptide [[Ala34]histone H2B-(29-35)], we found that lin benzoadenosine 5'-diphosphate (lin-benzo-ADP) was a competitive inhibitor of the enzyme with respect to ATP with a Ki (22 microM) similar to the Kd (20 microM) determined by fluorescence polarization titrations. The Kd for lin-benzo-ADP determined in the absence of the phosphorylatable peptide, however, was only 12 microM. ADP bound with lower affinity (Ki = 169 microM; Kd = 114 microM). With [Ala34]histone H2B-(29-35) as phosphoryl acceptor, the Km for lin-benzo-ATP was 29 microM, and that for ATP was 32 microM. The Vmax with lin-benzo-ATP, however, was only 0.06% of that with ATP as substrate [0.00623 +/- 0.00035 vs. 11.1 +/- 0.17 mumol (min.mg)-1]. Binding of lin-benzo-ADP to the kinase was dependent upon a divalent cation. Fluorescence polarization revealed that Mg2+, Mn2+, Co2+, Ni2+, Ca2+, Sr2+, and Ba2+ supported nucleotide binding to the enzyme; Ca2+, Sr2+, and Ba2+, however, did not support any measurable phosphotransferase activity. The rank order of metal ion effectiveness in mediating phosphotransferase activity was Mg2+ greater than Ni2+ greater than Co2+ greater than Mn2+. Although these results were similar to those observed with the cAMP dependent protein kinase [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], major differences in the Vmax with lin-benzo-ATP as substrate and the effect of peptide substrates on nucleotide (both lin-benzo-ADP and ADP) binding were observed. PMID- 3006743 TI - Sendai virus induced leakage of liposomes containing gangliosides. AB - Sendai virus induced liposome leakage has been studied by using liposomes containing a self-quenching fluorescent dye, calcein. The liposomes used in this study were prepared by a freeze and thaw method and were composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine (1:2.60:1.48 molar ratio) as well as various amounts of gangliosides and cholesterol. The leakage rate was calculated from the fluorescence increment as the entrapped calcein leaked out of the liposomal compartment and was diluted into the media. It was shown that the target liposome leakage was virus dose dependent. Trypsin-treated Sendai virus in which the F protein had been quantitatively removed did not induce liposome leakage, indicating that the leakage was a direct result of F-protein interaction with the target bilayer membrane. The activation energy of this process was approximately 12 kcal/mol below 17 degrees C and approximately 25 kcal/mol above 17 degrees C. Gangliosides GM1, GD1a, and GT1b could serve as viral receptor under appropriate conditions. Liposome leakage showed a bell-shaped curve dependence on the concentration of ganglioside in the liposomes. No leakage was observed if the ganglioside content was too low or too high. Inclusion of cholesterol in the liposome bilayer suppressed the leakage rate of liposomes containing GD1a. It is speculated that the liposome leakage is a consequence of fusion between Sendai virus and liposomes. PMID- 3006745 TI - Anti-nitroxide immunoglobulin G: analysis of antibody specificity and their application as probes for spin-labeled proteins. AB - Antibodies have been elicited to the nitroxide spin-label 4-maleimido-2,2,6,6 tetramethyl-piperidinyl-1-oxy conjugated, via protein sulfhydryl groups, to bovine serum albumin. Antibody-hapten cross-reactivity was demonstrated by double immunodiffusion and by a broadening of the nitroxide electron paramagnetic resonance spectrum. The specificity of the antibodies with respect to hapten structure was examined by means of a simple filter binding assay. Under these conditions, antibodies were shown to distinguish between the nitroxide and hydroxylamine derivatives and between spin-labels comprising either five- or six membered ring structures. In addition, protein-bound nitroxide spin-labels were detected at the nanogram level by immunoblotting. By use of this method, the specificity of the antibody-hapten reaction predicted by the filter binding assay procedure was utilized to differentially detect various types of bound spin label. Finally, antibodies were used to identify protein-bound nitroxide spin label of protein fractionated by gel electrophoresis. PMID- 3006746 TI - Functional properties of covalent beta-endorphin peptide/calmodulin complexes. Chlorpromazine binding and phosphodiesterase activation. AB - The 31-residue neuropeptide porcine beta-endorphin was shown to inhibit the Ca2+ dependent calmodulin activation of highly purified bovine brain cyclic nucleotide phosphodiesterase (3',5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17). Using a series of deletion peptides, the minimal inhibitory peptide sequence was found to correspond to beta-endorphin residues 14-25, confirming previously reported results for crude enzyme preparations. A correlation was found between the relative inhibitory potency of a particular beta-endorphin deletion peptide and the efficacy of cross-linking that peptide to calmodulin with bis(sulfosuccinimidyl) suberate, strongly implicating peptide binding to calmodulin as the mechanism of the observed inhibition. We found that relatively modest concentrations of chlorpromazine significantly reduced the efficiency of cross-linking beta-endorphin 14-31 to calmodulin. Chlorpromazine-Sepharose affinity chromatography of peptide/calmodulin adducts showed that a significant portion of the cross-linked beta-endorphin 14-31/calmodulin complex (stoichiometry of 1 mol/mol) retained the ability to interact with the immobilized phenothiazine in a Ca2+-dependent and calmodulin-displaceable manner. In contrast, the 2:1 (peptide:protein) product exhibited no affinity for the immobilized phenothiazine. The use of this affinity chromatographic step allowed preparation of homogeneous populations of both 1:1 and 2:1 beta-endorphin 13 31/calmodulin complexes and assessment of their functional characteristics. Equilibrium binding studies with chlorpromazine revealed that the covalent attachment of one peptide molecule to calmodulin perturbed all phases of Ca2+ dependent drug binding, but the adduct still bound significant quantities of chlorpromazine. The 2:1 complex, however, showed little detectable binding of the phenothiazine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006747 TI - Conversion of bovine cardiac adenosine cyclic 3',5'-phosphate dependent protein kinase to a heterodimer by removal of 45 residues at the N-terminus of the regulatory subunit. AB - The type II adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase from bovine heart, consisting of a dimeric regulatory subunit and two catalytic subunits, was converted to a heterodimer by limited tryptic digestion. Loss of the tetrameric structure was accompanied by proteolysis of the regulatory subunit to a form with an apparent molecular weight of 45 000 vs. 52 000 for the native subunit. The proteolyzed subunit behaved as a monomer, in contrast to the dimeric native subunit. Amino acid sequence analysis established that proteolysis removed 45 residues at the N-terminus, indicating that these 45 residues constitute the dimerizing domain of this protein. The kinetic properties of this heterodimer were indistinguishable from those of the native tetramer: half-maximal kinase activation occurred at 48 nM cAMP with a Hill coefficient of 1.45, the regulatory subunit bound 1.5 equiv of cAMP with half-maximal binding occurring at 33 nM, and kinetics for dissociation of bound cAMP were biphasic, indicating the presence of two different binding sites. These observations suggest that residues 1-45 function only in the formation of dimers and that dimerization has little influence on other functional properties of the regulatory subunit. More extensive proteolysis cleaved the monomeric fragment at Lys-311. The fragments resulting from this second cleavage did not dissociate, and the complex inhibited the catalytic subunit in a cAMP-dependent manner. PMID- 3006748 TI - Oxygenation of carbon monoxide by bovine heart cytochrome c oxidase. AB - Cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1), as the terminal enzyme of the mammalian mitochondrial electron transport chain, has long been known to catalyze the reduction of dioxygen to water. We have found that when reductively activated in the presence of dioxygen, the enzyme will also catalyze the oxidation of carbon monoxide to its dioxide. Two moles of carbon dioxide is produced per mole of dioxygen, and similar rates of production are observed for 1- and 2-electron-reduced enzyme. If 13CO and O2 are used to initiate the reaction, then only 13CO2 is detected as a product. With 18O2 and 12CO, only unlabeled and singly labeled carbon dioxide are found. No direct evidence was obtained for a water-gas reaction (CO + H2O----CO2 + H2) of the oxidase with CO. The CO oxygenase activity is inhibited by cyanide, azide, and formate and is not due to the presence of bacteria. Studies with scavengers of partially reduced dioxygen show that catalase decreases the rate of CO oxygenation. PMID- 3006749 TI - Spectroelectrochemical study of the cytochrome a site in carbon monoxide inhibited cytochrome c oxidase. AB - The reduction potential of the cytochrome a site in the carbon monoxide derivative of beef heart cytochrome c oxidase has been studied under a variety of conditions by thin-layer spectroelectrochemistry. The reduction potential exhibits no ionic strength dependence and only a 9 mV/pH unit dependence between pH 6.5 and 8.5. The weak pH dependence indicates that protonation of the protein is not stoichiometrically linked to oxidoreduction over the pH range examined. The temperature dependence of the reduction potential implies a relatively large standard entropy of reduction of cytochrome a. The measured thermodynamic parameters for reduction of cyctochrome a are (all relative to the normal hydrogen electrode) delta Go'(25 degrees C) = -6.37 kcal mol-1, delta Ho' = -21.5 kcal mol-1, and delta So' = -50.8 eu. When cytochrome c is bound to the oxidase, the reduction potential of cytochrome a and its temperature dependence are not measurably affected. Under all conditions studied, the cytochrome a site did not exhibit simple Nernstian n = 1 behavior. The titration behavior of the site is consistent with a moderately strong anticooperative interaction between cytochrome a and CuA [Wang, H., Blair, D. F., Ellis, W. R., Jr., Gray, H. B., & Chan, S. I. (1985) Biochemistry (following paper in this issue)]. PMID- 3006750 TI - Temperature dependence of the reduction potential of CuA in carbon monoxide inhibited cytochrome c oxidase. AB - The temperature dependence of the reduction potential of the CuA site in carbon monoxide inhibited cytochrome c oxidase has been measured with a spectroelectrochemical method adapted to the relatively weak near-infrared absorption of this copper ion. These measurements, together with parallel measurements on the 604-nm absorption due to Fea, indicate that an interaction between CuA and Fea causes the reduction potential for one of these sites to be decreased by approximately 40 mV upon reduction of the other. The temperature dependence of the CuA reduction potential indicates a relatively large and negative standard entropy of reduction of CuA (delta So' = -48.7 +/- 2.3 eu). Possible implications of the intersite redox interaction and the large standard entropy of reduction of the CuA site are discussed. PMID- 3006751 TI - Identifying regions of membrane proteins in contact with phospholipid head groups: covalent attachment of a new class of aldehyde lipid labels to cytochrome c oxidase. AB - A series of amine-specific reagents based on the benzaldehyde reactive group have been synthesized, characterized, and used to study beef heart cytochrome c oxidase reconstituted in phospholipid bilayers. The series contained three classes of reagents: lipid-soluble phosphodiesters having a single hydrocarbon chain, phospholipid analogues, and a water-soluble benzaldehyde. All reagents were either radiolabeled or spin-labeled or both. The Schiff bases formed by these benzaldehydes with amines were found to be reversible until the addition of the reducing agent sodium cyanoborohydride, whereas attachment of lipid-derived aliphatic aldehydes was not readily reversible in the absence of the reducing agent. The benzaldehyde group provides a convenient method of controlling and delaying permanent attachment to integral membrane proteins until after the reconstitution steps. This ensures that the lipid analogues are located properly to identify amine groups at the lipid-protein interface rather than reacting indiscriminately with amines of the hydrophilic domains of the protein. The benzaldehyde lipid labels attach to cytochrome c oxidase with high efficiency. Typically, 20% of the amount of lipid label present was covalently attached to the protein, and the number of moles of label incorporated per mole of protein ranged from 1 to 6, depending on the molar ratios of label, lipid, and protein. The efficiency of labeling by the water-soluble benzaldehyde was much less than that observed for any of the lipid labels because of dilution effects, but equivalent levels of incorporation were achieved by increasing the label concentration. Electron spin resonance spectra of a nitroxide-containing phospholipid analogue covalently attached to reconstituted cytochrome c oxidase exhibited a large motion-restricted component, which is characteristic of spin labeled lipids in contact with the hydrophobic surfaces of membrane proteins. The line shape and splittings were similar for covalently attached label and label free to diffuse and contact the protein molecules in the bilayer, providing independent evidence that the coupling occurs at the protein-lipid interface. The distribution of the benzaldehyde reagents attached to the polypeptide components of cytochrome c oxidase was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeling pattern observed for the lipid analogues was not affected by the presence of the nitroxide moiety on the acyl chains but was dependent on the molar ratio of labeling reagent to protein.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3006752 TI - Temperature dependence of rotational dynamics of protein and lipid in sarcoplasmic reticulum membranes. AB - We have investigated the relationship between function and molecular dynamics of both the lipid and the Ca-ATPase protein in sarcoplasmic reticulum (SR), using temperature as a means of altering both activity and rotational dynamics. Conventional and saturation-transfer electron paramagnetic resonance (EPR) was used to probe rotational motions of spin-labels attached either to fatty acid hydrocarbon chains or to the Ca-ATPase sulfhydryl groups in SR. EPR studies were also performed on aqueous dispersions of extracted SR lipids, in order to study intrinsic lipid properties independent of the protein. While an Arrhenius plot of the Ca-ATPase activity exhibits a clear change in slope at 20 degrees C, Arrhenius plots of lipid hydrocarbon chain mobility are linear, indicating that an abrupt thermotropic change in the lipid hydrocarbon phase is not responsible for the Arrhenius break in enzymatic activity. The presence of protein was found to decrease the average hydrocarbon chain mobility, but linear Arrhenius plots were observed both in the intact SR and in extracted lipids. Lipid EPR spectra were analyzed by procedures that prevent the production of artifactual breaks in the Arrhenius plots. Similarly, using sample preparations and spectral analysis methods that minimize the temperature-dependent contribution of local probe mobility to the spectra of spin-labeled Ca-ATPase, we find that Arrhenius plots of overall protein rotational mobility also exhibit no change in slope. The activation energy for protein mobility is the same as that of ATPase activity above 20 degrees C; we discuss the possibility that overall protein mobility may be essential to the rate-limiting step above 20 degrees C. PMID- 3006753 TI - Large-scale overproduction and rapid purification of the Escherichia coli ssb gene product. Expression of the ssb gene under lambda PL control. AB - We report a rapid procedure for the large-scale purification of the Escherichia coli encoded single-strand binding (SSB) protein, helix-destabilizing protein which is essential for replication, recombination, and repair processes in E. coli. To facilitate the isolation of large quantities of the ssb gene product, we have subcloned the ssb gene into a temperature-inducible expression vector, pPLc28 [Remaut, E., Stanssens, P., & Fiers, W. (1981) Gene 15, 81-93], carrying the bacteriophage lambda PL promoter. A large overproduction of the ssb gene product results upon shifting the temperature of E. coli strains which carry the plasmid and also produce the thermolabile lambda cI857 repressor. After 5 h of induction, the ssb gene product represents approximately 10% of the total cell protein. The overexpression of the ssb gene and the purification protocol reported here enable one to isolate SSB protein (greater than 99% pure) with final yields of approximately 3 mg of SSB protein/g of cell paste. In fact, very pure (greater than 99%) SSB protein can be obtained after approximately 8 h, starting from frozen cells in the absence of any columns, although inclusion of a single-stranded DNA-cellulose column is generally recommended to ensure that the purified SSB protein possesses DNA binding activity. The ability to easily purify 1 g of SSB protein from 300-350 g of induced cells will facilitate physical studies requiring large quantities of this important protein. PMID- 3006754 TI - Isolation of intercalator-dependent protein-linked DNA strand cleavage activity from cell nuclei and identification as topoisomerase II. AB - DNA intercalating agents such as 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) have previously been found to induce in mammalian cells the formation of protein-associated DNA single- and double-strand breaks. In the current work, an activity characterized by the production of DNA-protein links associated with DNA strand breaks and by stimulation by m-AMSA was isolated from L1210 cell nuclei and was shown to be due to topoisomerase II. Nuclei were extracted with 0.35 M NaCl, and the extract was fractionated by gel filtration, DNA-cellulose chromatography, and glycerol gradient centrifugation. A rapid filter binding assay was devised to monitor the fractionation procedure on the basis of DNA protein linking activity. The active DNA-cellulose fraction contained both topoisomerase I and topoisomerase II whereas the glycerol gradient purified material contained only topoisomerase II activity. The properties of the active material were studied at both stages of purification. m-AMSA enhanced the formation of complexes between purified topoisomerase II and SV40 DNA in which the DNA sustained a single- or double-strand cut and the enzyme was covalently linked to the 5' terminus of the DNA. This action was further enhanced by ATP, as well as by nonhydrolyzable ATP analogues. m-AMSA inhibited the topoisomerization and catenation reactions of topoisomerase II, probably because of trapping of the enzyme-DNA complexes. The activity showed a dependence on the type of DNA intercalators used, analogous to what was previously observed in intact cells. m AMSA had no effect on topoisomerase I.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006755 TI - Monomers, dimers, and minifilaments of vertebrate skeletal myosin in the presence of sodium pyrophosphate. AB - The self-assembly of myosin in the presence of sodium pyrophosphate was studied in the pH range between 7.0 and 8.5. As evidenced by sedimentation velocity (S0(20,w) = 6.30 S) and light-scattering measurements (molecular weight of 470 000; radius of gyration = 45 nm), myosin existed in a predominantly monomeric form in the presence of 5 mM sodium pyrophosphate at pH 8.5 and above. The concentration-dependent monomer-dimer equilibrium could be easily shifted toward dimeric species at pH 8.0 in the presence of 5 mM sodium pyrophosphate and 5 mM 2 [bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol. The estimated parameters of the dimeric particles were S0(20,w) between 10 and 11 S, molecular weight of 1.1 X 10(6), and radius of gyration = 52 nm. These results are consistent with a head to tail (parallel) arrangement of staggered myosin molecules in the dimer. At lower pH values (7.5), and in the presence of 10 mM sodium pyrophosphate, the monomer-dimer species were in dynamic equilibrium with myosin minifilaments. At pH 7.0, the minifilaments appeared to be the only detectable species present in solutions of myosin in 5 mM sodium pyrophosphate. The molecular parameters of these minifilaments, including sedimentation and viscosity coefficients, molecular weight, radius of gyration, and morphological appearance, were almost indistinguishable from those obtained for myosin minifilaments prepared in 10 mM citrate-tris(hydroxymethyl)aminomethane at pH 8.0 [Reisler, E., Smith, C., & Seegan, G. (1980) J. Mol. Biol. 143, 129-145]. The equilibrium polymerization reactions of myosin in sodium pyrophosphate are discussed in the context of minifilament assembly. PMID- 3006756 TI - Roles of the two copper ions in bovine serum amine oxidase. AB - With a view to obtaining information on the roles of the two copper ions in bovine serum amine oxidase (BSAO), spectroscopic and magnetic studies on several BSAO derivatives have been carried out. Cu-depleted BSAO (Cu-depBSAO) exhibits no enzyme activity and only a low absorption intensity at ca. 475 nm, which is the characteristic absorption maximum of the chromophore in BSAO. The binding of 1 mol of Cu to 1 mol of Cu-depBSAO slightly but definitely increases the enzyme activity and the absorptivity, although they are much lower than those of native BSAO. The incorporation of 2 mol of Cu into Cu-depBSAO gives rise to a similar high activity and absorptivity as those of the native enzyme. Electron paramagnetic resonance (EPR) spectra of the BSAO derivatives reveal that two copper ions in the enzyme molecule are environmentally identical. Titrations of BSAO, Cu-depBSAO, and Cu-half-depleted BSAO (Cu-half-depBSAO), containing 1 mol of copper per mole of protein, with phenylhydrazine (an inhibitor of BSAO) indicate that only 1 mol of phenylhydrazine reacts with 1 mol of the enzyme. In other words the enzyme possesses only one chromophore or one active site, though the molecule is composed of two electrophoretically identical subunits. The binding constants between phenylhydrazine and BSAO, Cu-depBSAO, or Cu-half depBSAO were estimated to be 5 X 10(6), 5 X 10(4), and 1 X 10(5) M-1, respectively. The binding of phenylhydrazine to the chromophore is assisted by the presence of two copper ions by a factor of 100.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006757 TI - Identification of distinct binding site subunits of mu and delta opioid receptors. AB - Iodinated human beta-endorphin was affinity-cross-linked to opioid receptors present in membrane preparations from bovine frontal cortex, bovine striatum, guinea pig whole brain, and rat thalamus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed covalently labeled peptides of 65, 53, 41, and 38 kilodaltons (kDa). The 65- and 38-kDa peptides were present in all four tissues. The 41-kDa peptide was seen only in bovine caudate and guinea pig whole brain while the 53-kDa peptide was absent in rat thalamus. All four labeled peptides were constituents of opioid receptors since their labeling was fully suppressed by the presence of excess opiates, such as bremazocine, during binding. The distribution and levels of the labeled species in the brain tissues examined and, in earlier work, in the neuroblastoma X glioma NG 108-15 cell line suggested that the 65-kDa peptide is a binding component of mu receptors while the 53-kDa peptide is a binding subunit of delta receptors. This result was strongly supported by the finding that the labeling of the 65-kDa peptide is selectively reduced by the presence of the highly mu-selective ligand Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol (DAMGE) during binding, while while the labeling of the 53-kDa peptide is selectively reduced or eliminated by the highly mu selective ligand [D-Pen2, D-Pen5]enkephalin (DPDPE). The labeling of the 41- and 38-kDa bands was reduced by either DAMGE or DPDPE. The relationship of these lower molecular weight opioid-binding peptides to mu and delta receptors is not understood. Several possible explanations are presented. PMID- 3006758 TI - Vasoactive intestinal peptide receptor on liver plasma membranes: characterization as a glycoprotein. AB - The receptor for vasoactive intestinal peptide (VIP) was identified in rat liver plasma membranes after covalent cross-linking to 125I-VIP by three different agents [disuccinimido dithiobis(propionate), disuccinimido suberate, and succinimido 4-azidobenzoate] and examined by sodium dodecyl sulfate-acrylamide electrophoresis. Regardless of the presence of reducing conditions, two molecular species of the putative VIP binding unit were identified as broad autoradiographic bands of 80,000 and 56,000 daltons (Da). Both the large and small species showed the same high affinity for 125I-VIP binding and subsequent cross-linking (half-maximal inhibition at 3 nM unlabeled VIP). The 80-kDa species was partially converted to the 56-kDa form by denaturing conditions and was extensively degraded when incubated at 20 degrees C for 30 min with 1 microgram/mL chymotrypsin, trypsin, or elastase to fragments that that migrated similarly to the 56-kDa unit. In contrast, the 56-kDa moiety was resistant to attack by serine proteases. Both the 80- and 56-kDa species were microheterogeneous due at least in part to the presence of carbohydrate chains, each species binding fractionally to wheat germ agglutinin (WGA)-agarose (approximately 50%). The WGA-bound fraction (eluted with N-acetylglucosamine) was relatively retarded on acrylamide gels as compared to the WGA-unbound fraction. Exposure of the 80- and 56-kDa species to endo-beta-acetylglucosaminidase F reduced the apparent molecular mass of each by 19 kDa, indicating the presence of complex N-linked carbohydrate chains. The receptor species do not appear to have high-mannose N-linked chains since they did not interact with concanavalin A and were not cleaved by endo-beta-acetylglucosaminidase H.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006759 TI - Dependence of the conformational state of the isolated adenine nucleotide carrier protein on the detergent used for solubilization. AB - The mitochondrial adenine nucleotide (AdN) carrier can assume two conformational states that are trapped by the specific inhibitors of AdN transport carboxyatractyloside (CATR) and bongkrekic acid (BA). When the AdN carrier protein was extracted from beef heart mitochondria by the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio)]-1-propanesulfonate (CHAPS) and purified in the same detergent, the fluorescence of the tryptophanyl residue(s) of the protein was partially quenched by ATP (or ADP), but not by nontransportable nucleotides; CATR, which alone was ineffective, was able in the presence of ATP (ADP) to further quench the fluorescence, and BA reversed the quenched fluorescence to the original level. With 3'-O-naphthoyl-ATP (N-ATP) as an extrinsic fluorescence probe, it was shown that BA could release bound N-ATP but that CATR was ineffective. These results indicate that the AdN carrier in CHAPS is able to react readily with BA, but not with CATR. The opposite situation occurs with the carrier solubilized and purified in (laurylamido)-N,N dimethylpropylamine oxide (LAPAO) [Brandolin, G., Dupont, Y., & Vignais, P.V. (1985) Biochemistry 24, 1991-1997]. These data taken together were interpreted to mean that the CATR and BA conformations of the isolated AdN carrier depend on the micellar structure in which it is embedded; the carrier in LAPAO is in the CATR conformation, and the carrier in CHAPS is in the BA conformation. For the transition between the CATR and BA conformations to occur in the carrier in CHAPS and in the carrier in LAPAO, ATP or ADP is required; nontransportable nucleotides were ineffective.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006760 TI - Acetylation of decarboxylated S-adenosylmethionine by mammalian cells. AB - Decarboxylated S-adenosylmethionine was found to be a substrate for the nuclear acetyltransferases that act on polyamines and on histones. The rate of acetylation of decarboxylated S-adenosylmethionine was more than twice that of spermidine at saturating substrate concentrations, and decarboxylated S adenosylmethionine was an active inhibitor of the acetylation of histones by nuclear extracts from rat liver. The acetylation of decarboxylated S adenosylmethionine occurred in vivo in SV-3T3 cells exposed to the ornithine decarboxylase inhibitor 2-(difluoromethyl)ornithine. The decline in putrescine and spermidine brought about by exposure to 2-(difluoromethyl)ornithine was found to be accompanied by a large rise in the content of both decarboxylated S adenosylmethionine and acetylated decarboxylated S-adenosylmethionine. These results indicate that decarboxylated S-adenosylmethionine is metabolized not only in the well-known reactions in which it serves as an aminopropyl donor for polyamine biosynthesis but also by acetylation in reaction with acetyl coenzyme A. Furthermore, the inhibition of histone acetylation by decarboxylated S adenosylmethionine could contribute to the biological effects brought about by inhibitors of ornithine decarboxylase. PMID- 3006761 TI - Replication intermediates formed during initiation of DNA synthesis in methotrexate-resistant CHOC 400 cells are enriched for sequences derived from a specific, amplified restriction fragment. AB - 1-beta-D-Arabinofuranosylcytosine (ara-C) inhibits nuclear DNA replication in Chinese hamster ovary cells by an efficient chain termination mechanism without affecting the rate at which cells traverse G1 and enter S [Heintz, N. H., & Hamlin, J. L. (1983) Biochemistry 22, 3557-3562]. Here we have employed ara-C to enrich for replication intermediates formed during initiation of DNA synthesis in synchronized CHOC 400 cells, a methotrexate-resistant derivative of Chinese hamster ovary cells that contains approximately 1000 copies of an early replicating 150-kb chromosomal domain. This highly amplified domain includes the gene for dihydrofolate reductase (DHFR). CHOC 400 cells were collected at the G1/S boundary of the cell cycle with aphidicolin prior to release into S in the presence of both [methyl-3H] thymidine and various concentrations of ara-C. Chromatographic fractionation of restriction endonuclease digests over benzoylated naphthoylated DEAE-cellulose (BND-cellulose) showed that high concentrations of ara-C inhibited the maturation of chromosomal replication intermediates containing ssDNA (replication forks) into dsDNA for up to 60 min. The effect of ara-C on the sequence complexity of replication intermediates formed during early S phase was determined by hybridizing purified intermediates labeled with 32P in vitro to Southern blots of genomic DNA derived from both methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells. In the absence of ara-C, 32P-labeled ssDNA BND-cellulose fractions from cultures released into S for 30-60 min hybridized to a spectrum of restriction fragments encompassing 40-50 kb of the amplified DHFR domain.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006762 TI - Preparation and characterization of a viral DNA molecule containing a site specific 2-aminofluorene adduct: a new probe for mutagenesis by carcinogens. AB - The synthetic oligonucleotide heptamer 5'-ATCCGTC-3' was reacted in vitro with N acetoxy-N-(trifluoroacetyl)-2-aminofluorene and the resulting product isolated by reverse-phase high-performance liquid chromatography (HPLC). This purified oligonucleotide, which was shown by chemical and enzymatic analysis to be a heptamer containing a single N-(deoxyguanin-8-yl)-2-aminofluorene adduct, was then used to situate the putatively mutagenic aminofluorene lesion within the genome of M13 mp9 by ligating it into a complementary single-stranded region located at a specific site in the negative strand of the duplex M13 mp9 DNA molecule. The presence of the adduct at the anticipated location was confirmed by taking advantage of the facts that AF adducts inhibit many restriction enzymes when located in or near their restriction sites and that the AF moiety should be contained within the HincII recognition sequence on M13 mp9 DNA. Upon attempted cleavage of the M13 DNA containing the site-specific AF adduct with HincII, we find that the large majority of the DNA remained circular, demonstrating the incorporation of the AF adduct in high yield into the DNA molecule at this location. This system should prove useful in vivo for the study of mutagenesis by chemical carcinogens and in vitro to study the interaction of purified DNA metabolizing proteins with a template containing a site-specific lesion. PMID- 3006763 TI - Lipid-protein interactions in cytochrome c oxidase. A comparison of covalently attached phospholipid photo-spin-label with label free to diffuse in the bilayer. AB - The aim of this study was to clarify the possible origins of the motion restricted electron spin resonance (ESR) spectral component observed in membranes. For this purpose, a phospholipid photo-spin-label was synthesized, characterized, and used to study lipid-protein interactions in beef heart cytochrome c oxidase. The probe was designed with a nitroaryl azide incorporated in the phospholipid head group, and a spin-label on the sn-2 side chain, and was radiolabeled. The resulting molecule, 1-palmitoyl-2-(14-proxyl [2-3H]stearoyl)-sn glycero-3-phospho-N-(4-azido-3-nitrophenyl)ethanolami ne, was stable under subdued light and during the procedures required to reconstitute cytochrome c oxidase in phospholipid bilayers. Upon photolysis, the photo-spin-label reacted with the protein in high yields (50% attached). There was no detectable destruction of the spin-label. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytochrome c oxidase after reaction with the photo-spin-label showed highest levels of attachment to bands I, III, and VII, with some labeling of other bands. The labeling pattern demonstrated a distribution of attachment sites, which was needed for the spin-labeling studies. ESR spectra of the attached labels at 25 degrees C indicated a constant fraction of motion restricted lipid chains, independent of the lipid to protein ratio. In contrast, a spin-labeled phosphatidylcholine and the prephotolyzed photo-spin-label, both free to diffuse in the bilayer, exhibited behavior in agreement with the multiple equilibria binding model. These results, as well as data obtained with membranes frozen at -196 degrees C, show how several situations that lead to a motion restricted ESR line shape can be distinguished. This study provides additional evidence that the fraction of lipids normally in contact with protein, and not aggregation artifacts, accounts for the observed motion-restricted component of ESR spectra of reconstituted cytochrome c oxidase in phospholipid bilayers. PMID- 3006764 TI - Identification of polypeptides of the phencyclidine receptor of rat hippocampus by photoaffinity labeling with [3H]azidophencyclidine. AB - Polypeptide components of the phencyclidine (PCP) receptor present in rat hippocampus were identified with the photolabile derivative of phencyclidine [3H]azidophencyclidine ( [3H]AZ-PCP). The labeled affinity probe was shown to reversibly bind to specific sites in the dark. The number of receptor sites bound is equal to those labeled by [3H]PCP, and their pharmacology and stereospecificity are identical with those of the PCP/sigma-opiate receptors. The dissociation constant of [3H]AZ-PCP from these receptors is 0.25 +/- 0.08 microM. Photolysis of hippocampus membranes preequilibrated with [3H]AZ-PCP, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed the existence of five major labeled bands of which a Mr 90 000 band and a Mr 33 000 band were heavily labeled. Inhibition experiments, in which membranes were incubated with [3H]AZ-PCP in the presence of various PCP analogues and opiates, indicate that labeling of both the Mr 90 000 band and the Mr 33 000 band is sensitive to relatively low concentrations (10 microM) of potent PCP/sigma receptor ligands, while similar concentrations of levoxadrol, naloxone, morphine, D-Ala-D-Leu enkephalin, atropine, propranolol, and serotonin were all ineffective. Stereoselective inhibition of labeling of the Mr 90 000 band and of the Mr 33 000 band was also observed by the use of dexoxadrol and levoxadrol. The Mr 33 000 band was not as sensitive as the Mr 90 000 band to inhibition by the selective PCP receptor ligands N-[1-(2-thienyl)cyclohexyl]piperidine and PCP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006765 TI - Kinetic studies suggest that light-activated cyclic GMP phosphodiesterase is a complex with G-protein subunits. AB - Cyclic GMP phosphodiesterase (PDE) in rod disk membranes has three subunits of molecular weight 88 000 (alpha), 84 000 (beta), and 13 000 (gamma). Physiological activation of the enzyme by light is mediated by a GTP binding protein (G protein). The enzyme can also be activated by controlled digestion with trypsin, which destroys the gamma subunit, leaving the activated enzyme as PDE alpha beta [Hurley, J. B., & Stryer, L. (1982) J. Biol. Chem. 257, 11094-11099]. Addition of purified gamma subunit to PDE alpha beta inhibited the enzyme fully. This suggested the possibility that G protein could also activate PDE by removing the gamma subunit and leaving the active enzyme in the form of PDE alpha beta. Should this be true, the properties of light- and trypsin-activated enzymes should be comparable. We found this not to be the case. The Km of light-activated enzyme for cyclic GMP was about 0.9-1.4 mM while that of trypsin-activated enzyme was about 140 microM. The cyclic AMP Km was also different for the two enzymes: 6.7 mM for light-activated enzyme and 2.0 mM for trypsin-activated enzyme. The inhibition of both enzymes by the addition of purified gamma subunit also differed significantly. Trypsin-activated enzyme was fully inhibited by the addition of about 200 nM gamma, but light-activated enzyme could not be fully inhibited even with 2600 nM inhibitor subunit. The Ki of the trypsin-activated enzyme for gamma was 15 nM and of the light-activated enzyme 440 nM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006766 TI - Photosystem I charge separation in the absence of centers A and B. II. ESR spectral characterization of center 'X' and correlation with optical signal 'A2'. AB - The Photosystem I electron acceptor complex was characterized by optical flash photolysis and electron spin resonance (ESR) spectroscopy after treatment of a subchloroplast particle with lithium dodecyl sulfate (LDS). The following properties were observed after 60 s of incubation with 1% LDS followed by rapid freezing. (i) ESR centers A and B were not observed during or after illumination of the sample at 19 K, although the P-700+ radical at g = 2.0026 showed a large, reversible light-minus-dark difference signal. (ii) Center 'X', characterized by g factors of 2.08, 1.88 and 1.78, exhibited reversible photoreduction at 8 K in the absence of reduced centers A and B. (iii) The backreaction kinetics at 8 K between P-700, observed at g = 2.0026, and center X, observed at g = 1.78, was 0.30 s. (iv) The amplitudes of the reversible g = 2.0026 radical observed at 19 K and the 1.2 ms optical 698 nm transient observed at 298 K were diminished to the same extent when treated with 1% LDS at room temperature for periods of 1 and 45 min. We interpret the strict correlation between the properties and lifetimes of the optical P-700+ A2 reaction pair and the ESR P-700+ center X- reaction pair to indicate that signal A2 and center X represent the same iron-sulfur center in Photosystem I. PMID- 3006767 TI - Cellular localization of sulfobromophthalein transport activity in rat liver. AB - The movement of sulfobromophthalein is measured in rat liver plasma-membrane vesicles by direct dual-wavelength spectrophotometry. The technique is based on the principle that the dye, when entering a more acidic compartment, changes its absorption in the visible region. From this study it may be concluded that, among the different cellular subfractions, only liver plasma-membrane vesicles can catalyze electrogenic transport of sulfobromophthalein. Plasma membranes from erythrocytes are unable to perform such a function. The movement follows the distribution pattern of (Na+ + K+)-ATPase and it is therefore concluded that this process occurs exclusively at the sinusoidal membrane level. Inhibition studies confirm that the process is catalyzed by bilitranslocase. PMID- 3006768 TI - Contamination of a cardiac sarcolemmal preparation with endothelial plasma membrane. AB - Preparation of sarcolemma from whole rabbit heart using the method of Jones et al. (Jones,L.R., Besch, H.R., Fleming, J.W., McConnaughey, M.M. and Watanabe, A.M. (1979) J. Biol. Chem. 254, 530-539) results in a 46-fold purification of the endothelial plasmalemma-specific marker angiotensin converting enzyme. This implies contamination of the sarcolemma with vascular endothelial plasmalemma. During preparation of sarcolemma from sheep heart, using the same method, angiotensin converting enzyme copurified with the general plasma membrane marker (Na+ + K+)-ATPase. The ratio of myocyte to endothelial plasma membrane in the final preparation is therefore similar to that in the whole heart homogenate. Ultrastructural analysis has shown that the myocyte/endothelial surface area is 70:30 in whole cardiac muscle. Comparison of angiotensin converting enzyme activity of an endothelial plasma membrane fraction with that of whole heart sarcolemma suggests an upper limit of 42% for endothelial contamination. Contamination by endothelial plasmalemma was dramatically reduced by preparing sarcolemma from myocytes produced by proteolytic disruption of whole hearts. Following disruption, myocytes were separated from non-muscle cells by sedimentation through 0.5 M sucrose. Sarcolemma prepared from sheep cardiac myocytes had approximately 15-fold less angiotensin converting enzyme activity than whole sheep heart sarcolemma but comparable ouabain-inhibitable (Na+ + K+) ATPase activity. PMID- 3006769 TI - Orientation of synaptic plasma membrane vesicles containing calcium pump and sodium-calcium exchange activities. AB - Sidedness of synaptic plasma membrane vesicles isolated from brain synaptosomes has been assessed by two distinct experimental approaches: first, analysis of (Na+ + K+)-ATPase, Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities before and after permeabilization of vesicles; second, analysis of Ca2+ fluxes via the Na+/Ca2+ exchanger, before and after modification of an imposed Na+ gradient by penetrating or nonpenetrating Na+ channel-modifying drugs. 0.05% saponin, which completely permeabilizes the vesicles, increases digitoxigenin-sensitive (Na+ + K+)-ATPase, basal Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities by 51.0, 47.4, and 83.6%, respectively. Saponin increases only the Vmax of the latter activity, the Km for Ca2+ (0.13 microM; the same as that for Ca2+-pumping) being unaltered by saponin. An increment of 20.5% in the Vmax of (Ca2+ + Mg2+)-ATPase activity with 10 microM A23187, reveals that the enzyme activity in nonpermeabilized vesicles is limited by the formation of a Ca2+ gradient. Thus, the saponin induced increment in (Ca2+ + Mg2+)-ATPase due only to exposure of occluded sites (as opposed to Ca2+ gradient dissipation) is actually 52%, which is similar to values for both other ATPases, and suggests that 32-35% of plasma membranes exist in an inverted orientation. Vesicle orientation was independently assessed by the differential actions of tetrodotoxin (a membrane impermeant blocker) and veratridine (a membrane permeant agonist) on Na+-channel opening measured indirectly by dissipation of an imposed Na+ gradient utilized to drive a large 45Ca2+ accumulation via the Na+/Ca2+ exchanger. Tetrodotoxin reverses 35-44% of veratridine-mediated Na+ gradient-dissipation, the relative membrane-permeability of the two channel modifiers, suggesting that 56-65% of sealed vesicles are inverted. The concurrence of these two independent measurements of vesicle orientation reinforces their validity. PMID- 3006770 TI - Insulin stimulates renal glomerular sodium-potassium adenosine triphosphatase activity. AB - The effect of insulin on total and ouabain-inhibited membrane-bound adenosine triphosphatase (ATPase) activity in renal glomeruli isolated from adult white rats was examined. In concentrations of 1-10 micrograms/ml, insulin significantly stimulated the ouabain-inhibited (Na+ + K+)-ATPase activity, without affecting total (composite) ATPase activity. These results, coupled with previous findings demonstrating that glomerular (Na+ + K+)-ATPase activity is reduced in acute streptozotocin diabetes, suggest that the renal glomerulus is a target tissue with respect to this biologic effect of insulin. PMID- 3006772 TI - Evidence for proteic water pathways in the luminal membrane of kidney proximal tubule. AB - The osmotic permeability of the apical membrane of proximal tubule cells was studied on rat brush-border membrane vesicles by following their rate of shrinkage with a stopped-flow device coupled to light transmission recording. The mercuric sulfhydryl reagent para-chloromercuribenzenesulfonic acid (PCMBS) reduced the water permeability of the membrane, in a time- and dose-dependent manner, to 35% of the control value. Mercuric chloride was a more potent inhibitor and decreased the osmotic water permeability of the brush-border membrane to 15% of the control. This inhibition was reversed by an excess of cysteine, while cysteine per se did not modify the rate of vesicle shrinkage. These results suggest that most of the osmotic water movements across kidney brush-border membranes are through polar pathways which involve the integrity of the membrane proteins. PMID- 3006771 TI - Protein blot analysis of virus receptors: identification and characterization of the Sendai virus receptor. AB - Receptors for Sendai virions in human erythrocyte ghost membranes were identified by virus overlay of protein blots. Among the various erythrocyte polypeptides, only glycophorin was able to bind Sendai virions effectively. The detection of Sendai virions bound to glycophorin was accomplished either by employing anti Sendai virus antibodies or by autoradiography, when 125I-labeled Sendai virions were used. The binding activity was associated with the viral hemagglutinin/neuraminidase (HN) glycoprotein, as inferred from the observation that the binding pattern of purified HN glycoprotein to human erythrocyte membranes was identical to that of intact Sendai virions. No binding was observed when blots, containing either human erythrocyte membranes or purified glycophorin, were probed with the viral fusion factor (F glycoprotein). Active virions competed effectively with the binding of 125I-labeled Sendai virions (or purified HN glycoprotein), whereas no competition was observed with inactivated Sendai virus. The results of the present work clearly show that protein blotting can be used to identify virus receptors in cell membrane preparations. PMID- 3006773 TI - Trifluoperazine inhibits Sendai virus-induced hemolysis. AB - Sendai virus-induced hemolysis, a manifestation of virus-red cell fusion, is inhibited by exposure of the virus to 50 microM and higher concentrations of trifluoperazine. Trifluoperazine does not disrupt the virus, since trifluoperazine-treated virus with no hemolytic activity sediments slightly faster than untreated virus on sucrose density gradients and contains viral proteins in proportions characteristic of untreated virus. Trifluoperazine affects the fusion protein to a greater extent than the hemagglutinin, since trifluoperazine-treated virus with no hemolytic activity is as active or nearly as active in agglutinating red cells. The partition coefficient of trifluoperazine between the virus membrane and buffer is lower at 4 degrees C than, but the same at 37 degrees C, as that between the red cell membrane and buffer. Nevertheless, virus-independent red cell lysis and inactivation of virus mediated hemolysis occur when the red cell and viral membranes, respectively, contain similar concentrations of trifluoperazine. Furthermore, 13-28% more trifluoperazine is necessary to achieve either effect at 4 degrees C or at 25 degrees C than at 37 degrees C. Changes in the surface activity of trifluoperazine do not explain these results, insofar as the critical micellar concentration of (0.75 mM) and maximal reduction in surface tension by (40 dyn/cm) trifluoperazine are the same at 25 degrees C and 37 degrees C. The fluorescence of viral tryptophan decreases by approx. 25% when viral hemolysis is inactivated by trifluoperazine, by trypsin treatment or by heating at 100 degrees C for 5 min. PMID- 3006774 TI - An unusual pattern of Na+ and K+ movements across the horse erythrocyte membrane. AB - Marked differences in the activities of three monovalent cation transport systems in horse versus human erythrocytes are reported. Whereas horse erythrocytes exhibit a 6-fold higher sodium-lithium countertransport, the unidirectional flux of potassium through the sodium pump is 3-4 times slower and the sodium-potassium cotransport system cannot be detected. In spite of this, horse and human cells are able to maintain similar Na+ and K+ gradients. PMID- 3006775 TI - DNA topoisomerase I from rat brain neurons. AB - The DNA topoisomerase I has been isolated from neurons of rat cerebral cortex. The most homogeneous fraction purified contains only one polypeptide of Mr approx. 100 000. The enzyme relaxes supercoiled DNA in the absence of ATP or Mg2+. The optimum monovalent cation concentration for the relaxation of superhelical DNA under conditions of DNA excess is found to be 175-200 mM. The neuron enzyme is similar to other mammalian type I DNA topoisomerases in that it links to the 3' ends of the broken DNA strands. Like calf thymus DNA topoisomerase I, the neuron topoisomerase can be selectively inhibited by poly(dG) but not by other homopolymerical deoxyribonucleotides. PMID- 3006776 TI - Purification of the Gin recombination protein of Escherichia coli phage Mu and its host factor. AB - Inversion of the G-segment of Escherichia coli phage Mu was studied in vitro. The reaction requires the Gin recombination protein, which was purified to near homogeneity from overproducing cells. Upon purification the protein lost activity, which was restored by addition of an extract from uninfected E. coli cells. The stimulatory host factor is a small heat-stable protein and was purified from E. coli cells. Full recombination required both proteins, but Gin alone promoted some recombination by itself, particularly at high concentrations. Relaxation of negative supercoils and recombination of a substrate with two recombination sites in an inverted orientation both have the same specificity for Gin and the host factor. The Gin-associated topoisomerase activity appears tightly coupled to its recombination activity. PMID- 3006777 TI - Binding to tubulin of an allocolchicine spin probe: interaction with the essential SH groups and other active sites. AB - EPR titration of tubulin with an allocolchicine spin probe showed more than one binding site: one high-affinity binding site (Kd = 8 microM), consistent with the Ki found for competition with colchicine, and one or more low-affinity site(s) (Kd higher than 50 microM). No disturbance of the EPR signal of the tubulin-bound allocolchicine spin probe could be observed at room temperature in the presence of other paramagnetic probes: Mn(II) for the binding site of Mg(II), Co(II) for the Zn(II) binding site and Cr(III)GTP for the binding site of the exchangeable GTP. Labelling of tubulin with both the allocolchicine and a SH-group spin probe also showed lack of interaction. The colchicine-binding site is thus sterically isolated from the binding sites for GTP, Mg(II), Zn(II) and the two essential SH groups. In the tubulin-colchicin complex, all SH-groups could still be labelled with an excess of the SH-reagent, N-ethylmaleimide. Furthermore, colchicine binding was only minimally influenced by the blocking of the two essential SH groups. However, the rate constant of the reaction of two equivalents of the SH reagent, a maleimide spin probe, with the tubulin-colchicine complex was only 50% of the rate constant found with uncomplexed tubulin. As direct steric interaction of the essential SH-groups with the colchicine-binding site can be excluded, we can now definitively decide that binding of colchicine to tubulin induces a conformational change, which affects the accessibility of the most reactive SH groups. PMID- 3006778 TI - Chemical modification of arginine residues of porcine muscle acylphosphatase. AB - Acylphosphatase (acylphosphate phosphohydrolase, EC 3.6.1.7) from porcine skeletal muscle is inactivated by phenylglyoxal following pseudo-first-order kinetics. The dependence of the apparent first-order rate constant for inactivation on the phenylglyoxal concentration shows that the inactivation is also first order with respect to the reagent concentration. Among the competitive inhibitors for the enzyme examined, inorganic phosphate and ATP almost completely, and Cl- partially, protect the enzyme against the inactivation. The dissociation constants for inorganic phosphate and ATP determined from protection experiments by these inhibitors agree well with those from inhibition experiments by them. These results support the idea that the modification occurs at the phosphate-binding site. The amino-acid analysis reveals the lack of reaction at residues other than arginine. Circular dichroism spectra of the modified enzymes show that the inactivation seems not to be due to denaturation of the enzyme resulting from the modification of the non-essential arginine residues. The relationship between the loss of the enzyme activity and the number of arginine residues modified in the presence and absence of ATP shows that one arginine residue is possibly responsible for the inactivation of acylphosphatase. PMID- 3006780 TI - Characterization of the light-induced increase in the Michaelis constant of the cGMP phosphodiesterase in frog rod outer segments. AB - Activation of cGMP phosphodiesterase in rod disk membrane in the light is thought to be an intermediary process of phototransduction. In various preparations of frog rod outer segments, the Michaelis constant (Km) of the phosphodiesterase was measured with pH assay method. On illumination, the Km increased from the value of the dark (130 microM) by about 8-fold (1 mM) in crude preparations, but did not change appreciably in purified disk membranes, confirming the previous finding by Robinson et al. (Robinson, P.R., Kawamura, S., Abramson, B. and Bownds, M.D. (1980) J. Gen. Physiol. 76, 631-645). The present work further showed that the light-induced Km increase is labile to various experimental manipulations such as sonication, freeze-thawing, etc. However, the Km in the light was relatively high in a crude disk membrane preparation and in a lyzed preparation. In addition, reconstitution experiments revealed that the Km increase does not require soluble components. Both proteolytic digestion and phospholipase treatment reduced the light Km of the phosphodiesterase in crude disk membranes. The above results suggest that there is a labile factor(s) responsible for the light-induced Km increase and that the factor is a membrane bound protein and requires structural integrity of the disk membrane to exert its function. The latency of the Km increase after light stimulation was less than 2 s. PMID- 3006779 TI - Galactose-specific lectin in the hemolymph of solitary ascidian, Halocynthia roretzi. Molecular, binding and functional properties. AB - Galactose-specific lectin isolated from the hemolymph of solitary ascidian, Halocynthia roretzi, has been further characterized. The hemagglutinating activity of the lectin is Ca2+-dependent. The lectin has a large molecular form as revealed by gel-permeation chromatography, sedimentation equilibrium and velocity measurement, and electron microscopic observation. The lectin is adsorbed to columns of blue-Sepharose and phenyl-Sepharose, and eluted with ethylene glycol, not with lactose or high concentration of NaCl. The lectin shows a stimulatory effect on the superoxide anion production by guinea-pig polymorphonuclear leukocytes, and the effect is inhibited, among various sugars, most strongly by melibiose. PMID- 3006781 TI - Purification of canine surfactant-associated glycoproteins A. Identification of a collagenase-resistant domain. AB - A procedure for purification of surfactant-associated glycoproteins A from canine surfactant was established utilizing preparative isoelectric focusing as a major purification step in absence of detergents. The proteins migrated as charge trains, isoelectric points 4.2-5.0. Unglycosylated forms of surfactant-associated protein A1 (26 kDa) and glycoproteins A2 and A3 (32-36 kDa) were identified by silver-staining and immunoblot analysis. These forms were demonstrated to be identical polypeptides by fingerprint analysis of 125I-labeled peptides generated by tryptic-chymotryptic digests of the iodinated proteins. Size and charge heterogeneity were related to varying amounts of N-linked complex carbohydrates, including sialic acid, which were sensitive to endoglycosidase F and neuraminidase but resistant to endoglycosidase H. A collagenase-sensitive region was demonstrated which was required for sulfhydryl-dependent oligomerization of the molecule. Collagenase treatment resulted in removal of approx. 10 kDa from the glycoprotein molecule. Collagenase-resistant fragments of 21-23 kDa migrated with carbohydrate-dependent size and charge heterogeneity and were reduced to 16 kDa by endoglycosidase F. Amino acid composition of the surfactant glycoproteins demonstrated high glycine content which was diminished after digestion with collagenase. Several glycine-rich tryptic peptides were isolated by reverse-phase chromatography. Partial sequence information shows Gly-X-Y repeat sequences containing hydroxyproline residues. The major canine surfactant-associated proteins, glycoproteins A contain complex-type N-linked carbohydrate. In addition, a separate collagen-like peptide domain is present and is required for sulfhydryl-dependent oligomerization. PMID- 3006783 TI - An ESR study of the effect of an electrostatic field on binding of divalent cations to the surface of serum low-density lipoproteins. AB - The ESR technique has been used to study binding of Mn(II) ions to low-density lipoprotein (LDL) in solutions of various electrolyte ionic strengths. A model of the binding has been proposed which describes all the observations in electrolytes of ten different concentrations in terms of two types of binding sites and two corresponding sets of intrinsic binding parameters (n1 = 8, Kd1 = 1.31 X 10(-3) mol X l-1 and n2 = 170, Kd2 = 5.71 X 10(-2) mol X l-1). These parameters, together with the values of the potential (phi 0) responsible for binding of the ions to specific charged sites on the surface, reproduce the observed binding curves well in all the systems studied. The phi 0 values are obtained as an appropriate solution of the Poisson-Boltzmann equation. PMID- 3006782 TI - The stoichiometry of oxygen uptake and conjugated diene formation during the dioxygenation of linoleic acid by the pure reticulocyte lipoxygenase. Evidence for aerobic hydroperoxidase activity. AB - Simultaneous measurements of oxygen uptake and conjugated diene formation (increase in the absorbance at 234 nm) during the dioxygenation of linoleic acid by the pure reticulocyte lipoxygenase gave a nearly theoretical stoichiometry of 1.1 in a temperature range from 5 to 30 degrees C and a wide range of concentrations of both oxygen and linoleic acid. At low concentrations of either oxygen or linoleic acid or both, secondary processes occurred such as linoleic acid-supported lipohydroperoxidase reactions leading to the disappearance of conjugated dienes and to the formation of oxodienes, linoleic acid dimers and epoxyhydroxy derivatives. Under these conditions marked deviations of the stoichiometry between oxygen uptake and conjugated diene formation appeared. The formation of conjugated oxodienoic fatty acids absorbing at 285 nm occurred only under conditions of high concentrations of linoleic acid and limiting oxygen supply. The results indicate that lipohydroperoxidase reactions catalyzed by the pure reticulocyte lipoxygenase do not only take place under strictly anaerobic conditions but also under conditions of limiting concentrations of either linoleic acid or oxygen or both. PMID- 3006785 TI - Specific saturable binding of rat high-density lipoproteins to rat kidney membranes. AB - The binding of rat 125I-labelled high-density lipoprotein (HDL) to rat kidney membranes was studied using HDL fractions varying in their apolipoprotein E content. The apolipoprotein E/apolipoprotein A-I ratio (g/g) in the HDL fractions ranged from essentially 0 to 1.5. All these HDL preparations showed the same binding characteristics. The saturation curves, measured at 0 degrees C in the presence of 2% bovine serum albumin, consisted of two components: low-affinity non-saturable binding and high-affinity binding (Kd about 40 micrograms of HDL protein/ml). Scatchard analyses of the high-affinity binding suggest a single class of non-interacting binding sites. These sites could be purified together with the plasma membrane marker enzyme 5'-nucleotidase. The binding of rat HDL to rat kidney membranes was not sensitive to high concentrations of EDTA, relatively insensitive to pronase treatment and influenced by temperature. The specific binding of rat HDL was highest at acid pH and showed an additional optimum at pH 7.5. On a total protein basis unlabelled rat VLDL competed as effectively as unlabelled rat HDL for binding of 125I-labelled rat HDL to partially purified kidney membranes. Rat LDL, purified by chromatography on concanavalin A columns and human LDL did not compete. Unlabelled human HDL was a much weaker competitor than unlabelled rat HDL and the maximal specific binding of 125I-labelled human HDL was only 10% of the value for 125I-labelled rat HDL. PMID- 3006784 TI - Effect of diabetes and Sorbinil treatment on phospholipid metabolism in rat glomeruli. AB - To explore the hypothesis that changes in membrane phospholipids accompany tissue myo-inositol depletion and reduced (Na+ + K+)-ATPase activity in diabetes, we examined phospholipid concentrations in glomeruli isolated from control and streptozotocin-diabetic rats and the effect of diabetes on myo-[3H]inositol incorporation in vitro into glomerular phosphatidylinositol. Since the aldose reductase inhibitor, Sorbinil, prevents the fall in myo-inositol and the decrease in (Na+ + K+)-ATPase activity associated with diabetes, phospholipid and phosphatidylinositol content were also examined in glomeruli isolated from Sorbinil-treated diabetic rats. Total phospholipids (microgram phosphorus/mg dry weight) did not differ in the three groups of animals. The concentration of phosphatidylcholine was elevated in preparations from diabetic rats, both untreated and Sorbinil-treated. Phosphatidylethanolamine was reduced in glomeruli from Sorbinil-treated rats. Neither acute experimental diabetes nor Sorbinil treatment produced detectable changes in the glomerular concentration of phosphatidylinositol. In vitro incubations with glomeruli isolated from control and diabetic animals resulted in increased levels of incorporation of myo [3H]inositol into phospholipids of diabetic glomeruli. The specific activity of [3H]phosphatidylinositol in glomeruli from diabetic rats was significantly greater than that in control samples. The findings do not support the postulate invoking correspondent changes in myo-inositol and phosphatidylinositol contents as contributory to diminished glomerular (Na+ + K+)-ATPase activity in diabetes, but are compatible with depletion of glomerular intracellular myo-inositol in diabetes. PMID- 3006787 TI - Growth hormone-binding proteins in high-speed cytosols of multiple tissues of the rabbit. AB - Soluble, specific binding protein(s) for growth hormone (GH) have been identified and partially characterized in high-speed cytosolic preparations from a number of rabbit tissues. The binding of 125I-labelled human GH to proteins in liver, heart, adipose tissue, skeletal muscle and kidney cytosols was dependent on time and cytosolic protein concentration. By Scatchard analysis, the binding affinities (KA: (2-7) X 10(9) M-1) were somewhat higher than those generally reported for membrane GH receptors. The binding proteins had a greater specificity for somatotrophic hormones than lactogenic hormones, although the kidney appeared to have, in addition, a lactogen-binding protein. By gel filtration, the Mr of the cytosolic GH-binding protein was approximately 100 000 in all tissues. None of the binding proteins was detectable by the poly(ethylene glycol) precipitation method used widely for soluble hormone receptors. The cytosolic GH-binding proteins also cross-reacted with a monoclonal antibody to the rabbit liver membrane GH receptor. These results indicate the ubiquitous presence of apparently naturally soluble GH-binding proteins in the cytosolic fractions of several tissues in the rabbit. Of great interest is their presence in muscle, where GH receptors or binding proteins have not previously been detected, despite muscle being recognized as a classical GH target tissue. PMID- 3006786 TI - A gel-electrophoretic analysis of protein ADP-ribosylation in polyoma virus transformed and non-transformed BHK-21/C13 fibroblasts. AB - We have examined a variety of conditions for solubilizing and electrophoresing cell proteins in order to define optimum conditions for studying proteins modified by ADP-ribosylation. We have identified conditions in which proteins can be quantitatively extracted from cells in an undegraded form with the protein ADPribose linkages intact. Effective measures include boiling cells briefly (4 min) in the presence of 2% SDS and 2 M urea at pH 6.8. Both SDS and urea were present in the 6-18% gradient polyacrylamide gel matrix used for electrophoresis. Under these conditions good resolution of proteins of a wide molecular-weight range is obtained. This system has been used to compare protein ADP-ribosylation in non-transformed and polyma virus-transformed baby hamster kidney (BHK) fibroblasts, since the latter cells have a greater NAD+ ADP-ribosyltransferase activity (measured in isolated nuclei and permeabilized cells). Addition of DNAase to permeabilized BHK cells over the range 10-150 micrograms led to a progressively greater activation of transferase compared with controls. When PyY cells were used, however, maximum activation was achieved with only 10 micrograms of DNAase, further additions producing a successively smaller activation relative to control cells without added nuclease. There were also differences between these cells in response to salt. Addition of NaCl (to about 0.3 M) to BHK cells resulted in various extents of transferase activation, whereas any addition of NaCl to the incubate of permeabilized PyY cells decreased transferase activity. These different enzyme activities between this transformed and non-transformed cell line are for the most part not reflected in the protein modification profiles seen on autoradiograms of acrylamide gels after electrophoresis 32P labelled proteins. A variety of proteins are modified and their molecular weights depend on the NA concentration in the permeabilized cell incubation. At 0.5 microM NAD+ there were two major acceptors with Mr values of 14 kDa and 30 kDa, and at 100 microM NAD+, three major acceptors, with Mr values of 19 kDa. 45 kDa and greater than 170 kDa. NAD concentrations of between 1 microM and 100 microM had no further effect on protein ADP-ribosylation profiles, except for the protein(s) of Mr greater than 170 kDa, pointing to a critical difference around 0.5-1.0 microM substrate. In some experiments, however, a difference was observed in the intensity of radioactivity in two bands. This may represent two different proteins, or a single protein modified to different extents.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3006788 TI - Comparative reactivity of carbamyl phosphate and glucose 6-phosphate with the glucose-6-phosphatase of intact microsomes. AB - The ability of glucose 6-phosphate and carbamyl phosphate to serve as substrates for glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase; EC 3.1.3.9) of intact and disrupted microsomes from rat liver was compared at pH 7.0. Results support carbamyl phosphate and glucose 6-phosphate as effective substrates with both. Km values for carbamyl phosphate and glucose 6-phosphate were greater with intact than with disrupted microsomes, but Vmax values were higher with the latter. The substrate translocase-catalytic unit concept of glucose-6-phosphatase function is thus confirmed. The Km values for 3-O-methyl-D-glucose and D-glucose were larger when determined with intact than with disrupted microsomes. This observation is consistent with the involvement of a translocase specific for hexose substrate as a rate-influencing determinant in phosphotransferase activity of glucose-6-phosphatase. PMID- 3006789 TI - Local changes in fractional saturation of cGMP- and cAMP-receptors in intestinal microvilli in response to cholera toxin and heat-stable Escherichia coli toxin. AB - Cyclic nucleotide modulation of electrolyte transport across intestinal brushborder membranes is initiated by binding of cGMP and cAMP to high-affinity receptors at the interior of the microvilli. Previously these receptors have been identified by photoaffinity-labelling techniques as regulatory domains of cGMP- and cAMP-dependent protein kinases. In the present study, the receptor concentration in isolated brushborder membrane vesicles and their fractional saturation in absorptive and secretory states of the tissue were estimated. In microvillous membrane vesicles isolated from rat small intestine in the absorptive state, about 10% of the total number of cGMP receptors (25.5 pmol/mg protein) and 40% of all cAMP receptors (28.7 pmol/mg protein) were occupied by endogenous cyclic nucleotides. Luminal exposure of the intestinal segments in vivo to heat-stable Escherichia coli toxin for 3-5 min increased the occupancy of cGMP receptors by about 5-fold without affecting receptor-bound cAMP levels. In contrast, incubation with cholera toxin for 2 h increased the fractional saturation solely of cAMP receptors by 2-fold. Addition of heat-stable E. coli toxin to cholera toxin-pretreated segments, again raising the cGMP levels by 5 fold, did not reduce the amount of receptor-bound cAMP. This finding argues against the concept that increased levels of cAMP during cholera would mimick cGMP effects on ion transport by low-affinity binding to microvillar cGMP receptors. This analysis of local changes in cyclic nucleotide levels at the microvillous level might help to explore the mechanism of action of other secretagogues or antidiarrhoeal agents and to delineate a possible compartmentation of cGMP and cAMP pools within the intestinal mucosa responding differently to external signals. PMID- 3006790 TI - Further comparison of calmodulin-dependent protein kinase II from brain and calmodulin-dependent glycogen synthase kinase from skeletal muscle. AB - Calmodulin-dependent protein kinase II was purified from rabbit brain and its properties were compared with those of calmodulin-dependent protein kinase II from rat brain and calmodulin-dependent glycogen synthase kinase from rabbit skeletal muscle. Rabbit brain calmodulin-dependent protein kinase II was clearly distinguished from rabbit skeletal muscle glycogen synthase kinase with respect to size, behavior on autophosphorylation, immunological cross-reactivity and peptide mapping, but was indistinguishable from rat brain calmodulin-dependent protein kinase II in all respects examined. Thus, differences between calmodulin dependent protein kinase II and glycogen synthase kinase appear not to reflect a species difference but to reflect a tissue difference. PMID- 3006791 TI - [Determination of the degree of collagen coiling by electron paramagnetic resonance]. AB - It has been shown that kinetics of the death of free radicals UV-induced at 77 degrees K in collagen is determined by two reactions having different rates. Such shape of the kinetic curve is substantiated by the spatial structure of macromolecules and permits to find easily the portion of peptide chains in the helical form and the portion of end peptides not incorporated in this structure. The degree of helical pattern of collagen from rat skin was shown to be 92%. PMID- 3006792 TI - [Synthetic Ca++-dependent proton carrier]. AB - Influence of 1,5-(3,3'-dimethylphosphate)diphenoxy-3-oxapentane (DDOP) on conductivity (G) of bilayers of common fabbit brain lipids is studied. It has been found that DDOP increases the bilayer conductivity in the presence of Ca++ and Mg++ (G-maximum at pH = 7.0) they do not act in the presence of K+, Na+. pK'DDOP, pK"DDOP values are equal to 1.2, and 7.7 respectively as determined by titration. Formation of "pseudomacrocyclic" DDOP structure is suggested. The role of Ca++, Mg++ ions seems to consist in lipophilisation of ionized forms of DDOP. PMID- 3006793 TI - [Mechanism of electron transfer between myoglobin derivatives and ferricytochrome C]. AB - Progress in the studies of the electron transport mechanism in biological systems is greatly hindered by the lack of detailed structural information about the components of these systems. That is why a study of electron transfer between protein molecules with the known spatial organization in model reactions in vitro is of great importance. In this respect the MbO2--Cyt C oxidation-reduction reaction offers unique possibilities. Studies of the effects of pH and ionic strength of the medium on the kinetics of this reaction in combination with chemical modification of single amino acid residues of Mb and Cyt C enabled us to identify those parts of the surface of haemoproteins where the molecules come into "active contact". A variation in the number or/and the arrangement of the charged groups at the "active sites" of the molecules induced by both changing the medium pH and chemical modification of some of these groups lowers markedly the probability of electron transfer in the system (e.g. His GH1 and His A10 in Mb) or blocks it entirely (acylation of Lys 72 (73) or Tyr 74 in Cyt C). Based on the results obtained and on the data of Mb and Cyt C X-ray analysis, the figures of spatial arrangement of the groups at the "active sites" of these molecules are presented. PMID- 3006794 TI - [Relation between the lipid composition and changes in the compressibility of phospholipid monolayers caused by substitution of H2O with D2O]. AB - Compressibility of monolayers of dipalmitoyl phosphatidyl choline, dimyristoyl phosphatidyl choline, egg lecithin, triolein, azolectine, phosphatidyl serine and total brain lipids formed on air--H2O or air--D2O interfaces is investigated. PMID- 3006796 TI - [Vasoactive intestinal peptide (VIP): a ubiquitous neuropeptide member of structural family of regulatory peptides]. PMID- 3006795 TI - [Effect of cholesterol on the physical state and function of lymphocyte membranes]. AB - Effect of gradual increase of cholesterol content in T-lymphocyte membranes on the structure and physical state of plasmic membrane lipids and activities of the membrane-bound enzymes was investigated. The increase in cholesterol content was shown to result in a two-phase change of luminescence parameters of the fluorescent probes dimethylaminochalcone and pyrene, which indicates heterogeneity of cholesterol in the membranes. With the growth of steroid content in the cell membranes, at first, we observed a sharp decrease in the lipid bilayer fluidity and inhibition of Na+, K+-ATPase activity, which at the molar ratio cholesterol/phospholipids 0.6 in thymocyte membranes, remains at the same level. With higher cholesterol concentrations ATPase activity did not change. The effect of cholesterol on ATPase activity was in a good agreement with the effect of membrane lipids on fluidity. It is suggested that two pools of cholesterol molecules exist in the membranes, differing in their effects of bilayer fluidity and functional activity of the membranes. PMID- 3006798 TI - A sequence from Drosophila DNA cross-hybridizing with a mouse class I H-2 gene: absence of relevant nucleic acid or amino acid sequence homology. AB - Following serological data (1) showing cross-reactivity of drosophila surface antigens with anti-H-2 and mouse beta 2-microglobulin (beta 2-m) antisera, we have looked for homologous sequences in the drosophila genome by low stringency hybridization with mouse H-2 and beta 2-m probes. A 240 bp drosophila DNA segment cross-hybridizing with a H-2 probe, was isolated and sequenced. No homology at the nucleotide or amino acid levels was found with the mouse probe which was used, except for a perfectly matching 16-mer containing 11 G-C, which might be responsible for the cross-hybridization. Therefore, our present data do not support the existence of class I H-2 and/or beta 2-m related gene sequences in the drosophila genome. PMID- 3006797 TI - Modification of HT 29 cell response to the vasoactive intestinal peptide (VIP) by membrane fluidization. AB - We used liposomes made with phospholipids of fatty acid chain length ranging from C12:0 to C16:0 to modify the cAMP dependent protein kinase (PK) activity of HT 29 cells induced by VIP or forskolin. Both VIP and forskolin effects were inhibited in dilauroylphosphatidylcholine (DLPC) treated cells. PK activity was slightly lowered when cells were treated by dimyristoylphosphatidylcholine (DMPC) liposomes. However neither VIP nor forskolin-induced PK activities were affected with dipalmitoylphosphatidylcholine (DPPC) liposomes. Furthermore, the binding of [125I]VIP to DLPC treated cells was drastically lowered whereas no change was observed when cells were incubated with DMPC or DPPC liposomes. On the other hand, the interaction of HT 29 cells with DLPC vesicles provoked a decrease in membrane cholesterol content with subsequent increase in membrane fluidity. These findings provide evidence that, in HT 29 cells, the mechanisms of VIP-receptor interaction and of adenylate cyclase activation is lipid dependent and is regulated by membrane fluidity. PMID- 3006799 TI - [Interaction of subunits of cAMP-dependent protein kinase with structural elements of the cell nucleus]. AB - Using the method of protein transfer from polyacrylamide gel to nitrocellulose filters with subsequent incubation of filter-adsorbed protein with [32P]DNA, it was found that the catalytic subunit of cAMP-dependent protein kinase from porcine brain is capable of interacting with DNA to form a stable complex. This complex is resistant even to 2 M NaCl. The ability of the catalytic subunit to interact with DNA depends on the degree of enzyme nativity. The regulatory subunit of cAMP-dependent protein kinase does not bind to DNA both in the presence and absence of cAMP. The 125I-labeled regulatory subunit can interact with some chromatin proteins, in particular, with histone H1 and core histones. An essential role in this binding belongs to electrostatic and hydrophobic interactions. PMID- 3006800 TI - [Effect of sheep haptoglobin on the hemoglobin molecule in the Hp-Hb complex]. AB - Sheep haptoglobin (HpC) binding hemoglobin increases the stability of the latter to acid denaturation and oxidation by atmospheric O2. HpC is also capable of binding methemoglobin (MetHb) denaturated at pH 3.5 to form a stable complex. This process is accompanied by partial reconstitution of the structural integrity and peroxidase activity of MetHb. Consequently, the formation of a HpC-MetHb complex leads to changes in the tertiary structure of the MetHb molecule. The increase in the peroxidase activity of MetHb at pH less than or equal to 4.0 after its binding to HpC is due to the stabilizing and stimulating activity of HpC. PMID- 3006801 TI - [Changes in cAMP reception in the cytosol of the developing rat kidney]. AB - The age-dependent changes in the cAMP-binding activity of renal papillary cytosol were studied in intact rats and in animals with experimental delay in development of the concentrating renal function induced by hydrocortisone injection in early postnatal period. The age dynamics of specific cAMP binding in the experimental group differ significantly from that in intact and control (sham injected) animals. It is suggested that the developmental changes in experimental animal kidneys are due to disturbances in hormonal regulation of renal tissue differentiation caused by the changes in intracellular receptors of cAMP. Another possible reason for the observed delay of development of the renal function is the weakening of the stimulating effect of endogenous cAMP on the genome function due to cAMP receptor deficiency in the cell. PMID- 3006803 TI - Renal bicarbonate handling in low birth weight infants during metabolic acidosis. AB - We studied the fractional excretion of bicarbonate (FE HCO-3) in 10 low birth weight infants aged 1-6 days during metabolic acidosis (base excess greater than or equal to -5 mEq/l) and during subsequent sodium HCO-3 infusion. The mean birth weight was 1,095 g; the mean gestational age was 29 weeks. The ability to decrease urine pH to less than 5.5 and FE HCO-3 to less than 1% during metabolic acidosis was not limited by low gestational age or birth weight. After HCO-3 therapy, all infants corrected their negative base excess, and plasma HCO-3 increased significantly. All infants with blood pH less than or equal to 7.22 or PaCO2 greater than or equal to 50 mm Hg had minimal or absent FE HCO-3. Infants with elevated PaCO2 and mild or absent acidosis also had complete HCO-3 tubular reabsorption. These results suggest that the HCO-3 tubular reabsorption is adequate during metabolic and/or respiratory acidosis in low birth weight infants. PMID- 3006802 TI - [Energy characteristics of the transport of monovalent cations in ascites tumor cells]. AB - The intensity of O2 adsorption by ascite tumour cells does not practically depend on the monovalent cation concentration gradient between the cells and the incubation medium, whereas the rate of glycolysis decreases simultaneously with the diminution of the concentration gradient. In synchronized cultures at the beginning of the mitotic cycle, the bulk of ATP resynthesized via glycolysis is utilized for the synthesis of biopolymers, whereas that at the end of the S-phase and in the G2-phase--for cation transport across plasma membranes. From 35 to 100% of the whole amount of ATP resynthesized via glycolysis is utilized for transport purposes. The experimental results and theoretical calculations suggest that in glucose-containing media Na+ transport increases from 0.75 to 1.78 pmol/hour on a per cell basis. The activation of Na+ transport is due to the exchange of protons formed via glucose conversion into lactate for Na+, i.e., to the stimulation of Na+/H+ antiport. The permeability of plasma membranes for K+ increases 2.75-fold, while the passive flux of Na+ diminishes. It is concluded that the observed increase in the Na+/K+ ratio in ascite tumour cells is connected with their enhanced ability to synthesize lactic acid. Presumably, glycolysis is one of regulatory mechanisms of intracellular ratios of monovalent cations. PMID- 3006804 TI - Desmethylimipramine treatment increases melatonin production in humans. PMID- 3006805 TI - Changes in serum prolactin concentrations and ovarian prolactin receptors during embryonic diapause in mink. AB - Experiments were conducted to determine if prolactin receptors were present in the mink ovary, and to examine the relationship between receptor numbers and serum levels of prolactin (PRL) during embryonic diapause and blastocyst reactivation. For analysis of the physicochemical properties of prolactin receptors, ovaries were obtained from anestrous mink. All binding determinations were made using 125I-ovine prolactin (125I-oPRL), and 20 micrograms of tissue protein from the 100,000 X g particulate fraction. To quantify prolactin receptors during gestation, 20 primiparous mink were mated twice on consecutive days between 4 and 10 March and assigned randomly to one of two groups. Mink in Group 1 (N=8) were killed on 13 March when blastocysts were completing their migration into the uterus and entering a state of diapause. Animals in Group 2 (N=10) were killed on 26 March during the period of blastocyst reactivation, just prior to implantation. To determine serum levels of prolactin during gestation, an additional 20 primiparous mink were similarly mated and bled every 4 days from 15 March to 8 April, and then every 7 days until 23 April. Prolactin concentrations were determined by a heterologous double antibody radioimmunoassay using porcine PRL for both tracer and standards. Optimum conditions for binding 125I-oPRL to ovarian membranes were attained at 25 degrees C after 12 h. Scatchard analysis revealed a single class of high affinity binding sites with a Kd of 6.14 X 10(-11) M. The total concentration of receptors during anestrus was 85 fmol/mg protein, which increased significantly during embryonic diapause to 484 fmol/mg protein, then declined to 16 fmol/mg during blastocyst reactivation. PMID- 3006806 TI - Changes in Leydig cell ultrastructure and function during pubertal development in the boar. AB - Changes in the ultrastructure of Leydig cells during pubertal development in the boar (40 to 250 days of age) were assessed using quantitative morphometric procedures, and the results were compared to the in vitro steroid-producing capacity and gonadotropin sensitivity of testicular tissue obtained from the same boars. Volume of individual Leydig cells declined through 100 days of age, increased rapidly to a peak at 130-160 days (i.e., puberty), and then declined to intermediate levels by 220-250 days of age. The pattern of change in the number of intracellular organelles per Leydig cell was very similar to the change that occurred in Leydig cell volume. Changes in the total intracellular volume occupied by each type of organelle were highly correlated with changes in Leydig cell volume (r = 0.40-0.99, p less than 0.01), and this was particularly true for the nucleus (r = 0.63), mitochondria (r = 0.88), smooth endoplasmic reticulum (SER; r = 0.97), and total cytoplasm (r = 0.99) of the boar Leydig cell. In vitro production of testosterone and estradiol, expressed per Leydig cell, also peaked at 130-160 days, and was highly correlated to average Leydig cell volume, volume of SER, and number and total volume of mitochondria (r = 0.63-0.84; p less than 0.01). Observations in the present study indicated that onset of puberty in boars coincides with a dramatic increase in average Leydig cell size and SER volume per Leydig cell, accompanied by an increase in number of other intracellular organelles, including mitochondria, lysosomes, and lipid droplets, and a peak in the steroid-producing capacity per Leydig cell. A decline in Leydig cell size, intracellular organelles, and sensitivity to gonadotropin stimulation occurred postpubertally. PMID- 3006807 TI - Neonatal release of gonadotropins is essential for development of ovarian follicle-stimulating hormone receptors. AB - In the rat, ovarian follicle-stimulating hormone (FSH) receptors increase markedly during the first two postnatal weeks, when serum gonadotropin levels are most elevated. This study was conducted to evaluate the hypothesis that these high gonadotropin levels, and in particular FSH, are involved in the acquisition of FSH receptors by the developing ovary. Gonadotropin release was suppressed by administration of several non-aromatizable androgens, among which dihydrotestosterone propionate (DHTP) was the most effective. In one series of experiments the steroids were administered from Days 5 to 11, and serum FSH and luteinizing hormone (LH) were measured on Day 12. Surprisingly, FSH receptor content was greater in rats with suppressed serum gonadotropins than in controls. The greatest increase in available receptors was observed in DHTP-treated rats in which serum FSH was reduced to 20% of control values and LH suppressed to undetectable values. DHTP failed to directly increase available FSH receptors in hypophysectomized immature rats. Magnesium chloride (MgCl2) treatment of ovarian membranes removed bound 125I-hFSH by 87% without affecting receptor viability. Exposure of control 12-day-old ovaries to MgCl2 increased available FSH receptors to a level similar to that of ovaries from DHTP-treated rats not exposed to MgCl2, suggesting that more receptors were available in DHTP-treated rats because serum FSH was suppressed. Earlier initation of DHTP treatment (postnatal Day 1) suppressed serum FSH and LH to undetectable values by Day 5 and decreased FSH receptor content below control values by Day 12. MgCl2 treatment only slightly increased available receptors in these DHTP-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006808 TI - Prepubertal changes in the hypothalamic-pituitary axis of Holstein bulls. AB - We investigated the nature and sites of changes in the hypothalamic-pituitary axis associated with the onset of high-frequency, high-amplitude discharges of luteinizing hormone (LH) in young bulls during the transition from the infantile to the prepubertal phase of development. Blood serum and neuroendocrine tissues from bulls killed at 1, 6, 10, 14, or 18 wk of age were evaluated. Concentrations of LH in serum from bulls 1 or 6 wk old averaged less than 0.25 ng/ml and only one episodic discharge of LH was detected for 10 bulls. At 10, 14, or 18 wk, 14 of 15 bulls had episodic discharges of LH. Concentrations of testosterone in serum were progressively higher at 10, 14, and 18 wk, but the concentration of estradiol was maximal at 6 wk. The concentrations of gonadotropin-releasing hormone (GnRH) in the anterior hypothalamus, posterior hypothalamus, or median eminence were not influenced by age. However, concentration of GnRH receptors in the anterior pituitary gland increased 314% between 6 and 10 wk and the concentration of LH increased 67%. Between 6 and 10 wk, concentrations of estradiol receptors in the anterior and posterior hypothalamus declined by 68% and 46%, but the concentration of estradiol receptors in the anterior pituitary gland increased by 103%. For most characteristics, there was no major change between 10 and 18 wk. We postulate that between 6 and 10 wk of age, there is 1) removal of an estradiol-mediated block of GnRH secretion and 2) an estradiol mediated, and possibly GnRH-mediated, increase in pituitary GnRH receptors. Together, these changes result in greatly increased stimulation of the anterior pituitary gland by GnRH between 6 and 10 wk of age and stimulation of the discharges of LH characteristic of bulls in the early prepubertal phase of development. PMID- 3006810 TI - Retention of the speract receptor by isolated plasma membranes of sea urchin spermatozoa. AB - Membrane vesicle preparations enriched in plasma membrane marker proteins, such as adenylate cyclase, were prepared from spermatozoa of the sea urchin, Lytechinus pictus. These membranes, prepared by nitrogen cavitation and subsequent sucrose gradient centrifugation, retained the capacity to bind [125I] Bolton-Hunter speract (nonspecific binding was less than 5% of specific binding). Speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), Tyr-Asp-Leu-Asn-Gly-Gly-Gly Val-Gly, Tyr-Asp-Leu-Thr-Thr-Gly-Gly-Gly-Val-Gly and Gly-Phe-Ala-Leu-Gly-Gly-Gly Val-Gly caused a 50% decrease in [125I]-Bolton-Hunter speract binding at 10, 600, 1260 and 3160 nM concentrations, respectively. One analogue (Phe-Asp-Leu-Asn-Gly Gly-Gly), which had no biological activity, failed to compete at concentrations as high as 10 microM. To demonstrate that the binding was due to the isolation of membranes with an intact receptor, the speract analogue (Gly-Gly-Gly-Gly-Tyr-Asp Leu-Asn-Gly-Gly-Gly-Val-Gly) was synthesized, radiolabeled with 125I at the position of tyrosine, and covalently cross-linked to the receptor with disuccinimidyl suberate. A single radiolabeled band at an apparent molecular weight of 77,000 was detected on Na X dodecyl X SO4 gels. These studies are the first to identify a receptor for egg-associated peptides in isolated spermatozoan membranes. PMID- 3006811 TI - Human haptoglobin adsorption by a total internal reflection fluorescence method. AB - Haptoglobin (Hp) is one of the major protein constituents of plasma. Three different forms are found in the population. The 1-1 and 2-2 forms adsorb similarly onto hydrophobic silica [treated with dimethyl dichlorosilane (DDS)] and onto clean silica, although the affinities on the silica surface are lower at 60 minutes contact time. The two forms desorb differently from silica, but desorb similarly from DDS-silica. Adsorption is less reversible on the hydrophobic surface. Due to its adsorbtion tendencies and its high concentration in plasma, the adsorption of Hp may be important in blood interaction at solid-liquid interfaces. PMID- 3006809 TI - Synergistic interactions of somatomedin-C with adenosine 3',5'-cyclic monophosphate-dependent granulosa cell agonists. AB - Recent studies have demonstrated the ability of somatomedin-C (Sm-C) to synergize with follicle-stimulating hormone (FSH) in the activation of cultured rat granulosa cell progesterone biosynthesis as well as the induction of luteinizing hormone (LH) receptors. Neither effect could be attributed to Sm-C-enhanced granulosa cell survival or replication, but could be accounted for, in part, by increased adenosine 3',5'-cyclic monophosphate (cAMP) generation. The present study was undertaken to determine if the synergistic property of Sm-C is FSH selective and hence limited in relevance to follicular maturation, as well as to clarify further the role of cAMP in Sm-C-amplified agonist action. To this end, the ability of Sm-C to modulate the hormonal action of a series of physiologic as well as pharmacologic granulosa cell agonists was examined in vitro using cultured granulosa cells from immature, hypophysectomized, diethylstilbestrol treated rats. Concurrent treatment with highly purified Sm-C (50 ng/ml) resulted in marked increases over controls in the LH-stimulated [1 ng human chorionic gonadotropin (hCG)]-and beta 2-adrenergic-stimulated (10(-6) M terbutaline) accumulation of cAMP (3.8- and 2.6-fold, respectively and progesterone (3.2- and 7.4-fold, respectively). Similarly, concurrent treatment with Sm-C also augmented the vasoactive intestinal peptidergic stimulation of granulosa cell cAMP generation (4.1-fold) and progesterone biosynthesis (2.1-fold). In contrast, Sm-C was incapable of enhancing progesterone accumulation in response to stimulation with rat prolactin, a cAMP-independent granulosa cell agonist.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006812 TI - Short and long range silicate release from doped bioglass into 199 medium. PMID- 3006814 TI - Hydrophobic ion interactions with membranes. Thermodynamic analysis of tetraphenylphosphonium binding to vesicles. AB - The thermodynamic properties for the interaction of the hydrophobic ion tetraphenylphosphonium (TPP+) with egg phosphatidylcholine vesicles were studied in detail by equilibrium dialysis and spin label techniques. A partition coefficient of beta = 4.2 + 0.4 x 10(-6) cm (K congruent to 100) was determined. Electrostatic saturation sets in at approximately 600 microM (about one absorbed TPP+ molecule per 100 lipids), and is not screened by salt. The temperature dependence of binding was determined, which reveals that the binding is entropy driven with a positive (repulsive) enthalpy of binding, a result to be compared with hydrophobic anions in which the binding enthalpy is negative. The membrane dipole potential may be responsible for this binding difference. Activity coefficients are determined and shown to be significantly different from those of most common salts, an important result that should be considered in all hydrophobic ion studies. Comparison of the TPP+ results with those of its anionic structural analogue, tetraphenylboron (TPB-), permits a general analysis of hydrophobic ion interactions with membranes. A theoretical model consistent with the entire set of data is developed in an accompanying article. PMID- 3006813 TI - Cold shock hemolysis in human erythrocytes studied by spin probe method and freeze-fracture electron microscopy. AB - When human erythrocytes are osmotically stressed or chemically treated, they hemolyze on cooling below 10 degrees C (called cold shock). We have studied the effects of osmotic stress and cooling on the state of membrane by the spin-probe method and freeze-fracture electron microscopy. At room temperature, the membrane fluidity detected by 12-doxyl stearate spin probe showed a steady decrease with osmolality in hypertonic NaCl solutions up to 900 mOsm/kg, above which it remained unchanged. In hypertonic sucrose solutions, the electron paramagnetic resonance spectra showed an additional pair of absorptions, indicating development of regions, in the membrane, further immobilized than in NaCl solutions. Mobility of a cholesterol analogue probe, androstane, did not show change by hypertonicity, but the spectral intensity dropped at 1,200 mOsm/kg, probably due to formation of loose aggregates in the cholesterol phase. On cooling the osmotically stressed cells in NaCl solution, the isotropic rotational correlation time vs. inverse temperature plot of 12-doxyl stearate probe exhibited a step-wise discontinuity at approximately 10 degrees C, suggestive of a drastic transition in the state of the membrane. At about the same temperature, the freeze-fracture pattern of osmotically stressed cells revealed the development of large wrinkles and aggregation of membrane particles, in contrast to the case of the cells in isotonicity. Significance of these findings in understanding cold shock hemolysis is discussed. PMID- 3006816 TI - A molecular mechanical study of netropsin-DNA interactions. PMID- 3006817 TI - AIDS and haemophilia. AB - Approximately 1% of all AIDS cases are haemophiliacs. LAV/HTLV-III is transmitted by blood and in factor VIII concentrates. Since 1981, increasing numbers of haemophiliacs have been infected, as indicated by detection of antibodies to LAV/HTLV-III. Up to 90% of haemophiliacs in some populations are now seropositive, but to date less than 1% have progressed to clinical AIDS. Immunological abnormalities, in particular reduced T-lymphocyte helper/suppressor ratios, are common in haemophiliacs treated with factor VIII. Such abnormalities do not necessarily indicate past infection by the AIDS virus, but they may predispose to infection following exposure to the virus. Blood Transfusion agencies are introducing screening tests for antibodies to LAV/HTLV-III to help prevent the spread of AIDS by blood products. Heat treated factor VIII concentrate is now available and appears not to transmit AIDS. Factor IX concentrate may also transmit LAV/HTLV-III, but less frequently. A few cases of AIDS have occurred in Haemophilia B (Christmas Disease) patients. PMID- 3006815 TI - Investigation of surface potential asymmetry in phospholipid vesicles by a spin label relaxation method. AB - In earlier work, Castle and Hubbell (1976) demonstrated the use of a spin-labeled amphiphile as a probe for the electrostatic potential at the outer surface of charged phospholipid vesicles. In recent experiments, we have shown that the hydrophobic anion tetraphenylboron (TPB) promotes transbilayer migration of the probe molecule. Relaxation data recorded following the rapid mixing of the probe with TPB-containing vesicle samples provides information about the electrostatic potentials at both the outer and inner vesicle surfaces. The measured potentials for both surfaces of asymmetrically screened vesicles were found to be in good agreement with theoretical values calculated using their known surface charge density. The method is also sensitive to transmembrane potentials as indicated by the response of the label to potentials created with the use of potassium concentration gradients and valinomycin. PMID- 3006818 TI - [LAV virus infections in children]. AB - The clinical signs and immunological abnormalities in ten babies with lymphadenopathy associated virus infections (LAV) are described. We have made this diagnosis based on the association of specific immunological abnormalities, serological tests or the positive isolation of the virus. In one very severe case, whose mother died of AIDS, the serology and attempts to isolate the virus were both negative. Excluding the two post-transfusion cases, the ethnic and familial history always suggested the diagnosis. The clinical characteristics were very close to those in adults, being hepatosplenomegaly and generalized lymphadenopathy. There was an associated retardation of growth and weight gain. A decrease of the absolute number of OKT4(+) lymphocytes and hypergammaglobulinaemia were the abnormalities most frequently observed. The degree of immunodeficiency, both humoral and cellular was very variable. The existence of LAV infection during the first few months of life suggests a maternal-foetal transmission, but a post-natal infection cannot be eliminated. It is difficult to decide the long-term prognosis; it depends mainly on the degree of the immune deficiency. PMID- 3006820 TI - C1r serine proteinase of human complement: a case of intramolecular autolytic activation. AB - This paper presents a short review of our contribution to the knowledge of the structure and function of human C1r, the activation unit of C1, the first component of the classical pathway of complement. On the basis of the domain structure of C1r, a model accounting for its autolytic activation mechanism is proposed. We suggest that this represents the basic mechanism of C1 function. PMID- 3006819 TI - HLA restriction fragment length polymorphisms associated with insulin-dependent diabetes mellitus and the seronegative spondyloarthropathies. PMID- 3006822 TI - Changes in topoisomerase I activity after irradiation of lymphoid cells. AB - The activity of topoisomerase I in nuclear extracts increased about three-fold 5 min after gamma-irradiation (840-2500 rads) of human peripheral blood lymphocytes or cultured lymphoblastoid cells. The change may reflect modification of the enzyme by nuclear ADP-ribosyl transferase, which is known to be activated by DNA breaks. PMID- 3006821 TI - Genes for the protein antigens of the tuberculosis and leprosy bacilli. AB - The lambda gt 11 expression vector permitted us to survey protein antigens of Mycobacterium leprae and Mycobacterium tuberculosis expressed in Escherichia coli. Using monoclonal antibodies, recombinant clones were detected producing three major antigens of M. tuberculosis and five major protein antigens of M. leprae. These recombinant antigens produced in E. coli should prove useful for diagnosis, epidemiology and possibly the development of recombinant mycobacterial vaccines. PMID- 3006823 TI - Rat carcinoma cells in long-term, serum-free culture provide a continuing supply of collagenase. AB - Neoplastic, epithelial cells derived from a spontaneously-arising rat mammary carcinoma have been cultured in a defined medium, in the absence of serum, continuously, for over 2 years. The medium is a mixture of Ham's F12 and Dulbecco's Modified Eagle's media supplemented with insulin, transferrin and bovine serum albumin. The cells have retained their potential to produce tumours and, in culture, a true vertebrate collagenase. This system provides a continuing supply of vertebrate collagenase through the application of recently developed methods. PMID- 3006825 TI - [Enkephalin and cyclic nucleotide content in the brain structures of rats at different stages of the formation and development of alcoholic dependence]. AB - Rats with increased alcohol motivation have been found to have a rise in enkephalin levels in limbic cortex and a decrease in met-enkephalin levels in the brain basal ganglia. Reduction of met-enkephalin to leu-enkephalin ratio in basal ganglia, limbic cortex and hypothalamus may serve as an index of increased inclination to ethanol in these animals. Alcohol dependence is characterized by reduced cAMP content in the majority of brain structures studied, sharply decreased met-enkephalin levels in limbic cortex and hypothalamus, and diminished cAMP and cGMP content in hypothalamus. In the third stage of experimental alcoholism the partial normalization of met-enkephalin and cAMP levels is observed in brain structures, with cGMP content increased in hypothalamus and considerably reduced in basal ganglia. PMID- 3006824 TI - [Hormonal activity of the ACTH(4-10) analog--a prolonged-action stimulant of learning]. AB - Possible hormonal activity of ACTH4-7, a long-acting ACTH4-10 analog (ProGly Pro) was studied. Unlike ACTH5-10 the peptide did not reveal steroidogenic and melanocyte-stimulating activity. PMID- 3006826 TI - [Interaction of thymosin fraction 3 with opiate receptors]. AB - Using the radioreceptor method thymosin fraction 3 was demonstrated to displace 3H-morphine and 3H-naloxone from opiate receptor of crude membrane fraction of the rat brain. The value of EC50 ratio in the presence and absence of 100 mM NaCl seems to indicate that thymosin contains one or several partial agonists of morphine. Furthermore, the peptide nature of these ligands is suggested on the basis of enzymatic treatment data. The possible role of opioid peptides in the realization of thymus endocrine functions is discussed. PMID- 3006827 TI - [2 types of GABA receptors in the intact olfactory bulb and primordial hippocampus of the frog: the pharmacological data]. AB - The in vivo effects of (+/-) baclofen (10(-6)-10(-4) M), muscimol (10(-5)-10(-4) M), and (+) bicuculline (10(-4)-10(-3) M) applications on evoked potentials in the olfactory bulb (OB) and primordial hippoccamp (PH) were studied in frogs. Baclofen was found to decrease drastically postsynaptic components of OB orthodromic potential and to slightly increase OB antidromic potential. Muscimol decreased only the second component of OB orthodromic potential and OB antidromic potential. Baclofen and muscimol decreased all the components of PH orthodromic potential, but for the first positive one. All the effects were reversible and dose-dependent. Bicuculline antagonized muscimol effects, without affecting baclofen effects in both structures under study. The results suggest the presence of two types of GABA-receptors in OB and PH of frogs. GAGAB-receptors are shown to be located on the primary olfactory afferents and to be responsible for presynaptic inhibition in OB. PMID- 3006828 TI - [Effect of dimedrol on the function of the neuromuscular synapse and the generation of action potentials by motor nerve fibers]. AB - Microelectrode registration of synaptic potentials in the frog cutaneous-pectoris muscle has shown dimedrol (7.9 X 10(-5) M) to act on synaptic transmission decreasing the quantal content, estimated by mean EPP amplitude to mean miniature EPP amplitude ratio, the quantal content calculated by variation coefficient of EPP amplitude being unaffected. The data suggest possible transmitter release and depletion of mediator stock. The experiments on isolated motor nerve fibers have demonstrated dimedrol to cause the increase in transmitter release probability by widening the action potentials in the terminals and thus enhancing Ca2+ influx. PMID- 3006829 TI - Monoclonal antibodies binding to the human neutrophil C3bi receptor have disparate functional effects. AB - Three monoclonal antibodies (MAb)--OKMI, 7C3, and 60.3--immunoprecipitated a common 170-kd neutrophil membrane antigen closely associated with, or identical to, the C3bi receptor (CR3). Despite binding to a common receptor, these antibodies displayed marked differences in their effects on C3bi-mediated neutrophil function as assessed by the binding and ingestion of opsonized zymosan and the subsequent triggering of the respiratory burst. Antibody 7C3 caused a time-dependent, irreversible inhibition of the neutrophil oxidative response to opsonized zymosan that correlated with capping of the bound antibody. In contrast, antibody 60.3 caused an immediate inhibition of the neutrophil oxidative response to opsonized zymosan that required the continuous presence of exogenous antibody to achieve the maximal inhibitory effect. Antibody OKMI demonstrated minimal inhibition of O2- release. Despite their functional differences, binding of either 7C3 or 60.3 led to up-regulation of new antigen, presumably from intracellular sites as previously described using OKMI. Crossed immunoprecipitations of radiolabeled neutrophil lysates indicated that each MAb bound to different antigens near or within the CR3 complex. Thus three MAb binding to the neutrophil CR3 receptor each caused receptor up-regulation but had markedly different functional effects on the cell. PMID- 3006830 TI - Activation of human granulocytes by arachidonic acid: its use and limitations for investigating granulocyte functions. AB - The effects of arachidonic acid on human granulocytes (polymorphonuclear neutrophil leukocytes, PMN) were examined with respect to early events associated with activation of the superoxide (O2-)-generating system and its reactivation with other stimuli after the addition of fatty acid-free bovine serum albumin. As with other stimuli, O2- production occurred after a lag that was dependent on the amount of arachidonic acid used. Although the lag, rate, and extent of O2- production were dose dependent, at all concentrations used, the duration of O2- production was four to six minutes. As previously described, when fatty acid-free albumin was added one minute after stimulation of PMN by arachidonic acid, O2- production ceased immediately. The O2(-)-generating system could then be reactivated by the addition of either phorbol myristate acetate (PMA), concanavalin A, or serum-treated zymosan. In previously activated cells, the lag time for reactivation was shorter and the rate of O2- production greater with PMA. PMN membrane potential was depolarized by arachidonic acid. This depolarization was not reversed with the addition of albumin. PMN cytoplasts were also capable of reversible activation by arachidonic acid in a manner identical to whole cells. Divalent cations were found to be necessary for the activation of PMN by arachidonic acid. In the absence of calcium, arachidonic acid caused rapid lysis of the cells, as manifested by the release of lactic acid dehydrogenase. We were unable to measure later events in PMN stimulated by arachidonic acid since even in the presence of divalent cations lactic acid dehydrogenase was released from the cells after five minutes of incubation. We conclude that, in addition to its ability to activate PMN, arachidonic acid is toxic to the cells and cannot be used to study late events in granulocyte activity, and, in the absence of calcium, it may be difficult to interpret its action as a stimulant of the oxidase. PMID- 3006832 TI - Use of oligonucleotide hybridization in the characterization of a beta zero thalassemia gene (beta 37 TGG----TGA) in a Saudi Arabian family. AB - Analysis of restriction site polymorphisms in the beta-globin gene cluster of a Saudi Arabian female with beta zero-thalassemia demonstrated that both of her beta-globin genes were missing a nonpolymorphic AvaII site in exon 2. Examination of the normal nucleotide sequence surrounding this AvaII site revealed that either of two nucleotide substitutions, TGG----TAG or TGG----TGA, could produce a nonsense codon at codon 37 and eliminate the AvaII site. Consequently, two oligonucleotides (19-mers spanning codons 36 through 41 and containing either TAG or TGA at codon 37) were synthesized and hybridized against genomic DNA of the proband and her family. Specific hybridization with one of the oligomers demonstrated that the patient's beta o-thalassemia was the result of homozygosity for the TGG----TGA mutation at codon 37. In certain cases, oligonucleotide hybridization using genomic DNA may obviate the need for gene cloning and sequencing in the characterization of point mutations. PMID- 3006831 TI - Cytomegalovirus infection after bone marrow transplantation: an association with acute graft-v-host disease. AB - Among 181 patients undergoing allogeneic bone marrow transplantation over a five year period (1978 through 1982), cytomegalovirus (CMV) infection was a frequent and often lethal complication. Recipient pretransplant serology was the most important predictor of posttransplant CMV infection. CMV infection occurred in 26/137 seronegative recipients and in 28/44 seropositive recipients (P less than .001). Among patients who developed CMV infection, the time to infection was identical in seronegative and seropositive patients (median, 71 days post transplant). Bone marrow donor CMV serology did not significantly influence CMV infection rate. CMV infection was strongly associated with acute graft-v-host disease (AGVHD), occurring in 34/81 patients with AGVHD and 20/100 without GVHD (P less than .001). AGVHD preceded CMV infection by 33.7 days (mean) in patients developing both complications. Patients who developed CMV infections had also received more cellular blood products post transplant. These data suggest that CMV infection may occur through reactivation of latent virus (in seropositive recipients) or through exogenous exposure, possibly through transfused blood products, but that duration of immunoincompetence may be more critical than route of exposure in timing of clinically evident CMV infection. Prophylaxis tailored to the likely infectious source and more effective GVHD prevention both may be critical in preventing CMV infection after bone marrow transplantation. PMID- 3006833 TI - Myeloperoxidase biosynthesis by a human promyelocytic leukemia cell line: insight into myeloperoxidase deficiency. AB - The biosynthesis and processing of myeloperoxidase (MPO), a cationic enzyme present in the azurophilic granules of human polymorphonuclear leukocytes (PMNs), were studied in the human promyelocytic leukemia cell line, HL-60. HL-60 cells produce large quantities of enzymatically active MPO that has the same electrophoretic behavior as MPO isolated from normal PMNs. Mature MPO is a glycoprotein of approximately 150,000 molecular weight (mol wt) composed of two heavy-light protomers (alpha 2 beta 2) with subunits of 59,000 and 13,500 mol wt, respectively, under reducing conditions. The primary translation product of MPO messenger RNA (mRNA) isolated from HL-60 cells was a single polypeptide of mol wt 80,000. In HL-60 cells labeled with [35S]-methionine, the labeled MPO isolated by immunoprecipitation had a mol wt of 89,000. Treatment of this 89-kilodalton (kDa) species with endoglycosidase H produced a 79-kDa peptide, suggesting that the 89 kDa protein contained high-mannose side chains. The 89-kDa species had no detectable peroxidase activity. During chase experiments some of the 89-kDa peptide was processed to smaller species of mol wt 39,000, 59,000, and 13,500, although a fraction of the 89-kDa peptide remained unprocessed after a chase of 100 hours. In addition, a small amount of the 89-kDa peptide appeared in the medium without any of the processed smaller peptides. These studies suggest that the primary translation product in MPO biosynthesis is an 80-kDa peptide that undergoes cotranslational cleavage of the signal peptide and glycosylation to produce an 89-kDa pro-MPO, that pro-MPO is a single polypeptide containing the alpha and beta subunits of MPO and contains endoglycosidase H-susceptible high mannose side chains, and that posttranslational modification of pro-MPO results in targeting to the lysosome and proteolytic maturation of pro-MPO to active enzyme. In light of the previous observation that MPO-deficient and normal PMNs contain an 89-kDa protein immunochemically related to MPO, these studies on MPO biosynthesis indirectly support the hypothesis that defective posttranslation processing by pro-MPO may underlie hereditary MPO deficiency. PMID- 3006834 TI - Deficient induction of leukotriene synthesis in human neutrophils by lipoxygenase deficient platelets. AB - The effect of human platelets with deficient lipoxygenase activities on leukotriene B4 (LTB4) synthesis by neutrophils was studied. When arachidonic acid (AA) metabolites obtained from the incubation of washed normal neutrophils and platelets with N-formylmethionylleucylphenylalanine (FMLP), cytochalasin B, and AA were analyzed by reversed-phase high-performance liquid chromatography, the synthesis of 5-lipoxygenase products, including LTB4, was remarkably stimulated by platelets, with their maximal effect at a ratio of platelets to neutrophils of 15:1. However, the use of lipoxygenase-deficient platelets obtained from four patients with myeloproliferative disorders instead of normal platelets showed the deficient production of 5-lipoxygenase-derived products, whereas platelets with normal lipoxygenase activities obtained from MPD patients stimulated the 5 lipoxygenase pathway similarly to the way in which normal platelets did. The addition of 12-hydroperoxyeicosatetraenoic acid (12-HPETE), a labile AA metabolite via the platelet lipoxygenase pathway, could activate the 5 lipoxygenase pathway in neutrophils incubated with FMLP, cytochalasin B and AA, but its stable end product, 12-hydroxyeicosatetraenoic acid, could not. Thus, it is suggested that lipoxygenase-deficient platelets did not sufficiently stimulate LTB4 synthesis during platelet-neutrophil interactions because of defective formation of 12-HPETE. This altered interaction between platelets and neutrophils through the lipoxygenase pathway might result in deficient responses at sites of thrombosis or inflammation in patients with deficient platelet lipoxygenase activities. PMID- 3006835 TI - Chemoattractant-mediated stimulation of the respiratory burst in human polymorphonuclear leukocytes may require appearance of protein kinase activity in the cells' particulate fraction. AB - Low doses of aliphatic alcohols produce divergent effects on the function of chemoattractant receptors on human polymorphonuclear leukocytes (PMNs) since they enhance chemotaxis but inhibit stimulation of superoxide production by chemoattractants. As such, alcohols can provide useful pharmacologic tools to probe the mechanisms of stimulus-response coupling in leukocytes. A role for protein kinase C has been implicated in the activation of the respiratory burst in PMNs. Although the vast majority of this enzyme activity is located in the cytosolic fraction of unactivated PMNs, protein kinase C activity appears in the particulate fraction of the cells when they are stimulated to produce superoxide by either chemoattractants or by phorbol myristate acetate (PMA). Doses of the alcohols that selectively inhibited stimulation of superoxide production by chemoattractants also inhibited the appearance of protein kinase C activity as well as an undefined protein kinase activity in the particulate fraction of the cells. In contrast, the alcohols did not affect either the ability of PMA to stimulate the production of superoxide in PMNs nor the appearance of protein kinase activity in the cells' particulate fraction. PMA is known to bind and activate protein kinase C directly, thus bypassing receptor-mediated events. These data suggest that alcohols inhibit the stimulation of the respiratory burst by chemoattractants in PMNs by blocking the ability of receptor occupancy to induce the appearance of protein kinase activity in particulate fractions. These results moreover suggest that the appearance of protein kinase activity in the particulate fraction may be required for activation of the respiratory burst in PMNs. PMID- 3006836 TI - Modulation of neutrophil oxidative responses to soluble stimuli by platelet activating factor. AB - The role of platelet activating factor (PAF) as a regulator of human neutrophil superoxide (O2-) generation in response to soluble and particulate stimuli was examined. At concentrations greater than 10(-7) mol/L, PAF alone induced a brief burst of O2- production. When cells were exposed to PAF and either the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP 10(-7) mol/L) or the tumor promoter phorbol myristate acetate (PMA 10 ng/mL), a marked synergistic augmentation of O2- release was noted when compared to control cells stimulated with FMLP or PMA alone. Mean percentage of enhancement by 10(-5) mol/L of PAF was 297% +/- 35% (n = 9) of control responses to FMLP and 185% +/- 16% (n = 3) of control responses to PMA. Consistent enhancement occurred with PAF concentrations of as low as 10(-9) mol/L. Enhancement could be demonstrated when neutrophils were exposed to PAF either at the same time as, or up to 60 minutes prior to, the second stimulus, and was neither reversed by removal of PAF from the medium prior to addition of FMLP or PMA nor dependent on the presence of extracellular divalent cations. Continuous recordings revealed that the enhancement was due to an increased maximal rate of O2- production. In contrast, PAF concentrations up to 10(-5) mol/L had only a minimal effect on the response to neutrophils to opsonized zymosan. Analysis of the enhancing properties of lipids structurally related to PAF revealed that the critical moiety was the saturated fatty acid at position 1. These results indicate the presence of a PAF mediated positive feedback loop whereby the oxidative burst induced by some soluble stimuli is augmented. Modulation of neutrophil O2- production by PAF may serve to amplify neutrophil oxidative responses at sites of inflammation. PMID- 3006837 TI - Leukopenic chronic T cell leukemia mimicking hairy cell leukemia: association with human retroviruses. AB - We report two cases of a T cell lymphoproliferative disease not previously described, with cytologic and clinical features similar to those associated with Galton's "prolymphocytic" leukemia (PL). Our patients, like those with Galton's PL, had massive splenomegaly and minimal or absent hepatomegaly and lymphadenopathy. In contrast, however, our patients had leukopenia, as well as low percentages of leukemic cells in the peripheral blood and in the bone marrow. In splenic imprints, the nuclear chromatin pattern of most of the leukemic cells was intermediate between those of mature lymphocytes and those of lymphoblasts, and the nuclei contained single, centrally located, conspicuous nucleoli. In sections of the spleen, the leukemic cells diffusely infiltrated the red pulp in a pattern strikingly similar to that of hairy cell leukemia; however, when the leukemic cells were studied cytochemically, the cytoplasmic acid phosphatase positivity was punctate and tartrate-sensitive. The leukemic cells were sheep erythrocyte rosette-positive and expressed T cell-associated antigens. Initially, both patients responded well to therapeutic splenectomy. One patient received combination chemotherapy after splenectomy and is alive and well 24 months after diagnosis. The other patient was in complete clinical remission for one year after splenectomy and received chemotherapy at relapse. He died, however, 23 months after splenectomy, with disseminated disease. IgG antibody titers against human T lymphotropic virus type I (HTLV-I) were detected in one patient and against HTLV-II in the other. The leukemia in these patients represents a distinct clinicopathologic entity within the spectrum of peripheral T cell lymphoproliferative diseases that includes Galton's PL of T cell derivation, T cell chronic lymphocytic leukemia, T cell hairy cell leukemia, and adult T cell leukemia/lymphoma. PMID- 3006838 TI - B lymphoblastoid cell lines with normal and defective O-glycosylation established from an individual with blood group Tn. AB - Individuals with the Tn blood group contain terminal serine/threonine-linked N acetylgalactosamine residues in their blood cells. This is due to lack of UDP-D galactose: D-N-acetyl galactosamine beta-D-galactosyl transferase from part of their red cells and probably from their leukocytes. We have established B lymphoblastoid cell lines from such an individual by in vitro infection of his lymphocytes with Epstein-Barr virus. The original line contained a mixture of cells reactive and nonreactive with Helix pomatia lectin (Hp). These cells were subcloned after staining with fluorescent Hp by a fluorescence-activated cell sorter (FACS) into homogeneous, phenotypically stable lines of Hp-positive (Hp+) and Hp-negative (Hp-) cells. The molecular differences between the membrane glycoproteins were characterized by carbohydrate-specific surface labeling techniques, Hp affinity chromatography, polyacrylamide slab gel electrophoresis and glycopeptide/oligosaccharide analysis. The major O-glycosidic membrane glycoprotein (GP105) was retained on Hp-Sepharose columns only from Hp+ cells, whereas the common leukocyte antigen (GP160-200) was partially retained on Hp columns from both lines. These proteins were isolated by immune precipitation with monoclonal antibodies and characterized. The results show that the GP105 glycoprotein from Hp+ cells contains terminal N-acetylgalactosamine residues but also more complex oligosaccharides. The common leukocyte antigen showed different electrophoretic mobilities in Hp+ and Hp- cells. UDP-galactose D-N-acetyl galactosamine beta-galactosyl transferase was almost absent in the Hp+ cells. These cell lines are useful for studies on the functional role and regulation of the biosynthesis of O-glycosidic carbohydrates. PMID- 3006839 TI - Mechanisms of adenosine 5'-monophosphate catabolism in human erythrocytes. AB - Uncertainties regarding the role of pyrimidine nucleotidase (PyrNase) in AMP catabolism were resolved by studies of erythrocytes from normal controls, controls with young mean cell ages, and patients with hereditary hemolytic anemia due to severe deficiency of PyrNase. Hemolysates from the latter exhibited undiminished capacity to dephosphorylate AMP over a broad range of pH, indicating that PyrNase was not directly involved. In each subject group, the rates of AMP dephosphorylation between pH 5.1 and 8.3 were indistinguishable from those of IMP, suggesting a potential role for AMP-deaminase, an erythrocyte enzyme that was stimulated by coformycin at pH 7.2. Quantitative analysis of catabolites in incubated hemolysates confirmed that AMP degradation preferentially occurred via deamination to IMP with subsequent dephosphorylation by another erythrocyte nucleotidase isozyme, deoxyribonucleotidase. Both AMP-deaminase and deoxyribonucleotidase have acidic pH optima with minimal activities at physiologic pH, suggesting that this pathway of AMP catabolism could accelerate depletion of the adenine nucleotide pool and thereby mediate the demise of senescent erythrocytes sequestered in the spleen. PMID- 3006840 TI - Deficient IFN alpha production in hairy cell leukemia. AB - Since the application of low doses of IFN-alpha is necessary to maintain remissions in Hairy Cell Leukemia (HCL) it is of interest whether peripheral blood mononuclear cells (MNC) of HCL patients can be induced in vitro to produce IFN-alpha. 9 patients suffering from advanced HCL were included in the study. The diagnoses were confirmed by characteristic findings in peripheral blood and bone marrow biopsies. For IFN treatment we initially used natural IFN-alpha (Bioferon) and switched later to recombinant IFN-alpha2 (Boehringer). MNC of 5 patients before IFN therapy and of 6 patients during IFN therapy (2-47 weeks) were induced by phytohemagglutinin (PHA), Corynebacterium parvum (C.p.), and sendai virus (SV). PHA is known to induce IFN-gamma. Both, C.p. and SV induced IFN-alpha but no IFN-gamma in MNC of healthy controls and of IFN treated breast cancer patients. In HCL patients normal antiviral activities could be induced by PHA. Zero or only low antiviral activities could be induced in MNC from 9 patients tested on 22 occasions. It is concluded that MNC from patients with advanced HCL can be induced to produce IFN-gamma but no IFN-alpha. Since IFN-alpha but not IFN gamma is produced by monocytes it is likely that reduced numbers of monocytes which were found in our HCL patients before and during IFN treatment account for the described deficiency of IFN-alpha production. PMID- 3006841 TI - Role of the parasympathetic nervous system and of cholinergic mechanisms in bronchial hyperreactivity. AB - The parasympathetic nervous system of the respiratory tract is involved in the control of airway calibre in three ways: through afferent nerve pathways (pulmonary reflexes); through efferent nerve pathways (reflexes, interaction between efferent vagus and mediators or modulating transmitter substances) and through cholinergic muscarinic receptors and postreceptor mechanisms in the target organ. To what extent do these mechanisms contribute to airway hyperreactivity? Pulmonary reflexes: Reflex bronchoconstriction has been established in divided lung experiments for histamine and in experiments involving an isolated segment of trachea for SO2. A reflex pathway is the most likely explanation for the heightened reactivity induced by aerosols of prostaglandin E2 and by maximal respiratory manoeuvers. Reflex bronchoconstriction is mediated through rapidly adapting ("irritant") receptors and through C-fibre endings. The influence of C-fibre endings is greater than hitherto suspected and many effects ascribed to irritant receptors are probably due to stimulation of C-fibre endings which outnumber myelinated fibres 3-4 to 1. From studies on the control of respiration (which reflect more directly the state of sensory receptors in the airways than studies of airway calibre) it appears that the activity of C-fibre endings increases during ozone-induced hyperreactivity. This could explain the increased bronchial reactivity seen in this condition. Interaction: Aerosols of serotonin cause bronchoconstriction when the vagus nerve is intact but have little effect during vagal block. This is an effect neither on afferent receptors nor on the end organ, but on the efferent nerve pathway. Interaction effects of this type ("cholinergic facilitation") are frequent and may be more important quantitatively than reflex effects. From data on serotonin and by analogy to other systems it appears that preganglionic and ganglionic sites are important points of interaction. Parasympathetic ganglia are located in the airway wall. Several transmitter substances have been identified in airway ganglia and in autonomic nerves, sensory and motor. Thus there seem to be convergent inputs capable of modulating transmission through the ganglia. An altered balance of converging ganglionic inputs may cause hyperreactivity. Receptors in airway smooth muscle may not be a homogeneous population. Muscarinic receptors are being classified into subgroups and the existence of at least 3 subtypes: M-1, M-2 and M-3 has been postulated. Their distribution depends on the tissue studied. They differ in agonist affinity messenger systems.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3006842 TI - Parasympathetic ganglia in the airways. AB - Catecholamines seem to inhibit transmission through airway parasympathetic ganglia by two mechanisms: Beta-2-adrenoceptor stimulation induces a ganglionic inhibition characterised by: slow onset, effect mainly on neural signals of high frequency, localisation to the presynaptic nerve terminal in the ganglion (inhibition of transmittor release). In vivo this mechanism could be operated upon by circulating adrenaline, which is active on beta-2-adrenoceptors. Alpha adrenoceptor stimulation induces a ganglionic inhibition characterised by: rapid onset, localisation to the presynaptic level (inhibition of transmittor release). This mechanism could be operated upon by noradrenaline released by sympathetic nerves supplying airway ganglia. In bronchial hyperreactivity the effect of catecholamines on transmission through airway ganglia could serve as a negative feed back mechanism reducing bronchoconstriction induced by neural reflexes. Tentatively, fascilitation of transmission through airway ganglia could contribute to bronchial hyperreactivity. However, such fascilitation has not yet been demonstrated for airway ganglia, although it has been found in sympathetic ganglia. PMID- 3006843 TI - The beta-adrenergic receptor and bronchial hyperreactivity. PMID- 3006844 TI - Receptors on human airway smooth muscle. PMID- 3006845 TI - Airway microvascular permeability to large molecules. PMID- 3006846 TI - Assessment of vitamin D sulphate in human milk using desorption chemical ionization mass spectrometry. AB - Vitamin D3 sulphate (SD3) identification in human milk was obtained using Desorption Chemical Ionization (DCI). The chemical ionization reagent gas used was nitrogen, molecules were ionized when the emitter was heated. SD3 was obtained from lactarium human milk and purified by high-performance liquid chromatography (HPLC). A selected ion monitoring (SIM) measurement was carried out with typical ions, m/z 366 for SD3 and m/z 384 for parent vitamin D3, the intensity ratio (I366/I384) greater than 1 being related to the presence of the sulphoconjugated form of vitamin D3 in the sample analysed. The detection of small quantities of SD3 in human milk is possible using this technique. PMID- 3006847 TI - Application of computer-assisted image analysis for studying viral structures. AB - The application of CAIE has been shown to be useful for analyzing the structures of RNA viruses. Critical assessment of this method is essential for the selection of the micrographs of viruses. In our experience this procedure was helpful for resolving some questions concerning virus morphology. PMID- 3006848 TI - Biochemical and ultrastructural findings in a lymphoid cell line from Niemann Pick disease type A. AB - A lymphoid cell line (LCL) established by Epstein-Barr Virus (EBV)-transformation of blood B-lymphocytes from a patient affected with Niemann-Pick disease (NPD) Type A exhibited a severe deficiency of sphingomyelinase activity (less than 10% residual activity). Ultrastructural investigation showed in LCL from NPD type A, the presence of numerous osmiophilic, electron-dense inclusions with myelin-like figures characteristic of the accumulation of sphingomyelin (and other amphiphilic lipids) similar to those observed in tissues of patients affected with NPD. PMID- 3006849 TI - Rat plasma kallikrein clearance by perfused rat liver. AB - Previous studies have shown that perfused rat liver in situ is able to clear recirculating rat plasma kallikrein (RPK) in two phases: an initial clearance lasting a few minutes, followed by a slow exponential phase. Using purified RPK preparations we now show that: RPK is a glycoprotein; clearance was inhibited by human serum against blood group B and 0.1 M melibiose but was not affected by human serum against blood group A, 0.1 M lactose, 0.1 M mannose, 0.05 M N-acetyl galactosamine, 0.05 M galactose or 15 microM asialofetuin. Prolonged incubation of RPK with alpha-galactosidase reduced RPK clearance. Oligosaccharide structures in RPK may have terminal galactose units since treatment of RPK with neuraminidase did not affect the clearance rate; RPK clearance occurs in the absence of added Ca2+, with either EDTA or EGTA in the perfusion fluid; the exponential phase is reversibly inhibited by the addition of NH4Cl or chloroquine to the perfusion fluid. This observation, along with experiments using liver homogenates, suggests that RPK catabolism is carried out by lysosomal enzymes, probably cathepsin B of possible hepatocyte origin. PMID- 3006850 TI - [Determination of cyanogenic compounds in "health foods" made of ume (Japanese apricot)]. PMID- 3006851 TI - Non involvement of gamma-aminobutyric acid in catechol-induced seizures. AB - The effects of certain anticonvulsant agents, namely, valproate, diazepam and phenobarbitone were investigated on catechol-induced spontaneous and evoked convulsions, in anaesthetized rats and mice. Valproate and diazepam significantly reduced the intensity of spontaneous convulsions and the frequency of occurrence of the longer-latency components (M2 and M3) of the evoked muscle response. Phenobarbitone significantly reduced spontaneous convulsions and the M3 component of the evoked muscle response. None of the drugs affected the short latency M1 component indicating a supra-spinal site of action of these drugs. Agents which modify gamma-aminobutyric acid (GABA)-mediated transmission were without effect on the frequency of occurrence of M1, M2 or M3. The results suggest that the convulsant action of catechol is not dependent on antagonism of GABA-mediated inhibition. PMID- 3006852 TI - Blockade of Ca-activated K conductance by apamin in rat sympathetic neurones. AB - Effects of apamin on rat sympathetic neurones were investigated by means of intracellular and extracellular recording. Apamin (50 nM) significantly shortened the after-hyperpolarization (AH) following the spike evoked by current injection and slightly decreased its peak amplitude without affecting the time course of the spike. The AH following the synaptically-evoked spike was also blocked by apamin. This effect was dose- and time-dependent (ID50 estimated by extracellular recording approximately 15 nM, 20 min after application) and poorly reversible. Transmission of a single volley was not affected by 50 nM apamin. Though a long depolarizing current caused one or two spikes in the cell, greater repetitive firing was observed in the presence of apamin. Spontaneous repetitive firing, however, was not observed except for anodal-break spikes. Resting potential and input membrane resistance were essentially unchanged by apamin. The maximum rate of rise of the Ca spike was not decreased by 50 nM apamin but the duration of the spike was lengthened by 60%. The AH following the Ca spike was also blocked by apamin. These results suggest that apamin suppressed the slow AH without any inhibition of the Ca flux into the cell and is useful as a blocker of GK(Ca) in the rat sympathetic neurone. PMID- 3006853 TI - Role of the endothelium on the facilitatory effects of angiotensin I and angiotensin II on noradrenergic transmission in the caudal artery of the rat. AB - Perfusion of carbogen gas through the lumen of the rat caudal artery abolished the dilator response to acetylcholine (1 mumol 1(-1) in artery segments which had been precontracted with noradrenaline (50 nmol 1(-1]. Hence, it was assumed that gas perfusion was effective in removing the vascular endothelium. Angiotensin I (30 nmol 1(-1] and angiotensin II (10 nmol 1(-1] enhanced the responses of artery segments to field stimulation of their sympathetic nerves (0.5 Hz, 10 s). In arteries with an intact endothelium the ability of each peptide to enhance responses to stimulation was the same whether applied through the lumen or to the adventitial surface. Removal of the endothelium, by gas perfusion, did not significantly alter the facilitatory effects of extraluminally or intraluminally applied angiotensin I or angiotensin II. The converting enzyme inhibitor enalaprilat was equally effective in inhibiting the facilitatory effect of angiotensin I in the presence and absence of an intact endothelium. It is concluded that in the rat caudal artery, conversion of angiotensin I to angiotensin II does not depend on an intact endothelium and that the facilitatory effect of angiotensin II on noradrenergic neuroeffector transmission is not modified by, or dependent on, an intact endothelium. PMID- 3006854 TI - Characterization of prostanoid relaxant/inhibitory receptors (psi) using a highly selective agonist, TR4979. AB - TR4979, an analogue of prostaglandin E1 (PGE1) was evaluated on respiratory and non-respiratory isolated tissues known to contain heterogeneous or homogeneous populations of the two classes of prostanoid (prostaglandins and thromboxanes) receptors. These receptors are classified as 'chi' the contractant/stimulant receptor with 'chi 1,2,3' being three subdivisions and 'psi' the relaxant/inhibitory receptor(s). On a respiratory tissue (cat trachea) containing predominantly 'psi'-receptors, TR4979 was 26 times less potent than PGE1 or PGE2. On other respiratory tissues known to contain mixtures of the 'chi 1,2,3'- and 'psi'-receptors (guinea-pig trachea and lung strip, cat lung strip and human bronchial muscle), TR4979 consistently acted as a potent relaxant whereas PGE2 and to a lesser extent PGE1 had significant contractant activities. Human pregnant uterus, guinea-pig and rat pseudo-pregnant uteri, rat colon and fundic strips and chick ileum are known to contain one or more of the three subclasses of the 'chi'-receptor. TR4979 (10(-9) -10(-5) M) was inactive on all these tissues whereas all of the reference prostanoids were contractants of varying potencies. PGE1 and histamine-induced contractions of the guinea-pig isolated ileum were both non-competitively antagonized by increasing concentrations of TR4979 suggesting that 'psi'-receptors also exist on this tissue. TR4979 is a highly selective agonist of prostanoid 'psi' (relaxant/inhibitory)-receptors which at present have been demonstrated to exist mainly in the lung. This prostaglandin analogue is a useful new selective pharmacological tool for revealing as yet unidentified prostanoid 'psi'-receptors and actions in a wide range of non-respiratory tissues/organs such as the guinea-pig ileum. PMID- 3006856 TI - A comparison of the acute inflammatory response in adrenalectomised and sham operated rats. AB - Carrageenin pleurisy was induced in adrenalectomised (ADX) and sham-operated (SHO) rats. The magnitude and duration of inflammation, as estimated by fluid exudation and cell migration, was greatly increased (approximately doubled) in ADX rats compared with that in their SHO controls. The content of eicosanoids (6 keto-prostaglandin F1 alpha (6-keto-PGF 1 alpha), thromboxane B2 (TXB2), and leukotriene B4 (LTB4] in inflammatory exudates from ADX rats was significantly (2 4 fold) greater than that of their SHO controls. Resident macrophages obtained from ADX rats produced more eicosanoids per cell per unit time when stimulated in vitro with zymosan, than did cells from the SHO controls. Administration of glucocorticoids blocked the inflammatory response and reduced the release of eicosanoids both in vitro and in vivo in both groups of rats. These data are consistent with the notion that physiological amounts of glucocorticoids exert a tonic inhibitory action on phospholipase activity in normal animals and that the increased secretion of these hormones during the inflammatory response serves to check and control the development of inflammation. PMID- 3006855 TI - Influence of the epithelium on responsiveness of guinea-pig isolated trachea to contractile and relaxant agonists. AB - The potency (pD2) and maximal contractile effect (Emax) of histamine, acetylcholine, carbachol and K+ were assessed from cumulative concentration effect curves in guinea-pig isolated tracheal ring preparations with and without an intact epithelium. Estimates of Emax were not significantly different in epithelium-denuded preparations compared with those measured in intact preparations; pD2 values for acetylcholine, carbachol and K+ were not significantly altered. In contrast, the potency of histamine was significantly increased by about 4 fold in preparations devoid of epithelial cells. Estimates of potency and Emax were also determined for the smooth muscle relaxants isoprenaline, forskolin and theophylline (which increase intracellular cyclic AMP) and for nitroglycerin (which increases cyclic GMP) in both intact and epithelium-stripped tracheal rings. The pD2 values for these relaxants were not significantly altered by the removal of the epithelium. However, with the exception of nitroglycerin, Emax values for these relaxants were significantly lower in stripped than in intact tracheal rings that had been maximally precontracted with carbachol. The autoradiographic localisation of binding sites for the non-selective beta-adrenoceptor ligand [125I]-iodocyanopindolol (I-CYP) showed that the epithelium of the guinea-pig trachea had a 75 +/- 16% greater density of beta-adrenoceptors than the smooth muscle. Removing the epithelium did not significantly alter either the density of smooth muscle binding sites or the affinity of I-CYP binding. It was concluded that the reduced functional response of guinea-pig trachea to isoprenaline was probably not due to smooth muscle beta adrenoceptor dysfunction. Results indicate that the epithelium plays an important role in the modulation of responsiveness of guinea-pig trachea to histamine and relaxants that mediate their effects by selectively increasing intracellular cyclic AMP levels. PMID- 3006857 TI - Kadsurenone distinguishes between different platelet activating factor receptor subtypes on macrophages and polymorphonuclear leucocytes. AB - The effects of the antagonist kadsurenone on the platelet activating factor (Paf) induced chemiluminescence of guinea-pig peritoneal macrophages and on pig peripheral blood leucocyte aggregation were compared. Linearity and slopes of unity of the Schild plots confirmed the competitive nature of the antagonism by kadsurenone. pA2 values indicated a 91 fold lower affinity of kadsurenone for leucocyte Paf receptors than for those in macrophages. It is concluded that these two types of Paf receptors are not identical and are provisionally designated Paf1 and Paf2 receptors, respectively. PMID- 3006859 TI - Receptor-responses in fresh human ciliary muscle. AB - The physiological and pharmacological properties of ciliary muscle isolated from fresh human eyes were investigated. The muscle exhibited no spontaneous activity. Concentration-dependent contractions in response to carbachol were competitively antagonized by atropine (pA2 = 8.95). The muscle, precontracted by carbachol (2.7 X 10(-4)M), responded to the application of isoprenaline by concentration dependent relaxation blocked by propranolol (3.5 X 10(-9)M to 3.5 X 10(-8)M; pA2 = 9.15). Angiotensin-evoked contractions were antagonized by 8-Ala-angiotensin II (4.5 X 10(-8)M) in a competitive manner, but were not inhibited by phentolamine or propranolol. Contractions generated by electrical stimulation of the muscle (30 ms, 20 Hz, 60 pulses) were antagonized by atropine (10(-7) M) and tetrodotoxin (6.3 X 10(-7) M). Phentolamine and propranolol did not influence these responses. An increase of the external potassium concentration ([K+]o) from 5.4 to 158.8 mM produced a mechanical response, antagonized by atropine, but not influenced by tetrodotoxin, phentolamine or propranolol. The human ciliary muscle appears to carry muscarinic and angiotensin receptors and beta 2-adrenoceptors. The estimate of Katropine for muscarinic receptors mediating carbachol-induced contractions agrees with estimates of Katropine reported for human and rabbit iris. PMID- 3006858 TI - B-HT 958 lowers blood pressure and heart rate in the rat through stimulation of dopamine receptors. AB - To investigate whether the hypotensive and bradycardiac effects of B-HT 958 (2 amino-6-(p-chlorobenzyl)-4H-5,6,7,8-tetrahydrothiazolo-(5,4-d) azepine) are due to the stimulation of peripheral prejunctional alpha2-adrenoceptors, the selective alpha2-adrenoceptor antagonist idazoxan was given either intravenously (i.v.) or intracerebroventricularly (i.c.v.) to anaesthetized rats before the administration of i.v. B-HT 958. Plasma noradrenaline was used as an approximate index of peripheral sympathetic nervous activity. B-HT 958 350 micrograms kg-1 i.v. caused significant falls in blood pressure and heart rate which were maximal 5 min after dosing (-29.25 +/- 3.2 mmHg and - 52 +/- 6.8 beats min-1 respectively, mean of all control animals). The hypotension and bradycardia were accompanied by significant falls in plasma noradrenaline concentration of 30-40%. Idazoxan 300 micrograms kg-1 i.v. caused a marked, but transient tachycardia and a large sustained rise in plasma noradrenaline concentration. Idazoxan 300 micrograms kg-1 and 1000 micrograms kg-1 i.v. did not prevent B-HT 958-induced falls in mean arterial pressure, heart rate and plasma noradrenaline concentration. Responses to B-HT 958 were unaffected by idazoxan 20 micrograms i.c.v. B-HT 958-induced falls in mean arterial pressure, heart rate and plasma noradrenaline concentration were significantly attenuated by i.v. administration of the dopamine receptor antagonist, sulpiride 300 micrograms kg-1. Sulpiride 10 micrograms and 50 micrograms i.c.v. caused inhibition of B-HT 958 hypotension and bradycardia similar to that of intravenous sulpiride. After i.c.v. sulpiride, B HT 958 did not cause a significant fall in plasma noradrenaline concentration. A combination of idazoxan 1000 micrograms kg-1 i.v. and sulpiride 300 micrograms kg 1 i.v. did not cause further significant inhibition of B-HT 958 hypotension and bradycardia compared with sulpiride 300 micrograms kg-1 i.v. alone. This combination however had a significantly greater effect in inhibiting B-HT 958 hypotension than had idazoxan 1000 micrograms kg-1 alone, and almost completely blocked the B-HT 958-induced fall in plasma noradrenaline concentration. These results suggest that in the anaesthetized rat the cardiovascular effects of B-HT 958 are due to stimulation of dopamine receptors, probably located within the central nervous system, and not to stimulation of peripheral prejunctional alpha2 adrenoceptors. PMID- 3006860 TI - Dementia and Parkinson's disease--pathological and neurochemical considerations. PMID- 3006861 TI - Cholinergic approaches to the treatment of Alzheimer's disease. PMID- 3006863 TI - Spatial EEG patterns, non-linear dynamics and perception: the neo-Sherringtonian view. AB - Spatial analysis with preamplifier arrays and computers offers fresh perspectives on brain function. Realization of its potential depends on development of appropriate procedures for data processing and display, experimental paradigms to serve as benchmarks, and theories of brain function to predict what to look for and how to distinguish valid results from artifacts. Measurement of EEGs from arrays of 64 electrodes chronically implanted on the olfactory bulbs of rabbits that are trained to discriminate odorant conditioned stimuli show that the odorants induce spatially distinctive amplitude patterns of neural activity. The odor-specific information density is inferred to be uniform over the whole main bulb. The neural dynamics that produce these activity patterns emerge from the synaptically interactive sheet of excitatory mitral and inhibitory granule cells with distributed input and output tracts and with static nonlinearities deriving from the nerve impulse mechanism. Excitatory synapses between mitral cells are subject to modification when odorants are paired with unconditioned stimuli, thus forming nerve cell assemblies. Odorant-specific information established by a stimulus locally in the bulbar unit activity is integrated with past experience by an assembly, disseminated over the entire bulb on the order of 100 mm2 in area in a time period of 2.5 ms, and sustained for a time period on the order of 0.1 s. An arbitrary spatial sample on the order of 20% of bulbar EEG activity captures the entire integrated information albeit at lesser resolution than the whole. This synaptic mechanism of local input and global output may be common to all of the cerebral cortex. The implications are discussed for neocortical sensory systems, motor pattern generators, and goal-directed behavior in the context of self-organizing non-linear dynamic systems. PMID- 3006862 TI - An early marker of fetal infection after primary cytomegalovirus infection in pregnancy. AB - Fourteen patients with primary cytomegalovirus infection diagnosed by serological screening at antenatal attendances were examined for their responses in the lymphocyte transformation test against cytomegalovirus. Tests were done during pregnancy, shortly after the diagnosis of primary infection. Eight women showed positive lymphocyte transformation responses and gave birth to uninfected babies. Six showed negative responses and four of the babies were born congenitally infected. Cellular immunity therefore plays a part in preventing intrauterine transmission of cytomegalovirus, and its depression after primary infection in the mother during pregnancy may be used as an early marker of fetal infection. PMID- 3006864 TI - Enzymology of DNA replication and repair in the brain. AB - A number of enzymes thought to be involved in DNA replication have been identified in the brain. These include single-stranded DNA-binding proteins, topoisomerases I and II, DNA polymerase alpha, a protein that binds Ap4A and might be classified as a DNA polymerase alpha accessory protein, RNase H, DNA polymerase beta, DNA ligase, an endo- and an exonuclease of unknown function, DNA methyl transferase and poly(ADPR) synthase. In contrast, little is known about the enzymology of DNA repair in brain. The few enzymes identified comprise uracil DNA glycosylase, DNA polymerase beta, DNA polymerase alpha (which in neurons is present only at immature stages), DNA ligase, poly(ADPR) synthase, and O6 alkylguanine-DNA alkyltransferase. In addition, an exonuclease acting on depurinated single-stranded DNA (tentatively listed here as 3'----5' exonuclease), an endonuclease of unknown function as well as ill-defined acid and alkaline deoxyribonucleases also occur in brain. PMID- 3006865 TI - Double-labeling of rat alpha-motoneurons for cytochrome oxidase and retrogradely transported [3H]WGA. AB - In this study, we have demonstrated the co-localization of a retrograde tracer and the reaction product of an oxidative enzyme within the same neurons in the same spinal cord section, using [3H] wheat germ agglutinin and cytochrome oxidase histochemistry. This approach allows unequivocal identification of the alpha motoneurons innervating specific muscles. We have determined that there is a positive correlation between the distribution of cytochrome oxidase reactivity in alpha-motoneurons and the muscles that they innervate. The degree of cytochrome oxidase reactivity within the labeled alpha-motoneurons appears to be independent of spinal cord level and cell size. PMID- 3006866 TI - Effect of 5,7-dihydroxytryptamine on the concentration of individual proteins in different areas of the rat brain. AB - Proteins which are apparently regulated in concentration in two different areas of the rat brain by the indole neurotransmitter serotonin were identified using two-dimensional gel electrophoresis combined with computerized scanning densitometry. Reduction in central serotonin levels produced a decrease in the concentration of 3 different proteins (2 in the parietal cortex, 1 in the hippocampus). Two proteins, both in the hippocampus, were elevated in concentration following serotonin depletion. These results demonstrate that there exist in the brain a limited number of proteins whose concentration is influenced by serotonin. PMID- 3006867 TI - Brain dopamine depletion by lesions in the substantia nigra attenuates the development of hypertension in the spontaneously hypertensive rat. AB - The involvement of brain dopamine in the development of hypertension in the spontaneously hypertensive rat (SHR) was studied. Intracerebroventricular (i.c.v.) injections of 6-hydroxydopamine (6-OHDA) in young SHR caused depletion of dopamine in frontal cortex and striatum and induced an attenuation of the development of hypertension in SHR. Depletion of noradrenaline and to a lesser extent of serotonin was found as well. The ratio of DOPAC and of HVA to dopamine was increased after 6-OHDA. Pretreatment with the dopamine re-uptake inhibitor GBR-12909 inhibited the effects of 6-OHDA on both blood pressure and brain dopamine content. The effect of 6-OHDA on noradrenaline and serotonin levels were not influenced by pretreatment with GBR-12909. Electrolytic lesions in the substantia nigra delayed the rise in blood pressure in SHR. Lesions in the ventral tegmental area (VTA) were ineffective. After substantia nigra lesions depletion of dopamine was found especially in the nucleus caudatus posterior and the dorsomedial nucleus. After lesions in the VTA substantial dopamine depletion was found in the nucleus accumbens, frontal cortex, lateral septal nucleus and zona incerta. These data suggest that brain dopamine systems play a role in the development of hypertension in SHR and that especially the nigrostriatal system is important in this respect. Moreover, the present results may help to explain the attenuating effect of prehypertensive treatment with 6-OHDA on the development of hypertension. PMID- 3006868 TI - Brain serotonin synthesis and Na+,K+-ATPase activity are increased postnatally after prenatal administration of L-tryptophan. AB - The effect of prenatal L-tryptophan supplementation on the serotonin (5-HT) synthesis and the activity of Na+,K+-ATPase in the cerebral cortex was studied during postnatal development, from birth up to day 30. A parallel and significant elevation of the serotonin content and the activity of tryptophan-5-hydroxylase was observed in the brain of infant rats born to mothers treated with L tryptophan, as related to non-treated controls. The activity of Na+,K+-ATPase was also significantly elevated at the different ages studied throughout the developmental period, as related to controls. These results suggest an important role of L-tryptophan in the early regulation of the serotonin-synthesizing machinery, which lasts postnatally. Elevation of ATPase activity seems to be associated to the elevation in the activity of the 5-HT system. PMID- 3006870 TI - Locus coeruleus neurons in culture have a developmentally transient alpha 1 adrenergic response. AB - Intracellular recordings from locus coeruleus neurons grown in explant cultures reveal age-related differences in the responses to iontophoretically applied noradrenaline. Cells grown in culture for more than 26 days exhibit a simple hyperpolarizing response while cells cultured for less than 26 days often have biphasic hyperpolarizing-depolarizing responses. Pharmacological studies with selective adrenergic agonists and antagonists show that the hyperpolarizations and hyperpolarizing parts of biphasic responses are mediated by alpha 2 adrenergic receptors while the depolarizing responses are mediated by alpha 1 receptors. These observations may reflect a population of neurotransmitter receptors which is transiently present during early postnatal development. PMID- 3006871 TI - Regional distribution of [3H]naloxone binding in the brain of a newborn rhesus monkey. AB - The distribution of opiate receptors in the brain of a newborn monkey (Macaca mulatta) was mapped by in vitro autoradiographic localization of [3H]naloxone binding to tissue sections. The autoradiographs of the newborn brain were compared to those from two adult brains. The distribution of opiate receptors appeared to be adult-like in subcortical structures (both limbic and nonlimbic) and allocortical areas. By contrast, all neocortical areas, except the primary visual cortex, lacked at birth the laminar specific patterns that characterize the adult. The results therefore suggest that, like many other aspects of neocortical maturation, such as dendritic growth, synaptogenesis, myelination and neurotransmitter concentrations, the distribution of opiate receptors continues to develop postnatally. PMID- 3006869 TI - Antagonist-induced opiate receptor upregulation in cultures of fetal mouse spinal cord-ganglion explants. AB - Chronic exposure of fetal mouse spinal cord-ganglion explants to the opioid antagonist naloxone (10 microM, 7 days) produced a pronounced upregulation of mu opioid receptors. The antagonist action was stereospecific, as it was produced by (-)-, but not by (+)-naloxone, and was dose-dependent. Half-maximal naloxone induced receptor upregulation occurred after two days; receptor density was maximal at 5 days. Exposure of the explant cultures to naloxone (10 microM) in the presence of the protein synthesis inhibitor, cycloheximide (1 microM; a concentration which blocks greater than 90% protein synthesis) resulted in receptor density changes that were similar to those observed in cultures exposed to naloxone alone. This finding suggests that antagonist-induced opiate receptor upregulation does not require the synthesis of new receptor molecules. PMID- 3006873 TI - In vivo and in vitro development of alpha-MSH and ACTH in the embryonic and postnatal rat brain. AB - The appearance of immunoreactive alpha-melanotropin (alpha-MSH) and adrenocorticotropin (ACTH) during development was studied in 3 areas of the rat brain--cerebral hemispheres, midbrain and hindbrain--from embryonic day (ED) 13 14 until day 21 postnatally. The alpha-MSH content in vivo was always highest in the midbrain; a peak content at birth was followed by a transient decline and a later, higher plateau from postnatal day 7 onwards. The alpha-MSH content in the cerebral hemispheres rose progressively after birth reaching a peak at day 21. Values in the hindbrain rose at day 3 and changed relatively sue taken at ED 15 16 showed a gradual increase in alpha-MSH content over the 20 days. The alpha-MSH content of hindbrain cultures remained at constant low levels, while no alpha MSH was detectable in cerebral hemisphere cultures. ACTH appeared in vivo earlier than alpha-MSH and was detectable in embryonic brains at ED 13-14. A transient rise was seen at ED 17-18 and major peaks at birth, day 2 and day 3, in the midbrain, hemispheres and hindbrain, respectively. In vitro, the ACTH content increased in all brain regions during the first 5 days in culture and showed no further change thereafter. Comparisons of the in vivo and in vitro development of alpha-MSH and ACTH demonstrate that (i) these two peptide systems are independent in respect to their localization and time of appearance; (ii) they undergo maturation both in vivo and in vitro; (iii) epigenetic factors, such as interactions with other neurotransmitter systems may modulate the developmental pattern of these two peptides. PMID- 3006872 TI - Spectrin does not redistribute with actin during dBcAMP-induced changes in astrocytes in vitro. AB - Cells of the astrocyte lineage obtained from mouse neopallium and grown in colony culture have been investigated for a correlation between distributions of F-actin and the common subunit of an erythrocyte actin binding protein, alpha-spectrin (brain fodrin). The cells of the astrocyte lineage at the astroblast stage have F actin organized in the form of prominent, linearly arranged microfilament bundles. We have demonstrated that spectrin in these cells forms a fine reticulum lining the cell cortex. During the dibutyryl cyclic (dBcAMP)-induced transition from astroblasts to reactive astrocytes, actin-containing microfilaments undergo the dramatic rearrangement from a predominantly linear to a predominantly circumferential spatial organization. remains in the form of a fine reticulum lining the cellular cortex. These remains in the form of a fine reticulum lining the cellular cortex. These findings support the recent notion that spectrin in non-erythroid cells is not essential for maintaining the organization and plasma membrane membrane anchorage of the prominent microfilament bundles. PMID- 3006874 TI - Ontogeny of the nerve growth factor receptor in primary cultures of neural crest cells. AB - Neural crest cells maintained as primary cultures for 4-7 days demonstrated specific binding of radioiodinated nerve growth factor ([125I]NGF); occasionally, significant binding of ([125I]NGF could be detected in 3-day cultures. Parallel cultures processed for indirect immunofluorescence revealed that the NGF receptor positive neural crest cells represented a subpopulation of the total cells in culture. Cultures aged 4-7 days demonstrated fluorescent cells which were often grouped as aggregates. Some 3-day cultures contained faintly fluorescent cells. One- and two-day cultures were non-fluorescent. Melanocytes did not appear to bind NGF. Preliminary flow cytofluorometric analysis indicated that ca. 28% of neural crest cells in 5-day cultures possessed specific receptors for NGF. PMID- 3006875 TI - Two simian virus 40 (SV40)-transformed cell lines from the mouse striatum and mesencephalon presenting astrocytic characters. II. Interactions with mesencephalic neurons. AB - In an accompanying paper we report the characterization on the basis of pharmacological and immunological criteria of two astrocytic cell lines originating from the rostral mesencephalon and the striatum of the embryonic mouse (F7-Mes and F12-Str). This report compares the interactions of primary mesencephalic neurons with the astrocytic clones to that displayed with either an SV40-transformed fibroblastic clone (BT2) or primary mesencephalic (G-Mes) and striatal (G-Str) astrocytes. We show that BT2 differs from all other cell types (F7-Mes, F12-Str, G-Mes and G-Str). Indeed, as opposed to these cells BT2 is a poor substratum for neuronal adhesion or neuritic growth. This was clearly demonstrated by morphological examination of cocultures of the tested cells with either mesencephalic explants or dissociated cells. In addition a statistical analysis is provided which only concerns the dopaminergic (DA) neurons visualized by autoradiography after specific uptake of [3H]DA. The number of DA cells attached, the total length of their neurites and the degree of branching behaviour were examined. With the help of these criteria we show that F7-Mes and F12-Str are very similar to primary astrocytes and differ highly significantly from BT2. However, although sharing the main astrocytic features, F7-Mes and F12 Str do not differ from one another in their ability to induce the branching of DA neurites as their non-transformed counterparts do. PMID- 3006876 TI - Two simian virus 40 (SV40)-transformed cell lines from the mouse striatum and mesencephalon presenting astrocytic characters. III. A light and electron microscopic study. AB - In two preceding papers we described the cloning of two astrocytic cell lines by simian virus 40 (SV40) transformation of embryonic mouse mesencephalon (F7-Mes) and striatum (F12-Str). The characterization of these lines as belonging to the astrocytic lineage is based on pharmacological, immunocytochemical and physiological data. Here we present quantitative and qualitative data on the morphological aspects of these two astrocytic clones observed under light and electron microscopy. We show that the clones present ultrastructural characters reminiscent of the morphology of young astrocytes. On one hand, they are rather similar to primary astrocytes in culture; on the other, they differ both from a clonal fibroblastic cell line (BT2) and from embryonic mouse fibroblasts in primary culture. These astroblastic clones display 4 morphologically different cell populations which we called types I, II, III and IV. Types II and III are very similar and represent the most predominant cells; their morphologies strongly remind of that of astroblasts. Type I corresponds to glioblasts and does not account for more than 15-20% of the total population. Type IV, which is very similar to differentiated velamentous astrocytes, normally represent ca. 5% of the cells. However, when the transformed cells are treated with mitomycin or mitomycin + dibutyryl cyclic AMP (dbcAMP), the proportion of type IV cells increases very much (up to more than 50% of the cells) while types I, II and III become less numerous. Morphological analysis therefore confirms that the two cell lines derived from the SV40 transformation of 14-day-old embryonic mesencephalic and striatal cells belong to the astrocytic lineage. Moreover, it seems that they can differentiate in vitro in cell culture conditions either spontaneously or under the action of pharmacological treatments known to enhance normal astrocyte maturation. PMID- 3006877 TI - Tetanus toxin binding to neuroblastoma cells differentiated by antimitotic agents. AB - We have examined the binding of tetanus toxin (TT) to surface receptors of neuroblastoma cells by flow cytometry following chemically induced differentiation. Cells were treated with mitomycin C, bromodeoxyuridine, prostaglandin E1 or cyclic adenosine monophosphate at different doses, alone or in combination for 4 days. Cells extended long neurites within 24 h in the presence of prostaglandin/cyclic AMP or mitomycin/bromodeoxyuridine treatment while single-drug treatment was less efficient in morphological differentiation of these cells. Cells exposed to the drug combinations stopped growing after 3 days while flow-cytometric analysis of DNA levels of each cell stained with propidium iodide indicated that at least 60% of these cells were arrested in phase G0/G1 of the cell cycle. Drug-treated cultures were stained for TT binding by immunofluorescence of cells in suspension and analyzed by flow cytometry. Chemically differentiated N2AB-1 cells were shown to bind significantly more TT than control cultures. Receptors for TT could be saturated by increasing doses of TT and differentiated cells bound twice as much toxin at saturation as did control cells. Immunofluorescence of TT binding to monolayers revealed staining in a stippled fashion along all neurites and cell bodies. These data support the concept that drugs which stimulate differentiation of neuroblastoma cells as determined by morphological and cell-cycle criteria also increase the presence of ganglioside receptors on the cell surface available for toxin binding. PMID- 3006879 TI - [Minimal carcinoma of the breast detected by secretion cytology]. PMID- 3006880 TI - [Methylxanthines and enzymes which degrade adenine nucleotides]. PMID- 3006878 TI - Quantitative autoradiography of 125I-[Sar1, Ile8]-angiotensin II binding in the brain of spontaneously hypertensive rats. AB - The brain contains its own angiotensin II (AII) system. To better understand the role of central AII in cardiovascular regulation, we used 125I-[Sar1, Ile8]-AII (125I-SI-AII), radioactive AII antagonist, to autoradiographically localize putative AII receptor binding in many parts of the central nervous system of the spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. With 125I-SI-AII binding on brain membrane preparations. Scatchard analysis indicated that Kd values were from 0.10 +/- 0.04 nM to 0.13 +/- 0.05 nM, whereas Bmax values (femtomol/mg protein) were found to be from 6.95 +/- 1.60 to 15.52 +/- 4.99 among brain regions studied. Various SI-AII receptor binding activities among brain regions revealed in this study were therefore most likely due to differences in AII receptor density with high affinity binding of 125I-AII. Using 125I-SI-AII, specific binding for SI-AII was found in the nucleus tractus solitarius (NTS), paraventricular hypothalamic nucleus (PVN), subfornical organ (SFO), suprachiasmatic nucleus (SCN), area postrema, the dorsal motor nucleus of the vagus (DMX), and the nucleus of spinal tract of the trigeminal system (NSV). With quantitative receptor autoradiography in conjunction with radioactive standards, we have observed that the NTS possesses the highest SI-AII binding, followed by the PVN, SFO, NTS, DMX, and NSV. No significant differences were observed between the SHR and WKY rats in the SI-AII binding within the SFO, PVN and NTS. However, SHR at early hypertensive (7 weeks) and established hypertensive (16 weeks) stages contained significantly higher SI-AII bindings in the NSV, as compared to age-matched WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006881 TI - Growth factors in cancer and their relationship to oncogenes. PMID- 3006882 TI - [Mitogenic action of factors secreted by avian erythroblastosis virus transformed cells]. AB - We describe here the capacity of erythroid LSCC HD3 cells, transformed with a ts mutant of avian erythroblastosis virus, to grow in a chemically defined medium without serum at 36 degrees C, but not at 41 degrees C. At this latter temperature the activity of v-erbB oncogene is suppressed. However, cell growth at 41 degrees C could take place either by addition of the medium derived from LSCC HD3 cells grown at 36 degrees C (conditioned medium), or by addition of fetal calf serum. These results show that LSCC HD3 cells, maintained under conditions in which the v-erbB oncogene is active, secrete growth factor(s) which exhibit a mitogenic effect similar to that observed with calf serum. PMID- 3006883 TI - [Benzodiazepine receptors in the hippocampus of suicides]. AB - The characteristics of benzodiazepine binding sites (distribution, number, affinity) were defined on frozen sections of suicide's hippocampus (death by hanging) labeled with 3H flunitrazepam and compared to data previously obtained on control brains. The study was carried out qualitatively by autoradiography (distribution) and quantitatively by a biochemical technique (number and affinity). As a whole, the characteristics of BZD binding sites were not modified in relation to controls, except for a very slight decrease in affinity of subtype I. PMID- 3006884 TI - [Epidemiologic surveillance of Japanese encephalitis]. PMID- 3006886 TI - [Identification of beta and M receptors in the lung tissue of guinea pigs and their changes during experimental allergic asthma]. PMID- 3006885 TI - [Determination of serum indices of iron metabolism and its clinical significance (with a report of 82 cases]. PMID- 3006887 TI - Molecular characterization of the EGF receptor and involvement of glycosyl moieties in the binding of EGF to its receptor on a clonal osteosarcoma cell line, UMR 106-06. AB - The epidermal growth factor receptor in cells of the UMR 106-06 clonal osteoblast line has been shown to be structurally similar to that previously characterized in other cell lines. A specific receptor component of approximately 165,000 185,000 Mr has been identified by polyacrylamide gel electrophoresis using the chemical crosslinker disuccinimidyl suberate to crosslink 125I-EGF to its receptor. Tunicamycin treatment of cells resulted in a dose-dependent loss of binding suggesting involvement of glycosyl moieties in EGF binding to its receptor. Competitive binding studies carried out using wheat germ lectin (WGL), concanavalin A (CON.A.), soybean lectin (SBL), and lentil lectin (ILL) to compete for binding of 125I-EGF revealed that CON A, WGL, and to a lesser extent LL could inhibit EGF binding; SBL was without effect. Treatment of the cells with neuraminidase which cleaves terminal sialic acid residues resulted in total loss of binding while alpha-glucosidase, beta-N-acetylglucosaminidase and alpha mannosidase were without effect. These data indicate a specific interaction of EGF with terminal sialic acid residues of the EGF receptor. However, it would seem that the mannose residues which appeared to modify EGF binding were not available for the action of the above enzymes due to the presence of sialic acid. PMID- 3006888 TI - Omeprazole, a specific inhibitor of H+-K+-ATPase, inhibits bone resorption in vitro. AB - Omeprazole has been previously shown to block gastric acid secretion by specific inhibition of gastric parietal cell membrane H+-K+-ATPase. It is now demonstrated that similar concentrations of omeprazole will inhibit PGE2- and PTH-stimulated 45Ca++ release from prelabelled neonatal mouse calvariae without affecting the viability of cultured calvaria explants. PMID- 3006889 TI - Reduced absorption of 45calcium from isolated duodenal segments in vivo in juvenile but not adult X-linked hypophosphatemic mice. AB - In juvenile X-linked hypophosphatemic (Hyp) mice, whole body calcium balances are significantly lower than in genetically normal mice. This is associated with low duodenal vitamin D-dependent calcium-binding protein and a failure of skeletal mineralization. To seek more specific evidence of an intestinal defect in these mice, absorption of 45Ca was measured in isolated duodenal segments in vivo in mice from 2-13 weeks of age. The duodenum was isolated by sutures and 45Ca was injected into the lumen in 150 mM NaCl and 2 mM CaCl2 at pH = 7.2. Absorption was measured by the amount of isotope remaining in the lumen and by the plasma isotope level. Hemizygous Hyp male and heterozygous Hyp female mice absorbed significantly less 45Ca at 4 and 7 weeks of age than genetically normal mice while Hyp mice at 2, 10, and 13 weeks of age were not significantly affected. At 4 and 7 weeks of age, the Hyp mice also had significantly reduced plasma radioactivity midway through the collection period as well as at the end of the period. To explore a possible mechanism for this malabsorption, 1,25(OH)2-vitamin D receptors were measured in cytosol prepared from 4-week-old normal and Hyp duodenum. There were non-significant differences between the normal and Hyp mice in both binding affinity, Kd, and the number of receptors, nmax. In conclusion, juvenile Hyp mice at 4 and 7 weeks of ages malabsorbed calcium from their duodenum. Hyp mice younger than this period were not affected nor were adult Hyp mice.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006890 TI - A 105 000 dalton antigen of transformed mouse cells is a stress protein. AB - Antisera prepared in mice against syngeneic spontaneously transformed AL/N cells (anti-TAL/N serum) identified a number of protein antigens synthesized by simian virus 40 (SV40) transformed cells, among which was a protein with a molecular mass of 105 000 daltons (p105). Of these transformed cell antigens which were immunogenic in a syngeneic system, only p105 was detected in primary mouse kidney cell cultures. p105 isolated from normal and transformed mouse cells was demonstrated to be identical by two-dimensional gel analysis. Relatively small amounts of p105 were synthesized in quiescent primary cultures, while the protein was actively synthesized in SV40-infected as well as in proliferating mouse kidney cells, and its synthesis in quiescent cells could be induced by subjecting the cultures to glucose starvation or heat-shock treatment. Immunofluorescent staining and cellular fractionation showed that p105 is normally localized to cytoplasmic structures. The results suggest that the expression of p105 is intimately associated with the metabolic state of the cell. PMID- 3006891 TI - Effects of severity of mitral stenosis on left and right ventricular function at rest and during exercise. AB - Few studies have assessed the effect of severity of mitral stenosis (MS) on ventricular function. Using equilibrium radionuclide ventriculography to measure ejection fraction and volume changes, 63 patients were studied during supine, symptom-limited exercise. To more carefully assess the 12 patients with MS and impaired left ventricular function, 2 groups of patients were formed. Group I (n = 51) had a normal (less than 50%) resting left ventricular (LV) ejection fraction (EF) and group II (n = 12) had an abnormally low (less than 50%) resting LVEF. Both groups were divided into mild (greater than 1.4 cm2), moderate (1.1 1.4 cm2) and severe (less than 1.0 cm2) MS. There were no differences in mean rest or exercise LVEF for group I. Exercise LVEF increased significantly (p less than 0.05) from rest with mild MS, but not with moderate or severe MS. The decrease in exercise LVEF was due to a decrease in exercise end-diastolic volume of 9 +/- 23% and 15 +/- 18% for moderate and severe MS, respectively. Exercise end-systolic volume decreased normally for all degrees of MS severity. Exercise right ventricular (RV)EF did not increase for any degree of MS severity due to an increase in end-systolic volume. All patients in group II had an RVEF of less than 40%. For this group, severity of MS had no effect on resting LVEF and the response to exercise was similar to group I. We conclude that in patients with MS, resting LVEF is unaffected by MS severity whereas exercise LVEF decreases with increased severity of MS due to impaired diastolic filling.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006892 TI - Calcium-dependent proteolysis and its inhibition in the ischemic rat myocardium. AB - Activity of calcium-dependent neutral proteinase and its specific inhibition was investigated in the acutely ischemic myocardium after ligation of the left descending coronary artery in anaesthetized open chest rats. In experiments where the mean arterial blood pressure could be maintained above 70 mm Hg the hearts were quickly removed 5 to 30 min after ligation and homogenized in ice-cold buffer. The activity of the calcium-dependent neutral proteinase and of its endogenous inhibitor were determined in the 10,000 g supernatant of ventricular and septal tissues. Hearts from normal anaesthetized and sham operated animals left on the respirator for 10, 20 and 30 min, were used as controls. Only traces of proteinase activity could be found in the supernatants obtained from normal controls, while in the sham-operated animals the specific activity of the Ca2+ dependent proteinase increased with time, reaching significantly elevated values after 30 min on the respirator. In the ischemic groups enzyme activity also increased with increasing duration of ischemia and was substantially elevated in the ventricular myocardium 20 min after ligation. The increased calcium-dependent proteinase activity was accompanied by significantly reduced activity of its endogenous inhibitor. The concomitant changes in the activities of the myocardial calcium-dependent neutral protease and its endogenous inhibitor suggest that interaction between the enzyme and its inhibitor play a role in the regulation of intracellular calcium-dependent proteolysis. PMID- 3006893 TI - The 1985 Upjohn award lecture. Tolerance, learning, and neurochemical adaptation. AB - Alcohol or drug tolerance has been viewed traditionally as a homeostatic response to a direct chemical action of the agent on the neuron. This concept has undergone major modification as a result of recent observations that behavioral and environmental factors can alter markedly the tolerance developed to the same drug regimen. Obligatory task performance under the influence of the drug, classical conditional stimuli in an environment habitually associated with drug administration, previous exposure to a tolerance-producing regimen, and environmental modification of the expression of the drug's effect can all influence dramatically the degree of tolerance produced by a given dosage. Attempts to identify possible cellular mechanisms of tolerance development are illustrated by a review of studies on the relations between ethanol tolerance and changes in the neuronal membrane Na+ -K+ ATPase and its interaction with ethanol and norepinephrine, hippocampal serotoninergic systems and their interaction with a vasopressin derivative, a membrane-bound calcium- and calmodulin- dependent kinase, and hypothalamic-hypophyseal endorphin-producing systems. None of these studies or other similar ones, whether correlational or interventional in nature, has yet provided full and credible explanations of the effects of behavioral and environmental factors on tolerance development. Finding such explanations is the major current challenge in the neurobiology of tolerance. PMID- 3006894 TI - Modulation of positive inotropy and metabolic coronary dilatation by myocardial alpha-adrenoceptors. AB - Cardiac hyperactivity and its consequent metabolically induced coronary vasodilation (MCD) were studied in isolated, perfused, electrically paced rat hearts. The alpha-adrenoceptor agonists, phenylephrine and methoxamine, produced a concentration-dependent inhibition of the inotropic responses to noradrenaline, dobutamine, isoprenaline, tyramine, and glucagon, while relatively potentiating their MCD reactions. This inhibition was unrelated to the alpha-agonists' known inotropic action and was not affected by catecholamine depletion of the heart. Withdrawal of the alpha-agonists or administration of the alpha-adrenoceptor antagonists phentolamine, phenoxybenzamine, or prazosin returned the inotropic and MCD reactions to normal. Neither the MCD response to electrically induced tachycardia nor the inotropic reactions produced by calcium chloride were affected by alpha-adrenoceptor agonists or antagonists. Alone, alpha-adrenoceptor antagonists were shown to potentiate the inotropic responses to noradrenaline and isoprenaline while the MCD was relatively diminished. The responses to glucagon were unaltered by alpha-antagonists. We postulate that myocardial reactivity to sympathetic stimulation can be modulated through alpha-adrenoceptors by the inhibition of processes that mediate cardiostimulation at post-beta-adrenoceptor sites, together with facilitation of those leading up to MCD. Accordingly, this modulation would act to prevent ischaemic damage to the heart by acting to limit the inotropic responses to increasing sympathetic stimulation while maximizing the blood supply to the myocardium. PMID- 3006895 TI - Effect of aldosterone on vascular angiotensin II receptors in the rat. AB - The effect of aldosterone on the density and affinity of binding sites for 125I labelled angiotensin II was investigated in a particulate fraction prepared from the rat mesenteric arteriolar arcades. The infusion of aldosterone 6.6 micrograms/h intraperitoneally via Alzet osmotic minipumps for 6 d produced an increase in the density of binding sites for 125I-labelled angiotensin II without change in affinity. After sodium depletion, mesenteric artery angiotensin II receptors were down-regulated as expected. An increase in the number of binding sites could be found when aldosterone was infused into sodium-depleted rats with no change in the elevated plasma renin activity. The intraperitoneal infusion of angiotensin II (200 ng X kg-1 X min-1 for 6 d) simultaneously with aldosterone resulted in down-regulation of vascular angiotensin II receptors, whereas after intravenous angiotensin II infusion (at 60 ng X kg-1 X min-1) the density of angiotensin II binding sites rose with aldosterone infusion. Plasma renin activity (PRA) was reduced and plasma angiotensin II increased in a dose dependent fashion after angiotensin II infusion. An aldosterone concentration of 3 ng/mL for 18 h produced an increase in the number of angiotensin II binding sites in rat mesenteric artery smooth muscle cells in culture. We conclude that increased plasma aldosterone may result in up-regulation of vascular angiotensin II receptors independently of changes in plasma renin activity, and may in certain physiological states effectively antagonize the down-regulating action of angiotensin II. PMID- 3006897 TI - Studies of angiotensin I converting enzyme: effects of kinins, bacitracin, gamma aminobutyric and epsilon-aminocaproic acids, and related compounds on substrate binding and catalysis in vitro. AB - Bradykinin and 22 of its analogs were evaluated for their abilities to inhibit the hydrolysis of [3H]hippurylglycylglycine by purified porcine kidney angiotensin I converting enzyme. The mean inhibitory concentration (IC50) for bradykinin was 1.2 +/- 0.2 X 10(-6) M. Except for Ile-Ser-bradykinin and [Sar4] bradykinin, none of the kinin analogs were more potent in this regard than bradykinin. Bacitracin, gamma-aminobutyric acid, epsilon-aminocaproic acid, and structurally related compounds were also tested. The IC50 value for bacitracin was 1.9 +/- 0.4 X 10(-4) M, gamma-aminobutyric acid, 83.4 +/- 7.2 mM, and for epsilon-aminocaproic acid, 7.0 +/- 1.4 mM. Compounds were also evaluated for their abilities to prevent 125I-labelled [Tyr1]-kallidin binding to angiotensin I converting enzyme inhibited by EDTA. The IC50 values for bradykinin, bacitracin, gamma-aminobutyric acid, and epsilon-aminocaproic acid were 1.6 +/- 0.3 X 10(-8) M, 2.6 +/- 0.9 X 10(-6) M, greater than 291 mM, and 13.2 +/- 3.9 mM, respectively. PMID- 3006896 TI - Effect of the calcium ionophore A23187 on superoxide generation in phorbol ester stimulated human neutrophils. AB - The calcium ionophore, A23187, and the tumor-promoting phorbol ester, phorbol 12 myristate 13-acetate (PMA), interacted synergistically to elicit an accelerated superoxide production response in human neutrophils. The lag period preceding PMA induced superoxide generation was decreased in a dose-dependent manner by A23187 at a concentration range from 1.0 X 10(-8) to 1.0 X 10(-5) M. Superoxide production rate, however, was subject to biphasic effects. While the rate was potentiated in a dose-dependent manner at A23187 concentrations below 1.0 X 10( 6) M, inhibitory influences became manifest at higher concentrations. Total superoxide production was subject to inhibitory effects, characterized by a mean inhibitory dose of 1.3 X 10(-6) M. The synergistic interaction of A23187 with PMA is consistent with a role for protein kinase C in neutrophil activation. Inhibition at high A23187 concentrations appeared to result from the effects of elevated intracellular Ca2+ levels on either NADPH oxidase itself, or some step in the transduction process linking protein kinase C to the oxidase complex. PMID- 3006898 TI - Activity changes of three nucleolytic enzymes during the life cycle of Saccharomyces cerevisiae. AB - Conditions were established for the assay of three nucleolytic enzymes: a Mg2+ independent endoribonuclease, a Mg2+-dependent endonuclease, and a Mg2+-dependent 5'-exonuclease in Saccharomyces cerevisiae cell extracts. The changes in the activities of these enzymes were determined throughout the life cycle of the organism. As the cells progressed from the exponential to the stationary growth phase, the specific activities of the Mg2+-independent endoribonuclease and of the Mg2+-dependent 5'-exonuclease increased, whereas the Mg2+-dependent endonuclease decreased. During sporulation the Mg2+-independent endoribonuclease and the Mg2+-dependent 5'-exonuclease increased several-fold over the first 10 h, but, since a similar increase was seen in nonsporulating control cells, the increases did not appear to be related to sporulation. However, the specific activity of the Mg2+-dependent endonuclease showed a sporulation-related increase during the first 3 h of sporulation, with a subsequent decline to very low levels. The specific activity of this enzyme increased again during germination to the levels seen in exponential phase cells. The Mg2+-independent endoribonuclease and the Mg2+-dependent 5'-exonuclease showed little change during germination of the ascospores. The high specific activity of the Mg2+ independent endoribonuclease during periods of nutrient deprivation is in agreement with the proposed role for this enzyme in the degradation of rRNA under these conditions. PMID- 3006899 TI - Trypsin-susceptible cell surface characteristics of Streptococcus sanguis. AB - The adherence of Streptococcus sanguis to saliva-coated hydroxylapatite was markedly reduced by treatment of the cells with trypsin. In Scatchard plots of adherence data, protease-treated S. sanguis did not exhibit the characteristic positive slopes, suggesting that trypsin prevented cooperative interactions between the cells and artificial pellicle. Trypsin also reduced the tendency of S. sanguis to bind to hexadecane and to octyl-Sepharose. When sodium dodecyl sulfate was used to elute S. sanguis from columns of octyl-Sepharose, it was observed that the elution profiles of trypsin-treated cells were more complex than those of control cells. Water and salts were incapable of removing the cells from octyl-Sepharose. The results suggest that adherence to saliva-coated hydroxylapatite, binding to hexadecane and to octyl-Sepharose depend on trypsin susceptible cell surface molecules. PMID- 3006900 TI - Effect of temperature on growth of guanidine-resistant mutants of poliovirus. AB - Guanidine-resistant (gr) mutants of poliovirus were previously categorized into four groups by electrophoretic properties and peptide maps of nonstructural virus protein 2C. Growth of mutants in the presence of guanidine depends upon temperature of incubation. The four groups of gr variants respond differently to temperature when guanidine is included in the culture medium. The data suggest clustering of gr mutations at several sites in the guanidine locus. PMID- 3006901 TI - Effects of cod-liver oil on intimal hyperplasia in vein grafts used for arterial bypass. AB - Cod-liver oil rich in eicosapentaenoic acid, an unsaturated fatty acid, has been shown to inhibit platelet aggregation. To determine the effect of this acid on vein-graft intimal hyperplasia, 46 segments of undistended external jugular vein were interposed between the bilaterally divided femoral arteries of 26 mongrel dogs. The animals received a 2% cholesterol diet for 1 week before and 6 weeks after the operation. Eight control animals received the diet alone, eight received cod-liver oil containing 1.8 g of eicosapentaenoic acid daily, for 1 week before and 6 weeks after operation, and seven animals received 1.8 g of eicosapentaenoic acid daily for 6 weeks after operation. Intimal thickness was measured at 6 weeks with a Zeiss computerized interactive image analysing system from multiple cross-sections of vein graft; 395 +/- 10 measurements were made from each graft. The intima measured 4 +/- 0.2 micron (SEM) before implantation and increased to 83 +/- 10 micron in the controls. Eicosapentaenoic acid administered before and after operation reduced intimal hyperplasia to 24 +/- 2.5 micron (p less than 0.001) and to 30 +/- 5 micron in animals receiving eicosapentaenoic acid after operation only (p less than 0.001). These results indicate that the acid inhibits intimal hyperplasia of canine vein grafts but that it is more effective when given before operation (p less than 0.01). PMID- 3006903 TI - The aging brain. AB - Changes in neurons, glial cells, synapse morphologic and electrophysiologic appearance, and neurotransmission mechanisms during normal aging are considered for review in this article. These processes not only are fundamental in the neurobiology of normal aging but also are implicated in the pathogenesis of aging. PMID- 3006902 TI - Clinical status of antiprogesterone steroids. PMID- 3006904 TI - Primary malignant pulmonary tumors in the older patient. AB - The incidence of lung cancer, the most common visceral malignancy, is increasing in the elderly patient. Careful preoperative preparation and postoperative care will allow some of these patients to have surgical resections. Radiotherapy and chemotherapy offer benefits for those patients who cannot have a curative surgical resection. PMID- 3006905 TI - Mesothelioma and mineral fibers. PMID- 3006906 TI - Astrocytoma as a second malignancy in patients with acute lymphoblastic leukemia. AB - Three cases of astrocytoma, two cerebral (grades II and III) and one spinal (grade II) occurring as second malignancies in patients with previously diagnosed acute lymphoblastic leukemia are described. All had received prophylactic cranial irradiation and intrathecal methotrexate. All were in remission at the time of development of the second malignancy. The time interval between central nervous system (CNS) prophylaxis and symptoms of CNS tumor was between 3 and 5 years. The possible causes of the combination of astrocytoma with acute lymphoblastic leukemia are discussed. PMID- 3006907 TI - Serum amyloid A in carcinoma of the lung. AB - Serum concentrations of serum amyloid A protein (SAA), peripheral blood lymphocytes (PBL) mitogenic response to phytohemagglutinin (PHA) and Concanavalin A (Con A), numbers of circulating T- and B-lymphocytes and length of survival after diagnosis were measured in 50 patients with cancer of the lung. SAA levels were significantly elevated when compared to 50 control subjects (P less than 0.001), but did not correlate with state of tumor spread at the time of diagnosis. Mitogenic responses of PBL from the cancer patients to PHA and Con A were significantly depressed (P less than 0.001), but also did not predict state of metastatic spread. The percentage of circulating T-lymphocytes was also significantly depressed in cancer patients when compared to controls. In six patients who survived tumor-free for greater than 2.5 years, SAA serum concentrations returned to normal. Statistical analysis showed a significant correlation between SAA serum concentrations and PBL mitogenic response to Con A. In addition, both high SAA concentrations and depressed PBL responses to Con A correlated with shortened survival. Therefore, these parameters may be of value in evaluating prognosis in patients with lung cancer. In addition, serial monitoring of SAA concentrations may be of value in evaluating recurrence or cure of lung cancer. PMID- 3006908 TI - Diagnostic value of high molecular weight alkaline phosphatase in detection of hepatic metastasis in patients with lung cancer. AB - High molecular weight alkaline phosphatase (HMW-ALP) was measured in the sera of 126 patients with lung cancer to determine its diagnostic value in the detection of hepatic metastasis. This isoenzyme was found in 21 of 24 patients with hepatic metastasis and in 27 of 102 patients without hepatic metastasis. When 10 U/L was used as a cut-off value, the sensitivity, specificity, and accuracy of this test were 71%, 89%, and 86%, respectively. From the standpoint of histologic type, this test was most useful in patients with small cell carcinoma. HMW-ALP was not detected in the sera of 15 controls. It is concluded that HMW-ALP is a useful marker for hepatic metastasis in patients with lung cancer. PMID- 3006909 TI - Wilms' tumor, overgrowth, and fetal growth factors: a hypothesis. AB - A hypothesis is advanced suggesting that the association between high birthweight, overgrowth features of certain congenital malformations, and Wilms' tumor may be due to the action of loci in addition to the putative Wilms' tumor locus on the short arm of chromosome #11. These genes include insulin, insulin like growth factor II and the c-Ha-ras 1 oncogene. The possible role of environmental factors in the oncogenesis of Wilms' tumor is discussed. PMID- 3006910 TI - Simultaneous presence of translocations t(14;18) and t(2;8) in a case of chronic lymphocytic leukemia. AB - We report a patient with classical chronic lymphocytic leukemia of IgM kappa phenotype and a stable clinical course, in which repeated chromosome analyses of blood lymphocytes revealed the coexistence of t(14;18), a marker often associated with follicular low grade lymphocytic lymphomas, and t(2;8), a variant of the t(8;14) typically seen in Burkitt's lymphoma. Both these translocations involve immunoglobulin gene regions, the t(2;8) being almost always found in patients with kappa light chain restriction. However, in an EBV-immortalized cell line of this patient, most karyotypes contained t(14;18) alone, without the t(2;8). This suggests that t(14;18) was the primary cytogenetic event, and that t(2;8) evolved subsequently. As a secondary cytogenetic event, the t(2;8) may not share the grave clinical consequences of a primary t(2;8) as seen in Burkitt's lymphoma and related disorders. PMID- 3006911 TI - Loss of alleles at polymorphic loci on chromosome 2 in uveal melanoma. AB - The loss of alleles at loci on specific chromosomes in some malignant tumors, such as retinoblastoma and Wilms' tumor, suggests that recessive mutations are important in their oncogenesis. We postulate that similar mechanisms may be involved in the formation of uveal melanomas. We studied alleles at autosomal loci in uveal melanoma cells and in the constitutional cells from 19 patients who developed the tumors. We observed loss of alleles only at loci on chromosome #2. This suggests that recessive alleles at some chromosome #2 locus may be important in the oncogenesis of uveal melanomas. PMID- 3006914 TI - Response of glucagonoma syndrome to lomustine. PMID- 3006915 TI - Solubility behaviour of whole human enamel. PMID- 3006912 TI - Search for possible antitumor promoters by inhibition of 12-O tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus activation; ursolic acid and oleanolic acid from an anti-inflammatory Chinese medicinal plant, Glechoma hederaceae L. AB - From an anti-inflammatory Chinese medicinal plant, Glechoma hederaceae L., two triterpene carboxylic acids, ursolic acid (UA) and oleanolic acid (OA) have been isolated as inhibitors of 12-O-tetradecanoylphorbol-13-acetate (TPA) induced Epstein-Barr virus (EBV) activation in Raji cells. Both acids significantly inhibited the activation at a 1000-fold molar ratio to TPA, and also teleocidin B 4. The dose responses of the acids were very similar to those of the antitumor promoters, retinoic acid (RA) and glycyrrhetinic acid (GA). However, a characteristic property that UA and OA possess, far higher cell viability to the Raji cells than RA to the Raji cells, has been pointed out. Furthermore, enhancement of the inhibitory activity was found in 3-keto derivatives of UA and OA, while either loss of oxygen functionality at C-3 position of UA or oxidation at C-3 of GA led to reduction of the activity. Binding assay suggested that the inhibitory activity should be exhibited by some event caused after binding of TPA to the receptor in the cells. PMID- 3006916 TI - [Antibody response to EB viruses in patients with infectious mononucleosis. Relation of the antibody response to the course of the disease]. PMID- 3006913 TI - Phase I-II evaluation of intra-arterial diaziquone for recurrent malignant astrocytomas. AB - Diaziquone (AZQ) is a lypophilic alkylating agent that crosses the blood-brain barrier and has shown broad activity in animal tumor models. Five of 12 patients with malignant astrocytoma treated with iv AZQ had clinical and/or radiographic improvement (Schold, Neurology 34:615, 1984). Intra-arterial administration of AZQ to patients with brain tumors should produce higher peak levels of drug in the tumor and should reduce systemic toxicity. Twenty-one patients with astrocytoma (grade II, four; grade III, 11; and grade IV, six), in all of whom irradiation and intra-arterial carmustine chemotherapy failed, received intra arterial AZQ as a single dose every 28 days. Two of 20 evaluable patients experienced partial responses of 5 and 8+ months, respectively. Four patients had disease stabilization of 3, 4, 5, and 8 months' duration, respectively, and one of these patients had tumor shrinkage (partial response) after seven courses of AZQ. The initial dose in the first three patients was 10 mg/m2, and doses in subsequent groups of three patients were begun at increases of 5 mg/m2. The within-group dose escalation was 5 mg/m2 per course if there was no hematologic toxicity. Dose-limiting toxicity was myelosuppression, which occurred at doses greater than 15 mg/m2. The maximum tolerated dose was 25 mg/m2. Intra-arterial AZQ appears to be of marginal effectiveness in patients refractory to carmustine and offers no advantage over iv AZQ in efficacy or toxicity. PMID- 3006917 TI - [New quinoline chemotherapy. DNA gyrase inhibitors]. PMID- 3006919 TI - Primary hepatocellular carcinoma presenting as haemobilia: report of a case. PMID- 3006918 TI - Cyclic AMP and Ca2+ uptake by mastocytoma mitochondria. AB - A thorough re-investigation was undertaken of a variety of factors that might explain the increased uptake of 45Ca2+ by mitochondria isolated from N6, O2' dibutyryladenosine-3',5'-cyclic monophosphate (DB cyclic AMP)--treated PY815 cells. This showed that mitochondria isolated from DB cyclic AMP treated cells take up 45Ca2+ at a 30 per cent faster rate than mitochondria from untreated cells, although both mitochondria eventually reduce the total external Ca2+ to the same levels. 45Ca2+ precharged mitochondria from DB cyclic AMP-treated cells also leaked 45Ca2+ more slowly than those from untreated cells when they were recovered by filtration. Thus an apparently greater uptake of 45Ca2+ by mitochondria from DB cyclic AMP-treated cells was a consequence of the filtration procedure. In fact, mitochondria from DB cyclic AMP-treated cells contained less total Ca2+ than those from untreated cells, while DB cyclic AMP-treated cells also contained less total Ca2+ than untreated cells. The results suggest that mitochondria do not play an important role in controlling the growth of DB cyclic AMP-treated PY815 cells through effects on cytoplasmic Ca2+ availability. PMID- 3006920 TI - Protein kinase C phosphorylation at Thr 654 of the unoccupied EGF receptor and EGF binding regulate functional receptor loss by independent mechanisms. AB - To test the functional consequence of phosphorylation of the EGF receptor at Thr 654 by protein kinase C, the normal Thr 654 human EGF receptor cDNA or a mutant encoding an Ala 654 were expressed in heterologous cells. In cell lines expressing both the Thr 654 and Ala 654 receptors, functional cell-surface Thr 654 receptors were reduced or were totally lost, but were not degraded, following activation of protein kinase C by phorbol esters (TPA), whereas Ala 654 receptors were unaffected. These data suggest that protein kinase C regulates ligand independent receptor binding and internalization via phosphorylation of Thr 654 of the EGF holoreceptor. Because EGF induces internalization and degradation of the Ala 654 EGF receptor, at least two independent mechanisms can serve to signal loss of functional EGF receptors. PMID- 3006921 TI - Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: a Ca2+-triggered molecular switch. AB - Calcium/calmodulin-stimulated autophosphorylation of a prominent brain calmodulin dependent protein kinase (Type II CaM kinase) produces dramatic changes in its enzymatic activity. These changes suggest a mechanism by which the kinase could act as a calcium-triggered molecular switch. Incorporation of 3-12 of a possible total of 30 phosphate groups per holoenzyme causes kinase activity toward exogenous substrates as well as autophosphorylation itself to become independent of calcium. Thus, kinase activity could be prolonged beyond the duration of an initial activating calcium signal. The calcium-independent autophosphorylation could further prolong the active state by opposing dephosphorylation by cellular phosphatases. PMID- 3006922 TI - Identification and sequence of a gene required for a developmentally regulated DNA excision in Anabaena. AB - Vegetative cells of the cyanobacterium Anabaena contain an 11 kb DNA element within the coding region of the nifD gene. This element is excised by site specific recombination between directly repeated 11 bp sequences at each of its ends during differentiation of nitrogen-fixing cells called heterocysts. Site specific recombination, leading to the same rejoined nifD gene, was observed during propagation in E. coli of a fragment containing the 11 kb element and flanking sequences. An assay for excision of the element in E. coli was developed, based on mini-Mu-lac transposition into the element. Since the 11 kb element lacks an origin of replication, its excision results in loss of lac and conversion of blue colony-forming cells to white on X-gal plates. Insertion and deletion mutagenesis identified a region of the element needed for excision. Mutations in this region could be complemented by a 6 kb fragment containing an open reading frame that runs counter to those of the nif genes, beginning 240 bp from the recombination site. PMID- 3006924 TI - Interactions between adenosine and oscillatory cAMP signaling regulate size and pattern in Dictyostelium. AB - We present evidence for the hypothesis that in multicellular structures of Dictyostelium, production of adenosine by hydrolysis of cAMP near the tip region prevents both generation of competing tips and differentiation of prespore cells near the tip, and thus establishes a "prestalk" region. We demonstrate that adenosine affects the immunological prespore specific staining pattern in slugs in a manner opposite to cAMP:cAMP induces an increase of prespore antigen; adenosine induces a decrease. When endogenous adenosine is removed from slugs, prespore vacuoles are synthesized throughout the prestalk region. Adenosine was found to inhibit the induction of prespore differentiation by cAMP in an apparently competitive manner. It was also found that adenosine specifically increased the amount of tissue controlled by one tip, probably by inhibiting generation of competing oscillators. Removing endogenous adenosine from slugs resulted in a decrease of tip dominance. PMID- 3006923 TI - Rous sarcoma virus transforming protein lacking myristic acid phosphorylates known polypeptide substrates without inducing transformation. AB - Mutagenesis of glycine 2 of p60src, the transforming protein of Rous sarcoma virus (RSV), yields a protein that is neither myristylated nor bound to cellular membranes. Although these mutant viruses retain full tyrosine protein kinase activity, they are transformation-defective. We examined in detail tyrosine phosphorylation of cellular polypeptides and the phenotype induced by infection with two such viruses. Infection failed to cause growth in agar, cytoskeletal reorganization, or changes in fibronectin synthesis and protease secretion. Strikingly, tyrosine phosphorylation of the known substrates of p60src was extensive, and differed from that found in wild-type transformed cells only quantitatively. There was no apparent correlation between the extent to which any of eight known protein substrates of p60src were phosphorylated and the phenotype of infected cells. We suggest that the phosphorylation of as yet unidentified proteins, which are probably found in cellular membranes, is essential for transformation by RSV. PMID- 3006925 TI - Sequence-specific interactions of nuclear factors with the insulin gene enhancer. AB - Insulin 5'-flanking DNA contains two elements controlling cell-specific expression, one a cell-specific enhancer. We show that factors in nuclear extracts derived from an insulin-secreting cell line interact with three distinct regions within the insulin enhancer. The 5' border of the farthest upstream protected region coincides with the 5' border of the enhancer element, indicating a functional role for this interaction in insulin gene transcription. This protected region covers 46 bp, including an enhancer core sequence. This region was not protected in nuclear extracts prepared identically from two heterologous cell lines, indicating at least a 3-fold lower level of interacting factors in these cells. The size of this protected region suggests that more than one factor is binding, possibly facilitating cell-specific interaction of a binding protein with the core enhancer nucleotides. The other two protected regions were observed in extracts prepared from one or both of the heterologous cells. PMID- 3006926 TI - Extensive unwinding of the plasmid template during staged enzymatic initiation of DNA replication from the origin of the Escherichia coli chromosome. AB - Early in the staged initiation of enzymatic replication of plasmids containing the unique origin of the E. coli chromosome (oriC), the plasmid is converted to a new topological form which is highly underwound, two to 15 times more than native supercoiled DNA. The underwinding reaction precedes priming of DNA synthesis and follows an initial complex formation, requiring ATP and proteins dnaA, dnaB, and dnaC; underwinding depends on the further addition of gyrase and SSB. DnaB protein as a helicase and gyrase as a topoisomerase drive the underwinding with the energy of ATP hydrolysis. The underwound template, extensively single stranded and complexed with proteins, is an active form for priming by primase and elongation by DNA polymerase III holoenzyme. PMID- 3006927 TI - Both DNA topoisomerases I and II relax 2 micron plasmid DNA in living yeast cells. AB - Measurements at various temperatures of the linking number of yeast 2 microns plasmid DNA in wild-type cells and in cells bearing mutations in the DNA topoisomerase I and II genes show that bulk 2 microns plasmid minichromosome are maintained in a relaxed state by the combined action of topoisomerases I and II. Bulk 2 microns minichromosomes are not under torsional stress in vivo and are not substrates for a putative gyrase-like topoisomerase. PMID- 3006928 TI - Phosphomannosyl receptor from bovine liver: effect of storage condition on the appearance of different molecular species. PMID- 3006929 TI - [Screening examination of the liver directed at the incidence of adenomas in women using hormonal contraception]. PMID- 3006930 TI - [The number of neonates treated with exchange transfusion for hemolytic disease due to Rh isoimmunization before and after the administration of anti-Rho (D) immunoglobulin prophylactically]. PMID- 3006931 TI - [Neuromuscular transmission and its disorders. Review]. PMID- 3006932 TI - [EMG diagnosis of myasthenia gravis]. PMID- 3006933 TI - Sensitivity of 115 strains of the genus Brucella to some antibiotics (cephalosporins, ureidopenicillins and aminoglycosides). AB - The Authors tested piperacillin, azlocillin, aztreonam, cefotaxime, ceftizoxime, ceftriaxone, cefotetan, moxalactam, gentamicin, sisomicin and dibekacin, on about 120 strains of the genus Brucella, using as control drugs tetracycline, streptomycin and rifampicin. Cefotaxime, ceftizoxime, gentamicin, and sisomicin exhibited antibrucella activity comparable to that of tetracycline and just a little superior to that of rifampicin. Among the aminoglycosides tested, gentamicin, sisomicin and dibekacin were, in decreasing order, more active than streptomycin. For all the other drugs tested there was a large variability among the brucella strains, with roughly a peak of activity on the strains which have a history of repeated in vitro passages. PMID- 3006934 TI - Formation of iminoxyl and nitroxide free radicals from nitrosonaphthols: an electron spin resonance study. AB - The possible metabolic activation of nitrosonaphthols, suspected carcinogens, was investigated by electron spin resonance (ESR) spectroscopy. Free radicals were found to be the primary metabolites formed during both the reduction and oxidation of these compounds. Whereas the one-electron oxidation of nitrosonaphthols is enzymatic and catalyzed by the peroxidase prototype, horseradish peroxidase, their one-electron reduction by reducing cofactors such as NADH or NADPH was not enhanced by rat liver microsomal enzymes. The ESR spectra of the radicals found during the oxidation of nitrosonaphthols were analyzed and characterized as iminoxyl free radicals. The reduction pathway leads to nitroxide free radicals with unusually low nitrogen hyperfine constants. PMID- 3006935 TI - Substituted polychlorinated dibenzofuran receptor binding affinities and aryl hydrocarbon hydroxylase induction potencies--a QSAR analysis. AB - The rat hepatic cytosolic receptor binding affinities of 8-substituted 2,3 dichlorodibenzofurans and 8-substituted 2,3,4-trichlorodibenzofurans have been measured. The EC50-value for each compound was determined by dose-response competitive displacement of 2,3,7,8-[3H] tetrachlorodibenzo-p-dioxin (TCDD). Multiple parameter linear regression analysis of the data using several substituent parameters [lipophilicity (pi), hydrogen bonding (HB), electronegativity (sigma op), STERIMOL (delta B5)] demonstrated that for both sets of ligands, the binding affinities were dependent on substituent pi-values. The equations derived for the 8-substituted 2,3,4-trichlorodibenzofurans (a) and 8-substituted 2,3-dichlorodibenzofurans (b) were (Formula: see text) remarkably similar, moreover the relatively bulky t-butyl substituent was treated as an outlier for both calculations. The in vitro induction of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells by both sets of ligands demonstrated that there was not a rank order correlation between induction potencies and receptor binding affinities for these compounds. Analysis of the data for the 8-substituted 2,3-dichlorodibenofurans demonstrated that the monooxygenase enzyme induction was dependent on substituent lipophilicity and a STERIMOL (delta B5) factor which is related to (Formula: see text) substituent width. In contrast, the equation for the 8-substituted 2,3,4 trichlorodibenzofurans also included a substituent sigma op Hammett constant. The results indicate that although the binding affinities of the two sets of ligands are dependent only on substituent pi-values, their enzyme induction activities are both substituent and chlorine substitution pattern-dependent. PMID- 3006936 TI - Synthesis and biological activity of 1 alpha-hydroxy-24,24-dimethyl-22E dehydrovitamin D3 and 1 alpha, 25-dihydroxy-24,24-dimethyl-22E-dehydrovitamin D3. PMID- 3006937 TI - Characterization of the benzodiazepine binding site (diazepam site) on human serum albumin. PMID- 3006938 TI - [Influence of nitrogenous fertilizer on the growth of Vinca minor]. PMID- 3006939 TI - [Cystosarcoma phyllodes of the breast in a 10 year-old child]. AB - A case of cystosarcoma phyllodes in a 10 year-old, prepubertal white female is reported. In spite of the common belief of hormonal dependency of this tumor, hormonal receptor assays were negative. The diagnosis, behaviour and treatment of this unusual lesion is discussed. PMID- 3006940 TI - Small cell carcinoma of the lung. AB - Small cell lung cancer is a common, usually fatal neoplasm. Although palliative therapy is available for the majority of patients, only a very small minority enjoy long-term survival. Ironically, this neoplasm is nearly entirely preventable and a successful antismoking program is desperately needed. Our efforts to understand the basic biology of this tumor should continue, and, hopefully, will eventually translate into improvements in therapy. In addition to following the leads provided by basic research, a concerted clinical research effort needs to continue to build upon the advances already achieved. PMID- 3006942 TI - Rapid electrophoretic determination of neuron-specific enolase isoenzymes in serum. AB - This assay procedure for each of the two neuron-specific enolases (alpha gamma and gamma gamma) and the non-neuronal enolase (alpha alpha) in serum involves two steps: electrophoretic separation of the three isoenzymes--alpha alpha, alpha gamma, and gamma gamma--on cellulose acetate, and bioluminescence measurement of total enolase activity. From these data, the activity concentrations (U/L) of the three isoenzymes in serum are calculated. Both measurement steps are based on the enzymatic activity of enolase and thus differ from the immunological methods currently in use, which require the availability of specific antibodies. The method is rapid (approximately 30 min for both steps) and requires only 10 microL of serum for the complete analysis. Studies of normal children and adults, and of patients suffering from neuroblastoma and small-cell lung cancer, show that it is suitable for clinical use. Furthermore, the fact that both neuron-specific isoenzymes of enolase can be systematically separated is an advantage over immunological techniques in determining isoenzyme patterns for pathological samples. PMID- 3006941 TI - Membrane antigen expression during hemopoietic differentiation. AB - The pattern of certain groups of antigens expressed on the surface of hemopoietic cells changes either during the course of differentiation from pluripotent stem cells to mature functional cells or as a function of the proliferative state of the cells. A map of these changes is emerging and is providing valuable information for selecting and purifying rare stem cells and for classifying the acute leukemias. This knowledge is also beginning to provide insights into physiological and pathological cellular interactions affecting the early stages of hemopoiesis, and is being exploited to remove T lymphocytes from allogeneic bone marrow grafts in order to prevent graft-vs.-host disease as well as leukemic cells from bone marrow before autologous reinfusion. In this article I will briefly review the cellular basis of hemopoiesis and then discuss the methods used to determine the presence of antigens on normal hemopoietic cells. I will then summarize the pattern of membrane antigens expressed during differentiation and conclude by discussing the biological and therapeutic implications. PMID- 3006944 TI - Distribution of paraoxon hydrolytic activity in the serum of patients after myocardial infarction. AB - The activity of paraoxonase in serum was found to be bimodally distributed, both in a control group and in a group of patients who had suffered myocardial infarction. Activity in the myocardial infarct group was significantly lower than in the control group. Low paraoxonase activity in serum may provide an indication of susceptibility to the development of coronary heart disease. PMID- 3006943 TI - A kinetic colorimetric procedure for quantifying magnesium in serum. AB - We have developed a kinetic colorimetric procedure for determination of magnesium in serum. The magnesium-dependent enzyme glycerol kinase is used to phosphorylate glycerol to glycerol 3-phosphate, the latter being oxidized to dihydroxyacetone phosphate and hydrogen peroxide by glycerophosphate oxidase. The generated hydrogen peroxide is then reduced by peroxidase with the simultaneous oxidative coupling of 4-aminoantipyrine and 2-hydroxy-3,5-dichlorobenzenesulfonate, producing a red reaction product with an absorption maximum at 510 nm. The rate of color production is proportional to the concentration of the Mg X ATP complex, which is, in turn, proportional to the magnesium concentration in serum. This method is rapid and precise, avoids the use of expensive instrumentation, is easily automated, and results compare well with those by the Du Pont aca and manual Magon sulfonate methods. PMID- 3006945 TI - In vitro inhibition of angiotensin-converting enzyme by prednisolone and methylprednisolone. PMID- 3006946 TI - Urinary pseudouridine as a tumor marker in patients with small cell lung cancer. AB - The urinary concentration of pseudouridine, primarily a degradation product of transfer ribonucleic acid, was determined by high-performance liquid chromatography in 22 patients with small cell lung cancer, 30 patients with non small cell lung cancer, 13 patients with pulmonary infectious diseases and 24 healthy controls. The concentration of pseudouridine in both groups of patients with lung cancer was on the average significantly higher than that in the patients with pulmonary infectious diseases or in healthy controls. Thirteen (59%) of the patients with small cell lung cancer and 8 (27%) of those with non small cell lung cancer had a urinary pseudouridine level above the mean value plus 2 for the healthy controls. In 11 patients followed up during chemotherapy, urinary pseudouridine levels changed almost in parallel with the changes in the clinical responses. PMID- 3006947 TI - Spindle activity in the waking electroencephalogram: report of a case with hemispheric glioblastoma. AB - In this paper, we reported a patient with a hemispheric glioblastoma extending into the lateral thalamus and the posterior limb of the internal capsule. The waking electroencephalogram showed spindle activity on the side ipsilateral to the tumor. Based on the topography of the tumor in our patient, we speculate that a disruption of the synaptic pathways within the thalamus-cortex-thalamus circuit was primarily involved in the pathogenesis of abnormal spindling. PMID- 3006948 TI - Pathogenesis of hypercalcaemia of malignancy. AB - The hypercalcaemia of malignancy is multi-factorial, even within individual tumours. In most cases, hypercalcaemia is due to a combination of increased bone resorption associated with decreased renal capacity to excrete the increased extracellular fluid calcium. In solid tumours such as carcinoma of the lung, tumour-derived growth factors are probably primarily responsible for the increased bone resorption, and a separate family of factors which interact with some parathyroid hormone (PTH) receptors cause increased renal tubular calcium reabsorption. PTH production by non-parathyroid tumours rarely if ever occurs. In contrast, haematological malignancies such as myeloma and T-cell lymphomas produce locally acting bone resorbing lymphokines in excessive amounts. Some T cell lymphomas in addition have the capacity to metabolize 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D. In myeloma, impaired glomerular filtration frequently contributes to the pathogenesis of hypercalcaemia by impairing renal compensatory mechanisms for maintaining normal serum calcium concentrations in the presence of increased bone resorption. PMID- 3006949 TI - ACTH response to CRF in ACTH deficient patients. PMID- 3006950 TI - Paracrine action of transforming growth factors. AB - Polypeptide growth factors form a class of regulatory molecules which exert their effects by binding to specific receptors present on the cell surface. Most of the time the exact role of these factors in the healthy body is unknown. Some, like PDGF and TGF beta, seem to be involved in wound healing. Others, like EGF, promote epithelial cell growth and differentiation. The site of synthesis of most polypeptide growth factors is unknown. Their target can be identified by detecting the cells which present the specific receptors at their surface. It is though that polypeptide growth factors have a paracrine mode of action. Many different cancerous cells produce polypeptide growth factors and the appropriate receptors. Thus, they are able to stimulate their own growth in an autocrine fashion. Recently, some polypeptide growth factors and receptor genes or cDNAs have been molecularly cloned. Growth factor genes and messengers are much more complex than would be expected from the size of the polypeptide. Some cDNAs have been introduced into bacterial expression vectors and large amounts of the factors have been produced by bacteria. New tools, such as molecular probes and specific antibodies, are thus now available to investigate the production of the growth factors and their receptors. The same tools will facilitate the identification and understanding of the molecular mechanism whereby cancerous cells produce the growth factors and the appropriate receptors simultaneously. The importance of growth factors and receptors in cancer is stressed by the finding that three oncogenes are in fact the genes coding for one growth factor and two receptors. Finally, the molecular probes and the specific antibodies raised against these molecules can be used to identify precisely the growth factor(s) and receptor(s) produced abnormally in cancers. Antibodies that inhibit specifically the interaction of this very growth factor with its receptor could then be developed, thus allowing human tumour cell growth to be controlled. PMID- 3006951 TI - Viral diarrhoea. AB - It is apparent from this review that great progress has been made over the past 10 years in defining the aetiology of viral diarrhoea. Rotavirus is a major cause of gastroenteritis in children, particularly during the winter months. However, if bacteriological and virological data are pooled, our current aetiological knowledge reveals that a pathogen is not detected in 20 to 30% of cases in most perennial investigations. Now that human rotavirus has been cultured, complete characterization may be possible. However, practical methods for cultivating many of the other possible viral pathogens are needed before they can be characterized completely. Meanwhile, electron microscopy, although time-consuming, cumbersome and expensive, is the only method for detecting many of the other potential viral pathogens. We have much still to learn about the epidemiology of these agents, particularly in the developing countries, their importance in causing chronic diarrhoea, how they are transmitted, and the immune responses to infection. The development of a potential rotavirus vaccine is exciting and creates the possibility of control for this devastating disease. PMID- 3006952 TI - Inhibition of Plasmodium falciparum growth by IgG antibody produced by human lymphocytes transformed with Epstein-Barr virus. AB - Supernatants from Epstein-Barr virus (EBV)--stimulated B lymphocytes obtained from two adult Gambians who were partially immune to malaria markedly inhibited the growth of Plasmodium falciparum in vitro (55-95% inhibition). When 22 separate colonies were derived by micromanipulation from one of these primary cultures and their supernatants assayed, the degree of inhibition correlated with levels of IgG fluorescent antibody and total IgG. The inhibitory anti-P. falciparum IgG immunoprecipitated an antigen of mol. wt 195,000, identified as the major schizont surface glycoprotein by dual biosynthetic labelling with 3H glucosamine or 35S-methionine. Other studies on the analogous schizont surface protein of rodent malarias have shown that this antigen stimulates protective immunity. Production of this inhibitory antibody by adult Gambians may therefore contribute to their immunity to malaria. Human antibodies produced by EBV stimulated B lymphocytes may be used to identify other important P. falciparum antigens and have potential applications for immunotherapy. PMID- 3006953 TI - Systemic lupus erythematosus sera antilymphocyte reactivity: detection of antibodies to Tac-antigen positive T cell lines. AB - Sera obtained from patients with systemic lupus erythematosus (SLE) were tested for their reactivity to cell lines derived from cutaneous T-cell lymphoma (CTCL), or adult T cell leukaemia (ATL), and with other cell lines, by indirect immunofluorescence method. Approximately one half of SLE sera reacted with the surface antigens of HUT-102 cells, a cell line from CTCL, which constitutively expresses Tac antigen. The titre tended to be higher in the active than in the inactive stage. These positive sera also reacted with other neoplastic or normal T cell lines having Tac antigen. SLE sera reacting with HUT-102 surface antigens were further examined for their reactivities to Tac antigen, the putative IL-2 receptor, using HUT-102 or ATL-2. Pretreatment with anti-Tac monoclonal antibody partially blocked the reactivities to HUT-102 surface antigens in nine of 15 SLE sera tested. The binding of 125I-labelled anti-Tac monoclonal antibody was displaced by the addition of sera from six of 15 SLE patients. In addition, nine of the 15 SLE sera could inhibit the binding of 125I-labelled IL-2 to ATL-2 cells. These results suggested that some of SLE sera contained antibodies against the IL-2 receptor. PMID- 3006956 TI - [Cystic pleomorphic adenoma underlying the clinical picture of a large maxillary cyst]. PMID- 3006954 TI - Tumors of the nail. AB - Tumors of the nail unit are frequently difficult to diagnose in a timely fashion. This article reviews the principal benign and malignant conditions the clinician may expect to encounter and stresses differential diagnosis. PMID- 3006955 TI - [Mandibular ridge augmentation using hydroxylapatite ceramic]. PMID- 3006957 TI - [Changes in indications for augmentation alveoloplasty]. PMID- 3006958 TI - Sports-related extraarticular wrist syndromes. AB - Extraarticular wrist syndromes are relatively common causes of underperformance or nonparticipation in sports. Many of these conditions may be caused by nonathletic activities. The spectrum of pathologic signs ranges from tendon inflammation, stenosis, and subluxation to neuropathies, vascular conditions, congenital anomalies, cysts, calcarea, and callosities. Appropriate treatment requires an accurate diagnosis and specific measures directed toward the condition. Moreover, many of these problems may be preventable by modification of sports equipment, proper conditioning, alteration of athletic techniques or habits, and various orthoses. PMID- 3006959 TI - Malignant fibrous histiocytoma of bone. AB - Malignant fibrous histiocytoma of bone is a highly malignant neoplasm similar to that found in soft tissues. It accounts for at least 5% of all primary malignant bone tumors. The majority of lesions are primary, but some arise in preexisting benign lesions. Definitive diagnosis is made on histologic examination, a storiform arrangement of spindly tumor cells being the classic finding. Radical resection combined with preoperative and postoperative chemotherapy is the treatment of choice. PMID- 3006960 TI - Osteonecrosis of bone associated with combination chemotherapy without corticosteroids. AB - Osteonecrosis (aseptic or avascular necrosis of bone) is an entity with many causes that can occur at a variety of sites. It is a known complication of corticosteroid therapy, either alone or combined with other drugs in the treatment of malignancy. Osteonecrosis associated with chemotherapy that does not include corticosteroids is rare; three such cases have been reported in the English literature. One received cyclophosphamide alone, another vinblastine and bleomycin, while the third received cyclophosphamide, methotrexate, and 5 fluorouracil. The authors report a 40-year-old woman who had a left radical mastectomy in 1978 and a right radical mastectomy in 1980 for infiltrating ductal adenocarcinoma of the breasts. She received melphalan following the first surgery and a combination of doxorubicin, cyclophosphamide, and 5-fluorouracil after the second operation. In 1984 she noted pain in both knees that slowly increased in severity. A bone scan revealed increased periarticular activity in the medial and lateral femoral condyles of both knees compatible with bilateral osteonecrosis. There was no evidence of metastatic carcinoma on the bone scan. The patient was treated surgically with drilling and autologous bone grafting. A bone biopsy at the time of surgery revealed osteonecrosis but no metastatic adenocarcinoma. PMID- 3006961 TI - Polydactyly and polysyndactyly of the fifth toe. AB - Classification and treatment of polydactyly and polysyndactyly of the fifth toe are described based on a study of 37 patients with 46 affected feet. Polydactyly was seen in 26.1% of duplicated toes, polysyndactyly in 28.3%, and polysyndactyly fused with the fourth toe in 45.7%. Thirty-three patients with 42 toes were surgically treated. The medial toe was removed in patients with the duplicated fifth toe fused with the neighboring fourth toe; if necessary, a free full thickness skin graft was performed on the fourth toe and not on the fifth toe. Either the lateral or the medial fifth toe was excised for better contour of the forefoot in patients with polysyndactyly without fusion with the fourth toe. The lateral digital ray, including the metatarsal, was excised in patients with polydactyly of the metatarsal type. The average age of patients at operation was 12.3 months (range, five days to five years). Reorganization of the foot was facilitated when the child was treated early or before it could walk. PMID- 3006962 TI - Characterization of cells from human giant cell tumors of bone. AB - In vitro cell culture techniques were used to identify and characterize the individual cell types present in human giant cell tumors of bone. Three major cell types were identified based on morphologic characteristics, patterns of specific cell surface antigens, presence of receptors for hormones, and cell products. One population consisted of mononuclear cells that expressed monocyte macrophage markers. These cells lacked receptors for skeletal hormones and did not persist in culture. Distinct from these cells was a population of mononuclear cells that proliferated in culture and most likely represented the neoplastic element of the tumor. These cells phenotypically resembled connective tissue stromal cells, i. e., they did not express macrophage surface antigens and they produced collagens (Types I and III). They also possessed receptors for parathyroid hormone. The final population of tumor cells consisted of multinucleated giant cells. These cells lacked monocyte-macrophage surface antigens and possessed receptors for calcitonin, a phenotypic marker for osteoclasts. These studies illustrate that in vitro cell culture techniques can be employed to identify and characterize the cell populations of tumors of skeletal tissues, and demonstrate the usefulness of these cell culture models for investigating the biology of bone tumors. PMID- 3006963 TI - Osteogenic sarcoma. AB - Guarded optimism could be clearly stated for the treatment of osteogenic sarcoma. Improved methods of diagnosis and staging have been made possible by the computed tomography (CT) scan and other new modalities. The advent of pre- and postoperative chemotherapy has significantly enhanced survival rates that are now approaching 85%. Improved concepts of en bloc resection and better reconstructive techniques suggest that limb salvage procedures are not only possible but can provide excellent functional results in the context of muscle loss. Continued work must be directed toward improving the reconstructive techniques to provide implants with permanence, particularly for young patients and to clearly define the indications for such procedures. Patients presenting with thoracic lesions, although difficult to treat, can expect a 30%--45% long-term survival following repetitive thoracotomies. PMID- 3006964 TI - Evaluation of radionuclide imaging and echography in the diagnosis of thyroid nodules. AB - Radionuclide imaging with both Tc-99m sodium pertechnetate and Tl-201 chloride was studied in 152 patients with thyroid nodules. Ultrasonography also was performed in 81 of those patients. Tc-99m sodium pertechnetate scans demonstrated nodules in 69.7% of 78 differentiated thyroid carcinomas (DC) and 72.2% of 54 thyroid adenomas (Ad). Tl-201 chloride was accumulated in 73.7% of DC and 53.6% of Ad. By combining the Tc-99m sodium pertechnetate and Tl-201 chloride scans, the detectability of the nodules was increased to 90.8% for DC and 88.9% for Ad, respectively. The Tc-99m sodium pertechnetate scans showed better visualization of cystic lesions than did the Tl-201 chloride imaging. The Tl-201 chloride images clearly demonstrated intrathoracic tumor invasions in six cases of carcinoma and two cases of Ad. The Tl-201 chloride scan was also of value in detecting regional lymph node involvement and the recurrence and metastasis after thyroidectomy. The detectability of space-occupying lesions by ultrasonography was 96.3% in 81 patients with thyroid nodules. Ultrasonography differentiated well between solid and cystic lesions. The presence and extent of nodular lesions were detected with radionuclide imaging and ultrasonography in 98.8% of patients. Radionuclide imaging combined with ultrasonography provides a rapid, convenient, and useful method for the localization and visualization of thyroid tumors. PMID- 3006965 TI - Evolution of acute epididymitis to testicular infarction. Scintigraphic demonstration. AB - A case of acute epididymitis evolved into testicular infarction. Scrotal scintigraphy identified the initial epididymitis and subsequent evolution into infarction. PMID- 3006966 TI - Simplified count-based estimates of left ventricular volume. AB - Accurate count-based radionuclide estimates of left ventricular volume without the use of a blood sample have not been well described. Resting gated blood pool scans were obtained within 24 hours of catheterization in 31 patients (group 1), and simultaneously with thermodilution cardiac outputs in 29 other patients (group 2) at rest and during an induced-volume change (intervention). End diastolic and end-systolic volumes were calculated from the single-plane angiogram in Group 1 and from the combination of thermodilution stroke volume and radionuclide ejection fraction in group 2. Excellent correlations were noted between scintigraphic counts and angiographic volumes (r = 0.964), between scintigraphic counts and thermodilution-derived volumes (r = 0.979), and between interventional scintigraphic counts and interventional thermodilution-derived volumes (r = 0.941). Thermodilution and scintigraphic volume changes during the intervention were well correlated (r = 0.85). Accurate count based estimates of left ventricular volume without a blood sample are feasible at rest and during an intervention. PMID- 3006967 TI - Dual tracer imaging for localization of parathyroid lesions. AB - During the period from July 1983 through October 1984, a group of 38 patients with elevated serum calcium, parathormone (PTH) and/or clinical suspicion of hyperparathyroidism were studied by TI-201 Tc-99m dual tracer parathyroid imaging (DTPI). Seventeen of 18 parathyroid lesions were identified correctly. There was one false-negative, and the size of the adenoma missed by DTPI was less than 1.0 cm in diameter (1.0 x 0.5 x 0.2). There was one true-negative case. The other fifteen with negative scans are being followed clinically. Because of the small population studied, statistical analysis was not ascertained. However, this simple, noninvasive procedure has become a very useful diagnostic tool for the detection and localization of parathyroid lesions causing hyperparathyroidism, and the DTPI should be used in conjunction with ultrasonography and CT scanning in the preoperative evaluation in primary and secondary hyperparathyroidism. PMID- 3006968 TI - Perspectives on copper biochemistry. AB - The biochemistry of the essential trace element copper has been outlined. Following absorption, Cu(II) is transported by serum albumin and transcuprein to the liver where it is incorporated into the plasma Cu-protein, ceruloplasmin, or, possibly, stored as Cu-metallothionein or as superoxide dismutase. Ceruloplasmin is the long-term copper transporter and carries Cu(II) to the tissues for the biosynthesis of key Cu(II) enzymes, especially cytochrome c oxidase, lysyl oxidase and others. The production of copper enzymes raises many new questions about the metabolism of copper. Since ceruloplasmin is the centerpiece of copper metabolism and function, we conclude with more details on its chemistry and multifunctions. This Cu-protein of 132,000 daltons has now been totally sequenced and the copper-containing active sites located. Finally, we have proposed seven possible functions for ceruloplasmin, and there is now good evidence for the existence of ceruloplasmin receptors to expedite some of these functions. PMID- 3006969 TI - Iron metabolism. AB - Major aspects of iron metabolism are reviewed including iron absorption, internal iron distribution and iron storage along with the development of laboratory tests for the evaluation of iron status. PMID- 3006970 TI - Potential role of in vitro iron bioavailability studies in combatting iron deficiency: a study of the effects of phosvitin on iron mobilization from pinto beans. AB - Iron deficiency and iron overload affect one billion people worldwide. Treatment of iron malnutrition can be enhanced by an understanding of iron bioavailability from the diet. We have focused on the development of in vitro methods for determining iron bioavailability in the hopes of providing both an understanding of the chemical basis leading to the inhibition or enhancement of iron absorption and the provision of methodologies which will allow nutritionists around the world to ascertain iron bioavailability of local foods and food combinations. The study reported here focuses on the effects of phosvitin, a suspected inhibitor of iron absorption found in egg yolks, on the chemistry of iron during the in vitro enzymatic digestion of pinto beans. Three basic types of information were obtained. First, the total soluble iron was determined during in vitro enzymatic digestion under simulated oral, gastric (pH 2) and duodenal (pH 6) conditions. Phosvitin was found to have a strong solubilizing effect at pH 6 and pH 2 when in the presence of ascorbate. Pyrophosphate also leads to high iron mobilization. A second approach is to determine the static Fe2+ and Fe3+ concentrations following in vitro enzymatic digestion of pinto beans at pH 2 and pH 6. Ascorbic acid enhanced the total soluble iron at both pH values, however, only at pH 2 was a large proportion of the iron found in the Fe2+ state and then only in the presence of phosvitin but not pyrophosphate. A third approach is to determine the amount of Fe2+ formed in the digestive supernatant during a 10-min incubation with ferrozine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006971 TI - HTLV-III status and abnormalities in T lymphocyte distribution in children with hemophilia A. AB - Children with hemophilia A are at risk for the acquired immunodeficiency syndrome (AIDS). Clinically asymptomatic hemophiliacs demonstrate many immune abnormalities that might represent exposure to the AIDS agent through blood products or be a natural reaction to their therapy. In this study, we examined lymphocyte subset distribution in children with hemophilia A who had been exposed to HTLV-III as determined by antibody seroconversion. Seroconversion to HTLV-III was confirmed using Western blot analysis. The lymphocyte subsets studied included T4+ and T8+ cells. The distribution of lymphocyte subsets in children with hemophilia A was independent of seroconversion to HTLV-III. Children with hemophilia A treated with commercial factor VIII concentrate had normal numbers of circulating T4+ lymphocytes and significantly increased numbers of circulating T8+ lymphocytes compared with their nontransfused age-matched counterparts. An increased number of T8+ lymphocytes was not observed, however, in children treated exclusively with cryoprecipitate. These results suggest that HTLV-III alone cannot account for changes in lymphocyte subsets in hemophiliacs. Higher antigenic protein loads in factor VIII concentrate or additional factors might account for the increased absolute numbers of T8+ lymphocytes and represent a natural response to therapy. PMID- 3006972 TI - A retrospective study of thirty-two patients with small-cell bronchogenic carcinoma and inappropriate secretion of antidiuretic hormone. AB - Thirty-two patients with inappropriate secretion of antidiuretic hormone (ADH) complicating small-cell carcinoma of the bronchus were identified from a total of 226 patients with small-cell carcinoma of the bronchus treated at the Christie Hospital, Manchester, between 1978 and 1984. Basic data were collected from patients' files concerning the extent of the tumour, symptoms of inappopriate secretion of antidiuretic hormone, biochemical findings, treatment, the course of the inappropriate secretion of ADH and that of the underlying tumour. The data were then analysed and compared with results of previous studies. The response of the inappropriate secretion of ADH to treatment was found to be a significant prognostic factor. Further data were collected to determine the reason for this but it was difficult to drawn any firm conclusions. PMID- 3006973 TI - The effect of sodium bicarbonate on the flow-dependency of urinary prostaglandin excretion in man. AB - The influence of oral water loading on the excretion rate of prostaglandin (PG) E was investigated in healthy human subjects in a control study where the urine was acidic (pH 5.7) and after oral sodium bicarbonate, which made the urine mildly alkaline (pH 7.2). PGE was immediately extracted from urine and measured by a radioimmunoassay technique. After sodium bicarbonate (5 g) the urinary PGE excretion rate was some three-fold higher (P less than 0.01) than in the control study, in the absence of any significant difference in the urine flow (approximately 80 ml/h). In the control study (urine pH 5.7) the urinary PGE excretion rate increased significantly (P less than 0.01) as the urine flow rose in response to the oral fluid load. However, after sodium bicarbonate, PGE excretion did not alter after the fluid load despite a 10-fold increase in urine flow. Since after bicarbonate administration PGE excretion is independent of urine flow, mildly alkaline urine may represent a condition under which renal PGE synthesis can be effectively assessed from measurements of urinary PGE excretion, in the presence of changes in urine flow. In addition, the results are compatible with the hypothesis that, in man, PGE may be passively reabsorbed in the distal nephron, and a reduction in this reabsorption could contribute to or be responsible for the dependency of the excretion rate of PGE on urine flow. PMID- 3006975 TI - Antihypertensive effects of S9490-3, a new converting enzyme inhibitor, in the conscious spontaneously hypertensive rat. AB - The acute and chronic cardiovascular effects of S9490-3, a new converting enzyme inhibitor, were studied by means of a computerized analysis of the intra-aortic blood pressure recorded for 48 consecutive hours, in the conscious, unrestrained adult spontaneously hypertensive rat (SHR). The software used allowed the simultaneous study of one control and one treated animal. A single intravenous dose of S9490-3 (2 mg/kg) induced a rapid fall in systolic and diastolic blood pressure, which remained significant for 11 h. This decrease was not associated with an elevation in heart rate. A chronic oral treatment with S9490-3 (1 mg/kg/24 h for 12 days) significantly lowered systolic and diastolic blood pressure and reduced the variability of systolic blood pressure. The heart rate was slightly increased at rest (day time) but significantly elevated in the freely exercising SHR (night time). However, it must be noted that this increase remained moderate. The baroreflex sensitivity of chronically treated animals was enhanced when measured after phenylephrine but not after nitroglycerine bolus. Chronically given S9490-3 significantly reduced the pressor effect of phenylephrine and decreased the left ventricle weight. PMID- 3006974 TI - Tissue specific modulation of beta-adrenoceptor number in rats with chronic hypoxia with an attenuated response to down-regulation by salbutamol. AB - The number of beta-adrenoceptors and their affinity for the radioligand 125I labelled cyanopindolol (125I-CYP) were measured in crude membrane preparations of left ventricle, spleen and lung from Wistar rats exposed to 28 days continuous hypoxia. beta-Adrenoceptor density in the left ventricle was not significantly altered after exposure to chronic hypoxia (binding site maxima, Bmax.: normoxic control 36 SEM 5, hypoxic 24.8 SEM 2 fmol/mg of protein). There was no change in beta-adrenoceptor number in the spleen in response to chronic hypoxia (Bmax.: normoxic control 76 SEM 19 fmol/mg of protein, hypoxic 80 SEM 15 fmol/mg of protein). Chronic hypoxia resulted in a significant increase in beta-adrenoceptor number in lung tissue (binding site maxima, Bmax.: normoxic control 406 (SEM 31) fmol/mg of protein; hypoxic 535 (SEM 30) fmol/mg of protein, P less than 0.01 without change in the dissociation constant (KD) of the radioligand. beta Adrenoceptor subtypes in lung homogenates were studied by establishing displacement curves for 125I-CYP by ICI 118551 (a selective beta 2-antagonist). A significant difference was seen in the proportion of beta 1-/beta 2-adrenoceptor subtypes after hypoxia (normoxic control 66 SEM 2.5%, hypoxic 79 SEM 2.4% beta 2 adrenoceptors, P less than 0.01). alpha 1-Adrenoceptor number in lung membranes was measured with 125I-labelled 2-[beta-(4 hydroxyphenyl)ethylaminomethyl]tetralone (125I-HEAT). No difference was seen in the number of alpha 1-receptors in normoxia and in chronic hypoxia [Bmax.: normoxic control 48 (SEM 3), hypoxic 48 (SEM 5) fmol/mg of protein].(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006976 TI - Monocytes and granulocytes in rheumatoid arthritis (RA): phagocytic activity and superoxide anion production. AB - It is suggested by many tests that phagocytic cells were implied in inflammation which occurred during rheumatoid arthritis (RA). Further, three subject populations were selected for this study: Rheumatoid arthritis patients diagnosed according to American Rheumatism Association criteria (ARA mean = 6) and treated with gold compounds. Control subjects treated with the same non-steroidal anti inflammatory drug (NSAID), diclofenac (75 mg per day). Normal subjects without disease or treatment. Blood granulocytes and monocytes were separately tested for ingestion of three different particle species (opsonized zymosan, immunoglobulin G sheep red cells, glutaraldehyde-treated sheep red cells) and stimulation of superoxide anion production by these particles. All phagocytic cells in RA patients have normal phagocytic response and superoxide anion production. Autologous serum does not inhibit the activity of these cells. In addition the NSAID (diclofenac) does not act upon phagocytosis and oxidative burst of control cells. PMID- 3006978 TI - The morphology of redox-dye-treated HbH-containing red cells: differences between cells treated with brilliant cresyl blue, methylene blue and new methylene blue. AB - The ultrastructure of redox-dye-treated HbH-containing red cells has been shown to be dependent on the nature of the redox dye used. Brilliant cresyl blue causes the formation of a large number of small inclusions which are invariably attached to the inner surface of the red cell membrane. Methylene blue and new methylene blue cause the formation of a much smaller number of larger inclusions only some of which appear to be membrane-bound. These ultrastructural differences are reflected in marked differences in the light microscope appearances of the supravitally-stained cells. PMID- 3006977 TI - Sciatic neuropathy at the popliteal fossa: clinical, ultrasonographic and computed tomographic diagnosis. AB - A 22-year-old female had been suffering from sciatica-like pain in the left leg for four years. Clinical findings strongly directed further investigations to the popliteal fossa. Ultrasonography located a hypoechogenic mass in the upper lateral popliteal space. Guided by these data, computerized tomography (CT) with vertical reconstructions made the tentative diagnosis of a common peroneal nerve tumor, which was confirmed at operation. Microscopic examination showed a neurinoma of the mixed neurilemmoma-neurofibroma type. In the presence of atypical features of sciatica, a high index of suspicion seems advisable. Emphasis is laid on the complementary contribution of ultrasonography and CT in this type of ill-defined lower limb pain. PMID- 3006979 TI - Follow-up of circulatory changes secondary to deep venous thrombosis with special regards to radionuclide tests. AB - The influence of circulatory changes, which are secondary to deep venous thrombosis (DVT) in the leg, on result of radionuclide tests was studied in eight patients. Strain gauge plethysmography, a radionuclide blood-pool test and phlebography were performed both in the acute phase and during recovery up to 6 months after the initial admission. Morphological and functional changes were correlated with results from repeatedly performed 99Tcm-plasmin tests, a test currently used for diagnosis of DVT. In the acute phase, the thrombotic leg showed an increase in pooled blood and, in the case of proximal thrombosis, also impaired venous outflow. During the 6-month follow-up complete recanalization was observed in three patients and partial recanalization in five. The circulatory changes were found to recover progressively and earlier than the morphological changes. The 99Tcm-plasmin test was pathological at admission in all patients. It was normalized in parallel with plethysmography and blood-pool test results, at a time when morphological recovery was still incomplete. These findings confirm that a positive 99Tcm-plasmin test reflects haemodynamic changes which are secondary to the DVT rather than a specific binding of the radiopharmaceutical to the thrombus. The 99Tcm-plasmin test was normalized from 1 to more than 26 weeks after an acute DVT. This finding is of practical importance when using radionuclide tests for evaluation of acute recurrent DVT. PMID- 3006980 TI - Beta-blockade and binding of digoxin to skeletal muscle. AB - The effect of beta-blockade and a 1-h bicycle exercise test on the digoxin concentration in skeletal muscle (thigh) and serum was studied in 10 healthy men, who had ingested 0.5 mg digoxin daily for 2 weeks. Each subject performed two exercise tests at 100-140 W during maintenance digoxin treatment and 24 h after the latest dose. They rested in the supine position for 2.5 h before the exercise. Sixty minutes before the start of the exercise 0.25 mg/kg b.w. propranolol or saline (control) were injected (single-blind). At the end of the exercise the mean heart rate was 30% lower with beta-blockade (P less than 0.001). During exercise the mean skeletal muscle digoxin concentration increased by 29% (P less than 0.01) in the control situation and by 12% (NS) with beta blockade. The results indicate that propranolol partly inhibits the exercise induced increase in skeletal muscle digoxin binding. This might be due to inhibition of a catecholamine-induced stimulation of Na+-K+ATPase during exercise. PMID- 3006981 TI - Effect of beta 1-selective and non-selective beta-blockade on work capacity and muscle metabolism. AB - Six well-trained men were studied while performing a maximal bicycle exercise. The seven experiments included in this study were randomized in a double-blind cross-over fashion. On each occasion the subjects were given either placebo or 40, 80, or 160 mg propranolol (non-selective blockade) or 25,50, or 100 mg atenolol (beta 1-selective blockade). After completion of the study each subject had performed once under each of the seven treatments. Heart rate, maximal oxygen uptake (Vo2max), blood lactate and performance time to exhaustion were measured. A muscle biopsy from vastus lateralis was taken at exhaustion after placebo, 80 mg propranolol and 50 mg atenolol trials, for analysis of ATP, creatine phosphate (CP), glucose-6-phosphate (G-6-P), glucose and lactate. The performance time was reduced (P less than 0.05-0.001) with both blockers compared to placebo. At an equal heart rate reduction, Vo2max was equally reduced by both blockers. Performance time, on the other hand, was reduced to a greater extent (P less than 0.05) with propranolol. ATP and CP levels were decreased (P less than 0.05) by both drugs. G-6-P, however, was lower (P less than 0.05) with propranolol than with either placebo or atenolol. No difference was observed between placebo and atenolol. In conclusion, both beta1-selective and non-selective blockade reduced short-term maximal exercise capacity. The major limiting factor seems to be the reduction in oxygen transport. The finding that at an equivalent reduction in Vo2max propranolol reduced performance time to a greater extent than atenolol suggests that beta 2-blockade may reduce performance by mechanisms additional to those that affect oxygen transport.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3006983 TI - Comparison of commercial sources of primary rabbit kidney and MRC-5 cell cultures for herpes simplex virus isolation. AB - Two commercial sources of MRC-5 and primary rabbit kidney cell cultures were compared for sensitivity of herpes simplex virus isolation and cytopathic effect detection time. Forty-six of 78 previously positive and 50 of 234 fresh clinical specimens were positive and could be evaluated. Herpes simplex virus recovery from frozen specimens positive in at least one cell culture ranged from 85% in MRC-5 cells (M.A. Bioproducts) to 89% in primary rabbit kidney cells (M.A. Bioproducts). Herpes simplex virus recovery from fresh clinical specimens ranged from 94% in primary rabbit kidney cells (Flow Laboratories), MRC-5 cells (Flow Laboratories), and MRC-5 (M.A. Bioproducts) cells to 100% in primary rabbit kidney (M.A. Bioproducts) cells. Among the 96 positive specimens evaluated, no significant difference was found between commercially prepared MRC-5 and primary rabbit kidney cell cultures in either sensitivity or time to first detectable cytopathic effect. Cells from M.A. Bioproducts and Flow Laboratories were equally sensitive. Herpes simplex virus detection was dependent upon inoculating at least two cell culture tubes rather than if the cells were MRC-5 cells or primary rabbit kidney cells. PMID- 3006982 TI - Acquired immune deficiency syndrome (AIDS). AB - The epidemiology, clinical and pathologic manifestations, identification of groups at high risk, results of laboratory analyses, and description of the modes of transmission of the putative etiologic agent HTLV-III/LAV have been summarized. Mechanisms of induction of T-cellular immunodeficiency have been described, including the role of HTLV-III/LAV, and possible roles of CMV and EBV as cofactors have been presented. Prospects for preventing and treating AIDS have been succinctly summarized. PMID- 3006984 TI - The mouse egg's receptor for sperm: what is it and how does it work? PMID- 3006986 TI - Molecular and genetic analysis of the hairy locus in Drosophila. PMID- 3006985 TI - Isolation and structural analysis of the extra sex combs gene of Drosophila. PMID- 3006987 TI - Localized maternal mRNAs in Xenopus laevis eggs. PMID- 3006988 TI - Embryonic pattern in Drosophila: the spatial distribution and sequence-specific DNA binding of engrailed protein. PMID- 3006990 TI - Structure and expression of two classes of mammalian homeo-box-containing genes. PMID- 3006991 TI - Human cDNA clones containing homeo box sequences. PMID- 3006992 TI - The Ac and Spm controlling element families in maize. PMID- 3006993 TI - Regulation of Tcl transposable elements in Caenorhabditis elegans. AB - C. elegans strains contain variable numbers of a 1.6-kb transposable genetic element. Activity of this element, which is denoted Tcl, shows regulation at at least two levels. At one level, excision of Tcl elements occurs in somatic cells at a frequency several orders of magnitude higher than in germ cells. Evidence is presented suggesting that this results from regulation at the level of trans acting functions that are required for excision or that repress excision. At the second level, germ line transposition of Tcl occurs at greater frequency in some strains than in others. The hypothesis is proposed that this is because Tcl is one component of a two-element system, the second element of which differs between strains. Evidence for a second putative transposable element family in C. elegans is presented. This family has properties that suggest a relationship to Tcl. This possibility is currently under investigation. PMID- 3006989 TI - Expression of murine genes containing homeo box sequences during visceral and parietal endoderm differentiation of embryonal carcinoma stem cells. PMID- 3006994 TI - Germ line specificity of P-element transposition and some novel patterns of expression of transduced copies of the white gene. PMID- 3006996 TI - Hybrid genes in the study of glue gene regulation in Drosophila. PMID- 3006995 TI - The 68C glue puff of Drosophila. PMID- 3006997 TI - Use of gene transfer to increase animal growth. PMID- 3006999 TI - Promoter sequences of murine alpha A crystallin, murine alpha 2(I) collagen or of avian sarcoma virus genes linked to the bacterial chloramphenicol acetyl transferase gene direct tissue-specific patterns of chloramphenicol acetyl transferase expression in transgenic mice. PMID- 3006998 TI - Elastase I promoter directs expression of human growth hormone and SV40 T antigen genes to pancreatic acinar cells in transgenic mice. PMID- 3007000 TI - Retroviruses and insertional mutagenesis. PMID- 3007001 TI - An insertional mutation in a transgenic mouse line results in developmental arrest at day 5 of gestation. PMID- 3007002 TI - Molecular analysis of Kruppel, a segmentation gene of Drosophila melanogaster. PMID- 3007003 TI - spoOH: a developmental regulatory gene for promoter utilization in Bacillus subtilis. PMID- 3007004 TI - A simple gene with a complex pattern of transcription: the alcohol dehydrogenase gene of Drosophila melanogaster. PMID- 3007005 TI - Identification of DNA sequences required for the regulation of Drosophila alcohol dehydrogenase gene expression. PMID- 3007006 TI - Sex-lethal, a link between sex determination and sexual differentiation in Drosophila melanogaster. PMID- 3007007 TI - Control of sexual differentiation in Drosophila melanogaster. PMID- 3007008 TI - Sexual differentiation is controlled by a protein kinase encoded by the ran1+ gene in fission yeast. PMID- 3007010 TI - Persistence and transmission in the mouse of autonomous plasmids derived from polyoma virus. PMID- 3007009 TI - A comparison of several lines of transgenic mice containing the SV40 early genes. PMID- 3007011 TI - The regulation of gene expression in murine teratocarcinoma cells. PMID- 3007012 TI - Conservation and divergence of RAS protein function during evolution. PMID- 3007013 TI - Viral enhancer activity in teratocarcinoma cells. PMID- 3007014 TI - Coordinate expression of myogenic functions and polyoma virus replication. PMID- 3007015 TI - Structure and regulated transcription of DIRS-1: an apparent retrotransposon of Dictyostelium discoideum. PMID- 3007016 TI - Regulation of cell-type-specific differentiation in Dictyostelium. AB - During development of Dictyostelium, there are at least a dozen discrete stages of differentiation that can be distinguished by the expression of specific genes. The early stages are triggered by amino acid starvation and are dependent on a small heat-stable effector secreted by the cells to indicate a critical cell density. After development has proceeded for 12 hours, late genes are expressed that are dependent on the conditions found in multicellular aggregates. Cells monitor these conditions and appear to respond by raising their internal cAMP levels to act as a second messenger. Multicellularity can be bypassed as an essential condition if high levels of cAMP are added to the environment. The EDTA resistant cell-adhesion mechanism that develops by 12 hours is not a required aspect of multicellularity. A final set genes can be induced in 18-hour-developed cells by lowering the pNH3. The spore coat proteins are well-characterized markers for prespore differentiation; their genes are first expressed at 12 hours. Prestalk cells do not express these genes. A small number of prestalk cells become redistributed in the posterior during slug migration and appear to undergo respecification when their position is changed. Prestalk genes become repressed in these "anterior-like" cells and prespore genes are activated. These results clearly indicate that a fieldwide system of positional information regulates cell-type differentiation in Dictyostelium. PMID- 3007017 TI - Two feedback loops may regulate cell-type proportions in Dictyostelium. PMID- 3007018 TI - cAMP receptors controlling cell-cell interactions in the development of Dictyostelium. PMID- 3007019 TI - Cell interactions govern the temporal pattern of Myxococcus development. PMID- 3007020 TI - Isolation and characterization of Cu,Zn superoxide dismutase of the shark Prionace glauca. AB - A Cu,Zn superoxide dismutase was purified for the first time from an elasmobranch species (Prionace glauca) and showed the following differences with respect to other animal superoxide dismutases. The enzyme displays a low isoelectric point. The enzyme activity is unusually independent of ionic strength. The isolated enzyme has 30% of its copper in the reduced state. PMID- 3007021 TI - Comparison of the spermicidal activity and acute toxicity of nonoxynol-9 and agent 741 [alkylphenoxy polyethoxy ethanol(10)]. AB - A spermicidal agent called "741" is used in the People's Republic of China as a substitute for nonoxynol-9 in vaginal contraceptive preparations. The relative spermicidal activity of these two agents was evaluated using dog, rabbit, monkey and human semen. Acute LD50 determinations in mice were also performed. The spermicidal activity and acute toxicity of both agents were either identical or very similar in all species. Since agent 741 is less expensive than nonoxynol-9 to manufacture and equally as potent in vitro, it is an alternative spermicide for vaginal contraceptive formulations. PMID- 3007022 TI - Current status of the role of parathyroid hormone in uremic toxicity. PMID- 3007023 TI - Adrenergic nervous system in hypertension. PMID- 3007024 TI - Protein translocation across and integration into membranes. AB - This review concentrates mainly on the translocation of proteins across the endoplasmic reticulum membrane and cytoplasmic membrane in bacteria. It will start with a short historical review and will pinpoint the crucial questions in the field. Special emphasis will be given to the present knowledge on the molecular details of the first steps, i.e., on the function of the signal recognition particle and its receptor. The knowledge on the signal peptidase and the ribosome receptor(s) will also be summarized. The various models for the translocation of proteins across and the integration of proteins into membranes will be critically discussed. In particular, the function of signal, stop transfer, and insertion sequences will be dealt with and molecular differences discussed. The cotranslational mode of membrane transfer will be compared with the post-translational transport found for mitochondria and chloroplasts. This review will conclude with open questions and an outlook. PMID- 3007026 TI - Marek's disease--a model for herpesvirus oncology. AB - The article will review the salient features of pathogenesis of Marek's disease in terms of sequential events which occur from the time of virus entry to the development of frank lymphomas. A hypothesis will be presented which will offer a credible explanation for this specific sequence of changes. Then, various factors which influence the incidence of neoplasms will be identified and the possible mechanisms by which they are influential will be discussed. These include the variable oncogenic properties of different virus strains, the influence of host genotype, and immune responses. PMID- 3007025 TI - Cardiopulmonary resuscitation in a pediatric ICU. AB - A 30-month, retrospective study of CPR was undertaken in a 10-bed, medical/surgical pediatric ICU (PICU). The 121 episodes of CPR reviewed represented 81 of 1357 admissions and 7537 cumulative days of PICU care. Of the 121 CPR attempts, 64% were initially successful, 48% were associated with at least 24-h survival, and 31% were followed by discharge from PICU. Unlike pediatric arrests outside the hospital or on general pediatric wards, PICU arrests were seldom unanticipated, were commonly nonrespiratory in origin, and generally occurred in spite of aggressive support. Of 118 PICU deaths during the study period, 45 (38%) were associated with CPR. In the 73 remaining PICU deaths, CPR had been withheld because of an order not to resuscitate. CNS status before arrest was the most important factor influencing outcome. In this pediatric population, 29% were noncomatose survivors 24 h after more than 30 min of resuscitation. PMID- 3007027 TI - Pharmacology of DMSO. AB - A wide range of primary pharmacological actions of dimethyl sulfoxide (DMSO) has been documented in laboratory studies: membrane penetration, membrane transport, effects on connective tissue, anti-inflammation, nerve blockade (analgesia), bacteriostasis, diuresis, enhancement or reduction of the effectiveness of other drugs, cholinesterase inhibition, nonspecific enhancement of resistance to infection, vasodilation, muscle relaxation, antagonism to platelet aggregation, and influence on serum cholesterol in experimental hypercholesterolemia. This substance induces differentiation and function of leukemic and other malignant cells. DMSO also has prophylactic radioprotective properties and cryoprotective actions. It protects against ischemic injury. PMID- 3007028 TI - Freezing injury in the yeast respiratory system. AB - The effect of freeze-thawing on the yeast respiratory system was studied at rapid rates of cooling. Freezing of whole cells with liquid nitrogen induced decrease of respiratory activity to under 20% of that of original cells. Mitochondria harvested from freeze-thawed cells have markedly decreased succinate oxidizing activity. Activity of succinate cytochrome c reductase was reduced significantly after freeze-thawing of whole cells while activities of succinate dehydrogenase and cytochrome c oxidase were reduced slightly. By spectrophotometric analysis it was found that about one-half the amount of cytochrome c + c1 was eluted from mitochondria to cytosol after freeze-thawing of cells. The activities of succinate oxidation in mitochondria from freeze-thawed cells were restored to normal levels by the addition of cytochrome c. Freeze-thawing of isolated mitochondria did not induce deactivation of succinate oxidizing activities and succinate cytochrome c reductase, and no elution of cytochrome c was observed. It was concluded that the decreased respiratory activities of yeast cells by freezing of cells with liquid nitrogen can be attributed primarily to the elution of cytochrome c from mitochondria. PMID- 3007030 TI - Association of changes in intracellular cyclic AMP with changes in phagocytosis in cultured rat pigment epithelium. AB - Papavarine, cholera toxin, and isoproterenol each elevated cyclic AMP in cultured rat retinal pigment epithelium and reduced the phagocytosis of isolated rod outer segments (38-66% of controls) during a 4 hour incubation. In addition, cultured pigment epithelium was preincubated with 25 mM exogenous cyclic AMP for two days and then incubated with outer segments for 4 hours either in the continued presence of exogenous cyclic AMP (condition A) or in the absence of exogenous cyclic AMP (condition B). Under condition B phagocytosis was 38% higher than under condition A. Intracellular cyclic AMP decreased under condition B but remained constant under condition A. These observations provide evidence that short term decreases in intracellular cyclic AMP are associated with increases in phagocytosis of outer segments by cultured rat pigment epithelium. PMID- 3007029 TI - Pigeonpea as an important food source. AB - Pigeonpea is an important source of proteins, carbohydrates, B-group vitamins, and certain minerals. India contributes over 90% of the pigeonpea production in the world where it is mostly consumed as dehusked splits or dhal. In African countries and Latin America, it is mainly consumed as canned peas. In this review, world production and distribution, genetic background, and biochemical and nutritional properties, storage and processing of pigeonpea are discussed. Future research needs to improve the utilization of pigeonpea as human food are also addressed. PMID- 3007031 TI - Recovery of a latent HSV-1 thymidine kinase negative strain following iontophoresis and co-cultivation in the ocularly-infected rabbit model. AB - Previous studies in the mouse and guinea pig have reported little or no colonization of sensory ganglia by strains of herpes simplex type 1 failing to express the enzyme, thymidine kinase (TK). The current study in the rabbit demonstrated trigeminal ganglionic colonization and reactivation of a latent thymidine kinase negative strain of HSV-1 by two independent methods: iontophoresis-induced ocular shedding and co-cultivation. Treatment with topical steroids during the acute infection did not enhance the latency rate. Following reactivation, back mutation with phenotypic reversion to thymidine kinase positive was demonstrated in a few recovered isolates. The current study also emphasized the importance of species differences to explain differing experimental results in studies of HSV-1 TK negative latency. PMID- 3007032 TI - Effect of autonomic mediators in recurrent shedding of herpes simplex in the rabbit eye. AB - After recovery from primary herpes simplex virus (HSV) infection of the eye in rabbits, recurrent shedding of virus in the external eye can be produced by the local application of 6-hydroxydopamine (6-HD) and epinephrine. Surgical sympathectomy did not prevent shedding of HSV with 6-HD/epinephrine so the source of the virus in the outer eye induced by adrenergic stimulation is not just the superior cervical ganglia (SCG). Chemical sympathectomy with 6-HD prior to HSV infection of the cornea led to decreased viral replication in the SCG during acute infection but did not interfere with uptake of the virus and latent or low grade infection of the ganglion. Shedding of virus in the outer eye induced by 6 HD/epinephrine was reduced by treatment with the beta-adrenergic blocker, timolol. These experiments strongly suggest that recurrent HSV shedding in the eye induced by catecholamines is due in part to effects on the peripheral (i.e. post synaptic) cells. While the peripheral neurons are the source of virus, it is possible that not all HSV detected in the external eye is due to release of preformed virus from nerve endings. PMID- 3007033 TI - On neuronal and glial differentiation of a pluripotent stem cell line, RT4-AC: a branch determination. PMID- 3007034 TI - Transdifferentiation of endocrine chromaffin cells into neuronal cells. PMID- 3007035 TI - Recurrent cutaneous leishmaniasis: successful treatment with sodium antimony gluconate. AB - The case of a 15-year-old boy with recurrent cutaneous leishmaniasis is reported. Species identification was based on results of serotyping and isoenzyme analysis. The patient did not respond to rifampin combined with isoniazid or to ketoconazole. Subsequent therapy with systemic sodium antimony gluconate resulted in complete regression of the lesions. PMID- 3007036 TI - Ectopic nucleolus organizer regions (NORs) in human testicular tumors. AB - Investigation of nucleolus organizer regions (NORs) in hematopoietic malignancies has indicated that the distribution and rearrangement of these regions may be more important in malignant tissues than is their number. In one of the few studies thus far reported on NORs in human solid tumors, we describe here Ag-NORs in a group of human testicular germ-cell tumors and the corresponding patients. Four of seven malignancies demonstrated consistent ectopic NORs; explanations could include chromosomal rearrangement (insertion?) or derepression of preexisting inactive NORs. PMID- 3007037 TI - Chromosomal mapping of enzyme loci in the domestic cat: GSR to C2, ADA and ITPA to A3, and LDHA-ACP2 to D1. AB - A panel of 42 rodent X cat somatic cell hybrids segregating individual cat chromosomes in different combinations was used to assign five isozyme structural loci to cat chromosomes. The feline homolog for glutathione reductase (GSR) was mapped to chromosome C2. Adenosine deaminase (ADA) and inosine triphosphatase (ITPA) were located on chromosome A3. Lactate dehydrogenase-A (LDHA) and acid phosphatase-2 (ACP2) were reassigned to chromosome D1. Localization of these genes increases the known feline genetic map and extends the known syntenic homologies between the cat and other mammalian species. PMID- 3007039 TI - A tale of two lipids. Cholesterol and eicosapentaneoic acid. PMID- 3007038 TI - Sublocalization of c-myb to 6q21----q23 by in situ hybridization and c-myb expression in a human teratocarcinoma with 6q rearrangements. AB - We have sublocalized the human proto-oncogene c-myb by applying two different techniques: in situ hybridization of metaphase spreads and chromosome spot hybridization of flow-sorted chromosomes. For this we used a teratocarcinoma cell line carrying specific chromosome translocations involving the two chromosomes 6 and one chromosome 11. The distribution of the c-myb gene copies on the different translocation chromosomes revealed that c-myb is located in the region 6q21--- q23. Because of the close proximity of the c-myb locus to the chromosomal breakpoints in the teratocarcinoma, we investigated whether c-myb was implicated in the development of this tumor. No rearrangement, deletion, or amplification of the gene was detected in the teratocarcinoma cells. Furthermore, the level of c myb expression was comparable to that of other cell lines of nonhematopoietic origin. These results suggest that c-myb was not affected by the translocation and played no significant role in the development of this teratocarcinoma. PMID- 3007040 TI - Tumor markers in patients with lung cancer. AB - The most examined tumor markers in lung cancer patients are CEA, hormonal peptides, and some neurogenic enzymes in small cell carcinoma. Calcitonin, ACTH, ADH, CEA, neurophysin, oxytocin, beta-endorphin, neuron-specific enolase, and CK BB are elevated in serum specimens in 25-75% of cases of small cell carcinoma. The level of these markers is related to the stage of the disease in groups of patients; elevated pretreatment levels decrease with tumor regression. Marker levels are not valid in defining the tumor load and the presence of disease in the individual patient. It has not yet been documented that the markers can be used for clinical decisions on antineoplastic therapy. A recent development is the finding that measurement of CSF and plasma concentrations of ADH, calcitonin, CK BB, bombesin, and neuron-specific enolase may contribute in the diagnosis of CNS metastases including meningeal carcinomatosis. PMID- 3007041 TI - Recent advances in the biology of small cell lung cancer. AB - Advances in the techniques for culturing human tumors in vitro, especially lung cancer cells, have greatly facilitated studies of the biologic properties of both small cell and non-small cell lung cancer cells. Detailed analysis has been done of well-characterized cell lines of both groups with respect to growth properties, biomarker and antigen expression, cytogenetics, and oncogene amplification and expression. Two major conclusions have emerged from these studies: (1) considerable heterogeneity exists within a given tumor type (eg, SCLC) in the expression of a given biomarker, and (2) overlap in the expression of biomarkers exists between cells of SCLC and non-SCLC, suggesting a common stem cell for all types lung cancer. In the future, clinical trials the impact of the biologic properties of cells on responses to therapy and survival will need assessment. PMID- 3007042 TI - Current chemotherapy of small cell lung cancer. AB - Since the advent of effective cytotoxic combinations in the early 1970s, results from chemotherapy for small cell lung cancer have improved very little. Maintenance chemotherapy appears of no benefit. Although attractive theoretically, "non-cross-resistant" combinations may not yet exist, and most data do not support alternating 1 regimen with another. Anticoagulant therapy with warfarin probably does not have a meaningful impact on survival, at least in extensive stage disease. To date the addition of VP-16, an active new agent, has not produced improvement in survival over earlier programs. The most promising leads to date involve dose escalation, especially with cyclophosphamide. Moderate "outpatient" escalation in limited disease induction therapy produced survival benefit in a randomized trial, and several studies indicate that the incidence of complete response can be increased by more intensive, inpatient "consolidation" with cyclophosphamide with or without other drugs after the induction period. Some form of local therapy, however, will be necessary to control disease in the chest, even with maximal dose intensification. PMID- 3007043 TI - Surgery of small cell lung cancer. AB - The role of surgical resection in the management of patients with small cell lung cancer remains to be defined. Some data suggest the potential benefit of resection in the few patients with very limited disease (peripheral T1N0 and T2N0 lesions), and there are chemotherapy regimens with 80-85% response rates in patients with more extensive but still localized disease. Interest has been reawakened in the role of adjuvant surgical resection in selected patients by 2 approaches: in patients with peripheral T1 or T2 lesions with negative mediastinal exploration, initial surgical resection followed by an adequate chemotherapeutic regimen and prophylactic cranial irradiation has resulted in an 80% disease-free survival at 30 months; initial chemotherapy in patients with only localized disease is followed by resection in the responders. Approximately 30% of the responders have undergone exploratory thoracotomy after completion of the chemotherapy. Local irradiation, as well as prophylactic cranial irradiation, generally has been used postoperatively. Early pilot studies suggest benefit of this approach in patients found to have T1-3 N0-1 disease but not in those with N2 disease. Prospective, randomized, clinical trials by the Lung Cancer Study Group in North America and its counterparts in Europe are now being carried out in hopes of supplying definitive data relative to this multi-modality therapy in small cell lung cancer. Unfortunately, no data are available to date. PMID- 3007045 TI - Advances in the biology of non-small cell lung cancer. AB - We have initiated an in-depth, comprehensive study of non-SCLC in the expectation that improvements in clinical management and diagnosis of lung cancer patients will follow. We have established and characterized over 30 such tumors in long term culture and as xenografts in athymic nude mice. These models usually retain the morphologic and other properties of the tumors from which they were derived. With the use of fully defined medium, most SCLC and adenocarcinomas can be established as long-term cultures. The growth conditions for squamous cell carcinomas have not been fully defined, and the success rate is low. In contrast to SCLC, there is considerable heterogeneity of non-SCLC tumors, with more than 12 phenotypes identified among the first 30 lines established. In addition, there is considerable overlap of properties, both within the non-SCLC types as well as between non-SCLC and SCLC. These findings indicate a common origin for all lung cancers. Radiation and drug sensitivity studies suggest that cell line data may correlate with clinical responses. Clinical protocols utilizing our ability to culture and drug test individual tumors are currently under way. They represent the first steps in a combined clinico-laboratory approach to the management of lung cancer. PMID- 3007044 TI - Radiotherapy for small cell lung cancer. AB - The role of radiation therapy in the primary management of small cell lung cancer is very much a matter of current debate. Its value in palliative treatment is unquestioned. Disappointment in the apparent inability to demonstrate improvement in survival in some randomized studies as a result of locoregional radiotherapy and prophylactic cranial irradiation may be due to the use of inappropriate study analysis. Recent studies using the end points of 2-year survival and local thoracic control do demonstrate improvements associated with locoregional thoracic radiotherapy. Factors such as total dose and radiation fraction size may be important. Large-field irradiation is also currently attracting interest, but its use should remain a research investigation. PMID- 3007046 TI - Non-small cell lung cancer. Role of radiation therapy. AB - The most important role of radiation therapy for carcinoma of the lung is its contribution to cure. Important technical advances in radiation therapy have been made that, along with advances in understanding the natural history of cancer of the lung, permit treatment with a higher expectation of cure than previously. Studies of fractionation have defined the doses required for control of intrathoracic tumors, and ancillary studies have better defined the treatment volumes necessary. Operable patients benefit from postoperative irradiation only when there is involvement of regional lymph nodes. In inoperable cases, the greatest survival benefit from radiation therapy is found among those who have a high performance status. Patients with adenocarcinoma and large cell carcinoma have a greater probability of long-term survival than those with squamous carcinoma. Investigations of altered fractionation may lead to improved results in radiation therapy for all histopathologic types of carcinoma of the lung. PMID- 3007048 TI - Radiotherapy in lung cancer. PMID- 3007047 TI - Surgical adjuvant therapy of non-small cell lung cancer. PMID- 3007049 TI - Innovative therapies (lung cancer). PMID- 3007050 TI - Research on normal values of serum angiotensin-I converting enzyme. Cooperative Group of Sarcoidosis Research. PMID- 3007052 TI - [Hepatoma in addition to active viral hepatitis. A simplified technic of right hepatectomy in the variety with a long portal pedicle. Zinc in hepatectomies. Apropos of a case]. PMID- 3007051 TI - [Chemosensitive metastatic cancer cured by surgery of the residual lesions]. PMID- 3007053 TI - Chronomodulatory infradian synchronization by placebo or ACTH 1-17 of Musca autumnalis mortality on shifted lighting regimens. AB - The face fly, Musca autumnalis, exposed to shifts of an LD16:8 lighting schedule at varying intervals, whether previously untreated or given placebo or ACTH 1-17 treatment, before the initiation of shifts, exhibits an infradian frequency response in mortality. At overall 50% mortality, a periodicity of approximately 4.5 days is found for flies exposed to placebo or ACTH 1-17 as a response to the shift interval. As compared to controls, the mortality of flies treated with placebo or ACTH 1-17 is delayed. Not all shift schedules are detrimental; some are actually beneficial. PMID- 3007054 TI - [Present status of nuclear cardiology: myocardial radionuclide imaging]. PMID- 3007055 TI - [Evaluation of blood sugar monitoring during an insulinoma operation]. PMID- 3007056 TI - [Diagnosis and treatment of insulinomas]. PMID- 3007057 TI - Effect of diet on intestinal xylose absorption in dogs. AB - The absorption of xylose at different levels of the intestine was compared in five dogs receiving diets containing either wheat bran, polyethylene particles (PE), or horse-bean hulls. The absorption was determined by serial collection of the interstitial fluid (ISF) in different parts of the small intestine and colon, and blood concentration after the administration of D-xylose as a solution (0.5 g/kg body weight) into the duodenal bulb. Xylose was mostly absorbed from the duodenum, and its concentration in the duodenal ISF and in plasma was reduced on a diet containing fiber, irrespective of the nature of fiber. In contrast, a negative linear relation between the mean retention time of digesta in the small intestine and the amount of xylose absorbed by duodenum was evidenced (r = 0.843). The results indicate that changes in transit linked to the presence of fiber in a diet are a major operative factor in the rate of carbohydrate absorption. They suggest that the absorption can be affected by a relatively minor change in the intestinal transit of digesta. PMID- 3007058 TI - Hepatocellular carcinoma associated with arteriohepatic dysplasia. AB - This report documents the occurrence of hepatocellular carcinoma in a 36-year-old man with arteriohepatic dysplasia and a noncirrhotic liver. This case represents one of the oldest patients reported to have arteriohepatic dysplasia and the first with the development of a hepatocellular carcinoma, of which I am aware. Although this tumor has been reported with other familial cholestatic syndromes, the absence of an underlying cirrhosis is of particular interest. PMID- 3007059 TI - The management of drop attacks. AB - From the preceding it is evident that drop attacks can result from a myriad of causes. As in all situations, the patient's history and the clinical picture are the most important factors in arriving at the appropriate diagnosis. However, understanding the neurophysiologic basis of posture should prove significantly helpful in this endeavor. PMID- 3007060 TI - A general method for retrieving the components of a genetically engineered fusion protein. AB - Escherichia coli expression vectors encoding an acid-labile aspartyl-proline (Asp Pro) dipeptide bridging two protein sequences were constructed and used to synthesize two different bovine growth hormone (bGH) fusion proteins. The codons GAT-CCX coding for Asp-Pro are provided by the recognition site for Bam HI (GGATCC). Treatment of the bGH fusion proteins at low pH in the presence of guanidine hydrochloride releases the bGH moiety from the fusion protein. The release of the bGH from the fusion protein specifically requires the Asp-Pro dipeptide linking the bGH sequence to the fusion protein. The bGH released from the fusion protein retains anti-bGH immunoreactivity as well as the ability to bind to growth hormone receptor in vitro. PMID- 3007062 TI - Enalapril: a new angiotensin converting enzyme inhibitor. AB - Enalapril maleate is a new angiotensin converting enzyme inhibitor marketed in the U.S. by Merck Sharp and Dohme. It has been demonstrated to actively interfere with the renin-angiotensin-aldosterone system. This is reflected by both hemodynamic (decreased blood pressure) and humoral (increased plasma renin, angiotensin I, and decreased angiotensin II) responses to enalapril therapy. Activity in the kallikrein-bradykinin system is still controversial. Enalapril maleate is a prodrug which is quickly absorbed, hydrolyzed by the liver to the active metabolite enalaprilic acid, and excreted 33 percent in the bile and 61 percent in the urine. The therapeutic dosage range is 10-40 mg/d, maximum of 40 mg, given once or twice daily. The onset and duration of action are dose related. Vertigo and headache have been the most commonly reported side effects. Clinical comparison of enalapril to hydrochlorothiazide, beta-adrenergic blockers, and captopril find it efficacious in the treatment of essential hypertension. Efficacy in treating congestive heart failure and hypertension secondary to renal artery stenosis has also been demonstrated for both angiotensin converting enzyme inhibitors. The overall efficacy and safety of enalapril and captopril appear equivalent when used at low doses in patients with uncomplicated hypertension. PMID- 3007061 TI - Isolation and characterization of the alpha 1-antitrypsin gene of mice. AB - We have characterized a genomic clone of the mouse equivalent of alpha1 antitrypsin (alpha1-antiprotease) a gene that is expressed in liver. There are at least three other genomic sequences similar to the gene expressed in mouse liver and all appear to be located on chromosome 12. The duplicated regions might include genes that are also expressed in the liver and/or in other tissues. Comparison of the sequences of the mouse alpha1-antitrypsin sequence expressed in liver with sequences of alpha1-antitrypsins from other mammals reveals great homology in the coding regions, particularly in the amino acids at the active site, including the neighboring methionine and serine thought to play a key role in the protease inhibition. PMID- 3007063 TI - Metoclopramide test for Addison's disease. PMID- 3007064 TI - [Clinical course of cytomegalovirus infection in adults]. AB - Between 1972 and 1984 73 patients with acute cytomegalovirus infection have been monitored. In 55 patients case history did not reveal any former disease. In 18 patients the cytomegalovirus infection was a sequela of cardiac surgery ("postperfusion syndrome"), transplantation or underlying malignant disease. After a prodromal stage which comprised unspecific symptoms in most cases various organ manifestations were noted in the course of disease. Typical cases presented hepatitis, myocarditis, Guillain-Barre syndrome with or without thrombocytopenia and (or) lympho-monocytosis. The organ symptoms were varying in degree and resolved completely within 4 to 12 weeks. Only one female patient died from congestive heart failure due to myocarditis after one year. PMID- 3007065 TI - [Increase in the prevalence of antibodies against LAV/HTLV III in drug addicts in West Germany]. AB - Sera obtained from 927 drug addicts in 1983 to 1985 were tested for antibodies against LAV/HTLV-III. There was a steadily rising proportion of positive results: 10.1% in 1983, 17.6% in 1984 and 23.9% in 1985. In each year the prevalence of anti-LAV/HTLV-III was higher among female than male addicts. No increased proportion of positive results was demonstrable in relation to age. Among 152 sera from 1983/84, hepatitis-B markers were found in 72 (43.7%), of whom 10 (14%) were also anti-LAV/HTLV-III positive. Among hepatitis-B marker-negative sera there were 8 (6%) which were also anti-LAV/HTLV-III positive. The prevalence of anti-LAV/HTLV-III in drug addicts in prisons, rehabilitation centres, hospitals and medical practices was similar. There is a danger that prostitution by addicts for obtaining drugs will cause a penetration of LAV/HTLV-III in the rest of the population. PMID- 3007067 TI - [Intestinal cryptosporidiosis in suspected acquired immune deficiency]. AB - In a 13-year-old boy--with haemophilia A, positive tests for HTLV-III antibodies, and lymphocytopenia--there occurred watery voluminous stools which persisted over many months. Cryptosporidia were demonstrated microscopically in the stool. Cryptosporidiosis should be considered in patients with acquired immune deficiency who have diarrhoea. PMID- 3007066 TI - [Bone marrow transplantation in severe aplastic anemia]. AB - Bone marrow transplantations were performed on 15 patients (aged 5-39 years) with severe aplastic anaemia. Twelve patients are alive 76-1930 days (median 668 d) after transplantation, with complete haematopoetic recovery. Total-body radiation with 3.6 Gy in four patients, cyclosporin A administration to ten patients and buffy-coat transfusion to nine patients entirely prevented early rejection. Two patients died of pneumonia (aspergillus; varicella-zoster virus), one patient died of bleeding from a splenic-artery aneurysm. In patients under the age of 40 years with severe aplastic anaemia bone marrow transplantation as early as possible after diagnosis is the treatment of choice if HLA-identical siblings are available as donors. In patients over 40 years treatment should at first be tried with antithymocyte globulin. PMID- 3007068 TI - Neuronal growth and shape. AB - Several aspects of neuronal growth related to the questions of shape acquisition, target attainment and target recognition are reviewed with an emphasis on how external cues and fields intervene in these developmental processes. PMID- 3007070 TI - Transpalatal approach to the skull base. PMID- 3007069 TI - Neoplasms and neoplasm-like lesions involving the skull base. PMID- 3007071 TI - The molecular relationship between HBV infection and hepatoma in Kenya. PMID- 3007072 TI - [Crystallochemical and micromorphologic studies of the enamel structure in odontodysplasia]. PMID- 3007073 TI - [Prostaglandins in jaw cysts. Elimination of pain during cystectomy]. PMID- 3007074 TI - [Surface characteristics of dental enamel, root cementum, and dentin after ultrasound treatment and after jet-spray of a water-sodium bicarbonate mixture]. PMID- 3007075 TI - [Coloring effect of chlorhexidine and a 2% erythrosine solution on new anterior dental filling materials]. PMID- 3007076 TI - [Postoperative hypoparathyroidism: calcium and phosphate metabolism and serum 25 hydroxycholecalciferol and the effectiveness of treatment with vitamin D3]. PMID- 3007077 TI - [Neutrophils of the peripheral blood and thyroid hormones]. PMID- 3007078 TI - [Antigenic properties, thyrotropin receptor and enzymatic activity of thyroid cell membranes exposed to the action of glutaraldehyde]. PMID- 3007079 TI - Mechanisms of gonadotropin-releasing hormone agonist action in the human male. AB - Agonist analogs of GnRH are being increasingly utilized to induce medical castration for treatment of a variety of hormonally responsive clinical disorders. However, the mechanism/s of their paradoxical antigonadal action in the human male remain poorly understood. Basal and integrated concentrations of immunoreactive LH after intermediate term (4-16 weeks) GnRH agonist treatment are only modestly decreased and cannot fully account for the far greater decline in serum testosterone (T) concentrations. Bioassayable LH concentrations, however, decrease markedly and parallel the fall in serum T suggesting secretion of qualitatively different LH species with diminished biological activity while the circulating concentrations of beta-subunit parallel the measured bioassayable LH concentrations, free alpha-subunit secretion remains persistently and disproportionately elevated during chronic GnRH agonist treatment. Cross reactivity of free alpha-subunits in the human LH RIA contributes to this disparity between the LH immuno and bioactivity. Chromatography of serum LH during GnRH agonist treatment suggests secretion of a qualitatively different LH species. Unlike the rat, in which the antifertility effects of the agonist are mediated predominantly by direct inhibition of testicular steroidogenesis, significant direct gonadal effects have not been demonstrated in man. Thus the bulk of evidence points to a predominant pituitary site of action in the human male. The molecular basis of the heterogeneity of LH during GnRH agonist, however, remains to be elucidated. The hypothesis that co- or posttranslational modification by the agonist may attenuate biologic activity of LH has not yet been directly tested. PMID- 3007080 TI - The molecular basis of gonadotropin-releasing hormone action. PMID- 3007081 TI - Pharmacokinetics of gonadotropin-releasing hormone and its analogs. PMID- 3007082 TI - Antioxidant enzyme activity in alveolar type II cells after exposure of rats to hyperoxia. AB - The activity of antioxidant enzymes were measured in alveolar type II cells isolated from control and 85% oxygen-exposed rats to determine if type II cells, an oxygen-resistant lung cell type had constitutively high enzyme activities and to measure the effect of hyperoxia on these antioxidant enzyme. Type II cells were isolated from lungs of control rats and rats exposed to 85% O2 for 7 days. In whole lungs of rats exposed to 85% oxygen there is an increase in activity (per lung or per mg lung DNA) in the antioxidant enzymes CuZn superoxide dismutase, Mn superoxide dismutase, catalase, glutathione peroxidase and glucose 6-phosphate dehydrogenase. Oxygen exposure significantly increased (p less than 0.05) all type II cell antioxidant enzyme activities when expressed per mg DNA. The protein content of oxygen exposed type II cells increased 25% from (63.9 +/- 4.8 micrograms/10(6) cells to 79.6 +/- 4.2 micrograms/10(6) cells, p less than 0.05). When type II cell enzyme activities were expressed in U/mg cell protein, only CuZn superoxide dismutase and Mn superoxide dismutase increased in activity following oxygen exposure (by 43% and 28% relative to air exposed lung type II cells, respectively, p less than 0.05). This suggested that most lung cell antioxidant enzymes increased in activity following oxidant stress in proportion to increased cell mass. CuZn and Mn superoxide dismutase increased activity to an extent greater than the increase in type II cell protein content after oxygen exposure. Alveolar macrophages lavaged from control and oxygen-exposed rats were also evaluated, and they had no significant change in CuZn and Mn superoxide dismutase activities. Type II cells accounted for 10% and 17% of alveolar cells in control and oxygen treated rats. By knowing the antioxidant enzyme activities in type II cells, the total enzyme activity of whole lung and the number of type II cells in control and oxygen exposed rats from morphometric data, we calculated the percent of whole lung enzyme activity accounted for by type II cells. Type II cells accounted for a high percentage of lung glucose-6-phosphate dehydrogenase (58% in control rats, 65% in oxygen exposed rats) but a low percentage of Mn superoxide dismutase (4% in control rats, 6% in oxygen exposed rats). PMID- 3007083 TI - Free-radical chemistry of cigarette smoke and its toxicological implications. AB - Cigarette smoke contains two very different populations of free radicals, one in the tar and one in the gas phase. The tar phase contains several relatively stable free radicals; we have identified the principal radical as a quinone/hydroquinone (Q/QH2) complex held in the tarry matrix. We suggest that this Q/QH2 polymer is an active redox system that is capable of reducing molecular oxygen to produce superoxide, eventually leading to hydrogen peroxide and hydroxyl radicals. In addition, we have shown that the principal radical in tar reacts with DNA in vitro, possibly by covalent binding. The gas phase of cigarette smoke contains small oxygen- and carbon-centered radicals that are much more reactive than are the tar-phase radicals. These gas-phase radicals do not arise in the flame, but rather are produced in a steady state by the oxidation of NO to NO2, which then reacts with reactive species in smoke such as isoprene. We suggest that these radicals and the metastable products derived from these radical reactions may be responsible for the inactivation of alpha 1-proteinase inhibitor by fresh smoke. Cigarette smoke oxidizes thiols to disulfides; we suggest the active oxidants are NO and NO2. The effects of smoke on lipid peroxidation are complex, and this is discussed. We also discuss the toxicological implications for the radicals in smoke in terms of a number of radical-mediated disease processes, including emphysema and cancer. PMID- 3007084 TI - Free-radical metabolites of acetaminophen and a dimethylated derivative. AB - The oxidation of acetaminophen (4'-hydroxyacetanilide) to the corresponding N acetyl-p-benzoquinone imines by plant and mammalian peroxidases is discussed. The acetaminophen free radical (N-acetyl-4-aminophenoxyl) has been reported as an intermediate. It is very reactive and forms melanin-like polymeric products. Application of a fast-flow system makes it possible to detect the transient species and clearly distinguish it from persistent paramagnetic melanin polymers. A model system, leading to more stable metabolites, can be obtained by introduction of methyl groups next to the oxygen, 3',5'-dimethylacetaminophen (3',5'-dimethyl-4'-hydroxyacetanilide). The ESR spectrum of the free radical formed could be completely analyzed and confirmed by deuterium substitution. The data are consistent with the assignment to a phenoxyl free radical (N-acetyl-2,6 dimethyl-4-amino-phenoxyl). Its formation is discussed in terms of substrate, hydrogen peroxide and enzyme concentration dependence. It is believed to be formed via a direct one-electron oxidation of 3',5'-dimethyl-4'-hydroxy acetanilide. The radical does not form polymers or react with nucleophiles. Its redox behavior is discussed. The possible reaction of these phenoxyl free radicals with oxygen is thought to be negligible. PMID- 3007085 TI - The tyrosyl free radical in ribonucleotide reductase. AB - The enzyme, ribonucleotide reductase, catalyses the formation of deoxyribonucleotides from ribonucleotides, a reaction essential for DNA synthesis in all living cells. The Escherichia coli ribonucleotide reductase, which is the prototype of all known eukaryotic and virus-coded enzymes, consists of two nonidentical subunits, proteins B1 and B2. The B2 subunit contains an antiferromagnetically coupled pair of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of ferric ions and a stable tyrosyl free radical. EPR studies show that the tyrosyl radical, formed by loss of an electron, has its unpaired spin density delocalized in the aromatic ring of tyrosine. Effects of iron-radical interaction indicate a relatively close proximity between the iron center and the radical. The EPR signal of the radical can be studied directly in frozen packed cells of E. coli or mammalian origin, if the cells are made to overproduce ribonucleotide reductase. The hypothetic role of the tyrosyl free radical in the enzymatic reaction is not yet elucidated, except in the reaction with the inhibiting substrate analogue 2'-azido-CDP. In this case, the normal tyrosyl radical is destroyed with concomitant appearance of a 2'-azido-CDP-localized radical intermediate. Attempts at spin trapping of radical reaction intermediates have turned out negative. In E. coli the activity of ribonucleotide reductase may be regulated by enzymatic activities that interconvert a nonradical containing form and the fully active protein B2. In synchronized mammalian cells, however, the cell cycle variation of ribonucleotide reductase, studied by EPR, was shown to be due to de novo protein synthesis. Inhibitors of ribonucleotide reductase are of medical interest because of their ability to control DNA synthesis. One example is hydroxyurea, used in cancer therapy, which selectively destroys the tyrosyl free radical. PMID- 3007086 TI - Chemistry and biology of spin-trapping radicals associated with halocarbon metabolism in vitro and in vivo. AB - The spin-trapping method is introduced and discussed. Some chemistry of nitroxides and nitrones is reviewed. Pattern recognition of ESR spectra of nitroxides is outlined. Factors controlling the magnitude of hyperfine splitting constants are mentioned. Methods of assigning spin adducts are listed. Review articles in the literature are referenced. Results in the electrochemical reduction of halocarbons are presented and some parallels with superoxide chemistry shown. Various speculative reactions are given. The in vitro and in vivo experiments where halocarbon radicals have been detected by spin trapping are reviewed and some new results reported. A comparison for different animals is added. PMID- 3007087 TI - Oxidative activation of benzidine and its derivatives by peroxidases. AB - Benzidine (4,4'-diaminobiphenyl) is a known human carcinogen; exposure to this substance resulted in an epidemic of bladder cancer among workers in the dye industry in Europe and North America. The chemical or enzymatic oxidation of benzidine proceeds via a racial cation detectable by electron spin resonance. Peroxidase-catalyzed oxidation of benzidine generates reactive electrophiles which readily form adducts with phenol and thiol compounds. The structures of these novel metabolites are described. Peroxidases, including prostaglandin synthase, catalyze benzidine binding to protein and nucleic acid; the nature of the resulting adducts is unknown. The relevance of these processes to benzidine carcinogenesis in vivo is the subject of research and debate. A central question remains: is benzidine activated in extra-hepatic target tissues such as bladder epithelium, or transported to these tissues following hepatic oxidative metabolism? PMID- 3007088 TI - Free radical-mediated activation of hydrazine derivatives. AB - Hydrazines are known to undergo oxidative activation in several enzymatic systems in vitro. Free radicals or carbonium ions have been proposed as active intermediates during such activation. The toxic effects elicited by hydrazines have also been linked to free radical-mediated activation. In this report, we have reviewed the identification of organic free radicals from hydrazines by direct ESR and ESR-spin trapping. PMID- 3007091 TI - Free radical generation by ultrasound in aqueous and nonaqueous solutions. AB - The physical principles underlying the oscillatory behavior of minute gas bubbles in liquids exposed to ultrasound are reviewed. Results from mathematical analyses suggest that these oscillations sometimes become unstable leading to transient cavitation in which a bubble violently collapses during a single acoustic half cycle producing high temperatures and pressures. The role that micronuclei, resonant bubble size, and rectified diffusion play in the initiation of transient cavitation is explained. Evidence to support these theoretical predictions is presented with particular emphasis on sonoluminescence which provides some non chemical evidence for the formation of free radicals. Acoustic methods for conducting sonochemical investigations are discussed. In aqueous solutions transient cavitation initially generates hydrogen atoms and hydroxyl radicals which may recombine to form hydrogen and hydrogen peroxide or may react with solutes in the gas phase, at the gas-liquid boundary or in the bulk of the solution. The analogies and differences between sonochemistry and ionizing radiation chemistry are explored. The use of spin trapping and electron spin resonance to identify hydrogen atoms and hydroxyl radicals conclusively and to detect transient cavitation produced by continuous wave and by pulsed ultrasound is described in detail. The study of the chemical effects of cavitation in organic liquids is a relatively unexplored area which has recently become the subject of renewed interest. Examples of the decomposition of solvent and solute, of ultrasonically initiated free-radical polymerization and polymer degradation are presented. Spin trapping has been used to identify radicals in organic liquids, in polymer degradation and in the decomposition of organometallic compounds. PMID- 3007089 TI - Semiquinone anion radicals of catechol(amine)s, catechol estrogens, and their metal ion complexes. AB - The characterization and identification of semiquinone radicals from catechol(amine)s and catechol estrogens by electron spin resonance spectroscopy is addressed. The use of diamagnetic metal ions, especially Mg2+ and Zn2+ ions, to detect transient semiquinone radicals in biological systems and to monitor their reactions, is discussed. A brief account of the identification and reactions of quinones is also presented. PMID- 3007092 TI - Generation of reactive species and fate of thiols during peroxidase-catalyzed metabolic activation of aromatic amines and phenols. AB - The horseradish peroxidase (HRP)-catalyzed oxidation of p-phenetidine and acetaminophen was investigated. Studies using the spin probe 2-ethyl-1-hydroxy 2,5,5-trimethyl-3-oxazolidine (OXANOH) suggested these oxidations involve the generation of substrate-derived free radicals. This was confirmed by using glutathione (GSH) in these incubations in the presence of the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO). DMPO-glutathionyl radical adducts were observed using EPR spectroscopy during HRP-catalyzed oxidation of both p phenetidine and acetaminophen. Investigations of oxygen uptake and oxidized glutathione (GSSG) formation during HRP-catalyzed oxidations of p-phenetidine and acetaminophen suggested that further reactions of the glutathionyl radical involve glutathione peroxysulfenyl radical and glutathione sulfenyl hydroperoxide production. Quinonoid products of the peroxidatic oxidations of p-phenetidine and acetaminophen, and their interaction with GSH via both conjugation and redox mechanisms are described. The relevance of these reactions of GSH with reactive species as detoxification mechanisms is discussed. PMID- 3007093 TI - Biochemical mechanism of oxidative damage by redox-cycling drugs. AB - Biochemical mechanisms of production of redox intermediates of redox-cycling drugs include: photochemical events, either photoionization process or electron transfer from photoexcited states; electron exchange of reduced form of a drug with the oxy state of oxygen-binding hemoproteins; oxidation by catalytic metal centers (oxidases, peroxidases, oxygenases) of the reduced forms of drugs; or electron transfer to the oxidized form of a drug from activated intracellular electron transfer chain (mitochondria, microsomes, etc.). Further reaction of these drug free radicals can lead to oxidative damage by either direct attack of biological macromolecules or via oxygen reduction, giving O2-, H2O2, and OH. The reaction pathway depends on the presence of metal ions, natural scavengers, enzymes that control relative concentrations of reactive species, and availability of oxygen in the environment. PMID- 3007090 TI - Free-radical-mediated DNA binding. AB - Free-radical metabolites can be generated metabolically by a one-electron reductase-catalyzed reaction or a "peroxidase" catalyzed oxidation or by photoactivation of a wide variety of aromatic xenobiotics. Radicals may also be generated during lipid peroxidation. Some radicals can react with DNA or bind covalently or noncovalently as a dismutation product or as a dimer, trimer or polymeric product. Modification to the DNA can result in single-strand breaks, loss of template activity, and crosslinking. The binding can prevent enzymic digestion. In some cases, the radicals react with oxygen, resulting before conversion to DNA reactive oxygen species. Most radicals probably do not interact with DNA. PMID- 3007094 TI - Reactions of hemoglobin with phenylhydrazine: a review of selected aspects. AB - It is well known that phenylhydrazine induces hemolytic anemia. This is thought to result from the reaction of phenylhydrazine with hemoglobin. The accompanying oxidation of phenylhydrazine leads to the formation of a number of products, including benzene, nitrogen, hydrogen peroxide, superoxide anion and the phenyl radical. The products formed depend critically on the conditions of the experiment, especially the amount of oxygen present. It is now known that oxyhemoglobin and myoglobin react with phenylhydrazine to yield a derivative of hemoglobin containing N-phenylprotoporphyrin in which the heme group is modified. The recent identification of sigma-phenyliron(III) porphyrins in phenylhydrazine modified metmyoglobin has aided elucidation of the mechanism of hemoglobin modification. Mechanistic schemes are proposed to account for product formation. PMID- 3007096 TI - Monosaccharide autoxidation in health and disease. AB - The reduction of oxygen by the ene-diol tautomer of simple monosaccharides produces hydrogen peroxide and alpha-oxoaldehydes. This process, termed monosaccharide autoxidation, occurs at physiological pH and temperature and may contribute to the development of several pathological processes. Enolization of the monosaccharide to an ene-diol tautomer is a prerequisite for the reaction of the monosaccharides with oxygen. The reaction kinetics suggest a two step process: the enolization of the monosaccharide to the ene-diol followed by the reaction of the ene-diol with oxygen. Free-radical reactive intermediates are formed by the reaction of the ene-diol with oxygen: superoxide, semidione, and 1 hydroxyalkyl radicals are formed under physiological conditions (hydroxyl radicals are also detected at high pH). The autoxidation of monosaccharides stimulates the oxidation of oxyhemoglobin in erythrocytes, producing methemoglobin and hydrogen peroxide, and the oxidation of reduced pyridine nucleotides NAD(P)H to the oxidized congener NAD(P)+ and enzymatically inactive nucleotide. This stimulates oxidative metabolism (via the hexose monophosphate shunt) and alpha-oxoaldehyde metabolism (via the glyoxalase system) in erythrocytes in vitro. The oxidative challenge is relatively mild even with very high concentrations (50 mM) of monosaccharide. However, crosslinking of membrane proteins by alpha-oxoaldehydes is enhanced; this effect may exacerbate ageing and decrease the lifetime of erythrocytes in circulation. In vivo, the autoxidation of monosaccharides is expected to be a chronic oxidative process occurring in biological tissue which utilises simple monosaccharides, e.g., in glycolysis and gluconeogenesis. Monosaccharide autoxidation is suggested to be a determinant in the control of cellular mitosis and ageing, providing physiological substrates for the glyoxalase system, and may contribute to the chronic disease processes associated with diabetes mellitus and the smoking of tobacco. PMID- 3007095 TI - Free radicals of benzo(a)pyrene and derivatives. AB - The evidence for biological involvement, the spectroscopic properties (especially EPR), and the reactions, of free radicals derived from benzo(a)pyrene and its methylated, hydroxylated, and fluorinated derivatives are reviewed. PMID- 3007097 TI - Free-radical production and oxidative reactions of hemoglobin. AB - Mechanisms of autoxidation of hemoglobin, and its reactions with H2O2, O2-, and oxidizing or reducing xenobiotics are discussed. Reactive intermediates of such reactions can include drug free radicals, H2O2, and O2-, as well as peroxidatively active ferrylhemoglobin and methemoglobin-H2O2. The contributions of these species to hemoglobin denaturation and drug-induced hemolysis, and the actions of various protective agents, are considered. PMID- 3007099 TI - Production of oxygen-centered radicals by neutrophils and macrophages as studied by electron spin resonance (ESR). AB - Neutrophils and macrophages undergo a respiratory burst and an increase in the activity of the hexose monophosphate pathway in response to particulate or soluble agents. The increase in oxygen consumption was found to be associated with the production of oxygen-centered radicals. The ESR technique of spin trapping showed that besides a superoxide spin adduct, a hydroxyl spin adduct is also produced. ESR is considered to be the least ambiguous technique for the detection of free radicals. The spin-trapping agents used for oxygen-centered radical detection are usually nitrones. The most commonly used nitrone is 5,5 dimethyl-1-pyrroline-N-oxide (DMPO), which reacts with O2-. to form 5,5-dimethyl 2-hydroperoxypyrroline-N-oxide (DMPO-OOH) and with OH. to form 5,5-dimethyl-2 hydroxypyrroline-N-oxide (DMPO-OH). Although spin-adduct formation is considered to be the most direct technique for the detection of free radicals, some disadvantages are encountered. There has been considerable interest in the isolation of the O2-. generating activity from phagocytic cells. The enzyme can be extracted with deoxycholate and gel filtration indicates that it is a high molecular weight complex. Maximum activity was between pH 7.0 and pH 7.5. The Km value was 15.8 microM for NADPH and 434 micron for NADH, indicating that NADPH is the preferred substrate. PMID- 3007100 TI - Model studies in cytochrome P-450-mediated toxicity of halogenated compounds: radical processes involving iron porphyrins. AB - Haloalkane toxicity originates from attack on biological targets by reactive intermediates derived from haloalkane metabolism by a hemoprotein, cytochrome P 450. Carbon-centered radicals and their peroxyl derivatives are most likely involved. The reactions of iron porphyrin--a model for cytochrome P-450--with various carbon-centered and peroxyl radicals generated by pulse radiolysis are examined. Competition between iron porphyrin and unsaturated fatty acids for attack by peroxyl radicals is pointed out. These kinetic data are used to derive a model for toxicity of haloalkanes with particular attention to carbon tetrachloride and halothane. The importance of local oxygen concentration and structural arrangement of fatty acids around cytochrome P-450 is emphasized. PMID- 3007101 TI - Involvement of free radicals in the mechanism of 3-methylindole-induced pulmonary toxicity: an example of metabolic activation in chemically induced lung disease. AB - 3-Methylindole (3-MI) is a metabolite of tryptophan which causes acute pulmonary edema and emphysema in ruminants when administered orally or intravenously. 3-MI is metabolized by mixed-function oxidases to a reactive intermediate which may play a role in 3-MI-induced pneumotoxicity. Electron spin-trapping techniques have been used to investigate the in vitro and in vivo formation of free radicals during 3-MI metabolism by goat lung. A nitrogen-centered free radical of 3-MI has been generated from 3-MI in goat lung microsomal incubations. Although a nitrogen centered free radical can be generated chemically from most of the indolic compounds, only the 3-MI free radical can be generated enzymatically. The formation of the nitrogen-centered 3-MI free radical was followed by the appearance of a carbon-centered lipid radical in microsomal preparations. The findings that an identical carbon-centered free radical was generated by FeSo4 in the microsomal system in the absence of 3-MI and that malonaldehyde formation is stimulated by 3-MI in microsomes led to the conclusion that 3-MI metabolism induces lipid peroxidation of microsomal membranes. The formation of 3-MI-induced lipid radicals was inhibited by vitamin E and glutathione. A carbon-centered radical was spin trapped in vivo in the lungs of goats infused with 3-MI. This radical had the same splitting constants as the carbon-centered lipid radical trapped in microsomal incubations containing 3-MI. This finding indicates that the metabolism of 3-MI in goat lung in vivo generates a lipid radical.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007098 TI - Physiological aspects of free-radical reactions. AB - Enzymes which catalyze the formation of free radicals in vitro will catalyze similar reactions in vivo. We believe that the formation of some kinds of free radicals has definite physiological meanings in metabolism. In this sense, the enzymes forming such free radicals are concluded to be in evolutionally advanced states. Elaborated structure and function of enzymes such as horseradish peroxidase and microsomal flavoproteins support the idea. Deleterious and side reactions caused by free radicals are assumed to be minimized in vivo by localizing the reactions, but this assumption should be verified by future studies. PMID- 3007103 TI - Relationship of silica and bacillus Calmette-Guerin exposures to humoral and cellular immune functions. AB - The consequences of silica preexposures were studied on subsequent bacillus Calmette-Guerin (BCG) pulmonary infections. Silica had a variable effect on colonization by BCG depending on organ and number of BCG treatments. Silica preexposures resulted in increased numbers of free alveolar cells subsequent to BCG infections, with an initially rapid increase of neutrophils. Both silica and two BCG treatments were capable of decreasing the animals' splenic response to stimulations with Escherichia coli aerosols, and BCG greatly enhanced antibody dependent cell-mediated cytotoxicity in silica preexposed mice. 5'-Nucleotidase activity was depressed by both silica and BCG in pulmonary washout cells indicating that inhalation of these substances may have a stimulating effect on the alveolar macrophages. PMID- 3007104 TI - Asbestos effects on superoxide production. An in vitro study of hamster alveolar macrophages. AB - Inhaled asbestos induces accumulation of alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) in lung. Asbestos-enhanced production of superoxide anion (O2-) by AM and/or PMN may be involved in the pathogenesis of asbestos-induced fibrosis, either through direct effects on collagen synthesis or via mediation of tissue injury and repair. In in vivo experiments, bronchoalveolar lavage (BAL) 3 to 8 weeks following intratracheal asbestos injections showed increases in both PMN and AM, with AM representing 78 to 82% of cells recovered. Inhalation models, generally regarded as more analagous to human exposures, have confirmed AM as the predominant component of the cellular response to inhaled asbestos. In this study, the in vitro effects of asbestos fiber on O2- production by AM have been determined in cell populations derived from the Syrian golden hamster. AM for in vitro study were obtained by BAL. O2- production was monitored as superoxide dismutase (SOD) - inhibitable cytochrome c reduction. Significant rises in O2- release by AM were noted in the presence of 0.4 mg/ml crocidolite (2.53 +/- 0.33 nmole cytochrome c reduced/10(6) cells/30 min, 37 degrees C; controls 1.13 +/- 0.18 nmole; P less than 0.02). Chrysotile induced levels of O2- release in AM which were similar to those evoked by crocidolite. PMID- 3007102 TI - Biochemical studies on the metabolic activation of halogenated alkanes. AB - This paper reviews recent investigations by Slater and colleagues into the metabolic activation of halogenated alkanes in general and carbon tetrachloride in particular. It is becoming increasingly accepted that free radical intermediates are involved in the toxicity of many such compounds through mechanisms including lipid peroxidation, covalent binding, and cofactor depletion. Here we describe the experimental approaches that are used to establish that halogenated alkanes are metabolized in animal tissues to reactive free radicals. Electron spin resonance spectroscopy is used to identify free radical products, often using spin-trapping compounds. The generation of specific free radicals by radiolytic methods is useful in the determination of the precise reactivity of radical intermediates postulated to be injurious to the cell. The enzymic mechanism of the production of such free radicals and their subsequent reactions with biological molecules is studied with specific metabolic inhibitors and free-radical scavengers. These combined techniques provide considerable insight into the process of metabolic activation of halogenated compounds. It is readily apparent, for instance, that the local oxygen concentration at the site of activation is of crucial importance to the subsequent reactions; the formation of peroxy radical derivatives from the primary free-radical product is shown to be of great significance in relation to carbon tetrachloride and may be of general importance. However, while these studies have provided much information on the biochemical mechanisms of halogenated alkane toxicity, it is clear that many problems remain to be solved. PMID- 3007105 TI - Comparative pathological aspects of chronic olivine and silica inhalation in mice. AB - A comparative study was done in mice on the effects of silica and olivine inhalation. The exposure periods in the dust chambers were for periods of 150, 300, and 570 days, with the longest time period covering a large portion of the predicted life span of the Balb/c mouse. Detailed necropsies including histology were done at the end of the exposures and at 30 and 150 days following removal of animals from the 150- and 300-day dust exposures. The results indicate that silica causes considerably more tissue damage in lungs and mediastinal lymph nodes, leading to granulomas. PMID- 3007107 TI - Solubility of asbestos and man-made mineral fibers in vitro and in vivo: its significance in lung disease. AB - Work on the solubility of asbestiform minerals and of man-made mineral fibers, both in vitro and in vivo, is reviewed. The significance of solubility in determining the pathogenic potential of these materials is also discussed. PMID- 3007106 TI - Effects of asbestos fibers on cell division, cell survival, and formation of thioguanine-resistant mutants in Chinese hamster ovary cells. AB - The ability of crocidolite fibers to induce point mutations and mitotic abnormalities in Chinese hamster ovary (CHO) cells was examined in cell cultures. The purpose has been to study the possibilities for establishing in vitro test methods to quantify genetic damage induced by asbestos and other mineral fibers. Results obtained with the CHO/hypoxanthine guanine phosphoribosyl transferase system indicated that crocidolite fibers per se do not significantly increase the number of thioguanine-resistant mutants. Crocidolite fibers also failed to potentiate the mutagenicity of benzo[a]pyrene. Time-lapse cinematography and microscopy showed that asbestos (crocidolite) fibers were markedly cytotoxic. Among surviving cells some underwent abnormal cell divisions which resulted in multi- and micronucleate cells. Many cells that contained a few asbestos fibers, however, underwent mitosis and successfully formed two mononucleate daughter cells capable of further divisions. Individual, fiber-containing cells were examined by time-lapse television recordings for 4-5 days. During this time period some cells underwent six divisions and generated an almost normal number of daughter cells. Cells which contained fibers that were longer or equivalent to the diameter of the mitotic cell (20 microns), showed different forms of mitotic abnormalities. The frequency of multinucleate cells was drastically increased following exposure to asbestos fibers. Only rarely, however, did these cells divide to produce viable daughter cells capable of continued cell multiplication. The frequency of multinucleate cells was dependent on the dose of exposure to asbestos fibers and could possibly be used as an index of the degree of mitotic disturbances induced by mineral fibers. PMID- 3007108 TI - One adenosine deaminase allele in a patient with severe combined immunodeficiency contains a point mutation abolishing enzyme activity. AB - We have cloned and sequenced an adenosine deaminase (ADA) gene from a patient with severe combined immunodeficiency (SCID) caused by inherited ADA deficiency. Two point mutations were found, resulting in amino acid substitutions at positions 80 (Lys to Arg) and 304 (Leu to Arg) of the protein. Hybridization experiments with synthetic oligonucleotide probes showed that the determined mutations are present in both DNA and RNA from the ADA-SCID patient. In addition, wild-type sequences could be detected at the same positions, indicating a compound heterozygosity. Studies with ADA expression clones mutagenized in vitro showed that the mutation at position 304 is responsible for ADA inactivation. PMID- 3007109 TI - Promoter sequences required for function of the human gamma globin gene in erythroid cells. AB - The human gamma- and beta-globin genes are expressed during the fetal and adult developmental periods, respectively. Differences in the sequences of their promoters may be relevant to their developmental regulation. The gamma globin gene promoter was found to be stronger than that of the beta gene. In HeLa cells co-transfected with plasmids containing the two genes, transcripts arising from the gamma promoter accumulated to a level 3-fold higher than those initiated from the beta-promoter. We have recently shown that deletion of the most distal of the conserved elements of the promoter (CACCC) reduces function to 25% of the wild type, whereas the removal of the proximal of the two duplicated (CCAAT) elements increases promoter function 2- to 4-fold in HeLa cells during transient gene expression. Both the wild-type promoter and the truncation and linker-scanning mutants from which one of the two duplicated "CCAAT' elements had been removed, exhibited regulated expression when stably integrated into chromosomes of mouse erythroleukemia (MEL) cells. Thus, duplication of the "CCAAT' element, the feature by which the gamma promoter differs most strikingly from the beta, is not essential for function in erythroid cells. PMID- 3007111 TI - Molecular analysis of the ecdysterone-inducible 2B5 "early' puff in Drosophila melanogaster. AB - The 2B5 region of the X-chromosome in Drosophila melanogaster plays a developmentally important role in the ecdysterone-triggered response of the late third instar salivary gland. Using a combination of transposon-tagging and chromosomal walking techniques, we have isolated 231 kb of contiguous genomic DNA sequences corresponding to this region. We have more precisely aligned this DNA to the 2B1,2 to 2B5-6 interval of the cytogenetic map by locating the position of three well-characterized chromosomal breakpoints by in situ hybridization and genomic DNA blotting experiments. Labeled cDNA, synthesized from poly(A)+ RNA isolated from hormone-induced salivary gland and imaginal disc tissues and hybridized to the cloned DNA, demonstrated that the ecdysterone-inducible sequences mapped to DNA segments corresponding to the 2B3,4 to 2B5-6 interval. Although some of these sequences were inducible in only one tissue type, many were found to be inducible in both salivary glands and imaginal discs. RNA blotting experiments have detected a major 4.5-kb RNA which is hormone inducible in the larval salivary gland and whose quantitative induction is not inhibited by cycloheximide. Thus, the 4.5-kb RNA represents at least one product from the ecdysterone-responsive 2B5 "early' puff. PMID- 3007110 TI - A new site of integration for mouse mammary tumor virus proviral DNA common to BALB/cf(C3H) mammary and kidney adenocarcinomas. AB - The BALB/cf/Cd substrain of mice, developed by inbreeding and selection, shows a 70% incidence of spontaneous kidney adenocarcinomas. Initially foster-nursed on C3H mothers, these mice no longer produce mammary tumors, but there is evidence that mouse mammary tumor virus (MMTV) is involved in the formation of these renal carcinomas. We identified a chromosomal region called int-41, representing a locus interrupted by the integration of an exogenous MMTV provirus in a BALB/c mammary tumor. We found a DNA rearrangement and transcriptional activation in the domain specified by the int-41 probe in one primary kidney adenocarcinoma. A 5.2 kb int-41 specific mRNA was detected in the kidney tumor cell line established from a transplantable renal adenocarcinoma. This mRNA hybridized with MMTV and int-41 specific probes suggesting that it is a hybrid molecule composed of MMTV LTR sequences covalently linked to host cell RNA. This mRNA was strongly stimulated by the presence of glucocorticoid hormone in the culture medium. Our data are compatible with the hypothesis that the int-41 chromosomal domain is involved in the neoplastic transformation of epithelial cells from different organs. PMID- 3007112 TI - In vitro transcription by Xenopus oocytes RNA polymerase III requires a DNA topoisomerase II activity. AB - Extracts of Xenopus laevis oocytes are able to assemble minichromosomes in vitro when they are supplemented with ATP and Mg2+. We have followed the time course of in vitro DNA supercoiling and transcription of polyoma virus closed circular DNA. The transcriptional activity increased with the assembly time of chromatin in the presence of both ATP and Mg2+, but only residual activity was detected in the absence of either of them. We also found that polyoma DNA as well as 5S RNA gene transcription were carried out mainly by an RNA polymerase III. On the other hand, polyoma RNA and 5S RNA synthesis were inhibited by novobiocin in a way that suggested the requirement of a DNA topoisomerase II or DNA gyrase activity for the initiation of transcription. PMID- 3007113 TI - The upstream operator of the Escherichia coli galactose operon is sufficient for repression of transcription initiated at the cyclic AMP-stimulated promoter. AB - Two operators are known to bind Escherichia coli galactose repressor with roughly equal affinity. A study of the control these two operators exert on the two overlapping gal promoters is reported. The experiments rest on a set of mutations specifically constructed to inactivate individual control units of the gal operon and on quantitation of gal promoter activities. Messenger RNAs initiated at one or other of the promoters in a cell-free transcription-translation system were determined by a primer extension assay with synthetic deoxyoligonucleotide primers. The main conclusions are: (i) the classical galactose operator O1, located upstream with respect to the two overlapping promoters is sufficient for negative control of the cAMP activated promoter P1; (ii) complete repression of the second promoter P2, on the other hand, needs the presence of both intact operators O1 and O2. Thus, the two overlapping gal promoters (with only 5 bp separating their respective transcriptional start sites) are both subject to negative control by the galactose repressor. This regulation, however, is exerted by two different mechanisms. PMID- 3007114 TI - Endonuclease VII resolves Y-junctions in branched DNA in vitro. AB - Endonuclease VII (gp 49 of phage T4) resolves four-way junctions in branched DNAs. We have extended our investigations of the specificity of endo VII and tested its activity with three-way junctions (Y-structures) constructed in vitro. Both 'closed' and 'open' Y-structures were made, absolutely identical in sequence but differing from each other by a single nick in one of the three arms. Pure Y structures were obtained on a preparative scale by annealing plus and minus strands from two M13mp strains. One strain has an inverted repeat of 2 X 31 nucleotides cloned into the single EcoRI site while in the other strain this repeat is absent. The structures were used in reactions with endo VII, which recognizes the branch point of both structures and introduces a characteristic number of nicks, 3' to the junction in each arm of the structure. Strong and weak sites could be distinguished and the cleavage pattern differed significantly between the two structures. The observed resolution of Y-junctions by endo VII in vitro is compatible with a model for the resolution of recombinant Y-branches in DNA. PMID- 3007115 TI - The structure of cruciforms in supercoiled DNA: probing the single-stranded character of nucleotide bases with bisulphite. AB - The single-stranded character of cytosine bases in three cruciform structures has been assessed by an examination of reactivity towards sodium bisulphite. Unpaired cytosine residues undergo deamination at C4 to give deoxyuracil, and propagation in an ung Escherichia coli host results in C-G----T-A transition mutations, detectable by restriction cleavage or sequence analysis. Very high frequencies of such mutations have been found at cruciform loops, confirming their unpaired character, with almost zero background mutation frequencies elsewhere. A low level of modification was observed at the four-way junction of a cruciform. The results indicate that the optimal cruciform loop size is four bases, with loose 'breathing' at the first base pair at the top of the cruciform stem at 37 degrees C, and little or no opening of base pairs at the four-way junction. PMID- 3007116 TI - Ultraviolet radiation inactivates SV40 by disrupting at least four genetic functions. AB - We have examined the relative sensitivities of different genetic functions to damage induced by u.v. light. For this purpose we have introduced defined amounts of damage into specific regions of the genome of SV40 and have determined the effect of the damage on the survival of transfected viral DNA. We found that the lethal effect of the damage depends on its location within the viral genome. The region most sensitive to damage contains the transcriptional promotors and enhancers for the early and late viral genes plus part of the origin of DNA replication. Lesions within this regulatory region are 3.2-fold more effective in inactivating viral DNA than is the same amount of damage randomly distributed throughout the viral genome. The region least sensitive to damage lies within the coding portion of the viral coat protein genes, which are expressed only late in infection and would therefore be transcribed from undamaged progeny viral genomes, provided DNA replication occurs. Damage within this region is only 45% as effective in inactivating viral DNA as are randomly distributed lesions. Thus there is a 7-fold difference in the lethal effect of DNA damage within the most sensitive and least sensitive regions of the viral genome. Intermediate sensitivities are observed within the transcribed portion of the viral A gene, coding for the T antigen whose expression is required early in infection, and in a region at the terminus of DNA replication. The sum of the individual sensitivities for all regions of the SV40 genome is equal to the total sensitivity of viral DNA subjected to random damage.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007117 TI - The URF 5 gene of Chlamydomonas reinhardtii mitochondria: DNA sequence and mode of transcription. AB - A gene homologous to unassigned reading frame (URF) 5 of the mammalian mitochondrial genome has been identified in the mitochondrial DNA of the unicellular green alga, Chlamydomonas reinhardtii. The algal URF 5 gene is closely flanked by the gene for subunit I of cytochrome oxidase (COI) and by an unidentified gene (ORF x). The URF 5 and ORF x genes are transcribed in the same direction, but opposite to that of the COI gene. Transcript analysis reveals a 1.9-kb mRNA whose major 5' terminus maps to the putative URF 5 initiation codon and whose 3' end abuts the 5' end of the ORF x transcript. Characterization of other C. reinhardtii mitochondrial RNAs suggests a general pattern of abutting transcripts and mature mRNAs having little or no 5' leader sequence. While this is reminiscent of post-transcriptional processing in animal mitochondria, different mechanisms must be employed in the two systems, since tRNA sequences (which appear to function as transcript processing signals in animal mitochondria) do not generally flank protein coding sequences in the C. reinhardtii mitochondrial genome. Nevertheless, characteristic secondary structure motifs do occur within the 3'-terminal regions of C. reinhardtii mitochondrial mRNAs, and their location close to mRNA termini suggests that such motifs may play a role in directing the precise endonucleolytic cleavage of long primary transcripts. PMID- 3007118 TI - Pertussis toxin inhibits thrombin-induced activation of phosphoinositide hydrolysis and Na+/H+ exchange in hamster fibroblasts. AB - Prior treatment with pertussis toxin of G0-arrested hamster fibroblasts (CCL39) results in a dose-dependent inhibition of two early events of the mitogenic response elicited by alpha-thrombin: accumulation of inositol phosphates in Li+ treated cells, and activation of the Na+/H+ antiport, measured either by the amiloride-sensitive 22Na+ influx or by the increase in intracellular pH. At 10( 1) U/ml of alpha-thrombin, the maximal inhibition was approximately 50% for these two early cellular responses, but the pertussis toxin effect was more pronounced at lower thrombin concentrations. In contrast, pertussis toxin does not affect the Na+/H+ antiport activation induced by phorbol esters or EGF, the action of which is not mediated by the phosphoinositide-metabolizing pathway in CCL39 cells. Therefore, our data suggest the following. A GTP-binding regulatory protein is probably involved in signal transduction between thrombin receptors and the phosphatidylinositol 4,5-bisphosphate-specific phospholipase C. This regulation does not seem to be exerted via modulations of cyclic AMP levels. The thrombin-induced activation of Na+/H+ antiport is, at least in part, mediated by the protein kinase C, as a consequence of stimulation of phosphatidylinositol turnover. PMID- 3007119 TI - A lysine in the ATP-binding site of P130gag-fps is essential for protein-tyrosine kinase activity. AB - The P130gag-fps transforming protein of Fujinami sarcoma virus (FSV) possesses tyrosine-specific protein kinase activity and autophosphorylates at Tyr-1073. Within the kinase domain of P130gag-fps is a putative ATP-binding site containing a lysine (Lys-950) homologous to lysine residues in cAMP-dependent protein kinase and p60v-src which bind the ATP analogue p-fluorosulfonylbenzoyl-5' adenosine. FSV mutants in which the codon for Lys-950 has been changed to codons for arginine or glycine encode metabolically stable but enzymatically defective proteins which are unable to effect neoplastic transformation. Kinase-defective P130gag-fps containing arginine at residue 950 was normally phosphorylated at serine residues in vivo suggesting that this amino acid substitution has a minimal effect on protein folding and processing. The inability of arginine to substitute for lysine at residue 950 suggests that the side chain of Lys-950 is essential for P130gag-fps catalytic activity, probably by virtue of a specific interaction with ATP at the phosphotransfer active site. Tyr-1073 of the Arg-950 P130gag-fps mutant protein was not significantly autophosphorylated either in vitro or in vivo, but could be phosphorylated in trans by enzymatically active P140gag-fps. These data indicate that Tyr-1073 can be modified by intermolecular autophosphorylation. PMID- 3007120 TI - In situ hybridization of immunoglobulin-specific RNA in single cells of the B lymphocyte lineage with radiolabelled DNA probes. AB - A method for in situ hybridization has been developed which detects immunoglobulin-specific mRNA transcripts in single murine B lymphocytes with radiolabelled, immunoglobulin gene-specific single-stranded DNA probes. The method has been applied to myeloma and hybridoma cells and to B lymphocytes at various stages of their maturation from small, resting B cells to Ig-secreting plasma cells. A critical step in the procedure is the treatment of the cells with pronase. The various cell types have been found to be differently susceptible to this treatment. Single-stranded DNA probes of different lengths, i.e., between 26 and 1000 bp, have been employed in the hybridization. The number of silver grains over a cell increases proportionally with the length of the probe and with its concentration in the hybridization reaction. The kinetics of the increase of mu heavy chain-specific RNA molecules in single cells and the appearance of 'switched', gamma-heavy chain-expressing cells are shown after stimulation of murine B cells with lipopolysaccharide. PMID- 3007121 TI - Specificity of immunoglobulin heavy chain switch correlates with activity of germline heavy chain genes prior to switching. AB - IgM+ cells cultured from the I.29 B cell lymphoma can be induced with lipopolysaccharide (LPS) or, to a greater extent, with LPS plus anti-idiotype antibody to switch to IgG2a, IgE or IgA expression. The isotype switch is accompanied by rearrangement of immunoglobulin (Ig) heavy (H) chain genes. Here we demonstrate that the commitment of the I.29 IgM+ cells to switch to IgA appears to be manifested by hypomethylation of the alpha constant region genes in IgM+ cells, and by the presence of small amounts of RNAs transcribed from non rearranged alpha gene(s) in IgM+ cells. The commitment to switch to IgE or IgG2a is also in accord with the presence of small amounts of RNA transcripts from the non-rearranged epsilon and gamma 2a genes, although the hypomethylation of the epsilon and gamma 2a genes is not as dramatic as that of the alpha genes. These results suggest that I.29 cells switch specifically to IgA, IgE or IgG2a due to the activation of the corresponding H chain constant region genes in IgM+ cells prior to the actual switch recombination event. PMID- 3007122 TI - Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Codistribution of unoccupied receptor with cytosolic marker enzymes during fractionation of mouse liver, rat liver and cultured Hepa-1c1 cells. AB - In early experiments Ah receptor appeared to be localized in cytosol when in its unoccupied state and it was thought that the receptor translocated into nuclei only when occupied by its ligands. However, a recent report [Whitlock and Galeazzi (1984) J. Biol. Chem. 259, 980-985] concluded that unoccupied Ah receptor in the intact cell was primarily located within the nucleus and that apparent 'cytosolic' Ah receptor was a redistribution artifact caused by fractionation of cells in large volumes of buffer. We examined the effect of buffer volume and ionic strength on apparent 'cytosolic' versus 'nuclear' distribution of unoccupied Ah receptor in liver from C57BL/6J mice and Sprague Dawley rats as well as Hepa-1c1c9 cells in culture. In all three systems the Ah receptor appears to shift out of the nuclear fraction and into the cytosolic fraction as the volume of buffer is increased or when the ionic strength of the buffer is increased. In each system, however, the distribution of the Ah receptor was identical to the distribution of each of three standard cytosolic marker enzymes: aldolase, glucose-6-phosphate dehydrogenase and lactate dehydrogenase. Co-distribution of unoccupied Ah receptor with these cytosolic marker enzymes during fractionation at varied buffer volumes and ionic strengths makes it seem unlikely that the unoccupied receptor is predominantly a 'nuclear' component in intact cells. Marker enzyme data favor an interpretation that unoccupied Ah receptor is primarily cytoplasmic or that this soluble protein is in equilibrium between cytoplasm and nucleus. PMID- 3007123 TI - Further study of hydroxyapatite high-performance liquid chromatography using both proteins and nucleic acids, and a new technique to increase chromatographic efficiency. AB - Previously developed hydroxyapatite high-performance liquid chromatography columns were tested further by using not only proteins but also nucleic acids. In both cases it was confirmed that the column can, in fact, discriminate subtle structural differences among molecules. Especially for protein mixtures a new technique was introduced to increase the efficiency of chromatography. PMID- 3007124 TI - Differential protein synthesis in the induction of thyroid cell proliferation by thyrotropin, epidermal growth factor or serum. AB - Protein synthesis in the G1 period of the cell cycle has been investigated using two-dimensional gel electrophoresis in primary cultures of dog quiescent thyroid cells, incubated in defined medium and induced to proliferate by the combined action of thyrotropin (TSH), epidermal growth factor (EGF) and serum or by each of these agents, acting alone. The analysis of the proteins, pulse-labeled for 3 h with [35S]methionine, in quiescent cells deprived of serum and in cells that had been stimulated for various periods of time by the addition of TSH, EGF and serum showed maximal modifications before entry into S phase: the labeling of at least ten proteins was enhanced while that of at least six proteins was decreased. The synthesis of one of these proteins (protein 1; Mr approximately equal to 81 000) was maximal 9-12 h after stimulation by the proliferative agents but began to decrease at 15-18 h and was still decreased at 29-32 h. The study of the effect of each of the proliferation agents alone on the labeling of these sixteen proteins showed that TSH specifically stimulated the labeling of eight polypeptides (proteins 2-9) and that, in contrast, EGF and serum specifically increased the labeling of two other proteins (proteins 1 and 10). The labeling of one protein was decreased by each of the different agents (protein 6') while TSH specifically decreased the labeling of four polypeptides (proteins 1'-4') and increased the labeling of one polypeptide (protein 5') whose synthesis was decreased by EGF and serum. The specific effect of TSH on one protein labeling (protein 7; Mr approximately equal to 39 000) was potentiated by EGF and serum while the specific effect of EGF and serum on another protein labeling (protein 1) was potentiated by TSH. There is thus a correlation between the level of synthesis of these two proteins and the proliferative state of the cells, which is much greater when the stimulating agents are acting together. The induction of protein 1 synthesis by EGF was no longer observed when the cells were no longer proliferating. In the same way, TSH no longer stimulated the synthesis of protein 7 in thyroid cells at confluence. In conclusion, the present study has identified some proteins (proteins 1 and 7) which, as judged by the peculiar stimulation and the kinetics of their synthesis, could be part of the final key events triggering DNA replication in thyroid cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3007125 TI - Processing of human choriogonadotropin and its receptors by cultured pig Leydig cells. Role of cyclic AMP and protein synthesis. AB - We have examined the process by which human choriogonadotropin/luteinizing hormone (hCG/LH) receptors are regulated in cultured porcine Leydig cells. Treatment of Leydig cells with human choriogonadotropin, cholera toxin, forskolin and cyclic 8-bromoAMP (8-BrcAMP) produced a loss of surface receptors without modification of the binding affinity. This negative regulation of the number of receptors mediated by maximal concentrations of hCG was higher than that induced by the other agents. The extent of receptor loss in cells treated with increasing concentrations of hCG was highly correlated with their capacity to stimulate cAMP production. However, there was little correlation between down-regulation and cAMP production of these cells treated by hCG plus forskolin or cholera toxin plus forskolin, where a synergistic cAMP production was obtained. Following exposure of Leydig cells to both hCG and 8-BrcAMP, the surface receptor disappearance began after an initial lag period of about 6-8 h. Thereafter a 50% loss of surface receptor was observed in the next 8-h incubation. Monensin with hCG shortens this lag period before initiation of receptor loss. Kinetic studies with 125I-hCG, in the presence or absence of monensin, showed that the half-life of the receptor-bound hormone complexes at the cell surface was 10.5 h and 8 h respectively. Therefore, the steady state of the surface receptor during the lag phase of 8 h is probably related to recycling of internalized receptors and/or translocation of performed receptors. Cycloheximide and actinomycin D inhibit hCG mediated and 8-BrcAMP-mediated down-regulation. Cycloheximide lengthens ligand receptor complexes at the surface by slowing down the rate of internalization (half-life of 20 h), but this mechanism is not enough per se to explain the effect of cycloheximide. Pulses of hCG or 8-BrcAMP for 4 h and 8 h sufficed to induce nearly maximal down-regulation. However, it was possible to attenuate this triggering effect by adding cycloheximide after pulse of the cells. Thus, even after removal of the triggering agent (hCG or 8-BrcAMP), the loss of surface receptor could be triggered by a protein-sensitive signal. Taken as a whole these results indicate that a coordinated interaction is involved in the cell-surface hCG/LH receptor regulation. The apparent steady state of the number of receptors during the first hours of stimulation passed through a reuptake of internalized receptors.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3007127 TI - Rate constant for capping of the barbed ends of actin filaments by the gelsolin actin complex. AB - The rate of capping of actin filaments by the gelsolin-actin complex was measured by inhibition of elongation of the barbed ends of actin filaments. Polymeric actin (0.1-1.0 microM) was added to 0.5 microM monomeric actin and various concentrations of the gelsolin-actin complex (0.08-2.4 nM) to induce nucleated polymerization. As under the experimental conditions (2 mM MgCl2, 100 mM KCl, 37 degrees C, actin monomer concentration less than or equal to 0.5 microM) actin filaments treadmilled, filaments elongated only at the barbed ends and the gelsolin-actin complex did not nucleate actin filaments to polymerize towards the pointed ends. The rate of nucleated actin polymerization in the presence of the gelsolin-actin complex was quantitatively analyzed. The rate constant for capping of the barbed ends of actin filaments by the gelsolin-actin complex was found to be about 10(7) M-1 s-1. PMID- 3007126 TI - Characterization of phosphatidylinositol kinase activity associated with the insulin receptor. AB - Various lipids were tested as substrates for the insulin receptor kinase using either receptor partially purified from rat hepatoma cells by wheat-germ agglutinin-Sepharose chromatography or receptor purified from human placenta by insulin-Sepharose affinity chromatography. Phosphatidylinositol was phosphorylated to phosphatidylinositol 4-phosphate by the partially purified insulin receptor. In contrast, phosphatidylinositol 4-phosphate and diacylglycerol were not phosphorylated. In some, but not all preparations of partially purified insulin receptor, the phosphatidylinositol kinase activity was stimulated by insulin (mean effect 33%). Phosphatidylinositol kinase activity was retained in insulin receptor purified to homogeneity. Insulin regulation of the phosphatidylinositol kinase was lost in the purified receptor; however, dithiothreitol stimulated both autophosphorylation of the purified receptor and phosphatidylinositol kinase activity in parallel about threefold. (Glu80Tyr20)n, a polymeric substrate specific to tyrosine kinases, inhibited the phosphatidylinositol kinase activity of the purified receptor by greater than 90% and inhibited receptor autophosphorylation by 67%. Immunoprecipitation by specific anti-receptor antibodies depleted by greater than 90% the phosphatidylinositol kinase activity in the supernatant of the purified receptor and the phosphatidylinositol kinase activity was recovered in the precipitate in parallel with receptor autophosphorylation activity. These characteristics of the phosphatidylinositol kinase activity of the purified insulin receptor and its metal ion preference paralleled those of the receptor tyrosine kinase activity and differed from bulk phosphatidylinositol kinase activity in cell extracts, which was not significantly inhibited by (Glu80Tyr20)n, stimulated by dithiothreitol or depleted by immunoprecipitation with anti-(insulin receptor) antibody. These results suggest that the insulin receptor is associated with a phosphatidylinositol kinase activity; however, this activity is not well regulated by insulin. This kinase appears to be distinct from the major phosphatidylinositol kinase(s) of cells. Its relationship to insulin action needs further study. PMID- 3007128 TI - Specific cleavage of the fibroblast receptor for platelet-derived growth factor by an endogenous Ca2+-dependent thiol protease. AB - Previous studies have shown that platelet-derived growth factor (PDGF) stimulates the phosphorylation of two components in membranes prepared from human fibroblasts in the presence of Ca2+. One of these represents the 185-kDa PDGF receptor, which undergoes autophosphorylation, and the other has an Mr of 130 000. We show in this communication that the 130-kDa component is derived from the 185-kDa receptor via proteolysis by an endogenous Ca2+-dependent protease, which is dependent on a reduced -SH group for its activity. The 130-kDa fragment contains several of the characteristics of the receptor, such as the PDGF-binding site and the major autophosphorylation sites. Furthermore, the cleaved receptor retains tyrosine kinase activity. PMID- 3007129 TI - Free energy coupling between H+-generating and H+-consuming pumps. Ratio between output and input forces. AB - The delta Gp/delta mu H ratio has been measured in mitochondria close to state 4 in the presence of various uncoupler or K+/valinomycin concentrations in media containing either 1 mM or 50 mM Pi. Care has been taken to control the factors affecting delta Gp and delta mu H which could lead to an artefactual increase of the delta Gp/delta mu H ratio above the highest accepted value for the H+/ATP stoichiometry (n = 4, synthesis + transport). In particular, to avoid overestimation of delta Gp due to inactivation of the ATPases at low delta mu H or to the presence of adenylate kinase, the static head state was approached from the side of net ATP synthesis and delta Gp was measured in a state close to static head but still maintaining a residual rate of aerobic phosphorylation. For each concentration of uncoupler or K+, the Pi concentration and/or the adenylate energy charge (EC) as a function of time have been measured as indicators of net ATP synthesis. Only the values of delta Gp measured during a decrease in Pi concentration and/or an increase in EC have been considered to be meaningful for calculations of delta Gp/delta mu H ratios. Both uncouplers and K+ transport cause a marked depression of delta mu H and a parallel depression of the rate of ATP synthesis. However the low rate of ATP synthesis taking place under conditions of low delta mu H eventually results, especially at high Pi concentrations, in a relatively large delta Gp. The delta Gp/delta mu H ratios obtained at the lower delta mu H values exceed 4 and approach 6. Although slightly higher delta Gp/delta mu H ratios are obtained with valinomycin-treated than with uncoupler-treated mitochondria, the pattern of the rise of the force ratio as delta mu H decreases is similar in both cases. An increase of the delta Gp/delta mu H ratio above 4, the maximal accepted H+/ATP stoichiometry is thermodynamically incompatible with the delocalized protonic coupling model. PMID- 3007130 TI - In vitro deacylation of lipopolysaccharide of Salmonella minnesota by Acanthamoeba castellanii enzymes. AB - Enzymatic deacylation of the lipopolysaccharide isolated from a Salmonella Rd mutant by a cell-free preparation from Acanthamoeba castellanii has been studied. The degradation was found to be dependent on the presence of a surface-active component (Triton X-100) in the reaction mixture. The lipid A part of the lipopolysaccharide was the primary target of the enzymes, which cleaved with high efficiency the ester-bound long-chain nonhydroxylated and 3-hydroxylated acyl residues, i.e. lauric, myristic, palmitic and 3-hydroxymyristic acid. The cell free preparation also exhibited amidase activity cleaving about 50% of the amide bound 3-hydroxymyristic acid residues. In addition the extract proved to possess phosphatase activity liberating ester-bound and glycosidically bound phosphate groups of lipid A. On the other hand, the glucosaminyl-beta 1,6-glucosamine disaccharide was not degraded and remained bound to the oligosaccharide part (heptose/3-deoxyoctulosonic acid) of the lipopolysaccharide. PMID- 3007131 TI - Chemical modification of NADPH-cytochrome P-450 reductase. Presence of a lysine residue in the rat hepatic enzyme as the recognition site of 2'-phosphate moiety of the cofactor. AB - Chemical modification of rat hepatic NADPH-cytochrome P-450 reductase by sodium 2,4,6-trinitrobenzenesulfonate (TNBS) resulted in a time-dependent loss of the reducing activity for cytochrome c. The inactivation exhibited pseudo-first-order kinetics with a reaction order approximately one, and a second-order constant of 4.8 min-1 X M-1. The reducing activities for 2,6-dichloroindophenol and K3Fe(CN)6 were also decreased by TNBS. Almost complete protection of the NADPH-cytochrome P 450 reductase from inactivation by TNBS was achieved by NADP(H), while partial protection was obtained with a high concentration of NADH. NAD, FAD and FMN showed no effect against the inactivation. 3-Acetylpyridine-adenine dinucleotide phosphate, adenosine 2',5'-bisphosphate and 2'AMP protected the enzyme against the chemical modification. Stoichiometric studies showed that the complete inactivation was caused by modification of three lysine residues per molecule of the enzyme. But, under the conditions where the inactivation was almost protected by NADPH, two lysine residues were modified. From those results, we propose that one residue of lysine is located at the binding site of the 2'-phosphate group on the adenosine ribose of NADP(H), and plays an essential role in the catalytic function of the NADPH-cytochrome P-450 reductase. PMID- 3007132 TI - Regulation of dehydrogenases/one-electron transferases by modification of flavin redox potentials. Effect of product binding on semiquinone stabilization in yeast flavocytochrome b2. AB - Spectroscopic and potentiometric measurements have been carried out, at room temperature, during anaerobic titrations of Hansenula anomala L-lactate cytochrome c oxidoreductase (or flavocytochrome b2) both in the presence and in the absence of pyruvate (the physiological reaction product). Under the same conditions, the flavin spectral contribution was estimated and the flavosemiquinone proportion was directly determined by electron paramagnetic resonance measurements. In the present study, we show the visible light absorption and paramagnetic characteristics of the flavin radical at 18 degrees C and also the dramatic effect of pyruvate on the redox potential of each monoelectronic couple of the flavin. Thermodynamic stabilization of the semiquinone form, in the presence of pyruvate, is interpreted as a mode of regulation of flavocytochrome b2 activity. Taking into account that analogous controls have been observed with two other flavoenzymes belonging to this class of dehydrogenases/one-electron transferases, we suggest that redox potential modulation could be a type of regulation effective for the whole class of enzymes in which a semiquinone is an obligate intermediate. PMID- 3007133 TI - The binding of porphyrin cytochrome c to yeast cytochrome c peroxidase. A fluorescence study of the number of sites and their sensitivity to salt. AB - Porphyrin c, the iron-free derivative of cytochrome c, is a reasonably good model for cytochrome c binding to cytochrome c peroxidase (CcP). It binds with the same stoichiometry but only one-quarter as tightly as cytochrome c. CcP (resting, FeIII) and CcP X CN can both bind up to two molecules of porphyrin c. The binding of the first porphyrin c is tight (kd = 1 X 10(-9) M, pH 6, ionic strength mu = 0, 4 degrees C) and results in quenching of the porphyrin c fluorescence. The binding is sensitive to ionic strength. The binding of the second porphyrin c is looser (Kd unknown) and does not result in quenching of the porphyrin fluorescence. The binding of porphyrin c to the cyano form and the resting forms of CcP cannot be distinguished by our methods. ES is the first acceptor of electrons from c(II) and can bind at least two molecules of porphyrin c. The binding of the first porphyrin c is extremely tight, results in substantial quenching and is insensitive to ionic strength. The binding of porphyrin c to the loose site (Kd = 2 X 10(-9) M, pH 6, 4 degrees C, mu = 0) results, unlike the resting and cyano forms, in quenching of fluorescence of the second porphyrin c. The binding of the second porphyrin c to ES is sensitive to ionic strength. The calculated distances between porphyrin c and the hemes of CcP(FeIII) and ES are approximately 2.5 nm. PMID- 3007134 TI - Determinants for protein translocation across mammalian endoplasmic reticulum. Membrane insertion of truncated and full-length prelysozyme molecules. AB - The translocation of fragments of prelysozyme lacking varying portions of the COOH terminus of the protein is studied in comparison to full-length prelysozyme using transcription-coupled capping of RNA and subsequent translation in a wheat germ cell-free system. The fragments are generated by restricting cloned lysozyme cDNA at selected sites. We found that fragments of 102 and 74 amino acid residues could still be translocated by mammalian endoplasmic reticulum. Addition of signal-recognition particles (SRP) to the cell-free system blocked the nascent chain synthesis. The SRP-depleted membrane by itself could neither process nor translocate the prepolypeptide chain. The presence of both components was essential for processing and translocation as well as the release of the nascent chain arrest induced by SRP. However, when the size of the fragment was limited to 51 amino acids, the SRP-induced arrest, the translocation and processing failed to take place. These results define minimum length and structural requirements for translocation of the nascent chain across mammalian endoplasmic reticulum. PMID- 3007135 TI - High expression of Bacillus licheniformis alpha-amylase with a Bacillus secretion vector. AB - A gene coding for the heat-stable alpha-amylase from Bacillus licheniformis ATCC14580 has been expressed with the aid of a B. amyloliquefaciens alpha-amylase based expression/secretion vector by joining the structural part of the gene to a pool of vectors after the B. amyloliquefaciens alpha-amylase promoter and signal sequence. The recombinant plasmids obtained were stably maintained in B. subtilis and the heat-stable alpha-amylase activity rose several hundred times from the level of the donor. Eight different constructions were further analyzed. Each of them had an intact B. amyloliquefaciens signal sequence, the only difference being in a few nucleotides beyond the C terminus of the signal peptide. This, however, was enough to cause up to fourfold differences in protein yield. Possible reasons for the variation in the production level are discussed. PMID- 3007136 TI - Complete structural analysis of globoseries glycolipids by two-dimensional nuclear magnetic resonance. AB - Combined two-dimensional proton nuclear magnetic resonance allowed the determination of complete oligosaccharide structures of glycolipids belonging to the globo series, without any other analytical methods. Although a chemical modification by peracetylation was required for the above purpose, the derivatization permitted facile assignment of the pyranose ring proton resonances of the oligosaccharide moiety. Two-dimensional chemical-shift-correlated spectroscopy of the acetylated glycolipid enabled us to elucidate the glycosidic positions from the chemical shifts of the protons at the substituted sites. The monosaccharide species were also identified from the characteristic splitting patterns of the methine protons on individual pyranose rings. The sequence of the monosaccharides was inferred from the interresidue connectivity across glycosidic linkages shown by two-dimensional nuclear Overhauser effect spectroscopy, which also gave intraresidue interaction on the pyranose rings. The linkage sites of long oligosaccharide chains having more than five monosaccharides, such as globopentaosylceramide, were analyzed by two-dimensional J-relayed coherence transfer, which yielded 1,3 interactions along with 1,2 interactions. PMID- 3007138 TI - Target size analysis of opioid receptors. No difference between receptor types, but discrimination between two receptor states. AB - Target size analysis of opioid receptor is complicated by the presence of multi exponential inactivation curves. Irradiation of intact frozen tissue proved essential to eliminate such artifacts, due to indirect irradiation effects. Upon irradiation condition, opioid binding activity was inactivated in a single mono exponential manner. Identical inactivation curves were obtained for mu, delta and kappa binding activities in brain membranes from rat, guinea-pig and frog and in NG 108-15 cells: the molecular mass obtained was 98 +/- 2 kDa. However, when opioid binding was assayed in the presence of Na+, Mg2+ and GTP, the molecular mass was found to be only 56 +/- 4.4 kDa. We suggest that the opioid recognition site comprises a unit of 56 kDa and that in the absence of Na+, Mg2+ and GTP an additional membrane component of 40-44 kDa is necessary for high-affinity opioid binding. PMID- 3007137 TI - Solubilization of the bovine cardiac sarcolemmal binding sites for calcium channel blockers. AB - Nonionic and ionic detergents were used to solubilize the bovine cardiac sarcolemmal binding sites for nimodipine and (-)desmethoxyverapamil in the absence of added ligand. Only Chaps, digitonin and sucrose monolauryl ester were able to solubilize the binding sites in a form that bound radioligands. About 45% of each of the membrane-bound high-affinity site was solubilized by 0.4% Chaps (w/v) in the presence of 48% (w/v) glycerol. The solubilized binding sites were destroyed by trypsin or by a 10-min incubation at 50 degrees C. Calcium stimulated nimodipine binding slightly at 0.3 mM and inhibited ( )desmethoxyverapamil binding completely with an IC50 of 1.2 mM. Nimodipine binding was reduced by 20% in the presence of EGTA. The solubilized receptors sedimented in sucrose density gradients with an apparent s20,w of 21 S. An identical sedimentation value was obtained for the cardiac sarcolemmal and skeletal transverse tubulus receptor which were prelabeled with nitrendipine and solubilized by digitonin. Solubilization reduced the affinity of nimodipine for its high-affinity site slightly from 0.35 nM to 1.2 nM and that for its low affinity site from 33 nM to 130 nM. Solubilization did not affect significantly the specific density of these sites. Binding of nimodipine to the low-affinity site was completely abolished by 0.1 microM nitrobenzylthioinosine. After solubilization only the high-affinity site for (-)desmethoxyverapamil could be measured with tenfold reduced affinity (Kd = 15.3 nM) but unchanged specific density. Binding to the solubilized high-affinity site for nimodipine and ( )desmethoxyverapamil was stereospecific and showed a similar rank order as the particulate binding sites. Binding of nimodipine was inhibited allosterically by phenylalkylamines. Similarly, (+)PN200-110 inhibited allosterically ( )desmethoxyverapamil binding. d-cis-Diltiazem stimulated nimodipine binding at 20 degrees C 1.2-fold, reduced the dissociation rate from 0.018 min-1 to 0.0083 min 1 and had no effect on the association rate (0.173 min-1. nM-1). The Kd calculated from the rate constants was 0.1 nM and in close agreement with the value of 0.49 nM measured under equilibrium conditions in the presence of nitrobenzylthioinosine. In contrast, desmethoxyverapamil increased the dissociation rate of nimodipine to 0.03 min-1. The association and dissociation rate constants for (-)desmethoxyverapamil were 0.024 min-1. nM-1 and 0.025 min-1, respectively.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3007139 TI - Hyperchromic effect of collagen induced by human collagenase. AB - Loss of the highly ordered triple-helix structure of native collagen on denaturation or enzymatic degradation involves a helix-to-coil transition, which can be seen as an increase at 227 nm in its ultraviolet difference absorption spectrum. We report here the successful use of this hyperchromic effect to quantify collagen in solution and to follow up the time-course of collagen degradation catalyzed by collagenase. Using 14C-labelled collagen substrate we show the excellent correlation between enzyme-induced increase in ultraviolet difference absorption and formation of specific cleavage products. The novel method was found to be suitable to characterize the enzymatic properties of human leukocyte collagenase. Activation of latent collagenase to the active enzyme could be followed continuously and an activation lag estimated. PMID- 3007140 TI - The protein phosphatases involved in cellular regulation. Evidence that dephosphorylation of glycogen phosphorylase and glycogen synthase in the glycogen and microsomal fractions of rat liver are catalysed by the same enzyme: protein phosphatase-1. AB - Glycogen synthase (labelled in sites-3) and glycogen phosphorylase from rabbit skeletal muscle were used as substrates to investigate the nature of the protein phosphatases that act on these proteins in the glycogen and microsomal fractions of rat liver. Under the assay conditions employed, glycogen synthase phosphatase and phosphorylase phosphatase activities in both subcellular fractions could be inhibited 80-90% by inhibitor-1 or inhibitor-2, and the concentrations required for half-maximal inhibition were similar. Glycogen synthase phosphatase and phosphorylase phosphatase activities coeluted from Sephadex G-100 as broad peaks, stretching from the void volume to an apparent molecular mass of about 50 kDa. Incubation with trypsin decreased the apparent molecular mass of both activities to about 35 kDa, and decreased their I50 for inhibitors-1 and -2 in an identical manner. After tryptic digestion, the I50 values for inhibitors-1 and -2 were very similar to those of the catalytic subunit of protein phosphatase-1 from rabbit skeletal muscle. The glycogen and microsomal fractions of rat liver dephosphorylated the beta-subunit of phosphorylase kinase much faster than the alpha-subunit and dephosphorylation of the beta-subunit was prevented by the same concentrations of inhibitor-1 and inhibitor-2 that were required to inhibit the dephosphorylation of phosphorylase. The same experiments performed with the glycogen plus microsomal fraction from rabbit skeletal muscle revealed that the properties of glycogen synthase phosphatase and phosphorylase phosphatase were very similar to the corresponding activities in the hepatic glycogen fraction, except that the two activities coeluted as sharp peaks near the void volume of Sephadex G-100 (before tryptic digestion). Tryptic digestion of the hepatic glycogen and microsomal fractions increased phosphorylase phosphatase about threefold, but decreased glycogen synthase phosphatase activity. Similar results were obtained with the glycogen plus microsomal fraction from rabbit skeletal muscle or the glycogen-bound form of protein phosphatase-1 purified to homogeneity from the same tissue. Therefore the divergent effects of trypsin on glycogen synthase phosphatase and phosphorylase phosphatase activities are an intrinsic property of protein phosphatase-1. It is concluded that the major protein phosphatase in both the glycogen and microsomal fractions of rat liver is a form of protein phosphatase-1, and that this enzyme accounts for virtually all the glycogen synthase phosphatase and phosphorylase phosphatase activity associated with these subcellular fractions. PMID- 3007141 TI - Active-site ionizations of papain. An evaluation of the potentiometric difference titration method. AB - The underlying assumption of the potentiometric difference titration method, as applied to the evaluation of the sulfhydryl-dependent ionizations in the active site of papain, is that the pKa of His-159 is independent of whether the neighboring sulfhydryl (Cys-25) is protonated or methylthiolated. That this idealized assumption may not strictly apply is indirectly indicated by the larger pKa of His-159 in S-methylpapain versus that in S-methylthiopapain, as determined from fluorometric titrations (delta pKa = 0.32 +/- 0.05, 25 degrees C). On the basis of the Wegscheider principle of the equivalence of protons and methyl groups, the potentiometric difference titration method will underestimate the concentration of thiolate-imidazolium ion pair in the active site versus that of the thiol-imidazole tautomer, provided that there is no significant H-bonding interaction in the latter species. PMID- 3007142 TI - Respiration of Trichomonas vaginalis. Components detected by electron paramagnetic resonance spectroscopy. PMID- 3007143 TI - Tissue-specific and species-specific distribution of -SH groups in cytochrome c oxidase subunits. AB - Cytochrome c oxidase was isolated from pig, bovine, rat and human tissues including liver, heart, diaphragm and kidney. The native and the sodium-dodecyl sulfate (SDS)-dissociated enzymes were labelled under optimal conditions with N ethyl-[2,3-14C]maleimide before and after reduction with dithiothreitol, separated into 13 subunits by SDS gel electrophoresis and the radioactive bands were visualized by fluorography. In some cases the radioactive bands were cut out and counted. All isozymes were labelled in subunits I, III, Va and VIIb, and in subunit II after reduction. Labelling of subunit Vb was equivocal, and in no case were subunits IV and VIc labelled. All other subunits were labelled tissue specifically and/or species-specifically. No differences were found between labelling of the native and SDS-dissociated enzyme. By relating the molar amount of bound N-ethylmaleimide to the known amount of cysteines in subunits of bovine heart cytochrome c oxidase, the percentage of -SH group reactivity was calculated. Only the cysteine of subunit Va was found to be 100% reactive. The distinct and different reactivity of subunit VIIb as compared to subunits VIIa and VIIc clearly establishes this polypeptide as an independent subunit of mammalian cytochrome c oxidase. PMID- 3007144 TI - Acute in vivo stimulation of low-Km cyclic AMP phosphodiesterase activity by insulin in rat-liver Golgi fractions. AB - A low-Km phosphodiesterase activity, which is acutely stimulated by insulin in vivo, has been identified in plasma membranes and Golgi fractions prepared from rat liver homogenates in isotonic sucrose. Within seconds after insulin injection (25 micrograms/100 g body weight) cAMP phosphodiesterase activity increases by 30 60% in Golgi fractions and by 25% in plasma membranes; activity in crude particulate and microsomal fractions is unaffected. The increase in activity is short-lived in the light and intermediate Golgi fractions, but persists for at least 10 min in the heavy Golgi fraction. It precedes the translocation of insulin and insulin receptors to these fractions, which is maximal at 5 min. The doses of insulin required for half-maximal and maximal activation are, respectively, 7.5 micrograms/100 g and 25 micrograms/100 g body weight. Golgi associated cAMP phosphodiesterase activity shows non-linear kinetics; a high affinity component (Vmax, 13 pmol min-1 mg protein-1; Km, 0.35 microM) is detectable. Insulin treatment increases the Vmax 60-70%, but does not affect the Km. Unlike the low-Km cAMP phosphodiesterase associated with crude particulate fractions, the Golgi-associated activity is not easily extractable by solutions of low or high ionic strength. On analytical sucrose density gradients, low-Km cAMP phosphodiesterase associated with the total particulate fraction equilibrates at lower densities than endoplasmic reticulum and lysosomal markers, but at a higher densities than plasma membrane, Golgi markers and insulin receptors. Insulin treatment increases the specific activity of the enzyme by 20 60% at densities below 1.12 g cm-3, and by 20-40% in the density interval 1.23 1.25 g cm-3. Such treatment also causes a slight, but significant shift in the distribution of phosphodiesterase towards lower densities. It is suggested that Golgi elements or physically similar subcellular structures are a major site of localization of insulin-sensitive cAMP phosphodiesterase in rat liver. However, internalization of the insulin-receptor complex is probably not required for enzyme activation. PMID- 3007145 TI - Human complement component C1s. Partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation. AB - Human C1s proenzyme (Mr 83 000) was isolated by a rapid two-stage method involving affinity chromatography of C1 on IgG-Sepharose and isolation of subcomponent C1s by ion-exchange chromatography on DEAE-Sephacel. Single-chain C1s proenzyme was activated to two-chain C1s with self-activated C1r. After reduction and S-carboxamidomethylation the heavy chain of C1s (Mr 57 000) was isolated by ion exchange chromatography on DEAE-Sephacel. Cleavage of C1s heavy chain with CNBr yielded five fragments whose N-terminal sequences were determined. The alignment of the fragments within the heavy chain was established by tryptic peptides containing methionine. C1s heavy chain comprises about 470 amino acid residues and 42% of its sequence was determined. An intrachain sequence homology and a homology to the alpha 2 chain of human haptoglobin were identified. The C-terminal CNBr fragment comprising 44 amino acid residues was completely sequenced. From BNPS-skatole cleavage of reduced and alkylated C1s proenzyme a fragment was isolated which overlaps the C1s heavy and light chain parts and which contains the peptide bond cleaved during activation. The results show that this is an Arg-Ile bond and that under standard conditions of activation no peptide material is liberated from this portion of the molecule. The sequence data and homology to two-chain serine proteases indicate a single interchain disulfide bond in C1s. PMID- 3007146 TI - Assay of mannose-6-phosphatase in untreated and detergent-disrupted rat-liver microsomes for assessment of integrity of microsomal preparations. AB - An accurate, precise, and convenient procedure was developed for measurement of the latency of the low-Km mannose-6-phosphatase activity for the purpose of assessment of the membrane permeability barrier in microsomes. This approach is based on previous work of Arion et al. [J. Biol. Chem. (1976) 251, 4901-4907] and consists of measurement of mannose-6-phosphatase activity in the untreated microsomal fraction and in the corresponding microsomes that are fully disrupted in order to eliminate the membrane permeability barrier. Complete disruption of rat liver microsomes was achieved by incubation for 60 min at 0 degree C in the presence of 4 mM zwitterionic detergent 3-[(3-cholamido-propyl)dimethyl-ammonio] 2-hydroxy-1-propane sulphonate (Chapso). That the microsomal membrane permeability barrier was eliminated under those conditions was suggested by the fact that the enzyme activation (up to 50-fold) produced by this pretreatment was at least as large as the effect of any other previously reported disruptive procedure. Disruption of the microsomes by Chapso or by ultrasonication markedly enhanced the thermolability of the mannose-6-phosphatase activity. In addition, exposure of the microsomes to high concentrations of Chapso produced enzyme inactivation that could be partially reversed by dilution of the detergent prior to assaying the enzymic activity. Investigation of these enzyme inactivation phenomena under various incubation conditions for disruption of the microsomes by Chapso and for subsequent assay of mannose-6-phosphatase activity in the presence of Chapso enabled us to define conditions under which instability of the enzyme was undetectable. Using these optimized procedures for disruption of microsomes and assay of hexose-6-P phosphohydrolase, we found that the low-Km mannose-6 phosphatase activity of untreated rat liver microsomes consistently was less than 5% of the total enzyme activity in the fully disrupted microsomes. Accurate and precise assay of the structural latency of mannose-6-phosphatase in membrane preparations must be performed under well-controlled conditions, with special attention to the marked thermolability of the enzyme in the presence of detergent, and is a prerequisite for using this approach for the purpose of assessing intactness of microsomal preparations. PMID- 3007147 TI - F-actin aggregates in transformed cells contain alpha-actinin and fimbrin but apparently lack tropomyosin. AB - Transformation-specific F-actin structures are examined in tumor cells after in vitro tumor cell growth alone or on an untransformed cell monolayer. In transformed cells F-actin aggregates near the ventral plasma membrane in close substrate adhesion areas contain the cytoskeletal proteins alpha-actinin and fimbrin but, unlike microfilament bundles, are not labeled with antibody against tropomyosin. By electron microscopy the dense ventral aggregates in transformed cells resemble stress fiber termini found at the membrane in normal cells. These transformed-cell cytoskeletal structures are not limited solely to substrate adhesion areas; they are also expressed at cell-cell contacts about 48 h after transformed cells are plated on untransformed cells. These specialized F-actin aggregates appear to be implicated in the processes of penetration of these transformed cells between adjoining untransformed cells in vitro. PMID- 3007148 TI - Ultrastructural studies of endothelial and platelet receptor binding of thrombin colloidal gold probes. AB - Endothelial cells and platelets are reported to have receptors for alpha thrombin. To visualize the binding of alpha-thrombin to these cells, we developed a method to label thrombin with colloidal gold. Formed by electrostatic adsorption of thrombin to the negatively charged gold, the resulting probe is stable for weeks and consists of approximately 30 thrombin molecules adsorbed to each 16.5 nm gold particle. The probe retained about 10% of the enzymatic activity (fibrinogen clotting) of the unlabeled native thrombin and 20% of the ability of the native thrombin to aggregate platelets in platelet-rich plasma (PRP). In PRP, approximately 90% of the observed probes were bound to fibrin strands, with the remaining probes (650 per cell) attached to activated platelets. In contrast, washed, paraformaldehyde-fixed human platelets exhibited a marked increase in probe density (4900 per cell). Time-dependent ultrastructural studies (2-240 min) of binding of the thrombin-gold probe to confluent cultures of porcine aortic endothelial cells revealed that the initial binding (7300 probes per cell) occurred randomly at the cell surface. A limited number (25%) of the probes clustered at coated-pit regions and were internalized (60-240 min). The probe induced a limited amount of cellular retraction similar to that achieved with unlabeled thrombin. These results suggest that the thrombin gold probe is suitable for investigations of the localization of thrombin receptors on cell surfaces and the interaction of thrombin with these receptors during thrombotic events. PMID- 3007149 TI - Receptor-mediated uptake of lipoproteins by cultured porcine granulosa cells. AB - Receptor-mediated uptake of low density lipoprotein (LDL) has been shown to provide a major source of cholesterol for a variety of cell types, particularly steroidogenic cells. In this study, the functional significance of lipoproteins in porcine ovarian granulosa cells and their mechanism of uptake by the cell was examined. Porcine LDL and high density lipoprotein (HDL) were isolated using a KBr density gradient, and the purity of both lipoproteins was confirmed by single corresponding bands on agarose gel stained for lipid and protein. Purified LDL and HDL were radioiodinated and labelled with colloidal gold for binding and tracer studies respectively. Both lipoproteins bind to cell surface and are internalized within 30 min at 37 degrees C. The cultured granulosa cells possess more HDL binding sites than LDL binding sites and are more responsive in progesterone production when supplemented with HDL. These results suggest that granulosa cells may preferentially utilize HDL over LDL as a source of cholesterol for steroidogenesis. PMID- 3007150 TI - Identification of a transformation-sensitive 110-kDa plasma membrane glycoprotein of rat hepatocytes. AB - Monoclonal antibodies were used to define cell surface antigens which are present on rat hepatocytes but are absent from hepatoma cells. One monoclonal antibody, referred to as Be 9.2, recognizes a major component of purified rat liver plasma membranes with a Mr of 110 000. This antigen (gp110) was not found in the transplantable Morris hepatoma 9121 and 7777 nor on two cultured hepatoma cell lines. Isoelectric focussing showed that gp110 is a very acidic membrane component with an isoelectric point of 3.6 to 3.8. Treatment with neuraminidase reduced the Mr to 95 000. Gp110 while bound to the membrane was resistant to trypsin, but sensitive to papain. The tissue distribution of gp110 was examined by indirect immunofluorescence in frozen sections. The antigen was found on the bile canalicular domain of hepatocytes, the microvillous zone of enterocytes of the small intestinal villi, the luminal plasma membrane of acinar cells in the submaxillary and extraorbital gland and of epithelial cells of the vesicular gland. Gp110 could not be detected in the stomach, pancreas, large intestine, kidney, thymus, spleen, heart, lung, muscle cells and fibers and in the brain. Identical results were obtained by the use of an antiserum raised against purified gp110. They confirm the transformation-sensitive character of this glycoprotein. A possible identity with dipeptidyl peptidase IV and aminopeptidase M, which have similar molecular weights and are also present in rat liver on the bile canalicular domains, could be excluded. The results suggest that the loss of gp110 might be regarded as a marker for transformation or dedifferentiation of hepatocytes. PMID- 3007151 TI - The processing and intracellular transport of myeloperoxidase. Modulation by lysosomotropic agents and monensin. AB - Myeloperoxidase, stored in azurophil granules of neutrophils, is synthesized in promyelocytes as a larger molecular weight precursor, which is processed to yield a transient Mr 82 000 intermediate and mature polypeptides with molecular weights of 62 000 and 12 000. We have tried to define subcellular sites for processing using metabolic labelling of the promyelocytic leukemia cell line HL-60 in combination with subcellular fractionation on a Percoll gradient. A reasonable separation was achieved between azurophil granules, Golgi elements and endoplasmic reticulum. The finding of almost exclusively fully processed myeloperoxidase in granules and a mixture of unprocessed and processed polypeptide in fractions enriched in Golgi elements suggests that processing occurred mainly in pregranular structures. Monensin, which exchanges protons for Na+, and the base chloroquine blocked processing probably by inhibition of transport through the Golgi apparatus. However, the lysosomotropic NH4+ cation did not inhibit processing or transport indicating that processing is not necessarily influenced by pH-dependent mechanisms. Results from digestion with endoglycosidase H, incubation with tunicamycin and metabolic labelling with [3H]mannose indicated that myeloperoxidase contained high mannose oligosaccharide side chains. Also [32P]phosphate incorporated into Mr 90 000 and Mr 62 000 myeloperoxidase was susceptible to endoglycosidase H indicating that oligosaccharide side chains are modified by phosphorylation as in lysosomal enzymes. Thus, even if myeloperoxidase contained mannose 6-phosphate residues, these may not necessarily be involved in directing transport to the azurophil granules. PMID- 3007153 TI - Comparison of five methods for detecting human rotavirus in stool specimens. PMID- 3007152 TI - Decreased bone density in severely handicapped children and adults, with reference to the influence of limited mobility and anticonvulsant medication. AB - Bone density and related biochemical parameters were investigated in institutionalised children and adults with severe handicaps, who were classified according to the degree of limited mobility (group 1, bed-ridden; group 2, capable of crawling; group 3, capable of walking) and according to whether or not they were receiving anticonvulsants. As determined by microdensitometric analysis of radiograms of the second metacarpal bone, bone width (D), bone pattern area (sigma GS) and bone salt density (sigma GS/D) were decreased in the patients, the decreases being most prominent in group 1, followed by groups 2 and 3, in that order. Significant decreases of sigma GS and sigma GS/D, but not of D, were found in patients on anticonvulsant treatment in comparison to patients without therapy. Serum alkaline phosphatase (Al-p) and parathyroid hormone (iPTH) as well as urinary calcium and cyclic adenosine-3',5'-monophosphate (cAMP) excretion were significantly increased in group 1. In comparison to patients without therapy, anticonvulsant-treated children showed significantly decreased levels of serum calcium (Ca), ionised Ca (Ca2+), 25-hydroxy vitamin D3 and urinary phosphate (PO4) excretion, and elevated levels of Al-p, iPTH and calcitonin (iCT). It is suggested that limited physical activity results in a mild hyperparathyroid state, which is aggravated in patients on anticonvulsant treatment. PMID- 3007154 TI - Coproantibody response to rotavirus in an outbreak in a day-care nursery. PMID- 3007155 TI - High-dose cyclophosphamide and high-dose VP 16-213 for recurrent or refractory small cell lung cancer. A phase II study. AB - In nine patients with recurrent or refractory small cell lung cancer a phase II study with high-dose cyclophosphamide and high-dose VP 16-213 with autologous bone marrow transplantation was performed. The regimen used was based on a previously reported phase I study. In eight of the nine evaluable patients a response was seen (six PR and two CR). One patient died of treatment related toxicity. Infection is the most important toxicity. The response duration was short. This combination is a suitable "late intensification' regimen for patients with minimal residual disease after standard dose induction chemotherapy. PMID- 3007156 TI - Leukocyte activation in advanced cancer as an explanation for absent leukocyte adherence inhibition to cancer extracts and chemoattractant. AB - Tube leukocyte adherence inhibition (LAI) measures human immunity by antigen binding leukocytes releasing chemoattractant-like mediators that are the ultimate inhibitors of adherence by bystander leukocytes. We determined whether the absent LAI responses to cancer extracts for patients with large body burdens of bladder cancer was related to a defect in antigen binding or chemoattractant responsiveness. Leukocytes from patients with small body burdens of bladder cancer gave positive LAI responses of a similar extent to either bladder cancer extracts or chemoattractant [n-formyl-L-methionylleucylphenylalanine (FMLP)]. Of the adherent leukocytes, about 20-30% became non-adherent with a positive LAI response: monocytes, neutrophils and lymphocytes responded. In the control tubes, leukocytes from patients with large body burdens of bladder cancer were more non adherent and about 15% less adherent than leukocytes from controls or patients with early cancer. They showed no further decrease in adherence, or conversely increase in non-adherence, with either extracts of bladder cancer or FMLP. The leukocytes also failed to transduce transmembrane signals to the same stimuli. The defect was reversible since PGE2 restored the adherence of leukocytes to normal, and subsequently they exhibited membrane potential changes and about 34% non-adherence to either bladder cancer extracts or FMLP. From these results we conclude that chemoattractant LAI-responsive leukocytes from patients with large body burdens of bladder tumor are activated in vivo, probably by mediators from inflammatory cells. PMID- 3007157 TI - The effects of retinoid treatment and antiestrogens on the growth of T47D human breast cancer cells. AB - The ability of all-trans-retinoic acid, 13-cis-retinoic acid, the free acid of etretinate (RO 10-1670), the 'arotinoid' RO 13-6298 and its free acid RO 13-7410 to affect the growth of T47D human breast cancer cells in vitro was investigated. The growth of T47D cells was inhibited by all of the retinoids tested, with the arotinoids being up to 100 times more effective than all-trans-retinoic acid. The presence of cellular retinoic acid binding protein (cRABP) was indicated by the cellular uptake of [3H]all-trans-retinoic acid. Maximum binding was 460 fmol/micrograms DNA. All of the retinoids with a polar terminal free carboxyl group readily competed for the binding sites, but none of the retinoids competed for the estrogen or progesterone receptor. Co-treatment of the T47D cells with 0.1 microM all-trans-retinoic acid and either tamoxifen (1 microM) or hydroxytamoxifen (10 nM or 0.1 microM) produced an additive effect on growth inhibition. No such additive effect was observed when T47D cells were co-treated with arotinoids and antiestrogens. The results showed that the T47D cells can serve as a useful model in vitro to test the effects of the synthetic retinoids and antiestrogens on steroid receptor-positive human breast cancer. PMID- 3007158 TI - Production of leukotrienes and prostaglandins by human ascites cells. AB - Ascites was collected from six patients with liver cirrhosis and the cells isolated. These cells, mainly macrophages, were labelled with 14C-arachidonic acid and stimulated with the calcium ionophore A23187. The metabolites formed were separated by HPLC. The main substances formed by the ascites cells were leukotriene B4, 5-hydroxy-6,8,11,14 eicosatetraenoic acid and leukotriene C4. Smaller amounts of thromboxane B2, 12-hydroxy-5,8,10 heptodecatrienoic acid and 6 keto-prostaglandin F1 alpha were isolated. Human peritoneal macrophages are therefore capable of producing leukotrienes and prostaglandins. Production of these substances might play a role in some of the complications of patients with liver cirrhosis and ascites. PMID- 3007159 TI - Evidence that epinephrine acts preferentially as an antilipolytic agent in abdominal human subcutaneous fat cells: assessment by analysis of beta and alpha 2 adrenoceptor properties. AB - Investigations were carried out to demonstrate the function and the possible advantage of the interplay between beta 1 and alpha 2 adrenoceptor sites in the regulation of human subcutaneous fat-cell lipolysis. alpha 2 and beta adrenoceptor binding studies were conducted with antagonist radioligands and revealed that alpha 2-adrenoceptors ([3H]yohimbine and [3H]rauwolscine binding sites) are more numerous than beta 1-adrenoceptors ([3H]dihydroalprenolol and [3H]CGP-12177 binding sites) in human fat-cell membranes. Physiological agonists epinephrine and norepinephrine competed with [3H]-ligand sites with a higher affinity for alpha 2 sites than for beta 1 sites. Epinephrine exhibited a higher affinity than norepinephrine for the alpha 2 sites; the two amines had the same affinity for beta 1 sites. In lipolysis studies conducted in the absence of adenosine deaminase the beta lipolytic action of the biological amines predominated; after alpha 2-adrenoceptor blockade by yohimbine or idazoxan, the amines exhibited an intrinsic activity similar to that of isoproterenol. When adenosine was prevented from accumulating in the incubation medium by inclusion of adenosine deaminase, low concentrations of epinephrine and norepinephrine preferentially exerted an antilipolytic action. We conclude that: he lipolytic response in abdominal human subcutaneous fat cells to physiological amines results from the interplay between beta 1-and alpha 2-adrenoceptor stimulation; alpha 2 adrenoceptors, with their higher number and higher affinity for the physiological amines, and the adrenoceptor population involved at the lowest (i.e. physiological) concentrations of the amines.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007160 TI - On the mechanism of renin release during experimental myocardial ischaemia. AB - An increase in plasma renin activity (PRA) following experimental coronary occlusion has previously been demonstrated in anaesthetized and conscious dogs. The purpose of the present study was to analyse the mechanism of this renin release. In two distinct models of myocardial ischaemia in anaesthetized dogs- i.e. occlusion of the left-anterior descending coronary artery (model A, n = 21) and atrial pacing in the presence of stenosis of the left-anterior descending coronary artery (model B, n = 23), an increase in arterial PRA was found from 1.68 +/- 0.43 to 3.06 +/- 0.63 ng ml-1 h-1 (model A, mean +/- SEM, P less than 0.025) and from 9.87 +/- 3.59 to 14.96 +/- 4.06 ng ml-1 h-1 (model B, P less than 0.05), respectively. The increase in PRA following coronary occlusion was not blunted by adrenergic beta-receptor blockade with propranolol (3 mg kg-1 i.v.; n = 4). Coronary sinus PRA was lower than arterial PRA and the increase in PRA did not occur in nephrectomized dogs (n = 5). The data suggest that myocardial ischaemia induces a release of renin from the kidney which is not mediated by adrenergic beta receptors. PMID- 3007161 TI - Platelet alpha 2 adrenoceptors in Parkinson's disease: decreased number in untreated patients and recovery after treatment. AB - [3H]-yohimbine binding sites were quantified in platelets from Parkinsonians with no clinical signs of dysautonomia. Never-treated Parkinsonians had a lower specific binding than control subjects. This alteration was associated with decreased epinephrine-induced platelet aggregation. Treatment with dopaminergic agents induced a significant increment of [3H]-yohimbine binding sites. These results show that Parkinson's disease is associated with a reduced number of peripheral alpha 2 adrenoceptors and that dopaminergic agents induce partial recovery. PMID- 3007163 TI - Effect of metoprolol and propranolol on platelet aggregation and cAMP level in hypertensive patients. AB - Ten patients with uncomplicated moderate essential hypertension were recruited to evaluate the effect of the non-selective beta-blocker propranolol and the beta 1 selective beta-blocker metoprolol on platelet aggregation and cAMP formation. Five patients began treatment with propranolol 80 mg b.i.d. and 5 with metoprolol 100 mg b.i.d., and after 2 weeks the treatments were exchanged. ADP- and adrenaline-induced platelet aggregation and the basal level of platelet cAMP were measured at the end of each treatment period. Platelet aggregation was tested turbidometrically, using the threshold value for irreversible aggregation, and cAMP measurements were performed using a protein-binding assay. Both ADP and adrenaline threshold values were significantly lower after propranolol than after metoprolol. The basal cAMP level was lower during propranolol than metoprolol treatment. The results indicate that platelet aggregation and basal cAMP level are influenced by beta-blockers in proportion to their affinity to different beta adrenoceptors. This may be of value in the beta-blocker treatment of patients at high thrombotic risk. PMID- 3007162 TI - Comparison of enalapril and propranolol in essential hypertension. AB - In 40 patients with essential hypertension, enalapril was compared with propranolol as an antihypertensive agent in a double-blind study. The patients were randomly given either enalapril 5-10-20 mg bid or propranolol 40-80-120 mg bid in a treatment consisting of step-by-step increases in dosage. When the diastolic blood pressure remained greater than 90 mm Hg on the highest dosage, hydrochlorothiazide was added. Both enalapril and propranolol reduced blood pressure, although the patients tended to achieve lower blood pressures while on enalapril. More patients on propranolol required additional diuretic therapy than patients on enalapril. Propranolol reduced heart rate; with enalapril there were no changes in heart rate. Both drugs increased serum potassium and urea. Plasma renin substrate was reduced by enalapril, but raised by propranolol. Enalapril increased plasma renin activity and angiotensin I, while propranolol reduced both. Converting enzyme activity was lowered with enalapril but was unchanged with propranolol. Both drugs reduced angiotensin II. Plasma aldosterone concentration was more suppressed with propranolol than with enalapril. PMID- 3007164 TI - In vitro effect of fansimef on human neutrophil and monocyte function. AB - The effect of Fansimef, a recently registered triple combination of mefloquine, sulfadoxine and pyrimethamine (10:25:1) on human peripheral blood polymorphonuclear and mononuclear leucocyte function was studied. At clinically obtainable concentrations Fansimef had no effect on leucocyte viability, it did inhibit neutrophil chemiluminescence and it had no effect on other major functions, such as chemotaxis, NBT reduction, superoxide production, phagocytosis and the bactericidal activity of phagocytic cells. PMID- 3007165 TI - Phorbol ester enhances both interleukin 2 receptor expression and immunoglobulin secretion in human Epstein-Barr virus-immortalized B cells. AB - The enhanced expression of interleukin 2 (IL2) receptors by 12-O tetradecanoylphorbol 13-acetate (TPA) on sublines of an Epstein-Barr virus immortalized human B17 B cell line (M. Steinitz et al., Immunobiology 1979. 156: 41.) was studied by immunofluorescence using the anti-Tac monoclonal antibody and by binding studies with purified radiolabeled IL2. These studies show that TPA at a final concentration of 5 ng/ml greatly increased Tac antigen expression on a number of sublines of B17. IL2-binding studies revealed that TPA induced an increase in not only the number of IL2 receptors per cell but also the affinity of the receptors for IL2. The number and affinity of IL2 receptors on the C76 subline treated with TPA appear to be similar to those of activated normal human peripheral T cells. Furthermore, TPA-induced differentiation of these B cell lines was measured by induction of immunoglobulin secretion using an enzyme linked immunosorbent assay. The capacity of TPA to induce differentiation in human B cells and the biological significance of IL2 receptor expression by activated B cells are discussed. PMID- 3007166 TI - A protective human monoclonal IgA antibody produced in vitro: anti-pneumococcal antibody engendered by Epstein-Barr virus-immortalized cell line. AB - Human lymphocytes that produce anti-pneumococcal antibodies were separated and immortalized by Epstein-Barr virus and then cloned. One clone (NAD-Sel) produces an IgA, kappa antibody which is specific for the polysaccharides of type 8 pneumococcus, while not reactive with any of the polysaccharides derived from 24 other pneumococcal strains. The antibody, which is present in the cell supernatant as monomer and polymer, binds to protein A and does not fix complement. When incubated in vitro with type 8 pneumococci, it induces direct killing and increases the opsonization of these bacteria by mouse macrophages. PMID- 3007167 TI - Induction of murine T cell lymphoma expressing specific cytotoxic activity. AB - The present work reports the establishment of an antigen-specific cytotoxic T cell lymphoma line after immortalization with a murine leukemia virus. Lymph node cells from mice bearing a transplanted syngeneic MCA sarcoma were infected in vitro with radiation leukemia virus and injected intrathymically into cogeneic recipient mice. Some lymphomas of donor origin were established as permanent continuous cell lines in vitro. One of them, NS8, expressed Thy-1.2, Lyt-1, Lyt-2 and peanut agglutinin surface markers. These cells were cytotoxic in vitro for the tumor cell line corresponding to the immunizing MCA sarcoma. No significant cytolytic activities against other syngeneic or allogeneic sarcomas or lymphomas, nor against the mastocytoma P815 were observed. After several months of in vitro propagation, the specificity of the cytotoxic activity had degraded and the level of Lyt-2 or peanut agglutinin receptors dropped. These characteristics were restored with a single in vivo passage. Thus, murine leukemia virus can be used to immortalize antigen-specific cytotoxic T cells. PMID- 3007169 TI - Clonidine-evoked selective P1-purinoceptor antagonism of contraction of guinea pig urinary bladder. AB - The effect of clonidine on P1- and P2-purinoceptors of guinea-pig urinary bladder was compared to that of alpha, beta-methylene ATP, a selective P2-purinoceptor desensitizer. After, alpha, beta-methylene ATP, 10 microM, vesical contraction produced by ATP was eliminated while that caused by acetylcholine was unaffected. Clonidine, however, failed to antagonize ATP-induced contraction of the segment even at 100 microM. Electrically evoked contraction of the bladder was partly attenuated by 0.3 microM atropine and the remainder was markedly reduced by 3-30 microM alpha, beta-methylene ATP, suggesting an important role of ATP as an excitatory transmitter in this tissue. This stimulus-evoked contraction was also suppressed by adenosine, a P1-purinoceptor agonist, in a concentration-dependent fashion, and the suppression was greatly antagonized by 50 microM clonidine. These results suggest that the antagonistic property of clonidine is substantially selective for presynaptic P1-purinoceptors in contrast with that of alpha, beta-methylene ATP for postsynaptic P2-purinoceptors. PMID- 3007168 TI - Repeated treatment with clenbuterol produces desensitization of rat brain beta- and alpha 2-adrenoceptors without changes of alpha 1-adrenoceptors. AB - The effects of single and repeated administrations of the beta-agonist, clenbuterol (0.5 mg/kg i.p., twice daily for 4 or 7 days) were measured in the open-field test as responsiveness of rats to beta- (clenbuterol), alpha 2 (clonidine) and alpha 1-(phenylephrine) adrenergic stimulation. Furthermore, the effects of such treatment on beta- and beta 1-adrenoceptor binding in the rat brain cortex were studied. Repeated administration of clenbuterol failed to change the exploratory activity, in contrast to the acute sedative effect. Repeated treatment with clenbuterol resulted in a decrease in [3H]dihydroalprenolol binding to cortical beta-adrenoceptors. A single dose of clenbuterol reduced the clonidine-induced sedation and repeated treatment of clenbuterol abolished this sedation. Clenbuterol did not affect the action of phenylephrine nor did it change the binding of [3H]prazosin. These results indicate that repeated administration of a beta-agonist produces a rapid appearance of beta- and alpha 2-adrenoceptor subsensitivity, which it not followed by alpha 1-adrenoceptor changes. PMID- 3007170 TI - Stereotyped behavior correlates better than ataxia with phencyclidine-receptor interactions. AB - The interaction of phencyclidine, dexoxadrol, their analogs and stereoisomers with phencyclidine receptors was compared to their ability to induce stereotyped behavior and ataxia after i.c.v. administration to rats. The order of potency for binding to phencyclidine receptors revealed that among the stereoisomers of phencyclidine derivatives, the (+) isomer was more potent than the (-) isomer. A similar order of potency of phencyclidine derivatives and degree of stereoselectivity was seen in the assays for stereotyped behavior and phencyclidine receptor interactions, which resulted in a good correlation between the relative potencies for binding to phencyclidine receptors and inducing stereotyped behavior. However, the order of potency for induction of ataxia and the stereoselectivity was different than that seen in the assays for phencyclidine receptor interactions and stereotyped behavior. A comparison of relative potencies for binding to phencyclidine receptors to induction of ataxia still resulted in a good fit to a straight line, but the line did not intersect the origin, indicating that a non-phencyclidine receptor component is also involved in mediating ataxia. Dextrorphan and 2-methyl-3,3-diphenyl-3-propylamine were equipotent as phencyclidine in phencyclidine receptor and behavioral assays. The order of potency for interacting with phencyclidine receptors and inducing phencyclidine-like behavior by the isomers of cyclazocine were opposite to that of other phencyclidine analogs. Also, the order of potency for induction of ataxia by the isomers of N-allylnormetazocine was opposite to that for phencyclidine receptor interactions. Ethylketocyclazocine did not induce any stereotyped behavior or ataxia, indicating that it is unlikely that a kappa opioid receptor interaction plays a role in mediating ataxia. Furthermore, stereotyped behavior and ataxia were not due to interactions with mu, kappa or delta opioid receptors as naloxone did not antagonize the behavioral effects of phencyclidine, (-)cyclazocine or (-)N-allylnormetazocine. These results indicate that stereotyped behavior is mediated by phencyclidine receptors, whereas ataxia is mediated by more than just phencyclidine receptors. PMID- 3007171 TI - Changed sensitivity of alpha 2-adrenoceptors mediating a decrease in protein kinase inhibitor activity in the brain of vasopressin-hypertensive rats. AB - Clonidine produced an increase of cGMP content and a decrease of the endogenous type II inhibitor of protein kinase in rat hypothalamic slices. When administered to rats, the effect of clonidine on type II inhibitor activity in the hypothalamus and brain-stem depended on the dose. Low doses (10-50 micrograms X kg-1 i.p.) produced an increase, probably by stimulating presynaptic alpha 2 adrenoceptors, whereas large doses (200-1000 micrograms X kg-1 i.p.) produced a decrease of type II inhibitor activity by stimulating postsynaptic receptors. The development of vasopressin hypertension was associated with a gradual reduction of the response of the type II inhibitor to low and high doses of clonidine. In vasopressin-hypertensive rats neither small nor large doses of clonidine were able to induce changes in type II inhibitor activity suggesting subsensitivity of pre- and postsynaptic alpha 2-adrenoceptors. However, clonidine appeared to be equally effective in blocking electrically stimulated [3H]noradrenaline release from hypothalamic slices of vasopressin-hypertensive and control, normotensive rats. Reduced reactivity of postsynaptic alpha 2-adrenoceptors seems to be of great importance since treatment of vasopressin-hypertensive rats with 6 hydroxydopamine resulted in a decrease of blood pressure and reappearance of the sensitivity of postsynaptic alpha 2-adrenoceptors to clonidine. PMID- 3007172 TI - Cyclic GMP as the mediator of molsidomine-induced vasodilatation. AB - The mode of action of the in vitro active metabolites SIN-1 and SIN-1A of the vasodilator prodrug molsidomine was studied in bovine coronary artery strips. Both compounds increased cyclic GMP levels in close association with, but prior to their relaxing action. Relaxation and rises in cyclic GMP by SIN-1 were potentiated by M & B 22,948, an inhibitor of cyclic GMP phosphodiesterase and attenuated by methylene blue, a dye that inhibits activation of guanylate cyclase by SIN-1 and various nitrovasodilators. A single significant correlation between rises in cGMP and relaxation was obtained for both SIN compounds and various nitrovasodilators. Relaxation by SIN-1A was independent of the presence of endothelium and was not affected by various inhibitors of arachidonic acid metabolism. In contrast to nitroglycerin, SIN-1 did not induce substantial tolerance nor were its actions reduced in artery strips that were tolerant to nitroglycerin. The results indicate that SIN-1A relaxes coronary smooth muscle by a direct stimulant effect on soluble guanylate cyclase in vascular smooth muscle cells. PMID- 3007174 TI - D-1 dopamine receptor stimulation elevates plasma prolactin levels. AB - SKF 38393, a D-1 dopamine receptor agonist, produced dose-dependent elevations in plasma prolactin concentrations. Following the administration of SCH 23390, a D-1 antagonist, plasma prolactin concentrations tended to decrease; and low doses of SCH 23390 completely prevented the SKF 38393-induced elevations in plasma prolactin. These observations suggest that D-1 receptor stimulation can promote prolactin secretion. PMID- 3007173 TI - Evidence for prejunctional GABAB receptors mediating inhibition of ovarian follicle contraction induced by nerve stimulation. AB - The motor effects of gamma-aminobutyric acid (GABA) on the bovine ovarian follicle were studied in vitro using strips from follicle walls. Electrical field stimulation of nerves in the preparation, secured by tetrodotoxin blockade, caused a contraction that was almost totally abolished by phentolamine and only slightly affected by atropine. This mainly adrenergic neurogenic response was inhibited by GABA in a dose-dependent way. The GABAA-receptor antagonists, bicuculline and picrotoxin, did not affect the GABA action whereas the GABAB receptor antagonist, homotaurine, significantly inhibited the GABA effect. The GABAA-receptor agonist, muscimol, did not affect the contractile response while the GABAB-receptor agonist, baclofen, imitated the action of GABA. On the other hand, GABA had no direct contractile or relaxing effect on the follicle strips nor did it interfere with the contractile response induced by noradrenaline or acetylcholine. The findings suggest that activation of prejunctional GABAB receptors inhibits transmitter release from mainly adrenergic nerves associated with the follicle, thereby affecting nerve-mediated tension in the follicle wall. PMID- 3007175 TI - Classification of presynaptic opioid receptors in the rabbit ear artery by competitive antagonists. AB - Electrical field stimulation (5 impulses at 5 Hz every min) evoked vasoconstriction in the rabbit isolated ear artery. The vasoconstriction was depressed by [Leu5]enkephalin (IC50 = 47 nM) and ethylketocyclazocine (IC50 = 80 nM). Naloxone 300 nM antagonized the effect of both agonists with a similar potency; the respective KB values were 41 and 64 nM. ICI 174864 1000 nM antagonized only the effect of [Leu5]enkephalin (KB = 126 nM) but not that of ethylketocyclazocine. In contrast, MR 2266 100 nM was a more potent antagonist of ethylketocyclazocine (KB = 8 nM) than of [Leu5]enkephalin (KB = 45 nM). The results suggest that the rabbit ear artery contains both presynaptic delta- and kappa-receptors. PMID- 3007176 TI - Forskolin effects on longitudinal myometrial strips from the pregnant rat: relationship with membrane potential and cyclic AMP. AB - The effects of forskolin on tension, membrane potential and cyclic AMP accumulation were studied in longitudinal myometrial strips from pregnant rats. 0.1 microM forskolin reduced the amplitude of spontaneous contractions by decreasing the frequency of action potential discharge without a change in resting potential or cyclic AMP accumulation. Forskolin, 1.0 microM, abolished contractions and action potentials, hyperpolarized the membrane and increased cyclic AMP accumulation. Ouabain, 1 mM, depolarized the muscle and increased resting tension. Ouabain reduced potential change produced by forskolin but did not prevent the relaxation or cAMP accumulation. Therefore changes in membrane potential are not prerequisite for the inhibitory actions of forskolin. The cyclic AMP-related relaxation may result primarily from intracellular events that remove calcium from the contractile elements. PMID- 3007177 TI - Beta 1-adrenoceptor antagonism by urapidil prior to and after the alpha 2 antagonist rauwolscine in anesthetized dogs. AB - The purpose of this investigation was to evaluate the in vivo beta-adrenoceptor antagonistic properties of urapidil, a new antihypertensive alpha 1-adrenoceptor blocking agent. In dogs anesthetized with pentobarbital, the cardioaccelerator nerve was isolated, decentralized and stimulated electrically to elicit adrenergically mediated increases in heart rate. Dobutamine was administered (3 30 micrograms/kg i.v.) to elicit increases in heart rate by the direct stimulation of beta-adrenoceptors. Urapidil 2 mg/kg i.v. had no significant effect on cardioaccelerator nerve-induced tachycardia, but 5 mg/kg i.v. decreased the response by 27%. Heart rate responses to dobutamine were suppressed slightly by the low dose of urapidil and to a greater degree by the high dose of urapidil, whereas the pressor response to dobutamine was markedly attenuated at both dose levels. Neurally or dobutamine-elicited tachycardia remaining after urapidil treatment was eliminated by propranolol (1 mg/kg i.v.). When the selective alpha 2-antagonist rauwolscine was administered (1000 micrograms/kg i.v.), tachycardic responses to nerve stimulation increased 49% above control, and urapidil (5 mg/kg) given subsequently, caused a 48% reduction in the response. These data indicate that urapidil has weak beta 1-blocking activity more clearly seen after blockade of alpha 2-adrenoceptors. PMID- 3007178 TI - Accumulations of inositol phosphates and cyclic AMP in brain slices: synergistic interactions of histamine and 2-chloroadenosine. AB - 2-Chloroadenosine, 5'-N-ethylcarboxamidoadenosine, N6-cyclohexyladenosine and other adenosine analogs enhance histamine-elicited, but not norepinephrine- or carbamylcholine-elicited accumulations of inositol phosphates in [3H]inositol labeled guinea-pig cerebral cortical slices. The adenosine analogs alone have no effect on accumulations of inositol phosphates. The effect of 2-chloroadenosine is blocked by the adenosine receptor antagonists theophylline and 1,3-dialkyl-8-p sulfophenylxanthines. The rank order of activity of the six adenosine analogs with respect to augmentation of histamine-elicited accumulation of inositol phosphates in guinea-pig cerebral cortical slices is different from the rank order at an A2-adenosine receptor that mediates synergistic accumulations of cyclic AMP by adenosine analogs and histamine in guinea-pig cerebral cortical slices. PMID- 3007179 TI - Converting enzyme inhibitor-induced changes of plasma angiotensinogen concentration in the rat. AB - Chronic treatment with converting enzyme inhibitors induces a fall of plasma angiotensinogen concentration, which is thought to result from increased consumption by renin. As this explanation cannot account for all the observations, we reexamined this effect. Rats received captopril (50 mg/kg per day), enalapril (10 mg/kg per day) or Hoe 498 (1 mg/kg per day) for 7 days. Plasma angiotensinogen (by indirect assay) fell to 34, 37 and 43% of its initial values, respectively. Total immunoreactive angiotensinogen (by direct radioimmunoassay, which also measures des-AI-angiotensinogen) was 76, 70 and 95% of the initial values, respectively. This suggests that a major part of the fall of intact angiotensinogen can be attributed to increased cleavage by renin, but an additional factor is likely to be involved. Experiments with isolated hepatocytes indicated that converting enzyme inhibitors in high concentrations may have a direct effect on angiotensinogen secretion. Whether this can account for the fall in total immunoreactive angiotensinogen in vivo remain unclear. PMID- 3007180 TI - Stage and tissue-specific expression of a collagen gene during Drosophila melanogaster development. AB - Based on data from developmental RNA profiles and in situ hybridization, we report a direct examination of the expression of one collagen gene (Dcg1) during drosophila melanogaster life cycle. These studies show, for the first time, that the expression of a collagen gene is both differential and tissue-specific during the course of development. Moreover, they demonstrate that the connective tissues in Drosophila do contain a collagen type synthesized by mesodermal tissues. Indeed the accumulation of Dcg1 transcripts was located mainly within the second instar fat bodies, the third instar lymph glands, and over adepithelial cells associated with third instar imaginal discs. In addition, these results seem to confirm the interpretation that wandering hemocytes released by the lymph glands could contribute in extracellular matrix composition in some tissues in the larva. PMID- 3007181 TI - Possible role of calpain I and calpain II in differentiating muscle. AB - The variable distribution of the 80-kD subunit of two calcium-activated proteases, calpain I and calpain II, has been examined in L8 and L6 myoblasts, and their non-fusing variants, fu-1 and M3A using non-cross-reacting monoclonal antibodies to both subunits. Immunofluorescence results have shown that while the 80-kD subunit of calpain I is localized in the cytoplasm of all the myoblasts, the 80-kD subunit of calpain II appears to be predominantly associated with the plasma membranes of L8 and L6 myoblasts. The distribution of the 80-kD subunit of calpain II in non-fusing myoblasts, fu-1 and M3A, is generally cytoplasmic and diffuse. Immunoblot analysis of membrane and cytosol fractions of all the myoblasts using the monoclonal antibodies described above essentially confirms the immunofluorescence findings. Because calpain II exhibits a peripheral distribution in cells which are fusion-competent, L6 and L8 myoblasts, but not in fu-1 and M3A myoblasts, we suggest that calpain II may play a role in the Ca2+ mediated fusion events of differentiating (prefusion) myoblasts. PMID- 3007182 TI - Protein phosphorylation and oocyte maturation. I. Induction of starfish oocyte maturation by intracellular microinjection of a phosphatase inhibitor, alpha naphthylphosphate. AB - Oocyte maturation (meiosis re-initiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Following hormonal stimulation of the oocyte, an intracellular Maturation Promoting Factor (MPF) appears in the cytoplasm which triggers nuclear envelope breakdown and maturation divisions. alpha Naphthylphosphate (alpha-NP), a widely used phosphatase inhibitor/substrate, was found to induce oocyte maturation when microinjected intracellularly (50% maturation of 3.5 mM; 100% above 6mM, final intracellular concentration) into oocytes of Marthasterias and Asterias but not of Astropecten. As 1-MeAde, alpha NP triggers a complete maturation, i.e. germinal vesicle breakdown, extrusion of the two polar bodies and formation of the female pronucleus. The kinetics of alpha-NP-induced maturation (35-45 min) is, however, longer than the kinetics of 1-MeAde-induced maturation (18-20 min). The addition of alpha-NP externally to oocytes does not trigger maturation. Among several reported phosphatase inhibitors, including two natural protein phosphatase inhibitors and several products structurally related to alpha-NP, only alpha-NP was found capable of inducing maturation when microinjection into oocytes. alpha-NP triggers the appearance of MPF activity in the cytoplasm of oocytes into which it has been injected. Although alpha-NP-induced maturation is insensitive to inhibitors whose action is known to be restricted to the hormone-dependent period (such as the protease inhibitor leupeptin), it is blocked by inhibitors of MPF action (such as nicotinamide and lithium). Finally it was found that alpha-NP-induced maturation is inhibited by simultaneous microinjection of protein phosphatase-2A; also, alpha-NP, classically used as an inhibitor of acid and alkaline phosphatases, is able to inhibit protein phosphatases, is able to inhibit protein phosphatases 1 and 2 A. The addition of alpha-NP to oocytes increases the level of phosphorylated proteins. These results constitute direct evidence that an elevated level of phosphorylated proteins is sufficient to trigger MPF activity and to induce maturation. PMID- 3007183 TI - Protein phosphorylation and oocyte maturation. II. Inhibition of starfish oocyte maturation by intracellular microinjection of protein phosphatases 1 and 2A and alkaline phosphatase. AB - Oocyte maturation (meiosis re-initiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Following hormonal stimulation of the oocyte, an intracellular Maturation Promoting Factor (MPF) appears in the cytoplasm which triggers nuclear envelope breakdown and maturation divisions. Microinjection of pure preparations of the catalytic subunits of protein phosphatases 1 and 2A inhibits 1-MeAde-induced maturation in a dose-dependent manner. Calmodulin dependent protein phosphatase 2B is inefficient. Maturation induced by mimetics of 1-MeAde, such as dithiothreitol (DTT), methylglyoxal-bis(guanylhydrazone) (MGBG), 8-hydroxyeicosatetraenoic acid (8 HETE) and arachidonic acid (AA) is also inhibited by these protein phosphatases. In all cases inhibition can be reversed by increasing the concentration of 1-Me-Ade or of mimetic. Alkaline phosphatase also inhibits maturation in a dose-dependent way and in a reversible manner. Microinjection of protein phosphatase is still effective when preformed long after the end of the hormone-dependent period, and can even be effective a few minutes before the breakdown of the nuclear envelope. No detectable MPF activity is found in 1-MeAde-treated phosphatase-injected oocytes. However, microinjection of phosphatase 2A simultaneously with MPF (obtained from 1-MeAde-treated donors) does not result in inhibition. These results constitute direct evidence for the necessity of an elevated level of phosphorylated proteins for MPF activity and maturation. The mode of action of 1-MeAde in inducing starfish oocyte maturation is discussed in relation to protein phosphorylation. PMID- 3007184 TI - An expressed beta-tubulin gene, TUBB, is located on the short arm of human chromosome 6 and two related sequences are dispersed on chromosomes 8 and 13. AB - The chromosomal assignments of an expressed beta-tubulin gene and two related sequences have been determined by Southern blot analysis of DNA from a panel of human x Chinese hamster somatic cell hybrids cleaved with Hind III or EcoRI. Probes containing the 3' untranslated regions of the expressed gene M40 and of pseudogene 21 beta were used to localize the M40 sequence (gene symbol TUBB) to chromosome 6 region 6p21----6pter, the 21 beta pseudogene (TUBBP1) to chromosome 8 region 8q21----8pter and a third related sequence (TUBBP2) to chromosome 13. Asynteny of expressed genes and related processed pseudogenes has now been demonstrated for several gene families. PMID- 3007186 TI - SV40 large T-antigen and transformation related protein p53 are associated in situ with nuclear RNP structures containing hnRNA of transformed cells. AB - The localization of SV40 large T-antigen (T-Ag) and the cellular protein p53 in the nuclei of mouse and human SV40-transformed cells and of a methylcholanthrene transformed mouse cell line, was studied. Their detection by ultrastructural immunocytochemistry with specific monoclonal antibodies employed two complementary methods used in parallel. These consisted of indirect immunoperoxidase labelling carried out before embedment on Triton-permeabilized cells, or indirect immunogold labelling applied to thin sections of cells embedded in Lowicryl K4M. The results indicate that in SV40-transformed cells both proteins are chiefly localized on peri- and interchromatin RNP fibrils. This shows that they occur in structures involved in the synthesis and processing of hnRNA. The nucleoli and chromatin did not appear to be labelled. In methylcholanthrene-transformed cells the protein p53 (in the absence of large T Ag) was also detected on peri- and interchomatin fibrils. Taken together with recent results which demonstrated that, during lytic infection, T-Ag was associated chiefly with cellular chromatin (Harper, F, Florentin, Y & Puvion, E, Exp cell res 161 (1985) 434) [33], our experiments provide evidence that the transforming function of SV40 large T-Ag is dissociable from its function in SV40 lytic infection in terms of its subnuclear distribution. PMID- 3007187 TI - Primitive series embryonic chick erythrocytes express the transferrin receptor. AB - A monoclonal antibody specific for the chicken transferrin receptor was used to study receptor expression on circulating red cells from chick embryos of different ages. The use of indirect immunofluorescence with this antibody showed that all circulating immature reticulocytes and primitive series erythrocytes- but not erythrocytes from the definitive series--expressed the receptor. In all cells, the protein was synthesized as a 90-95-kD form. The retention of the transferrin receptor (and another proliferation-dependent cell surface protein) contrasted with the behaviour of a series of other developmentally regulated antigens which are lost during maturation of both primitive and definitive series erythroid cells. PMID- 3007185 TI - Epidermal growth factor, its receptor, and related proteins. AB - An interesting aspect of the developments forthcoming from the study of cell growth control by growth factors is the structural and functional homologies that have been found to exist between growth factors or their receptors and other molecules. The epidermal growth factor (EGF) system has been particularly fruitful in this regard. The information in this article is meant to summarize the relationships that have been recently described between EGF and other EGF like molecules and between the EGF receptor and related macromolecules. PMID- 3007188 TI - Generation of lipoxygenase products in the avascular tissues of the eye. AB - We have examined the ability of the avascular cornea and lens to generate lipoxygenase products from arachidonic acid. The cornea and lens were obtained from rabbit and frog eyes, and their ability to metabolize exogenous [14C] arachidonic acid determined in vitro. The cornea of rabbits and frogs formed lipoxygenase products from arachidonic acid. Both rabbit and frog corneas were found to generate considerable quantities of 12-HETE, although other lipoxygenase products were also produced. In contrast, no lipoxygenase products were detected following incubations of rabbit or frog lens with arachidonic acid. Thus, the cornea and lens, the two avascular tissues in the eye, exhibit marked differences in their ability to metabolize arachidonic acid via lipoxygenase enzymes. PMID- 3007189 TI - Morphology, response properties, and collateral projections of trigeminothalamic neurons in brainstem subnucleus interpolaris of rat. AB - Intracellular recording, electrical stimulation and horseradish peroxidase (HRP) injection techniques were used to delineate the structural and functional characteristics of trigeminothalamic projection neurons in subnucleus interpolaris of the trigeminal brainstem nuclear complex in rat. Eleven such neurons were successfully characterized and recovered. All were medium to large multipolar neurons in the ventral part of interpolaris and all except one also projected to the superior colliculus. Six of these cells also sent axon collaterals to subnucleus principalis and the medullary parvicellular reticular formation and had local collaterals within interpolaris. None of these trigeminothalamic cells were antidromically activated from the cerebellum. All but one of the recovered cells were responsive to deflection of any one of a number (4-19) of vibrissae. The remaining cell was discharged by displacement of mystical guard hairs. Analysis of electrophysiological and anatomical data revealed significant correlations between receptive field size and dendritic area, thalamic conduction latency and axon diameter, and number of targets innervated and axon diameter. PMID- 3007190 TI - Pathways mediating descending control of spinal nociceptive transmission from the nuclei locus coeruleus (LC) and raphe magnus (NRM) in the cat. AB - We have previously reported that electrical stimulation in LC or NRM when tested on the activity of a multireceptive neurone in the spinal cord produced similar inhibitory actions. The present study aimed to define the pathways that mediate this descending inhibitory action in the spinal cord by pharmacological means and by making surgical lesions in the spinal cord or NRM. Attempts to differentiate pathways pharmacologically did not succeed since the i.v. administration of the 5 HT antagonists, methysergide and cinnanserin failed to antagonise descending inhibition evoked from either NRM or LC. Lesions involving a part or whole of the ipsilateral ventral quadrant reduced the inhibition produced from LC to a greater extent than that from NRM in 24 multireceptive neurones. In seven of these neurones stimulation in LC was without any effect after the lesion. In 23 multireceptive neurones recorded after making lesions that spared the ipsilateral ventral quadrant the effects of LC stimulation were unchanged. NRM effectiveness was reduced by an ipsilateral dorsolateral funiculus (DLF) lesion but required a bilateral DLF lesion for an almost complete abolition. Similar results were obtained when the effect of the various lesions were studied on the dorsal root potentials (DRPs) generated from LC or NRM. Lesions in the midline raphe complex, that included NRM, did not block the inhibitory action of LC stimulation. The inhibition produced from both these nuclei was additive whereas excitation was not. We conclude that LC actions in the spinal cord are mediated primarily through a pathway in the ipsilateral ventral quadrant whereas those from NRM are mediated through bilateral projections in DLF. Furthermore, although NRM plays no part in mediating LC actions and separate and independent pathways mediate their spinal action yet these apparently independent pathways have plenty of scope for interaction in the dorsal horn of the spinal cord itself. PMID- 3007191 TI - Enhancement by serotonin of tonic vibration and stretch reflexes in the decerebrate cat. AB - The effects of pharmacological manipulation of serotonergic systems on spinal reflexes were determined in the unanesthetized decerebrate cat. The prolonged motor output that continues after cessation of high frequency longitudinal tendon vibration was strongly enhanced by the serotonin reuptake blocker fluoxetine and the serotonin precursor 5-hydroxytryptophan, and was decreased by the serotonin receptor antagonist methysergide. In addition, both dynamic and static stretch reflex stiffness was markedly increased by fluoxetine and 5-hydroxytryptophan, while methysergide produced a decrease in stretch reflex stiffness. These powerful effects on tonic vibration and stretch reflexes could not be explained by drug-induced alterations in muscle spindle primary afferent discharge. In light of other recent results on serotonin-mediated effects on motoneurons, we believe that the effects of these agents result from modification of an intrinsically mediated prolonged depolarization of spinal neurons. However, the possibility that these drugs modify longlasting discharge in associated interneuronal pathways cannot be ruled out. PMID- 3007193 TI - Effects of neonatal monocular enucleation on the number of GAD-positive puncta in rat visual cortex. AB - Rats that had one eye removed on the day of birth were examined at various postnatal ages with immunocytochemical methods to determine the effect on the development of the GABAergic axonal plexus in the visual cortex. The monocular segment of visual cortex contralateral to the enucleated orbit had 20-30% fewer GABAergic axon terminals than the monocular segment of visual cortex contralateral to the normal eye. Other cortical areas did not show any significant changes. These findings suggest that sensory deprivation of the visual cortex interferes with the normal development of GABAergic neurons. PMID- 3007192 TI - Intracellular electrophysiology of CA1 pyramidal neurones in slices of the kainic acid lesioned hippocampus of the rat. AB - Intracellular recordings were made from hippocampal CA1 pyramidal cells in slices where the CA3/CA4 region had been lesioned using intracerebroventricular kainic acid. In 55% of the cells studied orthodromic excitation evoked bursts of action potentials. This bursting activity was associated with a decrease in or loss of the early phase to the hyperpolarisation which normally follows orthodromically evoked action potentials. The recurrent inhibitory post-synaptic potential produced by antidromic activation of pyramidal cells was also reduced or absent. A late phase to the orthodromic hyperpolarisation was reduced in cells from lesioned slices. However, in normal slices treated with bicuculline this potential showed an apparent increase. The afterhyperpolarisation which follows a short current evoked burst of action potentials was reduced in bursting cells from lesioned slices. In addition, a silent period in the firing pattern produced by long depolarising current pulses was reduced or absent in these cells. These results together with observations made with bicuculline suggest that the bursting activity in lesioned slices is largely due to a loss of inhibition mediated by gamma-aminobutyric acid. It is proposed that the kainic acid-lesioned in vitro hippocampus may be a suitable preparation for studying the electrophysiology of temporal lobe epilepsy. PMID- 3007194 TI - The relationship between GABA immunoreactivity and labelling by local uptake of [3H]GABA in the striate cortex of monkey. AB - An antiserum to GABA was used in the macaque monkey to determine whether neurons that accumulate exogenously applied [3H]GABA in vivo are also immunoreactive for GABA. Following the injection of [3H]GABA into different laminae of striate cortex in two untreated animals and in one animal treated with amino-oxyacetic acid, selective accumulation of the labelled amino acid was demonstrated in perikarya by autoradiography. Radiographically labelled neurons (n, 519) and their unlabelled neighbours were tested in consecutive 0.5 micron thick sections by immunocytochemistry for GABA immunoreactivity. Injection of [3H]GABA did not increase the number of neurons showing GABA immunoreactivity. On the contrary many of the cells that accumulated [3H]GABA were immunonegative. These neurons were mostly located in layers IVC and VA following [3H]GABA injection into layers II-III, and in layers upper III and II following injection into layers V and VI. A comparison of the position of these neurons with known local projection patterns in the striate cortex of monkey suggests that GABA-immunonegative neurons may nevertheless become labelled by [3H]GABA if most of their local axon terminals fall within the injection site. The interlaminar projection of GABA immunopositive neurons, which probably contain endogenous GABA, could be deduced from the position of the [3H]GABA injection site that leads to their autoradiographic labelling. Although the present study confirmed our previous results on the interlaminar connections of neurons that accumulate [3H]GABA, it demonstrated that [3H]GABA labelling alone may not be a sufficient criterion to assess the GABAergic nature of neurons in the striate cortex of monkey. PMID- 3007195 TI - Long-term exposure of cortical cell cultures to clonazepam reduces benzodiazepine receptor binding. AB - To determine if reduced drug efficacy after long-term exposure to clonazepam may be a consequence of benzodiazepine receptor alterations, cerebral cortical cell cultures were exposed to the drug (200 nM) for 14 days. Receptor binding was assayed on living cells in situ. After drug exposure, binding in experimental cultures differed markedly from controls with respect to total, specific, and clonazepam-displaceable (neuronal) benzodiazepine binding (60%, 53%, and 6% of control values, respectively) but recovered within 96 h of drug removal. RO5-4864 displaceable (nonneuronal) binding was modestly reduced at 0 time (72% of control), but returned to control values in 24 h. The differences in binding could be attributable to a relatively reduced affinity of the high-affinity binding site (Kd approximately 18 nM for controls and approximately 30 nM for drug-exposed cultures) but not to changes in the low-affinity binding site or to reduced numbers of receptors. PMID- 3007196 TI - Babesia bovis: purification and concentration of merozoites and infected bovine erythrocytes. AB - Babesia bovis merozoites, externalized by removal of infected erythrocytes from ordinary culture conditions, were completely separated from red blood cells and stroma by centrifugation in a Percoll gradient. A merozoite band formed at a point corresponding to about 1.087 g/ml specific density. Infected red blood cells were concentrated approximately fourfold to obtain greater than 49.0% parasitemia after centrifugation in Percoll. Most highly enriched fractions positioned between 1.121 and 1.123 g/ml specific density. Full parasite viability was retained. PMID- 3007197 TI - Suitability of urethane anesthesia for physiopharmacological investigations in various systems. Part 2: Cardiovascular system. AB - Urethane produces a level of surgical anesthesia characterized by preservation of a number of cardiovascular reflexes. When the proper route of administration is used, and the use of unnecessarily high doses is avoided, urethane anesthesia appears to be suitable for a number of investigations at cardiovascular level. However in certain types of studies involving pharmacological stimulation of peripheral adrenoceptors urethane affects markedly the magnitude of the response under study. PMID- 3007198 TI - Gametes contain angiotensin converting enzyme (kininase II). AB - The localization of angiotensin converting enzyme (ACE) in the gonads of the normal rabbit was studied by immunofluorescence and immunoelectron microscopy. The enzyme is present in the cytoplasm of testicular spermatids and of epididymal and ejaculated spermatozoa, and on the surface of follicular and tubal oocytes. These findings support the hypothesis that ACE has a role in gamete maturation and in fertilization. PMID- 3007199 TI - A note on the use of protease inhibitors during chromatin fractionation on hydroxyapatite columns. AB - It was found that NaHSO3 present in the eluents enabled full and reproducible recovery of chromosomal proteins from a hydroxyapatite column. Another protease inhibitor, PMSF, did not have that effect. PMID- 3007200 TI - Phospholipid composition of cardiac (Na+ + K+)-ATPases from various species. AB - There is a difference in phospholipid composition of cardiac (Na+ + K+)-ATPase preparations between species which are sensitive to ouabain and those which are not. Sphingomyelin is higher and phosphatidylcholine is lower in the enzymes from sensitive species than in those from insensitive ones. Lysophosphatidylcholine is detectable only in the latter preparations. PMID- 3007201 TI - Lithium lowers renal, cardiac and splenic ornithine decarboxylase activity in mice. AB - A single i.p. injection of lithium chloride (5-7.5 mumoles/g b.wt) in mice caused a 70-80% decrease in renal, cardiac and splenic ornithine decarboxylase (ODC) activity within 1 h, whereas pulmonary ODC activity was unaffected. Lithium chloride did not have any effect on ODC activity in vitro when added to homogenates of the tissues studied. We suggest that the effect of lithium on ODC activity is not direct, but mediated via e.g. hormonal or nervous influence. PMID- 3007203 TI - Reduced tuberoinfundibular dopaminergic neuronal function in rats after long-term withdrawal of estrogen treatment. AB - Hypothalamic fragments from female rats treated repeatedly with estradiol valerate (EV) and bearing prolactin (PRL)-secreting tumors contained, seven months after the last EV injection, lower concentrations of dopamine (DA) than age-matched controls. Depolarizing concentrations of K+ (35 mM) and amphetamine (50 microM) evoked in PRL-secreting tumor bearing rats an endogenous DA release significantly lower than in controls. PMID- 3007202 TI - VIP and histamine H2 receptor activity in human fetal gastric glands. AB - Vasoactive intestinal peptide (VIP, EC50 = 6.4 X 10(-10)M) and histamine (EC50 = 3 X 10(-6)M) activated the cyclic AMP generating system in gastric glands isolated from two human fetuses at 23 weeks gestation. Histamine antagonism by the H2 receptor blockers cimetidine (Ki = 0.35 X 10(-6)M) and ranitidine (ki = 0.51 X 10(-7)M) clearly characterized the histaminic activation as being of the H2 type. It is suggested that these two vasoactive hormones may operate as neurocrine/paracrine regulators of the differentiation and/or function of the human gastric mucosa in utero. PMID- 3007204 TI - A daily rhythm in hCG binding to ovarian follicles of the cyclic hamster. AB - On each day of the estrous cycle hCG binding to follicle increased from 09.00 to 21.00 h; then hCG binding was static until 09.00 h of the next day. FSH binding did not exhibit rhythmicity. This pattern of hCG binding may be related to the pulsing of LH on each cycle day. PMID- 3007205 TI - Acetylcholine receptors in the gastrulating chick embryo. PMID- 3007206 TI - Two-electron reduction of cytochrome c oxidase triggers a conformational transition. AB - The slow increase of a cyanide-induced optical change at 437 nm following rapid cyanide inhibition of cytochrome oxidase has been followed as a function of the number of electrons donated from ferrocytochrome c to cytochrome a and CuA. The initial rate of optical change is a parabolic function of this number. The results have been analyzed in terms of a model where addition of electrons causes a conformational transition allowing rapid cyanide binding. The binding is followed by a slow intramolecular responsible for the optical change. The analysis demonstrates that only molecules with both cytochrome a and CuA reduced can undergo the conformational change, which is suggested to be involved in the proton-pump mechanism of the oxidase. PMID- 3007207 TI - In vitro study of the interaction of the LexA repressor and the UvrC protein with a uvrC regulatory region. AB - The in vitro interaction of the LexA repressor with a regulatory region of the uvrC gene has been studied by polyacrylamide gel electrophoresis. Although the uvrC promoter region shows some homology with the canonic LexA binding site, no specific binding of the repressor to this DNA sequence could be observed, but only a cooperative nonspecific binding. By the same technique we show that the UvrC protein does not bind specifically to this regulatory DNA sequence either, although the protein is able to bind nonspecifically and cooperatively to the double-stranded DNA fragment. PMID- 3007208 TI - Influence of lipid peroxidation on beta-adrenoceptors. AB - The peroxidation of lipids in biological membranes is a destructive phenomenon that can be elicited in various ways. Surface receptor molecules that allow cells to respond to hormones are possibly inactivated during lipid peroxidation. Effects of lipid peroxidation on receptors have not been extensively examined thus far. This investigation shows that there is a decrease in beta-adrenoceptor density (measured as specific (-)-[125I]iodocyanopindolol binding) during lipid peroxidation, in both lungs and erythrocytes of the rat. To this end, lung membranes (containing both beta 1- and beta 2-adrenoceptors) and intact erythrocytes (containing a homogeneous beta 2-adrenoceptor population) were pretreated with cumene hydroperoxide (lung membranes with 0.1 mM and erythrocytes with 1 mM) and Fe2+ (1 X 10(-5) M) for 60 min which resulted in extensive lipid peroxidation measured as malondialdehyde formation. The ration beta 1-:beta 2 adrenoceptor density in lung membranes after treatment with cumene hydroperoxide did not change and remained at 30%:70%. A single injection (i.p.) with the herbicide paraquat (50 mg/kg, 24 h), which is known to cause lung damage via lipid peroxidation, resulted in similar alterations in receptor density to those caused by cumene hydroperoxide in the in vitro experiments. PMID- 3007210 TI - Intracellular protein catabolism: state of the art. PMID- 3007209 TI - Phospholipase C in rat liver plasma membranes. Phosphoinositide specificity and regulation by guanine nucleotides and calcium. AB - Phospholipase C activity against phosphoinositides in isolated rat liver plasma membranes has been examined using exogenous substrates. The enzyme hydrolyzed phosphatidylinositol 4,5-bisphosphate 30-40-times faster than phosphatidylinositol 4-monophosphate, while phosphatidylinositol was not a substrate. Maximum activity was observed with 1.1 mM phosphatidylinositol 4,5 bisphosphate at pH 5.0. The enzyme was stimulated by micromolar concentrations of Ca2+. The GTP analogue guanylyl (beta,gamma-methylene)diphosphonate enhanced phospholipase C activity at and above 0.3 microM Ca2+, but was inhibitory at 0.1 microM Ca2+. This supports the suggestion that plasma membrane phospholipase C is regulated by guanine nucleotide-binding protein, but indicates a regulatory mechanism different from that of other enzymes regulated by such proteins. PMID- 3007211 TI - Phosphorylase a is an allosteric inhibitor of the glycogen and microsomal forms of rat hepatic protein phosphatase-1. AB - The dephosphorylation of glycogen synthase by protein phosphatase-1 in hepatic glycogen and microsomes was inhibited by nanomolar concentrations of phosphorylase a. The I50 for phosphorylase a was 1000-fold lower than its Km as a substrate, while tryptic digestion increased the I50 1000-fold without affecting Km. Protein phosphatase-1 from skeletal muscle and protein phosphatase-2A from liver were only inhibited at 1000-fold higher concentrations. Protein phosphatase 1 became desensitized to phosphorylase a when released from hepatic microsomes, but sensitivity was partially restored by readdition of the solubilized enzyme to the microsomes. The results demonstrate that phosphorylase a is a potent allosteric inhibitor of hepatic protein phosphatase-1 and suggest that inhibition may be conferred by a novel phosphorylase a-binding subunit. PMID- 3007212 TI - Treatment of rabbit neutrophils with phorbol esters results in increased ADP ribosylation catalyzed by pertussis toxin and inhibition of the GTPase stimulated by fMet-Leu-Phe. AB - The effects of pretreatment of rabbit neutrophils with phorbol 12-myristate 13 acetate on the ability of pertussis toxin to catalyze ADP-ribosylation and of fMet-Leu-Phe to activate a high-affinity GTPase in these cell homogenates were examined. The addition of phorbol 12-myristate 13-acetate, but not 4 alpha phorbol 12,13-didecanoate, to intact cells was found to stimulate by more than 100% the pertussis toxin-dependent ribosylation of a 41 kDa protein (either the alpha-subunit of the 'inhibitory' guanine nucleotide-binding protein N or a closely analogous protein) and to inhibit by more than 60% the activation by fMet Leu-Phe of the GTPase of the neutrophil homogenates. The addition of fMet-Leu-Phe to intact cells increases the ADP-ribosylation catalyzed by pertussis toxin of the 41 kDa protein. On the other hand, the exposure of neutrophil homogenates to fMet-Leu-Phe results in a decreased level of ADP-ribosylation. This decreased ribosylation reflects a dissociation of the GTP-binding protein oligomer that is not followed by association, possibly because of the release of the alpha-subunit into the suspending media. The implications of these results for the understanding of the mechanism of inhibition of cell responsiveness by phorbol esters and the heterologous desensitization phenomenon are discussed. Prominent among these are the possibilities that (i) the rate of dissociation of the Ni oligomer is affected by the degree of its phosphorylation by protein kinase C, and/or (ii) the dissociated phosphorylated alpha-subunit (the 41 kDa protein) is functionally less active than its dephosphorylated couterpart. PMID- 3007213 TI - An osmotic model for the fusion of biological membranes. AB - A molecular model for fusion-fission reactions in membranes is proposed that is based on data from studies on artificially induced cell fusion and on the behaviour of phospholipid bilayers: it is put forward as a framework for further investigations into this fundamental property of biological systems. PMID- 3007214 TI - Atrial natriuretic factor regulation of cyclic GMP levels and steroidogenesis in isolated fasciculata cells of rat adrenal cortex. AB - Isolated fasciculata cells of rat adrenal cortex, when incubated with atrial natriuretic factor (ANF), stimulated the levels of cyclic GMP and corticosterone production in a concentration-dependent manner without a rise in the levels of cyclic AMP. The ANF-dependent elevation of cyclic GMP was rapid, with a detectable increment in 30 s. ANF also stimulated the particulate guanylate cyclase. These results not only indicate the coupling of cyclic GMP and corticosterone production with ANF signal, but also demonstrate that, like the ACTH signal, cyclic AMP is not the mediator of ANF-induced adrenocortical steroidogenesis. PMID- 3007215 TI - Characterisation of a high-affinity VIP receptor in human lung parenchyma. AB - A method is described for preparing human lung parenchymal membranes essentially free of carbon contamination. Using this technique, a high-affinity 125I-VIP binding site has been characterised. The receptor density is approx. 200 fmol/mg protein, and the Kd of 125I-VIP by saturation binding is 200 pM. The dissociation kinetics are complex and cannot be described by first-order kinetics. Several VIP related peptides displace 125I-VIP from this binding site with a rank order of potency: VIP greater than rat GRF greater than PHM greater than PHI greater than human GRF greater than secretin greater than glucagon. Displacement curves of these peptides exhibited slope factors significantly less than unity with the exception of human GRF. PMID- 3007216 TI - Isolation of the haemopexin-haem receptor from pig liver cells. AB - Isolated pig liver plasma membranes interact specifically with the haemopexin haem complex (Kd 4.4 X 10(-7) M). Affinity chromatography was used to isolate a membrane component which binds this complex with high affinity. Pig serum haemopexin was first isolated by affinity chromatography on haemin-Sepharose followed by HPLC gel filtration. Liver membranes solubilized with Triton X-100 were incubated with haemin-Sepharose saturated with haemopexin, and as a control, with affinity gel lacking haemopexin. SDS-poly-acrylamide gel electrophoresis of the eluted protein indicated that from the haemin-Sepharose emerglow-molecular mass haemin-binding proteins whereas the eluate from haemopexin-haemin-Sepharose contained an additional 71 kDa protein, which did not bind free haemin. This protein appears to represent the haemopexin-haem receptor or a part of it. Haem from the haemopexin complex, as also free haemin, was accepted by a binder in the plasma membrane, which in gel filtration behaved like an 80 kDa molecule. This component probably represents a second functional subunit of the haemopexin-haem receptor. PMID- 3007217 TI - Ferritin, a physiological iron donor for microsomal lipid peroxidation. AB - In the process of lipid peroxidation of microsomes induced either by oxygen radicals generated by xanthine oxidase or by NADPH, ferritin is able to donate the necessary iron. The amount of ferritin necessary to catalyze the process of lipid peroxidation is in the physiological range. In contrast to the finding with phospholipid liposomes, catalase hardly stimulates the lipid peroxidation of microsomes. PMID- 3007218 TI - Retroperitoneal lymphadenectomy in clinical stage I nonseminomatous germinal testis cancer. AB - Thirty-six consecutive patients underwent retroperitoneal lymphadenectomy for clinical stage I nonseminomatous germinal testis cancer from January 1980 to August 1981. Retroperitoneal lymphnode metastases were pathologically documented in 8 cases (22.2%). No patient received adjuvant therapy following surgery. The disease relapsed in 4 patients (11.1%) always in the lung, from 5 to 7 months after lymphadenectomy. The pulmonary disease was minimal in 3 cases and bulky (1 lung nodule greater than 2 cm) in 1 patient. All the relapsed patients entered continuous complete remission with cisplatin, vinblastine and bleomycin (plus surgery in one case). All the 36 patients in this series are alive and disease free after a follow-up period of 40-60 months from lymphadenectomy. Antegrade ejaculation was lost by 10 out of 13 cases who had undergone the bilateral lymphadenectomy (77%) and by 2 of the 21 adult patients who had had the unilateral dissection (9.5%). Unilateral retroperitoneal lymphadenectomy is recommended in patients with negative intra-operative findings who undergo surgery for clinical stage I nonseminomatous testis cancer. PMID- 3007219 TI - Surgical treatment of 43 retroperitoneal sarcomas. AB - Forty-three cases of primary retroperitoneal sarcomas, observed and treated from 1970 to 1983 at this Institute, were analysed. The series consisted of 16 liposarcomas (37%), 10 leiomyosarcomas (23%), 7 rhabdomyosarcomas (16%), 5 fibrosarcomas (12%), 2 malignant histiocytomas (5%), 2 sarcomas NOS (5%) and 1 mesenchymoma (2%). All the patients underwent surgery. In order to evaluate the results of surgery, the patients were divided into three groups, according to the type of operation performed, that is open biopsy, resection and excision. Survival of the patients in the first group never exceeded 24 months. The symptom free period for the patients treated by incomplete removal of the tumour (second group) lasted 3-24 months. Further surgery, in three cases for this group, did not result in a useful control of the disease. As regards radically treated cases, local recurrence was observed in 3 of the 7 liposarcomas, 2 of the 5 leiomyosarcomas, 1 of the 2 rhabdomyosarcomas and 1 fibrosarcoma. Out of 16 cases of the third group, regularly followed up, only 3 patients (liposarcomas) were alive and free of disease at 5 years from first operation. Overall 5-year survival for this group was 31.9%; disease-free survival was 18.7%. For the whole series of 43 cases, overall 5-year survival was 11.4%. As far as histology was concerned, liposarcomas showed the highest operability rate: out of 16, 7 were resected and 9 radically excised. There is a lack of convincing evidence for the utility of post-operative chemotherapy. On the contrary, post-operative radiotherapy seems to be worthwhile for liposarcomas, especially after non radical operations. PMID- 3007220 TI - Long-term survival in bronchogenic carcinoma with a solitary metastasis. AB - Partial resection of a huge anaplastic large cell carcinoma of the upper lobe of the right lung was performed in a 47-year-old patient in order to relieve symptoms of pulmonary hypertrophic osteoarthropathy. Several months later a solitary metastasis was noted in the muscles of the right forearm. The metastasis was resected and the forearm irradiated. The patient was further treated with injections of autologous tumour cell vaccine and BCG. Today, 7 years later, the patient is alive, without any signs of neoplastic disease. PMID- 3007221 TI - Angiotensin II receptors in the kidney. AB - Angiotensin II (AngII) receptors have been localized in rat kidney by using the high-affinity agonist analog 125I-labeled [Sar1]AngII as a probe for in vitro autoradiography. Receptors were associated with four morphologically distinct patterns of distribution. First, a high density of receptors occurs in glomeruli. These are diffusely distributed, consistent with a mesangial localization. AngII receptor density shows a cortical gradient, which is highest in superficial and midcortical glomeruli and lowest in juxtamedullary glomeruli. Receptors associated with both superficial and deep glomeruli show down-regulation during low-sodium intake. Second, low levels of tubular AngII binding were seen in the outer cortex. Third, a very high density of AngII receptors occurs in longitudinal bands in the inner zone of the outer medulla in association with vasa recta bundles. Receptors in this site also show down-regulation during low dietary sodium intake. Fourth, a moderate density of receptors occurs diffusely throughout the inner zone of the outer medulla in the interbundle areas. These results suggest that AngII exerts a number of different intrarenal regulatory actions. In addition to the known vascular, glomerular, and proximal tubular effects of AngII, these findings focus attention on possible actions of AngII in the renal medulla where it could regulate medullary blood flow and thereby modify the function of the countercurrent concentrating system. PMID- 3007222 TI - Effects of locally formed angiotensin II on renal hemodynamics. AB - The kidney produces angiotensin II (AngII) by conversion of both locally formed and systemically delivered angiotensin I (AngI). The latter may be physiologically significant because the kidney can convert 20-25% of systemically delivered AngI. To determine possible differences between the effects of circulating and locally converted AngII, we compared the renal responses to renal arterial infusions of AngI and AngII in equiconstrictor doses. Both reduced the renal blood flow and increased the filtration fraction; it is important that the AngI infusions consistently reduced glomerular filtration rates (GFR), which indicates effects proximal to or at the glomerulus. Micropuncture experiments revealed that AngI infusions reduced proximal tubular and peritubular capillary pressures and the single-nephron GFR; glomerular capillary pressure was not altered significantly. AngI infusions increased both pre- and postglomerular resistances and reduced the glomerular filtration coefficient. In other studies designed to estimate net intrarenal AngII generation, it was determined that the kidney degrades about 90% of arterially delivered AngII. Thus, most of the AngII in renal venous blood was formed intrarenally. Local production of AngII was enhanced, in association with increased renin release, after reductions in renal arterial pressure. Such increases in intrarenal AngII production may contribute to the AngII-dependent changes in renal vascular resistance that occur in conditions where the renin-angiotensin system is stimulated. PMID- 3007224 TI - Type IV collagenolytic activity in human preovulatory follicular fluid. AB - During normal ovulation, the two basement membrane layers of the ovarian follicle are degraded locally. The main component of basement membranes is type IV collagen, which is specifically cleaved by type IV collagenase. According to our results, type IV collagenolytic activity is present within follicular fluid and increases toward ovulation, decreasing rapidly as the follicles rupture. These results suggest that importance of type IV collagenolytic activity in the ovulatory process. PMID- 3007223 TI - Immunostaining of transferrin and transferrin receptor in human seminiferous tubules. AB - Transferrin (TF) and transferrin receptor (TFr) were studied in human testicular biopsy specimens with the use of immunostaining techniques. A polyclonal antibody to human TF (obtained in goat), a murine monoclonal antibody (B3/25) to human TFr, and antisera antigoat IgG and antimouse IgG, both labeled with peroxidase, were used. In seminiferous tubules of subjects with normal spermatogenesis, TF was found mainly in Sertoli cells and, in lesser amounts (probably related to the presence of receptor-TF complexes), in spermatocytes and early spermatids. TFrs were found only in spermatocytes and early spermatids. In patients with spermatogenetic disorders, TF was always found in Sertoli cells, whereas TFrs were found in spermatocytes only when they were present. These results seem to demonstrate that in human seminiferous tubules, Sertoli cells are devoted to the production and/or storage of TF, whereas spermatocytes and early spermatids use TF. PMID- 3007225 TI - [Various membrane mechanisms of disorders on cardiac function of immune origin]. PMID- 3007226 TI - Acquired immune deficiency syndrome (AIDS): an update for nurses. PMID- 3007227 TI - ANA urges use of CDC guidelines for care of AIDS patients. PMID- 3007229 TI - Invasive mole. AB - A case of persistent trophoblastic disease with resistance to chemotherapy is presented. The value of continued and frequent serum hCG measurements in such cases is discussed as well as the indications for performing hysterectomy. PMID- 3007228 TI - [Quartz-induced scleroderma. Scleroderma-like syndrome or true progressive scleroderma?]. PMID- 3007231 TI - Purification and characterization of phosphodiesterase from the venom of Trimeresurus mucrosquamatus. AB - Phosphodiesterase was isolated from the venom of Trimeresurus mucrosquamatus from Taiwan using gel filtration on a Sephadex G-100 column, followed by anion or cation exchange chromatography. Phosphodiesterase was homogeneous as established by a single band on acrylamide gel electrophoresis and immunodiffusion. Phosphodiesterase activity was inhibited by ethylenediamine tetraacetic acid (EDTA), o-phenanthroline, thioglycolic acid or p-chloromercuribenzoate (PCMB) but not by soybean trypsin inhibitor (SBTI) or benzamidine. The molecular weight of this enzyme was determined to be approximately 140,000 and the isoelectric point was found to be pH 7.4 by isoelectric focusing with carrier ampholyte. The Michaelis constant (Km) of this enzyme for p-nitrophenyl thymidine-5'-phosphate and inhibition constant (Ki) for PCMB were found to be 5.6 X 10(-3) and 7.6 X 10( 4) M, respectively. PMID- 3007230 TI - Hydrocortisone promotes the neodifferentiation of Kirsten murine sarcoma virus transformed human skin fibroblasts to adipose cells: relevance to oncogenic mechanisms. AB - Here we have demonstrated that transformation of human skin fibroblasts (SF) by the Kirsten murine sarcoma virus (KiMSV) is associated with their neodifferentiation into preadipose cells. Hydrocortisone (HC) promotes the transformation/neodifferentiation of such preadipocytes into mature fat cells. The effects of HC on the expression of adipocyte-containing foci and on the total number of transformed foci present in KiMSV-treated cultures appeared to be dose dependent and was optimal at a concentration of about 500 ng/ml, or 1.25 X 10(-6) M. Although increasing serum concentrations (2-15%) increased the total number of transformed foci, it had no effect on the expression of adipocyte-containing foci in the presence of HC. The virus-induced preadipocytes undergoing partial conversion in the presence of HC were capable of clonal expansion and extensive proliferative activity. In contrast, mature adipocytes were terminally differentiated and as such have lost their ability to proliferate. The results suggest a role for a ras oncogene and HC in the transformation/neodifferentiation of human cells that might ultimately lead to cancer in some fraction of such cells. PMID- 3007232 TI - Enzymic properties of a Ca2+-dependent protease in rat ventral prostate: differences in distribution between lobes of the prostatic complex. AB - Soluble extracts of rat ventral prostate contain a calcium-dependent, neutral thiol protease which is separated from an endogenous inhibitor by DEAE-cellulose chromatography. The Ca2+-dependent protease had a high calcium requirement (half maximal activation at 0.19 mM CaCl2), a pH optimum in the neutral range (pH 7-8), and it was inhibited by increased ionic strength (30% inhibition at 0.2 M NaCl). Leupeptin and antipain were strong inhibitors of the enzyme. Ca2+-activated protease activities of the coagulating gland (anterior prostate) were about 40% of those of the ventral prostate and were not detectable in the dorsolateral prostatic lobe. There was no difference in specific activities of this enzyme in chromatographed extracts of prostatic lobes from young sexually mature adults and 12 month old retired breeders. In addition, Ca2+-dependent protease activity was not detectable in chromatograms of rat ventral prostate and coagulating gland secretions. Therefore, the Ca2+-activated protease does not appear to be a secretory protein and probably acts at some intracellular site(s). PMID- 3007233 TI - The effect of ouabain on basal and thyroid hormone-stimulated muscle oxygen consumption. AB - In vivo triiodothyronine treatment (T3) increased soleus muscle oxygen consumption (QO2) when measured in vitro. Ouabain significantly decreased T3 induced muscle QO2, but not basal muscle QO2. Ouabain-sensitive metabolic processes in muscle, which essentially represents Na-K-ATPase activity, accounted for approximately 85% of the increased energy utilization by muscles that was caused by T3 treatment. PMID- 3007234 TI - Electron-transfer processes in photosynthetic reaction centres. PMID- 3007235 TI - Phosphoprotein B-50 and phosphoinositides in brain synaptic plasma membranes: a possible feedback relationship. PMID- 3007236 TI - Chloroplast genes for photosynthetic membrane components of higher plants. PMID- 3007237 TI - Inhibition of prolactin release and blockade of adenohypophyseal cell cyclic AMP accumulation are two dissociable effects of dopaminergic and non-dopaminergic drugs. AB - The secretion of PRL by the anterior pituitary gland is under a tonic inhibitory control exerted by dopamine (DA). However, the mechanism(s) involved in the inhibition of PRL secretion is not clearly defined. Several recently published papers supported the hypothesis that DA inhibits the release of PRL through blockade of the pituitary adenylate cyclase-cyclic AMP system. We have recently demonstrated that sodium ions are essential for dopaminergic inhibitory action on PRL secretion. The present paper reports the effects, in the presence or in the absence of Na+, of either DA, bromocriptine, apomorphine or 2 anticalmodulin drugs, penfluridol and W-7, on cyclic AMP accumulation by rat adenohypophyseal cells in primary culture. Studies with dopaminergic agonists show that in the presence of Na+ inhibition of both PRL and cyclic AMP is obtained at 15 and 30 min, while in the absence of the ion a dissociation exists between the inhibition of PRL release which is completely abolished, and that of cyclic AMP content which is still present. Dose-response studies done in the presence of Na+ show the existence of a good correlation between hormone and nucleotide effects of dopaminergic agonists while, in the absence of Na+, a dissociation is observed between the inhibition of PRL release, which is completely suppressed, and that of cyclic AMP accumulation which is slightly or not at all decreased. The inhibitory effects of penfluridol after 15 and 30 min of incubation were not suppressed by Na+ removal, although its hormonal actions were slightly decreased.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007238 TI - A non-responsive alpha-secreting thyrotropic tumor contains T3 receptors and a TSH beta gene. AB - We have recently described a mouse pituitary tumor line, MGH 101A which is derived from a TSH-producing thyrotropic tumor line and now produces only the alpha-subunit of the glycoprotein hormones. In these studies, we have investigated the mechanism for the lack of TSH beta subunit expression in MGH 101A, as well as the failure of triiodothyronine (T3) to regulate alpha-subunit. Southern blot analysis of restriction endonuclease-digested DNA from MGH 101A tumors indicates the presence of a TSH beta gene and an alpha-subunit gene indistinguishable from those in a TSH-producing tumor (TtT 97). In MGH 101A tumors, however, TSH beta gene transcription was minimal (4 +/- 2 ppm) relative to alpha-subunit (283 +/- 29 ppm) and there was no significant difference in transcription after T3 treatment. In contrast, TtT 97 tumors had nearly equal rates of alpha-subunit (375 +/- 25 ppm) gene transcirption, and T3 respectively. The MGH 101A suppressed the transcription of alpha-subunit and TSH beta genes by 76% and 87%, respectively. The MGH 101A tumor contained T3 receptors with a binding affinity (1.54 X 10-10M) similar to receptors on TtT 97 tumors (1.78 X 10 10M), but at a lower concentration (2800 vs. 4000 sites/cell). We conclude that the absence of TSH beta production in MGH 101A tumors is not due to the absence of the TSH beta gene, but perhaps to some other modification of the gene structure. This could also explain the failure of MGH 101A tumors to respond to T3, since they do contain T3 receptors of normal affinity. PMID- 3007240 TI - Genetic and molecular analysis of fs(1)h, a maternal effect homeotic gene in Drosophila. AB - Mutations at the Drosophila melanogaster locus female sterile (1) homeotic (fs(1)h) result in segmental abnormalities including missing organs and homeotic transformations in the progeny of mutant mothers. Homeotic transformations are enhanced when the zygotes carry one of several third chromosome mutations, specifically alleles or deficiencies of the trithorax (trx) locus, also called Regulator-of-bithorax, and some alleles of bithorax complex (BX-C) genes. These observations suggest that maternally derived fs(1)h+ product is required, in interaction with trx and BX-C genes, for normal segment specification. The fs(1)h gene and an adjacent gene, lethal (1) myospheroid (l(1)mys), have been cloned by chromosomal walking. Mutations of fs(1)h were found within a 13-kb stretch of DNA. Poly(A)+ RNAs migrating as a doublet at 7.6 kb and a single band at 5.9 kb, which are homologous to the fs(1)h+ chromosomal region, are found in ovaries and early embryos. The largest RNAs are derived from a 20-kb chromosomal region encompassing the sites of all mapped fs(1)h alleles. PMID- 3007239 TI - Differential responses of the dopa decarboxylase gene to 20-OH-ecdysone in Drosophila melanogaster. AB - The dopa decarboxylase gene (Ddc) of Drosophila melanogaster responds to 20-OH ecdysone in the mature larval epidermis and in imaginal discs (presumptive adult epidermis) in a tissue-specific manner. Exposure of the mature larval epidermis to 20-OH-ecdysone caused a rapid accumulation of DDC transcripts. In the absence of protein synthesis, transcript accumulation was substantially reduced suggesting an indirect hormonal effect on DDC transcription (and/or RNA turnover). By contrast, neither DDC activity induction nor transcript accumulation was detected in imaginal discs cultured in the continuous presence of the hormone. However, when discs were exposed to 20-OH-ecdysone and then cultured in its absence, DDC activity and DDC transcript levels started to increase 6 hr after hormone withdrawal. A Northern analysis failed to reveal any novel transcripts in discs making the utilization of an alternative promotor an unlikely explanation for the very different responses of the Ddc gene in the two epidermal tissues. The results demonstrate that the Ddc gene in the larval epidermis responds rapidly to an increase in hormone titer. In imaginal discs a fall in hormone titer is required before DDC transcripts accumulate. PMID- 3007242 TI - Analysis of the transcription unit adjacent to the 3'-end of the dopa decarboxylase gene in Drosophila melanogaster. AB - Using strand specific RNA probes, we have ascertained that the transcription unit immediately downstream of the dopa decarboxylase (DDC) gene in Drosophila, is oriented in the opposite direction to DDC. The 3'-termini of these two transcription units lie within 550 bp of one another, and the mature transcripts actually may overlap. We show here that the inclusion of part of the 3'-flanking transcript on a genomic DNA clone originally assumed to include just DDC, accounts for most of the hybridization seen in our previous Northern analysis of very early embryonic RNA. Thus, while DDC-specific transcripts are detectable in the early embryonic stages, they are much less prevalent than we have previously reported. PMID- 3007241 TI - Drosophila melanogaster Ddc gene transcripts are not expressed at high levels during early embryogenesis. PMID- 3007244 TI - The specificity of the cAMP receptor mediating activation of adenylate cyclase in Dictyostelium discoideum. AB - In Dictyostelium discoideum amoebae, binding of cyclic AMP (cAMP) to surface receptors elicits numerous responses including chemotaxis, cyclic GMP (cGMP) accumulation, and activation of adenylate cyclase. The specificity of the surface cAMP receptor which mediates activation of adenylate cyclase and cAMP secretion was determined by testing the relative effectiveness of a series of 10 cAMP analogs. Each of the 10 analogs elicited cAMP secretion, chemotaxis, and cGMP accumulation in the same dose range. The order of potency for eliciting these responses (cAMP greater than 2'-H-cAMP greater than N1-O-cAMP greater than cAMPS(Sp) greater than 6-Cl-cAMP greater than cAMPN(CH3)2(Sp) greater than 3'-NH cAMP greater than 8-Br-cAMP greater than cAMPS(Rp) greater than cAMPN(CH3)2(Rp] matches that for binding to the major cell surface cAMP binding sites and differs from that of the cell surface phosphodiesterase and the major intracellular cAMP binding protein. PMID- 3007243 TI - Changes of surface glycoproteins after retinoic acid-dibutyryl cAMP-induced differentiation of teratocarcinoma stem cells. AB - Retinoic acid induces differentiation of embryonal carcinoma F9 cells into parietal endoderm. The surface proteins of F9 cells from induced and control cultures were labeled with the 125I-lactoperoxidase system and analyzed by two dimensional gel electrophoresis. Their quantitative comparison has shown an 11 fold increase of protein p220 of apparent MW 220,000 and isoelectric point 5.6. Among other enhanced surface proteins, 3.5-fold increases of p50, p45, and p40 of MW 50,000-40,000 and isoelectric point 5.1-5.3 were observed. Simultaneously another surface protein, p70 of MW 70,000 and isoelectric point 6.1-6.3, disappeared. The quantitative changes of surface proteins produced after treatment with retinoic acid were enhanced in the presence of dibutyryl cAMP. Analysis of lectin-binding proteins demonstrated that increasing proteins p220, p50, p45, and p40 have an affinity for concanavalin A, whereas p70, which decreases, has an affinity for wheat germ agglutinin. Antibodies raised against p70 from undifferentiated cells have shown a specific immunoreaction with p220 from differentiated cells and also with the subunit B of purified laminin. The electrophoretic mobilities of p220 and of the B subunit of laminin are similar. It is suggested that p70, p220, and laminin B subunit share structural homology. PMID- 3007245 TI - Length polymorphism in rDNA indicates somatic alterations in the genome of Triturus vulgaris. AB - We have analyzed the EcoRI restriction pattern of ribosomal genes (rDNA) isolated from several organs of single individuals of the newt Triturus vulgaris by Southern blotting and hybridization with corresponding Xenopus probes (r11 and r12). Using length polymorphism of rDNA spacers as a molecular marker, it became evident that in individual newts the pattern of ribosomal genes is not always constant but varies between different tissues of the same animal suggesting the occurrence of genome alterations during ontogenesis. PMID- 3007246 TI - Insulin decreases H4IIE cell PEPCK mRNA by a mechanism that does not involve cAMP. AB - Insulin is thought to influence some metabolic events by decreasing the intracellular concentration of cyclic AMP (cAMP). To test whether this explains the repression of hepatic phosphoenolpyruvate carboxykinase (PEPCK) by insulin we measured intracellular cAMP, cAMP-dependent protein kinase, mRNAPEPCK, and PEPCK gene transcription in cultured Reuber H4IIE hepatoma cells treated with forskolin with and without insulin. In untreated cells, the concentration of cAMP was 2.9 pmol/mg of protein. Forskolin at 1, 10, and 50 microM increased the level of cAMP to 9.2, 35.8, and 115 pmol/mg of protein, respectively; 5 nM insulin had no significant effect on these cAMP concentrations. In untreated cells, the activity ratio of cAMP-dependent protein kinase was 0.43, and 50 microM forskolin increased this to 0.96; insulin had no effect on this ratio at times from 15-180 min. In untreated cells mRNAPEPCK bound 15 cpm of a 32P-labeled cDNA probe per microgram of total cellular RNA. Forskolin, at 1, 10, and 50 microM increased this to 48, 96, and 115 cpm/microgram RNA. Insulin (5 nM), in combination with 0, 1, 10, and 50 microM forskolin, decreased the concentration of mRNAPEPCK to 5, 8, 23, and 29 cpm/micrograms RNA, respectively. Finally, the rate of transcription of the PEPCK gene was 85, 168, 630, 823, and 884 parts per million (ppm) in H4IIE cells treated for 30 min with 0, 1, 5, 10, and 50 microM forskolin, respectively, while the corresponding rates in the presence of 5 nM insulin were 49, 45, 84, 85, and 136 ppm.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007247 TI - The role of transferrin receptors and iron delivery in mouse embryonic morphogenesis. AB - The iron-carrying serum protein transferrin is required for the proliferation and differentiation of embryonic tissues in culture. We studied the expression and role of transferrin receptors in two model systems using a monoclonal antibody against the transferrin receptor of mice. The addition of 20-100 micrograms/ml antibody to a chemically defined culture medium containing transferrin (10 micrograms/ml) inhibited morphogenesis and cell proliferation in kidneys and teeth. However, the antibody did not inhibit development when iron was delivered to the cells by a lipophilic iron chelator i.e., by-passing the receptor-mediated pathway. Hence, the binding of the receptor antibody to the receptor apparently did not affect cell proliferation, and the antibody was not toxic to the tissues. Our results suggest that the antibody to the transferrin receptor inhibits development by blocking the normal endocytotic route of iron delivery. Cells derived from embryonic kidneys and teeth expressed the transferrin receptor when cultured as monolayers. However, using immunofluorescent techniques, we were unable to detect the receptor in frozen tissue sections. It is possible that the seeding of cells in monolayer cultures affects the expression of the transferrin receptor, since it is known that all types of cells require transferrin for continued proliferation in culture. Organ-cultured kidney mesenchymal cells are not initially responsive to transferrin, but they acquire responsiveness as a consequence of an inductive tissue interaction. Although it remains unknown as to whether the acquisition of transferrin responsiveness is directly related to the expression of transferrin receptors, our results suggest that transferrin and its receptors play a role in embryonic morphogenesis. PMID- 3007248 TI - Changes in the activity and subcellular distribution of cyclic-AMP-dependent protein kinases with retinoic-acid-induced differentiation of embryonal carcinoma cells. AB - We investigated changes in the activity and subcellular distribution of cyclic AMP-dependent protein kinases (cAMP-PKs) in response to treatment with retinoic acid in three different embryonal carcinoma cell lines derived from the same teratoma 6050. After retinoic-acid treatment, F9 and PCC4 cells gave rise to parietal-like endoderm, while PC13 cells differentiated into visceral endoderm. Retinoid treatment of F9 and PCC4 cells caused an increase in cAMP-PK activity as measured by histone phosphorylation, as well as increases in the amount of the RI and RII regulatory subunits of the cAMP-PKs, as quantitated by photoaffinity labeling with 8-azido-cyclic-32P-AMP, in both the soluble and plasma-membrane fractions. The increases in membrane cAMP-PK activity and RI and RII levels reached their maximum within 18 h of retinoid treatment, and then dropped to intermediate levels after 3 days of treatment. The cytosolic activity and the levels of the regulatory subunits exhibited a progressive increase during the 3 days of exposure to retinoic acid. The relative RI/RII ratios in the cytosol and membrane fractions of the treated cells were comparable to those found in established PYS-2 parietal-endoderm cells. PC13 stem cells had high levels of cAMP-PK activity and cAMP binding to the regulatory subunits in both the cytosol and plasma membranes, while also exhibiting very low levels of type-II cAMP-PK. Retinoid treatment induced a progressive increase in cAMP-PK activity in the cytosol, and a decrease in activity at the membrane level.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007249 TI - Cytoskeletal differences between human neuroendocrine tumors: a cytoskeletal protein of molecular weight 46,000 distinguishes cutaneous from pulmonary neuroendocrine neoplasms. AB - The cytoskeletons of various human neuroendocrine (NE) tumors were analyzed immunohistochemically using antibodies against intermediate-filament (IF) proteins as well as by two-dimensional gel electrophoresis of proteins from microdissected tissue samples. All of the tumors studied were found to contain cytokeratin filaments and are therefore referred to as 'NE tumors of the epithelial type'. In addition, neurofilaments were found in most cutaneous and some pulmonary NE tumors, as well as in medullary carcinomas of the thyroid and in pancreatic islet cell tumors. The neurofilament staining was frequently concentrated in cytoplasmic IF aggregates. Gel-electrophoretic analyses showed that all NE tumors examined synthesize 'simple epithelium-type' cytokeratin polypeptides, cytokeratins nos. 8 and 18 being the most prominent ones, whereas cytokeratin no. 19 was found in variable and usually minor amounts. A new cytoskeletal protein, designated IT protein, with a relative molecular weight of 46,000 and an isoelectric pH value of approximately 6.1 (in 9.5 M urea) was detected in all 9 cases of cutaneous NE tumors ('Merkel-cell carcinomas'), including 2 lymph-node metastases, but was not found in any of the 17 cases of pulmonary NE tumors. In addition, 2 medullary carcinomas of the thyroid, 2 islet cell tumors of the pancreas, and 1 intestinal carcinoid tumor also seemed to lack this protein. A protein indistinguishable from IT protein by electrophoresis and tryptic peptide mapping was found in cytoskeletal preparations of mucosal cells of human intestine and in cultured human colon carcinoma cells of line HT-29. A possible relationship between IT protein and the type-I subfamily of cytokeratin polypeptides is discussed. Our study shows that the co-expression of cytokeratin filaments and neurofilaments may provide a criterion which is useful for the recognition of some NE tumors but which does not distinguish between NE tumors of different types and origins. In contrast, IT protein seems to be present specifically in cutaneous NE tumors, but absent in pulmonary NE tumors. The implications of these findings for the elucidation of the histogenesis of cutaneous NE tumors and for the histopathological differential diagnosis of NE tumors of cutaneous and pulmonary origin are discussed. PMID- 3007250 TI - [The relation between myocardial oxygen consumption and ischemia during exertion in patients with angina pectoris: effect of propranolol]. AB - Patients with coronary artery disease exhibit a reduced coronary vasodilator reserve in response to exercise testing. Drugs which block coronary beta adrenergic receptors could exacerbate this abnormality leaving the vasoconstrictor alpha tone unopposed and/or counteracting the beta 2-mediated vasodilation elicited by the increase in myocardial oxygen demand. To test this hypothesis we administered propranolol 40 mg qid and placebo, using a cross over randomized single blind protocol, to 14 patients each with effort angina and critical coronary stenosis (greater than or equal to 75%). We performed computer assisted multistage bicycle ergometer testings (25 W increments at 2 min intervals) after 2 weeks open label placebo (control) and at 2 week intervals following daily administration of propranolol and placebo. Compared to placebo, propranolol reduced significantly (p less than 0.001) peak heart rate (x +/- SD: 114 +/- 6 vs 150 +/- 11 beats/min) and rate pressure product (20.1 +/- 2.1 vs 28.0 +/- 3.9 X 10(-3)) and increased exercise duration (462 +/- 91 vs 355 +/- 85 sec). Conversely 0.1 mV ST segment depression was observed at lower heart rate (106 +/- 9 vs 127 +/- 8 beats/min, p less than 0.001) and rate pressure product (16.9 +/- 3.6 vs 22.4 +/- 2.4 X 10(-3), p less than 0.001). No significant differences were found between placebo and control. Moreover, we assessed the regression lines of the relationship between ST segment depression (ST) and heart rate (HR) during exercise. These have been shown to be shifted to the right after surgical revascularization and are an indirect measure of coronary reserve.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007251 TI - [Analogs of pyrimidine nucleosides with selective antiherpetic activity]. PMID- 3007252 TI - [Possible inhibitors of experimental viral replication against cytomegalovirus. Results and prospectives]. PMID- 3007253 TI - [Direct and mediated effects of the interferon system on human retroviruses]. PMID- 3007254 TI - [Ifosfamide and adriamycin after induction with cisplatin and etoposide (VP-16) in pulmonary microcytoma. Preliminary results]. PMID- 3007255 TI - [Mitomycin C, adriamycin and vinblastine after induction with cisplatin and VP-16 in the treatment of non-small cell lung carcinoma]. PMID- 3007256 TI - [Biological aspects of brain tumors: therapeutic implications]. PMID- 3007257 TI - [Toward a simplification of the diagnostic strategy in hilar cholangiocarcinoma?]. PMID- 3007259 TI - [The intestinal immune system. The VIP receptor on T lymphocytes: a new marker of differentiation?]. PMID- 3007258 TI - [Hepatocellular carcinoma and the alpha 1-antitrypsin phenotype]. AB - To determine whether or not alpha 1-antitrypsin phenotype PiMZ is a predisposing factor for hepatocellular carcinoma alpha 1-antitrypsin phenotype was studied in 83 French patients with hepatocellular carcinoma compared with 1,030 blood donors. In 66 patients, alpha 1-antitrypsin phenotype was determined by isoelectric focusing which allows the distinction between subtypes of phenotype M. No difference has been shown between the two groups for alpha 1-antitrypsin alleles or subtypes of allele Pi*M. The authors conclude that phenotype PiMZ is not a significant predisposing factor for hepatocellular carcinoma in French people. As well, a review of literature suggests that, in contrast with the conclusion of previous reports, the link between alpha 1-antitrypsin phenotype PiMZ and hepatocellular carcinoma is either nonexistent or very weak. PMID- 3007260 TI - Fine needle aspiration biopsy in malignant obstructive jaundice. AB - Percutaneous cytodiagnosis of malignancy in patients with biliary tract obstruction is often useful in planning subsequent therapy. Of 121 patients presenting for percutaneous transhepatic cholangiography and biliary drainage, 45 had fine needle aspiration biopsies. Forty-one patients had malignant obstruction of the biliary tree, while benign disease was present in 4 patients. Neoplasia was diagnosed in 12 of 13 patients with bile duct carcinoma, 16 of 22 patients with pancreatic cancer, and 3 of 6 patients with other malignancies. Radiologic biopsy sensitivity was only slightly inferior to surgical biopsy sensitivity in the same patient population. A scheme for biliary cytodiagnosis is presented, which uses a percutaneous approach for patients with suspected pancreatic carcinoma and a transcatheter approach for patients with suspected bile duct carcinoma. The utility of this procedure and the low complication rate are stressed. PMID- 3007261 TI - Serum procollagen type III peptide as a marker of hepatic fibrogenesis in alcoholic hepatitis. AB - To evaluate if serum procollagen type III peptide levels reflect the extent of liver fibrosis and hepatic collagen synthesis, we have studied 19 patients with histologically proven alcoholic hepatitis and 9 chronic alcoholics with normal liver histology or minimal steatosis. Serum procollagen peptide type III was measured at the time of liver biopsy, and determination of hepatic prolyl hydroxylase activity, as an index of collagen synthesis, was performed in all liver samples. Hepatic prolyl-hydroxylase activity and serum procollagen peptide levels were significantly higher in patients with alcoholic hepatitis (959 +/- 115 cpm/mg and 33.2 +/- 5.3 ng/ml, respectively) than in alcoholics from the control group (537 +/- 62 cpm/mg and 10.9 +/- 1.5 ng/ml, respectively) (p less than 0.05 and p less than 0.01, respectively). All patients with alcoholic hepatitis had fibrosis (10 mild and 9 severe). Prolyl-hydroxylase activity and procollagen peptide levels were significantly higher in alcoholic hepatitis patients with severe fibrosis than in those with mild fibrosis (1208 +/- 154 cpm/mg vs. 734 +/- 138 cpm/mg, p less than 0.05 and 49.1 +/- 8.8 ng/ml vs. 20.4 +/- 2.6 ng/ml, p less than 0.01). Furthermore, a close correlation was found between the hepatic prolyl-hydroxylase activity and the serum level of procollagen peptide (r = 0.76, p less than 0.001). We conclude that the serum procollagen peptide level is a good marker of hepatic fibrogenesis in alcoholic hepatitis; thus, its serial measurement could be useful in identifying patients in progress to cirrhosis and in assessing the therapeutic efficiency of antifibrogenic drugs. PMID- 3007262 TI - Aberrant insulinoma in the duodenum. AB - A rare case of aberrant insulinoma in the duodenum is described. Hyperinsulinemia with typical hypoglycemic symptoms was induced by prolonged fasting. Selective angiography demonstrated a tumor supplied from the first branch of the jejunal artery, and duodenoscopy revealed a submucosal tumor at the third portion of the duodenum. An increase in venous plasma immunoreactive insulin concentration was evident in the vein draining from the tumor, by sampling through percutaneous transhepatic catheterization. Hypoglycemia was ameliorated after the removal of the submucosal tumor of the duodenum. Histologic and immunocytochemical characterization of the tumor showed an insulinoma, predominantly composed of cells with typical B-cell-like granules. The acid extract of the tumor contained 1.2 U/g of insulin, and this insulin, analyzed by reverse-phase high-pressure liquid chromatography, revealed that it had the same amino acid structure as that of human insulin. PMID- 3007263 TI - Liver adenomatosis. PMID- 3007264 TI - [Treatment of urinary urgency symptoms in the female with the beta-2 sympathomimetic clenbuterol]. AB - Aside from parasympatheticolytis, calcium antagonists, musculotropic relaxants and tricyclic antidepressants, beta 2-sympatheticomimetics are also used in the treatment of urgency. The beta 2-sympatheticomimetic Clenbuterol, which has relatively slight cardiological side effects, was administered per os to 30 women. Prior to and six weeks after the commencement of daily medication with 2 x 20 micrograms of Clenbuterol, the therapeutic effect of the drug was tested by means of a urogynecological examination (history, gynecological findings, cystometrography, urethrocystometrography, uroflowmetry and pretherapeutic urethrocystometrography). An improvement rate of 65% was found. The parameters of urethrocystometrography and uroflowmetry were not altered under medication with Clenbuterol. Cystometrographic parameters, on the other hand, improved (minimal desire to urinate, maximal desire to urinate, compliance). The results of this study are discussed with reference to the literature. For the beta 2 sympatheticomimetic Clenbuterol, a good level of efficacy can be established in women patients on whom parasympatheticolytics alone failed to have a satisfactory effect. PMID- 3007265 TI - [Proof of diaplacental transmission of HTLV III/LAV before the 20th week of pregnancy]. AB - Tissue samples were taken from a 20-week old foetus in a mother with positive LAV/HTLV-III reaction without clinical or immunological signs of AIDS. These samples were used for cocultivation of LAV/HTLV-III. This resulted in inducing in MT-2 cells a cytopathic effect characteristic for LAV/HTLV-III infections, induced by thymus, spleen, lungs and brain tissue of the foetus after cocultivation with umbilical cord lymphocytes; in H-9 and MT-2 cells this led to the expression and release of antigens reacting with LAV/HTLV-III-specific anti bodies. This observation shows that intrauterine infection of the foetus must have occurred already before the 20th week of gestation; the same target cells are involved via uterine transmission as via postnatal infection, i.e. cells of the lymphatic system and of the CNS. PMID- 3007266 TI - In vitro study of frog (Rana ridibunda Pallas) interrenal function by use of a simplified perifusion system. VIII. Structure-activity relationship of synthetic ACTH fragments and gamma-MSH. AB - The present study was undertaken to determine the structure-activity relationships of ACTH analogs on corticosteroid production by frog adrenal gland. Rana ridibunda interrenal dice were perifused with amphibian culture medium for 10 hr. Corticosterone and aldosterone concentrations were measured in the effluent perifusate using sensitive and specific radioimmunoassay methods. Perifusion of interrenal fragments with increasing concentrations of synthetic human ACTH 1-39 (ranging from 6.25 X 10(-11) to 10(-9) M) led to a linear log dose increase in both corticosterone and aldosterone secretion. Thus, this model made it possible to compare the steroidogenic potency of several ACTH analogs. Synthetic alpha-MSH and its des-N alpha-acetyl derivative were found to be approximately equipotent, and 5 X 10(3) times less active than authentic ACTH. The short-chain analog ACTH 1-10 was 2 X 10(4) times less potent than ACTH whereas ACTH 4-10 was totally inactive. A fragment of the N-terminal region of the proopiomelanocortin molecule, gamma 3-MSH, caused a dose-related stimulation of steroid secretion. However, in contrast to what has been observed in the rat, gamma 3-MSH did not potentiate the corticotropic action of ACTH on frog interrenal gland. Since processing of proopiomelanocortin in frog intermediate lobe generates high amounts of alpha-MSH and des-N alpha-acetyl alpha-MSH, these results suggest that in amphibians, several peptides other than ACTH may be involved in the control of corticosteroidogenesis. PMID- 3007267 TI - The interrelationship of cortisol, gill (Na + K) ATPase, and homeostasis during the Parr-Smolt transformation of Atlantic salmon (Salmo salar L.). AB - Serum cortisol concentrations were measured in juvenile Atlantic salmon (Salmo salar L.) undergoing the parr-smolt transformation in fresh water, at either 1 year (S1 population) or 2 years (S2 population) after hatching. Serum cortisol levels were generally low (less than 10 ng ml-1), but during smoltification became significantly elevated in both populations. In addition, the S2 population showed a small cortisol peak in the autumn prior to smoltification. Simultaneous measurement of gill (Na + K) ATPase activity and serum cortisol concentrations in S2 salmon juveniles revealed that both features rose during smoltification in fresh water. The rise in gill (Na + K) ATPase activity was independent of cortisol levels, and preceded the rise in cortisol titer by approximately 1 month. After seawater transfer, gill enzyme levels remained high while cortisol titers fell sharply. Serum cortisol levels, but not gill (Na + K) ATPase activities, were progressively reduced by acclimation of smolts to increasing salinities. Linear regression studies indicated that, at any one level of gill (Na + K) ATPase, cortisol titer increased with increasing surface area: volume ratio. Extracellular fluid volume (sodium space) was found to decline with increasing gill (Na + K) ATPase activity, and to increase with serum cortisol titers. These results indicate that high serum cortisol levels represent a secondary response caused by the development of hypoosmoregulatory ability while still resident in fresh water. Cortisol does not appear to directly stimulate gill (Na + K) ATPase activity in Atlantic salmon smolts. PMID- 3007268 TI - Lack of glycosilation of pro-opiomelanocortin might account for the periodic acid Schiff-negative reaction in ACTH cells of teleost fishes. AB - Unlike tetrapod ACTH cells, teleost ACTH cells do not react with the periodic acid-Schiff method (PAS). To find an explanation for this unique feature, chromatographic fractions obtained after filtration of pituitary extracts of Prochilodus platensis in Sephadex were immunologically analyzed. A high-molecular weight protein which was identified as pro-opiomelanocortin (POMC) was detected. When this POMC was submitted to affinity chromatography in concanavalin binding, it was not detected. Furthermore, pituitaries incubated in media containing [3H]glucosamine or [3H]fucose did not incorporate these amino acids to the newly synthesized POMC. The results obtained strongly suggest an inability of the fish to glycosilate POMC, and this failure could account for the PAS-negative reaction in the ACTH cells. PMID- 3007269 TI - Circadian rhythm of interrenal activity in Xenopus laevis. AB - Young specimens of Xenopus laevis were kept under constant environmental conditions (artifical light from 600 to 1800 hr, feeding between 800 and 900 hr) and the concentrations of aldosterone and corticosterone in the serum were measured every 3 hr. Furthermore, the kidneys containing the interrenals were removed and their corticosteroid release under stimulation by the pars distalis and mammalian ACTH was determined. Under these conditions, the corticosteroid levels in the serum were maximal from 900 to 1200 hr (corticosterone, 7.7 +/- 0.47 ng/ml; aldosterone, 2.8 +/- 0.27 ng/ml) and minimal during the night (corticosterone, 5.2 +/- 0.43 ng/ml; aldosterone, 1.7 +/- 0.18 ng/ml). The basal secretion rate of the interrenals in vitro showed the opposite course (corticosterone, 24 +/- 3 to 72 +/- 8 pg/min/tissue; aldosterone, 46 +/- 5 to 68 +/- 9 pg/min/tissue). Stimulation by the pars distalis and mammalian ACTH clearly increased the secretion rate. After both types of stimulation the original rhythm was lost for aldosterone but still present for corticosterone. The ratio of the amounts of corticosterone/aldosterone was greater than 1.0 in the serum but less than 1.0 in the incubation fluid. It decreased significantly after stimulation in vitro by pars distalis or ACTH. PMID- 3007270 TI - Specific gonadotrophin binding sites in the bullfrog testis. AB - We demonstrated the presence of specific binding sites for bullfrog luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in a testicular crude plasma membrane fraction of the bullfrog, Rana catesbeiana. The equilibrium analysis of the binding showed that a straight line was fitted to the Scatchard plot of LH and a curvilinear line to that of FSH, suggesting the presence of single-type binding sites for LH and the presence of multiple-type binding sites or negative cooperactivity of the binding for FSH. Competition experiments showed that bullfrog LH could replace the specific binding of bullfrog FSH and vice versa. Binding affinity of bullfrog LH to radioiodinated bullfrog FSH binding sites was as high as that of intact bullfrog FSH to the same sites. In contrast, binding properties of bullfrog FSH to radioiodinated bullfrog LH binding sites were not simple: the competition curve obtained with FSH against the binding of bullfrog LH had an extremely low slope value and was not parallel to that obtained with LH. These results suggest that differentiation of LH and FSH receptors is not complete in this species, although it has been reported that these hormones have separate actions on the testis. Competition experiments further showed that bullfrog testicular LH and FSH receptors possessed higher affinities for gonadotrophins of homologous or closely related animal species compared with phylogenically distant groups. Species specificity of the hormone-receptor interaction may cause species specificity of the gonadotrophin action. PMID- 3007271 TI - Alpha- and beta-adrenergic mechanisms mediate blood pressure control by norepinephrine and angiotensin in ducks. AB - Adrenergic mechanisms for the pressor actions of blood-borne L-norepinephrine (NE) and fowl angiotensin II (ANG II) were studied in barbiturate-anesthetized adult ducks (Anas platyrhynchos). NE (1.5-6.0 nmol X kg-1) or ANG II (0.4-1.6 nmol X kg-1) injected iv caused dose-dependent increases in mean arterial pressure (Pa) and pulse pressure (Pp) but slowed cardiac frequency (fH); higher doses of ANG II increased Pa, Pp, and fH X beta-Adrenergic blockade by propranolol lowered baseline Pa, completely blocked cardiovascular responses to isoproterenol, augmented the bradycardic effect of NE, and inhibited the stimulation of Pp by ANG II. However, the tachycardiac effect of high-dose ANG II persisted during beta-blockade. alpha-Adrenergic blockade following iv prazosin completely blocked the pressor effect of methoxamine, diminished the pressure response to NE, and decreased Pa sensitivity to ANG II injections. Combined alpha and beta-adrenergic blockade decreased both the sensitivity and the maximal Pa response to ANG II. We conclude that (i) beta-adrenergic mechanisms predominate in the maintenance of resting Pa, (ii) NE increases Pa principally by alpha adrenergic action while beta-adrenergic stimulation buffers the consequent bradycardia, and (iii) although the positive chronotropic effect of high doses of ANG II probably is not mediated by catecholamines, low doses of ANG II elevate Pa and Pp by alpha- and beta-adrenergic mechanisms. PMID- 3007272 TI - Immunocytochemical identification and localization of the different cell types in the pituitary of the seabass (Dicentrarchus labrax). AB - Antisera raised against chum salmon prolactin (PRL), trout growth hormone (GH), mammalian adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), and alpha-melanophore-stimulating hormone (alpha-MSH) were used to localize PRL, GH, ACTH, gonadotropic, TSH, and MSH cells in the hypophysis of the teleost Dicentrarchus labrax using the unlabeled peroxidase anti-peroxidase method. In the rostral pars distalis, ACTH cells stained very intensively with anti-ACTH; so did the MSH cells in the pars intermedia. The prolactin cells stained very specifically with anti-prolactin without staining the growth hormone cells. In the proximal pars distalis anti-GH, anti-TSH beta, and anti-LH stained selectively the corresponding cells; with these antisera no cross-reaction with any other cell type was observed. Anti-alpha-MSH only stained cells in the pars intermedia. Some cells in the pars intermedia did not react at all; these could correspond to the PAS-positive cells. A characteristic feature was positive staining with anti-LH in some cell groups encircling the pars intermedia, indicating the fact that in the seabass some cells of the proximal pars distalis surround the pars intermedia. PMID- 3007273 TI - Comparative effects of corticotropin-releasing factor, arginine vasopressin, and related neuropeptides on the secretion of ACTH and alpha-MSH by frog anterior pituitary cells and neurointermediate lobes in vitro. AB - The ability of corticoliberin (CRF), urotensin I, sauvagine, arginine-vasopressin (AVP), and mesotocin to stimulate ACTH release by frog anterior pituitary cells and alpha-melanotropin (MSH) by frog neurointermediate lobe was studied in vitro using a perifusion technique. CRF and AVP were found to be potent stimulators of ACTH secretion, whereas urotensin I and sauvagine were totally inactive. In opposition to recent findings in the rat. CRF did not modify alpha-MSH secretion by the frog neurointermediate lobe. Mesotocin, which is present in the parenchymal cells of the frog pars intermedia, had no effect on alpha-MSH release in vitro. No potentiation of CRF-induced ACTH release was observed when anterior pituitary cells were incubated with a combination of AVP and CRF. Together with the recent elucidation of a CRF-like molecule in the frog diencephalon, these results suggest that, in Amphibia, CRF and AVP exert their stimulatory action specifically on distal lobe corticotrophs. PMID- 3007274 TI - Localization of action of the IS50-encoded transposase protein. AB - The movement of the bacterial insertion sequence IS50 and of composite elements containing direct terminal repeats of IS50 involves the two ends of IS50, designated O (outside) and I (inside), which are weakly matched in DNA sequence, and an IS50 encoded protein, transposase, which recognizes the O and I ends and acts preferentially in cis. Previous data had suggested that, initially, transposase interacts preferentially with the O end sequence and then, in a second step, with either an O or an I end. To better understand the cis action of transposase and how IS50 ends are selected, we generated a series of composite transposons which contain direct repeats of IS50 elements. In each transposon, one IS50 element encoded transposase (tnp+), and the other contained a null (tnp ) allele. In each of the five sets of composite transposons studied, the transposon for which the tnp+ IS50 element contained its O end was more active than a complementary transposon for which the tnp- IS50 element contained its O end. This pattern of O end use suggests models in which the cis action of transposase and its choice of ends is determined by protein tracking along DNA molecules. PMID- 3007276 TI - Beta-glucuronidase mutants of the nematode Caenorhabditis elegans. AB - Using a screening procedure that is based on a histochemical stain for the enzyme beta-glucuronidase, we have isolated several mutants of the nematode Caenorhabditis elegans affected in beta-glucuronidase activity. All of the mutations fall into one complementation group and identify a new gene, gus-1, which has been mapped on the right arm of linkage group I (LG I), 1.1 map units to the left of unc-54. The mutants have no visible phenotype, and their viabilities and fertilities are unaffected. Linked revertants of two of the mutations have been isolated. They restore enzyme activity to almost wild-type levels; the beta-glucuronidase that one of the revertants produces differs from that of the wild type. We propose that gus-1 is the structural locus for beta glucuronidase. PMID- 3007275 TI - Homologous recombination in Escherichia coli: dependence on substrate length and homology. AB - We studied the in vivo recombination between homologous DNA sequences cloned in phage lambda and a pBR322-derived plasmid by assaying for the formation of phage plasmid cointegrates by a single (or an odd number of) reciprocal exchange. (1) Recombination proceeds by the RecBC pathway in wild-type cells and by low levels of a RecF-dependent pathway in recBC- cells. The RecE pathway appears not to generate phage-plasmid cointegrates. (2) Recombination is linearly dependent on the length of the homologous sequences. In both RecBC and RecF-dependent pathways there is a minimal length, called the minimal efficient processing segment (MEPS), below which recombination becomes inefficient. The length of MEPS is between 23-27 base pairs (bp) and between 44-90 bp for the RecBC- and RecF dependent pathways, respectively. A model, based on overlapping MEPS, of the correlation of genetic length with physical length is presented. The bases for the different MEPS length of the two pathways are discussed in relationship to the enzymes specific to each pathway. (3) The RecBC and the RecF-dependent pathways are each very sensitive to substrate homology. In wild-type E. coli, reduction of homology from 100% to 90% decreases recombinant frequency over 40 fold. The homology dependence of the RecBC and RecF-dependent pathways are similar. This suggests that a component common to both, probably recA, is responsible for the recognition of homology. PMID- 3007277 TI - Molecular and cytogenetic characterization of a metallothionein gene of Drosophila. AB - A chromosomal DNA segment containing the metallothionein gene was isolated from a genomic library of Drosophila melanogaster using a previously characterized cDNA of this species as a probe. A segment of 1543 base pair (bp) was sequenced and found to include the cDNA sequence interrupted by one small intron. Several lines of evidence indicate that there is a single copy of the metallothionein gene (Mtn) in Drosophila; any other related genes, if they occur, must be sufficiently different that they are not detectable by our probe, even under hybridization conditions of reduced stringency. According to in situ hybridization and deletion mapping, Mtn is located in the right arm of the third chromosome in region 85E10 15. Within 300 bases upstream of the apparent site of transcription initiation, there are several short intervals very similar to the 12-bp segments considered to be responsible for metal regulation in mammalian systems. PMID- 3007278 TI - DNA repair dependence of somatic mutagenesis of transposon-caused white alleles in Drosophila melanogaster after treatment with alkylating agents. AB - DNA repair-defective alleles of the mei-9, mei-41, mus-104 and mus-101 loci of Drosophila melanogaster were introduced into stocks bearing the UZ and SZ marker sets. Males with the UZ marker set, z1 (zeste allele) and w+(TE) (genetically unstable white allele presumably caused by a transposable element), or the SZ marker set, z1 and w+R (semistable white allele caused by partial duplication of the w+ locus plus transposon insert), were exposed to EMS at the first instar. After emergence, adult males bearing red spots on lemon-yellow eyes were scored as flies with somatic reversions of w+(TE) or w+R. The relative mutabilities (relative values of reversion frequency at an equal EMS dose) of either w+(TE) or w+R in a repair-proficient strain and in mei-9, mei-41, mus-104 and mus-101 strains were 1: approximately 1.2:0.3:0.3:0.7, despite the fact that w+(TE) reverted two to three times as frequently as w+R under both the repair-proficient and repair-deficient genetic conditions. Similarly, after treatment with MMS, MNNG and ENNG, w+(TE) was somatically more mutable in the mei-9 strain and less mutable in the mei-41 and mus-104 strains than in the repair-proficient strain. From these results, we propose that mutagenic lesions produced in DNA by treatment with these chemicals are converted to mutant DNA sequences via the error-prone repair mechanisms dependent on the products of the genes mei-41+ (mei 41 and mus-104 being alleles of the same locus) and mus-101+, whereas they are eliminated by mei-9+-dependent excision repair. In contrast to the approximately linear responses of induced reversions of w+(TE) with ENNG in the repair proficient, mei-9, and mei-41 strains, seemingly there were dosage insensitive ranges for induced reversion with MNNG in the repair-proficient and mei-41 strains, but not for reversion in the mei-9 strain; w+(TE) in the mus-104 strain was virtually nonmutable with MNNG and ENNG. These results suggest that O6 methylguanine (O6MeG) produced in DNA with MNNG, but not O6-ethylguanine produced with ENNG, is almost completely repaired in a low dose range by constitutive activity of DNA O6MeG transmethylase. From the distribution of clone sizes of spontaneous revertant spots and other data, we propose that both w+(TE) and w+R have a similar tendency to spontaneously revert more frequently at early rather than at later developmental stages probably reflecting a common property of their inserted transposons. PMID- 3007280 TI - On the identification of the rosy locus DNA in Drosophila melanogaster: intragenic recombination mapping of mutations associated with insertions and deletions. AB - DNA extracts of several rosy-mutation-bearing strains were associated with large insertions and deletions in a defined region of the molecular map believed to include the rosy locus DNA. Large-scale, intragenic mapping experiments were carried out that localized these mutations within the boundaries of the previously defined rosy locus structural element. Molecular characterization of the wild-type recombinants provides conclusive evidence that the rosy locus DNA is localized to the DNA segment marked by these lesions. One of the mutations, ry2101, arose from a P-M hybrid dysgenesis experiment and is associated with a copia insertion. Experiments are described which suggest that copia mobilizes in response to P-M hybrid dysgenesis. Relevance of the data to recombination in higher organisms is considered. PMID- 3007279 TI - Complex cis-acting regulators and locus structure of Drosophila tissue-specific ADH variants. AB - Diverse patterns of tissue-specific expression of alcohol dehydrogenase (ADH) among species of the grimshawi subgroup of Hawaiian picture-winged Drosophila suggests control by complex or multiple, independently acting regulatory elements. These elements act by controlling Adh mRNA accumulation in individual tissue types. Restriction mapping of the Adh loci from these species reveals several insertion/deletion differences, one of which lies just outside the 5' end of the structural sequences and correlates with differences in larval patterns of ADH expression. No tissue-specific rearrangement of Adh sequences was observed. PMID- 3007281 TI - A diphenol oxidase gene is part of a cluster of genes involved in catecholamine metabolism and sclerotization in Drosophila. II. Molecular localization of the Dox-A2 coding region. AB - Mutations at the Dox-A2 (2-53.9) locus alter the A2 component of diphenol oxidase, an enzyme having an important role in cuticle formation. This locus is in the dopa decarboxylase, Df(2L)TW130 region, which contains a cluster of at least 14 genes involved in catecholamine metabolism and the formation, sclerotization and melanization of cuticle in Drosophila. The region is subdivided by deficiencies, and localization of breakpoints in cloned DNA reveals a dense subcluster of six genes in the 23 kb proximal to Ddc. Five lethal loci distal to Ddc comprise a second such subcluster. The proximal breakpoints of deficiencies Df(2L)hk18 and Df(2L)OD15 define a 14.3- to 16.8-kb region containing Dox-A2 and l(2)37Bb, and those of Df(2L)OD15 and Df(2L)TW203 define a 9.3- to 12.1-kb region containing l(2)37Ba, l(2)37Bc and l(2)37Be. Southern blots show two of the Dox-A2 mutations are small deletions (0.1 and 1.1 kb). The Dox-A2 locus mRNA is 1.7 kb. cDNA clones indicate that the 3' end is centromere proximal and that the coding region contains at least one small intron. The Dox-A2 locus is within 3.4 to 4.4 kb of the Df(2L)OD15 breakpoint, placing four of the vital loci within a maximum of 15.5 kb. The location of Dox-A2 in a cluster of genes affecting cuticle formation is discussed. PMID- 3007283 TI - [Two loci in the genome of the transposable phage B39 of Pseudomonas aeruginosa affecting the process of integration. I. Study of the properties of phages with the Pde- phenotype and mapping of the rms site]. AB - Mutants and recombinants of transposable Pseudomonas aeruginosa bacteriophage B39 with a specific phenotype Pde- (pleiotropic developmental effect) were studied. Pde- phages produce clear minute plaques on lawns of P. aeruginosa PAO1 and fail to grow in cells of PAO1 harbouring Rms 163 (Inc P5) plasmid. Pde+ character is under control of the two loci in phage genome which were designated pdeX and pdeY. In hybrid phages the pdeX and pdeY loci originating from different transposable phages (pdeX from B39 and pdeY from PH132) do not accomplish their function and, as a result, the hybrid phages have the Pde- phenotype. The frequency of integration (f.o.i.) of Pde- phages into bacterial chromosome is lower than f.o.i. for Pde+ phages, as well as the frequency of stable lysogenization of infected bacteria; lytic development of the Pde- phages is also limited. The great difference among the transposable phages in their reaction to the presence of Rms163 plasmid is caused by some differences in the specific rms site in the phage genome. The site is located inside the interval 1.1-3.9 kb of the physical genome map, being closely linked to cI gene of phage B39. The growth of Pde- phages in cells with Rms163 can be restored, due to additional mutations in phage genes affecting lysogenization. PMID- 3007284 TI - [Enhancer-like structures in moderately repetitive sequences of eukaryotic genomes]. AB - The results of contextual analysis of 25 different middle repetitive DNA sequences are presented. It was shown that each of these repetitive DNA sequences contains at least one enhancer-like structure homologous to real enhancers, as well as to their consensus. The enhancer-like structures have been also revealed in the replication origin of some prokaryote genomes. The results are discussed in the light of a possible role of middle repetitive DNA sequences in the modulation of gene expression. Some aspects of genomes' evolution, in relation to enhancers, are also considered. PMID- 3007282 TI - The molecular through ecological genetics of abnormal abdomen. II. Ribosomal DNA polymorphism is associated with the abnormal abdomen syndrome in Drosophila mercatorum. AB - Restriction endonuclease cleavage analyses of cloned and genomic DNA samples indicate that the structure of the DNA encoding the large cytoplasmic RNAs (rDNAs) is altered in Drosophila mercatorum lines which exhibit an abnormal abdomen (aa) phenotype. In a majority of the rDNA repeat units from aa flies, the 28S coding sequence is interrupted by a large [5-6 kilobase pairs (kbp)] insert. A subclone containing this inserted DNA (ins 3) hybridizes primarily to rDNA containing sequences in in situ and genomic blot hybridization experiments. Additionally, genomic nitrocellulose blot hybridization analyses show that ins- containing rDNA repeat units are clustered in a spontaneously arising aa mutant. This rDNA alteration in D. mercatorum flies with the aa phenotype more closely resembles the bobbed (bb) defect of D. hydei than the bb defect of D. melanogaster, which involves alterations in rDNA copy number. By analogy with the other Drosophila systems, we propose that the altered D. mercatorum rDNA repeat units are defective in rRNA production at a critical stage. The lowered levels of rRNA ultimately would limit the concentration of ribosomes needed to produce large quantities of a protein (in these cases, juvenile hormone esterase) needed for normal development. PMID- 3007285 TI - [Structure of long terminal repeats of transcriptionally active and inactive copies of the mobile dispersed gene MDG3 in Drosophila melanogaster]. AB - The nucleotide sequences of long terminal repeats (LTRs) and adjacent regions are determined in the transcribed and non-transcribed variants of a mobile dispersed genetic element MDG3. MDG3 is similar to other mdg elements. A 4 bp duplication of the host DNA is generated upon its integration. MDG3 is flanked by a 5 bp inverted repeat. The length of the LTRs varies in different MDG3 variants, the difference being connected mainly with duplications of certain sequences in U3 and R regions. The copies with 267 bp LTR are the most abundant ones and perfectly conservative in their primary structure. They are transcribed in 67J25D cell culture and are not transcribed in Kc cell line, where another variant with LTR length 293 bp is transcriptionally active. S1-mapping of transcription initiation and termination sites has demonstrated that in both MDG3 variants they are situated in the same positions, and the LTR itself may be subdivided into U3, R and U5 regions, like retroviral LTRs. Possible factors involved in the regulation of mdg transcription are discussed. PMID- 3007286 TI - Universal restriction endonucleases: designing novel cleavage specificities by combining adapter oligodeoxynucleotide and enzyme moieties. AB - Class IIS restriction endonucleases cleave double-stranded (ds) DNA at precise distances from their recognition sequences. A method is proposed which utilizes this separation between the recognition site and the cut site to allow a class IIS enzyme, e.g., FokI, to cleave practically any predetermined sequence by combining the enzyme with a properly designed oligodeoxynucleotide adapter. Such an adapter is constructed from the constant recognition site domain (a hairpin containing the ds sequence, e.g., GGATG CCTAC for FokI) and a variable, single stranded (ss) domain complementary to the ss sequence to be cleaved (at 9 and 13 nucleotides on the paired strands from the recognition sequence in the example of FokI). The ss sequence designated to be cleaved could be provided by ss phage DNA (e.g., M13), gapped ds plasmids, or supercoiled ds plasmids that were alkali denatured and rapidly neutralized. Combination of all three components, namely the class IIS enzyme, the ss DNA target sequence, and the complementing adapter, would result in target DNA cleavage at the specific predetermined site. The target ss DNA could be converted to the precisely cleaved ds DNA by DNA polymerase, utilizing the adapter oligodeoxynucleotide as primer. This novel procedure represents the first example of changing enzyme specificity by synthetic design. A practically unlimited assortment of new restriction specificities could be produced. The method should have many specific and general applications when its numerous ramifications are exploited. PMID- 3007287 TI - Conversion of the FokI endonuclease to a universal restriction enzyme: cleavage of phage M13mp7 DNA at predetermined sites. AB - Endonuclease FokI belongs to class IIS of restriction enzymes, for which the DNA cut points lie outside the enzyme-recognition sites. This permitted conferring new cleavage specificities by combining FokI with tailored oligodeoxynucleotide adapters. Such adapters carry a single-stranded (ss) target-recognition domain, complementary to the selected ss target DNA, and a double-stranded (ds) enzyme recognition site. Neither enzyme nor adapter alone has endonucleolytic activity toward phage M13mp7 ss DNA, whereas the enzyme-adapter complex cleaves this ss target DNA at the particular sites foreordained by the sequence of the ss domain of the adapter. Two kinds of adapters (32 and 34 nucleotides long), with opposing orientations of the asymmetric FokI recognition site, were constructed and shown to direct specific cleavage under a variety of conditions. In addition to FokI, other class IIS enzymes, HphI, MboII and BbvI, which alone do not cleave ss DNA, are suitable for construction of tailored enzyme-adapter complexes with predictable cleavage specificities. This report provides a preliminary experimental confirmation for the proposal of Szybalski [Gene 40 (1985) 169-173] for the design of adapter-enzyme complexes with novel and predictable specificities. Theoretically, using this approach any sequence could be precisely cleaved at a predetermined point. PMID- 3007288 TI - "ATG vectors' for regulated high-level expression of cloned genes in Escherichia coli. AB - A plasmid cloning vector system has been constructed that allows for the production of large quantities of foreign proteins or fragments thereof, in an unfused state. These vectors provide strong regulated trp-lac fusion promoters and the lacZ ribosome-binding site (RBS) followed by an ATG translation initiation codon at an appropriate distance from the RBS. The ATG codon is located within a unique NcoI restriction site (CCATGG). Digestion with NcoI exposes the ATG for fusion. Gene fragments lacking a prokaryotic RBS and/or ATG start codons can be inserted in several ways. Expression experiments using a truncated cI gene of bacteriophage lambda or a large portion of the coding region of the Herpes simplex virus type 1 glycoprotein D gene have been performed. The results of these studies show that the vectors are useful for the high-level expression of prokaryotic and eukaryotic genes in Escherichia coli. PMID- 3007289 TI - New vectors for construction of recombinant high-copy-number yeast acentric-ring plasmids. AB - Yeast acentric-ring plasmid 1 (YARp1), comprising 1453 bp of entirely yeast chromosomal DNA, is maintained in Saccharomyces cerevisiae as a high-copy, relatively stable plasmid. To determine the feasibility of using YARp1 as a yeast cloning vehicle, we subcloned the GAL1-10 promoter and the URA3 gene into YARp1 at different locations. To facilitate these constructions, a class of permuted YARp1 construction vectors was generated which enabled us to use various restriction sites in YARp1 as insertion points. Transformation frequencies, plasmid stabilities, and copy numbers of these YARp1 derivatives remained elevated, comparable to those of YARp1 itself. Also, when OMP decarboxylase was assayed using strains containing URA3-YARp's, specific activities of 100-300 times that of wild type were found. This evidence supports the use of YARp1 as a high-copy yeast-expression vector or for analyzing structural and regulatory DNA sequences. PMID- 3007290 TI - Cloning, sequencing and transcription of an inactivated copy of Bacillus amyloliquefaciens extracellular ribonuclease (barnase). AB - The gene for Bacillus amyloliquefaciens extracellular RNase (barnase) has been cloned in an inactive form in Escherichia coli following insertional mutagenesis by transposon Tn917. The nucleotide (nt) sequence of the gene was determined and the deduced amino acid (aa) sequence found to correspond almost precisely to the previously determined sequence. An open reading frame (ORF) of 72 codons precedes the mature sequence. The probable translation start site is 46 or 47 codons before the N-terminal alanine of the mature protein, 11 (or 14) bp from a putative ribosome-binding site (RBS). Within this leader sequence is a hydrophobic 15 aa core preceded by three basic residues which is characteristic of a secretory signal sequence. A pro-barnase protein with four extra aa at the N terminus has been detected extracellularly indicating that the signal peptidase cutting site lies before the mature protein. An inverted repeat that may act as a transcription terminator was found at the 3' end of the gene. The gene is maintained in E. coli with a short inverted repeat from the termini of Tn917 inserted into the coding sequence. Northern blot analysis of RNA from B. amyloliquefaciens shows an approx. 780-nt transcript produced during exponential and stationary growth phases. The inactive cloned gene produces an approx. 480-nt transcript in E. coli and two transcripts of approx. 480 and 780 nt in Bacillus subtilis. PMID- 3007291 TI - Molecular characterization and genetic mapping of two clusters of genes encoding chlorophyll a/b-binding proteins in Lycopersicon esculentum (tomato). AB - We have constructed a tomato genomic library in the gamma Charon 4 phage vector. The library was screened with a pea cDNA probe encoding a chlorophyll a/b-binding protein (CAB), and several recombinant phages containing tomato CAB genes were isolated and characterized by restriction mapping, heteroduplex analysis and nucleotide sequencing. Two phages with overlapping segments of the tomato genome contain a total of four CAB genes, all arranged in tandem. A third phase contains three CAB genes, two arranged in tandem and one in opposite orientation, and an additional, truncated CAB gene. Genetic mapping experiments showed that the four CAb genes on the first two phages belong to a locus, previously designated Cab-1, on chromosome 2. The CAB genes from the third phage belong to the Cab-3 locus on chromosome 3. Complete sequence determination of two CAB genes, one from each locus, and additional sequence determination of about 50% of each of the other five CAB genes showed that each gene within a CAB locus is more similar to other CAB genes in the same locus than it is to the CAB genes from the second locus. Furthermore, the polypeptides encoded by Cab-1 genes diverge significantly from those encoded by Cab-3 genes in the domains of transit peptide and the N terminus of the mature polypeptide but are essentially identical in the rest of the sequence. PMID- 3007292 TI - Analysis of cosmids using linearization by phage lambda terminase. AB - A group of cosmid clones was isolated from the region of the mouse t complex and analysed by a rapid restriction mapping protocol based on linearization of circular cosmid DNA in vitro. A plasmid capable of producing high levels of phage lambda terminase was constructed and procedures for in vitro cleavage of cosmid DNAs were optimised. After linearization, the cosmids were partially digested with restriction enzymes, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence, a step which we have described previously for clones in phage lambda (Rackwitz et al., 1984). High-resolution restriction maps derived by this method were used to identify and align the cosmids, to localise the position of repetitive sequences, and to interpret the results of electron microscopy heteroduplex experiments. PMID- 3007293 TI - Nucleotide sequence and genome organization of bacteriophage S13 DNA. AB - The complete sequence of bacteriophage S13 DNA has been determined. The molecule has 5386 nucleotides and differs from phi X174 by 87 transitions and 24 transversions. All the proteins, A,A*,B,C,D,E,F,G,H, J and K found in phi X174 are also present in S13. Due to changes in the H/A intergenic region of S13, the start of an additional protein, A', has been identified. Genes F and H coding for the capsid and spike proteins, respectively, are the least conserved in comparison to phi X174. Many of the silent changes, as well as some amino acid changes, are found in the same nucleotide sequence positions in phage G4, confirming the interrelationship between the three phages. PMID- 3007294 TI - One strand of ars189 from the maxicircle of Crithidia fasciculata transforms Saccharomyces cerevisiae more efficiently than its complementary strand as a single stranded DNA. AB - An autonomously replicating element (ars189) has been isolated from the maxicircle DNA of an insect trypanosomatid Crithidia fasciculata. This 189-bp fragment contains two copies of the yeast consensus ARS sequence of (A/T)TTTATPuTTT(T/A), has an A + T composition of 79.4%, and shows a large asymmetry in the distribution of adenine and thymine residues between the two strands. The complementary strands of ars189 have been cloned into an M13 vector containing the URA3 gene of Saccharomyces cerevisiae. When these circular single stranded (ss) DNAs were used to transform yeast spheroplasts, the M13 chimeric DNA carrying the strand of ars189 rich in adenine generated approximately four times more yeast Ura + transformants than the construct containing the thymine rich strand. In contrast, both strands of yeast ARS1 cloned into an M13 vector transformed yeast at an equivalent level. The conversion of ARS-containing ss DNAs to duplex forms in vivo and their subsequent autonomous replication have been verified by Southern hybridization analysis of extracts from yeast transformants. PMID- 3007296 TI - Rapid and reliable dideoxy sequencing of double-stranded DNA. AB - We report a simple and reliable protocol for nucleotide sequencing using the Sanger dideoxy technique on linearized double-stranded DNA molecules with specific oligonucleotide primers. The method is demonstrated for restriction fragments cloned into the plasmid vectors pSP64 and pSP65 using two vector specific primers, the M13 reverse primer and a new SP6 primer, flanking the multiple cloning site. Template DNA may be prepared by a rapid alkaline lysis procedure. Mild linearization conditions with the appropriate restriction endonuclease avoid the appearance of artifact bands. PMID- 3007295 TI - The complete sequence of the Bacillus phage phi 29 right early region. AB - We have sequenced the rightmost 2216 bp of the Bacillus phage phi 29 genome. This region encompasses the right early region and completes the sequence of the phi 29 early functions. The sequence of gene 17, an early gene implicated in the replication process, is presented. From these results we predict that gene 17 encodes a 19.1-kDal protein. Further analysis of the sequence revealed five previously undetected potential genes, encoding 12.6-, 12.4-, 15.2-, 6.2- and 4.6 kDal proteins. The biological efficacies of some of these putative genes were demonstrated using an Escherichia coli in vitro transcription-translation system. We also examined the transcriptional and translational signals present on this region of the genome. PMID- 3007297 TI - Formation of small circular DNA molecules via an in vitro site-specific recombination system. AB - The Cre-lox site-specific recombination system of bacteriophage P1 has been used to investigate the role of DNA flexibility in recombination. We have determined that a minimal distance of 82 bp must separate two loxP sites located on the same DNA molecule to allow these sites to undergo intramolecular recombination with one another. As a result of recombination, DNA circles as small as 116 bp have been produced. IN addition, we have demonstrated that the nuclease BAL 31 recognizes distortions in the DNA helix resulting from the formation of small DNA circles whose length is not a multiple of the helical repeat. PMID- 3007298 TI - The sequence of foot-and-mouth disease virus RNA to the 5' side of the poly(C) tract. AB - The nucleotide sequence of foot-and-mouth disease virus (FMDV) RNA to the 5' side of the poly(C) tract (S fragment) has been determined for representatives of the A and O serotypes of the virus. The two S fragments differ in length by five nucleotides (nt), with 367 nt for O1 compared with 362 nt for A10, due to a number of insertions/deletions. However, the two sequences show 86% homology. There are no conserved open reading frames (ORFs). Secondary structure predictions reveal a high degree of potential base-pairing, such that the entire S fragment sequence can be folded into a hairpin structure. PMID- 3007299 TI - Molecular characterisation of the Stc- mutation of Escherichia coli K-12. AB - The previously described Stc- (suppressor of TolC) mutation modifies the phenotype of tolC mutants from OmpF- to OmpF+. Restriction mapping of chromosomal DNA from Stc+ and Stc- strains was performed to investigate the nature of the mutation which was shown to be a deletion, upstream of the ompC gene. DNA from the region of the deletion was cloned into pUC18 and a 650-bp PstI-EcoRI fragment was sequenced. The deletion started 49 bp upstream of the AUG start codon of the ompC gene, thus removing part of the ompC promoter and the whole of the micF gene. We suggest that the deletion of micF gives rise to the Stc- phenotype since the effect of micF expression is assumed to reduce ompF expression, and the Stc- phenotype involves increase in ompF expression. PMID- 3007300 TI - Chimeric vector construction for higher-plant transformation. AB - A chimeric vector pKR612B1 was developed containing the neomycin phosphotransferase (APH) gene from the Tn5 transposon under the control of the gene VI promoter of cauliflower mosaic virus (CaMV), and was used to transform higher plant protoplasts. Plasmid pDOB612, the parental vector of pKR612B1, has two unique restriction sites, SmaI and BamHI, positioned just downstream of the CaMV gene VI promoter sequence. These unique cloning sites can be used for any kind of gene insertion into this vector. Using the polyethylene glycol transformation procedure, a large number of turnip and tobacco protoplasts were transformed and proved to be resistant to kanamycin (Km). From tobacco protoplasts whole Km-resistant plants were regenerated and shown to contain the integrated foreign gene. APH activity was detected in both transformed calli and in regenerated plants. DNA from transformed clones was analysed by Southern blot hybridization, showing the presence of the Tn5-derived gene. PMID- 3007302 TI - Selection for the transfer of phenotypically nonselectable chromosomal mutations to recombinant plasmids through introduction of an altered restriction site. AB - We describe a simple method to select for transfer of mutant alleles from the Escherichia coli chromosome to a plasmid which formerly carried the wild-type (wt) allele. The wt allele on the plasmid is modified by introduction of a unique restriction site (e.g., XhoI) and transformed into a rec+ strain carrying the mutant allele on the chromosome. Upon homogenotization, the efficiency of which was increased by UV irradiation of the transforming plasmid [Chattoraj et al., Gene 27 (1982) 213-222], plasmids carrying the mutant allele are formed which are resistant to XhoI. These plasmids are selected from the population by resistance to XhoI digestion coupled with the low transformation efficiency of linear DNA molecules in recA- strain. The method is efficient and rapid and has particular advantages in situations where the mutant allele is difficult to detect by its phenotype. PMID- 3007301 TI - Isolation and characterization of the yeast aspartyl-tRNA synthetase gene. AB - A yeast genomic library in Escherichia coli, constructed by insertion of Sau3A restriction fragments into the hybrid Saccharomyces cerevisiae-E. coli plasmid pFL1, was screened by a radioimmunoassay (RIA) for colonies expressing yeast aspartyl-tRNA synthetase (AspRS). Four clones were isolated by this technique. Data obtained by Southern and restriction analysis of the inserts showed a common 3.8-kb BamHI restriction fragment which, when inserted into the plasmid pFL1, gave a positive RIA. Several controls showed that this 3.8-kb insert codes for the entire AspRS: (i) S. cerevisiae transformed by the PFL1 plasmid carrying the 3.8-kb fragment overproduces AspRS activity by a factor of ten compared to the wild-type yeast strain; and (ii) a new protein with electrophoretic behaviour similar to AspRS and immuno-reactive toward anti-AspRS appears in crude extracts of transformed yeast and E. coli. PMID- 3007303 TI - Geriatric diabetes: latest research on the role of dietary fiber. PMID- 3007304 TI - [Experimental data in evaluation of the preventive effectiveness of various inhalation methods in silicosis]. PMID- 3007305 TI - [Relation between the biological effect and kinetics of quartz dust in the body]. PMID- 3007306 TI - [Regional disorders of blood flow in the microcirculatory bed of the pulmonary circulation in acute purulent pneumonia in children]. PMID- 3007308 TI - Separation of CFU-GM from normal human bone marrow mononuclear cells on density gradients of polyvinyl pyrrolidone coated silica gel (Percoll). PMID- 3007307 TI - Assessment of oxidative metabolism in adults with hepatocellular carcinoma in the Sudan. AB - The hypothesis that an increased rate of oxidative metabolism may be an initiator or promoter of hepatocellular carcinoma was tested in vivo. Elimination of antipyrine (phenazone) was used as an index of the activity of microsomal mixed function oxidative enzymes. Plasma antipyrine kinetics were examined in 10 patients with hepatocellular carcinoma and in 10 normal Sudanese adults. The half life, volume of distribution and clearance of antipyrine in patients were 18.8 +/ 7.9 hours (mean +/- SD), 33.8 +/- 7.7 litres and 23.7 +/- 10.1 ml/min, respectively; and in normal adults were 20.3 +/- 8.8 hours, 40.1 +/- 10.4 litres and 25.7 +/- 12.0 ml/min, respectively. These differences were not significant. Antipyrine plasma clearance when corrected for weight was similar in the two groups. This study suggests that in a population at risk for hepatocellular carcinoma, the overall activity of mixed function oxidative enzymes is not an important determinant in selectively increasing this risk. PMID- 3007309 TI - [Effect of sodium fluoride on magnesium-activated ATPase from human erythrocyte membranes]. PMID- 3007310 TI - [Protein phosphorylation and inositol phospholipid metabolism in activated rat mast cells]. AB - The interactions of rat mast cells with a variety of stimuli results in the secretion of a number of mediators through intracellular biochemical events. Calcium and cyclic AMP appear to be two major biochemical factors for the control of cellular activities and are known to activate protein kinases. Another line of evidence indicates that extracellular stimuli induce phospholipid turnover in their target cells. in this article, the author reviews protein phosphorylation and phospholipid metabolism, especially that of inositol phospholipid, during mediator release from stimulated rat mast cells. PMID- 3007311 TI - [Epinephrine uptake and release and presynaptic alpha 2-adrenoceptic regulation in the guinea pig hypothalamus]. AB - The uptake and the release of [3H] epinephrine ([3H] E) and the release of endogenous E in slices of guinea pig hypothalamus were investigated. [3H]E rapidly accumulated in the slices incubated in Krebs-Ringer solution containing [3H]E. Kinetic analysis indicated two components of E accumulation, one representing a high (Km1, 7.7 X 10(-8) M and Vmax1, 0.13 pmoles/mg/10 min) and the other a low (Km2, 1.8 X 10(-6) M and Vmax2, 1.4 pmoles/mg/10 min) affinity uptake system. Endogenous E released in response to electrical stimulation was estimated using gas chromatography and mass spectrometry. The electrical stimulation produced a release of both [3H]norepinephrine ([3H]NE) and [3H]E from hypothalamic slices preloaded with [3H]NE. With electrical stimulation of the slices, there was an efflux of [3H]E from tissues preloaded with [3H]E, in a current- and frequency-dependent manner. Electrically stimulated release of [3H]E from the slices was inhibited by tetrodotoxin (10(-6) M) and by a calcium-free medium containing EGTA (10(-4) M), in cases of up to 1 mA of intensity of electrical stimulation. The release of [3H]E induced by electrical stimulation was enhanced by yohimbine, and this effect was suppressed by clonidine. These results provide strong evidence for the neurotransmitter role of this catecholamine in the hypothalamus and suggest the possible existence of a presynaptic regulatory mechanism of E release, through the presynaptic alpha 2 receptors on the E nerve terminals. PMID- 3007312 TI - A study of somatostatin receptors in amygdaloid-kindled rat brain. AB - The present study indicates that in kindled rats there are no differences in the total number or affinity of the binding sites in the temporal cortex and a slight increase in the total number of binding sites in the cortex when compared with controls. These results, in view of our other observations, suggest that in the kindled rat brain there may be an increased release of SRIF but no down regulation of SRIF receptors in the temporal cortex and cortex. There appears to be a significant decrease in the number of SRIF receptors in kindled hippocampus. The mechanism by which this occurs remains unclear. PMID- 3007313 TI - Isolated myocarditis as a cause of sudden death in the first year of life. AB - A series of three cases of isolated myocarditis, presenting as sudden death in infancy, occurred over a period of 3 months. This prompted a review of the autopsy records of the Children's Hospital of Winnipeg. Over a period of 40 years, 24 cases of isolated myocarditis were traced from 3196 autopsies. Most (21 of 24) cases of isolated myocarditis occurred in infants less than 12 months of age. In 16 of the infants there were either no antecedent clinical signs (sudden deaths), or a short clinical history of less than 24 h duration. Heart weights, however, were greater than the 99th percentile of published normals in three infants and above the 95th percentile in a further 16 infants. Areas of hypertrophied fibres were seen even in infants with a short history. These latter findings suggest that a latent phase of myocarditis may exist. The responsible pathogens were identified very rarely, due to a lack of suspicion of the existence of myocarditis, and it is suggested that samples of myocardium should be submitted for virologic examination in all cases of sudden death in the first year of life. PMID- 3007314 TI - [Thyroid diagnosis with image producing procedures. 2: Technic and results of modern thyroid scintigraphy]. PMID- 3007315 TI - [Long loop reflexes--a clinically relevant method]. AB - Late reflex potentials have been know for a long time. On the upper limb it has been proven that these potentials have a transcortical pathway. The electrical stimulation of nerve trunks is easily applicable in clinical practice and produces clear long-loop responses. The typical results can be reproduced for extrapyramidal, cerebellar and pyramidal lesions by this method. The long-loop reflex is sensitive to lesions in the course of the pyramidal tract. PMID- 3007316 TI - The relation between hepatitis B virus infection and hepatocellular carcinoma. PMID- 3007317 TI - Assessment of HBV replicative status by receptors for polymerized human albumin. AB - Receptors for polymerized human albumin (pHSA-r) are expressed on HBsAg in larger numbers during complete viral replication. We have evaluated the usefulness of pHSA-r as a marker of high-level infection by a comparison with serum HBeAg, liver HBcAg and serum HBV-DNA. One-hundred-and-fifty-seven patients with HBsAg positive chronic liver disease were tested for HBeAg and pHSA-r titre. In 73 of them liver HBcAg was tested by immunofluorescence, while in the remaining 84 serum HBV-DNA was determined. Seventy-three subjects were HBeAg positive, 20/73 were HBcAg positive, and 60/84 had circulating HBV-DNA. The best cut-off for pHSA r was found at 1:204,800 (sensitivity of 67.6% and specificity of 95.7% in detecting viral replication). At this cut-off the concordance rate between pHSA-r and viral replication (as assessed by HBV-DNA or liver HBcAg) was 83%, a figure similar to that of HBeAg (88%). Three HBeAg negative subjects who were actually complete replicators were identified by the pHSA-r test. PMID- 3007318 TI - Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis. AB - Neutrophil functions of phagocytosis and intracellular killing of bacteria were examined in 40 patients with alcoholic cirrhosis of whom 18 had a superimposed acute alcoholic hepatitis. In 65% of these, defective neutrophil phagocytosis was demonstrable, and in 62.5% there was a defect of intracellular killing of either Staphylococcus aureus or Escherichia coli. Studies of the patients' serum failed to reveal inhibitors of neutrophil function. Additional assays of superoxide (O2 ) and hydrogen peroxide production, hexose monophosphate shunt activity, degranulation and cellular levels of granule enzymes and glutathione revealed that these neutrophil defects are caused by both reduced production of superoxide and defects of degranulation. The hydrogen peroxide/superoxide molar ratio was raised in patients' neutrophils, and the strong inverse correlation found between the value of this ratio and intracellular levels of reduced glutathione would be consistent with the hypothesis that the neutrophils from patients with cirrhosis are unable to detoxify hydrogen peroxide effectively and that this is a result of reduced levels of glutathione in the cells. The consequent increase in oxidant stress, both intra- and extracellularly, may be the cause of phagocytic and degranulation defects. The reduced responses of patients' neutrophils may be caused by previous exposure of the cells to activating stimuli in circulation, as evidenced by depleted intracellular levels of granule enzymes and glutathione. Neutrophils from the patients with a superimposed acute alcoholic hepatitis had depressed phagocytosis in the early stages of incubation but, on the whole, neutrophils from these patients had a greater capacity for ingestion and killing of bacteria than neutrophils from patients with cirrhosis alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007319 TI - Models of hepatic elimination. PMID- 3007320 TI - Immunohistochemical demonstration of p24 HTLV III major core protein in different cell types within lymph nodes from patients with lymphadenopathy syndrome (LAS). AB - The p24 protein is the major core protein of LAV/HTLV III which is the putative agent of the lymphadenopathy syndrome. By the use of an anti-p24 monoclonal antibody we have studied the presence of LAV/HTLV III infected cells in 20 lymph nodes obtained from lymphadenopathy syndrome patients: 14 lymph nodes were characterized by prominent follicular hyperplasia consistent with the early phase of the syndrome and six lymph nodes presented marked regressive changes. Cells positive for p24 were detected in 8/14 lymph nodes with hyperplastic changes and in 1/6 lymph nodes with regressive changes. Positive cells were most often located in germinal centres and were mainly characterized by a lymphoid morphology. However, immunoreactivity for p24 protein was also occasionally observed in some histiocytic-like cells and in high endothelial cells of post capillary venules, suggesting that these 'accessory cells' also play a role in the early phases of the lymphadenopathy syndrome. PMID- 3007321 TI - Immunocytochemical characterization of 10 pancreatic tumours, associated with the glucagonoma syndrome, using antibodies to separate regions of the pro-glucagon molecule and other neuroendocrine markers. AB - Histological diagnosis of neuroendocrine tumours can be hampered by their lack of peptide or amine immunoreactivity. In order to assess the usefulness of a range of specific and general markers of neuroendocrine differentiation, 10 pancreatic endocrine tumours, associated with high levels of circulating glucagon, were studied using histology, histochemistry, immunocytochemistry and electron microscopy. All cases showed immunoreactivity for one or other of the peptides derived from pro-glucagon, although only seven were found to contain immunoreactive pancreatic glucagon. The presence of secretory granules in eight of the tumours was demonstrated by electron microscopy, argyrophilia or chromogranin immunoreactivity. Not only was neuron specific enolase positively immunostained in all the tumours, thereby revealing their neuroendocrine nature, but also the intensity of the immunostain was higher in four of the five malignant ones than in the rest of the cases. Pancreatic polypeptide was present in non-glucagon cells in six out of 10 cases. Our results emphasize the importance of the use, not only of general histochemical and immunocytochemical tests but also antibodies to all possible derivatives of the precursor form of the active tumour product in the diagnosis of possible endocrine tumours. In this way, any abnormal molecular forms of the peptide synthesized by tumour cells with altered synthetic and secretory mechanisms may be detected. PMID- 3007322 TI - Comparative study of the histopathology of breast cancer in a screened and unscreened population investigated by mammography. AB - The histopathology of 360 surgical resections for breast cancer in a consecutive series of patients aged 45-69 years from 1979-1983 is described. Two hundred and seventeen patients who were offered screening (screened patients) were compared with 143 patients who were referred as out-patients with breast problems and were not offered screening (unscreened patients). Both groups were investigated by mammography. Comparisons between tumour staging, grading and quadrant involvement are reported. In the screened patients 31% were in the in-situ stage or had an invasive carcinoma less than 1.0 cm in maximum diameter, compared with 7% in the unscreened patients. Conversely 26% of the screened patients had cancers greater than 2.0 cm compared with 52% in the unscreened group. The percentage of cancer patients with lymph node metastases was comparable in both 1.0-2.0 cm and greater than 2.0 cm groups of invasive carcinoma. There were more multiquadrant cancers in the unscreened patients (32%) than screened patients (17%) and this was mainly due to differences in the incidence of carcinomas 1.0-2.0 cm in diameter. This suggests that invasive tumours of comparable size are more likely to produce symptoms leading to detection if multiquadrant. Multicentric cancers were more common in unscreened patients. Differences in the histological grading related to tumour size were found within the screened and unscreened groups but not between the two groups. PMID- 3007323 TI - Primary neuroectodermal tumour of the testis. AB - A 51-year-old male presented with metastasis of a small cell carcinoma of unknown origin in a right inguinal lymph node. Clinical and laboratory studies failed to locate the primary tumour. After three years, a swelling appeared in the right testis, which was removed. Histological examination revealed a proliferation of small tumour cells forming irregular masses or nests that occupied most of the testicular parenchyma. At the periphery of the testicular parenchyma a few seminiferous tubules could be observed, showing a low and incomplete seminiferous epithelium and numerous tumour cells in the lumen. Most of the tumour cells showed a euchromatic nucleus with small nucleoli and scanty cytoplasm. Among these cells, larger binucleate or trinucleate cells as well as small cells with pyknotic nuclei were also observed. Mitoses were numerous. Electron microscopy revealed some tumour cells with 80 to 100 nm vesicles containing electron-dense granules. Some cells displayed dendrite-like prolongations with numerous intermediate filaments and electron-dense vesicles. This tumour is compatible with a primary neuroectodermal tumour of the testis. PMID- 3007324 TI - Federal Government intensifies its efforts in the mental health aspects of AIDS. PMID- 3007325 TI - Immunohistologic detection of estrogen receptors in paraffin-embedded breast cancers: correlation with cytosol measurements. AB - Paraffin-embedded sections of 76 human breast tissue specimens were analyzed for estrogen receptors (ER) and endogenous bound estrogen (ER-E). Preincubation of sections with polyestradiol phosphate was followed by stabilization of the complex with glutaraldehyde. The bound hormone was then visualized by the peroxidase-antiperoxidase (PAP) technique with antiestradiol as the primary antiserum. Normal breast tissue and benign proliferations were consistently positive for ER and ER-E. All specimens were examined for free and bound receptors in cytoplasm and nuclei. Among the carcinomas examined, a high correlation was found between the presence of ER by the PAP method and by the biochemical analysis of cytosol preparations. The PAP method, requiring no special preparation of surgical specimens, overcomes many of the disadvantages of the cytosol method and adds the advantage of independent evaluation of nuclear and cytoplasmic estrogen binding sites. PMID- 3007326 TI - Human papillomaviruses of different types in precancerous lesions of the uterine cervix: histologic, immunocytochemical and ultrastructural studies. AB - In a prospective study of 34 women with abnormal Papanicolaou smears, biopsy and cervicovaginal lavage specimens were analyzed for the presence of human papillomaviruses (HPVs) by Southern blot analysis and probes for HPVs 6, 11, 16, and 18. In 22 of the 23 patients with cervical lesions (96%), HPV DNA was identified in one or more specimens. All patients in whom HPV DNA was found had either koilocytotic or dysplastic lesions on biopsy or Papanicolaou smear. Immunocytochemical demonstration of HPV in biopsy samples was associated with the presence of large amounts of HPV DNA and with the ultrastructural identification of viral particles. The presence of HPV DNA in cervical biopsy specimens was limited to discrete geographic areas of the cervix with histologic abnormalities. Although HPV 16 and other related HPV types were found in all cases of severe cervical intraepithelial neoplasia, the type of HPV present in a given specimen could not be predicted on the basis of morphologic, immunocytochemical, or electron microscopic findings. It is concluded that virtually all dysplastic lesions of the cervix contain HPV DNA, that HPV is thus likely to be a major etiologic agent in the pathogenesis of cervical dysplasia, and that histopathologic features are not predictive of HPV type. PMID- 3007327 TI - Beta A and beta thal DNA haplotypes in Sicily. AB - A close association between specific restriction fragment polymorphism patterns and specific mutations in Mediterranean people with thalassemia has been demonstrated by Kazazian et al. (1984). This finding is useful to characterize the number and types of mutations in each ethnic group for setting up prenatal diagnosis in the first trimester of pregnancy by the oligonucleotide technique. For this reason we studied 99 beta thal and 46 beta A chromosomes in the Sicilian population. We found seven different cleavage patterns, not considering two new haplotypes so far uncharacterized. Many of the patients (68.3%) were genetic compounds for different haplotypes while only 31.7% were haplotype homozygotes. They may still be thalassemia compound heterozygotes. These findings confirm the molecular basis of the heterogeneity of beta thalassemia in Sicily. PMID- 3007328 TI - X-linked ichthyosis, due to steroid sulphatase deficiency, associated with Kallmann syndrome (hypogonadotropic hypogonadism and anosmia): linkage relationships with Xg and cloned DNA sequences from the distal short arm of the X chromosome. AB - We report a large Italian pedigree in which five out of six males are affected by a syndrome, following an X-linked inheritance pattern, characterized by ichthyosis, hypogonadotropic hypogonadism, and anosmia. The concurrence of features of X-linked ichthyosis (XLI) with those of Kallmann syndrome, another disease often inherited as an X-linked trait, prompted us to perform biochemical, cytogenetic, and molecular studies in relation to the short arm of the X chromosome (Xp). Steroid sulphatase (STS) activity was found to be completely deficient in all affected members of the family. Prometaphase chromosome analyses of two obligate heterozygous women and one affected male showed normal karyotypes. Xg blood group antigen analysis and molecular studies employing cloned DNA sequences from the distal segment of the Xp (probes RC8, 782, dic56, and M1A), did not provide evidence for deletions or rearrangements of the X chromosome. The linkage analysis showed no crossovers between the disease, Xg, and DXS143, the locus defined by probe dic56, thus suggesting the possibility of a linkage between these two markers of the distal segment of Xp and the X-linked ichthyosis, hypogonadism, and anosmia syndrome. PMID- 3007329 TI - Assessment of small polymorphisms in defined human collagen gene segments. AB - Restriction fragment length polymorphisms (RFLPs) greater than 1.5 kb in size have been identified in all but one of the human fibrillar collagen genes. However, the number of informative RFLPs for these genes is still limited. Here we present the conjunct use of two techniques for the assessment of small length variations within defined segments of the genome. This strategy has the potential to be used for the allelic exclusion of cloned genomic fragments prior to sequencing. Moreover it can be a useful and sensitive tool to determine if genetic linkage exists between abnormal phenotypes and loci where conventional Southern blotting analysis has failed to detect informative RFLPs. PMID- 3007330 TI - The Alu I-induced bands in metaphase chromosomes of orangutan (Pongo pygmaeus). Implications for the distribution pattern of highly repetitive DNA sequences. AB - Restriction endonucleases have been recently proved to be active on fixed chromosomes, thus they are useful in chromatin structure studies. Within this class of enzymes, Alu I is able to detect the presence and localization of highly repetitive DNA sequences in human and in other mammalian and dipteran species. In this paper the pattern obtained on fixed metaphase chromosomes of orangutan (Pongo pygmaeus) by Alu I digestion and Giemsa staining is shown. The results are discussed in the light of the distribution, in this species, of the I-IV human satellite DNAs. It is also suggested that in Pongo some highly repetitive sequences, different from the major human satellites, are present. PMID- 3007331 TI - Recording from the Aplysia abdominal ganglion with a planar microelectrode array. PMID- 3007332 TI - A peripheral nerve information transducer for amputees: long-term multichannel recordings from rabbit peripheral nerves. PMID- 3007333 TI - Autocrine models of B-lymphocyte growth. II. Interleukin-1 supports the proliferation of transformed lymphoblasts but not the stimulation of resting B cells triggered through their receptors for antigen. AB - Purified, monocyte-derived interleukin-1 (IL-1) was found to provide growth support for Epstein-Barr virus (EBV) transformed B-lymphocytes seeded at densities below which their own autostimulatory factors were limiting. By contrast, highly purified resting B cells triggered via their receptors for antigen failed to respond to identical preparations of IL-1 by DNA synthesis. That successful priming of the B cells had occurred was evidenced by a transient rise in RNA synthesis and the ability of the cells to respond to T-cell supernatants by DNA synthesis. The findings indicate that while IL-1 might perform an autostimulatory function in B lymphocyte proliferation it is not by itself sufficient to provide growth support for resting B cells activated through their receptors for antigen. The implications of these observations for autocrine models of B-cell growth are discussed. PMID- 3007334 TI - Human immune responses to herpes simplex virus, varicella-zoster and cytomegalovirus in vitro. AB - The cell principally responsible for lymphocyte proliferation to herpes simplex virus (HSV), varicella-zoster (VZ) and cytomegalovirus (CMV) has been shown to be a T cell of helper phenotype. Lymphocytes from a proportion of proliferation positive normal individuals produced anti-viral antibody in vitro. Although in some cases, and at some time-points, the antibody was specific for the priming virus, in others, antibodies to more than one virus were detected. Similarly, some T-cell clones proliferated specifically to the priming virus, whereas others were not specific for the virus used in the priming culture. Two clones helped the production of HSV-specific antibody, one by autologous, the other by both autologous and allogeneic non-T cells. PMID- 3007335 TI - Production of an antibody-like factor in the sea star Asterias rubens: involvement of at least three cellular populations. AB - Cells from the axial organ of sea stars stimulated in vivo with TNP coupled to polyacrylamide beads and subsequently cultured in vitro were able to produce an antibody-like factor which induced the lysis of mammalian red cells sensitized with TNP. The axial organ cells were fractionated in two populations, adherent and non-adherent to nylon wool. The release of the antibody-like factor required the contact of both populations. When the adherent cells were disrupted by sonication the factor was not produced, but the non-adherent cells could be substituted by their membranes. Destruction by silica of the phagocytic cells present in the adherent population inhibited the production of the factor. The addition of mercaptoethanol to the cultures was essential and did not neutralize the effect of silica. It is concluded that at least three types of cells are involved in the production of the antibody-like factor adherent and non-adherent cells, and phagocytes. PMID- 3007336 TI - Non-adherent, low-density cells from human peripheral blood contain dendritic cells and monocytes, both with veiled morphology. AB - Dendritic cells (DC) from human peripheral blood, known to adhere transiently and to become non-adherent by 16 hr, can be separated in the low-density interface on hypertonic Metrizamide gradients. Many more low-density cells (5.8% of the mononuclear cells separated on Ficoll) were obtained from the population that was non-adherent after only 90 min. Over 95% of these low-density cells had veiled morphology. A proportion were monocytes by phenotypic and phagocytic properties. One-third of the cells (on average) were DC on the basis of lack of monocyte phenotype and of potency as stimulators in the mixed lymphocyte reaction. Including both the 16 hr and 90 min non-adherent cells, over 2% of the mononuclear cells isolated from human peripheral blood may be DC. PMID- 3007337 TI - Characterization of a novel Burkitt's lymphoma-associated antigen: GP70. Reactivity of anti-GP70 antibodies with various malignant and non-malignant cell lines. AB - A glycoprotein of 70 kDa (GP70) was isolated from sera of Burkitt's lymphoma (BL) patients and used to immunize rabbits. Anti-GP70 antibodies at a high titer were obtained and used for screening of cancer cells of various origin by the indirect immunofluorescence test. Thus, 66% of BL-cell lines tested were positive to GP70. On the other hand, all lymphoblastoid cell lines tested were negative. Moreover, all peripheral blood cells and mononuclear cells from tonsils were negative, indicating specificity of antibodies to malignant transformation. Comparison between positively stained BL-cell lines indicated no correlation between the presence of GP70 and EBNA. Positive stain (1-5%) obtained with bone marrow cells might indicate that anti-GP70 antibodies are directed against a surface membrane differentiation glycoprotein. PMID- 3007338 TI - Anti-Epstein-Barr virus memory T-cell response in Chediak-Higashi syndrome patients. AB - Memory cytotoxic T-cell responses to Epstein-Barr virus transformed B cells were studied in two EBV seropositive Chediak-Higashi syndrome patients who were manifesting the terminal lymphoproliferative stage of the syndrome. The number of cells required for 50% regression of EBV-infected autologous lymphoblastoid cell lines was similar to that in the parents and healthy subjects. This indicates adequate memory cytotoxic T-cell control of EBV-infected B lymphocytes. Defective mechanisms other than those mediated by circulating cytotoxic cells, might be responsible for the chronic active EBV infection observed in these patients. PMID- 3007339 TI - The role of the transferrin receptor in lymphocyte activation. AB - Blockade of the transferrin receptors whose expression is induced in lymphocytes incubated with the mitogenic lectin phytohaemagglutinin (PHA) does not affect the initial stimulation of protein synthesis but does strongly and progressively inhibit the subsequent induction of DNA synthesis. When the effects of transferrin receptor blockade on the induction of the enzymes uridine kinase (whose induction begins early in G1 phase of the cell cycle) and thymidine kinase (whose induction is closely associated with DNA synthesis) were examined, both enzymes were found to be induced normally. This indicates that the function of the transferrin receptor is directly to provide a component essential for DNA synthesis itself (probably iron) rather than to act as the receptor for a general signal required to initiate entry into S-phase. PMID- 3007341 TI - Cytolytic T lymphocyte-defined retroviral antigens on normal cells: encoding by the Akv-1 proviral locus. AB - We previously described the generation and specificity of H-2-restricted cytolytic lymphocytes (CTL) directed against tumors induced by AKR/Gross murine leukemia viruses (MuLV). Such anti-AKR/Gross virus CTL demonstrated type specificity; only tumors induced by endogenous MuLV that expressed the Gross cell surface antigen were lysed. These CTL and their precursor also recognized normal spleen cells from AKR-H-2b, but not AKR-H-2b, Fv-1b mice, however, suggesting that N-ecotropic, retrovirus-associated antigens were primarily involved. Here, expression of these CTL-defined retroviral antigens by H-2b-positive AKR X C57L recombinant inbred strains was examined by using normal spleen cells as stimulators in the generation of specific anti-AKR/Gross virus CTL. Analysis of the strain distribution pattern of stimulation indicated that a single proviral locus, Akv-1, was primarily, if not entirely, responsible for CTL-defined retroviral antigen expression. The lack of correlation with two other well defined proviral loci was interesting. Whereas Akv-3 is known to encode a defective virus, Akv-4 has been shown to code for an infectious virus thought to be very similar or identical to that of Akv-1. Although quantitative differences cannot be formally excluded, dose response experiments argued against this possibility and suggested that Akv-1 and Akv-4 may exhibit qualitative differences germane to antiviral CTL recognition. PMID- 3007340 TI - Rearrangement of 21-hydroxylase genes in disease-associated MHC supratypes. AB - Human cDNA probes for 21-hydroxylase (21-OH) and for complement component C4 are used on restriction digests of the members of several families with interesting supratypes. The presence of two Taq I fragments of 3.7 kb and 3.2 kb in size with a 21-OH probe is confirmed in most individuals who show no evidence of C4 deletions or 21-OH deficiency. Most individuals also show a doublet of weakly hybridizing bands at approximately 2.5 kb, the smaller of which is part of the 21 A gene. The arrangement of the 21-OH genes on disease-associated supratypes was examined, and it is shown that copies of the same supratype from unrelated individuals are usually identical. Evidence is provided for deletions of 21A on the B8, C4AQ0, C4B1, BfS, DR3 and B18, C4A3, C4BQ0, BfF1, DR3 supratypes and a duplication of 21A on the B14, C4A2, C4B1/B2, BfS supratype. Gene rearrangements may be relevant to diseases such as juvenile onset diabetes mellitus. PMID- 3007342 TI - Correlation between an HLA-DQ alpha length polymorphism of messenger RNA and serologically defined specificities (DQw1, DRw53, DR3+5). AB - mRNAs for the two chains of the HLA-DQ molecule were analyzed, in particular the DQ alpha mRNA whose polymorphism had previously been suggested (Schenning et al. 1984). Northern blot transfers of the mRNA of 12 LCLs and of B lymphocytes from a healthy donor were carried out. We report that a length polymorphism of DQ alpha mRNA exists, and we show that it can be correlated with serologically defined specificities (DQw1, DRw53, DR3+5). This correlation could be explained by a linkage disequilibrium, as these specificities are considered to be different from those carried by the DQ molecule (except for the DQw1 specificity). PMID- 3007343 TI - HLA-DQ beta chain DNA restriction fragments can differentiate between healthy and narcoleptic individuals with HLA-DR2. PMID- 3007344 TI - HLA-DR2-associated Dw subtypes correlate with RFLP clusters: most DR2 IDDM patients belong to one of these clusters. AB - Two variants of the serologically defined HLA-DR2 specificity have been reported: DR2 long and DR2 short. Distinct HLA-DR2-associated Dw subtypes have been described at the cellular level. In the Israeli population, DR2 individuals may be grouped into three clusters: DR2/Dw2, DR2/Dw12, and DR2/Dw"AZH". A new approach for the study of the polymorphism of HLA class II genes is to investigate restriction endonuclease fragments obtained from genomic DNA with specific class II cDNA probes. Previous analysis of DQ beta restriction endonuclease fragments subdivided the DR2 haplotypes into two subsets: a DQR1 positive subset and a DQR2.6-positive subset. These two subsets behave in the population as alleles that split HLA DQw1. In the present study, we have analyzed class II DQ alpha, DQ beta, and DR beta restriction fragment length polymorphism (RFLP) in HLA-DR2/Dw-typed healthy, unrelated Israeli individuals, as well as in 11 French HLA-DR2 insulin-dependent diabetes mellitus (IDDM) patients and 11 French DR-matched controls. Three DQ beta allelic clusters (DQR2.6, DQR1, and DQR12) were observed among the DR2 haplotypes and clearly correlated with Dw2, Dw"AZH", and Dw12, respectively. The vast majority of the DR2 IDDM patients (9 out of 11) fit into the DQR1 cluster which correlates with Dw"AZH", while only two patients (2 out of 11) belong to the DQR2.6 cluster (Dw2-like). In contrast, among 11 DR-matched healthy controls, 9 belonged to the DQR2.6 cluster and only 2 belonged to the DQR1 cluster. These studies establish the correlation between the DR2-associated Dw subtypes with specific RFLPs, and indicate that the frequency of the DQR1 subset which correlates with Dw"AZH" is increased in DR2 IDDM patients. PMID- 3007345 TI - HLA-JY328: mapping studies and expression of a polymorphic HLA class I gene. AB - The JY328 clone was identified in a human genomic library using cDNA corresponding to mRNA for HLA-B7 as a probe. The L/328 cell line was established by cotransformation of mouse Ltk- cells with the herpes thymidine kinase gene and clone JY328. On Northern blots, RNA from L/328 strongly hybridized to an HLA class I probe, and an antigen was recognized by an anti-HLA class I framework antibody on the cell surface. A DNA probe corresponding to a segment of intron 7 was developed by comparing the nucleotide sequence of clone JY328 with that of other HLA class I-type genes. Using the radiolabeled probe to screen Southern blots of DNA from families with siblings exhibiting intra-HLA recombinations, a restriction fragment length polymorphism was revealed--a 1.4 kb BstE II band not present in all individuals. A corresponding fragment was apparent in the base sequence of clone JY328. The occurrence of this band on Southern blots established that JY328 maps distinct from and centromeric to the HLA-C locus and near to the HLA-B locus. Antibody absorption studies and cytotoxicity tests indicated that the JY328 gene product was not an HLA-B antigen but that it did specifically absorb CW7-specific antibody. In sum, these results suggest a novel, polymorphic HLA class I gene which expresses a product serologically similar to HLA-Cw7 but which does not map within the corresponding locus. PMID- 3007347 TI - An isolated beta 1 exon next to the DR alpha gene in the HLA-D region. AB - A cosmid clone containing the DR alpha gene and a beta 1 exon of a DR beta related gene was isolated from a human cosmid clone bank made from the consanguineous DR7 cell line MANN. No other DR beta-related exons were found on this clone. The beta 1 exon was located about 15 kb away from the DR alpha gene in a tail-to-tail (3' to 3') orientation. The exon contained several deleterious mutations: a defective splice site at the 5' end, two translational frame shifts (a 1 bp deletion and a 1 bp insertion), and three extra cysteine residues. Nucleotide and amino acid sequence comparisons of the beta 1 exon indicated that although it is substantially different from other class II beta-chain genes, it is slightly more related to DR than to any other class II gene. The DR beta related sequence was on a DNA fragment which showed no polymorphism on a panel of cell lines with Eco RI or Pst I. These Southern blots, however, revealed a related, polymorphic sequence in the human genome. Nucleotide sequences in the intron flanking the beta 1 exon shared greater sequence homology than the beta 1 exon itself when compared with the DR beta genomic sequence. The exon may play a role in the generation of variation in expressed class II beta-chain genes and it may be a relic of a different subset of class II products. PMID- 3007346 TI - Xenotropic virus-related restriction patterns of non-H-2 histocompatibility mutant mice strains. AB - The relationship between alteration in the number of xenotropic virus-related sequences and non-H-2 histocompatibility (H) mutations in mice was investigated. Mutant classifications included gain, loss, and loss-gain mutations. Genomic DNA from a panel of non-H-2 H mutant strains on the C57BL/6 and BALB/c backgrounds was digested with a set of restriction enzymes with varying numbers of sites within endogenous xenotropic-related sequences. The digested DNA was then resolved on agarose gels. Southern blots of digested DNA were hybridized with the pXenv probe specific for the env sequence of xenotropic viral sequences. The number of hybridizing bands varied from 7 to 19, depending on the restriction enzyme and inbred background. Most mutant strains were identical in their restriction patterns to the respective background strains. However, two B6 mutant strains, KH84 and HZ54, differed from C57BL/6 at a single band which appeared to be inherited from BALB/c in the derivation of the two congenic strains. The HZ43 strain lacked a male-specific band shared by both C57BL/6 and BALB/c; this loss was evidently independent of the original mutation which was observed to be autosomal. However, the KH148B and KH84 strains on the C57BL/6 background lacked single B6 bands. Both mutants were classified as gain mutants. An examination of previous reciprocal graft rejection patterns and retrovirus linkage to non-H-2 H loci indicated a strong inverse relationship between a linked retroviral sequence and presentation of a non-H-2 H antigen. This inverse correlation is consistent with reports of gene inactivation following retroviral insertion. PMID- 3007349 TI - Life span and 'immortalization' of mammalian cells. PMID- 3007348 TI - DQ alpha and beta RFLP reveals the composition of the DQ molecule recognized by T cell clones. AB - Pst I RFLP, revealed with DQ alpha and DQ beta probes, was compared with Taq I RFLP using a panel of DR-homozygous cell lines and HLA-typed family members. Taq I patterns, characteristic for each DR-associated DQ alpha and beta allelic forms, were recognized in the homozygous state and then proven to segregate in the heterozygous members of informative families. The presence of both specific alpha and beta chains was found to be necessary to form the type of DQ molecule specifically recognized by two alloreactive T-cell clones. Particular alpha and beta associations also seem to be responsible for some Dw splits of the DRw6 positive cells. Taq I RFLP analysis may be more complex than the Pst I analysis, but is certainly more informative and complete, considering the type of information we were seeking by performing these types of experiments. PMID- 3007350 TI - Age-related changes in immunologic and hormonal activities. AB - The immune system has been a focus of intensive gerontological research, because it appears to be involved in many deleterious diseases associated with ageing, including cancer, and because the likelihood of successful therapeutic strategies for altered immune functions in the elderly is high. Research efforts have been centred on two levels of biological complexity--the cell and the organism. At the cellular level, emphasis has been on the nature of the alteration in immune functions with age, and, at the level of the organism, whether the diseases associated with immunological dysfunction of elderly individuals can be controlled by immunomodulation. We discuss age-related changes at the cellular level with emphasis on qualitative changes in the antigen/mitogen responsive cells with respect to production of and response to hormones. We then consider whether ageing 'hot spots' exist along the differentiation vector of antigen/mitogen responsive cells; i.e., whether immune cells are vulnerable to ageing only at certain stages of their differentiation processes. We conclude by considering the use of tissue grafting and chemical treatment, the two most encouraging methods of immunopotentiation at the level of the organism, as an option in modulating resistance of old individuals to cancer. PMID- 3007351 TI - Use of the converting enzyme inhibitor enalapril in renovascular hypertension. Effect on blood pressure, renal function, and the renin-angiotensin-aldosterone system. AB - Thirteen patients were entered into a protocol to assess the safety and efficacy of enalapril (MK 421), 5 to 20 mg b.i.d., and hydrochlorothiazide, 50 to 100 mg daily, for the treatment of renovascular hypertension. Specifically monitored were the effects of therapy on blood pressure and pulse, renal function, and the renin-angiotensin-aldosterone axis. Enalapril and hydrochlorothiazide therapy produced excellent control of blood pressure with no adverse side effects. After approximately 8 weeks of therapy, renal vascular resistance was decreased and no adverse effects on glomerular filtration rate or renal blood flow were noted, except in one patient with a functional unilateral stenotic kidney. Patients receiving enalapril and hydrochlorothiazide showed stimulation of plasma renin activity and suppression of plasma angiotensin II, although the initial degree of suppression was not sustained in all patients during prolonged therapy. Although plasma aldosterone concentration was initially suppressed, the degree of suppression was not sustained. Nine patients have been followed for an additional 6 months; none have experienced further progression of renal disease, as assessed by repeated measurements of glomerular filtration and effective renal plasma flow. These results suggest that combined enalapril and hydrochlorothiazide therapy is safe and effective in the medical management of renovascular hypertension and that blood pressure control may be achieved in the absence of sustained interruption of the renin-angiotensin-aldosterone system. PMID- 3007352 TI - Renal alpha 2-adrenergic receptors multiply and mediate sodium retention after prazosin treatment. AB - Renal nerve stimulation-induced antinatriuresis normally is mediated through post synaptic alpha 1-adrenergic receptors; however, prazosin-induced alpha 1 adrenergic receptor blockade is associated clinically with sodium retention and not natriuresis. To study whether alpha 2-adrenergic receptors mediate renal nerve stimulation-induced antinatriuresis after chronic prazosin treatment, Sprague-Dawley rats were pretreated for 3 days with prazosin (3 mg/kg/day i.p. plus 0.15 mg/ml drinking water) or vehicle (untreated). In isolated perfused (Krebs-Henseleit; Ficoll, 3.5 g/dl, + albumin, 1.0 g/dl at 36 degrees C) kidneys from untreated rats, subpressor levels of renal nerve stimulation (approximately 1 Hz, 10 V, 1 msec) decreased (p less than 0.05) sodium (from 4.50 +/- 0.42 to 1.71 +/- .23 muEq/min) and urinary excretion rate (from 87.2 +/- 4.1 to 57.9 +/- 3.9 microliter/min). Adding prazosin (30 nM) to the perfusate completely (approximately 90%) reversed this effect (p less than 0.05), while alpha 2 adrenergic receptor blockade with yohimbine (300 nM) had no effect. In perfused kidneys from prazosin-treated rats, renal nerve stimulation decreased (p less than 0.05) sodium (from 3.24 +/- .40 to 1.32 +/- .27 muEq/min) and urinary excretion rate (from 78.7 +/- 5.0 to 54.1 +/- 5.3 microliter/min). However, adding prazosin (100 nM) to the perfusate produced only a slight, insignificant reversal of these effects; prazosin plus yohimbine were required to completely reverse the effects. These results suggest that renal nerve stimulation-induced sodium reabsorption was activated by alpha 1-adrenergic receptors in untreated rats and in part by alpha 2-adrenergic receptors in rats pretreated for 3 days with prazosin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007353 TI - Degradation of hyaluronic acid by polymorphonuclear leukocytes. AB - Degradation (depolymerization) of hyaluronic acid is readily accomplished by superoxide-ion-generating systems, especially those which beget secondary free radicals. It has been presumed, but not confirmed, that this is the mechanism by which neutrophils might alter synovial fluid viscosity. We have demonstrated, in a neutrophil (PMN) superoxide system, physical disruption of the hyaluronate macromolecule using column chromatography and by measurement of intrinsic viscosity. In addition, comparison of calibrated free radical fluxes between a cell-free superoxide system and a neutrophil system revealed very close parallels in iron requirement, inhibition by free radical scavengers, and magnitude of effect. It is concluded that oxygen-derived free radicals are probably the major, if not sole, mechanism by which neutrophils might degrade hyaluronate. PMID- 3007354 TI - Calmodulin-dependency of human neutrophil phosphodiesterase. AB - A recent study has reported that the phosphodiesterases of human neutrophils are calmodulin-insensitive (Smolen and Geosits, Inflammation 8:193-199, 1984). In the present study, two forms of human neutrophil phosphodiesterase were separated by chromatography on DEAE-52. Peak I phosphodiesterase is activated 2.3-fold by calcium and calmodulin but is not stimulated by either calcium or calmodulin alone. Calmodulin-dependent activation of the phosphodiesterase is blocked by both 20 microM trifluoperazine and 20 microM W-7. Peak II is not stimulated by calmodulin. These findings suggest that calmodulin may play an important role in regulating alterations in cyclic nucleotide metabolism that accompany neutrophil activation. PMID- 3007355 TI - Human polymorphonuclear leukocytes release leukotriene B4 during phagocytosis of Staphylococcus aureus. AB - We studied release of leukotriene B4 (LTB4) by human polymorphonuclear leukocytes (PMNs) during phagocytosis of staphylococci in the presence or absence of arachidonic acid. The 12 X 10(7) PMNs incubated with 3 X 10(9) opsonized S. aureus and 50 microM arachidonic acid released 1.45 +/- 0.42 nmol LTB4. No LTB4 was detected after stimulation of PMNs with S. aureus or arachidonic acid by themselves. However, by increasing the concentration of arachidonic acid to 200 or 400 microM, 1.22 +/- 0.45 and 1.98 +/- 0.49 nmol LTB4, respectively, was released by PMNs. The effect of different bacteria-PMN ratios on LTB4 production was also studied. LTB4 varied from 0.3 to 2.0 nmol when bacteria/PMN ratios increased from 5 to 50 (respectively) in the presence of 50 microM arachidonic acid. Thus, phagocytizing PMNs produce LTB4 in the presence of arachidonic acid, and its production is dependent on the number of bacteria phagocytized. PMID- 3007356 TI - Modulation of phospholipid methylation in rabbit leukocytes by indomethacin. AB - Methylation of membrane phospholipids has been implicated as an early biochemical signal for the chemotactic migration of phagocytic cells into inflammatory sites. In this study, the ability of indomethacin to modulate phospholipid methylation as one of its mechanisms of antiinflammatory action was investigated. Nontoxic doses of indomethacin (10(-4) M and 10(-5) M) were found to retard the methylation of phosphatidylmonomethylethanolamine (PMME) to phosphatidyldimethylethanolamine (PDME) and phosphatidylcholine (PC) in rabbit leukocytes, suggesting inhibition of methyltransferase II activity. Indomethacin was, however, without effect on the uptake of L-[methyl 3H]methionine by rabbit leukocytes. It is suggested that the inhibition of membrane phospholipid methylation could result in the suppression of inflammatory responses such as prostaglandin and leukotriene synthesis, generation of reactive oxygen radicals, and chemotaxis. PMID- 3007357 TI - Antioxidant action of natural health products and Chinese herbs. AB - The scavenging effects of reactive oxygen species (ROS) by two natural health products, antioxidant analogs (AOA), and green magma (GM), and 16 medical Chinese herbs were investigated in two in vitro ROS-generating systems, activated neutrophils and xanthine-xanthine oxidase. Native, unheated AOA and GM products significantly reduced ROS levels, while unheated Chinese herbs had a negligible effect on ROS levels. In contrast, heat-extracted Chinese herbs and AOA markedly, and GM mildly, suppressed the levels of ROS in both systems. The ROS scavenging activity of these native, unheated products was unaffected by dialysis, but that of heated products was markedly diminished by dialysis. Further, the incubation of these products with gastric juice obtained by a gastric tube from healthy volunteers revealed results comparable to those induced by heat treatment with or without dialysis. Although the antioxidant activity of these natural products appears to be partly due to enzymes such as superoxide dismutase (SOD), the predominant factor seems to be low-molecular-weight ROS scavengers that are liberated or activated by gastric juice digestion as observed after heat treatment. PMID- 3007358 TI - Mechanisms of killing of Toxoplasma gondii by rat peritoneal macrophages. AB - Rats are resistant to Toxoplasma infection, and macrophages are thought to mediate this resistance. We performed a series of experiments to investigate the mechanism of the anti-Toxoplasma activity of resident rat peritoneal macrophages. Resident rat peritoneal macrophages killed more than 90% of ingested Toxoplasma gondii in vitro. This capacity was reduced progressively with the prolongation of culturing of macrophages in vitro before challenge with T. gondii. Exhaustion of the respiratory burst of macrophages with phorbol myristate acetate impaired their ability to kill and limit the replication of T. gondii. Histidine and diazabicyclooctane, presumed scavengers of singlet oxygen, were the only members of a battery of scavengers of metabolites of the respiratory burst that impaired the anti-Toxoplasma activity of macrophages. Ingestion of heat-killed Candida albicans by macrophages reduced large amounts of intracellular Nitro Blue Tetrazolium dye, whereas little dye was reduced by the ingestion of T. gondii. Challenge of macrophages with T. gondii released no detectable superoxide anion, as measured by the reduction of ferricytochrome c, whereas stimulation of macrophages with phorbol myristate acetate or ingestion of heat-killed Candida by macrophages released abundant superoxide anion. These data are consistent with the contributions of oxygen-dependent and oxygen-independent mechanisms to the anti-Toxoplasma activity of rat peritoneal macrophages. In addition, neonatal rats are known to be susceptible to Toxoplasma infection in vivo. However, resident neonatal rat peritoneal macrophages ingested and killed T. gondii to the same extent as did adult macrophages. Thus, the susceptibility of neonatal rats to Toxoplasma infection probably resides in other aspects of macrophage function or the immune response. PMID- 3007359 TI - Identification and characterization of mouse small intestine mucosal receptors for Escherichia coli K-12(K88ab). AB - Adhesion of 3H-labeled Escherichia coli K-12(K88ab) to CD-1 mouse small intestine mucus and brush border preparations, immobilized on polystyrene, was studied. E. coli K12(K88ab) was shown to adhere readily to either crude mucus or brush border preparations, but not to bovine serum albumin. In contrast, the nearly isogenic E. coli K-12 strain, i.e., lacking the K88ab plasmid, did not bind well to either mucus, brush borders, or bovine serum albumin. The adhesion of E. coli K 12(K88ab) to both mucus and brush borders required pilus expression (i.e., growth at temperatures greater than 18 degrees C) and was inhibited by pretreatment of either mucus or brush borders with trypsin, pronase, or sodium metaperiodate and by the presence of D-galactosamine. Crude mucus was fractionated by gel filtration, and the proteins in receptor-containing fractions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Separated proteins were Western blotted to nitrocellulose. Adhesion of 35SO4-labeled E. coli K 12(K88ab) and 35SO4-labeled E. coli K-12 to Western blots followed by autoradiography revealed two E. coli K-12(K88ab)-specific mucus receptor proteins (57 and 64 kilodaltons). Brush borders contained the same two receptor proteins present in mucus and an additional 91-kilodalton receptor protein. PMID- 3007361 TI - Ontogeny of macrophage function to release superoxide anion in conventional and germfree mice. AB - To determine whether the presence of bacterial flora contributes to the ontogenic development of macrophage function, the ability of macrophages to release superoxide anion (O2-) in response to stimulation with phorbol myristate acetate was compared in conventional and germfree mice of various ages after birth. One week-old conventional mice showed a very low level of O2- release by their macrophages, and gradual increases were observed in 2-, 3-, and 4-week-old mice in an age-dependent manner. Macrophages from germfree mice always showed a significantly lower level of O2- release compared with conventional mice of the same age; however, age-dependent functional development was seen also in the germfree group. The poor level of O2- release by macrophages from adult germfree mice could be restored to more than the level by conventional mice when the mice were conventionalized for 3 weeks. These results suggested that the ontogenic development of macrophage function is not controlled by the presence of bacterial flora but that the full-scale expression of function at each age is under the influence of microflora. PMID- 3007360 TI - Antibodies to porin antigens of Salmonella typhi induced during typhoid infection in humans. AB - Immunoglobulin G (IgG)- and IgM-specific antibody titers against Salmonella typhi Ty2 porins have been measured in 30 paired typhoid sera by enzyme-linked immunosorbent assay. These studies have found that IgG serum titers of acute and convalescent sera were 625 and 5,000 times higher, respectively than the control serum titers. The same typhoid sera were titrated with S. typhi Ty2 flagellin and S. typhi lipopolysaccharide. The titers against these antigens were considerably lower than those against the porins. The highest IgM-specific titer has also been found against porins in convalescent-phase sera. However, the largest increase in IgM-specific titer compared with the control group titer was obtained against flagellin during the acute phase of typhoid. The lowest increases in antibody titer were obtained with the IgM-specific anti-lipopolysaccharide in both types of sera. This may be because many normal individuals in endemic areas already have IgM titers against lipopolysaccharide. This study has provided good evidence that porins are excellent antigens and that IgG-specific antiporin titers may be of diagnostic value in typhoid infections in endemic areas. PMID- 3007362 TI - Molecular cloning and expression in Escherichia coli K-12 of the gene for a hemagglutinin from Vibrio cholerae. AB - Using antiserum to the purified soluble hemagglutinin we isolated an Escherichia coli K-12 clone expressing the gene for a hemagglutinin from Vibrio cholerae 569B. The plasmid present in this clone was designated pPM471. By deletion analysis with both specific restriction endonucleases and Bal 31 nuclease, we localized the gene, to a 0.72-kilobase region of DNA, implying a molecular weight of less than 27,000 for the protein. Analysis in E. coli K-12 minicells of plasmids containing the cloned gene and deletion derivatives of these plasmids identified a protein of 24,000 daltons correlating with hemagglutinating activity. Using the cloned gene as a probe, we demonstrated the presence of homologous DNA in a variety of V. cholerae strains including both biotypes. Furthermore, by screening gene banks in E. coli K-12 of V. cholerae El Tor O17, we isolated several El Tor clones containing this region of DNA and also expressing hemagglutinating activity. PMID- 3007363 TI - Transposon mutagenesis as a tool to study the role of hemolysin in the virulence of Listeria monocytogenes. AB - The role of hemolysin in the virulence of Listeria monocytogenes was studied by using transposon mutagenesis. The 26-kilobase conjugative transposon Tn1545, originally found in Streptococcus pneumoniae, was transferred to a hemolytic virulent strain of L. monocytogenes. The frequency of transfer was estimated to be about 10(-8) per recipient. This allowed us to isolate a nonhemolytic mutant which most likely harbors a single copy of Tn1545. Loss of hemolysin production was associated with loss of virulence. The 50% lethal dose of the mutant was assessed to about 10(9.6) bacteria per mouse after intravenous challenge. Nonhemolytic bacteria were unable to grow in host tissues and were rapidly eliminated from the spleen and liver of infected mice. Virulence was restored in hemolysin-producing revertant obtained by spontaneous loss of transposon Tn1545. These results strongly suggest that hemolysin is a major virulence factor implicated in the intracellular growth of L. monocytogenes. PMID- 3007364 TI - Variables which affect suppression of the immune response induced by Pseudomonas aeruginosa exotoxin A. AB - Pseudomonas exotoxin A has been shown previously to induce suppression of the murine immune response. In the present study, various parameters were examined which may have an effect on immunosuppression. The addition of 10(-4) ng of exotoxin A induced suppression of the immune response to trinitrophenylated Ficoll from days 3 to 10, while 10 ng of toxin exerted no suppressive effect over the same examination periods. When the toxin was administered 1 or 2 days before antigen stimulation, suppression of the response was observed with both 10 and 10(-4) ng. Priming splenocytes with toxin either in vivo or in vitro for 1 or 2 days suppressed the response of fresh cultured splenocytes to antigenic stimulation. Heated toxin, photoaffinity-labeled toxin, or preincubation of the toxin with rabbit anti-exotoxin A antiserum eliminated the toxin-induced suppression. These results suggest that Pseudomonas exotoxin A can generate multiple biological effects. PMID- 3007366 TI - [Clinical pharmacology of ofloxacin: a new chemotherapeutic agent belonging to the gyrase inhibitor group]. AB - Ofloxacin (HOE 280, DL 8280, OFX) is a new broad-spectrum chemotherapeutic agent belonging to the group of the gyrase inhibitors. The tolerability and pharmacokinetics have been investigated for the dose range from 100 mg to 2 X 600 mg. The substance has proved to be well tolerated; investigation of the effects on renal enzymes after multiple dosing with 300 mg b.i.d. showed that the risk of nephrotoxicity is negligible. Ofloxacin is rapidly and almost completely absorbed. Cmax and AUC are dose-dependent. The favourable half-life--between 6 and 7 h, irrespective of the dose--results in prolonged serum concentrations. Food interaction is slight and of no clinical relevance. The penetration into tissue and body secretions is rapid, and high levels are reached. The main route of elimination is the kidneys. The urinary concentrations are dose-dependent; but the proportion of the dose excreted via the kidneys remains approximately constant, 80% or more of the dose being recovered as unchanged ofloxacin. The degree of metabolisation in humans is small and of no clinical relevance. The glucuronide of ofloxacin found in the bile together with the two metabolites detected in the urine account for at most 5% of the dose given. The favourable kinetic profile of ofloxacin means that the daily regimen required is two doses at most. This is confirmed by clinical findings to date. PMID- 3007365 TI - Adrenal function in paracoccidioidomycosis: a prospective study in patients before and after ketoconazole therapy. AB - The adrenal gland functional reserve was studied in a group of 22 patients with active paracoccidioidomycosis before therapy and in 18 of the same patients after termination of six months of ketoconazole treatment. 22 control subjects were also tested. Serum cortisol was measured before and after i.v. infusion of 250 micrograms of corticotropin given over a period of two hours. Basal cortisol levels were subnormal in only one patients before treatment and in four of 18 patients after therapy. Overt Addison's disease was found in 14% of the patients before treatment. However, corticotropin stimulation revealed diminished adrenal reserve in 23% of patients before, and in 44% of the patients after treatment. Although decreased adrenal cortex function after therapy may be influenced by ketoconazole, more studies are needed to determine the role of this agent after prolonged use. The high frequency of subclinical adrenal failure in paracoccidioidomycosis should alert clinicians in charge of such patients, should they face physiological stress. PMID- 3007367 TI - [Biotransformation of selected gyrase inhibitors]. AB - The common structure of the gyrase inhibitors norfloxacin, ciprofloxacin, pefloxacin, and ofloxacin is 3-carboxy-4-oxo-6-fluoro-7-(1-piperazinyl)-1,4 dihydro-quinolone. Several biotransformations of these substances are reported in the literature, mostly in animals and partly in humans: 1. Conjugation of the carboxylic acid to glucuronic acid (formation of an O-methyl ester of norfloxacin was found only in the rat); 2. Oxidation of the piperazine ring to the oxo derivative and subsequent metabolisation (or degradation) of the piperazine ring to several intermediates and finally to elimination of the side chain; 3. Substitution of the piperazine side chain to the 4-N-acetyl or 4-N-formyl derivative (norfloxacin, ciprofloxacin); 4. Methylation of the 4-methyl piperazine side chain (pefloxacin, ofloxacin). 5. N-oxidation of the 4-methyl piperazine side chain (pefloxacin, ofloxacin). The glucuronides are microbiologically inactive. The activity of metabolites with a modified piperazine side chain varies from high (oxo derivatives) to low (after splitting of the ring). Quantitative data on the formation of the described transformation products in humans are presently still incomplete. The oxo derivative appears to be the main metabolite of norfloxacin and ciprofloxacin. Additional metabolites to the described ones are likely to be detected in the near future. PMID- 3007368 TI - Induction of autoimmune diseases with adjuvants: separation of delayed hypersensitivity and antibody formation from diseases in experimental thyroiditis and aspermatogenesis with Legionella adjuvant. AB - The induction of autoimmune diseases in animals was studied with Legionella and mycobacteria as adjuvants, emulsified in oil with antigen extracts of thyroid, testis, spinal cord, and peripheral nerve. Both adjuvants were equally effective in inducing delayed hypersensitivity and humoral antibody to the tissue antigens. The Legionella adjuvant, however, induced little or no thyroiditis and aspermatogenesis, whereas the mycobacterial adjuvant induced thyroiditis and aspermatogenesis. Both adjuvants caused allergic encephalomyelitis and peripheral neuritis. The results indicated that delayed hypersensitivity by itself may not be sufficient to cause thyroiditis and aspermatogenesis. Legionella adjuvant apparently lacked the ability to induce certain immune factor(s) which caused the disease in experimental thyroiditis and aspermatogenesis. The differential properties of Legionella adjuvant and mycobacterial adjuvant in inducing immunity to autoantigens could provide a useful means to study the pathogenic and immunoregulatory mechanisms of some experimental autoimmune diseases. PMID- 3007369 TI - Lithium intoxication. PMID- 3007370 TI - Presence of human papillomavirus type-16 and type-18 DNA sequences and their expression in cervical cancers and cell lines from Japanese patients. AB - Southern blot analyses of surgical specimens of cervical carcinoma from Japanese patients showed that 3/9 samples contained human papillomavirus (HPV) type-16 DNA sequences, and 2 contained HPV type-18 DNA sequences. By Northern blot analyses, RNA transcripts of HPV DNA sequences were demonstrated in some of the tissues containing HPV type-16 or HPV type-18 DNA sequences. Two cell lines established from cervical cancers of Japanese patients also contained HPV type-18 genomes and these cell lines contained HPV type-18 transcripts. Two other cervical cancer cell lines from a Japanese patient were found to contain HPV type-16 DNA sequences and their RNA transcripts. PMID- 3007371 TI - Molecular cloning of two new HPV types (HPV 37 and HPV 38) from a keratoacanthoma and a malignant melanoma. AB - Several benign and malignant skin tumors were analyzed for the presence of human papillomavirus (HPV) DNA. By hybridization with different HPV DNA probes under non-stringent conditions (Tm -40 degrees C), two tumors were found to contain HPV specific DNA sequences in high copy numbers: (1) a keratoacanthoma from a patient who also suffered from a basalioma; (2) a superficial spreading malignant melanoma of an immunosuppressed patient. For further analysis of these DNA sequences genomic libraries from both tumor DNAs were constructed and, out of these, 4 different HPV DNA types have been cloned. By cross-hybridization experiments and restriction map analysis HPV 9 DNA was identified in the keratoacanthoma whereas HPV 17a DNA could be cloned from the malignant melanoma. From each tumor one additional HPV-type not identical to other known HPV-types was cloned. These isolates are closely related to HPV 9, 15, 17, 22 and 23. A physical map of both HPV DNAs was constructed. Size (7.8 kbp), co-linear alignment to HPV 16, cross-hybridization with other HPV-types under conditions of low stringency and monomeric episomal state of the HPV molecules indicate that these two DNA probes represent new HPV types that have been tentatively designated as HPV 37 (keratoacanthoma) and HPV 38 (malignant melanoma). None of these two HPV types could be found in any other of 231 tumor DNAs originating from different tissues. PMID- 3007373 TI - Morphology of bronchogenic carcinoma in workers formerly exposed to crocidolite at Wittenoom Gorge in Western Australia. AB - Cytology and histology material from 46 bronchogenic carcinomas occurring in ex workers from the Wittenoom crocidolite mine and mill in Western Australia and a matched random sample of 234 other bronchogenic carcinomas occurring in Western Australia over the same period were reviewed by a single histopathologist without knowledge of asbestos exposure status. Squamous-cell carcinomas formed 45.7% of the cancers in the asbestos-exposed group but only 32.5% of the cancers in the comparison group. This difference could not be explained by differences in smoking history between the two groups of lung cancer patients or in the type of histopathological material available for review. The excess of squamous-cell cancers was observed in subjects both with and without parenchymal asbestosis. PMID- 3007372 TI - Comparison of risk factors for cervical cancer in different populations. AB - The incidence of cervical cancer has been found to vary between populations. Risk factors of cervical cancer include early age at first marriage, multiple marriages and antibodies to herpes simplex virus type 2 (HSV-2). The interrelatedness of these risk factors was examined by comparing data collected from 428 cancer cases and 947 control women selected from 6 populations having standardized cervical cancer incidence rates varying from 9.3 to 85.1 per 100,000. Logistic regression analysis revealed that multiple marriages, early age at first marriage or pregnancy and HSV-2 antibodies were all associated with significant risk when all 3 factors were entered into the model. Cervical cancer incidence rates were best predicted by the occurrence of HSV-2 antibodies among control women. To further assess the relation between cervical cancer rates and HSV-2 antibody, 2,306 additional sera representing an 0.8% random sample of females over 9 years of age residing in the Republic of Panama were assayed for antibodies to the virus, and the occurrence of antibodies was correlated with invasive cervical cancer rates specific to each Province. Data from both the random sample and the other study populations yielded a linear relation between the occurrence of HSV-2 antibodies and the incidence of cervical cancer. An exception was found for women living in Herrera Province, Republic of Panama, who had a higher cancer rate than predicted by HSV-2 antibody occurrence. The data suggested that infection with HSV-2 is a co-variable of venereal factors, although a role for the virus in the genesis of a certain proportion of cervical cancers is not excluded. PMID- 3007374 TI - Virus production and surface marker expression in human lymphocytes immortalized following dual infection with human T-cell leukemia virus and Epstein-Barr virus. AB - By co-cultivating peripheral blood lymphocytes from normal male donors with cells from a line designated Lma-66VP, established from a female donor and simultaneously producing both HTLV-I and EBV, 2 continuous culture lines were obtained. Normal male lymphocytes were considered to be immortalized by co cultivation because they were of male karyotype and lacked the 3q+ chromosome that was observed in all Lma-66VP cells. These immortalized cultures were designated Co-culture 1 and Co-culture 2. After prolonged cultivation, chromosome abnormalities characteristic of each co-culture line were observed in all cells examined, indicating their clonal origin. Immortalized cells initially produced both HTLV-I and EBV. Although production of EBV was not seen in the co-culture lines after prolonged cultivation when all cells examined had the characteristic chromosomal abnormality indicating their clonality, EBNA+ cells persisted. Thus, clonal cells were doubly infected by HTLV-I and EBV. While the majority of clonal cells in Lma-66VP, Co-culture 1, and Co-culture 2 lines expressed a surface marker of B-cells, small numbers of cells expressed a T-cell surface marker. These findings demonstrate that human lymphocytes immortalized following dual infection by HTLV-I and EBV exhibit chromosome rearrangement, resulting in expression of surface markers inconsistent with their differentiation lineage. PMID- 3007375 TI - Detection of DNA cross-links in tumor cells with the ethidium bromide fluorescence assay. AB - Until now the fluorescence assay with ethidium bromide has only been used on pure DNA. This assay depends on the difference in fluorescence between single- and double-stranded DNA (dsDNA). Cross-links in DNA are measured by the return of fluorescence of dsDNA after heat denaturation at pH 12. Under these conditions denatured DNA gives very low fluorescence. In the present study this assay was applied to tumor cells. The mouse Ehrlich ascites tumor cell line (EAT) and a human small-cell carcinoma line (GLC4) were incubated for 4 hr at 37 degrees C, with the cross-linking agent cis-diamino-dichloro platinum (cDDP). The samples of whole cells were thereafter resuspended in potassium phosphate buffer with 10 mM EDTA, 4M NaCl, 0.1% Sarkosyl pH 7.2, for 16 hr at 37 degrees C. Measurements were performed with a spectrofluorometer with excitation wavelength 525 nm, emission wavelength 580 nm. There was a linear relationship for cDDP concentrations of 0 150 microM and the extent of DNA cross-links in EAT (r = 0.958). In GLC4 there was a linear relationship at low cDDP concentrations of 0-50 microM (r = 0.968) while between 50 and 150 microM a plateau was reached. RNase added to the lysate of whole cells had no influence on the extent of cross-links. This assay was compared with the alkaline elution assay, and results were identical. PMID- 3007376 TI - Mode of transmission of human T-cell leukemia virus type I (HTLV I) in a human promyelocytic leukemia HL60 cell. AB - HTLV-I propagated in IMR90 human diploid fibroblasts was transmitted to human myeloid leukemia HL60 cells at a low efficiency. After co-cultivation for 3 months, the viral genome was detected in 14/48 HL60 cell clones. Among the 14 HTLV-I-infected clones, 8 contained subgenomic fragments alone or in addition to the complete HTLV-I genome. The frequency of deleted proviruses (9/24 total proviruses) was unexpectedly high. Hirt's supernatant of some of the clones harboring complete HTLV-I genome(s) in the chromosome contained both linear and circular HTLV-I proviral DNAs. The circular DNAs were composed of one LTR and 2 LTR closed circular proviruses. These clones produced infectious HTLV-I constitutively, which was proved by transmission of the viral genome into fresh IMR90 cells by co-cultivation. However, in these clones, re-integration of extrachromosomal provirus into their own chromosomes was not observed. PMID- 3007377 TI - Incidence of bovine leukemia virus-specific antibodies in West African cattle. AB - A sero-epidemiological survey of bovine leukemia virus (BLV) infection in indigenous West African cattle was undertaken using a radioimmunoassay and enzyme linked immunosorbent assay. A high incidence of anti-BLV antibodies was found in the breeds tested and their crosses, whatever the test used. More extensive studies are needed to establish the prevalence of BLV infection in other parts of Africa; these may provide additional data on the similarities found between human and bovine leukemia viruses. PMID- 3007378 TI - Aging and cardiac function. PMID- 3007379 TI - Enalapril in congestive heart failure: acute and chronic invasive hemodynamic evaluation. AB - Following hemodynamic evaluation using invasive and noninvasive methods, 73 patients were treated in an open, uncontrolled, multicenter study with single oral doses of enalapril maleate 1.25 to 40 mg until the optimal dose for each patient (based upon hemodynamic response) was achieved. Diuretics were withheld and reinstituted only if necessary. Hemodynamic measurements were made at 0 (predrug), 1, 2, 3, 4, 6, 8, 10, 12 and 24 hours postdrug. Patients were discharged on their optimal dose, treated 1 to 4 months and then rehospitalized for repeat hemodynamic measurements. The optimal enalapril single dose was associated with the following mean peak responses: increased cardiac index 42% (SE = 6) and decreased pulmonary capillary wedge pressure 40% (SE = 3), systemic vascular resistance 39% (SE = 2), and mean arterial pressure 23% (SE = 1.5). These changes persisted during chronic therapy. Chronic treatment with enalapril also improved exercise capacity 40% (P less than 0.01), ejection fraction 18% (P less than 0.05) and clinical status (N.Y.H.A. functional class, P less than 0.01). Ten and 20 mg/day, taken as once- or twice-daily regimens, were the most commonly effective doses. PMID- 3007380 TI - Hormonal and metabolic effects following a football match. AB - Hormonal and metabolic parameters were studied for two teams after a football match. Blood samples were collected before the start, at half-time, at the end, and 45 and 90 min after the end of the game. In the first team, ACTH, cortisol, and lactate levels increased significantly during the whole match to resume basal levels in the fourth sample (45 min after the end of play). HGH, prolactin, and blood glucose were found to be increased only at half-time. The second team had an intense and long warm-up period before the match and the lactate concentrations were already elevated in the first sample. All the other parameters, except ACTH and glucose, displayed a pattern similar to that of the first team. The differences in the time courses of the hormonal and metabolic values are discussed. PMID- 3007381 TI - Human papillomavirus and cervical neoplasia: epidemiological considerations. AB - Recent clinical research using new diagnostic and laboratory techniques indicates that human papillomavirus (HPV) infection may play a key role in the development of cervical neoplasia. Cervical HPV infection is found in 1-3% of routine Papanicolaou smears, and preliminary data suggest that women with cervical HPV infection have about a tenfold greater risk of developing cervical intraepithelial neoplasia (CIN) compared with those with no HPV infection. This paper discusses how epidemiological studies can reduce misclassification of cervical HPV infection and CIN through selective use of research advances in cytologic, histologic and colposcopic techniques. These studies can also assess whether certain potential cocarcinogens interact synergistically with HPV. Since an estimated 2-10% of women with cervical HPV lesions develop associated CIN within a year, it appears feasible to conduct prospective studies of the risk of malignant transformation of cervical HPV lesions. PMID- 3007382 TI - The thermogenic role of adipose tissue in the dog. AB - Brown adipose tissue was clearly present in neonatal dogs. In the adult the tissue was superseded by a tissue with the gross characteristics of white adipose. However despite their appearance adult adipose tissue depots may contribute to non-shivering thermogenesis. Regional blood flow measurements using injected radioactive microspheres indicated large increases in blood flow to adipose depots during infusion of noradrenaline. Coupled with blood flow estimations, measurement of arteriovenous differences in dissolved oxygen across the bladder fat depot demonstrated a quantitative increase in oxygen extraction by the depot during noradrenaline infusion. Acute activation of non-shivering thermogenesis in the dog was not associated with increased mitochondrial GDP binding in adipose tissue. However chronic treatment with a beta-stimulant (LY79730) which increased capacity for non-shivering thermogenesis was associated with increased mitochondrial GDP-binding and cytochrome oxidase activity in peri renal adipose tissue. PMID- 3007383 TI - Epstein-Barr virus related central nervous system lymphoma in a child after renal transplantation. AB - In this report, we describe the development of a rapidly progressive Epstein-Barr virus (EBV) related cerebral lymphoma in an 11 year old girl, eight months after renal transplantation. No serological evidence for a persistent EBV infection was found, but Epstein-Barr nuclear antigen (EBNA) could be demonstrated in the tumor. The clinical course of our patient was different from EBV-related syndromes in renal transplanted patients described in previous reports. Furthermore, pathological investigations of the biopsy specimen and tumor cells obtained at necropsy revealed a discrepancy in light chain expression. The possibility that lymphoproliferative disorders represent multiclonal B cell lymphomas is discussed. PMID- 3007385 TI - Brief report: susceptibility of Filipino nurses to the varicella-zoster virus. AB - Following an outbreak of varicella, 18% of a group of 174 young female Filipino nurses ranging in age from 20 to 25 years and working at the American University of Beirut Medical Center (AUMC) were found susceptible to the varicella-zoster virus; as compared to 3% of a matched group of 133 of their Lebanese colleagues. The level of antibody was determined by the Enzyme Linked Immunosorbent Assay (ELISA). Those susceptible were assigned duties in low-risk areas to varicella zoster in the hospital. PMID- 3007384 TI - Radiolysis of human gastric glycopolypeptides in aqueous solution. AB - The degradation of human gastric glycopolypeptides by hydroxyl radicals formed in irradiated N2O-saturated aqueous solution has been investigated. Gel exclusion chromatography shows the formation of lower molecular weight degradation products after irradiation and the appearance of unsaturated carbonyl-containing products which absorb in the ultra-violet. The radiation-induced destruction of individual monosaccharides in three human glycopolypeptides having different oligosaccharide chains has been measured. The results indicate that the structure of the oligosaccharide chain determines the extent of destruction of each type of monosaccharide present. PMID- 3007386 TI - The risks of transmission of the HTLV-III and hepatitis B virus in the hospital. PMID- 3007387 TI - The molecular biology of antigenic variation in trypanosomes: gene rearrangements and discontinuous transcription. PMID- 3007388 TI - [Age-related changes in sensitivity to drugs]. PMID- 3007389 TI - Diradical nitroxyl spin label contrast agents for magnetic resonance imaging. A comparison of relaxation effectiveness. AB - The proton relaxation enhancement characteristics of seven potential MRI contrast agents containing two nitroxyl spin labels per molecule (diradicals) were compared with eight similar agents with only one spin label per molecule (monoradicals). Diradical nitroxyls were evaluated to test the hypothesis that multiple paramagnetic centers in one molecule will result in stronger proton relaxation enhancement characteristics, allowing effective contrast enhancement at lower molar concentrations and thus a reduced osmotic load and greater safety. The acute toxicity of these agents is believed to be largely related to osmotic load. Five of seven diradical nitroxyls tested had spin-lattice relaxivities that were substantially greater than all eight of the monoradicals tested. The spin spin relaxation properties of these agents and other pertinent characteristics are favorable for contrast enhancement. The results indicate that diradical nitroxyl spin labels may be used advantageously for the design of safer, more effective MRI contrast agents. PMID- 3007390 TI - Influence of paramagnetic ions and pH on proton NMR relaxation of biologic fluids. AB - The extent to which various concentrations of the paramagnetic metal ions [gadolinium (III), manganese (II), chromium (III), iron (III), nickel (II), copper (II), and cobalt (II)] affect proton magnetic relaxation times of distilled water, 4% human serum albumin (HSA), and dog plasma was studied in vitro. The pH of water and HSA varied from 4 to 8. Nuclear magnetic resonance relaxation parameters, T1 and T2, were measured at 10.7 MHz using inversion recovery and spin-echo radiofrequency sequences, respectively. The presence of Mn(II), Gd(III) and Cr(III) in water significantly reduced T1, while Fe(III), Ni(II), Cu(II) and Co(II) had only a minimal effect. In 4% HSA and dog plasma Mn(II) and Cu(II) had the greatest effect on T1. At neutral pH, Gd(III) and Cr(III) had little effect on T1, while Mn(II) induced a large shortening of T1. All of the metal ions changed T2 less than T1. These differences in proton relaxation enhancement caused by the various ions in the three solutions studied are due to variations in the effective magnetic moment, the degree of binding of the ions to protein, and the chemical form of the ion associated with changes in pH. Thus, it is impossible to predict the effect of metal ions on proton relaxation in vivo based solely on in vitro studies, because of the complexity of various biologic fluids in vivo. PMID- 3007391 TI - Enhancement of red blood cell proton relaxation with chromium labeling. AB - Nuclear medicine has utilized chromium (Cr) for decades to label red blood cells (RBCs). The purpose of this project was to determine whether sufficient paramagnetic Cr could be bound to red cells to influence proton relaxation significantly. We demonstrated that the T1 and T2 of RBCs can be substantially shortened by labeling them with paramagnetic Cr. Proton relaxation enhancement occurs when red cells are incubated with sodium chromate (VI) over a concentration range of 0.10 mM to 31.6 mM. Labeling with Cr at a concentration of 31.6 mM shortened the T1 of packed cells from 714 msec to 33 msec, and the T2 from 117 msec to 24 msec, as compared with nonlabeled red cells. In vitro hemolysis was significantly increased after labeling at 31.6 mM, but not at lower concentrations. Cr-induced proton relaxation enhancement varied with RBCs from different species, temperature, pH, and length of incubation. T1 values of kidneys containing labeled red cells (303 msec), or labeled cells diluted 10-fold with nonlabeled cells (479 msec), were decreased compared with kidneys containing only nonlabeled cells (600 msec). Finally, preliminary data indicate that the signal intensity of perfused renal tissue is significantly influenced in vivo by infusion of Cr-labeled RBCs. This study demonstrated that Cr labeling of RBCs sufficiently enhances red cell proton relaxation to provide excised organs containing red cells, of which 10% have been Cr-labeled, with shorter T1 and T2 values than organs containing nonlabeled cells. In addition, the ability of labeled cells to alter signal intensity in vivo suggests that Cr may have the potential to become an MRI contrast agent. PMID- 3007393 TI - Immune thrombocytopenia in a heterosexual male hemophiliac. PMID- 3007392 TI - Pilot study of the efficacy of spent grain dietary fiber in the treatment of constipation. AB - Spent grain is the crude fiber obtained by decanting the fermented distillate of barley. The spent grain was processed to yield dietary fiber composed of: cellulose and hemicellulose 65.6% (by weight), lignin 5.2%, pectin 2.2%, protein 10.9% and lipid 8.0%. Biscuits and scones were prepared by 25% substitution of wheat flour by fiber, yielding 7 to 8 g fiber per biscuit/scone. Nineteen ambulatory patients with chronic, laxative-dependent constipation were treated in a pilot study for 4 weeks with 20 to 25 g fiber daily. Fifteen patients (79%) showed improvement in some or all of five factors, while four patients were largely unresponsive to fiber. Specific symptoms improved as follows: bowel movement frequency in 15 patients (79%), flatulence in 12 (63%), abdominal pain in 10 (53%), stool consistency in 8 (42%) and laxative dependence in 14 (74%). A 4-week post-treatment follow-up showed a return to prefiber status in 11 of 13 improved subjects. This preliminary study suggests a role for spent grain fiber in the treatment of constipated patients, and a comparative study with placebo and wheat fiber is now warranted. PMID- 3007394 TI - The influence of an electrostatic precipitator and a mechanical filter on Rn decay products. AB - In the present work the effect of an HEPA filter and an electrostatic precipitator on the behaviour of Rn decay products has been studied in laboratory conditions. Both filters were found to decrease the equilibrium factor of progeny and increase the unattached fraction of decay products. In a clean air they also decreased the activity of unattached progeny. An electrostatic precipitator was found to produce condensation nuclei which were observable when no other particle sources were present. The ozone produced by the corona discharge of the filter probably has a great effect on the production of these submicron particles. The impurities of the air were found to grow the particles and thus their influence on the behaviour of Rn decay products became in some cases significant. PMID- 3007395 TI - Randomization of grab-sampling strategies for estimating the annual exposure of U miners to Rn daughters. AB - Periodic grab sampling in combination with time-of-occupancy surveys has been the accepted procedure for estimating the annual exposure of underground U miners to Rn daughters. Temporal variations in the concentration of potential alpha energy in the mine generate uncertainties in this process. A system to randomize the selection of locations for measurement is described which can reduce uncertainties and eliminate systematic biases in the data. In general, a sample frequency of 50 measurements per year is sufficient to satisfy the criteria that the annual exposure be determined in working level months to within +/- 50% of the true value with a 95% level of confidence. Suggestions for implementing this randomization scheme are presented. PMID- 3007396 TI - Identification of hemoglobin D Punjab by gene mapping. PMID- 3007398 TI - Greig cephalopolysyndactyly syndrome. Report of a sporadic case. AB - A female newborn of healthy parents demonstrates the combination of postaxial hexadactyly type B of the hands (X-ray investigations show in addition duplicated terminal phalanges of the thumbs), preaxial hexadactyly of the toes, partial and total syndactyly of the fingers and toes. These findings are compatible with the diagnosis of Greig cephalopolysyndactyly syndrome. Dyscrania can not be demonstrated. In addition, our patient exhibits genu recurvatum on the left side resulting from intrauterine deformation. Sonography of the head reveals normal formation of the corpus callosum. The neurological status of our baby is inconspicious, and the psychomotor development up to the age of 2 5/12 years normal. The differential diagnosis of this characteristic combination of pre- and postaxial polydactyly has to consider the acrocallosal syndrome. In sporadic cases of Greig cephalopolysyndactyly syndrome it is important to investigate and follow the neurological status of the patients and in particular to use sonography to document the intact corpus callosum. Patients with Greig cephalopolysyndactyly syndrome have a normal development, children with the acrocallosal syndrome are retarded. PMID- 3007397 TI - Lymphadenopathic form of Kaposi's sarcoma (KS) in an African child. No evidence for acquired immunodeficiency syndrome (AIDS). AB - A nine-year-old boy from the Central African Republic presented with an enlarged cervical lymph node. Biopsy revealed Kaposi's sarcoma. Lymphocyte subsets and reactivity to all antigens tested were normal, and the patient's serum contained no antibodies against LAV or HTLV3, the viruses presumably responsible for AIDS. We conclude that in this case, the lymphadenopathic form of Kaposi's sarcoma was not related to AIDS. PMID- 3007399 TI - Scirrhous endocrine cell carcinoma of the stomach with synchronous production of six polypeptide hormones and glycoproteins. A case report. PMID- 3007400 TI - Long-term influence of a wheat-bran supplemented diet on secretion of gastrointestinal hormones and on nutrient absorption in healthy man. AB - Responses of gastric inhibitory polypeptide (GIP), gastrin and vasoactive intestinal polypeptide (VIP) to a test meal and also nutrient absorption were measured in five healthy men before and after 1, 3 and 7 weeks of daily ingestion of 20 g of wheat bran added to a normal balanced diet. Basal levels of the three hormones were not affected by bran ingestion. Bran ingestion induced a progressive decrease of GIP response to the test meal which became significant after 7 weeks at 30 min (373.9 +/- 71.4 vs 231.1 +/- 47.8 pg/ml, mean +/- s.e.m., P less than 0.05) and at 180 min (389.4 +/- 43.9 vs 262.2 +/- 37.9 pg/ml, P less than 0.01). Gastrin release did not change except for a slight but not significant decrease after 3 weeks. There was no VIP secretion after meal ingestion and addition of bran caused no change. Blood glucose response decreased with time with the greatest effects during the third week with fibre at 30 min (5.00 +/- 0.50 vs 7.38 +/- 0.05 mmol/l before bran), and at 60 min (3.88 +/- 0.34 vs 5.94 +/- 0.27 mmol/l before bran, P less than 0.05). Wet and dry weight of faeces increased by at least 60 per cent from the first week with bran onwards. Faecal nitrogen and fat also increased from 1.77 +/- 0.16 to 2.44 +/- 0.13 g/d for nitrogen (P less than 0.02) and threefold for fat (from 3.78 +/- 0.58 to 10.35 +/- 0.67 per cent dry weight, P less than 0.005) at the third week. Fat and nitrogen contents remained higher until the end of the experiment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007401 TI - A new look at dietary fibre. PMID- 3007403 TI - [Neuralgias in the area of the head and neck from the neurosurgical viewpoint]. PMID- 3007404 TI - [Indications for venous digital subtraction angiography in otorhinolaryngologic diseases]. AB - Intravenous DSA was performed on 45 patients for diagnosis of otorhinolaryngologic diseases. Hypervascular glomus tumours can be accurately diagnosed. Furthermore, vascular architecture and topography can be analysed to allow the planning of tumour embolization or surgery. Hypovascular tumours are inadequately demonstrated by intravenous DSA. A specific and detailed analysis of thin vessel architecture in malignant tumours is impossible by intravenous DSA. PMID- 3007402 TI - Enzyme cytochemical and immunocytochemical studies of flask cells in the amphibian epidermis. AB - The localization of oxidoreductases and transport enzymes in flask cells of the amphibian epidermis was studied at the light-microscopic level. In these cells, the deposition of cytochemical reaction products was very similar to that found in fish epidermal ionocytes, thus demonstrating histochemical similarities between these two types of cells. The present histochemical results revealed high levels of activity of alkaline phosphatase (ALPase), potassium-dependent nitrophenylphosphatase (K+-p-NPPase) and carbonic-anhydrase isozymes (CA-I and CA II) in the apical region of the flask cells, indicating that enzyme zonation may be the main site of the ion pumping. PMID- 3007405 TI - HLA-DR beta gene DNA polymorphisms revealed by Taq I correlate with HLA-DR specificities. AB - Human genomic DNA digested with restriction endonuclease Taq I was hybridized with cDNA probes for DR beta and DQ beta, for correlation of restriction fragment length polymorphisms with HLA-DR specificities. DR beta Taq I RFLPs were distinctive for DR serological types 1 to w9, with the exception of DR3 and w6, and were markedly consistent within DR specificity. Some common variants in RFLPs did emerge within serological type; DR3, e.g., was associated with two different fragment patterns, one of which occurred on the A1.B8.DR3 haplotype and was linked with a DR alpha Bgl II variant, and the other on the B18.DR3 haplotype. In the Pacific specimens examined, RFLPs were, with few exceptions, similar to those seen in Caucasoids sharing the same DR specificity. This study indicates that genomic hybridization is a useful adjunct to serological and cellular class II typing and should be particularly informative in identifying new HLA-DR alleles in populations serologically less well-defined than Caucasoids. PMID- 3007406 TI - DNA restriction fragment length polymorphisms characteristic for Dw subtypes of DR2. AB - DNA restriction fragment length polymorphisms (RFLP) can be easily demonstrated in DNA of cells expressing different DR specificities when class II cDNA probes are used for hybridization. Previous studies of DR4+ homozygous typing cells (HTCs) carrying different Dw subtypes, however, detected no RFLP correlating with subtypes. In contrast, we report here Southern blotting studies of DR2+ HTCs carrying different subtypes which showed RFLP patterns characteristic for each subtype, using both DR beta and DQ beta probes and several restriction enzymes. The RFLP between subtypes of DR2 was, however, appreciably lower than that found between DR specificities. PMID- 3007407 TI - Neurotoxicity in long-term survivors of small cell lung cancer. AB - Chronic central nervous system neurotoxicity was studied in 38 long-term survivors (greater than or equal to 3 years) of small cell lung cancer who were treated at the University of Texas M. D. Anderson Hospital and Tumor Institute at Houston between 1971 and 1980. All but one patient received combination chemotherapy with or without chest irradiation. Twenty-four patients received whole brain irradiation (Group I), 22 for "elective" and two for therapeutic purposes, while 14 did not (Group II). Abnormalities in computed tomographic (CT) scans of the brain were more frequently observed in Group I than in Group II (70% vs. 0%, p less than 0.01). Clinical central nervous system neurotoxicity developed in three patients in Group I, while none developed in patients in Group II (p less than 0.05). Patients who received methotrexate and procarbazine after whole brain irradiation were at a higher risk for clinical central nervous system neurotoxicity (p less than 0.05), and for development of periventricular white matter changes in CT brain scans (p less than 0.05) than were patients in Group II. Impaired methylation of the myelin sheath is proposed as a possible underlying pathogenic mechanism. PMID- 3007409 TI - Elective brain irradiation for small cell anaplastic lung cancer. PMID- 3007408 TI - Neurologic dysfunction in patients treated for small cell carcinoma of the lung: a clinical and radiological study. AB - The neurologic dysfunction in 7 patients treated for small cell carcinoma (SCC) of the lung by combination chemotherapy and prophylactic brain irradiation was evaluated. The disease appeared to be a diffuse encephalopathy frequently affecting the higher cortical functions. Five out of seven patients had progressive dysfunction leading to death in 1 to 26 months; one patient had stabilization of symptoms followed by death in 21 months, probably from the neurologic disease as well as SCC; one patient's symptoms improved. The clinical course of the neurologic disorder seemed different from the known reactions to brain irradiation and from the other neurologic syndromes associated with lung cancer. The relative contributions of cranial irradiation and treatment with chemotherapeutic agents in producing the neurotoxicity are not known. Computed tomographic (CT) brain scans done after the onset of symptoms did not show any focal signs or necrosis. However, there was a suggestion of progressive increase in intracranial fluid volume on the scans. The incidence of the disorder, 10.2% among a group of 49 patients, suggests the need for prospective studies to evaluate the problem. PMID- 3007411 TI - Chemotherapy of synovial cell sarcoma in a dog. AB - A synovial cell sarcoma of the tarsus in a 6-year-old dog was treated with doxorubicin HCl and cyclophosphamide. The tumor regressed after treatment. There was no recurrence 3 years after initiation of treatment. Previously, synovial cell sarcoma has not been reported to be responsive to chemotherapeutic agents, and the treatment of choice has been amputation. PMID- 3007410 TI - Nuclear angiography in a dog with congestive cardiomyopathy. AB - Nuclear angiography was used as a diagnostic aid and in monitoring the clinical course of a case of congestive cardiomyopathy in a dog. Serial examinations revealed progressively deteriorating values for left ventricular ejection fraction before the dog's death. This noninvasive technique can be an alternative to echocardiography for the evaluation of cardiac performance. PMID- 3007413 TI - Inactivation of lymphocyte-transforming activity of human T-cell leukemia virus type I by heat. AB - Peripheral blood lymphocytes from 2 normal individuals seronegative for human T cell leukemia virus type I (HTLV-I) were co-cultured with HTLV-I-producing MT-2 cells that had been heated at 56 degrees for 30 min or exposed to 10,000 rad of X irradiation. HTLV-I-induced lymphocyte transformation was consistently achieved by co-culture with irradiated MT-2 cells but not by co-culture with heated MT-2 cells. The heat treatment was found to be lethal to both MT-2 cells and the virus. These findings are discussed in terms of their potential clinical application for preventing the transmission of HTLV-I. PMID- 3007412 TI - Dietary sodium bicarbonate as a treatment for exertional rhabdomyolysis in a horse. AB - A 3-year-old mare repeatedly had clinical signs of rhabdomyolysis on mild exertion. Serum creatine kinase and aspartate transaminase activities were high at rest. Responses to dietary sodium bicarbonate were tested through 7 alternating periods of supplementation of a basal ration of timothy hay and oats. Physical signs; venous blood pH and gases; blood glucose and lactate; serum electrolytes, enzymes, and creatinine; and urine pH were monitored before and after exercise. Dietary sodium bicarbonate raised resting venous blood pH and bicarbonate slightly and significantly increased urine pH from pH 7.46 to 8.2 (P less than 0.001). An exercise test included 5 minutes at the walk followed by 20 minutes at the trot. The exercise induced gait stiffness, muscle fasciculations, and muscle induration when the diet was not supplemented, but not when it was supplemented with sodium bicarbonate. Myoglobin was present in 16 of 21 urine samples after exercise during nonsupplemented periods, but only in 3 of 28 urine samples during supplemented periods (P less than 0.0001). Bicarbonate supplementation significantly decreased the responses of blood lactic acid, serum creatine kinase, and aspartate transaminase to exercise. Supplementation of the diet was associated with higher venous blood pH and bicarbonate ion concentrations throughout exercise. Dietary sodium bicarbonate apparently mitigated or prevented physical, chemical, and enzymatic characteristics of exertional rhabdomyolysis in this mare, possibly through its enhancement of buffering capacity in muscle tissue fluids. PMID- 3007416 TI - Novenamine is the active moiety in novobiocin. AB - Novobiocin inhibits semiconservative DNA replication in procaryotes and more specifically DNA gyrase, an enzyme essential for DNA replication. Chemically, novobiocin consists of three distinct entities: the sugar noviose, a coumarin residue and a benzoic acid derivative. The subentity consisting of noviose plus the coumarin residue is referred to as novenamine; the subentity consisting of the coumarin plus the benzoic acid derivative is referred to as novobiocic acid. These subentities as well as noviose and the benzoic acid residue are essentially devoid of inhibitory activity against whole bacterial cells. These fragments were tested for their ability to inhibit DNA replication in a permeabilized Escherichia coli cell system and as potential inhibitors of DNA gyrase. Of all the fragments tested, only novenamine was found to inhibit DNA replication and DNA gyrase. The potency of novenamine essentially equaled that of novobiocin. This subunit thus represents the minimal structural entity necessary to interact with DNA gyrase. PMID- 3007414 TI - Glycyrrhetic acid inhibits tumor-promoting activity of teleocidin and 12-O tetradecanoylphorbol-13-acetate in two-stage mouse skin carcinogenesis. AB - Glycyrrhetic acid suppressed tumor promoter-induced effects in vitro, such as stimulation of 32Pi-incorporation into phospholipids of cultured cells and down regulation of the epidermal growth factor receptor. Glycyrrhetic acid inhibited the promoting activity of both 12-O-tetradecanoylphorbol-13-acetate (TPA) and teleocidin on skin tumor formation in mice initiated with 7,12 dimethylbenz[a]anthracene (DMBA). The percentage of tumor-bearing mice in the group treated with DMBA plus teleocidin was 88% at week 18, whereas that in the group treated with DMBA plus teleocidin and glycyrrhetic acid (10 mumol/painting) was 6%. Similarly, the percentage of tumor-bearing mice of the group treated with DMBA plus TPA was 97% at week 20, whereas that of the group treated with DMBA plus TPA and glycyrrhetic acid was 40%. Therefore, glycyrrhetic acid was proved to inhibit the activity of two different tumor promoters, teleocidin and TPA, in mouse skin. PMID- 3007415 TI - Distinct isozyme patterns of cyclic nucleotide phosphodiesterase in human neuroblastoma and ganglioneuroma; a possible marker of differentiation of neural crest-derived tumors and Schwann cells. AB - By DEAE-cellulose chromatography, the 30,000g supernatants of human neuroblastoma (n = 7), ganglioneuroma (n = 5), sympathetic ganglia (n = 3), and Schwannoma (n = 2) were found to contain three major peaks of adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase (PDE) activity, which were termed peaks I, II, and III in the order of their elution from the column. Peak I isozyme was calmodulin-dependent, and had two different Km values for cAMP (32 and 2.3 microM) and a low Km for guanosine 3',5'-cyclic monophosphate (cGMP) (2.9 microM). Peak II isozyme had a high Km for both cAMP, 76 microM, and cGMP, 32 microM, and peak III isozyme was a cAMP-PDE with Km of 1.8 microM. The peak II and III isozymes were calmodulin-independent. The activity ratio of peak I isozyme to peak III isozyme (I/III isozyme ratio) was significantly different (P less than 0.001) in neuroblastoma and in ganglioneuroma/sympathetic ganglia, i.e., 0.23 +/- 0.11 for neuroblastoma vs. 0.79 +/- 0.20 for ganglioneuroma and 0.51 +/- 0.08 for sympathetic ganglia. Schwannoma showed the highest value of 1.05 (P less than 0.05). These results suggest that the I/III isozyme ratio of cAMP-PDE could be a useful marker in studies on the differentiation of neural crest-derived tumors and Schwann cells. PMID- 3007417 TI - The structure of an epidermal growth factor-receptor kinase inhibitor, erbstatin. PMID- 3007418 TI - Elevation of serum testosterone during chronic LHRH agonist treatment in the bull. AB - The objective of this study was to try to depress serum testosterone (T) in bulls by prolonged treatment with a potent luteinizing hormone-releasing hormone (LHRH) agonist. Eight sexually mature bulls (325 to 475 kg) were assigned to treatment or control groups. Treatment consisted of 150 micrograms nafarelin acetate 6-D-2 naphthyl-alanine-LHRH (LHRH-A) injected im every 6 h for 15 d. Bovine serum albumin (BSA, .01%) in a carrier solution was injected at the same times in control bulls. Serial 15-min blood samples were collected via jugular cannula during the initial 36 h of treatment and during 6-h windows on d 4, 8 and 14. Bulls were slaughtered and pituitaries and testes collected on d 15. Serum luteinizing hormone (LH), follicle stimulating hormone (FSH) and T were elevated after initial injection of LHRH-A, but returned to basal concentrations by 12, 5 and 17 h, respectively. Prolonged LHRH-A treatment prevented pulsatile LH and T secretion compared with control bulls. Mean serum LH did not differ from that of controls on d 4, 8 and 14 of LHRH-A treatment, while serum T was elevated (P less than .01) during the same time periods. Oscillating patterns and mean concentrations of serum FSH were not different between control and LHRH-A-treated bulls. Fifteen days of LHRH-A treatment depressed pituitary LHRH receptor numbers (P less than .05) and pituitary LH (P less than .01) and FSH (P less than .05) concentrations. Testicular LH receptor numbers were elevated (P less than .01), but testicular FSH receptor numbers were not altered.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007419 TI - Effect of supplemental sodium bicarbonate on nutrient digestibilities and ruminal pH measured continuously. AB - A technique was used to monitor continuously ruminal pH using a strip-chart recording pH meter. Ruminal pH measurements were made in four ruminal-cannulated crossbred wether lambs (ag initial weight, 42.5 kg). For l.5 h daily, lambs were given ad libitum access to 50% concentrate-50% chopped orchardgrass hay diets supplements with 0, l.5, 3.0 and 4.5% sodium bicarbonate (NaHCO3). A split-plot Latin-square design was used to evaluate NaHCO3 level and day of adaptation on the percentage of time (%T) that ruminal pH was less than 6.6, 6.2, 5.8, 5.4 and 5.0. No effect due to day of adaptation existed for ruminal pH measurements (P more than .10), while the effect of dietary NaHCO3 level was quadratic (P more than .01) for the %T that ruminal pH was less than 5.4. To evaluate the effects of NaHCO3 on nutrient digestion, the same diets were fed to eight wether lambs (avg initial weight, 38 kg) at 85% of their ad libitum intake in a replicated 4 x 4 Latin-square digestion trial. Digestibilities of dry matter (DM), organic matter, N and fiber fractions were not different due to level of NaHCO3 (P greater than .10). Ash digestibility increased with increasing levels of dietary NaHCO3 (P less than .01). Four ruminal-cannulated mature Hereford steers were also given ad libitum access to the diets in a split-plot Latin-square trial to evaluate effects of dietary NaHCO3 level on ruminal pH and in situ digestion of soybean meal N and orchardgrass DM. During incubation of the dacron bags for 3, 6, 9, 12, 24 and 36 h also increased linearly with increasing level of NaHCO3. Ruminal solid and liquid dilution rates were not affected by level of supplemental NaHCO3 (P greater than .10). The results of these trials suggest that increasing level of dietary NaHCO3 greatly increases the proportion of time ruminal pH is above critical levels for ruminal protein and dry matter digestion, but does not affect total tract nutrient digestion when 50% concentrate diets are fed. PMID- 3007420 TI - Evaluation of prostaglandin E2 as a regulator of lipolysis in bovine adipose tissue. AB - Effects of exogenous prostaglandin E2 (PGE2) on rates of lipolysis in sections of subcutaneous adipose tissue biopsied from fed and fasted Holstein steers were determined. The interaction of PGE2 with several exogenous effectors of lipolysis and of the adenylate cyclase-cAMP system also was measured. Epinephrine increased basal (nonstimulated) lipolysis approximately one-fold. Prostaglandin E2 had no effect on either basal or epinephrine-stimulated lipolysis. Dibutyryl cAMP increased rate of lipolysis .4-fold, whereas theophylline increased lipolysis more than one-fold. Theophylline had an additive effect on epinephrine-stimulated lipolysis. Dibutyryl cAMP increased theophylline-stimulated lipolysis but not epinephrine-stimulated lipolysis. Prostaglandin E2 had no effect on epinephrine-, dibutyryl cAMP- or theophylline-stimulated lipolysis. Fasting decreased basal lipolysis by 40%. Furthermore, lipolysis in tissue incubated with PGE2, epinephrine or PGE2 plus epinephrine decreased from 30 to 50% upon fasting. As also shown with tissue from fed steers, PGE2 did not alter basal or epinephrine stimulated lipolysis in tissue from fasted steers. Influences of exogenous effectors on lipolysis in adipose tissue from fed and fasted steers indicate that PGE2 does not control the adenylate cyclase-cAMP system that regulates lipolysis in bovine adipose tissue. PMID- 3007421 TI - Ovarian follicular growth, function and turnover in cattle: a review. AB - Studies in cattle assessing changes in number and size of antral follicles, concentrations of estradiol, androgens and progesterone in serum and follicular fluid, and numbers of gonadotropin receptors per follicle during repetitive estrous cycles and postpartum anestrus are reviewed. The rate of growth of small follicles (1 to 3 mm) into larger follicles increases as the estrous cycle progresses from d 1 to 18 (d 0 = estrus). Size of the largest antral follicle present on the ovary also increases with advancement of the estrous cycle. Most large follicles (greater than 10 mm) persist on the ovarian surface for 5 d or more between d 3 and 13 of the bovine estrous cycle. After d 13, most of these large follicles are replaced more frequently by new growing follicles (turnover) with an increased probability for recruitment of the ovulatory follicle after d 18. More research is needed to determine the time required for growth of bovine follicles from small to large antral size and evoke recruitment of the ovulatory follicle. Factors that regulate selection of the ovulatory follicle are unknown but may involve increased frequency of LH pulses in blood, altered blood flow and(or) changes in intrafollicular steroids and proteins. Quantitative evaluation of ovarian follicles indicated occurrence of consistent short-term changes in fluid estradiol and numbers of luteinizing hormone receptors in cells of large follicles only during the pre-ovulatory period. Presumably, low concentrations of follicular estradiol found during most of the estrous cycle are not due to a lack of aromatizable precursor or follicle-stimulating hormone receptors. Follicular fluid concentrations of progesterone increase only near the time of ovulation. Little is known about changes in follicular growth, turnover and function during postpartum anestrus in cattle. However, preliminary data suggest that the steroidogenic capacity of large follicles changes markedly during the postpartum period. PMID- 3007422 TI - Comparison of Keyes technique vs. traditional periodontal therapy. PMID- 3007423 TI - Reference material for the evaluation of a standard method for the detection of salmonellas in foods and feeding stuffs. AB - To evaluate a standard salmonella isolation method a reference material consisting of 0.2 g spray-dried milk inoculated with Salmonella typhimurium and contained in gelatin capsules was prepared. The organisms were distributed homogeneously between capsules, and their numbers were stable for 120 d when the capsules were stored in dry conditions at 4 degrees C. Addition of these capsules with or without food samples to pre-enrichment broth gave low and reproducible levels of Salm. typhimurium contamination without altering the pre-enrichment and without influencing the other bacterial flora present. As a result of an interlaboratory trial, the reference material indicated that the food and/or its competitive flora may have a negative influence on the detection of salmonellas. PMID- 3007424 TI - Prediction of the keeping quality of pasteurized milk by the detection of cytochrome c oxidase. AB - The keeping quality (KQ) of pasteurized milk samples stored at 5 degrees C and 10 degrees C was satisfactorily predicted after 18 h pre-incubation with 0.05% benzalkonium chloride at 20 degrees C, by estimating the numbers of Gram-negative psychrotrophic bacteria using the simple, cheap and rapid (5 min) assay of cytochrome c oxidase. Correlation coefficients for the relationship between cytochrome c oxidase activity at 20 degrees C and KQ at 5 degrees C or 10 degrees C of -0.89 and -0.84 respectively were obtained. The method correctly predicted the KQ of more than 89% of the samples of pasteurized milk. The assay was not satisfactory for use on samples after pre-incubation at 30 degrees C. PMID- 3007425 TI - Influence of angiotensin-converting enzyme inhibitor, foroxymithine, on dynamic equilibrium around the renin-angiotensin system in vivo. AB - To understand the in vivo actions of angiotensin-converting enzyme (ACE) inhibitors, a prolonged study was performed in rabbits over a half year, using one of such inhibitors, foroxymithine. During the initial 2 months of the inhibitor administration, the serum level of ACE was suppressed. Thereafter, probably triggered by the consequent sharp rise in the plasma renin activity (PRA) level, the ACE level regained its initial value. Thus the close correlation between the levels of PRA and ACE seen in the control animal was entirely broken by this inhibitor. A multivariate study indicated that the inhibitor drastically changed the normal networks of peptide metabolism in vivo. These results are compatible with the notion that the ACE inhibitor blocks the regulatory mechanisms of the renin-angiotensin system in vivo. PMID- 3007426 TI - Methacholine sensitivity and cAMP protein kinase in tracheal smooth muscle. AB - We studied regional variation in canine trachealis smooth muscle sensitivity and responsiveness to methacholine as well as basal and methacholine-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) and cAMP-dependent protein kinase activity. The trachea between the cricoid cartilage and the carina was divided into three segments of equal length (designated cervical, middle, and thoracic regions), each consisting of approximately 12-14 cartilage rings. Smooth muscle strips from each of the three regions were exposed to cumulative half-log increments of methacholine chloride. The sensitivity (-log EC50) and responsiveness (force per cross-sectional area and force per milligram protein) of the smooth muscle to methacholine in each region was determined from these data. Smooth muscle strips from cervical and thoracic regions were frozen before and after exposure to cumulative half-log increments of methacholine up to each region's previously determined EC50. Frozen samples were assayed for cAMP content or cAMP-dependent protein kinase activity. The relationship between resting tension and methacholine sensitivity and responsiveness were studied. For the size strips we used, 4 g resting tension set the average cervical and thoracic strips at 96 and 101% of their optimal length, respectively. The methacholine EC50 was not affected by a variation in resting tension. Sensitivity to methacholine was 7.1, 6.8, and 6.5 for cervical, middle, and thoracic regions, respectively. The responsiveness of the cervical and thoracic smooth muscle to methacholine was 16.4 and 16.3 g force/mm2, respectively, at an EC50 methacholine. Basal cAMP was lower in cervical smooth muscle than in thoracic. cAMP-dependent protein kinase activity ratios under both basal and EC50 methacholine-stimulated conditions were lower in cervical smooth muscle than in thoracic. We have observed in trachealis smooth muscle an inverse relationship between methacholine sensitivity and either cAMP or cAMP-dependent protein kinase activity. We suggest that cAMP and cAMP-dependent protein kinase play a role in the regulation of airway smooth muscle sensitivity to cholinergic agonists. PMID- 3007427 TI - Suppression of pulmonary and systemic vascular histamine H2-receptors in allergic sheep. AB - We have previously demonstrated a depression of airway H2-receptor function in sheep allergic to Ascaris suum antigen. To investigate whether this is a generalized defect, we studied the H1- and H2- histamine receptor functions in the pulmonary and systemic circulations of allergic and nonallergic sheep. Pulmonary arterial pressure, and cardiac output were measured for calculation of pulmonary vascular resistance (PVR) and systemic vascular resistance (SVR) before and immediately after a rapid intrapulmonary infusion of histamine (10 micrograms/kg), with and without pretreatment with H1- (chlorpheniramine) and H2- (metiamide) antagonists. Histamine alone increased mean PVR to 435 and 401% of base line and decreased mean SVR by 51 and 54% in the nonallergic and allergic sheep, respectively (P less than 0.001). In the nonallergic sheep following pretreatment with chlorpheniramine (selective H2 stimulation) or metiamide (selective H1 stimulation), histamine decreased SVR by 18 and 36%, respectively, suggesting that approximately two-thirds of the vasodepressor response was mediated by H1-receptors and one-third by H2-receptors. Combined H1- and H2 antagonists completely blocked the histamine response. In allergic sheep the histamine-induced decrease in SVR was primarily mediated by H1-receptors, because the response was blocked by H1-antagonist, chlorpheniramine, and the H2 antagonist, metiamide, had no effect. In the pulmonary circulation selective H1 stimulation caused a similar increase in PVR in allergic (365%) and nonallergic sheep (424%), whereas selective H2-stimulation caused a significant decrease in PVR in the nonallergic group (14%) but not in the allergic group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007428 TI - Hyperoxia reduces plasma membrane fluidity: a mechanism for endothelial cell dysfunction. AB - To evaluate the relative contributions of three possible mechanisms that can be advanced to explain the observation that hyperoxia decreases serotonin uptake by endothelial cells, we examined the effect of high O2 tensions on Na+-K+-ATPase activity, ATP content, and plasma membrane fluidity in cultured endothelial cells. Confluent monolayers of pulmonary artery and aortic endothelial cells were exposed to 95% O2 (hyperoxia) or 20% O2 (controls) in 5% CO2 at 1 ATA for 4-42 h. Exposure to high O2 tensions had no effect on Na+-K+-ATPase activity or ATP content in pulmonary artery or aortic endothelial cells in culture. However, hyperoxia decreased the fluidity of the plasma membrane of pulmonary artery and aortic endothelial cells in culture, and the time course for the decrease in fluidity parallels that of the hyperoxic inhibition of serotonin transport. These results indicate that hyperoxia decreases fluidity in the hydrophobic core of the plasma membranes of cultured endothelial cells. Such decreases in plasma membrane fluidity may be responsible for hyperoxia-induced alterations in membrane function including decreases in transmembrane transport of amines. PMID- 3007430 TI - F plasmid ccd mechanism in Escherichia coli. AB - The ccd mechanism specified by the ccdA and ccdB genes of the mini-F plasmid determines fate of plasmid-free segregants in Escherichia coli (Jaffe et al., J. Bacteriol. 163:841-849, 1985). The killing function in plasmid-free segregants by the ccd mechanism did not affect cell growth of coexisting cells in the same culture. Elongated cells and anucleate cells caused by the ccd mechanism were clearly detected by flow cytometry in cultures of bacterial strains harboring Ccd+ Sop- mini-F plasmids defective in partitioning. This indicates that the defect in correct partitioning of plasmid DNA molecules into daughter cells also induces the ccd mechanism to operate. PMID- 3007429 TI - Effects of pheochromocytoma on cardiovascular alpha adrenergic receptor system. AB - We have examined the in vivo consequences of prolonged stimulation of the cardiovascular alpha-adrenergic receptor system in a rat model harboring pheochromocytoma. New England Deaconess Hospital rats with transplanted pheochromocytomas developed systolic hypertension and their plasma norepinephrine concentrations were approximately 60-fold greater than controls. Alpha 1 adrenergic receptors were quantitated in hearts from controls and rats with transplanted pheochromocytoma using the alpha 1-receptor selective antagonist [3H]prazosin. Down-regulation of alpha 1-receptors was found in the hearts of pheochromocytoma rats (33.0 vs. 23.0 fmol/mg protein) without any significant change in the affinities of these receptors for the circulating catecholamine, norepinephrine. Furthermore, the responsiveness of the blood vessel to the alpha adrenergic stimulation was assessed using in vitro contractile experiments. Aortic rings from pheochromocytoma animals showed an eight fold decrease in sensitivity (EC50) and a 74% decrease in maximal contractility (Emax) to norepinephrine as compared with controls. Similarly, mesenteric artery rings prepared from the same animals showed a five fold loss of EC50 but no decrease in Emax to phenylephrine as compared with controls. In addition, serotonin EC50 and Emax of these mesentery preparations remained unaltered. Coupled with our previous findings [9], the present study suggests that rats with pheochromocytoma secreting large amounts of norepinephrine provide a valuable model system for studying in vivo desensitization of the cardiovascular alpha-receptor systems as well as the beta-adrenergic receptor system. PMID- 3007431 TI - Identification of the pleiotropic sacQ gene of Bacillus subtilis. AB - The sacQ gene of Bacillus subtilis, a pleiotropic gene affecting the expression of a number of secreted gene products, has been identified as a small 46-amino acid polypeptide. The increased expression of this polypeptide in strains carrying the sacQ36 allele, or in strains carrying the sacQ gene on a high copy plasmid, appears to be responsible for the phenotype of higher levels of proteases seen in these strains. A deletion of the sacQ gene had no apparent phenotype, indicating that it is not an essential gene. PMID- 3007433 TI - Subdivision of flagellar genes of Salmonella typhimurium into regions responsible for assembly, rotation, and switching. AB - Three flagellar genes of Salmonella typhimurium (flaAII.2, flaQ, and flaN) were found to be multifunctional, each being associated with four distinct mutant phenotypes: nonflagellate (Fla-), paralyzed (Mot-), nonchemotactic (Che-) with clockwise motor bias, and nonchemotactic (Che-) with counterclockwise motor bias. The distribution of Fla, Mot, and Che mutational sites within each gene was examined. Fla sites were fairly broadly distributed, whereas Mot and Che sites were more narrowly defined. Local subregions rich in sites of one type were not generally rich in sites of another type. Among Che sites, there was little overlap between those corresponding to a clockwise bias and those corresponding to a counterclockwise bias. Our results suggest that within the corresponding gene products there are specialized subregions for flagellar structure, motor rotation, and control of the sense of rotation. PMID- 3007432 TI - Physical and genetic analysis of the ColD plasmid. AB - The plasmid ColD-CA23, a high-copy-number plasmid of 5.12 kilobases, encodes colicin D, a protein of approximately 87,000 daltons which inhibits bacterial protein synthesis. Colicin D production is under the control of the Escherichia coli SOS regulatory system and is released to the growth medium via the action of the lysis gene product(s). A detailed map of the ColD plasmid was established for 10 restriction enzymes. Using in vitro insertional omega mutagenesis and in vivo insertional Tn5 mutagenesis, we localized the regions of the plasmid responsible for colicin D activity (cda), for mitomycin C-induced lysis (cdl), and for colicin D immunity (cdi). These genes were all located contiguously on a 2,400 base-pair fragment similar to a large number of other Col plasmids (A, E1, E2, E3, E8, N, and CloDF). The ColD plasmid was mobilizable by conjugative transfer by helper plasmids of the IncFII incompatibility group, but not by plasmids belonging to the groups IncI-alpha or IncP. The location of the mobilization functions was determined by deletion analysis. The plasmid needs a segment of 400 base pairs, which is located between the mob genes and the gene for autolysis, for its replication. PMID- 3007434 TI - Vegetative expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis. AB - Bacillus thuringiensis subsp. kurstaki total DNA was digested with BglII and cloned into the BamHI site of plasmid pUC9 in Escherichia coli. A recombinant plasmid, pHBHE, expressed a protein of 135,000 daltons that was toxic to caterpillars. A HincII-SmaI double digest of pHBHE was then ligated to BglII-cut plasmid pBD64 and introduced into Bacillus subtilis by transformation. The transformants were identified by colony hybridization and confirmed by Southern blot hybridization. A 135,000-dalton protein which bound to an antibody specific for the crystal protein of B. thuringiensis was detected from the B. subtilis clones containing the toxin gene insert in either orientation. A toxin gene insert cloned into a PvuII site distal from the two drug resistance genes of the pBD64 vector also expressed a 135,000-dalton protein. These results suggest that the toxin gene is transcribed from its own promoter. Western blotting of proteins expressed at various stages of growth revealed that the crystal protein expression in B. subtilis begins early in the vegetative phase, while in B. thuringiensis it is concomitant with the onset of sporulation. The cloned genes when transferred to a nonsporulating strain of B. subtilis also expressed a 135,000-dalton protein. These results suggest that toxin gene expression in B. subtilis is independent of sporulation. Another toxin gene encoding a 130,000- to 135,000-dalton protein was cloned in E. coli from a library of B. thuringiensis genes established in lambda 1059. This gene was then subcloned in B. subtilis. The cell extracts from both clones were toxic to caterpillars. Electron microscope studies revealed the presence of an irregular crystal inclusion in E. coli and a well-formed bipyramidal crystal in B. subtilis clones similar to the crystals found in B. thuringiensis. PMID- 3007437 TI - Influence of gyrA mutation on expression of Erwinia chrysanthemi clb genes cloned in Escherichia coli. AB - Erwinia chrysanthemi clb genes cloned into Nals Escherichia coli allowed growth on cellobiose, arbutin, or salicin. In contrast, Nalr isogenic strains grew only on cellobiose. It is proposed that expression of cloned E. chrysanthemi clb genes is reduced by the E. coli chromosomal gyrA (Nalr) mutation, resulting in apparent segregation of the Clb and Arb Sal characters. PMID- 3007436 TI - Inversion of aerotactic response in Escherichia coli deficient in cheB protein methylesterase. AB - Mutants of Escherichia coli and Salmonella typhimurium that were deficient in protein methylesterase activity encoded by cheB had an inverted response to oxygen; they were repelled by concentrations of oxygen that attract wild-type bacteria. Normal responses to oxygen and phosphotransferase substrates were observed in mutants that were deficient in protein methyltransferase (CheR) and the methyl-accepting transducing proteins (Tsr, Tar, Trg). However, the methylation-independent response to oxygen was modified by the loss of esterase activity. The inversion was apparently effected by the amidated Tsr protein present in cheB tsr+ mutants because aerotaxis was normal in cheB tsr strains. Chemotaxis to phosphotransferase sugars was normal in cheB mutants provided the extreme clockwise bias of the flagellar motors was modified to increase the probability of counterclockwise rotation. PMID- 3007435 TI - Nucleotide sequence of the Escherichia coli motB gene and site-limited incorporation of its product into the cytoplasmic membrane. AB - The motB gene product of Escherichia coli is an integral membrane protein required for rotation of the flagellar motor. We have determined the nucleotide sequence of the motB region and find that it contains an open reading frame of 924 nucleotides which we ascribe to the motB gene. The predicted amino acid sequence of the gene product is 308 residues long and indicates an amphipathic protein with one major hydrophobic region, about 22 residues long, near the N terminus. There is no consensus signal sequence. We postulate that the protein has a short N-terminal region in the cytoplasm, an anchoring region in the membrane consisting of two spanning segments, and a large cytoplasmic C-terminal domain. By placing motB under control of the tryptophan operon promoter of Serratia marcescens, we have succeeded in overproducing the MotB protein. Under these conditions, the majority of MotB was found in the cytoplasm, indicating that the membrane has a limited capacity to incorporate the protein. We conclude that insertion of MotB into the membrane requires the presence of other more hydrophobic components, possibly including the MotA protein or other components of the flagellar motor. The results further reinforce the concept that the total flagellar motor consists of more than just the basal body. PMID- 3007438 TI - Transposonlike elements in Caedibacter taeniospiralis. AB - We report that the 1.5- and 7.5-kilobase-pair (kbp) transposonlike sequences present in the R-body-coding plasmids of Caedibacter taeniospiralis share homology. The R-body-coding plasmids of two new strains of C. taeniospiralis, derived from strains 169 and A30, carry the 7.5- and 1.5-kbp elements, respectively, inserted at new positions. Sequences homologous to the 7.5-kbp sequence from C. taeniospiralis 47 were detected in the chromosomes of three other strains of C. taeniospiralis. PMID- 3007439 TI - Temperature sensitivity of a nifA-like gene in Enterobacter cloacae. AB - Nitrogen fixation (nif) genes of Enterobacter cloacae, a rhizosphere diazotroph of rice plants, were identified by using cloned Klebsiella pneumoniae nif gene fragments as probes for molecular hybridization. The product of a nifA-like gene of E. cloacae appeared less temperature sensitive than the K. pneumoniae nifA gene product. This result correlates with the fact that E. cloacae can fix nitrogen at 39 degrees C, while K. pneumoniae cannot. PMID- 3007440 TI - Identification of a specific membrane-particle-associated DNA sequence in Bacillus subtilis. AB - After the Bacillus subtilis nucleoid was dissected with restriction endonucleases, a specific DNA sequence from the purA region was isolated in a particulate form that probably originated from the cell membrane. Precise definition of the binding region within this sequence was achieved by a novel procedure based on a previously reported observation that additional copies of the binding region, introduced into the chromosome using an integrative plasmid, were also predominantly particle bound. Subsections of the original plasmid insertion were cloned into the integrative plasmid and introduced into B. subtilis, in which they became tandemly reiterated under appropriate selective conditions. HaeIII sites in the vector, flanking each insertion, were used to excise the latter for subsequent tests of particle association. Examination of 10 strains containing subsections of the original 5.2-kilobase-pair region showed that the binding region was confined to 283 base pairs. This was confirmed by dissection in vitro of a larger, isolated, particle-bound sequence. The nucleotide sequence of a 1,300-base-pair region that contained this site was determined. The entire region had a notably high A + T content and was deficient in open reading frames for transcription. PMID- 3007442 TI - Identification of the nahR gene product and nucleotide sequences required for its activation of the sal operon. AB - The product of the nahR gene, a salicylate-dependent activator of transcription of the nah and sal hydrocarbon degradation operons of the NAH7 plasmid, was identified and characterized after synthesis in Escherichia coli maxicells. The nahR gene product had a subunit molecular weight of 36,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas gel filtration analysis of the nondenatured nahR protein indicated a molecular weight in excess of 250,000. However, DNase I treatment of this high-molecular-weight complex shifted the apparent molecular weight of the nahR protein to 40,000. Various upstream portions of the sal operon promoter were transcriptionally fused to the E. coli galactokinase gene. Fusion plasmids containing the sal promoter sequence from --83 to 27 (relative to the transcription start site) showed salicylate inducible expression of galactokinase in the presence of the cloned nahR gene, while expression of galactokinase from a fusion plasmid containing the sal promoter sequence from --45 to 27 was not induced by the nahR gene and salicylate. Results suggest that the nahR gene product is a 36-kilodalton polypeptide which exerts its salicylate-dependent activation of transcription of the sal operon by interacting with the promoter sequence in the region of --83 to --45 base pairs before the transcription start site. PMID- 3007441 TI - Mechanism of delta pH maintenance in active and inactive cells of an obligately acidophilic bacterium. AB - The acidophilic bacterium PW2 possessed a delta pH of ca. 1.9 and a delta psi of 0 mV, corresponding to a proton motive force (delta p) of--114 mV. Protonophore treated cells possessed little delta p but a delta pH of ca. 1.5, as measured by salicylic acid distribution or pH measurement of cell lysates. Starving PW2 cells continued to possess a delta pH of ca. 1.7, but exhibited converse changes in delta psi and delta p, with the former rising to +80 to +100 mV and the latter dropping essentially to 0; progressive loss of respiration, cellular ATP, and culture viability accompanied these changes. Thus, the protonophore-treated or starving PW2 cells attained an H+ electrochemical equilibrium. Net H+ influx resulting from declining respiration probably accounted for the increased delta psi in these cells; indeed, when respiration was progressively inhibited in active cells, there was increasing transient H+ influx and a proportional increase in delta psi. This transient H+ influx was sufficient to lethally acidify the cytoplasm, but for a buffering capacity of 85 nmol of H+/mg of protein per pH unit. Thus, the linkage of the transient H+ influx with the rise in the delta psi and the cytoplasmic buffering capacity play central roles in acidophilism, and it is conceivable that the same impermeant cellular macromolecule(s) accounts for both. If so, the delta psi would be a Donnan potential that in active cells is offset by energy-dependent H+ extrusion. PMID- 3007443 TI - Modulation of ethanol intake by serotonin uptake inhibitors. AB - The most commonly prescribed agents for decreasing ethanol intake are alcohol sensitizing drugs; however, their efficacy is unproven, they are associated with toxicity, and there are several contraindications for use. A program to identify and test new drugs to decrease ethanol intake has focused on drugs that enhance central serotonergic neurotransmission and consistently attenuate ethanol consumption. Animal studies have shown consistent findings with direct and indirect serotonin (5-HT) agonists. Ethanol intake decreased after the administration of 5-HT precursors, 5-HT uptake inhibitors, intracerebral 5-HT, and postsynaptic 5-HT agonists; in contrast, destruction of serotonin-containing neurons with 5,6- or 5,7-dihydroxytryptamine increased ethanol intake. Administration of zimelidine (200 mg/day p.o.) to 16 healthy alcohol abusers was associated with a significant increase in number of abstinent days and a decrease in number of drinks consumed. Approximately 50% of the subjects were responders, 35% were partial responders, and 10%-15% were nonresponders. In a recent double blind crossover study, citalopram, an even more selective serotonin uptake inhibitor, produced similar results. Because serotonin uptake inhibitors acted rapidly and subjects were not clinically depressed, this action is distinct from antidepressant effects. These drugs most likely interfere with the neurobiologic mechanisms regulating ethanol intake and provide an innovative approach for modulating the use of alcohol in problem drinkers. PMID- 3007444 TI - Inactivation of calpain I and calpain II by specificity-oriented tripeptidyl chloromethyl ketones. AB - Three new tripeptidyl chloromethyl ketones, Leu-Leu-XCH2Cl, with X representing Phe, Tyr, or Lys, were synthesized and their potencies to inactivate calpains I and II were compared. They were designed to fulfil the specificity requirement of calpains established recently. When compared in terms of the dose for 50% inactivation, Leu-Leu-PheCH2Cl was the strongest inactivator, being 500-600 times more effective than tosyl-PheCH2Cl and 5-14 times more than N-[N-(L-3-trans carboxyoxiran-2-carbonyl)-L-leucyl]agmatine (E-64). The potency toward calpain, either I or II, decreased in the order Phe greater than Tyr greater than Lys derivatives greater than E-64, whereas that toward papain was E-64 greater than Lys greater than Phe greater than Tyr derivatives. From the determined kinetic parameters, the Phe derivative was 18.3 and 16.6 times more effective than E-64 on calpains I and II, respectively. Likewise, the rate of the alkylation reaction by these chloromethyl ketones with calpain I was 2-4 times greater than that with calpain II. Leu-Leu-PheCH2Cl and its N-dansylated product should be useful for highly selective affinity labeling of calpains I and II. PMID- 3007445 TI - Mapping and sequencing of RNAs without recourse to molecular cloning: application to RNAs of the Sabin 1 strain of poliovirus and its defective interfering particles. AB - Complementary DNA to the genome of the Sabin 1 strain of poliovirus was prepared by reverse transcription with oligo(dT)10 as a primer and separated into six classes of DNA by their size. Each class of the DNA, after digestion with restriction endonuclease HaeIII, was analyzed by two-dimensional polyacrylamide gel electrophoresis. Comparison of the patterns of the restriction fragments led us to compose a possible arrangement of the restriction fragments on the viral genome. Sequence analysis of these fragments indicated that the arrangement was consistent with the known total nucleotide sequence of the genome. In the determined sequences, two bases were observed to differ from those of a cloned complementary DNA of the Sabin 1 genome. This suggested that the sequence of the cloned DNA reflected that of a mutated virus genome that was a minor component in the virus inoculation stock. The genomes of defective interfering particles generated from the Sabin 1 strain were also analyzed by this technique. The results suggested that the RNAs lacked an internal region of the Sabin 1 RNA encoding viral capsid proteins. The location of the deletion was further confirmed by determination of the nucleotide sequence of a cloned complementary DNA copy of the defective interfering particle RNA. Thus, the method described here is useful for mapping and sequencing of RNAs and for knowing whether cloned cDNAs represent the major population of RNA molecules or not. PMID- 3007446 TI - Purification and characterization of alpha-galactosidase from watermelon. AB - An alpha-galactosidase [EC 3.2.1.22] was isolated from the fruit of the watermelon, Citrullus battich. The enzyme was purified by procedures including extraction, ammonium sulfate precipitation, and chromatographies on DEAE Sephadex, CM-Sephadex and Sephadex G-100. The final preparation was found to be fairly homogeneous on disc and SDS-polyacrylamide gel electrophoresis, and sufficiently free from other glycosidase activities. The molecular weight of the enzyme was estimated to be 45,000 by Sephadex G-100 column chromatography and SDS polyacrylamide gel electrophoresis. The enzyme was most active at pH 4.5 for natural substrates and at 5.9 for artificial substrates. The enzyme liberates the alpha-galactose units from oligosaccharides of the raffinose series and ceramide trihexoside, and the hemagglutination-inhibiting activities of human ovarian cyst B-glycoprotein and blood group B-type ghosts were abolished by the enzyme. PMID- 3007447 TI - Two non-identical heads of myosin molecules. AB - Four different preparations of skeletal subfragment-1, denoted in this report as S1(Aa), S1(Ab), S1(Ba), and S1(Bb), and two different preparations of cardiac subfragment-1, denoted as S1(A) and S1(B), were obtained as described in our recent report (J. Biochem. 97, 965, 1985). (i) The four preparations were obtained from chicken breast myosin trinitrophenylated with 2,4,6-trinitrobenzene sulfonate in the absence of inorganic pyrophosphate (-PPi), and they were all shown to be trinitrophenylated. Addition of PPi caused change in the absorption spectra of trinitrophenyl(TNP)-S1(Aa) and TNP-S1(Ba), but not in those of TNP S1(Ab) and TNP-S1(Bb). (ii) The two preparations of S1 were obtained from cardiac myosin trinitrophenylated either in the absence (-) or presence (+) of PPi. S1(B) was trinitrophenylated, whereas S1(A) was not. Specifically emphasized is the observation that the yield of cardiac S1(A) was practically equal to that of cardiac S1(B). On the basis of these results, we propose the hypothesis of "two iso-myosins with non-identical heads," which is essentially a combination of the hypothesis of isoenzymes and that of non-identical heads. PMID- 3007448 TI - Differential labeling of the subunits of respiratory complex III with [3H]succinic anhydride, [14C]succinic anhydride, and p-diazobenzene [35S]sulfonate. AB - Exposure of antimycin-treated Complex III (ubiquinol-cytochrome c reductase) purified from bovine heart mitochondria to [3H]succinic anhydride plus [35S]p diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [3H]succinic anhydride. In contrast, relative labeling by [35S]DABS was similar to [3H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex II depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [3H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by 14C- and 3H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7,8, and 9. Two additional polypeptides of molecular masses 23 and 12kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of 14C/3H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in 14C/3H labeling ratios of core proteins I and II, cytochrome c1, and a polypeptide of molecular mass 13kDa identified as an antimycin-binding protein. PMID- 3007449 TI - Effect of trypsin on the kinetic properties of reconstituted beef heart cytochrome c oxidase. AB - Isolated beef heart cytochrome c oxidase was reconstituted in liposomes by the cholate dialysis method with 85% of the binding site for cytochrome c oriented to the outside. Trypsin cleaved specifically subunit VIa and half of subunit IV from the reconstituted enzyme. The kinetic properties of the reconstituted enzyme were changed by trypsin treatment if measured by the spectrophotometric assay but not by the polarographic assay. It is concluded that subunit VIa and/or subunit IV participate in the electron transport activity of cytochrome c oxidase. PMID- 3007450 TI - Morphology of proteoliposomes containing fluorescein-phosphatidylethanolamine reconstituted with native and subunit III-depleted cytochrome c oxidase. AB - Beef heart cytochrome c oxidase was reconstituted in asolectin liposomes containing the pH indicator fluorescein-phosphatidylethanolamine (FPE) by the cholate-dialysis procedure. The influence of FPE on the asolectin liposome size and of the removal of subunit III from the complex on its incorporation into liposomes was analyzed by freeze-fracture electron microscopy. Samples were frozen without the addition of cryoprotectants. The vesicle size distribution of native enzyme reconstituted into asolectin liposomes was homogeneous, 84% of the population having a diameter of 14-37 +/- 7.5 mm. The preparation containing FPE had a similar vesicle size distribution, but with bigger diameter range (20-50 nm). In all three different types of proteoliposome preparations the majority of particles containing vesicles was found to have 1 particle (42-81%). The absence of subunit III did not influence the incorporation of the enzyme into the liposomes and was as good as the preparation with native enzyme (greater than 99%). Therefore we conclude that the suppression of the proton pump activity was due to the intrinsic properties of subunit III and not to defective incorporation into artificial membrane systems. PMID- 3007451 TI - Identification of a plasma gelatinase in preparations of fibronectin. AB - Preparations of fibronectin purified from human plasma according to conventional methods was found to contain a latent gelatinolytic activity. The protease was activated by exposure to trypsin or electrophoresis in sodium dodecyl sulfate. Zymography of the enzyme under nonreducing conditions gave an estimated Mr of 72,000. Reducing agents destroyed the activity of the enzyme. The gelatinase co purified with fibronectin in chromatography on Sepharoses conjugated with gelatin, arginine, and heparin but could be separated from fibronectin by gel filtration in a physiological buffer. This protease was found to be a normal constituent of plasma and was probably not derived from the blood cells since the 72-kDa protease was not detected in lysates of these cells. PMID- 3007452 TI - Components responsible for transport between successive Golgi cisternae are highly conserved in evolution. AB - Transport of a glycoprotein between compartments of the Golgi has been reconstituted in an in vitro system (Balch, W. E., Dunphy, W. G., Braell, W. A., and Rothman, J. E. (1984) Cell 39, 405-416). Cytosolic components and ATP are absolutely required for transport. Here, we have tested the acceptor activity of Golgi fractions and of cytosolic fractions prepared from a variety of organisms. All mammalian Golgi fractions can act as "acceptor" in the in vitro assay. Similarly, the cytosol fractions obtained from plants as well as animals and a lower eukaryote substitute for the homologous CHO cytosol normally used. Moreover, a cytosol subfraction prepared from wheat germ complements a different cytosolic fraction obtained from bovine brain. Apparently, the essential components involved in the post-translational protein transport are remarkably conserved between plants, animals, and lower eukaryotes. PMID- 3007453 TI - Two different forms of cytochrome c oxidase can be purified from the slime mold Dictyostelium discoideum. AB - Cytochrome c oxidase was purified from mitochondria of Dictyostelium discoideum cells harvested at different phases of the vegetative stage. Comparison of the preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the smallest enzyme subunit present during exponential growth is replaced by a larger polypeptide component in the stationary phase. The two polypeptides are structurally and immunologically unrelated. PMID- 3007454 TI - The murine transforming growth factor-beta precursor. AB - Transforming growth factor-beta (TGF-beta) is a homodimeric polypeptide which can act, often in cooperation with other growth factors, as a mitogenic factor for a variety of cells. TGF-beta can also exert growth inhibitory activity on many other cell lines. We have isolated cDNAs coding for the murine TGF-beta cDNA precursor. The deduced amino acid sequence localizes the 112-amino acid long TGF beta monomer to the C terminus of the precursor. Two areas of the precursor exhibit a marked degree of homology to the human counterpart. One of these regions comprises the mature TGF-beta monomer, while the other corresponds to the NH2 terminus of the precursor and suggests an important biological function for this area. Northern hybridization results identify a major 2.5-kilobase TGF-beta mRNA and several minor TGF-beta mRNA species. PMID- 3007455 TI - Do human neutrophils make hydroxyl radical? Determination of free radicals generated by human neutrophils activated with a soluble or particulate stimulus using electron paramagnetic resonance spectrometry. AB - Using electron paramagnetic resonance spectrometry and the spin trap 5,5-dimethyl 1-oxide (DMPO), neutrophil free radical production in response to phorbol myristate acetate and opsonized zymosan was investigated. Using phorbol myristate acetate and zymosan (3 mg/ml), the superoxide spin-trapped adduct 2-2-dimethyl-5 hydroperoxy-1-pyrrolidinyloxyl (DMPO-OOH) and the hydroxyl spin-trapped adduct 2 2-dimethyl-5-hydroxy-1-pyrrolidinyloxyl (DMPO-OH) were detected. Only DMPO-OH was observed with zymosan (0.5 mg/ml). Hydroxyl radical production in the presence of dimethylsulfoxide (Me2SO) and DMPO yields 2,2,5-trimethyl-1-pyrrolidinyloxyl. The only 2,2-trimethyl-1-pyrrolidinyloxyl detected following neutrophil stimulation was that expected from DMPO-OOH degradation. Superoxide dismutase but not catalase inhibited generation of all three spin-trapped adducts. These data indicate that DMPO-OH arose from DMPO-OOH degradation and does not represent hydroxyl radical production. Under certain conditions DMPO-OH is the predominant spin-trapped adduct resulting from neutrophil superoxide production, perhaps due to cellular bioreduction of DMPO-OOH to DMPO-OH. Cytochalasin B, which prevents phagosome closure, inhibited zymosan-stimulated neutrophil oxygen consumption and electron paramagnetic resonance superoxide detection. No hydroxyl radical was detected. Spin trapping with DMPO appears to detect intraphagosomal free-radical formation. PMID- 3007456 TI - Inhibition of ACTH action on cultured bovine adrenal cortical cells by 2,3,7,8 tetrachlorodibenzo-p-dioxin through a redistribution of cholesterol. AB - The conversion of cholesterol to cortisol by cultured bovine adrenal cortical cells is stimulated 6-fold by adrenocorticotropin and is limited by the movement of cholesterol to the mitochondria (DiBartolomeis, M.J., and Jefcoate, C.R. (1984) J. Biol. Chem. 259, 10159-10167). Exposure of confluent cultures to the potent environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10( 8)M), for 24 h prior to adrenocorticotropin (ACTH) addition decreased the rate of ACTH-stimulated steroidogenesis but did not affect the basal rate. TCDD was more effective against stimulation at 10(-11) M ACTH (4-fold) than at 10(-7) M ACTH (10%), consistent with an increase in EC50 for ACTH. Stimulation of bovine adrenal cortical cells by cAMP was similarly decreased by TCDD. In both cases the effectiveness of TCDD increased with time of exposure to the stimulant. The transfer of cholesterol to mitochondria in intact cells was quantitated by means of the 2-h accumulation of mitochondrial cholesterol in the presence of aminoglutethimide, an inhibitor of cholesterol side chain cleavage. Although cholesterol accumulated in the presence of ACTH (13 to 28 micrograms/mg), pretreatment of cells with TCDD caused a decrease in mitochondrial cholesterol (13 to 8 micrograms/mg). The effect of TCDD was produced relatively rapidly (t1/2 approximately 4 h). In absence of TCDD, the mitochondria of ACTH-stimulated cells also eventually lose cholesterol (after 2 h). It is concluded that TCDD pretreatment may increase the presence of a protein(s) that cause mitochondrial cholesterol depletion when the cells are stimulated by ACTH or cAMP. TCDD enhanced cholesterol efflux from mitochondria diminishes cholesterol side chain cleavage when mitochondrial cholesterol is sufficiently depleted (after 2-4 h). PMID- 3007457 TI - The elongation factor G carries a catalytic site for GTP hydrolysis, which is revealed by using 2-propanol in the absence of ribosomes. AB - In the absence of ribosomal particles, elongation factor G (EF-G) promotes very little GTP hydrolysis. After the addition of some aliphatic alcohols to EF-G, the rate of nucleotide cleavage was significantly increased and GTPase activity was easily detectable. The highest stimulation, nearly 16-fold, occurred with 2 propanol at a 20% (v/v) concentration. The reaction showed the characteristics of an enzymatic catalysis, but the rate was three orders of magnitude lower than that of the ribosome-dependent EF-G GTPase activity. Striking similarities between the two activities indicated that the catalysis stimulated by the alcohol was due to EF-G itself. We found that EF-G GTPase activity in the presence of 2 propanol displayed an absolute specificity for GTP as in the presence of ribosomes; the two activities copurified to a constant ratio and exhibited coincident chromatographic and electrophoretic patterns; the temperature for the half-inactivation of EF-G was 59.3 degrees C for both GTPase systems, as well as the kinetic constant for the thermal inactivation process which was found to be 0.05 min-1; and the Km for the GTP in the presence of 2-propanol (59 microM) was similar to that found in the presence of ribosomes. These results indicate that the EF-G molecule carries a catalytic site for GTP hydrolysis, which in the absence of ribosomal particles is activated by an appropriate alcohol/water surrounding medium. PMID- 3007459 TI - Polyphosphate kinase from Propionibacterium shermanii. Demonstration that the synthesis and utilization of polyphosphate is by a processive mechanism. AB - The mechanism of synthesis of inorganic polyphosphate by polyphosphate kinase (EC 2.7.4.1) from Propionibacterium shermanii is shown to be processive. Analysis of the synthesized polyphosphate on polyacrylamide gels, which resolve on the basis of molecular weight, proves that the elongation reaction occurs without dissociation of intermediate sizes of the polymer from the enzyme. As a consequence, only high molecular weight polyphosphates are synthesized. The mechanism is processive both in the presence and absence of basic protein. It has been shown previously that basic proteins stimulate the synthesis of polyphosphate (Robinson, N.A., Goss, N.H., and Wood, H.G. (1984) Biochem. Int. 8, 757-769). In addition, using a similar method, it is shown that the reverse reaction, the utilization of polyphosphate to phosphorylate ADP, occurs by a processive mechanism. Accordingly, polyphosphates formed by polyphosphate kinase in the cell would be entirely high molecular weight. PMID- 3007458 TI - Polyphosphate glucokinase from Propionibacterium shermanii. Kinetics and demonstration that the mechanism involves both processive and nonprocessive type reactions. AB - Polyphosphate glucokinase (EC 2.7.1.63, polyphosphate glucose phosphotransferase) has been partially purified (960-fold) from Propionibacterium shermanii. Throughout the purification, the ratio of polyphosphate glucokinase activity to ATP glucokinase activity remained approximately constant at 4 to 1. It is considered that both activities are catalyzed by the same protein. The mechanism of utilization of polyphosphate by polyphosphate glucokinase was investigated using polyphosphates of limited sizes that were isolated following gel electrophoresis of commercial heterogeneous polyphosphates. The results show that with long chain polyphosphates, the reaction proceeds by a processive type mechanism, and with short polyphosphates, it is nonprocessive. The Km for polyphosphate of chain length 724 is 2 X 10(-3) microM and increases with a decrease in chain length to 3.7 X 10(-2) microM at chain length 138. Subsequently, there is a very rapid increase of Km and at chain length 30 the Km is 4.3 microM. The rapid change in Km coincides with the shift in mechanism from the processive type mechanism in which there apparently is successive phosphorylation prior to release from the enzyme to a nonprocessive process in which the polyphosphate is released from the enzyme after each transfer. During the nonprocessive process, there is preferential utilization of the longer species. The Vmax is relatively constant with shorter polyphosphates but decreases with chain lengths longer than 347. In the cell, as a consequence of the low Km, the long chain polyphosphates probably are used preferentially to phosphorylate glucose. PMID- 3007460 TI - The mechanism of inactivation of dopamine beta-hydroxylase by hydrazines. AB - Dopamine beta-hydroxylase is inactivated by phenyl-, phenethyl-, benzyl-, and methylhydrazine, but not by hydrazine itself. With phenyl-, methyl-, and phenethylhydrazine, the rate of inactivation decreases in the presence of ascorbate and increases in the presence of tyramine. Reduction of the enzyme bound copper occurs with all of the hydrazines tested. In the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone the carbon-centered radicals generated from each compound are trapped. This is consistent with reduction of the enzyme-bound copper by the hydrazine-containing compounds, resulting in formation of the hydrazine cation radical. Homolytic cleavage of the carbon-nitrogen bond then generates a carbon-centered radical which reacts with the enzyme, resulting in inactivation. Inactivation with [14C]phenylhydrazine results in the incorporation of 0.94 molecule of label per enzyme subunit. Benzylhydrazine behaves as a mechanism-based inhibitor of the enzyme. Both benzyl and phenethylhydrazine are substrates for dopamine beta-hydroxylase. The second order rate constant for inactivation of dopamine beta-hydroxylase by benzylhydrazine in the presence of ascorbate is increased about 4-fold when the benzylic hydrogens are replaced with deuterium. The apparent Vmax shows an observed deuterium kinetic isotope effect of 13 +/- 2. The partition ratio for product formation versus inactivation is 11-fold less for alpha,alpha-d2 benzylhydrazine. These results are interpreted in terms of a model where inactivation is due to abstraction of an electron from nitrogen instead of abstraction of a hydrogen atom from the benzylic carbon. PMID- 3007461 TI - Identification of an essential sulfhydryl group in the ouabain binding site of (Na,K)-ATPase. AB - Ellman's reagent 5,5'-dithiobis-(2-nitrobenzoic acid) inhibits sodium- and potassium-stimulated ATPase, p-nitrophenyl phosphatase activity, and [3H]ouabain binding to lamb kidney (Na,K)-ATPase. The inactivation of [3H]ouabain binding follows pseudo-first order reaction kinetics at pH values less than or equal to 8.2. The inactivation of [3H]ouabain binding, but not of enzymatic activity, can be blocked by preincubation with ouabagenin, a rapidly reversible aglycone derivative of ouabain. The reduction in [3H]ouabain binding is due to a decrease in the number of binding sites rather than an alteration of the affinity of the enzyme for ouabain. Differential labeling at pH 8.2 with 1.0 mM 5,5'-dithiobis-(2 nitrobenzoic acid), preincubated with or without 5 microM ouabagenin, followed by tryptic digestion and reverse-phase high performance liquid chromatography of the generated soluble peptides reveals a single peptide labeled by the sulfhydryl probe that is protected by ouabagenin. From these results it is concluded that there is a single sulfhydryl group, essential for ouabain binding, presumably located in the ouabain binding site of lamb kidney (Na,K)-ATPase. PMID- 3007462 TI - Na+/H+ exchange in Ehrlich ascites tumor cells. Regulation by extracellular ATP and 12-O-tetradecanoylphorbol 13-acetate. AB - The effects of extracellular ATP and/or the phorbol ester 12-O tetradecanoylphorbol-13-acetate (TPA) on the intracellular pH of Ehrlich ascites tumor cells were measured using both distribution of [14C]5,5-dimethyloxazolidine 2,4-dione, and the fluorescent indicator 5(6)-carboxyfluorescein. Micromolar concentrations of extracellular ATP induce a biphasic change in the intracellular pH characterized by a rapid acidification of 0.04 pH units followed by an alkalinization of 0.11 pH units. Concurrently with the alkalinization, an increase in the total cellular [Na+] from 37.5 to 45.0 mM is observed. The pH change is half-maximally activated by 0.5-2.5 microM extracellular ATP. The intracellular alkalinization, but not the initial acidification, phase requires extracellular Na+, with half-maximal alkalinization in the presence of 24-32 mM Na+, and is inhibited by amiloride. Exposure of Ehrlich ascites tumor cells to TPA alone produces a slight alkalinization of approximately 0.04 pH units. Conversely, preincubation of the cells with TPA partially inhibits the ATP induced changes in intracellular pH. Under identical conditions TPA also inhibits the ATP-induced increase in the cytosolic [Ca2+]. The half-maximal dose for both effects is produced by 3-10 nM TPA. These data indicate that extracellular ATP triggers the activation of Na+/H+ exchange. Furthermore, activation of protein kinase C mediates at least part of the Na+/H+ exchange, although a second mechanism may also exist. PMID- 3007464 TI - Leucine-proton cotransport system in Chang liver cell. AB - The stimulatory effect of an inward H+ gradient on the Na+-independent L-leucine uptake by the plasma membrane vesicles from Chang liver cells (Mohri, T., Mitsumoto, Y., and Ohyashiki, T. (1983) Biochem. Int. 7, 159-167) has been shown to be due to the increase of the Km value without changing the Vmax value in the transport kinetics. The uptake of leucine by the vesicles is accompanied by intravesicular acidification, and a stimulated uptake of leucine by the countertransport with a high concentration of leucine in the vesicles enhances the acidification. All of these uptakes of leucine and proton and their stimulations are amplified by imposing an inward proton gradient. These results suggest appreciably different affinities of proton for the leucine transport carrier in the inner and outer sides of the plasma membrane. A rapid decrease in the cytoplasmic pH was observed only in the first minute of incubation of intact cells with leucine in Na+-containing medium. But the leucine-dependent decrease of the cytoplasmic pH persisted longer when either Na+ in the medium was replaced by choline or amiloride was present along with Na+. Addition of amiloride to Na+ containing medium was inhibitory on the leucine uptake of cells, without effect on the early phase of glycine uptake. We conclude that Chang liver cells are provided in their plasma membrane with an amino acid-H+ cotransport system, and this is coupled to the amiloride-sensitive Na+/H+ exchange system. PMID- 3007463 TI - The formation of a novel free radical metabolite from CCl4 in the perfused rat liver and in vivo. AB - Electron spin resonance spectroscopy has been used to monitor free radicals formed during CCl4 metabolism by perfused livers from phenobarbital-treated rats. Livers were perfused simultaneously with the spin trap phenyl N-t-butylnitrone and with either 12CCl4 or 13CCl4. Perfusate samples and CHCl3:CH3OH extracts of perfusate and liver samples were analyzed for phenyl N-t-butylnitrone radical adducts of reactive free radicals. In the organic extracts, hyperfine coupling constants and 13C isotope effects observed in the ESR spectra indicated the presence of the radical adduct of the trichloromethyl radical. Surprisingly, an additional free radical signal about two orders of magnitude more intense than that of the phenyl N-t-butylnitrone/CCl.3 radical adduct was observed in the aqueous liver perfusate. This adduct was also detected by ESR in rat urine 2 h after intragastric addition of spin trap and CCl4. This radical adduct had hyperfine coupling constants and 13C isotope effects identical with the radical adduct of the carbon dioxide anion radical (CO2-.). Analysis of the pH dependence of the coupling constants yielded a pK alpha of 2.8 for the CO2-. radical adduct formed either in the perfused liver or chemically. Carbon tetrachloride is converted into CCl.3 by cytochrome P-450 through a reductive dehalogenation. The trichloromethyl free radical reacts with oxygen to form the trichloromethyl peroxyl radical, CCl3OO., which may be converted into .COCl and then trapped. This radical adduct would hydrolyze to the carboxylic acid form, which is detected spectroscopically. Alternatively, the carbon dioxide anion free radical could form through complete dechlorination and then react with the spin trap to give the CO2-. radical adduct directly. PMID- 3007466 TI - Expression of the Proteus mirabilis lipoprotein gene in Escherichia coli. Existence of tandem promoters. AB - The Ipp gene from Proteus mirabilis was cloned onto pBR322 and expressed in Escherichia coli. The P. mirabilis lpp gene is unique in that it has two tandem promoters transcribing two mRNAs that differ in length by approximately 70 nucleotides at their 5'-ends. The two mRNAs thus encode the identical lipoprotein. The P. mirabilis prolipoprotein has a 19-amino acid signal peptide and a 59-amino acid lipoprotein sequence. In spite of the substantial differences in the amino acid sequence from the E. coli prolipoprotein, the P. mirabilis prolipoprotein is normally modified and processed in E. coli, and the resultant lipoprotein is assembled in the E. coli outer membrane as is the E. coli lipoprotein. PMID- 3007465 TI - The role of the essential sulfhydryl group in assimilatory NADH: nitrate reductase of Chlorella. AB - Incubation of the complex metalloflavoprotein, assimilatory nitrate reductase with N-ethylmaleimide, or a spin-labeled analog, 4-maleimido-2,2,6,6 tetramethylpiperidinooxyl, resulted in a time-dependent inactivation of NADH:nitrate reductase and NADH: cytochrome-c reductase activity with no effect on reduced methyl viologen:nitrate reductase activity. Inactivation of the enzyme, which could be prevented by incubation in the presence of NADH, was achieved following modification of a single sulfhydryl group determined from [3H]N-ethylmaleimide incorporation and quantitation of the EPR spectrum of the spin-labeled enzyme. Sulfhydryl group modification precluded reduction of the enzyme by NADH and NAD+ binding. The EPR spectrum of the spin-labeled enzyme revealed the presence of a single species with the nitroxide retaining substantial motional freedom. Cleavage of the spin-labeled enzyme using corn inactivating protease and separation into its flavin and molybdenum/heme domains followed by EPR spectroscopy revealed the modified sulfhydryl group to be associated with the latter fragment suggesting a close interaction of these domains in the region of the nucleotide-binding site. PMID- 3007467 TI - The exchange of Fe3+ between pyrophosphate and transferrin. Probing the nature of an intermediate complex with stopped flow kinetics, rapid multimixing, and electron paramagnetic resonance spectroscopy. AB - A detailed study of the exchange of Fe3+ between pyrophosphate and human serum transferrin was undertaken to test the hypothesis of a generalized reaction route for exchange of Fe3+ between transferrin and chelators. The initial rate of Fe3+ transfer from pyrophosphate to apotransferrin-CO2-3 is highly sensitive to the pyrophosphate to iron ratio with a maximal rate being observed at a ratio of 3:1, consistent with the presence of slowly reactive polymeric species at ratios less than 3:1 as revealed by EPR and kinetic measurements. At a ratio of 4:1 the reaction is distinctly biphasic. The rapid first phase results in the formation of an intermediate postulated as a mixedligand complex of the type PPi-Fe3+ transferrin-CO2-3. The intermediate has a distinct EPR spectrum and an absorption spectrum similar to that of Fe3+-transferrin-CO2-3, but with a spectral maximum at 450 nm rather than 465 nm. The second phase principally arises from the slow reaction of polymeric iron-pyrophosphate with the apoprotein and has contributions from the breakdown of the intermediate formed in the first phase. The rate of formation of the intermediate shows a hyperbolic dependence on NaHCO3 and apotransferrin concentrations, the latter suggesting a rate-limiting labilization of Fe3+(PPi)3, perhaps to form species of the type Fe3+(PPi)2, prior to attack by apotransferrin-CO2-3. Multimixing stopped flow spectrophotometry was employed to test the chemical reactivity of the Fe3+ to reduction at various times during the first phase. Surprisingly, a diminution of reactivity of 1000 fold was noted after only 2% of the first phase was completed, indicating a fast initial reaction which is not observed by normal rapid flow spectrophotometry. This initial reaction may involve the binding of iron-pyrophosphate to allosteric sites on the protein. The kinetics of iron removal from Fe3+-transferrin-CO2-3 by PPi are consistent with a rate-limiting conformational change in the protein as proposed earlier. PMID- 3007468 TI - Activation of a novel KpnI transcript by downstream integration of a human T lymphotropic virus type I provirus. AB - A cDNA library was constructed from the HUT102 cell line established from a patient with adult T-cell leukemia/lymphoma and screened for cDNA clones that contain (i) cellular sequences abundantly expressed in HUT102 cells and not in the virus-negative T-cell line HUT78, and (ii) viral long terminal repeat (LTR) sequences either in the 5' end or in the 3' end. One such cDNA clone, KT1, was isolated and its nucleotide sequence was determined. It contains three regions: a KpnI repeat, a unique cellular region (UCR), and the U3 + R sequence of the human T-lymphotropic virus type I LTR. The arrangement of this clone suggests that its RNA transcript was activated by provirus integration in cis, possibly by the activity of a downstream provirus enhancer. Analysis of HUT102 DNA shows that one allele of the KT1 UCR is rearranged. The expression of the KT1 UCR is unique to HUT102. These data are consistent with the idea that the human T-lymphotropic virus type I LTR contains an enhancer which can activate upstream sequences in cis. The possible significance of this finding is discussed. PMID- 3007470 TI - Histone H1 kinase in exponential and synchronous populations of Chinese hamster fibroblasts. AB - Nuclear histone kinase activity, specifically histone H1 phosphotransferase activity, was shown to increase in synchronous Chinese hamster cells from the G1/S boundary to late G2/early M phase. Chromatin extracts purified by DEAE Sephacel chromatography showed a cAMP-independent kinase activity that demonstrated cell cycle dependence and high specificity for histone H1 as the phosphate acceptor in the presence of [gamma-32P] ATP. This activity was purified approximately 40-fold. Using as substrates calf thymus histone H1 subfractions resolved by Bio-Rex 70 ion exchange chromatography, phosphorylation by the nuclear histone H1 kinase indicated that 32P incorporation into H1-2 was at least twice that for H1-1 and H1-3 subfractions. Both amino- and carboxy-terminal fragments generated by N-bromosuccinimide cleavage were phosphorylated. Phosphoamino acid analysis showed phosphothreonine to be approximately twice as abundant as phosphoserine. Histone H1 kinase activity was not activated by cyclic nucleotides, nor inhibited by cAMP-dependent protein kinase inhibitors or regulatory subunits. There was no effect on activity by Ca2+ alone or in the presence of calmodulin or diacylglycerol. Kinase activity was inhibited by nonhydrolyzable analogs of ATP such as adenyl-5'-yl imidodiphosphate, by 5'-p fluorosulfonylbenzoyladenosine which binds to the ATP binding site of the enzyme, and by quercetin. Column fractions enriched in histone H1 kinase were labeled with 5'-p-fluorosulfonylbenzoyl[8-14C]adenosine, and peptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band, Mr 67,000, was specifically labeled and may represent the H1 kinase catalytic subunit. PMID- 3007469 TI - Coordinate induction of multiple cytochrome c mRNAs in response to thyroid hormone. AB - Because of the well documented influence of thyroid hormones in the general control of oxidative metabolism and their putative role in the transcriptional activation of specific genes, changes in the levels of multiple cytochrome c mRNAs were monitored in thyroidectomized rats after administration of 3,5,3' triiodo-L-thyronine. In contrast to normal animals where the concentration of cytochrome c mRNAs in the polyadenylated RNA fraction is approximately 4- to 5 fold higher in kidney than in liver, hypothyroid animals displayed an equivalent low level of all three mRNAs (1400, 1100, and 700 nucleotides) in both tissues. Following the establishment of chronic hyperthyroidism, the levels of the three mRNAs were coordinately elevated by about 4- to 5-fold resulting in their restoration to approximately normal amounts for kidney but to a level substantially above the normal for liver. Only modest induction of mRNA is detected in the first 12 h following a single intravenous injection of 3,5,3' triiodo-L-thyronine. The major increase occurs between 12 and 24 h with the plateau level attained between 24 and 48 h. The magnitude of the response is in excess of the general increase in total cellular RNA mediated by the hormone. Induction of the cytochrome c mRNAs is coincident with an elevation in gene transcription of comparable magnitude detected using nascent RNA chains synthesized by isolated nuclei. The kinetics of these responses are similar to those observed for the increase in respiratory activity mediated by the hormone. PMID- 3007471 TI - A re-examination of the 5' termini of mouse dihydrofolate reductase RNA. AB - Using primer extension and nuclease S1-mapping techniques we have re-examined the 5' termini of RNA transcribed from the mouse dihydrofolate reductase gene. We characterize a previously undescribed transcription initiation site at position 55 relative to the AUG codon, in addition to the previously identified start site at position -115. Differences in the 5' noncoding regions of these two transcripts with respect to their length and relative G + C content result in their differential ability to form stable hybrids with the DNA probe used in previous analyses of these transcripts and thus precluded the detection of transcripts initiated at -55. We show that changes in the temperature of the hybridization reaction result in the ability to detect the RNA having a shorter noncoding region and a lower G + C content. That position -55 represents an authentic transcription start site is confirmed by use of a DNA probe with which the two transcripts can form S1-resistant hybrids of equal stability and by primer extension analysis using an oligonucleotide primer that hybridizes near the AUG codon. These analyses also demonstrate that the transcript with a 5' end mapping near position -55 accounts for the majority of cellular dihydrofolate reductase RNA. PMID- 3007472 TI - Insulin activation of insulin receptor tyrosine kinase in intact rat adipocytes. An in vitro system to measure histone kinase activity of insulin receptors activated in vivo. AB - We have studied the effect of incubation of intact cells with insulin on insulin receptor kinase activity. Following exposure of rat adipocytes to insulin, cells were solubilized and insulin receptors purified by specific immunoprecipitation or by insulin affinity chromatography. Kinase activity of the receptors, as measured by phosphorylation of histone 2B, was then determined. Insulin treatment of the cells resulted in a 10-20-fold increase in histone kinase activity of the subsequently isolated insulin receptors. The insulin effect was half-maximal at 3 s and maximal within 15 s of exposure, was dose-dependent (EC50 = 21 ng/ml), and was rapidly reversible following dissociation of insulin from the cells. The insulin effect in intact cells on insulin receptor kinase activity could be partially reversed in vitro by dephosphorylation of the isolated receptors by alkaline phosphatase. It is proposed that: in intact cells, insulin causes alterations in insulin receptors, such that their kinase activity toward non receptor substrates increases; increased insulin receptor kinase activity following insulin stimulation in intact cells is, at least in part, the result of an increased phosphate content of the receptors; and effects of insulin on insulin receptors in intact cells can be preserved during receptor isolation and thus can be measured in a cell-free system. PMID- 3007473 TI - The Cpx proteins of Escherichia coli K12. Immunologic detection of the chromosomal cpxA gene product. AB - Previous studies described lacZ'- and cat'-'cpxA fusion genes whose expression restored to normal all the phenotypic defects associated with cpxA mutations (Albin, R., and Silverman, P. M. (1984) Mol. Gen. Genet. 197, 272-279). Here, we show by DNA nucleotide sequence analysis that the fusion genes encode 241 carboxyl-terminal amino acids of the CpxA polypeptide. Using this information, we constructed a fusion gene containing the same 241 cpxA codons preceded by 1007 codons of beta-galactosidase. The resultant hybrid polypeptide was purified and used to raise an anti-(CpxA polypeptide) antiserum. Using the antiserum, we have identified the chromsomal Escherichia coli K12 cpxA gene product as a 52-kDa polypeptide. The polypeptide showed temperature-sensitive accumulation in a strain carrying both the cpxA2[Ts] and cpxB1 alleles and accumulated to a level higher than normal in cells that carried a high-copy number, cpxA+ plasmid. Immune precipitates of in vitro transcription-translation reactions with cpxA+ plasmids as template also contained a 52-kDa polypeptide, indistinguishable in electrophoretic mobility from the immunoreactive polypeptide synthesized in vivo. Two regions of amino acid sequence at the carboxyl-terminus of the CpxA polypeptide are significantly homologous to corresponding regions of the E. coli K12 EnvZ polypeptide, an inner membrane component that, like the CpxA polypeptide, is required to maintain the protein composition of the cell envelope. The cpxA coding sequence is followed by two repetitive extragenic palindrome sequences in opposite orientation. PMID- 3007474 TI - The Escherichia coli dnaB replication protein is a DNA helicase. AB - Genetic and biochemical analyses indicate that the Escherichia coli dnaB replication protein functions in the propagation of replication forks in the bacterial chromosome. We have found that the dnaB protein is a DNA helicase that is capable of unwinding extensive stretches of double-stranded DNA. We constructed a partially duplex DNA substrate, containing two preformed forks of single-stranded DNA, which was used to characterize this helicase activity. The dnaB helicase depends on the presence of a hydrolyzable ribonucleoside triphosphate, is maximally stimulated by a combination of E. coli single-stranded DNA-binding protein and E. coli primase, is inhibited by antibody directed against dnaB protein, and is inhibited by prior coating of the single-stranded regions of the helicase substrate with the E. coli single-stranded DNA-binding protein. It was determined that the dnaB protein moves 5' to 3' along single stranded DNA, apparently in a processive fashion. To invade the duplex portion of the helicase substrate, the dnaB protein requires a 3'-terminal extension of single-stranded DNA in the strand to which it is not bound. Under optimal conditions at 30 degrees C, greater than 1 kilobase pair of duplex DNA can be unwound within 30 s. Based on these findings and other available data, we propose that the dnaB protein is the primary replicative helicase of E. coli and that it actively and processively migrates along the lagging strand template, serving both to unwind the DNA duplex in advance of the leading strand and to potentiate synthesis by the bacterial primase of RNA primers for the nascent (Okazaki) fragments of the lagging strand. PMID- 3007475 TI - Phosphorylation of rhodopsin by protein kinase C in vitro. AB - Calium/phospholipid-dependent protein kinase (protein kinase C) was purified from bovine retinae rod outer segments (ROS). In the presence of 0.1-2 microM calcium protein kinase C binds tightly to ROS and phosphorylates rhodopsin in the absence or presence of illumination. This property of protein kinase C contrasts with that of rhodopsin kinase, which in vitro phosphorylates only bleached rhodopsin. Peptide maps of rhodopsin phosphorylated by protein kinase C or rhodopsin kinase were compared using limited Staphylococcus aureus V8 protease digestion or complete tryptic digestion. Phosphorylation sites map to serine and threonine residues on the cytoplasmic carboxylterminal domain of rhodopsin for both kinases. The functional consequence of protein kinase C phosphorylation of rhodopsin was a reduced ability to stimulate the light-dependent rhodopsin activation of [35S]guanosine 5'-O-(thiotriphosphate) binding to transducin, the GTP-binding regulatory protein present in ROS. Properties of the calcium stimulated interaction of protein kinase C with membranes and in vitro phosphorylation of intrinsic proteins are discussed based upon the findings. PMID- 3007476 TI - 17O electron nuclear double resonance characterization of substrate binding to the [4Fe-4S]1+ cluster of reduced active aconitase. AB - To characterize the binding of substrate to aconitase, we have made 17O electron nuclear double resonance (ENDOR) measurements on reduced active ([4Fe-4S]1+) beef heart aconitase, both in H216O and H217O, in the presence of substrate and the inhibitors, tricarballylate, trans-aconitate, and 1-hydroxy-2-nitro-1, 3 propanedicarboxylate, referred to here as nitroisocitrate; the hydroxyl of the latter also was isotypically labeled with 17O. The hydroxyl oxygen of citrate and isocitrate is exchanged with solvent water by aconitase, but the hydroxyl of nitroisocitrate is not. Thus, the isotopic composition of nitroisocitrate can be chemically controlled, allowing direct identification of any 17O ENDOR signal associated with it. 17O ENDOR signals were observed from Hx17O (mean = 1 or 2) bound to the [4Fe-4S]1+ cluster in samples prepared with trans-aconitate and unlabeled nitroisocitrate. 17O-Labeled nitroisocitrate in H216O bound to the cluster showed a signal from the 17OH group; in H217O it showed 17O ENDOR resonances due to both Hx17O and 17OH of substrate. This result demonstrates that the cluster participates in substrate binding and can simultaneously coordinate the hydroxyl of a substrate (or analogue) and water (or hydroxyl). The sample with citrate in H217O showed only the Hx17O signal, although aconitase exchanges the hydroxyl of substrate with solvent water. The mechanism of action of aconitase is discussed in light of this observation. Comparison shows the ENDOR study to be in agreement with previous Mossbauer and EPR spectroscopic results. PMID- 3007477 TI - Regeneration of native bacteriorhodopsin structure following acetylation of epsilon-amino groups of Lys-30, -40, and -41. AB - The chymotryptic fragment of bacteriorhodopsin, C-2 (residues 1-71), has been acetylated completely at its three lysines (residues 30, 40, and 41) by treatment with acetic anhydride. The triacetylated C-2 fragment is able to reassociate with fragment C-1 (residues 72-248) and the complex binds all-trans-retinal to form a native bacteriorhodopsin-like chromophore, which is essentially identical with that formed from fragments C-2 and C-1. Further, the kinetics and pH dependence of chromophore regeneration and the proton pumping of the reconstituted triacetylated C-2 and C-1 complex are indistinguishable from that of the unmodified C-2 and C-1 complex. However, the extent of regeneration of the chromophore from triacetylated C-2 and C-1 is less than that from fragments C-2 and C-1, suggesting that the acetylated C-2 fragment is less stable than unacetylated C-2 in the reconstitution medium. We conclude that the amino groups in Lys-30, -40, and -41 do not contribute to the stabilization of the folded bacteriorhodopsin structure and are not required for proton translocation. PMID- 3007478 TI - Sequences of Escherichia coli uvrA gene and protein reveal two potential ATP binding sites. AB - We have determined the nucleotide sequence of the uvrA gene of Escherichia coli. The coding region of the gene is 2820 base pairs which specifies a protein of 940 amino acids and Mr = 103,874. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the UvrA protein: the sequence of the first 7 NH2-terminal amino acids as well as the amino acid composition of the pure protein agreed with those predicted from the nucleotide sequence. By comparing the sequence of UvrA protein to the amino acid sequences of other ATPases, we found that two regions in the UvrA protein, separated from one another by about 600 amino acids, have the highly conserved G-X4-GKT(S)-X6-I(V) sequence found at the active sites of many, but not all, ATPases. Our findings suggest that UvrA protein may have two ATP binding sites. PMID- 3007479 TI - Cyclic AMP-dependent protein kinase promotes glucocorticoid receptor function. AB - Murine lymphoma cell lines such as WEHI-7 exhibit a cytolytic response to both cAMP and glucocorticoids. We have exploited this behavior to ask if cyclic AMP dependent protein kinase plays a role in regulating glucocorticoid receptor function. We have found that cAMP-resistant cell lines containing a defective cAMP-dependent protein kinase activity give rise to spontaneous steroid-resistant variants at a high frequency (approximately 10(-7)) relative to wild type cells (less than 10(-10)). Unlike previous results with wild type cells, nearly complete loss of glucocorticoid receptor function was observed in a single selection using unmutagenized cAMPr derivatives of WEHI-7. Thus, the initial selection of the cAMPr phenotype serves as a permissive step toward the acquisition of glucocorticoid resistance in WEHI-7. In addition, cAMP was found to increase the levels of steroid binding in these cell lines, and the dose response was dependent upon the phenotype of the cyclic AMP-dependent protein kinase. The results demonstrate an important role for cAMP in regulating glucocorticoid receptor activity and strongly suggest that this novel two-step selection scheme leads to the isolation of new forms of glucocorticoid resistance. PMID- 3007480 TI - Muscarinic receptor enhancement of nicotine-induced catecholamine secretion may be mediated by phosphoinositide metabolism in bovine adrenal chromaffin cells. AB - Bovine adrenal chromaffin cells possess both nicotinic and muscarinic cholinergic receptors, but only nicotinic receptors have heretofore appeared to mediate Ca2+ dependent exocytosis. We have now found that muscarinic receptor stimulation in bovine adrenal chromaffin cells leads to enhanced inositol phospholipid metabolism as evidenced by the rapid (less than 1 min) formation of inositol trisphosphate (IP3) and inositol bisphosphate (IP2). Muscarinic receptor-mediated accumulation of IP3 and IP2 continues beyond 1 min in the presence of LiCl and is accompanied by large increases in inositol monophosphate. Muscarinic receptor stimulation was also found to enhance nicotine-induced catecholamine secretion by 1.7-fold if muscarine was added 30 s before nicotine addition. Moreover, since the muscarinic antagonist atropine reduces acetylcholine-induced secretion, we conclude that muscarinic receptor stimulation somehow primes these cells for nicotinic receptor-mediated secretion, perhaps by causing small nonstimulatory increases in cytosolic free Ca2+ mediated by IP3. Furthermore, we show that small depolarizations of these cells with 10 mM K+, which themselves do not affect basal secretion, also enhance nicotine-induced secretion. Thus, small increases in cytosolic free Ca2+ produced either by physiologic muscarinic receptor stimulation or by small experimental depolarizations with K+ may prime the chromaffin cells for nicotinic receptor-mediated secretion. PMID- 3007481 TI - Structures of O-linked oligosaccharides present in the proteoglycans secreted by human mammary epithelial cells. AB - The structures of O-glycosidically linked oligosaccharides present in the heparan sulfate and chondroitin sulfate proteoglycans isolated from the culture medium of a normal (HBL-100) and a malignant (MDA-MB-231) human mammary epithelial cell line have been determined. Both proteoglycan types from the two cell lines contain a series of O-linked oligosaccharides ranging in size from di- to hexasaccharide. Cells were grown in the presence of either [3H]glucosamine or [3H]galactose and Na2 35SO4, and the proteoglycans were isolated as described (Gowda, D. C., Bhavanandan, V. P., and Davidson, E. A. (1986) J. Biol. Chem. 261, 4926-4934). The O-linked oligosaccharides were released from the proteoglycans by alkaline borohydride treatment and purified by a combination of gel filtration and high voltage paper electrophoresis. The structures of two neutral and seven acidic oligosaccharides were established based on sugar composition, the results of periodate oxidation, sequential exoglycosidase treatment, and methylation analysis. Periodate oxidation, taking advantage of tritium label at specific positions of constituent sugars, proved to be a valuable tool in establishing the structure of isomeric components in the mixture. The structures of the oligosaccharides were assigned as follows: (Formula: see text) The oligosaccharide containing both sialic acid and ester sulfate is novel and has not been reported previously. PMID- 3007482 TI - Redox modification of sodium-calcium exchange activity in cardiac sarcolemmal vesicles. AB - Na-Ca exchange activity in bovine cardiac sarcolemmal vesicles was stimulated up to 10-fold by preincubating the vesicles with 1 microM FeSO4 plus 1 mM dithiothreitol (DTT) in a NaCl medium. The increase in activity was not reversed upon removing the Fe and DTT. Stimulation of exchange activity under these conditions was completely blocked by 0.1 mM EDTA or o-phenanthroline; this suggests that the production of reduced oxygen species (H2O2, O2-.,.OH) during Fecatalyzed DTT oxidation might be involved in stimulating exchange activity. In agreement with this hypothesis, the increase in exchange activity in the presence of Fe-DTT was inhibited 80% by anaerobiosis and 60% by catalase. H2O2 (0.1 mM) potentiated the stimulation of Na-Ca exchange by Fe-DTT under both aerobic and anaerobic conditions; H2O2 also produced an increase in activity in the presence of either FeSO4 (1 microM) or DTT (1 mM), but it had no effect on activity by itself. Superoxide dismutase did not block the effects of Fe-DTT on exchange activity; however, the generation of O2-. by xanthine oxidase in the presence of an oxidizable substrate stimulated activity more than 2-fold. Hydroxyl radical scavenging agents (mannitol, sodium formate, sodium benzoate) did not attenuate the stimulation of activity observed with Fe-H2O2. Exchange activity was also stimulated by the simultaneous presence of glutathione (GSH; 1-2 mM) and glutathione disulfide (GSSG; 1-2 mM). Neither GSH nor GSSG was effective by itself and either 0.1 mM EDTA or o-phenanthroline blocked the effects on transport activity of the combination of GSH + GSSG. Treatment of the GSH and GSSG solutions with Chelex ion-exchange resin to remove contaminating transition metal ions reduced (by 40%) the degree of stimulation observed with GSH + GSSG. Full stimulating activity was restored to the Chelex-treated GSH and GSSG solutions by the addition of 1 microM Fe2+; Cu2+ was less effective than Fe2+ whereas Co2+ and Mn2+ were without effect. In the presence of 1 microM Fe2+, GSH alone produced a slight increase in transport activity, but this was markedly enhanced by the addition of Chelex-treated GSSG. The results indicate that stimulation of exchange activity requires the presence of both a reducing agent (DTT, GSH, O-.2, or Fe2+) and an oxidizing agent (H2O2, GSSG, and perhaps O2) and that the effects of these agents are mediated by metal ions (e.g. Fe2+).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3007483 TI - Scavenger receptor for aldehyde-modified proteins. AB - This paper describes an unexpectedly broad ligand specificity of a scavenger receptor of sinusoidal liver cells that is responsible for endocytic uptake of formaldehyde-treated bovine serum albumin (f-Alb). Binding of 125I-f-Alb to the isolated cells was effectively inhibited by bovine serum albumin (BSA) modified with aliphatic aldehydes such as glycolaldehye, DL-glyceraldehyde, and propionaldehyde whereas albumin preparations modified by aromatic aldehydes such as pyridoxal, pyridoxal phosphate, salicylaldehyde, and benzaldehyde did not affect this binding process. Binding of 125I-glycolaldehyde-treated BSA to the cells exhibited a saturation kinetics with an apparent Kd = 3.3 micrograms of the ligand/ml. This binding process was inhibited by unlabeled f-Alb as well as by the antibody raised against the f-Alb receptor. Indeed, 125I-glycolaldehyde treated BSA underwent a rapid plasma clearance (t1/2 approximately 2 min) which was markedly retarded by unlabeled f-Alb. Upon treatment by these aldehydes, other proteins such as ovalbumin, soybean trypsin inhibitor, and hemoglobin were also converted to active ligands for the f-Alb receptor, while no ligand activity was generated with gamma-globulin and RNase A. These results clearly show that the f-Alb receptor, originally described as being specific for f-Alb, exhibits a broad ligand specificity in terms of both aldehydes and proteins and, hence, should be described as a scavenger receptor for aldehyde-modified proteins. PMID- 3007484 TI - Response of three enzymes to oleic acid, trypsin, and calmodulin chemically modified with a reactive phenothiazine. AB - Calmodulin was covalently modified with 10-(1-propionyloxysuccinimide)-2 trifluoromethylphenothiazine++ + to stoichiometries between 0 and 2 mol/mol in the presence of Ca2+. The modified calmodulins, oleic acid, and trypsin were assayed for their ability to activate pea plant NAD kinase, bovine brain 3',5' cAMP phosphodiesterase, and human erythrocyte Ca2+-ATPase. All modified calmodulins activated both phosphodiesterase and Ca2+-ATPase; at the highest concentration assayed, calmodulin modified with 2 mol of reagent/mol activated phosphodiesterase and Ca2+-ATPase to 53% and 100%, respectively, of the activation obtained with unmodified calmodulin. However, higher concentrations of the modified calmodulins were required to observe the same activation; at least 900-fold and 100-fold higher concentrations were required for the two enzymes, respectively. NAD kinase was not activated by any calmodulin labeled to a stoichiometry greater than 1 mol/mol even when a concentration equal to 17,000 times the apparent dissociation constant of calmodulin for NAD kinase was assayed. Therefore, the modified protein (and not some fraction resistant to labeling) is active toward the mammalian enzymes but inactive toward plant NAD kinase. The different response of the three enzymes to the chemical modification suggests that the enzymes may utilize different binding domains on calmodulin. NAD kinase also was not activated by other known activators of the two mammalian enzymes, namely lipids and limited proteolysis. In parallel experiments using the same agents on each enzyme, NAD kinase was the only enzyme of the three that was not activated by oleic acid and several other lipids or by limited trypsin digestion. These results show that NAD kinase possesses several attributes which would not be predicted by current models of the mechanism of activation of enzymes by calmodulin. PMID- 3007486 TI - Transforming growth factor-beta and retinoic acid modulate phenotypic transformation of normal rat kidney cells induced by epidermal growth factor and platelet-derived growth factor. AB - In this study we have investigated the ability of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) together with retinoic acid (RA) at saturating concentrations to induce phenotypic transformation of normal rat kidney (NRK) cells in a growth factor defined medium. This medium contains serum in which all growth factor activity has been chemically inactivated, thereby eliminating the effects of growth factors from serum in the assay. It is shown that neither TGF eta nor a ligand binding to the EGF receptor is essential for phenotypic transformation of NRK cells, since anchorage-independent growth is also induced by EGF in combination with RA and by PDGF in combination with RA and TGF beta. Our data indicate strong similarities between TGF beta and RA in their ability to act as modulators for phenotypic transformation. In addition, both agents enhance the number of EGF receptors in NRK cells, without affecting the number of PDGF receptors. On the other hand, TGF beta has mitogenic effects on a number of non-transformed cell lines, such as Swiss 3T3 fibroblasts, particularly when assayed in the absence of insulin, whereas RA is mitogenic for these cells only in the presence of insulin. These data demonstrate that phenotypic transformation of NRK cells requires specific combinations of polypeptide growth factors and modulating agents, but that this process can be induced under many more conditions than previously described. Moreover, our data point toward both parallels and differences in the activities of TGF beta and RA. PMID- 3007485 TI - A guanine nucleotide-dependent phosphatidylinositol 4,5-diphosphate phospholipase C in cells transformed by the v-fms and v-fes oncogenes. AB - The metabolism of phosphatidylinositol (PtdIns) was studied in a mink lung epithelial cell line and its subclones transformed by feline sarcoma viruses containing either the v-fms or v-fes oncogenes. The transformed cell lines had a higher rate of PtdIns turnover but did not have elevated levels of phosphorylated PtdIns species or PtdIns kinase activity. Significantly higher specific activities of a guanine nucleotide-activated PtdIns-4,5-diphosphate phospholipase C were detected in both transformed cell lines (F3CL7(v-fes), 55 pmol/min/mg of protein and G2M(v-fms), 18 pmol/min/mg of protein) as compared to the nontransformed parental cell line (CCL64, 2 pmol/min/mg of protein). The guanine nucleotide-stimulated phospholipase C activity was specific for PtdIns-4,5 diphosphate, and the water-soluble hydrolysis product was inositol 1,4,5 triphosphate. Both GTP and nonhydrolyzable GTP analogs activated the phospholipase C, whereas ATP was weakly effective and GDP was inactive. The phospholipase C activity was maximally active in the presence of 9 mM sodium cholate, had a sharp pH optimum of pH 6.5, and was not activated by calcium although hydrolysis was inhibited by high concentrations of EDTA. These data point to enhanced production of diacylglycerol and inositol 1,4,5-triphosphate second messengers in transformed cells due to the activation of guanine nucleotide-dependent PtdIns-4,5-diphosphate-specific phospholipase C and suggest that the generation of aberrant hormonally independent signals is associated with cell transformation by oncogenes encoding tyrosine-specific protein kinases. PMID- 3007487 TI - Oxidation of glutathione to its thiyl free radical metabolite by prostaglandin H synthase. A potential endogenous substrate for the hydroperoxidase. AB - The oxidation of glutathione to a thiyl radical by prostaglandin H synthase was investigated. Ram seminal vesicle microsomes, in the presence of arachidonic acid, oxidized glutathione to its thiyl-free radical metabolite, which was detected by ESR using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide. Oxidation of glutathione was dependent on arachidonic acid and inhibited by indomethacin. Peroxides also supported oxidation, indicating that the oxidation was by prostaglandin hydroperoxidase. Glutathione served as a reducingcofactor for the reduction of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid to 15-hydroxy 5,8,11,13-eicosatetraenoic acid at 1.5-2 times the nonenzymatic rate. Although purified prostaglandin H synthase in the presence of either H2O2 or 15 hydroperoxy-5,8,11,13-eicosatetraenoic acid oxidized glutathione to a thiyl radical, arachidonic acid did not support glutathione oxidation. Glutathione also inhibited cyclooxygenase activity as determined by measuring oxygen incorporation into arachidonic acid. Reverse-phase high pressure liquid chromatography analysis of the arachidonic acid metabolites indicated that the presence of glutathione in an incubation altered the metabolite profile. In the absence of the cofactor, the metabolites were PGD2, PGE2, and 15-hydroperoxy-PGE2 (where PG indicates prostaglandin), while in the presence of glutathione, the only metabolite was PGE2. These results indicate that glutathione not only serves as a cofactor for prostaglandin E isomerase but is also a reducing cofactor for prostaglandin H hydroperoxidase. Assuming that glutathione thiyl-free radical observed in the trapping experiments is involved in the enzymatic reduction of 15-hydroperoxy 5,8,11,13-eicosatetraenoic acid to 15-hydroxy-5,8,11,13-eicosatetraenoic acid, then a 1-electron donation from glutathione to prostaglandin hydroperoxidase is indicated. PMID- 3007488 TI - Regulation of VL30 gene expression by activators of protein kinase C. AB - The mouse genome contains a retrovirus-like sequence, designated VL30, which is expressed at high levels in transformed cells and which can be induced by exogenously supplied epidermal growth factor (EGF). Binding of EGF to the EGF receptor produces changes in intracellular calcium levels and phospholipase activity which indirectly lead to activation of protein kinase C. We treated AKR 2B cells, Swiss 3T3 cells, and the 3T3 variants NR6 (EGF receptorless) and TNR9 (phorbol ester nonresponsive) with various phorbol ester tumor promoters and with the synthetic diacylglycerol sn-1,2-dioctanoylglycerol. Tumor-promoting phorbol esters (e.g. 12-O-tetradecanoyl phorbol acetate (TPA] increased the level of VL30 expression. Stimulation with either TPA or EGF produced a similar time course of VL30 expression. TPA induced VL30 expression in the EGF-receptorless NR6 cell line, indicating that neither EGF ligand-receptor binding nor phosphorylation of the EGF receptor was required for induction of VL30 expression. Protein synthesis was not required for the TPA-mediated increase in VL30 expression, as pretreatment with cycloheximide did not block or reduce the TPA effect. VL30 expression was also stimulated by treatment with sn-1,2-dioctanoylglycerol, an analog of a probable endogenous activator of protein kinase C. These results suggest that activation of protein kinase C plays a direct role in regulating VL30 expression. PMID- 3007489 TI - Kinetics of estrogen-dependent modulation of apolipoprotein A-I synthesis in human hepatoma cells. AB - Apolipoprotein (apo) A-I is the principal protein component of high density lipoproteins and the major in vivo activator of lecithin-cholesterol acyltransferase. We have used the human hepatoma cell line, HepG2, as a model to examine the ability of estrogen to modulate hepatic synthesis of apo-A-I. Primer extension studies have demonstrated that the major transcriptional initiation site of the apo-A-I gene utilized in the hepatoma cells is the same as that used in human liver. The kinetics of induction of high- and low-affinity estrogen binding sites, rates of secretion of apolipoproteins, and apo-A-I mRNA levels were examined following treatment of the cells with estrogen. Initial concentrations of 20 nM 17 beta-estradiol resulted in a 14-15-fold increase in the levels of high-affinity nuclear estrogen-binding sites within 8 h, while the level of low-affinity sites increased by only 10%. During the same period, the levels of apo-A-I mRNA and the rate of accumulation of the secreted protein increased by 55 and 50%, respectively. New steady state levels of apo-A-I mRNA and rates of accumulation of protein, approximately twice those in control cultures, were established within 24-48 h of exposure to hormone. Experiments with a 50-fold higher concentration of estrogen resulted in only an additional 10% increase in mRNA levels. The increase in mRNA levels following estrogen treatment was adequate to account for 85-90% of the elevation observed in the rate of accumulation of secreted apo-A-I. Comparison of the apo-A-I mRNA levels in HepG2 cells with those present in human liver revealed that the concentration of the mRNA was approximately 3-fold lower than that found in vivo. PMID- 3007490 TI - The role of cholesterol in the activity of reconstituted Ca-ATPase vesicles containing unsaturated phosphatidylethanolamine. AB - The effect of cholesterol on the activity of sarcoplasmic reticulum Ca-ATPase, reconstituted in proteoliposomes containing soybean phosphatidylethanolamine (PE), egg phosphatidylcholine (PC), and cholesterol, was examined. The protein incorporation efficiency increased with PE content but appeared to be independent of cholesterol content. At low cholesterol, PE stimulated calcium uptake. The coupling efficiency of the proteoliposomes increased with an increase in cholesterol content at each PC/(PC + PE) ratio and was more pronounced for those proteoliposomes containing high PE. Dynamic fluorescence measurements of the incorporated lipophilic probe, diphenyl-1,3,5-hexatriene, revealed a decrease in the motion and an increase in the order of the phospholipid fatty acyl chains in proteoliposomes with high cholesterol content. A complementary observation was made using electron spin resonance of the spin label, 2,2-dimethyl-5-dodecyl-5 methyloxazolidine N-oxide. Freeze-fracture electron microscopy studies on proteoliposomes containing 0.20 molar ratio of PC/(PC + PE) and cholesterol revealed predominantly vesicular structures with occasional bilayer defects at high cholesterol content. It is postulated that the cholesterol-induced enhancement of the Ca-transport function of the Ca-ATPase is related to the hydration-related bilayer-destabilizing characteristic of the cholesterol molecule as revealed by 31P NMR. PMID- 3007491 TI - The purification and characterization of CTP:phosphorylcholine cytidylyltransferase from rat liver. AB - We have purified CTP:phosphorylcholine cytidylyltransferase from rat liver cytosol 2180-fold to a specific activity of 12,250 nmol/min/mg of protein. The purified enzyme was stable at -70 degrees C in the presence of Triton X-100 and 0.2 M phosphate. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide electrophoresis. Separation by sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that the purified enzyme contained subunits with Mr of 39,000 and 48,000. Gel filtration analysis indicated that the native enzyme was a tetramer containing two 39,000 and two 48,000 subunits. The purified enzyme appeared to bind to Triton X-100 micelles, one molecule of tetramer/micelle. Maximal activity was obtained with 100 microM phosphatidylcholine-oleic acid vesicles (8-10-fold stimulation). Phosphatidylglycerol produced a 4-5-fold increase in activity at 10 microM. The pH optimum and true Km values for CTP and phosphorylcholine were similar to those reported previously for crude preparations of cytidylyltransferase. The overall behavior of cytidylyltransferase during purification and subsequent analysis suggested that it has hydrophobic properties similar to those exhibited by membrane proteins. PMID- 3007492 TI - The role of single-strand breaks in the catenation reaction catalyzed by the rat type I topoisomerase. AB - The type I topoisomerase from rat cells produces true catenanes from circular SV40 DNA in a reaction which is dependent on the presence of a single-strand break in at least one member of a pair of reacting molecules. The role of the single-strand break in the reaction was examined. Molecules containing a nick with a 3'-hydroxyl and 5'-phosphate or a nick with a 3'-phosphate and 5'-hydroxyl and molecules with single-stranded gaps were all found to be equally effective in the catenation reaction. It was found that the enzyme could, at a low frequency, break DNA by acting opposite a pre-existing single-strand break. Thus, incubation of nicked circular DNA in the presence of the topoisomerase, polynucleotide kinase, and [gamma-32P]ATP led to the production of a low level of labeled linear molecules containing covalently attached protein. Nicked linear molecules treated with topoisomerase in the absence of polynucleotide kinase generated fragments of sizes consistent with breakage in the opposite strand near the pre-existing nick. Based on these results, we propose that the catenation reaction may involve the transient production of linear intermediates by the action of the topoisomerase opposite a pre-existing nick in the DNA. Rejoining of the two ends by the enzyme could lead to the interlocking of two or more circular DNAs. In addition, these results suggest a possible role for the type I topoisomerase in illegitimate recombination. PMID- 3007493 TI - Proteolytic cleavage of phospholamban purified from canine cardiac sarcoplasmic reticulum vesicles. Generation of a low resolution model of phospholamban structure. AB - Purified phospholamban isolated from canine cardiac sarcoplasmic reticulum vesicles was subjected to proteolysis and peptide mapping to localize the different sites of phosphorylation on the protein and to gain further information on its subunit structure. Five different proteases (trypsin, papain, chymotrypsin, elastase, and Pronase) degraded the oligomeric 27-kDa phosphoprotein into a major 21-22-kDa protease-resistant fragment. No 32P was retained by this protease-resistant fragment, regardless of whether phospholamban had been phosphorylated by cAMP-dependent protein kinase, Ca2+/calmodulin dependent protein kinase, or protein kinase C. Phosphoamino acid analysis and thin-layer electrophoresis of liberated phosphopeptides revealed that 1 threonine and 2 serine residues were phosphorylated in phospholamban and that 1 of these serine residues and the threonine residue were in close proximity. Only serine was phosphorylated by cAMP-dependent protein kinase, whereas Ca2+-calmodulin dependent protein kinase phosphorylated exclusively threonine. The results demonstrate that phospholamban has a large protease-resistant domain and a smaller protease-sensitive domain, the latter of which contains all of the sites of phosphorylation. The 21-22-kDa protease-resistant domain, although devoid of incorporated 32P, was completely dissociated into identical lower molecular weight subunits by boiling in sodium dodecyl sulfate, suggesting that this region of the molecule promotes the relatively strong interactions that hold the subunits together. The data presented lend further support for a model of phospholamban structure in which several identical low molecular weight subunits are noncovalently bound to one another, each containing one site of phosphorylation for cAMP-dependent protein kinase and another site of phosphorylation for Ca2+/calmodulin-dependent protein kinase. PMID- 3007494 TI - NADPH binding component of neutrophil superoxide-generating oxidase. AB - The 2',3'-dialdehyde derivative of NADPH was used as an affinity labeling reagent of a solubilized NADPH-dependent superoxide-generating oxidase preparation of pig neutrophils. The analogue served as both an electron donor and a competitive inhibitor of the NADPH oxidase against NADPH. The apparent Michaelis constant (Km) for the derivative (31 microM) was essentially the same as that for NADPH (33 microM). The activity of the superoxide formation in the presence of 2',3' dialdehyde NADPH was about a half of that in the presence of NADPH. Incubation of the enzyme with the derivative inactivated the superoxide-generating activity and the inactivation was prevented by the addition of NADPH. We performed the labeling of the oxidase preparation with 2',3'-dialdehyde NADPH and sodium cyanoboro[3H]hydride, based on the above results. A protein of 66,000 daltons was selectively labeled among more than 20 bands in sodium dodecyl sulfate polyacrylamide gel electrophoresis which were visualized with Coomassie Brilliant Blue. The protein was not labeled when the oxidase preparation was pretreated with p-chloromercuribenzoate or it was labeled in the presence of excess NADPH. The protein is suggested to be the NADPH binding component of the neutrophil superoxide-generating oxidase system. PMID- 3007495 TI - A possible role for glucose metabolites in the regulation of inositol-1,4,5 trisphosphate 5-phosphomonoesterase activity in pancreatic islets. AB - Rat pancreatic islets demonstrate inositol-1,4,5-trisphosphate 5 phosphomonoesterase activity which is 3 times higher than that in the exocrine pancreas. This enzyme has several features in common with the erythrocyte and hepatocyte enzymes: it is located primarily in the plasma membrane, it has a similar Km for inositol trisphosphate (IP3) (16 microM), and it requires Mg2+. The activity of the islet enzyme is inhibited by several diphosphorylated glucose metabolites: 2,3-bisphosphoglycerate, fructose 1,6-bisphosphate, fructose 2,6 bisphosphate, and glucose 1,6-bisphosphate. Monophosphorylated and unphosphorylated metabolites have little or no effect on its activity. Several reports show that stimulation of islets with glucose raises the concentrations of various glucose metabolites including fructose 1,6-bisphosphate, glucose 1,6 bisphosphate, and 2,3-bisphosphoglycerate to concentrations that are in the range that inhibit the islet inositol-1,4,5-trisphosphate 5-phosphomonoesterase. Other reports show that IP3 mobilizes calcium when added to permeabilized insulin secreting cells. It is possible that the increase in cytosolic calcium known to occur during glucose-induced insulin secretion may be sustained in part by higher IP3 levels resulting from the inhibition of inositol-1,4,5-trisphosphate 5 phosphomonoesterase by some of the diphosphorylated glucose metabolites. PMID- 3007496 TI - Thyrotropin-releasing hormone (TRH) elevation of inositol trisphosphate and cytosolic free calcium is dependent on receptor number. Evidence for multiple rapid interactions between TRH and its receptor. AB - Thyrotropin-releasing hormone (TRH) increases rapidly two potential intracellular signals, inositol trisphosphate (IP3) and free cytosolic calcium ([Ca2+]i), for stimulated prolactin release and synthesis in GH4C1 rat pituitary cells. We have examined the temporal relationships between TRH-enhanced formation of inositol phosphates and TRH-elevated [Ca2+]i. TRH-enhanced IP3 content was closely paralleled by the initial phase of TRH-elevated [Ca2+]i. To investigate receptor effector coupling for these rapid actions of TRH, we examined their dependence on receptor number in five GH4C1 variant strains containing 0-2.6 X 10(5) receptor sites/cell. We found that receptor number (up to 1.7 X 10(5)/cell) was limiting for TRH-enhanced IP3 formation as well as for both the initial burst and plateau phases of TRH-elevated [Ca2+]i. The ED50 for rapid (5 s) TRH-stimulated IP3 formation was higher than for other sustained TRH actions in these cells, and we postulated that the initial TRH receptor interactions occur with rapid dissociation kinetics. To test this hypothesis, we performed rapid dilution experiments following a 1-s stimulation and found that TRH-stimulated IP3 formation decreased within 4 s of dilution and disappeared within 60 s at which time fresh TRH could restimulate IP3 formation. We conclude that receptor occupancy is the limiting step for TRH-stimulated IP3 formation and elevated [Ca2+]i and that maximal TRH action requires multiple rapid interactions between TRH and its receptor. PMID- 3007497 TI - [mono[125I]iodo-Tyr10,MetO17]-vasoactive intestinal polypeptide. Preparation, characterization, and use for radioimmunoassay and receptor binding. AB - Vasoactive intestinal polypeptide (VIP) was labeled with sodium [125I]iodide using the chloramine-T method and subsequently purified by reverse-phase high performance liquid chromatography. Three main 125I-labeled peaks designated A, B, and C resulted from the radioiodination and purification procedures. They were characterized by electrophoresis of tryptic fragments; Edman degradation (for Peaks A and C); enzymatic digestion to amino acids by leucine aminopeptidase, carboxypeptidase Y and Pronase; and treatment with cyanogen bromide. Peak A corresponds to VIP monoiodinated on Tyr10 and with the Met17 residue oxidized to methionine sulfoxide. This [mono[125I]iodo-Tyr10,MetO17]VIP displays the following characteristics. 1) It constitutes quantitatively the major product of the iodination procedure (62.5%); 2) it is well resolved from other labeled and unlabeled products; 3) it is stable (2 months at -20 degrees C); 4) it possesses a high specific activity (2050 Ci/mmol); 5) it maintains the biological activity of native VIP; and 6) it binds to antibody and membrane recognition sites in a specific, saturable, and reversible manner. Reduction of [mono[125I]iodo-Tyr10, Met-O17]VIP to [mono[125I]iodo-Tyr10]VIP does not improve the performance of the tracer in a radioimmunoassay. The method described in this article is simple and rapid and yields a molecular form of 125I-labeled VIP that has been fully characterized and is suitable for use in biological studies. PMID- 3007499 TI - Two apparent human endothelial cell growth factors from human hepatoma cells are tumor-associated proteinase inhibitors. AB - Two polypeptides from secretory products of human hepatoma cells were isolated and characterized on the basis of their stimulation of maintenance and growth of human endothelial cells in serum-free cell culture. Both factors were purified to homogeneity by a combination of reverse-phase, ion exchange, and molecular filtration high performance liquid chromatography. One factor (endothelial cell growth factor (ECGF-2a) had Mr approximately 6,500 and pI near 6. The second (ECGF-2b) had Mr = 27,000 and a pI below 4.0. Both ECGF-2a and ECGF-2b exhibited single NH2-terminal sequences. The first 25 NH2-terminal residues of ECGF-2a and the first 49 residues of ECGF-2b were determined by gas-phase microsequencing. All clearly determined residues of ECGF-2a were identical with human pancreatic secretory trypsin inhibitor. All assignable residues of ECGF-2b were identical with urinary glycoprotein proteinase inhibitor (HI-30/EDC1). Both proteins are absent or at low levels in normal plasma and urine, but appear during acute inflammatory disease and cancer. Amino acid composition of ECGF-2a and ECGF-2b was also similar to human pancreatic secretory inhibitor and HI-30/EDC1, respectively. Both ECGF-2a and ECGF-2b inhibited bovine pancreatic trypsin (2 micrograms/ml) by 50% at 750 ng/ml. ECGF-2a and ECGF-2b stimulated endothelial cell number at a half-maximal dose of 50 ng/ml (8 nM) and 80 to 130 ng/ml (5 to 9 nM) protein, respectively. When assayed under identical conditions, no effect of either factor on human smooth muscle cells, human hepatoma cells, or human, rat, and mouse fibroblasts could be detected. PMID- 3007498 TI - A new de novo pathway for the formation of 1-alkyl-2-acetyl-sn-glycerols, precursors of platelet activating factor. Biochemical characterization of 1-alkyl 2-lyso-sn-glycero-3-P:acetyl-CoA acetyltransferase in rat spleen. AB - 1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC, platelet activating factor (PAF] can be biosynthesized either by acetylation of alkyllyso GPC through a remodeling pathway or by the transfer of phosphocholine to alkylacetyl-sn-glycerol (alkylacetyl-G) via a putative de novo pathway involving a dithiothreitol-insensitive cholinephosphotransferase. However, the relevance of the de novo pathway in the biosynthesis of PAF depends on the existence of enzymes that can directly synthesize alkylacetyl-G from 1-alkyl-2-lyso-sn-glycero 3-P (alkyllyso-GP) or some other source. In this study, we demonstrated that microsomal preparations of rat spleen can synthesize alkylacetyl-GP by an alkyllyso-GP:acetyl-CoA acetyltransferase and that this intermediate is subsequently dephosphorylated by an alkylacetyl-GP phosphohydrolase to generate alkylacetyl-G. The properties of alkyllyso-GP:acetyl-CoA acetyltransferase were characterized under conditions where the contaminating activity of alkylacetyl-GP phosphohydrolase was minimal; this was accomplished by inhibiting the phosphohydrolase with the addition of sodium vanadate and sodium fluoride to the assay mixtures and incubating at relatively low temperatures (23 degrees C). Alkyllyso-GP:acetyl-CoA acetyltransferase had a pH optimum of 8.4 at 23 degrees C and was located in the microsomal fraction. The apparent Km for acetyl-CoA under these conditions was 226 microM and the optimal concentration of alkyllyso-GP ranged between 16 and 25 microM. Based on pH optima, substrate inhibition studies, and sensitivities to preincubation temperatures of the microsomes, it appears that alkyllyso-GP:acetyl-CoA acetyltransferase differs from the acetyltransferase responsible for the transfer of acetate from acetyl-CoA to alkyllyso-GPC to form PAF. A variety of tissues had high activities of alkyllyso GP:acetyl-CoA acetyltransferase, which indicates that this pathway is operational in many cell types. Our results document the existence of a complete de novo biosynthetic pathway for the assembly of PAF, and this route could be responsible for maintaining physiological levels of platelet activating factor for normal cell function. PMID- 3007500 TI - Tumor necrosis factor receptors in HeLa cells and their regulation by interferon gamma. AB - Incubation of HeLa cell cultures with human interferon-gamma (IFN-gamma) increased the binding of radioiodinated human tumor necrosis factor (TNF) to specific cell surface receptors (TNF-R). IFN-gamma also produced a proportionate increase in receptor-mediated endocytosis of TNF. TNF-R expression was significantly increased after 6 h of exposure to IFN-gamma (100 units/ml), and it remained elevated in the continuous presence of IFN-gamma for at least 20 h. Incubation of cells with IFN-gamma in the presence of cycloheximide, followed by treatment with actinomycin D and reversal of the inhibition of protein synthesis, also resulted in increased TNF-R expression as compared to cultures subjected to the same treatments in the absence of IFN-gamma. These results suggest that IFN gamma can directly stimulate accumulation of the mRNA for TNF-R and that TNF-R is among the cellular proteins whose synthesis is increased by IFN-gamma. PMID- 3007501 TI - Metabolism of leukotriene B4 in isolated rat hepatocytes. Identification of a novel 18-carboxy-19,20-dinor leukotriene B4 metabolite. AB - Isolated rat heptocytes were found to metabolize leukotriene B4 (LTB4) to a number of products which could be separated by reverse phase high performance liquid chromatography (HPLC). After incubation of LTB4 with hepatocytes for 15 min, the known omega-oxidized metabolites, 20-hydroxy- and 20-carboxy-LTB4, were identified by HPLC retention time and gas chromatography-mass spectrometry. An early fraction corresponding to 15% of the initial LTB4 was structurally characterized as a novel metabolite, 18-carboxy-19,20-dinor-LTB4, by ultraviolet spectroscopy and gas chromatography-mass spectrometry of the derivatized and derivatized, reduced metabolite. The short HPLC retention time of this metabolite was consistent with its reduced lipophilicity. An additional minor metabolite was tentatively identified as 3-hydroxy-LTB4. These two novel metabolites provide evidence for beta-oxidation as an important route of hepatic biotransformation of LTB4 and 20-hydroxy-LTB4. PMID- 3007502 TI - Parathyroid hormone increases sodium/calcium exchange activity in renal cells and the blunting of the response in aging. AB - Na+-dependent Ca2+ efflux was demonstrated in cells isolated from the rat renal cortex, suggestive of the presence of a Na+/Ca2+ exchange carrier in the cells. Parathyroid hormone, when incubated with the cells in vitro, increased Na+ dependent Ca2+ efflux about 60%. The effect of the hormone was specific for biologically active parathyroid hormone analogs and could be mimicked by cyclic nucleotides and forskolin. The effects of parathyroid hormone concentration on Ca2+ efflux and cyclic AMP formation were similar. These findings would be consistent with the view that the cyclic nucleotide might act as the intracellular messenger to increase Na+/Ca2+ exchange activity. Cells isolated from parathyroidectomized rats had decreased Na+-dependent Ca2+ efflux. When these cells were treated in vitro with parathyroid hormone, Na+-dependent Ca2+ efflux was enhanced to the same rate as found with cells from sham-operated animals. Parathyroid hormone-sensitive Na+/Ca2+ exchange activity was markedly blunted in cells from senescent (24 months) rats. Basal Na+-dependent Ca2+ efflux and Na+-independent Ca2+ efflux were not altered in the aged animal. Parathyroid stimulated adenylate cyclase was also decreased in aging. In contrast, forskolin stimulated Na+-dependent Ca2+ efflux and adenylate cyclase did not change with senescence. These findings would be compatible with a mechanism of desensitization that occurred at the level of the receptor or hormone-receptor coupling to adenylate cyclase. These results may be of physiological significance in understanding calcium homeostasis and the imbalances in mineral metabolism associated with old age. PMID- 3007503 TI - Characterization of the proteoglycans recovered under nondissociative conditions from normal articular cartilage of rabbits and dogs. AB - Pretreatment of articular cartilage with a highly purified collagenase in the presence of selected protease inhibitors allowed the extraction under nondissociative conditions of 65% of the tissue hexuronate. Extracted proteoglycans were purified by two successive equilibrium centrifugations in Cs2SO4 and CsCl, respectively, and then characterized by their sedimentation properties. The use of labeled proteoglycan preparations demonstrated that no detectable degradation was introduced by the new extraction procedure. When applied to growth cartilage of rachitic rats the sedimentation profile of the purified proteoglycans was practically identical to that of the proteoglycan molecules recovered by micropuncture-aspiration. Proteoglycans were extracted from normal articular cartilage of rabbits and dogs with either the new procedure or 4.0 M guanidine HCl. The purified aA1 and A1 preparations were characterized by their sedimentation properties. The aA1 contained a higher proportion of aggregates which sedimented as two distinctive populations of molecules. This bimodal distribution of the aggregates was never observed in the A1 preparations even when the dissociative extraction was performed after collagenase pretreatment of cartilages. The two extraction procedures, however, extracted the same proteoglycan monomers since the aA1-D1 and A1-D1 preparations had similar biochemical composition and g(s) distribution functions. These observations and additional in vitro aggregation studies suggested that the differences in the size and proportion of aggregates between the aA1 and A1 preparations result from a more efficient recovery of link glycoproteins in nondissociative extractions that could have determined two structurally different hyaluronate molecules. PMID- 3007504 TI - Control of phosphorylase kinase in the isolated glycogen particle by Ca2+-Mg2+ synergistic activation and cAMP-dependent phosphorylation. AB - The isolated glycogen particle provides a means to examine the regulation of glycogen metabolism with the components organized in a functional cellular complex. With this system, we have studied the control of phosphorylase kinase activation by Ca2+ and cAMP. Contrary to a previous report (Heilmeyer, L. M. G., Jr., Meyer, F., Haschke, R. H., and Fisher, E. H. (1980) J. Biol. Chem. 245, 6649 6656), phosphorylase kinase became activated during incubation of the glycogen particle with MgATP2- and Ca2+. Part of this activation could be attributed to the action of the cAMP-dependent protein kinase; however, it was not possible to quantitatively correlate activation with phosphorylation in the presence of Ca2+ and Mg2+ due to a large, but uncertain, contribution of synergistic activation caused by these ions. This latter activation had properties similar to those described by King and Carlson (King, M. M., and Carlson, G. M. (1980) Arch. Biochem. Biophys. 209, 517-523) with the purified enzyme, and its occurrence also explains why phosphorylase kinase activation in the glycogen particle was not observed previously. The cAMP-dependent activation of phosphorylase kinase in the glycogen particle has been characterized. It occurred in a similar manner when either the cAMP-dependent protein kinase or cAMP was added, thus indicating that the phosphorylation sites of phosphorylase kinase complexed in the glycogen particle were accessible to endogenous or exogenous enzyme. In the glycogen particle, both the alpha and beta subunits were phosphorylated by the cAMP dependent protein kinase, but the alpha subunit dephosphorylation appeared to be preferentially regulated by Ca2+. The activity of phosphorylase kinase in the glycogen particle is regulated by the phosphorylation of both the alpha and beta subunits. PMID- 3007505 TI - Depletion of cellular ATP inhibits Na+/H+ antiport in cultured human cells. Modulation of the regulatory effect of intracellular protons on the antiporter activity. AB - The metabolic energy dependency of Na+/H+ exchange activity was investigated in cultured human A431 carcinoma cells and foreskin fibroblasts. Following the activation of Na+/H+ exchange which results from loading cells with Na+ or Li+, depletion of cellular ATP by incubation with metabolic inhibitors causes an inhibition of Na+/H+ exchange activity. This inhibition is reversible and correlates with the extent of the reduction in the ATP pool. On the other hand, reduction of the intracellular pH (pHi) to approximately 6.0-6.2 results in a similar effective activation of Na+/H+ exchange in both control and ATP-depleted cells. Na+/H+ exchange activity in ATP-depleted cells that either have or have not been loaded with Na+ shows a steep dependency on pHi, being essentially abolished above pHi of 6.4-6.5, whereas control cells show a considerable activity also at more alkaline pHi values. Thus, Na+/H+ exchange activity in ATP depleted cells shows an increased dependency on intracellular protons. These findings suggest that although metabolic energy is not required as a driving force for Na+/H+ antiport, depletion of metabolic energy substrates inhibits the antiporter activity due to a modulation of an intracellular proton-dependent regulatory mechanism. PMID- 3007507 TI - Effects of protein-protein and protein-lipid interactions on heme site conformation in the mitochondrial b cytochromes. AB - Removal of lipid from detergent-solubilized succinate cytochrome c reductase by a mild method leads to a series of changes in the optical and EPR spectra of the b cytochromes. This culminates in a state that resembles purified b cytochromes from the same source and bisimidazole ferriheme model complexes. Reconstitution of the lipid-depleted complex with phospholipid restores the native spectra in a significant fraction of the complexes in the early stages of lipid depletion. Once the final state has been reached, however, reconstitution has so far been incapable of restoring described in this communication can be related to a model for integral membrane cytochromes. PMID- 3007506 TI - Rabbit C-reactive protein. Biosynthesis and characterization of cDNA clones. AB - To study the biosynthesis of rabbit C-reactive protein (CRP), a cDNA library was constructed from CRP mRNA-enriched polysomal poly(A) RNA. Four recombinant plasmids, designated pCX9, pCX23, pCX28, and pCX39, from 39 positive clones were sequenced and found to represent overlapping clones. DNA sequencing of CRP cDNA and primer extension of the 5'-end of CRP mRNA have demonstrated that the complete length of rabbit CRP mRNA consists of 2331 nucleotides and a terminal poly(A) segment. Analysis of the resulting sequence indicated that rabbit CRP mRNA contained a 5'-noncoding region of 107 nucleotides, a leader sequence encoding 20 amino acids, a coding region covering 205 amino acids, and a 3' noncoding region of 1549 nucleotides. The 3'-noncoding region contained a consensus AAUAAA sequence that is 105 nucleotides upstream from the 3'-terminal poly(A) segment. Using an in vitro translation system, we have confirmed that CRP is synthesized as a precursor polypeptide (Mr approximately equal to 26,000) which undergoes processing to form the mature polypeptide (Mr approximately equal to 23,500). The CRP precursor failed to display a calcium-dependent affinity for phosphorylcholine ligand as demonstrated by mature CRP, suggesting that the phosphorylcholine-binding site of CRP only formed after processing. Northern blot analysis suggested that following induction with turpentine, liver was the only site where CRP mRNA synthesis could be demonstrated and that the change in mRNA concentration correlated with the course of CRP production. Southern blot analysis of liver genomic DNA indicated a single gene copy for CRP. PMID- 3007508 TI - Cytochemical identification of the regulatory subunit of the cAMP-dependent protein kinase by use of fluorescently labeled catalytic subunit. Examination of protein kinase dissociation in hepatoma cells responding to 8-Br-cAMP stimulation. AB - Homogeneous catalytic subunit from the cAMP-dependent protein kinase, when derivatized with a fluorophore, was used as a cytochemical probe to locate intracellular sites of the protein kinase regulatory subunit. After conjugation, the fluoresceinated catalytic subunit (F:C), derivatized to a stoichiometry of approximately 1 mol/mol, retained near full activity as judged by specific activity and by titration against either regulatory subunit or Inhibitor Protein of the protein kinase. With this molecular probe the dissociated regulatory subunit was localized by direct cytochemistry in Reuber H-35 hepatoma cells that had been exposed, while intact, for 0-120 min to 10(-4) M 8-Br-cAMP. After stimulation, cultures were fixed and washed and then incubated for 16 h with F:C. Following 8-Br-cAMP stimulation, extensive binding of the probe to both cytoplasmic and nucleolar sites was observed. This binding was diminished but not eliminated when 50 microM cAMP was present during the incubation of the fixed cells with F:C that was eliminated by a 40-fold molar excess of underivatized catalytic subunit but not by heat-denatured catalytic subunit, and was not reduced by a 20-fold molar excess of cGMP-dependent protein kinase, examined plus or minus cGMP. Collectively, the results allow the conclusion that the F:C probe binds free regulatory subunit. The time course of its change with 8-Br-cAMP (measured as the difference between binding in the presence or absence of cAMP during the postfixation treatment) mirrors that previously reported for changes in the catalytic subunit in these cells, also identified cytochemically (Byus, C. V., and Fletcher, W.H. (1982) J. Cell Biol. 93, 727-734). The binding of the F:C probe, detected when cAMP is present during postfixation treatment, may possibly represent binding to free Inhibitor Protein of the cAMP-dependent protein kinase. If so, it was at a level of approximately 20% of the maximal level of detectable regulatory subunit, and it also showed cytosolic and nucleolar localization. PMID- 3007510 TI - Mouse glandular kallikrein genes. Identification, structure, and expression of the renal kallikrein gene. AB - Glandular kallikreins are a highly homologous subfamily of serine proteases encoded by 25 genes in the mouse. Several of the gene products have been implicated as having specific roles in growth factor biosynthesis, suggesting a general role for members of this gene family in the generation of biologically active peptides (Mason, A.J., Evans, B.A., Cox, D.R., Shine, J., and Richards, R.I. (1983) Nature 303, 300-307). Here we describe the identification, structure, and expression of the renal kallikrein gene, the major kinin-generating member of the family. Kinins are potent vasodilators implicated in regulation of local blood flow and ion transport. A gene-specific oligodeoxyribonucleotide probe was designed and chemically synthesized from the renal kallikrein gene sequence to distinguish this gene's transcripts from those of other members of the gene family. Differential expression phenotypes were found between the renal kallikrein gene and other members of the kallikrein gene family. PMID- 3007509 TI - Rat liver NAD(P)H:quinone reductase. Construction of a quinone reductase cDNA clone and regulation of quinone reductase mRNA by 3-methylcholanthrene and in persistent hepatocyte nodules induced by chemical carcinogens. AB - We have used polysomal immunoabsorption techniques to purify rat liver quinone reductase mRNA (NAD(P)H:quinone oxidoreductase, EC 1.6.99.2, formerly called DT diaphorase). Using the purified mRNA as template, cDNA clones complementary to quinone reductase mRNA have been constructed. One cDNA clone, pDTD55, has a 1900 base pair insert which has been demonstrated by hybrid-select translation experiments to be complementary to quinone reductase mRNA. Clone pDTD55 has been used in RNA and DNA blot hybridizations to show that quinone reductase mRNA is approximately 1900 nucleotides in length and is encoded by a gene which spans approximately 7000-8000 base pairs. We have also shown that quinone reductase mRNA is markedly elevated by 3-methylcholanthrene administration and in persistent hepatocyte nodules induced by chemical carcinogens. The elevation of quinone reductase mRNA in persistent hepatocyte nodules is not due to either gene amplification of DNA rearrangement. Rather, the quinone reductase gene is hypomethylated in persistent hepatocyte nodules compared to the gene in either liver tissue surrounding the nodule or normal liver. These data suggest that hypomethylation of specific gene sequences occurs at early stages during chemical carcinogenesis. PMID- 3007511 TI - The nucleotide sequence of the rat liver fatty acid-binding protein gene. Evidence that exon 1 encodes an oligopeptide domain shared by a family of proteins which bind hydrophobic ligands. AB - We have determined the nucleotide sequence of the gene encoding rat liver fatty acid-binding protein (L-FABP). Previous structural studies have shown that L-FABP belongs to a family of low molecular weight cytosolic proteins which bind hydrophobic ligands. Rat L-FABP is the first member of this family whose genomic organization has been defined. The L-FABP transcription unit spans 3790 nucleotides and contains four exons (115, 173, 93, and 121 base pairs) interrupted by three introns (1454, 1224, and 610 base pairs). No other abundant mRNAs appear to be transcribed from sequences located within 4 kilobases 5' or 6.5 kilobases 3' of this gene. Sequence analyses have detected the presence of several related amino acid sequence blocks within L-FABP which may serve similar structural roles. A variety of computational techniques were used to compare the oligopeptides specified by each exon with other known members of the protein family. The results indicate that only the amino acid sequence present in the first exon is conserved among the homologous proteins. This suggests that the first exon may encode a shared structural and functional domain important in the interaction of these proteins with their ligands. PMID- 3007512 TI - Isolation of genes active during hormone-induced morphogenesis in Drosophila imaginal discs. AB - Mass-isolated Drosophila imaginal discs cultured in vitro undergo morphogenesis (evagination) in response to the insect steroid hormone 20-hydroxyecdysone. In vitro translation of mRNA isolated from evaginating discs shows accumulation of at least five new mRNA transcripts that are present only in membrane-bound polysomal RNA and presumably encode imaginal disc membrane or secreted proteins. Using a modified differential hybridization screen employing a competition step, six different hormone-inducible membrane protein gene sequences were isolated. These genes constitute a new set of 20-hydroxyecdysone responsive loci that may encode gene products specifically required for imaginal disc morphogenesis. PMID- 3007513 TI - Purification and characterization of two inactive/latent protein phosphatases from pig brain. AB - Two inactive/latent protein phosphatases termed LP-1 (Mr 260,000) and LP-2 (Mr 350,000) were identified and purified from pig brain. Examination of molecular structures indicated that LP-1 has three subunits with molecular weights of 69,000, 55,000, and 34,000, respectively, whereas LP-2 contains only one subunit, with molecular weight of 49,000. When using phosphorylase a as a substrate, LP-1 was completely inactive and could be dramatically activated by freezing and thawing in 0.2 M 2-mercaptoethanol, whereas LP-2 contained some basal activity but could also be stimulated 40-fold by the same treatment. Kinetic analysis further indicated that both LP-1 and LP-2 enzymes dephosphorylate histone 2A, myelin basic protein, and phosphorylase a at a rather comparable rate, but the dephosphorylation of histone 2A and myelin basic protein seems to be spontaneously active. This, together with the results that trypsinolysis could specifically knock off phosphorylase phosphatase activity but caused no effect on the associated myelin basic protein/histone phosphatase activities, supports the notion that a two-site mechanism may possibly be involved in the regulation of substrate specificity of LP-1 and LP-2 enzymes in the central nervous system. PMID- 3007514 TI - Immunological characterization of sn-1,2-diacylglycerol and sn-2-monoacylglycerol kinase from pig brain. AB - Rabbit antisera were raised against diacylglycerol kinase purified from pig brain cytosol. Upon immunoblot analysis, the antibody was specifically reactive with the kinase (Mr = 79,000-80,000). Pig brain cytosol, microsomal, and synaptosomal fractions all contained the immunoreactive Mr = 80,000 polypeptide, thus showing that the same enzyme is present in the soluble as well as membrane fractions of the brain. The antibody could precipitate only 60% of the kinase activity present in the crude cytosol. Further, the antibody exhibited very little or no cross reactivity toward liver cytosolic enzymes obtained from different animals including pigs. Immunostaining of brain tissues demonstrated that neurons, in particular, their nuclei, were positively stained, whereas glial cells were not stained. It is likely that there exists a tissue-and/or cell-dependent immunological multiplicity of diacylglycerol kinase. The enzyme activities phosphorylating sn-1 and sn-2 monoacylglycerols were co-precipitated by the antibody, indicating their identity with diacylglycerol kinase. The enzyme activity toward sn-1 monoolein was much lower than that obtained with sn-2 monoolein. Enzymic as well as chemical analyses of acyl isomers of the reaction products showed that even tested with pure (greater than 95%) sn-1 monoolein, about 70% of the formed lysophosphatidate was of the sn-2 acyl type. The results show that diacylglycerol kinase phosphorylates almost exclusively the sn-2 acyl type of monoacyl-glycerol. PMID- 3007516 TI - A missense mutation in the oxi-3 gene of the [mi-3] extranuclear mutant of Neurospora crassa. AB - We have determined the DNA sequence of the oxi-3 gene and its 5' flanking region in the extranuclear [mi-3] mutant of Neurospora crassa. The oxi-3 gene encodes subunit 1 of cytochrome c oxidase, a protein known to be altered in the [mi-3] mutant (Bertrand, H., and Werner, S. (1979) Eur. J. Biochem. 98, 9-18). When the sequence from [mi-3] was compared to previously published sequences of the same region of mtDNA from wild-type N. crassa, a total of five differences was found. Four of these differences can be accounted for as either genetic polymorphisms or previous errors in DNA sequence determination. The remaining difference is a G/C to T/A transversion that changes a codon specifying an aspartic acid residue (GAC) to one that would specify tyrosine (TAC) at amino acid 448 of the 555 amino acid mature subunit 1 protein. This alteration was also found in the mtDNA of two separate heterokaryotic strains that had acquired the [mi-3] phenotype after repeated subculturing of heterokaryons forced between an [mi-3] strain and a strain containing a wild-type cytoplasm. The particular aspartic acid residue that would be affected by the mutation observed in [mi-3] is conserved in a diversity of species as either aspartic acid or glutamic acid, suggesting that an acidic residue at this position is important for the correct function of the subunit 1 protein. For these reasons, we consider it likely that the observed missense mutation is responsible for the [mi-3] phenotype. PMID- 3007515 TI - Photoaffinity labeling of alpha 1-adrenergic receptors of rat heart. AB - The photoaffinity probe [125I]aryl azidoprazosin was used to examine structural aspects of rat left ventricular alpha 1-adrenergic receptor. Autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins from photoaffinity-labeled membranes revealed a specifically labeled protein of mass 77 kDa. Adrenergic drugs competed with the photoaffinity probe for binding to the receptor in a manner expected of an alpha 1-adrenergic antagonist. Because the autoradiographic pattern was unaltered by incubating labeled membranes in gel sample buffer containing high concentrations of reducing agents, the binding component of the cardiac alpha 1-adrenergic receptor appears to be a single polypeptide chain. The photoaffinity probe specifically labeled a single protein of approximately 68 kDa in membranes of cardiac myocytes prepared from rat left ventricles. The role played by sulfhydryls in receptor structure and function was also studied. Dithiothreitol (DTT) inhibited [3H]prazosin binding to left ventricular membranes and altered both the equilibrium dissociation constant and maximal number of [3H]prazosin-binding sites but not the ability of the guanine nucleotide guanyl-5'-yl imidodiphosphate to decrease agonist affinity for the receptors. When photoaffinity-labeled membranes were incubated with 40 mM DTT for 30 min at room temperature, two specifically labeled proteins of 77 and 68 kDa were identified. The DTT-induced conversion of the 77-kDa protein to 68 kDa was irreversible with washing, but the effect of DTT on [3H]prazosin binding was reversible. Both 77- and 68-kDa proteins were observed with liver membranes even in the absence of reducing agent. We suggest that the DTT-induced conversion of the 77-kDa protein to 68 kDa is due to enhancement in protease activity by the reductant. These results document that the cardiac alpha 1-adrenergic receptor is a 77-kDa protein, similar in mass to the receptor in liver and other sites. Proteolysis likely accounts for lower Mr forms of this receptor found in cardiac myocytes and in previous publications on hepatic alpha 1-receptors. PMID- 3007517 TI - Basic and acidic fibroblast growth factors interact with the same cell surface receptors. AB - Despite quantitative differences, the activity of basic and acidic fibroblast growth factors (FGF) on a wide variety of normal diploid cells derived from neuroectoderm and mesoderm is intrinsically similar. This suggests that they bind to the same cell surface receptors. This was investigated using a baby hamster kidney cell line (BHK-21) as a model. BHK-21 cell membrane components that interact with basic and acidic FGF have been identified by covalent cross-linking to their respective 125I-labeled ligands. Under appropriate conditions, basic and acidic 125I-FGF were cross-linked, using disuccinimidyl suberate, to two receptor species with apparent molecular masses of 145,000 and 125,000 daltons, respectively. The labeling of those receptors is inhibited when either native basic or acidic FGF are present in excess during incubation of cells with either acidic or basic 125I-FGF. Competition of basic 125I-FGF with increasing concentrations of native acidic FGF results in a preferential decrease in the labeling of the 125,000-dalton species, whereas competition of acidic 125I-FGF with increasing concentrations of native basic FGF leads to a preferential decrease in the labeling of the 145,000-dalton species. The data suggest that qualitatively both mitogens interact with the same 145,000- and 125,000-dalton receptor species. The different affinities displayed by acidic and basic FGF toward their common receptor molecules could explain why acidic FGF, depending on the cell type considered, is 20-100-fold less potent than basic FGF. PMID- 3007518 TI - Secreted forms of human neutrophil collagenase. AB - Collagenase in human neutrophils is found within intracellular granules which can be stimulated to be secreted with phorbol myristic acetate. This extracellular secreted form of neutrophil collagenase was isolated by immunoaffinity chromatography using a monoclonal antibody previously shown to specifically recognize neutrophil collagenase. The enzyme efficiently bound to this column and was eluted with NaSCN as three major species of 75, 57, and 22 kDa, respectively. These proteins were closely related immunologically since, after radiolabeling and separation by gel filtration, each of the three proteins was precipitated by the monoclonal antibody. Also, the 75- and 57-kDa proteins exhibited collagenase activity after elution from polyacrylamide gels run under nondenaturing conditions. Further, the 57-kDa protein autodegraded into a 22-kDa protein with time. Polyclonal antibody, prepared to the 57-kDa enzyme, also recognized the 75- and 22-kDa proteins using an immunoblot technique. When crude neutrophil supernatants containing latent collagenase were immunoblotted, both the 75- and the 57-kDa enzymes were present. Our immunoaffinity purified active enzymes, although activated during the course of purification, resemble the latent enzymes in crude neutrophil supernatants. The multiple forms of secreted collagenase from degranulated leukocytes may resemble more closely that seen in inflammation. PMID- 3007519 TI - Overproduction and purification of the B2 subunit of ribonucleotide reductase from Escherichia coli. AB - The nrdB gene of Escherichia coli, coding for the B2 protein of ribonucleotide reductase, has been cloned in a runaway-replication vector. The runaway derivative pBEU17 carries the promoter-proximal portion of the E. coli alanyl tRNA synthetase gene and proved useful for expressing cloned genes lacking their native transcription initiation signals. The alaS promoter is located approximately 500 base pairs upstream of a single BamHI restriction endonuclease cleavage site utilized in the construction of an expression recombinant plasmid, pBS1, for the nrdB product. After 5-h thermal induction of cells carrying the runaway recombinant pBS1, protein B2 constituted 40% of the soluble protein fraction of the cells. The high concentration of protein B2 in crude extracts of induced cells has enabled a simplified purification scheme to be developed for production of homogeneous and concentrated B2 preparations. Protein B2 produced from pBS1 is identical to the chromosomally encoded nrdB product of E. coli as regards molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme activity, tyrosine radical content, and structure of the binuclear iron center. Amino acid sequence analysis showed that the two polypeptide chains of protein B2 are identical. They start with an alanine residue, and the first 30 residues confirmed the amino acid sequence predicted from the nucleotide sequence of the nrdB gene, apart from an NH2-terminal processing removal of the initiator methionine. PMID- 3007520 TI - Cloning of the bacteriophage T4 uvsX gene and purification and characterization of the T4 uvsX recombination protein. AB - The bacteriophage T4 uvsX gene is a nonessential gene required for normal levels of DNA repair, recombination, and replication. We demonstrate that plasmids containing the T4 DNA approximately 300-2900 base pairs upstream of T4 gene 41 express a biologically active uvsX protein. This uvsX protein imparts increased survival to UV-irradiated T4 uvsX- phage and decreases the T4 uvsX- mutant suppression of a conditionally lethal T4 mutant in the gene 49 recombination nuclease. The uvsX protein purified from cells with a uvsX+ plasmid catalyzes ATP hydrolysis to ADP and AMP and, in the presence of the T4 gene 32 helix destablizing protein, ATP-dependent strand exchange between homologous circular single-stranded and linear duplex DNA. These results agree with the recent characterization of uvsX protein from T4-infected cells by Yonesaki et al. (Yonesaki, T., Ryo, Y., Minagawa, T., and Takahashi, H. (1985) Eur. J. Biochem. 148, 127-134) and by Formosa and Alberts (Formosa, T., and Alberts, B.M. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 363-370). In addition, we find that under some reaction conditions strand exchange is catalyzed by uvsX protein in the absence of 32 protein. The level of the uvsX protein expressed by the uvsX+ plasmids is high and independent of the orientation of the T4 DNA within the vector. This suggests that transcription promoter(s) lie upstream of the uvsX gene on the cloned T4 DNA. In vitro transcription of T4 DNA restriction fragments reveals two tandem promoters whose transcripts initiate approximately 500 and 600 nucleotides upstream of the uvsX gene and extend through the gene. PMID- 3007521 TI - An evaluation of some in vivo immunization strategies for the production of monoclonal antibodies to insulin and ACTH. AB - Immunization schedules for the production of monoclonal antibodies to bovine insulin and porcine adreno-corticotrophic hormone (ACTH) have been investigated. Specifically, prime dose, prime route, pre-fusion boost dose and immune status have been evaluated for their effect on both the number of hybrids observed after fusion, and the proportion of wells containing antibody which bound to the immunogen. Although a single optimum protocol was not identified, the results indicate that spleen cells from mice primed at multiple sites should be used for fusion after the peak of the primary antibody response. Excessive hyperimmunization should be avoided. Dose regimes combining a low prime amount and a high pre-fusion boost amount or a high prime amount and a low pre-fusion boost amount (except in the presence of circulating antibody) were favoured. Monitoring of the immune response of animals used in fusion experiments is of paramount importance. PMID- 3007523 TI - Functional and biochemical modifications of lung beta-adrenoreceptors after in vivo desensitization: prevention by indomethacin. AB - Desensitization of lung beta-adrenoreceptors induced by 4 day in vivo isoprenaline administration to rats has been investigated both from a functional a biochemical viewpoint. Chronic isoprenaline treatment significantly reduced the relaxing activity of the beta-agonist when tested ex vivo in lung parenchymal strips and also impaired the adenylate-cyclase system. Moreover, the desensitization procedure decreased by about 30% beta-adrenoreceptor number. In vivo indomethacin treatment prevented the loss of pharmacological responsiveness of the tissue to isoprenaline and restored basal adenylate-cyclase activity. These data indicate that in vivo isoprenaline administration actually leads to pulmonary beta-adrenoreceptor desensitization. The involvement of arachidonic acid metabolites in this phenomenon is also discussed. PMID- 3007522 TI - Selectivities of some agonists acting at alpha 1- and alpha 2-adrenoreceptors in the rat vas deferens. AB - In this paper, estimates of the selectivities of a series of twelve sympathomimetic agents acting at postjunctional alpha 1- and prejunctional alpha 2-adrenoreceptors were investigated, using epididymal and prostatic segments of the rat vas deferens. The relative order of potency for the twelve agonists at prejunctional alpha 2-adrenoreceptors mediating inhibition of field-stimulation induced contractions in the prostatic segment of the vas deferens was: clonidine greater than (-)-adrenaline greater than xylazine greater than or equal to (-) noradrenaline greater than (+)-adrenaline greater than dopamine greater than or equal to phenylephrine greater than or equal to metaraminol greater than or equal to (+)-noradrenaline greater than (-)-isoprenaline greater than methoxamine greater than (+)-isoprenaline. The relative order of potency for the agonists at postjunctional alpha 1-adrenoreceptors mediating contraction of smooth muscle in epididymal segments of the vas deferens was: (-)-adrenaline greater than or equal to (-)-noradrenaline greater than phenylephrine greater than clonidine greater than or equal to (+)-adrenaline greater than or equal to methoxamine greater than or equal to (+)-noradrenaline greater than or equal to metaraminol greater than or equal to dopamine greater than or equal to (-)-isoprenaline greater than or equal to xylazine; (+)-isoprenaline was inactive. (+)-Noradrenaline, the stereoisomers of adrenaline and isoprenaline, dopamine, clonidine, xylazine and metaraminol displayed alpha 2-selectivity whereas phenylephrine and methoxamine displayed alpha 1-adrenoreceptor selectivity. (-)-Noradrenaline possessed a similar potency at both alpha 1- and alpha 2-adrenoreceptors thus making it non selective by the criteria used in this study.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007524 TI - The postganglionic excitatory innervation of the mouse urinary bladder and its modulation by prejunctional GABAB receptors. AB - Field stimulation produced reproducible contractions of the mouse isolated urinary bladder whose amplitude was frequency-related. These contractions were partially sensitive to atropine (3 microM), unaffected by hexamethonium (10 microM) and almost abolished by tetrodotoxin (0.5 microM). Atropine (3 microM) suppressed contractions produced by exogenous acetylcholine thereby indicating atropine-resistance of the nerve-mediated contractions. Nerve-mediated contractions of the mouse urinary bladder were enhanced by physostigmine (0.1-0.5 microM) and inhibited by hemicholinium-3 (0.5 mM) thus confirming the presence of a cholinergic component in the excitatory postganglionic innervation. Atropine (3 microM) inhibition of the nerve-mediated contractions increased with increasing duration and strength of the train of stimulation. The nerve-mediated contractions of the mouse bladder were unaffected by phentolamine (0.2 microM), propranolol (0.3 microM) or indomethacin (5 microM). ATP (1mM) the major candidate for the role of nonadrenergic-noncholinegic (NANC) excitatory neurotransmitter in the mammalian urinary bladder produced a contraction of the mouse isolated bladder. Exposure to the stable ATP analogue alpha, beta-methylene ATP (APCPP) or beta, gamma-methylene ATP (APPCP) produced a partial desensitization of the nerve-mediated response which, for APCPP, was greater in the presence than in the absence of atropine (3 microM). In the presence of atropine (3 microM) and after APCPP desensitization the amplitude of the response to field stimulation amounted to about 20% of the original response and was sensitive to tetrodotoxin, indicating that it is nerve-mediated. GABA (0.001-0.3 mM) inhibited the amplitude of field stimulation induced contractions of mouse urinary bladder. This effect was mimicked by the selective GABAB receptor agonist, (+/-)-baclofen, but not by the selective GABAA receptor agonist, homotaurine. GABA and (+/-)-baclofen exhibited cross-desensitization. The GABA-or (+/-)-baclofen-induced inhibition of the nerve-mediated contractions were reduced by previous exposure to homotaurine (1 mM) or to 5-aminovaleric acid (2 mM), two GABAB receptor antagonists. On the other hand the inhibitory effects of GABA or (+/-)-baclofen were unaffected by picrotoxin (0.1 mM), a selective GABAA receptor antagonist. The inhibitory effect of GABA on nerve-mediated contractions was reduced in the presence of atropine or hemicholinium-3 as well as following desensitization of P2-purinoreceptors.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3007526 TI - Hydroxyapatite deposition in articular cartilage by intra-articular injections of methylprednisolone. A histological, ultrastructural, and x-ray-microprobe analysis in rabbits. AB - The mineral deposits in rabbit articular cartilage induced by intra-articular injections of glucocorticoid were studied by light and electron microscopy, using histochemical techniques and x-ray-probe microanalysis. This study demonstrated that the mineral deposits consisted of hydroxyapatite crystals. The initial deposition of hydroxyapatite crystals was seen around degenerating chondrocytes, where a halo-like pericellular space contained a large amount of electron-dense amorphous material. The initial precipitation of the crystals with a low ratio of calcium to phosphorus and the subsequent growth of crystals were seen only on or within the electron-dense amorphous material until the crystals formed mature, calcified nodules. The electron-dense amorphous material frequently coexisted with proteoglycans and degenerated collagen fibers. Digestion studies using chondroitinase ABC, papain, or chloroform and methanol suggested that the electron-dense amorphous material consisted of some protein and a small amount of lipid. Matrix vesicles were rarely seen in the calcifying areas. In addition, there was a correlation between sulphur, calcium, and phosphorus in the calcifying areas, where the relative element concentrations were: S (estimation counts of sulphur) = -0.862 X (calcium counts) + 1.472 X (phosphorus counts) + 102.146. This study demonstrated that electron-dense amorphous material, proteoglycans, and degenerated collagen fibers are present in loci where the hydroxyapatite crystals are formed in articular cartilage. PMID- 3007525 TI - Aging and the cardiovascular system. AB - What has preceded has been largely a listing of contradictory data: what follows is an attempt to gleam a pattern from all of this. In general, responsiveness is either reduced or unchanged by aging; reduced responsiveness has been demonstrated most frequently for cardiac beta-adrenoreceptors, cardiac muscarinic receptors, vascular beta-adrenoreceptors and vascular alpha 2-adrenoreceptors. A reduced ability of the amine uptake system has been demonstrated in some but not all studies: any reduction in re-uptake would tend to potentiate the effects of NA and counteract the reduced receptor responsiveness (or vice versa). This may explain why the most consistent cardiovascular alteration reported in the elderly is an increased plasma NA. While there are clinical reports that beta-blockers and converting enzyme inhibitors are less effective in elderly hypertensives at lowering blood pressure, this may reflect more the pathological development of hypertension with time rather than a true aging phenomenon. Overall, it appears that resting function of the cardiovascular system is near normal in the aged, but since the mode of control is somewhat altered, in particular with a blunted baroreflex, perturbations in the system produced by drugs may cause a higher incidence of adverse effects. PMID- 3007527 TI - Idiopathic hyperphosphatasia with dermal pigmentation. A twenty-year follow-up. AB - Hyperphosphatasia, or hereditary bone dysplasia with hyperphosphatasaemia, is a rare genetic disorder which is characterised by failure to transform woven into lamellar bone. Clinical, radiological and histological features establish the diagnosis, fractures, deformities, diffuse sclerosis on radiographs and high serum alkaline phosphatase being characteristic. We report the case of a 27-year old man with follow-up at the same hospital for 20 years. Attempts at treatment with calcitonin and disocium etidronate (EHDP) failed, but stapling of the growth plates at the knee was successfully performed. Transverse "brittle" fractures of the humerus, lower leg and ribs healed normally, but internal fixation and late bone grafting were required for a subtrochanteric stress fracture of the femur at the age of 24 years. At present the patient has no clinical problems and leads a normal life. PMID- 3007528 TI - An agent or agents produced by virus-transformed cells cause unregulated ruffling in untransformed cells. AB - KNRK cells (a normal rat kidney [NRK] cell line transformed by Kirsten murine sarcoma virus) in sparse culture exhibit a highly ruffled morphology, but the cause of this ruffling is unknown. In this study, we have demonstrated that the continuous, excess ruffling on KNRK cells is caused by one or more soluble agents secreted by the KNRK cells themselves. To do this study, an assay for ruffling responses in live cell cultures was defined, and its reproducibility was demonstrated. This assay permitted observation of the kinetics of ruffling responses (percentage of cells ruffled as a function of time after stimulation). This method was used to compare the kinetics of ruffling induced by insulin, epidermal growth factor, fibroblast growth factor, glucose, and KNRK cell conditioned medium (CM). Ruffling was elicited on NRK cells by each of the polypeptide mitogens and nutrients, but, in each case, this ruffling subsided spontaneously within an hour. CM from KNRK cells also caused ruffling movements on untransformed NRK cells, but this ruffling continued for at least 20 h. This response was largely blocked by premixing the KNRK cell CM with rabbit IgG against rat transforming growth factor, type alpha, (TGF-alpha). KNRK cells made quiescent (ruffle free) by a pH shift (from 7.4 to 8.4) responded to insulin, glucose, and KNRK cell CM with kinetics similar to those observed for each of these factors in NRK cells. The unusual feature for the ruffle-inducing agent(s) produced by KNRK cells was that this activity was not subject, in either NRK or KNRK cells, to the cellular off-regulation that limits the responses to insulin or glucose. Thus, the continuous ruffling of KNRK cells is caused by their own unregulated ruffle-inducing agent or agents, which appear to include TGF-alpha. This work also demonstrates that kinetic analysis of cellular responses to exogenous factors can provide new insights into the regulatory mechanisms involved in the normal limitation of these responses. PMID- 3007529 TI - Cell-mediated co-action of transforming growth factors: incubation of type beta with normal rat kidney cells produces a soluble activity that prolongs the ruffling response to type alpha. AB - Intense, continuous ruffling is a characteristic of many transformed cells, but untransformed cells ruffle intensely only briefly after exposure to growth factors. We reported previously that cells of a normal rat kidney (NRK) cell line transformed by Kirsten murine sarcoma virus secrete their own ruffle-inducing agent(s) that cause sustained ruffling in either themselves or untransformed NRK cells. In the present study, we examined the roles of the transforming growth factors TGF-alpha and TGF-beta in the induction and maintenance of ruffling in untransformed NRK cells and observed the following: TGF-alpha caused a transient epidermal growth factor (EGF)-like response, which could be blocked by prior exposure of cells to EGF or by antiserum directed against the COOH-terminus of TGF-alpha. TGF-beta caused no ruffling and did not itself prolong TGF-alpha ruffling. A new, buffer-soluble (transferable) mediator activity produced by incubation of TGF-beta with NRK cells for 6-h extended the duration of maximal TGF-alpha-induced ruffling by several-fold. This study demonstrates that TGF alpha alone causes an EGF-like, transient ruffling response, but neither TGF alpha or TGF-beta alone, nor the two together, cause transformation-associated sustained ruffling. Rather, TGF-alpha acts in concert with a new, TGF-beta dependent activity. This new activity appears to inhibit normal cellular off regulation of TGF-alpha-induced ruffling. Inhibition of the cellular off regulation of a growth factor response could play a key role in the unregulated growth associated with malignancy. PMID- 3007530 TI - Sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells. AB - Previous studies (Rindler, M. J., I. E., Ivanov, H. Plesken, and D. D. Sabatini, 1985, J. Cell Biol., 100: 136-151; Rindler, M. J., I. E. Ivanov, H. Plesken, E. J. Rodriguez-Boulan, and D. D. Sabatini, 1984, J. Cell Biol., 98: 1304-1319) have demonstrated that in polarized Madin-Darby canine kidney cells infected with vesicular stomatitis virus (VSV) or influenza virus the viral envelope glycoproteins G and HA are segregated to the basolateral and apical plasma membrane domains, respectively, where budding of the corresponding viruses takes place. Furthermore, it has been shown that this segregation of the glycoproteins reflects the polarized delivery of the newly synthesized polypeptides to each surface domain. In transfection experiments using eukaryotic expression plasmids that contain cDNAs encoding the viral glycoproteins, it is now shown that even in the absence of other viral components, both proteins are effectively segregated to the appropriate cell surface domain. In transfected cells, the HA glycoprotein was almost exclusively localized in the apical cell surface, whereas the G protein, although preferentially localized in the basolateral domains, was also present in lower amounts, in the apical surfaces of many cells. Using transfected and infected cells, it was demonstrated that, after reaching the cell surface, the G protein, but not the HA protein, undergoes interiorization by endocytosis. Thus, in the presence of chloroquine, a drug that blocks return of interiorized plasma membrane proteins to the cell surface, the G protein was quantitatively trapped in endosome- or lysosome-like vesicles. The sequestration of G was a rapid process that was completed in many cells by 1-2 h after chloroquine treatment. The fact that in transfected cells the surface content of G protein was not noticeably reduced during a 5-h incubation with cycloheximide, a protein synthesis inhibitor that did not prevent the effect of chloroquine, implies that normally, G protein molecules are not only interiorized but are also recycled to the cell surface. PMID- 3007531 TI - The lateral mobility of the (Na+,K+)-dependent ATPase in Madin-Darby canine kidney cells. AB - Fluorescence microphotolysis (recovery after photobleaching) was used to determine the lateral mobility of the (Na+,K+)ATPase and a fluorescent lipid analogue in the plasma membrane of Madin-Darby canine kidney (MDCK) cells at different stages of development. Fluorescein-conjugated Fab' fragments prepared from rabbit anti-dog (Na+,K+)ATPase antibodies (IgG) and 5-(N hexadecanoyl)aminofluorescein (HEDAF) were used to label the plasma membrane of confluent and subconfluent cultures of MDCK cells. Fractional fluorescence recovery was 50% and 80-90% for the protein and lipid probes, respectively, and was independent of developmental stage. The estimated diffusion constants of the mobile fraction were approximately 5 X 10(-10) cm2/s for the (Na+,K+)ATPase and approximately 2 X 10(-9) cm2/s for HEDAF. Only HEDAF diffusion showed dependency on developmental stage in that D for confluent cells was approximately twice that for subconfluent cells. These results indicate that (Na+,K+)ATPase is 50% immobilized in all developmental stages, whereas lipids in confluent MDCK cells are more mobile than in subconfluent cells. They suggest, furthermore, that the degree of immobilization of the (Na+,K+)ATPase is insufficient to explain its polar distribution, and they support restricted mobility of the ATPase through the tight junctions as the likely mechanism for preventing the diffusion of this protein into the apical domain of the plasma membrane in confluent cell cultures. PMID- 3007532 TI - Heterologous transmembrane and cytoplasmic domains direct functional chimeric influenza virus hemagglutinins into the endocytic pathway. AB - Chimeric genes were created by fusing DNA sequences encoding the ectodomain of the influenza virus hemagglutinin (HA) to DNA coding for the transmembrane and cytoplasmic domains of either the G glycoprotein of vesicular stomatitis virus or the gC glycoprotein of Herpes simplex virus 1. CV-1 cells infected with SV40 vectors carrying the recombinant genes expressed large amounts of the chimeric proteins, HAG or HAgC on their surfaces. Although the ectodomains of HAG and HAgC differed in their immunological properties from that of HA, the chimeras displayed the biological functions characteristic of the wild-type protein. Both HAG and HAgC bound erythrocytes as efficiently as HA did and, after brief exposure to an acidic environment, induced the fusion of erythrocyte and CV-1 cell membranes. However, the behavior of HAG and HAgC at the cell surface differed from that of HA in several important respects. HAG and HAgC were observed to collect in coated pits whereas wild-type HA was excluded from those structures. In the presence of chloroquine, which inhibits the exit of receptors from endosomes, HAG and HAgC accumulated in intracellular vesicles. By contrast, chloroquine had no effect on the location of wild-type HA. HAG and HAgC labeled at the cell surface exhibited a temperature-dependent acquisition of resistance to extracellular protease at a rate similar to the rates of internalization observed for many cell surface receptors. HA acquired resistance to protease at a rate at least 20-fold slower. We conclude that HAG and HAgC are efficiently routed into the endocytic pathway and HA is not. However, like HA, HAG was degraded slowly, raising the possibility that HAG recycles to the plasma membrane. PMID- 3007533 TI - Specific binding sites for albumin restricted to plasmalemmal vesicles of continuous capillary endothelium: receptor-mediated transcytosis. AB - The interaction of homologous and heterologous albumin-gold complex (Alb-Au) with capillary endothelium was investigated in the mouse lung, heart, and diaphragm. Perfusion of the tracer in situ for from 3 to 35 min was followed by washing with phosphate-buffered saline, fixation by perfusion, and processing for electron microscopy. From the earliest time examined, one and sometimes two rows of densely packed particles bound to some restricted plasma membrane microdomains that appeared as uncoated pits, and to plasmalemmal vesicles open on the luminal front. Morphometric analysis, using various albumin-gold concentrations, showed that the binding is saturable at a very low concentration of the ligand and short exposure. After 5 min, tracer-carrying vesicles appeared on the abluminal front, discharging their content into the subendothelial space. As a function of tracer concentration 1-10% of plasmalemmal vesicles contained Alb-Au particles in fluid phase; from 5 min on, multivesicular bodies were labeled by the tracer. Plasma membrane, coated pits, and coated vesicles were not significantly marked at any time interval. Heparin or high ionic strength did not displace the bound Alb-Au from vesicle membrane. No binding was obtained when Alb-Au was competed in situ with albumin or was injected in vivo. Gold complexes with fibrinogen, fibronectin, glucose oxidase, or polyethyleneglycol did not give a labeling comparable to that of albumin. These results suggest that on the capillary endothelia examined, the Alb-Au is adsorbed on specific binding sites restricted to uncoated pits and plasmalemmal vesicles. The tracer is transported in transcytotic vesicles across endothelium by receptor-mediated transcytosis, and to a lesser extent is taken up by pinocytotic vesicles. The existence of albumin receptors on these continuous capillary endothelia may provide a specific mechanism for the transport of albumin and other molecules carried by this protein. PMID- 3007535 TI - Hexose transport in L6 rat myoblasts. II. The effects of sulfhydryl reagents. AB - The importance of sulfhydryl groups for hexose transport in undifferentiated L6 rat myoblasts was investigated. N-ethylmaleimide (NEM) and p chloromercuribenzenesulfonic acid (pCMBS) inhibited 2-deoxy-D-glucose (2-DOG) transport in a time and concentration-dependent manner. The inhibition produced by both reagents was virtually complete within 5 min, although neither reagent inhibited transport more than 70-80% regardless of the concentrations or incubation times used. Furthermore, the inhibition of 2-DOG transport by pCMBS or NEM could not be prevented by simultaneous preincubation of cells with 20 mM D glucose or 20 mM 2-DOG. This suggests that sulfhydryl groups required for transport are separate from the hexose binding and transport site. By comparing the effects of the membrane impermeant pCMBS to those of the membrane permeant NEM, cell surface sulfhydryl groups were shown to be essential for hexose binding and transport. In contrast to the inhibition of 2-DOG transport, pCMBS and NEM had much less of an effect on 3-O-methyl-D-glucose (3-OMG) transport. For example, 1 mM NEM inhibited 2-DOG transport by 66%, whereas 3-OMG transport was inhibited by only 7%. This supports the suggestion that these hexose analogues may be transported by different carriers. Kinetic analysis of transport shows that treatment of cells with 1 mM NEM or 1 pCMBS results in inactivation of the high affinity 2-DOG transport system, whereas the low affinity transport system is unaffected. 3-OMG is preferentially transported by the low affinity system. PMID- 3007534 TI - Sequential expression of chicken actin genes during myogenesis. AB - Embryonic muscle development permits the study of contractile protein gene regulation during cellular differentiation. To distinguish the appearance of particular actin mRNAs during chicken myogenesis, we have constructed DNA probes from the transcribed 3' noncoding region of the single-copy alpha-skeletal, alpha cardiac, and beta-cytoplasmic actin genes. Hybridization experiments showed that at day 10 in ovo (stage 36), embryonic hindlimbs contain low levels of actin mRNA, predominantly consisting of the alpha-cardiac and beta-actin isotypes. However, by day 17 in ovo (stage 43), the amount of alpha-skeletal actin mRNA/microgram total RNA increased more than 30-fold and represented approximately 90% of the assayed actin mRNA. Concomitantly, alpha-cardiac and beta-actin mRNAs decreased by 30% and 70%, respectively, from the levels observed at day 10. In primary myoblast cultures, beta-actin mRNA increased sharply during the proliferative phase before fusion and steadily declined thereafter. alpha Cardiac actin mRNA increased to levels 15-fold greater than alpha-skeletal actin mRNA in prefusion myoblasts (36 h), and remained at elevated levels. In contrast, the alpha-skeletal actin mRNA remained low until fusion had begun (48 h), increased 25-fold over the prefusion level by the completion of fusion, and then decreased at later times in culture. Thus, the sequential accumulation of sarcomeric alpha-actin mRNAs in culture mimics some of the events observed in embryonic limb development. However, maintenance of high levels of alpha-cardiac actin mRNA as well as the transient accumulation of appreciable alpha-skeletal actin mRNA suggests that myoblast cultures lack one or more essential components for phenotypic maturation. PMID- 3007536 TI - Alteration in cyclic adenosine 3':5'-monophosphate binding proteins during the phenotypic change of mouse macrophages transformed by a temperature-sensitive mutant (tsA640) of SV40. AB - By using a photoaffinity ligand, cell extracts from transformed macrophages that were established by infection with temperature-sensitive mutants (tsA640) of simian virus 40 (SV40) were examined for cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins. At the nonpermissive temperature for SV40 large T antigen, 39.0 degrees C, no significant cAMP-binding proteins could be detected, such as primary mouse macrophages. At the permissive temperature of 33.0 degrees C, cAMP-binding proteins appeared later than SV40 T antigen expression and cellular DNA synthesis. The profile of cAMP-binding proteins was similar to that of resting, but not proliferating, mouse clonal fibroblasts (BALB/c 3T3). These and previous results suggest that SV40 T antigen influences the expression of cAMP-binding proteins in tsA640-transformed macrophages; the large/small T antigen converts the profile of cAMP-binding proteins from macrophage to fibroblastic cells. PMID- 3007537 TI - EGF induces cell cycle arrest of A431 human epidermoid carcinoma cells. AB - The human carcinoma cell line A431 is unusual in that physiologic concentrations of epidermal growth factor (EGF) inhibit proliferation. In the presence of 5-10 nM EGF proliferation of A431 cells is abruptly and markedly decreased compared to the untreated control cultures, with little loss of cell viability over a 4-day period. This study was initiated to examine how EGF affects the progression of A431 cells through the cell cycle. Flow cytometric analysis of DNA in EGF-treated cells reveals a marked change in the cell cycle distribution. The percentage of cells in late S/G2 increases and early S phase is nearly depleted. Since addition of the mitotic inhibitor vinblastine causes accumulation of cells in mitosis and prevents reentry of cells into G1, it is possible to distinguish between slow progression through G1 and G2 and blocks in those phases. When control cells, not treated with EGF, are exposed to vinblastine, the cells accumulate mitotic figures, as expected, and show progression into S, thus diminishing the number of cells in G1. In contrast, no mitotic figures are found among the EGF-treated cells in the presence or absence of vinblastine, and progression from G1 into S is not observed, as the number of cells in G1 remains constant. These results suggest that there are two EGF-induced blocks in cell cycle transversal; one is in late S and/or G2, blocking entry into mitosis, and the other is in G1, blocking entry into S phase. After 24 hours of EGF treatment, DNA synthesis is reduced to less than 10% compared to untreated controls as measured by the incorporation of [3H]thymidine or BrdU. In contrast, protein synthesis is inhibited by about twofold. Although inhibition of protein synthesis is less extensive, it occurs 6 hours prior to an equivalent inhibition of DNA synthesis. The rapid decrease in protein synthesis may result in the subsequent cell cycle arrest which occurs several hours later. PMID- 3007538 TI - Further studies on the enhancing factor and its possible mechanism of action. AB - In this study the nature of binding of enhancing factor (EF) and its mode of action are examined. EF binds to A431 cells through its own receptor, which is distinct from the receptor for epidermal growth factor (EGF). EF binds to the cell membrane and in turn provides a binding site for EGF. Data analyzed from Scatchard plots show that prior treatment of formalin-fixed A431 cells with EF for 30 minutes results in an increase in the number of binding sites for 125I EGF. 3H-Thymidine incorporation studies, using the EGF-receptorless cell line NR 6, indicate that neither EF nor EGF alone stimulates the cells to synthesise DNA, but when both are added together the cells show 3H-thymidine incorporation. The role of EF may be to trap EGF and make it available to the cells through its own receptors even in the absence of EGF receptors. EF also induces anchorage independent growth of normal fibroblasts in soft agar only in the presence of EGF. PMID- 3007540 TI - Norepinephrine decreases EGF binding in primary rat hepatocyte cultures. AB - Norepinephrine (NE) produced a dose-dependent inhibition of 125I-epidermal growth factor (EGF) binding to adult rat hepatocytes in primary culture. This effect was maximal after 1 hr of incubation with NE and could be blocked by the presence of an alpha 1-specific adrenergic receptor antagonist. The inhibition of binding correlates with the ability of NE to enhance hepatocyte DNA synthesis in the presence of EGF and appears to be mediated by a reduction in EGF receptor number, without a significant change in receptor affinity. PMID- 3007539 TI - Multilineage, non-species specific hematopoietic growth factor(s) elaborated by a feline fibroblast cell line: enhancement by virus infection. AB - In studies designed to determine the role of feline leukemia virus (FeLV) in the pathogenesis of marrow failure in the cat, we tested medium conditioned by uninfected and FeLV-infected feline embryonic fibroblasts (FEA) for its effect on hematopoietic colony growth in culture. As opposed to an inhibitory effect, we found that the conditioned medium (CM) from FEA or FEA/FeLV increased the in vitro growth of multiple hematopoietic progenitor cell types including erythroid burst-forming cells (BFU-E), granulocyte/macrophage colony-forming cells, megakaryocytic colony-forming cells, and mixed-cell colony-forming cells. Furthermore, CM enhanced the growth of progenitors in cultures of mouse or human marrow cells, as well as cat marrow cells. Stimulation of feline BFU-E was most marked with an increment in growth of 400% over control. The human burst promoting activity (BPA) of the CM was equivalent or better than other CM available in our laboratory. The evidence suggest that the growth-promoting activity is a constitutive product(s) released by FEA which was enhanced eightfold with virus infection. Studies with non-adherent and T-lymphocyte depleted human marrow cells and human peripheral blood cells suggest that the growth factor(s) acts directly on progenitor cells and not through readily identified accessory cells. These findings are consistent with the concept that mesenchymal cells such as fibroblasts have the capacity to release hematopoietic growth factor(s) capable of acting on primitive hematopoietic progenitors. The results provide an example of how injury of such cells, through virus infection, may enhance growth factor(s) release and influence the hematopoietic microenvironment. PMID- 3007541 TI - Binding and second messengers of prostaglandins F2 alpha and E1 in primary cultures of rabbit endometrial cells. AB - Several factors and hormones are thought to play a role in the growth control of endometrial cells. We have shown that prostaglandin F2 alpha (PGF2 alpha) is a growth factor for primary cultures of rabbit endometrial cells grown in serum free, chemically defined medium and that prostaglandin E1 (PGE1) antagonizes the PGF2 alpha induction of growth (Orlicky et al., 1986). [3H]PGF2 alpha binds to whole cells in a time (optimal approximately 30 min)- and temperature-dependent (optimal 37 degrees C), disassociable (90% disassociable within 30 min), saturable (Kd1 = 4.9 X 10(-8) M, n1 = 1.2 X 10(5) molecules/cell; Kd2 = 2.6 X 10( 7) M, n2 = 3.0 X 10(5) molecules/cell), and specific manner. [3H]PGE1 binds in a time-dependent (optimal 25 min), disassociable (90% disassociable within 10 min), saturable (Kd = 6.4 X 10(-8) M, n = 1.2 X 10(5) molecules/cell), and specific manner. This specific binding of [3H]PGF2 alpha and [3H]PGE1 is down-regulatable by prior treatment of the cultures with unlabeled ligand, and up-regulatable by prior treatment of the cultures with indomethacin to inhibit endogenous PG synthesis. Proteolytic enzyme treatment for 2 min reduces the specific binding of PGF2 alpha by 75%. PGE1 stimulates intracellular cAMP synthesis and accumulation in a time (optimal 10 min)- and concentration (half-maximal stimulation at 10(-6) M)-dependent manner but has no effect on intracellular cGMP. PGF2 alpha has no effect on either intracellular cAMP or cGMP in this system. We describe here for the first time the analysis at a biochemical level of the interaction between two prostaglandins, antagonistic to each other in terms of growth regulation. PMID- 3007543 TI - DNA-mediated transfer of cAMP resistance in CHO cells. AB - Chinese hamster ovary (CHO) strain 10215 carries a dominant mutation which confers resistant to cAMP by virtue of an altered catalytic subunit of the cAMP dependent protein kinase (Evain et al., 1979). This mutation was transferred to wild-type CHO cells by DNA-mediated gene transfer. Based on the absence of cAMP growth inhibition, seven transformant colonies were isolated. One of these, 11586, was studied in detail. This transformant showed the same phenotype as the mutant, including resistance to the morphological changes and growth inhibitory effects of 1 mM 8-Br-cAMP, reduced total cAMP dependent protein kinase activity and lowered sensitivity of the kinase to cAMP activation. When the cAMP-dependent protein kinase was fractionated on a DEAE-cellulose column, the transformant was lacking in type II cAMP dependent protein activity, to the same degree as the mutant. The transformant and mutant, but not wild-type cells, also failed to phosphorylate a 52,000-dalton protein in a cAMP-dependent manner. These characteristics support the conclusion that the gene for the mutant cAMP dependent protein kinase has been transferred. The ability to transfer this gene by DNA-mediated transfer suggests that this methodology may be useful for the molecular isolation of the gene encoding the catalytic subunit of cAMP-dependent protein kinase. PMID- 3007542 TI - Evidence for a pool of non-recycling transferrin receptors in peripheral sheep reticulocytes. AB - Sheep reticulocytes from phlebotomized animals have a total transferrin binding potential that may exceed by an order of magnitude the surface binding capacity. Steady state uptake of transferrin at 37 degrees C is generally less than 50% of the total transferrin binding capacity. During long-term incubation of the reticulocytes, all transferrin binding ability is lost, the ability to internalize being lost most rapidly. The loss in ability to bind transferrin during long-term incubation is independent of the number of surface transferrin binding sites, since removal of surface receptors with pronase does not affect the rate of loss of the internal pool of receptors during long-term incubation. Moreover, after removing surface receptors with pronase, only a fraction of the original number of receptors is restored to the surface, despite the presence of a large pool of internal receptors. These data suggest that only a fraction of the internal pool of receptors is capable of recycling to the cell surface in sheep reticulocytes. PMID- 3007545 TI - Cleft hands: classification and treatment. AB - "Atypical" cleft hand should be classified under symbrachydactyly because its etiology and clinical and radiologic findings are quite different from those of typical cleft hand. The operative treatment also differs considerably. For severe forms of cleft hand, the procedure described by Snow and Littler is recommended. PMID- 3007544 TI - Congenital hand anomaly: etiology and associated malformations. AB - Congenital malformations of the hand may be present as part of syndromes. The recognition of these syndromes directly influences the surgical care of the hand anomaly. The natural history of the disorder may be predicted. The associated malformations may affect surgical timings and the indications for surgical correction. Various schema are presented here for evaluating the common abnormalities of the hand--radial club hand, ulnar defects, syndactyly, and polydactyly. PMID- 3007546 TI - Antibodies specific for the carboxy-terminal region of the major surface glycoprotein of simian rotavirus (SA11) and human rotavirus (Wa). AB - Antibodies specific for the major outer capsid protein (VP7) of the simian rotavirus SA11 were obtained by immunization of rabbits with a synthetic peptide, Ser-Ala-Ala-Phe-Tyr-Tyr-Arg-Val, corresponding to the eight carboxy-terminal amino acids of the viral protein predicted from the nucleotide sequence of the gene segment 9 of the SA11 genome. As the carboxy-terminal region of the VP7 of human rotavirus Wa has an identical sequence, cross-reactivity of the raised antibodies was observed with this strain. PMID- 3007547 TI - Glucose transport across the blood-brain barrier in normal human subjects and patients with cerebral tumours studied using [11C]3-O-methyl-D-glucose and positron emission tomography. AB - The kinetics of the regional cerebral uptake of [11C]3-O-methyl-D-glucose ([11C]MeG), a competitive inhibitor of D-glucose transport, have been studied in normal human subjects and patients with cerebral tumours using positron emission tomography (PET). Concomitant measurement of regional cerebral blood volume and blood flow enabled corrections for the contribution of intravascular tracer signal in PET scans to be carried out and regional unidirectional cerebral [11C]MeG extractions to be determined. A three-compartment model containing an arterial plasma and two cerebral compartments was required to produce satisfactory fits to experimental regional cerebral [11C]MeG uptake data. Under fasting, resting conditions, normal controls had mean unidirectional whole-brain, cortical, and white matter [11C]MeG extractions of 14, 13, and 17%, respectively. Mean values of k1 and k2, first-order rate constants describing forward and back transport, respectively, of tracer into the first cerebral compartment, were similar for [11C]MeG and [18F]2-fluoro-2-deoxy-D-glucose (18FDG), a second competitive inhibitor of D-glucose transport. k3, a rate constant describing FDG phosphorylation, was 20 times higher for cortical FDG uptake than the k3 fitted for [11C]MeG cortical uptake. Glioma [11C]MeG extractions ranged from normal levels of 12% to raised levels of 30%. Transport of [11C]MeG in and out of contralateral cortical tissue was significantly depressed in patients with gliomas. It is concluded that under fasting, resting conditions, regional cerebral glucose extraction remains relatively uniform throughout normal brain tissue. Gliomas, however, may have raised levels of glucose extraction. The nature of the second cerebral compartment required to describe [11C]MeG uptake is unclear, but it could represent either a useless phosphorylation dephosphorylation cycle or nonspecific tracer uptake by a cerebral subcompartment. PMID- 3007548 TI - [Absorbable material in surgery]. AB - Synthetic absorbable sutures, copolymers of sugars absorbed slowly by hydrolysis within defined periods of time and well tolerated by tissues, are resistant to infection and in biological fluids. They represent, after 15 years of use, more than 50% of suture material employed in surgery, and although limited for a long time to thread they are now available as trellis, clips, visceral prostheses and staples in all surgical disciplines. Future perspectives are discussed. PMID- 3007549 TI - The pathophysiology of acute pain. PMID- 3007550 TI - Post-stroke mood disorders. PMID- 3007551 TI - Two-step purification of cytochrome P-450 from rat liver microsomes using high performance liquid chromatography. AB - Cytochrome P-450 from rat liver microsomes treated with phenobarbital (PB) was separated into six fractions, as was cytochrome P-450 treated with 3 methylcholanthrene (MC), by high-performance liquid chromatography (HPLC) with an anion-exchange column. PB and MC induced three forms and one form of cytochrome P 450, respectively. The major forms induced by PB and by MC were further purified to apparent homogeneity based on sodium dodecyl(lauryl)sulphate--polyacrylamide gel electrophoresis by HPLC using a hydroxyapatite column. These new HPLC techniques are simple, rapid and useful for the purification of major forms of cytochrome P-450 from solubilized microsomes. PMID- 3007552 TI - Corticotropin-releasing hormone (CRH) stimulation in Nelson's syndrome: response of adrenocorticotropin secretion to pulse injection and continuous infusion of CRH. AB - Nelson's syndrome develops in 10-15% of patients with Cushing's disease who undergo bilateral adrenalectomy. Whether the pituitary tumors of Nelson's syndrome are autonomous or are regulated by hypothalamic signals or glucocorticoids is controversial. We, therefore, compared the plasma ACTH responses to synthetic ovine corticotropin-releasing hormone (CRH) in 11 patients with Nelson's syndrome, 1 patient with Cushing's disease who had had bilateral adrenalectomy and did not have Nelson's syndrome, 14 patients with Cushing's disease, and 27 normal subjects. The plasma ACTH response to CRH in 10 patients with Nelson's syndrome was markedly increased and prolonged compared to the responses of normal subjects or patients with Cushing's disease. In 4 patients with Nelson's syndrome, plasma ACTH and cortisol concentrations also were determined at frequent intervals for 10-24 h during continuous infusions of 0.15 M saline or CRH (1 microgram/kg X h). There was no desensitization of ACTH secretion during short term continuous infusion of CRH. Exogenous cortisol inhibited CRH-stimulated ACTH secretion. These findings suggest that the ACTH response to CRH of patients with ACTH-secreting tumors of Nelson's syndrome differs from the response of those who have the microadenomas of Cushing's disease in two ways: the magnitude is greater, and the response is prolonged. These differences can be explained by the greater size of the tumor and the reduced glucocorticoid feedback in adrenalectomized patients with Nelson's syndrome. PMID- 3007553 TI - Beta-endorphin attenuates the serum cortisol response to exogenous adrenocorticotropin. AB - Controversy surrounds the issue of whether beta-endorphin affects adrenal steroidogenesis. Recent work has both supported and refuted the claim that beta endorphin stimulates a rise in serum aldosterone. We investigated the role of beta-endorphin in adrenal steroidogenesis by examining its potential modulation of the response of serum cortisol to exogenous ACTH (Cosyntropin). Four of five normal men received: 1) synthetic beta-endorphin (1 microgram/kg X min) for 30 min, followed by a bolus dose of 0.2 micrograms ACTH; 2) beta-endorphin (100 micrograms, iv), followed by 0.2 micrograms ACTH iv; 3) 0.2 micrograms ACTH iv; and 4) beta-endorphin (100 micrograms iv) alone. The integrated cortisol response to exogenous ACTH, calculated as the area under the cortisol response curve, was significantly less when the ACTH infusion was preceded by the 30-min beta endorphin infusion than when administered alone [163 +/- 50 (SE) microgram/dl X min vs. 282 +/- 51 micrograms/dl X min, respectively; P less than 0.01]. By contrast, there was no difference between the integrated cortisol response to exogenous ACTH alone and exogenous ACTH after the bolus dose of beta-endorphin (282 +/- 51 vs. 293 +/- 39 micrograms/dl X min, respectively). Beta-Endorphin (30 min infusion or 100-micrograms bolus dose alone) caused no change in serum aldosterone, dehydroepiandrosterone, or PRA. Serum PRL levels, however, were raised significantly (P less than 0.05) by the 30-min infusion of beta-endorphin. The infusion and bolus doses of beta-endorphin raised plasma beta-endorphin levels to over 100,000 pg/ml and 5,000 pg/ml, respectively. We conclude that very high plasma levels of beta-endorphin may influence the response of cortisol to ACTH through a direct effect on the adrenal cortex. However, even in disease states such as Addison's and Nelson's diseases, such levels of plasma beta endorphin are not known to be achieved. PMID- 3007554 TI - Pulsatile administration of human corticotropin-releasing hormone in patients with secondary adrenal insufficiency: restoration of the normal cortisol secretory pattern. AB - Human corticotropin-releasing hormone (hCRH) was administered in a pulsatile fashion to eight patients with secondary adrenal insufficiency. These patients were selected on the basis of a normal or exaggerated plasma ACTH response to exogenous ovine CRH, suggesting sparing of the corticotrophs. A continuous 48-h iv infusion of ACTH to restore the adrenal glands to an ACTH-responsive state preceded hCRH administration. Eight 1 microgram/kg bolus injections of hCRH were administered in a 24-h period. The time intervals between hCRH injections were selected to resemble the frequency of spontaneously occurring secretory episodes of plasma ACTH and cortisol. Four of the patients underwent a second study, of identical design, in which normal saline injections were administered instead of hCRH. Pulsatile hCRH treatment resulted in a secretory pattern of ACTH and cortisol similar to that in normal subjects. ACTH and cortisol levels during saline administration were low and had no circadian variation. These findings indicate that exogenous CRH is able to restore normal basal ACTH and cortisol secretory patterns when given in an appropriate manner. It is possible that the pulsatile administration of hCRH may prove to be a more physiological technique for restoring adrenal function of patients with corticotroph-sparing secondary adrenal insufficiency and may avoid some of the complications of glucocorticoid administration. PMID- 3007555 TI - Adrenergic receptors in human liver plasma membranes: predominance of beta 2- and alpha 1-receptor subtypes. AB - Adrenergic receptors in human liver plasma membranes were characterized by radioligand binding assays. The binding of [3H]dihydroalprenolol [( 3H]DHA) to partially purified membranes was rapid, of high affinity, saturable, and stereospecific. The binding of [125I]iodocyanopindolol to the same membranes was also saturable and stereospecific, but extremely slow, and at 37 C required about 6 h for equilibration. The maximum number of binding sites from six livers determined with these two beta-receptor ligands was 36-83 fmol/mg protein. Catecholaminergic agonists competed for these binding sites in the order typical for beta 2-adrenergic receptors. IPS 339 [(tertiarybutylamino-3-ol-2 propyl)oximino-9-fluorene hydrochloride], a beta 2-selective antagonist, was at least 3 orders of magnitude more potent in inhibiting the binding of [3H]DHA than the beta 1-antagonist, atenolol. Computer-aided analysis of the competition curves as well as Hofstee transformations of the binding data indicated the predominance of the beta 2-subtype. The GTP analog guanyl-5'-yl-imidodiphosphate, decreased the binding affinity of the agonist, l-isoproterenol, indicating the modulation of agonist-promoted coupling of the receptors to guanine nucleotide regulatory proteins. The maximum number of binding sites for the binding of [3H]prazosin and [3H]dihydroergocryptine were the same (60-70 fmol/mg protein), indicating that the majority of the alpha-receptors are of the alpha 1-subtype. Competition experiments with prazosin and yohimbine confirmed the predominance of the alpha 1-receptor subtype, although the presence of alpha 2-receptors cannot be completely ruled out. These results indicate that adrenergic receptors in human liver plasma membranes are predominantly of the beta 2- and alpha 1 subtypes. PMID- 3007556 TI - Characterization of corpora lutea in monkeys after superovulation with human menopausal gonadotropin or follicle-stimulating hormone. AB - The objective of this study was to characterize the corpora lutea (CL) of superovulatory follicles, which form in nonhuman primates after treatment with exogenous gonadotropins. Adult female rhesus monkeys (n = 15) with amenorrhea or irregular menstrual cycles received im injections of either human menopausal gonadotropin [hMG; equivalent amounts (37.5 IU) of hFSH and hLH] or human FSH (37.5 IU) twice daily for 6 or 9 days. One day later, hCG (1000 IU) was administered to induce ovulation. Serum estradiol levels rose rapidly in hMG treated monkeys. In contrast, estradiol levels did not rise in FSH-treated animals for 3-4 days, but ultimately reached concentrations comparable to or greater than those in hMG-treated monkeys. Serum progesterone levels were low in all groups before hCG injection, but rose thereafter. Peak progesterone levels were greater (P less than 0.05) in 9- vs. 6-day treatment groups. Serum concentrations of hCG peaked within 24 h of injection and declined to undetectable levels 6-7 days later. The mass of luteinized tissue removed 7 days after hCG injection was markedly (P less than 0.01) increased in hMG- and FSH treated monkeys compared to that of the active CL of the natural menstrual cycle (n = 6). However, the protein content of luteal tissue from FSH-treated monkeys was less (P less than 0.05) than that in hMG-treated groups or in the CL of the natural cycle. Luteal particulate fractions from all treatment groups had [125I]human LH binding sites, with the Kd for LH interaction comparable to that in the CL of the natural cycle. However, the LH-binding capacity in hMG-treated groups was less (P less than 0.05) than that in the CL of the cycle, when normalized per mg tissue wt or protein. Notably, the binding capacity in FSH treated groups was comparable to that in the CL cycle when expressed per mg protein. Nevertheless, only after 6 days (not 9 days) of FSH treatment or 9 days (not 6 days) of hMG treatment did tissues have a LH-sensitive (activation constant) or LH-responsive adenylate cyclase comparable to that in the CL of the cycle. Thus, properties of the primate CL after superovulation varied markedly with the type and length of gonadotropin treatment employed for follicular stimulation. The findings support the concept that gonadotropin-regulated events in the developing follicle(s) are important determinants of the subsequent character of the primate CL. PMID- 3007558 TI - Epidermal growth factor inhibits the proliferation of a human endometrial carcinoma cell line. AB - Specific epidermal growth factor (EGF) receptors were measured in RL95-2 human endometrial carcinoma cells. At 37 C, binding of 125I-labeled EGF was associated with marked ligand internalization. Maximal cell surface binding occurred within 10 min. Bound [125I]EGF was partially degraded to low mol wt products, and this degradation was blocked by the lysosomotropic compound ammonium chloride. At 4 C, maximal cell surface binding occurred at 3 h. Scatchard analysis of data obtained after 3 h at 4 C revealed a single order of binding sites with a Kd of 1.8 nM and approximately 150,000 surface receptors/cell. EGF, at a concentration of 0.83 nM, inhibited the proliferation of RL95-2 cells. Inhibition was reversible and was associated with morphological changes leading to a fusiform appearance of the growth-arrested cells. These findings indicate that RL95-2 human endometrial carcinoma cells bind, internalize, and degrade EGF in a manner similar to that described for other target cells for EGF action. The ability of EGF to inhibit the growth of RL95-2 cells supports the hypothesis that this type of inhibition is dependent on postreceptor events, rather than on the presence of an overabundance of EGF receptors. PMID- 3007557 TI - Adrenal androgen hyperresponsiveness to adrenocorticotropin in women with acne and/or hirsutism: adrenal enzyme defects and exaggerated adrenarche. AB - To determine the adrenal contribution to elevated plasma androgens in 31 young hyperandrogenemic women with acne and/or hirsutism, we compared their responses to ACTH with those of 14 normal women. Each subject was given a low dose (10 micrograms/m2) of synthetic ACTH-(1-24) (Cortrosyn) after administration of 1.5 mg dexamethasone the night before the test. Thirty and 60 min responses of plasma 17 alpha-hydroxypregnenolone (17-Preg), 17 alpha-hydroxyprogesterone, (17-prog), dehydroepiandrosterone (DHEA), androstenedione, 11-deoxycortisol, and cortisol were measured. Eighteen (58%) patients had increased responses of at least one 17 ketosteroid or adrenal androgen precursor. All patients had cortisol responses within the range of those of the 14 normal subjects. Nine patients (29%) had evidence of steroid biosynthetic enzyme deficiencies, either mild congenital adrenal hyperplasia or the heterozygote state; after ACTH, 4 of these patients had elevated 17-prog in the range of values in heterozygote carriers of 21 hydroxylase deficiency, 2 had elevated levels of 11-deoxycortisol compatible with 11 beta-hydroxylase deficiency, and 3 had elevated levels of 17-Preg and DHEA, suggestive of 3 beta-hydroxysteroid dehydrogenase deficiency. Another 9 subjects (29%) had 17-ketosteroid (DHEA and/or androstenedione) hyperresponsiveness to ACTH with associated elevated 17-Preg responses. As a group, their patterns suggested relatively deficient 3 beta-hydroxysteroid dehydrogenase and relatively hyperactive C lyase without impairment of cortisol secretion. This pattern resembles exaggerated adrenarche, and we postulate that these 9 patients have hyperplasia of the zona reticularis. Neither basal levels of plasma androgens (free testosterone and DHEA sulfate) nor menstrual history predicted which patients would have abnormal ACTH responses. Although 5 of 11 (45%) patients with acne alone had abnormal responses to ACTH, 10 of 14 patients with acne and hirsutism (71%) had abnormal responses to ACTH. We conclude that an adrenal contribution is found in about half of hyperandrogenemic women with acne and/or hirsutism. This adrenal androgen hyperresponsiveness is heterogeneous. Some patients may have mild forms of congenital adrenal hyperplasia. However, functional androgenic hyperresponsiveness to ACTH, which resembles an exaggeration of adrenarche, is the most common abnormality found. Such findings may provide an explanation for the clinical observation of exacerbations of acne with stress. PMID- 3007559 TI - Pituitary stimulation by combined administration of four hypothalamic releasing hormones in normal men and patients. AB - Ten normal young men (22-28 yr of age), within 10% of their ideal body weight, were given the four releasing hormones (TRH, 200 micrograms; GnRH, 100 micrograms; ovine corticotropin-releasing hormone, 50 micrograms; GH-releasing hormone, 80 micrograms) iv on separate days and then in combination on the same day. Plasma TSH, PRL, FSH, LH, cortisol, ACTH, and GH were measured by RIA in samples collected from 20 min before to 120 min after injection. There were no significant differences in responses to the separate and combined tests for FSH, LH, cortisol, ACTH, and GH. The plasma TSH (0.001 less than P less than 0.01) and PRL (P less than 0.001) responses were significantly higher after the combined test. The tolerance was identical to that of TRH alone. In eight patients studied after pituitary surgery, combined administration provided results comparable to those obtained after separate administration of TRH, GnRH, and insulin. PMID- 3007560 TI - Evidence for the secretion of an antimineralocorticoid in congenital adrenal hyperplasia. AB - Plasma extracts from patients with congenital adrenal hyperplasia were found to contain substances that competed with aldosterone for mineralocorticoid receptor binding sites in a rat kidney cytosol system. In normal subjects and patients with other disorders, the mineralocorticoid receptor-binding activity in such extracts could be entirely accounted for by the sum of the contributions of the steroids known to bind to the mineralocorticoid receptor. The secretion of these binding substances in patients with the C-21 hydroxylation defect was ACTH dependent. While these substances could be either mineralocorticoid agonists or antagonists, the latter is more likely. Production of mineralocorticoid antagonists would account for the compensatory hyperaldosteronism that occurs in the simple virilizing form, in which there is minimal impairment of aldosterone secretory reserve, and for the tendency to Addisonian crisis in patients with the salt-losing form, who have a more severe defect in aldosterone biosynthesis. PMID- 3007561 TI - Alpha-human atrial natriuretic polypeptide inhibits steroidogenesis in cultured human adrenal cells. AB - The ability of synthetic alpha-human atrial natriuretic polypeptide-(1-28) (alpha hANP) to alter steroidogenesis by human adrenal glands was investigated in primary human adrenal cell cultures. alphahANP (10(-9)-10(-7) M) inhibited basal and ACTH (10(-8) M)-stimulated aldosterone, cortisol, and dehydroepiandrosterone (DHEA) secretion in a dose-dependent manner. alpha hANP inhibited aldosterone (IC50, 1.3 X 10(-8) M) and cortisol (IC50, 0.7 X 10(-8) M) secretion more potently than it did DHEA (IC50, 7.5 X 10(-8) M) secretion. ACTH dose-dependent (10(-10)-10(-8) M) increases in aldosterone, cortisol, and DHEA secretion were significantly inhibited by alpha hANP (10(-8) M). In addition, alpha hANP enhanced the accumulation of intracellular cGMP in a dose-dependent manner. As aldosterone, cortisol, and DHEA secretion from cultured human adrenal cells was inhibited by alpha hANP, the site of inhibition of steroidogenesis by alpha hANP is probably localized in the early pathway of steroidogenesis in human adrenal cells. The results also suggest a link between inhibitory effects of alpha hANP and accumulation of intracellular cGMP. PMID- 3007562 TI - Gene conversion in salt-losing congenital adrenal hyperplasia with absent complement C4B protein. AB - Two of four siblings expressed the salt-losing form of congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH) and had identical human lymphocyte antigen (HLA) and complement C4 (fourth component of complement) types (HLA-A3,C4,B35,C4A3,C4BQO,DR1/A2,C-,B18,C4A3, C4BQO,DR6). The father and one unaffected sibling were heterozygous carriers of CAH, as determined by a 30-min iv ACTH stimulation test and HLA typing. In addition, the iv ACTH stimulation test revealed that the mother and the other unaffected sibling also carried an allele for an attenuated form of CAH. Restriction endonuclease digests of genomic DNA obtained from members of this family and from normal unrelated subjects were hybridized with cDNA probes encoding human 21-hydroxylase and C4. With the 21 hydroxylase probe, Southern blots prepared from control DNA samples revealed two major restriction fragments in each of four restriction endonuclease digests; TaqI produced major bands at 3.7 and 3.2 kilobases (kb), KpnI at 4.0 and 2.9 kb, EcoRI at 18 and 13 kb, and BglII at 15 and 12.5 kb. Southern blots prepared from DNA of the two patients lacked the 3.7-kb TaqI and 2.9-kb KpnI fragments, but had increased hybridization intensity (relative to control DNA samples) in the 3.2-kb TaqI and 4.0-kb KpnI fragments. By contrast, blots with EcoRI or BglII had two large hybridization fragments not different from control DNA samples. These data indicate the presence of two different 21-hydroxylase genes. Additional mapping studies revealed that the two genes had the restriction pattern of the inactive 21-hydroxylase gene. When genomic DNA that had been isolated from all members of this family and from normal subjects was hybridized with the human C4 cDNA probe, the restriction fragment hybridization patterns for all four endonuclease digests were similar in the two groups. Hence, our results suggest that the 21 hydroxylase deficiency of our patients is due to conversion of the active 21 hydroxylase gene to the inactive gene. This gene conversion was associated with absence of functional C4B protein, without any detectable alterations in the restriction fragment pattern of the C4 genes. PMID- 3007563 TI - Altered immunity in hemophilia correlates with the presence of antibody to human T-cell lymphotropic virus type III (HTLV-III). AB - Twenty-nine heterosexual patients with hemophilia were investigated with histories, physical examinations, laboratory evaluations of immune function, delayed hypersensitivity skin tests, and assays for antibody to human T-cell lymphotropic virus type III (HTLV-III). Sixteen patients were HTLV-III antibody positive and 13 were HTLV-III antibody negative. No patient had the acquired immune deficiency syndrome (AIDS). Patients who had antibody to HTLV-III had received significantly more units and lots of factor concentrates in the preceding 5 years than those who did not have antibody. HTLV-III antibody positive patients had significantly fewer total T cells (Leu-1 positive) and significantly fewer helper T cells (Leu-3 positive) than HTLV-III negative patients. Antibody-positive patients also had increased amounts of IgG and decreased thymidine incorporation in response to concanavalin A in vitro. There were no differences in in vitro lymphocyte responses to phytohemagglutinin (PHA), pokeweed mitogen, Candida, tetanus, or purified protein derivative (PPD), no significant impairments of gamma interferon or interleukin-2 (IL-2) production, and no anergy. Ten patients with antibody to HTLV-III had immunologic studies repeated 1 year after the original evaluation. A significant increase was seen in suppressor (Leu-2-positive) T cells but not in total T-cell or helper T-cell numbers, helper/suppressor ratios, or T-cell functional assays. We conclude that the immune abnormalities in hemophiliacs are the result of contact with HTLV-III but that these abnormalities may remain stable over prolonged periods. PMID- 3007564 TI - Inhibition of interleukin-2-induced T-cell proliferation by sera from patients with the acquired immune deficiency syndrome. AB - Sera from 22 patients with either lymphadenopathy syndrome (LAS), acquired immune deficiency syndrome (AIDS)-related complex (ARC), or acquired immune deficiency syndrome were examined for their effect on the interleukin-2 (IL-2)-induced proliferative response of an IL-2-dependent cytotoxic T-cell line, CTL-20. All of the patient sera included in this study were positive for the presence of antibodies against human T-cell lymphotropic virus type III (HTLV-III) as determined by an HTLV-III-specific enzyme-linked immunosorbent assay (ELISA). Eighteen of the 22 patient sera examined (81.8%) exhibited at least a modest suppressive effect on the proliferative response of CTL-20 cells. The inhibitory effect was dose-dependent and varied in intensity for each individual serum. In many cases, the magnitude of suppression was absolute in that it totally abrogated IL-2-induced DNA synthesis. Normal human serum (NHS) exerted no suppressive influence on the IL-2-induced proliferative response of identical control cultures. This same panel of 22 patient sera exhibited no significant inhibitory effects on the levels of protein synthesis in cultures of a non-IL-2 dependent human T-cell line, CCRF-HSB-2, indicating that the suppressive effect was not mediated by nonspecific serum cytotoxicity. The inhibitory effect of patient sera in the IL-2-dependent target cell assay correlated with the ability of these same sera to suppress the mitogen-induced proliferative response of normal human peripheral blood lymphocytes (PBL). These observations are particularly striking in view of the recognized defects of IL-2-dependent effector T-cell functions in AIDS. PMID- 3007565 TI - Adsorption, purification, and growth characteristics of hepatitis A virus strain HAS-15 propagated in fetal rhesus monkey kidney cells. AB - A human fecal isolate of hepatitis A virus strain HAS-15 was adapted to rapid growth in FRhK-4 cells by more than 20 7-day passages. A cell culture-derived inoculum of strain HAS-15 was used at a multiplicity of infection of 80 radioimmunofocus-forming units per cell, and a one-step growth curve was determined. Both intracellular production and supernatant release of infectious virions were evaluated. Detection of virus release into the medium directly corresponded to intracellular production of infectious virions. A classical eclipse period was not observed during the growth curve determinations; however, detectable infectious virion production was absent for approximately 20 h after infection. This 20-h period was immediately followed by a 4-day logarithmic phase of virus production. A maximum intracellular virus titer of 10(9) radioimmunofocus-forming units per ml was achieved, and this level remained essentially constant for up to 14 days after infection. The infectious virus and viral antigen produced during the growth cycle were ascertained by a radioimmunofocus assay and by a radioimmunoassay, respectively. Cell culture supernatants were negative for viral antigen as determined by the radioimmunoassay, even though as many as 10(8) hepatitis A virus radioimmunofocus forming units per ml were found. An adsorption study was also performed with strain HAS-15 by using FRhK-4 cells. More than 99.9% of the infectious virus was adsorbed at 25 degrees C in less than 20 min. PMID- 3007566 TI - Three adenovirus type 8 genome types defined by restriction enzyme analysis: prototype stability in geographically separated populations. AB - Adenovirus type 8 strains were collected over a 19-year period from eye specimens from patients with keratoconjunctivitis. These strains were divided by restriction enzyme analysis with the endonucleases SalI, HindIII, SacI, KpnI, and SmaI into three genotypic subgroups. The prototype strain (Trim) was found throughout the United States from 1966 through 1985 and also in Taiwan and Greece in the early 1980s. Genotype 8C was identified in an arc from Maryland to Missouri to Alabama from 1971 through 1974. Genotype 8D was found only in an epidemic of eye disease among Vietnamese refugees being resettled in northwest Florida in 1975. Genotypes 8C and D were distinct from genotypes 8A and B described from Japan in 1975 through 1981. Hemagglutination tests with a battery of avian and mammalian erythrocytes did not distinguish between the genotypic subgroups. Similarly, the genotypes could not be differentiated by hemagglutination inhibition or serum neutralization tests with reference prototype antisera. The long-term prevalence of the Trim strain suggests that adenovirus type 8 has greater genetic stability than the other adenovirus types studied to date. PMID- 3007567 TI - Clinical and epidemiological features of acute infantile gastroenteritis associated with human rotavirus subgroups 1 and 2. AB - During a prospective 1-year study rotavirus isolates from 169 children with gastroenteritis were investigated by polyacrylamide gel electrophoresis. A total of 118 (70%) of the strains analyzed contained sufficient viral nucleic acid to give visible electrophoretic patterns; 36% were identified as strains belonging to subgroup 1 (short patterns), and 64% were identified as strains belonging to subgroup 2 (long patterns). The two subgroups cocirculated at equal frequencies during the first 7 months of the year, after which subgroup 1 rotavirus completely disappeared. Subgroup 2 rotavirus occurred throughout the year. No significant differences between the subgroups in relation to age or sex distribution were observed. Fever and temperatures exceeding 39 degrees C were significantly more frequent in children who shed rotavirus subgroup 1. Diarrhea and vomiting occurred at similar rates in both groups of patients, but were more pronounced in children who shed rotavirus subgroup 2. One of three dominant electropherotypic variants of subgroup 2 rotavirus was found to be associated with more intense symptoms, higher rates of hospitalization, and a significantly higher frequency of respiratory symptoms; the clinical picture may indicate that this rotavirus electropherotype has higher virulence. PMID- 3007568 TI - Latex agglutination test for detecting feline panleukopenia virus, canine parvovirus, and parvoviruses of fur animals. AB - A latex agglutination (LA) test for the detection of parvoviruses of fur animals, cats, and dogs was developed, and its sensitivity and specificity were compared with those of hemagglutination (HA) and the enzyme-linked immunosorbent assay (ELISA). Tissue culture isolation was used to confirm the specificity results. Fecal samples from various sources were tested, including specimens from raccoon dogs and mink which were experimentally infected with parvoviruses by oral exposure. LA compared favorably with the other tests. The ELISA was the most sensitive. When it was considered as a reference test, the corresponding sensitivities for HA and LA were 96 and 91%, respectively. The specificities were 93% for the ELISA, 95% for the HA test, and 92% for the LA test. LA seems to be a suitable technique for screening animals in the field and in laboratories in which sophisticated techniques are not available. PMID- 3007569 TI - Dot-enzyme immunoassay for visual detection of antibodies to pseudorabies virus in swine serum. AB - A modified solid-phase enzyme immunoassay (EIA) is described for the visual detection of anti-pseudorabies virus (anti-PRV) antibody in porcine serum. Dots of PRV antigens were adsorbed to nitrocellulose paper (hence the name dot-EIA), and the remaining nonspecifically reactive sites were blocked with bovine serum albumin or skim milk powder. After immersion in test serum, bound antibodies were reacted with a peroxidase-conjugated anti-porcine immunoglobulin G (H & L). Positive reactions were easily visualized as brown dots after enzyme degradation of a substrate containing hydrogen peroxide and diaminobenzidine. The dot-EIA was comparable to the serum neutralization test and the standard microtiter EIA in its ability to detect antibody in the sera of pigs 9 days after experimental infection and 12 days after contact with infected pigs. The sensitivity and specificity of the dot-EIA relative to the serum neutralization test and the standard EIA were determined from the testing of 856 field sera from the United Kingdom, the United States, and Canada. In all comparisons, both the relative sensitivity and specificity of the dot-EIA were in the order of 98 to 99%. The dot EIA appears to have potential application as a rapid and economical field test in the diagnosis of PRV infection. PMID- 3007570 TI - Elimination of nonspecific cytomegalovirus immunoglobulin M activities in the enzyme-linked immunosorbent assay by using anti-human immunoglobulin G. AB - Direct enzyme-linked immunosorbent assay methods offer several advantages in assessing past or recent exposure to cytomegalovirus (CMV) infection, but there persist many pitfalls in the use of these methods for determining specific immunoglobulin M (IgM). The efficiency of absorption of sera by IgG-coated latex beads, aggregated human IgG, or Staphylococcus aureus, i.e., for removing nonspecific CMV IgM activities, was evaluated in comparison with the effect of an anti-human IgG hyperimmune serum. Large routine series comprising serum samples from patients of various clinical groups and healthy individuals were examined. The CMV IgM-positive samples were at first treated with latex or aggregated IgG, but these absorptions left too many CMV IgM-positive individuals. S. aureus increased the nonspecific activity of some sera and, in other cases, removed or impaired specific IgM activities. The anti-IgG treatment caused the disappearance of nonspecific CMV IgM activities that had resisted the other treatments, whereas specific activities remained intact. Utilizing this method, only 1.03% of the routine series patients remained CMV IgM positive by the enzyme-linked immunosorbent assay, a figure in good agreement with a mean probability of CMV antibody acquisition of 0.33% for the population living in Belgium. On the other hand, in a series of patients who were investigated for serological response to several viruses, eight individuals displayed multiple IgM activities after anti IgG treatment. In these cases, most IgM activities were found in patients who had IgG toward the related antigen for a long time before transient IgM was detected. This result implies that to assess a diagnosis of primary infection, it is necessary to examine serial specimens for IgG acquisition accompanying specific IgM. PMID- 3007571 TI - Molecular epidemiology of non-O1 Vibrio cholerae and Vibrio mimicus in the U.S. Gulf Coast region. AB - Ten toxigenic Vibrio cholerae non-O1 and V. mimicus strains isolated from clinical and environmental sources in the U.S. Gulf Coast region were examined for genetic relatedness. Restriction digest patterns of chromosomal DNA and Southern blot analysis with a cholera toxin gene probe revealed that the strains exhibited greater genetic divergence than the highly conserved V. cholerae O1 strains isolated from clinical and sewage samples in this region. PMID- 3007573 TI - Peripheral neuropathy and monoclonal IgM with antibody activity against peripheral nerve myelin; effect of plasma exchange. AB - Serum IgM antibodies directed against peripheral nerve myelin were demonstrated using enzyme-linked immunosorbent assay, mixed haemagglutination and indirect immunofluorescence in 3 patients with chronic polyneuropathy and monoclonal serum IgM. Isoelectric focusing followed by antigen immunofixation and autoradiography showed that the antimyelin antibodies co-migrated with the monoclonal IgM. Plasma exchange alone, without chemotherapy, proved beneficial in 2 patients. In one patient, plasma exchange was discontinued because of low IgG levels. Serum IgM and antimyelin antibodies decreased during plasma exchange and no increase beyond initial levels was noted after cessation of treatment. PMID- 3007572 TI - Molecular epidemiology of human rotavirus infection in children in Hong Kong. AB - Rotavirus was identified in 256 (28.5%) of 899 hospitalized children with diarrhea during a 12-month period in Hong Kong. Fourteen electropherotypes were identified, and the appearance of each occurred in a sequential manner during the study period. One patient was shown to be mixedly infected with two prevalent electropherotypes. Sequential stool specimens from another case showed the appearance of an extra RNA band during the course of the diarrheal episode. PMID- 3007574 TI - A simple 'free 3H-ligand' assay for rapid screening of hybridoma monoclonal antibodies against neurotransmitter analogs and anti-idiotype antibodies. AB - Effective, rapid screening of hybridoma supernatants for monoclonal antibodies against the dopaminergic antagonists pimozide and haloperidol, and the serotonergic antagonist ketanserin was performed using a 'free 3H-ligand' assay. Anti-mouse Ig-coated microtiter plates were incubated with hybridoma supernatants prior to incubation with excess 3H-ligand. After removal of free 3H-ligand, bound 3H-ligand was eluted with acid for liquid scintillation counting. With minor modification, the assay can be used to screen hybridomas for anti-anti-ligand (anti-idiotypic) antibodies. PMID- 3007575 TI - Defective postbinding lysis underlies the impaired natural killer activity in factor VIII-treated, human T lymphotropic virus type III seropositive hemophiliacs. AB - We investigated the diminished natural killer (NK) activity in human T lymphotropic virus type III (HTLV-III) seropositive hemophiliacs. Despite normal percentages of NK cells, lymphocytes from five hemophiliacs showed impaired NK activity against K-562 tumor cells in 4-h chromium release microcytotoxicity assays. For example, at an effector-to-target cell ratio of 10:1, cells from patients caused 21.7 +/- 2.5% lysis of tumor targets compared with 47.9 +/- 5.1% lysis by cells from controls (mean +/- SEM, P less than 0.005). Cells from patients were as cytotoxic in 18 h as were cells from controls in 4 h. Binding to tumor targets was not impaired since 11.0 +/- 1.5% of cells from patients and 11.1 +/- 1.3% of cells from controls bound to K-562 cells. Patients' binding cells, however, showed defective killing of attached tumor cells at all time points tested from 0 to 18 h. At 4 h, for example, patients' cells had lysed 10.9 +/- 2.1% of attached tumor cells compared with 26.3 +/- 3.3% lysis by controls' cells (P less than 0.005). The percentage of lymphocytes which were active NK cells (i.e., cells that bound and lysed a tumor cell) was always lower for patients than for controls (1.17 +/- 0.25% vs. 2.82 +/- 0.33%, P less than 0.005). Two methods for estimating recycling of effector cells against multiple target cells demonstrated that active NK cells from patients could recycle as well as those from controls (approximately 3-4 times in 4 h). Mixing experiments showed no evidence for cellular suppression of NK activity. The lytic function of NK cells from HTLV-III seropositive hemophiliacs is thus heterogeneous. This is characterized by a defect in post-binding lysis, with relative sparing of binding capability and recycling capacity. PMID- 3007576 TI - Stimulation of gonadal steroid synthesis by chronic excess of adrenocorticotropin in patients with adrenocortical insufficiency. AB - Analysis of 24-h urinary steroid excretion was performed by capillary gas chromatography in six patients (five men, one woman) with adrenocortical insufficiency. Ten healthy subjects (five men, five women) served as controls. A complete absence of all 21-hydroxylated steroid metabolites was seen in patients with adrenocortical insufficiency, whereas the excretion of several steroids lacking hydroxylation in the 21-position (pregnenolone, pregnenetriol, and 11 ketoandrosterone) was markedly increased. In addition, the presence of 11 beta hydroxyandrosterone was confirmed by mass-spectrometry in the urine of three patients. This pattern of steroid excretion was unchanged in patients with adrenocortical insufficiency, both after stimulation by 1-24 adrenocorticotropin (ACTH) and after short-term (3-d) suppression with dexamethasone. We conclude that patients with adrenocortical insufficiency present a pattern of steroid excretion characterized by the absence of 21-hydroxylated metabolites. In the absence of functional adrenocortical tissue, long-term pathologically elevated concentrations of ACTH apparently stimulate early steps of steroid synthesis, most likely in the gonads. In addition, the presence of 11-hydroxylated steroid metabolites (11-ketoandrosterone, 11 beta-hydroxyandrosterone) in the urine of patients with adrenocortical insufficiency demonstrates that chronic ACTH excess in this disorder may induce some activity of 11 beta-hydroxylase, an enzyme not found in the gonads under physiological conditions. PMID- 3007580 TI - The mode of action of cardiac glycosides. AB - Throughout the 200 years during which the cardiac glycosides have been in therapeutic use, they have challenged the understanding of clinician and scientist alike. For the clinician the task has been to define the circumstances in which drugs of this class are indicated and to tread the narrow line between pharmacological activity and serious toxicity; neither problem has been entirely solved. For the scientific investigator, a curious paradox has emerged: whereas early workers sought to identify the precise site of action of digitalis, recent research has revealed that cardiac glycosides specifically inhibit a ubiquitous membrane transport process and this makes the apparent tissue specificity of these drugs the more surprising. The gap between the empirical knowledge of clinical experience and the scientific insights derived from laboratory biochemistry and electrophysiology has not yet been closed. PMID- 3007577 TI - Mechanism of action of glucocorticosteroids. Inhibition of T cell proliferation and interleukin 2 production by hydrocortisone is reversed by leukotriene B4. AB - The mechanism whereby glucocorticosteroids are immunosuppressive is unknown. One potential mechanism of action of these compounds is inhibition of arachidonic acid metabolism. We found that the inhibition of lymphocyte proliferation by hydrocortisone or dexamethasone was mimicked by nonspecific lipoxygenase inhibitors and also by a specific 5-lipoxygenase inhibitor, but not by a specific cyclooxygenase inhibitor. Mitogen-stimulated cultures of T cells produce approximately 5 X 10(-9) M leukotriene B4 (LTB4) in 24 h. This production of LTB4 is completely inhibited by concentrations of hydrocortisone or lipoxygenase inhibitors that inhibit mitogen-induced [3H]thymidine incorporation. The inhibition of lymphocyte proliferation by either hydrocortisone or by the 5 lipoxygenase inhibitor was totally reversed by LTB4 but not by leukotriene C4 or leukotriene D4. LTB4 had no effect on the inhibition of lymphocyte proliferation by noncorticosteroids such as prostaglandin E2, histamine, or gamma-interferon. The inhibition of interleukin 2 (IL-2) production by hydrocortisone or dexamethasone was also completely reversed by exogenous LTB4. LTB4 alone did not cause IL-2 production or cell proliferation when added to resting lymphocytes. Thus, endogenous LTB4 production appears to be necessary but not sufficient for phytohemagglutinin-induced IL-2 production and lymphocyte proliferation. Glucocorticosteroids inhibit IL-2 production and lymphocyte proliferation by inhibiting endogenous LTB4 production. PMID- 3007578 TI - von Willebrand factor interaction with the glycoprotein IIb/IIa complex. Its role in platelet function as demonstrated in patients with congenital afibrinogenemia. AB - We have studied three afibrinogenemic patients, who had only trace amounts of plasma and platelet fibrinogen as measured by radioimmunoassay, and demonstrate here that the residual aggregation observed in their platelet-rich plasma is dependent upon von Willebrand factor (vWF) binding to the platelet membrane glycoprotein (GP)IIb/IIIa complex. The abnormality of aggregation was more pronounced when ADP, rather than thrombin, collagen, or the combination of ADP plus adrenaline was used to stimulate platelets. With all stimuli, nevertheless, the platelet response was completely inhibited by a monoclonal antibody (LJP5) that is known to block vWF, but not fibrinogen binding to GPIIb/IIIa. Addition of purified vWF to the afibrinogenemic plasma resulted in marked increase in the rate and extent of aggregation, particularly when platelets were stimulated with ADP. This response was also completely blocked by LJP5. Addition of fibrinogen, however, restored normal aggregation even in the presence of LJP5, a finding consistent with the knowledge that antibody LJP5 has no effect on platelet aggregation mediated by fibrinogen binding to GPIIb/IIIa. Two patients gave their informed consent to receiving infusion of 1-desamino-8-D-arginine vasopressin (DDAVP), a vasopressin analogue known to raise the vWF levels in plasma by two- to fourfold. The bleeding time, measured before and 45 min after infusion, shortened from greater than 24 min to 12 min and 50 s in one patient and from 16 min to 9 min and 30 s in the other. Concurrently, the rate and extent of ADP induced platelet aggregation improved after DDAVP infusion. The pattern, however, reversed to baseline levels within 4 h. The concentration of plasma vWF increased after DDAVP infusion, but that of fibrinogen remained at trace levels. We conclude that vWF interaction with GPIIb/IIIa mediates platelet-platelet interaction and may play a role in primary hemostasis. PMID- 3007579 TI - Relationship of superoxide production to cytoplasmic free calcium in human monocytes. AB - Calcium has been proposed as an intracellular second messenger for activation of secretion, phagocytosis, and the oxidative burst of neutrophils. We have examined the role of calcium in human monocyte activation. Concanavalin A (Con A) stimulated monocytes displayed an increment in cytoplasmic ionized calcium at 31 +/- 6 s and the onset of superoxide production at 61 +/- 9 s. The increase in cytoplasmic calcium invariably preceded the onset of superoxide production. If the external calcium concentration was reduced to less than 28 nM by the addition of 10 mM EGTA, superoxide production was not diminished at 5 min; however, superoxide production decreased thereafter. The Con A-evoked increment in cytoplasmic ionized calcium was blunted upon the addition of EGTA and decreased further with time. Both the production of superoxide and the Con A-evoked increment in cytoplasmic ionized calcium displayed a 50% inhibition after 15 min of calcium depletion and were completely inhibited after 60 min. Total cell calcium fell from 0.7 to 0.5 fmol/cell, and the basal level of ionized calcium fell from 83 to 30 nM after 60 min. Histidine, a strong chelator of divalent cations other than calcium and magnesium, had no effect on monocyte superoxide production or on ionized calcium concentrations, indicating that EGTA inhibition was due to cell calcium depletion. In calcium-depleted cells, Con A did not evoke superoxide production until calcium was restored to the incubation medium. The restoration of calcium to Con A-treated, calcium-depleted monocytes permitted a rapid rise in the cytoplasmic ionized calcium, and the production of superoxide within 9 s. These data suggest that an increase in ionized cytoplasmic calcium is necessary for the activation of monocyte superoxide production by Con A. The rise in ionized calcium in response to Con A results, in part, from an internal redistribution of calcium, which is sufficient to permit superoxide generation. PMID- 3007581 TI - Restriction enzyme analysis of cytomegalovirus DNA to study transmission of infection. AB - Restriction enzyme analysis of cytomegalovirus deoxyribonucleic acid (DNA) has been used to characterise virus isolates and has provided information on patterns of viral transmission. It was shown that virus isolated from a congenitally infected infant was unlikely to have originated from the 13 congenitally infected children with whom the mother, a nurse, had been in contact. Of nine mother and infant pairs, from whom cytomegalovirus was isolated, seven yielded strains that were indistinguishable for mother and child; one pair showed minor differences and one was clearly distinguishable. Virus isolates from seven children attending a day nursery were typed, and only siblings were excreting similar strains of cytomegalovirus. Further examples of the application of this technique to studies of cytomegalovirus in a family environment are given. It is concluded that characterisation of virus strains by restriction analysis of DNA is a valuable epidemiological tool. PMID- 3007583 TI - Pharmacokinetics and pharmacodynamics of pentopril, a new angiotensin-converting enzyme inhibitor in humans. AB - In a single, ascending-dose tolerance study, nine healthy volunteers were given oral pentopril 50 to 750 mg (CGS 13945) in groups of three each. Disposition characteristics of pentopril and its active metabolite (CGS 13934) were determined using plasma concentration and urinary excretion data. The drug was absorbed rapidly following zero-order kinetics. The drug has an apparent volume of distribution of 0.83 L/kg and an oral clearance of about 0.79 L/hr/kg. Urinary excretions, calculated after 125- and 250-mg doses, showed a dose proportional urinary recovery of 21% (+/- 5%) for pentopril and 40% (+/- 5%) for CGS 13934. In the multiple-dose study of 125 mg orally q12h in six healthy subjects, the plasma concentrations for both drug and metabolite showed no appreciable accumulation of either compound, which was expected from their short pharmacokinetic half-lives (pentopril, less than 1 hr; CGS 13934, approximately 2 hr). In a separate pharmacodynamic study, drug and metabolite concentrations were evaluated against angiotensin-I (AI)-induced changes in blood pressure and plasma angiotensin converting-enzyme (ACE) activity in healthy volunteers after single oral doses (range, 10-500 mg). The pharmacodynamic half-life for plasma ACE inhibition increased with the dose (10 mg, 1.5 hr; 500 mg, 9.8 hr). There was a close relationship between the plasma level of the metabolite and the inhibition of plasma ACE activity and AI-induced pressor response. A hyperbolic function adequately described the dependence of plasma ACE activity on plasma metabolite concentration with a concentration at half-maximal inhibition of 53 ng/mL. PMID- 3007582 TI - Anti-HTLV-III positive laboratory reagents. PMID- 3007584 TI - Quantitative autoradiographic analysis of mu and delta opioid binding sites in the rat hippocampal formation. AB - The distributions of mu and delta opioid binding sites were studied in rat hippocampal formation by using quantitative in vitro autoradiography. Mu binding sites, labeled with 125I-FK-33824, showed a highly organized laminar distribution. Binding was greatest at the foot of the obliterated hippocampal fissure in stratum lacunosum-moleculare of CA3. Stratum pyramidale and stratum lacunosum-moleculare of CA2 and stratum pyramidale of CA3 were next highest in mu binding, followed by stratum oriens and stratum radiatum of CA2, stratum oriens of CA3, and stratum pyramidale of CA1. The distribution of delta binding sites, labeled with 125I-D-ala2-D-leu5-enkephalin in the presence of the unlabeled mu receptor ligand PL-032, was similar to the distribution of mu binding in that binding within each region was greatest in a band centered over stratum pyramidale and in stratum lacunosum-moleculare. Over all, delta binding was greatest in CA2 followed by CA3 and then CA1. Compared to mu binding, delta binding was relatively enriched in stratum moleculare of the dentate gyrus. These laminar distributions correlate reasonably well with the distribution of enkephalin immuno-reactivity in hippocampal formation, although binding was surprisingly low in stratum lucidum, an area rich in dynorphin and enkephalin immunoreactivity. PMID- 3007586 TI - Differential effect of visual deprivation on cytochrome oxidase levels in major cell classes of the cat LGN. AB - Cytochrome oxidase histochemistry was used to examine the effects of visual deprivation on the development of neurons in the lateral geniculate nucleus of the kitten. Early postnatal monocular suture results in a decrease in reactivity within the neuropil of visually deprived binocular laminae A, A1, magnocellular C, and medial interlaminar nucleus. Within these regions, monocular suture has a greater effect on the relative numbers of, and the growth of darkly reactive (normally large), presumed Y-cells than on other less reactive geniculate neuronal classes. The decreases in the reactivity of the neuropil may be attributed to the decreases in the number of mitochondria, the number of darkly reactive mitochondria, and/or the number of darkly reactive mitochondria localized within dendrites. Although all classes of dendrites appear to be adversely affected, the decrease in C.O. reactivity was most dramatic within the presumed proximal dendrites of class 1 Y-cells. These dendrites were identified by the type of synaptic contacts they formed with retinal terminals (Rapisardi and Miles, '84, J. Comp. Neurol. 223:515-534; Wilson et al., '84, Proc. R. Soc. Lond. [Biol.] 221:411-436). As with Y-cells, the effects of monocular suture on the large darkly reactive cells were not as dramatic at sites where binocular interactions were either absent or where they had been experimentally eliminated. Based on the present and previously reported findings from several laboratories, it is likely that the selective physiological and morphological effects of monocular suture on Y-cells are accompanied by metabolic deficits involving both dendrites and perikarya. These effects appear to be due more to binocular interactions than to visual deprivation per se. PMID- 3007585 TI - Freeze-fracture study of the large myelinated club ending synapse on the goldfish Mauthner cell: special reference to the quantitative analysis of gap junctions. AB - The large myelinated club endings (LMCEs) of primary eighth nerve afferents form mixed synapses on the lateral dendrite of the giant Mauthner cell. The double replica freeze-fracture technique was employed to examine the intramembrane fine structure of these LMCE synapses. Morphological correlates of both chemical and electrical transmission were found at the LMCE synapses. Electrical synaptic junctions, or gap junctions, were located over much (10-20%) of the synaptic contact. These were seen in both pre-and postsynaptic membrane as tightly packed P face particle aggregates and corresponding aggregates of E face pits. Specializations characteristic of chemical synaptic junctions were most prominent at the periphery of the synaptic contact. These specializations consisted of postsynaptic E face particle aggregates which were subjacent to presynaptic active zones. The active zones were distinguishable as regions with an increased density of large particles and vesicle attachment sites represented by P face depressions and E face protuberances. Quantitative analysis of gap junction particle (connexon) number at five LMCEs revealed 24,000-106,000 connexons per LMCE. Comparison with data from electrophysiological studies of single LMCEs indicates that only a small fraction of the connexon channels are open at any given time during electrotonic transmission at an LMCE synapse. PMID- 3007588 TI - Nevus anemicus. AB - The results of three cases of nevus anemicus studied by mechanical, histologic, pharmacologic, and electron microscopic technics are presented. The proposed pathogenesis and the differential diagnosis of this congenital disorder are discussed. PMID- 3007587 TI - Bowenoid papulosis of the male and female genitalia: risk of cervical neoplasia. AB - Sixteen patients with bowenoid papulosis (eleven male patients with bowenoid papulosis of the penis and five female patients with bowenoid papulosis of the vulva) were studied clinically, histologically, and virologically and followed up from 12 months to 5 years. In eleven of sixteen cases of bowenoid papulosis, molecular hybridization disclosed the presence of human papillomavirus type 16. In four cases we found new, not fully characterized human papillomavirus, and in two cases, we found both human papillomavirus 16 and new human papillomavirus (double infection). The mean age of male patients was 33 years and of female patients, 31 years. The mean duration of the disease was 2.4 and 3.6 years, respectively. The lesions cleared or did not recur in eight of eleven male patients after repeated partial excisions and in two of five female patients after conservative surgery. Cervical intraepithelial neoplasia (severe dysplasia) was present in three of five female patients, and human papillomavirus infection of the cervix was present in five of six sexual partners of male patients available for examination. Thus bowenoid papulosis presents a high risk for cervical neoplasia both for female patients and for sexual partners of male patients. PMID- 3007590 TI - Phosphorylation of salivary proteins by salivary gland protein kinase. AB - Human saliva contains a number of phosphorylated acidic proline-rich proteins (APRP). Monkey parotid saliva contains a similar protein with the same phosphorylated sequences as the human proteins. A crude protein kinase was prepared from Macaca fascicularis parotid glands which phosphorylated human APRP. The enzyme was activated by Mg2+, it had a pH optimum between pH 7.0 and 7.5, the Km for ATP was 78 mumol/L, and for APRP it was 85 mumol/L. Phosphorylation of APRP was independent of cAMP and calmodulin. Phosphate was incorporated as phosphoserine, and the kinase phosphorylated the same residues in dephosphorylated APRP which are phosphorylated in the secreted protein. In addition, the enzyme preparation also phosphorylated dephosphorylated and native APRP in a region which is not phosphorylated in the secreted protein. There was no difference in the rate of phosphorylation of APRPs and their tryptic peptides. The kinase also phosphorylated other dephosphorylated salivary phosphoproteins. An enzyme was demonstrated in the human salivary gland which gave the same pattern of phosphorylation of APRP as did the simian kinase. More than one kinase may be necessary for the observed phosphorylation. PMID- 3007589 TI - Proteinases of psychrotrophic bacteria: their production, properties, effects and control. PMID- 3007591 TI - Micro-Raman line broadening in synthetic carbonated hydroxyapatite. AB - Using a Raman microspectrometer, we have recorded Raman spectra of synthetically produced hydroxyapatite samples with varying carbonate contents. The apatites were produced from aqueous solutions at about 40 degrees C. From line-broadening values of the symmetric phosphate stretch, it was concluded that the carbonate substitution in these lattices has a maximum of 4.5 wt %. Both phosphate and hydroxyl ions seem to be involved in the carbonate substitution process. PMID- 3007592 TI - Cross-resistance of erythrosin B-resistant house fly, Musca domestica (Diptera: Muscidae), to insecticides. PMID- 3007593 TI - Contact sensitivity in rats induced by tolylene diisocyanate (TDI). PMID- 3007594 TI - Development of auditory-evoked potentials in the cat. I. Onset of response and development of sensitivity. AB - Auditory-evoked potentials, originating from the brain stem and the forebrain, were studied in 30 unanesthetized kittens during the first 3 months of postnatal life, and from a smaller set of animals before and after surgical exposure of their tympanic membranes. In intact animals, responses to 135-dB peak SPL clicks were first reliably discernible on the seventh postnatal day; when stimuli were presented directly to the exposed tympanic membrane, responses were observed several days earlier. Responses progressed through three stages during maturation: an early period of gross insensitivity during which responses are evoked only by high-intensity stimuli and whose response thresholds remain essentially constant (week 1); a middle period characterized by rapid acquisition of sensitivity to near-adult values (week 2); and a late period during which adult thresholds and latencies are acquired. A sequential stage model of threshold maturation is proposed, in which thresholds decline linearly during stage two and exponentially during stage three. It is hypothesized that mechanical reorganization of the cochlea during the first 2 to 3 postnatal weeks and development of the stria vascularis are primarily responsible for the linear stage, and that neural factors primarily underlie the exponential stage and account for the gradual acquisition of adult thresholds. Rates of maturation for brain stem responses are frequency dependent, with responses to high frequencies achieving adult thresholds earlier than those to low frequencies. PMID- 3007595 TI - Development of auditory-evoked potentials in the cat. II. Wave latencies. AB - Brain stem and forebrain auditory-evoked potentials were studied parametrically during the first 90 postnatal days in unanesthetized kittens using tonal and click stimuli. This paper describes changes that occur in transmission time through the auditory pathway during development by analyses of the maturational time courses of latencies associated with waves of both auditory brain stem responses (ABR's) and late-occurring auditory-evoked potentials (AER's), recorded subdermally from the vertex. In response to click stimuli, ABR latencies were found to decay rapidly early in postnatal life and more slowly after the third postnatal week. Those trends were modeled as a two-stage sequential process, with a linear stage occurring between 7 and 18 postnatal days followed by an exponential stage during which adult latencies were achieved. AER latencies changes during development were less complicated, and followed a single-stage exponential time course. When threshold influences were taken into account--that is, when data were adjusted so that sensation level (SL) was constant across age- the latency-maturation curves associated with all ABR waves were adequately described by a single exponential, and latencies recorded from young animals were substantially shorter than latencies associated with the same aged animals when analyses were carried out with constant sound-pressure level (SPL) stimuli across age. In addition, the difference function, generated when isoasymptotic SPL and SL latency versus age functions were subtracted from one another, was also represented by an exponential curve, suggesting that at least two processes underlie the latency decay that occurs during postnatal development. Evoked responses to tonal stimuli throughout development were consistent with the basoapical developmental gradient that is observed anatomically. PMID- 3007596 TI - Development of auditory-evoked potentials in the cat. III. Wave amplitudes. AB - Amplitudes of auditory-evoked brain stem response (ABR) and late-occurring auditory-evoked potential (AER) components were recorded from kittens between birth and 90 postnatal days. All ABR and AER wave amplitudes increased during the first postnatal month. Wave amplitudes exhibited nonmonotonic growth with increasing age, attaining a maximum at 40-60 days of age, after which amplitudes decreased. Amplitudes of waves originating in the auditory nerve matured somewhat faster than waves originating in the brain stem and forebrain, and the order in which waves reached maturity was roughly the reverse order of the latencies of their peaks. Input-output curves for ABR and AER waves displayed nonmonotonic behavior that varied as a function of postnatal age. Wave amplitudes recorded from adult cats increased between threshold and 70 dB SPL, then decreased between 70 and 100 dB SPL, and rapidly increased above 100 dB SPL. The intensity corresponding to the change from increasing to decreasing amplitudes was higher for younger animals and achieved adult values during the first postnatal month. PMID- 3007597 TI - Effects of prednisolone on acute viral myocarditis in mice. AB - The effect of prednisolone on viral myocarditis was studied in BALB/c mice with encephalomyocarditis virus myocarditis. Prednisolone was injected intramuscularly, 10 mg/kg once a day, on days 4 to 13 (experiment 1) and on days 8 to 17 (experiment 2). The control mice in each experiment received injections of distilled water. In experiment 1, myocardial virus titers were maximal but neutralizing antibodies were rarely present on day 4, and viral titers were still elevated and antibody titers were high on day 8. The survival rate of the prednisolone group was significantly lower (p less than 0.05) than that of the control group on days 21, 22 and 23. On day 10, the antibody titers of the prednisolone group were significantly lower (p less than 0.01) than those of the control group, and viral titers of the prednisolone group remained significantly elevated (p less than 0.01), whereas viruses were rarely isolated in the control group. In experiment 2, the survival rate and antibody titers were not significantly different in the prednisolone and control groups. In both experiments, no viruses were isolated on day 14. The present study suggests that corticosteroids given in the early stage aggravate the course of acute viral myocarditis, and that they may not have detrimental effects if given when neutralizing antibody titer levels are high, although they are not expected to have a beneficial effect. PMID- 3007598 TI - Malignant fibrous histiocytoma complicating mitral valve replacement. AB - A 67 year old woman developed a fatal febrile illness 8 years after mitral valve replacement for rheumatic valvular heart disease. The final disease persisted for 1 year and was characterized clinically by weakness, weight loss, congestive heart failure and multiple embolic events to the central nervous system and abdominal organs. The source of the emboli was a tumor, malignant fibrous histiocytoma of the left atrium, originating from the anulus fibrosus around the covered base of the prosthetic valve. This unique case suggests the possibility that chronic exposure to materials, such as Dacron, covering prosthetic valves may induce local malignant tumors. PMID- 3007599 TI - Ontogeny of adrenoceptor-mediated contractile responses of Muller's smooth muscle in the spontaneously hypertensive rat: role of impulse activity. AB - Contractile responses of Muller's smooth muscle to alpha-adrenoceptor stimulation were evaluated in anesthetized Spontaneously Hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats on postnatal days 1-80. Force of contraction elicited by methoxamine, tyramine and electrical stimulation of the preganglionic nerve in SHRs was significantly less than that of WKY rats throughout development. alpha-Adrenoceptor sensitivity, as evaluated by methoxamine ED50, was comparable at 10 and 20 days of age, but receptor sensitivity was decreased in SHRs at 40 and 80 days. Elimination of impulse activity by decentralization of the superior cervical ganglion on postnatal day 3 resulted in a decrease in force of contraction of about 25% in both strains, and responses in SHRs remained significantly below those of WKY rats. Decentralization increased adrenoceptor sensitivity in both groups, and differences between strains were eliminated. It is concluded that the smaller adrenoceptor-mediated contractions in SHRs are an intrinsic property of the muscle while adrenoceptor subsensitivity is a consequence of elevated sympathetic nerve activity in this strain. PMID- 3007600 TI - A specific immune response to purified HA antigen (HAAg) demonstrated by leukocyte migration inhibition in patients recovering from viral hepatitis A. AB - The mechanism responsible for liver cell necrosis in patients with hepatitis A is not known. Since the type of hepatic lesions and the clinical presentation of acute hepatitis B are similar and are probably related to the cell-mediated immune response to a viral antigen located in the liver cell, it is possible that a similar mechanism is involved in hepatitis A. In the present paper, immune reactivity to hepatitis A antigen (HAAg) was measured in 13 patients at the time of recovery from hepatitis A, by using the leukocyte migration inhibition test (LMIT) under agarose with purified HAAg as antigen. Eleven normal subjects without history of hepatitis and 4 patients convalescent from hepatitis B were used as controls. Inhibition of leukocyte migration by HAAg was found in 11 of the 13 patients, with an average migration index (MI) of 77.0% (SEM 3.5). No such inhibition was found in any of the controls: MI = 100.8% (SEM 1.0), P less than 0.0001. These findings show that, like for HBsAg in hepatitis B, an immune response specific for HAAg can be demonstrated by the LMIT after HAV infection. This response could perhaps be related to the liver injury associated to hepatitis A. PMID- 3007601 TI - Central haemodynamics, baroreceptor sensitivity and alpha 1-adrenoceptor-mediated vascular reactivity during weight-stable sodium restriction in obese men with hypertension. AB - Ten obese men (20-40% overweight) with previously untreated arterial hypertension (WHO stages I and II) were examined before and during sodium-restricted isocaloric diets. The mean (+/- s.d.) daily sodium excretion was reduced from 199 +/- 65 to +/- 25 mmol/24 h. Intra-arterial blood pressure (BP), cardiac output (CO), plasma volume, circulating and urinary noradrenaline (NA), plasma renin activity (PRA) and urinary aldosterone were measured. Vascular reactivity was assessed with intravenous bolus injections of 50, 100 and 200 micrograms phenylephrine, and baroreflex sensitivity was assessed with the R-R interval response to pressure elevations on electrocardiogram. Significant reductions in systolic BP from 163 +/- 18 to 147 +/- 17 mmHg and in diastolic BP from 97 +/- 7 to 88 +/- 9 mmHg occurred during salt restriction. Blood pressure reductions were correlated with changes of urinary sodium excretion (r = 0.71; P less than 0.05). No significant changes in CO, heart rate (HR) or stroke volume (SV) were observed; therefore, BP reduction was secondary to the fall in total peripheral resistance (TPR) from 21.8 +/- 4.1 to 19.0 +/- 4.1 units (P = 0.05). Plasma volume, as well as total blood volume, was not affected by the moderate sodium restriction, but PRA rose from 0.71 +/- 0.1 to 0.87 +/- 0.1 micrograms angiotensin 1/ml per h (P less than 0.05). Urinary aldosterone was increased from 32 +/- 12 to 54 +/- 9 nmol/24 h. No change in venous or arterial concentrations of NA or of urinary NA was observed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007602 TI - Concentration and molecular forms of active and inactive renin in human fetal kidney, amniotic fluid and adrenal gland: evidence for renin-angiotensin system hyperactivity in 2nd trimester of pregnancy. AB - In a study of 38 fetuses total kidney renin was significantly correlated with gestational age (r = 0.63). Although whole fetal kidney renin specific activity was found to decrease with gestational age (r = -0.65), the mean value of the specific activity was about 20 times greater than in normal adult cortex and double that in tissue from patients with renal artery stenosis, suggesting renin angiotensin system hyperactivity. In approximately 40% of fetal kidneys examined, evidence for an inactive (trypsin-activatable) renin precursor was found. The molecular weight of this form was indistinguishable from active renin (45 000 daltons) by Sephadex chromatography. Amniotic fluid from nine cases (100%) contained angiotensin (ANG) 1, angiotensin converting enzyme (ACE), renin substrate, active and inactive renin (both 45 000 daltons). Five of the 38 (13%) fetal adrenal glands contained renin, but no evidence for trypsin-activatable forms. Aldosterone was present in low concentration in the earliest adrenals examined, and a positive correlation existed between total tissue aldosterone and gestational age (r = 0.73). These findings suggest that the fetal renin angiotensin system has an important role to play in the maintenance of extracellular fluid volume and blood pressure in the developing fetus. PMID- 3007603 TI - Altered calcium homeostasis in Dahl hypertensive rats: physiological and biochemical studies. AB - Abnormal calcium (Ca) homeostasis has been reported in essential hypertension and in the Okamoto-Aoki strain of spontaneously hypertensive rats. These abnormalities include increased urinary excretion of calcium and decreased ionized serum calcium (Ca2+). To pursue these abnormalities we studied the chronology of urinary excretion of electrolytes in a genetically homogeneous strain of hypertensive rat, the Dahl/Rapp salt sensitive (S) and resistant (R) rat (at ages 3, 5, 7, 9, 12, 20 and 32 weeks). We also characterized the renal adenylate cyclase-cAMP system by measuring urinary cAMP excretion and adenylate cyclase response to membrane receptor agonists in renal membranes from S and R rats at day 2 and at 6 and 28 weeks of age. Urinary calcium excretion was higher in S than in R at 3, 5 and 7 weeks (0.48 +/- 0.04 versus 0.24 +/- 0.01 mg/mg creatinine at 7 weeks, P less than 0.01). Sodium and phosphorous excretion were lower in S than in R rats at 5, 7, 9, and 12 weeks, and at 5, 7, 9, 12, 20 and 32 weeks, respectively. Potassium excretion was similar in the two groups. Plasma ionized calcium was lower in S than in R rats (3.9 +/- 0.1 versus 4.5 +/- 0.1 mg/dl, P less than 0.01) only at 7 weeks of age. Plasma parathyroid hormone (PTH) was not different between S and R rats. Cyclic AMP excretion and the renal adenylate cyclase response to PTH when referenced to basal activity was lower in S than in R rats at all ages.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007604 TI - The effects of enalapril on blood pressure and the kidney in normotensive subjects under altered sodium balance. AB - Eight healthy volunteers received both oral enalapril (EN; MK-421) 20 mg and placebo (PL) under stable conditions of sodium repletion, 300 mmol sodium/day (HS) and sodium depletion, 10 mmol sodium/day (LS). During PL therapy, fivefold increases in the plasma concentrations of renin and aldosterone were observed when measurements under LS were compared with those under HS conditions. Basal blood pressure (BP) readings were consistently higher and the hypotensive response to EN greater under LS compared with HS conditions. After EN, renal plasma flow increased significantly over the first 4 h, while the glomerular filtration rate, measured both by inulin and creatinine clearances, did not change. A significant natriuretic response was observed within 2 h; further natriuresis between 6 and 12 h after EN was accompanied by increased phosphate excretion. PMID- 3007605 TI - Unchanged pressor effect of norepinephrine in normal man following the oral administration of two angiotensin converting enzyme inhibitors, captopril and HOE 498. AB - The norepinephrine - (50, 100 and 200 ng/kg per min) induced rise in blood pressure (BP) was determined in six health male volunteers following angiotensin converting enzyme (ACE) inhibition by either captopril (100 mg orally) or HOE 498 (10 mg orally). In terms of absolute BP measurements both compounds induced a reduction in basal and norepinephrine stimulated BP, whereas norepinephrine induced increments of BP above individual basal levels were unchanged by either converting enzyme inhibitor. It is concluded that attenuation of the pressor response to norepinephrine does not contribute to the hypotensive action of ACE inhibitors in healthy man. PMID- 3007606 TI - 1 alpha,25-Dihydroxyvitamin D3-binding macromolecules in human B lymphocytes: effects on immunoglobulin production. AB - Previous studies have indicated that upon in vitro activation with mitogenic lectins, human peripheral blood T lymphocytes express receptors for the steroid hormone 1 alpha, 25-dihydroxyvitamin D3(1,25(OH)2D3). Furthermore, the hormone can inhibit interleukin 2 production by the activated cells. In this investigation, we report that human peripheral B lymphocytes activated in vitro with the B lymphotropic Epstein-Barr virus (EBV) also express 1,25(OH)2D3 receptor-like macromolecules. These receptors are localized in the cell nucleus and exhibit properties similar to those found in classical target tissues for 1,25(OH)2D3. They sediment on sucrose gradients at 3.3 S, display a dissociation constant (Kd) of 4 X 10(-10) M, and can bind to DNA. In addition to the 1,25(OH)2D3 receptors, however, EBV-activated lymphocytes express a second class of 1,25(OH)2D3-binding proteins that appear to occur mainly in the cell cytosol and exhibit distinct biochemical properties from the receptor, including higher sedimentation coefficients (3.7 S to 4 S) and the lack of ability to bind to DNA. The addition of 1,25(OH)2D3 to cultures of EBV-infected cells inhibited the production of IgM and IgG by the B cells. The vitamin D3 analog 24,25(OH)2D3 did not inhibit Ig production, thus suggesting that the effect is probably mediated through the high affinity receptor macromolecule localized in the nucleus. Because the EBV-induced Ig production is independent of T cell participation, the data also suggest that the effects of 1,25(OH)2D3 are exerted directly on the B cell. The present results add to the evidence of the importance of 1,25(OH)2D3 as an immunoregulatory hormone. PMID- 3007608 TI - Tumor necrosis factor induction by Sendai virus. AB - Supernatants of peripheral blood mononuclear leukocytes (PBMC) treated with Sendai virus were found to exert significant cytotoxic effects mediated by leukocyte-produced proteins distinct from interferon. Fractionation of the PBMC into adherent and nonadherent cells indicated that these virus-induced cytotoxins (CTX) were produced primarily in the mononuclear phagocytes. Cells of the monocyte-like U937 line pretreated with 4 beta-phorbol-12-myristate-13-acetate could also be induced with Sendai virus to produce CTX. The nonadherent mononuclear cells of the peripheral blood responded poorly to the virus with regard to CTX production, even though they could be induced to produce CTX with phytohemagglutinin (PHA). With the use of monospecific antibodies to tumor necrosis factor (TNF) and to lymphotoxin (LT), it was found that TNF is the major CTX produced by PBMC and by the U937 cells after 24 hr stimulation by the virus, whereas LT is not induced under these conditions to any measurable extent. TNF was also found to be produced in significant amounts together with LT upon stimulation of the nonadherent fraction of the PBMC by PHA. These findings indicate that besides bacterial lipopolysaccharides, other biological agents including viruses can be effective inducers of tumor necrosis factor, suggesting implications regarding the physiologic role of this protein. PMID- 3007607 TI - Monoclonal antibody-mediated inhibition of interferon-gamma-induced macrophage antiviral resistance and surface antigen expression. AB - A monoclonal antibody (MAb) with specificity for murine interferon-gamma (IFN gamma) was used as a probe for studying the effect of recombinant IFN-gamma (rIFN gamma) on antiviral activity, Fc receptor expression, and Ia antigen induction in macrophages. Cultures of C3H/HeJ peritoneal exudate macrophages were used to allow direct comparison of all three functions in the same target cell system. Our data provide two major findings: the efficacy of the MAb is very different depending on whether murine fibroblasts or macrophages are used as the target cell in the antiviral assay, i.e., greater than 20 to 100 times more MAb was required to block antiviral activity in macrophage cultures; and 10 to 50 times more MAb was required to inhibit Fc receptor vs Ia antigen expression in response to rIFN-gamma. These latter findings confirm and extend previous observations, which indicate that the induction pathways of two important differentiation markers by IFN-gamma may be dissociable. PMID- 3007609 TI - Defective production of anti-herpes simplex virus antibody by neonatal mice. Reconstitution with Ia+ macrophages and T helper lymphocytes from nonimmune adult syngeneic mice. AB - Both neonatal humans and mice are exquisitely susceptible to severe HSV infection. We have now documented a profound defect in the ability of neonatal C57BL/6 mice to produce anti-HSV ADCC antibody. This ability is acquired over the first 2 to 4 wk of life. Reconstitution of neonatal mice by i.p. injection of peritoneal cells from adult nonimmune syngeneic mice both affords dose-dependent protection against lethal HSV infection and reconstitutes the antibody-production defect. By cell-separation techniques (adherence, nylon wool column purification, B cell panning) and cell ablation techniques (silica treatment, irradiation, anti T cell, anti-Ia, anti-Lyt-1.2 and anti-Lyt-2.2 monoclonal antibodies plus complement treatment) the subpopulations involved in the antibody production reconstitution of neonatal mice by adult cells were identified. These include both an Ia+, radioresistant, adherent, silica-sensitive macrophage population and a nylon wool column-purified, radiosensitive, anti-T, anti-Lyt-1.2-sensitive helper T cell population. The latter cell may be substituted for by concanavalin A-stimulated lymphokine-containing spleen cell supernatants or human recombinant IL 2. In addition to reconstitution of ADCC antibody production, the same cell populations, or cells plus lymphokine-containing supernatants or IL 2, protected the newborn mice from lethal HSV infection. Further characterization of this system and of soluble replacement factors has implications for therapy or immunoprophylaxis of human neonates with, or at risk of, HSV infection. PMID- 3007610 TI - Antibody mediated suppression of secondary IgM response in nude mice against vesicular stomatitis virus. AB - Nude mice immunized with either of the two serotypes of vesicular stomatitis virus (VSV), VSV Indiana (VSV-Ind) or VSV-New Jersey (VSV-NJ), showed an early T cell independent immunoglobulin (Ig) M antibody response comparable with normal euthymic mice. Unlike euthymic mice, however, nude mice reinjected with the homologous serotype were unable to mount a second measurable serum neutralizing (SN) antibody response; a second injection with the heterologous serotype induced a normal primary type of SN antibody response. The serotype-specific refractoriness to a second challenge recovered at about 10 wk after primary infection. When antibody responses were assayed by enzyme-linked immunoabsorbent assay (ELISA), suppressive effects by previous immunization could be observed even after challenge with the heterologous serotypes; this finding probably reflects the known existence of common nonneutralizing determinants shared between the two serotypes. A weak 2-mercaptoethanol (2-ME)-resistant anti-VSV IgG SN antibody response was noticed during the primary response in nude mice and was also found in ELISA; after second infections, this 2-ME-resistant response did not develop. Serum transfer studies in nude and +/+ mice confirmed that the serotype-specific transitory refractoriness of a second response in nude mice was mediated through the anti-VSV-specific IgM antibodies. PMID- 3007611 TI - Restriction fragment-length polymorphisms of class II gene sequences in mice expressing minor structural variants of I-Ak and I-Ap. AB - Serologic and structural analyses of the I-A molecules expressed among a large collection of wild mouse-derived H-2 haplotypes has led to the definition of "families" of I-A alleles which encode antigenically similar molecules that are identical in more than 90% of their tryptic peptides. Two of these families, denoted the I-Ak and I-Ap families, consist of 10 I-A alleles which encode I-A molecules whose structures are closely related to either I-Ap or I-Ak. The evolutionary relationships of the I-A alleles in these families were assessed by a molecular analysis of their genomic structures. The A alpha and A beta alleles within these I-A families were compared by analysis of restriction fragment length polymorphisms (RFLP) detected at high stringency by Southern blot hybridization with DNA probes specific for either A alpha or A beta. The polymorphic restriction enzyme sites detected in this survey were distributed over more than 7 kb of genomic DNA surrounding each gene. Because both A alpha and A beta are encoded by about 700 bp of exon DNA, the majority of the restriction enzyme sites assayed by this RFLP analysis reflect polymorphisms in noncoding regions. The DNA sequence homologies of these alleles were estimated from the RFLP results with seven restriction endonucleases by calculating the fraction homologous value as defined previously. The results indicate that evolutionarily dissimilar I-A alleles can encode I-A molecules with very similar structures. The five I-A alleles in the I-Ak family could be divided into two discrete groups, denoted K1 and K2, on the basis of their restriction fragment (RF) genotypes. The RF genotypes of alleles within each group shared more than 80% of the restriction fragments for both A and A beta. In contrast, the RF genotypes of alleles in group K1 differed extensively from those in group K2, indicating that alleles in these separate groups may not be evolutionarily closely related. These observations suggest that gene conversion or intragenic recombinational events may have been involved in the evolution of groups K1 and K2 in the I-Ak family. The RF genotypes of alleles in the I-Ap family demonstrated a close evolutionary relationship among all but two of the alleles. These two alleles encoded I-A molecules whose structures were the least related to I-Ap of any of the alleles in the I-Ap family.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3007612 TI - Wheat germ agglutinin activates human T lymphocytes by stimulation of phosphoinositide hydrolysis. AB - Recently, it has become evident that stimulated phosphoinositide (PI) hydrolysis plays a crucial role in early T lymphocyte activation. We have investigated the effects of the nonmitogenic lectin wheat germ agglutinin (WGA) on several parameters associated with PI hydrolysis in human T cells. It was found that WGA was as effective as anti-T3 antibody and PHA in producing a rise in cytosolic free Ca++ ((Ca++)i) in blood T cells and in cells of the T cell line CCRF-CEM. It was inferred that identical cells within the blood T cell preparation responded to each of the three agents, refuting the contention that WGA only stimulated a subfraction of circulating mature T lymphocytes. WGA-induced, but not PHA-induced rises in (Ca++)i could be blocked completely by N-acetyl-D-glucosamine, demonstrating that the sugar-binding characteristics of the lectin dictate its action on T lymphocytes. Anti-T3 antibody, PHA, and WGA all initiated inositol phosphate formation in blood T cells, indicating that each of the agents stimulated PI hydrolysis. The combination of WGA with nonmitogenic amounts of phorbol-12-myristate-13-acetate resulted in strong mitogenicity. It is concluded that WGA, like anti-T3 antibody and PHA, is a pan-T activator of PI hydrolysis. PMID- 3007613 TI - Phenotype, frequency, and EBV responsiveness of human marrow B and pre-B cells. AB - We have purified subpopulations of B lineage cells from human adult (rib) bone marrow by cell sorting and panning. Limiting dilution analysis was then used for a clonal analysis of cells able to secrete IgG, IgA, or IgM spontaneously or after infection with EBV. Nonproliferating, high rate IgG or IgA producers occurred at frequencies of about one per 1000 marrow mononuclear cells. Their frequency and Ig production was unaffected by EBV, and they appeared not to express EBNA after exposure to EBV. These cells were Ia+, B1+, and over 85% expressed sIg of the IgM/D (up to 75%) and/or IgG/A isotypes (40 to 60%). B cells committed to the secretion of IgM represent 2 to 10% of marrow B lymphocytes. They were found to be Ia+/B1+/B2+/CALLA- and C3b receptor (CR3)-cells, and most (greater than 90%) required infection with EBV and proliferation to develop into IgM-producing lymphocytes. Thirty to 40% of these cells did not express Ig (H or L chain) on their surface, and therefore resembled pre-B cells at the beginning of the 4- to 5-wk culture period. Proliferating pre-B cells from adult human marrow have been described, but their conversion into IgM-producing cells has not been formally demonstrated. Although EBV induces IgM production, the expression of EBNA, and several rounds of cell division in these cells, the induction of stable (greater than 5 wk) growth transformation represents a rare event in these pre-B cells: in several thousand limiting dilution wells, not a single culture of sIg-cells showed stable growth transformation. The dichotomy between EBV-induced high-rate IgM responses and absent growth transformation discriminates activation and transformation as distinct aspects of EBV-induced B cell "responses", and suggests that cellular properties play critical roles for viral transformation. We propose a model in which cellular target genes for transforming sequences in the EBV genome are transiently expressed during B cell differentiation. PMID- 3007615 TI - Interleukin 1 is present in normal human epidermis. AB - We investigated the presence of interleukin 1 (IL 1)-like molecules in normal unstimulated human epidermal tissue. Epidermis from 21 healthy individuals that was prepared by two different methods showed prostaglandin E2 (PGE2) and collagenase stimulating activity for human dermal fibroblasts. All epidermal extracts tested were positive for thymocyte comitogenic activity (lymphocyte activating factor; LAF). Removal of the horny layer decreased epidermal IL 1-like activity. In contrast to epidermal tissue, freshly isolated peripheral blood mononuclear cells (PBMC) contained no detectable PGE2 stimulatory activity. They could, however, produce PGE2 stimulatory activity after culture and stimulation with phytohemagglutinin (PHA) and concanavalin A (Con A). Little membranous IL 1 like activity could be detected in epidermal extracts when using a method that has previously rendered membranous IL 1 from murine proteose peptone-elicited peritoneal macrophages. Gel filtration chromatography yielded double peaks at m.w. approximately 30,000 and approximately 17,000 for all three activities. High pressure liquid chromatography (HPLC) analysis identified two species with a m.w. of approximately 17,000, and one approximately 30,000 species nondissociable in detergent, all having superposable PGE2 and collagenase stimulatory as well as LAF activity. These results establish the existence of IL 1-like molecules, together with a possible precursor, in normal human epidermis. The release of these preformed epidermal IL 1 stores might be important in vivo. PMID- 3007614 TI - Modulation of IgM anti-lymphocyte antibody-reactive T cell surface antigens in systemic lupus erythematosus. AB - Cold-reactive lymphocytotoxic autoantibodies are present in the serum of most patients with active systemic lupus erythematosus (SLE) and may be important for the development of the lymphopenia and T cell dysfunction characteristic of this disorder. Neither the mechanisms of autoantibody action in this regard, nor the nature of the relevant T cell membrane target molecules have been defined, however. In the present investigation, preincubation of T cells with SLE serum at 37 degrees C reduced their reactivity with SLE IgM anti-lymphocyte autoantibodies, as demonstrated by indirect immunofluorescence and complement dependent cytotoxicity. Modulation was restricted to SLE IgM autoantibody reactive antigen; monoclonal antibody staining of various T cell differentiation and activation antigens remained unchanged. Loss of antigen from the surface membrane was rapid, but transient. A nadir was reached after approximately 120 min of 37 degrees C incubation, followed by essentially complete reexpression of antigen several hours later. Although modulation occurred spontaneously at 37 degrees C in the absence of SLE serum, loss of antigen was enhanced by IgM anti lymphocyte autoantibodies, despite their low thermal amplitude. Modulation was inhibited by sodium azide, by fixation of cells with paraformaldehyde, and by low incubation temperatures. Colchicine and cytochalasin D had no effect on this process, suggesting that the integrity of the cytoskeleton was not essential. Cycloheximide did not prevent loss of antigen, but inhibited its reexpression. In experiments to determine the fate of modulated antigen, both intracytoplasmic accumulation and shedding from the cell surface were demonstrated. Only shedding was increased by the presence of anti-lymphocyte antibodies, however. These studies delineate modulation of T cell membrane antigen as a new mechanism for anti-lymphocyte autoantibody action in SLE. The occurrence of modulation at physiologic temperatures in vitro suggests that a similar phenomenon of potential relevance to T cell dysfunction may obtain in patients with this disorder. PMID- 3007616 TI - Effect of T3 modulation on pokeweed mitogen-induced T cell activation: evidence for an alternative pathway of T cell activation. AB - Modulation of the T3 molecule on human T cells with monoclonal anti-T3 antibodies has been shown to result in the disappearance of the T3-Ti complex from the membrane and to preclude subsequent T cell activation by various mitogenic and antigenic stimuli. We have examined the effect of T3 modulation on pokeweed mitogen (PWM)-induced T cell activation. T3 modulation was accomplished by incubating peripheral blood mononuclear cells (PBMC) or mixtures of T cells and non-T cells at 37 degrees C for 18 hr in the presence of UCHT-1, a mouse IgG1 anti-T3 monoclonal antibody. Only donors whose PBMC were unresponsive to the mitogenic activity of this antibody were selected. Although T3 modulation resulted in complete to substantial inhibition of T cell proliferation induced by low PWM concentrations of 5 or 50 ng/ml, it had no effect on T cell proliferation when PWM was added at a concentration of 0.5 and 5 micrograms/ml. The results demonstrate that the higher doses of PWM can induce T cell proliferation via an alternative pathway that does not involve participation of the T3-Ti complex. In contrast, irrespective of the PWM dose added, T3 modulation almost totally inhibited PWM-induced interleukin 2 (IL 2) production. The differential effect of T3 modulation on IL 2 production and on T cell proliferation induced by high doses of PWM suggests that this alternative pathway of T cell proliferation is IL 2 independent. This suggestion was additionally substantiated by the lack of effect of anti-Tac, and anti-IL 2 receptor antibody, on PWM-induced proliferation of T3-modulated T cells. In conclusion our data demonstrate that high doses of PWM can induce T cells to proliferate via an alternative pathway that does not involve perturbation of the T3-Ti complex. PMID- 3007617 TI - Immunomodulation by neutrophil myeloperoxidase and hydrogen peroxide: differential susceptibility of human lymphocyte functions. AB - The coexistence of activated polymorphonuclear leukocytes and lymphocytes in tumor masses and inflammatory tissues suggests the possibility of interaction between secreted neutrophil products and nearby lymphocytes. To test this hypothesis, we examined the effects of neutrophil myeloperoxidase and H2O2 on lymphocytes. Human peripheral blood mononuclear leukocytes were exposed to myeloperoxidase, an H2O2-generating system (glucose + glucose oxidase), and a halide, and were then tested for functional activities. Natural killer activity against K562 cells, lymphocyte proliferation in response to mitogens, and generation of immunoglobulin-secreting cells were all susceptible to oxidative injury by myeloperoxidase and H2O2. The degree as well as the mechanism of suppression was dependent on the glucose oxidase concentration (i.e., the rate of H2O2 delivery). At low H2O2 flux, myeloperoxidase was essential for induction of lymphocyte suppression; as the rate of H2O2 generation increased, suppression became myeloperoxidase-independent and was mediated by H2O2 alone. Various lymphocyte functions were differentially susceptible to oxidative injury by myeloperoxidase and H2O2. The proliferative response to poke-weed mitogen was the least sensitive, whereas antibody formation was the most sensitive. Proliferative responses to concanavalin A and phytohemagglutinin as well as natural killer activity displayed intermediate degrees of susceptibility. In all assays, lymphocyte viability was greater than 90%. Removal of monocytes from mononuclear leukocytes by adherence to glass increased susceptibility of lymphocytes to oxidative injury. Monocytes in proportions within the range present in peripheral blood mononuclear leukocytes protected lymphocyte functions against oxidative injury by myeloperoxidase and H2O2. This study demonstrates a differential susceptibility of various immune functions to oxidative injury by the neutrophil products myeloperoxidase and H2O2, and shows, in addition, that monocytes can modulate these interactions. PMID- 3007619 TI - Metabolic comparison between basophils and other leukocytes from human blood. AB - Basophilic granulocytes were purified from the blood of normal individuals by successive isopyknic centrifugation and elutriation centrifugation. Starting with the leukocyte-rich fraction of 500 ml of blood, we recovered 31 to 80% (mean 51%, n = 20) of the basophils in 45 to 87% purity (mean 69%, n = 23). The contaminating cells were mainly lymphocytes. The basophils were greater than 98% vital (exclusion of ethidium bromide and hydrolysis of fluorescein diacetate). The histamine content of the basophils was 1.1 to 2 pg/cell (mean 1.6 pg/cell, n = 22). With anti-IgE, 30 to 50% of the histamine was released; with phorbol myristic acetate (PMA) or the calcium ionophore A23187, 70 to 100% of the histamine was released. Serum-opsonized zymosan (STZ) did not induce histamine release. Reactions with monoclonal antibodies revealed that the basophils expressed the C3bi receptor (CR3) and the leukocyte function-associated antigen 1 (LFA1), but not the gp 150,95 antigen, the C3b receptor (CR1), or the low avidity Fc gamma receptor. Basophils carry class I but not class II HLA antigens. During incubation of the basophils with serum-opsonized Staphylococcus aureus or Escherichia coli, these bacteria were neither phagocytized nor killed. STZ, PMA, A23187, or anti-IgE did not initiate an "oxidative burst" in the basophils. This was tested with oxygen consumption, cytochrome c reduction, NBT reduction, chemiluminescence, and release of hydrogen peroxide. Moreover, we did not detect cytochrome b558, superoxide dismutase, catalase, or peroxidase in the basophils. Of the typical granule-associated enzymes lysozyme, Vitamin B12-binding protein, and beta-glucuronidase, only beta-glucuronidase was present in the basophils in detectable amounts. This enzyme was released, together with histamine, on incubation of the cells with PMA, A23187, or anti-IgE, but not with STZ. We conclude that basophils from normal human blood are not phagocytes and are probably not involved in the oxidative defense of the host against foreign antigens. PMID- 3007618 TI - In vitro binding of an IgE protein to human platelets. AB - Bronchoconstriction in extrinsic asthma is initiated by mediators released from IgE-sensitized leukocytes after contact with polyvalent antigen. Because platelets also contain soluble mediators that can cause bronchoconstriction, platelet activation and release of the contents of platelet granules may play a role in IgE-mediated responses under some circumstances. We therefore sought to determine if platelets are capable of binding IgE and if cross-linking this cell bound IgE initiates secretion of platelet granule contents. Platelets from 10 normal donors were studied by using automated fluorescence analysis and fluorescence microscopy. We detected binding of a purified myeloma IgE protein to 24.1 +/- 9.6% (mean +/- 2 SD) of the gel-filtered platelets from these normal individuals. Although we could detect the binding of IgE and anti-IgE to a minority of cells, every normal individual had a population of platelets that bound IgE. The amount of IgE that bound to normal platelets appeared to be distributed heterogeneously among the IgE-positive platelet population. Platelets from two individuals with type II Glanzmann's thrombasthenia bound normal amounts of heat-aggregated IgG, but less than 3% of the platelets bound detectable IgE. Moreover, a combination of monoclonal antibodies to glycoproteins IIb and IIIa inhibited the binding of the IgE protein to normal platelets but did not affect the binding of aggregated IgG. Thus, the binding of IgE to human platelets appeared to require the presence of the glycoprotein IIb-IIIa complex. Binding of monomeric IgE to platelets, by itself, did not initiate either platelet aggregation or release of 14C-serotonin. However, both aggregation and secretion of serotonin followed the addition of anti-IgE to IgE-sensitized platelets. These studies indicate that human platelets can bind an IgE myeloma protein in vitro and that cross-linking of surface-bound IgE with anti-IgE initiates aggregation and secretion. If platelets have a similar capacity to bind normal IgE in vivo, it is possible that platelets may participate directly in several atopic or inflammatory disorders in man mediated by this class of antibody. PMID- 3007620 TI - Mechanism of action of glucocorticoid-induced immunoglobulin production: role of lipoxygenase metabolites of arachidonic acid. AB - Glucocorticoids stimulate polyclonal immunoglobulin (Ig) production in cultures of human peripheral blood lymphocytes. The mechanism of action of glucocorticoids in this system, and indeed in any physiologic system, is unknown. Because glucocorticoids stimulate the production of phospholipase A2-inhibitory glycoproteins, we investigated whether glucocorticoids stimulate polyclonal Ig production by inhibition of arachidonic acid metabolism. Nonspecific lipoxygenase/cyclooxygenase inhibitors stimulate polyclonal Ig production in a manner similar to the effect of glucocorticoids, whereas specific cyclooxygenase inhibitors actually inhibit Ig production. Two specific 5-lipoxygenase inhibitors, with little or no activity against cyclooxygenase or other lipoxygenases, also stimulate Ig production. The dose-response effect of all of these drugs on Ig production was similar to the dose response of inhibition of 5 lipoxygenase. Leukotriene B4 (LTB4) added in low concentrations (10(-10)M) on days 1, 2, and 3 of a culture eliminated the stimulatory effect of glucocorticoids or 5-lipoxygenase inhibitors, whereas LTC4, LTD4, prostaglandin E, or 5-hydroxyeicosatetraenoic acid had no effect. These results suggest that the relevant action of glucocorticoids in stimulating Ig production might be in preventing endogenous arachidonic acid metabolism, perhaps the endogenous production of LTB4. PMID- 3007622 TI - Molecular basis of a unique tumor antigen of radiation leukemia virus-induced leukemia B6RV2: its relation to MuLV gp70 of xenotropic class. AB - Hybridomas secreting monoclonal antibodies that reacted with the B6 radiation leukemia virus (RadLV)-induced leukemia B6RV2 were produced by fusion of BALB/c NS-1 myeloma cells with spleen cells from (BALB/c X B6)F1 mice immunized with B6RV2. By direct and absorption analyses with 28 B6 and BALB/c leukemias, the monoclonal antibodies NU7-4 and NU7-99 were shown to react only with B6RV2, indicating that they recognized an individually distinct antigen on B6RV2 that was identified previously with conventional (BALB/c X B6)F1 anti-B6RV2 serum. Another monoclonal antibody, NU1-132, showed relatively restricted reactivity with B6 RadLV leukemias. These three monoclonal antibodies all precipitated material of approximately 80,000 daltons, which is the same size as that precipitated by anti-xenotropic MuLV gp70 serum. Sequential immunoprecipitation analysis revealed that the molecules precipitated by NU7-4 were not removed by pretreatment of NU7-99 or NU1-132 and that the molecules precipitated by NU7-99 were not removed by NU7-4 or NU1-132. The molecules precipitated by NU1-132 were partially removed by pretreatment with NU7-4, but not with NU7-99. The molecules precipitated by these three monoclonal antibodies were removed by pretreatment with anti-xenotropic gp70. These results suggested heterogeneity of the xenotropic MuLV gp70-related molecules expressed on B6RV2 and a possible relation between serologically defined unique tumor antigens and gp70-related molecules. PMID- 3007621 TI - Polypeptides coded for by the region pre-S and gene S of hepatitis B virus DNA with the receptor for polymerized human serum albumin: expression on hepatitis B particles produced in the HBeAg or anti-HBe phase of hepatitis B virus infection. AB - There are four polypeptides coded for by the region Pre-S and gene S on DNA of hepatitis B virus that carry the receptor for polymerized human serum albumin (poly-HSA), i.e., P31 and P39, as well as their glycosylated counterparts P35 and P43. With the use of monoclonal antibodies directed to Pre-S(1) sequence and Pre S(2) sequence (bearing the receptor for poly-HSA), the content of these polypeptides, as well as their expression on the surface, was determined for hepatitis B particles of various categories. P39 and P43, carrying both Pre-S(1) and Pre-S(2) sequences, were contained abundantly in Dane and tubular particles, and to a much lesser extent in small spherical particles, all of which were purified from plasma containing hepatitis B e antigen (HBeAg). P31 and P35, carrying Pre-S(2) but not Pre-S(1) sequence, were contained comparably in these three categories of hepatitis B particles. In remarkable contrast, small spherical particles derived from plasma containing antibody to HBeAg were very low in the content of any Pre-S polypeptides. P31 and P39 showed higher activities for poly-HSA receptor than their glycosylated versions. When Dane particles were digested with trypsin, the poly-HSA receptor was deprived in parallel with the loss of antigenicity for Pre-S(2) sequence. The antigenicity for Pre-S(1) sequence was much less affected, and that for the product of gene S was virtually unchanged by the digestion. PMID- 3007624 TI - Specific enzyme immunoassay for the detection of antibody to HTLV-III using rheumatoid factor-coated plates. AB - A 1-step method was developed to detect IgG antibody to human T-cell lymphotropic virus type III (HTLV-III). The supernatant of T-lymphocyte cultures infected with HTLV-III was incubated for several days with peroxidase-labelled anti-HTLV-III F(ab')2 antibody fragments. During this incubation period an enzyme-labelled immune complex (ELIC) was formed which could be stored at -20 degrees C. For the detection of anti-HTLV-III antibody only a single incubation step was required: ELIC and serum specimens were mixed in the wells of a microtitre plate coated with rheumatoid factor (RF). In the presence of HTLV-III specific antibody additional immune complexes form which are selectively bound to the immobilized RF. Compared with other routine procedures the test was able to detect antibody to HTLV-III with improved sensitivity and specificity. PMID- 3007623 TI - Measurement of antibody-mediated binding of human polymorphonuclear leukocytes to HSV-1 infected anchorage fibroblasts. AB - A method for the quantitation of effector cell binding to anchorage fibroblast monolayers infected with HSV-1 is described. Human peripheral blood polymorphonuclear leukocytes (PMN) as effector cells were labeled with chromium 51. Fetal human lung fibroblasts were grown to confluency in microtiter plates, infected with HSV-1 and loaded with anti-HSV antibody. The amount of radiolabeled PMN adhering to the monolayer was determined after appropriate incubation and washings. The effector binding assay was shown to be dependent on specific anti HSV antibodies, antibody concentration, HSV viral expression, and inoculation time. This assay system is especially useful for the evaluation of effector to target cell conjugate formation when applied to anchorage target cells. PMID- 3007626 TI - Further studies on the ELISA-spot technique. Its application to particulate antigens and a potential improvement in sensitivity. AB - Applications of the ELISA-spot technique to particulate antigens (sheep erythrocytes and E. coli) are reported; sheep erythrocytes were used to ensure a rigorous comparison of the spot assay and the haemolytic plaque assay. The spot technique was also applied to soluble antigens (dextran and trinitrophenyl lipopolysaccharide) to assess the putative occurrence of non-haemolytic antibodies. In most instances, the spot assay disclosed higher numbers of antibody-secreting cells than the plaque assay. An examination of the kinetics of spot formation demonstrated that spot development was most rapid during the first and second hours of enzyme activity and slowed thereafter, although the numbers of spots at 16 h were higher than those at 2 h. To shorten the assay time a redox reaction (which yields an insoluble formazan) was coupled to the enzymatic reaction. In duplicate assays, this improved technique gave larger numbers of spots in a shorter time, than the conventional assay. PMID- 3007625 TI - Factors important for the measurement of chemiluminescence production by polymorphonuclear leukocytes. AB - Chemiluminescence (CL) production by phagocytosing polymorphonuclear leukocytes (PMNLs) was measured by an automatic photoluminometer with built-in mixing and temperature controls. Agitation of the vials with PMNLs and opsonized zymosan particles influenced both the lag time and the CL production. Maximal production was obtained by continuous mixing of the samples, the reaction peak occurring within 6 min. Increasing the temperature from 20 to 40 degrees C also increased the CL production, and in further experiments 37 degrees C was used. Aggregation of the PMNLs was avoided by washing the cells in PBS containing gelatin 1 g/l. Glucose, Ca2+ and Mg2+ in the final reaction mixture were necessary for maximal CL responses. The measurements of CL per s up to 4 min, the peak CL value, or the integral below the CL curve up to 6 min were all linearly proportional to the number of PMNLs in the reaction mixture. Since the lag time and the time before reaching peak CL may vary, the integral below the curve up to 6 min was chosen as the mode of CL measurement. On repeated measurements the coefficient of variation was 6.3%. The mean CL integral value for PMNLs from 14 healthy individuals was 205 +/- 19 mVs, indicating a good reproducibility of the standardized assay. PMID- 3007627 TI - Purification, identification and characterization of chicken C1q, a subcomponent of the first component of complement. AB - A component, having the equivalent haemolytic activity to that of human complement subcomponent C1q, was purified by a combination of precipitation with EGTA, gel filtration, ion exchange and adsorption chromatography from chicken serum. Yields ranged from 8 to 15 mg/litre of serum. The finally purified preparation generates full Cl haemolytic activity when assayed with human complement subcomponents C1r and C1s, and have been identified as chicken C1q. The molecular weight of undissociated C1q, as estimated on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS), is 504,000. Under dissociating but non-reducing conditions, the C1q was shown to consist of 2 subunits having molecular weights of 52,700 and 51,200 in a molar ratio of 2:1. On reduction, the 52,700 molecular weight subunit gave chains with molecular weights of 25,900 and 24,800 in equimolar ratio, and the 51,200 molecular weight subunit decreased to 24,800. The C1q contains hydroxyproline, hydroxylysine, a high percentage of glycine and approximately 7% carbohydrate. Collagenase digestion of C1q caused a rapid loss of haemolytic activity and produced much smaller peptide fragments. PMID- 3007629 TI - The isolation of Moellerella wisconsensis from stool samples in the U.K. AB - Three strains of Moellerella wisconsensis were isolated from a total of 400 stool specimens screened for this organism by means of a new selective medium developed in this laboratory. This is the first report of the isolation of this organism in the U.K. The exact role of M. wisconsensis in causing diarrhoea remains to be elucidated. PMID- 3007628 TI - Rapid diagnosis of viral infections by an alkaline phosphatase immunocytochemical method. AB - The use of alkaline phosphatase immunocytochemical staining was explored for the rapid diagnosis of poliovirus, adenovirus, herpes simplex virus and cytomegalovirus infections in cell cultures. In this test, viral antigens treated with their relative antibody were incubated with alkaline phosphatase-labelled antisera. The enzyme label was developed with a naphthol salt in the presence of a diazonium salt (Fast Blue) in order to obtain a blue coloured precipitate at the site of the enzyme. It is suggested that this immunocytochemical technique is valuable in the detection of viral infections and would be an appropriate test to use when rapid diagnosis is required. PMID- 3007630 TI - Rotavirus infection among children in hospital in Nigeria. AB - Faecal samples of 139 Nigerian infants and young children admitted to hospital for gastro-enteritis and of 169 admitted for various other illnesses were tested for rotaviruses by an ELISA technique. Rotaviruses were detected significantly more often in those with gastro-enteritis (20.1%) than in those with other illnesses (3.6%). By contrast, in a representative sample of the population from which the patients had been derived no difference was observed between two similar groups in either isolation or detection rates of recognised enteric bacterial pathogens or intestinal parasites. Hence, as elsewhere, rotaviruses are the most significant enteric pathogens associated with gastro-enteritis among infants and young children in this locality. PMID- 3007631 TI - Potential route of transmission of HTLV-III. PMID- 3007632 TI - [Germinal tumors of the testis]. AB - A well documented and clear report briefly analyzes frequency of these tumors, their lymph node and metastatic spread and their classification. The advantages of assay of biological markers are emphasized for determining extratesticular localization and evaluation of treatment efficacy. Clinical examinations and complementary investigations are summarized and surgical, radiotherapeutic and chemotherapeutic modalities discussed, as well as their indications as a function of the histopathologic variety and developmental stage of the tumor. Finally, complications arising from the different treatments proposed are outlined. PMID- 3007633 TI - Action of Bacillus thuringiensis subsp. israelensis delta-endotoxin on the ultrastructure of the house fly larva neuromuscular system in vitro. PMID- 3007634 TI - Calcification of nerves in leprosy--report of three cases. AB - Three cases of leprosy who showed evidence of calcification of nerve trunks on radiological examination are reported. Two of these had calcified ulnar nerve at elbow and in one lateral popliteal nerve was calcified at the knee level. PMID- 3007636 TI - [Numerical analysis for spread of the infection with Japanese encephalitis virus]. PMID- 3007635 TI - Clinical evaluation of Durapatite submerged-root implants for alveolar bone preservation. AB - A study was undertaken to evaluate the safety and efficacy of Durapatite cones as an immediate submerged-root implant in the mandibular symphysis region. The study involved 30 patients of which 15 received 96 implants and the other 15 served as controls. The clinical and radiographic results revealed the implants to be well accepted by alveolar bone. No evidence of rejection or major complications were observed. The principal problem which occurred was dehiscence of mucosa over some implants. This problem was attributed to operative technique. There was significantly less vertical bone loss and contour change in the anterior part of mandible in the implant group than in the control group. PMID- 3007637 TI - [Analysis of pregnancy outcome after chemotherapy of trophoblastic disease]. AB - The pregnancy outcome of 57 women who had been treated for trophoblastic disease, between 1975 and 1982, was studied. All patients had regular menstrual cycle within six months after the last chemotherapy. Frequency of secondary infertility is 5% (3/60). No correlation between the amount of chemotherapy and secondary infertility was apparent. Of eighty-three pregnancies in 57 women, 55 pregnancies or 66.3% terminated in full term deliveries, 4 or 4.8% terminated in premature deliveries. Eleven of the 83 or 11.3% ended in spontaneous abortions and 2 or 2.4% in recurrent hydatiditorm mole. Only one infant (1.7%) had congenital malformation. There was no increase in fetal wastage or congenital anomaly when compared with pregnancies prior to chemotherapy or after untreated molar patients. Among the 59 live born infants, 4 or 6.8% infants belong to light for date, 49 or 83.1% to appropriate for date and 6 or 10.2% to heavy for date. Chemotherapic agents, mainly MTX and Act-D, appeared to have no effect on fetal growth. The incidence of choriocarcinoma subsequent to pregnancy in treated patients was 1.8% (1/57) and was 0.6% (2/365) in untreated molar patients (n.s.). PMID- 3007638 TI - Measurement of human prolactin messenger RNA in decidual tissues using complementary DNA probe cloned in M13mp9 bacteriophage. AB - A human prolactin (PRL) cDNA clone was digested with restriction enzyme Pst I and the resultant fragments were cloned into bacteriophage M13mp9. Single stranded recombinant DNAs having a coding strand of the PRL cDNA were selected by hybridization with 125I-labeled PRL mRNA obtained from human prolactinoma tissue. One of the single stranded recombinant DNAs was purified by agarose gel electrophoresis and labeled with 125I to a specific activity of 1.4 X 10(8) cpm per microgram of DNA. The probe could be successfully used in RNA dot hybridization. Analysis of poly (A) RNAs from prolactinoma, liver and placental tissues revealed that this probe was specific to PRL mRNA sequence. Hybridization of poly (A) RNA from decidual tissue to this probe revealed the presence of PRL mRNA sequence. However, PRL mRNA in decidua was at least 20,000 times less than that in pituitary prolactinoma. PMID- 3007639 TI - [Photoaffinity labeling of hCG receptor on rat luteal cells]. PMID- 3007640 TI - [Clinical evaluation of the tumor marker CA125 in ovarian tumor (a cooperative study among 8 institutions)]. PMID- 3007641 TI - [Pathogenesis of iron accumulation in bone marrow plasma cells. Correlation with parenchymal iron overload]. PMID- 3007642 TI - [Postnatal development of synaptic transmission in rat jaw-opener motoneurons and electrical properties of their membrane]. PMID- 3007643 TI - Malignant transformation of a benign cutaneous mixed tumour. AB - Whilst benign cutaneous mixed tumour is common, malignant cutaneous mixed tumour is rare. There are only eleven accepted cases of the malignant counterpart in the literature. In none was there residual benign tumour tissue present to suggest that they arose from malignant transformation of the benign tumour. We report a very rare case of a malignant transformation of a benign cutaneous mixed tumour in an eighty-four year old female. Other unusual features in this case included considerable involvement of bone in the primary lesion and the histological picture of extreme pleomorphism and active mitoses, not seen in other reported cases of the malignant tumour. PMID- 3007644 TI - Malignant fibrous histiocytoma of the second metacarpal. AB - A case of low grade malignant fibrous histiocytoma (MFH) arising from a metacarpal bone in a 21 year old African female is presented. The rarity of this disorder is emphasized with review of the relevant literature. PMID- 3007645 TI - Na-K-adenosine triphosphatase and cation content in the erythrocyte in essential hypertension. AB - We measured ouabain-sensitive and ouabain-resistant Na-K-adenosine triphosphatase (ATPase) activity in the red cells of 25 normotensive (average mean blood pressure [BP] 90.2 +/- 1.27 mm Hg) and 29 hypertensive subjects (average mean BP 115.3 +/- 2.45 mm Hg). Intracellular Na and K content were measured in 13 of the normotensive and 19 of the hypertensive subjects. All subjects were male, black, of comparable weight, and had not received antihypertensive medications for at least 30 days. Ouabain-sensitive ATPase activity was found significantly lower in the hypertensive than in the control group (140.2 +/- 11 and 97.6 +/- 7.6 nmol/mg/hr, respectively, P = 0.0008), whereas no significant difference in ouabain-insensitive ATPase was observed. Intracellular Na concentration was higher (9.47 vs. 7.24 mmol/L, P = 0.0144), and intracellular K concentration lower (82.8 vs. 88.8 mmol/L, P = 0.0425) in the hypertensive subjects. These results are consistent with diminished activity of the Na-K pump in black males with essential hypertension who have received no treatment. PMID- 3007647 TI - Characterization and localization of neutral sphingomyelinase in bovine adrenal medulla. AB - Homogenates of bovine adrenal medullae hydrolyzed exogenous sphingomyelin at 4.3 +/- 1.6 nmol X mg-1 X min-1 and 97% of this sphingomyelinase activity was sedimentable at 110,000 g. The sphingomyelinase had a broad pH optimum centered at pH 7. Enzymatic activity was maximal with 80 microM added Mn2+; Mg2+ supported less than half maximal activity and both Ca2+ and EDTA inhibited activity. No activity was detected in the absence of Triton X-100. Response to detergent was biphasic with dose-dependent stimulation from 0.02% to 0.05% Triton X-100 followed by inhibition with increasing concentrations of detergent. Activity in response to detergent was also modulated by protein concentration. Sphingomyelinase activity was associated with a plasma membrane-microsomal fraction. Phosphatidylcholine was not hydrolyzed under optimal conditions for sphingomyelin hydrolysis and a variety of other conditions. Neutral-active sphingomyelinase activity in adrenal medulla was similar in magnitude to that observed in other non-neural bovine tissues. This study demonstrates the presence of a potent neutral-active sphingomyelinase in a plasma membrane-microsomal fraction of bovine adrenal medulla. This enzyme may be involved in membrane fusion and lysis during catecholamine secretion through its ability to alter membrane composition. PMID- 3007646 TI - Computed tomography of vascular middle ear masses. PMID- 3007648 TI - Non inflication with LAV/HTLV III in Thai workers. PMID- 3007649 TI - Intradental sensory units: physiological and clinical aspects. PMID- 3007650 TI - Dental observations in vitamin D-resistant rickets with special reference to periapical lesions. PMID- 3007651 TI - Information transfer during embryonic induction in amphibians. AB - Neural induction and differentiation has been studied using Concanavalin A, cyclic AMP, tunicamycin and calcium ionophore A 23187. Competent ectoderm of Xenopus laevis treated with Concanavalin A differentiates into neural (archencephalic) structures. Binding studies with gold-labelled ConA indicate that the superficial ectodermal layer contains fewer ConA-sensitive sites (alpha D-mannoside and alpha-D-glucoside residues of glycoproteins) than the inner ectodermal layer. The small number of ConA-sensitive sites can be correlated with the fact that the isolated superficial ectoderm layer, in contrast to the inner layer, does not differentiate into neural structures. The gold-ConA particles bound to inner ectoderm are quickly (within 30 minutes) internalized, presumably by receptor-mediated endocytosis. However, endocytosis is not a prerequisite for neural induction. On the contrary ConA apparently must be bound to the plasma membrane for a certain period to initiate neural induction. The rapid internalization of ConA could explain why neural inductions are evoked only if ectoderm is incubated in ConA-containing medium for longer than 30 minutes. On the other hand cyclic AMP or calcium ionophore A 23187 does not elicit neural inductions. On the contrary calcium ionophore A 23187 apparently inhibits neural and mesodermal differentiation. This effect could be correlated with an increase of intracellular calcium level of the ectodermal target cells, which could influence the permeability of gap junctions resulting in a loss of cell communication, followed by a change of differentiation and pattern formation. PMID- 3007653 TI - [In vitro diagnosis and anti-LAV/HTLV III]. PMID- 3007652 TI - Octopaminergic modulation of the membrane potential of the Schwann cell of the squid giant nerve fibre. AB - The actions of octopamine on the Schwann cells of the giant nerve fibre of the tropical squid are described. The pharmacology of the receptors mediating the actions of octopamine has been investigated in terms of stereospecificity, amine specificity and interactions with a range of agonists and antagonists. The receptors are maximally activated by D(-)-octopamine and share many of the characteristics of OCTOPAMINE2 class receptors in other preparations. The octopamine receptors appear to mediate their actions by increasing the intracellular levels of cyclic AMP in the Schwann cells. Low concentrations of octopamine potentiate the actions of the nicotinic cholinergic activation system of the Schwann cells. The results are discussed in terms of the possible physiological role of octopamine in the modulation of Schwann cell activity during stressful conditions when the giant axon system is likely to be used at a high frequency to facilitate the escape response of the squid. PMID- 3007654 TI - Transfection of Corynebacterium lilium protoplasts. AB - A protoplast transfection system has been developed for a lysine-producing bacterium, Corynebacterium lilium, using the DNA of phage CL31. Phage CL31 is lytic and specific to C. lilium and has a genome of approximately 48 kb. The transfection procedure involves a polyethylene-glycol-mediated introduction of the DNA into lysozyme-treated cells and has a maximum efficiency of 3 X 10(4) transfectants per microgram DNA. PMID- 3007655 TI - Cyclic AMP and the stimulation of trehalase activity in the yeast Saccharomyces cerevisiae by carbon sources, nitrogen sources and inhibitors of protein synthesis. AB - Addition of glucose to acetate-grown cells of Saccharomyces cerevisiae caused a rapid transient increase in the cAMP level followed by a 10-fold, transient increase in the activity of trehalase. Ethidium bromide and acridine analogues inhibited both glucose-induced responses in a similar way, confirming the role of the cAMP signal as the second messenger in the sugar-induced activation of trehalase. When nitrogen sources or protein synthesis inhibitors were added after the transient glucose-induced increase in the trehalase activity, a rapid reactivation of trehalase occurred. In this case, however, there was only a very small increase in the cAMP level, which appeared to be insignificant. When the nitrogen source or the protein synthesis inhibitor was added together with glucose, the trehalase activity remained high for a much longer time also without a significant effect on the cAMP level. When a membrane depolarizing agent was added together with the glucose, both the trehalase activity and the cAMP level remained high. Reversibility experiments in which trehalase was activated to different degrees also showed that for high sugar-induced trehalase activation a high cAMP level is needed, while nitrogen sources stimulate trehalase activity without affecting cAMP levels. In cell extracts, both cAMP and cGMP were able to activate trehalase, the latter however only at 10-fold higher concentrations. The cGMP level in vivo was about 10-fold lower than the cAMP level and was not significantly affected by nitrogen sources or protein synthesis inhibitors. Hence, neither cAMP nor cGMP seem to be involved as the second messenger in the stimulating effect of nitrogen sources and protein synthesis inhibitors on trehalase activity in yeast. Since all other evidence obtained here and before strongly points to regulation of trehalase by a 'cAMP-dependent' protein kinase, we suggest that the presence of a nitrogen source in the growth medium of yeast induces the rapid synthesis of an alternative second messenger able to activate this or another protein kinase. PMID- 3007656 TI - Molecular analysis of an antibiotic resistance plasmid, pAV5, and its derivative plasmids in Acinetobacter calcoaceticus. AB - The non-conjugative plasmid pAV5 specifies resistance to kanamycin/neomycin (KmR) and tetracycline (TcR). Physical evidence is presented to show that pAV5 gives rise to two plasmids, pAV51 (KmR) and pAV52 (TcR), which are formed by deletion of apparently non-overlapping segments of pAV5. Expression of TcR has been obtained in Escherichia coli and is associated with a 1.9 kb HindIII fragment found in pAV5 and in pAV52. Expression of KmR has been obtained in E. coli and is associated with a 1.3 kb PstI fragment found in pAV5 and pAV51. Evidence is presented that the KmR gene is flanked by inverted repeat sequences and is therefore tentatively identified as a transposon, designated Tn4411. The KmR gene specifies an aminoglycoside 3'-phosphotransferase-type I (APH(3')-I) enzyme. PMID- 3007657 TI - Evolutionary comparisons of the S segments in the genomes of herpes simplex virus type 1 and varicella-zoster virus. AB - The genomes of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) consist of two covalently joined segments, L and S. Each segment comprises an unique sequence flanked by inverted repeats. We have reported previously the DNA sequences of the S segments in these two genomes, and have identified protein coding regions therein. In HSV-1, the unique sequence of S contains ten entire genes plus the major parts of two more, and each inverted repeat contains one entire gene; in VZV, the unique sequence of S contains two entire genes plus the major parts of two more, and each inverted repeat contains three entire genes. In this report, an examination of polypeptide sequence homology has shown that each VZV gene has an HSV-1 counterpart, but that six of the HSV-1 genes have no VZV homologues. Thus, although these regions of the two genomes differ in gene layout, they are related to a significant degree. The analysis indicates that the inverted repeats are evidently capable of large-scale expansion or contraction during evolution. The differences in gene layout can be understood as resulting from a small number of recombinational events during the descent of HSV-1 and VZV from a common ancestor. PMID- 3007658 TI - Genetics of natural resistance to herpes simplex virus type 1 latent infection of the peripheral nervous system in mice. AB - The genetics of natural resistance to the development of latent infection in the trigeminal ganglia of mice inoculated in the lip with herpes simplex virus type 1 (HSV-1) was examined. Based on coefficients of a logistic regression relating latency to strain and HSV-1 concentration, inbred strains of mice formed a continuum of resistance ranging from most resistant (C57BL/6J) to most susceptible (PL/J). When these results were analysed along with latency data derived from studies employing a non-fatal concentration of HSV-1, three subpopulations were identified among these strains: resistant (C57BL/10J, BALB/cByJ, C57BL/6J), moderately resistant (DBA/2J, SWR/J, A/J, AKR/J, DBA/1J) and susceptible (PL/J, LP/J, CBA/J). Results from F1 hybrids between resistant and moderately resistant strains (B6D2F1/J, B6AF1/J) and between resistant and susceptible strains [(C57BL/6J X CBA/J)F1, (C57BL/6J X LP/J)F1)] indicated that resistance is dominant. Data from both inbred and congenic strains failed to show an association between H-2 and resistance to the development of a latent infection. Studies of mortality also indicated that a continuum was present, with C57BL/10J, C57BL/6J and DBA/1J being most resistant and PL/J mice most susceptible. When inbred strains were categorized on the basis of resistance to the development of latent infection and mortality, five groups could be identified. Group A are strains resistant to both mortality and latency (C57BL/6J, C57BL/10J, DBA/1J) while group B consists of one strain (BALB/cByJ) intermediate in resistance to mortality but resistant to latency. Group C are strains intermediate in resistance to mortality and susceptible to latency (LP/J, CBA/J) while Group D are strains susceptible to mortality and intermediate in susceptibility to latency (AKR/J, SWR/J, DBA/2J). Group E consists of one strain (PL/J) susceptible to both mortality and latency. These results indicate that host factors play an important role in the establishment of latent infection in vivo. PMID- 3007660 TI - Characterization of a herpes simplex virus type 2-specified glycoprotein with affinity for N-acetylgalactosamine-specific lectins and its identification as g92K or gG. AB - Extracts from herpes simplex virus type 2 (HSV-2)-infected cells were subjected to affinity chromatography with gel-bound Helix pomatia lectin (HPA). Only one HSV-2-specified glycoprotein was isolated by this procedure and the glycoprotein had an apparent molecular weight of 130 000 (130K). The HPA-binding glycoprotein was genetically mapped, using HSV-1 X HSV-2 intertypic recombinants into the short component of the HSV-2 genome. The mapping position, electrophoretic mobility and the antigenic properties of the HPA-binding protein indicated that it was unrelated to glycoprotein C (gC), which is the HPA-binding glycoprotein in HSV-1-infected cells, and distinct from gE and gD which map in the S component. The glycoprotein was almost quantitatively precipitated by monoclonal antibody AP1, specific for glycoprotein g92K and it also reacted with monoclonal antibody 1206-3, specific for the HSV-2 glycoprotein G previously described. It is concluded that the isolated glycoprotein is identical to g92K and consequently also to the HSV-2-specific glycoprotein G. PMID- 3007659 TI - Increased resistance to the anticellular effect of interferon in an ultraviolet light-resistant human cell line, UVr-1. AB - Interferon (alpha, beta and gamma) susceptibility was tested in a human cell line, UVr-1, a u.v. light-resistant variant of RSa cells; the latter have high sensitivity to both u.v. lethality and the cell proliferation inhibition (anticellular) effect of human interferon (HuIFN) preparations. UVr-1 cells were less sensitive than the parental RSa cells to the inhibitory effects of HuIFN preparations, as measured by cell proliferation and the incorporation of [3H]deoxythymidine and [3H]deoxyadenosine into acid-insoluble cellular material. Nevertheless, UVr-1 cells exposed to HuIFN showed almost the same enhanced levels of antiviral activity and pppA(2'p5'A)n synthetase activity as similarly treated RSa cells. Further, UVr-1 cells had much the same binding capacity for 125I labelled HuIFN-alpha A. Thus, it seems likely that the variant has an increased resistance to the anticellular effect but not to the antiviral effect of HuIFN preparations. UVr-1 cells showed no significant difference from RSa cells in u.v. induced DNA repair synthesis. However, when a comparison was made between the susceptibility of normal fibroblasts and fibroblasts from patients with Cockayne's syndrome, characterized by an altered u.v. sensitivity but no alteration of DNA repair replication synthesis, the Cockayne's syndrome fibroblasts, CCK-3 and CCK-4, were more susceptible to HuIFN-beta as judged by cell proliferation and deoxythymidine incorporation tests. PMID- 3007661 TI - Novel herpes simplex virus type 1 glycoproteins identified by antiserum against a synthetic oligopeptide from the predicted product of gene US4. AB - Gene US4 of herpes simplex virus type 1 (HSV-1) has been predicted, from DNA sequence analysis, to encode a protein of molecular weight 25237 and its properties suggest it to be a membrane-associated protein. We have investigated this protein by raising antiserum to a synthetic oligopeptide corresponding to a stretch of amino acids from an internal hydrophilic region of the predicted sequence. This antiserum immunoprecipitates three glycoprotein species of apparent mol. wt. 37 000, 48 000 and 56 000 from extracts of cells infected with HSV-1. These species are also specifically immunoprecipitated from purified virions. The in vitro translation product of gene US4 has an apparent mol. wt. of 35 000. Sequence comparisons of the short unique regions of the HSV-1 and HSV-2 genomes, in combination with published mapping data for glycoprotein G (gG) of HSV-2, has led to the conclusion that the product of gene US4 of HSV-1 is the equivalent of gG. PMID- 3007662 TI - Evidence that the 'active centre' of the herpes simplex virus thymidine kinase involves an interaction between three distinct regions of the polypeptide. AB - The nucleotide sequence of the coding region of the thymidine kinase gene from each of three mutant strains of herpes simplex virus type 1 and from the parental strain, SC16, has been determined. The mutants were known to express thymidine kinase enzymes with distinct substrate binding properties. Consideration of the lesions in the genes responsible for these altered biochemical properties. Consideration of the lesions in the genes responsible for these altered biochemical properties has led us to postulate a preliminary model for the active centre of the enzyme, involving the cooperation of three distinct regions of the polypeptide. PMID- 3007663 TI - Evidence that the major delayed-early DNA-binding proteins of herpesvirus saimiri are bound to DNA in vivo. AB - Associations of herpesvirus saimiri-specified proteins with nuclear fractions from cultures of infected cells were probed by nuclease digestion, detergent extractions and extractions and immunofluorescence microscopy using monoclonal antibodies to virus polypeptides. Nuclease digestion selectively released delayed early polypeptides with apparent mol. wt. of 110 000 (110K) and 51 000 (51K) from nuclei of infected cultures and the majority of each of these polypeptides partitioned with the insoluble fraction after detergent extraction of such nuclei. However, the nuclease-mediated release of both these proteins was specifically reduced when nuclei were isolated from cultures in which virus DNA synthesis had been inhibited with phosphonoacetic acid (PAA). In addition, the 110K polypeptide partitioned into the soluble fraction when nuclei from PAA treated cultures were extracted with detergent. Immunofluorescence microscopy revealed characteristic and distinctive subnuclear localizations of the 110K and 51K polypeptides in control cultures and these patterns of subnuclear accumulations were markedly altered in cultures treated with PAA. We conclude that the DNA-binding properties of the delayed-early 110K and 51K proteins of herpesvirus saimiri previously observed in vitro are likely to reflect their functions as DNA-binding proteins in vivo. PMID- 3007665 TI - Cloning and analysis of integrated hepatitis B virus DNA of the adr subtype derived from a human primary liver cell carcinoma. AB - A 10 kb genomic DNA fragment derived from a human primary liver cell carcinoma (PLC) and containing integrated hepatitis B virus (HBV) DNA was cloned and analysed. Physical mapping showed the viral DNA to comprise a linear sequence of at least 2.8 kb (87%) of the HBV genome and to be of the adr subtype. Integration appeared to have occurred in the region of the viral genome spanning the cohesive ends. The cellular flanking DNA sequences to one side of the integrated viral DNA contained repeats of the Alu family. The finding of no apparent rearrangements of the integrated HBV DNA sequences in this clone is in contrast to the situation in the huSP and PLC/PRF/5 PLC cell lines in which the integrated viral DNA sequences are greatly rearranged and suggests that such rearrangements may be atypical of solid PLCs. PMID- 3007664 TI - Strain-dependent virulence characteristics of bluetongue virus serotype 11. AB - Two strains of bluetongue virus (BTV) serotype 11, UC-2 and UC-8, were identified by the electrophoretic migration pattern of their genomic RNA segments on polyacrylamide gel electrophoresis. Significant differences in virulence of these two viruses could be demonstrated by subcutaneous inoculation of newborn mice. No signs of disease were observed in mice infected with UC-2. Mice infected with UC 8 died of a severe necrotizing encephalitis, which resembled lesions in bovine and ovine foetuses infected with BTV. PMID- 3007666 TI - Polyoma virus middle T gene can trigger malignant transformation of early passage rodent cells. AB - Previous studies on the tumourigenic conversion of early passage rat embryo cells by the polyoma virus early genes have suggested a multigenic control of tumourigenesis. Thus, the large T gene can immortalize early passage rat cells and can relieve the serum dependence of normal and transformed cells. The middle T gene alone cannot immortalize early passage cells; however, it can induce cells of established cell lines to become anchorage-independent and tumourigenic. Here we show that when linked to transcriptional enhancers, the polyoma virus middle T gene can trigger the complete malignant transformation of early passage rodent cells. Therefore, the polyoma virus middle T gene does not require a cooperating oncogene to induce malignant conversion of these cells. PMID- 3007667 TI - HVJ (Sendai virus) stimulates release of interferon from leukocytes used once for interferon production. AB - Leukocytes, subjected once to interferon (IFN) induction by HVJ (Sendai virus), were studied for their capability to produce IFN after a second similar stimulus. Substantial amounts of IFN (about 30 000 IU/ml) were recovered. Experiments using cycloheximide or actinomycin D and kinetic studies showed that this IFN originated mainly in IFN which resided within the cell as a result of the first induction and was released after the second stimulation. Increasing amounts of HVJ used for the second stimulus resulted in proportionally increased yields of IFN, reaching a plateau at the same dose of HVJ (1000 HAU/ml) as that which gave optimal yields after the first stimulation. Evidence is presented that the capacity of HVJ to trigger the production of a second IFN harvest is closely associated with its infectivity. PMID- 3007668 TI - Reaction products from platinum(IV) amine compounds and 5'-GMP are mainly bis(5' GMP)platinum(II) amine adducts. AB - The reaction products obtained from mixtures of 5'-GMP and platinum(IV) compounds with formula Pt(IV)Cl4(LL) and Pt(IV)Cl2(OH)2(LL) (LL representing two monodentate or one bidentate amine ligand) have been characterized by proton NMR spectroscopy. The amines used are NH3, H2N-CH2-CH2-NH2 (ethylenediamine, en), H2N CH2-C(CH3)2-CH2-NH2 (2,2-dimethyl-1,3-diaminopropane, dmdap), and HC(CH3)2-NH2 (isopropylamine, ipa). Conditions varied during the reaction are pH (values of 4, 7, and 10), effect of visible light, and addition of vitamin C as a reducing agent. In all cases, the major product appeared to be the bis(5'-GMP)(LL)Pt(II) compound. The pH effect is limited; i.e., at pH 4 the reactions proceed somewhat faster than at neutral pH, while at pH 10 slower reactions occur. The illumination with visible light also induces only slight differences in the yields of the products. On the other hand, when vitamin C is present, the reactions proceed quite rapidly, resulting in the same main product but in higher yields (up to 80%). The facts that apparently no Pt(IV) adducts with 5'-GMP can be observed under these conditions and that the major products are bis(5' GMP)(LL)Pt(II) compounds clearly support the hypothesis that the antitumor activity of certain platinum(IV) compounds is based upon in vivo reduction to the corresponding platinum(II) compounds. PMID- 3007670 TI - Separation of assembly-competent tubulin from brain microtubule protein preparations using a fast-performance liquid chromatography procedure. AB - Fast-performance liquid chromatography was used to purify assembly-competent tubulin from porcine brain microtubule protein prepared by two cycles of assembly disassembly. Microtubule protein (1-100 mg at 1.5-2.5 mg/ml) in buffer consisting of 0.1 M 2-(N-morpholino)ethanesulfonic acid, 0.5 mM MgCl2, 1 mM EGTA, 0.3 M KCl, and 0.02 mM GTP (pH 6.6) was applied to the Mono Q column (anion exchanger). The microtubule-associated proteins, GTP and GDP, eluted in the void volume. The tubulin fraction eluted at 0.45-0.50 M KCl with 65-80% recovery. The tubulin fraction contained trace enzymatic activities when compared with the starting microtubule protein, i.e., less than 1 versus 60 mU/mg/min of nucleoside diphosphate kinase, 0.2 versus 7.0 nmol/mg/min of Mg-ATPase at pH 6.6, and 0.2 versus 88 mU/mg/min of adenylate kinase. Both the Mono Q-purified tubulin and the pelleted microtubules that were assembled in 0.5 mM [3H]GTP contained 0.77 mol of labeled nucleotide/tubulin dimer. The Mono Q-purified tubulin fraction was competent to assemble, i.e., the critical concentration was 0.1 mg/ml in the presence of 0.03 mM taxol and 1 mM GTP at 37 degrees C. The Mono Q-purified tubulin fraction showed trace high-molecular-weight components, which were removed on Mono S (cation exchanger) columns. Alternatively, microtubule protein in buffer was applied to the Mono S column. Tubulin, trace nontubulin proteins, and several enzymatic activities came off in the void volume. A combination of Mono Q-Mono S or Mono S-Mono Q chromatography resulted in highly purified protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007669 TI - Metal binding to angiotensin converting enzyme: implications for the metal binding site. AB - Angiotensin converting enzyme interacts with the chelator, 1,10-phenanthroline (OP) to form an OP-Zn-ACE ternary complex, which subsequently dissociates to OP Zn and apoenzyme. The association and dissociation rate constants for the reaction OP + Zn-ACE in equilibrium OP-Zn-ACE have been determined and compared with those of known OP-metal complexes. Such constants were also used to calculate the rate constant for formation of the OP-Zn complex from OP-Zn-ACE. The rate of dissociation of zinc from ACE has been measured in the presence of EDTA (which acts only as a metal scavenger) as a function of chelator concentration, at different pH values, and with different buffers. The stability constant for the binding of zinc to apoACE log Kc = 8.2, determined by equilibrium dialysis using atomic absorption spectroscopy to assess metal concentration, is much smaller than that for Zn-carboxypeptidase A. Zn thermolysin, or Zn-carbonic anhydrase. This weak binding is attributable to the zinc dissociation rate constant of ACE, 7.5 X 10(-3) sec-1 at pH 7.0, which is much greater than that of the other zinc metalloenzymes. These results lead to inferences regarding the metal binding site of ACE. PMID- 3007671 TI - Potassium ion facilitation of phosphoinositide turnover activation by muscarinic receptor agonists in rat brain. AB - In rat hippocampal slices kept in Krebs-Henseleit medium, an increase of K+ ions to 12 mM potentiates the stimulation of phosphoinositide turnover elicited by carbachol and (+/-)-cis-methyldioxolane. Oxotremorine is inactive if tested in Krebs-Henseleit medium but it stimulates by 220% the phosphoinositide turnover when K+ is increased to 12 mM. The K+ facilitation of the carbachol stimulation of phosphoinositide turnover was blocked by pirenzepine, a muscarinic antagonist. This drug was equally potent in inhibiting the carbachol stimulation of phosphoinositide turnover both in normal and 12 mM K+ Krebs medium. This facilitatory effect of K+ appears to be preferential for muscarinic receptors, since it failed to increase the activation of phosphoinositide breakdown induced by norepinephrine and histamine. The K+ potentiation of the muscarinic stimulation of phosphoinositide turnover is not mediated by a release of one of the endogenous neurotransmitters stored in these slices because such a facilitation occurs in Ca2+-deprived Krebs-Henseleit medium and failed to occur following a depolarizing dose of veratrine. Our experiments excluded that K+ facilitates carbachol stimulation of phosphoinositide turnover because it modifies the binding characteristics of muscarinic receptors; however, they cannot exclude that K+ acts at the receptor transducer coupling. PMID- 3007672 TI - Characterization of the solubilized A1 adenosine receptor from rat brain membranes. AB - A1 adenosine receptors from rat brain membranes were solubilized with the zwitterionic detergent 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized receptors retained all the characteristics of membrane-bound A1 adenosine receptors. A high and a low agonist affinity state for the radiolabelled agonist (R)-N6-[3H]phenylisopropyladenosine([3H]PIA) with KD values of 0.3 and 12 nM, respectively, were detected. High-affinity agonist binding was regulated by guanine nucleotides. In addition agonist binding was still modulated by divalent cations. The solubilized A1 adenosine receptors could be labelled not only with the agonist [3H]PIA but also with the antagonist 1,3-diethyl-8 [3H]phenylxanthine. Guanine nucleotides did not affect antagonist binding as reported for membrane-bound receptors. These results suggest that the solubilized receptors are still coupled to the guanine nucleotide binding protein Ni and that all regulatory functions are retained on solubilization. PMID- 3007673 TI - CSF and plasma levels of pro-opiomelanocortin-related peptides in reversible ischaemic attacks and strokes. AB - Plasma and CSF beta-endorphin (beta-EP), beta-lipotropin (beta-LPH) and ACTH levels were studied in a group of 25 patients who underwent reversible ischaemic attacks or completed strokes. CSF beta-EP and beta-LPH in ischaemic patients were higher than those of the control population, independently of both clinical reversibility of the cerebral damage, and the time lapse sampling and the acute event. The presence of a CT demonstrable lesion was related to the highest CSF beta-EP levels. These data confirm an involvement of central opioid substances in the phenomena related to brain ischaemia. ACTH levels in the CSF did not differ from the controls; this finding further supports the concept of an independent central secretion of the different pro-opiomelanocortin-related peptides. The peripheral plasma concentrations of beta-EP, beta-LPH and ACTH, were, in contrast, within the normal range, confirming that CSF and plasma contents of pro opiomelanocortin-related peptides are differently controlled and originate from different sources. PMID- 3007674 TI - Peripheral neuropathy associated with the sicca syndrome. AB - Three patients with the sicca syndrome and chronic sensory neuropathy are described; in two of them neuropathy was the presenting feature of the disease. The sicca syndrome can give rise to a characteristic neurological syndrome comprising areflexia and asymmetrical sensory loss, particularly of proprioception, in the limbs. This is often associated with tonic pupils and trigeminal anaesthesia. PMID- 3007675 TI - Cisplatin neuropathy with Lhermitte's sign. AB - Following chemotherapy with cis-diamminedichloroplatinum II (cisplatin) three patients developed Lhermitte's sign and peripheral neuropathy. The toxic side effects did not become apparent until after treatment had ceased. Because of increasing use of cisplatin to achieve lasting remission in patients with malignant disease proprioceptive and myelopathic side effects may become increasingly common. PMID- 3007677 TI - Neurophysiological evaluation of associated demyelinating peripheral neuropathy and multiple sclerosis: a case report. AB - A case of combined multiple sclerosis and demyelinating neuropathy is presented. Percutaneous electrical stimulation of the cortex and spinal cord has shown that pyramidal tract conduction time was prolonged and conduction velocity in the cord was 4 m/s. Motor conduction velocity in proximal segments of peripheral nerves was slowed to the same extent as in distal segments. PMID- 3007676 TI - The role of myo-inositol in multiple sclerosis. AB - Myo-inositol was given orally to nine multiple sclerosis patients and nine healthy control subjects. Pattern reversal evoked potential testing was used to assess its effect. The principal positive wave increased in amplitude, duration and area in a dose-dependent manner in the multiple sclerosis group compared with controls. Cerebrospinal fluid concentrations of myo-inositol in multiple sclerosis and controls were similar. The significance of these observations is discussed in relation to recent discoveries in inositol phospholipid function. PMID- 3007678 TI - Severe sensorimotor neuropathy after cisplatin therapy. PMID- 3007679 TI - Effects of recombinant interleukin-2 on resting human T lymphocytes. AB - The effects of recombinant interleukin-2 on resting T lymphocytes were examined in vitro. T cell enriched populations were isolated from the peripheral venous blood of three normal individuals by Ficoll-Hypaque gradient separation and standard sheep erythrocyte rosetting techniques. Interleukin-2 at concentrations of 100 and 1,000 U/ml stimulated tritiated thymidine incorporation, Tac antigen expression, interferon production, and blast transformation in T cell cultures. These changes were observed in the absence of cell proliferation. The effects of interleukin-2 at a concentration of 100 U/ml were substantially blocked by the addition of a 1:1,000 dilution of anti-Tac antibody to the culture medium indicating that the effects observed were mediated by the Tac receptor. These results indicate that recombinant interleukin-2 can have demonstrable functional and morphologic effects on unstimulated human T cells. PMID- 3007680 TI - Purified interleukin-2 induces proliferation of fresh human lymphocytes in the absence of exogenous stimuli. AB - Highly purified interleukin-2 (IL-2) induced proliferation of fresh human peripheral blood mononuclear leukocytes (PBML) in the absence of identifiable exogenous mitogenic or antigenic stimuli. Dose dependent proliferation was observed with three IL-2 preparations, including two preparations purified from natural sources and a preparation produced by recombinant DNA techniques. Both T and non-T cells proliferated. Purified helper/inducer and suppressor/cytotoxic T cells cultured in the absence of non-T cells proliferated only weakly; the proliferative response to IL-2 was restored by the addition of irradiated non-T cells. Proliferation to IL-2 was not blocked by the monoclonal antibody anti-Leu 4, which reacts with a component of the T-cell receptor complex for antigen and blocks mitogen and antigen-induced T-cell responses. Monoclonal antibody to HLA DR also failed to significantly block the proliferation of resting cells to IL-2. The IL-2 induced proliferative response thus appears to be dependent on interactions between different subpopulations of PBML but probably does not simply reflect augmentation by IL-2 of antigen-driven or autoreactive processes. PMID- 3007681 TI - Cerebral granular cell tumor: immunohistochemical and electron microscopic study. AB - A rare intracerebral granular cell tumor (GCT) was studied by immunocytochemical and ultrastructural methods. The tumor was composed of two cell types--filament rich and granular cells. Granular cells contained PAS-positive, diastase resistant granules that ultrastructurally corresponded to autophagic cytosegresomes. Glial fibrillary acidic protein, the intermediate filament protein specific for astrocytes, was demonstrated in the filament-rich and, to a lesser extent, in the granular cells. Unlike noncerebral GCT, neither S-100 protein nor vimentin was detected in the tumor cells. On the other hand, both cerebral and noncerebral GCT were labeled immunocytochemically with peanut lectin (Arachis hypogaea). The results suggest that cerebral GCT share some features with noncerebral GCT, but differ in other respects. They further suggest that GCT may be derived from different cell types depending on the tissue of origin, and that cerebral GCT may be derived from astrocytes. PMID- 3007682 TI - WR-2721 protects against the hematologic toxicity of cyclophosphamide. PMID- 3007684 TI - Teniposide (VM-26), an overlooked highly active agent in small-cell lung cancer. Results of a phase II trial in untreated patients. AB - Teniposide, VM-26 (Vumon), was administered in a dose of 60 mg/m2 on days 1 to 5 every third week to 36 patients with histologically confirmed small-cell lung cancer. None had previously received chemotherapy or radiotherapy. The median age was 73 years (range, 52 to 79). Thirty-three patients were evaluable; 21 of these had local disease. Five patients had bone marrow metastases, four had liver involvement, and one CNS metastases. All patients had a performance status less than or equal to 2 before the start of treatment. Thirty patients obtained a response (90%), ten of whom had a complete remission (30%). The median duration of remission was 8+ months (range, 1.1 to 17+ months), whereas the median survival was 8.7 months (range, 1.9 to 20 months). Toxicity was primarily hematologic, with leukopenia the only dose-limiting effect. Besides alopecia, all other side effects were minimal including nausea and vomiting. We find these results provocative in regard to the response rate and the duration of response obtained as well as in reference to the dismal results that prior investigations in previously treated patients have shown. These data may indicate the need for reconsideration of the usual strategy for performing phase II trials. PMID- 3007683 TI - Effect of alternating combination chemotherapy on survival of ambulatory patients with metastatic large-cell and adenocarcinoma of the lung. A Southwest Oncology Group Study. AB - Using a randomized prospective trial design, chemotherapy with 5-fluorouracil, vincristine, and mitomycin C (FOMi) was compared with cyclophosphamide, doxorubicin, and cisplatin (CAP) and with FOMi alternating with CAP (FOMi/CAP) in 452 eligible patients with metastatic large-cell undifferentiated and adenocarcinoma of the lung. Objective responses were obtained in 26%, 17%, and 22% of patients treated with FOMi, CAP, and FOMi/CAP, respectively. The median survival was similar for FOMi, CAP, and FOMi/CAP therapies (20, 24, and 23 weeks, respectively), but the overall survival (log rank test), 1-year survival, and remission duration were longer for FOMi/CAP-treated patients. Survival was significantly longer for fully ambulatory FOMi/CAP-treated patients compared with either FOMi (P = .01) or CAP (P = .04). Younger patients treated with full doses of therapy responded more often than older patients receiving reduced drug doses (26% and 11%, respectively; P = .003). A prognostic factor regression analysis of all eligible patients indicates that sex, performance status, stage, and treatment assigned were important independent variables determining survival (P less than .05). Toxicity was comparable in each treatment group. PMID- 3007685 TI - Human T-lymphotropic virus type III infection in previously immunocompromised hosts. AB - The human T-lymphotropic virus type III (HTLV-III) is the primary etiologic agent of the acquired immunodeficiency syndrome (AIDS). HTLV-III infection in patients with prior underlying immune deficiency states such as cancer has not yet been studied. We report on the occurrence of clinically atypical opportunistic infections in previously immunocompromised patients that resulted from transfusion-acquired HTLV-III infection. Development of unusual infectious diseases in patients with neoplasms and other underlying immune deficiency disorders should lead to consideration of HTLV-III infection. Surveillance data should be obtained on these patients to accurately define the scope of HTLV-III infection. PMID- 3007686 TI - WR-2721 protects against the hematologic toxicity of cyclophosphamide: a controlled phase II trial. AB - WR-2721 S-2-(3-aminopropylamino) ethyl phosphorothioic acid, is an organic thiophosphate compound that in the animal model selectively protects against the hematologic toxicity of cyclophosphamide by factors of 1.5 to 2.0. Preliminary data from our controlled phase I trial of WR-2721 and cyclophosphamide suggested that WR-2721 protected against cyclophosphamide-induced granulocytopenia. Since variable drug doses and infusion rates were used in these early studies, we initiated a controlled phase II trial using constant drug doses to establish more precisely WR-2721's level of protection. Initially, 21 patients received 1,500 mg/m2 of cyclophosphamide alone and were retreated 4 weeks later after hematologic recovery was complete with 740 mg/m2 of WR-2721 before the same dose of cyclophosphamide. With WR-2721 pretreatment, 19 of 21 (90%) patients had improved WBC and granulocyte counts. The mean WBC increased from 1,760/mL with cyclophosphamide alone to 2,500/mL with WR-2721 pretreatment (P less than .0005). The mean granulocyte count increased from 541/mL on cyclophosphamide to 1,247/mL with WR-2721 and cyclophosphamide (P less than .0005). Following cyclophosphamide administration alone, neutropenic fevers developed in three patients. No patient experienced a febrile episode following WR-2721 and cyclophosphamide administration. Platelet nadirs below 100,000/mL were only noted in two patients treated with cyclophosphamide alone. Objective partial responses were observed in four of 19 (21%) patients with measurable or evaluable disease. These data suggest that WR-2721 provides significant protection against cyclophosphamide induced hematologic toxicity. PMID- 3007687 TI - T1 and T2 proton nuclear magnetic resonance (N.M.R.) relaxation times in vitro and human intracranial tumours. Results from 98 patients. AB - 137 samples of intracranial tumours have been studied in proton NMR spectroscopy. T1 and T2 relaxation times are above those of normal grey and white matter. Differential diagnosis between benign and malignant brain tumours does not seem feasible upon proton T1 and T2 alone. Histological correlations allowed us to specify secondary changes accounting for T1 and T2 variations (oedema, microcyst, stroma reaction, necrosis). PMID- 3007689 TI - Long-term facilitation alters transmitter releasing properties at the crayfish neuromuscular junction. AB - Synaptic transmission at the neuromuscular junction of the excitatory axon supplying the crayfish opener muscle was examined before and after induction of long-term facilitation (LTF) by a 10-min period of stimulation at 20 Hz. Induction of LTF led to a period of enhanced synaptic transmission, which often persisted for many hours. The enhancement was entirely presynaptic in origin, since quantal unit size and time course were not altered, and quantal content of transmission (m) was increased. LTF was not associated with any persistent changes in action potential or presynaptic membrane potential recorded in the terminal region of the excitatory axon. The small muscle fibers of the walking leg opener muscle were almost isopotential, and all quantal events could be recorded with an intracellular microelectrode. In addition, at low frequencies of stimulation, m was small. Thus it was possible to apply a binomial model of transmitter release to events recorded from individual muscle fibers and to calculate values for n (number of responding units involved in transmission) and p (probability of transmission for the population of responding units) before and after LTF. In the majority of preparations analyzed (6/10), amplitude histograms of evoked synaptic potentials could be described by a binomial distribution with a small n and moderately high p. LTF produced a significant increase in n, while p was slightly reduced. The results can be explained by a model in which the binomial parameter n represents the number of active synapses and parameter p the mean probability of release at a synapse. Provided that a pool of initially inactive synapses exists, one can postulate that LTF involves recruitment of synapses to the active state. PMID- 3007688 TI - Continuous human glioma-derived cell lines UC-11MG and UC-302MG. Morphologic, immunocytochemical and chromosomal characterization. AB - Two continuous human glioma-derived cell lines, UC-11MG and UC-302MG were established in our laboratory. Both cell lines persistently showed cytologic features similar to those of their respective original tumors. UC-11MG expressed glial fibrillary acidic protein (GFAP) and S-100 protein. The cell lines were negative for factor VIII related antigen (FVIII/RAg) and positive for fibronectin and neuronal specific enolase (NSE). Electron microscopic studies of UC-11MG revealed intermediate filament; filopodia and pinocytic vesicles were present in both lines. Dibutyryl cAMP (dB-cAMP) caused inhibition of growth and marked shift in the morphology of UC-302MG toward spindle cells. The cytologic appearance of UC-11MG treated with dB-cAMP was altered less, but cells showed a stronger GFAP expression, with 'cable' formation. Doubling time was 41.0 +/- 6.4 hours for UC 11MG and 43.7 +/- 6.6 hours for UC-302MG. The karyotypes of both cell lines were aneuploid with chromosomal derangement and markers characteristic for each line. PMID- 3007690 TI - Excitatory action of ATP on embryonic chick muscle. AB - It has been suggested that ATP might play a role in synaptic transmission at developing vertebrate neuromuscular junctions. To increase our understanding of the events underlying synapse formation, we have used intracellular recording and patch clamp recording to examine the response of chick myoblasts and myotubes to to ATP and other nucleotides, ATP, applied at micromolar concentrations, has a potent depolarizing action on chick myoblasts and myotubes. The ATP depolarization declines during prolonged application of ATP and shows no recovery for at least 20 min after the removal of ATP. The physiological event that underlies the ATP response has a reversal potential near O mV and is due to a conductance increase. However, contrary to our expectation, in a series of nearly 200 cell-attached and outside-out patch recordings, we did not detect single channel currents that were related to ATP. The myotube ATP receptor is pharmacologically distinct from putative ATP receptors in other systems. It is not activated by ADP, AMP, or adenosine. Furthermore, the nonhydrolyzable ATP analogs, AMP-PNP, alpha,beta-meATP, and beta,gamma-meATP (respectively, 5 adenylylimido diphosphate; alpha,beta-methylene adenosine 5'-triphosphate; and beta,gamma-methylene adenosine 5'-triphosphate), which are potent ATP agonists in other systems, have no depolarizing action on myotubes. The ATP receptor is also distinct from the nicotinic ACh receptor since responses to ATP are unaffected by the nicotinic antagonists d-tubocurarine and alpha-bungarotoxin. We therefore applied alpha-bungarotoxin to nerve-muscle co-cultures in the hope of uncovering an additional component of the postsynaptic potential, which might represent a synaptic action of ATP. Under these experimental conditions no evidence indicative of a postsynaptic action of ATP released from nerve terminals was observed. PMID- 3007691 TI - Characterization of pro-ACTH/endorphin-derived peptides in rat hypothalamus. AB - The proteolytic processing pattern of pro-ACTH/endorphin in rat hypothalamus is similar to the pattern in the pars intermedia; peptides the size of beta endorphin, gamma-lipotropin (gamma-LPH), corticotropin-like intermediate lobe peptide (CLIP), alpha-melanotropin (gamma-MSH), joining peptide, and glycosylated gamma 3-MSH all represent predominant end products. Equimolar amounts of beta endorphin-, alpha-MSH-, CLIP-, gamma-LPH-, and joining peptide-related immunoreactivity are found in hypothalamic extracts (approximately 3 pmol per hypothalamus). Although the proteolytic processing pattern in the hypothalamus is similar to that in the pars intermedia, a tissue-specific posttranslational processing pattern was detected. Ion-exchange analysis of beta-endorphin-sized immunoreactive material from hypothalamic extracts resolves three major forms, corresponding to beta-endorphin(1-31), beta-endorphin(1-27), and beta-endorphin(1 26). The alpha-N-acetylated forms of endorphin represent less than 10% of the total beta-endorphin immunoreactivity. Analyses of hypothalamic alpha-MSH-sized molecules with acetyl- and amide-directed alpha-MSH antisera suggest that hypothalamic alpha-MSH is fully amidated, but largely not alpha-N-acetylated. Fractionation by reverse-phase high-performance liquid chromatography (HPLC) confirms that greater than 85% of the alpha-MSH immunoreactivity corresponds to ACTH(1-13)NH2 or its sulfoxide, and less than 10% corresponds to alpha-MSH [alpha N-acetyl-ACTH(1-13)NH2] or its sulfoxide. Isoelectric focusing demonstrates that 83-93% of hypothalamic CLIP is phosphorylated. Isoelectric focusing suggests that the majority of the hypothalamic gamma-LPH-sized immunoreactive material is indistinguishable from gamma-LPH synthesized by pituitary melanotropes. The minor extent of alpha-N-acetylation of alpha-MSH and beta-endorphin, the limited carboxyl-terminal proteolysis of beta-endorphin, and the extensive phosphorylation of CLIP represent major differences between the posttranslational processing patterns of pro-ACTH/endorphin in the hypothalamus and pars intermedia. PMID- 3007692 TI - Teaching communication skills: effects of two methods of instruction and selected learner characteristics. AB - Performance outcomes were compared for groups of students (N = 147) randomly assigned to role play or lecture instruction for learning basic communication skills. These students also differed on the attributes of learning style and traditional-nontraditional status. Analysis of variance with age as a covariant was used in a factorial design to compare performance of the various subgroups on objective tests at two time intervals and on instructor ratings of students' skills on four dimensions of communication in process recordings of actual nurse patient interactions. Significant (p = less than .05) differences were demonstrated as follows: the field-independent learning style group had higher mean scores on objective tests than did the field-dependent group, and the nontraditional group (older with prior academic degrees and life experience) had higher mean scores on the initial objective test and on instructor rated process recordings for the dimensions of caring, concreteness and empathy than did the traditional BSN student group in the sample. Differences in student evaluation ratings of the instructional method which were significant (chi 2 p = less than .05) included: role-play students indicated greater interest, active involvement and preference for the method, and lecture students indicated greater confidence that the method met the objectives and that they understood the material although no differences in overall mean performances were demonstrated. Interactive effects and the implications of the study findings are discussed. PMID- 3007693 TI - Evaluation of a cardiac arrest simulation. AB - Prior to using a simulation strategy for educational and/or research purposes, it is essential to assess whether or not the simulation elicits responses one would expect in the "real" situation. The purpose of this study was to evaluate the stress-eliciting capacity of two variations of a simulated cardiac arrest situation. Twenty-seven senior baccalaureate students in nursing volunteered to participate in the study. Each subject was randomly assigned either to a simulation containing interpersonal stressors or a simulation containing environmental stressors. The simulations took place in a videotape studio equipped as an intensive care unit. All subjects were instructed via an audiotape recording to perform tasks similar to those undertaken by intensive care nurses in the context of a cardiac arrest situation. Pre-post measurements included: self-reported anxiety as measured by the State Trait Anxiety Inventory (A-State), pulse rate, and diastolic and systolic blood pressure. Task performance on cardiopulmonary resuscitation (CPR) and on a medication memory activity also were measured. Since no significant differences in the dependent measures were found between the two groups, data from both groups were analyzed together. Univariate analyses showed significant pre-post increases in pulse rate (p less than .0001), systolic blood pressure (p less than .03), and self-reported anxiety (p less than .0001). No significant change was noted in diastolic blood pressure. Ratings on CPR performance and a medication memory task were well below expected performance standards. The implications of these findings for educational and research purposes are discussed. PMID- 3007694 TI - Relationship of nursing program predictors and success on the NCLEX-RN examination for licensure in a selected associate degree program. AB - This ex post facto correlational study sought to determine the relationship of selected admission criteria and performance in the integrated nursing major didactic courses of an associate science in nursing degree program as predictors for performance on the licensing examination for registered nurses. A significant positive relationship at .01 with NCLEX was individually demonstrated with all of the seven ASN nursing courses and with SAT verbal scores. Not significant were age at graduation from the program, high school class rank percentile, and SAT math scores. Multivariate regression weights derived from an equation using course grades of 104 graduates were used to predict NCLEX scores. The predicted scores correlated strongly (n = .78551, p less than .001) with the actual scores. Ten predicted and actual scores were provided for demonstration. PMID- 3007695 TI - Current status of nursing process in ADN programs. PMID- 3007697 TI - Attribution theory and behavior change: ideas for nursing settings. PMID- 3007696 TI - The expanded community role: "jail" experience. PMID- 3007698 TI - Baccalaureate nursing education at extension sites: a survey. AB - The use of extension sites in baccalaureate nursing education has increased significantly since 1978. This survey found that the majority of extension sites were developed for RNs although large numbers of generic students are also served. The use of extension sites ranges from delivering selected courses away from the lead campus to delivering an entire program. Extension sites may be located on other university campuses or may be found in a store front setting or other community agency. Administrative control of extension sites emanates from the lead campus. Faculty participation in faculty activities, such as school of nursing or university committees, is expected. The degree to which this is accomplished, however, may vary. In order to maintain program integrity, the curriculum must remain the same regardless of where it is implemented. One of the primary ways of doing this is to use the same syllabi, texts and, in many cases, the same exams. Faculty may be stationary at established extended sites or may travel from the lead campus to teach, carrying with them educational materials. Extension sites are a phenomenon of the here and now. They provide a way of delivering baccalaureate nursing education to students who might otherwise be denied this level of education. Extension sites may be operationally cumbersome, challenging, and costly, but they are meeting a need. With the advent of more sophisticated telecommunications and the continued demand for baccalaureate level education, the possibility exists for even greater variation and potential for this type of program. PMID- 3007699 TI - Expanding the armamentarium for faculty development. AB - When peers teach in a faculty workshop, a wonderful opportunity is provided for a mutually rewarding experience. The biggest problem was a lack of time to fully develop all of the content with the participants. A full-day workshop was really needed. Evaluations immediately after the workshop were very positive. The four month follow-up evaluations were also positive; we had a return rate of 70% in the latter time period. We had hoped that the workshop would encourage other components within our nursing school to offer similar exchanges in their areas of expertise. This has not come to pass. All of us who came up with the idea, developed the content, and presented the material felt very good about the role we played in maximizing gerontology expertise and promoting positive attitudes about elderly in our school. Although peer workshops have limitations, especially the lack of depth gained over time in course work, the advantages are many. The time frame is rapid for the learners; the learning is relatively painless and allows for much independent follow-up through annotated bibliographies, discussion, handouts, and tools for teaching. Furthermore, presenters are being recognized for their expertise; preparation time is minimal because the teaching is within their area of expertise and many of the tools and literature are in constant use. It is economically feasible because of the numbers of faculty reached and the resources already available. The positive aspects of the peer workshop coupled with nursing facultys' natural interest and motivation outweigh the disadvantages. The peer workshop becomes an addition to our armamentarium of dynamic and innovative methods for expanding faculty growth and knowledge. PMID- 3007700 TI - Teaching an introductory research course on and off campus. PMID- 3007701 TI - Diversity and challenge in our student body. PMID- 3007702 TI - Baccalaureate education and NCLEX: the causes of success. AB - A systems framework was used to study the unusual failure rate on the National Council Licensing Examination (NCLEX) experienced by one-third of the 1983 graduates of a Northwestern Bachelor of Science Nursing (BSN) program. The study group included 176 graduates; 28 were nonsuccessful and 148 were successful candidates. All candidates experienced an integrated curriculum in the two-year nursing major. This comprehensive study addressed the conceptual and experiential areas of the curriculum as well as student academic potential and performance. It is one of the first research reports to include the new NCLEX as the dependent variable. Data were analyzed for correlational and causal relationships using regression analysis and tests of statistical inference. Results indicated that graduates who entered the program with low SAT scores, low cumulative and Science GPAs, who scored below the class mean on School of Nursing examinations and whose cumulative grade point averages drifted downward while in the School of Nursing were at a significantly high risk of failing the NCLEX. It was demonstrated that the bulk of the learners attracted to the University program fell in the B to C range of academic performance, some graduates were dissatisfied with faculty teaching skill and methods, as well as with the amount of hands-on experience available either in skills labs or clinical settings. The integrated curriculum used by the School of Nursing, threading concepts across practice areas, also presented a difficulty for students. Overall, the study indicated a concern about whether the avant-garde integrated curriculum and teaching methods used by the School of Nursing met the needs of average learners attracted to the program. PMID- 3007704 TI - Sympathetic tone and pressor response to noradrenaline during mineralocorticoid induced blood pressure rise in man. AB - To gain insight into the role of the sympathetic nervous system in the development of mineralocorticoid hypertension, we determined noradrenaline and adrenaline in plasma and urine before, during and after administration of the synthetic steroid, fludrocortisone, for a period of 6 weeks in normotensive volunteer subjects. In addition, pressor reactivity to exogenous noradrenaline, platelet alpha 2- and lymphocyte beta 2-receptors, and platelet intracellular free calcium were determined. Fludrocortisone induced a fall in free and sulpho conjugated plasma noradrenaline and after 6 weeks, a rise in free and sulpho conjugated noradrenaline excretion. The number of alpha 2- and beta 2-adrenergic binding sites decreased. A marked increase in platelet free intracellular calcium was found after the first week of fludrocortisone administration followed by a decrease in the following weeks. Reactivity to exogenous noradrenaline was found to be enhanced and this could be a factor contributing to the development of hypertension. Whereas the decrease in plasma noradrenaline observed would suggest a diminution in sympathetic tone, the finding of a rise in urinary noradrenaline excretion after 6 weeks of steroid administration in the presence of suppressed plasma levels points to an increased renal sympathetic drive. The decreased number of platelet alpha 2- and lymphocyte beta 2-receptors observed would also be consistent with the assumption of an increased sympathetic tone. PMID- 3007703 TI - Azo dye-induced alterations in vitamin B-6 metabolism and in pyridoxal 5' phosphate-binding proteins in rat liver. AB - The effects of the hepatocarcinogen, 3'-methyl-4-dimethylamino-azobenzene, on vitamin B-6 metabolism in rat liver were studied. The following parameters were measured: pyridoxal 5'-phosphate (PLP) concentrations in plasma, brain, liver and azo dye-induced hepatomas, as well as the activities of pyridoxine (PN) kinase, pyridoxine 5'-phosphate (PNP) oxidase, PNP phosphatase and PLP-dependent ornithine decarboxylase. Hepatomas more closely resembled fetal than normal adult rat liver with respect to their ability to convert vitamer forms such as PN to coenzymatically active PLP. Microtiter plate enzyme-linked immunosorbent analyses revealed that the absence of PNP oxidase activity in a dissectable hepatoma was attributable to the absence of enzyme protein. In addition, monoclonal antibodies to vitamin B-6 were used in a Western immunoblot technique to examine the effects of azo dye ingestion on the pattern of PLP-binding proteins in cytosolic extracts of liver and hepatomas. Nitrocellulose blots of electrophoretically resolved cytosolic extracts probed for PLP-binding proteins showed increasing complexity with development; hepatomas bore a striking resemblance to fetal liver. The data indicate that hepatomas lose the properties of terminally differentiated hepatic tissue and take on the properties of fetal hepatic tissue characterized by lower concentrations of PLP, selective use of the coenzyme, and a lowered-to-absent capability to convert precursor vitamer forms to PLP. Therefore, with respect to vitamin B-6 metabolism and use, it appears likely that azo dye-induced hepatocarcinogenesis involves proliferation of a stem cell type(s) having the phenotypic characteristics of fetal hepatic tissue. PMID- 3007706 TI - Role of drugs modifying sympathetic nervous system activity in the treatment of hypertension. AB - The major sites of action in the nervous system of the various types of drug used in the treatment of hypertension were considered, together with some of their advantages and disadvantages. Consideration was given to the way in which the circulation may be controlled, particularly in hypertension, and the difficulties of using various drugs to analyse controlling mechanisms. The assessment of sympathetic activity and over-activity was also considered, since this remains an area of great difficulty, both in man and in other animals. PMID- 3007705 TI - Adrenergic activity and myocardial anatomy and function in essential hypertension. AB - In hypertension, changes of cardiac anatomy and function are not just a simple consequence of the increased pressure load. The sympathetic nervous system activity is one of the factors which may influence the cardiac performance, and possibly also the cardiac anatomy of hypertensive patients. Several clinical studies have provided evidence of a subset of patients, usually with mild or borderline hypertension, with an increased cardiac performance, higher plasma catecholamine concentrations and/or greater response to beta-adrenergic stimulation. Animal studies have strongly suggested a possible role of adrenergic factors in the development of left ventricular hypertrophy (LVH). In man, plasma catecholamines are usually higher in hypertensive patients with LVH, and a correlation between left ventricular mass and plasma noradrenaline has also been observed. An impaired response to beta-adrenergic stimulation has been reported in hypertensive animals and in patients with LVH. Several studies have also suggested that reversal of LVH may be more easily induced by those antihypertensive drugs that reduce, or at least do not stimulate, the sympathetic activity, although exceptions to this statement may be observed. PMID- 3007708 TI - Acquired immunodeficiency syndrome: review of epidemiology. PMID- 3007709 TI - Acquired immunodeficiency syndrome: review of clinical aspects. PMID- 3007707 TI - Naloxone does not affect the cardiovascular, sedative or neurohormonal effects of clonidine in normal and hypertensive man. AB - The possibility that some of the cardiovascular, sedative or neurohormonal effects of clonidine are mediated by opiate receptors was investigated in normotensive and hypertensive subjects. In normal subjects intravenous (i.v.) clonidine lowered blood pressure, increased sedation and raised levels of plasma renin activity and growth hormone. Levels of other anterior pituitary hormones (prolactin, luteinizing hormone and follicle stimulating hormone) and of arginine vasopressin were unchanged. The effects of clonidine were similar after the administration of naloxone. In patients with essential hypertension clonidine lowered blood pressure, increased sedation and reduced plasma noradrenaline levels. There was an insignificant fall in levels of plasma renin activity. Prior administration of naloxone did not influence the effects of clonidine. It is concluded that the cardiovascular, sedative and neurohormonal effects of acutely administered clonidine are not dependent on opiate receptor activation in either normal or hypertensive man. PMID- 3007710 TI - Oral granular cell tumors: epithelial hyperplasia associated with Candida? PMID- 3007712 TI - A two-piece surgical splint to facilitate hydroxylapatite augmentation of the mandibular alveolar ridge. AB - Details of the design and fabrication of a two-piece splint that facilitates the HA augmentation of the mandibular alveolar ridge are presented. The use of the splint has eliminated migration of HA particles, reduced elevation of the vestibule, and increased the predictability of the outcome of the procedure. PMID- 3007713 TI - Dietary fiber and peptic ulcer. A review. PMID- 3007711 TI - Elastic fibers in pleomorphic adenomas of the minor salivary glands. PMID- 3007716 TI - Primary Epstein-Barr virus infections in children with AIDS. PMID- 3007714 TI - Pulmonary disease in children with acquired immune deficiency syndrome and AIDS related complex. AB - Two major pulmonary diseases were defined on the basis of lung biopsies in 15 children with acquired immune deficiency syndrome (AIDS) or AIDS-related complex. Pneumocystis carinii pneumonia was observed in eight children, and pulmonary lymphoid hyperplasia in six. One child had nonspecific interstitial pneumonitis. Children with P. carinii pneumonia had more severe hypoxemia, with higher alveolar-arterial oxygen gradients, and higher isomorphic elevations of serum lactate dehydrogenase. Clinically, children with pulmonary lymphoid hyperplasia were older, and had digital clubbing, parotid gland enlargement, and elevated serum IgG levels. Results of serologic assays and lung tissue analysis were suggestive of persistent Epstein-Barr virus infection exclusively in patients with pulmonary lymphoid hyperplasia. Recognition of the clinical and laboratory findings characteristic of each entity may assist in the differential diagnosis without the need of surgical biopsy. PMID- 3007715 TI - Hemophiliac immunodeficiency: influence of exposure to factor VIII concentrate, LAV/HTLV-III, and herpesviruses. AB - The relationship between hemophiliac immunodeficiency and exposures to factor VIII concentrate, LAV/HTLV-III retrovirus, and infection with Epstein-Barr virus and cytomegalovirus was examined. Exposure to factor VIII concentrate was significantly correlated with decreased percentages of T helper/inducer cells, decreased T helper/suppressor cell ratios, and decreased proliferative responses to plant mitogens. LAV/HTLV-III seropositivity was the primary predictor of increased percentages of HLA-DR-bearing mononuclear cells and decreased proliferative responses to pokeweed mitogen. Epstein-Barr virus and cytomegalovirus infections acted in a synergistic manner with LAV/HTLV-III to produce immunoregulatory defects. Increased percentages of T suppressor cells and decreased delayed cutaneous hypersensitivity skin test responses were observed in LAV/HTLV-III seropositive hemophiliacs infected with Epstein-Barr or cytomegalovirus. We conclude that hemophiliacs receiving commercial factor VIII concentrate experience several stepwise incremental insults to the immune system: alloantigens in factor VIII concentrate, LAV/HTLV-III infections, and herpesvirus infections. PMID- 3007717 TI - Gastrointestinal tract angiography in infants and children. AB - In the past 10 years since the development of newer imaging modalities, the method of evaluation of gastrointestinal diseases has changed to less invasive examinations. Angiography of the gastrointestinal tract and its accessory organs, which once was one of the primary procedures for hepatic tumors especially, is now considered nonessential except for occasional demonstration of vascular distribution prior to attempted surgery. As for gastrointestinal bleeding, although the incidence of ulcer disease is not as common in children as in adults, a child with bleeding who is at high risk for surgery will benefit occasionally from intra-arterial infusion of vasopression. As the major cause of gastrointestinal bleeding in children is from esophageal varices, though great anatomical detail of portal circulation can be seen in computed tomography and ultrasound, the coronal mapping of vascular anatomy of portal circulation is of utmost benefit prior to any attempt for surgery. Intra-arterial portography and splenoportography can be very helpful to delineate the anatomy and hemodynamics of portal hypertension, as well as for evaluation of suspected shunt thrombosis. Briefly, an update of information on digital subtraction angiography in gastrointestinal pathology will be given though the pediatric application has not been as popular as in adults. PMID- 3007718 TI - Enteropathogens associated with acute diarrhea in hospitalized infants. AB - Thirty-five infants of low socioeconomic status who were living in urban Santiago were hospitalized for acute diarrhea were prospectively evaluated for the presence of enteropathogens associated with the episode. Some degree of malnutrition was evident in 20 infants (57.1%); 15 of these (75%) were under 6 months of age. Mean duration of the hospital stay was 11.8 days for well nourished patients and 15.7 days for the malnourished patients. One or more enteropathogens were found in 60% of the cases studied: in 17 cases (48.6%) these were bacteria and in four cases (11.4%) it was rotavirus. Parasites were not detected. In three patients, two different pathogens were demonstrated. Among the bacteria, 12 isolates (34.3%) were enteropathogenic E. coli (EPEC) and two (5.7%) were Shigella. Campylobacter jejuni was also isolated from two different cases (5.7%) and Salmonella from one case (2.9%). The recovery of pathogens was independent of the nutritional status. Mean age of detection of EPEC was 3.2 months among well-nourished infants and 6.2 months among the malnourished (p less than 0.001). Half of the EPEC strains isolated were multiresistant to antibiotics. One of these strains transferred some of its resistance in vitro to E. coli K12. Ampicillin and kanamycin were the antibiotics to which EPEC showed the greatest resistance. The other bacterial pathogens were mostly sensitive to antibiotics. Campylobacter jejuni, together with Shigella, was the second most frequent pathogen isolated during episodes of diarrhea. Campylobacter should be included in the routine study of diarrheal episodes in our setting. PMID- 3007719 TI - Is Crohn's disease caused by a lymphotropic virus? PMID- 3007720 TI - A comparison of fibronectin and laminin binding to undemineralized and demineralized tooth root surfaces. PMID- 3007721 TI - Gingival crevicular fluid myeloperoxidase at periodontitis sites. PMID- 3007722 TI - Initial characterization of neutral proteinases from oral spirochetes. PMID- 3007723 TI - Bone formation within porous hydroxylapatite implants in human periodontal defects. AB - Tissue samples from three subjects who had periodontal defects treated with a porous hydroxylapatite implant were investigated using light microscopy and scanning electron microscopy. The 3-month specimen showed connective tissue infiltration through the pores and a narrow zone of bone formation present along the walls of the pores. At 4 months, continued evidence of bone deposition was present with osteocytes, osteoblasts and organization of collagen fibers apparent throughout the implant. The 6-month implant had further evidence of continued bone formation with lamellar bone being the major component within the pores. PMID- 3007724 TI - Ultrastructure of durapatite-periodontal tissue interface in human intrabony defects. AB - The ultrastructure of the interface between Durapatite (hydroxylapatite) and human periodontal tissues was examined. Durapatite was implanted into the intrabony periodontal defects during periodontal surgery. Reentry procedures were performed after 1 year and the tissues in the defects were biopsied and processed for transmission electron microscopy. Nineteen tissue blocks from four patients were examined, 17 contained Durapatite particles embedded in fibrous tissue and two contained particles encased in bone, all without inflammation. The fibrous connective tissue consisted of densely packed collagen fibrils surrounding the implant particles. The bone surrounding the Durapatite consisted in one case of relatively mature bone, and in the other of osteoid tissue. A granular, amorphous, collagen-free, electron-dense layer was routinely observed between implant and tissue. This layer was thicker in the bone-encased samples than in those surrounded by fibrous connective tissue. Except for the particle surrounded by mature bone, this layer was continuous with an organic meshwork located on the periphery of the implant spaces. The ultrastructural features of the interface are consistent with the existence of a mucopolysaccharide "bonding zone" described by other investigators. The organic meshwork appears to outline areas similar in size and shape to the individual crystallites of hydroxylapatite. This may indicate that the reactive surface of hydroxylapatite is much larger than merely the exterior surface of the implant, a finding which may explain the apparently good tissue adhesion to the implant. PMID- 3007725 TI - Effects of the inorganic salts sodium chloride, sodium bicarbonate, and magnesium sulfate upon the growth and motility of Treponema vincentii. AB - The use of inorganic salt solutions as chemotherapeutic agents in the control of periodontal disease has received considerable attention in the past few years. Although some research has been published on their clinical effectiveness, little is known about their therapeutic activity and bactericidal effects upon oral spirochetes. The present study investigated the effects of varied concentrations of NaCl, NaHCO3, and MgSO4 upon the in vitro growth and motility of Treponema vincentii. Growth determinations were performed using a turbidiometric analysis at 545 nm. Motility was qualitatively studied by direct examination of 200 treponemes in a wet mount specimen. Samples were taken at 24, 48, 72, and 96 hours following inoculation with the treponemes. Concentrations of 0.5 M NaCl, NaHCO3, or MgSO4 totally inhibited the growth and motility of T. vincentii over a 96-hour period. Salt concentrations less than or equal to 0.10 M had little if any effect upon growth and motility. The data support the hypothesis that the bactericidal and antimotility effects of these salts are related more to their concentrations than to the presence of a specific inorganic ion. They also suggest that motility may be a valid indicator of bacterial viability. Before the clinical significance of the results can be ascertained, human studies are needed to establish sulcular salt concentrations which can be achieved with local irrigation and to determine how long bactericidal concentrations can be maintained. PMID- 3007726 TI - Hamster and rat pineal gland beta-adrenoceptor characterization with iodocyanopindolol and the effect of decreased catecholamine synthesis on the receptor. AB - Rat and hamster pineal glands were used in binding studies to characterize their beta-adrenoceptors with a new specific antagonist ligand, iodocyanopindolol. The receptors were saturable, and the ligand was selective and demonstrated stereospecificity for both species. The rat pineal had a 20-fold greater density of beta-adrenoceptors, while the affinity was one-third that of the hamster pineal. utilizing this radioligand, we examined the effects of decreased sympathetic input to the pineal on beta-adrenergic receptors in both species. Decreased noradrenergic input to the pineal gland of the hamster was accomplished by superior cervical ganglionectomy, or by exposing the animals to continuous light for 36 hours. Parallel studies were conducted with hamster pineal gland in which catecholamine synthesis was measured. The results indicate that a selective decrease in catecholamine synthesis in the hamster pineal does not change the beta-adrenoceptor density or affinity. In contrast, a concomitant increase in beta-adrenoceptor density but not affinity occurs in the rat pineal gland after similar decreased sympathetic input. PMID- 3007727 TI - Pharmacological studies on the regulation of N-acetyltransferase activity and melatonin content of the pineal gland of the Syrian hamster. AB - Thus far, all attempts to stimulate melatonin synthesis by beta-adrenergic receptor agonists in the Syrian hamster pineal gland have failed. Neither a wide range of dosages of isoproterenol (0.5 mg/kg to 24 mg/kg), nor prolonged treatment with norepinephrine, the natural neurotransmitter, increased N acetyltransferase (NAT) activity or melatonin production. In the present study, the administration of isoproterenol at night was likewise ineffective in advancing or enhancing the normal nightly melatonin peak. Also, we did not find a delayed effect 7 or 8 h after the administration of the drug. Furthermore, we tested the idea of coneurotransmitters such as octopamine or dopamine being possibly necessary for stimulation, but could not find any effect of these substances on melatonin synthesis. In addition, a parasympatholytic agent, atropine, did not increase the responsiveness to sympathomimetic agents. Administration of a phosphodiesterase inhibitor was also ineffective in stimulating NAT activity. On the other hand, isoproterenol did retard the drop in NAT and melatonin after lights-on at night, indicating that beta-receptors are involved in maintaining elevated melatonin levels. PMID- 3007729 TI - Effects of some fatty acids on the serotonin N-acetyltransferase activity in cultured chick pineal glands. AB - Low concentrations of arachidonate, oleate, or palmitate significantly affected the cycle of NAT activity of cultured chick pineal glands in different ways. The effects observed were altered by change of the lighting conditions of culture. Effects of arachidonate were also shown to be altered by change of the serum component of the culture medium. Effects of premature exposure to light of glands cultured under diurnal conditions of illumination were changed markedly by substitution of the serum component of the medium or a supplement of ionophore A23187 and less markedly by supplements of fatty acids. PMID- 3007728 TI - Interdependent effects of the ionophore A23187 and serum on the serotonin N acetyltransferase activity of cultured chick pineal glands. AB - Marked effects of the ionophore A23187 on the cycle of N-acetyltransferase (NAT) activity in cultured chick pineal glands were observed under three conditions of illumination. However, the effects were qualitatively and quantitatively dependent on the batch of fetal calf serum used in the medium and time of explanation into culture. Ionophore increased the level of NAT activity remaining in glands exposed prematurely to light regardless of the serum used. The ionophore suppressed the "spike" in cyclic GMP content of glands cultured in the dark, and extended the period of maximum cyclic GMP content of glands under diurnal illumination. PMID- 3007731 TI - Antihypertensive aminotetralins related to labetalol and medroxalol. AB - A series of compounds was prepared in which the 1-methyl-3-phenylpropylamino moieties of the antihypertensive agents labetalol and medroxalol were replaced by 2-aminotetralins. Compounds containing a 6-methoxy and 6,7-methylenedioxy group in the aminotetralin were at least as active as labetalol in lowering the blood pressure of the spontaneously hypertensive rat (SHR). As determined by ligand binding, these compounds were comparable to labetalol as alpha 1-antagonists but were substantially weaker beta 1-antagonists. PMID- 3007730 TI - Responsiveness of pineal N-acetyltransferase and melatonin in the cotton rat exposed to either artificial or natural light at night. AB - In three separate experiments, the effect of acute exposure to either artificial or natural light during darkness of pineal N-acetyltransferase (NAT) activity and melatonin content was studied in the cotton rat (Sigmodon hispidus). The exposure of animals to an artificial-light irradiance of 160,000 microW/cm2 during darkness for either 1 s, 5 s, or 30 min was followed by a precipitous decline in pineal NAT activity and melatonin content when measured at either 15 or 30 min after light onset. When cotton rats were acutely exposed to light at night for 5 s, irradiances of either 3.2, 32, 320, and 3,200 did not suppress either pineal NAT or melatonin 30 min later; however, if the 5-s exposure had an irradiance of either 32,000 or 160,000 microW/cm2, the pineal enzyme activity and indole content were depressed. Moonlight, which had a maximal irradiance of 0.32 microW/cm2, was unable to suppress pineal NAT activity and melatonin content even when the animals were exposed to the moonlight for 30 min. The treatment of cotton rats with either norepinephrine or its agonist, isoproterenol, before their exposure to light at night retarded slightly the suppressive effect of light on the pineal constituents measured. Also, these drug treatments suppressed the pre-exposure levels of both NAT activity and melatonin content in the cotton rat pineal gland. PMID- 3007732 TI - Thermodynamic proton-ligand and metal-ligand stability constants of some drugs. AB - The thermodynamic proton-ligand (pKa) and metal-ligand stability constants of clioquinol, clofibrate, nitrofurazone, and tetracycline with Cu2+, Zn2+, Mn2+, Mg2+, and Ca2+ have been determined at 35 degrees C in 50% ethanol-water media. An empirical pH correction for mixed-aqueous media has been applied. The metal ligand stability constants were determined by following the Bjerrum Calvin titration technique as applied by Agrawal to mixed-aqueous solvents. The effect of the basicity of the ligand and the order of stability constants is discussed. The stability constants of the divalent metals follow the order: Cu2+ greater than Zn2+ greater than Mn2+ greater than Mg2+ greater than Ca2+ with all the drugs. PMID- 3007733 TI - Podiatric implications of acquired immunodeficiency syndrome. PMID- 3007734 TI - Catecholaminergic regulation of opiate-stimulated growth hormone secretion in the developing rat. AB - In adult rats, the noradrenergic system plays a role in pulsatile and opiate stimulated growth hormone (GH) secretion through stimulation of alpha-2 adrenergic receptors. The present studies examine catecholaminergic mechanisms which might regulate GH release in the neonatal rat. Catecholaminergic involvement in opiate-induced GH secretion was examined in 10-day-old and 30-day old rats. Rats were sacrificed 30 min after administration of methadone (2.5 mg/kg s.c.) and serum was analyzed for GH by radioimmunoassay. Reserpine (1 mg/kg s.c.) abolished methadone-induced GH release in neonatal rats. Haloperidol (1 mg/kg s.c.) but not cyproheptadine (5 mg/kg i.p.) attenuated methadone-stimulated GH release. The role of noradrenergic neurons in opiate-induced GH secretion was examined by pretreating rats with diethyldithiocarbamate (400 mg/kg i.p.) or phenoxybenzamine (10 mg/kg i.p.). Both treatments blocked methadone-induced GH release in neonatal rats. In 30-day-old rats, pretreatment with yohimbine (2.5 mg/kg i.p.), but not prazosin (2.5 mg/kg s.c.) blocked opiate-induced GH release. Although selective blockade by these alpha-1 and alpha-2 antagonists could not be demonstrated in neonatal rats, the alpha-2 agonist, clonidine, did stimulate GH release in a dose-related manner. Findings suggest that noradrenergic mechanisms participate in the regulation of GH secretion in both neonatal and adult rats. A tonically active alpha-2 system is demonstrable in neonates despite the absence of GH surges. The GH response to opiates appears to be mediated through this mechanism. Neural regulation of surging may develop as a separate pathway or become superimposed on the neonatal circuit. PMID- 3007735 TI - Vascular vasopressin receptors mediate inhibition of beta adrenergic receptor induced cyclic AMP accumulation. AB - Beta adrenergic receptor agonists and forskolin stimulated cyclic AMP (cAMP) accumulation in cultured rat aortic smooth muscle cells (A-10). Furthermore, these cells display a high density of vasopressin receptors of the vascular (V1) subtype. Addition of vasopressin to these cells inhibited beta adrenergic agonist and forskolin-stimulated cAMP accumulation by 30 to 40% and by 25 to 35%, respectively. The extent of inhibition was dependent on the concentration of vasopressin used. Half-maximal inhibition of cAMP accumulation by isoproterenol occurred at 8 X 10(-10) M vasopressin. Basal cAMP levels were not affected. The inhibition by arginine vasopressin was mediated by V1 receptors because the V2 renal receptor subtype selective agonists (1-deamino, 8-D-arginine)vasopressin and (1-deamino,4-valine,8-D-arginine)vasopressin were ineffective. Of the antagonists tested, the V1-selective antagonist [1-(beta-mercapto-beta,beta cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]vasopressin was more potent than the mixed V1/V2 antagonist [1-beta-mercapto--beta, beta cyclopentamethylenepropionic acid), 2-D-(O-ethyl)tyrosine,4-valine 8 arginine]vasopressin. The V2-selective antagonist [1-(beta-mercapto-beta,beta cyclopentamethylenepropionic acid),2-D-isoleucine,4-valine,8-arginine]vasopressin displayed minimal ability to block the vasopressin-mediated inhibitory effect. These data demonstrate that in rat aortic smooth muscle cells V1 receptors are negatively coupled to adenylate cyclase. The studies presented suggest that the vasoconstrictor activity of vasopressin might involve inhibition of beta adrenergic receptor-mediated vascular relaxation through inhibition of cAMP accumulation. PMID- 3007737 TI - Roles for Ca++ and cyclic AMP in mediating the cardiotonic actions of isomazole (LY175326). AB - The contractile state of cat papillary muscles was increased by isomazole in a concentration-dependent manner; inotropic effects of the drug were not altered by either prazosin, propranolol or cimetidine. Isomazole inhibited the peak III isozyme of dog heart phosphodiesterase with an IC50 of 100 microM; effects on isozymes I and II were less pronounced. In cat papillary muscles, carbachol (10( 5) M) shifted the relationship between contractility and concentration of isomazole to the right. These data suggest cyclic AMP (cAMP) is involved in the actions of isomazole. In order to assess the relative effects of isomazole on intracellular cAMP and Ca++, cAMP-dependent protein kinase and glycogen phosphorylase, respectively, were used as reporters of these two second messengers. The source of enzymes was either cultured cardiomyocytes or right ventricular biopsies obtained from anesthetized dogs. In the latter case, biopsies were obtained after i.v. administration of isomazole; the pure beta agonist, isoproterenol, was included for comparative purposes. A submaximal inotropic dose of isomazole (0.1 mg/kg i.v.) in dogs resulted in a pronounced increase in contractility that was associated with a 3-fold increase in phosphorylase activity (0.15 +/- 0.01 to 0.46 +/- 0.06, -5'-AMP: +5'-AMP, P less than .05); the activation state of protein kinase was not altered. By contrast, a comparably effective inotropic dose of isoproterenol (0.1 microgram/kg) caused less than a 2-fold increase in phosphorylase activity (0.15 +/- 0.01 to 0.26 +/- 0.02, -5'-AMP: +5'-AMP, P less than .05) and this was associated with a significant increase in the protein kinase activity ratio (0.36 +/- 0.01 to 0.51 +/- 0.04, -cAMP: +cAMP, P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007736 TI - Effects of d-amphetamine and dopamine synthesis inhibitors on dopamine and acetylcholine neurotransmission in the striatum. I. Release in the absence of vesicular transmitter stores. AB - The release of endogenous dopamine (DA) elicited by electrical stimulation and by d-amphetamine (AMPH) from superfused striatal slices of reserpine-pretreated rabbits was examined. Although reserpine pretreatment reduced tissue DA levels by greater than 95%, the basal efflux of DA and the DA metabolite dihydroxyphenylacetic acid (DOPAC) was slightly greater than that observed in untreated slices. DOPAC constituted the large majority of the basal efflux of endogenous compounds. No overflow of endogenous compounds was evoked by electrical stimulation (3 Hz, 3 min) after reserpine pretreatment. Superfusion with alpha-methyl-p-tyrosine (100 microM) abolished the efflux of endogenous DA and DOPAC. AMPH (0.3-10 microM) produced a concentration-dependent increase in the basal efflux of endogenous DA and a concomitant decrease in endogenous DOPAC efflux. The total efflux of endogenous compounds (DA + DOPAC) tended to be decreased by AMPH. No electrically evoked overflow of endogenous compounds was observed in the presence of AMPH. The increase in synaptic DA produced by AMPH was reflected by a concentration-dependent reduction in the electrically evoked overflow of [3H]acetylcholine (ACh). The ability of AMPH to increase DA efflux and inhibit [3H]ACh release was blocked by inhibition of DA synthesis with alpha methyl-p-tyrosine (100 microM) or by blockade of the DA neuronal uptake carrier with nomifensine (NOM) (10 microM) and was potentiated by inhibition of monoamine oxidase with pargyline (10 microM). NOM also blocked partially the ability of AMPH to reduce endogenous DOPAC efflux. NOM increased the basal efflux of endogenous DA and inhibited electrically evoked [3H]ACh release but these effects were quantitatively much less than those produced by AMPH. NOM had no effect on DOPAC efflux. Pargyline had little effect on endogenous DA efflux or electrically evoked [3H]ACh release but abolished DOPAC efflux and increased tissue DA levels measured at the end of superfusion. When given in combination, NOM and pargyline produced a similar degree of inhibition of [3H]ACh release as AMPH, although the increase in DA efflux produced by this drug combination was less than that produced by AMPH. These results suggest that in the absence of vesicular transmitter stores (reserpine-pretreatment): synthesis provides a continuous supply of DA which is metabolized rapidly within the neuron and is lost as DOPAC; AMPH facilitates the synthesis-dependent efflux of extravesicular DA probably by an accelerated exchange diffusion mechanism.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3007739 TI - Presynaptic alpha-2 adrenoceptor activation and coupling of the receptor presynaptic effector system in the perfused rat heart: affinity and efficacy of phenethylamines and imidazoline derivatives. AB - The right sympathetic nerves of perfused rat hearts were stimulated in the presence of inhibitors of neuronal and extraneuronal uptake and propranolol. The inhibition by alpha adrenoceptor agonists of stimulation-evoked (10 pulses, 0.1 Hz) [3H]norepinephrine (NE) overflow into the perfusate was taken as a parameter of presynaptic adrenoceptor activation. Under the present conditions, autoinhibition of NE release is not activated by endogenous NE as evident from ineffectiveness of adrenoceptor antagonists in facilitating evoked [3H]NE overflow. The potency (EC50, -log10), affinity (agonist-presynaptic receptor dissociation constant KA, -log10) and relative efficacies (RE) were determined for phenethylamines (NE or alpha-methylepinephrine) and for imidazoline derivatives. NE (-log EC50, 7.76) was 0.88 log units more potent than alpha methylepinephrine (-log EC50, 6.88) and about the same difference was observed for the -log KA values (5.92 vs. 4.75). RE were similar (NE, 100%; alpha methylepinephrine, 98%) and 22- to 50-fold higher than efficacies of imidazoline derivatives. Hydroxylations in positions 3 and 4 of the phenyl moiety of phenylaminoimidazoline (-log EC50, less than 5; -log KA, less than 5; RE, less than 1%) resulted in a marked increase in potency (-log EC50, 8.32) of the resulting dihydroxyphenylaminoimidazoline due to a high affinity (-log KA, 8.22) at a low efficacy (2% of NE). In contrast, hydroxylation in positions 3 and 4 of the phenyl ring of tolazoline (no agonist activity under the present conditions; antagonist affinity constant from the literature, 6.4-6.6) produced dihydroxytolazoline, a moderately potent agonist (-log EC50, 7.25) with an efficacy of 3.5% at an affinity (-log KA, 6.92) not much different from that of tolazine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007738 TI - Effects of d-amphetamine and dopamine synthesis inhibitors on dopamine and acetylcholine neurotransmission in the striatum. II. Release in the presence of vesicular transmitter stores. AB - The release of endogenous dopamine (DA) elicited by electrical stimulation and by d-amphetamine (AMPH) from superfused striatal slices of untreated rabbits was examined. AMPH (0.3-10 microM) produced a concentration-dependent increase in basal DA efflux (30-fold increase at 10 microM) and stimulation-evoked (SE) DA overflow (11-fold increase at 10 microM). Although AMPH had little effect on the basal efflux of dihydroxyphenylacetic acid (DOPAC), the drug was an effective inhibitor of the SE overflow of the DA metabolite (66% inhibition at 0.3 microM). AMPH increased significantly the total basal efflux of endogenous compounds (DA + DOPAC) only at high concentrations (3-10 microM) whereas the total SE overflow of total endogenous compounds was decreased at all concentrations of AMPH tested. AMPH inhibited SE [3H]acetylcholine (ACh) release in a concentration-dependent manner (71% inhibition at 10 microM). Inhibition of DA synthesis with alpha methyl-p-tyrosine (100 microM) or 3-iodotyrosine (100 microM) reduced both the basal efflux and SE overflow of endogenous DA and DOPAC; synthesis inhibition had greater effects on the SE overflow. Neither synthesis inhibitor altered SE [3H]ACh release. alpha-Methyl-p-tyrosine and 3-iodotyrosine reduced the absolute values of the basal efflux and SE overflow of DA elicited by AMPH by approximately 60%; however, the inhibition of SE [3H]ACh release produced by AMPH was attenuated only slightly (approximately 20%). Synthesis inhibitors also reduced tissue DA levels (approximately 30%). These results suggest that: basal efflux of endogenous DA from superfused rabbit striatal slices may derived both from DA newly synthesized in the cytoplasm and from spontaneous leakage of DA from storage vesicles. In addition, synthesis may provide a continuous supply of DA to vesicles that are used for exocytotic DA release during electrical stimulation. However, the depletion of tissue DA produced by synthesis inhibitors as well as other extraneous pharmacological actions of these drugs makes firm conclusions difficult. AMPH increases the synaptic concentration of DA by accelerating the basal efflux as well as the SE overflow of unchanged DA. At concentrations less than 1 microM AMPH has no effect on basal efflux of DA or DOPAC but reduces SE overflow of DOPAC via an unknown mechanism. At higher concentrations (greater than or equal to 1 microM) acceleration of carrier mediated DA efflux coupled with displacement of DA from vesicular stores, as well as interference with the uptake of exocytotically released DA produces a marked increase in synaptic DA which in turn inhibits SE ACh release.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3007740 TI - Inhibition of angiotensin-converting enzyme with quinapril (CI-906): investigation of antihypertensive mechanisms in spontaneously hypertensive rats. AB - Treatment for 8 days with a new nonsulfhydryl angiotensin-converting enzyme inhibitor, quinapril (CI-906), produced a marked and progressive reduction in the blood pressure of spontaneously hypertensive rats. Quinapril was given p.o. in a dose of 20 or 40 mg/kg once daily. Both doses increased plasma renin activity and decreased the urinary excretion of aldosterone. These results, together with a marked decrease in serum angiotensin-converting enzyme activity, indicate that the drug produced a considerable fall in circulating angiotensin II. The urinary excretion of vasopressin was not altered by the smaller dose of quinapril but was reduced by the larger dose, which increased water intake and urine excretion. Quinapril did not affect plasma kininogen or the urinary excretion of kallikrein. The urinary excretion of neither the prostacyclin metabolite 6-keto-prostaglandin F1 alpha nor the thromboxane metabolite thromboxane B2 were altered by the drug. However, quinapril did produce a temporary decrease in the excretion of prostaglandin E2, the effect passing off with the continuation of the treatment. These data indicate that vasodilatory prostanoids do not contribute to the blood pressure lowering effect of quinapril in spontaneously hypertensive rats. The inhibition of the renin-angiotensin system is probably the principal mechanism of the drug's antihypertensive action, but these results do not rule out the possibility that an increase in vasodilatory kinins may also be involved. PMID- 3007742 TI - Comparison of the effects of muscarine and vasopressin on inositol phospholipid metabolism in the superior cervical ganglion of the rat. AB - Both muscarine and vasopressin have been shown previously to increase the accumulation [3H]inositol phosphates in superior cervical ganglia in which the phospholipids were labeled with [3H] inositol. In this study, we have compared the effects of muscarine and of vasopressin on phospholipid metabolism in the ganglion. The effects of these agents on [3H]inositol phosphate accumulation are additive. The response to muscarine levels off after approximately 10 min, whereas the response to vasopressin increases for at least 30 min. The incorporation of [3H]inositol into phospholipids is enhanced in decentralized ganglia and in ganglia maintained in organ culture compared to freshly isolated ganglia. These treatments appear to potentiate the effect of muscarine on [3H]inositol phosphate accumulation, but do not affect the response of the ganglia to vasopressin. Muscarine and vasopressin also increase the incorporation of [3H]inositol into phospholipids in the ganglion. Autoradiographic techniques were used to localize the inositol-containing phospholipids in the ganglion. Muscarine increases phospholipid labeling primarily in the cell bodies of the principal ganglionic neurons, whereas vasopressin increases phospholipid labeling primarily in the neuropil. These data are consistent with the hypothesis that muscarine and vasopressin stimulate the hydrolysis of different pools of ganglionic phospholipids. PMID- 3007741 TI - Affinity of various purine nucleosides for adenosine receptors on purified myenteric varicosities compared to their efficacy as presynaptic inhibitors of acetylcholine release. AB - Isolated myenteric varicosities (autonomic synaptosomes)prepared from the guinea pig ileum were used as the substrate in competition experiments designed to study the properties of the adenosine receptor present on peripheral nerve endings. Competition experiments using [3H]-N6-[R-(-)-1-methyl-2-phenethyl] adenosine and [3H]-5'-N-ethylcarboxamidoadenosine as the labeled ligands permitted the affinities of several unlabeled adenosine analogs to be examined. In addition, the ability of theophylline, an antagonist at adenosine receptors, to compete for binding was determined. The relative affinities of the nucleosides were compared to their efficacies as inhibitors of acetylcholine release in the stimulated guinea-pig ileum preparation and an excellent correlation was obtained. The identity of the substrate used in the binding studies with the target in the bioassay system suggests that the isolated myenteric varicosity contains the adenosine receptor responsible for the observed biological activity. Similar affinities for theophylline as a competitor of the binding of both labeled ligands paralleled the establishment of similar PA2 values for the antagonist in the bioassay. These findings, together with the similarity of the biological efficacy of N6-[R-(-)-1-methyl-2-phenethyl]adenosine and 5'-N ethylcarboxamidoadenosine, suggest that the adenosine receptor present on myenteric nerve endings is unitary but do not permit its designation as an A1 or A2 subtype. PMID- 3007743 TI - U50,488, a highly selective kappa opioid: anticonvulsant profile in rats. AB - Subcutaneous or i.c.v. administration of U50,488, a highly selective kappa opioid agonist, resulted in a dose-and time-dependent anticonvulsant action in rats. The anticonvulsant effect was seizure-specific; thus, U50,488 protected against supramaximal electroshock seizures but failed to raise the threshold of flurothyl induced convulsions. The ED50 for s.c. U50,488 was 8.6 mg/kg, with a duration of action longer than 8 hr. In contrast, the ED50 for i.c.v. U50,488 was 103.8 micrograms, lasting approximately 1 hr. The anticonvulsant effect of U50,488 was partially antagonized by high (10.0 mg/kg), but not low (1.0 mg/kg), doses of s.c. administered naloxone. Results indicate that U50,488 is an efficacious, long acting anticonvulsant against supramaximal, but not chemical threshold, seizures in rats. Furthermore, the results with naloxone suggest that this effect of U50,488 is mediated by non-mu (probably kappa) binding sites. This structurally novel nonpeptide opioid may offer new insights into the development of therapeutically effective agents in the treatment of grand mal or partial epilepsies. PMID- 3007744 TI - Direct effect of ethanol on adrenocorticotropin (ACTH) release in vitro. AB - In order to determine whether the pituitary directly contributes to the stimulatory action of ethanol on adrenocorticotropin (ACTH) release, the response of rat pituitaries to ethanol was studied in vitro. Acute exposure of superfused rat pituitaries to ethanol (20-200 mg/dl) produced dose-related increases in ACTH which were significant at doses of 40, 80 and 160 mg/dl. The response to each dose was multiphasic, consisting of three peaks of ACTH in the 28-min sampling period after addition of ethanol. The dose-response profile of each peak revealed a maximally effective stimulatory dose of 40 mg/dl of ethanol and the direct stimulatory effect decreased with higher doses, resulting in a bell-shaped pattern. In contrast, ovine corticotropin-releasing factor, in doses of 1.0 to 10.0 nM, produced a single dose-dependent ACTH response, indicating a unitary mechanism for its action as opposed to the probable multiple mechanisms of ethanol's action on the corticotrophs. These results indicate that low doses of ethanol act directly on the pituitary to induce ACTH release, while higher doses may have their primary site of action on the hypothalamus. PMID- 3007745 TI - Effects of ethanol on stimulated inositol phospholipid hydrolysis in rat brain. AB - The effect of ethanol in vitro on inositol lipid metabolism in brain slices was investigated under nonstimulating and stimulating conditions. In cerebral cortical slices 100 microM norepinephrie (NE), 1 mM carbachol, 100 microM serotonin, 20 mM KCl, 1 mM glutamate and 30 microM A23187 stimulated inositide hydrolysis as measured by the release of [3H]inositol phosphates from [3H]myoinositol labeled slices. Ethanol (500 mM) inhibited nonstimulated inositide hydrolysis but had variable effects on stimulated inositide breakdown. NE-, KCl- and glutamate-stimulated [3H]inositol phosphate release was inhibited by 500 mM ethanol in the cortex. The inhibitory effect of ethanol on NE stimulated inositide hydrolysis was concentration dependent and significant at concentrations as low as 100 mM. Inhibition by ethanol appeared to be noncompetitive. A similar pattern of inhibition by ethanol was observed when KCl was the stimulant. In hippocampal and hypothalamic slices, similar to cortical slices. NE- and KCl-stimulated inositide breakdown was significantly inhibited by ethanol. However, in brain stem slices, only KCl-stimulated [3H]inositol phosphate release was inhibited. Striatal slices stimulated by carbachol, NE and KCl were sensitive to the inhibitory effects of ethanol on inositol lipid breakdown. These results suggest that ethanol in vitro has specific effects on inositol lipid metabolism depending on the brain region studied and the type of stimulation. Moreover, the differential sensitivity to ethanol of stimulated inositide hydrolysis in the brain may contribute, at least in part, to some of the pharmacological effects of ethanol in vivo. PMID- 3007746 TI - Excitation of neurones in the rat paraventricular nucleus in vitro by vasopressin and oxytocin. AB - Extracellular recordings were made from ninety-seven spontaneously firing cells in the paraventricular nucleus (p.v.n.) of the rat hypothalamic slice preparation. The spontaneously firing cells tested fired at 0.1-8 spikes/s but the majority showed a slow irregular firing pattern. The average firing rate of all ninety-seven cells was 2.2 +/- 0.2 spikes/s (mean +/- S.E. of mean). Six cells showed a phasic firing pattern. Following bath application of arginine vasopressin (AVP) 10(-7) M, sixty-four (66%) of ninety-seven p.v.n. cells showed excitatory responses and three (3%) cells inhibitory responses. Bath application of oxytocin (OXT) 10(-7) M excited thirty-nine (57%) of sixty-eight p.v.n. cells and inhibited two (3%) cells. Individual p.v.n. cells responded to application of both AVP and OXT, but the magnitude and threshold of the responses varied from cell to cell. Of the sixty-six cells tested with both peptides at 10(-7) M, sixteen showed similar responses to both and fifteen showed no response to either: twenty cells showed a greater response to AVP and fifteen a greater response to OXT. Of six phasic firing cells, two showed excitatory responses to AVP and all four cells tested did not show any response to OXT. The dose dependence of the response to AVP and OXT was tested in six p.v.n. cells. There was a direct relationship between peptide concentration and increased firing rate. The threshold concentration of the peptides ranged from 10(-8) to 10(-10) M. The cells responsive to the peptides were not located in particular areas of the p.v.n. but were diffusely distributed throughout the nucleus. After blocking synaptic transmission with a low Ca2+ and high Mg2+ medium, all tested cells (AVP, n = 15; OXT, n = 14) which had responded to applications of AVP or OXT in normal medium still showed responses to the peptides, although the effect was less marked in half the cells. However, in the absence of synaptic transmission two cells showed unimpaired responses to one of the peptides but greatly depressed responses to the other. The V1-receptor antagonist [1-(beta-mercapto-, beta-cyclopentamethylenepropionic acid)], 8-D-arginine-vasopressin (d(CH2)5DAVP) or V1/V2-receptor antagonist [1-(beta-mercapto-, beta cyclopentamethylenepropionic acid), 2-D-tyrosine,4-valine]arginine-vasopressin (d(CH2)5D-TyrVAVP) completely or partly blocked the AVP-induced responses, while the V2-receptor agonist 1-deamino-8-D-arginine-vasopressin (dDAVP) did not influence the spontaneous discharges of the cells.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3007747 TI - Distribution and properties of excitatory and inhibitory junction potentials in circular muscle of the guinea-pig stomach. AB - In the presence of guanethidine (10(-6)-5 X 10(-6) M), transmural nerve stimulation evoked an excitatory junction potential (e.j.p.) in the fundus region and an inhibitory junction potential (i.j.p.) in the antrum region of the circular muscle of the guinea-pig stomach. The e.j.p. was blocked by atropine (over 2 X 10(-7) M) while the i.j.p. was blocked by apamin (10(-7) M) but not by adrenergic or cholinergic receptor antagonists. Therefore the i.j.p. may be non adrenergic and non-cholinergic in nature. In the presence of atropine, nerve stimulation evoked the non-adrenergic, non-cholinergic i.j.p. in both regions of the stomach. In the antrum region, single stimuli enhanced the subsequent slow wave by 1.1-1.3 times, in comparison with that before the stimuli, and this effect was blocked by atropine (over 2 X 10(-7) M). The reversal potential for the e.j.p. was about -18 mV, while that for the i.j.p. was -87 mV in the atropinized fundus and -89 mV in the antrum region. In the fundus region, a pair of nerve stimulations with short intervals (1-4 s) reduced (depression) and with long intervals (5-20 s) enhanced (facilitation) the second e.j.p. or the i.j.p. After inhibition of acetylcholine esterase (AChE) by physostigmine or neostigmine, nerve stimulation evoked an enhanced e.j.p., and then produced a sustained depolarization with a duration of 3-5 s in the fundus region, while the amplitude of slow waves after nerve stimulation was further enhanced in the antrum region. These effects of anticholinesterases were blocked by atropine. Exogenously applied acetylcholine (ACh) depolarized the smooth muscle membrane; the threshold concentration of ACh was about 1000 times higher in the antrum region (10(-6) M) than in the fundus region (10(-9) M). It is concluded that in the guinea-pig stomach, regional differences in junction potentials may be due to different sensitivities of ACh receptor, and that nerve stimulation evokes a cholinergic e.j.p. in a high-sensitivity region (fundus) and a non-adrenergic, non-cholinergic i.j.p. in a low-sensitivity region (antrum). PMID- 3007749 TI - [Tetanic potentiation of miniature end-plate potential frequency at frog neuromuscular junction in manganese solutions]. AB - Using a sciatic nerve-sartorius muscle preparation of the frog, we have studied the effect of Mn on the increase in miniature end-plate potential (MEPP) frequency that occurs during tetanic stimulation (50 Hz, 2-3 min) of the nerve in nominally Ca2+-free, Mn2+ and Mg2+ solutions. During stimulation the frequency increased over the first minutes to reach an asymptote. The time course for the increase was analyzed following the model proposed by Barton, Cohen and Van der Kloot (1983). The ratio of the Ca2+ bound to the receptor at intervals during the tetanus, b, to the Ca2+ bound before stimulation was begun, b0, was calculated from MEPP frequencies. b/b0 indicates changes in intraterminal Ca2+ concentration produced by the tetanus. In solutions made with no added Ca2+ but containing 1 mM MgEGTA, the increase in b/b0 during stimulation showed a linear or convex time course. Similar time courses were obtained in solutions containing Mn2+ or Mg2+ as the sole divalent cation. On the other hand, when solutions contained Ca2+, the time course for the increase followed a sigmoidal curve. The present results suggest that Mn2+ enters the nerve terminal during stimulation and raises the intracellular Ca2+ concentration, which in turn promotes transmitter release. PMID- 3007748 TI - Inositol 1,4,5-trisphosphate activates pharmacomechanical coupling in smooth muscle of the rabbit mesenteric artery. AB - To clarify the nature of the noradrenaline (NA)-induced contraction, the effects of NA on inositol phospholipid metabolism and the actions of inositol 1,4,5 trisphosphate (InsP3) on skinned muscle of the rabbit mesenteric artery were investigated. NA, in concentrations over 1 nM, reduced the amount of phosphatidylinositol 4,5-bisphosphate (PI-P2) and increased the amount of phosphatidic acid (PA). The maximum reduction in the amount of PI-P2 and the maximum increase in the amount of PA were observed in the presence of 1 microM NA. With prolonged application of NA, the PI-P2 was gradually restored to near the control level, but with repeated applications of NA separated by rinses with Krebs solution, there was a consistent reduction of PI-P2. The NA-induced PI-P2 breakdown was inhibited by the alpha 1-adrenoceptor blocking agent, prazosin. After incubation of the tissue with radioactive inositol-containing solution, NA transiently increased the amount of radioactive InsP3 which was followed by increases in the amount of inositol 1,4-bisphosphate (InsP2) and inositol monophosphate (InsP). After accumulation of Ca by saponin-treated muscle cells of the dog mesenteric artery dispersed by collagenase, InsP3 released Ca stored in cells but InsP2 did not. A23187 (5 microM) but not InsP3 (up to 10 microM), depleted Ca accumulated in the presence of ATP. In saponin-treated skinned muscle tissues, InsP3 in concentrations over 0.3 microM, produced contraction following accumulation of Ca into the store site. InsP3 released Ca from the same source as caffeine. The release of Ca by InsP3 appeared in a concentration-dependent manner and this release also depended on the amount of Ca stored in cells (the median effective dose (ED50) was 3.0 microM in 0.1 microM-Ca and 1.0 microM in 0.3 microM-Ca). We concluded that NA activates alpha 1-adrenoceptors, thus hydrolysing PI-P2 and synthesizing InsP3. This product can release Ca stored in cells as estimated from the contraction in skinned muscle tissues, and also reduces the residual amount of Ca stored in skinned dispersed muscle cells. Contraction evoked by NA through pharmacomechanical coupling can be explained as a function of InsP3. PMID- 3007750 TI - Mandibular augmentation with hydroxyapatite. AB - This study investigated the success of hydroxyapatite mandibular augmentation in nine patients, the histologic response around hydroxyapatite, and the effect of collagen in localizing hydroxyapatite. It can be concluded that hydroxyapatite is biocompatible, causes minimal inflammatory response, and can increase denture retention. However, a large number of patients develop lip paresthesia, and hydroxyapatite migrates. Paresthesia might be avoided by altering the surgical technique to avoid manipulation of the mental nerve, and collagen might be a useful binder to confine hydroxyapatite particles. Further studies are in progress to develop new methods to place the hydroxyapatite and materials to confine it. PMID- 3007751 TI - Detection of Babesia bovis using DNA hybridization. AB - Plasmids containing inserts of Babesia bovis DNA were prepared and clones suitable for use in the diagnosis of B. bovis infections were isolated. Dot blot hybridization with DNA from these plasmids, which probably contain repetitive sequences, can detect after an overnight exposure 100 pg of B. bovis DNA, which corresponds to the amount of DNA present in 50 microliters of 0.01% parasitemic erythrocytes. No detectable cross-hybridization was observed with Babesia microti, Plasmodium falciparum, Plasmodium vivax, Boophilus, or cow DNA. A small amount of cross-hybridization was observed with 10 ng Babesia bigemina DNA. Use of these probes in a hybridization assay may be helpful in the diagnosis of babesiosis in cattle and ticks, in the confirmation of strain identities, and in correlating virulence with particular strains of Babesia. PMID- 3007752 TI - Sequence of the small subunit ribosomal RNA gene from the hypotrichous ciliate Euplotes aediculatus. AB - We have determined the complete nucleotide sequence of the coding region of the small subunit rRNA gene of the hypotrichous ciliate Euplotes aediculatus. It is 1882 nucleotides long and contains several inserts not present in the small subunit rRNA genes of the hypotrichs Oxytricha nova and Stylonychia pustulata. A comparison of the sequences suggests that E. aediculatus is much less closely related to these other two hypotrichs than they are to each other. Although the gene sequence of E. aediculatus is drifting more rapidly than those of these other two species, its faster evolutionary clock is not enough to account for the degree of difference between them. PMID- 3007753 TI - Alternative processing of sequences during macronuclear development in Tetrahymena thermophila. AB - DNA is eliminated during development of the somatic MACronucleus from the germinal MICronucleus in the ciliated protozoan, Tetrahymena thermophila. Facultatively persistent sequences are a class of sequences that persist in the MAC DNA of some cell lines but are eliminated from the MAC DNA of other cell lines. One cloned MAC fragment contains a persistent sequence as well as sequences normally retained in the MAC. When this cloned fragment was used to construct MAC restriction maps of this region in cell lines whose MAC DNAs do, or do not, contain the persistent sequence, extensive variation in the map flanking this region was observed. The different DNA rearrangements of this MIC segment are epigenetically determined during or soon after MAC development. Moreover, different rearrangements may occur among the 45 copies of this MIC segment as a MAC is formed, resulting in polymorphisms that are later resolved by phenotypic assortment. PMID- 3007754 TI - Retroperitoneal yolk-sac tumour. PMID- 3007755 TI - Recent techniques for the conservative management of tubal pregnancy. Surgery, laparoscopy and medicine. AB - Differentiation between radical and conservative surgery for the management of tubal pregnancies depended, in the past, upon whether an effort was made to preserve all or part of the affected fallopian tube. Recent techniques for conservative management include laparoscopic salpingotomy with either an electrical current or the carbon dioxide laser and the use of methotrexate. The latter method does not require surgery. In the near future any laparotomy approach for tubal pregnancy may well be considered nonconservative. PMID- 3007756 TI - The immunopathogenesis of rheumatoid arthritis. AB - Rheumatoid arthritis (RA) may represent a T cell dependent immune response to a restricted antigen(s) within the joint, with inflammatory pathways reflecting secondary recruitment. The nature of the inciting antigen is unknown--the stimulus could be an infectious agent or a host constituent. In patients with RA, the centrality of antiself reactivities and immunogenetic influences is apparent. Further clarification of these mechanisms would result in a potential for antigen specific immunosuppressive therapy. PMID- 3007757 TI - 1-Ethyl-7-[3-[(ethylamino)methyl]-1-pyrrolidinyl]-6,8-difluoro-1,4- dihydro-4-oxo 3-quinoline-carboxylic acid. New quinolone antibacterial with potent gram positive activity. PMID- 3007758 TI - Carbocyclic analogues of 5'-amino-5'-deoxy- and 3'-amino-3'-deoxythymidines. AB - The carbocyclic analogue of 5'-amino-5'-deoxythymidine was synthesized from the carbocyclic analogue of 2,5'-O-anhydrothymidine acetate. The carbocyclic analogues of 3'-amino-3'-deoxythymidine and of 1-(3'-amino-2',3'-di deoxylyxofuranosyl)thymine (an all-cis structure) were synthesized from the carbocyclic analogues of 5'-O-trityl-2,3'-O-anhydrothymidine and 5'-O-trityl-3'-O (methylsulfonyl)thymidine, respectively. The carbocyclic analogue of 5'-amino-5' deoxythymidine inhibited cytopathogenic effects (CPE) induced by a TK+ strain of type 1 herpes simplex virus replicating in L929 (mouse connective tissue) cells, but it did not inhibit CPE in Vero cells. In contrast, the all-cis-3'-azido-3' deoxythymidine analogue demonstrated modest inhibition of CPE in Vero cells, but not in L929 cells. PMID- 3007759 TI - Synthesis, structure, and antitumor and antiviral activities of a series of 5 halouridine cyclic 3',5'-monophosphates. AB - A series of potential prodrug 5-halouridine 3',5'-cyclic monophosphates (5-X cUMPs, X = F, Cl, Br, I, 1-4) has been prepared and tested for antitumor activity against murine leukemia L1210/0 and human lymphoblast Raji/0 cells and their deoxythymidine kinase deficient (TK-) counterparts, as well as for antiviral activity in primary rabbit kidney cells infected with herpes simplex virus type 1 or 2, vaccinia virus, or vesicular stomatitis virus. The 5-halopyrimidine bases, nucleosides (5-X-U), and 5'-monophosphates (5-X-UMP) were tested for comparison. 5-F-cUMP (1) showed reasonably potent inhibition of tumor cell proliferation (ID50 = 0.33-1.6 micrograms/mL), while the remaining diesters displayed ID50's ranging from 210 to greater than 1000 micrograms/mL. 5-F-cUMP was 70- to 300-fold less active than 5-F-dU in the same systems. With TK- L1210 cells, 5-F-cUMP was as potent as with the normal (L1210/0) line but was about fourfold less active with TK- Raji cells compared to Raji/0 cells. The 5-X-cUMPs showed little potency as antivirals. A single-crystal X-ray analysis of the ammonium salt of 5-I-cUMP confirmed its structure and showed the conformation of the phosphate ring to be the expected chair. The ribose pucker is near 3(4)T, and the torsion angle about the beta-glycosidic N(1)-C(1') bond is in the syn range (-84.8 degrees). PMID- 3007760 TI - N-substituent modulation of opiate agonist/antagonist activity in resolved 3 methyl-3-(m-hydroxyphenyl)piperidines. AB - A series of 3-methyl-3-(m-hydroxyphenyl)piperidines with N-substituent variations have been synthesized and resolved, and an X-ray crystal structure of one analogue was determined. The compounds have been characterized, pharmacologically, by detailed opiate receptor binding studies and determination of in vivo analgesia and opiate antagonism. The results indicate that all compounds bind with high selectivity and moderate affinity to mu-receptors with no qualitative difference between enantiomeric pairs. By contrast a striking difference in activities is found, with the (-) enantiomers being pure agonists and the (+) enantiomers having both agonist and antagonist activity. The effect of N-substituents on relative agonist and antagonist potency does not mimic that of fused ring opiates with the N-phenethyl compound, the most potent antagonist. These results together with the X-ray structure obtained suggest that agonist and antagonist activity is initiated by a bimodel binding of the compounds in two different orientations at the mu-receptor site. PMID- 3007761 TI - Geometrical correspondence between phenazocine and the enkephalins. AB - Calculations have been performed on phenazocine using Allinger's MM2 (molecular mechanics II) program with full energy minimization. The N-phenethyl group was found to have considerable flexibility with a number of low-energy conformers. The best N-phenethyl axial conformer was 1.6 kcal/mol higher in energy than the best equatorial one. Calculations were also performed on the beta isomer of phenazocine with the result that the energy difference between the best equatorial and axial conformers rose to a substantial 4.6 kcal/mol. The hypothesis that opiate agonism requires an N substituent in the axial position does not appear to be consistent with the increased potency of beta isomers in which axial N substituents are thermodynamically more unstable. Comparisons have also been made between the low-energy conformers of phenazocine and those that have been observed or proposed for the enkephalins. One conformation of the tyrosine portion of the enkephalins that was observed by X-ray crystallography by Karle et al. was found to be a good fit to morphine-like opiates. The backbone conformer suggested by Gorin et al. was found to be the best fit to the two phenyl rings of phenazocine. PMID- 3007763 TI - Purinergic regulation of basal and arginine vasopressin-stimulated hydraulic conductivity in rabbit cortical collecting tubule. AB - An extracellular adenosine responsive site that stimulates adenylate cyclase activity has been identified in several tissues. There is limited information on the presence and physiologic significance of adenosine receptors in well-defined segments of the mammalian nephron. We therefore examined the effect of adenosine and selected analogues on basal hydraulic conductivity in rabbit cortical collecting tubules (CCT) perfused in vitro. Adenosine and analogues with an intact ribose moiety produced a significant, sustained increase in hydraulic conductivity. No increase in hydraulic conductivity was seen in either time control CCT's or CCT's exposed to an adenosine analogue with an altered ribose moiety. These experiments are compatible with the presence of a functional adenosine receptor which requires an intact ribose moiety and acts to increase hydraulic conductivity in the mammalian CCT. An intracellular adenosine responsive site, termed the "P site," which inhibits adenylate cyclase activity, has also been described in several tissues. We therefore examined the effect of a P site agonist on hydraulic conductivity responses to arginine vasopressin, forskolin and cAMP. P site stimulation with 2'5' dideoxyadenosine inhibited the effect of AVP and of forskolin but not of cAMP to increase hydraulic conductivity. These results are compatible with a functional P site in the rabbit CCT which acts at the catalytic subunit of adenylate cyclase to inhibit hydraulic conductivity. Together, these results demonstrate purinergic modulation of basal and arginine vasopressin-stimulated water flux in the mammalian collecting tubule. PMID- 3007764 TI - Demonstration of pseudorabies virus DNA in the mouse inner ear by an in situ nucleic acid hybridization technique in plastic embedded bony material. AB - This investigation is concerned with the possibility of identifying viral DNA using the in situ DNA hybridization method in methylmethacrylate-embedded material. As an experimental model we chose viral labyrinthitis produced by intranasal infection of the mouse with pseudorabies virus. Fixation and embedding methods specially adapted to this procedure and bony histology preparation technique (specimens by grinding or micromilling) made it possible to identify viral DNA directly morphologically and virologically in the inner ear. Quantitative microphotometric analyses of trans-sagittal sections of the entire skull after in situ DNA hybridization are presented and discussed here as an explicit method of investigating the path of distribution of viral DNA in the brain and the inner ear. PMID- 3007765 TI - Initiation, attenuation and RNase III processing of transcripts from the Escherichia coli operon encoding ribosomal protein S15 and polynucleotide phosphorylase. AB - The rpsO gene of Escherichia coli, which encodes ribosomal protein S15 is located at 69 minutes on the chromosome. It is adjacent to the pnp gene, which encodes polynucleotide phosphorylase. The two genes are separated by 249 nucleotides and are transcribed in the same direction. We report here in vivo S1 nuclease mapping and in vitro transcription experiments that demonstrate that rpsO and pnp are cotranscribed from a promoter P1, located 108 nucleotides upstream from rpsO, and that another promoter P2, located between the two genes 158 nucleotides upstream from pnp, also directs the transcription of pnp. Transcription from P1 can either terminate at the terminator t1 identified in vivo and in vitro, 18 nucleotides downstream from rpsO, or transcribe through t1 and into pnp. Comparison of the transcripts synthesized in wild-type and RNase III-deficient strains of E. coli shows that all the P1 readthrough transcripts and P2 transcripts are cleaved by RNase III. Two specific cuts are made by RNase III in a double-stranded structure about 100 nucleotides upstream rpsO. We also found that some transcripts of this operon start 47 nucleotides downstream from rpsO, in the region of t1. No promoter has been identified in this region. This mRNA is attributed to an endonucleolytic cleavage of the polycistronic transcripts and the location of the cut is named M. The order of the transcription signals and of the maturation sites in relation to rpsO and pnp can be summarized as follows: P1, rpsO, t1, M, P2, RNase III-processing sites, pnp. The possible roles of mRNA processing events in the expression of rpsO-pnp operon are discussed. PMID- 3007762 TI - Transferrin receptor: its biological significance. PMID- 3007767 TI - Effects of cadmium inhalation on mitochondrial enzymes in rat tissues. AB - Pulmonary and extrapulmonary effects from a 2-h inhalation exposure to cadmium (850 micrograms Cd/m3) were studied in male rats. The effect of this chemical on mitochondrial enzyme activity in the lung, liver, kidney, and testis were investigated immediately after exposure and at 48, 144, and 336 h postexposure. In all tissues studied, mitochondrial citrate synthase activity was significantly increased immediately after the cessation of the exposure. This activity level began to decrease at 48 h postexposure. Succinic dehydrogenase activity was significantly decreased in the lungs and kidney at all periods tested, but increased activity was seen in the liver and testis. Cytochrome c oxidase activity in lungs and testis mitochondria was inhibited at all time periods studied. In the liver and kidney this activity was significantly increased immediately after the exposure ceased, and then a significant reduction began to appear at 48 h postexposure. This study demonstrates that inhaled cadmium, after deposition in the lungs, may alter various enzyme activities in other organs. PMID- 3007766 TI - Reduced in vitro 32P incorporation into phospholamban-like protein of sarcolemma due to myocardial ischaemia in anaesthetized pigs. AB - The mechanism of Ca2+ overload production in ischaemia-reperfusion of the heart is unclear. The present study was designed to examine whether loss of second messenger (Ca2+ and cyclic AMP) control of sarcolemmal Ca2+ transport systems occurs during ischaemia. Ischaemic (1, 2 and 3 h duration) and non-ischaemic tissue samples were taken from the coronary-ligated porcine heart and a membrane fraction, enriched in sarcolemmal vesicles, was isolated. The membranes were phosphorylated using [gamma-32P] ATP in the presence of either cyclic AMP or Ca2+ calmodulin. The in vitro 32P incorporation into the electrophoretically separated phospholamban-like protein, became markedly reduced depending on the duration of ischaemia. The reduction could neither be attributed to factors as ischaemia induced changes in membrane-bound kinase or phosphatase nor in situ phosphorylation of phospholamban. It is postulated that during ischaemia and reperfusion, a deficient control of the sarcolemmal Ca2+ pump by phospholamban like protein may serve as a source of intracellular Ca2+ overload. PMID- 3007768 TI - Lipid and glycolipid metabolism of cultured astrocytes: a time course study. AB - Primary cultures of astrocytes free of neurons and containing less than 1% of oligodendrocytes were examined for their ability to incorporate labeled precursors into lipids and glycolipids. At selected developmental stages cultures were double-labeled with either [3H]glycerol and [14C]acetate or with [3H]galactose and Na2[35SO4] for a total of 72 hr. Lipids were extracted with CHCl3/CH3OH, fractionated on a silicic acid column, and further resolved by two dimensional thin-layer chromatography. It was found that cultured astrocytes actively incorporate acetate and glycerol into various phospholipids; they have very limited ability to utilize galactose and virtually lack the synthetic machinery to use Na2SO4 for the synthesis of sulfated sphingogalactolipids; and their overall lipid metabolism is very distinct from that of oligodendrocytes. It was also found that cultured astrocytes have low levels of 2',3' cyclic phosphodiesterase and glycerol phosphate dehydrogenase activities; the latter is less than one fifth of that in oligodendrocytes. PMID- 3007769 TI - Theiler's virus replication in isolated Schwann cell cultures. AB - Theiler's murine encephalomyelitis viruses causing both fatal encephalitis (GDVII virus) and chronic demyelinating disease (WW virus) are capable of replicating in isolated Schwann cell cultures. Light microscopy combined with immunohistochemical staining of viral antigens revealed that large numbers of Schwann cells infected with the two viruses show cytopathic effect (rounding) and contain viral antigens. Electron microscopy of virus-infected Schwann cells shows that the morphological alterations that the cells undergo following infection by the two virus isolates are different. In the early stages of GDVII and WW virus infection, different inclusion bodies are formed in the cells cytoplasm. At late stages of the infection GDVII virions are found in all infected cells and are arranged in crystalline arrays around inclusion bodies. In contrast, in WW virus infected Schwann cells only in few cells virions were observed and they appeared aligned between two membrane units. PMID- 3007771 TI - Enhancing effect of preadministration of carbon tetrachloride on methylazoxymethanol acetate-induced intestinal carcinogenesis. AB - This study concerns the modifying effect of carbon tetrachloride (CCl4) on methylazoxymethanol acetate (MAM)-induced intestinal carcinogenesis in ACI rats of both sexes. Forty five animals were given CCl4 (0.5 ml/kg body weight) through a stomach tube, followed by an i.p. injection with MAM (25 mg/kg body weight) 24 hours after CCl4 treatment. The paired administrations were done once a week for 4 weeks and animals were observed until sacrifice 30 weeks later. Pretreatment with CCl4 caused not only early death from chemical toxicity of MAM but also an increase in small-bowel tumors. PMID- 3007770 TI - Mu-type calcium-activated neutral protease in the rat peripheral nerve. AB - Previously we reported results of an incubation experiment with neurofilaments that supported the existence of a mu-type of Ca2+-activated neutral protease in the rat peripheral nerve; it was active with microM order Ca2+ (mu-CANP). This time, we partially purified the mu-CANP from a crude CANP fraction of rat peripheral nerve by using a DE52 column and a Phenyl-Sepharose column followed again by DE52 column chromatography. The presence of mu-CANP was verified by an immunoblotting technique. The mu-CANP degraded the neurofilament triplet as previously reported; i.e., among the neurofilament triplet, the 160K component was most sensitive, and in the order of the 160K, 68K, and 200K components, respectively. PMID- 3007772 TI - Tumorigenesis and cystic lesion of the liver by N-bis(2-hydroxypropyl)nitrosamine in ddY mice. AB - The tumorigenesis and cystic lesion by a single intraperitoneal administration (ip) of N-bis (2-hydroxypropyl)nitrosamine (DHPN) for 52 weeks were studied in ddY mice. The amount of DHPN was 1000 mg/kg in group I, 500 mg/kg in group II, 250 mg/kg in group III, 125 mg/kg in group IV and 0 mg/kg in group V. The tumorigenesis of DHPN was found in the lung and liver. However, cystic lesion was observed only in the liver. Lung tumors were adenoma, adenocarcinoma and squamous cell carcinoma. As liver tumors, adenoma, hepatocellular carcinoma, cholangioma and hemangioma were observed only in the mice treated with DHPN. Incidence of cystic lesion in the liver was detected in all groups treated with DHPN. Histologically, cystic lesion of the liver showed four patterns of bile duct like, sinusoid-like, hepatocyte-like and mixed. PMID- 3007773 TI - Laminated cytoplasmic bodies in Schwann cells and phagocytes: an ultrastructural and cytochemical study in the normal and lead-damaged peripheral nervous system of the rat. AB - We studied the distribution and cytochemical characteristics of laminated bodies (LBs) in the peripheral nervous system of normal and lead-intoxicated rats. In normal rats, LBs were exclusively present in myelin-forming Schwann cells (SCs). Nerves from lead-intoxicated animals showed extensive demyelination and remyelination. In these nerves we found an increase of LBs in the SC cytoplasm, and also within phagocytes involved in myelin removal, but not in remyelinating SCs. Cytochemical studies revealed that LBs were positive for acid phosphatase, thus demonstrating the lysosomal nature of such inclusions. Taken together, these data suggest that LBs are autophagolysosomes derived from myelin catabolism, which may be enhanced in the lead-induced demyelinating neuropathy of the rat. The possible mechanisms underlying this phenomenon and their pathological relevance are discussed. PMID- 3007775 TI - Ultrastructural study of hemangiomas. 3. Specific endothelial granules in the cerebellar hemangioblastoma. AB - We report a case of cerebellar hemangioblastoma in a 45-year-old man. To our knowledge, only two papers have been reported previously on the cystic inclusions other than Weibel-Palade bodies in the endothelial cells, and this is the first reported case of a hemangioblastoma exhibiting large, cystic endothelial inclusions with microtubular subunits. The function of these inclusions is obscure. PMID- 3007776 TI - The fate of plasma membrane macrophage enzyme markers during endocytosis of Trypanosoma cruzi. AB - The enzyme activity of Na+-K+-ATPase, Mg++-ATPase, 5'-nucleotidase and adenylate cyclase was cytochemically detected, at the ultrastructural level, in normal mouse peritoneal macrophages Trypanosoma cruzi. Reaction product, indicative of the enzyme activity, was seen in association with the plasma membrane of the macrophage, showing an homogeneous distribution. No reaction product indicative of Na+-K+-ATPase, Mg++-ATPase or adenylate cyclase activity was seen within the cell. Reaction product, indicative of 5'-nucleotidase activity, was seen in cytoplasmic vacuoles. Macrophages incubated in the presence of T. cruzi ingest this parasite which can be seen within membrane-bounded cytoplasmic vacuoles (phagosomes). No activity of the plasma membrane-associated enzymes was seen in the membrane lining vacuoles containing parasites. This observation, together with others previously reported, suggest that the macrophage possesses mechanisms which determine the components of the plasma membrane which are interiorized during the process of endocytosis. PMID- 3007777 TI - Naltrexone. AB - Naltrexone (Trexan R), a long acting, orally affective narcotic antagonist was approved by the Food & Drug Administration in November, 1984 for use as an adjunct in the treatment of Opioid Addiction. This research capsule will explore what is known about naltrexone, and the most appropriate ways of using the drug. PMID- 3007774 TI - Sinusoids ultrastructure of human hepatocellular carcinoma. AB - Sinusoids ultrastructure was studied in a case of hepatocellular carcinoma developed in the non cirrhotic liver of a 40-year-old man. The surgical biopsy was perfusion-fixed with 1.5% glutaraldehyde. The number of Kupffer cells was very low. Endothelial cells with signs of hyperactivity were very irregular; digitations were often attached by numerous well identified junctional complexes to their own cell processes or to adjacent cell processes. Perisinusoidal cells without lipids resembled fibro-myofibroblasts. Discontinuous basement membranes underlaid endothelial cells and perisinusoidal cells. In addition numerous strands of short basement membranes segments were seen in the Disse space. Well organized bundles of collagen were not seen. The sinusoidal membranes of hepatocytes were flattened. The perfusion-fixation revealed to be a very useful technique in the identification of all these changes which have also been reported to some degree in benign liver cell adenoma and in cirrhosis; these two conditions are known to be associated with hepatocellular carcinoma. PMID- 3007778 TI - Structure of two-dimensional crystals of membrane-bound Na,K-ATPase as analyzed by correlation averaging. AB - The structure of two-dimensional crystals of membrane-bound Na,K-ATPase from rabbit kidney has been analyzed with a correlation averaging procedure. Two principally different crystal forms are observed with p1 and p21 symmetry, respectively. In the p1 form the averaged projection structure shows a triangular shaped protein domain interpreted as a protomer (alpha beta-unit) of Na,K-ATPase. In the p21-form the stain-deficient area is extended toward a twofold symmetry axis. The results are in good agreement with a previous analysis where Fourier methods were applied to well ordered crystals of pig kidney Na,K-ATPase and illustrate that the correlation averaging procedure can be used for the analysis of membrane crystals of Na,K-ATPase showing curved lattice lines. PMID- 3007779 TI - Congenital mesoblastic nephroma of infancy associated with hypercalcemia. AB - We report a case of hypercalcemia associated with congenital mesoblastic nephroma. Hypercalcemia was corrected preoperatively and a nephrectomy was performed. Postoperatively, the serum calcium returned to normal. The patient has been followed for more than 18 months, during which time he has remained free of disease with normal serum calcium. PMID- 3007780 TI - Mucinous adenocarcinoma of prostate: a case report and review of the literature. AB - Mucinous adenocarcinoma of the prostate is rare and its biological behavior is not well known. We report a case of mucinous adenocarcinoma of the prostate, which was treated successfully with castration. Positivity for prostatic specific antigen by immunohistochemistry confirmed the prostatic origin of this tumor. A review of the literature revealed 30 authentic cases. Prostatic mucinous adenocarcinoma has been said to be different clinically from ordinary prostatic adenocarcinoma. It is insensitive to hormonal therapy, rarely produces acid phosphatase and rarely metastasizes to the bone. However, our case, together with the frequent presence of coexisting acinar elements in mucinous adenocarcinoma, indicates no significant difference in the clinical behavior between mucinous and ordinary acinar carcinomas. PMID- 3007781 TI - Changes of Lex and dimeric Lex haptens and their sialylated antigens during development of human kidney and kidney tumors. AB - The carbohydrate antigen termed Lex (Gal beta 1----4[Fuc alpha 1----3]GlcNAc beta 1----R), its di- or trimeric form, and their sialylated antigens have been characterized as developmentally regulated, tumor-associated antigens in human gastrointestinal epithelia. In this paper, remarkable changes of these antigens, defined by respective monoclonal antibodies FH3, FH4, and FH6, in fetal kidney (mesonephros and metanephros) and other urogenital organs, as well as in various types of kidney tumors, have been investigated. During the development of each organ and tissue, the antigens were found to be maximally expressed at a defined period of organogenesis, and a shifting of expression from one locus to another was observed. Each antigen showed a slightly but clearly different stage of maximum expression. The following changes in metanephros development are of particular interest. Expression of the antigen defined by FH3 followed by the antigen defined by FH4 appeared only after six weeks of gestation in the convoluted tubuli at the central region of metanephros, and propagated rapidly into those at the peripheral cortex region with a simultaneous regression at the central region. The regression of FH4 antigen was more rapid than that of FH3. All three antigens were expressed in the medullar thin tubuli, which develop into the thin-limb of Henle's loop, in which only the antigens defined by FH3 and FH4 persisted and FH6 antigen disappeared. Well-differentiated, but not undifferentiated, renal adenocarcinomas strongly expressed the antigens defined by FH4 and FH6, although the antigen defined by FH6 was expressed in more differentiated tumor cells than the antigen defined by FH4. Well-differentiated cells organized into tubular structures showed a strong expression of these differentiation antigens. However, some tumor cells that were organized into tubular structures, but were characterized by undifferentiated cytomorphology (larger nucleus and smaller volume of cytoplasm), did not express FH4 and FH6 antigens. Thus, cytodifferentiation and histotypic differentiation proceed independently within kidney tumors. The fucosylated type 2 chain structures defined by these three monoclonal antibodies are useful markers that indicate the degree of tumor differentiation. PMID- 3007782 TI - Effects of pyruvate salts, pyruvic acid, and bicarbonate salts in preventing experimental oxalate urolithiasis in rats. AB - Sodium pyruvate, potassium pyruvate, pyruvic acid, sodium bicarbonate and potassium bicarbonate were added to a calcium-oxalate lithogenic diet (a glycolic acid diet) in order to determine their effects in preventing lithogenicity. Male Wistar-strain rats who had been fed the glycolic-acid diet developed marked urinary calculi within four weeks. Rats in the sodium and potassium pyruvate groups had, however, almost no stones in the urinary system. Rats in the bicarbonate and pyruvic-acid groups showed slightly less effect than those in the pyruvate groups. Urinary oxalate excretion was high in all the groups during the experiment. The urinary oxalate concentration was relatively higher in the sodium pyruvate group, and significantly higher in the potassium-pyruvate group, than in the glycolic-acid group. Urinary citrate excretion was high both in the pyruvate and bicarbonate groups; the urinary citrate concentration was, however, significantly higher in the pyruvate groups than in the bicarbonate groups at the fourth experimental week. The urinary calcium and magnesium concentrations were irrelevant to the diets administered. Therefore, it can be concluded that pyruvate salts inhibit urinary calculi formation, not by decreasing oxalate synthesis, but by increasing the urinary citrate concentration; bicarbonate salts work in the same manner, but a little less effectively. PMID- 3007783 TI - Oncogenes: a review with relevance to cancers of the urogenital tract. PMID- 3007784 TI - Primary or secondary extragonadal germ cell tumors? AB - We reviewed 16 patients treated for primary extragonadal germ cell tumors whose testes were initially negative for cancer at palpation. Residues compatible with an occult testicular primary, overlooked at the pretreatment examination, were found in 10 of 12 patients with retroperitoneal germ cell tumors, whereas the testes in all 4 patients with mediastinal germ cell tumors showed no pathological signs. Therefore, we conclude that mere palpation to exclude a testicular primary is not sufficient and the testes of patients with so-called extragonadal germ cell tumors should be examined by all available means, at least by high frequency ultrasound. Orchiectomy is advisable if a focal lesion is found. PMID- 3007785 TI - Sensitivity of 99mtechnetium-dimercaptosuccinic acid for the diagnosis of chronic pyelonephritis: clinical and theoretical considerations. AB - Radioisotopic renal imaging proved to be much more sensitive than excretory urography in diagnosing renal parenchymal damage in 6 children with acute febrile urinary tract infections. This increased sensitivity may affect clinical management. More importantly, it may change the interpretation of scientific studies evaluating the natural history and treatment of vesicoureteral reflux. PMID- 3007786 TI - Partial nephrectomy for Wilms tumor. AB - We report a case of a left Wilms tumor that was treated successfully by partial nephrectomy. The advantages and disadvantages of this approach are discussed. PMID- 3007787 TI - The current management of bilateral Wilms tumor. AB - The good prognosis of synchronous bilateral Wilms tumor seems inappropriate for the magnitude of the disease process. Our experience with 11 cases demonstrates the unusual tumor response to chemotherapy and limited preservative surgery. Although 2 patients died 2 with metastatic disease have responded to chemotherapy and are alive at 5 and 8 years after treatment. In addition, 1 patient has survived with biopsy only and no definitive surgery. Although all surgical options have been used, ranging from biopsy alone to bench surgery with autotransplantation to bilateral nephrectomy, our experience demonstrates the effectiveness of a conservative approach with initial biopsy, chemotherapy and subsequent partial nephrectomy if needed. Our survival data and the histological examination of the tumors after chemotherapy suggest a strong relationship of bilateral Wilms tumor to the nodular renal blastema-nephroblastomatosis complex, and a mechanism to explain the excellent tumor response to chemotherapy. PMID- 3007788 TI - Wilms tumor. PMID- 3007790 TI - Disinfection for HTLV-III: halogenated soaps. PMID- 3007789 TI - Human T-cell lymphotropic virus type III infection in a cohort of homosexual men in New York City. AB - Using blood samples collected since 1978, we investigated the epidemiology of human T-cell lymphotropic virus type III (HTLV-III), the etiologic agent of the acquired immunodeficiency syndrome, in a group of 378 homosexually active men who have resided in New York City since the acquired immunodeficiency syndrome epidemic began. The anti-HTLV-III prevalence was 6.6% in sera from 1978 or 1979, and the subsequent annual incidence of seroconversion among susceptible men ranged between 5.5% and 10.6%. The highest incidences were in recent years, even though these men reported a decrease in their sexual activity during this time. These data demonstrate the continuing risk of HTLV-III infections in the homosexual population studied and emphasize the need for more effective prevention of transmission. The year during which antibody was first present was the only factor identified that was associated with altered cell-mediated immunity in antibody-positive men. Men who became antibody positive in 1981 or earlier currently had significantly lower OKT4/OKT8 ratios than did those who seroconverted more recently. Further follow-up will be necessary to establish the reasons for this association. PMID- 3007791 TI - Spectrum of human T-cell lymphotropic virus type III infection in children. Recognition of symptomatic, asymptomatic, and seronegative patients. AB - This study was done to gain insight into the clinical spectrum and immunologic disturbances resulting from infection with the human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) in children. Serum antibody to p41 antigen of HTLV-III and/or direct evidence of HTLV-III in lymphocytes was considered indicative of HTLV-III/LAV infection. In 36 children with HTLV-III/LAV infection, a wide clinical spectrum was noted, ranging from asymptomatic (seven children) to symptomatic, the latter including 14 children with the acquired immunodeficiency syndrome. Microcephaly was noted in five symptomatic infants. Immunologic dysfunction was noted in all symptomatic and in two asymptomatic children. Panhypogammaglobulinemia was noted in one child. Two asymptomatic children who were HTLV-III antibody negative had virologic evidence of HTLV-III infection. All of 20 mothers who were studied were HTLV-III antibody positive and had immunologic abnormalities but only nine were symptomatic, indicating that apparently healthy women may transmit HTLV-III/LAV infection to their offspring. PMID- 3007792 TI - HTLV-III/LAV infection in hemodialysis patients. AB - Twenty-five (4.8%) of 520 hemodialysis patients were seropositive for antibody to human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV III/LAV) by enzyme immunoassay. Four had high reactivity on enzyme immunoassay and positive results of Western blot tests, and one of the four had a positive culture. The remaining 21 seropositive patients had low reactivity on enzyme immunoassay, negative results of Western blot tests, and negative cultures. All had received blood transfusions and 19 had antibodies to antigens associated with the H9 cell line used to propagate HTLV-III for serological tests. We found that HTLV-III/LAV was not transmitted in the dialysis centers. Frequent blood transfusion places dialysis patients at risk for HTLV-III/LAV infection, but may more commonly lead to false-positive results of enzyme immunoassay tests. PMID- 3007794 TI - Mechanism responsible for "down regulation" during successive beta-agonist administration in rat hearts. AB - The role of phospholipase (PLase) on the mechanism of the so called "down regulation", which decreases the number of beta-adrenergic receptors (beta-AR) of rat cardiac membranes during successive beta-agonist administration, was investigated. In vivo study: The rats were divided into 3 groups: (1) the control group, untreated; (2) the isoproterenol (ISP) one day group, ISP (10 mg/kg) was subcutaneously injected once; and (3) the ISP 6 days group, ISP (10 mg/kg) was injected once a day for 6 successive days. Binding assay using [3H] dihydroalprenolol and analysis of the content and the composition of fatty acids in phospholipids in cardiac membranes were conducted. The endogenous PLase activity in heart homogenate and tissue ATP levels were also determined. In the 6 days group, the equilibrium dissociation constant of beta-AR was not affected; however, the number of beta-AR decreased significantly when compared with the control group. Phospholipid content also decreased in parallel with the decrease in the number of beta-AR and the composition of fatty acids was altered. However, in the one day group, there were no significant changes in either the number of beta-AR or the phospholipid content. The PLase activity of heart homogenate increased significantly, and the tissue ATP levels decreased in both the one day and the 6 days group. In vitro study: Cardiac membranes prepared from intact rats were incubated with 0.03 and 0.1 unit of PLase A2. PLase A2 reduced the number of beta-AR dose-dependently.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3007793 TI - Serum levels of vitamin D metabolites in children receiving total parenteral nutrition. AB - Serum levels of 25-hydroxyvitamin D (25-OHD) and 1,25-dihydroxyvitamin D (1,25 (OH)2D) were measured on 19 occasions in seven children receiving total parenteral nutrition (TPN). The daily intakes of vitamin D3 ranged from 44 to 540 IU/day, and all serum samples were obtained after the same daily intake of vitamin D3 for more than 1 month. There was a significant positive correlation between serum 25-OHD levels and parenteral vitamin D3 intakes (r = 0.90, p less than 0.01). In this study, serum 25-OHD levels in all cases taking 200 to 360 IU/day of vitamin D3 were within the normal range. On the other hand, no significant correlation was found between serum 1,25-(OH)2D levels and vitamin D3 intakes, and serum 1,25-(OH)2D levels were normal or elevated in all cases. PMID- 3007795 TI - Mural thrombi in mice with acute viral myocarditis. AB - In an investigation of the relationship between mural thrombosis and congestive heart failure (CHF) in acute viral myocarditis, inbred BALB/c mice were inoculated intraperitoneally with the M variant of encephalomyocarditis (EMC) virus and sacrificed on days 7 (n = 33), 10 (n = 35) and 14 (n = 39). Myocarditis was found in 105 of 107 sacrificed mice (98.1%). Myocardial necrosis with cellular infiltration was evident after day 7 and was extensive in the ventricles and atria. In the 105 mice with myocarditis, 17 mice (16.2%) developed CHF after day 10, and 15 mice (14.3%) had thrombi as early as day 7. All thrombi were in the left and/or right atrium. The incidence of thrombi in mice with CHF was higher, but not significantly so, than that in mice without CHF (23.5% vs 12.5%). All 50 control mice had no myocardial lesions or thrombi. This study demonstrates that thrombus formation was not rare in the absence of CHF in the acute stage of viral myocarditis and suggests that clinically acute viral myocarditis has a risk of systemic and/or pulmonary embolism even when resting cardiac function is normal. PMID- 3007796 TI - Relationship between the myofibrillar ATPase activity of human biopsy material and hemodynamic parameters. AB - The myofibrillar ATPase activity and pyrophosphate gel electrophoretic pattern of native myosin of fresh human left ventricular papillary muscles were examined in 52 cases of mitral valve replacement. The myofibrillar ATPase activity of hypertrophied myocardium did not differ from that of non-hypertrophied myocardium (mean +/- SD, 36.2 +/- 8.7 vs 31.8 +/- 8.6 nmolPi/mg/min, ns) and there was no significant difference in myofibrillar ATPase activity as a function of left ventricular enddiastolic pressure. Pyrophosphate gel electrophoresis of myosin revealed the presence of two components. It is questionable whether the component of higher electrophoretic mobility (approximately 25-35% in concentration) is identical with rat ventricular myosin VM-1 because an increase in this component seems to correlate with a decrease of myofibrillar ATPase activity, its concentration was significantly higher in the hearts with left ventricular hypertrophy, high enddiastolic pressure, high aortic pressure or low cardiac index. From these results, it is not necessarily clear whether hemodynamic overload in valvular heart diseases can alter left ventricular myofibrillar ATPase activity, but it can be said that the overload influences the concentration of the two components of native myosin revealed by pyrophosphate gel electrophoresis. PMID- 3007797 TI - Vasodepressor effects of prazosin during insulin-induced hypoglycemia in hypertensive patients. AB - As both hormonal and hemodynamic alterations similar to those occurring during exercise can also be produced in humans by hypoglycemia, the present study explored changes in hemodynamic parameters during hypoglycemia and the effects of the alpha 1-adrenergic blocker, prazosin, on those responses in hypertensive patients. In the control group, which did not receive prazosin, plasma epinephrine, plasma norepinephrine and plasma renin activity (PRA) all increased along with a rise in blood pressure during hypoglycemia. On the other hand, the blood pressure decreased despite similar increases in plasma catecholamine levels and PRA in the prazosin treated group. The hemodynamic parameters, analyzed using M-mode echocardiography, changed in both the control and prazosin groups during hypoglycemia; stroke volume and cardiac output showed similar increases. However, while the total peripheral resistance did not change significantly in the control group, it decreased in the prazosin group during hypoglycemia. In accord with the changes in total peripheral resistance, the increment in mean-velocity of circumferential fiber shortening (m-Vcf) during hypoglycemia was greater in the prazosin group than in the control. These results suggest that: hypoglycemia stimulates the sympatho-adrenal axis which then releases catecholamines leading to a rise in blood pressure and tachycardia; In contrast, the blood pressure decreases during hypoglycemia in the prazosin group despite an increase in plasma catecholamines, because the alpha-receptors are blocked by prazosin and the unopposed beta-adrenergic effects of the catecholamines are pronounced enough to reduce the total vascular resistance. PMID- 3007798 TI - Histamine induced or enhanced delayed afterdepolarization and triggered activity in guinea-pig papillary muscles. A preliminary study. AB - We used standard microelectrode techniques to study the histamine induced or enhanced delayed afterdepolarization (DAD) and triggered activity (TA) of guinea pig papillary muscle superfused with low-potassium Tyrode's solution. Before histamine, a series of driven action potentials did not induce DAD and TA. Immediately after histamine (10(-5)M), DAD was induced and, finally, TA was induced after high rate pacing (150/min to 300/min). The effect of histamine was antagonized by cimetidine (5 X 10(-6) to 5 X 10(-5)M) but not by diphenhydramine. Also, the amplitude of DAD decreased after verapamil (10(-7) to 3 X 10(-6)M) and lidocaine (4 X 10(-5) to 8 X 10(-5)M). To investigate indirect evidence of increased cyclic AMP mediation in this histamine induced DAD, we studied the effects of a phosphodiesterase inhibitor (papaverine 10(-5)M) or activator (N methylimidazole 20 mM) on the histamine induced or enhanced DAD. The former enhanced and the latter depressed the histamine-induced (or enhanced) DAD. Thus, histamine may induce or enhance the DAD and TA by increasing the slow inward current. This mechanism may be mediated by histamine H2-receptors and the adenylate cyclase system in the cardiac ventricular muscle. PMID- 3007799 TI - [The cancer family syndrome--a case report]. AB - A family diagnosed as cancer family syndrome because endometrial cancer was found in a member of this colorectal cancer-prone family was reported. The fact that uterine (endometrial) cancer was found in a family member during the serial follow-up of this family shows that there is no more than a difference of time between familial colorectal cancer and cancer family syndrome. The clinical significance of the familial cancer aggregation would be an important clue in the early detection of another new cancer in the family. PMID- 3007800 TI - [Retrograde metastasis of hepatocellular carcinoma with an interesting lesion of the stomach--a case report]. AB - A 48-year-old man with hepatocellular carcinoma (HCC) showing tumor emboli in the portal vein and a typical retrograde metastasis via the portal vessels, is reported. Metastatic lesions were localized only in the veins of the lesser omentum, stomach, lower esophagus, pancreas, left hemidiaphragm and left adrenal gland, due to the hemodynamic alteration of the portal blood flow caused by liver cirrhosis and HCC. No metastatic lesion was found in the lung, Kidney, bone or intestine. As gastric metastasis Borrmann I, II, III and submusal tumor types were reported, but the present case revealed hard fold-like lesions, as it were, hard white varices. PMID- 3007801 TI - [Spinal cord compression complicating gynecological malignancy--report of four cases]. AB - Five episodes of spinal cord compression (SCC) in four patients with gynecological malignancy are described. Two patients had a recurrent cervical cancer, one had a recurrent uterine sarcoma, and one had a primary ovarian cancer. All presented with back pain, and the site of compression was at the thoracic spine. Three patient were treated with radiotherapy, and one underwent decompression laminectomy followed by radiotherapy. One patient improved, but the other three did not. SCC is an oncologic emergency, which should be borne in mind by all physicians who take care of cancer patients. PMID- 3007802 TI - [Thyroid hormones]. PMID- 3007804 TI - [Mutual interactions among various growth factors]. PMID- 3007803 TI - [Cyclic nucleotides]. PMID- 3007805 TI - [Growth factor receptor genes and oncogenes]. PMID- 3007806 TI - [Epidermal growth factor and an experimental model of psoriasis]. PMID- 3007807 TI - [Reverse transformation of neoplastic cells and phytoglycosides, with special reference to redifferentiation induction in cancer cells by ginsenosides]. PMID- 3007808 TI - [Protein and polypeptide growth factors--platelet-derived and macrophage-derived growth factors]. PMID- 3007809 TI - [Protein and polypeptide growth factors--somatomedin and insulin-like growth factor]. PMID- 3007811 TI - [Protein and polypeptide growth factors--adrenal cortex-stimulating hormone and thyroid-stimulating hormone]. PMID- 3007810 TI - [Protein and polypeptide growth factors--epidermal growth factor]. PMID- 3007812 TI - [Protein and polypeptide growth factors--prolactin]. PMID- 3007813 TI - [A colorimetric method for determination of guanase activity in serum based on superoxide anion with elimination of interference by endogenous xanthine]. PMID- 3007814 TI - [Late radiation effects of low doses from occupational exposure. Epstein-Barr virus-related antibody titers in radiological technologists]. PMID- 3007816 TI - [A case report of liver cell adenoma with difficulty of preoperative diagnosis]. PMID- 3007815 TI - [Localization of angiotensin converting enzyme (ACE) in granulomas of sarcoidosis of the skin]. PMID- 3007817 TI - [Two cases of lung cancer associated with chronic arsenic poisoning in an arsenic oxide (As2O3) refinery]. PMID- 3007819 TI - [Antinephritic effect of SA-446, an angiotensin I converting enzyme inhibitor, on crescentic-type anti-GBM nephritis in rats]. PMID- 3007818 TI - Genome type analysis of adenovirus type 4 isolates, recently obtained from clinically different syndromes in some areas in Japan. AB - DNA cleavage analyses with EcoRI, HindIII and BamHI were carried out to investigate genome types of recent Ad 4 isolates obtained from acute respiratory disease (8 strains), and ocular disease (11 strains) in Japan. DNA cleavage patterns of all 19 isolates studied were identical regardless of whether they were recovered from respiratory tract or conjunctiva, but were distinct from that of the prototype strain. PMID- 3007820 TI - [Effects of angiotensin I converting enzyme inhibitor on renal function and serum potassium in renal and renovascular hypertension]. PMID- 3007821 TI - [Studies on the modification of hormones in patients with renal failure and clinical evaluation--with reference to carbamylated ACTH and insulin]. PMID- 3007823 TI - [Preclinical evaluation of the 11C-Ro 15-1788 solution for injection as a radiopharmaceutical]. PMID- 3007822 TI - [Tumor imaging using Tc(V)-99m dimercaptosuccinic acid, a newly developed radiopharmaceutical: its clinical usefulness]. PMID- 3007824 TI - [Localization of technetium-99m methylene diphosphonate in hepatocellular carcinoma--a report of two cases]. PMID- 3007825 TI - [A study of the benzodiazepine receptor in the human brain using 11C-Ro 15-1788 and positron emission tomography]. PMID- 3007826 TI - Epstein-Barr virus-related antibody pattern in uveitis. AB - Epstein-Barr virus-related antibody titers were determined in 30 patients with anterior uveitis, 45 with pan-uveitis, including 19 with Behcet's disease, 11 with Vogt-Koyanagi-Harada's disease and 15 with unclassified pan-uveitis, and 144 age-matched healthy controls. The frequencies of anti-VCA (viro-capsid antigen) and anti-EA (early antigen) in uveitis patients and in controls were similar, but the frequency of sera with elevated titers (greater than or equal to 1:160) and the geometric mean titer of anti-VCA in the patients with anterior uveitis was significantly higher than in the controls (P less than 0.025, P less than 0.01) and in the patients with pan-uveitis (P less than 0.001). The frequency of sera with elevated titers of anti-EBNA (Epstein-Barr virus associated nuclear antigen) (greater than or equal to 1:160) in the patients with pan-uveitis was significantly lower than in the controls (P less than 0.02) and in the patients with anterior uveitis (P less than 0.01). Among the patients with pan-uveitis, there was no significant difference in the pattern of antibodies. These data are compatible with the interpretation that there may be both anatomical and immunological differences between anterior and pan-uveitis. PMID- 3007827 TI - Developmental alteration in adrenergic regulation of hepatic glycogen phosphorylase. AB - The effects of development upon adrenergic regulation of glycogenolysis were characterized using isolated hepatocytes from 3 different age groups of male rats (6 week-old, 8 week-old and 30 week-old). The phosphorylase a response in isolated hepatocytes to alpha-adrenergic stimulation decreased moderately with advancing age; whereas, that to beta-adrenergic stimulation declined more rapidly and almost disappeared at the age of 30 weeks. This developmental alteration in relative contribution of alpha- and beta-adrenergic regulation of phosphorylase was further confirmed by the experiments with specific antagonists. Also, the dramatic decrease of beta-adrenergic response on glycogen phosphorylase activity was found to be closely associated with a similar change of cAMP generation. In addition, the glucagon effect on cAMP production was found to be declined with advancing animal age. These results demonstrate that the glycogenolytic response of isolated rat hepatocytes to catecholamines can be mediated by different pathways according to the age of the animal; thus, juvenile male rats exhibit both the alpha- and beta-adrenergic mechanism for activation of phosphorylase and the maturation is associated with a modest decline of alpha receptor-mediated effect and a dramatic attenuation of a beta-adrenergic/cAMP response. PMID- 3007828 TI - Arsenic excretion after treatment of arsenic poisoning with DMSA or DMPS in mice. AB - The effects of 2,3-dimercaptosuccinic acid (DMSA) and 2,3-dimercaptopropane-1 sulfonic acid, Na salt (DMPS) on arsenic excretion in arsenic poisoning were studied using ICR mice. One group of mice was given arsenic trioxide (5 mg As/kg, s.c.) and another two groups were given DMSA or DMPS (100 mg/kg, i.p.) immediately after administration of the arsenic (5 mg/kg, s.c.). Arsenic excretion in urine and feces was determined by atomic absorption spectrophotometry. Results obtained showed a marked arsenic excretion in the urine collected at the first 12 hr in the group treated with DMSA. Further remarkable arsenic excretion in the feces was seen in the group treated with DMPS, suggesting that arsenic might have been excreted in the bile. PMID- 3007829 TI - Developmental alterations in maturing rats caused by chronic prenatal and postnatal diazepam treatments. AB - The post treatment effects of early prenatal, late prenatal, early postnatal or combined prenatal and neonatal treatment with diazepam on the development of pain sensitivity, acoustic startle responsiveness, and benzodiazepine receptors in the cerebral cortex were investigated in rats between 14 and 90 days of age. Tail flick latency was significantly decreased by combined prenatal and neonatal and by early prenatal diazepam treatment, but not by diazepam during the last half of gestation or during the neonatal period alone. Acoustic startle response was decreased by either late prenatal or neonatal diazepam treatment, but not by early prenatal treatment alone. Density of benzodiazepine receptors in the cortex was increased from postnatal day 1 to 21 by either early or late prenatal diazepam treatment. Neonatal diazepam treatment suppressed cortical benzodiazepine receptor or development until postnatal day 21; thereafter, receptor density increased to significantly higher values than in controls at 90 days of age. The results demonstrate that diazepam can alter development of pain sensitivity by actions during early gestation, startle responsiveness by actions late in pregnancy, and cortical benzodiazepine receptors by actions throughout gestation and the early postnatal period. PMID- 3007830 TI - Agonist and antagonist actions of buprenorphine on three types of opioid receptor in isolated preparations. AB - Both agonist and antagonist actions of buprenorphine on isolated preparations were studied. The Ke (equilibrium dissociation constant) values of both naloxone and Mr 2266 [(-)-2-(3-furylmethyl)-5, 9-diethyl-2'-hydroxy-6,7-benzomorphan] against buprenorphine and the ratio of IC50 (concentration of the drug to produce 50% inhibition of the twitch) value of buprenorphine after to before exposure of mouse vas deferens to beta-FNA (beta-fumarate methyl ester derivatives of naltrexone), an irreversible mu antagonist, suggest that buprenorphine acts as both a mu and kappa agonist on mouse vas deferens. The agonist effect of buprenorphine at relatively high doses on guinea-pig ileum and mouse vas deferens and the negative agonist effect on both rat and rabbit vas deferens indicate that buprenorphine acts as a partial agonist on isolated preparations. The Ke values of buprenorphine show that buprenorphine has about equal antagonist effectiveness against a mu and kappa agonist with approximately five-fold lower effectiveness against a delta agonist. The possible mechanisms for the several characteristic actions of buprenorphine on guinea-pig ileum such as the slow onset of action, the increased magnitude of inhibition after washing the tissue, the negative elimination of the inhibition by either washing the tissue or the naloxone administration, and the negative elimination of the antagonist action by washing the tissue were discussed. PMID- 3007832 TI - Surgical management of insulinoma: diagnosis of tumor location and high incidence of malignancy. AB - The findings in twenty-two patients with insulinoma were reviewed, as continuous efforts should be made to establish preoperative localization of the tumor. Superselective arteriography and percutaneous, transhepatic portal vein and pancreatic venous catheterization are highly recommended approaches. At the time of surgical intervention, a cautious exploration of the pancreas after thorough mobilization is most important. Recent use of intraoperative ultrasonography increases the likelihood of finding these occult tumors which locate deeply in the head of the pancreas. Apart from the diagnostic problems, we wish to emphasize the high incidence of malignancy (7/22, 31.8 per cent) in our series. Although patients with malignant insulinoma had a much better prognosis compared to those with a pancreatic ductal malignancy, pancreatic resection with regional lymphnode dissection seems to be a rational procedure. Enucleation can be done when intraoperative findings of the tumor and regional lymphnode indicate no malignant features and no multiple lesions. However, at the first operation, enucleation is still a procedure of choice, even for the malignant insulinoma in the head with a well-defined capsule and no metastatic lesions, the objective being to avoid a duodenopancreatectomy or total pancreatectomy. PMID- 3007831 TI - Bone lesions and dental caries after gastrectomy--evaluation of milk intolerance and operative procedure. AB - We investigated the development of bone lesions and dental caries in 350 patients who had undergone gastrectomy during the 11 year period from 1968 to 1978, on the basis of bone mineral content (BMC), decayed missing and filled (DMF) teeth and urinary c-AMP. We found that patients with low BMC and incidence of DMF teeth increased in number during the postoperative years; These abnormalities were, in comparison to the Billroth-I (B-I) and milk intake groups, significant in the Billroth-II (B-II) and non-milk intake groups. Among the gastrectomized patients, those with increased DMF teeth had significantly higher contents of urinary c AMP. The possibility that secondary hyperparathyroidism resulting from gastrectomy contributes to the development and incidence of dental caries has to be given consideration. PMID- 3007833 TI - [Cyclic nucleotides]. PMID- 3007834 TI - [Significance of angiotensin converting enzyme in respiratory diseases]. PMID- 3007835 TI - Opioids and CNS control of the gut. PMID- 3007836 TI - Characterization of receptors on isolated smooth muscle cells of the gut. PMID- 3007838 TI - Effects of various surgical procedures on gastric emptying of glucose solution. PMID- 3007837 TI - Action of isoproterenol and epinephrine on the guinea pig taenia coli studied by intracellular microelectrodes. PMID- 3007839 TI - A study of synaptic transmission and neuropeptide function in extramural colonic ganglia of the cat. PMID- 3007840 TI - Protection against bovine leukemia virus infection in sheep by active and passive immunization. PMID- 3007841 TI - Ascitic disease in ICR-nude mice due to mouse hepatitis virus. PMID- 3007842 TI - Diet and cancer of the colon and rectum: a case-control study. AB - In 1979-81, 419 patients with incident cases of colon and rectal cancer and 732 controls were questioned regarding diet and alcohol. Cancer cases were a population-based series reported to the South Australian Central Cancer Registry, were 30-74 years of age, and were residing in Metropolitan Adelaide. Controls were selected from the electoral roll and individually age- and sex-matched to cancer cases. The most consistent risk factor for colorectal cancer was dietary protein, which was associated with a twofold-to-threefold relative risk for colon cancer and for rectal cancer in women for all levels of consumption above the base line (i.e., the lowest consumption quintile). For male colon cancer the corresponding relative risk was similar; but for male rectal cancer, risk was elevated only at old ages. Total energy intake and, less clearly, meal frequency were also positively associated with increased risk. Total alcohol intake (but not specifically beer) was associated with increased risk of both colon and rectal cancer in women; in both sexes, there was an increased risk of colon and rectal cancer associated with spirits consumption. A reduced risk of rectal cancer was associated with vitamin C but not with vitamin A. The increased risk associated with high protein and total energy was confined to those consuming a low fiber diet, particularly among women; but some other aspects of the relationship between fiber consumption and risk of colorectal cancer were more complex. Some modifications and extensions of the current fat-to-bile acid-to fiber theory of bowel carcinogenesis were suggested. PMID- 3007844 TI - Serologic responses to murine mammary tumor virus (MuMTV) in MuMTV-exposed laboratory personnel. AB - A retrospective study was conducted to determine whether exposure to murine mammary tumor virus (MuMTV) induced MuMTV-specific serologic responses among intramural laboratory personnel. Results obtained with a panel of five purified structural proteins of the RIII mouse strain milk-derived MuMTV (gp55, gp34, p28, p18, and p12), by means of the enzyme-linked immunosorbent assay to assess antibody binding, established that MuMTV exposure resulted in highly significant increases in serologic responses to these test antigens as compared to age- and gender-matched controls without overt contact with MuMTV. Furthermore, immunoreactions to gp55 and gp34 were found not to be directed to the carbohydrate moieties of these glycoproteins. Similar results were obtained by assays of human immunoglobulin binding to Western blots of MuMTV proteins. These increased MuMTV-specific immunoreactivities, in general, were found to be related to degree and length of exposure to this virus. These results with MuMTV suggest the possibility of important human immune response differences between exposure to type B (MuMTV) and animal type C (leukemia-sarcoma) RNA tumor viruses, perhaps reflective of sensitivity to antibody dependence of complement-induced virolysis. PMID- 3007843 TI - Iron-binding proteins and risk of cancer in Taiwan. AB - The relationship of serum ferritin and transferrin levels to risk of cancer was examined in a population of 21,513 Chinese male government workers in Taiwan who have been followed prospectively since 1975. On the basis of a previous study in the Solomon Islands, increased ferritin and decreased transferrin levels were predicted for those men who developed cancer. The results were consistent with the prediction. The mean serum ferritin was higher at the start of the study in 192 men who had died of cancer or who had developed primary hepatocellular carcinoma (PHC) as of July 1983, as compared to their controls. The mean serum transferrin level was lower in men who had died of cancers other than PHC. The estimate of relative risk of cancer death for a man with 200 ng ferritin/ml and 200 mg transferrin/dl, as compared to a man with levels of 20 ng/ml and 400 mg/dl, respectively, is 2.9. These serum iron-binding protein levels are at the extremes of the "normal" range. Men who subsequently died of cancer had lower hemoglobin, lower hematocrit, lower albumin, and higher globulin levels at the start of the study than did the controls. These results are consistent with the hypothesis that increased iron stores increase the risk of cancer. However, direct assessment of iron stores prior to disease was not possible, and the same constellation of findings may be consistent with other explanations. PMID- 3007846 TI - Challenge and responsibility. PMID- 3007845 TI - Effect of hormones and epidermal growth factor on the growth of the hormone responsive 13762NF rat mammary tumor in collagen gel culture. AB - The hormone-responsive mammary tumor 13762NF of the F344 rat was cultured in a collagen gel matrix with the use of a serum-free medium supplemented with hormones and epidermal growth factor (EGF). Hydrocortisone (F) had the greatest effect on cell growth. EGF had no growth-promoting activity when used alone, but it had a significant effect when used with F. There was also a population of cells responsive to progesterone (P) and prolactin (PRL). P synergized with EGF as well as with PRL to promote growth. 17 beta-Estradiol alone or in combination with other hormones had no growth-promoting activity. Receptor levels in the tumor were high for glucocorticoids, intermediate for P and EGF, and low for estrogens. Metastasis in the lung and lymph node showed the same basic hormonal responses as the parent tumor. Cultured cells produced tumors with the same histopathology as the parent tumor when transplanted back into female rats; they had the same receptor levels and when placed back in culture showed a growth response to the same set of hormones. The tumor cells formed colonies that were spherical, which was different from the branching structures formed by normal mammary cells in collagen gel. PMID- 3007847 TI - AIDS in Kansas. PMID- 3007848 TI - [Cockayne syndrome--human papilloma virus and cervical cancer]. PMID- 3007849 TI - Renal kallikrein-kinin system. AB - In the last decade, our knowledge of the renal kallikrein-kinin system has been advanced significantly. More specific and sensitive methods for assessing its activity have been developed. Further, it has been found that in the kidney this system is localized in the distal nephron, where it appears to be linked to processes that control water and electrolyte excretion. Data indicate that the kallikrein-kinin system interacts with other renal hormonal systems such as the prostaglandin and renin-angiotension-kinin system may participate in the control of renal function and the pathophysiology of renal diseases. An increase in kallikrein excretion has been observed after administration of antihypertensive drugs. The kallikrein-kinin system may therefore participate in their mechanism(s) of action. Our current knowledge suggests that the renal kallikrein kinin system is an integral part of the intrarenal hormonal system that controls water and electrolyte excretion and participates in the regulation of blood pressure. PMID- 3007851 TI - Primary active sodium transport, oxygen consumption, and ATP: coupling and regulation. AB - Several metabolic aspects of primary active transport have been explored in this communication. One emphasized theme entailed the need to investigate the properties of the mitochondria and the active transport systems within the intact cell. Several methodological and conceptual approaches were described that permitted such an analysis. The answers provided were sometimes qualitative or quantitative. Qualitative information was provided regarding the cytosolic signal linking active transport with respiration, suggesting that the cytosolic ADP concentration was an important element in that link. The intact renal cell was found to work normally at 50 to 60% of its maximal respiratory capacity, indicating that sufficient reserve capacity was present for increased metabolic demands. Several examples were described in which a combination of qO2 measurements and/or optical techniques were used to differentiate between effects of agents which act primarily on transport or metabolic events. Finally, the control of transport by metabolism was discussed, primarily emphasizing the role of ATP and Pi. One of the overall conclusions from these studies is that, in general, the mitochondria and the transport systems seem to display similar properties in the intact cell as they do in isolated form. However, uncertainties concerning the cellular microenvironment surrounding the mitochondria and the plasma membrane transporters have produced some interesting surprises concerning their function in the intact cell. More quantitative information on the energy compartmentation of the renal cell would be helpful to clarify numerous aspects of metabolic function. PMID- 3007852 TI - Cellular mechanism of hormone action in the kidney: messenger function of calcium and cyclic AMP. PMID- 3007853 TI - Metabolism and sites of action of vitamin D in the kidney. PMID- 3007850 TI - Contributions of nuclear magnetic resonance to renal biochemistry. AB - 31P NMR as a descriptive technique is of interest to nephrologists. Particular contributions of 31P NMR to our understanding of renal function may be enumerated.: "Free" metabolite levels are different from those classically accepted; in particular, ADP and Pi are low with implications for the control of renal metabolism and Pi transport, and, via the phosphorylation potential, for Na+ transport. Renal pH is heterogeneous; between cortex, outer medulla, and papilla, and between cell and lumen, a large pH gradient exists. Also, quantitation between cytosol and mitochondrion of the pH gradient is now feasible. In acute renal failure of either ischemic or nonischemic origin, both ATP depletion and acidification of the renal cell result in damage, with increasing evidence for the importance of the latter. Measurements of renal metabolic rate in vivo suggest the existence of a prodromal phase of acute renal failure, which could lead to its detection at an earlier and possibly reversible stage. Human renal cancers show a unique 31P NMR spectrum and a very acidic environment. Cancer chemotherapy may alter this and detection of such changes with NMR offers a method of therapeutic monitoring with significance beyond nephrology. Renal cortex and medulla have a different T1 relaxation time, possibly due to differences in lipid composition. It seems that NMR spectroscopy has much to offer to the future understanding of the relationship between renal biochemistry and function. PMID- 3007854 TI - [The syndrome of enlarged orbits]. AB - Combined orbital fractures cause hematomas and subsequent rigidity of the ligament apparatus around the inferior rectus muscles, while the few true blow out fractures may lead to an adherence of connective tissues to the floor. The authors describe a third mechanism: Enlargement of the orbit impairs motility, especially the elevation of the globe: Diagnostic pressing of the orbital, parabulbar contents improves elevation. In 9 such cases that were free of the two other knows mechanisms motility was improved by reducing orbital volume. This was performed by implants (4 cases), by instillation of hydroxylapatite (4 cases) or by re-resection of the bony frame (1). Subsequent muscle surgery, if at all necessary, gave truly satisfactory results. PMID- 3007855 TI - [Electron microscopy diagnosis of intraocular herpes infection with a case demonstration]. AB - The tentative diagnosis of herpetic anterior uveitis was confirmed by means of an analysis of the aqueous humor employing the electron-microscopic negative contrast method; Acyclovir therapy was subsequently administered. The case is described and routine application of the method is discussed. PMID- 3007856 TI - [Focus of epidemic keratoconjunctivitis]. PMID- 3007857 TI - [Hereditary neuropathy with dominant inheritance, giant axons and cardiac involvement]. AB - Showing the clinical, genetic and electrophysiological features, the diagnosis of hereditary motor and sensory neuropathy type II (HMSN) in a german kinship was questioned by unusual findings in 3 members of the family: they presented signs suggestive of cardiomyopathy and the sural nerve biopsy showed occasional giant axons with neurofilament accumulation. These findings may mean either a new type within the heterogeneous group of peroneal muscular atrophies or that these features have not been realized in HMSN II as yet. PMID- 3007858 TI - [Early diagnosis and early treatment of hypophosphatemic vitamin D-resistant rickets]. AB - This is a report on two children with hypophosphatemic vitamin D-resistant rickets born to affected mothers diagnosed at the age of one and three months, respectively. The most important diagnostic criterium is the reduced value of the transport maximum of phosphorus in relation to glomerular filtration rate. At the age of one and six months, respectively, therapy with 1.25 (OH)2D3 and phosphate was started with the result of normal growth of length, radiological healing of rickets and normalisation of alkaline serum-phosphatase, but persistently low fasting values of serum-phosphorus during the time of observation up to twelve and twenty-four months of age, respectively in both children. The therapy with 1.25 (OH)2D3 and phosphate has a positive effect on intestinal absorption of phosphorus, because the serum-phosphorus is significantly elevated to the age corrected normal range two hours after oral administration of phosphate. There were no negative effects during therapy. It seems, therefore, that early diagnosis and therapy with 1.25 (OH)2D3 and phosphate is successful in healing this rare form of rickets. PMID- 3007859 TI - Parathyroid hormone secretion by brain and pituitary of sheep. AB - The secretion of immunoreactive PTH by different brain regions and pituitary of sheep was studied in vitro. Separate tissue samples of gyrus, internal capsule, basal ganglia, cerebellum, and pituitary were incubated in culture medium with low, normal, and high Calcium (Ca)(0.7 mM, 1.2 mM and 2.4 mM) concentrations. PTH release in medium containing low Ca were observed by all samples. The concentrations increased in the fifth and sixth hour from 100% (1st hour basis value) up to 300%. The PTH release showed an inverse relationship to the Ca concentration in the culture medium. To induce any significant decrease in medium concentrations of PTH, 1.25(OH)2D (100 ng/ml) was added to the culture medium. This effect could be reversed by DB-cAMP. Our results indicate that secretion of immunoreactive PTH by brain and pituitary of sheep may occur in vitro. The secretion depends on the content of Ca, 1.25(OH)2D, and DB-cAMP. PMID- 3007860 TI - Isolation of megakaryocytes by a combination of density gradient centrifugation and velocity sedimentation, and centrifugal elutriation. PMID- 3007861 TI - Some comments concerning purification of bovine enterovirus. PMID- 3007862 TI - Attempts to label bovine enterovirus with 32P. PMID- 3007863 TI - Cerebrospinal fluid cytology using a cytocentrifuge in the early diagnosis of cause of meningitis. PMID- 3007865 TI - Varicella-like herpesvirus infections of nonhuman primates. AB - At least three distinct herpesviruses cause varicella like exanthematous diseases among nonhuman primates. Spontaneous epizootics have resulted in high morbidity and mortality rates among Cercopithecus aethiops, Erythrocebus patas and Macaca species in research colonies. Mild infections have been observed in infant chimpanzees and a gorilla. This group of diseases is of interest not only because they are a threat to primate colonies, but also because their pathologic similarity to progressive human varicella makes them a potentially valuable animal model of this disease. The nonhuman primate varicella-like exanthematous diseases are reviewed and compared to varicella in man. PMID- 3007864 TI - Association of SAIDS/RF-related signs with current or past SAIDS type 2 retrovirus infection in a colony of Celebes black macaques. AB - The 83 members of the Celebes black macaque (Macaca nigra) colony were screened for viremia with simian acquired immunodeficiency syndrome (SAIDS) type 2 retrovirus and antibodies against the retrovirus. On the basis of this screening, the Celebes colony was divided into four groups: retrovirus-positive/seropositive (virus+/Ab+); retrovirus-negative/seropositive (virus-/Ab+); retrovirus positive/seronegative (virus+/Ab-); and retrovirus-negative/seronegative (virus /Ab-). Monkeys in the virus+/Ab+ group displayed more major clinical signs and required medication more times than monkeys in the other groups. In contrast, monkeys in the virus-/Ab- group had fewer health problems than monkeys in the other groups. The five monkeys that had surgically confirmed retroperitoneal fibromatosis (RF), palpable abdominal masses, or both, were in the virus+/Ab+ group. Some of the monkeys in groups with current or past retrovirus infection were well clinically. There were no statistically significant differences in the mitogen reactivities of mononuclear cells obtained from monkeys of the different groups. PMID- 3007867 TI - 15th annual UCLA symposia. January 20-February 15, 1986. Abstracts. PMID- 3007866 TI - Protooncogene expression during the cell cycle. PMID- 3007868 TI - 15th annual UCLA symposium. Abstracts: Viruses and human cancer. PMID- 3007869 TI - A kinetic analysis of enzyme inactivation as applied to the covalent modification of Na+ + K+ -ATPase and Ca2+ -ATPase. AB - A kinetic analysis of enzyme inactivation due to the covalent binding of chemically modified ligands is presented. Reaction schemes similar to the Michaelis-Menten scheme have been studied as well as schemes with two states of the enzyme or two binding sites. The resulting kinetic equations lead to time courses of inactivation which can be represented by two exponential functions at least in a quasi-steady state approximation. These curves are frequently encountered in inactivation experiments. Since rapid methods for model selection and parameter estimation are desirable, but not available, a technique for a preliminary analysis of the experimental data is presented. A mere glance at the time courses shows what reaction schemes are inapplicable. For each family of inactivation curves, the construction of a line of intersections is proposed. This line contains essential kinetic information and can further be utilized for a rough parameter estimation. The technique is illustrated for three sets of experimental data where Na+ + K+ -ATPase and Ca2+ -ATPase have been inactivated by ATP-analogs. PMID- 3007870 TI - Evolution of bacterial surface exclusion against incompatible plasmids. AB - Many conjugative transferable plasmids exhibit surface exclusion against plasmids of the same incompatibility group. A mathematical model is developed to calculate under which conditions surface exclusion against incompatible plasmids can evolve. It appears that plasmids inducing surface exclusion can evolve and even replace non-excluding plasmids if the copy number is low and the transfer rate high provided that the cost of surface exclusion is small. They can more easily expel the non-excluding plasmids if the possession of a plasmid is not very harmful for a bacterium and if the rate at which plasmids are lost is small. PMID- 3007871 TI - A simple nonequilibrium thermodynamic description of some inhibitors of oxidative phosphorylation. AB - We propose a macroscopic description of some inhibitors of oxidative phosphorylation based on a simple modification of the phenomenological coefficients appearing in the constitutive equations of linear irreversible thermodynamics. In this theoretical model, we consider protonophores, some ATPase inactivators and some electron-chain inhibitors, and we provide quantitative expressions for their consequences on the protonmotive force, oxidation flux and phosphorylation flux as well as on heat generation. PMID- 3007872 TI - Detection of HTLV-I (human T-cell lymphotropic virus, type I) proviral DNA in leukemic cells from a French patient with Sezary syndrome. AB - Human T-lymphotropic type I (HTLV-I) proviral sequences were detected in leukemic cells of a patient living in Marseilles (south of France) and suffering from Sezary syndrome. He did not have any travel history outside France and did not receive blood transfusion or hepatitis B vaccination. This case of HTLV-I positive Sezary syndrome is the first one described outside the known endemic regions for HTLV-I. Moreover, this patient was found to be negative for viral antibodies. This observation should therefore stimulate new and thorough analysis of the association of this human retrovirus with leukemia and lymphoma in the Mediterranean region, both by seroepidemiological and molecular biology techniques. PMID- 3007873 TI - T-cell colony formation in patients with T-cell malignancies: growth factor requirements for in-vitro proliferation of peripheral blood T-cell colony-forming cells. AB - Peripheral blood T colony-forming cells (T-CFC) from patients with T-cell malignancies are capable of proliferation in methylcellulose, in the absence of added growth factors or mitogenic stimulation. We show here that based on the spontaneous plating efficiencies of their T-CFC, two groups of patients can be established: Group A (10 patients) with a high colony number (more than 100 colonies/10(5) seeded cells), and group B (12 patients) with less than 100 colonies/10(5) cells. The addition of interleukin 2-(IL2) containing PHA leukocyte conditioned medium enhanced colony growth from group B but not group A patients. Moreover, both biochemically purified and recombinant IL2 induced the colony growth from group B but not group A patients without any other stimulation. In addition, a monoclonal antibody (moAb) against the IL2 receptor (IL2-R; anti-Tac) inhibited the spontaneous colony formation from T-CFC of both groups of patients. These observations strongly suggest that IL2-R are involved in the spontaneous colony growth of T-CFC. To determine whether IL2 is also involved in the spontaneous colony formation, media conditioned (LCM) by unstimulated leukemic cells were tested for IL2 activity. A constitutive release of IL2 was detected in LCM from only 2 out of 10 patients tested, and some of them were capable to inhibit thymidine incorporation by IL2-dependent cells cultured in the presence of highly purified IL2. However, most of these LCM contained a T-cell colony-promoting activity (TCPA) inducing colony formation from both normal resting and PHA-stimulated E+ clonogenic cells without added IL2 or mitogenic stimulation. TCPA-containing LCM induced the expression of functional IL2-R and IL2 release by normal resting lymphocytes. TCPA was constitutively released by blast-enriched cell fractions suggesting that it is released by the leukemic cells. PMID- 3007874 TI - Transferrin induces maturation of neutrophil granulocyte precursors in vitro. AB - Previous studies showed that dialyzed serum from guinea pigs contained a factor which induces guinea pig granulocyte precursors to form mature neutrophil granulocytes in vitro. We show here that this maturation inducing activity of serum is associated primarily with transferrin. This activity is not species specific since both guinea pig and human transferrin serve equally well in this capacity. These findings raise the possibility that transferrin could serve as a physiological regulator of granulocyte maturation. PMID- 3007875 TI - Picture processing techniques applied to autoradiographic studies of visual cortex. AB - Picture processing techniques are applied to 2-deoxyglucose autoradiographs of sections from striate cortex and to patterns resulting from staining these sections for cytochrome oxidase. This procedure allows computer identification of deoxyglucose active and inactive regions in the autoradiographs and cytochrome active and inactive regions in the stain patterns. Subsequently, the topographical relationship between these patterns can be quantitatively analyzed by means of overlap and density distribution measures and can be displayed using color enhanced graphics. The processing techniques have been applied in studies of the functional organization of visual cortex in primates. Computer graphic techniques have allowed implementation of split-field presentations of stimuli in deoxyglucose experiments. An application of this split-field technique for presenting multiple-stimuli to distinct parts of the visual field is described and an autoradiograph from a split-field experiment is shown. PMID- 3007876 TI - The occurrence of cyclic AMP in aging cultures of the fungus Aspergillus ornatus. AB - The presence of endogenous cAMP was demonstrated in mycelial mats of Aspergillus ornatus Raper grown on cellulose xanthate membranes overlying a defined agar medium. Using a competitive binding assay cAMP was detected in 30 separate samples. Purification techniques increased the apparent cAMP concentration in excess of eight times the level observed in crude extracts. Cyclic AMP was only slightly detectable in extracts treated with phosphodiesterase. The concentration of endogenous cAMP as measured in purified extracts declined by approximately 60% in non-growing aging mycelial mats. Although there is an apparent age-related decline in endogenous cAMP levels and the activity of the tryptophan inducible enzyme, 2,3-dihydroxybenzoic acid carboxylyase (EC 4.1.1.46), (OPCA carboxylyase), the addition of 10(-3) M cAMP and/or 0.1% tryptophan to the culture medium did not increase OPCA carboxylyase activity in the fungus. PMID- 3007877 TI - Age-related change in adrenergic regulation of glycogen phosphorylase in rat hepatocytes. AB - The effects of development on adrenergic regulation of glycogenolysis were studied in rat liver. In isolated hepatocytes, the activation of glycogen phosphorylase by alpha-adrenergic stimulation decreased moderately with advancing age. However, activation by beta-adrenergic receptors more markedly declined and almost disappeared in isolated hepatocytes from 6-month-old rats. Furthermore, the ability to glucagon to stimulate phosphorylase activity and cAMP generation was found to decrease with increasing age. The age-related changes in the pattern of stimulation of glycogen phosphorylase by catecholamines in isolated hepatocytes were accompanied by parallels changes in the numbers of alpha 1- and beta-adrenoceptors in membranes prepared from the isolated hepatocytes. A progressive decrease in the total number of alpha 1-receptors measured with [3H] prazosin and beta-receptors measured with [125I]cyanopindolol (CYP) was found with increasing age. The loss of beta-adrenergic receptors was much more dramatic. Our results suggest that age-related alterations of hepatic glycogen phosphorylase activation by catecholamines may in part be explained by the changes in the expression adrenergic receptors. PMID- 3007878 TI - [Multiple enzymatic changes in T4 and T8 lymphocytes in primary hypogammaglobulinemia in the adult]. PMID- 3007879 TI - [Immunity and drug addiction: immunologic situation in relation to the drug, HTLV III infection and other associated infections]. PMID- 3007880 TI - [HTLV-III infection in risk groups]. PMID- 3007881 TI - [The AIDS virus]. PMID- 3007882 TI - [Seropositivity against antigens of the HTLV-III lymphotropic virus in different communities from the Barcelona area]. PMID- 3007883 TI - [Evidence of exposure to the virus of acquired immune deficiency syndrome in risk groups from the Seville area. Preliminary evaluation]. PMID- 3007884 TI - [Penetration of LAV/HTLV-III and infectious complications in parenteral drug addicts]. PMID- 3007885 TI - [Heterosexual transmission of HTLV-III/LAV]. PMID- 3007886 TI - [Human T-lymphotropic virus (HTLV-III) antibodies in intravenous drug addicts from the Valencia community]. PMID- 3007887 TI - [Changes in cellular immunity in parenteral drug addicts]. PMID- 3007889 TI - The clinical approach to the male homosexual patient. AB - A history of sexual orientation, practices, and life styles, a complete review of organ systems, and a thorough physical examination are unique but essential elements of the examination of homosexual men. Laboratory screening should take into account the incubation period for infections, the risk for infection based on sexual practices, and cost. Physicians should counsel homosexual men about all aspects of a healthy life style; for this advice to be accepted, however, physicians must develop a relation with their homosexual patients that is based on awareness, expertise, and mutual understanding. PMID- 3007888 TI - [Insulinoma: diagnosis of localization using the collection of transhepatic portal venous blood samples]. PMID- 3007891 TI - The acquired immunodeficiency syndrome: epidemiology and risk factors for transmission. AB - The acquired immunodeficiency syndromE (AIDS) is a very serious illness caused by a human T-lymphotropic retrovirus: human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV). It primarily affects young adults living in one of several major metropolitan areas. Most patients are homosexual men, but heterosexual men and women have also been affected. Heterosexual men and women who use intravenous drugs, who are hemophiliacs, or who are sex partners of AIDS patients appear to be at increased risk for developing the disease. PMID- 3007890 TI - Clinical approach to viral hepatitis in homosexual men. AB - The prevalence of both hepatitis A and hepatitis B is increased in homosexual men. On an annual basis, 5% to 7% of homosexual men will acquire hepatitis A. Risk factors for HAV infection include length of homosexual activity, number of sexual contacts, and oral--anal sexual contact. The HBsAg carrier rate of homosexual men is 5% to 6%, and another 50% have evidence of previous HBV infection with a positive anti-HBs. HBeAg is present in a higher precentage of HBsAg-positive homosexual men (38% to 75%) than in general population carriers (3% to 30%). The annual incidence for HBV infection in homosexual men is 16% to 28%, higher than that for hepatitis A. Transmission of HBV infection in homosexual men is facilitated by a large number of sexual partners, high HBsAg carrier rate, high infectivity of carriers (positive HBeAg), and the specific sexual practices of oral--anal and anal--genital contact with exposure to HBV on open mucosal surfaces. The prevalence of non-A, non-B and delta infection in homosexual men is probably somewhat increased, but the importance of these viruses in the development of hepatitis in this population remains uncertain. Prevention of hepatitis A and B in homosexual men will ultimately be achieved by vaccination of susceptible individuals, which currently is feasible only for hepatitis B. Appropriate use of immune globulins for postexposure prophylaxis and knowledge of specific sexual practices that transmit disease may reduce the incidence of hepatitis A and B. PMID- 3007893 TI - Kaposi's sarcoma and the acquired immunodeficiency syndrome. AB - This review has briefly summarized the place of KS in the AIDS epidemic, offering a framework for more critical evaluation of difficult therapeutic decisions. It should be expected (and it is hoped) that with the current rapid pace of AIDS research, many of these issues will be resolved shortly, and that therapy will become more rational. Until then, however, conventional approaches will be used. Given the hazards of these, it is incumbent upon the physician to remain up to date on developments in this rapidly evolving field. PMID- 3007892 TI - Immunologic aspects of the acquired immunodeficiency syndrome and male homosexuality. AB - The acquired immunodeficiency syndrome (AIDS) is the most common and best characterized disorder of T cells leading to enhanced susceptibility to infection. Current hypotheses hold that infection with human T-cell lymphotropic virus type III/lymphadenopathy virus (HTLV-III/LAV) is a necessary but not a sufficient condition for the development of AIDS, and that a variety of cofactors participate in the pathogenesis of the syndrome. This article reviews the immunologic aspects of AIDS and the AIDS-related syndromes, as well as concepts of etiology and pathogenesis. Predisposing factors for this disease in the homosexual or bisexual host are emphasized. PMID- 3007894 TI - Lymphadenopathy related to the acquired immunodeficiency syndrome in homosexual men. AB - Persistent generalized lymphadenopathy is a nonspecific physical finding common in AIDS and AIDS-related disorders. A subset of patients with a benign course and normal laboratory evaluation are at low risk for developing full-blown AIDS. Simple clinical and laboratory findings may predict progression from lymphadenopathy syndrome to more malignant manifestations of AIDS. PMID- 3007895 TI - [Occurrence, role and characteristics of human Enteroviruses in Poland 1973-1982. II. Polioviruses]. PMID- 3007896 TI - [Experimental viral-bacterial infections. I. Elaboration of the model of mixed infection with EMC virus and Staphylococcus aureus in mice]. PMID- 3007898 TI - [ACE inhibitors in pregnancy can be harmful to the fetus]. PMID- 3007897 TI - In vitro receptor occupancy allows to establish equieffective doses of beta blockers with different pharmacodynamic profiles in man. Investigations with propranolol and bufuralol. AB - The aim of the present study was to establish equieffective doses of propranolol (PROP) and bufuralol (BUF) in man. Both drugs compete for radioligand binding of 3H-CGP 12177 at beta-adrenoceptors of rat reticulocytes with Ki-values of 6.7 (PROP) and 19.6 (BUF) ng/plasma in the mean. In contrast to PROP, which is a pure antagonist, BUF shows partial agonism with an intrinsic activity around 5% (relative to isoprenaline) in vitro. After i.v. injection of cumulative doses of both drugs to 10 healthy volunteers, drug present in plasma occupies beta adrenoceptors in vitro in a dose-dependent manner up to 80%. In man the full beta adrenoceptor agonist isoprenaline (8 micrograms/min for 3 min) induces an increase in heart rate by 73%, an increase in stroke volume by 47%, a decrease in total peripheral resistance by 63% and a shortening of the preejection period by 70%. PROP exerts effects in man in the opposite direction of isoprenaline (at resting state), whereas BUF does not influence heart rate and stroke volume to a significant extent. Only the preejection period and total peripheral resistance are influenced in analogy to the isoprenaline effects. A direct comparison of the effects of PROP and BUF at 50% in vitro receptor occupancy by the respective plasma samples shows the clear-cut qualitative differences between the pharmacodynamics of both drugs in man as expected from the comparison of the full agonist isoprenaline and the antagonist propranolol. It is concluded that only on the basis of the respective receptor occupancy one may delineate pharmacodynamic differences of different drugs of the same class in man. PMID- 3007899 TI - [Temporal bone approach to the retromaxillary-infracranial space and the orbit in tumor surgery]. AB - The article gives a detailed description of a temporal approach to the retromaxillary infracranial space and to the orbit, developed by one of the co authors (H. Obwegeser) of this article. Case reports show the broad spectrum of indication in surgery of benign, and especially of malignant, tumours in this region. Surgery is performed via an extended type of incision in the scalp. The cheekbone and the coronoid process, or the upper portion of the ascending ramus of the lower jaw, can be osteotomized and transposed as muscular flaps. The great advantages of this surgical procedure are an excellent overview, absence of visible scarification, and preservation of all tumour-free parts of the facial skeleton, combined with the possibility of radical en-bloc removal of the neoplasm. PMID- 3007900 TI - Oculopharyngeal muscular dystrophy: recent ultrastructural evidence for mitochondrial abnormalities. AB - Oculopharyngeal muscular dystrophy is a rare, autosomal dominant disorder which has been traced through 11 generations of a French Canadian family. The classic presenting complaints are palpebral ptosis and oropharyngeal dysphagia. The dysphagia is usually progressive and leads to repetitive regurgitation, increased alimentation time, and weight loss. Immunologic studies generally reveal elevated levels of IgA and IgG, while manometry demonstrates poor pharyngeal contraction. The dysphagia is frequently relieved by cricopharyngeal myotomy. We present two case reports and also a new ultrastructural finding of abnormal metallic mitochondrial inclusions. This finding has not been previously described in this disorder. PMID- 3007901 TI - [Focal liver processes: additional diagnostic aspects as seen by nuclear magnetic resonance tomography?]. PMID- 3007903 TI - An endogenous clonidine-displacing substance from bovine brain: receptor binding and hypotensive actions in the ventrolateral medulla. AB - A substance has been isolated from bovine brain which displaces 3H-clonidine binding to rat brain membranes (clonidine-displacing substance; CDS). To determine whether CDS is similar to the antihypertensive agent clonidine, the in vitro binding properties of partially-purified CDS and its physiological action in the rostral ventrolateral medulla were examined. Like clonidine, CDS potently inhibited 3H-para-aminoclonidine binding to receptors in bovine ventrolateral medulla membranes (clonidine, IC50 = 24 +/- 8nM; CDS, IC50 = 0.30 +/- .10 Units), with highest affinity for non-adrenergic sites (clonidine, IC50 = 6 +/- 1nM; CDS, IC50 = 0.12 +/- .07 Units). CDS had no effect at beta-adrenergic or muscarinic cholinergic receptors. Like clonidine, CDS elicited a potent, reversible (less than 10 min) dose-dependent fall in arterial pressure (AP) and heart rate when microinjected specifically into the C1 area of the rostral ventrolateral medulla in the rat (maximum delta AP, -65 +/- 7 mm Hg). CDS represents an as-yet uncharacterized endogenous, physiologically-active agent in brain which may participate in cardiovascular control via non-adrenergic receptors in the rostral ventrolateral medulla. PMID- 3007902 TI - Effects of enprofylline and theophylline may show the role of adenosine. AB - It is well established that at low and clinically relevant concentrations theophylline (and caffeine) exerts antagonism at cell surface receptor sites for adenosine. However, it is not known which actions of theophylline are due to adenosine antagonism, because theophylline apparently activates other cellular mechanisms at the same low concentrations. Investigations into the actions of xanthines and their structure activity relationships have identified xanthine compounds like enprofylline (3-propylxanthine) that only has some actions in common with theophylline and that has a negligible ability to antagonize adenosine. Enprofylline is a more potent smooth muscle relaxant and antiasthmatic drug than theophylline but does not produce, e.g., theophylline-like diuretic effects, CNS-stimulant behavioural effects (restlessness - seizures), gastric secretory effects and release of free fatty acids. It is proposed that pharmacodynamic dissimilarities between enprofylline and theophylline may indicate physiological roles of adenosine. PMID- 3007904 TI - Functional compartmentalization of arginine-vasopressin-activated cyclic AMP in anterior pituitary gland: the presence of a compartment activated by prostaglandin E2. AB - AVP(10(-8)-10(-6)M) increased ACTH as well as PGE2 release from rat anterior pituitary quarters in vitro in a concentration dependent manner. IBMX (0.1 mM), a phosphodiesterase inhibitor, increased the ACTH response to AVP. The cAMP content in pituitary tissue was increased by AVP. Cyclooxygenase inhibition by indomethacin(1.4 X 10(-5) M) or diclofenac (1.8 X 10(-5)M) led to a potentiation of AVP-evoked ACTH secretion and to a decrease in AVP-stimulated cAMP formation. PGE2(10(-6)M) significantly increased pituitary cAMP content and indomethacin did not affect cAMP levels activated by PGE2. PGE2 attenuated the AVP-induced ACTH release. These results indicate that at least two functional compartments of AVP activated cAMP responses are involved in the AVP-induced ACTH release. One compartment is directly activated by AVP and participates in the propagation of AVP-induced ACTH release. The second compartment is activated by PGE2. The contribution of the second compartment to the regulation of ACTH secretion is not well understood since PGE2 shows an inhibitory effect on AVP-induced ACTH secretion. PMID- 3007905 TI - Epinephrine and norepinephrine stimulation of adenylate cyclase in bovine retina homogenate: evidence for interaction with the dopamine D1 receptor. AB - Dopamine, epinephrine and norepinephrine provoke dose-dependent stimulation of adenylate cyclase activity in bovine retina homogenates. The stimulatory effect of all three compounds is inhibited by the D1 antagonist SCH 23390 and, in a stereoselective manner by the cis-isomer of flupenthixol. The D2 antagonist haloperidol and alpha-and beta-adrenergic antagonists failed to block the catecholamine stimulation. These results evidence that, in bovine retina, not only dopamine but also epinephrine and norepinephrine interact with dopamine D1 receptors that are stimulatory coupled to an adenylate cyclase system. PMID- 3007907 TI - Identification of a ouabain-like compound in toad skin and plasma as a bufodienolide derivative. AB - An ouabain-like compound (OLC) was purified from toad skin. The purification procedure consisted of three sequential separations on HPLC using amino and reverse phase chromatography. Using UV, NMR and Mass spectroscopy the structure of the purified material is suggested to be mono-hydroxy-14,15-epoxy-20,22 dienolide glycoside (resibufogenin). Evidence is presented that this compound is also present in the toad plasma. It is suggested that the endogenous bufodienolide derivative participates in the physiological regulation of the Na+,K+-ATPase activity. PMID- 3007906 TI - Effects of cocaine on the electrical activity of single noradrenergic neurons from locus coeruleus. AB - The effects of intravenous (i.v.) cocaine HCl on single identified spontaneously firing noradrenergic neurons in the nucleus locus coeruleus (LC) were studied in rats in vivo. Cocaine (0.25-1 mg/kg) produced inhibition of spontaneously firing LC neurons, which was reversed by the administration of the selective alpha 2 adrenoceptor antagonist, piperoxane (250 micrograms/kg, i.v.). Procaine, a local anesthetic that is structurally related to cocaine, did not inhibit LC neurons in doses up to 4 mg/kg, i.v. These results suggest that cocaine in low doses has significant central sympathomimetic effects at the single noradrenergic neuron level and that the inhibition of spontaneous activity may be mediated by alpha 2 adrenoceptors. Our results also indicate that cocaine in pharmacologically relevant doses, can significantly affect central alpha 2-adrenoceptor regulatory processes. PMID- 3007908 TI - Purification and sequence determination of bovine atrial natriuretic factor. AB - We report the purification and the sequence determination of bovine atrial natriuretic factor (ANF) in acid extracts of bovine atrial appendages. The monitoring of the activity along the purification steps was performed with a radio-receptor assay using bovine adrenal cortex membranes sites and 125I ANF. Bovine ANF was separated by carboxymethyl agarose gel chromatography from catecholamines and major protein contaminants. It behaved as a 3 K dalton peptide on Sephadex G-50. The active fractions were then subjected to high performance liquid chromatography (HPLC) using a sulfopropyl cation exchange column. Subsequent purification steps by reverse phase mode on Ultrapore RPSC, Vydac TP 218 and uBondapak using acetonitrile gradients led to the obtention of a pure fraction which amino acid sequence was identical to that for human ANF. This confirms the high degree of homology of ANF structure among mammalian species and advocates the use of the radio-receptor assay based on bovine adrenal receptor for measuring human ANF. PMID- 3007909 TI - Antihypertensive substance in seeds of Areca catechu L. AB - Among various tannins tested, Areca II-5-C, a fraction isolated from seeds of Areca catechu L., showed the most potent angiotensin-converting enzyme (ACE) inhibitory activity in vitro. Its antihypertensive activity was therefore investigated in normotensive and spontaneous hypertensive rats (SHR) after both oral and intravenous (i.v.) administration. The activity was compared with that of captopril (D-3-mercapto-2-methylpropanoyl-L-proline), a potent ACE inhibitor. Oral administration of Areca II-5-C to SHR produced a lasting, dose-related antihypertensive effect, and the responses obtained with doses of 100 and 200 mg/kg were comparable to those of captopril at doses of 30 and 100 mg/kg. Intravenous administration of Areca II-5-C to SHR produced a rapid and marked reduction in blood pressure at doses of 10 and 15 mg/kg. The maximum antihypertensive effect of Areca II-5-C in SHR, at an i.v. dose of 15 mg/kg, was about 5 times as large as that of captopril at the same dose. Although the vasopressor response to norepinephrine and vasodepressor responses to bradykinin and acetylcholine were not appreciably changed by i.v. treatment with Areca II-5 C at a dose of 5 mg/kg, it did produce dose-related inhibition of the pressor responses to angiotensin I and II. It is suggested that Areca II-5-C has favorable properties as a hypotensive drug through its ability to inhibit the pressor responses to both angiotensin I and II. PMID- 3007910 TI - Specific [3H]-N-methyl scopolamine binding without cholinergic function in cultured adult skin fibroblasts. AB - Cultured adult skin fibroblasts were studied for binding and functional evidence of muscarinic receptors in order to assess their utility as a model of cholinergic function in affective illness. Saturable, specific, high affinity binding could be demonstrated in intact cells from some cell lines with [3H]-NMS, but not [3H]-QNB, presumably because of intracellular trapping of unbound [3H] QNB. [3H]-NMS specific binding indicated a single site with a KD of approximately 210 pM. [3H]-NMS was displaced by cholinergic agonists and antagonists with relative affinities similar to muscarinic receptors in brain. Many cell lines, however, showed no specific binding. No functional response to carbachol could be demonstrated with respect to inhibition of isoproterenol-stimulated cyclic AMP formation, stimulation of cyclic GMP formation or stimulation of phosphoinositide hydrolysis in any cell line regardless of either high or no specific [3H]-NMS binding. PMID- 3007911 TI - Re-evaluation of drug-interaction with alpha-adrenoceptors in vivo and in vitro using imidazole derivatives. AB - The critical spatial dimension requirements for drug interaction with alpha adrenoceptors were examined using imidazole derivatives MPV 295 and its semi rigid analogue MPV 305 T (= trans) or MPV 305 C (= cis). The ethenyl bridge bond between the phenyl and imidazole moieties of MPV 305 prevents it achieving the critical spatial dimensions of the phenethylamines (e.g. norepinephrine). MPV 295 (0.03-10 mg/kg i.v.) and the trans-extended MPV 305 T (0.01-1 mg/kg i.v.) were hypotensive and bradycardic in anesthetised rats. In pithed rats, MPV 295 and MPV 305 T induced vasoconstriction, the doses giving a 50 mmHg rise in mean arterial pressure being 34.5 and 11.5 ug/kg, respectively. The pressor activity of MPV 295 was antagonized by idazoxan (1 mg/kg i.v.) but not by prazosin (0.1 mg/kg i.v.), whereas that of MPV 305 T was antagonized by prazosin and to a greater extent by idazoxan. Both compounds inhibited the increase in heart rate produced by electrical stimulation of the cardioaccelerator sympathetic nerve fibres in the pithed rats. The doses which induced a 50% inhibition of sympathetic transmission were 49.0 and 38.0 ug/kg for MPV 295 and MPV 305 T, respectively. This peripheral sympatho-inhibitory action was antagonized by idazoxan. Both compounds inhibited the twitch response of electrically stimulated mouse vas deferens, the pD2 values being 7.59 and 7.89 for MPV 295 and MPV 305 T, respectively. In the rat anococcygeus muscle only MPV 305 T was active (pD2 4.84). The cis-folded MPV 305 C was practically inactive in pithed rats and in rat anococcygeus muscle. According to the results, the strengthening of the ethano bridge of MPV 295 to that of MPV 305 T, thus preventing it fitting into the proposed dimensions of alpha-agonists, does not lead to a decrease in alpha-adrenoceptor mediated activities. Therefore, the spatial dimension requirements among imidazoles are different from those among the phenethylamines, supporting the concept that imidazoles interact differently with alpha-adrenoceptors when compared to the phenethylamines. PMID- 3007912 TI - Subcellular distribution of dietary beta-carotene in chick liver. AB - Studies were conducted examining the subcellular distribution of beta-carotene (BC), alpha-tocopherol (E) and retinol (A) in livers of control and BC-fed male White Leghorn chicks. Chicks were fed Cornell B chick starter diet with or without the addition of 0.5 g BC/kg diet. A first study involved liver fractionation by differential centrifugation in 0.25 M sucrose followed by high performance liquid chromatographic (HPLC) analyses of all fractions for quantitation of BC, E and A. A second study employed both intravenous injection of Triton WR-1339 four days prior to sacrifice and centrifugation in 1.0 M sucrose to separate mitochondria from lysosomes more efficiently. Fraction purity was assessed by marker enzyme analyses. Results showed that chick liver accumulated BC; BC-fed chicks had higher concentrations of BC in all fractions relative to controls, and the mitochondrial fraction contained the highest concentration of BC, followed by lysosomes, microsomes and nuclei, respectively. Plasma BC increased more than fivefold in BC-fed chicks. Dietary BC increased A and E levels in liver and in the mitochondrial and lysosomal fractions while the plasma E level was decreased. Plasma A changed little with BC feeding. While dietary BC had no effect on fatty acid composition of subcellular fractions, the increase in E resulted in a large increase in the molar ratio of E to polyunsaturated fatty acids. The incorporation of BC and increased amounts of E into cellular membranes presumably would result in increased resistance to peroxidative damage. PMID- 3007913 TI - [Diagnostic importance of dynamic computerized esophageal scintigraphy]. AB - In healthy persons the mean time of the passage of water through the esophagus was 3.75 +/- 0.12 s, that of 10% test meal 6.15 +/- 0.34 s. The average rates of the passage of water and test meals were 5.35 +/- 0.25 cm/s and 3.25 +/- 0.62 cm/s, respectively. In postburn cicatricial esophageal stenosis the time of the passage of water ranged within 5-9 s depending on a degree of stenosis, that of the test meal within 12-20 s, and the rates of their passage were 2-4 cm/s and 1 1.7 cm/s, respectively. While in some cases complete esophageal obstruction was shown by x-ray, scintigraphy revealed the presence of a lumen of the esophagus. PMID- 3007914 TI - [Diagnostic potentials of osteoscintigraphy]. PMID- 3007915 TI - [Heavy charged particles and neutrons in oncology (a review of the foreign research)]. PMID- 3007916 TI - [Effect of radiation and drug therapy on the hormonal status of patients with breast cancer, taking into consideration the receptor level of the tumor]. AB - A study was made of the content of the steroid and peptide hormones in the blood of 115 patients with Stage III a,b,c breast cancer (54 patients at the reproductive age and 61 in the menopause) before treatment and during radio- and chemotherapy. A group of healthy women (28 with preserved menstruation and 20 in the menopause) was taken as controls. Data on the concentration of the steroid hormones in the patients' blood were compared with the presence of the respective receptors in tumor. Before treatment a significant rise of the estradiol concentration was noted in the blood of the menopause patients, that of prolactin both in the menopause patients and in the patients with preserved menstruation. A raised testosterone concentration was also noted in the patients with preserved menstruation. After radiotherapy the blood prolactin level, particularly in the patients with preserved menstruation, increased more than 2-fold. There was no correlation between the levels of the steroid and peptide hormones during therapy and its efficacy. The prolactin level can be used as a criterion of the efficacy of antitumor therapy, its stable rise in operated patients during therapy being an unfavorable prognostic sign. PMID- 3007917 TI - [Neurological disorders in alcoholism]. PMID- 3007918 TI - Proton magnetic relaxation in aqueous glycerol solutions containing manganese(II) ion. AB - The proton nuclear magnetic relaxation rate, 1/T1, is reported as a function of d5-glycerol concentration for aqueous manganese(II) ion solutions over a range of magnetic field strengths corresponding to proton Larmor frequencies between 0.01 and 38 MHz. The data are analyzed in terms of the Solomon, Bloembergen, and Morgan equations which are found to be sufficient to account for the relaxation data over the field and concentration ranges studied. These data provide a useful reference for the viscosity dependence of manganese(II)-ion-induced magnetic relaxation in aqueous systems and a useful comparison with macromolecule bound manganese(II)-ion-induced relaxation where the theory is less successful. PMID- 3007919 TI - Measurement of intracellular oxygen concentration using the spin label TEMPOL. AB - We have developed a noninvasive method with general applicability for measuring intracellular oxygen using the spin label TEMPOL (2,2,6,6,-tetramethypiperidine-N oxyl-4-ol) which has superhyperfine structure in its electron spin resonance spectra that is broadened in the presence of oxygen. This broadening is linear over a range of 1 to 6 ppm oxygen which covers the important physiological range of oxygen concentration. Viscosity, TEMPOL concentration, and instrument modulation intensity also can affect superhyperfine structure but the contributions from these effects can be determined. The TEMPOL distributes equally into the intra- and extracellular compartments but its intracellular signal can be studied selectively by addition of transition metal ions such as potassium ferricyanide and potassium tris(oxalato)chromiate, which broaden away the signal from extracellular TEMPOL and do not cross the cell membrane to affect the intracellular TEMPOL. Results with a cell culture line (mouse thymus-bone marrow) indicate that under our experimental conditions these cells may maintain an average intracellular oxygen concentration lower than the extracellular oxygen concentration, and that there is not a constant relationship between extracellular and intracellular oxygen concentrations. PMID- 3007920 TI - One- and two-dimensional EPR imaging studies on phantoms and plant specimens. AB - The advantages of electron paramagnetic resonance (EPR) imaging at L-band frequencies are discussed. The construction and calibration of a low-field L-band EPR imaging spectrometer is described with capillary phantoms containing aqueous nitroxides as paramagnetic imaging agents. The peak separation induced by the magnetic field gradient is related to the object separation. The linewidth of each (first-derivative) line is used to calculate the dimensions of each paramagnetic object, which is the sum of an intrinsic and a gradient-induced component. By extrapolating the linewidth back to zero field gradient, one obtains the intrinsic linewidth correction factor in computing image size. The nonuniformity caused by deterioration of biological substructure was examined with plant stems where one capillary vessel had become leaky. Spin-label destruction, particularly by biological reducing agents, was compared for three species of plant stems. PMID- 3007921 TI - [Conduction in the motor fibers of the ulnar nerve in cut-glass workers]. AB - The authors have estimated the motor conduction velocity of ulnar nerve motor fibres in 39 glass-cutters suspected to neuropathy originating from work performance. The conduction has been checked in two segments; wrist-elbow and below-above elbow (peripheral) and sulcus segments). Twenty-nine workers exhibited reduced conduction; in the sulcus segment--13-fold, in the peripheral segment--4-fold, and in both segments--12-fold. The nerve impairment was greater on the left side, especially in the sulcus segment. After 4 to 24 months another investigation was performed in 22 persons--10 carrying on the work and 12-who had abandoned it. The former group exhibited the impairment, whereas the latter--both improvement and progression. The authors suggest that an appropriate evaluation of the ulnar nerve lesion in glass-cutters but also people representing other professions causing the nerve pressure calls for an investigation of the conduction in the sulcus and peripheral segments. PMID- 3007922 TI - The interaction of MHC and Gm in liability to autoimmune thyroid disease. AB - In studies on immunogenetic factors in autoimmune thyroid disease, the association among Graves' disease, Hashimoto's disease, HLA and Gm haplotypes was investigated in 37 families in which two or more first degree relatives had Graves' disease. The results showed that two genes linked to HLA and Gm appeared to control susceptibility to Graves' and Hashimoto's disease, respectively, and that the individuals who did not have immunogenetic factors were very unlikely to develop Graves' or Hashimoto's disease. In the second study, the role of HLA-DR antigen expression on thyrocytes was investigated in 18 patients with Graves' disease. It was found that DR-positive thyrocytes increased. DR-positive T-cells (from thyroids and peripheral blood) increased in Graves' disease. Interferon gamma increased DR expression on thyrocytes. The results indicated that these changes may cause a vicious circle to produce and perpetuate autoimmune processes in Graves' disease. Finally, the correlation between thyroids and immunoglobulins was investigated in 11 untreated patients with Graves' disease. Thyroid tissues obtained from untreated patients were incubated in organ culture systems with autologous as well as allogeneic immunoglobulins to observe the release of hormones. The release of hormone was stimulated only by autologous immunoglobulins and it is, therefore, postulated that the role of self recognition, such as anti-idiotype antibody or anti-MHC antibody is crucial in the pathogenesis of Graves' disease. PMID- 3007923 TI - Cultured capillary endothelial cells from bovine adipose tissue: a model for insulin binding and action in microvascular endothelium. AB - Capillary endothelial cells were cultured from bovine adipose tissue. The endothelial nature of the cultures was documented by characteristic morphology, uniform presence of factor VIII antigen, and uptake of the endothelial cell marker Dil-Ac-LDL. The capillary cell cultures had specific, high affinity binding sites for insulin, demonstrating time and temperature dependence of binding, pH optimum, analog specificity, and inhibition of insulin binding by anti-insulin receptor antibodies. In both subconfluent and confluent cultures, insulin stimulated thymidine incorporation into DNA; significant stimulatory effects of insulin were observed at insulin concentrations of 1 ng/ml with maximal 8- to 10-fold increases at hormone concentrations of 1,000 to 10,000 ng/ml. Because of the ease of routine preparation, cell purity, presence of high affinity insulin binding sites, and insulin-sensitive metabolic responses, we suggest that the bovine capillary endothelial cultures could serve as a model cell system for the detailed study of insulin interactions with capillary endothelial cells. PMID- 3007924 TI - Alteration in activities of Na,K, ATPase, sugar transport, and insulin receptors in erythrocytes from hyperthyroid patients. AB - To investigate the selectivity of the reduction in Na,K, ATPase activity in erythrocytes from patients with hyperthyroidism, we have assessed cytochalasin B sensitive galactose uptake and insulin receptors as well as Na,K, ATPase-mediated rubidium uptake in erythrocytes from 11 patients with hyperthyroidism, 5 patients after treatment and 11 normal controls. There was a significant reduction in the Vmax values for rubidium uptake and the number of insulin receptors (23% and 20% below control, respectively), whereas there was a significant increase in the Vmax and Km values for galactose uptake (49% and 30% above control) and also in the average affinity of insulin receptors. The alterations both in rubidium and galactose transport activities were reversible with effective treatment of hyperthyroidism. The magnitude of alterations in the Vmax for rubidium uptake correlated inversely (r = -0.537 P less than 0.01, n = 16) and the Vmax and Km for galactose uptake correlated positively (r = 0.597 P less than 0.02 and r = 0.553 P less than 0.05, respectively) with serum T4 level. No correlation was found between the number of insulin receptors and serum IRI or T4 levels. These results suggest that the reduction in Na,K, ATPase activity observed in erythrocytes from hyperthyroid patients is not selective to this enzyme, but rather a reflection of widespread alterations of erythrocyte cell-surface proteins. PMID- 3007925 TI - Experimental diabetes mellitus impairs the function of the retinal pigmented epithelium. AB - The retinal pigmented epithelium (RPE), which influences the composition of the retinal extracellular fluid, is significantly affected in diabetes. Changes in RPE morphology, permeability, and electrophysiology in experimentally diabetic animals have been described. To facilitate the study of diabetes-related changes in RPE metabolism, we applied the techniques of quantitative histochemistry to pure samples of RPE and individual retinal layers from eyes of normal and alloxan diabetic rabbits. Glucose within the RPE approximated serum levels in both normal and diabetic animals. Other changes in diabetics included increased sorbitol, decreased myo-inositol, elevated total Na, and loss of measurable Na+-K+-ATPase activity within the RPE. The altered ion metabolism was associated with a progressive decrease in the amplitude of the RPE-generated c-wave of the electroretinogram. The deterioration of the c-wave was arrested by treatment of the diabetic animals with either myo-inositol supplementation or with sorbinil, an inhibitor of aldose reduction. Diabetic alterations in the RPE might impair the ability of the tissue to maintain normal transport functions. The subsequently altered composition of the extracellular environment of the retina may play an important role in the pathogenesis of diabetic retinopathy. PMID- 3007926 TI - Aldose reductase, glomerular metabolism, and diabetic nephropathy. AB - To explore a possible link between diabetic nephropathy and the enhanced activity of the polyol pathway known to occur in diabetes, we examined several pertinent metabolic parameters in glomeruli isolated from control and streptozotocin diabetic rats and assessed whether changes observed in diabetic glomeruli could be prevented by the oral administration of the aldose reductase inhibitor sorbinil. We found that glomerular polyol content is significantly increased in diabetes, whereas glomerular myo-inositol content is significantly reduced. The sorbitol accumulation and myo-inositol depletion were both completely prevented by sorbinil, which was given throughout the duration of diabetes. Activity of the membrane-bound sodium/potassium adenosine triphosphatase (Na-K-ATPase) was decreased in diabetic samples; this change was also completely prevented by sorbinil. Erythrocyte deformability is another factor that has been implicated in the pathogenesis of microangiopathic complications. The ability of red blood cells to undergo an adaptation in shape that permits passage through the smallest vessels is impaired in diabetes. Using blood from control, diabetic, and sorbinil treated diabetic rats, we found that erythrocyte deformability was decreased in diabetic samples and that sorbinil treatment significantly improved this parameter. Thus, if the glomerular consequences of sorbitol accumulation, myo inositol depletion, reduced Na-K-ATPase activity, and decreased erythrocyte deformability are pathogenetically implicated in diabetic nephropathy, the ability of sorbinil to impact on these changes suggests that it could favorably impact on the nephropathic process. PMID- 3007928 TI - Nuclear submicroscopy. PMID- 3007927 TI - Three-dimensional analysis of given cell structures: nucleolus, nucleoskeleton and nuclear inclusions. PMID- 3007929 TI - Role of the nuclear matrix during viral replication. PMID- 3007930 TI - The repeated sequences (incB) preceding the protein E gene of plasmid mini-F are essential for replication. AB - At the XhoI site (45.08F) of plasmid mini-F a deletion of 649 bp was generated employing exonuclease Bal31. By this deletion nucleotide sequences functioning as origin II and the four 19 bp direct repeats constituting the incB region in front of the E protein gene were removed from the plasmid. Analysis of proteins radioactively labelled in Escherichia coli mini-cells indicated that all mini-F encoded proteins are expressed. However, the plasmid carrying the deletion was not capable of replicating from the primary origin (origin I, 42.6F). Recently a smaller deletion at the XhoI site (45.08F) of about 300 bp, removing only the region functioning as origin II and replicating from origin I, was described by Tanimoto and Iino (1984, 1985). The data presented suggest that the incB repeats are essential for the initiation of replication from origin I, and possibly also from origin II, and seem not to be engaged in the autoregulation of E protein expression. PMID- 3007931 TI - The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system. AB - The DNA sequences of the mercuric resistance determinants of plasmid R100 and transposon Tn501 distal to the gene (merA) coding for mercuric reductase have been determined. These 1.4 kilobase (kb) regions show 79% identity in their nucleotide sequence, and in both sequences two common potential coding sequences have been identified. In R100, the end of the homologous sequence is disrupted by an 11.2 kb segment of DNA which encodes the sulfonamide and streptomycin resistance determinants of Tn21. This insert contains terminal inverted repeat sequences and is flanked by a 5 base pair (bp) direct repeat. The first of the common potential coding sequences is likely to be that of the merD gene. Induction experiments and mercury volatilization studies demonstrate an enhancing but non-essential role for these merA-distal coding sequences in mercury resistance and volatilization. The potential coding sequences have predicted codon usages similar to those found in other Tn501 and R100 mer genes. PMID- 3007932 TI - Achlya mitochondrial DNA: gene localization and analysis of inverted repeats. AB - Mitochondrial DNA from four strains of the oomycete Achlya has been compared and nine gene loci mapped, including that of the ribosomal protein gene, var1. Examination of the restriction enzyme site maps showed the presence of four insertions relative to a map common to all four strains. All the insertions were found in close proximity to genic regions. The four strains also contained the inverted repeat first observed in A. ambisexualis (Hudspeth et al. 1983), allowing an examination by analysis of retained restriction sites of the evolutionary stability of repeated DNA sequences relative to single copy sequences. Although the inverted repeat is significantly more stable than single copy sequences, more detailed analysis indicates that this stability is limited to the portion encoding the ribosomal RNA genes. Thus, the apparent evolutionary stability of the repeat does not appear to derive from the inverted repeat structure per se. PMID- 3007933 TI - Complete nucleotide sequences of Bacillus plasmids pUB110dB, pRBH1 and its copy mutants. AB - The deletion plasmids, pRBH1 (1.5 MDa, kanamycin resistance, Kmr) and pUB110dB (1.5 MDa, Kmr), were obtained from pTB913 (2.9 MDa, Kmr, isolated from a thermophilic bacillus) and pUB110 (3.0 MDa, Kmr, from Staphylococcus aureus), respectively. All the nucleotide sequences of these deletion plasmids were determined. Replication origin regions of pRBH1 and pUB110dB contained, respectively, 63 base-pair inverted repeat and a large open reading frame, encoding RepB protein (235 amino acid residues). The nucleotide sequences were identical to each other except for one base in the center of the inverted repeat. Two copy number mutant plasmids, pRBHC3 and pRBHC7, were obtained from pRBH1. The mutation points were located at different positions in the RepB protein coding region (Gly to Asp for pRBHC3 and Gly to Glu for pRBHC7). RepB protein was shown to be involved in the copy number control of these plasmids. PMID- 3007934 TI - Effect of base substitutions in the colicin E1 gene on colicin E1 export and bacteriocin activity. AB - Base substitutions have been introduced into the segment of the colicin E1 gene corresponding to the polypeptide region between the 404th and the 502nd residues which was considered to participate in colicin E1 export and bacteriocin activity. The methods used were in vitro localized mutagenesis with sodium bisulphite and in vivo mutagenesis using either nitrosoguanidine or ethyl methane sulphonate. Cells carrying mutagenized plasmids were screened by their inability to form a clear zone on a lawn of colicin E1 sensitive cells. Mutation sites were determined from the nucleotide sequence analysis and the altered amino acid residues were reduced. The mutant proteins were analysed for their ability to be exported to the periplasmic space and for their bacteriocin activity. Out of eight mutants obtained, three had a single amino acid replacement. Mutant proteins that had Ser and Glu in place of Pro-462 and Gly-502, respectively, showed a decrease in both the export and the bacteriocin activity. A mutant protein having Arg in place of Gly-439 showed a decrease only in the bacteriocin activity. These results suggest that the target region of colicin E1 contributes to the export as well as the bacteriocin activity but the two functions are supported in part by different amino acid residues of the protein. PMID- 3007935 TI - Genetic and physical characterization of putP, the proline carrier gene of Escherichia coli K12. AB - Two new mutants of E. coli K12, strains PT9 and PT32 were isolated, that were defective in proline transport. They had no high affinity proline transport activity, but their cytoplasmic membranes retained proline binding activity with altered sensitivity to inhibition by p-chloromercuribenzoate (pCMB). The lesion was mapped at the putP gene, which is located at min 23 on the revised E. coli genetic map (Bachmann 1983) as a composite gene in the proline utilization gene cluster, putP, putC, and putA, arranged in this order. The putC gene was shown to regulate the synthesis of proline dehydrogenase (putA gene product). Hybrid plasmids carrying the put region (Motojima et al. 1979; Wood et al. 1979) were used to construct the physical map of the put region. The possible location of the putP gene in the DNA segment was determined by subcloning the putP gene, genetic complementation, and recombination analyses using several proline transport mutants. PMID- 3007936 TI - Gene structure in the histidine operon of Escherichia coli. Identification and nucleotide sequence of the hisB gene. AB - The bifunctional enzyme imidazoleglycerolphosphate dehydratase and histidinolphosphate phosphatase is encoded by the hisB gene. The fourth gene of the histidine operon, hisB, was cloned and mapped on a 2,300 base pair DNA fragment. In the present study we report the complete nucleotide sequence of the hisB gene of Escherichia coli. The gene is 1,068 nucleotides long and codes for a protein of 355 amino acids with an apparent molecular weight of 39,998 daltons. The protein product(s) of the hisB region of both Salmonella typhimurium and E. coli were identified by subcloning and expression in an in vitro translation system. In both organisms the hisB gene directed the synthesis of a single protein with an apparent molecular weight of 40,500 daltons, consistent with the data derived from the nucleotide sequence analysis. PMID- 3007937 TI - The sigma-like product of sporulation gene spoIIAC of Bacillus subtilis is toxic to Escherichia coli. AB - The amino-acid sequence deduced from the nucleotide sequence of the spoIIAC gene of Bacillus subtilis has been shown to be homologous to that of the sigma subunit of the Escherichia coli RNA polymerase (Errington et al. 1985). I now describe results that indicate that this gene can be cloned in E. coli only under conditions in which it is not expressed. PMID- 3007938 TI - Regulation of exoprotein gene expression in Staphylococcus aureus by agar. AB - Insertion of the erythromycin-resistance transposon Tn551 into the Staphylococcus aureus chromosome at a site which maps between the purB and ilv loci has a pleiotrophic effect on the production of a number of extracellular proteins. Production of alpha, beta and delta hemolysin, toxic shock syndrome toxin (TSST 1) and staphylokinase was depressed about fifty-fold while protein A production was elevated twenty-fold. Hybridization analysis showed that the defect in expression of TSST-1 and alpha hemolysin was at the transcriptional level. Inability of the mutant strain to express either a cloned TSST-1 gene or the chromosomal gene indicates that the transposon has inactivated a trans-active positive control element. This element has been designated agr for accessory gene regulator. PMID- 3007939 TI - Isolation and characterization of the two structural genes coding for phosphofructokinase in yeast. AB - Yeast phosphofructokinase is an octamer composed of two different kinds of subunit. The genes coding for these subunits have been isolated by means of functional complementation in a pfk1 pfk2 double mutant. As a source of DNA the genomic library of Nasmyth and Tatchell (1980) constructed in the yeast multicopy vector YEp13 was used. Plasmids containing the information of one or the other gene were identified by back-transformation into pfk single mutants (pfk1 PFK2, PFK1 pfk2). Restriction maps of the respective insertions are provided. The genomic organization was confirmed by Southern analysis. Northern analysis showed hybridization to mRNAs of about 3.6 kb for both genes, corresponding to the molecular weight of the protein subunits. Transformation with one of the plasmids did not lead to an increase in phosphofructokinase activity. Subcloning of both genes in one multicopy vector (YEp13) and reintroduction into the yeast cell resulted in a 3.5-fold higher specific activity compared to the wild type. Overproduction of the protein subunits in this transformant was confirmed by SDS polyacrylamide electrophoresis of crude extracts stained with Coomassie-blue. This was not accompanied by an increased ethanol production. The sequences encoding the two subunits were shown to share homology. PMID- 3007940 TI - Isolation and molecular analysis of the phosphoglucose isomerase structural gene of Saccharomyces cerevisiae. AB - The PGI1 gene of Saccharomyces cerevisiae coding for the glycolytic enzyme phosphoglucose isomerase has been cloned by complementation of a mutant strain (pgi1) with a strongly reduced phosphoglucose isomerase activity. A genomic library constructed in the yeast multicopy vector YEp13 (Nasmyth and Tatchell 1980) was used. Four plasmids containing an overlapping region of 4.1 kb were isolated and characterized by restriction endonuclease mapping. Southern analysis of genomic digests prepared with different restriction enzymes confirmed the same pattern for the chromosomal sequences. Transformants with the isolated plasmids had a phosphoglucose isomerase activity increased by a factor of 7. The cloned sequence hybridized to a constitutively synthesized 2.2 kb RNA in Northern analysis. The coding region includes a 2.05 kb EcoRI fragment common to all four inserts. A fragment including part of the PGI1 region was subcloned into vector YRp7 and used to induce integration at the PGI1 locus. Genetical and Southern analysis of stable transformants showed that single as well as tandem integration took place at this locus. This showed that the PGI1 gene had been isolated. Finally, and in contrast to the results of Kempe et al. (1974a, b) who reported three isoenzymes in yeasts, only one copy of the PGI1 gene per genome was found in several laboratory strains tested by Southern analysis. PMID- 3007942 TI - [Genetic studies on cytochrome-c peroxidase in Saccharomyces cerevisiae: possible influence of cytoplasmic factors]. PMID- 3007941 TI - Plasmid pAO1 of Arthrobacter oxidans encodes 6-hydroxy-D-nicotine oxidase: cloning and expression of the gene in Escherichia coli. AB - The 160 kb plasmid pAO1 from Arthrobacter oxidans (Brandsch and Decker 1984) was subcloned in Escherichia coli with the aid of the plasmid vectors pUR222 and pBR322. Screening of the recombinant clones for enzyme activity revealed that the flavoenzyme 6-hydroxy-D-nicotine oxidase (6-HDNO), one of the enzymes of the nicotine-degradative pathway in A. oxidans, is encoded on pAO1. Immunoprecipitation of 35S-methionine-labelled E. coli cells with 6-HDNO-specific antiserum and expression of recombinant plasmid DNA in E. coli "maxicells" revealed that 6-HDNO is made as a 52,000 dalton protein, approximately 4,500 daltons larger than 6-HDNO from A. oxidans. The 6-HDNO activity was constitutively expressed in E. coli cells, possibly from an A. oxidans promoter, as shown by subcloning of the 6-HDNO gene in pBR322, using the expression vector pKK223-3 and the promoter probe vector pCB192. PMID- 3007943 TI - Pre- and postjunctional alpha-adrenoceptors at sympathetic neuroeffector junction in bovine mesenteric lymphatics. AB - We studied isolated bovine mesenteric lymphatics to elucidate the pharmacological characteristics of pre- and postjunctional alpha-adrenoceptors at the sympathetic neuroeffector junction. Cylindrical strips were incubated with [3H]-noradrenaline and mounted for superfusion. Electrical stimulation (2 Hz, 0.5 msec, 50 V) augmented the fractional release of labeled noradrenaline. Exogenous noradrenaline and clonidine caused a depression of the evoked tracer release. Phenoxybenzamine and yohimbine markedly enhanced the evoked overflow of adrenergic transmitter. Rings of lymphatic vessels were mounted for isometric tension recording in organ chambers filled with Krebs-Ringer bicarbonate solution. The vessels contracted when exposed to phenylephrine and clonidine. The ED50 of clonidine was significantly lower than that of phenylephrine. Prazosin caused a parallel shift to the right of the dose-response curve to phenylephrine. The antagonist, however, suppressed the magnitude of the maximum response to clonidine. Yohimbine caused parallel shift to the right of the dose-response curves to phenylephrine and clonidine, respectively. The Schild plots for yohimbine demonstrated that the drug was a competitive antagonist to phenylephrine and clonidine. The pA2 value of yohimbine to clonidine (7.6 +/- 0.4) was larger than that to phenylephrine (6.2 +/- 0.4). The pA2 value of prazosin to phenylephrine was 7.2 +/- 0.3. These results suggest that prejunctional alpha-adrenoceptors are involved in the negative feedback mechanism for autoregulation of noradrenaline release during postganglionic sympathetic nerve stimulation, and that both alpha 1- and alpha 2-like adrenoceptors do exist on lymphatic smooth muscle cells. PMID- 3007945 TI - HTLV-3 produces a broad range of illness. PMID- 3007944 TI - Neuromuscular transmission in bovine mesenteric lymphatics. AB - Neuromuscular transmission in bovine mesenteric lymphatics was investigated using the double sucrose-gap technique. Single pulses of 0.3 msec duration (35----90 V) elicited excitatory junction potentials (EJP's) with a time to peak of about 1 sec. The EJPs showed facilitation and at stimulus frequencies greater than about 0.25 Hz could summate to reach threshold for action potential firing. The action potential so produced was followed by a phasic contraction of an "all or none" type. Increasing the frequency of stimulation did not increase the force of contraction but the resulting sustained depolarization increased the probability of a second or third action potential (and thus contraction) being elicited. EJPs and their electrical and mechanical consequences could be blocked by tetrodotoxin (10(-6) M) and by phentolamine (5 X 10(-7) M) confirming their neural mediation and dependence on postjunctional alpha-receptors. PMID- 3007947 TI - [AIDS (acquired immune deficiency syndrome)]. PMID- 3007946 TI - Education is key to the battle against AIDS. PMID- 3007948 TI - Are the vascular complications of diabetes mellitus preceded by an altered thromboxane/prostacyclin plasmatic ratio? AB - Although many data regarding the biosynthesis of thromboxane A2 and prostacyclin in diabetes mellitus have recently appeared in the literature, it is not clear whether an imbalance between the generation of the two prostaglandins might be connected to the vascular complications of diabetes. In the present review we have tried to emphasize the most significant aspects of these studies and we have focused on alterations of platelet prostacyclin receptors and on the effects of circulating immune complexes on platelets of diabetics. It is likely that studies on the release of platelet derived growth factor as well as more precise definitions of its action on vessel wall cells leading to a massive release of prostacyclin, will permit us to ascertain whether an alteration in prostaglandin ratio is linked to the genesis of the vascular complications in diabetics. PMID- 3007949 TI - Pre-menstrual syndrome--are gonadotropins the cause of the condition? AB - It is proposed that premenstrual syndrome results from the action of elevated gonadotropin levels in various tissues of body other than their natural target organs. These levels are derived from an increased sensitivity to estrogen after pregnancy, childbirth, etc., particularly with respect to the positive feedback on gonadotropin release from the pituitary. Estrogen in conjunction with gonadotropin-releasing hormone (GnRH) releases excessive amounts of follicle stimulating hormone (FSH) and luteinizing hormone (LH) at ovulation and in the premenstrual phase (post-menopausal patients have greatly elevated gonadotropins and can also demonstrate cyclic symptoms). Gonadotropin action via adenylate cyclase in the adrenal cortex elevates cortisol, while antagonism of parathyroid hormone action on bone gives rise to hypocalcemia. The physiological and psychological symptoms may thereby be explained. PMID- 3007950 TI - Tonsillectomy as a co-factor in the development of AIDS. AB - Hypotheses regarding the factors that predispose individuals to developing AIDS after exposure to HTLV-III/LAV are beginning to emerge. It is suggested here that surgical removal of tonsils in childhood may increase the risk of developing opportunistic infections after infection with this newly discovered retrovirus. PMID- 3007951 TI - A robotic system to prepare samples for HTLV-III testing. AB - A robotic handling system was adapted to perform the sampling and dilution steps needed in an assay to detect antibodies to the HTLV-III virus, the causative agent of AIDS. The system reduced the labor required to prepare the samples and provided standardization and accuracy in the preparation of the samples. PMID- 3007952 TI - Arbovirus infections of humans in New South Wales. Seroepidemiology of the flavivirus group of togaviruses. AB - A seroepidemiological study of 16 842 human sera, collected in 1981 and 1982 from all health regions of New South Wales, was carried out using the haemagglutination-inhibition (HI) test and eight Australasian flaviviruses: Murray Valley encephalitis (MVE); Kunjin (KUN); Alfuy (ALF); Stratford (STR); Kokobera (KOK); Edge Hill (EH); Sepik (SEP); and Saumarez Reef (SRE). A limited survey was also carried out with two recently discovered flaviviruses, Gadgets Gully and CSIRO 946. Antibody prevalence rates were low on the coast and tablelands (around 2%-8%), moderate on the western slopes (6%-11%) and high on the western plains (26%-42%). Some centres had higher prevalence rates, Bourke being the highest at 78%. The survey indicated that SEP and SRE viruses are unlikely to infect humans in New South Wales. Similarly, there was no evidence for Gadgets Gully and CSIRO 946 infection of humans. HI antibody prevalence rates were highest to STR, MVE, KUN and ALF in that order, these agents being closely related antigenetically. Reactions to KOK and EH occurred less frequently. Serological tests of greater specificity will be required to identify the flaviviruses that elicit these HI antibodies. PMID- 3007953 TI - Bone-marrow transplantation for haematological malignancy in childhood. AB - Twenty-three children with haematological malignancies and a poor prognosis underwent bone-marrow transplantation. Thirteen children had acute lymphoblastic leukaemia, eight had acute nonlymphoblastic leukaemia, one had chronic myeloid leukaemia and one had malignant histiocytosis. One child was in relapse at the time of transplant and 22 were in first or subsequent remission. Before transplantation all patients received cyclophosphamide (60 mg/kg) on two consecutive days followed by total body irradiation given as a single dose of 10 Gy at 0.18 Gy/min (one patient) or 0.07 Gy/min (three patients), or as a fractionated dose of 10-12 Gy at 0.07-0.1 Gy/min (19 patients). One child with malignant histiocytosis also received two doses of etoposide (5 mg/kg). Methotrexate was given after transplantation to prevent or modify graft-versus host disease (GVHD). One patient who received a transplant in relapse died early from overwhelming bacterial sepsis. Twenty-two patients engrafted, and of these 11 developed acute GVHD; five developed chronic GVHD; seven developed interstitial pneumonitis, with four deaths; and five relapsed between three and 12 months after transplantation, with three deaths. Fifty-nine per cent (13/22) of patients who received a transplant during remission remain in continuous complete remission and 68% (15/22) have survived for a median of 18 months (range, four to 73 months). Bone-marrow transplantation that is undertaken during remission of disease offers a prolonged disease-free survival in selected childhood malignancies. PMID- 3007954 TI - The painful swollen calf. A comparative evaluation of four investigative techniques. AB - Complications of popliteal cysts may closely mimic the clinical features of a deep venous thrombosis. We assessed the sensitivity and specificity of the non invasive procedures of radionuclide venography and popliteal space ultrasound examination compared with those of contrast venography and arthrography, respectively, and then prospectively studied 23 non-surgical patients with acutely painful, swollen calves to determine the utility of these techniques. The cause of this symptom was popliteal cyst complications in 10 patients, deep venous thrombosis in seven patients, and both conditions in two patients. Radionuclide venography was highly reliable and ultrasound examination was specific but only moderately sensitive in these studies. The painful, swollen calf may be investigated adequately in most cases by means of noninvasive invasive techniques; contrast venography and arthrography should be reserved for only a minority of patients. PMID- 3007955 TI - Pharmacology of opioids. Part 1. Basic aspects. PMID- 3007956 TI - A polyoma-like virus associated with an acute disease of fledgling budgerigars (Melopsittacus undulatus). AB - A virus previously isolated from fledgling budgerigars (Melopsittacus undulatus) suffering from an acute disease, has been purified and the structural characteristics have been determined. The virions with a buoyant density of 1.34 g/ml are non-enveloped icosahedral particles with a diameter of about 46-48 nm. Their DNA genome has a molecular weight of about 3.3 X 10(6) d, and exists as supericoiled circular, relaxed circular, and linear molecules. There are eight structural proteins, the most abundant of which has a molecular weight of about 42,000 d. Empty capsid shells with buoyant densities of 1.31 g/ml are similar in size and shape, but lack DNA and histone-like polypeptides. Virus replication in chicken embryo cells results in cytopathic changes characterized by rounding and enlargement of the nucleus, and formation of intranuclear inclusion bodies. All these properties justify classification of the virus as polyoma-like. PMID- 3007958 TI - Anesthetic consideration in a patient with acquired immune deficiency syndrome. PMID- 3007959 TI - Genesis of polarity in renal tubular cells. AB - Using enveloped RNA viruses as probes to study the biogenesis of surface polarity in epithelial cell lines, evidence has been obtained that, as shown for secretion in exocrine cells, the delivery of plasma membrane proteins to the cell surface is polarized. Divergence between the pathways of apical and basolateral plasma membrane proteins occurs at the level of the distal Golgi apparatus. Presumably, carrier vesicles with affinity for specific receptors in the apical or in the basolateral regions of the plasmalemma participate in the transport between the Golgi apparatus and the cell surface. Agents which disrupt actin filaments and microtubules do not alter significantly this process of vectorial exocytosis. PMID- 3007960 TI - Functional properties of glomerular cells in culture. AB - With the development of techniques to isolate and propagate homogeneous cultures of glomerular cell types, numerous investigations have been initiated to study the functional characteristics of cultured glomerular cells. Since much of the work to date has been performed on glomerular mesangial cells, a good deal of this discussion will be about this cell type. Glomerular mesangial cells together with the surrounding matrix material form the glomerular mesangium. These cells contain contractile microfilaments as well as receptors for vasoconstrictor substances such as angiotensin II. Therefore, one proposed function of this cell type is the regulation of glomerular perfusion and filtration by contraction. Cultured mesangial cells contract in response to angiotensin II and arginine vasopressin and, in addition, produce prostaglandins which may function to regulate contraction. In this article, we will review the evidence that has accumulated concerning the contractile nature of mesangial cells. Since prostaglandins may influence mesangial cell contraction, the prostaglandin synthetic capabilities of glomerular epithelial and mesangial cells will be discussed. We will conclude by discussing how glomerular cell culture can be used to study the pathobiology of certain glomerular diseases. PMID- 3007957 TI - Bacillus thuringiensis and related insect pathogens. PMID- 3007961 TI - Characteristics of renal collecting tubule cells in primary culture. AB - Isolated nephron segments have been suitable for many types of kidney experiments. Nevertheless, the quantity of cells easily obtained is insufficient for many biochemical analyses. The advent of tissue culture has provided an alternative which allows researchers to work with large numbers of a single cell type. Unfortunately, the parentage of many established cell lines is uncertain. Madin-Darby canine kidney cells, for instance, while most like epithelial cells of distal origin, also retain characteristics of other kidney epithelial cells. Established cell lines have undergone dedifferentiation. For those biochemical experiments in which a closer link to 'physiological relevance' was desired, it was necessary to develop the technology to isolate large numbers of a single identifiable kidney cell type. In this review, some of the characteristics of an isolated population of one particular cell type, the collecting tubule cell, will be discussed. PMID- 3007963 TI - Use of cultured renal tubular cells in the study of cell injury. AB - The use of various types of cultured mammalian renal tubular epithelial cells in the study of cell injury has been reviewed. Permanent cell lines, primary explant cultures, monolayers from individually microdissected tubules, isolated cells and organ cultures have been used. In the majority of studies, cultured cells of normal tissue origin have been treated with a noxious agent and alterations in growth, morphology, biochemical and immunological properties studied. Earliest studies examined infection by parasites and bacteria and the effects of plant and bacterial toxins, carcinogens, metabolic and transport inhibitors, cytoskeletal perturbants, general inhibitors of protein, glycoprotein, DNA and RNA synthesis. More recent studies have concentrated on the effects of specific nephrotoxins, such as heavy metals and aminoglycoside antibiotics and of ischemia which have bearing on the pathogenesis of acute renal failure. An additional approach has been to culture diseased renal epithelia of cystic, diabetic or tumor origin and compare their properties with those of normal cultured tubular epithelia. Future studies using cultured renal tubular cells will be valuable in elucidating the cellular and subcellular mechanisms of renal epithelial cell injury in disease. PMID- 3007962 TI - Vasopressin receptor-adenylate cyclase interactions. Studies in an intact cultured renal epithelial cell line (LLC-PK1). AB - The antidiuretic action of vasopressin is mediated by activation of adenylate cyclase and generation of cyclic AMP. Radioligand binding and radiation inactivation studies have been done using a pig kidney epithelial cell line in culture to define the sequence of subunit interactions involved in the activation of adenylate cyclase by vasopressin. Based on the results of these studies and the known biochemical properties of the individual adenylate cyclase subunits, a hormonal activation model is proposed. According to this model, activation results from the sequential steps of dissociation of the alpha beta subunits of the guanyl nucleotide regulatory unit followed by the binding of GTP to dissociated alpha. The development of this model would not have been possible without the availability of the intact cultured cell system. PMID- 3007964 TI - The role of metabolic acidosis in causing uremic hyperphosphatemia. AB - Metabolic acidosis was corrected within 1 h in 7 chronic anuric uremic patients on maintenance hemodialysis and, in the course of a week, in 11 other patients on conservative therapy. These patients were on a diet supplying constant amounts of phosphate throughout the duration of the study. In the former group the intravenous infusion of sodium bicarbonate was used, and, in the latter, the oral administration of sodium citrate. In both groups serum phosphate was found to decrease significantly. In the patients on conservative therapy, phosphaturia also had significantly decreased while no changes occurred in their daily fecal loss of phosphate that was measured in 3 patients. These findings indicate that metabolic acidosis is one of the causes of hyperphosphatemia in chronic uremic patients. PMID- 3007965 TI - Intact ability to lower urine pH in nonacidotic adrenalectomized rats. AB - Distal acidification was assessed in adrenalectomized (ADX) rats in which the development of acidosis was prevented by oral supplementation with NaHCO3, with or without glucocorticoid replacement. Totally corticosteroid-deficient nonacidotic rats were capable of lowering their urine pH in response to Na2SO4 infusion from a baseline of 7.47 +/- 0.22 to 4.83 +/- 0.1 (p less than 0.001). A similarly intact ability to lower the urine pH was also demonstrated in glucocorticoid-replaced mineralocorticoid-deficient rats. Absolute ammonium excretion was lower in ADX animals compared to controls (0.79 +/- 0.08 vs. 0.46 +/- 0.06 microEq/min, p less than 0.01) but when corrected for the difference in GFR, ammonium excretion was the same in ADX and adrenal-intact rats. During bicarbonate loading and at similar blood and urine pH, and bicarbonate concentrations, the U-B pCO2 gradient was similar in mineralocorticoid-deficient and adrenal intact rats (44 +/- 5.1 vs. 36 +/- 2.6 mm Hg, respectively). Amiloride administration to mineralocorticoid-deficient rats led to a reduction in the U-B pCO2 gradient from 30 +/- 4.5 to 10 +/- 3.0 mm Hg (p less than 0.002). These results indicate that the ability to lower the urine pH and raise the urine pCO2 is intact in the nonacidotic ADX rat; ammonium excretion in this model is reduced in proportion to the observed reduction in GFR, and amiloride administration inhibits acidification in ADX rats. The data strongly suggest the presence of a major site of aldosterone-independent, sodium-dependent acidification mechanism likely located at the level of the cortical collecting tubule. PMID- 3007966 TI - Differential biological effects of calcitonin and parathyroid hormone on isolated perfused bone. AB - These studies examine the release of 3',5'-cyclic adenosine monophosphate (cyclic AMP) from isolated perfused canine bones in response to synthetic bovine parathyroid hormone (syn b-PTH 1-34) and human calcitonin (hCT). Bones for perfusion were obtained from three groups of dogs: control (n = 11); thyroparathyroidectomized (n = 7), and mithramycin-treated thyroparathyroidectomized animals (n = 5). The results indicate that PTH causes a greater release of cyclic AMP from adult bones than CT; the addition of the two hormones simultaneously results in a synergistic rather than an additive effect; mithramycin inhibits the cyclic AMP response to calcitonin; and thyroparathyroidectomy decreases the cyclic AMP release in response to CT stimulation, suggesting that the cells (osteoclast-like) that respond to CT have an impaired response in the absence of PTH; acute TPTX does not affect the response of the cell populations of adult bones to PTH. PMID- 3007967 TI - [Use of hydroxyapatite (Calcitite) in the morphofunctional restoration of severe bone defects]. PMID- 3007968 TI - Life after a mastectomy: a message to nurses. PMID- 3007969 TI - Public health guidelines for enhancing diabetes control through maternal- and child-health programs. PMID- 3007970 TI - Recommendations for preventing transmission of infection with human T lymphotropic virus type III/lymphadenopathy-associated virus during invasive procedures. PMID- 3007971 TI - Safety of therapeutic immune globulin preparations with respect to transmission of human T-lymphotropic virus type III/lymphadenopathy-associated virus infection. PMID- 3007972 TI - [Study on glycosaminoglycans contents in scirrhous carcinoma of the stomach]. PMID- 3007973 TI - [Cytotoxicity of interleukin 2-induced lymphocytes and effects of serum immunosuppressive factors]. AB - IL2-induced lymphocytes (IIL) obtained from normal subjects were examined as to a possibility for induction of tumor-specific killer cells. Moreover, effects of serum on their cytotoxic activity were observed. The IIL which were pre sensitized with mitomycin C-treated MKN-28 (IILM), showed a markedly increased cytotoxic activity against MKN-28. The IIL which were either pre-cultured with PHA or not, showed augmentation of cytotoxic activity against various human cancer cell lines, although their cytotoxic activity against MKN-28 was lower than that of IILM. The cytotoxic activity of IILM was suppressed by addition to the medium of non-specific immunosuppressive factors (IAP, ferritin, alpha fetoprotein) or of various sera obtained from gastric cancer patients, in whom histological diagnosis was tub1, por or sig, but the suppression to the IILM activity was significantly milder than that to the activity of peripheral lymphocytes. The degree of suppression by tub1 serum was significantly higher than that by the serum from por or sig. When tub1 serum was added to the medium during pre-sensitization, the cytotoxic activity of IILM against MKN-28 was reduced significantly. However, addition of por or sig serum showed no significant reduction in cytotoxic activity of IILM. PMID- 3007974 TI - [Mitotic activity of cancer cells and survival of gastric cancer patients]. AB - An in vivo stathmokinetic method was used to analyze the mitotic activity of cancer cells from 43 gastric cancer patients. The mitotic activity was distributed between mitotic index (MI) 40.0% and MI 127.0%. The patients were classified into 3 groups; low mitotic activity (L-MA) group (MI less than 70.0%), middle mitotic activity (M-MA) group (70.0%) less than or equal to MI) 90% and high mitotic activity (H-HA) group (MI greater than 90.0%). Survival curve and average survival period were examined and compared with each of 3 mitotic activity groups. The survival curve of L-MA group was better than that of M-MA group (generalized Wilcoxon test, Z = 1.815, p less than 0.1), and the latter was significantly better than that that of H-MA group (generalized Wilcoxon test, z = 2.048, p less than 0.05). Average survival period (mean +/- S.D.) of dead patients from cancer recurrence was 24.0 months in L-MA group (n = 1), 16.1 +/- 2.4 months in M-MA group (n = 9) and 7.4 +/- 2.4 months in H-HA group (n-13). Patients with gastric cancer of high mitotic activity died earlier than patients with that of low mitotic activity in the identical histologic type, identical degree of cancer invasion and the identical stage of cancer. The results suggested that the mitotic activity of cancer cells was utilized as a new prognostic parameter of gastric cancer. PMID- 3007975 TI - Reactive-oxygen formation and its relationship to prostaglandin and cyclic AMP production by zymosan-treated rat peritoneal macrophages. AB - Addition of zymosan (20 particles/cell) to suspensions of resident rat peritoneal macrophages caused an increase in the concns of prostaglandins and cyclic AMP. Preincubation of the cells with inhibitors of arachidonate metabolism led to inhibition of prostaglandin, but not of cyclic AMP, formation, which suggested that the two processes may occur independently of each other in phagocytosing cells. The luminol-dependent chemiluminescence associated with the addition of zymosan to the cells consisted of a minor, Ca2+-dependent, glucose-independent component and a major, glucose-dependent, Ca2+-independent component. Only the minor, Ca2+-dependent component appeared to be related to the lipoxygenation of arachidonic acid. Close examination of the production of prostaglandins and cyclic AMP and of chemiluminescence after zymosan addition, indicated that the expanded pool of endogenous cyclic AMP was probably not a negative modulator of the other two processes, although they remained susceptible to inhibition by exogenously-added cyclic AMP analogues or PGE2. The events induced by zymosan may be relevant to the physiological roles of prostaglandins during the inflammatory response. PMID- 3007977 TI - The interaction of 1-anilino-8-naphthalene sulphonate with human C1q. AB - C1q has 12 binding sites for 1-anilino-8-naphthalene sulphonate (ANS), two per peripheral subunit. This number increases to 18 upon weak-acid-induced conformational transition in the globular heads. One ANS binding site is present in each C gamma 2 domain of human IgG. ANS is bound by C1q with a higher affinity (Ka = 2.07 X 10(6) M-1) than by the Fc fragment (Ka = 9.07 X 10(4) M-1) of human IgGl. Hence the inhibitory capacity of C1q binding to IgG immune complexes of ANS probably reflects its preferential binding to the globular heads of C1q. The characteristics of ANS-C1q binding may in part explain the hydrophobic component of the C1q-IgG interaction. It is suggested that an ionic-hydrophobic two-step process is involved in the contact between C1q and IgG. PMID- 3007976 TI - Co-operative interaction of subcomponents of the first component of complement with IgG: a functional defect of dimeric Facb from rabbit IgG. AB - By following dissociation kinetics of radiolabelled C1q from rabbit IgG antibody sensitized sheep red blood cells (SRBC) before and after its incorporation in the C1 complex, it was demonstrated that the binding stability is markedly enhanced by the presence of the C1r2-C1s2 subunit of C1 which by itself exhibits no significant binding capacity to immune complexes. The dissociation of C1q was decreased by up to 95%, the extent of decrease being pronounced as the cell surface IgG antibody density increased. However, such a stabilizing effect of C1r2-C1s2 was largely abolished when SRBC sensitized with the dimeric fragment F(acb)2 lacking C gamma 3 was used as the C1 binder, whereas the dissociation rate of uncomplexed C1q from F(acb)2-sensitized cells was similar to that from whole IgG-sensitized cells. It was also shown that, although the C1r2-C1s2 subunit is dissociated selectively from C1 bound to either IgG- or F(acb)2 sensitized cells in the presence of EDTA, it is held on much longer by the former cells than the latter cells. These results were taken to indicate that, although the C1 fixation by immune complexes of IgG is undertaken primarily by the interaction between C1q and the C gamma 2 domain, it is also strengthened by the secondary interaction between the C1r2-C1s2 subunit of C1 and the C gamma 3 domain or a structure which is dependent on the pair of C gamma 3 domains. PMID- 3007978 TI - Lack of UV-induced respiration shutoff in a recF strain of Escherichia coli: temperature conditional suppression at 30 degrees C by the sfrA mutation. AB - A mutation in the recF gene of Escherichia coli results in a radiation-sensitive strain. The RecF pathway and the RecBC pathway account for nearly all of the conjugative recombination occurring in E. coli. recBC cells are radiation sensitive and carry only out a small amount of recombination but these deficiencies are suppressed by an sbcB as recombination is shunted to the RecF pathway. A recBC sbcB recF strain is very radiation-sensitive and is devoid of recombination ability. These deficiencies are suppressed by the srfA mutation; srfA is a recA allele. UV-induced respiration shutoff is a recA+, lexA+ and recBC+ dependent. We report in this paper that respiration does not shutoff in a recF strain at 37 and 30 degrees C. an srfA mutation suppresses this lack of respiration shutoff effect in a recF srfA mutant at 30 degrees C but not at 37 degrees C; no suppression by this mutation occurs at either temperature in a recF recBC sbcB strain. An srfA strain also does not shut off its respiration at 37 degrees C and shows a temperature conditional UV-induced respiration shutoff response at 30 degrees C. The srfA mutation is thought to cause an altered RecA protein to be produced and we suggest that at 37 degrees This altered protein is temperature sensitive. We conclude from the results in this paper that the recF gene product is required for UV-induced respiration shutoff and that the RecA protein plays a special role in the induction process. PMID- 3007980 TI - CDC collect neonatal herpes simplex virus data. PMID- 3007979 TI - The nature of mutants induced by ionising radiation in cultured hamster cells. III. Molecular characterization of HPRT-deficient mutants induced by gamma-rays or alpha-particles showing that the majority have deletions of all or part of the hprt gene. AB - DNA from 58 independent HPRT-deficient mutants of V79 hamster cells induced by ionising radiation was analysed by Southern blot hybridization to a full-length hamster hprt cDNA. About half of the gamma-ray-induced mutants (20/43) were apparently total gene deletions, because they lacked all functional hprt gene sequences hybridizing to the cDNA probe. Another 10 mutants showed various partial deletions and/or rearrangements of the hprt gene. The remaining 13 mutants showed no detectable change in comparison to the structure of the normal gene, which correlated well with previous characterization of these mutants indicating that most carry point mutations in the hprt gene. However, it is probable that some of these point mutations occurred spontaneously rather than being radiation-induced. A smaller number of alpha-particle induced mutants gave similar results: out of a total of 15 mutants, 6 appeared to be total gene deletions, 5 had partial deletions and/or rearrangements, and 4 had no detectable changes. Thus, 70% or more of radiation-induced HPRT-deficient mutants arise through large genetic changes, especially deletions of all or part of the hprt gene. This result is to be contrasted with data published previously by ourselves and others indicating that the majority of spontaneous and ethyl methanesulphonate-induced mutations of hprt and similar genes arise by point mutation. PMID- 3007981 TI - Structure and organization of the histidine-rich protein gene of Plasmodium lophurae. AB - Recombinant cDNA clones representing the carboxy-terminal portion of the histidine-rich protein of Plasmodium lophurae and the 3' untranslated region of the mRNA have been sequenced. Histidine accounts for 78% of the predicted amino acid sequence. The DNA and protein sequences in this region differ significantly from published sequences deduced from cloned genomic DNA of P. lophurae. Sequence data from two independent cDNA clones, comparison of restriction endonuclease sites present in genomic DNA, genomic and cDNA clones, gene titrations, S1 nuclease digestion of cDNA-mRNA hybrids and comparison of predicted and published data for the amino acid composition of the histidine-rich protein all suggest that P. lophurae contains one histidine-rich protein gene and that the sequence of the 3' coding region of this gene has been correctly deduced from the cDNA clones. PMID- 3007982 TI - In vivo indexes of platelet and vascular function during fish-oil administration in patients with atherosclerosis. AB - Populations that consume a diet rich in marine lipids may have a lower risk of atherosclerotic disease. Fish oil contains the N-3 polyunsaturated fatty acid eicosapentaenoate, and the biosynthesis of thromboxanes and prostacyclins from eicosapentaenoate (thromboxane A3 and prostaglandin I3), rather than from the usual precursor arachidonate (thromboxane A2 and prostaglandin I2), may help to reduce the risk. To examine this hypothesis, we studied the effect of eicosapentaenoate supplementation (10 g per day) for one month on the synthesis of thromboxanes and prostacyclins, as assessed by urinary metabolite excretion, in six patients with peripheral vascular disease and seven normal controls. Supplementation markedly increased the eicosapentaenoate content of phospholipids from red cells and platelets. Synthesis of the platelet agonist thromboxane A2, which was elevated in the patients at base line, declined by 58 percent during supplementation but did not reach normal values. The decline in thromboxane A2, which is synthesized from arachidonate, coincided with the formation of the inactive thromboxane A3, which is synthesized from eicosapentaenoate. A lower dose of eicosapentaenoate (1 g per day) was not sufficient to maintain the changes in thromboxane A2 synthesis. Platelet function was only moderately inhibited during eicosapentaenoate supplementation, consistent with incomplete suppression of thromboxane A2 synthesis. These studies show that a high dose of eicosapentaenoate alters the pattern of synthesis of thromboxanes and prostacyclins. However, effects comparable to those of aspirin require long-term administration in high doses. Whether other properties of fish oil might render it a more attractive antithrombotic therapy remains to be determined. PMID- 3007983 TI - A long-term follow-up study of patients with post-poliomyelitis neuromuscular symptoms. AB - A "post-polio" syndrome characterized by new neuromuscular symptoms, including muscle weakness, may develop years after recovery from acute paralytic poliomyelitis. We studied 27 patients (mean age, 50.6 years) in whom new muscle weakness developed a mean of 28.8 years after recovery from acute polio. We reevaluated these patients during a mean follow-up period of 8.2 years (range, 4.5 to 20) after they were originally studied at the National Institutes of Health. The total mean follow-up period after the onset of new weakness was 12.2 years (range, 6 to 29). The patients were assessed with quantitative muscle testing, muscle biopsy, electromyography, and virologic and immunologic examination of the cerebrospinal fluid. Muscle strength had declined in all patients. The rate of decline averaged 1 percent per year. The decrease was irregular, with subjective plateau periods that ranged from 1 to 10 years. None of the patients had amyotrophic lateral sclerosis. Oligoclonal bands (IgG) were found in the cerebrospinal fluid of 7 of 13 patients studied, but no specific elevation of antibodies to poliovirus was observed in the cerebrospinal fluid. The newly affected muscles that were evaluated longitudinally with follow-up muscle biopsies and electromyography showed signs of chronic and new denervation. Groups of atrophic muscle fibers (group atrophy) and "neurogenic jitter" were not present. New post-polio muscle weakness is not a life-threatening form of motor neuron deterioration. It appears that this weakness is not due to a loss of whole motor neurons, as in amyotrophic lateral sclerosis, but that it is due to a dysfunction of the surviving motor neurons that causes a slow disintegration of the terminals of individual nerve axons. PMID- 3007984 TI - Cytomegalovirus immune globulin and seronegative blood products to prevent primary cytomegalovirus infection after marrow transplantation. AB - In an attempt to prevent primary cytomegalovirus infection after marrow transplantation, we randomly assigned 97 patients who were seronegative for antibody to cytomegalovirus before transplantation to receive one of the following: (1) both intravenous cytomegalovirus immune globulin and seronegative blood products (23 patients); (2) seronegative blood products alone (28 patients); (3) globulin alone (22 patients); or (4) neither treatment (24 patients). Patients not assigned to receive seronegative blood products received unscreened blood products from random donors. The incidence of cytomegalovirus infection according to study group among patients in the study for at least 62 days was 5 percent, 13 percent, 24 percent, and 40 percent, respectively. Among 57 patients with seronegative marrow donors, those who received seronegative blood products had significantly less infection (1 of 32) than those who received standard blood products (8 of 25, P less than 0.007). In contrast, the use of seronegative blood products did not appear to prevent cytomegalovirus infection among patients with seropositive marrow donors. The possibility that cytomegalovirus immune globulin as used in this study can prevent cytomegalovirus infection or ameliorate cytomegalovirus disease was not confirmed, and it cannot be recommended for routine use without additional study. PMID- 3007985 TI - Protecting confidentiality in epidemiologic investigations by the Centers for Disease Control. PMID- 3007988 TI - More on HTLV-III antibody testing in New York City. PMID- 3007987 TI - The calcium messenger system (2). PMID- 3007986 TI - Secretion of chromogranin A by peptide-producing endocrine neoplasms. AB - Chromogranin A, the protein that is co-stored and co-released with catecholamines from the adrenal medulla, has recently been identified in a variety of human endocrine tissues, both normal and neoplastic. We investigated the secretion of chromogranin A by peptide hormone-producing human tumors in studies of patients with the following neoplastic disorders: pheochromocytoma, parathyroid adenoma, primary parathyroid hyperplasia, medullary thyroid carcinoma, thyroidal C-cell hyperplasia, carcinoid tumor, oat-cell lung carcinoma, pancreatic islet-cell tumor, and aortic-body tumor. All these patient groups had elevated concentrations of plasma chromogranin A. We distinguished different forms of immunoreactive plasma chromogranin A by size with the use of gel filtration. Plasma chromogranin A levels were not elevated in patients with diverse "control" conditions--both benign and malignant and both endocrine and nonendocrine--in which peptide hormones are not produced. The sensitivity and specificity of plasma chromogranin A elevations in the diagnosis of peptide-producing endocrine neoplasms were 81 and 100 percent, respectively. The elevation of plasma chromogranin A in our subjects suggests that their neoplasms co-release chromogranin A along with the usual resident hormone of the tumor, that these neoplasms could be characterized as "chromograninomas," and that measurement of plasma chromogranin A may be a useful diagnostic procedure in subjects with endocrine tumors, especially multiple endocrine neoplasia. PMID- 3007989 TI - Bovine leukaemia virus and multiple sclerosis. PMID- 3007990 TI - Origins of human T-lymphotropic viruses. PMID- 3007991 TI - Endogenous retrovirus in multiple sclerosis? PMID- 3007993 TI - No help on AIDS. PMID- 3007992 TI - Genomic diversity correlates with clinical variation in Ph'-negative chronic myeloid leukaemia. AB - The Philadelphia chromosome (Ph') is found in the blood cells of about 90% of patients with chronic myeloid leukaemia (CML) and usually results from the reciprocal chromosome translocation t(9;22). This translocation relocates the proto-oncogene c-abl, normally found on chromosome 9q34, to within the breakpoint cluster region (bcr) on chromosome 22q11 (refs 3-8). The juxtaposition of c-abl and the 5' portion of bcr appears to be the critical genomic event in CML and results in a novel 8-kilobase (kb) fused abl/bcr transcript and a c-abl-related protein of relative molecular mass 210,000 (ref.11). About 10% of adult patients diagnosed as CML lack the Ph' chromosome; they represent a heterogeneous group of disorders which are difficult to diagnose precisely. We have examined five patients with CML whose leukaemic cells have a normal karyotype. We report here that two of the patients showed the same genomic change as occurs in Ph'-positive CML, but the change resulted from a mechanism other than chromosomal translocation. The remaining three patients showed no genomic rearrangement. This genomic diversity correlated with the clinical differences between the patients. PMID- 3007994 TI - Spatial restriction in expression of a mouse homoeo box locus within the central nervous system. AB - A common feature of Drosophila homoeo box genes appears to be their spatially restricted expression patterns during morphogenesis. Using Northern blot analysis and in situ hybridization to mouse tissue sections, the spatially restricted expression of a newly identified mouse homoeo box locus, Hox-3, within the central nervous system of newborn and adult mice has been demonstrated. PMID- 3007995 TI - The trans-activator gene of HTLV-III is essential for virus replication. AB - Studies of the genomic structure of human T-lymphotropic virus type III (HTLV III) and related viruses, implicated as the causal agent of acquired immune deficiency syndrome (AIDS), have identified a sixth open reading frame in addition to the five previously known within the genome (gag, pol, sor, env and 3'orf). This gene, called tat-III, lies between the sor and env genes and is able to mediate activation, in a trans configuration, of the genes linked to HTLV-III long terminal repeat (LTR) sequences. We now present evidence that the product of tat-III is an absolute requirement for virus expression. We show that derivatives of a biologically competent molecular clone of HTLV-III, in which the tat-III gene is deleted or the normal splicing abrogated, failed to produce or expressed unusually low levels of virus, respectively, when transfected into T-cell cultures. The capacity of these tat-III-defective genomes was transiently restored by co-transfection of a plasmid clone containing a functional tat-III gene or by introducing the TAT-III protein itself. As HTLV-III and related viruses are the presumed causal agents of AIDS and associated conditions, the observation that tat-III is critical for HTLV-III replication has important clinical implications, and suggests that specific inhibition of the activity of tat-III could be a novel and effective therapeutic approach to the treatment of AIDS. PMID- 3007996 TI - Molecular biology. Transposon tricks revealed. PMID- 3007998 TI - Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. AB - Endothelium-derived vascular relaxing factor (EDRF) is a humoral agent that is released by vascular endothelium and mediates vasodilator responses induced by various substances including acetylcholine and bradykinin. EDRF is very unstable, with a half-life of between 6 and 50 s, and is clearly distinguishable from prostacyclin. The chemical structure of EDRF is unknown but it has been suggested that it is either a hydroperoxy- or free radical-derivative of arachidonic acid or an unstable aldehyde, ketone or lactone. We have examined the role of superoxide anion (O-2) in the inactivation of EDRF released from vascular endothelial cells cultured on microcarrier beads and bioassayed using a cascade of superfused aortic smooth muscle strips. With this system, we have now demonstrated that EDRF is protected from breakdown by superoxide dismutase (SOD) and Cu2+, but not by catalase, and is inactivated by Fe2+. These findings indicate that O-2 contributes significantly to the instability of EDRF. PMID- 3007997 TI - A new acute transforming feline retrovirus and relationship of its oncogene v-kit with the protein kinase gene family. AB - A new acute transforming feline retrovirus, the Hardy-Zuckerman 4 feline sarcoma virus (HZ4-FeSV), has been isolated from a feline fibrosarcoma. The viral genome of HZ4-FeSV contains a new oncogene designated v-kit, has the structure 5' delta gag-kit-delta pol-delta env 3' and specifies a gag-kit polyprotein of relative molecular mass 80,000. The predicted kit amino-acid sequence displays partial homology with tyrosine-specific protein kinase oncogenes. HZ4-FeSV appears to have been generated by transduction of feline c-kit sequences with feline leukaemia virus. PMID- 3007999 TI - Dioxin exposure. CDC study still at square one. PMID- 3008000 TI - Postsynaptic hyperpolarization during conditioning reversibly blocks induction of long-term potentiation. AB - Activity-induced changes in the efficacy of synaptic transmission between neurones are central to several prominent theories of learning. In both in vivo and in vitro preparations of the hippocampus, a conditioning high-frequency stimulus delivered to afferent fibres results in a long-term potentiation of synaptic transmission at those inputs. Evidence has been provided supporting both presynaptic and postsynaptic sites as loci where critical events occur in the development of potentiation. In this study we report that long-term potentiation is reversibly blocked by intracellular injection of hyperpolarizing current in the postsynaptic cell during the conditioning high-frequency stimulus, suggesting the involvement of a voltage-dependent postsynaptic mechanism. PMID- 3008002 TI - Expression of AIDS virus envelope gene in recombinant vaccinia viruses. AB - Acquired immune deficiency syndrome (AIDS) is an infectious disease characterized by severe impairment of the patient's cell-mediated immune system. Several lines of evidence have indicated that the aetiological agent of AIDS is a group of T lymphotropic retroviruses, variously known as lymphadenopathy-associated virus (LAV), human T-lymphotropic virus type III (HTLV-III) and AIDS-associated retrovirus (ARV). Serological surveys have indicated that as many as one million people in the United States may have been infected by LAV/HTLV-III, and the spread of AIDS has become a global concern. The need for a better understanding of the viral immunology and for a vaccine against AIDS is self-evident. To this end, we have constructed recombinant vaccinia viruses containing the envelope (env) gene of LAV, and demonstrate here that cells infected with these viruses express immunoreactive proteins similar to those present on LAV virions. Experimental animals infected with these recombinant viruses elicited antibodies that specifically recognized LAV envelope proteins. PMID- 3008001 TI - Expression of the HTLV-III envelope gene by a recombinant vaccinia virus. AB - The discovery that the aetiological agent of acquired immune deficiency syndrome (AIDS) is a retrovirus, referred to as human T-lymphotropic virus type III (HTLV III) or lymphadenopathy-associated virus (LAV) (for review see ref. 1), has raised the possibility of developing a vaccine. In this regard, the envelope (env) proteins of murine retroviruses can induce protective immunity in mice. The HTLV-III env gene specifies a primary polypeptide of approximately 860 amino acids that is glycosylated to form a precursor of relative molecular mass (Mr) 160,000 (gp160), which gives rise to mature membrane-associated proteins of Mr 120,000 (gp120) and 41,000 (gp41). The HTLV-III env gene has been expressed in Escherichia coli and by simian virus 40 (SV40) vectors but formation of the authentic proteins has not been demonstrated. Here, we describe the expression of the complete env gene by a vaccinia virus vector. Evidence is presented that synthesis, glycosylation, processing and membrane transport of the env polypeptide occurred without other HTLV-III gene functions; the env protein was recognized by sera from unrelated AIDs patients; and a single vaccination with the infectious recombinant vaccinia virus induced antibodies to gp120 in mice. PMID- 3008003 TI - Viral particles induce Ia antigen expression on astrocytes. AB - Recent studies have shown that gamma-interferon (IFN-gamma) induces the expression of Ia antigen on astrocytes. This observation is of immunological significance because such activated astrocytes can act as antigen-presenting cells, as demonstrated with myelin basic protein for antigen-specific encephalitogenic T-cell lines. However, the lack of lymphatic drainage in brain and the presence of the so-called blood-brain barrier restricting traffic of cells and macromolecules suggests that IFN-gamma may not be readily available, at least during the initial phases of viral infections. The question therefore arises as to whether astrocytes can be induced to express Ia antigens by other signals directly related to viral infection and possibly independent of IFN gamma. In the present report we demonstrate that a neurotropic murine hepatitis virus induces expression of Ia antigen on astrocytes in tissue culture without infection, rendering these brain cells competent to participate directly in the immune response to a viral infection. PMID- 3008004 TI - A chromosome 14 inversion in a T-cell lymphoma is caused by site-specific recombination between immunoglobulin and T-cell receptor loci. AB - Specific chromosomal aberrations are associated with specific types of cancer (for review see ref. 1). The distinctiveness of each association has led to the belief that these chromosomal aberrations are clues to oncogenic events or to the state of differentiation in the malignant cell type. Malignancies of T lymphocytes demonstrate such an association characterized most frequently by structural translocations or inversions of chromosomes 7 and 14 (refs 7-9). Analyses of these chromosomally marked tumours at the molecular level may therefore provide insight into the aetiology of the cancers as well as the mechanisms by which chromosomes break and rejoin. Here we report such an analysis of the tumour cell line SUP-T1 derived from a patient with childhood T-cell lymphoma carrying an inversion of one chromosome 14 between bands q11.2 and q32.3, that is, inv(14) (q11.2; q32.2). These are the same chromosomal bands to which the T-cell receptor alpha-chain (14q11.2) and the immunoglobulin heavy chain locus (14q32.3) have been assigned. Our analysis reveals that this morphological inversion of chromosome 14 was mediated by a site-specific recombination event between an immunoglobulin heavy-chain variable region (Ig VH) and a T-cell receptor (TCR) alpha-chain joining segment (TCR J alpha). S1 nuclease analysis shows that this hybrid gene is transcribed into poly(A)+ RNA. PMID- 3008006 TI - [Intersensory interactions in the CNS of Helix lucorum]. AB - Hair cell responses to adequate stimulation of statocyst, eye and chemoreceptors of the optic tentacle bulb were studied by means of intracellular and extracellular recordings. Hair cells increased firing frequency to titling of the statocyst. Displacement of the statocyst caused a depolarizing wave with superimposed spikes in the hair cell. This depolarizing wave or generator potential increased in proportion to magnitude of displacement. The onset of a light flash and the illumination of eye photoreceptors caused a short-latency (0.3-2 s) increase in the firing frequency of hair cells. The latency of the response was in reversed proportion to the light intensity. Adequate stimulation of chemoreceptors of optic tentacle bulb caused a long-latency (20-40 s) discharge response in the hair cell. In some cases adequate stimulation of photoreceptors and chemoreceptor pathway caused inhibitory responses in 2 of above 50 hair cells. PMID- 3008005 TI - [No indications for LAV/HTLV-III in non-drug-using prostitutes in Amsterdam]. PMID- 3008008 TI - [Effect of synaptic activation and serotonin application on the calcium current in Helix neurons]. AB - Changes in inward calcium current observed on the background of postsynaptic currents have been studied in snail neurons under voltage clamp conditions. It is shown that when inhibitory or excitatory postsynaptic current developed, the calcium current reversibly decreased. Application of serotonin to the neuronal soma has also led to a reversible block of the calcium current. Possible cellular mechanisms involved in the changes under study and their putative physiological role are discussed. PMID- 3008007 TI - [Paired facilitation of the summated extracellular synaptic potentials of individual retino-tectal fibers in the frog]. AB - The paired-pulse facilitation of the EEG quanta (the extracellular monosynaptic PSPS of all synapses of one axon) was examined in layer F of the frog tectum at normal and at increased concentrations of external Ca or Mg ions. The maximum values of facilitation were observed with intervals between stimuli of about 5 ms. Under normal conditions the amount of maximal facilitation was different for separate EEG quanta and varied from 1.4 to 2.4. Distribution of the EEG quanta by the amount of maximal facilitation had two modes at the values fmax = 1.65 and 1.95. The time course of facilitation could be approximated by two exponential components with the time constants of decays tau 1 = 5-6 ms and tau 2 = 140-150 ms for more facilitated EEG quanta, and tau 1 = 6-8 ms and tau 2 = 60-70 ms for the others. Nonequal degree of facilitation of separate EEG quanta and experiments with increased external Ca2+ and Mg2+ concentrations have suggested the dependence of paired-pulse facilitation on the quantum content of the transmitter released in retinotectal synapses. Two types of terminal arbors of axons whose synapses differ in quantum content are supposed to exist in the tectum layer F. PMID- 3008009 TI - [Electrophysiologic study of non-pyramidal neurons of the stratum radiatum in slices of guinea pig hippocampus]. AB - The activity of 67 nonpyramidal neurons of str. radiatum-moleculare (NSRM) and of 8 presumed interneurons of str. oriens-pyramiidale (NSOP) was recorded extracellularly in guinea pig hippocampal slices. In comparison with high frequency grouped discharges characteristic of NSOP, NSRM had low frequency background activity consisting of single (77%) and grouped (23%) spikes. The level of the background activity of NSRM decreased with an increase of the distance of their location from str. pyramidale. Electrical stimulation of dentate fascia usually evoked 1-2 spike discharges in NSRM, while bursts of spikes were evoked in NSOP. The thresholds of responses in NSRM were not different or higher than those of pyramidal neurons, while in NSOP they were significantly lower. The period of suppression of the spontaneous activity usually followed the evoked spike discharge in NSRM. During evoked synchronous epileptiform discharges of pyramidal neurons the simultaneous excitation of NSRM was observed. Excitatory influence of pyramidal neurons on NSRM and participation of the latter in dendritic inhibition of pyramidal neurons is suggested. PMID- 3008010 TI - Flow cytometry in brain tumors. I. Ploidy abnormalities. AB - Flow cytometry (FCM) was introduced for estimation of DNA content in 75 brain tumors. Among these astrocytomas (38 cases) and meningiomas (16 cases) predominated. Astrocytomas were histologically subdivided into 3 groups of malignancy grade. Aneuploid cell populations were found in 1 out of 4 cases of astrocytoma grade I, in 11 out of 20 astrocytomas grade II and in 10 out of 14 astrocytomas grade III of malignancy. DNA index (DI) in most aneuploid astrocytomas is in the range between 1 and 2. More than one cell population with aneuploid value of DNA was found in 36% of all aneuploid tumors. PMID- 3008011 TI - Are recent findings on 1,25-dihydroxycholecalciferol metabolism relevant for the pathogenesis of uremia? PMID- 3008012 TI - The glioblastoma multiforme: a lifelong challenge to the neurosurgeon. AB - During a period of 34 years 1316 gliomas of the brain have been treated, among them 508 glioblastomas (= 39%). In addition to the operative removal and postoperative X-ray therapy of glioblastomas various attempts have been undertaken to prevent recurrences: Intracerebral application of Cobalt60 (6 patients); locally applied antimitotic agents (76 patients); bacterial liquefaction induced by intracarotid administration of Clostridium butyricum M 55 spores ("Oncolysis", 67 patients); circumscribed heating of the extirpation cavity with metal and high-frequency electromagnetic field (85 patients); vaporization of the tumour bed with the defocussed CO2- or Neodymium-YAG-Laser beam (177 patients). Permanent cure has been attained only in single cases so that the problem does not yet seem to be definitely solved by any of these methods. However, bacterial oncolysis in combination with the periodic postoperative generation of heat locally in the excision cavity of the tumour, might justify cautious optimism about future developments. PMID- 3008013 TI - A comparison of methods for removal of endogenous GABA from brain membranes prepared for binding assays. AB - Two commonly used procedures for removing endogenous GABA from brain homogenates were evaluated by measuring residual GABA using high performance liquid chromatography (HPLC). The effect of these treatments on [3H]muscimol binding to the GABA receptor was also determined. Membranes subjected to osmotic lysing and eight washes with Tris-citrate buffer contained significant quantities of residual GABA whereas lysing and incubation with Triton X-100 followed by three buffer washes resulted in GABA levels below the limits of detection. The apparent affinity for [3H]muscimol was significantly higher in the Triton X-100 treated membranes and this was probably a result of the lower amount of GABA present in these membranes. The effect of Triton treatment or buffer washing on residual levels of glutamate, glutamine, aspartate, and taurine were also determined. PMID- 3008014 TI - GABA receptor binding site "induction" in rabbit retina after nipecotic acid treatment: changes during postnatal development. AB - The development of the GABA system in the rabbit retina was studied. The number of high- and low-affinity GABA receptor binding sites increased in a sigmoidal manner, with the curve for the low-affinity sites lagging 2-3 days behind that for the high-affinity sites. The KD for both high- (17.5 nM) and low-affinity (138.0 nM) sites remained constant during development. Treatment of isolated eyecups with the uptake blocker nipecotic acid resulted in an increase in the Bmax for high-affinity sites in developing tissue with the maximum sensitivity around eye opening; mature tissue exhibited a decrease in Bmax. In contrast, a gradual decrease in sensitivity to stimulation of the low-affinity sites occurred. These data indicate that the "trophic" action of GABA is limited to the time when the tissue is developing. PMID- 3008015 TI - Modulation of the GABA-benzodiazepine receptor complex by taurine in rat brain membranes. AB - The interactions of taurine and its precursor hypotaurine with the GABA benzodiazepine receptor complex were studied by investigating their effects on GABA and flunitrazepam binding in rat brain membranes. Taurine, and to a lesser degree also hypotaurine, displaced the high- and low-affinity GABA binding. The maximal binding capacities of both sites were decreased in the presence of taurine, while the binding constants remained the same, suggesting noncompetitive interactions. Taurine and hypotaurine affected flunitrazepam binding only at a very high concentration (50 mmol/l), whereas GABA (within the concentration range of 0.1-100 mumol/l) significantly enhanced the binding. Taurine inhibited the GABA-stimulated binding dose-dependently. These modulatory effects of taurine on the GABA-benzodiazepine receptor complex could result from interactions with the GABA recognition site but not from direct actions on the benzodiazepine site. PMID- 3008016 TI - [Clinical aspects of herpes simplex meningoencephalitis]. AB - On the basis of the observed 10 cases the authors discuss the diagnostic, clinical and therapeutic problems of herpes encephalomeningitis. The diagnosis in early phase of the disease but already after appearance of psychic and neurological symptoms and signs is based on clinical criteria and also on the result of examination of the cerebrospinal fluid and rapid serological method ELISA. In view of the already available possibilities of virostatic treatment early diagnosis is of utmost importance, for beginning treatment (preferably with Vidarabine) before the development of extensive necrotic brain lesions. PMID- 3008017 TI - Characterization of receptors for angiotensin-induced drinking and blood pressure responses in conscious rats using angiotensin analogs extended at the N-terminal. AB - Angiotensin II analogs with N-terminal extensions were synthesized to examine their effects on the brain and vascular angiotensin II (Ang II) receptors of the rat. Ang II, Crinia-Ang II, Thr.Ala.Gly-Ang II and Val. Ser.Leu.Thr.Ala.Gly-Ang II were all found to elicit drinking and raise blood pressure when given into the cerebrospinal fluid (CSF), and elevate blood pressure when given intravenously. When given intracerebroventricularly, the order of potency of the peptides in eliciting blood pressure and drinking responses was: Ang II (100%) = Crinia-Ang II (100%) greater than Thr.Ala.Gly-Ang II (10% blood pressure, 15% drinking) greater than Val.Ser.Leu.Thr.Ala.Gly-Ang II (5%). The order of pressor potency did not change when the Ang II analogs were given intravenously, but compared with the central effects, there was a marked difference in the relative potencies of the peptides. The potencies were: Ang II (100%) greater than Crinia-Ang II (80%) greater than Thr.Ala.Gly-Ang II (60%) greater than Val.Ser.Leu.Thr.Ala.Gly Ang II (20%). Blood pressure and drinking responses produced by all of these peptides were markedly attenuated by the Ang II receptor antagonist, [Sar1,Thr8] Ang II. These findings indicate a difference in the Ang II receptors present in the brain and the periphery. However, no differences were noted between the central Ang II receptors mediating the pressor responses and the central Ang II receptors stimulating drinking behavior. PMID- 3008018 TI - Interaction between corticosterone and alpha-2-noradrenergic system of the paraventricular nucleus in relation to feeding behavior. AB - Food consumption elicited by injection of norepinephrine (NE) into the paraventricular nucleus (PVN) of satiated rats has been shown to be dependent on the glucocorticoid corticosterone. To determine the specific nature of this dependence of NE-induced feeding on corticosterone, the efficacy of PVN-injected NE and its interaction with peripherally administered corticosterone was examined in adrenalectomized rats. NE, at doses ranging from 10 to 160 nmol, failed to elicit a reliable feeding response in these adrenalectomized animals. This loss of noradrenergic responsiveness developed at least as early as 4 h after adrenalectomy and continued until the end of the test sequence 6-8 weeks post surgery. Single subcutaneous injections of corticosterone, administered to adrenalectomized rats, significantly restored the NE feeding response when injected 15-120 min prior to NE PVN injection, but not when administered 5 min before. Corticosterone was effective at doses of 0.5-4.0 mg/kg. Tests with other steroid hormones, namely, the synthetic glucocorticoid dexamethasone, the mineralocorticoid deoxycorticosterone, and the gonadal hormones testosterone and progesterone, were generally ineffective in restoring the sensitivity of the PVN to noradrenergic stimulation. Radioimmunoassay of circulating corticosterone in adrenalectomized rats, as a function of dose and time after injection of corticosterone, indicated that physiological levels of the hormone, at least 3 micrograms%, are required for a patent behavioral effect. These findings demonstrate a specific, sensitive, and rapid dependence of the PVN alpha 2 noradrenergic eating response on circulating corticosterone. PMID- 3008019 TI - Sex differences in [3H]-estradiol binding in brain and pituitary after acute dopaminergic treatment. In vivo studies in the rat. AB - Responses to estrogen differ between the sexes, yet sex differences in specific binding of estradiol (E2) to its receptor are not observed consistently. Dopaminergic treatment has been shown to increase binding of 3H-E2 in selected brain areas and anterior pituitary in the female rat, and the dopaminergic system is sexually differentiated. In order to determine whether or not dopaminergic stimulation might induce sex differences in 3H-E2 binding, male and female gonadectomized-adrenalectomized rats were pretreated either with bromocriptine, a dopamine agonist, or with diethyldithiocarbamate (DDC), an inhibitor of dopamine beta-hydroxylase. DDC was used in order to increase endogenous release of dopamine. After such acute dopaminergic treatment, specific binding of 3H-E2 in nuclear and extranuclear fractions of 6 brain areas and pituitary in vivo was determined 1 h after intravenous injection of 3H-E2 (1 microgram/kg body weight). Administration of either bromocriptine or DDC increased specific 3H-E2 binding to nuclear and extranuclear fractions of basal hypothalamus and anterior pituitary from female but not from male rats, thus inducing sex differences in binding in these two tissues. Bromocriptine also increased specific binding in the pineal in females. Total binding was increased in a crude membrane fraction (P2) from pituitary of female but not of male rats after administration of DDC, but the percent of extranuclear specific binding found in the P2 fraction was decreased after DDC in both males and females. The findings suggest that dopaminergic stimulation may induce sex differences in 3H-E2 binding by increasing binding in some brain areas and anterior pituitary in females but not in males. PMID- 3008020 TI - Different opioid mechanisms are involved in the modulation of ACTH and gonadotrophin release in man. AB - Both the pituitary-adrenal axis and the pituitary-gonadal axis are under the tonic inhibitory control of endogenous opioid peptides in man. However, the precise opioid receptor involved in the modulation of these hormones remains unknown. The effect of a dose of intravenous naloxone on serum levels of luteinising hormone (LH), follicle-stimulating hormone (FSH) and plasma cortisol was therefore investigated in ten normal subjects. In the male subjects, naloxone at a dose of 25 micrograms/kg caused a significant increase in serum LH and FSH; no increase in response was seen at the two higher doses (100 micrograms/kg and 250 micrograms/kg). The lowest dose (6 micrograms/kg) caused no change in serum LH and FSH. In the female subjects, tested in the early follicular phase of their cycles, no dose of naloxone significantly increased circulating gonadotrophins. In both male and female subjects, naloxone only stimulated a rise in serum cortisol at the highest dose (250 micrograms/kg). A second study in six normal subjects demonstrated that the rise in cortisol with the highest dose of naloxone was secondary to a rise in plasma ACTH. It is concluded that the opioid receptor(s) controlling gonadotrophin release in man are naloxone-sensitive, and are probably epsilon-receptors; the naloxone insensitivity of the pituitary adrenal axis suggests that these responses are modulated by kappa- or delta receptors. PMID- 3008022 TI - The distribution of glycine receptors in the human brain. A light microscopic autoradiographic study using [3H]strychnine. AB - Glycine receptors were localized autoradiographically in postmortem human brain material using [3H]strychnine as a ligand. Slide mounted tissue sections were labeled in vitro by incubation with [3H]strychnine and autoradiograms obtained using [3H]Ultrofilm. Receptor densities were quantified by computer assisted microdensitometry. No specific binding of [3H]strychnine was observed in any of the forebrain areas studied. Low densities were seen in the midbrain except for dorsal and lateral parts of the periaqueductal grey matter and the oculomotor nuclei. In pons, medulla oblongata and upper cervical cord high densities of [3H]strychnine binding sites were associated with some nuclei including the motor and sensory trigeminal nuclei, the facial and the hypoglossal nuclei. The highest densities of grains were associated with the substantia gelatinosa of the trigeminal nucleus in the medulla oblongata. A peculiar spotty distribution of [3H]strychnine binding sites were found in the gracilis and cuneatus nuclei. The distribution of glycine receptors in the human brain is comparable to that seen in the rat brain, although densities are much higher in the rat. The distribution of glycine receptors in the human brain provides an anatomical substrate for the understanding of the effects of drugs acting in these receptors, particularly strychnine. PMID- 3008021 TI - Disturbed oxidative metabolism in subacute necrotizing encephalomyelopathy (Leigh syndrome). AB - Several disorders of oxidative metabolism have been described in association with subacute necrotizing encephalomyelopathy (SNE) or Leigh syndrome. We present an eight-year-old girl with a mild spastic paraparesis and clinical deterioration on intercurrent infections. One sib died of SNE proven by autopsy. Biochemical examination of muscle tissue points to a disturbance in the process of oxidative phosphorylation due to a disturbed oxidation of NADH. The biochemical disorders associated with SNE are reviewed. The relation of SNE to the concepts of encephalomyopathy and mitochondriopathy is discussed. PMID- 3008023 TI - Different developmental schedules of dopaminergic and noradrenergic neurons in dissociation culture of fetal rat midbrain and hindbrain. AB - The development of dopaminergic and noradrenergic neurons in dissociation cultures of mesencephalon and rhombencephalon obtained from 18-day-old rat fetuses was characterized by their capacity to take up and release catecholamines. In both types of cultures, uptake of [3H]dopamine and [3H]noradrenaline was obtained which could be inhibited by reserpine. Autoradiographic studies demonstrated an almost exclusive neuronal localization of the labeled catecholamines. The transmitters could be released by depolarization with K+ in a Ca2+-dependent manner during the entire cultivation period. In contrast, catecholamine uptake by cultures of neocortex was minimal, could not be inhibited by reserpine, and the accumulated radioactivity could not be released upon depolarization. These points provide evidence for an active accumulation of the exogenous transmitters and for the presence of stimulus secretion coupling in a distinct population of neurons of both brain stem cultures. Striking differences between the two brain stem cultures concerned their sensitivity to desmethylimipramine and benztropine as well as the time course of the development of the uptake capacity. Desmethylimipramine inhibited the uptake of both catecholamines in rhombencephalic, but not in mesencephalic cultures. The reverse was true for benztropine. It is concluded that cultures of rhombencephalon contain predominantly noradrenergic, and those of mesencephalon dopaminergic cells. Comparison of the uptake behaviour suggested that noradrenergic neurons mature considerably later than dopaminergic neurons. The results show that dissociation cultures of mid- and hindbrain, inspite of their heterogeneous composition, can serve as valuable models for the study of development and function of dopaminergic and noradrenergic neurons, respectively. PMID- 3008024 TI - Distinct sites of functional interaction between dopamine, acetylcholine and gamma-aminobutyrate within the neostriatum: an electromyographic study in rats. AB - In order to study the functional interaction between dopamine, acetylcholine and gamma-aminobutyrate within the rat neostriatum, we investigated the effect of intrastriatal injection of different drugs acting on these transmitter systems on muscle tone measured as tonic activity in the electromyogram of the gastrocnemius muscle. Bilateral injection of haloperidol (500 ng) into the rostral neostriatum (rostral injection: A8920-9650(46] induced tonic activity in the electromyogram, whereas injection into the intermediate part (intermediate injection; A7020 7890(46] was ineffective. Muscimol (25 ng) induced tonic activity in the electromyogram, when injected into the intermediate part and not into the rostral part, while bethanechol (1 microgram) was effective when injected into either site. Haloperidol-induced tonic activity in the electromyogram was prevented by coadministration of apomorphine (500 ng) or scopolamine (1 microgram), but not of bicuculline (300 ng). Haloperidol-induced tonic activity in the electromyogram was also reduced by subsequent intermediate injection of scopolamine or bicuculline, while apomorphine was ineffective. Tonic activity in the electromyogram induced by rostral injection of bethanechol was prevented by coadministration of scopolamine, but not of apomorphine. Intermediate injection of scopolamine or bicuculline reduced the tonic activity in the electromyogram after rostral or intermediate injection of bethanechol. Tonic activity in the electromyogram induced by intermediate injection of muscimol was prevented by coadministration of bicuculline, but not of scopolamine. Rostral injection of apomorphine or scopolamine failed to alter the tonic activity in the electromyogram induced by intermediate injection of bethanechol or muscimol. These results point to the existence of: a functional interaction between dopamine and acetylcholine in the rostral neostriatum; a functional interaction between acetylcholine and gamma-aminobutyrate in the intermediate neostriatum, and a functional flow of information from the rostral to the intermediate neostriatum. PMID- 3008025 TI - Single brain metastases: surgery plus radiation or radiation alone. AB - We reviewed the records of patients treated for single brain metastases from non small-cell lung cancer for 1978 through 1982. Forty-three patients received surgical treatment, including 37 who had surgery plus postoperative whole-brain radiation therapy and 6 patients who had surgery after failing to respond to radiation therapy. The surgically treated patients were matched with 43 patients treated with radiation therapy alone. The combined therapy group had significantly longer survivals than those treated with radiation therapy alone (19 months versus 9 months). The rates of local recurrence and neurologically related deaths were significantly higher in the radiation therapy-alone group. Patients treated with combined therapy survived longer, and the increased survival was due to lower recurrence of brain metastases after surgery and fewer neurologically related deaths. PMID- 3008026 TI - Thromboxane, prostacyclin, and leukotrienes in cerebral ischemia. AB - Our understanding of the biochemistry and biologic actions of AA metabolites has been greatly expanded in recent years. The discoveries of TXA2, PGI2, and LTs have fostered new concepts of the pathophysiology of cerebral ischemia. New approaches to treatment of ischemia include seeking an optimal dose of aspirin, developing drugs that selectively inhibit or antagonize TXA2 or LTs, and administering PGI2 or its analogues. Altering the dietary content of essential fatty acids for prophylaxis is also being studied. Though the results of this thrust are still preliminary, the exploration of these therapeutic strategies in cerebrovascular disorders based on further understanding of the pathophysiologic roles of TXA2, PGI2, LTs and probably other AA metabolites is anticipated with some optimism. PMID- 3008027 TI - Progressive multifocal leukoencephalopathy: JC virus detection by in situ hybridization compared with immunohistochemistry. AB - In four cases of progressive multifocal leukoencephalopathy (PML), we compared biotin-labeled DNA:DNA in situ hybridization with peroxidase immunohistochemistry for the detection of JC virus (JCV). The localization of JCV DNA and JCV capsid protein was compared in formalin-fixed, paraffin-embedded brain tissues. Infected oligodendrocytes showed both JCV DNA and JCV protein. However, bizarre astrocytes demonstrated JCV capsid protein less often than JCV DNA. In situ hybridization with a biotinylated probe was as sensitive and specific as immunohistochemistry for diagnosis on formalin-fixed tissue. The presence of both JCV DNA and viral capsid protein in bizarre astrocytes suggests that these cells are neither truly transformed nor permissively infected, but are distinctively altered by JCV. PMID- 3008028 TI - Polymyositis in an immunodeficiency disease in monkeys induced by a type D retrovirus. AB - Fifty percent of primates with acquired immunodeficiency caused by a well characterized type D retrovirus (SAIDS D) developed clinical, laboratory, and histologic features of polymyositis. By use of specific antisera and immunochemical techniques, we found the virus in the lymphoid cells surrounding muscle fibers and invading the endomysia septa. SAIDS D virus was isolated from the involved muscles and infected myotubes of normal muscle in tissue culture. These results suggest that retroviruses, a group of viruses increasingly associated with human diseases, can cause polymyositis with immunodeficiency in nonhuman primates and could play a role in human polymyositis. PMID- 3008029 TI - Pathophysiology of hemifacial spasm. PMID- 3008030 TI - Brain fine structure in Creutzfeldt-Jakob disease with plaques and tangles. I. Neuritic plaques. PMID- 3008031 TI - A non-invasive approach to the diagnosis of amyloid cardiomyopathy in elderly patients. PMID- 3008033 TI - [Adenoid cystic carcinoma of the Bartholin's gland. Presentation of a case and review of the literature]. PMID- 3008032 TI - [Vulvovaginitis. Etiopathogenesis and microbiological diagnosis]. PMID- 3008034 TI - [Cervical intraepithelial neoplasms and viral infections. Cytological, colposcopic and histological study]. PMID- 3008035 TI - Cholinergic innervation displays strikingly different laminar preferences in several cortical areas. AB - A new rabbit polyclonal antiserum against choline acetyltransferase (ChAT) reveals that cholinergic innervation of the cortex varies strikingly among different cytoarchitecturally defined areas in the rat neocortex. These findings suggest that cholinergic transmission may be integrated differently into the local circuitries of various regions of the cerebral cortex. In addition, the pattern of staining observed with acetylcholinesterase histochemistry, which has been used for many years to demonstrate putative cholinergic fibers, only partially matches the staining pattern obtained with the more specific cholinergic marker, ChAT. PMID- 3008036 TI - Properties of binding sites for [3H]cyclohexyladenosine in the hippocampus and other regions of rat brain: a quantitative autoradiographic study. AB - The properties of binding sites for the adenosine receptor ligand, [3H]cyclohexyladenosine ([3H]CHA), were investigated in rat brain using quantitative autoradiography. Scatchard analysis of the binding data showed that there were no significant differences between Kd values for [3H]CHA in any of the regions investigated. The highest concentrations of [3H]CHA binding sites were found in the cerebellum (molecular layer) and the stratum oriens and stratum radiatum of the hippocampus (CAI region). Displacement curves obtained using N ethylcarboxamidoadenosine (NECA) and the R- and S-diastereoisomers of phenylisopropyl adenosine (PIA) showed the [3H]CHA binding sites to have the pharmacological properties of A1-adenosine receptors, i.e. the order of potency for these derivatives was R-PIA greater than NECA greater than S-PIA, in all regions tested. Further, [3H]CHA binding was in all cases attenuated by the guanosine triphosphate derivative, beta, gamma-imidoguanosine triphosphate. These results indicate that [3H]CHA binding sites throughout the central nervous system have the properties of A1-adenosine receptors and that these are in all regions associated with guanine nucleotide regulatory proteins. PMID- 3008038 TI - Novel synaptic responses mediated by dopamine and gamma-aminobutyric acid in neuroendocrine cells of the intermediate pituitary. AB - Neuroendocrine cells of the pituitary pars intermedia contain pro opiomelanocortin and secrete mainly alpha-melanocyte-stimulating hormone and, in lesser amounts, corticotropin and endorphin. We have recorded intracellularly from rat pars intermedia cells in vitro and analysed the synaptic inputs to these cells. Electrical stimulation of the pituitary stalk evoked a biphasic postsynaptic response in pars intermedia cells. An initial inhibitory postsynaptic potential was most likely mediated by gamma-aminobutyric acid (GABA) because there was a conductance increase, and it was blocked by bicuculline, a GABA antagonist. A more prolonged hyperpolarization was also observed which could last 10-30 s and was associated with a decreased whole cell conductance. This late hyperpolarization was blocked by the dopamine antagonists, chlorpromazine and domperidone, and was therefore most likely dopamine-mediated. PMID- 3008037 TI - The formation of new neuronal circuit between transplanted nigral dopamine neurons and non-immunoreactive axon terminals in the host rat caudate nucleus. AB - Using immunoelectron microscopic techniques, whether or not host neuronal elements newly form synaptic contact with the grafted dopamine (DA) neurons in the caudate nucleus of the rat with unilateral lesion in the nigrostriatal DA pathway was examined. Tyrosine hydroxylase (TH) was used as a marker for DA containing structures. Motor imbalances after the lesion and before or after the transplantation were assessed by the amount of circlings after the injection of Met-amphetamine. In animals which recovered well from motor imbalance, non immunoreactive axon terminals made synaptic contact with grafted TH-positive cell bodies and their dendrites. Since the incidence of these synapses was quite low in poorly recovered animals, the formation of a new neuronal circuit may be one of the important bases for behavior recovery. PMID- 3008039 TI - Kappa-opiate binding to rat brain and guinea pig cerebellum: sensitivity towards ions and nucleotides. AB - Sodium ions and guanine nucleotides markedly affect the binding to mu and delta sites. Using the guinea pig cerebellum, a tissue whose opioid binding sites are almost exclusively kappa, we have examined the sensitivity of [3H](-) ethylketocyclazocine ([3H]EKC) binding to ions and guanine nucleotides. Unlike mu and delta binding, sodium ions and guanine nucleotides had little effect on the [3H]EKC binding in the guinea pig cerebellum. On the other hand, calcium and manganese at 1 mM lowered [3H]EKC binding in the guinea pig cerebellum by approximately 25%. Binding in rat brain, which was primarily mu and delta, was unaffected. These results point out additional biochemical differences between kappa sites and the classic mu and delta binding sites. PMID- 3008040 TI - The effect of presynaptic receptor stimulation on adenosine 3',5'-cyclic monophosphate concentrations in rat cortical synaptosomes. AB - Changes in adenosine 3',5'-cyclic monophosphate (cAMP) concentration were measured in cortical synaptosomes. Preincubation with adenosine deaminase reduced cAMP concentration by 45%. Oxotremorine, clonidine, gamma-aminobutyric acid (GABA) and baclofen produced no change in basal concentration. 2-Chloroadenosine and noradrenaline (NA), acting at beta-adrenoceptors, both caused a dose dependent increase in cAMP; the NA-stimulated increase was depressed by GABA and by baclofen. PMID- 3008041 TI - Central noradrenaline depletion attenuates amphetamine-induced locomotor behavior. AB - Male rats were given 6-hydroxydopamine-induced lesions of the locus coeruleus (LC) or the dorsal noradrenergic bundle (DNAB), prior to the measurement of locomotor and rearing activity induced by D-amphetamine. The increased locomotor activity induced by D-amphetamine (1.8 mg/kg) was significantly attenuated by both the LC and the DNAB lesions. The stimulatory effect of the 7.2 mg/kg dose of amphetamine was attenuated by the LC lesion, whereas the DNAB lesion potentiated this effect. The LC lesion also attenuated rearing induced by the 7.2 mg/kg dose of amphetamine. These results suggest some involvement of central noradrenergic neurons in the activity induced by amphetamine in the rat. PMID- 3008042 TI - Thyrotropin-releasing hormone receptors in gut tissues resemble pituitary receptors. AB - The relative order of activity of thyrotropin-releasing hormone (TRH) and various analogs in contracting the isolated guinea pig antrum and duodenum correlated with their potencies in activating thyroid-stimulating hormone (TSH) release. The action of TRH in both tissues was selectively antagonized by the putative pituitary TRH receptor antagonist, chlordiazepoxide (10 microM). The data indicate that the contractions produced by TRH in these gut tissues are mediated by TRH receptors with similar characteristics as the pituitary TRH receptors responsible for TSH release. PMID- 3008043 TI - 2-Amino-7-phosphonoheptanoic acid (2-APH) infusion into entopeduncular nucleus protects against limbic seizures in rats. AB - Motor limbic seizures occur following a systemic injection of pilocarpine (380 mg/kg) in rats. Focal injection of the selective N-methyl-D-aspartate receptor antagonist, (+/-)-2-amino-7-phosphonoheptanoic acid (2-APH, 5-20 pmol), bilaterally into the entopeduncular nucleus (EP) prior to pilocarine blocks these seizures. Muscimol (50 pmol), a potent gamma-aminobutyric acid receptor agonist, injected bilaterally into EP produces a similar protection against pilocarpine induced seizures. Thus by blocking excitatory neurotransmission or facilitating inhibition within the EP, the severity of limbic seizures can be reduced. PMID- 3008044 TI - Cholecystokinin octapeptide depolarizes guinea pig inferior mesenteric ganglion cells and facilitates nicotinic transmission. AB - Cholecystokinin octapeptide (CCK-8) applied either by superfusion (0.1-10 microM) or by pressure ejection elicited a slow depolarization in a portion of inferior mesenteric ganglion cells studied in vitro. The depolarization which persisted in a low Ca2+/high Mg2+ solution, or solution containing cholinergic antagonists, was often associated with a small to moderate increase in neuronal input resistance, and the response was reduced by conditioning hyperpolarization. Nicotinic excitatory postsynaptic potentials were consistently augmented during the course of CCK-8-induced depolarization. Our results, together with findings of the presence of CCK-immunoreactive fibers in the prevertebral ganglia, suggest that the peptide may serve to facilitate nicotinic transmission. PMID- 3008045 TI - Giant multivesicular bodies in the rat hippocampal pyramidal cells after chronic alcohol consumption. AB - Multivesicular bodies (MVBs) with diameters up to 4.5 microns were observed in the hippocampal pyramidal cells of rats submitted to chronic alcohol consumption. A significant increase in the volumetric density (Vv) of these organelles was found in CA1 pyramidal cells. Transitional forms of MVBs towards lysosomes were seen. A failure in MVB's enzymatic hydrolytic mechanisms, due to the prolonged alcohol aggression, could underlie its formation. PMID- 3008046 TI - The effects of ischemia and CDPamines on Na+, K+-ATPase and acetylcholinesterase activities in rat brain. AB - Cerebral ischemia produced a decrease in Na+, K+-ATPase activity in striatum and cortex; acetylcholinesterase activity was not affected in either region. Pretreatment of the animals with CDPcholine and CDPethanolamine did not prevent the decline in ATPase activity, suggesting that the accumulation of free fatty acids associated with ischemia is not responsible for these changes. Addition of exogenous diacylglycerols to the ATPase assay mixture produced an inhibition of the enzyme similar in magnitude to that observed in tissue samples from ischemic brain. These results support our hypothesis that the local accumulation of diacylglycerols following ischemia is involved in the observed changes in enzymatic activity. PMID- 3008047 TI - Refitting denture bases with a visible light cured denture base resin. PMID- 3008048 TI - Radionuclide evaluation of left ventricular synergic pumping efficiency during acute myocardial infarction. AB - The clinical value of an index of left ventricular synergic pumping efficiency (EFF) was evaluated in 50 patients with uncomplicated myocardial infarction in which radionuclide ventriculography was performed within 72 h of the onset of the infarction and between 7 and 13 days post infarction. EFF was defined as the ratio of global left ventricular stroke volume to the magnitude of all intraventricular blood pool variations measured on a pixel by pixel basis. EFF correlated better with a subjectively evaluated wall motion index (r = 0.84) than did the ejection fraction (EF) measurements (r = 0.69). In all patients, but most significantly in patients with inferoposterior infarctions, EFF measurements in the acute phase were a better predictor of the predischarge ventricular performance than the EF measurements. The left ventricular EFF is a reliable parameter to quantitate the effect of regional wall motion disturbances on global ventricular function in a standardized manner. PMID- 3008049 TI - Use of perchlorate to block gastric uptake of free 99Tcm in the investigation of gastrointestinal bleeding. AB - The use of perchlorate to block gastric uptake of free 99Tcm-pertechnetate after in vivo labelling of red cells was investigated. In 19 out of 20 cases there was no evidence that previous administration of perchlorate adversely affected red cell labelling using commercial stannous agents. Adequate scintigraphic images of the vascular system could be obtained for up to 24 h after the cells were labelled. The technique was found to be of value in the investigation of sites of gastrointestinal bleeding. PMID- 3008050 TI - Quantitation of renal function with 99Tcm-DMSA. A comparison with creatinine clearance in children with single kidney. AB - To study the accuracy of renal function quantification with 99Tcm-DMSA we compared DMSA renal uptake and creatinine clearance in 16 cases of children with single kidney. The age of the patients ranged from two months to fourteen years. Creatinine clearance was normalized to 1.73 m2. DMSA uptake was measured 7 h after intravenous injection. Background subtraction was used and soft tissue attenuation was taken into account. The uptake was normalized in percentage of the injected activity. A significant correlation was found between creatinine clearance and DMSA uptake (rt = 0.866, p less than 0.01). Normal creatinine clearance range in children (80 to 120 ml min-1/1.73 m2) allowed determination of normal uptake range (36 to 60%). This study indicates that in case of asymmetrical renal impairment renal uptake will reflect split renal creatinine clearance. Since the former is much easier to measure, DMSA should play an important role in the evaluation of differential renal function. PMID- 3008051 TI - Tumor-associated antigen Ca 125 before and during the treatment of ovarian carcinoma. AB - Serum concentrations of Ca 125, a tumor-associated antigen of epithelial ovarian cancer, were measured in 29 ovarian cancer patients before cytoreductive surgery and in 112 patients during and after treatment. Ca 125 levels were increased (greater than 30 IU/mL) in 89.8% of patients with clinically demonstrable ovarian tumors and were negative in 92.1% of clinically disease-free patients. Low levels of Ca 125 were associated with early clinical stages or a minimal tumor burden, and predicted a successful response to treatment and a low recurrence rate. High values indicated advanced disease and a poor response to cytotoxic chemotherapy. In 77% of patients the operation was explorative, with a preoperative Ca 125 level higher than 1000 IU/mL, whereas all the patients with values less than 100 IU/mL could be operated radically. Serum levels of Ca 125 were increased in similar frequency in epithelial, sex cord, and germ cell ovarian malignancies. The assay of Ca 125 seems to be a reliable noninvasive method for monitoring the presence and clinical behavior of ovarian cancer. Preoperative values have prognostic significance in predicting operability and response to chemotherapy. PMID- 3008053 TI - Changes in the frequency of genital herpes recurrences as a function of time. AB - To obtain objective information regarding changes in the frequency of recurrent genital Herpes simplex infections, the data from two consecutive pregnancies in 22 women with culture-proved genital Herpes simplex infections were reviewed. The pregnancies studied were separated by a mean of 2.0 years. When only culture proved recurrences were considered, nine women had fewer recurrences in their second pregnancy than in their first, four had more recurrences in their second than in their first, and nine had the same number of recurrences in both pregnancies. The mean interval between culture-proved recurrences was 58.5 +/- 36.1 (SD) days in first pregnancies and 51.7 +/- 28.6 days in second pregnancies. Mean duration of viral shedding during 14 recurrences in first pregnancies was 4.6 +/- 3.4 days, and 3.2 +/- 2.2 days in 14 recurrences in second pregnancies (differences not significant by Mann-Whitney). Cervical Herpes simplex shedding in asymptomatic women occurred in four of 200 (2.0%) of first pregnancy cultures and zero of 167 second pregnancy cultures (NS). During culture-positive recurrent vulvar infections, 18 of 55 (32.7%) cervical cultures in first pregnancies were positive compared with four of 50 (8%) cervical cultures in second pregnancies (P less than .025). Route of delivery was very similar in the first and second pregnancies with vaginal delivery in 63.6% of first pregnancies and 72.7% of second pregnancies. Overall there was no appreciable difference in the frequency or severity of recurrent genital Herpes simplex infections over time, but more data are needed. PMID- 3008052 TI - Role of Ca 125 as tumor marker in ovarian carcinoma. AB - Thirty-three healthy women (group 1), 20 patients with a history of ovarian carcinoma but no manifest disease at the time of the study (group 2), and 45 patients with surgically demonstrable ovarian cancer (group 3) were studied to establish guidelines for the use of the ovarian cancer antigen Ca 125 in monitoring the course of ovarian carcinoma. Ninety-nine percent of all Ca 125 titers of patients in groups 1 and 2 were less than or equal to 25 U/mL. By contrast, 96% of patients with manifest ovarian cancer had Ca 125 levels greater than 25 U/mL. Ca 125 values rising from the normal range to greater than 25 U/mL predicted recurrent disease in all of ten patients, provided benign causes (four cases) for titer elevations such as bowel obstruction could be ruled out. Seven of ten patients with recurrent cancer had elevated antigen levels two to five months before the diagnosis could be made clinically. In patients with Ca 125 values greater than 25 U/mL, titer changes of greater than or equal to 50% compared with reference values predicted tumor response or progression in 41 of 43 patients (95%) with antigen positive tumors. Antigen levels less than or equal to 25 U/mL did not exclude the presence of tumor at second look operation in six of 13 patients (46%). It is concluded that the Ca 125 is useful for the detection of persistent and recurrent disease, and for the evaluation of treatment responses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008054 TI - Efficacy of human lymphoblastoid interferon in the therapy of resistant condyloma acuminata. AB - The efficacy and tolerance of human lymphoblastoid interferon (Wellferon) were studied in an open label trial of 17 patients with resistant and persistent condyloma acuminata. Patients were treated intramuscularly with 5 X 10(6) U (5 MU)/m2 daily for 28 days followed by thrice weekly injections for two weeks. Sixteen patients were considered evaluable; eight experienced complete clearance, seven had significant reduction (greater than 50%) in lesion size, and one showed no response during the course of this trial. Biologic side effects of interferon occurred in all patients during initial dosing and diminished during thrice weekly therapy. Intramuscular injections and associated side effects were tolerated well. This study shows that systemic human lymphoblastoid interferon is active in treating severe recurrent genital warts in women with a history of recalcitrant disease. PMID- 3008055 TI - Effects of prophylactic chemotherapy for persistent trophoblastic disease in patients with complete hydatidiform mole. AB - Seventy-one patients with complete hydatidiform mole were prospectively randomized into two groups: one group (39 patients) was treated with a single course of methotrexate and citrovorum factor rescue as chemoprophylaxis; the other group (32 patients) was not treated. After molar evacuation, four patients from the treated group (10.3%) and ten patients from the untreated group (31.3%) developed persistent trophoblastic disease. The time interval from evacuation of the mole to diagnosis of persistent trophoblastic disease was longer in the treated group than in the untreated group (9.5 +/- 2.4 weeks versus 5.1 +/- 1.6 weeks, P less than .05). Among high-risk patients, there was a lower incidence of persistent trophoblastic disease in the treated group than in the untreated group (14.3 versus 47.4%, P less than .05). Among low-risk patients there was no difference between the groups (5.6 versus 7.7%, P greater than .05). All 14 patients with persistent trophoblastic disease achieved complete remission with therapeutic chemotherapy. More courses of chemotherapy were required until complete remission in the treated group than in the untreated group (2.5 +/- 0.5 versus 1.4 +/- 0.5, P less than .005). These findings suggest that even though chemoprophylaxis reduces the incidence of persistent trophoblastic disease in patients at high risk, it increases tumor resistance and morbidity. Although prophylactic chemotherapy with methotrexate and citrovorum factor rescue may be helpful for high-risk patients who cannot be followed or whose compliance is in question, careful follow-up remains the most important way to identify patients who should receive chemotherapy. PMID- 3008057 TI - [The history and development of follow-up medical care in Berlin]. PMID- 3008056 TI - Mechanical properties of coated absorbable multifilament suture materials. AB - The mechanical properties of coated polyglactin 910 (Vicryl) and coated plyglycolic acid (Dexon) were defined. Scanning electron microscopy of 0 gauges of both materials showed some differences. Coated polyglycolic acid had minimal surface aberrations. Unknotted tensile strength measurements of gauges 1, 0, 00, and 000 demonstrated no statistically significant difference between the two materials. Both materials reached knot security at the 2=1=1=1 configuration. The in vivo tensile strength measurement of 0 gauges showed polyglactin 910 to be significantly stronger at 21 days. Histologic evaluation revealed no statistically significant difference between the materials. PMID- 3008058 TI - [Role of brain noradrenergic mechanisms in disturbing the circadian rhythm of the functioning hypothalamo-hypophyseo-adrenal cortex system in white rats in early ontogeny]. AB - The effects of hormone action and disturbance in catecholamine synthesis in the early postnatal ontogenesis on the circadian rhythm in the hypothalamic hypophysial-adrenocortical system function were compared in the adult albino rat males. Injection of prednisolone on the 17-19th days of life blocked completely the diurnal rhythm of the corticosterone basal level in blood, the rhythm of adrenocortical response to an emotional stressor and to injection of noradrenaline into the brain lateral ventricle in 3-4 month old animals. Injection of an inhibitor of tyrosine hydroxylase, alpha-methyl-p-tyrosine, at the same period resulted in disappearance of the diurnal rhythm of the corticosterone basal level in adult animals, although the rhythm of response to an emotional stressor or injection of noradrenaline into the brain remained unchanged. A conclusion has been reached that disturbances in catecholamine synthesis in the early postnatal period induces long-term changes of predominantly tonic corticosterone secretion, while the hormone action on the circadian rhythm of the corticosterone basal level and stress response is only partly due to changes in noradrenergic regulation of the hypothalamic-hypophysial adrenocortical system. PMID- 3008059 TI - Isolation and identification of a South African lentivirus from jaagsiekte lungs. AB - In the course of attempts to grow the jaagsiekte retrovirus in cell culture, a typical lentivirus was isolated for the first time in South Africa from adenomatous lungs. Morphologically the virus could not be distinguished from other lentiviruses, but serologically it was shown to be more closely related to visna virus than to caprine arthritis-encephalitis virus. However, a preliminary restriction enzyme analysis of the linear proviral DNA of this new lentivirus (SA DMVV) revealed that it is significantly district from visna virus and CAEV and therefore may represent a third type of lentivirus. Antibodies to the virus were demonstrated in a number of sheep in various parts of the country, but a direct link to a disease condition was not found. Attempts to produce lung lesions by intratracheal injection of the virus have been unsuccessful to date but a transient arthritis was produced by intraarticular inoculation. Viral replication seems to be enhanced in jaagsiekte lungs. PMID- 3008060 TI - Discharge planning: an ongoing function of quality care. PMID- 3008061 TI - Skull-base surgery: operative refinements. AB - Advances in microsurgical techniques, with the use of the laser and the ultrasonic suction aspirator, have paved the way for even more aggressive surgical approaches to large skull-base lesions, such as glomus tumors, adenocarcinomas of the temporal bone, and cranial nerve neuromas. Postoperative morbidity of multiple cranial nerve dysfunction has been aggravated in the past by cerebrospinal fluid leakage and/or gastro-intestinal dysfunction. At the Otology Group, P.C., in the course of dealing with more than 86 skull-base tumors, we have developed a standardized, multispecialty approach to such extensive skull-base lesions, including tracheotomy, gastrostomy, "ventriculoatrial" shunt placement, and rotated temporalis muscle flap closure. This article presents cases that illustrate the evolution of the current, "standard skull-base" operative procedure and the rationale for incorporation of the shunt and the muscle flap closure as operative refinements. Their beneficial effects upon postoperative morbidity are detailed. PMID- 3008062 TI - Intrathecal D-Ala2-D-Leu5-enkephalin (DADL) restores analgesia in a patient analgetically tolerant to intrathecal morphine sulfate. AB - DADL was administered to a patient who was analgetically tolerant to continuously infused, intrathecal morphine sulfate. DADL restored analgesia without respiratory depression but opiate withdrawal syndrome was not prevented. Somnolence, not responsive to naloxone but completely reversed with morphine, was seen as an idiosyncratic mu receptor opiate withdrawal symptom. Clonidine hydrochloride and decreasing doses of oral morphine were used to successfully treat the withdrawal syndrome, including somnolence. Further research is indicated to verify the findings of this one patient and investigate the efficacy of DADL to provide analgesia for morphine-tolerant patients. PMID- 3008063 TI - Epidural steroid injections for low back pain and lumbosacral radiculopathy. AB - Non-surgical treatments of back pain may have prolonged and lasting benefit. Epidural steroid injections is one of the non-operative managements of back pain. These injections are recommended in patients with signs and symptoms of nerve root irritation. Relief of pain is attributed to the anti-inflammatory effect of the steroid. Patients with acute radiculopathy have better response compared to patients with chronic symptoms. Improvement may not be noted until 6 days after the injection. The depression of the hypothalamic-pituitary-adrenal (HPA) axis lasts 3 weeks. While complications have been reported, these are rare. Intrathecal steroid injection is not advisable since polyethylene glycol, the vehicle used in depot steroid preparations, may cause arachnoiditis. PMID- 3008064 TI - [Fine-needle aspiration biopsy in the diagnosis of kidney tumors]. PMID- 3008065 TI - [Epidemiology of breast tumors histologically diagnosed in 19 Italian hospitals in 1983]. PMID- 3008066 TI - [Morphologic and morphometric parameters of infiltrating duct carcinoma of the breast and receptor state in fluorescence: correlations in 104 cases (1983 case survey)]. PMID- 3008067 TI - [Morphometric evaluations in breast carcinoma]. PMID- 3008068 TI - [Correlations between receptor state and various clinico-morphological parameters: evaluation of circa 2000 cases of breast tumors]. PMID- 3008069 TI - [Comparison and correlations of results obtained in the study of steroid receptors using fluorescent histochemical and radiometric methods in 300 cases of breast neoplasms]. PMID- 3008071 TI - [Importance of the study of hormonal receptors in the treatment of breast carcinoma (case report)]. PMID- 3008070 TI - [Follow-up of patients with receptor state determined by radiometric and histochemical methods]. PMID- 3008072 TI - [Prevalence of human cytomegalovirus infection in the city of Lublin and in villages in the Lublin area]. PMID- 3008073 TI - [Liver cirrhosis in congenital generalized cytomegalic inclusion disease]. PMID- 3008075 TI - Quinolones: pharmacology. AB - The quinolones are synthetic antibiotics chemically related to nalidixic acid. Since its introduction, several structural analogues have been synthesized. A fundamental breakthrough was the addition of a fluorine atom. The quinolones interfere with bacterial DNA transcription by inhibiting the enzyme DNA gyrase, that so far has only been found in bacteria. The nature of the activity of the quinolones on DNA gyrase makes it highly unlikely that resistance is carried on plasmids. PMID- 3008074 TI - [Galactose 1-phosphate level in children with various types of hexosephosphate uridylyltransferase deficiency]. PMID- 3008076 TI - Physical properties of the rat renal brush border membrane during growth. AB - The biophysical properties of rat renal brush border membranes were examined during neonatal development and following unilateral nephrectomy by sensitive fluorescence techniques. Differences in the fluorescence anisotropy of 1,6 diphenyl-1,3,5-hexatriene (DPH) following unilateral nephrectomy were not apparent; by contrast, the luminal membrane becomes more rigid as animals age. Although growth of the kidneys is common to both states examined, fundamental differences are apparent at the level of the luminal membrane. PMID- 3008077 TI - Adaptation of electrolyte transport in rat large intestine after proximal resection. I. Cecum and colon after 60% jejunoilectomy. AB - Electrolyte transport was studied in rat cecum and colon adapting to 60% resection of the small intestine. Four weeks after surgery, the absorbing gross surface area, dry and wet weight, net absorption in vivo of sodium, chloride and volume, net potassium secretion, transmural electrical potential difference and mucosal Na-K-ATPase specific activity were determined. In the cecum, all tissue mass parameters were increased compared to sham-operated controls. Net transport was stimulated per organ but not per unit mass, and potential difference and Na-K ATPase remained unaffected. In the colon, surface area and wet weight increased slightly while dry weight and electrolyte transport were not influenced. Thus, by enlargement without a change in cell function, the cecum but not the colon contributes to electrolyte homoeostasis after 60% jejunoilectomy in the rat. Na-K ATPase specific activity and electrical potential difference were higher in the colon than the cecum, suggesting segmental heterogeneity of the larger bowel with respect to Na-K-ATPase-linked active sodium absorption. PMID- 3008078 TI - Adaptation of electrolyte transport in rat large intestine after proximal resection. II. Colon after 50% jejunoilectomy combined with cecectomy. AB - Electrolyte transport was studied in rat colon adapting to 50% small intestinal resection with cecectomy. Four weeks after surgery, colonic gross surface area, dry and wet weight were increased compared to sham-operated controls. Net absorption in vivo of sodium, chloride and volume was stimulated per organ and per unit tissue mass, and net potassium secretion was diminished. The electrical potential difference, mucosal Na-K-ATPase specific activity and cAMP concentration remained unaffected. In vitro, measurements of unidirectional fluxes and electrical parameters across isolated mucosa indicated enhanced electrically neutral sodium chloride absorption in the proximal and possibly diminished bicarbonate secretion in the distal colon. Thus, removal of the cecum in addition to partial jejunoilectomy induces profound adaptive changes in the colon, involving not only mucosal growth but also a functional reaction of the individual epithelial cell. PMID- 3008079 TI - [Digital subtraction angiography with scanning laser-stimulated luminescence; experimental and clinical evaluations and comparisons with digital fluorography and conventional angiography]. PMID- 3008080 TI - Restriction endonucleases HindII and TaqI cleave DNA with mismatched nucleotides within their recognition sequences. AB - Restriction endonucleases HindII and TaqI, but not SalI, were found to efficiently cleave synthetic hexadecanucleotide duplexes which contained either an A/C or a G/T mismatch within their respective restriction sites. Double stranded M13 DNAs with identical mismatches were also cleaved under the assay conditions. These results suggest that the distortion of the DNA duplex, caused by these purine/pyrimidine mismatches is not sufficiently large so as to interfere with the recognition and the subsequent cleavage of the DNA by these two enzymes. HindII and SalI, but not TaqI, were furthermore shown to hydrolyze the two strands of the duplex with different rates. The differences between the mode of recognition of their respective restriction sites by these three enzymes are discussed. PMID- 3008081 TI - A new restriction endonuclease from Spirulina platensis. AB - Three restriction endonucleases, Sp1I, Sp1II and Sp1III have been purified partially from Spirulina platensis subspecies siamese and named. Sp1I cleaves bacteriophage lambda DNA at one site, phi X 174 RF DNA at two sites, but does not cleave pBR322 DNA. This enzyme recognizes the sequence 5'CGTACG3' 3'GCATCG5' and cuts the site indicated by the arrows. Sp1II is an isoschizomer of Tth111I and Sp1III is an isoschizomer of HaeIII. PMID- 3008082 TI - Two unique restriction endonucleases from Neisseria lactamica. AB - Two new site-specific endonucleases, N1a III and N1a IV, have been isolated from Neisseria lactamica. N1a III recognizes the sequence, CATG, and cleaves 3' of the sequence to produce a four base 3' extension. N1a IV recognizes the sequence, GGNNCC, and cleaves between the two N's to produce blunt ended fragments. PMID- 3008083 TI - DNA sequence recognition by under-methylated analogues of triostin A. AB - Two new analogues of TANDEM (des-N-tetramethyl triostin A) have been synthesised in an effort to elucidate the molecular basis of DNA nucleotide sequence recognition in this series of compounds. Their binding preferences have been investigated by DNAase I footprinting and differential inhibition of restriction nuclease attack. The presence of a single N-methyl group on only one valine residue (in [N-MeVal4] TANDEM) abolishes the ability to recognise DNA, presumably because this antibiotic analogue has suffered an unfavourable conformational change in the depsipeptide ring. A bis-methylated analogue, [N-MeCys3, N MeCys7]TANDEM, was found to interact quite strongly with DNA and afforded binding sites, rich in AT residues, identical to those of TANDEM. Footprinting with various DNA fragments of known sequence showed that this analogue recognises sequences containing the dinucleotide TpA, although we cannot exclude the possibility that it binds to ApT as well. [N-MeCys3, N-MeCys7]TANDEM inhibits cutting by RsaI, a restriction enzyme that recognises GTAC but not by Sau3AI which recognises GATC. This provides further supportive evidence that the ligand (and, by extension, TANDEM itself) prefers binding to sequences containing the dinucleotide step TpA. PMID- 3008084 TI - Transcriptionally active SV40 minichromosomes are restriction enzyme sensitive and contain a nucleosome-free origin region. AB - A nucleosome-free region or gap containing the origin of replication and the transcriptional promoter elements is observed on 20 to 25% of the SV40 minichromosomes isolated at physiological ionic strength late in infection. We used the preferential sensitivity of the gapped minichromosomes to restriction enzymes to obtain sucrose gradient fractions containing 50 to 80% of gapped molecules. The same fractions are also enriched in RNA polymerase B (II) molecules engaged in transcription. Using electron microscopy, we demonstrate here that the transcriptional complexes are preferentially sensitive to restriction enzyme digestion, which indicate that they represent a subpopulation of the gapped minichromosomes. PMID- 3008085 TI - Novobiocin inhibits RNA polymerase III transcription in vitro by a mechanism distinct from DNA topoisomerase II. AB - The role of DNA topoisomerases in eucaryotic class III gene transcription in vitro has been studied through the use of inhibitory drugs and antisera to DNA topoisomerases I and II. The DNA topoisomerase II inhibitors, novobiocin and coumermycin AI, were found to inhibit transcription of cloned 5S and tRNA genes. Novobiocin acts by interfering with an ATP-requiring step in the pathway to stable preinitiation complex formation. However, it is unlikely that this step reflects the enzymatic action of DNA topoisomerase II since a specific inhibitor of this enzyme (VM-26) and anti-DNA topoisomerase II antibodies fail to inhibit transcription under conditions where topoisomerase II enzymatic activity is inhibited. Similarly, a specific inhibitor of DNA topoisomerase I (camptothecin) and anti-DNA topoisomerase I antibodies fail to inhibit class III gene transcription. These results argue against a role for either DNA topoisomerase in 5S or tRNA gene transcription in vitro. PMID- 3008086 TI - Methidiumpropyl-EDTA-iron(II) cleavage of ribosomal DNA chromatin from Dictyostelium discoideum. AB - We have used methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)] in parallel with micrococcal nuclease to investigate the chromatin structure of the extrachromosomal palindrome ribosomal RNA genes of Dictyostelium. Confirming our earlier results with micrococcal nuclease (1,2), MPE.Fe(II) digested the coding region of rapidly transcribing rRNA genes as a smear, indicating the absence or severe disruption of nucleosomes, whereas in slowly transcribing rRNA genes, a nucleosomal ladder was produced. In the central non-transcribed spacer region of the palindrome, MPE.Fe(II) digestion resulted in a normal nucleosomal repeat, whereas micrococcal nuclease gave a complex banding pattern. The difference is attributed to the lower sequence specificity of MPE.Fe(II) compared to micrococcal nuclease. In the terminal region of the palindrome, however, both substances gave a complex chromatin digestion pattern. In this region the DNA appears to be packaged in structures strongly positioned with respect to the underlying DNA sequence. PMID- 3008088 TI - The MURFI linker for multiple reading frame insertion of a sense or nonsense codon into DNA. AB - Blunt-end palindromic DNA linkers with a central restriction site have been designed for the multiple reading frame insertion (abbreviated MURFI) of a sense or nonsense codon into DNA. We have utilized an amber MURFI linker, 5'CTAG TCTAGA CTAG3' to disrupt the lacZ gene, yielding truncated beta-galactosidase proteins. Conditional disruption of the tetr gene in E. coli has also been demonstrated. Nonsense codon MURFI linkers permit conditional fusion of multiple gene products while sense codon linkers can add structural elements (e.g. beta-turn, cationic segment, hydrophobic segment) or a desired amino acid to a protein (e.g. methionine, cysteine). Shotgun or alternatively site-directed insertion of the symmetric linkers is possible. The over-all length of the linker may be adjusted to retain the original reading frame, matching nucleotide additions or subtractions at recipient DNA sites. If a linker restriction site occurs elsewhere in the target DNA, single linker copies may still be inserted using non phosphorylated linkers. PMID- 3008087 TI - Sequence characterization of Tetrahymena macronuclear DNA ends. AB - Tetrahymena is a ciliated protozoan which has two nuclei: a micronucleus, which maintains the genetic continuity of the cell, and the macronucleus which is derived from the micronucleus after sexual conjugation. A macronuclear DNA library was constructed to contain DNA ends. A probe containing C4A2 repeats which are known to be present at macronuclear DNA ends (1) was used to screen the library. Three clones were characterized by sequencing, restriction enzyme mapping and Bal 31 digestion. The data indicate that these three clones represent macronuclear DNA ends which were generated by DNA fragmentation during macronuclear formation. The sequencing data at the C4A2 repeat junction show a conserved sequence of five nucleotides, TTATT. Sequences further away show no obvious homologies except that they are highly enriched in AT. This structure is quite different from the subtelomeric sequences of other organisms. PMID- 3008089 TI - Palindromic oligonucleotides containing 7-deaza-2'-deoxyguanosine: solid-phase synthesis of d[(p)GG*AATTCC] octamers and recognition by the endodeoxyribonuclease EcoRI. AB - Octadeoxynucleotides with the sequence d[(p)GG*AATTCC] have been prepared by solid-phase synthesis employing regular and base-modified phosphoramidites. These oligomers which contain an isosterically altered recognition sequence of the endodeoxyribonuclease Eco RI form duplexes under appropriate salt conditions. Since G* can represent 7-deaza-2'-deoxyguanosine the oligomers were used as probes to study their cleavage by the endodeoxyribonuclease Eco RI. The enzymatic hydrolysis of the modified octamer was strongly decreased compared to the regular DNA-fragment. This shows that guanine N-7 located at the cleavage site is important for the recognition process by the enzyme. The residual enzymatic activity is discussed on the basis of reduced specificity towards the recognition fragment. The fact that this cleavage occurs already under regular conditions indicates that the process described here bases on an intrinsic property of the oligomer and is different from the star activity. PMID- 3008090 TI - Cloning and expression of cDNA encoding mouse tyrosinase. AB - We have isolated a pigment cell-specific cDNA clone from a B16 mouse melanoma cDNA library by differential hybridization. The mRNA of isolated cDNA is highly expressed in B16 melanoma cells and in black mouse (C57BL/6) skin, but is not detectable in mouse neuroblastoma cells nor in K1735 mouse amelanotic melanoma cells. The protein sequence deduced from the nucleotide sequence of the cloned cDNA shows significant similarity to the entire region of Neurospora tyrosinase. To know the identity of cDNA, we transfected K1735 amelanotic melanoma and COS-7 cells with the cDNA carried in a simian virus 40 vector (pKCRH2). We confirmed that the isolated cDNA encodes mouse tyrosinase by immunofluorescence staining of transfected cells using two different anti-T4-tyrosinase monoclonal antibodies. Tyrosinase is composed of 513 amino acids with a molecular weight of 57,872 excluding a hydrophobic signal peptide of 24 amino acids. PMID- 3008091 TI - DNA methylation and transcriptional controls of proviral DNA in avian sarcoma virus-transformed mammalian cells. AB - Restriction mapping has been used to study the integration state of the single provirus present in the DNA of two subclones, RS2/3 and RS2/6, of hamster cells transformed in vitro by Rous sarcoma virus, but differing markedly in their level of proviral transcription which was higher in RS2/3 cells. It was observed that both proviruses are complete and located at the same integration site in each DNA. However, the RS2/6 provirus and its flanking cellular sequences were found to be hypermethylated, although a very short region was hypomethylated at about 1 kb upstream of the src gene. A low level of methylation was observed in RS2/3 cells, in the proviral region. Northern analysis of viral RNA detected only the src mRNA in RS2/6 cells, whereas the two other viral mRNA were found in RS2/3 cells, however their levels were very low compared to that of the src mRNA. These findings suggest a correlation between the methylation state and the transcriptional control of the proviral genes. Sequences responsible for such a control by methylation should lie within both the provirus and its 5' flanking cellular sequences. PMID- 3008093 TI - The presence of intact mitochondrial DNA in HeLa cell nuclei. AB - Restriction analysis of DNA labelled with [32P]dCTP in an in vitro replication system with isolated nuclei from early S phase cells showed preferential labelling of restriction fragments derived from mitochondrial DNA (mtDNA) by a replication machinery distinct from that responsible for bulk nuclear DNA replication. Use of restriction nucleases with one recognition site in mtDNA gave rise to 16.5 kbp long fragments corresponding to full-length linearized mtDNA, indicating the presence of intact mtDNA in the isolated cell nuclei. Incorporation of dNTPs into mtDNA was not restricted to the S phase of the cell cycle. We were unable to increase the labelling of mtDNA by the addition of purified mitochondria or mtDNA to the nuclear replication system. These and other results presented is evidenced that the presence of mtDNA in the isolated nuclei was not due to uptake during preparation, thus indicating its presence in the cell nucleus in vivo. PMID- 3008092 TI - Sequence comparison of alleles of the fourth component of complement (C4) and sex limited protein (Slp). AB - cDNA clones specific for the fourth component of complement (C4) and its androgen regulated isotype, sex-limited protein (Slp), have been isolated from two mouse haplotypes (H-2d and H-2w7) that show differential C4 activity and differential regulation of Slp. Clones were first isolated using a cDNA probe enriched by subtractive hybridization. Subsequent screening has resulted in cDNAs spanning the entire C4d mRNA, as well as much of C4w7, Slpw7 and a short region of Slpd. The cDNAs for C4 and Slp show extensive sequence homology, but can be distinguished using oligonucleotide probes synthesized to regions of greatest sequence divergence. Sequence differences between C4 and Slp indicate structurally important features of C4 that have been altered in Slp such that Slp is unable to participate in the complement pathway. Of the few nucleotide differences between C4d and C4w7, a single base change resulting in one less glycosylation site in the C4w7 alpha chain could account for its 4-fold reduced hemolytic efficiency. Sequence comparison of multiple alleles of C4 and Slp indicates that possible gene conversion events occurred in the H-2w7 strain that has multiple Slp genes. PMID- 3008094 TI - An Epstein-Barr virus transcription unit is at least 84 kilobases long. AB - We have studied the structure of the Epstein-Barr virus mRNAs expressed in B95-8, a productively-infected Marmoset cell line established from in vitro-infected B lymphocytes. We constructed a cDNA library from the cytoplasmic polyadenylated RNAs of B95-8 in the lambda gt10 bacteriophage. We present here the analysis of a 3.5 kbp cDNA containing exons transcribed from the US, IR and UL regions of the viral genome. The corresponding transcription unit is at least 84 kbp long. Two exons are transcribed from the US region, five from the IR region and two from the UL region. The exons from the IR region consist of two tandem repeats of a unit containing two exons, 66 and 132 nucleotides, and of a third copy of the 66 nucleotide exon. The exons from the UL region contain an open reading frame coding for a 944 amino acid polypeptide. The C-terminal end of this polypeptide harbors three types of repeated sequences. The corresponding mRNA is the second described of a family of mRNAs produced by alternative splicing of exons transcribed from the US, IR and UL regions. PMID- 3008095 TI - Negative regulatory sequences in the EIa-inducible enhancer of the adenovirus-2 early EIIa promoter. AB - The adenovirus type 2 early EIIa (EIIaE) transcriptional control region exhibits an EIa-dependent enhancer activity (Imperiale et al., 1985, Proc. Natl. Acad. Sci. USA 82, 381-385). We have determined the sequence requirements for this enhancer activity by analysing the enhancing capacity of the entire EIIa promoter region, or portions of it, when inserted approximately 400 bp upstream of the rabbit beta-globin gene. Globin-specific transcription efficiency from the resulting recombinants was measured after transfection into HeLa cells, both in the presence and absence of the EIa products. It was found that the minimal EIIa element with bidirectional, EIa-dependent enhancer activity extends between -111 and -27 relative to the EIIaE major startsite (+1). Furthermore an extensive deletion analysis revealed, within this element, three functionally distinct regions: a central region between about -90 and -70, corresponding to an essential EIIaE upstream promoter element, and two flanking control elements (about 20 bp each) which, in the absence of the EIa products, exert a negative effect on the enhancer activity. Deletion of either one of these control elements renders the EIIaE enhancer activity constitutive, suggesting that the EIa products stimulate the EIIaE enhancer by relieving the negative control mediated by these sequences. PMID- 3008096 TI - Primary structure of leader RNA and nucleoprotein genes of the rabies genome: segmented homology with VSV. AB - We have determined the nucleotide sequence of the 3'region of the rabies genome (PV strain). This work is a first step in a project aimed at establishing the complete primary structure. From the 3'nucleotide sequence of the RNA genome, an octadecanucleotide complementary to the 3'extremity was constructed and used to prime cDNA synthesis. Two overlapping recombinant cDNA clones hybridizing with the nucleoprotein mRNA (NmRNA) were isolated and sequenced. The 1500 first nucleotides of the rabies genome cover two transcriptional units: the leader RNA and the NmRNA which was shown to be initiated around residue 59 by S1 nuclease protection experiments. Comparison between rabies PV and CVS strains up to residue 180 suggests a rapid evolution in the leader region. Studies of the sequence relationships between the 3'regions of two Rhabdoviruses, rabies virus and Vesicular Stomatitis Virus (VSV), demonstrate that there is a segmented homology. Stretches of highly conserved amino acids possibly involved in the interaction with the RNA genome were observed in the N protein, despite a wide divergence in the remaining sequence. In addition, the high homology between the transcription start and stop signals reflects the conservation of a similar transcriptional mechanism in these two non segmented negative strand RNA viruses. PMID- 3008098 TI - Molecular cloning and sequence analysis of human Na,K-ATPase beta-subunit. AB - We have isolated a cDNA clone for the beta-subunit of HeLa cell Na,K-ATPase, containing a 2208-base-pair cDNA insert covering the whole coding region of the beta-subunit. Nucleotide sequence analysis revealed that the amino acid sequence of human Na,K-ATPase exhibited 61% homology with that of Torpedo counterpart (Noguchi et al. (1986) FEBS Lett. in press). A remarkable conservation in the nucleotide sequence of the 3' non-coding region was detected between the human and Torpedo cDNAs. RNA blot hybridization analysis revealed the presence of two mRNA species in HeLa cells. S1 nuclease mapping indicated that they were derived from utilization of two distinct polyadenylation signals in vivo. Total genomic Southern hybridization indicated the existence of only a few, possibly one set of gene encoding the Na,K-ATPase beta-subunit in the human genome. PMID- 3008097 TI - Analysis and comparison of the internal promoter, pE, of the ilvGMEDA operons from Escherichia coli K-12 and Salmonella typhimurium. AB - It was previously determined that the distal portion of the ilvGMEDA operon was expressed despite the insertion of transposons into ilvG and ilvE. This observation suggested the existence of internal promoters upstream of ilvE (pE) and ilvD (pD). The internal promoter pE, responsible for part of ilvEDA expression, has been analyzed both in vivo and in vitro. Our results indicate that: pE exists in both E. coli K-12 and S. typhimurium; pE is located in the distal end of the ilvM coding sequence; the pE sequence is highly conserved in the two bacteria; the amino acid sequence of the ilvM gene product is 93% homologous between the two bacteria; transcription from pE can be demonstrated both in vivo and in vitro; the efficiency of pE is essentially equivalent in the two bacteria. PMID- 3008099 TI - Structure of the uvrB gene of Escherichia coli. Homology with other DNA repair enzymes and characterization of the uvrB5 mutation. AB - The complete nucleotide sequence of the Escherichia coli uvrB gene has been determined. The coding region of the uvrB gene consists of 2019 nucleotides which direct the synthesis of a 673 amino-acid long polypeptide with a calculated molecular weight of 76.614 daltons. Comparison of the UvrB protein sequence to other known DNA repair enzymes revealed that 2 domains of the UvrB protein (domain I = 6 amino acids, domain II = 14 amino acids) are also present in the protein sequence of the uvrC gene. We show that the structural homologies between UvrB and UvrC are as well reflected by the cross-reactivity of anti-uvrB and anti uvrC antibodies with UvrC and UvrB protein respectively. In the N-terminal part of UvrB, domain III (17 amino acids) shows a strong homology with one part of the AlkA gene product. Adjacent to domain III, an ATP binding site consensus sequence is found in domain IV. The uvrB5 mutant gene from strain AB1885 has been cloned on plasmid pBL01. We show that the uvrB5 mutation is due to a point deletion of a CG basepair and results in the synthesis of an 18 kD protein composed of the 113 N-terminal amino acids of the wild type uvrB gene and a 43 amino acid long tail coded in the -1 frame. PMID- 3008100 TI - DNA sequence of Rhizobium trifolii nodulation genes reveals a reiterated and potentially regulatory sequence preceding nodABC and nodFE. AB - The Rhizobium trifolii nod genes required for host-specific nodulation of clovers are located on 14 kb of Sym (symbiotic) plasmid DNA. Analysis of the nucleotide sequence of a 3.7 kb portion of this region has revealed open reading frames corresponding to the nodABCDEF genes. A DNA sequencing technique, using primer extension from within Tn5, has been used to determine the precise locations of Tn5 mutations within the nod genes and the phenotypes of the corresponding mutants correlate with their mapped locations. The predicted nodA and nodB genes overlap by four nucleotides and the nod F and nodE genes overlap by a single nucleotide, suggesting that translational coupling may ensure the synthesis of equimolar amounts of these gene products. The nodABC and nodFE genes constitute separate transcriptional units and each is preceded by a conserved 76-bp sequence which may be involved in the regulation of expression of these genes. PMID- 3008101 TI - Isolation and characterization of a Ty element inserted into the ribosomal DNA of the yeast Saccharomyces cerevisiae. AB - The yeast Saccharomyces cerevisiae has about 30 to 50 copies of a transposable element Ty. Most of these elements are located at the 5' ends of protein coding sequences and are flanked by a 5 bp duplication. We report below an insertion of a Ty element into one of the repeated ribosomal RNA (rRNA) genes of yeast. The element is located between the 3' ends of the divergentally transcribed 37S and 5S rRNA's and is not flanked by a 5 bp duplication. In addition, one end of the Ty insertion is contiguous with a 306 bp deletion of the sequences of the rRNA gene. We find that this insertion, unlike most Ty insertions, is mitotically unstable. PMID- 3008102 TI - High frequency excision of Ty elements during transformation of yeast. AB - Yeast (Saccharomyces cerevisiae) transposons (Ty elements) are excised from up to 20% of supercoiled plasmids during transformation of yeast cells. The excision occurs by homologous recombination across the direct terminal repeats (deltas) of the Ty element, leaving behind a single delta in the transforming plasmid. Only the initial transforming plasmid is susceptible to excision, and no high frequency excision is observed in plasmids that have become established in transformed cells or in plasmids that are resident in cells undergoing transformation. High frequency excision from plasmids during yeast transformation is not specific for Ty elements and can be observed with other segments of plasmid DNA bounded by direct repeats. The frequency of Ty excision from supercoiled plasmids is greatly reduced when the host yeast cells contain the rad52 mutation, a defect in double-strand DNA repair. When linear or ligated linear plasmid DNAs containing a Ty element are used for transformation, few or no excision plasmids are found among the transformant colonies. These results suggest that when a yeast cell is transformed with a supercoiled plasmid, the plasmid DNA is highly susceptible to homologous recombination for a short period of time. PMID- 3008104 TI - Three distinct activities possibly involved in mRNA splicing are found in a nuclear fraction lacking U1 and U2 RNA. AB - A nuclear extract from HeLa cells was fractionated by DEAE-Sepharose chromatography, and the fractions were assayed for the binding activity for a small RNA transcript carrying a splice junction or branch point sequence. The binding activity for the RNA carrying a 5' splice junction was localized in the small nuclear ribonucleoprotein (snRNP) fraction together with binding activity for the RNA carrying a 3' splice junction or branch point sequence. However, stronger binding activities for the 3' splice junction RNA and for the branch point RNA were discovered in the flow-through fraction where no small nuclear RNA were detected. When the flow-through fraction was added to purified U1 ribonucleoprotein, the binding activity for the 5' splice junction RNA was markedly enhanced. We propose that the factors responsible for the three types of activities found in the flow-through fraction play a role in the splicing of mRNA precursor. PMID- 3008103 TI - A tandemly-oriented late gene cluster within the vaccinia virus genome. AB - The nucleotide sequence of a 5.1 kilobase-pair fragment from the central portion of the vaccinia virus genome has been determined. Within this region, five complete and two incomplete open reading frames (orfs) are tightly-clustered, tandemly-oriented, and read in the leftward direction. Late mRNA start sites for the five complete orfs and one incomplete orf were determined by S1 nuclease mapping. The two leftmost complete orfs correlated with late polypeptides of 65,000 and 32,000 molecular weight previously mapped to this region. When compared with each other and with sequences present in protein data banks, the five complete orfs showed no significant homology matches amongst themselves or any previously reported sequence. The six putative promoters were aligned with three previously sequenced late gene promoters. While all of the nine are A-T rich, the only apparent consensus sequence is TAA immediately preceeding the initiator ATG. Identification of this tandemly-oriented late gene cluster suggests local organization of the viral genome. PMID- 3008105 TI - Isolation, physical characterization and expression analysis of the Saccharomyces cerevisiae positive regulatory gene PHO4. AB - The Saccharomyces cerevisiae PHO4 gene, which positively controls the expression of phosphatase genes, has been isolated by complementation of a pho4 mutation. The isolated DNA directed integration at the chromosomal PHO4 locus. The nucleotide sequence of PHO4 has a coding region of 930 nucleotides, flanked by sequences with typical transcription initiation and termination signals. The 5' region has characteristics of low-expression promoters and carries several uncommon elements, whose significance is not known. The predicted primary structure of the PHO4 protein, of 309 residues, does not show sequence elements typical of DNA-binding proteins. The transcription of PHO4 is independent of inorganic phosphate. Like other regulatory genes, PHO4 is transcribed at a very low level and the translation of its message uses preferentially several codons which are not employed for highly expressed genes. PMID- 3008106 TI - The organization of the human HPRT gene. AB - The organization of the X-linked gene for human hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8.) has been determined by a combination of restriction endonuclease mapping, heteroduplex analysis and DNA sequence analysis of overlapping genomic clones. The entire gene is 42 kilobases in length and split into 9 exons. The sizes of the 7 internal exons and the exon intron boundaries are identical to those of mouse HPRT gene. The 5' end of the gene lacks the prototypical 5' transcriptional regulatory sequence elements but contains extremely GC-rich sequences and five GC hexanucleotide motifs (5'-GGCGGG 3'). These structural features are very similar to those found in the mouse HPRT gene and to some of the regulatory signals common to a class of constitutively expressed "housekeeping" genes. Several transcriptional start sites have been identified by nuclease protection studies. Extensive sequence homology between the mouse and human genes is found in the 3' non-coding portion of the gene. PMID- 3008107 TI - Conservation in the 5' region of the long interspersed mouse L1 repeat: implications of comparative sequence analysis. AB - A clone of 7.1kb corresponding to the mouse L1 interspersed repeat family was selected for homology to a human interspersed repeat. This clone fairly represents mouse genomic members. Mapping of the clone revealed one common element at both the 5' and 3' ends in a head to tail arrangement, suggesting that at least some long L1 family members are tandemly arranged; genomic studies confirmed the unexpected tandem arrangement of a minor proportion of L1 members. A short SmaI tandem repeat appears to define the 5' end of most L1 family members. SmaI repeats may maintain, via a recursive regulatory function, the transcriptional viability of L1 members after retroposition events. A 2.5kb portion of the mouse L1 repeat that has not been previously sequenced is presented. It is 55-70% homologous to a corresponding portion of the human KpnI repeat family. Comparative sequence analysis revealed that one common open reading frame may conserve potential coding function across species. A second open reading frame bears an asymmetric distribution of codon replacements unlike both genes and pseudogenes. This latter feature could be consistent with a proposed chromosome organization function that is unrelated to peptide expression. PMID- 3008109 TI - [The nitroblue tetrazolium reduction test in patients with lung cancer during radiotherapy]. PMID- 3008108 TI - Evidence for transcription and potential translation of the human 1.9 kb HindIII repetitive element. AB - Recombinant cDNA clones corresponding to the human 1.9kb HindIII repetitive element have been isolated from a cDNA library of liver cytoplasmic polyadenylated RNA. These cDNAs share 95% homology with the reported genomic DNA sequence and a similar amount of homology at the amino acid level with putative coding sequences (see preceding article by Mottez et al). They were isolated as two of four false positives from a human cDNA library in lambda gt11 and were selected with an antibody to an unrelated enzyme. These results provide direct evidence that this repetitive element is transcribed to form poly(A)+ RNA which could be translatable. Also, these observations may add to our understanding of the sources of false positives which are frequently observed in screens of cDNA libraries with antibodies as probes. PMID- 3008111 TI - Hospital-acquired infection. Counting the cost of infection. PMID- 3008112 TI - AIDS: boys and girls come out to play? PMID- 3008110 TI - Fear and loathing. PMID- 3008113 TI - Ethical dilemmas: licensed to kill? PMID- 3008114 TI - Treatment of juvenile periodontitis with microbiologically modulated periodontal therapy (Keyes technique). PMID- 3008116 TI - Behavioural and spectrum power effects of opioid peptides in chicks. AB - The effects of intracerebroventricular administration of beta-endorphin, dermorphin, D-ala2-methionine-enkephalinamide (DALA) and pentazocine, on behaviour, electrocortical activity and ECoG spectrum power in young chicks were studied. Beta-endorphin, dermorphin and DALA produced dose-dependent behavioural sedation and sleep accompanied by electrocortical high voltage slow wave activity and increase in total voltage power with a predominant increase in the lower bands of ECoG spectrum. Dermorphin was found significantly more potent than beta endorphin and DALA since it was active at much lower doses. Behavioural and ECoG sleep evoked by opioid peptides were rapidly reversed by naloxone showing that these effects are mediated through an activation of specific opioid receptors. Doses of pentazocine equimolar with doses of dermorphin and beta-endorphin producing sleep did not affect behaviour and spectrum power whereas higher doses produced opposite effects, i.e., behavioural stimulation, pecking and increase in locomotor activity. PMID- 3008115 TI - Bombesin in human neuroendocrine (NE) neoplasms. AB - Bombesin is a 14 amino acid peptide isolated from amphibian skin which was found to have stimulatory effects upon gastric and pancreatic secretions, release of gastrointestinal hormones, gallbladder contraction and bronchoconstriction. It is present in amphibian gastric endocrine cells, avian proventriculus endocrine cells and avian brain. In mammals it is present mainly in nerve cells and fibers. The only mammalian endocrine cell shown to date to have bombesin is the P-cell in fetal lung. Bombesin is also found in mammalian brain, with its highest concentration in the hypothalamus. We examined several groups of human neuroendocrine neoplasms for the presence of bombesin by immunohistochemistry. Our findings indicate that bombesin is present 68% of bronchial carcinoids, 65% of pulmonary neuroendocrine carcinomas, 62% of neuroendocrine carcinomas of the skin, 5-10% of pheochromocytomas and extraadrenal paragangliomas and 35% of gastrointestinal carcinoids and neuroendocrine carcinomas. Parallel studies in a wide variety of non neuroendocrine neoplasms failed to reveal the presence of bombesin. We conclude that bombesin is a highly specific marker of neuroendocrine differentiation and thus a valuable tumor marker. Furthermore, its specificity compares favorably with another neuroendocrine marker, neuron specific enolase, an enzyme thought to be present only in neural tissues and neuroendocrine cells but recently found in non neural human tissues and non neuroendocrine neoplasms. PMID- 3008117 TI - Central pharmacological activities and opiate receptor binding studies of some dermorphin analogs. AB - A series of dermorphin-like compounds were injected intracerebroventricularly in the rat to assess in vivo their effects on intestinal motility and analgesia. In vitro they were tested by binding assay using 3H-naloxone as radioligand or by guinea pig ileum bioassay. The synthetic peptides were less potent than dermorphin in inhibiting intestinal transit and in producing analgesia, or even inactive up to doses 30 times the dermorphin ED50. This reduction in pharmacological activity was coupled with a decrease in binding potency. The 3H naloxone binding studies in the absence or presence of Na+ indicated that Na+ reduced the interaction of dermorphin and its analogs with brain opiate receptors. Only the dibenzyl derivative was slightly affected by sodium, suggesting a dual action for this peptide, as confirmed by preliminary data from guinea pig ileum bioassay. PMID- 3008118 TI - The effects of dermorphin on the endocrine system in man. AB - This paper summarizes the results of our recent studies in a group of healthy subjects on the endocrine effects of the new potent opioid peptide, dermorphin (H Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2), originally isolated from amphibian skin. Intravenous infusion (5.5 microgram/kg/min for 30 min) of dermorphin (D) significantly increased plasma levels of prolactin (PRL), growth hormone (GH), thyrotropin (TSH) and renin activity (PRA), but decreased plasma levels of cortisol. D produced a small decrease in ACTH, and a small increase in plasma aldosterone. Pretreatment with the opioid receptor antagonist naloxone (N) suppressed the PRL and TSH response to D, blunted the D-induced GH and PRA increase, and completely prevented the D-induced plasma cortisol decrease, but enhanced plasma cortisol and ACTH levels. These data indicate that the action of D is mediated through opioid receptors, and are consistent with the conclusion that: (1) D, a new opioid peptide, can stimulate PRL, GH and TSH release in humans; (2) D increases PRA levels, perhaps via activation of the sympathetic nervous system, providing evidence that opioid peptides may exert an influence on renin secretion; (3) D suppresses plasma cortisol levels, by affecting ACTH secretion, corroborating previous observations that opioid peptides might affect the function of the pituitary-adrenocortical axis. PMID- 3008119 TI - Novel peptides from the calcitonin gene: expression, receptors and biological function. AB - Calcitonin gene products include calcitonin and its carboxyl-terminal flanking peptide (in man PDN-21), and calcitonin gene-related peptide (CGRP). Alternative splicing of the initial gene transcripts results in the production of two distinct messenger RNA encoding precursors of CGRP and of calcitonin. CGRP messenger RNA is the predominant transcription product of the calcitonin gene in neural tissues, but it is also present in the pituitary and the C-cells of normal thyroid glands and in medullary thyroid carcinoma. Immunoreactive CGRP has, moreover, been recognized around blood vessels of the heart. Calcitonin and PDN 21 are cosecreted from thyroid C-cells, but they are also found in the brain and pituitary. CGRP receptors are present in the brain and the heart, and calcitonin receptors in bone and kidney cells and in the hypothalamus. Calcitonin administered peripherally and in vitro inhibits bone resorption and stimulates renal 1.25-dihydroxycholecalciferol production. CGRP used in the same manner has potent cardiovascular effects (vasodilation, hypotension, positive chronotropic and inotropic action in the heart). Intracerebroventricular administration of CGRP raises the blood pressure, and both CGRP and calcitonin inhibit gastric acid secretion and food intake. The distinct but overlapping effects of calcitonin and CGRP raise important regulatory and functional issues. PMID- 3008120 TI - Studies on the antinociceptive effect of intrathecal salmon calcitonin. AB - The involvement of spinal opioid receptors and spinal monoaminergic systems, in the antinociceptive effect of intrathecal (IT) salmon calcitonin, has been evaluated by means of the hot plate test, in the rat. Intrathecal pretreatment with 40 micrograms MR 1452 and 40 micrograms ICI 154,129, purported selective antagonists respectively for kappa and delta opioid receptors did not modify sCT induced antinociception (2 micrograms IT). A delay in the development of IT sCT induced antinociception was observed in rats selectively depleted of cord serotonin (25 mg/kg IP desipramine plus 100 micrograms IT 5,7 dihydroxytryptamine), whereas the administration of serotonergic antagonists, methysergide and ketanserin, 30 micrograms IT, did not influence sCT effect. Cord catecholamine depletion (6-OHDA pretreatment) reduced significantly sCT antinociception. A similar reduction was produced by the dopaminergic antagonist haloperidol (15 micrograms IT), but not by the alpha-blocker phentolamine (15 micrograms IT). Findings of this study rule out an involvement of opioid peptidergic system in sCT-induced increase of hot plate latencies at spinal level; a possible involvement of cord dopaminergic receptors is suggested. PMID- 3008121 TI - Cholecystokinin and its receptors in vertebrates and invertebrates. AB - Recent studies have indicated that cholecystokinin (CCK) peptides have a long evolutionary history. However, whereas all vertebrates examined have been shown to contain CCK-like peptides, this has not been possible to demonstrate for all invertebrate groups. Immunostaining studies indicate that CCK peptides originate only in neurons in groups below the level of the protochordates. It seems likely that CCK gastrointestinal endocrine cells evolved first at the level of the protochordates, possibly from sensory gut neurons similar to those seen in the invertebrates. Immunochemical and biological studies of a few invertebrate CCK like peptides suggest that those molecules are substantially different in structure from vertebrate CCKs. Gastrin appears to have evolved from CCK at the level of the appearance of amniotes in vertebrate phylogeny. In mammals, central and peripheral CCK receptors differ in specificity for CCK- and gastrin-like peptides. Comparative studies reveal that this is true for birds as well, but reptiles, amphibia, and fish brain and pancreas CCK receptors exhibit nearly identical specificity patterns. This suggests that the lower vertebrate CCK receptor is ancestral to the distinct brain and pancreas CCK receptors seen in birds and mammals. PMID- 3008122 TI - Evolutionary relationship between nonmammalian and mammalian peptides. AB - An hypothesis has been developed to rationalize the evolution of regulatory peptides. In order to account for critical relationships involving peptide regulators, their receptors, and peptide processing enzymes, the following generalizations will be supported: (1) peptides arose from protein precursors as proteolytic digestion by-products and acquired hormonal status during the course of natural selection; (2) initially, peptides served primarily nutritional roles, thereby permitting increased growth rates and reproductive advantages for recipient cells; (3) specific peptide sequences were conserved during evolution and were associated with biological activities which were essential for survival of species as divergent as unicellular organisms, amphibians, and mammals; and (4) regulatory peptides probably arose simultaneously with their membrane oriented, macromolecular receptor sites. In support of the conservation of sequence information or function, or both, during evolutionary development, evidence has been obtained to indicate that peptide sequences which occur in two classes of amphibian peptides appear to be extensively conserved in mammals. Studies with an antiserum directed against the N-terminal sequence of amphibian physalaemin have permitted the recognition of a mammalian octapeptide which exhibits 80% homology with residues 1-5 in that region. Another study with an antiserum directed against the midregion (sequence 5-8) of amphibian bombesin has indicated the existence of milk peptides which mimic bombesin in several pharmacological bioassays. These studies indicate that radioimmunoassays can be powerful tools in facilitating recognition of peptide sequences conserved throughout evolution. PMID- 3008123 TI - Structure-function studies on the bradykinin potentiating peptide from Chinese snake venom (Agkistrodon halys Pallas). AB - A bradykinin potentiating peptide (BPP) was purified from the Chinese snake venom (Agkistrodon halys Pallas). The amino acid sequence of this BPP was determined to be pyroGlu-Gly-Arg-Pro-Pro-Gly-Pro-Pro-Ile-Pro-Pro. Removal of the N-terminal residue with pyroglutamate aminopeptidase enhanced two-fold the activity of BPP, the resulting despyroGlu-BPP gradually lost its activity on further Edman degradation. However, around 90% of the original activity was still present in the C-terminal tripeptide Ile-Pro-Pro. Some analogs of this tripeptide were synthesized by the conventional method, and investigated by two biological assays, i.e., potentiating response on bradykinin (BK) and inhibitory activity on angiotensin converting enzyme (ACE). It was shown that the two biological activities inherent in the synthetic analogs were not parallel to each other. In addition, the isolated guinea pig ileum strips treated with chelating agent to irreversibly inactivate kininase (the same enzyme ACE) still responded to BPP. Consequently the potentiating effect of BPP on BK in vitro bioassay might be due to its influence on the binding receptor for BK rather than the inhibitory effect on kininase. PMID- 3008124 TI - The fish neuropeptide urotensin I: its physiology and pharmacology. AB - Significant structural and biological homologies between urotensin I (UI), ovine hypothalamic corticotropin releasing factor (oCRF) and the frog skin peptide sauvagine (SVG) have been investigated and compared in fishes and mammals. In mammals, urotensin and the related peptides exert uniquely selective mesenteric vasodilatation, oCRF having approximately equal to 4% the activity of the other two. All three peptides are equipotent in stimulation of ACTH secretion in the rat in vivo and in vitro. UI is significantly more potent than the other two related peptides in stimulation of ACTH secretion in the goldfish pituitary. Immunocytochemical demonstration of UI not only in the caudal spinal cord but also in the brain, mainly in the lateral tuberal region and of an oCRF-like substance in the preoptic nucleus and pituitary, suggests that ACTH secretion in fishes may be controlled by two similar but distinct UI- or oCRF-like peptides. PMID- 3008125 TI - Peptides as modifiers of Na+-induced pinocytosis in starved Amoeba proteus. AB - Low concentrations of six peptide hormones; glucagon, vasoactive intestinal peptide, substance P, angiotensin II, lysine-vasopressin, arginine-vasopressin, and the chemotactic peptide fMet-Leu-Phe, activated the capacity for pinocytosis in starved Amoeba proteus. Competitive inhibitors of the chemotactic peptide in leucocytes inhibited activation by fMet-Leu-Phe, suggesting that its action in the amoeba is mediated by specific receptors. The opioid peptides, beta endorphin, dynorphin (1-13) and leu-enkephalin abolished through a naloxone sensitive mechanism activation by hormones and several other activating agents. Also, low concentrations of beef and pork insulin inhibited activation by peptide hormones. An insulin analogue of low potency in mammalian cells was inactive in the amoeba. These results support the hypothesis that besides opioid receptors, there may be insulin receptors and possibly receptors for several other peptide hormones in Amoeba proteus. PMID- 3008126 TI - Mammalian bombesin-like peptides: neuromodulators of gastric function and autocrine regulators of lung cancer growth. AB - Peptides corresponding closely in structure to the biologically active carboxyl terminal region of the amphibian peptide bombesin have now been isolated from several mammalian species, including man. Two principal forms have been found: one contains 27 amino acids and exhibits variations in amino acid sequence in the amino terminal region; the other is the carboxyl terminal decapeptide and probably does not vary among mammals. These peptides exhibit full immunoreactivity with most bombesin antisera and account for "bombesin-like immunoreactivity" that has been described in mammalian brain, sympathetic ganglia, and nerve fibers in the gut as well as in fetal lung endocrine cells and certain lung tumors, especially small cell lung carcinoma. The name gastrin releasing peptide (GRP) was given to the porcine and avian heptacosapeptides by McDonald and Mutt. The larger and smaller mammalian peptides now often are called GRP27 and GRP10. Both forms exhibit the full spectrum of activity shown by bombesin. Evidence has been obtained that neural release of mammalian bombesin like peptides is physiologically important in regulation of gastrin release from the stomach. Lung tumors that produce bombesin-like peptides also have receptors for bombesin. These receptors appear to be involved in the autocrine regulation of tumor cell proliferation. PMID- 3008127 TI - [Population studies of phosphoglycollate phosphatase in the Bydgoszcz region]. PMID- 3008128 TI - [Omeprazole--a drug-blocking proton pump of the parietal cells of the stomach]. PMID- 3008129 TI - Malignant phyllodes tumor following irradiation of the breast. AB - This is a case report of malignant phyllodes tumor (cystosarcoma phyllodes) which appeared 15 years following medical irradiation of the breast for presumable carcinoma which had not been histologically or cytologically confirmed prior to treatment. Histology of the phyllodes tumor disclosed remnant of fibroadenoma in one area, and it is believed that the latter gave rise to the malignant phyllodes tumor within the field of irradiation. In view of recent popularity of the limited surgery and postoperative irradiation in treatment of breast carcinoma the possibility of malignant transformation of fibroadenoma left in situ is raised. PMID- 3008130 TI - Preliminary pharmacological investigation on 38 aminophosphonic acids and their derivatives. AB - Central pharmacological properties of 38 aminophosphonic acids and their derivatives, mostly newly synthesized, were investigated on mice and rats. Acute toxicity, neurotoxic activity, influence on spontaneous locomotor activity, body temperature, electrogenic and pentetrazol convulsions, on cerebral GABA level were tested. The most active compounds were (in a decreasing order of activity): 2-amino-7-phosphonoheptanoic, 2-amino-5-phosphonovaleric, 2-amino-8 phosphonooctanoic, 2-amino-2-methyl-3-methylphosphonopropionic, and 3-amino-3 hydroxy-5-phosphonovaleric acid. PMID- 3008131 TI - Parathion-methyl effect on the activity of hydrolytic enzymes after single physical exercise in rats. AB - Rats were subjected to physical exercise in the form of a single race in a treadmill. Unexercised animals and those immediately or 1 h after the exercise were given oil or oily solution of parathion-methyl in a dose of 10 mg/kg by stomach gauge. At 1 h after pesticide administration the activity of cholinesterase (ChE) was determined in the serum, of paraoxonase--in the serum and liver, and that of beta-glucuronidase (beta-gluc)--in the serum, liver and intestine. Single physical exercise increased ChE activity in the serum, inhibited paraoxonase activity in the serum and liver; on the other hand, it did not affect significantly beta-gluc activity in the serum, but inhibited that enzyme in a transient manner in the liver and activated it in the intestine. In acute poisoning with parathion-methyl it was observed that ChE and paraoxonase activities were inhibited, while beta-gluc activity was enhanced in the serum. Single physical exercise either diminished or had no effect on enzymatic changes observed in acute poisoning with parathion-methyl. PMID- 3008132 TI - The effect of chronic physical exercise on the activity of hydrolytic enzymes in acute poisoning with parathion-methyl in rats. AB - Parathion-methyl, 10 mg/kg p.o., was given to unexercised rats, rats trained for 3 weeks and resting for 2 days, and trained rats subjected to a single physical exercise before treatment. The activity of cholinesterase (ChE) in the serum, of paraoxonase--in the serum and liver, and of beta-glucuronidase (beta-gluc)--in the serum, liver and intestine were determined 1 h after the treatment. Repeated physical exercise increased beta-gluc activity in the serum and liver and inhibited it in the intestine, while a single race after repeated exercise inhibited paraoxonase activity in the serum. Parathion-methyl inhibited ChE and paraoxonase activities and an increased beta-gluc activity in the serum. Repeated physical exercise and a single race, applied 2 days after the end of training, affects the activity of parathion-methyl in a significant yet diversified manner, dependent upon the examined biochemical parameters. PMID- 3008133 TI - Action of antidepressant neuroleptics, chlorprothixene and levomepromazine, on the central noradrenergic system: comparison with other antidepressants. AB - In contrast to imipramine, chlorprothixene (CPX) and levomepromazine (LMZ) given chronically did not affect clonidine-induced hypothermia and depression of noradrenaline synthesis rate, while similarly to imipramine and citalopram depressed the density of cortical 3H-clonidine binding sites and increased their affinity. Two week treatment with the antidepressants did not affect the parameters of 3H-dihydroalprenolol binding sites, while spiroperidol administration increased their density, without changing KD values. The data suggest that changes in alpha 2 adrenoceptor population parameters may be a general characteristics of antidepressant treatment, but not necessarily followed by changes in clonidine effects. PMID- 3008134 TI - Possible relationship of the locus coeruleus--hippocampal noradrenergic neurons to depression and mode of action of antidepressant drugs. AB - Recent studies performed in our laboratory suggest involvement of locus coeruleus (LC) and hippocampal noradrenergic (NE) neurons in the mechanism of depression and mode of action of antidepressants. Both electrolytic and 6-OHDA lesions to the LC abolished desipramine action in forced swim test in rats. The action of desipramine was also reduced in rats pretreated with alpha 1 adrenolytic drugs -- phenoxybenzamine and prazosin. Electrical stimulation of the LC produced, like desipramine, activating effect in forced swim test, the phenomenon never observed in phenoxybenzamine-pretreated animals. Chronic (but not acute) administration of desipramine potentiated activatory effects of intrahippocampal injections of NE and phenylephrine but not isoprenaline in both open field and forced swim test. Depressant effect of intrahippocampal clonidine was reversed by chronic desipramine (in the open field test). The effect of desipramine was partially shared by citalopram. PMID- 3008135 TI - A role of some central neurotransmitter systems in the mechanism of electroconvulsive shock action. AB - Behavioral effects of electroconvulsive shock (ECS) on the neurotransmitter systems were studied in relation to the mechanism of action. Repeated ECS, applied to rats, enhanced the behavioral response to dopaminergic, serotonergic and opiate agonists and attenuated the behavioral effects to GABA-ergic antagonists. The same ECS schedule did not significantly alter the behavioral response to alpha 2- and beta-adrenergic agonists and apomorphine-induced stereotypy but significantly potentiated haloperidol-induced catalepsy. PMID- 3008136 TI - The responsiveness of alpha 2-adrenergic receptors in hypothalamus of vasopressin hypertensive rats. AB - Administration of clonidine produced biphasic changes in the type II inhibitor activity (endogenous inhibitor of protein kinases which specifically regulates phosphorylation mediated by cGMP-dependent system). Small doses of clonidine (10 50 micrograms/kg) produced an increase while large doses (200-1000 micrograms/kg) induced a decrease in the type II inhibitor activity. The both actions of clonidine were greatly reduced in vasopressin hypertensive rats suggesting subsensitivity of alpha 2-adrenergic receptors. Subsensitivity of postsynaptic alpha 2-receptors was also observed in anterior hypothalamus in in vitro experiments. Incubation of anterior and posterior hypothalamic slices with clonidine resulted in concentration-dependent increase in cGMP content and a decrease in the type II inhibitor activity. The clonidine action in anterior hypothalamus of vasopressin-hypertensive rats was greatly reduced. In contrast, the clonidine action in posterior hypothalamus was the same in vasopressin hypertensive as in the control rats. PMID- 3008137 TI - Chemotherapy in adult cancer. When is it most useful? AB - Much has been accomplished in the cure of cancer with chemotherapy, but much remains to be done. The primary care physician's responsibility is to know which cancers are curable and in which a significant portion of patients may benefit from chemotherapy. Patients with a malignancy in a relatively resistant organ site represent the greatest challenge to the "art" of medicine. Even if more than half of patients with a given malignancy are cured by chemotherapy, the remainder deserve the right to participate in research protocols, an undertaking that might further improve these otherwise substantial gains. As research studies show improved cure rates with antineoplastic agents, the need to reduce both short- and long-term side effects will remain a challenge. PMID- 3008138 TI - Acute exanthems in children. Clues to differential diagnosis of viral disease. AB - The numerous viral skin diseases that affect children present a diagnostic challenge to the clinician. Most of these diseases may be conveniently grouped according to the clinical appearance of the exanthem as maculopapular, petechial, papular, or vesicular. In some situations, viral infection may be difficult to differentiate clinically from nonviral disease; thus, extensive laboratory evaluation is sometimes necessary to pinpoint the virus involved. Nonviral disease should be considered in the differential diagnosis of a patient with skin eruptions, especially of the maculopapular type. Common nonviral causes include Kawasaki disease, toxic shock syndrome, and drug reactions. PMID- 3008140 TI - In vivo responsiveness of the broilers' adrenocortical system. AB - The possible interaction of circadian rhythms in serum concentrations of corticosterone (B) with the response of broiler cockerels to heat stress (HS) and adrenal manipulation with adrenocorticotropic hormone (ACTH) and dexamethasone acetate (DA) was investigated. Adrenocortical response, as indicated by corticosterone concentrations, was greater early in the photoperiod in cockerels exposed to HS or injected with ACTH. A single injection of DA tended to be more inhibitory on B when administered later in the photoperiod. PMID- 3008139 TI - Lumbar disk disease. Clinical presentation, diagnosis, and treatment. AB - Back pain is a very common disorder, and low back pain has many causes. The clinical features of a single identifiable cause of back pain, lumbar disk disease, are summarized in this article. The hallmark of disk disease is single nerve root involvement, which often produces back pain and usually causes even more intense leg pain. Physical findings include sensory loss in a specific nerve root dermatome, weakness and atrophy of the muscle supplied by that nerve root, and reflex changes appropriate to the specific nerve root. Diagnosis is usually confirmed by one or several corroborative tests, the most frequently used of which are computed tomography, myelography, and electrodiagnostic studies. PMID- 3008141 TI - [Neurochemical characteristics of opioid peptides]. AB - Opioid peptides seem to be involved in an increasing number of cerebral functions. It appears from the most recent studies that these peptides are divided into three families: pro-opio-melanocortin, pro-enkephalin and prodynorphin. Unlike conventional neurotransmitters, these substances are fraught with extremely complex problems of specific biosynthesis and degradation. Finally, the multiplicity of opiate receptors and the search for specific endogenous or synthetic ligands raise major fundamental questions concerning the clinical relevance of studies performed on opioid peptides. PMID- 3008142 TI - [Neurotropic action of adrenocorticotropic hormone]. AB - The adrenocorticotropic hormone (ACTH) is produced within the cell body of hypothalamic neurons by proteolytic cleavage of its large precursor molecule, pro opiomelanocortin. These neurons distribute ACTH-containing nerve endings throughout the central nervous system. ACTH is able to evoke motor and behavioural responses and to modify neuronal metabolism. Since ACTH has been shown to regulate glucose uptake and utilization, its implication in the adaptative response to stress situations, such as cerebral hypoxia, deserves further investigations. PMID- 3008143 TI - [Infected retroperitoneal hemolymphangioma in an adult with the Klippel-Trenaunay syndrome. Ultrasonic diagnosis]. PMID- 3008144 TI - [Does adrenaline play a role in essential arterial hypertension?]. PMID- 3008145 TI - [Anemia and dysmyelopoiesis with marker chromosome and transferrin receptor anomaly]. AB - A 64-year old man presented with microcytic hypochromic aplastic acquired anemia without iron depletion. His bone marrow was hypercellular with dyserythropoiesis and no stainable iron deposits. 59 Fe incorporation by erythroblasts was reduced, and the karyotype revealed an aneuploidy with marker chromosome. After study of transferrin receptor with specific antibody, we conclude that the receptor presents a functional defect. PMID- 3008146 TI - [Role of human parvovirus in the erythroblastopenic crisis of chronic hemolytic anemia]. PMID- 3008147 TI - [Tropical spastic paraparesis in Martinique. High prevalence of anti-HTLV-I antibodies]. AB - Owing to the frequent occurrence in tropical countries of subacute spinal cord diseases of unknown origin, a nosological entity called tropical spastic paraparesis has been individualized. Twenty-two cases have been observed in Martinique. The presence in the serum of antibodies directed against human T-cell leukaemia/lymphoma virus (HTLV-I) in 15 of these 22 patients suggests that this lymphotropic virus or a related one might also be neurovirulent. PMID- 3008148 TI - v-abl activates embryonic globin gene expression in mouse erythroleukemia cells. AB - The Philadelphia chromosome translocation, which is present in 90-95% of chronic myelogenous leukemia patients, involves translocation of the c-abl protooncogene to chromosome 22 and is accompanied by activation of embryonic globin gene expression in the K562 chronic myelogenous leukemia cell line. To test directly if the protein products of the translocated c-abl protooncogene can activate embryonic globin gene expression, we transfected the v-abl oncogene (which shares the property of autophosphorylation with the translocated c-abl protooncogene) into mouse erythroleukemia cells. v-abl-transfected mouse erythroleukemia cells, which contained multiple copies of the v-abl transgenome, exhibited activation of mouse embryonic globin gene expression. These results suggest that the translocated c-abl protooncogene of the Philadelphia chromosome translocation is central to the pathogenesis of chronic myelogenous leukemia and that it may result in the activation of embryonic globin genes in some chronic myelogenous leukemia cell lines. PMID- 3008149 TI - A mutant of Escherichia coli fumarate reductase decoupled from electron transport. AB - The terminal electron-transfer enzyme fumarate reductase of Escherichia coli is a complex iron-sulfur flavoenzyme composed of four nonidentical subunits organized into two domains: FrdA and -B (a membrane-extrinsic catalytic domain) and FrdC and -D (a transmembrane anchor domain). We have identified a mutation within the membrane-intrinsic domain that alters the electron transfer properties of the iron-sulfur and flavin redox centers of the catalytic domain. Functional electron flow from the quinone analog 2,3-dimethyl-1,4-naphthoquinone or from the electron transport chain is impaired. However, the mutant enzyme can be reduced normally by single-electron donors such as the dye benzyl viologen. The mutant phenotype results from a single A----G transition changing His-82, within the second transmembrane alpha-helix of the FrdC anchor sequence, to an arginine. The mutation, physically located within the anchor domain, is manifested by altered catalytic properties, indicating that the intrinsic and extrinsic domains are conformationally connected. These results confirm the important role of the anchor subunits in functional electron transport and have implications for communication between intrinsic and extrinsic domains of membrane proteins. PMID- 3008150 TI - The active site structure of Na+/K+-transporting ATPase: location of the 5'-(p fluorosulfonyl)benzoyladenosine binding site and soluble peptides released by trypsin. AB - When the dog kidney Na+/K+-transporting ATPase (EC 3.6.1.37, formerly EC 3.6.1.3) was labeled with an ATP analogue, 5'-(p-fluorosulfonyl)benzoyladenosine (FSBA), there was a concomitant loss of ATPase activity. The presence of ATP protected the enzyme from both labeling and inactivation. The ATP-sensitive incorporation of FSBA is associated only with modification of the alpha subunit from which two labeled tryptic peptides were purified and sequenced. To establish any regions of the enzyme protruding from the membrane, the native Na+/K+-transporting ATPase from the electric ray, Torpedo californica, was treated with trypsin; and four peptides, which were released into the water phase, were purified and sequenced. A comparison of the peptide sequences with the deduced amino acid sequences of the DNA coding for the alpha subunit of T. californica and sheep kidney reveal the following. (i) FSBA-labeled peptides from the dog kidney enzyme are located in the central hydrophilic domain and show almost complete sequence homology with the same region in the alpha subunit from the electric ray and sheep kidney. Furthermore, the sequence homology of one of the two labeled peptides can be extended to the sarcoplasmic Ca2+-transporting ATPase and B subunit of Escherichia coli K+-transporting ATPase. (ii) Three trypsin-exposed peptides are found in the central hydrophilic domain, and one peptide is in the hydrophilic segment near the C terminus of the alpha subunit. (iii) The active center of Na+/K+-transporting ATPase is likely to be constructed from at least four different stretches in the primary sequence and, irrespective of the different specificity of cations, the various cation transport ATPases that form phosphorylated enzyme appear to have a common structure at the catalytic site for ATP hydrolysis. PMID- 3008151 TI - Purification of a factor from human placenta that stimulates capillary endothelial cell protease production, DNA synthesis, and migration. AB - A protein that stimulates the production of plasminogen activator and latent collagenase in cultured bovine capillary endothelial cells has been purified 10(6)-fold from term human placenta by using a combination of heparin affinity chromatography, ion-exchange chromatography, and gel chromatography. The purified molecule has a molecular weight of 18,700 as determined by NaDodSO4/PAGE under both reducing and nonreducing conditions. The purified molecule stimulates the production of plasminogen activator and latent collagenase in a dose-dependent manner between 0.1 and 10 ng of protein/ml. The purified protein also stimulates DNA synthesis and chemotaxis in capillary endothelial cells in the same concentration range. Thus, this molecule has all of the properties predicted for an angiogenic factor. PMID- 3008152 TI - Ethanol regulation of adenosine receptor-stimulated cAMP levels in a clonal neural cell line: an in vitro model of cellular tolerance to ethanol. AB - The acute and chronic neurologic effects of ethanol appear to be due to its interaction with neural cell membranes. Chronic exposure to ethanol induces changes in the membrane that lead to tolerance to the effects of ethanol. However, the actual membrane changes that account for tolerance to ethanol are not understood. We have developed a model cell culture system, using NG108-15 neuroblastoma-glioma hybrid cells, to study cellular tolerance to ethanol. We have found that adenosine receptor-stimulated cAMP levels increased markedly upon acute exposure to ethanol. However, the cells became tolerant to ethanol, since chronically treated cells required ethanol to maintain normal adenosine stimulated cAMP levels. Moreover, the cells appeared to be dependent on ethanol, as evidenced by reduced adenosine-stimulated cAMP levels in the absence of ethanol. Recovery occurred after ethanol was withdrawn. These cellular changes appear to parallel the clinical events of acute ethanol intoxication, tolerance, and dependence. PMID- 3008153 TI - Transforming activity of human papillomavirus type 16 DNA sequence in a cervical cancer. AB - A genomic DNA sample from cervical cancer tissue, containing human papillomavirus (HPV) type 16, was found to induce malignant transformation of NIH 3T3 cells when it was tested by transfection assays using the calcium phosphate coprecipitation technique. The primary and secondary transformants contained the HPV type 16 DNA sequences and human specific Alu family sequences. To the best of our knowledge, it has not been reported previously that HPV type 16 DNA sequences in total genomic DNA from a cervical cancer have transforming activity. PMID- 3008154 TI - Three novel genes of human T-lymphotropic virus type III: immune reactivity of their products with sera from acquired immune deficiency syndrome patients. AB - Human T-lymphotropic virus type III or lymphoadenopathy associated virus (HTLV III/LAV) is the cause of acquired immune deficiency syndrome (AIDS). In addition to the conventional retroviral genes involved in virus replication, namely, gag, pol, and env genes, DNA sequence analysis of HTLV-III genome predicted two additional open reading frames termed by us short open reading frame (sor) and 3' open reading frame (3' orf). Furthermore, functional analysis revealed another gene with transactivating function, termed tat. We have now structurally identified and functionally characterized these HTLV-III specific genes by way of cDNA cloning. DNA sequence analysis of the clones shows that the tat and 3' orf genes contain three exons and their transcription into functional mRNA involves two splicing events and that the sor gene contains at least two exons. In vitro transcription and translation of the cloned spliced sequences show that the sor, tat, and 3' orf genes code for polypeptides with apparent mobility of 24-25 kDa, 14-15 kDa, and 26-28 kDa, respectively. All three polypeptides are immune reactive and are immunogenic in the natural host. The results demonstrate that the three extra open reading frames of HTLV-III, two of which are unique to HTLV III, are in fact genes that function in vivo and further allow the identification of three new and previously unrecognized HTLV-III antigens with differential immunogenicity in individuals with acquired immune deficiency syndrome and related disorders. PMID- 3008155 TI - Synaptogenesis of cultured striatal neurons in serum-free medium: a morphological and biochemical study. AB - Striatal neurons were cultured from the fetal mouse brain and maintained in serum free medium for 14-21 days in vitro (DIV). Pretreatment of the culture dishes successively with a polycation followed by fetal calf serum resulted in rapid neuron attachment and neurite proliferation. After 9-10 DIV, electron microscope observations revealed the presence of vesicles in axon terminals forming mature synapses with axons and perikarya of adjacent neurons and in varicosities along extended axons. Synapsin I, a synaptic vesicle-specific protein, was present only in neuronal perikarya after 3 DIV, in perikarya and in varicosities along extended axons after 6 DIV, and in varicosities and contact points between axon terminals and adjacent axons or perikarya after 11-14 DIV. Neurotransmitter stimulated intracellular formation of cAMP decreased markedly during neuronal differentiation. Inositol phosphate formation in response to neurotransmitters, however, increased significantly throughout the period of striatal neuronal development. K+ (56 mM) depolarization resulted in a 2-fold increase in endogenous gamma-aminobutyric acid (GABA) release from striatal neurons, 50% of which was Ca2+-dependent, between 3 and 11 DIV. Between 11 and 14 DIV, subsequent to synapse formation (as revealed by electron microscope observations), GABA release evoked by 56 mM K+ increased up to 5-fold, 75% of which was Ca2+ dependent. It appears that the complete differentiation of striatal neurons in serum-free medium may provide a suitable model for the study of the physiological and regulatory mechanisms involved in nerve cell development. PMID- 3008158 TI - Effects of eicosapentaenoic acid (20:5 omega 3) on stress reactivity in rats. AB - This study examined the effects of eicosapentaenoic acid (EPA) on cardiovascular responses to isolation stress in male rats. Group-reared rats, on a fat-free diet, were given olive oil (OL), or EPA in OL (1.47 X 10(-7) mol/hr) via 8 week osmotic pumps, or a dummy pump (DUM), 2 weeks prior to a 4 week isolation period. Blood pressure (BP), heart rate, and body weight were monitored weekly and pressor responses to i.a. norepinephrine and angiotensin were assessed at the end of the study. BP increased during stress in all animals vs. pre-stress conditions, but was attenuated by EPA (p less than 0.001). Heart rate also increased during stress in all groups, but was greater in the EPA group (p less than 0.001). In contrast, body weight gain during stress was similar in DUM and EPA groups, but depressed by OL (p less than 0.001). Vascular response to norepinephrine was enhanced by EPA vs. DUM and OL, whereas the response to angiotensin was similar in EPA and DUM groups, but reduced by OL. These data suggest that EPA may attenuate cardiovascular responses to psychological stress. PMID- 3008156 TI - Binding of calmodulin to the neuronal cytoskeletal protein kinase type II cooperatively stimulates autophosphorylation. AB - The kinetics of autophosphorylation of the cytoskeletal form of the neuronal calmodulin-dependent protein kinase type II were studied as a function of calmodulin binding under the same conditions. Whereas calmodulin binding was noncooperative with respect to calmodulin concentration (Hill coefficient = 1), the activation of autophosphorylation and the phosphorylation of exogenous substrates showed marked positive cooperativity (Hill coefficient greater than or equal to 1.6). Reduction of the active calmodulin concentration by the addition of the calmodulin antagonist trifluoperazine confirmed the cooperative nature of enzyme activation, because autophosphorylation was more sensitive to the drug than was binding at high concentrations of calmodulin. At intracellular levels of calmodulin the binding and activation of autophosphorylation were cooperative functions of magnesium and calcium concentration. The calmodulin-dependent cooperative activation seems to be a unique feature of the cytoskeletal, but not the soluble, form of the protein kinase and may result from the supramolecular organization of the cytoskeletal enzyme. These observations suggest that interactions among the subunits of the oligomeric cytoskeletal calmodulin dependent protein kinase regulate enzyme activation, enhancing the sensitivity of the enzyme to small changes in the intracellular calcium levels that may be particularly relevant to signaling at the synapse. PMID- 3008157 TI - JC papovavirus large tumor (T)-antigen expression in brain tissue of acquired immune deficiency syndrome (AIDS) and non-AIDS patients with progressive multifocal leukoencephalopathy. AB - Progressive multifocal leukoencephalopathy (PML) is a JC papovavirus infection of the central nervous system in immunocompromised patients. It is well established that demyelination in PML is caused by JC virus infection of oligodendroglia, but whether the nonstructural regulatory protein, large tumor (T) antigen, is detectable in infected human tissue was not known. Using a modification of the peroxidase-antiperoxidase technique, we found T antigen expressed in the nuclei of cells in virus-infected sites in five cases of PML studied, including two with acquired immune deficiency syndrome (AIDS). PML occurs in AIDS at a much higher frequency than in other immunosuppressive disorders, and PML in AIDS may represent a more severe form of JC virus infection of the central nervous system. PMID- 3008159 TI - Atrial natriuretic peptide inhibits the stimulation of aldosterone secretion by ACTH in vitro and in vivo. AB - Previous studies have shown that atrial natriuretic peptide (ANP) inhibits the secretion of aldosterone by isolated adrenal glomerulosa cells stimulated by angiotensin II, ACTH and potassium in vitro and by angiotensin II in conscious unrestrained rats. In this study we investigated further the effects of synthetic ANP on the dose-response curve of aldosterone secretion stimulated by ACTH in vitro. ANP displaced the dose-response curve of aldosterone to ACTH to the right with a significant change in EC50. A similar effect of ANP was reproduced in vivo in conscious unrestrained rats. There was no significant effect of ANP on the corticosterone response to ACTH in vivo. ANP is a potent regulator of aldosterone secretion which may modulate the effects of ACTH on the adrenal in vitro and in vivo. PMID- 3008160 TI - HTLV-III and the etiology of AIDS. PMID- 3008161 TI - Clinical symptomatology of the acquired immunodeficiency syndrome (AIDS) and related disorders. PMID- 3008163 TI - Immunologic abnormalities in the acquired immunodeficiency syndrome. PMID- 3008162 TI - Acquired immunodeficiency syndrome in infants and children. PMID- 3008164 TI - T cell proliferation and ablation. A biologic spectrum of abnormalities induced by the human T cell lymphotropic virus group. PMID- 3008165 TI - Human retrovirus-associated lymphoproliferative disorders in homosexual men. PMID- 3008166 TI - The role of Epstein-Barr virus in lymphomas of homosexual males. PMID- 3008167 TI - The involvement of cytomegalovirus in acquired immune deficiency syndrome and Kaposi's sarcoma. PMID- 3008168 TI - FeLV-induced feline acquired immune deficiency syndrome. A model for human AIDS. PMID- 3008169 TI - The epidemiology of the acquired immunodeficiency syndrome. PMID- 3008170 TI - Antiviral activity towards VSV and Mengo virus of a chemically stabilized 2-5A analog upon microinjection into HeLa cells. PMID- 3008171 TI - The interaction of recombinant HuIFN-gamma 1 and recombinant HuIFN-alpha 2 on the 2-5A system in two distinct cell types. PMID- 3008172 TI - Persistent elevation of 2-5A synthetase and prognosis in the AIDS-related complex (ARC). PMID- 3008173 TI - 2'5' Oligoadenylate synthetase assay in human diseases. Its clinical value in viral liver diseases. PMID- 3008174 TI - 2-5A analogs as mechanistic probes of the 2-5A system: stereoconfiguration of phosphorothioate analogs of 2-5A markedly influences metabolic stability. AB - The stereoconfiguration of the diester bond of phosphorothioate analogs of 2-5A strongly influenced lability to enzymatic hydrolysis by cellular and serum phosphodiesterases. Bonds containing the Sp configuration were extremely resistant to hydrolysis compared to the corresponding Rp configuration linkages. The rate of hydrolysis of the diester bond was influenced by chain length of the adenylate oligomer, with a stability ranking of dimer greater than trimer greater than tetramer; as well as by the stereo-configuration of the diester bond adjacent to the one undergoing hydrolysis. The anti-proliferative and anti-viral activities of these various phosphorothioate 2-5A core analogs were assessed. The most stable analogs possessed the greatest biological activities (at 25-50 microM), which were not readily attributable to 2-5A degradation products or endonuclease activation. A 5'-triphosphate analog of 2-5A containing a phosphorothioate substituent in the gamma-position was obtained in good yield by enzymatic synthesis from ATP-gamma S. This gamma-thio 2-5A analog showed full biological activity. PMID- 3008175 TI - 2'5' Oligoadenylate analogues:synthesis, biological activities and intracellular delivery. PMID- 3008176 TI - Pathology of germ cell tumors of testes. AB - We have presented a brief discussion of WHO International Histological Classification of Testicular Germ Cell Tumors. This classification divides the tumors into those of one histologic type and those of more than one type. Seven basic histological categories are recognized: seminoma, spermatocytic seminoma, embryonal carcinoma, yolk sac tumor, polyembryoma, choriocarcinoma and teratoma. Comparison of the WHO classification with the Pugh-Cameron classification reveals that except for seminoma and spermatocytic seminoma comparability between the 2 classifications is impossible. The relationship of various cell types to each other has been discussed. The origin of each cell type from malignant transformation of germ cell has been emphasized. The WHO classification has been demonstrated to correlate well with clinical tumor markers. The Pugh-Cameron classification can not be correlated with markers. More importantly, by specifying the individual components that are present in a tumor, the WHO classification provides exact information as to the possible structure of metastasis so that the urologist is in a much better position to properly manage the patient with a metastatic testicular tumor. PMID- 3008178 TI - Trauma and testis cancer. PMID- 3008177 TI - Interest of the dosage of the H.C.G. in the spermatical blood at the moment of the castration for a testicular tumor (about 28 cases 1978-1984). PMID- 3008180 TI - Current randomized trials on adjuvant chemotherapy in stages II A and II B. PMID- 3008179 TI - Diagnostic and therapeutic significance of retroperitoneal lymph node dissection in low stage and localized disease: point of view. PMID- 3008181 TI - Surgical treatment for thoracic metastases from malignant germ cell tumors of the testes. PMID- 3008182 TI - A phase I-II trial of intensive chemotherapy (CT) with autologous bone-marrow transplantation (ABMT) in metastatic malignant germ cell tumors of the testis (NSGCTT). PMID- 3008184 TI - Nonseminomatous germ cell tumours. PMID- 3008183 TI - Treatment of non-seminomatous testicular germ cell tumors in Denmark since 1979. PMID- 3008185 TI - Non-seminomatous germ cell testis tumors--how do you manage clinical state IIA? PMID- 3008186 TI - Stage III non-seminomatous germ cell tumours of the testis--patients treated at the Centre Leon Berard. AB - The study of the survival of a series of patients with stage III non-seminomatous germ cell tumours of the testis enables us to confirm two points: the use of cis platinum has multiplied the survival rate for stage III patients by 3, the concept of stage III includes various metastatic states with different prognoses. The use of a sub-classification, like that proposed by Samuels, allows for a better definition of the natural history of these lesions. PMID- 3008187 TI - Extragonadal germ cell tumours. PMID- 3008188 TI - Bilateral testicular germ cell tumors. AB - Patients with testicular tumors have an increased risk of a second tumor in the opposite testicle. The surgeon should ensure careful follow-up, possibly with testicular biopsies if the opposite testicle is maldescended or atrophied. In addition, the patient should examine the testicle carefully and regularly. Hormonal substitution is mandatory. PMID- 3008189 TI - Testicular cancer in a renal transplant patient. PMID- 3008190 TI - Germinal cell tumors of the testis (GCTT) in renal transplanted patients (RTP): report of two cases. PMID- 3008191 TI - Anatomo-surgical aspects after chemotherapy for metastatic germ-cell tumors. PMID- 3008192 TI - Molecular characterization of somatic gene mutations arising in vivo in humans. PMID- 3008193 TI - In vivo mutant frequency of technicians professionally exposed to ionizing radiation. PMID- 3008194 TI - Long-term culture of human granulocytes and granulocyte progenitor cells. PMID- 3008195 TI - Monoclonal antibodies: convergence of technology and application. PMID- 3008196 TI - [Synthesis of carbonyl-bisamino acid derivatives as potential ACE inhibitors]. PMID- 3008197 TI - Micro- and submicro determination of antimony(III) in antibilharzial compounds by oxidation with N-bromosuccinimide. PMID- 3008198 TI - Effect of CGS-8216 on high-dose flurazepam convulsive threshold. AB - Flurazepam, a clinically proven and widely accepted sedative hypnotic, has been shown by a number of investigators to produce convulsions at toxic doses. In the present study, CGS-8216, a benzodiazepine receptor antagonist, reduced the dose of flurazepam required to produce convulsions. This suggests that the convulsant action of FLZ is exerted at a site other than the benzodiazepine receptor. PMID- 3008199 TI - Catecholamine excretion and beta-adrenergic responsiveness in estrogen-treated rats. AB - Chronic treatment with an estrogenic agent is known to reduce the responsiveness to beta-adrenergic stimulation in rats. To assess the possibility that endogenously produced catecholamines may play a role in this phenomenon, female rats were treated either with implanted Silastic tubes containing estradiol benzoate (26 micrograms/kg/day) or with empty tubes. After 12 weeks of treatment, the dipsogenic, colonic temperature and tail skin temperature responses to acute administration of the beta-adrenoceptor agonist, isoproterenol, were blunted compared to controls. Each of these responses was negatively correlated with serum estradiol concentration measured by radioimmunoassay. Urinary norepinephrine output was elevated significantly in treated rats during a 24-hour basal period. During a 2-hour exposure to air at 4 degrees C, urinary outputs of norepinephrine, epinephrine, and dopamine were elevated significantly above those of controls. There were significant positive correlations between plasma estradiol concentration and the outputs of each of the above catecholamines during the 24-hour basal period and during cold exposure, with norepinephrine being the most strongly correlated. Dipsogenic and metabolic (tail skin and colonic temperature) responses to administration of isoproterenol were negatively correlated with catecholamine excretion during both the basal 24-hour period and during cold exposure. These results suggest that reduced beta-adrenergic responsiveness in estrogen-treated rats may have resulted from down-regulation of beta-adrenoceptors associated with increased secretion of catecholamines induced by chronic estrogen treatment. PMID- 3008200 TI - Effects of the benzodiazepine receptor ligands midazolam, Ro 15-1788, and Ro 5 4864, alone and in combinations, on platelet serotonin uptake. AB - The benzodiazepine (BZ) agonist midazolam, the BZ antagonist Ro 15-1788, and the peripheral BZ binding site ligand Ro 5-4864 have been assessed, separately and in combinations, as to their effect on the active uptake of serotonin (5HT) in human blood platelets in vitro, in an artificial, protein-free medium. Midazolam had a moderate noncompetitive (or mixed competitive/noncompetitive) inhibitory effect on the uptake. This effect was not influenced by Ro 15-1788, which in itself had only a very weak inhibitory effect. Ro 4-5864 showed in several independent experiments (but not always) a biphasic inhibitory effect, with a moderate but significant inhibition in the concentration range of about 10(-8) to 10(-6) M, and a stronger, noncompetitive inhibitory effect above 10(-6) M. When Ro 5-4864 was tested in the presence of a fixed concentration of midazolam (4 X 10(-6) M), the inhibitory effect of the former was markedly reduced (at higher concentrations), eliminated (at intermediate concentrations), or even reversed into a relative stimulatory effect (at low concentrations). Thus, low concentrations of Ro 5-4864 reduced the inhibitory effect of midazolam. A possible explanation of this interaction is that low concentrations of Ro 5-4864 have both an inhibitory and a stimulatory effect on platelet 5HT uptake (often seen to vary in relative strength between experiments), and that the stimulatory component is unmasked in the presence of a drug that already has an inhibitory effect, probably mediated by the same site.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008201 TI - Instrumentation and light dosimetry for intra-operative photodynamic therapy (PDT) of malignant brain tumours. AB - A technique is described for uniform light irradiation of the tumour cavity after radical subtotal malignant brain tumour resection in patients whose tumours have been photosensitized by intravenous injection of hematoporphyrin derivative. The cavity is maintained by an inflatable rubber 'balloon' filled with a light scattering liquid, within which is placed a single-strand optical fibre coupled to an argon/dye laser operating at 630 nm. Details of the construction of the applicator are given, together with measurements of the uniformity of irradiation and the light loss within the scattering medium. The device has been assessed in nine patients in a phase I trial. In two of these patients, the penetration depth of 630 nm light in brain tissue has also been measured in vivo using an optical fibre probe, indicating that the penetration depth of 630 nm light in human brain is greater than that found in vitro. PMID- 3008202 TI - Central and peripheral thermal control of effectors in homeothermic temperature regulation. PMID- 3008203 TI - Liver blood flow (LBF) estimated by Kupffer cells clearance in health and disease. AB - Liver blood flow (LBF) in normal state and in diseases suspected to impair liver perfusion has been estimated by an indirect method, based on the removal by Kupffer cells of the colloidal particles from the circulation. 150 microCl 198Au have been used and the results have been expressed through percentual decrease from the theoretical value. In chronic liver disease there is a progressive reduction of the Kupffer cells clearance and of the LBF corresponding to the severity of the liver impairment. There is a mild decrease in LBF in heart failure. In liver tumours and collagenoses, the values are like in normals. PMID- 3008204 TI - Effect of bicuculline on sexual activity in castrated male rats. AB - The GABA antagonist (+) bicuculline methiodide (30 ng/cannula) was injected in the medial preoptic-anterior hypothalamic area of castrated rats treated with subthreshold dosages of testosterone propionate (150 micrograms/kg/day). The treatment resulted in a facilitation of sexual activity suggesting a role of GABA as a neurotransmitter in neural processes determining sexual arousal. PMID- 3008205 TI - Restraint stress stimulation of prolactin and ACTH secretion: role of brain histamine. AB - The possible role of brain histamine in the release of prolactin, ACTH and corticosterone following acute restraint, was pharmacologically evaluated in adult male rats. Fifteen min of restraint caused marked increases in the plasma levels of these hormones. alpha-Fluoromethyl histidine (FH), a histidine decarboxylase inhibitor which depleted hypothalamic histamine, inhibited the enhancement of plasma prolactin levels. In contrast, plasma ACTH levels were not modified. FH treatment decreased plasma corticosterone concentrations in animals submitted to stress or in rest; this suggests a direct action of FH on the adrenal. Intraventricular (IVT) injection of ranitidine (H2 antagonist) blunted the prolactin response to restraint stress whereas its systemic administration had no effect. On the contrary, pyrilamine (H1 antagonist) given systemically decreased slightly, but significantly, the prolactin rise but when injected IVT it was ineffective. Pyrilamine was also unable to affect the ranitidine action. ACTH and corticosterone levels in plasma of restrained rats were not modified by the histamine antagonists. It is concluded that histamine is involved, mainly through central H2 receptors, in the enhancement of plasma prolactin levels produced by an acute stress. The failure of both antihistaminic compounds and a histamine depletor to alter the ACTH stimulation suggest that histamine has no participation in the hypophysio-corticoadrenal response to acute restraint. PMID- 3008206 TI - [Thermographic studies with fluid crystals in peripheral nerve damage]. AB - Common peripheral neurogenic lesion in various locations and of different origins were examined thermographically and the results were compared with those obtained by clinical and electromyographic methods. Lesions in the regions of the N. ulnaris and N. medianus can, except in the case of carpal tunnel syndrome, be identified without difficulty by thermography. The results obtained in the case of paresis of the radialis are non-specific, and thermographic examination of radicular lesions are also sometimes ambiguous. Damage in the plexus-brachialis and root region yield impressive results which, however, are not particularly useful. PMID- 3008207 TI - Human plasma melatonin is elevated during treatment with the monoamine oxidase inhibitors clorgyline and tranylcypromine but not deprenyl. AB - Melatonin was measured in plasma collected between 8:00 and 8:30 a.m. from 27 depressed patients studied before and after 21- to 24-day treatment with three monoamine oxidase (MAO) inhibitors. Baseline plasma melatonin concentrations determined by radioimmunoassay were 4.0 +/- SD 4.7 pg/ml. Tranylcypromine, a nonselective MAO inhibitor given in doses of 20-40 mg/day for 3 weeks, significantly elevated plasma melatonin to 10.6 +/- SD 2.0 pg/ml. Clorgyline, given in doses of 15-30 mg/day for 3 weeks, produced a significant, approximately three-fold increase in plasma melatonin (13.6 +/- SD 13.5 pg/ml). This clorgyline dose was selective for MAO type A inhibition, as MAO-B activity measured in platelets from the same blood samples was unaffected by clorgyline. In contrast, the selective MAO-B inhibitor deprenyl (10-30 mg/day for 3 weeks) led to a 96 +/- 4% inhibition of platelet MAO-B activity but no significant change in plasma melatonin (5.1 +/- SD 4.2 pg/ml). As both serotonin and norepinephrine are preferentially metabolized by MAO-A rather than MAO-B, an increased availability of serotonin (the precursor of melatonin) or enhanced noradrenergic function might mediate the melatonin changes observed to follow MAO-A but not MAO-B inhibition. PMID- 3008208 TI - Effect of ascorbic acid on pituitary weight during stress. PMID- 3008209 TI - The effects of compounds related to gamma-aminobutyrate and benzodiazepine receptors on behavioural responses to anxiogenic stimuli in the rat: extinction and successive discrimination. AB - In a first set of experiments rats were trained to run in a straight alley for food reward on a continuous reinforcement schedule and the running response was then extinguished. On the last 2 days of training and daily throughout extinction different groups of animals were injected IP with saline, 5 mg/kg chlordiazepoxide, 0.75 mg/kg picrotoxin, chlordiazepoxide + picrotoxin, chlordiazepoxide + 1.5 mg/kg bicuculline, 0.00125 or 0.25 mg/kg muscimol, 1 mg/kg baclofen, chlordiazepoxide + baclofen, or 0.00125 mg/kg muscimol + baclofen. Chlordiazepoxide increased resistance to extinction, a well-known anxiolytic effect. This effect was blocked by both picrotoxin and bicuculline. Picrotoxin on its own reduced resistance to extinction (an anxiogenic-like effect). Whether given alone or in combination with other drugs, muscimol and baclofen had no effect. In a second set of experiments rats were trained in a successive operant discrimination (signalled by a flashing or steady light) between components in which sucrose reward was available on a variable-interval schedule for barpressing and components in which no reward was given. Chlordiazepoxide at 10 mg/kg increased responding in both rewarded and nonrewarded components, but more in the latter than could be accounted for by change in the former. This effect is as expected with an anxiolytic drug. It was not altered by administration of bicuculline at 1.5 or 1.75 mg/kg; at 2 mg/kg bicuculline acted synergistically with chlordiazepoxide. Picrotoxin (1 and 1.5 mg/kg) also acted synergistically with chlordiazepoxide, enhancing the latter's rate-increasing effects, but only during rewarded components. Neither muscimol (0.00125 and 0.25 mg/kg) nor baclofen (0.01 mg/kg) affected response rates, whether given alone or in combination. However, baclofen in a dose of 1 mg/kg, provided it was given to rats also injected with muscimol (0.00125 or 0.25 mg/kg) at other times, significantly reduced responding during nonrewarded components (an apparently anxiogenic effect). The results of the two sets of experiments are discussed in relation to the hypothesis that anxiolytic drugs affect behaviour by increasing GABAergic inhibition. PMID- 3008210 TI - The peripheral benzodiazepine receptor ligand Ro 5-4864 induces supraspinal convulsions in rabbits. Reversal by the central benzodiazepine antagonist Ro 15 1788. AB - The peripheral BDZ receptor ligand Ro 5-4864 was administered to rabbits in doses ranging from 0.2 to 7 mg/kg IV. Changes in electrocortical activity appeared within 1 min after administration, characterized by trains of slow waves in the posterior sensorimotor and optic cortices (0.6-2 mg/kg) and by grand mal seizures (2-10 mg/kg). The low doses also induced alterations in the basic rhythms both of the hippocampus (reduced amplitude and spike-like waves) and of the nucleus ventralis of thalamus (trains of slow waves), not associated with observable behavioural changes. The paroxysmal EEG activity observed at higher doses of the drug was first recorded in the cortical areas and then spread to the subcortical structures. No change in electrical activity could be observed in the spinal cord. The paroxysmal activity was associated with tonic-clonic convulsions and scialorrea. The EEG and behavioural manifestations were inhibited by administration of Ro 15-1788. This drug at doses of 0.6 and 6 mg/kg antagonized the effects of Ro 5-4864 at doses of 0.6-5 mg/kg and 6-7 mg/kg, respectively. This effect began 1-3 min after administration of the antagonist, and led to EEG synchronization. These data suggest that in rabbits the convulsant effect of Ro 5 4864 is due to interference of the drug at the GABA-BDZ-picrotoxin receptor oligomeric complex. Such an effect seems to be mediated at least in part by central BDZ receptors. PMID- 3008211 TI - [Use of computed tomography for quantifying Thorotrast and detecting Thorotrast induced liver tumors]. AB - 242 Thorotrast patients were examined by CT. The mean CT-density-values of the whole liver correlate well (0.72) with the measurements of the radioactivity by total-body-counting. Twenty-one of 24 primary liver tumors were correctly diagnosed by CT. The smallest tumor measured 3 cm in diameter. In 8 patients surgical treatment was possible. The sensitivity of CT for the detection of liver tumors, is higher than the sensitivity of echography. PMID- 3008212 TI - Detection of hepatic metastases: analysis of pulse sequence performance in MR imaging. AB - Forty-three patients with liver metastases were imaged using 14 different pulse sequences (average, 7.5 sequences per patient) to allow direct comparison of their performance. "T2-weighted" spin-echo (SE) images, "T1-weighted" inversion recovery (IR) images, and "T1-weighted" SE images were obtained using a wide range of timing parameters. Pulse sequence performance was quantitated by measuring liver signal-to-noise (S/N) ratios and cancer-liver signal difference to-noise (SD/N) ratios. Data were standardized to reflect a constant imaging time of 9 minutes for all pulse sequences. The SE 2,000/120 (TR [repetition time]/TE [echo time]) sequence resulted in the greatest SD/N ratio of the T2-weighted SE sequences but also yielded the low S/N ratios, poor anatomic resolution, and motion artifacts common to all T2-weighted SE images. IR sequence images were also sensitive to motion artifacts because of the use of a long TR (1,500 msec). Short TR/TE T1-weighted SE sequences (SE 260/18) had the greatest SD/N ratio (P less than .05), S/N ratio, and anatomic resolution. Furthermore, extensive signal averaging appears to be a powerful solution to all types of motion artifacts in the abdomen. PMID- 3008213 TI - Diagnosis of small hepatocellular carcinoma: correlation of MR imaging and tumor histologic studies. AB - Magnetic resonance (MR) images of the liver were used to study 43 patients with relatively small hepatocellular carcinomas (HCCs) and 36 with other hepatic mass lesions. In 27 HCC patients, histologic findings were available. All focal lesions detectable by CT without contrast media were delineated with greater contrast by MR imaging. The rate of detection depended on tumor size, being 97.5% for HCCs greater than 2 cm in the longest axis and 33.3% for those less than 2 cm. MR imaging demonstrated the ring sign characteristic of encapsulated HCC twice as frequently as CT scans. Inversion recovery (IR) images depicted the internal structure of the HCC better than T2-weighted spin-echo images. Lesions were classified into four patterns of intensity: low, iso, high, and mixed. The latter three were relatively characteristic of HCC and related closely with steatosis of cancer tissue. HCCs with fibrosis tended to have long T1 values; those with steatosis had short T1 values. T1 and T2 relaxation times were useful in the differential diagnosis. PMID- 3008215 TI - Structural requirements for chemotactic activity of leukotriene B4 (LTB4). AB - LTB4 (5s, 12R dihdroxy-6, 14-CIS-8, 10-trans-eicosatetraenoic acid) formed in activated neutrophils by lipoxygenation of arachidonic acid is an extremely potent chemotaxin. We examined structural requirements for chemotactic and aggregatory activity of the ligand using synthetic LTB4 and several of its isomers. Additionally we examined the potency of two analogs, nor- and homo-LTB4. Dose response curves for neutrophil chemotaxis to these compounds were obtained using a modified Boyden chamber. The mean distance cells moved into the filter was determined after 30 minutes. Peak chemotactic activity of LTB4 was at 10( 7)M. At higher concentrations, chemotactic activity was decreased. The shape of the dose response curve was similar to that of FMLP except that maximum chemotaxis to LTB4 was consistently greater than chemotaxis to FMLP. A mixture of the two epimers at c-5 and c-12 shifted the response curve to the right but did not lower maximum activity. Increasing or decreasing the chain by one carbon between the first hydroxyl group and the carboxyl group also shifted the response curve to the right without lowering maximal activity. Changing the 6 double bond from cis to trans has a greater effect. Activity was only detectable at high concentrations and maximum activity achieved was less than 50% that of LTB4. Thus the chain length between the carboxyl and C-5 hydroxyl groups, the c-5 and c-12 absolute stereochemistry and the stereochemistry of the delta6 double bond are all important structural features for chemotactic activity with delta6 stereochemistry apparently having the greatest contribution. The relative potencies of these compounds in inducing aggregation were comparable to their chemotactic potencies.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008214 TI - On the mechanism of acetylcholine release. PMID- 3008216 TI - Analysis of leukotriene B4 in human lung lavage by HPLC and mass spectrometry. AB - Although leukotrienes are believed to mediate symptoms of human lung disease, there is little direct evidence of their existence in the lung. This is due to the difficulty in obtaining lung samples, the small amounts of leukotrienes typically present in such samples and the problems associated with purifying and analyzing leukotrienes in complex biological samples. In this study, lung lavagates were collected and analyzed for leukotrienes. The methods in this analysis included solid phase extraction using a C-18 reverse phase cartridge followed by HPLC using a new photodiode array detector which provides full UV spectra of eluting compounds. Lung lavage fluid from a patient with chronic pulmonary disease contained a compound with a UV spectra of LTB4 which was found to elute with synthetic [3H]-LTB4. This compound was confirmed as LTB4 using gas chromatography/mass spectrometry in the negative ion-chemical ionization mode. The inclusion of oxygen-18 LTB4 as an internal standard allowed approximate quantitation of the amount of LTB4 present in this 5 ml lung lavagate as 40-50 ng. PMID- 3008217 TI - [Epidemiology and etiology of lymphocytic cerebrospinal meningitis in Szczecin in the second half of 1982]. PMID- 3008218 TI - [Viruses transmitted by blood-sucking arthropods. I. Bunyaviridae and Reoviridae]. PMID- 3008219 TI - [Viral conjunctivitis]. PMID- 3008220 TI - [Occurrence of enteroviruses and adenoviruses in the Warsaw district 1973-1982]. PMID- 3008221 TI - [Calcified gastric cancer: traditional study, CT and differential diagnosis]. PMID- 3008222 TI - The significance of mammographic calcifications in early breast cancer detection. AB - 3,126 medical reports on women sent to mammary biopsy following breast examination at the Florence Center for the Study and Prevention of Cancer in November 1978-July 1982 were reviewed in order to assess the diagnostic significance of mammographic microcalcifications. All mammographies were examined in order to assess the presence and morphological aspects of the microcalcifications on the bioptic site. Microcalcifications were classified on the basis of the following morphological criteria: spatial disposition (isolated, clustered, diffuse); total number; number per cm; morphological aspect (dot-like, stick-like or ramified); shape (regular or irregular); radiological density; association with mammographic opacity; maximum and average diameter. Microcalcifications were encountered in 19.7% of 157 breast cancer diagnosed in a mammographic screening programme conducted on the asymptomatic population in 19.5% of 953 breast cancers diagnosed in self-referring women (most with symptoms). Among cases where subsequent histological examination revealed a benign pathology, microcalcifications were more frequent in the cases deriving from the screening programme (14.5% of 198 cases) than among self-referred cases (4.5% of 1818 cases). The presence of microcalcifications is in itself a predictive sign of the presence of a carcinoma (positive predictive value = 66.2%) but this radiological sign is only present in 20% of breast cancers. Among the various parameters considered in assessing the diagnostic significance of microcalcifications, irregular shape was the most indicative of carcinoma with a predictive value of 80% and presence in 88% of carcinoma with microcalcifications. Other microcalcification parameters with a particular predictive significance are diffuse spatial disposition, total number (over 10) and number per cm (over 50), site contiguous with a mammographic opacity and a mean diameter of 0.6-0.9 mm. Unfortunately these latter parameters are only 24% of tumour cases with microcalcifications. The incidence of microcalcifications in cancer does not vary according to age, but is strongly correlated with the tumour stage. In particular microcalcifications are found in about 1/3 of in situ carcinomas. In invasive cancers, the presence of microcalcifications tends to increase with the diameter of the lesion. No correlation was found in breast cancers between the presence of microcalcifications, lymph node condition and histological type.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3008224 TI - [Thrombocytopenic purpura in cytomegalovirus infection]. PMID- 3008223 TI - [Hepatic resection in primary malignant tumors of the liver]. PMID- 3008225 TI - [Percutaneous sclerotherapy as primary treatment of testicular insufficiency in idiopathic varicocele]. AB - Among 1217 percutaneous retrograde phlebographies of the left renal/testicular vein 1069 were performed for primary treatment of left-sided idiopathic varicocele. In 989 of these patients (92.5%) an insufficient testicular (internal spermatic) vein with reflux to the pampiniform plexus could be shown phlebographically, in 802 cases percutaneous sclerotherapy could be performed. This amounts 75.0% of patients with varicocele or 81.1% of the cases with a phlebographically proven testicular vein insufficiency. In this group percutaneous sclerotherapy has proven to be a safe and effective treatment of idiopathic varicocele on an outpatient basis and has replaced surgical ligation as method of choice. However, with anatomical and technical obstacles to sclerotherapy, especially in patients with varicocele but without phlebographic evidence of an insufficient testicular vein, surgery remains an essential means of therapy. PMID- 3008226 TI - [Persistent varicocele following high ligation of the internal spermatic vein. Phlebography and vaso-occlusion with ethibloc]. AB - The importance of phlebography and vaso-occlusion using ethibloc in diagnosis and treatment of the persistent varicocele following ligation ("Bernardis' technique") is demonstrated. We performed 280 surgical ligations of the internal spermatic vein. In 30 patients persistence of the varicocele was found. In 9 patients following phlebography embolisation of a long segment including smaller collaterals was performed using ethibloc. The other 21 patients were treated surgically because of unusual draining of the collaterals into renal capsular veins, into the hemiazygos vein and into renal segmental veins. In all cases following embolisation complete resolution of the varicocele was observed within 3 months. PMID- 3008227 TI - [Pancreas divisum as a possible cause of misinterpretation in ERCP, computed tomography, sonography and MDP]. AB - In 488 patients endoscopic retrograde pancreatography (ERP) revealed a pancreas divisum in 21 (4.3%): in 17/21 patients we found a complete, in 4/21 an incomplete separation of the pancreatic ducts. The pancreas divisum is caused by a malfusion of the ductal system. On examination by ultrasound, computed tomography or hypotonic duodenography this variant can suggest an inflammation or tumour of the head of the pancreas. A definite diagnosis is possible by ERP only. Since the small ventral duct can be confused with an alteration caused by inflammation or by a tumour, to much of contrast medium can be injected. Pancreas divisum is often associated with a chronic pancreatitis which can be demonstrated via ERP of the dorsal duct through the accessory papilla. PMID- 3008229 TI - [Chronic obstructive respiration disorders imaged by inhalation scintigraphy. Differential diagnosis and early detection using radio-aerosols]. AB - Inhalation radioaerosol lung imaging was performed in 86 patients with chronic obstructive lung disease (COLD) and in 45 asymptomatic smokers with small airways dysfunction. Within certain limits analysis of abnormal aerosol scans helped distinguish emphysematous from brochitic and asthmatic types of disease. Patients with asthma often responded to bronchodilator treatment and abnormal aerosol images reverted to near normal patterns. Besides a slight aerosol distribution inhomogeneity in the lung periphery, patients with small airways dysfunction showed significant hypodeposition of inhaled particles distal to the ciliated airways. Compared with small airways dysfunction, COLD was associated with an even more decreased aerosol penetrance to the alveoli. PMID- 3008228 TI - [Magnetic resonance tomography of focal liver lesions in comparison with computed tomography and sonography]. AB - The results of magnetic resonance tomography (MRT) performed in 37 patients with predominantly solitary intrahepatic space-occupying masses are presented. Examinations were carried out with a superconducting magnet operating at a field strength of 0.35 Tesla. The majority of intrahepatic masses exhibited a well distinguishable signal amplification compared to normal hepatic parenchyma when T2 weighted sequences were used. For a further differentiation of the lesion, however, T1 and T2 weighted sequences have to be performed. In all cases diagnostic value of MRT is compared to that of sonography and computed tomography. The assessment of magnetic resonance tomograms of focal hepatic lesions is based on the sequence-depending on the signal behaviour of the mass - especially with regard to morphological criteria. The high soft-tissue contrast of magnetic resonance tomography compensates for the low spatial resolution, thus resulting in a sensitivity of focal lesions comparable to that of sonography and computed tomography. The good visualisation of vessels is an advantage. With the exception of haemangioma, however, MRT does not seem to improve diagnosis of the type of focal hepatic lesions. PMID- 3008230 TI - [Preoperative staging of bronchial carcinoma. Value of magnetic resonance in comparison with computed tomography]. AB - The results of MR and CT examinations for tumour staging were compared in 30 patients with bronchial carcinomas. They were also related to operability. The findings have been compared with the data in the previous literature. The results have shown both methods to be of equal value. The differentiation between tumour tissue and vascular structures is somewhat better shown by magnetic resonance, but peripheral and bone involvement is better shown on CT. These results require further examinations as well as operative and histological confirmation. PMID- 3008231 TI - [Necessity for ECG triggering in digital subtraction angiography. Construction of a simple trigger device]. AB - Movement unsharpness during digital subtraction angiography of the aortic arch and supra-aortic vessels can be avoided by means of ECG gating. The usual techniques resulted in exposures during the phase of least cardiac movement. The relatively long exposure time of about 60 to 300 msec caused marginal blurring during the exposure. An ECG-triggered device is described which produces short exposure times for the mask and filled frame during identical cardiac phases. An example with and without ECG gating shows marked improvement in detail rendering. The design of an ECG-triggered device with digitally adjustable delay and freedom from interference is described. PMID- 3008232 TI - [X-ray diagnosis of thoracic aorta ruptures]. AB - One hundred and twenty-two patients were examined in order to diagnose, or for follow-up of, acute or chronic traumatic rupture of the thoracic aorta; sixty-two had arterial angiography, thirty-three had intravenous digital subtraction angiography, thirty-one had computer tomograms and seventeen were examined by ultrasound. In addition, plain films were evaluated in forty-two cases. The value of these methods in diagnosing acute or chronic aneurysms is analysed and discussed. Arterial angiography was the best method for acute aneurysms, whereas in the sub-acute and chronic stages CT and DSA are indicated. PMID- 3008233 TI - [3-dimensional multi-echo magnetic resonance tomography: principle and clinical application to the brain]. AB - This paper presents the clinically most efficient MRI method at the present stage of development. The method combines a multi-echo sequence with TEs between 30 and 240 ms with an isotrope or anisotrope data acquisition mode known as 3DME-MRI. Both conventional head or whole body receiver coils can be used, or, for a better spatial resolution, surface coils. The theoretical background of this method is discussed and results of application in the region of the brain are presented. PMID- 3008234 TI - [Magnetic resonance tomography of intracranial tumors: use of contrast media versus T2-weighted tomograms]. AB - Nuclear magnetic resonance tomography was performed on 38 patients with intracranial tumours, before and after the administration of contrast, using a 0.35 T Magnetom. The MR examinations included various plain spin-echo sequences (SE 400/35, 800/35, 1600/35, 1600/70, 1600/105, 1600/120) as well as examinations after the iv-administration of gadolinium-DTPA (SE 800/35). On all occasions, the abnormalities were visible without contrast. Differentiation of tumour and its surroundings was possible in 19 out of 38 cases without contrast. Delineation of expansively growing tumours (eg. meningiomas) was possible in twelve out of 14 cases, but in only seven out of 24 cases with infiltrating tumours (eg. glioblastomas). On the other hand, clear differentiation between tumour and adjacent edema and normal brain, respectively, was possible in 35 out of 38 cases after Gadolinium-DTPA. PMID- 3008235 TI - [Results of nuclear spin tomography of facial bone tumors]. AB - We examined 21 patients with tumours of the mouth, one with tumour involvement of the facial skeleton and three with lymph node involvement in the upper neck, by nuclear tomography. Twenty patients with carcinomas of the mouth were also examined by CT. Nuclear tomography was superior to CT in showing the extent of the tumour; in ten patients the extent of the tumour could not be accurately determined, since there was no clear demarcation from normal tissue. Amongst our patients, nuclear tomography was also superior for showing lymph node metastases. Computed tomography is better at showing bone infiltration. Our results indicate that nuclear tomography is better than CT for showing the tumour and lymph nodes. If there is a suspicion of bone involvement, CT should be used in addition. PMID- 3008236 TI - Densities and sizes of the main masticatory muscles in computed tomography compared with clinical findings related to temporomandibular joint (TMJ) dysfunction. AB - Computed tomography (CT) has a good resolution capacity and is an excellent method for measuring tissue densities. The aim here was to compare the densities and sizes of the main masticatory muscles, the masseter, medial pterygoid and lateral pterygoid muscles, as obtained with CT, with clinical findings in patients with TMJ dysfunction (25) and controls (29). The densities of the masseter muscles and the medial pterygoid muscles showed no statistical difference between the patients and controls, while the lateral pterygoid muscles of the patients had densities that were only almost significantly higher than those of the controls. The masseter muscles on the patients who more often had signs of bruxism in their dentition were statistically significantly thicker than in the controls, but the widths of the medial pterygoid muscles gave no statistical difference between the groups. The results seem to indicate that in addition to the bone density changes associated with functional disorders of the masticatory system, there may be also density and size changes in the masticatory muscles which are detectable by CT. PMID- 3008237 TI - [Diagnosis of hamartoma of the tuber cinereum]. AB - Hamartomas of the tuber cinereum are tumour-like collections of normal tissue in abnormal location. They are benign lesions with slow or absent growth and without any tendency to neoplastic evolution. Due to their neurosecreting properties they usually cause precocious puberty. Further neuroendocrine disturbances, seizures, or psychoneurological symptoms may be associated in some cases. Cisternography and CT are the most conclusive radiologic procedures in all cases. The typical feature is a well circumscribed round-shaped isodense soft tissue mass without contrast enhancement. Usually the tumour is small, rarely exceeding 2 cm. in diameter. If CT diagnosis is not conclusive, examination in the coronal plane or CT cisternography are recommended. Although CT does not permit a histological diagnosis the clinical and radiological features together are sufficient to make a highly suggestive diagnosis. The treatment of choice is medical therapy. Surgery should be restricted to those tumours which damage surrounding structures by their size and cause other symptoms than precocious puberty. PMID- 3008238 TI - [Results of percutaneous transluminal dilatation of cerebral vascular stenoses]. AB - The present paper is a review of 37 successful catheter dilatations of supra aortic vascular stenoses. There were sixteen patients with a total of 21 stenoses of the internal carotid, vertebral artery or common carotid artery and sixteen patients with subclavian stenoses. Amongst the patients with stenoses of the cerebral vessels, there were ten with multiple lesions and six with a single stenosis. Three patients had successful dilatations of bilateral stenoses. The indications, technique, and complications of catheter dilatation of lesions of the cerebral vessels are described and discussed. PMID- 3008239 TI - [Results of real-time sonography and raster mammography of 200 breast cancers]. AB - The article reports on 200 patients with histologically verified carcinomas of the breast in whom both real time mammasonography and grid mammography were performed. The indications for additional use of real time sonography in the diagnosis of malignant diseases of the female breast were a palpable node or a suspicious finding in mammography. All patients were examined with a high resolution realtime scanner with a 7.5 MHz transducer. The diagnostic criteria of sound deep to the mass (type I-IV), the regularity of the conteur of the mass, and the presence or absence of internal echoes. The typical sonographic features of histologically different types of carcinoma of the female breast are demonstrated by means of case examples. Mammasonography offers a high degree of accuracy in preoperative differential diagnostic assessment of tumour status in malignant processes of the breast. The combined mammographic-sonographic evaluation is the method of choice in clinical evaluation of masses of the female breast. PMID- 3008240 TI - [Sonography of the irradiated breast]. AB - Sonography was carried out in 30 patients several weeks and up to ten years after conservative surgery followed by irradiation of the breast. The changes due to radiotherapy generally lead to a diffuse increase in echogenicity, as compared with the normal breast. The changes are most marked in the early phase after treatment. They include widening of the cutaneous reflex. Sometimes there is a double contour, indicating oedema of the skin, increased echo from the subcutaneous fat and masking of Cooper's ligaments. Sometimes the increased echoes may obscure detail in the deeper glandular components. Localise recurrences are easily distinguished from the highly echogenic tissues. Bearing in mind the limitations of the method, ultrasound can be used with advantage in addition to clinical examination and mammography for follow-up of breast tumours after radiotherapy as well as in primary diagnosis. PMID- 3008241 TI - The use of iohexol in meconium obstruction in the newborn. PMID- 3008242 TI - [Aneurysmal dilatation of the portal vein]. PMID- 3008243 TI - [Sonography and nuclear spin tomography of hourglass neurinomas]. PMID- 3008244 TI - [Computed tomographic diagnosis of a bone lipoma]. PMID- 3008245 TI - [The lunate-triquetral coalition]. PMID- 3008246 TI - [Bilateral tibial aplasia]. PMID- 3008247 TI - [Magnetic resonance tomography (MRT) and computed tomography (CT) of bronchial carcinoma. Comparison of the value of both study methods for preoperative staging]. AB - Fifty patients with histologically proven bronchial carcinomas were examined by CT and MRT for preoperative T-staging and N-staging. CT and MRT provided the same classification in 87% of the T2 tumours, 78% of T3, 71% of N0, 74% of N1 and 100% of N2 tumours. MRT had advantages for demonstrating tumours at the apices, for central tumours and for demonstrating hilar and some mediastinal lymph node enlargement. CT was better at demonstrating small pulmonary metastases, small basal pleural effusions and also in showing pulmonary structure and bone lesions. PMID- 3008248 TI - [Pleural infiltration by peripheral bronchial carcinoma. Is computed tomography reliable?]. AB - Pre-operative computed tomograms were obtained in 52 patients with histologically confirmed peripheral bronchial carcinomas and possible involvement of the pleura and thoracic wall was analysed. The results were compared with the operative and histological findings. Thickening of the pleura and of the sub-pleural fat line was observed in 24 cases. Amongst these patients there was only one case with histologically confirmed pleural infiltration. In 17 patients the sub-pleural fat line was obliterated, or could not be defined. Amongst these, 11 showed tumour extension to the pleura or soft tissues of the chest wall. It is concluded that the most suspicions sign of pleural tumour extension is absence of the sub pleural fat line. The more frequently observed pleural thickening cannot be considered as a reliable sign for pleural involvement. PMID- 3008250 TI - [Suitability and comparison of compensating filters for thoracic diagnosis]. AB - The effect of three types of filter on the quality of radiographs of the chest was compared. These filters improve visualization of mediastinal structures without significantly reducing the quality of the pulmonary image. In practice the Du Pont filter proved best; the quality of the central and peripheral portions of the lung image is equal to that of an ordinary radiograph and visualization of the mediastinum is improved. The Agfa-Gevaert filter showed no significant disadvantages compared with the ordinary techniques but the improvement in mediastinal visualization is not that marked. The 3 M filter yields poor images of the central portions of the lung and its type of construction prevents the retrocardiac structures from being pictured as well as with the other filters. PMID- 3008249 TI - [X-ray diagnosis of invasive thymoma]. AB - Between January 1981 and September 1985, invasive thymomas were diagnosed in 13 patients attending the municipal hospital at Koln-Merheim. All these patients were examined radiologically and the tumour removed at thoracotomy, and irradiated in our Radiotherapy Clinic. Invasive thymomas appear as space occupying lesions on chest radiographs taken in two planes, or on computed tomography. Infiltration by the thymoma in stages III and IV can be demonstrated by computed tomography. PMID- 3008252 TI - [Computed tomographic diagnosis and differential diagnosis of malignant paranasal sinus tumors]. AB - The CT appearances of malignant tumours of the paranasal sinuses are illustrated on the basis of 15 patients, and the differential diagnosis discussed. Malignant soft tissue tumours in the paranasal sinuses are characterised on CT by their non homogeneous structure; they may destroy the bony margins of the sinus and infiltrate neighbouring regions in certain preferred directions, and they may enhance following the administration of contrast. Precise definition of the malignant tumour by CT permits their exact staging, may help to determine therapy and is valuable for serial observation. It remains to be seen, however, whether the improved radiological diagnosis results in improved prognosis of malignant tumours of the paranasal sinuses. PMID- 3008251 TI - [3-dimensional display of computed tomographic studies of craniofacial anomalies]. AB - Craniofacial anomalies are conventionally investigated by cephalometry using ordinary radiographs and by computed tomography. Both methods have the major disadvantage of trying to demonstrate a complex three-dimensional structure, such as the skull, in two dimensions and they therefore cannot display a true spatial image. We present the principle underlying a three-dimensional display derived from computer tomographic studies and discuss the clinical application in the diagnosis of craniofacial anomalies. PMID- 3008253 TI - [Cranial computed tomography in maple syrup urine disease]. AB - Cranial computed tomography in the initial stage of the intermediate phenotype of maple syrup urine disease (MSUD) demonstrates diffuse, symmetric hypodensities in white and grey matter, which show a complete return to normal after early introduction of an adequate protein-restrictive diet. If diagnosis of this disease is missed or delayed, progressive global (end-stage) atrophy will take place over several years. A decrease in density values correlates well with the total cerebral lipid and water content (closely related to myelinisation), whereas progression and grade of atrophy show a relationship with the severity of pathological white and grey matter changes that are not demonstrable with computed tomography but can be proven histologically. Analysis of both morphological parameters corresponds well with clinical-neurological outcome and therapeutic success. PMID- 3008255 TI - [High-resolution 7.5-/10-MHz B-scan sonography for the localization of hyperparathyroid tumors]. AB - Apart from computed tomography, sonography is the most valuable clinical method for the pre-operative diagnosis of parathyroid tumours. Eighty-six patients were examined by high resolution ultrasound (7.5 and 10 MHz) and in 63 of these the sonographic findings could be compared with the results of surgery. Sensitivity of 67.5% was highest amongst 40 patients with primary hyperparathyroidism. Patients who had had previous operations on the neck showed a slightly lower sensitivity of 65%. In secondary and tertiary hyperparathyroidism, sensitivity was 54%. The main causes of error were small size of the adenoma, changes in the thyroid gland and previous surgery to the neck. The value of sonography for diagnosis and surgical planning is discussed. PMID- 3008254 TI - [Computed tomographic determination of thyroid iodine concentration in an endemic goiter area]. AB - We determined the CT density of the thyroid gland in 60 patients with normal thyroids and 176 patients with various thyroid disorders. The density was 75 +/- 6.2 H.U. in normal thyroids, which was markedly higher than in goiters (66 +/- 6.0 H.U.), while there was a considerable further decrease in patients with immunogenic hyperthyroidism, the density being 48.5 +/- 7.9 H.U. The thyroids of patients with nonimmunogenic hyperthyroidism differed from these by virtue of a significantly greater density, 79.8 +/- 12.5 H.U. The clinical importance of CT investigation of the thyroid is its ability to distinguish rapidly between immunogenic hyperthyroidism without ocular symptoms and the (mostly iodine induced) non-immunogenic form. There is a linear correlation between CT density and iodine concentration in the thyroid tissue; this was determined in surgical specimens from 17 patients. Iodine concentration in the thyroid, as well as the iodine content of the whole gland, can thus be calculated from the measured CT density at any time by estimating the volume of the gland with ultrasound and combining this value with the measured iodine concentration. The results correlate well with those found using the x-ray fluorescence method. PMID- 3008257 TI - [Computed tomographic and sonographic detection of renal and perirenal changes following shockwave lithotripsy]. AB - The finding of a major haemorrhage after lithotripsy induced us to look at 42 patients undergoing extracorporeal lithotripsy by CT and by sonography, both before and after the procedure, in order to determine the frequency of bleeding. In 37 patients, significant bleeding was found in the perirenal tract and in Gerota's capsule. There were six small sub-capsular haematomas which were not found by sonography and two extensive renal haematomas. PMID- 3008256 TI - [Imaging of prostatic cancer by 1.5 Tesla nuclear resonance tomography]. AB - Twenty-two patients with histologically confirmed carcinomas of the prostate were examined by nuclear magnetic resonance, using a 1.5 Tesla magnet (stage T1: one patient, T2: eight patients, T3: six patients, T4: seven patients). In 19 out of the 21 patients in stages T2 to T4, the tumour showed a specific signal intensity. In 12 cases, the tumour signal was more intense than from a normal prostate when using medium repetition and echo delay times; in 19 cases, multi echo sequences with increasing echo delay time (30 to 240 ms) and long repetition times (usually 1600 ms) showed less reduction in signal intensity than surrounding structures (except urine). Unlike computed tomography, 1.5 Tesla MR is able to demonstrate carcinomas confined to the prostate. Demonstration of infiltration is possible with MR with great accuracy because of the ability to obtain images in three planes and because of the accurate rendering of soft tissue detail. In particular, MR differentiates between stages T2 and T3 more clearly than does CT. The best demonstration of anatomical structures in the true pelvis is achieved with a repetition time of 800 ms and an echo delay time of 30 ms, the best demonstration of tumour with corresponding 1600 ms and 120 ms. The effect of catheters in the bladder, or previous transurethral resection on the MR images is discussed. PMID- 3008258 TI - The use of subtraction arthrography in total hip arthroplasties. AB - The results of plain film radiography and subtraction arthrography in 24 patients prior to revision surgery for a loosened total hip arthroplasty (T.H.A.) were compared with operative findings. Loosening of both the acetabular and the femoral components was evaluated. In plain film radiography the overall accuracy for evidence of loosening in 22 acetabular and 23 femoral components was 58%. The overall accuracy with arthrography was 93%. Three results were false-negatives; arthrography showing no evidence of loosening, while the arthroplasty was found to be loose on surgical evaluation. The results of this study are compared with findings reported in the literature. Arthrography was performed by a lateral puncture technique. There were no complications. The use of the puncture technique has not been described previously. The extent of contrast leakage into the interfaces is described and discussed. PMID- 3008259 TI - [Computed tomographic determination of the anteversion angle. Premises and possibilities]. AB - Thirty-two macerated femora were examined by CT in order to determine the degree of anteversion and to relate this to the position of the femur and to the various reference lines quoted in the literature. The accuracy of CT is the same as that of the Rippstein method, provided the following conditions are met: 1. Position of the femur with its long axis perpendicular to the image plane. 2. Demonstration of the maximal configuration of the femoral condyles to enable one to construct a tangent to the dorsal aspect of the condyle. 3. Demonstration of the head and neck by a plane which divides the neck into approximately equal portions and sections the femoral head. These conditions are more easily met, even in immobile patients, than the requirements for the Rippstein method. PMID- 3008260 TI - [Diagnosis and course of synovial sarcoma. Comparison of x-ray diagnosis and bone scintigraphy]. AB - The unfavourable prognosis of malignant synoviomas makes it essential to arrive at an early diagnosis. The early clinical symptoms and radiological appearances may be minimal and sometimes absent. It is therefore advisable to obtain bone scintigrams with perfusion and early images as well as the radiographs. The local extent of the tumour can be evaluated by ultrasound and CT. Angiography is required only if the relationship to the vessels cannot otherwise be ascertained, or if intra-arterial therapy is being considered. The final diagnosis depends on a biopsy. For subsequent observation, both scintigrams and radiographs should be obtained. It is essential to perform a three-phase scintigram. This improves the recognition of recurrences and of soft tissue or bone metastases. Most bone metastases are visible on scintigraphy, but a normal scintigram in the presence of an osteolytic lesion on a radiograph may be obtained. Pulmonary metastases can only be demonstrated radiologically. PMID- 3008262 TI - [Perforated ischemic colitis following vascular surgery]. PMID- 3008261 TI - [Nuclear resonance tomography of the breast--diagnosis, differential diagnosis, problems and solutions. I. Study procedures]. AB - The experience gained from performing nuclear resonance tomography on the breast, using a special surface coil, is described. The technical problems, causes of artefacts and the advantages of tomography in three planes are discussed. The procedure is slow. Images obtained by various repetition and echo delay times in several planes are illustrated. Problems in quantifying T1 and T2 images and methods for improving the technique are described in detail. During the course of 85 examinations on 72 patients, it has been possible to achieve technical improvements. PMID- 3008263 TI - [Computed tomography of pseudomyxoma peritonei]. PMID- 3008264 TI - [Unusual computed tomographic findings following shunting of hydrocephalus internus permagnus]. PMID- 3008265 TI - [Diagnosis of neurofibromatosis by nuclear resonance tomography]. PMID- 3008266 TI - CT in the diagnostic work-up of hypogenetic lung syndrome (HLS) in homozygotic twins. PMID- 3008267 TI - [Evaluation of 4 years of surveillance of enteroviruses in sewage. Correlation with human pathology]. AB - This survey is concerned with isolations of enteroviruses from sewage and stools of children admitted to pediatric wards of the Clermont-Ferrand hospital during 4 years (from january 1, 1980, to december 31, 1983). Some epidemics of different serotypes (Coxsackies B1, B4, B5, Echovirus 33) occurred, mainly between june and october. In some instances, virus isolation in sewage occurred a few weeks before epidemic of the same virus, giving some predictive value to pediatric pathology. This survey allowed us to adapt immunologic diagnosis of enteroviruses in children and limit the use of expensive reagents. PMID- 3008268 TI - [Acute leukemia secondary to small-cell bronchial cancer]. PMID- 3008269 TI - [The autonomous nervous system and pulmonary circulation. Stimulation of chemoreceptors and ventilation/perfusion ratios]. AB - The existence of neurogenic pulmonary vasomotrocity and of vasomotor reflexes elicited by stimulation of peripheral chemoreceptors has been demonstrated in different animal species. There are efferent noradrenergic and cholinergic nerve endings in the walls of musculo-pulmonary arteries. Stimulation of sympathetic efferents and exogenous noradrenaline cause vasoconstriction of pulmonary arteries when initial vasomotor tone is normal. There are also beta-adrenergic and cholinergic vasodilation pathways whose effects oppose the above mentioned vasoconstrictor ones, are blocked by the corresponding inhibitors, and can be demonstrated when initial vasomotor tone is high. The role of pulmonary vasomotor tone in the distribution of ventilation perfusion ratios is unknown. The stimulation of peripheral chemoreceptors causes a rise in pulmonary vascular resistance. Inversely, the stimulation of peripheral chemoreceptors by hypoxia prevents the local vasoconstrictor effect of localised alveolar hypoxia in sheep. There are thus theoretical reasons to think that the effect on regional pulmonary resistance of nervous stimulation may differ in cases of inhomogeneous lung disease, according to the local state of the pulmonary vessels. Consequently, it is not possible to anticipate the effect of stimulating chemoreceptors on the distribution of ventilation/perfusion ratios. PMID- 3008270 TI - [Liver transplant: clinical indications]. PMID- 3008271 TI - [Aspects of vocational reintegration following myocardial infarct in relation to disease severity--follow-up of patients with after-care treatment]. AB - Medical examination data were compared with psychosocial questionnaire findings in a retrospective study of 536 myocardial infarction patients. The average observation period was four years. On the basis of cardiac catheterization results, obtained during participation in post-clinical rehabilitation, four diagnostic groups were formed in order of disease severity. Return to work, early resumption of working following post-clinical rehabilitation, and the subjective perception of cardiac complaints did not show any clear-cut association with severity of the cardiac findings. The two conclusions drawn are that in addition to the somatic factors involved, psychosocial ones will have to be given greater emphasis in the rehabilitation process, and successful rehabilitation should not be determined exclusively on the criterion of vocational resettlement. PMID- 3008272 TI - Renal autoregulatory efficiency during angiotensin-converting enzyme inhibition in dogs on a low sodium diet. AB - Autoregulatory efficiency of renal blood flow (RBF) and glomerular filtration rate (GFR) was evaluated in 12 anesthetized dogs that had been maintained on low sodium diet during control conditions and following infusion of an angiotensin converting enzyme inhibitor (captopril). Converting enzyme inhibition (CEI) decreased systemic blood pressure by 15.5 +/- 3.5%, increased RBF by 36.3 +/- 6.5%, and increased GFR by 25.9 +/- 10.7%. In response to reductions in renal arterial pressure, RBF was efficiently autoregulated and did not change significantly until the 89- to 75-mm Hg range during the control period and the 74- to 54-mm Hg range during CEI. Overall GFR autoregulatory efficiency was generally well maintained during CEI; however, evaluation of the coupled autoregulatory efficiency of RBF and GFR indicated that during angiotensin blockade, there was a greater incidence of a dissociation between RBF and GFR autoregulatory efficiency. Six of the 12 dogs showed reduced GFR autoregulatory efficiency at renal arterial pressures where RBF was still well maintained. Thus, while the data indicate that blockade of the renin-angiotensin system does not abolish the basic capability of the kidney to autoregulate either RBF or GFR efficiently, more subtle influences on the coupling of RBF and GFR autoregulatory efficiency were observed at the lower level of the autoregulatory range. PMID- 3008274 TI - Comparative antidotal effects of diethyldithiocarbamate, dimercaptosuccinate, and diethylenetriaminepentaacetate against cadmium-induced testicular toxicity in mice. AB - The compounds diethyldithiocarbamate (DDTC, Dithiocarb), 2,3-dimercaptosuccinic acid (DMSA), and diethylenetriaminepentaacetic acid (DTPA) were evaluated for effectiveness in protecting mouse testes following administration of an LD100 dose of CdCl2 X 2.5 H2O (Cd). Toxicologic responses were assessed by light microscopic techniques. DTPA gave the most effective testicular protection of the three compounds when given within 30 minutes prior to Cd, but only DDTC was effective when given 30 minutes or longer after Cd. Degenerative changes were minimal when DDTC was given as late as three hours after Cd, and were mild following a four-hour interval. However, the changes were marked, but less severe than in mice receiving Cd only, when DDTC was administered five hours after Cd. PMID- 3008273 TI - Maternal plasma concentrations of catecholamines and cyclic nucleotides during labor and following delivery. AB - In plasma obtained from seven mothers before, during, and after normal labor and delivery, catecholamine and cyclic nucleotide concentrations were investigated. Dopamine concentration showed a significant elevation on admission to hospital in labor and there were marked increases in norepinephrine and epinephrine concentrations during labor at 10 cm cervical dilatation and immediately after delivery, respectively. No significant change in dopa, cAMP, or cGMP was found during the experimental period. However, since the van Beaumont quotient (J.Appl. Physiol. 34, 102-106) for cAMP did not follow the reduction in plasma volume, the concentration appeared to rise during labor. Positive correlations were observed between epinephrine on one hand, and heart rate and systolic blood pressure on the other, as well as between norepinephrine and cAMP, respectively, during labor. The diminution of epinephrine on the fourth day postpartum might reflect a reduction of emotional stress concomitant with labor. cAMP was found to be quickly cleared from the bloodstream within 2 hours after delivery. These results suggest that plasma concentrations of epinephrine and cAMP, especially epinephrine, are indices of maternal psychological and physiological stress during labor and following delivery. PMID- 3008275 TI - A single radial hemolysis technique for the measurement of influenza virus antibody in cattle serum. AB - A single radial hemolysis technique (SRH) was used to measure swine influenza virus antibody in calf serum. Heating at 56 C for 60 minutes was necessary to prevent non-specific hemolysis. A significant association was found between the mean diameter of the hemolysis zone obtained with the SRH test and the geometric mean hemagglutination inhibition (HI) titer in sera of 5 calves inoculated with the virus after treatment with periodate (r = 0.92, P 0.01) and receptor destroying enzyme (r = 0.94, P 0.01). Although the SRH technique is not affected by the presence of non-specific inhibitors it is no more sensitive than the HI test. Some disadvantages of the technique included several biological variables difficult to control. PMID- 3008276 TI - In vitro interaction of organic copper (II) compounds with soluble glutathione S transferases from rat liver. AB - The in vitro interaction of organic copper (II) compounds with rat liver glutathione S-transferases (GST) was studied using reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. The inhibition of the GST activity was dose dependent. The different GST isoenzymes were inhibited to different degrees. Kinetic studies never revealed competitive inhibition. Titration of remaining GSH in appropriate incubation mixtures with organic copper (II) compounds revealed no GST catalyzed conjugation of these compounds with GSH. These experiments showed a spontaneous conjugation of the copper (II) compounds with GSH, explaining the parabolic inhibition observed in the kinetic studies with GSH as the variable substrate. Both organic and inorganic copper (II) are spontaneously conjugated with GSH, but interact with GST by direct binding to these proteins. This binding could have a protective function against copper (II). PMID- 3008277 TI - 3 alpha, 3 beta and 17 beta-hydroxysteroid dehydrogenase activities in the cytoplasmic and microsomal fractions of human liver. AB - Optimized assay systems for the determination of cytoplasmic and microsomal 3 alpha-, 3 beta-, and 17 beta-hydroxysteroid dehydrogenase (HSDH) activities in human liver have been developed. Using these methods the activities of cytoplasmic 3 alpha-, 17 beta-, and microsomal 3 alpha- and 3 beta-HSDH were measured in cell fractions prepared from liver biopsies from 17 patients who showed no signs of endocrinologic disturbances. The means and standard deviations (SD) of these four activities (nmol/min/mg protein) were as follows: cytoplasmic 3 alpha-HSDH, 1.03 +/- 0.34; cytoplasmic 17 beta-HSDH, 0.365 +/- 0.129; microsomal 3 alpha-HSDH, 2.07 +/- 1.16; microsomal 3 beta-HSDH, 8.95 +/- 5.20. No dependence on sex or age was detected, but this may have been due to the heterogeneity of the sample material rather than a genuine absence of such correlations. PMID- 3008278 TI - Alternating chemotherapy, prophylactic cranial irradiation and late local thoracic irradiation for small-cell lung cancer. AB - 28 consecutive patients with small-cell lung cancer (SCLC) aged 48-78 years (with exclusion of 4 patients over 80 years) were treated with combination chemotherapy in the schedule AAA-BBB-AAA-BBB, where A consisted of cyclophosphamide 1,000 mg/m2, adriamycin 50 mg/m2, and etoposide 100 mg/m2 X 3, and B of cyclophosphamide 1,000 mg/m2, methotrexate 50 mg/m2 and vincristine 1 mg/m2 X 2. Patients in complete remission after 3 courses received prophylactic cranial irradiation, and thoracic irradiation was given after completion of chemotherapy. There were 3 toxic deaths. Of the patients with limited disease, 71% reached complete remission and 24% partial remission; in extensive disease these percentages were 36 and 45%, respectively. Three patients survived more than 2 years, 1 with recurrence of squamous cell carcinoma after 125 weeks. It is concluded that this scheme of combination chemotherapy is as effective as those reported earlier in remission rate and survival in SCLC. However, the addition of thoracic irradiation failed to prevent local relapse in 83% of the patients. PMID- 3008279 TI - Hemofiltration in septic ARDS. The artificial kidney as an artificial endocrine lung. AB - Twenty-four patients with high microvascular permeability pulmonary edema were initially treated by means of conventional supportive therapy for 1-12 days. Continued deterioration was treated by predilutional hemofiltration and induced a dramatic improvement in 22/24 patients. Survival was 92%. Sieving coefficients for autacoids and middle molecular weight vasoactive peptides involved in the development of high microvascular permeability pulmonary edema were higher than 0.88 indicating that clearing from blood of these peptides during one pass through the hemofilter is similar to that obtained during one pass through the pulmonary normal microvasculature. Hemofiltration seems to be a significant breakthrough in the treatment of ARDS secondary to severe sepsis. PMID- 3008280 TI - Phenytoin's value as a neuroplegic drug in brain preservation. PMID- 3008281 TI - Cardiopulmonary effect of halothane concentration during pulmonary air embolism in dogs. AB - The purpose of this study was to determine the effect of different halothane concentrations on the hemodynamic consequences of pulmonary air embolism in dogs. The animals were studied in two groups. Group A (N = 11) at a halothane concentration of 0.5%, and Group B (N = 8) at a halothane concentration of 1.5%. Air was injected into a central vein in a bolus of 0.5 ml/kg, 1 ml/kg, and 2 ml/kg. With the lower halothane concentration, sudden loading to the right heart and pulmonary artery occurred with air embolism. Recovery in this group was faster in comparison to the higher concentration of halothane. Survival was not effected with air embolism to 2 ml/kg. PMID- 3008282 TI - Immunohistochemical localization of glutathione peroxidase in the brain of the rat. AB - The distribution of glutathione peroxidase (GSH-PO) in the brain of rats was studied by using the peroxidase-anti-peroxidase (PAP) immunohistochemical method employing highly specific antibodies raised in rabbits to GSH-PO. The purity of the antigen and the specificity of the antibodies were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transblot assay, respectively. The specificity of the immunohistochemical staining was confirmed by using a pre-immune serum, eliminating the first to third antibodies in the PAP method and absorption test. Under pentobarbital anesthesia, male Wistar rats were perfused with a mixture of fixatives (paraformaldehyde, glutaraldehyde and picric acid) via the left cardiac ventricle. The brain was immediately removed en masse and fixed in a similar solution but lacking glutaraldehyde, and then thin-sectioned with a cryostat. The sections were stained by the PAP immunohistochemical method. The immunoreactive products were observed chiefly in the nuclei of some nerve cells in the following areas: layers II, IV, VI in the cerebral cortex; CA2, CA1 and CA4, CA3 (listed in descending degree) in the hippocampus; granular and molecular layers in the cerebellar cortex. Few immunoreactive products were observed in the pyramidal cells in layers III, V of the cerebral cortex, and not at all in the Purkinje cells of the cerebellum. The nerve cells where lacking GSH-PO well coincided with the cells vulnerable to hypoxia. During or following hypoxia, lipid peroxides will be generated in the tissues and do harm when they exceed some amount.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008283 TI - [Inorganic inhalants as one of the etiologic agents in idiopathic interstitial pneumonia]. PMID- 3008284 TI - Intrapulmonary and extrapulmonary shunt in ducks. AB - We measured shunt in seven anesthetized, mechanically ventilated Pekin ducks by the multiple inert gas elimination technique (MIGET) and by the oxygen method during 100% O2 breathing (O2 shunt). MIGET shunt measures only intrapulmonary shunt but O2 shunt measures intra- and extrapulmonary shunt (e.g. bronchial drainage). O2 shunt was calculated from oxygen contents of blood estimated from measured PO2 and standard O2 equilibrium curves at appropriate pH and hematocrit measured in 4 other ducks. In normal lungs MIGET shunt was 1.3 +/- 0.4 (mean +/- SEM) percent of cardiac output and O2 shunt was 6.3 +/- 1.3%. O2 shunt exceeded MIGET shunt by 5.4 +/- 1.4% which we attribute to extrapulmonary shunts. These include part of the bronchial circulation, thebesian veins and vertebral venous pulmonary circulation connections. Both MIGET and O2 shunts increased when water was introduced into lungs. Overall, the relationship between % MIGET shunt and % O2 shunt was described by the equation: O2 shunt = 0.86 X MIGET + 5.67 (r = 0.96, n = 24). PMID- 3008285 TI - Effect of phrenic nerve stimulation on neural transmission and diaphragmatic force generation in the dog. AB - The effect of supramaximal bilateral phrenic nerve stimulation on neural transmission and diaphragmatic force generation was studied in anesthetized dogs. Different combinations of duty cycle and stimulation frequency were examined during intermittent stimulation (pacing) for 15 min. The effect of different stimulation frequencies was also examined during continuous stimulation for 60 sec. Force declined more with increasing stimulation frequency and duty cycle with intermittent stimulation, and with increasing stimulation frequency with continuous stimulation. Neural transmission decreased with increasing stimulation frequencies. The changes were always greater with continuous than with intermittent stimulation. We found no unique relationship between changes in neural transmission and changes in force generation, suggesting that if neural transmission failure is causally responsible for fatigue, it does so by a very complex mechanism. PMID- 3008286 TI - Respiratory responses to chemical activation of left ventricular receptors. AB - Respiratory responses to chemical activation of receptors in the left ventricle were measured in anesthetized cats. Application of capsaicin (10 micrograms) or bradykinin (500 ng) to the serosal surface of the left ventricle caused increases in phrenic nerve activity and in arterial pressure. These responses persisted after cervical vagotomy and after bilateral transection of the carotid sinus nerves. Bilateral stellate ganglionectomy abolished the respiratory responses to capsaicin and bradykinin. In addition, stellate ganglionectomy prevented the arterial pressure response to bradykinin; however, a slight increase in arterial pressure occurred with application of capsaicin. It is concluded that activation of sympathetic afferents originating in the left ventricle causes an increase in respiratory output. PMID- 3008287 TI - [Increasing the detection of primary hyperparathyroidism. Critical analysis of the usefulness of the laboratory]. PMID- 3008288 TI - [Reactions of amino acids with ninhydrin and their applications]. PMID- 3008290 TI - Effectiveness of transcutaneous electrical neural stimulation in the treatment of pain. Recommendations for use in the treatment of sports injuries. PMID- 3008289 TI - [Drug-induced hepatic lesions]. PMID- 3008291 TI - [The climacteric syndrome and its management]. PMID- 3008292 TI - Autoimmunity to cell membrane receptors. PMID- 3008293 TI - The role of calcium in cell injury and repair: a hypothesis. PMID- 3008294 TI - [Meningoradiculitis caused by a spirochete (Borrelia burgdorferi) after arthropod bite]. AB - Eight cases of meningoradiculitis (Garin-Bujadoux-Bannwarth's syndrome) are presented; the first case followed an "unidentified insect" bite and erythema chronicum migrans, whereas the second and third cases were not preceded by any documented insect bite or erythema; they occurred during summer in 1984 and 1985 and were characterized by cranial or radicular neuritis, lymphocytic meningitis, positive serology by immunofluorescence against Borrelia Burgdorferi and a good response to Penicillin (20 000 000 U during 14 days I.V.). Five other cases were observed in the same area as the first and second cases (Walloon Brabant) during the preceding summers; in two, serological proof of Borrelia Burgdorferi infection was obtained retrospectively. Lyme disease and Garin-Bujadoux-Bannwarth syndrome are both tick-born spirochetosis, due to two slightly different subtypes of Borrelia Burgdorferi. Their early neurological manifestations differ mainly by focalised pain on the bitten region in Garin-Bujadoux-Bannwarth's syndromes. This could be due to direct aggression of the peripheral nerve in Garin-Bujadoux Bannwarth syndrome. PMID- 3008295 TI - [Hallervorden-Spatz disease with Lewy bodies]. AB - At the age of 6 years a patient developed disorders of character, intellectual deterioration, tremor, falls and epileptic seizures. This was followed by extrapyramidal and pyramidal disorders with a fatal outcome at age 21. There was no family history. Histopathology showed evidence of Hallervorden-Spatz disease, remarkable by the diffusion of spheroids into the central nervous system gray matter and by the presence of innumerable Lewy bodies in the substantia nigra and locus coeruleus. Similar findings have been reported in only 3 other cases of typical Hallervorden-Spatz disease. They suggest a preferential affection of monoaminergic neurons. PMID- 3008296 TI - [Characterization of the binding of 125I-TSH to its receptor in low-uptake thyroid hyperplasias]. PMID- 3008297 TI - [Management of mammillary discharge. Apropos of 38 cases]. AB - The authors offer a diagnostic approach in cases of mammillary discharge and present a study of 38 cases of spontaneous discharge. The diagnosis is based on clinical findings combined with supplementary examinations, beginning with a cytological examination of the discharges, either bilateral or, in most cases, unilateral. The supplementary investigations are standard: mammography, galactography, with more and more frequent recourse to ultrasonography. If warranted on sufficient grounds, biopsy by sectorectomy is carried out to provide a histological diagnosis. The observed results show epithelial vegetation with (38%) and without (37.5%) atypical cells, the histological interpretation of which is: benign papilloma: 50 percent, diffuse papillomatosis: 17 percent, galactophoric carcinoma: 1 case and galactophoric ectasias: 22 percent. This study is compared to the results of Mouriquand et al [10] on induced discharges, where the frequency of papilloma and papillomatosis is slightly inferior by about 5 percent. PMID- 3008298 TI - [But what do I have?]. PMID- 3008299 TI - [Hypoglycemic insular tumor or insulinoma]. PMID- 3008300 TI - [AIDS: duties of the Transfusion Service and Hematology Clinic]. PMID- 3008301 TI - [Laboratory results in the evaluation of anti HTLV-III in subjects at risk]. PMID- 3008303 TI - Understanding AIDS. PMID- 3008302 TI - [Acute polyradiculoneuropathy of Guillain-Barre. Critical review of potential etiopathogeneses]. AB - The authors go over the etiopathogenetic hypothesis suggested, in the last years, a propos of the Guillain-Barre syndrome. They point out that about two/thirds of cases are preceded by infectious symptoms of the upper respiratory and alimentary tracts, with prevailing viral etiology. The nervous lesion, which characterize the histology of the illness, seems related to immunological change. In particular one must emphasize the possibility that the Guillain-Barre syndrome is an autoimmune disorder of delayed hypersensivity, the laboratoristics model of which is constituted from experimental allergic neuritis. PMID- 3008305 TI - Vasotocin release from the pineal gland of newborn mammals: the involvement of GABA mechanisms. AB - The arginine vasotocin (AVT) levels were bioassayed in the pineal extracts from 20-day-old kittens and from 10-day-old rat pups and in the cerebrospinal (CSF) samples taken from 20-day-old kittens. The animals received daily (between the postnatal day 2 and the day of sacrifice) the selective GABA receptor antagonist picrotoxin (5 X 10(-7) mg) or the specific GABA agonist valproic acid (10(-5) mg). Picrotoxin was found to produce a decrease of the pineal AVT levels in newborn kittens and rats, and an increase of the AVT amount of the CSF samples from kittens. Valproic acid had opposite effects, producing an increase of pineal AVT in both species and a decrease of AVT levels in the kittens CSF samples below the sensitivity limit of the bioassay method. The result suggests that not only in adult, but also in newborn mammals, the inhibitory feed-back loop between the brain and the pineal gland is GABA-mediated and that the GABA-ergic mechanisms are involved in regulating the release of AVT from the pineal gland. PMID- 3008306 TI - [Classification of angiodysplasias and vascular tumors]. AB - Numerous vascular dysplasiae belong to the group of genodysplasiae. Most arteriovenous dysplasiae (cirsoid, racemosum aneurysms,...) are the substratum of various regional "angiomatosis". Stenotic or ectatic arterial dysplasiae can be associated with genodysplasiae. In addition, lymphatic dysplasiae (congenital elephantiasis, etc...) also exist. Among benign tumours, angiomas are sometimes hardly distinguishable from dysplasiae. Some tumors with intermediate malignancy have an uncertain prognosis: chemodectoma, hemangiopericytoma. As for malignant vascular tumours, they fall into 3 varieties: angiosarcoma, Kaposi's sarcoma and sarcoma of vascular walls. PMID- 3008307 TI - Outbreak of poliomyelitis in Finland in 1984. Description of nine cases with persisting paralysis. AB - Finland has been free of poliomyelitis since 1964 following a high coverage regular immunization programme using the Salk-type trivalent inactivated poliovirus vaccine. In late 1984 an outbreak of poliomyelitis with widespread circulation of poliovirus type 3 throughout the country was found. A thorough surveillance revealed 9 sporadic cases with acute persisting paralysis, all shown to be caused by poliovirus type 3. The preceding 20-year period free from poliomyelitis contributed to some diagnostic problems. PMID- 3008304 TI - [GABAergic theory of epilepsy]. PMID- 3008309 TI - Differences in EBV-specific antibody patterns at onset of infectious mononucleosis. AB - The EBV-specific antibody patterns of infectious mononucleosis (IM) patients were analyzed in relation to the onset of symptoms and clinical parameters during the acute phase of the disease. The antibody patterns varied considerably on admission. Three groups of patients were identified: one had not yet attained peak antibody titers, the second was at the peak and the third had passed the peak pattern. Patients with a "peak" current pattern had significantly higher lymphocyte counts, ASAT, ALAT, serum IgG and serum IgA concentrations than patients of the third group. Unexpectedly, there was no difference between the groups with regard to duration of sore throat and general malaise before admission. It thus seems that the lymphocyte proliferation during IM closely parallels the course of the EBV-specific antibody responses, whereas the onset of IM does not closely correlate to a specific stage of the antibody pattern. PMID- 3008308 TI - Cross-sectional seroepidemiologic study of the prevalence of cytomegalovirus and herpes simplex virus infection in a Canadian Inuit (Eskimo) community. AB - The prevalence of antibody to cytomegalovirus (CMV) and herpes simplex virus (HSV) was determined, using enzyme-linked immunosorbent assay techniques, in a cross-sectional serologic survey of an isolated northern Canadian Inuit (Eskimo) community. The population studied included 155 Inuit and 11 Caucasian residents. By 6 years of age, 80% of the Inuit population were seropositive for CMV and 100% for herpes simplex virus. While only 7/63 Inuit greater than 20 years were seronegative for CMV, 5/11 Caucasian residents were seronegative (p = 0.01). For the Inuit population, no association between seropositivity for CMV and seropositivity for hepatitis A or hepatitis B was observed. This prevalence survey shows a serologic profile for infection with CMV and HSV in this northern Inuit community with an early age of acquisition and high prevalence of infection characteristic of socioeconomically deprived populations throughout the world, and is distinct from that observed in many other North American populations. PMID- 3008310 TI - Alterations in the human oropharyngeal microflora related to therapy with aztreonam, moxalactam and ampicillin plus sulbactam. AB - 33 patients undergoing colorectal surgery were treated prophylactically with aztreonam (19 patients), moxalactam (10 patients) and ampicillin plus sulbactam (4 patients). The effect on the normal oropharyngeal microflora and new colonization of oropharynx were studied during and after the treatment. Aztreonam had minor effect on the normal flora but induced colonization with staphylococci. Moxalactam suppressed gram-negative aerobic and anaerobic bacteria and promoted colonization with Candida albicans. Ampicillin plus sulbactam had marked effect on the normal flora and suppressed aerobic as well as anaerobic bacteria. New colonization with enteric gram-negative rods or fungi was observed in all patients. PMID- 3008311 TI - Oral administration of antibiotics and intestinal flora associated endotoxin in mice. AB - The contribution of aerobic and anaerobic gram-negative intestinal bacteria to the release of endotoxin in the intestinal tract was investigated during oral administration of various nonabsorbable antimicrobial drugs to C3H/Law mice. The intestinal endotoxin release was studied by determination of the endotoxin concentration in faecal supernatants with the Limulus amebocyte lysate assay. Selective elimination of aerobic gram-negative bacteria by oral treatment with polymyxin, aztreonam or temocillin resulted in a reduction of the endotoxin concentration of faecal supernatants to 10% of the untreated control. Further decrease of the endotoxin concentration to 1% was achieved by total decontamination of the intestinal tract by oral cephalothin/neomycin treatment. Endotoxin determination with the Limulus amebocyte lysate assay appeared to be unaffected by the antibiotics present in the faecal supernatants after oral treatment. On basis of these experiments, it is concluded that in mice 90% of the faeces derived endotoxin can be ascribed to release of endotoxin by intestinal aerobic gram-negative bacteria. PMID- 3008313 TI - Incidence of cytomegalovirus-infection after renal transplantation and first experiences with prophylactic hyperimmunoglobulin. AB - Incidence and clinical symptomatology of CMV-infection was investigated in 83 patients, who received cadaveric renal transplants in 1982 and 1983. CMV-antibody status was determined using an ELISA-technique, 43 of the 83 patients (52%) were seronegative, and 40 (48%) were seropositive for CMV before transplantation. Seroconversion (i.e. primary CMV-infection) or an increase in titre (i.e. reactivation or reinfection) was found in 18 cases (42% and 45%, respectively) in both groups. 89% of all infections occurred within the first 3 months. Clinical symptomatology was much more severe in the group with primary CMV-infection; all cases with atypical pneumonia (n = 8) and both fatal cases belonged to this group. Preformed CMV-antibodies thus appeared to prevent severe syndromes associated with CMV-infection. Therefore a randomized controlled study was started in 1984 in order to investigate the efficacy of an i.v. applicable CMV hyperimmunoglobulin passively administered prior to the transplant procedure. The passive immunization was not capable of preventing CMV-infection in every case: 2 (late) seroconversions did occur, but in both cases subclinical infections were diagnosed, whereas in the control group CMV-infections were regularly associated with typical clinical complications (prolonged fever, liver damage, leucopenia etc.). Thus prophylactic CMV-hyperimmunoglobulin seems to be capable of preventing the occurrence of severe CMV-syndromes post transplantation. The study is being continued. PMID- 3008312 TI - Cytomegalovirus (CMV) infections in renal transplant recipients. Preliminary results of prophylaxis by an intramuscular human hyperimmune CMV IgG. AB - Infectious diseases are the most serious complications in immunosuppressed patients and the major cause of death in renal transplant recipients (23, 44). Besides bacterial, fungal and protozoan infections, viruses of the human herpes group (herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus) are known to be of major importance (21, 37). The most common and clinically relevant virus of this group is the cytomegalovirus (CMV). PMID- 3008314 TI - Infectious diseases under prophylactic ALG treatment and their prevention in a prospectively randomized trial. PMID- 3008315 TI - The incidence of cytomegalo- and herpes simplex virus infections in renal allograft recipients treated with high dose recombinant leucocyte interferon: a controlled study. PMID- 3008316 TI - Clinical experiences with phosphonoformate (foscarnet) treatment of viral diseases following renal transplantation. AB - The effect of the antiviral drug phosphonoformate (Foscarnet) was studied in 32 patients following renal transplantation. Viral diagnosis was verified in 29 of 33 episodes of suspected clinical virosis. The clinical effect of Foscarnet was good in 12 of 14 primary cytomegalovirus infections. In 5 of 7 varicella-zoster virus, in 4 of 6 secondary cytomegalovirus and in 2 herpes simplex virus (type 1; type 2) infections the effect of the treatment was clinically judged as good. The beneficial effect could in these patients be overestimated as the natural courses of the viral infections were unknown. No clinical side effects of Foscarnet were found. Clinically unimportant changes in s-calcium were noted in 6 patients. The dosage of Foscarnet was increased during the study. At present an initial bolus dose followed by 14 days of parenteral infusions can be recommended. PMID- 3008317 TI - Cytomegalo and herpes simplex virus infections in renal transplant recipients. AB - In a prospective study data were obtained from renal transplant recipients with respect to the incidence and source of infections by CMV and HSV. The incidence of infections with CMV and HSV appeared to be related to the immune suppression regimen used. Especially the high incidence of reinfection with CMV (88.0%) and HSV (81.0) at high prednisolone dosage compared to the lower incidence (CMV 54.5%, HSV 44.1%) at low prednisolone dosage was apparent. Even in very moderate immune suppressed patients infections occurred in at least 40% of them. These infections are known to be potentially harmful to the recipient or to the graft. CMV can be transmitted by the graft in both seronegative and seropositive recipients as demonstrated by restriction enzyme analysis. Therefore CMV related disease has to be considered in all recipients with grafts from seropositive donors. For CMV infection the diagnostic value of serology (CF test) appeared high, while for the diagnosis of HSV infection virus-isolation was of more value. PMID- 3008318 TI - Cyclosporin A inhibits Epstein-Barr virus induced gamma-interferon production in vitro. PMID- 3008319 TI - IgM immune complexes, lymphocytotoxins, and rheumatoid factors in renal transplant recipients with CMV disease. AB - In studying 127 consecutive adult recipients of cadaver renal transplants, we found that the 23 patients who developed CMV disease produced IgM immune complexes as measured by a polyethyleneglycol precipitation (PEG) assay which coincided with symptoms of their illness. In addition to anti-CMV antibodies, PEG precipitated apparently non-specific antibodies such as lymphocytotoxins and rheumatoid factor (RF). The lymphocytotoxins were IgM antibodies that were not directed against HLA antigens and lysed granulocytes as well as lymphocytes but not platelets at 22 degrees C. Lymphocytotoxin production was correlated with HLA DR 3 and 7 and with graft dysfunction during the CMV disease. The RF also were predominantly IgM antibodies that were detectable for only 3-8 weeks. The production of RF coincided with the initial rise in IgG anti-CMV antibody activity and some reacted with the Fab fragments of IgG raising the possibility that they could modulate the cellular or humoral immune response to CMV. Patients with RF tended to have severe CMV infections with pneumonia and graft dysfunction. PMID- 3008320 TI - Acute myelogenous leukemia of unfavourable prognosis treated with retinoic acid, vitamin D3, alpha-interferon and low doses of cytosine arabinoside. PMID- 3008321 TI - Effect of diclofenac sodium, tolfenamic acid and indomethacin on the production of superoxide induced by N-formyl-methionyl-leucyl-phenylalanine in normal human polymorphonuclear leukocytes. AB - The effect of three non-steroidal anti-inflammatory drugs, diclofenac, indomethacin and tolfenamic acid, on the production of superoxide (O2-), by normal human polymorphonuclear leukocytes (PMNL) was studied, in vitro. The cells were activated with N-formyl-methionyl-leucyl-phenylalanine (FMLP) and O2- production was measured as superoxide dismutase inhibitable cytochrome c reduction. Cell viability was checked with assays of liberated LDH. Concentrations of the drugs considerably higher than those of therapeutic plasma were required to inhibit O2- production. The drug concentrations producing a 50% inhibition (IC50) of total O2- production were: diclofenac 2.7 X 10(-4) M, indomethacin 4.0 X 10(-4) M and tolfenamic acid 4.2 X 10(-4) M. At drug concentrations causing a significant suppression of O2- generation, diclofenac showed a slight and tolfenamic acid a marked inhibition of [3H]FMLP binding to its cellular receptor; indomethacin has earlier been shown to inhibit FMLP binding slightly. No dismutating activity of the drugs could be demonstrated. It is concluded that the inhibition of O2- production is due to a combined effect on FMLP binding and on cellular O2- metabolism. Because of the high drug concentrations required to inhibit O2- production, this phenomenon is obviously of little significance for the anti-inflammatory effect obtained with therapeutic doses of the drugs studied. PMID- 3008322 TI - Effects of allyl chloride on occupationally exposed subjects. AB - Effects of allyl chloride on occupationally exposed subjects were studied in two factories manufacturing sodium allyl sulfonate. Twenty-six subjects in factory A and 27 workers in factory B were exposed to allyl chloride at levels of 2.6-6 650 mg/m3 for 2.5 months--6 years and 0.2-25.13 mg/m3 for 1-4.5 years, respectively. Most subjects of factory A had weakness, paresthesia, and numbness in extremities with sensory impairment in the glove-stocking distribution, as well as reduced ankle reflexes. Electroneuromyography showed neurogenic abnormalities in 10 of the 19 subjects examined, the prevalence of neuropathy therefore being 52.6%. Similar symptoms of workers in factory B were clinically much milder, and there were few abnormal neurological signs--yet electromyographic findings indicating mild neuropathy were found in 13 of the 27 subjects. No significant abnormalities of other organs were noted. Possible etiologic factors other than exposure to allyl chloride were excluded. All the evidence obtained indicates that chronic exposure to allyl chloride mainly causes toxic polyneuropathy. The neurotoxicity of allyl chloride has also been confirmed by experimental neuropathological studies. PMID- 3008324 TI - Enalapril following captopril-induced nephrotic syndrome. AB - The development of proteinuria, and more rarely nephrotic syndrome, has been seen with the use of the first orally active converting-enzyme inhibitor captopril. Both of these side effects appear to occur more frequently when the drug is used at higher dose, particularly in the presence of renal impairment. We have used enalapril, a new orally active converting-enzyme inhibitor in the treatment of a patient with drug-resistant hypertension and renal impairment who previously developed nephrotic syndrome with captopril. Recurrence of the nephrotic syndrome was not seen in this patient during a period of 20 months on enalapril. On the contrary, urinary protein excretion over the same period was reduced to around 1 g in 24 hours. Our experience would suggest that enalapril may usefully be substituted for captopril in the treatment of hypertensive patients in whom the latter has caused proteinuria or nephrotic syndrome. PMID- 3008323 TI - [Transfusion-associated LAV/HTLV-III infections in Switzerland. Report of 2 cases]. AB - Two patients developed signs of LAV/HTLV-III infection one and two years respectively after a blood transfusion. The leading symptom in one patient was generalized lymphadenopathy, while the other patient presented with Candida stomatitis. In both cases, blood transfusions administered in 1983 were found to be the only possible source of infection; one blood donor of each patient was anti-LAV/HTLV-III positive. Family members and sexual partners of the two were negative for antibodies against LAV/HTLV-III. Our findings document the first two cases of LAV/HTLV-III associated disease most probably related to blood transfusions in Switzerland. PMID- 3008325 TI - HTLV-III legend correction. PMID- 3008326 TI - Research on mental illness and addictive disorders. PMID- 3008327 TI - A new twist in AIDS patent fight. PMID- 3008328 TI - Occurrence of peptide and clavine ergot alkaloids in tall fescue grass. AB - Evidence is presented that ergot alkaloids are ubiquitous in tall fescue pastures infected with the clavicipitaceous fungal endophyte Sphacelia typhina (or Acremonium coenophialum). Ergopeptide alkaloids, predominantly ergovaline, constituted 10 to 50 percent of the total ergot alkaloid concentration, which was as high as 14 milligrams per kilogram in sheaths and 1.5 milligrams per kilogram in blades. Ergot alkaloid concentrations were substantially increased by application of large amounts (10 millimoles per liter) of potassium nitrate or ammonium chloride to infected plants in the greenhouse. The results indicate that ergot alkaloids are probably responsible for the toxicity to cattle of this common pasture and lawn grass and that ergotism-like toxicoses may be caused by clavicipitaceous fungi other than Claviceps. PMID- 3008329 TI - Receptor-associated resistance to growth hormone-releasing factor in dwarf "little" mice. AB - Anterior pituitaries from the dwarf mouse strain "little" did not release growth hormone or accumulate adenosine 3',5'-monophosphate (cyclic AMP) in response to human and rat growth hormone-releasing factor (GRF). Dibutyryl cyclic AMP, as well as the adenylate cyclase stimulators forskolin and cholera toxin, markedly stimulated growth hormone (GH) release. The basis of the GH deficiency in the little mouse may therefore be a defect in an early stage of GRF-stimulated GH release related either to receptor binding or to the function of the hormone receptor complex. PMID- 3008330 TI - Calcium antagonist receptors in cardiomyopathic hamster: selective increases in heart, muscle, brain. AB - The Syrian cardiomyopathic hamster has a hereditary disease in which a progressive myocardial necrosis mimics human forms of cardiac hypertrophy. Lesions are associated with calcium overload and can be prevented with the calcium antagonist verapamil. Numbers of receptor binding sites for calcium antagonists in heart, brain, skeletal muscle, and smooth muscle were markedly increased in cardiomyopathic hamsters. The uptake of calcium-45 into brain synaptosomes was also increased in cardiomyopathic hamsters. The increase in calcium antagonist receptors and related voltage-sensitive calcium channels may be involved in the pathogenesis of this cardiomyopathy. PMID- 3008332 TI - Crystal structure analysis of deamino-oxytocin: conformational flexibility and receptor binding. AB - Two crystal structures of deamino-oxytocin have been determined at better than 1.1A resolution from isomorphous replacement and anomalous scattering x-ray measurements. In each of two crystal forms there are two closely related conformers with disulfide bridges of different chirality, which may be important in receptor recognition and activation. PMID- 3008331 TI - Gene transfer and molecular cloning of the human NGF receptor. AB - Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor. PMID- 3008333 TI - Protection of cattle against foot-and-mouth disease by a synthetic peptide. AB - A chemically synthesized peptide consisting essentially of two separate regions (residues 141 to 158 and 200 to 213) of a virus coat protein (VP1) from the O1 Kaufbeuren strain of foot-and-mouth disease virus was prepared free of any carrier protein. It elicited high levels of neutralizing antibody and protected cattle against intradermolingual challenge by inoculation with infectious virus. Comparative evaluation of this peptide with a single-site peptide (residues 141 to 158) in guinea pigs suggests the importance of the VP1 carboxyl terminal residues in enhancing the protective response. PMID- 3008334 TI - A mutation in the R body-coding sequence destroys expression of the killer trait in P. tetraurelia. AB - This report describes a mutant strain of Caedibacter taeniospiralis 169 that does not produce refractile (R) bodies or kill sensitive paramecia, but still renders its host resistant to killing by wild-type strains of Caedibacter taeniospiralis. The mutation is due to insertion of a 7.5-kilobase, transposon-like element into the R body-coding region of the plasmid pKAP169. The results provide strong evidence that R body synthesis is required for expression of the killer trait. PMID- 3008335 TI - Human immunodeficiency viruses. PMID- 3008336 TI - AIDS virus has new name--perhaps. PMID- 3008337 TI - The structure, function, and expression of interleukin-2 receptors on normal and malignant lymphocytes. AB - Antigen or mitogen-induced activation of resting T cells induces the synthesis of interleukin-2 (IL-2) as well as the expression of specific cell surface receptors for this lymphokine. Failure of the production of either IL-2 or its receptor results in a failure of the T-cell immune response. The receptor is composed of a 33,000-dalton (251-amino acid) peptide precursor that is post-translationally glycosylated into the mature 55,000-dalton form. In contrast to resting T cells, human T-cell lymphotrophic virus I (HTLV-I)-associated adult T-cell leukemia cells constitutively express large numbers of IL-2 receptors. Because IL-2 receptors are present on the malignant T cells but not on normal resting cells, clinical trials have been initiated in which patients with adult T-cell leukemia are treated with a monoclonal antibody that binds to the IL-2 receptor. PMID- 3008338 TI - Activation of the AIDS retrovirus promoter by the cellular transcription factor, Sp1. AB - The nature and position of transcriptional control elements responsible for the expression of genes encoded by the retrovirus associated with acquired immune deficiency syndrome (AIDS) have not been precisely defined. In this study it is shown that the mammalian Sp1 transcription factor binds to promoter sequences within the AIDS retrovirus long terminal repeat (LTR) and activates RNA synthesis five- to eightfold in reconstituted reactions in vitro. Experiments in which regions of DNA were protected from added reagents by specifically bound proteins (footprinting) indicated that the upstream promoter region of the AIDS virus LTR lies between -45 and -77 (relative to the RNA start site, +1) and contains three tandem, closely spaced SP1 binding sites of variable affinity. Base-substitution mutations targeted to one or all three Sp1 binding sites were found both to eliminate the binding of Sp1 and to cause up to a tenfold reduction in transcriptional efficiency in vitro. These findings suggest that one important component of the AIDS virus transcriptional control region interacts with a cellular transcription factor, Sp1, and that this factor must function in conjunction with transcriptional elements located downstream of the RNA cap site to mediate the response of the LTR to viral trans-activation. PMID- 3008340 TI - Measurement of brain deoxyglucose metabolism by NMR. PMID- 3008341 TI - Pitfalls of gastrointestinal bleeding studies with 99mTc-labeled RBCs. PMID- 3008339 TI - Identification and characterization of the protein encoded by the human N-myc oncogene. AB - The human N-myc gene is related to the c-myc proto-oncogene, and has been shown to have transforming potential in vitro. Many studies have reported amplification of N-myc in human neuroblastoma and retinoblastoma cell lines. In primary tumors, amplification of the gene was found to correlate directly with behavior of the tumor. Specific restriction fragments of a partial complementary DNA clone of N myc from LA-N-5 human neuroblastoma cells were placed into a bacterial expression vector for the purpose of producing antigens representative of the N-myc protein. Rabbits immunized with these antigens produced antisera that recognized a protein of 62-64 kilodaltons in neuroblastoma cells. By several criteria, this protein appears to be part of the same proto-oncogene family as the c-myc protein. Moreover, the antisera to fragments of this protein were capable of histochemically identifying malignant cells in clinical specimens. PMID- 3008342 TI - Applications of molecular biology to perinatal medicine. PMID- 3008343 TI - Magnetic resonance imaging of lesions of synovial origin. AB - Three patients with histologically differing lesions of synovial origin and two with synovial cysts, one of which was a dissecting popliteal cyst, were examined by magnetic resonance imaging (MR) and computerized tomography (CT). The three histologically proven synovial lesions were synovial sarcoma, diffuse giant cell tumor of tendon sheath, and synovial chondromatosis. In two of the five patients MR provided better anatomic and morphologic appreciation than CT, while in the others they were of equal value. CT demonstrated calcification in two of the lesions while on MR calcification could be identified in only one patient where it outlined the mass. MR did not demonstrate calcification in the substance of the diffuse giant cell tumor of tendon sheath. Coronal, transverse, and sagittal images of magnetic resonance graphically demonstrated the extent of the soft tissue masses and their relationship to bone, vessels, and soft tissue structures. Synovial sarcoma had a shorter T1 than diffuse giant cell tumor of tendon sheath (these two lesions being of comparable size) and also had a uniformly longer T2. The dissecting popliteal cyst showed the most intense signals on the T1 weighted images, while the uncomplicated synovial cyst showed a long T1. On the T2 weighted images, each type of cyst showed a long T2. The variance and overlap of intensity of MR signals suggest limited specificity in predicting the histologic nature of the synovial lesion. PMID- 3008344 TI - Cost-effectiveness of the expanded programme on immunization in the Ivory Coast: a preliminary assessment. AB - A preliminary calculation was made of the cost-effectiveness of the measles component of the Expanded Programme on Immunization (EPI) in the Ivory Coast. The calculation is based on existing data (program budgets, coverage surveys, counts of vaccinations provided and subjective estimates) and applies to the first three demonstration and training zones (Abidjan, Abengourou and Korhogo) with a combined population of 1.75 million people. The average annual cost of the measles program (assumed to be 75% of all EPI costs, including supplies, personnel and equipment) in these three zones was $527,000 at 1980 prices. Having achieved an average coverage rate of 61%, the cost per vaccine was moderately high, $12. Yet, vaccinees are a sufficiently small part of the population that the cost per capita is only $0.30. The program is estimated to prevent 38,000 cases of measles and 1100 deaths per year in these three zones. Thus, the cost per measles case averted is $14, and the cost per death averted is $479. This means that the measles component of the EPI Program is highly effective in preventing deaths for the sums expended compared to many alternative health programs in developing countries. PMID- 3008345 TI - [Synacthen retard]. PMID- 3008346 TI - [Sarcoma in the zone of irradiation after radiation therapy of cancer of the uterine cervix]. PMID- 3008347 TI - [Radiomodifiers in the radiation and combined therapy of patients with cancer of the rectum]. PMID- 3008348 TI - Transfer of antibiotic resistance genes between yeast and mammalian cells under conditions favoring cell fusion. AB - Antibiotic resistance to G418 has been transferred into Chinese hamster cell lines via a plasmid vector. The same plasmid, which also contained the Leu2 gene, has been used to transform Leu2- yeast (strain MC16) to leucine prototrophy. Subsequent fusion between transformed yeast and untransformed hamster cells demonstrated that plasmid DNA could be transferred and its genes expressed within the mammalian cell genome. The fusion of transformed hamster cells with untransformed MC16 yeast cells demonstrated that DNA integrated within the mammalian cell genome could be transferred to correct the Leu2 deficiency and also confer G418 resistance on some yeast colonies. PMID- 3008349 TI - An established avian fibroblast cell line without mitochondrial DNA. AB - An established avian fibroblast cell line (LSCC-H32) has been found to be inherently resistant to the growth-inhibitory effect of ethidium bromide, when supplied with exogenous uridine. After long-term exposure to ethidium bromide (90 days), the cell population has been transferred to drug-free medium for 60 days, and then seeded at low cell density. Three clones have been isolated and propagated in drug-free medium for 5, 6, and more than 12 months, respectively. It was found that none of these cell lines had detectable cytochrome c oxidase activity and that they were virtually devoid of cytochromes aa3 and b. Mitochondrial DNA was quantitated by DNA-DNA reassociation kinetics with a probe of chicken liver mitochondrial DNA. A mean number of 300 copies of mitochondrial DNA per cell was found in LSCC-H32 cells. Analysis of DNA extracted from cell populations exposed to ethidium bromide for 90 days and then transferred to drug free medium for long periods of time revealed no mitochondrial DNA molecules by reassociation kinetics or by Southern blot hybridization of HindIII-or AvaI digested total cellular DNA. PMID- 3008350 TI - Stability of DNA methylation of the human hypoxanthine phosphoribosyltransferase gene. AB - Methylation sensitive restriction enzymes were used to evaluate the methylation level of several restriction sites near human hypoxanthine phosphoribosyltransferase (HPRT) genes on active and inactive X chromosomes. DNA samples from leukocytes, from clonally derived fibroblasts, and from independent mouse-human hybrid lines isolated from the fusion of A-9 cells and these clonally derived human cells were studied. Comparison of the methylation patterns shows that restriction sites may show variable or constant methylation among tissues and clones, and heritability of methylation is also different among restriction sites. Methylation is more stable at sites whose methylation status correlate well with HPRT activity. Our results suggest that the methylation of certain cytosine residues may critically affect gene activity and that the methylation pattern of these sites is stably inherited. PMID- 3008351 TI - Fractionation of large mammalian DNA restriction fragments using vertical pulsed field gradient gel electrophoresis. AB - A new design for pulsed field gradient (PFG) gel electrophoresis of large (greater than 50 kb) DNA fragments is described. The method eliminates distortion of migration of DNA because of the geometry of the applied electric field, requires a single power supply and a simple switching device, and is extremely simple to use. Parameters investigated include variation in pulse time, conditions of restriction enzyme digestion and DNA concentration, and the use of different restriction endonucleases. The method has been applied to restriction enzyme digested mammalian DNA from Chinese hamster and human/Chinese hamster hybrid sources and has allowed examination of the minimum separation of two DNA markers known to be on the same human chromosome, chromosome 21. PMID- 3008352 TI - Mapping of haploid expressed genes: genes for both mouse protamines are located on chromosome 16. AB - Mouse spermatozoa contain two protamines with different amino acid sequences. By hybridizing Southern blots of a series of mouse-hamster somatic cell hybrids containing subsets of mouse chromosomes and a complete set of hamster chromosomes with 32P-labeled cDNAs for each mouse protamine, we assign the two mouse protamine genes to chromosome 16. This report presents the first evidence for chromosomal linkage of two sperm-specific, haploid regulated gene products. PMID- 3008353 TI - [Mixed mesodermal tumors of the uterus]. PMID- 3008354 TI - [Neurogenic tumors of the mediastinum]. PMID- 3008356 TI - Human papillomavirus infection related to oesophageal carcinoma in black South Africans. A preliminary study. AB - Oesophageal specimens derived from 70 patients with established invasive squamous cell carcinoma of the oesophagus were histologically reviewed with special reference to the morphological manifestations of human papillomavirus (HPV) infection. Epithelial changes fulfilling the criteria for HPV infection were noted in 23 cases (33%). The presence of HPV antigens was demonstrated by immunohistochemical staining in 7 of these 23 cases. Although acceptable for routine diagnostic purposes, histological typing and immunoperoxidase staining methods are not entirely conclusive of HPV infection. Electron microscopy for detection of viral particles and a molecular hybridization technique have to be used for absolute confirmation and viral subtyping. The results of this pilot study will be used for prospective studies to determine the role of HPV infection in the aetiology of oesophageal carcinoma. PMID- 3008355 TI - Langerhans' cells, papillomaviruses and oesophageal carcinoma. A hypothesis. AB - A hypothesis linking the concept of mucosal immune surveillance (Langerhans' cells and intra-epithelial lymphocytes) with recent evidence of human papillomavirus (HPV) infection of the oesophagus in a sequence of events which leads to squamous dysplasia and invasive carcinoma is presented. It is believed that the essential pathway to squamous carcinoma involves an aberration of the Langerhans' cell/lymphocyte network and its symbiotic relationship with squamous cells as a result of persistent HPV infection in the epithelium. Neoplastic transformation may occur when this 'at-risk' mucosa is exposed to one or several co-carcinogenic factors present in the environment. This hypothesis is supported by morphological changes present in acanthotic lesions of the oesophagus and by immunocytochemical evidence of HPV infection of the mucosa. In addition, this viewpoint offers a possible explanation for the regional variation of oesophageal carcinoma, the rising incidence of the disease, and the association with multifocal squamous carcinomas of the upper aerodigestive tract. PMID- 3008357 TI - Effects of an oats fibre tablet and wheat bran in healthy volunteers. AB - The daily intake of total dietary fibre of a group of 18 healthy volunteers was raised from a mean of 22.1 g to 32 g by supplementing their diet with either 23 g wheat bran or 15 g oats fibre tablets in a cross-over design for two 3-week periods with a wash-out period of 4 weeks in between. Both fibre supplements improved mean glucose tolerance, although not significantly. During the first period, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and very-low-density lipoprotein cholesterol were significantly lowered by both fibre preparations. During the second period significant reductions in TC and LDL-C were obtained only in the group taking the oats fibre tablets. This could probably be explained as an effect of the gel-forming fibre components in oats fibre. High-density lipoprotein cholesterol concentrations remained unchanged. The oats fibre tablet also proved easier to take and caused fewer side-effects. This study shows that if dietary fibre concentrates are used to increase fibre intake in Western societies, better results will probably be obtained by using a dietary fibre concentrate or mixture of concentrates that contain both soluble and insoluble components. PMID- 3008358 TI - [The acquired immunodeficiency syndrome (AIDS)]. PMID- 3008360 TI - [The century's catastrophe? (2) How contagious--no infection with AIDS virus?]. PMID- 3008359 TI - [Comparison of the computed tomographic changes and the clinical course following the irradiation of intracranial tumors and metastases]. AB - The authors compared the clinical courses and the CT courses recorded at the same time in patients with primary or secondary cerebral tumors. 69 patients improved their neurologic state under external radiotherapy, 35 patients remained unchanged, and 8 patients had a deteriorated condition. The changes in computed tomography and the modifications of the clinical state of patients corresponded only in 55% of all cases. The reasons are discussed. Some patients suffered from fatigue and weak concentration about three months after the end of radiotherapy, in some cases even the neurologic state was deteriorated. These manifestations known as "early delayed reaction" were reversible. PMID- 3008361 TI - [Glycoside poisoning: some aspects of the problem]. PMID- 3008362 TI - [Importance of radionuclide methods in the diagnosis of stenocardia at the outpatient stage]. PMID- 3008363 TI - [Bone marrow suppressor cells of patients with viral and alcoholic liver cirrhoses]. AB - A study was made of bone marrow suppressor cells potencies in 12 viral and alcoholic liver cirrhosis patients and 6 healthy persons using a method of the registration of bone marrow suppressor cells activity in the inhibition of xenogenic target cells proliferation. The activity of bone marrow suppressor cell in viral and alcoholic liver cirrhosis was significantly lowered as compared to that of the healthy donors. Besides, the spontaneous bone marrow cells proliferation level both in viral and alcoholic liver cirrhosis exceeded essentially that of the healthy donors. It might be due either to the reduced number or decreased function bone marrow suppressor cells in liver cirrhosis. These patients' peripheral blood lymphocytes under in vitro conditions as well as the healthy donors' lymphocytes did not produce any suppressive effect. PMID- 3008364 TI - [Antibodies to the AIDS virus: methods of detection and clinical significance]. PMID- 3008365 TI - Specific desensitization in the cytoskeletal assembly of platelet by receptor dependent activations. AB - Cytoskeletal assembly induced by receptor-dependent or independent activation of bovine platelets was investigated. When platelets were preactivated with receptor dependent stimulus, ADP or thrombin, cytoskeletal assembly was not induced repeatedly by the same agonist. However, in the case of receptor-independent stimulus, cryo-activation, the assembly was induced not only by receptor dependent but also by independent stimulus. The desensitization, therefore, lies in the transmission of stimuli from receptors to cytoskeletal proteins. PMID- 3008367 TI - Effect of tranexamic acid on neutrophil superoxide anion generation. PMID- 3008366 TI - Specific association of thrombin-antithrombin complexes with a human hepatoma cell line. AB - The clearance of thrombin-antithrombin (TAT) complexes from blood by the liver is through a receptor mediated pathway. We have used the established human hepatoma cell line, Hep G2, to determine if these hepatocytes have the capacity to bind this enzyme-inhibitor complex. The TAT complex was bound to the cells in a time and temperature dependent manner, reaching an apparent steady state at 90 minutes at both 4 and 37 degrees C. Binding at 4 degrees C was 5-7-fold less extensive than at 37 degrees C. The bound TAT was structurally similar to the added ligand. This interaction was specific, as it was inhibited by nonlabeled TAT but not by 50-fold molar excesses of unrelated proteins or by the individual constituents, thrombin or antithrombin. Factor Xa- antithrombin complex inhibited the binding reactions slightly. Specific binding isotherms at 37 degrees C were subjected to Scatchard plots. The apparent dissociation constant was 247 +/- 74 nM, and the number of TAT molecules bound per cell was 5.19 +/- 0.89 X 10(5). Bound TAT complexes did not undergo degradation at 4 or 37 degrees C for up to 2.5 hr, as greater than 85% of the bound ligand was acid precipitable during the time course of binding. Internalization of the TAT complex was compared with transferrin, a molecule known to be internalized by Hep G2 cells, by resistance of the cell bound ligands to degradation by trypsin or pronase. In contrast to transferrin, most of the TAT complexes remained cell-surface associated for at least 2 hr at both 4 degrees and 37 degrees C, indicating that TAT was not substantially internalized by the Hep G2 cells. PMID- 3008368 TI - Characterization of U46619 binding in unactivated, intact human platelets and determination of binding site affinities of four TXA2/PGH2 receptor antagonists (13-APA, BM 13.177, ONO 3708 and SQ 29,548). AB - The binding of U46619 and the inhibition of this binding by four TXA2/PGH2 receptor antagonists (13-APA, BM 13.177, ONO 3708 and SQ 29,548) were studied in unactivated, intact human platelets. Washed platelets were equilibrated with [3H] U46619 (5 nM) and the time course of binding determined. The receptor-specific binding reached equilibrium within 2-4 minutes, and could be displaced by addition of excess unlabelled ligand. Saturation of this binding was achieved at 750 nM. Scatchard transformation of the saturation binding curve yielded a single class of binding site with a Kd of 108 nM and Bmax of 360 fmole/10(8) platelets. When [3H]-U46619 (4 nM) was incubated with platelets in the presence of increasing concentrations of the antagonists, binding of U46619 was inhibited in a dose dependent manner. The potency series for inhibition of U46619 binding was: SQ 29,548 (IC50 = 7.9 nM) greater than ONO 3708 (IC50 = 38 nM) greater than BM 13.177 (IC50 = 0.91 microM) greater than 13-APA (IC50 = 6.2 microM). These findings are consistent with the notion that these compounds all act as competitive antagonists at the level of the platelet TXA2/PGH2 receptor. PMID- 3008369 TI - A functional assay of protein C in human plasma. AB - A functional assay for protein C in plasma is described in which barium eluates of plasma are incubated with bovine thrombin and rabbit thrombomodulin to activate protein C. The activated protein C solution is added to an activated partial thromboplastin time (APTT) system containing normal plasma and an APTT reagent (Dade ActinR). The prolongation of coagulation time after recalcification in this system is taken as a measure of the anticoagulant activity of protein C. When expressed as per cent of the value in pooled normal plasma, the results obtained by this method in 34 normal controls and in 3 untreated patients with protein C deficiency were very similar to those obtained by radioimmunoassay of protein C. In 2 patients with protein C deficiency and 23 patients without, all on dicoumarol or warfarin treatment, the anticoagulant activity of protein C was less than its antigen concentration. The day to day analytical coefficient of variation (SD/mean) was 12% at the 100% level (n = 12), and 10% at the 25% level (n = 12). PMID- 3008370 TI - Microsomal CA2+ uptake in human platelets is stimulated by calmodulin. AB - Calcium accumulating vesicles were prepared from washed human platelets. Ca2+ uptake could be stimulated with calmodulin. The stimulation of Ca2+ uptake was not abolished by the adenylate cyclase inhibitor 2',5'-dideoxyadenosine, thus excluding enhanced cAMP formation as a possible cause for the calmodulin effect. Our findings suggest that, in addition to cAMP, calmodulin is also involved in the regulation of platelet free Ca2+ concentrations, i.e. Ca2+ homeostasis in platelets is under dual control. PMID- 3008371 TI - Analysis of thromboxane B3 converted from eicosapentaenoic acid in human platelet rich plasma by gas chromatography/mass spectrometry. AB - Gas chromatographic mass spectrometric determination of thromboxane B3 (TXB3) synthesized from platelet is described. Eicosapentaenoic acid (EPA) was added to human platelet rich plasma and after the reaction the exstraction was carried out. Plasma thromboxanes were run through an Amberlite XAD-2 and SEP-PAK silica cartridge, and then chromatographed using silicagel thin layer plate to remove interfering materials, such as 6-keto-prostaglandin F1 alpha. Extracted thromboxanes were converted into the methoxime-dimethylisopropylsilylmethyl ester derivatives and they were measured by gas chromatography/ammonia chemical ionization mass spectrometry. Three peaks were obtained on the gas chromatogram which were presumed to be 3-series metabolite product TXB3 and their related substances. Results indicates the human platelet may easily convert EPA to TXB3 by adding EPA to PRP without adding arachidonic acid. PMID- 3008372 TI - Leukotrienes potentiate the effects of epinephrine and thrombin on human platelet aggregation. AB - A cooperation between leukocytes and platelets relative to metabolism of arachidonic acid has been observed in animal studies. To determine potential stimulatory effects of leukotrienes (LTs) on human platelets, LTs were incubated with platelet rich plasma followed by addition of subthreshold concentration of aggregatory stimulus. LTs (LTE4 LTD4 LTC4) alone had no direct effect on platelet aggregation, but potentiated the effects of subthreshold concentrations of epinephrine and thrombin and caused complete platelet aggregation. This potentiation was similar in citrated or heparinized blood and was unaffected by exogenous CaCl2. LTs did not induce secondary wave of aggregation in aspirin or selective TXA2-synthetase blocker OKY-046-treated platelets. In addition, LTs stimulated TXA2 biosynthesis by platelets in the presence of subaggregatory concentrations of epinephrine, but not when platelets had been pretreated with OKY-046. These data indicate that LTs potentiate epinephrine-induced platelet aggregation by modulating TXA2 synthetase activity. PMID- 3008373 TI - [Polyneuropathy. 9 years of case material]. PMID- 3008374 TI - [Myelopathy and peripheral neuropathy after radiotherapy]. PMID- 3008376 TI - Application of immune adherence hemagglutination test in the screening of blood donors carrying a high titer antibody to varicella-zoster virus. AB - The immune adherence hemagglutination (IAHA) test was compared with the complement fixation (CF) test and fluorescent antibody (FA) test to measure the antibody titer to varicella-zoster virus (VZV) in 70 sera of convalescents 2 to 8 weeks after herpes zoster onset. The IAHA titer correlated with the FA titer, but not with the CF titer which was lower than the IAHA and FA titers. Then, the IAHA test was used for screening of blood donors with a high titer (higher than 64 units) of antibody to VZV from 2,592 blood donors. About 14% of the sera from the donors aged 16 to 19 years had an antibody titer higher than 64 units, about 10% of sera from the donors aged 20 to 49 years, and 5.6% of sera from the donors older than 50 years. An average positive rate was about 9%, indicating that an enough VZV immune plasma with a high titer of antibody might be obtained from blood donors. PMID- 3008375 TI - Endotoxemia in liver diseases: detection by a quantitative assay using chromogenic substrate with perchloric acid pretreatment. AB - With a quantitative blood endotoxin assay using a chromogenic substrate with a perchloric acid pretreatment (PCA-LCT), endotoxemia in various liver diseases was studied. With PCA-LCT, recovery of added endotoxin in human plasma was nearly 90%, as evidenced by an intra- and inter-assay coefficients of variation of 5.7% and 11%, respectively. Because the recovery of endotoxin was not affected in severely icteric plasmas, PCA-LCT proved to be applicable to patients with liver diseases where various degree of jaundice exist. In none of the plasmas from patients with chronic hepatitis, acute hepatitis without hepatic failure or liver cirrhosis without ascites did the endotoxin level exceed the normal range of less than 5 pg/ml. With the presence of ascites, however, endotoxemia became detectable, but at low levels and not in all cases. At the stage of hepatic failure complicated with renal failure or disseminated intravascular coagulation, endotoxemia was more frequent and endotoxin concentration greater. It is uncertain, at present, whether endotoxemia itself is deteriorating factor in hepatic failure or is merely concomitant phenomenon resulting from Kupffer cell failure. PMID- 3008377 TI - Toxicological evaluation of quartz and silica contents in studded tire-generated dust in the city of Sendai. AB - Roadside dust samples were collected at six locations in the City of Sendai at the end of the winter season of 1980-1981, and analyzed for quartz and silica contents. X-ray diffraction and ICP analyses revealed that the mean quartz and silica contents in dust samples were 14.6% (ranging from 12.2 to 19.4%) and 56.3% (53.7 to 59.7%), respectively. From these results in connection with current dust levels and estimated duration of exposure, it appears likely that the risk of silicosis among the citizens may not be high if appropriate measures are taken to protect the atmosphere. PMID- 3008378 TI - Modulation of ovarian LH receptor and serum hormone levels in rats with hyperprolactinemia induced by administration of ovine prolactin or sulpiride. AB - Hyperprolactinemia was experimentally produced in rats by administration of ovine prolactin (oPRL) and sulpiride, and tried to evaluate the effect of hyperprolactinemia on ovarian receptor for luteinizing hormone (LH) as well as that on serum gonadotropin and steroid hormone levels. Wistar-Imamichi strain mature female rats showing 4-day estrous cycles were treated with various doses of oPRL or sulpiride twice a day for 4 days from diestrus. They were killed on the fifth day. Binding of ovarian LH receptors was reduced by a small dose of oPRL (0.1 IU) or sulpiride (0.25 mg) and restored to normal by larger doses of oPRL. However, larger doses of sulpiride (50 or 100 mg) increased the receptor bindings beyond the control level (4.39 +/- 0.40 ng/mg homogenate protein). Serum prolactin levels decreased in rats treated with larger doses of oPRL, but increased with larger doses of sulpiride. Serum LH levels increased with both agents. Although the ovaries treated with either oPRL or sulpiride suggested the lack of ovulation, there were no significant changes of steroid hormones in oPRL groups. In contrast, sulpiride treatment resulted in a reduction of estradiol and an increase of progesterone secretion, suggesting the prolonged effect of the drug. Thus, prolactin appeared to act on the rat ovarian LH receptors in two different manners in hyperprolactinemia, depending on the amount of this hormone or a ratio of prolactin to LH. PMID- 3008379 TI - Late preventive effects of trifluoperazine on carbon tetrachloride-induced hepatic necrosis. AB - As a very preliminary test for a possible role of calmodulin in CCl4-induced hepatic injury, we studied the effects of the anticalmodulin drug trifluoperazine (TFP) on several deleterious actions of CCl4 on the liver. TFP administrated 30 min before or 6 or 10 hr after CCl4 significantly prevented hepatic necrosis induced by the hepatotoxin at 24 hr but not at 72 hr. TFP did not modify the CCl4 concentrations reaching the liver, or the intensity of the covalent binding of CCl4-reactive metabolites to hepatic microsomal proteins or lipids or the CCl4 induced cytochrome P-450 and glucose 6 phosphatase destruction. TFP administration decreased body temperature between 0 and 1 degree C in controls and between 1.2 and 3.5 degrees C in CCl4-treated animals during the 24-hr observation period. When TFP-treated CCl4-poisoned animals were kept normothermic, protective effects were eliminated. One possibility is that the protective effect of TFP might be due to a nonspecific action related to decreased body temperature. Alternatively, prevention might result from TFP inhibition of a late-occurring process critical for CCl4-induced cell necrosis requiring calmodulin participation. If this alternative were in operation, protective consequences of this inhibitory effect of TFP should be either canceled or counteracted in the normothermic TFP + CCl4-treated animal. PMID- 3008380 TI - Triorganotin-induced cytotoxicity to rat thymus, bone marrow and red blood cells as determined by several in vitro assays. AB - To further investigate the immunotoxic effects of tri-n-propyltin chloride (TPTC), tri-n-butyltin chloride (TBTC) and triphenyltin chloride (TPhTC) several cytotoxicity tests with a series of trialkyltin chlorides and TPhTC were carried out, using isolated rat thymocytes as target cells. Thymocytes, cultured in a serum-supplemented medium, were exposed to organotin concentrations ranging from 0.01 to 10 microM for periods up to 30 h. Parameters such as cell count, trypan blue exclusion, chromium release, thymidine incorporation and cyclic AMP production were used to evaluate the cytotoxicity of these compounds. The more lipophilic compounds TPTC, TBTC, tri-n-hexyltin chloride (THTC) and TPhTC appeared most cytotoxic, reducing thymidine incorporation at concentrations as low as 0.05-1 microM. Membrane damage as determined by trypan blue exclusion and chromium release occurred at higher levels (1-10 microM). The water soluble homologue trimethyltin chloride (TMTC) was least effective in all test models. When phosphate-buffered saline supplemented with glucose was used as incubation medium, TBTC appeared more cytotoxic to thymocytes. Using this medium in 5-h incubations the cytotoxicity of TBTC to thymus, bone marrow and red blood cells was compared. Bone marrow cells were slightly less sensitive than thymocytes, while red cells were relatively resistant. In conclusion, of the triorganotin compounds especially the lipophilic homologues are cytotoxic in vitro. PMID- 3008381 TI - [Cellular immunologic parameters in maxillofacial tumors with special reference to T-lymphocytes and skin tests]. PMID- 3008383 TI - Substrate-specific stimulation by glucagon of isolated murine brain mitochondrial oxidative phosphorylation. AB - Glucagon has been shown to increase further the enhanced tolerance for hypoxia of mice with elevated blood ketones and to stimulate ketone utilization by rat brain slices, suggesting that glucagon may affect brain metabolism. In addition to stimulating gluconeogenesis, glucagon alters the metabolism of mitochondria isolated from liver and heart. This study was designed to test whether glucagon can act directly and selectively on brain mitochondrial substrate oxidation. Mitochondria were isolated from normal murine brains using differential centrifugation through Ficoll gradients. Glucagon (3.6 microM) stimulated respiration in the presence of glutamate, and glutamate plus beta hydroxybutyrate, but not in the presence of glutamate plus malate, succinate or beta-hydroxybutyrate alone. With glutamate as the substrate the hormone significantly increased State 3 oxygen consumption rates from control values of 91 mol O2/mol of cytochrome aa3/min to 117 mols O2/mol/aa2/min (p less than 0.0001), and also increased State 4 rates slightly but significantly. Glucagon did not change mitochondrial respiratory control ratios, but increased estimated rates of ATP synthesis from 434 (control) to 597 mols ADP consumed/mol aa3/min (p less than 0.0001). The data indicate that in vitro glucagon has a direct and substrate-specific stimulatory effect on isolated brain mitochondria. These substrate-specific effects were not altered when respiration was studied in the presence of postmitochondrial supernatant or exogenous 3',5'-cyclic AMP, indicating that glucagon, in addition to an in vivo action via activation of membrane-bound adenylate cyclase, can act, at least in vitro, directly and selectively on brain mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008382 TI - Characterization of beta adrenergic receptors in human cerebral arteries and alteration of the receptors after subarachnoid hemorrhage. AB - The nature of beta adrenergic receptors in human cerebral arteries was characterized and alteration of these receptors after subarachnoid hemorrhage was examined using a radioligand binding assay. The specific 3H-dihydroalprenolol, a beta adrenergic antagonist, binding to human cerebral arteries was saturable and of high affinity (KD = 12.3 nM) with a Bmax of 790 fmol/mg protein. Ki values and Hill coefficients of adrenergic agents for 3H-dihydroalprenolol were as follows; propranolol, 4.1 X 10(-8)M, 1.01; isoproterenol, 1.7 X 10(-6)M, 0.80; epinephrine, 8.3 X 10(-6)M, 0.48; norepinephrine, 2.3 X 10(-5)M, 0.45; metoprolol, 6.8 X 10(-8)M and 7.9 X 10(-6)M, 0.62; butoxamine, 2.2 X 10(-8)M and 2.1 X 10(-6)M, 0.43. The analysis of inhibition of specific 3H-dihydroalprenolol binding by these adrenergic agents suggests that human cerebral arteries contain a high density of beta adrenergic receptors and that the receptors are classified into two types, namely beta 1 and beta 2 adrenergic receptors. The calculated beta 1/beta 2 ratio from Hofstee plots was approximately 4/6. KD and Bmax of 3H dihydroalprenolol binding to the cerebral arteries after subarachnoid hemorrhage were compared with those of control group. KD and Bmax of 3H-dihydroalprenolol binding of subarachnoid hemorrhage group were 13.9 nM and 1140 fmol/mg protein, respectively. The calculated beta 1/beta 2 ratio was approximately 6/4. These data suggest that the density of total beta adrenergic receptors increased without any significant change in the affinity after subarachnoid hemorrhage and that the increase of beta 1 adrenergic receptors was dominant. PMID- 3008384 TI - Presynaptic inhibitory action of adenosine on neuromuscular transmission in the canine cavernous carotid artery. AB - We investigated the effect of adenosine on neurogenic contraction of the canine cavernous carotid artery, using an isometric tension recording device and transmural nerve stimulation. Adenosine, in concentrations under 10(-5)M, had no relaxing effect on the contractions produced by high [K]o solution or 10(-5)M norepinephrine. Transmural nerve stimulation (stimulus: 1 msec duration, 100V intensity) evoked a frequency-dependent contraction, which was abolished by 3 X 10(-7)M tetrodotoxin. Adenosine in concentrations of 10(-6)M and 10(-5)M, inhibited the neurogenic contraction at each frequency, more so in the low frequency range. This inhibitory effect of adenosine was significantly antagonized by 10(-5)M theophylline. Pretreatment with 2 X 10(-8)M dipyridamole had no effect on neurogenic contractions, but augmented the inhibitory effect of adenosine. 10(-5)M theophylline did not augment the neurogenic contractions. The findings that both dipyridamole and theophylline failed to affect the neurogenic contractions in the absence of adenosine suggests that the presynaptic autoinhibition mechanism of adenosine may not be involved in neuromuscular transmission in this tissue. These results suggest that there is a presynaptic adenosine receptor in the nerve terminal which inhibits the release of neurotransmitter in canine cavernous carotid artery. It is also probable that the vasodilating effect of adenosine in the cavernous carotid artery is mainly due to its inhibitory effect on neurotransmission rather than to a direct relaxing effect on smooth muscle. PMID- 3008385 TI - [Multicentric alveolar carcinomatosis of the lungs as a cause of sudden death]. PMID- 3008387 TI - Recombinant DNA technology. AB - The advent of new techniques in the study of DNA has generated great interest among clinicians in their application to genetic disorders. These new techniques are outlined, and their application in the study of X-linked disorders, in particular X-linked retinitis pigmentosa (RP), is described. This concerns chiefly the use of probes in studying DNA sequences on a given chromosome and the basic principles which govern their application. PMID- 3008386 TI - Use of silica gel with aqueous eluent for simultaneous high performance liquid chromatographic assay of disopyramide and mono-N-dealkyldisopyramide. AB - Disopyramide and its pharmacologically active metabolite, mono-N dealkyldisopyramide, were determined simultaneously in serum by high performance liquid chromatography using a bare (unbonded) silica gel with aqueous eluents. p Chlorodisopyramide was used as the internal standard. Separations of all compounds, which contain amine moiety in their chemical structures, were easily accomplished with a good peak symmetry on silica. The method is sufficiently precise, sensitive, and specific. Analytical recoveries of all compounds were greater than 96%; CVs for reproducibility were less than 6.5% for disopyramide and mono-N-dealkyldisopyramide; the lower detection limits of both analytes were 0.05 mg/L. Comparison of the present method with a fluorescence polarization immunoassay for disopyramide gives a good correlation (r = 0.992). PMID- 3008388 TI - Serum and liver enzymes of collagen synthesis in hepatic murine schistosomiasis mansoni. AB - Serum galactosylhydroxylysyl glucosyltransferase (S-GGT), liver galactosylhydroxylysyl glucosyltransferase (L-GGT), liver hydroxylysyl galactosyltransferase (L-GGal-T) and liver prolyl hydroxylase (L-PH) activities were measured in CF1 female mice each week for 10 weeks after infection with Schistosoma mansoni (50 cercariae per mouse). None of the enzyme activities measured were significantly increased in the first six weeks following the infection. However, the activities increased thereafter reaching a maximum of seven-fold for S-GGT, five-fold for L-GGT, three-fold for L-GGal-T and ten-fold for L-PH compared with control animals. The increase in S-GGT activity was positively correlated with L-GGT (r = 0.788), L-GGal-T (r = 0.774) and L-PH (r = 0.800). The present data suggest that during the first six weeks of the development of murine hepatic schistosomiasis mansoni infection, few eggs bombard the liver. The biochemical changes during this period are mild. During weeks 7 to 10, which is the active fibrotic stage, the liver biochemical markers of collagen synthesis increase rapidly and lead to fibrotic morphological features that are the chronic irreversible hepatic changes characteristic of this infection. The data reported here indicate that the aggressive fibrotic changes in liver during weeks 7 to 10 of murine schistosomiasis infection are predicted by S-GGT activity. PMID- 3008389 TI - The impact of cyclosporine therapy on the occurrence of infection in the renal transplant recipient. PMID- 3008390 TI - New immunosuppressive drug combinations for mismatched related and cadaveric renal transplantation. PMID- 3008391 TI - Density of Culex vishnui and appearance of JE antibody in sentinel chicks and wild birds in relation to Japanese encephalitis cases. AB - In the District of Burdwan, a rural area of West Bengal State, India, Japanese encephalitis (JE) virus is endemic. In one village a longitudinal survey was conducted in order to find out whether associations could be established between the density of the vector mosquito Culex vishnui in two types of resting places, the incidence of infections in sentinel chicks exposed at monthly intervals, the prevalence of antibodies in wild birds and the occurrence of clinical infections in the human population. The experiment lasted from August 1981 till August 1982. Meteorologically a summer season (March-June), a rainy season (July-October) and a winter season (November-February) are distinguished. In the sentinel chicks infections were observed in all three seasons; in wild birds antibodies were prevalent throughout the year; these observations suggest perennial transmission of the virus in its maintenance cycle. Human infections were observed periodically with, in August 1982, a pronounced peak; this may point to fluctuations in the level of circulation of the virus in its maintenance cycle and spillover to the human population at times of peak circulation. The peaks may be related to the influx of young non-immune birds and newborn mammals into the animal population in summer. Further studies, including virus isolation attempts from mosquitoes and nestling birds, are required to prove this hypothesis. PMID- 3008392 TI - [Waldenstrom's disease with renal, neurologic and bone complications]. PMID- 3008393 TI - Formation of tubuloreticular inclusions in mitogen-stimulated human lymphocyte cultures by endogenous or exogenous alpha-interferon. AB - Tubuloreticular inclusions (TRI) were induced in normal blood lymphocytes after incubation with Staphylococcus aureus Cowan 1 (STA), but they were not induced by pokeweed mitogen (PWM), as we reported previously. TRI were also induced in Raji cells when grown in the medium of STA culture. Alpha-interferon (alpha IFN) was detected only in the medium of STA culture and not in PWM culture. The cells of PWM cultures formed TRI when exposed to various concentrations of human leukocyte alpha IFN. The incidences of TRI-positive cells in the presence of 50-500 IU/ml of alpha IFN were 3-5% on day 2 and increased to 10% on day 7. On days 5-7 of the PWM cultures, plasmacytoid cells containing TRI were seen not infrequently. In the presence of a high concentration of alpha IFN (10,000 IU/ml), which was sufficient to inhibit cell growth and differentiation, the growth of the TRI region was not altered and the incidence of TRI-positive cells was 9% on day 2 and increased to 15% on day 7. Our observations suggest that the TRI formation in STA culture is attributable to the alpha IFN produced endogenously by STA stimulated cells and that some relationship might exist between the incidences of TRI-positive cells in these mitogen-stimulated cultures and the biologic functions of IFN. PMID- 3008394 TI - Human osteogenic sarcoma: fine structural localization of adenosine triphosphatase. AB - The localization of ATPases in 7 osteogenic sarcomas of osteoblastic, chondroblastic and fibroblastic type was investigated at the fine structural level using two types of substrates: one with lead as capturing ion and one with strontium (the latter presumed to reveal sites of Na+-K+-dependent transport ATPase). Reaction product with the lead-ATP medium was located on the plasma membrane and the membranes bordering subjacent vesicles and vacuoles in all the various types of osteoblastlike and fibroblastlike cells and also in types 1 and 3 chondroblastlike cells, and multinucleated giant cells believed to be neoplastic. Furthermore, deposits of reaction product were demonstrated in lysosomelike organelles in all the aforementioned cells. Except in the case of chondroblastlike cells, precipitates marking the localization of enzyme were confined to areas of the plasma membrane where adjacent cells were closely applied (the free surface lacked precipitates). In chondroblastlike cells the reaction product was usually deposited along the whole plasma membrane. Presence of L-Homoarginine or L-Tetramisole in the incubation medium in concentrations that have been shown to completely abolish alkaline phosphatase activity did not affect the occurrence of the reaction product with ATP as substrate indicating that the enzyme hydrolysing ATP was substrate-specific. Reaction product marking sites of Na+-K+-dependent ATPase was confined to plasma membranes and lysosomes of cells in vessel walls. The observations strengthen the notion obtained in studies on the localization of alkaline phosphatase, namely that osteoblastlike, chondroblastlike, and fibroblastlike cells in osteogenic sarcomas are histogenetically related to one another and to those multinucleated giant cells that presumably are of a neoplastic nature. PMID- 3008395 TI - Random glycogen-ER complexes in small cell chest wall tumor. PMID- 3008396 TI - [Malignant fibrous histiocytoma located on the left index finger]. PMID- 3008397 TI - [Femoral nerve neuropathy--a neurological complication after gynecologic laparotomy]. PMID- 3008398 TI - Silica stones in humans. AB - Twenty cases of silica stones (including a personal one, the first case ever observed--April 1952) are reviewed. Almost all of the patients had been taking magnesium trisilicate for several years, one up to 40 years. The average age of the patients was 54 years. There were 9 males and 1 female. The patients came from the following locations, given in chronological order: Beirut, Lebanon (1952); Stockholm, Sweden (1953, 1962); Houston, Tex., USA (1958, 1961); New York, N.Y., USA (1960); Johannesburg, South Africa (1964); London, UK (1973, 1982); Osaka, Japan (1978); Madrid, Spain (1978, 1981); and Torrance, Calif., USA (1984). 14 patients passed out the stones spontaneously. In 3 patients, the stone was formed in the left kidney. Bilateral renal stones were found in 2 patients. In 2 patients, they were removed from the left ureter and in 2 patients they were found in the bladder. The size of the stones varied between 2 mm and 3 cm, the weight from 8 mg to 3.6 g. The silica stone is of a relatively low radio density. Our case is the only one in whom the level of urinary silica was determined; it was of the order of 0.006% i.e. 1 mmol/l. PMID- 3008399 TI - Indolent nonseminomatous germ cell tumor of testis. Prolonged survival of patient with persistent metastatic disease. AB - The complete response rate of disseminated nonseminomatous germ cell tumors (NSGCT) of the testes with current aggressive chemotherapy and surgical resection of residual disease is between 70 and 80 per cent. Those patients who do not attain complete response tend to have short survivals. A case is presented of a forty-one-year-old white man who has had nearly continuous evidence of metastatic embryonal carcinoma for more than eleven years. Although NSGCTs are characterized by rapid proliferation, early metastasis, high response rate to chemotherapy, and rapid death if uncontrolled, this case demonstrates an indolent form of disease with poor response to chemotherapy and yet prolonged survival in spite of uncontrolled disease. This is the first reported case of indolent metastatic germ cell neoplasm with survival of more than ten years. PMID- 3008400 TI - Malignant fibrous histiocytoma of prostate. AB - A prostatic tumor removed suprapubically in a sixty-three-year-old man was found to be a malignant fibrous histiocytoma. Various aspects of prostatic sarcomas and malignant fibrous histiocytomas are considered. PMID- 3008402 TI - [Clinical picture and diagnosis of Barre-Masson disease]. AB - Based upon an experience with the treatment of 13 patients with arteriovenous glomus tumors of Barre-Masson the author describes the clinical course of the disease. Errors and problems of diagnostics of the disease are discussed. In none of the cases the primary diagnosis was correct. The method of examination and recommendations for the improvement of diagnostics are given. PMID- 3008401 TI - [Microcirculatory characteristics of the blood in a focal allergic reaction in patients with herpetic keratitis]. PMID- 3008403 TI - [Induction of biological protection in pigs against infection with Aujeszky disease virus by vaccination with large doses of live or inactivated vaccines]. AB - Pigs were inoculated against the Aujeszky's disease twice in a four-week interval. The dose of the live vaccine was 10(6) TKID50 and the titres of neutralizing antibodies were 1 : 16 to 1 : 128 in blood serum. Two weeks later the pigs were exposed to contact infection. Primary multiplication of the virus was observed on the mucous membranes of the nose and oropharynx and the virus was detected on the nasal mucous membrane within one to five days, the maximum infection titre values being 10(1.3) TKID50, and on the oropharyngeal mucous membrane within seven days, the maximum titres being up to 10(3.5) TKID50. In another group the pigs were inoculated with the same dose of attenuated virus or re-vaccinated with a dose of 10(8.5) TKID50, with neutralizing antibody titres of 1 : 256 to 1 : 1024 in blood serum. No viruses were detected on the nasal mucous membrane after contact infection and only trace amounts of the virus were found in the oropharynx within one to five days. Six piglets were inoculated in the same way but the infection was intranasal. The infective virus was detected on the nasal mucous membrane of only one piglet; however, trace amounts of the virus were found in the oropharynx of all the six piglets within three to nine days after infection. The nasal mucous membrane and oropharynx of the noninoculated control piglets exposed to intranasal infection were infectious until death and those of the contact-infected piglets remained so until the 14th day. At the intranasal infection of the piglets infected twice with a live or inactivated vaccine and slaughtered the 1st to 14th day after intranasal infection, the virus was replicated only in the place of primary multiplication without penetrating into the CNS and the internal organs. The intranasal infection of susceptible control piglets resulted in the dissemination of the infection via the neurogenic and lymphohaematogenic routes. PMID- 3008404 TI - Ultrastructure and frequency of mastitis caused by ovine progressive pneumonia virus infection in sheep. AB - Twenty-five sheep, experimentally (n = 15) or naturally (n = 6) infected with ovine progressive pneumonia virus and noninfected controls (n = 4), were evaluated for histological and ultrastructural lesions of mastitis. Histologically, nine of 15 experimentally infected sheep and all six naturally infected sheep had lympho-plasmacytic mastitis. Severity of the lesion increased with length of time after infection. Periductal lymphatic nodules were seen in five sheep experimentally infected for 2.8 years or longer and in five naturally infected sheep that were 3.7 years old or older. Ultrastructurally, responses to ovine progressive pneumonia virus were diffuse lympho-plasmacytic infiltrates in glandular interstitium, lymphocytic and occasional plasmacytic infiltrates in ductal walls and lumens, lymphoblasts surrounded by small lymphocytes in glandular interstitium, and degeneration of epithelium releasing cells and cellular debris into the lumen. Based on the prevalence of lesions, the mammary tissue was more susceptible to ovine progressive pneumonia virus than other target organs: lung, brain, and synovium. Lesions did not differ between breeds of sheep. Ovine progressive pneumonia virus was not seen in the mammary tissue but was isolated from 15 of 17 mammary glands. PMID- 3008405 TI - Prevalence of herpesvirus infection in British red deer and investigations of further disease outbreaks. AB - A serological survey of the prevalence of a new herpesvirus isolated from red deer (Cervus elaphus), tentatively designated herpesvirus of Cervidae type 1 (HVC 1), has shown that the virus is widespread in free-living and farmed red deer. Neutralising antibodies were detected in hill deer culled at three different locations in the north of Scotland, in farmed deer on five of eight Scottish farms and in four of 12 groups of English farmed or park deer. Fifty-eight of 145 (40 per cent) hill deer, 67 of 203 (33 per cent) Scottish farmed deer and 26 of 172 (14 per cent) English deer had antibody, the overall prevalence being 29 per cent. Further outbreaks of ocular disease in farmed red deer calves caused by HVC 1 were investigated. Deer sent to an auction from one farm were found after sale to have been incubating the disease and it was thus spread to seven deer farms. Despite a high incidence of clinical disease in the calves from the original farm, few in-contact deer showed clinical signs. PMID- 3008406 TI - Field observations on serological responses to an oil emulsion vaccine against avian infectious bronchitis. PMID- 3008407 TI - Bovine herpes mammillitis therapy. PMID- 3008408 TI - Cr mordanted plant cells in chicken food studies. PMID- 3008409 TI - Possible immunoenhancement of persistent viremia by feline leukemia virus envelope glycoprotein vaccines in challenge-exposure situations where whole inactivated virus vaccines were protective. AB - Kittens immunized with purified native FeLV-gp70 or -gp85 envelope proteins developed ELISA, but not virus neutralizing, antibodies in their serum to both whole FeLV and FeLV-gp70. Kittens vaccinated with envelope proteins and infected with feline sarcoma virus (FeSV) developed smaller tumors than nonvaccinates, but a greater incidence of persistent retroviremia. Similarly, FeLV-gp70 and -gp85 vaccinated kittens were more apt to become persistently retroviremic following virulent FeLV challenge exposure than nonvaccinates. Kittens vaccinated with inactivated whole FeLV developed smaller tumors after FeSV inoculation and had a lower incidence of persistent retroviremia than nonvaccinates. The protective effect of inactivated whole FeLV vaccine against persistent retroviremia was also seen with FeLV challenge-exposed cats. Protection afforded by inactivated whole FeLV vaccine was not associated with virus neutralizing antibodies, although ELISA antibodies to both whole FeLV and FeLV-gp70 were induced by vaccination. PMID- 3008410 TI - Feline leukemia vaccine: efficacy, contents and probable mechanism. AB - An effective subunit vaccine against feline leukemia virus infection and related diseases has been developed. The source of the vaccine immunogen is feline retrovirus persistently infected cells that continuously synthesize and shed virus polypeptides. Western blot analysis identifies FeLV-gp70, p27, p15, p12, and p10 in the subunit vaccine preparation. Cats immunized with the vaccine developed antibodies to a 70,000 MW protein of the vaccine that seems to be distinct from, but related to the FeLV-gp70 polypeptide. PMID- 3008411 TI - Virological investigations in adults with acute pneumonia. AB - Virological investigations (immunofluorescence reactions and isolation attempts with pharyngeal exudate specimens, as well as serological tests) were performed in 110 adult patients with pneumonia. Viral or inframicrobial agents were involved in 70 (63.7%) of the cases, either alone (27 cases) or in association with bacteria (43 cases). Parainfluenza and adenoviruses were most frequently encountered both in the cases with mixed (viral + bacterial) and in those with strictly viral pneumonia. Mycoplasma pneumoniae accounted for 11% of the cases; the role of chlamydial and rickettsial germs was insignificant. PMID- 3008412 TI - Surveillance of enterovirus circulation in children communities. AB - In three children communities covered by a virological study enteroviruses could be isolated from 19.3% of the stool samples, 52.3% of the sewage samples and 9.7% of the samples taken from different objects and surfaces. The isolates belonged to 14 enterovirus serotypes, Coxsackie viruses being predominant. There was a good concordance between the serotypes of enteroviruses shed in the stools and those isolated from the environment. The high epidemiological potential of enteroviruses calls for a surveillance of their circulation in children communities, preferably by the virological monitoring of sewage. PMID- 3008413 TI - The 10K virion phosphoprotein encoded by gene US9 from herpes simplex virus type 1. AB - Gene US9 of herpes simplex virus type 1 has been predicted, from DNA sequence analysis, to encode a protein of mol wt 10,026, designated 10K (D.J. McGeoch, A. Dolan, S. Donald, and F.J. Rixon (1985). J. Mol. Biol. 181, 1-13). We have investigated this protein by using a synthetic peptide corresponding to the 11 amino acids adjacent to the amino-terminal methionine and rasing antisera in rabbits. One antiserum was able to precipitate at least 12 electrophoretically distinct polypeptide species from extracts of BHK cells infected with HSV-1. The estimated molecular weights of these polypeptides ranged from 12K to 20K and immunoblotting showed them to be related proteins. The primary translation product has an apparent mol wt of 13K. The various forms of 10K differ in their relative abundance in the infected cell and also in their degree of phosphorylation. Lower molecular weight forms of the 10K protein can be precipitated from NP-40 extracts of HSV-1 virions, suggesting that these forms of 10K are contained in the virion tegument or envelope. An association between this protein and nucleocapsids has also been observed in the nuclei of infected cells by immunoelectron microscopy. These observations imply that the product of US9 is a tegument protein which becomes associated with nucleocapsids at, or soon after, their formation in the nuclei of infected cells. PMID- 3008414 TI - Influence of amino acids encoded in the 3' open reading frame of the SV40 early region on transformation and antigenicity of large T antigen. AB - The mutant dlA2414 bears a frame-shift deletion of nucleotides 2936-2927 in the coding sequence for the simian virus 40 (SV40) large T antigen. Based on its nucleotide sequence, this mutant should produce a T antigen containing the first 627 authentic large T antigen amino acids followed by 97 amino acids encoded in the alternate open reading frame at the 3' end of the early region. This protein resembles the hypothetical T* protein that would be translated from an early SV40 mRNA if it were spliced to permit utilization of the open reading frame. We show that stable mouse cell lines can be generated that express the T antigen produced by dlA2414 and that this T antigen has an altered carboxy terminus. In addition, the expected tryptic peptides were missing from the large T antigen and replaced by more hydrophobic peptides. The T*-like protein produced by dlA2414 was apparently less stable than wild-type T antigen and did not stably complex with the cellular phosphoprotein p53. This protein retained the ability to immunize mice against a challenge of syngeneic SV40-tumor cells. The dlA2414 T antigen was expressed at the surface of cells as shown by in vitro lymphocyte-mediated cytotoxicity assay. The results presented here also showed that the expression of a T*-like protein at the cell surface is not likely to be essential for tumorigenesis of cells transformed by SV40. PMID- 3008415 TI - T1 pip: a mutant which affects packaging initiation and processive packaging of T1 DNA. AB - The pip mutation of phage T1 is located between the tar (gene 2.5) and am6 (gene 3) mutations in the region of the T1 genome which codes for early functions. The tar and pip mutations are additive in increasing the efficiency of transduction by T1. When T1 carries the pip mutation the initiation of DNA packaging by the phage at the non-T1, esp-lambda site is more efficient than when the phage is pip+; the small average burst size of 8 to 10 by T1pip suggests that pip causes a reduction in the efficiency with which T1 utilizes pac, the normal packaging initiation site of the phage. The presence of the BglII-D fragment (cut at one end at pac and the other by BglII) after digestion of T1pip DNA by BglII shows that T1pip continues to initiate DNA packaging at pac. The increased molarity of BglII-D coupled with the absence of the BglII-C fragment (which contains DNA on both sides of pac and can only be cut from processively packaged genomes) shows that T1pip packages only genomes which are initiated at pac and is defective in processive packaging. PMID- 3008417 TI - Intracellular K+ and the expression of transformation parameters by chick cells transformed with the Bryan strain of Rous sarcoma virus. AB - As normal chick embryo (CE) cells entered quiescence the intracellular concentrations of both Na+ and K+ declined. Comparable decreases in intracellular concentrations of Na+ and K+ were not observed in CE cells transformed by either the Schmidt-Ruppin (SR) or the Bryan (B) strain of Rous sarcoma virus (RSV). Intracellular concentrations of Na+ were higher in SR-RSV-transformed CE cells than in B-RSV-transformed cells and uninfected CE cells at all times after plating. In contrast, intracellular concentrations of K+ were higher in B-RSV transformed CE cells than in SR-RSV-transformed cells. Uninfected CE cells incubated in medium containing an elevated concentration of K+ (an increase from 5 to 30 mM) exhibited several, but not all, of the transformation parameters expressed by B-RSV-transformed CE cells. PMID- 3008416 TI - The effects of 2-deoxyglucose and tunicamycin on the biosynthesis of the murine mammary tumor virus proteins, and on the assembly and release of the virus. AB - The role of glycosylation in the biosynthesis, processing, and shedding of the murine mammary tumor virus (MuMTV) glycoproteins and in virus production was investigated in a clonal mammary tumor cell line, GR-3A, using two inhibitors of protein glycosylation, 2-deoxyglucose (2-DG) and tunicamycin (TM). It was found that both 2-DG and TM completely inhibited the synthesis of the MuMTV envelope precursor polyprotein, Pr70env, and, as a consequence, the synthesis of the viral glycoproteins gp52 and gp36. By contrast, the synthesis of Pr73gag, the polyprotein precursor of the internal structural proteins of the virus, was only inhibited by 10-15% by 2-DG and TM. Although 2-DG and TM blocked the synthesis of Pr70env, a new polypeptide, related to gp52 and gp36, with a mol wt of 60,000 (P60env) was found to be synthesized in the treated cells. The P60env molecules appeared to be degraded intracellularly since they were not found to (1) undergo site-specific cleavage; (2) accumulate inside the cell or on the cell surface; (3) be secreted into the culture medium; and (4) be incorporated into the virions produced during the drug treatment. In spite of the lack of gp52 and gp36 synthesis in the presence of TM and 2-DG, mature MuMTV particles containing the characteristic surface projections known to be composed of gp52 and gp36 continued to be assembled and released at a reduced rate for at least 30 hr. In addition, the buoyant density and the polypeptide composition of the particles were found to be identical to virions produced by untreated cells. Thus, the virions assembled and released during 2-DG and TM treatment were not defective. Our investigations into the origin of gp52 and gp36 in these particles revealed that both molecules were synthesized prior to 2-DG and TM treatment and continued to be incorporated, along with the newly synthesized viral core proteins, into budding virions during the drug treatment. Furthermore, we found that gp52 and P75env (an aberrant form of Pr70env) that were not incorporated into virions continued to be shed normally from the cell during drug treatment. In conclusion, our results suggest that MuMTV assembly is not dependent on the synchronized synthesis of the viral core and envelope polypeptides, and that the assembled virions contain the correct ratio of these polypeptides, even when their ratio in the cell varies. PMID- 3008418 TI - Nucleotide sequence of the vaccinia virus hemagglutinin gene. AB - Vaccinia virus hemagglutinin (HA) is expressed at late time of infection cycle, and it is nonessential for virus growth. Location of the HA structural gene was determined by hybrid-arrested and hybrid-selected translation methods at the right terminus of the HindIII A fragment. The position of the HA gene was confirmed by the production of the complete HA protein in the cells transfected with the plasmid containing that region. Examination of this nucleotide sequence revealed the positions of cleavage sites for a number of restriction endonucleases. The deduced amino acid sequence revealed that the HA protein is a member of typical surface membrane glycoproteins. Comparison of the nucleotide sequence upstream of the HA coding region with corresponding region of other late genes suggested the existence of the consensus decanucleotides TTCATTTa/tGT between 34 to 18 bp upstream to the initiation codon followed by a cluster of A or T, a unique feature of the late genes of vaccinia virus. These results in conjunction with the ease of isolating HA- mutants provide a basis for a new site suitable for inserting foreign genes. PMID- 3008420 TI - The predicted primary structure of the measles virus hemagglutinin. AB - The complete nucleotide sequence of cloned cDNAs corresponding to the full length of the mRNA encoding the measles virus hemagglutinin (H) protein has been determined. the mRNA contains a single large open reading frame which is capable of encoding a protein of 617 amino acids with a molecular mass of 69,250 Da. The deduced amino acid structure of the protein indicates that the only major hydrophobic region of sufficient length to anchor the molecule in membranes is located near the amino terminus. Comparison of the amino acid structure of the measles virus H protein with that of the hemagglutinin-neuraminidase (HN) molecules of Sendai virus and simian virus 5 (SV5) reveals little homology. However, 11 of the 13 cysteine residues found in the measles H protein can be aligned with cysteines in the Sendai virus HN protein in similar positions relative to one another. Five potential N-linked glycosylation sites are present in the measles H protein sequence. These are relatively closely grouped between amino acids residues 168 and 240 in the amino terminal half of the molecule. No obvious structural features are present in the measles H protein amino acid sequence which might explain the reported absence of neuraminidase activity associated with the molecule. PMID- 3008419 TI - Localization of P, NP, and M proteins on Sendai virus nucleocapsid using immunogold labeling. AB - The distribution of NP, P, and M proteins on Sendai virus nucleocapsids purified from cells and virions were studied by immunogold staining using monoclonal antibodies. NP molecules were found uniformly along the entire length of both cytosol and virion derived nucleocapsids. This observation is in accord with the earlier proposals that NP molecules maintained the structural integrity of the nucleocapsid. The distribution of P in nucleocapsids derived from the cytosol differed from the distribution in those originating from virions. In nucleocapsids derived from the cytosol, P molecules occurred in 4 to 10 discreet clusters at varying locations along the length of the nucleocapsid. In contrast, on nucleocapsids derived from virions, P molecules were uniformly distributed over the entire length of the nucleocapsid. These observations suggest that the distribution of P depends on the functional state of the nucleocapsid. The occurrence of P clusters at different locations on intracellular nucleocapsids indicates that P is a mobile molecule; this suggestion is consistent with P's role in viral RNA synthesis. The distribution of the matrix (M) protein also depended on where the nucleocapsids were derived from. Large quantities of M protein were found along the entire length of nucleocapsids derived from the cytosol, while in virion nucleocapsids, many fewer molecules of M were observed. The large amounts of M on the nucleocapsids originating from the cytosol supports the hypothesis that M protein mediates the recognition between the nucleocapsid and the envelope glycoproteins. PMID- 3008421 TI - Relationship between the cellular resistance to Friend murine leukemia virus infection and the expression of murine leukemia virus-gp70-related glycoprotein on cell surface of BALB/c-Fv-4wr mice. AB - Fv-4r is a dominant resistance gene which controls resistance of mice to exogenous infection with ecotropic murine leukemia viruses. Cell lines with various degrees of resistance to ecotropic Friend murine leukemia virus infection were established from BALB/c-Fv-4wr mice which are partially congenic with BALB/c. The degree of resistance of these cell lines correlated well to the amount of glycoprotein with mol wt 80 K on cell surface. The resistance of cells was reduced by the treatment of glycosylation inhibitors, tunicamycin and 2-deoxy D-glucose. The results indicate that the Fv-4 resistance is ascribed to the unique glycoprotein on cell surface. PMID- 3008422 TI - Herpes simplex virus type 1 DNA sequences which direct spread of virus from cornea to central nervous system. AB - The virulence of a herpes simplex virus (HSV) intertypic recombinant possessing HSV-1 DNA sequences from map units 0.31 to 0.44 and HSV-2 sequences from map units 0 to 0.30 and 0.45 to 1.0 were compared with the virulence of the two parental strains. Following ocular inoculation, both the intertypic recombinant and the HSV-1 parent replicated at the infection site and spread to the peripheral and central nervous system (CNS) to produce fatal encephalitis. The HSV-2 parent also replicated at the infection site but failed to progress to the CNS. However, when inoculated intracerebrally, the HSV-2 strain was as lethal as the HSV-1 parent. Furthermore, the HSV-2 strain could produce thymidine kinase at 37 and 39 degrees in levels comparable to the HSV-1 strain. The results indicate that transfer of the HSV-1 DNA sequences imparted to the recombinant virus the necessary genetic information to spread from the cornea into the central nervous system. PMID- 3008424 TI - Phosphorylation of pp60v-src by the TPA receptor kinase (protein kinase C). AB - Incubation of pp60v-src with the purified 12-O-tetradecanoyl-13-acetate (TPA) receptor kinase (protein kinase C) resulted in an increase in the phosphorylation of pp60v-src. Two-dimensional tryptic phosphopeptide mapping showed that three major phosphoserine containing peptides were labeled which were localized within the amino terminal 18,000 Da of the src protein. Based on comparative tryptic mapping, one of these major phosphopeptides was identical to the peptide labeled on pp60v-src immunoprecipitated from cells labeled with [32P]orthophosphate and treated with TPA. These data suggest that a direct interaction takes place between protein kinase C and pp60v-src. PMID- 3008423 TI - The polyomavirus early promoter: role of proximal promoter elements in the formation of 5' termini in vivo. AB - Analysis of polyomaviruses with altered early promoter sequences indicates that in vivo (1) the polyomavirus early ATA motif and nearby upstream sequences dictate homogeneity of 5' termini, in contrast to what has been observed in vitro, where the cap site was reported to affect homogeneity of 5' termini (P. Jat, V. Novak, A. Cowie, C. Tyndall, and R. Kamen, 1982, Mol. Cell. Biol. 2, 737 751). (2) Substitution of the polyomavirus ATA motif by the adenovirus 2 major later promoter suppresses minor early 5' termini between the origin and the early cap sites, indicating that these termini are not the result of DNA replication. PMID- 3008425 TI - Partial N-terminal amino acid sequences of three nonstructural proteins of two flaviviruses. AB - Partial N-terminal amino acid sequences for the three largest nonstructural proteins of two flaviviruses, yellow fever virus and St. Louis encephalitis virus, have been obtained. The determined sequences of these proteins exhibit significant amino acid sequence homology, and allow the positioning of these three nonstructural proteins in the polyprotein sequence deduced from the nucleotide sequence of yellow fever virus (C. M. Rice, E. M. Lenches, S. R. Eddy, S. J. Shin, R. L. Sheets, and J. H. Strauss, 1985, Science 229, 726-733.) The deduced start points support the hypothesis that the N terminus of nonstructural glycoprotein NS1 results from cleavage by signalase, whereas the N termini of NS3 and NS5 result from cleavages following double basic residues that are flanked by amino acids with short side chains. PMID- 3008426 TI - Sindbis virus mutant ts20 of complementation group E contains a lesion in glycoprotein E2. AB - A technique has been devised to readily obtain the entire structural protein region of Sindbis virus cloned into a plasmid vector. This method uses the fact that the nearest site for restriction enzyme HindIII to the 3' terminal poly(A) occurs at nucleotides 6266-6271 in the genomic RNA. Inserts extending from the poly(A) tract to this HindIII site are 5438 nucleotides long (excluding the poly A tract) and contain the entire 4106-nucleotide structural protein region. Using an oligo(dT)-tailed vector as a primer for first strand cDNA synthesis such clones could be obtained in high yield. We were interested in a precise determination of the mutation responsible for the temperature-sensitive phenotype of ts20, a mutant belonging to complementation group E which has a defect in the function of glycoprotein E2 at the nonpermissive temperature. Using this technique we have cloned and sequenced the structural protein region of ts20 and of several revertants and concluded that the mutation was a change from histidine to leucine at amino acid 291 of E2. Reversion to temperature insensitivity occurred by same site reversion to the parental nucleotide, restoring the original histidine as amino acid 291. Thus, complementation group E of Sindbis virus results from changes in glycoprotein E2 and together with previous results from our laboratory (Arias et al., 1983; Hahn et al., 1985) demonstrates that the three RNA+ complementation groups of Sindbis virus, C, D, and E, result from changes in the three structural proteins of the virus, capsid, glycoprotein E1, and glycoprotein E2, respectively. PMID- 3008427 TI - The nucleotide sequence and genome organization of human papilloma virus type 11. AB - The complete nucleotide sequence of human papilloma virus type 11 (HPV11) DNA (7931 bp) was determined. HPV11 DNA which has been isolated from laryngeal papillomas and from genital warts (condylomata acuminata) shows a high degree of sequence homology to HPV6b (82%). The arrangement of open reading frames is very similar to HPV6b, the homology of the deduced amino acid sequences varies between 58 and 92%. Characteristic features of the noncoding region between the L1 and E6 open reading frames is an AT-rich domain of about 200 bp with extended stretches of alternating thymine-purine bases and a 12-bp inverted repeat element ACCG NNNN CGGT arranged in tandem upstream of the putative early promoter TATA box. PMID- 3008428 TI - Polyproteins containing a domain encoded by the V-SKI oncogene are located in the nuclei of SKV-transformed cells. AB - SKV-transformed nonproducer clones were isolated from infected quail and chicken embryo cells. Analysis of intracellular viral RNAs by the Northern technique revealed that each clone contained a single SKV genome (either 5.7 or 8.9 kb) but no genome of the helper virus. Analysis of intracellular viral proteins containing gag determinants revealed that each clone contained a single species of either 55, 110, or 125 kDa. The intracellular location of these proteins was determined by indirect immunofluorescence employing either monoclonal antibodies (anti-p19gag) or conventional antiserum against gag proteins. All three of the SKV-specific proteins were localized to the nuclei of the transformed cells. PMID- 3008430 TI - An infectious cDNA clone of the poliovirus Sabin strain could be used as a stable repository and inoculum for the oral polio live vaccine. AB - Viruses were recovered from HeLa S3 cells and African green monkey kidney (AGMK) cells transfected with an infectious cDNA clone of poliovirus vaccine Sabin 1 strain. The viruses recovered from the different DNA-transfected cells were tested for the biological characteristics of temperature sensitivity (rct marker), plaque size, and bicarbonate concentration dependency (d marker). The results revealed that the above properties were similar to those obtained from tests on the Sabin 1 vaccine reference strain. The recovered viruses and the vaccine reference virus were passaged in AGMK cells at an elevated temperature of 37.5 degrees, and the passaged isolates were tested for the rct marker. The virus recovered from AGMK cells had the most stable rct phenotype while the virus from HeLa S3 cells had a similar stability to that of the reference virus, suggesting that the virus from AGMK cells would be more suitable as a vaccine strain than the other two viruses. Furthermore, an infectious cDNA clone of high specific infectivity, constructed by introducing SV40 large T antigen into the plasmid, was used for production of high titers of virus after transfection. The results of in vitro biological tests on the recovered virus suggested that virus produced in the transfected AGMK cells also had the high quality that is desirable in vaccine stocks. Monkey neurovirulence tests performed with these recovered viruses revealed that the recovered viruses were weakly neurovirulent, similar to the vaccine reference virus. The infectious cDNA clone of the poliovirus vaccine strain could therefore be used to generate a possible inoculum of the oral polio live vaccine. Our findings strongly suggest that an infectious cDNA clone of poliovirus RNA may be used to preserve the constancy and quality of the present seed viruses of the Sabin 1 vaccine strain. PMID- 3008429 TI - Nucleotide sequence comparison of the 3' regions of avian retroviruses NY203 and NTRE-2. AB - We have been characterizing molecular clones of two subgroup E avian retroviruses (NTRE-2 and NY203RAV-60) that produce different proliferative diseases after inoculation into susceptable K28 chickens. Both viruses arose by recombination between exogenous and endogenous viral genomes. To further characterize regions of these viruses that are important for the production of disease, we have determined the nucleotide sequence of a 1.2-kb EcoRI fragment extending from the carboxyl end of gp85 through 150 bases of the U3 region of the LTR. From the sequence data it is possible to precisely define one point where recombination occurred between PrRSV-B and RAV-0 to produce NTRE-2. We suggest a hypothesis, based on the core enhancer consensus sequence, for the higher incidence of disease when chickens are infected with viruses bearing the LTR of NY203RAV-60. PMID- 3008431 TI - A new class of retrovirus present in many murine leukemia systems. AB - A new class of MuLV has been detected and isolated from normal and leukemic AKR, C58, SJL, and NFS.AKV mice as well as from NFS mice inoculated with Friend or Moloney ecotropic viruses. These new viruses are XC negative and serologically cross-react with MCF env antigens but are ecotropic in host range, being able to only infect mouse cells to varying degrees and unable to infect mink or other cells infectable by MCF or xenotropic viruses. Viruses of this type from AKR mice cross-interfere with Moloney ecotropic and MCF viruses in SC-1 cells and appear to have properties similar to those of the SL3-2, GPA-V2, and R-XC- isolates. Analysis of their genomes by restriction endonuclease mapping of proviral DNA indicates structures similar to class II MCFs with the 5' half of the genome being like ecotropic viruses and the env region exhibiting restriction sites characteristic of MCF viruses. In normal AKR mice, these ecotropic recombinant like viruses are found in spleen and bone marrow as early as 1 week of age, but first appear in the thymus at 3-4 months of age. These viruses have not been detected in mice with no or low expression of ecotropic viruses (NFS, NZB, DBA/2, BALB/c, C57BL/6). Because of their apparent recombinant structure and ecotropic host range we have provisionally designated them ecotropic recombinant virus (ERV) to distinguish them from the MCF class of recombinant MuLV. PMID- 3008432 TI - Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene. AB - The 3' end of the 20-kb genome of the Purdue strain of porcine transmissible gastroenteritis coronavirus (TGEV) was copied into cDNA after priming with oligo(dT) and the double-stranded product was cloned into the PstI site of the pUC9 vector. One clone of 2.0-kb contained part of the poly(A) tail and was sequenced in its entirety using the chemical method of Maxam and Gilbert. Another clone of 0.7 kb also contained part of the poly(A) tail and was sequenced in part to confirm the primary structure of the most 3' end of the genome. Two potential, nonoverlapping genes were identified within the 3'-terminal 1663-base sequence from an examination of open reading frames. The first gene encodes a 382-amino acid protein of 43,426 mol wt, that is the apparent nucleocapsid protein on the basis of size, chemical properties, and amino acid sequence homology with other coronavirus nucleocapsid proteins. It is flanked on its 5' side by at least part of the matrix protein gene. The second encodes a hypothetical 78-amino acid protein of 9101 mol wt that is hydrophobic at both ends. A 3'-proximal noncoding sequence of 276 bases was also determined and a conserved stretch of 9 nucleotides near the poly(A) tail was found to be common among TGEV, the mouse hepatitis coronavirus, and the avian infectious bronchitis coronavirus. PMID- 3008433 TI - Generation of packaging-defective DNA molecules of equine adenovirus. AB - Equine adenovirus (EAd) DNA prepared from infected bovine kidney (MDBK) cells contained additional sequences of about 100 to 700 bp at the left-hand end of the genome. These aberrant viral genomes were produced even after the first passage of the wild type EAd in MDBK cells and their relative amounts did not change significantly during serial passage. The left terminal fragments of two defective viral DNAs were cloned into the plasmid vector pBR322 and the nucleotide sequences of their terminal regions were analyzed. The data indicate that one viral DNA contained a duplication of the inverted terminal repetition (ITR) and the other contained 270 bp of additional sequences derived from the right terminal region of EAd genome added to the left-terminal, ITR. While the former DNA was packaged into virions, the latter was not, presumable due to the alteration of the distance from the left terminus to the putative DNA packaging signal, reported to be located between 290 and 390 bp (Hammarskjold and Winberg, 1980). The possible mechanism for the generation of these defective DNAs is discussed. PMID- 3008434 TI - [Multiple primary cancer of the lung and larynx]. AB - An experience of treatment of 43 cases of primary multiple cancer of the lung and larynx (synchronous--9, metachronous--31 and metachrono-synchronous--3) is discussed. Patients with lung and laryngeal cancer should undergo thorough examination. Primary multiple malignancies of the said organs make the case for surgery. PMID- 3008435 TI - [Hormones in the milk]. PMID- 3008436 TI - [Characteristics of the interaction of the influenza and vesicular stomatitis viruses with the splenocytes of inbred mice]. AB - The reproduction activity of many viruses in cells of human and animal immune system has not been studied sufficiently on the body level. It is especially important to determine exactly the sensitivity of the main triad of the immune system cells to a certain virus from the parameters of its intracellular reproduction. Complete elimination of extracellular influenza A/PR8/34 virus from in vivo pre-infected cells of the immune system of mice revealed no reproduction of this virus. Under similar conditions, permanent reproduction of vesicular stomatitis virus has been demonstrated. PMID- 3008439 TI - [Varying nature of the cell receptors for influenza and para-influenza viruses]. AB - A comparative study of receptors for influenza virus, fowl plague virus, and human parainfluenza type 3 virus was carried out. Natural receptors of guinea pig erythrocytes were destroyed with neuraminidase, and individual gangliosides GM1, GD1a, and GT1b were inserted into their membranes. The labeled virus was adsorbed on the erythrocytes modified in this manner, and the degree of restoration of the receptor activity of erythrocytes lost after neuraminidase treatment was determined. Two gangliosides, GD1a and GT1b, were found to be capable of functioning as specific receptors for influenza virus. Both gangliosides restored completely the virus adsorption on erythrocytes. In contrast, none of the three gangliosides used did not restore parainfluenza virus adsorption. It is concluded that the nature of influenza and parainfluenza virus receptors is different. PMID- 3008438 TI - [Long-term persistence of coxsackieviruses in familial foci of infectious myocarditis patients]. AB - Numerous examinations during 12-36 months of 16 children suffering from myocarditis and their 37 relatives revealed long-term persistence of Coxsackie A13/A18 viruses in the familial foci of infection in 13 cases. In addition, 9 families were found to have foci of Coxsackie B virus infection of which 2 were also persistent (Coxsackie B1 and B3) and 7 transitory. In 8 families, Coxsackie B viruses circulated simultaneously with Coxsackie A13/A18. PMID- 3008437 TI - [Comparison of serous meningitis morbidity and the concentration of the Coxsackie B virus in sewage]. AB - The paper shows that the frequency of detection and concentration of Coxsackie B virus in sewage correlate with the number of patients and asymptomatic virus excretors. PMID- 3008440 TI - [Structural organization and proteins of the human type-3 para-influenza virus]. AB - The structure of human parainfluenza type 3 virus was studied by electron microscopy and virion fractionation by treatment with a detergent and high ionic strength. The protein spectrum of the virus was studied. The presence of 6 structural proteins was revealed of which two, HN and F, are glycoproteins. Intracellular cleavage of F0 protein into F1+2 proteins was demonstrated in a pulse-chase experiment. A tighter binding of HN protein than of F protein with the virus lipoprotein membrane was observed which may be useful for obtaining purified F protein preparations. PMID- 3008441 TI - [Clinico-epidemiological characteristics and diagnosis of viral non-A, non-B hepatitis with fecal and oral mechanisms of transmission of the infection]. AB - The paper deals with observations of the patients with non-A-non-B hepatitis (NANBH) transmitted by the fecal-oral mode. The disease was diagnosed by ruling out other similar diseases of the liver, primarily viral A (HA) and B (HB) hepatitides, using clinical and epidemiological data and highly sensitive methods of laboratory diagnosis of HA and HB. Cases of NANBH occurred in one of Central Asia regions in the period of the usual seasonal rise of incidence. The disease was more frequent in adults, running a mild course in most patients, although there were also severe forms with fatal outcomes observed only in pregnant women in the second half of pregnancy. The lethality among the pregnant women was 15.7%. Immune electron microscopy of fecal specimens collected from the patients in the early days of jaundice revealed virus-like particles of 27-30 nm in diameter, morphologically similar to HA virus but forming no immune complexes upon treatment with blood sera containing antibody to HA virus antigen. PMID- 3008443 TI - [Determination of specific hepatitis A markers by a radioimmunological method with autoradiographic recording of the results]. AB - The possibility of determining specific markers of hepatitis A by a simple variant of radioimmunoassay on a polyethylene film with autoradiographic recording of the results was demonstrated. The high sensitivity of the method, an extremely simple procedure, no necessity of special radiometric apparatuses, visual demonstration and reliability of the results recommend it for hepatitis A diagnosis. PMID- 3008444 TI - Hepatocellular carcinoma--screening and latency period. PMID- 3008442 TI - [A macrophage migration-inhibiting factor in preparations of alpha- and gamma interferon]. AB - Native preparations of human alpha- and gamma-interferon synthesized in stationary, suspension or roller leukocyte cultures regularly showed high levels of migration inhibition factor (MIF) activity a significant portion of which was lost in the course of interferon purification and concentration. Minimal losses of MIF activity were achieved when medicinal preparations of interferon were made by the method of negative immunoabsorption on immobilized antibodies to inductor antigens and chromatography on Sephadex G-25. PMID- 3008445 TI - [Myeloperoxidase activity in the neutrophils of patients with simple psoriasis]. PMID- 3008446 TI - [Benign form of pancreatic insuloma of 23 years' duration]. PMID- 3008447 TI - [Diagnosis and therapy of pneumocystis carinii pneumonia in a patient with AIDS]. AB - The clinical, immunological and histological findings in a patient suffering the acquired immunodeficiency syndrome and Pneumocystis carinii pneumonia are discussed. At the time of diagnosis HTLV III antibodies were not demonstrable. Treatment with trimethoprim sulfamethoxazole achieved a remission lasting 7 months so far. HTLV III antibodies became demonstrable after 5 months; the markedly decreased Helper/Suppressor ratio of 0.30 remained unchanged. PMID- 3008448 TI - [Effect of bile on intestinal calcium and vitamin D absorption. Animal experiment studies in swine]. AB - In the human system calcium is the major constituent of bone and the regulator of important bioelectric and biochemical effects. Calcium homeostasis is underlying exact control mechanisms in which vitamin D is a predominant factor. Cholecalciferol (VD3) is metabolized and the active form 1 alpha, 25 dihydroxycholecalciferol (1,25(OH)2D3) is formed by the kidney. 1,25(OH)2D3 acts on the cell nuclei and on the luminal membrane of the intestinal mucosal cell. It enhances intestinal Ca absorption and the Ca transport to the blood system. VD3 metabolism and mechanisms of action are reported in the introduction. Early reports have described the important influence of bile for the intestinal Ca absorption. Up to now conclusive investigations are missing and became major topics as new regulator mechanisms were described recently. One of the main questions arising is, whether VD3 and other vitamin D metabolites can be absorbed in the absence of the biliary system and which effect on the enterocyte can be observed. Intestinal Ca absorption and transport was estimated in piglets using triple lumen tube system and duodenal perfusion. 4 untreated animals and 3 experimental animals with bile deprivation for a period of 5 (7) days were studied. Ductus choledochus ligation and concommittant cholecysto-colic anastomosis was applied for this purpose. The effect of vitamin D metabolites was estimated on 3 experimental animals applying a daily dosage of 600.000 I.E. VD3 orally, measuring Ca absorption 5 days afterwards; 3 animals in addition were administered a daily dosage of 2 micrograms 1,25(OH)2D3, measuring the Ca absorption 5 (7) days afterwards. Electrolytes, bilirubin, transaminases, total protein, albumin, triglycerides, Ca, phosphate, alkaline phosphatase, parathormone (PTH), 25 hydroxycholecalciferol (25OHD3) and 1,25(OH)2D3 were measured in all the animals before and after the experimental procedure. Data were calculated statistically. VD3 absorption was measured in 3 untreated control animals and 3 animals with bile deprivation, absorption of 1,25(OH)2D3 in 2 animals with bile deprivation. The basis for the evaluation of the experimental model was given by the laboratory values after bile deprivation. Changes in electrolyte and intermediary metabolism were observed postoperatively only and are assigned to the surgical treatment, thus ruling out severe metabolic disorders, which means that the experimental model should be appropriate for our purpose to look for Ca homeostasis.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3008449 TI - Regulation of collagen biosynthesis by ascorbic acid: a review. AB - L-ascorbic acid is an essential cofactor for lysyl hydroxylase and prolyl hydroxylase, enzymes essential for collagen biosynthesis. In addition, L-ascorbic acid preferentially stimulates collagen synthesis in a manner which appears unrelated to the effect of L-ascorbic acid on hydroxylation reactions. This reaction is stereospecific and unrelated to intracellular degradation of collagen. The effect apparently occurs at a transcriptional or translational level, since L-ascorbic acid preferentially stimulates collagen-specific mRNA. In addition, it stimulates lysyl hydroxylase activity but inhibits prolyl hydroxylase activity in human skin fibroblasts in culture. PMID- 3008450 TI - Thirty-five years of progress in the study of MSH. AB - In this paper, initial work on MSH at Dr. Lerner's laboratory in Portland, Oregon, from 1952 to 1954 is presented. The development of an in vitro bioassay method enabled us to show increased urinary excretion of MSH in Addison's disease. The ability of MSH to increase skin pigmentation in man was also demonstrated. Subsequent work on MSH during the past thirty years is reviewed, such as characterization of alpha- and beta-MSH and their precursors in the pituitary gland and localization of MSH-like peptides in various regions of the brain. Finally there are presented the characterization of gamma-MSH, the hypothermic effect of intracisternal administration of gamma-MSH, the effect of corticortropin releasing factor on increased secretion of alpha-MSH from rat pituitary, and the effect of arginine vasopressin on secretion of alpha-MSH from pituitary adenoma. PMID- 3008451 TI - Studies on the Cloudman melanoma cell line as a model for the action of MSH. AB - A review of the studies done at Yale on the role of MSH in regulating pigmentation and growth of Cloudman (S91) melanoma cells is presented. The areas covered include the isolation and analyses of mutant cell lines unresponsive to MSH; the role of cyclic AMP, cyclic AMP-dependent protein kinases, and protein phosphorylation reactions in the response of MSH; new regulators of the melanogenesis pathway; the cytotoxicity of melanin precursors; the development of methodology for synthesizing 125I-beta-MSH; the use of this ligand to study receptors for MSH; and the chemical and biological properties of phosphorylated isomers of L-dopa, a new class of compounds exhibiting potent bio-activity toward melanocytes. All of the experiments described were carried out in the Department of Dermatology at the Yale University School of Medicine during the tenure of Dr. Aaron B. Lerner as chairman. PMID- 3008453 TI - [A further therapeutic possibility in sudden deafness]. PMID- 3008452 TI - Development of affinity resins for isolation of angiotensin receptors. AB - The development of affinity resins for the isolation of angiotensin II receptors from adrenal fasciculata cells is described. The approach is based on the avidin biotin interaction. The advantages of the technique are delineated. PMID- 3008454 TI - [Correlation between some physiochemical parameters and the biologic activity of industrial dusts]. PMID- 3008455 TI - [New knowledge about signal transmission through the cell membrane and mechanisms of action of growth factors]. AB - Growth factors are proteins or peptides which evoke the replication of the nuclear DNA and thus cell division. A survey is given of the structure, the importance and the mechanism of action of the biochemically exactly characterized growth factors. The epidermal growth factor consists of 53 amino acids and is made over outwardly by proteolysis from a large precursor molecule in the membrane: it is attached to a receptor of the cell membrane consisting of three large parts, by means of which the intracellularly lying part of the receptor becomes effective as proteinkinase and phosphorylizes the parts of the receptor as well as adjacently lying proteins. This acts as a signal for the beginning of the phase of the DNA-synthesis (replication) in the chromosomes. The receptor for the epidermal growth factor has a high degree of structure similarity with the insulin receptor which is also active as proteinkinase after insulin binding. The importance of the decomposition of phosphatidylinosite and of its phosphate esters in binding of certain ligands to receptors is shown. PMID- 3008456 TI - [Detection and significance of HBsAg associated receptors for polymerized human serum albumin in acute hepatitis B virus infection]. AB - Receptors for polymerized human serum albumin (R-pHSA) may play a role in the attachment of hepatitis B virus (HBV) to hepatocytes. Therefore, we evaluated the incidence and prognostic value of R-pHSA in acute hepatitis B virus infection. High titers of R-pHSA were found in 12/12 patients with HBeAg positive acute hepatitis B (log2-Titer: 8.5 +/- 1.5). Titers for R-pHSA and HBsAg correlated closely. Furthermore, R-pHSA occurred (log2-titer: 6.5 +/- 1.5) in 10/10 patients with HBeAg positive chronic active hepatitis B (CAH-B). In the course of HBV infection, R-pHSA were eliminated earlier than HBsAg. 4/20 patients which were asymptomatic HBsAg carriers had high titers of R-pHSA in correlation to the histological findings of CAH-B. Asymptomatic HBsAg carriers which were R-pHSA negative had a normal liver histology. R-pHSA were found to be an early prognostic marker in acute hepatitis B infection. PMID- 3008457 TI - [Direct involvement of the central nervous system by HTLV III]. PMID- 3008458 TI - MTD approach to quantitative structure-activity relationships for cardiotonic steroids. AB - A minimal topological difference (MTD) approach is made to describe quantitative structure-activity relationships (QSAR) for the Na+, K+-ATPase inhibitory activity of cardiotonic steroids. The calculations take into account 20 derivatives of digitoxigenin, digoxigenin, and gitoxigenin with small substituents at different sites of the steroid backbone. A multiple correlation coefficient of r = 0.916 is obtained using the MTD and an indicator variable for the presence of a 15 beta substituent. The corresponding receptor map reveals receptor wall vertices in the C11, C12, C15, and C22 regions. Both 3 beta and 16 beta substituents are found to contain receptor cavity vertices. The MTD results are discussed with respect to lactone-ring conformational investigations presented and they are compared with findings of previous structure-activity studies. PMID- 3008459 TI - Response of adult human volunteers to oral administration of bovine and bovine/human reassortant rotaviruses. AB - Small groups of adult volunteers, in sequence, were inoculated orally with inactivated purified bovine rotavirus of strain NCDV, with live NCDV purified or unpurified and with two different NCDV X human rotavirus reassortant viruses. One of five volunteers given 200 micrograms of ultravioletinactivated NCDV developed a virus-neutralizing (VN) and a binding antibody response detected by enzyme linked immunosorbent assay (ELISA). Four of 10 volunteers given from 1 X 10(6) to 1 X 10(8) p.f.u. of live NCDV developed VN antibody, but nine of 10 responded when ELISA, HAI and radioimmuno-precipitation tests for serum antibody were also considered. Two different NCDV X human serotype 1 Wa strain virus reassortants, each containing Wa gene segment 9 and the serotype 1 neutralization phenotype, were administered orally in doses up to 10(6) p.f.u. The reassortants were relatively ineffective in eliciting a serum antibody response at the dosage level employed. PMID- 3008460 TI - Trial of inactivated Japanese encephalitis vaccine in children with underlying diseases. AB - Two shots of inactivated Japanese encephalitis (JE) vaccine were given to children, 139 with underlying diseases and 42 healthy, and their antibody responses were studied by the neutralization test. Before vaccination, most of the vaccinees did not have antibody against JE virus. One month after the second vaccination, they were all seroconverted and showed considerably high neutralizing titres. One healthy child developed fever on the day of vaccination without any severe symptoms afterwards, and no side reactions were observed in the handicapped children. These results suggest that the current JE vaccine is safe and can induce a strong immune response even in handicapped children. PMID- 3008461 TI - Intramuscular and intravaginal vaccination of pregnant cows with thymidine kinase negative, temperature-resistant infectious bovine rhinotracheitis virus (bovine herpes virus 1). AB - To test the safety and efficacy of a thymidine kinase-negative (TK-), temperature resistant (TR) mutant of bovine herpes virus-1 (BHV-1) in pregnant cows, seronegative cows, 2-5 months pregnant, were vaccinated intramuscularly (i.m.) or intravaginally (i.vag.) with this candidate vaccine virus. I.m. vaccinated cows did not shed virus i.vag. or intranasally (i.n.), but i.vag. vaccinated cows replicated virus i.vag. for 8-9 days postvaccination (p.v.) Some of the cows were challenge exposed i.n. at 46 days p.v. with virulent TK+ BHV-1(Cooper). Vaccinated cows showed no clinical disease signs p.v. or postchallenge and responded anamnestically postchallenge. All cows delivered live calves. Pre colostrum sera of the calves were negative for BHV-1 antibodies. PMID- 3008462 TI - [Consultation 7--here everything turns around the AIDS virus]. PMID- 3008463 TI - [Radiation exposure and risk of radon in the room air of Swiss houses]. AB - The radioactive noble gas radon, a member of the natural decay chains of uranium and thorium, enters the indoor environment in regionally highly diverging amounts. Subsoil of dwellings, building materials and drinking water are the main sources. In Switzerland and in many other countries, exposure of the lung tissue to the short lived radon decay products is the most important component of the radiation dose of the general public. Annual doses in areas with crystalline rock of high uranium content may reach the limits set up for occupational exposure. However, a clear link between cumulative exposure to radon daughters and elevation of the lung cancer incidence exists only for underground miners. The majority of human epidemiological studies point to a linear dose effect relationship. The indoor radon levels are determined by geology, building materials and techniques, climate and behaviour of the occupants. Experiences from Scandinavia and the Northern parts of America clearly indicate the possibility of cost-efficient remedial measures to reduce indoor radon levels. PMID- 3008464 TI - An unusual case of Wilms' tumour in a horseshoe kidney. AB - An unusual case of Wilms' tumour arising in a horseshoe kidney gave conflicting appearances on ultrasound and intravenous urography and difficulties in preoperative diagnosis. PMID- 3008465 TI - The neonatal development of benzodiazepine receptor subtypes in the rat cerebral cortex and cerebellum. AB - The development of benzodiazepine receptor subtypes in the rat cerebral cortex and cerebellum was studied. The proportions of Type I (CL218872 sensitive binding sites) and Type II (CL218872 insensitive binding sites) in both regions of the neonatal brain were quite different from those of adult. In one day old rat cortex and cerebellum, about 95% of the benzodiazepine receptor were Type II receptor, while these receptors comprised about 50% and 15% of the total in the adult cortex and cerebellum, respectively. From 21 days onward, these ratios in both brain regions did not significantly change through adult age (over 55 days old). The proportion of receptor subtypes in the 21 day cortex (50% type I, 50% type II) was approximately equivalent to that seen in the cerebellum at the 9th day of age. The 3H-flunitrazepam specific binding sites in both brain regions increased markedly after birth, although Type II receptor binding sites did not show any notable gain. Type II receptor specific binding sites in the cortex were not significantly different between neonatal and adult, while these sites in the cerebellum to decrease after birth. These results suggest that Type I and Type II receptors seemed to develop independently during their embryogenesis. PMID- 3008466 TI - [Glycogen content and glucose-6-phosphatase activity in the tissues of the adult frog and tadpole]. AB - Studies have been made on the content of glycogen and the activity of glucose-6 phosphatase in tissues of adult frog Rana temporaria (liver, brain, pia mater, n. ischiadicus, fast and slow muscles) and tadpoles (liver, tail muscles). It was found that the enzymic activity is somewhat higher in the liver of tadpoles than that in the liver of adult frogs, being low in the tail muscles. Pia mater and n. ischiadicus exhibit higher activity of glucose-6-phosphatase as compared to the liver. In tadpoles, glycogen content of the liver increases from the 40th stage of metamorphosis and decreases in the tail muscles to the 42nd-50th stages. Glycogen content in tissues other than liver of adult frogs is higher than in similar tissues of mammals. PMID- 3008467 TI - [Progressive sequelae of cranio-cerebral injuries]. AB - Progressively increasing pathological phenomena, those of a vegetovascular character in the main, develop often in the late period after closed craniocerebral trauma even if it was of a mild degree. Impaired immune response, mosaicly manifested disorders of regional volumetric cerebral blood flow and local reactivity of the cerebral vessels, and other changes are always revealed in such patients. Experimental studies showed disorders of the mechanisms of self regulation of brain cells to be the underlying factors of these posttraumatic changes. They are reflected in particular by stable changes of fermentative reactions determining the synthesis and catabolism of cyclic nucleotides. PMID- 3008469 TI - [Distribution of antibodies against the human T-cell leukemia virus in healthy Venezuelan adults]. PMID- 3008468 TI - Endothelial cell seeding of synthetic vascular prostheses. PMID- 3008470 TI - Preoperative aspiration cytology of breast tumors. AB - During a ten-year period, 1,942 aspirations of 1,906 mammary tumors in 1,874 patients were performed before excisional biopsy or mastectomy. The cytology findings were categorized as positive (1,107 cases), suspicious (152 cases), atypical (183 cases), benign (166 cases) and unsatisfactory (298 cases). All cytologically positive cases with follow-up were confirmed histologically or by clinical observation. Follow-up showed that 96% of the cases in the suspicious category, 86% of the cases in the atypical category, 51% of the cases in the benign category and 72% of the cases in the unsatisfactory category had malignant neoplasms. Aspiration cytology diagnosed 1,031 of 1,539 primary malignant mammary neoplasms (67%) and 19 of 28 neoplasms (68%) metastatic to the breast; if unsatisfactory cases are excluded, these figures become 1,031 of 1,365 cases (75%) and 19 of 25 (76%), respectively. If those cases reported as suspicious are included with the positive cases and those reported as atypical are included with the negative cases, aspiration cytology would have a sensitivity of 84% for the presence of carcinoma, a specificity of 97% for the absence of carcinoma, a predictive value of 99% for a positive diagnosis and a predictive value of 56% for a negative diagnosis; the diagnostic efficiency would be 86%. Our findings reaffirmed that the cytologic diagnosis of mammary carcinomas is reliable but that negative or inconclusive cytologic findings should not be regarded as a definitive diagnosis if there is clinical suspicion of a malignant neoplasm. PMID- 3008471 TI - Megakaryocytes in a hemorrhagic pleural effusion caused by anticoagulant overdose. AB - Sudden widespread hemorrhage, including a hemorrhagic pleural effusion, developed in a patient due to an overdose of anticoagulant. The pleural fluid contained megakaryocytes. Necropsy did not reveal myeloid metaplasia or any myeloproliferative disorder. Since megakaryocytes are normally found in pulmonary parenchyma, their presence in the pleural fluid was attributed to the hemorrhagic condition of the lung, which enabled megakaryocyte-containing blood to enter the pleural cavity. PMID- 3008472 TI - Viral changes versus those due to electrocoagulation therapy. PMID- 3008473 TI - Multinucleation versus "cell-in-a-cell" pattern. PMID- 3008474 TI - Cytomorphology and marker expression of malignant neuroendocrine cells in pleural effusions. AB - Three cases of pulmonary neuroendocrine carcinoma with malignant pleural effusions were retrospectively studied to determine if cellular morphology and expression of neuroendocrine markers were the same in the fluid as in the solid milieu. In fluids, changes were noted in cell grouping, shape and cytoplasm. Neuroendocrine markers expressed in both solid and dispersed tumors were neuron specific enolase (NSE) in all cases and leu-enkephalin in one case. Vasoactive intestinal polypeptide (two cases) and serotonin (one case) were detected only in the solid tumor. ACTH, bombesin and calcitonin were not expressed. We tentatively conclude that, in effusions, neoplastic neuroendocrine cells may alter their cytostructure, growth patterns and marker expression capabilities. NSE appears to be the most reliable neuroendocrine marker for use in small samples and with varying preparatory methods. PMID- 3008475 TI - ACTH and prolactin deficiency. AB - A 35 year old woman suffering from ACTH and prolactin (Prl) deficiency is described. Her symptoms of adrenal insufficiency appeared gradually after her first pregnancy in 1970; however, she conceived twice more and delivered healthy babies in 1972 and 1974, which she could not breast feed due to lack of milk. During an episode of pneumonia in 1977 she suffered acute adrenal insufficiency, after which she began treatment with hydrocortisone. Her pituitary reserve for TSH, GH, LH and FSH was normal, but her ACTH and Prl levels were undectable and did not respond to acute iv challenges of corticotrophin-releasing factor (CRF) and TRH, respectively. Autoantibodies, including antilactotroph titres, were negative, except for a positive pituitary immunofluorescence to ACTH. There was also no ACTH stimulation to a prolonged infusion of CRF followed by an acute iv bolus. These results, together with the gradual onset of symptoms which worsened after each pregnancy, suggest a possible autoimmune aetiology of her pituitary ACTH and Prl deficiencies. PMID- 3008476 TI - An intrasellar pituitary tumour producing metastases in liver, bone and lymph glands and demonstration of ACTH in the metastatic deposits. AB - We report the history, laboratory and histological findings in a man who presented with Cushing's disease. Despite removal of the primary pituitary tumour, his disease progressed and after bilateral adrenalectomy, he became pigmented and plasma ACTH levels remained elevated. He had further pituitary surgery and radiotherapy, to relieve compression of the optic chiasma. Plasma ACTH levels remained elevated. He developed liver, bone and lymph gland metastases and after an acute paraparesis due to spinal metastases he died. Immunoperoxidase staining techniques demonstrated ACTH in the pituitary recurrence and metastases. The combination of bone, liver and lymph node metastases has not previously been reported, nor has ACTH been reported before in metastases from a primary intrasellar tumour. PMID- 3008477 TI - Priming procedure and cell isolation for study on luteal cell response to peptide hormone in the rat. AB - The objective of this study was to develop a method of isolating luteal cells from the ovaries of immature rats pretreated with pregnant mare serum gonadotrophin (PMSG). After the ovaries were digested by collagenase and trypsin, the corpora lutea were obtained from the tissues, gently pressed in a test tube, and then placed on a sucrose density gradient. The two bands that appeared in the tube after centrifugation were designated S1 (top band) and S2 (bottom band). Progesterone and 20 alpha-dihydroprogesterone (20 alpha-DHP) secreted by the isolated cells during short-term incubation were measured by radioimmunoassay (RIA). A larger amount of progesterone, i.e., 60 to 260 ng/10(5) cells, was secreted by S1 cells than by S2 cells during the 18-h incubation. These results suggest that this simple procedure for isolation of luteal cells may provide a suitable model for in vitro studies of the luteal function. PMID- 3008478 TI - The effect of oestrogen and relaxin on uterine and cervical enzymes: collagenase, proteoglycanase and beta-glycuronidase. AB - Relaxin (Rlx) classically causes uterine quiescence during pregnancy and cervical dilatation prior to parturition. Its actions involve major changes in the components of the extracellular matrix of these tissues. The activities of three enzymes, collagenase, proteoglycanase and beta-glucuronidase, major determinants of the integrity of the extracellular matrix have been measured in the rat uterus and cervix in different reproductive states. The results show that there are marked differences in the changes of these enzymes occurring in the uterus and cervix during the course of pregnancy and the puerperium. It was not possible to directly relate these changes to a single hormonal event over this period of major endocrine fluctuations. Two models have therefore been used in an attempt to delineate the effects of oestrogen and Rlx on the tissue enzyme levels or their secretion into culture medium. In the first model cyclic animals were treated with oestrogen alone or oestrogen followed by Rlx and tissue enzyme levels measured. The addition of Rlx treatment reversed an inhibiting effect of oestrogen alone on both uterine and cervical collagenase and proteoglycanase activities, at the same time as completely obliterating the stimulating effect of oestrogen on uterine and cervical beta-glucuronidase activity. A second model used in vivo oestrogen priming of cyclic rats followed by in vitro Rlx treatment and measurement of the enzymes secreted into the culture medium over 7 days. The results showed as in the first model that Rlx treatment could in particular overcome the inhibiting effect of oestrogen on uterine proteoglycanase secretion without affecting beta-glucuronidase levels. In contrast, the effect of Rlx on the cervix was to decrease collagenase and proteoglycanase secretion whilst not affecting the beta-glucuronidase levels. PMID- 3008479 TI - Isolation, purification and culture of Sertoli cells from immature piglet testes. AB - Several studies suggest a role of Sertoli cells in the control of Leydig cell steroidogenesis. In order to verify this hypothesis, we have developed a system for the purification of pig Sertoli cells. These cells were then characterized by their morphological appearance in light and electron microscopy, their ability to bind [125I]follicle stimulating hormone (FSH) and their functional capacity as evaluated by adenosine 3',5' monophosphate (cAMP) accumulation and lactate production when in primary culture under basal and FSH-stimulated conditions. Crude Sertoli cell suspensions from immature porcine testes were fractionated on discontinuous Percoll gradients (densities 1.025, 1.039, 1.055, 1.080 g/ml). Highly purified Sertoli cells were contained in the second band (d: 1.039) generated on the gradient. These cells demonstrated morphological and functional integrity as evidenced by binding specifically [125I] FSH and by responding to FSH stimulation (by an increased production of cAMP and lactate after 3 days in primary culture), but not to human chorionic gonadotrophin (hCG). This preparation represents a useful model for the study of Sertoli cell functions and their interation with Leydig cells in the regulation of testicular steroidogenesis. PMID- 3008480 TI - Studies on shrew (Suncus murinus) epidermal growth factor. AB - The shrew SMG contains a very high level of EGF. By heterologous radioreceptor assay, the EGF content was determined to be about 300 pmol/mg wet weight in the male. The content is sex dependent; in the female gland, it is only 4% of the male gland level. EGF level and EGF receptor content in other tissues showed a reciprocal distribution pattern with the parotid gland containing the second highest level of EGF and the liver containing the largest amount of EGF receptor. Shrew EGF is heat stable. It could induce early eyelid opening and incisor eruption in neonatal rat pups at a very low dose level. There is apparently no high molecular weight form of EGF in shrew tissues and body fluids. However, gel chromatography revealed that the foetus contained multiple forms of EGF. The contribution of a high EGF level to the active life in adult shrews and the exceptionally high growth rate in shrew pups are discussed. PMID- 3008481 TI - Late-onset 3 beta-ol-hydroxysteroid-dehydrogenase deficiency: prevalence, clinical presentation and endocrine pattern. AB - 3 patients with late-onset 3 beta-HSD deficiency are described. They presented increased DHEAs/delta 4 ratio either in basal conditions or after ACTH stimulation. All patients were hirsute but only one had increased serum values of delta 4 and total testosterone. However, all patients presented increased serum levels of free testosterone. Two patients had normal menstrual cycles and in one case a secondary PCO was found. PMID- 3008483 TI - [Incidence of paraneoplastic syndromes in a series of 204 hepatocellular carcinomas]. PMID- 3008484 TI - [Anatomo-pathologic aspects of malignant tumors of the liver]. PMID- 3008485 TI - Prevention of hepatocellular carcinoma. PMID- 3008482 TI - [Clinical, biological and epidemiologic characteristics of hepatocarcinoma in the Brussels region]. PMID- 3008486 TI - Liver tumours associated with contraceptive hormonal treatment. PMID- 3008487 TI - [Primary tumors of the liver in the child]. PMID- 3008488 TI - Acquired immunodeficiency syndrome (AIDS). PMID- 3008489 TI - [Crystalline inclusions of the mouse thyroid. Effect of chronic treatment with lithium gluconate]. AB - Intracytoplasmic crystalline bodies of various sizes are found in thyroid cells of 10-month-old mice and in younger animals under chronic lithium treatment. They are frequently surrounded by small microvesicles and dense bodies or enclosed in larger vesicles having a dense content. The crystalline skeleton is a network of protein fibers assembled in a characteristic axis with a periodicity of 8 nm. A deficiency of thyroid cell metabolism related to aging or lithium gluconate treatment would lead to an accumulation of substances of a crystalline pattern. PMID- 3008490 TI - Characteristic inclusions in the kidney of canine globoid cell leukodystrophy. AB - The kidney of a 7-month-old male Cairn terrier with globoid cell leukodystrophy (GLD) was investigated with light and electron microscopes. A few tubular epithelial cells in the inner medulla as well as some exfoliated cells in the lumina revealed PAS-positive cytoplasm in which needle-like structures were to be seen on occasion. At the ultrastructural level, characteristic inclusions of GLD were found in these cells. This observation indicates that in addition to our previous report in the kidney of murine GLD (Takahashi et al. 1984), kidney in canine GLD also is a site of abnormal storage of galactosylceramide, although so far no morphological or biochemical evidence of galactosylceramide storage was demonstrated in human GLD. PMID- 3008491 TI - Characterization of amyloid deposits in biopsies of 15 with "sporadic" (non familial or plasma cell dyscrasia amyloid polyneuropathy. AB - Review of the clinical and laboratory findings of 39 patients with amyloid polyneuropathy (AP) showed 12 cases to be hereditary and 12 to be associated with plasma cell dyscrasia (PCD). The remaining 15, termed "sporadic" AP, had neuropathy clinically indistinguishable from the other two groups but without a clinicopathologically identified PCD or positive family history. In an attempt to identify the type of amyloid in "sporadic" AP, the immunoreactivity of amyloid deposits was investigated using specific antisera raised against the following different chemical types of amyloid fibril proteins: variable regions of amyloid light chains kappa (A kappa) and lambda (A lambda), amyloid protein AA, and prealbumin. It was found that the amyloid in "sporadic" AP had A lambda antigenic determinants in ten cases, A kappa in one and prealbumin in three; in one case, the A lambda nature of amyloid was confirmed biochemically on the extracted amyloid fibrills. Thus, the most common type of AP in our population appears to be the "sporadic" form. In "sporadic" AP, the amyloid is most commonly of immunoglobulin light chain origin, even in the absence of overt PCD, and it can be rapidly categorized immunocytochemically to determine therapeutic directions or provide genetic guidance. PMID- 3008493 TI - A prospective study of rotavirus infections in neonatal and maternity wards. AB - The occurrence and symptomatology of rotavirus infections was studied at three maternity wards and one neonatal unit. Rotavirus was identified in 12.7% of 553 infants and 1.3% of 542 mothers at the maternity wards. Infections were more frequent in a mixed obstetric/gynecology ward than in the pure obstetric wards. Only 10% of the infants had symptomatic infections. Subgroups of rotavirus was determined in 41 infants: 22 of subgroup I and 19 of subgroup II, which is the subgroup accounting for the majority of childhood gastroenteritis. Rotavirus was found in faecal samples from 37% of the infants at the neonatal unit during an eight-month survey. A seasonal variation with most infections during colder months was seen. Subgroup determination was possible in 29 cases, 14 subgroup I and 15 subgroup II. Fifteen per cent of the infections demonstrated diarrheal symptoms. No significant difference among other clinical data registered was seen among rotavirus infected compared to the non-infected infants. We conclude that neonatal rotavirus infections occur as an endemic infection at our maternity wards possibly combined with infections due to external sources of virus in mixed wards and neonatal units. PMID- 3008492 TI - Immunocytochemical binding of serum IgG from a patient with oat cell tumor and paraneoplastic motoneuron disease to normal human cerebral cortex and molecular layer of the cerebellum. AB - Serum from a patient with oat cell (OC) tumor and paraneoplastic upper and lower motoneuron syndrome showed binding of IgG but not IgM to normal human cerebral cortex (CC), molecular (M), and Purkinje (P) cell layers of the cerebellum, anterior horn cells (AHC), and dorsal root ganglia (DRG). There was no binding to glial and granular cells, white matter, peripheral nerve, or OC. Sera from three patients with OC tumor without neurologic deficit, three patients with non-OC lung cancer and peripheral neuropathy, and five healthy subjects were used as controls. While none of the control sera showed binding to the CC or M layer, the three controls with OC showed 50% reactivity with AHC, and other controls showed weak staining of PC, AHC, or DRG. Absorption of the patient's serum with cerebral or cerebellar tissue, but not with liver or spinal cord, resulted in elimination of the immunostaining. Staining of the CC and M layer could not be blocked by a monoclonal IgG to a glioma cell line, but partial blocking occurred by preincubating the tissue with monoclonal IgG (MF 491) to gastric carcinoma and cross-reacting with OC and several neural elements. The results suggest specific binding of the patient serum IgG to the CC and M layer; however, the relationship of this antibody to the pathogenesis of the paraneoplastic syndrome remains elusive. PMID- 3008494 TI - Severe pancreatitis and fatty liver progressing to cirrhosis associated with Coxsackie B4 infection in a three year old with alpha-1-antitrypsin deficiency. AB - A 3-year-old girl in whom severe acute pancreatitis was associated with evidence of Coxsackie B4 virus infection was alpha-1-antitrypsin deficient. Lack of this modulator of proteolysis may have been responsible for her severe course. Fatty liver at presentation progressed to cirrhosis in the ensuing 18 months. PMID- 3008495 TI - Malignant fibrous histiocytoma with widespread metastasis and pulmonary cancer. AB - An autopsy case of malignant fibrous histiocytoma (MFH) with widespread metastases and lung carcinoma in a 64-year-old Japanese woman is reported. The initial signs were cough and sputum, followed by hemosputum. A chest X-ray photo showed a right pleural tumor, which could not be identified from a biopsy specimen, but was identified as MFH by light and electron microscopic studies on biopsy specimens of tongue tumors. Autopsy examination revealed metastases of the MFH to the brain, lung, liver, kidney, adrenal, pancreas, retroperitoneum, and some bones, and pulmonary adenocarcinoma. PMID- 3008496 TI - A Japanese Burkitt's lymphoma with t(2;8) and EBNA. AB - A 56-year-old Japanese man was admitted to the hospital with a large mass in his right axilla. Histological investigation revealed that it was a Burkitt's lymphoma. Ultrastructures of the tumor cells showed immature lymphoid features with frequent lipid droplets within the cytoplasms. Virological studies before the treatment revealed that the lymphoma was closely related to EB virus infection, being positive for EBNA (more than 95% of all tumor cells) and IgG antibodies to VCA (X 5,120), EA (X 640), and EBNA (X 160). The tumor cells exhibited low levels of cytoplasmic IgM and other properties of B cells. They were positively stained with L26, L27, Leu 14, and HLA-DR MAb. In cultured tumor cells, L25 and CALLA antigens were demonstrated, but no surface Ig was shown. In contrast, the tumor cells were negative for T cell markers including AcP, E receptor, and Leu 1, 2, and 3. Cytogenetic studies demonstrated that the karyotype of the tumor cells was 46, XY, dup (1q), t(2;8)/46, X, -Y, t(2;8), +mar. VEMP therapy was immediately conducted. However, following a two-month partial remission, a relapse with bone marrow infiltration occurred. Thus, a case of Japanese Burkitt's lymphoma with EBNA (+) and t(2;8) properties is described, and the relationships among primary sites, phenotypes, and genotypes are discussed. PMID- 3008498 TI - Adrenocortical adenoma with Cushing's syndrome in culture. AB - Two adrenocortical adenomata with Cushing's syndrome were examined employing cell culture methods. The results revealed that the clear-type cells changed into compact-type cells, and that the compact cells played a role in the production and secretion of steroid hormones. Furthermore, it is likely that they underwent fragmentation and produced collagen fibrils. PMID- 3008497 TI - Immunohistochemical expression of mammary tumor virus antigens in mammary glands of virgin mice, in relation to Mtv genes. AB - MTV antigens in the resting mammary glands of GRS/A, SHN, C3H, and Balb/c virgin mice were detected by immunoperoxidase techniques using antiserum against MTV whole virion, gp 52 or p 25 to differentiate the expression between endogenous and exogenous MTV. Balb/c mice were crossed to infect MTV into each reciprocal hybrid or fosternursed inbred. Immunohistochemical stainings of gp 52 in the formol-fixed sections were almost the same as those of the whole MTV virion, and the results on various cases were as follows: In the mice with endogenous GR-MTV, positivity was first observed at the age of 14 days, while the first expression of exogenous GR-MTV was delayed to the age of 140 days. The mice with endogenous and/or exogenous SHN-MTV showed the first antigen appearance at the age of 65 days, and those with exogenous C3H-MTV did at the age of 80 days. The virgins with only endogenous C3H-MTV came to express the antigen after the age of 200 days. Staining of p 25 in Carnoy-fixed sections of MTV-positive mammary glands was found in the supranuclear cytoplasm and apical surface of the glandular cells and the lumen, all of which are the site of A and B particles. By means of preembedding method for gp 52, the reaction products were ultrastructurally detected not only on the MTV-budding apical surface, together with the intraluminal B particles, but also on the MTV-free apical cell membrane of the glandular cells in the mammary gland of the GR virgins. PMID- 3008499 TI - Cystosarcoma phyllodes. A review of 19 cases with emphasis on the occurrence of associated breast carcinoma. AB - The clinico-pathological aspects of 19 cases of cystosarcoma phyllodes (CP) were reviewed with special attention to the occurrence of associated breast carcinoma (BC). Twelve women had histologically benign, 4 had borderline and 3 malignant CP. Recurrent CP was diagnosed in 9 women within 1 to 15 years after initial treatment. Of the 19 women, 5 had associated in situ or invasive BC, 2 with the primary CP and 3 with its recurrences. In 1 case the BC was located within the confines of the CP, whereas in 4 women the CP and BC were separate lesions. The frequency of incidentally found BC in this group of women with CP does not support the hypothesis of an increased risk of BC development in women with CP. PMID- 3008500 TI - Is a relay mechanism of antioxidant effect of tocopherols valuable for membrane systems? AB - The efficiency of the inhibitory action of 3 tocols derivatives--alpha-tocopherol (alpha-T), 2, 2, 5, 7, 8-pentamethylchromane (PMC) and alpha-tocopherylacetate (alpha-TA) on lipid peroxidation (LPO) in rat liver microsomes was compared. LPO was induced by O2-generating systems (Fe2+ + NADPH, Fe2+ + ascorbate) or by ROO generating system (Fe2+ + tert-butyl hydroperoxide). It was found that PMC was much more potent LPO inhibitor than alpha-T, whereas alpha-TA was ineffective in all LPO-initiating systems used. The protective effect against cytochrome P-450 (cyt. P-450) degradation induced by LPO-products was also maximal for PMC and minimal for alpha-TA. The data obtained suggest that the presence of phytyl radical is not necessary for antioxidant activity of tocols and contradict the hypothesis about a relay mechanism of antioxidant action of tocopherols in biomembranes. PMID- 3008501 TI - On the role of taurine in the cerebellar cortex: a reappraisal. AB - Certain amino acids are now widely accepted as constituting one of the major groups of neurotransmitters in the mammalian central nervous system. However, although the available data suggest that taurine may well be involved in central synaptic transmission, its precise neurohumoral role in many areas is still poorly understood. Some of the prerequisites for the identification of chemical transmitters have been fulfilled by taurine in various central structures, including the cerebellum, but data are still inconclusive concerning its possible role either as a classical neurotransmitter or as a modulator of neuronal excitability. The synaptic role of taurine in the cerebellum is supported by: 1) its high levels associate with the synaptic fraction, 2) the identification of a high-affinity uptake mechanism, 3) its inhibitory effects exerted upon Purkinje cells, and 4) the calcium-dependence of its stimulus-induced release. However, the criterion of identity of action has not been demonstrated and a specific taurine receptor has not yet been identified. Furthermore, the observed calcium dependence of taurine efflux may be explained as a secondary result of the release of endogenous glutamate by an inhibitory feed-back mechanism acting through autoreceptors. Therefore, although the present data support a possible involvement of taurine in central neurotransmission, its precise synaptic role remains to be established. PMID- 3008502 TI - Estradiol binding and phosphatase activity in the uterus after castration and chronic diabetes: effect of molybdate and estrogen replacement. AB - We have studied the cytosolic estrogen receptor in uterus of rats after castration and diabetes induction with Streptozotocin, and the relationship of estradiol (E2) binding with phosphatase activities. Ovariectomy (OVX) and diabetes produced a significant reduction in Type I and II binding sites, but did not affect the equilibrium dissociation constants. The reduction of receptor levels was partially reversed by homogenization and incubation with 20 mM molybdate (MoO4) and also by chronic treatment with E2. Considering the possibility that the increase in E2 binding in the presence of MoO=4 was due to phosphatase inhibition, the activities of these enzymes hypothetically involved in receptor dephosphorylation and inactivation were determined in uterine homogenate and cytosol from intact, OVX, and diabetic rats with and without E2 treatment. Chronic OVX and diabetes induced stimulation of alkaline, acid and neutral phosphatase activities. On the contrary, the increment of estrogenic receptors due to E2 treatment was not correlated with changes in phosphatase activity. It is possible that this effect was due to the protection of the receptor or to the induction of receptor synthesis by E2. Molybdate inhibited acid and neutral phosphatases and increased alkaline phosphatase, which suggest that neutral and acid phosphatases are identical and that they were responsible for the receptor inactivation. However, it is unclear at present the relationship between the increment of alkaline phosphatase and the reduction of receptors. PMID- 3008503 TI - The effect of alkalosis on the glucose-mediated insulin release by the rat. AB - Bicarbonate loaded (alkalotic) rats had reduced plasma phosphate and ionized Ca concentrations and increased urinary cAMP excretion, phosphate clearance and pancreatic tissue uptake of extracellular calcium, all known effects of increased parathyroid hormone (PTH) secretion. Total insulin secretion after glucose challenge was enhanced in these animals. The response of alkalotic thyroparathyroidectomized rats, on the other hand, suggested exhaustion or inhibition of insulin secretion. The hypothesis is advanced that PTH, enhancing the permeability to calcium of the beta-cell membrane, compensated the effect of decreased calcium concentration in the extracellular fluid caused by alkalosis. PTH appears instrumental for homeostatic adjustments of insulin secretion. PMID- 3008504 TI - Vagally induced non-adrenergic, non-cholinergic contractions in the feline small intestine--an involvement of opioid receptors? PMID- 3008505 TI - The effect of hypoprotein nutrition upon granular cationic proteins and myeloperoxidase and lactic dehydrogenase enzyme activities in rat peripheral blood granulocytes. Study I. PMID- 3008506 TI - Organ distribution of asialo-red blood cell ghosts: an attempt at targeting to the liver. AB - We investigated the organ distribution of four types of red blood cells (RBC) preparations: native RBC, asialo-RBC, native ghosts and asialo-ghosts. Intravenously injected asialo-ghosts were rapidly removed from the blood stream and accumulated mainly in the liver 120 min after the injection. Our results suggest that asialo-ghosts are a simple and effective carrier for targeting of drugs to the liver. PMID- 3008507 TI - Diagnostic contribution of scanning in focal ischemia of the cerebral hemispheres. PMID- 3008508 TI - Multicomponent analysis of reflection spectra from the guinea pig heart for measuring tissue oxygenation by quantitative determination of oxygen saturation of myoglobin and of the redox state of cytochrome aa3, c, and b. PMID- 3008509 TI - Myocardial ischemic injury and beta-adrenergic receptors in perfused working rabbit hearts. PMID- 3008510 TI - An upper bound on the minimum PO2 for O2 consumption in red muscle. PMID- 3008511 TI - Skeletal muscle capillarity in hyperthyroid and hypothyroid rats. PMID- 3008512 TI - Reticulocytosis and bone marrow cAMP level in rats following physical exercises. PMID- 3008513 TI - Cerebral bioenergetics and in vivo cytochrome c oxidase redox relationships. PMID- 3008515 TI - Non-invasive, near infrared monitoring of cellular oxygen sufficiency in vivo. PMID- 3008514 TI - The near infrared (NIR) absorption band of cytochrome aa3 in purified enzyme, isolated mitochondria and in the intact brain in situ. PMID- 3008516 TI - Monitoring of cerebral oxygenation in the intensive care nursery. PMID- 3008517 TI - Monitoring cerebral oxygen sufficiency in anesthesia and surgery. PMID- 3008518 TI - Near infrared optical monitoring of intact skeletal muscle during hypoxia and hemorrhagic hypotension in cats. PMID- 3008519 TI - Near infrared spectrophotometry: potential role during increased intracranial pressure. AB - Two experiments were conducted to assess the feasibility of near infrared spectrophotometry (niroscopy) to directly monitor the effects of increased intracranial pressure on brain metabolism. Intracranial pressure (ICP) was increased in cats by subarachnoid infusion of a "mock" CSF solution. Cytochrome a,a3 redox state, oxyhemoglobin, deoxyhemoglobin and cerebral blood flow were non invasively and continuously monitored by niroscopy. The results of both experiments indicated that untreated increases in ICP correlated with a reduction in cytochrome a,a3 redox state (p less than 0.01), a decrease in the quantity of oxyhemoglobin and cerebral blood flow (p less than 0.01), and an increase in deoxyhemoglobin. This study suggests that niroscopy has the potential for providing noninvasively and continuously data assessing brain metabolic activity. The excellent correlations obtained with simultaneous direct measurements of intracranial pressure make this an attractive method for eventual application to humans at risk for increased intracranial pressure. The value of niroscopy is even more evident in Exp. II where it can be seen that knowledge only of ICP would give the physician a false sense of security, whereas direct, non-invasive, continuous assessment of brain perfusion and oxygenation may well prove to be more appropriate parameters to monitor. PMID- 3008520 TI - Continuous non invasive monitoring of human brain by near infrared spectroscopy. PMID- 3008521 TI - The molecular genetics of components of complement. AB - Rapid progress has been made in establishing linkages and in chromosome allocation of the genes of some 9 complement components. In the MHC, C2, Factor B, and two C4 or C4 related genes have been placed in some detail in both man and mouse. The gene coding for the cytochrome P-450 21-hydroxylase has been shown to be duplicated and immediately 3' to the two C4 genes, though it appears to be functionally and structurally unrelated to the complement components. Thus six genes have been mapped to this region where particular haplotypes are associated with increased susceptibility to a number of diseases, some of which are autoimmune in character. The complete gene structure of Factor B has been solved in man and rapid progress is being made with the C2 and C4 genes. The structural basis of the polymorphisms of these genes is being established. In C4, the polymorphism is exceptionally complex with varying numbers of loci and probably more than 50 allotypes occurring in man. A structural basis has also been found for the big differences in the biological activity of some of the C4 allotypes in man. Apart from the genes in the MHC, linkage has been found between the genes coding for C4bp, CR1, and Factor H. Remarkably there are sequence homologies between these proteins and C2 and Factor B, probably related to the ability to bind to one or other of the structurally similar proteins C3b and C4b. The complete cDNA sequences of C3 and C4 in mouse and man have given much information on the many posttranslational modifications of these proteins. A partial structure has been obtained for the C3 gene and the homology shown between C3, C4, C5, alpha 2-macroglobulin, and pregnancy zone protein. Although the amount of detailed information in the molecular genetics of complement components is accumulating rapidly, there appears to be a reasonable prospect that linkages and homologies will classify the data into a comprehensible form. PMID- 3008522 TI - Human lymphocyte hybridomas and monoclonal antibodies. PMID- 3008523 TI - The role of avian retroviral LTRs in the regulation of gene expression and viral replication. PMID- 3008524 TI - Marek's disease virus. PMID- 3008525 TI - Seasonal changes in the biochemical indices of vitamin D deficiency in the elderly: a comparison of people in residential homes, long-stay wards and attending a day hospital. AB - The seasonal changes in the biochemical indices of vitamin D nutrition have been measured in elderly people with differing requirements for institutionalized care. Residents of local authority homes (LAH) showed an increase in serum 25 hydroxyvitamin D3 [25(OH)D3] between spring and autumn (means 14-17 nmol/l, P less than 0.002). No significant seasonal changes were seen in patients on long stay wards [(GW) serum 25(OH)D3 9.5 and 9.5 nmol/l] and in day-hospital attenders [(GDH) 25 and 26.8 nmol/l]. Significant differences (P less than 0.02 to P less than 0.0001) were found between the mean serum 25(OH)D3 amongst the three groups. A significant linear relationship (r = 0.84, P = 0.036) was found between mean serum 25-hydroxyvitamin D2[25(OH)D2] and dietary vitamin D2. The intake of vitamin D was suboptimal in all groups. The incidence of 25-hydroxyvitamin D deficiency [25(OH)D less than 12.5 nmol/l] varied from 11.7% of residents in LAH in autumn to 47% of GW patients in spring; but hypocalcaemia occurred less often (LAH 1.3% in autumn, GW 4.7% in spring). The diet assumes a greater role in protecting against vitamin D deficiency when the total 25(OH)D is low. Because most diets contain insufficient amounts of vitamin D, elderly institutionalized people will remain at high risk of developing vitamin D deficiency unless specific preventative measures are adopted. PMID- 3008526 TI - Vasoregulin, a glucocorticoid-inducible vascular permeability inhibitory protein. AB - A vascular permeability inhibitory protein 'Vasoregulin' was induced by dexamethasone and other glucocorticoids in Namalva cells. Precipitates produced by trichloroacetic acid from cultured supernatant (crude released vasoregulin) suppressed serotonin-induced paw edema of mice (ID30 = 750 micrograms/mouse) when dosed 30 min. before serotonin. Carrageenan paw edema of rats was also suppressed (ID30 = 9 mg/rat, s.c. and i.p., not by oral dosing) when the compound was injected at the same time as carrageenan and the edema determined after 3 hrs. The possible effect of glucocorticoid itself being contained in the vasoregulin preparation was excluded. The presence of Cu, Zn-superoxide dismutase (SOD), spermidine and spermine also gave good yields of vasoregulin in culture; SOD protected the inactivation of vasoregulin from superoxide radical in vitro. Vasoregulin partially purified by Sephadex G-75 columns had no malonaldehyde formation inhibitory capacity. Thus, vasoregulin is a glucocorticoid-inducible anti-inflammatory protein which differs from lipocortin. PMID- 3008529 TI - Determinants of inflammation test selection: in vitro or in vivo? PMID- 3008527 TI - Multiple control of inflammation by glucocorticoids. PMID- 3008528 TI - Glucocorticoid induction of angiotensin converting enzyme. AB - Angiotensin converting enzyme (ACE) converts angiotensin I (Angio I) to angiotensin II (Angio II) and inactivates bradykinin (BK). Glucocorticoids in the physiological range increase ACE in rabbit alveolar macrophages and bovine endothelial cells in culture. Since Angio I and BK are cleaved by ACE catalysis during passage through the pulmonary vasculature we have studied the steroid modulation of ACE in the rat lung. The conversion of Angio I to Angio II by isolated lungs from normal or adrenalectomized male Wistar rats has been evaluated. The initial conversion of Angio I to Angio II in lungs from normal rats was about 60%. In contrast the initial converting activity in lungs from adrenalectomized rats was about 30%. In both groups the converting activity progressively decreased. After 3 h it was about 30% in normal lungs and virtually undetectable in lungs from adrenalectomized rats. Dexamethasone infusion (1 microgram/ml) prevented the decrease in ACE activity observed in normal lungs and induced a gradual enhancement of converting activity in lungs from adrenalectomized animals up to the control level. The effect of dexamethasone was abolished by simultaneous infusion of cycloheximide (1 microgram/ml). These results demonstrate that glucocorticoids induce ACE synthesis in the rat lung. By this induction glucocorticoids promote the increase of both Angio II formation and BK degradation. Thus ACE induction may represent a possible mechanism whereby glucocorticoids might control vascular tone and permeability according to the general mode of action of steroid hormones. PMID- 3008530 TI - Synthesis of eicosanoids by tissues of the synovial joint during the development of chronic erosive synovitis. PMID- 3008531 TI - Selective inhibition of leukotriene B4 formation by Ebselen: a novel approach to antiinflammatory therapy. PMID- 3008532 TI - The cellular immune response in thermal injured patients. PMID- 3008533 TI - Production of leukotriene B4 by polymorphonuclear leukocytes during phagocytosis of Staphylococcus aureus. PMID- 3008534 TI - Changes in guinea pig lung beta-adrenoceptor function by Haemophilus influenzae and its mediation by specifically stimulated pulmonary macrophages. PMID- 3008535 TI - The involvement of reactive hydroxyl radicals in Haemophilus influenzae-induced deterioration of guinea pig lung beta-adrenergic receptor function. PMID- 3008536 TI - Radioisotopic evaluation of adenocarcinoma and solitary cyst of the kidney. PMID- 3008537 TI - [Studies on the endocrinological metabolism of the parathyroid. I. The production of renal calcinosis by cyclic AMP injection in rat]. AB - We found that a few patients with urolithiasis had normal parathyroid hormone levels but high cyclic AMP excretion. The purpose of this paper was to study the endocrinological mechanism. Male rats were given intraperitoneally dibutyryl cyclic AMP (DBcAMP), a derivative of cyclic AMP, per 100 gm of body weight for 50 days. Feed and water were supplied ad libitum. Crystal formation or calcification in mainly the dystal tubules and collecting system were found in 3 out of 10 rats, and renal calcium stones in 2 rats. The cyclic AMP of the renal parenchyma, especially the renal medulla, was elevated by more than 100 times after DBcAMP administration. Serum calcium levels, urinary calcium and phosphate excretion, and the adrenaline levels of the renal parenchyma were significantly increased. Serum parathyroid hormone was slightly enhanced, but vitamin D and the noradrenaline levels of the renal parenchyma were not changed. Based on these findings, it is suspected that stone formation in rats injected DBcAMP occurs through the action of DBcAMP on the renal tubules to increase urinary calcium excretion and to make renal stones as a form of primary hyperparathyroidism. PMID- 3008538 TI - [Studies on the endocrinological metabolism of the parathyroid. II. Influence of ACTH on parathyroid function and calcium metabolism]. AB - Parathyroid hormone (PTH) is strongly concerned with the pathogenesis of urinary stones. PTH is mainly regulated by the serum calcium concentration and not by other hormones, as is usually the case. We studied whether PTH is also regulated by adrenocorticotrophic hormone (ACTH) or not. ACTH (0.25 mg) was injected intravenously to 17 patients with primary hyperparathyroidism PHP, 7 patients with urolithiasis, 7 patients with malignant hypercalcemia, and 6 control subjects. Serum calcium was significantly increased in only PHP. The serum calcium increase rate showed a significant positive correlation with serum alkaline phosphatase, and a negative correlation with the preinjected serum calcium. PTH was slightly increased in all four groups. Serum cortisol and ACTH concentrations were not significantly different among the groups. PTH concentration in a culture medium of parathyroid tissues increased after ACTH addition. Serum calcium was significantly increased after ACTH injection in an adrenalectomized rat, and decreased in a parathyroidectomized rat. From our data and those of others, it appears that ACTH acts on the adrenal glands to decrease the serum calcium concentration, and might act directly on the parathyroid gland or bones to increase it. PMID- 3008539 TI - [Epidemiological and therapeutic studies of gonorrheal infection--clinical efficacy of sultamicillin]. AB - We conducted an epidemiological study including analyses of background factors of 192 male and 13 female patients with gonorrheal infection in the Sapporo area and at the same time, investigated the therapeutic efficacy of sultamicillin, an ester linked prodrug of ampicillin and beta-lactamase inhibitor sulbactam in the treatment of these patients. The percentage of infections in Sapporo was rather high in the young generation, being as high as 13.5% in teen-age boys and 30.8% in teen-age girls, which were higher than the 6.1% and 6.3% of corresponding groups in Honshu island. The source of infections was so-called special public bath-ouse which accounted for about 31.8% of all cases which however, was lower than the 50% in Honshu island. By contrast, the percentage of their friends or so called pick-up friends as a source of infection in Sapporo was as high as 46.9% which was significantly higher than the 19.9% in Honshu. Juveniles who had nonprostitutes of the other sex as a source of infection are a characteristic of the patients in Sapporo. The isolation rate of PPNG was 13.8%. The MIC (10(6) CFU/ml) of sultamicillin ranged from 0.05 to 0.39 micrograms/ml in beta-lactamase non-producing strains and from 0.20 to 1.56 micrograms/ml in beta-lactamase producing strains showing no trend of higher MIC against beta-lactamase producing strains. There was almost no difference in the efficacy of sultamicillin between a daily dose of 750 mg (2 tablets) and 1125 mg (3 tablets) nor in side effects. The eradication rate (efficacy rate) of gonococcus following a 3-day therapy was 96.2% (38.9% excellent cure rate) in male patients and 83.3% (8.3%) in female patients. In 31% of the male patients who underwent a 7-day therapy, residual serous secretion was found though some inaccuracy is involved in this data since dropouts were not counted. This suggests the need of concurrent therapy with other appropriate drugs in consideration of possible mixed infection involving Chlamydia trachomatis or other microorganisms. PMID- 3008540 TI - Hepatocellular carcinoma presenting as primary extrahepatic mass on CT. AB - Seven cases of hepatocellular carcinoma (hepatoma) (HCC) presenting as primary extrahepatic masses on CT are reported. All cases were diagnosed at the time of percutaneous biopsy, surgical resection, or autopsy. In none of the cases was the final diagnosis of HCC prospectively suspected on the basis of clinical and radiologic findings. Although three of the patients were at higher risk for development of HCC because of their medical histories, the absence of an elevated serum alpha-fetoprotein level and the extrahepatic location of the masses by CT suggested other disease. This variable pattern of radiologic presentation of HCC should be kept in mind during the evaluation of patients with suspected HCC, or when the findings on abdominal/pelvic CT of mass and presumed hepatic metastases are at variance with the clinical presentation. PMID- 3008541 TI - Clinical value of blood pool radionuclide venography. AB - Blood pool radionuclide venography and contrast venography were carried out in 50 patients in whom deep vein thrombosis was suspected. The two procedures were compared on the basis of 198 individual vessel segments. The overall concordance between radionuclide and contrast venography was 92%. Using contrast venography as the standard, the sensitivity of radionuclide venography was 90% and specificity 96%. The predictive accuracy of a positive test was 77% and of a negative test was 97%. The performance of radionuclide venography improved if the calf veins were excluded from analysis. The main limiting factor affecting the performance of radionuclide venography was the moderately poor resolution of the technique. Technical manipulations to improve resolution were time consuming and added little to the accuracy of the test. Radionuclide will not replace contrast venography but may well be used to complement contrast venography when it is technically unsatisfactory or unequivocal, in patients with a history of intolerance to contrast media, and in bedbound patients. PMID- 3008542 TI - MRI of Wilms' tumor: promise as the primary imaging method. AB - The magnetic resonance appearance of Wilms' tumor in 14 patients is described and its clinical utility is evaluated. In all cases, magnetic resonance was correlated with surgical and pathologic findings to assess accuracy. Magnetic resonance accurately identified the primary tumor and its renal origin in all cases, and tumor margins and local extension were accurately demonstrated. Tumor margins were smooth and well defined in nine of 12 cases. Local extension and size were accurately assessed, but because capsular invasion could not be predicted, four surgically proven instances of capsular invasion were missed. Metastatic spread into the liver and inferior vena cava was well documented in four cases and excluded in 10. Magnetic resonance was sensitive for identifying lymph-node enlargement in five of 14 cases, but could not predict the etiology of the enlargement. All Wilms' tumors had signal intensities consistent with prolonged T1 and T2 relaxation times. Signal intensity was highly variable, mainly because of necrosis and hemorrhage within the tumor. Magnetic resonance based on signal intensity could not distinguish Wilms' tumor from other solid renal tumors. Magnetic resonance has the potential for providing the same information as computed tomography, sonography, liver spleen radionuclide scanning, and excretory urography. Although expensive, magnetic resonance will be cost-effective if it can replace all the above techniques. This limited study indicates that magnetic resonance has promise as the primary imaging technique for Wilms' tumors. PMID- 3008543 TI - Chemical shift imaging: a review. AB - Chemical shift is the phenomenon that is seen when an isotope possessing a nuclear magnetic dipole moment resonates at a spectrum of resonance frequencies in a given magnetic field. These resonance frequencies, or chemical shifts, depend on the chemical environments of particular nuclei. Mapping the spatial distribution of nuclei associated with a particular chemical shift (e.g., hydrogen nuclei associated with water molecules or with lipid groups) is called chemical shift imaging. Several techniques of proton chemical shift imaging that have been applied in vivo are presented, and their clinical findings are reported and summarized. Acquiring high-resolution spectra for large numbers of volume elements in two or three dimensions may be prohibitive because of time constraints, but other methods of imaging lipid of water distributions (i.e., selective excitation, selective saturation, or variations in conventional magnetic resonance imaging pulse sequences) can provide chemical shift information. These techniques require less time, but they lack spectral information. Since fat deposition seen by chemical shift imaging may not be demonstrated by conventional magnetic resonance imaging, certain applications of chemical shift imaging, such as in the determination of fatty liver disease, have greater diagnostic utility than conventional magnetic resonance imaging. Furthermore, edge artifacts caused by chemical shift effects can be eliminated by certain selective methods of data acquisition employed in chemical shift imaging. PMID- 3008544 TI - Thorotrast-induced hepatosplenic neoplasia: CT identification. AB - Despite discontinuation of its use in the 1950s, the consequences of Thorotrast usage continue to be recognized. In a review of plain film and CT findings of 20 cases of Thorotrast exposure, 15 of 17 patients demonstrated Thorotrast accumulation in the liver and spleen on plain films. Typically, this appeared as regions of trabeculated increased density within the liver. The spleen was of normal or decreased size and often demonstrated a finely punctate pattern of opacification. Only two malignancies could be suggested on plain film alone: one hepatic and one splenic. Five patients with hepatic malignancies underwent CT examinations: three with cholangiocarcinoma, one with angiosarcoma, and one with hepatocellular carcinoma. No specific criteria could be established to distinguish among these lesions, as each neoplasm appeared as relatively low density mass(es). Two cases of splenic angiosarcoma appeared as low-density filling defect(s) in the otherwise opaque spleen, one case primary and the other metastatic from the liver. CT was superior to plain radiography in detecting and characterizing Thorotrast distribution and any superimposed malignancy. In addition to periodic liver function tests, screening CT of patients exposed to Thorotrast might detect hepatic neoplasms at an operable stage. PMID- 3008546 TI - Current surgical options in carcinoma of the breast. PMID- 3008545 TI - Leukotrienes and their possible significance for the pathogenesis of asthma. AB - Six years ago the structure of slow reacting substance of anaphylaxis (SRS-A), a strongly bronchoconstrictive substance, has been unravelled SRS-A proved to be a mixture of different closely related compounds, now denominated as sulfidopeptide Leukotrienes. Leukotrienes possess a conjugated triene system and one or more oxygen functions. They are formed from membrane derived arachidonic acid by an initial oxygenation by the enzyme lipoxygenase. Sofar the following leukotrienes have been characterized leukotriene A4, B4, C4, D4, E4 and F4. Leukotrienes possess important biological properties. Leukotriene B4 is strongly chemotactic for leukocytes, whereas the sulfidopeptide leukotrienes C4, D4 and E4 are strongly spasmogenic. In this review the formation and the different biological activities of leukotrienes and the possible role of leukotrienes in the asthmatic process will be discussed. PMID- 3008547 TI - Effect of hypertonic sodium bicarbonate on encainide overdose. PMID- 3008548 TI - Studies on cardiovascular actions of Salvia miltiorrhiza. AB - Cardiovascular actions of S. miltiorrhiza (SM) were studied on systemic blood pressure in the rat. Langendorff cardiac preparation in the guinea pig, and four types of vasculature in the dog, including coronary, renal, femoral, and mesenteric arteries. SM induced dose-related hypotension without changing heart rate. The hypotension was antagonized by atropine, propranolol, and chlorpheniramine plus cimetidine. In the isolated whole-heart preparation, SM increased coronary blood flow significantly for 15 min and positive inotropic action for 3 min after pulse injection. SM relaxed all arteries at low concentration (3.0 mg/ml) and contracted all but the coronary artery at higher concentration (10.0 mg/ml). The coronary artery relaxed at all doses of SM tested. PMID- 3008549 TI - Failure to thrive in children with hyperphosphatasia. PMID- 3008550 TI - Transcatheter arterial embolization for spontaneous rupture of hepatocellular carcinoma. AB - For the last 6 yr, we had 47 cases of spontaneous rupture of hepatocellular carcinoma causing hemorrhage into the peritoneal cavity. Thirty-three cases were treated by supportive care and 14 cases by emergency transcatheter arterial embolization. Fourteen cases underwent emergency transcatheter arterial embolization and abdominal pain developed in 71.4%, shock in 50.0% and bloody ascites in all cases and the hematocrit of bloody ascites ranged between 1.0% and 40%. On hepatic angiogram, the mean tumor extent rate was 47.9% and the extravasation of the contrast material was noted in 35.7%. The mean survival time of 14 cases that underwent emergency transcatheter arterial embolization was 98.5 days and 13.0 days in 33 cases with supportive care. The former was significantly longer than the latter. PMID- 3008552 TI - Fiber size and number in workers exposed to processed chrysotile asbestos, chrysotile miners, and the general population. AB - We analyzed chrysotile and chrysotile-associated amphibole (largely tremolite) asbestos fibers in 21 workers exposed to various types of processed (milled) chrysotile ore, 20 long-term chrysotile miners, and 20 members of the general population (controls). Significantly greater amounts of both chrysotile and tremolite were found in processed-ore workers and miners than in controls. On average, the mean fiber lengths and aspect ratios for the mining and processed ore-exposed workers were similar and were significantly greater than the values seen in the controls; within the processed-ore group, there was a marked variation in these parameters, and some workers appeared to be exposed to fairly long, thin fibers. It was found empirically that the fiber size data, and to a lesser extent the concentration data, could be used to classify workers accurately into those with processed-ore exposure and controls. We conclude that fiber sizes in the lungs of processed-ore-exposed workers are similar to those of chrysotile miners and are considerably longer than those found in the general population; some processed-ore workers have longer fibers which might be responsible for higher disease incidences in certain working groups; tremolite accompanies chrysotile in a variable proportion of workers exposed to processed chrysotile products and might be important in the genesis of mesothelioma in such workers; and mineralogic analysis will usually detect exposure even when chrysotile has largely disappeared from lung tissue. PMID- 3008551 TI - Rises in titers of antibody to human coronaviruses OC43 and 229E in Seattle families during 1975-1979. AB - Sequential serum specimens were obtained every four months during 1975-1979 from 44 children and adults of 10 Seattle families. The 419 specimens were tested for antibody to human coronaviruses OC43 and 229E by enzyme-linked immunosorbent assay (ELISA). Antibody titers were found to increase with age, and titers as well as frequency of rises were greater for OC43 than for 229E virus in all age groups. Significant antibody rises were most frequent in specimens bracketing the winter interval, but some also occurred in the spring-summer and summer-fall intervals. Concurrent significant antibody rises to OC43 virus in different members of the same family were observed in 15 instances, to 229E virus in seven instances, and to OC43 virus in some members and 229E virus in others in eight instances. Significant antibody rises to OC43 or 229E virus indicating reinfections were frequently observed throughout the three-year period but were always separated by at least two four-month intervals. Concurrent significant antibody rises to both 229E and OC43 viruses were seen only in three persons. Finally, the frequency of significant antibody rises in children, about one per person-year, was almost three times higher than in adults. PMID- 3008553 TI - Exposure to fluoride in the chemical industry. AB - Industries that use fluoride-containing materials are usually aware of their toxicity and adopt adequate medical measures. It has been found that workers in factories who have not been considered as subjected to fluorine hazard and therefore have not been controlled had significantly elevated urinary fluoride levels. Most workers in a medically controlled fertilizer plant had normal urinary levels. Maintenance workers were found to have higher values, up to 12 mg/liter. PMID- 3008554 TI - Clinically silent congenital adrenal hyperplasia masquerading as ectopic adrenocorticotropic hormone syndrome. AB - A 64-year-old man with an asymptomatic pulmonary mass discovered on routine chest roentgenography was found to have substantial bilateral adrenal enlargement by abdominal computed tomography. Percutaneous adrenal aspiration biopsy showed cytologically normal adrenal glands. A diagnosis of subclinical 21-hydroxylase deficiency was established by stimulation testing with adrenocorticotropic hormone. The adrenal size and appearance by computed tomographic scanning in congenital adrenal hyperplasia and particularly in its subclinical form have not been well defined. This case demonstrates that marked adrenal enlargement can occur and may provide the only clue to the diagnosis in an asymptomatic patient without other clinical stigmata of adrenal hyperplasia. PMID- 3008555 TI - Granular cell myoblastoma: rare localization in the trachea. Report of a case and review of the literature. AB - Granular cell myoblastoma is an uncommon tumor in the respiratory tract. It usually occurs in the tongue, skin, breast, or subcutaneous tissue. When it occurs in the respiratory tract, it is usually located in the bronchus or larynx. Primary tracheal location is rare with only nine such cases reported in the literature. This report describes a 26-year-old woman with granular cell myoblastoma of the trachea. She presented with a four-year history of bronchial asthma. The tumor was surgically excised by tracheal resection and reconstruction. The patient has remained well and free of obstructive airway symptoms, without recurrence of the tumor for more than one year. PMID- 3008556 TI - Subclavian artery supply disruption sequence: hypothesis of a vascular etiology for Poland, Klippel-Feil, and Mobius anomalies. AB - A hypothesis is presented to explain the pathogenesis of the Poland, Klippel Feil, and Mobius anomalies, isolated absence of the pectoralis major with breast hypoplasia, isolated terminal transverse limb defects, and the Sprengel anomaly. We propose that these conditions are the result of an interruption of the early embryonic blood supply in the subclavian arteries, the vertebral arteries and/or their branches, and hypothesize that the occlusions occur at specific locations in these vessels during or around the sixth week of embryologic development and produce predictable patterns of defects. The term subclavian artery supply disruption sequence (SASDS) is suggested for the group of birth defects represented by the above conditions. Possible causes for interruption of embryonic blood supply are discussed. PMID- 3008557 TI - Use of two different deoxyribonucleic acid probes to detect Y chromosome deoxyribonucleic acid in subjects with normal and altered Y chromosomes. AB - Four experiments evaluated the sensitivity and specificity of molecular techniques to detect human Y chromosome deoxyribonucleic acid. In experiment I, electrophoretic separation of normal male deoxyribonucleic acid fragments after digestion with endonuclease Hae III revealed two male-specific bands of 3.4 and 2.1 kilobase (kb). These bands were not visible if the fraction of male deoxyribonucleic acid in mixed samples was less than 0.3. In experiment II, by means of a repetitive copy Y deoxyribonucleic acid probe (pS4) mapped to Yq12, a male-specific 2.3 kb band was detectable in mixtures of 2.5 ng of male deoxyribonucleic acid and 997.5 ng of 45,X female deoxyribonucleic acid. In experiment III, hybridization with the pS4 probe was performed on the deoxyribonucleic acid of 20 subjects with a normal or a variant Y chromosome. In experiment IV, deoxyribonucleic acid from the same subjects was hybridized to a single copy probe (4B-2) mapped to the Yq11 region. Deoxyribonucleic acid from category A subjects (n = 8) with cytologically normal Y chromosomes hybridized to both deoxyribonucleic acid probes. Deoxyribonucleic acid from category B subjects (n = 2), including a variant Y chromosome that was negative for Q-banding but positive for C-bands, hybridized with the distal pS4 and proximal 4B-2 probes. Deoxyribonucleic acid from category C subjects (n = 10) with variant Y chromosomes uniformly negative for Q- and C-bands, did not hybridize with the pS4 probe. Deoxyribonucleic acid from three of the 10 category C subjects did hybridize to the more proximal sequence-detecting 4B-2 probe. Deoxyribonucleic acid from the remaining seven subjects in category C did not hybridize with either of the deoxyribonucleic acid probes. PMID- 3008558 TI - The relation of angiotensin-converting enzyme to the pregnancy-induced hypertension-preeclampsia syndrome. AB - Angiotensin-converting enzyme, the polypeptide that converts angiotensin I to angiotensin II, was measured in the serum of 114 pregnant women who had normal blood pressure, pregnancy-induced hypertension-preeclampsia, and chronic hypertension with or without pregnancy-induced hypertension. Angiotensin converting enzyme levels were unrelated to weeks of gestation. The angiotensin converting enzyme levels were similar in normotensive women (21.1 +/- 6.9 units/ml), women with chronic hypertension without pregnancy-induced hypertension (23.1 +/- 2.7 units/ml), and patients with pregnancy-induced hypertension where magnesium sulfate (22.6 +/- 8.7 units/ml) had been administered prior to angiotensin-converting enzyme assay, but these values were significantly less than those in patients with pregnancy-induced hypertension with no magnesium sulfate (29.1 +/- 6.5 units/ml) therapy and in women with chronic hypertension with superimposed pregnancy-induced hypertension (30.7 +/- 4.4 units/ml) (p less than 0.005). Maternal venous and umbilical venous and arterial angiotensin converting enzyme levels were as follows: The maternal venous level was less than the cord venous level and greater than the cord arterial value. Neither neonatal size nor twin gestation influenced the angiotensin-converting enzyme levels. Patients with diabetes mellitus had variable angiotensin-converting enzyme values regardless of the status of the blood pressure. The physiologic theories of blood pressure control in pregnant women are discussed in relation to the renin angiotensin, bradykinin, and prostaglandin systems. PMID- 3008559 TI - A rapid method of culturing bloody amniotic fluid for chromosome analysis. PMID- 3008560 TI - Antiviral therapy for cytomegalovirus retinitis in AIDS with dihydroxy propoxymethyl guanine. AB - Six patients (all male, five homosexual and one bisexual, 23 to 48 years old) with the acquired immune deficiency syndrome (AIDS) who had cytomegalovirus retinitis were treated with a new antiviral drug as a part of a prospective open labeled trial for serious cytomegalovirus infections. The drug, 9-[2-hydroxy-1 (hydroxymethyl)ethoxymethyl] guanine (referred to as dihydroxy propoxymethyl guanine), a new acyclic nucleoside antiviral agent similar in structure to acyclovir, produced positive results. These patients treated with dihydroxy propoxymethyl guanine (2.5 mg/kg of body weight every eight hours) showed regression and often disappearance of the lesions of cytomegalovirus retinitis during and for several weeks after therapy, usually with concomitant resolution of viral shedding. The cytomegalovirus retinitis recurred in four patients (the other two were lost to follow-up), but retreatment usually led to remission. Adverse drug toxicity (reversible granulocytopenia) occurred in two patients. PMID- 3008561 TI - In vitro inhibition of human sarcoma cells' invasive ability by bis(5-amidino-2 benzimidazolyl)methane--a novel esteroprotease inhibitor. AB - Bis(5-amidino-2-benzimidazolyl)methane (BABIM) is a synthetic aromatic amidine compound which has a number of important biochemical effects, including inhibition of a family of esteroproteases (trypsin, urokinase, plasmin) previously linked to the complex process of tumor invasion. Previous work has suggested that exogenous natural protease inhibitors can block invasion of tumor cells across basement membranes (BM) in vitro. The authors studied the effect of BABIM on the human cell line HT-1080 with the use of a quantitative in vitro amnion invasion assay system. They have verified the ability of these cells to grow in nude mice and metastasize via the lymphatics or blood vessels on the basis of the route of administration of the inoculum. Cells which were able to actively cross the entire BM were trapped on filters and counted by both brightfield microscopy and by beta scintillation counting of cells whose DNA was labeled with tritiated thymidine. In agreement with either counting technique, BABIM, at a concentration of 10(-4) M, significantly inhibited invasion (P less than 0.005) over the 7-day course of the experiments. Under these conditions, the inhibitor was nontoxic and did not alter the attachment of the cells to the amniotic membrane. Furthermore, a highly significant inhibition of invasion (P less than 0.001) was also demonstrated across a variation in molar concentration of BABIM of more than 2 orders of magnitude. Most remarkably, cells were initially inhibited in their ability to invade in the presence of between 10(-9) and 10(-3) M BABIM. Measurement of Type IV specific collagenase in media from these cells shows a significant inhibition of activity in the presence of BABIM. These results suggest two, not necessarily exclusive, alternative interpretations: first, that inhibition of the proteolytic steps along the pathway of activation of basement membrane degrading enzymes results in inhibition of invasion; second, that arginine directed esteroproteases may work in concert with cellular collagenolytic metalloproteinases in the process of invasion by human tumor cells through native matrix barriers. PMID- 3008562 TI - HTLV-III/LAV viral antigens in lymph nodes of homosexual men with persistent generalized lymphadenopathy and AIDS. AB - The presence of core antigens of retrovirus HTLV-III/LAV, referred to as "AIDS related virus" (AV), has been sought in lymph node samples of patients with persistent generalized lymphadenopathy (PGL, 28 patients), prodromal AIDS (1 patient) and AIDS with Kaposi sarcoma (3 patients). In 30 patients the deposition of viral antigens, detected by monoclonal antibodies to HTLV-III and LAV, could be observed within the germinal centers (GCs) primarily within the extracellular network of immune complexes, and the two patients who were negative were atypical. No AV could be found in normal tonsil or in samples with follicular hyperplasia of unknown etiology (20 cases). These findings, taken together with the ultrastructural identification of typical retrovirus particles in all 9 PGL and 2 AIDS cases studied, indicates that the network of follicular dendritic (FD) cells is an important reservoir of AV virus antigen at this site. The persistence of this retrovirus inside the GCs helps explain how the follicular hyperplasia affecting FD cells and B blasts in PGL may in progressive cases be accompanied by destruction of FD cells and gradual development of T4+ lymphopenia. T4+ T cells may circulate through the GCs and become infected with AV there. In addition, the identification of retrovirus antigen in situ may be of diagnostic value. PMID- 3008564 TI - Na+-K+-ATPase in vascular smooth muscle. AB - Na+-K+-ATPase has been isolated and characterized from canine aortic tissue. The ouabain-sensitive enzyme activity was 24 mumol X mg protein-1 X h-1, and the remaining Mg2+-ATPase activity was 54 mumol X mg protein-1 X h-1. The ratio of Na+-K+-ATPase to ouabain-sensitive K+-phosphatase was 13 to 1, similar to other more homogeneous preparations from other tissues. The dissociation characteristics of the enzyme-glycoside complex of this aortic preparation were the same as for cardiac preparations in that it was stabilized by K+. These data suggest that the nature of both the ATP hydrolytic site of Na+-K+-ATPase and the ouabain binding site are the same in preparations from vascular smooth muscle as in preparations from other tissues. PMID- 3008563 TI - Intrinsic, apparent, and effective affinities of co- and countertransport systems. AB - Solutions to kinetic schemes for the simple carrier, the countertransporter (antiport, exchanger), and the rapid equilibrium cases of the cotransporter (symport) and co-chemiporter (cation-dependent ATPase) are listed. A distinction is made between the intrinsic, apparent, and effective affinities of the transporters for their substrates. Effective pumping requires that the active transporter binds the pumped substrate, at high affinity, realized at the "whence side" (from which pumping takes place) and, at low affinity, at the "whither side" (to which pumping takes place). It is demonstrated how effective pumping might be achieved by appropriate design of the transporter or chemiporter in terms of the energies of the intrinsic binding sites, the energies of the conformation changes that the pump protein undergoes, the dissociation constant of the chemical reaction that drives the co-chemiport, and the order of binding of the cosubstrates, appropriate at different prevailing levels of the driving substrate. PMID- 3008565 TI - Small vessel membrane potential, sympathetic input, and electrogenic pump rate in SHR. AB - Comparative measurements of transmembrane potential (Em) were made in situ in vascular smooth muscle cells (VSM) of mesenteric small principal arteries and veins with innervation and circulation intact. Vessels were in an externalized, topically suffused jejunal loop in 4- to 5-wk-old (initial hypertension) and 12- to 15-wk-old (established hypertension) anesthetized, spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive control rats. Comparable in vitro measurements of Em were also made in VSM of isolated intact small mesenteric vessel segments (from the 12- to 15-wk-old animals) maintained at their in situ lengths and suffused with physiological salt solution (PSS). During suffusion in situ with control PSS, VSM of both small veins and arteries in older (but not younger)SHR were less polarized than in WKY. Local chemical sympathetic denervation in situ (with 6-hydroxydopamine) hyperpolarized VSM of both vessel types in older (but not younger) SHR to the same Em levels measured in situ in respective WKY vessels. After local denervation, VSM of small arteries (but not veins) of both SHR and WKY remained less polarized in situ than in vitro, suggesting the presence of one or more circulating factors with a specific depolarizing action on the arterial side in both animal types. In vitro, VSM of both small arteries and veins from WKY but not SHR were depolarized immediately by 10(-3) M ouabain. In contrast, reduction of the PSS suffusate temperature to 16 degrees C caused a significantly greater depolarization in VSM of SHR vessels.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008566 TI - Role of protein kinase C in inhibition of renin release caused by vasoconstrictors. AB - It was the aim of the present study to get insight into some of the intracellular mechanisms by which the vasoconstrictor hormones angiotensin II (ANG II), arginine vasopressin (AVP), and norepinephrine (NE) inhibit renin release from renal juxtaglomerular cells. To this end a primary cell culture from rat renal cortex was established that consisted of 50% juxtaglomerular cells. The cultured juxtaglomerular cells contained prominent renin granules closely resembling those in the intact kidney and responded to a number of stimuli of renin release. By using these cultures, we found that ANG II (10(-7) M), AVP (10(-6) M), and NE (10(-5) M) inhibited renin release and increased the calcium permeability of the plasma membrane of the cultured cells. Both the effects on renin release and on calcium permeability could be diminished or even be abolished by the calcium channel blocker verapamil (Vp) (10(-5) M). ANG II, AVP, and NE led to an increased formation of diacylglycerol (DAG), a well-known stimulator of protein kinase C (PKC). Moreover, a direct stimulation of PKC by 12-O tetradecanoylphorbol-13-acetate (TPA) (10(-8)-10(-6) M) also inhibited renin release and increased the calcium permeability of the cell membrane. Similar to ANG II, AVP, and NE, the effects of TPA on calcium permeability and renin release could be diminished by Vp. In conclusion, these results point toward a common mechanism by which vasoconstrictors inhibit renin release from renal juxtaglomerular cells: ANG II, AVP, and NE activate a phospholipase C, which generates DAG.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008567 TI - Effect of prostaglandin E1 on DNA synthesis in vascular smooth muscle cells. AB - The effect of prostaglandin E1 (PGE1) on DNA synthesis was determined using cultured vascular smooth muscle cells. It was found that when PGE1 was added to synchronous, quiescent (growth-arrested) cells, it enhanced DNA synthesis. This was in contrast to the effects of PGE1 on asynchronous, cycling (growing) cells. In these cells, when PGE1 was added, it functioned as an antiproliferative agent. In both cases the effects of PGE1 could be mimicked by 6 alpha-carbaprostacyclin (stable prostacyclin analogue) or by 8-bromo adenosine 3',5'-cyclic monophosphate [a permeable adenosine 3',5'-cyclic monophosphate (cAMP) analogue]. In addition PGE1 was shown to cause an elevation in cellular cAMP levels. On the basis of these studies it is hypothesized that the ultimate effect of addition of PGE1 to vascular smooth muscle cells is dependent on the phase of the cell cycle in which it is added. PMID- 3008569 TI - By 95 days of gestation CRF increases plasma ACTH and cortisol in ovine fetuses. AB - Plasma adrenocorticotropin (ACTH) and cortisol (F) responses to 15-min intravenous infusions of synthetic ovine corticotropin-releasing factor (CRF) were measured by radioimmunoassay in three groups of chronically cannulated ovine fetuses. Fetuses (n = 7) in group I were 107 +/- 2 days gestation (0.72 G) and fetuses (n = 7) in group II were 126 +/- 2 days gestation (0.86 G). Group III fetuses (n = 5) were studied at 5-day intervals during the last 2 wk of gestation. Resting fetal plasma ACTH levels were not significantly different among the three experimental groups. Infusions of CRF (50 ng X kg-1 X min-1) provided similar, significant increases in fetal plasma ACTH concentrations in the three groups. In the young fetuses (group I) the ACTH responses to 500 ng X kg-1 X min-1 infusions of CRF were greater than responses to 50 ng X kg-1 X min-1 infusions P less than 0.001. In group II both doses of CRF produced equivalent increases in ACTH that were comparable with the responses to 50 ng X kg-1 X min-1 infusions in Group I. This suggests that fetal ACTH responsiveness to large doses of CRF decreases between 0.72 and 0.86 G. Resting fetal plasma F levels increased during gestation (P less than 0.01). There was an F response (P less than 0.001) to CRF in all groups with greater responses observed in the older fetuses. This suggests that fetal adrenal responsiveness to ACTH or other peptides released by CRF increases during gestation. PMID- 3008568 TI - Inhibition of hepatic glucose production by insulin in vivo in rats: contribution of glycolysis. AB - The action of insulin on hepatic glucose production (HGP) has been studied in fed anesthetized rats during a euglycemic hyperinsulinemic clamp. At the end of the clamp, the liver was rapidly removed, frozen, and enzyme activities and metabolites were measured. When insulin totally suppressed HGP, it did not modify glycogen phosphorylase or synthase activity, nor did it "spare" or increase glycogen content. Insulin decreased glucose 6-phosphate while increasing glycolytic intermediates (fructose 1,6-bisphosphate, alpha-glycerophosphate, lactate, and pyruvate) as well as fructose 2,6-bisphosphate, the potent effector of 6-phosphofructo-1-kinase. Insulin also increased pyruvate kinase activity of low substrate concentration. Lipogenesis measured with 3H2O incorporation into fatty acids was increased four-to fivefold by insulin. The data suggest that in normal rat liver, when glycemia is maintained at constant basal level, insulin promotes no change in glycogen metabolism, whereas the hormone stimulates the glycolytic pathway. This action contributes to the suppression of hepatic glucose production observed after the addition of the hormone. PMID- 3008571 TI - Pituitary-dependent and -independent secretion of CS caused by bacterial endotoxin in rats. AB - Injection of bacterial endotoxin [lipopolysaccharide (LPS)] or immobilization stress increased serum levels of ACTH with a concomitant increase in the levels or corticosterone (CS) of rats. LPS also caused a significant increase in the serum CS levels of hypophysectomized rats. In contrast, immobilization stress induced CS release was abolished completely in these rats. Injection of histamine, a possible mediator of LPS-induced CS secretion, provoked a significant increase in the serum CS levels of hypophysectomized rats. Neither histamine nor LPS had any appreciable effect on production or release of CS by cultured adrenal cells. These results suggest that LPS-induced CS secretion is largely dependent on hypophysial ACTH release but it also depends, in part, on an extrapituitary mechanism and that the LPS-induced pituitary-independent secretion of CS is mediated by histamine produced in the peripheral tissues. On the other hand, stress-provoked CS secretion is absolutely dependent on the pituitary adrenocortical system. PMID- 3008572 TI - Cluster analysis: a simple, versatile, and robust algorithm for endocrine pulse detection. AB - Endocrine signaling provides one critical means of physiological communication within an organism. Many endocrine signals exhibit an episodic or pulsatile configuration. In an effort to provide a versatile and statistically based algorithm for investigating the regulation of endocrine pulse signals, we have formulated a computerized algorithm in which a pulse is defined as a statistically significant increase in a "cluster" of hormone values followed by a statistically significant decrease in a second cluster of values. The increase or decrease is judged in relation to the actual experimental error expressed by the replicates in the presumptive nadir and peak data clusters. The program permits the operator to specify the cluster sizes of test peaks and pre- and postpeak nadirs. This method is largely insensitive to unstable base-line hormone concentrations and is not adversely affected by varying pulse amplitudes, widths, or configurations within the endocrine series. In addition, the simple statistical basis for this algorithm renders it minimally dependent on explicit or a priori assumptions about rates of hormone secretion or disappearance. The program has been validated for false-positive errors against a wide range of intraseries coefficients of variation (4-52%). We have illustrated its performance for profiles of luteinizing hormone, follicle-stimulating hormone, growth hormone, prolactin, adrenocorticotropic hormone, and cortisol and compared these episodic patterns with those of stable serum constituents (total serum protein and calcium), which do not exhibit pulsatile fluctuation. PMID- 3008570 TI - Effects of sodium on iodide transport in primary cultures of turtle thyroid cells. AB - Iodide uptake by primary cultures of turtle thyroid cells decreased linearly with reduction of Na+ concentration in the medium, but changes in medium Cl- concentration did not affect iodide uptake. Ouabain, furosemide, monensin, and perchlorate all decreased 125I-uptake by cultured thyroid cells, whereas amiloride and triamterene did not. Ouabain, monensin, perchlorate, and amiloride depolarized the membrane of cultured cells, whereas furosemide and triamterene had no effect. Ouabain and perchlorate increased intracellular Na+ and Cl- and decreased K+ activities; furosemide and monensin reduced all three ions, but triamterene had no effect. Amiloride decreased intracellular Na+ and increased intracellular Cl- activities, however, its effect on K+ activity could not be determined because of interference by this compound of the K+ ion exchanger. All the agents, except furosemide, inhibited Na+-K+-ATPase activity. These experiments demonstrate that 1) Na+-I- cotransport is responsible for most iodide accumulation in thyroid cells; 2) Na+-I- cotransport system is linked to the Na+ K+ pump; 3) active iodide transport does not always correlate with Na+-K+-ATPase activity; 4) a perchlorate-sensitive iodide transport system is present in thyroid cells; 5) transport processes, not involved in active iodide transport (Na+-Cl- cotransport and Na+-H+ counter transport), are also present in cultured thyroid cells. PMID- 3008573 TI - Intestinal interaction of bile acids, phospholipids, dietary fibers, and cholestyramine. AB - Binding of bile acids and phospholipids to a number of dietary fibers and cholestyramine (CH) within the small intestine was determined. The fibers used were cellulose, wheat bran, oat bran, guar gum (GG), and lignin (LG). GG, LG, and CH bound significant quantities of bile acids. However, only the CH reduced the bile acid concentration within the aqueous phase of the intestinal contents. Significant phospholipid binding was found only with CH. None of the test substances significantly reduced the quantity of solubilized lipid. Multiple regression analysis indicated that the total quantity of bile acids and phospholipids in the aqueous phase of the intestinal contents was a significant predictor of the quantity of lipid solubilized within the contents (r2 = 0.67). The failure of GG and LG to significantly decrease the amount of solubilized lipid suggests that the hypocholesterolemic effect of these fibers is due more to their bile acid binding capacity than to an effect on lipid solubilization. PMID- 3008574 TI - Intestinal anaphylaxis in the rat: jejunal response to in vitro antigen exposure. AB - In previous studies we showed that rats sensitized to egg albumin respond to in vivo intraluminal antigen with decreased net absorption of Na+, Cl-, and water. These abnormalities are associated with high serum levels of immunoglobulin E (IgE) antibodies and mucosal mast cell degranulation. In the present in vitro study electrical parameters, unidirectional fluxes of Na+ and Cl-, and levels of cAMP were determined in jejunum from sensitized and control rats during a basal period and after antigen addition. In Ussing chambers potential difference and short-circuit current increased significantly in tissue from sensitized rats after addition of 100 micrograms/ml of egg albumin to both mucosal and serosal surfaces. These changes were accompanied by a reversal of net Cl- absorption to net Cl- secretion. The presence of doxantrazole, a mast cell-stabilizing agent, in the buffer prevented these abnormalities. No changes occurred in response to antigen challenge in tissue from controls. In a further series of experiments the antigen was added only to the mucosal side of the tissue in Ussing chambers. In these studies short-circuit current increased after a lag period of approximately 25 min and was significantly increased (P less than 0.025) at 35 min. cAMP levels increased significantly in jejunal slices from sensitized rats exposed to antigen for 2 min. Our findings suggest that the in vivo transport abnormalities induced by IgE-mediated mucosal reactions to a food protein are related to antigen stimulation of a Cl- secretory process. PMID- 3008576 TI - Role of CCK in pancreatic exocrine response to amino acids and fats. PMID- 3008575 TI - Na+-H+ and Cl(-)-OH-(HCO3-) exchange in gastric glands. AB - The pH-sensitive, fluorescent, cytoplasmic-trapped dye 2,7-bis(carboxyethyl)-5(6) carboxyfluorescein (BCECF) has been used to measure intracellular (pHi) and pH electrode to measure extracellular pH (pHo) in suspensions of gastric glands isolated from rabbit stomachs. The fluorescence of BCECF-loaded glands was calibrated in terms of pHi by equilibrating pHo and pHi using ionophores or digitonin and titrating pHo to different values. An APPENDIX is included that covers details of dye calibration and interpretation of fluorescence signals. Glands incubated in NaCl Ringer solution had pHi 7.11. Na+-free Ringer solution caused pHi to decrease reversibly to 6.80. Na+-dependent alkalinization of pHi followed a similar time course to the acidification of pHo. These changes were blocked by 1 mM amiloride. When gland cells were acidified (using two different techniques) realkalinization was completely Na+ dependent but was independent of the presence of Cl-; also, neither high extracellular K+ concentration ([K+]o) nor high [K+]o plus 10(-5) M valinomycin affected the rates of Na+-dependent alkalinization. A neutral Na+-H+ exchanger was implicated. Glands also exhibited Cl(-)-dependent changes of pHi that were blocked by 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid (2 X 10(-4) M). A Cl(-)-OH-(HCO3-) exchanger was indicated. Other studies showed that intracellular buffering capacity was approximately 45 mM (pH-1) and that the apparent proton conductance of gland cell membranes was small. PMID- 3008578 TI - In vitro autoradiographic localization of ANP receptors in rat kidney and adrenal gland. AB - The radioligand 125I-ANP-(99-126) was used to map receptors for atrial natriuretic peptide (ANP) in the rat kidney and adrenal gland using in vitro autoradiography and computerized densitometry. In the kidney a very high density of receptors was found overlying glomeruli; these sites had a binding affinity constant of 0.48 +/- 0.06 X 10(-9) M-1 and a site concentration 818 +/- 108 fmol X mg protein-1. A moderate density of receptors was seen in the inner renal medulla; these sites had a binding constant of 0.9 +/- 0.2 X 10(9) M-1 and a receptor concentration of 204 +/- 44 fmol X mg protein-1, and moderate receptor density was also seen in the inner stripe of the outer medulla overlying vasa recta bundles. Diffuse low-density binding was also detected in the outer cortex. In all cases these patterns were abolished by 1 micron ANP-(101-126) or 1 microM atriopeptin III but not by a range of unrelated peptides. In the adrenal, a high density of binding was found overlying the zona glomerulosa, whereas a moderate density occurred in the zona fasciculata. Binding was not detected in the adrenal medulla. These results provide evidence for several classes of ANP receptor distribution in kidney and adrenal and suggest multiple roles of the peptide in fluid and electrolyte homeostasis. PMID- 3008577 TI - Phosphorylation of type II cAMP-dependent protein kinase in renal brush border membranes. AB - We have previously demonstrated cAMP-dependent 32P phosphorylation and dephosphorylation of a 62,000 relative molecular weight (Mr) protein in autoradiograms of sodium dodecyl sulfate polyacrylamide gels originating from canine renal brush border membranes. In the current studies 32P phosphorylation of the 62,000 Mr protein that was independent of cAMP was noted in the presence of Zn2+. Under these conditions, cAMP inhibited the 32P phosphorylation of this protein. Concentration-dependent photoaffinity labeling of a band with Mr 60,000 in autoradiograms of gels resulted from incubation of membranes with cyclic 8 azidoadenosine-3',5'-monophosphate (8-N3-[32P]cAMP) followed by exposure to light. In the presence of Zn2+ and ATP, an apparent shift in the Mr of a portion of the photoaffinity-labeled band to 62,000 was seen. The 62,000 Mr phosphoprotein in detergent-solubilized supernatants of brush border membranes was immunoprecipitated with antibodies directed against the regulatory subunit of type II cAMP-dependent protein kinase. Our observations strongly suggest that the 62,000 Mr protein is the regulatory subunit. PMID- 3008579 TI - Proteolysis of the platelet surface: dissociation of shape change from aggregation. AB - Stimulation of intact platelets by ADP results in a shape change followed by aggregation in the presence of fibrinogen. ADP was found to induce a shape change in chymotrypsin-treated platelets that was similar in extent and initial velocity to that of intact (untreated) platelets. Scanning-electron microscopy verified an ADP-induced shape change in chymotrypsin-treated platelets. This shape change could be completely blocked by stimulators of platelet adenylate cyclase (forskolin, prostaglandin E1, and prostacyclin). On the other hand, the aggregation of chymotrypsin-treated platelets by fibrinogen was not dependent on the presence of ADP and could not be blocked by forskolin, prostaglandin E1, or prostacyclin, even though the levels of cyclic AMP (cAMP) formed in chymotrypsin treated platelets were comparable to levels that completely inhibited the ADP induced aggregation of intact platelets. This lack of inhibition of platelet aggregation was not due to degradation of the adenylate cyclase or prostaglandin receptors, since chymotrypsin-treated platelets were found to have a functional adenylate cyclase system that could be stimulated by forskolin, prostaglandin E1, and prostacyclin and inhibited by ADP and epinephrine, similar to that of intact platelets. These results provide direct evidence that cAMP does not interact with fibrinogen binding sites once they have become permanently exposed on the surface of platelets. Pretreatment of platelets with chymotrypsin therefore appears to be a useful tool that allows for the dissociation of platelet shape change from aggregation, without inhibiting either response. PMID- 3008581 TI - [Role of the beta-adrenoreceptor inhibitory mechanism in regulating the contractile activity of the human uterus]. PMID- 3008580 TI - Prevalence of antibody to LAV/HTLV-III among homosexual men in Seattle. AB - The prevalence of antibody to LAV/HTLV-III among homosexual men attending a community clinic and a sexually transmitted disease clinic in Seattle, Washington in early 1985 was 42 per cent and 32 per cent, respectively. Seropositivity was apparently not related to age or number of sexual partners. The high prevalence of LAV/HTLV-III seropositivity in an area where overt AIDS (acquired immune deficiency syndrome) is still relatively uncommon suggests that public health measures to prevent acquisition and transmission of LAV/HTLV-III should be a high priority even in areas with low incidences of AIDS. PMID- 3008582 TI - [Pituitary-adrenal cortex system of mother and fetus during pregnancy and labor]. PMID- 3008583 TI - Growth hormone, somatomedin and prolactin--relationship to brain function. AB - In recent years it has been found that the brain of several mammals including have receptors for somatomedin both IGF-I and II and measurable hGH has been identified in human brain tissue. IGF-II has been determined in CSF. The presence of these hormones in brain tissue seems to be of developmental and functional importance as experimental studies in frogs, tadpoles and rats showed that injection of growth hormone enhanced brain growth and increased the ratio of neurons to glia. In man early initiation of hGH therapy to children with hGH (who have a less than normal head circumference) induced a fast catch-up growth of the head and improved their IQ. The data available seems to indicate that growth hormones and/or the somatomedins play an important role in the early brain development, maturation and function. In case of hereditary or congenital GH-RH, hGH or somatomedin deficiency, the effectiveness of therapy seems age limited similar to hypothyroidism. The finding of prolactin receptors in human brain and the report of a child with congenital hypoprolactinemia who had mild mental retardation raises the possibility that also prolactin plays a role in brain function. PMID- 3008584 TI - Suppressive action of ACTH on growth hormone secretion in patients with infantile spasms. AB - The changes in insulin-induced growth hormone secretion and in serum cortisol level were studied in 3 cases of West syndrome. The ACTH therapy consisted of an eight weeks course with gradual tapering every two weeks. Daily administration of 12.5 or 25.0 micrograms per kg ACTH for two weeks suppressed an insulin-induced rise in serum GH. The patients who showed sharply suppressed responses as to serum GH had been exposed to high cortisol levels of over 50 micrograms per dl serum. When they were examined before starting the therapy and 72 or 96 hours after the last ACTH injection, all the subjects showed a normal rise in the level of serum GH. The clinical implications of the findings were discussed in terms of the possible adverse effect on the developing brain. PMID- 3008585 TI - The effect of 5,5'-dithiobis(1-methyltetrazole) on cytoplasmic aldehyde dehydrogenase and its implications for cephalosporin-alcohol reactions. AB - Cephalosporin antibiotics with a 1-methyltetrazole-5-thio side chain have the ability to cause an unpleasant flushing reaction if they are taken some time before the drinking of alcohol. It is proposed that the explanation for this is that the side chain becomes liberated in vivo and oxidized to 5,5'-dithiobis(1 methyltetrazole) or to a mixed disulfide analogue which then inactivates aldehyde dehydrogenase. Support for this proposal is given by the results below concerning the interaction in vitro between the disulfides and sheep liver cytoplasmic aldehyde dehydrogenase. 5,5'-Dithiobis(1-methyltetrazole) has a rapid and pronounced inactivatory effect, very similar in many ways (though not identical) to that of disulfiram, to which it has a structural similarity. (Disulfiram is widely used therapeutically to deter alcoholics from drinking.) 1-Methyl-5 methylthiotetrazole (which is a simple model of the antibiotics) and the free 1 methyltetrazole-5-thiol have no effect on the enzyme in vitro, but methyl 5-(1 methyltetrazolyl) disulfide is a potent inactivator; this also supports the proposed pathway. PMID- 3008586 TI - [Recent ideas on the mechanism of insulin secretion]. PMID- 3008587 TI - Isolation of ADP-ribosyltransferase by affinity chromatography. AB - An affinity adsorbent for ADP-ribosyltransferase (EC 2.4.2.30) has been synthesized by coupling 3-aminobenzamide to Sepharose 4B. Using this material, ADP-ribosyltransferase from human placenta has been purified from crude extract to homogeneity within a few hours. The enzyme has an apparent Km for NAD+ of 52 microM. Its molecular mass is 115,000 as determined by gel electrophoresis. The enzyme is DNA dependent and stimulated by histone, its temperature optimum is at 25 degrees C, and its pH optimum is around pH 9. alpha-NAD+, thymidine, caffeine, theophylline, theobromine, 3-methoxybenzamide, and nicotinamide inhibit the enzyme. Purification of ADP-ribosyltransferases from horse, rat, and chicken liver was also achieved with the method described. PMID- 3008588 TI - The purification of alphavirus virions and subviral particles using ultrafiltration and gel exclusion chromatography. AB - Conventional methods of virus purification using ultracentrifugation frequently result in distorted particles with low levels of biological activity, and are thus unsuitable for preparing samples for high-resolution techniques such as neutron scattering, X-ray scattering in solution, and X-ray crystallography. Moreover, in the event of an instrument failure, ultracentrifugation can also pose a significant hazard when preparing pathogenic viruses or subunits derived from them. By employing exclusively ultrafiltration and gel exclusion chromatography, a method has been developed to prepare highly purified, intact alphavirus particles retaining high levels of biological activity. These procedures have also been adapted to prepare aggregates of viral envelope protein with a defined immunogenic content. PMID- 3008589 TI - An assay for inorganic pyrophosphate in chondrocyte culture using anion-exchange high-performance liquid chromatography and radioactive orthophosphate labeling. AB - A method is described for determination of inorganic pyrophosphate (PPi) in cell culture medium and in rabbit articular chondrocytes grown in the presence of radioactive orthophosphate (32Pi). Intra- and extracellular 32PPi formed was measured using high-performance liquid chromatographic (HPLC) separation of the PPi from orthophosphate (Pi) and other phosphate-containing compounds. The chromatographic separation on a weak anion-exchange column is based on the extent to which various phosphate compounds form complexes with Mg2+ at low pH and the rate at which such formation occurs. These complexes are eluted more readily than the uncomplexed compounds. Best results were obtained using a simultaneous gradient of Mg2+ ions and ionic strength. In this case separation of small amounts of PPi from a large excess of Pi was possible without prior removal of Pi or extraction of the PPi fraction. The assay is also useful for measurement of inorganic pyrophosphatase activity. The sensitivity of the assay depends on the specific activity of the added 32Pi and on the culture conditions, but is comparable with the most sensitive of the enzymatic assays. Sample preparation, particularly deproteinization, proved to be of importance. The losses of PPi which occur during procedures of this sort due to hydrolysis and coprecipitation were quantitated. PMID- 3008590 TI - Purification of the iron-sulfur protein, ubiquinone-binding protein, and cytochrome c1 from a single source of mitochondrial complex III. AB - By detergent-exchange chromatography using a phenyl-Sepharose CL-4B column, Complex III of the respiratory chain of beef heart mitochondria was efficiently resolved into five fractions that were rich in the iron-sulfur protein, ubiquinone-binding protein, core proteins, cytochrome c1, and cytochrome b, respectively. Complex III was initially bound to the phenyl-Sepharose column equilibrated with buffer containing 0.25% deoxycholate and 0.2 M NaCl. An iron sulfur protein fraction was first eluted from the column with buffer containing 1% deoxycholate and no salt after removal of phospholipids from the complex by washing with the buffer for the column equilibration, as reported previously (Y. Shimomura, M. Nishikimi, and T. Ozawa, 1984, J. Biol. Chem. 259, 14059-14063). Subsequently, a fraction containing the ubiquinone-binding protein and another containing two core proteins were eluted with buffers containing 1.5 and 3 M guanidine, respectively. A fraction containing cytochrome c1 was then eluted with buffer containing 1% dodecyl octaethylene glycol monoether. Finally, a cytochrome b-rich fraction was eluted with buffer containing 2% sodium dodecyl sulfate. The fractions of the iron-sulfur protein and ubiquinone-binding protein were further purified by gel chromatography on a Sephacryl S-200 superfine column, and the cytochrome c1 fraction was further purified by ion-exchange chromatography on a DEAE-Sepharose CL-6B column; each of the three purified proteins was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PMID- 3008591 TI - The separation of o-phthalaldehyde derivatives of amino acids by reversed-phase chromatography on octylsilica columns. AB - Amino acids were reacted with o-phthalaldehyde and 2-mercaptoethanol and were separated using a simple linear gradient from 10 to 65% methanol over 15 min on an octyl silica (C8) column by reversed-phase chromatography. The separation obtained was found to be sensitive to the pH, ionic strength, and tetrahydrofuran concentration of aqueous solvent A [THF: sodium acetate (45 mM), pH 5.7, (4:96)]. These effects were characterized and used to design a rapid (17 min) separation of the amino acids commonly found in acid hydrolysates of proteins. A more involved procedure was used to separate the more complex mixture of amino acids that are found in enzymatic hydrolysates of proteins or in physiological fluids. The simplicity of the methods allows their use on different chromatographic systems with little or no alteration. PMID- 3008592 TI - A radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies. AB - A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approximately 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D3 and should be useful for the detection of antibodies to ligand-binding proteins in general. PMID- 3008593 TI - Fiber type and non-endplate acetylcholinesterase in normal and experimentally altered muscles. AB - The non-endplate (sarcoplasmic) acetylcholinesterase (AChE) was investigated in eight different muscles of the rat. Serial consecutive sections were stained for AChE, myofibrillar ATPase (after alkaline and acid preincubation), and cytochrome C-oxidase. The following general correlation could be established: within a given muscle the sarcoplasmic AChE was highest in type IIB fibers, lowest in type I and intermediate in type IIA. Additionally, the intensity of the reaction was directly proportional to the size of the type IIA fibers. The distribution of sarcoplasmic AChE was correlated to the ATPase fiber types but was complementary to the cytochrome C-oxidase staining pattern. In single fiber preparations, accumulation of AChE at the myotendinous junction was found to occur in "cap like" form exclusively in fibers with very low or absent sarcoplasmic AChE. To study the role of innervation in the expression of the sarcoplasmic AChE, we cross-reinnervated the extensor digitorum longus (EDL) muscle with the soleus (SOL) nerve and vice versa (X-EDL, X-SOL). In the X-EDL the sarcoplasmic AChE was transformed to that of a normal SOL as were also the ATPase and the cytochrome oxidase. Surprisingly, in the X-SOL the high AChE activity typical for a normal EDL was present after 3 weeks but decreased steadily to very low levels lacking any correlation with ATPase and cytochrome oxidase. The results suggest that the cytoplasmic AChE of the SOL muscle depends more on the load-bearing function of the muscle than on the imposed impulse pattern. There is additional evidence for a retrograde effect of the X-SOL upon its motoneurons. PMID- 3008594 TI - Naloxone attenuates hypoxic depression of ganglionic transmission. AB - The effect of the opioid antagonist naloxone on hypoxia-induced blockade of synaptic transmission in the superior cervical ganglion (SCG) of the rat was studied in vitro. Naloxone (6 X 10(-6) M or more) attenuated the hypoxia-induced blockade of synaptic transmission in the SCG. In addition, in the concentrations studied, naloxone itself had a blocking effect on ganglionic transmission that involved terminal sites of the preganglionic axons. These data suggest that the protective effect of higher doses of naloxone on the hemodynamic responses to hypoxemia or ischemia may originate at least in part from the attenuating effect of naloxone on the hypoxia-induced blockade of ganglionic transmission. PMID- 3008595 TI - Physiology of alfentanil-induced rigidity. AB - The authors investigated the hemodynamic, metabolic, electroencephalographic (EEG), and electromyographic (EMG) characteristics of narcotic-induced rigidity during induction of anesthesia with alfentanil (175 micrograms/kg) in 10 patients. Thiopental (4 mg/kg) was administered to a ten-patient control group. Rigidity was quantified in eight muscle groups (sternocleidomastoid, deltoid, biceps, forearm flexors, intercostal, rectus abdominus, vastus medialis/lateralis, and gastrocnemius). Marked rigidity was observed in all muscle groups in all patients receiving alfentanil and in none receiving thiopental. Central venous pressure increased with onset of rigidity, while mean arterial pressure and cardiac index remained unchanged. Manual ventilation was extremely difficult during alfentanil-induced rigidity. Arterial oxygen tension decreased more rapidly during rigidity than during the same time interval in the control group, while patients experiencing rigidity were more acidotic, as reflected by greater increases in base deficit. The EEG demonstrated an anesthetic state without seizure activity. The immediate increase in central venous pressure with the onset of rigidity, along with occasional simultaneous parallel variations in central venous pressure and the EMG, strongly suggest a mechanical mechanism for the change in central venous pressure. The metabolic changes during rigidity may be partly related to the absence of the normal cardiovascular reflexes that are reported to occur during voluntary isometric muscle contractions. A neurochemical mechanism of narcotic-induced rigidity is briefly reviewed. PMID- 3008596 TI - Dual effect of local anesthetics on the function of excitable rod outer segment disk membrane. AB - The effects of local anesthetics and a divalent cation, Ca2+, on the function of rhodopsin were estimated from the measurements of light-induced proton uptake. The light-induced proton uptake by rhodopsin in the rod outer segment disk membrane was enhanced at lower pH (4) but depressed at higher pHs (6 to 8) by the tertiary amine local anesthetics lidocaine, bupivacaine, tetracaine, and dibucaine. The order of local anesthetic-induced depression of the proton uptake followed that of their clinical anesthetic potencies. The depression of the proton uptake versus the concentration of the uncharged form of local anesthetic nearly describes the same curve for small and large dose of added anesthetic. Furthermore, a neutral local anesthetic, benzocaine, depressed the proton uptake at all pHs between 4 and 7. These results indicate that the depression of the proton uptake is due to the effect of only the uncharged form. It is hypothesized that the uncharged form of local anesthetics interacts hydrophobically with the rhodopsin in the disk membrane. The dual effect of local anesthetics on the proton uptake, on the other hand, suggests that the activation of the function of rhodopsin may be caused by the charged form. There was no significant change in the light-induced proton uptake by rhodopsin when 1 mM of Ca2+ was introduced into the disk membrane at varying pHs in the absence or presence of local anesthetics. This fact indicates that Ca2+ ion does not influence the diprotonating process of metarhodopsin; neither does it interfere with the local anesthetic-induced changes in the rhodopsin molecule. PMID- 3008597 TI - [Effect of hyperbaric oxygenation on changes in the functional activity of beta adrenoreceptors in experimental fecal peritonitis]. PMID- 3008599 TI - Acute arthritis in caprine arthritis-encephalitis virus challenge exposure of vaccinated or persistently infected goats. AB - Goats vaccinated with inactivated caprine arthritis-encephalitis virus (CAEV) developed more severe arthritis after infectious CAEV challenge exposure than did goats vaccinated with tissue culture medium. Arthritis also developed more rapidly in the group vaccinated with inactivated virus. In another experiment, goats with persistent CAEV infection developed acute arthritis after at least 2 injections of infectious CAEV at monthly intervals. In this experiment, the control group consisted of goats with persistent CAEV that were given tissue culture medium. Seemingly, the immune response to CAEV is an important cause of the CAEV-induced arthritis. PMID- 3008598 TI - [Possibilities and limitations of Doppler flowmeters in the study of angiodysplasias of the extremities]. PMID- 3008600 TI - Enzyme-linked immunosorbent assay to detect subgroup-specific antibodies to avian leukosis viruses. AB - An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies against avian leukosis viruses (ALV), using antigens extracted from Rous associated virus-inoculated chicken embryo fibroblast (CEF) cells by Nonidet P-40 treatments. The antigens reacted strongly to sera of chickens immunized with antigenically homologous viruses, but weakly to those of chickens immunized with heterologous viruses. Antigens extracted from noninoculated CEF cells by the same procedures did not react to either of the immune sera. Normal control sera did not react to any of the antigens. Reactivities of immune sera were decreased markedly by the sera adsorbing with homologous Rous-associated virus-inoculated CEF cells, but not with heterologous CEF cells. The ELISA-specific optimal doses (the differences between the optimal doses with antigens from ALV-inoculated and noninoculated CEF cells) were correlated strongly with the virus-neutralization titers (r = 0.876, P less than 0.01). Examination of the antibody response from ALV-inoculated chickens revealed that ELISA detected antibodies at the same time or several weeks earlier than did the virus-neutralization test. PMID- 3008601 TI - Safety of a temperature-sensitive vaccine strain of bovine viral diarrhea virus in pregnant cows. AB - Safety tests were conducted in 78 pregnant cows vaccinated with a commercial preparation of a temperature-sensitive vaccine strain of bovine viral diarrhea (BVD) virus. After vaccination, seroconversion was detected in 33 (97%) of 34 cattle that did not have antibodies against BVD virus. Overall, 43 (91%) of 47 cows with prevaccination titers less than or equal to 4 seroconverted. During the test period, cows did not become naturally infected with BVD virus, and BVD associated reactions to the vaccine were not observed in vaccinated cows. Calves born to vaccinated cows did not have clinical signs of fetal BVD. Precolostral blood samples collected from the progeny of cows that were seronegative at vaccination were free of antibody against BVD virus. Bovine viral diarrhea virus was not isolated from the cattle evaluated in the present study. PMID- 3008602 TI - Scintigraphic diagnosis of primary hepatocellular carcinoma in the woodchuck (Marmota monax). AB - Hepatic imaging with 99mTc-sulfur colloid was used to diagnose primary hepatocellular carcinoma (PHC) in woodchucks infected with woodchuck hepatitis virus (WHV). Based on imaging results, 6 of 12 WHV-infected woodchucks had space occupying hepatic lesions, and all 6 had PHC. Of the remaining woodchucks, 2 did not have PHC, 2 had discrete tumors (less than 1 cm diameter), 1 had miliary small tumors, and 1 had tumors located near the great vessels. Hepatic imaging was a valuable technique for diagnosis of PHC in WHV-infected woodchucks. PMID- 3008603 TI - Plasmid DNA of virulent Alcaligenes faecalis. AB - Alcaligenes faecalis strains originating from chickens and from epizootics of coryza in turkeys were screened for antibiotic susceptibility and for the presence of plasmid DNA. Seven of 35 strains contained plasmid DNA ranging in size from 10.5 to approximately 32 megadaltons. All of the strains isolated from turkeys were virulent in turkey poults, but only the plasmid-containing strains were resistant to sulfonamides and streptomycin. Four of the plasmid-containing strains were also resistant to tetracycline. Five different plasmids representing at least 2 different incompatibility groups were identified in the 7 plasmid bearing A faecalis strain. PMID- 3008604 TI - Inoculation of lambs with ovine adenovirus 5 (Mastadenovirus ovi 5) strain RTS 42. AB - Twelve 1-week-old colostrum-deprived lambs inoculated with the RTS-42 strain of Mastadenovirus ovi 5 were killed and necropsied (2 lambs/day) on postinoculation days (PID) 2, 4, 6, 8, 12, and 21. Four noninoculated lambs were killed and necropsied (2/day) on PID 6 and 12. Virus was isolated from nasal secretions and feces on PID 1 to PID 6, from tracheal fluids and lung tissue of lambs necropsied on PID 2, 4, and 6, and from lung tissue from 1 lamb necropsied on PID 8. Virus was not recovered from liver, kidney, or small intestine of inoculated lambs or samples from noninoculated lambs. Serum antibody was first detected on PID 6 in the inoculated lambs. Noninoculated lambs remained seronegative. None of the lambs in the study developed clinical signs of infection although lesions were produced in the respiratory tract. PMID- 3008605 TI - Detection of bovine herpesvirus-specific nucleic acids by in situ hybridization with biotinylated DNA probes. AB - Biotin-labeled DNA probes for bovine herpesvirus type 1 (BHV-1) were used to detect viral nucleic acids in infected cell cultures and clinical specimens by in situ hybridization. Hybridization signal was detected 2 hours after inoculation in the cytoplasm of infected cells, presumably representing input virus. Hybridization was first detected in the nucleus at 4 hours after inoculation; by 10 hours after inoculation, hybridization signal was detected in both nucleus and cytoplasm of almost 50% of the cells. By 15 hours after inoculation, 95% of the cells were positive. Treatment of specimens with ribonuclease or deoxyribonuclease before hybridization allowed clear differentiation of virus specified DNA and RNA within infected cells. The BHV-1 nucleic acid sequences were detected in nasal epithelial cells obtained from inoculated calves. Since in situ hybridization provides a rapid technique for the detection of BHV-1 specified nucleic acid sequences, it should facilitate studies on the replication, pathogenesis, and diagnosis of BHV-1 infections. PMID- 3008606 TI - Effect of recombinant DNA-derived bovine and human interferons on replication of bovine herpesvirus-1, parainfluenza-3, and respiratory syncytial viruses. AB - Antiviral effects of recombinant DNA-derived bovine (Bo) and human (Hu) interferons (IFN) on the replication of bovine herpesvirus-1, parainfluenza-3, and respiratory syncytial viruses were studied. Bovine monolayer cultures were treated with recombinant DNA-produced Bo IFN-alpha 1, Bo IFN-beta 2, Hu IFN-alpha A, or Hu IFN-alpha A/D and then challenge exposed with bovine herpesvirus-1, bovine parainfluenza-3 virus, bovine respiratory syncytial virus, or vesicular stomatitis virus. Treatment with each IFN reduced the viral yield for each of these viruses, compared with that of control cultures. PMID- 3008607 TI - Virion polypeptide specificity of immune complexes and antibodies in cats inoculated with feline infectious peritonitis virus. AB - Immune complexes purified from sera and ascitic fluids of cats after inoculation with feline infectious peritonitis (FIP) virus contained proteins and proteolytic fragments of the peplomer, nucleocapsid, and envelope polypeptides; in addition, host proteins were demonstrated in the immune complexes. Free (uncomplexed) antibodies against the 3 classes of virion polypeptides were detected and quantitated; the weakest and latest response was directed against the peplomer protein. Immunofluorescence titers showed the best correlation with the antibody response directed against the envelope polypeptides. Differences in reactivity were not found between sera and ascitic fluids from the same animals and between seropositive healthy cats and cats which had died of FIP. Humoral antibody and hypergammaglobulinemia showed a linear correlation, but the wide variation in antiviral titers at a given concentration of gamma-globulin indicated that additional (autoimmune) reactions occur during the pathogenesis of FIP. PMID- 3008608 TI - Genetic stability in calves of a single strain of bluetongue virus. AB - Newborn calves were inoculated IV with highly plaque-purified bluetongue virus (BTV), serotype 10. The electrophoretic migration patterns of RNA segments and proteins of viruses isolated from calves at intervals after inoculation were compared. In addition, sera collected from calves at intervals after inoculation were compared for their abilities to neutralize several virus isolates from the same calf. Viremia persisted in calves for up to 56 days. Differences were not detected in the electrophoretic migration pattern of RNA segments or proteins of any of the BTV isolates. All calves produced high titers of neutralizing antibody to the original BTV inoculum by 28 days after inoculation, and significant (greater than or equal to 4-fold) differences were not detected in the neutralizing titers of sera to viruses collected at intervals after inoculation. The plaque-purified strain of BTV appeared to be stable genetically in infected calves, and failure to demonstrate antigenic variation among isolates indicated that antigenic shift was not the mechanism that allowed viremia to persist in BTV infected calves. PMID- 3008609 TI - Prevalence of feline leukemia virus infection among adult cats at an animal control center: association of viremia with phenotype and season. AB - The overall frequency of feline leukemia virus infection among 555 cats tested from the Hillsborough County Florida Animal Control facility, using a commercial enzyme-linked immunosorbent assay, was 9.4%. Among male cats, the frequency was 13.8%, which was statistically higher (P = 0.003) than that for females (5.9%). There was no statistical evidence to associate frequency of viral infection with hair length or coloration. There was an association with color distribution. The frequency of viral infection in cats with a solid color in their coat, excluding tabby, calico, and tortoise, was higher (12.2%) than the frequency in the remainder of the cats (5.5%; P = 0.011). Finally, there was a difference in frequency related to season. For the 10 months of the study, cats collected in the 5 cooler months (January, February, March, April, and October) had a frequency of 14.6%; cats obtained in the 5 warmer months (May, June, July, August, and September) had a frequency of 7.2% (P = 0.038). PMID- 3008610 TI - Enterotoxin activity of a Salmonella typhimurium of equine origin in vivo in rabbits and the effect of Salmonella culture lysates and cholera toxin on equine colonic mucosa in vitro. AB - To determine whether a strain of Salmonella typhimurium (UCD 1755) of equine origin had enterotoxin activity, 2 ml of a cell-free culture lysate of strain UCD 1755 and approximately 10(9) viable strain UCD 1755 organisms were inoculated into ligated small intestinal segments of rabbits. Intestinal segments inoculated with viable strain UCD 1755 organisms and those inoculated with a cell-free culture lysate of strain UCD 1755 had significant (P less than 0.05) accumulations of fluid 10 hours after inoculation when compared with ligated intestinal segments either inoculated with sterile brain-heart infusion broth or left empty. There was not a statistically significant difference between fluid accumulation of intestinal segments inoculated with viable strain UCD 1755 and that of segments inoculated with a cell-free culture lysate of strain UCD 1755. The responses of equine colonic mucosa to culture filtrates of 2 strains of salmonella typhimurium (UCD 1755 and SL 1027) and purified cholera toxin were studied in vitro. Isolated smaples of colonic mucosa were incubated for 4 hours at 37 C in Krebs-Henseleit bicarbonate buffer (KHB) alone, KHB plus culture lysate of strain UCD 1755, KHB plus culture lysate of strain SL 1027, and KHB plus 1 microgram of cholera toxin/ml. Cyclic adenosine monophosphate (cAMP) content of each sample was determined.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008611 TI - Seroepizootiology of types 3 and 7 adenoviruses and bovine viral diarrhea virus infections of beef cattle from birth to first parturition. AB - Twenty-nine female Angus cattle were monitored from birth until 24 months of age for infection with types 3 and 7 adenoviruses (AV3, AV7) and bovine viral diarrhea (BVD) virus by virus isolation and neutralization tests. Twenty-seven animals remained in the study for 450 days, whereas 16 animals remained at the conclusion of the tests. Viruses were not isolated from test animals. Seroepizootiologic examination indicated that there were 28 infections with AV3, 33 infections with AV7, and 13 infections with BVD virus. Of these infections, 5 with AV3, 6 with AV7, and 2 with BVD virus were reinfections. All but 1 primary AV3 infection had occurred between 180 and 360 days after parturition. Infection with AV7 occurred earlier than that with AV3, as shown by 6 seropositive animals by 180 days. Most adenovirus infections were documented after animals had left confinement and were turned out to pasture. Most BVD virus infections occurred within the first 90 days after parturition. At completion of the study, only 1 animal had not shown evidence of AV3 infection, 2 animals had not been infected with AV7, and 7 cows had remained free of BVD virus infection. PMID- 3008612 TI - Evaluation of terminal deoxynucleotidyl transferase expression in bone marrow of clinically normal and feline leukemia virus-exposed cats. AB - Bone marrow cells from 8 specific-pathogen-free and 11 feline leukemia virus exposed cats were examined for the expression of the nuclear antigen terminal deoxynucleotidyl transferase (TdT). Using a standard indirect immunofluorescence technique, feline leukemia-exposed cats had increased expression of TdT in bone marrow aspirates (2.0% to 29.0%) when compared with that in bone marrow cells from specific-pathogen-free cats (2.5% to 6.0%). Seemingly, TdT can be used as an antigenic marker in leukemogenesis of FeLV-infected cats. PMID- 3008613 TI - Chicken embryonal vaccination with avian infectious bronchitis virus. AB - A commercial infectious bronchitis virus (IBV) vaccine of the Massachusetts 41 strain was injected in embryonating chicken eggs on embryonation day (ED) 18. The IBV vaccine was pathogenic for embryos, and it was passaged in chicken kidney tissue culture to reduce the pathogenicity. At the 40th tissue culture passage (P40-IBV), the virus became apathogenic for the embryos. Maternal antibody positive or -negative chicks hatching from eggs injected with P40-IBV developed antibody to IBV and were protected against challenge exposure at 4 weeks of age with virulent Massachusetts 41 IBV. Although P40-IBV protected chicks when administered on ED 18, this virus did not protect chicks well if given at hatch. When combined with the turkey herpesvirus (HVT), P40-IBV given on ED 18 did not interfere with the protection against challenge exposure with virulent Marek's disease virus, nor did the presence of HVT interfere with protection by P40-IBV. Thus, under laboratory conditions, IBV vaccine could be combined with HVT to form a bivalent embryonal vaccine. PMID- 3008614 TI - Equine endothelial cells in vitro. AB - Certain in vitro culture conditions were determined for equine endothelial cells obtained from the aorta and pulmonary arteries. Cells were enzymatically isolated from the vessel lumen, using clostridial collagenase (2.5 mg/ml of Hanks's balanced salt solution) incubated at 37 C for 30 minutes. Cells were cultured in alpha minimum essential medium supplemented with plasma-derived and nonplasma derived bovine fetal sera, endothelial cell-growth supplement, heparin, and antibiotics. Smooth muscle cell growth was not inhibited with nonplasma-derived animal sera, plasma-derived equine serum, or heparin. Heparin and a serum replacement were toxic to the cells used in the present study. Statistically significant differences were not found between the various media supplements. PMID- 3008615 TI - On the interaction of small molecules with hemoglobin: orientational effects of hydration layers in protein crystal. AB - The spin label TEMPO does not show a binding to hemoglobin molecule in solution. In the crystal however the spin label is bound and a considerable anisotropy in the ESR spectra is observed similar to that with covalent spin labeling. Since TEMPO is a small spherical molecule the anisotropy should be a consequence of the ordering in the crystal packing observed for the hydration layers. Besides the anisotropic sites (one per asymmetric unit) an isotropic signal is apparent. The population of these sites is sensitive to the temperature and above around 30 degrees C a transition is observed of the label from the anisotropic site to the isotropic one. This is consistent with a change in the hydration structure above this temperature so that the spin label is sensitive to the reorganization of water or crystallization. Results of simulations based on the relaxation theory in liquids are compared for different hemoglobin physical states: crystal, powder and solution. It is shown that the ESR parameters obtained in the crystal are very different from those used for spectral stimulation at low temperature. PMID- 3008616 TI - Chronic Epstein-Barr virus infection associated with fever and interstitial pneumonitis. Clinical and serologic features and response to antiviral chemotherapy. AB - Two patients developed fever, interstitial pneumonitis, and pancytopenia associated with extremely high titers of antibody to replicative antigens of the Epstein-Barr virus. In contrast to most patients seropositive for Epstein-Barr virus, neither patient had an antibody response to the Epstein-Barr nuclear antigen K polypeptide. In addition, virus isolated from one patient had a deletion of the B95-8 type in the EcoRI C region of the genome. An etiologic relation between Epstein-Barr virus replication and the clinical manifestations of this syndrome is further shown by the response of each patient to acyclovir therapy. These patients have a new Epstein-Barr-virus-associated syndrome and provide additional evidence that acyclovir may play a role in therapy for selected patients with Epstein-Barr virus infection. PMID- 3008617 TI - Hepatic veno-occlusive disease associated with renal transplantation and azathioprine therapy. AB - Four patients with renal transplants developed hepatic veno-occlusive disease after immunosuppressive therapy with azathioprine. Severe progressive portal hypertension developed in all patients, with the clinical presentation varying from a mild viral-like syndrome to rapidly fulminant liver failure and death. The disease was associated with cytomegalovirus infection but not with the dose of azathioprine, the type or duration of transplant, or the type of underlying kidney disease. In view of the high mortality rate associated with veno-occlusive disease (a combined 55% in our four patients and in five reported in the literature) and wide spectrum of clinical presentation in patients with renal transplants, a high index of suspicion is required and aggressive intervention indicated. PMID- 3008618 TI - Chronic leukemias: oncogenes, chromosomes, and advances in therapy. PMID- 3008619 TI - Complications of the acquired immunodeficiency syndrome. PMID- 3008620 TI - Hydrazine toxicity, pyridoxine therapy, and peripheral neuropathy. PMID- 3008621 TI - [Value of dietary fiber in nutritional and gastroenterologic therapy]. PMID- 3008623 TI - Post-translational processing of procollagens. PMID- 3008622 TI - [Biology of states of burnout]. PMID- 3008624 TI - Collagenase in recessive dystrophic epidermolysis bullosa. PMID- 3008625 TI - Biochemical and immunohistochemical studies on the scirrhous carcinoma of human stomach. AB - Tumor mass of the stomach from patients with scirrhous carcinoma was analyzed biochemically and immunohistochemically to elucidate whether or not infiltrating carcinoma cells are directly responsible for overproductions of collagen in the lesion. Collagen content per unit transverse section of the tumor was two to four times higher than the normal. Of particular interest was that the contents of hyaluronic acid and chondroitin sulfate were five to ten times higher than the normal, suggesting that cells in the lesion of the tumor are in an actively proliferating stage. Immunohistochemical observations using type-specific anti collagen antibodies and anti-carcinoembryonic antigen antibody revealed that type IV collagen was diffusely distributed through the tumor stroma of submucosa and fragmented regions of muscle layer, along with dense fibrous components composed of type I and type III collagens. Stroma cells in the lesion were often stained with antibody to type IV collagen. In contrast, carcinoma cells were with antibody to type I collagen, but not with antibodies to type III and type IV collagen. Quantitative analysis of the collagen production by isolated stroma cells and undifferentiated (KATO-III) and highly differentiated (MKN-28) carcinoma cells in culture in the presence and absence of a combination of the conditioned medium of these cells has shown that the scirrhous carcinoma of stomach results from the "stroma reaction" of stroma cells induced by infiltrating malignant epithelium. PMID- 3008626 TI - Role of the extracellular matrix in cancer. PMID- 3008628 TI - Clinicopathologic correlation of ocular and neurologic findings in AIDS: case report. AB - The protean ocular manifestations of patients with the acquired immunodeficiency syndrome (AIDS) have been well documented both clinically and histopathologically. Since 1978, many case reports have emphasized the opportunistic ocular infections in patients with AIDS. We report the clinicopathologic findings in a patient with AIDS who demonstrated focal necrosis of the ciliary processes and cerebellum due to cytomegalovirus (CMV). The former explained the clinical manifestations of uveitis and the latter masqueraded as drug toxicity. PMID- 3008627 TI - Collagenase in rheumatoid arthritis. PMID- 3008629 TI - Diffuse cutaneous hypersensitivity reaction after dexamethasone/polymyxin B/neomycin combination eyedrops. AB - Localized cutaneous hypersensitivity reactions to antibiotic eyedrops are not unusual. To our knowledge, however, a diffuse cutaneous reaction to eyedrops containing dexamethasone/polymyxin B/neomycin has never been reported. We describe the diffuse skin changes noted in a 72-year-old patient five days after starting eyedrop therapy. PMID- 3008630 TI - Collagenase in human head and neck tumors and rat tumors and fibroblasts in monolayer cultures. AB - Invasive tumors must release collagenase to break down the surrounding host connective tissues. The cellular origin of this enzyme is still unclear. We used anticollagenase antibodies to localize collagenase in the human head and neck tumor and rat tumor tissues. Collagenase appeared to be localized in the tumor connective tissue stroma but not in tumor cells. The rat skin fibroblasts in monolayer culture treated with the rat tumor cell-conditioned medium demonstrated marked extranuclear and particulate staining. Fibroblasts without treatment showed no staining. Tumor cells in the culture also showed no staining. Assay of culture media demonstrated that only fibroblasts with the addition of the tumor cell-conditioned medium produced collagenase. These findings suggest the following: collagenase is produced by fibroblasts which are harbored in the connective tissue stroma, but not by tumor cells; cellular interaction between tumor cells and fibroblasts appears to be involved in breakdown of the host connective tissue for tumor cell invasion; and tumor cells release soluble factors which stimulate production of collagenase by fibroblasts. PMID- 3008631 TI - [Aglossia-adactylia. Apropos of a case. Review of the literature]. PMID- 3008632 TI - Hepatic toxicity of nickel chloride in rats. AB - Enhanced lipid peroxidation was observed in livers of rats killed 24 hr after sc injection of nickel chloride (NiCl2) (750 mumol per kg), as evidenced by 13-fold increase of conjugated dienes in microsomal lipids and 4-fold increase of thiobarbituric acid (TBA) chromogens in hepatic cytosol. Histologic examination of livers from rats killed one to three days after NiCl2 injection (500 mumol per kg) showed microvesicular fatty metamorphosis, mild hydropic degeneration, and foci of inflammation. Microvesicular steatosis of hepatocytes was confirmed by electron microscopy. Dose-related increases of serum aspartate aminotransferase (ALT) activity (up to 7-fold vs controls) and alanine aminotransferase (ALT) activity (up to 3-fold vs controls) were observed 24 hr after injection of NiCl2 (125 to 750 mumol per kg); diminished serum alkaline phosphatase activity (up to 72 percent reduction vs controls) was seen at NiCl2 dosages from 375 to 750 mumol per kg. Diethyldithiocarbamate did not influence the effects of NiCl2 on TBA chromogens in liver homogenates or on serum AST and ALT activities but acted synergistically with NiCl2 to diminish serum alkaline phosphatase activity and to increase serum bilirubin concentration. This study demonstrates that parenteral administration of NiCl2 to rats produces acute hepatic toxicity, with enhanced lipid peroxidation, microvesicular steatosis, and increased serum AST and ALT activities. PMID- 3008633 TI - Imaging techniques for myocardial inflammation. AB - Dilated cardiomyopathy (DC) represents a heterogeneous group of disorders which results in morbidity and mortality in young individuals. Recent evidence suggests that a subset of these patients have histologic evidence of myocarditis which is potentially treatable with immunosuppression. The identification of myocardial inflammation may therefore lead to development of therapeutic regimens designed to treat the cause rather than the effect of the myocardial disease. Ultimately, this may result in improvement in the abysmal prognosis of DC. The currently accepted technique for identification of active myocardial inflammation is endomyocardial biopsy. This technique is not perfect, however, since pathologic standards for the diagnosis of myocarditis have not been established. Furthermore, focal inflammation may give rise to sampling error. The inflammation avid radioisotope gallium-67 citrate has been used as an adjunct to biopsy improving the yield of myocarditis from 7 percent to 36 percent. Serial imaging correlates well to biopsy results. Future studies are designed to study the applicability of lymphocyte labelling techniques to myocardial inflammatory disease. PMID- 3008634 TI - Review of pyridoxal phosphate and the transaminases in liver disease. AB - In vitro supplementation with the active form of vitamin B6, pyridoxal-phosphate (PLP), increases measurements of both serum aminotransferase enzymes, L aspartate: 2-oxoglutarate amino transferase, EC 2.6.1.1 (AST) and L-alanine: 2 oxoglutarate aminotransferase, EC 2.6.1.2 (ALT). The plasma PLP level in normal individuals clearly relates inversely to the degree of stimulation of serum AST and ALT. PLP added in vitro increases the reference values but does not decrease the biological variability of AST measurements in healthy individuals. Since B6 deficiency is observed in alcoholics, in some significant percentage of hospitalized patients and in apparently healthy people over age 64, these individuals will show PLP stimulation of their serum amino-transferase enzymes. Patients with liver disease show lesser activation with PLP of AST activity but not ALT activity than patients with heart disease (myocardial infarction). AST isoenzyme measurements in the form of a mitochondrial AST/total AST ratio may discriminate alcoholic hepatitis from all other hepatic diseases. In renal dialysis patients including transplant patients, it may be desirable to measure the aminotransferases with added PLP in order to reflect better the cytolytic state of the liver. While unconfirmed studies suggest the combination of PLP activation and AST isoenzyme measurements may aid in the diagnosis of hepatoma, PLP activation per se does not provide clear cut improved diagnostic value of AST and ALT in liver diseases. However, in view of PLP incorporation into the IFCC reference methods for AST and ALT, and the National Reference System for the Clinical Laboratory, it is recommended that PLP be included in all AST and ALT measurements. PMID- 3008635 TI - Genetic basis of trimethoprim and O/129 resistance in Vibrio cholerae. AB - Because of its important consequences on prophylaxis and therapy of cholera and on bacterial identification, we have studied the genetic basis of cross resistance to trimethoprim and O/129 of strains of Vibrio cholerae O1 independently isolated in Africa. Two classes of bacteria were found. In the first class, the strains were also resistant to ampicillin and kanamycin and to high levels of streptomycin by synthesis of a 3"- or 6-aminoglycoside phosphotransferase. The strains hybridized weakly with a Tn7 probe and all the resistance characters were transferable en bloc to Escherichia coli. The second class included strains which, in addition to trimethoprim and O/129, were resistant to moderate levels of streptomycin and spectinomycin by production of a 3",9-aminoglycoside-aminocyclitol adenylyltransferase. The resistance characters were not self-transferable to E. coli and the host strain hybridized strongly with Tn7. It therefore appears, that both plasmids and transposons are responsible for the dissemination of resistance to trimethoprim and O/129 in Vibrio. PMID- 3008636 TI - Monoclonal antibody analysis of mononuclear cells in myopathies. III: Immunoelectron microscopy aspects of cell-mediated muscle fiber injury. AB - We have previously obtained light microscopical immunocytochemical evidence for cell-mediated muscle fiber injury and destruction in polymyositis and inclusion body myositis. To evaluate further interactions of the different cell phenotypes with each other and with the muscle fibers, the T8, T4, and Leu 7 markers in 7 cases of polymyositis and in 9 cases of inclusion body myositis were localized by immunoelectron microscopy. In the early stages of the cell-mediated process, T8+ cells and macrophages are apposed against, and/or send spikelike processes into, nonnecrotic muscle fibers. Leu-7+ cells penetrate fibers infrequently, and T4+ cells do not penetrate muscle fibers. Subsequently, an increasing number of T8+ cells and macrophages traverse the basal lamina; focally replace, displace, or compress the fiber; and spikes from these cells honeycomb the adjacent muscle fiber regions. The macrophages contain only few heterophagic vacuoles and therefore act in a cytotoxic rather than a phagocytic capacity. The integrity of the muscle fiber surface membrane facing the invading cells is maintained, but the possibility also exists that the membrane is damaged and rapidly repaired, or that the damage cannot be detected by electron microscopy. Nearby fiber regions often show either degenerative or regenerative changes. Ultimately, segments of the entire muscle fiber are replaced by the invading cells. PMID- 3008638 TI - Dysglobulinemic neuropathy: absence of immunoglobulin within myelin sheaths. PMID- 3008637 TI - Phenytoin neuropathy: structural changes in the sural nerve. AB - Phenytoin has been implicated as a causative agent in peripheral neuropathy, although structural changes in nerve have not been characterized. A 47-year-old man was seen with clinical and electrophysiological signs of peripheral neuropathy after 30 years of phenytoin administration. Despite a modest dose of phenytoin (300 mg/day) blood levels were 31 to 38 micrograms/ml. A sural nerve biopsy showed a loss of large myelinated nerve fibers and a nonrandom clustered distribution of segmental demyelination and remyelination. The latter findings were accompanied by axonal shrinkage. Sixteen months after phenytoin was stopped, the patient's clinical and electrophysiological findings reflected improvement. These data indicate that long-term phenytoin administration can cause a reversible neuropathy characterized by axonal shrinkage and secondary demyelination. PMID- 3008639 TI - Cytoskeletal protein abnormalities in neurodegenerative diseases. AB - The nervous system is a rich source of filamentous proteins that assume critical roles in determining and maintaining neuronal form and function. Neurons contain three major classes of these cytoskeletal organelles: microtubules, intermediate filaments, and microfilaments. They also contain a variety of proteins that organize them and serve to connect them with each other. Such major neurodegenerative diseases as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, as well as a variety of toxic neuropathies, are characterized pathologically by intraneuronal filamentous inclusions. Recent studies using biochemical and immunocytochemical techniques have established that these abnormalities represent disorganized states of the neuronal cytoskeleton and have determined some of the specific molecular constituents of these inclusions. This knowledge has led to new ways of thinking about their origins. PMID- 3008640 TI - Fatal Pneumocystis pneumonia from ACTH therapy for infantile spasms. PMID- 3008641 TI - [Molecular genetic analysis of the determinant of gentamicin resistance in Enterobacter cloned from a clinical strain]. AB - Resistance of clinical strains of Enterobacteriaceae to aminoglycoside antibiotics and in particular to gentamicin was studied. The data on conjugation and plasmid DNA transformation of a clinical strain 10171 of E. cloacae, as well as the data on P1 transduction showed that its gentamicin resistance was due to plasmids. The gentamicin resistance determinant was cloned from the plasmid DNA with the use of the restriction sites of EcoR I, Hind III and Pst I into the vector plasmid pUC 19. Plasmids pAA1, pAA2, pAA3 and pAA4 carrying the gmr determinant were isolated. Their physical maps were constructed. The restriction analysis supplemented by the data on DNA-DNA hybridization was indicative of the identical 2.0 kb sequences in all the plasmids carrying the gmr determinant. PMID- 3008642 TI - [Status and prospects in the use of biocatalysis in the synthesis of beta-lactam antibiotics]. AB - The present world status of using biocatalysts for production of beta-lactam antibiotics and their semiproducts is analyzed and the biocatalytic and chemical methods for production of antibiotics are compared. The requirements to the quality of biocatalysts used in production of drugs are formulated and the criteria of the efficiency of biocatalytic production processes are presented. Interrelation between the requirements to the biocatalyst quality and the efficiency of the biocatalytic production processes is examined. The general principles of development of efficient enzymatic processes including biocatalytic processes in production of beta-lactam antibiotics as closed-loop and practically wasteless systems are discussed. The main advantages of the developed production processes are illustrated by an example of large-scale processes use in manufacturing of the basic products for synthesis of beta-lactam compounds. PMID- 3008644 TI - Efficacy of 5-methoxymethyl-2'-deoxyuridine in combination with arabinosyladenine for the treatment of primary herpes simplex genital infection of mice and guinea pigs. AB - The relative efficacy of 5-methoxymethyl-2'-deoxyuridine (MMdUrd), arabinosyladenine (ara-A) and the combination of MMdUrd and ara-A in the treatment of experimental genital herpes (GH) was investigated using mouse and guinea pig models. The infection was initiated by intravaginal inoculation using either HSV-2, strain X-265 or HSV-2, strain MS. Treatment was initiated 3 h post virus inoculation. The parameters used to evaluate efficacy were: percent mortality; mean day of death; virus yield from the vaginal secretions; and mean lesion score. The simultaneous application of 5% MMdUrd and 5% ara-A was an effective treatment for controlling primary GH in both animal models. Combination chemotherapy was also effective in preventing recurrence of infection as well as the emergence of drug resistant virus. At 20% concentration, ara-A was effective in providing protection against GH. However, lesions due to recurrent GH appeared after cessation of treatment and the virus isolated from vaginal secretions of ara-A treated animals required higher concentration of drug for inhibition of virus replication in cell culture. 20% MMdUrd was only partially effective in controlling GH. The production of infectious virus particles (virus yield) in cell culture after exposure to either ara-A of MMdUrd alone or in combination was determined. When MMdUrd and ara-A were used together, a substantially lower amount of each drug was needed to inhibit virus production completely and removal of drugs did not result in an increase in virus yield. PMID- 3008643 TI - On the complex nature of the antiviral activity of coumermycin A1: its interference with the replication of herpes simplex virus type 1. AB - The mechanism of inhibition of the replication of herpes simplex virus type 1 (HSV-1) by coumermycin A1 (CA1), an inhibitor of bacterial DNA gyrase, has been investigated. Concentrations of antibiotic slightly higher than those needed for 50% inhibition of viral growth were able to inhibit viral DNA synthesis in infected cells. This effect was accompanied by a depressed synthesis of viral polypeptides. Protein synthesis was also inhibited in uninfected cells, especially after long exposure to the drug, but not in a cell-free system. In vitro assays of highly purified HSV-1 DNA polymerase in the presence of the drug, provided evidence that the enzyme was a target of CA1. The viral polymerase was in fact inhibited by the antibiotic to an extent comparable to that of viral DNA synthesis in intact cells. In contrast, DNA polymerase alpha, the enzyme involved in chromosomal DNA replication, was relatively insensitive to CA1. The drug was also shown to bind to protein and to viral and cellular DNA. PMID- 3008645 TI - Selective inhibition of virus multiplication by new acylated 1,2,4-triazole derivatives. AB - Six out of 99 new acylated 1,2,4-triazole derivatives specifically inhibited rubella virus replication in RK 13 cell cultures. These are the following: 3 methylthio-5-(2-chlorobenzamido)-1H-1,2,4-triazole; 3-methylthio-5-(2 bromobenzamido)-1H-1,2,4-triazole; 3-methylthio-5-(2-methylbenzamido)-1H-1,2,4 triazole; 3-methylthio-5-(2-nitrobenzamido)-1H-1,2,4-triazole; 3-methylthio-5-(2 methylthiobenzamido)-1H-1,2,4-triazole and 3-ethylthio-5-(2-methylbenzamido)-1H 1,2,4-triazole. The compounds did not directly interfere with the infectivity of the rubella virus particles and the antiviral effect was demonstrable only within cells infected with rubella virus. The active compounds did not inhibit the replication of herpes simplex virus type 1, influenza virus and adenovirus in cell culture systems. Structure-activity relationships are discussed. PMID- 3008646 TI - Comparative efficacy of broad-spectrum antiviral agents as inhibitors of rotavirus replication in vitro. AB - Several nucleoside analogues which have previously been established as broad spectrum antiviral agents, i.e. ribavirin, vidarabine, pyrazofurin, tubercidin, carbodine, (S)-9-(2,3-dihydroxypropyl)adenine [(S)-DHPA], carbocyclic 3 deazaadenosine (C-c3 Ado), (RS)-3-adenine-9-yl-2-hydroxypropanoic acid [(RS) AHPA] isobutyl ester and neplanocin A were compared for their potency and selectivity as inhibitors of human rotavirus (strains Wa, KUN and MO) replication in vitro. As the most efficacious inhibitors emerged (S)-DHPA, C-c3 Ado, (RS) AHPA isobutyl ester and neplanocin A, with a minimum inhibitory concentration of 60, 1.4, 1.2 and 0.2 micrograms/ml, and a selectivity index of greater than 3, 70, 80 and greater than 20, respectively. As has been postulated for their antiviral action in general, these adenosine analogues probably owe their anti rotavirus activity to inhibition of S-adenosylhomocysteine hydrolase, a key enzyme in regulating methylations including those that are required for the maturation of viral mRNA. PMID- 3008647 TI - Cloning and constitutive expression of the N-acetylneuraminate lyase gene of Escherichia coli. AB - The N-acetylneuraminate (NANA) lyase (EC 4.1.3.3) gene from Escherichia coli was self-cloned in E. coli. Transformants were selected by complementation of a NANA lyase-deficient E. coli strain. One clone was found to produce NANA lyase, and it contained a recombinant plasmid, pNAL1, with a 9.0-kilobase HindIII insert. The cloning of the NANA lyase gene resulted in the change from inducible to constitutive production of the enzyme. The level of expression of the NANA lyase gene in E. coli(pNAL1) clones was two- to three-fold higher than that in the fully induced wild-type strains. PMID- 3008648 TI - Electron microscopic heteroduplex study and restriction endonuclease cleavage analysis of the DNA genomes of three lactic streptococcal bacteriophages. AB - Three lactic streptococcal bacteriophages were compared with one another by electron microscopic analysis of heteroduplex DNA molecules. The phages were almost identical in morphology and had been isolated over a period of 10 years on different strains of Streptococcus cremoris from cheese plants situated in different parts of New Zealand. There was a high degree of homology between the DNAs, in agreement with Southern blot hybridization data reported earlier. There were, however, distinct regions of nonhomology, mostly between 0.45 and 1.71 kilobases in length, suggestive of the occurrence of block recombination events. A deletion of 2.23 kilobases in the two more recently isolated phages, or an insertion in the first isolate, was found. All three phage DNAs showed differences in restriction endonuclease cleavage sites. Alignment of the restriction endonuclease maps with the heteroduplex maps showed that differences in cleavage sites occurred most frequently in regions of nonhomology. However, differences in cleavage sites in regions of apparent homology were also detected, indicating that point mutations may have occurred in addition to block recombination events. PMID- 3008649 TI - Protoplast transformation in coryneform bacteria and introduction of an alpha amylase gene from Bacillus amyloliquefaciens into Brevibacterium lactofermentum. AB - The goal of this study was to investigate the likelihood of developing useful transformation systems for coryneform bacteria. Two species of coryneform bacteria, Brevibacterium lactofermentum and Corynebacterium lilium, were transformed with chimeras constructed from pUB110 and a cryptic coryneform plasmid (pGX1901). C. lilium protoplasts were also efficiently transfected with phage CS1 DNA. High transformation and transfection frequencies were obtained after only 2 min of lysozyme treatment of lysozyme-sensitive mutants. A series of experiments was also conducted to determine whether DNA from other species of important industrial microbes from the genus Bacillus could be expressed in coryneform bacteria. Evidence of restriction of Bacillus subtilis DNA by B. lactofermentum was observed but could be overcome. A Bacillus amyloliquefaciens alpha-amylase gene (amyEBamP) was subcloned onto a plasmid able to replicate in B. lactofermentum. B. lactofermentum transformants for this plasmid expressed amylase activity and produced material cross-reactive to amylase antibody. PMID- 3008650 TI - Aggregation and proton release of purple and white membranes following cleavage of the carboxyl-terminal tail of bacteriorhodopsin. AB - Our results indicate that the previously reported decrease in proton release by proteolyzed purple membrane sheets was due merely to the aggregation state of these preparations and not to the loss of the carboxyl-terminal tail. Changes in H+/M412 ratios obtained for purple and white membrane preparations correlate with the measured aggregation. White membrane preparations consistently exhibit H+/M412 ratios more than twice those measured for native purple membranes under the same conditions. Quasi-elastic light scattering was used to characterize the size of isolated purple and white membrane sheets before and after proteolysis. The results clearly show that native purple membrane preparations are larger in size than would be expected and that, following trypsin treatment, they are on average more than an order of magnitude larger. Negative staining electron microscopy showed that the purple membrane became aggregated in stacked arrays. Bleaching and reconstitution with retinal also affect aggregation, but iodination or nitration of purple membrane does not affect the measured size. The average size of white membranes is smaller; this is consistent with results of electron microscopy and the size increase is much less than that of purple membranes following trypsin treatment. No size change occurs with retinal reconstitution. In aggregated purple membrane preparations, protons and other cations are unable to exchange freely with the aqueous medium, explaining why proteolysis lowers the proton release from purple membrane sheets in suspension. PMID- 3008651 TI - In vitro inhibition of carrot chalcone synthase by 3'-nucleotidase: the role of the 3'-phosphate group of malonyl-coenzyme A in flavonoid biosynthesis. AB - In mixing experiments with extracts derived from two cell lines of Daucus carota tissue cultures with and without chalcone synthase activity, strong inhibition of chalcone synthase (CHS) became obvious. This inhibition was due to the presence of a heatlabile protein in extracts from cells devoid of CHS activity. This protein was partially purified and identified as 3'-nucleotidase (EC 3.1.3.6). Inhibition was also observed in the presence of purified 3'-nucleotidase from Lolium multiflorum. The phosphate group in the 3'-position of adenosine, a part of the CoA thioester substrates of CHS, was hydrolyzed by this enzyme. The dephosphorylated form of malonyl-CoA was no longer a substrate, whereas 4 coumaryl-3'-dephospho-CoA as well as 4-coumaryl-CoA was still able to act as a primer for the CHS reaction. Further studies showed that malonyl-3'-dephospho-CoA was an efficient CHS inhibitor. On the other hand, CoA-SH lost its inhibitory activity after dephosphorylation in the 3'-position. These results are discussed with respect to the mechanism of chalcone synthesis. PMID- 3008652 TI - Proteolysis of the calcium-dependent protease inhibitor by myocardial calcium dependent protease. AB - Bovine heart peak II calcium-dependent protease was capable of hydrolyzing its specific inhibitor protein at high molar ratios of protease to inhibitor. The proteolysis was inhibited by leupeptin and required millimolar calcium. Thus, it appeared to be attributable to the calcium-dependent protease and not to possible contaminating proteases in the purified preparations of inhibitor or calcium dependent protease. Incubation of the purified inhibitor with the calcium dependent protease produced a discrete pattern of inhibitor fragments on Western blots developed with an inhibitor-specific monoclonal antibody. Traces of similar or identical lower molecular weight immunoreactive material could be observed in Western blots of bovine heart extracts, and the immunoreactivity present as these lower molecular weight forms could be increased by incubation of the extracts with calcium ion. These results suggest that the inhibitor can be proteolyzed to low molecular weight forms which can be detected in cardiac tissue extracts, and that calcium-dependent protease(s) may be responsible for this phenomenon. PMID- 3008653 TI - Phosphorylated threonine as the covalent intermediate in snake venom phosphodiesterase. AB - The covalent intermediate of snake venom phosphodiesterase has been isolated using thymidine 5'-[alpha-32P]triphosphate as substrate. Phosphoamino acid analysis of the labeled enzyme demonstrates that threonine is the active site residue forming the covalent intermediate. 5'-Nucleotide phosphodiesterase is the first enzyme reported to have an active site threonine forming a covalent intermediate. PMID- 3008654 TI - Rat liver uridine diphosphate glucose dehydrogenase: dual mechanism of regulation in vivo. AB - Rat liver UDPglucose (UDPG) dehydrogenase activity was observed to be decreased after fasting and could be restored to normal levels after refeeding glucose. This could be prevented by prior injection of puromycin, suggesting de novo protein synthesis. Administration of insulin to normal rats on stock diet did not influence the enzyme activity. However, the enzyme activity was decreased in the diabetic condition. Intraperitoneal injection of insulin caused an enhancement of the enzyme activity in diabetic animals. Hepatic UDPG dehydrogenase activity was observed to be decreased on ascorbic acid feeding or intraperitoneal injection of the same. The intraperitoneal injection of either insulin or cAMP to ascorbic acid-treated rats resulted in an increase in enzyme activity reaching normal levels. The insulin-mediated increase could not be prevented by prior injection of puromycin, suggesting a post-translational effect. These results indicate two distinct mechanisms for in vivo regulation of hepatic UDPG dehydrogenase. PMID- 3008655 TI - Protein rotational mobility in thylakoid membranes of different polypeptide composition in the wild type and mutant strains of Chlamydomonas reinhardtii. AB - The rotational mobility of thylakoid membrane proteins labeled with a paramagnetic analog of N-ethylmaleimide was investigated by saturation transfer electron spin resonance. In the wild type strain of Chlamydomonas reinhardtii two polypeptides are prominently labeled. They correspond to the 19-kDa subunit of the reaction center I protein and to the 30-kDa subunit of the light harvesting complex. Several polypeptides, most of which are either trypsin or alkaline sensitive, are also labeled. In order to circumvent the lack of specificity during the labeling, we have compared the rotational mobilities of labeled proteins in thylakoid membranes from several mutant strains which lack in photosystem I., ATPase or light harvesting complexes. Comparison of the saturation transfer electron spin resonance spectra obtained with these mutant membranes as well as with trypsin- and alkaline-treated membranes allowed us to characterize the rotational contribution of some of the labeled proteins to the overall protein dynamics observed in the wild type strain. The reaction center I protein undergoes slow rotation as compared to the other labeled proteins. The rotational characteristics of the labeled light harvesting complexes are those of a peptide fragment in the complex which is in rapid motion in unstacked membranes. Stacking of the thylakoid membranes upon Mg2+ addition is accompanied by a marked change in shape of the saturation transfer spectra, and corresponds to the appearance of highly immobilized nitroxides. We interpret these changes as arising mainly from the hindrance upon membrane appression, of the labeled fragment of the light harvesting complexes which protrude at the thylakoid outer surface. PMID- 3008656 TI - Phosphatidylcholine homeostasis in phosphatidylethanolamine-depleted Tetrahymena. AB - The relative contributions of the two pathways of phosphatidylcholine biosynthesis, phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17) and diacylglycerol: CDP-choline cholinephosphotransferase (EC 2.7.8.1), are altered in the ciliate protozoan Tetrahymena thermophila whose phospholipid composition has been modified by culturing the organism in the presence of one of several aminophosphonic acids, as determined by measuring the incorporation of [methyl 3H]choline and [methyl-14C]methionine into phosphatidylcholine in vivo. In control cells the phosphotransferase pathway provides about 40% of the phosphatidylcholine, while in cells grown with 2-aminoethylphosphonate (AEP), 3 aminopropylphosphonate (APP), and N,N,N-trimethylaminoethyl-phosphonate (TMAEP) the contribution of the phosphotransferase pathway to phosphatidylcholine formation is 75, 90, and 26%, respectively. In AEP- and APP-grown cells, in which 80% of the phosphatidylethanolamine has been replaced by the corresponding phosphonolipid, the methyltransferase is less active since the level of the substrate phosphatidylethanolamine is reduced and neither of the phosphonolipids is a substrate for the enzyme. In TMAEP-grown cells, TMAEP competes with and reduces the incorporation of phosphocholine by the phosphotransferase pathway, leading to a smaller contribution of the pathway to phosphatidylcholine biosynthesis. The relative amounts of the two different radioactive labels incorporated into diacylphosphatidylcholine vs alkylacylphosphatidylcholine are also altered in the phosphonate-grown cells. The exogenous AEP induces a change in the glyceryl ether content of the 2-aminoethylphosphonolipid--33% in the AEP grown cells compared to 70% in the control cells--indicating that the exogenous AEP is entering the phospholipids by the ethanolamine-phosphotransferase pathway rather than by the route of the endogenous AEP. PMID- 3008657 TI - Intracellular location of unoccupied 1,25-dihydroxyvitamin D receptors: a nuclear cytoplasmic equilibrium. AB - In order to investigate the subcellular distribution of unoccupied 1,25 dihydroxyvitamin D3 receptors, highly purified cytoplasts and nucleoplasts were prepared from two kidney cell lines (PK1 and MDBK). This was accomplished utilizing the technique of enucleation by cytochalasin B and density gradient centrifugation. Unoccupied 1,25-dihydroxyvitamin D3 receptors were found in both the nuclear and cytosolic compartments, with approximately 70% of the receptors localized in the cytoplasm. When cells were pretreated with 1,25 [3H]dihydroxyvitamin D, prior to enucleation, it was found that 90% of the receptor-hormone complex was associated with nucleoplasts, thus demonstrating that cytochalasin B treatment does not alter the high-affinity association of the receptor-hormone complex with the nucleus. The ratio of unoccupied receptor/protein was found to be the same in whole cells, cytoplasts, and nucleoplasts for both cell types. The ratio of unoccupied receptor/DNA was highest in cytoplasts and lowest in nucleoplasts. Taken together, these data indicate that the unoccupied 1,25-dihydroxyvitamin D receptor is generally associated with cell proteins and not specifically associated with cell DNA. We therefore propose, at least for these cells, that the unoccupied 1,25 dihydroxyvitamin D receptor exists in equilibrium between the nuclear and cytosolic compartments of the whole cell, and receptor-hormone binding shifts this equilibrium to favor nuclear localization. PMID- 3008658 TI - Pigeon liver phosphoprotein phosphatase: an effective activator of pyruvate dehydrogenase in tissue homogenates. AB - A fluoride-insensitive, non-metal-requiring pyruvate dehydrogenase phosphatase has been purified 730-fold from pigeon liver acetone powder and proven to be a convenient reagent for studies of pyruvate dehydrogenase complex and its activation (phosphorylation) state in brain and other tissues. This phosphatase is a cytoplasmic enzyme (Mr = 80,000), and fits the functional definition of a type 1 phosphoprotein phosphatase. The pigeon liver phosphatase can be used to activate pyruvate dehydrogenase complex in vitro in brain and other crude tissue homogenates. Addition of the cytoplasmic pigeon liver phosphatase to a homogenate from rat or mouse brain frozen in situ activated pyruvate dehydrogenase to levels comparable to that found in ischemic brain. The fluoride insensitivity of this phosphatase was used to develop a convenient technique for stopping the pyruvate dehydrogenase activation state in situ in cultured skin fibroblasts and then fully activating the complex in vitro in 5 min. The use of this phosphatase as a reagent can facilitate the study of pyruvate dehydrogenase activation defects in mammalian tissues including cultured cells in normal and disease states. PMID- 3008659 TI - Regulation of rat and bovine adrenal metabolism of polycyclic aromatic hydrocarbons by adrenocorticotropin and 2,3,7,8-tetrachlorodibenzo-p-dioxin. AB - The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on polycyclic aromatic hydrocarbon (PAH) metabolism and steroidogenesis in primary cultures of bovine adrenal cortical (BAC) and rat adrenal cortical (RAC) cells have been examined. Remarkably TCDD is an ineffective inducer (15-50%) of PAH metabolism in confluent BAC cells and completely antagonizes a 5-fold induction by benz[alpha]anthracene (BA). In the same concentration range (EC50 5 X 10(-11) M) TCDD suppresses steroidogenesis through an effect on cholesterol metabolism. Adrenocorticotropin (ACTH) and cAMP also suppress PAH metabolism at concentrations which stimulate steroidogenesis (10(-7) M). In RAC cells ACTH potently induces PAH metabolism (7 fold) at a comparable concentration to the stimulation of steroidogenesis. Parallel stimulation of PAH metabolism and steroidogenesis by cAMP suggest that ACTH induction of PAH metabolism is mediated by cAMP. TCDD induces PAH metabolism (2.8-fold, EC50 8 X 10(-11) M) at similar concentrations to the inhibitory effect in BAC cells and this action is additive with ACTH induction. In male rats in vivo TCDD induces adrenal microsomal PAH metabolism (72%) and is more effective in this respect than 3-methylcholanthrene (3MC). Rabbit antibodies against rat liver cytochrome P-450c (the major TCDD-inducible liver form) inhibited the TCDD induced adrenal metabolism of 7,12-dimethylbenz[alpha]anthracene (DMBA), which also exhibited regioselectivity typical of metabolism by P-450c. Constitutive adrenal microsomal metabolism, which exhibited regioselectivity of DMBA metabolism comparable to the ACTH-sensitive cellular metabolism, was not affected by anti-P-450c. It is concluded that ACTH and TCDD induce distinct forms of cytochrome P-450 in RAC cells and that the latter represents a typical Ah receptor mediated response. The anomalous effect on PAH metabolism in BAC cells that parallels inhibition of steroidogenesis may derive from repression of a distinct adrenal form of P-450 by the TCDD-Ah-receptor complex. PMID- 3008660 TI - Calmodulin inhibits the phosphorylation of spectrin in vitro. AB - In vitro phosphorylation of purified spectrin dimer was studied in the presence of Ca2+-calmodulin (CaM). CaM inhibited autophosphorylation of the beta subunit of spectrin. The inhibitory effect (65% at a 32-fold molar excess) appeared to be due to a weak interaction of CaM with spectrin. CaM was similarly effective in a phosphatase-stimulated autothiophosphorylation of the beta subunit with [gamma 35S]ATP. Hence, its inhibitory effect was not due to stimulation of a spectrin associated phosphatase activity. Phosphorylation of spectrin by the catalytic subunit of a cAMP-dependent protein kinase occurred in both subunits (1984, FEBS Lett. 169, 323). CaM selectively inhibited a cAMP-dependent phosphorylation of the alpha subunit of spectrin to 30% at two CaM per spectrin. It was ineffective on the cAMP-dependent phosphorylation of the beta subunit up to a 32-fold molar excess. These results yield functional evidence for a CaM-spectrin interaction. They further suggest that CaM can regulate the extent of a cAMP-dependent phosphorylation of the alpha subunit of spectrin. PMID- 3008661 TI - Covalent crosslinking of thyrotropin to thyroid plasma membrane receptors: subunit composition of the thyrotropin receptor. AB - The subunit composition of the thyrotropin (TSH) receptor has been characterized using the bifunctional crosslinking agent, disuccinimidyl suberate (DSS), to covalently link [125I]TSH to its receptor. Purified thyroid membranes were labeled with [125I]TSH, and the hormone-receptor complex was crosslinked by incubation with 0.1 mM DSS. Analysis of this crosslinked complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions indicated the presence of a specifically labeled hormone-receptor complex, corresponding to a Mr of 68,000 +/- 3000 before correction for the relative molecular mass of TSH. When reducing agents were absent during SDS solubilization, the mobility of the band increased slightly, suggesting the presence of intramolecular disulfide bonds. The labeling of the 68,000 band was specifically inhibited by TSH, but not by other glycoprotein hormones. Specific labeling occurred only in thyroid, and not in liver or muscle plasma membranes. Protease-free immunoglobulin G, isolated from sera of patients with Graves' disease and capable of competing with TSH for binding to its receptor, inhibited the labeling of the 68,000 complex. When the hormone-receptor complex was crosslinked with higher concentrations of DSS (greater than 0.3 mM), a second specifically labeled band was observed, with a Mr of 80,000 +/- 5000. This complex exhibited hormone, tissue, and immunologic specificities similar to those of the 68,000 band. Continuous sucrose density gradient analysis indicated that the intact solubilized receptor possessed a sedimentation coefficient of 10.5 S prior to correction for detergent binding. However, this value increased to 16 S when determined under conditions which took into account the change in hydrodynamic properties attributable to bound Triton X-100. These data suggest that the 80,000 and 68,000 bands represent binding components of the TSH receptor and that the receptor molecule most likely contains multiple subunits, linked by noncovalent forces. PMID- 3008662 TI - Properties of the 23,000-Da phosphoproteins in cardiac sarcolemma and sarcoplasmic reticulum. AB - The calmodulin- and cAMP-dependent protein kinase-mediated phosphorylations of isolated sarcolemma and sarcoplasmic reticulum vesicles have been compared. Similarities in the calmodulin-mediated phosphorylation of the sarcolemma and sarcoplasmic reticulum 23,000-Da phosphoproteins included their Mg2+, Na+, Ca2+, and calmodulin sensitivities, as well as the size of their dissociated subunits. In contrast, a number of differences between these phosphoproteins were indicated in their sensitivity to detergents (Triton X-100 and sodium dodecyl sulfate) and calmodulin antagonists (R24571 and trifluoperazine). Furthermore, in contrast to the sarcoplasmic reticulum phosphoprotein, the sarcolemma phosphoprotein could not be affinity labeled with 125I-calmodulin. While these results indicate the probable chemical similarity of the sarcolemma and sarcoplasmic reticulum 23,000 Da phosphoproteins, they also indicate there are differences in the lipid/phosphoprotein interactions in these two membranes. PMID- 3008663 TI - [Hormone production and abnormalities in gene expression in tumors]. AB - In order to elucidate the mechanism responsible for ectopic hormone production in tumors, biosynthesis of ACHT and related peptides was studied in the pituitary and tumors at levels. In vitro biosynthesis of the ACTH/beta-LPH precursor directed by mRNA extracted from the pituitary and tumors showed no difference in translation products. It is highly likely, therefore, that different final products produced in the pituitary and tumors are caused by different posttranslational processing, such as proteolysis and glycosylation of translation products. The RNA blot analysis of tumors revealed mRNA identical to that of the pituitary. In certain tumors, however, there were larger or smaller mRNA hybridized with an ACTH/beta-LPH precursor probe, in addition to mRNA of normal size. Further studies with endonuclease S1 mapping have provided evidence suggesting that the larger one was probably produced by abnormal splicing of RNA precursor and that the smaller one was resulted possibly from aberrant transcription of the gene. The Southern blot analysis revealed no difference in restriction DNA fragments between the pituitary and tumors, indicating no evidence of gene rearrangement. From these studies, it is conceivable that ectopic ACTH production is resulted from abnormalities in the regulatory mechanism of gene expression. To further study the mechanism regulating the expression of the ACTH/beta-LPH precursor gene, human gene was transfected to mouse pituitary ACTH-producing adenoma cells (AtT-20) and fibroblasts (L-cell). The introduced human ACTH/beta-LPH precursor gene was expressed in AtT-20 cells and suppressed by glucocorticoids to an extent similar to the suppression of mouse gene. On the other hand, possible aberrant transcripts were observed in mouse L cells. It is likely, therefore, that there is a regulatory mechanism, probably "trans" acting, in the pituitary ACTH-producing producing cells and similar mechanism, though not identical, could be exerted in ectopic ACTH producing tumors. PMID- 3008664 TI - [Regulation of EGF receptor by tumor promoters]. AB - Regulation of the EGF receptor system by tumor promoters Nobuyoshi Shimizu (Dept. of Mol. Biol., Keio Univ. Sch. Med.) EGF inhibits the growth of several tumor cell lines that possess extremely high numbers of EGF receptors. The growth of some of these cell lines is also inhibited by the tumor promoter 12-O tetradecanoyl phorbol 13-acetate (TPA) while others are insensitive to TPA. We have isolated variants from these cells that are no longer affected by EGF and/or TPA. Initial characterization of these cell variants is presented with regard to the TPA-directed regulation of the EGF receptor system. PMID- 3008666 TI - [Regulation of cAMP cascade by yeast RAS genes]. AB - In the yeast, Saccharomyces cerevisiae, cell proliferation is regulated by cAMP through cAMP-dependent protein kinase. Cyclic AMP-dependent phosphorylation is involved in the G1 phase of the cell cycle, conjugation, and the post-meiotic stage of sporulation, but inhibition of cAMP-dependent protein phosphorylation is required in order to induce meiotic division. The yeast RAS gene, which is homologous to the H-ras gene, is one of several genes which are related to cAMP cascade, and its product seems to be a regulatory protein of adenylate cyclase. PMID- 3008665 TI - [Possible modes of action of growth factors and tumor promoters in the activation of the c-myc gene in Swiss 3T3 fibroblasts]. AB - It has been suggested that the c-myc gene may play an important role in the regulation of cell proliferation. We have investigated the transmembrane signaling mechanisms of various growth factors and tumor promoters in Swiss 3T3 fibroblasts and have examined the causal relationship between these mechanisms and c-myc gene activation. Platelet-derived growth factor and FGF (fibroblast growth factor) induced the activation of diglyceride-protein kinase C and Ca2+ systems through phosphoinositide turnover, resulting in the activation of the c myc gene. Epidermal growth factor did not activate these two systems but stimulated gene activation. Prostaglandin E1 elicited Ca2+ mobilization and cyclic AMP generation followed by c-myc gene activation. In contrast to these growth factors, tumor-promoting phorbol esters induced the direct activation of protein kinase C which led to c-myc gene expression. Bile acids, which are known to be colon tumor promoters, were inactive by themselves but enhanced FGF-induced diglyceride formation and thereby potentiated protein kinase C activation. It has not yet been examined whether bile acids potentiate FGF-induced activation of the c-myc gene. The growth factors described above and the phorbol esters stimulated DNA synthesis in the presence of insulin, whereas the bile acids potentiated FGF induced DNA synthesis. These results strongly suggest that three messenger systems, diglyceride, Ca2+ and cyclic AMP, may be involved in c-myc gene activation which may be implicated in DNA synthesis in Swiss 3T3 cells. PMID- 3008667 TI - [Role of inositol phospholipids metabolism in the signal transduction of erbB gene product]. AB - We have observed the enhanced metabolism of inositol phospholipids in erbB transformed chicken fibroblasts. This increased metabolism seemed to be due to the activation of PI-kinase, DPI-kinase and DG-kinase activities in these cells. And also, C-kinase activity in the transformed cells was observed to be sustained as an active form. These results suggest the signal transduction system through the enhanced metabolism of inositol phospholipids was activated in the transformed cells. When the normal cells were treated with the combination of TPA, which is an activator of C-kinase, and Ca2+-ionophore A23187, the stimulation of DNA synthesis was detected. However, such treatment caused the only inhibitory effect on the cell growth of the transformed cells. On the other hand, the erbB gene product by itself dose not have significant activity of PI kinase, DPI-kinase and DG-kinase. Therefore, erbB gene product may indirectly enhance inositol-phospholipids metabolism and induce abnormal growth of transformed cells. PMID- 3008668 TI - [The role of protein kinase C in cell surface signal transduction and tumor promotion]. AB - Information from certain extracellular signals, including a group of peptide hormones, some neurotransmitters and many other biologically active substances flows from the cell surface into the cell interior by means of two mechanisms, Ca2+ mobilization and protein kinase C activation. These two signal pathways are activated by the receptor-mediated hydrolysis of phosphatidylinositol-4, 5 bisphosphate. Both protein kinase C activation and Ca2+ mobilization are essential for short-term responses as well as long-term responses. However, additional receptor occupation by growth factor is necessary to induce full activation of cell proliferation, and the signal pathway through protein kinase C appears to be separate from and synergistic to that via growth factors. Several functional proteins in many tissues have been reported to serve as substrates of protein kinase C. The phosphorylation of some of these proteins is apparently related to down-regulation or negative feedback control of cellular function activation. It is possible that protein kinase C has a dual action in the positive as well as the negative phase of regulation depending on the function of each target substrate protein. This article summarizes the possible roles of this unique protein kinase system. PMID- 3008669 TI - [Evaluation of postoperative immunochemotherapy in lung cancer]. AB - A study of postoperative adjuvant chemotherapy according to cell type, combined with immunotherapy using PSK and OK-432 was conducted in 178 lung cancer patients who had undergone resection. BRM (PSK or OK-432) was selected by randomization. Chemotherapy was mainly performed with VCR, MMC, and MTX except for small cell carcinoma. Of the total number of lung cancer cases, 113 were evaluable. Four year survival rates were 36.7% for the OK-432 group, 55.9% for the PSK group and 34.7% for the control group, with significant differences among these three groups. Survival rates for stage III showed significant differences between immunochemotherapy groups, (OK-432 group, PSK group) and the chemotherapy group. Postoperative administration of immunomodulators is therefore considered to contribute to the improved survival of patients in lung cancer. PMID- 3008670 TI - [Proton beam therapy]. AB - Proton beam therapy has been developed for about the last ten years as one of the most reasonable treatment techniques in cancer therapy, based upon the beneficial features of proton beams. Because of the favorable clinical results obtained further progress in this form of treatment is expected. The fundamental and clinical features of this technique and also the future plans being studied at the moment are discussed here, based on the clinical results obtained mainly at the Particle Radiation Medical Science Center of Tsukuba University. The fundamental features of a proton beam are the possibility of selective irradiation of the tumor lesion and minimal irradiation of the surrounding normal tissues due to its fine dose distribution and favorable biological response which is similar to that of 60Co gamma rays. Due to these beneficial features of proton beams, tumor lesions have been successfully irradiated with sufficiently large doses, avoiding irradiation of important organs close to the lesions. Treatment results revealed sufficient control of lesions resistant to conventional radiotherapy, such as locally advanced lesions, lesions with large volume or those with low sensitivity. These results suggested that proton beam therapy could be applied to radical treatment of lesions over a much wider indication range, especially in deep-seated organs, compared with conventional radiotherapy. Investigations of proton beam treatment are to be further continued with the development of techniques to delives precisely controlled irradiation to lesions in moving organs and of a treatment unit installable in cancer hospitals. PMID- 3008671 TI - [A phase II study of intravenous VP-16-213 in small cell and non-small cell carcinoma of the lung]. AB - A phase II clinical trial of VP-16-213 was carried out in 71 patients with small cell and non-small cell carcinoma of the lung. Forty-eight evaluable cases consisted of 36 small cell carcinomas, 7 epidermoid carcinomas, 4 adenocarcinomas and one unclassified carcinoma. VP-16-213 was administered by drip infusion at dosages of 60-100mg/m2/day for 5-consecutive days at 3-4 week intervals. Twelve of 36 (33.3%) small cell carcinomas had partial responses, while no responses were obtained in non-small cell carcinomas. Median duration of responses was 46 days (range 31-133 days). The dose limiting toxicity was leukopenia. Median number of days to nadir was 14 days and median numbers of days for recovery was 11 days. Nausea (38%), vomiting (12%), anorexia (45%) and alopecia (74%) were major clinical toxicities although these were mild or reversible. We concluded that VP-16-213 was useful in the treatment of small cell lung cancer and the dose schedule used in this study was recommendable with small dose reduction for further trial of combination chemotherapy. PMID- 3008672 TI - Rotavirus vaccines--achievements and prospects. PMID- 3008673 TI - Monoclonal antibody B72.3 as a diagnostic adjunct in fine needle aspirates of breast masses. AB - Fine needle aspirations of breast masses were evaluated to establish a cytologic diagnosis. In 45 cases, the sensitivity was 73%, specificity 100%, and diagnostic accuracy 84%. Fifty cases (27 malignant and 23 benign) were evaluated by immunoperoxidase staining of the needle aspirates, using the monoclonal antibody B72.3. Twenty-six of 27 malignant cases stained positively, and 22 of 23 benign cases were negative. The sensitivity was 96%, specificity was 96%, and diagnostic accuracy was 96%. All cases considered malignant cytologically were confirmed by immunocytochemistry. In addition, the B72.3 monoclonal antibody appears to be a valuable diagnostic adjunct in atypical and suspicious cases, particularly those in which hypocellularity and/or cellular monomorphism preclude a diagnosis of malignancy by routine cytologic examination. PMID- 3008674 TI - Factors influencing survival after resection for ductal adenocarcinoma of the pancreas. AB - Twenty-three patients with pancreatic cancer who survived greater than or equal to 3 years after surgical treatment and 56 who survived less than 12 months were studied. The association of steatorrhea with long survival was significant (p less than 0.05), and the association of back pain with short survival showed a trend toward significance (p = 0.06). Other presenting symptoms, as well as the age, sex, or past medical history of the patients; the gross morphology of the tumor and regional lymph nodes; the operations performed; and the use of postoperative adjuvant therapy had no significant influence on survival. Certain histopathologic characteristics of the resected specimens were significantly associated (p less than 0.05) with a poor prognosis: malignant infiltration of the pancreatic capsule, proximity of the tumor to lymphatic and blood vessels, a round-cell infiltrate at the tumor margin, and epithelial atypia in the uninvolved pancreatic ducts. The association of Broders' grades 3 and 4 in the primary tumor and metastases to lymph nodes showed a trend toward significance with short survival. Multivariate analysis confirmed that the associations of Broders' grades 3 and 4 in the primary tumor, a round-cell infiltrate at the tumor margin, and atypia of the pancreatic ductal epithelium with short survival were statistically significant. PMID- 3008675 TI - Evaluation of dopaminergic and alpha adrenergic activities of TL-99 in peripheral tissues in vitro. AB - The dopaminergic and alpha adrenergic activities of TL-99 were investigated using in vitro preparations including cat right atria, guinea-pig atria, rat vas deferens and rabbit aortic strip. TL-99 was found to be as potent as clonidine as a stimulant of presynaptic alpha-2 adrenoceptors of noradrenaline nerve endings in guinea-pig atria and rat vas deferens. In the cat right atria, TL-99 preferently stimulates presynaptic dopamine receptors. The involvement of presynaptic alpha-2 adrenoceptor stimulation in TL-99-induced inhibition of tachycardic response to transmural stimulation in cat right atria is not clear. TL-99 is one thousand times less active as a stimulant of alpha-1 adrenoceptors of rabbit aorta than as a stimulant of presynaptic alpha-2 adrenoceptors of guinea-pig atria and rat deferens. Data from this manuscript as well as reports in the literature strongly suggest that TL-99 may interact with both alpha 2 adrenoceptors as well as dopamine receptors. The selectivity for receptor-type depends on test conditions, species variations and organ selectivity. The significance of these findings with TL-99 is discussed. PMID- 3008676 TI - Postsynaptic alpha-adrenoceptor subtypes in the internal carotid, mesenteric, splenic, renal and femoral vascular beds of the dog. AB - Pressor effects of noradrenaline, phenylephrine and alpha-methylnoradrenaline and the inhibition of these effects by prazosin or yohimbine (or both) were studied in vivo in the renal, splenic, femoral, anterior mesenteric and internal carotid vascular beds of the dog. In all the vascular beds noradrenaline was more potent than alpha-methylnoradrenaline and alpha-methylnoradrenaline was more potent than phenylephrine. However, the ratios between the ED50 for phenylephrine and the ED50 for alpha-methylnoradrenaline in the mesenteric, in the femoral, in the splenic and in the renal circulations was 2.19, 1.89, 1.48, and 1.33, respectively, showing that the relative potency of phenylephrine increased in that order. In the internal carotid vascular bed it was not possible to determine any value. Furthermore, in the renal vascular bed, prazosin (100 micrograms/kg) inhibited pressor responses to all agonists more readily than yohimbine (250 micrograms/kg) and yohimbine was more potent than prazosin against all the agonists in the mesenteric and in the femoral vascular beds. Our results show that: there are both postsynaptic alpha 1- and alpha 2-adrenoceptors in the four vascular beds; the contribution of alpha 2-adrenoceptors to pressor responses to sympathomimetic agents is maximal in the mesenteric followed by the femoral, the splenic and the renal vascular bed, whereas the participation of alpha 1 adrenoceptors is maximal in the renal and progressively smaller in the splenic, in the femoral and in the mesenteric vascular bed, where it reaches the lowest value. PMID- 3008677 TI - Facilitation of acetylcholine-evoked catecholamine release by cyclic AMP on isolated perfused dog adrenal glands. AB - The effects of cyclic nucleotides on catecholamine (CA) release and Ca2+ efflux were determined on isolated dog adrenals perfused with 1.3 mM Ca2+ containing fluid except indicated. Dibutyryl cyclic AMP (DBcAMP, 50 microM--1 mM) substantially enhanced CA release evoked by acetylcholine (ACh) and slightly increased the spontaneous CA release. Dibutyryl cyclic GMP (100-500 microM) narrowly increased the basal CA release. DBcAMP, 200 microM also significantly facilitated CA release caused by nicotine, bethanechol and excess K+, and by caffeine in the absence of extracellular Ca2+. 45Ca efflux evoked by ACh and by caffeine in the absence of extracellular Ca2+ from prelabelled adrenals with 45Ca was enhanced significantly by DBcAMP, 200 microM. These results suggest that cAMP may function as a facilitatory modulator in CA release from the adrenal medulla via in part the effect on Ca2+ flux including alterations in the function of systems for intracellular Ca2+ homeostasis. PMID- 3008678 TI - Antibody to the retrovirus associated with the acquired immunodeficiency syndrome (AIDS). Presence in presumably healthy San Franciscans who died unexpectedly. AB - We performed autopsies and serologic tests in 189 subjects (152 men and 37 women) between 20 and 50 years of age with no history of immunosuppression who died unexpectedly and whose bodies were referred to the San Francisco coroner's office. Forty-eight of the 88 single men for whom addresses were available lived in areas of the city with a high incidence of the acquired immunodeficiency syndrome (AIDS). In addition, 36 of the subjects (30 men) were intravenous drug abusers. Antibody to the retrovirus associated with AIDS was present in 23 (18%) of the 121 subjects whose sera were tested. However, neither pathologic nor laboratory manifestations of AIDS were present in any of the 189 subjects who underwent autopsy. These results suggest that antibody to the retrovirus is common but subclinical manifestations of AIDS are uncommon in San Francisco, a city where the incidence of clinical AIDS is high. PMID- 3008680 TI - HTLV-III antibody in intravenous drug abusers of metropolitan Barcelona, Spain. PMID- 3008679 TI - Chemotherapy-induced complete remission of a malignant fibrous histiocytoma of bone. An 11-year follow-up. AB - In a young woman with multiple malignant fibrous histiocytoma of bone and soft tissue with a rapidly progressive course, long-term chemotherapy with prednisone and cyclophosphamide resulted in a complete remission still lasting after an 11 year follow-up. PMID- 3008681 TI - Plasma renin activity, aldosterone, and cortisol responses to a short-acting intravenous adrenocorticotropic hormone test. PMID- 3008682 TI - Pseudo-Addison's disease. Isolated corticotropin deficiency associated with hyporeninemic hypoaldosteronism. AB - A 60-year-old man presented with loss of weight and appetite, eosinophilia, and hyperkalemia consistent with a diagnosis of Addison's disease. Adrenal responsiveness to exogenous corticotropin was normal, but endogenous corticotropin and cortisol responses to insulin-induced hypoglycemia were both absent. Pituitary function was otherwise intact. Renin and aldosterone levels were subnormal and did not respond to postural change. To our knowledge, this is the first reported case of isolated corticotropin deficiency and hyporeninemic hypoaldosteronism together mimicking primary adrenocortical failure. PMID- 3008684 TI - The protective effect of pollen extracts against allyl alcohol damage of the liver. AB - In male Wistar rats the hepatoprotective effect of pollen extracts (Cernitins) agains. orally introduced 1% allyl alcohol (0.4 ml per 100 g body weight) was investigated Cernitins were applied orally at 0.6, 24 and 30 h after allyl alcohol administration. After 48 h an autopsy was performed and blood was collected for biochemical tests. Liver damage was evaluated by measurement of aminotransferases (AspAT, AlAT) and alkaline phosphatase activity, total bilirubin level in the blood serum as well as by histological examination of the livers. Cernitins significantly reduced the serum enzymes elevations induced by allyl alcohol. The hepatoprotective properties of Cernitins were confirmed by histopathological studies. PMID- 3008683 TI - [Treatment of visceral leishmaniasis]. AB - A better knowledge of the mechanisms of resistance of leishmanias to drugs, of their survival strategy and the investigation of the immune mechanisms developed by the host against these parasites would be of the highest importance in the therapeutic approach of the disease, for a better use of available drugs, to try to stop the infectious process and to develop and immunotherapy and an efficient protection against the disease. PMID- 3008686 TI - [Antiviral agents. 27. The aminomethinylation of 5-oxo-2-pyrazoline-3-carboxylic acid derivatives]. PMID- 3008685 TI - Wilms' tumor in a 'lump' kidney associated with sacral agenesis. AB - Many cases of Wilms' tumor developing in a horseshoe kidney have been previously reported. We present a case of Wilms' tumor developing in a "lump" kidney, associated with sacral agenesis. We discuss the implications for staging and make recommendations for follow-up in these cancer-prone children. PMID- 3008687 TI - Biodegradation of polyglycolic acid in bone tissue: an experimental study on rabbits. AB - The biodegradation of polyglycolic acid (PGA) was investigated in cortical bone of 21 rabbits and in cancellous bone of 15 rabbits. The follow-up times were 3, 6, and 12 weeks. Radiographical, histological, microradiographical, and oxytetracycline labeling studies were done. PGA was biocompatible and was degraded to a great extent in the cancellous bone and partly in the cortical bone in 12 weeks without any sign of inflammation or foreign body reaction. The biodegradation of PGA started peripherally in the area of the implant and continued with subsequent replacement by new bone. PMID- 3008689 TI - A rapid, sensitive and quantitative high-performance liquid chromatographic determination of catecholestrogens in human serum. PMID- 3008688 TI - [The biology of the inner ear]. PMID- 3008691 TI - Antibody response to the herpes simplex virus type 2 AG-4 antigen in guinea pigs with genital herpes infections. AB - Female guinea pigs, inoculated intravaginally with a herpes simplex virus-type 2 (HSV-2) strain M1, developed typical symptoms of a primary and recurrent genital herpes infection. Sera from HSV-2 infected guinea pigs taken during the primary or recurrent stages of the genital infection contained complement (C)-fixing antibody which reacted with an apparent type specificity to an early 4 h HSV-2 infected cell extract (AG-4) when compared to a 4 h HSV-1 infected cell extract. This C-fixing anti-AG-4 activity was shown to be associated with the IgG2 subclass and directed primarily against HSV-2 infected cell polypeptide (ICP)6 and ICP8. Furthermore, C-fixing anti-AG-4 levels remained constant after the primary infection and during recurrences for over 6 months. Thus, while the anti AG-4 response in guinea pigs was of the IgG type, it varied from that found previously in human genital HSV-2 sufferers which was of the IgM type. Also, while an IgG anti-AG-4 response in human genital HSV-2 sufferers is associated with a reduction in the number of recurrences, this reduction is not apparent in the guinea pig model. PMID- 3008690 TI - Preventive initiatives in medicine and surgery. The importance of nutrition and tranquillity. PMID- 3008693 TI - Encephalomyocarditis virus infection and pig disease in Queensland. PMID- 3008692 TI - Attempted conversion of twin to singleton pregnancy in two mares with associated changes in plasma oestrone sulphate concentrations. AB - The removal of one of twin embryos was attempted by infusion of 24% (w/v) saline into the gestation sac in 2 mares by laparotomy. The treatment was successful in one mare (Case 1) and the untreated embryo remained viable. However, neither foetus survived in the second mare (Case 2). Plasma oestrone sulphate (E1S) concentrations fell immediately after treatment in both mares but recovered to approximately 50% of pretreatment levels in Case 1. In Case 2 plasma E1S concentrations declined steadily and were less than 1 ng/ml within 6 days of treatment. These preliminary results suggest that the method may be useful for selective removal of one of twin embryos in mares. Furthermore, plasma E1S concentrations may be a useful indicator of embryonic viability. PMID- 3008694 TI - Some epidemiological features and effects on reproductive performance of endemic porcine parvovirus infection. AB - The time of development of demonstrable antibody to porcine parvovirus (PPV) was determined for 661 gilts entering the breeding herd in a 2800 sow intensive piggery; 13.2% of these gilts did not have detectable antibody to PPV when first introduced into the breeding herd at 25 to 26 weeks of age. Exposure to PPV was found to vary in different sheds and even in different areas within a shed. Gilts that developed antibody to PPV during the first third of pregnancy were not adversely affected. Those that developed antibody during the middle third of pregnancy had fewer piglets born alive, more stillborn piglets and more mummified foetuses. PMID- 3008695 TI - Interaction of low-pathogenicity reoviruses and low levels of infection with several coccidial species. AB - Various combinations of reoviruses and coccidia were studied to see if interactions would occur. Two reoviruses were used: virus 2035, a moderate to low pathogen, and virus 2177, a nonpathogen. Coccidia used were Eimeria acervulina, E. mitis, and E. maxima at dosages of 10(3) or 10(4) sporulated oocysts/chick and E. brunetti at 10(4) sporulated oocysts/chick. In Hubbard-Hubbard cockerels, a combination of virus 2035 and E. acervulina (10(4) oocysts/chick) or E. maxima (10(3) oocysts/chick) significantly (P less than or equal to 0.05) increased the frequency of stunting (% of chicks with body weight less than 80% of controls) and further depressed weight gain over that seen with either virus or coccidia alone. Conversely, virus 2177 ameliorated the same effects in Shaver-Arbor Acre cockerels given 10(4) oocysts/chick of E. mitis or E. maxima. The interaction could not be attributed to changes in the degree of coccidial infection based on oocyst production. Reovirus did not generally change the effect of coccidia on levels of plasma pigment and plasma protein. In Hubbard-Hubbard cockerels, coccidia-induced effects were not ameliorated by virus 2177, suggesting that breed difference in interaction can be expected. PMID- 3008696 TI - An enzyme-linked immunosorbent assay for detection of antibodies to exogenous avian leukosis virus. AB - A microplate enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to avian leukosis virus (ALV) of subgroups A and B in infected chickens was developed with the use of Rous-associated virus (RAV)-1 (subgroup A) and RAV-2 (subgroup B) antigens purified by sucrose-gradient centrifugation. The antigen was used for ELISA after treatment with Triton X-100. In the ELISA, the subgroup viral antigen reacted strongly with homologous antiserum but also reacted with heterologous antiserum. Tests with serum absorbed with purified homologous and heterologous virus and tests for antigen-blocking by group-specific antibodies to ALV revealed that the reaction was caused mainly by subgroup-specific antibodies. The ELISA was 8 to 32 times more sensitive than the virus-neutralization (VN) test and detected antibodies to ALV earlier than the VN test in chickens infected experimentally with RAV-1 and RAV-2. In field application of the ELISA, 44.2% of 484 chicken sera were positive for RAV-1 and/or RAV-2 antigen, and 80.4% of flocks were positive. These findings indicate that ELISA is superior to the VN test in sensitivity, simplicity, rapidity, and applicability for large-scale field surveys for ALV infection. PMID- 3008697 TI - Epidemiology of reticuloendotheliosis virus in broiler breeder flocks. AB - Six broiler breeder flocks from two companies in Mississippi were tested at intervals for reticuloendotheliosis (RE) virus infection. Virus was isolated and antibody demonstrated in all six flocks. Infection was first detected at ages ranging from 13 to 47 weeks. Studies showed that neither congenital transmission from grandparent flocks nor treatment with contaminated vaccines was a likely source of infection; thus, exposure to RE virus was assumed to come from the environment. Virus was isolated from litter samples from two of the flocks, but no specific environmental infection source was identified. Infection rates of flocks differed between the two companies. Although adequate controls were lacking, no performance problems due to RE virus infection were apparent in breeder or broiler progeny flocks. However, the RE viruses isolated from these flocks were immunosuppressive and oncogenic when inoculated into day-old chicks. A moderate (3-16%) incidence of neoplasms was induced by contact exposure to these field isolates in the laboratory. PMID- 3008698 TI - Embryo vaccination with infectious bursal disease virus alone or in combination with Marek's disease vaccine. AB - Studies with specific-pathogen-free chickens revealed that chicks hatching from eggs inoculated at the 18th day of embryonation with infectious bursal disease (IBD) vaccine viruses of low virulence (isolates TC-IBDV and BVM-IBDV) developed antibody against IBD virus (IBDV) and resisted challenge with virulent IBDV at 3 weeks of age or older. Embryo vaccination did not adversely affect hatchability of chicks or survival of hatched chicks. Chicks embryonally vaccinated with TC IBDV had transient histologic lesions in the bursa of Fabricius at hatch. Similar but milder lesions were also noted in chickens that received TC-IBDV at hatch. The level of protection following embryo vaccination with TC-IBDV and BVM-IBDV was similar to that following vaccination with the same vaccines at hatch. Vaccine viruses of moderate virulence (isolates BV-IBDV and 2512-IBDV) were not suitable as vaccines in embryos lacking maternal antibody to IBDV, because the vaccinated chicks developed acute IBD after hatch. Isolate 2512-IBDV was not pathogenic for embryos bearing maternal antibody to IBDV. Maternal antibody against IBDV interfered with efficacy of embryo vaccination with BVM-IBDV but not with 2512-IBDV. Embryo vaccination with a mixture of vaccines against IBD and Marek's disease resulted in protection of hatched chicks against challenge with virulent IBDV and Marek's disease virus. PMID- 3008699 TI - Immunogenicity and antigenicity of infectious bursal disease virus serotypes I and II in chickens. AB - Chickens were inoculated with infectious bursal disease virus serotype I or serotype II to determine if their immune system can distinguish between the two serotypes. Chickens had neutralizing antibodies to only serotype I viruses following exposure to serotype I viruses, and chickens had antibodies to only serotype II viruses following exposure to serotype II viruses. No cross-reactions were observed between antisera prepared to each of these two serotypes using a cross-virus-neutralization assay. Signs of disease were detected only in birds exposed to a virulent serotype I isolate. Chicks exposed to the serotype II viruses were not protected from challenge with a virulent serotype I isolate. In one experiment, antibodies to a serotype II isolate, which were detected before challenge, did not protect chicks from challenge with a virulent serotype I isolate. PMID- 3008700 TI - Protein concentrations and immunosuppressive properties of serum in chickens experimentally infected with avian tumor viruses. AB - Sera from chickens affected by Marek's disease or developing Rous sarcoma were investigated. There were changes in the protein fractions, and the amount of alpha and beta fractions was consistently increased. At the same time, immunosuppressive factors were found to inhibit the number of plaque-forming cells in the spleen of mice immunized with sheep red blood cells. PMID- 3008701 TI - Gastrointestinal transit times in normal and reovirus-inoculated turkeys. AB - Brilliant pigment powder was used to measure minimum gastrointestinal transit times (GTT) in control and cloned-reovirus-inoculated turkey poults. Compared with controls, inoculated poults had longer GTT at 120 hr (P less than 0.05) but not at 24 and 72 hr after receiving a single oral dose of cloned reovirus. Reovirus inoculation was associated with failure to eat following a fasting period. Feces passed by reovirus-inoculated turkeys were more fluid and less well formed than normal. PMID- 3008702 TI - Pathological changes of tracheal mucosa in chickens infected with infectious laryngotracheitis virus. AB - Six-week-old chickens were inoculated via the posterior thoracic air sac with infectious laryngotracheitis virus. Chickens were sacrificed on various days through day 16 postinoculation (PI), and the trachea was examined by scanning electron microscopy (SEM) and light microscopy (LM). The pathological changes observed on day 1 PI were hypertrophy and hyperplasia of goblet cells. From day 3 PI, the epithelial cells protruded collectively and fused to form syncytia, which contained many intranuclear inclusion bodies. Subsequently, epithelial syncytia desquamated, one after another, and connective tissues were exposed in places. Serofibrinous exudate and detritus were abundant on the surface of the exposed connective tissues and seemed to form a pseudomembrane. On day 5 PI, the remaining epithelial cells began to repair the devastated mucosa just under the pseudomembrane. On day 6 PI, microvillus-rich regenerating epithelial cells were arranged like paving stones. On day 8 PI, the epithelial cells proliferated extensively and formed folds with cyst-like structures. By day 16 PI, the tracheal epithelium was covered with cilia and regained its normal histologic appearance. PMID- 3008704 TI - Current concepts of ridge augmentation. PMID- 3008705 TI - Disruption of neophobia, conditioned odor aversion, and conditioned taste aversion in rats with hippocampal lesions. AB - Previous studies have implicated the hippocampus in the acquisition of conditioned taste aversions. However, the effect of hippocampal (HPC) lesions on the acquisition of conditioned aversions to the distal olfactory cue has not been investigated. In this study rats with bilateral electrolytic hippocampal lesions were given access to an odor conditioned stimulus (CS) alone or a compound odor taste CS, followed by an injection of LiCl or saline. The results indicated that HPC lesions attenuated the neophobic response to both CSs, and disrupted conditioned odor and taste aversions, relative to sham-operated controls. Furthermore, the disruption in conditioned odor aversions could not be attributed to attenuation of neophobia in lesioned subjects nor to prolonged neophobia in sham-operated controls. The results are consistent with pharmacological studies in suggesting that the hippocampus is involved in the formation of conditioned odor aversions. PMID- 3008703 TI - 1985 Armstrong lecture. Cardiovascular receptors and fluid volume control. AB - Experiments with negative pressure breathing led Gauer and Henry to the discovery of left atrial receptors that affected the release of ADH and the concept of a cardiovascular basis for fluid volume control. Since then, neural mechanisms involving cardiac receptors controlling the release of renin have been described, thus giving strong support for the role of cardiac receptors in fluid volume control. The original experimental work in the dog has been confirmed. The available pool of patients that have undergone heart transplant operations provide the opportunity for more definitive studies on the role of cardiac receptors in the control of cardiovascular function. PMID- 3008706 TI - Characterization of stable Epstein-Barr (EB) virus transformed cell lines and mouse-human hybridomas producing a large quantity of anti-tetanus toxoid (TT) monoclonal antibody. AB - Antigen-specific human monoclonal antibodies (hMoAb) were investigated with particular emphasis on the stability and scale-up of production. Peripheral blood lymphocytes from a healthy donor previously vaccinated with tetanus toxoid (TT) were resensitized in vitro with the antigen in the presence of pokeweed mitogen (PWM). Three days after the stimulation, lymphocytes were infected with Epstein Barr (EB) virus. Among EB virus transformants, several lymphoblastoid cell clones producing anti-TT hMoAb were established. One of these clones, 3G6, produced about 10 micrograms/ml of IgM (K) in the culture supernatant. Furthermore, 3G6 cells were fused with murine myeloma cells (X63-Ag8 X 653) and three heterogeneous hybridoma clones (HE719, HE810 and HE811) were obtained after careful hybrid selection in hypoxanthine-aminopterin-thymidine-ouabain containing medium and cloning by limiting dilution technique. These hybrid clones contained human HLA and mouse H-2d antigens together with mouse-human mixed karyotype. When these mouse-human hybridomas were injected i.p. into pristane-treated nude mice of BALB/c origin, ascites were found and a large amount of anti-TT hMoAb up to 0.5 mg/ml were produced there. These hybridomas have retained their anti-TT antibody-producing capabilities over 10 months. The approach described here has a potential application for the production of other antigen-specific hMoAb. PMID- 3008707 TI - Characteristics of site variation among clones of the 340-base pair, tandemly repeated EcoR1 family of human DNA. AB - Twenty-four clones of EcoR1-restricted, 340-base pair (bp) DNA derived from human DNA have been sequenced and compared to a published consensus sequence for this family. No two clones were found to have identical sequences; the clones studied differed from the consensus sequence in as few as 1 or as many as 41 sites. On the average in these clones, a 5.2% divergence from exact homology was found, with 1 of 10 of the site variations being "nonrandom," i.e., cases in which five or more clones had the same nucleotide substitution at that site (viz., 53, 124, 126, 138, 152, and 157). At site 157, for example, 16 of the 24 clones differed from the reference sequence. Positions and their respective changes, as compared to the consensus sequence, are summarized. Variations are discussed with relation to possible functions for these sequences. PMID- 3008709 TI - Signal transfer in cardiac muscle. Alteration of the beta-adrenoceptor adenylate cyclase system in the hypertrophied myocardium. AB - The beta-adrenoceptor adenylate cyclase complex (beta ACR), located in the sarcolemmal membrane, is one of the most effective signal transduction systems regulating function and metabolism of heart muscle primarily via cyclic AMP. It is thought to play an important role in adaptive mechanisms of the heart as to pressure load and stressful stimuli. Present knowledge about composition and function of beta ARC enable us to clear up which of the single components of this system contributes to pathophysiological alterations of heart function. Cardiac beta ARC was investigated in three experimental groups: I) adult rats of WKY strain (WKY), II) adult rats of WKY strain cardiac hypertrophy in which was induced by aortic constriction (WKYAC), and III) adult spontaneously hypertensive rats (SHR). Quantitative assessment of beta-adrenoceptor number (beta AR) as revealed by [3H]dihydroalprenolol binding studies showed a significant reduction to 30% and 35% of control beta AR in membrane preparations of WKYAC and SHR, respectively. The diminished density of myocardial beta AR was accompanied by a reduction of the maximum stimulatory effect of 1(-)adrenaline on adenylate cyclase (AC). Evidence was obtained for a close correlation between the diminished response of beta ARC to beta AR-mediated stimuli and the heart index as measure of cardiac hypertrophy. No correlation between graduated states of hypertrophy and activation of AC has been observed by NaF and Gpp(NH)p. The results indicate that in rat hearts severe hypertrophy of which was induced by pressure-load, mainly the beta AR component of the beta ARC complex contributes to the reduction of beta ARC-mediated responsiveness of the hypertrophied myocardium. PMID- 3008708 TI - Satellite DNA-correlated nucleosomal proteins in Drosophila virilis. AB - Three major satellite DNAs comprise 40-45% of the genome of Drosophila virilis. Since these satellites are not substrates for most restriction enzymes, we were able to digest D. virilis nuclei with HaeIII and micrococcal nuclease and isolate chromatin fractions containing variable levels of satellite DNA. Electrophoretic analysis of these chromatin fractions revealed that the level of the acid-soluble chromosomal protein, cp17.3, was directly related to the percentage of satellite DNA in chromatin. The correlation between cp17.3 and satellite DNA abundance suggests that cp17.3 is involved in the heterochromatic condensation of satellite DNAs. cp17.3 occurs at a frequency of one molecule per 10-20 nucleosomes. It is detected in an electrophoretically distinguishable class of mononucleosomes, provisionally identified as MN1uH2A, which contains ubiquitinated histone H2A (uH2a) but lacks histone H1. It is not detected in MN1, a second class of mononucleosomes, which lacks uH2A and H1. Since cp17.3 is correlated with satellite DNAs and present in nucleosome cores, it might be a histone variant specifically associated with satellite DNAs. PMID- 3008710 TI - Oscillation of cyclic AMP with the heart cycle of the canine myocardium in situ. AB - Oscillations of cyclic AMP with the cardiac cycle were demonstrated in the canine heart in situ. For tissue sampling an ECG(R-wave)-triggered, automatically working push-freeze-drill apparatus was employed which allowed intraventricular cryobiopsies from the left ventricular muscle of anaesthetized open-chest dogs. The nucleotide cyclic AMP oscillated with the cardiac cycle during normal working conditions, the higher cyclic AMP values occurring during systole. Cyclic GMP was assayed to be without oscillatory changes during the contraction-relaxation cycle of the heart. PMID- 3008711 TI - Metabolic and contractile changes in ischaemic rat hearts after isoproterenol administration: effect of reperfusion. AB - The effect of isoproterenol and of ischaemia followed by reperfusion was studied in isolated perfused rat heart. Whereas after an interval of 5 min of ischaemia the mechanical and biochemical responses to catecholamine were drastically reduced, reperfusion for 1 min normalized the cardiac reactivity. After prolonged ischaemia and reperfusion the hormone-induced rise in cyclic AMP and activation of cyclic AMP dependent protein kinase remains reduced. It is suggested that these changes might be of importance for cardiac protection during a transient ischaemic period. PMID- 3008712 TI - Developmental changes of Ca++ transport systems in chick heart. AB - Sarcolemma (SL) Na+/Ca++ exchange, binding of the Ca++ channel antagonist [3H]nitrendipine and sarcoplasmic reticulum (SR) Ca++ uptake were studied in crude membranes from developing chick heart. Energy-linked Ca++ uptake of mitochondria (MT) was measured in tissue homogenates. When reckoned per unit of heart mass Na+/Ca++ exchange increases linearly (20-fold) from embryonic day 4 to postnatal day 10. These changes correlate strongly with developmental variations of (Na+, K+)ATPase activity. The density of high-affinity [3H]nitrendipine receptors increases in parallel, while the specific affinity does not change SR Ca++ uptake rises steadily during embryogenesis and increases steeply (3-fold) at the time of hatching. Hearts of 10-day-old chickens exhibit 50-fold higher SR Ca++ transport activities than those of 4-day-old embryos. Between the latter stage and postnatal day 10 a more than 100-fold increase of MT Ca++ uptake occurs. The results suggest developmental variations in the contribution of single Ca++ transporting systems in cardiac Ca++ control. PMID- 3008713 TI - Indirect technique for the estimation of cAMP-dependent and Ca2+/calmodulin dependent phospholamban phosphorylation state in canine heart in vivo. AB - An indirect technique was employed to estimate the in vivo phosphorylation state of phospholamban in preparations from dog hearts depleted from catecholamines and from dog hearts treated with isoproterenol. This method allows the separate detection of cAMP-dependent and Ca2+/calmodulin-dependent phospholamban phosphorylation. The data obtained demonstrate the phosphorylation of both the cAMP-dependent and the Ca2+/calmodulin-dependent phosphorylatable site of phospholamban in response to the beta-adrenergic agonist isoproterenol in canine heart in vivo. PMID- 3008714 TI - The cultured myocardial cells as a model in cardiac research. AB - Cultured myocardial cells have become a standard tool in experimental cardiology. Advantages of this model are pointed out, and some of the results obtained demonstrate the usefulness of this model for investigations of contractile, biochemical, and electrophysiological parameters. The first report on cultivated heart cells was published 75 years ago by BURROWS. He described the independent pulsatory activity of single cells grown out of a tissue explant. This observation confirmed the old view that the cell and not a syncytium acts as the elementary structural and functional unit of heart muscle. More than 40 years later RINALDINI (1954) and CAVANOUGH (1955) succeeded in isolating discrete single cells from embryonic avian heart tissue and HARARY (1963) from postnatal mammalian heart muscle by means of proteolytic enzymes (for review see 1). Today myocardial cells in primary culture can be kept alive and physiologically active for several weeks, but there exist not yet a permanent line of cardiac muscle cells with specific attributes of excitability and contractility. Cultivated myocardial cells have become a standard tool in experimental cardiology during the last 10 to 15 years. They provide a model system to study problems of development, differentiation, adaptation, and regulation of metabolism and function. This model affords unique advantages as compared to intact myocardium for solving some of these problems. PMID- 3008715 TI - Isolation and characterization of two 70 kDa modulator-complexes from rabbit skeletal muscle. AB - The activation as well as the inactivation of the ATP,Mg-dependent protein phosphatase has been shown to be totally dependent upon the presence of the modulator subunit. This modulator (inhibitor-2) is a heat stable protein and its isolation in pure form (32 kDa) always includes a boiling step. The boiled modulator fractions are known to be inhibitory to the phosphatase activity. Unboiled rabbit skeletal muscle preparations do not contain "free modulator", but two higher molecular weight complexes (70 kDa) can be isolated which have the 32 kDa modulator together with a 38 kDa protein. One complex is the already characterized inactive ATP,Mg-dependent phosphatase [FCM] while the second one, [MX], although seemingly of identical composition, does not exhibit phosphatase activity when measured under the usual conditions. The MX-complex does not inhibit the phosphatase activity unless subjected to a boiling step which dissociates the modulator subunit. The unboiled [MX] exhibits the activation as well as the inactivation characteristics of the free modulator. PMID- 3008716 TI - Amino acid sequence homology among fructose-1,6-bisphosphatases. AB - The hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate is a key reaction of carbohydrate metabolism. The enzyme that catalyzes this reaction, fructose-1,6-bisphosphatase, appears to be present in all forms of living organisms. Regulation of the enzyme activity, however, occurs by a variety of distinct mechanisms. These include AMP inhibition (most sources), cyclic AMP dependent phosphorylation (yeast), and light-dependent activation (chloroplast). In the present studies, we have made a comparison of the primary structure of mammalian fructose-1,6-bisphosphatase with the sequence of peptides isolated from the yeast Saccharomyces cerevisiae, Escherichia coli, and spinach chloroplast enzymes. Our results demonstrate a high degree of sequence homology, suggesting a common evolutionary origin for all fructose-1,6-bisphosphatases. PMID- 3008717 TI - Arachidonic acid strongly stimulates prostaglandin I3 (PGI3) production from eicosapentaenoic acid in human endothelial cells. AB - Eicosapentaenoic acid (EPA) is a prominent polyunsaturated fatty acid in fish oil which inhibits blood platelet aggregation and thromboxane A2 formation but not prostacyclin-like material generation from vascular endothelium. In this study we investigated interaction between EPA and arachidonic acid (AA) during their oxygenation by cultured endothelial cells. As measured by gas chromatography-mass spectrometry (GC-MS), AA increased markedly prostaglandin I3 (PGI3) production from EPA while that of PGI2 from AA was decreased by EPA. However, increasing the ratio AA/EPA over one almost suppressed the inhibition of PGI2 formation by EPA, and the stimulation of PGI3 production by AA was even higher. The effect of AA on EPA conversion to minor prostaglandins like PGE3 and PGF3 alpha was similar then confirming the stimulating effect and suggesting it is occurring at the cyclooxygenase instead of the prostacyclin synthase level. Altogether these data indicate that, in certain nutritional states where the liberation of EPA from endothelial cells will be accompanied with that of endogenous AA, substantial amounts of PGI3 could contribute to the prostacyclin-like activity of the vessel wall in addition to PGI2. PMID- 3008719 TI - Protein kinase C and non-functional EGF receptor in K-ras transformed cells. PMID- 3008718 TI - The effect of nalidixic acid on expression from related E. coli promoters. AB - The effect of the DNA gyrase inhibitor, nalidixic acid, on expression from E. coli promoters was studied using the pKO-1, galactokinase expression vector system. Expression from a series of related hybrid promoters, tet promoter variants and the trp promoter flanked by oligonucleotide blocks was measured after incubation with nalidixic acid. Expression from the pBR322 tet promoter and tet promoter mutants within the -10 region was reduced after the drug treatment. The lacUV5, trp, and tettrp promoters were essentially unaffected while the trplac and the trptet promoters were stimulated. Studies of the trp promoter flanked by upstream or downstream oligonucleotide blocks revealed similar responses to the trp promoter parent control plasmids. PMID- 3008720 TI - Oxidation of lysine side-chains of elastin by the myeloperoxidase system and by stimulated human neutrophils. AB - Exposure of [3H]-lysine labeled elastin to either purified myeloperoxidase plus H2O2 and halides or human neutrophils plus phorbol myristate acetate resulted in oxidation of lysine side chains quantitated as 3H2O release. In both the enzyme and cell system oxidation was blocked by azide, cyanide or catalase, but not by beta-aminopropionitrile, an inhibitor of lysyl oxidase. Myeloperoxidase-deficient neutrophils were ineffective unless exogenous myeloperoxidase was added. These data provide a biochemical basis for inflammatory changes in connective tissue proteins mediated by oxidant secretory products of neutrophils. PMID- 3008721 TI - Chlorpromazine induces accumulation of inositol phosphates in C6 glioma cells. AB - The capacity to modify the incorporation of [2-3H]myo-inositol into inositides and inositol phosphates was different for three psychotropic cationic amphiphilic drugs. Chlorpromazine, desmethylimipramine and propranolol were able to increase the labeling of inositol-containing lipids, but only chlorpromazine dramatically increased the incorporation into inositol phosphate, -bisphosphate and trisphosphate. The increase was 10- to 50-fold in 60 min as compared with controls. This effect is not due to stimulation of lipid labeling, because in chase experiments radioactivity in inositol phosphates increased to a greater extent than in their parent lipids. It is possible that the alteration of phosphoinositide catabolism is related to the neuroleptic activity of the drug. PMID- 3008722 TI - Selective covalent modification of newly synthesized activation-induced sialoglycoconjugates on the T lymphocyte membrane. AB - Resting human lymphocytes were oxidized by periodate and subsequently reduced with borohydride to block the aldehydes formed. The cells were then incubated for 24 or 48 hours with (or without) the mitogenic lectin phytohemagglutinin. A second oxidation with periodate at the indicated times resulted in strong surface aldehyde formation in samples incubated with the mitogen, compared to control samples which exhibited very low quantities of aldehydes. The data show that this elevation in aldehyde formation was strictly dependent on protein synthesis, similarly to the appearance of Tac antigen in these cells. Cell surface aldehydes were detected in flow cytometry with a fluoresceinated hydrazide molecule and electrophoretically with biocytin hydrazide in conjunction with 125I streptavidin. The proposed method for the elimination of the chemical reactivity of carbohydrates from the surface of resting lymphocytes thus enabled the selective covalent modification of newly synthesized sialoglycoconjugates formed upon a mitogenic trigger. The data also suggest the existence of a very low turnover rate of sialoglycoconjugates on the resting lymphocyte membrane. PMID- 3008723 TI - Identification and solubilization of atrial natriuretic factor receptors in human placenta. AB - Specific high affinity 125I-atrial natriuretic factor binding sites have been identified in human placental membranes. Using the nonionic detergent, Triton X 100, these binding sites were quantitatively solubilized and retained binding activity. In the solubilized preparation, the macromolecular component that binds atrial natriuretic factor is a 160,000 dalton protein as shown by covalently cross-linking it to 125I-atrial natriuretic factor with the bifunctional chemical crosslinker, disuccinimidyl suberate, followed by gel electrophoresis and autoradiography. On Sephadex G-200 gel filtration in the presence of detergent, the hormone-receptor complex elutes in the molecular weight range of 140,000. These observations suggest strongly that a 140- 160,000 dalton protein present in human placental membranes is the receptor for specific recognition of atrial natriuretic factor. PMID- 3008724 TI - Generation of hybridomas producing human monoclonal antibodies against herpes simplex virus after in vitro stimulation. AB - Hybridomas producing human monoclonal antibodies against herpes simplex virus were generated by in vitro antigen stimulation before cell fusion. The cell fusion with tonsillar lymphocytes which were stimulated with antigen and/or pokeweed mitogen generated many hybridomas producing human IgG against the virus. A combination of antigen and pokeweed mitogen synergistically enhanced the generation of virus-specific hybridomas. Furthermore, the higher the antibody response of the tonsil, the more virus-specific hybridomas were generated by the cell fusion. These results suggest that cell fusion with in vitro stimulated lymphocytes can be applied to a variety of clinically relevant viruses. PMID- 3008725 TI - Chronic treatment with phenobarbital decreases the expression of rat liver EGF and insulin receptors. AB - In male F-344 rats fed phenobarbital 0.05% with chow the binding of EGF to microsomal and Golgi liver fractions decreased after 2 weeks, dropped by more than 60% after one month and remained low for the following five months of the experiment. The binding of insulin remained stable for 2 weeks after which it decreased by half. In both cases the receptors decreased in number without changes in their affinity. Autophosphorylation of the receptors showed changes parallel to those of the binding of the ligands. PMID- 3008726 TI - Phorbol 12, myristate 13, acetate potentiates the respiratory burst while inhibits phosphoinositide hydrolysis and calcium mobilization by formyl-methionyl leucyl-phenylalanine in human neutrophils. AB - It is widely believed that the transduction pathway in the activation of the NADPH oxidase by formyl-methionyl-leucyl-phenylalanine (FMLP) in neutrophils involves the stimulation of phosphoinositide hydrolysis, the increase in [Ca2+]i and the activity of the Ca2+ and phospholipid dependent protein kinase C. The results presented here show that the activation of the respiratory burst by FMLP can be dissociated by the stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate and Ca2+ changes. In fact, in neutrophils pretreated (primed) with non stimulatory doses of phorbol myristate acetate the respiratory burst by chemotactic peptide is greatly potentiated while the increase in [3H] inositol phosphates formation and in [Ca2+]i are depressed due to the inhibition of phospholipase C. This finding indicates that FMLP can trigger also a sequence of transduction reactions for the activation of the NADPH oxidase different from that involving the formation of the second messengers diacylglycerol and inositol phosphates and the increase in free Ca2+ concentration. PMID- 3008727 TI - Selective antimitochondrial agents inhibit calmodulin. AB - Certain cationic-lipophilic compounds are known to selectively accumulate in tumor mitochondria and inhibit energy production. Since these substances bear a structural resemblance to known inhibitors of calmodulin, we studied whether rhodamine-123 or a bis-4-aminoquinaldinium could antagonize the action of calmodulin. Rhodamine-123 (IC50 = 58 microM) and dequalinium (IC50 = 1 microM) inhibited the activity of a calmodulin-stimulated cyclic nucleotide phosphodiesterase. Propylinium, a compound similar to dequalinium except for having a 3 rather than 10 carbon alkyl bridge connecting two non-substituted quinoline rings, had no inhibitory effect. Kinetic analysis showed that dequalinium competitively inhibited calmodulin's activation of phosphodiesterase. We also studied the antiproliferative effects of the compounds on the C6 astrocytoma cell line. Rhodamine-123 and dequalinium inhibited the proliferation of this cell line while propylinium had no effect. These studies demonstrate that rhodamine-123 and dequalinium are calmodulin-antagonists and inhibit cellular proliferation. PMID- 3008728 TI - Formation of superoxide and hydroxyl radicals from 1-methyl-4-phenylpyridinium ion (MPP+): reductive activation by NADPH cytochrome P-450 reductase. AB - Formation of free radical intermediates from 1--methyl-4-phenylpyridinium ion(MPP+) has been studied using spin-trapping techniques. Incubation of MPP+ with purified NADPH cytochrome P-450 reductase and NADPH under anaerobic conditions failed to produce any detectable radical intermediates. However, in the presence of air and a spin-trap, a significant stimulation of superoxide and hydroxyl radicals was detected. Formation of these toxic radicals from MPP+ was inhibited by superoxide dismutase, catalase, and ethanol. Under identical conditions, however, considerably less of these radicals were formed with MPP+ in comparison to paraquat, a lung toxin containing two pyridinium moieties. PMID- 3008729 TI - Estrogen receptor is associated with protein and phospholipid kinase activities. AB - The ability of immunopurified estrogen receptor from MCF-7 cells, incubated with [Y-32P]-ATP, to conduct its own phosphorylation and to phosphorylate phosphatidylinositol and 1,2-diacylglycerol was investigated. SDS gel electrophoresis analysis of the phosphorylated products revealed three major labeled phosphopeptides with molecular weights of 57, 47 and 43 kilodaltons. Other polypeptides were detected although in much smaller amounts. When the phosphorylation system was supplemented in vitro with phosphatidylinositol, phosphatidylinositol-4-P, or 1,2-diacylglycerol and the 32P-lipids were analyzed by thin layer chromatography, PI, but not 1,2-diacylglycerol, was converted in part to 32P-phosphatidic acid and phosphatidylinositol-4-32P. Phosphatidylinositol-4-P was also phosphorylated to phosphatidylinositol-4,5-32P. An estrogen receptor-negative cell line (MDA) produced negligible 32P polypeptides and 32P-lipids under identical conditions used for MCF-7 cells. PMID- 3008730 TI - An investigation on reduction process of cucumber ascorbate oxidase. AB - Reduction process of cucumber ascorbate oxidase with L-ascorbate was investigated in detail through absorption and electron paramagnetic resonance (EPR) spectra under anaerobic condition. One of the three type I coppers (the type I copper which is oxidized rapidly (Sakurai, T. et al. (1985) Biochem. Biophys. Res. Commun. 131, 647-652)) and a pair of type III coppers only which contribute to the absorption at 330 nm were reduced faster than other two type I and the other pair of type III coppers, respectively. The principal active site of ascorbate oxidase was confirmed to be comprised of one type I, one type II and a pair of type III coppers. Type II copper seemed to be reduced after all type I and type III coppers have been reduced. PMID- 3008731 TI - Effect of polymyxin-B on T-lymphocyte protein synthesis. AB - The role of protein kinase-C (PK-C) protein phosphorylation on the mitogen triggered responses of T-lymphocytes was examined by observing the effect of polymyxin-B (an inhibitor of PK-C) on mitogen induced protein and DNA synthesis. Polymyxin-B inhibited 3H-thymidine incorporation by PHA activated T-lymphocytes over a range of PHA concentrations. 3H-leucine incorporation by PHA activated T lymphocytes was inhibited by polymyxin-B in a dose dependent manner. A partially purified PK-C fraction from polymyxin-B treated PHA activated T-lymphocytes demonstrated less than 25% of the phosphorylating activity of untreated lymphocytes. These results suggest that protein synthesis during the T-lymphocyte activation process is dependent on PK-C activity. PMID- 3008732 TI - Diet fat alters the structure and function of the nuclear envelope: modulation of membrane fatty acid composition, NTPase activity and binding of triiodothyronine. AB - Mice were fed a diet either high or low in P/S ratio to determine the effect of altering dietary lipid on the fatty acid composition of liver nuclear envelopes and thereby on functions of the nuclear envelope. Mice fed the high P/S diet exhibited higher levels of C18:2 omega 6 and unsaturates in liver nuclear envelopes, higher specific activity of NTPase and specific binding for L triiodothyronine at 15 degrees C and 22 degrees C compared with the low P/S diet fed group. These observations indicate that diet-induced differences in the fatty acid composition of nuclear envelope lipid affects functions of the nuclear envelope. PMID- 3008733 TI - Arachidonic acid inhibits 5-lipoxygenase in human T cells. AB - The data on whether T cells produce leukotrienes or other 5-lipoxygenase metabolites of arachidonic acid is conflicting. We report that exogenous arachidonic acid added to phytohemagglutin-stimulated human T cells profoundly inhibits leukotriene B4 production, with 90% inhibition caused by 10(-6) M arachidonic acid. The 12- and 15-lipoxygenase pathways were also inhibited by arachidonic acid. Recent reports that human T cells produce no 5-lipoxygenase metabolites of arachidonic acid might be explained by the fact that the studies used greater than or equal to 10(-5)M arachidonic acid in the incubation media. PMID- 3008734 TI - Isolation of fumarate reductase from Desulfovibrio multispirans, a sulfate reducing bacterium. AB - Fumarate reductase was isolated and purified 100-fold to homogeneity from Desulfovibrio multispirans, a new species of sulfate-reducing bacteria. The enzyme contained 1 mol of non-covalently bound FAD and four subunits with Mr 45,000, 32,000, 30,000 and 27,000. EPR spectroscopy showed the existence of two iron-sulfur clusters. The absorption spectrum showed a broad region of high absorbance from 450 nm to 300 nm with a protein peak at 278 nm. The ratio of A278:A400 was 2.60. The specific activity was 110 mumoles H2/mg of protein. The Km for fumarate was 2.5 mM. The activation energy was 8.7 kcal/mol. Electron transport from H2 to fumarate in intact cells was inhibited by 2-heptyl-4-hydroxy quinoline-N-oxide, a quinone inhibitor, indicating the participation of quinone (probably menaquinone) in fumarate reduction. PMID- 3008735 TI - Solubilization and further chromatographic purification of highly purified, membrane-bound Na,K-ATPase. AB - Highly purified membrane-bound Na,K-ATPase from pig kidney outer medulla was dissolved in the non-ionic detergent C12E8. Chromatography of the dissolved material on a DEAE matrix yielded enzymatical material having a ouabain-binding capacity of 6.9 nmoles per mg protein (measured according to Lowry et al., with bovine serum albumin as standard). This material, which after addition of lipids had the same K+-phosphatase turnover as the membrane-bound enzyme, could consist entirely of live molecules with a molecular weight of 145 kDa, a value close to that expected for alpha beta-promoters of Na,K-ATPase. PMID- 3008736 TI - Reduced rhodopsin phosphorylation during retinal dystrophy. AB - During inherited retinal dystrophy in Irish Setter dogs, decreased activity of cGMP phosphodiesterase (PDE) results in high cGMP levels and retinal degeneration (1-3). This defect could be in PDE itself, or in its interactions with other proteins of the rod outer segment. We report herein that when retinas from 8-week old dogs were phosphorylated with gamma-32P-ATP, and separated on SDS-PAGE, phosphorylation of rd dog rhodopsin was reduced. When rd retinas were mixed with normal dog retinas, phosphorylation of the latter was inhibited. Since rd mediated inhibition was prevented by 1 mM NaF, the results suggest that the cause of reduced rd phosphorylation is increased phosphatase activity. Together, these results demonstrate that decreased phosphorylation of rhodopsin due to increased phosphatase activity is a fundamental biochemical change which may partially account for the degenerative process and loss of visual acuity during inherited retinal dystrophy. PMID- 3008737 TI - Primaquine can mediate hydroxyl radical generation by Trypanosoma cruzi extracts. AB - Primaquine increases the NAD(P)H dependent oxygen consumption and hydroxyl radical generation by extracts of Trypanosoma cruzi, the causative agent of Chagas' disease. Spin-trapping studies show that hydroxyl radical yield is completely inhibited by catalase and slightly increased by SOD, indicating that radical generation is dependent on the pair primaquine-NAD(P)H and their interaction product, H2O2. Primaquine effects upon Trypanosoma cruzi extracts are compared with those obtained with nifurtimox, a compound effective in the treatment of Chagas' disease. The observed similarity suggests that hydroxyl radical may be involved in the antiprotozoan activity of primaquine. PMID- 3008738 TI - Rat atrial natriuretic factor suppresses proopiomelanocortin-derived peptides secretion from both anterior and intermediate lobe cells and growth hormone release from anterior lobe cells of rat pituitary in vitro. AB - Synthetic rat atrial natriuretic factor (ANF) was found to attenuate, in a dose dependent manner, basal and corticotropin-releasing factor-induced secretion of proopiomelanocortin-derived peptides from cultured anterior and intermediate lobe cells of rat pituitary. ANF was also found to suppress basal and growth hormone releasing factor-stimulated secretion of growth hormone from anterior lobe cells of rat pituitary. These results, together with reports of the existence of ANF positive neurons in the hypothalamus and ANF-positive fibers in the median eminence, suggest that hypothalamic ANF is probably involved in the regulation of pituitary hormone secretion, especially that of proopiomelanocortin-derived peptides and growth hormone. PMID- 3008739 TI - In vitro transformation of androgen receptor from murine skeletal muscle by cAMP. AB - Sedimentation constants and DNA-cellulose-binding of cytosolic androgen receptor from murine skeletal muscle were determined in presence of cyclic nucleotides. Without cAMP, two testosterone-binding fractions of similar amount at 4-5S and 8 9S were obtained. With 3 microM cAMP the receptor sedimented predominantly at 4 5S. Binding of testosterone-receptor-complexes to DNA-cellulose was enhanced by increasing cAMP-concentrations and reached a maximum at 20-90 nM cAMP depending on the DNA-concentrations in the test. A similar DNA-binding characteristic was obtained after partial purification of the receptor by affinity chromatography. cGMP had no effect. We conclude that the androgen receptor is transformed in vitro by cAMP. PMID- 3008740 TI - Prolactin receptor regulation by LH in the rat mammary gland. AB - The aim of this study was to investigate hormonal factors responsible for the huge increase in PRL receptors on the day of estrus in the rat mammary gland. For this purpose, ovariectomized rats were primed with E2 so as to reach a physiological serum concentration of E2 (21.5 +/- 1.2 pg/ml) and high PRL serum values (72.8 +/- 21.9 ng/ml). In these conditions, PRL specific binding and capacity were respectively 22.8 +/- 8.3%/mg protein and 96 +/- 29 fm/mg protein. An injection of either LHRH (500 ng/rat) or LH (60 micrograms LH-RP1/rat) was capable of increasing significantly both PRL specific binding and capacity. Capacity reached the values of 498 +/- 103 and 507 +/- 240 fm/mg protein for LHRH and LH respectively. LHRH action appeared to be mainly mediated through LH secretion, since no difference was found between LHRH and LH. LHRH and LH injections alone were unable to modify PRL binding, suggesting that they only potentiate E2 and PRL action. These results show for the first time that LH is involved in the regulation of PRL receptors in the rat mammary gland. PMID- 3008741 TI - Inhibitory action of cyclic GMP on secretion, polyphosphoinositide hydrolysis and calcium mobilization in thrombin-stimulated human platelets. AB - The effect of cyclic GMP (cGMP) on human platelet activation was investigated, using its metabolically stable analogue, 8-bromo cGMP (8-bcGMP). Thrombin-induced serotonin secretion was inhibited by pretreatment with 8bcGMP in a dose-dependent manner. Production of inositol trisphosphate (IP3), a Ca2+ releaser was inhibited by 8bcGMP pretreatment of platelets. Preincubation of platelets with 8bcGMP was without effect on the basal level of cytosolic free Ca2+, measured by fluorescent indicator quin2, but suppressed its thrombin-induced enhancement independently of extracellular Ca2+. These results indicate that cGMP may be implicated in phospholipase C activation and Ca2+ mobilization (both influx through the plasma membrane and efflux from internal stores) in thrombin-activated human platelets. PMID- 3008744 TI - The protein kinase C inhibitors H-7 and H-9 fail to inhibit human neutrophil activation. AB - The protein kinase C inhibitors 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) were examined for their ability to inhibit human neutrophil activation. At concentrations up to 100 micromolar, these compounds failed to inhibit either respiratory burst or the secretory response of neutrophils stimulated with particulate (serum-opsonized zymosan) or soluble (A23187, FMLP, PMA) stimuli. In contrast, the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) inhibited both oxygen radical generation and lysosomal enzyme release in response to the same stimuli. These results suggest that calmodulin-dependent enzymes, rather than protein kinase C, may be essential for neutrophil activation. PMID- 3008743 TI - Dual activation of the inositol-triphosphate-calcium and cyclic nucleotide intracellular signaling systems by adrenocorticotropin in rat adrenal cells. AB - Hormones which primarily utilize cAMP as their intracellular "second messenger" are generally not thought to activate the IP3-Ca++ signalling system. Presently, we show that ACTH, at certain concentrations, can activate both the cAMP and IP3 Ca++ intracellular signalling systems. PMID- 3008742 TI - Purified tyrosine kinases, the EGF receptor kinase and the src kinase, can catalyze the phosphorylation of the band 3 protein from human erythrocytes. AB - The band 3 glycoprotein from human erythrocytes was found to be phosphorylated on tyrosine residues by the purified EGF receptor kinase and the purified src kinase in vitro. Kinetic analysis revealed that Km of the band 3 protein phosphorylation by the EGF receptor kinase was 0.17 microM and 0.65 microM in the absence and presence of EGF (3 X 10(-7)M), respectively, and that in the case of the src kinase it was 0.4 microM. From these data the band 3 protein can be regarded as one of the best substrates common for the EGF receptor kinase and the src kinase in vitro. PMID- 3008745 TI - Inhibition of [3H]captopril binding by peptide analog angiotensin converting enzyme inhibitors. AB - Ketomethylene containing peptide analogs, modeled after a snake venom pentapeptide, have been shown to be potent angiotensin converting enzyme inhibitors. Although the most potent compounds are up to five times more potent than captopril in inhibiting angiotensin converting enzyme activity, they are relatively weak inhibitors of [3H]captopril binding to membrane bound angiotensin converting enzyme. This indicates that inhibition of [3H]captopril binding and enzymatic activity is due to binding to distinct sites. These results suggest that the inhibitors bind to an additional site on the enzyme distinct from the captopril binding site. PMID- 3008746 TI - Complete dissociation between the activation of phosphoinositide turnover and of NADPH oxidase by formyl-methionyl-leucyl-phenylalanine in human neutrophils depleted of Ca2+ and primed by subthreshold doses of phorbol 12,myristate 13,acetate. AB - Evidences have been provided by many laboratories that the activation of the NADPH oxidase in neutrophils by formyl-methionyl-leucyl-phenylalanine (FMLP) is strictly linked to a transduction pathway that involves the stimulation, via GTP binding protein, of the phosphoinositide turnover and the increase in [Ca2+]i. The results presented in this paper demonstrate that FMLP can activate the NADPH oxidase by triggering a transduction pathway completely independent of phosphoinositide turnover and Ca2+ changes. In fact: i) Ca2+-depleted neutrophils do not respond to FMLP with the activation of phosphoinositide hydrolysis and NADPH oxidase. Both the responses are restored by the addition of exogenous Ca2+. ii) In Ca2+-depleted neutrophils phorbol-myristate-acetate (PMA) activates the NADPH oxidase. iii) The pretreatment of Ca2+-depleted neutrophils with non stimulatory doses of PMA restores the activation of the NADPH oxidase but not of the turnover of phosphoinositides by FMLP. This priming effect of PMA and the role of this phosphoinositide and Ca2+-independent pathway for the stimulation of the NADPH oxidase by receptors mediated stimuli are discussed. PMID- 3008747 TI - Pertussis toxin does not prevent alpha adrenergic stimulated breakdown of phosphoinositides or respiration in brown adipocytes. AB - The role of Ni in mediation of alpha adrenergic stimulated respiration and breakdown of phosphatidylinositol 4,5-P2 in brown adipocytes was examined using pertussis toxin. Phenylephrine stimulation of respiration and breakdown of PtdIns 4,5-P2 was still present in adipocytes harvested from hamsters treated with pertussis toxin although toxin modification of Ni appeared complete as judged from the absence of incorporation of [32P] from [32P]-NAD into a 41 KD protein in membranes. These data suggest that alpha-1 receptors on brown adipocytes are not coupled to inositide hydrolysis through Ni. PMID- 3008748 TI - Phosphofructokinase and fructosebisphosphatase from muscle can interact at physiological concentrations with mutual effects on their kinetic behavior. AB - Phosphofructokinase (PFK) and fructosebisphosphatase (FBPase) from muscle studied at physiological concentrations have been found to influence the kinetic behavior of each other. Under these conditions PFK can be activated up to ca. 4-fold by FBPase, while the latter can be inhibited up to ca. 3-fold by the former. Diluted enzymes did not interact with each other; nevertheless, they did so in the presence of polyethylene glycol. Equimolar amounts of either glucosephosphate isomerase or aldolase had no effect on concentrated PFK. The kinetic interactions between PFK and FBPase should be taken into account for fuller understanding of their regulatory behavior in vivo. PMID- 3008749 TI - Methylation analysis of a plasmid containing a mammalian cell cycle regulatory sequence after transient transfection into the host cell. AB - A hamster genomic sequence containing a cell cycle regulatory sequence derived from a histone H3.2 gene was transfected into K12 hamster fibroblasts in the form of plasmid DNA prepared from dam+ E. coli. Analysis of the plasmid DNA recovered 72 hr after the transfection revealed that it was resistant to Mbol but was sensitive to Dpn 1 enzyme digestion, suggesting that this plasmid had retained its dam E. coli methylated sites and was therefore unable to undergo replication following transfection into homologous host cells. These results were discussed with relation to a previously described yeast cell cycle regulatory sequence which was functionally linked to an autonomous replicating sequence and was located near a yeast H2B gene. PMID- 3008750 TI - A possible model of hemoprotein-peroxide complexes formed in an iron tetraphenylporphyrin system. AB - A ferric low-spin species with an anomalously small g3-g1 separation generated by the reaction of Fe(III)tetraphenylporphyrin with t-butylhydroperoxide in the presence of tatramethylammonium hydroxide was characterized by ESR spectroscopy. The reaction kinetics, investigated by monitoring ESR intensity, indicated that this low-spin complex is highly reactive and easily changed to non-heme type iron complexes. The rhombic parameters of this complex are very similar to those of heme-peroxide adducts such as [Fe(II)-hemoglobin-O2]- and [Fe(II)-horseradish peroxidase-O2]-. PMID- 3008751 TI - Diazepam inhibits potassium-induced aldosterone secretion in adrenal glomerulosa cell. AB - In an attempt to elucidate a possible role of peripheral benzodiazepine receptor in adrenal glomerulosa cell, effect of diazepam on potassium-induced aldosterone secretion was studied using isolated bovine adrenal glomerulosa cell. Diazepam inhibited aldosterone secretion stimulated by 8mM potassium in a dose dependent manner. The ID50 was approximately 14 nM. Although diazepam inhibited potassium action effectively, forskolin-induced aldosterone secretion was not affected by diazepam. These results indicate that peripheral benzodiazepine receptor may have an active role in regulating aldosterone secretion. The voltage dependent calcium channel may be a possible site of benzodiazepine action in this tissue. PMID- 3008752 TI - Changes of fluorescence spectra of thymine in aqueous solution by radiation. AB - Aqueous solution of thymine (5 X 10(-4) M, buffered at pH 7.0) was irradiated with 60Co gamma-rays under four different atmospheric conditions. In the presence of t-BuOH-N2, there was little increase in the fluorescence intensity as was previously reported in the radiolysis of cytosine. Under O2 there was also no significant increase differing from the case of cytosine. The fluorescence intensity was found to increase appreciably under N2O but it was less under N2 indicating that OH radical is mainly responsible for the formation of the highly fluorescent products. However, the fluorescence yields under these conditions were much lower in thymine radiolysis than cytosine radiolysis. PMID- 3008753 TI - Delayed alteration of membrane fluidity in intact cultured B-16 melanoma cells affected by ultraviolet irradiation. AB - Lipid peroxidation in the plasma membrane has been reported to decrease membrane fluidity. We examined membrane fluidity in relation to lipid peroxidation processes after UV-B exposure of cultured B-16 melanoma cells. UV exposure promptly increased TBA-positive material(s), but alteration of membrane fluidity was delayed. Plasma membrane fluidity increased significantly 6 hours after exposure when the TBA-value(s) had become under the control level. To examine the direct effect of lipid peroxides on the fluidity, tert-butyl hydroperoxide was added to B-16 melanoma cells. Similar results were obtained with respect to membrane fluidity. These results suggest that lipid peroxidation at UV doses maintaining cell viability does not directly induce a significant alteration of membrane fluidity, but may influence the fluidity either during metabolizing processes of UV-induced lipid peroxides or during repair processes following oxidative cell membrane damage. PMID- 3008754 TI - Drug targeting to the lungs. PMID- 3008755 TI - Beta-adrenergic receptors on human suppressor, helper, and cytolytic lymphocytes. AB - Using the radioligand beta-adrenergic blocker [125I]cyanopindolol (ICYP), we have characterized the beta-adrenergic receptors on Leu 3+ (T helper [TH]), Leu 2+, 9.3- (T suppressor [Ts]) and Leu 2+, 9.3+ (T cytolytic [Tc]) subsets of human lymphocytes. Peripheral blood T cells were isolated by rosetting, and then subsets were purified by their affinities to monoclonal antibodies against their Leu 3 and 9.3 markers. ICYP binding to the subsets was saturable with time and with concentration; the binding was stereoselective and reversible by beta adrenergic antagonists. A biological response produced by beta agonists increased intracellular concentrations of cAMP and corresponded to the number of binding sites. Each subset of cells had a number of binding sites, which was characteristic for the given subset. The data indicate that the density of distribution of beta-adrenergic receptors was not homogeneous on the precursors of phenotypically and functionally distinct T cells (Ts approximately 2900, Tc approximately 1800 and TH approximately 750 binding sites). The displacement studies using beta-adrenergic agonists were performed on the cytolytic and suppressor T cell subsets, suggesting that the receptors were mainly of the beta 2 type. The immunobiological significance of such selective distribution of numbers and subtypes of beta-adrenergic receptors on distinct T cell subsets is under investigation. PMID- 3008756 TI - Studies on the effect of reserpine on the peroxidase activity of submaxillary gland. AB - In an earlier report we have described how the administration of reserpine (0.5 mg/kg) stimulates the peroxidase activity of rat and mouse submaxillary gland [R. Chakraborty, B. Mukherjee and R. N. Hati, Life Sci. 35, 1913 (1984)]. Further studies have been carried out in this area. This stimulation of the enzyme activity started from 0.02 mg reserpine/kg body wt. The effect of reserpine was found in all subcellular fractions of submaxillary gland. Iodinating activity of the enzyme preparation was also increased by reserpine treatment. Kinetic studies indicate that reserpine increased the Vmax of the enzyme without altering Km. Protein synthesis inhibitors inhibited the increase of the enzyme activity by reserpine. Dibutyryl c-AMP, theophylline or both drugs together did not alter the peroxidase activity. The effect of reserpine was also found in experiments using slices and single cell preparations of submaxillary gland, suggesting that the drug is probably acting directly on the acinar cells. PMID- 3008757 TI - Enhanced formation of ethane and n-pentane by rat hepatocytes in the presence of dimethyl sulfoxide. AB - Incubation of rat hepatocytes with 14 mM dimethyl sulfoxide (DMSO) produced an increase in the formation of ethane, measured by capillary column gas chromatography, to 18.0 pmoles/hr/10(7) cells from 11.2 pmoles/hr/1-(7) cells from 5.6 pmoles/hr/10(7) cells in control hepatocytes. This was about one-third the stimulation of ethane and n-pentane formation produced by incubation of hepatocytes with 13 mM carbon tetrachloride. DMSO-stimulated ethane and n-pentane formation was inhibited up to 63% by 0.1 microM alpha-tocopherol and up to 89% by N2. Formation of dimethylsulfide from DMSO by hepatocytes was the same in air and N2. DMSO increased methane production by hepatocytes to 31.3 pmoles/hr/10(7) cells from 6.9 pmoles/hr/10(7) cells in control hepatocytes. Although DMSO apparently stimulated lipid peroxidation by hepatocytes, as measured by ethane and n-pentane formation, there was no increase in the formation of thiobarbituric acid reactive material. DMSO was not toxic to hepatocytes, measured by release of cytosolic lactate dehydrogenase, over a 2-hr incubation. Possible mechanisms for the increase in alkane formation by DMSO are discussed. PMID- 3008759 TI - Termini generated at the site of the DNA breakage mediated by photoexcited promazines. AB - Promazine derivatives are known to be able to photoinduce, in vitro, direct single-strand breaks into DNA (Decuyper et al., Biochem. Pharmac. 33, 4025-4031 (1984]. Using [32P]end labeled DNA fragments, it is demonstrated that this DNA breakage occurs almost regardless of the nucleotide sequence of the DNA. Using 3' [32P]end or 5'-[32P]end labeled oligonucleotide and enzymatic digestion of the fragments generated, it is demonstrated that the termini generated at the site of the breakage are 5'-phosphate, 3'-phosphate and 3'-termini which are presumed to be 3'-phosphoglycolate. This is consistent with an attack of the sugar moeity of the sugar-phosphate backbone of the DNA by the reactives species generated upon near-u.v. irradiation of promazine derivatives. PMID- 3008758 TI - Redox activities of antitumor anthracyclines determined by microsomal oxygen consumption and assays for superoxide anion and hydroxyl radical generation. AB - To explore the structural characteristics of various derivatives of the anticancer drugs, doxorubicin and daunorubicin, for exhibiting redox activities believed to be associated with toxic radical production, we tested over fifty derivatives in a rapid screening procedure for augmenting oxygen consumption by rat liver microsomes. Measurement of parent drug disappearance and of metabolite appearance for fourteen anthracyclines with a broad range of activities for augmenting oxygen consumption indicated that a single reaction, conversion to the 7-deoxyaglycone, occurred. Multiple tests of selected compounds showed that the liver microsome system exhibited saturation kinetics, and calculated values of Vmax/Km gave the same relative order of activities as did the screening test. The liver microsome system was not found to be stereoselective. Measurements of the abilities of a number of the anthracycline derivatives after chemical activation by reduction with sodium borohydride to convert oxygen to superoxide anion, or to the hydroxyl radical, were also made. The reactivities of the anthracyclines in these latter two assays were positively related to the activities obtained in the rat liver microsome screening test, suggesting that all three tests were measuring various steps in the sequence from anthracycline semiquinone radical formation through oxygen activation and radical formation. Superoxide anion generation from chemically reduced anthracyclines was inhibited by the addition of calf thymus DNA, and the extent of inhibition was positively correlated with the measured DNA association constants of the anthracyclines. However, the DNA association constants were unrelated to superoxide anion generation in the absence of DNA or to the augmentation of oxygen consumption in liver microsomes. Half-wave potentials were negatively correlated with both the results of the microsomal oxygen consumption test and the production of superoxide anion in the chemical test system. No relationships were discerned among the DNA association constants, half-wave potentials, or reoxidizabilities of the anthracyclines tested. Comparisons of the relatively low activities of certain of the anthracyclines in the biochemical and chemical tests for oxygen activation with their known high activities against murine tumors in vivo, but low cardiotoxicities in animal model systems, suggest that the separation of the cytotoxic antitumor and cardiotoxic actions of these derivatives may have been achieved. PMID- 3008760 TI - Relationship between 5-fluoro-2'-deoxyuridylate, 2'-deoxyuridylate, and thymidylate synthase activity subsequent to 5-fluorouracil administration, in xenografts of human colon adenocarcinomas. AB - 5-Fluorouracil (FUra) has been administered to mice bearing xenografts of human colon adenocarcinomas. In two tumor lines, HxGC3 and HxVRC5, intrinsically resistant to FUra, 2'-deoxyuridylate (dUMP) accumulated 13.4- and 23.9-fold above basal levels. In HxELC2 xenografts, which demonstrated some sensitivity to FUra, there was a decrease in dUMP concentration after drug administration. Maximal intratumor levels of 5-fluoro-2'-deoxyuridylate (FdUMP) were found at 1 hr, but decreased in all tumor lines by 4 hr after administration of FUra. Data derived in tumor cytosols suggested that FdUMP levels in situ were not rate-limiting for formation of covalent ternary complex, but that accumulation of dUMP would retard the rate of complex formation. Subsequent to administration of FUra, thymidylate synthase activity was reduced greater than 75% in all tumors, but it recovered rapidly in tumors resistant to FUra. In addition, the pretreatment level of activity of thymidylate synthase was 12.7-fold greater in HxVRC5 tumors than in HxELC2 tumors. This elevated activity in HxVRC5 tumors appears not to be a consequence of gene amplification. Formation of FdUMP or the accumulation of dUMP did not correlate with the activity of phosphatases measured at pH 5.8 or pH 9.2 in each tumor line. Further, inhibition of phosphatase activity did not alter, significantly, the net rate of dissociation of the FdUMP-thymidylate synthase [6R]-CH2-H4PteGlu complex. PMID- 3008762 TI - Selective lysosomal uptake and accumulation of the beta-adrenergic antagonist propranolol in cultured and isolated cell systems. AB - The beta adrenoreceptor antagonist propranolol is rapidly taken up and accumulated in various cultured cell lines. When incubated in the presence of low concentrations of propranolol (10(-9) M), Hela (non-differentiated epithelia), BC3H1 (smooth muscle) and MDCK (differentiated kidney epithelia) cell cultures take up (t1/2 = 4-10 min) and accumulate the drug such that the intracellular concentration is over 1000 times that in the incubation medium. The release of propranolol from the cells was slower (t1/2 = 22 min) than the rate of uptake but the dissociation was stimulated by the addition of 1 microM propranolol to the external medium (t1/2 = 9 min). Uptake, which is non-stereoselective, is dependent on pH and is inhibited by the lysosomotropic agents, NH4Cl, methylamine and chloroquine. At higher concentrations (greater than 10(6) M), uptake is accompanied by a visual swelling of intracellular acidic vesicles staining with acridine orange. These results suggest that propranolol, a basic amphiphilic amine, is accumulated within the lysosomes of these cells. Uptake was confined to these cultured cell systems with no chloroquine-sensitive propranolol uptake, being found in isolated rabbit ventricular myocytes, red blood cells or blood platelets. Although alprenolol and cyanopindolol competed with propranolol for uptake, isoprenaline, adrenaline, noradrenaline, phenylephrine, atenolol, practolol and salbutamol were not effective inhibitors. The possible consequences of this uptake and accumulation of propranolol by certain tissues is discussed in relation to the known actions of the drug, particularly during or after abrupt withdrawal from chronic applications. PMID- 3008761 TI - Effect of disulfide-bond reducing agents on the specific binding of growth hormone to microsomal membrane preparations from rabbit liver. AB - The effect of the disulfide-bond reducing agents, mercaptoethanol (ME) and dithiothreitol (DTT), on the specific binding of 125I-labeled human growth hormone (hGH) to microsomal membrane preparations from rabbit liver was investigated. The presence of ME or DTT caused a time- and dose-related inhibition of [125I]hGH binding to both particulate and solubilized somatotrophic receptors of rabbit liver membranes. Maximum inhibition was 20-30%. Disulfide bond reduction also caused a marked increase in the extent of reversibility of [125I]hGH binding. These effects were not due to effects on the GH, itself, but appeared to be directed at the receptor. Scatchard analysis showed that ME and DTT caused a change in the nature of the binding interaction, with at least partial conversion of receptors into sites with reduced affinity. These data together suggest that the partial effect of reducing agents on somatotrophic receptors of rabbit liver may reflect two types of receptors within the microsomal membrane preparations--one (approximately 30% of total receptors) involving disulfide bonding and the remaining type (70%) being independent of disulfide bonds--or, alternatively, that the state of reduction affects a single class of receptors in a rather more complex manner. These studies provide further intriguing insights into the overall mechanism(s) involved in the interaction of GH with its target cell receptors. PMID- 3008763 TI - [Primary structure of the 3'-terminal region of the cloned DNA of the bovine leukemia virus]. AB - The nucleotide sequence of the 3'-terminal region of the cloned bovine leukaemia virus cDNA (1474 bp) was elucidated using both Sanger and Maxam-Gilbert techniques. This DNA region contains U3 and R parts of the BLV LTR and an upstream sequence with four open reading frames (ORF) of unknown function. The comparison of the nucleotide substitutions in these ORF with the two variants of proviral BLV DNA suggests that the only pX1 ORF possesses a coding function. The role of the pX1 protein is discussed. PMID- 3008764 TI - Effect of warfarin sodium therapy on excretion of 4-carboxy-L-glutamic acid in scleroderma, dermatomyositis, and myositis ossificans progressiva. AB - The effect of warfarin sodium on excretion of calcium, phosphorus, and 4-carboxy L-glutamic acid (Gla) was studied in 5 patients with ectopic calcification (2 with scleroderma, 1 with dermatomyositis, and 2 with myositis ossificans progressiva). Warfarin reduced urinary excretion of Gla in all patients, but no changes in calcium and phosphorus excretion or in objective parameters of calcinosis were observed during 6-36 months of treatment. Two patients experienced hemorrhagic complications during therapy, emphasizing a hazard of long-term anticoagulation treatment. Since ectopic calcium deposits contain Gla rich protein, suppression of Gla synthesis by warfarin sodium over a longer period may prevent deposition and allow removal of existing calcinosis deposits. PMID- 3008765 TI - Oxygen radicals as effectors of cartilage destruction. Direct degradative effect on matrix components and indirect action via activation of latent collagenase from polymorphonuclear leukocytes. AB - Degradation of intact cartilaginous tissue (bovine nasal cartilage) by oxygen derived free radicals (ODFR) generated enzymatically by xanthine oxidase and hypoxanthine was studied. The degree of tissue destruction was determined by measuring the indentation under a defined compression force as well as by the loss of uronic acid- and hydroxyproline-containing matrix components. Cartilage slices altered by prior elastase treatment were more susceptible to oxygen radical attack than were intact tissue specimens. Degradation of cartilage matrix by ODFR was strongly inhibited by superoxide dismutase or catalase. Coincubation of latent collagenase from polymorphonuclear leukocytes with the ODFR-generating system led to activation of collagenolytic activity, resulting in marked degradation of the bovine cartilage slices. In further studies, activated polymorphonuclear leukocyte-collagenase was shown to degrade intact human articular cartilage to a degree of mechanical insufficiency. Thus, our assay system serves as an in vitro model of tissue damage, which may be relevant to pathophysiologic states such as rheumatoid arthritis. PMID- 3008766 TI - Scalloped pupils in familial amyloid polyneuropathy. PMID- 3008767 TI - Pressure erosions: rheumatoid arthritis or calcium pyrophosphate dihydrate crystal deposition disease? PMID- 3008768 TI - Selective inhibition of replication of the AIDS-associated virus HTLV-III/LAV by synthetic D-penicillamine. AB - A new antimetabolite approach to block the replication of the AIDS (acquired immunodeficiency syndrome)-associated virus is described. In this communication the effect of a synthetic D-penicillamine (Trolovol) and L-penicillamine on the replication of HTLV-III/LAV was studied. The replication of HTLV-III/LAV in H9 cells was determined by measuring the expression of HTLV-III proteins p15 and p24 using monoclonal antibodies in an immunofluorescence assay system. A concentration dependent inhibition of HTLV-III replication by D- and L penicillamine was observed. At lower concentrations L-penicillamine was more effective than D-penicillamine. However, at concentrations above 20 micrograms/ml the inhibitory response of both isomers is similar. To obtain a total inhibition a drug concentration of 40 micrograms/ml was needed for both isomers. Cytotoxicity of the compounds in uninfected H9 cells was observed only at high concentrations; e.g., at 500 micrograms/ml a 32% inhibition was found for D penicillamine and a 24% inhibition for L-penicillamine. In the presence of D- or L-penicillamine there was a significant increase in cell density of infected H9 cell cultures, indicating that both the compounds have a protective effect on H9 cells. In view of the low cytotoxicity, a selective antiviral activity against HTLV-III/LAV, and a T-cell protective effect of D-penicillamine, the evaluation of D-penicillamine for clinical treatment of AIDS is implicated. PMID- 3008770 TI - [Cloning of Coxsackie virus B 3]. PMID- 3008769 TI - Effect of suloctidil on cerebral circulation patterns of conscious rabbits. AB - Suloctidil (SUL) produces calcium antagonistic and antispasmodic effects on peripheral and pial arteries. The present studies were performed with the aim of evaluating the action of SUL on cerebral blood flow (CBF), which was taken as an index for evaluating the cerebral circulation. The drug was administered by rapid intravenous injection to groups of unanaesthetized rabbits at doses of 100-200 micrograms/kg and by intravenous infusion at doses of 10-20 micrograms/kg/min. In other experiments, SUL was chronically administered p.o. to normal rabbits and to rabbits receiving Kritchevsky's atherogenic diet; the daily dose of the drug was about 16 mg/kg. Cerebral blood flow and its compartmental distribution were determined in unanaesthetized animals by the intracarotid 133Xe clearance method. The data demonstrate that the atherogenic diet brings about a significant impairment of CBF; SUL is inactive in normal rabbits, while in the atherosclerotic rabbits it induces a pronounced increase in cerebral blood flow in the grey matter and an enhancement of the corresponding circulatory compartment. These changes are less evident in the white matter. PMID- 3008772 TI - Hypertension and atherosclerosis in cholesterol-fed rabbits. Part 1. Mild, two kidney, one-clip Goldblatt hypertension treated with enalapril. AB - Ten groups of New Zealand white rabbits were used to study the effects of mild, chronic two-kidney, one-clip hypertension (HT) and long-term antihypertensive therapy on atherogenesis. Five groups were fed a normal diet (ND) over the 8 month study period; 2 groups, one of which was given enalapril, remained normotensive (NT) throughout the study. Of the 3 HT groups, one was hypertensive for 7 months; the blood pressures of the other groups were normalized after 2 months with enalapril, or by removal of the clipped kidney. The other 5 groups were similar except that they were fed at 0.1% cholesterol diet (CD). The results showed that: neither mild chronic HT nor abrupt, short-term HT exacerbated atherogenesis in the CD-animals; although fibromuscular vascular lesions were present in the aorta of normal-diet, HT animals no atheroma was observed; enalapril therapy had no effect on atherogenesis; enalapril therapy reduced the total weight and the cholesterol and triglyceride content of the aorta of the ND groups regardless of blood pressure history; the aortic triglyceride content, but not the cholesterol content, of the CD group, was reduced by enalapril; and although heart size was unaffected by either diet or blood pressure levels, the mitochondria volume per unit volume of the left ventricle was reduced in both NT ND and HT-CD groups treated with enalapril. PMID- 3008771 TI - Pharmacological profile of tazasubrate, a new cholesterol-lowering agent. AB - The properties of tazasubrate (2-phenyl-2-[(6-ethoxy-2 benzothiazolyl)thio]propionic acid), a potent hypocholesterolemic agent, were studied in rats. Tazasubrate was found to be a reliable and highly effective hypocholesterolemic agent. There was a marked and reproducible reduction of serum cholesterol in various rat models differing in age, sex and diet, an improvement of the pathological lipoprotein pattern, slight but variable effects on serum triglycerides and phospholipids, no accumulation of intermediates of cholesterol biosynthesis, no inhibition of phospholipid metabolism (i.e. no induction of phospholipidosis), no interaction with the thyroid gland, and in contrast to fibrates, only minimal induction of peroxisomes in hepatocytes. PMID- 3008774 TI - [Sarcoma phyllodes of the breast. Apropos of a case withan ultrastructural and histoenzymological study]. AB - One case of cystosarcoma phyllodes is reported. Histologically, the neoplasm was composed of two elements: benign epithelial cells and malignant stromal cells. The stromal cells, spindle shaped, grow in solid sheets or in sparcely cellular, myxomatous and alcianophilic areas. Scanning electron microscopy demonstrates a mucous secretion, showing porelike openings and mucous droplets on the surface of tumoral cells.By transmission electron microscopy, sarcomatous cells look like very polymorphous. Secretory cells (showing dilated granular endoplasmic reticulum and abundant lysosomes) are mixed with primitive mesenchymal cells and with intermediate, myoepithelial cells. The myoid origin of these cells is demonstrated by the bundless of myofilaments terminated in marginal plaques on the plasma membrane, the numerous pinocytic vesicles and basal lamina investing irregularly the cells. Histoenzymology corroborates these findings, disclosing a high activity of alkaline phosphatase. Discovery of ambiguous myoepithelial cells associated with undifferentiated cells, likewise reported in epitheliomas, is of a great histogenetic interest. It suggests development of mammary neoplasm from stem cells coming under local of general influences, unknown today, but perhaps responsible of epithelial or conjunctive differentiation. The presence of a contralateral carcinoma in our case is consistent with this hypothesis. PMID- 3008775 TI - [Silicone inclusions in the spleen in a patient under chronic hemodialysis]. AB - A 67-year-old woman with chronic renal failure, in periodic hemodialysis, presented an hemolytic anemia in relation to splenic sequestration. Light Microscopic examination of the spleen revealed multiple granulomatous reaction. Numerous macrophages contained inclusions which were non-birefringent and non staining with routine stains. Electron microscopy, scanning electron microscopy and energy dispersive X-Ray analysis were performed and revealed silicone. Its origin was silicone tubing of the hemodialysis equipment. Pathogenic effects of silicone are discussed. PMID- 3008773 TI - [Hepatocellular carcinoma in a non-cirrhotic liver. Anatomo-clinical study of 22 cases of hepatectomy. Histo-prognostic factors]. AB - The present clinicopathological study concerns 22 cases of hepatocellular carcinoma in non cirrhotic liver; they all underwent hepatectomy performed in the same surgical unit, and this group is clinically homogeneous. This cancer shows higher frequency in males (17/22), and in ages between 40 and 72. No etiologic relevance is shown. An abdominal mass is detected as first clinical symptom. Macroscopically: the tumor most often appears as a chief nodule 2-4 cm in diameter surrounded by smaller ones, always in close proximity. Histologically: the tumors are usually well or rather well differentiated hepatocarcinomas; 4 of them are fibrolamellar. Because of these macroscopical features and because of the absence of metastasis at the time of laparotomy even in case of recurrence, partial hepatectomy seems justified. A rich reticulin framework and its even distribution seem to be the best morphological signs indicating good prognosis. Long lasting survival, exceeding 11 years for one patient, concerns chiefly fibrolamellar tumors. PMID- 3008776 TI - [Small-cell bronchial cancers. Apropos of their subtyping]. AB - Histopathological diagnosis of small cell carcinoma may be rather uneasy, due to merely technical reasons. We believe that a careful preparation of the sections' thickness and smearing would reduce the percentage of non routinely classifiable tumors. PMID- 3008777 TI - Massive degloving injury of the trunk. AB - A case of massive degloving injury of the perineum, thigh, and buttocks is presented. Hemostasis was achieved with a pneumatic anti-shock garment (PASG), followed by direct suturing of bleeding areas. A colostomy was performed. Initial conservative debridement was followed in ten days by multiple skin grafts. The patient was treated with sodium bicarbonate and mannitol to preclude myoglobinuric renal failure. Intravenous hyperalimentation was also utilized. PMID- 3008780 TI - The hippocampal memory indexing theory. AB - The hippocampal formation (comprising the hippocampus proper, the dentate gyrus, and the subiculum) has been repeatedly implicated in information storage models of the mammalian brain. The precise nature of the hippocampal role in the storage of information has, however, remained elusive. Here it is proposed that the role of the hippocampus is to form and retain an index of neocortical areas activated by experiential events. The hippocampal index, thus, represents those unique cortical regions activated by specific events. The neuronal mechanism underlying the memory index is hypothesized to be long-term potentiation. It is asserted that the reactivation of the stored hippocampal memory index will serve to also reactivate the associated unique array of neocortical areas and thus will result in a memorial experience. This hippocampal reactivation of a neocortical array may also be involved in establishing a cortically based memory trace. PMID- 3008779 TI - Stimulation of the ventral tegmental area (A10 region) enhances the hypothalamic attack behavior in the cat. AB - The influence of the A10 region of the ventral tegmental area (VTA) on the quiet biting attack evoked by stimulation of the lateral hypothalamic nucleus (LH) was studied. The latency of the biting was considered as reference value and measured with stop-watches; it remained constant when hypothalamic stimulation was performed with the same parameters. Simultaneous activation of the A10 neuron group induced a facilitation of the aggression in the form of a decrease in the biting latency or a display of the attack pattern when LH was stimulated with parameters below the threshold for biting appearance. The facilitatory effect of the A10 neurons of the VTA is discussed. PMID- 3008778 TI - Gamma aminobutyric acid mediates ventromedial hypothalamic mechanisms controlling the execution of lordotic responses in the female rat. AB - To study the participation of gamma-aminobutyric acid (GABA) in the control of sexual behavior in the female rat, the effect of GABA and picrotoxin injections in the ventromedial hypothalamic nucleus (VMN) upon lordosis frequency and multiunit spike activity (MUSA) was determined. Infusion of 100 micrograms GABA into conscious rats reduced lordotic responsiveness within 15 min after injection; with a similar time course, the same dose markedly reduced MUSA in urethane anesthetized rats. Forty-five min after injection lordotic responsiveness recuperated to preinjection levels; at this time MUSA showed a rebound increase in neuron firing frequency. The possible relation between ventromedial hypothalamic neuronal activity and capacity for lordotic responses was further tested with injections of a local anesthetic: 1 microliter of 2% Xylocaine infused into the VMN produced similar results, suppressing MUSA and lordotic responsiveness for ca. 45 min beginning immediately after injection. Microinjections of GABA antagonist picrotoxin had the opposite effects: 0.1 microgram increased MUSA and lordotic responsiveness at 5 and 45 min; however at 20 min, when MUSA was at its highest, lordosis frequency was not elevated. Injections of solvent had no consistent effects on either measure. Two conclusions many be tentatively drawn from these data: (a) the VMN is the origin of a neural signal which exerts a moment-to-moment gating control on the execution of lordosis, and (b) the generation and/or the output of this signal is under the control of a GABAergic hypothalamic mechanism which normally exerts an inhibitory effect on the display of lordotic responses. PMID- 3008781 TI - Reinstatement of orienting behavior by d-amphetamine in rats with superior colliculus lesions. AB - The involvement of the nigrotectal pathway in the expression of visual orienting behavior was assessed by a combination of superior colliculus (SC) lesions and increased dopamine transmission produced by administration of d-amphetamine. Orienting behavior elicited by apparently moving or stationary light displays, its habituation, and recovery were observed. In intact animals, amphetamine injections had a small but reliable effect on the habituation of orienting behaviors. Rats with SC lesions did not orient to the lights. Amphetamine injected rats with SC lesions did orient, and the topography of their orienting behavior, rate of habituation, and recovery of orienting with changes in the light display were comparable to those of the intact animal. These results suggest a view of SC-lesion-impaired orienting behavior as a disturbance of sensory attention and emphasize the interaction of the SC and other neural systems in processes mediating the direction of attention. PMID- 3008783 TI - Kinetic characterization and partial purification of the membrane-bound inorganic pyrophosphatase from Rhodopseudomonas palustris. AB - A membrane-bound inorganic pyrophosphatase from Rhodopseudomonas palustris has been studied by kinetic analysis. The enzymatic activity was stimulated by Mg2+, and the (Mg-PPi) complex is regarded to be the functional substrate. Free Mg2+ revealed a significant influence on the membrane-bound PPiase activity. Kinetic data were determined at various fixed concentrations of free Mg2+. Mg2+ is proposed to act as an activator in two ways. It may interact with the enzyme directly, and may combine with PPi to yield the functional substrate Mg-PPi. Ca2+ revealed a non-competitive type of inhibition on the Mg2+-activated enzyme. The membrane-bound PPiase activity was firmly attached to the chromatophore membrane. To achieve an almost entire solubilization, both, Triton X-100 and high concentrations of Mg2+, had to be applied. An enrichment method along with stepwise lowering the concentrations of Triton X-100 and Mg2+ after the solubilization has been established. The solubilized and partially purified enzyme was stimulated by phospholipids while the influence of free Mg2+ was lost. Three different energies of activation as a function of temperature were derived from Arrhenius plots for the membrane-bound as well as for the solubilized PPiase activity. PMID- 3008782 TI - Purification and properties of a soluble inorganic pyrophosphatase from Rhodopseudomonas palustris. AB - A soluble inorganic pyrophosphatase from photolithoautotrophically grown Rhodopseudomonas palustris was purified to a state of apparent homogeneity applying high resolving liquid chromatography steps. Values of 65 500 and 64 500 were calculated for the relative molecular mass under non-dissociating conditions employing gel filtration and high-performance liquid chromatography, respectively. Dissociation sodium dodecyl sulfate gel electrophoresis resulted in a value of 32 000, indicating that the enzyme is composed of two subunits of equal molecular mass. Isoelectric focusing revealed a pI value of 4.7. The purified enzyme was specific for PPi and the activity was modified by divalent cations. Ca2+, Mn2+, Mg2+ and Co2+ were potent activators at a concentration ratio of [Me2+]/[PPi] less than 1. Ca2+ turned out to be the most potent activator. Free Me2+ was inhibitory on the PPiase activity. The (Me-PPi) complex is regarded as the functional substrate. Km and Ki values of the metal activation and inhibition were determined. An activation energy of Ea = 14.4 kJ/mol was derived from Arrhenius plots for the enzymatic reaction. PMID- 3008784 TI - Studies of enzyme-ligand complexes using dynamic fluorescence anisotropy. I. The substrate-binding site of malate dehydrogenase. AB - The substrate binding site of pig mitochondrial malate dehydrogenase was characterized using complexes of the enzyme with the substrate analogue 8 hydroxypyrene-1,3,6-trisulfonate with and without the addition of coenzymes. The rotational mobility of the fluorescent dye within the binding site was examined with the aid of a multi-frequency phase-fluorimeter. Together with absorption, circular dichroism and fluorescence spectroscopy, conformational changes of the substrate binding site could be defined. The dye was generally found to be immobilized in the binding site. Addition of NADH to the binary complex caused strengthening of a hydrogen bond and further loss of mobility, whereas NAD enlarged the space available for motion of the dye with concomitant loss of the hydrogen bridge. PMID- 3008785 TI - Identification of the catalytic subunit of the ATP diphosphohydrolase by photoaffinity labeling of high-affinity ATP-binding sites of pancreatic zymogen granule membranes with 8-azido-[alpha-32P]ATP. AB - Photoaffinity labeling has been performed on pancreatic zymogen granule membranes using 8-azido-[alpha-32P]ATP (8-N3-ATP). Proteins of 92, 67, 53, and 35 kdaltons (kDa) were specifically labeled. ATP (100 microM) inhibited very strongly the labeling with 8-N3-ATP, while ADP was much less potent, AMP and cAMP being inefficient. The apparent constants for 8-N3-ATP binding were in the micromolar concentration range for the four labeled proteins. Without irradiation, 8-N3-ATP was a competitive inhibitor (Ki = 2.66 microM) for the hydrolysis of ATP by the ATP diphosphohydrolase. The optimal conditions for the photolabeling of the 92- and 53-kDa proteins were pH 6.0 in presence of divalent cations. On the other hand the 67- and 35-kDa proteins required an alkaline pH and the addition of EDTA in the photolabeling medium. No proteins could be labeled on intact zymogen granules, showing that all the high-affinity ATP-binding sites of the membrane were located at the interior of the granule. Both the 92- and 53-kDa glycoproteins could bind to concanavalin A-Sepharose and be extracted in the detergent phase in the Triton X-114 phase separation system. These latter properties are typical of integral membrane proteins. In addition, the 53-kDa labeled protein was sensitive to endo-beta-N-acetylglucosaminidase digestion. Photolabeling with 8-N3-ATP of two different preparations of purified ATP diphosphohydrolase also led to the labeling of a 53-kDa protein. Thus among the four proteins labeled with 8-N3-ATP on the pancreatic zymogen granule membrane, the 53-kDa integral membrane glycoprotein was shown to bear the catalytic site of the ATP diphosphohydrolase. PMID- 3008786 TI - Structure of the O-chain polysaccharide of the phenol-phase soluble lipopolysaccharide of Escherichia coli 0:157:H7. AB - The phenol-phase soluble lipopolysaccharide isolated from Escherichia coli 0:157 by the hot phenol-water extraction procedure was shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis, periodate oxidation, methylation, and 13C and 1H nuclear magnetic resonance studies to be an unbranched linear polysaccharide with a tetrasaccharide repeating unit having the structure: (formula; see text) The serological cross-reactivity of E. coli 0:157 with Brucella abortus, Yersinia enterocolitica (serotype 0:9), group N Salmonella, and some other E. coli species can be related immunochemically to the presence of 1,2-glycosylated N-acylated 4 amino-4, 6-dideoxy-alpha-D-mannopyranosyl residues in the O-chains of their respective lipopolysaccharides. PMID- 3008787 TI - Chemical DNA sequencing in the opposite direction to the dideoxy chain termination method, using a single preparation of M13 single-stranded DNA. AB - A strategy has been developed allowing the use of a single preparation of single stranded DNA clones for chemical DNA sequencing in the opposite direction to the classical dideoxy chain termination method. Oligonucleotide complementary to the 5'-end of the multipurpose cloning sequence, with the proper restriction enzyme, is used to cleave specifically the molecules to expose a unique 5'-end, upstream to the inserted DNA, for the kinase labeling reaction. No further treatments are necessary before Maxam-Gilbert chemical sequencing reactions. PMID- 3008788 TI - A simple immunological detection method for the direct screening of genes from clone banks. AB - A simple immunoassay has been developed which can be used in the isolation of particular gene(s) from a clone bank of recombinant plasmids. A clone bank of the DNA is constructed with a plasmid vector and maintained in Escherichia coli. The recombinant clones were filtered onto a hydrophobic grid membrane and grown up into individual colonies, and a replica was made onto nitrocellulose paper. The bacterial cells were then lysed with chloroform and the proteins were immobilized onto the nitrocellulose paper. The nitrocellulose paper is then reacted with a rabbit antibody preparation made against the particular antigenic product to detect the recombinant clone which carries the corresponding gene. The bound antibodies can be detected easily by a colorimetric assay using goat anti-rabbit antibodies conjugated to horseradish peroxidase. Positively reacting clones can be recovered from the master hydrophobic grid membrane filter for further characterization. We proposed to call this method "colony ELISA blot" and described the isolation of the genes coding for the soluble antigens of Pasteurella haemolytica using this method. PMID- 3008789 TI - [Effect of alpha-1-thymosin on intracellular cyclic AMP in mouse thymic subpopulations]. PMID- 3008790 TI - The granulomatous reaction in herpetic stromal keratitis: immunohistological and ultrastructural findings. AB - A granulomatous reaction was identified in six corneal buttons obtained from patients with herpetic stromal keratitis. The inflammation, characterized by lymphocytes, plasma cells, macrophages and multinucleate giant cells, was associated with Descemet's membrane in five cases and with a break in Bowman's membrane in one case. The ultrastructural changes were documented by scanning electron microscopy in all cases. Transmission electron microscopy, performed in four cases, failed to disclose the presence of viral inclusions. Immunoperoxidase stains utilizing antibodies to the herpes simplex virus were done on all cases. Positivity (the presence of herpes simplex virus antigen or herpes simplex-like antigen) was detected within epithelium (three cases), within stroma (three cases), and within some inflammatory cells of the granulomatous reaction at Bowman's membrane. Positive staining was not detected in cells of the granulomatous reaction at Descemet's membrane. These results suggest that the reaction at Descemet's membrane, which has been found in a variety of corneal conditions, may be due to a poorly understood alteration in the antigenicity of Descemet's membrane. PMID- 3008791 TI - The science of ophthalmology. PMID- 3008792 TI - [Superior protective activity of phenytoin against hypoxia in pharmacological screening test]. AB - In the treatment of cerebral infarction, it is important to select drugs for inhibiting development of the pathology. In order to select markedly effective drugs among many drugs, it is possible to grasp the outline of drug effects by means of an experimental system with simplicity and regularity--a screening test. In the present study, we induced a hypoxic load by exposing mice to a gas mixture consisting of 4% O2 and 96% N2, and investigated the effects of various kinds of drugs with reference to the survival time(ST: mean +/- SE), the time until the respiratory arrest of the mice. In the control group (n = 110), ST was 170 +/- 6 sec, with a normal distribution, and no mice that survived for eight minutes or more after the load were found. In contrast, ST was 1,833 +/- 487 sec in the suloctidil-treated group (12.5 mg/kg, n = 11), 1,160 +/- 342 sec in the vitamin E group (200 mg/kg, n = 15), 602 +/- 74 sec in the pentobarbital group (50 mg/kg, n = 12) and 2667 +/- 452 sec in the phenytoin group (100 mg/kg, n = 10). On the other hand, no prolongation of ST was found in the groups to which vitamin C, coenzyme Q, cytochrome c, betamethasone, mannitol or germanium-132 were administered. The ratio of the mice that survived for one hour or more after the load was 0% in the control group, 45.5% in the suloctidil group (12.5 mg/kg), 23.5% in the vitamin E group (200 mg/kg), 0% in the pentobarbital group (50 mg/kg) and 70% in the phenytoin group (100 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008793 TI - [Effect of long-term prior antihypertensive treatment on cerebral ischemia induced by bilateral common carotid artery ligation in SHRSR]. AB - The susceptibility to cerebral ischemia was studied in stroke-resistant spontaneously hypertensive rats (SHRSR) treated by a long-term antihypertensive treatment, and compared with untreated SHRSR and Wistar rats (WR). Male SHRSR, aged 8 weeks, were divided into two groups and a long-term antihypertensive treatment for 4-6 weeks was started on one group (treated SHRSR: T-SHR) while the other group was left untreated as control (untreated SHRSR: U-SHR). The changes of blood pressure were checked on these rats. The prior treatment of hypertension was achieved by administration of hydroflumethiazide (120 mg/kg/day) and captopril (15-30 mg/kg/day) orally for 4-6 weeks by mixing in drinking water. All the experiments were performed at the age of 12-16 weeks and WR of similar age served as normotensive untreated control. Cerebral ischemia was induced by bilateral common carotid artery ligation (BLCL) and blood pressure was always checked before BLCL. The survival ratio was observed from 1 hour to 24 hours after BLCL. The regional cerebral blood flow (rCBF) were measured before and 4 hours after BLCL periodically. The brain energy metabolites were measured 4 hours after BLCL. rCBF were measured at the thalamus by the hydrogen clearance method. ATP concentrations were determined by luciferine-luciferase method, c-AMP was measured by RIA and lactate by enzymatic method. The brain water content was measured by freeze-dry method.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008794 TI - [Detection of free radicals generated in NADPH-dependent lipid peroxidation in ischemic brain homogenates--use of the spin trapping technic]. AB - Spin trapping technique has been applied to the detection of free radicals generated in NADPH stimulated lipid peroxidation process in ischemic brain homogenate. Using male Wistar rats, complete cerebral ischemia for 30 min, 60 min or 120 min was produced by decapitation followed by preservation of the heads at 37 degrees C. Global cerebral ischemia of 30 min or 60 min duration was induced by occlusions of three vessels (bilateral common carotid and basilar artery) in the ventilated rats. In some animals, bilateral carotid occlusions were released for 30 min following 30 min of ischemia to study postischemic event. Two reaction mixtures containing of brain homogenate, NADPH, Fe-EDTA and spin trapping reagent, phenyl-t-buthylnitrone (PBN), were prepared from each brain sample--one to be incubated in air and the other to be incubated in nitrogen gas. After the incubation for 20 min at 37 degrees C, free radical adducts of PBN were measured by electron spin resonance (ESR). In preliminary experiments, no ESR signals were obtained from the reaction mixtures without the addition of NADPH and Fe-EDTA. And the dependence of ESR signal intensity upon the NADPH concentration was observed. The six-line signals (triplet of doublets), which hyperfine splitting constants were AN = 16.2-16.5 G and A beta H = 3.6-3.8 G, were obtained from both ischemic models. These signals were dependent upon the presence of oxygen in the reaction systems, as evidenced by the fact that the signal intensity obtained from aerobic incubation was consistently stronger than that obtained from anaerobic incubation in each brain sample.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008795 TI - Parathyroid hormone-stimulated development of osteoclasts in cultures of cells from neonatal murine calvaria. AB - Evidence was sought for the presence of osteoclasts or preosteoclasts in cells prepared from neonatal murine calvaria by sequential enzymatic digestion. Freshly isolated cells released late in the digestion process resorbed both living and devitalized calvarial bone matrix in response to PTH, accompanied by the development of osteoclasts. Light and scanning electron microscopy of these cells after 1 to 2 days in culture revealed the presence of round cells (10 to 15 mu in diameter) with minimal surface microvilli in addition to the larger osteoblastic cells. Few cells contained tartrate-resistant acid phosphatase (TRAPase). If initially seeded at confluent density, more cells positive for TRAPase developed during subsequent culture, and these cells retained the ability to resorb calvarial bone matrix upon treatment with parathyroid hormone (PTH). These cultured cells responded to PTH with increased secretion of TRAPase into the medium and increased numbers of TRAPase positive cells. These were 20 to 50 mu in diameter, sometimes multinucleated, and some were spread 100 to 200 mu in length. Observations of living cells that took up neutral red showed that, upon treatment with calcitonin (CT), surface filopodia of some but not all of the labeled cells retracted within 30 minutes. Loss of resorptive response to PTH, as well as PTH stimulated development of TRAPase-positive cells, occurred if the cells were initially seeded at low density and grown to confluence before exposure to hormone. This correlated with the loss of most of the 10 to 15 mu diameter round cells. These observations suggest that preosteoclasts are present among these small cells that can give rise to osteoclasts upon treatment with PTH. PMID- 3008796 TI - Effect of hypophysectomy on the occupied and unoccupied binding sites for 1,25 dihydroxyvitamin D3 in rat intestine. AB - It is not known why the intestinal active transport of calcium per unit of mucosal mass is not affected by hypophysectomy (HX) even though serum 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] and intestinal calcium-binding protein are decreased. In order to study the effect of HX on the quantity of intestinal receptor of 1,25(OH)2D3 and its binding characteristics, the intestinal total occupied and unoccupied binding sites for 1,25(OH)2D3 were measured, by the use of the mercurial reagent mersalyl, in the intestine of HX and age-matched control rats. In addition, the effect of bovine growth hormone (bGH) replacement on the quantity of both binding states was examined in the HX rats. Results of Scatchard analysis and sucrose density gradients showed that the 3.5S receptor of the HX rat intestine was not distinguishable from that seen in the intact rat intestinal cytosol. Under vitamin D-supplemented conditions, HX was shown to reduce the levels of occupied receptors when the data were expressed on the basis of cytosolic protein. The reduced occupied sites could, in fact, have resulted from the reduction in plasma 1,25(OH)2D3 levels. No synthesis rates were determined. The unoccupied and total binding sites for 1,25(OH)2D3 per length of intestine were lower in the HX group than in the intact group. Administration of bGH resulted in an increase of endogenously occupied binding sites without affecting the total binding activity. Under vitamin D-depleted (-D) conditions, the total binding activity (intestinal) for 1,25(OH)2D3 was increased in the intact but not in the -DHX rats. Administration of bGH to the -DHX rat resulted in no effect on the binding levels of 1,25(OH)2D3 receptor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008797 TI - The effect of enalapril on baroreceptor mediated reflex function in normotensive subjects. PMID- 3008798 TI - Enalapril and baroreflex function in normotensive subjects. PMID- 3008799 TI - Inhibition of the growth of human hepatocellular carcinoma in vitro and in athymic mice by a quinazoline inhibitor of thymidylate synthase, CB3717. AB - Two human primary liver cancer cell-lines, PLC/PRF/5 and Hep 3B, grown both in vitro and as xenografts in nude mice were used to evaluate the chemotherapeutic potential of a new quinazoline antifolate CB3717. Xenograft growth rate was followed both by serial serum alphafoetoprotein (AFP) measurement and direct volume estimation. A dose regime of 200 mg CB3717 kg-1 body wt day-1 for 5 days caused a significant reduction in growth rate, as measured by relative serum AFP, of both xenografts; PLC/PRF/5 derived xenograft growth was also inhibited by 125 mg CB 3717 kg-1 day-1 for 5 days. Cell culture experiments showed that the ID50 for the cell lines fell within the range of serum CB3717 concentration achieved by a dose of 300 mg m-2 given to patients. Treatment with CB3717 stimulated the incorporation of exogenous thymidine into DNA by the tumour cells, presumably because of inhibition of the de novo pathway and reduction of endogenous thymidine triphosphate pools. These results suggest that CB3717 may be a useful new therapeutic agent in human primary liver cell carcinoma and that blocking the salvage pathway may further increase efficacy. PMID- 3008800 TI - The significance of electron spin resonance of the ascorbic acid radical in freeze dried human brain tumours and oedematous or normal periphery. AB - The ESR spectrum, attributed to the ascorbic acid (ascorbyl) radical and obtained by exposing freeze dried material to air, can not be used as proof for the occurrence of in vivo free radical reactions. Depending on the method of freeze drying, the content of blood or hemolyzed blood is the dominant factor in creating higher than normal ESR signals in brain or related tissue. These findings explain why the signal, though larger in many human brain tumours than in their surroundings, is not indicative of malignancy. No differences are seen between oedematous and normal tissue. The ascorbyl radical is definitely not stable in aqueous solution, which indicates that fresh tissue sections can also not be used to study in vivo radicals by ESR. PMID- 3008801 TI - Epidermal hyperproliferation following the induction of microabscesses by leukotriene B4. AB - The percentage of non-diploid epidermal cells was determined by flow cytometry following application of leukotriene B4 (LTB4) to human skin. Doses in the range 35-500 ng were shown to cause a marked increase in proliferation, the non-diploid cells reaching a maximum between 72 and 96 h after LTB4 application. No difference was observed between the response of healthy controls and the uninvolved skin of psoriatic patients. We suggest, therefore, that the hyperproliferation is a consequence of the physical disruption of the stratum corneum accompanying the rupture of microabscesses. PMID- 3008802 TI - Early warning skin signs in AIDS and persistent generalized lymphadenopathy. AB - Distinctive patterns of skin disease other than Kaposi's sarcoma have been in patients with AIDS, in others with persistent generalized lymphadenopathy (PGL) and in a group at high risk of developing AIDS. We found a chronic acneiform folliculitis on the face, back, chest and buttocks, extensive cutaneous fungal infections and a striking neck and beard impetigo. These skin diseases were not present in asymptomatic male homosexual control subjects, 32% of whom were found to have antibodies to human T cell lymphotropic virus type III (HTLV-III). We regard these dermatoses as early warning signs of AIDS. PMID- 3008803 TI - Two cases of adult T-cell leukaemia. AB - A 70-year-old male and a 46-year-old female, both living in Japan, were seen because of multiple erythematous papules and plaques on the trunk and extremities. The peripheral white blood cell counts were increased to 30,000 80,000/mm3. The majority of white cells had characteristic lobulated and deeply indented nuclei. The patients and most of their family members had anti-ATLV antibody. Using an OKT series of monoclonal antibodies the tumour cells were found to be T-cells. Trisomy 21 was detected in the first case and ATL virus proviral DNA in the second. PMID- 3008805 TI - Dietary factors in the aetiology and treatment of constipation during pregnancy. AB - Using a 7-day weighed-intake method, dietary factors implicated in the aetiology of constipation were examined by comparison between the nutrient intakes of nine women with constipation in the third trimester of pregnancy who had not altered their diets, and a matched group of nine women who had not suffered constipation at any time during pregnancy. No significant differences were found for intakes of major nutrients, including dietary fibre, between the constipated and non constipated groups, indicating that inappropriate diets do not appear to be the primary cause of constipation during pregnancy. The nutrient intakes of 40 women with constipation in the third trimester of pregnancy were examined to assess dietary changes made in attempts to treat constipation. No difference in intakes of dietary fibre were found between those who claimed to have increase their intakes, and those who had not altered their diets. Both were well below the level of dietary fibre intake known to be successful in treating constipation. PMID- 3008804 TI - Six cases of hereditary spherocytosis revealed by human parvovirus infection. AB - Recent research has shown that the human parvovirus is a causative agent of aplastic crisis in hereditary haemolytic anaemias. We report six cases--four children and two adults--of hereditary spherocytosis revealed by aplastic crisis due to human parvovirus infection. PMID- 3008806 TI - Screening for antibodies to HTLV-III amongst the semen donors of an AID programme. AB - This study suggests that, at the present time, AID is not likely to be a significant risk factor in the transmission of AIDS in Britain, particularly in centres which are already practising careful screening of semen donors. New facilities which are becoming available for HTLV-III screening should help to provide reassurance that semen donations are free from the virus. PMID- 3008807 TI - Random urinary cyclic 3',5' guanosine monophosphate in epithelial ovarian cancer: relation to other prognostic variables and to survival. AB - Random urinary cyclic guanosine monophosphate (cyclic GMP) has been measured in a control group of 83 women and in 92 women with histologically proven epithelial ovarian cancer. Forty-eight cancer patients (53%) had levels greater than the mean +2SD of the control population. There was no correlation between urinary cyclic GMP and tumour histology, degree of differentiation or stage. Patients with gross residual disease after laparotomy had higher postoperative levels. This degree of sensitivity in addition to poor specificity for ovarian cancer renders this marker unsuitable for screening purposes. While residual disease state after surgery was found to be the best single predictor of survival, an elevated urinary cyclic GMP level before chemotherapy was found to be an independent adverse prognostic variable. PMID- 3008808 TI - Sequential antibody changes following ulcerative herpetic keratitis. AB - Blood and tear levels of immunoglobulins against herpes simplex virus (HSV) were examined in 28 patients with dendritic keratitis over a period of 28 days. By means of an indirect micro-immunofluorescent technique blood and tear HSV IgG were detected, but neither circulating HSV IgM nor local HSV IgA were found. Over a four-week interval non-diagnostic fluctuations of HSV IgG occurred in most patients, though seven (25%) developed a rising blood IgG titre. Tear IgG appeared to be an exudate from blood. HSV was isolated from 68% of corneal swabbings and 11% of conjunctival swabbings. This study provides guidelines for laboratory testing in recurrent herpetic keratitis. PMID- 3008809 TI - Cryptophthalmos: surgical treatment of the congenital symblepharon variant. AB - A case of the congenital symblepharon variant of cryptophthalmos is reported. Surgical treatment is described. The ophthalmic features of cryptophthalmos and its systemic associations are reviewed. PMID- 3008810 TI - Molecular structure and polymorphic map of the human phenylalanine hydroxylase gene. AB - Human phenylalanine hydroxylase is a liver-specific enzyme that catalyzes the conversion of phenylalanine to tyrosine. Absence of enzymatic activity results in phenylketonuria, a genetic disorder that causes development of severe mental retardation in untreated children. In this paper we report the cloning and structure of the normal human phenylalanine hydroxylase gene, which was isolated in four overlapping cosmid clones that span more than 125 kilobases (kb) of the genetic locus. The peptide coding region of the gene is about 90 kb in length and contains 13 exons, with intron sizes ranging from 1 to 23 kb. Exons at the 3' half of the gene are compact, whereas those at the 5' half are separated by large introns. The human phenylalanine hydroxylase gene codes for a mature messenger RNA of approximately 2.4 kb, and its noncoding to coding DNA ratio is one of the highest among eukaryotic genes characterized to date. The map positions of nine polymorphic restriction sites identified within the locus were established by restriction enzyme mapping of the cloned gene fragments. Two clusters of polymorphic sites were demonstrated: (1) BglII, PvuII(a), and PvuII(b) at the 5' end of the gene and (2) EcoRI, XmnI, MspI(a), MspI(b), EcoRV, and HindIII at the 3' end. The polymorphic site distribution within this gene is a useful tool for prenatal diagnosis and carrier detection of the genetic disorder, while knowledge of normal gene structure is a prerequisite for future characterization of mutant alleles. PMID- 3008811 TI - Mucidin and strobilurin A are identical and inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain at the same site as myxothiazol. AB - Mucidin and strobilurin A, antifungal antibiotics isolated from the basidiomycetes Oudemansiella mucida and Strobiluris tenacellus, respectively, inhibit electron-transfer reactions in the cytochrome bc1 complex of the mitochondrial respiratory chain. The two compounds have identical effects on oxidation-reduction reactions of the cytochromes b and c1 in isolated succinate cytochrome c reductase. They inhibit reduction of cytochrome c1 by succinate but do not inhibit reduction of cytochrome b. When added in combination with antimycin, either inhibitor blocks reduction of both cytochromes b and c1. Mucidin and strobilurin A differ from antimycin in that they inhibit, rather than promote, oxidant-induced reduction of cytochrome b. They also differ from antimycin in that they do not block reduction of cytochrome b by succinate when cytochrome c1 is previously reduced by ascorbate and they do not inhibit oxidation of cytochrome b by fumarate. These effects of mucidin and strobilurin A are, however, qualitatively identical with those of myxothiazol, an antibiotic that inhibits respiration by binding to cytochrome b [Von Jagow, G., Ljungdahl, P. O., Graf, P., Ohnishi, T., & Trumpower, B. L. (1984) J. Biol. Chem. 259, 6319 6326]. Mucidin and strobilurin A have identical UV and mass spectra, and they elute together on high-pressure liquid chromatography. We thus conclude that these antibiotics, although isolated from different bacteria, are structurally identical. Our results indicate that strobilurin A and mucidin inhibit electron transport at the same site as myxothiazol and not at the antimycin site, as previously reported [Subik, J., Behren, M., & Musilek, V. (1974) Biochem. Biophys. Res. Commun. 57, 17-22]. PMID- 3008812 TI - Characterization of electron-transfer and proton-translocation activities in bovine heart mitochondrial cytochrome c oxidase deficient in subunit III. AB - The electron-transfer and proton-translocation activities of cytochrome c oxidase deficient in subunit III (Mr 29 884) prepared by native gel electrophoresis [Ludwig, B., Downer, N. W., & Capaldi, R. A. (1979) Biochemistry 18, 1401-1407] have been investigated. This preparation has been depleted of 82-87% of its subunit III content as quantitated by Coomassie Brilliant Blue staining intensity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and [14C]dicyclohexylcarbodiimide labeling. The maximum rate of electron transfer of the subunit III deficient enzyme at pH 6.5 is 383 s-1, 78% of control enzyme. Neither the high-affinity site (Km = 10(-8) M) nor the low-affinity site (Km = 10(-6) M) of the cytochrome c kinetic interaction with cytochrome c oxidase is affected by the removal of subunit III. Subunit III deficient cytochrome c oxidase retains the ability to bind cytochrome c in both the high- and low affinity sites as determined in direct thermodynamic binding experiments. Liposomes containing this preparation exhibit a respiratory control ratio [Hinkle, P. C., Kim, J. J., & Racker, E. (1972) J. Biol. Chem. 247, 1338-1341] of 3.9, while liposomes containing control enzyme exhibit a ratio of 4.3, suggesting that they have a similar proton permeability. Vectorial proton translocation initiated by the addition of ferrocytochrome c in liposomes containing subunit III deficient enzyme is decreased by 64% compared to those containing control enzyme. When the proton-translocated to electron-transferred ratio is measured in these phospholipid vesicles at constant enzyme turnover, removal of subunit III from the enzyme decreases the ratio from 0.52 to 0.21, a 60% decrease.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008813 TI - Variations in the oxidation-reduction behavior of liganded species of Pseudomonas cytochrome oxidase. AB - In an effort to determine the steady-state redox properties of Pseudomonas aeruginosa cytochrome cd1, changes in absorption spectra after the addition of excess reductant (ascorbate, ferrous ethylenediaminetetraacetic acid) were monitored for degassed unliganded enzyme and samples in the presence of CO and CN at pH 6.0, 8.0, or 10.0. Plots of [c2+]/[c3+] vs. [d2+]/[d3+] indicate that a "pseudoequilibrium" was reached for all samples at pH 8.0. Calculated values of delta Ed-c, the difference in reduction potential between the heme c and heme d moieties, at pH 8.0 were -25 +/- 5 (unliganded), -10 +/- 5 (enzyme-CO), and -25 +/- 5 mV (enzyme-CN). Relative rates of heme c and heme d reduction were found to be dependent upon type of ligand, reductant, and pH. Evidence for a cooperative heme c-heme d interaction is discussed. PMID- 3008814 TI - Correlation between acetylcholine receptor function and structural properties of membranes. AB - Protein-lipid interactions were studied by using Torpedo californica acetylcholine receptor (AChR) as a model system by reconstituting purified AChR into membranes containing various synthetic lipids and native lipids. AChR function was determined by measuring two activities at 4 degrees C: (1) low to high agonist affinity-state transition of AChR in the presence of an agonist (carbamylcholine) in either membrane fragments or sealed vesicles and (2) ion gating activity of AChR-containing vesicles in response to carbamylcholine. Sixteen samples were examined, each containing different lipid compositions including phosphatidylcholine, cholesterol, phosphatidic acid, phosphatidylethanolamine, asolectin, neutral lipid depleted asolectin, native lipids, and cholesterol-depleted native lipids. Phosphatidylcholines with different configurations of fatty acyl chains were used. The dynamic structures of these membranes were probed by incorporating spin-labeled fatty acid into AChR containing vesicles and measuring the order parameters. It was found that both aspects of AChR function were highly dependent on the lipid environment even though carbamylcholine binding itself was not affected. An appropriate membrane fluidity was necessarily required to allow the interconversion between the low and high affinity states of AChR. An optimal fluidity hypothesis is proposed to account for the conformational transition properties of membrane proteins. In addition, the conformational change was only a necessary, but not sufficient, condition for the AChR-mediated ion flux activity. Among membranes in which AChR manifested the affinity-state transition, only those containing both cholesterol and negatively charged phospholipids (such as phosphatidic acid) retained the ion gating activity. PMID- 3008815 TI - Generation of superoxide free radical by neocarzinostatin and its possible role in DNA damage. AB - Spectroscopic analysis of the reduction of both nitro blue tetrazolium and ferricytochrome c induced by neocarzinostatin shows that superoxide free radical is produced during the spontaneous degradation of the antibiotic. The amount of superoxide free radical produced from neocarzinostatin is not affected by the presence of thiol, although earlier work has shown that DNA damage is stimulated at least 1000-fold by thiol. Transition metals are not involved in this reaction. Although superoxide dismutase inhibits the reduction of nitro blue tetrazolium and cytochrome c induced by neocarzinostatin, neither it nor catalase interferes with the action of neocarzinostatin on DNA, whether or not drug has been activated by thiol. The pH profiles for spontaneous base release and alkali labile base release (a measure of nucleoside 5'-aldehyde formation at a strand break) do not correspond with that for the generation of superoxide free radical from neocarzinostatin. The same holds for supercoiled DNA cutting by neocarzinostatin chromophore in the absence of a thiol, which is an acid-favored reaction. These results indicate that the generation of superoxide free radical by the drug does not correlate with DNA damage activity, whether or not thiol is present. Furthermore, the failure of hydroxyl free-radical scavengers to inhibit drug-induced single-strand breaks in supercoiled DNA in the absence of thiol also indicates that a diffusible hydroxyl free radical is most probably not involved in this reaction. PMID- 3008816 TI - Hexokinase receptor complex in hepatoma mitochondria: evidence from N,N' dicyclohexylcarbodiimide-labeling studies for the involvement of the pore-forming protein VDAC. AB - In rapidly growing, highly glycolytic hepatoma cells as much as 65% of the total cell hexokinase is bound to the outer mitochondrial membrane [Parry, D.M., & Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912]. In this paper, we describe the purification to apparent homogeneity of a mitochondrial pore-forming protein from the highly glycolytic AS-30D rat hepatoma cell line. The purified protein shows a single 35 000-dalton band in sodium dodecyl sulfate polyacrylamide gel electrophoresis, an amino acid composition slightly more hydrophobic than that of the rat liver pore protein (also known as VDAC or mitochondrial porin), and a channel-forming activity of 136 channels min-1 (microgram of protein)-1. In addition to displaying the properties characteristic of VDAC (single-channel conductance, voltage dependence, and preference for anions), we observe that the AS-30D VDAC protein is one of only three mitochondrial proteins that bind [14C]dicyclohexylcarbodiimide (DCCD) at relatively low dosages (2 nmol of DCCD/mg of mitochondrial protein). Significantly, treatment of intact mitochondria isolated from either rat liver or the AS-30D hepatoma with DCCD results in an almost complete inhibition of their ability to binding hexokinase. Fifty percent inhibition of binding occurs at less than 2 nmol of DCCD/mg of mitochondrial protein. In contrast to DCCD, water soluble carbodiimides are without effect on hexokinase binding. These results suggest that the pore-forming protein of tumor mitochondria forms at least part of the hexokinase receptor complex. In addition, they indicate that a carboxyl residue located within a hydrophobic region of the receptor complex may play a critical role in hexokinase binding. PMID- 3008817 TI - Inhibition of angiotensin converting enzyme by aldehyde and ketone substrate analogues. AB - Three classes of carbonyl-containing substrate analogues and partial substrate analogues have been tested for their ability to inhibit angiotensin converting enzyme. (4-Oxobutanoyl)-L-proline is proposed to occupy the S1' and S2' subsites on the enzyme, thus locating its aldehyde carbonyl group at the position of the active site zinc atom. This aldehyde is 70% hydrated in aqueous solution and could mimic a tetrahedral intermediate occurring during enzyme-catalyzed substrate hydrolysis, but its Ki is only 760 microM. Carbobenzoxy-L-isoleucyl-L histidyl-L-prolyl-L-phenylalaninal is proposed to occupy the S1 through S4 subsites on the other side of the zinc atom. Its weak Ki of 60 microM is nearly equipotent to its parent peptide terminating in phenylalanine. However, ketoace, (5RS)-(5-benzamido-4-oxo-6-phenylhexanoyl)-L-proline [Almquist, R.G., Chao, W.R., Ellis, M.E., & Johnson, H.L. (1980) J. Med. Chem. 23, 1392-1398], one of the third class of inhibitors proposed to occupy subsites S1 through S2' on both sides of the zinc atom, has a Ki of 0.0006 microM under our assay conditions, orders of magnitude more potent than its parent peptide. The carbonyl carbon of ketoace is less than 3% hydrated in aqueous solution as determined by carbon-13 nuclear magnetic resonance spectroscopy. If the hydrate is the species bound to converting enzyme, its Ki must be less than 18 pM. Ketoace is a slow-binding inhibitor of converting enzyme, but its overall Ki is dependent on its concentration and therefore prevents calculation of kinetic constants for slow binding.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008818 TI - 1H NMR investigation of cytochrome cd1: complexes with electron-donor proteins. AB - Mixtures of the dissimilatory nitrite reductase cytochrome cd1 from Pseudomonas aeruginosa and potential electron-donating proteins were prepared in both fully oxidized and fully reduced states and examined by 1H NMR spectroscopy. The relatively narrower lines of the donor proteins enabled them to be clearly observed in spectra in the presence of significant amounts of the high molecular weight cd1. Mixtures of the physiological donor (Pseudomonas ferrocytochrome c 551) and ferrocytochrome cd1 showed specific line-broadening effects on the resonances of c-551 that depended on the mole ratio of c-551 to cd1. The experimental broadening fit a model in which c-551 is in intermediate or fast exchange between free solution and a complex with cd1, with an association constant for the complex in excess of 10(4) M-1. The model yields a minimum estimate for the forward bimolecular rate constant of 5 X 10(7) M-1 s-1 and suggests that the actual value may be much larger. The complexation was independent of pH in the range of 6-8, was independent of ionic strength over a salt concentration range of 20-1000 mM, and possessed a low thermal activation barrier. Mixtures of ferricytochrome c-551 and ferricytochrome cd1 showed no observable NMR perturbations, indicating that any hypothetical complex involving the oxidized forms must follow different dynamical and/or equilibrium conditions. No observable NMR perturbations existed in spectra of mixtures of cd1 and mammalian cytochrome c or Pseudomonas azurin in either oxidation state. PMID- 3008819 TI - Two-dimensional 1H NMR studies of cytochrome c: assignment of the N-terminal helix. AB - The 1H resonances of 11 sequential amino acids in the N-terminal helix of horse ferrocytochrome c were studied by two-dimensional nuclear magnetic resonance techniques. All the main-chain protons from Lys-5 through Ala-15 and many of the side-chain protons were assigned. J-Correlated spectroscopy (COSY) was used to distinguish protons on neighboring bonds and to recognize amino acid types. Nuclear Overhauser effect spectroscopy (NOESY) was used to define spatially contiguous protons and to determine amino acid sequence neighbors. The relayed coherence experiment (relay COSY) was used to resolve many ambiguities in intraresidue J-coupled connectivities and interresidue NOE connectivities. This required no explicit knowledge of the solution structure. The pattern of NOEs found is consistent with a regular alpha helix between glycine-6 and lysine-13; H bonding continues at least through alanine-15 [see Wand, A.J., Roder, H., & Englander, S. W. (1986) Biochemistry (following paper in this issue)]. Chain disorder occurs at the N-terminus. There is no indication of significant spin diffusion among the backbone amide and alpha-protons of this 12.4-kilodalton protein even at the longest NOE mixing time used (140 ms). PMID- 3008820 TI - Two-dimensional 1H NMR studies of cytochrome c: hydrogen exchange in the N terminal helix. AB - The hydrogen exchange behavior of the N-terminal helical segment in horse heart cytochrome c was studied in both the reduced and the oxidized forms by use of two dimensional nuclear magnetic resonance methods. The amide protons of the first six residues are not H bonded and exchange rapidly with solvent protons. The most N-terminal H-bonded groups--the amide NH of Lys-7 to Phe-10--exhibit a sharp gradient in exchange rate indicative of dynamic fraying behavior, consistent with statistical-mechanical principles. This occurs identically in both reduced and oxidized cytochrome c. In the oxidized form, residues 11-14, which form the last helical turn, all exchange with a similar rate, about one million times slower than the rate characteristic of freely exposed peptide NH, even though some are on the aqueous face of the helix and others are fully buried. These and similar observations in several other proteins appear to document local cooperative unfolding reactions as determinants of protein H exchange reactions. The N terminal segment of cytochrome c is insensitive to the heme redox state, as in the crystallographic model, except for residues closest to the heme (Cys-14 and Ala-15), which exchange about 15-fold more slowly in the reduced form. The cytochrome c H exchange results can be further considered in terms of the conformation of the native and the transiently unfolded forms and their free energy relationships in both the reduced and the oxidized states. PMID- 3008821 TI - Inhibition of myofibrillar and actomyosin subfragment 1 adenosinetriphosphatase by adenosine 5'-diphosphate, pyrophosphate, and adenyl-5'-yl imidodiphosphate. AB - Adenosine 5'-diphosphate (ADP), inorganic pyrophosphate (PPi), and adenyl-5'-yl imidodiphosphate (AMPPNP) act as competitive inhibitors of the ATPase of myofibrils and actomyosin subfragment 1 (acto-S1). At I = 0.2 M, pH 7, and 15 degrees C, the inhibition constants for rabbit myofibrils are 0.17, 3, and 5 mM, respectively; the values for frog myofibrils at 0 degrees C are very similar, being 0.22, 1.5, and 2.5 mM. The inhibition constant of AMPPNP is about 2 orders of magnitude larger than the reported dissociation constant for fibers [Marston, S. B., Rodger, C. D., & Tregear, R. T. (1976) J. Mol. Biol. 104, 263-276]. A possible reason for this difference is that AMPPNP binding results in the dissociation of one head of each myosin molecule. The inhibition constants for rabbit acto-S1 cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide measured under the same conditions were 0.12, 2.6, and 3.5 mM for ADP, PPi, and AMPPNP, respectively. The inhibition of cross-linked and native acto-S1 was compared at low ionic strength and was found to be similar. The value for ADP is very similar to reported values of the dissociation constant whereas the inhibition constants for AMPPNP and PPi are an order of magnitude weaker [Greene, L. E., & Eisenberg, E. (1980) J. Biol. Chem. 255, 543-548]. PMID- 3008822 TI - Inhibition of hydroxyapatite crystal growth by bone-specific and other calcium binding proteins. AB - Mineralization of bone matrix may be influenced by the presence of specific, noncollagenous bone proteins. The quantitative influence of two bone-specific proteins--bone gamma-carboxyglutamic acid (Gla) protein and osteonectin--and other proteins that decreased the rate of crystal growth was measured by adding seed crystals of hydroxyapatite to a solution of CaCl2 and KH2PO4, pH 7.4 at 37 degrees C. The molar concentrations of proteins needed to inhibit the rate of crystal growth by 50% were as follows: osteonectin, 0.15 microM; bone Gla protein, 0.8 microM; prothrombin, 0.9 microM; prothrombin fragment 1, 1.0 microM; soybean trypsin inhibitor, 3 microM; prethrombin 1, 9 microM; cytochrome c, 30 microM. Calmodulin and parvalbumin were found to be less active than prothrombin fragment 1 and had no activity in the micromolar range. The combination of two inhibitors resulted in a mixture with an inhibitory activity that was the sum of the two inhibitors. Decarboxylation of bone Gla protein significantly reduced its inhibitory activity. These results indicate that the inhibitory activity of a protein does not correlate with Ca2+-binding affinity under these conditions, that the mixture of inhibitors has an additive effect, and that gamma carboxyglutamic acid residues enhance the ability of a protein to inhibit hydroxyapatite-seeded crystal growth. PMID- 3008823 TI - Photosensitive DNA cleavage and phage inactivation by copper(II)-camptothecin. AB - Upon irradiation with 365-nm light, copper(II)-camptothecin significantly produced single- and double-strand breaks of DNA and also induced a marked inactivation of bacteriophage. The nucleotide sequence analysis exhibited considerably random DNA cleavage. The DNA strand scission by the camptothecin Cu(II)-UV light system, as well as the phage inactivation, was strongly suppressed by bathocuproine and catalase, indicating participation of cuprous species and hydrogen peroxide in the reaction. The present results suggest that (1) Cu(II) ion may play an important role as a cofactor in antitumor action of camptothecin and (2) the combination of copper-camptothecin plus long-wave ultraviolet light is useful against certain cancer treatment as a new photochemotherapy. PMID- 3008825 TI - Cytochrome c peroxidase compound ES is identical with horseradish peroxide compound I in iron-ligand distances. AB - X-ray absorption studies of compound ES of cytochrome c peroxidase show a short iron-oxygen distance of 1.67 +/- 0.04 A, an iron-histamine distance of 1.91 +/- 0.03 A, and an iron-pyrrole nitrogen average distance of 2.02 +/- 0.02 A. This is identical within the error with the reported structure of horseradish peroxidase compound I [Chance, B., Powers, L., Ching, Y., Poulos, T., Yamazaki, I., & Paul, K. G. (1984) Arch. Biochem. Biophys. 235, 596-611]. Comparisons of the structures of myoglobin peroxide [Chance, M., Powers, L., Kumar, C., & Chance, B. (1986) Biochemistry (preceding paper in this issue)], compound ES, and the intermediates of horseradish peroxidase reveal the possible mechanisms for the stabilization of the free radical species generated during catalysis. The proximal histidine regulates the structure and function of the pyrrole nitrogens and the heme, allowing for the formation and maintenance of the characteristic intermediates. PMID- 3008824 TI - DNA sequence specificity of the pyrrolo[1,4]benzodiazepine antitumor antibiotics. Methidiumpropyl-EDTA-iron(II) footprinting analysis of DNA binding sites for anthramycin and related drugs. AB - Anthramycin, tomaymycin, and sibiromycin are members of the pyrrolo[1,4]benzodiazepine [P(1,4)B] antitumor antibiotic group. These drugs bind covalently through N2 of guanine and lie within the minor groove of DNA [Petrusek, R. L., Anderson, G. L., Garner, T. F., Fannin, Q. L., Kaplan, D. J., Zimmer, S. G., & Hurley, L. H. (1981) Biochemistry 20, 1111-1119]. The DNA sequence specificity of the P(1,4)B antibiotics has been determined by a footprinting method using methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)], and the results show that each of the drugs has a two to three base pair sequence specificity that includes the covalently modified guanine residue. While 5'PuGPu is the most preferred binding sequence for the P(1,4)Bs, 5'PyGPy is the least preferred sequence. Footprinting analysis by MPE.Fe(II) reveals a minimum of a three to four base pair footprint size for each of the drugs on DNA with a larger than expected offset (two to three base pairs) on opposite strands to that observed in previous analyses of noncovalently bound small molecules. There is an extremely large enhancement of MPE.Fe(II) cleavage between drug binding sites in AT rich regions, probably indicating a drug-induced change in the conformational features of DNA which encourages interaction with MPE.Fe(II). In the presence of sibiromycin or tomaymycin the normally guanine-specific methylene blue reaction used in Maxam and Gilbert sequencing cleaves at other bases in defined positions relative to the drug binding sites. Finally, modeling studies are used to rationalize the differences and similarities in sequence specificities between the various drugs in the P(1,4)B group and their reactions with DNA. PMID- 3008826 TI - Predicted membrane topology of the coronavirus protein E1. AB - The structure of the envelope protein E1 of two coronaviruses, mouse hepatitis virus strain A59 and infectious bronchitis virus, was analyzed by applying several theoretical methods to their amino acid sequence. The results of these analyses combined with earlier data on the orientation and protease sensitivity of E1 assembled in microsomal membranes lead to a topological model. According to this model, the protein is anchored in the lipid bilayer by three successive membrane-spanning helices present in its N-terminal half whereas the C-terminal part is thought to be associated with the membrane surface; these interactions with the membrane protect almost the complete polypeptide against protease digestion. In addition, it is predicted that the insertion of E1 into the membrane occurs by the recognition of the internal transmembrane region(s) as a signal sequence. PMID- 3008827 TI - Radioactive probes for adrenocorticotropic hormone receptors. AB - Our attempts to develop adrenocorticotropic hormone (ACTH) analogues that can be employed for ACTH receptor identification and isolation began with the synthesis of ACTH fragments containing N epsilon-(dethiobiotinyl)lysine (dethiobiocytin) amide in position 25 to be used for affinity chromatographic purification of hormone-receptor complexes on Sepharose-immobilized avidin resins. Because labeling ACTH or ACTH fragments by conventional iodination techniques destroys biological activity due to oxidation of Met4 and incorporation of iodine into Tyr2, we have prepared [Phe2,Nle4]ACTH1-24, [Phe2,Nle4,biocytin25]ACTH1-25 amide, and [Phe2,Nle4,dethiobiocytin25]ACTH1-25 amide by conventional synthetic techniques. The HPLC profiles and amino acid analyses of the final products indicate that the materials are of a high degree of purity. The amount of tertiary butylation of the Trp residue in the peptides was assessed by NMR and was found to be less than 0.5%. All three peptides are equipotent with the standard ACTH1-24 as concerns their ability to stimulate steroidogenesis and cAMP formation in bovine adrenal cortical cells. Iodination of [Phe2,Nle4]ACTH1-24, with iodogen as the oxidizing agent, has been accomplished without any detectable loss of biological activity. The mono- and diiodo derivatives of [Phe2,Nle4]ACTH1 24 have been prepared, separated by HPLC, and assayed for biological activity. Both peptides have the full capacity to stimulate steroidogenesis and cAMP production in bovine adrenal cortical cells. PMID- 3008828 TI - A novel catecholamine-activated adenosine cyclic 3',5'-phosphate independent pathway for beta-adrenergic receptor phosphorylation in wild-type and mutant S49 lymphoma cells: mechanism of homologous desensitization of adenylate cyclase. AB - Virtually all known biological actions stimulated by beta-adrenergic and other adenylate cyclase coupled receptors are mediated by cAMP-dependent protein kinase. Nonetheless, "homologous" or beta-adrenergic agonist-specific desensitization does not require cAMP. Since beta-adrenergic receptor phosphorylation may be involved in desensitization, we studied agonist-promoted receptor phosphorylation during homologous desensitization in wild-type S49 lymphoma cells (WT) and two mutants defective in the cAMP-dependent pathway of beta-agonist-stimulated protein phosphorylation (cyc- cannot generate cAMP in response to beta-adrenergic agonists; kin- lacks cAMP-dependent kinase). All three cell types demonstrate rapid, beta-adrenergic agonist-promoted, stoichiometric phosphorylation of the receptor which is clearly not cAMP mediated. The amino acid residue phosphorylated is solely serine. These data demonstrate, for the first time, that catecholamines can promote phosphorylation of a cellular protein (the beta-adrenergic receptor) via a cAMP-independent pathway. Moreover, the ability of cells with mutations in the adenylate cyclase cAMP-dependent protein kinase pathway to both homologously desensitize and phosphorylate the beta-adrenergic receptors provides very strong support for the notion that receptor phosphorylation may indeed be central to the molecular mechanism of desensitization. PMID- 3008829 TI - Kinetics of electron transfer between cytochromes c' and the semiquinones of free flavin and clostridial flavodoxin. AB - Rate constants have been measured for the reactions of a series of high-spin cytochromes c' and their low-spin homologues (cytochromes c-554 and c-556) with the semiquinones of free flavins and flavodoxin. These cytochromes are approximately 3 times more reactive with lumiflavin and riboflavin semiquinones than are the c-type cytochromes that are homologous to mitochondrial cytochrome c. We attribute this to the greater solvent exposure of the heme in the c'-type cytochromes. In marked contrast, the cytochromes c' are 3 orders of magnitude less reactive with flavodoxin semiquinone than are the c-type cytochromes. We interpret this result to be a consequence of the location of the exposed heme in cytochrome c' at the bottom of a deep groove in the surface of the protein, which is approximately 10-15 A deep and equally as wide. While free flavins are small enough to enter the groove, the flavin mononucleotide (FMN) prosthetic group of flavodoxin is apparently prevented by steric constraints from approaching the heme more closely than approximately 10 A without dynamic structural rearrangements. Most cytochromes c' are dimeric, but a few are monomeric. The three-dimensional structure of the Rhodospirillum molischianum cytochrome c' dimer suggests that the heme should be more exposed in the monomer than in the dimer, but no relationship is observed between intrinsic reactivity toward free flavin semiquinones and the aggregation state of the protein. Likewise, there is no evidence that the spin state or ligand field of the iron has any effect on intrinsic reactivity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008830 TI - Coenzyme Q analogues reconstitute electron transport and proton ejection but not the antimycin-induced "red shift" in mitochondria from coenzyme Q deficient mutants of the yeast Saccharomyces cerevisiae. AB - Mitochondria isolated from coenzyme Q deficient yeast cells had no detectable NADH:cytochrome c reductase or succinate:cytochrome c reductase activity but contained normal amounts of cytochromes b and c1 by spectral analysis. Addition of the exogenous coenzyme Q derivatives including Q2, Q6, and the decyl analogue (DB) restored the rate of antimycin- and myxothiazole-sensitive cytochrome c reductase with both substrates to that observed with reduced DBH2. Similarly, addition of these coenzyme Q analogues increased 2-3-fold the rate of cytochrome c reduction in mitochondria from wild-type cells, suggesting that the pool of coenzyme Q in the membrane is limiting for electron transport in the respiratory chain. Preincubation of mitochondria from the Q-deficient yeast cells with DBH2 at 25 degrees C restored electrogenic proton ejection, resulting in a H+/2e- ratio of 3.35 as compared to a ratio of 3.22 observed in mitochondria from the wild-type cell. Addition of succinate and either coenzyme Q6 or DB to mitochondria from the Q-deficient yeast cells resulted in the initial reduction of cytochrome b followed by a slow reduction of cytochrome c1 with a reoxidation of cytochrome b. The subsequent addition of antimycin resulted in the oxidant induced extrareduction of cytochrome b and concomitant oxidation of cytochrome c1 without the "red" shift observed in the wild-type mitochondria. Similarly, addition of antimycin to dithionite-reduced mitochondria from the mutant cells did not result in a red shift in the absorption maximum of cytochrome b as was observed in the wild-type mitochondria in the presence or absence of exogenous coenzyme Q analogues.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008831 TI - Electron spin resonance and magnetic relaxation studies of gadolinium(III) complexes with human transferrin. AB - A human serum transferrin complex was prepared in which Gd(III) was substituted for Fe(III) at the two metal-binding sites. Characteristic changes upon metal binding in both the UV absorption of ligated tyrosines and the solvent proton longitudinal magnetic relaxation rates demonstrated 2/1 metal stoichiometry and pH-dependent binding constants. Binding studies were complicated both by binding of Gd(III) to nonspecific sites on transferrin at pH less than or equal to 7 and by complexation of the Gd(III) by the requisite bicarbonate anion at pH greater than or equal to 6.0. A unique Gd(III) electron spin resonance spectrum, with a prominent signal at g = 4.96, was observed for the specific Gd(III)-transferrin complex. The major features of this spectrum were fit successfully by a model Hamiltonian which utilized crystal field parameters similar to those determined for Fe(III) in transferrin [Aasa, R. (1970) J. Chem. Phys. 52, 3919-3924]. The magnetic field dependence of the solvent proton relaxation rate was measured as a function of both pH and metal ion concentration. An observed biphasic dependence of the relaxation rate on metal concentration is attributed to either sequential metal binding to the two iron-binding sites with different relaxation properties or random binding to two sites that are similar but show conformationally induced changes in relaxation properties as the second metal is bound. The increase in the solvent proton relaxation rate with pH is consistent with a model in which a proton of a second coordination sphere water molecule is hydrogen bonded to a metal ligand which becomes deprotonated at pH 8.5. PMID- 3008832 TI - Measurement of the proton-motive stoichiometry of the respiratory chain of rat liver mitochondria: the effect of N-ethylmaleimide. AB - The apparent proton-motive stoichiometry as measured by the oxygen-pulse technique in KCl medium is depressed by the rapid uptake of inorganic phosphate, unless endogenous phosphate is depleted or uptake is inhibited. In sucrose or choline chloride media, where the internal pH is more acid than in KCl media, uptake may be greatly diminished. In the absence of significant phosphate uptake, the observed stoichiometry of around 8, obtained with no added substrate or respiratory inhibitors, appears to be characteristic of NADH oxidation without significant participation of the proton-translocating NAD(P) transhydrogenase. A mechanistic stoichiometry of at least 8 is indicated. PMID- 3008833 TI - Tryptic modification of red-cell sodium pump behaviour. AB - Inside-out membrane vesicles derived from human red cells were used to probe the effects of controlled tryptic digestion on the sodium pump as it exists in situ. Digestion of the enzyme in its E1 conformation resulted in several alterations which are generally similar to those reported for the purified kidney enzyme, namely (i) greater loss in overall hydrolytic activity compared to level of phosphoenzyme intermediate and (ii) cleavage of the alpha-subunit by trypsin as well as chymotrypsin at the cytoplasmic surface to yield a fragment of approx. 78 kDa. Tryptic digestion effected similar rates of inactivation of pump-mediated Na+-K+(Rb+) exchange, (ATP- plus ADP)-dependent Na+-Na+ exchange and, in the absence extracellular alkali cation, 'uncoupled' Na+ flux (Na+/0 flux). Alteration in the Na+:Rb+(K+) stoichiometry following trypsin cleavage could not be detected. The conformational transitions of phosphoenzyme and dephosphoenzyme are affected similarly by trypsin, as evidenced by similar inactivation rates of reactions through the 'forward' sequence involving the E1P to E2P transition as well as through the 'reserve' sequence involving the E1 to E2 transition. PMID- 3008834 TI - Triton solubilization of proteins from pig liver mitochondrial membranes. AB - Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X 100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined. PMID- 3008835 TI - Cation sites, spermine, and the reaction sequence of the (Na+ + K+)-dependent ATPase. AB - Spermine, at 0.3 mM, inhibits the K+-nitrophenyl phosphatase activity of a dog kidney (Na+ + K+)-ATPase preparation, increasing the K0.5 for K+, reducing the Km for substrate, and affecting little the inhibition by Na+. These actions can be attributed, in a model of the phosphatase reaction, to parallel decreases in affinity for K+ and Na+ at their cytoplasmically accessible sites. In the (Na+ + K+)-ATPase reaction, spermine increases the K0.5 for Na+ and, to a lesser degree, the K0.5 for K+ as activators. With spermine, the double-reciprocal plots of velocity vs. ATP concentration (in the range 0.3-3 mM), at fixed levels of K+ (from 1 to 10 mM), remain parallel but are rotated clockwise and spread somewhat, reflecting stimulation at low ATP concentrations and inhibition at high ATP but low KCl concentrations. These actions can be attributed, in a steady-state ping pong model of the ATPase reaction, solely to decreased rates of binding of Na+ and K+ to their sites, with major effects at the cytoplasmically accessible sites for Na+ (acceptance) and K+ (discharge), and with a lesser effect at the extracellularly accessible sites for K+ (acceptance). On these grounds, spermine is a highly specific and potentially valuable reagent for studying the reaction. Furthermore, the model for K+-ATP interactions not only supports a specific reaction sequence (K+ addition, Pi release, ATP addition, K+ release) but also argues against the availability of low-affinity substrate sites except during sharply restricted segments of the reaction sequence, thereby favoring proposals that the low-affinity substrate sites are transformed into high-affinity substrate sites with the E2 to E1 conformational change. PMID- 3008836 TI - Demonstration of plasma-membrane adenosine diphosphatase activity in rat lung. AB - The adenosine diphosphatase (ADPase) activity of rat lung has been investigated. Subcellular fractionation of lung tissue homogenates by sucrose density gradient centrifugation has shown the ADPase activity to be associated with the plasma membrane. ADPase was solubilised from the membranes and fractionated by ammonium sulphate precipitation to separate a specific, low-Km ADPase from non-specific alkaline phosphatase activity. The solubilised ADPase has a Km of 50 microM at pH 7.5 and appears to be distinct from ATPase. PMID- 3008837 TI - Changes in membrane polypeptides, polyphosphoinositides and phosphatidate in dense fractions of sickle cells. AB - When sickle erythrocytes were fractionated on discontinuous isotonic stractan gradients the denser fractions, which were rich in irreversibly sickled cells contained less polyphosphoinositides and more phosphatidate than either lighter sickle cell fractions or normal cells. These changes could be due to activation of a polyphosphoinositide phosphodiesterase in the denser cells. Membrane polypeptide analysis of the denser fractions also showed a marked depletion of band 4.1 and a protein of molecular mass about 110 kDa but an increased amount of a 180 kDa polypeptide which might be a breakdown product of ankyrin. These biochemical alterations could be consequences of Ca2+ accumulation in the denser sickle cells and may contribute to the structural alterations which give rise to irreversibly sickled cells. PMID- 3008838 TI - Inhibition of phosphate transport in human erythrocytes by 7-chloro-4-nitrobenzo 2-oxa-1,3-diazole (NBD-Cl). AB - Treatment of intact human erythrocytes with 7-chloro-4-nitrobenzo-2-oxa-1,3 diazole (NBD-Cl) leads to inhibition of anion transport as measured by [32P]phosphate exchange for intracellular chloride. Inhibition is rapid at 37 degrees C (80% inhibition, 1.7 mM NBD-Cl, 3 min, pH 6.9) and not reversed by washing the cells with 1% bovine serum albumin in isotonic sucrose citrate buffer. Pretreatment of cells with N-ethylmaleimide and p chloromercuribenzenesulfonic acid enhanced transport inhibition by NBD-Cl. Transport inhibition caused by brief incubations of erythrocytes with NBD-Cl could be almost completely reversed with dithiothreitol or beta-mercaptoethanol. Prolonged incubation (60 min, 37 degrees C, pH 6.4, sucrose-citrate buffer) following NBD-Cl treatment leads to partial reversal of transport inhibition. The residual inhibition is then only partially reversed by dithiothreitol treatment. Reversal of transport inhibition of dithiothreitol or beta-mercaptoethanol may be prevented by incubation of the erythrocytes with sodium dithionite. Phosphate transport was readily inhibited by other tyrosine-directed reagents, tetranitromethane (55% inhibition, 1.6 mM, 3 min, 37 degrees C, pH 8.3 in sucrose citrate medium) and p-nitrobenzene sulfonyl fluoride (31% inhibition, 1.8 mM, 3 min, 37 degrees C, pH 8.1 in sucrose-citrate medium) but not by N-acetylimidazole (10% inhibition, 37.5 mM, 30 min, 37 degrees C, pH 7.5). These results suggest that NBD-Cl inhibits anion exchange by two mechanisms; a rapid inhibition reversible by sulfhydryl reagents, possibly due to modification of a tyrosine residue(s), and a slower irreversible inhibition due to modification of an essential amino group in the transporter. PMID- 3008839 TI - A comparison of brush-border membranes prepared from rabbit small intestine by procedures involving Ca2+ and Mg2+ precipitation. AB - Brush-border membranes were isolated from rabbit small intestine by procedures involving precipitation of undesired membranes with either 10 mM MgCl2 or 10 mM CaCl2. The membranes were compared on the basis of marker enzyme content and lipid composition. Ca2+-prepared membranes displayed a greater enrichment of alkaline phosphatase and sucrase activity compared to homogenate than did the Mg2+-prepared membranes. The former also displayed an impoverishment of (Na+ + K+)-ATPase activity, the specific activity of which increased several-fold in Mg2+-prepared membranes. Membranes prepared with Ca2+ were characterized by a lower phosphoacylglycerol-protein ratio and a higher phosphatidylethanolamine phosphatidylcholine ratio. Although lysophosphoacylglycerols accounted for about 6% of the total phospholipids in these membranes compared to 2% in Mg2+-prepared membranes, the free fatty acid content was similar in both types of membranes. It was concluded that Ca2+ prepared membranes were less contaminated by basolateral membranes than were Mg2+-prepared membranes and the use of Ca2+ did not notably enhance degradation of endogenous lipids by brush-border membrane phospholipase A. PMID- 3008840 TI - Preparation and characterization of F-protein vesicles isolated from Sendai virus by means of octyl glucoside. AB - We have demonstrated that Triton X-100 is always present in F-protein vesicles at concentrations that can provoke cell lysis. In order to avoid any misinterpretation of the fusogenic capacity of this protein, we solubilized the Sendai virus using octyl glucoside, which can be totally removed from the F protein preparation in less than 16 h by dialysis in the presence of absorbent beads. F-glycoprotein preparations preserved their ability to lyse erythrocytes in the presence of lectins and to induce cell-vesicle fusion as demonstrated by ESR studies. These vesicles were characterized by electron microscopy and SDS polyacrylamide gel electrophoresis. Lipid analysis of these preparations by thin layer chromatography indicated that they had the same proportion of lipids as virus envelopes, with slight variations in the sphingomyelin content and the cholesterol/phospholipid molar ratio. F-protein vesicles of different sizes can be obtained by adding exogenous lipids before detergent removal. The hemolytic activity of the vesicles was retained over a large range of lipid concentrations. We conclude that F-protein vesicles prepared with octyl glucoside are convenient tools for studying the fusogenic mechanism of this protein and improving the fusion process between liposomes and cells. PMID- 3008841 TI - Effects of the ATP, ADP and inorganic phosphate on the transport rate of the Na+,K+-pump. AB - (Na+ + K+)-ATPase from kidney outer medulla was incorporated into artificial dioleoylphosphatidylcholine vesicles. In the reconstituted system the pump can be activated by adding ATP to the external medium. ATP-driven potassium extrusion by the Na+,K+-pump was studied using a voltage-sensitive dye in the presence of valinomycin. ADP strongly reduced the turnover rate of the pump with a concentration for half-maximal inhibition of cD,1/2 = 0.1 mM. cD,1/2 was found to be virtually independent of ATP concentration, indicating that the inhibition is non-competitive with respect to ATP. The non-competitive inhibition by ADP can be explained on the basis of the Post-Albers reaction cycle of the Na+,K+-pump, assuming that the main action of ADP is the reversal of the phosphorylation step. A similar 'product inhibition' was observed with inorganic phosphate, but at much higher concentrations (cP,1/2 = 14 mM). PMID- 3008842 TI - Solubilized (Na+ + K+)-ATPase from shark rectal gland and ox kidney--an inactivation study. AB - The bi-exponential time-course of detergent inactivation at 37 degrees C of C12E8 solubilized (Na+ + K+)-ATPase from shark rectal glands and ox kidney was investigated. The data for shark enzyme, obtained at detergent/protein weight ratios between 2 and 16, are interpreted in terms of a simple model where the membrane bound enzyme is solubilized predominantly as (alpha-beta)2 diprotomers at low detergent concentrations and as alpha-beta protomers at high C12E8 (octaethyleneglycoldodecylmonoether) concentrations. It is observed that the protomers are inactivated 15-fold more rapidly than the diprotomers, and that the rate of inactivation of both oligomers is proportional to the detergent/protein ratio. Inactivation of kidney enzyme was biexponential with a very rapid inactivation of up to 40% of the enzyme activity. The observed rate of inactivation of the slower phase varied with the detergent/protein ratio, but the inactivation pattern for the kidney enzyme could not readily be accommodated within the model for inactivation of the shark enzyme. The rates of inactivation at 37 degrees C were about the same in KCl and NaCl, i.e., in the E2(K) and E1 X Na forms, for both enzymes. PMID- 3008843 TI - Identification of the sites on rabbit skeletal muscle protein phosphatase inhibitor-2 phosphorylated by casein kinase-II. AB - Inhibitor-2 was phosphorylated by casein kinase-II in vitro at a rate similar to that of glycogen synthase, a physiological substrate of this protein kinase. The major phosphorylation sites were identified as serines-86, -120 and -121, the peptide containing serines-120 and -121 being labelled about 2.5-fold more rapidly than that containing serine-86. The 13 residues C-terminal to serine-121 (SGEEDSDLSPEERE) contain seven acidic amino acids, while the six residues following serine-86 (SDTETTE) contain three. These results are consistent with the known specificity requirements of casein kinase-II. The three serines are C terminal to the threonine (residue 72) whose phosphorylation by glycogen synthase kinase-3 is potentiated by prior phosphorylation with casein kinase-II. This reinforces the view that a C-terminal phosphoserine residue is important for the specificity of glycogen synthase kinase-3. Identification of the residues phosphorylated by casein kinase-II will facilitate further studies on the in vivo phosphorylation state of inhibitor-2. PMID- 3008844 TI - X-ray studies of nucleotide binding and pyridoxal phosphate labeling of the gene 5 DNA unwinding protein. AB - X-ray diffraction studies have been carried out using difference Fourier methods to evaluate the reaction or interaction of an affinity label and 5'-phosphate nucleotides with the gene 5 DNA binding protein in the crystalline state. In the first case the crystalline protein was reacted with pyridoxal phosphate. Pyridoxal phosphate, which has served as an affinity label for nucleotide binding sites on other enzymes, demonstrated a major site of substitution at the center of the protein's DNA binding cleft adjacent to lysine 46 as well as two other reaction sites near residues implicated in DNA binding. Difference Fourier maps of crystals exposed to 5'-dAMP, 5'-dCMP and 5'-dTMP indicated that phosphate groups were associated with most lysine and arginine side-chains on the surface of the protein but that the nucleoside portion of the ligands were generally disordered. In several cases, however, more specific binding of the nucleotides appeared to have occurred and these sites were primarily within the proposed DNA binding cleft of the protein. In particular, binding was observed near tyrosine 34, phenylalanine 73 and within the curl of the DNA binding loop containing tyrosine 26. PMID- 3008845 TI - The effects of salts and amino group modification on the iron binding domains of transferrin. AB - The origins of the effects of salts on the properties of the iron binding sites of transferrin have been investigated. The chaotropically distinct salts NaCl and NaClO4 each induce characteristic changes in the EPR lineshapes of the N- and C terminal Fe3+ binding domains, respectively. To a good approximation the perturbed EPR spectrum of diferric transferrin in the presence of salts is the sum of the EPR spectra of the N- and C-terminal monoferric proteins. Acetylation of amino groups causes spectral and kinetic changes in the protein similar to those induced by NaClO4. Thus, both acetylation and NaClO4 cause a loss of structure in the g' = 4.3 EPR signal of the N-terminal domain, and both retard iron removal from this domain. In contrast, iron removal from the C-terminal domain is accelerated by acetylation or the presence of NaClO4. These observations are ascribed to charge effects of lysine residues which are probably in the vicinity of the iron binding sites. PMID- 3008846 TI - Evidence that centre 2 in Escherichia coli fumarate reductase is a [4Fe 4S]cluster. AB - Redox titrations of the iron-sulphur clusters in fumarate reductase purified from Escherichia coli, monitored by ESR spectroscopy, identified three redox events, similar to those observed in other fumarate reductases and succinate dehydrogenases: Centre 1, a [2Fe-2S] cluster, at g = 2.03, 1.93, appeared on reduction with Em = -20 mV. Centre 3, probably a [3Fe-xS] cluster, at g = 2.02 appeared in the oxidized state with Em = -70 mV. Centre 2 has been observed as an increase in the electron-spin relaxation of Centre 1. It titrates as an n = 1 species with Em = -320 mV, but in our hands did not appear to contribute significant intensity to the g = 2.03, 1.93 signal. It therefore appears to be an additional centre which undergoes spin-spin interaction with Centre 1. The reduction of Centre 2 coincided with the appearance of an extremely broad ESR spectrum, observed at temperatures below 20 K, with features at g = 2.17, 1.9, 1.68. The broad signal was observed in both soluble and membrane-bound preparations. Its midpoint potential was -320 mV. Its integrated intensity was approximately equal to that of Centre 1, if its broad outer wings were taken into account. Consideration of the ESR properties of this signal, together with the amino acid sequence of the frdB subunit of the enzyme, indicates that Centre 2 is a [4Fe-4S] cluster which, in its reduced state, enhances the spin relaxation of the [2Fe-2S] Centre 1. PMID- 3008847 TI - Inhibition of angiotensin-converting enzyme by des-Leu10-angiotensin I: a potential mechanism of endogenous angiotensin-converting enzyme regulation. AB - Des-Leu10-angiotensin I is a nonapeptide generated from angiotensin I by the action of carboxypeptidase-like activities residing in the human platelet and mast cell. This nonapeptide was found to inhibit rabbit lung angiotensin converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) with a Ki of 3.1 X 10(-7) M. The mechanism of inhibition was competitive. Inhibition of human serum angiotensin-converting enzyme by des-Leu10-angiotensin I was comparable in magnitude to inhibition by bradykinin and angiotensin III. These results suggest that limited proteolysis of angiotensin I by cells resident in vascular tissue may result in the generation of an endogenous inhibitor of angiotensin-converting enzyme. Such pathways may play roles in controlling levels of vasoactive peptides at local vascular sites. PMID- 3008848 TI - The superoxide dismutase activity of myeloperoxidase; formation of compound III. AB - The reaction of superoxide anions with myeloperoxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7), which results in the formation of Compound III of myeloperoxidase, was investigated. It is shown that myeloperoxidase has a high affinity for superoxide anions because formation of Compound III was only partially inhibited by high concentrations of superoxide dismutase. Furthermore, when superoxide anions were generated in a mixture of both cytochrome c and myeloperoxidase in the absence of Cl-, only Compound III was formed and reduction of cytochrome c was not observed. In the presence of Cl-, Compound III was also formed and reduction of cytochrome c was inhibited. From the results described in this paper we conclude that Compound III is able to react with superoxide anions, probably resulting in formation of an intermediate (Compound I) which is catalytically active in the oxidation of Cl- to yield hypochlorous acid (HOCl). Because Compound III of myeloperoxidase is formed in phagocytosing neutrophils (Winterbourn, C.C., Garcia, R.C. and Segal, A.W. (1985) Biochem. J. 228, 583-592) we propose that, in vivo, myeloperoxidase also acts as a superoxide dismutase, and via formation of Compound I uses superoxide anions in the formation of HOCl. PMID- 3008849 TI - Pinocytosis and phagocytosis: the effect of size of a particulate substrate on its mode of capture by rat peritoneal macrophages cultured in vitro. AB - Both phagocytosis (of particles) and pinocytosis (of solutes) occur in macrophages. It is not known, however, whether particles, if they are small enough, can enter by pinocytosis, nor whether there is a minimum size of particle capable of triggering phagocytic uptake. These questions have been investigated by studying, in vitro, the uptake by rat peritoneal macrophages of particles ranging in diameter from 30 nm to 1100 nm. Percoll (30 nm diameter) and polystyrene beads (100, 300, 600, 800 or 1100 nm diameter) were 125I-iodinated and their uptake by macrophages was measured in the absence or presence of metabolic and cytoskeletal inhibitors. Since uptake, expressed as an Endocytic Index (microliter/10(6) cells per h), increased steadily with the duration of incubation and was inhibited by low temperature or metabolic inhibitors, it was concluded that true endocytosis, and not a superficial cell-association, was being measured. Rates of clearance increased with increasing particle diameter. The rate of uptake of Percoll was 10-times, and of 100 nm polystyrene beads 100 times, the rate of fluid-phase pinocytosis, as measured by the uptake of 125I labelled polyvinylpyrrolidone. Polystyrene beads of 1100 nm diameter were captured at 700-times this rate. The differential effects of colchicine and cytochalasin B on the uptake of 125I-labelled polyvinylpyrrolidone and of 1100 nm polystyrene beads were taken as indicators of their effects on pinocytosis and phagocytosis respectively. It is concluded that Percoll, although particulate, is captured by pinocytosis. The pattern of inhibition of uptake of polystyrene particles suggests that there is no radical discontinuity between pinocytic and phagocytic uptake, but that the contribution of phagocytosis steadily increases with increasing particle diameter. The results are discussed. PMID- 3008850 TI - Alterations of the microsomal glucose-6-phosphatase system evoked by ferrous iron and haloalkane free-radical-mediated lipid peroxidation. AB - Alterations of catalytic activities of the microsomal glucose-6-phosphatase system were examined following either ferrous iron- or halothane (CF3CHBrCl) and carbon tetrachloride (CCl4) free-radical-mediated peroxidation of the microsomal membrane. Enzyme assays were performed in native and solubilized microsomes using either glucose 6-phosphate or mannose 6-phosphate as substrate. Lipid peroxidation was assessed by the amounts of malondialdehyde equivalents formed. Regardless of whether the experiments were performed in the presence of NADPH/Fe3+, NADPH/CF3CHBrCl, or NADPH/CCl4, with the onset of lipid peroxidation, mannose-6-phosphatase activity of the native microsomes increased immediately, while further alterations in catalytic activities were only detectable when lipid peroxidation had passed characteristic threshold values: above 2 nmol malondialdehyde/mg microsomal protein, glucose-6-phosphatase activity of the native microsomes was lost, and at 10 nmol malondialdehyde/mg microsomal protein, glucose-6-phosphatase and mannose-6-phosphatase activity of the solubilized microsomes started to decline. It is concluded that the latter alterations are due to an irreversible damage of the phosphohydrolase active site of the glucose 6-phosphatase system, while the changes observed at earlier stages of microsomal lipid peroxidation may also reflect alterations of the transporter components of the glucose-6-phosphatase system. Virtually no changes in the catalytic activities of the glucose-6-phosphatase system occurred under anaerobic conditions, indicating that CF3CHCl and CCl3 radicals are without direct damaging effect on the glucose-6-phosphatase system. Further, maximum effects of carbon tetrachloride and halothane on lipid peroxidation and enzyme activities were observed at an oxygen partial pressure (PO2) of 2 mmHg, providing additional evidence for the crucial role of low PO2 in the hepatotoxicity of both haloalkanes. PMID- 3008851 TI - Defective chemiluminescence response in differentiated HL60 cells due to impaired degranulation. AB - In the presence of dimethyl sulfoxide, the promyelocytic leukemic cell line, HL60, differentiates into apparently mature polymorphonuclear leukocytes. When correlating the superoxide production from HL60 cells with the number of phagocytozing and NBT-positive cells, no difference was observed in comparison with normal peripheral blood leukocytes. In contrast, the luminol-dependent chemiluminescence was greatly impaired in the differentiated HL60 cells. Analysis of degranulation, i.e., release of myeloperoxidase and N-acetyl-beta glucosaminidase- and myeloperoxidase-mediated iodination by HL60 cells, suggested that the defective chemiluminescence response observed in HL60 cells may be due to impaired release of myeloperoxidase from azurophilic granulae. This may lead to impaired microbicidal activity in these cells. PMID- 3008852 TI - Human leukemic K562 cells: suppression of hemoglobin accumulation by a monoclonal antibody to human transferrin receptor. AB - The receptor for transferrin plays an important role both in tumor cell growth and in hemoglobin synthesis. In this paper, we demonstrate that the monoclonal antibody 42/6 to human transferrin receptor inhibits iron uptake in the human leukemic K562 cell line and suppresses hemoglobin accumulation in K562 cells induced to erythroid differentiation by butyric acid. In contrast, only slight inhibitory effects were observed on cell proliferation of both uninduced and erythroid-induced K562 cells treated with the 42/6 monoclonal antibody. In addition, the 42/6 monoclonal antibody to human transferrin receptor does not inhibit butyric acid-induced accumulation of gamma-globin mRNA. The effect of the 42/6 monoclonal antibody on hemoglobin synthesis appears to be restricted to human cell lines, as murine Friend erythroleukemic cells undergo erythroid differentiation when cultured in the presence of hexamethylenebisacetamide plus the 42/6 monoclonal antibody. The findings reported in this paper suggest (a) a dissociation of iron transport and accumulation of heme molecules from the expression of globin genes and (b) a different requirement of iron uptake by different iron-dependent functions such as cell proliferation and hemoglobin expression. PMID- 3008853 TI - Possible involvement of angiotensin I-converting enzyme in the inactivation of bradykinin by intact macrophages. AB - As intact macrophages inactivated bradykinin, the subcellular localization of the bradykinin-inactivating activity was studied using guinea-pig macrophages. The bradykinin-inactivating activity was found to be present in membrane and cytosol fractions but not in granular and nuclear fractions. The bradykinin-inactivating activity of the membrane fraction was inhibited by captopril, a specific inhibitor of angiotensin I-converting enzyme, whereas that of the cytosol fraction was hardly inhibited by various proteinase inhibitors used. Angiotensin I-converting enzyme activity was located predominantly in the membrane fraction and its activity was inhibited by captopril. Angiotensin I-converting enzyme activity measured with a synthetic substrate was competitively inhibited by bradykinin, suggesting that bradykinin is a possible substrate for macrophage angiotensin I-converting enzyme. When macrophages were modified chemically by diazotized sulfanilic acid, a poorly permeant reagent, both the bradykinin inactivating activity and the angiotensin I-converting enzyme activity of macrophages decreased significantly without any inhibition of the cytosol bradykinin-inactivating activity. These findings seem to suggest that the angiotensin I-converting enzyme would be responsible for the inactivation of bradykinin in intact macrophages. PMID- 3008854 TI - Interaction of epidermal growth factor with specific binding sites of enterocytes isolated from rat small intestine during development. AB - Isolated enterocytes from rat small intestine were characterized for their specific binding of epidermal growth factor (EGF). Intestinal epithelial cells were isolated at 4 degrees C to minimize the loss of receptor sites during the isolation procedure. 125I-labelled EGF binding to enterocytes from adult rats was found to be specific, saturable, temperature dependent and trypsin sensitive. Binding performed in the presence of a lysosomotropic agent (NH4Cl) increased the time required to reach maximal binding at 25 degrees C. NH4Cl had no significant effect on the time-course of EGF binding at 4 degrees C and 37 degrees C. A Scatchard plot showed a curvilinear relationship indicating that EGF binds to enterocytes with more than one binding site. Developmentally, enterocytes from fetuses and pups showed characteristic temperature dependence and trypsin sensitivity, but with different levels of binding to EGF. Specific EGF binding was demonstrably higher in enterocytes from small intestine of term fetuses. EGF binding to isolated enterocytes declined rapidly after birth, and the level stayed fairly constant thereafter. Pretreatment of enterocytes from fetal intestine with mature rat milk led to a dose-dependent decrease in EGF binding. These results suggest the presence of endogenous milk factors that modify EGF binding and account for, at least partly, the observed rapid decrease of EGF binding after birth. PMID- 3008855 TI - Effects of [1-N alpha-trinitrophenylhistidine, 12-homoarginine]glucagon on cyclic AMP levels and free fatty acid release in isolated rat adipocytes. AB - [1-N alpha-Trinitrophenylhistidine, 12-homoarginine]glucagon (THG) stimulated, in a concentration-dependent fashion, lipolysis (2-fold) and cyclic AMP accumulation (50% over basal) in isolated rat adipocytes, but was much less effective than glucagon, which stimulated lipolysis 4-fold and cyclic AMP accumulation 10-15 fold. THG displaced to the right the concentration-response curves for glucagon and diminished in a concentration-dependent fashion the effects of a fixed concentration of glucagon. The data indicate that THG is a mixed agonist antagonist (partial agonist) in isolated rat fat cells. PMID- 3008857 TI - [The effect of highly-dispersed zinc powder on the functional state of blood components and rat liver mitochondria]. AB - The functional state of blood components and liver mitochondria have been investigated by means of ESR method and atomic adsorption spectroscopy. The experimental data indicated that injection of highly dispersed Zn-powder in the concentration of 5 mg/kg of weight did not result in disturbance of mitochondria bioenergetic functions, although the functional state of blood components and the level of metal ions in it were changed. PMID- 3008856 TI - [Conformation and reduction-oxidation properties of cytochrome c incorporated into reversed micelles of a surfactant in an organic solvent]. AB - Changes observed in CD- and absorption spectra of cytochrome c solubilized in reversed micelles AOT showed significant structural transformations of protein in the region of the active centre and particularly revealed a replacement of the sixth ligand of heme iron. These changes also affected the redox properties of cytochrome c. PMID- 3008859 TI - [Changes in the levels of paramagnetic centers in the mouse liver and a mathematical model of synchronized auto-oscillations]. AB - Investigations of circadian rhythms in paramagnetic particles (free radicals and metal complexes) (PP) concentration variations in tissues are under continuation. A mathematical model advanced considers PP biorhythms as self-induced oscillations subjected to weak synchronization by an external source, by geomagnetic field intensity daily variations for example. By computation parameters of the model were obtained giving good agreement between the theory and experimental data. Due to the mathematical model the amplitude of external synchronizer influence is ten times less than the self-amplitude of PP biorhythm. The experiment was performed on the normal mouse liver. ESR signals g = 1.94, 2.00 and 2.25 were studied. PMID- 3008858 TI - [The role of the electromechanical and reaction-diffusion system of intraneuronal information processing in brain function]. AB - The experimental data of previous papers are considered as a basis for the hypothesis about intraneuronal system controlled by cyclic nucleotides and changing the membrane permeability upon creating the generatory potential. This system is suggested to be an extremal molecular regulator in which the price of action per single operation approximates the physical limit. The electro mechanical intraneuronal system is capable of solving multidimensional physical problems by means of molecular "digital" hypersound holo-gram coded by DNA molecular text which is an image of functions of target search. PMID- 3008860 TI - [Identification of electron spin resonance signals of ribonucleotide reductase in animal tissues with high proliferative activity]. AB - A new ESR signal (doublet with hyperfine splitting of 20 gauss and g = 2.005) was found for animal tissues having high level of proliferative activity. This signal is shown to be due to radical enzyme ribonucleotide reductase. The results reported for spleen development suggest that there is a correlation between changes in the enzyme activity during the first week of neonatal development and intensity of ESR doublet signal. PMID- 3008861 TI - [Mechanism of the activating effect of detergents and chelating agents on the Na, K-ATPase activity of erythrocyte ghosts]. AB - The reasons for differences in the Na,K-ATPase activity in rat erythrocyte ghosts obtained by hypoosmotic hemolysis in 10 mM Tris-HCl buffer pH 7.6 in the absence ("Tris-ghosts") and presence ("EDTA-ghosts") were investigated. Structurally different detergents (Triton X-100, Tween-20 and sodium deoxycholate) taken at optimal concentrations increased the enzyme activity in a similar way, i. e., 4 fold in "Tris-ghost" and by 30% in "EDTA-ghosts", the absolute activity of Na,K ATPase in both preparations being levelled out. In the absence of EDTA, only 50 60% of the maximal enzyme activity could be revealed. Thus, in non-nuclear erythrocyte ghosts the maximal Na,K-ATPase activity can be revealed only upon a combined use of a detergent and chelator. It is concluded that the activating effect of the detergents consisting in the increase of the membrane permeability is realized on the outer surface of the membrane, whereas that of EDTA is localized on its inner surface, which is probably due to the disintegration of the cytoskeleton as a result of attachment of membrane-bound Ca2+. PMID- 3008864 TI - [Characteristics of sulfhydryl groups of histone H3 from the calf thymus using mercury-containing nitroxyl radicals]. AB - The interaction between total histone and deoxyribonucleoprotein (DNP) preparations from calf thymus with mercury-containing nitroxyl radicals in low ionic strength solutions, 2 M NaCl and urea was investigated. It was found that the label is rapidly incorporated into the SH-groups of histone H3 to produce characteristic EPR signals. Titration of SH-groups within DNP demonstrated that in low ionic strength solutions only one SH-group (presumably, the SH-group of the cysteine residue in position 110) is accessible to the reagents. After dissociation by 2 M NaCl, two SH-groups become titrable; however, the EPR spectra point to differences in the conformational state of these two groups. In 4 M urea, these differences are compensated for by structural disintegration. The spin labels may be used for the analysis of SH-groups under different conditions and at different functional states of nucleoproteins. PMID- 3008862 TI - [Characteristics of an enzyme hydrolyzing the covalent bond between RNA and protein VPg of the encephalomyocarditis virus]. AB - The enzyme termed by us as uridilylpolynucleotide-(5'P----O)-tyrosine phosphodiesterase (Y-pUpN PDE) was isolated from mouse ascites Krebs II cells by ion-exchange and affinity chromatography. The enzyme was found to specifically split the natural covalent bond between VPg and EMC or polio viral RNAs. The enzyme is completely inactivated at 55 degrees C and partially by EDTA. The enzyme preparation isolated by the above-mentioned procedure is not homogeneous and contains inhibiting admixture(s). Possible role of the enzyme in living cells is discussed. PMID- 3008863 TI - [Dependence of hormonal stimulation of adenylate cyclase on the fraction of plasma membrane accessible to the lateral transport of adenylate cyclase complex proteins]. AB - Hormonal activation of the adenylate cyclase complex is associated with lateral mobility of proteins constituting this complex. Modification of interaction of the adenylate cyclase complex proteins due to variations in the "fluid" lipid fraction in cell membranes was studied. The decrease of percentage of "fluid" lipids in rat reticulocyte plasma membranes resulted in a decrease (up to a full stop) of interaction of beta-adrenoreceptors with regulatory N-proteins. The interaction of N-proteins with catalytic proteins was also blocked. On the other hand, an increase in the "fluid" lipid fraction led to a more intensive interaction. The observed phenomena do not result from functional damages of interacting proteins. Analysis of experimental results within the framework of the percolation theory suggests that hormonal activation of the adenylate cyclase complex depends on the "fluid" lipid fraction in the membrane and that the interacting proteins diffuse at distances comparable with the size of the cell membrane. The intrinsic activity of the beta-agonist isoproterenol changes from 1 to 0, depending on the "fluid" lipid fraction. The experimental data also suggest that there are no beta-receptors precoupled with N-proteins in rat reticulocyte membranes in vitro. PMID- 3008865 TI - Differential responsivity of verbal and visual recognition memory to physostigmine and ACTH. PMID- 3008866 TI - The role of carbohydrates in sperm-egg interaction in rats. AB - The first step in fertilization is the interaction of the capacitated sperm with the zona pellucida. It has been proposed that the initial interaction, as in other types of cell adherence, is due to complementary interacting sites on the opposing surfaces of the gametes. This work intended to investigate the role of carbohydrates in sperm-egg binding in rats. Ejaculated sperm was collected from uterine horns of mated females. The sperm was suspended in Rat Fertilization Medium at final concentrations of 3-7 times 10(5) sperm/ml. After 5 1/2 h of sperm incubation, eggs were added to the sperm suspensions concomitantly with various carbohydrates to achieve a final concentration of up to 50 mM. The eggs were separated after 30 min, and the number of sperm bound to the zona pellucida was counted. Among a variety of monosaccharides tested at 50 mM concentration, it was found that alpha-methyl-mannoside was the most potent inhibitor (producing 80% inhibition); less potent was D-mannose and even less, L-fucose. A combination of alpha-methyl-mannoside and L-fucose showed a synergistic effect. Mannan was not more effective as an inhibitor than the monosugar mannose, while fucoidin was extremely potent, causing over 90% inhibition of binding at 0.1%. We assume the presence of macromolecules containing sugars on the zona pellucida because inhibition of sperm binding to this layer was observed: a) after preincubation of mannan or fucoidin with sperm, but not with the eggs; and b) after pretreatment of the egg with specific enzymes. The results obtained in this study in the rat are consistent with the hypothesis that carbohydrates are critical for the sperm egg interaction. PMID- 3008867 TI - Blockade of first ovulation in pubertal rats by delta-9-tetrahydrocannabinol: requirement for advanced treatment due to early initiation of the critical period. AB - Successful blockade of ovulation in pubertal rats by delta-9-tetrahydrocannabinol (THC) required earlier treatment during proestrus than was required in adults under the same conditions. Only 1 of 8 adult rats ovulated after treatment with THC (10 mg/kg body weight, i.p.) at 1400 h proestrus, whereas 77% of pubertal rats released full sets of ova following similar treatment during proestrus of the first or second vaginal cycle. When treatment of pubertal rats was advanced to 1300 h, only 2 of 10 THC-treated rats exhibited full ovulation, an incidence significantly lower than the 80% ovulation rate observed in vehicle-treated animals (p less than 0.05). To determine whether the requirement for earlier THC treatment in pubertal rats was related specifically to THC or reflected possible age-associated differences in timing of the critical period, the ovulation blocking efficacy of atropine sulfate (ATR) was tested in pubertal rats for comparison with that of THC. The serum concentrations of luteinizing hormone (LH) during the first proestrus (1200-1900 h) were determined in pubertal rats that remained untreated. The incidence of ovulation in rats treated with ATR (350 mg/kg, s.c.) at 1400 h proestrus was not significantly reduced from that in vehicle-treated rats; however, after ATR treatment at 1300 h, only 2 of 11 animals released full sets of ova whereas all vehicle-treated rats ovulated (p less than 0.025). The mean serum LH concentration in untreated pubertal rats was not significantly increased over baseline at 1300 h proestrus, but was markedly elevated by 1400 h (1009 +/- 375 ng/ml; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008868 TI - Dolichol kinase activity in the developing rat testis. AB - Dolichyl phosphate concentrations, a primary factor in regulating the rate of N glycosidically linked glycoprotein synthesis, are dependent upon a cytidine triphosphate (CTP)-dependent dolichol kinase. This study examines dolichol kinase in rat testicular microsomes and defines assay conditions. As with dolichol kinases from other tissues, addition of 2-mercaptoethanol increased activity 60%. Inclusion of NaF, an inhibitor of testicular dolichyl phosphate phosphatase activity, also resulted in a 38% increase in activity. Triton X-100 was necessary for phosphorylation of both endogenous and exogenous dolichol; however, concentrations of detergent in excess of 0.25-0.35% were inhibitory. A 2- to 5 fold stimulation of kinase activity was obtained by addition of 50-100 microM exogenous dolichol. The high level of nucleoside triphosphatase activity in testicular microsomes mandated the inclusion of high levels of uridine triphosphate (UTP) to protect the [gamma-32 P] CTP. Increasing UTP concentrations up to 50 mM resulted in increased product formation. A clear requirement for divalent cations was observed; 5 mM ethylenediaminetetraacetate (EDTA) abolished activity. The following order of cation effectiveness was observed: Mn greater than or equal to Ca greater than Cd greater than Zn much greater than Mg. Ten mM optima were established for Ca2+ and Mn2+; the presence of UTP, however, results in significantly reduced concentrations of free Ca2+. Ion combination studies demonstrated interactive inhibitory effects between Ca2+ and other stimulatory divalent cations. Addition of 2 microM brain calmodulin, in the presence of 10 mM Ca2+, resulted in a 75-100% stimulation of activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008869 TI - Gonadotropins and cyclic adenosine 3',5'-monophosphate (cAMP) alter the morphology of cultured human granulosa cells. AB - Morphological changes in human granulosa cells in culture were observed by phase, fluorescent, scanning electron and transmission electron microscopy following the addition of human chorionic gonadotropin (hCG), luteinizing hormone (LH), 8 bromocyclic adenosine 3',5'-monophosphate (cAMP) and cytochalasins B and D. In response to these agents, polygon-shaped granulosa cells with granular cytoplasm became rounded, leaving fingerlike processes attached to the substratum and adjacent cells. The changes in cell shape were accompanied by a centripetal movement of mitochondria and lysosomes to a perinuclear location. The morphological alterations appeared to be mediated by cyclic AMP and to be the result of a dismantling and reorganization of microfilament-containing stress fibers. Follicle-stimulating hormone (FSH), prolactin (PRL), growth hormone (GH), and human placental lactogen (hPL) did not provoke cell shape changes. We conclude that tropic hormones capable of stimulating progestin secretion by luteinized granulosa cells cause a change in cell structure in vitro which leads to a redistribution of organelles involved in steroid synthesis. The possible relationship of the cytoskeleton to steroidogenesis is considered. PMID- 3008870 TI - Isolation and characterization of three forms of luteinizing hormone from the pituitary gland of the horse. AB - Three isoforms of equine luteinizing hormone (eLH-A, eLH-B and eLH-C) have been isolated from horse pituitary glands. Separation was achieved on the basis of charge heterogeneity by ion-exchange chromatography. These charge differences were apparent after final purification, as determined by electrophoretic mobility on polyacrylamide disc gels (RF = 0.14, 0.19 and 0.26 for eLH-A, -B and -C, respectively). Apparent size differences were also noted between the isohormones by gel filtration on Sephadex G-100. Ve/Vo ratios for eLH-A, -B and -C were 1.72, 1.54 and 1.47, respectively. All 3 isoforms were found to contain an equivalent amount of hexose (9.0-9.2%). Isohormones eLH-B and eLH-C, however, possess more sialic acid than eLH-A (6.6-6.7%, vs. 4.5%). The eLH-A and eLH-B preparations contain a similar amount of hexosamine, which is slightly lower than the amount of eLH-C (8.8-9.1% vs. 11.2%). No differences were noted between the isohormones by rat Leydig cell LH bioassay, equine testis LH radioreceptor assay (RRA) or calf testis follicle-stimulating hormone (FSH) RRA. Slight, but nonsignificant, variations were noted between preparations in an eLH radioimmunoassay (RIA). Although chemical variations were detected between the eLH isoforms, no significant differences were observed in in vitro biological and immunological activities. The differences detected in sialic acid content raises the possibility that differences in in vivo clearance rates may exist. PMID- 3008871 TI - Complete analysis of the cytochrome components of beef heart mitochondria in terms of spectra and redox properties. The c1-cytochromes. AB - Using newer techniques for conducting and analyzing potentiometric titrations, we have studied the thermodynamic and spectral properties of cytochrome c1 in beef heart mitochondria. We find two species of cytochrome c1, both with n = 2 values for the number of electrons involved in their oxidation or reduction. One has an Em approximately 210 mV and a spectral peak near 555 nm and the other has an Em approximately 255 mV and a spectral peak nearer 553 nm. These Em values are pH independent in the range of pH 6 to 8. The Em and n values of these two components are indistinguishable from those of two species of cytochrome aa3 (i.e. spectral feature of 605 nm). PMID- 3008872 TI - Complete analysis of the cytochrome components of beef heart mitochondria in terms of spectra and redox properties. Cytochromes aa3. AB - Using newer techniques of data collection that accumulate entire spectra at a series of discrete voltages and newer techniques of analysis that utilize the additional data, we have re-examined the redox behavior and corresponding difference spectra of redox centers responsible for the alpha absorbance features of cytochromes aa3 in beef heart mitochondria. Our analysis reveals three Nernstian components with Em values of 200, 260, and 340 mV with n values of 2, 2, and 1, respectively. The maximum alpha absorbance in the difference spectra for each of these species is located at 602, 605, and 607 nm respectively. Titrations in the presence of carbon monoxide led to the identification of the lowest voltage species as cytochrome a3. The Em of the carbon monoxide-liganded species was not raised. This is contrary to the result expected when a ligand has a much stronger affinity for the reduced form of a redox couple than the oxidized form. It is, however, consistent with a proton-pumping model of cytochrome oxidase in which the binding of ligand results in the dissociation of protons. PMID- 3008873 TI - Analysis of the spectra and redox properties of pure cytochromes aa3. AB - The findings in the current studies with pure cytochrome aa3 confirm the findings in an accompanying paper pertaining to cytochrome aa3 in mitochondria (Reddy et al., 1985). In both cases, three Nernstian titrations are seen with Em values near 200, 260, 340 mV with n values of 2, 2, and 1. Similarly, the alpha absorption features of the difference spectra in both cases were centered near 602, 605, and 607 mn. The component with Em approximately 200 mV was identified as heme a3 on the basis of experiments conducted in an atmosphere of carbon monoxide, and in both cases, the carbon monoxide-liganded species did not display an elevated Em. In the current studies, unique Soret absorbance features are added to the difference spectra for the three Nernstian transitions. Specifically, absorption peaks at 429, 446, and 448 nm go with the alpha peaks seen respectively at 602, 605, and 607 nm. Evidence was presented to support the hypothesis that the redox state of heme alpha may control the redox potential of heme a3. PMID- 3008874 TI - Whither brown fat? PMID- 3008875 TI - Structural alterations of the human erythrocyte membrane upon influenza virus attachment. AB - Molecular events on the human erythrocyte membrane subsequent to influence virus binding were investigated by electron spin resonance (ESR) measurements after spin labeling of the cell membrane at different positions. Virus binding affected the glycocalyx structure as well as the physical state of the cytoskeleton at the inner leaflet, but not the lipid phase. A lateral reorganization of spin-labeled glycophorin was not indicated after virus attachment. PMID- 3008876 TI - [Mechanism of the suppression of the spinal pain syndrome by serotonin derivatives]. AB - The ability of serotonin derivatives to stimulate cAMP accumulation in isolated nerve terminals and lumbar enlargement of the spinal cord of normal rats was compared. The effect of the compounds on the intensity of spinal pain syndrome was also assessed. It has been established that substitutes injected into NH2 group of serotonin in 5-OH position attenuate the ability to stimulate cAMP accumulation in synaptosomes, with the effect more pronounced with substitutes of larger volume. A certain correlation between the ability of serotonin derivatives to stimulate adenylate cyclase in vivo and in vitro, on the one hand, and their analgetic effect, on the other hand, is suggested. PMID- 3008877 TI - [Interrelation between the natural antioxidant content and lipid viscosity in normal organelle membranes]. AB - Two systems regulating lipid peroxidation (LP) natural antioxidants and structural organization were studied in the liver mitochondria, microsomes and nuclear membranes, using chemiluminescent and spin labelling techniques. Mitochondrial lipids were most protected against LP and were characterized by the maximum content of effective antioxidants and high viscosity. Nuclear lipids were the least protected against the systems studied and had lower antioxidant efficiency and viscosity. Direct correlation between the content of natural antioxidants and lipid viscosity was established. PMID- 3008878 TI - [Nonuniform effect of prolonged haloperidol administration on the GABA(A) and benzodiazepine receptors in different parts of the brain]. AB - The experiments on male mice and rats have revealed reversed behavioral effects of muscimol and Ro 15-1788 after 15 days of haloperidol (0.25 mg/kg, twice daily) treatment. Muscimol (0.75 mg/kg), which depressed motor activity in saline pretreated mice, stimulated it after discontinuation of long-term haloperidol administration. Ro 15-1788 stimulating effect in saline-pretreated rats gave way to sedative effect following haloperidol withdrawal. Simultaneously, the number of 3H-muscimol and 3H-flunitrazepam binding sites was decreased in forebrain, but increased in hindbrain. It was suggested that GABAA and benzodiazepine receptors in forebrain and hindbrain play opposite (inhibiting and stimulating, respectively) functional roles in the regulation of behaviour. PMID- 3008879 TI - [Effect of leukotriene E4 on the central hemodynamics and vascular contractile activity]. AB - The effect of leukotriene E4 (LTE4) on the cardiac output and peripheral vascular resistance was studied. It was shown to damage the heart pump function and to cause spasms of peripheral resistive vessels. LTE4 was also found to induce constriction of heart and cerebral human arteries due to calcium entering myoplasm from the environment and intracellular depot. Low LTE4 concentrations enhanced vascular wall sensitivity to different vasoactive agents, including thromboxane A2. Prostacyclin appeared to be LTE4 antagonist, preventing its vasoconstrictive action. The role of LTE4 as a systemic vasoconstrictive agent is discussed. PMID- 3008880 TI - [Detection of the pro-opiomelanocortin peptide fragments--beta-endorphin and ACTH -in the adrenals of rats and mice by immunohistochemistry]. AB - Independent peptide fragments of pro-opiomelanocortin molecule, beta-endorphin and ACTH, have been detected immunohistochemically in the adrenal glands of rats and mice. Immunoreactive beta-endorphin and ACTH have been revealed in the adrenal medulla and reticular zone of the adrenal cortex. beta-endorphin and ACTH distribution patterns in adrenal sections were identical, which is indicative of the linked synthesis of these peptides in the adrenal gland. The data obtained suggest the existence of pituitary-independent mechanisms regulating corticosteroidogenesis in the adrenal gland, involving adrenal pro opiomelanocortin fragments. PMID- 3008882 TI - [Isolation and identification of normal killers from Syrian hamsters]. AB - Natural killers (NK cells) possessing cytotoxic activity were isolated from the blood, spleen and bone marrow of Syrian hamsters in discontinuous Percoll density gradient. These cells were morphologically identified as granular lymphocytes (GL). The largest amount of GL was isolated from the fraction containing 52-55% of Percoll. Cytotoxic activity of cells in this fraction was the highest, as compared to nonfractionated control or cells in other Percoll fractions. PMID- 3008881 TI - [Piracetam correction of deficient GABA-ergic inhibition due to early postnatal exposure to cycloheximide]. AB - Cycloheximide, administered to 7-day-old rats, caused the delay of CNS activity changes in grown-up rats. The animals showed impaired memory function, expressed in the decrease of habituation in the "open field" test, and the alteration of passive avoidance reflex. The increased number of intersignal reactions due to hypermotility was found during elaboration of avoidance reflex. The analysis of evoked potential recovery revealed the deficiency of GABA-ergic inhibition in the neocortex. Piracetam was shown to prevent completely behavioural disturbances and deficiency of GABA-ergic processes in grown-up animals. PMID- 3008883 TI - [Dinucleosome as a product of initial chromatin cleavage by endogenous nucleases]. AB - The ability of nucleic endonucleases to recognize dinucleosomal level of chromatin structure was studied. Rat liver chromatin endonucleases were shown to be capable of DNA cleavage at dinucleosome linkers. The cleavage was observed mainly at initial stages of chromatin autohydrolysis, i.e. up to 10-20th min of incubation in the medium containing 5 mmol of magnesium chloride and 2 mmol of calcium chloride. Thus, mononucleosomal DNA in dinucleosomes and other even chromatin subunits was larger and more homogeneous than in odd subunits. The cleavage of autodigested chromatin by bovine spleen and snake venom exonucleases and by nuclease S1 has shown that dinucleosomal structure is independent of differences in the length of internal and external dinucleosome linkers. The initial endonucleolysis appears to be characterized by different accessibility of the linkers for chromatin nucleases. PMID- 3008884 TI - [Identification of the nucleotide sequences specific for the 5'-flanking regions of the genes regulated by glucocorticoids by a computer analysis method]. AB - Nucleotide sequences of 5'-flanking regions of 11 glucocorticoid-regulated genes and 14 genes non-regulated by these hormones were studied using context computer analysis. Consensus TGTTCT, previously found in DNA fragments protected by glucocorticoid-receptor complexes from DNAase I digestion, was shown to be nonspecific for glucocorticoid-regulated genes. However, the analysis of sequences flanking the TGTTCT consensus has revealed that only glucocorticoid regulated genes contain four regularly distributed cytosine residues, one of them belonging to TGTTCT consensus. Three of cytosine residues are separated by 8-10 bp, which provides their close neighbourhood at one side of DNA double helix; the fourth extreme cytosine residue is located 6 bp from the nearest one and therefore, the complementary guanine residue is adjacent to the consensus in DNA helix. It is suggested that the consensus itself and two flanking cytosine and one guanine residues form a specific site for the interaction with glucocorticoid receptor complex. PMID- 3008885 TI - Characterization of murine peritoneal macrophage receptors for fibrin(ogen) degradation products. AB - The binding of human fibrinogen degradation fragments D1, E, X, and Y, as well as fibrin fragment D1 dimer, to mouse peritoneal macrophages was examined. A Scatchard plot of fragment D1 binding was biphasic, suggesting two classes of receptors. Fragments D1, D1 dimer, X, and Y in low concentrations bound to macrophages with high affinity (Kd = 23 to 73 X 10(-11) mol/L). Fragment E bound specifically but at a much lower level than the other fragments. Fragment D1 was able to compete for the binding of radiolabeled fragments X and Y but not radiolabeled fragment E. These studies indicate that fragments D and E are recognized by separate receptor systems but that all of the fibrinogen degradation products that contain the D domain are recognized by the same receptor system. PMID- 3008886 TI - Human macrophage maturation and heterogeneity: analysis with a newly generated set of monoclonal antibodies to differentiation antigens. AB - We have analyzed the expression of late differentiation antigens during terminal in vitro maturation of human macrophages (M phi) from blood monocytes (MO) in comparison to their distribution among mature M phi residing in various tissue sites. By immunizing mice with M phi derived from blood MO by culture on hydrophobic Teflon foils, monoclonal antibodies (mAbs) were developed (MAX.1, MAX.2, MAX.3, MAX.11) that reacted with lineage-restricted differentiation antigens. These antigens were expressed exclusively on M phi or were markedly increased after in vitro differentiation. The only overlap to another hemopoietic cell lineage was observed with MAX.3, which is shared by platelets and megakaryocytes. In the course of M phi maturation in vitro, the MAX.1 and MAX.3 antigens are detected within the cytoplasm two days before they appear on the cell surface. In contrast, the MAX.11 antigen is expressed simultaneously in the cytoplasm and at the cell surface, is found in varying degrees on a minor portion of blood MO and U937 cells, and is expressed rapidly at high density during early M phi differentiation in vitro. Among conventional mAbs that do not react with MO we found those against the transferrin (TF)-receptor, the BA-2, and the PCA1 antigen to label M phi. M phi matured in vivo and isolated from body fluids were positive with some but not all MAX mAbs. Distinctive patterns were observed with pulmonary M phi, exudate M phi from pleural and peritoneal effusions, synovial fluids, and early lactation milk. M phi from the alveolar space, for example, constantly expressed the MAX.2 antigen but not the MAX.3 antigen. Pleural effusion M phi, however, did not react with the MAX.1 mAb, but in most cases, it did react with the MAX.3 mAb. The detection of novel differentiation antigens, all expressed on monocyte-derived M phi but differently expressed on site specific M phi in situ, underlines the remarkable heterogeneity among human M phi. The expression of these antigens is flexible because those MAX antigens that were not expressed in situ could be induced if cells from distinct tissue sites were cultured in vitro for several days. MAX mAbs may be of potential value to study both the sequential stages of maturation within the M phi lineage as well as differential developments induced by various culture conditions in parallel to environmental factors in vivo. PMID- 3008888 TI - A combination of a T cell-derived lymphokine differentiation-inducing activity and a physiologic concentration of retinoic acid induces HL-60 to differentiate to cells with functional chemotactic peptide receptors. AB - The human acute promyelocytic leukemia cell line HL-60 is induced by retinoic acid (RA) and N,N-dimethylformamide (DMF) to differentiate into cells having many of the functional and morphologic characteristics of mature granulocytes. With normal human phagocytic cells there is both superoxide anion (O2-) production and chemotaxis in response to chemoattractants such as N-formyl-methionyl-leucyl phenylalanine (FMLP). We have now found that although HL-60 cells induced with RA alone produce O2- in response to 12-0-tetradecanoyl-phorbol-13-acetate (TPA) they are deficient in FMLP-stimulated O2- production and chemotaxis. In contrast, HL 60 induced either with DMF or with a combination of 10 nmol/L RA and a T cell derived lymphokine, differentiation-inducing activity (DIA), produce O2- and exhibit chemotaxis in response to FMLP. The basis for these results appears to be the concentration of cell surface chemotactic peptide receptors. Thus, untreated HL-60 and HL-60 induced with either RA alone or DIA alone do not have measurable levels of FMLP receptors, whereas HL-60 induced with a combination of RA and DIA has 5,400 receptors per cell. HL-60 induced with RA and DIA plus 1 mumol/L dexamethasone have 25,000 receptors per cell and have greater chemotactic activity than HL-60 induced with the combination of RA and DIA. Thus, differentiation of HL-60 to cells with many properties of normal phagocytes can be induced in vitro by physiologic substances. PMID- 3008887 TI - Inhibition of neutrophil cytolysin production by target cells. AB - Neutrophils, triggered by heat-aggregated human IgG (Agg.IgG), were found to lyse chicken red blood cells (CRBC) as determined by a 51Cr release method. The lysis was inhibited by azide, catalase, chloride-free medium and amino acids, suggesting the requirement for myeloperoxidase (MPO), hydrogen peroxide (H2O2), chloride ions (Cl-), and hypochlorous acid (HOC1), respectively. These results indicate that neutrophils lyse CRBC through an HOCl-(ie, MPO-H2O2-Cl-) dependent process. Although HOCl can react with neutrophil-derived nitrogenous (N-) compounds to yield chloramines, the main and well-characterized chloramines did not play a direct role in the lysis of CRBC in our model system. Thus, it appears that lysis is due either to HOCl or to an unknown compound derived from and with characteristics similar to HOCl. When CRBC were replaced with HRBC targets, no lysis could be observed. Treatment of HRBC with carmustine, to inhibit the glutathione cycle, did not affect the cell resistance to lysis by neutrophils. Conversely, the inhibition of HRBC catalase activity with aminotriazole (AT) made the cells susceptible to neutrophil-mediated HOCl-dependent lysis: this suggests that HRBC escape lysis by neutrophils through an AT-inhibitable, ie catalase dependent, process. Through an identical catalase-dependent process, HRBC were capable of efficiently preventing the H2O2 and HOCl recovery from Agg.IgG triggered neutrophils, tested under experimental conditions similar to those used for cytolytic assays. Together, these data suggest that HRBC targets, endowed with high catalase activity, escape neutrophil-mediated lysis by consuming (by catalase) neutrophil-derived H2O2, so that HOCl cannot be produced in amounts sufficient to promote lysis. Parallel experiments, performed with AT-treated CRBC, showed that these cells, endowed with a relatively low catalase content, only partially limit neutrophil cytolytic efficiency by a process qualitatively similar to that observed with HRBC targets. The results provide evidence that target cells can restrain neutrophil cytolytic efficiency by interfering with the MPO-H2O2-Cl system through their catalase activity. PMID- 3008889 TI - Separation of human megakaryocytes by state of differentiation on continuous gradients of Percoll: size and ploidy analysis of cells identified by monoclonal antibody to glycoprotein IIb/IIIa. AB - Human bone marrow was separated on continuous Percoll density gradients to analyze the distribution of cells of the megakaryocytic lineage. Megakaryocytes were identified by indirect immunofluorescence using a monoclonal antibody (LJP4) specific to the glycoprotein IIb/IIIa (GPIIb/IIIa) complex of platelets. Neither endothelial cells nor monocytes expressed the epitope identified by this antibody. Simultaneous measurement of size and ploidy were made on 2,359 GPIIb/IIIa-positive cells. The smallest cells were located in the most dense fractions where 81% of all 2N and 66% of 4N cells were found at densities greater than or equal to 1.050 g/mL. The largest cells were detected in the least dense fractions, with 70% of 16N, 78% of 32N, and 100% of 64N cells found at densities less than 1.050 g/mL. Ninety-four percent of all GPIIb/IIIa-positive cells less than 14 micron in diameter were found at densities greater than 1.050 g/mL. An exception to this inverse relationship was observed in the uppermost gradient fractions where an anomalous distribution of size and ploidy was found. Megakaryocytic viability was identified as being greater than 90% in all fractions. The data show that megakaryocytic differentiation as assessed by size and ploidy varies inversely with Percoll density. Separation of marrow on continuous Percoll gradients may be a useful method to separate megakaryocytes at different stages of differentiation. PMID- 3008890 TI - Mapping of distinct von Willebrand factor domains interacting with platelet GPIb and GPIIb/IIIa and with collagen using monoclonal antibodies. AB - We have used monoclonal antibodies (M Abs) and proteolytic fragmentation to localize structurally the functional sites of human von Willebrand factor (vWF) responsible for interaction with membrane glycoproteins GPIb, GPIIb/IIIa, and with collagen. SpII (215 kd) and SpIII (320 kd), the S aureus V-8 protease homodimeric fragments representing the carboxy-terminal and amino-terminal segments of the vWF subunit, competitively inhibited the binding of multimeric vWF to thrombin-stimulated or ristocetin-stimulated platelets, respectively. Specific saturable binding of each fragment was observed to stimulate platelets appropriately and was inhibited only by selected M Abs that both bound to the specific fragment and inhibited the corresponding function. M Ab 9, which blocks thrombin-induced binding of vWF to platelets, inhibited binding of SpII to platelets and bound to SpII as well as to a dimeric, 86-kd thermolysin fragment composed of 42-kd and 23-kd subunits, each possessing the epitope. Binding of SpII was also inhibited by a M Ab to GPIIb/IIIa. Thus, it appears that a portion of the carboxy-terminal end of vWF contains the ligand site for the GPIIb/IIIa receptor. In contrast, M Ab H9, which blocks ristocetin-induced binding of vWF to platelets, inhibited binding of SpIII to platelets and bound to SpIII as well as to monomeric 33-kd and 28-kd subtilisin fragments. Binding of SpIII to platelets was also inhibited by a M Ab to GPIb. Thus, it appears that a small segment of the amino-terminal part of vWF contains the ligand for the platelet GPIb receptor. The collagen binding site of vWF was localized with M Ab B203, which inhibits vWF interaction with collagen. This M Ab also bound to SpIII as well as to monomeric 26-kd and 23-kd subtilisin fragments. Thus, the third functional site responsible for collagen binding appears to be localized on the amino terminal portion of vWF, in a linear sequence different from those responsible for interaction with either of the platelet receptors. These assignments of functional sites should facilitate the localization of structural defects of vWF in the various forms of vWD and support the role of vWF as an adhesive protein with multiple interactive sites. PMID- 3008891 TI - Human parvovirus B19-induced epidemic acute red cell aplasia in patients with hereditary hemolytic anemia. AB - From March to August 1984, 26 patients with hereditary hemolytic anemia in northeastern Ohio developed acute, profound red cell aplasia. The patients included 14 males and 12 females 2 to 23 years old, with sickle cell anemia (20 cases), hemoglobin SC-disease (4 cases), sickle-beta-thalassemia (1 case), or hereditary spherocytosis (1 case). All had an acute onset of severe reticulocytopenia and anemia and prodromal symptoms of illness including fever, abdominal symptoms, headache, and arthralgias. Twenty-two received transfusions. Reticulocytosis occurred spontaneously within 2 to 14 days of presentation. In five acute-phase sera, 10(8) to 10(12) viral particles/mL were detected by electron microscopy. Human parvovirus B19 DNA was demonstrated in high concentration by hybridization in the same five acute-phase sera and in low concentration in sera of eight additional patients. The five highly viremic sera inhibited erythroid colony formation in vitro. B19-specific IgM was detected in sera of 24/26 patients, and B19-specific IgG in 21 of 22 patients tested. Our results indicate that human parvovirus B19 was the etiologic agent in this large epidemic of life-threatening acute red cell aplasia in patients with hereditary hemolytic anemia. PMID- 3008892 TI - Analysis of the peptide subunits of human neutrophil myeloperoxidase. AB - Myeloperoxidase is a cationic protein present in the azurophilic granules of human neutrophils and constitutes 2% to 5% of neutrophil protein by weight. Despite extensive characterization of the functional importance of myeloperoxidase in the microbicidal activity of neutrophils, there is no consensus regarding the subunit composition of this enzyme or the presence of genetic polymorphism. Using isoelectric focussing and peptide mapping of chymotryptic digests of myeloperoxidase subunits under reducing and nonreducing conditions, we found two different heavy chain peptides that have overlapping but distinctive chymotryptic digest maps. One of these heavy chains has a reducible intrapeptide disulfide bond not found in the other. These results suggest that the model proposed for the structure of myeloperoxidase as a symmetric molecule with two identical heavy-light protomers may not be correct. PMID- 3008893 TI - Use of a BamHI polymorphism in the factor IX gene for the determination of hemophilia B carrier status. AB - A BamHI polymorphism has been identified in the human factor IX gene. This polymorphism, which occurs in approximately 6% of X chromosomes, has been used to determine the carrier status of a female in a family with a history of hemophilia B. This family was uninformative for the previously reported TaqI and Xmnl polymorphisms in the factor IX gene. PMID- 3008894 TI - Myeloperoxidase-deficient polymorphonuclear leucocytes. Longitudinal study during the preremission--and the remission phase in acute myeloid leukaemia. Comparison to neutrophil alkaline phosphatase (NAP) activity. AB - Serial determinations of MPO and NAP activities in granulocytes were performed during the preremission phase and the remission phase in patients with AML. Of 18 patients examined during the preremission period, 9 showed an increased number of MPO deficient PMN. Complete remission was attained in 4 of these, in 3 the number of abnormal granulocytes changed to normal 7, 7 and 14 days before and in 1 simultaneously with the attainment of complete remission. In the other patients no changes in granulocyte MPO activity occurred during the preremission period. All 20 patients examined during complete remission showed a normal MPO activity in granulocytes. Of eight patients, who at diagnosis had shown abnormal granulocyte MPO activity, three developed relapse. In two of these, an increased number of MPO deficient PMN reappeared two and eight months prior to and in one simultaneous with clinical and laboratory suspicion of relapse. A statistically significant relation between low NAP scores and an increased number of MPO deficient PMN was found (P = 0.011). Serial determinations of MPO activities in PMN, although restricted to cases of AML with initially abnormal values, may prove helpful in predicting achievement of complete remission and may furthermore prove to be useful as an indicator of early relapse. PMID- 3008895 TI - Expression of cytolytic functions in HL-60 leukaemic cells after induction of polymorphonuclear leukocyte differentiation. AB - Mature polymorphonuclear leukocytes (PMN) are capable of mediating phorbol myristate acetate (PMA)- and antibody (A)-dependent cellular cytotoxicity (DCC) against ox red blood cells (ORBC) by using oxidative means. The purpose of the present study was to investigate the acquirement of these cytotoxic functions during PMN ontogeny, using the promyelocytic HL-60 cell line as a model for PMN differentiation. HL-60 cells were induced to differentiate along the PMN pathway by exposure to dimethyl sulfoxide (DMSO). Uninduced HL-60 cells were found to be completely devoid of PMA-DCC and ADCC activity. DMSO-induced cells progressively acquired the capacity to kill ORBC and to undergo the activation of oxidative metabolic burst when triggered by PMA. Despite approximately 40% of them also were capable of binding IgG-sensitized ORBC, no ADCC activity and respiratory burst activation was observed: this finding indicates that maturing HL-60 cells require a more complete maturation than that induced by DMSO to actually exert ADCC. Together the results suggest that: a. the acquirement of both PMA-DCC and ADCC potential is a post-promyelocytic event; b. the cytotoxicity activating stimuli, PMA and IgG-coated targets, follow different post-receptor transductional pathways to trigger the effector cell lytic systems: only the PMA receptor-linked pathway develops during DMSO-driven differentiation of HL-60 cells. PMID- 3008896 TI - Giant malignant cystosarcoma phylloides with hepatic metastases. PMID- 3008897 TI - Ultrastructural classification of tumor cells. PMID- 3008898 TI - Responses of quail, pheasants, and sparrows to one oral dose of dimethoate and to consumption of dimethoate treated bran baits. PMID- 3008899 TI - Early correction of the thoracic deformity of Poland's syndrome in children with the latissimus dorsi muscle flap: long term follow-up of two cases. AB - For treatment of the costo-sternal ridge that is sometimes caused by the missing pectoral muscles in Poland's syndrome, the latissimus dorsi muscle can be transposed as a functional substitute. This transposition should be carried out in early childhood as soon as the deformity becomes visible. Two patients have been observed after this procedure for 13 and 10 years respectively and show no evidence that the transposition of the latissimus dorsi caused any skeletal changes in the spine or impaired shoulder motion. The procedure is very efficient in correcting the chest wall deformity. PMID- 3008900 TI - Calcified gastric cancer: report of a case and review of literature. PMID- 3008901 TI - Primary oat cell carcinoma of the urinary bladder. PMID- 3008902 TI - Prognostic factors in breast cancer and the development of a prognostic index. AB - A number of different factors are known to be correlated with survival of patients with breast cancer. Among these are lymph node status, tumour size, oestrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR) status. The purpose of this study was to investigate the relative significance of these factors and use this information to construct a prognostic index capable of predicting survival. These factors together with age and menopausal status were studied and correlated with prognosis in 796 women with primary breast cancer. The data were analysed in a stepwise manner by the Cox proportional hazards regression technique. Statistically, greater than 3 nodes involved gave the worst prognosis (P less than 0.001). This was followed by ER if less than 10 fmol/mg cytosol protein (P less than 0.001), PR if less than 10 fmol (P less than 0.01), greater than 0 lymph nodes involved (P less than 0.01) and the number of years over age 65 (P less than 0.01). When these factors were accounted for, tumour size, menopausal status and AR did not significantly improve prediction of survival. The significant factors were incorporated into a prognostic index: I = N + E + P + A, where N = 0 if no nodes involved, 13 (if 1-3 nodes involved and 31 if greater than 3 nodes involved, E = 15 if ER less than 10 fmol, P = 12.5 if PR less than 10 fmol and A = number of years over 65. Using this index five year survival curves were constructed corresponding to groups of patients with widely differing prognoses. Predicted five year survival ranged from 96 to 12 per cent. PMID- 3008903 TI - Two genetic markers closely linked to adult polycystic kidney disease on chromosome 16. AB - The genetic locus for autosomal dominant adult polycystic kidney disease was recently assigned to chromosome 16 by the finding of genetic linkage to the alpha globin gene cluster. Further study showed that the phosphoglycolate phosphatase locus is also closely linked to both the locus for adult polycystic kidney disease and the alpha globin gene cluster. These findings have important implications for the prenatal and presymptomatic diagnosis of adult polycystic kidney disease and for a better understanding of its pathogenesis. PMID- 3008904 TI - Vegetable consumption and acute appendicitis in 59 areas in England and Wales. AB - Rates of acute appendicitis in 59 areas of England and Wales were correlated with consumption of different foods per caput, measured from household food purchases. There was a statistically significant positive correlation with potato consumption and a negative correlation with non-potato vegetables. This negative correlation depended mainly on green vegetables and tomatoes. There was no consistently significant correlation with any other main food group. In particular the correlations with cereal foods, cereal fibre, and total dietary fibre were small and not significant. Green vegetables and tomatoes may protect against appendicitis, possibly through an effect on the bacterial flora of the appendix. PMID- 3008906 TI - Absence of seroconversion for HTLV-III in haemophiliacs intensively treated with heat treated factor VIII concentrate. PMID- 3008905 TI - HTLV-III: should testing ever be routine? PMID- 3008907 TI - Ontogenesis of unit activity in the raphe dorsalis of the behaving kitten: its relationship with the states of vigilance. AB - The relationship between the spontaneous unit activity in the raphe dorsalis (RD), and the sleep-wakefulness cycles, was analyzed in the cat from birth to 40 days of age. Electrodes for polygraphic sleep monitoring were implanted under anesthesia, and unit recordings were obtained from bundles of microwires positioned in the RD area in kittens of different ages. Attention was paid only to units with slow firing in wakefulness (W) (1-6 spikes/s), and two types of discharge patterns during this state were obtained: a 'regular' type, whose discharge in W had the same characteristics of regularity as those described for the adult under the same conditions, was always found inside the RD. An 'irregular' type was always found in sites outside the RD. Injections of different doses of 5-methoxy-N,N-dimethyltryptamine (5-MeODMT i.m.) induced a transient decrease in the firing rate of the regular type of cells, and no change for the units of the irregular type, suggesting that the regular neurons were of serotoninergic nature. Whereas the cells of the irregular type exhibited an increase of discharge frequency in active or paradoxical sleep (AS-PS), those which fired in a clock-like manner during W exhibited a rate of discharge which progressively decreased in quiet or slow wave sleep (QS-SWS) and even more in AS PS. Such a pattern was qualitatively close to the adult one at all ages, but the discharge rate in AS was significantly higher during the first and second weeks of life than later on. The observation that these serotoninergic neurons exhibited at birth an adult-like pattern of discharge during W, indicates that during ontogenesis there was no direct relationship between the RD activity and the behavioral output. It is proposed that the RD neuronal discharge would be largely under genetic influences, and that the maturation of sleep regulations at the brainstem and mesencephalic levels is achieved only after the second week of postnatal age. PMID- 3008908 TI - Release of [3H]norepinephrine: alteration by early developmental exposure to diazepam. AB - The effect of prenatal exposure to diazepam (over gestational days 13-20) on the release of tritiated norepinephrine [( 3H]NE) from selected brain regions was analyzed to determine mechanisms whereby such exposure could disrupt functioning in specific NE neurons, as previously observed. Pregnant rats were administered diazepam (DZ) once daily at doses of 1.0, 2.5 or 10.0 mg/kg and the offspring studied as adults at 70-90 days of age and during development at 14, 21, 35 and 56 days of age. Release of [3H]NE was measured during in vitro incubation using 25 mM potassium as the depolarizing stimulus. As noted previously, prenatal exposure to DZ induced an effect only on NE neurons innervating the hypothalamus, sparing the NE innervation to the hippocampus and cerebellum. Prenatal exposure to DZ had no effect on the depolarized release of [3H]NE in the hypothalamus until after 35 days of age, a developmental pattern previously observed with respect to endogenous NE levels. In adult rat offspring, however, the depolarization-induced release of [3H]NE from the hypothalamus decreased 28%, 32% and 64% (relative to uninjected control values) in animals prenatally exposed to DZ at 1.0, 2.5 or 10 mg/kg/day respectively. Concurrent exposure of the pregnant dam to benzodiazepine antagonists (Ro 15-1788 or ethyl-beta-carboline-3 carboxylate) prevented the effects of DZ (2.5 mg/kg/day) on [3H]NE release, demonstrating again the importance of the benzodiazepine binding site to the effects induced by the early DZ exposure. The initial accumulation of the [3H]NE was not altered by the prenatal exposure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008909 TI - Autoradiographic localization of beta 1 and alpha 1-adrenoceptors in the midbrain and forebrain of normal and reeler mutant mice. AB - The distribution of alpha-1 and beta-1 adrenoceptors has been studied in the midbrain and forebrain of normal and reeler mutant mice, using autoradiographic visualization of radioiodinated HEAT and ICYP, respectively. All cortical structures and nuclear groups of the murine forebrain and midbrain bind ICYP and HEAT. For each ligand, there is substantial regional variation in binding density and these variations tend to observe boundaries between nuclei or cortical regions or the stratification of cortical regions. Regional variations in binding densities are generally different for ICYP and HEAT. Binding sites for ICYP are distributed densely throughout all fields of the neocortex (particularly, layers I-III greater than VI) and paleocortex, the striatum, pallidum, substantia nigra and superficial strata of the superior colliculus. Dense concentrations of binding sites for HEAT in cortical structures, by contrast, are limited to frontal (all layers except IV) and anterior cingulate regions of the neocortex and, as with ICYP, the stratum lacunosum-moleculaire of the regio superior of the hippocampal formation. In subcortical structures, again in contrast to the pattern with ICYP, binding density is greatest in the principal nuclei of the dorsal thalamus and the septal nuclei. The regional binding patterns of both ICYP and HEAT in the reeler brain are identical to those in the normal animal. Differential laminar binding patterns within the neocortex are approximately inverted in the two genotypes, however. Thus, binding of ICYP is densest in an inner zone of the mutant, but in the outer 3 layers of the normal neocortex. Binding of this ligand is of relatively lower density in an outer zone of the mutant and in the inner 3 layers of the normal neocortex. Similar inversions are characteristic of the laminar binding patterns of HEAT in the frontal, primary sensory and associational cortical regions of the two genotypes where densest binding is encountered superficially in reeler but at deeper levels of the normal neocortex. PMID- 3008910 TI - Nipecotic acid, an uptake blocker, prevents fading of the gamma-aminobutyric acid effect. AB - In rats under urethane, iontophoretic applications of GABA (30-60 nA) in the str. pyramidale of CA1, showed a rapidly fading inhibitory effect. By contrast, GABA had a well-maintained inhibitory effect in str. radiatum. During iontophoresis of nipecotic acid (30-85 nA) identical applications of GABA in str. pyramidale caused a more prominent depression without fading, which suggests that removal of GABA, by uptake, can at least in part account for 'fading'. Nipecotic acid also prolonged the paired-pulse inhibition, presumably by prolonging the duration of inhibitory postsynaptic potentials. PMID- 3008911 TI - 5-Hydroxytryptamine (serotonin) causes a reduction in the afterhyperpolarization following the action potential in lamprey motoneurons and premotor interneurons. AB - The actions of 5-hydroxytryptamine (5-HT) on single neurons in the lamprey spinal cord have been investigated by intracellular recordings in vitro. Administration of 5-HT either by pressure ejection or by perfusion in the bath caused a reversible reduction of the late phase of the afterhyperpolarization (AHP) following the action potentials. This effect was antagonized by methysergide. A reduction of the late AHP was observed in lateral interneurons, motoneurons and some unclassified cells. PMID- 3008912 TI - Adenosine inhibits locus coeruleus neurons: an intracellular study in a rat brain slice preparation. AB - Intracellular recording was used to study the effect of adenosine (3-100 microM) on rat locus coeruleus (LC) neurons in a brain slice preparation. Bath application of adenosine (100 microM) reduced the rate of spontaneous firing in 88% of LC neurons. In some LC neurons adenosine also caused membrane hyperpolarization (2-10 mV) and reductions in input resistance of 9-24%. Adenosine effects were dose-dependent and antagonized by the adenosine receptor antagonist theophylline. PMID- 3008913 TI - Supernumerary locus coeruleus neurons as a determinant of inherited epilepsy in the convulsive mutant mouse quaking. AB - In quaking mice (a genetic model of epilepsy with an increased number of noradrenergic neurons) bilateral electrolytic coagulation of locus coeruleus (LC) in adult mice inhibited the convulsions elicited by somatic stimulations while neonatal 6-hydroxydopamine (6-OHDA) treatment remained ineffective upon the convulsions. Biochemical effects of the two treatments differed only in the brainstem where electrolytic lesion decreased while 6-OHDA treatment increased noradrenaline (NA) and 3-methoxy 4-hydroxyphenylethyleneglycol (MHPG) levels. Our results suggest that supernumerary LC neurons mediate the convulsions of the mutants through an action presumably restricted to the brainstem. PMID- 3008914 TI - Intracellular recordings from rat nucleus accumbens neurons in vitro. AB - Intracellular recordings were obtained from rat nucleus accumbens (NAC) neurons in brain slice preparations. Local stimulations evoked depolarizing postsynaptic potential (DPSP). Injections of low intensity depolarizing currents decreased the amplitude of the DPSP and reversed a later portion of the DPSP into a hyperpolarizing potential. Superfusion of pentobarbital facilitated the reversal of this later portion of DPSP and bicuculline abolished this polarity reversal. These data suggested that the DPSP evoked by local stimulation consisted of a combination of an excitatory and an inhibitory postsynaptic potential, and that the latter was probably mediated by gamma-aminobutyric acid. PMID- 3008915 TI - Regional brain homovanillic acid following delta 9-tetrahydrocannabinol and cocaine. AB - delta 9-Tetrahydrocannabinol (10 mg/kg) increased homovanillic acid in rat prefrontal cortex and olfactory tubercle. This dose did not affect homovanillic acid in the caudate. Higher doses increased homovanillic acid in all 3 regions. Cocaine (20, 30, or 50 mg/kg) did not affect homovanillic acid in any of these brain regions. PMID- 3008916 TI - Alterations in the noradrenergic projection to the cerebellum of the dystonic (dt) rat. AB - The genetically dystonic rat (dt) has elevated resting levels of cerebellar norepinephrine (NE) in comparison with phenotypically normal littermates. This difference is not secondary to cerebellar hypoplasia. Increased NE is observed as early as postnatal day 12, when clinical symptoms have become evident. The elevation in cerebellar NE levels in the dt rat involves all cerebellar areas, but is not generalized to all terminal fields of the locus coeruleus. Elevations in cerebellar NE are followed developmentally by a reduction in sensitivity to the NE-depleting effects of reserpine, a change which is also confined to the cerebellum. The effects of amphetamine and the tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine were similar in normal and dt rats. Levels of the major cerebellar metabolite of NE, 3-methoxy-4-hydroxyphenylglycol, did not differ between mutant and normal animals. Nor were any changes noted in the number or affinity of beta-adrenergic receptors. These data indicate that there is a regional alteration in NE storage. Cerebellar morphology appears normal in the dt rat, except for a decrease in Purkinje cell size. This change and other evidence of biochemical abnormalities in the Purkinje cells suggest that the alterations in cerebellar NE in the dt mutant may be a secondary response to a functional change in the target neuron for this system, the Purkinje cell. PMID- 3008917 TI - Denervation-like postsynaptic supersensitivity to dopamine agonists induced by microinjection of colchicine into the substantia nigra pars compacta. AB - Circling behavior induced by dopamine (DA) agonists following microinjection of colchicine or 6-hydroxydopamine (6-OHDA) into the substantia nigra pars compacta (SNC) or electrolytic lesions of the SNC was investigated. Methamphetamine produced a contralateral circling behavior 3, 7 and 14 days following injection of colchicine into the SNC. Apomorphine produced an ipsilateral circling behavior followed by a contralateral rotation 3 and 7 days after the infusion of colchicine, whereas only an ipsilateral circling behavior was produced by apomorphine on day 14. 6-OHDA lesions of the SNC produced a contralateral circling behavior to apomorphine and an ipsilateral circling to methamphetamine, whereas both apomorphine and methamphetamine induced ipsilateral circling behaviors in rats with electrolytic lesions of the SNC. The possible mechanisms of action of colchicine are discussed in relation to the known effects of colchicine on axoplasmic transport. PMID- 3008919 TI - Endocrine cells do not exist in rat Brunner's glands. An electron microscopic study. AB - It was revealed by light microscopic experiments that in Brunner's glands and the epithelium of the human and mammalian duodenum, cells were present, with basally lying granulations. These granules could be identified at the ultrastructural level as accumulations of pleomorphe electron-dense granules ('argentaffin', 'chromaffin' and 'osmiophilic' granules). Depending on the light and electron microscopic staining techniques used, these cells were classified as 'mucous', 'serous', or 'endocrine' ('enteroendocrine', 'paracrine' and 'enterochromaffin') cells. In the present study, the following ultrastructural morphological criteria of Brunner's gland cells were correlated: size, shape and electron density of their nuclei and nucleoli with morphological changes of their cytoplasmic and intranuclear organelles, which serve the synthesis of mucopeptides. This proved that the synthesis of mucopeptides occurred in 3 phases. The first phase served the intranuclear synthesis of organelles, which latterly were used for the cytoplasmic mucopeptide synthesis. This phase was characterized by the appearance of an electron-lucent and euchromatic nucleus, which also contained an euchromatic and electron-lucent nucleolus, and a thin electron-dense nucleus membrane (euchromatic phase). Only a few freely lying ribosomes, membrane-bound ribosomes (rough endoplasmic reticulum, RER) and mitochondria were dispersed throughout the cytoplasm. Therefore, the cytoplasm appeared electron-lucent and nearly empty. The second phase was characterized by a round but heterochromatical nucleus containing also a heterochromatic nucleolus, and the appearance of receptosome- (endosome, endocytotic vesicle) and lysosome-like structures near to the cytoplasmic membrane of the basal cell portion, as well as in the basal and lateral perinuclear region (early heterochromatic phase). The receptosome-like structures were round-oval and contained a single electron-lucent inclusion vesicle, which had an empty space or an electron-opaque granule. Both inclusion structures were located eccentrically on the inner surface of the membrane of the receptosome-like structure. Here and there, it was possible to visualize great receptosome-like structures (multivesicular bodies) containing numerous membranous electron-lucent and electron-opaque inclusion vesicles. Electron-dense pleomorphe lysosome-like structures, bearing 1-4 electron-lucent inclusion vesicles with faint grains in their spaces, were also recognized in the basal cell portion, as well as in the basal and lateral perinuclear region.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3008918 TI - The acute effects of 6-hydroxydopamine treatment on noradrenergic function in the rat hippocampus in vitro. AB - The electrophysiological consequences of in vitro treatment with 6 hydroxydopamine (6-OHDA) were examined in the CA1 region of the rat hippocampal slice. In control slices, norepinephrine (NE) increased the amplitude of the population spike response elicited by synaptic stimulation of hippocampal pyramidal neurons with a threshold of approximately 5 microM. When hippocampal slices were pretreated with 500 microM 6-OHDA for 10 min, perfusion with a subthreshold concentration of NE (0.5 microM) produced responses similar to those observed with a 10-fold higher concentration of NE in untreated slices. Baseline electrophysiological responses were unchanged following the 6-OHDA exposure. The potentiation of the response to NE by in vitro pretreatment with 6-OHDA was accompanied by a greater than 40% decrease in NE content and greater than 90% decrease in [3H]NE accumulation. In vivo treatment with 6-OHDA or N-(2 chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP4) also potentiated the electrophysiological response to NE in a manner similar to that observed with acute in vitro 6-OHDA pretreatment. This action does not appear to be due to development of beta-adrenergic receptor supersensitivity, because the apparent potency of isoproterenol in increasing the population spike amplitude was unaffected. These data suggest that the increase in the potency of NE in slices pretreated with 6-OHDA is due to the rapid disruption of the high-affinity NE uptake mechanism characteristic of noradrenergic nerve terminals. PMID- 3008920 TI - Postsynaptic potentials evoked in spiny neostriatal projection neurons by stimulation of ipsilateral and contralateral neocortex. AB - Postsynaptic potentials were evoked in neostriatal neurons by stimulation of the ipsilateral and contralateral medial agranular frontal cortical field (AGm) in the rat. This cortical region is known to project bilaterally to the dorsal lateral head of the caudate-putamen of rats. Ipsilateral stimulation of AGm should excite all types of corticostriatal neurons projecting to neostriatal neurons in the corresponding area in neostriatum, while stimulation of the same cortical area on the side contralateral to the recording should evoke synaptic potentials from a more restricted subpopulation of crossed corticostriatal neurons. Neostriatal neuronal responses were recorded intracellularly and spiny projection neurons identified by intracellular staining with horseradish peroxidase. The initial EPSP response to contralateral stimulation was similar to that evoked from the ipsilateral side, except for the absence of a relatively small short latency component responsible for the earliest part of the response to ipsilateral cortical stimulation. Comparison with previous findings indicated that this earliest EPSP component was due to activation of fast-conducting descending cortical efferents with collateral projections exclusively to the ipsilateral neostriatum. Stimulation of contralateral neostriatum evoked responses identical to those obtained using stimulation of contralateral neocortex. Analyses of these responses indicated that both EPSPs arise from activation of the same population of fibers. Stimulation of the contralateral internal capsule just caudal to neostriatum was not effective in evoking the EPSP. Chronic hemidecortication did not change the shape of the EPSP evoked from the intact contralateral side, but reduced its amplitude by approximately one half. These observations indicate that contralaterally projecting corticostriatal neurons in the rat project bilaterally in neostriatum, have axonal branches to the contralateral cerebral cortex as well as neostriatum, and converge onto neostriatal neurons that also receive input from the corresponding cortical region on the ipsilateral side. PMID- 3008921 TI - Ethanol-stimulated endorphin and corticotropin secretion in vitro. AB - Although acute administration of ethanol in vivo results in increased plasma glucocorticoid concentration, it is unclear whether this effect is mediated by corticotropin (ACTH) from the anterior pituitary. Secretion of beta-endorphin like (BE-IR) and corticotropin-like (ACTH-IR) immunoreactivity from perifused, dispersed mouse adenohypophyseal cells was used to evaluate the effect of 17 mM ethanol on secretion of pituitary peptides. Cells were also exposed to 10 nM synthetic corticotropin-releasing factor (CRF), 1 microM vasopressin, 54 mM KCl, 100 nM corticosterone, and calcium-free medium, separately and in combination. Secretion of BE-IR and ACTH-IR were markedly sensitive to low concentrations of ethanol. Exposure to 17 mM ethanol produced 3-fold stimulation of the rate of hormone release. This represented one-third to two-thirds that of the rate of maximum stimulation by CRF. Unlike CRF-stimulated secretion, ethanol-stimulated secretion was transient. Further, a second ethanol exposure 1 h after the first did not stimulate peptide secretion. Similar to CRF-stimulation, ethanol stimulated peptide secretion required extracellular calcium and was inhibited by the glucocorticoid corticosterone. We suggest that this system is a useful model for investigation of the actions of low concentrations of ethanol at the cellular level. PMID- 3008922 TI - Evidence for neuronal interactions by electrical field effects in the CA3 and dentate regions of rat hippocampal slices. AB - During population spikes in slices of rat hippocampus, transmembrane differential recordings (intracellular minus extracellular) revealed electrical field-effect depolarizations in CA3 pyramidal and dentate granule neurons. The field-effect depolarizations were not seen in single-ended recordings, and were consistently shown to increase neuronal excitability. Therefore, as previously shown for the CA1 area, large population spikes from CA3 pyramidal and dentate granule cells are associated with transmembrane depolarizations that increase neuronal excitability. PMID- 3008923 TI - Intracellular records of theta rhythm in hippocampal CA1 cells of the rat. AB - In the urethane-anesthetized rat, intracellular recordings from hippocampal CA1 cells, some of them identified as projection (probably pyramidal) cells, showed oscillations of the resting membrane potential in the theta frequency range ('intracellular theta rhythm') which is phase-locked to the extracellularly recorded theta rhythm. Current injection or acetate ion diffusion, which reversed an inhibitory postsynaptic potential (IPSP) evoked by alvear stimulation, inverted the phase relationship between intracellular and extracellular theta rhythms. A high correlation was also found between amplitudes of the intracellular theta and the evoked IPSP at different membrane potentials. These results indicate that hippocampal theta rhythm in the urethane-anesthetized rat is predominantly caused by a rhythmic modulation of impinging IPSPs on pyramidal cells. PMID- 3008924 TI - Glucocorticoid modulates the sensitivity of the GABAA receptor on primary afferent neurons of bullfrogs. AB - With intracellular and voltage-clamp recording techniques, we have demonstrated that the glucocorticoids, prednisolone and hydrocortisone at a concentration of 5 microM to 1 mM, reversibly depressed gamma-aminobutyric acid (GABA)-induced responses on primary afferent neurons of bullfrogs. An analysis with dose response curves revealed that the glucocorticoids decreased the sensitivity of the GABAA receptor in a non-competitive manner. We suggest that glucocorticoids act as an antagonist of the GABAA receptor on primary afferent neurons, probably by reducing the number of functional GABAA receptor ionic channel complexes. PMID- 3008925 TI - Loss of inhibition in the CA1 region of the kainic acid lesioned hippocampus is not associated with changes in postsynaptic responses to GABA. AB - Somatic and dendritic responses to gamma-aminobutyric acid (GABA) were recorded intracellularly from CA1 pyramidal cells in slices of the hippocampus ipsilateral and contralateral to a unilateral kainic acid lesion of the CA3 region. Ipsilateral CA1 cells show a loss of GABA-mediated synaptic inhibition. However, somatic GABA responses and the sensitivity of cells to GABA were very similar in ipsilateral and contralateral cells. This was also true for dendritic applications of GABA. PMID- 3008926 TI - Synergistic effects of 5'-nucleotides on rat taste responses to various amino acids. AB - Synergism between 5'-nucleotides and L-amino acids in the rat taste nerve responses was investigated. The synergism was examined between low concentration of the nucleotides which induce only a negligibly small response and various concentrations of amino acids. A marked synergism was found between 5' nucleotides and all the amino acids examined. Purine 5'-nucleotides such as GMP, deoxy GMP, GDP, GTP, AMP or IMP exhibited the synergistic effect on the responses to amino acids, while pyrimidine 5'-nucleotides such as CMP or UMP exhibited practically no synergistic effect. The presence of GMP led to a shift of the concentration-response curves for amino acids to a lower concentration region without affecting the response of the saturation level. These curves were analyzed under the assumption that there exist two types of receptor sites with different dissociation constants for each amino acid. The results suggest that GMP leads to a decrease in dissociation constants, especially for high affinity sites without affecting maximal responses. Monosodium glutamate (Glu) itself, which is known as a typical 'flavor potentiator', exhibited no enhancing effect on the responses to 4 primary taste stimuli. The mechanism of how Glu and the 5' nucleotides 'potentiate' flavor of food is discussed. PMID- 3008927 TI - Evidence for opiate action at the brain receptors for thyrotropin-releasing hormone. AB - The effect of drugs acting on mu-, delta-, kappa- and sigma-opiate receptors on the binding of [3H-3-MeHis2]thyrotropin releasing hormone ([3H-Me]TRH) to rat brain membranes was determined. The drugs used included morphine and naloxone (mu ligands), N,N-bisallyl-Tyr-Gly-Gly-psi-(CH2S)-Phe-Leu-OH (ICI 154, 129, delta ligand), ethylketocyclazocine (EKC, kappa-ligand), and N-allylnormetazocine (SKF 10,047, sigma-ligand). [3H-Me]TRH bound specifically to brain membranes at a single high-affinity site with a Bmax value of 50 fmol/mg protein and a Kd of 5 nM. A concentration of 2 nM of [3H-Me]TRH was used for in vitro interaction studies. The binding of [3H-Me]-TRH was unaffected by morphine or naloxone up to and including 1 mM concentration, but was inhibited by ICI 154,129 (IC50, 8 X 10( 5) M), EKC (IC50, 3 X 10(-4) M), SKF 10,047 (IC50, 1 mM), and TRH (IC50, 2 X 10( 7) M). In the presence of an IC25 concentration of IDI 154, 129 or EKC, there was a 25% decrease in the Bmax values of [3H-Me]TRH but the apparent dissociation constant, Kd value did not change. The results demonstrate for the first time that ligands acting at the delta- and kappa-opiate receptors interact with the brain TRH receptors; sigma-receptor ligands have weak interaction and mu-receptor ligands do not interact with brain TRH receptors. These results may help in explaining the observed pharmacological interactions between opiates and TRH.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008928 TI - Dihydropyridine calcium channel antagonists binding in non-mammalian vertebrates: characterization and relationship to 'peripheral-type' binding sites for benzodiazepines. AB - The densities of dihydropyridine calcium antagonist binding sites were examined in brains and cardiac issues of representative species from 4 classes of non mammalian vertebrates (aves, pigeon; amphibia, frog; reptilia, chameleon; and osteichthyes, trout) previously shown to have low or undetectable levels of 'peripheral-type' binding sites for benzodiazepines. Dihydropyridine binding sites were present in brain and cardiac tissue of these 4 classes of non mammalian vertebrates. The apparent dissociation constants for [3H]nitrendipine in non-mammalian vertebrates were comparable to those found in mammalian (rat) tissues. The densities of [3H]nitrendipine binding sites in brain and cardiac tissues were highest in the pigeon, with lower densities in tissues from chameleon, frog and trout. The densities of dihydropyridine binding sites in the latter tissues were comparable to those observed in the rat. Thus, dihydropyridine binding sites are phylogenetically diverse yet have similar kinetic characteristics in mammals and non-mammalian vertebrates. Despite the pharmacologic evidence that links dihydropyridine binding sites and peripheral binding sites for benzodiazepines in mammals, the earlier evolutionary appearance of the former sites suggest that even though they may share one or more common effector mechanisms, they are discrete entities. PMID- 3008929 TI - MPTP causes a non-reversible depression of synaptic transmission in mouse neostriatal brain slice. AB - MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) causes a Parkinson's disease like syndrome. The mechanism of MPTP's neurotoxicity is unknown; however, one hypothesis is that MPP+ (1-methyl-4-phenylpyridinium), a product of MPTP's oxidation, is the neurotoxic agent. Using a mouse brain slice preparation we studied the effects of MPTP and MPP+ on synaptic transmission. We found MPTP caused a decrease in amplitude of an excitatory synaptic response not reversed by washing. This non-reversible action of MPTP was prevented by GBR-32 and pargyline. MPP+s caused a decrease in synaptic transmission, but this decrease was reversed by washing. The results suggest that the toxic effect of MPTP on synaptic transmission is not accounted for by the action of MPP+. PMID- 3008930 TI - 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) inhibition of [3H]N ethylcarboxamidoadenosine (NECA) binding allows the visualization of putative non A1 adenosine receptors. AB - The binding of the adenosine receptor agonists, [3H]N-ethylcarboxamidoadenosine (NECA) and [3H]cyclohexyladenosine (CHA) to membrane preparations and to cryostat sections of the rat brain was examined. The xanthine derivative, 1,3-dipropyl-8 cyclopentylxanthine (DPCPX) was ca. 500-fold more effective at A1 than at A2 sites. [3H]CHA binding to A1 adenosine receptors was virtually eliminated by the inclusion of DPCPX (50 nM), while [3H]NECA binding was only partially inhibited. The pattern of DPCPX-insensitive [3H]NECA binding sites was strikingly different from that of A1 receptors and is believed to represent an association with A2 type adenosine receptors and perhaps another or several, previously undescribed non-A1 sites. PMID- 3008932 TI - Facilitation of lumbar monosynaptic reflexes by locus coeruleus in the rat. AB - The present study was initiated to delineate whether species difference exists between cats and rats in the descending influence of locus coeruleus (LC) on spinal motoneuronal activity. In male Sprague-Dawley rats anesthetized with chloral hydrate (400 mg/kg, i.p.), localized activation of LC promoted an exclusive facilitation of lumbar spinal extensor and flexor monosynaptic reflexes (MSRs). Such LC-evoked potentiations may vary in degree (37.5-147.4%), duration (70.6-72.9 ms) and latency (3.0-5.5 ms) among different animals. While minimally affecting the control MSRs, the alpha 1-adrenoceptor blocker prazosin (20 micrograms/kg, i.v.) significantly antagonized the enhancing effect of the LC on MSRs, suggesting the participation of noradrenergic neurotransmission in the process. Since these results are in general agreement with previous observations from our laboratory on the cat, we conclude that the LC exerts similar facilitatory actions on both extensor and flexor motoneuron activity of the hindlimb in at least two animal species, rat and cat. PMID- 3008931 TI - Impact of circulating corticosterone on alpha 1- and alpha 2-noradrenergic receptors in discrete brain areas. AB - The impact of adrenalectomy (ADX) and subsequent corticosterone(CORT) replacement, on the binding of [3H]p-aminoclonidine to alpha 2-noradrenergic receptors and [3H]prazosin to alpha 1-noradrenergic receptors, was studied in 8 discrete hypothalamic and 5 extra-hypothalamic areas of rats. With little change in extra-hypothalamic receptors, ADX produced a large CORT-reversible decrease in alpha 2-receptor binding specifically within the hypothalamic paraventricular nucleus (PVN) and a CORT-reversible increase within the supraoptic nucleus. alpha 1-Noradrenergic receptors, in contrast, were generally unaffected by ADX. This and other evidence leads us to propose a potential modulatory influence of circulating CORT on hypothalamic alpha 2 receptors and a specific function for this CORT-alpha 2 receptor interaction specifically within the PVN, in the control of eating behavior. PMID- 3008933 TI - Circadian corticosterone rhythm did not develop in rats seven weeks after destruction with 5,7-dihydroxytryptamine of the serotoninergic nerve terminals in the suprachiasmatic nucleus at the age of 16 days. AB - In order to study the role of the serotoninergic innervation of the hypothalamic suprachiasmatic nucleus (SCN) in the development of the circadian rhythm of corticosterone 5,7-dihydroxytryptamine (5,7-DHT) selectively destroying the serotoninergic structures was injected into the cell group of 16-day-old male rats prior to the appearance of the corticosterone rhythm. Rats treated with 5,7 DHT did not show circadian fluctuations in plasma corticosterone concentrations 3, 5 and 7 weeks after the injection of the neurotoxin. Their hypothalamo pituitary-adrenal system responded to ether stress. Immunocytochemistry revealed that only a very few serotonin immunoreactive elements were visible in the SCN of the 5,7-DHT-treated rats. The results indicate that serotoninergic innervation of the SCN is essential for the development of the corticosterone rhythm. PMID- 3008935 TI - The effects of 4-aminopyridine on the spinal cord: rhythmic discharges recorded from the peripheral nerves. AB - The effects of an intravenous injection (20 mg/kg) of 4-aminopyridine (4-AP) were initially investigated in acute low spinal cats (Th 13), in which L-DOPA had induced fictive locomotion after paralysis. 4-AP first accelerated the locomotor rhythm and could also change markedly the pattern of activation of some muscle nerves. Shortly after, the locomotor activity was replaced by synchronous rhythmic discharges (2.5-8.5 Hz) in flexor and extensor muscle nerves of the same limb girdle. Similar rhythmic activity was recorded after 4-AP alone (5-20 mg/kg) in the acute decerebrate spinal cat. Whilst the mean rate of the rhythmic activity could differ in the two limb girdles, discharges generated in one girdle appeared to be strongly influenced by those generated in the other. After a complete section of the spinal cord (Th13), the activity persisted in both the rostral and caudal segments although the interactions between the two disappeared. The persistence of the rhythmic activity caudal to the section underscores its spinal origin. In the chronic spinal rat, such rhythmic activity could still be induced in the lumbo-sacral cord despite degeneration of descending pathways. It appears that large doses of 4-AP exert potent effects on the spinal cord which can override other patterns of activity and synchronize the electrical activity of many neuronal elements. PMID- 3008934 TI - Involvement of the midbrain reticular formation in self-injurious behavior, stereotyped behavior, and analgesia induced by intranigral microinjection of muscimol. AB - Bilateral microinjection of muscimol (60 ng), a gamma-aminobutyric acid (GABA) agonist, into the central region of the substantia nigra (pars reticulata) produced self-injurious behavior (SIB), stereotyped behavior and analgesic-like effects in rats. Bilateral electrolytic lesions of the midbrain reticular formation ventrolateral to the periaqueductal gray matter completely blocked the SIB but had little effect on stereotyped behavior produced by intranigral muscimol. Lesions of the midbrain reticular formation reduced the antinociceptive effect of intranigral muscimol on the tail-flick but not on the hot-plate test. Bilateral microinjection of muscimol (10-100 ng) into the midbrain reticular formation produced intense stereotyped behavior and had an analgesic-like effect on the hot-plate test but not on the tail-flick test. Stereotyped behavior appeared to interfere with the paw-lick response on the hot-plate test. These data suggest that the antinociceptive effect of intranigral muscimol on the tail flick test is mediated by fibers that project to or pass through the midbrain reticular formation and that analgesia may play an important role in muscimol induced SIB. The midbrain reticular formation does not appear to be involved in the stereotyped behavior produced by intranigral muscimol. PMID- 3008936 TI - Plasticity of catecholaminergic terminals in rat paraventricular hypothalamic nucleus after 6-hydroxydopamine lesion: an emphasis on bouton sizes and synaptic frequency. AB - Catecholaminergic (CA) nerve terminals in the paraventricular hypothalamic nucleus (PVN) of adult rats were studied at 4, 21, 56 and 180 days after a single injection of 6-hydroxydopamine (6-OHDA) neurotoxin into the right lateral ventricle of the brain. We previously described and quantified the extent of CA terminal sprouting in the PVN after 6-OHDA lesions. For this communication we studied parameters, specifically the bouton sizes and the synaptic frequencies of CA terminals during the renewal process, and evaluated how changes of these parameters are related to axonal sprouting. The CA boutons were identifiable in the electron microscope by exhibiting small granular vesicles (SGVs) after central administration of 5-hydroxydopamine (5-OHDA) marker. The marked CA boutons were measured and further categorized according to whether or not they were associated with distinct synaptic specializations at various post-lesion stages. The average sizes of CA boutons were strikingly similar in their diameters (1.0 micron) for both control and experimental tissues. However, CA boutons larger than 2.1 micron were rare and seen more often in the experimental tissues with 6-OHDA lesion and were sustained up to 180 days after lesions. Catecholaminergic profiles with ultrastructural features of growth cones were also seen in the PVN following the 6-OHDA lesions, indicating that there is growth activity in the PVN after 6-OHDA lesion. There were 33% of CA boutons in the PVN from the control tissues that appeared to have synaptic contacts.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3008937 TI - Corticotropin releasing factor (CRF) receptor antagonist blocks activating and 'anxiogenic' actions of CRF in the rat. AB - The effectiveness of a recently synthesized corticotropin releasing factor (CRF) antagonist, alpha-helical CRF9-41, in reversing the locomotor activating and proconflict effects of CRF was evaluated. The CRF receptor antagonist (50, 100 and 200 micrograms, i.c.v.) produced a dose-related attenuation of the response suppressing effects of CRF in a conflict model of anxiety. The antagonist also effectively suppressed the marked locomotor activation produced by CRF. No discernible intrinsic effects on behavior were noted when the antagonist was administered alone. These results suggest that the behavioral effects of CRF are receptor-mediated phenomena and point to the potential usefulness of a CRF antagonist in understanding the function of endogenous CRF in mediating responses to stressful stimuli. PMID- 3008938 TI - Identification of glycinergic synapses in the cochlear nucleus through immunocytochemical localization of the postsynaptic receptor. AB - The distribution and morphology of glycinergic synapses in the cochlear nucleus were investigated using monoclonal antibodies against the glycine receptor. Glycine receptor immunoreactivity was seen on somas and proximal processes of most cells in all divisions of the cochlear nucleus; distribution of label in neuropil was denser in the dorsal cochlear nucleus and granule cell cap than in the ventral cochlear nucleus. At the ultrastructural level, glycine receptor immunoreactivity was specifically distributed postsynaptically to terminals that contained flattened vesicles in the guinea pig anteroventral cochlear nucleus. These studies show that the immunocytochemical localization of the glycine receptor can provide a means of identifying and characterizing glycinergic synapses throughout the central nervous system. PMID- 3008939 TI - Mesolimbic dopamine projection modulates amygdala-evoked EPSP in nucleus accumbens neurons: an in vivo study. AB - In urethane anesthetized rats, excitatory postsynaptic potential (EPSP) recorded intracellularly from nucleus accumbens neurons following stimulation of the amygdala was attenuated by repetitive stimulation of the ventral tegmental area (VTA). VTA stimulation also depolarized the resting membrane potential of accumbens neurons. Attenuation of the EPSP and membrane depolarization were frequently dissociated but both were blocked by haloperidol, a dopamine antagonist. PMID- 3008940 TI - Characterization of hypothalamic noradrenaline receptors in the supraoptic nucleus and periventricular region of the paraventricular nucleus of mice in vitro. AB - In an attempt to determine the basis for apparently conflicting reports of the effects of noradrenaline (NA) on the neurohypophyseal system and its effects on the parvocellular periventricular region of the paraventricular nucleus (PVN), recordings were made from the neurons in the supraoptic nucleus (SON) and the periventricular region in the mouse hypothalamic slice preparation. Of 47 SON neurons, 43 (91%) were excited and two (4%) were inhibited by NA. Seven SON neurons increased the firing rate with increase of NA concentration (10(-7)-10( 4) M). Both the alpha 1-agonists phenylephrine and methoxamine also increased the activity of all SON neurons tested whereas application of the alpha 2-agonist clonidine and the beta-agonist isoproterenol had weak and inconsistent effects. While the alpha 2-antagonist yohimbine had no consistent influence, the alpha 1 antagonist prazosin blocked or reversed the effects of NA. Another group of 37 neurons in the periventricular region of the PVN was also tested; 13 (35%) were excited and 22 (59%) inhibited by application of NA (10(-5) M). When tested with phenylephrine or methoxamine, 6 of the 7 neurons were excited and one inhibited but all the 4 neurons tested were excited by isoproterenol. Clonidine strongly depressed the activity of all 12 neurons tested. The NA-induced excitatory effects were suppressed or reversed by pre-application of prazosin and the beta antagonist propranolol while the inhibitory ones were suppressed or reversed by yohimbine. Synaptic blockade did not affect the excitatory responses of SON cells to NA nor the inhibitory responses of periventricular neurons to NA or clonidine. We conclude that SON neurons receive adrenergic excitatory effects mainly through alpha 1-receptors. The periventricular neurons receive the excitatory effects through alpha 1- or beta-receptors and receive the inhibitory effects through alpha 2-receptors. PMID- 3008941 TI - Chemoarchitectonic zonation of the monkey cerebellum. AB - The modular organization of the monkey cerebellum is revealed in normal material by histochemical methods for demonstrating the presence of the enzymes acetylcholinesterase (AChE) and cytochrome oxidase (CO). In the cerebellar white matter, AChE- and CO-rich fibers are distributed in longitudinal compartments, which are aligned with AChE- and CO-rich bands in the granule cell layer, and with narrower AChE-rich strips in the molecular layer. The number and disposition of zones demonstrated histochemically are consistent with, and extend, the basic scheme of cerebellar zonation established by Voogd and Voogd and Bigare. PMID- 3008942 TI - Effect of aging on in vitro dopamine biosynthesis in the median eminence of rat hypothalamic slices. AB - The rates of basal and cyclic AMP-dependent DOPA accumulation in the median eminence of hypothalamic slices were not different between ovariectomized young and aged rats. However, the rate of Ca2+-dependent, depolarization-induced DOPA accumulation was smaller in aged rats, suggesting that the Ca2+ system in the regulation of dopamine biosynthesis in tuberoinfundibular dopaminergic neurons is altered by aging. PMID- 3008943 TI - Benzodiazepine- and barbiturate-interactions with GABAA receptor responses on lactotrophs. AB - Modulation of the biphasic effect of muscimol on prolactin secretion by benzodiazepines and secobarbital was investigated, using an in vitro superfusion system. The stimulatory effect of low concentrations of muscimol was potentiated by both classes of drugs, and the effect of benzodiazepines appeared to be mediated by central-type benzodiazepine receptors. Neither benzodiazepines nor secobarbital affected the inhibitory response to muscimol. Clonazepam reduced the potency of bicuculline methiodide as an antagonist of the stimulatory effect, but did not alter the potency of picrotoxinin. These results demonstrate a selective potentiation of one component of the GABAA receptor effect on lactotrophs by benzodiazepines and barbiturates and provide evidence for a functional effect of these drugs at a site without the CNS. PMID- 3008944 TI - Evidence for the functional role of monosialoganglioside GM1 in synaptic transmission in the rat hippocampus. AB - The hippocampal slices were incubated with compounds which hydrolyze, modify or bind with sialic acid containing molecules. The efficiency of synaptic transmission was tested in the presence of these compounds. The size of the evoked extracellularly recorded potential following Schaffer collateral stimulation was used as an indicator of synaptic transmission efficiency. Sodium periodate (10 mM) and sodium perchlorate (59.2 mM) evoked a reversible (after washout) decrease in the size of the population spike. Higher concentration of sodium periodate (60 mM) abolished the size of the population spike, which was only poorly reversible after washout. Tetanus toxin, which binds to polysialogangliosides, and neuraminidase from Vibrio cholerae (an enzyme which splits off sialic acid from polysialogangliosides, leaving GM1 intact, and splits off sialic acid from sialoglycoproteins) had no influence on the size of the population spike. Cholera toxin, which binds to GM1, slightly reduced the size of the population spike. Incubation of the slices with neuraminidase from Arthrobacter ureafaciens (an enzyme which splits off sialic acid from all gangliosides, including GM1, and from sialoglycoproteins) abolished the population spike after 5 h. GM1 antiserum abolished the potential after approximately 100 min. The conclusion is drawn that of all gangliosides only GM1 is necessary to support synaptic transmission in Schaffer collateral-pyramidal cell synapses. PMID- 3008946 TI - Effect of single and repeated doses of acrylamide and bis-acrylamide on glutathione-S-transferase and dopamine receptors in rat brain. AB - The effect of single and repeated doses of acrylamide (a neurotoxin) and N,N' methylene-bis-acrylamide (a non-neurotoxic analogue of acrylamide) on glutathione (GSH), glutathione-S-transferase (GST) and dopamine receptors has been studied in rat brain. In vitro, both acrylamide and bis-acrylamide decreased brain GSH content in a concentration-dependent manner. At equimillimolar concentrations (2 10 mM) bis-acrylamide was more effective than acrylamide in lowering GSH levels. In vitro, GST activity was also inhibited as a function of acrylamide concentration. A single dose of either acrylamide or bis-acrylamide depleted GSH content of rat brain in a concentration-dependent manner without inhibiting GST activity. Repeated administration of either acrylamide or bis-acrylamide in rats (50 mg/kg X 10 days) decreased GSH content in the brain but GST activity was inhibited only by acrylamide and not by bis-acrylamide. Single or repeated injections of acrylamide but not of bis-acrylamide increased brain dopamine receptors ([3H]spiroperidol binding) in a concentration-dependent manner. PMID- 3008945 TI - Noradrenaline-stimulated inositol phospholipid breakdown in rat dorsal lateral geniculate nucleus neurones. AB - Noradrenaline-stimulated inositol phospholipid breakdown in matched vibratome sections through the rat dorsal lateral geniculate nucleus (dLGN). The response was measured as a large accumulation of [3H]inositol labelled inositol monophosphate and was mediated via activation of alpha 1-adrenergic receptors. Accumulation of [3H]inositol phosphates was reduced in kainic acid-lesioned animals, indicating that this response occurred within dLGN neurones and not afferent terminals. The results implicate inositol phospholipid breakdown as part of the mechanism of noradrenergic neurotransmission within the dLGN. PMID- 3008947 TI - Increased delta, but not mu, opiate receptor binding in the medulla oblongata of Long-Evans rats following 5-day water deprivation. AB - Opiate receptors of the mu type were labeled with [125I]D-Ala2,N-Me-Phe4,Met-(O)5 ol-enkephalin (FK-33824). delta receptors were labeled with [125I]D-Ala2-D-Leu5 enkephalin (DADLE) in the presence of excess (N-Me-Phe3,D-Pro4)-morphiceptin (PL017). Since DADLE binds mu and delta receptor sites, and PL017 blocks mu receptors, this protocol improves specific labeling of delta receptors. Quantitative autoradiography showed that chronic dehydration causes no changes in mu receptor binding in the medulla oblongata of Long-Evans rats. However, there is increased delta receptor binding in the solitary, hypoglossal and gracilis nuclei, and the spinal nucleus of trigeminal system of dehydrated animals, suggesting that delta opiate receptors participate in the physiological response to dehydration. PMID- 3008948 TI - Localization of GABAA and GABAB receptor subtypes on serotonergic neurons. AB - The effect of selective destruction of serotonin (5-HT)-containing neurons with 5,7-dihydroxytryptamine (5,7-DHT) on [3H] muscimol and (-)-[3H]baclofen binding was investigated in various rat brain regions. Ten days after intracerebroventricular 5,7-DHT, serotonin levels and [3H]imipramine binding were markedly decreased. 5,7-DHT reduced [3H]muscimol binding only in the mesencephalon, and (-)-[3H]baclofen binding was unmodified in all the areas considered. These results suggest that except in the mesencephalon GABA receptors may not be localized on serotonergic nerve terminals. PMID- 3008949 TI - Dibutyryl cyclic guanine monophosphate inhibits natural but not limb amputation induced motoneurone death in the chick embryo. AB - The number and distribution of motoneurones in the lateral motor columns of normal chick embryos have been compared with embryos treated daily with dibutyryl cyclic guanine monophosphate and subject to unilateral limb bud amputation. Treatment with cyclic nucleotide rescued about 17% of the motoneurones compared with controls by Stage 37, but did not prevent amputation-induced motoneurone death. The role of cyclic nucleotides in motoneurone survival is therefore permissive, rather than instructive. PMID- 3008950 TI - Effect of postnatal light deprivation on the ontogenesis of dopamine neurons in rat retina. AB - In rats raised from birth under a regular dark-light cycle, retinal dopamine (DA) levels were low at 12 days, increased progressively and steeply at 16, 20, 30 and 60 days and peaked at about 75 days postnatally. Dark-rearing from birth suppressed the magnitude of the normal developmental pattern of DA concentrations in retina and at 20, 30 and 60 days of age they were markedly smaller compared with those in age-matched controls grown under regular cyclic illumination. In adult rats reared up to the age of 60 days in regular cyclic lighting and then transferred to complete darkness for additional periods of 7-60 days, there were marked reductions in retinal DA levels. In rats reared in total darkness for 16, 20 or 30 days after birth and then transferred to regular dark-light conditions for additional periods of up to 30 or 60 days, retinal DA levels were always higher than those in animals with continuous light deprivation. They were similar to or even higher than those in rats grown under regular dark-light cycle. Findings suggest that light stimulation is essential for the postnatal ontogenetic increases of synthesis and storage of DA within retinal DAergic neurons. Suppressed DA accumulation in retina induced by dark-rearing from birth is not irreversible and can be completely corrected by re-exposure to regular lighting conditions regardless of the durations of postnatal light deprivation. PMID- 3008951 TI - Early appearance of inhibition in the neonatal rat olfactory bulb. AB - The functional development of inhibition in the rat olfactory bulb was examined in the present study. Inhibition of presumed mitral cell spontaneous activity following stimulation of the lateral olfactory tract was present by postnatal day 5, the youngest age tested. The duration of this inhibition was greatest in young animals, decreasing after postnatal day 15. Possible mechanisms of this enhanced inhibition in neonates were discussed. PMID- 3008952 TI - Plasticity in the phenotypic expression of brain opioid receptors: differential response of forebrain and hindbrain cultures to chemical depolarization. AB - Potassium chloride at a depolarizing concentration has a differential effect on the expression of opioid receptors in aggregating neuronal cultures prepared from the rat forebrain or hindbrain. The mu-selective enkephalin analogue DAGO was used to indicate that the effect of potassium chloride is not uniform on all subtypes of opioid receptors. These results suggest for the first time that chemical depolarization may modulate the phenotypic expression of opioid receptors. PMID- 3008953 TI - Cyclic nucleotide content of ciliary and dorsal root ganglia during embryonic development in the chick. AB - The concentration of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) was measured in developing chick ciliary (CG) and dorsal root ganglia (DRG) as a function of embryonic age from day 8 through day 18 using radioimmunoassays. The concentration of cAMP and cGMP increased in both ganglia from day 8 through day 14. cAMP levels were nearly two-fold higher in DRG than in CG. Normalization of the data for ciliary ganglia to the number of cells per ganglion and calculation of their molar concentrations indicates a 48% increase in cGMP and a 3.2-fold increase in cAMP during the developmental process of natural neuronal cell death. PMID- 3008954 TI - Interactions between the duration of stimulation and noradrenaline on cholinergic transmission in the myenteric plexus-smooth muscle preparation. AB - Output of acetylcholine (ACh), electromyogram (EMG) recordings and contractions of myenteric plexus-longitudinal muscle strip preparations from the guinea-pig ileum were studied during stimulation by single impulses or by trains (30 Hz; 2 to 128 impulses) under control conditions and in the presence of noradrenaline (NA). During supramaximal stimulation NA (2.5 microM) inhibited both contractions of the smooth muscle and the release of ACh evoked by single impulses more effectively than those evoked by train stimulation so that in a train of 4 impulses the output of ACh per impulse after the 2nd to 4th impulses was 69 to 104% higher than the output after the 1st impulse. During submaximal stimulation, contractions and ACh release evoked by single impulses were almost completely inhibited by NA. The neurogenic EMG, a direct consequence of the localized action of released transmitter (ACh), was recorded in the longitudinal muscle 4 and 10 mm aborally from the focal stimulation site. The incidence of the neurogenic response was much higher at the proximal (4 mm) than at the distal (10 mm) site and was proportional to the number of impulses in a train (100 Hz). NA inhibited propagation of the neurogenic response evoked by single impulses whereas its effect during train stimulation was less. It is concluded that in the course of train stimulation, sites of transmission more distant from the stimulation focus was recruited, and consequently the secretion of ACh in succeeding impulses was enhanced. NA could interfere with this process; it might inhibit the invasion by action potentials of cholinergic nerve terminal varicosities, thereby reducing the release of ACh. PMID- 3008956 TI - Circling from unilateral VTA morphine: direction is controlled by environmental stimuli. AB - Crystalline morphine was applied unilaterally to the ventral tegmental area of rats. Contralateral circling was observed in traditional test chambers and inside the walls of an elevated square enclosure. When tested outside the walls of the same enclosure, the animals turned in the opposite direction, maintaining the same flank in the position nearest the wall. This dopamine-dependent circling thus reflects a more complex interaction between the side of activation and the direction of circling than is implied in current views of dopamine function. PMID- 3008955 TI - Autoradiographic localization of receptors in the cochlear nucleus of the mouse. AB - Light microscopic autoradiography of bound radiolabeled ligands was used to describe the distribution of six receptor types in the dorsal and ventral mouse cochlear nuclei: Glycine receptor ([3H]strychnine); GABA receptor ([3H]muscimol); benzodiazepine receptor ([3H]flunitrazepam); adenosine receptor ([3H]cyclohexyladenosine); muscarinic ACh receptor ([3H]quinuclidinyl benzilate); histamine receptor ([3H]mepyramine). The most intense [3H]strychnine labeling was observed in the deep region of the dorsal cochlear nucleus (DCN), with slightly lower levels in the molecular and pyramidal layers. Highest density of [3H]muscimol binding sites was observed in the granule cell layer of the posterior ventral nucleus (PVCN) and in the pyramidal layer of the DCN. Diffuse [3H]flunitrazepam labeling was distributed over all laminar regions of the DCN; the highest grain density was observed over the granule cell layer of the PVCN. Intense [3H]cyclohexyladenosine labeling was seen over the molecular layer, possibly extending into the pyramidal layer, of the DCN. The granule cell layer of the PVCN was also densely labeled. High concentrations of [3H]quinuclidinyl benzilate sites were seen in the molecular layer, possibly extending into the pyramidal layer, of the DCN. A thin band of high grain density was also visible over the granule cell layer of the PVCN. Moderate, diffuse [3H]mepyramine labeling was visible throughout the DCN, with slightly higher grain density over the molecular, and possibly the pyramidal layers, than over the deep region of the DCN. PMID- 3008957 TI - Glucose regulates [3H](+)-amphetamine binding and Na+K+ ATPase activity in the hypothalamus: a proposed mechanism for the glucostatic control of feeding and satiety. AB - Binding sites for [3H](+)-amphetamine in the hypothalamus may mediate the anorectic actions of amphetamine and related phenylethylamines. To investigate further the role of these sites in the central control of appetite, the binding of [3H](+)-amphetamine to the hypothalamus and brainstem was measured following food deprivation and refeeding, the onset of genetic obesity, or the administration of 2-deoxy-D-glucose. Food deprivation for 24 to 72 hours reduced the Bmax for [3H](+)-amphetamine binding in the hypothalamus and brainstem but not in other brain areas or peripheral tissues. The decrease in hypothalamic and brainstem [3H](+)-amphetamine binding observed following food deprivation was time-dependent and rapidly reversed by brief refeeding with either rat chow or a 10% glucose solution. Moreover the changes in [3H](+)-amphetamine binding were highly correlated to corresponding alterations in blood glucose concentration. Furthermore, D-glucose, but not L-glucose increases the number of hypothalamic [3H](+)-amphetamine binding sites when administered in vivo or when added to hypothalamic slices in vitro. These data suggest that the [3H](+)-amphetamine binding site in the hypothalamus and (or) brainstem may be coupled to a central "glucostat." PMID- 3008958 TI - [Study of acupuncture meridians using radioactive tracers]. PMID- 3008959 TI - [Molecular aspects of bioelectrogenesis: application to epilepsy]. PMID- 3008961 TI - [Chromosome anomalies of human cells transformed by the Rous sarcoma virus]. PMID- 3008960 TI - [Viral aspects of nasopharyngeal cancer]. PMID- 3008963 TI - [Epidemic periodicity of adenovirus type 3 and 7 pneumonia in infants and children]. PMID- 3008962 TI - [Superoxide dismutase and free radical pathology]. AB - Different defense systems against oxidative damage leading to pathological conditions are described. The superoxide radical plays a primary role in initiating and sustaining biological damage and is responsible for the production of other free radicals and lipoperoxides. Certain pathologies are associated with these events such as post-irradiation necrosis, or the Spanish Toxic Oil Syndrome. Superoxide dismutase has been used clinically with considerable success, particularly the liposomal form, to treat various diseases in which it has been shown that the superoxide radical plays an important role. Although the mechanism of the enzymic reaction catalysed by superoxide dismutase is now well defined, a complete explanation of the anti-inflammatory properties in vivo of the enzyme has not yet been established. PMID- 3008964 TI - [Demonstration of latent infection by herpes simplex virus type 2 (HSV-2) in the spinal cord of the guinea pig]. PMID- 3008965 TI - [Inhibitory effect of tai-ding-an on DNA and protein synthesis of herpes simplex virus type 2 (HSV-2)]. PMID- 3008966 TI - [Diagnosis of hepatic tumor by histogram pattern on the ultrasound image]. PMID- 3008967 TI - Nurses and AIDS: do we know the facts? PMID- 3008968 TI - Alterations in alpha 1-adrenoceptor stimulation of isolated atria from experimental diabetic rats. AB - This purpose of this investigation was to determine the influence of experimental diabetes (3 months) on the responsiveness of rat isolated atria to alpha 1 adrenoceptor stimulation by phenylephrine. Diabetes was chemically induced with streptozotocin (65 mg/kg i.v.) in 42- to 43-day-old, nonfasted male Sprague Dawley derived rats. Chronotropic (right atria) and inotropic (left atria) indices were recorded in response to alpha 1-adrenoceptor stimulation by phenylephrine. These experiments were performed in the presence of beta adrenoceptor antagonism (timolol). Isolated right atria from diabetic rats demonstrated a greater increase in heart rate in response to phenylephrine than did corresponding control atria. Left atria were supersensitive (decrease in EC50 values) and hyperresponsive to alpha 1-adrenoceptor stimulation by phenylephrine when compared with stimulation of control left atria. Diabetic left atria in response to phenylephrine were observed to exchange more radioactive calcium (45Ca2+) than control left atria, whereas both diabetic and control left atria exchanged the same amount of 45Ca2+ during basal contractile conditions. Phenylephrine had no effect on 45Ca2+ efflux from either diabetic or control atria. These results indicate that 3 months of uncontrolled experimental diabetes in the rat produces an enhancement of alpha 1-adrenoceptor activation of isolated atria, and that there is an alteration in Ca2+ mobilization which may contribute to the enhanced receptor activation. PMID- 3008969 TI - Steroid metabolism by cells from human chorion laeve isolated on Percoll gradients. AB - Collagenase-dispersed cells from human chorion laeve were examined on Percoll gradients. The 3 beta-hydroxysteroid dehydrogenase (a trophoblast marker) and steroid sulfatase activities of the cells were measured and a system was developed to isolate enriched preparations of the trophoblast cells. No cells were found to sediment at Percoll concentrations greater than 50%, and using continuous gradients of Percoll there appeared to be cells with different 3 beta hydroxysteroid dehydrogenase (3 beta HSD): steroid sulfatase ratios sedimenting in different regions of the gradient. Cells with a high ratio were found in the denser region of the gradient. Continuous gradients provided inadequate separations of distinct populations of cells, thus to obtain a more reproducible system to isolate cells, discontinuous gradients of Percoll were studied. A discontinuous gradient composed of 5, 20, 40, and 60% Percoll was developed and three bands of cells were found sedimenting at the 20, 40 and 60% interfaces, respectively. The number and appearance of cells at the 20 and 60% interfaces varied from tissue to tissue. In contrast, the cells sedimenting at the 40% interface were less variable, a substantial number was found to be present in every tissue studied, they were similar in appearance to the trophoblast cells and had high 3 beta HSD:sulfatase ratios. PMID- 3008970 TI - Incidence of antibiotic resistance and characterization of plasmids in Campylobacter jejuni strains isolated from clinical sources in Alberta, Canada. AB - Antibiotic susceptibilities of 382 strains of Campylobacter jejuni isolated in Alberta, Canada, in 1980 and 1981 were determined. Although none were resistant to erythromycin or gentamicin, 5.4 and 22% of strains were resistant to ampicillin in 1980 and 1981, respectively. Tetracycline resistance was noted in 6.8% of strains in 1980 and in 8.6% in 1981. Moreover, resistance to high-level tetracycline (32-128 micrograms/mL) was always mediated by a plasmid of 45-50 kilobases. Three transmissible plasmids from the Alberta strains were compared with the prototype plasmid pMAK175 by restriction enzyme analysis and some minor differences in restriction sites were noted between pMAK175 and pUA183. The two other plasmids pUA142 and pUA143 were 4 kilobases larger than pMAK175 and contained additional restriction sites. However, in all plasmids examined, the HincII and AccI fragments where the tetracycline-resistance determinant was located were shown to be conserved. PMID- 3008971 TI - Susceptibility of gram-negative bacteria to the synergistic bactericidal action of serum and polymyxin B nonapeptide. AB - Polymyxin B nonapeptide was able to sensitize Escherichia coli strains and strains of Salmonella typhimurium, Klebsiella spp., Enterobacter cloacae, Pseudomonas aeruginosa, and Haemophilus influenzae to the bactericidal action of fresh normal human serum. The degree of sensitization varied significantly within the strains. Strains of Proteus mirabilis, Neisseria gonorrhoeae, and N. meningitidis remained resistant. PMID- 3008973 TI - Threshold exposure level for chrysotile. PMID- 3008972 TI - A guide to the investigation and treatment of patients with AIDS and AIDS-related disorders. PMID- 3008975 TI - The rapid onset of cutaneous angiosarcoma after radiotherapy for breast carcinoma. AB - Malignant neoplasms known to develop following external beam radiation include squamous cell carcinoma, osteosarcoma, chondrosarcoma, malignant fibrous histiocytoma, mixed mullerian tumors, malignant schwannoma, myelogenous leukemia and angiosarcoma. Latency periods of many years characterize the onset of these tumors following the exposure. Cutaneous angiosarcoma following radiotherapy for breast carcinoma has been rarely documented, occurring up to 13 years postirradiation. Two cases of this entity are reported occurring 37 months postradiotherapy at the site of mastectomy performed for mammary duct carcinoma. PMID- 3008976 TI - Active uptake of testosterone by androgen receptors of hepatocellular carcinoma in humans. AB - Hepatocellular carcinoma (HCC) is more prevalent in males than it is in females, which has often been explained by the fact that alcoholism and chronic hepatitis B virus infection are more prevalent among males. The current studies, using biochemical and autoradiographic methods, verified that HCC contains higher concentrations of androgen receptors than the surrounding liver parenchyma and that extrinsically given testosterone are actively taken up by such tumors. These results may suggest that HCC is an androgen-dependent tumor and that, therefore, this tumor is more prevalent in males than it is in females. PMID- 3008977 TI - Serum beta-2 microglobulin levels in homosexual men with AIDS and with persistent, generalized lymphadenopathy. AB - In order to investigate the nature of the immune disorders associated with the acquired immune deficiency syndrome (AIDS) and the AIDS-related condition of persistent, generalized lymphadenopathy (PGL), serum beta-2 microglobulin (beta 2 M) levels were determined in patients with AIDS and PGL and in asymptomatic homosexual and heterosexual controls. Sixteen of 20 (80%) patients with AIDS exhibited elevated beta 2-M levels. In contrast, 20 of 44 (45%) patients with PGL, 4 of 20 (20%) asymptomatic homosexuals, and only 3 of 46 (7%) heterosexuals had increased serum beta 2-M levels (P less than 0.001). When considering mean levels of beta 2-M, only the asymptomatic control individuals had normal values. AIDS patients had significantly higher mean beta 2-M levels when compared to all other groups (P less than 0.05). The mean level for PGL patients was greater than that in the homosexual and heterosexual controls (P less than 0.05). No relationship was found between presence of antibody to human T-lymphotropic retrovirus (HTLV-III) and beta 2-M levels in the patients with AIDS or PGL. The authors conclude that beta 2-M is elevated in patients with AIDS and PGL, suggesting an increased turnover of a certain subpopulation of lymphocytes in these patients. Beta 2-M levels also appear to parallel disease activity, as well as immune dysfunction, with the greatest elevation occurring in patients with AIDS, followed by those with PGL, and asymptomatic homosexuals. Beta 2-M levels may be a useful confirmatory marker in AIDS and its related disorders. PMID- 3008979 TI - Response of cranial nerve abnormalities in nasopharyngeal carcinoma to radiation therapy. AB - Eighteen of 36 patients (50%) with the diagnosis of nasopharyngeal carcinoma had cranial nerve deficits before definitive radiotherapy. Within this group of 18 patients, there were 34 cranial nerve abnormalities and four Horner's syndromes. Overall, 62% of cranial nerve deficits recovered completely (CR) and 32% recovered partially (PR), for a total response rate of 94% to definitive radiotherapy. The magnitude of response (complete versus partial) depended upon the individual cranial nerve and the pretreatment duration of the abnormality. All of the responses except one occurred within 1 month after the completion of therapy. Complete responses were not obtained when deficits had existed longer than 2 months. However, PRs were obtainable. Seven of seven cases of posttreatment new or recurrent cranial nerve deficits were caused by recurrent tumor. The actuarial 5-year disease-free survival for this group of 18 patients was 31%. The results indicate that patients with cranial nerve deficits will respond to definitive radiotherapy and long-term disease-free survival can be achieved in some patients. PMID- 3008978 TI - Malignant fibrous histiocytoma of soft tissue in childhood. AB - Seven children aged 6 months to 11 years with malignant fibrous histiocytoma, a type of sarcoma of soft tissues, have been treated at the Children's Hospital of Philadelphia from January 1975 through July 1983. The primary tumor arose in the head and neck region in three patients, the chest wall in two patients and the pelvis or buttock in one patient each. Operative management consisted of complete tumor removal in the two patients with chest wall tumors, and biopsy only in the remaining five children. Afterward, all seven patients were treated with a multiple-agent chemotherapy program consisting of vincristine, dactinomycin, and cyclophosphamide for two years, with or without Adriamycin (doxorubicin). The five patients with residual tumor also received radiation therapy (RT) in doses of 1500 to 5500 rad. The two children with localized, completely excised sarcoma are continuously free of tumor at 1.4 and 9 years after initiation of treatment. Of the five with residual sarcoma, three had a complete response to radiation and chemotherapy, and two of them are free of recurrence at 4 and 5 years, respectively. In the three remaining children, the tumor spread regionally into the central nervous system or distantly into the lungs, subcutaneous tissues, and liver. Childhood malignant fibrous histiocytoma of soft tissue appears to be similar to childhood rhabdomyosarcoma in its modes of spread and response to management. Operative removal is the key to successful therapy. The roles of multiple-agent chemotherapy and RT remain to be defined. Adriamycin appears to be the most promising single agent. In the absence of concrete data, it seems prudent to follow the same guidelines for irradiation as those used for other soft tissue sarcomas of childhood. PMID- 3008974 TI - Pharmacophysiology of atopic dermatitis. AB - Atopic dermatitis is clearly characterized by altered cutaneous physiologic responses. There is a tendency to acral vasoconstriction. Rubbing causes skin pallor and white dermographism. Vascular instability is demonstrated by responses to cholinergic agents, histamine, and nicotinates. Psychophysiologic studies demonstrate exaggerated vasodilator responses to emotional stress with consequent pruritus and scratching. The itch threshold is low, duration is prolonged, and nighttime scratching movements may be frequent or almost continuous. Regardless of the inciting trigger factors, the scratching causes the damage and the severe dermatitis. Thermal as well as emotional stimuli to sweating cause severe itching in AD, yet the concept of a miliaria-type, poral occlusion mechanism remains unproven. Some studies suggest actually increased sweating along with erythema and pruritus during acute flares of AD. The concept of sweat-borne allergens causing skin reactions during sweating is interesting but has never been proven. Studies of sweat responses to pharmacologic agents have produced conflicting data, and attempts to link these responses to Szentivanyi's beta-adrenergic blockade theory are not convincing. The numerous variables of climate, season, sex, age, and habitus affect sweating greatly. Future studies must carefully control for each of these factors before pharmacologically induced sweat responses can be interpreted clearly. A number of lines of evidence suggest involvement of histamine and other mediators in the evolution of erythema, pruritus, and scratching in AD. Flares of the condition have been reproducibly evoked by only two incitants: experimental emotional stress interviews and specific food challenge in selected sensitive individuals. In the latter, increased plasma histamine has been demonstrated, presumably generated by antigen/IgE stimulated degranulation of mast cells in the gut and/or skin. The demonstrated increased histamine releasability of basophils from atopic individuals may be the result of defective cellular regulatory mechanisms. Recent studies have demonstrated increased cyclic AMP-phosphodiesterase activity in leukocytes from atopic individuals. The resultant decreased intracellular cyclic AMP removes an inhibitory factor, which in turn causes net cellular hyperresponsiveness. This effect has been shown to account, at least in part, for increased histamine release from leukocytes of patients with AD. These and other studies focused upon cell functional regulation are providing better understanding of basic biochemical abnormalities and may lead to improved diagnostic and therapeutic approaches in managing atopic disease. PMID- 3008980 TI - Malignant fibrous histiocytoma induced by intra-articular injection of 9,10 dimethyl-1,2-benzanthracene in the rat. Pathological and enzyme histochemical studies. AB - Malignant fibrous histiocytoma (MFH) was produced by injection of 9,10-dimethyl 1,2-benzanthracene (DMBA) into the rat knee joint. The tumor was observed in or around the knee in nearly all the animals 13 to 36 weeks after the initial DMBA administration. Histologically, these lesions were of the storiform-pleomorphic type (39/58, 67.2%), myxoid type (9/58, 15.5%), or giant cell type (8/58, 13.8%). Six cell types reported in human MFH were confirmed and phagocytosis of 0.81 micron latex particles by histiocyte-like cells was noted by electron microscopic examination. Acid phosphatase, beta-glucuronidase, and alpha-naphthyl acetate esterase were positive in enzyme histochemical examinations. Acid phosphatase activity was electron microscopically noted primarily in the lysosomes and the Golgi apparatus of the histiocyte-like cells. Cells from the storiform pleomorphic (M1) and myxoid (M2) type tumors were serially transplanted subcutaneously in the back of the rats, and are now at the thirtieth and fortieth passage, respectively. They also were studied by enzyme histochemical and electron microscopic techniques. Our observations suggested an undifferentiated mesenchymal cell origin of MFH. Transplantable MFH can be produced in rats by intra-articular injection of DMBA, and lesions thus produced are a useful experimental model for the investigation of the histogenesis and the effect of chemotherapy of MFH. PMID- 3008981 TI - Malignant fibrous histiocytoma of soft tissue. Correlation between clinical variables and histologic malignancy grade. AB - The prognosis for malignant fibrous histiocytoma (MFH) of soft tissue is correlated to histologic malignancy grade but also has been related to tumor size, depth, and location. In a consecutive series of 61 patients with MFH we found that large, deep-seated and thigh tumors were more often highly malignant histologically. This finding suggests that the prognostic significance which has been attributed to the clinical variables may be a function of their covariation with histologic malignancy grade. PMID- 3008982 TI - Intraluminal crystalloids in prostatic adenocarcinoma. Immunohistochemical, electron microscopic, and x-ray microanalytic studies. AB - Histochemical, immunohistochemical, electron microscopic, and x-ray microanalytic studies were performed on crystalloids within glandular lumina of adenocarcinomas of the prostate. In a review of light microscopic sections of 343 prostatic adenocarcinomas, unequivocal crystalloids were identified in 35 cases (10.2%). Immunohistochemical and ultrastructural studies revealed distinct differences between these crystalloids and the Bence Jones crystals of multiple myeloma: anti kappa and anti-lamda immunostaining was negative, and the characteristic lattice like architecture of Bence Jones crystals was not seen. Differences from corpora amylacea also were demonstrated. X-ray microanalysis did not elucidate the nature of the prostatic crystalloids, and their biochemical composition and mode of formation remain uncertain. Detection of the crystalloids in light microscopic sections nevertheless can aid in the diagnosis of prostatic adenocarcinoma, particularly when the tissue is distorted by crushing artifact, or if the tumor is so well-differentiated that it can be confused with atypical hyperplasia or inflammatory atypia. When intraluminal crystalloids are detected in prostatic glands that appear histologically benign or atypical, study of additional levels or a repeat biopsy should be undertaken. PMID- 3008983 TI - The in vivo inhibition of mouse brain protein kinase-C by retinoic acid. AB - The intracisternal injection of either all-trans-retinoic acid or [alpha] difluoromethylornithine (DFMO) into the brain of 9-day-old mice blocked (greater than 90%) phorbol ester-induced ornithine decarboxylase (ODC, EC 4.1.1.17) activity in a concentration-dependent fashion; this inhibition was not evident with the use of the biologically impotent furyl analog of retinoic acid. In a similar manner, retinoic acid reduced the soluble protein kinase-C (PK-C) activity by 60% as well as total EGTA-sensitive kinase activity (66%) associated with the plasma membrane. Sixty-six percent of the retinoic acid-induced loss of PK-C activity in the soluble fraction could be accounted for by the translocation of PK-C to the plasma membrane as measured by the specific binding of 12-O [3H]tetradecanylphorbol-13-acetate (TPA). DFMO and furyl-retinoic acid were not effective in altering PK-C activity or TPA binding to PK-C. In the presence of retinoic acid, however, there was a 2.3-fold increase in specific [3H]TPA binding in the plasma membrane fraction, which was 3.4-fold greater than that lost from the cytosol. Because retinoids do not directly affect TPA binding to PK-C, the data suggest that (i) the presence of retinoic acid results in the exposure of heretofore cryptic TPA-binding sites in the membrane, where this binding is most likely related to the alteration of membrane structure and (ii) de novo ODC induction is not required for retinoid-dependent inhibition of PK-C, although the TPA induction of PK-C appears to be necessary with regard to ODC induction. PMID- 3008984 TI - Increased number of beta-adrenoceptors in hepatocytes from rats treated with 2 acetylaminofluorene. AB - Treatment of rats with chemical carcinogens, including 2-acetylaminofluorene (2 AAF), leads to a strong increase in the hepatic catecholamine-sensitive adenylate cyclase activity. The present study was undertaken to investigate the mechanism for the development of this increase. We report that hepatocytes isolated from rats which had been fed 2-AAF (0.025% w/w) for 8-12 weeks had an increased number of beta-adrenoceptors, as determined by [3H]dihydroalprenolol binding to whole cells and [125I]iodocyanopindolol binding to washed particles. For both ligands the number of binding sites was about 4-fold higher in hepatocytes from 2-AAF treated rats than in those from controls. The adenylate cyclase activity of the carcinogen-fed animals showed both a general increase manifested in the basal level (2-fold) and in the activities obtained by stimulation with guanine nucleotides (2-3-fold), cholera toxin (1.5-fold), and glucagon (1.3-fold) and a selective, larger increase in the beta-adrenoceptor-linked activity (7-fold increment of the isoproterenol-sensitive activity). The results indicate that the number of hepatocyte beta-adrenoceptors increases during 2-AAF carcinogenesis. This may, at least in part, explain the rise in catecholamine-sensitive adenylate cyclase activity. PMID- 3008985 TI - Induction of myeloid differentiation of HL-60 cells with naphthalene sulfonamide calmodulin antagonists. AB - The naphthalene sulfonamide calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1 naphthalenesulfonamide and N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, both induce limited myeloid differentiation of the human promyelocytic cell line, HL-60. In addition, these inhibitors augment the differentiation observed when HL 60 cells are induced with retinoic acid, dimethyl sulfoxide, or dibutyryl cyclic adenosine monophosphate. The dose-response curve for HL-60 differentiation was consistent with the published 50% inhibitory dose for inhibition of calmodulin activated phosphodiesterase and with the calmodulin drug-binding potential of N (6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and N-(4-aminobutyl)-5-chloro-2 naphthalenesulfonamide and their less active congeners, N-(6-aminohexyl)-1 naphthalenesulfonamide and N-(4-aminobutyl)-2-naphthalenesulfonamide. These effects, of the naphthalene sulfonamide calmodulin antagonists, are consistent with a regulatory role for calmodulin in cell differentiation, but parallel effects on protein kinase C cannot be excluded. PMID- 3008986 TI - Inhibition of DNA synthesis in rat hepatocytes by platelet-derived type beta transforming growth factor. AB - Platelet-derived type beta transforming growth factor (TGF beta) is a potent inhibitor of DNA synthesis in primary monolayer cultures of adult rat hepatocytes. TGF beta induced a 50% inhibition of epidermal growth factor (EGF) mediated DNA synthesis at approximately 5 X 10(12) M. This inhibition did not appear to be due to a delay in the peak of DNA synthesis or a toxic action, nor could it be overcome by increasing concentrations of the mitogens EGF, insulin, or glucagon. Inhibition was observed either when TGF beta and EGF were continuously present together in the culture medium or when TGF beta was added to the hepatocyte cultures after removal of the EGF stimulant. This observation together with a lack of an inhibitory effect of TGF beta on the binding of 125I labeled EGF to hepatocytes in culture, suggests that the inhibitory action of TGF beta was not caused by a direct competition with EGF at the cell surface. TGF beta could not inhibit DNA synthesis once it had begun; however, the inhibitory action of TGF beta could be partially overcome by increasing amounts of conditioned medium produced by normal hepatocytes. Specific saturable receptors for TGF beta were found on the normal rat hepatocytes, but specific binding could not be detected on hepatocytes from regenerating liver. TGF beta is thus a potent inhibitor of EGF-induced DNA synthesis in adult rat hepatocytes. Its significance for growth control in vivo has yet to be assessed. PMID- 3008987 TI - Expression of myeloid and major histocompatibility antigens on small cell carcinoma of the lung cell lines analyzed by cytofluorography: modulation by gamma-interferon. AB - Recent reports have described the expression of myeloid cell surface antigens on cells of small cell carcinoma of the lung (SCCL). In order to confirm and extend these findings, we have examined by cytofluorography a large panel of well characterized cell lines derived from SCCL tumors for the expression of several myeloid cell-associated antigens, some of which were not examined in previous reports. In addition, we have studied the expression of Classes I and II HLA antigens on these SCCL cell lines. Finally, we examined the effect of gamma interferon on the expression of several cell surface markers and on proliferation of SCCL cells. We have found that several SCCL cell lines expressed a Mr 55,000 polypeptide antigen, My23, previously found only on monocytes and monocytic leukemia cells. In addition, the cell lines studied expressed another antigen, defined by monoclonal antibody AML-1-99, which is associated with monocytes and hematopoietic stem cells. We confirmed previous studies that the Leu-7 antigenic determinant is expressed on SCCL cells but observed only minimal or absent binding of monoclonal antibody OKM1 to most cell lines. Class I HLA antigens were present on eight of nine lines examined while Class II HLA was expressed on three of nine lines. Gamma-Interferon decreased the proliferative rate of all lines examined. However, this lymphokine was capable of inducing Class I HLA on several lines. The effect of gamma-interferon on other cell surface antigens was variable. These studies confirm that some myeloid cell-associated antigens are expressed on cultured SCCL lines and, additionally, show that their expression can be modulated by immune interferon. Determining the significance of finding myeloid cell-associated antigens on SCCL cells will require further study. PMID- 3008989 TI - Correlation between the induction of leukemic cell differentiation by various retinoids and modulation of protein kinases. AB - During retinoic acid induced differentiation of the human promyelocyte leukemia cell line HL60 and the human myeloblast cell line RDFD along the myeloid pathway, there is marked modulation of both the cyclic adenosine 3':5'-monophosphate dependent protein kinase and the phospholipid-sensitive calcium dependent protein kinase. In order to further assess whether these kinases are intimately associated with the differentiation process, we have correlated the modulation of these enzymes and phosphorylations of their substrates with the extent of differentiation induced by various retinoid derivatives. We observed that there was a direct relationship between the degree of differentiation of the two leukemic cell lines and the elevation of the cyclic adenosine 3':5'-monophosphate dependent protein kinase and phospholipid sensitive calcium dependent protein kinase activities. In addition, the increased phosphorylation of the various substrates of these enzymes also correlates with the degree of differentiation. These observations support the hypothesis that modulation of these protein kinases and phosphorylation of their substrates are necessary steps in the differentiation process. PMID- 3008988 TI - Glucocorticoid inhibition of c-myc, c-myb, and c-Ki-ras expression in a mouse lymphoma cell line. AB - Glucocorticoid hormone treatment of certain T-cell-derived lymphomas and leukemias and of immature thymocytes results in cell death. Furthermore, glucocorticoid-mediated killing of S49 mouse lymphoma cells appears to involve the control of the cell cycle; i.e., S49 cells treated with glucocorticoids are arrested in G1 of the cell cycle when events controlling cell proliferation occur. The protooncogenes, c-myc, c-myb, and c-Ki-ras may be involved in cell cycle regulation and proliferation state in both normal and neoplastic cells. We report here that the steady state mRNA levels of c-myc, c-myb, and c-Ki-ras in S49 cells are dramatically and rapidly decreased after glucocorticoid treatment. Minimal expression is observed after 9 h, remaining at a constant low level at 11 h. Flow cytometry reveals no significant alteration in the cell cycle distribution of S49 cells up to 12 h after treatment. These findings suggest that glucocorticoids suppress the expression of these protooncogenes and that this may be the mechanism whereby glucocorticoids inhibit cell cycle progression in T lymphoid cell lines. PMID- 3008990 TI - Growth and spread in nude mice of Epstein-Barr virus transformed B-cells from a chronic lymphocytic leukemia patient. AB - Subcutaneous inoculation of Epstein-Barr virus (EBV) transformed peripheral blood B-lymphocytes (PBL) from an untreated chronic lymphocytic leukemia (CLL) patient produced progressively growing lethal tumors in 4 of 11 whole body irradiated (440 rads) nude mice. In one tumor bearing mouse there was splenomegaly and generalized enlargement of lymph nodes. Chromosomal analysis and membrane immunofluorescence revealed that cells in all the 4 s.c. tumors and a proportion of cells in the enlarged spleen and lymph nodes had human chromosomes and contained human kappa or lambda chains demonstrating that these were polyclonal human B-cells. Epstein-Barr virus associated nuclear antigen could be detected in 100% of cells in all the 4 EBV transformed B-cell lines in vitro and aliquots of cells from several s.c. tumors and metastatic lesions examined. Successful serial transplantation into irradiated nude mice was possible for at least 3 generations with one of the 4 s.c. tumors. During serial transplantation, spread of tumor cells to the spleen and lymph nodes could be detected in all the 3 passage mice investigated; however, there was no evidence in any mouse of dissemination of tumor cells into the bloodstream or into any organ other than lymph nodes and spleen. s.c. tumors also developed in a proportion of irradiated nude mice after inoculation of cells from two other s.c. tumors and the metastatic spleen and lymph nodes, but all these tumors regressed during the first or second transplant passage. Two % of PBL from the untreated patient and 4% of EBV transformed PBL maintained in vitro were found to have trisomy of chromosome 12 which is the most frequently reported anomaly associated with human CLL B-cells. It is highly probable that the cells with trisomy were derived from the leukemic clone of this patient. Cells with this trisomy predominated in most metastatic sites compared to the parent s.c. tumors. Inoculation of irradiated nude mice with EBV transformed PBL from this patient after chlorambucil therapy (100% metaphase plates with 46,XY,11q+ karyotype) or with EBV transformed PBL from 2 normal adults failed to produce any progressively growing tumor in a total of 12 irradiated animals observed greater than 300 days. Although there are several reports of EBV induced immortalization of CLL B-cells in vitro, we have not seen any previous report on the successful serial transplantation and dissemination of EBV transformed CLL B-cells in nude mice. PMID- 3008992 TI - Superinfection of epithelial hybrid cells (D98/HR-1, NPC-KT, and A2L/AH) with Epstein-Barr virus and the relationship to the C3d receptor. AB - Three Epstein-Barr virus (EBV) genome-positive epithelial/hybrid cell lines (D98/HR-1, NPC-KT, and A2L/AH) were superinfected with EBV derived from P3HR-1 (HR-1), NPC-KT, and B95-8 cells. The HR-1 virus is lytic and induces early antigen in superinfected Raji cells; the virus is not capable of transforming B lymphocytes. Virus preparations from NPC-KT cells have both transforming and early antigen-inducing properties, while B95-8 virus can only transform B lymphocytes. It was possible to demonstrate EBV antigens after superinfection of D98/HR-1 cells with both HR-1 virus and NPC-EBV. The NPC-KT hybrid cells, which were originally prepared by fusing human adenoid epithelial cells (Ad-AH) and EBV genome-positive NPC explanted epithelial cells, were susceptible to superinfection with HR-1 virus but not to NPC-EBV. The A2L/AH hybrid cells, which were prepared by fusion between Ad-AH cells and lymphocytes transformed by B95-8 virus, could not be superinfected with any of the virus preparations. In order to further investigate the nature of the EBV receptor as it relates to other cell membrane components, we examined cell surface markers on Ad-AH, NPC-KT, A2L/AH, and D98/HR-1 cells using monoclonal antibodies and by rosette formation. We found that the NPC-KT, A2L/AH, and Ad-AH cell lines express the OKB2 antigen and that the common acute lymphoblastic leukemia antigen is expressed on the A2L/AH cells. We also found that NPC-KT parental cells and a clone of NPC-KT cells express erythrocyte antibody complement b and erythrocyte antibody complement d, as determined by rosette formation, but were negative for C3b and C3d when monoclonal antibodies against these two markers were used. The D98/HR-1 cells were also confirmed to be negative for C3b and C3d. The data suggest that the C3d receptor may be part of the EBV receptor but that the C3d receptor, by itself, is not the only receptor to which EBV can bind. PMID- 3008991 TI - Identification of hepatocarcinogenesis-associated aldehyde dehydrogenase in normal rat urinary bladder. AB - An aldehyde dehydrogenase (ALDH) with properties identical to those of the NADP+ dependent, tumor-associated aldehyde dehydrogenase appearing during rat hepatocarcinogenesis has been identified in normal rat urinary bladder. Like the tumor-associated aldehyde dehydrogenase, bladder NADP+-ALDH is cytosolic and preferentially oxidizes benzaldehyde-like aromatic aldehydes. Bladder ALDH is also extremely sensitive to the aldehyde dehydrogenase inhibitor disulfiram. Additionally, the electrophoretic mobility of bladder ALDH is identical to that of the NADP+-dependent, tumor-associated aldehyde dehydrogenase. Finally, antibodies to the tumor-associated ALDH cross-react with bladder aldehyde dehydrogenase. Histochemically, bladder aldehyde dehydrogenase is localized to the very active epithelial lining and to the inner and outer smooth muscle layers. The observation that normal urinary bladder possesses an enzyme activity very similar to one expressed during hepatocarcinogenesis, but not in normal liver, is consistent with the hypothesis that derepression of a gene normally repressed in liver is responsible for expression of the tumor-associated aldehyde dehydrogenase phenotype. PMID- 3008993 TI - Altered excretion of modified nucleosides and beta-aminoisobutyric acid in subjects with acquired immunodeficiency syndrome or at risk for acquired immunodeficiency syndrome. AB - Urinary excretion of modified nucleosides and beta-aminoisobutyric acid, subsequently referred to as markers, was determined in populations of patients with acquired immunodeficiency syndrome (AIDS) or at risk for development of AIDS. Our results show that asymptomatic adult male homosexuals excreted elevated amounts of markers as compared to male heterosexuals. This aberrant excretion was more pronounced in asymptomatic adult male homosexuals with antibodies to HTLV III. Significantly greater excretion of 1-methylinosine, N4-acetylcytidine, and N2-methylguanosine was observed in asymptomatic adult male homosexuals with antibodies to HTLV-III than in asymptomatic male homosexuals without antibodies to HTLV-III. Increased amounts of markers were also excreted by subjects with the generalized or chronic lymphadenopathy syndrome, AIDS related complex (ARC), or AIDS. In these subjects, the most pronounced differences between groups were between subjects with chronic lymphadenopathy syndrome and those with ARC; subjects with ARC excreted greater amounts of seven of the ten urinary markers. There were few differences between subjects with ARC and those with AIDS, Kaposi's sarcoma, or AIDS with opportunistic infections. This observation may be useful for identifying subjects who are at risk of developing AIDS. A prospective study to test this hypothesis is under way. PMID- 3008994 TI - Anti-neurofilament antibodies in the sera of patients with small cell carcinoma of the lung and with visual paraneoplastic syndrome. AB - The sera of patients with small cell carcinoma of the lung (SCCL) and an associated visual paraneoplastic syndrome (VPNS) have high titer immunoglobulins that react with retinal ganglion cells and with cloned lines of the SCCL. The immunoglobulins in the sera of two patients with SCCL and VPNS reacted with at least one common antigen shared by neural cells and cloned lines of the SCCL. The molecular weights of the predominant neural and tumor antigens were 205,000, 145,000, 65,000, and 20,000-24,000 as determined by Western blots. Three of the antigens from neural tissue copurify and comigrate electrophoretically with neurofilament proteins. Polyclonal antibodies prepared against authentic neurofilament proteins react with antigens having molecular weights identical to those of proteins that react with immunoglobulins from the SCCL-VPNS patients. Polyclonal antibodies that were prepared against isolated retinal ganglion cells and that were shown previously to cause the immunoablation of the ganglion cells in vivo reacted most intensely with the Mr 205,000 antigen and weakly with the Mr 145,000 and Mr 70,000 antigens. Treatment of the Western blots with alkaline phosphatase from Escherichia coli did not affect the immunoreactivity between the immunoglobulins and the purified neurofilament proteins. It is proposed that the immunoglobulins in the sera of patients with SCCL-VPNS may be involved etiologically in the development of the VPNS. PMID- 3008995 TI - Characterization of growth factors in human ovarian carcinomas. AB - Epidermal growth factor (EGF)-like factors with EGF competing and cell growth stimulating activity were investigated in malignant and nonmalignant tissues. About 37% of ovarian carcinomas present an increased factor activity between 9.0 and 19.3 ng EGF units/mg protein. In one tumor 175.0 ng EGF units/mg protein were found. In extracts of nonmalignant tissues, the factor concentration was about 1.0-6.4 ng EGF units/mg protein. Isoelectric focusing was performed to characterize these factors. In normal ovaries and ovarian carcinomas, factors with EGF competing activity focus at pH 8.0-9.0, pH 5.7-6.3, and pH 3.6-4.9. In ovarian carcinomas, an additional peak with EGF competing and cell growth stimulating activity was found between pH 6.5 and 7.2. Similar results could be achieved in other malignant tissues investigated. These data indicate the presence of EGF-like factors. EGF itself focuses at pH 4.6 (G. Carpenter and S. Cohen, Annu. Rev. Biochem., 48: 193-216, 1979). Specific EGF binding was determined in 12 ovarian carcinomas. In five of these cases EGF receptors could be detected. In the EGF receptor positive carcinomas, the content of EGF-like growth factors varied between 0 and 9 ng EGF units/mg protein. In EGF receptor negative cases, the content of EGF-like growth factors varied between 0 and 19.3 ng EGF units/mg protein. The clinical data of 19 patients are also demonstrated. PMID- 3008996 TI - Quantitative and qualitative characterization of human cancer-associated serum glycoprotein antigens expressing fucosyl or sialyl-fucosyl type 2 chain polylactosamine. AB - The quantity of tumor-associated antigens carrying type 2 chain polylactosamines with four types of fucosyl determinants, LeX (X-hapten), poly-LeX, sialyl LeX, and LeY (Y-hapten), present in sera of patients with various malignant and non malignant disorders, as well as the qualitative chemical properties of the carrier molecules in sera, have been investigated using four monoclonal antibodies, each of which defines one of these determinants. The following findings are of particular importance: the serum levels of LeX defined by antibody FH2 and poly-LeX defined by ACFH18 in patients with cancer were occasionally high (incidence about 10%); however, the majority of patients did not show elevated levels; the serum level of the antigen, defined by monoclonal antibody FH6 (termed sialyl LeX-i since this determinant is carried by i antigen), was significantly high in patients with cancers originating from organs from which adenocarcinomas often develop. For example, among various types of lung cancer, only adenocarcinoma but not squamous cell carcinoma, small cell carcinoma, or large cell carcinoma showed a high level of sialyl LeX-i antigen in sera. The incidence of high antigen levels in sera of patients with adenocarcinomas of lung was as high as 76% of the observed cases; the serum level of Ley (Y-hapten) was frequently high in patients with hepatoma (incidence, 34%); sialyl LeX-i antigen was separated on gel filtration as a glycoprotein with an average molecular weight greater than 10(6). It was characterized by its susceptibility to basehydrolysis, Pronase digestion, and sialidase and endo-beta galactosidase treatment and is assumed to be a high molecular weight mucin-type glycoprotein; sialyl LeX-i antigen expressed in sera of patients with cancer was soluble in perchloric acid, while the same antigen in sera of patients with noncancerous diseases and normal subjects was mostly insoluble in perchloric acid. LeX, a poly-LeX, and essentially all LeY antigens in sera of patients with cancer were perchloric acid-insoluble. PMID- 3008998 TI - Epidermal growth factor binding and protein kinase C activities in human breast cancer cell lines: possible quantitative relationship. AB - Quantitative polyacrylamide gel electrophoresis analysis of Ca2+, phospholipid dependent protein kinase (PKC) of human mammary tumor cell lines (MCF-7, ZR-75, T 47-D, MDA-MB-231, BT-20, and HBL-100) revealed that 80% of the total cellular PKC resided in the cytosol. The tumor cells with no detectable levels of estrogen receptors (MDA-MB-231, HBL-100, and BT-20 cells) exhibited significantly larger (P less than 0.001) cytosolic PKC activities than those cells that contained estrogen receptors (MCF-7, T-47-D, and ZR-75 cells). In addition, in estrogen receptor-negative cell lines, relatively high levels of specific low-affinity (apparent Kd = 700 pM) epidermal growth factor (EGF) binding activities were found as compared with estrogen receptor-positive cells with significantly (P less than 0.001) lower levels of specific high-affinity (apparent Kd = 90 pM) EGT binding. A significant positive correlation (P less than 0.01) was observed between the number of EGF receptor (Rs = 0.50) and/or the EGF receptor dissociation constants (Rs = 0.78) with the cytosolic PKC activity levels. These data indicate that, in human breast cancer cells, a positive relationship may exist between PKC activity, estrogen, and EGF receptors. PMID- 3008997 TI - Heterogeneity of cell surface antigen expression of human small cell lung cancer detected by monoclonal antibodies. AB - Using immunohistochemistry, radiobinding, and indirect immunofluorescence assays, seven distinct cell surface antigens, detected by monoclonal antibodies, were analyzed for the degree of homogeneity or heterogeneity of antigen expression on a panel of human small cell lung cancers. The panel included 7 tumors taken directly from patients, 21 established cell lines (9 of which were derived from different metastatic sites of 3 patients), and 33 clonal derivatives of 3 lines. With all assays, considerable heterogeneity of antigen expression between tumors from different patients was observed. In both fresh tumors and in cell lines, as well as in cell lines established from different metastatic sites in an individual patient, we observed intratumor heterogeneity finding antigen positive and negative cells and variation in antigenic density, by immunohistochemistry and indirect immunofluorescence assays. Antigenic expression was not cell cycle dependent. In addition, when cell lines or patient samples expressing antigen positive and antigen negative tumor cells were cloned, heterogeneity of antigenic expression was still present in the clonal lines. This suggests that either the expression of the antigen was not heritable and/or the ability to regenerate antigenic heterogeneity is an intrinsic property of the tumor cells. The heterogeneity of antigen expression on lung cancer cells has significant implications for the use of these and other monoclonal antibodies in the study and therapy of lung cancer. PMID- 3008999 TI - Different type of DNA damage caused by three aziridinyl substituted cyclophosphazenes in a human small cell lung carcinoma cell line. AB - Aziridinyl substituted cyclophosphazenes are a new group of inorganic chemical agents with in vitro and in vivo cytotoxic activity. We investigated the mode of action on DNA of three different compounds, 1,3,3,5,5-pentakis (1-aziridinyl)-1 lambda 6,2,4,6,3 lambda 5,5 lambda 5-thiatriazadiphosphorine -1-oxide (SOAz), trans-1,3-bis(1-aziridinyl)-1,3,5,5-tetrakis (methylamino)-2,4,6,1 lambda 5,3 lambda 5,5 lambda 5-triazatriphosphorine (AZP), and 1,trans-5-bis(1-azaridinyl) gem-1,3,3'-cis-5,7,7'-hexakis (methylamino)-2,4,6,8,1 lambda 5,3 lambda 5,5 lambda 5,7 lambda 5 -tetraazatetraphosophocine (AZM), of this group in the Ehrlich ascites tumor cell line (EAT) and a human small cell carcinoma cell line. The DNA damage was evaluated by alkaline elution and ethidium bromide fluorescence assay. Each compound gave a different pattern of DNA damage. SOAz caused neither single strand breaks nor cross-links, AZP gave cross-links, and AZM gave single strand breaks and cross-links in both cell lines after drug incubation for 6 h. The range of concentrations leading to cytoxicity of AZP and AZM in the clonogenic assay coincided with the concentrations leading to DNA damage. Cell kill occurred with SOAz in the same range of concentrations, however, without detectable evidence of DNA damage. It was concluded that cyclophosphazenes are probably a heterogeneous group as far as their mode of action as cytostatic agents is concerned. PMID- 3009000 TI - Selective protection against cis-diamminedichloroplatinum(II)-induced toxicity in kidney, gut, and bone marrow by diethyldithiocarbamate. AB - Diethyldithiocarbamate (DDTC) has been shown to inhibit nephrotoxicity induced by cis-platinum (DDP) without inhibition of tumor response in the rat. We report here that DDTC at doses of 25-300 mg/kg inhibits DDP-induced nephrotoxicity and bone marrow toxicity in C57BL/6 X DBA/2F1 (hereafter called B6D2F1) mice, F344 rats, and beagle dogs and is also antiemetic in the dog. DDTC doses which afford excellent protection do not decrease median survival time following DDP treatment in L1210 and P388 leukemias, B16 melanoma, and Lewis lung and colon 26 carcinomas in B6D2F1 mice when DDTC is given 2 h after DDP. Preliminary experiments indicate that DDTC does not alter median survival time after treatment of P388 leukemia with the platinum analogues diammine(1,1-cyclobutanedicarboxylato)platinum(II) and cis-diisopropylamine-cis-dichloro-trans-dihydroxyplatinum(IV ). Maximum blood urea nitrogen levels after DDP treatment are reduced significantly by DDTC in all species; blood urea nitrogen elevation, total kidney platinum, weight loss, and leukopenia correlate with DDP-DDTC interval in the rat and indicate optimum protection at 2 h, the shortest interval examined. Bone marrow toxicity was assessed by peripheral white blood cell counts in all species and by marrow cellularity in the mouse. White blood cell nadirs were higher and bone marrow recovered more rapidly after DDTC compared with DDP given alone. DDP reduced marrow cellularity 50-60% in the mouse; administration of DDTC 2 h after DDP afforded no protection to the lymphocytes in the marrow but maintained the granulocyte + precursor population near control levels. DDTC plasma pharmacokinetic values have been determined after s.c., i.p., and i.v. administration in the mouse, rat, and dog. Peak plasma levels of 0.3-1.2 mM are observed after a 250-mg/kg dose, with a plasma half-life of 10-20 min. Our data indicate that DDTC may provide protection against most clinically significant toxicities arising from cis-platinum at doses which do not inhibit tumor response. PMID- 3009001 TI - Heme enzyme patterns in genetically and chemically induced mouse liver tumors. AB - Chemically induced rat hepatocyte nodules and hepatomas have repeatedly been shown to be deficient in Phase I drug-metabolizing enzymes. Some of these reduced activities are attributable to a diminution of the heme-containing terminal electron acceptor, cytochrome P-450. We recently demonstrated that spontaneous mouse liver tumors exhibit the same deficiency. Therefore, chemically induced and spontaneous liver tumors share common metabolic alterations which are likely to represent intrinsic characteristics of the tumorigenic process and are independent of its etiology. To determine whether the cytochrome P-450 deficit was the result of an altered heme metabolism, we quantitated four heme-containing proteins in normal mouse liver, spontaneous mouse liver tumors, and those induced by a single injection of diethylnitrosamine: cytochrome P-450; cytochrome b5; tryptophan 2,3-dioxygenase (EC 1.13.11.11); and catalase (EC 1.11.1.6). The amounts of these components in spontaneous tumors relative to normal liver were 0.35, 0.68, 0.76, and 0.51, respectively. Similar values were obtained with chemically induced tumors. The enzymes delta-aminolevulinic acid synthase (EC 2.3.1.37), the rate-limiting enzyme in the heme synthetic pathway, and heme oxygenase (EC 1.14.99.3), a degradative enzyme, were also quantitated. The amounts of these enzymes in spontaneous tumor relative to liver were 0.49 and 1.51, respectively. Again, similar values were observed for the chemically induced tumors. Alteration of the latter two enzyme activities may be sufficient for the altered hemoprotein patterns seen in mouse liver tumors. Further, this pattern of metabolic alteration is common to both chemically induced and spontaneous tumors. Thus, tumor resistance to cytotoxic agents activated by the monooxygenase system is not necessarily induced by exposure to these agents, nor as a result of selection. PMID- 3009002 TI - DNA methylation patterns of the calcitonin gene in human lung cancers and lymphomas. AB - Generalized hypomethylation of the genome and of specific genes has been described in human tumors. We now report that in human lung cancers, especially in the most aggressive form, small cell lung carcinoma, and in lymphomas, the 5' region of the calcitonin (CT) gene exhibits methylation of increased numbers of CCGG sites in comparison with normal adult tissues. These unusual methylation patterns are found much less frequently in other tumor types examined. In the spectrum of the four major types of lung cancer (small cell, adeno-, squamous, and large cell carcinomas), the frequency of occurrence of hypermethylation in the 5'-region of the CT gene parallels that for presence of the neuroendocrine related biochemistry which characterizes small cell lung carcinoma. In medullary thyroid carcinoma, a tumor which expresses high levels of CT gene mRNA, the 5' region of the CT gene is hypomethylated. Our findings provide a potential new molecular marker for two important human cancers (lung cancer and lymphomas) and suggest that there is a close relationship between abnormal CT gene methylation and developmental events for these tumors. PMID- 3009003 TI - Pancreatic cancer in the Syrian hamster induced by N-nitrosobis(2-oxopropyl) amine: cocarcinogenic effect of epidermal growth factor. AB - Because epidermal growth factor (EGF) is rapidly bound and internalized into rat pancreas, stimulates uptake of tritiated thymidine, and increases pancreatic weight, a cocarcinogenic effect on pancreatic cancer seemed likely. Pancreatic adenocarcinomas were induced in 70 female Syrian hamsters by 19 weekly s.c. injections of N-nitrosobis(2-oxopropyl)amine (BOP) (10 mg/kg). From Wk 5 through Wk 8 of BOP injections, additional s.c. injections of EGF (5 micrograms every 3 days for 10 injections) were given to 45 animals, while 25 received saline solution. An additional group of 10 received EGF alone, and another 10 animals received saline solution alone (controls). Eleven wk later, the mean body weight of EGF-treated animals increased by 29% as compared with that of controls, and their mean pancreatic weight relative to body weight increased by 44% as compared with controls. The mean body weight of EGF + BOP-treated animals increased by 10%, and their pancreatic weight relative to body weight increased by 22% as compared with that of animals treated with BOP alone. The incidence of pancreatic cancer in the EGF + BOP-treated animals was 75% versus 44% in those treated with BOP alone (P = 0.016). No tumors developed in either animals treated with EGF alone or control animals. EGF augments pancreatic carcinogenesis induced by BOP. The incidence of bronchial carcinomas doubles. PMID- 3009004 TI - Southern blot analysis of DNA extracted from formalin-fixed pathology specimens. AB - We have developed a method for the extraction of DNA from formalin-fixed, paraffin-embedded pathology specimens. High-molecular-weight DNA was recovered from well-fixed nonautolyzed samples of viable tissue. DNA recovered from samples exposed to picric acid or mercuric chloride containing fixatives was not intact. Increasing the formalin fixation time decreased the amount of intact DNA available. When these limitations were taken into consideration, the procedure allowed for the removal of degraded and chemically modified DNA from the preparation, and the final product was suitable for quantitative and qualitative analysis by Southern or dot blotting techniques. Digestion with methylation sensitive restriction endonucleases showed that DNA methylation patterns were not altered after formalin fixation. PMID- 3009005 TI - Antigens associated with human squamous cell lung carcinoma defined by murine monoclonal antibodies. AB - A panel of 12 monoclonal antibodies that preferentially react with human squamous lung carcinoma cells has been produced. All are reactive with fresh frozen sections of squamous cell lung carcinoma tissues in immunoperoxidase assays and are unreactive with lymphoblastoid cells, red blood cells, and fibroblasts in enzyme-linked immunosorbent assay. At least eight of these antibodies interact with cell surface components. These reagents can be subdivided into four groups based upon their reactivities. Groups 1 to 3 are unreactive with normal liver, lung, kidney, colon, spleen, and pancreas in immunoperoxidase assays. Group 1 antibodies (PF1/A, PF1/B, PF1/C, PF1/D, and PF1/E) are all of IgG3 subclass and immunoprecipitate nonsulfated glycoprotein components with molecular weights of 80,000 and 180,000 and a nonglycosylated polypeptide with a molecular weight of 38,000. Group 1 antibodies are also reactive with some lung adenocarcinomas and, with the exception of PF1/E, stain certain differentiated strata within normal adult plantar and fetal epidermis. Group 2 antibodies (PF2/A and PF2/B) react also with breast, gastric, and colonic adenocarcinomas and some tumors of neuroectodermal origin. Group 2 antibodies, which are both of IgG3 subclass immunoprecipitate a nonglycosylated Mr 24,000 polypeptide. Group 3 antibodies (PF3/A, an IgG1; PF3/B, an IgGM; and PF3/C, an IgG2a) react additionally with certain other tumors, as well as with normal adult and fetal epidermis. Group 4 antibodies (PF4/A, an IgG2a; and PF4/B, an IgG1) are less specific than those of the preceding groups, as they react with some normal tissues, including pancreatic islets and pneumocytes, as well as with a variety of adenocarcinomas and tumors of neuroectodermal origin. PF4/A and PF4/B immunoprecipitate Mr 100,000 and 95,000 glycoproteins, respectively. PMID- 3009006 TI - C-type virus particles in spontaneous and virus-induced leukemia and malignant lymphomas in mice and rats. AB - The presence and numbers of C-type virus particles in an animal model consisting of mice and rats with either spontaneous or virus-induced leukemia and lymphomas were studied, in order to determine the relation of the appearance of virus particles to viral etiology of such neoplasms. The numbers and distribution of C type virus particles in organs from 13 mice with spontaneous leukemia and lymphomas were compared with the presence of virus particles in organs of 13 mice with leukemia and lymphomas induced by passage A (Gross) virus inoculation. C type virus particles were present in organs of all mice with either spontaneous leukemia or leukemia and lymphomas induced by virus inoculation. However, the number of particles observed was significantly higher in those mice in which leukemia was induced by virus inoculation. Virus particles were also observed, but in substantially smaller numbers, in organs of 13 of 25 untreated, healthy mice of the nonleukemic C3H(f) inbred line. In contrast to mice, C-type, or any other virus particles, were not found in organs of 10 Sprague-Dawley, Long-Evans or Sprague-Dawley X Long-Evans F1 hybrid rats with spontaneous leukemia. However, C-type virus particles were consistently present in organs of 11 Sprague-Dawley rats with leukemia induced by rat-adapted passage A (Gross) mouse leukemia inoculation. The virus particles appeared in the organs of the inoculated rats 5 days after i.p., and 11 days after s.c. inoculation. Virus particles were not found in organs of 10 healthy untreated Sprague-Dawley rats. The implications of these observations are discussed. PMID- 3009007 TI - Effect of adeno-associated virus on transformation of NIH 3T3 cells by ras gene and on tumorigenicity of an NIH 3T3 transformed cell line. AB - Transfection of NIH-3T3 cells with the plasmid pJ234, containing DNA from the human bladder carcinoma T24 cell line (ras gene), results in their transformation. Adeno-associated virus did not affect significantly the number of the transformed foci when different multiplicities of infection were used and when the virus was added to the cultures at different time intervals before or after transfection. A transformed cell line was derived following transfection of NIH 3T3 cells by the ras gene. Infection of these cells with adeno-associated virus resulted in a decrease in their growth rate and cloning efficiency. These infected cells showed a dose-dependent reduction in the frequency and an increase in the latent period for tumor appearance in nude mice. PMID- 3009008 TI - Liver as a tumor cell killing organ: Kupffer cells and natural killers. AB - Sinusoidal rat liver cells spontaneously kill tumor cells in vitro. They have the same preferences as do spleen cells for certain types of tumor cells, YAC-1, P815, BSP73Asml, BSP73As, EB, EsB, and L5222. Metastasizing tumor cells are less sensitive than their nonmetastasizing counterparts. Not all effector cells are Kupffer cells. These nonmacrophage killer cells share some features with classical natural killers: (a) fast reactions (4 h); (b) high toxicity against YAC-1 cells; (c) sensitivity to anti-asialo GM1 globulin; (d) similar age dependency; (e) short biological halflife (approximately 1 day) (deduced from radiation experiments); (b) silica particle insensitivity; and (g) nonadherence. The natural killing potency of the liver is higher than that of the spleen. The reduction of tumoricidal capacity of the liver in germ-free animals suggests environmental influences. Tumoricidal capacity (organ capacity) is increased in rats chronically fed thioacetamide, carbon tetrachloride (CC14), dimethylaminoazobenzene, and N-nitrosomorpholin. PMID- 3009010 TI - Morphology and in vivo growth characteristics of an atypical murine proliferative osseous lesion induced in vitro. AB - Mandibular condyles of late embryonic NMRI mice were used to study the effect of the FBR murine osteosarcoma virus in an in vitro tissue culture system. Chondroprogenitor cells and chondroblastic cells present in the condylar tissue normally undergo rapid differentiation in vitro which results in an advanced stage of bone formation. The infection of condyles with FBR murine osteosarcoma virus induced the transformation of bone progenitor cells and the formation of an atypical proliferative osseous lesion. In markedly disorganized tissue many spindle-like cells, giant cells, and pleomorphic cells were seen together with the formation of large bone spicules and the heavy mineralization of osteoid-like material and of the remaining cartilage. Fibroblast-like cells were found to penetrate from the perichondrial zone into the condylar mass and also into the underlying collagen sponge. The in vivo growth characteristics of FBR murine osteosarcoma virus-infected condyles after 3 days in culture were studied via s.c. transplantation into syngeneic mice. Control condyles developed normal trabecular bone, whereas the infected condyles induced a strong cellular response with the presence of atypical cells and newly formed connective tissue and bone in situ. These observations raise the possibility of a novel approach for further investigations related to numerous aspects of virus-induced osteosarcomagenesis. PMID- 3009009 TI - Altered DNA topoisomerase II activity in Chinese hamster cells resistant to topoisomerase II inhibitors. AB - Most DNA intercalators and epipodophyllotoxins inhibit mammalian topoisomerase II by trapping the enzyme within DNA cleavage complexes that can be detected in cells as protein-associated DNA strand breaks. We have characterized previously a line of Chinese hamster cells (DC3F/9-OHE cells) the resistance of which to the cytotoxic effect of intercalators and etoposide is associated with a reduced formation of protein-associated DNA strand breaks. In the present study, topoisomerases of these cells were compared to those of the parental sensitive cells (DC3F). NaCl extracts (0.35 M) of isolated DC3F/9-OHE nuclei did not form 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced DNA-protein linking, whereas DC3F nuclear extracts did. In addition, DC3F/9-OHE nuclear extract had an unusually high level of DNA linking activity in the absence of 4'-(9 acridinylamino)methanesulfon-m-anisidide. Topoisomerases II from DC3F/9-OHE and DC3F nuclei appeared similar qualitatively. DC3F/9-OHE nuclear extract had approximately twice less topoisomerase II molecules than did DC3F nuclear extract but similar topoisomerase II activity. Topoisomerase I activities appeared also similar in sensitive and resistant cells. However, part of DC3F/9-OHE topoisomerase I copurified with a DNA linking activity which was not present in DC3F nuclei. This unusual DNA linking activity was not sensitive to the stimulatory effect of 4'-(9-acridinylamino)methanesulfon-m-anisidide. PMID- 3009012 TI - Vincristine neurotoxicity enhanced in combination chemotherapy including both teniposide and vincristine. AB - A high incidence of severe peripheral neuropathy occurred during the pilot study of a new regimen for the treatment of non-Hodgkin's lymphoma. The clinically observed incidence and severity of vincristine-induced peripheral neuropathy was considerably enhanced by the sequential use of vincristine and teniposide in this combination chemotherapy. PMID- 3009011 TI - Effects of antioxidants and hyperbaric oxygen in ameliorating experimental doxorubicin skin toxicity in the rat. AB - Doxorubicin, an antineoplastic drug, can cause severe ulceration if extravasated when iv injected. In this study, the effects of hyperbaric oxygenation (HBO) and the antioxidants butylated hydroxytoluene (BHT) and beta-carotene were tested on such ulcers using female Sprague-Dawley rats. It was found that HBO and vitamin A did not greatly ameliorate the ulcers produced by doxorubicin, but BHT prefed for 1 week before doxorubicin was injected was able to significantly reduce lesion size (P less than 0.05). Doxorubicin with HBO was a lethal combination, with an 87% mortality among the animals by the fourth week after injection. This was probably due to doxorubicin and HBO both promoting the formation of free radicals which are highly destructive to cells. BHT, when prefed (and to a lesser extent, beta-carotene), demonstrated a protective effect by lowering the death rate (P less than 0.05), probably due to their ability to scavenge free radicals. This experiment also tested more conventionally recommended treatments such as sodium bicarbonate (NaHCO3), hydrocortisone, and ice. NaHCO3 and hydrocortisone decreased lesion size although only at a significance of P less than 0.10. Ice did not aid in the healing of the doxorubicin-induced ulcers and even proved deleterious. Multiple injections of hydrocortisone or NaHCO3 appeared to deepen ulceration. Of all the treatments tested, free radical scavengers appear to most significantly reduce skin toxicity of doxorubicin. PMID- 3009014 TI - Hepatic cyst associated with ventriculoperitoneal shunt in a child with brain tumor. AB - A 12-year-old boy with a thalamic grade IV astrocytoma and ventriculoperitoneal (VP) shunt developed epigastric pain and symptoms of increased intracranial pressure. The SGOT and alkaline phosphatase levels were markedly elevated and the radiological studies showed a cyst in the right lobe of the liver, extending to the porta hepatis. Simple repositioning of the shunt resulted in complete resolution of clinical findings and disappearance of the cyst. Although abdominal pseudocysts associated with VP shunts have been reported, this is the first report of a cyst involving liver and causing hepatic dysfunction. PMID- 3009013 TI - Antiplatelet pyrimido-pyrimidines and metastasis. AB - The role of antiplatelet drugs in relation to their potential antimetastatic activities has been reviewed and the effects of two pyrimido-pyrimidine derivatives (RX-RA69 and RX-RA85) with strong antiplatelet activities investigated in metastasizing tumour models. The routes of administration and drug dosages were always chosen in such a way that good antiplatelet activities were obtained. RX-RA69 (20 mg/kg/day) given in the drinking water had no effect on spontaneous metastasis of Lewis lung carcinoma. RX-RA85 (20 mg/kg/day) did not influence spontaneous metastasis of B16 melanoma. On the other hand, giving RX RA85 (8 mg/kg) daily i.p. to Lewis lung carcinoma bearing mice significantly increased the number of lung metastases but had no significant effect on primary tumour implant growth. Pretreating mice orally with 20 mg/kg RX-RA85 1 h before i.v. injection of B16 melanoma cells had no significant effect on lung colony number or distribution of extrapulmonary tumours while injecting the same dosage of RX-RA85 i.v. 1-2 h before tumour-cell injection decreased lung colony formation, but increased extrapulmonary tumour burden. This investigation like many others does not support the importance of platelets in metastasis formation. PMID- 3009015 TI - Nuclear magnetic resonance of the liver, spleen, and pancreas. AB - This review includes the initial experience with NMR imaging of the liver, spleen, and pancreas at the University of California, San Francisco, using a prototype 0.35 Tesla system. This experience shows great promise for detection of hepatic metastases using T1-weighted pulse sequences. T2-weighted pulse sequences appear sensitive for detecting cavernous hemangioma of the liver and may allow tissue specific discrimination of the benign lesion from cancer. NMR is also suitable for evaluating diffuse metabolic alterations and is sensitive and specific for the diagnosis of iron overload. Detection of fatty liver requires use of chemical shift techniques as conventional NMR imaging pulse sequences are relatively insensitive. Motion artifacts and lack of an effective bowel contrast agent limits imaging of the pancreas and retroperitoneum, where CT remains the procedure of choice. The normal spleen has longer T1 and T2 relaxation times than liver or pancreas and NMR has not been successful in diagnosing splenic metastases or lymphoma on a routine basis. We conclude that NMR imaging will be valuable in the diagnosis of focal liver disorders; until fast scan techniques and effective magnetic contrast agents are available for oral and/or intravenous use, other abdominal applications will remain limited. PMID- 3009016 TI - Spectroscopic applications of magnetic resonance to biomedical problems. PMID- 3009017 TI - Current concepts of small-cell lung cancer. PMID- 3009018 TI - Herpes simplex virus genital infections. PMID- 3009019 TI - Immunocytochemical localization of a rhodopsin-like protein in the lipochondria in photosensitive neurons of Aplysia californica. AB - Polyclonal antibodies directed against squid opsin were used in immunocytochemical and immunoblot experiments to identify a rhodopsin-like protein in photosensitive neurons of Aplysia. Aldehyde-fixed abdominal and cerebral ganglia were embedded in paraffin for peroxidase anti-peroxidase analysis or used whole for immunofluorescence studies. Ganglia were embedded in Lowicryl K4M for electron-microscope immunocytochemistry. In both the cerebral and abdominal ganglia, light-microscope immunocytochemical results showed reaction product deposited around the neuronal cell periphery corresponding in position to the lipochondria. In the abdominal ganglion, the giant cell R2, located in the right rostral quarter, and neurons in the right caudal quarter were consistently labeled with anti-opsin. Electron-microscopic studies demonstrated ferritin-labeling of the lipochondria in R2 and other immunoreactive neurons. Immunoblot analysis of R2 and cerebral neuron extracts was used to identify two prominent immunoreactive protein bands at 85 000 and 67 500 molecular weight. PMID- 3009021 TI - [Determination of polio-neutralizing antibody by the micro-tissue culture technic]. PMID- 3009020 TI - An ultracytochemical investigation of ouabain-sensitive p-nitrophenylphosphatase in chick osteoclasts. AB - Cytochemical methods for the demonstration of p-nitrophenylphosphatase (p-NPPase) at the electron-microscopic level were applied to avian osteoclasts to elucidate some of the functional differences among ruffled border, other membrane systems, and cellular organelles. The localization of p-NPPase activity occurred in mitochondria, in lysosomes, and on the cytoplasmic side of the ruffled-border membrane. Enzymatic activity in the ruffled-border membrane was sensitive to ouabain and was partially dependent on potassium. Thus, Na+,K+-ATPase, the proposed sodium pump, appears to be present in the osteoclast ruffled border. The activity in ruffled border occurred only when osteoclasts were attached to bone. Following calcitonin treatment many osteoclasts were detached from the bone surface and lacked reaction product along the ruffled-border membrane. The lysosomal p-NPPase activity was not sensitive to ouabain and was distinct from that of other lysosomal phosphatases. The p-NPPase activity in mitochondria was not inhibited by ouabain but was sensitive to duramycin. The mitochondrial p NPPase may, therefore, represent the mitochondrial proton pump. PMID- 3009022 TI - The intracellular signal for induction of resistance to alkylating agents in E. coli. AB - The E. coli ada gene positively controls its own expression and that of other genes (alkA, alkB, aidB) involved in repair of DNA alkylation damage. The cloned ada and alkA genes and purified Ada protein have been used in cell-free systems to identify the inducing signal. Self-methylation of the Ada protein by transfer of a methyl group from a phosphotriester in alkylated DNA to a cysteine residue in the protein converts it to an activator of transcription. The covalently modified Ada protein binds specifically to promoter regions containing the sequence d(AAANNAAAGCGCA) immediately upstream of the RNA polymerase binding sites. This is apparently the first example of conversion of a regulatory gene product to a transcriptional activator by a posttranslational modification event. PMID- 3009023 TI - Defective propeptide processing of blood clotting factor IX caused by mutation of arginine to glutamine at position -4. AB - Blood clotting factor IX is synthesized as a precursor polypeptide that would be expected to be proteolytically cleaved in at least two positions during maturation to remove the prepeptide and propeptide regions. We show that a point mutation causing hemophilia B changes the amino acid at position -4 in the propeptide region of factor IX from an arginine to a glutamine, which results in the expression of a stable longer protein with 18 additional amino acids of the N terminal propeptide region still attached. This suggests that in the normal maturation of factor IX the signal peptidase cleaves the peptide bond between amino acid residues -18 and -19, generating an unstable profactor IX intermediate. Further proteolytic processing to the mature factor IX depends on the arginine residue at -4. The significance of the homologous arginine residue in other processed proteins is discussed. PMID- 3009024 TI - v-erbA cooperates with sarcoma oncogenes in leukemic cell transformation. AB - The v-erbB, v-src, v-fps, v-sea, and v-Ha-ras oncogenes induce avian erythroid progenitor cells to self-renew in an erythropoietin-independent manner. These transformed erythroblasts retain both their capacity to differentiate into erythrocytes and their requirement for complex growth media. However, previous studies showed that erythroblasts transformed by v-erbB plus v-erbA (which by itself is not oncogenic) are blocked in differentiation and grow in standard media. Here we show that the introduction of v-erbA into erythroblasts transformed with v-src, v-fps, v-sea, or v-Ha-ras likewise induces a fully transformed phenotype. It also reduces the capacity of ts sea- and ts erbB transformed erythroblasts to differentiate terminally in an erythropoietin dependent manner after a temperature shift. Cooperativity involving v-erbA also occurs in vivo since chicks infected with a retroviral construct encoding v-erbA and v-src develop both acute erythroblastosis and sarcomas. PMID- 3009025 TI - Determinants for receptor interaction and cell killing on the avian retrovirus glycoprotein gp85. AB - The virion envelope glycoprotein gp85 confers a high degree of subgroup specificity for interaction with distinct cell receptors. Specific subgroups of gp85 have been associated with a cytopathic virus-cell interaction, most likely resulting from reduced resistance to superinfection, which allows the buildup of excessive amounts of viral DNA. Previous nucleotide sequence analysis of the gp85 coding region identified small regions of variable amino acid sequence within a conserved framework. To define the role of these variable regions we constructed a series of molecular clones carrying novel combinations of variable regions from viruses. Analysis of rescued virus shows that receptor binding is determined by the interaction of two major regions and one minor region in the middle of gp85. Cytopathogenicity is not associated with any specific variable region but rather with the ability to recognize the subgroup B receptor on chicken cells. PMID- 3009026 TI - In vitro protein translocation across the yeast endoplasmic reticulum: ATP dependent posttranslational translocation of the prepro-alpha-factor. AB - The in vitro synthesized precursor of the alpha-factor pheromone, prepro-alpha factor, of Saccharomyces cerevisiae was translocated across yeast microsomal membranes in either a homologous or a wheat germ cell free system. Translocated prepro-alpha-factor was glycosylated, sedimented with yeast microsomal vesicles, and was protected from digestion by added protease, but was soluble after alkaline sodium carbonate treatment. Thus prepro-alpha-factor was properly sequestered within yeast microsomal vesicles, but was not integrated into the lipid bilayer. In marked contrast to protein translocation across mammalian microsomal membranes, translocation of prepro-alpha-factor across yeast microsomal membranes could occur posttranslationally. This reaction required protein components in the yeast microsomal fraction that could be inactivated by alkylation or proteolysis, was ATP-dependent, and was insensitive to the presence of a variety of uncouplers and ionophores. PMID- 3009028 TI - Subperiosteal tissue expanders for ridge augmentation. PMID- 3009027 TI - The SV40 enhancer is composed of multiple functional elements that can compensate for one another. AB - We present evidence that the SV40 enhancer consists of three functional units, A, B, and C, each of which can cooperate with the others or with duplicates of itself to enhance transcription. We show that, when element C, containing the core consensus sequence, is inactivated by point mutations, revertants with restored enhancer function contain duplications of either one or both of the elements A and B. To search for additional elements, we isolated revertants of a mutant with the three elements mutated. These revertants do not identify any other elements; instead, enhancer function is effectively restored by "double duplications," in which the first duplication event either partially or entirely recreates one of the three elements A, B, and C and the second duplication then creates two copies of the newly created sequence. PMID- 3009029 TI - Hydroxylapatite reconstruction of the alveolar ridge. PMID- 3009030 TI - [The role of herpes simplex viruses in the etiology of acute respiratory diseases]. PMID- 3009031 TI - The behavior of 1,4-benzodiazepine drugs in acidic media. IV. Proton and carbon 13 nuclear magnetic resonance spectra of diazepam and fludiazepam in acidic aqueous solution. PMID- 3009032 TI - Angiotensin I converting enzymes in rat epididymis. PMID- 3009033 TI - [Study on the etiology of hand, foot and mouth disease (HFMD) in China]. PMID- 3009034 TI - [A study on the pathophysiologic changes in different types of patients with essential hypertension based on traditional Chinese medical classification]. PMID- 3009035 TI - [3H-TdR lymphocyte transfer value and plasma cAMP and cGMP levels in patients with chronic gastric diseases due to splenic deficiency and its clinical significance]. PMID- 3009036 TI - [Effect of disodium cantharidate and injectable herbal medicine on energy and cyclic nucleotide metabolism in hepatoma 22 cells and liver tissues of tumor bearing mice]. PMID- 3009037 TI - [Typology of acute leukemia according to symptom differentiation and observations on its relation to blood, bone marrow and cyclic nucleotides in serum and urine]. PMID- 3009038 TI - [Pharmacological studies of the extract of Equisetum pratense on the tolerance to myocardial hypoxia in animals]. PMID- 3009039 TI - Ca2+-translocation activities of phosphatidylinositol, diacylglycerol and phosphatidic acid inferred by quin-2 in artificial membrane systems. AB - Ca2+-translocating activities of phosphatidylinositol, diacylglycerol and phosphatidic acid were investigated in phosphatidylcholine liposomes. Using a fluorescent indicator of Ca2+ concentration, quin-2, release of encapsulated Ca2+ from egg yolk phosphatidylcholine liposomes containing 2 mol% of one of these lipids was measured at 37 degrees C. The rate of Ca2+ translocation across the liposomal membrane mediated by phosphatidic acid was about 3-fold larger than those mediated by phosphatidylinositol and diacylglycerol. The result implies that phosphatidic acid has Ca2+-ionophore activity in the agonist dependent metabolism of inositol phospholipids. The ionophoretic activity depended on the degree of unsaturation of the fatty acyl chains. The Ca2+ translocation rate was smallest in dipalmitoylphosphatidic acid, and it increased in the order of dioleoyl-, dilinoleoyl- and dilinolenoyl-phosphatidic acid. Ca2+ mobilization of a stimulated cell is discussed in the light of Ca2+-ionophore activity of phosphatidic acid converted from inositol phospholipids. PMID- 3009040 TI - [Epidemiological aspects and prevalence of Neisseria gonorrhoeae in genital infections in Abidjan: analysis of 1742 samples]. AB - The authors report a study about the investigations of N. gonorrhoeae in 1 742 samples from genital. Epidemiological aspects and the prevalence of gonococcal infections are precised: 10% in vulvovaginitis; 73% in acute anterior urethritis. PMID- 3009041 TI - Nucleotide sequence of exon I of the rat c-K-ras gene. AB - We have isolated a DNA fragment containing exon I of rat cellular proto-oncogene of K-ras and sequenced the exon I. The obtained sequence was compared with that of the corresponding region in viral oncogene of Kirsten strain of murine sarcoma virus (Ki-MSV). The results showed that the exon I sequences of cellular K-ras genes in rat and human cells could encode the same amino acid sequence, while the viral K-ras gene could code for two different amino acids corresponding to the 12th and 37th positions from the N-terminus of K-ras gene product. The amino acid substitutions found between viral and cellular K-ras genes are discussed in relation to the transforming ability of Ki-MSV. PMID- 3009043 TI - Small cell lung cancer: results of a phase II study of 1,2,4 triglycidylurazol. AB - Fourteen patients with small cell lung cancer (SCLC) received treatment with 1,2,4 triglycidylurazol (TGU) 600 mg/m2 or 800 mg/m2 as an IV bolus every 4 weeks. Twelve patients had received previous chemotherapy consisting of a five drug regimen given for the short duration of only 9 weeks. All had measurable disease. Following TGU 11 patients had progressive disease and 3 patients had stable disease. The most frequent toxicity was nausea and vomiting, which occurred in all patients but was generally mild. Myelosuppression was common with a median white blood count nadir of 2.5 X 10(9)/l (range 0.9-7.4 X 10(9)/l) and median platelet count nadir of 76 X 10(9)/l (range 5-173 X 10(9)/l. Alopecia, thrombophlebitis, and hepatic or renal toxicity were not observed. In this study, TGU had no activity in SCLC, and the dose-limiting toxicity was myelosuppression. PMID- 3009044 TI - Hematopoietic neoplasias of the RFM/Un mouse contain somatic re-integration of the restriction endogenous ecotropic provirus. AB - We have examined the spleen DNA of individual mice of the RFM/Un strain for evidence of re-integration of the endogenous ecotropic provirus in radiation induced and spontaneous neoplasms. The ecotropic env specific probe detects only a single 19 kb EcoRI or a single 7.0 kb HindIII fragment in all DNA preparations from normal tissues of RFM mice, corresponding to the endogenous provirus. Additional DNA restriction fragments containing the ecotropic virus (eco) specific sequence, corresponding to somatically acquired provirus, are detected in two out of five spleen DNA samples from animals with myeloid leukemia and one of three with thymic lymphoma. In addition somatically acquired eco-specific fragments are also detected in greater than 85% of DNA samples from reticulum cell sarcomas, a late occurring spontaneous hematopoietic neoplasm in this mouse strain. These results are consistent with a 'promoter/enhancer insertion' model of leukemogenesis involving the endogenous ecotropic provirus and are of particular interest since the RFM/Un mouse possesses a locus that restricts exogenous infection of cells by the endogenous virus. PMID- 3009042 TI - Cross-linking between histones and DNA following treatment with a series of dimethane sulphonate esters. AB - The cross-linking of the nucleohistone to the associated DNA by a series of methanesulphonates of the busulphan series (CH3SO2O(CH2)nOSO2CH3) from n = 4-9 has been studied by gel electrophoresis, by the use of 35S labelling and by pyrenemaleimide competition. It has been shown that all the dimethanesulphonates react with the Cys 114 of histone H3, and that octamethylene dimethanesulphonate (n = 8) cross-links histone H3 with DNA via the sulphydryl group, as well as forming H3-H3 dimers. PMID- 3009045 TI - Proliferation-dependent regulation of base excision repair in normal and SV-40 transformed human cells. AB - The effect of SV-40 viral transformation of human cells on the proliferation dependent regulation of base excision repair was examined. Normal human diploid WI-38 fibroblasts and SV-40 transformed WI-38 cells were used to examine whether the process of transformation perturbed the regulation of base excision repair. The regulation of excision repair enzymes in each cell type was examined by quantitating: the specific activities of the base excision repair enzymes uracil DNA glycosylase and hypoxanthine DNA glycosylase as a function of cell growth; and immunological analysis of the uracil DNA glycosylase using monoclonal antibodies to human uracil DNA glycosylase. In each cell type, both DNA glycosylase activities were increased as a function of cell proliferation; the extent of enhancement being greater in the transformed cells. As defined by ELISA and immunoblot analysis, the induced uracil DNA glycosylases in both cell types share the same determinants. Although other repair processes are altered upon SV 40 transformation, these results suggest that the proliferation-dependent regulation of these base excision repair enzymes is retained in SV-40 transformed human cells. PMID- 3009046 TI - The Inter-Regional, Epidemiological Study of Childhood Cancer (IRESCC): case control study in children with germ cell tumours. AB - In 1980-1983 members of IRESCC interviewed parents of 555 children with newly diagnosed cancer on topics of possible aetiological significance. Identical questions were asked of the parents of 1100 control children chosen from hospital admissions and general practitioner lists. Medical information was confirmed whenever possible by cross-checking with NHS records. Data for the 41 children with germ cell tumours and their 82 controls are reported here. The cases had more major congenital malformations than controls, including one neural tube defect. More case than control mothers and fathers reported occupational exposure to chemicals. Nine close relatives of cases had multiple primary tumours, which were often benign or of low-grade malignancy, compared with 1 hospital control and 3 general practitioner control relatives. Cases and controls differed with respect to birth weight and paternal age. No case-control differences were shown for: birth rank, maternal age, chronic illnesses and smoking, mothers' reproductive histories and oral contraceptive usage. In index pregnancies there were no case-control differences for maternal illness, infections, alcohol intake and X-ray and ultrasound exposure. There was no difference between cases and controls for the frequency of twinning the families. PMID- 3009047 TI - Increased hydroxyl radical production in liver peroxisomal fractions from rats treated with peroxisome proliferators. AB - Electron spin resonance (e.s.r.), using the spin trap 5,5-dimethyl-1-pyrroline-N oxide (DMPO), has been employed to measure hydroxyl radical production in liver peroxisome-enriched fractions isolated from male Alpk/Ap rats administered chemicals known to cause peroxisome proliferation. The DMPO-OH adduct was found to decay to an e.s.r. silent species so rapidly in the presence of the native peroxisome-enriched fraction as to preclude any measurements in this system. All of the experiments were therefore carried out in the presence of cyanide in order to visualise the DMPO-OH adducts, although a consequence of this was the inhibition of the peroxisomal catalase activity. The DMPO-OH adduct was identified in fractions from both control and treated animals in the presence of palmitoyl CoA as substrate and was found to be present at 3-4 times the control value in animals orally administered di(2-ethylhexyl)phthalate (2000 mg/kg), clofibrate (200 mg/kg) or methyl clofenapate (25 mg/kg) for 9 days. The rate of production of hydroxyl radicals was also greater in fractions from treated animals. The fatty acyl CoA oxidase system of liver peroxisome-enriched fractions has now been shown to produce increased levels of hydrogen peroxide and hydroxyl radicals in the presence of a suitable substrate. Despite such evidence from in vitro enzyme systems, evidence of genotoxicity in vivo is still required to confirm the hypothesis linking such reactive oxygen species to the carcinogenicity observed in rodents with certain peroxisome proliferators. PMID- 3009048 TI - Differential effects of chronic alcohol administration to rats on the activation of aromatic amines to mutagens in the Ames test. AB - Male Wistar albino rats were maintained on alcohol-containing liquid diets for 4 weeks. Hepatic post-mitochondrial preparations derived from these animals were more efficient than control in activating 4-aminobiphenyl and 2-aminofluorene to mutagens in the Ames test. The alcohol-induced enhancement in mutagenicity was not inhibited by dimethylsulphoxide indicating that the generation of hydroxyl radicals is not involved. The activation of 2-naphthylamine was not affected by the treatment with alcohol but the mutagenicities of 2-aminoanthracene, benzo[a]pyrene and 3-methylcholanthrene were inhibited. The same treatment markedly increased hepatic microsomal aniline p-hydroxylase and ethoxyresorufin O de-ethylase activities and to a lesser extent benzphetamine N-demethylase and microsomal levels of total cytochromes P-450. It is concluded that chronic alcohol administration to rats modulates the metabolic activation of pre carcinogens to their reactive intermediates presumably by causing the redistribution of cytochrome P-450 isozymes. PMID- 3009049 TI - The truth about AIDS. PMID- 3009051 TI - Prevalence of antibodies to HTLV-III in quality-assurance sera. AB - We tested 146 clinical-laboratory quality-assurance sera for antibodies to human T-lymphotropic virus III (HTLV-III). Of 127 human-based samples, 39 (31%) were positive by an immunoenzymometric assay (IEMA). Samples of human origin that gave IEMA reactivity included four of 10 ethylene glycol-based (liquid) samples, two of six in-house pools of fresh sera, and 32 of 111 lyophilized samples. All 19 bovine-based samples were negative. Antibodies to HTLV-III in 16 samples were remeasured by a second IEMA and the Western blot technique. All assays detected antibody reactivity in four of the 16 samples; however, results of the second IEMA and the Western blot agreed best. We report large discrepancies between assay results when laboratory reagents are tested for HTLV-III antibodies, and find that many quality-assurance samples containing human sera have measurable IEMA reactivity for HTLV-III antibody. Reactivity in these assays indicates the presence of antibody, not viral infectivity. PMID- 3009052 TI - Measurement of urinary free 20 alpha-dihydrocortisol in biochemical diagnosis of chronic hypercorticoidism. AB - Using liquid chromatography, we estimated the urinary excretion of 20 alpha dihydrocortisol (20-DH) and urinary free cortisol (UFC) in normal subjects and in 40 patients with Cushing's syndrome of different etiologies. The median normal excretion rate (nmol/24 h) was 174 for 20-DH and 68 for UFC, the 20-DH/UFC ratio thus being 2.55. For patients with Cushing's syndrome, the excretion rate was 1798 for 20-DH and 298 for UFC, the ratio 6.03. We evaluated the effect of acute stimulation of adrenal secretion on 20-DH and UFC by administering corticotropin to six normal subjects. After such stimulation, the excretion rate was 566 for 20 DH and 1238 for UFC (ratio 0.45). Whereas 20-DH excretion rate exceeded the normal range in all patients, six patients had normal or even below-normal values for UFC excretion. Evidently, measurement of urinary 20-DH is a better test for chronic hypercorticoidism than is measurement of urinary UFC, and chronic hypercorticoidism can be differentiated from the acute state by the 20-DH/UFC ratio. PMID- 3009050 TI - Abrupt withdrawal of beta-blockade therapy in patients with myocardial infarction: effects on infarct size, left ventricular function, and hospital course. AB - The effects of abrupt withdrawal or continuation of beta-blockade therapy during acute myocardial infarction were evaluated in 326 patients participating in the Multicenter Investigation of the Limitation of Infarct Size (MILIS). Thirty-nine patients previously receiving a beta-blocker and randomly selected for withdrawal of beta-blockers and placebo treatment during infarction (group 1) were compared with 272 patients previously untreated with beta-blockers who were also randomly assigned to placebo therapy (group 2). There were no significant differences between the two groups in MB creatine kinase isoenzyme (15.8 +/- 10.9 vs 18.2 +/- 14.4 g-eq/m2, respectively) estimates of infarct size, radionuclide-determined left ventricular ejection fractions within 18 hr of infarction (0.44 +/- 0.15 vs 0.47 +/- 0.16) or 10 days later (0.42 +/- 0.14 vs 0.47 +/- 0.16), creatine kinase determined incidence of infarct extension (13% vs 6%), congestive heart failure (43% vs 37%), nonfatal ventricular fibrillation (5% vs 7%), or in-hospital mortality (13% vs 9%). Patients in group 1 had more recurrent ischemic chest pain (p = .002) within the first 24 hr after infarction, but not thereafter. However, this did not appear to be related to a rebound increase in systolic blood pressure, heart rate, or double product. In a separate analysis, 20 propranolol eligible group 1 patients randomly selected for withdrawal of beta-blockade (group 3) were compared with 15 patients randomly selected for continuation of prior beta-blockade therapy (group 4). This comparison yielded similar results. These data indicate that the beta-blockade withdrawal phenomenon is not a major clinical problem in patients with acute myocardial infarction. beta-Blockade therapy can be discontinued abruptly during acute myocardial infarction if clinically indicated. PMID- 3009053 TI - Liquid-chromatographic determination of isoenzymes of alkaline phosphatase in serum and tissue homogenates. AB - We describe a new method for separating alkaline phosphatase (AP) isoenzymes by means of "high-performance" liquid chromatography. Isoenzymes are eluted from the column (Mono Q HR 5/5, a strong anion-exchanger) with a stepwise gradient of LiCl. The isoenzymes originating from small intestine, bone, liver, and bile were identified by use of tissue homogenates, pathological sera, and heat inactivation. PMID- 3009054 TI - Automated kinetic assay of angiotensin-converting enzyme in serum. AB - We have developed and validated an automated kinetic method for angiotensin converting enzyme (EC 3.4.15.1) on the Olli C + D analyzer, modified from that of Ronca-Testoni (Clin Chem 1983;29:1093-6) with N-[3-(2-furyl)-acryloyl]-L phenylalanylglycylglycine used as substrate. We have determined appropriate reaction conditions for the assay, verified the principal analytical reliability criteria (repeatability, reproducibility, sensitivity), and established normal reference intervals (mean +/- SD) for the enzyme's activity, using serum of normal adults (100 +/- 35 U/L, n = 150), newborns (130 +/- 27 U/L, n = 10), women taking oral contraceptives (103 +/- 30 U/L, n = 10), smokers (109 +/- 38 U/L, n = 27), and patients with sarcoidosis (220 +/- 48 U/L, n = 15). PMID- 3009055 TI - Should plasma beta-endorphin be measured in patients with disorders of the hypothalamic-pituitary-adrenal axis? PMID- 3009056 TI - The use of dyes in reagents for radioimmunoassays. PMID- 3009057 TI - Changes in renal enzyme activities following the administration of gentamicin to unilateral nephrectomized rats. AB - The effects of unilateral nephrectomy and the impact of gentamicin administration on renal tissue enzyme activities in adult Wistar rats were investigated. Gentamicin 200 mg/kg body wt. or an equivalent volume of saline to control rats was administered subcutaneously on three consecutive days, followed by unilateral nephrectomy. Rats were killed on day 3, 7 or 14 following nephrectomy. Alkaline phosphatase, predominantly a proximal tubular brush border enzyme, rose in both the experimental and control groups, however, significantly less in the gentamicin treated rats. Aspartate aminotransferase activity, an enzyme participating in renal glucogenesis, increased transiently in the control but remained unchanged in the experimental group. No difference in glucose-6 phosphate dehydrogenase activity between the two groups was observed, probably reflecting the localization of this enzyme to distal tubular segments, a site unaffected by gentamicin. Significant and similar increases in Mg2+ and Na+ K+ ATPase were observed on day 14 in both groups. The administration of the drug resulted in a marked reduction in oxygen consumption, with a higher oxidation to phosphorylation ratio (P/O). Serum creatinine concentration was significantly higher on days 3 and 7 in the experimental group reverting to control values on the 14th day. Urea concentration increased significantly on days 3 and 7, decreasing on the 14th day to values slightly, but significantly, higher than those of the controls. PMID- 3009059 TI - Number of loci responsible for the inheritance of high and low activity of paraoxonase. AB - On the basis of a Danish family material of Eiberg & Mohr (1981), especially 416 matings with 1595 children where the one parent has high activity of paraoxonase and the other low activity, and the 185 matings with 684 children where both parents have high activity, it is, by consideration of a two-loci model and other alternatives besides the earlier suggested one-locus model, confirmed that segregation into high and low activity is largely or exclusively due to a one locus system; in case any secondary locus is involved, its most common allele would probably have a frequency above 0.95. PMID- 3009058 TI - CA 125 (ovarian tumour-associated antigen) in ascitic liver diseases. AB - The presence of Ca 125, an ovarian cancer-associated antigen, was assessed in serum from patients with liver diseases with (n = 26) and without (n = 26) ascites. Abnormal levels of serum CA 125 were observed in all patients with ascites and in 4 patients without ascites (15%). We conclude that CA 125 is a non specific marker of ascites whatever the origin: ovarian carcinoma, cirrhosis or peritoneal inflammatory process. PMID- 3009060 TI - A family study of the X-linked lymphoproliferative syndrome: evidence for a B cell defect contributing to the immunodeficiency. AB - The X-linked lymphoproliferative syndrome (XLPS) is an immunodeficiency characterized by severe primary infection with the Epstein Barr (EB) virus, which is often fatal. This has been attributed to failure to generate T lymphocytes which are specifically cytotoxic for EB virus-infected B lymphocytes, and which develop in all normal individuals following primary infection. We have studied a kindred which carried the defective gene for XLPS and have confirmed that the pattern of serum antibody responses to EB viral antigens can be used to detect affected males and female carriers. Furthermore, an EB virus genome-carrying B cell line which grew spontaneously from a culture of bone marrow cells from a male child with XLPS at the time of primary infection showed a decreased sensitivity to MHC-restricted, EB virus-specific killing by a T cell clone when compared to its in vitro EB virus-transformed counterpart. From these results we suggest that a subset of EB virus-infected B lymphocytes which were resistant to EB virus-specific killing existed in this child, and may have contributed to the overwhelming EB virus infection and its fatal outcome. PMID- 3009061 TI - Studies of EBV-lymphoid cell interactions in two patients with the X-linked lymphoproliferative syndrome: normal EBV-specific HLA-restricted cytotoxicity. AB - Two X-linked lymphoproliferative syndrome (XLP) patients with the hypogammaglobulinemia phenotype were investigated at a time remote from their primary infection with the Epstein-Barr virus (EBV). The lymphoblastoid cell lines derived from these patients expressed the phenotypic markers characteristic of normal mature B lymphocytes and produced normal levels of immunoglobulins (Ig). These observations imply that at least some of their B cells are phenotypically normal. The natural killer (NK) activity of the two patients was low. In one patient, activated lymphocyte killer (ALK) activity was inefficient. These two XLP patients expressed a normal EBV-specific, HLA-restricted cytotoxic activity. It thus appears, from the present findings and those in cases published previously (6/11 patients expressing normal EBV-specific cytotoxic activity), that the notion of poor specific T cell memory for EBV may not be as pivotal ass suggested or, alternatively, that this defect may not be common in hypogammaglobulinemic survivors. PMID- 3009062 TI - Normal and certain leukaemic B cells express IL-2 receptors without in vitro activation. AB - IL-2 receptor expression by B cells has previously been considered to be confined to activated normal B cells and, among the B cell leukaemias, to the hairy cells (HC) of hairy-cell leukaemia. In the present paper, using alpha-Tac monoclonal antibodies in a highly sensitive indirect rosette method, we show that both normal and certain leukaemic B cells other than HC express IL-2 receptors. The density of these receptors is low since they were not detectable by indirect immunofluorescence. Various controls excluded non-specific-reagent or exogenous receptor binding and blocking studies with recombinant IL-2 confirmed the presence of the IL-2 receptors. The significance of the findings is discussed and it is suggested that B cell IL-2 receptor expression without in-vitro activation may be a function of B cell maturity. PMID- 3009063 TI - Abnormalities of in vitro immunoglobulin production in apparently healthy haemophiliacs: relationship with alterations of T cell subsets and with HTLV-III seropositivity. AB - The pokeweed mitogen (PWM)-induced immunoglobulin (Ig) production by cultures of peripheral blood mononuclear cells (PBMC) was reduced in healthy haemophiliacs treated with commercial factor VIII (or IX) concentrate, whereas the spontaneous IgG synthesis in vitro was enhanced. PWM-induced Ig production was lower in those who had received greater amounts of concentrate, in those with inverted T4/T8 lymphocyte ratios and in those with antibody to HTLV-III. The spontaneous IgG production in vitro was higher in haemophiliacs who had received larger amounts of concentrate, in those with inverted T4/T8 ratio and in those with antibody anti-HTLV-III. However, some patients with normal T4/T8 ratio and some with HTLV III antibody also had raised spontaneous IgG production. PMID- 3009065 TI - The Epstein-Barr virus-induced production of IgE by human B cells. AB - B cells, isolated from the blood of healthy individuals and patients allergic to pollen, produced IgE when exposed to the human B-cell polyclonal activator, Epstein-Barr virus (EBV) in vitro and placed in culture. Secreted IgM and IgE were measured using immunoenzymatic assays. No difference was seen between healthy donors and allergic patients in the amount of IgE (or IgM) secreted. Cells were placed in limiting dilution cultures in order to determine the frequency of cells producing IgE or IgM (total and pollen specific) on exposure to EBV. Again, no significant differences in EBV-driven, B-cell precursor frequencies (PF) were seen between normal and allergic individuals. EBV-driven B cell PF for total IgM and IgE, and pollen-specific IgM and IgE secretion, were 1/450, 1/6500, 1/83,000, and less than 1 per 2,500,000, respectively, for cells from healthy donors, and 1/140, 1/4000, 1/56,000 and less than or equal to 1 per 2,000,000, respectively, for cells from allergic patients. We propose that the increased IgE levels seen in atopic individuals result solely from regulatory defects, rather than an increase in the frequency of B cells committed to the secretion of IgE. PMID- 3009064 TI - Suppression of in vitro human antithyroglobulin antibody secretion by private and cross-reactive anti-idiotypic antibodies. AB - This is a report on suppression of in vitro human antithyroglobulin antibody secretion in Epstein-Barr (EB) virus-transformed B lymphocytes by private and cross-reactive anti-idiotypic antibodies. Two polyclonal anti-idiotypic antibodies, private anti-Yo-Id and cross-reactive anti-Uc-Id antibodies, were raised in rabbits. Human IgG, lambda antithyroglobulin antibody-producing cell line (Yo3) and human IgG-producing cell line (Yo5), which did not contain the antithyroglobulin activity were established by EB virus transformation of peripheral lymphocytes of patient Yo with chronic thyroiditis. Binding of anti-Yo Id and anti-Uc-Id antibodies to IgG F(ab')2 antithyroglobulin derived from patient Yo was significantly inhibited by human thyroglobulin and affinity purified IgG antithyroglobulin of Yo3 cell line. Both private anti-Yo-Id and cross-reactive anti-Uc-Id antibodies suppressed the antithyroglobulin antibody secretion in Yo3 cell line. But these anti-idiotypic antibodies did not suppress the IgG secretion in Yo5 cell line. These results suggested that interactions between idiotype and anti-idiotype may play a role in the immune system of human chronic thyroiditis. PMID- 3009066 TI - High dose chemotherapy in solid tumours in adults. AB - The available evidence suggests that if benefit is to be obtained from high dose chemotherapy regimens, it will be in patients whose tumours are either untreated or still responding to conventional therapy. In each of the diseases discussed in this chapter the optimum timing of the treatment regimen has still to be determined. Effective regimens have been found but it is probable that further improvements can be made. In small cell lung cancer initial high dose therapy followed by non-cross-resistant regimens may prove effective. In glioma studies with high dose therapy before irradiation are awaited and may offer the best means of exploiting this approach to treatment. In breast cancer some impressive responses have occurred but the category of patient likely to benefit has not yet been defined. In melanoma high dose treatment is likely to benefit only those patients with probable minimal disease after surgery. PMID- 3009067 TI - Effect of stimulation of left atrial receptors on the concentration of adrenocorticotropic hormone (ACTH). AB - The effect of stimulating atrial receptors on the concentration of ACTH in jugular venous blood was examined in dogs anaesthetized with chloralose. The atrial receptors were stimulated by stretching the pulmonary vein-atrial junctions and the atrial appendage. In 13 experimental runs in 9 dogs, it was found that the concentration of ACTH fell in 11 and remained unchanged in 2. The average concentration during control periods was 76.5 +/- 13.6 (pg/ml) and that during stimulation was 60.0 +/- 11.8 (pg/ml). This difference was statistically significant (p less than 0.05; 2 tailed t test). Cutting (2 dogs) or cooling the cervical vagi (3 dogs) abolished this response. It is concluded that stimulation of left atrial receptors reduces the concentration of ACTH in plasma. PMID- 3009068 TI - Characterization of peptides derived from pro-opiomelanocortin in the biological fluids of a patient with Nelson's syndrome. AB - Molecular forms of immunoreactive adrenocorticotropin (ACTH), beta-lipotropin (beta-LPH) (beta-endorphin (beta-END), human NH-2-terminal (hNT) of pro opiomelanocortin (POMC), and gamma-3-melanotropin (gamma-3-MSCH) were studied in plasma, CSF and urine of a patient with Nelson's syndrome by molecular sieving and concanavalin A (Con A)-sepharose chromatography. In the culture tumor medium of the tumor cells, and in the plasma and CSF, these compounds were found mainly in forms corresponding in molecular weight to the authentic peptides, with the exception of gamma-3-MSH. Stimulation of the pituitary tumor by synthetic ovine corticotropin-releasing factor (CRF 1-41) caused a 171-468% increase in vivo (60 min) and 453-953% increase in vitro (3h incubation) in the levels of POMC derived peptides; it increased the relative amount of beta-END in vivo, and that of beta LPH in vitro. Molecular sieving chromatography of urine samples revealed that beta-LPH and hNT are extensively degraded by the kidney. By contrast, ACTH showed no significant renal degradation before the removal of the pituitary adenoma. However, following pituitary surgery, only smaller fragments of immunoreactive (IR) ACTH were detected in the urine. These results suggest no major abnormal metabolic pathway for POMC in Nelson's syndrome, although the proportions of various peptides derived from the precursor could be different in vivo from those after in vitro incubation under basal conditions and during CRF stimulation. The results also indicate differences in the renal handling of ACTH in POMC hypersecretory states. PMID- 3009069 TI - [MAO-containing unmyelinated fibers in human biopsied sural nerve--an electron microscopic study]. PMID- 3009070 TI - [A case of hereditary hypertrophic interstitial neuropathy accompanied with cataract, neurogenic deafness and hand tremor]. PMID- 3009071 TI - [Review of clinical records and therapeutic trials to familial amyloidotic polyneuropathy--study of 50 cases in Kumamoto (1967-1984)]. PMID- 3009072 TI - [Two siblings of familial "oculocraniosomatic neuromuscular disease with ragged red fibers" due to a partial cytochrome c oxidase deficiency]. PMID- 3009073 TI - Visual determination of differential renal function. AB - Forty patients (43 studies) referred for determination of differential renal function were imaged 24 hours after intravenous administration of Tc-99m-2, 3 DMSA. Visual assessment of relative renal uptake was estimated independently by three observers at three different hospitals from analog images on standard x-ray film. The results were compared with the relative DMSA uptake obtained by summing counts in computer-assisted regions of interest placed over each kidney. There was excellent correlation between the visual estimates of each observer and the computer-generated values (r = 0.98, 0.96, and 0.98, respectively). If a computer is not available, good visual estimates of differential uptake still may be obtained when static imaging agents such as DMSA are administered. PMID- 3009074 TI - Congenital anomalies--retrospective and contemporary treatment. AB - Attention has been increasingly directed toward the correction of anomalies of the fingers, hands, and forearm that appear at the time of birth and early development. There are so many variations that it would be difficult to list each separately. However, during the last 50 years, a pattern of general principles and procedures has evolved that is directed to all problems in surgery of the hand. These give a broader application for steps needed for infants with just one problem solution. Now much more can be done. Instruction must be given to the family and child with a congenital anomaly that it is the basic growth pattern of the limb that is altered. This must be watched over a period of time to help modify changes that develop. Much has been done to improve the condition that comes with birth. No doubt, with time, we may have some insight into the factors that cause these changes. PMID- 3009075 TI - Peripheral nerve surgery--today and looking ahead. AB - The trend in peripheral nerve surgery is toward earlier definitive treatment of the lesion, based on the optimal use of preoperative and intraoperative electrodiagnostic techniques. Newer diagnostic tools include computed tomography (CT) and thermography. Knowledge is still being gained about the technology and limitations of the autogenous nerve grafts that are being used to overcome nerve gaps. The technique of nerve anastomosis is undergoing rapid improvement, and better methods have been developed for identifying motor and sensory fascicles at the time of operation. Research activity into the problem of nerve damage produced at the time of trimming nerve stumps promises to change to the technology of nerve repair in the near future. For benign nerve sheath tumors (schwannoma, neurofibroma), the trend is away from nerve excision and in the direction of tumor enucleation. Histologic methods for diagnosing malignant nerve tumors have been improved, making it possible to embark on radical excision with less hesitation. The pain syndromes (causalgia, phantom limb pain, and stump pain) that may follow nerve injury continue to present a problem in management, but steady progress is being made toward a rational program of management. A more distant prospect is for pharmacologic and electrophysiologic methods to accelerate axonal regeneration. PMID- 3009076 TI - Microbiological assessment of ultraviolet sterilization of dental handpieces. PMID- 3009078 TI - Adenosine receptors in papilla of human kidneys. AB - Adenylate cyclase activity in homogenates of human papillae was stimulated by adenosine agonist compounds. The rank order of agonist potencies for stimulation was 5'-N-ethylcarboxamidoadenosine greater than L-N6-phenylisopropyladenosine greater than 2-chloroadenosine greater than N6-cyclohexyladenosine greater than D N6-phenylisopropyladenosine. This order of agonist potencies is similar to that observed at adenylate cyclase-coupled A2-adenosine receptors in many different tissues. The methylxanthines 8-phenyltheophylline and 1,3-diethyl-8 phenylxanthine acted as competitive antagonists, with 1,3-diethyl-8 phenylxanthine being approximately 10 times more potent than 8 phenyltheophylline. This study demonstrates the presence of A2-adenosine receptors in the papilla of human kidney. These receptors are similar to adenosine receptors previously observed in the renal papillae of the rat in terms of both agonist and antagonist potencies. PMID- 3009077 TI - Reduced glycemic response to beet-fibre meal in non-insulin-dependent diabetics and its relation to plasma levels of pancreatic and gastrointestinal hormones. AB - Standardized breakfasts with or without beet-fibre were given, in random order, to non-insulin-dependent diabetics. The blood glucose levels were monitored continuously and hormonal responses were determined at regular intervals for 3 hr. After the beet-fibre breakfast including 10.8 g dietary fibre from the sugar beet, the glucose plateau level and the area below the curve were lower than after the control meal. The rate of glucose decrease was also slower after the beet-fibre meal. There were no notable differences with regard to the plasma levels of insulin, C-peptide or glucagon. The gastric inhibitory polypeptide response was greater during the first part of the curve, while the somatostatin response after the beet-fibre meal displayed a significantly larger total area below the curve. The results suggest that the diminished glycemic response after the beet-fibre meal is associated with an increased response of somatostatin, giving a reduced glucose absorption and a delayed gastrointestinal transit time. PMID- 3009080 TI - [Acute metabolic effects induced by the addition of dietary fiber to a test meal in patients with type I and II diabetes mellitus]. PMID- 3009079 TI - Mitochondrial damage and the subcellular distribution of 2 oxoglutarate:glyoxylate carboligase in normal human and rat liver and in the liver of a patient with primary hyperoxaluria type I. AB - The subcellular distribution of 2-oxoglutarate:glyoxylate carboligase was investigated in a normal human liver, a liver from a patient with pyridoxine resistant primary hyperoxaluria type I and rat livers subjected to various degrees and types of trauma. On continuous sucrose gradients most of the carboligase fractionated with a peak equilibrium density of 1.19-1.20 g/cm3 and paralleled the distribution of the major peaks of monoamine oxidase, glutamate dehydrogenase and cytochrome oxidase and can be considered to be mitochondrial. Various proportions of the carboligase and mitochondrial marker enzymes were found to be 'extramitochondrial' (at or near the top of the sucrose gradients), depending on the liver source and the severity of trauma to which they were subjected. Carboligase, monoamine oxidase (outer membrane marker) and glutamate dehydrogenase (matrix marker) were released from mitochondria by the homogenization and centrifugation procedures, to the extent of 19.9%, 32.4% and 11.5% respectively in hyperoxaluric liver, 12.5%, 17.9% and 8.2% in normal human liver and 3.0%, 4.9% and 3.8% in control rat liver. The proportion of extramitochondrial cytochrome oxidase (inner membrane marker) was virtually undetectable in both human and rat livers. However, sonication of rat liver homogenates or the addition of the detergent Triton X-100 caused a massive release of all four enzymes. The extramitochondrial carboligase was probably in the form of a free protein of very high molecular weight or aggregate, rather than associated with a mitochondrion-derived organelle. Subfractionation of a rat liver mitochondrial preparation indicated that most of the carboligase activity paralleled activities of 2-oxoglutarate decarboxylase, citrate synthase and glutamate dehydrogenase and was probably located in the matrix.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009081 TI - [Polyneuropathy in chronic uremia. Clinical and electroneurologic study]. PMID- 3009082 TI - Laboratory diagnosis of herpes simplex virus infections. Principles guiding the development of rapid diagnostic tests. AB - While the incidence of many bacterial sexually transmitted diseases appears to be decreasing, the complications and frequency of viral sexually transmitted diseases in developed countries has increased over 10-fold in the last decade. Since 1975 genital herpes simplex virus infections have increased at a rate of 12% in the United Kingdom. Significant advances in our understanding of the epidemiology, natural history, and therapy of symptomatic genital herpes has occurred in the last 5 yr. In order to properly utilize this information, however, more widespread availability of laboratory diagnostic testing for herpes simplex virus is needed. The availability of tissue culture isolation facilities for herpes simplex virus has expanded to many community hospitals. More importantly, rapid assays to detect herpes simplex virus using monoclonal and polyclonal antibodies in enzyme-linked immunosorbent assay and immunofluorescent assays and/or detection of herpes simplex virus deoxyribonucleic acid by hybridization methods have also been developed. Recent studies indicate that these assays approach the sensitivity of viral isolation when samples from mucocutaneous lesions are taken. These rapid assays have also allowed clinicians to more rapidly diagnose serious herpes simplex virus infection such as neonatal herpes and to institute antiviral therapy earlier in the course of disease. Although the specificity of these assays in high prevalence populations appears excellent, few studies have evaluated these assays in populations where the prevalence of herpes simplex virus is low. Rapid assays for herpes simplex virus also appear to have decreased sensitivity (less than 60%) in detecting asymptomatic excretion of herpes simplex virus from the lower genital tract.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009084 TI - Immunoassays for the diagnosis of viral enteric pathogens. AB - The accurate diagnosis of infectious diseases is important for the optimal management of infected patients as well as for the prevention of disease transmission to susceptible individuals. Because viral gastroenteritis constitutes an important cause of morbidity in children living in developed countries and of mortality in children living in developing countries, there has been a great deal of interest in the development of effective methods for the diagnosis and study of this disease. While there are a number of assay systems that are capable of accurate detection of the agents of viral gastroenteritis in the intestinal contents of infected individuals, solid phase enzyme immunoassays have been used widely for this purpose. The widespread utilization of enzyme immunoassays is based on the advantages inherent in the use of enzymatic markers. These advantages include the stability of enzyme-immunoglobulin conjugates, the high degree of sensitivity inherent in the magnifying nature of enzyme-substrate interactions, and the low cost of assays performed in simple reaction formats. Enzyme immunoassays have thus been developed and widely used for the detection and epidemiologic study of rotaviruses, adenoviruses, coxsackie viruses, hepatitis A virus, and other agents that replicate in the human gastrointestinal tract. In addition, latex agglutination assays and nucleic acid hybridization techniques have been applied to the rapid detection of enteric viruses. It is likely that the application of a number of assay systems will promote accurate identification of a wide range of viral pathogens under different clinical, epidemiologic, and laboratory situations. PMID- 3009083 TI - Rapid diagnosis of respiratory virus infections in patients with acute respiratory disease. AB - Viral respiratory infections represent a significant segment of the total respiratory disease spectrum; however, until recently the laboratory diagnosis of viral respiratory infections was relatively inefficient. Development of new and improved immunologic assay systems has paved the way for accurate and reliable rapid diagnostic tests that detect viral antigens in clinical specimens. We conducted a careful and elaborate study in which radioimmunoassay for antigen detection was compared with a battery of tissue culture systems for viral isolation and identification. Using a fine plastic catheter, a specimen of mucus was aspirated from the nasopharynx of patients with clinical signs and symptoms of acute viral upper respiratory tract infections. Each specimen was divided into two portions; one was used to inoculate a variety of tissue culture cell lines and the other was used for radioimmunoassay tests for influenza A and B, adenovirus, parainfluenza 1, 2, and 3, and respiratory syncytial virus. Radioimmunoassay results compared very favorably with the tissue culture data with only one exception--adenovirus. Essentially this degree of accuracy and reproducibility was obtained with an enzyme-linked immunosorbent assay test, which has replaced radioimmunoassay. Tissue cultures are still used for backup, but with a rapid antigen detection system in place, coupled with a modern computer program to facilitate the laboratory data to the clinician, considerable strides have been made, and will continue to be made, in the diagnosis and therapy of viral respiratory tract infections. PMID- 3009085 TI - Rapid identification of coxsackie B viruses after immunoprecipitation and nucleic acid hybridization. AB - To circumvent the difficulties in concentrating sufficient virus from a clinical or environmental sample for detection and identification, we have used immunoprecipitation to rapidly concentrate coxsackie B viruses from both large and small sample volumes. Antiviral serum and killed Staphylococcus aureus cells as a protein A source were used to bind and collect the virus. Radioactively labeled viral nucleotide sequences were used to identify the collected virus by nucleic acid hybridization. The technique is applicable to the rapid concentration of dilute viruses from extremely large sample volumes as well as the samples from which virus isolation can be difficult. the process requires less than 2 days to complete and should be adaptable to virus identification by cell culture or other standard means as well. PMID- 3009086 TI - Pain and symptom control: I. PMID- 3009087 TI - A comparison of proteoglycan from chick cartilage of different types and a study of the effect of vitamin D on proteoglycan structure. AB - Normal chick growth cartilage contains mainly large proteoglycans that interact well with hyaluronic acid. In contrast normal articular and sternal cartilages contain mainly smaller molecules that interact poorly with hyaluronic acid. Similar smaller proteoglycans are present in all the cartilages in the rachitic state. The smaller proteoglycans all appear to be synthesized as such, and not to arise from degradation of larger molecules unless such degradation occurs extremely rapidly after synthesis. Supplementation of the vitamin D-deplete animals with calcium resulted in the production of large proteoglycans by the growth cartilage, but did not affect the size of the proteoglycan produced by the articular or sternal cartilages. It would appear that in these chicks the chondrocytes of the growth cartilage may be unique in their ability to produce large aggregating proteoglycans, and in their response to plasma calcium levels. PMID- 3009089 TI - Control of cellular and viral transcription during adenovirus infection. AB - The control of transcription initiation is an issue central to the regulation of eukaryotic gene expression, and as such, the elucidation of the mechanisms of control of initiation frequency is critical. The study of adenovirus transcription control has provided insights into these mechanisms. Transcription of the early viral genes is activated by the product of the viral E1A gene. Possibly of greater importance is the fact that this activation does not appear to be "viral specific". Rather, the E1A protein effects a general activation of transcription in the cell, resulting in the stimulation of transcription of at least one cellular gene in addition to the viral genes. Furthermore, there appears to be a cellular activity that functions in a manner analogous to E1A. Recent experiments also suggest a role for E1A in negative regulation of transcription, mediated through enhancer elements, that may be one aspect of gene control during cellular differentiation. Therefore, the study of E1A action may well contribute to an understanding of cellular transcription control. Finally, other mechanisms of transcription control in adenovirus infected cells such as genome replication-dependent gene activation and transcription termination control will likely contribute to the overall understanding of the control of mammalian cell gene expression. PMID- 3009088 TI - Limitation on sensitivity of measuring collagen degradation: studies with cultured liver cells. AB - Intracellular degradation of newly synthesized collagen is studied by incubating cells with (14C)proline and measuring the hydroxy(14C)proline in a low molecular weight fraction relative to total hydroxy-(14C)proline synthesized. This communication describes a fundamental limitation on the precision with which the measurement can be made; the limitation derives from an irreducible uncertainty in estimating the background radioactivity contributed by the isotope used for metabolic labeling. The problem is illustrated by experiments with cultured liver cells. In rat hepatocytes, the hydroxy-(14C)proline in the low molecular weight fraction was barely detectable above background; the degradation was 29%, however the uncertainty was +/- 28%. In contrast, significant amounts of hydroxy(14C)proline were detected in fractions from human and rat hepatoma cells; and while the levels of degradation were approximately the same as in rat hepatocytes, the measurements could be made with substantially greater precision. PMID- 3009090 TI - Computed tomography findings in gastrointestinal involvement by opportunistic organisms in acquired immune deficiency syndrome. AB - The computed tomography findings in two cases of gastrointestinal superinfection by opportunistic organisms in acquired immune deficiency syndrome are presented. Findings included thickening of the mucosal folds and the bowel wall in the small intestine and colon associated with cytomegalovirus and cryptosporidiosis. These cases indicate that it is important to look carefully at the gastrointestinal tract when performing computed tomography in this group of patients. Unsuspected bowel pathology may be demonstrated, or disease may be confirmed in clinically suspected cases. PMID- 3009091 TI - Comparison of cold storage and perfusion of dog livers on function of tissue slices. AB - We compared how two methods of hypothermic preservation affect physiological functions of tissue slices of dog liver. Livers were preserved by either (i) cold storage (CS) in Collins' solution or (ii) continuous perfusion (P) with a perfusate, containing hydroxyethyl starch, sodium gluconate, adenosine, and potassium phosphate, recently developed in our laboratory. Livers were cold stored for 6 to 8, 24, or 48 hr, and perfused for 24 or 72 hr. Tissue slices of preserved livers were incubated at 30 degrees C and analyzed for volume control, electrolyte-pump activity (K and Na), and adenine nucleotide concentration. Also, mitochondria were isolated after preservation to quantify respiratory activity. Slice functions of livers preserved for short periods (6 to 8 hr by CS and 24 hr by P) were similar to those for control livers. After normothermic incubation, the mean (+/- SD) water content of tissue (expressed per unit dry mass of tissue) was 2.3 +/- 0.3 kg/kg for control, 2.6 +/- 0.4 kg/kg for 6- to 8-hr CS, and 2.5 +/- 0.5 kg/kg for 24-hr P. Longer periods of preservation resulted in cell swelling, and water content was 3.3 +/- 0.4 kg/kg for 24- to 48-hr CS and 2.8 +/- 0.3 kg/kg for 72-hr P. The mean (+/- SD) K/Na ratio was nearly normal for livers preserved for short periods: 3.7 +/- 0.5 for control, 4.1 +/- 0.2 for 6- to 8-hr CS, and 3.3 +/- 0.4 for 24-hr P.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009093 TI - Studies on the circadian rhythm of IOP in rabbits: correlation with aqueous inflow and cAMP content. AB - We investigated the relationship of aqueous humor inflow rate and cyclic AMP concentrations to the spontaneous and dramatic changes in IOP associated with onset of darkness in our previously described model of circadian rhythm of intraocular pressure. After onset of darkness, rabbits entrained in an environment with a daily alternating cycle of 12 hours light and 12 hours darkness (12L:12D) showed an 85% increase in outflow pressure, a nearly 60% increase in aqueous inflow rate and an 80% increase in aqueous cAMP. Animals desynchronized by an unpredictable light cycle showed no increase in IOP or inflow rate when measured at the same time intervals as were the entrained animals. Thus, the IOP, aqueous inflow rate and aqueous cAMP are all seen to change in the same direction in a pharmacologically unperturbed rabbit eye. Previous pharmacological studies in rabbits have correlated an increase in cAMP with a decrease in IOP and aqueous inflow. PMID- 3009092 TI - Adrenergic receptors. PMID- 3009094 TI - Quantitation and kinetics of induced HSV-1 ocular shedding. AB - Iontophoresis of 6-hydroxydopamine (6-HD) to the rabbit eye, followed by topical instillation of 2% epinephrine, induces ocular shedding of herpes simplex virus type 1 (HSV-1) reliably and with a high frequency in latently infected rabbits. Rabbit eyes inoculated with HSV-1 (McKrae strain) showed dendritic lesions indicative of acute HSV infection and subsequently shed virus spontaneously at least once during days 20 to 39 postinoculation (P.I.). Two iontophoretic conditions were employed. Group A (3 rabbits, 60.3 days P.I.) received iontophoresis of 1.0% 6-HD at 0.75 mAmp for 3 min. Group B (three rabbits, 67.3 days P.I.) received iontophoresis of 0.1% 6-HD at 0.5 mAmp for 8 min. Following iontophoresis, 2% epinephrine was instilled topically once on the day of iontophoresis and twice daily for four consecutive days. Tear film was collected on Dacron swabs and titered on African green monkey kidney cells by a plaque assay procedure. In group A, 100% (6/6) of the eyes shed virus, and the average duration of shedding was 4.0 days. The titers ranged from 2.0 to 7.7 X 10(4) plaque-forming units (PFU) per eye. The highest daily average titer, 9.89 X 10(3) PFU/eye, occurred on day 5 following iontophoresis. In Group B, 100% (6/6) of the eyes also shed virus, and the average duration of shedding was 5.3 days. The viral titer of the tear film ranged from 5.0 to 1.4 X 10(5) PFU/eye. The highest daily average titer, 4.68 X 10(3) PFU/eye, also occurred on day 5 following iontophoresis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009096 TI - Mixed small cell and non-small cell lung cancer. AB - Seventeen (10 percent) of 176 patients with small-cell carcinoma of the lung seen at this hospital since 1976 proved to have mixed small-cell and non-small-cell tumors. The presence of a mixed lung cancer was established prior to chemotherapy or irradiation in nine patients. Eight were initially diagnosed as pure small cell carcinoma but proved to have a mixed tumor at either surgery or autopsy. Of the 17 patients, eight received chemotherapy, and four had a partial response. Six of the 40 autopsies performed on patients with small-cell lung cancer demonstrated intrathoracic tumor which was histologically mixed. Extrathoracic metastases in these patients were heterogeneous and included pure small-cell, pure non-small-cell, and mixed histologic type. We conclude that mixed small-cell and non-small-cell lung cancers are relatively frequent and carry important prognostic and therapeutic implications. Clinical management of patients with small-cell lung cancer should therefore be flexible and tailored to the potential for histologic diversity. Mixed lung cancer in previously untreated patients suggests a common endodermal origin for small-cell and non-small-cell pulmonary tumors. PMID- 3009095 TI - Malignant solid tumors of childhood. PMID- 3009098 TI - Leukotriene-associated toxic oxygen metabolites induce airway hyperreactivity. AB - The effect of toxic oxygen metabolite scavengers was examined in a guinea pig trachealis model of leukotriene (LTD4)-induced synergism upon histamine contractures. Under both physiologic (2.5 mM) and low (OmM) extracellular calcium conditions, LTD4 (10(-7) to 10(-9) M) potentiated histamine isometric tension responses. This LTD4-induced histamine hyperresponse was inhibited by pretreatment with superoxide dismutase. Inhibition of LTD4 receptor binding by FPL 55712 (10(-5) M) also aborted this interaction. Actual trachealis superoxide anion (O2-) generation by LTD4 was observed with a maximal release of 15 nM O2 /CPK unit X 10(-2) over 60 min. Phorbol myristate acetate (PMA) also generated O2 in this preparation. Trachealis muscle hyperreactivity to histamine induced by 10(-8) M LTD4 assayed in OmM (Ca++)E was not induced by PMA. It is concluded that exogenous LTD4 activates toxic oxygen metabolites which interact to induce an acquired hyperreactivity to agonist histamine in trachealis smooth muscle. PMID- 3009099 TI - Sarcoidosis developing during therapy for breast cancer. AB - Two women with breast carcinoma developed bilateral hilar adenopathy, with pulmonary infiltrate in one, during treatment for breast carcinoma. There was strong suspicion of metastatic disease from breast carcinoma. However, biopsy of a mediastinal node in first patient and transbronchial specimen biopsy in the second patient proved the diagnosis to be sarcoidosis. In one patient improvement was noted without therapy, and in the other improvement was noted with steroid treatment. PMID- 3009097 TI - Increased circulating activated T-cells in lung cancer. AB - T-cell activation (Tac) antigens, which are closely associated with the receptors for interleukin 2 (IL 2) and expressed on activated human T-lymphocytes, are found on a small percentage of normal peripheral T-cells. Elevated levels of Tac antigen-positive (Tac+) cells were observed in a high proportion of patients with untreated primary lung cancer assessed by using monoclonal anti-Tac antibody. The mean percentage of Tac+ cells in peripheral blood lymphocytes was 13.1 +/- 6.4 percent in patients with primary lung cancer (n = 67), as compared with 4.3 +/- 1.9 percent in normal controls (n = 30) (p less than 0.001). No significant differences were observed among the cell types of lung cancer examined (adenocarcinoma and squamous and small cell carcinoma). The stages of the disease also showed no significant differences in the development of Tac+ cells. Our results suggest that T-cell-mediated active immune mechanisms against malignant cancer cells are operative in patients with lung cancer, resulting in an increase in activated T-cells in the peripheral blood, although it remains to be elucidated whether these activated T-cells exert a favorable or unfavorable effect on their host. PMID- 3009100 TI - An assessment of thymosine in the treatment of patients suffering from myocarditis with decreased cell-mediated-immunity. PMID- 3009101 TI - Beta-thalassemia in Chinese--analysis of polymorphic restriction site haplotypes in the beta-globin gene cluster. PMID- 3009103 TI - [Intraneural cystic ganglion of the external sciatic-popliteal nerve. Presentation of a case]. PMID- 3009104 TI - [Sex hormone studies of ovarian tumors in postmenopausal women]. PMID- 3009102 TI - [Neoadjuvant treatment of osteosarcoma and malignant fibrous histiocytoma of the bones of the extremities: preliminary results in 76 cases treated preoperatively with a methotrexate and cisplatin combination]. PMID- 3009106 TI - [CT-pathologic study of the specimen of hepatocellular carcinoma]. PMID- 3009105 TI - A transcribed satellite DNA from the bullfrog Rana catesbeiana. AB - We have cloned and sequenced a 360 bp repeated sequence from genomic DNA of the bullfrog Rana catesbeiana. This sequence, which we call satellite 1, makes up 0.7% of the genome (1.6 X 10(5) repeats) and is scattered throughout the length of all the chromosomes. In situ hybridization with strand-specific probes demonstrated that transcripts from both strands of satellite 1 occur on lamp brush chromosome loops. We suggest that these transcripts arise by readthrough from upstream structural gene promoters. Sequences that cross-react with satellite 1 were found in genomic DNA of four out of five other Rana species that we tested, but were absent from the DNA of Xenopus, Bombina, and Acris. PMID- 3009107 TI - [Observation of motor nerve conduction velocity in renal insufficiency]. PMID- 3009108 TI - Colorectal cancer in patients under 40 years of age. AB - In a review of 1037 patients with colorectal cancers, there were 32 patients below the age of 40 years (3 percent). Rectal bleeding and abdominal pain were the most common presenting symptoms. The average delay between the onset of symptoms and treatment was 6.5 months. An analysis of tumors according to Dukes' staging revealed no significant difference between young and elderly patients. The younger patients had a greater frequency of mucinous and poorly differentiated carcinoma. When compared by clinical staging, however, the young patient did as well or better than his older counterpart. Clinical staging was the most important prognostic factor, irrespective of age. No inherent difference was found in the virulence of the cancer in the young, and five-year survival rates were not significantly different in young and old patients (59 percent vs. 49 percent). PMID- 3009109 TI - Survival and prognostic indicators in compensated and decompensated cirrhosis. AB - Six-year survival of cirrhosis was assessed in a series of 1155 consecutive patients (751 men, 404 women). Among the men, 33% were alcoholics and 18% were HBsAg positive; corresponding figures for the women were 15% and 6% respectively. Features of decompensation at first presentation were observed in 63% of the patients. Six-year survival was 54% in compensated and 21% in decompensated patients. No significant differences in survival were found between alcoholics and nonalcoholics. Leading causes of death were liver failure (49%), hepatocellular carcinoma (22%), and bleeding (13%). The prognostic role of 21 variables was evaluated separately in compensated and decompensated patients by the Cox's regression model. The following variables were found to be significant predictors of death risk in compensated patients: male sex, HBsAg positivity, age, prothrombin time prolongation, and esophageal varices. In decompensated disease the significant indicators of death risk were: hepatocellular carcinoma, encephalopathy, hemorrhage, SGOT, esophageal varices, gamma globulins, prothrombin time prolongation, continued abuse of alcohol, HBsAg positivity, gamma glutamyl transpeptidase, and cholinesterase. A simple prognostic index based upon the relative risk coefficient of the significant variables is suggested. PMID- 3009110 TI - Exocrine pancreatic function of children from the Ivory Coast compared to French children. Effect of kwashiorkor. AB - One hundred nineteen children, either French or from the Ivory Coast, aged 1-8 years, were submitted to pancreatic function testing by duodenal aspiration. Trypsin, chymotrypsin, lipase, phospholipase, amylase, volume, bicarbonate, chloride, and calcium were estimated before and after an intravenous injection of 1 CU secretin + 3 CHR units pancreozymin per kilogram of body weight. Sixty-two patients were normal European children, and 11 were normal African children. Twenty-five African children presented with kwashiorkor and 10 African children had presented with kwashiorkor but had recovered at the time of the test. Three cases of recurrent kwashiorkor are also included. In the normal group of African children, phospholipase concentration, volume, and bicarbonate were significantly decreased but chymotrypsin and trypsin concentrations were not, when compared to the normal European population. In kwashiorkor patients, lipase, amylase, phospholipase, and chymotrypsin concentration were significantly decreased compared to normal Africans. Trypsin, volume, and bicarbonate were not affected. These modifications disappeared after refeeding. In cases of recurrent kwashiorkor, all enzymes, including trypsin, were decreased. Calcium was never modified. These modifications were very different from those observed in chronic alcoholic and hypercalcemic pancreatitis. In a two-year study, chronic calcifying pancreatitis (CCP) was diagnosed in 14 patients (13 males), hospitalized in Abidjan. The mean age at onset of the disease was 41 years (SD 12.71), which is very similar to European cases. The most frequent cause was alcoholism, as in Occidental countries. The nutrition of the population was low in protein, calories being provided mostly by manioc, but no apparent symptoms of malnutrition were observed in the parents of our patients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009112 TI - Insulinoma in a 94-year-old woman: long-term therapy with verapamil. AB - Several drugs can be used to control hypoglycemia caused by insulin-secreting pancreatic tumors but none are reliably efficacious or free of side effects. We report the case of a woman with an insulinoma who refused surgical intervention and was successfully treated with the Ca2+-channel blocker verapamil. PMID- 3009111 TI - Effect of diet on fecal occult blood testing in patients with colorectal polyps. AB - To determine the effect of a red-meat-free, high-fiber diet, patients performed fecal blood testing before colonoscopy for suspected polyps. One hundred twenty nine patients were allowed an unrestricted diet, and 159 consumed the special diet. Without dietary restrictions, 31 of 77 patients with adenomas, five of eight with cancer, one of seven with nonneoplastic polyps, and one of 37 with normal colonoscopy had positive tests. With the special diet, 30 of 97 patients with adenomas, eight of ten with cancer, one of six with nonneoplastic polyps, and one of 46 with normal colonoscopy had positive tests. There are no statistical differences between the groups. Comparing all patients with cancer to those with normal colonoscopy, the sensitivity of fecal blood testing is 72%, the specificity 98%, and the positive and negative predictive value 87% and 94%, respectively. The sensitivity for fecal blood testing in patients with adenomas (compared to those with normal endoscopy) is 35%, the specificity is 98%, and the positive and negative predictive values are 97% and 42%, respectively. Combining all patients with adenomas, those with multiple polyps, larger polyps, or polyps located distal to the splenic flexure had positive fecal blood tests more frequently. Fecal blood testing does not detect a large number of colorectal adenomas. This study does not indicate any benefit on the sensitivity or specificity of fecal blood tests from the red-meat-free, high-fiber diet currently recommended. PMID- 3009114 TI - [Nucleotide sequence and transcription of the highly repetitive EcoRI-sequences in the rat DNA]. PMID- 3009113 TI - Recent developments in viral hepatitis. PMID- 3009115 TI - [Species specificity of the restriction cleavage of DNA repetitive sequences in the Far Eastern salmon of the genus Oncorhynchus]. PMID- 3009116 TI - [Study of the regulation of cytokinesis in early embryos of the sea urchin using the electric breakdown of a membrane]. PMID- 3009118 TI - [Thyroid hormone receptor of cancer cells--a transforming growth factor]. PMID- 3009117 TI - [Permeability and fusion of membranes initiated by the products of phosphoinositide metabolism]. PMID- 3009119 TI - [Specific conformation changes in RNA polymerase from Escherichia coli during formation of open promoter complexes on T7-DNA]. PMID- 3009120 TI - [Possibility of biosynthesis of leukotrienes B4 by double oxygenation of arachidonate during catalysis by reticulocyte lipoxygenase]. PMID- 3009121 TI - [Human Na+,K+-ATPase genes. Nucleotide sequence encoding the C-terminal region of the alpha-subunit]. PMID- 3009122 TI - [Effect of light, ATP and GTP on the binding of cGMP to rod outer segment membranes in the frog retina. Possible mechanism of receptor stimulation]. PMID- 3009123 TI - [Experimental proof of the existence of reverberation in the central nervous system]. PMID- 3009124 TI - [Regulation of electrotonic synaptic conduction between snail neurons by fragments of the active site of the ACTH molecule]. PMID- 3009126 TI - [Nuclear medical detection of gastrointestinal hemorrhage]. PMID- 3009125 TI - Possible risk of invasive pulmonary aspergillosis with marijuana use during chemotherapy for small cell lung cancer. AB - Bacterial and fungal contaminants have been identified in marijuana samples and thus are a potential risk factor in the immunocompromised patient using it as an antiemetic. We describe the development of an invasive pulmonary aspergillosis in a patient using illicitly obtained marijuana as an antiemetic during combination antitumor therapy for small cell lung cancer. Although this patient had multiple risk factors implicated in the development of invasive pulmonary aspergillosis, the infectious potential of inhaled marijuana must be recognized. Further study of this potential health risk in needed. PMID- 3009127 TI - [Morphological findings in the lymphadenopathy syndrome (LAS) and acquired immunodeficiency syndrome (AIDS)]. PMID- 3009128 TI - [Detection of attenuated virus latency in infectious laryngotracheitis in the trigeminal ganglion of the chicken]. PMID- 3009129 TI - [Effects of blood group markers (B allele), immunoglobulins and leukosis infections on the development of Marek's disease]. PMID- 3009130 TI - [Detection of avian adenovirus antigens in chicken cell cultures]. PMID- 3009132 TI - [Properties of the causative agent of avian infectious anemia (chicken anemia agent, CAA) in vitro]. PMID- 3009131 TI - [Prevention of Marek's disease by various vaccination viruses following test infection at various ages, study of PD50]. PMID- 3009133 TI - [Interaction of growth factors and oncogenes in the tumorous transformation of cells]. AB - Different aspects of the interaction of polypeptide growth factors with their receptors are reviewed. The problem of structural and functional interactions of the normal and transforming growth factors as well as the protein products of oncogenes during transmittance of a mitogenic signal and the proliferation regulation of normal and tumour cells is discussed. PMID- 3009134 TI - [Binding of tritiated imipramine to human platelets. 1 or several binding sites?]. AB - Using Langers' method (10) under their laboratory conditions, the authors determined the characteristics of the high-affinity imipramine-binding site on human platelets in thirty-five normal subjects, equally divided between the sexes and distributed by decade from twenty to sixty years of age and above. Bmax was 440 +/- 121 fmol./mg. protein, Kd 0.96 +/- 0.62, 10(-9) M. Protein concentration was held within a range of 0.2 to 0.55 mg./ml. in order to avoid nonlinear bias with Bmax, expressed in fmol./ml. of incubate. The characteristics of the site did not co-vary with sex or age in this experimental series. However, subject to additional investigation, the curve of the Scatchard graph would seem to indicate the possible existence of a distinct, low-affinity imipramine-binding site. PMID- 3009135 TI - Thyroid hormone receptors in neuronal and glial nuclei from mature rat brain. AB - To elucidate the mechanism of thyroid hormone action in the mature brain, we analyzed nuclear T3 receptors (NT3R) in neuronal and glial nuclei. Neuronal and glial nuclear fractions were prepared from mature rat brains with about 80% and 98% purity, respectively. Results from Scatchard analyses showed that NT3R capacity in neurons was 684.2 +/- 95.4 pg T3/mg DNA (mean +/- SD) in isolated nuclei and 345.6 +/- 77.6 pg T3/mg DNA in nuclear protein extracted with 0.4 M KCl. Glial NT3R had only one eighth the capacity of neuronal receptors. Displacement studies with several T3 analogs showed highly selective affinity of the receptors for L-T3. The relative affinities for several analogs were similar to those of liver NT3R. In addition, the elution profiles of the nuclear extracts through HPLC using gel filtration or diethylaminoethyl ion exchange columns exhibited similarity between neuronal and hepatic NT3R. The receptors in neuronal and glial nuclear fractions were analyzed in three groups of rats with different T3 levels: T3 (20 micrograms/100 g BW daily, for 3 days)-injected hyperthyroid rats, intact rats, and thyroidectomized rats. There were no significant alterations in capacity or affinity of the receptors among groups. The present studies demonstrate that numerous NT3R, which seem identical to hepatic NT3R, exist in neuronal nuclei. This raises the possibility that thyroid hormone acts through binding to NT3R in the cerebral cortex of the mature rat brain. PMID- 3009136 TI - Impaired parathyroid hormone-stimulated adenosine 3',5'-monophosphate release by isolated perfused bones obtained from vitamin D-deficient rats. AB - The present studies were designed to explore the mechanism underlying skeletal refractoriness to PTH in a vitamin D-deficient animal by assessment of PTH stimulated cAMP release from isolated perfused bone. In vitamin D-deficient (-D) rats both basal and PTH-stimulated cAMP release were markedly diminished, compared with that in vitamin D-replete (+D) rats. Isolated perfused bones from D rats that had undergone parathyroidectomy 2 days before death still showed reduced cAMP release in response to PTH, compared with +D bones. To investigate which factors in terms of Ca, endogenous PTH, or vitamin D might primarily be responsible for the impaired PTH-stimulated cAMP release from -D bones, some -D rats were switched to a diet identical to the vitamin D-deficient diet but with high Ca content (4%) for 2 or 5 weeks before death. This schedule maintained normocalcemia despite vitamin D deficiency. PTH-stimulated cAMP release in these rats was increased to a level intermediate between that in -D rats and +D rats, indicating partial restoration of the impaired response to PTH in -D rats. These data indicate that skeletal refractoriness to PTH in vitamin D-deficient animals might, in part, be due to the impaired activation of adenylate cyclase, which cannot be explained entirely by hypocalcemia or associated secondary hyperparathyroidism. Vitamin D deficiency per se, therefore, may play a key role in the impaired cAMP response to PTH. PMID- 3009137 TI - The preovulatory increase in ovarian collagenase activity in the rat is independent of prostaglandin production. AB - In the present study, we have examined the role of gonadotropins and prostaglandins in the preovulatory increase of ovarian collagenase activity in the rat. Whole ovaries of immature PMSG-primed rats (20 IU) were removed before and 8 h after the rats were treated with human (h) CG, Nembutal, and/or indomethacin. The ovaries were homogenized in a solution containing Triton X-100 (0.25%) and centrifuged. Collagenase was extracted by resuspending the pellets in buffer containing 100 mM CaCl2, heating to 60 C for 6 min, and centrifuging. The supernatants were treated with dithiothreitol (2 mM) and iodoacetamide (5 mM) to inactivate collagenase inhibitors. Collagenase activity was measured as the percent digestion of 3H-type I collagen/100 microliters aliquot of ovarian sample. At zero time (52 h after PMSG), ovarian collagenase activity was 4.2 +/- 1.2% digestion (mean +/- SEM, n = 3). In ovaries collected 8 h after the endogenous LH surge or 8 h after the administration of 10 IU hCG at time zero, collagenase activity rose to 19.6 +/- 2.1 (n = 6) and 22.5 +/- 1.7% digestion (n = 11), respectively. Indomethacin (1.5 mg/100 g BW) administered 30 min after hCG, produced no change in collagenase activity (24.8 +/- 2.5% digestion, n = 7) although the expected increase in ovarian prostaglandin E after hCG treatment was blocked. When the endogenous LH surge was blocked with Nembutal (3 mg/100 g BW), collagenase activity in 8-h ovaries was 6.8 +/- 1.1% digestion (n = 10). The Nembutal block of the preovulatory collagenase increase was overcome by administration of hCG (8-h ovarian enzyme activity = 22.7 +/- 3.2% digestion, n = 8). These observations demonstrate that hCG stimulates ovarian collagenase activity and that this stimulation is not dependent on prostaglandin synthesis. PMID- 3009138 TI - The effect of trypsinization on the plasma membrane binding and action of 3,5,3' triiodothyronine in rat thymocytes. AB - We have previously demonstrated that in the rat thymocyte T3 produces a prompt, extranuclear effect to increase cellular cAMP concentration and 2-deoxyglucose (2 DG) uptake. We have also demonstrated the presence of specific receptors for T3 in the rat thymocyte plasma membrane. From these and other observations we have suggested that T3 initiates these actions by binding to its receptors on the surface of this cell. To test this hypothesis further we have now examined whether a decrease in the binding of T3 to the thymocyte plasma membrane, induced by mild trypsinization, attenuates the effect of T3 on thymocyte cAMP concentration and 2-DG uptake. Mild trypsin treatment of rat thymocytes in cell suspension did not cause appreciable nonspecific damage to the cells, since it did not change the ability of the cells to exclude either trypan blue or mannitol, a marker of extracellular water. Further, treatment with trypsin did not alter the ability of thymocytes to bind concanavalin A. It did produce, however, a dose-related decrease in the binding of T3 to its plasma membrane binding sites owing to a decrease in their number, but not their affinity. This was associated with a progressive decrease in the T3-induced increase in both 2 DG uptake and cAMP concentration. Thus, low concentrations of trypsin had no significant effect on 2-DG uptake in the absence of T3, but decreased significantly the increase in sugar uptake induced by T3, whereas a higher concentration of trypsin (5 mg/ml) decreased basal 2-DG uptake by only 23%, but abolished the response to T3 altogether. T3, epinephrine, and trypsin alone induced dose-related increases in cellular cAMP concentration, the response to trypsin exceeding that to T3 or epinephrine. Trypsinization also resulted in a progressive decrease in the response of cellular cAMP to both hormones, so that at a trypsin concentration of 5 mg/ml no further increase was produced by either hormone. The possibility that a maximum activation of adenylate cyclase by trypsin could explain these results was rendered unlikely by experiments with prostaglandin E1 (PGE1). The increase in cellular cAMP concentration induced by this agent was vastly greater than that produced by trypsin alone. Although the response to PGE1 was greatly inhibited by trypsin, cellular cAMP concentration in thymocytes incubated with trypsin (5 mg/ml) plus PGE1 was much higher than that in cells treated with trypsin alone.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3009139 TI - Binding of growth hormone to rat liver during experimental chemical hepatocarcinogenesis. AB - Male F-344 rats (180-200 g) received either a single injection of diethylnitrosamine (DEN) or continuous feeding with 2-acetylaminofluorene (AAF). DEN induced a decrease in the binding of human GH (hGH) to its hepatic Golgi receptors in a dose-dependent manner; the changes were due to a decrease in the number of hGH receptors without significant changes in their affinity. Thirty days after DEN, the binding of hGH returned to normal. After the administration of AAF, the binding of hGH increased owing to the greater number of binding sites, and this effect persisted for the 30-day period of the continuous AAF feeding. In three separate hepatocellular carcinomas, the hGH binding to the Golgi fraction of the tumors was only one quarter of the binding to the peritumorous tissues. We conclude that DEN and AAF, administered acutely for a short time, affect hepatic hGH receptors in a different way, but that hepatocellular carcinomas bind much less hGH than peritumorous or normal tissues. The results show that hepatocarcinogenesis encompasses changes in receptors belonging to different classes. PMID- 3009140 TI - Regulation of rat granulosa cell differentiation by extracellular matrix produced by bovine corneal endothelial cells. AB - The effect of bovine corneal extracellular matrix (ECM) on gonadotropin-primed rat granulosa cells in vitro was studied by examining the following parameters: 1) rate of cell attachment to culture dishes; 2) modulation of cell morphology; 3) specific binding of [125I]human(h)CG to LH/hCG receptors; 4) cAMP response to hCG stimulation; and 5) basal and hCG stimulated progesterone production. Attachment of cells to culture dishes occurred significantly earlier on ECM, as compared with uncoated dishes (6 h vs. 24 h). Cells grown on ECM were epitheloid and organized in multilayer aggregates, closely resembling their organization in the intact wall of the ovarian follicle. In contrast, cultures on uncoated dishes grew as a monolayer of markedly flattened cells. A 2-fold increase in number of LH/hCG receptors occurred on ECM within 48 h, probably due to de novo synthesis. Scatchard analysis revealed no change in hormone affinity to the receptor during the culture period [association constant (Ka) = 2.5 X 10(10)M-1 for hCG]. Cells grown on ECM had a parallel increase in cAMP responsiveness to hCG stimulation. Cells grown in serum-free medium on ECM-coated dishes preserved only 50% of LH/hCG receptors and cAMP responsiveness after 48 h. Cells cultured on ECM showed a marked elevation in progesterone production even in the absence of gonadotropin stimulation, whereas cells grown on uncoated dishes almost completely lost their ability to produce progesterone both in the presence and absence of hCG. These results indicate that ECM plays a substantial role in the maintenance and further propagation of granulosa cell differentiation in vitro. PMID- 3009141 TI - Regulation of tetrahydrobiopterin biosynthesis in cultured adrenal cortical tumor cells by adrenocorticotropin and adenosine 3',5'-cyclic monophosphate. AB - Y-1 adrenal cortical tumor cells in culture, which contain substantial amounts of tetrahydrobiopterin [6R-(L-erythro-1',2'-dihydroxypropyl)5,6,7,8 tetrahydropterin] (BH4) and GTP cyclohydrolase (GTP-CH), were used to study the regulation of BH4 biosynthesis by ACTH and cAMP. ACTH produced a dose-dependent increase in steroidogenesis, BH4 levels and GTP-CH activity. Maximal stimulation of BH4 biosynthesis occurred at the same concentration of ACTH that caused maximal stimulation of steroidogenesis. ACTH-(1-24) was more potent than ACTH-(1 39). The stimulation of BH4 biosynthesis by ACTH was dependent on cell density, being greater at lower cell densities, but was independent of time in culture. The lack of stimulation by ACTH at higher cell densities was due to an increase in the specific activity of GTP-CH in the control cells as density increased. This increase may be due in part to the increased release of steroids, since exogenous steroids added to low density cultures also resulted in an increase in the specific activity of the enzyme. Addition of steroids had no effect on ACTH dependent stimulation of BH4 biosynthesis at low cell densities. (Bu)2cAMP, 8 bromo-cAMP, and forskolin all produced time- and dose-dependent increases in BH4 levels, GTP-CH activity, and steroidogenesis. Maximum increases in GTP-CH and BH4 occurred at concentrations similar to those required for maximal stimulation of steroidogenesis. In the Kin-8 mutant of Y-1 cells, which has a type 1 cAMP dependent protein kinase with an altered regulatory subunit, ACTH was unable to increase BH4 levels or GTP-CH activity at a concentration that produced maximal stimulation of BH4 and steroid biosynthesis in the parent Y-1 line. These studies indicate that Y-1 cells in culture are useful for studying the regulation of BH4 biosynthesis in the adrenal cortex. PMID- 3009142 TI - Interaction of peptide YY with rat intestinal epithelial plasma membranes: binding of the radioiodinated peptide. AB - High affinity binding sites for peptide YY (PYY) have been identified and characterized in plasma membranes prepared from rat jejunal epithelium by studying the kinetics, stoichiometry, and chemical specificity of the interaction of 125I-labeled PYY with membranes. Binding of [125I]PYY was rapid, saturable, reversible, specific, and depended on temperature, pH, and ionic strength. In optimized steady state conditions of binding (2 h of incubation at 15 C), the degradation of both [125I] PYY and binding sites did not exceed 20%. The concentration dependence of PYY binding, determined by adding increasing concentrations of [125I]PYY, indicated that specific binding saturated at 2-3 nM peptide. Scatchard analysis revealed a single class of binding sites with a dissociation constant (Kd) of 434 +/- (SE) 56 pM and a binding capacity of 336 +/ 41 fmol/mg protein (n = 11). Identical results were obtained when increasing concentrations of unlabeled PYY were added to a fixed concentration of [125I]PYY, indicating that the radioiodinated peptide has the same apparent affinity as native PYY. Peptides structurally unrelated to PYY, such as members of the vasoactive intestinal peptide family, insulin, or cholecystokinin octapeptide, were unable to compete with [125I]PYY for binding to membranes. Rat, human, and avian pancreatic polypeptides, which display, respectively, 42%, 47%, and 53% homology with PYY, did inhibit [125I]PYY binding but with an approximate or equal to 100,000-fold lower potency than PYY, indicating the strict structural requirement for recognition by PYY binding sites. In contrast, natural or synthetic neuropeptide Y, which has 25 out of 36 amino acids in common with PYY, retained a high affinity for PYY binding sites [only 4.7 +/- 1.2 (n = 5) times lower than that of PYY]. Specific [125I]PYY binding was particularly high in the upper small intestine and could not be detected in stomach, large intestine, or liver. These findings indicate that rat small intestinal epithelium expresses specific binding sites for the candidate gut hormone PYY that also binds the neuropeptide Y with high affinity, suggesting that the two peptides may regulate the function of small intestinal epithelium, through interaction with a common receptor site. PMID- 3009143 TI - Properties and regulation of the thyrotropin receptor in the FRTL5 rat thyroid cell line. AB - Despite extensive use of FRTL5 cells in studies of responses to TSH and anti-TSH receptor antibodies, almost nothing is known of the properties of their TSH receptors, possibly because binding of TSH by these cells is negligible when studied in their usual culture medium. In the present studies, we have demonstrated that specific binding of TSH can readily be demonstrated in confluent monolayers of FRTL5 cells if their culture medium is replaced by Krebs Ringer bicarbonate (KRB) buffer. In keeping with previous observations concerning the effects of cations on the binding of TSH in other thyroid systems, binding of TSH to FRTL5 was far greater when the medium used was a modified KRB in which an isosmotic substitution of sucrose for NaCl had been made. Kinetic studies of TSH binding in both types of medium suggested the presence of two binding sites, one with a higher affinity and lower maximum binding capacity than the other. The influence of NaCl was to decrease the capacity of both sites, that of the low affinity site to a greater extent than that of the high affinity site, whereas the affinities of the two sites remained unchanged. Correlative studies indicated that physiological responses to TSH were associated mainly with occupancy of the higher affinity sites. Experiments in which TSH binding was studied in cells grown to confluence in the presence of TSH from which TSH was then withdrawn and in cells maintained in the absence of TSH to which TSH was then added demonstrated the occurrence of up-and down-regulation, respectively, of receptor concentrations without a change in their affinities. The reduction in maximum binding capacity induced by TSH was proportionately greater in the case of the high affinity than the low affinity receptor. Down-regulation by TSH was concentration dependent and was demonstrable at a TSH concentration of 10(-11) M, considered to be physiological. Further, maximum down-regulation was induced by 10(-9) M TSH, the approximate concentration at which other responses to TSH in these cells reach their peak. Therefore, down-regulation of TSH receptors can be considered to be one of the physiological responses that TSH elicits. PMID- 3009144 TI - Two species of adenylate cyclase-stimulating activity in a murine squamous carcinoma model of humoral hypercalcemia of malignancy. AB - PTH receptor-stimulating proteins may be a common mediator of humoral hypercalcemia of malignancy (HHM). Such proteins exhibit adenylate cyclase stimulating activity (ACSA) in PTH-sensitive assays, and ACSA has been used to follow their purification. Acid/urea tumor extracts from a murine squamous carcinoma model of HHM were previously shown to have very high ACSA, which was partially, but incompletely, inhibited by the PTH antagonist Nle8,18,Tyr34-bovine PTH-(3-34) amide. ACSA from murine tumor extracts has now been further purified using solvent fractionation and reverse phase HPLC. Approximately half of the ACSA is attributable to a family of three proteins (peaks IA, IB, and IC) with properties characteristic of the PTH receptor-stimulating protein extracted from rat Leydig cell and human HHM tumors. The ACSA in these three peaks of murine tumor extract elutes in the same region as human tumor ACSA on reverse phase HPLC, has a dose-response curve parallel to that of PTH, and is fully inhibited by the PTH-(3-34) antagonist in both the renal cortical and rat osteosarcoma (ROS) adenylate cyclase assays. The remaining half of the ACSA from murine tumor extracts elutes as a single peak (peak II) at a higher acetonitrile concentration on reverse phase HPLC. In the renal cortical assay, its dose-response curve differs from that of PTH, its ACSA is not affected by the PTH-(3-34) antagonist, and it potentiates PTH- or peak I-stimulated adenylate cyclase activity. In the PTH-sensitive intact cell ROS assay, peak II exhibits no ACSA. We conclude that the potent ACSA of murine tumor acid/urea extract results in large part from amplification of the PTH-specific ACSA (peak I) by peak II. Peak II is a distinct protein, not previously reported in tumor extracts, that may act as a postreceptor step in the adenylate cyclase system. PMID- 3009145 TI - Estradiol suppression of luteinizing hormone (LH)/human chorionic gonadotropin receptors and LH-sensitive adenylyl cyclase without decreased adenosine 3',5' monophosphate content in rabbit corpora lutea. AB - It has been observed that elevated concentrations of estradiol, the principle luteotropin in the rabbit, reduce LH-stimulated adenylyl cyclase activity in corpora lutea during midpseudopregnancy without suppressing serum and tissue progesterone concentrations. If LH modulates intraluteal cAMP levels in this species, this suggests that in the presence of exogenous estradiol, progesterone synthesis may be independent of cAMP. To test this possibility and to investigate the physiological significance of LH in regulating the rabbit corpus luteum, the concentrations of cAMP and progesterone and the activity of adenylyl cyclase were measured in luteal homogenates, the numbers of LH/hCG receptors were estimated in crude membrane preparations, and the concentrations of progesterone and estradiol were measured in serum on days 2-12 of pseudopregnancy in rabbits treated with or without estradiol. Throughout days 2-12 of pseudopregnancy, estradiol treatment increased LH-stimulated adenylyl cyclase activity on day 2, but reduced its activity after day 4 by up to 50%, reduced the number of LH/hCG receptors after day 2 by up to 50%, and had no effect on the activities of basal, epinephrine stimulated, or NaF-stimulated adenylyl cyclase. Tissue cAMP levels were not altered by estradiol treatment, nor were serum progesterone concentrations (except for an increase on day 2). Since LH receptors and LH-stimulated adenylyl cyclase were both reduced by day 5 of pseudopregnancy without a concomitant decrease in luteal cAMP or serum progesterone concentrations, our data suggest that LH is not a physiological regulator of luteal cAMP or serum progesterone during days 4-12 of pseudopregnancy. We propose that basal adenylyl cyclase activity, which is not reduced by estradiol treatment, may play a more significant role than LH-stimulated adenylyl cyclase activity in regulating tissue cAMP levels and the progesterone synthetic capacity of the rabbit corpus luteum. PMID- 3009147 TI - Modification of luteinizing hormone secretion by activators of Ca2+/phospholipid dependent protein kinase. AB - We investigated the role of Ca2+/phospholipid-dependent protein kinase (protein kinase C) in LH secretion using rat anterior pituitary pieces obtained at known stages of the estrous cycle and superfused in vitro. Secretagogues were administered as 10-min (LHRH) or 30-min (all others) pulses. Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) results 2 h later in an amplification of LHRH-induced LH secretion in a concentration (1 nM to 1 microM)-and protein synthesis-dependent manner in proestrous, but not estrous, pituitaries; the diacylglycerol analog 1-oleoyl-2-acetylglycerol (OAG) also augments subsequent LHRH-induced secretion. At 1 microM, PMA alone increases the LH secretory rate, but with a pattern different from that induced by LHRH; the characteristics of the PMA response are affected by prior exposure to LHRH, estrous cycle stage, and cycloheximide. Pretreatment with either 8-bromo-cAMP or forskolin results in augmentation of subsequent LHRH-induced secretion without affecting baseline secretion. If the cells are exposed simultaneously to forskolin and OAG, but not 8-bromo-cAMP and OAG, the augmentation is dampened. This preliminary result suggests a possible interaction between protein kinase C and cAMP-dependent protein kinase in LH secretion regulation. We conclude that, regarding initiation of LH release, protein kinase C appears to be but one of a complex of mediators required for the secretory response to LHRH. Regarding the amplification of LHRH-induced release, activation of protein kinase C may be a component of the LHRH self-priming response. PMID- 3009146 TI - Desensitization to growth hormone-releasing factor (GRF) is associated with down regulation of GRF-binding sites. AB - The time course, concentration dependence, and mechanism of rat anterior pituitary desensitization to GRF were studied. Chronic stimulation of cultures of rat anterior pituitary cells with rat GRF (rGRF) resulted in desensitization to a subsequent challenge with the peptide. Despite a slight enhancement of GH synthesis, prolonged exposure to GRF caused substantial depletion of cellular GH pools. As a result, acute secretory responses were markedly blunted. Depletion was accompanied by a time-dependent decrease in sensitivity to rGRF; GRF EC50 values for GH release of 0.5 nM rGRF-pretreated cells were 24.8 +/- 6 (+/- SEM) pM after 2 h, 46.2 +/- 2.4 pM after 4 h, and 154.7 +/- 31 pM after 8 h compared to 9.2 +/- 0.6 pM for control cells. The process of desensitization was complete within 8 h, as cells pretreated for 24 h exhibited sensitivity to rGRF comparable to that of cells pretreated for 8 h. Desensitization was associated with a time dependent decrease in rat anterior pituitary cell GRF-binding capacity; a 48% loss of binding sites was evident after a 2-h pretreatment with 0.5 nM rGRF, with a maximum loss occurring after 8 h. The dose of rGRF required to produce an attenuation of responsiveness did not completely correlate with the dose requirement for down-regulation of binding sites. The decrease in GRF-binding sites was not associated with any alteration of apparent Kd values, which were 0.36 (0.18-0.72) nM in control and 0.1 (0.01-0.82) nM after 8 h of exposure to 0.5 nM rGRF. Both the reduction in GRF-binding capacity and decreased sensitivity to GRF were reversible after 24 h, although cellular GH pools were not restored to control levels. These results suggest that rat anterior pituitary cells become desensitized to rGRF after chronic stimulation with a maximal concentration of the peptide. One mechanism for this decrease in apparent sensitivity to rGRF may be the pronounced reduction or down-regulation of GRF-binding sites. PMID- 3009148 TI - Glucocorticoid treatment facilitates cyclic adenosine 3',5'-monophosphate dependent protein kinase response in parathyroid hormone-responsive osteogenic sarcoma cells. AB - Late passage cultures of a clonal osteogenic sarcoma line (ROS 17/2.8) failed to respond to PTH with activation of cAMP-dependent protein kinase isoenzymes despite showing a sensitive and dose-dependent increase in cAMP after treatment with the hormone. When cells were treated with hydrocortisone or dexamethasone, protein kinase responsiveness to PTH was readily demonstrated; such treatment also resulted in enhanced cAMP production. Forskolin preincubation resulted in a cAMP response to PTH of similar magnitude to that seen with hydrocortisone but no activation of cAMP-dependent protein kinase occurred. Thus, the effect of glucocorticoid cannot be explained merely by the increased amplitude and sensitivity of the cAMP response which developed with glucocorticoid treatment in these cells. The data indicate that cellular activation of cAMP-dependent protein kinase does not automatically follow cAMP generation and that information transfer can be restored by pharmacological means. PMID- 3009149 TI - Adrenocorticotropin-induced changes in ovine pituitary gonadotropin secretion in vitro. AB - In vitro pituitary perifusion experiments were conducted to examine the effect of ACTH and related peptides on basal and GnRH-stimulated gonadotropin release. Treatments of 5 X 10(-7) M ACTH-(1-39), ACTH-(1-24), or ACTH-(18-39) were examined for their ability to influence basal gonadotropin secretion and the subsequent response to a 10(-9)- or 10(-8) M GnRH challenge. Administration of the 1-39 or 18-39 peptide sequences of ACTH similarly stimulated the release of LH and FSH (P less than 0.01). ACTH-(1-24) had no effect on basal gonadotropin secretion. Pretreatment with ACTH-(1-39) inhibited the LH and FSH responses to 10(-9) and 10(-8) M GnRH (P less than 0.05). Suppression of the LH response to 10(-8) M GnRH (P less than 0.05) and the FSH response to 10(-9) M GnRH (P less than 0.05) was observed after ACTH-(1-24) treatment. The administration of ACTH (18-39) had no significant effect on GnRH-induced gonadotropin release. PRL concentrations were not affected by any of the ACTH peptides. Exposure to 10(-10) M GnRH or 5 X 10(-7) M synthetic ACTH-(1-39) produced an equivalent stimulation of LH secretion. GnRH pretreatment enhanced (P less than 0.05), while ACTH-(1-39) diminished (P less than 0.05), the subsequent response to GnRH. The GnRH receptor antagonist [D-pGlu1, D-Phe2, D-Trp3,6]GnRH attenuated the LH and FSH responses to GnRH and ACTH-(1-39) (P less than 0.05). The results obtained in this study indicate that certain portions of the ACTH molecule may affect gonadotropin secretion, perhaps by interacting with the GnRH receptor. PMID- 3009150 TI - Inhibition of luteinizing hormone release by morphine and endogenous opiates in cultured pituitary cells. AB - Morphine sulfate was found to have a direct inhibitory effect on both basal and GnRH-stimulated LH release by cultured rat pituitary cells. The inhibitory effect of morphine on LH release was prevented by the opiate antagonist naltrexone, and treatment of cells with naltrexone or beta-endorphin antiserum significantly increased basal LH release. Also, incubation of pituitary cells with CRF caused a significant decrease in basal LH release, an effect that was reversed by naltrexone. Saturable opiate-binding sites were demonstrated in enriched gonadotrophs by [3H]etorphine binding studies. The ability of morphine to inhibit gonadotropin secretion through a direct action on pituitary opiate receptors suggests that long term exposure to exogenous opiates may suppress reproductive function at the hypophyseal level. In addition, the converse effects of CRF and naltrexone or beta-endorphin antiserum on LH release indicate that intrapituitary opioid peptides could exert a paracrine inhibitory action on the gonadotroph. PMID- 3009151 TI - Diminished pituitary responsiveness to growth hormone-releasing factor in aging male rats. AB - The pattern of GH secretion undergoes substantial changes in the aging rat, resulting in decreased daily secretion of GH. In this study, the pituitary responsiveness to GH-releasing factor (GRF) was examined in young (2- to 5-month old) and aging (14- to 18-month old) male rats. In vivo studies were performed under sodium pentobarbital anesthesia. After injection of 250 ng GRF/100 g BW, young rats experienced more GH secretion [peak level, 544.5 +/- 209.5 (+/- SEM) ng/ml] than did 18-month-old rats (89.3 +/- 13.7 ng/ml). To investigate the locus of this insensitivity to GRF, anterior pituitary cells from young and aging rats were dispersed and placed in primary culture. While basal GH secretion from the cultured pituitary cells was similar in the two groups (49.7 +/- 3.5 vs. 47.8 +/- 2.7 ng/ml X 4 h for the 2- and 18-month old rats, respectively), the GH-releasing ability of GRF was partially but significantly impaired in cells derived from both 14- and 18-month old rats; 100 nM GRF stimulated the release of 96.7 +/- 1.8 ng/ml X 4 h in the 18-month old rats as opposed to 115.0 +/- 6.0 (P less than 0.05) ng/ml X 4 h in the 2-month-old rats. Since GRF stimulates GH release through the activation of adenylate cyclase, intracellular cAMP levels were measured in the cultured pituitary cells. GRF stimulated 65% less intracellular cAMP accumulation in the 18-month-old rats. In 14-month-old rats, the ability of forskolin and (Bu)2 cAMP to release GH was impaired, while phorbol ester-elicited GH secretion was unchanged. In conclusion, the GH response to GRF is blunted in aging rats. While much of the insensitivity to GRF may be mediated by the increased somatostatin tone reported in aging rats, a diminished pituitary cAMP response to GRF may also be an important etiological factor in the hyposomatotropinemia of the aging male rat. PMID- 3009152 TI - hCG-induced TSH receptor activation and growth acceleration in FRTL-5 thyroid cells. AB - Our previous studies have indicated specificity cross-over between LH/hCG and the TSH receptor. We have now analyzed the ability of the TSH-dependent Fisher Rat thyroid cell line (FRTL-5) to proliferate in a differentiated state under the influence of highly purified hCG (hCG-CR121). TSH receptor activation and growth induction were observed after 7 days suspension from a TSH-induced growth phase. The effect of 10 ug hCG-CR121 was equivalent to 500 +/- 43 (mean +/- SEM) uIU of human TSH (2nd IRP, 80/558) with reference to growth stimulation, as judged by 72 h [3H]-thymidine uptake and to approximately 15 +/- 35 uIU human TSH with reference to receptor activation as judged by cyclic AMP accumulation. These data demonstrate specificity cross-over by hCG for the TSH-dependent FRTL-5 cell line and suggest that hCG has greater growth-stimulating than thyroid-stimulating potential when compared with human TSH. PMID- 3009153 TI - Purification and characterization of adrenocortical adenosine 3',5'-monoposphate dependent protein kinases. AB - In this manuscript we describe in detail the purification and biochemical and immunological characterization of cAMP-dependent protein kinases in bovine adrenal cortex, rat adrenal gland, and isolated fasciculata cells of the rat. DEAE-cellulose chromatography of bovine adrenal cortex extract yielded two major (type I and type II) cAMP-dependent protein kinase peaks and one minor cAMP binding peak. The minor peak (peak A) eluted at 30-80 mM NaCl and corresponded to the typical type I tetrameric structure of the holoenzyme. Peak B, eluting at 80 130 mM NaCl, comprised 10-15% of the total cAMP-binding activity and was identified as dimeric type I cAMP-binding regulatory subunit of the enzyme. Peak C (major peak) eluting at high salt (130-220 mM NaCl), was different from the typical type II holoenzyme; its mol wt was relatively low (123,000), and its cAMP binding subunit was type I rather than type II. The native enzyme contained dimeric cAMP-binding regulatory subunit and suggested the presence of only a single catalytic subunit. Based on these results and on the reduced activation of its kinase activity by cAMP, we suggest a type I trimeric structure, R I2 C, of this enzyme. Most of the bovine adrenocortical extracts (62 of 68) did not contain type II cAMP-binding regulatory subunit of the enzyme. When present, its concentration (free or part of the holoenzyme) was less than 15% of the total cAMP-dependent protein kinases. These results were further supported by the studies with rat adrenal glands and isolated fasciculata cells derived from these glands, where only the type I cAMP receptor was found. We, therefore, conclude that in contrast to the current notion, adrenal cortex contains little, if any, enzyme containing type II cAMP-binding receptor. The predominant form of the holoenzyme contains a typical type I cAMP-binding receptor, but possesses an anomalous type II-like high salt elution pattern. We suggest that the trimeric structure of this enzyme contains a typical dimeric type I cAMP-binding subunit and a single catalytic subunit, R I2 C. PMID- 3009154 TI - Estradiol treatment decreases the lipolytic responses of hamster white adipocytes through a reduction in the activity of the adenylate cyclase catalytic subunit. AB - After 5 days of daily administration of 10 micrograms estradiol to 6-week-old male hamsters, the in vitro maximal lipolytic and cAMP responses of white adipocytes to isoproterenol, epinephrine, ACTH, and theophylline were reduced by one half, with no change in the sensitivity of these responses. In contrast, the antilipolytic response to the alpha 2-adrenergic agonist clonidine was unimpaired. beta-Adrenergic receptor number and affinity, assessed in intact cells with [3H]CGP-12177 binding, showed no difference between control and estradiol-treated hamsters. In adipocyte membranes from estradiol-treated hamsters, maximal adenylate cyclase responses to Mn2+, GTP alone or in combination with isoproterenol, ACTH, or fluoride were all decreased by 30-40% below the values found in controls, but the sensitivity of these responses was unaltered. The maximum velocity (Vmax) of adenylate cyclase was reduced by one half in estrogen-treated animals, but the Michaelis-Menten constant (Km) of the enzyme for ATP was unchanged. Finally, complementation of adipocyte membranes with solubilized human erythrocyte Ns failed to restore to control values the maximal adenylate cyclase response to isoproterenol plus guanosine 5'-[beta, gamma-imido]-triphosphate in the estradiol-treated hamsters. These results indicate that a defect in the catalytic subunit of adenylate cyclase is one of the mechanisms through which estradiol treatment reduces the lipolytic response of hamster white adipocytes. PMID- 3009155 TI - Inhibitory action of epidermal growth factor on progesterone biosynthesis in hen granulosa cells during short term culture: two sites of action. AB - The acute effect of epidermal growth factor (EGF) on progesterone biosynthesis by hen granulosa cells in short term culture was investigated. Pretreatment of cells for 5 h with EGF at concentrations of 1000-4000 ng/ml inhibited LH-stimulated progesterone production by 54%. Shorter EGF pretreatment times of 1 and 3 h caused 25% and 35% inhibition of LH-stimulated progesterone production, respectively. In additional experiments, EGF was found to inhibit progesterone production in response to 8-bromo-cAMP (1 mM) and forskolin (100 microM) by 34% and 35%, respectively. EGF had no effect on the conversion of 25-hydroxy cholesterol or pregnenolone to progesterone, indicating that one site at which EGF inhibits progesterone biosynthesis is distal to cAMP generation, but before the side-chain cleavage step. EGF also inhibited LH-stimulated cAMP production by 32%, but had no effect on forskolin-stimulated cAMP accumulation. This indicated that there was a second site of EGF action in these cells, probably at the level of LH receptor coupling to the adenylate cyclase. Nerve growth factor (4000 ng/ml) had no effect on progesterone production, but fibroblast growth factor (4000 ng/ml) facilitated LH-stimulated progesterone production. The results demonstrate that the acute inhibitory effect of EGF on LH-stimulated progesterone biosynthesis in hen granulosa cells is due to its action at two sites: one at a site before the production of cAMP and the other at a step beyond cAMP generation. PMID- 3009156 TI - Catecholestrogen production by porcine ovarian cells. AB - Catecholestrogens, formed locally, have been proposed to serve as local regulators of ovarian function. However, to date little or no activity of the critical biosynthetic enzyme, estrogen-2/4-hydroxylase, has been identified in the ovary. In the present study we have established the presence of this enzyme in porcine ovarian cells and characterized some of its biochemical features as well as its intraovarian distribution during the reproductive cycle. Homogenates of ovarian follicular tissue converted [3H]-17 beta-estradiol to the catecholestrogens 2-and 4-hydroxyestradiol (2-OH-E2 and 4-OH-E2) in a linear fashion for up to 60 min, with protein concentrations of equal to or less than 1 mg. The principal product, 2-OH-E2, was stable under the conditions of the assay. The reaction exhibited a dependence on nicotinamide cofactors, a pH optimum of 7.8, and an apparent Michaelis constant (Km) of approximately 10 microM for the production of 2-OH-E2 and 4-OH-E2. The activity of different ovarian preparations varied dramatically as a function of the reproductive cycle. Assayed at saturating substrate concentrations, immature follicular tissue and luteal tissue produced 50 or less pmol 2-OH-E2/mg protein X 40 min, while preovulatory follicles produced approximately 600 pmol 2-OH-E2/mg protein X 40 min. Even within the population of large presumptively preovulatory follicles, a variation in activity of more than 10-fold was encountered. Estrogen-2/4-hydroxylase activity of large preovulatory follicles correlated with the concentration of 17 beta-estradiol in the same follicles (r = +0.89, P less than 0.001). In large preovulatory follicles enzyme activity was present in both granulosa and theca layers. However, approximately 80% of the follicular activity was localized in the membrana granulosa. Under all conditions tested and in all ovarian compartments the formation of 2-OH-E2 was favored over that of 4-OH-E2. These studies show, for the first time, significant estrogen-2/4-hydroxylase enzyme activity within ovarian tissue. The striking increase in activity in the preovulatory follicle suggests physiological control of catecholestrogen synthesis. In conjunction with other data demonstrating stimulatory actions of catecholestrogens on ovarian cells, these observations are consistent with an intraovarian autocrine or paracrine regulatory function for these metabolites. PMID- 3009157 TI - Gonadotropin-mediated desensitization in a murine Leydig tumor cell line does not alter the regulatory and catalytic components of adenylate cyclase. AB - MLTC-1 cells, derived from a murine Leydig tumor, contain a gonadotropin responsive adenylate cyclase that became desensitized to hCG. Prior exposure to hCG reduced the ability of MLTC-1 cells to accumulate cAMP by approximately 50%, but caused only a small reduction in hCG receptor number. Membranes isolated from desensitized cells showed a similar reduction in hCG-stimulated adenylate cyclase activity. Desensitization was time, temperature, and dose dependent. Elevating intracellular cAMP levels by incubating the cells with (Bu)2cAMP or cholera toxin failed to cause desensitization. Desensitization did not depend on protein synthesis. Desensitization caused no change in the dose response of adenylate cyclase to hCG or GTP. hCG receptor affinity for hCG was not affected by desensitization or guanine nucleotides. The stimulatory regulatory component of adenylate cyclase (Ns) from MLTC-1 cells was used to reconstitute S49 cyc- membranes, which lack Ns. Ns from control and desensitized MLTC-1 cells were equally effective in reconstitution of the beta-adrenergic-sensitive adenylate cyclase of cyc-. beta-Adrenergic receptors from cyc- membranes were also transferred to MLTC-1 membranes by fusion with polyethylene glycol to produce a beta-adrenergic-responsive adenylate cyclase. Isoproterenol-stimulated activity was similar, regardless of whether membranes from control or desensitized MLTC-1 cells were used. We conclude that neither Ns nor the catalytic subunit of the adenylate cyclase in MLTC-1 cells is the site of lesion in desensitization. Most likely, the hCG receptor itself may be affected when MLTC-1 cells are desensitized by hCG. PMID- 3009159 TI - Parathyroid hormone stimulates the proliferation of cells derived from human bone. AB - Despite its acute inhibitory effect on bone formation in vitro, PTH has been shown to have an anabolic effect on bone in vivo and to stimulate cell proliferation in osteoblastic cell lines and organ cultures. We have examined the effects of PTH on cells derived from human trabecular bone and compared these effects with those on human skin fibroblasts. Human bone cells have the capacity to synthesize type I collagen and osteocalcin, and to respond to 1,25 dihydroxyvitamin D3 with an increase in the synthesis of osteocalcin and alkaline phosphatase. PTH stimulated adenylate cyclase activity at both low and high cell density. However, the same concentrations of hormone stimulated the proliferation of these cells only when they were cultured at a high cell density. The effect of PTH was bone cell specific in that no proliferative effect of PTH was detected in cultures of human skin fibroblasts obtained from the same donor and cultured under the same conditions. The effect of PTH on DNA synthesis by human bone cells may be important in the generation of a long term anabolic response to PTH. PMID- 3009158 TI - Biosynthesis of a novel thyroxine-binding protein (27K protein) in human hepatoma (Hep G2) cells. AB - Human hepatoma (Hep G2) cells were shown to synthesize and secrete a novel T4 binding protein, called 27K protein for its apparent mol wt on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The mRNA coding for this protein was characterized by immunoprecipitation of [125I]T4 bound to 27K protein secreted into the medium of oocytes injected with total Hep G2 RNA. Sucrose gradient fractionation of RNA from Hep G2 cells showed that TBG mRNA and 27K mRNA had different sizes, indicating that TBG and 27K protein are two distinct proteins. In vitro translation of RNA in a rabbit reticulocyte lysate demonstrated that the translation product immunoprecipitated by anti-27K serum had the same mol wt as the immunoprecipitated protein from whole cells labeled with [35S]methionine, thus suggesting that 27K protein is neither derived from TBG nor synthesized through a larger mol wt precursor, and also that it does not contain carbohydrates. The absence of carbohydrates was further supported by the observation that [3H]mannose was not covalently bound to the 27K protein when Hep G2 cells were labeled with [3H]mannose, nor was there a shift in apparent mol wt when the cells were treated with the glycosylation inhibitor tunicamycin. The kinetics of secretion of 27K protein were similar to those of albumin and faster than those of TBG, which is also in keeping with the nonglycoprotein nature of 27K protein. PMID- 3009160 TI - Iodine suppression of iodide uptake in FRTL-5 thyroid cells. AB - Exposure of FRTL-5 cells to iodide (I-) in excess of 3 microM suppresses the concentrative uptake of I-. The depression of I- uptake measured at the steady state is due to decrease in the rate of I- influx and not to an effect on I- efflux. Exposure to NaI is associated with decreased T4 secretion and also depressed Na+-dependent amino acid accumulation. The depression in I- and amino acid transports increases proportionately with the duration of exposure and concentration of I- used but is not associated with alterations in FRTL-5 cell cAMP levels. The I- suppression effect is blocked, however, when methimazole is present during the incubation with NaI. In agreement with studies in vivo, I- suppression in FRTL-5 cells appears to depend on an intermediate in the organification process and to be independent of a TSH-induced cAMP-mediated action. PMID- 3009161 TI - Intestinal mucosa is a target tissue for pancreatic polypeptide. AB - Studies were carried out to identify mammalian tissues capable of specifically binding mammalian pancreatic polypeptide (PP). Bovine PP (bPP) radiolabeled with 125I was purified by HPLC to yield [125I]iodo-(Tyr-27) bPP. The label was injected into three pairs of fasted littermate dogs and allowed to circulate for 5 min. One of the dogs was a control which received an excess of unlabeled porcine PP to provide competition for receptor binding. Unbound bPP was removed by perfusion with Krebs-Ringer bicarbonate and the tissue fixed in situ with Karnovsky's fixative. Tissue samples from various organs were removed, weighed, and counted. The entire gastrointestinal tract demonstrated high levels of 125I after injection of the labeled peptide. The duodenum, jejunum, ileum, and colon were the only tissues to exhibit specific binding of bPP. These tissues (mucosal and muscle layers) from experimental animals exhibited 31-76% higher binding than the corresponding tissues from the control animals. Sections of the gastrointestinal tract were scraped to separate the mucosal layer from the underlying muscle layer. The mucosal layer of the duodenum, jejunum, and ileum exhibited 145-162% increases in binding compared to the control animals. The muscle layer of these tissues demonstrated no significant increase. These findings demonstrate that mucosal layer of the small intestine is a target tissue for mammalian PP. PMID- 3009162 TI - Dexamethasone effects on beta-adrenergic receptors and adenylate cyclase regulatory proteins Gs and Gi in ROS 17/2.8 cells. AB - Treatment of ROS 17/2.8 cells with dexamethasone (dex) increases (-)isoproterenol (ISO)-, PTH-, cholera toxin-, guanine nucleotide-, NaF-, and forskolin-stimulated adenylate cyclase activity. Enhanced hormone stimulation was first apparent 12 h after dex addition. (-)-[3H]Dihydroalprenolol binding, displaceable by ISO, increased up to 2-fold in dex-treated cells. This effect depended on protein synthesis and closely paralleled the extent and time course of the increase in adenylate cyclase stimulation. In dex-treated cells there was also an increase in the maximum velocity of guanyl-5'-yl imidodiphosphate-stimulated adenylate cyclase, a decrease in the lag time for guanyl-5'-yl imidodiphosphate enzyme activation in the presence of ISO from 3 to 1 min, increased stimulation of adenylate cyclase by cholera toxin, and increased labeling of 47,000 and 42,000 mol wt proteins by [32P]NAD in the presence of cholera toxin. [32P]NAD ribosylation in the presence of pertussis toxin resulted in the labeling of 40,000 mol wt protein, which was also increased by 20-50% in dex-treated cells. However, pertussis toxin treatment did not augment or reduce the effect on hormone stimulation, although it increased the cAMP response to PTH and (-)ISO. These findings suggest that dex increases (-)ISO stimulation of adenylate cyclase in ROS 17/2.8 cells by jointly increasing the number of hormone receptors and the abundance of Gs, the guanine nucleotide binding regulatory protein. PMID- 3009163 TI - Repopulation of Leydig cells in mature rats after selective destruction of the existent Leydig cells with ethylene dimethane sulfonate is dependent on luteinizing hormone and not follicle-stimulating hormone. AB - After selective destruction of Leydig cells in mature rats with ethylene dimethane sulfonate (EDS), repopulation of Leydig cells occurs. This repopulation process was studied in normal and sterile (prenatally irradiated) rats using morphological and histochemical techniques and by measuring hormone concentrations. Three days after administration of EDS to normal rats, extensive Leydig cell degeneration had occurred, testosterone concentrations were decreased to less than 10% of the normal value, and no 3 beta-hydroxysteroid dehydrogenase activity or pregnenolone production could be detected in isolated interstitial cells. Seven days after EDS administration, no cells with the appearance of Leydig cells were observed, and steroidogenic activities were still absent. After 14 days, single or paired Leydig cells were present again in the interstitium, but only after 21 days an increase in the plasma testosterone concentration and LH-dependent pregnenolone production was observed. On day 35, numerous Leydig cells were present, and testosterone levels were restored to normal. The depletion and repopulation of Leydig cells after administration of EDS to sterile rats showed a somewhat different pattern. Three days after administration of EDS, testosterone concentrations were decreased to less than 10% of the normal value, and isolated interstitial cells showed no steroidogenic activities as in normal rats, but a small number of Leydig cells was still present. A similar picture was observed between 4 and 9 days after EDS administration. This indicates that some Leydig cells from sterile rats, unlike Leydig cells from normal rats, were resistant to EDS. The repopulation of Leydig cells in sterile rats was faster than in normal rats. After 14 days, many groups of Leydig cells were present in the interstitium, and the plasma testosterone concentration and pregnenolone production in vitro were significantly increased. Normal plasma testosterone levels were restored on day 21. Serum LH and FSH were decreased immediately after EDS administration, but during the next days a sharp rise was observed in both normal and sterile rats. The rise in LH correlated with the decrease in testosterone, and restoration of LH levels took place when testosterone levels increased. FSH levels changed similarly, but were delayed, in comparison to LH. In rats with testosterone implants that suppressed LH levels to less than 2 ng/ml and maintained normal FSH levels, ranging from 150-340 ng/ml, as well as in hypophysectomized rats, no repopulation of Leydig cells could be observed until 35 days after EDS treatment.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3009164 TI - Gonadotropin-releasing hormone (GnRH) rapidly stimulates the formation of inositol phosphates and diacyglycerol in rat granulosa cells: further evidence for the involvement of Ca2+ and protein kinase C in the action of GnRH. AB - GnRH provokes a phospholipid response in rat granulosa cells that has been characterized by increased incorporation of radioactive precursors into phosphatidic acid and phosphatidylinositol, and by depletion of 32P-prelabeled polyphosphoinositides. In this report, rat granulosa cells from mature Graafian follicles were incubated with GnRH under various conditions to follow the hydrolysis of phosphoinositides and the generation of the metabolic byproducts of phospholipase C action. Granulosa cells were prelabeled for 3 h with myo[2 3H]inositol. GnRH provoked rapid (10 sec) and sustained (up to 60 min) increases in the levels of inositol monophosphates, inositol bisphosphates, and inositol trisphosphates (IP3). Time-course studies revealed that IP3 was formed more rapidly than inositol bisphosphate and inositol monophosphate after GnRH treatment. The response to GnRH was concentration dependent (maximal at 10 ng/ml) and was prevented by a specific GnRH antagonist. Lithium chloride (1-10 mM) greatly enhanced the GnRH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. No changes were observed in the levels of free [3H] inositol and [3H]phosphatidylinositol in GnRH-treated cells. However, treatment with both lithium and GnRH for 30 min significantly reduced the levels of free [3H]inositol and [3H] phosphatidylinositol. In the presence of lithium, the rate of hormone stimulated inositol phosphate formation was not altered by 30 min of prior treatment with GnRH, indicating that phospholipase C activity is not readily desensitized. GnRH also increased the formation of diacylglycerol (DAG), another product of phospholipase C action. In cells prelabeled with [3H] arachidonic acid, GnRH significantly increased levels of DAG in incubations lasting 2-5 min. Concomitant increases in [3H] phosphatidic acid were also observed in GnRH treated cells. In conjunction with these studies, intracellular free Ca2+ levels were measured by Quin 2 fluorescence. GnRH and its agonistic analog rapidly increased (5 sec) cytosolic free Ca2+ levels (approximately double). The results demonstrate that an early event in the action of GnRH is the hydrolysis of phosphoinositides by a phospholipase C-dependent mechanism. The products resulting from this action of GnRH, i.e. IP3 and DAG, may serve as intracellular mediators for the mobilization of intracellular calcium, or the activation of protein kinase C and arachidonic acid release. PMID- 3009165 TI - An improved method for measuring angiotensin I converting enzyme activity using a highly sensitive angiotensin II radioimmunoassay. AB - A highly sensitive assay for angiotensin I converting enzyme has been developed by using angiotensin I as a substrate. Angiotensin II generated in the reaction mixture was measured by a newly developed specific radioimmunoassay. To protect against angiotensin II destruction, bestatin, an inhibitor of renin, was also used to inhibit plasma renin activity. The reaction was stopped by adding EDTA and MK-521, inhibitors of angiotensin I converting enzyme. The specificity of the antiserum used for the angiotensin II radioimmunoassay was very high. The cross reactivity with angiotensin I was less than 0.5% and none of the proteolytic enzyme inhibitors crossreacted in the assay. The inhibitory effect of pepstatin on plasma renin activity was very high (more than 80%) under the standard assay conditions employed. Serum angiotensinase activity was completely inhibited by the addition of bestatin. An excellent correlation was obtained between this new method and the spectrophotometric method using a synthetic substrate, Hip-His Leu. The generation of as little as 12 pM of Angiotensin II can be detected. Such low concentration have not been measurable with the usual spectrophotometric method. This new method will facilitate clinical and experimental studies on this unique enzyme, since very low levels of activity can be determined by this highly sensitive radioimmunoassay for angiotensin II. PMID- 3009167 TI - Studies on prolactin in human milk. AB - A possible physiological role of prolactin (PRL) in human milk was studied. The concentrations of milk PRL were 36.5 +/- 5.8 ng/ml on the first day after delivery and increased rapidly on the second day. Higher levels continued up to the fifth day. The levels of milk PRL showed no significant correlation with milk yield. Milk PRL was capable of binding to its receptors, suggesting its biological activities. In conclusion, human milk contains significant amounts of immunoreactive PRL. Milk PRL appears to be biologically potent, which raises the possibility that it may have certain physiological significance for the newborn. PMID- 3009166 TI - Calcitonin-responsive clonal cell line from porcine kidney (PK(15)) in serum-free medium. AB - PK(15), a homogeneous epithelial cell line from porcine kidney which was originally established through single cell cloning from PK-2a, was found to respond to [Asu1,7]eel calcitonin with an increase in adenosine 3':5'-cyclic monophosphate (cAMP) content, but do not respond to parathyroid hormone or arginine vasopressin. These cells were able to grow in a synthetic medium (a 1:1 mixture of Dulbecco's modified Eagle's MEM and Ham's F12 medium) without any supplementary factor. The medium supplemented with selenous acid, transferrin, and insulin permitted a growth rate equivalent to those in serum containing medium. When grown in the serum-free defined medium, these cells showed an increase in cAMP content in response to [Asu1,7]eel calcitonin to approximately the same degree as in the serum containing medium (10% fetal calf serum). Our present study first indicates that PK(15) cells are capable of growing in the serum-free defined medium retaining the calcitonin responsiveness of the original cells. PMID- 3009168 TI - [In vivo stimulation of adenyl cyclase in rats]. PMID- 3009169 TI - [Nle4, D-Phe7]-alpha-MSH: a superpotent melanotropin that "irreversibly" activates melanoma tyrosinase. AB - The superpotent and ultraprolonged melanotropic properties of an alpha melanotropin analog, [Nle4, D-Phe7]-alpha-MSH, were investigated in a Cloudman S91 (CCL 53.1) melanoma cell line. [Nle4, D-Phe7]-alpha-MSH is 100-fold more effective than the native hormone, alpha-melanocyte stimulating hormone (alpha MSH), in stimulating melanoma cell tyrosinase activity, as determined from their minimum effective doses (10(-11)M and 10(-9)M, respectively). [Nle4, D-Phe7] alpha-MSH also exhibits a more sustained effect than alpha-MSH on tyrosinase after removal of the melanotropins from the incubation medium. When cells were exposed to alpha-MSH (10(-7)M) for 24 h, residual activity after removal of the hormone was minimally significant. In contrast, under the same experimental conditions [Nle4, D-Phe7]-alpha-MSH treatment induced tyrosinase activity 2-3 fold above basal level, and maintained remarkable stimulatory effects up to 72 h following melanotropin removal. When the exposure time to melanotropins was reduced to 4 h, alpha-MSH failed to elicit significant tyrosinase activity, whereas [Nle4, D-Phe7]-alpha-MSH stimulated significant tyrosinase activity during the first 24 h subsequent to melanotropin removal. Interestingly, this stimulation by the analog increased at 48 h, reached a maximum at 72 h following removal of the melanotropin analog, and remained significantly stimulated for 6 consecutive days in the absence of the analog. PMID- 3009170 TI - The roles of cAMP and cAMP-dependent protein kinase in forskolin's actions on Y1 adrenocortical tumor cells. AB - The effects of forskolin on the regulation of steroidogenesis and growth were examined in the Y1 adrenocortical tumor cell line, and the roles of cAMP and cAMP dependent protein kinase in these actions of forskolin were evaluated. Forskolin, like corticotropin, stimulated steroidogenesis 3-fold and inhibited growth by 90%. In mutants of the Y1 cell line harboring specific defects in cAMP-dependent protein kinase activity, the responses to forskolin were attenuated. The resistance of the protein kinase mutants to the diterpene was closely correlated with their resistance to corticotropin and with impaired responses of their protein kinases to cAMP. These results indicate that cAMP and cAMP-dependent protein kinase are obligatory components of forskolin's actions on Y1 adrenal cells. Forskolin, at concentrations which were approximately 100-times greater than those required to stimulate steroidogenesis, caused cAMP to accumulate. Apparently, only a small fraction of the cAMP generated in response to forskolin was required to stimulate steroidogenesis or inhibit growth. PMID- 3009171 TI - Conformational analysis of erythrosine B (FD&C Red No. 3) and its comparison with thyroid hormone structures. AB - Erythrosine B, also known as FD&C Red No. 3, is a tetraiodofluorescein dye that is widely used as a biological stain and color additive in food and drugs. Recent data show that erythrosine B and Rose Bengal, its polychlorophenyl derivative, are potent inhibitors of both 5'-T4 and 5-T4 monoiododeiodinase activity. However, fluorescein, the nonhalogenated parent compound, has no effect on deiodinase activity. The X-ray crystal structure of erythrosine B was determined to elucidate the structural basis for its competition with T4 for its hormone protein binding sites. These structural results show that the dye crystallizes as a free acid-ethanol solvate. The relative orientation of the benzoic acid and xanthine moieties is nearly perpendicular, similar to that observed in the structure of fluorescein. As frequently noted in thyroid hormone structures, there are short I...I and I...O contact distances in this structure. Because of the symmetric iodophenolic substitution pattern of the xanthine ring, there will always be one iodophenolic ring that is not homologous with the thyroid hormone structure. Therefore, this analysis suggests that the best conformational homology is achieved when the dye phenolic ring is matched with that of a skewed thyroid hormone structure. PMID- 3009172 TI - Use of 125I-triiodothyroacetic acid to measure nuclear thyroid hormone receptor. AB - 125I-Triac was employed to measure hepatic thyroid hormone nuclear receptor (RT) in the rat. The binding properties of 125I-Triac and 125I-T3 were compared in a 0.4 M KCl extract of a liver nuclear preparation. The order in which the stable compounds, Triac, T3, T4 and rT3, competed for 125I-Triac and 125I-T3 binding in liver nuclear extract was similar (Triac greater than T3 greater than T4 greater than rT3), suggesting association of both radioligands with RT. Scatchard plot analysis of specific 125I-Triac and 125I-T3 binding in nuclear extract gave approximately equal estimates of the maximum binding capacity (MBC). However, the binding affinity, as represented by the equilibrium association constant (KA), was higher for 125I-Triac than for 125I-T3 (7-10 X 10(9)M-1 vs 1-3 X 10(9)M-1). To determine the effect of contaminating serum proteins on estimates of MBC and KA, a small amount of dilute rat serum was added to the same nuclear extract preparation. Addition of serum decreased the KA value and markedly increased the MBC values estimated by analysis of 125I-T3 binding data. In contrast, KA and MBC values derived from 125I-Triac binding data were not influenced appreciably by the addition of serum. These data indicate that: 1) both 125I-Triac and 125I-T3 bound to RT in rat liver nuclear extract, 2) the affinity of RT for 125I-Triac is appreciably greater than for 125I-T3, and 3) estimates of RT concentration (MBC) made with 125I-Triac are less sensitive to serum protein contamination than those made with 125I-T3. These properties of 125I-Triac may be useful in efforts to demonstrate RT in tissues that have low RT levels and/or when serum contamination is present. PMID- 3009173 TI - Urinary cyclic AMP:creatinine ratio and nephrogenous cyclic AMP as indicators parathyroid functional status. AB - The clinical utility of the urinary cyclic AMP:creatinine ratio in assessing parathyroid function was evaluated in 33 hypercalcemic patients and compared this with the determination of the renal component of urinary cyclic AMP. We found the discriminatory value of urinary cyclic AMP:creatinine ratio to be slightly superior and to have additional advantages. Not only did the urinary cyclic AMP:creatinine ratio show empirically somewhat better discrimination between normals and patients with primary hyperparathyroidism, but it is technically simpler than the determination of the nephrogenous cyclic AMP. Our urinary cyclic AMP excretion data show 90% discrimination of primary hyperparathyroid subjects from normals. Among all hypercalcemic patients studied who had both elevated urinary cyclic AMP and elevated parathyroid hormone (PTH) levels by radioimmunoassay (RIA), 77% had primary hyperparathyroidism, and 23% had malignancy-associated hypercalcemia. Of those patients with malignant tumors and hypercalcemia, half had elevated urinary cyclic AMP and two thirds had elevated PTH by RIA. These data suggest that these tests have little discriminatory value in differentiating primary hyperparathyroidism from malignancy-associated hypercalcemia. No hypercalcemic patient who had both serum PTH and urine cyclic AMP in the normal range was found to have primary hyperparathyroidism. This suggests that further observation and evaluation is indicated in such patients before exploratory surgery is undertaken. PMID- 3009174 TI - Characterization of membrane-bound adenosinetriphosphatase activity of sarcolemma enriched fraction from vascular smooth muscle. AB - Membrane-bound adenosinetriphosphatase (ATPase) activities of the sarcolemma enriched fraction from bovine aorta were characterized. The membranes, isolated by a sucrose density gradient method, were enriched about 31-fold in sodium- and potassium-stimulated, magnesium-dependent ATPase (Na,K-ATPase) activity, and about 8-fold in 5'-nucleotidase activity compared to the homogenate, suggesting that the isolated membranes were substantially enriched with the sarcolemma. The membranes exhibited about 31, 33 and 42 mumol Pi/mg protein/h of Na,K-ATPase, magnesium-dependent ATPase and calcium-dependent ATPase activities, respectively, in the presence of 4 mmol/l ATP. The sarcolemma-enriched membranes required considerably high concentrations of well-known inhibitors for Na,K-ATPase such as vanadate (more than 1 mumol/l), lanthanum (more than 1 mmol/l) and calcium (10 mmol/l), to induce a significant inhibition in the Na,K-ATPase activity. Treatments of the membrane with physical disruptions and sodium dodecyl sulfate or deoxycholate reduced the total Na,K-ATPase activity, and did not expose fully the ouabain sensitivity of the Na,K-ATPase. These results indicate that there are marked differences in the properties of the ATPase between vascular smooth muscle sarcolemma and cardiac sarcolemma. PMID- 3009175 TI - Skeletal muscle damage: a study of isotope uptake, enzyme efflux and pain after stepping. AB - We have studied the occurrence of skeletal muscle uptake of 99mtechnetium pyrophosphate (Tc-PYP), creatine kinase (CK) release and muscle pain in normal subjects after exercise. Five subjects stepped on and off a high bench in such a way that one leg stepped up and the other down. Pain only developed in the muscles used for descending: quadriceps, adductors and gluteal muscles of one leg and the calf muscle of the other. A large rise in plasma CK occurred in four subjects but no increased Tc-PYP muscle uptake was seen in the quadriceps. In the four subjects with high CK effluxes, increased isotope uptake was seen in the thigh adductors used when stepping down; in the two subjects with the largest CK effluxes there was extensive uptake into the gluteal muscles. Muscle pain preceded and was not well correlated with either the magnitude of the enzyme release or the amount and distribution of increased muscle isotope uptake. We conclude that delayed onset muscle pain, the cause of which remains unknown, is a poor indicator of muscle damage as indicated by circulating muscle enzymes and muscle isotope uptake. Tc-PYP uptake by skeletal muscle can provide useful information about the localisation and time course of muscle damage. PMID- 3009176 TI - Beta-endorphin and ACTH levels in peripheral blood during and after aerobic and anaerobic exercise. AB - Beta-endorphin (beta-End) and adrenocorticotrophic hormone (ACTH) were determined in the peripheral blood of 14 human volunteers exercising on a bicycle ergometer. After 1 h of submaximal work below anaerobic threshold (AT), defined as the 4 mmol X l-1 lactic acid level in arteriolar blood (Kindermann 1979; Mader 1980), beta-End and ACTH levels did not change from control conditions. Eleven of the same 14 subjects performed an uninterrupted graded exercise test on the same bicycle ergometer until exhaustion. This time beta-End and ACTH levels increased concomitantly with exercise of high intensity: at each moment, during and after this maximal test, a highly significant correlation (P less than 0.0001) was noted between the levels of beta-End and ACTH. The peak values of these hormones were reached within 10 min after stopping maximal exercise, and coincided with lactic acid peak levels. A rise in lactic acid levels above the anaerobic threshold always preceded the exercise-induced rise in beta-End and ACTH. Within the population tested, two subgroups could be distinguished: one comprising individuals whose hormonal response nearly coincided with the rise in lactic acid (rapid responders) and a second group composed of subjects whose normal response appeared delayed with respect to the lactic acid rise (slow responders). These results support the view that beta-End and ACTH are secreted in equimolar quantities into the blood circulation in response to exercise, and suggest that metabolic changes of anaerobiosis play a key role in the regulation of stress hormone release.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009177 TI - Release of endotoxin from bacteria exposed to ciprofloxacin and its prevention with polymyxin B. AB - An in vitro system for studying the phenomenon of antibiotic-induced release of endotoxin from gram-negative bacteria is described. Endotoxin was measured by a quantitative limulus lysate microassay. Using a strain of Escherichia coli, ciprofloxacin was shown to cause an immediate and sustained increase in endotoxin release compared to control cultures whereas gentamicin did not, despite an equally rapid bactericidal effect. The potential value of the anti-endotoxin effect of polymyxin B was examined using a polymyxin B resistant strain of Klebsiella aerogenes. Ciprofloxacin alone caused endotoxin release, but when given with polymyxin B significantly less endotoxin was detected. These results indicate that in antibiotic-induced endotoxin release, the rate of cell death is less important than the site of antibiotic effect, and suggest that ciprofloxacin may act directly on the cell wall as well as on DNA gyrase. It is possible that polymyxin B could be used to reduce the extent of endotoxin release, and that this would be of clinical benefit. PMID- 3009178 TI - Molecular genetics of exopolysaccharide production by mucoid Pseudomonas aeruginosa. PMID- 3009179 TI - Structure and expression of mouse aldolase genes. Brain-specific aldolase C amino acid sequence is closely related to aldolase A. AB - Brain-specific aldolase C amino acid sequence (greater than 75% of the coding region) was determined for the first time. Two cDNA clones, pAM1 and pAM2, were identified, from a mouse brain library, by using human aldolase B cDNA as a probe. The larger one, pAM2, identified as a cDNA for aldolase C, has been completely sequenced and covers the 5'-untranslated region of the mRNA and the codons for amino acids 1-227 of the protein. The sequence indicates that aldolase C is more akin to aldolase A than to aldolase B. A cDNA library from mouse muscle was also screened, allowing the identification of clones pAM3 and pAM4, which contain cDNAs for aldolase A. The sequence obtained from pAM3 covers 70% of the coding sequence (amino acids 99-355) from the -COOH part of the protein. The cDNAs for the three aldolases, A, B and C, have been hybridized to RNA from various rat tissues. The results confirm the tissue specificity of the expression of the mRNA for the different isoenzymes and support the hypothesis that aldolase C expression, as aldolase A and B, is regulated at the transcriptional level or, in any case, via mRNA concentration. PMID- 3009180 TI - Characterization of the DNA-binding properties of the receptor for 2,3,7,8 tetrachlorodibenzo-p-dioxin. AB - The DNA-binding properties of the receptor for 2,3,7, 8-tetrachlorodibenzo-p dioxin (TCDD) were investigated using chromatography on DNA-cellulose columns. A maximal binding of about 40% of the total receptor complex to DNA-cellulose was observed. In order to interact with DNA, the receptor must first bind TCDD. A heat-activation step followed by gel permeation chromatography using Sephadex G 25 increased the binding of the cytosolic receptor to DNA. The DNA-binding ability of the receptor was almost lost following mild proteolysis using trypsin or alpha-chymotrypsin, although these treatments did not reduce its ligand binding capacity and had no apparent effect on its size. Furthermore, pre treatment of the DNA-cellulose column with an intercalating drug, ethidium bromide, resulted in inhibition of the binding of the TCDD-receptor complex to DNA, indicating that not only electrostatic interactions but also the configuration of DNA are of importance in receptor-DNA interactions. PMID- 3009181 TI - Purification and characterization of a novel, oligomeric, plasminogen kringle 4 binding protein from human plasma: tetranectin. AB - Purification of alpha 2-plasmin inhibitor (alpha 2PI) from human plasma by affinity chromatography on plasminogen-Sepharose resulted in copurification of a contaminating protein with Mr 17,000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. This contaminating protein could not be removed from the purified alpha 2-PI preparation by several types of gel chromatography applied. The use of the kringle 1-3 part of plasminogen, K(1 + 2 + 3), bound to Sepharose for affinity chromatography, instead of plasminogen Sepharose, resulted in an alpha 2PI preparation without this contaminant. The contaminating protein was found to interact specifically with the kringle 4 part of plasminogen (K4) and not with K(1 + 2 + 3) or miniplasminogen. The K4-binding protein was purified by ammonium sulphate precipitation, affinity chromatography on K4-Sepharose, ion-exchange chromatography and gel filtration on AcA 34. The relative molecular mass of the protein (Mr 68 000) was estimated by gel filtration. This suggests a tetrameric protein composed of four subunits (Mr 17,000), that are dissociated by 1% sodium dodecyl sulphate. Dissociation into subunits was also demonstrated by gel filtration in the presence of 6 M guanidine hydrochloride. A specific antibody was raised in rabbits against the purified protein and this antibody was shown not to react with any known fibrinolytic components. The pI of the K4-binding protein was found to be 5.8. The first three N-terminal amino acids were determined to be Glu-Pro-Pro. The concentration of the protein in plasma was estimated to be 0.20 +/- 0.03 microM (15 +/- 2 mg/l). The electrophoretic mobility of the K4-binding protein was shown by crossed immunoelectrophoresis to be influenced by the presence of Ca2+, EDTA and heparin. The protein was found to enhance plasminogen activation catalyzed by tissue-type plasminogen activator (t-PA) in the presence of poly(D-lysine). The protein appeared to be a novel plasma protein tentatively called 'tetranectin'. PMID- 3009182 TI - Persistence of cruciform structure and preferential location of nucleosomes on some regions of pBR322 and ColE 1 DNAs. AB - Inverted repeats of pBR322 and ColE 1 DNAs have been analyzed for the presence of cruciform structures upon formation of nucleosomes, using S1, P1 and restriction enzyme analysis. In both cases the fraction of molecules showing nuclease sensitive sites is unaffected by the DNA relaxation, owing to the formation of nucleosomes. A kinetic mechanism, based on the freezing of cruciform structures on the nucleosome surface or nearby, is proposed. This hypothesis is supported by a preferential location of nucleosomes at the DNA sequences containing the nuclease-sensitive sites, as indicated by restriction enzyme analysis and electron microscopy visualization after psoralen cross-linking. PMID- 3009183 TI - Different micrococcal nuclease cleavage patterns characterize transcriptionally active and inactive sea-urchin histone genes. AB - The micrococcal nuclease cleavage sites have been mapped in the H2A coding and flanking regions of the sea-urchin histone DNA chromatin. A hypersensitive area, centered around - 100 base pairs from the H2A starting site, is found only in embryos actively transcribing the alpha-subtype histone genes. In mesenchyme blastula embryos, upon inactivation of the H2A gene, this region becomes protected while two other areas, near the transcription starting site and in the proximity of the 3' palindromic sequence, become preferential targets for the enzyme. Analysis of the pattern of micrococcal nuclease cleavage on the same region of the histone gene cluster in sperm and late blastula chromatin and on the corresponding segment of protein-free DNA indicates that distinct nucleosomal arrangements characterize the histone genes in the two cell populations. PMID- 3009184 TI - Expression of polyoma virus middle-T antigen in Saccharomyces cerevisiae. AB - The polyoma middle-T gene, lacking its intron, was inserted into a yeast expression plasmid containing the phosphoglycerate kinase promoter. Such plasmids transformed yeast at low frequency and these transformants expressed middle-T antigen at a level of approximately 0.1% cell protein. Furthermore, expression of this protein was frequently lost during growth in liquid culture and this loss of middle-T was accompanied by a twofold increase in the rate of growth. The spontaneous production of a truncated middle-T antigen, lacking the C terminus, was also observed; the expression of this protein did not inhibit the growth rate of the cells. Recovery and analysis of the expression plasmids encoding the truncated molecule showed that a single C X G base pair had been deleted from a run of nine consecutive C X G base pairs (Pyr nucleotide 1239--1247) within the middle-T coding region. This frame-shift mutation results in premature termination of the protein and loss of the strongly hydrophobic region of the molecule believed to be responsible for the membrane association of middle-T antigen. PMID- 3009185 TI - Characterisation of two forms of phosphoribulokinase isolated from the green alga, Scenedesmus obliquus. AB - Two forms of phosphoribulokinase from the alga, Scenedesmus obliquus, have been purified to homogeneity by DEAE-cellulose, Ultrogel AcA34 and hydroxyapatite chromatography. An active form of the enzyme is a dimer of identical 42,000-Mr subunits. A latent form of phosphoribulokinase, requiring incubation with dithiothreitol for activity, is of Mr 470,000 and apparent subunit composition X8Y4. The subunits X and Y are of Mr 39,000 and 42,000 respectively. The latent form of phosphoribulokinase is lost during DEAE-cellulose chromatography but this is prevented by NAD. Depolymerisation of the latent phosphoribulokinase to give the low-Mr form of the enzyme accompanied its activation by dithiothreitol. An algal protein with all the properties of thioredoxin stimulates activation of the latent phosphoribulokinase when incubated with low concentrations of dithiothreitol. The latent form of phosphoribulokinase predominates in the heterotrophically grown algae whilst under photoheterotrophic conditions equal amounts of both enzyme forms are present in algal extracts. This is consistent with the suggestion that light activation of phosphoribulokinase in vivo is also due to depolymerisation of the large-Mr latent form of the enzyme. PMID- 3009186 TI - Measurements of the proton motive force generated by cytochrome c oxidase from Bacillus subtilis in proteoliposomes and membrane vesicles. AB - Cytochrome c oxidase from Bacillus subtilis was reconstituted in liposomes and its energy-transducing properties were studied. The reconstitution procedure used included Ca2+-induced fusion of pre-formed membranes. The orientation of the enzyme in liposomes is influenced by the phospholipid composition of the membrane. Negatively charged phospholipids are essential for high oxidase activity and respiratory control. Analyses of the proteoliposomes by gel filtration, density gradient centrifugation and electron microscopy indicated a heterogeneity of the proteoliposomes with respect to size and respiratory control. Cytochrome c oxidase activity in the proteoliposomes resulted in the generation of a proton motive force, internally negative and alkaline. In the presence of the electron donor, ascorbate/N,N,N',N'-tetramethyl-p phenylenediamine/cytochrome c or ascorbate/phenazine methosulphate, the reconstituted enzyme generated an electrical potential of 84 mV which was increased by the addition of nigericin to 95 mV and a pH gradient of 32 mV which was increased by the addition of valinomycin to 39 mV. Similar results were obtained with beef-heart cytochrome c oxidase reconstituted in liposomes. The maximal proton motive force which could be generated, assuming no endogenous ion leakage, varied over 110-140 mV. From this the efficiency of energy transduction by cytochrome c oxidase was calculated to be 18-23%, indicating that the oxidase is an efficient proton-motive-force-generating system. PMID- 3009188 TI - Cycle of the vasoactive intestinal peptide and its binding site in a human adenocarcinoma cell line (HT 29). AB - The disappearance of vasoactive-intestinal-peptide (VIP) binding sites at the cell surface of a cultured target cell, originating from a human colonic adenocarcinoma (HT 29 cell line), was studied, after preexposition of the cell to the peptide, as a function of time, VIP concentration and temperature. Maximum effect (60-80% loss of binding capacity) was obtained after a 5-10 min exposure of the cells at 37 degrees C with a VIP concentration of 100 nM. The t1/2 of maximum disappearance was less than 2 min and the concentration of native VIP giving half-maximum decrease in 125I-VIP binding was 6 nM. The affinity of remaining binding sites for VIP was not affected compared to that of control cells (Kd = 0.3 nM). Disappearance of VIP binding sites was specific since, with the same conditions of preincubation, the specific binding of 125I-labeled epidermal growth factor to HT 29 cells was not modified. The phenomenon was reversible and 90% of binding capacity could be restored in less than 60 min by incubating cells in VIP-free medium. Correlatively we showed, by two independent experimental procedures, that 125I-VIP, initially bound to HT 29 cells, was maximally internalized after 10 min of incubation at 37 degrees C. All the data strongly suggest that: internalization of VIP is receptor-mediated; upon exposure to native VIP, VIP receptors are down-regulated or at least sequestered within HT 29 cells. PMID- 3009187 TI - Specificity of Bacillus thuringiensis var. colmeri insecticidal delta-endotoxin is determined by differential proteolytic processing of the protoxin by larval gut proteases. AB - The native crystal delta-endotoxin produced by Bacillus thuringiensis var. colmeri, serotype 21, is toxic to both lepidopteran (Pieris brassicae) and dipteran (Aedes aegypti) larvae. Solubilization of the crystal delta-endotoxin in alkaline reducing conditions and activation with trypsin and gut extracts from susceptible insects yielded a preparation whose toxicity could be assayed in vitro against a range of insect cell lines. After activation with Aedes aegypti gut extract the preparation was toxic to all of the mosquito cell lines but only one lepidopteran line (Spodoptera frugiperda), whereas an activated preparation produced by treatment with P. brassicae gut enzymes or trypsin was toxic only to lepidopteran cell lines. These in vitro results were paralleled by the results of in vivo bioassays. Gel electrophoretic analysis of the products of these different activation regimes suggested that a 130-kDa protoxin in the native crystal is converted to a 55-kDa lepidopteran-specific toxin by trypsin or P. brassicae enzymes and to a 52-kDa dipteran toxin by A. aegypti enzymes. Two-step activation of the 130-kDa protoxin by successive treatment with trypsin and A. aegypti enzymes further suggested that the 52-kDa dipteran toxin is derived from the 55-kDa lepidopteran toxin by enzymes specific to the mosquito gut. Confirmation of this suggestion was obtained by peptide mapping of these two polypeptides. The native crystal 130 kDa delta-endotoxin and the two insect specific toxins all cross-reacted with antiserum to B. thuringiensis var. kurstaki P1 lepidopteran toxin. Preincubation of the two activated colmeri toxins with P1 antiserum neutralized their cytotoxicity to both lepidopteran and dipteran cell lines. PMID- 3009189 TI - Cardiac sarcoplasmic reticulum contains a low-affinity site for phenylalkylamines. AB - The distribution of the bovine cardiac binding sites for the organic calcium channel blockers was studied. Crude microsomal membranes were separated into three fractions, which contained mainly membranes derived from sarcolemma, 'junctional' sarcoplasmic reticulum containing transversal tubuli, and free sarcoplasmic reticulum. The high-affinity binding site for the dihydropyridines, determined in the presence of nitrobenzylthioinosine, was enriched 12-fold and 17 fold in sarcolemma and junctional sarcoplasmic reticulum. The binding sites for the phenylalkylamines, determined with [3H]verapamil or [3H]( )desmethoxyverapamil, were enriched 1.5-3.4-fold in sarcolemma and junctional sarcoplasmic reticulum but 6-10-fold in free sarcoplasmic reticulum. The phenylalkylamine-binding site, present in free sarcoplasmic reticulum, was partially destroyed by chymotrypsin or phospholipase A2 and C treatment. Specific binding was proportional to the concentration of the added membrane protein. The binding of (-)desmethoxyverapamil was half-maximally inhibited by 6.5 mM calcium chloride and was optimal in the presence of 5 mM EGTA. In three out of five preparations (-)desmethoxyverapamil bound to a single site with an apparent Kd value of 191 +/- 42.8 nM and a density of 34.5 +/- 7.7 pmol/mg protein. In two out of five preparations an additional high-affinity site (Kd approximately 0.67 nM) was detected. The low-affinity site bound other phenylalkylamines, but stereospecific binding of phenylalkylamines was not observed. Binding of phenylalkylamines to the low-affinity site was inhibited by some but not all calmodulin 'antagonists'. Furthermore dihydropyridines did not affect the binding of (--)desmethoxyverapamil suggesting that the low-affinity site differs considerably from the high-affinity sarcolemmal site. These results suggest that free sarcoplasmic reticulum contains a binding site for phenylalkylamines at a relative high density, which is not related to the high-affinity site present in the voltage-dependent calcium channel. PMID- 3009192 TI - Ecto-protein kinase activities in normal and transformed cells. AB - The incubation of intact uninfected and Rous sarcoma virus (RSV)-transformed chicken cells (SR-RSV-A) with micromolar amounts of [gamma-32P]ATP under physiological conditions resulted in the radioactive phosphorylation of a variety of proteins. According to the experimental protocol the detectable phosphorylation was restricted to ATP utilization at the cell surface and was catalyzed by surface located protein kinase (PK). Serine- and to a lesser extent, threonine residues were phosphorylated. With respect to this enzyme the cells under investigation showed upon incubation with phosvitin the release of surface (phosvitin) kinase into the incubation medium. Based on immunochemical analysis and PK-assays using antisera from RSV-tumor bearing rabbits (TBR-serum) the pp60v src with its associated tyrosine kinase activity was likewise detected in appreciable amounts at the outside of RSV-transformed chicken and mammalian cells. There was no cross reactivity of TBR-serum with phosvitin kinase. Phosvitin was not phosphorylated by the immunoprecipitated pp60v-src. Whereas phosphorylation catalyzed by pp60v-src was blocked with 10 to 20 microM diadenosine 5',5''-P1P4 tetraphosphate (Ap4A) the phosvitin phosphorylation was far less sensitive towards inhibition by Ap4A, similar to the cellular pp60c-src kinase activity in uninfected cells. The functional significance of the PK activities in uninfected and RSV-transformed cells observed at their surface or in cell-free form as well as the nature of their substrates remain to be established. PMID- 3009190 TI - Isolation and characterization of peroxisomes from the renal cortex of beef, sheep, and cat. AB - The isolation and characterization of highly purified and structurally well preserved peroxisomes from the renal cortex of different mammalian species (beef, sheep, and cat) is reported. Renal cortex tissue was homogenized and a peroxisome enriched light mitochondrial fraction was prepared by differential centrifugation. This was subfractionated by density-dependent banding on a linear gradient of metrizamide (1.12-1.26 g/cm3) using a Beckman VTi 50 vertical rotor. Peroxisomes banded at a mean density of 1.225 cm3. Ultrastructural morphometric examination revealed that peroxisomes made up 97 to 98% of the isolated fractions. By biochemical analysis the contamination with marker enzymes of mitochondria and lysosomes was extremely low. The specific activity of catalase was enriched, depending on the species, between 28- and 38-fold over the homogenate. Peroxisome preparations from all three species exhibited a high but varying level of activity for cyanide-insensitive lipid beta-oxidation. In beef and sheep preparations a small amount of esterase activity cosediments with peroxisomes. These peroxisomes show distinct structural membrane associations with smooth elements of ER. Urate oxidase, a marker enzyme for rat liver peroxisomes, is found only in peroxisomes prepared from beef kidney cortex, with sheep and cat preparations being negative. This correlated with the occurrence of polytubular inclusions in the beef kidney peroxisomes. The large size and the angular shape of isolated peroxisomes as well as the presence of paracrystalline matrical inclusions imply that the majority of peroxisomes are derived from the epithelial cells of the proximal tubule of the kidney cortex. The significant differences found in the characteristics of the renal peroxisomes in three different species investigated, demonstrate the remarkable adaptability and plasticity of this organelle. PMID- 3009191 TI - The endocytosis of asbestos by mouse peritoneal macrophages and its long-term effect on iron accumulation and labyrinth formation. AB - The effect of a single intraperitoneal injection of crocidolite asbestos fibres on the peritoneal cell population were studied. Attention was paid to the changes in the proportions taken by the various types of cell in this population after peritoneal stimulation as well as the handling of asbestos fibres by the peritoneal cells and the formation of asbestos bodies. Intraperitoneal administration of crocidolite led to an influx of inflammatory cells into the peritoneal cavity. The asbestos fibres were phagocytosed and gradually cleared from the peritoneal cavity. Long before this clearance was completed, the peritoneal cell population had returned to the steady state. The stimulated peritoneal macrophages showed increasing concentrations of iron in both lysosomes and the cytoplasm. At later time points, residual bodies containing iron and asbestos fibres were seen frequently in macrophages, but asbestos bodies were not found. As a reaction to the administration of crocidolite asbestos, macrophages from the peritoneal cavity develop tubular systems (labyrinths) that increase in number and size. PMID- 3009193 TI - Interactions of rat hepatocytes with type IV collagen, fibronectin and laminin matrices. Distinct matrix-controlled modes of attachment and spreading. AB - We have examined the interaction of adult rat hepatocytes in primary culture, to type IV collagen, fibronectin, and laminin, the major basement membrane proteins of normal rat liver. Culture substrata consisted of glass coverslips, which were covalently derivatized with individual purified basement membrane constituents at varying densities of protein. The attachment of freshly prepared hepatocytes was examined after incubation at 37 degrees C for 30 min as a function of the amount of protein on the coverslips. For each of the three types of substratum under study, distinct modes of cell attachment were observed, with the apparent affinity of hepatocytes for type IV collagen being three-fold greater than for fibronectin and ten-fold greater than for laminin. Cell attachment exhibited saturation on all substrata. Hepatocyte spreading was measured by scanning electron microscopy of cells incubated at 37 degrees for 2 h on similarly prepared coverslips. A five-fold greater surface density of type IV collagen was required for maximal spreading compared with attachment. For cells on fibronectin or laminin the maximal cell spreading reached on type IV collagen did not occur even at coverslip protein densities 10 to 20 times those providing for maximal cell attachment. A very similar qualitative pattern of cell proteins was secreted within a few hours of plating on the various substrata and further studies failed to reveal any evidence that attachment and spreading was mediated by endogenously produced matrix molecules.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009194 TI - Intracellular localization and serological identification of a HSV-2 protein in cervical cancer. AB - Exfoliated atypical cells from US and Italian patients with cervical intraepithelial neoplasia or invasive cancer stained in indirect immunofluorescence with a monoclonal antibody to a high MW HSV-2 protein (ICP10). The proportion of staining cells increased as a function of the severity of the cervical anaplasia. In the majority of cases with mild or moderate dysplasia staining was restricted to the cytoplasm while atypical cells from 6 of 7 cases with marked dysplasia and 7 of 7 cases with invasive cancer evidenced also nuclear staining. Normal exfoliated cells did not stain even when obtained from tissue adjacent to the tumor mass. Patients with ICP10 positive atypical cells had antibody to ICP10 as determined by the Western blot assay. Serologic analyses indicated that there was a good correlation between: (i) the Western blot and the CF assay previously used to detect antibody to AG-4 (Aurelian, et al, Cancer, 48, 455, 1981) and (ii) cervical anaplasia and ICP10 seropositivity. A similar correlation with cervical anaplasia was not observed with respect to antibody to two other viral proteins (ICP12/14 and 68K). PMID- 3009195 TI - Malignant trophoblastic neoplasias. Clinical experiences. AB - The Authors describe a treatment program for malignant trophoblastic disease based on a combination chemotherapy. The results of their clinical experience concerning 41 cases confirms the high cure rate for low risk metastatic disease (100%) with the only failures limited to the high risk patients (64.7% cure rate). PMID- 3009196 TI - Endodermal sinus tumor. AB - Three cases of Endodermal Sinus Tumor are presented and its difference from tumors of the mesonephroma type and associations with the embryoma are pointed out. The three cases were "pure" types and the patients died within 5 months in spite of surgical treatment and chemotherapy. Autopsy showed extensive involvement of peritoneum, diaphragm and pleura. PMID- 3009198 TI - Pathogenesis of experimental thyrotoxic myopathy. AB - Focal degenerative changes of skeletal muscle fibers (decrease in mean diameter, excessive axonal branching and a decrease in the mean diameter of motor end plates together with a reduction of their acetylcholinesterase levels) were found by means of the experimental model thyrotoxic myopathy in mice compared to controls. A decrease in protein kinase affinity to cAMP and an increase in the number of nucleotide binding sites were revealed with a simultaneous decrease in cAMP level. The weakening of hormonal control of cAMP-dependent processes is probably the basic cause of muscular weakness and structural changes in skeletal muscles in thyrotoxic myopathy. PMID- 3009197 TI - The role of thyroid scanning in hyperthyroidism. AB - Radionuclide thyroid imaging was performed in 872 consecutive patients with hyperthyroidism. Of these, 84% were found to have diffuse toxic hyperplasia (Graves' disease), while 12% had autonomously functioning nodules (Plummer's disease), 3% had Graves' disease developing in a multinodular gland, and in the remaining 1%, either a clear diagnosis could not be established or the hyperthyroidism was due to thyroiditis or the Jod-Basedow phenomenon. It was found that a thyroid scan seldom provides additional diagnostic information in patients with Graves' disease when a diffuse goitre is present. However, if patients are to be treated with radioiodine (131I), thyroid imaging with tracer quantitation can replace a 24-h 131I uptake measurement, this having the advantages that the patients are required to attend only once, and that the gland size can be measured. In addition, visual confirmation of tracer uptake by the thyroid is obtained and patients with thyroiditis will not receive inappropriate therapy. When single or multiple thyroid nodules are palpated, a thyroid scan is crucial in establishing an accurate diagnosis, as it is not otherwise possible to differentiate between Plummer's disease and Graves' disease developing in a multinodular gland. Indeed, in 20 of our 63 patients (32%) with single autonomously functioning nodules, the initial clinical assessment had been incorrect. PMID- 3009199 TI - Progressive myoclonic epilepsy (Unverricht type) with atypical Lafora bodies. Case report. AB - A patient with advanced progressive myoclonic epilepsy (Unverricht type) with Lafora bodies is presented. Although the clinical history and symptoms were classical, the regional distribution of the cerebral involvement differed from the classical picture: the corpora mamillaria, the nucleus subthalamicus, and the nucleus ruber, which are normally reported to be spared, contained multiple Lafora bodies, whereas the lateral geniculate body, which is usually involved, was intact. The number of inclusions per cell, up to 25, was extremely high and correlated with the marked cortical atrophy and the prolonged clinical course. Using electron microscopy, type I and type II Lafora bodies were found, but the latter lacked the typical filamentous ultrastructure in the peripheral zone. The lack of visceral Lafora bodies in this case suggests that liver, muscle, and skin biopsies, which are widely used for the diagnosis, may lead to false negative results and cannot always replace a stereotactic brain biopsy. The differential diagnosis on polyglucosan bodies is emphasized. PMID- 3009200 TI - Increase in type I and type IV collagenolytic activity in primary cultures of keratoconus cornea. AB - The collagenolytic activities were assayed in fibroblast cultures established from ten keratoconus and six control corneal explants. The assays included those for both type I and type IV collagenase. The amount of free, directly assayable type I collagenase was highly increased in the media of keratoconus primary cell cultures. Also the activity of type IV collagenase was increased significantly in these cultures but was detectable only after proteolytic activation. The total hydroxyproline content of keratoconus corneas was decreased when compared to controls. However the activity of the prolyl-4-hydroxylase, reflecting the biosynthesis of collagen, was higher in the keratoconus corneal tissue, either expressed per DNA, soluble protein or hydroxyproline content of corneas. The enhancement in collagenolytic processes could explain the pathological tissue destruction in keratoconus corneas and this enhancement is maintained still in primary cultures of keratoconus corneal fibroblasts. PMID- 3009201 TI - Macrophage activation by trehalose dimycolate requirement for an expression signal in vitro for antitumoral activity; biochemical markers distinguishing primed and fully activated macrophages. AB - It was possible to define the effects of trehalose dimycolate (TDM), a glycolipid extracted from Mycobacterium tuberculosis, on mouse peritoneal macrophages more precisely using endotoxin-free culture conditions. TDM-elicited macrophages, when assayed in vitro in the absence of endotoxin, were unable to limit tumor growth; however, after a short treatment (4 h) with low doses of lipopolysaccharide (LPS; 1-10 ng/ml), they exhibited a strong cytostatic capacity against P815 mastocytoma cells. Thus, TDM injected in vivo did not activate macrophages fully but it primed them to respond in vitro to low doses of LPS, which provided the final stimulus for activation to antitumor competence. Macrophages elicited by an injection of killed group C Streptococci were also in a primed state; in contrast, thioglycollate-elicited macrophages were in a nonreceptive state. Besides LPS, concanavalin A (5 micrograms/ml), MDP (0.2-1 microgram/ml) and the ionophore A23187 (5 microM) can deliver the activation signal to TDM-primed macrophages. Primed macrophages were found to express several biochemical markers previously described as specific for activated macrophages (low levels of alkaline phosphodiesterase and beta-galactosidase, for example) and, although they were not cytotoxic for tumor cells, they had the capacity to release large amounts of H2O2. However, when pulsed by LPS or MDP, primed macrophages responded by further modifications in their metabolism: the rate of glucose consumption and the labeling of glycoproteins by D-[2-3H]mannose were greatly increased and the secretion of a polypeptide of 22 kDa was enhanced. The activation-associated biochemical markers are thus acquired in two steps. The ability to produce activated oxygen species is expressed earlier than the antitumoral activity. PMID- 3009202 TI - Human natural killer cell lysis of virus-infected cells. Relationship to expression of the transferrin receptor. AB - Natural killer (NK) cells lyse tumor and virus-infected cells yet the nature of the target structure they recognize is unknown. A normal host cell glycoprotein, the transferrin receptor (TfR), has been proposed as a target structure on tumor cells. We therefore investigated whether changes in the number or physiological recycling of the TfR, consequent on virus infection, were related to the differential susceptibility of virus-infected cells to NK lysis. There was a direct correlation between TfR expression, susceptibility to NK lysis and ability to act as cold target competitors, for human fibroblasts infected with RNA and DNA viruses (cytomegalovirus, herpes simplex, polio, vaccinia and Semliki Forest virus). The NK lysis of human cytomegalovirus-infected fibroblasts was studied in more detail. NK lysis was increased coincident with human cytomegalovirus early antigen expression and this susceptibility to lysis was associated with increased total and recycling TfR but only a slight increase in surface TfR expression. In addition, susceptibility of uninfected human fibroblasts to NK lysis directly correlated with TfR number. However, we were unable to inhibit NK lysis by either excess iron-saturated Tf or affinity-purified TfR. We conclude that there is a direct correlation between total TfR expression and susceptibility to NK lysis of human virus-infected cells; however, the NK target structure on virus-infected cells is probably not the TfR itself. PMID- 3009203 TI - Antigen presentation is a function of all B cell subpopulations separated on the basis of size. AB - Purified splenic B cells from nonimmune mice were separated by counterflow centrifugal elutriation into 6 subpopulations containing cells of discrete sizes ranging from 119 to 200 micron3. B cells of each subpopulation were competent to process and present a native globular protein antigen, cytochrome c, to a cytochrome c-specific T cell hybrid. In all cases, the B cells' antigen presenting function was radiation sensitive and did not require T cells or T cell products, since B cells fixed with paraformaldehyde effectively presented a carboxyl-terminal peptide fragment of cytochrome c containing the T cell determinant. Furthermore, the antigen-presenting function of B cells of each subpopulation was augmented by treatment with submitogenic doses of the F(ab')2 fragment of rabbit anti-mouse Ig antibodies, in that 10-30-fold fewer B cells were required and higher maximal T cell responses were achieved, indicating that B cells of all sizes are capable of being regulated in their antigen presentation function through their surface Ig. In addition, B cells of each subpopulation responded to soluble factors present in the supernatants of activated T cells as evidenced by an increase in volume and by the uptake of [3H]thymidine. These results indicate that B cells, regardless of size, are able to participate in at least two essential phases of T cell-dependent antibody responses, initiating the interaction by processing and presenting antigen to helper T cells and responding to soluble helper factors secreted by activated T cells. PMID- 3009204 TI - Rearrangements of T cell receptor loci can be found only rarely in B lymphoid cells. AB - We have studied the rearrangement status of the T cell receptor genes in 64 B lymphoid cell lines, and we found that, unlike the immunoglobulin heavy chain genes in T lymphocytes, T cell receptor beta and related gamma chain genes are almost always in germ-line configuration in B lymphoid cells. The only exception was a myeloma MOPC511 (IgA, chi) which contained all T cell receptor genes, beta 1, beta 2, gamma 1, gamma 2, gamma 3 and alpha, in rearranged configuration in both homologous chromosomes. This exception supports the concept that all immunoglobulin and T cell receptor genes exploit the same recombinase to build their complete variable regions. Obviously, in MOPC511 cells the regulation, which confers the tissue specificity i.e. T vs. B lymphocytes, has failed. PMID- 3009205 TI - Human monoclonal antibody to purified protein derivative of tuberculin produced by hybrids constructed with Epstein-Barr virus-transformed B lymphocytes and mouse myeloma cells. AB - A method of producing human monoclonal antibody by combining somatic cell hybridization technology with the capability of Epstein-Barr virus (EBV) to transform human B lymphocytes is described. Peripheral blood lymphocytes from a donor with positive tuberculin skin test reaction were transformed by EBV and then tested for antibody production to mycobacterial purified protein derivative (PPD) by an enzyme-linked immunosorbent assay. Two EBV-transformed lymphoblastoid cell lines making IgM antibodies to PPD were obtained. One of these cell lines was fused by polyethylene glycol with a murine hypoxanthine-guanine phosphoribosyl transferase-deficient myeloma cell line that had been selected for resistance to ouabain. The human-mouse hybrids were selected in ouabain containing HAT medium and 11 heterohybridomas producing IgM antibody to PPD were obtained. One of these was cloned by limiting dilution with efficiency at least 20-fold higher than parent EBV-transformed cell line. Heterohybridoma subclones reached levels of IgM antibody as high as 75.0 micrograms/ml of culture medium, whereas IgM production of EBV-transformed B cell clones ranged between 3.0 and 4.0 micrograms/ml. PMID- 3009207 TI - Receptor-mediated inositol phospholipid hydrolysis in astrocytes. AB - Astrocyte-enriched cultures of the neonatal rat cortex were incubated for 24 h with [3H]inositol to prelabel the membrane inositol phospholipids. Exposure of the cultures to either noradrenaline or carbachol in the presence of Li+ produced a time- and dose-dependent accumulation of intracellular [3H]inositol phosphates. The separation of the individual inositol phosphates formed in response to receptor stimulation revealed that the major 3H-metabolite accumulated under these conditions was inositol monophosphate but that at least some of this was due to the initial formation of inositol trisphosphate. The use of selective receptor antagonists showed that noradrenaline- and carbachol-induced [3H]inositol phosphate accumulation was the result of the activation of alpha 1 adrenoceptors and muscarinic acetylcholine (probably of the M1 subtype) receptors respectively. Agonist-evoked [3H]inositol phosphate accumulation were found to be additive but the simultaneous addition of agonists and the Ca2+ ionophore A23187, which also promoted inositol phospholipid hydrolysis, was not. Agonist-induced [3H]inositol phosphate accumulation was only partially dependent on extracellular Ca2+, whilst that elicited by A23187 was entirely Ca2+-dependent. The results suggest that alpha 1-adrenoceptors and muscarinic acetylcholine receptors in these cultures are present either on the same cells and linked to separate inositol lipid pools or associated with different subpopulations of astrocytes in these cultures. Moreover, inositol lipids other than phosphatidylinositol 4,5 bisphosphate may be hydrolysed in response to agonist stimulation. PMID- 3009206 TI - Chronic treatment with nifedipine does not change the number of [3H]nitrendipine and [3H]dihydroalprenolol binding sites. AB - Possible receptor changes occurring after withdrawal of chronic nifedipine treatment or chronic propranolol treatment were examined by administering nifedipine (100 mg/kg per day) or propranolol (45 mg/kg per day) to rats for 2 weeks and then withdrawing treatment [3H]Nitrendipine and [3H]DHA binding were measured in membrane fragments of the ventricle. In propranolol-treated rats, 8 h after the last administration, the maximum binding for [3H]DHA was significantly increased from 79.9 +/- 8.0 to 139.8 +/- 12.8 fmol/mg protein (mean +/- S.E.); the dissociation constant was significantly increased from 4.9 +/- 0.7 to 10.7 +/ 1.2 nM. On the other hand, in nifedipine-treated rats, 12 and 48 h after the last administration, [3H]nitrendipine binding and [3H]DHA binding had not changed significantly. These results indicate that the mechanism of the calcium antagonist withdrawal syndrome may be different from that of beta-blocker withdrawal syndrome. PMID- 3009208 TI - Effects of sodium loading on antihypertensive responses to an angiotensin converting enzyme inhibitor in spontaneously hypertensive and renal hypertensive rats. AB - Sodium loading significantly attenuated the antihypertensive potency of an angiotensin-converting enzyme inhibitor, SA446, in 2-kidney, 1-clip renal hypertensive rats, but the potency of SA446 remained unchanged in spontaneously hypertensive rats. Basal levels of plasma renin activity of both types of hypertensive rats were suppressed by sodium loading. From these results, it appeared that a different mechanism was involved in the maintenance of hypertension in 2-kidney, 1-clip renal hypertensive rats and in spontaneously hypertensive rats. It also appeared that the mechanism of the antihypertensive action of angiotensin-converting enzyme inhibitors was not explainable only by the suppression of the plasma renin-angiotensin system. PMID- 3009209 TI - Effects of chemical sympathectomy with 6-hydroxydopamine on alpha- and beta adrenoceptors and muscarinic cholinoceptors in rat kidney. AB - The autonomic receptors in the rat kidney were characterized using the radioligands [3H]prazosin, [3H]clonidine, [3H]dihydroalprenolol (DHA) and [3H]quinuclidinyl benzilate (QNB). The specific binding of [3H]prazosin, [3H]clonidine, [3H]DHA and [3H]QNB to rat kidney membranes was saturable and of high affinity, and showed a pharmacological specificity as well as stereospecificity which characterized renal alpha 1-, alpha 2- and beta adrenoceptors and muscarinic cholinoceptors, respectively. There was a relatively greater density of alpha-adrenoceptors than beta-adrenoceptors or muscarinic cholinoceptors in the rat kidney. Chemical sympathectomy of rats with 6 hydroxydopamine X HBr (6-OHDA, 50 X 2 mg/kg i.v., 24 h interval) caused a significant increase (21-56%) in the Bmax values for renal [3H]prazosin, [3H]clonidine and [3H]DHA binding at 1 and 2 weeks following the treatment, without a change in the Kd values. 6-OHDA treatment had no significant effect on the Kd and Bmax values for [3H]QNB binding at 1-3 weeks after the treatment. The norepinephrine (NE) concentration was reduced (68-76%) in the 6-OHDA-treated rat kidney. In conclusion, the present study provides biochemical evidence for the possible localization of postsynaptic alpha 1-, alpha 2- and beta-adrenoceptors and muscarinic cholinoceptors in the rat kidney and also for the regulation of these adrenoceptors by the sympathetic nervous system. PMID- 3009210 TI - Feedback control of the inositol phospholipid response in rat brain is sensitive to ACTH. PMID- 3009211 TI - Irresectable bronchial carcinoid with a 32-year natural history. A report of two cases treated with Neodymium-YAG-laser, initially misinterpreted as small cell lung cancer. AB - Two patients are described with bronchial carcinoid. In both, the initial diagnosis was small cell lung carcinoma (SCLC). This diagnosis was discordant with the clinical course; a second evaluation yielded the diagnosis of carcinoid. Good palliation was achieved with YAG-laser therapy. PMID- 3009212 TI - Toluene diisocyanate-induced asthma without airway hyperresponsiveness. AB - Six workers with occupational asthma due to toluene diisocyanate (TDI) were studied. For each worker a detailed clinical and occupational history was taken, and lung function measurement and skin intradermal tests for common allergens were carried out. Methacholine inhalation challenge was performed before TDI inhalation, and 8 h after TDI inhalation. Methacholine challenge was within normal limits when performed before TDI inhalation, but went into the asthmatic range after TDI inhalation. These cases provide evidence that asthma can be induced by toluene diisocyanate in the absence of airway hyperresponsiveness. They further demonstrate that an isolated negative methacholine inhalation test cannot be used to exclude sensitization to TDI. Screening and follow-up studies on workers exposed on TDI require serial measurements of airway responsiveness and of variable air-flow obstruction. PMID- 3009214 TI - Elastase, collagenase, and cathepsin D activities in the aortas of spontaneously hypertensive and renal hypertensive rats. AB - In an attempt to clarify the roles of proteases in the developmental mechanisms of hypertensive vascular lesions, changes in activities of aortic elastase, collagenase, and cathepsin D in spontaneously hypertensive rats (SHR) and renal hypertensive rats were biochemically investigated. In SHR, elastase activity initially showed a significant increase, once two-fold higher than that in the control; but the activity tended to decrease earlier than that in the control. In both SHR and normotensive control rats collagenase activities tended to increase with advancing age. The activity in SHR was two-fold higher than that in the control at all ages examined. In both younger SHR and normotensive rats cathepsin D activities proved to be increased with advancing age, while in old rats the activities tended to decrease. The activity in SHR was three- to fivefold higher than that in the control at all ages examined. In renal hypertensive rats, the activities of elastase, collagenase, and cathepsin D increased gradually with increasing blood pressure, at levels significantly higher than those in the control. These findings suggest that the metabolisms of proteins such as elastin and collagen, expressed by these enzyme activities, are accelerated under hypertensive conditions. PMID- 3009213 TI - Human alveolar macrophage subpopulations isolated on discontinuous albumin gradients. Cytological data in normals and sarcoid patients. AB - Cells recovered by bronchoalveolar lavage (BAL) from ten subjects (five normals and five sarcoid patients) were centrifuged on discontinuous albumin gradients to study alveolar macrophage (AM) heterogeneity. The gradient was made with nine bovine albumin concentrations (from 8 to 30%). After centrifugation (4000 X g) cells were recovered from the interfaces. The majority of AM was recovered in upper and medium layers with AM purity reaching 90-100% in upper layers, while lymphocytes and granulocytes were found in lower layers. Alveolar macrophage morphology was different along the gradient. Large cytoplasmic vacuoles were found in AM from upper layers. Alveolar macrophage cytoplasmic diameters decreased from the top to the bottom of the gradient, but AM from sarcoid patients appeared larger than AM from normals. Alveolar macrophage nucleo cytoplasmic ratios increased from top to bottom, but were lower in sarcoid patients than in normals. Alveolar macrophage peroxidase activity was rare (0.5 2% of AM) but reached a higher proportion in upper and lower layers. PMID- 3009216 TI - Kinetic alterations of cytochrome-c oxidase in cystic fibrosis. AB - We compared the kinetics of cytochrome-c oxidase (cytochrome-c:oxygen oxidoreductase, EC 1.9.3.1) in fibroblasts derived from normal and cystic fibrosis individuals. The Km of the enzyme for reduced cytochrome c was significantly increased in CF cells; the change, however, was observed only at temperatures above 25 degrees C. The Vmax values were comparable in both types of individuals. PMID- 3009215 TI - Protease gene structure and env gene variability of the AIDS virus. AB - The protease gene structure and the env gene variability have been precisely compared between the AIDS virus and members of the HTLV/BLV family. The conserved amino acid sequence (LVDT) which is repeated in the proteases of the HTLV/BLV family is not repeated in AIDS virus. Comparative analysis of the env gene sequences reveals the striking fact that the env gene of AIDS virus is 8-12-times more variable than those of the HTLV/BLV family. Within the AIDS virus env gene, the surface glycoprotein region is more liable to vary than is the transmembrane region; unexpectedly, however, this liability is not a characteristic feature of the AIDS virus because it is more prominent in other retroviruses including members of the HTLV/BLV family. PMID- 3009217 TI - Stereospecific antibodies to propranolol. AB - The beta-adrenergic antagonist propranolol was activated through its side chain, coupled to bovine serum albumin, and injected into BALB/c mice. After fusion of the splenocytes from these immunized mice with the NS-1 myeloma cell line, two hybridomas, producing monoclonal anti-propranolol antibodies, were isolated. Clone P-49 was monospecific for propranolol, with a significant preference for the 1-stereoisomer, as compared to the d form. On the other hand, clone P-28 cross-reacted with alprenolol as well as some other beta-antagonists. Both classes of antibodies competed with A431 epidermoid carcinoma beta 2 adrenoceptors for the binding of [3H]propranolol. When ascites cells from clone P 28 were fixed with glutaraldehyde, the anti-propranolol monoclonal antibody became cell bound. These cell-bound P-28 antibodies bind propranolol and other beta-adrenergic ligands with a similar ranking order to the soluble monoclonal antibody. The cell-bound antibody displayed a 5-fold higher affinity towards 1 propranolol than the soluble monoclonal antibody. The practical implications of these findings are discussed. PMID- 3009218 TI - A hybrid protein between IFN-gamma and IL-2. AB - The complementary DNAs encoding human interferon-gamma (IFN-gamma) and human interleukin-2 (IL-2), two different proteins involved in the same immune system, were fused to code a hybrid protein, which was expressed in E. coli to investigate the interactions of these two proteins at the molecular level. Through immunoprecipitation analysis, this protein was revealed to be of about 31 kDa, which was expected from nucleotide sequencing, and to have the antigenicities of both IFN-gamma and IL-2. The extract from bacteria expressing this hybrid protein showed at least two biological activities: an antiviral activity derived from IFN-gamma and the ability to support the growth of natural killer (NK) cells derived from IL-2. Comparing the enhancement of NK cell activity of this hybrid protein with IFN-gamma and IL-2, this hybrid protein appears to conserve each activity almost completely without diminishing the other. PMID- 3009219 TI - Autoradiographic localisation of VIP receptors in human lung. AB - Localisation and pharmacological properties of the VIP receptor in human lung sections are described. The receptor density determined by saturation analysis using 125I-VIP is approx. 100 fmol/mg protein, with a Kd of approx. 600 pM. Inhibition of 125I-VIP binding with VIP and related peptides gives a rank order of potency: VIP greater than peptide histidine methionine greater than secretin. Light microscope autoradiography reveals specific VIP binding sites, with a high density over the pulmonary artery smooth muscle and the alveolar walls and with a lower density over the bronchial epithelium. PMID- 3009220 TI - Purification of ovine placental lactogen (oPL) using high-performance liquid chromatography. Evidence for two forms of oPL. AB - After initial purification of ovine placental lactogen (oPL) using the procedures described previously [(1976) Endocrinology 98, 65-75], the oPL preparation was further purified by high-performance liquid chromatography (HPLC) using an anionic exchange column (Bio-Sil TSK DEAE-2-SW). Two forms of oPL with different relative mobilities on HPLC were isolated and designated oPL-I and oPL-II. Subsequent analysis by polyacrylamide gel electrophoresis containing SDS revealed that oPL-I and oPL-II are nearly homogeneous (greater than 90% pure) and are identical in apparent Mr (approx. 22 000-23 000). Like human growth hormone (hGH), oPL-I and oPL-II are equally active in the radioreceptor assays for growth hormone-like activity (RRA-GH) and for prolactin-like activity (RRA-RRL). In the radioimmunoassay of oPL, both oPL-I and oPL-II are immunologically similar. Analysis of amino acid composition revealed that oPL-I and oPL-II consist of 199 and 196 residues, respectively, and have almost identical residues except that oPL-I has a higher content of glycine. Furthermore, both oPLs have a general similarity in amino acid composition to oGH and oPRL except for a lower content of methionine and leucine but with a higher content of lysine. Our studies demonstrated the presence of two similar forms of oPL. Whether these two similar forms of oPL share identical primary structure remains to be determined. PMID- 3009221 TI - Partial purification of a high density lipoprotein-binding protein from rat liver and kidney membranes. AB - The existence of a cell receptor which recognises plasma high density lipoprotein (HDL) has been suggested from studies which demonstrate specific binding of HDL3 to cultured cells derived from various tissues in the body. This study provides evidence of a specific HDL-binding protein in crude plasma membranes prepared from rat kidney and liver. Following separation of solubilised membrane proteins on polyacrylamide gel slabs and 'Western' blotting, one major band was identified which bound HDL3, or apo AI or apo AII. The protein, which was present in both liver and kidney membranes, was partially purified by repetitive preparative SDS polyacrylamide gel electrophoresis and although accompanied by considerable loss of binding activity, could still be detected by the ligand-blotting procedure used initially to detect its presence in cell membranes. PMID- 3009222 TI - A [3H]amine congener of 1,3-dipropyl-8-phenylxanthine. A new radioligand for A2 adenosine receptors of human platelets. AB - A xanthine amine congener (XAC), an amine-functionalized derivative of 1,3 dipropyl-8-phenylxanthine, is an antagonist ligand for A2 adenosine receptors of human platelets. XAC inhibited 5'-N-ethylcarboxamidoadenosine (NECA)-induced stimulation of adenylate cyclase activity with a KB of 24 nM. [3H]XAC exhibits saturable, specific binding with a Kd of 12 nM and a Bmax of 1.1 pmol/mg protein at 37 degrees C. [3H]XAC binding in platelets is the first example of labeling of A2 adenosine receptors in which the potencies of adenosine agonists and antagonists in inhibiting binding are commensurate with their potencies at these receptors in functional studies. Furthermore, [3H]XAC is the first antagonist radioligand with high affinity at A2 adenosine receptors. PMID- 3009224 TI - The complete amino acid sequence of 3-dehydroquinate synthase of Escherichia coli K12. AB - The complete amino acid sequence of the Escherichia coli 3-dehydroquinate synthase has been determined by a combined nucleotide and direct amino acid sequencing strategy. E. coli 3-dehydroquinate synthase is 362 amino acids long and has a calculated Mr of 38 880. Analysis of the aroB nucleotide sequence and its 5'- and 3'-flanking regions has identified the aroB promoter elements and a possible 3'-terminator site. PMID- 3009223 TI - Protein that induces cell differentiation causes nicks in double-stranded DNA. AB - The growth and differentiation of myeloid hematopoietic cells are regulated by different macrophage and granulocyte inducing proteins, those that induce growth and others that induce differentiation. The proteins that induce differentiation but not those that induce growth bind to double-stranded DNA. We now report that purified myeloid cell differentiation-inducing protein causes single strand breaks (nicks) in double-stranded DNA. This DNA nicking may initiate the changes in gene expression that are required for differentiation. PMID- 3009225 TI - Fructose 2,6-bisphosphate and the control of glycolysis in bovine spermatozoa. AB - Epididymal bovine sperm contain fructose-1,6-bisphosphatase activity which is inhibited by AMP and by fructose 2,6-bisphosphate. Sperm phosphofructokinase displays kinetic characteristics that are typical of the F-type and it is stimulated by fructose 2,6-bisphosphate. The concentration of sperm fructose 2,6 bisphosphate remained unaffected at 1-2 microM when the glycolytic rate was either increased by glucose, caffeine or antimycin, or decreased by alpha chlorohydrin or 6-chloro-6-deoxyglucose. PMID- 3009226 TI - Solid-phase peptide synthesis of human(Nle-27)-oxyntomodulin. Preliminary evaluation of its biological activities. AB - Oxyntomodulin is a peptide isolated from porcine intestine which consists of the whole glucagon sequence extended at its C-terminal part by a basic octapeptide. The analogue (Nle-27)-oxyntomodulin of the human sequence has been synthesized by solid-phase peptide synthesis, purified by HPLC and identified. Its biological activities are the same as those of the natural hormone. PMID- 3009227 TI - Expression of the cloned subunits of Escherichia coli transhydrogenase from separate replicons. AB - The pntA and pntB genes of Escherichia coli, encoding the alpha- and beta subunits of the pyridine nucleotide transhydrogenase, were cloned individually in two different compatible plasmids into Escherichia coli mutants lacking transhydrogenase activity. Energy-linked and non-energy-linked transhydrogenase activities were produced only in cells carrying both plasmids thus showing that the products of both genes are required for the formation of an active enzyme. ATP-energized transhydrogenase activity was not increased in cells containing amplified levels of the transhydrogenase when the cell membrane ATPase was also amplified. It is suggested that the excess transhydrogenase is effectively uncoupled from the ATPase by compartmentalization in the cell. PMID- 3009228 TI - Requirement for bivalent cations in the actions of insulin and sodium nitroprusside on metabolism in rat adipocytes. AB - The requirement for Ca2+ and Mg2+ in the actions of insulin and sodium nitroprusside on rat adipocyte metabolism was investigated: sodium nitroprusside, but not insulin, increased cGMP levels in cells incubated in the absence of Ca2+ and/or Mg2+; sodium nitroprusside and insulin are unable to increase the incorporation of [14C]glucose into triglycerides and [14C]leucine into proteins in the absence of Ca2+ and Mg2+; sodium nitroprusside and insulin showed antilipolytic actions in Ca2+- and Mg2+-free medium. We conclude that in the absence of Ca2+ and Mg2+, sodium nitroprusside and insulin have very similar regulatory properties on triglyceride, protein synthesis and adrenaline stimulated lipolysis, but not on cGMP levels in rat adipocytes. This could provide evidence that omission of bivalent cations was inhibitory at more than one site, or that sodium nitroprusside mimics insulin's actions by another mechanism that does not involve cGMP. PMID- 3009229 TI - Effect of ionic strength on production of cAMP- and Ca2+-independent protein kinase from rat liver plasma membrane. AB - Production of cAMP- and Ca2+-independent protein kinase was stimulated when rat liver plasma membrane was incubated with increasing concentrations of NaCl. This protein kinase release was diminished by addition of protease inhibitor. The molecular mass of this enzyme was approx. 50 kDa and a high concentration of Mg2+ was required for whole histone phosphorylation. These properties are similar to those of the protease-activated form of protein kinase C. The NaCl effect could be replaced by other salts such as LiCl and NaHCO3. These results suggest that membrane-bound protein kinase C is activated by limited proteolysis corresponding to an increase in ionic strength. PMID- 3009231 TI - Isolation and amino acid sequence of the 9.5 kDa protein of beef heart ubiquinol:cytochrome c reductase. AB - The 9.5 kDa protein of beef heart ubiquinol:cytochrome c reductase was isolated by a series of chromatographic steps involving dissociation of the complex by urea and guanidine. A clear distinction between the 9.5 kDa protein and the 9.2 kDa protein described earlier [(1982) J. Biochem. 91, 2077-2085] by SDS-PAGE was only achieved when the electrophoresis was performed according to Schagger et al. [(1985) FEBS Lett. 190, 89-94; (1986) Methods Enzymol. 126, 22] because in this gel system the apparent molecular mass of the 9.5 kDa protein is shifted to 11 kDa. The amino acid sequence was determined by solid-phase Edman degradation of the whole protein up to amino acid residue 80 and of the proteolytic cleavage fragments. The protein consists of 81 amino acid residues; its Mr was calculated to be 9507. Structure predictions have been made from average and sided hydropathy profiles. The 9.5 kDa protein is either bound to the core proteins within a 9.5 kDa-core protein subcomplex or else it aggregates easily with the core proteins during the isolation procedure. PMID- 3009230 TI - Glucose reduces both Rb+ influx and efflux in pancreatic islet cells. AB - Microdissected, beta-cell-rich pancreatic islets from ob/ob mice were used in studies of 86Rb+ transport. D-Glucose (20 mM) induced a biphasic reduction in 86Rb+ efflux. The reduction stabilized within 10 min at 34% of the efflux rate at zero glucose. The initial 86Rb+ uptake (5 min) was dose-dependently reduced by ouabain with maximum inhibition at 1 mM. D-Glucose (20 mM) did not affect the ouabain-sensitive 86Rb+ influx but markedly reduced (48%) the ouabain-resistant isotope influx. The results suggest that D-glucose does not affect the Na+/K+ pump in pancreatic beta-cells and that the glucose-sensitive K+-transporting modalities (K+ channels) in the beta-cells can mediate both inward and outward K+ flux. PMID- 3009232 TI - Lipoprotein-binding proteins in the human platelet plasma membrane. AB - The binding of homologous plasma lipoproteins to specific receptor proteins in the plasma membrane of human blood platelets was studied by ligand blotting techniques. HDL3, HDL2 and LDL showed saturable binding to three bands of 156, 130 and 115 kDa, respectively. This binding was not markedly affected by the presence or absence of Ca2+ nor by covalent modification of lysine and arginine residues of the apoprotein moieties. However, it can be almost completely reversed by the addition of heparin or suramin. PMID- 3009233 TI - Dynamic testing in reproductive endocrinology. PMID- 3009234 TI - The effect of spironolactone on aromatase activity. AB - Spironolactone, an aldosterone antagonist, causes decreased plasma testosterone (T) levels and gynecomastia in men and is clinically useful in the treatment of hirsutism in women. Many mechanisms of action for spironolactone have been proposed. It has been suggested that one cause for low plasma T levels may result from an increased metabolic clearance rate of T due to increased extraglandular aromatization to 17 beta-estradiol. The purpose of this investigation was to determine the effect of spironolactone on the rate of activity of aromatase in human fetal liver (hFL) cells. The activity of aromatase was assayed by determining the rate of incorporation of [1-3H]androstenedione into [3H]water in hFL cells exposed to spironolactone (10(-10) to 10(-4) M) for 24 or 72 hours. The aromatase activity in control hFL cells remained constant during the study. Dibutyryl cyclic adenosine monophosphate significantly stimulated aromatase activity from 63 to 257 pmol X mg-1 protein X 2 hours-1 after 24 hours and 72 hours exposure, respectively. In contrast, when hFL cells were exposed to spironolactone (10(-10) to 10(-5) M) for up to 72 hours, the activity of aromatase did not differ significantly from that in control hFL cells. It is concluded that spironolactone in therapeutic concentrations does not stimulate aromatase activity in hFL cells maintained in vitro. PMID- 3009235 TI - RU486-induced menses in cynomolgus monkeys: uniformity of endometrial sloughing. AB - A progesterone (P) antagonist (RU486) was administered to cynomolgus monkeys in the midluteal phase (cycle day 21 to 24), alone or in combination with P. Vehicle only was administered to other monkeys that served as controls. On cycle day 25, hysterectomy was performed on all of the monkeys. The endometrium was studied histologically in fundal, medial, and isthmic regions. RU486 induced menses in all treated monkeys; but the amount of residual endometrium was slightly higher in primates that received RU486 plus P. The effect of RU 486 on the endometrium was surprisingly homogeneous throughout the uterus. The findings demonstrate that administration of RU486 in the luteal phase induced a nearly uniform and homogeneous sloughing of the endometrium. Whereas concurrent P treatment did not prevent induction of menstruation, the depth of endometrial shedding was less than with RU486 alone. PMID- 3009237 TI - [Monosynaptic activation of motor neurons of the nucleus of the facial nerve evoked by stimulation of midbrain structures]. AB - Synaptic inputs of the midbrain structures to the facial nucleus motoneurons were studied in acute experiments in cats by means of intracellular recording. Stimulation of periaqueductal gray and pretectal area was found to evoke mono- and polysynaptic activation of facial motoneurons. Functional significance of the midbrain structures as an intermediate link transmitting descending signals to the facial nucleus, is discussed. PMID- 3009236 TI - [Synaptic responses of neurons of the red nucleus of the alert cat to stimulation of the sensomotor area of the cortex and the nucleus interpositus of the cerebellum]. AB - Postsynaptic potentials of the red nucleus neurons evoked by stimulation of the cortical sensomotor region and the cerebellar nucleus interpositus were studied in chronic experiments on awake cats by means of intracellules revording. Rubro spinal neurons identified by their antidromic activation in response to the stimulation. A decrease of the critical depolarization level was a specific feature for mono- and polysynaptic responses of the red nucleus neurons in alert animals. Isolated EPSPs, therefore, were rarely recorded as well as a great number of polysynaptic potentials. Burst discharges of rubro-spinal neurons and intensive activity of the red nucleus interneurons, were observed. PMID- 3009238 TI - [Effect of excess and deficit of hormones of the pituitary-adrenal system on drinking behavior in the rat]. AB - In rats, effects of hormonal disbalance of the pituitary-adrenal axis on formation, extinction and subsequent restoration of conditioned drinking response were studied. The hypophysectomia accelerated learning of this response whereas administration of ACTH retarded its extinction. In 45 days after extinction, a significant deficit of the habit retrieval was observed in hypophysectomized rats. The time of the conditioned response actualization increased in animals treated with ACTH or dexamethasone. The findings are discussed in the light of the hypothesis suggesting that hormonal balance of pituitary-adrenal system is important for the processes of consolidation and for retention of conditioned responses. PMID- 3009239 TI - Genetic structure of the endogenous proviruses and expression of the gag gene in Brown Leghorn chickens. AB - Seven loci of endogenous proviruses were detected in the genome of Brown Leghorn chickens. Sets of endogenous proviruses in DNA of the chicken embryos examined were identified by blot hybridization with 32P-labelled DNA of RSV and EcoRI restriction endonuclease digestion. Comparison of the results showed that only one locus (A) of endogeneous provirus was associated with a gs+ phenotype as determined by the immunoperoxidase reaction and antibodies against gag gene products of RSV. Restriction endonuclease analysis with HindIII, BamHI and SacI revealed that proviruses A and F in Brown Leghorn chickens correspond to loci ev 3 and ev-6, respectively, in White Leghorn chickens. Other loci (B, C, D, E, and X) were designated ev-22, ev-23, ev-24, ev-25, ev-26, respectively. None of these loci expressed infectious virions. The structure of most of the endogenous proviruses examined is considerably different from the genome of the endogenous chicken virus RAV-O. The difference in structure may be one possible cause of the absence of endogenous provirus expression. PMID- 3009241 TI - [Responses of plasma bradykinin and the renin angiotensin axis to angiotensin II in hyperthyroid patients]. AB - The present investigation was undertaken to elucidate the possible interplay between the circulating kinin(s) and the renin angiotensin axis in hyperthyroidism. The responsiveness of plasma aldosterone (p-Ald), kinin (p-BK), plasma renin activity (PRA) and serum angiotensin converting enzyme activity (ACEA) to infusion of angiotensin II at a dose of 4, 8 and 16 ng/kg.min. was asessed in 15 hyperthyroid patients and 10 euthyroid controls. There was impaired angiotensin II induced response of blood pressure in hyperthyroid patients, and basal concentrations of p-Ald were 7.7 +/- 3.8 ng/dl in euthyroid controls and 12.6 +/- 3.1 ng/dl in hyperthyroid patients (p less than 0.05). As compared to the euthyroid controls, the hyperthyroid patients showed a reduced response of plasma aldosterone to angiotensin II infusion. Angiotensin II infusion increased p-BK from basal levels of 19.1 +/- 8.2 pg/ml to 31.0 +/- 7.8 pg/ml (p less than 0.05) only in hyperthyroid patients and did not increase ACEA in either group. Next, the effects of a single administration of captopril (50 mg p.o.) on blood pressure and p-BK in hyperthyroid patients and euthyroid controls were studied. In the two groups blood pressure was not changed by captopril, but p-BK increased significantly. The present results do not support the view that there may be a direct linkage between the kallikrein kinin system and the renin angiotensin axis mediated by kininase II or angiotensin converting enzyme in human peripheral blood. Also it is unlikely that kinin may play a role in the mechanism of reduced responsiveness of aldosterone and blood pressure to angiotensin II in hyperthyroidism. PMID- 3009240 TI - Methylation of endogenous proviruses of Brown Leghorn chickens. AB - Among the 7 endogenous proviruses we have detected in Brown Leghorn chickens none encodes the production of virions and only one, ev-3, expresses the gag gene. To study the possible role of DNA methylation in the inhibition of provirus expression, we performed blot hybridization and restriction endonuclease analysis with EcoRI and SmaI, is sensitive to methylation. Of the six endogenous proviral loci examined (ev-3, ev-6,, ev-22, ev-23, ev-24, ev-25), two loci, ev-23 and ev 24, were methylated at all SmaI restriction sites, in both the DNA from erythrocytes of adult chickens and the DNA from 10-day embryos. Since both these viruses are closely related to the genome of RAV-O, DNA methylation might be the cause of the absence of gene expression. PMID- 3009242 TI - Reconstruction of the alveolar ridge with hydroxyapatite. AB - Diagnosis, classification, surgical and prosthetic techniques, supporting research, and clinical trials are described for reconstruction of the deficient alveolar ridge with dense, nonresorbable hydroxyapatite. The use of particulate and solid forms for preservation of the alveolar ridge, facial reconstruction, and orthognathic surgery are discussed. PMID- 3009244 TI - Retroviruses and the dermatologist. PMID- 3009243 TI - Advances in subperiosteal implant reconstruction. AB - Recent advances in subperiosteal implant surgery of the mandible and maxilla are described in terms of changes in implant design, use of particulate autogenous marrow cancellous bone in conjunction with subperiosteal implant to structurally rebuild marked deficient mandibles at the time the implant is placed, and the of computerized tomography to develop a three-dimensional model for the construction of implant framework, eliminating the direct bone impression surgery. The latter procedure has revolutionized the use of subperiosteal implants. These techniques are outlined, and areas of future research in use of implants in structurally deficient areas are described. PMID- 3009245 TI - Coexistence of pemphigus vulgaris, myasthenia gravis and hepatocellular carcinoma. PMID- 3009246 TI - Comparison between starvation and consumption of a high protein diet: plasma insulin and glucagon and hepatic activities of gluconeogenic enzymes during the first 24 hours. AB - Plasma insulin, glucagon, glucose, free fatty acids and glycerol, hepatic cyclic AMP and glycogen, and liver phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-bisphosphatase (FBPase), glucose 6-phosphatase (G6Pase) and alanine amino transferase (AAT) activities were examined in adult rats during the first 24 h of either starvation or consumption of a high protein, carbohydrate-free (HP) diet. Under both nutritional conditions, plasma insulin fell within 12 h and remained constant thereafter. Glucagon increased 12 h after the start of the experiment and peaked between 18-24 h. The insulin: glucagon ratio was lower during the last 12 h of the experiment. In both experimental groups, liver cyclic AMP increased progressively and peaked between 15-24 h, but it increase was higher on HP diet than on starvation. Whereas plasma glucose remained low on starvation for 24 h, it returned to normal on consumption of the HP diet. In both groups, liver glycogen fell within 12 h and remained low until the end of experiment. FBPase, G6Pase and AAT did not change on starvation, while they increased toward the end of 1 d HP consumption. During starvation or consumption of the HP diet, PEPCK increased progressively and peaked between 15-24 h, but the increase was greater with the HP diet than with starvation. These findings suggest that in the first 24 hours, the adaptative response of hepatic gluconeogenesis is higher with a HP diet than upon starvation. PMID- 3009247 TI - Relationships between the GTP content and the TSH-stimulated adenylate cyclase activity of cultured thyroid cells. AB - The adenylate cyclase activity of a crude membrane fraction derived from cells cultured for 4 days in the presence of TSH (0.1 mU/ml), when acutely stimulated with 25 mU/ml, is 5-8 times higher than that derived from control cells. It has been suggested that changes in the intracellular content of GTP resulting from TSH chronic treatment were the cause of the modified responsiveness of the cyclase. To investigate this hypothesis, a method for GTP determination was developed. The steady-state concentration of GTP in 4-day TSH-treated cells is 2 3 times higher than in 4-day control cells. The increase in GTP content is concentration dependent between 5 and 500 microU/ml TSH in the culture medium. It presents a maximum on day 4 of culture, but remains elevated up to day 5. Nevertheless the GTP content is not the only factor controlling the cyclase activity, indeed the addition of 0.1 mM GTP to membranes from control cells does not increase the response up to the level reached by membranes from TSH-treated cells. Treatment of the cells with virazole, a drug inhibiting the biosynthesis of guanyl nucleotides, greatly decreases the GTP level, but is unable to suppress the positive effect of the TSH chronic treatment on adenylate cyclase activity. These results show that the increase in GTP level resulting from culture of the cells in the presence of minute amounts of TSH is not exclusively linked to adenylate cyclase responsiveness to TSH. PMID- 3009249 TI - Somatostatin receptors in the adrenal. AB - Somatostatin receptors have been visualized in the adrenal by autoradiography using the iodinated Tyr3 derivative of the somatostatin octapeptide analog SMS 201-995 (H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr(ol), Sandostatin*). In the rat adrenal gland somatostatin receptors are not exclusively restricted to the glomerulosa cell layer, but can also be found in the adrenal medulla, particularly after in vitro incubation. The receptor sites in the adrenal are saturable and have affinity constants of 0.42 nM in the adrenal cortical and 0.15 nM in medullary membranes. The distribution of somatostatin receptors in the adrenal visualized in vitro is strongly species specific. Whereas only the adrenal medulla is labelled in the hamster, mouse and rhesus monkey, solely the glomerulosa layer shows somatostatin receptor sites in the cow and the guinea pig. The rat is the only species with a high density of somatostatin receptor sites in both the glomerulosa cell layer and the adrenal medulla. PMID- 3009248 TI - The Brattleboro rat: normal growth hormone secretion, decreased hepatic growth hormone receptors and low plasma somatomedin activity. AB - Three-month-old male Brattleboro rats with hereditary diabetes insipidus (DI) present a growth defect; Brattleboro rats were studied together with age-matched Long-Evans (LE) rats. Pituitary growth hormone (GH) content was comparable in both groups of rats. Pulsatile GH release and mean 6 h GH plasma levels did not appear significantly different in chronically catheterized DI and control animals. In parallel with the growth defect, the plasma somatomedin bioactivity was significantly lower in DI than in LE rats. The specific binding of [125I]iodo hGH to liver microsomal membranes of DI rats was 59.7% that of controls. The number of the GH binding sites rather than the affinity of the binding was decreased. The specific binding of [125I]iodo-insulin was oppositely affected by the DI state: it was 1.5 times higher in liver membranes of DI rats than in membranes of LE rats. These findings make a non-specific effect of the DI state on liver membrane proteins unlikely. The Brattleboro rats present a growth failure without reduction of their GH secretion. The decreased number of the hepatic GH receptors and the subsequent low plasma somatomedin activity could explain the growth retardation of the DI rats. PMID- 3009250 TI - Translocation of cAMP-dependent protein kinase to the nucleus during development of Dictyostelium discoideum. AB - The distribution of the catalytic and regulatory subunits of the cAMP-dependent protein kinase between cytoplasm and nucleus was determined during the development of Dictyostelium discoideum. In vegetative amoebae approximately 2% of the subunits were in the nucleus. During development there was an approximately 5-fold increase in total soluble cAMP-dependent protein kinase and a 15- to 30-fold increase of enzyme in the nuclear fraction. There was a reverse translocation from nucleus to cytoplasm, when Tipped Aggregates were disrupted and the resultant amoebae incubated in single-cell suspension. The addition of cAMP to these single-cell suspensions brought about the reentry of the subunits into the nucleus. The findings are discussed in relation to the potential role of the cAMP-dependent protein kinase in the regulation of mRNA and protein synthesis. PMID- 3009251 TI - Environmentally induced analgesia: ontogeny of a nonopioid system. AB - Previous research has demonstrated that exposure to 90 sec of hind paw shock activates an endogenous pain control system that involves cholinergic sites. The purpose of the present investigation was to examine the development of function of this nonopioid analgesic system. Research on the ontogeny of the cholinergic system suggests that this receptor system exhibits an extended period of postnatal development, with various neurochemical indexes reaching maturity between 30 to 50 days of age. Results revealed that exposure to hind paw shock produced very low levels of analgesia in 10- and 28-day-old rats. The analgesic response was more evident in 2-month-old rats, but the degree of hind paw shock induced analgesia was not at its maximum until 3 months of age. Systemic injection of naltrexone had no effect on the degree of analgesia induced by hind paw shock, while a systemic injection of scopolamine significantly attenuated the analgesia displayed by the 28-day-old and 2- and 3-month-old rats. Thus, the neurochemical indexes of cholinergic development over-estimated the degree of functional maturity of the endogenous analgesia system activated by hind paw shock. PMID- 3009252 TI - Adrenal involvement in the diabetes-induced loss of growth hormone and prolactin receptors in the livers of female rats. AB - Streptozotocin-induced diabetes causes a decrease in growth hormone and prolactin receptors in the livers of female rats, and in the serum concentration of somatomedin-C/insulin-like growth factor-I, concomitantly with an increase in the serum testosterone levels. In this study, a possible role for adrenal androgens in the loss of receptors was examined. Rats were adrenalectomised bilaterally 3 days after the induction of diabetes with streptozotocin (100 mg/kg intravenously), and livers were removed 3 days later. Adrenalectomy had no effect on binding of ovine prolactin or bovine growth hormone to liver microsomal membranes from non-diabetic rats, but in diabetic rats it entirely abolished the 56% decrease in prolactin binding and significantly reversed the 66% decrease in growth hormone binding and the parallel fall in serum levels of somatomedin C/insulin-like growth factor-I (p less than 0.05). Adrenalectomy also prevented the diabetes-induced rise in serum testosterone. Daily injection of testosterone to normal and diabetic rats for 12 days significantly reduced both prolactin and growth hormone binding (p less than 0.001), with the effect of diabetes being additive upon the testosterone effect. Implantation of testosterone-filled silastic capsules at the time of adrenalectomy (i.e. for 3 days) did not prevent the adrenalectomy-induced restoration of both growth hormone and prolactin receptors. The resulting high serum testosterone level did not reduce binding to growth hormone receptors in control rats over the 3 day period, and caused no further decrease in diabetic rats. However, binding to prolactin receptors was reduced by 47% in control animals with no further loss in diabetic animals (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009253 TI - Induction of renal mixed function oxidases in the rat and mouse: correlation with ultrastructural changes in the proximal tubule. AB - Rats and mice were pretreated with beta-naphthoflavone (BNF) at 100 mg/kg/day, ip, for 4 days, or polybrominated biphenyl (PBB) as a single ip dose at 150 mg/kg, and the temporal changes in renal mixed function oxidase (MFO) activity and ultrastructural changes in the proximal tubule were examined. Rat renal cytochrome P-450 (P-450), ethoxycoumarin-O-deethylase (ECOD), and ethoxyresorufin O-deethylase (EROD) were increased 5 days following the first dose of inducer. P 450 content and enzyme activity peaked on Day 5 to Day 10 and returned to control values by Day 15. BNF and PBB caused proliferation of smooth endoplasmic reticulum (SER) in only the S3 segment of the rat proximal tubule. Histologic analysis indicated that there was a close correlation between the temporal changes in renal MFO activity and proliferation of SER in the S3 segment of the proximal tubule. In contrast to the rat, mouse renal P-450 and ECOD were not induced by either BNF or PBB nor was there any significant proliferation of SER in any segment of the proximal tubule. Mouse renal EROD was slightly increased on Day 5 but not Day 10 or 15 by BNF; PBB had no effect. In addition to proliferation of SER, these inducers also caused proliferation of peroxisomes in the rat and mouse proximal tubule. These results demonstrate a temporal relationship between induction of rat renal MFOs and proliferation of SER in specific sections of the proximal tubule and thus suggest that induction of these renal enzymes may also be restricted to specific cell populations of the kidney. A species difference apparently exists since proliferation of SER was not observed in the mouse. PMID- 3009254 TI - Disposition of [14C]dimercaptosuccinic acid in mice. AB - Dimercaptosuccinic acid labeled with 14C ([14C]DMSA) was administered to mice iv; the mice were frozen by immersion in dry ice/hexane at 6 and 20 min and 1, 3, 9, and 24 hr after injection. The frozen mice were sectioned and processed for whole body autoradiography for soluble substances. The radioactivity was highly localized in extracellular fluids such as the subcutaneous, intrapleural, intraperitoneal, and periosteal spaces. There was a pronounced accumulation in the periosteal fluid above that in other fluids during the first hour after injection. Most of the radioactivity was eliminated by the kidney and liver. Pretreatment of a mouse with HgCl2 subcutaneously 1 hr before [14C]DMSA produced an increase in radioactivity in the liver and decrease in lung. A high concentration of radioactivity was seen at the subcutaneous site of injection of the HgCl2. The results are interpreted to indicate that most of the DMSA is in the extracellular space but that it can cross cellular membranes to some extent. The pronounced accumulation in periosteal fluid may be an interaction of DMSA with Ca2+ in this space. No tissue had a pronounced retention of the compound, but lung retained more than most other tissues. PMID- 3009256 TI - [Gastric transepithelial potential difference: constitutive elements and physiological significance]. PMID- 3009255 TI - [Randomized trial of elliptinium acetate and tamoxifen in the treatment of inoperable hepatocellular carcinoma]. PMID- 3009257 TI - Superoxide anion generating capacity of polymorphonuclear cells in patients with liver cirrhosis. AB - The superoxide anion generating capacity of polymorphonuclear cells (PMNs) in patients with liver cirrhosis and the effect of lipopolysaccharide on rat PMNs were examined. Superoxide anion generating capacity of PMNs was measured as luminol-dependent photon emission (chemiluminescence) during phagocytosis of peptide in vitro. Chemiluminescence of PMNs from patients with liver cirrhosis was significantly enhanced compared with normal healthy volunteers, and endotoxemia was detected in 3 out of 20 cases of liver cirrhosis by the limulus gelatin test. Serial studies revealed that chemiluminescence of PMNs and endotoxin in plasma decreased after administration of polymyxin B (3 X 10(6) u/day). Chemiluminescence of rat PMNs was also markedly enhanced after the injection of lipopolysaccharide, and persisted for more than 8 days even though endotoxemia was not detected. These results indicate that the enhancement of chemiluminescence by PMNs is related to endotoxins spilling over from the liver in patients with liver cirrhosis. PMID- 3009258 TI - Japanese Gastroenterology Society. Proceedings of the 71st general meeting. Sapporo, Japan, May 22-24, 1985. Abstracts. PMID- 3009259 TI - Hepatic microsomal function in rats with chronic dietary iron overload. AB - We determined whether alterations in hepatic microsomal function occur in association with iron-induced lipid peroxidation in vivo in rats with chronic dietary iron overload. In rats fed a 2.0% carbonyl iron diet for a period of 20 wk, there was no significant microsomal conjugated diene formation (evidence of microsomal lipid peroxidation) or difference in cytochrome P450 concentration found at mean (+/- SEM) hepatic iron concentrations of 1210 +/- 92 micrograms/g liver (wet wt) or 2730 +/- 100 micrograms/g. At a hepatic iron concentration of 4090 +/- 245 micrograms/g, however, there was significant conjugated diene formation (p less than 0.001) and a 56% decrease in the cytochrome P450 concentration (p less than 0.001). In rats fed a 2.5% carbonyl iron diet for 10 wk, achieving a liver iron concentration of 4820 +/- 420 micrograms/g, there was significant microsomal conjugated diene formation (p less than 0.001), a 35% reduction in cytochrome P450 (p less than 0.005), and a 16% reduction in aminopyrine demethylase activity (p less than 0.025), but only an 8% reduction in glucose-6-phosphatase activity (p = not significant). Finally, in rats fed a 3.0% iron-supplemented diet for 7 wk, achieving a liver iron concentration of 2730 +/- 205 micrograms/g, there was a 23% reduction in cytochrome P450 (p less than 0.025), a 28% reduction in cytochrome b5 (p less than 0.001), and a 47% increase in heme oxygenase activity (p less than 0.025) (heme oxygenase activity measured in this group only). We conclude that oral iron loading can produce microsomal lipid peroxidation in vivo that is associated with selective decreases in microsomal hemoprotein concentrations and cytochrome P450-dependent enzymes. PMID- 3009260 TI - Unusual esophageal ulcers containing enveloped viruslike particles in homosexual men. AB - Eight homosexual men presented with odynophagia. Five had a maculopapular rash and 2 had oral ulcers. Upper panendoscopy revealed the presence of multiple discrete ulcers measuring 3 mm-1.5 cm in diameter in the esophagus of each patient. The intervening mucosa appeared normal. Endoscopic brushings and biopsy specimens taken from the ulcer margins and examined by light microscopy showed no inclusion bodies or giant cells. Fungal stains were negative. Biopsy specimens examined by electron microscopy revealed enveloped virus-like particles 100-140 nm in diameter, exhibiting morphologic features consistent with retroviruses. No virus was isolated after incubation in either rhesus monkey kidney or human foreskin fibroblast culture. Serology for cytomegalovirus and herpes simplex virus was consistent with past infection. In each patient the esophageal ulcers healed completely. In summary, an illness characterized by the presence of esophageal ulcers is described in 8 homosexual men, 5 of whom also had a skin rash and 2 of whom had oral ulcers. The cause of the esophageal ulcers is likely to be the enveloped viruslike particles observed at electron microscopy. PMID- 3009261 TI - Phytohemagglutinin from red kidney bean (Phaseolus vulgaris) inhibits sodium and chloride absorption in the rabbit ileum. AB - Phytohemagglutinin (PHA), derived from red kidney bean (Phaseolus vulgaris), can induce malabsorption and diarrhea when fed to rats. In this study, we determined the effect of PHA on ion transport in the rabbit ileum in vitro. Compared with control tissues, PHA (1 mg/ml) added to the mucosal solution increased short circuit current (1.1 +/- 0.2 microEq/cm2 X h, p less than 0.001), decreased net Na (-1.0 +/- 0.5 microEq/cm2 X h, p less than 0.02) and Cl (-1.2 +/- 0.6 microEq/cm2 X h, p less than 0.025) absorption, and decreased tissue conductance (-1.8 +/- 0.5 mS/cm2, p less than 0.001). Serosal addition of PHA had no effect on the short-circuit current or tissue conductance. Mucosal PHA did not increase mucosal levels of cyclic adenosine monophosphate or cyclic guanosine monophosphate. Removal of serosal calcium did not affect the increase in short circuit current induced by mucosal PHA. Utilizing fluorescent microscopy, rhodamine-labeled PHA was found to bind to the luminal border of villus cells, but not to crypt cells, in the ileum. In the descending rabbit colon, PHA did not affect either the short-circuit current or conductance, and rhodaminated PHA did not bind to the epithelial surface. Using the increase in short-circuit current as an indicator of absorption, PHA did not affect Na-coupled glucose or amino acid absorption in the ileum. This study suggests that dietary lectins may play a role in regulating intestinal fluid and electrolyte transport. PMID- 3009262 TI - Significance of calcium for the prostaglandin E2-mediated secretory response to 5 hydroxytryptamine in the small intestine of the rat in vivo. AB - 5-Hydroxytryptamine (5-HT) has been claimed to mediate intestinal secretion in morphine withdrawal diarrhea through stimulation of local prostaglandin formation without involving cyclic adenosine monophosphate. Therefore, experiments were performed to study (a) the effects of exogenous 5-HT and the cyclic adenosine monophosphate-dependent secretagogue, vasoactive intestinal polypeptide, on intestinal prostaglandin E2 (PGE2) formation and (b) the involvement of calcium in the secretory response to close intraarterial infusion of 5-HT, PGE2, or vasoactive intestinal polypeptide in tied-off loops of rat jejunum in vivo. 5 Hydroxytryptamine and vasoactive intestinal polypeptide reversed fluid absorption to net secretion (p less than 0.01), but only 5-HT caused an increase in luminal PGE2 output (p less than 0.01). Indomethacin and d,l-verapamil prevented only the secretory effect of 5-HT. Exogenous PGE2 (1.6-160 ng/min) reversed absorption to secretion (p less than 0.01) in a dose-dependent manner, irrespective of whether the rats were pretreated with indomethacin or not. Racemic and l-verapamil, but not d-verapamil, markedly reduced (p less than 0.01) the secretory effect of physiologically low doses of PGE2 (1.6 and 16 ng/min), whereas high doses of PGE2 (160 ng/min), which caused a significant increase in mucosal cyclic adenosine monophosphate (p less than 0.005), were not inhibited by verapamil. These data suggest that PGE2 may be an important intermediate in the transduction mechanism leading to 5-HT-induced intestinal secretion, and that physiologic doses of PGE2 may act by facilitating calcium entry, rather than by increasing intracellular calcium through activation of the adenylate cyclase. PMID- 3009264 TI - [Breast sonography with high-frequency short-focusing transducers]. AB - Since 1984 two high-frequency short-focussed linear transducers have been used for breast sonography at Innsbruck University Gynaecological Clinic. In the study reported here 121 patients with pathologic change in the breast were examined. A significant improvement in diagnosis was achieved: on the one hand because of the improved resolution and distinction of the lesions, and on the other because of the manageability of the transducers and the individually adjustable breast compression. Both perforation of the physiological mammary strata as well as clearly distinguishable tumor ends without echoproof halos were considered reliable indicators of the malignancy of the respective changes. PMID- 3009263 TI - Preparation and characterization of a new cholecystokinin receptor probe that can be oxidatively radioiodinated. AB - Oxidative iodination is the simplest, most efficient, and least expensive method for radiolabeling peptide hormones. It has not been applied to cholecystokinin (CCK), however, because this peptide lacks an unsubstituted tyrosine residue and oxidation abolishes its biologic activity. We report the synthesis, purification, and characterization of the first derivative of CCK that can be radioiodinated with an oxidative method while maintaining full biologic activity and receptor binding affinity. Boc-Tyr-[(Thr28, Nle31)CCK-25-33] was synthesized, iodinated using a solid-phase oxidant, and purified on reverse-phase high-pressure liquid chromatography to a specific activity of 2125 Ci/mmol. Both native and iodinated Boc-Tyr-[(Thr28, Nle31)CCK-25-33] exhibited efficacy and potency for stimulation of in vitro pancreatic enzyme secretion that were identical to CCK-8. Binding of Boc-125I-Tyr-[(Thr28, Nle31)CCK-25-33] to rat pancreatic plasma membranes was rapid, reversible, temperature-dependent, saturable, and specific. The abilities of various molecular forms of CCK to compete for this binding paralleled their potencies for stimulation of pancreatic secretion (Kd: CCK-8, 0.8 nM; CCK-33, 3 nM; CCK-8DS, 1 microM; CCK-4, 50 microM), whereas structurally unrelated pancreatic ligands (1 microM) demonstrated no significant competition. These findings suggest that Boc-125I-Tyr-[(Thr28, Nle31)CCK-25-33] interacts with the same high-affinity pancreatic receptor as the CCK receptor probes previously reported, i.e., the Bolton-Hunter-labeled CCK-33 and CCK-8. This probe also possesses the major advantages of simplicity, efficiency, and cost of the labeling procedure, and the stability and relative oxidation-resistance of the product. Boc-125I-Tyr-[(Thr28, Nle31)CCK-25-33] will be useful for the characterization of binding interactions between CCK and its target tissues. PMID- 3009265 TI - [Transitory hypothyroidism of the newborn infant following amniofetography with an iodine-containing contrast medium]. AB - In the foetus and neonate the thyroid follicular cells are immature and hence incapable of protecting the organism against an iodide-induced inhibition of hormone synthesis (Wolff-Chaikoff effect). Thus, an iodide excess during pregnancy can lead to transient hypothyroidism in the foetus and neonate. Transient hypothyroidism due to iodide toxicity following amniofoetography in the newborn period is described. PMID- 3009266 TI - GABA A receptor mediated neurogenic inhibition of motility in the small intestine of urethane-anaesthetized rats. AB - Intravenous GABA (0.1-3 mg/kg) induced transient relaxation of the duodenum in urethane anaesthetized rats. The effect of GABA was mimicked by homotaurine and antagonized by bicuculline, suggesting the involvement of GABA A receptors in this type of response. Duodenal relaxation induced by GABA was unaffected by i.v. hexamethonium, phentolamine propranolol or 6-hydroxydopamine but was prevented in preparations pretreated with topical tetrodotoxin thus indicating its neurogenic origin. The ability of GABA to induce neurogenic relaxation decreased rapidly when increasing the distance from the duodenum while relaxations induced by DMPP or noradrenaline were observed throughout the whole rat small intestine. PMID- 3009267 TI - Interaction of vasoactive intestinal peptide (VIP) with human peripheral blood lymphocytes: specific binding and cyclic AMP production. AB - VIP binding sites and cyclic AMP production by the peptide have been studied in human blood mononuclear cells before and after selective depletion of or enrichment for T-lymphocytes, B-lymphocytes-K-NK cells and monocytes. The specifically bound 125I-labelled VIP correlated significantly with the presence of B-lymphocytes and/or cells of K-NK system. The stoichiometric data were compatible with the existence of two classes of binding sites. T-lymphocytes and monocytes did not show binding of the tracer. The cyclic AMP production stimulated by VIP correlated significantly with the presence of B-lymphocytes and/or K-NK cells. PMID- 3009268 TI - The GC clusters of the mitochondrial genome of yeast and their evolutionary origin. AB - We have studied the primary and secondary structures, the location and the orientation of the 196 GC clusters present in the 90% of the mitochondrial genome of Saccharomyces cerevisiae which have already been sequenced. The vast majority of GC clusters is located in intergenic sequences (including ori sequences, intergenic open reading frames and the gene varl which probably arose from an intergenic spacer) and in intronic closed reading frames (CRF's); most of them can be folded into stem-and-loop systems; both orientations are equally frequent. The primary structures of GC clusters permit to group them into eight families, seven of which are related to the family formed by clusters A, B and C of the ori sequences. On the basis of the present work, we propose that the latter derive from a primitive ori sequence (probably made of only a monomeric cluster C and its flanking sequences r* and r) through (i) a series of duplication inversions generating clusters A and B; and (ii) an expansion process producing the AT stretches of ori sequences. Most GC clusters apparently originated from primary clusters also derived from the primitive ori sequence in the course of its evolution towards the present ori sequences. Finally, we propose that the function of GC clusters is predominantly, or entirely, associated with the structure and organization of the mitochondrial genome of yeast and, indirectly, with the regulation of its expression. PMID- 3009269 TI - Expression of immunogenically reactive diphtheria toxin fusion proteins under the control of the pR promoter of bacteriophage lambda. AB - The tox228 gene encoding the non-toxic, immunologically cross-reactive CRM228 mutant diphtheria toxin (DT) has been cloned downstream of the PR promoter and the cro translational initiation region of bacteriophage lambda carried by plasmid pCQV2 (Queen, 1983). Efficient transcription but no appreciable amount of a translational product corresponding to complete DT could be detected in Escherichia coli hosts. Deletion of 320 bp from the C-terminal region of the B fragment of DT, and fusion of the truncated tox228 gene to lacZ yielded several hybrid beta-galactosidases (beta Gal) in an E. coli lon- strain in addition to beta Gal. The various DT fragments fused to beta Gal were immunologically reactive and were identified with antibodies specifically directed against the A- or the B-fragment of DT. Antibodies raised against the DT-beta Gal fusion proteins in guinea pigs cross-reacted with wild-type DT and its B-fragment and protected Vero cells in tissue culture against the lethal action of DT. Immunized guinea pigs survived upon injection of a five-fold lethal dose of wild-type DT. PMID- 3009270 TI - Improved vector, pHSG664, for direct streptomycin-resistance selection: cDNA cloning with G:C-tailing procedure and subcloning of double-digest DNA fragments. AB - A plasmid cloning vector, pHSG664, has been constructed which is suitable for the direct-selection of transformed cells with recombinant DNAs. The plasmid contains the replicon and ApR-gene region of pUC9 ligated to the strA+ (rpsL+) gene derived from pNO1523 [Dean, Gene 15 (1981) 99-102]. The vector contains ten unique restriction sites: EcoRI, HpaI, PvuII, SphI, PstI, SmaI(XmaI), BamHI, SalI(AccI, HincII), XbaI and HindIII. Five sites (bold-face lettering) are located within the coding region of the strA+ gene. Any insertion at the five bold-faced sites or any nucleotide replacement involving the strA+ gene region and the other unique sites can be selected by Ap and Sm double selection in a strA- (SmR) strain such as E. coli HB101. Thus, this vector is useful for cDNA cloning at either the SphI or the PstI site with the G:C-tailing procedure, as well as for the cloning of double-digest DNA segments. PMID- 3009271 TI - Ultraviolet irradiation of DNA: a way of generating partial digests for rapid restriction site mapping. AB - UV-irradiation of DNA can inhibit the activity of certain restriction endonucleases because of thymine dimer formation within the enzyme recognition sequence. The number of sites affected depends upon the dose of UV, thus making it easier to control the extent of enzyme digestion than by either limiting the digestion time, or the amount of enzyme. Restriction-site maps of bacteriophage lambda recombinants are readily produced by labelling DNA using a radioactive oligonucleotide that is complementary to either the left or right cohesive end of lambda, irradiating the DNA with UV light, limit digesting with the appropriate enzyme, and calculating the size of the fragments detected after gel electrophoresis and autoradiography. PMID- 3009273 TI - Complete nucleotide sequence of an EcoRI-1.35-kb repeated element of mouse: homology with the cellular flanking region between two intracisternal A-particle genes. AB - The complete DNA sequence (1369 bp) of an EcoRI-1.35-kb repeated element (ER-1) of the mouse BamHI family has been determined. Analysis of this sequence revealed that a portion of the 3' end (positions 1277-1369) of ER-1 was found to share 91% homology with the flanking cellular sequence between two adjacent intracisternal A-particle (IAP) genes, IAP-19A and IAP-19B. PMID- 3009272 TI - Analysis of Tn3 sequences required for transposition and immunity. AB - Tn3 is a 5-kb transposon (Tn) with 38-bp inverted terminal repeats (ITR). The two 38-bp terminal sequences are required in cis for Tn3 transposition. In this study, the role of the ITR in Tn3 transposition has been further dissected by the use of various mini-Tn3 Tn's. The transposition frequency of these mini-Tn's demonstrate that Tn3 contains no sequence other than the ITR sequences that are necessary for the first step in transposition; the two terminal repeats must be oriented as ITR for transposition to occur; the outside 34 bp of the ITR are required for transposition; and reducing the distance between the terminal sequences does not affect transposition frequency. Moreover, mutant copies of the ITR sequences that cannot function in transposition do not confer transposition immunity. PMID- 3009274 TI - Plasmid pKUN9, a versatile vector for the selective packaging of both DNA strands into single-stranded DNA-containing phage-like particles. AB - A versatile vector plasmid, pKUN9, has been constructed which, simply by infecting cells harboring this plasmid with either bacteriophage IKe or Ff (M13, fd, and fl), permits the selective packaging of both of its DNA strands into, single-stranded (ss) DNA-containing, phage-like particles. The plasmid, which is a derivative of plasmid pUC9 [Vieira and Messing, Gene 19 (1982) 269-276], contains in opposite orientations the replication origins and contiguous packaging signals of the distantly related filamentous phages IKe and Ff. As a result of the selective packaging, both strands of a DNA fragment cloned in pKUN9 can be obtained in a single-stranded form and can be sequenced by the dideoxy method using commercially available (+) and (-) sequencing primers. In addition, plasmid pKUN9 possesses all unique properties incorporated in the M13mp phages and the pUC plasmids. PMID- 3009275 TI - Expression of a bean storage protein 'phaseolin minigene' in foreign plant tissues. AB - Using the phaseolin gene and its cDNA counterpart we constructed a mutant phaseolin gene lacking the five introns but retaining its natural 5' and 3' plant regulatory sequences. This mutant phaseolin gene (minigene) was inserted into the Ti-plasmid of Agrobacterium tumefaciens strain 15955 which allowed its transfer and integration into the tobacco genome. Full-length and correctly initiated phaseolin mRNA was found among the poly(A)+RNA isolated from plant callus transformed with the minigene construction by using RNA-DNA hybridization and S1 nuclease mapping techniques. The presence of phaseolin polypeptides in soluble protein extracts from transformed tobacco tissues was confirmed by immunological methods. These results demonstrate that phaseolin gene introns and intron splicing are not a necessary requirement for biogenesis of stable phaseolin mRNA and that no alternative splice site was introduced by the removal of five introns. PMID- 3009277 TI - [Incidence and therapeutic control of gestational trophoblastic disease]. PMID- 3009276 TI - Mammalian expression-and-transmission vector derived from Akv murine leukemia virus. AB - A mammalian transmission-expression vector has been constructed based on the plasmid pBR322 and using the transcriptional signals from the Akv murine leukemia virus (AkvMuLV) to control the expression of the neo gene. The transmission vector pL psi PLneo, when transfected into the psi 2 cell line, confers G-418 resistance on recipient cell clones which produce viral particles encapsidating the transcripts of the vector. Cultures of such clones produce viral particles of titers up to 10(5) cfu/ml. The pL psi PLneo vector has two unique restriction sites which can be used for the insertion of new DNA material. PMID- 3009278 TI - [Relation of the toxicity of urea formaldehyde-based complex polymeric fertilizers to their structure]. PMID- 3009279 TI - [Role of papilloma virus (HPV) infection in the development of precancerous conditions and cancer of the cervix uteri in women]. PMID- 3009280 TI - Anticomplement receptor activity in the serum of patients with primary biliary cirrhosis. AB - Patients with primary biliary cirrhosis have a defect in the receptor mediated clearance of complement coated erythrocytes by fixed macrophages of the reticuloendothelial system. To investigate the probable mechanism of this defect peripheral blood monocytes were isolated from nine patients with primary biliary cirrhosis and seven control subjects and the ability of these cells to form rosettes with complement coated, IgM-sensitised sheep erythrocytes was assessed. Primary biliary cirrhosis peripheral blood monocytes formed rosettes to the same extent as control peripheral blood monocytes (71.0 +/- 7.1% [SEM] versus 73.3 +/- 4.3%) suggesting normal complement receptor function of primary biliary cirrhosis peripheral blood monocytes. When primary biliary cirrhosis or control peripheral blood monocytes were preincubated with primary biliary cirrhosis serum, however, the per cent of peripheral blood monocytes that formed rosettes was decreased: 2.4 +/- 0.8 and 3.1 +/- 1.3 fold respectively. To study this phenomenon further, fractions containing IgG or IgM synthesised by cultures of control or primary biliary cirrhosis lymphocytes were prepared. Rosette formation was not affected by exposure to fractions containing control or primary biliary cirrhosis IgG or control IgM, but was markedly inhibited (6.0 +/- 4.8 fold) by exposure to fractions containing primary biliary cirrhosis IgM. Similar results were obtained when freshly isolated peripheral blood monocytes or peripheral blood monocytes that had been cultured for 7-10 days--that is, macrophages, were used. Assuming that one can draw inferences concerning the status of fixed macrophages from data obtained using peripheral blood monocytes, the results of this study suggest that the complement specific defect in reticuloendothelial system clearance function in primary biliary cirrhosis is not caused by abnormality in the functional status of complement receptors on fixed macrophages but rather by a factor present in the serum of patients with primary biliary cirrhosis that has the capacity to inhibit the adherence of complement coated erythrocytes to complement receptors present on the surface of fixed macrophages. This serum factor does not appear to be a complement component but rather a product of peripheral blood mononuclear cells, other than IgG. PMID- 3009282 TI - Transcatheter embolization of pelvic vessels to stop intractable hemorrhage. AB - Six patients with severe vaginal bleeding were treated with transcatheter embolization of selected pelvic vessels. Three patients had Stage III(b) carcinoma of the cervix, one with dysfunctional uterine bleeding and two patients had gestational trophoblastic disease (GTD) with bleeding from vaginal metastases. Bleeding stopped in four of the six cases. Reasons for failure in the other two cases are given. No other reports of bleeding from vaginal metastases in metastatic GTD treated in this way have been seen in the literature. PMID- 3009281 TI - Recurrent typhoid in an HTLV-III antibody positive man. AB - A case of recurrent typhoid fever in a homosexual man with antibodies to human T cell lymphotropic virus-III (HTLV-III) and impaired cell mediated immunity is reported: we believe the first report of Salmonella typhi infection in association with HTLV-III disease. PMID- 3009283 TI - An unusual case of malignant cystosarcoma phyllodes of the breast. AB - A unusually large tumor of the left breast diagnosed as a cystosarcoma phyllodes with multiple malignant sarcomatous changes of the stroma, consisting of liposarcoma, myxoid fibrosarcoma, anaplastic, and giant cell sarcoma is described. The weight of 6200 g (13.5 lb) seems to be the largest so far presented in the literature reviewed by the authors. PMID- 3009284 TI - [Benign adnexal tumors of the skin]. PMID- 3009285 TI - [Inflammatory fibrous colonic histiocytoma with leukemoid reaction, and ileal carcinoid]. PMID- 3009286 TI - Investigation of sandflies (Diptera, Phlebotomidae) in an endemic focus of visceral leishmaniasis in Yugoslavia. AB - The paper presents the results of faunistic, ecological and viral investigations concerning phlebotomine sandflies in an endemic focus of visceral leishmaniasis in Yugoslavia. These investigations were carried out in the period from 1969 to 1981. PMID- 3009287 TI - [Studies on the diuretic effect of haloperidol in adult rats]. AB - An increase in urinary flow has been observed in rats during cataleptic response to haloperidol. The present experiment was carried out to study the mechanism of haloperidol-induced diuresis. Wistar-Imamichi adult female rats were injected i.p. with haloperidol in a dose of 0.1, 1 or 10 mg/kg, and the time course of changes in urine volume was observed. The dose-dependent diuretic effect of 1 or 10 mg/kg haloperidol was significant from 4 hr afterward, and the haloperidol induced diuresis was prevented by pretreatment with phenoxybenzamine, prazosin or yohimbine. Chlorpromazine but not spiperone and pimozide induced a significant increase in urine volume, though the effect of chlorpromazine was less marked as compared with that of haloperidol. Clonidine in a dose of 0.125-1.0 mg/kg enhanced urine flow markedly from 30 min, and the same alpha-adrenergic blockers were also effective in blocking the diuretic effect of clonidine. Urinary osmolarity in 1 mg/kg haloperidol- and 0.125 mg/kg clonidine-treated rats decreased significantly, whereas only clonidine stimulated urinary Na and K excretion. Plasma osmolarity and negative free water clearance did not change in both haloperidol- and clonidine-treated rats. The present results suggest that the haloperidol-induced diuretic effect could be due to the central alpha adrenoceptor blocking action of haloperidol. PMID- 3009289 TI - [Studies on the antibody to albumin receptor (AR) of HBs-antigen in anti-HBs antibody positive serum]. PMID- 3009288 TI - [Stimulation of pituitary-adrenocortical system by cepharanthine]. AB - We observed that cepharanthine might exert its anti-allergic action by stimulating the secretion of corticosterone. The present experiments were carried out to investigate stimulation of the pituitary-adrenocortical system by cepharanthine. Administration of cepharanthine to rats produced increases in plasma and adrenal corticosterone levels. Administration of cepharanthine to propranolol pretreated rats also produced increases in plasma and adrenal corticosterone levels and plasma ACTH level. The elevation of corticosterone level induced by cepharanthine was considered to be the specific effect of cepharanthine. Cepharanthine did not increase plasma corticosterone level in rats in the state of dexamethasone suppression of the pituitary-adrenocortical system, in which the level was lowered. Administration of cepharanthine to Bordetella pertussis vaccine induced beta-adrenergic blocked rats also produced increases in plasma and adrenal corticosterone levels. The production and release of corticosterone from an adrenal cell suspension were not influenced by cepharanthine in vitro. These results suggest that cepharanthine stimulates the pituitary-adrenotropic function. PMID- 3009290 TI - [IFN production by Epstein-Barr virus in human mononuclear leukocytes and suppression of the virus-induced transformation by the endogenous interferon]. AB - This study was undertaken to see whether or not interferon (IFN) is produced in infection with Epstein-Barr virus (EBV) and whether or not the IFN suppresses the transformation by EBV. When mononuclear leukocytes were infected with B95-8 EBV, IFN level reached a maximum 24 h after exposure to the virus, and a gradual decrease followed. There were apparent correlations between EBV inoculum size and IFN titer and between cell density and IFN titer. IFN was also induced by UV inactivated EBV and heat-inactivated EBV, but not by neutralized virus. IFN inducibility varied among donors, but there was no significant difference between EBV-seropositive and -seronegative groups. Cord blood leukocytes, however, responded lower than adults. EBV induced IFN in not only EBV-susceptible B cells but also non-susceptible T and NK cells. The activities of all IFN in this study were stable to acid and heat and exclusively neutralized by anti-human IFN-alpha. IFN production was probably generated by non-specific contact of EBV with the surface of mononuclear leukocytes in a dose-dependent fashion. The efficiency of EBV-transformation was significantly increased by neutralization of the endogenous IFN-alpha with anti-human IFN-alpha. However, there was no difference in appearance of EBV nuclear antigen (EBNA) positive cells at 24 h after EBV infection on incubation with or without IFN-alpha. The transformation of EBV seropositive adult B cell fraction was not suppressed even by a high dose of exogenous IFN-alpha as compared with whole mononuclear leukocytes. These data indicate that endogenous IFN suppresses the EBV-transformation not by direct anti neoplastic activity to B cells but by augmentation of T cell and NK cell activities. PMID- 3009292 TI - Acute effects of desipramine and clomipramine on pituitary-adrenal axis in man. AB - Brain neurotransmitters play an essential role in central regulation of hypothalamic factors which stimulate or inhibit the secretion of pituitary hormones. Insight in this complex system might be obtained by analysing changes in pituitary and peripheral hormone secretion following the administration of neuroactive drugs which influence the action of neurotransmitters. Desipramine is well-known to inhibit presynaptic norepinephrine reuptake, clomipramine on the other hand interferes with the serotoninergic system. In 15 male volunteers, the effects of single-dose administration of each drug were studied in comparison to placebo. Basal concentrations of ACTH and cortisol, as well as the rise of both hormones following insulin-induced hypoglycemia, were studied. Basal cortisol values and the response to hypoglycemia were not affected by either pharmacon in this study. Slight differences could be seen in the ACTH responses, which were however not significant. PMID- 3009291 TI - [Experimental viral labyrinthitis--an immunohistochemical investigation of the cochlear lesion]. AB - Pathogenesis of viral labyrinthitis is still poorly understood despite the dimension of clinical problem. This study was undertaken in order to elucidate the pathogenesis of viral labyrinthitis by the immunohistochemical investigations of infected cochleas. HVJ (Sendai virus) and mumps virus have been used to create an animal model of viral labyrinthitis. The cochleas of adult guinea pigs with intralabyrinthine inoculation of virus through the round window membrane and with intravascular injection of virus were investigated by histopathological and immunohistochemical techniques (immunofluorescent and Avidin-Biotin-Peroxidase Complex method). Control animal received only the culture medium. Histopathologically, fibrosis of scala tympani, vacuolization of stria vascularis, cell infiltration in perilymph were observed in the animals with intralabyrinthine inoculation, whereas no significant changes were observed in those with intravascular injection. Immunohistochemical studies revealed that viral antigen of HVJ was found in stria vascularis (6/11), Reissner's membrane (5/11), Organ of Corti (2/11) and viral antigen of mumps was found in stria vascularis (3/11), Reissner's membrane (1/11), Organ of Corti (1/11) in inoculated side of cochlea. In animal with intravascular injection, viral antigen of HVJ was found in stria vascularis (4/6), Organ of Corti (1/6) and viral antigen of mumps was found in stria vascularis (5/18), Organ of Corti (2/18). Cochleas of control animal showed no evidence of viral antigen. Viral antigen of both viruses were found in stria vascularis, Reissner's membrane, and Organ of Corti. Of these, Stria vascularis showed stronger affinity to these viruses. The results of intravascular injection indicate that endolymphatic labyrinthitis may ensure as a result of viremia. PMID- 3009293 TI - Disparate uptake of 99mTcO4- and 125I- in thyroid cells in culture. AB - Pertechnetate (99mTcO4-) is a large anion that is thought to be trapped but not organified by the thyroid. In order to determine its usefulness in discriminating trapping from organification in thyroid cell culture, the uptake of 99mTcO4- has been compared to that of 125I- both in sheep thyroid cells and in FRTL5 cells, a functional rat thyroid cell line. We found that uptake of both isotopes was dependent on TSH or agents that raised intracellular cAMP levels and was displaceable with iodide in both cell types. However in rat cell line 99mTcO4- uptake was up to eight-fold higher than 125I- uptake, and 99mTcO4- but not 125I- uptake was doubled by methimazole treatment. A considerable proportion of 99mTcO4 but not 125I- taken up by the FRTL5 cell but not the sheep cells was precipitable with TCA. This proportion was increased by methimazole treatment. We also found marked differences in sensitivity to ouabain between the two cell types. These results illustrate the differences between species in 125I- and 99mTcO4- transport and support the concerns expressed by Socolow and Ingbar (1967) in using 99mTcO4- as a direct and precise indicator of iodide transport activity. PMID- 3009294 TI - Human T-cell leukemia virus: causative roles in development of adult T-cell leukemia and provirus integration into leukemic cell DNA. PMID- 3009295 TI - Familial disposition of adult T-cell leukemia and lymphoma. AB - Sixteen families, each with two or more cases of adult T-cell leukemia or lymphoma were found in the Nagasaki district. Eight of the families had a parent with lymphoma. In the other eight families siblings were involved. Four families with sibling cases are presented in detail. Antibody titres to adult T-cell leukemia associated antigen (ATLA) in cases of ATL, CTL, T-CLL, and pre-ATL cases in Nagasaki were all positive. Of the non-leukemic T-cell malignant lymphoma 62.5 per cent were positive for antibody. The positive rate in healthy spouses and siblings of ATLL patients for ATLA antibody was high (67.5 per cent and 40 per cent respectively). The possibility of ATLV infection through spouses or from mother to child and the meaning of the high familial incidence of ATLL is discussed. PMID- 3009296 TI - Molecular approach to adult T-cell leukemia. PMID- 3009297 TI - Malignant lymphomas in Japan: epidemiological analysis on adult T-cell leukemia/lymphoma. AB - According to the Vital Statistics Japan Series in 1950-1981, the age-adjusted death rate for malignant lymphomas (ICDs, 200-202) in Japan has increased in recent years. A geographical clustering of malignant lymphomas in the Kyushu districts was observed both in the period 1969-1971 and more recently in 1979 1981. The excessive rate of malignant lymphomas in Kyushu was due to the high incidence of adult T-cell leukemia/lymphoma (ATLL). The distribution of healthy carriers of ATLV, a human retrovirus which is almost identical to HTLV, was closely related to that of patients with ATLL in Japan. Epidemiological features of the infectious mode of ATLV suggested two main routes of natural transmission, one from mother to child and the other from husband to wife. ATLV is considered to be a main causative agent of ATLL from virological and epidemiological evidence, but infection by this virus alone may not result in ATLL because the incidence of ATLL among ATLV carriers was apparently not very high even in endemic areas of ATLL. Thus, some risk factors other than ATLV, such as environmental and genetic factors, may contribute to the development of ATLL. PMID- 3009298 TI - Lymphoma type adult T-cell leukemia--a clinicopathologic study of HTLV related T cell type malignant lymphoma. AB - The clinical and pathological features of T-cell type malignant lymphoma related to human T-cell leukemia virus (HTLV) were investigated in eight patients presenting lymphadenopathy. Biopsy of lymph nodes showed an histology of diffuse non-Hodgkin's lymphoma. All patients were positive for anti-ATLA antibody and HTLV proviral DNA in the lymph node cells. Most patients showed pronounced hypercalcemia and high serum levels of lactic dehydrogenase. All patients died between 3 and 17 months (mean 8 months) after the onset of disease. HTLV-related malignant lymphoma should be added to the spectrum of ATL, being classified as a lymphoma type ATL. PMID- 3009299 TI - Virus associated adult T-cell leukemia (ATL) in Japan: clinical, histological and immunological studies. AB - Virus associated adult T-cell leukemia/lymphoma (ATLL), which includes both adult T-cell leukemia (ATL) and its non-leukemic counterpart (NLATL) was studied clinically, histologically, and immunologically. The disease usually occurred in the sixth decade in both sexes equally. The patients had a rapid clinical course with frequent leukemic changes, lymphadenopathy, hepatomegaly, and occasional skin rash. Bone marrow involvement with mild infiltration and hypercalcemia were more frequent in ATL than in NLATL. Histologically the disease was categorized as malignant lymphoma, diffuse pleomorphic type with cerebriform nuclear giant cells. The lymphoma was characterized by diffuse proliferation of tumor cells with irregular nuclear configurations, varying in size and shape, and the presence of giant cells with highly convoluted cerebriform nuclei. The giant cells seemed to be a diagnostic marker. Immunologically, the tumor cells usually possessed the surface antigens recognized by OKT 3, 4, Leu 8 and anti-Tac antibodies, indicating that they were lymphomas of helper/inducer peripheral T cells with the receptor for interleukin 2, but they demonstrated no helper/inducer functions. The patients often died of opportunistic infections due to T-cell dysfunction caused by the disease itself and strong chemotherapy. PMID- 3009300 TI - Molecular characterization of HbH disease in the Cuban population. AB - Molecular characterization of the alpha-thalassemia mutations present in nine HbH subjects from Cuba was achieved by digestion with Bam HI, Bgl II, and Apa I and hybridization with alpha- and zeta-specific probes. The results show that the molecular basis of the genetic defect is quite homogeneous, all the subjects carrying the - alpha 3.7 type I/--SEA genotype. Variations are observed in the size of the zeta polymorphic fragments. PMID- 3009301 TI - Phenotypic variation in the phosphotransferase activity of human red cell acid phosphatase (ACP1). AB - Red cell acid phosphatase (ACP1) catalyses the transfer of phosphate from phosphate ester substrates to suitable acceptor alcohols such as methanol and glycerol. The rate of substrate turnover in the presence of acceptors is increased by the increment of the phosphotransferase reaction, thus allowing this activity to be measured. There is specificity with regard to acceptors: (a) polyols (e.g., glycerol) are better acceptors than the corresponding n-alcohols, and (b) polyol configuration and chain length determine acceptor activity. Ribitol was the most efficient acceptor found. Each of the three common ACP1 alleles is represented electrophoretically by two isozyme bands; the phosphotransferase activity of the anodal isozyme was found to be more than twice that of the cathodal isozyme. The extent of phosphotransferase activity is also genotype dependent. In the presence of 2 M glycerol, the relative phosphotransferase efficiencies for the three homozygote types were: ACP1 B = 3.7, ACP1 A = 3.4, and ACP1 C = 2.5. This pattern of B greater than A greater than C is the same as found for the modulation of ACP1 by purines and folates. PMID- 3009302 TI - Assignment of the gene for dyskeratosis congenita to Xq28. AB - Dyskeratosis congenita is an X-linked recessive disorder with diagnostic dermatological features, bone marrow hypofunction, and a predisposition to neoplasia in early adult life. Linkage analysis was undertaken in an extensive family with the condition using the Xg blood group and 17 cloned X chromosomal DNA sequences which recognise restriction fragment length polymorphisms (RFLPs). No recombination was observed between the locus for dyskeratosis congenita (DKC) and the RFLPs identified by DXS52 (St14-1) (Zmax = 3.33 at theta max = 0 with 95% confidence limits of 0 to 14cM). Similarly no recombination was observed for the disease locus and F8 (Zmax = 1.23 at theta max = 0) nor for DXS15 (Zmax = 1.62 at theta max = 0), but both of these markers were only informative in part of the family whereas DXS52 was fully informative. DXS52, DXS15, and F8 are known to be tightly linked and have previously been assigned to Xq28. Thus the gene for dyskeratosis congenita can be assigned to Xq28. These DNA sequence polymorphisms will be of clinical value for carrier detection and prenatal diagnosis. PMID- 3009304 TI - Six month evaluation of particulate Durapatite in extraction sockets for the preservation of the alveolar ridge. PMID- 3009303 TI - Use of T cell-specific anti-idiotypes to immunize against viral infections. PMID- 3009305 TI - UDPglucose pyrophosphorylase from Ehrlich ascites carcinoma cell--purification and characterization. PMID- 3009306 TI - Heme-mediated effect of cAMP on mitochondriogenesis during glucose repression derepression in Saccharomyces cerevisiae. PMID- 3009307 TI - Effect of wheat bran fibre on tissue lipids in diabetic rats. PMID- 3009308 TI - Cloning and expression of EcoRI specific restriction modification system. PMID- 3009310 TI - Fructose-1,6-bisphosphatase: Part I--Purification, properties and proteolytic modification of fish liver enzyme. PMID- 3009309 TI - Initiation of sporulation in Bacillus subtilis by cyclic-guanosine-3',5' monophosphate under condition of catabolite repression. PMID- 3009311 TI - Fructose-1,6-bisphosphatase: Part II--Purification and properties of the fish brain enzyme. PMID- 3009312 TI - Structure-Activity relationship study on angiotensin-converting enzyme inhibitors -investigation of hydrophobic interaction in inhibition mechanism. PMID- 3009314 TI - Roles of internally and externally supplied cyclic AMP in the growth response of Escherichia coli to NaCl. PMID- 3009313 TI - Separation of early chick embryonic cells by Percoll density gradient centrifugation and their characterization. PMID- 3009315 TI - FSH binding to granulosa cells of normal and atretic follicles of mouse ovary in vivo and in vitro. PMID- 3009316 TI - Tests of association of immunoglobulin allotype genes and viral oncogenesis in chickens. AB - Chickens from Regional Poultry Research Laboratory (RPRL) inbred line 6(3) are resistant to virally-induced Marek's disease (MD) and lymphoid leukosis (LL) and are relatively strong regressors of virally-induced Rous sarcomas. In contrast, RPRL line 100 chickens are highly susceptible to MD and LL and are weaker regressors of Rous sarcomas than line 6(3). RPRL lines 100 and 6(3) differ for alleles at the IgG-1 (G-1) allotype locus, but have identical IgM-1 (M-1) allotype alleles. To test the possible association of the G-1 locus with variations in resistance to virally-induced tumors, homozygous and heterozygous genotypes among F3 crosses were infected. F3 chickens with different G-1 types were comparable in their resistance to MD tumors following inoculation with the JM strain of the MD virus, and for their ability to regress Rous sarcoma tumors induced by the Rous sarcoma virus (RSV) RAV-1. However, following RAV-1 virus infection a smaller proportion of G-1a/G-1aF3 or F4 birds developed LL tumors than G-1a/G-1e and G-1e/G-1e birds. Genes determining immunoglobulin heavy chains were therefore associated with a recessive resistance to B-cell lymphomagenesis in chickens. PMID- 3009318 TI - C4B gene polymorphism detected in a human cosmid clone. PMID- 3009317 TI - Polymorphism of the HLA DR1 haplotype in the Israeli population investigated at the serological, cellular, and genomic levels. AB - In the present report, we used serological, cellular, and restriction fragment length polymorphism (RFLP) to investigate the DR1 haplotype in the Israeli population. We describe an Israeli homozygous typing cell (HTC), HLA-Dw"LVA", which defines a new lymphocyte-activating determinant associated with Bw65, DR1 and distinct from Dw1. The parents of this donor, non-Ashkenazi Algerian Jews, are first cousins and share HLA-Cw8,Bw65,BfS,DR1,DQw1,DPw4. No specificity could be assigned to HLA-Dw"LVA" using the 91 Ninth Workshop HTCs. Two families and forty unrelated DR1 individuals were studied with Dw"LVA" and a panel of DR1/Dw1 HTCs. HLA-Dw"LVA" showed segregation as a single determinant within families. This new specificity was present in 24 out of 40 (60%) unrelated DR1 individuals, indicating that in the Israeli population Dw"LVA" is the main lymphocyte-defined determinant associated with the serologically defined DR1 specificity, in contrast to non-Jewish Caucasoids where DR1 is significantly associated with Dw1. The vast majority of Dw"LVA"-positive carriers were also Bw65 carriers, indicating that Bw65,DR1, Dw"LVA" may represent a typical allele combination in the Israeli population. The RFLP analysis established the correlation of certain RFLPs with Dw1 and Dw"LVA". In addition, we describe a cluster of FRLPs that may correspond to a new Dw subtype associated with DR1, for which no serological and cellular reagents have been described so far. PMID- 3009319 TI - HLA and gastrointestinal disorders. PMID- 3009320 TI - Enterovirus-70 antigen in spinal cord cells of patients with poliomyelitis-like illness. PMID- 3009321 TI - Chromosomal aberrations & sister chromatid exchanges in relation to herpes simplex virus antibody in women with cervical dysplasia. PMID- 3009322 TI - Malignant fibrous-histiocytoma of bone. A case report. PMID- 3009323 TI - A multiple malformation syndrome with ankyloblepharon filiforme adnatum, with cleft lip and palate. PMID- 3009325 TI - Dopamine selectively inhibits aldosterone responses to angiotensin II in humans. AB - Previous studies have suggested that dopamine may have an important role as an inhibitor of aldosterone secretion in humans. Recent studies have also suggested that the adrenergic nervous system may have an important role in controlling aldosterone secretion. The present study investigated the effects of dopamine on aldosterone secretion in response to angiotensin II, with and without pretreatment with propranolol, and to adrenocorticotropic hormone, another known stimulator of aldosterone secretion. Nine normal subjects in balance at 10 mEq sodium intake received dopamine (4 micrograms/kg/min) or vehicle for 270 minutes on 2 consecutive days on three separate occasions. After 120 minutes of dopamine infusion, the subjects received a 30-minute intravenous infusion of angiotensin II (in cumulative doses of 0.5, 1, 2, 4, and 6 pmol/kg/min), angiotensin II after oral pretreatment with propranolol, or adrenocorticotropic hormone (in cumulative doses of 0.5, 1, 2, and 5 U/hr). Aldosterone responses to 2, 4, and 6 pmol/kg/min of angiotensin II (without propranolol) were greater in vehicle-treated than in dopamine-treated subjects (p less than 0.05), as was the slope of the angiotensin II-vehicle dose-response curve (0.46, p less than 0.05). Propranolol suppressed the aldosterone response to angiotensin II, but dopamine still inhibited the response. Aldosterone and cortisol secretion were stimulated equally by adrenocorticotropic hormone in dopamine-treated and vehicle-treated groups. These results suggest that dopamine selectively inhibits the aldosterone response to angiotensin II and that this response is not mediated by teh activity of dopamine at beta-adrenergic receptors. PMID- 3009324 TI - Adrenocorticotropin responses to corticotropin releasing factor and vasopressin in spontaneously hypertensive rats. AB - The effects of exogenous corticotropin releasing factor and arginine vasopressin were evaluated in 6- and 11-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Basal adrenocorticotropic hormone (ACTH) and vasopressin levels did not differ between SHR and WKY, but basal corticosterone level was higher in 6-week-old SHR (p less than 0.01). To block endogenous corticotropin releasing factor secretion and nonspecific systemic responses, both groups were pretreated with chlorpromazine, morphine, and sodium pentobarbital anesthesia before measurement of ACTH responses to corticotropin releasing factor and vasopressin infusion. Basal ACTH level was lower in anesthetized 6-week-old SHR than in age-matched WKY (p less than 0.01), but no difference was seen between 11-week-old WKY and SHR. The ACTH response to corticotropin releasing factor in 6-week-old WKY was significantly greater than that in age-matched SHR (p less than 0.01), whereas in 11-week-old SHR and WKY the response was similar. Compared with responses in WKY, SHR showed an increased ACTH response to high doses of vasopressin (0.25 micrograms/100 g body weight) at both ages (p less than 0.05). These results indicate that the ACTH response to corticotropin releasing factor is blunted in the early stages of hypertension in SHR but later recovers. These abnormal responses to corticotropin releasing factor and vasopressin may be related to the development of spontaneous hypertension. PMID- 3009327 TI - Nonpharmacological approaches to the control of high blood pressure. Final report of the Subcommittee on Nonpharmacological Therapy of the 1984 Joint National Committee on Detection, Evaluation, and Treatment of High Blood Pressure. AB - This report reviews a variety of nonpharmacological approaches used to control arterial blood pressure. Of all the modalities considered, only three had sufficient scientific support to warrant recommendation for inclusion in hypertension treatment programs. Each of these three modalities--weight control, alcohol restriction, and sodium restriction--was found to be capable not only of independently controlling blood pressure (particularly in patients with mild hypertension) but also of reducing the number and dosage of prescribed pharmacological agents, should their prescription be indicated. Weight reduction was found to reduce the risk from elevated arterial pressure as well as overall cardiovascular morbidity and mortality. However, because the rate of recidivism was exceedingly high in these studies, close and continuous patient follow-up is considered necessary. Excessive alcohol intake is associated in many studies with proportionally higher arterial pressures and an increased prevalence of hypertension. Therefore, the recommendation of moderation in alcohol consumption to less than 2 oz of ethanol daily for patients with hypertension is supported. Restriction of dietary sodium to less than 2 g/day was the only other nonpharmacological approach with sufficient support to be recommended as a treatment for hypertension. Although long-term studies are sorely lacking, sodium restriction has been shown to be manageable and safe and probably will benefit those hypertensive patients who are sodium-sensitive. PMID- 3009326 TI - Effects of converting enzyme inhibition on split renal function in renovascular hypertension. AB - The effects of captopril on effective renal plasma flow and glomerular filtration rate were studied using a noninvasive radioisotopic method on individual kidneys in eight patients with renovascular hypertension and 12 patients with essential hypertension with various renin levels. Four patients with renovascular hypertension had unilateral while three had bilateral renal artery stenosis. The effective renal plasma flow and glomerular filtration rate were determined by using 131I-iodohippurate sodium and 99mTc-diethylenetriamine pentaacetic acid, respectively. Glomerular filtration rate and effective renal plasma flow were significantly reduced in the stenotic kidneys of patients with renovascular hypertension compared with values in nonstenotic kidneys (p less than 0.01). Treatment with captopril, 37.5 to 75 mg/day for 1 to 48 weeks, further reduced the glomerular filtration rate only in stenotic kidneys, and effective renal plasma flow increased in both kidney types. In two of the three renal hypertensive patients with bilateral renal artery stenosis, captopril produced a reversible azotemia that was unrelated to the fall in blood pressure, as evidenced by the lack of azotemia seen after a moderate blood pressure reduction induced by other antihypertensive medications. These results indicate that endogenous angiotensin II is essential in maintaining the glomerular filtration rate in stenotic kidneys and suggest that a reduction in glomerular filtration rate during captopril administration could indicate the presence of renal artery stenosis. PMID- 3009328 TI - Cytomegalovirus disease. PMID- 3009330 TI - Immunity to heat-labile enterotoxins of porcine and human Escherichia coli strains achieved with synthetic cholera toxin peptides. AB - Antiserum to a synthetic peptide, CTP3, consisting of residues 50 to 64 of the B subunit of cholera enterotoxin (CT), a sequence which is conserved in heat-labile enterotoxins from Escherichia coli strains of human (H-LT) or porcine (P-LT) origin, cross-reacted with and neutralized all three enterotoxins. It also cross reacted with genetically engineered chimeric B subunit proteins in which H-LT amino acids had been substituted for the corresponding residues in P-LT. Antisera against another CT peptide, CTP1, consisting of residues 8 to 20, were weakly cross-reactive with H-LT but not with P-LT, whereas antisera against four other CT peptides were not cross-reactive with the heterologous enterotoxins. A single dose of CTP3-linked tetanus toxoid, by itself nonimmunogenic, primed rabbits to a vigorous immune response to subsequent administration of single, small, subimmunizing doses of any of the three enterotoxins. PMID- 3009329 TI - Molecular studies of Pseudomonas exotoxin A gene. AB - A 2.7-kilobase DNA fragment carrying the entire exotoxin A (ETA) structural gene was divided into three nonoverlapping probes. Two probes covering the ETA structural gene were used in colony hybridization experiments to determine whether sequences homologous to the ETA gene could be detected in genera other than Pseudomonas or in Pseudomonas species other than Pseudomonas aeruginosa. The majority of strains examined other than the P. aeruginosa strains failed to react in the colony hybridization assays. Some Pseudomonas spp. other than P. aeruginosa and some Bordetella spp. did react in colony hybridization assays with the probes. However, additional studies in which we used Southern hybridization methods indicated that these reactions were apparently nonspecific and that the ETA gene is limited to P. aeruginosa. Studies in which we used all three ETA related probes in Southern hybridization experiments to analyze the ETA gene and surrounding sequences in P. aeruginosa strains isolated from diverse sources revealed the following: (i) the incidence of the ETA gene in P. aeruginosa is approximately equal to 95%; (ii) there are strains which have been isolated from human infections that do not carry the ETA structural gene; (iii) there is a maximum of one copy of the ETA gene per genome in any given strain; (iv) sequences within and 4 to 5 kilobases downstream of the ETA structural gene appear to be well conserved in different strains of P. aeruginosa; and (v) in contrast, sequences immediately upstream of the ETA structural gene are considerably rearranged from strain to strain. A multicopy plasmid carrying the entire cloned ETA gene was transferred to a tox- P. aeruginosa strain. This strain synthesized and secreted mature, full-length ETA, but the amount produced was small considering the multicopy nature of the plasmid. Synthesis of toxin in this strain was only minimally affected by iron. Our data suggest that the synthesis of ETA is positively regulated. Finally, we found that the presence of the ETA gene is independent of the ability of P. aeruginosa to produce several other recognized virulence factors, supporting the concept of the multifactorial nature of P. aeruginosa virulence. PMID- 3009331 TI - Detection of IgG and IgA antibodies to Epstein-Barr virus membrane antigen in sera from patients with nasopharyngeal carcinoma and from normal individuals. AB - IgG and IgA antibodies to Epstein-Barr virus (EBV) membrane antigen (MA) were detected in sera from 96 NPC patients and normal individuals by the indirect immunofluorescence test. For MA/IgG antibody, 100% of NPC patients were positive with a GMT of 1:439.7 and 97.9% of normal individuals were positive with a GMT of 1:94.7. In contrast, for MA/IgA antibody, 58.3% of NPC patients were positive with a GMT of 1:7.3 and none of the normal individuals were positive. There was no difference in the detection of antibodies to EBV MA when other P3HR-1 or B95-8 cell lines, differing in their major membrane antigen, were used. PMID- 3009332 TI - Biochemical regulation of adenylate cyclase in murine melanoma clones with different metastatic properties. AB - The regulation of adenylate cyclase in murine melanoma tumor cell clones with different metastatic capacities has been studied in intact cells and isolated membrane preparations. Analysis of the responses of intact cells from a number of B16 melanoma clones revealed that treatment with melanocyte-stimulating hormone (MSH) or the diterpene, forskolin, produced significantly greater accumulation of intracellular cyclic adenosine 3',5' monophosphate (cAMP) in strongly metastatic clones than in weakly metastatic tumor cell clones. In contrast, in isolated membranes from the same panel of clones, the extent of activation by forskolin but not by MSH correlated with metastatic capacity. Sodium fluoride and 5'-guanyl beta-gamma-imidodiphosphate [Gpp(NH)p] also stimulated adenylate cyclase in isolated membranes but the extent of activation did not correlate with the metastatic behavior of the donor cells. A combination of forskolin and Gpp(NH)p proved to be a sensitive prospective indicator for identifying differences in the metastatic capabilities of individual B16 melanoma clones. Adenylate cyclase in membrane preparations from strongly metastatic B16 clones displayed synergistic activation but stimulation of the enzyme from weakly metastatic clones was less than additive. To test the generality of these findings, similar investigations were performed on B16-BL6 melanoma cells, a highly invasive subline of the B16 melanoma, and the K1735, an ultraviolet-light-induced murine melanoma arising in a different mouse strain (C3H). Consistent with their high metastatic potential, clones derived from the B16-BL6 melanoma displayed elevated levels of hormonally stimulated adenylate cyclase, thereby confirming, for this tumor system, a close association between hormonal responsiveness and metastatic capacity. In contrast, K1735 melanoma cell clones exhibited significant interclonal variation in adenylate cyclase activity and metastatic performance, but no consistent relationship between the two traits was detected. Differences in the regulation and/or the intrinsic catalytic capacity of adenylate cyclase may account, at least in part, for the variation in hormonal responsiveness observed among B16 clones with distinct metastatic properties and suggest that cAMP-dependent molecular processes may be required for the expression of B16 melanoma experimental metastatic potential.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3009333 TI - Transformation of hamster spleen lymphocytes by human T-cell leukemia virus type I. AB - Co-cultivation of spleen cells of Syrian golden hamsters with lethally irradiated MT-2 cells harboring human T-cell leukemia virus type I (HTLV-I) resulted in the establishment of lymphoid cell lines, HCT-1 and HCT-2, which exhibited the normal karyotype of golden hamsters. Cells of both the HCT-1 and HCT-2 lines lacked surface immunoglobulins and reacted with a monoclonal antibody (MAb) specific for hamster T cells. Some were positive for OKIa1. None of them expressed HTLV structural antigens (p19 and p24) or virus particles, but they contained HTLV-I proviral DNA monoclonally. By immunochemical analysis of the labelled cell antigens, sera from adult T-cell leukemia (ATL) patients reacted with the two polypeptides, p37 and p40, which may not be viral structural proteins and still remain to be characterized. HCT-1 and HCT-2 cells were transplantable into newborn hamsters, pre-treated with anti-hamster thymocyte serum and non-treated, respectively, producing diffuse malignant lymphoma. These findings indicated that HTLV-I not only immortalized but also transformed hamster T cells non productively. PMID- 3009336 TI - Unilateral expression of multiple glomus tumors. An unusual occurrence. PMID- 3009334 TI - Overview of drugs used in treating drug-induced dependence: a treatise interrelating existing hypotheses in order to attain maximal therapeutic benefits. AB - Despite the fact that we do not yet completely understand the physiology of physical dependence, many treatment modalities are available. In this overview, an attempt is made to discuss available treatments and mechanisms to possibly attain a better understanding of the complex interactions that occur between systems. An attempt is also made to understand interrelations between drug misuse and mental disorders as one aspect of the treatment process--the ultimate goal being more efficacious and safer therapy. PMID- 3009335 TI - Malignant fibrous histiocytoma. PMID- 3009337 TI - Epidermolysis bullosa. PMID- 3009339 TI - Phosphatidylserine synthesis in adult Dirofilaria immitis females. PMID- 3009338 TI - Epidermolysis bullosa. Clinical aspects, pathology, and recent advances in research. PMID- 3009340 TI - Cystamine-enkephalin dimer. Syntheses and biological activities of enkephalin analogs containing cystamine and cysteamine. AB - A cystamine-enkephalin dimer, containing two molecules of [D-Ala2, Leu5] enkephalin cross-linked at the COOH-terminal leucine residue with cystamine, (NH2 CH2-CH2-S-)2, has been synthesized in order to examine directly the dimerization effect of an enkephalin molecule on the opiate receptor interactions. In a comparison of potencies against [3H]-[D-Ala2,D-Leu5] enkephalin (3H-DADLE) and [3H]-[D-Ala2,MePhe4,Gly-ol5] enkephalin (3H-DAGO) as delta and mu tracers, respectively, enkephalin dimer showed a very high affinity, especially for the delta opiate receptors. Dimer was almost threefold more potent than DADLE, which is one of the most utilized delta ligand to date. When the binding affinity of cystamine-dimer was compared with that of its reduced thiol-monomer, namely [D Ala2,Leu5,cysteamine6] enkephalin, the increment in affinity was four to fivefold for both delta and mu receptors. The results strongly indicate that the dimeric enkephalin is more potent presumably due to the simultaneous interaction with the two binding sites of the opiate receptors. PMID- 3009341 TI - Total synthesis of S-carbamoylmethyl bovine apocytochrome c by segment coupling. AB - Three peptide segments corresponding to the complete sequence of the 104 amino acid protein bovine apocytochrome c were synthesized by the solid-phase method. The peptides Ac-[Cys(Cam)14,17, GlyS23]-apocytochrome c-(1-23) (I), CF3CO [GlyS60]-apocytochrome c-(24-60) (II), and CF3CO-apocytochrome c-(61-104) (III) were purified by chromatography on CM-cellulose, partition chromatography and/or HPLC. Each of the peptides was reacted with citraconic anhydride to block all of the lysine side chains, and the 61-104 peptide was treated with 10% hydrazine to remove the trifluoroacetyl group, to give the corresponding peptides Ia, IIa, and IIIa. Peptides IIa and IIIa were coupled together by reaction with silver nitrate/N-hydroxysuccinimide to give the 24-104 sequence. After removal of the trifluoroacetyl group from the amino terminus, peptide Ia was also coupled. Treatment of the peptide mixture with aqueous acetic acid removed the citraconyl groups, and purification by chromatography on CM-cellulose and HPLC gave a 0.6% yield of [Cys(Cam)14,17]-apocytochrome c. The synthetic product was shown to be identical to a sample derived from native bovine cytochrome c by paper or gel electrophoresis, HPLC and by chymotryptic or tryptic map. PMID- 3009342 TI - Membrane effects of ionizing radiation and hyperthermia. AB - Results of numerous studies demonstrate that membranes are important sites of cell damage by both ionizing radiation and hyperthermia. Modification of membrane properties (mainly lipid fluidity) affects the cellular responses to radiation and hyperthermia but former concepts that membrane rigidification sensitizes cells to radiation while membrane fluidization potentiates hyperthermic damage have now been seriously challenged. It seems that the effects of membrane fluidity on cell responses to hyperthermia and radiation are due to an indirect influence on functional membrane proteins. The major role of lipid peroxidation in radiation damage to membranes has also been questioned. The existing evidence makes it unlikely that the interaction between radiation and hyperthermia is determined by the action of both agents on the same membrane components. PMID- 3009344 TI - OH-induced free radicals in purine nucleosides and their homopolymers: e.s.r. and spin-trapping with 2-methyl-2-nitrosopropane. AB - Free radicals produced by X-irradiation of N2O-saturated aqueous solutions of purine nucleosides (2'-deoxyadenosine, adenosine, 2'-deoxyguanosine, 3' deoxyadenosine, guanosine and inosine) and the corresponding homopolymers (poly A and poly I) have been investigated by the technique of spin-trapping and e.s.r. spectroscopy. 2-Methyl-2-nitrosopropane was used as a spin-trap. For 2' deoxyadenosine and 2'-deoxyguanosine, the resulting spin-adducts were separated by Bio-Gel P-2 column chromatography and analysed by e.s.r. spectroscopy. For homopolymers, e.s.r. spectra were recorded at 50 degrees C after enzymatic digestion to obtain signals with narrower line width. The e.s.r. signal consisting of only a primary triplet without further splittings, which is consistent with assignment to the trapping of an H-abstraction radical at the C4' position of the sugar moiety, was observed in all cases. For 2'-deoxyguanosine an e.s.r. signal consisting of a secondary triplet was observed. Examinations using other spin-trapping reagents such as PBN, 4-PyOBN and DMPO provided no positive evidence supporting the proposal that this was due to an alpha-nitrogen. The e.s.r. signal consisting of a secondary doublet which further splits into a doublet was observed for 2'-deoxyadenosine, adenosine, 3'-deoxyguanosine, 2' deoxyguanosine, and inosine, and tentatively associated with a radical centered in the sugar moiety. PMID- 3009343 TI - The effects of benzamide ADP-ribosyl transferase inhibitors on cell survival and DNA strand-break repair in irradiated mammalian cells. AB - We have recently shown that 3-acetamidobenzamide (3-AAB), a highly effective inhibitor of ADP-ribosyl transferase (ADPRT), can act as a post-irradiation (electrons) sensitizer on the mouse lymphoma cell lines L5178Y R and S. We have now shown that this compound sensitizes human derived skin fibroblasts but to a lesser extent. Fibroblasts derived from normal, Friedreich's ataxia, and ataxia telangiectasia individuals were equally sensitized by 3-AAB to electron radiation. 3-AAB was also effective in sensitizing the mouse lymphoma lines to fast neutron irradiation. In addition DNA strand break repair was retarded as had been found after electron irradiation. 3-Nitrobenzamide is structurally a potentially dual action radiation sensitizer with electron affinic and ADPRT inhibitory properties. It is a weaker inhibitor of ADPRT compared to 3-AAB, and results in a smaller sensitization of mouse lymphoma cells in air. However, a much greater sensitization is achieved in anoxia. This greater sensitization appears to be a synergistic rather than an additive combination of its electron affinic and ADPRT inhibitory properties. PMID- 3009345 TI - Alterations in biodistribution of [3H]Ro 15-1788 in mice by acute stress: possible changes in in vivo binding availability of brain benzodiazepine receptor. AB - The biodistribution of [3H]Ro 15-1788 in control and stress-loaded mice (forced swimming) was compared. In control mice, carrier-free [3H]Ro 15-1788 was selectively and highly distributed in Bz receptor rich brain regions, while radioactivity in the brain was very low following administration of carrier-added tracer, which suggested that in vivo non-specific binding of this tracer was very low. Significant changes in biodistribution of carrier-free [3H]Ro 15-1788 were observed in stress-loaded mice, which strongly indicated that in vivo binding availability of Bz receptor in the brain was rapidly and reversibly reduced by acute stress. The degree of these changes was very dependent upon the stressful conditions, such as swimming duration and water temperature, and a significant alteration in biodistribution of [3H]Ro 15-1788 was particularly observed in the cerebral cortex. Simplified Scatchard analysis of in vivo binding of this tracer was performed, and results suggested that these alterations were mainly caused by changes in the Kd value rather than the Bmax value. PMID- 3009346 TI - Comparative aspects of the production of oxygen radicals by phagocytic cells, and aspects of other effector substances. AB - The author reviews the information gleaned from a great variety of sources on the factors influencing the production of oxygen radicals by phagocytic cells, with reference to inflammation. He emphasizes the value of comparative studies, mentioning particularly the effect of leukocyte perturbants, fatty acids and somnogenic leukocyte modulators. PMID- 3009347 TI - CDC Guideline for Handwashing and Hospital Environmental Control, 1985. PMID- 3009349 TI - Intravascular death of disseminated cancer cells mediated by superoxide anion. AB - The release of radiolabelled Lewis lung carcinoma cells arrested in the vasculature of the major organs of mice after intravenous injection was significantly inhibited in animals treated with the oxygen radical scavenger, superoxide dismutase. There was a parallel increase in the numbers of tumor nodules which developed after injection of non-radiolabelled cells into superoxide dismutase-treated mice. The results indicate that superoxide anion plays an important role in the lethal release of arrested cancer cells, either directly since the radical is cytotoxic or possibly indirectly since its metabolites are also highly toxic. It is suggested that a major source of toxic oxygen species is the polymorphonuclear leukocyte. PMID- 3009348 TI - Hepatocarcinogenesis in the rat: the effect of promoters and carcinogens in vivo and in vitro. PMID- 3009350 TI - Refractile bodies in the inner segments of cones in the aging human retina. AB - Refractile inclusion bodies (approximately 0.80 micron in size) were found in the inner segments of cone photoreceptors in the aging human retina. They were easily resolved with the light microscope. They were never seen in rods, and occurred primarily in retinas from eye donors older than 40 years of age. The incidence of these inclusion bodies is related significantly to age (they occur more frequently with increasing age) and to sex (they are more likely to occur in the aging female than in the aging male). They were often smaller in size and fewer in number in the cones of males compared with females, and in males, fewer cones contained the RB than in females. Electron microscopy revealed that these inclusions are membrane-bound organelles having granular, fibrous, and tubular subcomponents. The occurrence of the RB appears to be unrelated to specific disease processes, medications in use at the time of enucleation, or specimen preparation times. PMID- 3009352 TI - Inhibition of Banzi viral RNA synthesis in BHK-21 cells by Banzi virion matrix protein. AB - Treatment of BHK-21 cells with purified matrix (M) protein isolated from Banzi virions resulted in decreased production of Banzi virus and in reduced levels of virus-specific positive-strand and negative-strand RNA. The M protein had no effect on the uncoating of the viral genome. This inhibition was virus-specific since the replication of unrelated togaviruses in BHK-21 cells was not affected. Purified M protein inhibited the in vitro activity of the Banzi viral RNA polymerase; prior treatment of the M protein with either trypsin or antiviral serum blocked this inhibition. PMID- 3009351 TI - Monoclonal antibodies to genus- and type-specific papillomavirus structural antigens. AB - Monoclonal antibodies against SDS-disrupted bovine papillomavirus type 1 (BPV1) were obtained from hybridomas prepared by fusing mouse myeloma cell line P3X63Ag8U1 with spleen cells from immunized BALB/c mice. Six hybridoma cell lines were obtained after testing supernatant fluids for positivity by the enzyme linked immunosorbent assay using the immunogen as antigen and by indirect immunofluorescence (IF) on frozen sections of BPV1-induced bovine fibropapillomas. Monoclonal antibodies (AU1-AU6) from these hybridomas were then tested for reactivity by IF tests on BPV2-induced fibropapillomas and on human plantar warts and vulvar condylomas and by avidin-biotin complex tests on sections of formalin-fixed cervical dysplasias. One monoclonal antibody (AU1) was reactive with all tissues, four (AU3-AU6) were reactive with both BPV1 and BPV2 fibropapillomas, and the remaining antibody (AU2) was only reactive with BPV1 induced fibropapillomas. All monoclonal antibodies reacted with the major capsid protein (mol. wt. 54,000) of BPV1, whereas five (AU1, AU3-AU6) reacted with the major capsid protein of BPV2. These results indicate that papillomavirus genus specific, cross-reactive, and type-specific antigenic determinants are located on the major capsid protein of BPV1. PMID- 3009353 TI - Administration of antithymocyte serum modifies the response of Calomys musculinus to junin virus infection. AB - Approximately 80% of Calomys musculinus inoculated with an attenuated strain of Junin virus (JV) developed a lethal encephalitis. Antithymocyte serum, a potent suppressor of T-cell-mediated immunity, was studied for its effect on JV pathogenicity. Early administration of an anti-C. musculinus thymocyte serum (ACTS) to neonatal animals significantly diminished clinical disease and death and abrogated brain damage, which is usually associated with viral presence in the brain. Late ACTS administration did not modify the pattern of JV infection. These results suggest that immune mechanisms participate in the pathogenesis of JV infection for its main natural host. PMID- 3009354 TI - Inhibition of human neutrophil function by 6-aminonicotinamide: the role of the hexose monophosphate shunt in cell activation. AB - Stimulation of polymorphonuclear leukocytes with phorbol myristate acetate (PMA) or chemotactic factors such as f-Met-Leu-Phe (fMLP) activates a membrane oxidase which results in the generation of the superoxide anion (O2-) and the oxidation of NADPH to NADP+. The subsequent reduction of NADP+ to NADPH is believed to be directly dependent upon activation of the hexose monophosphate shunt (HMPS). To further understand the role of the HMPS in the oxidative burst, we examined the kinetics of HMPS activation by fMLP and PMA. Both of these agents stimulate an increase in HMPS activity that parallels their production of O2-. To examine the role of the HMPS in cell activation, we treated polymorphonuclear leukocytes with the specific HMPS inhibitor, 6-aminonicotinamide. This pretreatment inhibited fMLP- and PMA-stimulated HMPS activity and O2- release by 80% and 60% respectively with a 50% inhibitory dose (ID50) of 5 X l0(-7)M. Measurement of reduced NADPH using 350 nm ultraviolet light-stimulated fluorescence and flow cytometry indicated that 6-aminonicotinamide had no effect on resting levels of NADPH fluorescence but significant inhibited the fluorescence recovery following stimulation with fMLP or PMA. In contrast, PMA- and fMLP-stimulated membrane depolarization measured with the carbocyanine dye 3,3'-dihexyloxacarbocyanine iodide and chemotaxis to fMLP were unaffected by 6-aminonicotinamide treatment. On the contrary, fMLP- or PMA-stimulated myeloperoxidase release by fMLP or PMA was enhanced by 30% and 150%, respectively, following treatment with 6 aminonicotinamide, suggesting a decreased oxidative inactivation of myeloperoxidase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009355 TI - [Hereditary bullous epidermolyses. Recent aspects of diagnosis and therapy]. AB - The introduction of antibodies specific for distinct basement membrane zone components is helpful in the diagnosis of hereditary mechanobullous diseases. In addition, these reagents have implications for investigation of the pathogenesis of these disorders, which might help to establish the classification based on molecular defects. Antibodies by means of indirect immune fluorescent microscopy can play a key role in the differential diagnosis of junctional and dystrophic types of epidermolysis bullosa. In epidermolysis bullosa hereditaria dystrophica, a disturbance in the regulation of collagenase activity has already been well documented, and several therapeutic approaches have been carried out based on the inhibition of collagenase activity. Concomitant inhibition of collagenolytic activity and stabilization of collagen fibrils might furthermore be helpful in the treatment of these patients. There are already several reports on the therapeutic effects of collagenase inhibitors, and stabilization of collagen fibrils in vitro has been demonstrated by (+)-cyanidanol-3. The preliminary results on therapeutic application of this drug seem to be promising. PMID- 3009357 TI - Viral respiratory disease in the intensive care unit. PMID- 3009356 TI - [Therapeutic and pathogenetic aspects of porphyria cutanea tarda]. AB - Seventy-three patients with porphyria cutanea tarda (PCT) were treated with chloroquine phosphate, which was administered in doses of 250 mg twice a week for more than 1 year on average. Of these patient, 49% did not have any associated ("inducing") factors. After treatment, serum transaminases and gamma glutamyltransferase (gamma-GT) always returned to normal values. However, gamma GT did not reach the normal range in the group without associated factors ("idiopathic group"). Of the 66 patients studied, 25 achieved an approximately physiological distribution of the pattern of urinary porphyrin fractions. Control biopsies were performed in 66 patients. Of them, 48 patients showed an improvement in liver damage. A statistically significant correlation was observed between a lasting increase in urinary prophyrin content and unchanged pathological liver morphology. A pathogenesis concept for PCT is discussed as a disease between inheritance (genetic enzyme defect) and environment ("inducing factors"). Even in the "idiopathic group" the improvement of pathological liver changes after therapeutic removal of the pathological prophyrin content in the liver cells shows that porphyrins could also cause liver damage. PMID- 3009358 TI - Different fixative-resin combinations and the immunocytochemical demonstration of immunoglobulins (kappa light chains) in semi-thin sections. AB - The effect of six different epoxy resins on the immunostaining of kappa light chains in tonsilar tissue which had been fixed by one of three fixing variants is described. The results show that not only may a specific resin influence the reaction but this may be enhanced by adopting a particular fixative-resin combination. PMID- 3009359 TI - Effects of tetrahydrocannabinol on rat preovulatory follicles: a quantitative cytochemical analysis. AB - Using a microdensitometric histochemical assay, delta 5-3 beta-hydroxysteroid dehydrogenase activity and glucose-6-phosphate dehydrogenase Types I and II hydrogen generation were measured in preovulatory follicles from normal rats, and in follicles from rats given tetrahydrocannabinol for three days prior to sacrifice. Hydroxysteroid dehydrogenase and Type I hydrogen generation are involved in steroidogenesis, whereas Type II hydrogen generation is involved with general cellular metabolism. All ovaries were removed on pro-oestrus, frozen, sectioned and the sections reacted with the appropriate media. Enzyme activity was measured in the theca and in three regions of the membrana granulosa; peripheral, antral and corona radiata. Compared to control animals, the hydroxysteroid dehydrogenase activity was significantly reduced in all follicular regions in rats exposed to tetrahydrocannabinol. Type I hydrogen generation was significantly less in the theca and peripheral region of preovulatory follicles from rats given tetrahydrocannabinol, but the same in the antral region and corona radiata. In all follicular regions examined, Type II hydrogen generation was unchanged following tetrahydrocannabinol administration. Thus, only the enzymes specifically associated with follicular steroidogenesis were affected by administration of the drug. PMID- 3009361 TI - [Malignant clear cell hidradenoma of the external auditory canal]. AB - A patient with a clear cell (malignant) hidradenoma of the external auditory canal and a detailed review of the literature are presented. Tumours of the ceruminous glands are rare. A sub-classification into distinct histological tumour types is useful with respect to therapy and prognosis. About two-thirds of all these neoplasms are malignant and the frequency of recurrence is very high (50%). Most of the adenomas are not encapsulated. The well differentiated adenocarcinomas (low grade malignancy) of the ceruminous glands can only be distinguished histologically from adenomas by their tendency to invade and infiltrate. Therapy of the ceruminous adenomas is wide excision; the treatment of carcinoma is radical block resection. PMID- 3009362 TI - [AIDS in ENT practice--case report and review]. AB - We present a case of AIDS with Kaposisarcoma in the oropharynx. In this case chemotherapy and radiotherapy were used with good response. Because of the clinical features of the disease otolaryngologists will be involved more and more in the management of AIDS disease. The diagnosis, therapy, and prognosis of the following three stages are described: serological identification of antibodies against HTLV-III without clinical symptoms, AIDS-related complex, full-blown AIDS. There is no inevitable transition between these stages. The mode of infection is similar to serum-hepatitis. PMID- 3009363 TI - Major histocompatibility complex haplotypes and the cell physiology of peptide hormones. PMID- 3009360 TI - Lectin binding to elastic fibres and associated components during development of the human aorta. AB - A panel of peroxidase-conjugated lectins was used to stain frozen sections of human foetal, neonatal, infant and adult aortae. Two main distinctive staining patterns were observed at the light microscope level, depending on the lectin employed. Lectins from Concanavalin A, Triticum vulgaris, Arachis hypogaea and Glycine max resulted in staining of the surfaces of aortic elastic lamellae and the interlamellar areas. With lectins from Dolichos biflorus and Bandeiraea simplicifolia staining was seen predominantly in the interlamellar areas. The findings indicate the presence of several different glycoconjugates at various sites within the aorta. In addition, there were alterations in the lectin binding affinities of the aortae that were dependent upon age. The findings are interpreted as indicating sequential changes in the composition of glycoprotein or proteoglycan moieties in the development of the human aorta. PMID- 3009364 TI - Speculations on sequence homologies between the fibronectin cell-attachment site, major histocompatibility antigens, and a putative AIDS virus polypeptide. AB - The core of the fibronectin cell-attachment site has been shown to be the tetrapeptide sequence Arg-Gly-Asp-Ser (RGDS). This peptide as well as its inverted analogue Ser-Asp-Gly-Arg (SDGR) efficiently inhibit fibronectin-mediated cell attachment in vivo and in vitro. Homology searches in protein data banks revealed the presence of the peptide SDGR in the alpha 2 domain of MHC class I antigens, and a variant of RGDS, Arg-Phe-Asp-Ser (RFDS), was found highly conserved in MHC class I (alpha 1 domain) and class II antigens (beta 1 domain). Three-dimensional models of MHC class I antigens suggested that the two tetrapeptide sequences may be located at the surface of the molecule, readily available for intermolecular contacts. We propose that fibronectin-mediated and MHC-mediated cell-cell interactions have similar molecular bases and that the RGDS-like sequences participate in specific cell adhesion between lymphoid cells. The RFDS tetrapeptide was also found in the sequence of a putative polypeptide chain encoded by the HTLVIII/LAV retrovirus family, the causative agent of AIDS. These amino acid sequence homologies suggest a common molecular basis for specific interactions between the MHC class II antigens, or the AIDS virus, and the T -cell specific T4 glycoprotein. PMID- 3009365 TI - Steroid 21-hydroxylase deficiency and the major histocompatibility complex. AB - Steroid 21-hydroxylase (21-OHase) deficiency is an HLA-linked recessive disorder of cortisol biosynthesis that can occur in several forms which differ in severity. Because they are in genetic linkage disequilibrium with different HLA antigens, the inheritance of these forms is consistent with the existence of several alleles at a single locus. When severe 21-OHase deficiency occurs in association with the HLA haplotype A3;Bw47;DR7, there is a simultaneous null allele at one of the C4 loci. This was hypothesized to result from a single deletion or rearrangement affecting the 21-OHase and C4 loci and perhaps the HLA B gene as well. To test this hypothesis and identify the 21-OHase gene, a cDNA clone was isolated that encoded the cytochrome P450 specific for steroid 21 hydroxylation in the bovine adrenal gland. This clone hybridized to two genes in normal human DNA, but to only one gene in DNA from an individual homozygous for A3;Bw47;DR7. All individuals heterozygous for A3;Bw47;DR7 carry a heterozygous deletion of a cytochrome P450 gene. Cosmid clones have been used to locate the 21 OHase genes both in man and mouse. In both species, there are two 21-OHase genes each located immediately 3' of one of the two C4 genes, and oriented in the same direction as the C4 genes. In man, the gene located 3' of the C4B gene is deleted in 21-OHase deficiency on the Bw47 haplotype, but the gene 3' of the C4A gene is deleted in hormonally normal individuals on the A1;B8;C4AQ0, C4B1;DR3 haplotype. Thus the 21-OHase B gene is normally active in man, but the 21-OHase A gene is not. PMID- 3009366 TI - Predictive biochemical assays for late radiation effects. AB - Surfactant precursors or other products of Type II pneumocytes have the potential to be the first biochemical marker for late radiation effects. This is particularly clinically important in the combined modality era because of the frequent occurrence of pneumonitis and pulmonary fibrosis secondary to radiation or chemotherapy. Accordingly, correlative studies have been pursued with the Type II pneumocyte as a beginning point to understand the complex pathophysiology of radiation pneumonitis and fibrosis. From our ultrastructural and biochemical studies, it is evident that Type II pneumocytes are an early target of radiation and the release of surfactant into the alveolus shortly after exposure persists for days and weeks. Through the use of lavaging techniques, alveolar surfactant has been elevated after pulmonary irradiation. In three murine strains and in the rabbit, there is a strong correlation with surfactant release at 7 and/or 28 days in vivo with later lethality in months. In vitro studies using cultures of type II pneumocytes also demonstrate dose response and tolerance factors that are comparable to the in vivo small and large animal diagnostic models. New markers are being developed to serve as a predictive index for later lethal pneumonopathies. With the development of these techniques, the search for early biochemical markers in man have been undertaken. Through the use of biochemical, histological, and ultrastructural techniques, a causal relationship between radiation effects on type II pneumocytes, pulmonary cells, endothelial cells of blood vessels, and their roles in the production of pneumonitis and fibrosis will evolve. PMID- 3009368 TI - Impact of tumor control on survival in carcinoma of the lung treated with irradiation. AB - The long-term results in tumor response, intrathoracic tumor control and survival are reported in patients with medically inoperable or unresectable non-oat cell and small cell carcinoma of the lung. In 376 patients with stages T1-3, NO-2 carcinoma of the lung tumors, accessioned to a Radiation Therapy Oncology Group (RTOG) randomized study to evaluate different doses of irradiation, a higher complete response rate (24%), intrathoracic tumor control (67%) and three year survival (15%) was observed with 6000 cGy, compared with lower doses of irradiation (4000 or 5000 cGy). Increased survival was noted in patients with complete tumor response. Three year survival in complete responders was 23%, in partial responders, 10%, and in patients with stable disease, 5%. Patients treated with 6000 cGy had an overall intrathoracic failure rate of 33% at 3 years, compared with 42% for those treated with 5000 cGy, 44% for patients receiving 4000 cGy with split course, and 52% for those treated with 4000 cGy continuous course (p = 0.02). Patients surviving 6 or 12 months exhibited a statistically significant increased survival when the intrathoracic tumor was controlled. Patients treated with 5000-6000 cGy, showing tumor control, had a three year survival of 22%, versus 10%, if they had intrathoracic failure (p = 0.05). In patients treated with 4000 cGy (split or continuous), the respective survival was 20% and 10%, if the intrathoracic tumor was controlled (p = 0.001). In patients surviving 12 months after treatment with 5000-6000 cGy, on whom the intrathoracic tumor was controlled, the median survival was 29 months, in contrast to 18 months, if they developed intrathoracic failure (p = 0.05). In patients treated with 4000 cGy, the median survival was 23 months with control and 18 months without control of the intrathoracic tumor [corrected] (p = 0.008). In another RTOG study for patients with more advanced tumors (T4 or N3), those with local tumor control at 12 months had a three year survival rate of 25%, compared with 5% for those with thoracic failures. These differences are statistically significant (p = 0.006). Higher doses of irradiation yield a greater proportion of complete response, higher intrathoracic tumor control and better survival in non-oat cell medically inoperable or unresectable carcinoma of the lung.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3009367 TI - The effect of combined modality therapy on local control and survival. AB - The systemic component of combined modality therapeutic programs has influenced both the selection of the approach to local control and survival in a number of tumor types. The more effective systemic therapy is against metastatic cancer by itself, the greater the impact on local control and survival. This observation is consistent with the invariable inverse relationship between curability and tumor cell number. For some common cancers, local control is good, but survival remains poor because of the inability to deal effectively with micrometastases. Improved systemic treatment is likely to have an impact on survival may shift local control measures, in some cases, to radiation therapy or lesser surgery without radiation therapy. There remains a substantial number of tumor types where both local control and survival is poor. In these tumors, improvement in local control by itself is not likely to improve survival because of the presence of micrometastases, but such improvements must occur before we can have a true evaluation of the systemic treatment of micrometastases in these tumors. The recent understanding that the metastatic process is under genetic control and the cloning of metastases genes offers a substantial opportunity to control this process and influence both local control and survival. PMID- 3009369 TI - Local control and survival in soft tissue sarcomas of the limbs, trunk walls and head and neck: a study of 113 cases. AB - One hundred thirteen patients with soft tissue sarcomas of the limbs, trunk walls, and head and neck have been treated at the Centre Francois Baclesse since 1972. Of these, 89 histologically confirmed patients were treated with a multimodality treatment protocol. Treatment policy was designed to use each treatment method as efficiently, economically and conservatively as possible: preoperative irradiation at moderate dose to a large volume (6.5 Gy, 2 sessions, 48 hr interval); surgery 48 hr after the last preoperative irradiation; surgical excision was guided and verified intra operatively by the pathologist (with frozen sections); postoperative irradiation aimed at sterilizing all residual isolated and radiosensitive tumor cells, possibly scattered throughout the anatomical region. The total dose is brought to the equivalent of 50 Gy (preoperative dose included). This dose was increased to 60 or even 70 Gy to a restricted volume, when limb conservation was sought, but tumor foci too large for total resection without amputation; actinomycin was added to the first five postoperative irradiations. The results at 5 years were as follows: local recurrence rate, 13.6%; metastatic rate, 28%; survival rate, overall (113 patients,) 65.6%, curative series (89 patients), 75%. When the surgical excision of the primary tumor was histologically complete (54 patients) the local recurrence rate was 1.9%, the metastatic rate 11.6%, and the survival rate 89.6% at 5 years. PMID- 3009371 TI - Nonweightbearing lameness secondary to synovial sarcoma in a young dog. AB - Synovial sarcoma was diagnosed in the right carpus of a 2 1/2-year-old mixed breed dog. The dog had developed a right forelimb lameness before one year of age. The lameness was progressive for nearly 2 years, resulting in severe disuse atrophy of the right forelimb musculature and pronounced osteopenia. A definitive diagnosis was not made until the dog was referred after 2 years of conservative treatment was ineffectual. Right forelimb amputation was done and the dog survived an additional 15 months. The protracted clinical course before definitive diagnosis underscores the need for aggressive pursuit of a diagnosis when conservative treatment of a lameness is not efficacious. PMID- 3009372 TI - High murine mammary tumor virus expression in milk in a low mammary tumor mouse strain and high incidence of mammary tumor in hybrids produced by crossing with another low mammary tumor mouse strain. AB - Large amounts of murine mammary tumor virus (MMTV) were expressed in milk from a low mammary tumor mouse strain called II-TES, established by crossbreeding DBA/2 mouse strain with two strains of Japanese pet mouse origin. The reciprocal hybrids of the II-TES strain and OZ-F strain, another low mammary tumor strain of Japanese pet mouse origin, developed early-appearing mammary tumors at a high rate, and large quantities of MMTV were expressed in the milk. These findings suggest that different regulatory genes control MMTV expression and mammary tumorigenesis. PMID- 3009370 TI - Distribution of radiation sensitivities for human tumor cells of specific histological types: comparison of in vitro to in vivo data. AB - The radiosensitivities of human tumor cell lines, grouped into 6 histological categories, have been studied using data from the published literature. The parameters alpha, beta, n, D0, D, and the surviving fraction to 2 Gy (S2) and 8 Gy (S8) were calculated. Only the two parameters mainly derived from the initial part of the survival curve, alpha and D, together with S2, provided data which were correlated with the clinical radioresponsiveness of each histological group. Thus, there are intracellular factors which influence clinical radioresponsiveness whose relative importance varies from one histological cell type to another. The value of D gave the most precise characterization of the average group radiosensitivity. It was possible to compare the in vivo radiosensitivities of non-severely hypoxic cells with those of tumor cells irradiated in vitro for 7 tumor lines grown as xenografts in mice. The average radiosensitivity was 1.9 times less in vivo than in vitro. This difference indicates that, in addition to the intrinsic factors of radioresistance demonstrated in vitro, and independently of severe hypoxia, there are other factors which specifically reduce radiosensitivity in vivo. PMID- 3009373 TI - Mitomycin C-augmented production of I-A antigen-positive macrophages in tumor vaccine-primed mice. AB - Intraperitoneal inoculation of L1210 murine leukemia cell vaccine increased I-Ad antigen-positive and -negative macrophages in the peritoneal cavity of histocompatible mice. Mitomycin C, a poor producer of I-Ad antigen-positive macrophages, selectively augmented the production of I-Ad antigen-positive macrophages in L1210 vaccine-primed mice, since it did not augment the production of non-macrophage cells. Since our previous study showed that the priming of mice with L1210 vaccine and mitomycin C induced augmented antitumor response, we tested the feasibility of the association of mitomycin C-augmented I-Ad antigen positive macrophages with the augmented antitumor response. Prostaglandin E2 suppressed the antitumor response in L1210 vaccine- and mitomycin C-primed mice and, consistent with this, it inhibited the production of I-Ad antigen-positive macrophages, although it inhibited the production of non-macrophage cells as well. This hypothesis was further tested by the use of silica. When L1210 vaccine and mitomycin C-primed mice were further given silica on the day of and one day after live L1210 inoculation, their antitumor response was strongly suppressed, while a kinetic analysis of the cellularity of peritoneal cells showed that administration of silica resulted in a decrease of I-Ad antigen-positive macrophages, but not I-Ad antigen-negative macrophages or non-macrophage cells in these mice. These results strongly suggest, though they do not prove, the association of mitomycin C-augmented production of I-Ad antigen-positive macrophages with the antitumor response in L1210 vaccine- and mitomycin C-primed mice. PMID- 3009374 TI - Separation of sarcoma growth inhibitor from avian sarcoma virus-transformed rat cells. AB - A cell growth inhibitor was isolated from an avian sarcoma virus-transformed rat cell line (77N1), from which we had previously obtained a novel transforming growth factor, TGF gamma 2. The growth inhibitor (named SGI) was found in cell extract of 77N1 cells and was separable from TGF gamma 2 by means of ion exchange chromatography. SGI inhibited the DNA synthesis of serum-starved, TGF gamma 2 stimulated BALB3T3 cells as well as the spontaneous and TGF-induced colony formation of various cells in semisolid agar. We present evidence that SGI is distinct from the ubiquitous growth modulator TGF beta, and we discuss the possible role of SGI in the neoplastic cell proliferation. PMID- 3009375 TI - Assignment of the gene for human DNA polymerase beta (POLB) to chromosome 8. AB - A cloned cDNA segment of 442 bp which contains the region coding the N-terminal sequence of rat DNA polymerase beta polypeptide was used as a probe in Southern hybridization analysis to identify the gene for this enzyme. This probe hybridized with DNA from mouse and human in addition to rat DNA, and the BamHI fragment containing DNA polymerase beta gene sequence was unique in size in each species: 15.8 kb for rat DNA, 8.7 kb for mouse DNA and 10.1 kb for human DNA. DNA extracted from a panel of 12 independent human-mouse somatic cell hybrids was analyzed by Southern blot hybridization with the cloned cDNA probe. The results indicate that the gene for the human DNA polymerase beta sequence (POLB) is located on chromosome 8. PMID- 3009376 TI - Monoclonal antibody NCC-pX-1G reactive with gene products coded from X regions of human T-cell leukemia virus. AB - Monoclonal antibody NCC-pX-1G (IgG1, kappa) was obtained by the hybridoma technique by immunization of mice with the C-terminal 54 amino acids of the pX protein, made from the 3'-terminal portion of the pX gene conjugated with bovine growth hormone gene transfected in E. coli. NCC-pX-1G recognized 41 kilodalton pX protein of HTLV-I, and immunocytochemically stained HUT102 and other HTLV-I infected cell lines, but no reaction was observed with non-HTLV-I-infected cell lines or normal human tissue cells. PMID- 3009377 TI - Amplifications of both c-Ki-ras with a point mutation and c-myc in a primary pancreatic cancer and its metastatic tumors in lymph nodes. AB - Activated c-Ki-ras with a point mutation (GGT to CGT) at codon 12, resulting in the substitution of arginine for glycine, was found in DNA from metastatic pancreatic adenocarcinoma in a lymph node. By means of restriction endonuclease length polymorphism with SacI digestion, we were able to demonstrate that the same point mutation of c-Ki-ras was present in the primary tumor and in metastases in lymph nodes. DNA from the normal spleen of the patient did not have this type of point mutation. Moreover, amplifications of 3- to 6-fold of the activated c-Ki-ras and 50-fold of c-myc were found in the primary tumor and the metastases in the two lymph nodes, indicating that point mutation had occurred at a relatively early stage of the tumor development, before amplification of the gene. This is the first clear demonstration of amplification of activated c-Ki ras accompanied by amplification of c-myc in both primary and metastatic human tumors in vivo. PMID- 3009378 TI - K-4, a novel inhibitor of angiotensin I converting enzyme produced by Actinomadura spiculosospora. AB - A novel inhibitor of angiotensin I converting enzyme (ACE), named K-4, was isolated from the culture broth of Actinomadura spiculosospora nov. sp. K-4. The K-4 was an oligopeptide containing L-phenylalanine with (R)-1-amino-2-(4 hydroxyphenyl)ethylphosphonic acid as the C-terminal residue. The compound proved to be a specific and reversible inhibitor of ACE with the inhibition constant (Ki) of 0.18 microM, and inhibited ACE non-competitively by use of hippuryl-L histidyl-L-leucine (HHL) as a substrate. When administrated intravenously to rats, K-4 inhibited the pressor response to angiotensin I. PMID- 3009379 TI - Anterior pituitary concentrations of gonadotropins, GnRH-receptors and ovarian characteristics following early weaning in beef cows. AB - Endocrine changes in the hypophyseal-ovarian axis associated with early calf removal were investigated in anestrous beef cows. Tissues were collected and analyzed from multiparous beef cows slaughtered at O (n = 8), 36 (n = 8) or 72 h (n = 8) after calf removal during the fifth week after calving. Cows that exhibited estrus; had postmortem signs of a recent ovulation or had serum concentrations of luteinizing hormone (LH) indicative of an ovulatory surge, were excluded from the analysis. Five control cows that were not slaughtered exhibited estrus from 30 to 84 h after calf removal. Seven additional cows were continuously kept with their calves and did not exhibit estrus until 72 +/- 9 d after calving. Serum concentrations of estradiol-17 beta (estradiol) averaged 8.2 +/- 1.7, 7.5 +/- 2.0 and 9.1 +/- 1.5 pg/ml at 0, 36 and 72 h, respectively, but they averaged 22.8 +/- 4.7 pg/ml prior to estrus in control cows. Therefore, observations were assumed to represent events that occur prior to the rise in serum concentrations of estradiol that occurs during proestrus. Volume of fluid from the largest ovarian follicle tended to be greater (P less than .10) at 72 h (1.5 +/- .2 ml) than at 0 h (1.1 +/- .1 ml) or 36 h (1.0 +/- .1 ml). Follicular fluid concentrations of estradiol, but not progesterone, were positively correlated (P less than .01) with follicular volume. However, numbers of small (less than 100 microliters), medium (100 to 400 microliters) and large follicles (greater than 400 microliters) as based on fluid volume, as well as follicular fluid concentrations of estradiol and progesterone, did not differ among treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009381 TI - The identification of the aminoglycoside-phosphorylating enzymes APH(2'') and APH(3') from the characterization of their reaction products by high performance liquid chromatography. AB - A method is described for the identification of the aminoglycoside phosphorylating (APH) enzymes APH(2'') and APH(3') by the high performance liquid chromatographic (HPLC) identification of their products of reaction with kanamycin and ATP. Twelve reference strains which produce either APH(2'') or APH(3') were examined by both this method and the radioenzymatic profile method; correct enzyme identification was made with 12/12 of the strains by the HPLC method and 11/12 of the strains by the radioenzymatic profile method. Reaction products of APH(2'') or APH(3') with ATP and butirosin, geneticin, lividomycin, ribostamycin, sissomicin or tobramycin were also characterized by HPLC. Conditions for their chromatography are given. PMID- 3009380 TI - Anovulation in postpartum suckled beef cows. II. Associations among binding of 125I-labeled gonadotropins to granulosa and thecal cells, and concentrations of steroids in serum and various sized ovarian follicles. AB - To determine if specific binding of 125I-labeled gonadotropins to granulosa and thecal cells, or concentrations of steroids in ovarian follicles change during the postpartum anovulatory period, 21 suckled beef cows were slaughtered on d 7, 14, 28, 42 or 56 after parturition (n = 4 to 6 per d). After slaughter, 10 to 15 follicles were dissected from each pair of ovaries and categorized by diameter: small (1.0 to 3.9 mm), medium (4.0 to 7.9 mm) or large (greater than or equal to 8 mm). Progesterone (221 to 612 ng/ml), androstenedione (48 to 94 ng/ml) and estradiol (2.7 to 23.9 ng/ml) did not change (P greater than .10) in fluid of small or medium follicles from d 7 to 42 to 56 after parturition. Similarly, specific binding of human chorionic gonadotropin (125I-hCG) or follicle stimulating hormone (125I-oFSH) to homogenates of small, medium or large follicles did not change (P greater than .05). In contrast, progesterone in fluid of large follicles increased (P less than .05) 3.4-fold between d 7 and 14, but decreased (P less than .05) 55% between d 14 and 28. Concentrations of androstenedione in fluid of large follicles did not change (P greater than .10) from d 7 to 42 to 56. Concentrations of estradiol in fluid of large follicles remained constant between d 7 and 14, but increased (P less than .05) 4.2-fold between d 14 and 28. We conclude that during the postpartum anovulatory period, there is no change in steroidogenic capabilities of small or medium follicles, both of which predominantly produce progesterone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009383 TI - Pharmacokinetics, safety and preliminary clinical experiences using foscarnet in the treatment of cytomegalovirus infections in bone marrow and renal transplant recipients. AB - Fifty seven episodes of severe cytomegalovirus (CMV) infection were treated with iv foscarnet in 13 bone marrow and 33 renal graft recipients. The ranges of the daily dose, duration, average steady state level and total dose were 23-268 mg/kg, 2-46 days, 42-400 mg/l and 2-399 g, respectively. Adverse effects, such as decreased haemoglobin, decreased renal function and increased serum calcium, were observed in a few patients only. Increased liver enzymes, hallucinations and tremor were seen in one uraemic patient and coincided with foscarnet plasma concentrations above 400 mg/l. Among 25 patients evaluated for clinical efficacy, 12 died. Improvements, such as eradication of CMV (8/14 assessable patients), resolution of fever (11/22), and improved laboratory values (13/23) were noted in 17/24 (70%). Controlled trials are warranted on the basis of this study. PMID- 3009382 TI - Reversible binding of polymyxin B and neomycin to the solid part of faeces. AB - Previous studies have shown that polymyxin B and neomycin were bound to the solid part of human faeces. In the present study, the faecal binding of polymyxin B and neomycin was studied, using a wide range of concentrations of drugs and faecal dilutions. Up to 25 mg of polymyxin B and 15 mg of neomycin could bind to 1 g of faeces but the binding was found to be reversible. With faeces of rats it was found that polymyxin B was mainly bound to the non-bacterial part of faeces, whereas neomycin was bound to the bacterial as well as the non-bacterial part. The implications of the reversible binding of antibiotics to intestinal contents for selective decontamination are discussed. PMID- 3009384 TI - A non-comparative study of parenteral ampicillin and sulbactam in intra-thoracic and intra-abdominal infections. AB - Fifty-four patients were treated with intravenous ampicillin and sulbactam in an open study of intra-thoracic and intra-abdominal infection. Thirty-one were treated with 500 mg each of the combination 6-hourly while 23 patients were given 1 g of ampicillin and 500 mg of sulbactam, 6-hourly. Thirteen of fourteen (93%) patients with severe respiratory tract infection and 22/26 (85%) patients in the intra-abdominal infection group responded clinically and bacteriologically. Seven patients with clinical sepsis (but not confirmed bacteriologically) improved on therapy. 50/55 (91%) clinical isolates from this study were eliminated. An increase in MIC was found in two cases. There were minimal side effects, pain at site of injection being the commonest complaint. PMID- 3009385 TI - Impaired pulmonary conversion of angiotensin I to angiotensin II in rats exposed to chronic hypoxia. AB - The effects of exposing rats to hypoxia at normal atmospheric pressure for periods of 21-24 days on intrapulmonary conversion of angiotensin I (ANG I) to angiotensin II (ANG II) were examined using an isolated rat lung preparation perfused at constant flow. 125I-ANG I (160 fmol) was injected alone and with graded doses (0.1, 1.0, and 100 nmol) of unlabeled ANG I into the pulmonary artery, and the effluent was collected for measurement of ANG I, ANG II, and metabolites. At low doses of injected ANG I (125I-ANG I alone or with 0.1 or 1.0 nmol unlabeled ANG I), the percent conversion of ANG I to ANG II was 67.5 +/- 2.1 (SE), 65.1 +/- 2.0, and 62.5 +/- 1.6 in 21-day hypoxia-exposed animals and 83.8 +/- 2.7, 81.4 +/- 3.9, and 79.6 +/- 2.3 (P less than 0.01) in control rats maintained under normoxic conditions. At the highest dose (100 nmol) of injected ANG I, percent conversion was reduced in both hypoxic and control groups to 46.8 +/- 5.0 and 64.0 +/- 6.0, respectively (P less than 0.05). Mean transit times of labeled material through the pulmonary circulation were not significantly different in hypoxic vs. normoxic lungs at any ANG I load, suggesting that the decreased conversion seen in hypoxic lungs was not related to altered kinetics of substrate exposure. Thus chronic hypoxia is associated with significant inhibition of transpulmonary ANG I conversion that is independent of perfusate flow. We postulate that this phenomenon is due to alterations at the endothelial membrane level. PMID- 3009387 TI - Granulocyte-mediated airway edema in guinea pigs. AB - To determine the role of polymorphonuclear leukocytes (PMNs) in the airway edema that accompanies airway inflammation, we studied the effects of a 1-h exposure to 2 ppm toluene diisocyanate (TDI) on tracheal extravasation of Evans blue dye and on the concentration of PMNs in the tracheal wall. Tracheal Evans blue content was significantly increased by TDI exposure (53.6 +/- 8.0 micrograms/g tracheal tissue (mean +/- SE) for animals exposed to TDI and 16.3 +/- 2.0 for animals exposed to air, P less than 0.0025) as were both the intravascular and extravascular concentration of PMNs in tracheal sections (intravascular PMNs were 28.0 +/- 8.4 X 10(3) cells/mm3 for TDI and 1.5 +/- 1.5 X 10(3) for air, P less than 0.025, extravascular PMNs were 10.9 +/- 4.5 X 10(3) for TDI and 0 for air, P less than 0.05). PMN depletion with vinblastine or with hydroxyurea abolished both the increase in tracheal Evans blue extravasation and the increase in the concentration of intravascular and extravascular PMNs in animals exposed to TDI. PMN depletion with hydroxyurea did not significantly inhibit the increase in tracheal Evans blue extravasation caused by intravenous histamine. Administration of donor PMNs to animals depleted of PMNs with hydroxyurea reconstituted the TDI induced increase in tracheal Evans blue extravasation (80.4 +/- 17.3 micrograms/g tissue (mean +/- SE) in animals exposed to TDI vs. 21.3 +/- 2.9 in animals exposed to air, P less than 0.025) and in the intravascular concentration of PMNs in tracheal sections [18.5 +/- 3.4 X 10(3) cells/mm3 (mean +/- SE) in animals exposed to TDI vs. 1.3 +/- 1.3 X 10(3) in animals exposed to air, P less than 0.0025].(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009388 TI - Effect of maternal exercise on fetal liver glycogen late in gestation in the rat. AB - To determine the effect of maternal exercise on fetal liver glycogen content, fed and fasted rats that were pregnant for 20.5 or 21.5 days were run on a rodent treadmill for 60 min at 12 m/min with a 0% grade or 16 m/min up a 10% grade. The rats were anesthetized by intravenous injection of pentobarbital sodium, and fetal and maternal liver and plasma samples were collected and frozen. Fetal liver glycogenolysis did not occur as a result of maternal exercise. Fetal blood levels of lactate increased 22-60%, but glucose, plasma glucagon, and insulin were unchanged during maternal exercise. Maternal liver glycogen decreased as a result of exercise in all groups of rats except the fasted 20.5-day-pregnant group. Plasma free fatty acids increased in all groups and blood lactate increased in fed (20.5 days) and fasted (21.5 days) pregnant rats. Maternal glucose, glucagon, and insulin values remained constant during exercise. The fetus appears to be well-protected from metabolic stress during moderate intensity maternal exercise. PMID- 3009386 TI - Atriopeptin II relaxes and elevates cGMP in bovine pulmonary artery but not vein. AB - Atriopeptin II, a 23-amino acid synthetic peptide fragment of atrial natriuretic factor, caused an endothelium-independent relaxation of isolated precontracted rings of bovine intrapulmonary artery that was accompanied by the concomitant accumulation of guanosine 3',5'-cyclic monophosphate (cGMP) but not adenosine 3',5'-cyclic monophosphate. In contrast, rings of intrapulmonary vein were unaffected by atriopeptin II whether or not endothelium was present. Whereas methylene blue, an inhibitor of soluble guanylate cyclase, abolishes endothelium dependent and independent arterial relaxation and cGMP accumulation in response to acetylcholine and glyceryl trinitrate, respectively, methylene blue failed to alter these responses to atriopeptin II. Similarly, the effects of atriopeptin II were unaltered by propranolol, indomethacin, or atropine. These results indicate that relaxation elicited by atriopeptin II may be selective for arterial smooth muscle receptors, does not require endothelial cells, and does not involve the soluble form of guanylate cyclase, although cGMP accumulation is stimulated. PMID- 3009389 TI - Intensity and duration effects of exercise on heart cAMP, phosphorylase, and glycogen. AB - Glycogen, phosphorylase, and adenosine 3',5'-cyclic monophosphate (cAMP) were determined in rat heart following an acute exercise bout. Intensity and duration of exercise were varied to gain further insights into the mechanism regulating myocardial glycogenolysis during exercise. Groups of rats were run at either 15 or 30 m/min for 0, 5, 10, 15, or 30 min and immediately killed. Heart glycogen degradation was influenced by intensity and duration of exercise and was independent of cAMP levels and activation of phosphorylase to its a form. cAMP levels were increased in the heart, dependent on intensity and duration of exercise. Phosphorylase in the a form increased at the onset of exercise, independent of intensity, and remained elevated throughout the exercise despite little or no glycogenolysis. Absolute phosphorylase a activity was also increased with exercise and was independent of intensity of exercise. Compared with resting levels, total phosphorylase activity was decreased at all times at the lower exercise intensity, whereas total phosphorylase activity declined at the higher intensity only after glycogenolysis had occurred. These data suggest that myocardial glycogen degradation during exercise can occur independently of cAMP and that the percentage of phosphorylase in the a form is not a good indicator of glycogenolytic rate. PMID- 3009390 TI - Hyperoxia alters effect of calcium on rat alveolar macrophage superoxide production. AB - The effect of hyperoxia on the Ca2+ dependence of stimulated superoxide anion radical (O2-.) production (the respiratory burst) of rat alveolar macrophages was investigated. Enhancement of the concanavalin A (con A)-stimulated respiratory burst by extracellular Ca2+ was suppressed by O2 exposure. Similarly, the inhibitory effect of verapamil on the con A-stimulated respiratory burst was reduced by O2 exposure. O2 exposure also inhibited con A stimulation that was independent of Ca2+ entry. Exposure to O2 also caused a decline in O2-. production stimulated by either A23187 or phorbol myristate acetate (PMA). With A23187 stimulation, extracellular Ca2+ was essential for either air-exposed (control) or O2-exposed cells. With PMA, stimulation was independent of extracellular Ca2+ for either air or O2-exposed macrophages and verapamil did not inhibit. Free intracellular Ca2+ concentration ([Ca2+]i) was measured in control and O2-exposed alveolar macrophages. Hyperoxic exposure did not alter [Ca2+]i in unstimulated cells. In controls, con A stimulated an immediate increase in [Ca2+]i followed by a rapid decrease and a second rise and fall. The second elevation was suppressed by verapamil or ethyleneglycol-bis (beta aminoethylether)-N,N'-tetraacetic acid or O2 exposure. The results of both the respiratory burst assays and measurement of con A-stimulated changes in [Ca2+]i suggest that Ca2+ entry involved in stimulus-response coupling is suppressed in cellular O2 toxicity. PMID- 3009391 TI - RNase H-defective mutants of Escherichia coli. PMID- 3009392 TI - Nucleotide sequences of the R1-19 plasmid transfer genes traM, finP, traJ, and traY and the traYZ promoter. AB - The complete nucleotide sequences of the R1 drd-19 (R1-19) plasmid transfer genes traM, finP, traJ, and traY and the region encoding the traYZ promoter were determined. The traM protein from R1-19 was similar to the 127-amino-acid traM product from the conjugative plasmid F; only 28 residues were not identical. finP, a negative regulatory element of the traJ gene, contained a 12-base-pair inverted repeat identical to that found in the F plasmid, but differed in the 7 base pairs found between the repeats. The traJ gene and the traYZ promoter (the site of transcriptional stimulation by the traJ product) were completely different from the equivalent sequences in plasmid F. Galactokinase fusion studies of the traYZ promoter indicated that the R1-19 and F plasmids have analogous but not homologous traYZ promoter strengths and regulation. The traY protein from R1-19 was 44 residues shorter than the traY product from plasmid F, but there was some homology within the C-terminal halves of the traY gene products. The predicted translational start codon for the traY gene is GUG. PMID- 3009393 TI - Cloning and expression of the genes specifying Shiga-like toxin production in Escherichia coli H19. AB - Some strains of Escherichia coli produce a protein which is cytotoxic for Vero cell and HeLa cell monolayers. This toxin is very similar to the toxin of Shigella dysenteriae 1 and has been named verotoxin or E. coli Shiga-like toxin. It has been shown that toxin conversion is due to a group of bacteriophages, one of which has been designated H-19B. In this study we report hybridization experiments showing that part of the H-19B genome is homologous to phage lambda. We have cloned a 1.7-kilobase BalI-BglII fragment from the genome of H-19B into pUC18. The recombinant plasmid confers the ability to produce high levels of Shiga-like toxin on transformed E. coli cells. We demonstrate using an in vitro transcription/translation system that the cloned fragment specifies the two verotoxin subunit peptides which have masses of 31 and 5.5 kilodaltons. The identity of peptides was confirmed by immunoprecipitation with verotoxin antiserum and protein A-Sepharose beads. PMID- 3009394 TI - Lysogenic conversion of staphylococcal lipase is caused by insertion of the bacteriophage L54a genome into the lipase structural gene. AB - Staphylococcus aureus PS54 manifests no lipase (geh) activity. This is due to the insertion of bacteriophage L54a DNA into the geh structural gene. The nucleotide sequence of this 2,968-base-pair DNA fragment was determined. Lipase deduced from the nucleotide sequence is a polypeptide of 690 amino acids which extends from nucleotide 706 to 2776. PMID- 3009396 TI - Preparation of the FhuA (TonA) receptor protein from cell envelopes of an overproducing strain of Escherichia coli K-12. AB - A rapid and simple method for purification of the FhuA receptor protein from cell envelopes of a FhuA-overproducing strain of Escherichia coli K-12 was developed. The overproduction of FhuA was programmed by the thermoamplifiable plasmid pHK232, which carried the fhuACD genes of pLC19-19 of the Clarke and Carbon collection. At low temperature (27 degrees C), pHK232 specified the overproduction of FhuA to levels comparable to those of major outer membrane proteins OmpF, OmpC, and OmpA. The amount of these proteins in the outer membrane was reduced along with overproduction of FhuA. Upon runaway replication of pHK232 at 37 degrees C, the precursor of the FhuA protein, proFhuA, was also accumulated in the cell envelope in amounts similar to FhuA. For extraction of the FhuA protein, crude cell envelopes were washed with 2% Triton X-100-6 M urea to remove less tightly bound proteins. Then FhuA but not proFhuA was solubilized by treating Triton X-100-urea-washed membranes with 1% octylglucoside-1 mM EDTA. This procedure yielded FhuA protein free from other membrane proteins. The amount of lipopolysaccharide and phospholipids was low and ranged from 5 to 15% and 10 to 25% of the weight of the FhuA protein, respectively. As shown by direct inactivation and by competition assays, the isolated FhuA protein retained receptor activity for ferrichrome, albomycin, colicin M, and phages T5 and T1. PMID- 3009395 TI - Cloning of a gene cluster encoding biphenyl and chlorobiphenyl degradation in Pseudomonas pseudoalcaligenes. AB - A gene cluster encoding biphenyl- and chlorobiphenyl-degrading enzymes was cloned from a soil pseudomonad into Pseudomonas aeruginosa PAO1161. Chromosomal DNA from polychlorinated biphenyl-degrading Pseudomonas pseudoalcaligenes KF707 was digested with restriction endonuclease XhoI and cloned into the unique XhoI site of broad-host-range plasmid pKF330. Of 8,000 transformants tested, only 1, containing the chimeric plasmid pMFB1, rendered the host cell able to convert biphenyls and chlorobiphenyls to ring meta cleavage compounds via dihydrodiols and dihydroxy compounds. The chimeric plasmid contained a 7.9-kilobase XhoI insert. Subcloning experiments revealed that the genes bphA (encoding biphenyl dioxygenase), bphB (encoding dihydrodiol dehydrogenase), and bphC (encoding 2,3 dihydroxybiphenyl dioxygenase) were coded for by the 7.9-kilobase fragment. The gene order was bphA-bphB-bphC. The hydrolase activity, which converted the intermediate meta cleavage compounds to the final product, chlorobenzoic acids, and was encoded by a putative bphD gene, was missing from the cloned 7.9-kilobase fragment. PMID- 3009397 TI - Determination of the chromosomal locations of four Bacillus subtilis genes which code for a family of small, acid-soluble spore proteins. AB - The chromosomal locations of four genes which code for small, acid-soluble spore proteins (SASP) in Bacillus subtilis have been determined. Although these four genes code for extremely homologous small, acid-soluble spore proteins (greater than 65% sequence identity), the genes are not clustered but are located at 70 degrees (adjacent to glyB [sspB gene]), 115 degrees (between metC and pyr cluster [sspD gene]), 180 degrees (between metB and kauA [sspC gene]), and 260 degrees (between ilvC and aroG [sspA gene]) on the B. subtilis genetic map. PMID- 3009398 TI - Cloning and nucleotide sequencing of genes for three small, acid-soluble proteins from Bacillus subtilis spores. AB - Three Bacillus subtilis genes (termed sspA, sspB, and sspD) which code for small, acid-soluble spore proteins (SASPs) have been cloned, and their complete nucleotide sequence has been determined. The amino acid sequences of the SASPs coded for by these genes are similar to each other and to those of the SASP-1 of B. subtilis (coded for by the sspC gene) and the SASP-A/C family of B. megaterium. The sspA and sspB genes are expressed only in sporulation, in parallel with each other and with the sspC gene. Two regions upstream of the postulated transcription start sites for the sspA and B genes have significant homology with the analogous regions of the sspC gene and the SASP-A/C gene family. Purification of two of the three major B, subtilis SASPs (alpha and beta) and determination of their amino-terminal sequences indicated that the sspA gene codes for SASP-alpha and that the sspB gene codes for SASP-beta. This was confirmed by the introduction of deletion mutations into the cloned sspA and sspB genes and transfer of these deletions into the B. subtilis chromosome with concomitant loss of the wild-type gene. PMID- 3009399 TI - Molecular cloning and characterization of scrB, the structural gene for the Streptococcus mutans phosphoenolpyruvate-dependent sucrose phosphotransferase system sucrose-6-phosphate hydrolase. AB - A DNA fragment encoding the sucrose-6-phosphate hydrolase component of the Streptococcus mutans phosphoenolpyruvate-dependent sucrose phosphotransferase system has been recovered from a plasmid-based genomic library of strain GS5. The locus, designated scrB, was found to reside within a 2.9-kilobase-pair restriction fragment present on the chimeric molecule pVA1343 (7.3 kilobase pairs). Minicell analysis of pVA1343-directed translation products revealed that the scrB product synthesized in Escherichia coli V1343 was a single peptide of Mr 57,000. This polypeptide was reactive with antiserum prepared against S. mutans intracellular invertase, which has been previously shown to have an Mr of 43,000 to 48,000. The basis of this difference in Mr was not established but may represent a posttranslational proteolytic event which occurred in S. mutans but not in recombinant V1343. Sucrose-6-phosphate hydrolase purified to homogeneity from V1343 exhibited Michaelis constants of 180 mM for sucrose and 0.08 mM for sucrose-6-phosphate. Deletion analysis of pVA1343 facilitated the assignment of a coding region for the hydrolase within the insert, as well as an orientation for the transcription of scrB. scrB-defective strains of S. mutans constructed by additive integration of an insertionally inactivated scrB locus exhibited the sucrose sensitivity characteristic of this mutant class. Similar loci were detected by DNA-DNA hybridization in additional strains of S. mutans and two strains of Streptococcus cricetus, but not in single strain representatives of S. rattus, S. sobrinus, S. sanguis I and II, S. salivarius, or S. mitis. PMID- 3009400 TI - Tn5-induced mutations affecting sulfur-oxidizing ability (Sox) of Thiosphaera pantotropha. AB - Mutants of Thiosphaera pantotropha defective in chemolithoautotrophic growth were obtained by transpositional mutagenesis with Tn5 coding for kanamycin resistance. The suicide vehicle for introducing Tn5 to T. pantotropha was pSUP5011 harbored by Escherichia coli. Kanamycin-resistant isolates were screened for the inability to grow with reduced sulfur compounds (Sox-). Four classes of Sox- mutants were obtained. Three were of different pleiotropic phenotypes: (i) unable to grow with formate, nitrate, and xanthine; (this class strongly suggested the involvement of a molybdenum cofactor in inorganic sulfur-oxidizing ability); (ii) no growth with hydrogen; (iii) slight growth with hydrogen and formate. Two plasmids, pHG41 (about 450 kilobase pairs) and pHG42 (110 kilobases), were identified in lysates of T. pantotropha. In one Sox- mutant pHG41 could not be detected. Revertant analysis suggested that pHG41 and pHG42 were not involved in the Sox character. PMID- 3009401 TI - Isolation of Bacillus subtilis genes transcribed in vitro and in vivo by a major sporulation-induced, DNA-dependent RNA polymerase. AB - As a means of determining the function of sigma 29, a sporulation-essential sigma factor, we have isolated and begun to characterize genes that require sigma 29 for their expression. RNA transcribed in vitro from total Bacillus subtilis DNA by using sigma 29-containing RNA polymerase (E-sigma-29) was hybridized to a bank of B. subtilis DNA fragments that had been cloned into bacteriophage lambda. Approximately 0.25% of the cloned B. subtilis DNA fragments displayed detectable hybridization with our RNA probe. Five DNA fragments that had strong in vitro template activity for E-sigma-29 were selected for further study. The DNA fragments which contained in vitro sigma 29 promoter activity encoded RNAs that were synthesized by B. subtilis during sporulation. Mutant B. subtilis that failed to synthesize sigma 29 (spoIIA, spoIIE) made less RNA that could hybridize to these cloned DNAs than did a mutant (spoIIC) which did synthesize sigma 29 but was blocked at a similar stage in development. A detailed analysis of several of the cloned DNAs demonstrated that they encoded RNAs that were transcribed from approximately the same start site in vivo that E-sigma-29 initiated transcription in vitro. These particular transcripts were present only during the period of sigma-29 abundance (2 to 4 h after the onset of sporulation) in sporulating cells which carried a wild-type allele of the sigma-29 structural gene (spoIIG). We conclude that the isolation procedure used in this study identified genes that are transcribed by E-sigma 29, not only in vitro but also in vivo. Preliminary characterization of the cloned genes indicate that they encoded multiple overlapping RNAs which were each synthesized at unique times during growth or sporulation. This result implies that sigma 29 does not activate a unique population of genes with a novel function in sporulation but rather that it has a temporal role in spore gene control, transcribing those genes required to be active during its period of abundance regardless of their specific function. PMID- 3009402 TI - Cloning and expression of a Saccharomyces diastaticus glucoamylase gene in Saccharomyces cerevisiae and Schizosaccharomyces pombe. AB - A recombinant plasmid pool of the Saccharomyces diastaticus genome was constructed in plasmid YEp13 and used to transform a strain of Saccharomyces cerevisiae. Six transformants were obtained which expressed amylolytic activity. The plasmids each contained a 3.9-kilobase (kb) BamHI fragment, and all of these fragments were cloned in the same orientations and had identical restriction maps, which differed from the map of the STA1 gene (I. Yamashita and S. Fukui, Agric. Biol. Chem. 47:2689-2692, 1983). The glucoamylase activity exhibited by all S. cerevisiae transformants was approximately 100 times less than that of the donor strain. An even lower level of activity was obtained when the recombinant plasmid was introduced into Schizosaccharomyces pombe. No expression was observed in Escherichia coli. The 3.9-kb BamHI fragment hybridized to two sequences (4.4 and 3.9 kb) in BamHI-digested S. diastaticus DNA, regardless of which DEX (STA) gene S. diastaticus contained, and one sequence (3.9 kb) in BamHI-digested S. cerevisiae DNA. Tetrad analysis of crosses involving untransformed S. cerevisiae and S. diastaticus indicated that the 4.4-kb homologous sequence cosegregated with the glucoamylase activity, whereas the 3.9-kb fragment was present in each of the meiotic products. Poly(A)+ RNA fractions from vegetative and sporulating diploid cultures of S. cerevisiae and S. diastaticus were probed with the 3.9-kb BamHI fragment. Two RNA species, measuring 2.1 and 1.5 kb, were found in both the vegetative and sporulating cultures of S. diastaticus, whereas one 1.5-kb species was present only in the RNA from sporulating cultures of S. cerevisiae. PMID- 3009403 TI - Activity of T-DNA borders in plant cell transformation by mini-T plasmids. AB - By using a binary vector system, we examined the requirements for border sequences in T-DNA transformation of plant genomes. Mini-T plasmids consisting of small replicons with different extents of pTiT37 T-DNA were tested for plant tumor-inducing ability in Agrobacterium tumefaciens strain LBA4404 containing helper plasmid pAL4404 (which encodes virulence genes needed for T-DNA transfer). Assays of these bacteria on carrot disks, Kalanchoe leaves, and SR1 Nicotiana tabacum plantlets showed that mini-T plasmid containing full length T-DNA including left and right borders was highly virulent, as were mini-T plasmids containing all onc (oncogenicity) genes and only the right border. In contrast, mini-T plasmids containing all onc genes and only the left border induced tumors only rarely, and a mini-T plasmid containing all onc genes but no T-DNA borders was completely avirulent. Southern hybridization analyses of tumor DNA showed that T-DNA border sequences delimited the extent of the two-border mini-T plasmid transferred and integrated into the plant genome. When only one T-DNA border was present, it formed one end of the transferred DNA, and the other end mapped in the vector sequences. The implications of these results for the mechanism of T DNA transfer and integration are discussed. PMID- 3009404 TI - Does secA mediate coupling between secretion and translation in Escherichia coli? AB - An amber mutation in the secA gene of Escherichia coli causes a pleiotropic decrease in the synthesis of secreted proteins, including maltose-binding protein (MBP) and alkaline phosphatase. Reversal of the inhibition of MBP synthesis in secA(Am) strains by signal sequence mutations in the malE gene has been reported. These results suggest a coupling between secretion and translation which involves an interaction between the signal sequence of nascent polypeptides and a cellular secretion machinery. Further analysis reported here indicated that signal sequence mutations of MBP or alkaline phosphatase did not selectively overcome the inhibition of MBP or alkaline phosphatase synthesis in secA(Am) strains. Rather, at a given time in parallel experiments there was substantial variability among closely isogenic secA(Am) strains in the magnitude of the synthesis block; this variability could account for the earlier results. Further experiments suggested that the inhibition of MBP synthesis in secA(Am) strains was caused by depletion of cyclic AMP, leading to decreased transcription of the malE gene. However, the secretion defects in secA(Am) strains were not affected by cyclic AMP levels. Therefore, we conclude that the reduction in MBP synthesis was a secondary consequence of the primary export defect in the secA(Am) strains. PMID- 3009405 TI - Cloning and molecular characterization of csm mutations allowing expression of catabolite-repressible operons in the absence of exogenous cyclic AMP. AB - The cyclic AMP (cAMP) suppressor mutation (csm) of Escherichia coli has been cloned from strain NCR30 in the HindIII-EcoRI site of pBR322. This mutation has been mapped in or near the crp gene. Wild-type crp DNA hybridized to recombinant plasmids pGM5 and pGM25 containing the cloned csm mutation. These recombinant plasmids encoded a protein product of identical molecular weight and charge as that of the wild-type cAMP receptor protein. Transformants of cya crp deletion strains harboring pBM5 or pGM25 exhibited phenotypic characteristics common to strain NCR30. These included the expression of catabolite-repressible enzymes, such as arabinose isomerase, tryptophanase, beta-galactosidase, and threonine deaminase; the expression of chemotactic and motility genes; cAMP sensitivity; and the accumulation of toxic levels of methylglyoxal. DNA sequence analysis indicated that the Csm suppressor phenotype was attributable to the insertion of a guanosine residue 17 base pairs downstream from the termination codon of the crp structural gene. The guanosine insertion is located in the stem region of the presumed transcriptional termination loop. This stem region contained a unique BssHII restriction site which was used to construct an in vitro deletion in the wild-type crp insert in plasmid pHA7. The resulting plasmid, pGM459, renders transformants having a phenotype common to that conferred by the chromosomal or cloned csm mutation. Our results indicate a novel role for the 3' flanking region of the crp structural gene in the expression of the cAMP receptor protein. PMID- 3009406 TI - Isolation of ntrA-like mutants of Azotobacter vinelandii. AB - A number of chlorate-resistant mutants of Azotobacter vinelandii affected in a general control of nitrogen metabolism were isolated. These mutants could not utilize dinitrogen, nitrate, or nitrite as a nitrogen source. The reason for this inability is that they were simultaneously deficient in nitrogenase and nitrate and nitrite reductase activities. They were complemented by a cosmid carrying a DNA fragment of A. vinelandii able to complement ntrA mutants of Escherichia coli, so they seemed to be ntrA-like mutants. PMID- 3009407 TI - Deletion analysis of the Klebsiella pneumoniae nitrogenase promoter: importance of spacing between conserved sequences around positions -12 and -24 for activation by the nifA and ntrC (glnG) products. AB - The nitrogen fixation promoters of Klebsiella pneumoniae are atypical procaryotic promoters lacking the usual -10 and -35 elements, requiring instead conserved sequences around -12 and -24 for transcriptional activation. By constructing a set of five deletions between the -12 and -24 elements in the nifH promoter, the spacing between the conserved GC and GG motifs at -12 and -24, respectively, has been reduced from the wild-type 10 bases to 9, 8, 6, 5, and 4 bases. The deletion of a single nonconserved nucleotide was sufficient to eliminate transcriptional activation by either nifA or ntrC (glnG). All deletions relieved the multicopy inhibition of chromosomal nif expression normally shown by the nifH promoter. These results demonstrate a stringent requirement for the 10-base spacing found in ntr-activated promoters. In addition, specific sequences around the invariant GG at -24 were shown to be necessary for activation by either nifA or ntrC, with a minimal requirement for nucleotides through to position -27 for this activation. PMID- 3009408 TI - Nodulation inhibition by Rhizobium leguminosarum multicopy nodABC genes and analysis of early stages of plant infection. AB - During analysis of early events in the infection and nodulation of Vicia hirsuta roots inoculated with normal and mutant strains of Rhizobium leguminosarum and strains containing cloned nodulation (nod) genes, a number of novel observations were made. (i) Alternating zones of curled and straight root hairs were seen on roots of V. hirsuta inoculated with the wild-type strain of R. leguminosarum. This phasing of root hair curling was not seen if plants were grown under continuous light or continuous dark conditions. (ii) Reduced nodulation and delayed nodule initiation was observed with a strain carrying a Tn5 mutation in the nodE gene. In addition the phased root hair curling was absent, and root hair curling was observed along the length of the root. (iii) The nodABC genes cloned on a multicopy plasmid in a wild-type strain inhibited nodulation but induced a continuous root hair curling response. Those few nodules that eventually formed were found to contain bacteria which had lost the plasmid carrying the nodABC genes. (iv) With a strain of Rhizobium cured of its indigenous symbiotic plasmid, but containing the cloned nodABCDEF genes, continuous root hair curling on V. hirsuta was observed. However, no infection threads were observed, and surprisingly, it did appear that initial stages of nodule development occurred. Observations of thin sections of these early developing nodules indicated that early nodule meristematic divisions may have occurred but that no bacteria were found within the nodules and no infection threads were observed either within the nodule bumps or within any of the root hairs. It was concluded that for normal infections to occur, precise regulation of the nod genes is required and that overexpression of the root hair curling genes inhibits the normal infection process. PMID- 3009409 TI - Cloning and characterization of livH, the structural gene encoding a component of the leucine transport system in Escherichia coli. AB - The physical location of the genetically defined livH gene was mapped in the 17 kilobase plasmid pOX1 by using transposon Tn5 inactivation mapping and further confirmed by subcloning and complementation analysis. These results indicated that the livH gene maps 3' to livK, the gene encoding the leucine-specific binding protein. Moreover, the nucleotide sequence of the livH gene and its flanking regions was determined. The livH gene is encoded starting 47 base pairs downstream from the livK gene, and it is transcribed in the same direction as the livK gene. The livK-livH intergenic region lacks promoter sequences and contains a GC-rich sequence that could lead to the formation of a stable stem loop structure. The coding sequence of the livH gene, which is 924 base pairs, specifies a very hydrophobic protein of 308 amino acid residues. Expression of livH-containing plasmids in minicells suggested that a poorly expressed protein with an Mr of 30,000 could be the livH gene product. PMID- 3009410 TI - Cloning and expression of the exfoliative toxin B gene from Staphylococcus aureus. AB - Exfoliative toxin type B is produced by bacteriophage group II strains of Staphylococcus aureus and is a causative agent of staphylococcal scalded-skin syndrome. In addition to exfoliative toxin B, most isolates also produce a bacteriocin and are immune to the action of the bacteriocin. These phenotypes, as well as resistance to cadmium, were lost after elimination of a 37.5-kilobase plasmid, pRW001, from S. aureus UT0007. Transduction and transformation showed that pRW001 carries the structural genes for four phenotypic characteristics of S. aureus UT0007: (i) exfoliative toxin B production, (ii) bacteriocin production, (iii) bacteriocin immunity, and (iv) resistance to Cd(NO3)2. The exfoliative toxin B structural gene (etb), which is located on a 1.7-kilobase HindIII fragment of pRW001, was cloned in the plasmid pDH5060 and transformed into phage group III S. aureus RN4220. Transformant clones produced extracellular exfoliative toxin B that was biologically active in the neonatal mouse assay. In the Escherichia coli genetic background, the exfoliative toxin B gene was expressed only after being cloned into the positive selection-expression vector pSCC31. The structural gene for cadmium resistance was also isolated on an HindIII fragment of pRW001 cloned in pDH5060. The loci for the exfoliative toxin B gene and the cadmium resistance gene(s) were identified on a restriction map of plasmid pRW001. PMID- 3009411 TI - Isolation and complementation analysis of 10 methanol oxidation mutant classes and identification of the methanol dehydrogenase structural gene of Methylobacterium sp. strain AM1. AB - A method has been developed for the direct selection of methanol oxidation mutants of the facultative methylotroph Methylobacterium sp. strain AM1 (formerly Pseudomonas sp. strain AM1). Using this direct selection technique, we have isolated mutants of Methylobacterium sp. strain AM1 that are no longer capable of growth on methanol but retain the ability to grow on methylamine. These methanol oxidation (Mox) mutants were complemented with a genomic clone bank of this organism constructed in the broad-host-range cosmid pVK100, and subcloning and Tn5 mutagenesis experiments have assigned the Mox mutants to 10 distinct complementation groups. Using an open reading frame beta-galactosidase fusion vector and antibodies specific for Methylobacterium sp. strain AM1 methanol dehydrogenase, we have identified the methanol dehydrogenase structural gene and determined the direction of transcription. The results suggest that the synthesis and utilization of an active methanol dehydrogenase in this organism requires at least 10 different gene functions. PMID- 3009412 TI - Phenotypic characterization of 10 methanol oxidation mutant classes in Methylobacterium sp. strain AM1. AB - Twenty-five methanol oxidation mutants of the facultative methylotroph Methylobacterium sp. strain AM1 have been characterized by complementation analysis and assigned to 10 complementation groups, Mox A1, A2, A3, and B through H (D. N. Nunn and M. E. Lidstrom, J. Bacteriol. 166:582-591, 1986). In this study we have characterized each of the mutants belonging to the 10 Mox complementation groups for the following criteria: phenazine methosulfate dichlorophenolindophenol dye-linked methanol dehydrogenase activity; methanol dependent whole-cell oxygen consumption; the presence or absence of methanol dehydrogenase protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; the absorption spectra of purified mutant methanol dehydrogenase proteins; and the presence or absence of the soluble cytochrome c proteins of Methylobacterium sp. strain AM1, as determined by reduced-oxidized difference spectra and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this information, we have proposed functions for each of the genes deficient in the mutants of the 10 Mox complementation groups. These proposed gene functions include two linked genes that encode the methanol dehydrogenase structural protein and the soluble cytochrome cL, a gene encoding a secretion function essential for the synthesis and export of methanol dehydrogenase and cytochrome cL, three gene functions responsible for the proper association of the pyrrolo-quinoline quinone prosthetic group with the methanol dehydrogenase apoprotein, and four positive regulatory gene functions controlling the expression of the ability to oxidize methanol. PMID- 3009414 TI - Effects of K+ on the proton motive force of Bradyrhizobium sp. strain 32H1. AB - In previous studies, respiring Bradyrhizobium sp. strain 32H1 cells grown under 0.2% O2, conditions that derepress N2 fixation, were found to have a low proton motive force of less than -121 mV, because of a low membrane potential (delta psi). In contrast, cells grown under 21% O2, which do not fix N2, had high proton motive force values of -175 mV or more, which are typical of respiring bacteria, because of high delta psi values. In the present study, we found that a delta psi of 0 mV in respiring cells requires growth in relatively high-[K+] media (8 mM), low O2 tension, and high internal [K+]. When low-[O2], high-[K+]-grown cells were partially depleted of K+, the delta psi was high. When cells were grown under 21% O2 or in media low in K+ (50 microM K+), the delta psi was again high. The transmembrane pH gradient was affected only slightly by varying the growth or assay conditions. In addition, low-[O2], high-[K+]-grown cells had a greater proton permeability than did high-[O2]-grown cells. To explain these findings, we postulate that cells grown under conditions that derepress N2 fixation contain an electrogenic K+/H+ antiporter that is responsible for the dissipation of the delta psi. The consequence of this alteration in K+ cycling is rerouting of proton circuits so that the putative antiporter becomes the major pathway for H+ influx, rather than the H+-ATP synthase. PMID- 3009413 TI - IS1-dependent generation of high-copy-number replicons from bacteriophage P1 Ap Cm as a mechanism of gene amplification. AB - Mutant P1 Ap Cm lysogens were isolated in which the drug resistance genes resident on the plasmid prophage P1 Ap Cm are amplified by a novel mechanism. The first step required for amplification is IS1-mediated rearrangement of the P1 Ap Cm prophage. The drug resistance genes are amplified from the rearranged P1 Ap Cm prophage by the formation of a plasmid (P1dR) which contains the two resistance genes. The P1dR plasmid is an independent replicon about one-half the size of P1 Ap Cm that can be maintained at a copy number eightfold higher than that at which P1 Ap Cm can be maintained. It contains no previously identified replication origin and is dependent on the Rec+ function of the host. PMID- 3009415 TI - Bacteriophage involvement in group A streptococcal pyrogenic exotoxin A production. AB - Lysogenic conversion has been suggested as a mechanism of control of group A streptococcal pyrogenic exotoxin type A production. Digestion of DNA from two converting bacteriophages, 3GL16 and T12, with a variety of restriction endonucleases yielded identical DNA fragments upon electrophoresis in agarose gels. Several known A toxin-positive strains that did not appear to produce converting phage upon induction were analyzed for toxin and phage DNA. Strains, including NY5, 594, and C203S, were shown by hybridization studies to carry the A toxin gene (speA) adjacent to chromosomally inserted phage fragments, homologous to phage T12 DNA, which may represent defective converting phages. The phage T12 att site mapped adjacent to speA. These data suggest that phage T12 acquired the A toxin gene from the bacterial genome. All streptococcal strains tested that were A toxin negative by Ouchterlony immunodiffusion failed to show any hybridization to speA-specific probes. PMID- 3009416 TI - Genetic locus in Rhizobium japonicum (fredii) affecting soybean root nodule differentiation. AB - A genetic locus in fast-growing Rhizobium japonicum (fredii) USDA 191 (Fix+ on several contemporary soybean cultivars) was identified by random Tn5 mutagenesis as affecting the development and differentiation of root nodules. This mutant (MU042) is prototrophic and shows no apparent alterations in its surface properties. It induces aberrant nodules, arrested at the same early level of differentiation, on all its host plants. An 8.1-kilobase EcoRI fragment containing Tn5 was cloned from MU042. In USDA 191 as well as another fast-growing strain, USDA 201, the affected locus was found to be unlinked to the large symbiotic plasmid and appears to be chromosomal. An analogous sequence has been shown to be present in Bradyrhizobium japonicum (J. Stanley, G.G. Brown, and D.P.S. Verma, J. Bacteriol. 163:148-154, 1985) as well as in R. trifolii and R. meliloti. MU042 was complemented for effective nodulation of soybean by a cosmid clone from USDA 201, and the complementing locus was delimited to a 6-kilobase EcoRI subfragment. An R. trifolii strain (MU225), whose indigenous symbiotic plasmid was replaced by that of strain USDA 191, induced more highly differentiated nodules on soybean than did MU042. This suggests that the mutation in MU042 can be functionally substituted by similar loci of other fast-growing rhizobia. Leghemoglobin and nodulin-35 (uricase II) were present in the differentiated Fix- nodules induced by MU225, whereas both were absent in MU042 induced pseudonodule structures. PMID- 3009418 TI - Cloning and analysis of the sfrB (sex factor repression) gene of Escherichia coli K-12. AB - The sfrB gene of Escherichia coli K-12 and the rfaH gene of Salmonella typhimurium LT2 are homologous, controlling expression of the tra operon of F and the rfa genes for lipopolysaccharide synthesis. We have determined a restriction map of the 19-kilobase ColE1 plasmid pLC14-28 which carries the sfrB gene of E. coli. After partial Sau3A digestion of pLC14-28, we cloned a 2.5-kilobase DNA fragment into the BamHI site of pBR322 to form pKZ17. pKZ17 complemented mutants of the sfrB gene of E. coli and the rfaH gene of S. typhimurium for defects of both the F tra operon and the rfa genes. pKZ17 in minicells determines an 18 kilodalton protein not determined by pBR322. A Tn5 insertion into the sfrB gene causes loss of complementing activity and loss of the 18-kilodalton protein in minicells, indicating that this protein is the sfrB gene product. These data indicate that the sfrB gene product is a regulatory element, since the single gene product elicits the expression of genes for many products for F expression and lipopolysaccharide synthesis. PMID- 3009417 TI - Structural genes encoding the thermophilic alpha-amylases of Bacillus stearothermophilus and Bacillus licheniformis. AB - The genes encoding the thermostable alpha-amylases of Bacillus stearothermophilus and B. licheniformis were cloned in Escherichia coli, and their DNA sequences were determined. The coding and deduced polypeptide sequences are 59 and 62% homologous to each other, respectively. The B. stearothermophilus protein differs most significantly from that of B. licheniformis in that it possesses a 32 residue COOH-terminal tail. Transformation of E. coli with vectors containing either gene resulted in the synthesis and secretion of active enzymes similar to those produced by the parental organisms. A plasmid was constructed in which the promoter and the NH2-terminal two-thirds of the B. stearothermophilus coding sequence was fused out of frame to the entire mature coding sequence of the B. licheniformis gene. Approximately 1 in 5,000 colonies transformed with this plasmid was found to secrete an active amylase. Hybridization analysis of plasmids isolated from these amylase-positive colonies indicated that the parental coding sequences had recombined by homologous recombination. DNA sequence analysis of selected hybrid genes revealed symmetrical, nonrandom distribution of loci at which the crossovers had resolved. Several purified hybrid alpha-amylases were characterized and found to differ with respect to thermostability and specific activity. PMID- 3009419 TI - Characterization of genetic transformation in Streptococcus mutans by using a novel high-efficiency plasmid marker rescue system. AB - We developed a marker rescue system for study of competence development and genetic transformation in Streptococcus mutans. The system involved the recombinational rescue of a tetracycline resistance (Tcr) determinant by a homologous, inactive locus (Tcs because of a small deletion). Streptococcal cells harboring this in vitro-prepared Tcs construct (pVA1208) were restored to Tcr when plasmid (pVA981) DNA was used as donor material. pVA981 contained the intact streptococcal Tcr locus and was unable to autonomously replicate in streptococci. Marker rescue with this system followed first-order kinetics and occurred at a frequency 8- or 160-fold higher than did transformation with homologous chromosomal or plasmid DNA, respectively. By using the rescue system, we were able to confirm that competence of S. mutans appeared to be inducible. This was indicated by a sequential increase and then decrease in Tcr transformation frequencies during growth in complex medium. Also, donor DNA binding was not sequence specific, since the recovery of Tcr transformants was reduced by increasing the concentrations of heterologous DNA. We investigated the fate of donor DNA and the kinetics of plasmid establishment in the transformation of S. mutans with plasmid DNA. Monomeric plasmid molecules transformed S. mutans as a second-order process, whereas multimeric plasmid DNA and chromosomal markers were recovered as a first-order process. Approximately 50% of the initially bound donor plasmid DNA was found to remain in a trichloroacetic acid-insoluble form. Our results suggested that molecular cloning in S. mutans would be conducted most efficiently by using helper plasmid systems or shuttle vectors and that gene transfer by transformation of S. mutans occurred in a manner similar to that observed in Streptococcus sanguis. PMID- 3009421 TI - Unrelated conjugative plasmids have sequences which are homologous to the leading region of the F factor. AB - Conjugative plasmids from various incompatibility groups which carry DNA homologous to the ssb gene of the F factor were found to have additional homology with the F factor. This region homologous with F was located on both sides of the ssb gene and occupied a considerable part of the leading region, i.e., the 12.9 kilobase portion of F transferred first during conjugation. This region was the only region of the F factor which has a homologous counterpart on many plasmids. PMID- 3009420 TI - Phosphate starvation regulon of Salmonella typhimurium. AB - Several phosphate-starvation-inducible (psi) genetic loci in Salmonella typhimurium were identified by fusing the lacZ gene to psi promoters by using the Mu d1 and Mu d1-8 bacteriophages. Although several different starvation conditions were examined, the psi loci responded solely to phosphate deprivation. A regulatory locus, psiR, was identified as controlling the psiC locus. The psiR locus did not affect the expression of the Escherichia coli phoA locus or any of the other psi loci described. PMID- 3009422 TI - Analysis of the products of the Myxococcus xanthus frz genes. AB - The frizzy (frz) genes of Myxococcus xanthus control the ability of cells to reverse direction of gliding motility. The orientation of the frz genes was studied by isolating transcriptional fusions with the transposon derivative Tn5 lac. The frz genes were then cloned in the proper orientation in an expression vector. By using maxicell experiments, we were able to identify several labeled bands which were plasmid encoded. To identify the labeled proteins and their respective genes, we constructed deletion plasmids in which various regions of the insert DNA had been removed. The plasmid-encoded proteins were then labeled in maxicell experiments, and the bands which correspond to the frzCD, frzE, and frzF gene products were identified. The sizes of the gene products agreed with the genetic and physical map of the cloned DNA. PMID- 3009423 TI - Successful plasma exchange for a patient with chronic demyelinating polyneuropathy and cold agglutinin disease due to anti-Pra. PMID- 3009425 TI - Barley leaf peroxidase: purification and characterization. AB - Peroxidase was prepared from extracts of barley leaves and separated into seven components, different in pI. The purification procedure comprised two parts. The first part was based on the fact that all the components had practically the same molecular weights. It consisted of fractionations with acetone and ammonium sulfate, ion-exchange chromatographies on CM-cellulose and DEAE-Sepharose CL-6B, and molecular-sieve chromatography on Ultrogel AcA44; the components were all purified together to near homogeneity on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, and the procedure resulted in 1,200-fold purification with a yield of 39%. The ion-exchange chromatographies were carried out under conditions such that the components would not be adsorbed. In the second part, the enzyme preparation was separated into the seven components by repeating isoelectric electrophoresis. Their isoelectric points (pI) were 6.3, 6.8, 7.4, 8.3, 8.5, 8.7, and 9.3. The components other than the pI 6.3 and 6.8 components were each purified to homogeneity in the electrophoresis. The seven components thus prepared were the same in molecular weight on SDS-gel electrophoresis (44,000) and showed absorption maxima at the same wave-lengths (403, 496, and 534 nm), RZ (A403/A275) ranging from 2.09 to 2.81. Their protoheme IX contents were 0.81-1.07 mol/mol, and their true sugar contents 15-26% (g/g). The amino acid compositions suggest that the five components described above are not real isoenzymes, but exhibit different pI values due to differences in glycosyl residue. The pI 9.3 component was crystallized in spite of its high sugar content. PMID- 3009424 TI - Comparison of inhibition by a tumor promoter (12-O-tetradecanoylphorbol-13 acetate) of expression of the differentiated phenotype of chondrocytes in rabbit costal chondrocytes in culture with inhibition by retinoic acid. AB - Both retinoids and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit expression of the differentiated phenotype by rabbit costal chondrocytes in culture, as judged by morphological changes and decreased sulfation of glycosaminoglycans (GAG). However, the inhibition of the differentiated phenotype of chondrocytes in TPA-treated cells is restored by parathyroid hormone (PTH), while the inhibition by retinoids is not [Takigawa et al. (1982) Mol. Cell. Biochem. 42, 145-153; Takigawa et al. (1983) Cell Differ. 13, 283-291]. In the present study, we examined the difference between TPA-treated chondrocytes and retinoic acid-treated chondrocytes to determine the mechanism of the restoration of the differentiated phenotype in de-differentiated cells treated with TPA. PTH increased the activity of ornithine decarboxylase [ODC; EC 4.1.1.17], a rate limiting enzyme of polyamine biosynthesis, and proteoglycan synthesis in chondrocytes pretreated with TPA as well as in normal chondrocytes. The maximal stimulations of ODC activity and GAG synthesis were observed 4 h and 24-36 h, respectively, after addition of PTH. The dose-response curve for ODC induction by PTH was parallel to that of PTH-stimulated proteoglycan synthesis both in TPA treated chondrocytes and in normal chondrocytes. PTH also increased the intracellular cyclic AMP level after 2 min in TPA-treated cells as in normal cells. Addition of dibutyryl cyclic AMP (DBcAMP) induced ODC and restored proteoglycan synthesis in TPA-treated cells. The dose-response curve for induction of ODC by DBcAMP was parallel to that of DBcAMP-stimulated proteoglycan synthesis in both TPA-treated chondrocytes and normal chondrocytes. On the other hand, the increases by PTH in the intracellular cyclic AMP level, ODC activity, and proteoglycan synthesis were inhibited in chondrocytes pretreated with a combination of TPA and retinoic acid as well as in those pretreated with retinoic acid alone. TPA stimulated the syntheses of DNA and RNA in chondrocytes but did not increase the cyclic AMP level or ODC activity. PTH and DBcAMP inhibited the syntheses of DNA and RNA both in TPA-treated cells and in normal cells. These results suggest that ODC induction mediated by elevation of cyclic AMP plays an important role in re-differentiation of de-differentiated cells pretreated with these agents. PMID- 3009426 TI - Crystallographic study of cytochrome c553 from Desulfovibrio vulgaris Miyazaki. AB - Cytochrome c553 from the sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki, has been crystallized. The combination of microdialysis and vapor diffusion allowed successful crystallization. The crystals were of good quality, and useful data were obtained that extended to the nominal resolution of 1.3 A. The space group is P4(3)2(1)2 with cell dimensions of a = b = 42.7 A, c = 103.4 A. More than twenty heavy-atom reagents were screened with the isomorphous replacement technique, and only the mersalyl derivative could be used for the phase determination. The single isomorphous replacement method combined with the anomalous scattering effect of the Hg-atom in mersalyl and the Fe-atom of the heme group was used for the phase determination. PMID- 3009429 TI - Alterations in local chromatin structure accompany thyroid hormone induction of growth hormone gene transcription. AB - The chromatin structure of the growth hormone gene and flanking DNA was analyzed in GC rat pituitary tumor cells, which appropriately express and regulate the gene. Thyroid hormone induced the DNase I hypersensitivity of a chromatin domain spanning the transcriptional initiation site from approximately -200 to +150. Three inducible hypersensitive sites were discerned in this region. One of these sites was in the first intron of the gene. The second site was within a region of 5' flanking DNA which promotes accurate, thyroid hormone-regulated transcriptional initiation. Centered between these two sites was a third hormone inducible hypersensitive site mapping at the position of the TATA sequence. The hormone responsiveness of these hypersensitive sites suggests that occupied 3,5,3'-triiodo-L-thyronine receptor interacts with sequences flanking the growth hormone promoter, perhaps facilitating the binding or activation of a transcription factor at the TATA homology. Three hormone-independent hypersensitive sites were identified near or within intron or flanking DNA sequences similar to Chinese hamster ovary type 2 repetitive DNA. Two additional hormone-independent hypersensitive sites were located more than 1000 nucleotides up-stream of the growth hormone promoter. These sites were present in the cloned genomic DNA and were the only distinct sites found in rat spleen and cultured rat liver cells. Thyroid hormone treatment of GC cells appeared to increase the moderate DNase I sensitivity of the growth hormone gene region beyond that found in deinduced or glucocorticoid-treated cells. Dexamethasone had no discernible effect on the chromatin structure of the gene or flanking DNA in these studies. PMID- 3009428 TI - The microcrystal alignment in human tooth enamel from carious and non-carious teeth in relation to caries susceptibility. AB - The degree of microcrystal alignment in the intact enamel from carious and non carious upper incisors, lower incisors and upper canines was determined by means of electron paramagnetic resonance. The results show that a low degree of alignment is not a generally valid indication for caries-susceptibility. Upper canines have a high degree of crystallite alignment whereas lower incisors have a relatively low one. With the exception of the upper central incisors no differences in microcrystal alignment are found between the intact enamel from carious and non-carious lower incisors and upper canines. These results indicate that a possible caries-susceptibility of tooth enamel reflected by low R-values is not solely determined by the degree of microcrystal alignment. PMID- 3009430 TI - Red blood cells contain a pathway for the degradation of oxidant-damaged hemoglobin that does not require ATP or ubiquitin. AB - It is generally accepted that ATP is required for intracellular protein breakdown. Reticulocytes contain a soluble ATP-dependent pathway for the degradation of highly abnormal proteins and for the elimination of certain proteins during cell maturation. Reticulocytes and erythrocytes also selectively degrade proteins damaged by oxidation. When these cells were exposed to oxidants, such as phenylhydrazine or nitrite, they showed a large increase in protein breakdown. This oxidant-induced proteolysis was not inhibited in cells depleted of ATP. However, ATP depletion did prevent the degradation of pre-existent cell proteins. In reticulocyte extracts, phenylhydrazine-treated hemoglobin is also degraded rapidly by an ATP-independent process, unlike endogenous proteins and many exogenous polypeptides. This lack of an ATP requirement means that the degradation of oxidant-damaged proteins does not require ligation to ubiquitin (even though phenylhydrazine treatment does make hemoglobin a very good substrate for ubiquitin conjugation). In many respects, the pathway for breakdown of oxidant-treated hemoglobin differs from the ATP-dependent process. The latter has a much higher activation energy than the degradation of oxidized proteins. The ATP-dependent process is inhibited by hemin, 3,4-dichloroisocoumarin, diisopropylfluorophosphate and N-ethylmaleimide. The ATP-independent pathway is less sensitive to N-ethylmaleimide, hemin, and 3,4-dichloroisocoumarin and is not affected by diisopropylfluorophosphate. In addition, only the ATP-dependent proteolytic process is inactivated by dilution or incubation at 37 degrees C in the absence of nucleotides. Reticulocytes thus contain multiple soluble systems for degrading proteins and can rapidly hydrolyze certain types of abnormal proteins by either an ATP-independent or ATP-dependent process. Erythrocytes lack the ATP-dependent process present in reticulocytes; however, erythrocytes retain the capacity to degrade oxidant-damaged hemoglobin. These two processes probably are active in the elimination of different types of abnormal proteins. PMID- 3009427 TI - Regulation of respiration and ATP synthesis in higher organisms: hypothesis. AB - The present view on the regulation of respiration and ATP synthesis in higher organisms implies only Michaelis-Menten type kinetics and respiratory control as regulatory principles. Recent experimental observations, suggesting further regulatory mechanisms at respiratory chain complexes, are reviewed. A new hypothesis is presented implying regulation of respiration and ATP synthesis in higher organisms mainly via allosteric modification of respiratory chain complexes, in particular of cytochrome c oxidase. The allosteric effectors, e.g., metabolites, cofactors, ions, hormones, and the membrane potential are suggested to change the activity and the coupling degree of cytochrome c oxidase by binding to specific sites at nuclear coded subunits. Recent results on the structure and activity of cytochrome c oxidase, supporting the hypothesis, are reviewed. PMID- 3009431 TI - Reversible inactivation of vasopressin and angiotensin II binding to hepatocyte membranes by a calcium-dependent, cytosolic protein. AB - A cytosolic protein is described which inhibits the binding of vasopressin and angiotensin to their rat liver receptors in the presence of calcium. The binding of insulin and transferrin was unaffected. Inhibition was temperature-dependent; it was maximal in 10 min at 37 degrees C, but required longer incubation times at lower temperatures. The pH optimum was 7.4. Inhibition also required the presence of calcium, with half-maximal inhibition at 6-8 microM calcium, but did not require any other low molecular weight cofactors. Inhibition could be reversed by washing the membranes at pH 5.5, but not by incubation with EGTA. Sephacryl S-300 chromatography showed that activity eluted in two peaks with approximate molecular weights of 70,000 and 150,000. In the presence of calcium, the inhibitory activity eluted at 150,000; in the absence of calcium, most of the inhibitory activity eluted at 70,000. A radiolabeled cytosolic protein with a molecular weight of 70,000 was eluted from inhibited rat liver membranes at pH 5.5 as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We propose that vasopressin and angiotensin II, which both mobilize calcium in hepatocytes via phosphatidylinositol turnover, can, by this same mechanism, activate a protein(s) which reduces further binding to their receptors. PMID- 3009433 TI - Mobilization of intracellular calcium by alpha 1-adrenergic receptor activation in muscle cell monolayers. AB - The fluorescent chelating agent quin 2 has been employed to monitor alterations of intracellular free Ca2+ concentrations ([Ca2+]i) in response to alpha 1 adrenergic receptor activation in adherent BC3H-1 cells. To correlate the kinetics of [Ca2+]i changes with transmembrane fluxes of this ion, continuous monitoring of [Ca2+]i has been undertaken on a monolayer of cells. Previous measurements of the transmembrane efflux of Ca2+ show a distinct lag in the response over a range of phenylephrine concentrations. By contrast, the elevation of [Ca2+]i is rapid (t1/2 approximately 2 s) and maintained for 30 s before it begins to decline to basal concentrations. The differences in kinetics indicate that the temporal delay in cellular Ca2+ efflux results from either activation of the transport system for Ca2+ extrusion or translocation of free Ca2+ to the transport site. The decline of [Ca2+]i with continued agonist exposure parallels both the efflux kinetics from the cell and the decline of total cellular Ca2+. At a time when free [Ca2+]i approaches the resting concentration, total cellular Ca2+ is reduced to a steady state value of 60% of that seen prior to stimulation. The Kact for phenylephrine-stimulated elevation in [Ca2+]i on the monolayer is 0.51 microM, which is similar to the Kact of 0.90 microM observed for phenylephrine-activated 45Ca2+ efflux. Addition of phentolamine subsequent to phenylephrine addition immediately reverses the agonist-stimulated Ca2+ mobilization, initiating a rapid return of [Ca2+]i to resting levels. A comparison of the kinetics of Ca2+ mobilization with its transmembrane flux suggests that the agonist augments the rate of recycling of intracellular Ca2+ between the free and bound states rather than causing release as a single bolus from the bound stores. PMID- 3009432 TI - Protein secretion from Saccharomyces cerevisiae directed by the prepro-alpha factor leader region. AB - The Saccharomyces cerevisiae secretory process was studied by evaluating secretion efficiency, processing efficiency, and the efficiency of protein folding for hybrid proteins containing the yeast prepro-alpha-factor leader region. Secretion of three proteins, beta-endorphin, calcitonin, and a consensus alpha-interferon (IFN-Con1), were compared in terms of secretion efficiency into the culture medium, beta-Endorphin and calcitonin, both small proteins, were found to be efficiently secreted from logarithmically grown cells. In contrast, the larger IFN-Con1 accumulated in the periplasmic space and cell wall. The glycosylated, unprocessed prepro-alpha-factor/IFN-Con1 fusion protein was also found to be secreted into the culture medium. The presence of (Glu-Ala) dipeptides in the alpha-factor spacer peptide increased the efficiency of cleavage at Lys-Arg in the prepro-alpha-factor/IFN-Con1 protein fusion. Purified secreted IFN-Con1 was structurally characterized to determine the effect of passage through the yeast secretory pathway on the fidelity and efficiency of protein folding. The disulfide structure of the secreted protein was found to be identical with that reported for the native human alpha-interferons. PMID- 3009434 TI - Isolation of a rat parvalbumin gene and full length cDNA. AB - The complete sequence of the rat parvalbumin mRNA was determined by sequencing a near full length cDNA clone and a primer extension product of the 5' untranslated region of this clone. The parvalbumin sequence contained 72 nucleotides of the 5' untranslated region, 333 nucleotides of the coding sequence, and 183 nucleotides of the 3' untranslated sequence in the case of the shorter parvalbumin messenger RNA or 556 nucleotides in the case of the larger parvalbumin messenger RNA. The two RNAs were derived from a single primary transcript since two clones were sequenced which were identical except for the site of polyadenylation. The polyadenylation signal of the shorter, more abundant RNA was AATAAA while the putative signal for the larger RNA was GATAAA. A 32-base pair sequence comprising part of the 5' untranslated region and the first seven codons of the parvalbumin cDNA was 81% homologous to a region of the chicken calmodulin cDNA encoding part of the first Ca2+-binding domain. This homology indicates that this portion of the parvalbumin mRNA may have evolved from a primordial first domain. A parvalbumin genomic clone which hybridized to probes derived from both the coding and 3' untranslated region of the cDNA was cloned from a rat genomic library. Partial sequencing identified an uninterrupted stretch of 585 base pairs identical to the 3' end of the parvalbumin cDNA. Two introns were localized in the genomic clone. Their position with respect to the parvalbumin amino acid sequence corresponded to the intron locations found in genes of several other Ca2+-binding proteins. This result indicates that, despite the deletion of one Ca2+-binding domain, the remainder of the parvalbumin gene has maintained the structural pattern of the Ca2+-binding protein gene family. PMID- 3009435 TI - Diadenosine 5',5'''-P1, P4-tetraphosphate alpha, beta-phosphorylase from yeast supports nucleoside diphosphate-phosphate exchange. AB - Homogeneous diadenosine 5',5'''-P1,P4-tetraphosphate alpha, beta-phosphorylase (Ap4A-phosphorylase), the enzyme recently found in yeast (Guranowski, A., and Blanquet, S. (1985) J. Biol. Chem. 260, 3542-3547) catalyzes an exchange reaction between the beta-phosphate of nucleoside diphosphate (NDP) and orthophosphate from the medium (Pi). The common purine and pyrimidine ribonucleoside diphosphates as well as ADP analogs modified either in aglycone, sugar, or at the anhydride bond beta-position are substrates. The Km and rate values for the NDP Pi exchange reaction were estimated at pH optima. These optima are 6.5 for UDP, 7.0 for ADP or CDP, and 8.0 for GDP. In the presence of 10 mM K2HPO4, 0.1 mM EDTA, and 100 mM Hepes/KOH (pH 7.0), the Km for ADP is 0.7 mM with a rate constant at saturating ADP of 96 s-1. The Km value for orthophosphate is 2 mM. In the NDP-Pi exchange reaction, phosphate can be substituted with arsenate and apparent arsenolysis of NDPs yields corresponding nucleoside monophosphates. The same pH optimum of 6.5 is found for arsenolysis of ADP, GDP, and CDP. Whereas the Ap4A phosphorylase sulfhydryl groups are essential for catalyzing the Ap4A phosphorolysis, the NDP-Pi exchange reactions, and the arsenolysis of NDPs, the divalent metal ions (Mg2+, Mn2+, Ca2+, Co2+, and Cd2+), which had been shown to be essential cofactors of the former reaction, are not required for the two latter ones. Used at concentrations which are optimum for Ap4A phosphorolysis, the cations (particularly Mg2+ and Cd2+) inhibit the NDP-Pi exchange and the arsenolysis of NDPs. Interestingly, the Ap4A phosphorylase exhibits higher specificity for adenosine 5'-phosphosulfate (APS) than for any other NDP tested. The V/Km ratio is almost 5-fold higher with APS than with ADP. However, in the presence of orthophosphate, the APS is irreversibly converted to ADP. Thus, the enzyme displays a property already attributed to ADP-sulfurylase (EC 2.7.7.5), (Grunberg-Manago, M., Del Campillo-Campbell, A., Dondon, L., and Michelson, A. M. (1966) Biochim. Biophys. Acta 123, 1-16; Nicholls, R. G. (1977) Biochem. J. 165, 149-155). PMID- 3009437 TI - Accessibility of the cytochrome a heme in cytochrome c oxidase to exchangeable protons. AB - The comparison of the resonance Raman spectrum of cytochrome a2+ from cytochrome oxidase in deuterated buffers to that in protonated buffers reveals many lines that have different frequency or intensity. Some of the frequency differences are very large, e.g. on the order of 10 cm-1. From these differences in the Raman spectra, we infer that the heme pocket is readily accessible to protons and that labile groups are either on the heme or interact strongly with it. These data suggest the possibility of direct participation in proton translocation and/or oxygen protonation by the heme of cytochrome a. PMID- 3009436 TI - Mechanism of DNA cleavage induced by sodium chromate(VI) in the presence of hydrogen peroxide. AB - Reactivities of chromium compounds with DNA were investigated by the DNA sequencing technique using 32P 5'-end-labeled DNA fragments, and the reaction mechanism was investigated by ESR spectroscopy. Incubation of double-stranded DNA with sodium chromate(VI) plus hydrogen peroxide or potassium tetraperoxochromate(V) led to the cleavage at the position of every base, particularly of guanine. Even without piperidine, the formation of oligonucleotides was observed, suggesting the breakage of the deoxyribose phosphate backbone. ESR studies using hydroxyl radical traps demonstrated that hydroxyl radical is generated both during the reaction of sodium chromate(VI) with hydrogen peroxide and the decomposition of potassium tetraperoxochromate(V), and that hydroxyl radical reacts significantly not only with mononucleotides but also with deoxyribose 5-phosphate. ESR studies using a singlet oxygen trap demonstrated that singlet oxygen is also generated both by the same reaction and decomposition, and reacts significantly with deoxyguanylate, but scarcely reacts with other mononucleotides. Furthermore, ESR studies suggested that tetraperoxochromate(V) is formed by the reaction of sodium chromate(VI) with hydrogen peroxide. These results indicate that sodium chromate(VI) reacts with hydrogen peroxide to form tetraperoxochromate(V), leading to the production of the hydroxyl radical, which causes every base alteration and deoxyribose phosphate backbone breakage. In addition, sodium chromate(VI) plus hydrogen peroxide generates singlet oxygen, which subsequently oxidizes the guanine residue. The mechanism by which both hydroxyl radical and singlet oxygen are generated during the reaction of sodium chromate(VI) with hydrogen peroxide was presented. Finally, the possibility that this reaction may be one of the primary reactions of carcinogenesis induced by chromate(VI) is discussed. PMID- 3009438 TI - The involvement of cellular ATP in receptor-mediated internalization of epidermal growth factor and hormone-induced internalization of beta-adrenergic receptors. AB - Beta-Adrenergic receptors and epidermal growth factor receptors are both expressed on the cell surface of human astrocytoma cells. Incubation with a catecholamine or epidermal growth factor results in rapid internalization of the respective receptor. The internalized receptors co-migrate in light fractions on sucrose gradients. Astrocytoma cells maintain a constant ATP concentration by either glycolytic or mitochondrial ATP production. When cells are incubated in a medium depleted of substrates for glycolysis and gluconeogenesis, addition of inhibitors of mitochondrial ATP synthesis causes a rapid reduction in cellular ATP content. An immediate return to control ATP levels occurs upon addition of an appropriate nutrient, such as glucose. Decreasing the cellular ATP content to less than 10% of control markedly inhibits internalization of beta-adrenergic receptors and epidermal growth factor. The inhibition of endocytosis is reversed as soon as the intracellular ATP content is restored. Previous work by others (Clarke, B.L., and Weigel, P.H. (1985) J. Biol. Chem. 260, 128-133) suggested that ATP is not required for internalization (per se) of asialoglycoprotein in hepatocytes but was required for recycling of the asialoglycoprotein receptor. In contrast, our results indicate that in astrocytoma cells the process of internalization of epidermal growth factor and beta-adrenergic receptors, per se, is highly ATP dependent. PMID- 3009439 TI - Platelet ADP receptor and alpha 2-adrenoreceptor interaction. Evidence for an ADP requirement for epinephrine-induced platelet activation and an influence of epinephrine on ADP binding. AB - The nucleotide affinity analog 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) is a potent irreversible inhibitor of ADP-mediated platelet activation. Utilizing this compound, the role of ADP in epinephrine-mediated platelet activation was evaluated. Pretreatment of platelets with FSBA under conditions producing covalent incorporation was able to completely block epinephrine-stimulated aggregation of human platelets. In addition, the exposure of latent fibrinogen binding sites by epinephrine was also inhibited in platelets modified by FSBA. The inhibition of epinephrine-mediated activation of the cells was time dependent, reflecting the need for covalent modification of the ADP receptor by FSBA. The inhibitory effect of FSBA was not due to effects on the affinity of binding methyl [3H]yohimbine or the number of platelet alpha 2-adrenergic receptors. Studies of the effect of epinephrine on the ability of ADP to protect against FSBA incorporation demonstrated that epinephrine can increase the affinity of ADP for its receptor 10-fold without affecting the total amount of FSBA covalently bound. This effect of epinephrine is mediated through the alpha 2 adrenoreceptor since the effect can be reversed by the competitive antagonist, methyl yohimbine. These results suggest that promotion of platelet aggregation and the exposure of fibrinogen receptors by epinephrine is dependent on ADP. The mechanism by which epinephrine renders low concentrations of ADP effective appears to be mediated by an increased avidity of the ADP receptor for the nucleotide. PMID- 3009440 TI - Stereoconfiguration markedly affects the biochemical and biological properties of phosphorothioate analogs of 2-5A core, (A2'p5')2A. AB - The diester bonds of phosphorothioate trimer analogs of (A2'p5')2A (2-5A core) of the Sp stereoconfiguration were found to be extremely stable to hydrolysis by both serum and cellular phosphodiesterases. The corresponding Rp isomers, although still more stable than parent ppp(A2'p5')2A (2-5A), were significantly more susceptible to enzymatic hydrolysis than were the Sp isomers. Utilization of these novel 2-5A trimer isomers containing various combinations of Sp or Rp configurations at the internucleotidic phosphorothioate linkages revealed a further specificity of this enzymatic hydrolysis. Thus, the stereoconfiguration of the bond adjacent to the one undergoing hydrolysis influenced the rate of enzymatic hydrolysis, as well as did the chain length of the oligomer. The most stable trimer analog, which contained both internucleotide phosphorothioate linkages of the Sp configuration, had a half-life of 30 days in serum, which is a 1500-fold increase over that of parent 2-5A core. This is the first report on biochemical stability of an oligonucleotide containing more than one phosphorothioate linkage of the Sp configuration and is the first demonstration that a phosphorothioate internucleotide bond of the Sp configuration can increase the enzymatic stability of an adjacent phosphorothioate bond. In marked contrast to all previous 2-5A core analogs of increased stability, the activity (antiproliferative and antiviral) of the stable phosphorothioate 2-5A core analogs was obtained with the intact trimer, i.e., it was not attributed to antimetabolite degradation products. PMID- 3009441 TI - The NADPH:O2 oxidoreductase of human neutrophils. Stoichiometry of univalent and divalent reduction of O2. AB - The ratio of superoxide production to oxidation of NADPH affected by the NADPH:O2 oxidoreductase of human neutrophils is strongly influenced by pH, NADPH substrate concentration, aging of the enzyme, or its exposure to excess deoxycholate. Freshly prepared enzyme exhibited a Km for NADPH of 52 microM as determined by assaying NADPH oxidase activity, or approximately 33 microM by measurement of superoxide formation. In the range of 100-150 microM NADPH at pH 7.6 and in the presence of 0.06% deoxycholate, the univalent flux of electron equivalents given up by NADPH to O2 was 99%. Following storage of the oxidoreductase overnight on ice, its Km for NADPH rose to 125 microM as determined by monitoring oxidation of NADPH but was unaltered when measured in terms of superoxide production. Concomitantly, its capacity to affect univalent reduction of O2 fell approximately 20-30% under the same assay conditions. Univalent flux rates of less than 40% were observed with exposure of the enzyme to concentrations of deoxycholate in excess of 0.1% or to pH values below 6.0 or above 8.0. The capacity of the enzyme to carry out univalent reduction fell with increasing NADPH concentrations in a manner resembling that previously reported with increasing concentrations of xanthine in the case of xanthine oxidase (Fridovich, I. (1970) J. Biol. Chem. 245, 4053-4057). The reduced form of the neutrophil oxidoreductase, like xanthine oxidase, thus appears to be capable of conducting both 1- and 2-electron transfer steps in reducing O2. Its capacity to affect univalent reduction of O2 depends upon the concentration of electron donor (NADPH) supplied as well as conditions of storage and assay of the native enzyme. PMID- 3009442 TI - Characterization of Haemophilus influenzae nucleotide pyrophosphatase. An enzyme of critical importance for growth of the organism. AB - A nucleotide pyrophosphatase isolated from Haemophilus influenzae was purified to electrophoretic homogeneity and characterized with respect to molecular weight, substrate specificity, pH profile, thermal stability, functional group involvement, and effectiveness of selective inhibition. The enzyme catalyzes the hydrolysis of NAD to NMN and AMP and appears located appropriately to facilitate the internalization of NAD needed to satisfy the V-factor growth requirement of the organism. In the processing of NAD and structurally related substrates, the enzyme exhibited negative cooperativity. Structural alterations in the purine moiety of these dinucleotide substrates had pronounced effects on the negative cooperativity of the enzyme. AMP, ADP, and several related nucleotides were observed to be effective substrate-competitive inhibitors of the enzyme. Several of the dinucleotides serving as substrates for the nucleotide pyrophosphatase were evaluated with respect to substituting for NAD in supporting growth of the organism. AMP and ADP inhibited growth of the organism when NAD served as V factor, and this inhibition correlated well with the inhibitory effects of these nucleotides on the purified nucleotide pyrophosphatase. PMID- 3009444 TI - Photodynamic guanine modification by hematoporphyrin is specific for single stranded DNA with singlet oxygen as a mediator. AB - Photodynamic modification of DNA by hematoporphyrin (Hp) was characterized by the DNA sequencing technique using 32P-labeled DNA fragments, and the reaction mechanism was investigated by ESR spectroscopy. Mild photodynamic treatment of single-stranded DNA with Hp induced an alteration of guanine residues, and subsequent treatment with piperidine led to chain cleavages at each guanine residue. On the other hand, methylene blue plus light modified the guanine residues in both single-stranded and double-stranded DNA. ESR studies using 2,2,6,6-tetramethylpiperidine and 2,2,6,6-tetramethyl-4-piperidone as singlet oxygen traps demonstrated that Hp plus light produced almost the same amount of singlet oxygen as methylene blue plus light and that the photochemically generated singlet oxygen reacts significantly with guanylate but only slightly with other mononucleotides. An ESR spin destruction method revealed that photoexcited Hp generated porphyrin radical, but guanylate did not react with this radical. These results indicate that photoexcited Hp reacts with oxygen to generate singlet oxygen which oxidizes the guanine residues of single-stranded DNA and that the difference in photoreactivities of DNA with Hp and methylene blue may be explained in terms of the structural difference in their intercalating abilities. PMID- 3009443 TI - Variant surface glycoprotein gene expression site switches in Trypanosoma brucei. AB - In Trypanosoma brucei bloodstream forms, transcription of variant surface glycoprotein (VSG) genes occurs at only one of several possible expression sites at any given time. Activation and inactivation of some expression sites are correlated with recombinational events that alter their chromosomal position (Van der Ploeg, L. H. T., Cornelissen, A. W. C. A., Michels, P. A. M., and Borst, P. (1984) Cell 39, 213-221). We present evidence that a 430-kilobase pair (kb) chromosome, containing the 1.8 expression-linked copy, is taken up in a reciprocal recombination when the 1.8 gene is inactivated. As a result, the 430 kb chromosome is reduced in length either to 140 kb (in variant 118b') or to 350 kb (in variant MITat 1.2000) and the 1.8 expression-linked copy is moved to a larger chromosome in both cases. The subsequent activation of the telomeric 118 VSG gene in variant 118b', located on a 2000-kb chromosome, occurs without detectable recombinations while, for variant MITat 1.2000, still another expression site is activated. We discuss a model that explains the occurrence of these apparently random recombinational events at expression site switching and antigenic variation. PMID- 3009445 TI - Structure of RNA polymerase II promoters. Coordinate conformational alteration of the upstream activator of the TATA- and RNA-initiation sequences under moderate torsional stress. AB - Different conformations of the circular DNA domains containing the intergenic region of the Saccharomyces cerevisiae GAL1-GAL10 divergent genes (914 base pairs) were modified by in vitro ligation in various conditions. The effect of increasing torsional stress on the conformation of the composing elements was determined by analysis of the sensitivity to the single strand-specific S1 endonuclease and it was observed that the sites of conformational alterations correspond to the positions relevant for promoter function (upstream activator sequence, TATA sequence, and RNA initiation site). PMID- 3009446 TI - Mosaic evolution of prepropancreatic polypeptide. AB - Pancreatic polypeptide, a 36-amino acid peptide hormone, is synthesized in pancreatic islets of Langerhans and acts as a regulator of pancreatic and gastrointestinal functions. We isolated cDNA clones encoding rat pancreatic polypeptide precursor from an islet cDNA library and determined their nucleic acid sequences. Rat pancreatic polypeptide was found to be flanked on the amino terminus by a putative signal peptide and on the carboxyl terminus by Gly-Lys-Arg followed by a 30-amino acid peptide. Nucleotide and amino acid sequences of the signal peptide and the pancreatic polypeptide of the rat were highly homologous to those of the human (Boel, E., Schwartz, T. W., Norris, K. E., and Fill, N. P. (1984) EMBO J. 3, 909-912). On the other hand, the rat carboxyl-terminal peptide differed markedly from the corresponding domain of the human precursor and did not contain any sequence similar to the icosapeptide, which has so far been known to be a second stable product from mammalian pancreatic polypeptide precursors (Schwartz, T. W., Hansen, H. F., Hakanson, R., Sundler, F., and Tager, H. S. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 708-712). The mosaicism of sequence conservation and divergence in prepropancreatic polypeptides may be a unique example in the evolution of prohormones. PMID- 3009447 TI - Growth factors activate the bumetanide-sensitive Na+/K+/Cl-cotransport in hamster fibroblasts. AB - alpha-Thrombin, a potent mitogen for the hamster fibroblast cell line CCL 39, stimulates by approximately 3-fold 86Rb+ uptake in a mutant lacking the Na+/H+ antiport activity (PS 120). The major component of this stimulated 86Rb+ (K+) uptake is a bumetanide-sensitive flux (IC50 = 0.4 microM), which accounts for 50% of total K+ uptake in Go-arrested cells and is approximately 4-fold stimulated by maximal thrombin concentrations (EC50 = 5 X 10(-4) units/ml). This bumetanide sensitive 86Rb+ uptake represents a Na+/K+/Cl- cotransport, as indicated by its dependence on extracellular Na+ and Cl- and by the existence in PS 120 cells of a bumetanide-sensitive K+-dependent 22Na+ uptake. The stimulation reaches its maximum within 2 min, is reduced at acidic intracellular pH values (half-maximal at pHi = 6.8), and can also be induced, to a lesser extent, by EGF and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, the effects of which are nearly additive. In contrast, preincubation with 12-O-tetradecanoylphorbol 13 acetate results in inhibition of thrombin- and EGF-induced stimulations, suggesting that activated protein kinase C might exert a feedback inhibitory control. This study clearly demonstrates that the growth factor-induced activation of the Na+/K+/Cl- cotransport is separated from the activation of the Na+/H+ antiport. However, activation of this cotransporter does not seem to play a major role in the mitogenic signaling pathway since its complete inhibition with bumetanide reduces only by 25-30% reinitiation of DNA synthesis. PMID- 3009449 TI - sn-1,2-Diacylglycerol kinase of Escherichia coli. Mixed micellar analysis of the phospholipid cofactor requirement and divalent cation dependence. AB - Efficient delivery of hydrophobic water-insoluble substrates and cofactors to membrane-bound enzymes is a recurring problem which has impeded kinetic analyses. Kinetic analysis of the Escherichia coli sn-1,2-diacylglycerol kinase, an extremely hydrophobic integral membrane protein of 122 residues, was facilitated by the development of a mixed micellar assay. beta-Octyl glucoside micelles quantitatively solubilized diacylglycerol kinase from membranes of strains which overproduced the enzyme up to 250-fold and provided an effective method to disperse and deliver the hydrophobic water-insoluble substrate, sn-1,2 dioleoyglycerol. Diacylglycerol kinase was active in mixed micelles containing octyl glucoside and dioleoyglycerol. Several phospholipids stimulated activity up to 6-fold, suggesting a cofactor function. Activation by phospholipids was not stereospecific and was mimicked partially by fatty acids. Half-maximal activation was observed at 1 mol % cardiolipin, suggesting that a small number of phospholipids are sufficient to activate the enzyme. Activity was dependent on the mole fractions of dioleoylglycerol and phospholipid in the mixed micelles, but independent of micelle number. Several lines of evidence indicated that the transfer of diacylglycerol between micelles was much more rapid than its utilization by the enzyme. Diacylglycerol kinase exhibited Michaelis-Menten kinetics with respect to diacylglycerol and MgATP. A second Mg2+ ion (in addition to MgATP) was required for activity. When Mg2+ was excluded from the assay, Mn2+, Zn2+, Cd2+, and Co2+ supported activity to lesser extents. These data establish a suitable system for in-depth kinetic analysis of the E. coli diacylglycerol kinase and its phospholipid cofactor requirements. PMID- 3009448 TI - Triphasic reduction of cytochrome b and the protonmotive Q cycle pathway of electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain. AB - Reduction of cytochrome b in isolated succinate-cytochrome c reductase is a triphasic reaction. Initially, there is a relatively rapid, partial reduction of the cytochrome b, the rate of which matches the rate of reduction of cytochrome c1. This is followed by partial or complete reoxidation of the b, which is then followed by slow rereduction. At very low concentrations of succinate, the initial partial reduction of b is followed by reoxidation, but the third (rereduction) phase is absent, owing to insufficient substrate to rereduce the cytochromes. If antimycin is added at various times during the triphasic reaction, it inhibits the reoxidation and also inhibits the rereduction phase. Antimycin does not inhibit the initial phase of b reduction and, if added before or during this phase, it causes reduction of b to proceed to completion as a monophasic reaction. Myxothiazol inhibits the first phase of b reduction and the subsequent reoxidation, but does not inhibit the third, slow phase of b reduction. The resulting monophasic reduction of b which is observed in the presence of myxothiazol is slower than that in the presence of antimycin. The combination of both inhibitors, whether added together or successively during the triphasic reaction, completely inhibits b reduction. The triphasic reduction of cytochrome b is consistent with electron transfer by a protonmotive Q cycle in which there are two pathways for cytochrome b reduction. One pathway allows the initial phase of cytochrome b reduction by a myxothiazol-sensitive reaction in which reduction of b by ubisemiquinone is linked to reduction of iron-sulfur protein and cytochrome c1 by ubiquinol. In the second phase of the triphasic reaction, the b cytochromes are reoxidized by ubiquinone or ubisemiquinone through an antimycin-sensitive reaction. If oxidation of ubiquinol by iron-sulfur protein is blocked, either by myxothiazol or by reduction of iron-sulfur protein and cytochrome c1, the b cytochromes can be reduced by reversal of the antimycin sensitive pathway, thus accounting for the third phase of b reduction. PMID- 3009451 TI - Phosphorylation and degradation of exogenous phosphatidylinositol incorporated into Friend erythroleukemic cells. AB - Phosphatidyl[2-3H]inositol (PtdIns) obtained from rat skeletal muscle and yeast was introduced into Friend erythroleukemic cells by use of the PtdInstransfer protein or by spontaneous route. The mammalian PtdIns incorporated by the transfer protein appeared metabolically inert while the spontaneously incorporated PtdIns was both phosphorylated to PtdIns-4-phosphate (i.e. 30% of the total PtdIns incorporated) and converted into lyso-PtdIns (i.e. 20% of the total PtdIns incorporated); formation of PtdIns, 4,5-bisphosphate was minimal. The extensive metabolism of the spontaneously incorporated PtdIns strongly suggests that this PtdIns does not rapidly equilibrate with the endogenous PtdIns pools. A similar spontaneous incorporation of yeast PtdIns was accompanied by a negligible degree of phosphorylation and hydrolysis. Evidence is provided that this difference in metabolism reflects the absence of arachidonate in the yeast PtdIns. PMID- 3009450 TI - Conformational transitions in fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles. AB - Fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles has been used to study conformational transitions. Addition of K+ or Na+ to the vesicle medium induces fluorescence changes characteristic of the E2(K) or E1Na states of fluorescein-labeled (Na,K)ATPase (Karlish, S.J.D. (1980) J. Bioenerg. Biomembr. 12, 111-136). The cation effects are exerted from the cytoplasmic surface of inside-out-oriented pumps. Equilibrium cation titrations and measurements of rates of conformational transitions have led to the following observations. 1) The rate of E2(K)----E1Na or E2(T1)----E1Na is 4-6-fold faster and E1K----E2(K) is about 2-fold slower in vesicles compared to enzyme. In equilibrium titrations the K0.5 for K+ is higher and that for Na+ is lower for vesicles compared to enzyme. The conformational equilibrium E(1)2K----E2(2K) is apparently shifted toward E(1)2K in vesicles compared to enzyme. 2) Diffusion potentials, positive-outside, induced with valinomycin or Li+ ionophore AS701, do not affect the rates of E2(T1)----E1Na or E1K----E2(K), or equilibrium cation titrations. This demonstrates that the conformational transitions E(1)2K--- E2(2K) are voltage-insensitive steps, confirming a prediction based on transport experiments. 3) In vesicles containing choline, K+, Na+, or Li+, the rate of E2(T1)----E1Na increases in the order given. Vesicles with reconstituted fluorescein-labeled (Na,K)ATPase provide a convenient system for correlating directly properties of conformational transitions with cation transport. PMID- 3009452 TI - Sequence analysis and characterization of a maize gene encoding a high-sulfur zein protein of Mr 15,000. AB - We have isolated a gene coding for a sulfur-rich zein protein from a maize genomic library. The nucleotide sequence of this gene predicts a protein composed of 180 amino acids, including a 20-amino acid signal peptide. As is true of other zeins, there are no intervening sequences in the gene. Comparison of the nucleotide sequence of this gene with that of a homologous cDNA clone revealed only a single difference resulting in a valine/alanine substitution. The Mr 15,000 zein contains no repetitive nucleotide sequences and shows no homology with genes encoding the Mr 22,000 and 19,000 zeins. Circular dichroism analysis of the Mr 15,000 zein protein revealed that it is composed primarily of beta and turn structures. This gene has a short region of nucleotide sequence homology to the cysteine-rich domain of the Mr 27,000 zein, as well as the cysteine containing barley B-hordein. PMID- 3009454 TI - Photoaffinity labeling of the Ah receptor. AB - A series of halodibenzo-p-dioxins with the photolabile aryl azide functional group were synthesized and screened as potential photoaffinity labels for the Ah receptor, and 2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin was selected for radiosynthesis with 125I (specific activity 2176 Ci/mmol, equilibrium dissociation constant, KD = 0.76 nM). Following incubation of this 125I-labeled photoaffinity ligand with the protamine sulfate-precipitated fraction of C57BL/6J mouse liver cytosol, and irradiation with long wavelength ultraviolet light, the radiolabeled macromolecules were precipitated with acetone and analyzed by denaturing gel electrophoresis and autoradiography. Among the labeled products, two peptides with apparent molecular masses of 95,000 and 70,000 daltons had the following properties: 1) they were selectively labeled at low ligand concentrations; 2) they were labeled in approximately a 1:1 ratio; 3) co incubation with receptor agonists inhibited the photoaffinity labeling of both peptides to a similar extent, and structure activity relationship for inhibition of labeling by these agonists corresponded to that for their binding affinity to the Ah receptor; 4) upon nondenaturing chromatographic separation of photoaffinity labeled cytosol on high performance liquid chromatography size exclusion and anion exchange columns, the 95- and 70-kDa peptides coelute; 5) the migration of these peptides upon denaturing electrophoresis is the same in the presence or absence of a thiol reducing agent; and 6) proteolysis of the 95- and 70-kDa peptides produces a similar pattern of cleavage peptides. The simplest structure of the Ah receptor in mouse liver cytosol, appears to be a dimer composed of two noncovalently linked subunits of apparent molecular masses of 95 and 70 kDa, which have homologous structure and similar ligand binding sites, but other possibilities are discussed. PMID- 3009453 TI - Leukotriene A4. Enzymatic conversion into 5,6-dihydroxy-7,9,11,14 eicosatetraenoic acid by mouse liver cytosolic epoxide hydrolase. AB - Mouse liver homogenates transformed leukotriene A4 into a 5,6-dihydroxy-7,9,11,14 eicosatetraenoic acid. This novel enzymatic metabolite of leukotriene A4 was characterized by physical means including ultraviolet spectroscopy, high performance liquid chromatography, and gas chromatography-mass spectrometry. After subcellular fractionation, the enzymatic activity was mostly recovered in the 105,000 X g supernatant and 20,000 X g pellet. Heat treatment (80 degrees C, 10 min) or digestion with a proteolytic enzyme abolished the enzymatic activity in the high speed supernatant. A purified cytosolic epoxide hydrolase from mouse liver also transformed leukotriene A4 into a 5,6-dihydroxyeicosatetraenoic acid with the same physico-chemical characteristics as the compound formed in crude cytosol, but not into leukotriene B4, a compound previously reported to be formed in liver cytosol (Haeggstrom, J., Radmark, O., and Fitzpatrick, F.A. (1985) Biochim. Biophys. Acta 835, 378-384). These findings suggest a role for leukotriene A4 as an endogenous substrate for cytosolic epoxide hydrolase, an enzyme earlier characterized by xenobiotic substrates. Furthermore, they indicate that leukotriene A4 hydrolase in liver cytosol is a distinct enzyme, separate from previously described forms of epoxide hydrolases in liver. PMID- 3009455 TI - Phosphonocarboxylic acids as specific inhibitors of Na+-dependent transport of phosphate across renal brush border membrane. AB - We investigated interactions of phosphonoformic acid (PFA), phosphonoacetic acid (PAA), and other phosphonyl derivatives with the Na+ gradient [Na+ extravesicular greater than Na+ intravesicular; Nao+ greater than Na+i]-dependent transport system for phosphate (Pi) in renal cortical brush border membrane vesicles (BBMV). PFA and PAA inhibited in a dose-dependent manner the Na+ gradient [Na+o greater than Na+i]-dependent uptake of Pi by rat kidney BBMV. PFA was a more potent inhibitor than PAA while phosphonopropionic acid, hydroxymethylphosphonic acid, and phenylphosphonic acid had no effect on Pi transport. The inhibitory effect of PFA was competitive (Ki approximately equal to 4.6 X 10(-4) M) and reversible upon dilution. The uptake of Pi by BBMV in the absence of Na+ gradient [Nao+ = Na+i] was also inhibited by PFA. The PFA had no effect on uptake of L [3H]proline, D-[3H]glucose, or 22Na+ by BBMV nor did it alter intravesicular volume of BBMV. The relative (%) extent of inhibition by PFA was not altered by changes in the extravesicular pH or changes in the steepness of the Na+ gradient [Nao+ greater than Na+i]. The inhibition of PFA was analogous in renal BBMV from rats, mice, rabbits, or dogs. Unlike other known inhibitors of brush border membrane (BBM) transport of Pi, e.g. arsenate, NAD, and ethane-1-hydroxy-1,1 diphosphonate, PFA and PAA had no inhibitory effect on BBM-bound or solubilized alkaline phosphatase. Also, PFA did not interfere with the activity of renal cortical (Na-K)ATPase. Administration of PFA (0.5 g/kg/day, intraperitoneally) to thyroparathyroidectomized rats fed a low Pi diet elicited an increase in urinary excretion of Pi, but did not change the excretion of Na+, K, and Ca2+. The results show that the PFA, and to a lesser degree PAA, are specific competitive inhibitors of the Na+-Pi cotransport in renal cortical BBM and are suitable probes for studies of this transport system. PMID- 3009457 TI - Cloning, sequencing, and species relatedness of the Escherichia coli cca gene encoding the enzyme tRNA nucleotidyltransferase. AB - The Escherichia coli cca gene which encodes the enzyme tRNA nucleotidyltransferase has been cloned by taking advantage of its proximity to the previously cloned dnaG locus. A series of recombinant bacteriophages, spanning the chromosomal region between the dnaG and cca genes at 66 min on the E. coli linkage map, were isolated from a lambda Charon 28 partial Sau3A E. coli DNA library using recombinant plasmids containing regions between dnaG and cca as probes. Two of the recombinant phage isolates, lambda c1 and lambda c4, contained the cca gene. A BamHI fragment from lambda c1 was subcloned into pBR328, and cells containing this recombinant plasmid, pRH9, expressed tRNA nucleotidyltransferase activity at about 10-fold higher level than the wild type control. The cca gene was further localized to a 1.4-kilobase stretch of DNA by Bal31 deletion analysis. The nucleotide sequence of the cca gene was determined by the dideoxy method, and revealed an open reading frame extending for a total of 412 codons from an initiator GTG codon that would encode a protein of about 47,000 daltons. Southern analysis using genomic blots demonstrated that the cca gene is present as a single copy on the E. coli chromosome and that there is no homology on the DNA level between the E. coli cca gene, and the corresponding gene in the Bacillus subtilis, Saccharomyces cerevisiae, Petunia hybrida, or Homo sapiens genomes. Homology was found only with DNA from the closely related species, Salmonella typhimurium. These studies have also allowed exact placement of the cca gene on the E. coli genetic map, and have shown that it is transcribed in a clockwise direction. PMID- 3009456 TI - Evolution of the apolipoproteins. Structure of the rat apo-A-IV gene and its relationship to the human genes for apo-A-I, C-III, and E. AB - We have determined the nucleotide sequence of the rat apolipoprotein (apo-) A-IV gene and analyzed its structural and evolutionary relationships to the human apolipoprotein A-I, E, and C-III genes. The rat A-IV gene is 2.4 kilobases in size and consists of three exons (142, 126, and 1157 base pairs) interrupted by two introns (277 and 673 base pairs). The 5'-nontranslated region and most of the signal peptide are encoded by the first exon. Thus, the apo-A-IV gene lacks an intron in the 5'-nontranslated region of its mRNA in contrast to all other known apolipoprotein genes. Sequences coding for amphipathic docosapeptides span both the second and third exons of the rat A-IV gene. We demonstrate that this is also true for the human apolipoprotein genes. This gene family seems to have evolved by the duplication of an ancestral minigene that resulted in the formation of two exons. Thereafter, evolution of these sequences was dominated by intraexonic amplification of repeating units coding for amphipathic peptides. Sequence divergence of these repeats resulted in the functional differentiation of the apolipoproteins. However, conservation of the fundamental amphipathic pattern allowed members of this protein family to retain their lipid-binding properties. PMID- 3009458 TI - Regulation of human neutrophil chemotaxis by intracellular pH. AB - The relationship of N-formyl-methionyl-leucyl-phenylalanine-stimulated Na+/H+ exchange to the chemotactic responsiveness of human neutrophils was investigated. The pHi changes, measured from the equilibrium distribution of 5,5 dimethyloxazolidine-2,4-dione, were correlated with the migratory behavior of the cells as assessed by the leading front method. Exposure of cells to 10 nM FMLP caused activation of Na+/H+ exchange, leading to a rise in pHi from approximately 7.25 to approximately 7.75. This intracellular alkalinization was inhibited by amiloride and by three more potent analogues. All four compounds reduced the chemotactic response to FMLP with apparent Ki values similar to those for inhibition of the pHi transients, thereby suggesting that the blocking effect of the drugs on directed cell migration was related to inhibition of Na+/H+ exchange. The effect was specific for stimulated cell locomotion: FMLP-induced chemotaxis and chemokinesis were inhibited in parallel, whereas random motility was unimpaired. The relationship of pHi to function was also studied as the pHi of FMLP-activated cells was varied between 6.8 and 8.6 by altering the chemical gradients for Na+ and H+ across the cell membrane. There was a direct, positive correlation between the pHi value attained following FMLP-stimulation and the locomotor response to a chemotactic gradient. These results indicate that the motile functions of human neutrophils can be regulated by their pHi. PMID- 3009459 TI - Inhibition of ubiquitin-dependent proteolysis by non-ubiquitinatable proteins. AB - The effect in reticulocyte lysates of proteins with blocked amino groups on the ATP-dependent degradation of casein and serum albumin was studied in order to assess the extent to which proteins with blocked and with free amino groups share common paths of proteolytic degradation. Completely acetylated or succinylated casein and acetylated or succinylated serum albumin (reduced and carboxymethylated), in addition to other amino-modified proteins, inhibited the ATP-dependent proteolysis of both casein and reduced carboxymethylated serum albumin. Inhibition of serum albumin degradation by acetylated serum albumin was competitive, whereas inhibition of casein degradation by acetylated casein was largely competitive with evidence of mixed kinetics. The different amino-blocked proteins studied, which were largely unfolded under assay conditions, were similarly effective as inhibitors on a weight basis, with Ki values in the range 0.2-0.6 mg/ml; there was no correlation between the ability of the blocked proteins to serve as proteolysis substrates and their effectiveness as inhibitors. Studies of the effects of acetylated proteins on the conjugation of ubiquitin to serum albumin and casein demonstrated that the acetylated proteins blocked formation of ubiquitin-albumin conjugates and of selected casein conjugates; the steady state concentration of selected conjugates of endogenous lysate proteins was increased in the presence of amino-blocked proteins. The results suggest that proteins with blocked amino groups, which cannot serve as substrates for ubiquitin conjugation, can compete for binding to those ubiquitin conjugation factors that recognize and ubiquitinate potential substrates of the ubiquitin pathway. The similar inhibitory properties of the different blocked proteins in turn suggest that a common factor in binding to these conjugation factors may be recognition of the polypeptide backbone. PMID- 3009460 TI - Membrane-surfactant interactions. The role of surfactant in mitochondrial complex III-phospholipid-Triton X-100 mixed micelles. AB - Complex III (ubiquinol-cytochrome c reductase) was purified from beef heart mitochondria in the form of protein-phospholipid-Triton X-100 mixed micelles (about 1:80:100 molar ratio). Detergent may be totally removed by sucrose density gradient centrifugation, and the resulting lipoprotein complexes retain full enzyme activity. In order to understand the role of surfactant in the mixed micelles, and the interaction of Triton X-100 with integral membrane proteins and phospholipid bilayers, both the protein-lipid-surfactant mixed micelles and the detergent-free lipoprotein system were examined from the point of view of particle size and ultrastructure, enzyme activity, tryptophan fluorescence quenching, 31P NMR, and Fourier transform infrared spectroscopy. The NMR and IR spectroscopic studies show that surfactant withdrawal induces a profound change in phospholipid architecture, from a micellar to a lamellar-like phase. However, electron microscopic observations fail to reveal the existence of lipid bilayers in the absence of detergent. We suggest that, under these conditions, the lipid:protein molar ratio (80:1) is too low to permit the formation of lipid bilayer planes, but the relative orientation and mobility of phospholipids with respect to proteins is similar to that of the lamellar phase. Protein conformational changes are also detected as a consequence of surfactant removal. Fourier transform infrared spectroscopy indicates an increase of peptide beta structure in the absence of Triton X-100; changes in the amide II/amide I intensity ratio are also detected, although the precise meaning of these observations is unclear. Tryptophanyl fluorescence quenching by acrylamide shows that a significant fraction of the Trp residues sensing the quencher become less readily available to it in the absence of surfactant. The temperature dependence of enzyme activity (expressed in the form of Arrhenius plots) is also different in the presence and absence of detergent. The effects of surfactant removal do not appear to be readily reversible upon readdition of Triton X-100. PMID- 3009461 TI - Superoxide perturbation of the organization of vascular endothelial cell membranes. AB - Cultured porcine thoracic aorta endothelial cells were covalently labeled with 4 maleimido-2,2,6,6-tetramethylpiperidinooxyl. Electron paramagnetic resonance spectrometry revealed two major binding environments representing strongly and weakly immobilized species. The disorder parameter of weak/strong, determined from the respective peak amplitudes, was irreversibly elevated following incubation of endothelial cells with a superoxide-generating system, indicating increased membrane fluidity. The rate of increase in membrane disorder was dependent upon superoxide generation rates. Incorporation of the spin-label at concentrations less than 250 microM had no effect on cell viability. The cellular proteins reacting with the spin-label were predominantly membrane proteins, characterized by immunoblotting using a rabbit anti-4-maleimido-2,2,6,6 tetramethylpiperidinooxyl IgG, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophorectic transfer to nitrocellulose. PMID- 3009462 TI - Acute stimulation of steroidogenesis in corpus luteum and adrenal cortex by peptide hormones. Rapid induction of a similar protein in both tissues. AB - Two-dimensional electrophoresis was used to detect a protein (ic) synthesized in rat corpus luteum cells in response to acute stimulation by human chorionic gonadotropin or dibutyryl cyclic AMP. This induced protein ic is isoelectric at pH 6.5 (isoelectric focusing) and has an apparent molecular weight of 28,000 (sodium dodecyl sulfate electrophoresis). The human chorionic gonadotropin or dibutyryl cyclic AMP dose response and time course of synthesis of the protein parallel those of progesterone synthesis in stimulated luteal cells. Additionally, cycloheximide, which inhibits the increase in progesterone formation caused by human chorionic gonadotropin or cAMP, also inhibits the synthesis of ic. Proteolytic polypeptide mapping suggests that ic has a very similar primary structure to another protein (pc), which has the same molecular weight as ic, differs from ic in pI, and is synthesized only in unstimulated cells. These polypeptide maps also demonstrate the close similarity of pc and ic to two proteins p and i, synthesized in control and in adrenocorticotropic hormone-stimulated rat adrenal cortex cells, respectively (Krueger, R. J. and Orme-Johnson, N. R. (1983) J. Biol. Chem. 258, 10159-10167). In both adrenal cortex and corpus luteum, binding of a tissue-specific polypeptide hormone acts via cAMP to cause increased steroidogenesis and induction of the synthesis of protein i (ic), with the same time course and hormone dose dependence. Also in both tissues, inhibition of protein synthesis at the level of translation (e.g. by cycloheximide addition) causes inhibition of i (ic) synthesis and of stimulated steroid production. This close correlation between the two different tissues in conditions which cause induction of the synthesis of these proteins suggests that the proteins may be common intermediaries in the control by polypeptide hormones of steroidogenesis in endocrine tissues. PMID- 3009463 TI - Human fibroblast collagenase. Complete primary structure and homology to an oncogene transformation-induced rat protein. AB - We have determined the complete sequence of the cDNA clone representing the full size human skin collagenase mRNA. Collagenase is synthesized in preproenzyme form, Mr 54,092, with a 19 amino acid long signal peptide. The primary secretion products of the enzyme consist of a minor glycosylated form, Mr 57,000, and a major unmodified polypeptide of predicted Mr 51,929. Proteolytic activation of human skin procollagenase results in removal of 81 amino acid residues from the amino-terminal portion of the proenzyme. Both potential N-glycosylation sites are contained within the proteolytically activated form of the enzyme. The primary structure of the coding region of the presented clone is homologous to an oncogene-induced rat protein whose function is still unknown, although preliminary observations suggest that it is not rat skin collagenase. PMID- 3009464 TI - The chicken myosin heavy chain family. AB - We have used a myosin heavy chain gene fragment to probe two chicken genomic libraries. The fragment, derived from a gene expressed in the fast-white fibers of adult chickens, contains 1400 bases upstream from the translational start site and 1600 bases downstream from the initiation codon. Thirty-one unique nonoverlapping clones were isolated. All of the genes showed homologies in the nucleotide sequences which code for the globular head portion of the myosin protein while no extensive homologies were detected in the 5'-flanking sequences. The relationships between the genes were studied using oligomeric sequences as probes. The hybridization patterns showed that seven of the genes fall within a well defined subgroup. The exon containing a domain of the nucleotide (ATP) binding site was sequenced for all seven of these genes and shown to be, except for 2 nucleotides in one of the genes, completely conserved. The lack of sequence conservation in the 5'-nontranslated portions of the genes was exploited in the preparation of transcript-specific probes. We have used these probes to show that two of the isoforms are expressed in a tissue-specific and developmental stage specific manner in the chicken. PMID- 3009465 TI - A canonical sequence organization at the 5'-end of the myosin heavy chain genes. AB - The sequences encoding the 5'-ends of three chicken fast-white myosin heavy chain (MHC) genes have been determined. When compared with the sequences of two other MHC genes it is apparent that both the exon and intron positions are conserved. All exon sequences are highly conserved; there is absolute amino acid conservation in the second and third exons. In addition, while the first and third introns diverge among the genes, the second intron is highly conserved between the five. This intron contains a 24-bp sequence that is repeated twice in one of the introns and once in the other four. Analyses indicate that this sequence, which is partially homologous to 7SL RNA, appears to be largely restricted to the MHC gene family. Analysis of the 5'-flanking sequences show that while small homologies are present between some of the genes, they have extensively diverged in this region. PMID- 3009466 TI - Exonuclease activity associated with a multiprotein form of HeLa cell DNA polymerase alpha. Purification and properties of the exonuclease. AB - The DNase that is associated with a multiprotein form of HeLa cell DNA polymerase alpha (polymerase alpha 2) has two distinct exonuclease activities: the major activity initiates hydrolysis from the 3' terminus and the other from the 5' terminus of single-stranded DNA. The two exonuclease activities show identical rates of thermal inactivation and coincidental migration during chromatofocusing, glycerol gradient centrifugation, and nondenaturing polyacrylamide gel electrophoresis of the DNase. Moreover, the purified DNase shows a single protein band of Mr 69,000 following nondenaturing polyacrylamide and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 3'----5' exonuclease activity hydrolyzes only single-stranded DNA substrates and the products are 5' mononucleotides. This activity recognizes and excizes mismatched bases at the 3' terminus of double-stranded DNA substrates. The 3'----5' exonuclease does not hydrolyze 3' phosphoryl terminated single-stranded DNA substrates. The 5'----3' exonuclease activity also only hydrolyzes single-stranded DNA substrates. The rate of hydrolysis, however is only about 1/25th the rate of the 3'----5' exonuclease. This exonuclease activity requires a 5' single-stranded terminus in order to initiate hydrolysis and does not proceed into double-stranded regions. The products of hydrolysis by 5'----3' exonuclease are also 5' nucleoside monophosphates. PMID- 3009467 TI - Internal structural features of E. coli glycyl-tRNA synthetase examined by subunit polypeptide chain fusions. AB - In contrast to most aminoacyl-tRNA synthetases which are monomers or oligomers of a single polypeptide, Escherichia coli glycyl-tRNA synthetase has an alpha-2, beta-2 structure. The enzyme requires both subunits for catalysis of either adenylate or aminoacyl-tRNA synthesis. The head-to-tail arrangement of the alpha- and beta-chain coding regions in the genome suggests that the two-subunit protein may be tantamount to a single chain. We fused the carboxyl terminus of the alpha chain to the amino terminus of the beta-chain, through a short peptide linker. Five different amino acid substitutions were placed in the linker. In all instances, the fusion polypeptide is stable in maxicell extracts. In a glyS null strain, a gene encoding any of the fusion proteins substitutes for the wild-type gene. Assays confirm that, in vitro, the engineered polypeptide fusion is active to within 2- to 3-fold of the wild-type, unfused chains. Oligomers of the fusion protein are observed and may be required for activity. Because the creation and limited manipulation of the artificial peptide linker region does not destroy the activity, we also conclude that the C-terminal part of the alpha-chain and the amino-terminal part of the beta-chain are not important for activity. PMID- 3009468 TI - Alpha 1 type IV collagen gene evolved differently from fibrillar collagen genes. AB - Type IV collagen is a major structural component in basement membranes. It is considerably different from the fibrillar collagens, types I-III. For example, unlike fibrillar collagens, the triple helical domain of type IV collagen is frequently interrupted by nonhelical regions. In this report, we demonstrate several overlapping genomic clones which cover most of the mouse alpha 1(IV) chain. Electron microscopic analysis of R-loops revealed that there were at least 28 exons within 35 kilobases of the gene segment. The sizes of six exons were determined by DNA sequence analysis to be 81, 178, 134, 73, 129, and 213 base pairs. These sizes do not appear to be related to the 54-base pair coding unit which is characteristic of fibrillar collagen exons, suggesting that the alpha 1 type IV collagen gene evolved differently from the fibrillar collagen genes. PMID- 3009469 TI - Metal ion complexes of apoferritin. Evidence for initial binding in the hydrophilic channels. AB - Metal binding to the iron storage protein apoferritin is the first step in the process by which iron accumulates within the protein shell. In the present study, the stoichiometry of metal binding to apoferritin in solution has been examined using the probe ions Mn(II), VO(IV), and Cd(II) in conjunction with EPR spectroscopic and cadmium ion selective electrode measurements. Binding studies were carried out with the individual ions, in competition with one another, and in competition with Fe(II), Fe(III), and Tb(III). All three probe ions show binding stoichiometries near 0.3 and 0.7 metal ion per subunit, close to the theoretically predicted values of 0.33 and 0.67 for the binding of one and two metal ions, respectively, per three subunits. These results in conjunction with other data are consistent with the binding of one, and possibly two, metal ions within each of the eight hydrophilic channels which are located on 3-fold axes leading to the interior of the protein. Pairs of cadmium binding sites have been located in these channels by x-ray crystallography (Rice, D. W., Ford, G. C., White, J. L., Smith, J. M. A., and Harrison, P. M. (1983) Adv. Inorg. Biochem. 5, 39-49). The possibility that some metal binding occurs elsewhere on the protein is not precluded by the present data, however. In competition experiments between various metal ions, approximately 0.3 metal ion per subunit is readily displaced implying common binding sites in the channels for all of them. The stoichiometry of Mn(II) displacement by Fe(II) is less clear. Oxidation of Fe(II) to Fe(III) by molecular oxygen in the presence of Mn(II) regenerates some Mn(II) binding on the protein, suggesting migration of iron(III) to other protein sites, or perhaps to core. PMID- 3009470 TI - Purification of wheat germ RNA ligase. I. Characterization of a ligase-associated 5'-hydroxyl polynucleotide kinase activity. AB - An RNA ligase that catalyzes the formation of a 2'-phosphomonoester-3',5' phosphodiester bond in the presence of ATP and Mg2+ was purified approximately 6000-fold from raw wheat germ. A 5'-hydroxyl polynucleotide kinase activity copurified with RNA ligase through all chromatographic steps. Both activities cosedimented upon glycerol gradient centrifugation even in the presence of high salt and urea. RNA ligase and kinase activities sedimented as a single peak on glycerol gradients with a sedimentation coefficient of 6.2 S. The purified polynucleotide kinase activity required dithiothreitol and a divalent cation for activity and was inhibited by pyrophosphate and by ADP. The kinase phosphorylated a variety of 5'-hydroxyl-terminated polynucleotide chains including some that were substrates for the RNA ligase (e.g. 2',3'-cyclic phosphate-terminated poly(A)) and others that were not ligase substrates (e.g. DNA or RNA containing 3'-hydroxyl termini). RNA molecules containing either 5'-hydroxyl or 5'-phosphate and 2',3'-cyclic or 2'-phosphate termini were substrates for the purified RNA ligase activity. The rate of ligation of 5'-hydroxyl-terminated RNA chains was greater than that of 5'-phosphate-terminated molecules, suggesting that an interaction between the wheat germ kinase and ligase activities occurs during the course of ligation. PMID- 3009471 TI - Purification of wheat germ RNA ligase. II. Mechanism of action of wheat germ RNA ligase. AB - The mechanism of action of purified wheat germ RNA ligase has been examined. ATP was absolutely required for the ligation of substrates containing 5'-OH or 5'-P and 2',3'-cyclic P or 2'-P termini. Ligation of 1 mol of 5'-P-2',3'-cyclic P terminated poly(A) was accompanied by the hydrolysis of 1 mol of ATP to 1 mol each of AMP and PPi. Purified RNA ligase catalyzed an ATP-PPi exchange reaction, specific for ATP and dATP, and formed a covalent enzyme-adenylate complex that was detected by autoradiography following incubation with [alpha-32P]ATP and separation of the products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A protein doublet with a molecular weight of approximately 110 kDa, the major product detected by silver staining, was labeled in these reactions. Isolated E-AMP complex was dissociated by the addition of ligatable poly(A), containing 5'-P-2',3'-cyclic P termini, to yield AMP and by the addition of PPi to yield ATP. The unique feature of the reactions leading to an exchange reaction between ATP and PPi and to the formation of an E-AMP complex was their marked stimulation (up to 400-fold) by the addition of RNA. This property distinguishes the wheat germ RNA ligase from other known RNA and DNA ligases which catalyze ATP-PPi exchange reactions and form E-AMP complexes in the absence of substrate. Thus, RNA appears to function in two capacities in the wheat germ system: as a cofactor, to stimulate the reaction of the enzyme with ATP, and as an authentic substrate for ligation. PMID- 3009473 TI - Oxidizing intermediates in the reaction of ferrous EDTA with hydrogen peroxide. Reactions with organic molecules and ferrocytochrome c. AB - The reaction between hydrogen peroxide and ferrous EDTA generates an oxidizing intermediate (I1) which is not the hydroxyl radical. It oxidizes ferrocytochrome c and also reacts with hydrogen peroxide (k5 = 3.2 X 10(3) M-1 S-1) to form a second oxidizing transient (I2). I1 is not scavenged by t-butyl alcohol whereas I2 is. I1 is found to be significantly less reactive than the hydroxyl radical toward benzoate ion, t-butyl alcohol, acetate ion, arginine, and serine, but is scavenged by compounds with readily oxidizable functional groups such as ethanol and isopropyl alcohol. This indicates that I1 does not undergo the characteristic reactions of the hydroxyl radical but shows a pattern of reactivity more associated with a metal ion oxidant like a ferryl (FeO2+)-EDTA complex. PMID- 3009472 TI - Structures of two HaeIII-type genes in the human salivary proline-rich protein multigene family. AB - Two members of the human salivary proline-rich protein (PRP) multigene family have been isolated and completely sequenced. These PRP genes, PRH1 and PRH2, are of the HaeIII-type subfamily and code for acidic PRP proteins. Both genes are approximately 3.5 kilobase pairs (kb) in length and contain four exons. Exon 3 encodes the proline-rich part of the protein and includes five 63-base pair (bp) repeats. CAT and ATA boxes and several possible enhancer sequences occur in a 1 kb region 5' to exon 1. Two sets of repeats occur in the sequenced region in addition to the 63-bp repeats: one pair of about 140 bp flanks 500 bp of DNA in the first intervening sequence, and the other pair of 72 bp is tandemly repeated 1.4 kb 5' to the PRH1 gene. The 4-kb region of sequenced DNA from PRH1 differs by an average of 8.7% from the same region in PRH2, but the nucleotide sequences of the exon 3 of the two genes differ by only 0.2%. This result suggests the occurrence of a recent gene conversion event. The regions containing the 5-fold repeated sequences of 63 bp are identical in the two genes, PRH1 and PRH2. A comparison of the human HaeIII and BstNI subfamily repeats and a comparison of the human, mouse, and rat repeats suggest that the individual repeats have evolved in a concerted fashion within each gene and within the PRP gene family as a whole. PMID- 3009474 TI - Resonance Raman studies of lactoperoxidase. AB - Resonance Raman (RR) spectra obtained at three excitation wavelengths are reported for various ferric, ferrous, and ferryl derivatives of bovine lactoperoxidase. The RR spectra of the ferric derivatives show the full complement of the vinyl stretching and scissor modes indicating that the two vinyls in the protoporphyrin IX prosthetic group are present in unmodified forms. The cysteine thiol complex exhibits a RR spectrum identical to that of the native enzyme, an observation which strongly suggests a nonheme binding site for the thiol substrates. The different ferrous complexes of lactoperoxidase which result from heme reduction at slightly alkaline and acidic pH gave identical low frequency RR spectra. Differences are observed, however, in the high-frequency region. Reduction in the presence of cyanide, however, yields two time-resolved complexes. Changes in the ligand field during the conversion to the final form of the cyanoferrous complex are proposed based on the changes observed in the low frequency vibrational spectrum. Comparisons are made between the low-frequency RR spectra of the limiting form of the cyanoferrous and the nitric oxide lactoperoxidase complexes. The similarity between the RR spectra of these two complexes in the 150-500 cm-1 region supports the assignment of structures for these complexes where the six-coordinate heme iron is displaced from the heme plane and away from the proximal histidine ligand. PMID- 3009475 TI - Molecular structure of the human albumin gene is revealed by nucleotide sequence within q11-22 of chromosome 4. AB - The human albumin gene spans 16,961 nucleotides from the putative "Cap" site to the first poly(A) addition site. It is split into 15 exons by 14 intervening sequences which are symmetrically placed within the three domains of albumin. The 5' region is highly conserved up to position -250 and contains the putative TATA (-32) and CAT (-88) boxes. A consensus 5' splice sequence reads /GTAGAGT while the 3' splice sequence is pyrimidine rich and contains CTAG/ at the splice junction. The gene contains three polyadenylation signals, and this 3' region presumably arose by triplication of a shorter fragment prior to mammalian radiation. The albumin gene exhibits a high degree of DNA polymorphism and appears to have been recently invaded by Alu repetitive sequences. PMID- 3009476 TI - Glycerated hemoglobin, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine. A new post-translational modification occurring in erythrocyte bisphosphoglyceromutase deficiency. AB - A new minor Hb fraction initially designated Hbx, has been found in the hemolysate of an erythremic patient that we have previously described with a complete erythrocyte bisphosphoglycerate mutase (EC 5.4.2.4) deficiency. Hbx (3.5% of the total) was detected by isoelectric focusing and exhibited electrophoretic and chromatographic properties similar to those of several variants of the Hb central cavity. By density fractionation of red cells, it was demonstrated that Hbx was an aging hemoglobin as in the case of glycated Hb A1c. Functional studies revealed a low oxygen affinity and almost complete inhibition of the allosteric effect of the organic phosphate effectors. Structural studies demonstrated an absence of tryptic cleavage between the peptides beta T9 and beta T10 suggesting the presence of an adduct on Lys beta 82 or on a neighboring residue. Fast atom bombardment mass spectrometry and a specific enzymatic assay with glyoxylate reductase demonstrated that the beta 82 adduct was a glycerate moiety. It was concluded that Hbx was a glycerylated Hb, alpha 2A beta 2(82) (EF6) N epsilon-glyceryllysine, to our knowledge the first example of glycerylated protein. The mechanism of formation of glyceryl Hb, which was found in the four studied subjects with a bisphosphoglyceromutase deficiency, remains to be determined. PMID- 3009477 TI - Phosphoribosylpyrophosphate synthetase of Escherichia coli. Properties of the purified enzyme and primary structure of the prs gene. AB - Phosphoribosylpyrophosphate (P-Rib-PP) synthetase of Escherichia coli has been purified to near homogeneity from a strain harboring the prs gene, encoding P-Rib PP synthetase, on a multicopy plasmid. Analysis of the enzyme showed that it required inorganic phosphate for activity and for stability. Magnesium ions were required both as a complex with the substrate ATP and as a free cation. P-Rib-PP synthetase activity was inhibited strongly by ADP. Kinetic analysis indicated multiple sites of action of ADP. In addition apparent substrate inhibition was exerted by ribose 5-phosphate in the presence of ADP. The nucleotide sequence of the E. coli prs gene has been determined and the coding segment established. The deduced amino acid sequence of P-Rib-PP synthetase contained 314 amino acid residues and the molecular weight was calculated as 34,060. The initiation site of transcription was determined. This site was preceded by well conserved -10 and -35 consensus sequences (pdT-dA-dG-dA-dA-dT and pdT-dT-dG-dA-dT-dG, respectively). The transcription initiation site preceded the potential translation initiation site by 302 nucleotides. Transcription terminated approximately 35 nucleotides downstream from the UAA translation stop codon, within a Thy-rich region following an inverted repeat sequence, indicative of an rho-independent transcription terminator. PMID- 3009478 TI - An endonuclease from Chlamydomonas reinhardtii that cleaves the sequence TATA. AB - An endodeoxyribonuclease, designated CreI, was purified 16,000-fold from zygotes of the eukaryote Chlamydomonas reinhardtii. CreI preferentially attacks the sequence TATA producing double strand breaks with 3'-phosphomonoester and 5' hydroxyl termini. The endonuclease has an Mr = 27,000 and requires Ca2+ at pH 7.5 for optimal activity. PMID- 3009479 TI - Functional states of the luteinizing hormone/choriogonadotropin-receptor complex in rat Leydig cells. AB - Three classes of gonadotropins with different ratios of stimulating to binding activities (S/B ratio) in rat Leydig cells have been identified. An S/B ratio of 1 was observed for rat luteinizing hormone (LH), porcine LH, and equine choriogonadotropin (CG) (class I), whereas ovine and equine LH exhibited and S/B ratio of 10-20 (class II) and human CG (hCG) (class III) an S/B ratio of 60. We coined the term "superactivity" to designate this particular behavior. This phenomenon was further studied by comparing the competitive activities of porcine LH (pLH) and hCG in radioreceptor assays using rat Leydig cell membranes and either radiolabeled oLH or hCG as the tracer, in the presence or absence of 150 mM NaCl. At equilibrium, both native hormones were equipotent in competing with 125I-oLH binding, but hCG was 4-fold more potent than pLH when 125I-hCG was used. Moreover, the binding rates of both hormones were considerably diminished in the presence of NaCl, but hCG binding at equilibrium was not affected, whereas that of oLH was almost completely abolished. From these results and previous data on the binding and internalization of these hormones, we suggest the existence of two interconvertible functional states of the hormone-receptor complex: (formula; see text). The equilibrium constant k3/k4 would be extremely high for hCG and lower and lower for the hormones in class II and class I, respectively. The equilibrium constant k1/k2 would be the one affected by the presence of NaCl and seems to be similar for all the hormones tested. The normal activity or superactivity of gonadotropins would thus be primarily dependent on the equilibrium between HR1 and HR2. PMID- 3009480 TI - Mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. Complete primary structures and homology with pulmonary surfactant apoprotein. AB - Preparations of mannose-binding protein isolated from rat liver contain two distinct but homologous polypeptides. The complete primary structures of both of these polypeptides have been determined by sequencing of peptides derived from the proteins, isolation and sequencing of cDNAs for both proteins, and partial characterization of the gene for one of the proteins. Each polypeptide consists of three regions: (a) an NH2-terminal segment of 18-19 amino acids which is rich in cysteine and appears to be involved in the formation of interchain disulfide bonds which stabilize dimeric and trimeric forms of the protein, (b) a collagen like domain consisting of 18-20 repeats of the sequence Gly-X-Y and containing 4 hydroxyproline residues in several of the Y positions, and (c) a COOH-terminal carbohydrate-binding domain of 148-150 amino acids. The sequences of the COOH terminal domains are highly homologous to the sequence of the COOH-terminal carbohydrate-recognition portion of the chicken liver receptor for N acetylglucosamine-terminated glycoproteins and the rat liver asialoglycoprotein receptor. Each protein is preceded by a cleaved, NH2-terminal signal sequence, consistent with the finding that this protein is found in serum as well as in the liver. The entire structure of the mannose-binding proteins is homologous to dog pulmonary surfactant apoprotein. PMID- 3009481 TI - Patterns of nuclease protection during strand exchange. recA protein forms heteroduplex DNA by binding to strands of the same polarity. AB - recA protein, in the presence of ATP, polymerizes on single-stranded DNA (plus strand) to form a presynaptic nucleoprotein filament that pairs with linear duplex DNA and actively displaces the plus strand from the recipient molecule in a polarized fashion to form a new heteroduplex molecule. The interaction between recA protein and DNA during strand exchange was studied by labeling different strands and probing the intermediate with pancreatic deoxyribonuclease I (DNase I) or restriction endonuclease. The incoming single strand was resistant to DNase I in the original nucleoprotein filament and remained resistant even after extensive strand exchange had occurred. Both strands of the parental duplex molecule were sensitive to DNase I in the absence of joint molecule formation; but as strand exchange progressed following homologous pairing, increasing stretches of the parental plus strand became resistant, whereas the complementary parental minus strand remained sensitive to DNase I throughout the reaction. Except for a region of 50-100 base pairs at the end of the newly formed heteroduplex DNA where strand exchange was initiated, the rest of the heteroduplex region was resistant to cleavage by restriction endonucleases. The data suggest that recA protein promotes strand exchange by binding both the incoming and outgoing strands of the same polarity, whereas the complementary strand, which must switch pairing partners, is unhindered by direct contact with the protein. PMID- 3009482 TI - The human tissue plasminogen activator gene. AB - The nucleotide sequence of the human tissue plasminogen activator (t-PA) gene has been established. A total of 36,594 base pairs (bp) was sequenced; this included 32,720 bp from the site of initiation of transcription to the polyadenylation site, in addition to 3,530 and 344 bp of 5' and 3' flanking DNA, respectively. Thirteen intervening sequences divide the gene into 14 coding regions; the size range for exons is 43-914 bp, while that for introns is 111-14,257 bp. The gene and 5' flanking region contain 28 copies of Alu repetitive DNA and a single KpnI repeat. The transcription initiation site was identified by S1 nuclease, exonuclease VII, and primer extension analysis as an A residue; "TATA" and "CAAT" boxes are located in the expected positions upstream of this proposed site. Results of the analysis of the gene sequence and its comparison with data banks are described. The protein and gene structures of tissue and urokinase plasminogen activator are compared; based on these features the evolutionary relationship of the two human plasminogen activators appears to be close. PMID- 3009484 TI - Peptidoglycan synthetic activities in membranes of Escherichia coli caused by overproduction of penicillin-binding protein 2 and rodA protein. AB - Penicillin-binding protein (PBP)-2 and the RodA protein are known to function in determining the rod shape of Escherichia coli cells. Peptidoglycan biosynthetic reactions that required these two proteins were demonstrated in the membrane fraction prepared from an E. coli strain that overproduced both of these two proteins and which lacked PBP-1B activity (the major peptidoglycan synthetase activity in the normal E. coli membranes). The cross-linked peptidoglycan was synthesized from UDP-N-acetylmuramylpentapeptide and UDP-N-acetylglucosamine in the presence of a high concentration of cefmetazole that inhibited all of PBPs except PBP-2. The peptidoglycan was synthesized via a lipid intermediate and showed up to 30% cross-linking. The cross-linking reaction was strongly inhibited by the amidinopenicillin, mecillinam, and by other beta-lactam antibiotics that have a high affinity for PBP-2, but not by beta-lactams that had very low affinity for PBP-2. The formation of peptidoglycan required the presence of high levels of both PBP-2 and the RodA protein in the membranes, but it is unclear which of the two proteins was primarily responsible for the extension of the glycan chains (transglycosylation). However, the sensitivity of the cross-linking reaction to specific beta-lactam antibiotics strongly suggested that it was catalyzed by PBP-2. The transglycosylase activity of the membranes was sensitive to enramycin and vancomycin and was unusual in being stimulated greatly by a high concentration of a chelating agent. PMID- 3009485 TI - Site-specific OsO4 modification of the B-Z junctions formed at the (dA-dC)32 region in supercoiled DNA. AB - OsO4 in the presence of pyridine specifically modifies the structural distortions of the primary helix of supercoiled pRW777 near the (dA-dC)32 sequence. Modification occurs at the same negative superhelix density value as required for formation of the Z-helix within the polymer block. Fine mapping of the distorted regions, which are probably the B-Z junctions, is presented. OsO4 reactions provide a powerful and sensitive chemical approach to study DNA polymorphism in solution. PMID- 3009483 TI - Attenuation of sn-1,2-diacylglycerol second messengers by diacylglycerol kinase. Inhibition by diacylglycerol analogs in vitro and in human platelets. AB - Diacylglycerol kinase is though to play a central role in the metabolism of diacylglycerol second messengers in agonist-stimulated cells. A series of diacylglycerol analogs were tested for their ability to act as substrates or inhibitors of diacylglycerol kinase with the goal of determining the substrate specificity of the enzyme, and of discovering inhibitors. Screening of these compounds was performed using a partially purified diacylglycerol kinase from pig brain. Modified assays for this enzyme using co-sonicated mixtures of diacylglycerol and anionic phospholipids were developed. This enzyme was found to be quite specific for sn-1,2-diacylglycerol (KM 24 microM for dioctanoyl glycerol). Among the analogs investigated, only 1,2-dioctanoyl-2-amino-1,3 propanediol was utilized at a significant rate. Two analogs, dioctanoylethylene glycol (KI 58 microM) and 1-monooleoylglycerol (KI 91 microM), were potent inhibitors in vitro. These compounds were tested for effects on diacylglycerol formation and metabolism in thrombin-stimulated human platelets. Dioctanoylethylene glycol inhibited diacylglycerol phosphorylation in platelets (70-100% at 100 microM) leading to a longer-lived diacylglycerol signal. This compound may be a useful tool for studies of diacylglycerol kinase in other cell types. 1-Monooleoylglycerol treatment elevated diacylglycerol levels up to 4-fold in unstimulated platelets and up to 10-fold in thrombin-stimulated platelets. The implications with regard to the pathways of diacylglycerol metabolism in human platelets are discussed. PMID- 3009487 TI - Determination of DNA single strand breaks and selective DNA amplification by N nitrodimethylamine and analogs, and estimation of the indicator cells' metabolic capacities. AB - N-nitrodimethylamine is metabolized oxidatively to N nitrohydroxymethylmethylamine, which decomposes to yield formaldehyde and N nitromethylamine. All four compounds and N-nitromethylamine were tested for their ability to induce DNA single strand breaks in hepatocytes and in SV 40 transformed Chinese hamster embryo cell lines. Only the two monoalkylnitramines were positive. They induced single strand breaks in hepatocytes, but were not effective in the other cells. Formaldehyde and N-nitrohydroxymethylmethylamine were toxic to the cells. None of the compounds tested was able to induce selective DNA amplification in the two transformed cell lines. Enzymes involved in drug metabolism were assayed in the hamster cell lines. The activity of UDP glucuronosyltransferase and cytosolic epoxide hydrolase were not detectable. N nitrodimethylamine demethylation was low. The content of reduced glutathione and the activities of glutathione transferase and membrane bound epoxide hydrolase were comparable to values obtained in the rat liver. PMID- 3009486 TI - Use of diethyldithiocarbamate as a probe to detect stable intermediates during the decomposition of several mutagenic and nonmutagenic N-nitroso compounds. AB - By showing that methyldiethyldithiocarbamate is formed from the reaction of methylnitrosourea and disulfiram, we demonstrated in previous experiments that one of the anticarcinogenic/antimutagenic mechanisms of disulfiram is the scavenging of reactive species. We propose that this reaction may be employed additionally as a model for elucidating the following: (a) possible reactions between alkylating species and nucleophilic sites within the cell, and (b) the existence of stable intermediates during the metabolism of N-nitroso compounds. With structurally related pairs of nitrosoureas (n-propyl/isopropyl; cyclopropyl/allyl; 2-phenylethyl/l-phenylethyl), for which each alkylating group of the first compound can spontaneously rearrange to form the alkylating group of the second isomer, we investigated whether the alkylation proceeds via a monomolecular (sn1) or a bimolecular substitution (sn2). For this, we comparatively determined the relative mutagenic activities of each isomer in Salmonella typhimurium TA 1535, as well as their reactivities towards diethyldithiocarbamate (DDTC) by identifying the reaction products. These studies were aimed at revealing the possible formation of a free carbonium ion in the decomposition of several nitrosoureas in the rat liver supernatant fraction. Our system showed that DDTC reacts by two competing mechanisms: attack at the diazonium ion and at the free carbonium ion. Therefore the striking differences which were observed in the mutagenic potency of cyclopropylnitrosourea and N nitrosoallylurea as well as of N-nitroso-2-phenylethylurea and N-nitroso-1 phenylethylurea cannot be explained only by the different electrophilic reactivities of the respective intermediates. PMID- 3009489 TI - In situ measurements of external pH and optical density oscillations in Dictyostelium discoideum aggregates. AB - In situ measurements of extracellular pH by means of microelectrodes and in situ measurements of optical density were performed on aggregating cells of Dictyostelium discoideum. Early aggregation stage AX2 cells showed sinusoidal pH oscillations, which could be inhibited by the specific relay inhibitor caffeine, indicating that they were coupled to cAMP oscillations. Sometimes biphasic pH oscillations were found, which can be explained by the superposition of two harmonic pH oscillations. These harmonic oscillations might arise by gating of the cAMP signal; a part of the cells respond to every cAMP signal and another subpopulation to every second cAMP pulse. Late aggregation-stage cells showed complex changes of the extracellular pH, which could be inhibited by caffeine. Optical density measurements of wave propagation in aggregation streams of HG220 also revealed gating behavior. In addition to sinusoidal optical density oscillations, biphasic and still more complex oscillations were observed. PMID- 3009488 TI - Biological activity of N-nitrosodiethanolamine and of potential metabolites which may arise after activation by alcohol dehydrogenase in Salmonella typhimurium, in mammalian cells, and in vivo. AB - The potent carcinogen N-nitrosodiethanolamine (NDELA) which is nonmutagenic in standard modifications of the S. typhimurium/mammalian microsome assay, can be activated effectively by alcohol dehydrogenase/NAD (ADH/NAD) to intermediates which are directly mutagenic in strains TA 98 and TA 100. The expected metabolites N-nitroso-2-hydroxymorpholine (NHMor), N-nitroso-(2-hydroxyethyl) glycine (NHEG), N-nitrosoiminodiacetic acid (NIDA), and glycolaldehyde were assayed for their direct mutagenic activities in S. typhimurium TA 1535, TA 98, and TA 100. All compounds were clearly mutagenic in TA 100, but different specificities were observed for the other strains. NDELA and its putative mutagenic metabolites were also tested for induction of genotoxic activities by determination of DNA single strand breaks in primary rat hepatocytes. In these cells, NDELA and NHMor were clearly genotoxic, whereas NHEG and NIDA were inactive. In contrast, when assayed for the induction of selective DNA amplification NDELA and its metabolites were not found to induce SV40 DNA synthesis in SV40-transformed Chinese Hamster cells. The compounds were also assayed for induction of DNA single strand breaks in the liver after a single oral application to rats. NDELA and NHMor were about equally active in this in vivo test, whereas NHEG, NIDA and glycolaldehyde were inactive. Differences in biological activity in the cultivated cells, as compared to hepatocytes or to the in vivo situation may most probably be due to differences in metabolism and/or pharmacokinetics. PMID- 3009490 TI - Liposome delivery of cyclic AMP-dependent protein kinase inhibitor into intact cells: specific blockade of cyclic AMP-mediated adrenocorticotropin release from mouse anterior pituitary tumor cells. AB - Insertion of a crude preparation of cyclic AMP (cAMP)-dependent protein kinase inhibitor (PKI) into a cloned mouse anterior pituitary cell line (AtT-20/D16-16) blocked cAMP-mediated hormone release. This was accomplished by developing a technique to incorporate PKI into multicellular cultures. The technique involved the encapsulation of the PKI into liposomes coupled to Protein A (a bacterial protein that binds to the Fc portion of antibodies). Application of such liposomes to AtT-20 cells targeted by pre-treatment with an antiserum against neural cell adhesion molecule (a cell surface glycoprotein expressed by these cells) resulted in the attachment of the liposomes onto the cell surface followed by the delivery of the liposome content into the cells. The AtT-20 cells respond to cAMP-promoting agents such as forskolin by secreting the hormone adrenocorticotropin (ACTH). Liposomes containing PKI and coupled to protein A specifically blocked cAMP-mediated ACTH release from cells treated with anti-N CAM antibodies. In contrast, the ACTH release response to K+ or phorbol esters does not appear to involve cAMP and was not reduced by such manipulations. The specificity of PKI to block hormone release initiated by one but not by other secretagogues directly links cAMP-dependent protein kinase with the ACTH release process but suggests that there are other mechanisms also involved in stimulus secretion coupling in corticotrophs. PMID- 3009492 TI - Mitogenic response of human SH-SY5Y neuroblastoma cells to insulin-like growth factor I and II is dependent on the stage of differentiation. AB - Human insulin-like growth factor I and II (IGF-I and IGF-II) in concentrations of 1-30 ng/ml, were shown to stimulate ornithine decarboxylase activity and [3H]thymidine incorporation in human SH-SY5Y neuroblastoma cells. Proliferation of these cells was also stimulated by IGF-I and II when added to RPMI 1640 medium, fortified with selenium, hydrocortisone, transferrin, and beta-estradiol. Labeled IGF-I and II bound to SH-SY5Y cells. The cross-reaction pattern of IGF-I, IGF-II, and insulin in competing with the binding of labeled IGF-I and IGF-II, respectively, indicated that SH-SY5Y cells express both type I and type II IGF receptors. Treatment of SH-SY5Y cells for 4 d with 12-O-tetradecanoylphorbol-13 acetate (TPA), which resulted in morphological and functional differentiation and growth inhibition, abolished the mitogenic response to both IGF-I and II. Concomitantly, the binding of IGF-II disappeared almost totally, which offers a possible explanation for the reduced biological response to IGF-II after TPA treatment. In contrast, the IGF-I binding in TPA-treated cells was only reduced to approximately 70% of the binding to control cells. It is therefore not excluded that the IGF-I receptor could be uncoupled by TPA, with persistent binding capacity for IGF-I. PMID- 3009493 TI - Cellular basis for the species differences in sensitivity to cardiac glycosides (digitalis). AB - The relative toxicity of numerous cardiotonic steroids (viz. ouabain, digitoxin, digoxin, convallatoxin, SC4453, bufalin, gitaloxin, digoxigenin, actodigin, oleandrin, digitoxigenin, gitoxin, strophanthidin, gitoxigenin, lanatosides A, B and C, alpha- and beta-acetyl digoxin, alpha- and beta-methyl digoxin) and related compounds towards a number of independent cell lines established from human, monkey, mouse, Syrian hamster, and Chinese hamster have been determined. All cardiac glycosides and their genins, as well as the cardiotonic alkaloid cassaine, exhibited greater than 100-fold higher toxicity towards cultured human and monkey cells in comparison to the cell lines of mouse, Syrian hamster, and Chinese hamster origins. These differences are species-related as all cell lines (both normal as well as transformed) from any one species, as well as cells from the closely related species (e.g., man and monkey or mouse, Chinese hamster, and Syrian hamster), showed similar sensitivity towards these drugs. The failure to see any significant differences in cellular toxicity for a larger number of other compounds which either bear limited structural resemblance to cardiac glycosides (viz. estradiol 17-beta-acetate, testosterone propionate, 21-acetoxy pregnenolone, beta-estradiol, digitonin, tigogenin, and tomatine) or interact with the Na+/K+ ATPase in a different manner (viz. veratridine, sanguinarine nitrate, penicillic acid, vanadium pentoxide, harmaline-HCI,5,5'-diphenyl hydantoin, quindonium bromide, and methyl quinolizinum bromide) provides strong evidence that the observed species-related differences are highly specific for cardiotonic steroids. Studies on the binding of [3H]ouabain show that, in comparison to human and monkey cell lines, no significant binding of the drug is observed in cells derived from the resistant species (i.e., mouse and Chinese hamster). The Na+/K+ ATPase from cells of the resistant species is inhibited at much higher concentrations of ouabain and digitoxin in comparison to the enzyme from human cells, and a good correlation is observed between these concentrations and those reported for inhibition of the enzyme from isolated heart muscles of the same species. These results provide strong evidence that the species-related differences in sensitivity to digitalis have a cellular basis and that the cultured cells from various mammalian species provide a useful model system for investigating the mechanism of action of cardiac glycosides. PMID- 3009491 TI - Redistribution and shedding of flagellar membrane glycoproteins visualized using an anti-carbohydrate monoclonal antibody and concanavalin A. AB - Two carbohydrate-binding probes, the lectin concanavalin A and an anti carbohydrate monoclonal antibody designated FMG-1, have been used to study the distribution of their respective epitopes on the surface of Chlamydomonas reinhardtii, strain pf-18. Both of these ligands bind uniformly to the external surface of the flagellar membrane and the general cell body plasma membrane, although the labeling is more intense on the flagellar membrane. In addition, both ligands cross-react with cell wall glycoproteins. With respect to the flagellar membrane, both concanavalin A and the FMG-1 monoclonal antibody bind preferentially to the principal high molecular weight glycoproteins migrating with an apparent molecular weight of 350,000 although there is, in addition, cross-reactivity with a number of minor glycoproteins. Western blots of V-8 protease digests of the high molecular weight flagellar glycoproteins indicate that the epitopes recognized by the lectin and the antibody are both repeated multiple times within the glycoproteins and occur together, although the lectin and the antibody do not compete for the same binding sites. Incubation of live cells with the monoclonal antibody or lectin at 4 degrees C results in a uniform labeling of the flagellar surface; upon warming of the cells, these ligands are redistributed along the flagellar surface in a characteristic manner. All of the flagellar surface-bound antibody or lectin collects into a single aggregate at the tip of each flagellum; this aggregate subsequently migrates to the base of the flagellum, where it is shed into the medium. The rate of redistribution is temperature dependent and the glycoproteins recognized by these ligands co redistribute with the lectin or monoclonal antibody. This dynamic flagellar surface phenomenon bears a striking resemblance to the capping phenomenon that has been described in numerous mammalian cell types. However, it occurs on a structure (the flagellum) that lacks most of the cytoskeletal components generally associated with capping in other systems. The FMG-1 monoclonal antibody inhibits flagellar surface motility visualized as the rapid, bidirectional translocation of polystyrene microspheres. PMID- 3009494 TI - Superoxide anion release by human endothelial cells: synergism between a phorbol ester and a calcium ionophore. AB - In order to study the signal transduction mechanism of human endothelial cells (EC), the regulation of superoxide anion (O2-)release in EC has been investigated using the calcium ionophore A23187 and phorbol myristate acetate (PMA), a potential activator of the Ca2+ activated, phospholipid-dependent protein kinase, designated "protein kinase C." PMA enhanced O2- release from EC, and this enhancement occurred regardless of the presence or absence of extracellular Ca2+. A similar increase was produced by A23187; omission of extracellular Ca2+ prevented this increase. Simultaneous stimulation with PMA and A23187 produced a large increase in O2- release at submaximal concentrations of these agents, which, when added separately, caused minimal effects. These findings indicate that the activation of protein kinase C and mobilization of Ca2+ evoked by PMA and A23187 respectively are synergistically effective for eliciting a full physiological response of EC in the generation and release of O2-. PMID- 3009495 TI - Regulation of hexose transporters in chicken embryo fibroblasts: stimulation by the phorbol ester TPA leads to increased numbers of functioning transporters. AB - As has been observed with many types of cultured cells, chicken embryo fibroblasts (CEF) when exposed to the tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA) develop a 3- to 4-fold increase in hexose transport activity in 4 h. This increase in transport activity occurred despite a modest decline of 20% in [3H]leucine incorporation into acid insoluble fractions. Cycloheximide largely, but not completely, blocked the increase in transport activity during TPA exposure. The effects of TPA were somewhat similar to those of glucose starvation induced enhancement of hexose transport activity. Furthermore, with TPA there was no additive effect to that produced by glucose starvation. Plasma membrane enriched fractions were prepared from CEF treated with or without TPA. Membranes prepared from TPA exposed cells had a two-fold enhancement of stereospecific D-glucose transport activity as well as D-glucose inhibitable [3H]cytochalasin B binding as compared to the membranes from control CEF. There was no effect on transport when membranes were exposed to TPA in vitro. These results provide strong evidence that TPA exposure leads to an increase in the number of functioning transporters, an effect largely requiring protein synthesis. PMID- 3009497 TI - Forskolin, phosphodiesterase inhibitors, and cyclic AMP analogs inhibit proliferation of cultured bovine aortic endothelial cells. AB - The role of cyclic AMP on endothelial cell proliferation was investigated, since these cells can be exposed to high concentrations of physiological and pharmacological agents that alter cyclic AMP metabolism. Cloned bovine aortic endothelial cells were plated at 25,000 cells/35mm dish and grown for 5 days in the presence of phosphodiesterase (PDE) inhibitors, forskolin, or cyclic AMP analogs. The PDE inhibitors dipyridamole, ZK 62 711, isobutylmethylxanthine (IBMX) and theophylline inhibited cell growth in a concentration-dependent manner. Dipyridamole produced a 30% and a 50% inhibition at 5 microM and 12.5 microM, while higher concentrations were cytotoxic. At its therapeutic plasma concentration range (50-100 microM) theophylline inhibited cell proliferation by 15-25%, while IBMX and the highly specific cyclic AMP phosphodiesterase inhibitor, ZK 62 711 inhibited growth by 60-80% and 40-50%, respectively. Forskolin (5 microM) increased cyclic AMP levels and cyclic AMP-kinase activity ratios by 2.5-fold and 2-fold. In the absence of PDE inhibitors forskolin produced a 20% growth inhibition at 0.5 microM and a 60% inhibition at 10 microM. The forskolin dose-response curve was not altered by theophylline, but was shifted to the left by approximately 10-fold with dipyridamole and ZK 62 711 and 5-fold with IBMX. Forskolin (5 microM), by itself produced a 1.8-fold increase in cyclic AMP. In the presence of 5 microM theophylline, dipyridamole, IBMX, and ZK 62 711, cyclic AMP was increased by forskolin 2.0, 2.6, 3.5, and 6.6-fold, respectively. 8-Bromo cyclic AMP and dibutyryl cyclic AMP produced a 55% and 60% growth inhibition at 100 microM. The cyclic GMP analogs were less effective inhibitors of growth (15-30%). Our results demonstrate that cyclic AMP analogs and pharmacological agents that elevate intracellular cyclic AMP levels inhibit cell growth and suggest that cyclic AMP may be an important endogenous regulator of endothelial cell proliferation. PMID- 3009496 TI - Retinoids stimulate the release of superoxide by neutrophils and change their morphology. AB - All-trans-retinal stimulated the release of superoxide by human and guinea pig neutrophils 63 +/- 14 SD and 53 +/- 5 SD nmol of O2-/min/10(7) cells, respectively. Superoxide release by unstimulated cells was negligible. All-trans retinal also induced morphological changes (i.e., evaginations) in these cells. Other retinoids were effective in instigating these phenomena. The similarities of these effects to those instigated by cis-unsaturated fatty acids (Badwey, J.A., et al., 1984, J. Biol. Chem., 259:7870-7877) are discussed in light of possible mechanisms. PMID- 3009499 TI - Accumulation of cells with 4N DNA content at nonpermissive temperature in rat embryo diploid cells transformed by tsA mutant of simian virus 40. AB - Primary rat embryo cells were transformed by a tsA mutant (tsA640) of simian virus 40 (SV40). Proliferation of all four independent diploid transformants was suppressed at a nonpermissive temperature (40.3 degrees C), being accompanied by a marked increase in the fraction of cells with a 4N DNA content (a 4N peak in the flow cytofluorogram). However, in this case, the fraction of cells with a 2N DNA content (a 2N peak in the flow cytofluorogram) was preserved. Both effects (suppression of proliferation and increase in the 4N peak) diminished when transformed cells were superinfected with wild-type SV40. The increased 4N peak was preserved, albeit not completely, for at least 24 hours, when cells were further incubated in the presence of hydroxyurea at the nonpermissive temperature. On the other hand, the preserved 2N peak all but disappeared within 24 hours, when cells were further incubated in the presence of colcemid at the nonpermissive temperature. These results suggest that the thermolabile large T antigen of SV40 directly or indirectly induces an accumulation of cells with a 4N DNA content, at the nonpermissive temperature, by prolonging the G2 (and/or late S) period. PMID- 3009498 TI - Regulation of amino acid uptake by phorbol esters and hypertonic solutions in rat thymocytes. AB - Growth factors, mitogens, and malignant transformation can alter the rate of amino acid uptake in mammalian cells. It has been suggested that the effects of these stimuli on proliferation are mediated by activation of Na+/H+ exchange. In lymphocytes, Na+/H+ exchange can also be activated by phorbol esters and by hypertonic media. To determine the relationship between the cation antiport and amino acid transport, we tested the effects of these agents on the uptake of alpha-aminoisobutyric acid (AIB), methyl-AIB, proline, and leucine in rat thymocytes. Both 12-O-tetradecanoylphorbol-13-acetate (TPA) and hypertonicity stimulated amino acid uptake through system A (AIB, proline, and methyl-AIB). In addition, TPA, but not hypertonicity, also elevated leucine uptake. The stimulation of the Na+ -dependent system A was not due to an increased inward electrochemical Na+ gradient. The effects of TPA and hypertonic treatment were not identical: Stimulation of AIB uptake by TPA was observed within minutes, whereas at least 1 hr was required for the effect of hypertonicity to become noticeable. Moreover, stimulation by hypertonicity but not that by TPA, was partially inhibited by cycloheximide, suggesting a role of protein synthesis. That stimulation of Na+/H+ exchange does not mediate the effects on amino acid transport is suggested by two findings: 1) the stimulation of AIB uptake was not prevented by concentrations of amiloride or of 5-(N,N-disubstituted) amiloride analogs that completely inhibit the Na+/H+ antiport and 2) conditions that mimic the effect of the antiport, namely, increasing [Na+]i or raising pHi failed to stimulate amino acid uptake. Thus, in lymphocytes, activation of Na+/H+ exchange and stimulation of amino acid transport are not casually related. PMID- 3009500 TI - Effect of 1,3,6-triaminohexane and 1,4,7-triaminoheptane on growth and polyamine metabolism in SV-3T3 cells treated with 2-difluoromethylornithine. AB - The possibility that one or both of the synthetic triamines, 1,3,6-triaminohexane and 1,4,7-triaminoheptane, could substitute for the naturally occurring polyamines in the growth of SV-3T3 cells was investigated. It was found that these triamines did lead to a restoration of growth in cells in which spermidine content had been depleted by exposure to the ornithine decarboxylase inhibitor 2 difluoromethylornithine. This resumption of a normal growth rate occurred prior to the reduction in the content of cellular decarboxylated S-adenosylmethionine, suggesting that this nucleoside (which increases in concentration several hundred fold in cells treated with 2-difluoromethylornithine) does not cause the reduction of cell growth. However, unlike the increase in cell growth brought about by spermidine, which continued indefinitely, the increase produced by 1,3,6 triaminohexane or 1,4,7-triaminoheptane was transient. Cell growth in the presence of 2-difluoromethylornithine and these triamines stopped after about three or four population doublings. This corresponded to the time at which the intracellular spermine content of the cells was reduced to values less than 20% of normal. It is suggested that the increased growth rate of spermidine-depleted cells in response to these triamines is due to their uptake into the cell and ability to displace spermine from intracellular sites, thus making spermine available to fulfill the polyamine function(s) essential for growth. These results indicate that the naturally occurring polyamines spermidine or spermine are essential for continued cell growth and cannot be replaced by analogues containing only primary amino groups. PMID- 3009501 TI - 1,2-Diacylglycerols mimic phorbol 12-myristate 13-acetate activation of the sea urchin egg. AB - Phorbol diesters have been reported to stimulate the Na+/H+ antiport of a variety of cells including sea urchin eggs. Since stimulation of the Na+/H+ antiport is necessary for metabolic derepression during fertilization and protein kinase C is a target of phorbol diesters, enhanced Na+/H+ exchange during fertilization may be a result of protein kinase C activity. Protein kinase C is probably physiologically activated by diacylglycerols, which are derived from hydrolysis of phosphatidylinositol. Treatment of sea urchin eggs with 1,2-diacylglycerols was found to stimulate the Na+/H+ antiport. The 1,3-isomers were without effect. Further, the effects of 1,2-diacylglycerol and phorbol diester are not additive with respect to Na+/H+ exchange. While a direct participation of protein kinase C activity during fertilization remains to be demonstrated, these data support the hypothesis that protein kinase C activity plays a role in fertilization. However, the cytotoxic effect of protein kinase C activators suggests effects associated with their pleiotropic nature. PMID- 3009502 TI - Problems and complications encountered in replantation surgery. AB - The surgical techniques involved in a discipline as comparatively new and complex as replantation might be expected to result in a significant rate of complications. However, serious complications related to replantation surgery remain relatively infrequent. Meticulous attention to detail helps to avoid predicaments, minimizes potential problems, and allows the surgeon to correct the occasional complication that arises. PMID- 3009503 TI - Copper ions and hydrogen peroxide form hypochlorite from NaCl thereby mimicking myeloperoxidase. AB - Sea urchins have elaborated multiple defenses to assure monospermic fertilization. In this work, we have concentrated on a study of the mechanism(s) by which hydrogen peroxide (H2O2) prevents polyspermy in Arbacia punctulata. We found that it is not H2O2 but probably hypochlorous acid/hypochlorite (HOCl/OCl-) derived from H2O2 that is toxic to the supernumerary sperm. The spermicidal activity of H2O2 is potentiated by at least one order of magnitude by cupric ions (Cu2+). This increased toxicity is not due to the formation of hydroxyl radicals (.OH) because .OH scavengers did not counteract the activity of Cu2+. Moreover, substitution of Cu2+ by ferrous ions (Fe2+), which are known to cause formation of .OH from H2O2, had no effect on fertilization even at 10(2)-10(3) times higher concentrations. In contrast, 3-amino-1,2,4-triazole (AT), and HOCl/OCl- scavenger, totally reversed the toxic effects of Cu2+. Furthermore, we found that HOCl/OCl- is generated in solutions of H2O2 and Cu2+ in the presence of 0.5 M NaCl and that its accumulation is abolished by AT. Thus it is possible that the antifertility properties of copper are due to its ability to mediate formation of HOCl/OCl-. HOCl/OCl- generated by Cu2+ from H2O2 and Cl-, a low concentration of exogenously added HOCl/OCl-, or increased concentrations of H2O2 has similar inhibitory effects on the fertilization process in sea urchins. Therefore, we suggest that polyspermy is prevented by the action of a myeloperoxidase that affects the formation of HOCl/OCl- from the Cl- present in sea water through reaction with H2O2 generated by the newly fertilized egg. PMID- 3009505 TI - TMB-8 inhibits respiration and cyclic GMP formation in Dictyostelium discoideum. AB - The putative inhibitor of intracellular calcium mobilization, TMB-8 was found to be a powerful inhibitor of respiration in amoebae of Dictyostelium discoideum. Consequently, the previously reported effects of this drug on cyclic GMP formation induced by chemoattractants were reassessed. It was found that TMB-8 abolished both folate and cyclic AMP-mediated accumulation of cyclic GMP in D. discoideum amoebae and that addition of Ca2+ completely restored this response. The Ca2+ chelating agent EGTA did not mimic the effect of TMB-8. The effect on cyclic GMP formation, however, occurred only at a concentration of TMB-8 that was ten times that causing maximal inhibition of respiration, and inhibition of cyclic GMP formation was completely restored by addition of excess Ca2+, whereas inhibition of respiration was only partially restored. The data suggest that TMB 8 has more than one inhibitory action, and because of the differential sensitivity of respiration and cyclic GMP formation to this drug, and the differential antagonism of excess Ca2+, we conclude that the effect of TMB-8 on the cyclic GMP response is probably due to its effect on Ca2+ mobilization, rather than indirectly via its effects on respiration. However, we advise caution in interpretation of data using this inhibitor where the responses measured are prolonged, are energy-requiring or are not freely reversible by excess Ca2+. PMID- 3009504 TI - Demonstration of a relationship between talin and P235, a major substrate of the calcium-dependent protease in platelets. AB - Talin is a 225,000-Dalton protein we have purified from smooth muscle. In chick embryo fibroblasts talin is found in adhesion plaques (focal contacts), areas where the cell is closely apposed to the substratum. In comparison with other cytoskeletal proteins, we found talin to be unusually susceptible to proteolysis and have identified a 190,000-Dalton proteolytic fragment of talin in the immunoblots of many tissues. These observations raised the possibility that the cleavage of talin to this fragment has physiological relevance. One system that we have investigated in which significant proteolysis occurs is platelets. During platelet activation several high-molecular-weight proteins are cleaved to lower molecular-weight forms. Here we demonstrate that talin is closely related to one of these platelet high-molecular-weight proteins, P235. The purification of talin is comparable to that developed for P235, and the two proteins have similar biophysical properties. In addition, antibodies raised against chicken gizzard talin recognize P235 in purified form as well as in crude platelet extracts. The platelet protein also resembles smooth-muscle talin in its susceptibility to endogenous proteolysis: P235 is rapidly cleaved to a 190-200 kD polypeptide by a calcium-activated protease found in platelet extracts. Moreover, partial proteolysis of P235 and talin with chymotrypsin, elastase, or trypsin also generates remarkably similar one-dimensional peptide maps. Because of their similar biophysical properties, immunological crossreactivity, and similar one dimensional partial peptide maps, we conclude that P235 is the platelet form of talin. PMID- 3009506 TI - Management of breast cancer with hormones and drugs. PMID- 3009507 TI - AIDS: what is now known. I. History and immunovirology. PMID- 3009508 TI - High-performance liquid chromatographic analysis of basic compounds on non modified silica gel and aluminium oxide with aqueous solvent mixtures. AB - A comparison is made between the use of aluminium oxide and non-modified silica gel as cation-exchange materials for the separation of basic drugs (amines) with aqueous solvent mixtures. The retention behaviour of the amines is studied and appears to be controlled predominantly by the pH and the concentration and nature of the modifier; the nature and concentration of the competing ions and the buffer components of the mobile phase also exert some influence on the retention. Preparations with imidazoline and tetracycline derivatives have been analysed as examples of the application of these ion-exchange systems on non-modified silica gel and aluminium oxide in the analysis of pharmaceutical formulations. PMID- 3009509 TI - High-performance liquid chromatographic determination of dimethyldithiocarbamate residues in some agricultural products. AB - Dimethyldithiocarbamates are widely used in agriculture as active fungicides. The degradation of dimethyldithiocarbamates (ferbam, thiram) confirmed the fact that they are not stable and decompose very rapidly. The aim of this work was to apply the results obtained in high-performance liquid chromatographic quantitative analysis of residues of dithiocarbamate fungicides in some agricultural products (strawberries, maize, tobacco). The developed method enables very simple control analysis of low concentrations of dimethyldithiocarbamate residues in very short time. All limits of detection correspond with the criteria of FAO (Codex Alimentarius). PMID- 3009510 TI - Radioimmunoassay of adjuvant-associated porcine parvovirus using a monoclonal antibody in a nitrocellulose membrane system. AB - A quantitative and simple indirect radioimmunoassay (IRIA) was developed for porcine parvovirus (PPV), employing a monoclonal antibody directed against PPV adsorbed to nitrocellulose membrane. The IRIA was equally sensitive to live or inactivated PPV. There was a linear relationship between membrane-bound radioactivity and PPV quantity within a range of 10-80 hemagglutinating (HA) units of virus. Two commercially used adjuvants, aluminum hydroxide (AH) and carboxyvinyl polymer (CP), reduced bound radioactivity in a concentration dependent manner. At fixed adjuvant concentrations, there were, nevertheless, linear relationships between bound radioactivity and HA units of PPV. Known amounts of PPV were prepared in adjuvants according to commercial vaccine formulations. Using these standards, the PPV content of 16 commercial PPV vaccines was estimated by IRIA. The IRIA may be one practical method of in vitro estimation of antigenic mass in adjuvanted vaccines. PMID- 3009511 TI - A comparison of the susceptibility of three human gut tumour-derived differentiated epithelial cell lines, primary monkey kidney cells and human rhabdomyosarcoma cell line to 66-prototype strains of human enteroviruses. AB - The growth of prototype strains of 31 serotypes of ECHO, 3 polio, 6 Coxsackie B, 24 Coxsackie A and enterovirus serotypes 70 and 71 were tested in parallel in primary monkey kidney cells (PMK), RD cells and three gut tumour-derived differentiated epithelial cell lines (HRT-18 HT-29 in SKCO-1). All 31 serotypes of ECHO viruses grew in HT-29, 27 and SKCO-1, 5 in HRT-18, 29 in PMK and 29 in RD. There was good growth of poliovirus serotypes in all five cell types. Coxsackie B viruses grew well in all the cell lines except RD. Fifteen of the Coxsackie A viruses grew in SKCO-1, 4 in HT-29, 3 in HRT-18 and 7 in RD. Enterovirus serotypes 70 and 71 grew only in RD cells after 3 serial passages. These results showed that 2 of the gut tumour-derived cell lines, HT-29 and SKCO 1 had a markedly wider susceptibility, with comparable or wider sensitivity, for enteroviruses, than PMK and RD. While their use for field isolation from clinical samples is not yet fully established HT-29 and SKCO-1 would appear to be ideal for a variety of laboratory manipulations of the majority of enteroviruses. PMID- 3009512 TI - Coxsackie B-2 virus infection in rat beating heart cell culture. AB - Beating rat heart cultures were prepared in vitro and infected with Coxsackie B-2 virus. The cells were evaluated in the post-infected period for changes in cardiac enzymes, alterations in beating frequency and cytotoxicity as measured by chromium 51 (51Cr) release. The cardiac enzymes, lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) were measured in infected and uninfected controls over a period of 120 h. Enzyme levels in the infected cells remained essentially the same for the first 42 h as compared to the controls. At this time, the LDH levels increased rapidly reaching 116 +/- 24.8 U/l while the controls remained at 46.9 +/- 9.7 U/l. Aspartate aminotransferase levels increased at a slower rate and obtained a level of 104 +/- 20.2 U/l compared to 66.6 +/- 13.2 U/l in the control. Visual evidence of cellular damage as measured by decreased beating frequencies and the appearance of cytopathic effect was first noted at 42 h post-infection. Complete loss of cardiac beats and maximal viral cytopathic effect occurred at 96 h post-infection. Cardiac cellular damage as measured by cytotoxicity assay was found to parallel those changes seen in cardiac enzymes. No significant changes in cytotoxicity were observed for the first 24 h; however, at 48 h increased release of 51Cr was noted and visual evidence of viral replication also was present. The cardiac enzyme changes noted in beating rat heart cells appear to be similar to those changes reported in patients with viral-induced myocardial disease.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009514 TI - Detection of human cytomegalovirus-induced DNA-binding proteins on Western blots. AB - DNA-binding proteins have been isolated from human embryonic fibroblast cells infected with human cytomegalovirus strain AD169 by native and denatured DNA cellulose affinity chromatography and Polymin P precipitation. The DNA-binding proteins separated by polyacrylamide gel electrophoresis and electrophoretically transferred to a nitrocellulose sheet were identified by a modified ELISA reaction. Six DNA-binding proteins were found in infected cells, their molecular weights range from 52K to 18K. PMID- 3009515 TI - Envelope proteins of avian infectious bronchitis virus: purification and biological properties. AB - Immunoadsorbents, made with monoclonal antibodies, were used to purify the spike and membrane proteins of infectious bronchitis virus (IBV). The purified proteins were inoculated into rabbits to produce antisera. The rabbit anti-spike sera neutralized the infectivity of the virus whereas the anti-membrane sera did not. IBV-infected chickens produced antibodies to both the spike and membrane proteins. Both these antibodies were at their highest concentration about 9-11 days after inoculation, whereas neutralizing antibodies were present only at very low concentrations at that time. Neutralizing antibodies were at their highest concentration 21 days after inoculation. A second inoculation of virus at 42 days induced an anamnestic antibody response to the spike and membrane proteins and also for the neutralizing antibodies. The neutralizing, anti-spike and anti membrane antibodies all reached highest concentrations 7-11 days after this inoculation. The advantages of purifying viral proteins using affinity chromatography with monoclonal antibodies are discussed. PMID- 3009513 TI - Use of the nitrocellulose-enzyme immunosorbent assay for rapid, sensitive and quantitative detection of human enteroviruses. AB - A modified enzyme immunosorbent assay (EIA) employing nitrocellulose (NC) membrane as a high-capacity solid phase was successfully employed for the sensitive and rapid detection of human enteric viruses, poliovirus and Coxsackievirus B-5. The sensitivity of the NC-EIA ranged from 7 to 70 pg of viral antigen diluted in phosphate-buffered saline. When virus was added to crude supernatants of mollusc tissue homogenates prepared by the standard procedure for the recovery of viruses in molluscs, the sensitivity was reduced by approximately 10-fold. The sensitivity of the NC-EIA for the detection of viral antigens was 10 to 100-fold higher than conventional EIA using polystyrene microtiter plates as solid phase supports. This simple, rapid and sensitive assay using minimal amounts of antibodies should prove to be a useful and practical diagnostic tool to augment infectivity assays currently employed by various virus monitoring procedures. The method also may be applicable for the detection of difficult to grow and/or non-cultivatable enteric viruses which may be present in sewage contaminated environments. PMID- 3009517 TI - Isolation of human pathogenic viruses from clinical material on CaCo2 cells. AB - The use of the epidermal-like cell line CaCo2 and the efficient propagation of viruses from clinical material for diagnostic purposes are reported. Virus replication was observed by cytopathic effects and/or immunofluorescence. The following viruses replicate in CaCo2 cells: enteroviruses (coxsackie B1-B6, poliovirus types 1-3, most echoviruses and coxsackie A viruses), adenoviruses, herpes simplex types 1 and 2, measles, respiratory syncytial, parainfluenza type 2 viruses, and to a lesser extent rubella and mumps viruses. CaCo2 cells were used in neutralization tests for the diagnosis of enteroviral infections. PMID- 3009516 TI - A rapid chemiluminescent enzyme-linked immunosorbent assay for cytomegalovirus immunoglobulin G antibodies using instant photographic film. AB - A rapid and convenient chemiluminescent enzyme-linked immunosorbent assay (ELISA) for IgG antibodies to cytomegalovirus has been developed which uses low cost equipment. Assays were carried out on transparent microtitre plates and used an anti-human IgG horseradish peroxidase conjugate. Bound peroxidase was detected chemiluminescently using a p-iodophenol-luminol-peroxide reagent. Light emission from the wells of the microtitre plate was detected on instant photographic film (ASA 20,000) held in a specially designed shutter type camera. The semi quantitative technique was tested in a routine laboratory for a period of 7 wk and the results obtained compared well (95.3% agreement) with those obtained by a conventional colorimetric ELISA using an alkaline phosphatase label. PMID- 3009518 TI - Heat inactivation of serum may interfere with tests for antibodies to LAV/HTLV III. PMID- 3009519 TI - Genetics of the insulin receptor defect in a patient with extreme insulin resistance. AB - A patient with extreme insulin resistance (leprechaun/Ark-1) had an 80-90% decrease in the number of insulin receptors on her circulating monocytes. In contrast, while a normal number of insulin receptors was expressed on the surface of Epstein-Barr (EB) virus-transformed lymphocytes from the patient, the receptors had decreased sensitivity to changes in temperature and pH. The father, who had a moderate degree of insulin resistance, resembled the patient in that his monocytes had a 60-80% decrease in the number of insulin receptors. Binding to the father's EB virus-transformed lymphocytes was normal. The mother was normally sensitive to insulin and had a normal number of insulin receptors on her circulating monocytes. In contrast, insulin receptors on the mother's EB virus transformed lymphocytes were qualitatively abnormal, closely resembling the daughter's cultured cells. These observations suggest that each parent has transmitted a different genetic defect to the patient. When both mutations coexist in the same individual, they fail to complement, but, rather, result in extreme insulin resistance. PMID- 3009520 TI - Glucocorticoid receptors in Epstein-Barr virus-transformed lymphocytes from patients with glucocorticoid resistance and a glucocorticoid-resistant New World primate species. AB - Members of a previously reported family with glucocorticoid resistance and several New World primates have high plasma cortisol concentrations without any signs of glucocorticoid excess. The glucocorticoid receptor in circulating leukocytes and cultured skin fibroblasts from these patients and the animals is characterized by a decreased affinity for dexamethasone. On the other hand, the cell content of receptor is similar to that of corresponding tissues of normal humans. Detailed biochemical-biophysical studies of the glucocorticoid receptor in this familial syndrome and animal model became possible with the use of Epstein-Barr virus-transformed lymphocyte lines. Cell lines from patients with this syndrome and from the marmoset (Saguinus oedipus) contained decreased amounts of glucocorticoid receptors with concomitant decreases in nuclear receptor content compared to cultured Epstein-Barr virus-transformed lymphocytes from normal human subjects. This may reflect diminished induction of glucocorticoid receptor during viral transformation of cells from the patients and the animal model. Receptors from a severely affected glucocorticoid-resistant patient and the marmoset had decreased affinity for dexamethasone. Evidence for a mild affinity defect of the glucocorticoid receptor in a patient with asymptomatic glucocorticoid resistance was obtained by increased hormone-receptor dissociation at an elevated temperature. Thermal stability, mero-receptor formation, thermal activation of cytosolic receptor, and mol wt of receptors from all cell lines were normal. Only the receptors of the severely affected patient had a discernible defect in temperature-induced activation of intact cells. We conclude that the major detectable change in the receptor in both the patients and the animal model is the decreased affinity for glucocorticoid. Viral receptor induction is decreased in both patient and marmoset cells. The physiological relevance of this phenomenon is not known. Gross receptor molecule changes or changes in its stability at higher temperatures were not found. Mixing studies did not show involvement of cytosolic modifiers or inhibitors. Mutation(s) of the receptor molecule leading to low affinity for the hormone is the most likely explanation of the isolated glucocorticoid resistance in the patients. The glucocorticoid resistance of the New World primate, which is part of generalized steroid hormone resistance, appears to be a result of more complex changes. PMID- 3009522 TI - Characteristics of human erythrocyte insulin-like growth factor I receptors. AB - The scarcity of purified materials has prevented studies of the mechanism of insulin-like growth factor I (IGF-I) action. Recently, an IGF-I analog, Thr59-IGF I, was synthesized by recombinant DNA technology. We found that this analog had binding characteristics similar to those of natural IGF-I in the radioreceptor assay system. We used this analog to characterize human erythrocyte IGF-I receptors as follows. Erythrocytes from 33 normal subjects specifically bound 6.9 +/- 0.7% (mean +/- SD)/2.8 X 10(9) cells/ml. Scatchard analysis of the binding data showed that the total number of receptors per erythrocyte was 13, and the affinity constant was 1.26 X 10(9) M-1, similar to that of other human IGF-I receptors previously reported. We also examined IGF-I receptors in patients with insulin receptor abnormalities. Although the specific values of IGF-I binding to erythrocytes were decreased or increased in parallel with those of insulin, the degrees of decrease or increase were much smaller. This suggests that the expression of insulin receptors and that of IGF-I receptors are discordant. PMID- 3009521 TI - Reversible hypopituitarism in patients with large nonfunctioning pituitary adenomas. AB - Detailed pituitary function studies were conducted on 26 patients with large nonfunctioning pituitary adenomas before and 2-3 months after transsphenoidal adenomectomy. Basal serum PRL, GH, TSH, LH, FSH, and ACTH levels were measured, and dynamic studies of their secretion were made. Preoperatively, GH deficiency was found in all 26 patients (100%), hypogonadism in 25 patients (96%), hypothyroidism in 21 patients (81%), and adrenal insufficiency in 16 patients (62%). Serum PRL levels were low (1.5-4 ng/ml) in 5 patients, normal (5-20 ng/ml) in 9 patients, and mildly elevated (21-53 ng/ml) in the remaining 12 patients. After selective adenomectomy, variable improvement in pituitary function occurred in 17 patients, worsening in 1 patient, and persistence of hypopituitarism in 8 patients. After surgery, normal thyroid function was documented in 12 of the 21 patients (57%) who were hypothyroid preoperatively. Similarly, 6 of the 16 patients (38%) with adrenal insufficiency recovered normal adrenal function, and 8 of the 25 patients (32%) with hypogonadism recovered normal gonadal function. GH deficiency persisted in all but 4 patients (15%). Serum PRL levels decreased in all patients, and only 5 had midly elevated levels after surgery. The presence of a normal or mildly elevated serum PRL level before surgery in these patients was of value in predicting possible recovery of pituitary function after surgery; none of the 5 patients with low preoperative serum PRL levels had any improvement in pituitary function after surgery. A rise in serum TSH levels after TRH administration before surgery also was helpful in predicting possible recovery from hypopituitarism. Most patients who had a rise in serum TSH level in response to TRH stimulation preoperatively recovered some pituitary function after adenomectomy. In contrast, no improvement in pituitary function occurred in patients who had blunted responses to TRH preoperatively. Improvement in pituitary function occurred more often in patients with tumors measuring 25 mm or less than in those with larger tumors. In conclusion, significant improvement in pituitary function may occur after surgical adenomectomy for nonsecreting pituitary tumors. A rise in serum TSH levels in response to TRH stimulation preoperatively suggested the presence of viable pituitary tissue in these patients with hypopituitarism. The presence of a normal or mildly elevated serum PRL level before surgery also suggested the presence of functioning pituitary lactotrophs. These observations suggest that compression of the portal circulation is a possible mechanism for hypopituitarism in this setting.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3009523 TI - Thyroid iodine content and serum thyroglobulin: cues to the natural history of destruction-induced thyroiditis. AB - Twenty-eight patients with destructive thyroiditis were followed to study the natural history of healing of thyroid gland injury. All had sequential measurements of thyroidal iodine [127I] content by fluorescent scanning (normal mean, 10.1 mg), 17 had serial serum thyroglobulin (Tg) measurements (normal, less than 21 ng/ml), and 13 had perchlorate discharge studies during the recovery phase. Seventeen patients had painful subacute thyroiditis (SAT), 9 had painless thyroiditis with thyrotoxicosis (PTT), and 2 had postpartum thyroiditis with thyrotoxicosis (PPT). Thyroidal iodine content decreased from a mean of 9.8 to a nadir of 3.8 mg in patients with SAT and from 8.5 to a nadir of 3.5 mg in patients with PTT. Mean serum Tg concentrations were highest (approximately 165 ng/ml) in both groups 1-3 months after the onset of symptoms. Abnormalities in both 127I content and Tg levels persisted for 2 or more yr in some individuals. No patient had detectable Tg antibodies by hemagglutination, but low titers were detected intermittently by sensitive RIA in 5 PTT patients. Microsomal antibodies were positive in only 1 of 16 SAT patients, but in 4 of 7 PTT patients and in both PPT patients. Three patients had positive perchlorate discharge tests (2 of 8 with SAT, 0 of 4 with PTT, and 1 of 1 with PPT). Permanent hypothyroidism occurred in 3 patients (2 with PTT; 1 with SAT and positive antibodies), but did not correlate with perchlorate results. HLA typing and serum immunoglobulin measurements were not useful for predicting the clinical course. These data indicate that several years may be necessary for complete resolution of destructive thyroiditis; many patients have evidence of thyroid injury persisting long after serum thyroid hormone and TSH levels become normal. PMID- 3009524 TI - Cortisol secretion by an incidentally discovered nonfunctional adrenal adenoma. AB - We describe a middle-aged man with late-onset multiple sclerosis and an incidentally discovered asymptomatic adrenal mass. He had no symptoms or signs of hypercortisolism. A 24-h profile revealed fluctuating serum cortisol values (between 15.1 and 4.7 micrograms/dl) and inappropriately low plasma ACTH values. Urinary cortisol excretion was 89 and 106 micrograms/day on two occasions. After a 4-h ACTH infusion, serum cortisol rose from 6.3 to 108 micrograms/dl. The serum dehydroepiandrosterone level, 33 ng/dl before ACTH stimulation, did not change. During dexamethasone administration, the lowest daily urinary cortisol excretion was 37 micrograms/day, and 17-ketosteroid excretion was 8 mg/day. The response to metyrapone showed a rise of serum 11-deoxycortisol to 25.6 micrograms/dl and of ACTH to 169.5 pg/ml. After removal of the tumor, most likely an adenoma, the circadian pattern of cortisol and ACTH was normal. During a 4-h ACTH infusion, the serum cortisol level rose from 10 to 27 micrograms/dl, and dehydroepiandrosterone rose from 62 to 90 ng/dl. During dexamethasone administration, daily urinary cortisol excretion decreased to 12 micrograms/day, and 17-ketosteroid excretion dropped to 3.9 mg/day. These data show that while the tumor appeared clinically to be nonfunctional, it was producing cortisol and possibly androgens autonomously, albeit at levels too low to cause complete suppression of the pituitary-adrenal axis. PMID- 3009525 TI - Rapid dot-immunobinding assay on nitrocellulose for viral antibodies. AB - A procedure is described for the routine laboratory diagnosis of viral serum antibodies. Antigens are dotted on nitrocellulose strips or sheets, and sera are applied on absorbent paper strips. Antigen-antibody complexes are detected with enzyme-conjugated antiglobulin and development of a colored, insoluble substrate product. The test allows processing of multiple sera in one 3- to 5-h operation and is equal to or more sensitive than serum neutralization, hemagglutination inhibition, and fluorescent antibody assays. Highly infectious viruses inactivated with a psoralen derivative and long-wavelength UV light irradiation can be used as antigens, allowing the study of human pathogens. Although the test detects cross-reacting, group-specific herpesvirus antigens, the intensity of the antibody reaction is greatest with type-specific antigens. Preliminary data suggest that the technique will be useful for the rapid typing of viruses from clinical specimens. PMID- 3009527 TI - Simultaneous infections with different enteric and respiratory tract viruses. AB - Infants and young children with rotavirus (RV) or visualized adenovirus in their stools were tested for the simultaneous presence of a respiratory viral pathogen in their upper respiratory tract. Overall, at least 10.7% of 484 study subjects had such dual infections, including 8.3% of 385 RV-positive gastroenteritis patients and 24.3% of 37 RV-positive respiratory disease patients. Respiratory syncytial virus was present in 34.1% of 41 dual infections with RV and at least 40% of the 12 to 15 dual infections with visualized fecal adenovirus. Other pathogens found in the respiratory tract of patients with RV or visualized fecal adenovirus infections included influenza viruses, adenoviruses, parainfluenza viruses, rhinoviruses, and a cytomegalovirus. PMID- 3009526 TI - Complex-specific immunoglobulin M antibody patterns in humans infected with alphaviruses. AB - Sera from humans with serologically confirmed eastern equine encephalitis, western equine encephalitis, Pogosta (Ockelbo), Mayaro, Ross River, and chikungunya virus infections were tested by immunoglobulin M (IgM) antibody capture enzyme immunoassay. Diagnostically useful IgM antibody titers were detected, and selected sera with high IgM antibody titers were tested for IgM antibody with nine heterologous alphaviruses. The results provide evidence for the complex specificity of IgM antibody and indicate the usefulness of this test in both individual cases and epidemic situations. PMID- 3009528 TI - Flying squirrel-associated Rickettsia prowazekii (epidemic typhus rickettsiae) characterized by a specific DNA fragment produced by restriction endonuclease digestion. AB - The DNA from flying squirrel-associated Rickettsia prowazekii was characterized by using a specific DNA fragment produced by digestion with the enzyme BamHI. The DNA fragment was cloned into a plasmid vector and used to readily distinguish between available human- and flying squirrel-associated R. prowazekii DNAs derived from crude cytoplasmic extracts. PMID- 3009531 TI - Detection of rotavirus with a new polyclonal antibody enzyme immunoassay (Rotazyme II) and a commercial latex agglutination test (Rotalex): comparison with a monoclonal antibody enzyme immunoassay. AB - A total of 176 human fecal specimens were examined for the presence of rotavirus by four different assays: a monoclonal antibody enzyme immunoassay; the original polyclonal antibody enzyme immunoassay marketed by Abbott Laboratories, North Chicago, Ill. (Rotazyme I); a modification of this assay which is now commercially available (Rotazyme II); and a latex agglutination test (Rotalex) recently introduced by Medical Technology Corp., Somerset, N.J. In addition, selected specimens were examined for the presence of rotavirus by electron microscopy, immune electron microscopy, and RNA gel electrophoresis. A total of 40 specimens were positive in the monoclonal antibody enzyme immunoassay, and 136 were negative. Using the results obtained with this procedure as the reference standard, we found the sensitivities of the Rotazyme I, Rotazyme II, and Rotalex tests to be 97.4, 100, and 81.6%, respectively. The specificities of these three procedures were 88.8, 83.9, and 100%, respectively. PMID- 3009532 TI - Relationship between performance in three of the Centers for Disease Control microbiology proficiency testing programs and the number of actual patient specimens tested by participating laboratories. AB - The performance of laboratories enrolled in three of the Centers for Disease Control microbiology performance evaluation programs was studied in relation to the number of actual patient specimens tested by the laboratories. Laboratories were grouped according to the number of specimens tested weekly to compare performance among the groups. Results of the study showed that laboratories, as a group, that tested large numbers of specimens performed better than laboratories that tested small numbers. The infrequency of performing certain tests or identifying certain microbial species can be an important factor in laboratory error. One strategy for laboratories that cannot resolve internal problems associated with low testing volumes would be to limit their testing to procedures that are done well. PMID- 3009530 TI - Quantitative estimation by a standardized enzyme-linked immunosorbent assay of human T-cell lymphotropic virus type I antibodies in adult T-cell leukemia and acquired immune deficiency syndrome. AB - Sera from patients with adult T-cell leukemia and asymptomatic carriers of human T-cell lymphotropic virus type I (HTLV-I) from widely separated areas of the world reacted strongly in a standardized quantitative enzyme-linked immunosorbent assay procedure with HTLV-I viral antigen prepared from a strain isolated in the United States. There was a sharp differentiation of the values seen in the patients as compared with a normal population. Of the 35 acquired immune deficiency syndrome patients with Kaposi's sarcoma, only 2 were positive for HTLV I antibodies in this test, and the distribution of the negative assay values in the other acquired immune deficiency syndrome patient sera was similar to that seen in the normal sera. Sera which contained extremely high levels of antibodies to other unrelated viruses (rubella virus, cytomegalovirus, and herpes simplex virus) all showed negative anti-HTLV-I results, in a pattern similar to the normal sera. Sera from patients with several autoimmune disease (systemic lupus erythematosus, rheumatoid arthritis, thyroiditis) as well as those with infectious mononucleosis or myeloma all also showed the normal distribution of negative results, in spite of the presence of very high levels of the autoantibodies, etc., associated with their illnesses. PMID- 3009529 TI - Role of keto acids and reduced-oxygen-scavenging enzymes in the growth of Legionella species. AB - Keto acids and reduced-oxygen-scavenging enzymes were examined for their roles in supporting the growth of Legionella species and for their potential reactions between the chemical components of the media. When grown in an experimental ACES (2-[(2-amino-2-oxoethyl)-amino] ethanesulfonic acid)-buffered chemically defined (ABCD) broth, the presence of keto acids shortened the lag periods, increased the rates of growth, and gave maximum cell yields. In addition, keto acids affected the specific activities of reduced-oxygen-scavenging enzymes determined during growth. The specific activities of superoxide dismutase of Legionella pneumophila (Knoxville) and L. dumoffii (TEX-KL) were increased three- to eightfold, while that of L. bozemanii (WIGA) was not affected. All strains appeared to be equally sensitive to the effects of superoxide anion (O2-) generated by light-activated riboflavin, and all were equally protected by the presence of keto acids in the ABCD broth. Production of trace amounts of acetate and succinate in pyruvate- and alpha-ketoglutarate-containing media exposed to light suggested that hydrogen peroxide was formed. Pyruvate and alpha-ketoglutarate were products of growth on amino acids, and there was no quantitative evidence that these keto acids were metabolized when they were added to the medium. The rate of cysteine oxidation in ABCD broth was increased by the presence of ferric ion or by exposure to light or by both, and keto acids reduced the rate of this oxidation. ACES buffer was a substrate for the production of O2- in the presence of light, and the combined addition of Fe2+ ions, cysteine, and either keto acid to the medium strongly inhibited the production of O2-. Thus, keto acids inhibited the rate of cysteine oxidation, they stimulated rapid growth by an unknown process, and, in combination with added Fe2+ ions and cysteine, they reversed the toxic effects of light by inhibiting O2- production. PMID- 3009533 TI - Polypeptide specificity of the antibody response after primary and recurrent infection with bovine herpesvirus 1. AB - The polypeptide specificities and defense mechanisms of the humoral immune response to bovine herpesvirus 1 were analyzed. Sequential serum samples taken from cows which were experimentally infected with bovine herpesvirus 1 were tested for their reactivity with individual bovine herpesvirus 1 polypeptides by immunoprecipitation, immunoblotting, and enzyme-linked immunosorbent assay. All bovine immune sera reacted with each of the three major bovine herpesvirus 1 glycoproteins, GVP 6/11a/16, GVP 3/9, and GVP 11b, during primary as well as recurrent infection. Among these glycoproteins, GVP 6/11a/16 induced the earliest and most consistent immune response. The levels of antibody to GVP 3/9 and GVP 11b varied among the animals, and they were slightly lower than the level of antibody to GVP 6/11a/16. Antibodies to several nonglycosylated polypeptides and two additional glycoproteins were also detected with the immunoblot assay. However, these antibodies were usually apparent only during recurrent infection, whereas they were undetectable or low during primary infection. The antibodies in the sera from all animals mediated virus neutralization and destruction of virus infected cells by two immune mechanisms, e.g., antibody- and complement-mediated lysis and antibody-dependent cell-mediated cytotoxicity. PMID- 3009534 TI - Antibody levels for cytomegalovirus, herpes simplex virus, and rubella in patients with acquired immune deficiency syndrome. AB - Significantly higher proportions of patients with acquired immune deficiency syndrome (AIDS) or lymphadenopathy syndrome (LAS) were positive for antibodies to cytomegalovirus (CMV) and herpes simplex virus (HSV) compared with control groups of commercial blood donors. In contrast, no differences were found in the incidence of individuals positive for antibodies to rubella in these groups of subjects. Of those positive for antibodies to CMV and HSV in each group, the mean antibody levels were significantly higher in AIDS-LAS patients compared with the controls. The entire distribution of antibody concentrations to CMV and HSV in AIDS patients was shifted upward, so that significantly more patients showed high values and significantly fewer showed low values, indicating hyperactive humoral immune responses to these viruses. In sharp contrast, the AIDS patients with antibody levels for rubella showed the same distribution of antibody levels as did two groups of controls. No correlation was found between concentrations of CMV and HSV antibodies in individual AIDS-LAS patients. PMID- 3009535 TI - Specificity of immunoglobulin M and G antibody responses in humans infected with eastern and western equine encephalitis viruses: application to rapid serodiagnosis. AB - Paired sera from 20 humans with eastern equine encephalitis (EEE) virus infections and from 17 humans with western equine encephalitis (WEE) virus infections, all with previously demonstrated fourfold or greater rises or falls in hemagglutination-inhibiting, complement-fixing, or neutralizing antibody titers, were tested for immunoglobulin M (IgM) and IgG antibodies by an enzyme immunoassay. All individuals with EEE and 14 of 17 individuals with WEE had IgM antibody, some as early as 1 day after onset. Two of the three persons with WEE who did not develop IgM antibody died. IgM antibody declined but persisted for at least 3 months after the onset of illness in one individual each with EEE and WEE. IgG antibody was not detected until the middle of week 2 after onset. The sensitivity of the IgM antibody capture enzyme immunoassay described and the specificity, as shown by the absence of heterologous alphavirus reactivity, indicate that this is the test of choice for the rapid diagnosis of human infections caused by EEE and WEE viruses. PMID- 3009536 TI - Effect of using heat-inactivated serum with the Abbott human T-cell lymphotropic virus type III antibody test. AB - The Abbott enzyme immunoassay (Abbott Laboratories, North Chicago, Ill.) for human T-cell lymphotropic virus type III (HTLV-III) antibody was evaluated to determine the effect of using heat-inactivated (56 degrees C for 30 min) serum as the sample. Each of 58 nonreactive serum samples gave a higher A492 value when tested after heat inactivation. Ten of the samples became reactive after heating. Heat-inactivated serum should not be used in the current Abbott HTLV-III antibody test, because this can cause false-positive results. PMID- 3009537 TI - Studies on fastidious adenoviruses in Ontario: a distinct strain associated with gastroenteritis. AB - From 100 cases of gastroenteritis among children caused by adenovirus infection in Ontario, 33 virus isolates were divided into three categories according to their biological behavior in tissue cultures. So far, the results of neutralization tests, structural protein analysis, and DNA restriction patterns showed that the virus of category 1 was similar to adenovirus type 40. However, the adenovirus of category 2 was a distinct adenovirus which shared some similarities with adenovirus type 5. Viruses of category 3 are still under investigation. PMID- 3009538 TI - Rapid detection of herpes simplex virus in clinical specimens by centrifugation and immunoperoxidase staining. AB - The effect of immunoperoxidase staining and centrifugation on the sensitivity and rapidity of herpes simplex virus detection in mink lung cell cultures was determined with 730 clinical specimens. In standard tube cultures, the use of immunoperoxidase staining resulted in detection of 31 (91%) of 34 positive cultures after overnight incubation, compared with 25 (74%) detected without the stain (P less than 0.05). The effect of centrifugation of specimens onto the monolayer followed by overnight incubation and immunoperoxidase staining was studied with 431 specimens. Of 107 positive specimens, 103 (96%) were detected by this method, compared with 91 (85%) detected in standard cell cultures observed for 5 days (P less than 0.02). Standard cell cultures that were examined after overnight incubation detected only 62 (58%) of the 107 positive specimens (P less than 0.001). Centrifugation of clinical specimens onto cell monolayers followed by overnight incubation and immunoperoxidase staining is more rapid and sensitive than are standard cell culture techniques for the laboratory diagnosis of herpes simplex virus infection. PMID- 3009539 TI - Comparison of immune electron microscopy and genome electropherotyping techniques for detection of turkey rotaviruses and rotaviruslike viruses in intestinal contents. AB - Seventy-nine intestinal contents specimens from 65 turkey flocks were examined for rotavirus and rotaviruslike virus (RVLV) by immune electron microscopy (IEM) and genome electropherotyping. The IEM procedure was slightly more sensitive in detecting these viruses; 7 of 48 specimens (14.6%) positive for virus by IEM were negative by the genome electropherotyping technique. The genome electropherotyping technique more readily differentiated the rotaviruses and RVLVs than did the IEM procedure; 15 of 48 specimens (31%) positive for virus by IEM could not be differentiated into rotavirus of RVLV, whereas only 4 of the 41 specimens (9.7%) positive by genome electropherotyping produced incomplete genome electropherotypes and could not be differentiated. Thirty-one specimens negative by IEM were also negative by genome electropherotyping. Specimens determined to contain only rotavirus by IEM produced only rotavirus genome electropherotypes. Likewise, specimens determined to contain RVLV alone by IEM produced only RVLV genome electropherotypes. Three specimens contained viruses morphologically resembling rotaviruses that were not aggregated by either the anti-turkey rotavirus serum or the anti-turkey RVLV serum and possessed genome electropherotypes distinct from those of the turkey rotavirus and RVLV. These rotaviruses may represent a third, previously unrecognized serogroup of turkey rotaviruses. PMID- 3009540 TI - Evaluation of cell line 293 for virus isolation in routine viral diagnosis. AB - Cell line 293, a continuous line of transformed human embryonic kidney cells, has been recognized for its sensitivity in the isolation of adenoviruses, particularly the fastidious species 40 and 41, from stool specimens. To explore the possibility of using this cell line for the isolation of other viruses from clinical specimens, 293 cells were tested for their susceptibility to a variety of viruses including herpes simplex virus, parainfluenza viruses, respiratory syncytial virus, and the enteroviruses ECHO 11, coxsackie B5, and coxsackie B6. All of the viruses induced a cytopathic effect in 293 cells. Consequently, 293 cells were introduced into the diagnostic laboratory and used along with primary African green monkey kidney (AGMK) cell cultures for the inoculation of all respiratory and stool specimens. The study represents a retrospective analysis of the performance of 293 cells over a 22-month period. It was confirmed that 293 cells were more sensitive than AGMK cells for the isolation of adenoviruses from both respiratory and stool specimens. The 293 cells were also sensitive for the isolation of enteroviruses (untyped) but more so from stool specimens than from respiratory specimens. Parainfluenza virus and respiratory syncytial virus were only rarely isolated in 293 cells. Herpesvirus isolates were obtained with equal frequency in both 293 and AGMK cells. This retrospective analysis confirms the value of 293 cells for the isolation of adenoviruses and demonstrates that 293 cells are also useful for the isolation of certain enteroviruses from both respiratory and stool specimens. PMID- 3009542 TI - Simplified method of rotavirus RNA analysis: a word of caution. PMID- 3009541 TI - Group C rotaviruses in humans. AB - Atypical rotaviruses obtained from human feces from Australia, Brazil, and the United Kingdom were shown by a combination of techniques--immunoelectron microscopy, immunofluorescence, genome profile analysis, terminal fingerprint analysis of genome segments, and dot-blot hybridization--to be related to group C porcine rotaviruses. The prevalence of antibody to group C rotaviruses was found to be low in human sera and immunoglobulin pools from six countries. No signs of infection were obtained when one of the human viruses was inoculated into gnotobiotic piglets. We conclude that the atypical human viruses are the first examples of group C rotaviruses in humans. PMID- 3009543 TI - Plasma catecholamine modulation of alpha 2 adrenoreceptor agonist affinity and sensitivity in normotensive and hypertensive human platelets. AB - We measured alpha 2-adrenoreceptor density as well as affinity for and sensitivity to agonist on intact platelets of normotensive and hypertensive subjects before and after physiological increases in plasma catecholamines. In normotensives, posture-induced rises in plasma catecholamines correlated with reduced alpha 2-adrenoreceptor agonist affinity and fewer high affinity state receptors. Platelet aggregation and inhibition of adenylate cyclase by L epinephrine also was reduced. Hypertensive subjects had similar rises in plasma catecholamines with upright posture, but showed no change in receptor affinity or sensitivity. No change in platelet alpha 2-adrenoreceptor number occurred in these studies. In vitro incubation with L-epinephrine revealed that platelets from hypertensives had slower desensitization than those from normotensives. Binding studies at different temperatures and with varying sodium concentrations found no thermodynamic or sodium-dependent differences between normotensive and hypertensive groups. These studies demonstrate that platelets from hypertensive subjects exhibit a defect in the ability of physiological concentrations of agonist to desensitize the alpha 2-adrenoreceptor. PMID- 3009544 TI - Respiratory chain defects in the mitochondria of cultured skin fibroblasts from three patients with lacticacidemia. AB - The cultured skin fibroblasts from three patients with lacticacidemia were found to have low rates of 1-[14C]pyruvate oxidation in the face of normal pyruvate dehydrogenase activity. After incubation with 1 mM glucose, these three cell strains also exhibited lactate/pyruvate ratios which were three times greater than those of controls. In two of the patients, both ATP and oxygen consumption in fibroblast mitochondrial preparations was deficient with NAD-linked substrates but normal with succinate and ascorbate/N'N'N'N' tetramethyl phenylene diamine. In the third patient, ATP synthesis in mitochondrial preparations was deficient with all substrates tested. Measurement of Rotenone-sensitive NADH-cytochrome c reductase in mitochondrial preparations from skin fibroblasts showed that two of the patients had 14 and 18%, respectively, of control activity. In the third patient, cytochrome oxidase activity was 15% of that in controls. We conclude that respiratory chain defects can be demonstrated in cultured skin fibroblasts with consistency using a number of different techniques. PMID- 3009545 TI - Alterations in cytotoxic and helper T cell function after infection of T cell clones with human T cell leukemia virus, type I. AB - HTLV-I is a transforming human retrovirus that is an etiologic agent of adult T cell leukemia/lymphoma. To investigate the effects of this virus on T cell functions, two OKT3+, OKT4+, OKT8- cytotoxic clones (8.7 and 8.8) specific for allogeneic cells bearing DPw2, a class II histocompatibility antigen, were studied before and after infection with HTLV-I. The clones retained cytotoxic function for up to 70 d after exposure to HTLV-I, even without subsequent antigenic stimulation, but then lost their cytotoxic activity. Prior to infection with HTLV-I, clone 8.8 also lysed OKT3 hybridoma cells; after infection, cytotoxic activity against these OKT3-antibody bearing cells was lost in parallel with the loss of activity against DPw2-bearing target cells. In addition, expression of T3 surface antigen by HTLV-I-infected 8.8 cells was decreased at a time when they lost their cytotoxic activity, possibly contributing to the loss of cytotoxic function. Finally, clone 8.8 could provide help for nonspecific IgG production by autologous B cells when stimulated with irradiated DPw2-bearing non T cells. After infection with HTLV-I, this helper function became independent of DPw2-stimulation and persisted even when the cytotoxic activity was lost. An OKT4+ T cell clone thus could simultaneously manifest both cytotoxic and helper T cell activities, and these activities were differentially affected after HTLV-I infection. PMID- 3009547 TI - Altered growth, differentiation, and responsiveness to epidermal growth factor of human embryonic mesenchymal cells of palate by persistent rubella virus infection. AB - We previously demonstrated that human embryonic mesenchymal cells derived from the palate (HEMP cells) retain alkaline phosphatase (ALP) content and capacity for collagen synthesis after long-term culture, and their growth is markedly stimulated by epidermal growth factor (EGF). There was a dramatic decrease in ALP content and capacity to synthesize collagen in HEMP cells (HEMP-RV cells) persistently infected with rubella virus (RV). EGF increased ALP activity and decreased collagen synthesis in HEMP cells, whereas EGF showed no effect on these activities in HEMP-RV cells. Growth of HEMP-RV cells was slightly reduced compared with that of HEMP cells. EGF stimulated growth of HEMP cells and to a lesser extent of HEMP-RV cells. Binding of 125I-EGF to cell-surface receptors in HEMP-RV cells was, to our surprise, twice as much as that in HEMP cells. However, internalization of bound 125I-EGF in HEMP-RV cells was profoundly diminished. Thus, persistent RV infection causes not only changes in HEMP cell growth and differentiation but a decrease in or loss of HEMP cell responsiveness to EGF. The effects of persistent RV infection on palatal cell differentiation as well as growth may be responsible for the pathogenesis of congenital rubella. Furthermore, since HEMP cells appear to be closely related to osteoblasts, these results suggest a mechanism for RV-induced osseous abnormalities manifested in congenital rubella patients. PMID- 3009546 TI - Effects of calcium on vasopressin-mediated cyclic adenosine monophosphate formation in cultured rat inner medullary collecting tubule cells. Evidence for the role of intracellular calcium. AB - We explored the effects of alterations in extracellular and intracellular calcium concentration on arginine vasopressin (AVP)-stimulated cAMP formation in cultured rat inner medullary collecting tubule cells. cAMP formation remains constant at extracellular calcium concentrations between 0.5 and 4.0 mM, which did not change intracellular calcium. Maneuvers that alter intracellular calcium concentration are associated with marked changes in cAMP generation. EGTA decreases intracellular calcium and enhances AVP-stimulated cAMP formation, while increasing cellular calcium with 2 microM A23187 decreases AVP-stimulated cAMP formation in the presence, but not in the absence, of extracellular calcium. The changes in cAMP formation observed when intracellular calcium is altered are associated with reciprocal changes in prostaglandin E2 (PGE2) synthesis. Despite greater than 95% inhibition of PGE2 synthesis with 5 microM meclofenamic acid, the changes in cAMP formation accompanying alterations in intracellular calcium concentration are still evident. These studies suggest that intracellular calcium critically influences AVP-stimulated cAMP formation. It does so by a mechanism independent of PG that is probably mediated by a direct effect of the cation on the adenylate cyclase complex. PMID- 3009548 TI - Human monoclonal autoantibodies that react with both pancreatic islets and thyroid. AB - Transformation of human peripheral blood lymphocytes with Epstein-Barr virus and rapid screening on rat insulinoma cells by an enzyme-linked immunosorbent assay were used to identify monoclonal autoantibodies that reacted with human pancreatic islets. Six such monoclonal autoantibodies were isolated and cloned. All six also were found to react with human thyroid. It is concluded that lymphocytes able to make autoantibodies that react with both the pancreas and thyroid are common in the human B cell repertoire. PMID- 3009549 TI - Regulation of interferon receptor expression in human blood lymphocytes in vitro and during interferon therapy. AB - Interferons (IFN) elicit antiviral and antineoplastic activities by binding to specific receptors on the cell surface. The binding characteristics of IFN to human lymphocytes were studied using IFN alpha 2 labeled with 125I to high specific activity. The specific binding curves generated were analyzed by the LIGAND program of Munson and Rodbard to determine receptor numbers. The number of receptors in peripheral blood lymphocytes (PBL) and tonsillar B-lymphocytes (TBL) from normal individuals were 505 +/- 293 (n = 10) and 393 +/- 147 (n = 3) respectively. When these cells were preincubated in vitro with unlabeled IFN alpha 2, the receptor number decreased to 82 +/- 45 and 61 +/- 16 respectively. Receptor binding activities recovered gradually over a period of 72 h when the cells were incubated in IFN-free medium. This recovery of receptors could be blocked by the addition of actinomycin D to the incubation medium. A similar decrease in receptor expression was observed in vivo in PBL from patients being treated daily with 5 X 10(6) units/m2 per d of IFN alpha 2 by subcutaneous injection, for acute lymphoblastic leukemia or papilloma virus infections. Receptor numbers in PBL in vivo were further reduced concurrent with the progression of IFN therapy. Thus the reduction in IFN receptor expression observed in vitro can be demonstrated in vivo. These studies indicate that monitoring IFN receptor expression in vivo can provide information regarding the availability of IFN receptors at the cell surface for the mediation of IFN actions during the course of IFN therapy. PMID- 3009550 TI - Load dependence of proximal tubular fluid and bicarbonate reabsorption in the remnant kidney of the Munich-Wistar rat. AB - Studies were undertaken to characterize the pattern of proximal tubular fluid (APRH2O) and bicarbonate reabsorption (APRHCO3) in the remnant kidney of euvolemic Munich-Wistar rats. The remnant kidney rats were placed on a diet containing either low or normal protein. Collections were obtained in the early, mid-, and late proximal convoluted tubule. Single nephron glomerular filtration rate (SNGFR) increased from 40.2 nl/min in controls to 58.8 nl/min in low protein remnant kidney and 78.1 nl/min in normal protein remnant kidney rats. The filtered load of bicarbonate was 1,272, 1,641, and 2,013 pmol/min, in the three groups, respectively. APRH2O and APRHCO3 increased nearly in parallel. Most of the increase in reabsorption occurred in the early proximal tubule. Tubular hypertrophy could account for at least 20-40% of the increase in reabsorption, but the majority of the increase appeared to be a delivery-dependent response similar to that observed in normal rats after an acute increase in SNGFR. PMID- 3009551 TI - Long-term nocturnal calcium infusions can cure rickets and promote normal mineralization in hereditary resistance to 1,25-dihydroxyvitamin D. AB - We report the beneficial effects of calcium infusions in a child with hereditary resistance to 1,25(OH)2D and alopecia. This patient after transient responsiveness to vitamin D derivatives became unresponsive to all therapy despite serum 1,25(OH)2D concentrations maintained at levels approximately 100 fold normal. A 7-mo trial with calcium infusions led to correction of biochemical abnormalities and healing of rickets. Bone biopsies (n = 3) showed a normal mineralization and the disappearance of the osteomalacia. Cultures of bone derived cells demonstrated a lack of activation of 25-hydroxyvitamin D 24 hydroxylase and osteocalcin synthesis by 1,25(OH)2D3 (10(-9) and 10(-6) M). These results demonstrate that even in the absence of a normal 1,25(OH)2D3 receptor effector system in bone cells, normal mineralization can be achieved in humans if adequate serum calcium and phosphorus concentrations are maintained; and calcium infusions may be an efficient alternative for the management of patients with this condition who are unresponsive to large doses of vitamin D derivatives. PMID- 3009552 TI - 12-o-Tetradecanoyl-phorbol-13-acetate-differentiated U937 cells express a macrophage-like profile of neutral proteinases. High levels of secreted collagenase and collagenase inhibitor accompany low levels of intracellular elastase and cathepsin G. AB - Human monocytic tumor cells of the U937 cell line contain substantial quantities of two neutrophil neutral proteinases, elastase and cathepsin G, raising the question of whether their presence reflects an expression of transformation or whether normal monocytes undergo a developmental stage in which they produce certain neutrophil proteinases. To address this issue, we examined U937 cells for production of collagenase, since human alveolar macrophages release fibroblast like collagenase, an enzyme that is distinct from neutrophil collagenase. Using an immunoassay that utilized antibody to skin fibroblast collagenase, we found that U937 cells secreted barely detectable quantities of enzyme, 10-12 ng/10(6) cells per 24 h, under basal conditions. Upon incubation with 10 nM 12-o tetradecanoyl-phorbol-13-acetate (TPA), however, collagenase release increased 200-fold, comparable to the amount secreted by phorbol-stimulated human fibroblasts. Metabolic labeling and immunoprecipitation confirmed the enhanced synthesis of U937 cell collagenase upon TPA exposure. This enzyme activity further resembled fibroblast collagenase and differed from neutrophil collagenase by exhibiting preferential cleavage of monomeric type III collagen relative to type I. As previously observed with human alveolar macrophages, U937 cells also released a protein identical to the collagenase inhibitor produced by human skin fibroblasts, a molecule not associated with neutrophils. Release of this inhibitor increased 10-fold with TPA exposure. In contrast to collagenase and collagense inhibitor, TPA-treated U937 cells contained only 10-15% as much elastase and cathepsin G activities as control cells. Thus, TPA-induced differentiation modified the presence of these enzymes in the direction of their content in normal monocytes. Since the neutral proteinase profile of undifferentiated U937 cells resembles that of neutrophils and changes markedly after cellular differentiation to one that is characteristic of monocytes, these data suggest that neutrophilic proteinases may be produced by normal monocytes during the early stages of their differentiation. PMID- 3009554 TI - Bilateral internuclear ophthalmoplegia with absence of convergent eye movements. Clinicopathologic correlation. AB - A case of bilateral internuclear ophthalmoplegia of long duration with autopsy confirmation is reported. The main features of the syndrome included paresis of ocular adduction upon attempted lateral gaze, horizontal nystagmus in the abducting eye, and an absence of converging eye movements. Examination of Weil stained sections revealed multiple plaques of demyelination. The medial longitudinal fasciculus was demyelinated bilaterally in the upper pons-caudal midbrain. The oculomotor, trochlear, and abducens nuclei appeared relatively well preserved. The internal capsule was severely damaged. Large cyst-like structures were centered in the anterior limb bilaterally and extended caudally to the level of the genu. Plaques of demyelination were present bilaterally in the posterior limb. The left side of the internal capsule was more severely affected than the right. It is thought that convergence in this case may have been eliminated by interruption of fibers from the frontal eye fields and/or other cortical areas in their descent through the internal capsule. PMID- 3009553 TI - Pyrophosphohydrolase activity and inorganic pyrophosphate content of cultured human skin fibroblasts. Elevated levels in some patients with calcium pyrophosphate dihydrate deposition disease. AB - In calcium pyrophosphate dihydrate (CPPD) crystal deposition disease, metabolic abnormalities favoring extracellular inorganic pyrophosphate (PPi) accumulation have been suspected. Elevations of intracellular PPi in cultured skin fibroblasts from a single French kindred with familial CPPD deposition (19) and elevated nucleoside triphosphate pyrophosphohydrolase activity (NTPPPH), which generates PPi in extracts of CPPD crystal-containing cartilages (14) favor this suspicion. To determine whether NTPPPH activity or PPi content of cells might be a disease marker expressed in extraarticular cells, human skin-derived fibroblasts were obtained from control donors and patients affected with the sporadic and familial varieties of CPPD (CPPD-S and CPPD-F) deposition. Intracellular PPi was elevated in both CPPD-S (P less than 0.05) and CPPD-F (P less than 0.01) fibroblasts compared with control fibroblasts. Ecto-NTPPPH activity was elevated in CPPD-S (P less than 0.01) but not CPPD-F. Intracellular PPi correlated with ecto-NTPPPH (P less than 0.01). Elevated PPi levels in skin fibroblasts may serve as a biochemical marker for patients with familial or sporadic CPPD crystal deposition disease; ecto-NTPPPH activity further separates the sporadic and familial disease types. Expression of these biochemical abnormalities in nonarticular cells implies a generalized metabolic abnormality. PMID- 3009555 TI - Sudden infant death and cytomegalovirus inclusion disease. AB - Four infants, apparently thriving and without clinical evidence of disease, died suddenly at ages ranging from 2 to 6 months. Inclusions bearing cells pathognomonic of cytomegalovirus infection were shown microscopically in a small number of extraneural organs. In view of the lack of associated tissue destruction on microscopy and the apparent well being of the infants before death whether the function of these organs had been impaired to any important degree was questionable: such limited disease, consequently, could not have contributed substantially to the cause of death. The brainstem, on the other hand, consistently showed small numbers of glial nodules. Damage to strategically located neurones associated potentially with the organisation of vital function was a possible basis of sudden death. Alternatively, the small number of glial nodules may have represented a residue of previous more severe brainstem disease, which had possibly started while the baby was in the uterus. PMID- 3009557 TI - Immunohistochemical detection of ras oncogene p21 product in benign and malignant mammary tissue in man. AB - The monoclonal antibody RAP-5 generated against a synthetic peptide corresponding to amino acid positions 10-17 of the ras p21 protein was used in an immunohistochemical study of the expression of ras in normal, benign, and malignant breast epithelium in man. The staining intensity and intracellular distribution of RAP-5 was similar in the three epithelial populations and extended to other tissue elements including myoepithelial cells, smooth muscle, myelin, capillary endothelium, and stromal fibroblasts, as well as sebaceous glands and sweat glands overlying the breast. These results suggest that RAP-5 recognises a normal cellular component, the expression of which is not more enhanced in hyperplastic or neoplastic conditions. The detection of mutant forms of p21 exclusively expressed in malignant tumours requires that alternative reagents be developed. PMID- 3009556 TI - Scar adenocarcinoma of the lung: a light and electron microscopic study. AB - Five well differentiated peripheral adenocarcinomas of the lung were investigated, using light and electron microscopy. Each tumour contained a central nidus of fibrous tissue and fulfilled the criteria for "scar cancer." One tumour also had a focus of lamellated collagenous tissue, suggestive of an old tuberculous granuloma. Electron microscopy showed the features of Clara cells, with characteristic dense bodies in the apical cytoplasm and scattered microvilli on the luminal surface. It was concluded that this variant of scar cancer was a carcinoma of Clara cells, which was sufficiently distinctive in appearance to be recognised on light microscopy alone. It remains uncertain, however, whether the central fibrous area is a desmoplastic response to tumour growth or a pre existing scar. PMID- 3009558 TI - Wash off liver cytology: a complementary diagnostic tool to liver biopsy. AB - Parenchymal and non-parenchymal cells were harvested by washing the liver tissue core and needle after percutaneous biopsy. The cytological material obtained was suitable for morphological analysis, including showing the presence of surface and cytoplasmic antigens using labelled antibody techniques. This technique provides a combined cytological and histological approach to the diagnosis of liver disease. PMID- 3009560 TI - View from the Nation's Capital. NIH and ADAMHA FY 1986 appropriations. PMID- 3009559 TI - PLP fixation for combined routine histology and immunocytochemistry of liver biopsies. PMID- 3009561 TI - Stimulation-induced [14C]2-deoxyglucose labeling of synaptic activity in the central auditory system. AB - The relative contribution of active synapses and discharging neurons to [14C]2-DG labeling in film autoradiographs of the auditory system was studied in a series of three experiments, two in cat, one in chick. In the first, the lateral superior olive in cats was specially prepared so that its inhibitory afferents could be stimulated without concurrent stimulation of its excitatory afferents. The film autoradiographs showed clear 2-DG labeling in the vicinity of the activated inhibitory synapses. In the second experiment, the medial superior olive in cat was specially prepared so that it could be stimulated antidromically without concurrent orthodromic stimulation. The film autoradiographs showed little or no elevations in 2-DG labeling of the antidromically stimulated nucleus over its unstimulated contralateral control despite heavy labeling of nearby orthodromically stimulated nuclei. In the third experiment, the highly polarized nucleus laminaris of a chick was specially prepared so that one set of its excitatory afferents could be stimulated without concurrent stimulation of the other set. The film autoradiographs showed that the distribution of heavy 2-DG labeling matched the distribution of the activated synapses and not the distribution of discharging postsynaptic membrane. The outcomes of the three experiments taken together suggest that it is active synapses and not actively discharging neurons that dominate typical [14C]2-DG film autoradiographs, at least of the vertebrate central auditory system. It follows that [14C]2-DG labeling of central auditory system tissue is not necessarily evidence of local cell discharge but instead evidence of synaptic activity whether excitatory or inhibitory, and whether or not it is accompanied by significant levels of postsynaptic cell discharge. PMID- 3009563 TI - Observations on the ability of avian reovirus vaccination of hens to protect their progeny against the effects of challenge with homologous and heterologous strains. AB - Avian reovirus vaccines of the S1133 strain, used indirectly in the dams of challenged chicks, were found to confer protection against clinical signs and/or deaths resulting from the use of both homologous and heterologous challenge strains. There was some protection against every strain used, although this did vary in degree. It was difficult to relate this variation in protection to the differences in in vitro neutralization of the strains by S1133 antiserum, though there were some similarities. PMID- 3009562 TI - The distribution of adenosine A1 receptors and 5'-nucleotidase in the hippocampal formation of several mammalian species. AB - The distributions of adenosine A1 receptors, as demonstrated by 3H cyclohexyladenosine (3H-CHA) binding, and the adenosine-producing enzyme 5' nucleotidase were examined in the hippocampal formation of the rat, mouse, gerbil, cat, hamster, rabbit, and guinea pig. The enzyme and binding sites were restricted to subregions and often individual layers of this structure. The distribution of 3H-CHA binding was consistent among the species with the strata radiatum and oriens of fields CA1 and CA3 exhibiting the highest levels of binding. A distinct band of 3H-CHA binding was observed in the stratum moleculare of the dentate gyrus; and in most species, this band was restricted to the inner one-third of the stratum moleculare (i.e., proximal to the stratum granulosum). The strata pyramidale, granulosum, and lucidum were in general only weakly positive for 3H-CHA binding. The binding to the stratum lacunosum/moleculare (or the distinct strata lacunosum and moleculare in the rabbit and cat) was moderate. In contrast to the relative consistency of the patterns of 3H-CHA binding in these species, 5'-nucleotidase exhibited wide variations in both the absolute amount of activity and its localization. In all species, the strata granulosum and pyramidale appeared devoid of 5'-nucleotidase activity. The only clear exception to this rule was the CA3 region of the cat where activity was seen between the cell bodies of stratum pyramidale. The strata radiatum and oriens of CA1 were strongly positive in the rat and hamster but only low to moderately stained in the other species examined.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009564 TI - Changing manifestations of a chronic feline haematopoietic proliferative disease during immunotherapy with staphylococcal protein A. AB - A cat with feline leukaemia virus-associated malignant disease was treated by ex vivo immunoadsorption using staphylococcal protein A coated filters. During the 12-week course of treatment, the morphological manifestations of the haematopoietic disease showed a progressive transition from erythroid to myeloerythroid to myeloid predominance, and the disease was preceded by and associated initially and terminally with a blast transformation of lymphoblastic morphology. Necropsy revealed massive meningo-cerebral, as well as hepatic, renal, myeloid, lymphoid, peritoneal and pelvic infiltrations largely consisting of lymphoblastic cells. Evidence of myeloid and erythroid differentiation was present in all the infiltrates. The several possible bases for this shift of morphological expression are considered. PMID- 3009566 TI - Aortic body tumour with adjacent ectopic thyroid tissue in a dog. AB - A 12-year-old neutered male Husky dog had a neoplasm at the base of the heart which did not invade surrounding tissues. Microscopically, the neoplasm was composed of nests and sheets of polyhedral cells subdivided into lobules by trabeculae of fine fibrovascular stroma. Adjacent to the neoplasm was a rim of ectopic thyroid tissue that appeared histologically normal. The possible differential diagnoses for the neoplasm were aortic body tumour, ectopic thyroid tumour and ectopic parathyroid tumour; the ultrastructural characteristics revealed it to be an aortic body tumour. PMID- 3009565 TI - A comparison of the oxidative reactions of neutrophils from a variety of species when stimulated by opsonized zymosan and F-met-leu-phe. AB - Bovine, ovine and porcine blood neutrophils produced less superoxide and consumed less oxygen than human neutrophils when they were challenged with serum-treated zymosan. These differences were not related to methodological considerations or the origin of the serum used to treat the zymosan. When the two main methods of neutrophil isolation were applied to human neutrophils, no differences in the particle stimulated respiratory bursts were observed. The bacterial chemotactic peptide, F-met-leu-phe, which enhances superoxide production of human neutrophils whether or not they are challenged with serum-treated zymosan, did not increase superoxide production of neutrophils from cattle, sheep or pigs under either of these conditions. We conclude that, if our understanding of these host defence mechanisms in domesticated animals is to match that of man, further detailed studies of animal neutrophils are necessary. PMID- 3009567 TI - CT findings in paravertebral synovial sarcoma. AB - Two patients with synovial sarcoma in paravertebral location (cervical and lumbar areas) presented with soft tissue masses that exhibited calcifications on CT. In one patient an unusual pattern of metastasis was found in the pancreas. PMID- 3009568 TI - Reactivation of congenital cytomegalic inclusion disease in an infant with HTLV III associated immunodeficiency: a CT-pathologic correlation. AB - Serial CT findings in an infant with HTLV-III infection and cytomegalic inclusion virus encephalitis are presented. PMID- 3009570 TI - Desensitization of the mammalian beta-adrenergic receptor: analysis of receptor redistribution on nonlinear sucrose gradients. AB - The distribution of beta-adrenergic receptors in lysates from several mammalian cell lines was analyzed on nonlinear sucrose density gradients before and after desensitization by isoproterenol. On the nonlinear gradients, the receptors in lysates from untreated HeLa, A431, S49 cyc- and C6 cells were well resolved into light and heavy density membrane fractions. In contrast, with the former three cell lines, there was very poor or no separation of the two peaks of receptors on linear sucrose gradients. With C6 cells, resolution was better on the nonlinear than on the linear gradient. In all cases, successful separation of the two density fractions of the receptor was achieved only when cells had been treated with concanavalin A prior to lysis. Adenylate cyclase activity cosedimented with the heavy membrane fraction of the receptor, and no activity was detected with the light fraction. After desensitization of adenylate cyclase by isoproterenol, there was a redistribution of the receptors to the light density fraction. This shift of receptors, but not desensitization, was prevented when cells were pretreated at 37 degrees C with concanavalin A prior to exposure to isoproterenol. Thus, sequestration of beta-adrenergic receptors away from the plasma membrane and adenylate cyclase to a lighter density membrane fraction appears to accompany, but may not be a prerequisite for desensitization in mammalian cells. This receptor redistribution, however, can be readily detected on nonlinear sucrose gradients. PMID- 3009569 TI - Paget's disease of the ectopic breast with an underlying intraductal carcinoma: report of a case. AB - Paget's disease may occur at mammary and extramammary sites. Mammary Paget's disease typically involves the nipple and adjacent skin. Almost all such cases are associated with an underlying ductal carcinoma of the breast. A case of Paget's disease occurring at the site of an ectopic breast adjacent to a supernumerary nipple and associated with an underlying intraductal carcinoma is described. A search of the literature revealed no previous report of such cases. Detailed clinical history and histopathologic, histochemical, and follow-up information on this case are presented. The literature on mammary and extramammary Paget's disease is reviewed. PMID- 3009571 TI - Comparison of binding of 125I-iodopindolol to control and desensitized cells at 37 degrees and on ice. AB - Binding of 125I-iodopindolol (IPIN) to intact 1321N1 human astrocytoma cell B adrenergic receptors was measured at 37 degrees and on ice. Control cells showed a single component of IPIN binding on ice with the same total number of receptors as measured at 37 degrees. In desensitized cells (pretreated for 20 min with 1 microM isoproterenol) approximately 40% of IPIN binding on ice exhibited kinetics similar to those observed in control cells. The remaining 60% of receptors were labelled by IPIN at a much slower rate requiring the use of very high concentrations of IPIN. Sucrose density gradient fractionation was used to separately study the labelling of plasma membrane receptors and those associated with a light vesicle fraction. Labelling by IPIN on ice of the plasma membrane receptors of control cells was rapid, labelling of the light vesicle receptors of desensitized cells was slow, and labelling of the plasma membrane receptors of desensitized cells appeared to occur with both rapid and slow components. Selective labelling of the plasma membrane receptors of intact cells thus could be obtained by incubation with IPIN on ice under selected conditions. Similar results were obtained when broken cell preparations from control and desensitized cells were used. The decreased binding of IPIN on ice to B-adrenergic receptors in the light vesicle fraction not only provides further evidence consistent with sequestration of B-adrenergic receptors during desensitization, it also provides a convenient and inexpensive means to assay the sequestration reaction. PMID- 3009572 TI - Adenylate kinase increases adenylate cyclase activity in membranes from rat lung. AB - The adenylate cyclase activity of membranes prepared from rat lung, measured under standard assay conditions, was markedly increased by the presence of a crude supernatant fraction prepared from rat lung, liver, or brain. This was not due to an increase in the initial rate of cyclic AMP (cAMP) synthesis, but to the maintenance of a constant rate of cAMP synthesis for periods of at least 10 min. After incubating lung membranes in the cyclase reaction mixture until cAMP synthesis had virtually ceased (10 min), the addition of alpha-(32P)-ATP caused a marked increase in the activity of the enzyme. This was the only component of the original reaction mixture that supported re-initiation of cAMP synthesis. Re initiation also occurred when supernatant was added. This implies that substrate depletion occurs in the presence of membranes and that lung supernatant can catalyze rapid regeneration of substrate. Chromatographic analysis confirmed that ATP was rapidly hydrolyzed to AMP in the presence of the membranes, that this rapid destruction of ATP did not occur when supernatant was present, and that ATP was resynthesized from AMP when supernatant was added to a reaction mixture in which most of the ATP initially present had been destroyed. The effects of supernatant were mimicked by commercially available adenylate kinase. Addition of adenylate kinase did not affect adenylate cyclase activity measured in membranes prepared from brain, heart, or kidney, suggesting that lung membranes may contain more nucleotide pyrophosphatase and/or less endogenous adenylate kinase activity. Studies of soluble factors that affect adenylate cyclase must carefully control for differential substrate depletion in the presence and absence of tissue extracts. PMID- 3009573 TI - Effect of supplemental vitamin E on the immune system of calves. AB - The effect of vitamin E on immune responses of Holstein calves was investigated. Treatments were: 0,1400, and 2800 mg of dl-alpha-tocopheryl acetate given orally at weekly intervals or 1400 mg of dl-alpha-tocopherol weekly by injection. Calves were fed milk for 6 wk and then fed a complete calf starter ad libitum. Calves were on experiment until they were 12 wk of age. Lymphocyte stimulation indices were significantly higher for calves given the high amount of oral supplementation and for injected calves than for unsupplemented calves. There were no significant differences at any of the individual weeks between unsupplemented and orally supplemented calves. Injected calves showed significantly higher values than unsupplemented calves at wk 4 and than all other calves at wk 8. There were no significant differences in the concentrations of immunoglobulins G1 and G2 among treatments. Immunoglobulin M was significantly higher at wk 6 in calves given the high amount of oral supplementation than in all other calves. At wk 12, serum from calves given the high amount of oral supplementation and calves given injections inhibited infectious bovine rhinotracheitis viral replication in tissue cultures as compared with those of unsupplemented calves. In supplemental experiments serum alpha-tocopherol and lymphocyte stimulation indices of yearling heifers determined 7 d after a single injection of 2000 IU of dl-alpha-tocopherol were significantly higher than preinjection values. In vitro addition of vitamin E to lymphocyte cultures did not increase phytohemagglutinin-induced blastogenesis. PMID- 3009575 TI - Body composition of lactating and dry Holstein cows estimated by deuterium dilution. AB - In three experiments patterns of water turnover and body composition estimated by deuterium oxide were studied in Holstein cows. In the first experiment, four lactating cows were infused with deuterium oxide, and blood samples were taken during 4-d collection. Milking was stopped; cows were reinfused with deuterium oxide and resampled. Slopes of deuterium oxide dilution curves indicated lactating cows turned water over more rapidly than nonlactating cows. In the second experiment with the same four cows, during 4-d collection, deuterium oxide concentrations in milk, urine, and feces showed dilution patterns similar to deuterium oxide in blood. Sampling milk may be an alternative to sampling blood. In the third experiment, 36 Holstein cows were fed 55, 65, or 75% alfalfa, smooth bromegrass, or equal parts of each forage as total mixed rations; remaining portions of rations were a grain mixture. Body composition was estimated at -1, 1, 2, 3, 4, and 5 mo postpartum. Empty body water, protein, mineral, fat, and fat percentage decreased from prepartum to postpartum. First calf heifers contained less empty body water, protein, and mineral than older cows. Cows fed diets with 55% forage had more body fat than those fed diets with 75% forage. Cows fed alfalfa-based diets had more gastrointestinal fill regardless of grain than cows fed diets that contained alfalfa and smooth bromegrass. Gastrointestinal fill of cows increased from prepartum to 5 mo postpartum. PMID- 3009574 TI - Preparation of brush border membrane vesicles from fresh and frozen bovine intestine for nutrient uptake studies. AB - Purified brush border membranes were obtained from homogenized jejunal epithelial cells of cattle by divalent cation aggregation of nonbrush border membranes and differential centrifugation. Membrane marker enzyme assays determined effectiveness of the fractionation procedure. Compared with cellular homogenate, maltase and sodium+-potassium+-adenosine triphosphatase specific activities in the membrane fraction isolated from the interface of discontinuous (35 and 45% wt/wt) sucrose gradients increased 14.5- and 1.9-fold whereas enzyme recoveries averaged 20.2 and 2.4%. These data indicate significant enrichment in brush border membranes with minimal basolateral membrane contamination. Vesicles formed from this membrane fraction had a predominately (93%) luminal side out orientation. After incubating vesicles with radiolabeled substrates, vesicles and accumulated substrates were separated from the incubation buffer by filtration and substrate uptake quantified by liquid scintillation counting. Observed uptake was the result of substrate accumulation within an osmotically active intravesicular space and was not due to nonspecific binding of the substrate to vesicular membranes. Vesicles exhibited sodium-dependent and independent substrate uptake pathways and were able to discriminate between substrate stereoisomers for uptake. Major differences were not detected between results obtained with vesicles prepared from fresh or frozen intestines. These vesicles can be utilized to investigate nutrient uptake by the bovine small intestine. PMID- 3009576 TI - Responses of lactating cows to dietary sodium source and quantity and potassium quantity during heat stress. AB - Effects of heat stress and added dietary sodium bicarbonate (0 or 1.0% of dry matter), sodium chloride (0 or .73% of dry matter), and total dietary potassium (1.3 or 1.8% of dry matter) on acid-base status, production, and mineral metabolism of lactating Holstein cows were evaluated. Design was split-plot with 24 cows in shade or no shade environments; dietary treatments were arranged as a 2 X 2 X 2 factorial within each environment. Basal diet (38% corn silage:62% concentrate) contained .18% sodium; sodium bicarbonate and sodium chloride treatments were in addition. All dietary treatments were equal in chloride content. Cows in no shade exhibited signs of respiratory alkalosis during the hot part of the day. Daily feed intake was lower in no shade than shade but milk yield and percent milk fat were not affected by environment. Sodium bicarbonate addition increased actual and 4% fat-corrected milk yields and percent milk fat. Sodium chloride addition increased actual and 4% fat-corrected milk yield when adjusted for amount of feed intake, and 1.8% dietary potassium increased feed intake and actual milk yield. Increasing total dietary sodium from .18 to .55%, from either supplemental source, enhanced 4% fat-corrected milk production, but combination of sources (.88% total sodium) showed no additional benefit over .55%. PMID- 3009577 TI - Vitamin D3 and D3 metabolites in young goats fed varying amounts of calcium and vitamin D3. AB - Twenty-four male goats, 2 to 4 wk of age, were allotted to four dietary treatments in a 2 X 2 factorial design and, for 20 wk, were fed a milk diet at 12.5% body weight. Treatments varied in amounts of supplemental calcium and vitamin D3. Daily allowances per kilogram body weight were: 9.4 IU vitamin D3 (basal), 9.4 IU vitamin D3 plus 406 mg calcium carbonate (basal plus Ca), 940 IU vitamin D3 (basal plus D3), and 940 IU vitamin D3 plus 406 mg calcium carbonate (basal plus Ca plus D3). At the end of wk 7, a corn supplement was added to all diets at 1% body weight daily. Addition of vitamin D3 to the diet resulted in a dramatic increase in plasma concentrations of vitamin D3. Goats in the basal plus D3 and basal plus Ca plus D3 groups had nearly 100 X greater concentrations of vitamin D3 than goats in the basal and basal plus Ca groups. When greater amounts of vitamin D3 were fed, dietary calcium interacted to decrease plasma vitamin D3 concentrations. Plasma concentrations of 25-hydroxyvitamin D3 were unaffected by additional dietary calcium but were increased by dietary vitamin D3, increasing sixfold to seven-fold in the basal plus D3 and basal plus Ca plus D3 groups. Supplemental calcium resulted in decreased plasma 1,25-dihydroxyvitamin D3. No signs of vitamin D toxicity were noted. The physiological responses reported implicate the goat as a potential animal model for vitamin D research in dairy cattle. PMID- 3009578 TI - Effect of high crude protein on pituitary and ovarian function in Holstein cows. AB - Intact and ovariectomized cows were used to determine the effect of high amounts of dietary crude protein on pituitary regulation of luteinizing hormone. Estrus was synchronized with prostaglandin F2 alpha in 10 intact dry cows fed 15 or 25% crude protein, and serum luteinizing hormone profiles were evaluated during both follicular and luteal phases of the estrous cycle. Serum progesterone, pituitary luteinizing hormone content, and luteinizing hormone-releasing hormone receptors were also measured. Basal concentrations of serum luteinizing hormone tended to be lower during the follicular phase and were significantly higher during the luteal phase in cows fed 25% crude protein. Serum progesterone was not affected by dietary treatment. In 10 ovariectomized cows fed 24% crude protein, amplitude of luteinizing hormone response to luteinizing hormone-releasing hormone challenge tended to be smaller than that of 8 cows fed 16% crude protein, although total luteinizing hormone released in response to luteinizing hormone releasing hormone challenge was not different between dietary treatments. In both intact and ovariectomized cows, pulsatile luteinizing hormone patterns, pituitary luteinizing hormone content, and luteinizing hormone-releasing hormone receptors were not influenced by treatment. High dietary crude protein did not have a primary effect on luteinizing hormone or progesterone in nonlactating cows. PMID- 3009580 TI - Adsorption of benzoic acid on pure and cupric ion-modified hydroxyapatite: implications for design of a coupling agent to dental polymer composites. AB - The adsorption isotherms of benzoic acid on synthetic hydroxyapatite (containing about 1.5 monolayers of physisorbed water) were studied from ethanol, dimethylsulfoxide, p-dioxane, methylene chloride, and benzene to discern the role of solvent in the process. The adsorption is reversible from the first three solvents and follows the Langmuir plots. It is irreversible from the last two, and a constant amount of absorbent is removed from solutions above a certain concentration. The isotherms of potassium benzoate on the apatite from ethanol and dimethyl sulfoxide were reversible. The isotherms of the acid on cupric ion modified apatite surfaces from ethanol and benzene were identical with those obtained on the pure hydroxyapatite. This may demonstrate that any "surface chelation" with the cation may not be a significant factor for adsorption to occur. The adsorptive behavior seems to depend upon the interplay of hydrogen bonding among the solute, the solvent, and the hydrated apatite surface. The capability of a solvent to hydrogen-bond may determine whether adsorption from it will be reversible or irreversible. Based upon its compatibility with a solvent, the benzene ring is upright or lies flat on the surface. The adsorbed molecules rotate about the center of the carboxylate groups which are hydrogen-bonded to the surface. These factors should be considered in designing or selecting a suitable surface-active moiety for a coupling agent between tooth mineral and a restorative resin. PMID- 3009579 TI - The kinetics of dissolution of tooth enamel--a constant composition study. AB - The kinetics of dissolution of powdered bovine enamel and of human enamel, both untreated and extracted with either hypochlorite or chloroform, has been studied using a constant solution composition technique in undersaturated solutions of calcium phosphate (total molar calcium concentration, TCa = 0.3 to 13.1 X 10(-3) mol L-1, total molar phosphate, Tp = 0.18 to 7.9 X 10(-3) mol L-1) at an ionic strength of 0.15 mol L-1, and pH = 4.5. The kinetic equations describing the dissolution reactions suggest a surface dislocation mechanism, and the presence of fluoride ion markedly retarded the reaction. For human enamel, a fluoride level of only 0.5 ppm reduced the rate of dissolution ten-fold. In contrast, the dissolution of hydroxyapatite, HAP, is best interpreted in terms of a polynucleation process. PMID- 3009582 TI - NADPH oxidase and myeloperoxidase activity in psoriasis leukocytes. PMID- 3009581 TI - Erosive adenomatosis of the nipple. AB - Erosive adenomatosis of the nipple is a benign lesion that clinically mimics Paget's disease of the nipple but has the histologic features of syringocystadenoma papilliferum. Some cases have been mistaken for intraductal papilloma or well-differentiated adenocarcinoma, and unnecessary mastectomies have been performed. Recognition of this lesion is important because it is benign and conservative excision is curative. PMID- 3009583 TI - [Accumulation and destruction of free radicals in DNA solutions and cells irradiated at 77 K]. PMID- 3009584 TI - Lymphocyte beta-adrenergic receptor density of patients with recurrent affective illness. AB - The binding characteristics of (-)3-[125I]iodocyanopindolol, a high specific activity antagonist for the lymphocyte beta-adrenoceptor, were determined in a group of euthymic lithium-treated patients and controls. There was a statistically significant reduction (less than 20%) in the density of beta adrenoceptors in a group of 56 lithium-treated patients. Division of this group of patients into the unipolar and bipolar distinction indicated that the abnormality was based mainly in the bipolar group of patients. The unipolar patients with an endogenous form of the illness also showed this abnormality. Reduced beta-adrenoceptor density may be associated with endogenous affective illness. However, the results must be interpreted with caution since lymphocyte fractions prepared for beta-adrenoceptor assays can be contaminated with platelets and since platelets have been reported to have beta-receptors then a proportion of the total binding could be associated with platelet beta adrenoceptors rather than with lymphocyte beta-adrenoceptors. PMID- 3009585 TI - What's new in dentistry. PMID- 3009586 TI - Dietary advice for diabetics: a perspective from the United Kingdom. PMID- 3009587 TI - Carbohydrate in the diabetic diet. PMID- 3009589 TI - Neutrophil granules in health and disease. AB - The granules of the neutrophil, in addition to contributing to its distinctive morphologic appearance, are critical to its unique functions. Specific granules appear necessary for neutrophil recruitment to sites of inflammation, for upregulation of receptors important in the control of chemotaxis and the respiratory burst, for disaggregation, for bactericidal activity, and for chemoattractant generation. The azurophilic granules supply enzymes for digestive and bactericidal functions and supply MPO to the MPO-halide-hydrogen peroxide bactericidal system. Azurophilic granule contents also regulate inflammation by degrading inflammatory products. Both granules may play a role in intracellular calcium regulation. In addition to these activities that protect the host from infection, granules also, under certain circumstances, contribute to disease processes. For these reasons, greater knowledge about granule contents, control of degranulation, inactivation of toxic granule contents and products, and the role of granules in neutrophil membrane events and function has widespread implications for treatment of patients with neutrophil dysfunction syndromes and patients with multiple other systemic diseases. PMID- 3009588 TI - Nutritional management of diabetes mellitus: rationale, ethics and practicability. PMID- 3009591 TI - Alpha-adrenergic receptors in circulating cells. PMID- 3009590 TI - Studies on the biosynthesis of leukotrienes in guinea pig lung in vitro by use of high-performance liquid chromatography. AB - The biosynthesis of leukotrienes (LTs) and 5-hydroxy-6,8,-11,14(E,Z,Z,Z) eicosatetraenoic acid (5-HETE) in perfused guinea pig lung was studied by reversed phase-high-performance liquid chromatography (RP-HPLC). Perfusion of lungs with a 20 microM solution of ionophore A23187 for 25 minutes induced a sustained release of 5S-hydroxy-6R-S-cysteinylglycyl-7,9,11,14-(E,E,Z,Z) eicosatetraenoic acid (LTD4) and 5S,12R-dihydroxy-6,8,10,14(Z,E,E,Z) eicosatetraenoic acid (LTB4). The perfusion of 60 microM of arachidonic acid together with the ionophore only slightly increased the synthesis of LTD4 and LTB4 in comparison with the effect of the ionophore alone but markedly stimulated the release of 5-HETE that was not detectable under other experimental conditions. Immunologic challenge of the lung by continuous perfusion of ovalbumin to previously sensitized guinea pigs induced the release of 50 to 150 pM of LTB4 and 15 to 50 pM of LTD4 during a 25-minute period. Antigen was a less potent stimulus than the ionophore that caused the release of 500 to 900 pM of LTB4 and 100 to 250 pM of LTD4 during 25-minutes of perfusion. None of the above mentioned metabolites were detected when lungs were perfused with arachidonic acid alone, indicating that the synthesis of 5-lipoxygenase products required activation of the lung. In addition to 5-lipoxygenase products, the cyclooxygenase products 12-hydroxy-5,8,10-(Z,E,E)-heptadecatrienoic acid and 12 keto-5,8,10-(Z,E,E)-heptadecatrienoic acid were analyzed by RP-HPLC in guinea pig lung perfusates. HPLC analysis provided quantitative data on the release of several arachidonic acid metabolites by lungs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009592 TI - Ileoanal pull-through: a new surgical alternative to ileostomy and a new challenge in diet therapy. AB - Colectomy mucosal proctectomy, and endorectal ileoanal pull-through is presently considered the best alternative for many patients with chronic ulcerative colitis, familial polyposis, and Gardner's syndrome. Although continence is achieved in nearly all patients who undergo the operation, high stool frequency and poor stool consistency are not infrequently encountered, particularly in the early postoperative period. Dietary control plays a likely role in postoperative management. At present, however, there are no published data to support specific dietary interventions. On the basis of our clinical experience and some preliminary study results, we have developed a comprehensive dietary regimen which is outlined in Table 1. Many questions in regard to the optimal diet for this patient population are presently unanswered, thus opening an exciting new area of nutrition research. PMID- 3009593 TI - Projections from brainstem nuclei to the nucleus paragigantocellularis lateralis in the cat. AB - Afferent projections from the pons and medulla to the nucleus paragigantocellularis lateralis (PGL) have been mapped in the cat using retrograde transport of horseradish peroxidase (HRP). In the caudal medulla, the major sources of afferents were the medial and lateral divisions of the solitary nuclei complex and the contralateral trigeminal nucleus caudalis. Labelled cells were also present in the dorsal column nuclei, nucleus intercalatus and praepositus hypoglossi but this may have been due to uptake of HRP into fibres of passage. In the dorsolateral medulla and pons, neurones in the vestibular complex and in the parabrachial nucleus were labelled bilaterally. Nucleus raphe magnus and raphe obscurus were both found to send projections to the PGL and labelled cells were also present throughout the pontine and medullary reticular nuclei as well as in PGL on the side opposite to the injection of HRP. These findings are discussed in relation to the role of the PGL in cardiovascular regulation and in the control of pain. PMID- 3009594 TI - Projections of supraspinal structures to the phrenic motor nucleus in cats studied by a horseradish peroxidase microinjection method. AB - The activity of phrenic motor neurons is influenced by the cardiovascular control system of the supraspinal structures. In order to obtain the basic data for analyzing the anatomical relations between the cardiovascular and the pulmonary control system, supraspinal structures projecting to the phrenic motor nucleus of the cat spinal cord were studied using a horseradish peroxidase method. A double barrel coaxial electrode was employed. To determine the site of the phrenic motor neurons, the inner barrel electrode, filled with 3 M NaCl solution, was used for recording the activity of these neurons. The outer barrel electrode, filled with a 20% HRP solution, was used for injecting HRP iontophoretically into the phrenic motor nucleus. Out of 13 experiments, 5 showed that the HRP-injection sites were centered in and almost confined to the phrenic motor nucleus. Some 1798 HRP labeled cells were thus identified in the selected 5 experiments. They were distributed in the medulla oblongata (93.5%), pons (6.0%) and midbrain (0.5%). The majority were concentrated in the nucleus para-ambiguus (48.9%), nucleus tractus solitarii (21.5%) and in or around the nucleus retrofacialis (9.8%). A few labeled cells were scattered throughout the nucleus raphe (1.1%) and the parabrachial and Koelliker-Fuse nuclei (0.3%), suggesting that these nuclei may be, if any, only minor sources of input to the phrenic motor nucleus. PMID- 3009595 TI - Activities of deglycosylated thyrotropin at the thyroid membrane receptor adenylate cyclase system. AB - A bovine thyrotropin (bTSH) preparation was deglycosylated by treatment with anhydrous hydrogen fluoride (HF) in the presence of anisole. The resulting material consisted of TSH derivatives that exhibited different molecular sizes, all smaller than the native hormone. The majority (62%) of the deglycosylated TSH derivatives did not bind to the lectin concanavalin A, while 98% of the native TSH was able to bind. The deglycosylated TSH derivatives bound to the high affinity-high specificity TSH binding sites in human thyroid membranes with a potency more than twice that of equivalent immunological amounts of the native bTSH. Despite the enhanced binding affinity for the TSH receptor, the deglycosylated TSH derivatives were unable to stimulate adenylate cyclase fully. Maximal stimulation achieved with bTSH derivatives was only 9 to 17% of the maximal stimulation achieved with native bTSH. Further, the deglycosylated derivatives competitively inhibited stimulation of the thyroidal adenylate cyclase by native bTSH. We conclude that HF treatment of bTSH results in partially deglycosylated TSH derivatives that exhibit enhanced ability to bind to the TSH receptor and markedly diminished adenylate cyclase-stimulating activity. PMID- 3009597 TI - Increased DHEAs levels in PCO syndrome: evidence for the existence of two subgroups of patients. AB - In 49 patients affected by PCO syndrome the serum levels of dehydroepiandrosterone-sulphate (DHEAs) were determined and correlated with the clinical presentation and the endocrine pattern. Twenty-three patients (47%) had high DHEAs levels (h-DHEAs patients). They presented a milder clinical presentation (low incidence of amenorrhea) than PCO patients with normal DHEAs levels (n-DHEAs patients). In h-DHEAs patients the finding of a normal DHEAs response to ACTH and of slightly increased 170HP serum levels suggested that the elevation of serum DHEAs was not due to an adrenal enzymatic deficiency but to a tonic hyperstimulation of the adrenals. Two subgroups of h-DHEAs patients were identified: in the first subgroup, PRL and estrone levels were increased and probably explained the DHEAs hypersecretion; in the second subgroup, the endocrine pattern was very similar to that observed in n-DHEAs patients and a clear explanation for DHEAs increase was not found, although the possibility of an exaggerated secretion of some pituitary hormones with adrenal androgen stimulating activity must be considered. PMID- 3009596 TI - Dissociation between CSF and plasma B-endorphin in major depressive disorders: evidence for a different regulation. AB - Plasma and cerebrospinal fluid (CSF) levels of ACTH, B-lipotropin (B-LPH) and B endorphin (B-EP) were simultaneously measured in 10 patients with major depression (35-57 yr) with a disease history of 10-34 yr, 7 of them with recurrent episodes, and in 13 age-matched healthy controls. In patients, lumbar puncture was performed after a 10 days drug-free period. Plasma B-EP and B-LPH levels were measured by RIA after silicic acid plasma extraction and Sephadex G 75 column chromatography. Plasma ACTH concentrations were measured by IRMA. For CSF assays the extraction step was avoided. In depressed patients, plasma ACTH (16.2 +/- 6.9 fmol/ml, mean +/- SD), B-LPH (19.8 +/- 8.5) and B-EP (17.8 +/- 7.0) levels were significantly higher (p less than 0.01) than in controls. On the contrary, CSF levels of the three peptides were similar in the two groups. No correlations were found between plasma or CSF concentrations and duration of the disease or severity of the actual episode. These data add further evidence to the independent regulation between central and peripheral POMC-related peptides. They also reduce the possibility that peptides of pituitary origin, directly from the gland or through the peripheral circulation, could penetrate the CSF. PMID- 3009598 TI - HLA and hormonal studies in 5 patients with late-onset 21-hydroxylase deficiency syndrome (21OHDS). AB - Late-onset 21-hydroxylase deficiency (21OHD) presents biochemical evidence of 21OHD and virilization in peri-or postpubertal age; it has been demonstrated that late-onset 21OHD is linked to HLA system. We present the HLA typing, the baseline and the ACTH-stimulated hormonal levels in 5 patients with late-onset 21OHD and in their family members. We identified 3 HLA identical male sibs within their respective families, 2 sibs sharing one haplotype with the affected member and 2 homozygous normal sibs. We observed elevated baseline (greater than 4 ng/ml) and ACTH-stimulated 17-hydroxyprogesterone levels, increased baseline Androstenedione levels, slightly elevated or normal DHEA-S and Testosterone values and subnormal response of Cortisol levels to ACTH in patients and in the HLA-identical sibs, reduced SHBG levels in patients but not in their identical sibs. The heterozygous family members presented hyperresponsiveness of 17-hydroxyprogesterone but not of androgens after ACTH. We confirm that late-onset of 21OHD is an autosomal recessive disease linked to HLA-B; there is in fact biochemical evidence of mild 21OHD in patients and in their HLA identical sibs and 17-hydroxyprogesterone levels in the range of heterozygotes for classical 21OHD in parents and sibs predicted by HLA to be carriers. Thus HLA typing and hormonal data, particularly 17-hydroxyprogesterone, are useful, also in this form of congenital hyperplasia, in detecting heterozygotes. PMID- 3009599 TI - Low-renin primary hypertension in a young patient treated with dexamethasone. AB - We have studied a young untreated patient with low-renin hypertension. All the clinical tests were in normal range, except plasma renin activity, which was low. Also all traditional tests evaluating the adrenal function were normal. A test of supraphysiological ACTH stimulation revealed a partial enzymatic defect of adrenal 11-hydroxylase. The patient has been treated with dexamethasone (0.75 mg once daily), during 4 weeks. At the end of the treatment blood pressure reached normal values. The traditional adrenal function tests are not sufficient to exclude enzymatic abnormalities and their possible involvement in the pathogenesis of hypertension. PMID- 3009600 TI - [Human papillomaviruses in the epithelial cells of the cervix uteri: frequency of types 16 and 18. Preliminary results of a clinical, cytologic and viral study]. AB - Between April 1 and June 30 1984, cervical scrapes were taken from 381 women attending the Gynecology Department of the Anticancer Center Rene-Huguenin. The scrapes were examined for the presence of human papillomavirus (HPV) DNA, by a molecular hybridization method, at the Pasteur Institute. The four HPV types involved in genital pathology, HPV 6, HPV 11, HPV 16 and HPV 18, were studied. Twenty four specimens (6.3%) were found positive: 19 for HPV 16, 3 for HPV 18, 2 for HPV 6 or HPV 11. Results of molecular hybridization were compared with cytological findings. HPV 6 or HPV 11 were detected in cases of mild dysplasia. HPV 16 or HPV 18 were mainly detected in cases diagnosed as severe dysplasia or carcinoma in situ (9 out of 14, i.e. 64.3%), and invasive carcinoma (3 out of 5 cases). The results were further confirmed when virological data obtained with cervical scrapes were compared with the histological diagnosis on biopsies: 14 out of 15 cases of severe dysplasia or carcinoma in situ (93%) and 3 out of 6 cases of invasive squamous carcinoma had been found positive for HPV 16 or HPV 18. Interestingly, four "normal" women (1.3%) with a negative cytology were found positive for HPV 16 or HPV 18. The data obtained by this sensitive and reliable method are useful to the clinician to identify women presenting a high risk of subsequent cervical intraepithelial neoplasia or invasive carcinoma, and, thus, to adapt the treatment and the follow-up of these patients. PMID- 3009601 TI - [A rare tumor of the ovary: Krukenberg's tumor. Apropos of 11 cases]. AB - The authors present 11 cases of Krukenberg tumour and have reviewed the literature concerning this condition. They conclude that the expectation of cure is increased if the primary tumour is removed early and if chemotherapy, hormono therapy when indicated and immunotherapy are carried out, preferably together. No treatment at present seems to be specific for this condition. PMID- 3009602 TI - Lipid deposition after hypophysectomy and growth hormone treatment in the sheep fetus. AB - Fifteen sheep fetuses hypophysectomized around 115 days gestation (term 150 days) were treated with growth hormone (GH), adrenocorticotrophin (ACTH) or triiodothyronine (T3) by continuous intravenous infusion. At normal or caesarian delivery, about 149 days gestation, body weights and dissectible internal brown fat and subcutaneous white fat depots were compared with seven control hypophysectomized fetuses and four sham-operated controls, the latter groups receiving no hormone infusions. Hypophysectomy caused the appearance of a large persistent depot of subcutaneous fat which was unaffected by ACTH or T3 infusion. Treatment with ovine pituitary GH resulted in the disappearance of this depot which was also absent from the sham-operated controls. Although the GH source was not subjected to extensive purification, there was very little contamination with prolactin or TSH, suggesting that GH itself has a major role in the control of lipid metabolism in the fetus. PMID- 3009603 TI - Subcellular distribution of acid NADPase activity within the parenchymal cells of rat liver. AB - We examined the distribution of acid NADPase activity in rat hepatocytes by cytochemistry and subcellular fractionation. Cytochemical studies revealed strong NADPase activity in many lysosomes and Golgi saccules situated between the medial and trans aspect of the stack. Light, spotty deposits of reaction product also were observed frequently within elements of the Golgi-associated endoplasmic reticulum lysosome (GERL) system. Biochemical studies with liver homogenates indicated that NADPase activity was distributed across various subcellular fractions in a pattern resembling typical nonspecific acid phosphatase activity, as monitored using beta-glycerophosphate as substrate. Thus, by differential centrifugation, most cellular NADPase activity was found in the large granule (ML) fraction (61%). The microsomal (P) fraction showed about 17% whereas the nuclear (N) and cytosol (S) fractions each accounted for about 11% of the remaining NADPase activity, respectively. Separation of the ML fraction into endosomes and secondary lysosomes revealed most of the acid NADPase activity associated with lysosomes. An alternate one-step method for isolation of fractions indicated that 3% of the recovered acid NADPase activity, but 55% of the Golgi marker enzyme galactosyl transferase, was associated with a purified intact Golgi fraction. Cytochemical studies with ML and intact Golgi fractions indicated that bona fide reaction product was localized to lysosomes and to the same saccules that were reactive in vivo. We propose that saccules making up the trans half of the Golgi stack may represent a site for accumulation of a biogenetic precursor of the lysosomal acid NADPase in hepatocytes. PMID- 3009604 TI - Immunohistochemical localization of two angiotensin I-converting isoenzymes in the reproductive tract of the male rabbit. AB - The male reproductive tract contains two different isoenzymes of angiotensin I converting enzyme (ACE), i.e., pulmonary and testicular ACE. The present study shows selectively the cellular distribution of the ACE isoenzymes in the reproductive tract of male rabbit, using indirect immunofluorescence or immunoperoxidase methods. Testicular ACE was found in the seminiferous tubules of the testes in spermatocytes containing mature spermatids, and in spermatids within the epididymal tubular lumen in sexually mature, but not in immature, rabbits. Epididymal tubular cells contained pulmonary ACE. In the young rabbit, epididymal tissue contained more ACE than that in adult rabbit, since ACE was observed in principal cells in addition to basal cells. In mature rabbit, ACE was observed in basal cells only. Strong staining for pulmonary ACE was observed in cells of the vas deferens in both young and adult rabbit. Therefore, synthesis of epididymal ACE, unlike the testicular isoenzyme, was not stimulated by sexual maturation. Enzymatically active ACE in seminal fluid corresponds to the pulmonary isoenzyme. The present study indicates that this seminal fluid ACE may originate from cells of the epididymal tubules, particularly those of the vas deferens. Endothelial cells of blood vessels lying in the interstitium of both testicular and epididymal tissue contained the pulmonary isoenzyme. PMID- 3009605 TI - Characterization of epithelial membrane antigen expression in human mammary epithelium by ultrastructural immunoperoxidase cytochemistry. AB - Ultrastructural immunocytochemistry was used to analyze cell surface distribution and intracellular localization of milk fat globule membrane antigen (MFGM-A) in cryosections from human breast carcinomas and benign breast biopsy specimens. The specimens were fixed in formaldehyde and frozen. Cryostat sections were cut at 15 micron, incubated with mouse monoclonal antibody to MFGM-A, and then with a peroxidase-conjugated goat anti-mouse antibody. After glutaraldehyde fixation, the sections were incubated with diaminobenzidine-H2O2 and further processed for electron microscopy. MFGM-A was specific for epithelial cells. MFGM-A staining was strictly confined to the apical surface membrane of normal ductal epithelium, never involving basolateral membranes below the tight junctions. In normal epithelial cells, MFGM-A was readily detected in cisternae of the endoplasmic reticulum (ER), but only to a lesser extent in Golgi complexes and presumptive secretory vesicles. In carcinoma cells, surface staining for MFGM-A was either distributed in a non-polarized manner on the entire cell surface or else was totally absent. In some carcinoma cells without surface-associated MFGM-A, very pronounced intracellular MFGM-A staining was seen in the ER, in the nuclear envelope, and in annulate lamellae. The observations on MFGM-A expression were supported by studies on a cell culture model system. PMID- 3009606 TI - Variation in virulence of bovine rotaviruses. AB - Forty-six gnotobiotic calves aged less than 16 days or 42-116 days were infected with three strains of bovine rotavirus designated C3-160, CP-1 and PP-1. Each virus was passaged and cloned in cell culture (cloned viruses) but CP-1 and PP-1 were also used before culture (faecal viruses). Infection of calves aged less than 16 days with faecal or cloned CP-1 caused disease whereas cloned C3-160 and faecal or cloned PP-1 caused subclinical infections. The clinical signs of disease were change in faecal colour to pale yellow or cream, increase of 2- to 7 fold in the volume of faecal output and, usually, anorexia. With the virulent CP 1 virus and the avirulent C3-160, similar amounts of virus were excreted in the faeces for 4-6 days. Infection of calves aged 56-116 days with faecal CP-1 produced disease of similar severity to that seen in calves aged 7-10 days infected with the same virus. No differences in clinical signs, virus excretion or levels of convalescent antibody were seen between the two groups. With cloned CP-1, 5 of 8 older calves developed disease but 3 showed only mild signs of infection. It was concluded that two strains of rotavirus caused sub-clinical infections in young calves while a third was virulent in calves up to at least 116 days of age. PMID- 3009607 TI - Altered phagocytosis by monocytes from HLA-DR2 and DR3-positive healthy adults is Fc gamma receptor specific. AB - Fc gamma receptor-dependent mononuclear phagocyte system (MPS) clearance of opsonized erythrocytes is prolonged in healthy adults with the class II alloantigens HLA-DR2, DR3, or DQw1, despite normal receptor-specific Fc ligand binding by monocytes in these groups. To investigate the basis for the MPS dysfunction, we determined the phagocytic capacity of blood monocytes from 66 disease-free adults and analyzed the data according to the HLA type of the subjects. The data demonstrate decreased phagocytosis of IgG-sensitized erythrocytes (EA) by monocytes from HLA-DR2, DR3, or DQw1-positive subjects compared with normals without these B cell alloantigens (2.87 +/- 0.83 erythrocytes/monocyte vs 3.87 +/- 1.05, p less than 0.004; 3.01 +/- 0.94 vs 3.87 +/- 1.05, p less than 0.02; 3.18 +/- 0.89 vs 3.87 +/- 1.05, p less than 0.02, respectively). Because HLA-DR2 and DQw1 are in linkage disequilibrium, we compared EA phagocytosis in subjects with DQw1 alone to normals without HLA-DR2, DR3, or DQw1. Among subjects positive only for DQw1, no significant decrease in phagocytosis could be seen (3.46 +/- 0.95 vs 3.87 +/- 1.05, p = NS). To determine whether these differences represented an Fc receptor-specific dysfunction or a more generalized decrease in phagocytic activity, we compared the quantitative phagocytosis of EA with that of neuraminidase-treated erythrocytes (EN), which is Fc receptor independent and beta-glucan receptor mediated. No segregation of phagocytic capacity for EN by HLA class II phenotypes could be demonstrated (DR2, 2.68 +/- 1.30 erythrocytes/monocyte; DR3, 2.95 +/- 1.30; DQw1, 2.84 +/- 1.15; others, 3.06 +/- 1.14). Our data indicate that the decrease in phagocytosis by blood monocytes from normal individuals with HLA-DR2 or DR3, the class II alloantigens associated with systemic lupus erythematosus and other autoimmune diseases, is specific for the Fc receptor-mediated process. This alteration of Fc receptor function among immunogenetically defined individuals may contribute to their predisposition to autoimmune disease. These differences may also apply to other Fc receptor-initiated cellular functions. PMID- 3009608 TI - Release of lymphokines after Epstein Barr virus infection in vitro. I. Sources of and kinetics of production of interferons and interleukins in normal humans. AB - Infection of human lymphocytes with Epstein Barr virus (EBV) activates the release of lymphokines. Previous experiments have emphasized the ability of interferon-gamma (IFN-gamma) to prevent EBV-induced B cell transformation. However, the factors that regulate IFN-gamma synthesis and release during in vitro EBV infection are controversial. In the present investigation we have systematically evaluated the kinetics of production, cellular origins, and accessory cell requirements for IFN-alpha and IFN-gamma and for IL 1 and IL 2, after EBV infection. Our data indicate that IFN-alpha is released entirely by natural killer (NK) cells and B cells, in the absence of accessory cells, independently of the other lymphokines and within 24 hr of infection. In contradistinction, IFN-gamma secretion is exclusively of T cell origin, is absolutely dependent on the prior elaboration of IL 1 and IL 2, and is maximal 8 days after EBV infection. IL 2 secretion by T cells peaks on day 5 and requires the earlier release of IL 1. Both NK cells and monocytes are a source of IL 1. Secretion of IL 2 and IFN-gamma occurs in the presence of either one of these cell types but not in the absence of both. Antibody against IL 1 blocks EBV induced IL 2 and IFN-gamma generation, and antibody against IL 2 decreases production of IFN-gamma. Thus, the production of IFN-gamma, the lymphokine that prevents EBV-induced B cell transformation, is the final outcome of a cascade of lymphokine-mediated events that involve interactions between virus-infected B lymphocytes that serve as antigen-presenting cells, NK cells and monocytes as sources of IL 1, and T lymphoblasts. Dysfunctions of any or all of these cell types would be expected to impair the regulation of EBV transformation. PMID- 3009609 TI - Release of lymphokines after infection with Epstein Barr virus in vitro. II. A monocyte-dependent inhibitor of interleukin 1 downregulates the production of interleukin 2 and interferon-gamma in rheumatoid arthritis. AB - Epstein Barr virus (EBV)-infection of normal peripheral blood mononuclear cells (PBMC) in vitro induces IFN-alpha secretion from B cell and natural killer (NK) cell populations, and IFN-gamma secretion from T cells. IFN-gamma depends on prior elaboration of IL 2 and IL 1 that originates from monocytes and NK cells. PBMC from rheumatoid arthritis (RA) patients released moderately elevated levels of IFN-alpha (236 +/- 62 U/ml vs 168 +/- 34 in normals). In contrast, IFN-gamma was significantly lower in RA (88 +/- 34 U/ml vs 209 +/- 32) with an associated deficit in IL 2. A monocyte-dependent factor was shown to be responsible for this deficit, since monocyte depletion of RA cultures normalized the levels of IL 2 and IFN-gamma. Significantly lower levels of IL 1 activity were present in the supernatants of RA PBMC cultures as compared with normal cultures, and this was shown to be associated with presence of a nondialyzable IL 1 inhibitor. This inhibitor was capable of preventing the IL 1-dependent synthesis of IL 2 and IFN gamma by normal PBMC. Exogenous IL 1 or IL 2 restored the deficient IFN-gamma secretion in RA PBMC. Thus, the deficient ability of RA lymphocytes to control EBV infection may be secondary to impairment of a monocyte-T cell interaction at the level of IL 1. PMID- 3009610 TI - Enhancement by monokines of leukotriene generation by human eosinophils and neutrophils stimulated with calcium ionophore A23187. AB - Human blood eosinophils and neutrophils that had been incubated with the supernatants of cultures of lipopolysaccharide (LPS)-stimulated blood mononuclear cells demonstrated respective enhanced abilities to produce immunoreactive leukotriene C4 (LTC4) and immunoreactive leukotriene B4 (LTB4) after activation by the calcium ionophore A23187. Under optimal conditions, the enhancing effect was observed with the eosinophils (n = 21) and the neutrophils (n = 14) from all but one donor of each type of granulocyte. Enhancement was maximum when granulocytes were preincubated with a 1/3 dilution of LPS-stimulated mononuclear cell culture supernatants for 1 to 2.5 min and were then stimulated with 2.5 microM ionophore for 1 to 2 min (neutrophils) or 15 min (eosinophils). Maximal enhancement ranged from 20 to 4500% for LTC4 generation by eosinophils (geometric mean, 87%) and from 30 to 1600% for LTB4 generation by neutrophils (geometric mean, 105%). There was no enhancement of leukotriene biosynthesis when the LPS stimulated mononuclear cell culture supernatants and ionophore were added simultaneously to the granulocytes. The enhancing activity for LTC4 generation by eosinophils was removed by washing the cells after the addition of the LPS stimulated mononuclear cell culture supernatants and before the introduction of ionophore. This enhancing activity was produced by Ig-, Leu-1- adherent blood mononuclear cells, which are presumed to be monocytes; supernatants of adherent cells augmented A23187-induced LTC4 generation by eosinophils from 21 to 2300% (geometric mean, 402%) in 11 experiments and LTB4 generation by neutrophils from 7 to 200% (geometric mean, 60%) in 10 experiments. There was an inverse correlation between the percent enhancement and the LTC4 levels produced by stimulated eosinophils in the absence of the monokine(s) (r = -0.79, p less than 0.01), but not between percent enhancement and the LTB4 levels generated by ionophore-activated neutrophils in the control buffer. The activity of the monocyte-derived enhancing material on each type of granulocyte was relatively heat stable. Enhancement of eosinophil production of LTC4 was associated with an acidic group of monocyte-derived molecules having isoelectric points of 4.2 to 4.3, 4.5 to 4.6, and 4.9, and exhibiting marked heterogeneity in size.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3009611 TI - Human endothelial cells inhibit granulocyte aggregation in vitro. AB - Granulocyte aggregation in response to circulating or locally released inflammatory mediators may cause vascular injury. The factors that regulate the granulocyte aggregation response and prevent its occurrence are not defined. We found that primary monolayers of human endothelial cells (EC) derived from umbilical veins released products that inhibited granulocyte aggregation. When polymorphonuclear leukocytes (PMN) and EC were incubated together, the subsequent aggregation response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) was inhibited by 40 to 60%, depending in part on the duration of incubation and the concentration of the agonist. Suspension of the granulocytes in albumin containing buffer that had been rocked with EC monolayers had a similar effect, demonstrating that the EC release a soluble product that modulates the aggregation response. The fMLP concentration-response curve was shifted downward and to the right by EC. Incubation of the granulocytes with endothelial monolayers for various times indicated that the inhibition was maximal at 2 to 3 min, and the PMN responsiveness returned to control over the next 15 min. The inhibiting effect was not selectively directed against fMLP, because incubation of PMN with EC or suspending the PMN in supernatants from endothelial monolayers also inhibited aggregation stimulated by platelet-activating factor, leukotriene B4, and C5a desarg. Release of the inhibitory activity by EC was attenuated by indomethacin, suggesting that the activity is in part due to a cyclooxygenase pathway product. Prostacyclin (PGI2), an eicosanoid produced by EC via the cyclooxygenase pathway, inhibited granulocyte aggregation; however, PGI2 was much less potent as an inhibitor of PMN aggregation than of platelet aggregation. Furthermore, the concentration of PGI2 in buffer that had been incubated with EC was not sufficient to account for the magnitude of the PMN inhibition. The concentration of prostaglandin E2 (PGE2) was also insufficient to completely account for the inhibition. EC that had been treated with indomethacin or aspirin, which blocked the release of PGI2 and PGE2, retained the partial ability to release an activity that blunted granulocyte aggregation; this inhibiting activity was stable at 37 degrees C for 60 min. The results indicate that human EC have the biologic potential to modulate granulocyte aggregation stimulated by inflammatory mediators, and the activity is only partly due to PGI2 and other cyclooxygenase products of arachidonic acid.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3009612 TI - Correlation between enhanced oxidative metabolism and leishmanicidal activity in activated macrophages from healer and nonhealer mouse strains. AB - A test system that allows a precise monitoring of intracellular killing of Leishmania parasites was used to measure the leishmanicidal capacity of activated macrophages from different strains of mice. Activation was obtained by exposure to dilutions of lymphokine (LK)-rich medium in the presence of ng/ml amounts of E. coli lipopolysaccharide (LPS). Highest leishmanicidal activity was displayed by macrophages from the healer mouse strain CBA/T6, whereas cells from the nonhealer strains DBA/2 and BALB/c were less effective. C3H/HeJ macrophages from a healer but LPS-unresponsive mouse strain failed to destroy leishmanias under these conditions. Experiments were then performed to determine the level of respiratory burst activity in the various macrophage populations. Hexose monophosphate shunt stimulation was higher in CBA/T6 than in BALB/c or DBA/2 macrophages, and only marginal in C3H/HeJ cells, correlating with differing leishmanicidal activities of such macrophages under the present experimental conditions. Measurements of O2- and H2O2 secretion and of chemiluminescence led to similar findings, i.e., CBA/T6 macrophages released higher amounts of oxygen metabolites than BALB/c cells activated under the same conditions, whereas C3H/HeJ cells were the least active. The results obtained by the four assays of oxidative metabolism were consistent in that endotoxin itself (in the 10 to 30 ng/ml range) stimulated the oxidative response of macrophages to a level close to that achieved by using LPS and LK together, however, only in the latter situation were parasites destroyed. PMID- 3009613 TI - 5C6-F4, a novel 100,000-dalton rat lymphocyte activation antigen defined by monoclonal antibody. AB - Con A-activated rat thymocytes were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated 5C6-F4, reacted strongly with Con A-activated rat thymocytes and some LPS-activated rat spleen cells but not with normal thymocytes, spleen cells, or bone marrow cells of rat origin. The 5C6-F4 did not react with Con A-activated thymocytes of mouse origin. Immunoprecipitation of 5C6-F4 antigen from surface-iodinated Con A-activated rat thymocytes or LPS-activated rat spleen cells revealed its m.w. to be approximately 100,000. The kinetic studies of the expression of 5C6-F4 antigen revealed that 5C6-F4 antigen was detectable at 6 hr after Con A stimulation of rat spleen cells, whereas IL 2 receptor (IL 2R) was detectable at 12 hr. The appearance of 5C6-F4 antigen and IL 2R precede the onset of DNA synthesis of Con A-activated spleen cells. Thus, 5C6-F4 antigen is classified as early activation antigen. The 5C6-F4 inhibits the lymphocyte proliferation induced by mitogen and the IL 2-driven rat T cell proliferation. Sequential immunoprecipitation study as well as binding inhibition study indicated that the 5C6-F4 antigen is distinct from IL 2R molecule. The 5C6-F4 antigen appears to be a novel rat lymphocyte activation antigen that exhibits immunoregulatory function and also may serve as a useful marker of T cell activation. PMID- 3009614 TI - Phenotypic, functional, and molecular genetic comparisons of the abnormal lymphoid cells of C3H-lpr/lpr and C3H-gld/gld mice. AB - Lymph node cells from C3H mice homozygous for lpr and gld were compared for expression of cell surface antigens, lectin-binding sites, functional characteristics, expression of ecotropic MuLV, and organization of Ig and T cell receptor (TcR) beta-chain genes. The abnormal cells (Ly-2-/L3T4-) populating nodes of both mutant strains were specifically purified by using plate separation techniques. The purified abnormal cells were shown to express the beta-chain of the TcR, to exhibit rearrangements of the beta-chain genes, and to express TcR beta and alpha gene mRNA, demonstrating the T cell origin of these populations. FMF analyses of the separated abnormal cells showed them to be Thy-1+, Ly-1+, Ly 2-, L3T4-, Ly-5(B220)+, Ly-6+, Ly-22+, Ly-24+, sIg-, ThB-, Ia-, HSA-/+, and PC.1+ and to bind at high levels lectins that normally bind preferentially to B cells. These cells did not proliferate or generate CTL in response to stimulation with alloantigens, and supernatants of cells stimulated with Con A were devoid of IL 2. These characteristics do not correspond to those of any known immature or mature population of normal T cells. The findings that the abnormal T cells of lpr and gld homozygotes are indistinguishable for each parameter examined support the suggestion that these mutations may affect different enzymes in a common metabolic pathway of major importance to T cell differentiation and function. PMID- 3009615 TI - Incorporation of the purified Epstein Barr virus/C3d receptor (CR2) into liposomes and demonstration of its dual ligand binding functions. AB - The 145-kDa molecule that has been identified as the C3d receptor CR2 was isolated from lysates of Raji cells by affinity chromatography by using the monoclonal antibody (MoAb)HB-5. The purified protein was incorporated into 14C phosphatidylcholine liposomes by deoxycholate dialysis followed by flotation on discontinuous sucrose gradients. Incorporation of the receptor was verified by testing the gradient fractions for CR2 by an enzyme-linked immunosorbent assay. Liposomes were shown to be unilamellar vesicles ranging in diameter from 25 to 100 nm by electron microscopy. The external orientation of CR2 in the membranes was demonstrated by immunoelectron microscopy. The functional activities of liposomes containing CR2 and liposomes without protein were compared. CR2 liposomes bound to EC3d, but not to E, and this binding was inhibited by the anti CR2 MoAb OKB7 and by a MoAb specific for C3d. Control liposomes failed to bind to either E or EC3d. The ability of CR2 to function as a receptor for Epstein Barr virus (EBV) was tested in two ways. First, CR2 liposomes bound to B95-8, a cell line expressing EBV membrane antigens, but not to B95-8 cells treated with the viral DNA polymerase inhibitor phosphonoformic acid. Second, liposomes containing CR2 were shown by ultracentrifugal analyses to bind directly to purified EBV, and this binding was also inhibited by OKB7. Control liposomes did not bind to B95-8 cells or to EBV. These findings show that CR2 purified from detergent extracts of Raji cells can be reconstituted into lipid membranes with maintenance of its dual functions as a receptor for C3d and EBV. PMID- 3009616 TI - Biochemical and antigenic analysis of the Epstein Barr virus/C3d receptor (CR2). AB - Four monoclonal antibodies (OKB7, HB-5, AB-1, and anti-B2) that recognize a 145 kDa B cell-specific membrane structure have markedly different abilities to 1) inhibit C3d and EBV binding to B cells, 2) immunoprecipitate a 145-kDa B cell protein, and 3) stimulate B cell proliferation and differentiation into Ig secreting cells. This study was initiated to determine whether these four monoclonal antibodies (MoAb) react with the same protein; a related goal was to determine whether the structure(s) recognized by these antibodies constitutes an antigenically related family of structurally distinct molecules. In the studies presented here, the four MoAb were found to fully immunoprecipitate the purified 145-kDa B cell molecule isolated by immunoaffinity chromatography on either OKB7, HB-5, or AB-1 columns, findings that show conclusively that the antibodies all react with the same B cell protein. The variable ability to immunoprecipitate this B cell membrane protein was found to result from differences in exposure or accessibility of the relevant antigenic epitopes in the detergent extract. The 145-kDa molecule immunoprecipitated with the four MoAb was equivalently sensitive to endoglycosidase F and yielded the same banding pattern after digestion with endoglycosidase F and after partial digestion with either S. aureus V8 protease or with trypsin. Within the limits of the sensitivity of these techniques, therefore, there is no evidence for carbohydrate or protein differences in the EBV/C3d receptor (CR2) molecule recognized by the four MoAb. Additional studies showed that the four MoAb react with distinct and nonoverlapping antigenic epitopes on the 145-kDa molecule. The variable abilities of the four MoAb to inhibit CR2 function and EBV binding and to trigger B cell activation, together with the other findings noted above, indicates that the 145-kDa EBV/C3d receptor possesses discretely localized functional domains. PMID- 3009617 TI - Release of leukotrienes C4 and B4 and prostaglandin E2 from human monocytes stimulated with aggregated IgG, IgA, and IgE. AB - Purified human peripheral blood monocytes were stimulated with aggregated human myeloma proteins of different classes or the calcium ionophore A23187 and the release of leukotrienes C4 and B4 (LTC4, LTB4), and prostaglandin E2 (PGE2) into the supernatant was determined. The ionophore induced release of 10 +/- 5 ng LTC4/10(6) cells and 25 +/- 8 ng LTB4/10(6) cells. Aggregated IgG, IgA, and IgE, but not IgM or monomeric immunoglobulins (Ig), induced release of LTC4 and LTB4 that was approximately 10 to 20% of that induced by ionophore. In addition, IgG, IgA, and IgE, but not IgM, induced release of PGE2 (range 0.015 to 0.22 ng/10(6) cells). Aggregated Ig induced LTC4, LTB4, and PGE2 release in a dose-dependent manner; maximal leukotriene (LT) release was observed by 30 min, in contrast to PG release, which continued to increase up to 2.5 hr. Both ionophore- and Ig induced LTC4 and LTB4 release were completely inhibited by removal of calcium from the media and by preincubation of cells with nordihydroguaiaretic acid. Indomethacin inhibited Ig-induced PGE2 release by 80%. Phagocytosis of the Ig aggregates was not required for LT or PGE2 release, since release was not inhibited by cytochalasin B. Release of LTC4, LTB4, and PGE2 induced by IgG, IgA, and IgE, but not IgM, correlated with the presence or absence of monocyte Fc receptors (FcR) as determined by rosette assays. The data suggest that IgG, IgA, and IgE immune complexes mostly likely induce monocyte arachidonic acid metabolism via cross-linking of FcR. The ability of monocytes to release eicosanoids in the absence of phagocytosis suggests that interaction of monocytes with immobilized immune complexes, such as those deposited in blood vessel walls or glomerular basement membranes, could initiate metabolism of arachidonic acid by monocytes. Such a mechanism could contribute to inflammatory reactions characterized by mononuclear cell infiltrates. PMID- 3009618 TI - Modulation of the heterogeneous membrane potential response of neutrophils to N formyl-methionyl-leucyl-phenylalanine (FMLP) by leukotriene B4: evidence for cell recruitment. AB - Individual human neutrophils (PMN) isolated by Hypaque-Ficoll gradient sedimentation, dextran sedimentation, or buffy coat preparation were assessed for the effects of leukotriene B4 (5S,12R dihydroxy 6,14-cis-8, 10 trans eicosatetraenoic acid (LTB4)-pretreatment on N-formylmethionyl-leucyl phenylalanine (FMLP)-mediated membrane potential or oxidative responses by using flow cytometry and a lipophilic probe of membrane potential (di-pentyl oxacarbocyanine, di-O-C(5)3), or the nitroblue tetrazolium dye (NBT) reduction test, respectively. Although exposure to LTB4 (10(-7) M) had no effect on the membrane potential of resting PMN and little effect on oxidant production, pretreating PMN with LTB4 followed by FMLP (10(-6) M) demonstrated a significant enhancement in the proportion of depolarizing PMN over that seen with FMLP alone (p = 0.0014, N = 9). This recruitment of previously unresponsive cells by LTB4 was dose and time dependent, with the maximal relative increase in the proportion of depolarizing cells occurring at LTB4 concentrations of 10(-8) to 10(-7) M and within 1 min of LTB4 addition. The recruitment effect persisted despite vigorous washing of the cells. LTB4 also increased the proportion of NBT-positive PMN in response to FMLP. Although LTB4 alone did not depolarize PMN it did induce a light scatter shift indicative of cell activation. 3H-FMLP binding studied at 0 degree C comparing buffer and LTB4-treated PMN indicated no significant change in the number or affinity of FMLP binding. The data provide evidence for the recruitment of a greater proportion of cells into a FMLP-responsive state as a mechanism for the enhanced functional response of PMN pretreated with LTB4, as well as for a dissociation of the membrane potential and light scattering responses of cells to this pro-inflammatory LT. The mechanism of recruitment remains unclear, but it most likely involves the modulation of a post-FMLP binding step. PMID- 3009619 TI - Stimulation of neutrophils by tumor necrosis factor. AB - Human recombinant tumor necrosis factor (TNF) was shown to be a weak direct stimulus of the neutrophil respiratory burst and degranulation. The stimulation, as measured by iodination, H2O2 production, and lysozyme release, was considerably increased by the presence of unopsonized zymosan in the reaction mixture, an effect which was associated with the increased ingestion of the zymosan. TNF does not act as an opsonin but, rather, reacts with the neutrophil to increase its phagocytic activity. TNF-dependent phagocytosis, as measured indirectly by iodination, is inhibited by monoclonal antibodies (Mab) 60.1 and 60.3, which recognize different epitopes on the C3bi receptor/adherence-promoting surface glycoprotein of neutrophils. Other neutrophil stimulants, namely N-formyl methionyl-leucyl-phenylalanine, the Ca2+ ionophore A23187, and phorbol myristic acetate, also increase iodination in the presence of zymosan; as with TNF, the effect of these stimulants is inhibited by Mab 60.1 and 60.3, whereas, in contrast to that of TNF, their stimulation of iodination is unaffected by an Mab directed against TNF. TNF may be a natural stimulant of neutrophils which promotes adherence to endothelial cells and to particles, leading to increased phagocytosis, respiratory burst activity, and degranulation. PMID- 3009620 TI - Demonstration of NK cell-mediated lysis of varicella-zoster virus (VZV)-infected cells: characterization of the effector cells. AB - Infection with varicella-zoster virus (VZV) rendered RAJI cells more susceptible to lysis by non-adherent blood lymphocytes. At an effector to target ratio of 80:1 the mean percentage of 51Cr release of VZV-infected RAJI cells was 41 +/- 12%, whereas that of uninfected RAJI cells was 15 +/- 6%. The increased susceptibility to lysis was associated with increased effector to target conjugate formation in immunofluorescence binding assays. The effector cells cytotoxic for VZV-infected RAJI cells were predominantly Leu-11a+ Leu-4- granular lymphocytes as demonstrated by fluorescence-activated cell sorting. The effector cell active against VZV-infected RAJI cells appeared similar to those active against herpes simplex virus (HSV)-infected cells, because in cold target competition experiments the lysis of 51Cr-labeled VZV-infected RAJI cells was efficiently inhibited by either unlabeled VZV-infected RAJI cells (mean 71% inhibition, 2:1 ratio unlabeled to labeled target) or HSV-infected RAJI cells (mean 69% inhibition) but not by uninfected RAJI cells (mean 10% inhibition). In contrast, competition experiments revealed donor heterogeneity in the overlap between effector cells for VZV- or HSV-infected RAJI vs K-562 cells. PMID- 3009622 TI - Congenital dislocation of the hip: treatment in infancy to walking age. PMID- 3009621 TI - Characterization of four constant region genes of rabbit immunoglobulin-lambda chains. AB - A cDNA plasmid insert encoding the constant (C) region of a rabbit immunoglobulin lambda light chain was used as a probe for screening a rabbit liver genomic DNA cosmid library. This allowed the isolation and identification of four distinct C lambda genes, designated C lambda 1, C lambda 2, C lambda 3, and C lambda 4, which were shown to be widely separated from each other along chromosomal DNA. Their nucleotide sequences have been determined. No in-frame termination codons were found within the coding regions. The C lambda 1, C lambda 2, and C lambda 3 sequences are quite similar to each other, but share less homology with the C lambda 4 gene or the cDNA-C lambda sequence used as a probe. The C lambda gene coding for the cDNA sequence was not isolated. Translation of the C lambda 1, C lambda 2, and C lambda 3 sequences predicts a Cys-Pro carboxy-terminal amino acid sequence, as found so far only for horse lambda-chains. Compared to the other rabbit C lambda genes, the C lambda 3 sequence exhibits two deletions, one of 9 bp, the other of 3 bp. The latter occurs at the same position as in the mouse C lambda 2 and C lambda 3 genes. These two deletions are located in the loops between anti-parallel beta-pleated sheets of the C lambda domain. When the C lambda nucleotide sequences from man, mouse, and rabbit are compared, there is less divergence within the same species than for interspecies comparisons. Possible genetic implications of this finding are discussed. PMID- 3009623 TI - Quantitation of Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA) by a two-site enzyme immunoassay, in parallel with EBV-DNA. AB - A two-site enzyme (TSE) immunoassay was developed for the quantitation of the Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA) using a rabbit serum raised against a synthetic peptide derived from the BamHI K region of the viral genome. Comparison of 12 EBNA-positive and 3 negative cell lines proved that the test was EBV-specific. A dot-blot assay utilizing cloned and nick translated EBV DNA BamHI M fragment confirmed the EBV-carrier status of the EBNA-positive lines. The results obtained with both the TSE immunoassay and dot-blot assay were in agreement with published values. In contrast to earlier reports, we could not demonstrate any correlation between the content of EBNA and the number of viral genome copies. PMID- 3009624 TI - Application of hepatitis B core particles produced by human primary hepatocellular carcinoma (PLC/342) propagated in nude mice to the determination of anti-HBc by passive hemagglutination. AB - Human primary hepatocellular carcinoma (PLC/342), carried by nude mice, produces hepatitis B core particles as well as hepatitis B surface antigen particles. Core particles purified form PLC/342 tumors displayed epitopes of hepatitis B core antigen (HBcAg) but not epitopes of hepatitis B e antigen (HBeAg) on their surface, unlike core particles prepared from Dane particles, derived from plasma of asymptomatic carriers, that expressed epitopes of both HBcAg and HBeAg. Core particles obtained from PLC/342 tumors were applied to the determination of antibody to HBcAg (anti-HBc) by passive hemagglutination. The assay detected anti HBc not only in individuals with persistent infection with hepatitis B virus and in those who had recovered from transient infection, but also in patients with acute type B hepatitis, indicating that it can detect anti-HBc of either IgG or IgM class. A liberal availability of core particles from tumors carried by nude mice, taken together with an easy applicability of the method, would make the passive hemagglutination for anti-HBc a valuable tool in clinical and epidemiological studies, especially in places where sophisticated methods are not feasible. PMID- 3009625 TI - Mucinous adenocarcinoma of the colon in a child. PMID- 3009626 TI - Multiple primary malignancies. PMID- 3009627 TI - AIDS: a serious challenge to public health. PMID- 3009628 TI - Evaluation of antibodies to Epstein-Barr virus in Italian patients with nasopharyngeal carcinoma. AB - An association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma (NPC) has been established serologically in Italian patients. The finding of IgA antibodies to EBV-capsid antigen (VCA) and to early antigen (EA) showed a high degree of correlation for patients with NPC, thereby confirming previous reports. In addition, when considered together, IgA anti-VCA and IgG anti-EA antibody titres appeared to distinguish NPC-patients from those in control populations. Poorly differentiated and undifferentiated carcinomas with lymphoid cellular infiltrations showed the highest frequency of association with positive EBV serological tests. PMID- 3009630 TI - The acute exanthem associated with seroconversion to human T-cell lymphotropic virus III in a homosexual man. AB - An illness characterised by fever, arthralgia, myalgia, a macular erythematous rash, a sore throat and the appearance of atypical lymphocytes in the blood film is described in a 27-year-old homosexual man. There was serological evidence that this illness was due to the human T-cell lymphotropic virus Type III. PMID- 3009631 TI - The isolation of Streptococcus pneumoniae from bile. AB - Streptococcus pneumoniae was isolated from the bile of two previously healthy adults presenting with extrahepatic obstructive jaundice. The organism was recovered on two occasions from both patients over periods of at least 10 days indicating established biliary colonisation and at the time of surgery pneumococci were present at multiple sites throughout the biliary tract. In one patient, pneumococcal cellulitis necessitating treatment with benzyl penicillin arose post-operatively at the exit site of an external biliary drainage tube. Both strains of pneumococci exhibited typical colonial and microscopical morphology. They were susceptible to optochin and demonstrated typical solubility in normal human and ox bile as well as in solutions of sodium deoxycholate and sodium taurocholate. PMID- 3009629 TI - Antibody against hepatitis A in Saudi Arabians and in expatriates from various parts of the world working in Saudi Arabia. AB - The age-specific rate of exposure to hepatitis A virus (HAV) was studied in 1015 native Saudi Arabians (504 males, 511 females) from the Riyadh area. The relatively high prevalence of antibody to HAV (anti-HAV) (38.6%) in children between 1 and 4 years of age indicates that infection is acquired early in life in the Saudi Arabian population. The prevalence of anti-HAV was found to increase steadily so that by the age of 30 years 91.0% of Saudi Arabians have anti-HAV. The prevalence in adult Saudi Arabians was compared with that in expatriates from various parts of the world working in Saudi Arabia. It was lowest among Swedish (10.7-12.3%) and highest among Yemeni (94.5%) blood donors while British blood donors were intermediate same among Saudi Arabian, Yemeni, Egyptian and Filipino blood donors (91.0-94.5%). All the donors tested were of the same age group (20 35 years). PMID- 3009632 TI - Rotavirus: the major etiologic agent of severe infantile diarrhea may be controllable by a "Jennerian" approach to vaccination. PMID- 3009633 TI - Evaluation of rhesus rotavirus vaccine (MMU 18006) in infants and young children. AB - Orally administered rhesus rotavirus vaccine was evaluated in a placebo controlled study in young children and infants (ages, eight months to 61 months). Thirteen children received the rotavirus vaccine, and ten children served as the control group. The vaccine was well tolerated. There were no significant differences between the vaccine recipients and the control group in the number of child-days with temperatures greater than or equal to 37.8 C, vomiting, diarrhea, or cough. There were significantly more child-days of rhinorrhea among the vaccine recipients than there were among the control group. The vaccine recipients under two years of age passed a larger number of stools than did the children in the control group, and vaccine recipients had significantly more semiformed and unformed stools than did the children receiving the placebo. All twelve of the children tested were positive for viral shedding. Peak viral shedding occurred on days three and five postvaccination. On day eight, over one half of the children from whom a stool specimen was obtained were still shedding rotavirus. Children less than two years old shed more rotavirus in their stool than did children more than two years old. All 13 vaccine recipients had a fourfold or greater rise in titer of antibody as measured by plaque reduction, tube neutralization, complement fixation, and/or immune adherence hemagglutination. PMID- 3009634 TI - A comparative trial of rhesus monkey (RRV-1) and bovine (RIT 4237) oral rotavirus vaccines in young children. AB - Heterologous live, oral rotavirus vaccines of rhesus monkey (RRV-1) and bovine (RIT 4237) origin were tested for immunogenicity, excretion of virus, and clinical reactions in six- to eight-month-old infants. Antibody response, indicating infection with the vaccine virus, was detected in 21 (88%) of 24 children receiving the RRV-1 vaccine and in 18 (75%) of 24 receiving the RIT 4237 vaccine. Excretion of virus in the stools within one week after vaccination was demonstrable in 84% of the RRV-1 and in 21% of the RIT 4237 vaccinees. RRV-1 vaccination was associated with a febrile response (over 38 C) that clustered on days 3 or 4 postvaccination in 64% of the recipient children. In addition, 20% of the RRV-1 vaccinees had watery stools on days 4 or 5. Fever on days 3 and 4 and loose stools were not seen in the RIT 4237 vaccinees. We concluded that in young children the RRV-1 (rhesus monkey) rotavirus vaccine is more immunogenic than the RIT 4237 (bovine) rotavirus vaccine, but vaccination with RRV-1 is associated with significant adverse reactions. PMID- 3009636 TI - Human papillomavirus DNA associated with foreskins of normal newborns. AB - Foreskins from 70 unselected infants undergoing routine circumcision were analyzed by dot blot hybridization for the presence of DNA related to that of human papillomavirus (HPV) type 6, 11, 16, or 18. Three foreskins (4%) contained HPV DNA; two contained DNA related to HPV-16, and one contained DNA related to HPV-6. These results suggest that neonates exhibit evidence of a relatively high incidence of exposure to HPV. The outcome of exposure may be initially asymptomatic. A retrospective study of maternal charts showed six mothers with abnormal Pap smears, but no correlation could be identified between these mothers and the HPV-positive foreskins. PMID- 3009635 TI - Clinical and subclinical reactivations of varicella-zoster virus in immunocompromised patients. AB - The frequencies of reactivated disease due to varicella-zoster virus (VZV) in immunocompromised patients were determined by enzyme-linked immunosorbent assay for antibody and also by the lymphocyte proliferation response to VZV antigen. Subclinical reactivations were as common as classical herpes zoster in all patient groups. Among bone marrow transplant (BMT) recipients, 36% developed herpes zoster and 26%, a subclinical reactivation. The corresponding frequencies for patients with leukemia during induction therapy were 5% and 10%; in renal transplant recipients, 0% and 26%; and in patients with seminoma, 0% and 6%, respectively. Subclinical reactivation of VZV thus appears to be a common finding in severely immunocompromised patients. A regained lymphocyte proliferation response to VZV antigen is a sensitive indicator of subclinical reactivation of VZV in BMT recipients. None of 19 BMT recipients with subclinical disease due to VZV later developed clinical reactivation of VZV. Acyclovir given as prophylaxis against infection with herpes simplex virus reduced the number of clinical and subclinical reactivations of VZV during treatment in BMT recipients, but not thereafter. PMID- 3009637 TI - The histopathology of experimental trachoma: ultrastructural changes in the conjunctival epithelium. AB - Experimental acute conjunctivitis was produced in cynomolgus monkeys by ocular inoculation with serovar B of Chlamydia trachomatis. The cellular responses to chlamydial conjunctivitis infection were examined by light, transmission, and scanning electron microscopy. A self-limited, acute conjunctivitis resulted from a single primary inoculation. A moderate lymphocytic infiltrate was present in the conjunctiva. After repeated inoculation, a chronic conjunctivitis (trachoma) developed. Prominent lymphoid follicles with distinct germinal centers were present in these tissues. Scanning electron microscopy revealed patchy areas of cellular alteration and loss of microvilli. Intracellular injury, documented by transmission electron microscopy, included disruption of the plasmalemmal membranes and rupture of the cytoplasmic organelles. The inflammatory infiltrate consisted of plasma cells, polymorphonuclear cells, lymphocytes, eosinophils, and degranulating mast cells. The immune response following single and repeated chlamydial infections was characterized by immunoperoxidase staining with monoclonal antibodies to pan-leukocytes, macrophages, and B cells. The center of the follicle was comprised of B cells, with T cells in the cap region. Large macrophages were also found in the germinal center. Further study is required to determine which cellular mechanisms are involved in the histopathologic and immunologic alterations induced in the conjunctiva after chlamydial infection. PMID- 3009638 TI - Restricted IgG subclass responses to HTLV-III/LAV and to cytomegalovirus in patients with AIDS and lymphadenopathy syndrome. PMID- 3009639 TI - Familial human parvovirus infection associated with anemia in siblings with heterozygous beta-thalassemia. PMID- 3009640 TI - Interferon response to mitogens and viral antigens in elderly and young adult subjects. PMID- 3009641 TI - Disinfection and inactivation of HTLV-III/LAV. PMID- 3009642 TI - Diagnosis of poliomyelitis by demonstration of intrathecal synthesis of neutralizing antibodies. PMID- 3009643 TI - Genital reinfection after recovery from initial genital infection with herpes simplex virus type 2 in guinea pigs. AB - The natural history of genital reinfection with herpes simplex virus type 2 (HSV 2) three weeks to one year after initial infection was explored by using a guinea pig model. Intravaginal reinoculation with HSV-2 of animals that had recovered from initial genital infection produced by wild-type or thymidine kinase deficient HSV-2 produced asymptomatic genital reinfection characterized by high titered replication of HSV-2 in the vaginal vault. Reinoculation did not result in acute neural infection, as was seen during initial genital herpes, and no evidence of latent infection by the rechallenge strain could be demonstrated. The frequency of recurrences appeared to be established by the initial infection because reinfection did not alter the pattern of recurrent genital herpes. The present study suggest that asymptomatic reinfection may produce a reservoir of communicable virus and that initial infection with a less virulent mutant of HSV 2 may provide protection against subsequent reexposure to wild-type HSV-2. PMID- 3009644 TI - Enzootic bovine rotavirus is not a source of infection in Panamanian cattle ranchers and their families. AB - Vaccination of humans against rotavirus (RV) diarrhea may be accomplished by oral immunization with attenuated animal strains known to be antigenically very similar to human strains. To define better the degree of infectivity in nature of these animal strains for humans, we conducted surveillance for RV infection/diarrhea in 180 farm workers, their 161 family contacts, and the 566 animals (512 cattle, 35 pigs, and 19 sheep) on 14 farms in rural Panama. No correlation between the high infection rates in farm workers (72%) and their family contacts (78%) and in cattle (56%) could be demonstrated. Heads of families with four or more children with RV infection experienced a twofold greater rate of RV infection compared with heads of families of similar size without RV infection. Despite the close similarity between human and bovine RV, in Panama intrafamilial (particularly child-to-child or child-to-parent) rather than interspecies transmission appeared to be the most important route for the spread of this highly infectious virus. PMID- 3009645 TI - Enzyme-linked immunosorbent assay for the detection of IgM-class antibodies to cytomegalovirus. PMID- 3009646 TI - Persistent excretion of cytomegalovirus in heart transplant patients correlates with inversion of the ratio of T helper/T suppressor-cytotoxic cells. PMID- 3009647 TI - A novel 4.9-kilobase plasmid associated with an outbreak of penicillinase producing Neisseria gonorrhoeae. PMID- 3009648 TI - Classification of HTLV-III/LAV-related diseases in children. PMID- 3009649 TI - The mechanism of action of erythropoietin. AB - The proliferation and differentiation of committed erythroid progenitor cells is regulated by the glycoprotein hormone erythropoietin. Erythropoietin increases the number of developing erythroid precursors and accelerates the release of reticulocytes from the marrow without markedly altering the cell cycle length or number of mitotic divisions involved in the differentiation process. Although the hormone has been purified, molecularly cloned and sequenced, its secondary and tertiary structure and active site have not been defined. Erythropoietin has both mitogenic and differentiation functions, and whether an erythroid progenitor cell responds to the hormone by proliferating or differentiating appears to depend on its level of maturation. Erythroid progenitor cells are responsive to a variety of growth and developmental agents but only erythropoietin appears obligatory in vivo for terminal differentiation. Erythropoietin interacts with its target cells through specific high-affinity receptors and Ca2+ may be involved in the receptor ligand interaction. Ca2+ may also be involved in the induction of differentiation by erythropoietin. An increase in RNA synthesis due to activation of transcription is one of the earliest recognized effects of the hormone and appears not to require protein or DNA synthesis but the initial sequence of biochemical events triggered by erythropoietin is still undefined. PMID- 3009650 TI - Parameters affecting the in vitro maturation of human monocytes to macrophages. AB - In vitro maturation of human monocytes to macrophages was characterized by morphological criteria, cell size and lysosomal enzymes activity. Purified populations of monocytes were maintained in culture at either adherent or nonadherent conditions and their maturation to macrophages was observed in both cases. The addition of external factors such as hydrocortisone and vitamin D3 inhibited monocyte maturation. In the absence of external factors, nonadherent monocytes were inhibited in their maturation for up to 10 days when plated at crowded cell concentrations. In addition, the presence of human serum in the culture media had a higher inhibitory activity than similar concentrations of fetal calf serum. Supernates from crowded macrophages were also inhibitory for monocyte maturation. We suggest the possibility that cell crowding, as well as soluble factors found in the serum and probably secreted by macrophages, participate in the regulation of monocyte development by inhibiting their maturation. Once released from this inhibitory signal or environment, the monocytes mature to macrophages. PMID- 3009651 TI - [Rapid diagnosis of herpes simplex viruses by the fluorescent antibody test using monoclonal antibodies]. PMID- 3009652 TI - [Theoretical analysis of the spread of infection by Japanese encephalitis virus. II. Comparison between newly contracted infection rates in consecutive unit periods and seropositivity rates in swine with 2-mercaptoethanol-sensitive antibody]. PMID- 3009653 TI - The metabolic production of oxalate from xylitol: activities of transketolase, transaldolase, fructokinase and aldolase in liver, kidney, brain, heart and muscle in the rat, mouse, guinea pig, rabbit and human. PMID- 3009654 TI - Dietary control of brain 5-hydroxytryptamine synthesis: implications in the etiology of obesity. PMID- 3009655 TI - [Two heads of myosin molecules]. PMID- 3009656 TI - [Antagonists of platelet activating factor]. PMID- 3009657 TI - [Molecular pharmacology on intracellular calcium messenger system]. PMID- 3009658 TI - [A study on ultrasound imaging of invasive mole and choriocarcinoma with regard to clinical significance]. AB - In order to establish the value of ultrasonography in the diagnosis and management of trophoblastic tumor, we have performed ultrasonic examination on 32 patients. In 24 out of 32 cases, the focal echo could be detected within the uterus. The ultrasonically estimated volume of the focus was correlated with the level of urinary hCG. Concerning 17 hysterectomized cases, the ultrasound imaging of focuses was quite consistent with the post-operative findings. The echo patterns of these focuses could be classified into 4 types, that is, Type I, massive echogenic; Type II, multiple echo free; Type III, solitary echo free; and Type IV, subserous cystic. Type I echoes were macroscopically recognized to be clot, molar tissue and cancer tissue. In Types II, III and IV, multifarious hemorrhages were proven to be echo sources. The histopathological results showed that it is difficult to discriminate choriocarcinoma from invasive mole clearly by this echo pattern analysis. A notable decrease in focal size and change in echo pattern could be observed after chemotherapy, but these echographic changes were much slower than the rapid response in the urinary hCG value. PMID- 3009659 TI - [Current status of sexually transmitted diseases]. PMID- 3009660 TI - [Reactivity of two monoclonal antibodies (Troma 1 and CAM 5.2) on tissue sections -as a histological trophoblast marker in normal pregnancy and trophoblastic disease]. AB - In normal and molar pregnancy, non-villous trophoblasts which exist in the placental bed are thought to be difficult to be distinguished morphologically from the surrounding maternal cells. We examined the reactivities of two monoclonal antibodies (Troma 1 and CAM 5.2) by an immunoperoxidase technique and analyzed their usefulness as histological trophoblast markers. Materials were taken from 41 uteri of normal pregnancy, 7 uteri of hydatidiform mole, 2 uterine gestational choriocarcinoma, 1 tubal tissue of non-gestational choriocarcinoma and 5 non-gestational uteri. The reactivity of Troma 1 was observed on frozen sections and paraffin sections were employed in the analysis of CAM 5.2. The reactivities of these antibodies were the same. In nongestational tissue, they reacted with endometrial gland epithelia and endometrial covering epithelia. In placental bed of normal pregnancy, they reacted with villous and non-villous trophoblasts, besides endometrial gland epithelia. In molar sections, they reacted with gland epithelia, villous trophoblasts and also non-villous trophoblasts which proliferated towards maternal tissue. In choriocarcinoma, they reacted with carcinoma cells specifically. Since endometrial gland epithelia are easily identified histologically, it was concluded that these antibodies could work as histological trophoblast markers in normal pregnancy and trophoblastic disease. PMID- 3009661 TI - [Superoxide dismutase activity in Bacteroides gingivalis]. PMID- 3009662 TI - [Effect of culture supernatants of peripheral blood leukocytes on the bone mineral resorbing activity of human monocytes]. PMID- 3009664 TI - [Evaluation of intermittent hepatic arterial occlusion with infusion chemotherapy of unresectable hepatocellular carcinoma]. PMID- 3009665 TI - [Phase II study of etoposide in patients with lung cancer, with special reference to small cell carcinoma]. PMID- 3009663 TI - [Thermal structural analysis on fluorapatite formed in silica gel]. PMID- 3009666 TI - [Phase II study of VP-16 in patients with primary bronchogenic carcinoma, with special reference to small cell carcinoma--a multi-institutional cooperative study]. PMID- 3009667 TI - [Diagnostic significance of technetium-99m-pyrophosphate, technetium-99m methylene diphosphonate and gallium-67-citrate scintigraphy in amyloid heart disease--a study with AL amyloidosis and familial amyloid polyneuropathy]. PMID- 3009669 TI - Involution of residual juvenile nasopharyngeal angiofibroma (a case report). AB - The apparent tendency of juvenile nasopharyngeal angiofibroma to involute with age has been known for many years but firm evidence of this has been lacking. This case report uses computerized tomography to provide the first documented evidence of this involution. PMID- 3009668 TI - [A case of Cushing's disease with paradoxical response to dexamethasone]. PMID- 3009670 TI - Duct carcinoma of the minor salivary glands: a case report. AB - A tumour was removed from the oral vestibule of a 62-year-old woman. It showed histological features of both salivary duct carcinoma and papillary adenocarcinoma. The possibility that these entities might be but varieties of duct adenocarcinoma is suggested. PMID- 3009671 TI - Measurement of active phagocytosis by polymorphonuclear leukocytes by fluorescence liberation from phagocytized microspheres. AB - Phagocytosis by polymorphonuclear leukocytes (PMN) was determined by a newly developed technique based on measurement of liberation of a fluorescence substance from PMN phagosomes; 4-methylumbelliferyl-beta-D-glucuronide (4MUGL), which is a substrate of beta-glucuronidase in lysosome, was conjugated with a microsphere, and 4-methylumbelliferone (4MU) liberated from phagocytized 4MUGL microspheres was measured. The microspheres were composed of glyceryl methacrylate having a diameter of 2.0 micron. Liberating activity of six kinds of 4MUGL-microspheres containing various amounts of amino and carboxyl groups was compared. Among these six kinds of 4MUGL-microspheres, four kinds showed activity similar to that of morphological phagocytosis. These four kinds of 4MUGL microspheres liberated 4MU into the extracellular fluid from PMN during phagocytosis. Furthermore, they were recognized as a substrate of purified beta glucuronidase. 4MUGL-MS610 showed the highest liberating activity among the four kinds of microspheres. Optimal conditions for phagocytosis by PMN were determined using 4MUGL-MS610. Total liberation of 4MU from the microspheres increased almost linearly with incubation time with PMN from 0 to 60 min and was linear with 4MUGL MS in concentrations up to 4 X 10(8) microspheres/ml. This liberation was parallel to phagocytosis in a dose-dependent fashion. During 10-min incubation 20.4% of 4MU was liberated from 4MUGL-microspheres with phagocytosis. Seventy five percent of the liberated 4MU was distributed in the extracellular fluid. 4MU distributed in the extracellular fluid was not attributable to hydrolysis of unphagocytized microspheres by beta-glucuronidase extracellularly leaked from PMN by phagocytosis. Also phagocytized 4MUGL-MS610 by PMN was observed by scanning electron microscopy. These results indicate that 4MU was liberated from 4MUGL-MS by hydrolysis due to beta-glucuronidase released into phagosomes with phagocytosis by PMN. Sensitivity of this assay was limited to about 50 pmol/ml, being less than 0.5-1 microsphere phagocytized into one cell. PMID- 3009672 TI - Effect of inapparent murine hepatitis virus infections on macrophages and host resistance. AB - Inapparent infections of mice with murine hepatitis virus (MHV) altered host resistance to experimental infection with a second virus, encephalomyocarditis virus (EMC), reduced the protective effects of exogenously administered interferon against EMC infections, and it altered macrophage ectoenzyme phenotypes in two macrophage populations. Resident peritoneal macrophages from mice experimentally infected with one of two strains of MHV also demonstrated altered ectoenzyme phenotypes. These data demonstrate that inapparent infections with MHV alter several host resistance and macrophage parameters and directly demonstrate that effects of inapparent MHV infection on macrophage parameters can be reproduced experimentally. PMID- 3009673 TI - Malignant fibrous histiocytoma of the tongue. AB - Malignant fibrous histiocytoma (MFH) can occur as a rare mesenchymal neoplasm of the deep structures of the head and neck region. An unusual case of MFH of the tongue is described in a 61-year-old male. The primary tumour measured 90 X 50 X 30 mm. Treatment consisted of hemiglossectomy in continuity with a supra omohyoidal neck dissection. The patient has been free from disease for 2 years. The tongue as a primary site of MFH has not been reported to date. The literature of oral manifestations and treatment of this neoplasm is reviewed. PMID- 3009674 TI - Diagnosis of perineurial arachnoid cysts using computed tomography: technical and clinical considerations. AB - Perineurial arachnoid cysts are often multiple lesions that are usually found in the lumbosacral area. These cysts may cause compression of surrounding structures. Examination of the first, second and third sacral nerves should be performed on patients with prolonged symptoms of a herniated disc or cauda equina syndrome. Three cases are presented for which computed tomography provided accurate, noninvasive diagnosis. Perineurial cysts are often associated with asymptomatic herniated disc; thus, surgery on the disc with an underlying symptomatic cyst may result in a failed back syndrome. PMID- 3009675 TI - The mechanism of the prolonged stimulatory effect of corticotrophin on pregnenolone production by guinea-pig adrenocortical mitochondria. AB - The postulated prolonged stimulatory influence of ACTH on the adrenocortical mitochondrial synthesis of pregnenolone in response to ACTH was studied in adrenal mitochondria isolated from control guinea-pigs and from animals treated s.c. with 100 micrograms ACTH(1-24) twice daily on the day before the animals were killed. The animals from both groups were injected with 100 micrograms ACTH s.c. 30 min before killing. The mitochondrial production of pregnenolone (expressed in nmol per mg mitochondrial protein after 10-min incubation) increased from 1.52 +/- 0.46 (S.E.M.) in the control group to 4.50 +/- 0.59 for mitochondria from ACTH-treated animals, despite a similar free cholesterol content in the mitochondria, even when determined after a previous in-vivo treatment with aminoglutethimide to block further metabolism of cholesterol into pregnenolone. In addition, in the presence of an excess of exogenous cholesterol (100 mumol/l), the production of pregnenolone remained higher for mitochondria from ACTH-treated animals. In contrast, when the calcium concentration in the incubation medium was raised to 1 mmol/l, with subsequent enhancement in pregnenolone synthesis, the mitochondrial pregnenolone production became similar for both groups (8.28 +/- 1.11 nmol in the ACTH-treated group and 9.55 +/- 1.90 nmol in the control group), even in the presence of 100 mumol cholesterol/l (13.5 +/- 1.80 nmol in ACTH-treated animals and 14.8 +/- 1.93 nmol in controls). Cycloheximide treatment administered on the day before the animals were killed was without any effect on pregnenolone production in control animals (3.51 +/- 0.43 nmol before and 3.65 +/- 0.63 nmol after cycloheximide treatment).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009676 TI - Effects of LH and an LH-releasing hormone agonist on different second messenger systems in the regulation of steroidogenesis in isolated rat Leydig cells. AB - The stimulation of steroid production in Leydig cells by LH is accompanied by increased cyclic AMP levels, activation of protein kinase A, increased phosphorylation of at least six phosphoproteins and requires protein synthesis. However, an LH-releasing hormone agonist (LHRH-A) can stimulate steroid production without stimulation of cyclic AMP levels. In the present study we have shown that LH action involves calcium fluxes through the plasma membrane, in addition to activation of protein kinase A. The action of LHRH-A, in contrast, does not require calcium fluxes and is not potentiated by 1-methyl-3 isobutylxanthine, indicating that cyclic AMP is not involved. Extracellular calcium is required for the action of both LH and LHRH-A. An increase in intracellular calcium concentration due to the effect of ionophore A23187 did not stimulate steroidogenesis and had deleterious effects on intracellular adenosinetriphosphate levels. LH and 4 beta-phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C, both stimulated phosphorylation of proteins of 17 000 and 33 000 mol. wt, whereas LHRH-A had no effect. However, compared with the effect of LH, PMA had a much smaller effect on steroid production, indicating that even if protein kinase C may be activated by LH its role in the regulation of steroid production may be less important than the role of protein kinase A. Action of LHRH-A does not appear to be mediated by calcium fluxes, protein kinase C activation or active protein phosphorylation. PMID- 3009678 TI - Myosin light chain kinase and cyclic AMP-dependent protein kinase activities and calmodulin levels in placental and non-placental regions of the rabbit myometrium. AB - Myosin light chain kinase activity in the placental region of the rabbit myometrium on day 28 of gestation was 4.7 +/- 0.1 (mean +/- S.E.M.) nmol/min per mg protein, which was significantly higher than that (3.6 +/- 0.1 nmol/min per mg protein) in the non-placental region. The amount of calmodulin in the placental region was 4.2 +/- 0.1 micrograms/mg protein, which was significantly higher than that (3.2 +/- 0.1 micrograms/mg protein) in the non-placental region. In contrast, cyclic AMP-dependent protein kinase activities showed no difference between the two regions. These findings suggest that calcium- and calmodulin dependent protein phosphorylation is activated mainly in the placental region, and uterine contractions can occur more strongly in this part than in the non placental region. Such enzymatic phenomena may be related to the mechanism whereby the placenta separates from the myometrium after delivery of the fetus. PMID- 3009679 TI - Saccharide structures of the mouse embryo during the first eight days of development. Inferences from immunocytochemical studies using monoclonal antibodies in conjunction with glycosidases. AB - Monoclonal anti-carbohydrate antibodies have been used in conjunction with glycosidases in immunofluorescence studies to derive information about the structures and in situ distribution of saccharides of the mouse embryo during the first 8 days of development. The salient findings are as follows: Branched poly-N acetyllactosamine sequences of I-antigen type are detectable from the first day onwards and are widely distributed in cells of the endoderm, ectoderm and mesoderm. Linear poly-N-acetyllactosamine sequences of i-antigen type are detectable from the fifth day onwards in cells of all three lineages, but have a more restricted distribution than the sequences of I-type. Poly-N acetyllactosamine sequences that are susceptible to digestion with endo-beta galactosidase are the main carriers of the SSEA-1, C14 and the blood group B-like antigens, which have the following structures (Formula; see text) and are found in endoderm and ectoderm but not in mesoderm cells. In the trophoblast however, these antigens are borne on saccharides that are resistant to endo-beta galactosidase. A proportion of the poly-N-acetyllactosamine structures in the endoderm and the ectoderm of the 5- and 6-day embryos may contain the following novel structures: (Formula; see text) in which antigenicities of SSEA-1 and C14 determinants are masked. There are several types of sialyl-oligosaccharides: those reactive with anti-Gd, which has a specificity for NeuAc alpha 2-3Gal beta 1-4GlcNAc sequence in the extraembryonic mesoderm and the heart; those reactive with anti-Pr2 but not with anti-Gd, which may correspond to other N acetylneuraminic acid containing sequences such as NeuAc alpha 2-3Gal beta 1 3GalNAc or NeuAc alpha 2-6Gal in preimplantation embryos and in the yolk sac, neural ectoderm and mesenchyme of the 8-day embryo; those with other sialic acid forms or linkages that do not react with anti-Gd and Pr2; among these are sialosyl-i sequences in the extraembryonic ectoderm, and sialosyl-I sequences in most cell types during the first 8 days. The latter are the main poly-N acetyllactosamine structures in the neural ectoderm of the 8-day embryo. The sequence Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc/GlcNAc, or cross-reactive structures, which bind FC10.2 antibody occur in the extraembryonic endoderm and yolk sac. The roles of specific carbohydrate structures as receptors during embryonic development and cell growth are important topics of current research.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3009677 TI - Effect of testosterone on gonadotrophin-releasing hormone receptors in the castrated rat: preliminary evidence for a stimulatory effect of testosterone on gonadotrophin function in the male rat. AB - In the long-term castrated rat the negative feedback effect of testosterone is markedly reduced and the raised levels of plasma LH seen in the castrated animals are not suppressed by physiological concentrations of plasma testosterone. In this study we have measured pituitary gonadotrophin-releasing hormone (GnRH) receptor content as well as plasma and pituitary LH on days 1, 10 and 40 after castration and noted the effect of testosterone replacement on these parameters. We found that the negative feedback effect of physiological concentrations of testosterone on plasma and pituitary LH, pituitary GnRH receptor content and response to exogenous GnRH was attenuated 10 and 40 days after castration. It is suggested that the lack of effect of testosterone in the long-term castrated rat is due to its inability to reduce the pituitary GnRH receptor content. On increasing testosterone to supraphysiological levels, the negative feedback effect was reinstated. We also found that in rats 40 days after castration, physiological and subphysiological concentrations of testosterone significantly increased pituitary GnRH receptor content and this may explain the previous findings that low concentrations of testosterone can enhance the effect of GnRH and increase plasma LH levels. PMID- 3009680 TI - Oxygen-independent killing by alveolar macrophages. AB - We have found that normal alveolar macrophages can kill an intracellular parasite by a mechanism that does not involve toxic metabolites of oxygen. We studied the interaction between Toxoplasma gondii and rat alveolar macrophages in vitro. We were interested in Toxoplasma because it causes pneumonia in immunosuppressed patients but not in healthy individuals, and we chose the rat because it resembles immunocompetent human subjects in being resistant to T. gondii. Resident rat alveolar macrophages could kill large numbers of T. gondii. This occurred without a respiratory burst as judged by intracellular reduction of nitroblue tetrazolium and quantitative release of superoxide. Furthermore, scavengers of toxic oxygen metabolites had no effect on the toxoplasmacidal activity of the alveolar macrophages, nor did prior exhaustion of their respiratory burst with PMA. Whereas acid pH (e.g., 4.5-6.0) rapidly kills extracellular T. gondii, raising of the intralysosomal acid pH of rat alveolar macrophages by incubating them with weak bases did not inhibit their ability to kill T. gondii. Killing of Toxoplasma occurred within 1 h of initial exposure to the alveolar macrophages. However, there was no evidence that killing preceded ingestion; Toxoplasma attached to the surface of the cell appeared viable, and when phagocytosis was blocked with sodium fluoride the organisms survived. These results indicate that rat alveolar macrophages possess a powerful nonoxidative microbicidal mechanism, which is distinct from acidification of the phagolysosome but which probably involves phagosome formation. This mechanism may be clinically relevant, for we have recently observed that human alveolar macrophages also kill T. gondii by an oxygen-independent process. PMID- 3009681 TI - Conditioned aversions and their memories in 5-day-old rats during suckling. AB - Association learning during suckling was investigated. Five-day-old rats equipped with tongue cannulae placed either 2 mm rostral or 4-6 mm caudal to the intermolar eminence received sweet or salty solutions while suckling. This ingestion was followed by lithium chloride toxicosis. Pups with anterior cannulae took in considerably less fluid than control pups when tested 5 or 16 days later. A series of control groups demonstrated that this acquired aversion was associative in nature. Pups with posterior cannulae did not form the association. The failure of 5-day-old rats with posterior cannulae to form associations while suckling is not due to the prevention of conditioning by the act of suckling per se. Rather, the failure rests in the fluid's not reaching anterior taste receptors when injected into the posterior oropharynx, where the nipple normally empties its contents. These findings are discussed in terms of the transfer of information obtained during suckling prior to weaning, to feeding and drinking during and after weaning. PMID- 3009682 TI - The decline of catalytic enzyme activity concentration of in vivo ageing erythrocytes of the man, the dog and the rat. Approach to a quantitative diagnostic enzymology, IV. Communication. AB - Human, dog and rat erythrocytes were separated by centrifugation on a discontinuous buffered Percoll gradient into fractions of progressively increasing mean cell age to measure the in vivo decline in catalytic activity of eleven enzymes during the erythrocyte lifespan. Erythrocyte enzymes decline exponentially at different rates and also differ between the species. The maximal and minimal catalytic activities (erythrocyte catalytic activity at the beginning and at the end of the appropriate erythrocyte life-span for a given species) and the intracellular half-life of enzymes estimated. To test the hypothesis that circulating erythrocytes make a significant contribution to the normal catalytic activity in plasma it was assumed as a working hypothesis that the measured loss of catalytic activity in ageing erythrocytes is equivalent to the amount of the enzymes released in catalytically active form into plasma. This contribution was calculated. PMID- 3009684 TI - Efficacy of cryotherapy in the treatment of human papilloma virus infection of the uterine cervix. PMID- 3009683 TI - Sphingomyelinases and Niemann-Pick disease. AB - In the first part the properties of normal mammalian sphingomyelinases are reviewed: The lysosomal acid sphingomyelinase is a polymeric glycoprotein (subunit Mr between 28 000 and 70 000) which hydrolyses natural sphingomyelin, coloured and fluorescent semi-synthetic analogues (trinitrophenyl-aminolauryl sphingomyelin and pyrenedecanoyl-sphingomyelin) and the synthetic analogue 2-N hexadecanoylamido-nitrophenyl-phosphorylcholine. The suitability of these substrates and of synthetic fluorescent derivatives of methylumbelliferone is discussed. The effect of lipids, detergents and other effectors on the enzyme activity is also described. The neutral sphingomyelinase from brain tissue, localized in cell membranes, has a high Mr (160 000 and 600 000), is heat labile, hydrolyses sphingomyelin and its coloured and fluorescent analogues, but not 2-N hexadecanoylamido-nitrophenyl-phosphorylcholine. A new method is available for determining enzyme activity in the intact cell through the utilization of endogenous or exogenous sphingomyelin as substrate. In the second part of the review, the classification of Niemann-Pick disease, the characteristic features of each type and the biological tools used for the diagnosis are reported. Experimental models (animal models and cellular models in culture) are reviewed with a particular attention to a new model system, Epstein-Barr virus-transformed lymphoid cell lines. PMID- 3009685 TI - Dynamic responses of electrically coupled systems. AB - An identified pair of electrically coupled neurons in the buccal ganglion of the freshwater snail Helisoma trivolvis is an experimentally accessible model of electrical synaptic transmission. In this investigation, electrical synaptic transmission is characterized using sinusoidal frequency (Bode) responses computed by Laplace transforms and responses to brief stimuli. The frequency response of the injected neuron shows a 20-dB/decade attenuation and a phase shift from 0 degree at low frequencies to -90 degrees at high frequencies. The response of a coupled cell shows a 40-dB/decade attenuation and a phase shift from 0 degrees at low frequencies to -180 degrees at high frequencies. A simple mathematical model of electrical synaptic transmission is described that displays each of these crucial features of the measured frequency responses. Methods are described to estimate the frequency responses of coupled systems based on presynaptic measurements. The responses of the coupled system to brief pulses of current were computed using the principle of superposition. The electrical properties of coupled systems impose a minimum delay in reaching a peak in all postsynaptic responses. The delays in the postsynaptic responses to brief stimuli are related to the electrical and anatomical parameters of coupled networks. PMID- 3009686 TI - Stimulation of Na,K-activated adenosine triphosphatase and active transport by low external K+ in a rat liver cell line. AB - Exposure of ARL 15 cells, an established line from adult rat liver, to concentrations of external K+ below 1 mM caused a rapid fall in intracellular K+ and a corresponding rise in intracellular Na+ that became maximal within 12 h. Upon continued exposure to low external K+, these initial changes were followed by a striking recovery such that, by 24 h, intracellular Na+ and K+ concentrations approached their control values. Concomitant with this recovery, there was a substantial increase in Na,K-ATPase specific activity that was detectable at 12 h and maximal at 24 h. After restoration of the external K+ concentration, the elevated level of enzyme activity showed little change for at least 24 h. In contrast, restoration of external K+ resulted in a rapid rise in intracellular K+ and a fall in Na+ such that within 30 min the Na+/K+ ratio was lower than in control cells. This overshoot, together with a demonstrated increase in active 86Rb+ uptake under "Vmax" conditions, confirms that the enhancement in Na,K-ATPase specific activity in response to low external K+ represents an increase in functional Na,K pumping capacity. PMID- 3009687 TI - Some highlights of animal virus research in 1985. PMID- 3009688 TI - The product of gene US11 of herpes simplex virus type 1 is expressed as a true late gene. AB - The genes of herpes simplex virus type 1 (HSV-1) can be divided into at least three temporally regulated groups termed immediate early (IE), early and late. We have studied in detail the expression of a member of the late class of genes, US11, which encodes a polypeptide of apparent molecular weight 21K. Highly specific and sensitive probes were used to monitor US11 RNA and protein synthesis during HSV-1 infection of tissue culture cells in the presence and absence of phosphonoacetic acid, an inhibitor of viral DNA replication. The results were compared with a similar study of the products of a delayed early gene, US6, encoding glycoprotein D (gD). It was found that the patterns of RNA and protein synthesis from US11 were significantly different to those of gD. US11 products appeared later and accumulated until late in infection, while gD RNA was significantly reduced at late times. In the presence of the inhibitor of DNA synthesis, US11 gene expression was reduced 50- to 100-fold while gD expression was reduced five- to tenfold. We conclude that US11 behaves as a true late gene during HSV-1 infection. However, the use of sensitive assays, which allowed the detection of very low levels of US11 gene products under conditions designed to eliminate DNA replication, brings into question the absolute requirement for DNA replication for the expression of a true late HSV-1 gene. These results are discussed in terms of current models for the regulation of late gene expression. PMID- 3009689 TI - Logical description of bovine herpesvirus type 1 latent infection. AB - Description of the interactions between bovine herpesvirus type 1 (BHV-1) and cattle was performed by the method known as kinetic logic. This logical formalization uses variables with two possible values, 1 and 0, which tell whether an element is present or not at a significant level. To each variable is associated a function which tells if the element is being produced at a significant rate. The temporal relation between a variable and its associated function is given by specific time delays. The BHV-1 infection system is described by a set of five logical equations which tell in what conditions each function is on or off. The five functions are: V, development of viral multiplication; R, development of reactivation of the latent virus; A, development of an immune response; G, establishment of the viral genome; M, development of a memory of a first immune response. Several examples are detailed in a dynamic analysis, in connection with known experimental data. PMID- 3009690 TI - Antigenic and molecular properties of type 3 poliovirus responsible for an outbreak of poliomyelitis in a vaccinated population. AB - Virus isolated from an outbreak of poliomyelitis in Finland has been examined serologically and at the molecular level. The causative agent was an antigenically unusual strain of type 3 poliovirus, which was unrelated to the strains used to manufacture either live or killed poliovaccines. It is likely that the antigenic properties of the virus played a part in establishing a limited outbreak of poliomyelitis in a vaccinated population. PMID- 3009691 TI - Translational control in murine hepatitis virus infection. AB - High multiplicity infection of mouse fibroblast L-2 cells with mouse hepatitis virus (MHV) resulted, within 6 h, in a decline in total protein synthesis to about 7% of that observed in uninfected cells. The amount of intracellular total translatable RNA, however, increased approximately threefold, as a result of the accumulation of virus-encoded mRNAs. MHV-infected cells could be superinfected with vesicular stomatitis virus, demonstrating that MHV infection did not irreversibly alter the cellular translational machinery to the exclusion of non MHV mRNAs. Comparative polysome analysis from MHV-infected and uninfected L-2 cells showed that MHV infection resulted in an increase in single 80S ribosomes and in a shift from longer to shorter polysomes. These observations suggest first, that MHV infection inhibits total protein synthesis at a very early stage, as evidenced by the increase in 80S ribosomes, and, second, that the increased number of viral mRNAs produced after infection compete with cellular mRNAs for cellular ribosomes. In vitro translation of RNA extracted from MHV-infected and mock-infected cells suggested that levels of cellular mRNAs were decreased after infection. This suggestion was confirmed by demonstrating the loss of cellular actin mRNA, using a radiolabelled cDNA probe, as a consequence of MHV infection. PMID- 3009692 TI - Neutralizing secretory IgA and IgG do not inhibit attachment of transmissible gastroenteritis virus. AB - Secretory IgA (sIgA) and IgG from porcine milk and serum, respectively, [3H]uridine-labelled virus, swine testis and pig kidney cell lines were used to examine the neutralized virus-cell interaction. Transmissible gastroenteritis virus (TGEV), 99.99% neutralized by immunoglobulin, was able to attach to the cells. Moreover, sIgA enhanced virus attachment. However, the neutralized virus was unable to enter cells, as demonstrated by the action of proteinase K which removed it from the cell surface. It was also found that pre-attached virus was still neutralizable and that IgG and sIgA had similar TGEV-neutralizing capacities. PMID- 3009694 TI - Quantification of infection and neutralization of murine leukaemia virus by a microtitre enzyme-linked immunosorbent assay. AB - We have developed an enzyme-linked immunosorbent assay for murine leukaemia virus using methanol-fixed cells as antigen. Specific antisera clearly recognized viral antigens within cells adherent to the microtitre plates. This test was used to quantify infection, to monitor virus multiplication and to demonstrate virus neutralization by antisera. The ELISA was as sensitive as, and faster and easier to perform than, the XC plaque assay and could even quantify large amounts of virus. It can easily be adapted for assaying other viruses. PMID- 3009693 TI - Japanese encephalitis virus latency following congenital infection in mice. AB - Latent Japanese encephalitis virus (JEV) infection was shown in inapparently congenitally infected Swiss albino mice after their mothers had been given JEV intraperitoneally during pregnancy. Only one of 37 (2.7%) of the baby mice showed persistence of infectious virus at 5 weeks of age. Reactivation of JEV in Swiss albino mice was demonstrated by stimulation with allogeneic spleen cells from Parks strain mice at 21 weeks of age; reactivation was demonstrated in 41% of the inapparently infected mice. The spleen cells of congenitally infected mice had depressed [3H]thymidine uptake following stimulation with concanavalin A, and depressed ability to induce a graft-versus-host response. PMID- 3009695 TI - Nucleotide sequence of cDNA clones encoding the outer capsid protein, VP5, of bluetongue virus serotype 10. AB - The complete sequence of the RNA segment that codes for a major outer capsid protein (VP5) of bluetongue virus serotype 10 has been determined from overlapping cDNA clones inserted into pBR322. The segment 5 RNA of the virus (M5 RNA) is deduced to be 1638 base pairs long (1.05 X 10(6) daltons) and has an open reading frame in one strand capable of coding for a protein with a calculated size of 59 163 daltons (526 amino acids) and a net charge of -4.5 at neutral pH. PMID- 3009696 TI - Hepatitis B virus DNA in leukocytes of patients with hepatitis B virus-associated liver diseases. AB - In order to determine the relationship between hepatitis B virus (HBV) infection of human white blood cells and different forms of HBV-associated liver diseases, we tested for HBV DNA in the sera and leukocytes of 11 healthy individuals without any serological markers of HBV infection and 91 patients with HBV infection and other gastrointestinal and urinary diseases by dot and Southern blot hybridization. HBV DNA was found in leukocytes of chronic HBV carriers, in acute and chronic hepatitis, and in patients with liver cirrhosis and hepatocellular carcinoma. Between 27 and 50% of individuals in different categories of patients examined were positive for leukocyte HBV DNA. HBV DNA was also detected in the sera of some of these patients but was absent in others. Serum HBV DNA-positive rates seemed to be highest in hepatitis B e antigen positive asymptomatic carriers (8/10, 80%), and tended to drop to lower levels as the disease progressed to liver cirrhosis (0/8) while leukocyte HBV DNA-positive rates were highest in patients with cirrhosis (4/8, 50%). The results also show that in individuals who were serologically negative for hepatitis B surface antigen (HBsAg) and positive for antibodies to HBsAg and/or HBcAg, HBV DNA was absent in most of the sera (27/28, 96%) but it was present in leukocytes of some of these patients (7/28, 25%). In control experiments with 11 healthy individual, HBV DNA was not detected in either sera or leukocytes. In all the cases with leukocyte HBV DNA, the HBV DNA molecules were present in free forms with discrete sizes. The exceptions were a case of liver cirrhosis and a case of chronic hepatitis with possible HBV sequence integration into high molecular weight cellular DNA. Since HBV does infect human leukocytes, it may perhaps interfere with the immunological functions of the white blood cells, and thus play an important role in the pathogenesis of HBV-induced liver disease. PMID- 3009697 TI - Genome analysis of species 3 adenoviruses isolated during summer outbreaks of conjunctivitis and pharyngoconjunctival fever in the Glasgow and London areas in 1981. AB - Genome analysis was performed on 125 adenovirus isolates from conjunctival swabs of patients with conjunctivitis obtained in Glasgow between 1981 and 1984. A summer outbreak in 1981 was mainly due to species 3 adenoviruses, of which genotype 3GB and five different genotypic variants cocirculated. Three species 3 variants were also observed in 1982. The genome changes of variants were located on physical maps of the Ad3 reference strain and found to be clustered near the ends of the adenovirus DNA (including the fiber area), whereas the hexon coding region was unaltered. In contrast to the genome heterogeneity observed among the species 3 adenoviruses collected in Glasgow in 1981 it was found that all 69 Ad3 isolates obtained from an outbreak of pharyngoconjunctival fever in a boarding school near London during the summer of 1981 possessed the 3GB genotype. PMID- 3009698 TI - An in vitro reactivation system for investigation of herpes simplex virus latency. AB - An in vitro reactivation system for investigation of herpes simplex virus latency which is inexpensive, simple, and convenient to establish and operate is presented. The system is useful for investigation of compounds that may be involved in suppression or augmentation of herpes simplex virus reactivation. The uses of this model in exploring the mechanism of HSV reactivation, in screening for drugs which affect this process, and in testing their mode of action are discussed. PMID- 3009699 TI - Hepatitis A virus replication in tamarins and host immune response in relation to pathogenesis of liver cell damage. AB - Hepatitis A virus (HAV) shedding in the faeces, appearance of HAV-Ag (antigen) in the liver, and development of humoral immunity to HAV have been studied in experimentally infected tamarins. The appearance of liver damage measured by transaminase elevation and histology, in relation to the above variables, suggests that the virus is not cytopathic and the immune system contributes to the production of liver cell damage. Preliminary data suggest that HAV replication may occur in the mucosa of the small intestine and in the liver. PMID- 3009700 TI - Role of Calomys musculinus peritoneal macrophages in age-related resistance to Junin virus infection. AB - In nature, the cricetid Calomys musculinus is the principal host of Junin virus, the etiological agent of Argentine hemorrhagic fever. In the experimental infection, adult C. musculinus survived whereas newborns died after intraperitoneal inoculation with the XJ.Cl3 strain of Junin virus. The role of peritoneal macrophages in this age-related resistance was studied. Junin virus multiplied in cultivated macrophages from either neonatal or adult animals and, therefore, it was not possible to correlate the susceptibility of peritoneal macrophages to Junin virus infection with the age-dependent resistance. When adult and neonatal animals were treated with silica prior to Junin virus infection, deaths occurred in the adults, while a delay and decrease in the mortality rate were observed in neonatals. These results suggest that in neonatal C. musculinus macrophages could be permissive cells for Junin virus multiplication, whereas in adult cricetids, these cells would act as a barrier against viral infection by means of an extrinsic antiviral activity. PMID- 3009701 TI - Radioimmunoassay for detection of the Snow Mountain Agent of viral gastroenteritis. AB - The Snow Mountain Agent (SMA) is a Norwalk-like viral agent of acute gastroenteritis that has been detected only by immune electron microscopy (IEM). We established a solid phase microtiter radioimmunoassay (RIA) for SMA antigen employing pre-and post-challenge sera from volunteer studies as capture antibodies. SMA was detected in 12 of 67 stool samples from volunteers who were ill after oral challenge with SMA. All samples in which virus particles were detected by IEM were positive by RIA, but the RIA was 10-80 times more sensitive than IEM. To detect serum antibody to SMA, a blocking test was developed, employing diarrheal stool containing SMA as a standard source of antigen. Serum antibody rises were detected in eight out of nine volunteers with experimentally induced illness following challenge with SMA, as well as in three out of three naturally occurring cases. A preliminary sero-epidemiologic survey suggested that infection with SMA was common in the population surveyed. This RIA should permit large scale seroepidemiologic studies of SMA to be carried out, and should also facilitate characterization of this agent. PMID- 3009702 TI - Hepatitis A antibody in an isolated Amerindian tribe fifty years after exposure. AB - Antibody to hepatitis A in an Amerindian tribe was found in everyone over 50 years old but in no one younger. We suggest that the tribe had become infected with hepatitis A virus during the period, about 50 years earlier, when they engaged in raids on Luso-Brasilian settlers, that the virus failed to persist in the tribe when they withdrew into isolation, and that those who had been infected maintained antibody titers without boosting since that time. PMID- 3009703 TI - An inactivated hepatitis A viral vaccine of cell culture origin. AB - Hepatitis A virus (HAV) strain CR326, adapted to grow in LLC-MK2 cells, was highly purified, inactivated with formalin, adsorbed to alum, and tested for capacity to induce antibody to HAV in both mice and marmosets. The minimum dose of HAV antigen necessary to produce antibody in 50% of mice was 10 ng. As little as three doses of 1 ng each produced antibody in 50% of marmosets. Further, all marmosets with any detectable antibody to HAV, as a result of vaccination, were protected against virulent infection on challenge with HAV. Thus a highly efficacious, inactivated hepatitis A vaccine can be produced from virus grown in cell culture. Although LLC-MK2 cells are unacceptable for use in human vaccine preparation, HAV can also be prepared in a similar manner in MRC-5 cells, which are acceptable for human vaccine manufacture. PMID- 3009704 TI - Viral hepatitis markers in patients on haemodialysis in a hyperendemic area. AB - Antibody profiles for cytomegalovirus (CMV), hepatitis A virus (HAV), hepatitis B virus (HBV) and the delta-agent were determined on 55 serum samples drawn from 55 Saudi patients on maintenance haemodialysis for periods ranging from 1.5 months to 2 years. The exposure rates for CMV, HAV, and HBV were 100%, 100%, and 72.7%, respectively. There was no intersex difference in positivity for HBV surface antigen (HBsAg), antibody to HBsAg (anti-HBs), antibody to HBV core antigen (anti HBc); 15.4%, 65.4%, 3.8% in males and 6.9%, 55.2%, and 0% in females, respectively. Among six HBsAg carriers, one and three were positive for e antigen (HBeAg) and antibody to HBeAg (anti-HBe), respectively, with two negative for HBeAg and anti-HBe. The six carriers were also negative for anti-delta antibody. A comparison of the above antibody profile to the profile of voluntary blood donors and those seeking treatment for minor ailments in the local general hospital, obtained earlier using identical test procedures, revealed no difference for CMV and HAV exposure rate. The HBV exposure rate was higher in the haemodialysed patients (P less than 0.001). The epidemiological measures for preventing nosocomial viral hepatitis including immunisation of susceptibles, can be supplemented, among carriers, by interferon and acyclovir therapy for active viral replication. In HBV hyperendemic areas, haemodialysis patients exposed to HBV should be screened periodically for early signs of hepatocellular carcinoma. PMID- 3009705 TI - Experimental infection of Akodon molinae (Rodentia, Cricetidae) with Junin virus. AB - Experimental infection with the XJ-Clone 3 strain of Junin virus in laboratory bred Akodon molinae, a cricetid rodent inhabiting the borders of endemic Argentine hemorrhagic fever areas, was studied. Suckling animals inoculated intracerebrally proved sensitive and became chronically infected. Sixty percent of the rodents showed neurologic involvement, with mortality reaching 60%. Virus was recovered from the brain at 7, 15, 21, 37, and 57 days postinfection (pi). By immunofluorescence (IF), viral antigens were observed up to 182 days pi in cells of the central nervous system (CNS). Concurrently, immunoglobulin deposits were detected in infected CNS cells from 21 up to 182 days pi. These deposits increased with the progression of the immune response as measured by IF antibodies. The detection of immune complexes in brain cells of apparently healthy animals suggests that neither viral replication nor the development of a humoral immune response are necessary requisites for neurovirulence in this host. Infection of adult rodents by different routes failed to induce disease or mortality and virus could not be recovered from oral swabs, blood, or organs. Our data suggest that Akodon molinae could play a role in nature as an alternative reservoir of Junin virus in addition to the main reservoir, Calomys musculinus. PMID- 3009707 TI - Mononuclear subsets during cytomegalovirus disease in renal transplant recipients treated with cyclosporine and rabbit antithymocyte globulin. AB - The effect of cytomegalovirus (CMV) disease on mononuclear subpopulations of 49 renal transplant recipients treated with cyclosporine and prednisone are reported. Clinical overt CMV infection developed in 8/21 patients treated for rejection with rabbit antithymocyte globulin. They all showed true inversions of the T helper/T suppressor-cytotoxic (Th/Ts-c) ratio. A reduction of T helper cells and increase in T suppressor-cytotoxic cells preceded clinical symptoms of CMV disease by one week. None of the 26 patients without RATG anti-rejection treatment developed CMV disease and in only three of them an inversion of Th/Ts-c ratio was found. PMID- 3009706 TI - Junin virus access to CNS by extraneural rat inoculation. AB - This study was carried out to determine the pathways along which two strains of Junin virus (JV), the pathogenic XJV and the attenuated XJC13V, reach the CNS following IP inoculation of 2-day-old rats. A sequential study of infectivity and antigen distribution in peritoneal macrophages, spleen, and brain was performed. Mortality was 85% with the former strain, but only 15% with the latter. At 4-7 days PI, XJV-infected animals had viral antigen in 10% of peritoneal macrophages. Viremia and spleen virus lasted for 10-15 days. Low brain titers were detected at day 7, with a peak at day 15. Brain antigen correlated with virus titers. In contrast, XJC13V-infected rats, macrophage antigen appeared later and to a lesser degree (1% of cells). Viremia and spleen virus were transient, while both the titer of brain virus and the viral antigen proved lower. Antibody titers were over twofold higher for XJ-infected animals. It is suggested that the different replication rate at the inoculation site could account for the greater ability of the XJV strain to reach the CNS. A greater antigen mass and/or more numerous antigenic determinants presented by the macrophage could explain the higher antibody titers found in XJ-injected rats, which were unable, however, to prevent viral spread. PMID- 3009708 TI - Marijuana's effects on human cognitive functions, psychomotor functions, and personality. AB - Marijuana is complex chemically and not yet fully understood, but it is not a narcotic. Like alcohol, marijuana acts as both stimulant and depressant, but it lingers in body organs longer than alcohol. Smoking marijuana can injure mucosal tissue and may have more carcinogenic potential than tobacco. Research has indicated that marijuana intoxication definitely hinders attention, long-term memory storage, and psychomotor skills involved in driving a car or flying a plane. Expectations and past experience with marijuana have often influenced results more than pharmacological aspects have. Marijuana has triggered psychotic episodes in those more vulnerable. Psychological and some instances of physiological dependence on marijuana have been demonstrated. As a psychoactive drug, marijuana surely alters mental functioning. Although it is possible that chronic use of marijuana produces irreversible damage to mind or brain areas, this has not been determined by research. PMID- 3009709 TI - Actions of the beta-carboline ZK 93426 in an animal test of anxiety and the holeboard: interactions with Ro 15-1788. AB - The effects of the beta-carboline ZK 93426, a putative benzodiazepine receptor antagonist, were investigated in the social interaction test of anxiety and in the holeboard. Like the receptor antagonist Ro 15-1788, ZK 93426 (2.5-10 mg/kg) caused a specific reduction in social interaction (interpreted as an anxiogenic effect) and caused a significant elevation in exploratory head-dipping (5 mg/kg). When low (ineffective) doses of both compounds (1 mg/kg ZK 93426; 4 mg/kg Ro 15 1788) were administered together they significantly reduced social interaction. No further reductions in social interaction were observed when effective doses of both compounds (5 mg/kg ZK 93426; 10 mg/kg Ro 15-1788) were tested in combination; it is likely that this is due to almost total benzodiazepine receptor occupancy at effective doses of either compound. When doses of each compound (5 mg/kg ZK 93426; 10 mg/kg Ro 15-1788) that resulted in stimulation of head-dipping were examined in combination, the elevation in exploration was no longer observed. Since at higher doses of both compounds there is an attenuation of the elevation in head-dipping, it is again likely that the effects of the two compounds are additive. PMID- 3009710 TI - Equol and other compounds from bovine urine as monoamine oxidase inhibitors. AB - Equol, its methylated derivative, and a carbazole, all isolated from bovine urine, are relatively potent inhibitors of monoamine oxidase with IC50 values of 158, 28, and 16 microM respectively (using 83 microM tyramine as substrate). The probable dietary origin of these compounds suggests that "natural" monoamine oxidase inhibitors may be more widespread than had previously been suspected. PMID- 3009711 TI - What is the correct value for Na, K-ATPase activity in homogenates of cerebral cortex? AB - Activity of Na, K-ATPase in homogenates of fresh cerebral cortex of rats was compared with that of cortex frozen under different conditions. Activity yields after rapid in situ freezing of the exposed cerebral cortex were twice, higher (26.1 U) than in homogenates of the fresh cortex (13.3 U). Fresh brain kept on ice for 60 and 300 s and subsequently frozen in liquid nitrogen yielded activities comparable to those of the tissue frozen in situ (24.1 U and 24.9 U for 60 s and 300 s periods, respectively). Inhibition of Na, K-ATPase by 10(-7) M vanadate was significantly stronger (38%) in homogenates of the fresh brain then in those of the cortex frozen in situ (28%). High Na, K-ATPase activity (47.6 U) in suspensions of synaptosomal membranes (SM) prepared from fresh cortical homogenates was only slightly inhibited by 10(-7) M vanadate (12%). Various treatments of homogenates or SM suspensions, like increase of piston rotation speed, repeated freezing and thawing procedure or vigorous shaking did not significantly affect the enzyme activity. Mg-ATPase activity and its sensitivity to vanadate was also modified by tissue treatment but the effect was much less pronounced. PMID- 3009712 TI - Thiol-induced hydroxyl radical formation and scavenger effect of thiocarbamides on hydroxyl radicals. AB - The effects of thiols and thiocarbamides on hydroxyl radical (.OH) formation by the hypoxanthine(HYP)-xanthine oxidase(XOD)-Fe3+ .EDTA system were investigated in the range of 0.5-5 mM by colorimetrically measuring salicylate hydroxylation. Thiocarbamides powerfully inhibited the hydroxylation while thiols showed a paradoxical effect, enhancing it at low concentrations, but inhibiting it at high ones. Thiols in the presence of Fe3+ .EDTA generated superoxide anions (O2-.) and .OH during the oxidation, but thiocarbamides did not. A study of the effect of ergothioneine, a thiocarbamide present in mammals, on the .OH spin adduct of 5,5 dimethyl-1-pyrroline-N-oxide(DMPO) by EPR spectrometry showed that it effectively decreased the .OH spin adduct without causing the appearance of other signals. Reaction mechanisms are proposed for the O2-. evolution and .OH formation by the thiols themselves in the presence of Fe3+ .EDTA and .OH with thiols and thiocarbamides. PMID- 3009713 TI - Relatively long-lived chromium(V) species are produced by the action of glutathione on carcinogenic chromium(VI). AB - X-Band EPR studies on aqueous solutions of potassium dichromate and gamma-L glutamyl-L-cysteinylglycine (reduced glutathione) at pH 6-8 have shown the formation of several relatively long-lived chromium(V) species. The major species formed at high glutathione:Cr(VI) ratios is characterised by an EPR band at g = 1.995, but the dominant complex at equimolar ratios produces a signal at g = 1.985. PMID- 3009714 TI - Electroconvulsive shock and cyclic AMP signal transduction: effects distal to the receptor. AB - Chronic electroconvulsive shock (ECS) induced a significant decrease in noradrenaline- and forskolin-stimulated cyclic AMP production in rat cortical slices, whereas a single ECS had a much smaller effect. In a cortical membrane preparation, adenylate cyclase activity in response to stimulation by forskolin, guanosine-5'-(beta,gamma-imido) triphosphate, and Mn2+ ions was significantly increased in membranes derived from rats that had received chronic ECS, but was either unchanged or reduced in membranes from rats that received a single treatment only. The results are interpreted in terms of changes occurring at components of the adenylate cyclase enzyme distal to the receptor. PMID- 3009715 TI - Phosphorylation of synaptic membrane glycoproteins: the effects of Ca2+ and calmodulin. AB - Synaptic membranes were incubated with [gamma-32P]ATP, and glycoproteins were isolated by affinity chromatography on concanavalin A agarose. Glycoproteins accounted for 1.5-2.5% of the total 32P incorporated into synaptic membrane proteins. Ca2+ and calmodulin enhanced the phosphorylation of synaptic membrane glycoproteins approximately threefold. In the presence of Ca2+ and calmodulin, the rate of glycoprotein dephosphorylation was also increased three- to four fold. Gel electrophoretic analysis identified several synaptic membrane glycoproteins that incorporated 32P, with the most highly labeled glycoprotein under basal phosphorylating conditions having an apparent Mr of 205,000 (gpiii). Ca2+ and calmodulin produced a marked increase in the phosphorylation of a glycoprotein with an apparent Mr of 180,000 (gpiv) and lesser increases in the labeling of three other glycoproteins. Membranes that had been labeled with [gamma-32P]ATP were extracted with Triton X-100 under conditions that yield a detergent-insoluble residue enriched in postsynaptic structures. The Triton X-100 insoluble residue accounted for 20-25% of the 32P associated with synaptic membrane glycoproteins. Gpiv and other glycoproteins, the phosphorylation of which was stimulated by calmodulin, were located exclusively in the Triton X-100 insoluble residue, whereas gpiii and other calmodulin-insensitive glycoproteins partitioned predominantly into the Triton X-100-soluble fraction. Phosphopeptide maps and phosphoamino acid analysis of gpiv isolated from synaptic membranes and a postsynaptic glycoprotein of apparent Mr of 180,000 (gp180) isolated from synaptic junctions indicated that the former protein was identical to the previously identified postsynaptic-specific gp180. In addition to phosphoserine and phosphothreonine, gpiv also contained phosphotyrosine, identifying it as a substrate for tyrosine-protein kinase as well as for Ca2+/calmodulin-dependent protein kinase. PMID- 3009716 TI - GABAB receptor-mediated enhancement of vasoactive intestinal peptide-stimulated cyclic AMP production in slices of rat cerebral cortex. AB - Basal and vasoactive intestinal peptide (VIP)-stimulated accumulations of cyclic AMP were measured in slices of rat cerebral cortex. Neither gamma-aminobutyric acid (GABA) nor the selective GABAB receptor agonist (-)-baclofen stimulated basal cyclic AMP accumulation, whereas VIP caused a large dose-dependent increase in cyclic AMP levels. However, in the presence of 100 microM (-)-baclofen, the effects of VIP on cyclic AMP accumulation were significantly enhanced, with the responses to 1 microM and 10 microM VIP being approximately doubled. The enhancing effects of (-)-baclofen was dose related (1-1,000 microM), but an enhancing effect was not observed with 100 microM (+)-baclofen. In the presence of the GABA uptake inhibitor nipecotic acid (1 mM), GABA caused a similar dose related enhancement of the VIP response. The ability of either GABA or (-) baclofen to augment VIP-stimulated production of cyclic AMP was not mimicked by the GABAA, agonists isoguvacine and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3 ol (THIP) and was not antagonized by the GABAA antagonist bicuculline. The putative GABAB antagonist 5-aminovaleric acid (1 mM) significantly reduced the effect of (-)-baclofen. The ability of (-)-baclofen to enhance VIP-stimulated accumulation of cyclic AMP was observed in slices of rat cerebral cortex, hippocampus, and hypothalamus. These results indicate that GABA and (-)-baclofen can enhance VIP-stimulated accumulation of cyclic AMP in rat brain slices via an interaction with specific GABAB receptors. PMID- 3009717 TI - An assessment of the role of opioid receptors in the response to cannabimimetic drugs. AB - Cannabimimetic drugs have been shown to inhibit adenylate cyclase activity in N18TG2 neuroblastoma cells. This investigation examines the possible role of opioid receptors in the cannabimimetic response. Opioid receptors of the delta subtype were found on N18TG2 membranes using [3H]D-Ala2-D-Leu5-enkephalin. No mu or kappa receptors were detected using selective ligands for these sites. The delta binding affinity and capacity were unaltered by cannabimimetic drugs. To test if cannabimimetic drugs may modulate opioid effector mechanisms, cyclic AMP metabolism was determined in intact cells and in membranes. N18TG2 adenylate cyclase was inhibited by the cannabimimetic drugs delta 9-tetrahydrocannabinol and desacetyllevonantradol, and by the opioid agents morphine, etorphine, and D Ala2-Met5-enkephalinamide. The opioid inhibition was reversed by naloxone and naltrexone; however, the cannabimimetic response was unaffected. Both cannabimimetic and opioid drugs decreased cyclic AMP accumulation in intact cells, but opioid antagonists blocked the response only to the latter. Thus, cannabimimetic effects are observed even though opioid receptors are blocked by antagonist drugs. The interaction between desacetyllevonantradol and etorphine was neither synergistic nor additive at maximal concentrations, suggesting that these two drugs operate via the same effector mechanism. Other neuronal cell lines having an opioid response were also examined. The cannabimimetic inhibition of cyclic AMP accumulation in NG108-15 neuroblastoma X glioma cells was not as great as the response in N18TG2. N4TG1 neuroblastoma cells did not respond to cannabimimetic drugs under any conditions tested. Thus, the cannabimimetic inhibition of adenylate cyclase is not universally observed, and the efficacy of the cannabimimetic response does not correlate with the efficacy of the opioid response. PMID- 3009718 TI - Secretin stimulates cyclic AMP formation in the rat brain. AB - The effects of secretin on cyclic AMP levels in the rat brain were determined. Incubation of rat brain frontal cortex slices with secretin or the structurally related peptides peptide histidine leucine (PHI) or vasoactive intestinal polypeptide (VIP) in the presence of 10 mM theophylline resulted in a dose dependent increase in the cyclic AMP levels. The half-maximal increase in cyclic AMP occurred using a 1 microM dose of secretin or a 2 microM dose of PHI or VIP. Preincubation of slices with secretin-(5-27) produced a dose-dependent inhibition of the secretin but not VIP- or PHI-stimulated increase in the cyclic AMP content. Also, in receptor binding studies, secretin-(5-27) produced a dose dependent inhibition (Ki = 400 nM) of 125I-secretin but not of 125I-VIP binding to rat brain membranes. Guanyl-5'-yl imidodiphosphate decreased the affinity of radiolabelled secretin binding as a result of an increased rate of dissociation of bound 125I-secretin. These data suggest that secretin receptors in the rat brain may be coupled to adenylate cyclase in a stimulatory manner and that secretin-(5-27) may function as a central secretin receptor antagonist. PMID- 3009719 TI - Antimalarial agents, 2. Artesunate, an inhibitor of cytochrome oxidase activity in Plasmodium berghei. AB - The activity of the cytochrome oxidase, which is located in the plasma and the nuclear and the food-vacuole-limiting membranes as well as in the mitochondria of the trophozoites of Plasmodium berghei, was inhibited completely by sodium artesunate, an antimalarial drug, in vitro at 1 mM and in vivo at 100 mg/kg iv. This enzyme appears to be a target for the antimalarial mechanism of action of artesunate and qinghaosu. PMID- 3009720 TI - Granulocyte dysfunction and myotonic dystrophy. AB - A 52-year-old Caucasian male with typical features of myotonic dystrophy (MD) developed a lung abscess and was found to have a mild atypical cyclic neutropenia. Granulocyte function testing revealed a defect in phagocytosis, bactericidal activity and chemotaxis. The defects were less severe at the nadir of the granulocyte counts. Skin windows demonstrated that the granulocyte defects were not just an in vitro artifact. The patient was treated with lithium carbonate and improved. Mobilization into a skin window and clinical MD were unchanged. Studies of his 10 children and 2 siblings, including granulocyte function tests and complete neurological evaluations were obtained. The 4 children with abnormal parameters of granulocyte function all had definite evidence of MD. Two children had equivocal findings of MD and the others were normal. There was minimal evidence of granulocyte dysfunction in these children. Twelve of 19 unrelated patients with MD had evidence of impaired granulocyte function with the most consistent defect being chemotaxis in response to bacterial factor. Mild granulocyte dysfunction is frequently associated with MD, but severe dysfunction with many defects is uncommon but can occur, as in this family. There was a tendency for the more severely afflicted members of this family to have more pronounced granulocyte dysfunction. Longitudinal testing in this family may determine any relationship between the granulocyte dysfunction and the onset of MD, as well as any correlation with the progression of the disorder. MD patients who develop infection should have granulocyte function tests as part of their evaluation. PMID- 3009721 TI - Antibody against mouse liver 5'-nucleotidase immunostains white matter in the adult mouse central nervous system. AB - An antiserum against rat liver 5'-nucleotidase has been shown to immunostain myelinated fibers and oligodendrocytes in the rat CNS, consistent with evidence for 5'-nucleotidase activity in rat brain myelin and oligodendrocytes (Cammer, Sacchi and Kahn, Devel. Brain Res., 1985, 20: 89-96). However, in the mouse CNS, in which myelin also has 5'-nucleotidase activity, that antiserum stained only blood vessels. To obtain an antibody against the mouse enzyme, 5'-nucleotidase was partly purified from mouse liver membranes by detergent extraction, heat treatment, affinity chromatography, acidification, and ammonium sulfate fractionation. The preparation, which was enriched about 110-fold in 5' nucleotidase specific activity, compared to the starting extract, was electrophoresed on a preparative slab gel containing Triton X-100, a strip was stained histochemically for 5'-nucleotidase, and the material corresponding to the stained band was used to immunize a rabbit. The immune IgG fraction, but not the preimmune IgG, reacted with mouse brain homogenates. The immune serum gave consistently greater inhibition of 5'-nucleotidase activity in mouse liver homogenates, mouse brain myelin and mouse brain homogenates, but not rat brain or liver homogenates, compared to the preimmune serum. The immune serum, but not the preimmune serum, immunostained white matter in the normal adult mouse brain and spinal cord. The findings suggest that the mouse may have one isozyme of 5' nucleotidase similar to that in rat with respect to subunit sizes but differing in primary structure at one or more antigenic sites and support previous observations of 5'-nucleotidase activity in myelin from mouse brains and spinal cords. PMID- 3009722 TI - Immunological treatment of multiple sclerosis. II. PMID- 3009723 TI - Neuropathy of vasculitic origin in a case of Garin-Boujadoux-Bannwarth syndrome with positive borrelia antibody response. AB - A 42-year-old man suffered from erythema chronicum migrans on different parts of the body after repeated tick bites. A few months after the last tick bite he developed a painful neuropathy in both legs with patchy disturbance of sensibility, mild weakness of the feet and loss of the right ankle jerk. Repeated determinations of antibodies against borrelia spirochetes revealed increasing IgG titres. Biopsy of the left sural nerve, which was clinically and electrophysiologically affected, showed a vasculitis of epineurial vasa nervorum and severe angiopathic lesions of the perineurium and the neural parenchyma. Parenteral high-dose penicillin treatment resolved the clinical symptoms. PMID- 3009724 TI - Thalidomide-induced peripheral neuropathy. A prospective clinical, neurophysiological and pharmacogenetic evaluation. AB - Prospective clinical and electrophysiological follow-up was performed on nine patients under thalidomide treatment in order to detect the very beginning of possible drug-induced peripheral neuropathy. For neurophysiological assessment, nerve conduction measurements of the median, peroneal and sural nerves (7 conduction parameters) and needle EMG examination of the anterior tibial muscle were performed. The results of a first control after about 3 months of treatment were compared with the starting point examination, and the patients were then classified as "affected" or "not affected" according to clinical and neurophysiological criteria. At this point, three patients showed clinical and electrophysiological, and another two only electrophysiological alterations suggesting early neuropathy. This classification did not change after further clinical and electrophysiological controls. Without starting-point values, the early detection of neuropathy would not have been possible in all patients. No single reliable neurophysiological parameter for detection of thalidomide-induced neuropathy could be found. Pharmacogenetic classification with regard to hydroxylation and acetylation phenotypes was then performed in some patients and interpreted with relation to thalidomide neurotoxicity. A possible relationship between slow acetylators and development of thalidomide-induced neuropathy was found. PMID- 3009725 TI - Intensive 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) monochemotherapy and autologous marrow transplantation for malignant glioma. AB - Intensive monochemotherapy with carmustine (BCNU) (either 1,050, 1,200, or 1,350 mg/m2) and cryopreserved autologous marrow transplantation was administered to 36 patients with malignant glioma: 27 with progressive disease and nine without progression (adjuvant therapy group). Twelve (44%) of the patients with progressive disease responded; two remain disease free 84 and 60 months after BCNU treatment. In the adjuvant therapy group, three patients remain progression free at 70, 48, and 27 months after BCNU therapy. Tumor progression posttransplantation occurred in 25 patients; six others died of therapy-induced complications. In addition, late neurologic deterioration of unknown cause has developed in two surviving patients. Results from this and other series using intensive BCNU monochemotherapy and autologous marrow transplantation for progressive malignant glioma indicate that prolonged progression-free survival can be produced in an occasional patient, an extremely unusual result with conventional chemotherapy. Although intensive BCNU and autologous marrow transplant regimens are toxic, these results are encouraging. The treatment of patients in an adjuvant fashion with BCNU and other active agents may produce improved results. PMID- 3009726 TI - Surgical adjuvant therapy for stage II and stage III adenocarcinoma and large cell undifferentiated carcinoma. AB - The Lung Cancer Study Group randomized 141 patients with resected stage II and III adenocarcinoma and large-cell undifferentiated carcinoma to receive postoperative Cytoxan (Bristol-Meyers, Syracuse, NY), Adriamycin (Adria Laboratories, Columbus, Ohio), and cisplatin (CAP) chemotherapy or bacillus Calmette-Guerin (BCG) and levamisole immunotherapy. Careful intraoperative staging was performed on all patients. Before randomization, patients were stratified by stage, weight loss, cardiac arrhythmia, and institution. Prognostic variables such as stage, age, weight loss, and nodal involvement were equally distributed between the two groups. Disease-free survival was significantly prolonged in the group receiving chemotherapy. There was no evidence of a deleterious effect of the immunotherapy. This study indicates that postoperative CAP chemotherapy is effective in prolonging disease-free survival in these patients. PMID- 3009728 TI - Altered synaptic transmission in dentate gyrus of rats reared in complex environments: evidence from hippocampal slices maintained in vitro. AB - Pre- and postsynaptic responses to activation of medial perforant path (MPP) axons were examined in hippocampal slices taken from rats reared for 3-4 wk in relatively complex (EC) or individual cage (IC) environments. Three types of extracellular field potentials were recorded in the infrapyramidal blade of the dentate gyrus: 1) granule cell population spikes (PSs), which reflect the number and synchrony of discharging granule cells (2), 2) population excitatory postsynaptic potentials (EPSPs), which reflect the amount of excitatory synaptic current flow into dendrites (28), and 3) presynaptic fiber volleys (FVs), which reflect the number of activated axons (28). Stimulation of the MPP evoked significantly larger PSs in slices taken from EC rats. There was no significant effect of rearing environment on PS/EPSP relationships. The slopes of EPSPs recorded at the site of synaptic activation in the dentate molecular layer and at the major current source in the dentate granule cell layer were significantly greater in slices taken from EC rats. The presynaptic FV was recorded at the site of synaptic activation in the molecular layer. FV amplitude did not differ significantly as a function of rearing environment. To examine possible differences in tissue impedance, granule cells were activated by stimulating granule cell axons in the dentate hilus and recording the antidromic PS in the granule cell layer. Antidromic PS amplitude was not significantly affected by rearing environment. The relative permanence of the experience-dependent alterations in synaptic transmission was assessed by comparing slices taken from rats that had been reared for 4 wk in complex environments followed by 3-4 wk in individual cages with those from rats reared for 7-8 wk in individual cages. There were no significant differences in MPP synaptic transmission between these groups of animals. The results suggest that experience in a relatively complex environment is associated with greater MPP synaptic transmission arising from an increased synaptic input to granule cells; the greater MPP synaptic transmission associated with behavioral experience can occur independent of behavioral state, influences from extrahippocampal brain regions and intrahippocampal inhibitory activity; and the experience-dependent synaptic alterations in the dentate gyrus are transient, in contrast to experience-dependent morphological alterations described in occipital cortex. The possible relationship of these alterations to the phenomenon of long-term enhancement is discussed. PMID- 3009727 TI - Impaired in vitro polymorphonuclear function secondary to the chemotherapeutic effects of vincristine, adriamycin, cyclophosphamide, and actinomycin D. AB - The present study investigated the in vitro effect of four different chemotherapeutic agents, namely, cyclophosphamide (CTX), vincristine (VCR), Adriamycin (Adria Laboratories, Columbus, Ohio) (ADR), and actinomycin D (ACT-D) on human polymorphonuclear leukocyte (PMN) function. Human PMNs suspended in phosphate-buffered saline (PBS) at 1 X 10(7) cells/mL were incubated with increasing concentrations of CTX (0, 10(-5), 10(-4), 10(-3) mol/L) or VCR (0, 10( 7), 10(-6), 10(-5), 10(-4) mol/L), ADR (0, 10(-6), 10(-5), 10(-4), 10(-3) mol/L), or ACT-D (0, 5 X 10(-8), 1 X 10(-7), 5 X 10(-7), and 10(-6) mol/L). The cells were then tested for bacterial killing against Staphylococcus aureus, chemotaxis activity stimulated by Escherichia coli endotoxin, N-formyl-methionyl-leucyl phenylalanine (FMLP)-stimulated aggregation, and cytochalasin B (Cyto B)/FMLP stimulated superoxide production and enzyme degranulation. High concentration of CTX, an alkylating agent, showed a significant depression of PMN superoxide production, (124 +/- 13 v 161 +/- 15 nmol/10(7) cells, 5 minutes, P less than or equal to .025). ADR, an intercalating agent and membrane inhibitor, showed a significant depression of PMN degranulation and lysozyme release at 10(-4) and 10(-3) mol/L (15.3% +/- 1.7% v 24% +/- 7%, P less than .01; and 15.0% +/- 2.5% v 24% +/- 7%, P less than or equal to .025). VCR, a microtubule inhibitor, showed a significant depression of PMN aggregation at 10(-6), 10(-5), and 10(-4) mol/L (P less than .05), lysozyme release at 10(-4) mol/L (P less than .004), and beta glucuronidase release at 10(-4) mol/L (P less than .004). In addition, chemotaxis was inhibited by VCR in a dose-dependent manner at all concentrations (10(-7) mol/L, P less than .02; 10(-6) mol/L, P less than .007; 10(-5) mol/L, P less than .006, and 10(-4) mol/L, P less than .003). ACT-D showed no significant effect on the PMN functions tested. These studies conclude that chemotherapeutic agents have modulating in vitro effects on PMN function. Further in vivo studies are therefore needed to assess PMN abnormalities in patients receiving cancer chemotherapy to determine their role in infectious complications. PMID- 3009729 TI - Rat hippocampal neurons in culture: Ca2+ and Ca2+-dependent K+ conductances. AB - Rat hippocampal neurons grown in dissociated cell culture were studied in a medium containing 1 microM tetrodotoxin (TTX) and 25 mM tetraethylammonium (TEA), which eliminated the Na+ and K+ conductances normally activated by depolarizing current injections. In this medium depolarizing current pulses evoked depolarizing regenerative potentials and afterhyperpolarizations in most cells. Both of these events were blocked by close application of Co2+ or Cd2+. These events resemble Ca2+ spikes reported previously in hippocampal pyramidal cells. The membrane potential at which these Ca2+ spikes could be triggered and the rheobase current necessary were dependent on the potential at which the cell was conditioned: the more depolarized the holding potential, the more negative the absolute potential at which a spike could be triggered and the less rheobase current required. The duration of these Ca2+ spikes was also sensitive to the holding potential: the more depolarized the holding level, the longer the duration of the triggered spikes. The amplitude and duration of the Ca2+ spikes were enhanced in a reversible manner by 0.5-1.0 mM 4-aminopyridine (4-AP) delivered in the vicinity of the cell. Two-electrode voltage-clamp analysis of cells studied in TTX, TEA-containing medium revealed an inward current response that peaked in 25-50 ms during depolarizing commands. This response first became detectable during commands to -30 mV. It peaked in amplitude during commands to 10 mV and was enhanced in medium containing elevated [Ca2+]0. It was blocked by either 20 mM Mg2+, 0.2 mM Cd2+, 5 mM Co2+, or 5 mM Mn2+. These results have led us to identify this inward current response as ICa2+. 4-AP enhanced the magnitude and duration of ICa2+ independent of the drug's depressant effects on a transient K+ current also observed under these same experimental conditions. In many but not all cells the Ca2+ spike was followed by a long-lasting hyperpolarization associated with an increase in membrane conductance. This was blocked by Co2+. Under voltage clamp ICa2+ was followed by a slowly developing outward current response that was attenuated by Co2+ or Cd2+. These properties observed under current- and voltage-clamp recording conditions are superficially similar to those previously reported for Ca2+-dependent K+ conductance mechanisms (IC) recorded in these and other membranes. Long-lasting tail currents following activation of IC inverted in the membrane potential range for the K+ equilibrium potential found in these cells. PMID- 3009730 TI - Synaptic functions in rat sympathetic neurons in microcultures. II. Adrenergic/cholinergic dual status and plasticity. AB - This is the second in a series of papers that describes the use of a sensitive microculture procedure to investigate the transmitter status of sympathetic neurons. Cultured immature principal neurons, dissociated from the superior cervical ganglia of newborn rats, are known to be plastic with respect to transmitter status; under certain culture conditions, populations of neurons that display (at least) adrenergic properties at the outset can be induced to display a variety of cholinergic properties, including the formation of functional neuron neuron cholinergic synapses, as adrenergic properties decline. With the microculture procedure described in the preceding paper (Furshpan et al., 1986a), we have examined the transmitter status of individual neonate-derived neurons during this transition. Many such neurons secreted both norepinephrine and ACh (adrenergic/cholinergic dual function); examination of such neurons with the EM revealed a mixed population of synaptic vesicles. Direct evidence for a transition via this dual status was obtained by serial physiological assays of 14 neurons. The neonate-derived neurons were markedly heterogeneous in the rate of change of transmitter status. Principal neurons derived from adult superior cervical ganglia also displayed dual status, but the incidence was lower than in neonate-derived neurons cultured for similar periods. In preliminary serial assays of adult-derived neurons, many of the neurons did not acquire detectable cholinergic function, but in two cases evidence consistent with plasticity was obtained. While it is known that several types of neurons will form functional junctions in the presence of agents that block electrical activity, sympathetic principal neurons have apparently not been tested. In microculture, neuron-neuron synapses and junctions with cardiac myocytes were formed by sympathetic neurons grown chronically in the presence of blocking concentrations of TTX and hexamethonium. PMID- 3009731 TI - Purification of a human red blood cell protein supporting the survival of cultured CNS neurons, and its identification as catalase. AB - We have previously reported that red blood cells contain high levels of a protein that supports the survival of a variety of CNS neurons in vitro for 24 hr. Here we report the isolation of this trophic activity from human red blood cells. The active material, purified over 1000-fold by ion-exchange chromatography and isoelectric point, subunit molecular weight, and ability to degrade hydrogen peroxide. Commercially produced bovine liver catalase, lactoperoxidase, HRP, and vitamin E all mimic the ability of the purified human protein to support neuronal survival in vitro. Pharmacological inhibitors of peroxidase activity inhibit the trophic effects of both commercial catalase and the purified blood-derived protein. These results suggest that peroxidase activity mediates the neuronotrophic activity of these agents. The bioassay culture medium itself generates peroxides in the absence of cells. Removal of this toxic material may be the basis for the trophic effects of catalase. PMID- 3009732 TI - Response properties of horizontal cells in the isolated retina of wild-type and pearl mutant mice. AB - Intracellular recordings were made from axon bearing horizontal cells in isolated retinas (with retinal pigment epithelium removed) of normal and pearl mutant mice superfused with mammalian Ringer's solution. Cells were injected with Lucifer yellow and identified by their morphology and their response wave-forms. On impalement, we measured a dark, resting voltage of 25-35 mV. All cells had similar spectral sensitivities that were consistent with the CIE scotopic relative spectral luminosity function. For stimuli of high irradiance, two types of responses could be distinguished, based on their waveform at stimulus offset. One consisted of a rapid and wavelength-dependent repolarization followed by a small, slow hyperpolarization. The other consisted of a large, long-lived, and wavelength-independent after-hyperpolarization. The former were recorded from the somatic end and the latter from the axon terminal arborization. The spatial distribution of sensitivity was measured in over 100 locations within the receptive field using small stimuli. The area within which sensitivity of the soma was within 0.1 log unit of the maximal sensitivity was larger than that of the soma dendritic field, but the sizes were nearly equal for the terminal arborization. No secondary maximum of sensitivity was noted over the dendritic field of the unimpaled part of the cell. For the terminal arborization, the half saturating irradiance for diffuse 502 nm stimuli was about 180 photons micron-2 sec-1, about one-tenth that for the soma. These values are in good agreement with those for cat and rabbit.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009733 TI - Parallel antagonism of synaptic transmission and kainate/quisqualate responses in the hippocampus by piperazine-2,3-dicarboxylic acid analogs. AB - A new series of potent antagonists of excitatory neurotransmission in the rat hippocampus has been identified. These derivatives of piperazine-2,3 dicarboxylate (PzDA) include the most potent acidic amino acid antagonists yet described for Schaffer collateral-commissural EPSPs. These antagonists also effectively block excitatory synaptic responses recorded in the lateral and medial perforant pathways and in the mossy fiber pathway. The PzDA derivatives also block focal depolarizations produced by kainate, quisqualate, and N-methyl-D aspartate. N-methyl-D-aspartate responses are more susceptible to inhibition by PzDA derivatives, although the spectrum of antagonism of N-methyl-D-aspartate and synaptic responses by PzDA derivatives is not parallel. However, the antagonism of kainate and quisqualate responses by PzDA derivatives shows the same rank order of potency as synaptic responses. These data indicate that synaptic receptors in the hippocampus have a pharmacologic profile similar to that of kainate or quisqualate receptors. PMID- 3009735 TI - Maxillary progonoma of infancy. PMID- 3009734 TI - PCPP-260, a Purkinje cell-specific cyclic AMP-regulated membrane phosphoprotein of Mr 260,000. AB - The present study reports the existence of Purkinje cell-specific phosphoprotein, Mr 260,000 (PCPP-260), a neuronal membrane phosphoprotein, in cerebellar Purkinje cells. PCPP-260, which on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has an apparent molecular mass of 260,000 Da, has been found to be phosphorylated in particulate preparations by endogenous or added exogenous cyclic AMP-dependent protein kinase, but not by cyclic GMP-dependent, calcium/calmodulin-dependent or calcium/phospholipid-dependent protein kinases. The protein has been found in high concentrations in all mammalian cerebella so far analyzed, including human cerebellum. One-and two-dimensional electrophoretic and peptide mapping analyses of proteins in other brain regions show that a closely related 265,000 Da phosphoprotein also exists, albeit in low concentrations, outside the cerebellum. Analysis of cerebella from mutant mice, deficient in either Purkinje cells or in granule cells, indicates that PCPP-260 within the cerebellum is restricted to Purkinje cells. Furthermore, subcellular fractionation of rat cerebella indicates that the protein is an integral membrane protein. The cAMP-regulated phosphorylation of PCPP-260 is presumably involved in membrane functions important to Purkinje cells. PMID- 3009736 TI - Superoxide production in experimental brain injury. AB - The appearance of superoxide anion radicals in cerebral extracellular space during and after experimental fluid-percussion brain injury was investigated in anesthetized cats equipped with cranial windows. Superoxide was detected by demonstrating the presence of superoxide dismutase (SOD)-inhibitable reduction of nitroblue tetrazolium (NBT). The SOD-inhibitable rate of reduction of NBT was 3.52 +/- 0.72 nM/min/sq cm during brain injury and 4.11 +/- 0.74 nM/min/sq cm 1 hour after injury. No significant superoxide production was detected in control animals. The sustained arteriolar dilation and reduced responsiveness to the vasoconstrictor effects of arterial hypocapnia observed 30 minutes after brain injury were eliminated by after-treatment with topical SOD (60 U/ml) and catalase (40 U/ml). The results show that experimental brain injury causes the generation and appearance in extracellular fluid space of superoxide. Superoxide production continues for at least 1 hour following injury. The sustained dilation and abnormal responsiveness of cerebral arterioles after injury are due to the continued generation of superoxide and other radicals derived from it. These functional changes can be reversed by after-treatment with appropriate scavenging agents. PMID- 3009737 TI - Intracranial tumors in patients with facial pain. AB - Over the past 10 years, 2000 patients with facial pain have been evaluated at the Mayfield Neurologic Institute. Sixteen of these patients were found to harbor intracranial tumors. The presenting features of this group of patients are analyzed, and the literature reviewed. Peripherally placed tumors tend to cause atypical facial pain associated with sensory loss. Middle fossa tumors may present as trigeminal neuralgia, but usually cause severe pain of an atypical nature and a progressive neurological deficit. Posterior fossa tumors are most likely to cause trigeminal neuralgia; these neoplasms are usually accompanied by subtle neurological deficits and are readily detected by current diagnostic studies. PMID- 3009739 TI - Faculty assignments during curriculum changes. PMID- 3009740 TI - Educational preparation for clinical teaching: perceptions of the nurse educator. AB - Clinical teaching in nursing has been found to be as problematic as it is in other practice-oriented professions. Numerous studies have identified and classified both effective and ineffective clinical teaching behaviors. Absent from the literature are investigations seeking the perceptions of the teachers regarding the adequacy of their preparation for the task and the possible reason(s) for ineffective performance. During the 1983 NLN convention and an ADN workshop, 211 nurse educators responded to a questionnaire soliciting their perception about the adequacy of their graduate program in preparing them for clinical teaching responsibilities. In addition, recommendations were sought on content necessary to effective performance. The study data reveal some startling findings and raise serious questions about the priority graduate programs assign to the content desired by survey participants as necessary for effective clinical teaching. Specific recommendations to address the problem are presented. PMID- 3009738 TI - Stereotaxic surgery with a magnetic resonance- and computerized tomography compatible system. AB - Modern stereotaxic surgery is dependent upon compatible advanced imaging tools, including computerized tomography (CT) scanning and magnetic resonance (MR) imaging. The authors describe three cases in which the patients underwent stereotaxic surgery for mass lesions identified by both MR imaging and CT scans. Identical target coordinates were defined by both techniques, and accuracy was confirmed by intraoperative CT. In comparison to stereotaxic CT, MR provided superior contrast resolution, allowed direct multiplanar imaging and target determination, and permitted accurate correlation of the image with histological features. The operative set-up and technique are described. Stereotaxic surgery with MR imaging may permit more accurate histopathological definition of tumor margins and ultimately lead to better dosimetry for therapeutic procedures such as interstitial brachytherapy. PMID- 3009741 TI - The effects of a well older adult clinical experience on students' knowledge and attitudes. AB - Is a planned learning experience with well older adults a positive influence on a nursing student's attitude, level of gerontological knowledge, and willingness to work with older adults after graduation? Students in a baccalaureate program were divided into treatment or control groups based on their attitudes on the Kogan's Attitude Toward Old People Scale. Knowledge was measured by the Palmore's Facts on Aging Quiz (Palmore, 1977). Half of the students were given experiences with well elderly while the other group had no experience with this population. Differences in pre- and post-test scores were compared by analysis of variance. All students, regardless of planned experience with older adults, increased their knowledge. Students who initially had negative attitudes significantly improved their attitudes regardless of the type of clinical experience. The investigation failed to support the idea that experiences with well elderly would make a difference on attitude and knowledge base. This project suggests knowledge and attitude changes are not dependent upon a particular type of clinical learning activity. PMID- 3009742 TI - The interaction of cognitive style, teaching methodology and cumulative GPA in baccalaureate nursing students. PMID- 3009744 TI - Accreditation: does it control the curriculum? PMID- 3009743 TI - Theory derivation: application to nursing. AB - This article demonstrates an application of the theory derivation strategy to concepts thought to contribute to the development of the caring perspective within professional nurse role development. A theoretical structure and relational statements are developed to describe and explain the phenomenon and to lay the groundwork for hypothesis testing. PMID- 3009745 TI - Split images: compared perceptions of nursing faculty. AB - This pilot study confirmed the existence of wide differences between a group of nursing faculty and a group of staff RNs with regard to the way in which faculty and their roles are perceived. Fewer differences were evident with regard to which characteristics were viewed as important for nursing faculty to possess. We suggest that comparable discrepancies may be present in other settings similar to ours; furthermore, we believe that such differences may contribute to the gap between service and education, and we therefore recommend further study. PMID- 3009746 TI - Health-care economics and the Medicare Prospective Pricing System: implications for undergraduate nursing curricula. PMID- 3009747 TI - A locally-based baccalaureate nursing program for registered nurses. PMID- 3009748 TI - Pre-school screening: an interdisciplinary training experience for nursing and dental students. PMID- 3009749 TI - The nursing class of 1963 revisited. PMID- 3009750 TI - Effect of increasing levels of hard wheat fiber on fecal weight, minerals and steroids and gastrointestinal transit time in healthy young women. AB - Hard red wheat bran (HRWB) baked in a yeast-leavened bread was fed to 36 healthy young college women consuming a basal diet of traditional foods, which contained 15 +/- 3 g/d dietary fiber (DF). Three levels of HRWB were added supplying, respectively, 5.7, 17.1 and 28.5 g/d DF; an additional treatment group did not receive any HRWB. Fecal collections were carried out in the last 5 d of treatment. Fecal wet weight, fecal dry weight and fecal ash increased significantly for each increase in HRWB (P less than 0.05). Fecal dry matter percent changed significantly only at the highest level of HRWB (P less than 0.05). After accounting for the minerals in the HRWB, there was an increased fecal loss of Ca, but not of Zn, Cu, Fe or Mg compared to the women fed no HRWB. HRWB at a level of 17.2 g/d induced faster transit times (TT) than no HRWB and 66 g/d HRWB induced faster TT than either 17.2 or 39.6 g/d HRWB (P less than 0.05). Total daily fecal steroids were not altered by changes in HRWB. Daily total bile acid excretion increased significantly (P less than 0.05) at the two higher levels of HRWB due primarily to higher excretion of chenodeoxycholic acid. PMID- 3009751 TI - Effect of browned and unbrowned corn products intrinsically labeled with 65Zn on absorption of 65Zn in humans. AB - Experimental browned and unbrowned corn products were formulated and processed from unenriched, degermed yellow corngrits. The browned product (cornflakes) contained more insoluble dietary fiber and bound more zinc (in vitro) than the unbrowned product (corngrits). During processing some of the cornflakes and corngrits were combined with a small amount of yellow corn endospermhull intrinsically labeled with 65Zn. The intrinsically labeled corn products were fed, in a crossover design, as components of two breakfasts to six normal, unconfined volunteers. Each volunteer absorbed more 65Zn from the corngrits than from the cornflakes. The reduced 65Zn absorption from cornflakes was attributed to heating and toasting reaction products, possibly Maillard, which bound zinc and consequently made the zinc less available for absorption. PMID- 3009752 TI - Diet calcium carbonate, phosphorus and acidifying and alkalizing salts as factors influencing silica urolithiasis in rats fed tetraethylorthosilicate. AB - Three experiments were conducted to determine the effects of excess dietary calcium carbonate, phosphorus and urine acidifying and alkalizing salts on silica urolith formation in a model using rats fed dextrose-based diets containing 2% tetraethylorthosilicate (TES). Diets containing 2% TES lowered weight gains to 91 95% of gains made by rats fed non-TES diets. Urine silica concentrations of rats fed TES were generally in the range of 50-60 mg/dl. In experiment 1, rats fed TES with no additional dietary calcium carbonate had a silica urolith incidence of 35%. With additions of 1 and 2% calcium carbonate to the basal-TES diet, respective urolith incidences were 45 and 60% (r = 0.99, P less than 0.02). In experiment 2, monobasic sodium phosphate (MP) providing 0.2% additional phosphorus resulted in a mean urine pH of 6.42 and no uroliths. Dibasic sodium phosphate (DP) without and with 0.5% sodium bicarbonate (SB) resulted in respective urine pH values of 6.78 and 7.14 and urolith incidences of 15 and 20% (MP less than DP and DP + SB, P less than 0.05). However, the uroliths were small averaging less than 1 mg. In experiment 3, substitution of autoclaved egg albumin for casein, the protein source in experiments 1 and 2, resulted in urine pH of 7.45 and a silica urolith incidence of 46%. An equal-molar mixture of MP and DP providing an added 0.2% phosphorus resulted in a urine pH of 7.07 and reduced the urolith incidence to 4%, and 0.75% of dietary ammonium chloride either with or without the added 0.2% phosphorus gave urine acidification and complete protection from uroliths.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009753 TI - Effect of age and diet on cyclic nucleotide concentrations in the intestinal mucosa of developing rats. AB - Mucosa isolated from the proximal third of the small intestine of infant rats had much lower cyclic nucleotide concentrations (expressed both per unit wet weight and per unit DNA content) than those determined in the intestinal wall. The steady-state concentrations of both cyclic AMP and cyclic GMP in jejunum showed dramatic increases during the first 5 d post partum. Another increase in cyclic nucleotide concentrations was observed in the isolated mucosa between d 15 and 21. Starvation for 24 h always resulted in lower intestinal cyclic nucleotide concentrations than those of the fed littermates. This effect was more pronounced in younger animals and more evident for cyclic AMP values. Three-week-old rats fed a high carbohydrate diet for 24-48 h exhibited more pronounced elevations in the concentrations of cyclic nucleotides from the jejunal mucosa than did rats fed a high fat diet. PMID- 3009754 TI - Study of chest radiographs and pulmonary ventilatory function in perlite workers. AB - A review of chest films from 152 workers who had been employed five or more years in perlite mining or processing showed none with small opacities of profusion 1/0 or higher. There were 14 films with doubtful changes (0/1), but these showed no correlation with type or duration of employment. Pulmonary function was measured in 122 current employees from the same plants. Multiple regression analysis showed no significant association between years of employment in perlite and either forced vital capacity (FVC) or forced expiratory volume (FEV1). There was a significant association between pack-years of cigarettes and both measurements. In 66 workers tested in 1975 and again in 1983, there was an average annual decrease in FVC of 32 mL, with 26 mL predicted by the Knudson formula, which is based on nonsmokers. The average annual decrease in FEV1 was 24 mL with 26 mL predicted. Comparison of groups with differing smoking patterns showed that the decreases in both FVC and FEV1 were associated with smoking. The 28 men who had added four or more pack-years in the interval between tests showed decreases in FVC and FEV1 of 44 mL/year and 31 mL/year, respectively, with 26 mL/year predicted for both groups. Those with less than four added pack-years (which included 26 nonsmokers) had decreases in FVC and FEV1 of 23 mL/year and 19 mL/year with 26 mL/year and 27 mL/year predicted. PMID- 3009755 TI - Effect of ketanserin on the in vitro regulation of aldosterone. AB - Serotonin (5-HT) is known to be a potent stimulator of aldosterone release in vitro and, to a lesser degree, in vivo. Since ketanserin, a specific 5-HT2 receptor antagonist, decreases aldosterone levels in some hypertensive patients, we have performed in vitro experiments to elucidate the mechanism by which ketanserin interferes with aldosterone regulation. After collagenase dispersion, rat zona glomerulosa cells were either perfused and resuspended in Biogel or incubated in Earle's medium and bovine serum albumin. The cells were stimulated with serotonin, angiotensin II, potassium (K+) and adrenocorticotrophic hormone (ACTH) in the presence or absence of ketanserin. Ketanserin did not decrease baseline aldosterone secretion although it reduced the stimulatory capacity of serotonin and K+, and had a minimal effect on ACTH. Furthermore, we demonstrated that ketanserin is able to have a significant effect on the adrenal response to angiotensin II. These data indicate that antiserotoninergic agents may act directly at the level of the adrenal glomerulosa by interfering with specific serotoninergic receptors and modifying the adrenal sensitivity to angiotensin II and K+. The importance of these findings in the clinical use of these antihypertensive drugs remains to be established. PMID- 3009756 TI - Zygomatic augmentation with hydroxylapatite: a preliminary report. AB - This paper describes the use of hydroxylapatite in combination with microfibrillar collagen and blood to yield a cohesive mixture that can be sculptured to correct symmetry over the zygomatic bones. Eleven cases were studied retrospectively, and the augmentation was found to remain stable and esthetically pleasing. PMID- 3009757 TI - Augmentation of the atrophic mandible with interposed bone grafts and particulate hydroxylapatite. AB - The use of hydroxylapatite mixed with autogenous bone to augment the area posterior to the mental foramen is described. This method is combined with an interposed bone graft technique in the symphyseal area. The clinical implications are discussed in relation to potential nerve damage, height to be gained, and effects on lip-chin profile, and the results in 29 patients are reported. PMID- 3009758 TI - Malignant fibrous histiocytoma in the maxilla: review of literature and report of a case. AB - This case report demonstrates the difficulty involved in establishing a diagnosis of malignant fibrous histiocytoma. The histologic characteristics of this tumor as well as the important contributions of electron microscopy and special staining techniques in making the diagnosis, have been discussed. Review of 16 cases of MFH revealed the typical presentation of maxillary sinus malignancy. Treatment modalities vary, depending on the size of the tumor and the preference of the surgeon. PMID- 3009760 TI - Breast cancer metastatic to the tongue: report of a case. PMID- 3009759 TI - Cardiovascular, biochemical, and hormonal responses to intravenous sedation with local analgesia versus general anesthesia in patients undergoing oral surgery. AB - The effects of intravenous conscious sedation and general anesthesia on a number of stress variables were compared in healthy patients undergoing oral surgery. In the intravenous sedation group only the levels of plasma growth hormone and prolactin rose significantly. In the general anesthesia group significant rises were seen in heart rate; systolic blood pressure; mean arterial pressure; levels of plasma adrenaline, adrenocorticotropic hormone, cortisol, and prolactin; and levels of blood glucose, all indicative of a stress response. This study demonstrates that oral surgery under intravenous conscious sedation with local analgesia is associated with less patient stress than is oral surgery under general anesthesia. PMID- 3009761 TI - Changes in cAMP phosphodiesterase activity during oral mucosal regeneration. AB - Preliminary studies (Lineweaver-Burk, Ca2+ calmodulin sensitivity) suggest that oral mucosa contains at least two cyclic AMP phosphodiesterases, one a high affinity (low Km) enzyme and the other a low affinity (high Km) enzyme. Analysis of the distribution of both enzymatic forms during oral mucosal regeneration revealed that the low Km, and high Km cAMP phosphodiesterase activities were significantly elevated prior to the first wave of cAMP accumulation and during the second cAMP wave. Although the cAMP peaks declined between 20-24 h, both cAMP phosphodiesterases remained significantly elevated at the wound site. The apparent Km of the low Km form increased from 5.3 to 7.5 microM, while that of the high Km form remained essentially unaltered 20 h after wounding. The low Km, and high Km cAMP phosphodiesterase activities in normal rat oral mucosa were not affected by epinephrine or insulin; were slightly inhibited by glucagon, and significantly inhibited by methylprednisolone. Imidazole and histamine activated both forms and theophylline was inhibitory to the enzyme. The high Km cAMP phosphodiesterase was sensitive, and the low Km form insensitive to Ca2+ calmodulin stimulation. PMID- 3009763 TI - [A case of adult T-cell leukemia/lymphoma manifested as paranasal tumor]. PMID- 3009762 TI - Alterations in enamel mineral with concentrated fluoride treatment: an electron spin resonance study. PMID- 3009764 TI - [Effect of prostaglandins on AMP metabolism and proliferation and phenotypic expression of rabbit costal chondrocytes in culture]. PMID- 3009765 TI - The control of fibrogenesis: stimulation and suppression of collagen synthesis in the chick chorioallantoic membrane with fibrin degradation products, wound extracts and proteases. AB - The chick chorioallantoic membrane model (CAM) has previously been used to demonstrate cell proliferation, characteristic of both angiogenesis and fibrogenesis, after exposure to fibrin degradation products. This model has now been adapted for quantitative in vivo assay of collagen polypeptide synthesis and prolyl hydroxylase activity. The CAM exhibits oscillations in the level of labelled collagen, a pattern attributable to rapid intracellular degradation and proline recycling following a brief labelling period. Both collagen synthesis and prolyl hydroxylase activity are stimulated by fibrin degradation products (less than 50 000 MW). Such stimulation occurs by 3 h and precedes the rise in general protein synthesis. Extracts of healing mouse skin wounds, rich in proteases, inhibited collagen synthesis, as did pure plasmin. Conversely, stimulation was achieved when proteolytic activity was neutralized by soybean trypsin inhibitor. These findings help to explain the observation that fibroblasts and endothelial cells proliferate and migrate centrally in an inflammatory lesion without depositing collagen, whilst in a milieu of high proteolytic activity. More peripherally, where proteases are inactivated by antiproteases in inflammatory exudate, such cell movement ceases and collagen deposition is observed. PMID- 3009766 TI - Transferrin receptor expression in non-malignant and malignant human breast tissue. AB - The expression of transferrin receptor by normal, pregnant and benign hyperplastic breast lesions and by breast carcinomas has been investigated immunohistochemically using two monoclonal antibodies directed against the receptor. Unlike a previous immunohistological study in which staining was confined to malignant breast, transferrin receptor has been detected in pregnant breast and in benign lesions as well as in all carcinomas examined. The latter showed variable reactivity but with staining of most cells in 70 per cent of cases. Although the expression of transferrin receptor in non-malignant conditions may be related to cell proliferation, as has been suggested from studies of activated cells, the extent of reactivity of carcinomas has shown no correlation with tumour characteristics such as differentiation and local tumour spread. It is therefore suggested that the immunologically active transferrin receptor of breast carcinomas may have significance other than that relating to proliferation. The finding that with some carcinomas differences in staining occurred between the two antibodies is a further illustration of the complexities of the nature of transferrin receptor. PMID- 3009767 TI - Modulation of fibroblast activity by normal and silica-exposed alveolar macrophages. AB - Silica-induced pulmonary fibrosis is thought to involve fibroblast stimulation by a product of alveolar macrophages (AM) but various cell culture systems have given conflicting results. Macrophage-fibroblast interactions are now studied using an homologous system in which supernatants of rat AM after incubation with silica, are tested on fibroblasts isolated from the same animals to assess the effects on cell proliferation and collagen production. Fibroblast growth varied with initial seeding density and changes induced by AM supernatants varied depending on the proliferative rate. Normal AM supernatants inhibited [3H] thymidine incorporation into fibroblasts, especially in more rapidly dividing cells. Supernatants of silica-treated AMs also inhibited division of rapidly growing fibroblasts, whereas the same material stimulated growth of slowly dividing cells. Collagen synthesis increased with the length of time that fibroblasts were confluent and was inhibited by control AM supernatants. Silica treated AM supernatants increased collagen production by fibroblasts confluent for 3 days, whereas the same supernatants inhibited collagen synthesis by cells confluent for at least 8 days. The observation that a factor derived from silica exposed AM first stimulates them inhibits fibrogenesis, indicates a modulation of the normal macrophage-fibroblast control system. This suggests that other factors may be required in vivo to shift this cellular balance towards the fibrotic process. PMID- 3009768 TI - Treatment of the alcoholic family. PMID- 3009769 TI - Acute lower respiratory tract infections in nonhospitalized children. PMID- 3009770 TI - Digoxin-like immunoreactive substances in urine and serum from preterm and term infants: relationship to renal excretion of sodium. PMID- 3009772 TI - Pseudomonas aeruginosa: biology, mechanisms of virulence, epidemiology. AB - Pseudomonas aeruginosa is a gram-negative pathogen, versatile and opportunistic in terms of its genetics, metabolic potential, and mechanisms of virulence. This versatility enables it to respond to variable and frequently adverse environmental conditions. Considered by many to be an aerobic organism, it is capable of growing anaerobically if certain substrates are available, for example, nitrates or arginine. Diversity of mechanisms of genetic exchange, including transformation, transduction, and conjugation, help P. aeruginosa adapt to changing conditions by acquiring new genetic information. Genetic manipulations have been exploited in recent years to study the basic biology of this bacterial species and the roles of its numerous virulence factors. Recently, transposon mutagenesis techniques and recombinant DNA methods (cloning) have been used to study some of the virulence factors of P. aeruginosa. The pathogenesis of P. aeruginosa infections is multifactorial, as manifested by the numerous toxins, or virulence factors, it produces and the variety of diseases it causes. P. aeruginosa is invasive and toxigenic. Infections appear to occur in stages: bacterial adherence, colonization, invasion and dissemination, and systemic or toxemic disease. Virulence factors can contribute to one or several stages of pathogenesis. Surface factors, including pili, lipopolysaccharide, and polysaccharide slime (alginate), probably contribute to the first two stages. Polysaccharide slime and lipopolysaccharide may also contribute to other processes later in the course of infection. Toxins, including exotoxin A and phospholipase C (hemolysin), and proteases of P. aeruginosa may contribute to tissue damage and dissemination. They may also aid in the procurement of nutrients required by the bacteria in the early stages of infection. The significance of the different virulence factors probably depends on the infection. Alginate production and phospholipase C are likely to have special significance in respiratory infections, particularly in cystic fibrosis. PMID- 3009771 TI - Posttransfusion cytomegalovirus infection in neonates: role of saline-washed red blood cells. PMID- 3009773 TI - A new alternative in the field of cardiac glycosides: gitoxin. PMID- 3009775 TI - [Antiviral compounds. XV. Reaction of 5,6-diphenyl-1,2,4-triazine-3-thiol with alcohols and antiviral activity]. PMID- 3009774 TI - Structure-activity relationship of bufotoxins and related compounds for the inhibition of Na+, K+ -adenosine triphosphatase. AB - Forty kinds of bufotoxins and related compounds were tested for inhibition of Na+, K+ -adenosine triphosphatase from guinea pig heart, and the structure activity relationship has been discussed. The inhibitory activities of bufotoxins were dependent upon the dicarboxylic acid and amino acid components. The compounds having both the arginine and suberic acid moieties showed the higher inhibitory activities. The sulfates and glucuronides of cardiac steroids exhibited much less potency than the parent genins. The mode of inhibition was determined by means of the Dixon and Lineweaver-Burk plots. PMID- 3009776 TI - [A new inhalation apparatus and its application to screening of anti-influenza agents]. PMID- 3009777 TI - [Effects of nicergoline on cerebral energy metabolism in normal mice]. PMID- 3009778 TI - Epidermodysplasia verruciformis associated with type II human papilloma virus. PMID- 3009779 TI - Inhibition of guinea-pig oxyntic cell function by adenosine and prostaglandins. AB - Adenosine and prostaglandins (PGs) are known inhibitors of oxyntic cell function. Using quantitative cytochemistry of hydroxyl ion production (HIP) in guinea-pig oxyntic cells, we examined the effects of adenosine and PGs on secretagogue stimulated HIP. Adenosine (10(-6) M) inhibited the actions of histamine (10(-14) M) and gastrin (2.5 X 10(-12) M) by 69 and 67%, respectively, but not that of dibutyryl cyclic AMP (10(-16) M) or carbachol (10(-9) M). These observations suggest that adenosine does not influence the Ca++-dependent pathway of carbachol action and that the adenosine activity precedes the generation of cyclic AMP. Adenosine and related analogs, N6-L-phenylisopropyladenosine and 5-N ethylcarboxam-idoadenosine (10(-12) to 10(-14) M), inhibited histamine-stimulated HIP (10(-14) M) in the following order: N6-L-phenylisopropyladenosine greater than 5-N-ethylcarboxamidoadenosine greater than adenosin. The adenosine antagonist, 1,3-diethylphenylxanthine (10(-6) M), reversed the inhibitory effects of adenosine. Exogenous PGE2 (10(-6) M) also inhibited histamine- and gastrin stimulated HIP by 65 and 55%, respectively. Indomethacin (10(-6) M) and flurbiprofen (10(-6) M), PG synthesis inhibitors, potentiated the action of histamine by 175 and 159%, respectively. Adenosine was incapable of reversing this potentiated effect. These data indicate that adenosine and related analogs are inhibitors of oxyntic cell HIP and suggest that these biological properties are mediated by binding to a cell surface receptor and thereby regulating oxyntic cell adenylate cyclase activity. The more potent properties of N6-L phenylisopropyladenosine as compared to 5-N-ethylcarboxamidoadenosine are consistent with activity at the high-affinity surface adenosine receptor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009780 TI - Norepinephrine-sensitive, phenoxybenzamine-resistant receptor sites associated with contraction in rabbit arterial but not venous smooth muscle: possible role in adrenergic neurotransmission. AB - The possibility that not all contractions of rabbit blood vessels to norepinephrine (NE) are mediated through alpha adrenoceptors sensitive to phenoxybenzamine (PBZ) was investigated. Dose-response curves (DRCs) to NE were made in the absence and presence of PBZ pretreatment which minimized the contribution of alpha adrenoceptors. In all arteries studied (saphenous, renal, femoral and central ear arteries), after PBZ-treatment, NE produced biphasic DRCs. The initial component of these DRCs corresponded to doses of NE which in the absence of PBZ were supramaximal. Under conditions of our experimentation the plateau-phase usually occurred at between 5 and 40% of the pre-PBZ maximal response to NE. The second phase occurred with further additions of NE, and achieved a mean of 72 (+/- 4)% of the pre-PBZ maximal contraction to NE. The latter component presumably represented contractions mediated through low affinity sites for NE which were insensitive to doses of PBZ sufficient to alkylate alpha adrenoceptors. In veins (saphenous and inferior vena cava), we found no evidence for such sites. Our results are discussed in light of current ideas of adrenergic neurotransmission in vascular smooth muscle as proposed by Hirst and Neild (1980a) and others who suggest that response to high concentrations of neuronally released NE occur through PBZ-resistant receptors termed gamma adrenoceptors located exclusively at the postsynaptic membrane. We were able to demonstrate PBZ-resistant, low-affinity sites for NE contractions in the femoral artery, a vessel with very sparse adrenergic innervation, and conclude that such sites for NE are present in a number of arteries (and not veins) irrespective of their innervation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009781 TI - Metaphit, a proposed phencyclidine receptor acylator: phencyclidine-like behavioral effects and evidence of absence of antagonist activity in pigeons and in rhesus monkeys. AB - Metaphit, a derivative of phencyclidine (PCP), binds irreversibly to PCP sites and appears to act as an antagonist of PCP under some conditions and as a PCP like agonist under other conditions. To describe further these conditions, the authors investigated the behavioral effects of metaphit by using different routes of administration, behavioral procedures and species. In pigeons, metaphit induced PCP-like catalepsy after i.c.v. administration and, after i.m. administration, produced PCP-like discriminative stimulus effects and stereotyped operant responding. In rhesus monkeys, metaphit produced ataxia and convulsions but did not induce catalepsy, anesthesia or PCP-like discriminative stimulus effects. None of the effects of PCP-type drugs [i.e., PCP, ketamine or (+/-)-SKF 10,047] in pigeons or rhesus monkeys was antagonized by metaphit. Metaphit potentiated the discriminative stimulus effects of PCP and of SKF 10,047 in pigeons. These results suggest that metaphit acts not as an antagonist of PCP but as a PCP-like agonist under these conditions. Metaphit's potentiation of behavioral effects of PCP may be related to the presumed ability of metaphit to acylate PCP receptors. The extent to which metaphit produces PCP-like behavioral effects in part may be species dependent. PMID- 3009782 TI - Central opioid receptors and baroreflex control of sympathetic and cardiovascular function. AB - The effect of central opioid receptor activation and blockade on arterial baroreflex regulation of cardiovascular function was studied. Baroreceptor reflexes were elicited in urethane-anesthetized rats by graded electrical stimulation of the aortic nerve while mean arterial pressure, heart rate and sympathetic nerve activity were recorded simultaneously. Baroreflex response curves were constructed after intracisternal administration of saline vehicle, after intracisternal infusion of the relatively selective mu and delta opioid receptor agonists D-Ala2-MePhe4-Gly(ol)5 enkephalin (DAGO), or D-Ala2-D-Leu5 enkephalin (DADLE) respectively, and again after i.v. naloxone. Reflex reductions in mean arterial pressure, heart rate and sympathetic nerve activity elicited by aortic nerve stimulation were attenuated in a dose-related fashion by intracisternal DAGO. Opioid effects were greatest at low levels of baroreceptor activation and became progressively less marked as the frequency of aortic nerve stimulation was increased. Baroreflex impairment was reversed completely by i.v. naloxone. Centrally administered DADLE also attenuated baroreceptor reflexes, but was approximately 10- to 100-fold less potent than an equimolar amount of DAGO. The effect of DADLE was reversed by a lower dose of naloxone than was required to normalize baroreflexes after DAGO. These results suggest that the effect of DADLE on baroreflexes was mediated by activation of mu rather than delta opioid receptors. No evidence was obtained to suggest a role for endogenous opioid modulation of baroreflexes because i.v. naloxone was without effect. These results demonstrate that activation of central mu opioid receptors significantly impairs baroreflex control of sympathetic and cardiovascular function. PMID- 3009783 TI - Discriminative stimulus effects of the opioid antagonist diprenorphine in the squirrel monkey. AB - Squirrel monkeys were trained in a discrete-trial avoidance paradigm to discriminate i.m. injections of the opioid antagonist diprenorphine (0.1 mg/kg) from vehicle. When the monkeys could complete reliably at least 22 trials of a 25 trial session on the choice level appropriate for the substance injected before the session (i.e., diprenorphine or vehicle), tests of stimulus generalization to novel drug conditions were conducted. Mu receptor agonists (morphine, etorphine and buprenorphine) and kappa receptor agonists (ethylketocyclazocine, nalorphine and I-N-allylnormetazocine) produced dose-dependent diprenorphine-like discriminative effects. The dextrorotatory isomer of N-allynormetazocine was almost two-orders of magnitude less potent than the levorotatory isomer in this respect and phenycyclidine generalized to diprenorphine only partially, suggesting that the phencyclidine/sigma site does not have a prominent role in the discriminative effects of diprenorphine. Other nonopioid drugs (d amphetamine, mescaline and pentobarbital) also did not produce discriminative effects comparable to those of the training drug. The pure opioid antagonists, naloxone, naltrexone, and WIN 44,441-3 [(2-alpha-6 alpha, 11S)-(-)-1-cyclopentyl 5-(1,2,3,4,5,6-hexahydro-8-hydroxy-3,6,11-trim eth yl-2, 6-metheno-3-benzazocin 11-yl)-3-pentanone] occasioned responding primarily on the lever appropriate for vehicle. Naloxone (1.0 mg/kg) blocked surmountably the diprenorphine-like discriminative effects of the mu and kappa agonists, displacing generalization curves to the the right by 10- to 100-fold; however, naloxone failed to shift the curve for diprenorphine itself. Thus, in the squirrel monkey diprenorphine has discriminative stimulus effects in common with mu- and kappa-opioid agonists.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009784 TI - Xylazine emesis, yohimbine and motion sickness susceptibility in the cat. AB - The possible role of the alpha-2 adrenoceptors in xylazine-induced vomiting and in motion sickness was investigated. Cats were divided into two groups according to motion sickness susceptibility and were observed after s.c. injections of xylazine. The incidence of vomiting increased with the dose, and at each dose, the high susceptibility group had a greater emetic incidence than the low susceptibility group. In another experiment with cats divided into two groups according to motion sickness susceptibility, s.c. administration of yohimbine effectively antagonized the xylazine-induced emesis in both susceptibility groups. The cats in the latter experiment were then challenged with a motion sickness stimulus, after s.c. pretreatment with yohimbine. Yohimbine failed to prevent motion sickness but did occasion an unexplained variability in the incidence of vomiting. These findings suggest that the emetic effect of xylazine results from stimulation of alpha-2 adrenoceptors but that these receptors are not fundamental to feline motion sickness. The fact that susceptibilities to xylazine-induced emesis and to motion sickness are correlated suggests a point of interaction other than the area postrema, which is known to be essential for xylazine-induced vomiting but not for motion sickness in the cat. PMID- 3009786 TI - Effects of gamma-aminobutyric acid (GABA) receptor agonists on the neurotoxicity and anticonvulsant activity of barbiturates in mice. AB - The effects of gamma-aminobutyric acid (GABA) receptor agonists [4,5,6,7 tetrahydroisoxazolo(5,4-c)pyridin-3-ol THIP]; [progabide and baclofen] on the minimal neurotoxicity and anticonvulsant activity of pentobarbital and phenobarbital in mice were investigated. When either progabide, THIP or baclofen were administered with pentobarbital, the components of this combination interacted additively by the rotorod test. Combinations of pentobarbital and progabide or phenobarbital and progabide interacted additively when subjected to the pentylenetetrazol (PTZ) minimal threshold seizure (clonic) test. THIP, even at toxic doses, did not alter the anti-PTZ activity of either pentobarbital or phenobarbital. In contrast, baclofen at toxic doses potentiated the anti-PTZ activity of pentobarbital and phenobarbital. Combinations of progabide and pentobarbital or progabide and phenobarbital interacted additively by the maximal electroshock seizure (MES) test. THIP, even when given in toxic doses, had no effect on the anti-MES activity of pentobarbital and phenobarbital. However, baclofen at nontoxic doses potentiated the anti-MES activity of phenobarbital but not that of pentobarbital. These results suggest that 1) in vitro interactions between barbiturates and GABAa receptor agonists may not be the same in vivo, 2) GABAa receptors may play a minor role in the minimal neurotoxicity and anticonvulsant activity of barbiturates and 3) inhibition of glutamate-induced excitation by baclofen may be an important action in potentiating the anti-MES activity of phenobarbital. PMID- 3009785 TI - Cellular toxicity of bromobenzene and bromobenzene metabolites to rabbit proximal tubules: the role and mechanism of 2-bromohydroquinone. AB - An in vitro model using a suspension of rabbit renal proximal tubules was developed to investigate the mechanism of nephrotoxicity of bromobenzene. Using oxygen consumption, glutathione concentrations and retention of lactate dehydrogenase activity as markers of toxicity, the rank order of potency was bromobenzene (5 mM) less than 2-bromophenol (2 mM) less than 3-, 4-bromophenol (1 mM) less than 2-bromohydroquinone (0.1 mM). These data support in vivo results and are consistent with the hypothesis that 2-bromohydroquinone or a metabolite thereof is responsible for bromobenzene-induced nephrotoxicity. Inhibitors of cytochrome P-450 and the cyclooxygenase and peroxidase components of prostaglandin H synthase did not protect the proximal tubules from 2 bromohydroquinone-induced toxicity, suggesting that these enzymes do not play a role in 2-bromohydroquinone bioactivation. There is a specific sequence of events in 2-bromohydroquinone-induced toxicity. Early events include decreased glutathione levels and inhibited mitochondrial respiration, whereas an increase in plasma membrane permeability is a late event. PMID- 3009787 TI - Thromboxane agonist (U46619) potentiates norepinephrine efflux from adrenergic nerves. AB - The effect of the synthetic thromboxane/prostaglandin (PG) H2 agonist U46619 on the electrically stimulated rabbit isolated was deferens was examined to test for thromboxane influences on adrenergic nerves. U46619 effects on force generation, [3H] norepinephrine release and norepinephrine-induced contractions were assessed to determine the mechanism of action. U46619 maximally enhanced adrenergic force generation 135 +/- 24% at a concentration of 100 nM. U46619 potentiated maximal contractile effects of exogenously administered norepinephrine 16 +/- 4% and augmented [3H]norepinephrine release from electrically stimulated preparations 142 +/- 44%. A competitive thromboxane/PGH2 receptor antagonist, SQ29548, significantly shifted the concentration-response curve for U46619 to the right in a concentration-dependent manner and blocked U46619-induced tritium release. Thus, U46619 appears to potentiate neurotransmitter release by interacting with thromboxane/PGH2 receptors. Because SQ29548 did not prevent the potentiation of norepinephrine contractions by U46619, the postjunctional effect may be independent of thromboxane/PGH2 receptors. We interpret these results to be indicative of both pre- and postjunctional sites of action of U46619. The physiological importance of these thromboxane effects is unknown currently. PMID- 3009789 TI - Effect of ethosuximide alone and in combination with gamma-aminobutyric acid receptor agonists on brain gamma-aminobutyric acid concentration, anticonvulsant activity and neurotoxicity in mice. AB - The acute administration of an anticonvulsant dose of ethosuximide (150 mg/kg) had no effect on brain gamma-aminobutyric acid (GABA) concentration, whereas a toxic dose (400 mg/kg) increased significantly the concentration of brain GABA (1.23 +/- 0.05 vs. 1.92 +/- 0.14 mumol/g of wet tissue). The administration of 500 mg/kg/day of ethosuximide for 1, 2, 4, 6, 8 and 11 days induced neurotoxicity in 100, 100, 67, 0, 0 and 0% of animals, respectively, and increased brain GABA concentration 46, 38, 25, 14, 9 and 0%, respectively. These results imply that the tolerance that develops in response to the chronic administration of toxic doses of ethosuximide correlates well with the concentration of brain GABA. 4,5,6,7-Tetrahydroisoxazolo [5,4-c]pyridin-3-ol even in toxic doses had no effect on the anticonvulsant activity of ethosuximide. Combination studies with ethosuximide and progabide demonstrated that the antipentylenetetrazol activity of the individual components interacts additively. Likewise, combinations of either ethosuximide and 4,5,6-tetrahydroisoxazolo [5,4-c]pyridin-3-ol or ethosuximide and progabide showed an additive effect by the rotorod test. These results indicate that the antipentylenetetrazol activity of ethosuximide is unrelated to GABA function and that the increase in brain GABA concentration induced by toxic doses of ethosuximide contributes to its neurotoxicity. PMID- 3009788 TI - GABAergic modulation of inferior colliculus excitability: role in the ethanol withdrawal audiogenic seizures. AB - The role of the inferior colliculus and GABAeric transmission within this structure in the development of susceptibility to sound-induced seizures in ethanol-dependent rats was examined. Ethanol-dependent rats with bilateral electrolytic lesions which destroyed approximately 50.0 +/- 6.4% of the inferior colliculus failed to exhibit susceptibility to sound-induced seizures. However, comparable medial geniculate body lesions (82.7 +/- 2.7% complete) did not alter wild running, slightly reduced tonus and actually increased clonus susceptibility in rats treated similarly with ethanol. As reported previously, bilateral injection of either muscimol (43-263 pmol/site) or racemic baclofen (520-1580 pmol/site) into the inferior colliculus also suppressed seizure susceptibility. Other studies in ethanol-naive animals found that bilateral microinfusion of (+) bicuculline methiodide (2 or 20 pmol/min for up to 5 min) into the inferior colliculus induced wild running and clonus closely resembling sound-induced seizure responses in ethanol-dependent rats. Although similar microinjections of (+)-bicuculline methiodide (0.4 pmol/min for 5 min) into the inferior colliculus did not induce seizure activity directly, an increased susceptibility to sound induced seizures was observed. Electrolytic lesions of the medial geniculate body did not block wild running responses induced by (+)-bicuculline methiodide, but slightly reduced clonus. Five-minute infusions of picrotoxin (200 pmol/min), Ro5 3663 (2000 pmol/min), kainic acid (20 or 200 pmol/min), strychnine (2000 pmol/min) or carbachol 2000 pmol/min) into the inferior colliculus of ethanol naive rats all induced bicuculline-like seizures. Seizures induced by bicuculline methiodide, picrotoxin or Ro5-3663 occurred within 5 min after the start of infusions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009790 TI - Prostaglandin E2 inhibition of secretagogue-stimulated [14C]aminopyrine accumulation in rat parietal cells: a model for its mechanism of action. AB - Prostaglandin E2 (PGE2) differentially inhibited histamine and isoproterenol stimulation of [14C]aminopyrine accumulation in rat parietal cell preparations. Low concentrations of PGE2 decreased the maximum response to isoproterenol whereas higher concentrations increased the EC50 of histamine with only a modest effect on the maximum response. Also, PGE2 potentiated dibutyryl cyclic AMP stimulation of aminopyrine accumulation in either the absence or presence of carbachol. In contrast, PGE2 inhibited potentiation between carbachol and histamine due to its inhibitory effect on histamine and possibly also to an inhibitory effect on cholinergic activity. Islet activating protein prevented the inhibitory actions of PGE2. To account for these results a model is presented based on the recent proposal by Gilman (Cell 36: 577-579, 1984) of an interaction between components of adenylyl cyclase stimulatory and inhibitory guanine nucleotide binding proteins. PMID- 3009791 TI - Depression of contractile responses in rat aorta by spontaneously released endothelium-derived relaxing factor. AB - Removal of endothelial cells on rings of rat aorta increased the sensitivity to the selective alpha-1 adrenoceptor agonist phenylephrine, to the nonselective alpha adrenoceptor agonist norepinephrine and to the selective alpha-2 adrenoceptor agonist clonidine. In the case of the first two, which are strong agonists for the alpha-1 adrenoceptor-mediating contraction, removal of endothelium increased sensitivity 4- and 6-fold at the EC30 level, but produced little or no increase in maximum. In the case of clonidine, a partial agonist for the alpha-1 adrenoceptor, which gave only about 15% of the maximum given by phenylephrine on endothelium-containing rings, removal of the endothelium not only shifted the curve to the left but also increased the maximum to about 50% of that given by phenylephrine. The depression of sensitivity to these agonists in rings with endothelium appeared to be due to the vasodepressor action of endothelium-derived relaxing factor (EDRF), as hemoglobin, a specific blocking agent of EDRF, abolished this depression. It is unlikely that the endothelium dependent depression was due to stimulation of release of EDRF, because clonidine did not produce endothelium-dependent relaxation in precontracted rings even when its contractile action was blocked by the alpha-1 adrenoceptor antagonist prazosin. Further evidence against alpha adrenoceptor agents stimulating release of EDRF was that neither phenylephrine nor clonidine induced a rise in cyclic GMP in aortic rings, whereas acetylcholine, which does release EDRF, caused a large rise in cyclic GMP content. The possibility that the muscle cells of intact rat aortic rings were under the tonic influence of released EDRF was supported by the finding that, in the absence of any contractile agent, hemoglobin induced a fall in the basal level of cyclic GMP in endothelium-containing rings. Also consistent with EDRF being released spontaneously was the finding that contraction induced by 5-hydroxytryptamine, like that by alpha-adrenergic agonists, was also depressed in endothelium-containing rings of aorta. When the efficacy of phenylephrine as an alpha-1 agonist was reduced to about the initial efficacy of clonidine by irreversible inactivation of a very large fraction of alpha-1 adrenoceptors of the smooth muscle cells by pretreatment with dibenamine, the concentration-contraction curves for phenylephrine for both endothelium containing rings and for endothelium-denuded rings now became very similar to the corresponding curves obtained for clonidine before receptor inactivation.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3009792 TI - Pharmacological evidence that human intralobar airways do not contain different receptors that mediate contractions to leukotriene C4 and leukotriene D4. AB - Contractile responses to leukotriene (LT)C4, LTD4 and LTE4 were examined in intralobar airways from human lung obtained after surgical resection. In addition, the ability of the LT receptor antagonist FPL55712 to antagonize responses to LTC4 and LTD4 was quantified (by calculating -log molar KB values) under experimental conditions designed to minimize metabolic transformation of the LTs. In the absence of drug pretreatment, the three peptide LTs were approximately equipotent and produced similar maximum degrees of contraction. L Serine borate complex, 45 mM, used as an inhibitor of the degradation of LTC4 to LTD4 by the enzyme gamma-glutamyl transpeptidase, in paired airway segments (adjacent segments from the same branch), produced a small degree (about 3-fold) of shift to the right of the dose-response curve and reduction of the maximum response to LTC4. L-Cysteine, 3 mM, used as an inhibitor of the degradation of LTD4 to LTE4 by the enzyme aminopeptidase, in paired segments, did not alter the dose-response effects of LTD4 or appear to further alter the dose-response effects of LTC4 when applied together with L-serine borate complex in unpaired (nonadjacent) segments. The -log molar KB value for FPL55712 (about 6) was similar for antagonism of responses to both LTC4 and LTD4 in the absence or presence of treatment with the metabolic inhibitors L-serine borate complex, L cysteine or a combination of the two treatments. The results suggest that inhibition of the enzymes involved in the pathway from LTC4 to LTE4 has little consequence in human airways because the three peptide LTs are approximately equipotent.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009794 TI - Muscarinic cholinergic receptor binding sites differentiated by their affinity for pirenzepine do not interconvert. AB - Although it has been suggested by many investigators that subtypes of muscarinic cholinergic receptors exist, physical studies of solubilized receptors have indicated that only a single molecular species may exist. To test the hypothesis that the putative muscarinic receptor subtypes in rat forebrain are interconvertible states of the same receptor, the selective antagonist pirenzepine (PZ) was used to protect muscarinic receptors from blockade by the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard (PBCM). If interconversion of high (M1) and low (M2) affinity binding sites for PZ occurs, incubation of cerebral cortical membranes with PBCM in the presence of PZ should not alter the proportions of M1 and M2 binding sites that are unalkylated (i.e., protected). If, on the other hand, the binding sites are not interconvertible, PZ should be able to selectively protect M1 sites and alter the proportions of unalkylated M1 and M2 binding sites. In the absence of PZ, treatment of cerebral cortical membranes with 20 nM PBCM at 4 degrees C for 50 min resulted in a 69% reduction in the density of M1 binding sites and a 55% reduction in the density of M2 binding sites with no change in the equilibrium dissociation constants of the radioligands [3H]quinuclidinyl benzilate or [3H]PZ. The reasons for this somewhat selective effect of PBCM are not apparent. In radioligand binding experiments using cerebral cortical membranes, PZ inhibited the binding of [3H]quinuclidinyl benzilate in a biphasic manner.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009793 TI - Inhibition of high-affinity choline uptake in the rat hippocampus by in vivo injection of phenobarbital in the medial septum. AB - Barbiturates administered i.p. inhibit the activity in the septal-hippocampal cholinergic pathway as assessed by an inhibition of choline uptake into hippocampal synaptosomes in vitro. The present experiments were designed to determine where in the central nervous system the drugs act to have this effect. Barbiturates and other drugs were injected into selected sites in the awake, freely moving rat via previously implanted cannulas. It was found that phenobarbital, barbital and muscimol injected into the medial septal area inhibited choline uptake in the hippocampus. Pentobarbital was less effective than phenobarbital even when it was tested in the highest amounts that could be applied locally. The data suggest that the barbiturates rapidly leave the site of local injection and that, in the case of pentobarbital, sufficient local concentrations cannot be maintained to achieve a substantial effect. Phenobarbital had a greater effect in the medial than in the lateral septum. The inhibition of choline uptake by phenobarbital was blocked by coadministration of picrotoxin in the medial septum. In contrast, the stimulation of choline uptake in the hippocampus by picrotoxin occurred when it was injected in the lateral septum but not in the medial septum. These data suggest that GABAergic receptor complexes in the medial septal area do not have a tonic inhibitory effect on septal cholinergic neurons. They also suggest there may be other GABAergic receptor complexes in the lateral septum which are tonically active and may influence the medial septal cholinergic neurons by an unknown pathway. PMID- 3009795 TI - Characterization and autoradiographic visualization of (+)-[3H]SKF10,047 binding in rat and mouse brain: further evidence for phencyclidine/"sigma opiate" receptor commonality. AB - The binding specificity of (+)-[3H]N-allylnormetazocine, the dextrorotatory isomer of the prototypical sigma opiate SKF10,047, was determined in rat and mouse brain and the neuroanatomical distribution of its binding sites elucidated by quantitative autoradiography in sections of rat brain. Computer-assisted Scatchard analysis revealed an apparent two-site fit of the binding data in both species and in all rat brain regions examined. In whole rat brain, the Kd values were 3.6 and 153 nM and the maximum binding values were 40 fmol and 1.6 pmol/mg of protein for the apparent high- and low-affinity binding sites, respectively. (+)-SKF10,047, haloperidol and pentazocine were among the most potent inhibitors of 7 nM (+)-[3H]SKF10,047 binding to the higher affinity sites; rank orders of ligand potencies at these sites differ sharply from those that have been reported for the [3H]phencyclidine (PCP) site, or for eliciting PCP-like or SKF10,047-like behaviors. By contrast, rank orders of potency of sigma opiods, PCP derivatives and dioxolanes for displacement of 100 nM (+)-[3H]SKF10,047 from the more numerous lower affinity sites in the presence of 100 nM haloperidol agreed closely with their potencies in the [3H]PCP binding assay as well as their potencies in exerting PCP- or SKF10,047-like behavioral effects. In order to compare directly the anatomical localizations of PCP and (+)-SKF10,047 binding sites, quantitative light microscopy autoradiography utilizing tritium-labeled PCP and (+)-SKF10,047 was carried out in rat brain sections. (+)-[3H]SKF10,047 binding was observed to follow the regional pattern of [3H]PCP binding but also to bind in other regions not associated with PCP receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009796 TI - Study of a neuronal potassium current in different culture conditions. AB - The potassium current of neurons in explants cultured from quail mesencephalic neural crest were studied in voltage clamp, using the whole cell recording technique. Two voltage-dependent potassium currents were identified; they differed in their sensitivity to blocking agents and to sustained depolarizing voltages. The potassium current component most sensitive to 4-aminopyridine had fast activation kinetics and inactivated quickly at sustained depolarized voltages. By analogy with a current described in other preparations, this current was called IA. The current component most sensitive to tetraethylammonium had slower activation kinetics and inactivated more slowly than IA at sustained depolarized voltages. This current was called IK. The properties of IA and IK were examined in neurons cultured in a defined medium and in neurons co-cultured with striated muscle. The rate of inactivation of IA appeared to be increased when neural crest neurons were cultured in the presence of striated muscle. The change in the properties of IA could be due to a direct effect of the co-culture with muscle on the membrane current; another possibility could be that co-culture favors the survival of a neuronal population that does not survive well when cultured in a defined medium. PMID- 3009798 TI - Bilateral cystosarcoma phylloides with osteogenic sarcomatous stroma (a case report with review of literature). PMID- 3009797 TI - Three types of transmitter release from embryonic neurons. AB - Prior to the contact with their target muscle cells in culture, growth cones of many isolated Xenopus embryonic neurons release acetylcholine (ACh) spontaneously. Using patch clamp techniques, this release can be detected by an outside-out patch of muscle membrane placed near the growth cone. Intracellular recording from innervated muscle cells showed spontaneous miniature endplate potentials (MEPPs) of varying amplitudes. Amplitude histograms showed a skewed distribution with multiple peaks, suggesting the existence of subunits in either the quantal packages of ACh released by the nerve terminal or in the postsynaptic muscle response. In addition to the quantal ACh release reflected by MEPPs, nerve terminal also release a large amount of ACh in a non-quantal fashion. This non quantal ACh release is revealed by the hyperpolarization of the muscle membrane following extracellular application of curare or alpha-bungarotoxin, as well as by denervation of the muscle cell. PMID- 3009799 TI - Carbohydrate influences the immunogenic and antigenic characteristics of the ZP3 macromolecule (Mr 55 000) of the pig zona pellucida. AB - The glycoprotein molecule ZP3 of the pig zona pellucida (Mr 55 000) was deglycosylated by trifluoromethane-sulphonic acid. While gas chromatography established that deglycosylation resulted in greater than 91% carbohydrate removal, deglycosylated ZP3 (DG-ZP3) still retained significant immunogenic potential as shown by its ability to elicit antibody production in the rabbit. Several lines of evidence, however, demonstrated that deglycosylation produced significant modifications in the ZP3 macromolecule which profoundly altered its antigenicity: 125I-radiolabelling procedures using chloramine-T consistently resulted in 125I-labelled ZP3 with a higher specific activity than 125I-labelled DG-ZP3; in competitive binding radioimmunoassays (RIAs) using an 125I-labelled ZP3 vs anti-ZP3 system, unlabelled DG-ZP3 successfully displaced ZP3 in a dose related manner; in direct binding RIAs comparing the reaction of labelled ZP3 and DG-ZP3 versus 5 different zona antisera, higher titres were consistently achieved with 125I-labelled ZP3; as compared to ZP3, 2-dimensional immunoelectrophoresis using DG-ZP3 as antigen yielded significantly modified precipitin arc patterns; with electrophoretic blotting procedures, antisera to ZP3 or DG-ZP3 cross-reacted with the heterologous antigen; treatment of pig zonae with anti-DG-ZP3 serum produced a dense precipitation layer on the zona surface. When evaluated collectively, these results provide grounds for an important role for protein and carbohydrate in establishing the immunological characteristics of the Mr 55 000 macromolecule of the pig zona pellucida. PMID- 3009800 TI - Differential regulation by LH and prostaglandins of steroidogenesis in small and large luteal cells of the ewe. PMID- 3009801 TI - Ovarian oxytocin and the maternal recognition of pregnancy. AB - The secretion of oxytocin by the corpus luteum is thought to stimulate the episodic release of PGF-2 alpha from the uterus, thereby contributing to luteolysis. In pregnancy corpus luteum function is maintained, and secretion of oxytocin, or its actions on the uterus, appear to be inconsistent with the successful establishment of gestation. Protection against the effects of oxytocin is ensured by a number of mechanisms, including the cessation of luteal oxytocin secretion, which is evident by Day 20 after mating in sheep, and the maintenance of low levels of the oxytocin receptor in the uterus. PMID- 3009802 TI - Psychosocial aspects of gestational trophoblastic disease in Chinese residents of Hong Kong. AB - Gestational trophoblastic disease has several special aspects as compared with others tumors of the female genital tract. It occurs in young women who want to start a family and expect to have a normal pregnancy. It can be very effectively treated with chemotherapeutic agents, and the subsequent reproductive potential of these young women is not affected. In a survey of 105 Chinese residents of Hong Kong who had had the disease, it was found that their emotional reactions to the disease and treatment, the effects of such on their self-esteem, martial and sexual life, and their attitudes towards their physicians and future pregnancy were different from those of their Western counterparts. PMID- 3009803 TI - Hormonal measurements in patients with theca lutein cysts and gestational trophoblastic disease. AB - Concentrations of human chorionic gonadotropin (HCG), human placental lactogen (HPL), prolactin (PRL), follicle-stimulating hormone (FSH), estradiol (E2) and progesterone (P) were measured in serum and fluid from ovarian theca lutein cysts (TC) in patients with gestational trophoblastic disease (GTD). Either intact hydatidiform mole (HM) or persistent GTD was present. The values were compared to serum hormone concentrations in ten GTD patients whose ovaries were not enlarged. In the presence of intact HM and TC, significant elevations in the mean serum concentration of HCG, PRL, P and E2 were observed when compared to levels in GTD patients with normal-sized ovaries (P less than .01). Serum FSH and HPL were not elevated in either control or TC patients. The mean concentration of P in cyst fluid from patients with intact HM was higher than that in patients with persistent GTD (P less than .05). From both groups of GTD patients with TC, the mean concentrations of P in cyst fluid were higher than those in the sera. These findings suggest that besides the markedly elevated HCG levels generally seen in TC patients, other hormones, such as P, PRL and E2 are elevated and may be involved in the formation and/or maintenance of TC. PMID- 3009804 TI - Meigs's syndrome and ovarian thecoma in pregnancy. A case report. AB - Ovarian thecoma is a rare tumor, accounting for less than 1% of all ovarian tumors. Thecomas occur even more infrequently during pregnancy, as evident from the fact that there are only 16 such case reports. A 16-year-old, pregnant, black woman presented with ovarian thecoma, pleural effusion and ascites at 32 weeks' gestation. To our knowledge, this was the first reported case that fulfills the criteria of Meigs's syndrome during pregnancy. Ovarian tumors in pregnancy may have very subtle clinical manifestations, making the diagnosis a challenge. Delivery by cesarean section at term is recommended in these patients for the best maternal and fetal outcome. PMID- 3009805 TI - Cytomegalovirus infection and fetal death in a twin. A case report. AB - In a diamnionic-dichorionic male twin pregnancy, one twin was stillborn, with disseminated cytomegalovirus (CMV) identified morphologically, and the other was liveborn, without clinical or laboratory evidence of CMV infection. The placenta of the affected twin had chronic villitis; that of the liveborn was normal. Although the discordance went unexplained, it illustrates the clinical variability of congenital CMV infection. Dissimilar immune responses between the affected and unaffected twin or direct extension of uterine CMV infection to only one twin may have been responsible for the discordance. PMID- 3009807 TI - Lymphocyte responses to viral antigens in rheumatoid arthritis. PMID- 3009806 TI - Effects of gold compounds on leukotriene B4, leukotriene C4 and prostaglandin E2 production by polymorphonuclear leukocytes. AB - The effects of auranofin (AF) and sodium aurothiomalate (GSTM) on the production of specific arachidonic acid metabolites by chemotactic tripeptide activated polymorphonuclear leukocytes has been investigated using radioimmunoassay techniques. AF insignificantly enhanced the production of leukotrienes B4 and C4 at a concentration of 0.5 microgram Au/ml. However, at increasing concentrations, this drug suppressed the production of these metabolites in a dose dependent manner. In contrast, GSTM did not affect the production of either leukotriene at the concentrations tested. Of particular interest, prostaglandin E2 production was not affected by either gold compound. Both leukotrienes and prostaglandins are metabolized from arachidonic acid and are potent mediators of inflammation. The inhibition of leukotriene production may be another mechanism by which AF manifests its antiinflammatory effects in patients with rheumatoid arthritis. PMID- 3009808 TI - Neurological dysfunction following coronary artery bypass graft surgery. PMID- 3009809 TI - Haematuria due to urinary bladder metastases from small cell carcinoma of the bronchus. PMID- 3009810 TI - Structurally specific binding of halogenated biphenyls to thyroxine transport protein. AB - Prealbumin is a major thyroxine binding protein in blood that has been well studied crystallographically and has also been proposed as a model for the thyroxine nuclear receptor in tissue. The high-affinity T4 binding site in prealbumin gave a linear plot on Scatchard analysis. The interactions of selected polychlorinated biphenyls (PCBs) with prealbumin have been studied with use of computer graphics and predictions made regarding relative binding affinities for such structures. These modeling predictions were tested by using competitive binding experiments involving selected PCBs and hydroxylated derivatives as soluble structural probes. The results are in excellent agreement with the modeling predictions and demonstrated that these compounds can be highly effective (3-8 times better than thyroxine itself) competitive binding ligands for thyroxine specific binding sites in prealbumin. Laterally (3,3',5,5'-) substituted PCBs show the highest binding activity and further substitution on nonlateral (2,2',6,6'-) positions lowers binding activity. Lateral chlorine substitution was common to all PCBs studied that showed high binding affinities. The binding model may also suggest a preference for a linear and symmetrical molecular shape. These structural requirements for binding are substantially consistent with the structure-toxicity relationship for closely related compounds of environmental interest. These specific binding interactions are likely to modulate the distribution of certain PCBs and related compounds and alter hormone protein interactions that are responsible for the maintenance of normal thyroid status. Since prealbumin is also a model for the putative thyroxine nuclear receptor in tissue, our hypothesis that high toxicity of certain halogenated aromatic hydrocarbons is at least in part due to their thyromimetic properties is further supported. PMID- 3009813 TI - Probes for narcotic receptor mediated phenomena. 9. Synthesis of (+/-)-(3 alpha,6a alpha,11a beta)-1,3,4,5,6,11a-hexahydro-2-methyl-2H-3,6a- methanobenzofuro[2,3-c]azocin-10-ol, an oxide-bridged 5-(m-hydroxyphenyl)morphan. AB - The synthesis of racemic (3 alpha,6a alpha,11a beta)-1,3,4,5,6,11a- hexahydro-2 methyl-2H-3,6a-methanobenzofuro[2,3-c]azocin -10-ol (2d) is described. The route used acid-catalyzed ring closure of enamine 5 to yield the unsaturated phenylmorphan 6. Conversion of 6 to oxide-bridged 2d was accomplished in a multistep fashion that utilized the introduction of a bromine atom, followed by O demethylation of the phenolic methyl ethers and base-catalyzed intramolecular phenoxide displacement of the bromine. Compound (+/-)-2d represents an oxide bridged derivative of the potent 5-(m-hydroxyphenyl)morphan class of opioid analgesics 1. Unlike the 5-(m-hydroxyphenyl)morphans that have a freely rotating phenyl group, 2d has the phenyl ring conformationally restricted at an angle of 49 degrees relative to atoms 1, 3, 11a, and 12 of 2d. The low binding of (+/-)-2d to rat brain homogenate receptor preparations [IC50 = 1000 nM] may indicate that the phenyl angle of 49 degrees is not suitable for binding to opioid receptors. PMID- 3009812 TI - Conformational effects on the activity of drugs. 11. Stereostructural models for the direct activation of the alpha- and beta-adrenergic receptor. AB - Two kinds of cyclic analogues of norepinephrine (NE, 7) and isoprenaline (ISO, 8), in which the C(1)-C(2) side chain of these amino alcohols is incorporated in its preferred conformation in the ring of the 2-(3,4-dihydroxyphenyl)-morpholines 9 and 10 (2-DPMs) and in the ring of the 3-(3,4-dihydroxyphenyl)-3-piperidinols 11 and 12 (3-DPPs), respectively, were synthesized and assayed for their adrenergic activity on various isolated preparations. The 2-DPMs and the 3-DPPs showed an alpha- and beta-agonist activity comparable to that of NE and ISO and to that of the trans-2-amino-5,6-dihydroxytetrahydronaphthalen-1-ols 13 and 14 (2 ADTNs), which represent another kind of semirigid analogue of NE and ISO. Through a comparison of the stereo structures of the compounds examined and of their pharmacological properties, it was possible to suggest a spatial situation in which the pharmacophoric groups of the adrenergic drugs examined (aryl moiety, amine nitrogen, and alcoholic or ethereal benzylic oxygen) should interact at the receptor site. This spatial situation corresponds to the one found in the preferred conformation of NE and ISO. It was also possible to construct two theoretical three-dimensional molecular models that provide information about steric requirements for the direct activation of alpha- and beta-adrenoceptors, respectively. PMID- 3009811 TI - Synthesis and antiherpes virus activity of phosphate and phosphonate derivatives of 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine. AB - A series of phosphate esters of 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 1) were synthesized and evaluated for antiherpes virus activity. The cyclic phosphate esters were made by a new, efficient method utilizing stannic chloride as a solubilizing agent. Monophosphate 2 and bisphosphate 4 showed comparable activity to DHPG and probably acted as prodrugs of DHPG. On the other hand, the cyclic phosphate of DHPG 3 was taken up by cells and bypassed the virus-specified thymidine kinase. As a result, 3 was active against DHPG-resistant HSV mutants that lacked the viral-specified thymidine kinase and was more toxic than DHPG to uninfected cells. The phosphonate 5, the least toxic of the derivatives tested, was only marginally active against HSV but showed substantial activity against human cytomegalovirus in vitro. PMID- 3009814 TI - Angiotensin-converting enzyme inhibitors: new orally active 1,4-thiazepine-2,5 diones, 1,4-thiazine-2,5-diones, and 1,4-benzothiazepine-2,5-diones possessing antihypertensive activity. AB - The preparation of a series of 1,4-thiazepine-2,5-diones, 1,4-thiazine-2,5 diones, and 1,4-benzothiazepine-2,5-diones and their ability in inhibiting the activity of angiotensin-converting enzyme (ACE) in vitro and in vivo were examined. These compounds are assumed to act as prodrugs since they undergo rapid ring-opening reactions to give the corresponding biologically active free SH compounds when incubated with rat plasma or when treated with aqueous 0.1 N HCl or phosphate buffer (pH 7.4). The thiazepines 23-25 and 30 are potent inhibitors of ACE when administered po to rats and are comparable in potency to captopril (1). The most active thiazines in rats, po, were 42 and 45. Of the benzothiazepines studied, 22a was the most active in inhibiting ACE in the conscious normotensive rat, ID50 = 0.15 mg/kg, po. The acute antihypertensive effects of oral administration of a number of these compounds on mean arterial pressure and heart rate were studied in spontaneously hypertensive rats (SHR) maintained on a sodium-deficient diet. PMID- 3009815 TI - Synthesis and antiviral properties of 5-(2-substituted vinyl)-6-aza-2' deoxyuridines. AB - The following 5-(2-substituted vinyl)-6-aza-2'-deoxyuridines were synthesized: (E)-5-(2-bromovinyl) (2) (6-aza-BVDU), 5-(2-bromo-2-fluorovinyl) (a mixture of E and Z isomers) (3), (E)-5-(2-chlorovinyl) (4), (E)-5-[2-(methylthio)vinyl] (5), 5 (2,2-dibromovinyl) (6), and 5-(3-furyl) (7). The synthesis of 2-6 utilized Wittig type reactions on 5-formyl-1-(2'-deoxy-3', 5'-di-O-p-toluoyl-beta-D-erythro pentofuranosyl)-6-azauracil (16). 6-Aza-BVDU (and its alpha-anomer) was also synthesized from (E)-5-(2-bromovinyl)-6-azauracil (12) by using standard deoxyribosidation methodology. Compound 7 was prepared from 5-(3-furyl)-6 azauracil (33) via a ribosidation/deoxygenation sequence. An attempt to prepare the corresponding 5-(2,2-difluorovinyl) analogue afforded instead a mixture of the 5-[(2,2-difluoro-2-methoxy)ethyl] and 5-(2,2,2-trifluoroethyl) derivatives 29 and 30. Compounds 2-7, 29, and 30 were tested for in vitro activity against herpes simplex virus types 1 and 2 (HSV-1, HSV-2). 6-Aza-BVDU (2) exhibited ID50s of 8 micrograms/mL vs. HSV-1 and 190 micrograms/mL vs. HSV-2. BVDU (1) had ID50s of 0.015 and 1.6 micrograms/mL against HSV-1 and HSV-2, respectively. Compound 4 showed a similar profile of activity, but the other analogues were either weakly active or inactive. PMID- 3009816 TI - Enzymatic phosphorylation of the antiherpetic agent 9-[(2,3-dihydroxy-1 propoxy)methyl]guanine. AB - The antiherpetic agent 9-[(2,3-dihydroxy-1-propoxy)methyl]guanine (iNDG) is phosphorylated by HSV1 thymidine kinase, and its phosphorylated products inhibit DNA polymerase activity. iNDG exists in two enantiomeric forms, each with a primary and a secondary hydroxyl; thus, a number of possibilities for preferential phosphorylation exist, which were explored in this study. HSV1 thymidine kinase phosphorylates the primary hydroxyl of both the R and the S isomers of iNDG. This was established by comparison with analogues in which either the primary or the secondary hydroxyl was replaced by fluorine or hydrogen and also by a study of the NMR spectrum of the monophosphate. GMP kinase phosphorylates the R and the S monophosphates to the respective diphosphates. Further phosphorylation, however, is much more efficient with the S than with the R isomer. Furthermore, (S)-iNDG triphosphate is a more potent inhibitor of HSV1 DNA polymerase than (R)-iNDG triphosphate. These differences in the biochemical specificities of the two isomers account for the observed higher antiviral potency of (S)-iNDG as compared to that of (R)-iNDG. PMID- 3009818 TI - The role of plasmid genes in the pathogenicity of Salmonella dublin. AB - The virulence (expressed as LD50 values) for mice of two mutant strains of Salmonella dublin, both containing TnA insertions in the resident plasmid, was reduced by 10(4)-10(5) when infection was by the oral or intravenous or intraperitoneal route. When the plasmid was lost from one of the mutants no further decrease in virulence was observed. Results also suggested that plasmid genes are not involved in the ability of S. dublin to cross the gut wall. PMID- 3009817 TI - Investigation of the structural requirements for the kappa-selective opioid receptor antagonist, 6 beta,6 beta'-[ethylenebis(oxyethyleneimino)]bis[17 (cyclopropylmethyl)- 4,5 alpha-epoxymorphinan-3,14-diol] (TENA). AB - In an effort to determine whether or not the basic nitrogens in the spacer of the bivalent ligand 6 beta,6 beta'-[ethylenebis(oxyethyleneimino)]bis[17 (cyclopropylmethyl)4,5 alpha-epoxymorphinan-3,14-diol] (TENA, 1) is responsible for its selective kappa opioid antagonist activity, we have synthesized monovalent analogues 2-4 that contain a C-6 side chain with basic nitrogens. Analogue 2 behaved as a potent opioid agonist in the guinea pig ileum preparation (GPI) and possessed no significant kappa opioid antagonist activity (IC50 ratio = 1) relative to TENA (IC50 ratio = 20). The agonist activity of 3 and 4 interfered with the opioid antagonist assay and therefore did not permit evaluation of antagonist activity in a concentration range where TENA is effective. Although the results obtained with 2 are consistent with the requirement of a second opiate pharmacophore (rather than a second basic nitrogen in the spacer) for the kappa antagonist activity of TENA, the potent agonism associated with these monomers do not allow a firm conclusion in this regard. PMID- 3009819 TI - Rhesus monkeys (Macaca mulatta) as a model for calcium pyrophosphate dihydrate crystal deposition disease. AB - Calcium pyrophosphate dihydrate crystal deposition disease (CPDD) was recognized in 4 of 30 free-ranging rhesus macaques. By means of tissue radiography, focal radiodensities were noted in lumbar intervertebral discs, menisci, and articular cartilage. Crystal deposits were identified as calcium pyrophosphate dihydrate (Ca2P2O7 X 2H2O) by means of X-ray diffraction. The pathogenesis of calcium pyrophosphate dihydrate arthropathy in man remains elusive. However, with the recognition of this arthritis in a well defined population of aged nonhuman primates, a model now exists to facilitate the study of this disease. PMID- 3009820 TI - Monitoring ovulation and implantation in the cynomolgus macaque (Macaca fascicularis) through evaluations of urinary estrone conjugates and progesterone metabolites: a technique for the routine evaluation of reproductive parameters. AB - Investigations were undertaken to determine the applicability of recently reported specific radioimmunoassays for urinary estrone conjugates and progesterone metabolites for monitoring ovarian function in the cynomolgus macaque (Macaca fasciularis) and other macaque species. Mean estrone conjugate measurements appear to accurately reflect the preovulatory estrogen peak in both conceptive (n = 5) and nonconceptive (n = 6) cycles, as well as to indicate early pregnancy through increases which are significantly elevated by Day + 15 (p less than 0.049) post estrone conjugates peak. The mean luteal phase levels of these progesterone metabolites are significantly elevated by Day + 14 (p less than 0.012) in conceptive cycles when compared to the mean values for nonconceptive cycles. PMID- 3009823 TI - Amiloride-sensitive Na+ transport in human red cells: evidence for a Na/H exchange system. AB - The role of transmembrane pH gradients on the ouabain, bumetanide and phloretin resistant Na+ transport was studied in human red cells. Proton equilibration through the Jacobs-Stewart cycle was inhibited by the use of DIDS (125 microM) and methazolamide (400 microM). Red cells with different internal pH (pHi = 6.4, 7.0 and 7.8) were prepared and Na+ influx was measured at different external pH (pHo = 6.0, 7.0, 8.0). Na+ influx into acid-loaded cells (pHi = 6.4) markedly increased when pHo was raised from 6.0 to 8.0. Amiloride, a well-known inhibitor of Na+/H+ exchange systems blocked about 60% of the H+-induced Na+ entry, while showing small inhibitory effects in the absence of pH gradients. When pHo was kept at 8.0, the amiloride-sensitive Na+ entry was abolished as pHi was increased from 6.4 to 7.8. Moreover, measurements of H+ efflux into lightly buffered media indicated that the imposition of an inward Na+ gradient stimulated a net H+ efflux which was sensitive to the amiloride analog 5-N-methyl-N-butyl-amiloride. Furthermore, in the absence of a chemical gradient for Na+ (Nai+ = Nao+ = 15 mM, Em = +6.7 mV), an outward H+ gradient (pHi = 6.4, pHo = 8.0) promoted a net amiloride-sensitive Na+ uptake which was abolished at an external pH of 6.0. These findings are consistent with the presence of an amiloride-sensitive Na+/H+ exchange system in human red cells. PMID- 3009824 TI - Steric hindrance in immunolabelling. AB - In this paper we describe immunocytochemical detection of PhoE pore protein in the outer membrane of E. coli K-12 cells in dependence of a variety of labelling approaches. Immuno-gold labelling on ultrathin cryosections showed a uniform, dense labelling of the outer membrane of all cells. However, using immunofluorescence, whole-mount or freeze-etch labelling methods, labelling was limited to less than 5% of the cell population. In order to understand this phenomenon, immunoincubated cells exhibiting less than 5% labelling were processed for cryo-ultramicrotomy. Reincubation of the sections with antibody and probe resulted in labelling of all of the cells. If an E. coli Gal-U mutant strain, defective in the lipopolysaccharide (LPS) carbohydrate chain length, was used, each approach rendered 100% labelling. From these results it is concluded that the antigenic sites of the PhoE pore protein are not accessible in intact 'wild-type' cells due to steric hindrance caused by the LPS carbohydrate chains. In cryosections this steric hindrance is partly precluded since antigenic determinants are exposed at the section surface in transversely cut membranes. Our results emphasize the necessity to compare results obtained by means of several, basically different approaches. PMID- 3009825 TI - Structural and functional studies of insertion element IS200. AB - The nucleotide sequence of the insertion element IS200 has been determined partially, including the junctions between the element and the host chromosome at the insertion site. At most, two bases (A-A) are found repeated at the junctions and could be duplications of host sequences generated by the insertion of the element. No obvious sequence repeats, either direct or inverted, have been detected between the sequences just within the two ends of the element. The element is an extremely strong block to host transcription across the insertion site. A sequence similar to known transcription termination signals was found just within the element near the right end. Removal of less than 50 base-pairs at the right end of the element abolishes the transcription block. The putative terminator sequence is located within this 50 base-pair region. Genetic studies suggest that the element contains a promoter located more than 93 base-pairs from its left end. The proposed promoter and terminator are in proper orientation to form an internal transcription unit. PMID- 3009822 TI - Mechanisms of regulation of the Na+/H+ exchanger. PMID- 3009826 TI - Chromosome-specific subfamilies within human alphoid repetitive DNA. AB - Nucleotide sequence data of about 20 X 10(3) base-pairs of the human tandemly repeated alphoid DNA are presented. The DNA sequences were determined from 45 clones containing EcoRI fragments of alphoid DNA isolated from total genomic DNA. Thirty of the clones contained a complete 340 base-pair dimer unit of the repeat. The remaining clones contained alphoid DNA with fragment lengths of 311, 296, 232, 170 and 108 base-pairs. The sequences obtained were compared with an average alphoid DNA sequence determined by Wu & Manuelidis (1980). The divergences ranged from 0.6 to 24.6% nucleotide changes for the first monomer and from 0 to 17.8% for the second monomer of the repeat. On the basis of identical nucleotide changes at corresponding positions, the individual repeat units could be shown to belong to one of several distinct subfamilies. The number of nucleotide changes defining a subfamily generally constitutes the majority of nucleotide changes found in a member of that subfamily. From an evaluation of the proportion of the total amount of alphoid DNA, which is represented by the clones studied, it is estimated that the number of subfamilies of this repeat may be equal to or exceed the number of chromosomes. The expected presence of only one or a few distinct subfamilies on individual chromosomes is supported by the study, also presented, of the nucleotide sequence of 17 cloned fragments of alphoid repetitive DNA from chromosome 7. These chromosome-specific repeats all contain the characteristic pattern of 36 common nucleotide changes that defines one of the subfamilies described. A unique restriction endonuclease (NlaIII) cleavage site present in this subfamily may be useful as a genetic marker of this chromosome. A family member of the interspersed Alu repetitive DNA was also isolated and sequenced. This Alu repeat has been inserted into the human alphoid repetitive DNA, in the same way as the insertion of an Alu repeat into the African green monkey alphoid DNA. PMID- 3009827 TI - Binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid R6K. AB - The three replication origins of the antibiotic resistance plasmid R6K require for their activity in Escherichia coli a DNA segment containing seven 22 base pair direct repeats and a plasmid-encoded initiation protein (pi). The pi protein functions in the negative control of R6K replication, in addition to its requirement for the initiation of replication. Construction of a plasmid containing the pi structural gene (pir) downstream from the inducible pR promoter of bacteriophage lambda provided high levels of production of pi protein in E. coli. The pi protein was purified and shown to possess general DNA binding properties with a preference for DNA fragments containing the gamma origin of replication, the operator region of the pir gene and the R6K beta-origin region. Velocity sedimentation analysis indicates that the pi protein exists as a dimer in its native form. Agarose gel electrophoresis analysis of pi-gamma-origin complexes suggests that one pi dimer binds to each copy of the 22 base-pair direct repeats in the gamma origin region. Purified mutant pi protein obtained from a temperature-sensitive initiation mutant (pir 105-ts) exhibited temperature sensitive binding activity to the gamma-origin region, whereas two mutant proteins exhibiting a high copy number phenotype were unaltered (pir104-cop) or slightly reduced (pir1-cop) in binding activity. The patterns of DNase I protection and enhancement were similar for the wild-type and mutant proteins examined. PMID- 3009821 TI - The role of phosphoinositides in signal transduction. PMID- 3009828 TI - Conservation throughout mammalia and extensive protein-encoding capacity of the highly repeated DNA long interspersed sequence one. AB - We report an investigation of the structure, evolutionary history, and function of the highly repeated DNA family named Long Interspersed Sequence One (L1). Hybridization studies show, first, that L1 is present throughout marsupial and placental mammalian orders. Second, L1 is more homologous within these species than between them, which suggests that it has undergone concerted evolution within each mammalian lineage. Third, on the whole L1 diverges in accordance with the fossil record. This suggests that it arose in each lineage rather by inheritance from a common ancestral family, which was present in the progenitor to mammals, than by cross-species transmission. Alignment of 1.6 X 10(3) bases of primate and mouse L1 DNA sequences shows a predominance of silent mutations within aligned long open reading frames, indicating that at least this part of L1 has produced functional protein. The observation of additional long open reading frames in further unaligned DNA sequences suggests that a minimum of 3.2 X 10(3) bases or at least half of the L1 structure is a protein-coding sequence. Thus L1, which contains about 100,000 members in mouse, is by far the most repetitive family of which a subset comprises functional protein-encoding genes. The ability of the putative protein-encoding regions of mouse L1 to hybridize to L1 homologs throughout the Mammalia implies that these sequences have been subject to conservative selection upon protein function in all mammalian lineages, rather than in a few. L1 is therefore a highly repeated family of genes with both a widespread and an ancient history of function in mammals. PMID- 3009830 TI - Isolation and characterization of sarcomeric actin genes expressed in Xenopus laevis embryos. AB - A Xenopus laevis complementary DNA (cDNA) library prepared from messenger RNAs extracted from embryos has been screened for actin-coding sequences. Two cDNA clones corresponding to an alpha cardiac and an alpha skeletal muscle actin mRNA have been identified and characterized. From a genomic library, we have furthermore isolated the genes that correspond to the characterized cDNAs. In addition we have identified an actin processed gene which seems to be derived from a second type of skeletal muscle actin gene. Southern blot analysis of X. laevis DNA reveals that each of the three genes is present in at least two copies. In Xenopus tropicalis, a similar Southern blot analysis demonstrates that the three alpha actin genes exist as single copy. This result correlates with the genome duplication that has been proposed to have occurred recently in a X. laevis ancestor. A sequence comparison of the X. laevis cardiac and skeletal muscle actin cDNAs shows that the encoded peptides are highly conserved. Nevertheless, the numerous nucleotide changes at silent mutation sites suggest that the genes originated before the amphibia/reptile-bird divergence, more than 350 million years ago. Comparison of the promoters of the cardiac and skeletal actin genes, which are co-expressed in embryos, reveals a few common structural sequence elements. PMID- 3009829 TI - Partial nucleotide sequence of the Murray Valley encephalitis virus genome. Comparison of the encoded polypeptides with yellow fever virus structural and non structural proteins. AB - The sequence of 5400 bases corresponding to the 5'-terminal half of the Murray Valley encephalitis virus genome has been determined. The genome contains a 5' non-coding region of about 97 nucleotides, followed by a single continuous open reading frame that encodes the structural proteins followed by the non-structural proteins. Amino acid sequence homology between the Murray Valley encephalitis and yellow fever (Rice et al., 1985) polyproteins is 42% over the region sequenced. The start points of the various Murray Valley encephalitis virus-coded proteins have been assigned on the basis of this homology and a consistent set of potential proteolytic cleavage sites identified, the sequences of which are similar in Murray Valley encephalitis and yellow fever. The deduced Murray Valley encephalitis gene order is 5'-C-prM (M)-E-NS1-ns2a-ns2b-NS3-3'. The genome organization of Murray Valley encephalitis and yellow fever appears to be identical and the sizes of the predicted virus-coded proteins similar between the two viruses. Both viruses encode a basic capsid protein followed by three glycoproteins; the glycoproteins appear to have the conventional topology of N terminus outside with a C-terminal membrane-spanning domain. There are conserved glycosylation sites in prM, the precursor to the M protein of the virion, and in NS1, a non-structural protein of uncertain function. The glycosylation sites in E, the major envelope protein of the virion, are not conserved as to position. We predict the existence, in flavivirus-infected cells, of two small, hydrophobic peptides, ns2a and ns2b, which show only limited amino acid sequence homology. Finally, about half of the amino acid sequence of NS3 has been obtained; NS3 is a hydrophilic non-structural protein that shows 55% amino acid sequence similarity between Murray Valley encephalitis and yellow fever over the region sequenced and is probably involved in RNA replication. PMID- 3009831 TI - Transcription initiation of the Saccharomyces cerevisiae iso-1-cytochrome c gene. Multiple, independent T-A-T-A sequences. AB - The expression of the Saccharomyces cerevisiae CYC1 gene, which encodes iso-1 cytochrome c, produces a family of messenger RNAs whose 5' ends map in the region from position +7 to -93 relative to the first nucleotide at position +1 of the protein-coding DNA sequence. The mechanism of transcription initiation of the CYC1 gene has been examined by using linker-scanning deletions and gene fusions. The various CYC1 derivatives with mutations in the 5' non-coding region were constructed, reintroduced into yeast using a multicopy plasmid, and the mRNA starts mapped by primer extension. The results indicate that four, and possibly five T-A-T-A sequences are located within the 5' non-coding region of the CYC1 gene, and that each T-A-T-A is required for a specific subset of mRNA starts. This conclusion has been confirmed by oligonucleotide mutagenesis of a chromosomal CYC1 T-A-T-A sequence. A loose spatial relationship also exists between the T-A-T-A sequences and the mRNA start sites, and this distance relationship varies from 100 to 60 base-pairs (+/- 15 base-pairs). PMID- 3009832 TI - Splicing of the E2A premessenger RNA of adenovirus serotype 2. Multiple pathways in spite of excision of the entire large intron. AB - During the early period of infection, the 4.9 kb (kb = 10(3) bases) E2A premRNA of adenovirus serotype 2 is matured mainly into a 2.0 kb mRNA by excision of introns of 2233 and 626 nucleotides. In order to define all the possible steps of splicing occurring in vivo, we characterized splicing intermediates present after a limiting treatment of cells with cycloheximide. Three complementary methods of analysis were used: RNA transfer analysis, S1 nuclease mapping and complementary DNA-RNase assay. Our principal conclusions concerning the poly(A)+ species are as follows. The RNA intermediate family detected is more complex than expected, since two major RNA intermediates of 4.6 kb and 4.3 kb, two minor intermediates of 2.9 kb and 2.6 kb, and a 2.3 kb RNA, which represents a minor alternative mRNA form, are revealed. Despite its large size and the presence of multiple internal donor and acceptor signals, intron 1 is exclusively excised as a whole. Intron 2 is either primarily excised as a whole, removing the standard 626-nucleotide sequence, or a smaller sequence of 337 nucleotides is removed, generating the 2.3 kb alternative mRNA. Kinetics of the ligation reaction demonstrate that the minimal time for excision of intron 2 is no more than two minutes, indicating a high level of co-ordination of the multiple individual reactions occurring during excision of an intron. Besides the major pathway for E2A premRNA splicing, namely the excision first of intron 2, followed by the excision of intron 1 after a lag time of five minutes, a minor pathway (used with a frequency of 10%) can be detected where the order of intron excision was inverted. With the alternative variant of excision of intron 2, at least three different pathways are therefore used to mature the E2A premRNA. RNA intermediates resulting from the cleavage at the 5' end of introns and branching can be detected by S1 mapping experiments, but their low accumulative level (1% relatively to the initial premRNA) precluded their direction by RNA transfer experiments and their complete characterization. PMID- 3009835 TI - Evolution of cytochrome c genes and pseudogenes. AB - A statistical analysis of the nucleotide sequences of cytochrome c genes from four species of animals and two of yeast and of cytochrome c pseudogenes from rat, mouse, and human was conducted. It was estimated that animals and yeast diverged 1.2 billion years ago, that the two duplicated genes DC3 and DC4 in Drosophila diverged 520 million years ago, and that the two duplicated genes Iso 1 and Iso-2 in the yeast Saccharomyces cerevisiae diverged 200 million years ago. DC3 is expressed at a low level and has evolved 3 times faster than DC4. This observation supports the neutralist view that relaxation of functional constraints is a more likely cause of accelerated evolution following gene duplication than is advantageous mutation. All the rodent pseudogenes examined appear to be processed pseudogenes derived directly from the functional genes, and most of them apparently arose after the mouse-rat split. No event of gene conversion could be detected between any pair of the rodent pseudogenes. Our analysis suggests that the human cytochrome c gene has evolved at a rate comparable to the average rate for pseudogenes, whereas some human cytochrome c pseudogenes have evolved at an exceptionally low rate. PMID- 3009833 TI - Evidence of sequences resembling avian retrovirus long terminal repeats flanking the trout protamine gene. AB - Additional TATA boxes are present in the flanking regions of trout protamine genes. Their activity as promoters was assayed using an in vitro transcription system. These additional TATA boxes, together with polyadenylation signals that include the consensus AATAAA and CACTG sequences very close to the promoters, suggest that these sequences may be closely related to retroviral long terminal repeat (LTR) sequences. Other features of retroviral LTRs that are also present are short inverted repeats. The LTR-like sequences flanking the trout protamine gene show significant homology to the avian sarcoma virus LTR over a 40-bp region. The trout protamine gene falls into the relatively rare intronless class of eukaryotic genes. This suggests that the gene could have been derived from a processed gene introduced into the genome by reverse transcription of a mature mRNA. The protamine-mRNA-coding region is flanked by AACA... TGTT sequences, which might represent vestigial traces of past recombination events and whose presence supports the notion that the protamine gene sequence was of foreign origin. Recent attempts in this laboratory to transfer the protamine gene into mouse cells have resulted in a high frequency of deletions similar to those observed with constructs in which a retrovirus was used as a vector to transfect foreign DNA with promoters. The distribution of protamine genes in the animal kingdom is very sporadic, which suggests that protamine genes appeared relatively late in evolution. The nonuniform occurrence of the gene among lower vertebrates may have been the result of its horizontal transmission only to certain species, possibly by infection with retroviruses that acquired it from a different species. PMID- 3009834 TI - Mitochondrial DNA evolution in the melanogaster species subgroup of Drosophila. AB - Detailed restriction maps (40 cleavage sites on average) of mitochondrial DNAs (mtDNAs) from the eight species of the melanogaster species subgroup of Drosophila were established. Comparison of the cleavage sites allowed us to build a phylogenetic tree based on the matrix of nucleotide distances and to select the most parsimonious network. The two methods led to similar results, which were compared with those in the literature obtained from nuclear characters. The three chromosomally homosequential species D. simulans, D. mauritiana, and D. sechellia are mitochondrially very related, but exhibit complex phylogenetic relationships. D. melanogaster is their closest relative, and the four species form a monophyletic group (the D. melanogaster complex), which is confirmed by the shared unusual length of their mt genomes (18-19 kb). The other four species of the subgroup (D. yakuba, D. teissieri, D. erecta, and D. orena) are characterized by a much shorter mt genome (16-16.5 kb). The monophyletic character of the D. yakuba complex, however, is questionable. Two species of this complex, D. yakuba and D. teissieri, are mitochondrially indistinguishable (at the level of our investigation) in spite of their noticeable allozymic and chromosomal divergence. Finally, mtDNA distances were compared with the nuclear-DNA distances thus far established. These sequences seem to evolve at rather similar rates, the mtDNA rate being barely double that of nuclear DNA. PMID- 3009838 TI - [The Reilly syndrome in oral medicine--medulla DC-potential changes induced by trigeminal stimulation]. PMID- 3009836 TI - Effects of insecticides on GABA-induced chloride influx into rat brain microsacs. AB - The actions of different insecticides known to affect binding of ligands to gamma aminobutyric acid (GABA) receptors were studied on the function of GABAA receptors in rat brain as assayed by GABA-induced 36Cl- influx into membrane microsacs. This flux was inhibited by the competitive antagonist bicuculline and the noncompetitive antagonist t-butylbicyclophosphorothionate, and the GABA effect was potentiated by the tranquilizer flunitrazepam and the depressant pentobarbital, as expected for effects on a GABAA receptor. The GABA-induced 36Cl flux was inhibited by several cyclodienes and gamma-hexachlorocyclohexane (gamma BHC) with the following order of decreasing potency: endosulfan I greater than endrin greater than endosulfan II greater than dieldrin greater than heptachlor epoxide greater than gamma-BHC greater than heptachlor. The noninsecticidal beta BHC had no effect, while the IC 50 values for gamma-BHC and endrin were 1 and 0.2 microM, respectively. The four pyrethroids tested also inhibited the GABA-induced 36Cl- flux with the following decreasing potencies: 1R,cis,alpha S-cypermethrin greater than 1R,trans, alpha S-cypermethrin greater than fluvalinate greater than allethrin. Avermectin B1a was the only insecticide tested that, in the absence of GABA, stimulated 36Cl- flux in a dose-dependent manner, and this flux was inhibited by bicuculline. The stereospecific inhibition of the GABA-induced 36Cl- influx by the cyclodienes and gamma-BHC supports previously published data on their binding to mammalian brain GABAA receptor and suggests that these insecticides inhibit this receptor's function. It is also suggested that type II pyrethroids are potent inhibitors of the same receptor. However, avermectin B1a appears to act as a partial agonist of GABAA receptors. PMID- 3009837 TI - Pharmacological identification of retinal cells releasing taurine by light stimulation. AB - The effect of drugs blocking synaptic activity at different retinal levels was examined in this study, in an attempt to identify the origin of the light stimulated release of 3H-taurine from the chick retina. It was determined by autoradiography that the chick retina accumulates taurine in photoreceptors, in cells from the inner nuclear layer, and in processes of the inner plexiform layer. All these are possible sites for the release of taurine upon illumination. To discriminate among these possibilities, the effects of aspartate, tetrodotoxin, strychnine, picrotoxin, chlorpromazine, tubocurarine, atropine, glutamate diethyl esther, alpha-amino adipate and 2-amino-4-phosphonobutyrate were studied. Aspartate (10 mM), which is known to eliminate the light response of cells postsynaptic to photoreceptors, induced a marked increase of 150% in the resting efflux of 3H-taurine but did not decrease significantly the light stimulated release. Tetrodotoxin, which blocks amacrine cell responses, decreased 3H-taurine release stimulated by light by less than 20%. The efflux of taurine was unaffected by strychnine, picrotoxin, tubocurarine, atropine, chlorpromazine, and 2-amino-4-phosphonobutyrate, whereas it was increased by glutamate diethyl esther and alpha-amino adipate. These results, all together, point to photoreceptors as the cells releasing 3H-taurine in response to light. PMID- 3009839 TI - [The use of bone graft material in conservative dentistry. 2. Long-term studies after artificial bone grafting and bibliographical studies of traumatotherapy following periodontal surgery]. PMID- 3009840 TI - Evaluation of extratesticular findings in scrotal neoplasms. AB - The significance and incidence of skin thickness, hydroceles, and epididymal size were evaluated in cases of testicular neoplasm. Skin thickness is unreliable and should not be used to differentiate pathological conditions without standardizing technique. Small hydroceles are common in scrotal neoplasms. Epididymal enlargement was seen in many cases of testicular neoplasia and cannot be used alone as a criterion to differentiate malignant from nonmalignant states. PMID- 3009841 TI - Pharmacokinetics of lithium in the dog. AB - The pharmacokinetics of lithium were determined in eight adult dogs. The data were fitted to a two-compartment model. Single intravenous doses of lithium chloride, and single oral doses of lithium carbonate were used. The mean plasma lithium half-life (t1/2) following the single intravenous dose was 21.6 h, and the mean apparent specific volume of distribution of the central compartment (V'c) was 0.189 l/kg. Mean bioavailability was 78.8% following oral administration. PMID- 3009843 TI - Identification and characterization of a major early cytomegalovirus DNA-binding protein. AB - We characterized a DNA-binding protein with an approximate molecular weight of 129,000 (DB129) which is present in the nuclei of cytomegalovirus- (strain Colburn) infected cells, but not in virus particles. Results of two types of experiments demonstrated that DB129 is a member of the early class of herpesviral proteins. First, time course pulse-labeling experiments showed that its synthesis begins after that of the immediate-early protein IE94, but prior to the appearance of late viral proteins, and was reduced at late times. Second, in the presence of inhibitors of viral DNA replication, DB129 continued to be made and accumulated to elevated levels. A second set of experiments showed that DB129 bound to single-stranded DNA in vitro and was eluted by a NaCl gradient in two peaks, one at about 0.2 M and the second at about 0.6 M. A similar pattern of release was observed when infected-cell nuclei were serially extracted with increasing NaCl concentrations. In addition, treatment of nuclei with DNase I selectively released DB129, along with a small but significant fraction of another DNA-binding protein, DB51. These results suggest that DB129 is associated with DNA in vivo and that it interacts directly with single-stranded DNA. It was also shown that cells infected with human cytomegalovirus (strain Towne) contain a slightly larger counterpart to DB129, which was designated DB140. Similarities between these proteins and the major DNA-binding protein of herpes simplex virus are discussed. PMID- 3009842 TI - Transfer, by selective breeding, of the pathogenic Mtv-2 endogenous provirus from the GR strain to a wild mouse line free of endogenous and exogenous mouse mammary tumor virus. AB - The GR laboratory mouse strain has five endogenous proviral copies of the mouse mammary tumor virus (MMTV). One of these, Mtv-2, is unique because it causes mammary carcinomas in virtually 100% of breeding GR females prior to 1 year of age. To facilitate studies of this locus in particular, and mammary tumorigenesis in general, we genetically tailored a new mouse line, WXG-2, which bears Mtv-2 as its only endogenous MMTV provirus. The WXG-2 line was constructed by making hybrids between the GR strain and a wild mouse line free of both endogenous and exogenous MMTV, backcrossing to the MMTV-free line, and fixing the Mtv-2 locus in a population with the desired genotype. Mammary tumors were observed in 5 of the 20 hybrid females carrying the endogenous Mtv-2 provirus. The WXG-2 line represents a new model system for studying MMTV-induced mammary tumorigenesis in the absence of multiple endogenous proviruses. PMID- 3009844 TI - Parvovirus H-1 expression: mapping of the abundant cytoplasmic transcripts and identification of promoter sites and overlapping transcription units. AB - The 5.2-kilobase (kb) genome of the autonomous parvovirus H-1 was transcribed in the rightward direction, yielding steady-state polyadenylated transcripts of 4.8, 3.2, and 2.9 kb. Detailed mapping of these transcripts demonstrated that the H-1 genome contained two overlapping transcription units: the larger unit extended from 4 map units (5' end) to 96 map units (3' end), and the smaller unit extended from 40 map units (5' end) to 96 map units (3' end). The 4.8- and 3.2-kb transcripts were derived from the larger transcription unit and differed by a 1,500-nucleotide segment (10 to 40 map units) which was present in the 4.8-kb transcript but was spliced from the 3.2-kb transcript. The 2.9-kb transcript, the most abundant of the three known H-1 transcripts, was derived from the smaller transcription unit. The sequence at each initiation site was consistent with the presence of a class II (RNA polymerase II) promoter, and cell-free transcription of parvovirus H-1 restriction fragments containing either promoter resulted in transcription of the correct DNA strand and produced 5' ends identical to those seen in vivo. All three transcripts contained a small but heterogeneous splice at 45 to 47 map units. Minor differences in splicing at this site may result in the synthesis of different viral proteins. PMID- 3009845 TI - Molecular basis of the glycoprotein C-negative phenotypes of herpes simplex virus type 1 mutants selected with a virus-neutralizing monoclonal antibody. AB - Previously (Holland et al., J. Virol. 52:566-574, 1984; Kikuchi et al., J. Virol. 52:806-815, 1984) we described the isolation and partial characterization of over 100 herpes simplex virus type 1 mutants which were resistant to neutralization by a pool of glycoprotein C- (gC) specific monoclonal antibodies. The genetic basis for the inability of several of these gC- mutants to express an immunoreactive envelope form of gC is reported here. Comparative nucleotide sequence analysis of the gC gene of the six mutants gC-3, gC-8, gC-49, gC-53, gC-85, and synLD70, which secrete truncated gC polypeptides, with that of the wild-type KOS 321 gC gene revealed that these mutant phenotypes were caused by frameshift or nonsense mutations, resulting in premature termination of gC translation. Secretion of the gC polypeptide from cells infected with these mutants was due to the lack of a functional transmembrane anchor sequence. The six secretor mutants were tested for suppression of amber mutations in mixed infection with a simian virus 40 amber suppressor vector. Mutant gC-85 was suppressed and produced a wild-type sized membrane-bound gC. Nucleotide sequence analysis of the six gC deletion mutants gC-5, gC-13, gC-21, gC-39, gC-46, and gC-98 revealed that they carried identical deletions which removed 1,702 base pairs of the gC gene. The deletion, which was internal to the gC gene, removed the entire gC coding sequence and accounted for the novel 1.1-kilobase mRNA previously seen in infections with these mutants. The mutant gC-44 was previously shown to produce a membrane-bound gC protein indistinguishable in molecular weight from wild-type gC. This mutant differed from wild-type virus in that it had reduced reactivity with virus neutralizing monoclonal antibodies. Nucleotide sequence analysis of the gC gene of mutant gC-44 demonstrated a point mutation which changed amino acid 329 of gC from a serine to a phenylalanine. PMID- 3009846 TI - Isolation of a receptor protein involved in attachment of human rhinoviruses. AB - Human rhinoviruses can be classified into major and minor groups on the basis of receptor specificity. Recently, a mouse monoclonal antibody was isolated which selectively blocked the attachment of the major group of human rhinoviruses to cells. Using this monoclonal antibody, the cellular receptor for the major group of human rhinoviruses was isolated. A radioimmunoassay was developed by using the receptor antibody to specifically detect rhinovirus receptor during isolation. Solubilized receptor from detergent-treated HeLa cell membrane extracts eluted from gel filtration columns with an apparent molecular weight of 440,000. A cellular receptor protein, which had a molecular weight of 90,000 when analyzed on sodium dodecyl sulfate-polyacrylamide gels, was purified from solubilized extracts on an immunoaffinity column containing receptor antibody. Polyclonal rabbit antiserum, resulting from immunization with the isolated receptor protein, specifically blocked the attachment of the major group of human rhinoviruses and indicated that the 90-kilodalton protein plays a functional role in attachment. Prolonged exposure of HeLa cell monolayers with the receptor antibody showed no inhibition of cell growth and division. PMID- 3009847 TI - Structure of the hepatitis A virion: peptide mapping of the capsid region. AB - Milligram amounts of highly purified hepatitis A virus (HAV) were obtained from persistently infected cell cultures. The HAV polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for detection by an enzyme-linked immunotransfer blot procedure. The HAV nucleotide-derived amino acid sequence was subjected to computer analysis to identify potential immunogenic regions within the HAV capsid polypeptides. Synthetic peptides corresponding to selected regions of each of the larger putative capsid polypeptides were coupled to keyhole limpet hemocyanin and used to immunize rabbits. Four of six anti-HAV peptide sera were strongly reactive. Antipeptide serum generated against amino acids (a.a.) 75 through 82 reacted with the 27,000-molecular-weight (MW) polypeptide; serum against a.a. 279 through 285 reacted with the 29,000-MW HAV polypeptide; and sera against a.a. 591 through 602 and 606 through 618 reacted with the 33,000-MW HAV polypeptide. These reactions enabled the identification of the gene order of the larger HAV P1 region gene products. Our data indicate the following molecular weights: HAV VP2 or 1B, 27,000; HAV VP3 or 1C, 29,000; and HAV VP1 or 1D, 33,000. PMID- 3009848 TI - Association of recombinant murine leukemia viruses of the class II genotype with spontaneous lymphomas in CWD mice. AB - We determined the phenotype and genotype of murine leukemia viruses associated with the development of spontaneous nonthymic lymphomas in the high-leukemia mouse strain CWD/J. By T1 oligonucleotide fingerprint analysis of the viral RNA, the ecotropic viruses recovered from the spleen or thymus of preleukemic CWD/J mice were found to represent the progeny of the two endogenous ecotropic proviruses present in this strain. Polytropic murine leukemia viruses were produced by tissues from one-half of the leukemic mice, and fresh tumor cells from one of the two animals tested expressed recombinant envelope glycoproteins. The genomic structure of the recombinant viruses resembled those of class II polytropic viruses of NFS X Akv mice and differed from those of class I recombinant viruses that are commonly isolated from other high-leukemia strains such as AKR and HRS. Acquired retroviral sequences with the structural features of class II recombinant proviruses were detected in the DNA from each CWD/J tumor by the Southern blot technique. Finally, the injection of a mixture of CWD/J ecotropic and class II recombinant polytropic viruses into neonatal CWD/J mice accelerated the onset of lymphoma, whereas the endogenous ecotropic virus was inactive in these assays. PMID- 3009849 TI - Deletion mutants that affect expression of Epstein-Barr virus nuclear antigen in COS-1 cells after gene transfer with simian virus 40 vectors containing portions of the BamHI K fragment. AB - We have identified sequences that affect the efficient expression of Epstein-Barr virus nuclear antigen (EBNA 1) when the structural portion of its gene, found within the 2.9-kilobase-pair BamHI/HindIII fragment called Ilf, is expressed from a simian virus 40 vector. A set of nested deletions at the BamHI end of the fragment was constructed by using BAL 31 digestion, the addition of linkers, and ligation into pSVOd. The mutants were tested for their ability to express antigen in COS-1 monkey cells by using indirect immunofluorescence and immunoblotting. Deletion endpoints were determined by DNA sequencing of the 5' ends of the mutants. The deletion mutants could be subclassified into four groups based on their ability to express EBNA polypeptide. Mutants that retain more than 106 base pairs upstream from the start of the open reading frame in Ilf exhibit antigen expression indistinguishable from that of wild type. Mutants that invade the structural gene by 1,115 or more bases destroy antigen expression. Mutants that alter the splice acceptor site or invade the open reading frame by a short distance make antigen at a markedly lower frequency. There are three mutants, whose deletions map at -78, -70, and -44 base pairs upstream of the open reading frame, that make reduced levels of EBNA. Since these three mutants differ in the extent to which EBNA expression is impaired, the data suggest that there are several critical regions upstream of the open reading frame that regulate EBNA expression in COS-1 cells. It is not known whether these regulatory sequences, which would be located in an intron in the intact genome, play any role in the expression of EBNA in infected lymphocytes. PMID- 3009850 TI - Regulation of herpes simplex virus-specific cell-mediated immunity by a specific suppressor factor. AB - Our study was designed to investigate the nature of an antigen-specific suppressor factor generated by antigen-stimulated herpes simplex virus (HSV) immune splenocytes. Factor SF-200, a 90,000- to 100,000-dalton fraction obtained after Sephacryl gel filtration, suppressed the generation of HSV-specific cytotoxic T-lymphocyte and lymphoproliferative responses. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis of SF-200 indicated that it contained an I-J+, anti-idiotypic protein. It was possible to adsorb the suppressor activity of SF-200 to an anti-I-J immunoaffinity column. The suppressor activity could be eluted from the immunoaffinity column with a low-pH buffer. The acid-eluted material was determined to be both I-J+ and reactive with anti-HSV antiserum by Western blot analysis. Both SF-200 and the I-J+ suppressor activity suppressed only HSV-specific cell-mediated immunity responses. However, it was possible to generate nonspecific suppressor activity by incubating the I J+ suppressor factor with Lyt 1+ splenocytes from HSV-immune mice. The implication of these results with respect to the model for a suppressor cell circuit regulating HSV-specific cell-mediated immunity responses is discussed. PMID- 3009851 TI - Characterization of a pseudorabies virus glycoprotein gene with homology to herpes simplex virus type 1 and type 2 glycoprotein C. AB - A pseudorabies virus (Becker strain) glycoprotein gene was located in the UL region at map position 0.40. The gene was identified by using open reading frame Escherichia coli plasmid expression vectors and specific antibody reagents. A 1.55-kilobase unspliced transcript from the gene was detected in pseudorabies virus-infected tissue culture cells. The DNA sequence revealed a single open reading frame of 1,437 base pairs encoding 479 amino acids. The predicted primary translation product has a molecular weight of 50,860 and contains features of a typical herpesvirus glycoprotein. An E. coli expression plasmid was constructed that contained essentially all of the open reading frame for this gene. Antibodies raised in rabbits against the protein expressed in bacteria by this plasmid immunoprecipitated pseudorabies virus-specific glycoproteins of 92,000 and 74,000 daltons from infected cell extracts. It is likely that these two forms represent different glycosylation states of the protein. PMID- 3009852 TI - Genetic analysis of the attenuation phenotype of poliovirus type 1. AB - Seven different recombinant viruses from the virulent Mahoney and the attenuated Sabin parental strains of type 1 poliovirus were constructed in vitro by using infectious cDNA clones. Monkey neurovirulence tests (lesion score, spread value, and incidence of paralysis) using these recombinant viruses revealed that the loci influencing attenuation were spread over several areas of the viral genome, including the 5' noncoding region. In vitro phenotypic marker tests corresponding to temperature sensitivity of growth (rct marker), plaque size, and dependency of growth on bicarbonate concentration (d marker) were performed to identify the genomic loci of these determinants and to investigate their correlation with attenuation. Determinants of temperature sensitivity mapped to many areas of the viral genome and expressed strong but not perfect correlation with attenuation. Recombinant viruses with Sabin-derived capsid proteins showed a small-plaque phenotype, and their growth was strongly dependent on bicarbonate concentration, suggesting that these determinants map to the genomic region encoding the viral capsid proteins. Plaque size and the d marker, however, were found to be poor indicators of attenuation. Moreover, virion surface characteristics such as immunogenicity and antigenicity had little or no correlation with neurovirulence. Nevertheless, viruses carrying Sabin-derived capsid proteins had an apparent tendency to exhibit less neurovirulence in tests on monkeys compared with recombinants carrying Mahoney-derived capsid proteins. Our results suggest that the extent of viral multiplication in the central nervous system of the test animals might be one of the most important factors determining neurovirulence. Moreover, we conclude that the expression of the attenuated phenotype of the Sabin 1 strain of poliovirus is the result of several different biological characteristics. Finally, none of the in vitro phenotypic markers alone can serve as a good indicator of neurovirulence or attenuation. PMID- 3009853 TI - Specific hybridization probes demonstrate fewer xenotropic than mink cell focus forming murine leukemia virus env-related sequences in DNAs from inbred laboratory mice. AB - We have derived hybridization probes from analogous 100-base-pair segments located within the N-terminal region of gp70 coding sequences which differentiate xenotropic from mink cell focus-forming (MCF)-related murine leukemia virus (MuLV) DNAs. The MCF probe annealed to the integrated proviruses of all six MCF MuLV isolates tested; the xenotropic probe hybridized to the DNAs of all four xenotropic proviral isolates examined. No cross-hybridization was observed, and neither probe reacted with the env segments of amphotropic or ecotropic MuLV DNAs. Southern blot analysis of HindIII- or EcoRI-digested genomic DNAs from a variety of inbred laboratory mice demonstrated the presence of more MCF- than xenotropic MuLV-related segments in every strain tested. PMID- 3009855 TI - Formation of deletions after initiation of simian virus 40 replication: influence of packaging limit of the capsid. AB - Transfected DNA is frequently broken and rejoined in mammalian cells by recombination processes that depend on minimal nucleotide sequence homology. Although measurements of breakage and joining account reasonably well for the frequent formation of deletions during transfection, they are inadequate to explain the high frequency of deletion formation by simian virus 40 (SV40) genomes that are slightly larger than the packaging limit of the capsid. To investigate this anomaly, we constructed and transfected into CV-1 cells a series of modified SV40 genomes containing 136, 284, 460, and 656 extra base pairs in the intron of the gene encoding T antigen. These experiments indicate that the effective packaging limit of an SV40 capsid lies between 284 and 460 extra base pairs. Further analysis of these transfections suggests that molecules just above the effective packaging limit may be encapsidated and transmitted between cells at low efficiency, thereby allowing multiple rounds of replication and multiple opportunities to generate and package genomes that contain deletions. The junctional sequences in several such deletions were determined; they were similar to the junctions in deletions that were formed before replication began, suggesting that the enzymatic machinery responsible for both types of deletion may be similar. PMID- 3009854 TI - Expression and regulation of glycoprotein C gene of herpes simplex virus 1 resident in a clonal L-cell line. AB - Ltk- cells were transfected with a plasmid containing the entire domain of glycoprotein C (gC), a true gamma or gamma 2 gene of herpes simplex virus 1 (HSV 1) and the methotrexate-resistant mouse dihydrofolate reductase mutant gene. The resulting methotrexate-resistant cell line was cloned; of the 39 clonal lines tested only 1, L3153(28), expressed gC after infection with HSV-1(MP), a gC- mutant, and none expressed gC constitutively. The induction of gC was optimal at multiplicities ranging between 0.5 and 2 PFU per cell, and the quantities produced were equivalent to or higher than those made by methotrexate-resistant gC- L cells infected with wild-type (gC+) virus. The gC gene resident in the L3153(28) cells was regulated as a beta gene inasmuch as the amounts of gC made in infected L3153(28) cells exposed to concentrations of phosphonoacetate that inhibited viral DNA synthesis were higher than those made in the absence of the drug, gC was induced at both permissive and nonpermissive temperatures by the DNA mutant tsHA1 carrying a lesion in the gene specifying the major DNA-binding protein and which does not express gamma 2 genes at the nonpermissive temperature, and gC was induced only at the permissive temperature in cells infected with ts502 containing a mutation in the alpha 4 gene. The gC induced in L3153(28) cells was made earlier and processed faster to the mature form than that induced in a gC- clone of methotrexate-resistant cells infected with wild type virus. Unlike virus stocks made in gC- cells, HSV-1(MP) made in L3153(28) cells was susceptible to neutralization by anti-gC monoclonal antibody. PMID- 3009856 TI - Polyomavirus late leader region serves an essential spacer function necessary for viability and late gene expression. AB - All three polyomavirus late mRNAs contain multiple tandem copies of the same nontranslated 57-nucleotide sequence, the late leader, at their 5' ends. We show here that a polyoma variant (ALM) lacking 48 central bases of the 57-base leader unit is nonviable by plaque assay and by a new method for testing virus viability, an immunofluorescence burst assay. ALM is, however, unaffected in early gene expression as measured both by indirect immunofluorescence of large T antigen and by transformation levels of rat F-111 cells. DNA replication in mouse cells is also as wild type, and the defect in ALM is complemented by an early defective helper virus DNA. ALM does not make detectable levels of late viral proteins and is minimally 200-fold depressed in the accumulation of cytoplasmic polyadenylated late RNA. When the deleted leader sequence of ALM is replaced by a variety of procaryotic sequences, viability almost always returns. Some of the substituted leader variants produce plaques with the same apparent kinetics as wild-type viral DNA. The indication is that the sequence of the polyoma late leader is not important for late gene expression but that it has an essential spacer function on the RNA or DNA level. This spacer function is apparently necessary for late viral RNA transcription, processing, or stability. PMID- 3009857 TI - Restricted replication of mouse hepatitis virus A59 in primary mouse brain astrocytes correlates with reduced pathogenicity. AB - Temperature-sensitive (ts) mutants of mouse hepatitis virus A59 (MHV-A59) are drastically attenuated in their pathogenic properties. Intracerebral inoculation of mice with 10(5) PFU of mutant ts342 results in prolonged infection of the central nervous system, whereas 100 PFU of wild-type virus are lethal (M. J. M. Koolen, A. D. M. E. Osterhaus, G. van Steenis, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 125:393-402, 1983). In the Sac(-) cell line ts342 grows as well at 37 degrees C (the body temperature of mice) as at 31 degrees C (the permissive temperature). There is, however, a difference in primary cultures of mouse brain astrocytes. After infection with ts342, astrocytes produced low levels of infectious virus (5.2 +/- 3.7%) compared with virus yields after infection with wild-type virus. The fraction of wild-type virus- and ts342 infected cells was similar. Electron microscopy showed in wild-type virus infected cells abundant virions in smooth vesicles usually closely associated with a well-developed Golgi apparatus. In mutant-infected cells no mature ts342 virus particles were found. There was no difference between ts342 and wild-type virus regarding the intracellular virus-specific RNAs. In ts342-infected cells the viral glycoproteins E2 and E1 were not detectable or were barely detectable. Either the mRNAs for the glycoproteins are not translated or the proteins are rapidly broken down. Revertants of ts342 were isolated. They grew as well as wild type virus in astrocytes, indicating that they apparently produced sufficient amounts of E2 and E1, the ts defect itself rather than a second site mutation is responsible for the defect in replication, and the ts defect acts in unison with host-cell factors. The revertants also regained the lethal properties of wild type virus. PMID- 3009858 TI - Constitutive and retinoic acid-inducible expression of cytomegalovirus immediate early genes in human teratocarcinoma cells. AB - Human teratocarcinoma stem cells are nonpermissive for human cytomegalovirus (HCMV) but become permissive after being induced to differentiate by treatment with retinoic acid. We show that in uninduced teratocarcinoma stem cells, and also in transformed human 293 cells expressing adenovirus E1a gene products, the HCMV immediate-early (IE) 68,000-molecular-weight polypeptide (68K polypeptide) was not expressed, and consequently input viral genomes were not replicated. However, after differentiation of the teratocarcinoma cells, synthesis of the HCMV IE 68K polypeptide was induced, and viral DNA replication occurred. In contrast to our observations for HCMV, simian cytomegalovirus (SCMV) displayed constitutive expression of its analogous IE 94K polypeptide, and the input SCMV genomes were replicated in both uninduced stem cells and 293 cells. Since little, if any, HCMV IE RNA was detectable in human teratocarcinoma or 293 cells after infection under IE conditions, we suggest that a direct transcriptional block to permissivity occurs in these cells. The presence of tandemly repeated sequences which bind nuclear factor I protein in the promoter for the SCMV IE 94K polypeptide gene but not in the promoter for the HCMV IE 68K polypeptide gene may allow the expression of the simian but not of the human IE gene product in transformed cells. PMID- 3009859 TI - Expression of the protein product of the mouse mammary tumor virus long terminal repeat gene in phorbol ester-treated mouse T-cell-leukemia cells. AB - Exposure of C57BL/6 mouse EL-4 T-cell leukemia cells to phorbol ester (12-O tetradecanoylphorbol-13-acetate) (TPA) induced the synthesis of protein products encoded by the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) region. Analysis of TPA-treated EL-4 cells with antiserum raised against a synthetic peptide predicted by the MMTV LTR open reading frame sequence detected a polypeptide migrating in gels with an apparent molecular weight of 37,000 Mr, as well as three less prominent proteins with apparent molecular weights of 31,000, 34,000, and 39,000. Tryptic peptide analysis established the identity of the immunoprecipitated cellular proteins with the LTR proteins obtained from in vitro translation of MMTV genomic RNA. All four proteins were glycosylated and were derived from one initial nonglycosylated translation product of 21,000 Mr. The 21,000-Mr apoprotein could be detected after digestion with endoglycosidase F or pretreatment of cells with tunicamycin. Untreated EL-4 cells synthesized three species of MMTV mRNA: 35S, 24S, and 20S. TPA treatment resulted in an increased level of transcription of the three mRNAs and the appearance of a new 1-kilobase mRNA. At least 10 acquired MMTV proviruses are present in the EL-4 genome, and examination of the degree of proviral methylation revealed extensive demethylation. However, no qualitative differences in the state of proviral methylation were apparent between TPA-treated and untreated cells. PMID- 3009860 TI - The absence of myristic acid decreases membrane binding of p60src but does not affect tyrosine protein kinase activity. AB - We have constructed two point mutants of Rous sarcoma virus in which the amino terminal glycine residue of the transforming protein, p60src, was changed to an alanine or a glutamic acid residue. Both mutant proteins failed to become myristylated and, more importantly, no longer transformed cells. The lack of transformation could not be attributed to defects in the catalytic activity of the mutant p60src proteins. In vitro phosphorylation of the peptide angiotensin or of the cellular substrate proteins enolase and p36 revealed no significant differences in the Km or specific activity of the mutant and wild-type p60src proteins. However, when cellular fractions were prepared, less than 12% of the nonmyristylated p60src proteins was bound to membranes. In contrast, more than 82% of the wild-type protein was associated with membranes. Wild-type p60src was phosphorylated by protein kinase C, a protein kinase which associates with membranes when activated. The mutant proteins were not. This finding supports the idea that within the intact cell the nonmyristylated p60src proteins are cytoplasmic and suggests that this apparent solubility is not an artifact of the cell fractionation procedure. The myristyl groups of p60src apparently encourages a tight association between protein and membranes and, by determining the cellular location of the enzyme, allows transformation to occur. PMID- 3009862 TI - Characterization of the Snow Mountain agent of viral gastroenteritis. AB - Snow Mountain agent (SMA) is a 27- to 32-nm virus which is the etiologic agent of outbreaks of acute gastroenteritis in Colorado and Vermont. SMA is morphologically similar to but antigenically distinct from the Norwalk and Hawaii agents of viral gastroenteritis but, like those agents, has not been cultivated in vitro. We purified and characterized SMA directly from human stool specimens containing the virus. The density of the SMA virion was 1.29 g/cm3 and 1.21 to 1.22 g/cm3 on potassium tartrate-glycerol gradients and 1.33 to 1.34 g/cm3 on cesium chloride gradients. SMA had an S value of 170 to 183S on a sucrose velocity gradient. The purified virion was iodinated, immunoprecipitated with acute and convalescent sera from volunteers challenged with SMA, and analyzed on polyacrylamide gels. The virion contains one major structural protein of 62,000 molecular weight, which is similar in size to the 59,000-molecular-weight protein found in the Norwalk virion. The biophysical properties and single structural protein of SMA most closely resemble those of the calicivirus group. PMID- 3009861 TI - Palindromic structure and polypeptide expression of 36 kilobase pairs of heterogeneous Epstein-Barr virus (P3HR-1) DNA. AB - Among the Epstein-Barr virions (EBV) produced by the P3HR-1 (HR-1) cell line are a defective subpopulation with rearranged viral DNA designated heterogeneous DNA (het DNA). These defective virions are responsible for the capacity of HR-1 virus to induce early antigen in Raji c cells and for trans activation of latent EBV in X50-7 cells. Virions with het DNA are independent replicons which pass horizontally from cell to cell rather than being partitioned vertically. We analyzed the structure and defined several polypeptide products of het DNA to understand these remarkable biologic properties. A 36-kilobase-pair (kbp) stretch of het DNA was cloned (as two EcoRI fragments of 20 and 16 kbp) from virions released from a cellular subclone of HR-1 cells. The unusual aspect of the 20-kbp fragment was the linkage of sequences of BamHI-M and BamHI-B', which are not adjacent on the standard EBV genome. The 16-kbp fragment was a palindrome in which at least two additional recombinations on each side of the palindrome had linked regions of the standard EBV genome which are not normally contiguous. The 20-kbp het DNA fragment was attached to at least one and possibly both ends of the 16-kbp het DNA fragment. We identified antigenic polypeptides produced in COS 1 cells after gene transfer of various cloned het DNA fragments. The 20-kbp fragment encoded a cytoplasmic antigen of about 95 kilodaltons (kDa). The 16-kbp fragment encoded antigens located in the nucleus, nuclear membrane, and cytoplasm. These were represented by several polypeptides, the most prominent of which were about 55, 52, and 36 kDa. The 36-kDa polypeptide was localized to a 2.7-kbp BamHI fragment which had homology to standard BamHI-W and BamHI-Z. Another polypeptide of 50 kDa found in the nucleus was mapped to the 7.1-kbp BamHI het DNA fragment which spans the EcoRI site linking the 20- and 16-kbp fragments of het DNA. Thus, HR-1 het DNA encodes several discrete polypeptide products, one or more of which could be responsible for the unusual biologic properties of the virus. The composition, regulation, and ultimately the expression of some of these products relative to standard EBV is probably altered by the genomic rearrangements of het DNA. PMID- 3009864 TI - Frequent partial deletion of human adult T-cell leukemia virus type I proviruses in experimental transmission: pattern and possible implication. AB - Human T-cell leukemia virus type I (HTLV I) propagated in human diploid fibroblast IMR90 was transmitted to human promyelocytic leukemia HL60 cells by coculture. Of 14 provirus-positive HL60 clones, five harbored only defective proviruses, five had defective proviruses in addition to full-sized HTLV I, and four had full-sized proviruses integrated in their chromosomes. The frequency of defective proviruses was unexpectedly high (41% of total proviruses). Analysis of the genomic structure of these defective proviruses revealed polarity of deletion, that is, preferred conservation of the 3' end of the proviral genome (pX and the 3' long terminal repeat). The implication of these findings are discussed with reference to the replication and pathogenesis of HTLV I. PMID- 3009863 TI - Properties of intracellular bovine papillomavirus chromatin. AB - Episomal nucleoprotein complexes of bovine papillomavirus type 1 (BPV-1) in transformed cells were exposed to DNase I treatment to localize hypersensitive regions. Such regions, which are indicative for gene expression, were found within the noncoding part of the genome, coinciding with the origin of replication and the 5' ends of most of the early mRNAs. However, there were also regions of hypersensitivity within the structural genes. These intragenic perturbations of the chromatin structure coincide with regulatory sequences at the DNA level. One of these regions maps in close proximity to a Z-DNA antibody binding site which is located near the putative BPV-1 enhancer sequence. PMID- 3009865 TI - Cloning and characterization of oriL2, a large palindromic DNA replication origin of herpes simplex virus type 2. AB - An origin of replication within the long unique sequence of herpes simplex virus type 2 designated oriL2 has been identified in a position homologous to its type 1 counterpart, oriL1, between map coordinates 0.398 and 0.413. The difficulties encountered in previous attempts to clone both oriL2 and oriL1 in an undeleted form were surmounted by minimizing the growth of the host Escherichia coli, using a recBC sbcB E. coli host, and purifying the full-length plasmid from delected forms by using a novel method which exploits the ability of a palindrome containing plasmid to adopt a cruciform conformation, thereby decreasing its supercoiling. In a previously developed assay for functional origin activity, oriL2 was localized to a 241-base-pair ApaI-SstII fragment. DNA sequence analysis revealed a 136-base pair, almost perfect palindrome. Comparison with oriL1 showed a very high degree of conservation: the two origins differ in only 16 of the 144 base-pair oriL1 palindromic region. Most significantly, the differences between oriL1 and oriL2 mainly occur in pairs so as to generally preserve the potential for intrastrand base pairing. The central region of oriL2 is homologous with the shorter palindromic structures detected in origins located within the repetitive sequences of the short component of herpes simplex virus type 1 or 2. PMID- 3009867 TI - Structural domains of the avian erythroblastosis virus erbB protein required for fibroblast transformation: dissection by in-frame insertional mutagenesis. AB - Avian erythroblastosis virus (AEV) induces erythroblastosis and fibrosarcomas. The viral erbB protein is required for AEV-mediated oncogenesis. To explore the structural aspects of the v-erbB polypeptide necessary for its oncogenic function, we created a series of small in-frame insertions in different domains of the v-erbB oncogene. AEV genomes bearing lesions within the v-erbB kinase domain demonstrated a drastically decreased ability to transform avian fibroblasts, establishing a functional role for this structurally conserved oncogene domain. In contrast, mutations in the extracellular domain, between the transmembrane region and the kinase domain, or at the extreme C terminus of the v erbB protein had no effect on AEV-mediated fibroblast transformation. One lesion within the v-erbB kinase domain, a 10-amino acid insertion, produced a temperature-sensitive mutant capable of fibroblast transformation at 36 degrees C but not at 41 degrees C, suggesting that small in-frame insertions have general utility for the in vitro creation of conditional mutants. PMID- 3009868 TI - Novel RNA family structure of hepatitis B virus expressed in human cells, using a helper-free adenovirus vector. AB - Ad5-HBL is a type 5 adenovirus bearing the large BglII fragment (2.8 kilobases; 87% of the total genome) of hepatitis B virus (HBV), subtype adr. Eight HBV RNAs expressed in HeLa cells infected with Ad5-HBL were mapped by the nuclease S1 technique. Three major RNAs spanning 2.4, 2.0, and 0.7 kilobases of the HBV sequences cover the coding regions of "presurface" plus surface antigen, surface antigen alone, and "X" protein, respectively. The 5' segment of an RNA which could code for core antigen (HBcAg) was also detected. All major HBV RNAs initiate from mutually exclusive 5' ends, terminate at the unique 3' end within the HBcAg coding region (except readthrough species), and have no spliced deletion, forming a novel RNA family structure. No TATA box-like sequences were found near the 5' end of these RNAs, except in the case of the 2.4-kilobase RNA. About two thirds of total HBV RNA does not terminate at the mapped 3'-end position, suggesting the termination signal is functionally inefficient. Since the potential 5' end of HBcAg mRNA was mapped at the same position as the minus strand nick of HBV DNA previously reported, we propose a model that requires inefficient poly(A) addition to produce an RNA which serves both as HBcAg mRNA and as the putative RNA template of minus-strand DNA synthesis in the HBV life cycle. PMID- 3009869 TI - Nucleotide sequences that affect replicative and transcriptional efficiencies of Sendai virus deletion mutants. AB - Structural features of the genomes of virus deletion mutants (DI virions) influence their replication efficiency. Among nonsegmented negative-strand RNA viruses, substitution of the genomic 3' terminus by a complementary copy of the 5' terminus (so-called "copy-back" sequence) could enhance replication either because the new 3' end is a better promoter of RNA replication or because DI RNAs that possess this sequence are incapable of acting as templates for transcription. Here we provide evidence that both mechanisms operate in mixed infections with Sendai virus DI RNAs. RNAs incapable of transcription always outgrew RNA species that were transcribed. This was true even when the 3' terminal sequence of the untranscribed RNA was identical to the genomic 3' terminus, as in the case of an internally deleted DI genome (RNA Ra) rendered transcriptionally inert by point mutations of bases 47 and 51 at the 5' end of the positive-strand leader RNA template. Nevertheless, Ra was outgrown by a copy back DI RNA, indicating that the 3' genomic end of Ra is a less efficient site for replication initiation than the copy-back sequence. PMID- 3009866 TI - Replication and transformation functions of in vitro-generated simian virus 40 large T antigen mutants. AB - We used sodium bisulfite mutagenesis to introduce point mutations within the early region of the simian virus 40 genome. Seventeen mutants which contained amino acid changes in the amino-terminal half of the large T antigen coding sequence were assayed for their ability to replicate viral DNA and to induce transformation in the established rodent cell line Rat-3. The mutants fell into four basic classes with respect to these two biological functions. Five mutants had wild-type replication and transformation activities, six were totally defective, three were replication deficient and transformation competent, and two were replication competent and transformation deficient. Within these classes were mutants which displayed intermediate phenotypes, such as four mutants which were not totally deficient in viral replication or cellular transformation but instead showed reduced large T antigen function relative to wild type. Three large T mutants displayed transforming activity that was greater than that of wild type and are called supertransforming mutants. Of the most interest are mutants differentially defective in replication and transformation activities. These results both support and extend previous findings that two important biological functions of large T antigen can be genetically separated. PMID- 3009870 TI - Generation of an inverting herpes simplex virus 1 mutant lacking the L-S junction a sequences, an origin of DNA synthesis, and several genes including those specifying glycoprotein E and the alpha 47 gene. AB - The herpes simplex virus genome consists of two components, L and S, that invert relative to each other to yield four isomeric arrangements, prototype (P), inversion of the S component (Is), inversion of the L component (Il), and inversion of both components (Isl). Previous studies have shown that the 500-base pair a sequences flanking the two components contain a cis-acting site for inversion. In an attempt to insert a third copy of the alpha 4 gene, the major regulatory gene mapping in the repeats flanking the S component, a fragment containing the alpha 4 gene and an origin of DNA synthesis, was recombined into the thymidine kinase gene mapping in the unique sequences of the L component. The resulting recombinants showed massive rearrangements and deletions mapping in the S component and in the junction between the L and S components. One recombinant (R7023) yielded two isomeric DNA arrangements, a major component consisting of Is and a minor component consisting of Isl. In these arrangements, the genome lacked the gene specifying glycoprotein E and all contiguous genes located between it and the alpha 0 gene in the inverted repeats of the L component. Among the deleted sequences were those encoding an origin of viral DNA synthesis, the alpha 47 gene, and the a sequences located at the junction between the L and S components. The recombinant grew well in rabbit skin, 143TK-, and Vero cell lines. We conclude that the four unique genes deleted in R7023 are not essential for the growth of herpes simplex virus, at least in the cell lines tested, and that the b sequence of the inverted repeats of the L component also contains cis acting sites for the inversion of herpes simplex virus DNA sequences. PMID- 3009871 TI - VA RNAs from avian and human adenoviruses: dramatic differences in length, sequence, and gene location. AB - Human adenoviruses encode low-molecular-weight RNAs, so-called VA RNAs, which are transcribed by RNA polymerase III. These RNAs are required for an efficient translation of viral mRNAs late after infection. The genes for the VA RNAs in the genome of CELO virus were mapped and characterized. The results showed a number of surprising differences between CELO virus and human adenovirus type 2 (Ad2). Thus, the CELO virus genome encoded only one VA RNA species, in contrast to human Ad2, which encoded two distinct species. The VA RNA from CELO virus was much shorter than the Ad2 VA RNAs (90 nucleotides compared with 160 nucleotides), and there existed no detectable primary sequence homology between them. The predicted secondary structure of CELO virus VA RNA was, however, similar to that of the Ad2 VA RNAs, implying that the folding rather than the primary sequence was the important feature for biological activity. CELO VA RNA also stimulated translation in a transient expression assay, as did the Ad2 counterparts, albeit with a much lower efficiency. The location of the gene for CELO VA RNA also differed from all previously characterized serotypes, suggesting that the genome organization of avian and human adenoviruses are different. Finally, termination of CELO VA RNA transcription occurred in a TTATT sequence which is unique as a stop signal for RNA polymerase III transcription. PMID- 3009872 TI - Biological activity and electron microscopy of poliovirus 14S particles obtained from alkali-dissociated procapsids. AB - Highly purified 14S subunit particles were obtained from alkali-dissociated poliovirus type 1 procapsids (naturally occurring empty capsids in poliovirus infected cells) to compare their morphological and biophysical properties with those of naturally occurring 14S particles. Procapsid-derived 14S particles (PC 14S), like naturally occurring 14S particles, were capable of self-assembly into an empty shell in buffer or extracts from uninfected cells. These empty capsids always exhibited pIs more acidic than those of procapsids but were themselves distinguishable by their respective pIs. Nevertheless, if PC-14S or naturally occurring 14S particles were incubated with extracts made from poliovirus infected cells, procapsidlike empty shells were formed. This clearly showed that the 14S particle, however obtained, possesses the information to form an empty shell of correct dimensions but of improper conformation, unless a factor present in poliovirus-infected cells is present. With the electron microscope, the PC-14S subunit frequently was seen as a pentagonal structure with a diameter of 20.4 +/- 1.4 nm, a size somewhat larger than expected for a subunit composing 1/12th of the poliovirus surface. Upon self-assembly in vitro, the empty shell formed exhibited a diameter of 29 +/- 1 nm and a wall thickness of ca. 6 to 7 nm. It was necessary to avoid CsCl banding of procapsids in their preparation as this treatment altered both their pI and their sensitivity to alkali dissociation into 14S subunits. The relevance of these findings to the nature and role of procapsids and the requirement for a morphopoietic factor in poliovirus morphogenesis is discussed. PMID- 3009873 TI - Mammary tumorigenesis in feral Mus cervicolor popaeus. AB - A pedigreed breeding population of feral Mus cervicolor popaeus with a high incidence of mammary tumors, arising between 6 and 14 months of age, is described. These mice were chronically infected with a type B retrovirus which is distantly related to the mouse mammary tumor virus (MMTV) of inbred strains of Mus musculus. MMTV-induced mammary tumors in inbred mice frequently (80%) contained an insertion of the viral genome into the int-1 or int-2 loci of the tumor cellular genome. These two cellular genetic loci were also altered by viral insertion in 11 of 20 M. cervicolor popaeus mammary tumor cellular DNAs tested. Results of our study of mammary tumorigenesis in feral mice demonstrate that viral-induced rearrangement and activation of the int loci are not limited to the genetic background of inbred mice selected for highly infectious MMTV and a high incidence of mammary tumors. PMID- 3009875 TI - Mapping 5' termini of JC virus early RNAs. AB - Within its enhancer promoter region, the MAD-1 strain of JC virus (JCV) has two 98-base-pair tandem repeats, each containing a TATA box-like sequence. In the present study, polyadenylated early JCV mRNAs were isolated 5 or 29 days after infection of primary human fetal glial (PHFG) cells. By using S1 nuclease, the 5' termini of the early mRNAs were mapped to nucleotide position(s) (np) 122 through 125, which lies within an AT rich region (at np 113 through 127). In contrast, when JCV DNA was transcribed in vitro, we observed a single major cluster of 5' start sites at np 94 through 97, which is approximately 25 base pairs downstream from one of the TATA boxes. By day 5, the earliest time at which JCV RNA was detected, viral DNA replication had begun; it continued for at least an additional 20 days. Since more late than early RNA was present at 5 days postinfection, the early RNAs whose synthesis began at np 122 through 125 may be analogous to SV40 late early mRNA (Ghosh and Lebowitz, J. Virol. 40:224-240, 1981). However, we have not detected RNAs with 5' termini 25 to 30 bp downstream from the TATA box at earlier times. While JCV contains two identical TATA boxes, one in each of the 98-bp repeats, only the upstream TATA box functions as an early promoter element. PMID- 3009874 TI - Epidermodysplasia verruciformis-associated human papillomavirus 8: genomic sequence and comparative analysis. AB - Human papillomavirus (HPV) 8 induces skin tumors which are at high risk for malignant conversion. The nucleotide sequence of HPV8 has been determined and compared to sequences of papillomaviruses with different oncogenic potential. The general organization of the HPV8 genome is similar to that of other types. Highly conserved, genus-specific sequences were found in open reading frames (ORFs) E1, E2, and L1. In ORFs E6, E7, and L2, HPV8 is more distantly related, but it was possible to differentiate subgenera in which HPV8 belonged to the HPV1-cottontail rabbit papillomavirus group. Sequences within ORF E4 and part of ORF L2 are rather type specific. HPV8 stands out by several unique features: the considerably reduced size of the noncoding region (397 base pairs), with a seemingly low potential for forming complex secondary structures; a cluster of putative promoter elements in the 3' half of ORF E1; an RNA polymerase III promoter-like sequence close to the C terminus of ORF E2; and of particular interest, the homology between the putative protein encoded by ORF E4 and the Epstein-Barr virus nuclear antigen 2 protein, which may reflect similar mechanisms in virus-mediated transformation. PMID- 3009876 TI - Amplification of a tandem direct repeat within inverted repeats of Marek's disease virus DNA during serial in vitro passage. AB - DNA of the oncogenic strain BC-1 of Marek's disease virus contains three units of tandem direct repeats with 132 base pairs in the terminal repeat and internal repeat, respectively, of the long region of the Marek's disease virus genome, whereas the attenuated, nononcogenic viral DNA contains multiple units of the tandem direct repeats. PMID- 3009877 TI - Immune responses to isolated human cytomegalovirus envelope proteins. AB - A group of envelope proteins of human cytomegalovirus, gA protein (L. Pereira, M. Hoffman, M. Tatsuno, and D. Dondero, Virology 139:73-86, 1984; L. Pereira, p. 383 404, in B. Roizman, ed., The herpesviruses, vol. 3, 1985), and two protein mixtures (58,000-molecular-weight [58K]-66K and 130K-66K), separated by serial columns prepared with anti-gA immunoglobulin G from sera of immunized guinea pigs, induced neutralizing antibodies and a cellular immune response in the animals. The gA is a disulfide-linked protein complex consisting of high molecular-weight (greater than 200K), 130K-150K, and 55K-58K proteins. PMID- 3009879 TI - Epstein-Barr virus-specific DNA polymerase in virus-nonproducer Raji cells. AB - Virus-nonproducer Raji cells, when induced to early antigen synthesis by 12-O tetradecanoyl-phorbol-13-acetate and sodium butyrate, showed an increase in DNA polymerase activity. This enzyme has the characteristics of a typical Epstein Barr virus DNA polymerase with regard to chromatographical pattern and biological properties: it is eluted from DEAE-cellulose at 0.08 M NaCl, has a high salt resistance, is sensitive to phosphonoacetic acid and phosphonoformate, and shows a substrate preference for poly(dC)-oligo(dG12-18). The resistance of Epstein Barr virus polymerase activity to aphidicolin is a property distinct from that of HSV DNA polymerase. Viral DNA polymerase activity increases in the absence of Epstein-Barr virus DNA replication, indicating that this enzyme is an early viral protein. PMID- 3009880 TI - Transformation of primary human embryonic kidney cells to anchorage independence by a combination of BK virus DNA and the Harvey-ras oncogene. AB - Primary human embryonic kidney (HEK) cells were transformed by a focus assay with BK virus (BKV) DNA molecularly cloned at its unique EcoRI site. Both viral DNA sequences and viral tumor antigens were present and expressed in all the foci that we examined. However, cells isolated from foci were incapable of growth in soft agar. We then examined the transformation of HEK cells after their transfection with a combination of BKV DNA and either the normal or the activated form of the human Ha-ras oncogene (EJ c-Ha-ras-1). Only the cells transfected with a combination of BKV DNA and the activated form of Ha-ras were capable of growth in soft agar. Both BKV and Ha-ras DNAs were present in the transformed colonies. BKV tumor antigens and the Ha-ras p21 protein were also expressed. PMID- 3009878 TI - Sequence homology between cloned caprine arthritis encephalitis virus and visna virus, two neurotropic lentiviruses. AB - Caprine arthritis encephalitis virus (CAEV) is an exogenous, nononcogenic retrovirus which causes neurological disease and crippling arthritis in goats. A complete CAEV genome was cloned from unintegrated viral DNA in two fragments of 9.4 and 0.4 kilobases in length, respectively. The biological activity of these clones was tested by ligation of the fragments followed by transfection onto goat synovial membrane cells; infectious virus was recovered. Cloned CAEV and visna virus, a related neurotropic virus of sheep, were compared by heteroduplex and molecular hybridization analyses. These data demonstrated that the greatest overall conservation of nucleotide sequences occurred in the gag and pol gene regions and two smaller regions, sor and the putative tat gene. The region of greatest divergence occurred in the env gene and, in particular, was localized primarily in the region coding for the glycosylated outer membrane protein. These findings and the recently demonstrated genetic relationship of visna virus, CAEV, and human T-cell lymphotropic virus type III, the etiologic agent of the acquired immune deficiency syndrome, may have important implications concerning the biological properties of these related viruses for human and veterinary medicine. PMID- 3009881 TI - Expression of herpes simplex virus glycoproteins in polarized epithelial cells. AB - Members of the herpesvirus family mature at inner nuclear membranes, although a fraction of the viral glycoproteins is expressed on the cell surface. In this study, we investigated the localization of herpes simplex virus type 2 (HSV-2) glycoproteins in virus-infected epithelial cells by using a panel of monoclonal antibodies directed against each of the major viral glycoproteins. All of the HSV 2 glycoproteins were localized exclusively on the basolateral membranes of Vero C1008, Madin-Darby bovine kidney, and mouse mammary epithelial cells. Using a monoclonal antibody to HSV-2 gD which cross-reacts with HSV-1 strains, we could also localize HSV-1 gD on the basolateral membranes of Madin-Darby bovine kidney cells. These results indicate that these molecules contain putative sorting signals that direct them to basolateral membrane domains. PMID- 3009882 TI - Localization of temperature-sensitive transformation mutations and back mutations in the Rous sarcoma virus src gene. AB - Cloning and sequencing of two temperature-sensitive transforming mutations of Rous sarcoma virus reveal that their lesions are due to distinct but close single amino acid changes near the carboxy terminus of the v-src gene product. Back mutations to wild type result from second mutations at either nearby or distant sites. PMID- 3009883 TI - Enhanced rate of conversion or recombination of markers within a region of unique sequence in the herpes simplex virus genome. AB - Insertion mutants of herpes simplex virus type 1, containing a second copy of the sequences of BamHI fragment L (map coordinates 0.706 to 0.744) inserted in inverted orientation into the thymidine kinase gene (at map coordinate 0.315), have been further characterized. We reported previously that, as a result of intramolecular or intermolecular recombination between copies of the BamHI-L sequence at the normal locus and inserted locus, a high proportion of progeny genomes exhibited either inversions of the unique sequence flanked by these inverted repeats or other rearrangements. Now we report that a genetic marker (syn-1 or syn-1+) originally present only in the inserted copy of BamHI fragment L appears in progeny at both the normal and inserted loci, and vice versa, at high frequency. Because these phenomena have not been observed with other insertion mutants containing duplications of other sequences from unique regions of the genome, we conclude that BamHI fragment L contains an element that enhances the rate of homologous recombination in adjacent sequences, resulting in genome rearrangements and gene conversion-like events. PMID- 3009884 TI - Anti-VPg antibody precipitation of product RNA synthesized in vitro by the poliovirus polymerase and host factor is mediated by VPg on the poliovirion RNA template. AB - Antibody to the poliovirus genome-linked protein, VPg, specifically immunoprecipitated the product RNA synthesized in vitro by the poliovirus RNA polymerase and HeLa cell host factor when VPg-linked poliovirion RNA was used as a template. The largest product RNA that was immunoprecipitated was twice the size of the template RNA. The complete denaturation of the product RNA with CH3HgOH had no effect on the immunoprecipitation reaction. In contrast, CH3HgOH denaturation prevented the immunoprecipitation of the oligo(U)-primed product RNA. Immunoprecipitation of the product RNA synthesized in the host-factor dependent reaction was prevented if VPg was removed from the template RNA by pretreatment with proteinase K or if an RNA template without VPg was used in the reaction. The results support our previous evidence that a covalent linkage exists between the labeled negative-strand product RNA and the VPg-linked template RNA and suggest that the purified polymerase and host factor initiated RNA synthesis in vitro in the absence of VPg or a VPg-precursor protein. PMID- 3009885 TI - Allosteric control of simian virus 40 T-antigen binding to viral origin DNA. AB - Simian virus 40 (SV40) large tumor antigen (T antigen) possesses several biochemical activities localized in different domains of the protein. These activities include sequence-specific binding to two major sites, I and II, in the SV40 control region, ATPase, and nucleotide-binding activity. In the present communication, we present evidence that specific binding of immunopurified T antigen to SV40 DNA is markedly inhibited by low concentrations of ATP, dATP, GTP, and dGTP. The inhibition is reversible after removal of the nucleotide, suggesting that simple nucleotide binding rather than a covalent modification of T antigen in the presence of ATP is responsible for the inhibition. The results suggest that T antigen may assume two conformations, one active and one inactive in binding to the SV40 origin of replication. In the presence of purine nucleoside triphosphates, the inactive conformation is favored. PMID- 3009886 TI - Cross-linking of a polyomavirus attachment protein to its mouse kidney cell receptor. AB - We used photoaffinity cross-linking with the heterobifunctional cross-linker N hydroxysuccinimidyl 4-azidobenzoate (HSAB) to covalently link polyomavirus to a mouse kidney cell surface component. The virus-HSAB combination was adsorbed to the cells and then cross-linked and isolated in monopinocytotic vesicles from the cells after endocytosis. The cross-linked product was identified on sodium dodecyl sulfate-polyacrylamide gels by the presence of a new band carrying 125I labeled virion protein with a higher molecular mass than the normal virion protein bands. A single new band, with an apparent molecular mass of 120 kilodaltons (120 kDa), was identified by this procedure. This band was formed only in the presence of the HSAB cross-linker when virions were bound to the cells. The band also copurified with cross-linked virions when virion-containing vesicles were treated with detergent to remove the cell membrane. Antibody treatments that blocked up to 100% of virus binding and internalization also blocked cross-linking, as measured by the formation of the 120-kDa band. The 120 kDa band was characterized by preparation of antibody against the excised band from the gel. This antibody was shown to have the expected dual specificity for polyomavirus VP1 sequences and plasma membrane proteins, as analyzed on Western blots. The anti-120-kDa antibody was also shown by immunofluorescence to bind to the surface of mouse kidney cells. These data have demonstrated that molecules of possible biological significance in the binding of polyomavirus to mouse kidney cells have been cross-linked and that cell surface molecules have been identified that may be characterized further for possible receptor function in polyomavirus attachment. PMID- 3009888 TI - Internal initiation of translation on the vesicular stomatitis virus phosphoprotein mRNA yields a second protein. AB - In vitro translation of a mixture of the vesicular stomatitis virus (VSV) polyadenylated mRNAs yielded a previously undetected protein with a molecular weight of approximately 7,000 (7K protein). Hybrid-arrested translation demonstrated that both the 7K protein and the VSV phosphoprotein (P protein) were encoded by the P protein message. Immunoprecipitation of the 7K protein with monoclonal antiserum directed against the P protein indicated that the two products were encoded in the same open reading frame. A protein of approximately the same size was immunoprecipitated from cytoplasmic extracts of VSV-infected cells by both the polyclonal and monoclonal antisera, and it is likely that it was a previously unrecognized viral gene product. Translational mapping of the P protein mRNA in vitro indicated that the 7K protein was encoded in the 3' one third of the sequence. The synthesis of the 7K protein in vitro was unaffected by hybrid arrest conditions which blocked the 5' two-thirds of the mRNA and inhibited synthesis of the P protein. These results imply that the ribosomes bind and initiate translation internally on the P protein mRNA at a site located hundreds of nucleotides downstream from the capped 5' end. PMID- 3009887 TI - Synthesis of plus- and minus-strand RNA from poliovirion RNA template in vitro. AB - The poliovirus RNA polymerase, 3Dpol, was used to synthesize RNA in vitro in the presence of a host factor preparation from uninfected HeLa cells and poliovirion RNA as the template. The transcription products included molecules approximately twice the length of the template, apparently resulting from hairpin formation and template-directed elongation, as previously reported (D. C. Young, D. M. Tuschall, and J. B. Flanegan, J. Virol. 54:256-264, 1985). Other polyadenylated template RNAs also yielded products that were twice the length of the template. The polarity of the products synthesized from plus-strand poliovirus RNA template was analyzed by Southern blotting using labeled product RNA to probe single stranded poliovirus DNAs cloned into M13 vectors. The results demonstrated that host factor-mediated polymerase products contain newly synthesized plus-strand sequences as well as the expected minus-strand sequences. Polymerase products primed with oligo(U) were all of minus-strand polarity. PMID- 3009889 TI - Mapping of phosphorylation sites in polyomavirus large T antigen. AB - The phosphorylation sites of polyomavirus large T antigen from infected or transformed cells were investigated. Tryptic digestion of large T antigen from infected, 32Pi-labeled cells revealed seven major phosphopeptides. Five of these were phosphorylated only at serine residues, and two were phosphorylated at serine and threonine residues. The overall ratio of phosphoserine to phosphothreonine was 6:1. The transformed cell line B4 expressed two polyomavirus specific phosphoproteins: large T antigen, which was only weakly phosphorylated, and a truncated form of large T antigen of 34,000 molecular weight which was heavily phosphorylated. Both showed phosphorylation patterns similar to that of large T antigen from infected cells. Peptide analyses of large T antigens encoded by the deletion mutants dl8 and dl23 or of specific fragments of wild-type large T antigen indicated that the phosphorylation sites are located in an amino terminal region upstream of residue 194. The amino acid composition of the phosphopeptides as revealed by differential labeling with various amino acids indicated that several phosphopeptides contain overlapping sequences and that all phosphorylation sites are located in four tryptic peptides derived from a region between Met71 and Arg191. Two of the potential phosphorylation sites were identified as Ser81 and Thr187. The possible role of this modification of large T antigen is discussed. PMID- 3009890 TI - Nucleotide sequences of a feline leukemia virus subgroup A envelope gene and long terminal repeat and evidence for the recombinational origin of subgroup B viruses. AB - Molecular clones of the subgroup A feline leukemia virus FeLV-A/Glasgow-1 have been obtained. Nucleotide sequence analysis of the 3' end of the proviral genome and comparison with the published sequence of FeLV-B/Gardner-Arnstein showed that the most extensive differences are located within the 5' domain of the env gene. Within this domain, several divergent regions of env are separated by more conserved segments. The 3' end of env is highly conserved, with only a single amino acid coding difference in p15env. The proviral long terminal repeats are also highly conserved, differing by only eight base substitutions and one base insertion. Specific probes constructed from the FeLV-A or FeLV-B env genes were used to compare the env genes of various exogenous FeLV isolates and the endogenous FeLV-related proviruses of normal cat DNA. An FeLV-A-derived env probe showed no hybridization to normal cat DNA but detected all FeLV-A and FeLV-C isolates tested. In contrast, an FeLV-B env probe detected independent FeLV-B isolates and a family of endogenous FeLV-related proviruses. Our observations provide strong evidence to support the hypothesis that FeLV-B viruses have arisen by recombination between FeLV-A and endogenous proviral elements in cat DNA. PMID- 3009891 TI - Identification by antibody to a synthetic peptide of a protein specified by a diploid gene located in the terminal repeats of the L component of herpes simplex virus genome. AB - In the course of studies on the a sequences located at the termini of and at the junction between the L and S components of herpes simplex virus 1 DNA, J. Chou and B. Roizman (J. Virol. 57:629-637, 1986) noted that the a sequence acted as a gamma 1 promoter when fused to the structural sequence of the thymidine kinase gene, the b inverted repeat sequences located in the L component next to the a sequences contained an open reading frame predicted to encode the protein of 358 amino acids with a molecular weight of 37,054, and the transcription of an RNA homologous to the open reading frame initiated within the a sequence. The nucleotide sequence of the open reading frame predicted the presence of the triplet Ala-Thr-Pro repeated 10 times. To verify the existence of the predicted gene, designated gamma 134.5, a synthetic peptide consisting of the triplet Ala Thr-Pro repeated 10 times was synthesized and used to raise antibodies in rabbits. The results were as follows. The antiserum to the peptide reacted with a 43,500-apparent-molecular-weight protein present in lysates of cells infected with herpes simplex virus 1 but not present in mock-infected or herpes simplex virus 2-infected cells. We genetically engineered a recombinant virus containing a single copy of a truncated gene. Concordant with predictions, the antibody reacted with a faster-migrating protein in cells infected with this recombinant. The gamma 134.5 gene product was soluble, and it accumulated primarily in the cytoplasm late in infection. The overlap of the domain of the gamma 134.5 gene with the a sequence raises the possibility that it acts in trans on the a sequence and is associated with one of the functions currently ascribed to the a sequences. PMID- 3009892 TI - Establishment of a rat cell line inducible for the expression of human cytomegalovirus immediate-early gene products by protein synthesis inhibition. AB - Upon transfection of Rat-2-TK- cells with plasmid pES, containing the cloned 7.0 kilobase (kb) EcoRI-SalI fragment (0.063 to 0.089 map units) of the human cytomegalovirus genome, major immediate-early antigen expression was obtained in 1 to 2% of the nuclei of the transfected cells, as determined by immunofluorescence with the E3 monoclonal antibody. Cotransfection of pES with the cloned herpes simplex virus type 1 thymidine kinase gene resulted in the establishment of a hypoxanthine-aminopterin-thymidine-resistant cell line which expressed a major immediate-early antigen in approximately 1% of the cells at early passages, with expression gradually declining to less than 0.1% upon subculturing. Southern blot analysis of DNA extracted from this cell line revealed the presence of multiple integration events of pES DNA sequences into cellular DNA, including a head-to-tail tandem array of approximately 10 copies of pES. The integration pattern was stable for at least 80 passages. Metaphase chromosomes prepared from this cell line showed, upon in situ hybridization, a strong hybridization signal in both sister chromatids of a large submetacentric chromosome which is considered to have harbored the tandemly integrated pES molecules. Whereas in most cells of the population, immediate-early expression seemed to be repressed, this repression could be overcome by protein synthesis inhibition, resulting in a massive induction of human-cytomegalovirus-specific transcripts of 2.1 and 1.9 kb and a minor species of 2.9 kb. After release from protein synthesis inhibition, approximately 20% of the cells showed nuclear fluorescence when the E3 monoclonal antibody was used. PMID- 3009893 TI - Intracellular localization and processing of pp60v-src proteins expressed by two distinct temperature-sensitive mutants of Rous sarcoma virus. AB - The transforming protein of Rous sarcoma virus, pp60v-src, is known to be a tyrosine protein kinase, but the mechanism of cell transformation remains unclear. In further investigating pp60v-src structure and function, we have analyzed two temperature-sensitive (ts) Rous sarcoma virus src gene mutants, tsLA29 and tsLA32. The mutations in tsLA29 and tsLA32 map in the carboxy-terminal region and the amino-terminal half of pp60v-src, respectively, and encode mutant proteins with either temperature-labile (tsLA29) or -stable (tsLA32) kinase activities. Here we examined the intracellular processing and localization of these pp60v-src mutants and extended our characterization of transformation parameters expressed by cells infected by the Rous sarcoma virus variants. No obvious defects in functional integrity of the tsLA32 pp60v-src could yet be demonstrated, whereas the tsLA29 pp60v-src was perturbed not only in kinase activity, but also in aspects of protein processing and localization. Analysis of transformation parameters expressed by infected cells demonstrated the complete temperature lability of both mutants. PMID- 3009894 TI - A second protease of foot-and-mouth disease virus. AB - Foot-and-mouth disease virus (FMDV) genes are expressed as a polyprotein which is rapidly processed into the four primary cleavage products L, P1, P2, and P3. In secondary cleavage reactions, these are further processed into the mature proteins. The FMDV L protein is located at the N terminus of the polyprotein and is the first gene product released from the nascent polyprotein. For analysis of its biological function, the L gene was mutated by site-directed mutagenesis of cloned cDNA. In vitro translation of in vitro transcripts of these DNAs and expression studies in Escherichia coli showed that the L mutants affect the processing of the viral polyprotein. The mutants isolated were partially or totally defective in processing the polyprotein at the L/P1 junction. These mutants could, however, be processed in the presence of the wild-type L protein. Furthermore, an antiserum directed against the L protein inhibited processing at the L/P1 cleavage site, so that the release of the L protein from the polyprotein was blocked. These data reveal that the L gene product represents a viral protease which catalyzes its own release from the nascent polyprotein. PMID- 3009895 TI - Enhancive and suppressive effects of cytomegalovirus on human lymphocyte responses in vitro. AB - Virus-specific lymphocyte proliferation in the presence of cytomegalovirus (CMV) without and with monocytes was studied in healthy persons. Three categories of lymphocyte response could be distinguished: seropositive low responders, naturally high responders, and lymphocyte populations responding well to CMV antigen in the presence of added CMV-incubated autologous monocytes. This latter category could be identified by preincubating autologous monocytes with CMV. CMV seronegative persons were nonresponders. Early CMV antigens were produced in monocytes but not in lymphocytes by all CMV isolates. Infection of monocytes as detected by antibody to early viral protein did not appear to abort the antigen presenting ability. The virus-specific responding lymphocytes were mainly of the T4+ phenotype. In contrast, addition of CMV to polyclonal mitogens significantly suppressed total lymphocyte DNA synthesis. CMV thus may have an enhanced virus specific stimulatory effect on lymphocytes together with monocytes but a suppressive effect on the total lymphocyte population. PMID- 3009896 TI - Tumorigenicity of persistently infected tumors in nude mice is a function of both virus and host cell type. AB - Although BHK-21 cells persistently infected with wild-type vesicular stomatitis virus (VSV) are sensitive to natural killer (NK) cells and do not form tumors in athymic nude mice, BHK-21 cells persistently infected with a previously isolated mutant virus (VSV-P) are resistant to NK cells and form tumors in nude mice. We used this VSV-P mutant to persistently infect HeLa cells and mouse tumor cell lines. A mouse mastocytoma line (P815) persistently infected with VSV-P was similar to BHK-21 cells in that it was resistant to NK cell lysis and formed tumors in nude mice. However, neither HeLa cells nor mouse myeloma lines persistently infected with VSV-P were resistant to NK cell lysis in vitro, and neither formed tumors in nude mice. Rejection by nude mice of HeLa cells and mouse myeloma cell lines persistently infected with VSV-P could be ablated by rabbit antiserum to asialo-GM1, implicating NK cells in the in vivo rejection of these persistently infected tumors. These results suggest that NK cell recognition and killing of virus-infected cells in vivo and in vitro depend upon genetic contributions from both the virus and the host cell. PMID- 3009898 TI - Susceptibility of fetal guinea pig brain cell cultures to replicating guinea pig cytomegalovirus infection is increased with increasing fetal age: infection of astrocytes. AB - Guinea pig brain cell cultures were established from fetuses at 25, 31, and 37 days of gestation (DG). After 7 days in vitro, the cultures were infected with guinea pig cytomegalovirus (GPCMV). Based on cytopathic effect, immunofluorescence staining for GPCMV by using virus-specific antiserum, and the amount of virus recovered, cultures established from fetuses at 25 DG were least susceptible to replicating infection, and cultures established from fetuses at 37 DG were most susceptible. Using cell-type-specific markers, it was determined that the increase in susceptibility to replicating infection paralleled an increase in the number of differentiated cells. Astrocytes were the most abundant cell type identified and were susceptible to replicating GPCMV infection, whereas neurons were not. PMID- 3009897 TI - Molecular cloning and long terminal repeat sequences of human endogenous retrovirus genes related to types A and B retrovirus genes. AB - By using a DNA fragment primarily encoding the reverse transcriptase (pol) region of the Syrian hamster intracisternal A particle (IAP; type A retrovirus) gene as a probe, human endogenous retrovirus genes, tentatively termed HERV-K genes, were cloned from a fetal human liver gene library. Typical HERV-K genes were 9.1 or 9.4 kilobases in length, having long terminal repeats (LTRs) of ca. 970 base pairs. Many structural features commonly observed on the retrovirus LTRs, such as the TATAA box, polyadenylation signal, and terminal inverted repeats, were present on each LTR, and a lysine (K) tRNA having a CUU anticodon was identified as a presumed primer tRNA. The HERV-K LTR, however, had little sequence homology to either the IAP LTR or other typical oncovirus LTRs. By filter hybridization, the number of HERV-K genes was estimated to be ca. 50 copies per haploid human genome. The cloned mouse mammary tumor virus (type B) gene was found to hybridize with both the HERV-K and IAP genes to essentially the same extent. PMID- 3009899 TI - Human papillomaviruses in Buschke-Lowenstein tumors: physical state of the DNA and identification of a tandem duplication in the noncoding region of a human papillomavirus 6 subtype. AB - Six Buschke-Lowenstein tumors, i.e., highly differentiated squamous cell tumors of the genital region, were shown to contain human papillomavirus 6 (HPV 6) or HPV 11 genomes. The viral DNA was found in an episomal state, including a very small fraction of circular oligomers. HPV 6a and HPV 6d genomes were cloned from two of the tumors. Comparison with HPV 6b, cloned from a benign genital wart (E. M. de Villiers, L. Gissmann, and H. zur Hausen, J. Virol. 40:932-935, 1981) by restriction mapping and partial sequence analysis, revealed a very high degree of homology with the different HPV 6 subtypes. A tandem duplication of 459 base pairs within the noncoding region of the genome was found in the new subtype HPV 6d. This structural rearrangement in a region containing the putative control elements for early gene transcription might influence the biological potential of that virus. No evidence for rearrangement of this region was found in the HPV DNA from the five other tumors. PMID- 3009900 TI - Nuclease mechanism of the avian retrovirus pp32 endonuclease. AB - In vivo, the inferred circular retrovirus DNA precursor to the provirus contains two long terminal repeats (LTRs) in tandem. We studied the site-specific nicking of supercoiled DNA that contains tandem copies of avian retrovirus LTR DNA in vitro by using purified avian myeloblastosis virus pp32 endonuclease, Mg2+, and viral DNA substrates containing different LTR circle junction sequences. The results confirmed our previous observation that the pp32 protein generates two nicks, one in either viral DNA strand, each 2 nucleotides from the circle junction site. The specificity of nicking by pp32 was unchanged over an eight fold range of protein concentration and with different avian retrovirus LTR circle junction substrates. These data are consistent with models which propose a role for the endonuclease in removal of two nucleotides from the LTR termini on integration of viral DNA in vivo. PMID- 3009901 TI - Cloning of monomeric human papillomavirus type 16 DNA integrated within cell DNA from a cervical carcinoma. AB - We have molecularly cloned and characterized monomeric human papillomavirus type 16 DNA with flanking cell DNA sequences from a cervical carcinoma. Determination of nucleotide sequence around the junctions of human papillomavirus and cell DNAs revealed that at the site of integration within cell DNA the cloned viral DNA had a deletion between nucleotides 1,284 and 4,471 (numbering system from K. Seedorf, G. Krammer, M. Durst, S. Suhai, and W. G. Rowekamp, Virology 145:181-185, 1985), which includes the greater part of E1 gene and the entire E2 gene. In the remaining part of the E1 gene, three guanines were found at the location where two guanines at nucleotides 1,137 and 1,138 have been recorded. This additional guanine shifted the reading frame and erased an interruption in the E1 gene described by Seedorf et al. The data strongly suggest that, like other papillomaviruses, human papillomavirus type 16 has an uninterrupted E1 gene. PMID- 3009902 TI - Genome organization and nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer. AB - The 7,909-nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer, has been determined and used to deduce the corresponding genome arrangement. Extensive sequence homologies and other genetic features are shared with the related oncogenic virus, human papillomavirus type 16, especially in the major reading frames. A surprising difference was found in the noncoding region of human papillomavirus type 33 as, unlike all other sequenced papillomaviruses, it contains a perfect 78-base pair tandem repeat. PMID- 3009903 TI - Dacron inhibition of arterial regenerative activities. AB - These biocompatibility studies evaluate the effects of Dacron, absorbable polymeric, and compound prostheses containing both elements in various constructions on the migration, proliferation, and functional characteristics of regenerating endothelial and smooth muscle-like cells in the rabbit aorta model. Prosthesis/tissue complexes explanted after 2 weeks to 9 months were studied grossly, photographed, sectioned for light microscopy and scanning and transmission electron microscopy, and assayed for 6-keto-PGE1 alpha contents in inner capsular tissues. Polyglycolic acid, polyglactin 910, or polydioxanone prostheses elicited a transinterstitial migration and proliferation of primitive mesenchymal cells that differentiated into smooth muscle-like myofibroblasts and a surface repopulation of confluent endothelial-like cells paralleling the time course of macrophage-mediated prosthetic dissolution. Even small Dacron components (20%) woven into or surrounding the absorbable polymer significantly inhibited these processes, yielding significantly thinner, less cellular inner capsules with lower 6-keto-PGF1 alpha contents. These studies show the augmentation of clinically efficacious arterial regenerative activities by polymers phagocytosed by macrophages and the inhibition of these activities by Dacron. PMID- 3009905 TI - Prisons confront dilemma of inmates with AIDS. PMID- 3009904 TI - Vascular anomalies. PMID- 3009906 TI - Physicians and the mental illnesses: the nudge from ADAMHA. PMID- 3009907 TI - Leads from the MMWR. Perspectives in disease prevention and health promotion: public health guidelines for enhancing diabetes control through maternal- and child-health programs. PMID- 3009908 TI - Hyperkalemia with enalapril in advanced renal failure. PMID- 3009909 TI - Federal epidemiologists link ills to environs. PMID- 3009911 TI - [Therapeutic effect of ceftizoxime on infection in patients with lung cancer]. AB - Ceftizoxime (CZX) was given in daily doses of 4 approximately 6 g by intravenous drip infusion to 30 patients with infection accompanying lung cancer to investigate the usefulness of the drug for infectious disease: The rate of effectiveness (marked and moderate) was 73.3% (22/30 patients). Of the 30 patients, 2 had drug fever; 1, arthralgia; and 1, eosinophilia. These side effects improved after the drug was withdrawn. CZX is a very useful antibiotic with high effectiveness and safety in immunocompromised patients with infection accompanying advanced lung cancer. PMID- 3009910 TI - Prevalence of antibodies to HTLV-I, -II, and -III in intravenous drug abusers from an AIDS endemic region. AB - Antibody prevalences for human T-cell lymphotropic virus (HTLV) types I, II, and III were determined for 56 intravenous drug abusers from Queens, NY. While control serum samples lacked antibodies to all HTLV subgroups, seropositivity among drug users was 41% for HTLV-III, 18% for HTLV-II, and 9% for HTLV-I. Infection by HTLV-I and -II occurred independently of HTLV-III infection. Blacks had greater HTLV-III antibody prevalence than whites (54% vs 16%) and were more likely than whites to be seropositive for HTLV-I or -II (46% vs 11%). They exhibited a greater incidence than whites of double infection with HTLV-I or -II and HTLV-III (27% vs 0%), and 73% were seropositive for at least one of the viruses, compared with only 26% of the whites. The increased HTLV-I and -II infection seen in intravenous drug users suggests that once introduced into a population, these viruses may be transmitted by the same routes as HTLV-III. Transmission may have been restricted mainly to blacks in this study because of local drug use practices. PMID- 3009912 TI - [Ultrastructural cytochemical demonstration of glucose-6-phosphatase activity in erythroblasts from human bone marrow. The significance of glucose-6-phosphatase activity positive erythroblasts]. PMID- 3009913 TI - [Gene transplantation. Studies of individual oncogenesis with transgenetic mice]. PMID- 3009914 TI - [Virus-induced neoplasms]. PMID- 3009915 TI - [The role of oncogenes]. PMID- 3009916 TI - [The role of hormones in neoplastic transformation]. PMID- 3009917 TI - [Early detection of neoplastic cells]. PMID- 3009918 TI - Histologic factors influencing prognosis of adenoid cystic carcinoma of the head and neck. AB - Histologic specimens from 61 cases of adenoid cystic carcinoma of the head and neck were reviewed in order to determine prognostic factors of this peculiar neoplasm. After the tumor was reviewed and classified according to several criteria, follow-up information, which was available in 54 cases, was analyzed and compared with each histological subgroup. As for the histologic pattern, the prognosis of adenoid cystic carcinoma with a solid pattern was worse than that without a solid pattern in respect of 5- and 10-year survival rates. However, there was no difference in 15-year survival between the two groups. Tumors with severe cell atypia showed more malignant behavior than those with moderate or mild atypia (p less than 0.001). Increased mitotic activity of more than two cells in mitosis per high power field was also found to be a sign of poor prognosis (p less than 0.001). Presence of perineural invasion was not correlated with the final outcome of the disease. Cell atypia and mitotic activity as well as the histologic pattern should be evaluated for assessing the degree of malignancy of adenoid cystic carcinoma. PMID- 3009919 TI - Diagnostic significance of cancer antigen 125, pancreatic oncofetal antigen, and carcinoembryonic antigen in malignant and tuberculous pleural effusions. AB - Pleural fluid levels of cancer antigen 125 (CA 125), pancreatic oncofetal antigen (POA), and carcinoembryonic antigen (CEA) were determined in 56 patients with malignant pleural effusion and in 35 patients with tuberculous effusion. Malignant effusions had significantly higher pleural fluid CA 125 and CEA levels than those of tuberculous origin (p less than 0.005). No significant difference in POA titers was found between the two kinds of effusion. Assays of CA 125 and CEA in pleural fluid may be useful in separating malignant from tuberculous effusions. Concurrent measurement of CA 125 and CEA proved superior to determination of CEA alone in discriminating between the two groups. PMID- 3009920 TI - Medical treatment for inoperable insulinoma: clinical usefulness of diphenylhydantoin and diltiazem. AB - This report describes a 78-year-old woman with insulinoma, treated with a combination of diphenylhydantoin and a calcium antagonist. The effectiveness of 200 mg of diphenylhydantoin and 180 mg of diltiazem was evaluated by measuring the levels of plasma glucose, immunoreactive insulin and immunoreactive insulin/plasma glucose after fasting or by the oral glucose tolerance test, and by the appearance of hypoglycemic symptoms. The mean concentration of fasting plasma glucose increased significantly during the treatment. The levels of immunoreactive insulin/plasma glucose and C-peptide immunoreactivity/plasma glucose significantly decreased. Symptomatically, no episode of hypoglycemia was noted during the combined treatment. PMID- 3009921 TI - A case of pneumocytoma (so-called sclerosing hemangioma) with lymph node metastasis. AB - A case of "sclerosing hemangioma" (pneumocytoma) of the lung with lymph node metastasis is reported. A 22-year-old Japanese man was found to have a well defined round lesion in the right lung (S7), which increased in size slightly during a 2-year follow-up period. He underwent right lower lobectomy with a preoperative diagnosis of a benign lung tumor. The pulmonary tumor revealed histological features characteristic of "sclerosing hemangioma" of the lung, in addition to which there were many large polygonal foamy cells, forming tubular or papillary structures. These cells were found by electron microscopy to contain numerous cytoplasmic lamellar bodies and showed a positive reaction with anti surfactant apoprotein antibody immunohistochemically. Therefore, they were considered to be cells differentiating toward type II pneumocytes. Review of 21 typical "sclerosing hemangiomas" disclosed a few or some such foamy cells in 10 cases. A single hilar lymph node was the site of microscopic metastases, which consisted of "large clear foamy cells" and smaller polygonal or round cells with slightly eosinophilic cytoplasm, both of which were components of the pulmonary "sclerosing hemangioma." This case supports the theory that "sclerosing hemangioma" is a neoplasm of type II pneumocyte lineage. Although it is said to be benign, rare cases apparently show metastatic potential. PMID- 3009922 TI - [Changes in sensory nerve action potentials in ischemia]. PMID- 3009923 TI - [Host defense mechanism in virus infections]. PMID- 3009925 TI - [Emergency hepatic artery embolization--treatment of intraperitoneal hemorrhage due to hepatoma and traumatic liver laceration]. PMID- 3009926 TI - [A case report of multilocular cystic nephroma]. PMID- 3009927 TI - [Abnormal accumulation of 99mTc-pyrophosphate in a patient with amyloidosis: case report]. PMID- 3009924 TI - [Application of monoclonal antibody to laboratory tests--immunoassay]. PMID- 3009928 TI - [Clinical evaluation of gallium-67 scintigraphy for carcinoma of the lung--with special reference to prognosis]. PMID- 3009929 TI - [Recent advances in human papillomavirus research]. PMID- 3009930 TI - [Adult T-cell leukemia and HTLV-I]. PMID- 3009931 TI - [Clinico-pathological studies on herpes zoster; [I]. Changes in antibody titers on herpes zoster]. PMID- 3009933 TI - [Clinical evaluation of digital subtraction angiography on liver tumor]. PMID- 3009932 TI - Failure to detect murine leukemia virus antigens in immune-complex glomerulonephritis of graft-vs-host F1 mice. PMID- 3009935 TI - [Endocrine pancreas tumor producing the growth hormone-releasing factor with growth hormone hypersecretion: a case report]. PMID- 3009934 TI - [Clinical significance of plasma c-AMP response to glucagon for the assessment of the prognosis in liver cirrhosis and hepatocellular carcinoma]. PMID- 3009936 TI - [Effect of NaHCO3 infusion on the healing of acetic acid-induced gastric ulcer in rats]. PMID- 3009937 TI - [Scintiphotosplenoportography for assessing the effects of injection sclerotherapy in esophageal varices]. PMID- 3009938 TI - [Endoscopic lymphoscintigraphy with SPECT]. PMID- 3009940 TI - [Evaluation of neuron specific enolase (NSE) radioimmunoassay kit]. PMID- 3009941 TI - Effects of neurotensin on the non-adrenergic inhibitory neurotransmission in the guinea-pig duodenum. AB - The effects of neurotensin on the non-adrenergic inhibitory neurotransmission in the guinea-pig duodenum were investigated. The resting membrane potential of the duodenal smooth muscle cells was reduced by neurotensin at the concentration of 3 X 10(-8) M or more. The amplitude of the non-adrenergic inhibitory potentials was decreased by neurotensin (1-3 X 10(-8) M). In the Ca2+-free solution, the amplitude of the inhibitory potentials was also decreased. The increased amplitude of the non-adrenergic inhibitory potentials evoked in the high calcium solution (Ca2+, 5 mM) was decreased by neurotensin (10(-8) M). Neither atropine (1.4 X 10(-6) M) nor propranolol (3.4 X 10(-6) M) blocked the inhibitory action of neurotensin (10(-8) M) on the inhibitory potential. The frequency of the spontaneous action potentials of the duodenal smooth muscle cells was strongly increased with accompanying depolarization by neurotensin 8 X 10(-8) M. The tonic contraction of the duodenal smooth muscles was produced by neurotensin (3-8 X 10( 8) M). The results obtained suggest that neurotensin relates to the non adrenergic inhibitory pathway in the control mechanism on intestinal motility. PMID- 3009939 TI - [Salivary gland scintigraphy using subtraction technique: evaluation for salivary gland neoplasm of positive localization of 99mTcO4]. PMID- 3009942 TI - Action of partially purified ACTH-potentiating substance from rat serum on isolated rat adrenal cells. AB - A serum extract possessing ACTH-potentiating activity was partially purified by Sephadex G-100 gel filtration. Fractions from 9,000 to 40,000 in molecular weight (APS-Fr) potentiated the corticosterone production induced by synthetic adrenocorticotropic hormone (ACTH1-24). Approximately 5 micrograms (as protein) of APS-Fr in 0.5 ml medium showed the maximum activity to potentiate the response of isolated rat adrenal cells to ACTH1-24. Puromycin, cycloheximide and actinomycin D did not abolish the potentiation by APS-Fr of ACTH1-24-induced steroidogenesis. The log-dose response curve for ACTH1-24 was shifted toward the side representing lower doses of ACTH1-24 by APS-Fr, but the maximum response was not changed. In a typical experiment, the median effective dose (ED50) for ACTH1 24 was decreased from 5.2 X 10(-11) M to 3.0 X 10(-12) M. ACTH1-24-induced production of cyclic AMP was also increased by APS-Fr. Time courses of both corticosterone and cyclic AMP production induced by ACTH1-24 did not differ qualitatively in either the presence or absence of APS-Fr, although they showed a marked difference quantitatively. When dibutyryl cyclic AMP was used in place of ACTH1-24, APS-Fr did not potentiate steroidogenesis. These results suggest that APS-Fr acts at a step between the binding of ACTH to its receptor and the formation of cyclic AMP. ACTH1-24-induced steroidogenesis which was increased by increasing concentration of calcium in the medium, was further increased by addition of APS-Fr. The calcium requirement for the same degree of response was decreased by addition of APS-Fr, in a similar way to when a higher concentration of ACTH1-24 was used. PMID- 3009943 TI - Activities of ACTH-potentiating substances isolated from rat serum on steroidogenesis in isolated rat adrenal cells. AB - Peptides that potentiate the response of adrenal cells to ACTH1-24 were isolated from rat serum. ACTH-potentiating activity was found in fractions of 9,000-40,000 in molecular weight (APS-Fr) and of smaller molecular weight (SM-Fr) on Sephadex G-100 gel filtration of the serum extract. The peptides were isolated from APS-Fr by preparative acid polyacrylamide gel electrophoresis and named d1, d, d2, e, f and g. Their molecular weights, estimated by Sephadex G-75 gel filtration, were 41,000, 33,000, 24,000, 17,500, 17,500 and 16,000, respectively. All of these peptides were glycopeptides. The isoelectric point of peptide d was 8.45 and some of the others, such as f and g, were more basic. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis revealed that these peptides were decomposed into various fragments. ACTH-potentiating activity was highest in peptide d1 and lowest in peptide e. The maximum activity of peptide d was observed at 3 X 10(-8) M when steroidogenesis was induced by 9 X 10(-12) M ACTH1-24. While these peptides did not show any ACTH-like activity at any stage of isolation, the fractions of these peptides eluted from a Sephadex G-75 column showed more or less ACTH-like activity. These peptides therefore seemed to possess latent ACTH like activity. The molecular weight of SM-Fr was smaller than ACTH1-24. The ACTH potentiating activity of SM-Fr was low, and SM-Fr did not show any ACTH-like activity. SM-Fr therefore seems to be the smallest structure which has ACTH potentiating activity. The similarity of these peptides to proopiomelanocortin related substances was discussed. PMID- 3009944 TI - Biochemical and pharmacological analysis of 2-[(2-dimethylaminobenzyl)sulfinyl] benzimidazole (NC-1300), a new proton pump inhibitor. AB - Biochemical and pharmacological properties of a newly synthesized compound, 2-[(2 dimethylaminobenzyl)sulfinyl] benzimidazole (NC-1300), were studied. NC-1300, at pH 6.0, potently inhibited the activity of H+ K+ ATPase in the rabbit gastric mucosa, thereby classifying it as a proton pump inhibitor. The inhibitory efficacy of NC-1300 on the pump was much the same as that seen with omeprazole. NC-1300 had no effect on acetylcholine-stimulated ileum contraction in guinea pigs at 10(-5) M, but it non-competitively inhibited the contraction at 10(-4) M. NC-1300 had no effect on histamine-stimulated atrial beating frequency in guinea pigs at 10(-4) or 10(-5) M. NC-1300, given either intraduodenally or orally, had a potent and long-lasting (more than 24 hr) inhibitory effect on gastric secretion in pylorus-ligated rats. Pretreatment with NC-1300 dose-dependently protected the gastric mucosa from damage induced by pylorus ligation, water immersion stress, aspirin, and indomethacin, and the duodenal mucosa from damage induced by mepirizole in rats. We conclude that the antisecretory activity of NC 1300 appears to be mainly related to an inhibition of H+ K+ ATPase, while its antigastric and antiduodenal lesion activities are primarily related to an antisecretory effect. PMID- 3009946 TI - Stimulation-evoked cyclic nucleotides efflux from isolated perfused dog adrenals and possible involvement of calcium. AB - Nicotine and muscarine caused the transient increase in cAMP and cGMP efflux from dog adrenal glands followed by a small but lasting increase in the nucleotide efflux. The initial increase preceded catecholamine (CA) release and the latter slowly developed. Nicotine and muscarine caused the maximal increase of cAMP and cGMP levels in adrenal medulla 15 sec after the treatment, and these effects were antagonized by hexamethonium and atropine, respectively. Hexamethonium in combination with atropine, verapamil and an omission of Ca2+ in the medium prevented ACh from producing the increase in cyclic nucleotide efflux and CA release. Reintroduction of Ca2+ (1.3 mM) in fluid after perfusion with Ca2+ and Mg2+-free fluid caused the transient increase in cyclic nucleotide efflux and CA release. Adenylate cyclase activity in adrenal medulla was activated by Ca2+. These results may suggest that cAMP formation was increased by activation of adenylate cyclase as a result of increased influx of Ca2+ when adrenal medulla were stimulated. PMID- 3009945 TI - Analysis of ouabain-induced contractions in isolated coronary arteries. AB - Contractile responses to ouabain in helical strips of dog and monkey coronary arteries were investigated. Ouabain (5 X 10(-8) to 5 X 10(-6) M) caused a dose related contraction in dog and monkey arteries; the response of monkey coronary arteries was significantly greater. In dog coronary arteries, contractile responses to high concentrations of ouabain were potentiated by treatment with propranolol. In the arteries contracted with ouabain, the addition of phentolamine caused a relaxation. Contractile responses of dog coronary arteries to ouabain were markedly suppressed by exposure to Ca2+-free media or by treatment with verapamil. Reduction of external concentration of K+ or lowering the temperature of bathing media did not selectively influence the ouabain induced contraction. These results suggest that ouabain-induced contractions of dog coronary arteries are associated mainly with an increase in the Ca2+-influx, which does not result from an inhibition of the Na+, K+-activated ATPase nor from an activation of alpha adrenoceptors by noradrenaline released from adrenergic nerves. Ouabain in high concentrations seems to liberate noradrenaline from adrenergic nerves, which preferentially activates beta adrenoceptors in dog coronary artery. PMID- 3009947 TI - Effects of eperisone applied by microiontophoresis on neurons in the medial and lateral vestibular nuclei. AB - Electrophysiological studies were performed to elucidate the mechanism underlying the antivertigo action of eperisone, an antispastic drug, using cats anesthetized with alpha-chloralose. Iontophoretic application of eperisone up to 100 nA produced a dose-dependent inhibition of spike generation upon vestibular nerve stimulation in monosynaptic and polysynaptic neurons of the medial vestibular nucleus (MVN). The inhibition of neurons in the MVN was more prominent than that in the lateral vestibular nucleus. In addition, iontophoretically applied eperisone in doses of 50-100 nA inhibited the orthodromic spike elicited by vestibular nerve stimulation in the MVN monosynaptic neurons projecting to the abducens nucleus (ascending neuron), without affecting that in the MVN neurons projecting to the spinal cord (descending neuron). An inhibition of antidromic spike elicited by abducens nucleus stimulation in the MVN monosynaptic ascending neurons was observed in some cases during application of eperisone. These results suggest that eperisone predominantly inhibits synaptic transmission of the MVN ascending neurons. PMID- 3009949 TI - Actions of melanotropins on mouse melanoma cell growth in vitro. AB - Melanotropins induce melanogenesis in mouse Cloudman S91 melanoma cells by stimulating the activity of tyrosinase. In monolayer culture, alpha-melanocyte stimulating hormone and the superpotent analogue 4-norleucine, 7-D-phenylalanine alpha-melanocyte, which had prolonged effects on tyrosinase activity, did not inhibit the proliferation of melanoma cells even at concentrations that elicited maximal tyrosinase stimulation. In soft agar the melanotropins stimulated the formation of melanized colonies and increased the cloning and proliferative potentials of melanoma cells. Both melanotropins increased the number of small (42-104 microns in diameter) colonies at initial plating densities ranging from 625 to 7,500 cells/dish. The number of larger (greater than 104 microns in diameter) colonies was also increased except at densities 5,000 cells or more/dish, wherein the proliferative capacity was inhibited; yet the cloning efficiency was still increased. Therefore, in bilayer soft agar cultures, melanotropins stimulate the growth of the clonogenic S91 melanoma cell population under conditions that allow for optimal expression of the cloning and proliferative potentials of these cells. PMID- 3009948 TI - The inactivation of [Met5]-enkephalin by bestatin-sensitive aminopeptidase, captopril-sensitive peptidyl dipeptidase A and thiorphan-sensitive endopeptidase 24.11 in mouse vas deferens. AB - The enkephalin-hydrolyzing peptidases in mouse vas deferens were studied and compared with those in guinea pig ileum which had been characterized in the previous study. The present results showed that three distinct peptidases, bestatin-sensitive aminopeptidase, captopril-sensitive peptidyl dipeptidase A, and thiorphan-sensitive endopeptidase-24.11, played a critical role in the inactivation of enkephalin in mouse vas deferens, being consistent with the previous results obtained with guinea pig ileum. However, the data in both previous and present studies showed that the activity of the bestatin-sensitive aminopeptidase relative to that of either the captopril-sensitive peptidyl dipeptidase A or the thiorphan-sensitive endopeptidase-24.11 in guinea pig ileum was higher than that in mouse vas deferens, while the activity of either peptidyl dipeptidase A or endopeptidase-24.11 relative to that of aminopeptidase in mouse vas deferens was higher than that in guinea-pig ileum. In contrast to these three enzymes, both L-tyrosyl-L-tyrosine-sensitive dipeptidyl aminopeptidase and D phenylalanine-sensitive carboxypeptidase were suggested not to be involved significantly in the inactivation of enkephalin in mouse vas deferens as well as guinea pig ileum. PMID- 3009950 TI - Effects of phenobarbital and secondary bile acids on liver, gallbladder, and pancreas carcinogenesis initiated by N-nitrosobis (2-hydroxypropyl)amine in hamsters. AB - The effects of dietary administration of phenobarbital [(PB) CAS: 50-06-6] or the secondary bile acids, deoxycholic acid [(DCA) CAS: 83-44-3] and lithocholic acid [(LCA) CAS: 434-13-9], on tumorigenesis in the liver, gallbladder, and pancreas were investigated in male Syrian golden hamsters after carcinogenic initiation by N-nitrosobis(2-hydroxypropyl)amine [(BHP) CAS: 53609-64-6]. BHP [500 mg/kg (body wt)] was injected sc once weekly for 5 weeks. The animals were then maintained on a basal diet or a diet containing either 0.05% PB, 0.1% DCA, 0.5% DCA, or 0.5% LCA for 30 weeks. DCA enhanced the development of cholangiocarcinomas without influencing that of hepatocellular lesions. PB promoted the induction of hepatocellular carcinomas but not that of cholangiocarcinomas. LCA was without effect on the induction of either hepatocellular carcinomas or cholangiocarcinomas. DCA at a dose of 0.5% enhanced the induction of polyps in the gallbladder. Both DCA, at a dose of 0.1%, and LCA significantly enhanced the induction of pancreas carcinomas. PB had no effect on the induction of polyps in the gallbladder or of pancreas carcinomas. These data document that different tumors may be differentially promoted following initiation with a common carcinogen. PMID- 3009951 TI - [Dynamics of cyclase systems and hormonal indices in patients with ischemic heart disease in a state of emotional stress]. AB - A total of 64 male patients with varying forms of coronary heart disease (CHD), aged 43 to 65 years, and free of diabetes mellitus, obesity and arterial hypertension symptoms, were studied in conditions of emotional stress simulated, using the method of mental calculations with shifts of attention under time shortage. Pre- and post-exercise blood levels of cyclic nucleotides (cAMP and cGMP), the somatotropic hormone and immunoreactive insulin were measured. Stress induced decrease in platelet cAMP/cGMP ratios, indicative of further increase in the functional activity of platelets, was demonstrated in coronary patients with marked coronary atherosclerosis, as contrasted to normal subjects and patients with milder disease. They also showed a more considerable (sixfold) increase in the somatotropic hormone levels and a tendency to decreased levels of immunoreactive insulin under stress, apparently as a consequence of the prevailing activation of alpha-adrenoreceptor pathways. PMID- 3009952 TI - [Role of the cholesterol of platelet membranes in disorders of their structuro functional condition in angina pectoris patients]. AB - A study of 80 anginal patients showed their platelet molar cholesterol/phospholipid ratios, platelet membrane microviscosity, membrane release of calcium and fatty acids, and platelet aggregation capacity to be elevated. It is assumed that excessive platelet cholesterol has a significant effect on platelet aggregation, that is mediated by the intraplatelet processes examined, in anginal patients. PMID- 3009953 TI - [Diagnostic possibilities of radionuclide methods of studying patients with stable and unstable angina pectoris]. AB - Myocardial radiocardiography and scintigraphy with 99mTc-pyrophosphate was performed in 58 patients with stable angina and 60 patients with unstable angina. In patients with stable angina, positive scintigrams were mostly recorded after anginal attacks. Their central hemodynamic parameters deteriorated progressively as angina increased in severity. Patients with unstable angina typically showed myocardial accumulation of the label that was unrelated to anginal attacks and recordable by direct-projection scintigraphy. The assessment of myocardial radiocardiographic and scintigraphic data allows one to differentiate between stable and unstable angina. PMID- 3009954 TI - Nephron responses to converting enzyme inhibition in non-clipped kidney of Goldblatt hypertensive rat at normotensive pressures. AB - To evaluate the effects of angiotensin converting enzyme inhibition (SQ 20881, CEI) on superficial nephron function of the non-clipped kidney in Goldblatt hypertensive rats in the absence of alterations in renal arterial pressure, control renal arterial pressure (RAP) was reduced first to the range generally obtained during CEI (124 +/- 4 mm Hg). RAP was maintained during the CEI period by adjustment of a suprarenal aortic clamp. At the reduced RAP, whole kidney and single nephron glomerular filtration rates (GFR) were reduced from the hypertensive levels and were lower than the measurements in normotensive control rats. During CEI, whole kidney GFR and single nephron GFR increased by 55 and 42%, respectively. There were decreases in absolute as well as fractional proximal reabsorption rates. In the intermediate nephron segment, fractional reabsorption was decreased, but absolute fluid reabsorption increased in proportion to the increased delivery rate. Proximal tubule and peritubular capillary hydrostatic pressures increased significantly during CEI also. These results indicate that an increased activity of the renin-angiotensin system occurring in Goldblatt hypertensive rats subjected to aortic constriction exerts effects to lower GFR and increase proximal reabsorption rate. The concomitant superficial nephron and whole kidney GFR responses to CEI when arterial pressure was maintained suggests that the pre-existing levels of angiotensin exerted similar influences on the total nephron population. PMID- 3009955 TI - The renal response to chronic mineral acid feeding: a re-examination of the role of systemic pH. AB - It has been widely held that systemic acidemia represents the proximate event signaling the kidney to elicit its acidification response to chronic metabolic acidosis. However, a previous study from this laboratory has cast serious doubt on the validity of this conventional viewpoint. When a large acid load (7 mEq/kg/day) was fed chronically to dogs as HCl, H2SO4 or HNO3, net acid excretion increased similarly in all three groups of animals despite wide variability in the prevailing systemic acid-base composition. Marked or moderate hypobicarbonatemia and acidemia were observed in the HCl- or H2SO4-fed animals respectively, but strikingly, plasma [HCO3-] and pH did not change significantly from the control in the HNO3-fed animals. That study concluded that the renal response to chronic mineral acid feeding appears to be triggered, not by acidemia, but by the interplay of sodium delivery to and sodium avidity of the distal nephron as modulated by the reabsorbability of the "acid" anion. We have re-examined the above provocative conclusion in the light of the observation that the only evidence for a dissociation of the renal response from systemic acidemia in that study was derived from preprandial (8:00 a.m.) blood samples obtained some 23 hr after the ingestion of the daily acid load (administered at 9:00 a.m.). We investigated the diurnal variation of plasma acid-base composition in two groups of dogs fed chronically a large acid load (7 mEq/kg/day) as either HCl or HNO3. Both groups exhibited significant diurnal oscillations of plasma acid base composition.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3009956 TI - Cytomegalovirus glomerulopathy: a controversial lesion. AB - Seven autopsies performed on immunosuppressed bone marrow transplant recipients who died with documented herpes virus infection were reviewed. The kidneys were studied by light and electron microscopy and revealed no pathological findings, specifically no glomerulopathy or tubular interstitial nephritis. Seven renal biopsies performed on kidney transplant recipients in whom a diagnosis of cytomegalovirus glomerulopathy was entertained were also studied. These patients exhibited clinical parameters suggestive of cytomegalovirus infection. Three patients had subsequent nephrectomies and two showed severe acute vascular rejection. The one kidney without demonstrable acute vascular rejection was negative for cytomegalovirus on culture. Three additional patients improved or stabilized their renal function under therapy for rejection. Light, electron microscopic and immunofluorescent studies, although confirmatory of endothelial cell damage, did not substantiate active cytomegalovirus renal infection in these patients. An additional group of fifteen children with disseminated cytomegalovirus infection revealed no evidence of glomerulopathy. Finally, two kidney transplant recipients with proven cytomegalovirus infection (one with associated tubular interstitial nephritis) also showed no glomerular alterations. It is the author's opinion that the entity that has been considered as cytomegalovirus glomerulopathy probably represents rejection, either a peculiar anti-endothelial type of rejection or a protracted, early, or partially resolved acute vascular rejection without residual or identifiable acute vascular rejection changes in the tissue sampled. PMID- 3009957 TI - Model of aluminum-induced osteomalacia: inhibition of apatite formation and growth. PMID- 3009959 TI - [Acquired immunologic deficiency syndrome (AIDS) in an infant from Austria]. AB - Manuel born in June 1984 is presented for the first time at the age of 6 months with the following symptoms: Otitis purulenta, enlargement of lymph-nodes, swelling of liver and spleen, enteritis, failure to thrive and candida albicansdermatitis. Immunoglobulins and the total serumprotein are elevated. At the age of 9 months a new Hospital admission was necessary. The following symptoms are present: severely enlarged lymph-nodes at different sites, enlargement of liver and spleen, interstitial pneumonia and enteritis. Body weight is below the third percentile. Tine-Test negative. The baby was BCG vaccinated after birth and at 3 months the Tine-Test was positive. The serologic antibody tests (Elisa and Western Blot) for HTLV III are positive, the index helper: suppressor T cells is 1,1 (normal above 1,5). The antibody titers in the maternal blood are higher than in the child. The mother was a regular drug user. A vertical perinatal transmission from the affected mother to her baby is postulated. The start of clinical illness was the 5th month of life. AIDS was fully developed at 9 months of age. Cotrimoxazol treatment resulted in clinical improvement. PMID- 3009958 TI - Clinical and biochemical diagnostics of Niemann-Pick disease. AB - Niemann-Pick disease (NPD) is an inherited lysosomal storage disorder characterised biochemically by a deficiency of sphingomyelinase activity and massive accumulation of undegraded sphingomyelin. There are three main clinical types of the disorder (NPD type A, B and C). NPD type A and B is diagnosed biochemically on the basis of measuring sphingomyelinase activity in leukocytes or cultured fibroblasts. The diagnosis may be established with the chromatographic method by the high level of sphingomyelin concentration in liver samples. Because in NPD type C the decrease of sphingomyelinase activity is moderate and only occurs in fibroblasts the thin-layer chromatography of the total liver lipid extracts is necessary for establishing the diagnosis. The thin layer chromatography of the total liver lipids is sufficient for the diagnosis of all types of NPD. PMID- 3009961 TI - Blood pressure in essential hypertension correlates with the concentration of a circulating inhibitor of the sodium pump. AB - The influence of serum from patients with essential hypertension on the sodium efflux rate constants of human lymphocytes and on the activity of isolated (Na+ + K+)-ATPase was investigated. The ouabain-sensitive sodium efflux rate constant was significantly decreased (p less than 0.001) in the sera of 19 hypertensives (1.92 +/- 0.11 h-1) compared with the sera of 30 normotensives (2.44 +/- 0.07 h 1). The ouabain-insensitive sodium efflux was unaffected. These results corresponded with a significant difference (p less than 0.005) of (Na+ + K+) ATPase activity (1.03 +/- 0.04 mU/ml and 0.079 +/- 0.06 mU/ml), when an isolated (Na+ + K+)-ATPase was incubated with the sera of 22 normotensives or 18 hypertensives. Both the rate constant of ouabain-sensitive sodium efflux and the (Na+ + K+)-ATPase activity correlated significantly with the diastolic and systolic blood pressure (p less than 0.001). These data, therefore, demonstrated the close relationship between essential hypertension and the concentration of a circulating inhibitor of the sodium pump. PMID- 3009963 TI - Detection of antibodies to HTLV-III in commercially available immunoglobulin preparations. AB - Samples of 96 polyvalent and virus-specific immunoglobulin batches commercially available in West-Germany were tested by enzyme-linked immunoassay and immunoblot for the presence of anti-HTLV-III antibodies. 37% of the polyvalent and 87% of the virus-specific batches were positive. It was concluded that these preparations are still safe and because of their reportedly low titer neutralizing antibodies possibly beneficial in certain cases, such as newborns of HTLV-III positive mothers or after accidental exposure to infectious material in clinics or laboratories. PMID- 3009960 TI - [Alpha 1-antitrypsin deficiency: a review with special reference to the significance of heterozygous deficiency]. AB - Homozygous deficiency of alpha-1 antitrypsin is the most common inborn error or metabolism in Europe. Severe deficiency of this major protease inhibitor in serum is associated with chronic obstructive lung disease, chronic liver disease in adults and neonatal hepatitis. An overview is given of the role of heredity, and the diagnostic criteria and clinical and histological findings in this disorder. Emphysema seems to be caused by the free elastolytic activity of white cells, leading to the degradation of elastin. The pathophysiology of liver disease - less well understood - is discussed with special emphasis on the importance of heterozygous alpha-1 antitrypsin deficiency. Exogenous noxae seem to play an important role in the pathogenesis of heterozygous deficiency. In view of the 7% frequency of heterozygous alpha-1 antitrypsin deficiency in the European population and the role of noxae in the development of pulmonary and liver diseases, improved prophylaxis is mandatory. PMID- 3009962 TI - Crossover comparison of the antiemetic efficacy of nabilone and alizapride in patients with nonseminomatous testicular cancer receiving cisplatin therapy. AB - Twenty nonseminomatous testicular cancer patients not pretreated with emetogenic chemotherapy were included in a crossover study of antiemetic therapy. Patients were randomly assigned to receive either nabilone (2 X 2 mg/day) or alizapride (3 X 150 mg/day) prior to beginning low-dose cisplatin chemotherapy. Patients on nabilone had significantly fewer episodes of emesis than those on alizapride (medians, 1.1 vs 2.9; p less than 0.01). Nabilone was superior to alizapride in giving complete relief from nausea (medians, 65% vs 30%; p less than 0.01), and was more effective in shortening the duration of nausea (medians, 1.3 h vs 5.1 h; p less than 0.01); however, it caused more adverse effects. It is concluded that nabilone has greater antiemetic activity than alizapride in young patients receiving low-dose cisplatin chemotherapy. Nabilone dosage should be reduced to decrease the incidence and degree of adverse reactions while leaving the definite antiemetic activity unchanged. PMID- 3009964 TI - Observations on the effects of vibration and noise on plasma ACTH and zinc levels, pregnancy and respiration rate in the guineapig. AB - The responses of female guineapigs to vibration and noise were examined with the use of an apparatus designed to simulate transport. Peak levels of plasma ACTH and zinc concentrations were attained after 4 min of exposure to vibration and 80 90 dB of noise. The respiration rate, normally around 90/min, was increased to 103/min when the animals were moved (while still in their cages) to the experimental area; it rose to 129/min when the apparatus was switched on to expose the animals to vibration and noise. Those left adjacent to the apparatus and exposed to noise alone elevated their respiration rates to 115/min. Respiration rates returned to normal within 2.5 h. There was no apparent effect on the maintenance of pregnancy, gestation length, litter size or post-partum growth of the young born to guineapigs exposed to this vibration and noise for a period of 1 h at mid-term. PMID- 3009965 TI - Rapid diagnosis and management of parainfluenza I virus infection in common marmosets (Callithrix jacchus). AB - During 1983 a severe episode of respiratory infection occurred in a marmoset colony at these laboratories. Of 91 marmosets, 69 showed clinical signs of disease, one died and nine were so ill that euthanasia was necessary. Eight were examined post mortem and all showed consolidation of the lungs. Laboratory studies were carried out in an attempt to establish the cause of the outbreak and an interstitial pneumonia was found in seven animals which were examined histologically. Direct electron microscopy of nasal swabs and lung samples revealed the presence of a high titre of a paramyxovirus, and subsequent immunofluorescence studies established that the particular paramyxovirus involved was parainfluenza virus type I. Subsequent studies showed that surviving affected animals had seroconverted to parainfluenza I virus while animals that had not been implicated in the outbreak had not. PMID- 3009966 TI - The organ tropism of mouse hepatitis virus A59 in mice is dependent on dose and route of inoculation. AB - The organ tropism of MHV-A59, a murine coronavirus, was studied in 4-6 week-old C57BL/6 mice inoculated by different routes and with various amounts of virus. MHV-A59 caused hepatitis after intracerebral and intraperitoneal inoculation (two clearly artificial routes) and also after intranasal and intragastric inoculation (two routes more likely to mimic naturally acquired infection). For each route, the severity of hepatitis was dependent on the amount of virus inoculated. Significantly higher doses were needed to cause hepatitis by the intranasal or intragastric routes. We have shown previously that mice inoculated intracerebrally with MHV-A59 develop mild meningoencephalitis followed by chronic central nervous system (CNS) disease, characterized by primary demyelination (1). We extend these results here to show that acute CNS disease can be produced also by intranasal and intragastric inoculation, although much larger doses are needed as compared to intracerebral inoculation. Thus induction of demyelination, not only by the intracerebral route but also by the intranasal route, provides a useful model system to study virus-induced demyelination. PMID- 3009967 TI - Mediation of IgA induced lung injury in the rat. Role of macrophages and reactive oxygen products. AB - Acute lung injury in the rat has been induced by the instillation of affinity purified mouse monoclonal IgA antibody with specific reactivity to the hapten dinitrophenol coupled to albumin. As previously reported, this model of lung injury requires an intact complement system but is independent of neutrophils. In contrast to macrophages obtained by bronchoalveolar lavage from rats receiving IgA into the airways in the absence of intravenously injected antigen, macrophages obtained from the lungs of rats developing IgA immune complex-induced lung injury were significantly increased in number, showed greater spontaneous generation of O2.-, and demonstrated significantly enhanced O2.- responses in the presence of an added stimulus, phorbol ester. Inhibition studies in vivo suggested that the IgA-induced lung injury is mediated by oxygen radicals generated from lung macrophages. Pretreatment of animals with superoxide dismutase, catalase, the iron chelator, deferoxamine, or the hydroxyl radical scavenger, dimethyl sulfoxide, suppressed the development of lung injury. Morphologically the lungs of protected animals showed increased numbers of mononuclear cells within the alveolar compartment but little evidence of alveolar or capillary injury, in contrast to unprotected animals in which there was evidence of severe injury, both to microvascular interstitial endothelial cells as well as to alveolar lining epithelial cells. These studies suggest that acute lung injury in the rat induced by IgA immune complexes is mediated by oxygen radical formation and that the macrophage may be the principle effector cell, as compared to IgG immune complex induced lung injury, which is also oxygen radical mediated but in which the neutrophil is the effector cell. PMID- 3009968 TI - Expression of gamma-subunit of enolase, neuron-specific enolase, in human non neuroendocrine tumors and derived cell lines. AB - The gamma-subunit of 2-phospho-D-glycerate hydrolyase, E.C. 4.2.1.11 (enolase), neuron-specific enolase (NSE), is present at high concentrations in neurons and neuroendocrine cells and has therefore recently been introduced as a marker for neuroendocrine tumors. By the indirect methods, immunocytochemistry and radioimmunoassay, NSE has been detected also in some nonneuroendocrine tumors, a finding that could reflect technical artifacts or the capacity for NSE expression in nonneuroendocrine tumor cells. This paper reports on the expression of NSE in human neuroendocrine and nonneuroendocrine tumor specimens and in a panel of permanent human cell lines, by using a direct (enzymatic) and an indirect (radioimmunoassay) method for determination of NSE. We detected NSE in all tested tumor specimens and neuroendocrine tumor cell lines and in a majority (21 of 24) of the nonneuroendocrine tumor cell lines. In general, neuroendocrine tumor specimens and derived tumor cell lines contained more NSE than the nonneuroendocrine tumor specimens and cell lines. However, some of the cultured hematopoietic cell lines (T leukemia and Epstein-Barr virus immortalized B lymphoblastoid cell lines) had NSE levels comparable to those found in some neuroblastoma and small-cell lung carcinoma cell lines. We conclude that NSE is not exclusively expressed in neuroendocrine tumor cells. PMID- 3009969 TI - GC/MS and EMIT analyses for delta 9-tetrahydrocannabinol metabolites in plasma and urine of human subjects. AB - Ten male subjects smoked placebo marijuana cigarettes spiked with delta 9 tetrahydrocannabinol (THC) at 150 micrograms/kg body weight. Plasma and urine samples were collected for 22 hr after smoking. Total cross-reacting cannabinoids were measured by immunoassay (EMIT) and THC and major metabolites were identified and quantitated by gas chromatographic/mass spectrometric (GC/MS) procedures. In plasma, THC concentration provided the best indication of recent (less than 6 hr) smoking. In enzyme-hydrolyzed urine, 8 beta, 11-dihydroxy-THC at high concentration was identified in the earliest voidings, falling rapidly to the limit of detection before 22 hr in most subjects. Levels above 15 to 20 ng/mL were indicative of use within the previous 4 to 6 hr. PMID- 3009971 TI - Activation of protein kinase C potentiates cyclic AMP production and stimulates steroidogenesis in differentiated ovarian granulosa cells. AB - Gonadotropin-stimulated steroidogenesis in the differentiating ovarian granulosa cell is mediated through the activation of cAMP-dependent protein kinase, and is also modulated by calcium-dependent mechanisms. Granulosa cells contain calcium activated, phospholipid-dependent protein kinase (C kinase), and show an increase in phosphatidylinositol turnover in response to GnRH agonist analogs. To evaluate the role of C kinase in ovarian steroidogenesis, the potent phorbol ester, TPA, and the permeant diacylglycerol, OAG, were used to activate C kinase in granulosa cells from PMSG-treated immature rats. Both TPA and OAG caused dose-dependent stimulation of progesterone production without affecting intra- or extracellular cAMP levels. However, the maximum steroid responses to these compounds were less than those stimulated by cAMP. The ED50 for TPA-stimulated progesterone production was 3 nM, which is close to the known Km for activation of C kinase. Stimulation of steroidogenesis was only observed with biologically-active phorbol esters and permeant diacylglycerols such as OAG and DOG. Exposure of granulosa cells to phospholipase C also increased progesterone production in a dose dependent manner without changing the cAMP content. Although TPA and OAG did not increase basal cAMP production, both agents enhanced the cAMP responses stimulated by hCG and forskolin; likewise, phospholipase C alone did not change cAMP production but caused a dose-dependent increase in the cAMP responses to hCG and forskolin. These results demonstrate that activation of C kinase promotes steroidogenesis in ovarian granulosa cells, and potentiates the activation of adenylate cyclase by hCG and forskolin. Such findings support the possibility that the calcium, phospholipid-dependent enzyme could be involved in the regulation of progesterone production by hormonal ligands such as gonadotropins and GnRH. PMID- 3009970 TI - Pharmacology of an antiandrogen, anandron, used as an adjuvant therapy in the treatment of prostate cancer. AB - To improve the inhibition of prostate cancer growth obtained by surgical or chemical castration (estrogens or LHRH analogs), blockade of the action of residual androgens of adrenal origin has been proposed. Among antiandrogens acting through the androgen receptor (AR), the nonsteroid anandron (RU 23908) has several advantages over available compounds: megestrol acetate and cyproterone acetate, both steroids, bind substantially to other hormone receptors (progestin, gluco- and mineralocorticoid); and anandron binds only to AR. The nonsteroid flutamide is a prodrug converted to the active metabolite, hydroxyflutamide; anandron is well absorbed on oral administration of an active dose and intact compound disappears slowly from plasma. This may explain why, although in vitro anandron interacts very transiently with AR, in vivo a high level of untransformed anandron is present at the receptor site to induce its antiandrogenic activity. Animal experiments confirm that anandron can counteract the effect of adrenal androgens and inhibit the LHRH analog-induced initial increase in androgen ("flare-up"). Thus, in rats castrated either surgically or by buserelin or DES and supplemented with adrenal androgens (since endogenous adrenal secretion is very low in this species compared to man), anandron decreased prostate weight to control levels. The administration of buserelin to intact rats over 15 days resulted in a significant increase in prostate weight between Days 1 and 5. The addition of anandron to the buserelin inhibited this increase and, furthermore, led to a far greater decrease in prostate weight than that due to buserelin alone at 15 days, indicating a synergy of action. PMID- 3009972 TI - Regulation of testicular steroidogenesis by gonadotropin-releasing hormone agonists and antagonists. AB - Clinical and experimental studies are described on the effects of a gonadotropin releasing hormone (GnRH) agonist (A) and antagonist (Ant.) on testicular endocrine function. Testicular effects of long-term gonadotropin suppression by GnRH-A were assessed during treatment of prostatic cancer patients. The testis tissue removed after 6 months of A treatment had less than 5% of the testosterone(T)-producing capacity in comparison to testis tissue removed from untreated control patients. However, the LH receptors (R) and responsiveness of T output to LH stimulation in vitro were unchanged. FSH-R decreased by 70%. Hence, despite suppression of gonadotropins and testicular androgen production during long-term GnRH-A treatment the responsiveness to exogenous gonadotropins is maintained. The testicular effects of a gonadotropin suppression induced with GnRH-Ant. and testicular GnRH-R blockade were studied in rats. Besides decreases of gonadotropins and testicular T, systemic Ant. treatment decreased testicular Prl-R, but had no effect on LH-R or FSH-R. Bromocriptine-induced hypoprolactinemia, in contrast, decreased LH-R but had no effect on Prl-R. The results indicate reciprocal regulation of LH-R and Prl-R, and that testicular steroidogenesis and LH-R are under differential regulation, the former by LH, the latter by Prl. In another study, testicular GnRH-R, and consequently the action of a putative testicular GnRH-like factor, were blocked by unilateral intratesticular infusion of Ant. (1 week, Alzet osmotic pumps). The treatment resulted in 90% occupancy of testicular GnRH-R in the Ant.-infused testes, and this was associated with decreased levels of R for LH, FSH and Prl, and of T. The results indicated that the testicular GnRH-R have a physiological function in subtle stimulation of Leydig cell functions. PMID- 3009973 TI - Receptor-mediated endocytosis of GnRH analogs: differential processing of gold labeled agonist and antagonist derivatives. AB - The hypothalamic decapeptide, GnRH, stimulates LH and FSH release from pituitary gonadotrophs. Many synthetic peptide analogs, both agonist (GnRH-A) and antagonist (GnRH-AT), have been developed which bind specifically to the GnRH receptor. We have utilized highly potent GnRH-A and GnRH-AT analogs labeled with 18 nm colloidal gold to analyze ultrastructurally the events of binding and interiorization of these specific ligands by gonadotrophs in vitro. To examine internalization of GnRH-A-gold, gonadotrophs were cooled to 4 degrees C and equilibrated with the ligand for 1 h. Next, the cells were either fixed immediately or warmed to 37 degrees C for various times (5, 15 and 30 min) and prepared for electron microscopy. For GnRH-AT-gold, which binds slowly at 4 degrees C, the ligand was incubated with gonadotrophs at 37 degrees C for 15, 30 and 60 min, and the cells were processed for electron microscopy at each time point. In both cases, control gonadotrophs were also incubated in an excess of GnRH-A and GnRH-AT, respectively, in the presence of the gold-conjugated ligands. The results indicated that GnRH-A-gold was bound and rapidly internalized via a receptor-mediated endocytic pathway. GnRH-AT-gold was also bound but showed only limited entry into gonadotrophs; the percentage of intracellular GnRH-AT-gold in gonadotrophs was the same as in other pituitary cells contaminating the gonadotroph fraction and did not increase with time. In the gonadotroph, binding of the specific antagonist ligand to GnRH receptors does not stimulate its interiorization, in contrast to the rapid endocytosis and processing of the agonist ligand. These data suggest that specific ligand internalization requires prior receptor activation, and that GnRH-AT which does not activate the receptor remains bound at the cell surface for a prolonged period. PMID- 3009974 TI - Mechanism of gene regulation by steroid hormones. AB - A first understanding of the molecular events on the DNA level, underlying transcriptional regulation by steroid hormones, has been approached in the last 3 years by means of protein/DNA interaction studies, using purified receptors. This work summarizes our knowledge of how purified glucocorticoid and progestine receptors interact with their cognate regulatory elements associated with polymerase II dependent genes like mouse mammary tumour virus, the genes encoding human metallothionein IIA, chicken lysozyme, human growth hormone and rabbit uteroglobin. The resulting data agree with those of functional test systems, that have been gene-transfer experiments using stable transformants or transient expression. A consensus sequence for the regulatory element of the glucocorticoid receptor could be deduced that, in its three-dimensional representation, gives an impression of the steric mode of interaction. The regulatory elements of the progestine receptor overlap in two analysed cases with those of the glucocorticoid receptor, but are not identical. Furthermore, also a polymerase I transcribed gene encoding ribosomal RNA in the mouse could be shown to contain a glucocorticoid regulatory element that is functional in in vitro transcription experiments. Finally, the latest strategies are the cloning of the glucocorticoid receptor gene and the analysis of receptor-mediated topological effects. PMID- 3009975 TI - Importance of A-ring substitution of estrogens for the physiology and pharmacology of reproduction. AB - Estrogens substituted in the ortho-position of the phenolic hydroxy-group with an additional hydroxy- or methoxy-group are quantitatively important estrogen metabolites; first isolated and identified from the urine of man and rodents have been demonstrated in blood and different organs, e.g. the pituitary and hypothalamus. The physiological importance of the preeminent representatives of this group, the 2- and 4-hydroxyestrogens, the so-called catecholestrogens, is still equivocal. For example, numerous in vivo investigations in rodents have demonstrated that gonadotrophin secretion is influenced by these catecholestrogens. However, depending on the position of the A-ring substituent, major potency differences have been observed. The significant discrepancies between the quantitative and qualitative effects of catecholestrogens in in vitro and in vivo experiments have been presented and explained on the basis of different receptor affinities and the pharmacokinetics of catecholestrogens. An array of A-ring-substituted steroid model substances has been tested with respect to the effects of 2- and/or 4-substitution on stimulation or blockade of the estrogenic potency. PMID- 3009977 TI - On the mechanism of opioid-oestradiol interactions. AB - Characteristics of opioid binding and possible relationships between oestradiol and opioid binding sites were studied in rat oestrogen sensitive tissues(uterus, preoptic area-anterior hypothalamus, median eminence-basal hypothalamus). Naloxone (Nal) and oestradiol (Oe) bindings were assessed by in vitro saturation analyses. In 800 g supernatants of both uterine and hypothalamic tissues homogenates high affinity (Kd: 2-4 X 10(-9) M) and low capacity [3H]Nal binding sites were found. These binding sites were sedimented from 800 g supernatant by further centrifugation at 10(5) g for 1 h. In competition studies [3H]Nal binding was completely prevented by morphine, while met-enkephalin and leu-enkephalin caused only a partial inhibition. [3H]Nal binding was increased by ovariectomy and decreased by Oe treatment (10 micrograms/100 g b.wt) in both tissues. The cytoplasmic [3H]Oe binding in the studied tissues seems to be affected by the naloxone binding system. After in vitro saturation of naloxone binding sites by naloxone the [3H]Oe binding to low affinity sites (type II) in hypothalamus as well as in uterus has been increased by 8- and 2-fold, respectively. These results indicate the presence of specific [3H]Nal binding in rat uterus with similar properties to those found in the hypothalamus. Furthermore an interaction between opioid and oestradiol receptor systems could be also suggested. PMID- 3009976 TI - The role of corticosteroids in the homeostasis of the eye. AB - Three problems were studied on the human and rabbit eye: to what extent the mineralocorticoids contribute to the control of Na+ and K+ transport in the lens epithelium, how do the glucocorticoids influence the concentration of glucose in the aqueous humour and what is the effect of the pituitary-adrenal axis on the hemato-ocular barrier. Specific receptor-like proteins binding aldosterone were found in the lens epithelium. Na+ and K+ concentrations in the aqueous were influenced by both aldosterone and spironolactone administration. The aldosterone concentration in human cataracts was found to be higher in cases of cataracts complicated by arterial hypertension. In spite of some indication of anticataractogenous action of mineralocorticoids, aldosterone did not prevent the formation of cortisol-induced cataract in chick embryos. Glucose concentration in the aqueous was increased by glucocorticoid administration as well as by stimulation of their secretion by ACTH. Further, the contribution of the pituitary-adrenal axis to the breakdown of the hemato-ocular barrier was investigated by measuring the changes of the total protein content in the aqueous. ACTH1-24 caused a partial breakdown of the barrier, as well as ACTH4-10 or alpha-MSH. As the latter two peptides lack the stimulative effect on the corticoid secretion and glucocorticoids themselves fail to increase the protein content in the aqueous, the breakdown of the hemato-ocular barrier seems to be essential for ACTH-linked peptide fragments and is not mediated by corticoids. PMID- 3009978 TI - Isolation of new types of dexamethasone-resistant variants from a cAMP-resistant lymphoma. AB - We have developed a sequential selection procedure for the isolation of novel steroid-resistant variants of the murine thymoma WEHI-7. The first step involves the isolation of cell lines with an altered cAMP-dependent protein kinase (cAPK) activity by selection for resistance to dibutyryl cAMP (dbcAMP). The second step involves the selection for resistance to dexamethasone (dex) which results in the isolation of variants with decreased receptor function and a cAMPrdexr phenotype. The initial selection, to cAMPr, serves as a permissive step since isolation of spontaneous glucocorticoid resistance from wild-type WEHI-7 does not occur at a measurable frequency. The results demonstrate a potential role for cAPK in regulating the functional levels of glucocorticoid receptor and suggest that mutations in other cellular functions that affect receptor activity could lead to steroid resistance in lymphoid cells. PMID- 3009979 TI - Cross-reactivity of a monoclonal antiglucocorticoid receptor antibody BuGR1 with glucocorticoid and mineralocorticoid receptors of various species. AB - The reactivity of a monoclonal antibody BuGR1, raised against glucocorticoid receptors of rat liver, with glucocorticoid and mineralocorticoid receptors of mammalian (rabbit) and amphibian (A6 cells) origin was examined. The glucocorticoid receptors of rabbit kidney and liver and of A6 cells were labeled with tritiated dexamethasone. The mineralocorticoid receptors were labeled with tritiated aldosterone in the presence or absence of RU26988, depending on whether aldosterone was bound to glucocorticoid receptors (A6 cells) or not (rabbit kidney), in addition to its binding to mineralocorticoid receptors. BuGR1 did not recognize mineralocorticoid receptors of A6 cells and rabbit kidney. BuGR1 cross reacted with glucocorticoid receptors of rabbit liver and kidney but not of A6 cells, suggesting that the domain of glucocorticoid receptors recognized by BuRG1 could be present only in the mammalian species. The findings indicate that BuGR1 shows species differences as well as receptor class specificity. PMID- 3009980 TI - Urinary estrogen profile determination in young Finnish vegetarian and omnivorous women. AB - For a long time it has been postulated that diet may influence estrogen metabolism and in this way affect breast cancer risk. In order to investigate possible effects of variations of dietary fiber intake on estrogen metabolism, the urinary estrogen profile (13 estrogens), including the catecholestrogens, was determined in one 72-h summer and one winter sample collected in the midfollicular phase of the menstrual cycle by 11 lactovegetarian and 12 omnivorous young Finnish women. Urinary estrogens were purified by ion-exchange chromatography and the quantitative determination was carried out by capillary gas chromatography-mass spectrometry. Detailed records of the subjects' diet during one 5-day period in summer and one in winter were obtained and dietary fiber intake calculated. The mean difference with regard to intake of total fiber in the two dietary groups was 3 g/day in the summer (not significant) and 5 g/day in the winter (P less than 0.05), the mean (geometric) consumption being 23 and 19 g/day by the vegetarian and omnivorous women, respectively. Within the groups we found seasonal variation in fiber intake only for the omnivorous women. During winter, compared to summer, the omnivorous women consumed significantly less grain (P less than 0.001), vegetable (P less than 0.02) and total fiber (P less than 0.02). The excretion of 13 estrogens was remarkably constant in the omnivoric group but a significant seasonal variation of total and individual catecholestrogens and of estrone was observed in the vegetarians (P less than 0.05-0.005). The quantitatively most important estrogen was 2-hydroxyestrone, followed by estrone, estriol, 2-hydroxyestradiol, 4-hydroxyestrone and estradiol, the three latter being excreted in similar amounts. Between the dietary groups there were no significant differences in excretion of total or individual urinary estrogens in any season or between the mean values for both seasons. However, numerous significant (P less than 0.05-0.01) negative correlations between dietary intake of total or grain fiber/kg body weight and the excretion of individual estrogens were found. These correlations disappeared if the fiber intake was not related to body weight. We conclude that dietary fiber intake significantly affects estrogen metabolism by reducing estrogen excretion in urine and that grain fiber seems to be most important in that respect. One of the mechanisms involved is a partial interruption of the enterohepatic circulation of the estrogens, due to alterations of the intestinal metabolism and reabsorption of these steroids, caused by the fiber. PMID- 3009981 TI - Absence of LHRH effect on testosterone production by Leydig cells from fetal mouse testis. AB - The effect of LHRH and one of its agonist (des-gly10 (D-Ala6)-LHRH-ethylamide) on the functional activity (testosterone and progesterone production) of purified fetal mouse Leydig cells was examined in short-term primary culture and under dynamic conditions. The continuous presence of increasing concentrations of LHRH (10(-10) to 10(-6) M) for 3 days was unable to affect the hCG-stimulated testosterone production on any day of culture. Stimulated testosterone production progressively decreased from day 1 to day 3 of culture (P less than 0.001). Progesterone accumulation increased in both basal and hCG stimulated conditions during the same period (P less than 0.001) and was not altered by the presence of LHRH at all three concentrations tested. There was no effect of LHRH pretreatment either on the basal production or on the acute hCG stimulation studied during a subsequent 6 h incubation. Exposure of cells to hCG for 120 min enhanced testosterone accumulation. No change in kinetic characteristics was observed when LHRH (10(-6) M) was continuously present in the medium. These results show that LHRH does not have any detectable effect on the fetal population of Leydig cell in the mouse. PMID- 3009982 TI - On the control of HMG-CoA reductase, a key regulatory enzyme of adrenal cholesterol synthesis. AB - In this study we have determined the effect of ACTH on the activity of HMG-CoA reductase in microsomes of hamster adrenals. Cycloheximide was used to study the dependence of the increased enzyme activity by ACTH on de novo protein synthesis. Microsomes were prepared and preincubated with and without NaF and in the presence or absence of phosphorylase phosphatase in order to differentiate between expressed (McNaF) and total (McPP) activity. ACTH induced (after 120 and 180 min) significant increases in HMG-CoA reductase activity with a latent period of 60 min for both McNaF and McPP preparations. Cycloheximide alone decreased the activity of the reductase and the coadministration of cycloheximide + ACTH caused a greater loss of activity. Also, both treatments produced an accumulation of free cholesterol in adrenals suggesting an increased turnover of the reductase by these substances. Preincubation of microsomes at 37 degrees C enhanced per se HMG CoA reductase activity, but the relative increase produced by ACTH treatments or endogenous ACTH remained essentially the same. In conclusion, under experimental conditions used, the enhancement of HMG-CoA reductase activity produced by ACTH seem to be due to increased enzyme synthesis. PMID- 3009983 TI - Development of a simplified perifusion system of rat zona glomerulosa. Effect of cytochalasin B on spontaneous and ACTH-stimulated corticosteroidogenesis. AB - The aim of the present study was to develop a perifusion technique for rat adrenal glomerulosa slices as a model to study the kinetics of corticosteroid production in vitro. Perifusion of adrenal tissue with increasing concentration of ACTH (3.16 X 10(-12) to 10(-9) M) led to a dose-related stimulation of corticosterone and aldosterone secretion. Administration of cytochalasin B did not alter the basal secretion of corticosterone and aldosterone. In addition, cytochalasin B did not modify the response of glomerulosa tissue to ACTH. The results indicate that the perifusion model for glomerulosa fragments may provide valuable information, concerning the kinetics of steroid production and its regulation at the cellular level. PMID- 3009984 TI - Enhanced 17 alpha-hydroxylation of pregnenolone and increased androgen production by rabbit adrenocortical cells stimulated chronically with corticotropin. AB - The postulated chronic stimulatory effect of corticotropin (ACTH) on pregnenolone production and on 17 alpha-hydroxylase activity was evaluated on adrenocortical cells obtained from control and chronically ACTH-treated rabbits. The cells were incubated with various concentrations of ACTH added alone or together with trilostane, so as to inhibit further conversion of pregnenolone and dehydroepiandrosterone. The maximal steroidogenic effect of ACTH (determined in the absence of trilostane) was increased 2-fold in adrenocortical cells from ACTH treated animals; furthermore, cortisol production was increased whereas that of corticosterone decreased. While the generation of pregnenolone was of comparable magnitude for cells from both experimental groups, chronic in vivo treatment with ACTH was followed by a 40-fold enhancement in 17-hydroxypregnenolone production. Concomitantly, maximal DHEA production documented in the presence of ACTH and trilostane was enhanced more than 200-fold, from 0.45 +/- 0.20 pmol in control rabbits to 147 +/- 67 pmol in cells from ACTH-treated animals. The corresponding values of DHEA-sulphate production were 0.86 +/- 0.12 and 432 +/- 334 pmol, respectively. Thus, a prolonged stimulatory effect of ACTH on rabbit adrenocortical cells consists in an enhancement of the capacity to generate pregnenolone, and to convert this compound into 17-hydroxylated steroids. PMID- 3009985 TI - Compared effects of ACTH, angiotensin II and POMC peptides on isolated human adrenal cells. AB - Human adrenocortical tissue obtained, on eight occasions, at the time of nephrectomy for renal carcinoma (outside the adrenal pole) was treated by collagenase to dissociate the cells. These were hen submitted to a short, 2-h, incubation with the N-terminal fragment (16 K) of POMC, its derivative, gamma 3 MSH, beta-lipotropin and beta-endorphin, in parallel with ACTH 1-24 (Synacthen Ciba) and angiotensin II (AII, Hypertensin Ciba). Under the influence of ACTH (10(-10) M), and AII (10(-10) M), basal glucocorticoid output, including more than 80% cortisol, was increased by factors of 3 +/- 0.51 (SEM) and 1.35 +/- 0.12 (SEM), respectively. The corresponding aldosterone responses were 1.60 +/- 0.13 for ACTH and 1.38 +/- 0.09 for AII. With the exception of gamma 3-MSH, the POMC peptides under study had no steroidogenic effect. gamma 3-MSH (10(-9) M) and AII (10(-10) M) stimulated aldosterone production to approximately similar levels of, respectively, 1.23 +/- 0.05 and 1.38 +/- 0.09 times the basal production. In contrast to AII however, gamma 3-MSH showed no apparent effect on glucocorticoid output. Steroidogenic response to ACTH was potentiated by gamma 3-MSH at a concentration of 10(-10) M which, when used alone, proved ineffective. This potentiating effect was pronounced for the aldosterone response, whereas the glucocorticoid production was hardly affected. This action ceased to be visible when the cells reached maximal stimulation by ACTH. These findings suggest that gamma 3-MSH--a portion of the 16 K fragment--may have a possible role in aldosterone secretion. PMID- 3009986 TI - Carcinogenicity of catechol estrogens in Syrian hamsters. AB - Estradiol and other estrogens induce renal carcinoma in male Syrian hamsters. The mechanism of carcinogenesis still remains unclear. Activation of estrogens to catechol metabolites has in the past been postulated to play a role in estrogen induced carcinogenesis. Therefore, the carcinogenic activity of catechol estrogens was investigated. After 175 days of treatment, 4-hydroxyestradiol was found to be as carcinogenic as estradiol in male Syrian hamsters (4/5 and 4/5 animals with kidney tumors, respectively). Animals treated with 2 hydroxyestradiol (0/5) or 2-methoxyestradiol (0/6) did not develop renal carcinoma. The catechol estrogens failed to be mutagenic in the Ames test (reversions of his- S. typhimurium to histidine prototrophy in the TA 100 strain). The lack of carcinogenic activity of 2-hydroxyestradiol was not due to a failure to stimulate estrogen-dependent tumor growth. Growth of H-301 cells, an estrogen-dependent hamster kidney tumor cell line, was supported in vivo by estrogens in the following order: estradiol greater than 4-hydroxyestradiol greater than 2-hydroxyestradiol. Stimulation of tumor growth by 2 methoxyestradiol was not detected. It was concluded that the carcinogenic activity of 4-hydroxyestradiol was consistent with a role of catechol metabolites in estrogen-induced carcinogenesis. However, the intrinsic carcinogenic or hormonal activity of 2-hydroxyestradiol probably can not be assessed accurately in vivo because of its rapid methylation and metabolic clearance. PMID- 3009987 TI - Activation-inactivation of hormone binding sites of the oestradiol-17 beta receptor is a multiregulated process. AB - The calf uterus oestradiol-17 beta receptor exists in a hormone binding form, which is phosphorylated on tyrosine, and in a non-hormone binding form, which is dephosphorylated. Two enzymes regulate the number of hormone binding sites of the receptor: a kinase which has been purified from cytosol and a phosphatase purified from nuclei. Recent and new findings on the regulation of this activation-inactivation process are reported. In vitro only a fraction (30-60%) of the receptor binding sites are inactivated by the phosphatase. Evidence is given suggesting that this is due to the production during the inactivation process of a powerful inhibitor of the phosphatase. Ca2+-calmodulin stimulates the kinase activity with a parallel increase of phosphorylation on tyrosine and hormone binding sites of the receptor. Nanomolar concentrations of oestradiol-17 beta also stimulate the kinase to activate hormone binding sites. These results suggest that in intact cells inactivation-activation of the oestradiol receptor is a multiregulated process. PMID- 3009988 TI - Antagonism of glucocorticoid induction of Epstein-Barr virus early antigens by different steroids in Daudi lymphoma cells. AB - Four antiglucocorticoids, RU38486, RU5020, RU25055 and progesterone were found to antagonize the induction of latent Epstein-Barr virus (EBV) information by dexamethasone. The dose response studies show that the antagonization was more prominent with the synthetic steroids than with the natural hormone. Specific binding characteristics of dexamethasone measured in whole cells indicate the presence of glucocorticoid receptors. Total cellular receptor contents deduced from binding data give values similar to those reported for B-lymphoblasts. Competition experiments between dexamethasone and RU38436 strongly suggest that RU38486 binds to two distinct sites in the whole cell; one is the glucocorticoid receptor but the nature of the other site is unknown. Inhibition by antiglucocorticoids differs from antagonism by 12-O-tetradecanoyl-phorbol-13 acetate (TPA) since the latter does not compete for any sites interacting with RU38486. PMID- 3009989 TI - Hormonal control of SBP in human hepatoma cells. AB - Since it is currently believed that the biosynthesis of human sex steroid binding plasma protein (SBP) takes place in the liver, the secretion of this protein and its hormonal control were studied in a human hepatoma cell line. The human hepatoma-derived cell line, Hep G2, and a clone, H5A, isolated from Hep G2, were both found to secrete SBP-like protein. This protein had the same dihydrotestosterone binding parameters as plasma SBP, with a Kd ranging from 0.3 to 1 nM at 4 degrees C, and it cross-reacted with a monospecific goat anti-human SBP antiserum. In a chemically defined medium, SBP-like protein secretion was stimulated approx 2-fold by estradiol (1 microM) whereas a smaller concentration of estrogen (100 nM) has only a slight effect. A combined incubation with estradiol (100 nM) and triiodothyronine (10 nM) increased SBP-like protein secretion more than estradiol (1 microM) alone. In response to dexamethasone (100 nM) or tamoxifen (100 nM) treatment, a 3-fold increase is obtained. Therefore, these human parenchymal cells should provide a potent material for investigation of the hormonal regulation of SBP gene. PMID- 3009990 TI - Mineralcorticoid receptor from rat kidney. Interaction with heparin and purification to a CBG-free stage. AB - Purification of the mineralcorticoid receptor is a particularly challenging problem. This receptor is present in target tissues at concentrations lower and is less stable than any other steroid receptor. Addition of molybdate ions (20 mM) to rat kidney cytosol enhances stability of mineralcorticoid-specific binding sites: the inactivation rate at 0 degrees C decreases from 7.2 to 1.7% per hour in the absence of aldosterone, and from 1.8 to 0.3% per hour in the presence of hormone. Rates of inactivation in the presence of molybdate are thus compatible with purification procedures. Also, the corticosteroid-binding globulin (CBG) is an important contaminating component of kidney cytosol because it cannot be specifically blocked preliminarily to affinity chromatography. We show that when kidney cytosol is incubated with heparin covalently linked to Sepharose (Sepharose-heparin), after 30 min at 0 degrees C more than 80% of the mineralcorticoid-specific binding sites interact strongly with Sepharose-heparin while CBG is not bound at all. The mineralcorticoid-specific binding sites can be recovered from Sepharose-heparin by washing with heparin (2 mg/ml; recovery up to 90%), KCl (0.3 M; recovery up to 90%); and, less efficiently, with total liver RNA (2 mg/ml; recovery up to 55%) and dextran sulfate (2 mg/ml; recovery up to 40%); little or no recovery is achieved with chondroitin sulfate, sonicated DNA, pyridoxal-5-phosphate, dextran, d-glucosamine and d-glucuronic acid. With demonstration that also the mineral-corticoid receptor binds to heparin, this property has become a general hallmark of steroid receptors. If the "heparin" binding site of steroid receptors is of physiological significance it remains to be established. By application of the newly found property of the mineralcorticoid receptor, an overall 10-fold purified, CBG-free preparation of this receptor can be obtained from kidney cytosol with a single chromatography on Sepharose-heparin. PMID- 3009991 TI - Functional analysis of the purified glucocorticoid receptor. AB - Glucocorticoid-receptor complex (GR) has been purified from rat liver by differential affinity for DNA before and after activation, followed by ion exchange chromatography. The purified GR has mol. wt 94,000 dalton. The protein contains three functional domains: (A) a steroid-binding domain; (B) a DNA binding domain; and (C) a domain necessary for normal biological function. A second protein, with mol. wt 72,000 dalton, copurifies with the GR. This protein does not bind steroid, does not interact with antibodies raised against the GR and does not show the same susceptibility to limited proteolytic cleavage as the 94,000 dalton protein. Analysis of the specific interaction of the purified GR with the mouse mammary tumour virus gene, assayed by glycerol-gradient centrifugation, shows that one molecule of 94,000 dalton protein binds to each of the specific binding sites in the long terminal repeat region. Analysis of the fractions from the glycerol gradients show that the 72,000 dalton protein is associated to the binding species (94,000 dalton receptor protein) in about equimolar amounts. Analysis of the molybdate-stabilized non-activated receptor complex using monoclonal antibodies raised against the 94,000 dalton receptor protein indicates that the molybdate-stabilized complex is a hetero-oligomer. The hetero-oligomer consists of only one molecule of the 94,000 dalton receptor protein, in association with other non-steroid-binding proteins. PMID- 3009992 TI - Physicochemical properties of type I corticosteroid receptors from rat brain. AB - [3H]Aldosterone binds with high affinity to Type I corticosteroid receptors in cytosols from adrenalectomized rat forebrains. Physicochemical parameters of these receptors were determined in the presence of molybdate, which stabilized receptors and maintained them in a presumably untransformed state. The Stokes' radius of the molybdate-stabilized receptor was 8.1 nm, as determined by gel filtration on Sephacryl S-300. Its sedimentation coefficient was 9.1S in linear sucrose density gradients. The receptor is asymmetric, with an axial ratio of 8 10 and an apparent mol. wt of 303,000 dalton. The [3H]aldosterone-receptor complex is anionic and elutes from DEAE-Trisacryl in a single peak with a maximum at 160 mM KCl. Exposure to heat or salt in the absence of molybdate, conditions which transform other steroid receptors to smaller DNA-binding forms, causes marked instability of the [3H]aldosterone-receptor complex. The [3H]aldosterone binding protein of rat forebrain, which displays the binding characteristics of a renal Type I (mineralocorticoid) receptor, is similar in size, shape and charge to the molybdate-stabilized oligomeric forms of other steroid hormone receptors. PMID- 3009994 TI - Different metabolism by mammalian and horseradish peroxidases in vitro of steroidal estrogens and their catechol metabolites. AB - Radioactively labeled estradiol-17 beta and 17 alpha-ethynylestradiol and their 2 hydroxy derivatives were incubated with either horseradish peroxidase or mouse uterus peroxidase, and the formation of polar products, which could not be extracted with diethyl ether, and of ether-extractable metabolites was studied. Moreover, the extent of DNA binding was determined. The different peroxidases gave rise to different products, indicating that different pathways in the metabolism of these steroidal estrogens are catalysed by the two peroxidases. PMID- 3009993 TI - Localization and origin of the intestinal peroxidase--effect of adrenal glucocorticoids. AB - Peroxidase activity in rat intestine is stimulated two-fold after bilateral adrenalectomy and is reversed by dexamethasone (9-fluoro-11 beta,17,21-trihydroxy 16 alpha-methyl-1-4-pregnadiene-3,20-dione). The enzyme activity is inhibited on administration of various glucocorticoids of which dexamethasone acts as the most potent inhibitor of the enzyme in vivo. The change of enzyme activity results neither from alteration of the apparent Km of the enzyme nor from enzyme synthesis. Although a small amount of peroxidase is located in the intestinal epithelial cells, a large amount is present in the rest of the intestine. Histochemical studies indicate the presence of peroxidase in the lamina propria, the core of the intestinal villi which contains eosinophil. The peroxidase isolated from the epithelial cell-free intestine is similar to the peroxidase obtained from the pure eosinophil in terms of various physicochemical properties. Dexamethasone also inhibits the eosinophil peroxidase and decreases the number of both circulating and intestinal eosinophil. Studies indicate that a large part of the peroxidase of the intestine is contributed by invading eosinophil and dexamethasone inhibits the enzyme by sequestration of eosinophil both from intestine and blood possibly to the peripheral lymph nodes. PMID- 3009995 TI - Inhibition of corticosteroid 11 beta hydroxylation in bovine zona fasciculata cells by the potassium entry blocker 4-aminopyridine. AB - The potassium entry blocker, 4-aminopyridine has been used to evaluate the importance of this cation in several tissues including the adrenal cortex. Since it inhibited corticosteroidogenesis, an important role for potassium entry in the control of this process has been implied. In the present in vitro study, 4 aminopyridine inhibited cortisol and corticosterone production and, at high concentrations, that of 11-deoxycortisol and 11-deoxycorticosterone. However, at lower concentrations, 11-deoxycorticosteroid production increased suggesting that 4-aminopyridine acts as an inhibitor of 11 beta hydroxylation. Its slight resemblance to metyrapone may suggest that it acts, at least in part, by competing for cytochrome P450. However, its use as a potassium inhibitor in studies of the adrenal cortex may give ambiguous results. PMID- 3009996 TI - Evidence that long-term methionine-enkephalin administration stimulates rat adrenal zona fasciculata. AB - Short-term methionine-enkephalin (DALA) treatment did not induce evident changes in the adrenal zona fasciculata and in the basal corticosterone output of dexamethasone-treated rats administered with maintenance doses of ACTH. Conversely, prolonged (5 days) DALA treatment caused a notable hypertrophy of zona fasciculata cells, along with a significant increase in the plasma concentration of corticosterone. It is suggested that enkephalins exert a trophic action on the rat zona fasciculata. PMID- 3009997 TI - The in situ perfused spinal cord of the rat. Applicability of drugs and chemicals, sodium-lithium exchange, and calcium reduction to functional intact central nervous system tissue. AB - A new forequarters preparation, the in situ perfused cervical spinal cord of the adult rat, is described with respect to its suitability for pharmacological and toxicological investigations. The dose-dependent actions of strychnine, p chloromercuriphenylsulfonate, ouabain, dinitrophenol, bicuculline, and (-) nipecotic acid ethyl ester on mass reflex discharges were studied. The study was extended to sodium-lithium exchange and calcium reduction in the perfusion medium. With the intent to discriminate between primary central actions of substances and ion changes and their secondary metabolic effects, the flow rate, the sodium and potassium concentrations, the pH, the pO2, and the pCO2 of the perfusion medium were measured. The new perfused forequarters preparation, including the intact cervical spinal cord, has a number of advantages over an experimental design using an intact animal dependent on its physiological circulation. These advantages are: the composition of fluids supplying the preparation is under control; the oxygen supply to the cord is no longer dependent on the cardiovascular function, which may be impaired by the substance under study; a complete dose-response curve may be obtained from each animal; washout experiments may be performed; the action of substances can be studied in the presence of extreme concentrations of drugs, ions, etc. PMID- 3009999 TI - Diplopia and pneumoencephalocoele after chest wall resection and intraoperative radiation therapy. AB - The dural sheath may be transected in the course of a posterior thoracic procedure. If air is introduced into the subarachnoid space, a cranial nerve palsy may result. Computed tomographic scanning establishes the diagnosis. The condition resolves without treatment. PMID- 3009998 TI - Indications and technique for endoscopic laser resections in bronchology. A critical analysis based upon 2,284 resections. AB - Over a period of 6 years, we have treated 1,310 patients in 2,284 sessions using a neodymium-yttrium aluminum garnet laser. Indications are more often palliative than curative, with the primary goal to relieve an obstructed airway in a single treatment. The effectiveness of such resections is widely recognized, but indications for such a technique with its limitations deserve emphasis. The use of a rigid bronchoscope is important to provide satisfactory operating conditions and especially to manage hemorrhage rapidly while maintaining a satisfactory airway. PMID- 3010000 TI - Effect of acupuncture on the activities of phosphodiesterases in rat brain. PMID- 3010001 TI - Interleukin-1 gene (IL1) assigned to long arm of human chromosome 2. AB - A complementary DNA (cDNA) probe for the predominant (pI 7) form of human monocyte-derived interleukin-1 (IL1) and a collection of 30 human-mouse somatic cell hybrids were used to assign the IL1 gene to human chromosome 2 by Southern blot analysis of hybrid cell DNA digested with the restriction endonuclease BglII. In situ hybridization to human metaphase chromosomes localized the IL1 gene to the long arm of chromosome 2 at position 2q13-2q21 between two fragile sites. PMID- 3010002 TI - Mechanism of oxygen toxicity in rat lungs. PMID- 3010003 TI - Impaired essential fatty acid metabolism in latent tetany. AB - Because of the biochemical role of magnesium in the metabolism of polyunsaturated fatty acids, we measured the levels of polyunsaturated fatty acids in the plasma phospholipids of 40 patients with latent tetany (LT). The level of linoleic acid (18:2 n-6) was 25% higher in patients than in controls (p less than 0.001). In contrast, dihomogamma linoleic acid (20:3 n-6) was 34% lower, arachidonic acid (20:4 n-6) was 14% lower than in controls (p less than 0.01) and the ultrapolyunsaturates, 22:4 n-6 and 22:5 n-6, were reduced by 27 and 53%, respectively (p less than 0.001). Linolenic acid (18:3 n-3) was not significantly different from control levels, but its metabolite, eicosapentaenoic acid (20:5 n 3) was reduced by 43% (p less than 0.05). Since the levels of plasma phospholipid fatty acids are a reflection of hepatic metabolism, these findings suggest that patients with LT have impaired desaturation of 18:2 n-6 and possibly 18:3 n-3. The desaturase enzyme is Mg dependent, and impaired desaturation has been demonstrated in animals rendered Mg deficient. Possible consequences of this impairment in essential fatty acid (EFA) metabolism are an increase in membrane viscosity and a distortion in the availability of fatty acid precursors for prostaglandin synthesis. Either of these abnormalities may contribute to the pathogenesis of illness associated with LT. PMID- 3010005 TI - Investigations of a screening test for early diagnosis of hepatocellular carcinoma. PMID- 3010004 TI - Nucleoside diphosphatase activity in ethanol-pyrazole damaged hepatic cells. PMID- 3010006 TI - The different mechanisms for suppression of pituitary and testicular function. AB - The differential mechanisms reducing androgen secretion by LHRH agonists are discussed with relevance to clinical therapy. LH secretion can be desensitised by exposure to agonists using high doses, frequent injections or sustained release/constant infusion. The desensitized pituitary is refractory to hypothalamic stimulation. Pituitary receptor suppression is associated with depletion of pituitary gonadotrophin content, and a decline of LH and FSH secretion to a basal rate. Recovery of LH responsiveness to endogenous LHRH stimulation requires restitution of gonadotrophin content (about 7 days in rats). After long-term infusions in normal men, testosterone secretion recovers within 7 10 days. The binding capacity of testicular LH/hCG receptors is reduced in rats after supraphysiological gonadotrophin stimulation, by agonists or directly by hCG, concomitantly the steroidogenic capacity of the testis in vitro is impaired. Qualitative changes in androgen biosynthesis are a marked fall in testosterone production and dose-dependent enhancement of progesterone production. After 12 months of buserelin injections, the changes in hCG-stimulated rat testes are an increased ratio of progesterone/17-OH-progesterone (inhibition of 17 hydroxylase), a reduced capacity for secretion of androstenedione and testosterone (block of 17,20-desmolase), and increased 5 alpha-pregnane-3,20 dione (this steroid inhibits the 17,20-desmolase, similarly to progesterone). After treatment, Leydig cell function recovers completely. Leydig cell hyperplasia is observed as a result of the steroidogenic changes. These findings in rats have not been observed in dogs, monkeys or in humans.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010007 TI - Physiological role of putative testicular gonadotrophin releasing hormone (GnRH). AB - Evidence suggests that exogenous GnRH and agonist analogues have short-term stimulatory effects on rat Leydig cell function - when administered intratesticularly. Since rat Leydig cells possess GnRH receptors and their endogenous ligand has not yet been identified the physiological importance of the observations for testis function is unknown. To address this issue we have determined the consequences of blockade of testis GnRH receptors on Leydig cell function under both normogonadotrophic and hypogonadotrophic stimulation of the testis in vivo. A GnRH antagonist (ANT) was used to achieve receptor blockade but during continuous systemic infusion ANT occupied pituitary GnRH receptors and markedly reduced serum LH, FSH, testosterone, and intratesticular testosterone in adult and 30 d old immature male rats. These results were similar to those obtained by administration of a GnRH antiserum which did not bind to testis GnRH receptors. Thus, blockade of testis GnRH receptors during hypogonadotrophism did not produce additional inhibition of steroidogenesis by Leydig cells. However, direct continuous infusion of ANT into one testis produced greater than 90% occupancy of GnRH receptors while reducing GnRH receptors by only 50% in the contralateral testis. Unilateral intratesticular infusion did not reduce serum LH, FSH, Prolactin or testosterone levels despite 75% occupancy of pituitary GnRH receptors. Thus, both ANT infused and saline infused testes were exposed to the same gonadotrophic stimulants but in the former GnRH-R were essentially non existent. Compared to the control testis, the ANT infused testis showed a 20-30% reduction in LH, FSH, lactogen receptors and 30-40% fall in testosterone content. Identical results were obtained in adult and 30 d-old male rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010009 TI - Modulation and role of Ca2+ in LH and LHRH agonist action in rat Leydig cells. AB - The results of our recent studies on purified rat Leydig cells indicate that there are no major qualitative differences in the stimulating effects of LH and LHRH agonists on steroidogenesis via mechanisms that are dependent on calcium. This was demonstrated by using inhibitors of calmodulin and the lipoxygenase pathways of arachidonic acid metabolism. Using the fluorescent indicator quin-2, it was shown that LH and LHRH agonist increase intracellular calcium levels; LH was more potent than LHRH agonist (max increase in concentrations obtained were 500 nM and 60 nM respectively). This difference was probably the result of a direct effect of cyclic AMP (whose production is stimulated by LH but not by LHRH) because cyclic AMP analogues were as potent as LH in increasing calcium levels. These studies indicate a major role for calcium in the control of steroidogenesis in testis Leydig cells. PMID- 3010008 TI - Paracrine role of Sertoli cells. AB - Data from several experimental approaches strongly suggest that Sertoli cells exert a paracrine control of the two main testicular functions, androgen secretion and spermatogenesis. Further evidence supporting this role of Sertoli cells was obtained by coculture of Sertoli cells with other testicular cells. Coculture of pig or rat Sertoli cells with pig Leydig cells produces an increase in the hCG receptor number and an increase in the steroidogenic activity of Leydig cells. Pretreatment with FSH further increases the values of these two parameters. These biochemical changes were associated with ultrastructural changes in Leydig cells. The effects of Sertoli cells on Leydig cells depend upon the ratio of the two cells and on the substrate in which the cells are cultured. Moreover, Leydig cells produce an increase in the FSH receptor number and in the FSH stimulation of plasminogen activator production by Sertoli cells. Coculture of rat or pig Sertoli cells with rat germ cells, induces an increase in the RNA and DNA biosynthetic activities of germ cells. Most of the stimulatory effects seemed to be mediated by diffusible factors, secreted by Sertoli cells, but full expression of the stimulatory action was observed when germ cells were in contact with other cells. In this coculture system, a fraction of rat germ cells containing mainly mature forms of spermatocytes inhibited rat Sertoli cell RNA and DNA synthesis, but had no effect on pig Sertoli cells. On the contrary, a fraction of rat germ cells richer in spermatogonias and preleptotene spermatocytes, stimulated rat Sertoli cell DNA synthesis but was without effect on pig Sertoli cells. These results clearly show that the stimulatory effects of Sertoli cells on Leydig and on germ cells which are not species specific are mediated mainly by diffusible factors, the secretion of which is regulates by FSH. PMID- 3010011 TI - Nabilone and metoclopramide in the treatment of nausea and vomiting due to cisplatinum: a double blind study. AB - Thirty-two patients were entered into a double blind trial of antiemetic support in patients being treated with cisplatin. Patients were allocated to receive either oral nabilone (1 mg 8 hourly) or intravenous metoclopramide (1 mg kg-1 3 hourly) in random order over 4 courses. There was no difference between the two treatments in the overall incidence or severity of vomiting, although a subgroup of patients enjoyed a substantial reduction in episodes of vomiting whilst receiving metoclopramide. Side-effects were predictable from the pharmacology of the drugs. PMID- 3010010 TI - Effects of physiological and abnormally elevated prolactin levels on the pituitary-testicular axis. AB - Prolactin can influence testicular function both directly, and indirectly via altering release of gonadotropins from the pituitary. Although numerous effects of prolactin on male reproductive functions have been described, only a few were demonstrated in more than one species. These include effects on male accessory reproductive glands, on testicular luteinizing hormone receptors, on the release of gonadotropins and on sexual behavior. In rodents, prolactin appears to play a physiological role in the pituitary regulation of testicular function. This is especially pronounced in the golden hamster. In this seasonally-breeding species, alteration of prolactin release is one of the mechanisms mediating the effects of photoperiod on the testis. In the hamster, prolactin is required for the maintenance of luteinizing hormone and prolactin receptors in the testis and treatment with prolactin can completely reverse testicular atrophy induced by exposure to short photoperiod. In both men and rats, excessive release of prolactin (hyperprolactinemia) leads to suppression of sexual behavior and gonadotropin release. These effects appear to be due to the action of prolactin on the central nervous system. Most, if not all, of the effects of prolactin exhibit striking variability among species. Moreover, prolactin can exert differential effects on the same target tissue in the same species, depending primarily on the dose. PMID- 3010013 TI - Experimental and clinical studies on the effects of synthetic muramyldipeptide derivatives on viral and opportunistic infections. AB - The efficacy of strearoyl-MDP derivatives, L18-MDP and MDP-Lys-18 against bacterial and viral infections in mice was examined. Both L18-MDP and MDP-Lys-L18 stimulated nonspecifically host-resistance against opportunistic infection with C. kutscheri in hydrocrotisone-treated mice against Sendai virus infection in mice. PMID- 3010012 TI - [Polyneuropathy associated with the parenteral administration of pentazocine]. PMID- 3010014 TI - Characterization of immunotherapeutic agents: the example of imuthiol. AB - Immunotherapeutic (IMT) agents are intended to restore or reequilibrate a faltering immune system. These aims will be attained provided the agent fulfills minimum requirements: in vivo defined activities; lack of suppressive, sensitizing, or autoimmunity-inducing influence; no immunotoxicity and carcinogenicity. Known chemical structure, analytical procedure, toxicology and pharmacology are also prerequisites. A follow-up pattern of sequential and progressively more demanding assays through which IMT agents could be screened for therapeutic potential is suggested. The example of Imuthiol, tested accordingly, demonstrates the usefulness of the proposed procedure. PMID- 3010015 TI - Clinical characterization of imuthiol. AB - Imuthiol is a nontoxic agent recruting and regulating T cells. Phase III studies in chronic bronchitis and bronchiectasis showed that immune functions were restored to normal, or near normal values. Cure was obtained in rheumatoid arthritis, tuberculosis and chronic infections in the elderly. Imuthiol is an effective agent for the treatment of syndromes and disease states where the underlying defect is a T-cell deficiency or dysfunction. PMID- 3010016 TI - Vagal body tumors: diagnosis and treatment. AB - This presentation includes a report of four vagal body tumors bringing the total number in the English literature to approximately 80. The diagnosis and treatment of this lesion, its familial incidence and the propensity for multiple secreting or nonsecreting chemodectomas, and appropriate clinical and pathologic studies required will be reviewed in some detail. Appropriate differential diagnostic features will be illustrated by comparison of these tumors with other primary vagal tumors from our experience such as neurofibromas, neurilemmomas, and cystic degeneration of neurilemmomas. PMID- 3010017 TI - Enkephalin-like substance in aplysia nervous tissue and actions of leu-enkephalin on single neurons. AB - Extract from the abdominal ganglia of Aplysia was fractionated by high performance liquid chromatography. Several large uv absorbing peaks were found. One of these peaks from the ganglionic extract was analyzed by displacement assay with the use of Mytilus edulis membranes. The results revealed the substance in this peak was able to displace 3H-D-Ala2, met5-enkephalinamide. In electrophysiological studies of abdominal and cerebral neurons, depolarizing and hyperpolarizing responses were obtained from some neurons, including neurons in the identified cerebral B cell cluster. A smaller population of cells exhibited biphasic responses. Some of the responses could be depressed by prior naloxone treatment. In conclusion, an endogenous opioid system, using substance related to but distinct from the enkephalins, may exist in Aplysia. PMID- 3010018 TI - Para-azidoclonidine: a novel photoaffinity ligand for the alpha 2-receptor. AB - Reversible and irreversible interactions of the photoreactive clonidine analogue p-azidoclonidine (PAZC) with brain alpha 2-adrenergic receptors were examined. In the absence of light, PAZC displayed selective, high affinity, competitive interactions with sites labeled by the alpha 2-agonist 3H-p-aminoclonidine (3H PAC). Reversible binding characteristics resembled those of other alpha 2 agonists. Preincubation of bovine frontal cortex membranes with 100 nM PAZC followed by ultraviolet irradiation and thorough washing decreased alpha 2 receptor density 42% relative to controls receiving irradiation alone. The loss of receptors could be prevented by inclusion of 500 nM phentolamine in the preincubation medium. Alpha 1- and beta-adrenergic receptors were relatively unaffected. PAZC is a potential photoaffinity ligand for the alpha 2-adrenergic receptor. PMID- 3010020 TI - Long-lasting blockade of P2-receptors of the urinary bladder in vivo following photolysis of arylazido aminopropionyl ATP, a photoaffinity label. AB - In the present study the possibility of provoking an irreversible blockade of P2 receptors in vivo by photolyzing ANAPP3 was investigated. ANAPP3 administered as an intraarterial infusion produced a short-lived blockade of P2-receptor mediated contractions of cat urinary bladders in situ. However, irradiation of the bladders with visible light during the infusion of ANAPP3 resulted in a blockade of the response for at least 120 minutes. Thus, photoactivation of ANAPP3 in vivo produced an essentially irreversible blockade of P2-purinergic receptors. PMID- 3010019 TI - The role of benzodiazepine receptors in the discriminative stimulus properties of delta-9-tetrahydrocannabinol. AB - Twenty male Sprague-Dawley rats were trained to discriminate 3.0 mg/kg delta-9 tetrahydrocannabinol (THC) from its vehicle. Following acquisition of this discrimination animals were tested for generalization to 3.0 mg/kg diazepam. Thirteen animals showed a generalization from THC to diazepam, whereas the remaining seven animals did not. The generalization curve for diazepam was dose dependent from 0.1 to 10.0 mg/kg in the first group; the latter group showed no generalization from THC at any dose of diazepam in this range. No differences were found between these groups in the generalization curve for THC. The benzodiazepine antagonist Ro 15-1788 (2.0 mg/kg) antagonized the generalization to diazepam in the group that discriminated diazepam as THC. In contrast, Ro 15 1788 increased THC lever responding of 10 mg/kg diazepam in the group which did not generalize from THC. Ro 15-1788 did not alter the discriminability of THC in either group. THC also showed partial generalization to pentobarbital (1 to 10 mg/kg). The generalization was again complete in one subgroup and absent in another, but there was only a 43 percent overlap between the subgroups found with testing for generalization to diazepam. The percent THC lever responding with 3.0 mg/kg pentobarbital was increased by Ro 15-1788 in the group which generalized to diazepam, but not the other group. These data suggest that the discriminative stimulus properties of THC may have some commonality with the effects of diazepam in a subpopulation of rats trained to discriminate THC. These THC-like effects of diazepam are probably mediated by benzodiazepine receptors since they are antagonized by a specific benzodiazepine receptor antagonist. PMID- 3010021 TI - 3H-diprenorphine is selective for mu opiate receptors in vivo. AB - The displacement of 3H-diprenorphine from opiate receptors by mu-selective opiates was measured in the mouse striatum and thalamus in vivo. In addition, the regional distribution of opiate receptor binding using 3H-diprenorphine, 3H naloxone and 3H-lofentanil was measured. The displacement of 3H-diprenorphine by naloxone and carfentanil in vivo showed no differences in the striatum and thalamus suggesting that 3H-diprenorphine binds only to one opiate receptor subtype in vivo. This finding is substantiated by the observation that the mu selective ligands 3H-naloxone and 3H-lofentanil have the same in vivo distribution of receptor binding as 3H-diprenorphine. The implication of these findings for PET imaging of opiate receptor subtypes is discussed. PMID- 3010022 TI - The inositide cycle in bovine photoreceptor membranes. AB - Various enzymic steps in the inositide cycle were investigated in purified bovine retinal rod outer segments (ROS). Incubation of ROS with [gamma-32P]ATP resulted in a rapid labeling of phosphatidic acid and phosphatidylinositol 4-phosphate (PIP), while little radio-tracer was recovered from phosphatidylinositol 4,5 bisphosphate (PIP2). This can be explained by the relatively low activity of PIP kinase activity in ROS as compared to the remainder of the retina. Similarly, relatively little phosphodiesteratic activity degraded PIP2 and PIP in ROS when 32P labeled phosphoinositides in synaptic membranes (heat-treated to inactivate endogenous enzymes) were used. Although light exposure of ROS did cause rapid rhodopsin phosphorylation, no enzymic steps of the cycle were changed, even when ROS were obtained from retinas excised from cows dark-adapted by unilateral eye patching the day prior to kill. These studies do not support the view that light is an agonist of the inositide cycle in mammalian photoreceptors. PMID- 3010023 TI - Subclass specificity of anti-idiotypic anti-opiate receptor antibodies in rat brain, guinea pig cerebellum, & neuroblastoma x glioma (NG 108-15). AB - Receptor subclass recognition properties of affinity-purified rabbit polyclonal anti-idiotypic anti-opiate receptor antibodies in various membrane preparations have been determined. The anti-receptor immunoglobulins significantly decrease binding of 3H-[D-Ala2,-MePhe4,Gly-ol5]enkephalin, a highly selective mu agonist, in rat neural membrane. In the presence of a concentration of the unlabeled ligand sufficient to block existing mu sites, the antibodies compete, to a lesser degree with 3H-[D-Ala2,D-Leu5]enkephalin for delta site occupancy in both rat neural membrane, and neuroblastoma x glioma membrane preparations. The antibodies do not displace 3H-ethylketocyclazocine from rat brain or guinea pig cerebellum. PMID- 3010024 TI - Effect of thyrotropin on conversion of T4 to T3 in perfused rat liver. AB - The present study was undertaken to elucidate the direct effect of thyrotropin (TSH) on the conversion of thyroxine (T4) to 3,5,3'-triiodothyronine (T3) in the isolated perfused rat liver. The liver was perfused without recirculation with a synthetic medium containing 10 micrograms/dl T4 and the effect of constant infusion of bovine TSH (125 or 250 microU/ml) on the conversion of T4 to T3 was examined. T4 uptake in the perfused liver was not changed by the addition of TSH. The release of T3 (10.3 +/- 1.4 ng/g/30min, mean +/- SD), tissue T3 production (99.5 +/- 21.4 ng/g/30min), net T3 production (102.6 +/- 20.2 ng/g/30min), and the conversion rate of T4 to T3 (14.8 +/- 3.5%) in the liver perfused with 250 microU/ml TSH were significantly higher than those in controls (8.1 +/- 1.2 ng/g/30min, 69.0 +/- 6.8 ng/g/30min, 69.9 +/- 6.1 ng/g/30min, and 10.0 +/- 0.8%), respectively. These results suggest that TSH may directly enhance hepatic conversion of T4 to T3 in rats in vitro. PMID- 3010025 TI - Studies on the use of a novel affinity matrix, sepharose amine-succinyl-amine haloperidol hemisuccinate, ASA-HHS, for purification of canine dopamine (D2) receptor. AB - Haloperidol Hemisuccinate (HHS) was synthesized specifically as a ligand for an affinity chromatography matrix. The affinity chromatography matrix, ASA-HHS was developed which had high affinity and capacity for dopamine D2 receptors in solubilized canine striatal preparations. ASA-HHS also demonstrated nonspecific interaction with the D2 receptor. Two fractions, which bound 3H-spiroperidol specifically, with similar one dimensional SDS-PAGE patterns could be eluted successfully with 20 microM haloperidol in only 30% of the runs. Both fractions represented 300-400 fold purification. Two dimensional IEF-PAGE analysis of one of the fractions demonstrated coelution of beta and gamma actin, alpha and beta tubulin with the 3H-spiroperidol binding sites. The pattern of the proteins eluted from ASA-HHS and the inconsistent recovery of active D2 receptors are discussed. PMID- 3010026 TI - Properties of diacylglycerol kinase purified from bovine brain. AB - A nearly homogeneous but somewhat unstable diacylglycerol kinase (ca. MW 72,000 daltons) was purified from bovine brain by modification of the procedure of Kanoh et al. (Kanoh, H., Kondoh, H., and Ono, T. [1983] J. Biol. Chem. 258, 1767-1774). The purification consisted of four steps (brain cytosol isolation and successive chromatography on DEAE-cellulose, Sephadex G-25 for desalting and ATP-agarose) carried out in buffers stabilized with EDTA, ATP and dithiothreitol (DTT). Specific activities, determined within 4 hr of purification, ranged from 908-1857 nmol ATP incorporated/min/mg protein, with the variation reflecting the instability. Optimal activities required deoxycholate (0.1%), one of the phosphoglycerides [phosphatidylcholine (PC), phosphatidylethanolamine (PE) or phosphatidylserine (PS)] (0.025-0.25 mM), ATP (5 mM, apparent Km = 0.57 mM), 1,2 dioleoyl-rac-glycerol (5 mM, apparent Km = 1 mM) and Mg2+ (10 mM, apparent Km = 2.2 mM). Phosphatidylinositol (PI) was slightly less effective than PC, PE or PS and noninhibitory in combination with PC, PE or PS. Relative to PC phosphatidic acid (PA) (52%), sphingomyelin (48%), lyso-PC (1.5%) and lyso-PI (28.6%) were less effective activators. The sulfhydryl reagents, p-chloromercuribenzoic acid (PCMB) (1.0 mM), N-ethylmaleimide (NEM) (1.0 and 2.0 mM) and 5,5'-dithiobis-(2 nitrobenzoic acid) (DTNB) (1.0 mM), showed strong inhibition of activity which was prevented by 0.5 mM DTT. In contrast to other reports, this purified enzyme showed no monoacylglycerol kinase activity. Comparison of diacylglycerols of varying fatty acid composition indicated that the enzyme showed a preference for substrates with at least one unsaturated fatty acid, particularly in the 2 position.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010027 TI - Effects of pre- and postweaning undernutrition on polyphosphoinositide pools in rat kidney. AB - The effects of undernutrition on polyphosphoinositide levels in rat kidneys removed and frozen immediately after animal death or 10 min later were determined. Weanling (21-day-old) rats of dams fed a 5 or 22% protein diet and litters fed either normal or protein-deficient diets for an additional six wk were used. Nutritional deprivation lowered phosphatidylinositol-4-phosphate (PtdIns4P) preferentially (35-40%) but preserved phosphatidylinositol-4,5 bisphosphate (PtdIns4,5P2) at weaning. This effect was not completely reversed in animals nutritionally rehabilitated after weaning. Postweaning protein deficiency did not reduce the levels of these lipids. Postmortem loss was the same for all five groups, minimal for PtdIns4P and about one-third for PtdIns4,5P2. PMID- 3010029 TI - [Osteoarticular tuberculosis in the Ivory Coast. Apropos of 24 cases]. AB - 24 cases of osteoarticular tuberculosis have been observed in two years in the Regional Hospital of Bouake (Ivory-Coast). Multifocus and pluri-visceral localizations are frequent. Diagnosis is based on a network of evidence and on the research of an evolutive bacillary focus out of bones. The medical treatment used alone because of local conditions of practice, appears to be effective, even in advanced and complicated cases. But mid-term follow-up is still not very satisfactory. PMID- 3010028 TI - Differentiation of exercise-induced metabolic responses during selective beta 1- and beta 2-antagonism. AB - The effect of beta 1- or beta 2-antagonism on the plasma levels of glucose, lactate, triglycerides, and free fatty acids was studied in seventeen normal male volunteers. All subjects performed three graded and uninterrupted exercise tests until exhaustion. Prior to each exercise test they received in a randomized order during three consecutive days either placebo or a predominant beta 1-blocker (atenolol, 50 mg once per day) or a predominant beta 2-blocker (ICI 118,551, 20 mg t.i.d.), according to a double-blind cross-over study design. Atenolol increased the plasma level of glucose at rest but did not influence the rise in plasma glucose during exercise. ICI 118,551 did not change the resting plasma glucose level, but it prevented the exercise-induced rise in plasma glucose, observed during placebo. During beta 1-antagonism the plasma lactate concentration at rest and during or after exercise was not different from placebo. During beta 2-blockade the exercise-induced rise in plasma lactate tended to be suppressed, and during recovery the plasma lactate levels were significantly lower than during placebo. The serum triglycerides concentration at rest and exercise was not altered, either by beta 1- or by beta 2-antagonism. Atenolol and ICI 118,551 did not affect the serum level of free fatty acids at rest, but at moderate exercise the serum free fatty acids concentration was lower during beta 1-blockade than during placebo. Our results provide further evidence that beta 2-adrenergic receptors are involved in the regulation of the plasma levels of glucose and lactate during exercise. PMID- 3010030 TI - [Vaccination of adults against hepatitis B virus in a tropical African environment (Ivory Coast). Study of results according to the serologic status toward the virus]. AB - A total of 103 volunteers, from 18 to 55 years of age have received subcutaneous injection of the hevac B Pasteur 5 micrograms vaccine: one injection a month during three months and one booster injection after one year. The study of the anti-HBs reaction of the subjects, with regard to the serologicale status to the hepatitis B virus before inoculation, has shown that only 78.8% of the subjects, who are only positive towards the anti-HBc antibody, will develop an anti-HBs response of primary type with a relatively low value. On the contrary, all anti HBc and/or anti-HBs subjects, who were positive before inoculation, react with relatively high anti-HBs value immediately after the first injection. 93.3% of the seronegative subjects before inoculation will develop an anti-HBs seroconversion after the complete inoculation procedure. PMID- 3010032 TI - Preparation of 3-aminopyridine 1,N6-ethenoadenine dinucleotide and 3 aminopyridine 1,N6-ethenoadenine dinucleotide phosphate. PMID- 3010031 TI - Highly sensitive methods for assaying the enzymes of vitamin B6 metabolism. PMID- 3010033 TI - Spectrophotometric assay of NADase-catalyzed reactions. PMID- 3010034 TI - Snake venom NAD glycohydrolase: purification, immobilization, and transglycosidation. PMID- 3010035 TI - Sensitive radiosubstrate/high-performance liquid chromatography assays for flavocoenzyme-forming enzymes. PMID- 3010036 TI - Enzymatic synthesis of thiamin triphosphate. PMID- 3010038 TI - 1,25-dihydroxyvitamin D microassay employing radioreceptor techniques. PMID- 3010037 TI - Quantitation of vitamin D2, vitamin D3, 25-hydroxyvitamin D2, and 25 hydroxyvitamin D3 in human milk. PMID- 3010039 TI - Assay for 1,25-dihydroxyvitamin D using rabbit intestinal cytosol-binding protein. PMID- 3010040 TI - Cytoreceptor assay for 1,25-dihydroxyvitamin D. PMID- 3010041 TI - Monoclonal antibodies as probes in the characterization of 1,25-dihydroxyvitamin D3 receptors. PMID- 3010042 TI - Isolation of native and proteolytically derived ileal receptor for intrinsic factor-cobalamin. PMID- 3010043 TI - Purification and properties of the egg plasma membrane. PMID- 3010044 TI - Molecular analysis of mutant ompR genes exhibiting different phenotypes as to osmoregulation of the ompF and ompC genes of Escherichia coli. AB - Expression of the ompF and ompC genes coding for major outer membrane proteins is osmoregulated by solutes, such as sucrose and NaCl, in the growth medium. The OmpR protein, a positive regulator of these genes, is involved in the osmoregulation (Dairi et al. 1985; Nara et al. 1984). In the present work, five mutant ompR genes exhibiting different phenotypes of osmoregulation were cloned and sequenced. Three of them, ompR1, ompR2 and ompR20, were previously isolated mutants. The others, ompR3 and ompR4, were isolated in the present work. The ompR1 mutation resulted in the deletion of 19 amino acids near the C-terminus of the OmpR protein. The ompR3 and ompR4 mutations resulted in Arg15 to Cys and Arg71 to Thr conversions, respectively, at the N-terminal portion, whereas the ompR20 and ompR2 mutations resulted in Arg150 to Cys and Val207 to Met conversions, respectively, at the C-terminal portion. Based on these results, the structure and function of the OmpR protein are discussed in relation to the mechanism of osmoregulation. PMID- 3010045 TI - Genetic analysis of a relaxed substrate specificity aromatic ring dioxygenase, toluate 1,2-dioxygenase, encoded by TOL plasmid pWW0 of Pseudomonas putida. AB - Toluate 1,2-dioxygenase is the first enzyme of a meta-cleavage pathway for the oxidative catabolism of benzoate and substituted benzoates to Krebs cycle intermediates that is specified by TOL plasmid pWW0 of Pseudomonas putida. A collection of derivatives harbouring Tn1000 insertions and defective in toluate dioxygenase have been isolated from pPL392, a pBR322-based hybrid plasmid carrying the TOL plasmid meta-cleavage pathway operon. In parallel, a series of N methyl-N'-nitro-N-nitro-soguanidine-induced mutant plasmids defective in this enzyme activity were isolated from pNM72, a pKT231-based hybrid plasmid carrying the same operon. Pairs of mutant plasmids, consisting of one Tn1000 derivative and one nitrosoguanidine-induced derivative, were used for complementation analysis of toluate dioxygenase in Escherichia coli recA bacteria, in which the formation of 2-hydroxymuconic semialdehyde from benzoate was examined. Four cistrons for toluate 1,2-dioxygenase were thus identified. DNA fragments containing nitrosoguanidine-induced mutant cistrons plus the other meta-cleavage operon genes were cloned into pOT5, an R388-based vector, and complementation tests between different nitrosoguanidine-induced mutant cistrons were carried out in Pseudomonas putida cells, this time scoring for growth on p-toluate. This analysis also identified four cistrons. Examination of the products of these cistrons, by means of E. coli minicells containing pPL392 or its Tn1000 insertion derivatives, indicated that the first two cistrons of the operon comprise a single gene, xylX, which encodes a 57 kilodalton protein, and that the third cistron, xylY, encodes a 20 kilodalton protein. PMID- 3010046 TI - Identification of a DNA sequence associated with plasmid integration in Streptomyces coelicolor A3(2). AB - We identified a DNA element of length about 1 kb that is present in two copies in the chromosome of Streptomyces coelicolor A3(2) and is also present on the plasmid SCP1 which has been carefully defined genetically, but never isolated as extrachromosomal DNA. A copy of the element is close (within 5 kb) of a gene coding for an extracellular agarase in the chromosome of S. coelicolor A3(2) and in an NF strain, in which SCP1 has integrated into the chromosome, the agarase gene has been deleted. The element has properties reminiscent of Insertion Sequences in Escherichia coli, but it is not yet know if it can transpose. PMID- 3010048 TI - Mapping of mRNA encoding endoglucanase A from Clostridium thermocellum. AB - The size and location of the 5' end of celA mRNA encoding endoglucanase A of Clostridium thermocellum were investigated in C. thermocellum and in an Escherichia coli clone that carries and expresses the celA gene. In E. coli, the 5' end of celA mRNA was located 134 bp upstream from the initiation codon and 10 bp downstream from a sequence homologous to the consensus sequence of E. coli sigma 70 and Bacillus subtilis sigma 43 (formerly sigma 55) vegetative promoters. In C. thermocellum, a minor transcript appeared to start from the same site, but a major species started 57 bp upstream from the coding sequence. The 5' end of this mRNA was preceded by a sequence reminiscent of B. subtilis sigma 28 vegetative promoters. In both organisms, the size of the transcript suggested that celA belongs to a monocistronic unit of transcription. PMID- 3010047 TI - DNA methylation differentially enhances the expression of one of the two E. coli dnaA promoters in vivo and in vitro. AB - The promoter/regulatory region of the dnaA gene, whose gene product is required for the initiation of DNA replication in Escherichia coli K-12, contains an unusually large number of Dam methylation sites. In this paper we report that the expression of the dnaA gene is decreased in Dam- strains of E. coli. The decrease in the expression of dnaA was measured in vivo using a dnaA-lacZ gene fusion. In vivo S1 nuclease mapping demonstrated that the decrease was due to a differential decrease in expression from the more proximal of the two dnaA promoters, dnaA2P. Comparison of the strengths of the two dnaA promoters in an in vitro transcription system using methylated and unmethylated DNA templates suggests that the effect of methylation on dnaA2P is probably at the level of RNA polymerase/DNA interaction. We suggest that this effect of methylation may be important in controlling the expression of dnaA during the E. coli cell cycle. PMID- 3010049 TI - Transformation of Aspergillus nidulans with a cloned, oligomycin-resistant ATP synthase subunit 9 gene. AB - An allele (oliC31) of the A. nidulans oliC gene has been cloned using homology with the equivalent gene from N. crassa. OliC31 codes for an oligomycin resistant, triethyltin-hypersensitive form of subunit 9 of the mitochondrial ATP synthase complex. Direct selection for oligomycin-resistance was possible following transformation of A. nidulans with the oliC31 gene. The phenotypes of transformants cultured in the presence of oligomycin were indicative of the position of integration of the transforming plasmid within the genome. Subsequent recombination events involving the integrated oliC31 gene were also apparent from altered levels of resistance to oligomycin or triethyltin. This gene should prove useful as a marker for transformation of strains lacking auxotrophic lesions and in gene replacement or disruption experiments. PMID- 3010050 TI - Gene cloning in Aspergillus nidulans: isolation of the isocitrate lyase gene (acuD). AB - An Aspergillus nidulans gene library was constructed in a high-frequency transformation vector, pDJB3, based on the Neurospora crassa pyr4 gene. This gene library was used to isolate the structural gene for isocitrate lyase (acuD) by complementation of a deficiency mutation following transformation of A. nidulans. Plasmids rescued in Escherichia coli were able to transform five different A. nidulans acuD mutants. Transformation using plasmids containing the cloned fragment resulted in integration at the acuD locus in six of nine transformants. PMID- 3010051 TI - The fission yeast cell cycle control gene cdc2: isolation of a sequence suc1 that suppresses cdc2 mutant function. AB - A DNA fragment called suc1 has been found to rescue cells mutated in the cell cycle control gene cdc2 of the fission yeast Schizosaccharomyces pombe. The suppressing activity of suc1 is observed when it is present on a multicopy number plasmid. The gene does not hybridize to cdc2 and maps elsewhere in the genome. Its effect is cdc2 allele specific suggesting that it interacts directly with the cdc2 gene function. PMID- 3010053 TI - Excision of pyrimidine dimers from the DNA of Neurospora. AB - Germinated conidia of Neurospora have been monitored for their ability to excise pyrimidine dimers. Dimer concentration was measured in DNA extracted immediately after UV treatment, and it was compared to that of DNA from cells which had a post-UV incubation before extraction. Two methods were used to assay dimer level in DNA: measurement of the number of single-strand breaks (as revealed in alkaline sucrose gradients) produced by a dimer-specific endonuclease; monitoring the ability to compete for binding to dimer-specific antibodies in a radioimmunoassay. Both methods showed efficient excision of dimers by wild-type and by uvs-2, even though an earlier study had reported that uvs-2 was unable to excise dimers. UV-induced mutation shows a dose-rate effect: acute UV yields several times as many mutations as does the same dose of chronic UV. There is a parallel effect on dimer accumulation. The concentration of dimers at the conclusion of the UV treatment shows a strong correlation with the resultant mutation frequency. PMID- 3010054 TI - Reversion and immobilization of phage Mud1 cts (Apr lac) insertion mutations in Salmonella typhimurium. AB - Phage Mud1 cts (Apr lac), or Mud1, insertion mutations may be accompanied by adjacent deletion formation which can complicate use of lac fusions generated with this phage for gene regulatory studies. As for phage Mu insertion mutations, phage Mud1 insertions fail to revert at significant frequency (whether or not accompanied by an adjacent deletion). We describe isolation of revertible (X mutant) derivatives of phage Mud1 in Salmonella typhimurium. The X mutant derivatives allow use of reversion as a simple test to determine whether a Mud1 insertion has occurred precisely without an adjacent deletion that may have fused the lac genes to a promoter outside of the gene of interest. In addition, a simple method for stabilizing Mud1 generated lac fusions against subsequent transposition is described. PMID- 3010052 TI - Primary structure of wild-type and mutant alleles of the PET494 gene of Saccharomyces cerevisiae. AB - The product of the yeast nuclear gene PET494 is required specifically for the translation of the mitochondrially encoded subunit III of cytochrome c oxidase. We have determined the DNA sequence of a 1.9 kb fragment carrying PET494. The sequence contains a single long open reading frame of 489 codons. This open reading frame encodes the PET494 protein since the DNA sequence of the corresponding fragment derived from a strain with a known pet494 amber mutation contained an in frame UAG codon. The results of S1 nuclease protection experiments demonstrated that this region is transcribed and that the 5' ends of the major transcripts lie 30 to 40 base-pairs upstream of the first AUG codon in the PET494 reading frame. The predicted PET494 protein has a highly basic amino terminal domain of 66 amino acids followed by a stretch of 32 uncharged residues, half of which are hydrophobic. The remainder of the protein is not unusual in amino acid composition or distribution except that the carboxyterminal region is notably basic. The phenotype of mutations generated in vitro around codon 119 by exonuclease digestion and linker insertion indicated that this region is dispensable for function. A mutation caused by deletion of 101 bp of coding sequence behaved like a simple frameshift when inserted into the chromosome: it was partially suppressed by the recessive non-group specific frameshift suppressor suf13 and reverted to Pet+ phenotype by mutations linked to PET494. PMID- 3010055 TI - Ovarian function during oestrogen-progestin replacement treatment in pre menopausal women. AB - Thirty-five pre-menopausal women were treated for 3 mth with sequential combinations containing oestradiol valerate and levonorgestrel in two different doses. Three treatment cycles, and 2 cycles before and 2 after treatment were studied. Subjective symptoms were relieved in 62% of women during treatment. Normal secretory phase was observed in 25-53% before and 33-35% at the end of treatment. Serum FSH and the frequency of serum progesterone increase greater than 5 nmol/1 decreased, particularly during treatment with the higher dosage. Ovulations occurred during both treatments; these combinations cannot therefore be used for contraceptive purposes. In ultrasonic studies, the average volume of ovaries in which no follicles were found, was 7.6 cm3. The volume of ovaries with follicles was 10.5 cm3. The follicular growth was often defective, also in ovulatory cycles, in both treatment and control cycles. In ovulatory cycles, luteinized unruptured follicles occurred in 2% before treatment and in 5% during treatment. In anovulatory cycles, persistent follicles were observed in 13% before treatment and in 19% during treatment. The frequency of luteinized unruptured follicle (LUF) syndrome did not increase significantly in pre menopausal women during oestrogen-progestin treatment. However, persistent follicles were frequent during anovulatory cycles. Serum oestradiol concentrations correlated significantly with the diameter of the dominant follicle. No significant changes were found in uterine volume. Both of the tested regimens are useful for treatment of pre-menopausal symptoms. PMID- 3010056 TI - Binding of horse-spleen ferritin to group A streptococci. AB - Receptors for horse-spleen ferritin were found on group A streptococci. Both electron microscopic and chemical investigation of Streptococcus cells treated with ferritin showed that M + variants of group A streptococci were able to bind substantially more ferritin than M - variants of the same serotypes. Ferritin receptors were located on the tops of filamentous protrusions of Streptococcus cell walls and only on the outer surface of isolated cell walls. Trypsin treatment destroyed the ferritin-binding capacity of streptococci completely, while mild pepsin treatment left the ferritin receptors undisturbed, or uncovered additional ones. The ferritin receptors were not identical with receptors for the Fc-portion of swine IgG. The finding of ferritin receptors on bacteria necessitates careful interpretation of results obtained by immunoferritin localization techniques. PMID- 3010057 TI - Infantile autism: a dysfunction of the opioids? AB - We postulate that infantile autism may be caused by a disorder of the endogenous opioid system. Evidence is provided linking the features of this condition with opiatergic dysfunction. PMID- 3010059 TI - Evolutionary theory, regeneration and cancer. AB - Three central problems for biology that remain unsolved are: a complete understanding of macroevolution, the problem of morphogenesis, i.e., the regulation of differentiation during development (related to this is the problem of regeneration), and the sequential maladaptation of these developmental processes, neoplasia. Combined breakthroughs in molecular cytogenetics concerning the "transposable elements" in the evolving genone, and the cellular transforming genes ("oncogenes") implicated in certain neoplastic diseases, add dimension to the discussion about the occurrence of "fetal" or anestral products of anaplastic cells. It is theorized that hormones are a major factor in the non-random regulation of cellular heterochrony in tumourigenesis. What is offered here is a theoretical overview of some current trends in the evolutionary sciences. Some ideas (old and new) on regeneration research will be discussed, and an attempt will be made to integrate those notions underlying hormonal oncology which suggest that there is a common thread woven through these problems, creating the fabric of biological change. PMID- 3010058 TI - Hypertension and diabetes in obesity: a review and new ideas on the contributing role of ions. AB - Ion metabolism in obesity-associated hypertension is reviewed. A hypothesis is presented which proposes that ion imbalances in obesity may play an etiological role in obesity-associated diabetes mellitus as well. It is suggested that the rise in intracellular calcium--secondary to reduced sodium, potassium-activated adenosine triphosphatase (Na,K-ATPase) activity--may aid in the development of increased vascular tone and decreased glucose tolerance. PMID- 3010060 TI - An integrated perception of autoimmunity and cancer. PMID- 3010062 TI - Can juvenile scoliosis be corrected by circumscribed radium-irradiation of the spine? AB - In 1936 scoliosis was produced in growing animals by exposing one side of one or more vertebrae to radium action. The purpose of this treatment was to recurve existing curvatures of the spine on the same principle. No attempt has been made so far to correct congenital scoliosis of infants by this method. Hundreds of infants treated by X-ray for Wilms' tumor developed incidental spinal curvatures. In none of these cases was there any recorded mention of damage to the spinal cord or any other organ. Since the epiphyseal plate is much more sensitive to radiation than is the Wilms' tumor and radium radiation can be more easily controlled than X-radiation, it is suggested that scoliosis of genetic or other etiology might be successfully treated by radium or possibly other gamma rays. The choice of the rays and the dosage must be left to the experts. PMID- 3010061 TI - The action of fluoride in teeth and bone. AB - The beneficial effect of fluoride to tooth enamel and its potentially harmful effect on bone, may be explained by simple and similar mechanisms. In individuals whose skeletal tissues contain higher than normal levels of fluoride there is a possibility that during resorptive and remodeling processes, bone (and bone marrow) cells may be exposed to genotoxic and lethal levels of fluoride. The success of fluoride as a preventive against dental caries does not mean that unnecessary exposure to the element should be tolerated. Total daily fluoride intake from a multiplicity of possible everyday sources should be monitored; and the assumption that sodium fluoride is safe to use as an anti-caries agent, particularly for expectant mothers and children, should be reviewed. PMID- 3010064 TI - Enalapril for hypertension. PMID- 3010063 TI - GABA and circadian timekeeping: implications for manic-depression and sleep disorders. AB - Circadian rhythms, evident in a wide variety of physiological and behavioural parameters, are under the control of central neural pacemakers, the best characterized of which is the suprachiasmatic nucleus of the hypothalamus. The neurophysiological mechanisms involved in central pacemaker function are unknown. Recent biochemical, pharmacological and behavioural evidence suggests that the inhibitory transmitter gamma-aminobutyric acid (GABA), present in the small interneurones of the suprachiasmatic nucleus, plays an important role in circadian timekeeping. This has enabled the formulation of strategies for treatment of patients with manic depressive illness and certain sleep disorders in which disorders of circadian timekeeping may be fundamental. PMID- 3010065 TI - A prototype epithermal neutron beam for boron neutron capture therapy. AB - An epithermal neutron beam has been designed and tested at the Georgia Institute of Technology's 5-MW Research Reactor. The prototype facility consists of aluminum and sulfur disks in a tangential beam port for fast neutron filtration. A cadmium sheet at the port exit removes the thermal neutrons from the transmitted beam, leaving an intensely epithermal neutron beam spanning five energy decades, each contributing to the flux demanded by boron neutron capture therapy. The thermal neutron flux generated by the incident epithermal neutrons in a polyethylene head phantom peaks at a depth of 3 cm and remains above the incident thermal flux to a 7-cm depth. The beam thus provides the penetration required for treating deep-seated gliomas. Photon contamination in the prototype facility is high, and a number of basic modifications are proposed for reducing it to safer levels. PMID- 3010067 TI - Minnesota's Nurses' Week: "Choose nursing". PMID- 3010066 TI - The propagation of relativistic heavy ions in multielement beam lines. AB - We describe calculations of the energy loss, range, stopping power, multiple scattering, and other related properties of a high-energy heavy-ion beam at any one of a set of beam line elements. A beam line element (e.g., any beam modification, detection, or control device) is characterized by its thickness, areal density, aperture, and function. The loss of multiply scattered particles to any finite-aperture detector is calculated in the small-angle approximation, and the position of the Bragg peak, as given by particles stopping in the second of two ionization chambers used for Bragg curve measurements, is estimated. A general purpose computer program, PROPAGATE, has been written to allow addition, deletion, and modification of the beam line elements used in the calculation and to provide a convenient means of repeating such calculations for arbitrary beam lines. Calculations and experimental measurements are compared and found to be in satisfactory agreement. PMID- 3010068 TI - National Nurses' Day: "Choose a nurse". PMID- 3010069 TI - Aftercare: a patient-centered model of advocacy for relocated mental health patients. PMID- 3010070 TI - Human T-lymphotropic virus type III/lymphadenopathy-associated virus antibody testing at alternate sites. PMID- 3010071 TI - Prevention and control of influenza. PMID- 3010072 TI - Classification system for human T-lymphotropic virus type III/lymphadenopathy associated virus infections. PMID- 3010073 TI - Characterization of alpha 1-adrenergic receptor subtypes in rat brain: a reevaluation of [3H]WB4104 and [3H]prazosin binding. AB - [3H]Prazosin and [3H]WB4101 [2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4 benzodioxane] have both been proposed to label alpha 1-adrenergic receptors in the rat central nervous system. As many discrepancies between the binding of these two ligands have arisen, we conducted these studies in order to reevaluate their binding characteristics and resolve the similarities and differences in the pharmacological characteristics of their respective binding sites. [3H]Prazosin binding is characterized by a monophasic saturation isotherm. Prazosin, indoramine, and dihydroergocryptine competitions with [3H]prazosin are steep and monophasic, and model best to a single binding site. In contrast, phentolamine and WB4101 competition curves are shallow in rat cortex, exhibiting Hill coefficients significantly less than 1.0, and model to two binding sites of approximately equal proportions. The higher and lower affinity components are defined as alpha 1A and alpha 1B, respectively. [3H]WB4101 also labels two binding sites in rat cortex and hippocampus with picomolar and nanomolar affinity, respectively. However, the nanomolar binding site is serotonergic and not adrenergic. The picomolar site (KD = 150 pm) has characteristics of an alpha 1-receptor binding site: prazosin, WB4101, and phentolamine affinities for this [3H]WB4101 binding site correlate with their affinities for the highest affinity component (alpha 1A) of [3H]prazosin binding. In addition, the Bmax of this [3H] WB4101-labeled site is equal to one-half of the total [3H]prazosin Bmax. Agonist competitions with [3H]prazosin binding are multiphasic with pseudo-Hill slopes less than 1.0 and with a rank order of affinity of epinephrine greater than norepinephrine greater than phenylephrine. When binding to the alpha 1A component is blocked by a 30 nM phentolamine mask, the same rank order of agonist affinities is preserved. Although the affinities of epinephrine and norepinephrine at the two subtypes are identical, phenylephrine is weaker at the alpha 1B site. The ratio of the potency of phentolamine versus prazosin is about 4 at the alpha 1A component but about 80 at the alpha 1B binding site. We discuss these data in relation to the reported potencies of these antagonists in blocking alpha 1-receptor-mediated responses which may correlate with our designation of alpha 1A or alpha 1B binding sites. PMID- 3010074 TI - Characterization of the A2 adenosine receptor labeled by [3H]NECA in rat striatal membranes. AB - [3H]NECA (1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuronamide) is known to bind to both the A1 and A2 subtypes of adenosine receptor in rat striatal membranes. In order to study the putative A2 component of [3H]NECA binding, we examined several compounds for the ability to selectively eliminate the A1 component of binding; N6-cyclopentyladenosine was found to give the most satisfactory results. Binding of [3H]NECA in the presence of 50 nM N6 cyclopentyladenosine was characterized. The rank order of potency for inhibition of [3H]NECA binding was NECA much greater than 2-chloroadenosine greater than N6 [(R)-1-methyl-2-phenyl-ethyl]adenosine (R-PIA) greater than N6 cyclohexyladenosine greater than S-PIA, indicating that binding was to an A2 adenosine receptor. When affinities of compounds in [3H]NECA binding to A2 receptors were compared to their affinities in [3H]N6-cyclohexyladenosine binding to A1 receptors, N6-cyclopentyladenosine was the most A1-sensitive agonist (A1 Ki, 0.59 nM; A2 Ki, 460 nM; Ki ratio, 780), whereas the selective coronary vasodilator 2-(phenylamino)adenosine was the most A2-selective agonist (A1, 560 nM; A2, 120 nM; ratio, 0.21). The antagonist 8-cyclopentyltheophylline had considerable A1 selectivity (A1, 11 nM; A2, 1400 nM; ratio, 130), whereas alloxazine had slight A2 selectivity (A1, 5200 nM; A2, 2700; ratio, 0.52). [3H]NECA binding to A2 receptors was highest in striatum but was detectable at much lower levels in each of seven other brain areas. The regional distribution of [3H]NECA binding and the affinities of adenosine agonists and antagonists for inhibition of binding indicate that the site labeled by [3H]NECA belongs to the high affinity, or A2a, subclass of A2 receptor. PMID- 3010076 TI - Paraquat uptake into freshly isolated rabbit lung epithelial cells and its reduction to the paraquat radical under anaerobic conditions. AB - Uptake of paraquat (PQ; 10 microM) into lung cell fractions enriched in alveolar type II cells or Clara cells was linear with time, and after 60 min the intracellular concentration was approximately 10-fold higher than that in the medium. In contrast, alveolar macrophages were not able to accumulate PQ from the extracellular medium. PQ uptake in preparations of type II and Clara cells, but not alveolar macrophages, was inhibited by an equimolar concentration of putrescine or spermidine and by a combination of the metabolic inhibitors, potassium cyanide and iodoacetate (1 mM each). The reduction of PQ (1 mM) under anaerobic conditions was investigated in lung cells by ESR spectroscopy. The amplitude of the ESR signal of the PQ radical increased with time with intact or sonicated type II and Clara cell preparations, but with macrophages it increased only when the cells were sonicated. The signal in sonicated cells but not whole cells was decreased by addition of antibodies to NADPH-cytochrome P-450 reductase, suggesting that the PQ radical is generated intracellularly under these conditions. PMID- 3010075 TI - Forskolin enhances calcium-evoked prolactin release from 7315c tumor cells without increasing the cytosolic calcium concentration. AB - The 7315c prolactin-secreting tumor cell was used as a model of a normal pituitary cell in order to study the enhancement by adenosine 3',5'-cyclic monophosphate (cAMP) of calcium-evoked hormone release. Forskolin and, by implication, cAMP had little effect on basal hormone release during a 10-min incubation period. Ionomycin and a high potassium concentration, treatments which enhanced the cytosolic calcium concentration, increased hormone release. When cells were exposed to forskolin prior to and during a challenge with either ionomycin or high potassium, a synergistic effect on prolactin release was observed. 8-Bromoadenosine 3',5'-cyclic monophosphate mimicked forskolin in enhancing ionomycin-evoked prolactin release while having little effect of its own on hormone release. Forskolin did not alter the increase in cytosolic calcium concentration elicited by either ionomycin or high potassium, nor did it increase the potency of ionomycin in enhancing prolactin release. The calcium channel antagonist, D-600, did not alter ionomycin-induced release or its enhancement by forskolin; D-600 blocked potassium-induced prolactin release. Ionomycin had no effect on basal cAMP synthesis by tumor cells and inhibited slightly the forskolin-induced increase in nucleotide synthesis. The results suggest that cAMP acts, at a site distal to the entry of calcium into the cytosol, to enhance the amount of prolactin released in response to an increase in the cytosolic calcium concentration. PMID- 3010077 TI - Neurotensin-mediated inhibition of cyclic AMP formation in neuroblastoma N1E115 cells: involvement of the inhibitory GTP-binding component of adenylate cyclase. AB - The tridecapeptide, neurotensin, inhibited prostaglandin E1-stimulated cyclic AMP production in intact plated neuroblastoma N1E115 cells. The peptide effect was concentration dependent (EC50 = 2 nM) and maximal inhibition reached 55% with 100 nM neurotensin. Acetyl neurotensin (8-13) was as active as neurotensin whereas neurotensins (1-8), (1-12), and (10-13) were barely active in inhibiting cyclic AMP production, thus showing the requirement of the carboxy terminal hexapeptide sequence of neurotensin for biological activity. The inhibitory effect of neurotensin on cyclic AMP production was largely prevented by pretreatment of N1E115 cells with islet-activating protein (pertussis toxin). In contrast, pertussis toxin did not inhibit neurotensin-stimulated cyclic GMP production in neuroblastoma cells. In cell membranes, the toxin promoted the selective ADP ribosylation of a single protein having the same molecular weight (41,000) as the alpha-subunit of Ni, the inhibitory regulatory protein of adenylate cyclase. In membranes prepared from N1E115 cells, monoiodo[125I-Tyr3]neurotensin bound to a single population of receptors characterized, at 25 degrees and in the absence of monovalent cations and guanyl nucleotides, by a dissociation constant (Kd) of 56 pM and a maximal binding capacity (Bm) of 30 fmol/mg of protein. Na+ (10-100 mM) and GTP (0.1-100 microM) inhibited neurotensin binding in a concentration dependent manner. At 100 mM Na+ and 100 microM GTP, receptor affinity was decreased by 5- and 2-fold, respectively. Li+ and K+ were less effective than Na+, and the effect of GTP was shared by GDP and guanyl-5'-yl-imidodiphosphate, but not by GMP, ATP, ADP, or adenyl-5'-yl-imidodiphosphate. It is concluded that in N1E115 cells, neurotensin attenuates cyclic AMP production by exerting an inhibitory effect on adenylate cyclase through an interaction of the peptide receptors with the regulatory GTP-binding protein Ni. PMID- 3010078 TI - gamma-Aminobutyric acid agonists and antagonists alter chloride flux across brain membranes. AB - gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, increases membrane chloride conductance. Previously, we reported that GABA increases 36Cl- uptake by membrane vesicles (microsacs) prepared from mouse brain. Employing this technique, we found that the GABAA agonists, muscimol, isoguvacine, 4,5,6,7-tetrahydroisoxazolo(5,4-C)pyridine-3-ol, and 3 amino-1-propane sulfonate, all produced a concentration-dependent increase in 36Cl- influx, but baclofen, a GABAB agonist, failed to alter 36Cl- flux. Inhibition of GABA-dependent 36Cl- influx was produced by the convulsant drugs, bicuculline, picrotoxin, and pentylenetetrazole. Ion specificity was demonstrated by a failure of GABA agonists to stimulate influx of 45Ca2+, 86Rb+, 22Na+, or 35SO4(2). GABA-stimulated uptake of 36Cl- was largest in cortex and cerebellum and smaller in hippocampus and striatum. There was little difference in sensitivity to GABA among the areas. Analysis of subcellular fractions prepared from mouse brain demonstrated that the GABA-dependent 36Cl- influx was enriched in the synaptosomal fraction. The nonspecific (GABA-independent) uptake of 36Cl- was enriched in the myelin fraction. These experiments provide evidence for a functional coupling among GABA receptors and the chloride ionophore and suggest that the GABA-activated chloride channel is a site of action for several convulsant compounds. PMID- 3010079 TI - Isolation and characterization of bovine cardiac muscle cGMP-inhibited phosphodiesterase: a receptor for new cardiotonic drugs. AB - We have identified and highly purified a "low Km" cAMP phosphodiesterase from bovine cardiac muscle. This phosphodiesterase was inhibited by low concentrations of cGMP and has, therefore, been temporarily designated as cGMP-inhibited phosphodiesterase. After a 16,000-fold increase in specific activity, the highly purified enzyme had a specific activity of 6 mumol/min-mg and contained three major polypeptides. Initial data indicated that all of these polypeptides were derived from a single common precursor by proteolysis. We used this enzyme preparation to generate polyclonal antisera and monoclonal antibodies directed against the "low Km" phosphodiesterase. Immunoadsorption and immunoblot analysis allowed us to identify and isolate several molecular weight species of phosphodiesterase, including a larger form than previously reported for any purified low Km phosphodiesterase. This large form of the enzyme had a subunit molecular weight of approximately 110,000 and was the only one seen in fresh extracts of cardiac muscle. Full catalytic activity was recovered in the phosphodiesterase-antibody complex and enzyme prepared by immunoprecipitation exhibited Michaelis-Menten kinetics for cAMP hydrolysis and for inhibition by cGMP. The Km for cAMP hydrolysis was 0.15 microM and the Ki for cGMP inhibition of cAMP hydrolysis was 0.06 microM. This immunoprecipitation approach also allowed us to determine that the enzyme was phosphorylated on serine residues by cAMP-dependent protein kinase, and that the low Km, cGMP-inhibited phosphodiesterase was selectively inhibited by several new cardiotonic agents. Milrinone, amrinone, and fenoximone were highly selective inhibitors of this isozyme, and the relative affinities of these inhibitors were consistent with their order of potency as positive inotropic agents. These studies suggest that the cGMP-inhibited phosphodiesterase is a receptor for several new cardiotonic drugs. PMID- 3010080 TI - [Principles of integration of cell metabolism]. AB - The notion of the "primary blocks" of cellular metabolism (designated as "metabolic system") has been introduced. Metabolic system is defined as a metabolic pathway which corresponds to the really existing multienzyme complex. The complex of glycolytic enzymes which catalyzes the anaerobic reduction of glucose-6-phosphate with production of ATP may serve as an example of metabolic system (this complex does not contain hexokinase). The complex is formed on thin filaments of I-band of the muscle fibers or on dimers of band 3 protein embedded in the erythrocyte membranes. The fixation of the multienzyme complex to the support of biological nature provides the material basis for regulation of the metabolic system by chemical signals produced by the higher levels of metabolic control. Owing to interaction with anchor protein of the support the chemical signals exert the general control of functioning the multienzyme complex (switching on--switching-off of the metabolic system). It is assumed that the glycolytic system in skeletal muscles is stimulated by Ca2+ ions which interact with the anchor protein of the support (troponin C). PMID- 3010081 TI - [A preparation of short DNA chains synthesized in vivo after introduction into DNA from live cells of trioxsalen crosslinks is not enriched by replication initiation regions]. AB - The experiments undertaken in order to verify the trioxsalen-crosslinking method suggested by Russev and Vassilev for isolation of eukaryotic replication origins are described. It was found that the preparation of viral DNA isolated by the above mentioned method from CV-1 cells lytically infected with SV40 was not enriched in sequences including SV40 replication origin. The hybridization pattern of DNA preparation isolated by the trioxsalen-crosslinking procedure from chicken erythroblastosis cells with the cloned fragments of globin gene domain was found to be identical to those of the total DNA probe. The DNA fraction enriched in replication origins was isolated from the same cells with the aid of nascent DNA strand extrusion method by Zannis-Hadjopoulos et al. The hybridization pattern of this DNA fraction with the cloned fragments of chicken alpha-globin gene domain was different from those of total DNA. Taking together, the results of our experiments demonstrate that trioxsalen-crosslinking procedure does not lead to the isolation of replication origins from the objects studied in the present investigation. PMID- 3010082 TI - [Spatial organization of replicons in the eukaryotic nucleus: attachment of replication initiation regions to the nuclear skeleton]. AB - The DNA fraction enriched in replication origins was isolated from chicken erythroblastosis cells with the aid of nascent DNA strand extrusion method. In renaturation experiments, the sequence specificity of this DNA fraction (oriDNA) was compared to the sequence specificity of the nuclear matrix DNA. The oriDNA was shown to comprise a specific subset of unique genes all of which being represented also in the nuclear matrix DNA. The same unique DNA sequences were found to be depleted from the oriDNA and from the nuclear matrix DNA. A conclusion has been drawn that replication origins are located at the basements of DNA loops. The putative positions of replication origins within the domain of chicken alpha globin genes were mapped by hybridization experiments and were found to coincide with the positions of stable (not depending on the transcriptional state of globin genes) sites of DNA attachment to the nuclear skeleton. PMID- 3010083 TI - [Changes in the contractibility of the cytochrome c globule during redox transition]. AB - Changes of the volume and compressibility of cytochrome c molecule in solution during red-ox transition were investigated using differential measurements of density and ultrasound velocity. Small changes were obtained: intrinsic compressibility of the globule increases by (2.5 +/- 1)% and intrinsic volume increases by not more than 0.2%. The results are in contradiction with the recently reported data of Eden et al. claiming that oxidation of the protein is accompanied by a large, of about 40%, increase of compressibility. The validity of our results is verified by three different methods; by comparison of independently measured absolute values of apparent volumes and compressibilities of the oxidized and reduced protein (i); by differential densimetric and ultrasound velocimetric titrations of oxidized cytochrome with ascorbate (ii) and of reduced cytochrome with ferricyanide (iii). The obtained data lead to the conclusion that oxidation-induced-changes of the root mean square amplitude for intramolecular motion of atoms of cytochrome c globule is really 50-fold less than that estimated from X-ray data. PMID- 3010084 TI - [Evolutionary variants of the mos gene]. AB - The occurence of members of mos oncogene family in the vertebrates genome has been studied with the help of highly labeled single-stranded DNA probes. These included subgenic v-mos clones as well as the unique sequence--specific recombinants from mos-related human locus gp5 and the K51 locus from rat genome. The probe from gp5 (mos pseudogene) interacts only with DNA of primates and of rodents. On the other hand, mos gene and the gene from K51 locus are present in all vertberates tested. Recent duplication of the main mos gene in Artiodactyla and Perissodactyla orders of mammals was identified. The persistence of K51 and mos genes during evolution indicates their importance. The segregation of three mos-related genes in human-hamster hybrids points to their location on different human chromosomes. PMID- 3010085 TI - [EBV diseases]. AB - Epstein-Barr virus (EBV) infection in early childhood is usually asymptomatic or associated with symptoms common to many other viral infections, such as pharyngitis, bronchitis or enteritis. Typical infectious mononucleosis, as seen in young adult patients, is rare in early childhood. There is also a lower rate of heterophil antibody responses in young children. Many complications of the infection are known, and many atypical manifestations can be identified as EBV associated using modern EBV-specific serological methods. The problems of EBV infection during gestation and the neonatal period are described and the chronic and persistent infections, as well as the lymphoproliferative syndromes associated with EBV infection, are particularly taken into consideration. PMID- 3010086 TI - [Antiviral chemotherapy]. AB - After a discussion of the principles of antiviral chemotherapy, treatment and chemoprophylaxis of the following virus infections are reviewed in detail: the various manifestations of herpes simplex virus infections, varicella-zoster, cytomegalovirus infections, Epstein-Barr virus infections, laryngeal papillomas, and influenza A. Special reference is made to the treatment of immunocompromized patients. Acycloguanosine (acyclovir) has been found particularly useful in the treatment of herpes simplex virus and varicella zoster virus infections in immunocompromized patients and for herpesencephalitis. Varicella-zoster can also be treated effectively with bromovinyldeoxyuridine (BVDU). Toxicity of the currently used antiviral drugs is discussed as well as the problem of drug resistance. PMID- 3010087 TI - Effects of endogenous and exogenous opioids on splenic natural killer cell activity. AB - Previous work from our own and other laboratories has shown that electroshock induced neurohormonal changes in rodents could modify host-tumor interactions by both increasing the frequency and growth rate of transplanted tumors and decreasing the elimination rate of a radiolabelled natural killer (NK) cell sensitive tumor. To test whether such neurohormonal changes could affect NK activity we subjected mice to tail electrode shock (TES) and examined in vitro splenic NK activity. We found that between 30 and 60 min after TES there is a significant but transient suppression of their splenic NK activity. To determine whether TES-induced endogenous opioids might be involved in this suppression mice were given intraperitoneal injections of the opioid antagonists naloxone or naltrexone before or at the end of the TES session. These drugs prevented NK suppression. In a further test of the hypothesis that opioids alter NK activity mice were given a single intraperitoneal injection of morphine or [D-Ala2-Met5] beta-endorphin, a relatively stable analogue of beta-endorphin, an endogenous opioid. Contrary to expectations these opioids enhanced splenic NK activity. Our interpretation of these results is that shock-induced NK suppression may not be mediated by endogenous opioids and that the effects of naloxone and naltrexone on NK activity may not be related to their opioid antagonist properties. On the contrary, opioids may participate in a homeostatic rebound from suppression mediated by other neurohormonal mechanisms activated during TES. PMID- 3010088 TI - The macrocellular (large cell) carcinomas of the lung. A histopathological analysis of 880 diagnosed cases. PMID- 3010089 TI - Apocrine carcinoma of the breast. PMID- 3010090 TI - Morphoclinical considerations on a primitive pure ovarian carcinoid. PMID- 3010091 TI - Synovial sarcoma. PMID- 3010092 TI - Morphobiochemical correlations in experimental acute intoxication with Dinocton. PMID- 3010093 TI - Inclusion body myositis: case reports and a reappraisal of an underrecognized type of inflammatory myopathy. PMID- 3010094 TI - Rat and hamster hepatocyte-mediated induction of SCEs and mutation in V79 cells and mutation of salmonella by aminofluorene and dimethylnitrosamine. AB - Aminofluorene (AF) and dimethylnitrosamine (DMN) were examined for their ability to induce multiple genetic endpoints after rat and hamster hepatocyte metabolic activation. The endpoints measured included mutations at the Na+/K+-ATPase (ouabain resistance) and hypoxanthine-guanine-phosphoribosyltransferase (6 thioguanine resistance) loci, and sister-chromatid exchanges (SCEs) in Chinese hamster V79 cells, and mutation of Salmonella typhimurium strains TA98 and TA100. AF, with rat and hamster hepatocyte activation, induced only low levels of mutations at either loci in V79 cells but did induce SCEs. Mutation of Salmonella by AF after hepatocyte activation also occurred and was a sensitive endpoint for detecting this aromatic amine. DMN induced high levels of mutations at both loci in V79 cells in addition to SCEs in the presence of hepatocytes from both species. DMN was also mutagenic to Salmonella, but only with hamster hepatocytes. Salmonella did not respond as strongly to DMN as the V70 cells. Hamster hepatocytes were more active than rat hepatocytes in activating both carcinogens. The results indicate the variable sensitivity of the genetic endpoints and species differences in activation for two potent chemical carcinogens. PMID- 3010095 TI - Lethal and mutagenic effects of radiation and alkylating agents on two strains of mouse L5178Y cells. AB - In previous studies, the two closely related strains of L5178Y (LY) mouse lymphoma cells, LY-R and LY-S, have been shown to differ in their sensitivity to UV and ionizing radiation. Thus, in comparison to strain LY-R, strain LY-S has been found to be more sensitive to the lethal effects of ionizing radiation, more resistant to the lethal effects of UV radiation, but less mutable at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus by both UV and X radiation. In the present work, the lethal and mutagenic effects of ethyl methanesulfonate (EMS), methyl nitrosourea (MNU) and UV radiation (254 nm) were compared in the two strains. Mutability at the Na+/K+-ATPase locus as well as the HGPRT locus was determined. As previously reported, we found strain LY-S to be more resistant than strain LY-R to the lethal effects of UV radiation. In contrast, strain LY-S was more sensitive to the cytotoxic effects of the two alkylating agents. In spite of these differences in sensitivity, we found strain LY-S to be less mutable than strain LY-R by all 3 agents at the HGPRT locus. At the Na+/K+-ATPase locus, strain LY-S was also less mutable than strain LY-R by equal concentrations of EMS and UV radiation and by equitoxic concentrations of MNU. However, the difference between the strains was much more pronounced at the HGPRT locus than at the Na+/K+-ATPase locus. We have suggested that the interaction of unrepaired lesions in strain LY-S tends to cause an excess of deletions and multilocus effects, which in turn result in a locus-dependent decrease in the recovery of viable LY-S mutant cells. PMID- 3010096 TI - Isolation and partial characterization of virus-transformed cell lines representing the A, G and variant complementation groups of xeroderma pigmentosum. AB - We have established viral-transformed, apparently permanent (immortalized) cell lines from diploid fibroblasts representative of normal and xeroderma pigmentosum (XP) A, G and variant individuals. The XP-G and XP-variant cells represent complementation groups not previously available as permanent lines. All the new permanent cell lines exhibit SV40 T-antigen expression. They are also aneuploid and have growth characteristics typical of viral transformants. They have retained the phenotypes of UV sensitivity, reduced repair synthesis or defective 'postreplication repair' appropriate to the XP complementation group they represent. Additionally, the new cell lines are all transfectable with the selectable plasmid pRSVneo. The XP-G and XP-variant cell lines show enhanced transfection with UV-irradiated plasmid DNA; a phenomenon previously reported for normal immortalized cells and for immortalized cells from the A and F complementation groups of XP. PMID- 3010097 TI - A mammalian cell variant in which 3-aminobenzamide does not potentiate the cytotoxicity of dimethyl sulphate. AB - Variants of mouse leukaemia L1210 cells have been isolated in which cytotoxicity to dimethyl sulphate is not fully potentiated by ADP-ribosyl transferase inhibitor 3-aminobenzamide, as occurs in normal L1210 cells. These variants were selected after mutagenesis by growing the cells in dimethyl sulphate and 3 aminobenzamide. The characterisation of one of these variants is described. Variant 3 cells repair low doses of DNA damage in the presence of ADP-ribosyl transferase inhibitors. The Vmax of the ADP-ribosyl transferase enzyme in these cells is only increased 35% compared to normal wild-type L1210 cells. The basal DNA ligase I activity is increased 66% above wild-type whereas DNA ligase II activity appears to be unchanged. The most striking observation, however, is that the DNA ligase II activity is not increased after dimethyl sulphate treatment as occurs in wild-type L1210 cells. It seems that by increasing DNA ligase I levels these cells can survive DNA damage in the presence of 3-aminobenzamide. This variant (mutant) provides genetic evidence for our previously published hypothesis that (ADP-ribose)n biosynthesis is required for efficient DNA repair after DNA damage by monofunctional alkylating agents, because ADP-ribosyl transferase activity regulates DNA ligase activity. This variant is the first mammalian cell reported in which DNA ligase activity is altered, as far as we are aware. In yeast, a DNA ligase mutant has a cell division cycle (cdc) phenotype. Presumably, DNA ligase is essential for DNA synthesis, repair and recombination. The present variant provides further evidence that in mammalian cells, DNA ligase II activity is related to ADP-ribosyl transferase activity. PMID- 3010098 TI - Elevation of Alu I-induced frequencies of chromosomal aberrations in Chinese hamster ovary cells by Neurospora crassa endonuclease and by ammonium sulfate. AB - The frequencies of chromosomal aberrations induced by the restriction endonuclease Alu I (recognition site AG/CT) can be elevated to a similar extent by additional treatments with a single-strand-specific endonuclease from Neurospora crassa (EC 3.1.30.1), or with ammonium sulfate in which the Neurospora endonuclease is suspended. These data indicate that Alu I does not produce DNA single-strand breaks in the chromatin of living cells, which can be recognized by the Neurospora endonuclease. The salt may induce conformational changes in the chromatin which make more recognition sites available for Alu I. Experiments with recovery times between the treatments with Alu I and the salt indicate that Alu I can act in the nucleus for at least 40 min. PMID- 3010099 TI - The restriction endonuclease Alu I induces chromosomal aberrations and mutations in the hypoxanthine phosphoribosyltransferase locus, but not in the Na+/K+ ATPase locus in V79 hamster cells. AB - The restriction endonuclease Alu I induces chromosomal aberrations and mutations in the hypoxanthine phosphoribosyltransferase (HPRT) locus as measured by 6 thioguanine resistance (TGr) in V79 hamster cells. Alu I does not induce mutations in the Na+/K+ ATPase locus as measured by ouabain resistance (OUAr). The data are interpreted to mean that most if not all Alu I-induced TGr mutations represent chromosomal aberrations. PMID- 3010100 TI - Congenital myasthenia: further evidence of disease heterogeneity. AB - The findings in two cases of congenital myasthenia investigated by intercostal muscle biopsy are presented. The first case, a 16-year-old boy, showed reduced miniature endplate potential amplitude and normal 125I-alpha-bungarotoxin binding to postsynaptic acetylcholine receptors. Muscle biopsy and endplate ultrastructure were normal. Tubocurarine affinity, ion channel properties, and passive membrane properties were normal. Limited data showed reduced effectiveness of applied acetylcholine in opening ion channels. The second case was an 18-year-old girl with consanguineous parents. Type 2 muscle fiber atrophy was seen in both limb and intercostal muscle. Intercostal endplates were elongated, although ultrastructure was normal. Negligible postsynaptic alpha bungarotoxin binding suggested an abnormality of the acetylcholine receptor macromolecule. PMID- 3010101 TI - Isolation and characterization of sarcoplasmic reticulum from normal and dystrophic chicken. AB - Purified sarcoplasmic reticulum vesicles from normal and dystrophic chicken skeletal muscle have been isolated by a procedure employing pressure disruption of a microsomal suspension. The dystrophic sarcoplasmic reticulum (SR) exhibited reduced Ca++ transport and phosphoenzyme formation, but the Ca++-ATPase activity was normal. Normal and dystrophic SR showed similar lipid profiles, except for a significant increase in free fatty acids in the dystrophic SR. Investigations involving the interaction of oleic acid with normal SR showed fatty acids can induce conditions similar to those found in dystrophic SR. PMID- 3010104 TI - Pseudo-anterior interosseous nerve syndrome. PMID- 3010103 TI - Peripheral neuropathy following acute carbon monoxide poisoning. PMID- 3010102 TI - Targeting of lysosomal enzymes: N-acetylglucosamine-1-phosphotransferase during muscle development. AB - It has been previously shown by morphological techniques and measurements of lysosomal enzyme levels that the I cell mutation is expressed in myoblasts but not in myotubes or mature muscle fibers. These findings suggested the possibility of developmental regulation of the affected enzyme, UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-phosphotransferase. In this article, we examine this possibility by measuring the phosphotransferase activity at various stages of muscle differentiation in three different animal species (human, chick, rat). Although activity of the enzyme is consistently higher in myoblasts than in myotubes or mature muscle, the difference in the levels of activity at these three states of muscle differentiation varies widely in the three species examined. We further found that the phosphotransferase activity was absent in the muscle of an I cell patient, in spite of normal muscle morphology. This indicates the presence of a mannose-6-phosphate-independent mechanism for lysosomal enzyme targeting in muscle and other unaffected tissues. The existence of such a pathway cannot be explained by lack of the necessary enzyme, as the phosphotransferase is present at a comparable level in normal muscle of three different species (human, chick, rat). PMID- 3010105 TI - Herpesvirus infections of pregnancy. PMID- 3010106 TI - Nifedipine for intractable hiccups. PMID- 3010107 TI - The regulation of hemostasis: the protein C system. PMID- 3010108 TI - Responses to corticotropin-releasing hormone in the hypercortisolism of depression and Cushing's disease. Pathophysiologic and diagnostic implications. AB - Primary depression can be associated with substantial hypercortisolism, thus prompting some researchers to suggest that depression shares pathophysiologic features with Cushing's disease. Clinically, depression can be difficult or impossible to distinguish from mild or early Cushing's disease that is associated with depressive features. The purpose of this study was to evaluate whether the pituitary-adrenal responses to ovine corticotropin-releasing hormone could help to clarify the mechanism of hypercortisolism in depression and in Cushing's disease and to assist in the differential diagnosis of these disorders. As compared with controls (n = 34), depressed patients (n = 30) had basal hypercortisolism (P less than 0.001) that was associated with attenuated plasma ACTH responses to ovine corticotropin-releasing hormone (P less than 0.001). This indicates that in patients with depression, the corticotroph cell in the pituitary responds appropriately to the negative feedback of high cortisol levels. In contrast, patients with Cushing's disease (n = 29) had plasma ACTH hyperresponsiveness to ovine corticotropin-releasing hormone (P less than 0.001), despite basal hypercortisolism (P less than 0.001), which indicates a gross impairment of the mechanism by which cortisol exerts negative feedback on the pituitary. Less than 25 percent of the patients with depression or Cushing's disease had peak ACTH responses that overlapped. We conclude that the pathophysiologic features of hypercortisolism in depression and Cushing's disease are distinct in each of the disorders and that the ovine corticotropin-releasing hormone stimulation test can be helpful in their differential diagnosis. PMID- 3010109 TI - Abnormal hypothalamic-pituitary-adrenal function in anorexia nervosa. Pathophysiologic mechanisms in underweight and weight-corrected patients. AB - To study the pathophysiology of hypercortisolism in patients with anorexia nervosa, we examined plasma ACTH and cortisol responses to ovine corticotropin releasing hormone before and after correction of weight loss. We also studied patients with bulimia whose weight was normal, since this disorder has been suspected to be a variant of anorexia nervosa. Before their weight loss was corrected, the anorexic patients had marked hypercortisolism but normal basal plasma ACTH. The hypercortisolism was associated with a marked reduction in the plasma ACTH response to corticotropin-releasing hormone. When these patients were studied three to four weeks after their body weight had been restored to normal, the hypercortisolism had resolved but the abnormal response to corticotropin releasing hormone remained unchanged. On the other hand, at least six months after correction of weight loss their responses were normal. The bulimic patients whose weight was normal also had a normal response to corticotropin-releasing hormone. We conclude that in underweight anorexics, the pituitary responds appropriately to corticotropin-releasing hormone, being restrained in its response by the elevated levels of cortisol. This suggests that hypercortisolism in anorexics reflects a defect at or above the hypothalamus. The return to eucortisolism soon after correction of the weight loss indicates resolution of this central defect despite persistence of abnormalities in adrenal function. PMID- 3010110 TI - Screening to reduce transmission of sexually transmitted diseases in semen used for artificial insemination. AB - The practice of artificial insemination by donor semen is increasing in the United States. Many sexually transmitted organisms are found in semen, but screening procedures for the detection of these agents in donor semen have not been standardized. Sexually transmitted organisms have been transmitted during artificial insemination by donor, and such transmission can cause local, disseminated, or fatal disease in the recipient woman and may harm the fetus or newborn. Therefore, screening of both the donor and the donated semen is necessary to avoid infectious complications. Because semen samples cannot be evaluated completely on the day of donation, the use of fresh semen for artificial insemination should be discouraged. Until accurate, rapid diagnostic tests are available, only frozen semen that has been appropriately screened should be used. PMID- 3010111 TI - Isolation of HTLV-III virus from saliva in AIDS. PMID- 3010112 TI - HTLV-III seropositivity in 1971-1972 parenteral drug abusers--a case of false positives or evidence of viral exposure? PMID- 3010114 TI - Acquisition of donor strains of cytomegalovirus by renal-transplant recipients. AB - To determine the source of post-transplantation cytomegalovirus (CMV) infection in renal-transplant recipients, viral isolates were collected from pairs of patients who received kidneys from the same cadaver. Among 36 pairs of recipients, CMV viruria or viremia occurred in both members of 4 pairs and in one member of 11 pairs. Restriction-enzyme analysis of viral DNA revealed 15 distinct strains of CMV among viral isolates from these 19 patients. In all four pairs in which both members shed CMV, both recipients shed the same strain, suggesting that the virus was of donor origin. In three of these pairs, one member had been seropositive for CMV before transplantation. One seropositive recipient was simultaneously shedding two strains of CMV after transplantation; one strain was of donor origin. Two patients who had CMV viruria before receiving grafts from a seropositive donor shed a different CMV strain two months after grafting. These findings indicate that cadaveric grafts can transmit an identifiable strain of CMV to recipients, and that seropositive recipients can be reinfected by a new CMV strain from the donor after transplantation. PMID- 3010113 TI - Increased rate of cytomegalovirus infection among parents of children attending day-care centers. AB - We screened parents of children from three previously studied day-care centers where children have maintained high rates of cytomegalovirus (CMV) excretion, as well as parents of children not in day-care centers (controls), for antibody to CMV. Longitudinal serologic follow-up of seronegative parents revealed that 14 of 67 with children in the day-care centers acquired CMV, as compared with none of 31 controls (P less than 0.003). All 14 parents who seroconverted had a child who was shedding CMV in saliva or urine. Among the day-care group, acquisition of CMV occurred in 14 of 46 parents of children who shed CMV, as compared with none of 21 whose children did not excrete the virus (P less than 0.0001). There was no correlation between parental seroconversion and sex, race, age, number of years of education, marital status, occupation, or number of children in the home. CMV infection occurred in 9 of 20 parents (45 percent) with a child shedding CMV who was 18 months of age or less at enrollment. We conclude that children often transmit CMV to parents and could be an important source of maternal CMV infection during pregnancy. PMID- 3010117 TI - AIDS vaccine and the private sector. PMID- 3010118 TI - Comparative analysis of total proteins, amino acids, carbohydrates, fibers, lipids, macro and micro minerals contents in different types of Egyptian bread. AB - Seven types of Egyptian bread were collected from different rural and urban areas. The chemical composition including total proteins, amino acids, carbohydrates, fibers, lipids, macro and micro minerals contents were determined. Total proteins content was higher in shamssy bread than other types of bread. Crude fat has the highest value in bread made from mixture of cereals, especially when trigonella is found, while fiber content was highest when mixture of cereals containing sorghum is used. Ash content of bread made from maize + wheat showed a higher value than other types and the differences were highly significant. Ca, Mg, Cu, and Fe were higher in bread made from sorghum + trigonella blend than other types of bread. Variations in the amino acids content of the different types of bread were found; these differences due to the origin of different cereals, the method of bread processing and the differences in the extraction rates of the flour. PMID- 3010119 TI - On the digestibility and utilization of labelled protein of feeds and foods rich in dietary fibres. PMID- 3010116 TI - Isolated failure of autonomic noradrenergic neurotransmission. Evidence for impaired beta-hydroxylation of dopamine. PMID- 3010115 TI - More on partitioning and inactivation of AIDS virus in immune globulin preparations. PMID- 3010120 TI - A case-control study of trophoblastic diseases in the People's Republic of China. AB - A case-control study is currently under way in Beijing, People's Republic of China, involving approximately 165 patients with invasive moles or choriocarcinoma, 165 with hydatidiform moles, and 330 population controls, who were matched to the patients with invasive moles or choriocarcinoma on age and interval since last pregnancy. The interviews are focused on a number of suspected risk factors, including previous pregnancy outcomes, history of hydatidiform mole, medical factors, drug usage, family history, and diet. A brief background of the study and methods as established through a previous pilot study are given. PMID- 3010122 TI - Risk factors for hepatocellular carcinoma in Guangxi, People's Republic of China. AB - Fusui County in the Guangxi Autonomous Region of China is a high-risk area for hepatocellular carcinoma (HCC). In 1971-73, the average annual standardized (world population) mortality rate of HCC in Fusui was 20.09 and 111.75/100,000 person-years for females and males, respectively. Epidemiologic and pathologic studies of HCC have been conducted in Fusui since 1959. This paper describes some of the studies relating to the hepatitis B virus infection and aflatoxin contamination of foodstuffs. PMID- 3010121 TI - T-cell lymphoma. AB - Of a total of 3,366 cases of malignant lymphoma in China reviewed histopathologically, T-cell lymphomas accounted for an average of 26.1% among all non-Hodgkin's lymphomas. However, the proportion of lymphomas of T-cell lineage varied in different parts of China, with the highest proportion (30-40%) along the east coast. Immunologic typing of 41 non-Hodgkin's lymphomas by the avidin biotin-peroxidase complex technique revealed similar patterns. When serum antibodies to human T-cell leukemia virus were assayed in 462 normal men and 103 monkeys, 2-5% of the men and 8.4-15% of the monkeys were positive for antibodies to this virus. PMID- 3010123 TI - AIDS research. French virus in the picture. PMID- 3010125 TI - Concerted activation of two potential proto-oncogenes in carcinomas induced by mouse mammary tumour virus. AB - The induction of tumours by retroviruses lacking transduced oncogenes can involve the transcriptional or functional activation of cellular proto-oncogenes by an integrated provirus. Thus, the two cellular genes int-1 and int-2, identified as common targets for activation by mouse mammary tumour virus (MMTV), may constitute previously unrecognized oncogenes. In tumours, proviral insertion at these loci leads to expression of messenger RNAs which are undetectable in normal mammary glands. Here we report that in a survey of the two transcriptional activity and structural integrity of the two int loci in 30 BR6 mouse mammary tumours, around 50% of the tumours expressed both of these genes, in ostensibly monoclonal cell populations. Our data suggest that int-1 and int-2 may act cooperatively in the genesis of mammary carcinomas. However, because three tumours (10%) involved neither gene, and because in five cases activation occurred in the apparent absence of an adjacent provirus, it is clear that other loci and mechanisms contribute to tumorigenesis. PMID- 3010124 TI - Cachectin and tumour necrosis factor as two sides of the same biological coin. AB - In response to invasive stimuli macrophages secrete cachectin, a multipotent protein. Prominent among its biological effects is the ability to induce wasting (cachexia) as well as a lethal state of shock. The identity of cachectin and tumour necrosis factor has led to a new view of its therapeutic potential. PMID- 3010126 TI - The inositol tris/tetrakisphosphate pathway--demonstration of Ins(1,4,5)P3 3 kinase activity in animal tissues. AB - Recent advances in our understanding of the role of inositides in cell signalling have led to the central hypothesis that a receptor-stimulated phosphodiesteratic hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) results in the formation of two second messengers, diacylglycerol and inositol 1,4,5 trisphosphate (Ins(1,4,5)P3). The existence of another pathway of inositide metabolism was first suggested by the discovery that a novel inositol trisphosphate, Ins(1,3,4)P3, is formed in stimulated tissues; the metabolic kinetics of Ins(1,3,4)P3 are entirely different from those of Ins(1,4,5)P3 (refs 6, 7). The probable route of formation of Ins(1,3,4)P3 was recently shown to be via a 5-dephosphorylation of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), a compound which is rapidly formed on muscarinic stimulation of brain slices, and which can be readily converted to Ins(1,3,4)P3 by a 5-phosphatase in red blood cell membranes. However, the source of Ins(1,3,4,5)P4 is unclear, and an attempt to detect a possible parent lipid, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), was unsuccessful. The recent discovery that the higher phosphorylated forms of inositol (InsP5 and InsP6) also exist in animal cells suggested that inositol phosphate kinases might not be confined to plant and avian tissues, and here we show that a variety of animal tissues contain an active and specific Ins(1,4,5)P3 3-kinase. We therefore suggest that an inositol tris/tetrakisphosphate pathway exists as an alternative route to the dephosphorylation of Ins(1,4,5)P3. The function of this novel pathway is unknown. PMID- 3010127 TI - The signal sequence of nascent preprolactin interacts with the 54K polypeptide of the signal recognition particle. AB - Hydrophobic signal sequences direct the translocation of nascent secretory proteins and many membrane proteins across the membrane of the endoplasmic reticulum. Initiation of this process involves the signal recognition particle (SRP), which consists of six polypeptide chains and a 7S RNA and interacts with ribosomes carrying nascent secretory polypeptide chains. In the case of aminoterminal, cleavable signal sequences, in the absence of microsomal membranes it exerts a site-specific translational arrest in vitro. The size of the arrested fragment (60-70 amino-acid residues) suggests that elongation stops when the signal sequence has emerged fully from the ribosome. However, a direct interaction between the signal sequence and SRP has not previously been demonstrated and has even been questioned recently. We now show for the first time a direct interaction between the signal sequence of a secretory protein and a component of SRP, the 45K polypeptide (relative molecular mass (Mr) 54,000). This was achieved by means of a new method of affinity labelling which involves the translational incorporation of an amino acid, carrying a photoreactive group, into nascent polypeptides. PMID- 3010128 TI - What to call the AIDS virus? PMID- 3010129 TI - Adrenergic receptors: some similarities and surprises. PMID- 3010130 TI - What's in a name? PMID- 3010133 TI - Epidermal growth factor-dependent phosphorylation of lipocortin. AB - Lipocortin-like proteins are a family of steroid-induced inhibitors of phospholipase activity with potential anti-inflammatory activity. Related proteins have been detected in a variety of tissues and species. The best characterized form is a protein of relative molecular mass (Mr) approximately 40,000 (40K), which is phosphorylated in vivo by protein tyrosine kinases and by protein serine-threonine kinases. It has been proposed that the phospholipase inhibitory activity of lipocortin can be regulated by its phosphorylation. In the A431 cell line, a protein of approximately 35K is phosphorylated by the protein tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Here we report that human lipocortin is phosphorylated near its amino terminus by the EGF receptor/kinase. By peptide mapping and immunological analyses, we show that lipocortin and the endogenous 35K substrate for the EGF receptor/kinase from A431 cells are the same protein. PMID- 3010131 TI - Virus nomenclature: controversy over AIDS virus extends to name. PMID- 3010134 TI - Ha-ras hypervariable alleles in myelodysplasia. AB - The somatic mutation of one of the ras oncogenes is now considered to be a critical step in the pathogenesis of many tumours. Circumstantial evidence also suggests that some individuals may be genetically predisposed to malignancy and a general method used to analyse such disease susceptibility is the study of restriction fragment length polymorphisms (RFLPs) at particular loci. The Harvey ras (Ha-ras) locus includes a hypervariable region (HVR) which consists of a series of 28-base-pair (bp) tandem repeats 3' to the gene. This arrangement gives rise to alleles of a wide range of sizes, making such genetic analysis possible. A previous study reported that white blood cell DNA from cancer patients frequently showed allelic restriction fragments at the Ha-ras locus which were found only rarely in normal unaffected individuals, and it was concluded that the inheritance of such unusual alleles may be linked to a susceptibility to cancer. As this conclusion has major implications we sought to investigate whether this association could be confirmed in patients with myelodysplasia, a common haematological malignancy reported to have the highest frequency of rare alleles. The Ha-ras alleles were characterized in normal healthy individuals and compared with those found in patients with myelodysplasia (MDS). Our results, reported here, show that the distribution of Ha-ras alleles in myelodysplastic patients is not significantly different from that in normal individuals. PMID- 3010132 TI - Cloning of the gene and cDNA for mammalian beta-adrenergic receptor and homology with rhodopsin. AB - The adenylate cyclase system, which consists of a catalytic moiety and regulatory guanine nucleotide-binding proteins, provides the effector mechanism for the intracellular actions of many hormones and drugs. The tissue specificity of the system is determined by the particular receptors that a cell expresses. Of the many receptors known to modulate adenylate cyclase activity, the best characterized and one of the most pharmacologically important is the beta adrenergic receptor (beta AR). The pharmacologically distinguishable subtypes of the beta-adrenergic receptor, beta 1 and beta 2 receptors, stimulate adenylate cyclase on binding specific catecholamines. Recently, the avian erythrocyte beta 1, the amphibian erythrocyte beta 2 and the mammalian lung beta 2 receptors have been purified to homogeneity and demonstrated to retain binding activity in detergent-solubilized form. Moreover, the beta-adrenergic receptor has been reconstituted with the other components of the adenylate cyclase system in vitro, thus making this hormone receptor particularly attractive for studies of the mechanism of receptor action. This situation is in contrast to that for the receptors for growth factors and insulin, where the primary biochemical effectors of receptor action are unknown. Here, we report the cloning of the gene and cDNA for the mammalian beta 2AR. Analysis of the amino-acid sequence predicted for the beta AR indicates significant amino-acid homology with bovine rhodopsin and suggests that, like rhodopsin, beta AR possesses multiple membrane-spanning regions. PMID- 3010135 TI - Good week for Pasteur institute. PMID- 3010136 TI - First isolation of HTLV-III. PMID- 3010137 TI - Potentiation of synaptic transmission in the hippocampus by phorbol esters. AB - Protein kinase C (PKC), a calcium-dependent phospholipid-sensitive kinase which is selectively activated by phorbol esters, is thought to play an important role in several cellular processes. In mammalian brain PKC is present in high concentrations and has been shown to phosphorylate several substrate phosphoproteins, one of which may be involved in the generation of long-term potentiation (LTP), a long-lasting increase in synaptic efficacy evoked by brief, high-frequency stimulation. Since the hippocampus contains one of the brain's highest levels of binding sites for phorbol esters and is the site where LTP has been most thoroughly characterized, we examined the effects of phorbol esters on hippocampal synaptic transmission and LTP. We found that phorbol esters profoundly potentiate excitatory synaptic transmission in the hippocampus in a manner that appears indistinguishable from LTP. Furthermore, after maximal synaptic enhancement by phorbol esters, LTP can no longer be elicited. Although the site of synaptic enhancement during LTP is not clearly established, phorbol esters appear to potentiate synaptic transmission by acting primarily at a presynaptic locus since changes in the postsynaptic responses to the putative transmitter, glutamate, cannot account for the increased synaptic responses induced by phorbol esters. These findings, in conjunction with previous biochemical studies, raise the possibility that, in mammalian brain, PKC plays a role in controlling the release of neurotransmitter and may be involved in the generation of LTP. PMID- 3010139 TI - The role of co-transported sodium in the effect of indirectly acting sympathomimetic amines. AB - The adrenergic nerve endings of vasa deferentia of either untreated or reserpine (R) and/or pargyline (P) pretreated rats were loaded with 3H-noradrenaline; COMT was inhibited by U-0521 (U). After 100 min of wash-out with Ca2+-free solution, the efflux of tritium (and of 3H-noradrenaline) from the tissue was largely of neuronal origin and remained constant with time (when expressed as fractional rate of loss; FRL). After 110 min of wash-out the effect of inhibition of the Na+,K+-ATPase (by low K+ or ouabain) on basal and on sympathomimetic amine induced efflux of tritium (or 3H-noradrenaline, under the condition U) was studied in paired experiments. Inhibition of the Na+,K+-ATPase caused a time dependent increase in the efflux of tritium (or 3H-noradrenaline) which was inhibited by desipramine. Inhibition of the Na+,K+-ATPase also caused a time dependent reduction of the initial rate of neuronal uptake of 3H-noradrenaline. The effectiveness of the sympathomimetic amines tyramine and amphetamine in inducing "release" (i.e., outward-transport) of noradrenaline depended on the experimental condition: it was most pronounced under the condition RPU, followed by the condition PU and lowest under the condition U (i.e., in tissue of untreated rats). Inhibition of the Na+,K+-ATPase caused an early and transient enhancement of the "release" of noradrenaline induced by tyramine or amphetamine. This enhancement was seen already within the first min after inhibition of the ATPase, i.e., before a pronounced inhibition of uptake (of noradrenaline) and before a pronounced increase of the basal efflux was observed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010138 TI - Rudolf Buchheim lecture. Drugs as tools in research on adrenoceptors. PMID- 3010140 TI - Reserpine-induced depletion of neuropeptide Y from cardiovascular nerves and adrenal gland due to enhanced release. AB - The mechanisms underlying reserpine-induced depletion of neuropeptide Y-like immunoreactivity (NPY-LI) in relation to tissue content of noradrenaline (NA) and cardiovascular impairment were studied in guinea-pigs. Reserpine pretreatment (5 mg/kg SC) caused a 5-fold increase in plasma levels of NPY-LI with a maximum after 4 h. This was associated with a progressive fall in systemic arterial blood pressure and heart rate (to about 50% of basal values). The contents of NPY-LI and NA in nerves of the heart and quadriceps muscle were then reduced by about 75% and 85%, respectively. The adrenal content of NPY-LI was reduced by 40% 8 h after reserpine, while the adrenaline content was uninfluenced. Pretreatment with guanethidine depleted NA in the heart but did not influence plasma levels or tissue content of NPY-LI per se. The reserpine-induced increase in plasma NPY-LI and the depletion of NPY-LI in the heart and skeletal muscle was to a large extent prevented by guanethidine. The reserpine-induced bradycardia and hypotension were reduced after guanethidine pretreatment. Chlorisondamine pretreatment depressed heart rate, blood pressure and plasma levels of NPY-LI. Furthermore, chlorisondamine inhibited the reserpine-induced increase in plasma NPY-LI and prevented the reduction in tissue content of NPY-LI in the heart, skeletal muscle and adrenal.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010141 TI - Acute and chronic effects of lithium chloride on GABA-ergic function in the rat corpus striatum and frontal cerebral cortex. AB - The acute (1 h, i.p.) and chronic (14 days, p.o.) effects of LiCl treatment upon GABA-ergic neurons were studied in the rat corpus striatum and frontal cerebral cortex. One hour after a single injection of LiCl the activity of glutamic acid decarboxylase (GAD) was reduced by 29% in the striatum (2 meq/kg LiCl) and by 38% in the cerebral cortex (10 meq/kg LiCl). In contrast, striatal GAD was activated by 34% 1 h after the injection of 10 meq/kg of LiCl; this dose also reduced the endogenous striatal GABA level by 24%. After 14 days of oral LiCl administration (2 meq/kg/day): a) cortical GAD activity was enhanced by 50% and GABA concentration was decreased by 28%; b) no changes were observed in the striatum. These findings suggest that: LiCl administration stimulates GABA-ergic function in specific areas (depending on the dose and length of treatment) increasing both GAD activity and probably GABA release. This occurs in the striatum after acute treatment only with a high dose, and in the frontal cerebral cortex after chronic treatment with a low dose. PMID- 3010143 TI - [Benign liver tumors and oral contraceptives: diagnosis and treatment]. PMID- 3010142 TI - Effect of adenosine receptor agonists and other compounds on cyclic AMP accumulation in forskolin-treated hippocampal slices. AB - The effect of adenosine analogues and some putative neurotransmitters have been studied on cyclic AMP accumulation in rat hippocampal slices treated with the adenylate cyclase activator forskolin. The effects of PGE2 and histamine were potentiated by forskolin (0.1 microM). Isoprenaline and NECA had essentially additive effects with 0.1 microM forskolin and serotonin (above 10(-4) M) inhibited forskolin-stimulated cyclic AMP accumulation. The A1-adenosine receptor selective adenosine analogue R-PIA inhibited forskolin stimulated cyclic AMP accumulation in low doses and stimulated in high. NECA, adenosine and 2 chloroadenosine uniformly stimulated cyclic AMP accumulation. 2',5' dideoxyadenosine inhibited, but only at high concentrations. Both the stimulatory and the inhibitory effects of R-PIA were antagonized by 8-phenyltheophylline (10 microM). Enprofylline (100 microM) selectively inhibited the stimulatory effect. In the presence of enprofylline both 2-chloroadenosine showed an inhibitory effect on cyclic AMP accumulation. It is concluded that the forskolin-treated rat hippocampal slice is a useful preparation to study both stimulatory and inhibitory effects of transmitters and modulators on adenylate cyclase. The results also show that the rat hippocampus has both A1-receptors that are linked to inhibition of cyclic AMP accumulation and A2-receptors that are linked to stimulation. Furthermore, enprofylline is shown to selectively antagonize the stimulatory response, revealing inhibitory effects of compounds such as 2 chloroadenosine and adenosine. PMID- 3010144 TI - Effects of area postrema lesions on taste aversions produced by treatment with WR 2721 in the rat. AB - The conditioned taste aversion procedure was used to further assess some behavioral effects of treatment with the putative radioprotectant WR-2721 and the role of the area postrema in mediating the behavioral effects of treatment. Treatment with 40, 150 or 300 mg/kg WR-2721 produced dose-dependent changes in sucrose intake in both control rats and rats with area postrema lesions. The effectiveness of the lesion in disrupting the acquisition of an aversion varied as a function of the dose administered, with the lesions producing the greatest disruption of aversion learning at the lowest dose and little disruption at the highest dose tested. At all dose levels, sucrose intake was greater for the rats with area postrema lesions than for the sham-operated control rats. Treatment with WR-2721 also produced significant decreases in total fluid intake, particularly at the higher dose levels. The results are discussed as indicating that treatment with WR-2721 produces highly toxic effects on behavior and that the use of the compound as a radioprotectant for radiotherapy requires additional assessment of its effects on brain function and behavior. PMID- 3010145 TI - A high-performance liquid chromatography assay of brain adenylate cyclase using [3H]ATP as substrate. AB - A sensitive, reproducible assay for adenylate cyclase is described which separates labeled cyclic AMP from ATP and other nucleotides by high-performance liquid chromatography (HPLC) on reverse-phase columns. The technique utilizes [3H]ATP as substrate, and the principal compound contaminating the [3H]cyclic AMP peak, adenosine, is removed by incubation of assay tubes with small amounts of adenosine deaminase. The HPLC elution utilizes high resolution (3 microns) short (10 cm) C-18 columns for increased resolution and decreased flow rates. Since cyclic AMP elutes at 4 min following injection, this procedure can easily process large numbers of samples per day when combined with automated techniques of sample injection and collection. PMID- 3010146 TI - Proteolysis in quaking mouse brain and spinal cord. AB - Six proteolytic enzymes were assayed for activity in quaking CNS in examining the hypothesis that increased proteolytic activity contributes to the hypomyelination characteristics of this mutant. Cathepsin B-like enzyme, cathepsin D, neutral proteinase, calcium-activated neutral proteinase, prolyl endopeptidase, and diaminopeptidase II were assayed in whole homogenate of brain or spinal cord and each was found to have activity similar to that in normal mice. These results do not support a relationship between proteolysis and the genetic defect and suggest that other factors should be investigated to delineate the pathogenesis of this mutant. PMID- 3010147 TI - A comparison of the dodecyl sulfate-induced precipitation of the myelin basic protein with other water-soluble proteins. AB - The interactions of sodium dodecyl sulfate with a number of proteins were examined at a variety of pH values ranging from 4.8 to 11.6. The dodecyl sulfate induced precipitation of some of these proteins was observed within a relatively limited range of total dodecyl sulfate concentration. Most of the basic proteins precipitated at low pH but as the isoelectric point of the protein was approached the amount of protein that precipitated decreased. Bovine myelin basic protein was unique in that it precipitated at all pH values examined both above and below its isoelectric point. Thus, the dodecyl sulfate-induced precipitation of myelin basic protein appears to be different from the dodecyl sulfate-induced precipitation of most proteins. A comparison of protein precipitation at equivalent dodecyl sulfate:protein molar or weight ratios revealed very little difference in the precipitation behavior of the proteins studied. When the bovine myelin basic protein was cleaved at its single tryptophan residue, the N-terminal fragment (1-115) formed insoluble dodecyl sulfate complexes at pH values ranging from 4.8 to 9.2. The C-terminal fragment (116-169) precipitated almost completely at pH 4.8 but to a lesser extent at pH 7.4 and 9.2. Equimolar mixtures of the N- and C-terminal fragments precipitated in the presence of dodecyl sulfate at pH 7.4 and 9.2 to an extent greater than the C-terminal fragment alone but comparable to the N-terminal fragment alone or the whole basic protein. These results suggest: that the mechanism by which dodecyl sulfate induces the precipitation of myelin basic protein may be unique compared to other proteins and that the intact myelin basic protein is not necessary for its precipitation by dodecyl sulfate. PMID- 3010148 TI - Nicotinic and muscarinic agonists, phorbol esters, and agents which raise cyclic AMP levels phosphorylate distinct groups of proteins in the superior cervical ganglion. AB - The phosphorylation of proteins in the superior cervical ganglion of the rat was investigated. Ganglia were incubated with 32Pi, and the 32P-labeled proteins in the ganglion were separated by two-dimensional electrophoresis and visualized by autoradiography. Approximately 40 distinct phosphoproteins could be visualized by these methods. The most heavily labeled ganglionic protein was an acidic protein with an Mr of approximately 83,000. Tyrosine hydroxylase was identified as a doublet of two closely-migrating radioactive spots. Treatment of intact ganglia with depolarizing agents, nicotinic and muscarinic agonists, phorbol esters, and agents that increase the content of cyclic adenosine 3':5'-monophosphate in the ganglion stimulated the incorporation of 32Pi into distinct but overlapping groups of phosphoproteins. All of these agents increased the phosphorylation of tyrosine hydroxylase. In contrast, only phorbol esters and muscarinic agonists increased the phosphorylation of the 83,000 ganglionic phosphoprotein. Our data are consistent with the idea that the various classes of agonists may activate distinct protein kinases in the ganglion. PMID- 3010149 TI - Stability of neuronal and glial marker enzymes in post-mortem rat brain. AB - The enzymatic activities in post-mortem rat brain kept at 4 degrees C and at 25 degrees C were determined for a number of enzymes localized in specific cell types in the central nervous system. Choline acetyltransferase (CAT), glycerol-3 phosphate dehydrogenase (GPDH), glutamine synthetase (GS), lactate dehydrogenase (LDH) and 2',3'-cyclic nucleotide phosphohydrolase (CNPase) were found to be very stable at both 4 degrees C and 25 degrees C with only slight, if any, losses of activity being seen even at periods as long as 72 hr. Glutamic acid decarboxylase (GAD) activity was less stable than that of the other enzymes. In brains kept at 4 degrees C GAD activity was stable out to 24 hr after which it began to decline rapidly to 65% of control at 72 hr. In brains kept at 25 degrees C, GAD activity was stable for 6-8 hr and then began to steadily decline to 58% of control at 24 hr and 29% of control at 72 hr. Assuming that these enzymes have similar stabilities in post-mortem human brain, the effect of post-mortem delay in processing tissues may be of lesser significance than other factors with regard to the measured enzyme activities in human brain samples. PMID- 3010151 TI - [Osteoplastic laminectomy using hydroxyapatite ceramic implants]. AB - Cervical osteoplastic laminectomy using ceramic or apatite beads as implants was performed in 31 patients with symptomatic cervical bony canal stenosis. Patients with degenerative changes such as osteochondrosis or disc herniation are included in this study but those with spinal cord tumor, arachnoid cyst or ossification of the posterior longitudinal ligament are not. Initially foraminotomies were performed at the C5-8 levels in an attempt at preventing worsening of radicular signs caused by posterior displacement of the cord and resultant tethering effect on the nerve root. Connecting the medial borders of the foraminotomies, laminae were removed in a single piece. A small suboccipital craniectomy and C1-2 laminectomy were added when necessary. Laminae were replaced maintaining an increased anteroposterior diameter of the bony canal, by means of interposition of apatite or ceramic beads of appropriate size between the cut surfaces of the laminae. Results were excellent in 13 (42%), good in 10 (32%), fair in 4 (13%), no change in 2 (6.5%) and worse in 2 (6.5%). Literature on the osteoplastic laminectomy (laminoplasty) for cervical canal stenosis was reviewed and the advantage of the present method was briefly discussed. PMID- 3010150 TI - Heterogeneous localization of some purine enzymes in subcellular fractions of rat brain and cerebellum. AB - The activity of guanine deaminase (GAH, E.C.3.5.4.3) was lower in rat cerebellum soluble and microsomal fractions than in rat brain subfractions. Adenosine deaminase (ADA, E.C.3.5.4.4) activity was released in higher proportion than guanine deaminase, purine nucleoside phosphorylase (PNP, E.C.2.1.2.4), 5' nucleotidase (5'N, E.C.3.1.3.5), and lactate (LDH, E.C. 1.1.1.27) and malate (MDH, E.C. 1.1.1.37) dehydrogenase in press-juices of rat brain. Furthermore, nerve ending-derived fractions (synaptosomes and synaptic vesicles) showed an enrichment of adenosine deaminase and also of 5'-nucleotidase. The action of deoxycholate over the subfractions did not increase the activity of either enzyme. The contrary occurred with the remaining enzymes studied. Thus, it is possible that one set of enzymes are located on the surface of the particulate vesicles, whereas another set are located inside these vesicles, suggesting a compartmentation of purine catabolic enzymes in different areas of the central nervous system. PMID- 3010152 TI - [Intraoperative ultrasonography through a burr hole: clinical trial of ultrasound guided stereotactic surgery]. AB - Intraoperative ultrasound diagnosis through a burr-hole was performed in 59 cases using electronic sector-scanning transducers. The burr-hole was conically enlarged and the gap between the transducer tip and the dura mater was filled either with saline or sterile echojelly. The pathology was successfully imaged in 33 cases (56%), or 20 out of 28 cases (71%) using a 5 MHz transducer. Compared to transdural ultrasonography, the quality of image through a burr-hole was found less satisfactory. Six cases of biopsy and/or aspiration were successfully performed under ultrasonic guidance through a burr-hole. Two burr-holes were necessary in these stereotactic operations. A needle-guide apparatus and the guideline superimposed on the monitor CRT screen were advantageously utilized. In biopsy, both the tumor and the biopsy needle were visible on the same monitor CRT screen, and accurate biopsy was possible. In aspiration, the tip of the needle could always be kept inside the cyst or abscess cavity. Bleeding during these manipulations was easily detected on the real-time monitoring CRT screen, and if necessary, it could be immediately controlled. Our results suggest that the intraoperative ultrasonography through a burr-hole will make these operations safer and more accurate. PMID- 3010153 TI - Light and electron microscopical studies of the immunoperoxidase staining of multiple sclerosis plaques using antisera to a feline-derived agent and to galactocerebroside. AB - Antibodies have been raised to two agents (CCA147 and MV631) that were isolated from central nervous tissue of cats. The cells co-cultivated with these agents are characterized by the presence of cytoplasmic inclusions consisting of 16-18 nm diameter tubular elements morphologically similar to inclusions seen in a demyelinating condition in cats and to inclusions described as 'curved linear profiles' in multiple sclerosis (MS) plaques. The peroxidase-labelled antibodies to CCA147 and MV631 stain these inclusions in MS plaques as well as small virus like particles. The antisera do not stain normal white matter either in MS or non MS brain tissue. The staining reaction of one agent is blocked by pretreatment with antisera to the other agent and also by pretreatment with MS sera but not by normal human sera. Peroxidase-labelled antibody to galactocerebroside stains normal myelin and myelin debris within MS plaques but does not stain the 'curved linear profiles' that are stained by the labelled antibodies to the feline derived agents. The results show that the 'curved linear profiles' described in MS plaques are not myelin degradation products, but are comparable to the nucleocapsids of morbilliviruses. In addition, the small virus-like particles are morphologically similar to morbillivirus virions. The results are discussed with particular emphasis on the features of the morbilliviruses, canine distemper and measles viruses. PMID- 3010155 TI - Stress-specific modulation of ACTH secretion by oxytocin. AB - The role of oxytocin (OT) in regulating stress-induced ACTH secretion was investigated by immunoneutralization of endogenous OT with an antiserum raised against synthetic OT. Rats were subjected to one of three stresses: novel environment, tail-hang, or ether. In otherwise untreated rats, ACTH levels rose at least 3-fold in response to all three stresses whereas OT levels increased only in response to tail-hang and ether. Injection of a highly specific antiserum to OT 60 min before the experiment inhibited the ACTH response to tail-hang and ether by 64 and 56%, respectively, but had no effect on the ACTH response to novel environment stress. The data support a physiologic role for OT in the control of ACTH secretion but suggest that it is not until OT levels rise in response to stress that the effects of OT are expressed. PMID- 3010154 TI - Effects of naloxone on hypothalamo-pituitary-adrenocortical activity in the rat. AB - The influences of morphine and naloxone on hypothalamo-pituitary-adrenocortical (HPA) function were studied in the rat to investigate further the role of opioidergic mechanisms in the control of the secretion of corticotrophin and its hypothalamic releasing factor (CRF). Morphine not only caused rises in hypothalamic CRF content and plasma ACTH concentration but also potentiated the HPA response to stress. Its effects were antagonized by naloxone which, when given alone, did not influence basal plasma concentrations of ACTH and corticosterone but which inhibited, in a dose-dependent manner, the release of both of these hormones which normally occurs in response to stress. Naloxone also attenuated the exaggeration in stress-induced HPA activity but did not affect the increases in plasma ACTH concentration which followed adrenalectomy. The findings suggest that opioidergic mechanisms may be involved in the regulation of the HPA response to stress. PMID- 3010157 TI - Stimulatory and inhibitory effects of the opioids on gonadotropin secretion. AB - In order to gain additional information on the role played by the opioids in the control of the secretion of anterior pituitary gonadotropins, morphine (an opioid agonist) and naloxone (an opioid antagonist) have been injected intraventricularly (i.v.t.) into normal or castrated male rats. The animals were killed by decapitation at different time intervals after treatment and serum luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin were measured by radioimmunoassay. Animals injected i.v.t. with 0.9% saline solution and sacrificed at the same time intervals served as controls. When morphine (at the dose of 200 and 400 micrograms/rat) and naloxone (at the dose of 7.5 and 15 micrograms/rat) were injected i.v.t. into normal male rats, a significant increase of serum levels of LH was observed 10 and 20 min after injection. There was no effect at 5, 40 and 60 min. Lower doses of morphine (6.25, 12.5, 25, 50 and 100 micrograms/rat) given i.v.t. were ineffective. When morphine (200 micrograms/rat) and naloxone (either in the dose of 7.5 micrograms/rat or of 15 micrograms/rat) were given simultaneously, serum LH was significantly higher than in the saline-treated controls both at 10 and 20 min. However, the increases of serum LH levels induced by the combined treatment were in both instances lower than those produced by the administration of either drug alone. Morphine (200 micrograms/rat) when administered i.v.t. to normal male rats significantly enhanced prolactin release at 10 and 20 min, and this effect of morphine was blunted by the concomitant i.v.t. administration of naloxone (7.5 and 15 micrograms/rat).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010156 TI - Somatotroph responsiveness to low dose dopamine infusion in normal subjects and acromegalic patients. AB - We have investigated the effects of intravenous administration of a low dose of dopamine (DA) on plasma growth hormone (GH) concentrations in acromegalic patients and normal subjects with the aim of defining the somatotroph responsiveness to peripheral (i.e., outside the blood-brain barrier) specific dopaminergic stimuli. DA (0.02 micrograms/kg/min) was infused for 180 min into 12 acromegalic patients and 10 normal subjects. DA infusion discriminated between two groups of acromegalics. In group I (n = 7), the elevated plasma GH levels (64.1 +/- 29.9 ng/ml, mean basal value +/- SEM) decreased significantly (mean overall GH inhibition, 26% reduction from basal levels; range, 10-49), whereas in group II (n = 5) plasma GH levels (29.8 +/- 12.5 ng/ml) remained elevated (mean GH variation, 8% above baseline; range, 0-15). Plasma GH concentrations showed a significant rebound above baseline values after stopping DA infusion only in group I. In contrast, the responsiveness to TRH was not significantly different between the groups (percentage increase 767 +/- 317% in group I vs. 382 +/- 210% in group II) and they were also comparable with regard to sex, age, glucose tolerance, plasma prolactin (PRL) concentrations and adenoma size. However, the mean duration of the disease was significantly (p less than 0.02) longer in group I (12.8 +/- 2.6 years) than in group II (4.5 +/- 1.5 years). Further, 3 patients with previous radiotherapy for invasive adenomas were nonresponders to DA. In normal subjects, DA infusion had no significant effect on plasma GH levels. It is concluded that: somatotroph responsiveness to DA is not a constant feature of acromegaly.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010158 TI - Selective control by corticosterone of serotonin1 receptor capacity in raphe hippocampal system. AB - The effect of short-term bilateral adrenalectomy (ADX) and corticosterone or aldosterone replacement was investigated on serotonin1 (5-HT1) receptor density in rat brain regions. One hour after ADX the 5-HT1 receptor density was increased in subiculum, molecular layer of the dentate gyrus, substantia nigra, and dorsal raphe nucleus as shown by in vitro autoradiography and computerized densitometric analyses of the film images. In subiculum, dentate gyrus, and dorsal raphe nucleus the 5-HT1 receptor density was restored (decreased) towards the level observed in sham-operated rats after administration of a low dose of corticosterone (CORT) at the time of ADX. CORT replacement decreased the 5-HT1 receptor number also in hippocampal pyramidal cell layer CA1, presubiculum, and other dentate gyrus subregions. The 5-HT1 receptor density was not affected by ADX or CORT replacement therapy in cerebral cortex, central grey, CA3, ventral hippocampus, and median raphe nucleus. Aldosterone administered under the same experimental conditions did not change the 5-HT1 receptor number in any of the hippocampus or raphe regions. The dose of CORT (30 micrograms/100 g body weight, s.c.) gave physiological plasma levels and maintained almost complete occupation of CORT receptors (mineralo-corticoid-like receptors) in hippocampus which was also the case in the sham-operated control animals. CORT replacement did not maintain occupancy of glucocorticoid receptors in hippocampus. When the binding of the selective glucocorticoid agonist, 3H-RU 28362, was taken as 100% at 1 h after ADX, CORT replacement and sham ADX left 75 and 50% of these sites available respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010159 TI - Interactions between delta 9-tetrahydrocannabinol and cannabidiol as evaluated by drug discrimination procedures in rats and pigeons. AB - Animals (rats and pigeons) were trained to discriminate between the presence and absence of delta 9-THC; the training doses were, respectively: 0.56 mg/kg (pigeons) and 3.0 mg/kg (rats). Once the drug discrimination was mastered, the pigeons were tested repeatedly after a single intramuscular (i.m.) injection of delta 9-THC (0.56 mg/kg) at the following intervals 0.5, 1.5, 4.5 and 9 hr after the injection. These results were compared with data from a separate procedure, i.e. where the various intervals after injection were examined only once per injection and both procedures yielded essentially the same outcome. Thus, less than 50% appropriate responding to THC was observed at 0.5 and 9 hr after injection, whereas greater than 90% responding to THC occurred at 1.5 and 4.5 hr. The two procedures have previously been compared in rats (Jarbe, Swedberg and Mechoulam, 1981). The repeated tests procedure was then used to evaluate combinations of delta 9-THC and cannabidiol in both species. Cannabidiol prolonged the cue effects of 1 mg/kg of delta 9-THC (intraperitoneal route of administration) in rats but did not change the time-effect curve for delta 9-THC in pigeons (dose range examined: 0.10--0.56 mg/kg); the challenge doses of cannabidiol were, respectively: 30.0 mg/kg (i.p.) and 17.5 mg/kg (i.m.). The rate of responding did not differ in tests with combinations of delta 9-THC and cannabidiol as compared to delta 9-THC given alone in pigeons. Subcutaneously administered 3-PPP, a dopamine pre-synaptic blocker, did not induce responding appropriate for delta 9-THC in rats. PMID- 3010160 TI - Dihydroergocristine-induced stimulation of the 5-HT autoreceptor in the hypothalamus of the rat. AB - In slices of the hypothalamus of the rat, prelabelled with [3H]5 hydroxytryptamine ([3H]5-HT), dihydroergocristine (DHEC) decreased in a concentration-dependent manner (0.01-1 microM) the release of [3H]transmitter elicited by stimulation. In the presence of phentolamine (1 microM), sulpiride (1 microM), atropine (1 microM) or methiothepin (0.1 microM), the effect of DHEC remained unchanged. However, methiothepin at 1 microM and citalopram at 1 microM antagonized the inhibition induced by DHEC. Methiothepin at 0.1 microM has been shown to be sufficient to shift to the right the concentration-effect curve of D lysergic acid diethylamide (LSD) on the stimulation-evoked release of [3H]5-HT by a factor of 10. However, in the present experiments, 1 microM methiothepin was required to antagonize the effect of DHEC. Thus, the alpha adrenergic, dopaminergic and cholinergic activities of DHEC do not seem to be responsible for its effect on the release of 5-HT. The lower potency of methiothepin suggests, however, that stimulation of the 5-HT autoreceptor by DHEC may not fully explain the results. PMID- 3010161 TI - Anticonvulsant profile of drugs which facilitate GABAergic transmission on convulsions mediated by a GABAergic mechanism. AB - The effects of selected modulators of GABAergic transmission, either alone or in combination, were tested for their potency on the seizure pattern and mortality induced by convulsant drugs in rat. Pentobarbital and diazepam were effective against both tonic and clonic seizure components induced by bicuculline and picrotoxin. The anticonvulsant profile of ethanol closely resembled that of pentobarbital. Pentobarbital, diazepam and ethanol did not modify seizures induced by strychnine. In contrast, progabide, a central gamma-aminobutyric acid (GABA) receptor agonist, caused significant protection only against convulsions induced by strychnine and its lethality, but did not protect against seizures induced by bicuculline or picrotoxin. Data on interaction of drugs with subprotective doses of these agents clearly demonstrated potentiation of the anticonvulsant actions of these modulators. Thus, seizures induced by bicuculline were more sensitive to the inhibition by pentobarbital in combination with diazepam or ethanol, while pentobarbital with progabide was equally effective against convulsions induced by GABA antagonists. Diazepam, in combination with progabide, blocked only convulsions induced by picrotoxin. Ethanol, in combination with either pentobarbital or with diazepam, was effective against all the three convulsant drugs. These results are consistent with the concept that drugs which facilitate GABAergic transmission are effective against seizures related to an impairment of GABA transmission. Further, the present data indicate that tonic seizures are more susceptible to the actions of drugs than the clonic component. Smaller doses of these drugs, alone or in combination, modified the seizure patterns and mortality, whereas at larger doses, the possible involvement of a nonspecific depressant action cannot be ruled out. PMID- 3010162 TI - Pharmacological modulation of GABAergic transmission in cultured cerebellar neurons. AB - GABA activates specific ion channels in post-natal cerebellar neurons in primary culture generating Cl- currents that can be recorded with the whole-cell patch clamp technique. Evoked and spontaneous GABA-mediated synaptic activity can be recorded from cells kept in culture for a few days. Benzodiazepine and beta carboline derivatives which bind with high affinity to the domains for allosteric regulation of GABA receptors facilitated and inhibited directly applied GABA responses and synaptically evoked Cl- currents recorded under voltage-clamp. PMID- 3010163 TI - Do kappa opioids mimic sigma agonists as amino acid antagonists? AB - The kappa opioids tifluadom and U-50,488 H, when tested on spinal neurones of rats in vivo and frogs in vitro, had no selective effect on responses to microelectrophoretically or bath applied N-methyl-D-aspartate, quisqualate or kainate. In the same preparations ketamine selectively reduces responses to N methyl-D-aspartate. Amino acid antagonism by dissociative anaesthetics and sigma opioids is thus not mediated by that binding site which kappa and sigma opioids have been reported to have in common. PMID- 3010165 TI - Lesion of noradrenergic neurones with DSP4 does not modify benzodiazepine receptor binding in cortex and hippocampus of rat. AB - The effect of lesioning noradrenergic pathways on the benzodiazepine receptor has been studied using a novel neurotoxic agent, N(2-chloroethyl)-N-ethyl-2 bromobenzylamine (DSP4) which has better selectivity than the classical 6 hydroxydopamine (6-OHDA) towards noradrenergic neurones, and which has the added advantage of being injected systemically rather than intracerebrally. Three different radioligands were used: an agonist, [3H]flunitrazepam, an antagonist, [3H]Ro 15-1788, and a partial agonist [3H]RU 43028. Binding was measured using membrane homogenates from the cortex and hippocampus of the rat, two regions of the brain which receive an extensive noradrenergic innervation. In contrast to previous reports of a decrease in the binding of [3H]flunitrazepam following lesioning with 6-OHDA, no significant change was observed in either the affinity (KD) or the number of sites (Bmax) of any one of these ligands after lesioning with DSP4. While the reasons for this discrepancy are not clear, these results do not confirm that destruction of noradrenergic afferents to the cortex or hippocampus leads to any modification of the benzodiazepine receptor as demonstrated by ligand binding. PMID- 3010164 TI - Modulation of catecholamine transmission and sleep in man. AB - The effect of modulation of catecholamine transmission on sleep in man was studied using mianserin (20 and 40 mg) and nomifensine (50 and 100 mg). It is suggested that reduced wakefulness induced by mianserin during sleep is primarily related to postsynaptic antagonism of alpha adrenoceptors, though a possible synergistic effect with antagonism of histamine H1 receptors cannot be excluded, while increased wakefulness with nomifensine is related to inhibition of the uptake of catecholamines and/or direct or indirect dopaminergic activity. The reduction in rapid eye movement (REM) sleep with both drugs is believed to be a non-specific effect which arises from a disturbance of the balance between monoaminergic and cholinergic influences. PMID- 3010166 TI - Evidence against adenosine 3',5'-monophosphate as a mediator of fever in the brain. AB - The aim of this study was to test the hypothesis that increased concentrations of adenosine 3',5'-monophosphate (cyclic AMP) in the pyrogen-sensitive preoptic/anterior hypothalamic region (PO/AH) mediate fever. Micro-injection of N6-2'-O-dibutyryl adenosine 3',5'-monophosphate (db cyclic AMP) into the preoptic/anterior hypothalamic region in rats produced a dose-dependent fall in body temperature which is inconsistent with the proposal that the nucleotide mediates fever. Hyperthermia was observed in some rats in response to large doses of db cyclic AMP, but this response was associated with convulsions. Endogenous concentrations of cyclic AMP in the preoptic/anterior hypothalamic region, as well as in the cerebral cortex, liver, spleen, thymus, white fat and plasma were unaffected by the febrile response to the subcutaneous injection of yeast in rats. A rise in levels of cyclic AMP was observed in the skeletal muscle of rats treated with yeast. The data presented do not indicate that cyclic AMP is involved in the neuronal events mediating fever in the rat. PMID- 3010167 TI - Specific binding and uptake of opioids in brain slices. AB - Studies on slices from whole rat brain indicate that the opioid peptide/receptor, but not the opiate drug/receptor, complex is internalized by metabolically dependent processes. Opiate drugs displace 3H-etorphine from high affinity binding sites with potencies identical to those in brain homogenates. 3H metenkephalin (ME) binds to a high affinity (IC50 10 nM) and a low affinity (micromolar) site. Opiate drugs and beta-endorphin compete at the high affinity but not at the low affinity binding site for ME. The biological relevance of the low affinity ME-binding site, which like the high affinity site is internalized, remains to be determined. The slice technique should be useful in the characterization of receptor dynamics that may be altered during opiate tolerance and dependence. PMID- 3010168 TI - Sodium ions increase the binding of the antagonist peptide ICI 174864 to the delta-opiate receptor. AB - The ability of N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174864) to displace [3H] [D-Ala2, D-Leu5]enkephalin bound to the delta-opioid site is increased 8-16 fold by addition of 25mM NaCl. A smaller shift is observed for the related N,N-diallyl Tyr-Gly-Gly-psi-(CH2S)-Phe-Leu-OH (ICI 154129) but no significant shift is seen with either naloxone or diprenorphine. The results stress the importance of using the correct medium for binding assays, and suggest the changes in delta-receptor conformation induced by Na+ ions also increase peptide antagonist binding. PMID- 3010170 TI - Preservation of neuronal function during prolonged focal cerebral ischemia by ventriculocisternal perfusion with oxygenated fluorocarbon emulsion. AB - The left middle cerebral artery and both carotid arteries of 17 cats were occluded to evaluate the effects of oxygenated fluorocarbon emulsion on brain ischemia. Carotid and middle cerebral arteries were occluded concurrently for 2 hours, followed by occlusion of the middle cerebral artery only for another 24 hours. Six animals were treated with oxygenated fluorocarbon emulsion delivered by ventriculocisternal perfusion, 5 received ventriculocisternal perfusion with mock cerebrospinal fluid, and 6 were untreated. Perfusions were started 3 hours after the initial ischemic insult. Infarct size judged by tetrazolium staining and standard neuropathological stains was significantly smaller in the treated animals. The mechanism of protection is as yet unknown, but most likely reflects oxygen/nutrient diffusion into the ischemic middle cerebral artery zone from the ventricular fluorocarbon, or removal of harmful metabolites. The results imply that ventriculocisternal perfusion with fluorocarbon emulsion can preserve neuronal function during a major cerebral vessel occlusion. In the cat, therapy is effective if begun within 3 hours after ischemia starts. PMID- 3010169 TI - Corticotropin inhibits food intake in rats. AB - The synthetic corticotropin ACTH (1-24) (tetracosactide), injected into a brain lateral ventricle after a 24h starvation period or into the ventromedial hypothalamus during the nocturnal feeding phase, markedly inhibited food intake, in rats. In starved rats, the dose of 4 micrograms/rat was maximally effective and reduced food intake by 76.6% during the first hour after treatment. The same dose, injected into the ventromedial hypothalamus, significantly inhibited food intake also in normally fed rats during the nocturnal phase (58.6% reduction during the 90 minutes of observation). These findings suggest that corticotropin may play a role in the central control of appetite. PMID- 3010172 TI - Ipsilateral sensory symptoms. PMID- 3010171 TI - Pathophysiology of cerebrospinal fluid in head injury: Part 2. Biochemical markers for central nervous system trauma. AB - Many substances are released into the cerebrospinal fluid after head injury. The study of these substances and their relationship to the severity and outcome of head trauma has lead to the search for biochemical markers to aid in the quantification of the severity of the lesion and serve as a prognostic guide. The authors review the potential usefulness of biochemical markers, qualities of an ideal marker, and several potential enzymes that may be utilized as markers in central nervous system trauma. PMID- 3010174 TI - Development of plastic mechanisms related to learning at identified chemical synaptic connections in Aplysia. AB - In the marine snail Aplysia californica learned changes in behavior have been traced to alterations in synaptic efficacy. With the ability to raise the animals in the laboratory, we have explored the development of four types of plastic mechanisms at identified synapses: post-tetanic potentiation and pre-synaptic inhibition, which do not as yet have known behavioral functions, and homosynaptic depression, the cellular mechanism of short-term habituation, and heterosynaptic facilitation, the basis of short-term sensitization. Homosynaptic depression and pre-synaptic inhibition are present early in juvenile life. In contrast, post tetanic potentiation and heterosynaptic facilitation appear only later, after a discrete interval. The step-wise ontogeny of synaptic plastic mechanisms in Aplysia parallels the gradual emergence of behavior in successive developmental stages. Interestingly, senescence reverses the developmental sequence for habituation and sensitization mechanisms. To pursue further an understanding of the relationship between synapse formation and plasticity underlying learning it will be necessary to extend these studies to dissociated cell culture where mechanisms can be explored on the molecular level. Cell culture may also permit examination of the development of cellular mechanisms underlying classical and operant conditioning which may clarify differences between associative and non associative mechanisms. PMID- 3010173 TI - Hexokinase in cultured gliomas. PMID- 3010175 TI - Variability of quantal events in control solution and after cholinesterase blockade in frog. AB - Amplitudes of miniature end-plate currents (AMEPCs) as well as their time constants of decay (TMEPCs) contribute to the amplitudes of miniature end-plate potentials and hence determine their mean values and variability. Larger mean amplitudes of miniature end-plate potentials observed after cholinesterase blockade result from both greater mean AMEPCs and greater mean TMEPCs. However, enhanced variability of amplitudes of miniature end-plate potentials is a result mainly of greater variability of TMEPCs. This occurs almost exclusively because the amplitude dependence of TMEPCs becomes markedly enhanced. Although the scatter of TMEPCs about TMEPC vs AMEPC curve is also larger, it does not contribute much to the increased variability of TMEPCs. The marked amplitude dependence of TMEPCs indicates that repetitive binding strongly depends on the quantal size--the number of acetylcholine molecules released per quantum, and suggests that if a quantum is the result of a release of several vesicles, then they should be released very close to each other. Marked amplitude dependence of TMEPCs also shows that, with cholinesterase inhibited, the quantal size can be estimated not only from AMEPCs, but also from TMEPCs. Greater scatter of TMEPCs from the TMEPC vs AMEPC curve is of more obscure origin and probably results because repetitive binding occurs over enlarged "critical areas" with more variable density of acetylcholine receptors. PMID- 3010176 TI - In vitro neurotoxicity of excitatory acid analogues during cerebellar development. AB - The neurotoxic effects of the selective excitatory amino acid receptor agonists, quisqualate, kainate and N-methyl-D-aspartate, were studied in slice preparations of cerebellum from rats at different stages of postnatal development. With increasing age, (i) Purkinje cells became more vulnerable to kainate and quisqualate but remained insensitive to N-methyl-D-aspartate (up to 300 microM); (ii) Golgi cells became more sensitive to kainate, quisqualate and N-methyl-D aspartate; (iii) granule cells became more sensitive to kainate, less sensitive to N-methyl-D-aspartate and remained unaffected by quisqualate (up to 100 microM), and (iv) basket and stellate cells and, up to 14 days of age, neurones of the deep cerebellar nuclei, became more vulnerable to kainate and quisqualate, but their sensitivity to N-methyl-D-aspartate stayed the same. The neurotoxicity of N-methyl-D-aspartate, but not that of kainate in 8-day-old cerebellar slices was prevented by 2-amino-5-phosphonovaleric acid; tetrodotoxin did not affect the toxicity of the agonists in 8-day-old or adult slices. The results with kainate are consistent with other studies indicating an insensitivity of the immature brain to its neurotoxic effects, but suggest that this property is not a peculiarity of kainate. Alterations in excitatory potency can explain some of the observed developmental changes. However, other observations cannot readily be accounted for on the basis of either changes in excitatory potency, the functional maturation of cerebellar circuits, changes in synaptic density, or the developmental appearance of Ca2+ channels in susceptible cells, suggesting that additional factors play an important role in the neurotoxic effects of the excitants. PMID- 3010177 TI - Stimulation-associated changes in frog neuromuscular junctions. A quantitative ultrastructural comparison of rapid-frozen and chemically fixed nerve terminals. AB - The ultrastructural effects of stimulation and subsequent rest were measured in frog neuromuscular junctions preserved by rapid-freezing and freeze-substitution, a method that minimizes fixation-associated membrane rearrangements. The effects were compared to those measured in junctions preserved by aldehyde fixation in order to identify artifacts attributable to the method of preservation. Effects of stimulation previously observed in tissue preserved by aldehyde fixation were evident in both the rapid-frozen and aldehyde-fixed neuromuscular junctions in the present study. Synaptic vesicles were reduced in number and cisternal profiles were increased. However, the sizes and shapes of the cisternae differed with the method of preservation. In addition, it was found that mitochondria underwent a change in shape with stimulation. This was accompanied by swelling in the fixed preparations, but not in the rapid-frozen ones. Fixation after stimulation also produced swelling of the nerve terminals, a stimulation associated change not evident in preparations that were preserved by rapid freezing. After stimulation and 60 min of rest, nerve terminals showed recovery towards control morphology, evidence that the effects of the stimulation parameters used in the study were reversible. This study, utilizing rapid-frozen material, confirms previous reports based on chemically fixed tissue that stimulation reduces the number of synaptic vesicles and increases the number of cisternae. The findings are in accord with the hypotheses of exocytotic neurotransmitter release and local recycling of synaptic membrane. In addition, the study emphasizes that accurate quantitative assessments of membrane redistribution in active secretory systems cannot depend on chemically fixed tissues. It also shows that mitochondria are susceptible to radical distortion by aldehyde fixatives, and that the degree of susceptibility differs with the physiological state of the tissue. PMID- 3010178 TI - Autoradiographic localization of thyrotropin releasing hormone receptors in human brain. AB - We used quantitative autoradiography to localize thyrotropin releasing hormone (TRH) receptors in human brain. Highest concentrations of TRH receptors were localized within the cortical, basal, and lateral nuclei of the amygdala and the molecular layer of the hippocampus. Low levels were found in the cortex, diencephalon, and basal ganglia. The radioligand bound with similar affinity and pharmacology to pituitary gland as to brain. These data suggest that authentic TRH receptors in the hippocampus and amygdala may mediate the putative effects of TRH on the human brain. PMID- 3010179 TI - Electrophysiologic features of abetalipoproteinemia: functional consequences of vitamin E deficiency. AB - We performed electrophysiologic testing in 10 patients with abetalipoproteinemia (ABL). Peripheral nerve studies implied an axonal disorder. Visual evoked potentials demonstrated prolonged P100 latency in three patients and abnormal electroretinograms in six. Somatosensory evoked potentials indicated dorsal column dysfunction in eight patients. Brainstem auditory evoked potentials were normal. Findings were consistent with the known neuropathology of ABL and of experimental vitamin E deficiency. Stabilization or improvement in electrophysiologic findings occurred with vitamin E supplementation. Neurophysiologic tests document retinal, central somatosensory and peripheral nerve lesions in vitamin E deficiency and provide an objective indication of response to treatment. PMID- 3010180 TI - Experimental spinal cord injury: effects of trauma or ischemia on TRH and muscarinic receptors. AB - Traumatic spinal cord injury in rats and ischemic spinal cord injury in rabbits were associated with time-dependent, localized decreases in thyrotropin-releasing hormone (TRH) and muscarinic receptor binding. Changes were not evident in the first 24 to 48 hours, consistent with the hypothesis that secondary alterations in spinal cord may occur relatively late after injury. TRH receptor binding, but not muscarinic receptor binding, recovered by 3 weeks following trauma. Since TRH and acetylcholine may serve as excitatory neurotransmitters within the spinal cord, such changes could contribute to the functional neurologic impairment that follows injury. PMID- 3010181 TI - Adult-onset neuronal intranuclear hyaline inclusion disease. AB - We studied the clinical and pathologic features of two cases of neuronal intranuclear hyaline inclusion disease. The cases were unique in late onset, presentation with dementia, possible autosomal dominant pattern of inheritance (in one patient), predominance of inclusions in glial cells, and mineral deposits within some inclusions. Differences from other reported cases indicate that this is probably not a homogeneous entity. PMID- 3010183 TI - Unusual viral causes of transverse myelitis: hepatitis A virus and cytomegalovirus. AB - Twenty to 40% of cases of acute transverse myelitis are attributed to viral infections, although the specific viral etiology is only rarely identified. We studied two patients with transverse myelitis in association with acute hepatitis A virus (HAV) infection and acute primary cytomegalovirus (CMV) infection. This is the first well-documented report of an association between HAV infection and transverse myelitis, and only the fourth documented case of transverse myelitis in association with CMV infection in an immunocompetent adult. Both viruses should be considered as rare causes of transverse myelitis in immunologically normal adults. PMID- 3010182 TI - From neurosecretion to neural communication and beyond. The 26th annual Robert Wartenberg lecture. PMID- 3010184 TI - Case for diagnosis: AIDS. PMID- 3010185 TI - The psychophysiologic study of stress in military populations. PMID- 3010186 TI - Case for diagnosis. AIDS. PMID- 3010188 TI - [New developments in the technic for the preparation and isolation of hepatocytes]. PMID- 3010187 TI - HTLV III and hepatitis A and B serological markers among U.S. military venereal disease patients. PMID- 3010189 TI - [Hepatocarcinoma associated with hypoglycemia and LDL hypercholesterolemia]. PMID- 3010190 TI - High-performance liquid chromatographic analysis of 'specifically bound' label after [125I]angiotensin II binding to rat brain membranes. AB - The combined hypothalamus-thalamus-septum and anteroventral third ventricular region (HTSA) of the rat was examined for [125I]angiotensin II ([125I]AII) binding using two protocols: one that preserved synaptosomal structure and a second that did not. Although maximum binding (Bmax) and dissociation constants (Kd) were similar in both preparations, high-performance liquid chromatographic analyses revealed that [125I]AII made up the majority of specifically bound label in the synaptosome preserved preparation while [125I]tyrosine ([125I]Tyr) represented most of the specifically bound label in the disrupted preparation. These results indicate that [125I]Tyr accumulation occurred subsequent to binding and degradation of [125I]AII and are consistent with the notion that rapid internalization of the receptor-[125I]AII complex occurs in those preparations where the synaptosomal structure remains intact. PMID- 3010191 TI - Diminished alpha 2-adrenoceptor-mediated modulation of noradrenergic neurotransmission in the posterior hypothalamus of spontaneously hypertensive rats. AB - An azepine derivative, 6-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo-[4,5-d] azepine (B-HT 920; 100 nM), inhibited the evoked noradrenaline release from slices of rat posterior hypothalamus, and yohimbine (100 nM) potentiated the release from slices of rat anterior and posterior hypothalamus. In the posterior hypothalamus of 4- and 15-16-week-old spontaneously hypertensive rats (SHRs), as compared with age-matched Wistar-Kyoto rats (WKYs), the inhibitory effect of B-HT 920 and the facilitatory effect of yohimbine were decreased. In the anterior hypothalamus there was no significant difference in the yohimbine effect between WKYs and SHRs, at either age. It is concluded that alpha 2-adrenoceptor-mediated autoinhibition of noradrenergic neurotransmission is diminished in the posterior hypothalamus of SHRs. PMID- 3010192 TI - Effects of chronic dehydration on angiotensin II receptor binding in the subfornical organ, paraventricular hypothalamic nucleus and adrenal medulla of Long-Evans rats. AB - Angiotensin II (AII) is an important peptide known to regulate blood pressure and body fluid. In the present study we used a potent AII antagonist, 125I (Sar1,Ile8)-AII (125I-SI-AII), to study AII receptor binding in Long-Evans rats 5 days after water deprivation. Specific structures evaluated include the subfornical organ (SFO) and adrenal gland. With quantitative autoradiography, we have found that there is an increase of 125I-SI-AII binding in the SFO, whereas there is a decrease in AII binding in the adrenal medulla. These observations suggest that central and peripheral AII target tissues are affected differently by dehydration. The increase in SI-AII binding in the SFO may indicate participation of this structure during dehydration, as angiotensin stimulation of SFO causes thirst and vasopressin release. PMID- 3010193 TI - Calcium discriminates two [3H]kainate binding sites with different molecular target sizes in rat cortex. AB - Binding of [3H]kainate to the kainate subtype of excitatory amino acid receptors revealed two binding sites. The high-affinity site (Kd = 3.5 nM) was sensitive to calcium ions and has a molecular target size as determined by high-energy radiation inactivation analysis of 76,600 daltons. The low-affinity site (Kd = 65 nM) was insensitive to calcium ions and has a molecular target size similar to the quisqualate-subtype of excitatory amino acid receptors. The change in binding parameters of the two sites with radiation dose strongly suggests that low affinity, calcium-insensitive [3H]kainate binding sites are equivalent to quisqualate sites. PMID- 3010194 TI - Lack of evidence for impaired dopamine receptor function in experimental hepatic coma in the rabbit. AB - In order to determine whether hepatic coma is associated with an altered sensitivity of the dopamine (DA) receptor in the brain, the activity of DA sensitive adenylate cyclase was assayed in homogenates from the corpus striatum of normal rabbits and rabbits with galactosamine-induced fulminant hepatic failure by measuring cyclic adenosine monophosphate production from adenosine triphosphosphate radioimmunochemically. The kinetic properties of adenylate cyclase in control rabbits and rabbits with hepatic coma were similar (Km, 17 +/- 2.9 (S.E.M.) vs 20 +/- 6.4 mM; Vmax, 816 +/- 58 vs 1054 +/- 233 pmol/mg protein/5 min, respectively). Hepatic coma was not associated with any changes in the responses of the DA receptor-adenylate cyclase system to DA (maximum stimulation 60% vs 57%), sodium fluoride (maximum stimulation 104% vs 132%), or a D-2 DA receptor agonist (maximum inhibition 18% vs 12%). These findings make it unlikely that alterations of dopaminergic neurotransmission play an important role in the pathogenesis of hepatic coma. PMID- 3010196 TI - Acetylcholine receptors and acetylcholinesterase activity in soleus muscle of trembler dysmyelinating mutant: a cytochemical and biochemical analysis. AB - We performed comparative biochemical and morphological studies of trembler and control soleus muscles. In the mutant, small multiple endplates were observed on some muscle fibers. The acetylcholine receptor (AChR) concentration and the acetylcholinesterase (AChE) activity of the muscle were not modified in the mutant. Our results suggest that both AChR and AChE levels are similar in trembler and control soleus but that these molecules are localized differently in the sarcolemma of the mutant muscles. PMID- 3010195 TI - gamma-Aminobutyric acid-dependent motility induced by avermectin B1a in the isolated intestine of the guinea pig. AB - In the isolated ileum of the guinea pig, neurally mediated rhythmic longitudinal mechanical activity was induced by avermectin, a macrolide anthelmintic that releases gamma-aminobutyric acid (GABA) and modulates the GABAA-receptor ionophore complex. This avermectin-induced activity was dependent on GABA, being reduced or abolished by bicuculline, a GABAA-receptor antagonist, and by 3 mercaptopropionic acid which prevents neural GABA release. These results provide additional direct evidence that GABA is a functional neurotransmitter in the myenteric plexus of the guinea pig intestine, evidently involved in the regulation of intestinal motility. PMID- 3010198 TI - The effects of aging on physiological properties of fast and slow twitch motor units in the rat gastrocnemius. AB - The mechanical properties of individual motor units were studied in the medial gastrocnemius of old (26-30 months) rats anesthetized with urethane and chloralose. The data were compared with results from motor units in the same muscle of middle-aged (11-14 months) rats. In the old rats there was a significant increase in mean tetanic tension for type S (slow twitch) units, whereas there was a slight decrease for type F (fast twitch) units. Twitch contraction time after maximum post-tetanic potentiation was shortened in type F units, but not in type S units. Conduction velocity of motor axons decreased in both unit types, but more marked change was found in type F units. These findings suggest the differential effects of aging on type F and type S motoneurons. PMID- 3010197 TI - Aspartate-like immunoreactivity in mitral cells of rat olfactory bulb. AB - Antisera against aspartate (Asp) were prepared by immunizing rabbits with Asp conjugated to bovine serum albumin by glutaraldehyde, after which the antisera were purified using an Asp-immobilized epoxy-activated affinity column. The purified Asp antiserum showed no cross-reactivity, except for a 3% cross reactivity against D-Asp. Asp-like immunoreactivity in mitral cells of the rat olfactory bulb was demonstrated, using this purified Asp antiserum. PMID- 3010199 TI - Control of sensorimotor function by dopaminergic nigrostriatal neurons: influence on eating and drinking. AB - The literature on the effects of lesions of the lateral hypothalamic area (LHA) on eating and drinking is reviewed in an effort to understand the function of the neural substrate destroyed. The data suggest that damage to the dopaminergic nigrostriatal neurons that course through the LHA results in a decrease in sensorimotor facilitation; that is, an increase in the threshold for responding to stimuli that elicit orientation, approach and consumption. This increase results in decreased consumption of food and water. Evidence is also reviewed suggesting the possibility that striatal dopaminergic activity may mediate a negative feedback signal related to blood glucose level that influences responsiveness to food, and therefore eating. There is no evidence that the nigrostriatal system mediates a similar signal related to water balance and drinking. A second deficit associated with LHA lesions, caused by damage to the pallidofugal neurons that descend through this area, is a dysfunction of motor control of the head and mouth. This results in an increase in the effort required to consume food and water, also leading to decreased consumption. These two behavioral factors: an increased threshold for responding to the sensory properties of food and water and an increase in the effort required to eat and drink are used to explain the symptoms making up the lateral hypothalamic syndrome without postulating changes in physiological regulatory (set point) mechanisms. PMID- 3010200 TI - Epidemiology of dietary fibre and colorectal cancer: current status of the hypothesis. AB - The hypothesis that lack of dietary fibre in the diet is responsible for a variety of large bowel problems, including cancer, has stimulated much discussion and research over the past 15 years. However, the epidemiological examination of this hypothesis has been hampered by the absence of data on the fibre content of most of the world's foods. In Scandinavia and Britain where the consumption of the major chemical fraction of dietary fibre, the non-starch polysaccharides, has been measured using accurate methods, significant negative associations have been shown with colon cancer occurrence. These studies suggest that non-starch polysaccharides may be protective in populations at otherwise high risk of colon cancer from an excess of meat and fat. However, methodological problems in the assessment of non-starch polysaccharide consumption in individuals preclude the use of case control studies in verifying these associations within a single homogeneous population. PMID- 3010201 TI - A comparison of the ages of patients with cervical smears showing human papilloma virus, dysplasia and carcinoma in situ changes. PMID- 3010202 TI - Bee venom and arthritis: magic, myth or medicine? PMID- 3010204 TI - Second-look laparotomy in the management of malignant germ cell tumors of the ovary. AB - From 1970 to 1985, 53 patients with malignant nondysgerminomatous germ cell tumors of the ovary underwent second-look laparotomy after initial surgery and combination chemotherapy. Twenty-two patients had immature teratoma, 15 had endodermal sinus tumor, 15 had mixed germ cell tumor, and one patient had embryonal carcinoma. Thirty-one of the neoplasms were stage I, four were stage II, 17 were stage III, and one was stage IV. Two patients received a combination of actinomycin-D, 5-fluorouracil, and cyclophosphamide; four patients received vinblastine, bleomycin, and cisplatin; 44 patients received vincristine, actinomycin-D, and cyclophosphamide; and three patients received a combination of the last two regimens. Second-look findings were negative in 52 patients and positive in one patient who was subsequently salvaged with further chemotherapy. One patient with stage I endodermal sinus tumor relapsed nine months after a negative second-look laparotomy and died. Two patients with negative findings subsequently died of leukemia. Of 53 patients undergoing second-look laparotomy, three are dead (one of cancer and two of leukemia), and 50 patients are surviving without disease. Although the precise role of second-look laparotomy in patients with malignant germ cell tumors is yet to be established, possible indications are discussed. PMID- 3010205 TI - Bartholin gland carcinoma. AB - Bartholin gland tumors are rare and management recommendations have been based on limited information. This report summarizes a 30-year clinical experience involving Bartholin gland carcinoma in 36 patients whose five-year survival rate was 84%. FIGO stages of the 36 tumors were stage I, nine; stage II, 15; stage III, ten; and stage IV, two. Cell types were: squamous, 27 (three nonkeratinizing with areas of a transitional component); adenomatous, six; adenoid cystic, two; and adenosquamous, one. Fourteen of 30 (47%) patients with lymph node dissections had nodal metastases and 11 remain disease-free. Disease recurred in nine patients (six local, two distant, one local and distant) and four were treated successfully. One of 14 (7%) patients receiving radiation and six of 22 (27%) patients not receiving radiation developed local recurrences. Wide excision (often necessitating a radical hemivulvectomy), ipsilateral inguinal lymphadenectomy, and adjunctive irradiation to the vulva and regional lymph nodes produced excellent results. PMID- 3010203 TI - Cytoreductive surgery in ovarian carcinoma: feasibility and morbidity. AB - Between 1974 and 1984, 70 patients underwent primary cytoreductive surgery for ovarian carcinoma at the University of California at Los Angeles. During the period of January 1974 to December 1978, optimal cytoreduction was achieved in 56.4% of the patients. With increased experience, this figure improved to 87.1% in the period of January 1979 to December 1983. The most common morbidity associated with the procedure was fever and prolonged ileus. Bowel resection was required in 20% of the patients and was not associated with increased morbidity. More liberal use of the end-to-end anastomosis stapling device facilitated low colon reanastomosis without colostomy, which contributed to the improved patient acceptance. PMID- 3010207 TI - [The synthesis of monodispersed spherical silica as a filler to be used in the manufacture of composite resins]. PMID- 3010206 TI - Danger? Asbestos. PMID- 3010208 TI - Light and electron microscopic evidence for a direct retinal projection to the pulvinar in the Asiatic chipmunk, Tamias sibricus. PMID- 3010209 TI - Steroids and neuroendocrine hormones detected by the immunoperoxidase technique from malignant melanomas and nevi of the choroid and conjunctiva. AB - By the peroxidase-antiperoxidase technique, the authors studied 7 malignant choroidal melanomas, 7 conjunctival nevi and 1 malignant conjunctival melanoma with the aim to detect the presence of vasoactive intestinal polypeptide (VIP), adrenocorticotropic hormone (ACTH), gastrin, estradiol and testosterone. Positive staining reaction for VIP, estradiol and testosterone was observed in both malignant melanomas of the choroid and conjunctival nevi. The case of conjunctival melanoma was positive for VIP and ACTH but not for estradiol and gastrin. PMID- 3010210 TI - Congenital cataract and evolutive myopia. Relationship with hypophyseal-adrenal cortical axis function. AB - To see whether or not reduced light input in children with congenital cataract produces degenerative myopia and whether it is associated with an impairment of the hypophyseal-adrenal cortical axis, the authors studied biometric values and circadian rhythm and cortisol reserve in nine children with monolateral and ten children with bilateral cataract. 38.75% of patients had: refractions less than 10D, increased antero-posterior diameter of the eyeball, varying degrees of myopic chorioretinitis, showing that the lack of light input induces degenerative myopia only in a group of patients who are probably genetically predisposed. The negative correlation between cortisol reserve and ocular lesions seems to reveal a precocious negative effect of the lack of light input on the development of the hypothalamic-hypophyseal-adrenal cortical axis. The alteration of the circadian cortisol rhythm and the reduction of the cortisol reserve found exclusively in children with monolateral cataract must be studied further to be explained. The slight increase in plasma cortisol levels earlier described in adults with acquired degenerative myopia was not found in children with congenital cataract. This difference could be due to: special endocrine characteristics in children with congenital cataract, endocrine differences between childhood and adulthood, differing influences of hormonal factors on myopia, or vice versa, in childhood and adulthood. PMID- 3010211 TI - The incidence of oral herpes simplex virus infection in patients undergoing cancer chemotherapy. AB - Oral lesions in twenty-nine immunocompromised patients were evaluated for the incidence of herpes simplex virus (HSV) infections during either cancer chemotherapy or cancer chemotherapy plus bone marrow transplantation (BMT). Patients' HSV antibody titers were not determined, and positive diagnoses were based solely on the results of viral cultures. Fourteen patients (48%) were found to have herpetic infections, which is comparable with incidence rates of 50% to 90% in antibody-positive patients and 40% to 50% in mixed antibody populations reported in the medical literature. However, this finding is in conflict with the 10.7% to 15.1% incidence rate cited in the dental literature for patients undergoing cancer chemotherapy. This underestimation is believed to reflect insensitivity in the criteria used for diagnosis in these studies. PMID- 3010212 TI - Immunoperoxidase investigation of Regan isoenzyme of alkaline phosphatase in maxillary sinus carcinomas. AB - The Regan isoenzyme of alkaline phosphatase is known to be produced ectopically by various malignant tumors. To assess whether this isoenzyme is expressed in maxillary sinus carcinomas, immunostaining for Regan isoenzyme was carried out on 35 maxillary sinus carcinomas comprising 27 squamous cell carcinomas, 6 adenocarcinomas, and 2 adenoid cystic carcinomas. The enzyme was shown to be present in 11 of 27 cases of squamous cell carcinoma, 2 of 6 cases of adenocarcinoma, and 1 of 2 cases of adenoid cystic carcinoma. Regan isoenzyme was identified in the cytoplasm and/or cell membrane of squamous cell carcinoma cells, whereas it was demonstrated mainly on the luminal membrane and in the secretions of both adenocarcinoma and adenoid cystic carcinoma cells. These findings show that Regan isoenzyme does, indeed, occur in maxillary sinus carcinomas. PMID- 3010213 TI - Collagen--the predominant protein of the tectorial membrane. AB - Evidence is presented that, in contrast to the traditional view, a large proportion of the proteins of the tectorial membrane (TM) consists of collagen, primarily type II: characteristic amino acid composition of TM, including high levels of glycine, hydroxyproline and hydroxylysine; comigration of the main TM protein with appropriate collagen standards in two-dimensional gel electrophoresis; digestion of the main TM protein band following treatment with bacterial collagenase; one- and two-dimensional peptide mapping of cyanogen bromide digests of the TM exhibits patterns characteristic for collagen type II. PMID- 3010214 TI - Aminoglycoside-induced hearing loss: a molecular hypothesis. AB - A multi-step hypothesis of aminoglycoside ototoxicity is presented which is consistent with the data elaborated by our laboratory and others. The first step in the reaction sequence is an electrostatic interaction of the aminoglycoside with negatively charged components of the outer plasma membrane. The resulting displacement of calcium accounts for the acute effects of the drug action and is reversible and antagonized by cations. The drug is then transported into the cell by an energy-dependent process. The next and most crucial step is the binding of the drug to phosphatidylinositol bisphosphate, a physiologically important phospholipid. The formation of the drug-lipid complex prevents the hydrolysis of phosphatidylinositol bisphosphate and disturbs membrane integrity and structure resulting in nonspecific permeability changes of the membrane. Once inside the cell, the aminoglycosides may interfere with further intracellular reactions. This interference may be based on competition with divalent cations or polyamines or on binding to negatively charged compounds. PMID- 3010215 TI - [Results of roentgenotherapy of chemodectoma of the ear]. PMID- 3010216 TI - [Evaluation of the use of bronchial brushings after cytocentrifugation in the diagnosis of bronchial tumors (475 fibroscopies)]. AB - Since 1971, cytodiagnosis of lung tumors by association of the bronchial brushing and cytocentrifugation techniques has been carried out routinely at the University Hospital of Angers. Our study covers 475 cytodiagnoses of bronchial brushings. The results obtained enable an evaluation of the diagnostic efficiency of bronchial brushing in the detection of lung tumors. The technique is convenient for routine screening and in several cases contributes directly to diagnosis. PMID- 3010217 TI - [Changes in the properties of the monoamine receptors in the organs of tumor carriers]. PMID- 3010218 TI - [Polyfunctionality of bone marrow peptides]. PMID- 3010219 TI - [Acquired immunodeficiency syndrome]. PMID- 3010221 TI - [Demonstration of carcinoembryonic antigen (CEA) in breast carcinoma using an immunohistochemical method: morpho-functional evaluations and diagnostic uses]. PMID- 3010220 TI - [Leukotrienes and the cardiovascular system]. PMID- 3010222 TI - Hypertrophic osteoarthropathy in a young child with cytomegalovirus pneumonia and the bare lymphocyte syndrome. AB - A case of hypertrophic osteoarthropathy is reported in a 3-year-old Turkish girl. She had combined immunodeficiency, later shown to be the bare lymphocyte syndrome, and chronic pneumonia. Lung biopsy showed cytomegalovirus. The child developed painful elbow and knee joints and hypertrophic osteoarthropathy was demonstrated radiologically. PMID- 3010223 TI - Protective effect of subinhibitory polymyxin B alone and in combination with ampicillin for overwhelming Haemophilus influenzae type B infection in the infant rat: evidence for in vivo and in vitro release of free endotoxin after ampicillin treatment. AB - The potential endotoxin modifying effects of subinhibitory doses of polymyxin B were evaluated in an animal model of overwhelming septicemia. Five to six day old Sprague-Dawley rats were infected intraperitoneally with 10(6)-10(7) cfu of Haemophilus influenzae type b. At 12 h after infection, at which time mortality was 18%, subinhibitory doses of polymyxin b (0.0125 mg/kg X 3 q 3 h) either alone or in combination with 500 mg/kg ampicillin significantly increased survival at 17 and 20 h (p = 0.009, 0.01 and p = 0.003, 0.01) compared to animals treated with 0.5 mg/kg of ampicillin alone. Prolonged survival at 36 h (p = 0.009) was seen in animals receiving both ampicillin and low dose polymyxin compared to either ampicillin dose alone. Ampicillin significantly reduced the number of bacteria in blood of survivors (p less than 0.023 at 30 min) compared to untreated animals but increased the activity of free endotoxin at 30 min compared to controls (p = 0.006). In vitro endotoxin release from H. influenzae type b increased 5-fold after addition of 100 micrograms/ml of ampicillin, whereas a six fold reduction in endotoxin activity was measured after the addition of 7 micrograms/ml of polymyxin B. Subinhibitory doses of polymyxin B modulate the ethal effects of overwhelming H. influenzae type b infection in infant rats and might be beneficial as adjunct treatment in gram-negative septicemia. PMID- 3010224 TI - Role of aldosterone for control of colonic NaKATPase activity in weanling rats. AB - Experiments were performed to examine how aldosterone modulates colonic NaKATPase activity during the weaning period. NaKATPase activity was determined in proximal and distal colon in control rats aged 16, 20, and 40 days, in rats aged 20 and 40 days fed low sodium diet for 4 days and in 20-day-old rats adrenalectomized on day 16. In some protocols net sodium and water transport was determined with in vivo perfusion technique. In control rats colonic NaKATPase activity increased significantly between day 16 and 20. This increase was abolished by adrenalectomy but restored by aldosterone substitution, 5 micrograms/100 g body weight/12 h. No significant increase in NaKATPase activity occurred between day 20 and 40. Serum levels of both aldosterone and of corticosterone were low until day 14 and increased to peak level at day 18-20. In 20- and 40-day-old rats fed a low sodium diet, NaKATPase activity increased significantly in proximal and distal colon in both age groups but the increases were significantly greater in the 20- than the 40-day-old animals. A low sodium diet increased serum aldosterone, but not serum corticosterone levels in both age groups: also the low sodium diet significantly increased net sodium and water transport in 20- but not in 40-day-old rats. Aldosterone is of physiological importance for the regulation of NaKATPase activity in the colon at the time of weaning. The immature colon may have an enhanced sensitivity to aldosterone. PMID- 3010225 TI - Presence of respiratory viruses in middle ear fluids and nasal wash specimens from children with acute otitis media. AB - During a 28-month period, 84 children with acute otitis media were studied by viral and bacterial cultures of middle ear fluid and viral cultures of nasal lavage fluid. Viruses were isolated from the middle ear fluid of 17 (20%) patients. Evidence of viral infection was demonstrated by positive viral cultures of middle ear fluid and/or nasal lavage fluid in 33 (39%) patients. Rhinovirus in one patient and influenza b virus in another were the only pathogens isolated. Influenza virus, enterovirus, and rhinovirus were the most common viruses found in middle ear fluids. Parainfluenza virus, adenovirus, and respiratory syncytial virus were found less often. In 82% of cases, the virus isolated from middle ear fluid was also isolated from nasal lavage fluid, but only 44% of viruses found in nasal lavage fluid were also found in middle ear fluid. Mixed bacterial and combined viral-bacterial infections were common. Only 15% of patients had no pathogen isolated from middle ear fluids. Using tissue culture techniques, we demonstrated that enterovirus and rhinovirus are also common middle ear pathogens. Our data reemphasize the significance of viruses as etiologic agents of acute otitis media and propose several questions regarding the viral-bacterial interactions and the types of viruses involved in the pathogenesis of the disease. PMID- 3010226 TI - [New immunotherapy methods for cancer. II. Anti-tumour mechanisms of interferon (IFN)-gamma analysed in an adult T cell leukemia patient with remarkable tumor regression]. PMID- 3010228 TI - Processing in the external transcribed spacer of ribosomal RNA from Physarum polycephalum. AB - The rDNA of the myxomycete Physarum polycephalum is transcribed to give a 13.3 kb precursor of ribosomal RNA. At 1.7 kb downstream of the primary initiation site there is a processing site or a second initiation site. This site was studied by S1-mapping, DNA sequencing and electron microscopy. None of these methods could conclusively distinguish between the two formal possibilities. However, capping experiments indicate that rapid processing is taking place at this site rather than reinitiation. In addition, primary transcripts and processed molecules were assayed throughout the synchronous mitotic cycle. During all interphase stages newly initiated transcripts of rDNA and products of the first processing step are present in similar amounts, indicating control of initiation and not of maturation as being the main regulatory step for the accumulation of mature rRNAs. During the brief period of mitosis the level of newly initiated rRNA precursors is lowered. PMID- 3010227 TI - [Long-term embolization of the portal vein with isobutyl-2-cyanoacrylate in hepatoma]. PMID- 3010230 TI - Nucleotide sequence of an heterochromatic segment recognized by the antibodies to Z-DNA in fixed metaphase chromosomes. AB - The purpose of this work was to analyse at the molecular level the DNA recognized by the antibodies to Z-DNA in in situ experiments. Antibodies to Z-DNA interact strongly with R-band positive heterochromatic segments of fixed metaphase chromosomes of Cebus (Viegas-Pequignot et al., 1983). These segments are constituted of a satellite DNA the repeat unit of which is about 1520 base pairs long. The base sequence of the repeat unit has been determined. It contains a (AC)n rich region which, in vitro, adopts the Z conformation under topological constraints. Experiments with nuclei suggest that this sequence is not predominantly in the Z conformation in vivo. The polymorphic structure of the (AC)n rich region argues for an active recombination sequence. PMID- 3010229 TI - The gene for Spirodela oligorhiza chloroplast ribosomal protein homologous to E. coli ribosomal protein L16 is split by a large intron near its 5' end: structure and expression. AB - The nucleotide sequence of a Spirodela chloroplast DNA fragment, which directs the synthesis of a approximately 15 kD chloroplast ribosomal protein in an E. coli cell free system, has been determined. The deduced aminoacid sequence of the open reading frame shows extensive homology with E. coli ribosomal protein L16. Primer extension analysis, S1 nuclease mapping and nucleotide sequence analysis indicate that the chloroplast L16 gene (rpl16) is interrupted by a 1411 bp intron, which separates a short 5' exon from a large 3' exon. The shorter in vitro synthesized ribosomal protein results from an artificial initiation event at an internal ATG codon in the 3' exon. The sequences at the 5' and 3' splice sites of the intron are similar to consensus sequences described for other, class II intron containing, protein coding chloroplast genes. Northern hybridization experiments reveal 6 stable transcripts of rpl16 ranging from 500 b to greater than 4000 b. As determined by S1 nuclease mapping, the 3'-end of the smallest transcript maps exactly after the stem of a proposed termination signal. Finally, the implications of the finding of a cluster of several chloroplast ribosomal protein genes and possible polycistronic transcription of this chloroplast DNA region, are discussed in relation to the organization and expression of ribosomal protein genes found in the S10 operon of E. coli. PMID- 3010231 TI - The stiffness of dsRNA: hydrodynamic studies on fluorescence-labelled RNA segments of bovine rotavirus. AB - The sedimentation coefficients of dsRNA segments of bovine rotavirus were determined in the analytical ultracentrifuge. The eleven segments were separated by preparative gel electrophoresis, and isolated by elution from gel pieces. The RNA was labelled by the intercalating fluorescent dye ethidium bromide at a ratio bound dye per base pair between 0.003 to 0.018. The analytical ultracentrifuge was equipped with a fluorescence recording optics. Sedimentation coefficients could be determined with amounts of RNA as little as 8 ng. All sedimentation coefficients were extrapolated to zero-concentration, zero-dye binding, and zero impurities from the preparative gel electrophoresis. The hydrodynamic model of flexible cylinders was applied for the interpretation of the sedimentation coefficients. All dsRNA segments of rotavirus (663-3409 base pairs) and the dsRNA5 of cucumber mosaic virus (335 base pairs) fit the model of a "worm-like" or flexible cylinder with a persistence length of 1125 A and a hydrated diameter of 30 A. The results are compared with data from the literature on the persistence lengths of the B- and Z-forms of dsDNA and of viroids. PMID- 3010232 TI - Supercoil induced S1 hypersensitive sites in the rat and human ribosomal RNA genes. AB - Rat and human ribosomal RNA gene fragments in supercoiled plasmids were examined for S1 nuclease hypersensitivity. In the transcribed portion of genes the number and distribution of S1 sites were found to be species specific. No S1 sites were detected in the promoter regions. In the nontranscribed spacer (NTS), downstream of the 3' end of 28S RNA gene, S1 sites appear to be conserved in rat and human rDNAs. A rat NTS fragment (2987 nucleotides long), containing three S1 sites was sequenced and the S1 sites in this region were localized in polypyrimidine . polypurine simple repeat sequences. Other types of simple sequences, two type 2 Alu repeats and an ID sequence were also found in the sequenced region. The possible role of simple sequences and S1 sites in transcription and in recombination events of rDNA is discussed. PMID- 3010233 TI - The genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase are expressed differentially in petunia leaves. AB - We have isolated five members of the multigene family encoding the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase in petunia and examined their expression in petunia leaves. Of the five rbcS genes, two (ssu11A and ssu8) are expressed at high levels in petunia leaves. Northern analysis using gene specific oligonucleotide probes revealed that ssu11A accounts for 40% of the total rbcS transcripts in petunia leaves while ssu8 accounts for 4 to 5% of the total rbcS transcripts. Structural comparisons of ssu8 and ssu11A revealed that the coding sequence of ssu8 is interrupted by three introns, while the coding sequence of ssu11A is interrupted by two introns. The positions of the first two introns are identical, the third intron in ssu8 is located in a highly conserved region of the protein. The 5' and 3' flanking sequences of ssu11A are highly homologous to the 5' and 3' flanking sequences of ssu8. S1 nuclease mapping was used to locate the start of transcription of ssu8 and ssu11A and showed that ssu8 mRNA leader differs in sequence from the ssu11A mRNA leader. PMID- 3010234 TI - The chicken fast skeletal troponin I gene: exon organization and sequence. AB - The gene encoding the fast skeletal isoform of the chick troponin I (sTnI) protein has been sequenced and its organization into exons and introns established. The gene is approximately 4.5 kb in length and composed of 8 exons, the first of which contains solely 5' untranslated sequence. In addition to its major mRNA product, there is evidence that the sTnI gene encodes a second mRNA, present at low abundance levels in embryonic skeletal muscle. Sl nuclease protection and primer extension experiments indicate that the low abundance mRNA is initiated approximately 47 nucleotides upstream of the major transcriptional initiation site. Both mRNAs appear to encode identical sTnI polypeptides. A comparison of nucleotide sequence in the 5' flanking region of several muscle specific genes, including the sTnI gene, reveals a heptanucleotide consensus sequence, 5'-CATTCCT-3', which is conserved in the 5' flanking regions of many vertebrate contractile protein genes. PMID- 3010235 TI - Studies on HSAG, a middle repetitive family of genetic elements which elicit a leukemia-related cellular surface antigen. AB - HSAG is a family of genetic elements capable of eliciting, in transfected cells, a cellular surface antigen which is correlated with human chronic lymphocytic leukemia (CLL). Its prototype member, HSAG-1, was cloned as a 3.4 kb insert and contains numerous Alu-related elements, including its left hand 1.4 kb antigen eliciting end. These elements are present in mammalian cells with copy numbers varying from 7,000 to 200,000 per haploid genome, depending on how closely their sequence conforms to the Alu consensus sequence. They are present in the configuration found in HSAG-1, a 3.4 kb EcoRI fragment which is part of a larger unit of at least 12.7 kb, at a frequency of 20-50 per haploid genome, and dispersed around the genome. A second family member, HSAG-2, isolated using a functional assay, was cloned as a 9.5 kb insert and contained a 1.5 kb antigen eliciting left hand end. As in HSAG-1, the antigen-eliciting portion of the insert also contained Alu-like elements, unlike most of the remainder of the insert. A number of HSAG family members were cloned from a library of human CLL genomic DNA by sequence homology with the antigen-eliciting portion of HSAG-1. Most of these members were also shown to be capable of eliciting antigen. Their only sequence similarity with HSAG-1 appeared to be in their content of numerous Alu-like elements. The evidence thus supports the view that the HSAG functional family consists of clusters of Alu-like elements. PMID- 3010237 TI - DNA sequence of the region in the genome of herpes simplex virus type 1 containing the exonuclease gene and neighbouring genes. AB - We report the sequence of a 7800 base pair region of herpes simplex virus type 1 DNA, representing approximately 0.16 to 0.20 map units in the genome. This contains sequences transcribed into a leftward oriented set of five 3' coterminal mRNAs, together with two rightward transcribed flanking genes. One of the leftward genes encodes the virus's alkaline exonuclease, but the other gene products are uncharacterized. The amino acid sequence of one encoded protein suggested that it is a membrane embedded species. The DNA sequence is densely utilised, with two predicted out-of-frame overlaps of coding sequences, and probably six occurrences of promoter elements within coding sequences. Homologues of five of the genes were found for the distantly related Epstein-Barr virus, with a similar overall relative arrangement. PMID- 3010236 TI - The structure of HSAG-1, a middle repetitive genetic element which elicits a leukemia-related cellular surface antigen. AB - HSAG-1 is a cloned member of a heterogeneous middle repetitive family of genetic elements which is capable of eliciting a leukemia-related surface antigen detected with a monoclonal antibody after DNA transformation of mouse cells. HSAG 1 was originally isolated from a Chinese hamster-human leukemia hybrid cell gene library both by sib-selection for antigen producing activity and by hybridization with labelled human genomic human DNA. We show here that the human labelled site is at the right hand end of the insert, while the antigen-eliciting portion is included in a 1450 bp fragment at the left hand end of the insert. We also present the complete nucleotide sequence of the 3369 bp insert. The sequence contains 12 elements which bear a significant resemblance to accepted consensus sequences for Alu repetitive elements. The right hand end contains adjacent elements with close sequence similarity to portions of the human and hamster type I and type II Alu consensus sequences. All of the other Alu-related elements have diverged relative to the Alu consensus sequences by additions, long deletions and substitutions. The left hand portion of the insert which has the antigen producing activity contains four of these diverged elements representing a relatively high proportion (26%) of the nucleotide sequence. The sequence is thus consistent with our previous observations of a repetitive family with biological function. PMID- 3010238 TI - Role of thymidine residues in DNA recognition by the EcoRI and EcoRV restriction endonucleases. AB - We have synthesized a series of oligonucleotides containing the EcoRI (GAATTC) or EcoRV (GATATC) recognition site within which or adjacent to which thymidine was substituted by uridine or derivatives of uridine. The effects of these substitutions on the rate of the EcoRI and EcoRV catalyzed cleavage reaction were investigated. Our results show that most of the substitutions within the site are quite well tolerated by EcoRI, not, however, by EcoRV. We conclude that the thymin residues most likely are not directly involved in the recognition process of the EcoRI reaction. In contrast, they are major points of contact, between substrate and enzyme in the EcoRV reaction. The effects of substitutions in the position adjacent to the recognition site is also markedly different for EcoRI and EcoRV. Here, EcoRI seems to be considerably more selective than EcoRV. PMID- 3010239 TI - A 'hot-spot' for Ty transposition on the left arm of yeast chromosome III. AB - The small ring derivative of Saccharomyces cerevisiae chromosome III, which was formed by a cross-over between HML on the left arm and HMR on the right arm, contains three Ty elements. The class II element Ty 1-17 lies immediately centromere-distal to LEU2 on the left arm while two class I elements are tandemly arranged distal to PGK on the right arm. We have sequenced the regions of chromosome III surrounding Ty 1-17 and have defined a region where a number of transposition events have occurred. This region is flanked by the 5' ends of two tRNA genes, tRNA3Glu on the centromere distal side and tRNA3Leu immediately in front of LEU2. Close to the tRNA3Glu gene there is a region containing degenerate delta sequences organised in opposite orientations. Immediately distal to Ty 1-17 there are two complete solo delta elements, one inserted into the other. The sequence indicates that these two delta sequences were inserted into chromosome II by separate transposition events. A model is presented to explain how this structure arose and the role of solo delta elements in transposon propagation and maintenance is discussed. PMID- 3010240 TI - Sequences of three promoters for the bacteriophage SP6 RNA polymerase. AB - Fragments of SP6 DNA generated by cleavage with Hpa II or Taq I were cloned into the Cla I site of pBR322 and the recombinant plasmids were screened for the presence of SP6 promoter activity by transcription in vitro with purified SP6 RNA polymerase. Three plasmids having promoter activity and small inserts of SP6 DNA were characterized. Hybridization studies showed that all three cloned promoters arose from different regions of the SP6 genome. Comparison of the consensus promoter sequence (5' ATTTAGGtgGACACTATAGAAGgaG 3') with the consensus sequences of promoters recognized by the T3 and T7 RNA polymerases reveals a common core sequence (5'-CACTA-3') extending from -7 to -3, as well as other features that may be important in selective promoter recognition by the phage RNA polymerases. PMID- 3010241 TI - Multiple mechanisms generate extrachromosomal circular DNA in Chinese hamster ovary cells. AB - Seven cloned small circular DNA molecules from CHO cells were sequenced and examined for the presence of homologies to each other and to a number of other functional sequences present in transposable elements, retroviruses, mammalian repeat sequences, and introns. The sequences of the CHO cell circular DNA molecules did not reveal common structural features that could explain their presence in the circular DNA population. A gene bank was constructed for CHO chromosomal DNA and sequences homologous to two of the seven small circular DNA molecules were isolated and sequenced. The nucleotide sequences present at the junction of circular and chromosomal DNA suggest that a recombination process involving homologous pairing may have been involved in the generation of one, but not the other, of the two circular DNA molecules. PMID- 3010244 TI - Ptac-85, an E. coli vector for expression of non-fusion proteins. PMID- 3010242 TI - Mouse muscle nicotinic acetylcholine receptor gamma subunit: cDNA sequence and gene expression. AB - Clones coding for the mouse nicotinic acetylcholine receptor (AChR) gamma subunit precursor have been selected from a cDNA library derived from a mouse myogenic cell line and sequenced. The deduced protein sequence consists of a signal peptide of 22 amino acid residues and a mature gamma subunit of 497 amino acid residues. There is a high degree of sequence conservation between this mouse sequence and published human and calf AChR gamma subunits and, after allowing for functional amino acid substitutions, also to the more distantly related chicken and Torpedo AChR gamma subunits. The degree of sequence conservation is especially high in the four putative hydrophobic membrane spanning regions, supporting the assignment of these domains. RNA blot hybridization showed that the mRNA level of the gamma subunit increases by 30 fold or more upon differentiation of the two mouse myogenic cell lines, BC3H-1 and C2C12, suggesting that the primary controls for changes in gene expression during differentiation are at the level of transcription. One cDNA clone was found to correspond to a partially processed nuclear transcript containing two as yet unspliced intervening sequences. PMID- 3010243 TI - Expression of the major heat shock gene of Drosophila melanogaster in Saccharomyces cerevisiae. AB - A copy of the gene which encodes the major heat shock protein (hsp70) of D. melanogaster was integrated in both orientations into the genome of S. cerevisiae at the leu2 locus. The level of transcript from the D. melanogaster gene was measured under both normal conditions and conditions which are known to give rise to the heat shock response in S. cerevisiae. In both orientations the D. melanogaster gene gave rise to an abundant transcript in uninduced cells. The level of this transcript was increased transiently on heat shock, peaking after about 30 min at the elevated temperature. The average induction observed was around 5-fold. Although the D. melanogaster gene is heat inducible in S. cerevisiae, the transcripts are initiated at several sites which lie between 10 and 40 base pairs downstream of the initiation site in D. melanogaster. Thus, the transcriptional apparatus of S. cerevisiae appears to recognize the promoter and regulatory elements of the D. melanogaster major heat shock gene, although the manner in which transcription is initiated differs between the two species. PMID- 3010246 TI - Simplified preparation of unidirectional deletion clones. PMID- 3010245 TI - Improved transfection of CV-1 and COS 1 cells using reduced serum medium. PMID- 3010247 TI - Surgical management of cerebrovascular disease. AB - After careful evaluation to pinpoint abnormal vessels, surgery may be performed, either to improve blood flow or to repair leaky vessels. This approach can serve to prevent future stroke events. PMID- 3010248 TI - Dietary fiber and cancer: a supplement for intervention studies. AB - Dietary fiber is one of several variables being considered in the study of the relationship between diet and cancer. Intervention trials in which dietary fiber is increased are the most direct way of assessing the possible role of fiber in this disease. Two dietary snack products have been developed for use in a fiber intervention study: the high-fiber snack (HFS), which supplies 23 g of dietary fiber per day (mostly from wheat bran) and the low-fiber product (LFS), which provides 3.5 g. Over a 12-week period, 28 volunteers consumed the HFS for 6 weeks and the LFS for 6 weeks. Compliance, as assessed by reports, through recovery of a riboflavin marker in the urine and fecal fiber analysis, was good. The only adverse effects reported were mild abdominal discomfort and gas. Serum ferritin and calcium decreased in some subjects, indicating a need to supplement the products with these essential minerals. Consumption of the snacks did not affect total energy intake or the intake of the nutrients monitored. PMID- 3010249 TI - Dietary factors in colon cancer: international relationships. An update. PMID- 3010250 TI - Influence of wheat bran on some reductive and hydrolytic activities of the rat cecal flora. AB - For 30 days, male weanling rats were fed a semipurified, fiber-free diet or a diet that contained 5, 15, or 30% (wt/wt) wheat bran. The activities of four cecal microbial enzymes were determined. Wheat bran significantly increased the wet weight content of the cecum and total bacterial count per cecum at the intermediate- and high-treatment levels, but it had no effect on bacterial concentration per gram wet weight of cecal contents. Total beta-glucosidase and beta-glucuronidase activities per cecum were generally increased. Wheat bran decreased total nitrate reductase activity, but there was no change in total nitroreductase activity. Wheat bran significantly decreased enzyme activities for nitro-and nitrate reduction per gram of cecal contents but increased beta glucosidase activity. The activities of the enzymes per 10(11) bacteria followed a similar pattern to that noted per gram of cecal contents. Such fiber-dependent changes in enzyme activity may alter the steady-state concentration of toxic and genotoxic chemicals in the lumen of the hindgut. PMID- 3010251 TI - In vitro binding of steroid hormones by natural and purified fibers. AB - The in vitro binding of estrone, estradiol-17 beta, estriol, testosterone, dihydrotestosterone, and estrone-3-glucuronide by wheat, oat, and corn brans, oat hulls, cellulose, lignin, and cholestyramine resin was measured. The extent of steroid sequestration was characteristic and reproducible for each hormone. Cholestyramine bound an average of 90% of all the steroids tested, whereas cellulose bound the least (12%). Of the other substances tested, each bound the following percentage of unconjugated hormones: lignin, 87%; wheat and oat brans, 45% each; corn bran 44%; and oat hulls, 32%. The conjugated steroid was less likely to bind than the unconjugated steroids. Lignin appeared to be an important component in the interaction with steroid hormones. The results support the hydrophobic nature of adsorption and suggest that the components of fiber in diet should be considered separately when evaluating in vivo metabolic effects. PMID- 3010252 TI - [Value of determining angiotensin converting enzyme activity in the differential diagnosis of sarcoidosis]. PMID- 3010253 TI - [Effect of seasonal allergic exposure of patients with pollen-induced asthma on basophil susceptibility to the action of polymyxin B in vitro]. PMID- 3010254 TI - Ultrastructural alterations of the paraventriculo-infundibular corticotropin releasing factor (CRF)-immunoreactive neuronal system in long term adrenalectomized rats. AB - The corticotropin releasing factor (CRF)-immunoreactive paraventriculo infundibular neuronal system of long-term adrenalectomized and adrenalectomized short term dexamethasone treated rats was analyzed at the ultrastructural level using the preembedding peroxidase anti-peroxidase complex (PAP) immunohistological method. In both groups of animals, parvocellular neurons located in the medial and dorsal subnuclei of the paraventricular nucleus (PVN) showed CRF-like immunoreactivity. The perikarya contained hypertrophied rough endoplasmic reticulum (rER) with dilated cisternae, active Golgi-complexes and numerous neurosecretory granules. The majority of the neurosecretory granules measured 80-120 nm. Dendrites of CRF-immunoreactive neurons contained labeled vesicles, secretory granules, bundles of microtubules, a well-developed smooth endoplasmic reticulum (sER) complex and free ribosomes. Unlabeled terminal boutons of axons were observed to synapse on dendrites and somata of CRF-neurons. In addition, CRF perikarya were found in direct somato-somatic apposition with both CRF-immunopositive and immunonegative parvocellular cells. Retraction of glial processes and the existence of puncta adherentia between the cell membranes characterized these appositions. Varicose CRF axons within the median eminence contained hypertrophied sER, labeled vesicles and neurosecretory granules. The preterminal portions of the CRF-axons were dilated and possessed many labeled 80 120 nm diameter granules. CRF-terminals were greatly enlarged and established direct neurohemal contacts with the external limiting basal lamina of portal vessels without the interposition of tanycytic ependymal foot-processes. These tanycytes were not CRF immunopositive. CRF positive terminals contained clusters of microvesicles, labeled small vesicles and multivesicular bodies, but fewer granular elements than were observed within the the preterminals. Many of the labeled organelles were attached to tubules of sER. Occasionally, CRF-axons were observed within the pericapillary space adjacent to portal vessels. The ultrastructural features of CRF-neurons, obtained from adrenalectomized and adrenalectomized plus short-term dexamethasone treated rats did not differ significantly from each other. The hormone content of the entire CRF-neuron was greater in the steroid treated group. Adrenocorticotrophic hormone (ACTH) synthesizing cells in the pars distalis of adrenalectomized-dexamethasone treated rats also showed increased numbers of immunopositive secretory granules (150-320 nm in diameter).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3010255 TI - Synthesis and release of vasoactive intestinal polypeptide (VIP) by mouse neuroblastoma cells: modulation by cyclic nucleotides and ascorbic acid. AB - The mouse neuroblastoma cell line N18TG2 synthesizes and secretes a VIP-like immunoreactive material. The majority of this VIP-like material from both cell and media extracts elutes on HPLC in the same position as porcine or rat VIP. Several additional peaks which appear in the media extracts may represent variant forms or degradation products of VIP. The synthesis and release of VIP was significantly enhanced by agents which elevate cAMP levels directly (dbcAMP and forskolin) or through a receptor mediated process (secretin). These agents are also known to promote differentiation of these cells. The synthesis and release of VIP was also enhanced by ascorbate (thought to be a co-factor for the enzyme which amidates the carboxyl-terminal of VIP) [11]. In the presence of forskolin, ascorbate had a synergistic effect on the release of VIP, suggesting that forskolin and ascorbate are elevating VIP levels by different mechanisms; forskolin through a possible effect on VIP mRNA synthesis or translation, and ascorbate by increasing the rate of VIP processing. These results suggest that VIP synthesis and release is controlled by more than one process, whose rate can be altered with pharmacological agents. PMID- 3010256 TI - Covalent labeling of neurotensin receptors in rat gastric fundus plasma membranes. AB - Neurotensin receptors from plasma membranes of rat gastric fundus smooth muscle were specifically and covalently labeled either by using the photoreactive analogue 125I-labeled azidobenzoyl (Trp11)-neurotensin or by cross-linking (monoiodo-Tyr3)neurotensin to the membrane preparation by means of disuccinimidyl suberate. Analysis of plasma membranes by sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the same protein band with an apparent molecular weight of 110,000 was specifically labeled by both methods. This band consisted of a single chain protein since its apparent size was found to be the same with or without reduction of membrane samples before electrophoresis. Only neurotensin and its biologically active analogues were able to protect plasma membranes against specific labeling of the protein band of molecular weight 110,000. Comparison of these results with those obtained from rat brain synaptic membranes shows that although rat central and peripheral neurotensin receptors exhibit similar specificities towards a series of neurotensin analogues, their subunit structures are different. PMID- 3010257 TI - Interleukin 1 reduces opioid binding in guinea pig brain. AB - Interleukin 1 (IL1) is a macrophage-derived polypeptide which signals neurons in the preoptic-anterior hypothalamus to initiate fever and the acute-phase glycoprotein response. Recently, increases in cerebrospinal fluid and hypothalamic levels of beta-endorphin have been reported during endotoxin (LPS)- and IL1-induced fevers, suggesting that this opioid may participate in the modulation of IL1 effects in the CNS. In this study, we investigated whether purified (human) IL1 influences the specific binding of three prototypic opioid agonists (2-D-alanine-5-L-methionineamide, DAME; (-)-ethylketocyclazocine, EKC; dihydromorphine, DHM) and one antagonist (naloxone) to opioid receptor-enriched membrane preparations in cerebral cortex, hypothalamus, midbrain, pons, medulla, and cerebellum of guinea pig brain. IL1 reduced the binding of these ligands to their receptors during a 30-min incubation. The extent of IL1 inhibition of a given ligand for its binding sites varied according to the brain region; within some regions, the extent of this inhibition also varied with the four ligands tested. But in cortex the effect of IL1 on the specific binding of DHM is dose dependent. Similar results were obtained with crude homologous IL1. S. enteritidis endotoxin, suspended in pyrogen-free saline at concentrations from 4 to 36 micrograms/ml, did not inhibit the binding of these opioid ligands to their receptors in any brain region. These results indicate that IL1 interacts with the opiate receptors in guinea pig brain. This interaction, moreover, is not limited to the hypothalamus alone, the primary site of the pyrogenic action of IL1, but also occurs in other brain regions. PMID- 3010259 TI - Comparison of structural requirements of alpha-MSH and ACTH for inducing excessive grooming and pigment dispersion. AB - alpha-MSH and ACTH-like peptides are known to play an important role in the adaptation of many vertebrates to a new environment. These peptides induce pigment dispersion in amphibian melanophores through a receptor-mediated mechanism. In this study we compared the structural requirements of these peptides for melanotropic activity on Xenopus laevis melanophores with those for inducing excessive grooming in the rat. With the exception of ACTH1-24 there is a close resemblance in structure-activity relationships of the fragments and analogs tested in the two bioassays. [Nle4,-D-Phe7]-alpha-MSH is extremely active in both assays. Weak agonists such as [Leu9]-alpha-MSH did not possess antagonistic properties either in the melanophore assay or in the excessive grooming test. The data suggest that the mechanism of action of alpha-MSH-like peptides in rat brain is receptor-mediated like their action on melanophores. PMID- 3010258 TI - Effects of opiate receptor drugs injected intracerebrally into the normovolemic and hypovolemic monkey. AB - Systemic injection of naloxone (NAL), an opioid-receptor antagonist, significantly elevates systolic blood pressure (SBP) in anesthetized hypovolemic monkeys, providing indirect evidence that endogenous opioids contribute to cardiovascular depression during shock. The purpose of this study was to identify specific centrally located opioid receptor sites that participate in SBP regulation under normovolemic and hypovolemic conditions. In 6 monkeys, bilateral guide cannulae were stereotaxically implanted above areas ranging from the diencephalon to the lower medulla. Microinjections (1 microliter) of D- Ala2-Met enkephalinamide (DAME) (3.4-27.2 nM) into normovolemic unanesthetized monkeys reduced SBP by 10-65 mm Hg in a dose-related fashion. Subsequent injection of NAL (12.2 nM) attenuated this hypotensive response. Heart rate fell 20-40 bpm with DAME, but not in response to dose. In the anesthetized animal rendered hypotensive (SBP = 45 mm Hg) by hemorrhage. NAL injected into predetermined DAME sensitive sites failed to increase SBP more than 5 mm Hg. Even consecutive injections into multiple sites elevated SBP only 20 mm Hg. We conclude that the centrally located opioid-sensitive sites tested exert only a mild influence in mediating hemorrhagic hypotension. PMID- 3010260 TI - [Effect of naloxone on parathyroid hormone and calcitonin secretion and 25 hydroxyvitamin D levels in patients with acute renal failure]. PMID- 3010261 TI - [A case of renal tubular acidosis type 3 (mixed) with age-related decreased requirement for bicarbonates]. PMID- 3010262 TI - [Pathogenic retrovirus--current facts and hypotheses]. PMID- 3010263 TI - What's new in cerebral pathology in acquired immune deficiencies? AB - Over the last few decades, a new pathology has appeared, directly related to the modified immune status of the hosts. It presents several distinctive points. The central nervous system is particularly affected. The opportunistic pathogenic agents do not usually injure the brain parenchyma and are not known for their aggressiveness in normal adults (papovavirus). Diagnosis of these different diseases is often difficult, some biological tests being irrelevant because of alterations of the immune system (toxoplasmosis). The lesions may be exclusively located in the brain (tuberculosis, lymphomas) which is not usually affected by these agents. Response to therapy is frequently poor, the clinical course being rapid and fatal. However, therapy may be successful in some cases, justifying the use of somewhat aggressive procedures (biopsy) in order to obtain an accurate diagnosis. It is important to be aware of these data, and an understanding of them may help in managing these already difficult patients. They also make possible some interesting pathogenic hypotheses. PMID- 3010264 TI - Non epidermotropic cutaneous lymphoma ending in leukemia. A case of adult T cell lymphoma (ATL) in France? AB - A case of uncommon non Hodgkin malignant lymphoma with initial skin involvement is reported. The pathological pattern does not come under standard classifications. An aspect of non epidermotropic diffuse pleomorphic lymphoma is observed. In an immunological cell membrane study, the T-cell lineage of the malignant proliferation exhibited a very unusual pattern including the presence of the E rosette receptor and the expression of HLA-DR monomorphic determinant. This type of T cell malignant lymphoma can probably be included in the group of adult T cell leukemia/lymphoma (ATL) which is endemic in South Western Japan and occasionally observed in Western countries. The emergence of such cases in these countries raises important epidemiological questions. PMID- 3010265 TI - Causes of mortality in chickens that regressed a Rous sarcoma virus-induced tumor. AB - Rous sarcomas were induced in 6-week-old chickens of several genetically different stocks: inbred lines C, 6(1), 6(3), and 7(2); crosses of inbred lines (6(3) X 7(2)) F4 and (6(1) X 15(1)) F2 X 6(1); and reciprocal crosses (15(1) X 100) F1 X 15(1) and (15(1) X 100) F1 X 100. The resulting tumors were scored for size six times during a 10-week period. Females that had completely regressed their sarcomas were placed in individual laying cages and examined weekly for reappearance of a tumor. After death, the probable cause was determined by necropsy. The major causes of death in the pooled sample of 49 females were fatty liver hemorrhagic syndrome (24.6%), reproductive disorder (14.2%), Marek's disease (12.2%), and lymphoid leukosis (6.1%). Elapsed time between tumor regression and death from any cause ranged from 21 days to 1930 days (5.3 years). One tumor recurred, this in a bird which eventually died with a massive sarcoma in the left wingweb and Rous metastasis in liver tissue. These data provide evidence of specific resistance to neoplastic disease. PMID- 3010266 TI - Evaluation of broiler breeder pullet vaccination programs for preventing clinical reovirus infections in the progeny. AB - The efficacy of various reovirus vaccination programs in commercial broiler breeder pullets for preventing clinical reovirus infections in broiler progeny was studied in three experiments. In each, 70 1-day-old broilers from each of four different commercial poultry companies that used various combinations of live and killed reovirus vaccines in breeder pullets were used. Broiler progeny from each company were bled and serum analyzed for neutralization antibody to the S1133 reovirus. Twenty chicks from each company were challenged by foot pad with a virulent reovirus (S1133) and 20 chicks challenged with another reovirus (81-B) isolate to determine their susceptibility. An additional 20 chicks from each of the four groups were maintained as unchallenged controls. Chicks were examined for morbidity, mortality, and weight gain for a 3-week period. Three weeks after challenge, all chickens were necropsied for gross lesions. There was a direct correlation between the number of doses of live inactivated reovirus vaccines that pullets received to antibody titer and resistance to challenge in the day old progeny. Day-old progeny derived from breeder pullets that received one dose of live and two doses of inactivated reovirus vaccines had the highest numerical maternal antibody titer and best resistance to clinical infection following challenge. PMID- 3010267 TI - Effects of adding acid or base to the diet on semen of heat-stressed, aging broiler breeder males. AB - Males were fed breeder basal diet, basal diet + .67 g NaHCO3, + .43 g NH4Cl, or + .86 g NH4Cl/100 g basal and exposed to diurnal cyclic temperatures of 23.9 to 35 C in chambers from 24 to 54 weeks of age. Males fed basal diet + NaHCO3 or .43 g NH4Cl were significantly heavier in body weight by 32 to 39 weeks of age than males on basal diet. Males fed the NaHCO3 diet produced significantly more semen in the first 16 weeks than males on basal diet. No differences were found in percent packed sperm value (PSV), percent dead sperm, or percent abnormal sperm between semen of males fed NaHCO3 diet and basal diet. Spermatozoal motility was significantly higher for semen of males fed NaHCO3 or NH4Cl diets during the 32- to 39-week age period. PMID- 3010268 TI - First-trimester prenatal diagnosis of severe alpha-thalassemia. AB - By means of chorion biopsy together with restriction endonuclease analysis of fetal DNA, first trimester diagnoses were successfully made in 33 fetuses at risk for Bart's hydrops fetalis. Seven pregnancies with Hb H or hydrops fetalis were therapeutically terminated before 4 1/2 months of gestation. Of the 26 pregnancies intended to continue, 18 have come to term with normal deliveries; one with threatened abortion was terminated at the end of the first trimester and, seven are progressing normally. PMID- 3010269 TI - [Multiple recurrences of malignant fibrous histiocytoma with terminal histiocytic character and peritoneal sarcomatosis]. PMID- 3010270 TI - [Cell receptors]. PMID- 3010271 TI - [Morphologic ultrastructural criteria in breast cancer: prevalence and significance for the early disease course]. PMID- 3010273 TI - Purification and characterization of a cytosolic activator protein for the gastric H+,K+-ATPase system from dog fundic mucosa. AB - An endogenous protein activator (AF) responsible for the activation of the gastric H+,K+-ATPase system, identified recently as the biochemical mechanism for the transport of H+, has been purified to homogeneity and partially characterized. The purification procedure (at 0-4 degrees C) involves simultaneous concentration and dialysis of the cytosolic fraction from dog fundic cells under negative pressure, pH 4.8 precipitation and two consecutive gel filtration steps on sephacryl S-200 columns. The highly purified and active AF is a protein of 80 Kd consisting of two identical subunits of 40 Kd each. The AF not only stimulates the gastric H+,K+-ATPase activity but also greatly enhances the rate of ATPase dependent proton pumping inside gastric microsomal vesicles. The data clearly suggest an important regulatory role of the cytosolic AF in the gastric HCl secretory process. PMID- 3010272 TI - Large-scale purification of beta-adrenergic receptors from mammalian cells in culture. AB - S49 Mouse lymphoma wild-type cells were grown in spinner cultures of 40 liters to a density of approximately 3 million cells/ml. Growth of cells to high density (2 3 million cells/ml) required that the cell suspensions be bubbled with oxygen. Cells from 40 liter cultures were collected by centrifugation and disrupted by nitrogen cavitation. Highly purified membranes (0.35 g membrane protein) that were rich in beta-adrenergic receptor (0.4-0.7 pmol receptor/mg membrane protein) were prepared by differential centrifugation and then solubilized with the plant glycoside, digitonin (1.5% digitonin at 3 mg of membrane protein/ml). Beta adrenergic receptors were isolated and purified by sequential affinity chromatography, ion-exchange chromatography, and steric exclusion high-pressure liquid chromatography. The extract was subjected to affinity chromatography on a derivatized Sepharose-4B CL column to which the high-affinity, beta-adrenergic antagonist (-)alprenolol had been immobilized. Following extensive washing, the receptor bound to this matrix was eluted using a 0-100 micromolar linear gradient of (-)alprenolol. The receptor eluted as a sharp peak at 30 micromolar ligand and displayed a specific activity of 280 pmol receptor/mg of protein. Ion-exchange chromatography on DEAE-Sephacel increased the specific activity to 950 pmol/mg of protein. The final step in the purification, steric-exclusion high-pressure liquid chromatography on two TSK-3000 and one TSK-2000 columns, tandem linked, resulted in a beta-adrenergic receptor preparation with a specific activity of 6700 pmol/mg of protein (15,900-fold purification). Autoradiography of the radioiodinated pure receptor, the receptor photolabeled with [125I]iodoazidobenzylpindolol or silver-staining of chemical amounts of protein revealed that the Mr of the pure receptor is 66,000 upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate under reducing conditions. The receptor is a beta2-subtype adrenergic receptor. PMID- 3010275 TI - [Increase in angiotensin converting enzyme levels in schistosomiasis]. PMID- 3010274 TI - Histochemical localization of phosphatases in the pig placenta: II. Potassium dependent and potassium-independent p-nitrophenyl phosphatases at high pH; relation to sodium-potassium-dependent adenosine triphosphatase. AB - Histochemical localization by Mg2+ capture methods of K+-dependent, ouabain sensitive phosphatase activity in the pig placenta shows that strong Na+,K+ dependent adenosine triphosphatase (Na+,K+-ATPase) activity is restricted to the basal zone of the columnar epithelium covering the areolar chorionic villi. It is proposed that active Na+ absorption at this epithelium may be the source of the ouabain-sensitive, fetal-side-positive potential difference which can be measured across the placental membrane in vitro. The one-step procedure for Na+,K+-ATPase localization is unsatisfactory in this organ as any specific ATPase reaction is swamped by activity probably attributable to uteroferrin and other non-specific phosphatases. PMID- 3010276 TI - [Intradural metastasis of cerebral glioblastoma multiforme]. PMID- 3010277 TI - Imaging of brain activity and behavioral disorders. AB - Recent advances in brain imaging permit metabolic variables to be measured in psychiatric patients. These include regional cerebral blood flow, oxygen utilization, oxygen extraction and glucose utilization--all measures which reflect CNS energy utilization. The quantitative relationships between these metabolic parameters, neuronal activity, and higher cortical function are poorly understood. In evaluating the results of imaging studies, it is important to establish whether the reported changes correlate with episodic symptomatology or with a chronic disease process. Affective disorders exemplify conditions which are episodic, progress rapidly and respond to medication, making it difficult to identify consistent patterns of metabolic change. Thus unipolar subgroups have been described with both left and right metabolic predominance. On the other hand, imaging studies in dementia have consistently shown decreased glucose utilization, and more particularly poor verbal performance may go with reduced glucose uptake in the left temporal/parietal area. Although neuro-imaging studies are still at an early stage, the additional capacity to measure regional receptor distribution and kinetics using ligands tagged with positron emitters will open up new dimensions in the study of psycho-pathophysiology. PMID- 3010279 TI - Transcriptional "silencer" element in rat repetitive sequences associated with the rat insulin 1 gene locus. AB - The enhancer elements from either simian virus 40 or murine sarcoma virus activate the expression of a transfected rat insulin 1 (rI1) gene when placed within 2.0 kilobases or less of the rI1 gene cap site. Inclusion of 4.0 kilobases of upstream rI1 sequence, however, results in a substantial reduction in the enhancer-dependent insulin gene expression. These observations suggested that a negative transcriptional regulatory element was present between 2.0 and 4.0 kilobases of the rI1 sequence. To test this notion, we employed a heterologous enhancer-dependent transcription assay in which the simian virus 40 72-base-pair repeat is linked to a human beta-globin gene. Addition of the upstream rI1 element to this system decreased the level of enhancer-dependent beta-globin transcription by a factor of 5 to 15. This rI1 "silencer" element functions in a manner relatively independent of position and orientation and requires a cis dependent relationship to the transcription unit on which it acts. Thus, the silencer sequence seems to have a number of the characteristics of enhancer elements, and we suggest that it may function by the converse of the enhancer mechanism. The rI1 silencer sequence was identified as a member of a long interspersed rat repetitive family. Thus, a potential role for certain repetitive sequences interspersed throughout the eukaryotic genome may be to regulate gene expression by retaining transcriptional activity within defined domains. PMID- 3010278 TI - The chicken delta 1-crystallin gene promoter: binding of transcription factor(s) to the upstream G+C-rich region is necessary for promoter function in vitro. AB - There are two linked delta-crystallin genes in the chicken (5' delta 1-delta 2 3'). Only the delta 1 gene has been shown definitively to be active in the lens. Transcription of deletion mutants, reported here, shows that the sequences necessary for the functioning of the delta 1 promoter in a HeLa cell extract are located upstream from the RNA initiation site, between nucleotide positions -121 and -38. This region includes a number of G+C-rich motifs, including one hexanucleotide sequence, CCGCCC, that is repeated six times in the simian virus 40 (SV40) promoter. Competition experiments with purified fragments from the delta 1-crystallin gene promoter showed that binding of transcription factor(s) from the HeLa cell extract to this G+C-rich region is required for promoter activity in vitro. Further, competition experiments using three different fragments from the SV40 promoter suggest that the transcription factor(s) is similar to Sp1, which stimulates transcription by binding to the G+C-rich 21-base pair repeats of the SV40 promoter, and differs from that which interacts with the SV40 enhancer region. PMID- 3010280 TI - Superoxide mediates the toxicity of paraquat for Chinese hamster ovary cells. AB - The roles of superoxide and H2O2 in the cytotoxicity of paraquat were assessed in Chinese hamster ovary cells. Neither catalase nor superoxide dismutase inhibited the loss of ability to form colonies when added to the medium. When introduced into the cells, superoxide dismutase but not catalase inhibited the toxicity of paraquat. That superoxide dismutase acted by its known catalytic action is shown by the loss of inhibition when the enzyme was inactivated by H2O2 before being introduced into the cells. The lack of inhibition by catalase, by dimethyl sulfoxide, and by desferoxamine suggests that the toxicity is not mediated by a reaction between H2O2 and superoxide to engender the hydroxyl radical. Exposure of Chinese hamster ovary cells to paraquat may be a suitable means to determine the effects of superoxide anion in cultured cells and the ways in which cells can resist this toxic action. PMID- 3010281 TI - Activation of gene expression is adversely affected at high multiplicities of linked simian virus 40 enhancer. AB - We have cloned multiple copies of 72-base-pair (bp) repeat transcriptional enhancer element from simian virus 40 in plasmid vectors upstream from the bacterial chloramphenicol acetyltransferase gene under the control of the simian virus 40 early promoter. Two copies of the 72-bp repeat provided efficient activation of gene expression. Increasing the number of linked 72-bp units to four substantially improved the activation of gene expression, but further addition of enhancers diminished the activation of gene expression proportionally to the number of enhancers added. Enhancer regions containing 10 or more copies of the 72-bp sequence were very inefficient in gene activation. Plasmids containing such expanded enhancer regions also competed less efficiently in vivo for trans-acting enhancer-binding factors. These effects are specific for the enhancer element and are not produced by reiterations of the 21-bp promoter element. Multiple enhancer units placed upstream do not interfere with the accuracy of mRNA initiation directed by the simian virus 40 early promoter in these plasmid constructs but do severely inhibit the initiation of replication at the neighboring simian virus 40 origin of replication that overlaps the early promoter region. These results are consistent with the hypothesis that the structural alterations induced in the DNA by a large number of copies of the enhancer are not favorable for the activation of a linked gene or for the binding of factors believed to mediate the enhancement effect. PMID- 3010282 TI - Murine myb protooncogene mRNA: cDNA sequence and evidence for 5' heterogeneity. AB - We have sequenced two overlapping cDNA clones from a murine pro-B cell library to generate a composite sequence that includes 3413 bases of the murine c-myb mRNA. There is a single long open reading frame, beginning at the first base of this sequence, and continuing from the first methionine codon at nucleotide 265 to a TGA termination codon at nucleotide 2173. The predicted murine translation product contains 636 amino acid residues and is about 71 kDa long, which is in good agreement with the 75-kDa molecular size determined for the avian c-myb protein. The murine c-myb protein shows a striking 82% amino acid homology in the region (amino acids 71-444) where it can be compared to the published avian c-myb gene sequence. S1 nuclease protection analysis indicates extreme heterogeneity at the 5' end of steady-state murine c-myb mRNA. PMID- 3010283 TI - Activation of rat c-raf during transfection of hepatocellular carcinoma DNA. AB - Rat c-raf was found to be activated in a transformant obtained with DNA of a hepatocellular carcinoma induced by 2-amino-3-methylimidazo[4,5-f]quinoline. This activated c-raf was cloned in a cosmid vector and actively transforming clones were obtained. Comparison of the restriction maps of this activated c-raf and cloned normal rat c-raf revealed a recombination in the 5'-terminal region of the activated form of this gene. The recombined DNA was shown to be actively transcribed and possibly to form a fused mRNA with c-raf, which is slightly smaller than normal c-raf mRNA. Since this recombination was not detected in the original tumor by Southern blot analysis, it presumably occurred during transfection. PMID- 3010285 TI - Alpha 4, the major regulatory protein of herpes simplex virus type 1, is stably and specifically associated with promoter-regulatory domains of alpha genes and of selected other viral genes. AB - Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. However, the mechanism by which alpha 4 regulates gene expression remained unknown. We report that labeled DNA fragments containing promoter regulatory domains of three alpha (alpha 0, alpha 4, and alpha 27) and a gamma 2 gene form stable complexes with proteins from infected-cell lysates as detected by a gel electrophoresis binding assay. The protein(s) exhibits sequence specificity since autologous DNA fragments but not heterologous DNA fragments, synthetic polydeoxynucleotide chains, or salmon sperm DNA competitively displace the DNA probe from the complexes. Murine monoclonal antibody to alpha 4 protein added before or after DNA-protein complex formation further retarded the electrophoretic mobility of the complexes whereas monoclonal antibody to alpha 0, alpha 27, or to a viral glycoprotein had no effect. Complexes consisting of the promoter-regulatory domain of the beta-class thymidine kinase gene and infected cell proteins were low in abundance and could be detected only in the presence of antibody to alpha 4 protein. The alpha 4 protein, therefore, forms stable complexes with promoter-regulatory domains of alpha genes and of selected other herpes simplex virus type 1 genes either alone or in combination with other proteins. PMID- 3010284 TI - Transcriptional activation encoded by the v-fos gene. AB - We present evidence that the fos oncogene encodes a transcriptional trans activation function. trans-activation was assayed by cotransfection into NIH 3T3 mouse fibroblasts of v-fos DNA containing plasmids together with a plasmid containing a test promoter. Three v-fos DNAs were used: (i) pFBR-1, a plasmid containing the FBR proviral sequences; (ii) pFBJ-2, a plasmid harboring the FBJ proviral sequences; (iii) pMF-J, a plasmid containing the FBJ fos sequences linked to a mouse metallothionein promoter. Each of the three v-fos DNA plasmids stimulated the expression of a cotransfected chimeric gene consisting of a promoter segment of the mouse alpha 1(III) collagen gene linked to the gene for chloramphenicol transacetylase. In similar experiments the v-fos gene also stimulated the long terminal repeat promoter of Rous sarcoma virus (RSV) but neither the early promoter of simian virus 40 nor the beta-actin promoter. Evidence that the trans-activation function is specified by the v-fos coding sequences comes from the fact that a frameshift mutation in the v-fos coding sequence inhibits the trans-activation. Two mutations that map around nucleotide 100 in the RSV promoter do not respond to cotransfection with v-fos, whereas other mutations respond like the wild-type RSV promoter. These experiments suggest that the v-fos gene either encodes or induces an activator of transcription that recognizes specific sequences in promoters. PMID- 3010287 TI - A block in initiation of simian virus 40 assembly results in the accumulation of minichromosomes containing an exposed regulatory region. AB - The initiation of simian virus 40 assembly is blocked at the nonpermissive temperature in cells infected with the viral capsid protein VP1 mutant tsC219. Greater than 95% of the minichromosomes isolated from these cells are accessible to cleavage by Bgl I and Sph I, which recognize the sequences near the viral replication origin and in the transcription enhancer elements, respectively. The accessibility of the Ori region to Bgl I is considerably reduced when virion assembly is allowed to proceed in tsC219-infected cells at the permissive temperature. A reduced accessibility to Bgl I is also observed for chromatin isolated from cells infected with wt776, the wild-type parental strain of tsC219. For wt776 chromatin, variability to Bgl I sensitivity is observed and this can be correlated to the relative virion-to-chromatin yield. A similar correlation is not apparent for restriction endonucleases that recognize sequences within the coding region of simian virus 40 chromatin. These results, considered together, indicate that, when virion assembly initiation is blocked, nucleosomes are nonrandomly arranged with respect to the viral regulatory sequences. It appears that the open regulatory region in minichromosomes is established during replication and that a protected regulatory region is generated with the onset of virion assembly. PMID- 3010286 TI - Specific binding of human corticosteroid-binding globulin to cell membranes. AB - Specific binding sites for corticosteroid-binding globulin were detected on membranes prepared from human prostates. The binding sites are typical of membrane receptors: they are saturable and specific and have high affinity. There was little specific binding at 4 degrees C and 23 degrees C. Maximal specific binding was obtained at 37 degrees C. Scatchard analysis revealed the presence of a single set of binding sites with an apparent dissociation constant of 8.7 X 10( 7) M and a binding capacity of 22 pmol/mg of membrane protein. The sites were specific for corticosteroid-binding globulin; binding was not inhibited by human testosterone/estradiol-binding globulin, by albumin, or by transferrin. The density of specific binding sites in membranes obtained from several organs from the rhesus monkey is consistent with the hypothesis that corticosteroid-binding globulin is involved in the transport of steroid hormones into target tissues. PMID- 3010288 TI - Retroviral mutants efficiently expressed in embryonal carcinoma cells. AB - The myeloproliferative sarcoma virus (MPSV) is a unique member of the Moloney murine sarcoma virus family. Due to mutations in the U3 region of its long terminal repeat, MPSV has an expanded host range that includes cells of the hematopoietic compartment. Using a MPSV recombinant containing the gene for neomycin-resistance (NeoR-MPSV), we demonstrate that the host range of MPSV also includes undifferentiated F9 embryonal carcinoma cells. Transfer of G418 resistance with NeoR-MPSV to F9 cells is almost as efficient as G418-resistance transfer to fibroblasts, in contrast to G418-resistance transfer to PCC4 embryonal carcinoma cells, which is at least 3 orders of magnitude lower. To isolate NeoR-MPSV mutants that are efficiently expressed in PCC4 cells, G418 resistant PCC4 cell lines were induced to differentiate, and the provirus was rescued by superinfection with murine leukemia virus. Viral isolates (PCMV-5 and 6; PCMV = PCC4 cell-passaged NeoR-MPSV) were obtained and assayed for expression in embryonal carcinoma cells. The efficiency of NeoR transfer was equally as high in both F9 and PCC4 as in fibroblasts. mos oncogene expression was unaltered as judged by transformation capability. No gross alteration in the coding region and in the long terminal repeat was detectable by restriction enzyme analysis. NeoR MPSV and its mutants PCMV-5 and -6 can thus be utilized as vectors for the efficient transduction of genes into embryonic cells. PMID- 3010289 TI - Specific binding of the mononuclear phagocyte colony-stimulating factor CSF-1 to the product of the v-fms oncogene. AB - Cells transformed by the McDonough strain of feline sarcoma virus (SM-FeSV) express a v-fms-encoded glycoprotein whose expression at the cell surface correlates with the transformed phenotype. The mouse mononuclear phagocyte growth factor CSF-1 specifically binds to SM-FeSV-transformed cells at high-affinity sites indistinguishable from those detected on normal feline macrophages. A monoclonal antibody to a v-fms-encoded epitope competed for CSF-1 binding to SM FeSV-transformed cells, and chemical crosslinking demonstrated that murine CSF-1 bound to the v-fms gene product at the cell surface. Although SM-FeSV-transformed fibroblast lines were found to secrete CSF-1, the growth of transformed cells was not affected by antibodies to the v-fms gene product or to the growth factor. Tyrosine phosphorylation of the v-fms products in membranes was observed in the absence of CSF-1 and was not enhanced by addition of the murine growth factor. The data support the hypothesis that the c-fms protooncogene product is related, and possibly identical, to the CSF-1 receptor and suggest that the v-fms-encoded kinase functions in the absence of an exogenous growth factor. PMID- 3010290 TI - Rearrangement of chicken immunoglobulin genes is not an ongoing process in the embryonic bursa of Fabricius. AB - We report a molecular analysis of the chicken Ig loci in single bursal follicles from 3- to 7-week-old chickens. Each follicle contained between 10(5) and 3 X 10(5) cells. The Ig gene rearrangement patterns obtained were compared to the pattern observed with the corresponding total bursal DNA. The results obtained for the light chain locus imply that a very small number (two on average) of rearrangement events takes place in each follicle. For the heavy chain locus similar results were obtained, each follicle showing a more restricted pattern than the total bursa. These data favor a model in which each follicle is colonized by a very few prebursal stem cells that are committed to a particular Ig gene rearrangement at the very beginning of the development of the embryonic bursa. The role of the bursa as the organ in which such a committed stem cell population for the B-cell lineage arises is discussed. PMID- 3010292 TI - HLA-DR2, -DR5, and DRw6 associated Dw subtypes correlate with HLA-DR beta and -DQ beta restriction fragment length polymorphisms. AB - DNAs extracted from peripheral blood leukocytes of 24 individuals, selected for their HLA-DR types, -DR2, -DR5, and -DRw6, were analyzed with four restriction enzymes, BamHI, EcoRV, HindIII, and Taq I, using the Southern technique. This panel includes 16 individuals with homozygous typing cells and 8 heterozygous individuals who carry rare Dw subtypes or unusual DR-DQ associations. Eighty-five polymorphic fragments were detected and assigned to the DR or DQ gene families according to their hybridization signals. Thirty-eight fragments (DR or DQ) were found to correlate with single DR or Dw specificities or rare associations such as DRw14-DQw3. Forty-two fragments correlated with the association of immunologically defined specificities. In total, these 85 fragments constituted 44 different patterns, each comprising 1-9 fragments. For each homozygous typing cell a combination of patterns was observed. Fourteen different combinations of 10-20 patterns were found among the 16 individuals with homozygous typing cells, showing that Dw18, Dw19, Dw9, and Dw5 are heterogeneous at the genomic level whereas only the Dw2 individuals tested here are identical. PMID- 3010291 TI - Atrial natriuretic factor receptors in rat kidney, adrenal gland, and brain: autoradiographic localization and fluid balance dependent changes. AB - Mammalian atria contain natriuretic peptides designated atrial natriuretic factors (ANF). Using in vitro autoradiography with 125I-labeled ANF, we have localized high-affinity (Kd = 150 pM) ANF binding sites to the glomeruli of the kidney, zona glomerulosa of the adrenal gland, and choroid plexus of the brain. The numbers of sites in both kidney and adrenal are increased in rats deprived of water; increases are detectable within 72 hr of water deprivation in the kidney and within 24 hr in the adrenal gland. Receptor numbers decline in rats given 2.0% NaCl as drinking water and in diabetic rats. The discrete localizations and dynamic alterations of these receptors suggest that ANF regulates fluid balance through diverse but coordinated effects on receptors in numerous organs including the kidney, adrenal, and brain. PMID- 3010293 TI - Tissue tropism of a leukemogenic murine retrovirus is determined by sequences outside of the long terminal repeats. AB - Although it has been previously determined that the long terminal repeat (LTR) sequences of several murine retroviruses specify the major tissue tropism of leukemias they induce, data reported here show that the LTR is not responsible for tissue tropism in the case of all leukemogenic viruses. In an effort to determine whether LTR sequences of the acute erythroleukemia-inducing spleen focus-forming virus (SFFV), like those of the other murine leukemia viruses, are uniquely required to confer tissue specificity to the virus, we prepared recombinant SFFVs in which the LTR region containing promoter and enhancer functions was replaced with analogous LTR regions from Friend and Moloney ecotropic and mink cell focus-inducing viruses. It was found that all of the SFFV constructs, even those with a LTR derived from lymphoma-inducing viruses such as Moloney murine leukemia virus, transformed erythroid cells in vitro and induced exclusively an erythroid disease. These results demonstrate that sequences in SFFV that determine the tissue-specific nature of the disease reside outside the LTR. PMID- 3010294 TI - The qa repressor gene of Neurospora crassa: wild-type and mutant nucleotide sequences. AB - The qa-1S gene, one of two regulatory genes in the qa gene cluster of Neurospora crassa, encodes the qa repressor. The qa-1S gene together with the qa-1F gene, which encodes the qa activator protein, control the expression of all seven qa genes, including those encoding the inducible enzymes responsible for the utilization of quinic acid as a carbon source. The nucleotide sequence of the qa 1S gene and its flanking regions has been determined. The deduced coding sequence for the qa-1S protein encodes 918 amino acids with a calculated molecular weight of 100,650 and is interrupted by a single 66-base-pair intervening sequence. Both constitutive and noninducible mutants occur in the qa-1S gene and two different mutations of each type have been cloned and sequenced. All four mutations occur within the predicted coding region of the qa-1S gene. This result strongly supports the hypothesis that the qa-1S gene encodes a repressor. All four mutations are located within codons for the last 300 amino acids of the qa-1S protein. The mutations in three of the mutants involve amino acid substitutions, while the fourth mutant, which has a constitutive phenotype, contains a frameshift mutation. The two constitutive mutations occur in the most distal region of the gene, possibly implicating the COOH-terminal region of the qa repressor in binding to its target. The two noninducible mutations occur in a region proximal to the constitutive mutations, possibly implicating this region of the qa repressor in binding the inducer. PMID- 3010295 TI - Illegitimate recombination at the replication origin of bacteriophage M13. AB - Hybrids composed of phage M13 and plasmid pHV33 were used to study the formation of deletions in Escherichia coli. Eighty to ninety percent of the deletion endpoints were at the position of the nick introduced into the M13 replication origin by the phage gene II protein. This suggests the existence of a novel mechanism of illegitimate recombination. PMID- 3010296 TI - DNA linkage analysis of X chromosome-linked chronic granulomatous disease. AB - Chronic granulomatous disease (CGD) is a disorder of phagocytes that is usually inherited as an X chromosome-linked trait. Previous family studies suggested that the CGD locus resides on the distal short arm (Xp22-Xpter). Using cloned, polymorphic DNA probes we have performed a linkage analysis within CGD families that suggests a more proximal location (Xp21). In addition, the CGD locus is proximal to the Duchenne muscular dystrophy locus and lies within a broad region of Xp in which recombination appears to be greater than anticipated on the basis of physical distance between markers. Regional localization of the X chromosome CGD locus should facilitate molecular cloning of the CGD gene and molecular dissection of the phagocyte oxidase system. PMID- 3010297 TI - Chemically induced mutagenesis in a shuttle vector with a low-background mutant frequency. AB - We have developed a recombinant DNA shuttle vector that permits the molecular analysis of mutations induced in human cells by chemical or physical mutagens. The vector is able to replicate as a plasmid in Escherichia coli and in Epstein Barr virus (EBV)-transformed human lymphoblastoid cell lines and contains the herpes simplex virus type 1 thymidine kinase gene (HSV tk) as the target for mutagenesis studies. After introduction of the vector into an EBV-transformed lymphoblastoid cell line (LCL-721) by electroporation, approximately equal to 2% of the transfected cells expressed the vector-encoded gene for hygromycin resistance. Plasmid DNA isolated from cells immediately after selection for hygromycin resistance (10 population doublings posttransfection) contained mutations in the HSV tk gene at a frequency of 6 X 10(-5). Treatment of plasmid bearing LCL-721 cells with N-ethyl-N-nitrosourea resulted in a dose-dependent increase of up to 15-fold in the frequency of mutations in the HSV tk gene. The dose-response for the induction of mutations in the plasmid-encoded gene closely paralleled that for the induction of mutations in the cellular gene for hypoxanthine (guanine) phosphoribosyltransferase. PMID- 3010298 TI - Effect of atrial natriuretic peptide on gonadotropin release in superfused rat pituitary cells. AB - Cardiac atrial muscle cells produce a polypeptide hormone that plays a role in the control of water and electrolyte balance and blood pressure. The circulating form of this hormone is the atrial natriuretic peptide (ANP), which contains 28 amino acids. Various immunohistochemical studies have shown that ANP is present in many areas of the central nervous system, including the median eminence. In our studies, we investigated the effect of ANP in a superfused rat pituitary cell system. When ANP was administered at increasing concentrations (0.01 microM to 1 microM), it caused a significant dose-related stimulation of the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The lowest effective dose of ANP in our system was 0.03 microM. When ANP and LH-releasing hormone were administered together, the response was prolonged and had the characteristics of ANP-stimulated LH and FSH release. In contrast with some previous reports, ANP in high concentration (1 microM) consistently induced a small but significant stimulation of the release of corticotropin. ANP did not influence the basal release of prolactin, growth hormone, and thyrotropin. PMID- 3010299 TI - Restriction fragment length polymorphism caused by a deletion within the human c abl gene (ABL). AB - A restriction fragment length polymorphism at the human c-abl locus (ABL) has been detected in 67 unrelated individuals by agarose gel electrophoresis and Southern blot hybridization using 32P-labeled v-abl probes. This polymorphism is generated by the existence of two alleles, a and b, which are in Hardy-Weinberg equilibrium, with frequencies of 94.8% and 5.2%, respectively. The minor allele, b, is due to a deletion of about 500 base pairs in an intron located downstream of the codon for the phosphate-acceptor tyrosine residue of the c-abl gene product. PMID- 3010300 TI - Class I histocompatibility antigens and insulin receptors: evidence for interactions. AB - We provide evidence for an interaction between mouse class I major histocompatibility complex antigens and insulin receptors. Antibodies against class I but not class II major histocompatibility complex antigens immunoprecipitate photoaffinity-labeled hepatic insulin receptors. Haplotype specificity is demonstrated by reciprocal precipitation using anti-class I antibodies and three strains of mice. Antibodies against the 45-kDa products of either the H-2K or H-2D locus and rabbit anti-mouse beta 2-microglobulin antibodies were shown to precipitate insulin receptors. We also demonstrate the specific binding of 125I-labeled insulin and 125I-labeled epidermal growth factor, but not 125I-labeled glucagon or 125I-labeled atrial natriuretic factor, to solubilized plasma membranes immunoprecipitated with anti-H-2K antibody. These observations suggest a specific interaction between class I major histocompatibility complex antigens and certain hormone receptors. PMID- 3010301 TI - Prostaglandin E2, a seminal constituent, facilitates the replication of acquired immune deficiency syndrome virus in vitro. AB - Acquired immune deficiency syndrome (AIDS)-associated virus is thought to be transmitted effectively through semen during sexual activities from male to male or from male to female. Prostaglandin (PG) E2 is one of the immunosuppressive compounds present in high concentrations in human semen. We, therefore, investigated direct effects of PGE2 and other PGs on AIDS-associated virus infection and replication in vitro. First, type III human T-lymphotropic virus (HTLV-III) was used to infect a T-cell line (MT-4) in culture. PGE2 (10 nM to 10 microM) added to the culture medium enhanced the production of infectious virus in a dose-dependent fashion. In the presence of 5 microM PGE2, 2.5-fold more virus were released from the infected MT-4 cells as compared to untreated control cells on day 3 after infection. Second, when we used an HTLV-III continuous producer cell line (Molt-4/HTLV-III), PGE2 and PGD2 added to the culture medium increased the number of viruses released from Molt-4/HTLV-III cells. Other PGs such as PGF2 alpha and 13,14-dihydro-15-keto PGE2 did not affect the replication of HTLV-III in this system. These results indicate that some PGs including seminal PGs enhance the AIDS-associated virus replication in vitro. We propose that PGE2 in human semen might directly facilitate the infection of AIDS associated virus and cause the efficient transmission of the virus during sexual activities. PMID- 3010302 TI - Synaptophysin: a marker protein for neuroendocrine cells and neoplasms. AB - Synaptophysin is an integral membrane glycoprotein (Mr 38,000) that occurs in presynaptic vesicles of neurons and in similar vesicles of the adrenal medulla. By using a monoclonal antibody to this protein (SY38), we have found, by immunohistochemistry and immunoblotting, that an identical or similar protein is also expressed in neuroendocrine tumors of neural type, such as pheochromocytomas and paragangliomas. In addition, this protein occurs in certain neuroendocrine epithelial cells, such as pancreatic islet cells; in a variety of neuroendocrine epithelial tumors, including isletcell adenomas and carcinomas and several carcinoids and neuroendocrine carcinomas of the gastrointestinal and the bronchial tracts; and in medullary carcinomas of the thyroid. Our results show that synaptophysin, and the vesicles that contain it, can occur in normal and neoplastic neuroendocrine cells of neural type, as demonstrated by colocalization with neurofilaments, as well as in those of epithelial type, as shown by colocalization with cytokeratin filaments and desmoplakins. We conclude that synaptophysin is expressed independently of other neuronal differentiation markers and propose that it be used as a differentiation marker in tumor diagnosis. PMID- 3010304 TI - Short- and long-term interactions of endothelium and vascular smooth muscle in coculture: effects on cyclic GMP production. AB - In intact blood vessels, many vasodilators act by stimulating the release from endothelium of factor(s) that relax vascular smooth muscle and stimulate increases in cGMP. To investigate how endothelium regulates cGMP production in vascular smooth muscle, bovine aortic endothelial cells and rat aortic smooth muscle cells were cultured both separately and together in cocultures for 48 hr. Nitroprusside (1 mM) increased intracellular cGMP concentration 30-fold in smooth muscle cells (from a basal level of 103 +/- 54 fmol/mg of cell protein to 2920 +/ 1800 fmol/mg) but only 2-fold in endothelial cells (from 41 +/- 7 fmol/mg to 93 +/- 23 fmol/mg). When endothelial and smooth muscle cells were cocultured as a mixed cell population (1:1 cell ratio), both basal and nitroprusside-stimulated cGMP levels were significantly increased (550 +/- 250 and 13,240 +/- 9950 fmol/mg of total cell protein, respectively). The calcium ionophore A23187 (10 microM) caused no increase in cGMP concentration in either cell type cultured alone but produced a 6-fold increase in cocultures. Neither aspirin nor 5,8,11,14 icosatetraynoic acid influenced these results. No changes in cAMP levels were detected. Using cocultures in which one cell type was grown on microcarrier beads, we have shown that cGMP increased only in vascular smooth muscle cells and was not dependent upon the formation of junctions between endothelium and smooth muscle cells. In long-term (48-hr) mixed-cell cocultures, but not in short-term microcarrier cocultures, amplification of the nitroprusside-induced increase in cGMP was observed. These results show that responses associated with endothelium dependent relaxation can be reconstituted in cultured endothelial and vascular smooth muscle cells and that endothelium generates a humoral factor(s) that stimulates accumulation of smooth muscle cGMP and has a longer-term effect that amplifies guanylate cyclase stimulation by nitroprusside, a drug acting directly upon smooth muscle to stimulate formation of the cyclic nucleotide. Cultured cells provide a valuable model system for the study of endothelium-vascular smooth muscle interactions. PMID- 3010303 TI - Role of cholecystokinin in corticotropin release: coexistence with vasopressin and corticotropin-releasing factor in cells of the rat hypothalamic paraventricular nucleus. AB - Cholecystokinin-8 (CCK8)-containing cell bodies in the parvocellular region of the rat paraventricular nucleus (PVN) contain vasopressin and corticotropin releasing factor (CRF). The CCK8 and vasopressin in these cells can readily be visualized in adrenalectomized, but not in shamoperated animals. Furthermore, CCK8 levels as measured by RIA change in the PVN and in the median eminence in response to adrenalectomy. CCK8 has a stimulatory effect on corticotropin (ACTH) release from primary cultures of the anterior pituitary. This stimulation is additive with that produced by vasopressin; CCK8 plus vasopressin have an effect as great as CRF in stimulating ACTH release. Our results suggest that CCK8 may participate in the regulation of ACTH release under certain physiological conditions. PMID- 3010305 TI - Genetic uniformity in two populations of Drosophila melanogaster as revealed by filter hybridization of four-nucleotide-recognizing restriction enzyme digests. AB - A filter hybridization method is described for identifying restriction-site and insertion/deletion variation by using restriction enzymes that recognize four nucleotide sequences and denaturing polyacrylamide gels for separating fragments. Eighty-seven lines of Drosophila melanogaster representing two natural populations were surveyed over a 2.7-kilobase region encompassing the alcohol dehydrogenase locus. Fifty distinct haplotypes were identified from 17 restriction-site and 11 insertion/deletion polymorphisms and from one allozyme polymorphism. There was no evidence for genetic differentiation between an East Coast and a West-Coast (North American) sample. This technique has widespread applications in screening for DNA polymorphism. PMID- 3010306 TI - Exon/intron organization of the chicken type II procollagen gene: intron size distribution suggests a minimal intron size. AB - Overlapping genomic clones have been isolated that contain the alpha chain and COOH-terminal propeptide coding regions of the chicken type II procollagen gene. All type II procollagen exon sequences present in these clones have been identified and mapped by DNA sequencing. These include 43 exons coding for the alpha-chain triple helix, 1 exon coding for the junction between the COOH terminal propeptide and the alpha-chain region, and 3 exons coding for the COOH terminal propeptide and 3' noncoding sequences. With the exception of one additional intron between 2 exons coding for amino acids 568-585 and 586-603, exon-intron boundaries have been conserved when compared with genes for all other characterized genes for fibrillar collagens. The chicken type II procollagen gene differs from most other collagen genes in having introns of considerably smaller average size. The size distribution of the introns suggests that approximately equal to 80 base pairs may be a minimal functional size for introns in this gene. This size of intron may be necessary in a gene with a very large number of small exons to prevent aberrant splicing from removing exon sequence together with intron sequence. PMID- 3010307 TI - Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase. AB - Plasmids containing the entire cDNA sequence of poliovirus type 1 (Mahoney strain) under control of a promoter for T7 RNA polymerase have been constructed. Purified T7 RNA polymerase efficiently transcribes the entire poliovirus cDNA in either direction to produce full-length poliovirus RNA [(+)RNA] or its complement [(-)RNA]. The (+)RNA produced initially had 60 nucleotides on the 5' side of the poliovirus RNA sequence, including a string of 18 consecutive guanine residues generated in the original cloning and an additional 626 nucleotides of pBR322 sequence beyond the poly(A) tract at the 3' end. Such RNA, while much more infectious than the plasmid DNA, is only about 0.1% as infectious as RNA isolated from the virus. Subsequently, a T7 promoter was placed only 2 base pairs ahead of the poliovirus sequence, so that T7 RNA polymerase synthesizes poliovirus RNA with only 2 additional guanine residues at the 5' end and no more than seven nucleotides past the poly(A) tract at the 3' end. Such RNA has much higher specific infectivity, about 5% that of RNA isolated from the virus. The ability to make infectious poliovirus RNA efficiently from cloned DNA makes it possible to apply techniques of in vitro mutagenesis to the analysis of poliovirus functions and the construction of novel and perhaps useful derivatives of poliovirus. A source of variant RNAs should also allow detailed study of the synthesis and processing of poliovirus proteins in vitro. PMID- 3010308 TI - A molecular mechanism for sensory adaptation based on ligand-induced receptor modification. AB - Physiological responses mediated by cell-surface receptors frequently adapt or "desensitize" (i.e., terminate despite persistent occupancy of receptors by ligand). Binding of ligands to the external domains of a wide variety of surface receptors induces covalent modification of their cytoplasmic domains. A mechanism is presented in which the variety of receptor states generated by ligand binding and covalent modification act together to regulate physiological responsiveness. The development of the model is guided by observations of adaptation for chemotaxis in Escherichia coli and adenylate cyclase activation in Dictyostelium. The general features of the marked response and eventual exact adaptation predicted by the model match those observed in the experimental systems. PMID- 3010309 TI - Primary structure and cDNA cloning of human fibroblast collagenase inhibitor. AB - We report the primary structure and cDNA cloning of human fibroblast collagenase inhibitor, a glycoprotein that appears to play a central role in modulating the activity of a number of metalloendoproteases of connective tissue origin including collagenase, gelatinase, and proteoglycanase. Secreted human fibroblast collagenase inhibitor was purified and subjected to automated Edman degradation. The secreted protein consists of 184 amino acid residues; it contains two sites of N-linked oligosaccharide linkage and six disulfide bonds. Synthetic oligonucleotide probes based on selected amino acid sequences of the inhibitor were used to screen a lambda gt10 cDNA library from a human fibroblast line. Two overlapping cDNA clones were characterized to determine the complete coding and noncoding sequences of the specific mRNA. The amino acid sequence deduced from the nucleotide sequence agrees with that determined by protein sequencing. One clone appears to contain the complete 5' end and, in addition, the cDNA sequence predicts a 23-amino acid leader peptide. The other clone represents the 3' end of the mature message and includes a short poly(A)+ tract. This 3' sequence is remarkably similar to a reported cDNA encoding part of the protein derived from mouse fibroblast poly(A)+ RNA. However, this inhibitor has no substantial homology with previously sequenced protease inhibitors. PMID- 3010310 TI - Induction of c-sis mRNA and activity similar to platelet-derived growth factor by transforming growth factor beta: a proposed model for indirect mitogenesis involving autocrine activity. AB - Treatment of quiescent cultures of mouse embryo-derived AKR-2B cells with transforming growth factor beta resulted in an early induction of c-sis mRNA. The increase in c-sis mRNA was followed by a corresponding increase in protein similar to platelet-derived growth factor (PDGF) in the culture medium. In addition, PDGF-regulated genes (c-fos and c-myc) were stimulated by transforming growth factor beta with delayed kinetics relative to that seen in other cell systems with direct PDGF stimulation. A model is proposed in which the monolayer mitogenicity of transforming growth factor beta is mediated by the induction of c sis and PDGF and the subsequent autocrine stimulation of c-fos, c-myc, and other PDGF-inducible genes. PMID- 3010311 TI - cAMP increases junctional conductance and stimulates phosphorylation of the 27 kDa principal gap junction polypeptide. AB - Membrane-permeant cAMP derivatives (dibutyryl- and 8-bromo-cAMP) increase gap junctional conductance within minutes when applied to voltage-clamped pairs of rat hepatocytes. Glucagon also increases junctional conductances, but the response has a more rapid onset and is more rapidly reversible. The glucagon effect can be prevented by intracellular injection of the protein inhibitor of the cAMP-dependent protein kinase (Walsh inhibitor), indicating that the catalytic subunit of cAMP-dependent protein kinase is directly involved. The 27 kDa major gap junction polypeptide is phosphorylated when liver cells dissociated into small groups are incubated with 32P. Addition of 8-bromo-cAMP to cells increases the incorporation of 32P into the 27-kDa junctional protein. Serine is the amino acid residue that is phosphorylated. When isolated liver gap junctions are incubated in the presence of catalytic subunit of the cAMP-dependent protein kinase, the 27-kDa gap junction polypeptide is phosphorylated with low stoichiometry on serine. The rapid increases in gap junctional conductance caused by agents that elevate cAMP and phosphorylation of the gap junction protein by cAMP-dependent protein kinase suggest that cAMP-dependent phosphorylation of the gap junction channel modulates the conductance of liver gap junctions. PMID- 3010312 TI - A gene expressed in undifferentiated vegetative Dictyostelium is repressed by developmental pulses of cAMP and reinduced during dedifferentiation. AB - We describe the gene M4-1, whose unique pattern of developmental expression will allow us to study the molecular mechanisms controlling expression in undifferentiated cells in addition to repression in response to cAMP during development and reinduction during dedifferentiation. M4-1 is a Dictyostelium gene expressed in the undifferentiated cell. We have shown that M4-1 continues to be expressed very early during the developmental cycle but is repressed at a later stage of development, at a time coincident with the establishment of oscillations in the cAMP pool. Studies on the expression of the M4-1 gene in shaking culture, under conditions that mimic early development, have established that pulsatile stimulation of cells with cAMP is sufficient to repress M4-1 expression. Consistent with this, cells that are exposed to high levels of cAMP are unable to respond normally to cAMP oscillations and continue to express M4-1 at vegetative levels. These data indicate that low-level oscillations of cAMP are required for the repression of M4-1 expression rather than the continuous high levels of cAMP responsible for the regulation of a different class of Dictyostelium genes. We suggest that cAMP may mediate developmental expression of the Dictyostelium genome by different mechanisms. We also show that cell-cell interaction, a developmental event that occurs subsequent to the cAMP pulse, does not normally influence the regulation of M4-1. Finally, we have shown that when cAMP-pulsed cells are induced to dedifferentiate, M4-1 RNA sequences rapidly reappear in nuclei and cytoplasm, suggesting that regulation of M4-1 expression is primarily mediated at the level of transcription. PMID- 3010313 TI - Molecular cloning of the muscle gene unc-22 in Caenorhabditis elegans by Tc1 transposon tagging. AB - The previously described mutator system of Caenorhabditis elegans var. Bergerac has as one of its targets unc-22, a previously uncloned gene on chromosome IV important in assembly and function of the body wall musculature. By assuming that the mutator activity involved transposition of the repetitive element Tc1 into the unc-22 gene we have succeeded both in cloning the unc-22 gene and in demonstrating that Tc1 transposition is the principal basis of the mutator activity in the Bergerac strain. Although germ-line excision of Tc1 is sensitive to genetic background, somatic excision appears to be less so, suggesting that Tc1 movement is controlled differently in germ-line and somatic tissue. The availability of a transposon-based mutator system should aid in the cloning of additional genes in C. elegans, and the particular properties of this Tc1 system may provide information about the control of transposable element activity more generally. PMID- 3010314 TI - Primary antibody responses to a well-defined and unique hapten are not enhanced by preimmunization with carrier: analysis in a viral model. AB - We used a viral model to reexamine classical experiments showing that mice previously primed with a "carrier" molecule alone and then challenged with the carrier-hapten conjugate exhibited an enhanced antihapten antibody response. Mice were primed with live or UV-inactivated vesicular stomatitis virus (VSV) Indiana (Ind) serotype with or without complete Freund's adjuvant. After challenge with VSV New Jersey (NJ), these mice developed a secondary-type IgG response, measured by antibody binding in an ELISA, against both VSV-Ind and VSV-NJ. The same result was found for the reciprocal experiments where mice were primed with VSV-NJ. Similarly, when mice were primed with live VSV, UV-inactivated VSV, or purified VSV glycoprotein G of Ind or NJ serotype and later were challenged with dinitrophenyl (N2ph)-conjugated, UV-inactivated VSV or with N2ph-conjugated G protein of either serotype, they exhibited a secondary-type anti-N2ph antibody response as demonstrated by the binding of IgG to dinitrophenylated bovine serum albumin measured by ELISA. In contrast, when neutralizing antibody responses were monitored, VSV-Ind-primed mice challenged with VSV-NJ developed a strictly primary type of anti-VSV-NJ response and vice versa. We conclude that preexistent helper T cells specific for shared carrier determinants do not improve virgin B cell responses specific for "new," unique determinants that are the target for the biologically relevant neutralizing antibodies. These findings suggest that priming of B cells rather than of helper T cells may be of importance to induce protective immunity mediated by antibodies. PMID- 3010316 TI - Antiviral effect of an oligo(nucleoside methylphosphonate) complementary to the splice junction of herpes simplex virus type 1 immediate early pre-mRNAs 4 and 5. AB - Selective inhibition of regulatory immediate early (IE) genes of herpes simplex virus type 1 (HSV-1) should inhibit virus growth. Treatment of HSV-1-infected cells with the oligo(nucleoside methylphosphonate) d(TpCCTCCTG) (deoxynucleoside methylphosphonate residues in italic), which is complementary to the acceptor splice junction of HSV-1 IE pre-mRNA 4 and 5, before (1-24 hr) or at the time of infection caused a dose-dependent inhibition in virus replication. Virus titers were decreased 50% and 90% in cells treated with 25 microM and 75 microM oligomer, respectively; at 300 microM, a 99% reduction in virus production was observed. Viral DNA synthesis was reduced 70-75% and there was a 90% reduction in synthesis of viral proteins, including other IE species and viral functional (130 kDa major DNA-binding) and structural (glycoprotein gB) proteins. In the same concentration range, d(TpCCTCCTG) caused a minimal reduction (0-30%) in protein synthesis and growth rates (less than 40%) of uninfected cells. The data suggest that oligo(nucleoside methylphosphonate)s may be effective in antiviral chemotherapy. PMID- 3010315 TI - Expression of the three influenza virus polymerase proteins in a single cell allows growth complementation of viral mutants. AB - Transformed cell lines derived from murine C127 cells were constructed that express the influenza virus RNA-dependent RNA polymerase proteins (PA, PB1, and PB2). Cell lines that express only one or all three of the proteins were tested for their ability to complement temperature-sensitive viral mutants incubated at the nonpermissive temperature. Two cell lines were isolated that express all three polymerase genes and complement the growth of PB2 temperature-sensitive mutants at the nonpermissive temperature. One of these lines also complemented PA temperature-sensitive mutants. The viral titers obtained in these two cell lines were 12-fold to 1000-fold higher than the viral titers obtained upon growth of the corresponding temperature-sensitive mutant in C127 cells at the nonpermissive temperature. PMID- 3010317 TI - High-affinity antibodies to the 1,4-dihydropyridine Ca2+-channel blockers. AB - Antibodies with high affinity and specificity for the 1,4-dihydropyridine Ca2+ channel blockers have been produced in rabbits by immunization with dihydropyridine-protein conjugates. Anti-dihydropyridine antibodies were found to specifically bind [3H]nitrendipine, [3H]-nimodipine, [3H]nisoldipine, and [3H]PN 200-110 (all 1,4-dihydropyridine Ca2+-channel blockers) with high affinity, while [3H]verapamil, [3H]diltiazem, and [3H]trifluoperazine were not recognized. The average dissociation constant of the [3H]nitrendipine-antibody complex was 0.06 (+/- 0.02) X 10(-9) M for an antiserum studied in detail and ranged from 0.01 to 0.24 X 10(-9) M for all antisera. Inhibition of [3H]nitrendipine binding was specific for the 1,4-dihydropyridine Ca2+-channel modifiers and the concentrations required for half-maximal inhibition ranged between 0.25 and 0.90 nM. Structurally unrelated Ca2+-channel blockers, calmodulin antagonists, inactive metabolites of nitrendipine, and UV-inactivated nisoldipine did not modify [3H]nitrendipine binding to the anti-dihydropyridine antibodies. Dihydropyridines without a bulky substituent in the 4-position of the heterocycle were able to displace [3H]nitrendipine binding, but the concentrations required for half-maximal inhibition were greater than 800 nM. In summary, anti dihydropyridine antibodies have been shown to have high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers and to exhibit dihydropyridine binding properties similar to the membrane receptor for the 1,4-dihydropyridine Ca2+-channel blockers. PMID- 3010318 TI - Control of cytochrome P1-450 gene expression: analysis of a dioxin-responsive enhancer system. AB - We analyzed the function and sequence of a dioxin-responsive genomic element flanking the 5' end of the cytochrome P1-450 gene in high-activity variant mouse hepatoma cells. The element can regulate the mouse mammary tumor virus promoter. The element retains responsiveness to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) when the distance, the 5' or 3' position, and/or the 5' or 3' orientation with respect to the promoter are varied. The function of the element requires TCDD receptor complexes. The element remains responsive to TCDD when transfected into cells from either a heterologous mouse tissue or a heterologous species (human). The DNA element and TCDD receptors together constitute a dioxin-responsive enhancer system. PMID- 3010319 TI - Overlapping promoters and their control in Escherichia coli: the gal case. AB - Two overlapping promoters compete for RNA polymerase in the region that controls the expression of the galactose operon in Escherichia coli. Kinetics of open complex formation at P1 and P2 can be followed through the rate of formation of two specific abortive transcripts. The corresponding forward kinetic constants appear to be identical over a wide range of enzyme concentrations and temperatures, indicating that the two processes are strongly coupled. We propose a scheme accounting for our observations. In a first step, the competition between the two sites is a simple kinetic process, involving the "on" rate constants. In a second step, a slow reequilibration occurs, implicating the "off" rate constants and the conversion of one open complex to the other through a set of closed complexes. The first step is clearly affected when the complex between cyclic AMP and its receptor is bound at the activator site. An estimate of the various rate constants describing open complex formation at P1 and P2 is provided, as well as a qualitative description of the effect of the activator complex on these two pathways. PMID- 3010321 TI - Topological and modulated distribution of surface markers on endothelial cells. AB - The mobility and distribution of angiotensin-converting enzyme (peptidyl dipeptide hydrolase, EC 3.4.15.1) and a specific endothelial cell surface protein was assessed by fluorescein-conjugated monoclonal antibodies on bovine and murine endothelial cells grown on their extracellular matrix. The combination of data obtained from fluorescence recovery after photobleaching measurements and observations under epifluorescence and total internal reflection fluorescence reveals a restriction of these protein markers to the apical membrane of endothelial cell. This asymmetry is evident both when cells are grown at a sparse density or at confluence. When cells are brought into suspension, the fluorescein conjugated antibody is found over the entire cell surface. The fluorescence disappears from the basal part of the cell when the cells are again spread on coverslips coated with a layer of extracellular matrix. Conversely, cells spread on glass coverslips without extracellular matrix do not show this restriction phenomenon. It is suggested that the extracellular matrix provides the signal to induce the restricted topology of membrane protein markers on endothelial cells. PMID- 3010320 TI - Role of DNA polymerase alpha and DNA primase in simian virus 40 DNA replication in vitro. AB - The role of DNA polymerase alpha (pol alpha) and DNA primase has been investigated in the simian virus 40 (SV40) DNA replication system in vitro. Removal of pol alpha and primase activities from crude extracts of HeLa cells or monkey cells by use of an anti-pol alpha immunoaffinity column resulted in the loss of replication activity. The addition of purified pol alpha-primase complex isolated from HeLa cells or monkey cells restored the replication activity of depleted extracts. In contrast, the pol alpha-primase complex isolated from either mouse cells or calf thumus did not. Extracts prepared from mouse cells (a source that does not support replication of SV40) did not replicate SV40 DNA. However, the addition of purified pol alpha-primase complex isolated from HeLa cells activated mouse cell extracts. pol alpha and primase from HeLa cells were extensively purified and separated by a one-step immunoaffinity adsorption and elution procedure. Both activities were required to restore DNA synthesis; the addition of pol alpha or primase alone supported replication poorly. Crude extracts of HeLa cells that were active in SV40 replication catalyzed the synthesis of full-length linear double-stranded (RFIII) DNA in reaction mixtures containing poly(dT)-tailed pBR322 RFIII. Maximal activity was dependent on the addition of oligo(dA), ATP, and creatine phosphate and was totally inhibited by aphidicolin. Since pol alpha alone could not replicate this substrate and since there was no degradation of input DNA, we propose that other enzymatic activities associate with pol alpha, displace the non-template strand, and allow the enzyme to replicate through duplex regions. PMID- 3010322 TI - Regulation of cytoskeletal proteins involved in cell contact formation during differentiation of granulosa cells on extracellular matrix. AB - The organization and the expression of cytoskeletal proteins involved in determining cell contact and shape were analyzed in granulosa cells during their differentiation on extracellular matrix (ECM)-coated culture plates. Rat granulosa cells from preovulatory follicles displayed an epithelial shape on ECM and formed multilayered aggregates with numerous gap junctions between neighboring cells. These cells had few actin cables and often only a diffuse pattern of actin and a low amount of vinculin in very thin focal adhesion sites. In contrast, cells grown on plastic formed a monolayer of flat cells with a reduced number of gap junctions but with numerous stress fibers and abundant large vinculin-containing focal contacts. On ECM, the cells were stimulated to produce high levels of progesterone, while only trace amounts of the steroid accumulated in cells on plastic dishes. Two-dimensional gel electrophoretic analysis of [35S]methionine-labeled cells revealed a dramatic decrease in vinculin, alpha-actinin, and actin synthesis in cells grown on ECM, as compared to cells grown on plastic, while the synthesis of the tubulins and of the intermediate filament protein vimentin remained unchanged. RNA blot analysis showed a marked decrease in actin mRNA levels in cells from ECM plates, while the level of tubulin mRNA remained essentially unchanged. It is concluded that the differentiation of granulosa cells on ECM in vitro is associated with changes in cell shape and cell contacts and that such changes in cell morphology are accompanied by simultaneous alterations in the organization and expression of cytoskeletal proteins that are involved in determining these cellular structures. PMID- 3010324 TI - Gene encoding T-cell-activating protein TAP maps to the Ly-6 locus. AB - Recently we described two murine T-cell membrane proteins, TAP (T-cell-activating protein) and TAPa (TAP-associated protein). Previous experiments suggested that TAP is involved in physiologic T-cell activation. The subject of this report is a genetic analysis of these molecules. TAP and TAPa map to the Ly-6 locus. The relationship of these molecules to other antigens encoded in this locus is examined. Based on tissue distribution, molecular structure, and functional properties, TAP is distinct from any previously described Ly-6 antigen, whereas TAPa is probably identical to the 34-11-3 antigen. TAP and TAPa are coexpressed on all cell types examined so far. Moreover, comparative studies demonstrate a complex developmentally regulated pattern in the expression of molecules encoded in this locus. PMID- 3010323 TI - Isolation of a cDNA clone for the human lysosomal proteinase cathepsin B. AB - The cysteine proteinase cathepsin B is one member of the lysosomal acid hydrolases. Based on the peptide sequence of rat liver cathepsin B, an oligonucleotide mixture containing 128 different 17-mers was synthesized and used as a probe to screen adult and fetal human liver cDNA libraries. A recombinant clone with a 1540-nucleotide insert was identified from the fetal library, and DNA sequence analysis confirmed that this clone encodes human cathepsin B. The clone, designated pCB-1, has sequences for 81% of the coding region (for amino acid residues 50-252) together with approximately equal to 880 nucleotides of the 3' untranslated region of the mRNA. The DNA sequence also shows that the predicted carboxyl terminus of the coding sequence is longer than the mature protein by 6 amino acid residues. Southern blot analysis of restriction enzyme digests of human placental DNA revealed a simple pattern of hybridizing fragments using the cathepsin B coding sequence as probe. The result suggests that there is a single copy of cathepsin B gene per haploid genome. PMID- 3010325 TI - Purification and characterization of peptides with corticotropin-releasing factor activity from porcine hypothalami. AB - Ten polypeptides that stimulated the release of corticotropin from superfused rat pituitary cells and that are structurally related to porcine corticotropin releasing factor were isolated from porcine hypothalami. The purification was carried out by gel filtration followed by reversed-phase HPLC using trifluoroacetic acid or heptafluorobutyric acid as the ion-pairing agent in water/acetonitrile solvent systems. The purified peptides were homogeneous by chromatography and by sequence analysis. One major polypeptide was characterized. Its structure is -H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu Arg-Gl u-Val -Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg Lys -Leu-Met-Glu-Asn-Phe-NH2 [Patthy, M., Horvath, J., Mason-Garcia, M., Szoke, B., Schlesinger, D. H. & Schally, A. V. (1985) Proc. Natl. Acad. Sci. USA 82, 8762-8766]. This 41-amino acid sequence is thought to represent porcine corticotropin-releasing factor. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptides, amino acid analysis, and carboxypeptidase Y digestion, the other nine polypeptides were found to be structurally similar to this 41-amino acid sequence. Modifications of this structure include deamidation of glutamine at position 26 or 29, oxidation of methionine at positions 21 and/or 38, a blocked N terminus, and deletion of phenylalanine amide at the C terminus. Eight of these nine modified peptides retained significant corticotropin releasing factor activity as shown by the stimulation of corticotropin release from superfused rat and pig pituitary cells. Some of these peptides may be present in pig hypothalami, while the others could have been produced during the isolation. PMID- 3010327 TI - Effect of delta 9-tetrahydrocannabinol on herpes simplex virus type 2 vaginal infection in the guinea pig. AB - The present investigation was undertaken to determine whether delta 9 tetrahydrocannabinol (delta 9-THC) decreases host resistance to herpes simplex virus type 2 vaginal infection in the guinea pig. The guinea pig was selected as the host since it has been shown to express a spectrum of primary herpes genitalis which is similar to that in humans. Animals were administered delta 9 THC or vehicle intraperitoneally on Days 1-4, 8-11, and 15-18. Herpes simplex virus was introduced intravaginally on Day 2. Host resistance to virus infection was assessed by comparing frequency and severity of lesions, virus shedding, and animal mortalities. Virus-infected animals treated with drug at doses of 4 and 10 mg/kg exhibited significantly greater severity of genital disease during the 30 day period of study when compared to virus-inoculated vehicle controls. A direct relationship was noted between dose of delta 9-THC and cumulative mortalities on Day 14 following primary infection. These results indicate that delta 9-THC decreases host resistance to herpes simplex virus type 2 vaginal infection in the guinea pig. PMID- 3010326 TI - Acetylcholine raises excitability by inhibiting the fast transient potassium current in cultured hippocampal neurons. AB - The effects of acetylcholine on cultured hippocampal neurons were investigated by using the whole-cell version of the patch-clamp technique. The CA1 region of the hippocampus was excised from brain slices of young rats (12-19 day old), incubated in a papain solution, and dissociated. Neurons were plated on a glial feeder layer. The experiments were conducted mostly on neurons cultured for 2-6 days. Upon depolarization under voltage clamp, these cells exhibited a fast transient outward current (A-current), which was inhibited by 4-aminopyridine (2.5 mM). Acetylcholine (0.1 microM) also inhibited this A-current, as did the muscarinic agonists bethanechol and muscarine. As expected from their inhibition of the A-current, acetylcholine and 4-aminopyridine both increased the amplitude of the action potential and prolonged its duration. We conclude that the inhibition of the A-current constitutes a mechanism by which acetylcholine exerts its excitatory influence on hippocampal neurons. PMID- 3010329 TI - Suppressive effect of delta 9-tetrahydrocannabinol in vitro on phagocytosis by murine macrophages. AB - Incubation of normal mouse peritoneal cells consisting of over 90% phagocytizing macrophages with delta 9-tetrahydrocannabinol (THC) resulted in a inhibition of phagocytic function. The THC in a dose-related manner suppressed the percentage of macrophages per culture which ingested yeast and the average number of yeast particles ingested by the phagocytizing macrophages. The vehicle used to suspend the THC in vitro, i.e., DMSO, had no detectable effect on macrophage function. Suppression of phagocytosis with no effects on viability or cell number occurred with doses of 10 micrograms or less THC per milliliter culture medium. Measurable suppression also occurred after 24- to 48-hr treatment of the macrophages with the THC. This compound had little if any detectable effect on phagocytosis when added directly to the cultures shortly before testing for phagocytosis. Further studies concerning the effects of THC on macrophage function appear warranted. PMID- 3010328 TI - The effect of altered 5-hydroxytryptamine levels on beta-endorphin content in rat brain. AB - The purpose of the present study was to examine the effect of altering the concentration of 5-hydroxytryptamine (5-HT) on beta-endorphin (beta-Ep) content in the hypothalamus, thalamus, and periaqueductal gray (PAG)-rostral pons regions of the rat brain. The selective 5-HT reuptake inhibitor, fluoxetine (10 mg/kg), significantly lowered beta-Ep content in the hypothalamus and the PAG. Parachlorophenylalanine, which inhibits 5-HT synthesis, significantly elevated beta-Ep in all brain parts studied. Intracisternal injections of the neurotoxin, 5',7'-dihydroxytryptamine, with desmethylimipramine pretreatment, significantly increased beta-Ep content in the hypothalamus and the PAG. In adrenalectomized rats, fluoxetine significantly decreased beta-Ep levels in the hypothalamus and increased the levels in the PAG. The results indicate that 5-HT may modulate the levels of brain beta-Ep. PMID- 3010330 TI - The role of cyclooxygenase and lipoxygenase pathways in the adhesive interaction between bovine polymorphonuclear leukocytes and bovine endothelial cells. AB - The effects of arachidonic acid metabolites, analogues and cyclooxygenase/lipoxygenase inhibitors were tested on an "in vitro" bovine model of endothelial cell (EC)-polymorphonuclear leukocyte (PMN) adhesion. Arachidonic acid blocked adhesion at 10(-5)M, a dose which also induced aggregation of PMN. Lower doses did not affect either EC-PMN adhesion or PMN aggregation. Various cyclooxygenase pathway metabolites were inactive in the EC-PMN adhesion assay, with the exception of prostaglandin A2 and prostaglandin B2 which significantly suppressed adhesion at 10(-5)M. Of the synthetic analogs tested, 6 alpha carbaprostacyclin I2, (5Z)-9 beta-ethynyl-calcium salt (U-64,567E) was inhibitory at 10(-5)M. The cyclooxygenase inhibitors acetylsalicylic acid, indomethacin and ibuprofen were inactive. Products of the lipoxygenase pathways, leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoic acid (5-HETE) and 15 hydroperoxyeicosatetraenoic acid (15-HPETE) exhibited variable inhibitory activity at 10(-5)M only. Paradoxical effects were observed with the putative lipoxygenase inhibitors 4,7,10,13-eicosatetraynoic acid (4,7,10,13 ETYA), 5,8,11,14-eicosatetraynoic acid (5,8,11,14 ETYA) and nordihydroguairetic acid (NDGA), which also suppressed EC-PMN adhesion at 10(-5)M. The dual cyclooxygenase lipoxygenase inhibitor, BW755C was inactive. Bovine PMNs did not respond chemotactically to LTB4 although they were able to synthesize the 5-lipoxygenase products LTB4 and 5-HETE. PMID- 3010331 TI - Evidence for distinct systemic extravasation effects of platelet activating factor, leukotrienes B4, C4, D4 and histamine in the guinea pig. AB - The relative potencies of platelet-activating factor (PAF), leukotrienes B4 (LTB4), C4 (LTC4), D4 (LTD4) and histamine to induce hemoconcentration (HC) were evaluated in the guinea pig. The maximal hematocrit increase (MHI) from PAF, LTD4 and histamine occurred 5-7 min after i.v. injection, whereas the MHI of LTC4 occurred 13-15 min after injection. LTB4 (2.97-5.95 nmol kg-1) did not produce HC. The magnitude of PAF-induced MHI was 2-fold that of LTC4 or LTD4, regardless of the dose of leukotrienes used. The doses (nmol kg-1) needed to produce 30% HC were: 0.14-PAF, 0.71-LTD4 and 3.37-LTC4 and 2,400 histamine. The HC effects of LTD4 were markedly reduced by prior administration of FPL-55712. However, neither LTC4 or LTD4 HC effects were significantly reduced by prior i.v. injection of CV 3988 (3.4 mg kg-1), a competitive receptor antagonist to PAF which is 98% effective in abolishing HC response to 0.14 nmol kg-1 PAF. Diphenhydramine abolished histamine-induced hemoconcentration but was without effect on PAF or LTD4. These results suggest that the responses of PAF, leukotrienes and histamine differ in their potency and may involve separate vascular recognition sites related to acute increases in vascular permeability. PMID- 3010332 TI - Cysteinyl leukotrienes undergo enterohepatic circulation. PMID- 3010333 TI - Caffeine intensifies taste of certain sweeteners: role of adenosine receptor. AB - Caffeine, a potent antagonist of adenosine receptors, potentiates the taste of some but not all sweeteners. It significantly enhances the taste of acesulfam-K, neohesperidin dihydrochalcone, d-tryptophan, thaumatin, stevioside, and sodium saccharin. Adenosine reverses the enhancement. Caffeine has no effect on aspartame, sucrose, fructose, and calcium cyclamate. These results suggest that the inhibitory A1 adenosine receptor plays an important local role in modulating the taste intensity of certain sweeteners and that several transduction mechanisms mediate sweet taste. PMID- 3010334 TI - Opioid-receptor blockade reduces nose-poke self-stimulation derived from medial entorhinal cortex. AB - Rats were trained to nose-poke for intracranial self-stimulation (SS) with electrodes unilaterally implanted in the medial entorhinal cortex. The acute effects of naloxone (NX; 0.1-10 mg/kg, IP) on a continuous reinforcement schedule were determined. Reductions in the self-stimulation rates occurred only at moderate doses (median of individual changes = -36% at 1 and 5 mg/kg), whereas the high dose (10 mg/kg) was ineffective. None of the doses influenced operant behavior. These results are consistent with the hypothesis that endogenous opioid opiate receptor mechanisms play a modulatory role in SS reward. Considering that NX was administered systemically the action of the drug on reinforcement levels may be mediated by a site distinct from the locus of stimulation. PMID- 3010335 TI - Mu- and kappa-opiate agonists modulate ingestive behaviors in the slug, Limax maximus. AB - Administration of the prototypical mu opiate agonist, morphine sulphate, 1-10 mg/kg, produced over three hours a significant dose-dependent increase in the ingestive responses of free-feeding slugs, Limax maximus, although lower doses, 0.10 mg/kg, attenuated feeding. The mixed mu and kappa opiate agonist, ketocyclazocine hydrochloride, in the dose range 1.0-10 mg/kg, also induced significant increases in food consumption. With both of these opiates there was a latency of about 0.5 hr before initiation of feeding. The more specific kappa opioid agonist, U-50,488H, given over the dose range 0.10-1.0 mg/kg, produced a more potent increase in three hour food consumption by Limax, whereas a dose of 10 mg/kg produced a significant increase in ingestive responses for 3-4 hr after a 1-2 hr period of inactivity. The prototypic mu opiate antagonist, naloxone hydrochloride (1.0 mg/kg) blocked the feeding effects of morphine and ketocyclazocine and reduced the effects of U-50,488H. The delta antagonist, ICI 154,129, in a dose of 10 mg/kg, reduced the effects of morphine as well as decreasing food intake of free-feeding slugs. These results indicate that activation of differential opiate receptors in invertebrates has similar effects on feeding behavior as occur in mammals, suggesting early evolutionary development of opioid involvement in the control of feeding. PMID- 3010336 TI - Saccharin aversions in hamsters as a result of nicotine injections. AB - Golden Syrian hamsters (males, N = 70) showed dose-related conditioned taste aversion (CTA) when saccharin drinking was followed by delayed nicotine injections. Baseline consisted of measuring amounts consumed after 20 minutes of daily access to tap water. Measures were taken for five days. The hamsters were then conditioned by offering them saccharin solution (0.1%, w/v) for 20 minutes; afterwhich a 30 minute delay was imposed. Subsequent to the delay, groups of 10 animals were treated as follows: nicotine injection (1.0, 3.0, or 9.0 mg/kg, IP), saline injection, lithium chloride injection (2% body weight of a 0.15 M solution), sham injection, or left in their cages as handling/stress controls. Following two recovery days with plain water available for 20 minutes, all animals were tested for CTA by offering them saccharin solution. Dose-related CTA was demonstrated in the nicotine animals as measured by a decrease in saccharin consumption compared to drinking measures obtained from animals injected with saline. Lithium chloride produced the same degree of CTA as 9 mg/kg of nicotine, and the aversions had extinguished in all groups by the third test day. PMID- 3010337 TI - Effects of the potentiation of the GABAergic neurotransmission in the olfactory bulbs on mouse-killing behavior. AB - Intra olfactory bulb administration of three classes of GABA-mimetics (GABAa agonists, inhibitors of reuptake, inhibitors of GABA degradation) clearly inhibit mouse-killing behavior, without sedation. A linear correlation is observed between GABA levels increase in the olfactory bulbs and muricidal inhibition following local injection of valproic acid and gamma-vinyl GABA, two GABA-T inhibitors; the differences observed between these two compounds may be due to the differences in their mechanism of action on GABA-T activity and to the different pool of GABA on which they act. No diffusion to extra bulbar sites were observed after local administration of gamma-vinyl GABA. This evidence suggests an inhibitory role of GABA from olfactory bulbs in the modulation of mouse killing behavior. PMID- 3010338 TI - Genetic influences on GABA-related seizures. AB - Genetic differences in susceptibility to chemically induced seizures were examined in various populations of mice. Three inbred strains: C57BL, DBA, and C3H and a heterogenous stock (HS) of mice were tested for sensitivity to seizures induced by 3-mercaptopropionic acid (MP) and flurothyl. Dose response curves were constructed for each population of mice with each agent by quantitating latencies to specific stages of seizures. Significant strain and sex differences were observed in sensitivity to MP-induced seizures. Rank order from least sensitive to most sensitive was C57BL, HS, DBA, and C3H. Sensitivity to flurothyl-induced seizures was also strain dependent, but the rank order of sensitivity was different than for MP. The least sensitive strain was C57BL followed by HS, C3H, and DBA. Analysis of GABA receptors in seven brain regions obtained from C57BL and DBA using 3H-muscimol to measure high affinity GABA binding did not reveal significant differences in receptor number between these two strains. It thus appears that different genetic factors influence susceptibility to MP-induced seizures than to flurothyl-induced seizures. Furthermore, there is probably little correlation between the number of high affinity GABA receptors and sensitivity to seizures. PMID- 3010339 TI - Lordosis behavior and GABAergic neurotransmission. AB - gamma-Aminobutyric acid (GABA) (1.0 microgram/cannula) or muscimol (50 ng/cannula) was injected into the ventromedial hypothalamus or the lateral septi nuclei of ovariectomized rats brought to sexual receptivity by combined treatment of estrogen and progesterone. No inhibitory effects of GABA or muscimol were observed on the lordosis behavior. Furthermore, systemic (1.0 mg/kg) or intrahypothalamic (50 ng/cannula) picrotoxin administration was followed by a statistically significant increase in lordosis behavior in ovariectomized, estrogen-primed rats. No such effect was observed in ovariectomized adrenalectomized animals, indicating its dependence on adrenal secretions. Present results do not support the hypothesis of a GABAergic mechanism in the hormonal control of lordosis behavior. PMID- 3010340 TI - Cholinergic and opiate involvement in the antinociceptive effect of diisopropylfluorophosphate. AB - The organophosphate diisopropylfluorophosphate (DFP; 3 or 6 mg/kg, IP) caused a dose-related antinociception in mice which was antagonized by the muscarinic antagonist scopolamine. The opiate antagonist naloxone antagonized the antinociceptive effect of the highest dose of DFP, but did not affect the antinociception caused by 3 mg/kg DFP. Twenty-four hours after the administration of DFP, reaction time in animals which received a 3 mg/kg dose did not differ from control. However, reaction time was still significantly higher than control in mice administered 6 mg/kg DFP twenty-four hours earlier. This residual antinociception was antagonized by naloxone but not by scopolamine, suggesting that it was opioid in nature. These results suggest that antinociception induced by a low dose of DFP is primarily due to a cholinergic mechanism, while higher doses appear to affect also the opiate system. Since we have previously shown that DFP (6 mg/kg) increases met-enkephalin levels in brain, it is possible that high doses of DFP might interfere with enkephalin metabolizing enzymes. This conclusion cannot be extended to the organophosphate disulfoton, whose antinociception, even at high doses, appears to involve only an interaction with the cholinergic system. PMID- 3010341 TI - Protonophoretic activity of hypoglycemic sulfonylureas in black lipid membranes. AB - The ability of glibenclamide and of gliclazide to act as protonophores has been studied in black lipid membranes made of glycerol monoleate as a function of pH and drug concentration. Protons may effectively be transported across lipid membranes with a maximum at a pH close to the pKa of the sulfamide (pKa of glibenclamide = 5.4). It is suggested therefore that hypoglycemic sulfonylureas may act in part as Ca2+ inward and H+ outward ionophores through plasma membranes of pancreatic B cells. PMID- 3010342 TI - Postsynaptic alpha 1- and alpha 2-adrenoceptors mediating the action of the sympathetic system on muscle spindles, in the rabbit. AB - In anaesthetized and curarized rabbits, the cervical sympathetic nerve (CSN) stimulation induces in jaw elevator muscles a tension response which can be mimicked by the intravenous injection of adrenaline, noradrenaline and phenylephrine. This response, previously described and attributed to the contraction of muscle spindle fibres, is entirely mediated by alpha adrenoceptors. The administration of phenoxybenzamine (2.5-3.5 mg/kg) markedly inhibits the responses to the sympathetic stimulation and to the injection of adrenergic agonists. Rauwolscine (1 mg/kg) reduces the development of tension induced by both CSN stimulation and noradrenaline injection without significantly affecting the response to phenylephrine. These data suggest the presence of postsynaptic alpha 1- and alpha 2-adrenoceptors in intrafusal muscle fibres. Moreover, the possibility that alpha 2-adrenoceptors may also have an extrasynaptic location is entertained. PMID- 3010343 TI - Arachidonic acid metabolites and lung beta-adrenoceptor desensitization. PMID- 3010344 TI - Enhanced susceptibility to HPD-sensitized phototoxicity and correlated resistance to trypsin detachment in SV40 transformed IMR-90 cells. PMID- 3010346 TI - Microgravity changes in heart structure and cyclic-AMP metabolism. PMID- 3010345 TI - Effects of weightlessness on neurotransmitter receptors in selected brain areas. PMID- 3010347 TI - 1,25-Dihydroxyvitamin D3 receptors in space-flown vs. grounded control rat kidneys. PMID- 3010348 TI - Cholecystokinin octapeptide and lithium produce different effects on feeding and taste aversion learning. AB - The strengths of taste aversion induced by sulphated cholecystokinin 26-33 (CCK 8; 1,2,4 and 8 micrograms/kg IP) and lithium chloride (LiCl; 7.5, 15, 30 and 60 mg/kg IP) were determined in order to assess the relative aversiveness of the two compounds. All doses of LiCl induced strong aversion, but only the highest dose of CCK-8 induced aversion, which was mild. Effects of CCK-8 and LiCl on food intake were then compared in the hour (hr) following 8 hr of food deprivation; rats were on this food deprivation schedule for a relatively long time (78 days) throughout testing. All doses of CCK-8 reduced food intake significantly. Most doses of LiCl either did not affect or significantly increased food intake. Although 60 mg/kg LiCl did not affect food intake when administered 15 or 30 min before food presentation, it significantly increased food intake when administered 1, 2 or 3 hr before food presentation. Overeating of solid food may be an illness-induced behavior. Although a very high dose of LiCl (120 mg/kg) decreased food intake markedly, the rats were obviously distressed, not satiated. Failure of CCK-8 to affect feeding behavior like LiCl is indirect evidence that the reduction of food intake by CCK-8 is not merely the result of aversiveness, but is an extremely potent and specific behavioral effect. PMID- 3010349 TI - Nutrient control of cardiac rate in the infant rat: alpha-adrenergic mechanisms. AB - Changes in level of nutrient intake have been shown to exert a major influence on beta-adrenergic cardiac activity in 2 week old suckling rats. A series of experiments demonstrates that the alpha-adrenergic vasoconstrictor system is functional by 2 weeks postnatal age, and that alpha-adrenergic blockade with phenoxybenzomine (PBZ) does not affect the high heart rates of well-fed pups but prevents development of the bradycardia after cessation of suckling and fully reverses the low heart rates of nutrient-deprived pups in a dose-dependent manner. This last effect is dependent upon beta-adrenergic reflex cardiac pathways. After PBZ, milk feeding no longer produces cardiac acceleration in nutrient-deprived pups. Systolic and mean arterial pressure during PBZ administration and estimates of plasma volume change during nutrient deprivation are consistent with the inference that changes in peripheral resistance, mediated by the alpha-adrenergic vasoconstrictor system, accompany nutrient regulation of cardiac rate in the suckling rat. PMID- 3010351 TI - The natriuretic effect of 1-24 ACTH in rats: a comparison with the natriuretic oxytocin analogue nacartocin. AB - A slight but significant natriuretic action of 1-24 ACTH was confirmed both in trained conscious nonhydrated rats and in anaesthetized rats with sustained water diuresis. This action was compared with a strongly effective oxytocin analogue, nacartocin. PMID- 3010350 TI - Influence of jute (Corchorus olitorius) seed protein enriched diet on some enzymes and liver lipids of albino rats (Rattus norvegicus). AB - The effect of protein, isolated from Jute (Corchorus olitorius) seed was studied upon albino rats with respect to some of their serum, liver and intestinal enzymes and liver lipids. An increase in the body weight (including the weight of the liver) was noted in test animals after feeding with a Jute seed protein enriched diet. It was also observed that AST, ALT and total lipid of liver increased significantly whereas AST and ALT of serum were decreased. An increase in the concentration of lipids in the liver was found and this may be due to excess of the seed protein in the diet. An overall observation reveals that there is slight fatty infiltration in the liver of test animals. PMID- 3010352 TI - Molecular cloning and analysis of Staphylococcus aureus chromosomal aminoglycoside resistance genes. AB - Most of the aminoglycoside resistant Staphylococcus aureus strains isolated in France are resistant to all the antibiotics belonging to this family. Two aminoglycoside-modifying enzymes were detected in the wild-type strains studied: an APH3'III and an AAC6'-APH2". These strains also carry two types of streptomycin resistance: high-level resistance due to chromosomal mutation(s) affecting ribosome affinity and low-level resistance, the mechanism of which was not characterized. All the aminoglycoside resistance genes were located on the chromosome. DNA fragments of 1.5 and 1.95 kb carrying the aphA and aacA genes, respectively, were isolated, by cloning, from the cellular DNA of a clinical isolate. When these genes were introduced into Escherichia coli and Bacillus subtilis strains, the enzymes synthesized were indistinguishable from those produced by the S. aureus strains. When the cellular DNAs of wild-type and resistant strains were hybridized with the cloned fragments, sequences homologous to the fragment carrying the aphA gene were found to be located at the same chromosomal site, while those hybridizing with the fragment carrying the aacA gene were at different chromosomal sites. PMID- 3010353 TI - Analysis of the vegetative replication origin of broad-host-range plasmid RK2 by transposon mutagenesis. AB - A range of Tn1723 transposon mutants of the oriV region of broad-host-range plasmid RK2 have been isolated, and the internal EcoRI fragment of the transposon has been deleted from each to reduce the insertion size from 9.6 kb (Tn1723) to 35 bp (delta Tn1723). Sequencing from the delta Tn1723-derived EcoRI site has allowed the precise mapping of these insertions to various points dispersed through the origin region. Using these mutants we have determined which regions of oriV RK2 are of functional importance to plasmid establishment following transformation of the host species Escherichia coli, Pseudomonas putida, and P. aeruginosa. Insertions into an A/T-rich region, and a region containing five direct repeat sequences prevented successful transformation of each host species tested, but the continuity of sequences adjacent to the five repeats were essential only in E. coli and P. putida. The establishment and maintenance in E. coli of a mini-RK2 replicon was found to be inhibited by transcription from an inducible promoter positioned to read into oriV RK2 against the direction of replication. Assays of transcription emerging from Tn1723 demonstrated significant levels from one end of the transposon only. Four mutants with insertions downstream of oriV RK2 were unable to become established in E. coli, and contained Tn1723 in the orientation which would supply transcription toward the oriV RK2 region. These results demonstrate both that the sequence requirements for oriV RK2 function differ between host bacterial species, and that origin function may be further influenced by the genetic environment in which it lies. PMID- 3010354 TI - Vectors for the construction of gene banks and the integration of cloned genes in Schizosaccharomyces pombe and Saccharomyces cerevisiae. AB - We have constructed a variety of vectors for use in both budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe). Four of these, pDB262, pWH4, pWH5, and pMAK262, have positive selection for the insertion of cloned DNA, making them convenient for the construction of gene banks. pDB262, pWH4 and pWH5 contain the 2 mu ARS and the LEU2 gene from S. cerevisiae and can be used for gene isolation. They can also be converted into integration vectors for use in the genetic mapping of cloned sequences. pMAK262 contains only the LEU2 gene from budding yeast and can be used to screen for ARS elements or for gene integration. We also describe two other integration vectors, pDAM3 and pDAM6, which have a variety of restriction sites suitable for subcloning. PMID- 3010355 TI - Overproduction of proteins encoded by lambda replicon-integrated plasmid: effect of partial inactivation of cI repressor. AB - Composite plasmids containing the 7.2-kb fragment of bacteriophage lambda cI857cro27, essential for lambda DNA replication and its control, have been constructed. The plasmids contained, in addition, pBR322, a part of Co1E1, and the Escherichia coli pss gene, coding for phosphatidylserine synthase. When the plasmid-harboring cells were induced at different temperatures, the phosphatidylserine synthase production was maximum at 37 degrees C, and the specific activities at 37 degrees C continuously increased until the culture reached saturation. The results suggest that, when the cro gene is defective, only a transient temperature for inactivation of cI repressor provides both the increase in copy number of the plasmids and cellular activities that allow overproduction of the plasmid-encoded protein. PMID- 3010357 TI - [Thoughts on the neurobiological aspects of Parkinson syndrome]. AB - The Parkinson-syndrome is the most important syndrome under the extrapyramidal disease. The therapy with L-DOPA has a prominent place in the therapy of Parkinson disease since the introduction of oral effective drugs. Dopamin has an effect to the basalganglia as a neurotransmitter and perhaps an inhibition effect to specific synapses of brain. The inhibition or the optimal balance of the cholinergic systems is the effect of Dopamin and Noradrenalin. The therapy with low dosis of L-DOPA in combination with a decarboxylase-inhibitor will be prevent the side-effects of nerv-cells. PMID- 3010356 TI - The nucleotide sequence of pUB110: some salient features in relation to replication and its regulation. AB - For the study of DNA-membrane interaction and the regulation of replication initiation we have determined the total nucleotide sequence of pUB110. As previously reported, this plasmid replicates in B. subtilis at a copy number of 30-50 per cell, with a majority of plasmids (60-80%) bound to the membrane (type I binding). The type-I membrane binding is apparently necessary for pUB110 initiation of replication in vivo, but the membrane binding site is not known. Furthermore, four areas of the plasmid specifically bind to Bacillus subtilis membrane in an in vitro binding reaction (type II binding). These two types of membrane binding of pUB110 are different in that the in vivo binding (type-I) requires one (dnaBI) of the host initiation genes and is high-salt resistant, whereas the in vitro binding (type-II) does not require the dnaBI gene product and is high-salt sensitive. 7-mer double-strand sequence, TCAGCAA/AGTCGTT, or one base derivatives of this sequence are frequently (17 of 23 of the 7-mer sequences) found in or close to the type-II binding areas. One of them is found at a restriction enzyme recognition site of a binding area that destroys the type II membrane binding. These sequences may or may not have significance in type-II membrane binding. In addition to the neomycin resistance gene, the sequence data indicate two sizable open reading frames, ORF alpha and ORF beta, and two small ORF, gamma, and delta. All of these reading frames are in the same direction, which coincides with the direction of the replication. The open reading frame alpha (ORF alpha) corresponding to 334 amino acids close to the replication origin may be essential for the initiation of replication of PUB110. The putative protein alpha corresponding to this open reading frame contains a consensus sequence of the DNA binding sites which are found in a number of known DNA binding proteins. The consensus DNA binding site of protein alpha is flanked by two hydrophobic areas. These two observations suggest that the corresponding protein may have both an affinity to a specific site in pUB110, and an affinity to the membrane. PMID- 3010358 TI - [Description of sociodemographic and psychiatric data of 295 patients following attempted suicide by poisoning--inpatient treatment within the scope of a psychiatric liaison service at an internal medicine clinic of a large municipal hospital]. AB - 295 attempted suicides with intoxication are described by sociodemographic, biographic, and psychiatric characteristics. The patients were hospitalized for detoxification, and after clearing up interviewed by a psychiatrist working in the hospital as a liaison-psychiatrist. The patients did not differ essentially from patients treated in the emergency unit of another general hospital of the city in terms of basic social data. The patients--65% women, 35% men--are young (up to 40 years), 39% had already attempted suicide before the index-time. 41% came from "broken homes", 40% had one or more psychiatric disease(s) in their families. 56% were diagnosed as "psychiatrically ill", second diagnosis was in 33.5% abuse of alcohol/drugs. The referal offer to the patients for the time after discharge from emergency unit is described in detail. Just 12% of the patients were treated in a psychiatric hospital, after-care in an ambulatory setting seemed to be sufficient for most of the other patients. 20% were not offered any after-care. Patients with addiction and psychosis were sent to institutions specialized for these diagnoses. PMID- 3010359 TI - Influence of prenatal ethanol exposure on hormonal responses to clonidine and naloxone in prepubescent male and female rats. AB - The expression of sexually dimorphic behavior has been found to be altered in adult animals following prenatal alcohol exposure. The present study examined whether such exposure would alter the sexually dimorphic response of luteinizing hormone (LH) to clonidine and naloxone observed in normal prepubescent animals. Both LH and corticosterone (CS) were measured in 16 day old male and female rats 30 min after injection of naloxone (2 mg/kg) or clonidine (0.1 mg/kg). Prenatal alcohol exposure did not influence the LH response to either drug in females. An LH response to clonidine in normal males did not occur, but it was present in the males exposed to alcohol in utero and in the pair-fed controls. Prenatal alcohol exposure influenced the CS response to both drugs. CS levels were depressed in the naloxone-treated males prenatally exposed to alcohol compared to their saline injected counterparts. The CS levels of other groups following naloxone administration were unchanged compared to saline injection. Normal animals of both sexes exhibited an elevation in CS levels following clonidine. However, this stimulatory effect of clonidine on CS release was absent in both female and male animals prenatally exposed to alcohol. The results of this study indicate that prenatal alcohol exposure may alter noradrenergic and opioid modulation of corticosterone and possibly of LH in young animals. PMID- 3010360 TI - Corticotropin-releasing factor (CRF): stimulation in normal controls and in patients with Cushing's syndrome. AB - Synthetic ovine and human CRF were given as an i.v. bolus to six healthy volunteers in four and two different dosages, respectively (oCRF: 25, 50, 100 and 200 micrograms; hCRF: 50 and 100 micrograms). There was a significant increase of ACTH and cortisol after the injection of all dosages though the dose-response relationship was only significant between the 50 and 100 micrograms dose of oCRF. No significant differences between ACTH and cortisol secretion after oCRF and hCRF were observed. Repetitive stimulation by hCRF led to repetitive release of identical amounts of ACTH. The CRF test with the 100 micrograms dosage was used in patients with proven Cushing's syndrome (n = 30). Results showed that the CRF test is useful in making the differential diagnosis of established Cushing's syndrome. In patients with ACTH-dependent Cushing's disease (n = 21), normal or elevated basal ACTH levels were significantly higher after stimulation by CRF compared to normal controls, with one exception. The pattern of cortisol secretion after CRF administration corresponded to the pattern of ACTH secretion in these patients. In two patients with ectopic ACTH syndrome, extremely elevated ACTH and cortisol levels did not change or showed only a small increase after CRF administration. In patients with unilateral adrenal adenoma or carcinoma (n = 7), suppressed ACTH levels did not rise after CRF administration. In addition, no significant change in cortisol secretion could be observed. After surgical removal of cortisol-producing adrenal tumors, the ACTH response to CRF can be demonstrated when cortisol levels are still undetectable. Pulsatile administration of CRF in one patient after unilateral adrenalectomy revealed that ACTH responses to CRF normalize rapidly but cannot be sustained if CRF administration is withdrawn, suggesting that the cause of adrenal failure after unilateral adrenalectomy for Cushing's syndrome or with long-term corticoid therapy is due to hypothalamic CRF deficiency. The suppression of ACTH responses to CRF in glucocorticoid-treated patients correlated with the daily corticoid dosage. Since the ACTH hyper-response to CRF in six patients with Cushing's disease was suppressed by short-term dexamethasone treatment, the pituitary as a target site for feedback inhibition also was demonstrated. PMID- 3010361 TI - Down regulation of serotonin-S2 receptor sites in rat brain by chronic treatment with the serotonin-S2 antagonists: ritanserin and setoperone. AB - Ritanserin is a potent and selective serotonin-S2 antagonist which slowly dissociates from the receptor sites, while setoperone has potent serotonin and moderate dopamine antagonistic properties and dissociates rapidly from the receptor sites. Acute administration of ritanserin (1-10 mg/kg) produced a non competitive inhibition of 3H-ketanserin binding, measured ex vivo in washed frontal cortex membranes, which lasted for 12 h. This is in accordance with the slow dissociation of the drug from the receptor sites. Setoperone (1-10 mg/kg orally) also produced a partially non-competitive inhibition of 3H-ketanserin binding in washed membranes, which is unlike its rapid dissociation. In contrast, there was no inhibition of dopamine receptor binding in washed striatal membranes. Chronic oral administration of 10 mg/kg X day of the drugs significantly reduced the Bmax values of 3H-ketanserin, without changing the KD value when drug-free periods were longer than 1 day. The maximum reduction following 25 days' treatment with 14 mg/kg ritanserin was 50% at 1 day drug-free; the Bmax values gradually returned to the control value in about 12 days. The receptor half-life was calculated to be 3.5 days and the receptor synthesis rate 4 fmoles/mg tissue X day. Ritanserin treatment did not alter radioligand binding to serotonin-S1, alpha 1-, alpha 2- and beta-adrenergic, dopamine-D2, benzodiazepine and substance P sites. Chronic treatment with setoperone at 10 mg/kg X day, orally, significantly reduced the Bmax value of 3H-ketanserin binding in frontal cortex but treatment with 1 mg/kg X day did not. In contrast, a dose-dependent increase in the number of striatal dopamine-D2 sites was observed, in accordance with the moderate dopamine-antagonistic properties of setoperone. Dopamine-D2 receptor up regulation up to 150% of control values, was maintained at the same level for 9 days, it started to decline 12 days after stopping drug treatment. Following chronic treatment and drug withdrawal for more than 1 day, ritanserin and setoperone levels in whole brain homogenates were below detection level (less than 1 ng/g). The similar reduction in the Bmax values of 3H-ketanserin binding following chronic treatment with the rapidly dissociating setoperone and the slowly dissociating ritanserin, the absence of effect on the KD value, the slow reappearance of the receptor sites and the opposite effect on serotonin-S2 and dopamine-D2 receptors with setoperone suggest that real serotonin-S2 receptor down regulation occurs following antagonist treatment. The findings illustrate the difference in receptor regulation between the serotonergic and the dopaminergic system.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3010362 TI - Heroin increases the density and sensitivity of platelet alpha 2-adrenoceptors in human addicts. AB - The density of platelet alpha 2-adrenoceptors, quantified by means of the binding of [3H]clonidine, and the aggregation response induced by adrenaline, was investigated in ten heroin addicts. The number of binding sites for [3H]clonidine was significantly increased during heroin abuse. Concomitantly, there was a potentiation of the adrenaline-induced platelet aggregation, which suggested that continuous heroin use (opiate dependence) is associated with supersensitive platelet alpha 2-adrenoceptors in human addicts. Spontaneous heroin withdrawal further increased in the same addicts the density of platelet alpha 2 adrenoceptors. These results suggest that platelet alpha 2-adrenoceptors may be used as a model to study receptor mechanisms of opiate dependence and withdrawal in humans. PMID- 3010363 TI - A late-appearing benzodiazepine-induced hypoactivity that is not reversed by a receptor antagonist. AB - The activity of rats in a holeboard test is reduced 30, 90, and 240 min after treatment with a single dose of lorazepam. The administration of a benzodiazepine antagonist (RO 15-1788) 20 min before the holeboard test (i.e., 10, 70, or 220 min after lorazepam administration) reverses the hypoactivity of animals tested 30 min after treatment with lorazepam, partially reverses the hypoactivity of animals tested 90 min after receiving lorazepam, but is without effect on the hypoactivity observed 240 min after treatment with the benzodiazepine. If, however, RO 15-1788 is given at the same time as lorazepam then it reverses the hypoactivity seen 4 h later. The results of these experiments demonstrate that a benzodiazepine can exert a behavioral effect at a time when it no longer appears to be acting at central benzodiazepine receptors. PMID- 3010364 TI - Antinociceptive activity of beta-adrenoceptor agonists in the hot plate test in mice. AB - Clenbuterol, isoproterenol and salbutamol increased the latency in the licking response of mice on the hot plate at 56.5 degrees C. This effect was dose-related and obtained after IP administration. Clenbuterol was active at lower doses than isoproterenol and salbutamol, a result which is consistent with its better penetration in the central nervous system. Moreover, the effect of clenbuterol was stereospecific, the (-)-isomer being the active form. Our results suggest the implication of central beta-adrenoceptors in the modulation of the response to a painful stimulation. PMID- 3010366 TI - Tooth reimplantation following unintentional avulsion utilizing synthetic bone. PMID- 3010365 TI - External root resorption associated with placement of Durapatite--case report. PMID- 3010367 TI - Spin-label substitutions in cisplatin reduce toxicity and interaction with radiation. AB - A piperidinyl-labeled platinum(II) complex, cis-Pt[2,2,6,6-tetramethyl-4 aminopiperidine-N-oxyl]2dichloride , is an ESR-traceable analog of the antitumor agent, cis-diamminedichloroplatinum(II), DDP. The toxicity of the analog (PDN-1) in Chinese hamster ovary (CHO) cells is 22 times less than that of the parent compound: D0 (where D0 = slope-1 of the straight-line portion of the survival curve) = 0.11 mM versus 0.0053 mM for 1.5-h treatments with PDN-1 and DDP, respectively. At PDN-1 doses that cause 10% killing when used alone, no statistically significant increase in cell killing of hypoxic, irradiated cells was observed. PDN-1 does not have significant dose-modifying properties but may be a useful probe to study platinum in mammalian cells. PMID- 3010369 TI - [Endonucleolysis of chromatin in thymocytes of irradiated and non-irradiated rats]. AB - It was shown that in conditions optimal for Ca/Mg endonuclease, chromatin endonucleolysis in the nuclei and thymocytes occurs due to internucleosome fragmentation of DNA. Irradiation activates chromatin degradation in thymocytes washed by a buffer containing 0.25 M sucrose, 10 mM tris-HCl, pH 7.2, 3 mM MgCl2, and does not influence this process in thymocytes washed by 10 mM tris-HCl, pH 7.2, 3 mM MgCl2. PMID- 3010368 TI - A comparison of radioprotection from three neutron sources and 60Co by WR-2721 and WR-151327. AB - Two thiophosphoroate radiation protectors (WR-2721 and WR-151327) were assessed for their ability to modify the effects of neutron or gamma irradiation on the gastrointestinal tract. Three neutron sources (DOSAR, JANUS, and FERMILAB) were compared to the response obtained after 60Co irradiation. The end points studied were intestinal stem cell survival and LD50(6). DOSAR and JANUS, both fission spectrum neutrons, showed somewhat different gut sensitivities [LD50(6)] of about 240 and 400 cGy respectively. The intestinal LD50 obtained with FERMILAB neutrons (25 meV) was closer (875 cGy) to that obtained after 60Co (1068 cGy) irradiation. WR-151327 protected against the lethal effects of fission neutron (DOSAR and JANUS) to a greater degree (DMF = 2.2) than with lower LET sources such as FERMILAB neutrons (DMF = 1.7) or 60Co (DMF = 1.7). The results did not correlate with the intestinal stem cell assays where WR-2721 when compared to WR-151327 showed either similar (DOSAR; fission spectrum neutrons) or somewhat better (60Co and FERMILAB neutrons) protection. Possible explanations for the differing results are discussed. PMID- 3010370 TI - [Changes in the ratio of cyclic nucleotide phosphodiesterase forms in the thymocytes of irradiated mice]. AB - An increase in the activity of cyclic nucleotide phosphodiesterase was noted during the first hour following irradiation of mice with a dose of 11 Gy and after in vitro irradiation of the thymocyte suspension. Using the methods of gel filtration and sedimentation and in studying the kinetic characteristics it was shown that the effect of radiation changed the degree of the enzyme aggregation, i. e. the share of the dissociated forms, which possessed a higher catalytic activity, increased. PMID- 3010372 TI - [Cyclic adenosine-3',5'-phosphate and guanosine-3',5'-phosphate in mouse plasma during administration of chemical compounds with different degrees of radioprotective effectiveness]. AB - A study was made of seven radioprotective agents of different chemical classes (sulfur-containing, indolylalkylamines, and imidazol, urea and pyridasine derivatives) and also of their six structural analogs without radioprotective properties on the content of cyclic nucleotides in blood plasma and on the postirradiation survival of mice. There was a correlation between the ability of the preparations to increase the level of cyclic adenosine-3',5'-monophosphate and their radioprotective properties; with guanosine-3',5'-monophosphate, this correlation was absent. PMID- 3010371 TI - [The effect of gamma-hydroxybutyric acid on the biosynthesis of macromolecules and metabolic paramagnetic centers in hepatocytes in acute radiation injury]. AB - It has been shown, that the single injection of gamma-hydroxybutyric acid (GHBA) (100 mg/kg) to mice 30 min before irradiation (6 Gy) prevents whole-body irradiation-induced inhibition of DNA at early post-irradiation period. GHBA stimulates the biosynthesis of macromolecules at 1-2 days after irradiation. GHBA also prevents the increase in the degree of reduction of the mitochondrial and microsomal electron transport chains at early post-irradiation time (up to 2 h.), that takes place only under irradiation. It means, that GHBA inhibits the production of O2 radicals, which induce lipid peroxidation processes at post irradiation period. PMID- 3010374 TI - Hepatic perfusion abnormalities during CT angiography: detection and interpretation. AB - Twenty-seven perfusion abnormalities were detected in 17 of 50 patients who underwent computed tomographic angiography (CTA) of the liver. All but one of the perfusion abnormalities occurred in patients with primary or metastatic liver tumors. Perfusion abnormalities were lobar in nine cases, segmental in 11, and subsegmental in seven; 14 were hypoperfusion and 13 were hyperperfusion abnormalities. The causes for the abnormalities included nonperfusion of a replaced hepatic artery (n = 11), cirrhosis and nodular regeneration (n = 3), altered hepatic hemodynamics (e.g., siphoning, laminar flow) caused by tumor (n = 7), contrast media washout from a nonperfused vessel (n = 1), compression of adjacent hepatic parenchyma (n = 1), and unknown (n = 4). Differentiation of perfusion abnormalities from tumor usually can be made by comparing the morphology of the known tumor with the suspected perfusion abnormality, changes of each on delayed CTA scans, and review of initial angiograms and other imaging studies. PMID- 3010373 TI - [Effect of gaseous hypoxic mixture GHM-10 on the intestinal death of Wistar rats and Na+,K+-ATPase activity of the plasma membrane of the small intestine mucosa after irradiation]. AB - It was shown that gas hypoxic mixture containing O2 (10%) and N2 (90%) significantly decreases "intestinal" death of Wistar rats on the 5th day following irradiation and normalizes Na+,K+-ATPase activity of the small intestine mucosa plasma membranes. PMID- 3010375 TI - Uterine neoplasms: MR imaging. AB - Magnetic resonance (MR) studies were performed on 20 healthy volunteers and 41 patients with proved cervical and uterine neoplasms. MR imaging demonstrated normal uterine landmarks in all patients. On T2-weighted images, the normal uterine wall could be differentiated into three distinct layers: a central high intensity zone, a junctional low-intensity band, and a peripheral medium intensity area. While most of the normal cervices had only two distinct zones (central high-intensity zone and peripheral low-intensity zone), a small percentage had three layers of signal intensity, similar to the uterine body. Primary cervical and uterine neoplasms could be identified on MR images. In 18 of 22 patients with proved carcinoma, a mass with a signal intensity higher than that of normal cervical lips was seen on T2-weighted images. Endometrial carcinoma was most often identified as expansion of the central high-intensity area; discrete tumor nodules were visible in nine of 15 patients. Mixed mullerian sarcoma appeared as a large pelvic mass with complete obliteration of normal uterine landmarks. MR imaging delineates primary cervical and endometrial carcinoma better than computed tomography does. PMID- 3010376 TI - Dynamics of zonal hepatocyte heterogeneity. Perinatal development and adaptive alterations during regeneration after partial hepatectomy, starvation and diabetes. AB - The liver is the "glucostat" of the organism and serves at the same time as an "ammonia-sink and pH stat". The key enzymes involved in glucose uptake and release and in urea and glutamine formation are reciprocally distributed over the liver parenchyma: The glucogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK), fructosebisphosphatase (FBPase) and glucose-6-phosphatase (G6Pase) as well as the ureagenic enzyme carbamoylphosphate synthetase (CAPS) are predominant in the periportal zone. The glycolytic enzymes glucokinase (GK) and pyruvate kinase type L (PKL) as well as the glutaminogenic enzyme glutamine synthetase (GluNS) are prevalent in the perivenous zone. This heterogeneity appears to be a prerequisite for the normal "glucostat, ammonia-sink and pH-stat" function of the liver. After birth the liver is a gluconeogenic organ, only with weaning it becomes a "glycolytic/gluconeogenic" glucostat. In the rat zonation of PEPCK, G6Pase and CAPS developed gradually after birth and was completed before weaning, i.e. before it would be functionally required. After 2/3 partial hepatectomy the liver looses its normal glucostat function and becomes a gluconeogenic organ. With this change the zonation of PEPCK and PKL were also lost; it was restored only during the second week after operation. During starvation the liver also looses its glucostat function to become the major glucose supplier of the organism. Zonation of PEPCK and PKL were diminished to such an extent that the major function of the perivenous zone was altered from glucose uptake to release. In diabetes the liver does not loose its glucostat function; however, the function is severely impaired. Zonation of PEPCK was increased and that of PKL decreased in such a manner that the major function of the perivenous zone, glucose uptake, was not entirely changed but only diminished. It can be concluded that in the various physiological states studied the zonation of enzymes correlated well with the glucostat function of the liver. PMID- 3010378 TI - Nutritional aspects of colon cancer. AB - During the last decade, a substantial amount of progress has been made in the understanding of the relationship between the dietary constituents and the development of colon cancer in man. The information base is sufficiently convincing with respect to an enhancing effect as a function of total fat intake and a protective effect of certain dietary fibers in colon cancer. The populations with high incidence of colon cancer are characterized by consumption of high-dietary fat which may be a risk factor in the absence of factors that are protective, such as use of whole-grain cereals, high fibrous foods and vegetables mainly of cruciferous type. Application of the findings made thus far in colon cancer research for the general public is, therefore, to have a far-reaching impact on the major premature, killing diseases in the western world. PMID- 3010377 TI - The oxygen free radical system: a fundamental mechanism in the production of myocardial necrosis. PMID- 3010380 TI - Dietary fibre: consensus and controversy. AB - Technological advances have reduced and refined man's plant food intake and consequently brought about an unprecedented decline in his consumption of dietary fibre (DF). The emergence of certain diseases selectively in regions which have been affected the most by this dietary change has led to an enhanced awareness of the functions of DF. DF is a heterogeneous group of substances which resist digestion by the endogenous enzymes of the human gut, although they are fermented to a substantial extent by the bacterial flora of the large intestine. Chemically, DF essentially consists of nonstarch polysaccharides and lignin, and its major constituents are cellulose, hemicelluose, lignin and pectin. The physiological effects of DF are attributable largely to its physicochemical properties. DF primarily affects gastrointestinal (GI) function; its effects are observable at all stages from ingestion through defaecation. It restricts caloric intake, shows gastric and small intestinal transit, and affects the activity of digestive enzymes and release of GI hormones. Its overall impact is to reduce apparent digestibility of nutrients marginally but consistently. In the large intestine, DF accelerates transit, supports bacterial growth and serves to hold water. As a result, the faecal weight and water content increase, and the transit time generally becomes shorter. Secondary to its GI effects, DF attenuates postprandial glycaemia and has long term effects on glucose tolerance and lipoprotein metabolism. These effects have important implications in the aetiopathogenesis of constipation and its sequelae including diverticulosis, cholesterol gallstones, colorectal cancer, obesity, diabetes mellitus and atherosclerosis. DF has traditionally been used therapeutically for constipation; now its use in diabetes is also well established. Our appreciation of the role of DF in human nutrition has undergone a major change in the last two decades. From a redundant constituent of plant foods, it has now moved to the position of an essential nutrient, the deficiency of which seems to have serious consequences. PMID- 3010379 TI - Diet, nutrition, and cancer. AB - Evidence pertaining to the role of dietary factors in carcinogenesis comes from both epidemiological studies and laboratory experiments. In 1982, the Committee on Diet, Nutrition, and Cancer of the National Research Council conducted a comprehensive evaluation of this evidence. That assessment as well as recent epidemiological and laboratory investigations suggest that a high fat diet is associated with increased susceptibility to cancer of different sites, particularly the breast and colon, and to a lesser extent, the prostate. Current data permit no definitive conclusions about other dietary macroconstituents including cholesterol, total caloric intake, protein, carbohydrates and total dietary fiber. Specific components of fiber, however, may have a protective effect against colon cancer. In epidemiological studies, frequent consumption of certain fruits and vegetables, especially citrus fruits and carotene-rich and cruciferous vegetables, is associated with a lower incidence of cancers at various sites. The specific components responsible for these effects are not clearly identified, although the epidemiological evidence appears to be most consistent for a protective effect of carotene on lung cancer and less so for vitamins A and C and various cancer sites. The laboratory evidence is most consistent for vitamin A deficiency and enhanced tumorigenesis, and for the ability of various nonnutritive components in cruciferous vegetables to block in vivo carcinogenesis. The data for minerals and carcinogenesis are extremely limited, although preliminary evidence from both epidemiological and laboratory studies suggests that selenium may protect against overall cancer risk. Frequent consumption of cured, pickled, or smoked foods, possibly because they may contain nitrosamines or polycyclic aromatic hydrocarbons, appears to increase the risk of esophageal or stomach cancer, however, the specific causative agents in these foods are not clearly identified. Excessive alcohol consumption among smokers appears to be associated with an elevated risk of cancers of the oral cavity, esophagus, larynx, and respiratory tract. The mechanisms of action of dietary factors on carcinogenesis are poorly understood. The NRC committee, and more recently, the National Cancer Institute and the American Cancer Society have proposed interim dietary guidelines to lower the risk of cancer. These guidelines are consistent with general dietary recommendations proposed by U.S. government agencies for maintenance of good health.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3010381 TI - Influence of estrogen levels on anticholinergic activity of tricyclic antidepressives. AB - Anticholinergic activity of a number of tricyclic antidepressives and nomifensine was demonstrated and their order of affinity for the cholinergic receptors of rat jejunum was determined. The influence of ethinyl estradiol and a conjugated estrogen product, Premarin on the binding of several tricyclic antidepressives to the hepatic mixed function oxidase system was investigated. The influence of these steroids on the metabolism of the antidepressives was evaluated and ethinyl estradiol was shown to have a marked influence on the metabolism of the antidepressives studied, while conjugated estrogens were shown to have little effect. PMID- 3010382 TI - Abnormal ACTH and cortisol responses to ovine corticotropin releasing factor in patients with primary affective disorder. AB - To further explore hypothalamic pituitary adrenal regulation in patients with affective illness, we administered 1 microgram/kg of synthetic ovine corticotropin releasing factor at 2000h to 26 drug-free patients with this disorder and to 15 healthy controls. Compared to controls, depressed patients (N = 12) showed a significant elevation in baseline cortisol and significant reductions in the net ACTH and cortisol responses to corticotropin releasing factor. These findings were normal in manic (N = 6) and improved (N = 8) subjects. An additional finding was that baseline cortisol and net ACTH and cortisol responses to CRF were negatively correlated in the entire group of patients and controls as well as in the patients alone. These data indicate that the reduced ACTH and cortisol responses to CRF in depression reflect normal functioning of the pituitary corticotroph cell (i.e., that the negative feedback effect of cortisol on ACTH secretion in depression is physiologically intact, effectively serving as a brake on the ACTH response to exogenous CRF. Thus, the hypercortisolism of depression may be due to a hypothalamic defect, possibly involving hypersecretion of endogenous CRF. This possibility may be of particular interest in light of clinical observations that depression can often be precipitated by stress and by data in experimental animals that CRF may influence several processes known to be altered in the overall symptom complex of depression. PMID- 3010384 TI - Vehicular transmission of hepatitis A. PMID- 3010385 TI - Transcatheter arterial embolization (TAE) in treatment for hepatoma--analysis of three-year survivors. AB - Among 224 hepatomas treated with transcatheter arterial embolization, ten patients who survived for three years are presented. All cases had nodular lesions and the volume ratio of tumor to liver was less than 10% in nine cases. Eight cases had no large venous extension, however, one had 90% occlusion of the portal trunk and the other had tumor thrombus within the inferior vena cava. Laboratory data showed liver function was largely spared in eight cases, whereas the other two had severe liver cirrhosis. CT and/or angiography showed an increase in tumor volume in eight cases after three years of survival. Beneficial factors for long-term survival and the limitations of embolization are also discussed. PMID- 3010383 TI - Cyclazocine-induced sleep disruptions in nondependent addicts. AB - After one adaptation night, the sleep of seven male nondependent opiate addicts was studied following intramuscular cyclazocine (0.125; 0.25; 0.50 mg/70 kg) or placebo at weekly intervals in a randomized double-blind crossover design. Drug effects were measured on sleep stages and several episodic phenomena. Cyclazocine caused dose-related increases in sleep latency, REMS latency, percent spindle sleep, and a marked increase over placebo in wakefulness, drowsiness, and shifts in sleep-waking states. Cyclazocine produced a dose-related decrease in all measures of delta sleep, and some measures of REMS, and a marked decrease below placebo of sleep efficiency and total REMS. All doses of cyclazocine caused sustained periods of waking with little muscle tension. Cyclazocine (0.5mg) consistently caused urination during periods of extended arousal; urination has not been seen after morphine or other opioids of the mu type. These studies indicate that cyclazocine has effects on human sleep which are in some ways similar and other ways dissimilar to morphine type analgesics. The results are consistent with the concept that cyclazocine is a mixed agonist-antagonist of the opioid type with agonist actions at the kappa receptor. PMID- 3010387 TI - Evidence for different pre-and post-junctional receptors for neuropeptide Y and related peptides. AB - The effects of neuropeptide Y (NPY), peptide YY (PYY), desamido-NPY and five C terminal fragments of NPY or PYY were tested on different smooth muscle preparations in vitro. The fragments were NPY 19-36, NPY 24-36, PYY 13-36, PYY 24 36 and PYY 27-36. NPY and PYY appear to exert three principally different effects at the level of the sympathetic neuroeffector junction. Firstly, they have a direct post-junctional effect, leading to constriction of certain blood vessels; this was studied on the guinea-pig iliac vein. Secondly, they potentiate the response to various vasoconstrictors; this was studied on the rabbit femoral artery and vein, using noradrenaline and histamine, respectively, as agonists. Thirdly, NPY and PYY act prejunctionally in that they suppress the release of noradrenaline from sympathetic nerve endings upon stimulation; this was studied in the rat vas deferens. NPY and PYY were approximately equipotent in constricting the guinea-pig iliac vein, while desamido-NPY and the fragments were without effect. Desamido-NPY and the fragments were ineffective also in potentiating the response to noradrenaline in the rabbit femoral artery, nor did they potentiate the response to histamine in the rabbit femoral vein. NPY and PYY potentiated the response to noradrenaline in the artery, as well as the response to histamine in the vein. The NPY- and PYY-induced suppression of noradrenaline release from the prostatic portion of the rat vas deferens was reproduced by PYY 13-36 but not by the shorter fragments nor by desamido-NPY. In conclusion, a C terminal portion seems to be sufficient for exerting the prejunctional effect of NPY and PYY, while the whole sequence seems to be required for post-junctional (direct and modulatory) effects. An amidated C-terminal is crucial for maintaining the biological activity of NPY. Desamido-NPY and the fragments that were inactive as agonists also seemed inactive as antagonists. PMID- 3010386 TI - Abdominal recurrences in Wilms' tumours: a report from the SIOP Wilms' tumour trials and studies. AB - The Wilms' tumour trials and studies conducted from 1971 to 1980 registered 1042 patients. Of these, 82 patients developed an abdominal recurrence. Particulars of these were studied. Half of the recurrences occurred in stage III patients. Often several untoward prognostic factors could be identified, such as large tumour size, difficult operation, incomplete excision, peritoneal adhesions or metastases, tumour extending to renal vein or vena cava. A tumour rupture increases the chance for an abdominal recurrence, especially if appropriate radiotherapy is not given. In many of these cases, postoperative radiotherapy seems to have been insufficiently tailored to the operative findings. For stage III cases, a careful discussion between surgeon, radiotherapist, and pathologist should lead to the optimal radiotherapy field size and dose for each individual patient, so that the risk of abdominal recurrence can be reduced. PMID- 3010388 TI - [Activation of cellular oncogenes]. PMID- 3010390 TI - Alcoholic subtypes based on multiple assessment domains. Validation against treatment outcome. AB - Research in the area of subtype development within alcoholic populations has undergone a number of changes over the recent years. First, there has been a shift from subtype derivation based on a priori comparative approaches toward a more recent use of a posteriori correlational approaches such as cluster analysis. Second, there has been an increased multivariate focus in which multiple measures from a number of practically and/or theoretically relevant assessment domains are entered into the classificatory process. Third, there has been an increased emphasis on the external validation of derived clusters. The present chapter briefly reviews these issues, using examples of research from the Seattle Veterans Administration Medical Center to illustrate each of these shifts across a series of studies focusing on the development of alcoholic subtypes. Finally, the results of a recent subtype identification and validation study will be presented to illustrate the difficulties in and advantages of a multidomain cluster analytic strategy. PMID- 3010391 TI - Clinical neuroendocrinology and neuropharmacology of alcohol withdrawal. AB - A number of alcohol research groups have measured anterior and posterior pituitary hormones, the endogenous opiates, CNS peptides, and putative neurotransmitters during alcohol withdrawal. The data are often complex and contradictory, though a number of themes have emerged. Activity of the hypothalamic-pituitary-adrenal axis (HPA) is increased during chronic alcohol exposure and appears to remain altered for at least 2 to 4 weeks after cessation of drinking. There is increased turnover of norepinephrine and enhanced binding of CNS adrenergic receptors. By contrast, there are decreases in CNS activity of select endogenous opiates and GABA. Other CNS compounds that may play a role in alcohol withdrawal are prolactin, thyrotropin-releasing hormone (TRH), vasopressin, cyclic 3'5'-adenosine monophophate (cAMP), Delta-sleep-inducing peptide (DSIP), and iron. Despite many studies in humans and animals, the roles of CNS dopamine and serotonin in withdrawal remain unclear. A number of peptides, including cholecystokinin (CCK), neurotensin, and bombesin, have been shown to interact with the CNS actions of alcohol and may play a role in alcohol withdrawal. Inadequate work has been performed on acetylcholine (ACh), human growth hormone (HGH) and luteinizing hormone (LH). Studies of the recently identified GABA-benzodiazepine-barbituate receptor complex indicate that this system is likely to be involved in the pathophysiology of alcohol withdrawal. Perturbation studies with corticotropin-releasing factor (CRF) and TRH (with measures of ACTH and cortisol and TSH and prolactin, respectively), may identify patients with withdrawal-related autonomic dysfunction. PMID- 3010389 TI - The use of epidemiology, scientific data, and regulatory authority to determine risk factors in cancer of some organs of the digestive system. 4. Colon cancer. AB - Colon cancer accounts for over 10% of all cancers diagnosed from 1973 to 1976 in the United States. Many factors have been identified as having a role in its etiology, including the intestinal microflora, colonic mutagens, bile acids, cholesterol, estrogens, and diet. The evidence supporting or refuting the importance of each of these factors and others is discussed. Dietary factors appear to be the most important risk determinants for colon cancer. In particular, a high-fat, low-fiber diet has been most consistently incriminated as a promoting agent of this cancer. The incidence of colon cancer and consumption of total fat and animal protein are highly correlated internationally. However, further epidemiologic studies have given equivocal results. A high-fiber diet has been proposed as being protective against colon cancer although the mechanism of protection remains unclear. A major correlational study has shown that dietary fiber consumption was higher in areas with low incidence of colon cancer than in those areas with high incidence. A few case-control studies have been supportive of the fiber hypothesis but many more have shown no difference among cases and controls in their fiber consumption. More epidemiological studies are needed to clarify the role of diet in the etiology of colon cancer. Before a dietary change is recommended, the competing risks of other cancers and other diseases should be extensively researched. PMID- 3010392 TI - [Herpes genitalis]. PMID- 3010393 TI - [Acquired immunodeficiency syndrome (AIDS)]. PMID- 3010394 TI - [Vertical transmission of sexually transmitted diseases]. PMID- 3010395 TI - [Hemophagocytic histiocytosis as a "second hematologic event" in acute null lymphoblastic leukemia]. PMID- 3010396 TI - [Poland's syndrome. Presentation of a case]. PMID- 3010397 TI - [Anesthetic implications of the purinergic system: a review]. PMID- 3010398 TI - [Amiodarone and anesthesia. A dangerous interaction?]. PMID- 3010399 TI - [Analgesia produced by subarachnoid and epidural administration of opioids: site and mechanisms of action]. PMID- 3010401 TI - [Peripheral neuropathies during treatment with cisplatin: clinical and electrophysiologic study of 11 cases]. AB - Eleven patients with bronchial epidermoid carcinoma and undergoing treatment with cis-D.D.P. (II) were kept under electrophysiological and clinical surveillance. No other neurotoxic medication was added. The total dose of cis-D.D.P. was 300 mg/m2 over a period of three months: namely, three courses of 100 mg/m2 distributed over 5 days. Following the pretreatment check-up, the patients were divided into two groups: those without any electrophysiological abnormality (group A), and those without clinical abnormality but with a delayed latency H of the Hoffmann Reflex (group B). Patients in group A showed a slowing down of the motor nerve conduction velocity of the Median and Peroneal Nerves after a course of 100 mg, without accompanying worsening of the conduction velocity after 300 mg/m2, and prolongation of the distal latency of the sensory Median Nerve after 300 mg/m2; in group B, no significant change of electrophysiological clinical features were noted. In the two groups a non-significant reduction in amplitude of evoked responses were noted. These findings are more consistent with an axonal injury than with functional myelin injury. The authors review the existing literature and discuss the physiopathologic mechanisms of cis-D.D.P. peripheral neuropathies. PMID- 3010400 TI - [Neuropathies and chronic respiratory insufficiency: electrophysiologic study]. AB - In order to determine the frequency and the characteristics of neuropathies in chronic respiratory insufficiency, a systematic study of 43 patients affected by a chronic obstructive pulmonary disease was performed. In addition to neurological and electrophysiological examinations (including 6 nerve conduction studies on median, ulnar and peroneal nerves and EMG of 4 to 8 muscles), nerve and muscle biopsies (of the superficial peroneal nerve and lateral peroneus brevis muscle) were performed in 13 cases. Polyneuropathies were found in 74% patients: but mild in 39%, severe in 35%. Most were subclinical or poorly symptomatic. We determined the importance and distribution of abnormalities on the different nerves. From this we established that neuropathies are mixed but predominantly of the axonal type. Axonal degeneration and demyelination were confirmed by nerve biopsy; muscles presented neurogenic atrophy. Statistical analysis showed that the duration of hypoxemia was related to neuropathy. The pathogeny of these neuropathies is discussed. PMID- 3010403 TI - [Diagnosis and treatment of subclinical hepatocellular carcinoma]. PMID- 3010402 TI - [Electro-clinical monitoring of peripheral neuropathies in Waldenstrom disease treated with plasmapheresis]. AB - The aim of this study was to determine the effects of plasma exchange (PE) associated with Chlorambucil in the treatment of peripheral neuropathies in four patients suffering from Waldenstrom's disease (three cases with IgM kappa paraproteinemia and one case with IgM lambda). Before PE therapy, there was a severe slowing down of the motor and sensory conduction velocities (MCV and SCV) in three cases (IgM kappa) and a less severe slowing in one case (IgM lambda). In three cases, PE rapidly (as early as the third or the fourth administration) brought about a moderate improvement in the MCV and SCV, especially in the upper limbs, whereas in one case, no effect was noted. Clinically, the paraesthesias and dysaesthesias diminished, but the deep tendon reflexes remained absent or markedly depressed. Three months after the PE, nerve conduction velocities were again very reduced. The current literature on the physiopathological mechanisms of the peripheral neuropathies in the Waldenstrom's disease and the effects of the treatment by immunosuppressive agents and PE are reviewed. PMID- 3010404 TI - Modulation of juvenile rat ovarian adenylate cyclase activity by calcium and calmodulin. AB - The involvement of calcium and calmodulin in the regulation of juvenile rat ovarian adenylate cyclase activity was investigated. Both basal and LH-stimulated cAMP production were inhibited by adding Ca2+ to the incubation medium at concentrations higher than 10(-5) M. Conversely, up to 10(-3) M concentrations of EGTA increased cAMP production (basal, stimulated by LH, FSH, NaF and Gpp(NH)p); higher concentrations of the chelator led to an inhibition of cAMP formation. However, when the homogenates were previously deprived of Ca2+ by treatment with buffer containing EGTA, a biphasic response to LH and Gpp(NH)p stimulation was obtained in the presence of increasing concentrations of added Ca2+:cAMP production was first enhanced at low concentrations and then inhibited at higher concentrations. These observations suggest that the optimal concentration of Ca2+ needed to obtained maximal stimulation of the enzyme was much lower than the Ca2+ content in the homogenates and that a minimal concentration of Ca2+ was required to activate it. In the presence of micromolar concentrations of trifluoperazine and pimozide, two potent inactivators of calmodulin, LH-stimulated cAMP production was markedly decreased. Reactivation was obtained by adding exogenous calmodulin to the assay medium. The addition of Ca2+-free exogenous calmodulin (10(-6) M) caused a specific and significant enhancement of cAMP accumulation induced by an optimal dose of LH. These results suggest that calcium ions regulated the adenylate cyclase activity in the rat ovaries and had a dual effect that was first stimulatory at low concentration and mediated by calmodulin and then inhibitory at high (non-physiological) concentration. PMID- 3010405 TI - [Physico-chemical stimulation of dietary origin and digestive motility in the rabbit]. AB - The effects on gastrointestinal motility of two different diets, one containing dehydrated lucerne and the other dehydrated beet pulp (both being either coarsely or finely ground before pelleting) were studied in 16 unanesthetized 50-60-day old rabbits fed ad libitum. The rabbits fed with lucerne had better antroduodenal and ileo-caecal coordination, a higher level of electrical activity on the duodenum, and more frequent migrating myoelectric complexes on the jejunum and ileum than those fed with beet pulp. Furthermore, the rabbits fed the finely ground pellets showed weak electrical activity on the ileum and poor ileo-caecal coordination irrespective of fiber source, suggesting a unique effect of size per se on these portions of the digestive tract. PMID- 3010406 TI - Influence of substrate and microbial interaction on efficiency of rumen microbial growth. AB - Microbial N produced in the rumen and flowing to the duodenum (Ni) is related to the total amount of OM fermented or apparently digested in the rumen (OMf). This relationship, best expressed as microbial N yield (gNi/kgOMf), is affected mainly by the physical and chemical properties of feed carbohydrates and the amounts ingested. These factors influence yields at three levels of increasing complexity: Bacterial fermentation within one compartment following the continuous culture model. Fermentation pattern as such does not seem to affect yields. High fermentation rates are associated with lactate production, low methane production and transient polysaccharide synthesis. These effects induce acidification and lower yields, partly compensated by faster growth. Protozoal action, determined by the presence of sequestration spaces provided mainly by roughage diets. The presence of protozoa depresses microbial N yield but allows more complete fibre digestion. Compartmentation and differential passage. With roughage diets, optimal microbial N yield seems to require well developed microbial compartmentation, involving a large proportion of microbes in a large particle pool with a slow turnover, balanced by a small proportion in liquid, small-particle pools with a fast turnover. Such a situation is associated with long roughage feeding. It is hypothesized that microbial N yields in the rumen may vary between two extremes which are associated with the feeding of long roughage on the one hand or with concentrate (starch) feeding on the other. PMID- 3010407 TI - [Hormonal control of hepatic metabolism in ruminants]. AB - Insulin/glucagon control of hepatic metabolism, i.e. a endocrine-nervous system, is one of the general systems of integration in vertebrates. In this system, substrates coming from the digestive tract or from extrahepatic metabolism are important messenger molecules. Liver uptake of insulin and glucagon mainly accounts for high metabolic clearance rates of these hormones in both ruminants and non-ruminants. Glucagon infusion into ruminants results in an increase in the net hepatic uptake of glucose precursors and gluconeogenesis. Glucagon effects have also been demonstrated in isolated hepatocytes. Glucagon, through its effect on pyruvate carboxylase (EC 6.4.1.1.) may regulate gluconeogenesis. Insulin infusion induces hypoglycaemia. As a result, glucagon secretion increases and counterregulates insulin action. However, it has been shown that hepatic gluconeogenesis decreases during euglycaemic hyperinsulin clamp, mainly due to a decrease in the hepatic supply of glucose precursors following insulin action in extrahepatic tissues. Insulin fails to elicit any significant effect in vitro. Hepatocytes exhibit insulin and glucagon receptors. The apparent characteristics of hormone binding in vitro are similar in ruminants and non-ruminants, but the characteristics of postreceptor events are unknown in the former. Glucagon, which influences hepatic glucose synthesis, may be a major hormone in ruminants. PMID- 3010408 TI - In ovo interference of embryo non-lethal avian infectious bronchitis viruses (IBV) with velogenic Newcastle disease virus and embryo adapted IBV. AB - Avian infectious bronchitis virus (IBV) interfered with the lethal effects of velogenic Newcastle disease virus (NDV) and embryo adapted IBV in eggs previously inoculated with non-lethal IBV. Greater interference was noted in eggs superinfected with embryo adapted IBV than velogenic NDV. The interference could be eliminated by treating the initial IBV with homologous anti-IBV serum. PMID- 3010409 TI - Detection of avian encephalomyelitis virus. AB - Methods for the detection of two strains of avian encephalomyelitis virus (AEV) in chick embryo brain cell cultures and chickens were compared. It was found that the agar gel precipitin test (AGPT) and the enzyme-linked immunosorbent assay (ELISA) carried out on the serum of inoculated chickens were more sensitive than either the indirect fluorescent antibody test in cell cultures or the detection of clinical signs in chicks. On the basis of results obtained in this experiment the effects were then determined of routes and time of inoculation of chickens on the detection of AEV. It was found that birds infected at two weeks old produced higher antibody titres than one-day-old birds and the AGPT and ELISA detected comparable levels of antibody in them. It was recommended that the tests to detect the presence of AEV as a contaminant of vaccines be replaced by a serological test carried out on chicks inoculated intramuscularly at two weeks old. PMID- 3010410 TI - Haematological values and changes in blood chemistry in chickens with infectious bursal disease. AB - Haematological and blood serum chemical changes were studied in two groups of specific pathogen free chickens infected at five weeks old with different field isolates of infectious bursal disease virus. Blood and serum components were determined five days after infection. There were significant decreases (P less than 0.05) in the total erythrocyte count, packed cell volume, haemoglobin concentration, albumin, albumin: globulin ratio, uric acid and glucose. Serum components which increased significantly (P less than 0.05) were globulins and cholesterol. PMID- 3010411 TI - The gp135 of caprine arthritis encephalitis virus affords greater sensitivity than the p28 in immunodiffusion serology. AB - The 135,000 mw glycoprotein (gp135) and the 28,000 mw internal protein (p28) of caprine arthritis encephalitis virus are major viral constituents in precipitin lines formed between crude antigen preparations and sera from infected goats. In testing 307 goat and sheep sera, 118 samples were positive in a gp135 assay and only 82 were positive in a p28 assay. However, some goat sera were found which reacted only with the p28 and therefore testing for antibody against both proteins may be necessary to identify a maximum number of virus infected goats by immunodiffusion. PMID- 3010412 TI - Canine parvovirus: development of immunofluorescence and immunoperoxidase techniques. AB - Two methods of immunocytochemistry, immunofluorescence (IFA) and immunoperoxidase (PAP) were used to demonstrate canine parvovirus (CPV) antigens in sections of canine tissue. Specific staining using IFA and PAP was successful only in sections of fresh frozen tissue and formalin fixed/formol sublimate postfixed tissues respectively. A range of tissues was then taken at post mortem examination from a puppy which had been experimentally infected with CPV. Upon comparison, PAP staining gave high resolution, a permanent preparation and clear intracellular localisation of antigen. IFA resulted in less defined localisation of antigen but the technique was simpler and more easily controlled. PMID- 3010413 TI - Insect transmission of capripoxvirus. AB - Capripoxvirus was transmitted between sheep using Stomoxys calcitrans as a vector. Attempts to transmit capripoxvirus between sheep and between goats using biting lice (Mallophaga species), sucking lice (Damalinia species), sheep head flies (Hydrotaea irritans) and midges (Culicoides nubeculosus) were unsuccessful, although capripoxvirus was isolated from sheep head flies that had previously fed on infected sheep. PMID- 3010414 TI - Spread of bovine syncytial virus in a dairy herd over a two year period. AB - During a two year period the spread of bovine syncytial virus was monitored in a closed herd of 50 to 100 milking cows. Out of a nucleus of 49 nonpregnant and pregnant heifers, six were found to be infected with bovine syncytial virus. Virus was detected only in the progeny of infected cows and not in the progeny of uninfected animals. Nineteen progeny of the bovine syncytial virus infected cows were studied in detail and virus was isolated from only four. Horizontal spread of the virus did not occur. PMID- 3010415 TI - Potential for the transmission of foot-and-mouth disease virus from African buffalo (Syncerus caffer) to cattle. AB - Foot-and-mouth disease viruses of types SAT 1 and SAT 2 isolated from diseased cattle and carrier buffalo, either on the same farm or in the same ecological area within a short time of each other, were compared by T1 oligonucleotide mapping. No similarity was observed between the maps obtained, indicating that the different populations of virus were unique to each species and that no interspecies transmission had occurred. PMID- 3010416 TI - [Oxidants and antioxidants in the lung]. PMID- 3010417 TI - Role of beta adrenergic receptors in carotid body function of the goat. AB - Previous studies in anesthetized or decerebrate cats and rabbits and awake man have shown conflicting results regarding a potential role for beta-adrenergic receptors in carotid body function. Therefore, we sought to clarify the role of beta-adrenergic receptor activity in carotid body function by assessing: the ventilatory response to intravenous isoproterenol infusion in awake and anesthetized carotid body intact and carotid body denervated goats, the effect of propranolol on the ventilatory response to isocapnic hypoxemia, and carotid sinus nerve chemoreceptor discharge rate response to isoproterenol and hypoxia. Isoproterenol increased ventilation to a similar degree in carotid body intact and denervated awake and anesthetized goats. This ventilatory increase was blocked by propranolol. Propranolol did not alter the hypoxic ventilatory response. Although ventilation increased, carotid sinus nerve chemoreceptor discharge rate was not altered by isoproterenol infusion or bolus IV injection in anesthetized goats. Hypoxia did increase carotid sinus nerve discharge rate. In this study, beta-adrenergic stimulation of ventilation did not occur via the carotid body, and beta-adrenergic blockade did not affect the carotid body hypoxic ventilatory response. Therefore, we found no evidence of functional beta adrenergic activity within the carotid body of the goat. PMID- 3010418 TI - [Action of almitrine dimesylate on the oxidant activities of alveolar macrophages]. PMID- 3010419 TI - [Approach to the mechanism of action of almitrine dimesylate on peripheral receptors and the ventilation-perfusion ratio (VA/Q)]. PMID- 3010420 TI - [Hepatitis B virus and the delta agent: diagnostic and prognostic value of their biological markers]. PMID- 3010422 TI - [HTLV-III, virus hepatitis B and CMV in Chilean homosexuals]. PMID- 3010421 TI - [Erythroblastopenic crisis caused by human parvoviruses in Minkowski-Chauffard disease]. PMID- 3010423 TI - [Peripheral neuropathy during treatment with cimetidine]. AB - A case of peripheral neuropathy following cimetidine treatment is reported. Four days after beginning cimetidine (200 mg four times a day), the patient developed muscle pain and a symmetric motor neuropathy in all 4 limbs, predominant distally and in the lower limbs. Cimetidine was discontinued. Within seven days motor function began to return and within five months recovery was complete. Electrophysiological studies showed an axonal neuropathy. Morphometric studies revealed loss of large myelinated fibers in some fascicles while other fascicles were normal. Teasing studies showed predominant axonal lesions. Microvasculitis was present in the epineurium. Such findings suggest a role for small-vessel immune-complex vasculitis in the pathogenesis of this cimetidine-induced peripheral neuropathy. PMID- 3010424 TI - [Augmentation materials in surgery. Can ridge height be increased in edentulous patients?]. PMID- 3010426 TI - HLA-DR2 and Dw2 in narcolepsy and in other disorders of excessive somnolence without cataplexy. AB - Studies on HLA antigens were conducted in several patient populations with the following findings: (a) All 135 Japanese narcoleptic patients, eight of whom were considered to have "symptomatic" narcolepsy, were found to be HLA-DR2 and HLA DQw1 positive. All 17 members of a subgroup of the original population were also found to be HLA-Dw2-positive. It was concluded that HLA-DR2 is a prerequisite for the development of narcolepsy and that the diagnosis of narcolepsy can be excluded if HLA-DR2 or HLA-Dw2 is negative. The distinction between idiopathic and symptomatic narcolepsy needs to be reconsidered. (b) Haplotype studies in three families with narcoleptic members enabled detection of children at high risk for narcolepsy. (c) Of the 54 patients with disorders of excessive daytime sleepiness other than narcolepsy, those with essential hypersomnia (EHS) had a higher frequency of HLA-DR2; the others had a lower frequency. The DR2-positive EHS group could include members with an incomplete form of narcolepsy; the DR2 negative EHS group had disorders essentially different from narcolepsy, although both positive and negative groups manifested hypnagogic hallucinations, sleep paralysis, and sleep onset REMs. Two further studies were conducted in subgroups of the original narcoleptic population studied. In a subgroup of 30 patients who underwent lymphocyte subset studies, no T-cell abnormalities were detected; it is unlikely that an autoimmune mechanism is involved in the development of narcolepsy. In a subgroup of 33 narcoleptic patients, Southern's blot analysis of DNA using a DQ beta probe revealed three specific restriction fragments. Further studies are necessary to locate the DNA locus that carries the susceptibility gene for narcolepsy. PMID- 3010425 TI - Brain benzodiazepine receptor characteristics in canine narcolepsy. AB - A regional analysis of brain benzodiazepine (BDZ) receptors was conducted in narcoleptic and normal dogs to determine whether these sites are altered in narcolepsy. It was postulated that BDZ receptors play a role in the excessive sleepiness of narcolepsy because activation of these sites in freely behaving normal animals is hypnogenic. [3H]Flunitrazepam binding sites were assessed in 11 discrete areas of the forebrain and brainstem. No consistent or statistically significant group differences in either receptor densities (Bmax) or binding affinities (Kd) were found. These findings do not support the assertion that BDZ receptors are involved in the pathogenesis of canine narcolepsy. PMID- 3010427 TI - Serotoninergic reuptake mechanisms in the control of cataplexy. AB - Zimelidine, a selective inhibitor of serotonin (5-HT) reuptake in the CNS, was administered to narcoleptic patients. This medication has a potent anticataplectic action without improving daytime somnolence. These results suggest that 5-HT neuronal systems are involved in the physiopathology of cataplexy. Zimelidine, however, has no anticholinergic effect, so it is unlikely that cholinergic mechanisms thought to be important in animal cataplexy would play a major role in human cataplexy. In addition, zimelidine had no effect on nocturnal sleep patterns of these patients which is surprising considering the importance of 5-HT neuronal systems in sleep physiology. A 5-HT hypothesis of cataplexy is formulated, and the mechanisms of action of other anticataplectic agents are discussed. PMID- 3010428 TI - [Present status of granular cell tumors. Apropos of 8 new cases with tracheobronchial localization]. AB - The authors report 8 new cases of granular cell tumour of Abrikossoff's tumour located in the trachea or bronchus. After briefly recalling the generally accepted features of this disease, they report several new aspects: the obviously under-estimated frequency, the absence of progression, requiring therapeutic abstention and regular follow-up, the epidemiological problems posed by the association with chronic bronchitis or the coexistence with a malignant bronchial tumour, the uncertainties which still surround the histogenesis, which is partly mesenchymal and partly nervous tissue, more particularly Schwann cells, which can be supported by ultrastructural arguments. PMID- 3010429 TI - [Bone evaluation in microcellular cancer treated by chemotherapy]. AB - Bone surveys represent a particular problem in the investigation of secondary tumours, especially in the case of small cell cancer. This study was based on 70 patients in which qualitative criteria (clinical, radiographic, bone scan, bone aspiration-biopsy) replaced quantitative criteria (calcium, alkaline phosphatase, phosphorus). The histological (BPO) and clinical (pain) markers were determinant in the prognosis; in contrast, the repetition of the bone scan in the evaluation of response to treatment is only of very limited value. PMID- 3010430 TI - [Nontumoral nervous complications of bronchial cancer]. AB - The authors present 52 cases of neurological complications of bronchial cancer including 35 cases of non-tumoral manifestations. They stress the frequency and the severity of these complications which are dominated by cerebral vascular accidents and describe the pathogenic features of this particular complication. They define the complications accessible to treatment: iatrogenic pathology, deficiency disorders and certain paraneoplasic syndromes. PMID- 3010431 TI - [Adrenal metastases of bronchial cancer: value of x-ray computed tomography]. AB - Up until now, adrenal metastases from bronchial cancer have always been an autopsy diagnosis with a frequency as high as 45%. These metastases can now be diagnosed by means of computed tomography of the adrenal glands and most of the recent studies report an incidence of 10 to 15% of adrenal metastases independently of the histological type of bronchial cancer. The authors stress the need to perform this examination in the context of pre-treatment staging and for the follow-up of bronchial cancer. The outcome of these adrenal metastases is still poorly understood, but prospective studies could provide more information concerning their prognostic significance. In cases of operable bronchial carcinoma with an isolated adrenal metastasis, it is important to be able to detect this lesion which may be able to be excised prior to the thoracic surgery. PMID- 3010432 TI - [Artificial ripening of the cervix by local action of maternal leukocytes]. AB - The known failures and risks of inducing labour ought to provoke research for novel techniques. Comparison of the results of different methods of established harmlessness must comply with the strict rules found occasionally in the literature. The necessary maturation of the cervical tissue seems an indispensable requirement. Known physiological facts readily call to mind the role played by the granulocytes in collagenolysis. Clinical tests based on them give results identical with other techniques. PMID- 3010433 TI - Detection of astrovirus-like in diarrhoeic stool and its coexistence with rotavirus. PMID- 3010434 TI - Positive inotropic effects of an ACTH analogue (ACTH 1-17) on myocardial performance. AB - The aim of the present investigation was to study the effects of a single 100 micrograms i.v. administration of the synthetic heptadecapeptide [beta-Ala1 Lys17]ACTH1-17-4-amino-N-butylamide (ACTH 1-17) on the left ventricular performance. The systolic time intervals (STI) were recorded in 20 healthy adult young subjects (10 treated with ACTH 1-17 and 10 receiving placebo) before as well as 20, 40, 60 and 80 min after the i.v. ACTH 1-17 or placebo infusion. The STI were recorded immediately after blood withdrawal for measuring cortisol, aldosterone, adrenaline and noradrenaline plasma levels. A highly significant statistical difference was demonstrated for preejection period (PEP) and preejection period/left ventricular ejection time (PEP/LVET) ratio between subjects treated with ACTH 1-17 and subjects receiving placebo. As expected, a significant increase of cortisol and aldosterone plasma levels was observed in subjects treated with ACTH 1-17. The difference of adrenaline and noradrenaline plasma levels was statistically highly significant between subjects treated with ACTH 1-17 and those receiving placebo. The lack of increase in PEP and PEP/LVET ratio recorded in subjects treated with ACTH 1-17 is consistent with an increased left ventricular contractile performance. An increased plasma catecholamine release is postulated as the mechanism of this improvement. PMID- 3010435 TI - [AIDS, acquired immunodeficiency syndrome]. PMID- 3010437 TI - Comparison of endogenous lectins in human embryonic carcinoma and yolk sac carcinoma. AB - Salt and detergent extracts of xenografted human embryonic carcinoma and a human yolk sac carcinoma were fractionated on different sets of Sepharose columns covalently derivatized with lactose, asialofetuin, melibiose, mannan and fucose. Successive elution by chelating reagent and specific sugar resulted in isolation of endogenous Ca2+-dependent and Ca2+-independent carbohydrate-binding proteins, as analyzed by gel electrophoresis. The salt extracts of the embryonic carcinoma contained a Ca2+-dependent carbohydrate-binding protein at Mr66,000, that was undetectable in yolk sac tumor extracts. Also, a Ca2+-independent fucose-binding protein at Mr62,000 could be purified. In general, the pattern of carbohydrate binding proteins showed quantitative and qualitative differences as compared to normal tissue. The carbohydrate-binding proteins were assayable as agglutinin and showed no enzymatic activity. They can thus be defined as lectins. Their presence within certain stages of differentiation and developmental regulation may contribute to improvement of the classification, diagnosis and therapy of this tumor class. These new lectins may also be functionally related to the stage specific embryonic antigens like SSEA-1. PMID- 3010436 TI - Fibrogenic potential of intratracheally instilled quartz, ferric oxide, fibrous glass, and hydrated alumina in hamsters. AB - As a first step in the development of an animal model for determining the role of pulmonary fibrosis in the etiology and pathogenesis of lung cancer, the fibrogenic potential of quartz, quartz and ferric oxide administered together, fibrous glass, and hydrated alumina were studied by multiple intratracheal instillation in groups of male Lak:LVG Syrian golden hamsters. Dose-related decreases in survival were evident for the groups instilled with the two highest doses of quartz or quartz and ferric oxide. Instillation of quartz or quartz and ferric oxide induced the greatest pulmonary fibrosis in response to the materials tested. However, the dense fibrous tissue present in the lungs in classical human silicosis and in experimental silicosis of rats was not observed in this study. The results of this study indicate that the Syrian golden hamster is not a suitable species for studying the role of quartz-induced pulmonary fibrosis in pulmonary carcinogenesis. PMID- 3010438 TI - The gastric 99mTc clearance--a technique for the evaluation of the gastric blood flow. AB - The gastric mucosa blood flow was investigated using the 99mTc clearance. Besides the clearance the "R" ratio, total radioactivity of the gastric juice and the volume of gastric juice (ml/15 min) were also investigated. The main characteristics of the method are its reproducibility and the parallelism existing between: clearance, the "R" ratio and total radioactivity. The parameters investigated reach a maximum 30-45 min after administration of 99mTc; consequently, to evaluate gastric blood flow, it is sufficient to determine the parameters at this time interval. The 99mTc clearance represents an accessible method for the evaluation blood circulation in the gastric mucosa. PMID- 3010440 TI - Increased binding to ADP-stimulated platelets and aggregation effect of the dysfibrinogen Oslo I as compared with normal fibrinogen. AB - Interactions of the dysfibrinogen Oslo I with platelets were investigated. This fibrinogen is a B beta-chain variant with faster than normal fibrin monomer polymerization. Fibrinogen Oslo I acted more efficiently in ADP-induced platelet aggregation, and bound to gel-filtered platelets with a higher affinity constant than did normal fibrinogen. At all concentrations more fibrinogen molecules became bound per platelet with the dysfibrinogen than with normal fibrinogen, both when the fibrinogens were tested separately or as a mixture using 125I or 131I to label the two types. At high concentrations this was probably due to ligand polymerization of the dysfibrinogen. These observations indicate that the increased cofactor function in platelet aggregation may be related to the increased affinity of the dysfibrinogen for the platelets. PMID- 3010441 TI - Molecular defects in 2 examples of severe Hb H disease. AB - Severe Hb H disease presented in unexpected ways in 2 families of Greek origin. In 1, Hb H disease led to neonatal death. The underlying molecular defect was double-heterozygosity for the --Med/ alpha thalassaemia haplotype and a nondeletional alpha thalassaemia defect (alpha alpha T'Karditsa'/). The 2nd family requested antenatal diagnosis. The husband had mild nondeletional alpha thalassaemia. Initial investigations in the wife demonstrated unexpected gene mapping patterns. These have recently been shown to result from the (-alpha)Med 20.5/ haplotype. PMID- 3010442 TI - An OKT4+ T-cell population in Sezary syndrome: attempts to elucidate its lack of proliferative capacity and its suppressive effect. AB - We have previously reported a case of Sezary Syndrome (SS), in which an OKT4+ T cell population exhibited a defective response to non-specific mitogens, and an ability to suppress lectin-induced T-cell proliferation and pokeweed mitogen (PWM)-induced B-cell differentiation of normal donor peripheral blood mononuclear cells (PBMC). We report now that resting Sezary cells (SC) were essentially negative for activation antigens (Ag) detected by monoclonal antibodies (MoAb) B1.49.9, anti-Tac, OKT9, OKT10, and OKIa1. After phytohaemagglutin (PHA) stimulation, all these Ag were expressed with the notable exception of OKT10. Further investigations of SC functions indicated that no interleukin 2 (IL-2) biological activity was detected in culture supernatants of SC constimulated with PHA and phorbol myristate acetate (PMA). Interestingly, such stimulated SC exhibited a marked capacity to absorb exogenous IL-2 while remaining unable to proliferate. These data suggest that patient's unresponsiveness to PHA may be unrelated to IL-2 as an extracellular growth signal, but may instead be due to a failure in a later cellular activation event, subsequent to the binding of IL-2 to its receptors. Lack of T10 Ag expression may be involved as a cause or a consequence. Kinetic study of suppression of PHA-induced T-cell proliferation of normal PBMC revealed that inhibition occurred during the first 24 h; moreover we showed that it was not due to limitation of available IL-2 since it persisted in excess of IL-2; remarkably the growth of an IL-2-dependent murine cell line was unaffected by the presence of SC. Further, inhibition was also observed on IL-2 independent calcium ionophore A 23187-induced T-cell proliferation of normal PBMC. Taken together, the data suggest that the target of suppressor activity is probably an important obligatory intracellular event controlling DNA replication, which is common to both IL-2-dependent and IL-2-independent T-cell activation processes. Human T-cell leukaemia/lymphoma virus I (HTLV-I) related p.19 and p.24 Ag were absent on fresh and 30-day cultured SC, suggesting the absence of HTLV-I infection, although not ruling out a proviral integration in the SC DNA. PMID- 3010439 TI - Enzyme activities from eight small-sized oral spirochetes. AB - The purpose of the present investigation was to detect strains of small-sized oral spirochetes isolated from subgingival plaque for protease, peptidase, lipase, glycosidase, phosphatase, hyaluronidase and chondroitinsulfatase activities. The analyses were routinely carried out with cultures in the early stationary phase of growth after 4 days incubation. Both culture media and harvested spirochete cells were examined for the different enzyme activities. The enzymes were assayed by use of the API ZYM system, by p-nitroanilide derivatized peptides, and by hydrolyzing of mucopolysaccharides incorporated in solid bacterial medium. Relatively strong activities of trypsin-like enzymes, mainly bound to the cells, were observed in all strains. Similarly all strains showed acid phosphatases bound to the cells, too. Extracellular hyaluronidase- and chondroitinsulfatase activities were detected qualitatively in all strains after 7 days growth. The activities of the two mucopolysaccharide degrading enzymes almost disappeared after 10 subcultivations. Weak lipase (butyrate), higher lipase (caprylate), and weak phosphoamidase activities were observed in all cell pellets. No glycosidase activities were found. The observations are discussed by regarding the spirochetal enzymes as potential virulence factors for the development of marginal periodontitis. PMID- 3010444 TI - The role of the CD8-positive subset of T cells in proliferative responses to soluble antigens. II. CD8-positive cells are not responsible for DR-associated differences in responsiveness to mumps and Coxsackie B4. AB - The role of CD8 (T8, Leu 2)-positive T lymphocytes in the proliferative T lymphocyte response to mumps and Coxsackie B4 viral antigens in vitro was investigated. The frequency among enriched T-lymphocyte blasts of antigen reactive T lymphocytes (ARTL) restricted by different DR-associated elements was investigated, using antigenic restimulation with allogeneic antigen-presenting cells in a limiting dilution assay. A decreased frequency of DR3-restricted and an increased frequency of DR4-restricted mumps and Coxsackie B4 ARTL were seen in the limiting dilution assay, whether or not HLA class I determinants were shared in the antigenic restimulation. Removal of CD8-positive cells did not increase the primary in vitro responsiveness to mumps and Coxsackie B4 viral antigens, and did not change the DR-associated differences in the frequency of ARTL seen in the limiting dilution assay. PMID- 3010443 TI - The role of the CD8-positive subset of T cells in proliferative responses to soluble antigens. I. Studies of healthy subjects, type 1 diabetics, and coeliac disease patients. AB - Using magnetic monosized polymer particles (M 450) coated with a monoclonal mouse IgM anti-CD8 (ITI 5C2) antibody, we were able to selectively remove and isolate functionally active CD8+ T cells from human peripheral blood mononuclear cells. Isolated CD8+ cells did not respond by proliferation to soluble antigens, but proliferated in response to phytohaemagglutinin. However, in the presence of CD4+ T cells, CD8+ cells were able to mount a substantial proliferation when stimulated with soluble antigens. Depletion of CD8+ cells decreased rather than increased the T-cell responses to the antigens glyc-gli, Coxsackie B4, and mumps in healthy individuals. We therefore found no indication of involvement of functionally-active CD8+ suppressor cells in vitro. The T-cell responsiveness to these antigens has previously been shown to be influenced by HLA-DR-associated restriction elements, but the tendency for decreased responsiveness to these antigens by CD8 depletion seemed independent of the DR type of the cell donors. As in healthy subjects, CD8 depletion resulted in a decreased responsiveness to the gluten antigen glyc-gli in untreated and treated coeliac disease patients and to Coxsackie B4 and mumps antigens in Type 1 diabetics. PMID- 3010445 TI - Well-defined cell-surface receptors may be entry points for infectious agents. PMID- 3010446 TI - Nitroblue tetrazolium (NBT) reduction by neutrophilic granulocytes in patients with HTLV-III infection. AB - The NBT reduction of granulocytes was determined in 11 patients with HTLV-III infection. The reaction was measured in granulocytes both in resting state and after stimulation with Escherichia coli, as well as with and without addition of plasma. In 5 patients with AIDS, the NBT reduction was significantly decreased in all these types of experiments, when compared with 12 healthy controls. In 6 patients with the lymphadenopathy syndrome (LAS), the NBT reduction of resting granulocytes was significantly higher than that of the controls. The findings could in general not be explained by superinfections. The elevated NBT test during the long term LAS stage reflects a release of free oxygen radicals, against which treatment might be directed. PMID- 3010447 TI - IgA antibodies to Epstein-Barr virus in infectious mononucleosis. AB - The IgA anti-EBV (Epstein-Barr virus) response during the course of IM (infectious mononucleosis) was investigated. The IgA anti-VCA (viral capsid antigen) response was found not to be restricted to the early acute phase of the EBV infection as is the IgM anti-VCA response. Some patients with normal total serum IgA levels did not respond with measurable EBV specific IgA. These patients and those with low titers of IgA anti-VCA had shorter duration of sore throat than responders with high titers indicative of a strong correlation between the IgA anti-VCA titers and the duration of sore throat. In this way the EBV specific IgA response is unique since recent observations show that local oropharyngeal symptoms during IM appear poorly synchronized with the IgM and the IgG antibody responses. As EB virus is excreted into the oropharynx during IM, antigens are available for local EBV immunization. The results of the present study imply a possible local immunization process as a positive correlation was found between serum IgA anti-VCA and total salivary IgA. PMID- 3010449 TI - [Arthropathy due to pyrophosphate deposition as a tumor-simulating disease of the temporomandibular joint]. PMID- 3010448 TI - Deoxythymidine kinase, a possible marker for monitoring activity of cytomegalovirus infection after renal transplantation. AB - Measurement of deoxythymidine kinase activity (S-TK) in serum is used as a marker for cytomegalovirus (CMV) infection following transplant surgery. A case is presented where a kidney transplant patient suffered a fatal CMV infection. The new antiviral drug Foscarnet was used to treat the infection. Retrospective analysis of S-TK showed a good correlation with the course of the disease, its treatment, recurrence and final outcome. PMID- 3010450 TI - [Unusual abdominal tumor in a dog]. PMID- 3010451 TI - Fibronectins. PMID- 3010452 TI - A clone of hepatitis B virus (subtype adr) DNA with a new HindIII site. AB - We have cloned the HBV genome subtype adr through its HindIII site into plasmid pBR322. Twelve recombinant plasmids each with an insert of the HBV genome were obtained. The restriction map of one of the recombinants, plasmid pADR-H1, was analyzed. The location of the sites for BamHI, BglI, BglII, SstII, XbaI and XhoI in pADR-H1 were found to be the same as that in pADR-1, but the HindIII site of pADR-H1 is distinctly different from that of pADR-1. The BamHI-HindIII fragment is 316 bp long in the genome of pADR-1. However, it is only 82 bp in pADR-H1. No deletion of sequence between BamHI and HindIII sites has been found in the HBV genome of pADR-H1. The sequencing data around the HindIII site of pADR-H1 in both pADR-H1 and pADR-1 showed that there is a stretch of AAGTTT in pADR-1 compared to an AAGCTT in pADR-H1. In addition, other differences were found. There are three sites for AvaI, four for HincII, one for HpaI and none for SphI in the genome of pADR-H1 compared to four sites for AvaI, three for HincII, one for SphI and none for HpaI in pADR-1. The biological significance of base pair mutation in HBV DNA and the possibility of the existence of HindIII site in the genome of subtypes ayw and adw were discussed. PMID- 3010453 TI - A study on the receptive field of acupoints and the relationship between characteristics of needling sensation and groups of afferent fibres. AB - We have recorded the afferent unit discharges and using it as an index, measured the locations and the volumes of nine acupoints, such as Neiguan and Shaoshang, which are innervated by median nerve. We have also investigated the relationships between electric needling sensations and afferent fibres of different groups as well as manual needling sensations by means of analysing power-spectrum of the unit discharges with FFT. It has been found that the fibres of Group II and Group IV convey numbness and soreness, respectively. The fibres of Group III relates to the conduction of heaviness and distention closely. PMID- 3010454 TI - AIDS patent negotiations break down. PMID- 3010455 TI - Regulation of class III major histocompatibility complex gene products by interleukin-1. AB - Interleukin-1 (IL-1) is a product of mononuclear phagocytes that mediates changes characteristic of the response to inflammation or tissue injury (the acute-phase response). One of two structurally and functionally homologous major histocompatibility complex (MHC) class III genes encodes a positive acute-phase protein, complement factor B. The closely linked complement C2 gene is not affected during the acute-phase response. Purified human IL-1, pH 7.0, and recombinant-generated murine IL-1, pH 5.0, increased the expression of factor B and other positive acute-phase proteins in human hepatoma cells but decreased the expression of albumin, a negative acute-phase reactant. Furthermore, in a murine fibroblast L-cell line transfected with cosmid DNA bearing the human C2 and factor B genes, IL-1 mediated a reversible dose- and time-dependent increase in factor B expression in the transfected cells. Expression of the C2 gene was not affected by IL-1. The effect of IL-1 on factor B expression involves a mechanism acting at a pre-translational level as demonstrated by an increase in specific messenger RNA content and a corresponding increase in biosynthesis and secretion of factor B. The structural basis and mechanism for selective and independent regulation of these genes provides insight into the molecular control of the inflammatory response. PMID- 3010456 TI - Trans-activator gene of HTLV-II induces IL-2 receptor and IL-2 cellular gene expression. AB - The human T-lymphotropic viruses types I and II (HTLV-I and -II) have been etiologically linked with certain T-cell leukemias and lymphomas that characteristically display membrane receptors for interleukin-2. The relation of these viruses to this growth factor receptor has remained unexplained. It is demonstrated here that introduction of the trans-activator (tat) gene of HTLV-II into the Jurkat T-lymphoid cell line results in the induction of both interleukin 2 receptor and interleukin-2 gene expression. The coexpression of these cellular genes may play a role in the altering T-cell growth following retroviral infection. PMID- 3010457 TI - Quality of intramural research. PMID- 3010458 TI - Biochemical topology: applications to DNA recombination and replication. AB - Processes of DNA rearrangement such as recombination or replication frequently have as products different subsets of the limitless number of distinguishable catenanes or knots. The use of gel electrophoresis and electron microscopy for analysis of these topological isomers has made it possible to deduce physical and geometric features of DNA structure and reaction mechanisms that are otherwise experimentally inaccessible. Quantitative as well as qualitative characterization is possible for any pathway in which the fate of a circular DNA can be followed. The history, theory, and techniques are reviewed and illustrative examples from recent studies are presented. PMID- 3010459 TI - Associative induction of posttetanic and long-term potentiation in CA1 neurons of rat hippocampus. AB - Electrical stimulation of fibers in the stratum radiatum causes an excitatory postsynaptic potential in CA1 neurons of the hippocampus. Other excitatory inputs to or direct depolarization of these CA1 neurons during stimulation of the stratum radiatum caused a subsequent increase in the excitatory postsynaptic potential. This enhancement was characterized as a brief potentiation (2 to 3 minutes, similar to posttetanic potentiation) and a long-term potentiation (presumed to be involved in learning and memory). These potentiations are probably induced by an interaction of the postsynaptic cell or other presynaptic terminals with the test presynaptic terminals. PMID- 3010460 TI - Coronavirus infection induces H-2 antigen expression on oligodendrocytes and astrocytes. AB - Infection of the central nervous system by mouse hepatitis virus strain A59, a murine neurotropic coronavirus, induces class I major histocompatibility complex antigens on mouse oligodendrocytes and astrocytes, cells that do not normally express these antigens on their surfaces. This induction, which occurs through soluble factors elaborated by infected glial cells, potentially allows immunocytes to interact with the glial cells and may play a critical role in the pathogenesis of virus-induced, immune-mediated demyelination in the central nervous system. PMID- 3010461 TI - AIDS retrovirus (ARV-2) clone replicates in transfected human and animal fibroblasts. AB - A molecular clone of the AIDS-associated retrovirus (ARV-2) was transfected into human T lymphocyte and monocyte cell lines as well as mouse, mink, monkey, and human fibroblast lines. A replicating virus with cytopathic and biologic properties of ARV-2 was recovered from all the cell lines. The animal and human fibroblast cells are resistant to direct infection by ARV, and in these experiments virus production in the fibroblast lines, especially mouse, was reduced compared to human lymphocytes. However, human fibroblasts were more permissive to virus expression than mouse cells. These results show that, whereas the primary block to ARV infection in certain cells may occur at the cell surface, intracellular mechanisms can also participate in controlling virus replication. The results have relevance to vaccine development and encourage further work with modified molecular clones to examine regions of the ARV genome necessary for cytopathology and replication. PMID- 3010462 TI - AIDS-related brain damage unexplained. PMID- 3010463 TI - AIDS retrovirus induced cytopathology: giant cell formation and involvement of CD4 antigen. AB - The formation of multinucleated giant cells with progression to cell death is a characteristic manifestation of the cytopathology induced by the AIDS retrovirus in infected T lymphoid cells. The mechanism of giant cell formation was studied in the CD4 (T4/Leu 3) positive T cell lines JM (Jurkat) and VB and in variants of these lines that are negative for cell surface CD4 antigen. By means of a two color fluorescent labeling technique, multinucleated giant cells in infected cultures were shown to form through cell fusion. Antibody to CD4 specifically inhibited fusion, and uninfected CD4 negative cells, in contrast to uninfected CD4 positive cells, did not undergo fusion with infected cells, suggesting a direct role for the CD4 antigen in the process of syncytium formation. These results suggest that, in vivo, cell fusion involving the CD4 molecule may represent a mechanism whereby uninfected cells can be incorporated into AIDS virus infected syncytia. Because the giant cells die soon after they are formed, this process may contribute to the depletion of helper/inducer T cells characteristically observed in AIDS. PMID- 3010464 TI - Neutralization of HTLV-III/LAV replication by antiserum to thymosin alpha 1. AB - An antiserum prepared against thymosin alpha 1, a hormone secreted by the thymus gland, effectively neutralized the AIDS-associated virus [HTLV-III/LAV (clone BH 10)] and blocked its replication in H9 cells. Reverse transcriptase activity and expression of the HTLV-III/LAV antigens p15 and p24 were inhibited by purified immunoglobulin G preparations of antisera to thymosin alpha 1. The antiviral activity of the antiserum was found to be due to a region of homology between thymosin alpha 1 and p17, a product of the gag gene of HTLV-III/LAV. Comparison of the primary sequences of thymosin alpha 1 and the gag protein revealed a 44% to 50% homology in an 18-amino acid region, between positions 11 and 28 on thymosin alpha 1 and 92 and 109 on the gag protein. The effectiveness of the thymosin alpha 1 antiserum and of immunoglobulin G-enriched preparations in blocking replication of HTLV-III(BH-10) in H9 cells suggests a novel approach to the development of an AIDS vaccine. A vaccine directed against the gag protein might overcome the problem of genetic drift in the envelope region of the virus and be useful against all genetic variants of HTLV-III/LAV. PMID- 3010465 TI - Where is the AIDS virus harbored. PMID- 3010467 TI - Visual pigment homologies revealed by DNA hybridization. AB - A bovine rhodopsin complementary DNA probe was used to detect homologous visual pigment genes in a variety of species. Under stringent DNA hybridization conditions, genomic DNA from most vertebrate species carried a single homologous fragment. Additional homologies were detected in some vertebrates by reducing the hybridization stringency. Homologous fragments were also detected in DNA isolated from invertebrate species, a unicellular alga, and an archaebacterium; many of these fragments were homologous to a Drosophila opsin probe. These results suggest that photosensory pigments in a wide variety of species arose from a common precursor. PMID- 3010468 TI - Congenital disorders of platelet function. PMID- 3010466 TI - Deletion in cysteine-rich region of LDL receptor impedes transport to cell surface in WHHL rabbit. AB - The Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal with familial hypercholesterolemia, produces a mutant receptor for plasma low-density lipoprotein (LDL) that is not transported to the cell surface at a normal rate. Cloning and sequencing of complementary DNA's from normal and WHHL rabbits, shows that this defect arises from an in-frame deletion of 12 nucleotides that eliminates four amino acids from the cysteine-rich ligand binding domain of the LDL receptor. A similar mutation, detected by S1 nuclease mapping of LDL receptor messenger RNA, occurred in a patient with familial hypercholesterolemia whose receptor also fails to be transported to the cell surface. These findings suggest that animal cells may have fail-safe mechanisms that prevent the surface expression of improperly folded proteins with unpaired or improperly bonded cysteine residues. PMID- 3010469 TI - Platelet activation: role of an ADP receptor. PMID- 3010470 TI - Evaluation of platelet function. PMID- 3010471 TI - [Effects of carbamylcholine on the ventricular fibrillation threshold and its relation to the levels of cAMP and cGMP in rat ischemic ventricle]. PMID- 3010472 TI - Case report 359: gigantic benign fibrous histiocytoma (nonossifying fibroma). PMID- 3010473 TI - [Neuromuscular diseases: symptomatology and classification]. PMID- 3010474 TI - Chronic tension pneumothorax presumably due to a "ball valve" bronchopleural fistula. AB - We have described an unusual variant of bronchopleural fistula whose ball valve characteristics led to a chronic tension pneumothorax without fluid collection in a postpneumonectomy space. PMID- 3010475 TI - Cystosarcoma phyllodes. AB - We present four cases of cystosarcoma phyllodes in which mammograms revealed a small or large lobulated mass occupying the entire breast. Preoperative diagnosis was a benign lesion with a low probability of nodular carcinoma. Histologically, all tumors were classified as malignant. Although uncommon, cystosarcoma should be considered in the differential diagnosis of these masses, since local recurrence is frequent unless wide wedge resection or simple mastectomy is done. PMID- 3010476 TI - Detection and quantitation of fetomaternal hemorrhage. AB - Failure to administer additional doses of Rh immune globulin (RhIG) to patients with excessive fetomaternal hemorrhage (FMH) is one of the causes of continued Rh isoimmunization. We compared the fetal cell ratio (FCR) with the other laboratory methods currently used at our hospital to quantitate FMH, ie, fetal cell preparation (FCP), RhIG cross-match with maternal serum, and indirect Coombs' testing 24 to 48 hours after administration of RhIG. The incidence of excessive FMH as detected by the various methods was 3.6% (cross-match), 9.6% (Coombs'), 18% (FCP), and 66% (FCR). The methods currently used do not accurately quantify FMH. The FCR is the most sensitive test but it has a high false-positive rate and thus does not appear to be clinically useful. We suggest that repeating the indirect Coombs' test may provide a practical alternative for determining the need for additional doses of RhIG. PMID- 3010477 TI - [The regulatory function of prostaglandins in the development of the eczematous process]. PMID- 3010478 TI - Chemonucleolysis (discolysis) with collagenase. AB - Fifty-four patients with proven lumbar disc displacement were treated by intradiscal injection of collagenase (Nucleolysin) between April 1981 and January 1984. Follow-up by history and physical examination, phone survey, and/or mail survey shows 72% success rate (excellent, good, and fair) and 28% poor or unsuccessful results. No patient was made worse by the treatment. Success rate was greater in patients older than the age of 50 years, male patients, and patients with pain complaints of longer than 4 months' duration. The presence of a list and/or crossed straight leg raising and/or straight leg raising positive less than 30 degrees was associated with a poor prognosis. Discography prior to collagenase injection did not compromise the outcome of treatment. Postinjection changes in the CT scan correlated with a satisfactory outcome of treatment. Findings at surgery in patients who failed to obtain relief following collagenase injection did not show a specific deleterious effect of the enzyme, nor were the expected results from surgery compromised by a previous unsuccessful intradiscal collagenase injection. PMID- 3010480 TI - Primary hepatocellular carcinoma in pregnancy. A case report. AB - A case of primary hepatocellular carcinoma (HCC) in a 17-year-old pregnant primigravida diagnosed during the 32nd week of pregnancy is presented. A live infant was delivered after induction of labour during the 33rd week. The mother, although still alive 3 months postpartum, had ongoing spread of the malignancy. Only 7 cases of primary liver cancer in pregnancy have been reported to date, with 100% maternal mortality and only 3 live infants. The literature and the diagnosis and management are discussed. PMID- 3010479 TI - [Value of delayed computed tomography of the liver 4 hours after the administration of meglumine diatrizoate]. PMID- 3010481 TI - Intraoperative ultrasonography in surgery for liver tumors. AB - Intraoperative ultrasonography was used in 37 patients during surgery for suspected liver tumors. The size, number, and site of the lesions were determined together with the relationship of the tumor to the intrahepatic vessel, as well as possible small daughter lesions within the liver. Final diagnosis in these patients was hepatocellular carcinoma in 19 cases, metastases from colorectal cancers in 15 cases, and benign lesions in three cases. Previously undetected small tumors were revealed in one patient with sigmoid cancer and in five patients with liver cell carcinoma who had cirrhosis. Vascular tumoral infiltrations were easily displayed and the surgical approach modified accordingly: a more extended resection was performed in two cases of huge central hepatic metastases. Intraoperative ultrasonography revealed seven cases of small (2 to 3 cm) hepatocellular carcinomas in cirrhotic livers that were not visible or palpable, thus allowing a subsegmentary resection. Finally, in three cases of atypical tumors, an intraoperative echo-guided biopsy specimen was required to establish the benign nature of lesions and resection was avoided. Intraoperative ultrasonography facilitates the diagnosis of small liver tumors and can also aid the surgeon in his choice of technique, especially in cases of cirrhosis of the liver. A resection can be avoided altogether when multiple lesions are involved, or echo-guided subsegmentary resections can be performed in cirrhotic livers when a less extended resection is required. This technique makes it possible to establish the relationship between the tumor and intrahepatic vessels, thus preventing vascular injury and making radical hepatic resection safer. PMID- 3010482 TI - [Dietary fiber and the lithogenic potential of bile]. PMID- 3010483 TI - Pneumopericardium after pneumonectomy and lobectomy. AB - Pneumopericardium is a rare condition, most frequently reported in connection with prolonged artificial ventilation in infants with hyaline membrane disease. No reports of pneumopericardium after pulmonary surgery have been published. Two cases of pneumopericardium are reported, one of tension pneumopericardium after pneumonectomy and artificial ventilation and one that followed radical lobectomy and artificial ventilation. The radiographic findings included pneumopericardium and subcutaneous emphysema and the patient who had had a pneumonectomy had severe symptoms of cardiac tamponade. Prolonged artificial ventilation in patients after pulmonary surgery and in the presence of an intrathoracic air leak may be a hazard. The importance of prompt surgical intervention in cases of tension pneumopericardium is underlined; the treatment of choice is thoracotomy with pericardiotomy. PMID- 3010484 TI - Amiodarone pulmonary toxicity: functional and ultrastructural evaluation. AB - Pulmonary function, chest radiographic appearances, and the cellular composition of bronchoalveolar lavage fluid were assessed in 13 patients who were receiving amiodarone treatment. Eight of the patients had developed clinical and radiological evidence of lung disease and five were symptom free. The proportions of lymphocytes (mean 8.6 (SD 6.9)) and neutrophils (mean 3.4 (3.3)) obtained by bronchoalveolar lavage were similar in patients with and without lung complications. Electron microscopic examination of alveolar macrophages showed intralysosomal inclusion bodies in all subjects, regardless of clinical state. There was no significant difference in the mean number of inclusion bodies per macrophage transection between those with and those without lung disease. The differential cell count in bronchoalveolar lavage fluid and the presence of macrophage inclusion bodies were therefore not useful as markers of disease activity. Among those who developed clinical and radiological evidence of lung disease, the cumulative drug dose per kilogram of body weight and the duration of treatment (mean 16.5 (SD 9.0) months) were significantly correlated with the degree of lung restriction as measured by total lung capacity and forced vital capacity. It is concluded that, while the severity of the restrictive pulmonary defect that is induced by amiodarone is largely dose related, the development of lung toxicity is to some extent idiosyncratic. PMID- 3010485 TI - Chalk in the prime. AB - After observations of cloudiness in the perfusion circuit at open intracardiac operations, laboratory experiments showed a precipitate in a Hartmann's solution (compound sodium lactate solution, Ringer-lactate) and sodium bicarbonate based priming fluid used for cardiopulmonary bypass. The precipitate was found to consist of calcium carbonate crystals. The crystals were not dissolved by adding plasma proteins, nor were they sufficiently cleared from the extracorporeal circuit by a 40 microns filter in the arterial line. The crystals may embolise in microvascular beds and thus be a cause of postoperative morbidity. The practice of adding sodium bicarbonate to the pump prime may be unnecessary. PMID- 3010486 TI - Pulmonary clearance of vasoactive intestinal peptide. AB - Vasoactive intestinal peptide causes bronchodilatation when given intravenously but is less effective in both animals and man when given by inhalation. This difference may be due to poor transit of the peptide across the bronchial epithelium. To test this hypothesis pulmonary clearance of radiolabelled vasoactive intestinal peptide was measured in Sprague Dawley rats and compared with that of pertechnetate (TcO4-) and diethylene triamine pentaacetate (DTPA). Despite a molecular weight (MW) of 3450, iodinated vasoactive intestinal peptide was cleared rapidly from the lungs, with a mean half time (t1/2) of 19 minutes after an initial slower phase. This compares with a t1/2 of 10 minutes with TcO4- (MW 163) and a t1/2 of 158 minutes with DTPA (MW 492). The possibility that vasoactive intestinal peptide mediates a non-specific increase in permeability was discounted by the fact that the combination of vasoactive intestinal peptide and DTPA did not alter DTPA clearance significantly. Chromatography and radioimmunoassay of blood taken after intratracheal administration of vasoactive intestinal peptide demonstrated a metabolite but no unchanged peptide. An intravenous injection of the peptide disappeared on first pass through the lung. It is concluded that inhaled vasoactive intestinal peptide lacks efficacy as a bronchodilator not because of slow diffusion to airway smooth muscle but because it is metabolised at an early stage of its passage through the respiratory epithelium. PMID- 3010487 TI - Coextraction of thrombomodulin and tissue factor from human placenta: effects of concanavalin A and phospholipid environment on activity. AB - Thrombomodulin and tissue factor activities have been co-extracted from human placenta by several non-ionic detergents, n-octylglucoside and Triton X-100 being the most efficient ones. The n-octylglucoside placenta extract had a strong cofactor activity in the activation of human protein C by human alpha-thrombin. Treatment of the n-octylglucoside and Triton X-100 placenta extracts by phospholipases C and A2 revealed that an adequate phospholipid environment is necessary for maximal thrombomodulin activity, while it is well known that this is crucial for tissue factor activity. Soluble concanavalin A reversibly inhibited thrombomodulin and tissue factor activities to the same extent. Con-A Sepharose affinity chromatography of the Triton X-100 placenta extract resulted in the same proportion (30%) of these two activities bound to the lectin, which were subsequently eluted in the same fractions by a linear gradient of alpha methyl-D-glucoside. This observation suggests that thrombomodulin activity is associated to a glycoprotein component presenting the same degree of carbohydrate heterogeneity, involving alpha-D-mannosyl or alpha-D-glucosyl residues, as tissue factor apoprotein. Relipidation of fraction eluted by alpha-methyl-D-glucoside was essential to detect tissue factor activity, it was also necessary to recover full thrombomodulin activity. An antibody to human brain tissue factor apoprotein inhibited human placenta tissue factor activity, whereas thrombomodulin activity was unaffected, suggesting that these two cellular activities are related to distinct molecular entities sharing striking functional and structural similarities. PMID- 3010488 TI - Conjugal transmission of HTLV-III and lymphadenopathy in Christmas disease. PMID- 3010489 TI - A simplified PTT-based protein C activity assay using the thrombin-thrombomodulin complex. AB - A simplified assay for protein C activity in plasma is described which uses the ability of rabbit lung thrombomodulin to inhibit the procoagulant activity of thrombin while stimulating protein C activation. Barium eluates of plasma are activated for one hour at 37 degrees C by a mixture of human thrombin and rabbit lung thrombomodulin at concentrations which neutralize each other's effect on the kaolin-cephalin activated partial thromboplastin time (PTT). Protein C anticoagulant activity in the activated eluates is then measured directly in the PTT. The method is independent of protein S levels in the test samples, and is suitable for warfarinized and heparinized plasma. Protein C levels obtained with this method correlate closely with functional levels of vitamin K-dependent procoagulants as measured by the prothrombin and proconvertin time (P&P) in normal subjects and in patients receiving warfarin, indicating specificity for gamma-carboxylated protein C. The method has the potential to detect molecular variants defective in any of the interactions required for generation of anticoagulant activity in vivo. PMID- 3010490 TI - Comparison of the in vitro effect of eicosapentaenoic acid (EPA)-derived lipoxygenase metabolites on human platelet function with those of arachidonic acid. AB - Eicosapentaenoic acid (EPA) has been reported to have a potent anti-aggregatory activity and to be efficiently metabolized by 12-lipoxygenase, not by cyclooxygenase in platelets. In vitro effect of 12-lipoxygenase metabolites of EPA on platelet function was studied and compared with those of arachidonic acid (AA). The 12-lipoxygenase metabolites of AA and EPA; 12 hydroperoxyeicosatetraenoic acid (12-HPETE) and 12-hydroperoxyeicosapentaenoic acid (12-HPEPE), and their hydroxy derivatives, 12-hydroxyeicosatetraenoic acid (12-HETE) and 12-hydroxyeicosapentaenoic acid (12-HEPE) were prepared enzymatically using human platelet lysate. These compounds were purified by high performance liquid chromatography and identified by gas chromatography mass spectrometry. 12-HPETE and 12-HPEPE inhibited dose-dependently washed human platelet aggregation and serotonin (5-HT) release induced by AA and collagen. The potency of 12-HPEPE was almost equal to that of 12-HPETE. Their hydroxy derivatives, 12-HETE and 12-HEPE were less potent. 12-hydroperoxy derivatives of AA and EPA were the most potent in inhibiting platelet aggregation and 5-HT release among 5-, 12- and 15-hydroperoxy isomers of AA and EPA. The inhibitory effects of 12-HPETE and 12-HPEPE on platelet aggregation were additive. PMID- 3010491 TI - Activation of protein kinase C by the action of 9,11-epithio-11,12-methano thromboxane A2 (STA2), a stable analogue of thromboxane A2, in human platelets. AB - Incubation of human washed platelets with 9,11-epithio-11, 12-methano-thromboxane A2 (STA2), a stable analogue of thromboxane A2, caused the activation of protein kinase C and myosin light chain (MLC) kinase to the same extents as those induced by thrombin as judged by measuring the phosphorylation of a 40-kilodalton protein and MLC, respectively. However, STA2 stimulated much less phosphoinositide turnover than thrombin. Furthermore, the doses of STA2 necessary for protein kinase C activation and phosphoinositide turnover were higher than those necessary for MLC kinase activation, although the doses of thrombin necessary for these three reactions were nearly the same. These results suggest that protein kinase C may be activated at the Ca2+ concentrations higher than those required for MLC kinase activation by the action of STA2, presumably due to the inability of this agonist to produce diacylglycerol in an amount enough to increase the affinity of the enzyme for Ca2+. PMID- 3010492 TI - Influence of granulocyte elastase-like proteinase (ELP) on platelet functions. AB - The influence of granulocyte elastase-like proteinase (ELP) on platelet functions was investigated. ELP inhibited the platelet aggregations induced by a wide variety of agonists. The inhibition was marked in the case of receptor-mediated agonists such as thrombin, ristocetin, etc. It was moderate with the pervading agonist, arachidonic acid, and mild with the bypassing agonist, Ca2+ ionophore A23187. ELP inhibited the release of thromboxane A2 from platelets in the case of the platelet aggregation induced by thrombin. On the other hand, ELP did not inhibit the release of thromboxane A2 from platelets in the platelet aggregation induced by arachidonic acid or Ca2+ ionophore A23187. ELP suppressed the release of serotonin from platelets induced by thrombin, while it did not markedly suppress the release of serotonin induced by Ca2+ ionophore A23187. Treatment of platelets with ELP resulted in a slight increase of intraplatelet cAMP levels. These results suggest that ELP acts on receptors and inhibits platelet functions. As a results, ELP markedly inhibits the platelet functions such as aggregation or release of serotonin or thromboxane A2 stimulated by receptor-mediated agonists. ELP slightly elevates the cAMP level in the platelets, resulting in the mild inhibition of the platelet functions stimulated by the pervading agonist, arachidonic acid, or the bypassing agonist, Ca2+ ionophore A23187. PMID- 3010493 TI - Effect of intravenously injected collagenase on the concentration of circulating platelets in rats. AB - An enzymatically induced irritation of the vessel wall by intravenous injection of collagenase (1.5 U per kg body weight) into rats resulted in a transient decrease of the platelet concentration in the flowing blood. This may be caused by an occurrence of inter-endothelial gaps distributed throughout the total area of the circulatory system, as could be observed by electron-microscopic studies. Subendothelial structures, at the sites of those gaps, induce a formation of multilocal microthrombi of platelets. At some gaps within the endothelial layer a deposit of fibrin could also be observed hinting at a participation of the blood coagulation system in the sealing processes. PMID- 3010494 TI - Isolation and characterization of an anticoagulant proteinase, cerastase F-4, from Cerastes cerastes (Egyptian sand viper) venom. AB - An anticoagulant protease, Cerastase F-4, was isolated from the venom of Cerastes cerastes (Egyptian sand viper) by a combination of gel filtration, ion-exchange chromatography, and HPLC. Homogeneity of the purified anticoagulant was established by discontinuous polyacrylamide disc gel electrophoresis and by isotachophoresis. The anticoagulant enzyme is a single polypeptide chain without subunits having a molecular weight of 22,500. It consists of 28% aspartic acid residues and only 7% are basic amino acids. This agrees well with the fact that the anticoagulant is an acidic protein with an isoelectric point of 5.2. The anticoagulant is a proteolytic enzyme which hydrolyzes casein, fibrinogen and fibrin. The enzyme's optimum activity occurs around 55 degrees C. The anticoagulant showed no phospholipase A activity, low lethal activity, low hemorrhagic and capillary permeability activity, and no myotoxic activity. PMID- 3010495 TI - Aspirin and dazoxiben as inhibitors of platelet behaviour: modification of their effects by agents that alter cAMP production. AB - The effects of aspirin and dazoxiben were determined on platelet behaviour in platelet-rich plasma (PRP) from 20 volunteers. Dazoxiben prevented aggregation and the release reaction induced by arachidonic acid (AA) in nine of the samples; in the other eleven aggregation and the release reaction still occurred. Aspirin always prevented aggregation and release but higher concentrations were needed in some of the samples of PRP than with others. When the platelets were sensitive to dazoxiben they were relatively sensitive to aspirin; when they were insensitive to dazoxiben they were relatively insensitive to aspirin. The effects of agents that alter production of cAMP on the sensitivity of platelets to aspirin and dazoxiben were determined. Increasing the intracellular level of cAMP rendered platelets more sensitive to the inhibitory effects of both aspirin and dazoxiben; lowering the level of cAMP made the platelets less sensitive to both agents. PMID- 3010496 TI - [Congenital myasthenia]. PMID- 3010497 TI - [Ulcer-fiber-cabbage and vitamin U]. PMID- 3010498 TI - [The beta-receptor system. The structure, function, regulation and relation to heart disease]. PMID- 3010499 TI - Influence of plating density on individual cell growth, cell division and differentiation of neonatal rat heart primary cultures. AB - The influence of plating cell density of an originally enriched myocardial cell population has been studied in neonatal rat heart cells in culture. Low density (LDM) is defined as a density (24 h after plating) of 209 +/- 44 cells/mm2 (mean +/- SEM) and is compared with high density (HDM), 419 +/- 67 cells/mm2. Cell growth is evaluated by the total cell number, the percentage of myocardial cells (M) in culture (PAS method) and the protein content per cell. Some differentiation parameters such as beating rates, glycogen concentration, enzymatic activities (cytochrome C oxidase and glycogen phosphorylase) are studied with time in culture (48, 96 and 192 hr). High density was designed to yield a complete confluency of the cells within 24 hr after plating and to minimize cell division of the non-muscle cells (F). At high density, cell division of F cells is effectively limited, thus leading to a more stable model regarding the cell density per plate and the percentage of M cells: 85.7 +/- 4% and 33.4 +/- 6% in LDM cultures compared with 86.5 +/- 4.7% and 51.7 +/- 9.8% in HDM cultures at 24 and 192 hr (mean +/- SEM). Heart cells increase similarly in size with age in culture in both groups. In HDM cultures the spontaneous contractions begin sooner (24 hr) than in LDM cultures and are more rapidly synchronized. The beating rate is higher in HDM cultures between 48 and 96 hr; however, after this time it falls in HDM and does not fall in LDM. Thus the overgrowth of muscle cells by non-muscle cells is not responsible for loss of beating with time in culture but more likely high density could be a limiting factor for isotonic contraction. There is more glycogen per myocyte in LDM than in HDM cultures. The cell density influences the enzymatic activities of cytochrome C oxidase and glycogen phosphorylase. The cytochrome oxidase activity is higher in HDM cultures than in LDM cultures at 96 hr whereas glycogen phosphorylase activity is higher in LDM cultures at time 96 and 192 hr. In LDM cultures, the ratio cytochrome C oxidase/glycogen phosphorylase decreases with time in culture from 1.685 +/- 0.680 at 48 hr to 0.780 +/- 0.290 at 192 hr but not in HDM cultures (2.13 +/- 0.36 and 1.64 +/- 0.34 respectively). Thus plating density influences properties of heart cell cultures with regard to the overgrowth of the F-cell population and the differentiated state of M cells. PMID- 3010500 TI - Assessment of cardiac performance by first pass radionuclide angiocardiography in infants and children with normal heart and endocardial fibroelastosis. AB - Peak to peak time from the right and to the left ventricle (PPT), left ventricular ejection fraction, left ventricular peak ejection rate and left ventricular peak filling rate were measured by first pass radionuclide angiocardiography in 27 infants and children with normal heart and in 8 patients (18 studies) with endocardial fibroelastosis. In normal subjects, the PPT significantly correlated with heart rate (r = -0.87, p less than 0.001). A PPT corrected by the heart rate (cPPT) was calculated by rotation of the regression equation relating the variables: cPPT = PPT + 0.018 X (heart rate)-2.1. The cPPT averaged 3.0 +/- 0.0 (mean +/- S.E.) sec. Consequently, there was no significant correlation between the cPPT and heart rate, but the cPPT and body surface area are significantly correlated (r = 0.41, p less than 0.05). Left ventricular ejection fraction, peak ejection rate and peak filling rate averaged 68 +/- 2%, 4.2 +/- 0.3/sec and 4.8 +/- 0.3/sec, all of which were independent of the heart rate and body surface area. In patients with endocardial fibroelastosis, the cPPT was prolonged (4.7 +/- 0.4 sec), and left ventricular ejection fraction, peak ejection rate and peak filling rate were all reduced (28 +/- 4%, 1.7 +/- 0.2/sec and 1.8 +/- 0.2/sec). These results indicate that parameters obtained from the radionuclide angiocardiography are useful for evaluating cardiac performance in patients with endocardial fibroelastosis. PMID- 3010501 TI - Auer rods in mature granulocytes and monocytes. AB - In a 73-year-old man with acute myelomonocytic leukemia (M4 on FAB classification) Auer rods were demonstrated in mature granulocytes and monocytes in the blood as well as the bone marrow. Cytochemically, a significant number of leukemic blasts were positive for both alpha-naphthyl-acetate esterase and naphthol-AS-D-chloroacetate esterase. Auer rods in mature granulocytes were associated with immature cytoplasm and not infrequently with pseudo-Pelger anomaly. The significance of these observations is discussed. PMID- 3010502 TI - Inhibitory effects of selected antiviral compounds on newly isolated clinical varicella-zoster virus strains. AB - Nine anti-herpes compounds, idoxuridine (IDU), bromovinyldeoxyuridine (BVDU), bromovinylarabinofuranosylurasil (BVaraU), carbocyclic bromovinyldeoxyuridine (C BVDU), chloroethyldeoxyuridine (CEDU), acyclovir (ACV), dihydroxypropoxymethylguanine (DHPG), adenine arabinoside (Ara-A), and phosphonoformate (PFA) were examined for their inhibitory activities against the replication of twenty-six newly isolated clinical strains of varicella-zoster virus (VZV) in human embryonic fibroblast cell cultures. The order of (decreasing) efficacy was: BVaraU greater than BVDU greater than or equal to C BVDU greater than CEDU greater than or equal to IDU greater than ACV greater than Ara-A greater than DHPG greater than PFA. There was little variation in the susceptibility of the 26 VZV strains to each particular compound, except for one strain which was resistant to IDU. PMID- 3010504 TI - Stereospecific effect of naloxone hydrochloride on cyanide intoxication. AB - Cyanide intoxication in mice can be antagonized by the opiate antagonist, ( )naloxone HCl, alone or in combination with sodium thiosulfate and/or sodium nitrite. Potency ratios, derived from LD50 values, were compared in groups of mice pretreated with sodium nitrite (sc, 100 mg/kg), sodium thiosulfate (ip, 1 g/kg), and (-)naloxone HCl (sc, 10 mg/kg) either alone or in various combinations. These results indicate that naloxone HCl provides a significant protection against the lethal effects of potassium cyanide. The protective effect of sodium thiosulfate, but not sodium nitrite, was enhanced with (-)naloxone HCl. The combined administration of sodium nitrite and sodium thiosulfate was further enhanced with (-)naloxone HCl. This protective effect of naloxone HCl against the lethal effect of cyanide appears to be restricted to the (-)stereoisomer, as the (+)stereoisomer, the inactive opiate antagonist, is also inactive in protecting against the lethal effects of cyanide. The mechanism of antagonism is discussed. PMID- 3010503 TI - Enhanced ovarian gonadotropin receptors in the testosterone-induced polycystic ovary in rats. AB - To establish the role of hormone receptors in patients with polycystic ovary (PCO), PCO rats were prepared by treating with testosterone propionate (TP). Five day old immature female rats were subcutaneously injected with 1.25 mg TP in sesame oil. They were then killed at the age of 12 weeks. The ovarian receptors for LH and FSH as well as serum hormone levels were investigated in PCO rats and also in control rats at the various stages of the estrous cycle. The LH receptor binding in the TP-treated ovaries was elevated almost as high as that of proestrus control, and was observed to be higher than the other control. The FSH receptor binding of PCO rats was elevated to 173% of that of diestrus control, which showed the highest value throughout the cycle. Thus, the gonadotropin receptors in PCO rats appeared to be in an activated state. High levels of the receptor binding were due to an increase in receptor binding sites. Serum LH level was significantly higher than that of diestrus control but still remained lower than that of proestrus control. In contrast, FSH level was as low as that of diestrus control. Prolactin level was markedly elevated and 17- and 2-fold higher than that of diestrus and proestrus control, respectively. Estradiol level was higher than that of diestrus control, increasing to almost the same level during proestrus control. While progesterone level was largely depressed to 23 and 13% of that of diestrus and proestrus control, respectively, testosterone level was almost the same as that of diestrus control. From these results, it was suggested that tonic secretion of LH, low level of FSH, and markedly high levels of prolactin would increase the gonadotropin receptors and result in extremely low production of progesterone in rat ovaries. Clinically, elevated levels of the LH and FSH receptors may be a relevant occurrence in PCO patients. PMID- 3010505 TI - Toxicological significance of hepatic responses to furylfuramide(AF-2) in the rat. AB - In male rats fed furylfuramide [2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide, AF-2] at a dietary level of 0.1%, a marked increase in the liver weight was observed with a concomitant reduction of microsomal mixed function oxidase activity. To clarify the significance of these hepatic responses to furylfuramide, a series of biochemical parameters that reflect hepatotoxicity was measured. The most significant elevations were seen in hepatic glycogen and glucose 6-phosphate dehydrogenase levels over 29-day feeding interval. A marked decrease was observed in mixed-function oxidases and glucose 6-phosphatase activities. Moderate decreases were seen in several other parameters, especially in the early phase of feeding. Measurement of serum parameters showed significant elevation in cholesterol and a transient increase in serum glutamic pyruvic transaminase. A comparative study demonstrated small differences between furylfuramide and carbon tetrachloride in their effects on hepatic parameters. The furylfuramide-induced changes appear to be reversible within 14 days after withdrawal of furylfuramide from the diet. Reduction of mixed-function oxidase activity by furylfuramide might be caused partly by the elevation of heme degrading enzyme. The results indicate a dysfunction of drug-metabolizing activity and acute and slight toxication of the liver by furylfuramide. PMID- 3010506 TI - Effects of the spermicide nonoxynol-9 on the pregnant uterus and the conceptus of rat. AB - In a series of experiments the embryotoxic potential of nonoxynol-9 (N-9) was investigated in adult female rats given a single per vaginam application of 5 mg/100 g (0.1 ml/100 g) of this spermicide on day 3 (pre-implantation period) or 7 (postimplantation period) of gestation. Control rats were given physiologic saline (0.1 ml/100 g) intravaginally. The vulvar labia were apposed for 24 h by metallic clips to prevent leakage of the solution. Groups of dams treated on pregnancy days 3 and 7 were killed by CO2 inhalation on gestational days 6, 9, 12 and 15, and 8, 9, 10, 12 and 15, resorption and total resorption of the conceptus, embryonal and placental resorption and total resorption of the conceptus, embryonal and placental necrosis, placentitis, endometritis, multicystic endometrium, and diffuse or segmental dilatation of the uterine horns. Generally, the incidence of these lesions varied with the length of time after N-9 was administered and it was consistently higher in the females treated on pregnancy day 3 than in those treated on day 7. Acute vaginitis waned with time after the application of N-9. It was concluded that under the conditions of this study, N-9 is embryocidal/fetocidal in the rat if administered during the first week of gestation. The impairment of embryonal/fetal development was attributed to the N-9-induced changes in the endometrium, the placenta and/or the embryo. PMID- 3010507 TI - Evidence for the inductive effects of hexafluorobenzene on hepatic microsomal enzymes in the male rat. AB - Acute administration of hexafluorobenzene (HFB) (1200 mg/kg body wt i.p.), induced significant increases in male rat hepatic microsomal protein content (39% increase), microsomal cytochrome P-450 content (150% increase) and microsomal glucose-6-phosphatase activity (25% increase). The assays were carried out 24 h after treatment. Hexafluorobenzene also significantly shortened (by 22%) hexobarbitone-induced sleeping time. PMID- 3010508 TI - A note on the predicted secondary structures of the active chains of cholera and diphtheria toxins. AB - Both the A regions of diphtheria and cholera toxins contain the site of ADP ribosyl transferase activity which is responsible for the modification of specific target proteins in mammalian cell types. The secondary structure prediction for these A regions has been made on the basis of their recently reported primary structures. In the center of both toxin A chains, the beta structure and alpha-helix regions alternate in a manner similar to that reported for some NAD binding proteins. Other regions of alpha-helix in the A chains may be involved in the interactions with the toxin B chains. The lack of primary structure homology between these toxins indicates that the secondary structure homology is the result of convergent evolution of a NAD binding domain in each protein. PMID- 3010509 TI - Allogeneic bone marrow transplantation in children: Tokai experience 1982 to 1984. AB - Ten children between the ages of five and fifteen years old with leukemia (two with acute nonlymphocytic leukemia in first remission, four with acute lymphocytic leukemia in first or second remission, one with acute lymphocytic leukemia in relapse, and one with chronic myelocytic leukemia in chronic phase), malignant lymphoma (one) or severe aplastic anemia (one) were given transplants from HLA-matched or mismatched family members between March, 1982 and April, 1984. Two patients died of leukemia relapses on days 107 and 257 following transplantation. One patient died of cardiac failure on day 157. One patient who received HLA-mismatched marrow from his father died of pulmonary edema and acute graft versus host disease on day 32. Six are alive 268-843 days post transplantation. None of the ten patients developed interstitial pneumonia due to cytomegalovirus which is one of the major causes of death reported in other published studies. PMID- 3010510 TI - Intravenous hyperimmune globulin prophylaxis against cytomegalovirus interstitial pneumonitis after allogenic bone marrow transplantation. AB - In an attempt to reduce the incidence of lethal cytomegalovirus (CMV) interstitial pneumonitis after allogenic bone marrow transplantation 49 patients were randomized in a multicenter controlled study to receive either CMV hyperimmune globulin or a control immune globulin with low anticytomegalovirus titer. Immune globulin was administered intravenously 6 times with 20 days interval, starting on day 7 before transplantation. Patients receiving CMV hyperimmune globulin or control immune globulin were comparable with regard to age, diagnosis, pretransplant anti-CMV titer, incidence of graft-versus-host disease and transfusions. In each group, the incidence of histologically proven CMV interstitial pneumonitis during the first 110 days post BMT was recorded. Six of 23 patients in the control group versus 1 of 26 in the CMV hyperimmune globulin group died of CMV interstitial pneumonitis (p less than 0.05). No significant effect on idiopathic pneumonitis or survival was observed. PMID- 3010511 TI - Intratesticular steroid levels and their hormonal control. AB - Litter-mate adult male rats were treated with daily intramuscular injections of ACTH (10.5 micrograms), dexamethasone (2.0 mg), ethynyl estradiol (1.7 micrograms) and hCG (5 IU) for three consecutive days. The animals were sacrificed on the fourth day and the intratesticular and peripheral plasma steroid levels were analyzed. The steroids measured by radioimmunoassay included pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone, progesterone, 17 hydroxyprogesterone, androstenedione, testosterone and dihydrotestosterone. In addition, the sulphoconjugated forms of pregnenolone, dehydroepiandrosterone, testosterone and dihydrotestosterone were estimated in the peripheral blood. The administration of ACTH diminished the intratesticular levels of all steroids studied. Also dexamethasone and ethynyl estradiol treatment suppressed all intratesticular steroid levels, except that of pregnenolone (the former) and of 17-hydroxyprogesterone (the latter). The suppressive effect of ethynyl estradiol was strongest on the levels of the delta 5-steroids and that of dexamethasone on the delta 4-steroids; the latter was significantly stronger than the effect of ACTH. The stimulatory effect of hCG was limited to the metabolism of progesterone and was restricted to the sequence: 17-hydroxyprogesterone----androstenedione--- testosterone---- dihydrotestosterone. Dexamethasone-suppression, and hCG stimulation of the intratesticular levels of delta 4-steroids, was mirrored by corresponding changes in the peripheral plasma levels, with the exception of the plasma levels of androstenedione which were not influenced by any of the treatments studied. Also the suppression of intratesticular testosterone and dihydrotestosterone levels by ACTH, dexamethasone, or ethynyl estradiol was closely reflected by their plasma levels both in the unconjugated and sulphoconjugated forms. On the hand, the administration of ACTH diminished the intratesticular levels of pregnenolone and progesterone but significantly increased those in the plasma. Moreover, both ACTH and ethynyl estradiol reduced the levels of all delta 5-steroids in testicular tissue, but not in the peripheral plasma, although they decreased the circulating levels of pregnenolone sulphate and dehydroepiandrosterone sulphate. The data are interpreted as suggesting that the hormonal agents studied interfere with testicular steroidogenesis through different mechanisms. PMID- 3010513 TI - The role of catecholestrogens in placental steroidogenesis. AB - The effect of the catecholestrogen, 2-hydroxyestrone (2-OHE1), on placental steroidogenesis was studied by incubating 2-OHE1 with placental explants for 24 hours and measuring the output of estradiol (E2) and progesterone (P4). 2-OHE1 stimulated the accumulation of E2 and P4 in the media. This effect was inhibited by the alpha-adrenergic blocker, phenoxybenzamine, and the beta-adrenergic blocker, propranolol. We conclude that 2-OHE1 affects placental steroidogenesis and that this effect could possibly be mediated through adrenergic receptors. PMID- 3010514 TI - CDC guidelines for prevention of surgical wound infections, 1985. PMID- 3010512 TI - Correlation of concentrations of estriol-3-sulfate with those of potassium and sodium in human breast cyst fluid. AB - Estriol-3-sulfate (E3-3S) was assayed in 92 specimens of human breast cyst fluid (BCF) obtained by needle aspiration from women with fibrocystic disease. The concentrations of K+ and Na+ were determined in the same samples. The median concentration of E3-3S in the fluids from premenopausal women under 51 years of age (69 cases) was 4.4 ng/mL. Based on the K+ levels the samples were divided into two groups, above 50 mM (Type I) and below 50 mM (Type II). Correlations were made between the concentrations of the estrogen conjugate and the univalent ions. In the premenopausal women, Type I cysts were associated with above median E3-3S and Type II cysts with below median E3-3S (P less than 0.01). A K+/Na+ ratio of more than one was also related to elevated E3-3S (P less than 0.025). The BCF obtained from postmenopausal women and women older than 50 years tended to be low in E3-3S (median 1.64 ng/mL) and high in K+ but there were too few cases to permit statistical comparisons to be made. Since fibrocystic disease constitutes a risk factor for the development of breast cancer, it will be of interest to determine retrospectively whether any of the above subsets of BCF may be useful in identifying a patient at such risk. PMID- 3010515 TI - Comparison of three methods for detecting antibody to cytomegalovirus. AB - To determine the availability of cytomegalic inclusion virus (CMV) antibody negative blood among our local donor population, 1384 consecutive group O donors were tested for CMV antibody by the indirect hemagglutination assay (IHA); 51 percent (711) were seronegative (titer less than 8). The highest percentage of seronegative findings (60%) was among donors 18 to 35 years old. This age group represented 57 percent of the donors. IHA, enzyme immunoassay (EIA), and passive latex agglutination (PLA) methods were compared on 491 donor samples. The reference method was the anticomplement immunofluorescence test. Sensitivities were: IHA, 89 percent; EIA, 93 percent; and PLA, 90 percent. Specificities were: IHA, 90 percent; EIA, 95 percent; and PLA, 100 percent. All but two of the false negative samples contained antibody at low titer (less than or equal to 10), which is of doubtful significance relative to transfusion-transmitted CMV infections. All three methods were suitable for screening blood donors. The PLA was easy to perform and had a short processing time that would allow rapid assay of fresh (less than 7 days old) untested blood at the time of selection for transfusion. Thus, both the amount of pretested blood kept in stock and the cost of obtaining each CMV-seronegative unit could be reduced. PMID- 3010516 TI - Detection of acquired immunodeficiency syndrome (AIDS) retrovirus antibody by lymphadenopathy-associated virus (LAV) enzyme immunoassay in low- and high-risk populations. AB - Antibody to the acquired immunodeficiency syndrome (AIDS) retrovirus was evaluated by an lymphadenopathy-associated (LAV) enzyme immunosorbent assay (EIA) and compared with the standard human T-cell lymphotropic virus, Type III (HTLV III) screening test in two groups of people. The first group consisted of those at low risk for AIDS, including 1352 random volunteer donors, 1140 of whom were prospective donors and 212 of whom were retrospective donors, repeatedly reactive by HTLV-III EIA. The second group was composed of those at high risk for AIDS, including 54 hemophiliacs, one of whom had AIDS, seven AIDS-related complex (ARC), and one immune thrombocytopenia (ITP). Of the 1140 prospective donors, one was repeatedly reactive by LAV EIA and four by HTLV-III EIA; none was positive by Western blot. Of the 212 retrospective donors, six were repeatedly reactive by LAV EIA and 212 by HTLV-III EIA; only six (the six LAV EIA positive) were positive by Western blot. Of the 54 hemophiliacs, 46 were repeatedly reactive by both LAV EIA and HTLV-III EIA, and all 46 were positive by Western blot. Both LAV EIA and HTLV-III EIA were positive in all hemophiliacs with AIDS, ARC, and ITP. The marked reduction in HTLV-III EIA repeatedly reactive, Western blot nonreactive samples by the LAV EIA system suggests that this assay may be as sensitive but more specific than the standard HTLV-III EIA in low-risk populations. The strong correlation between the LAV EIA and the Western blot assay further suggests that this assay may provide an efficient screening test for AIDS antibody in the donor population. PMID- 3010517 TI - Cytomegalovirus-induced diabetes mellitus in a renal allograft recipient. PMID- 3010518 TI - [Dependence of Na, K-ATPase enzyme activity on the surface properties and charge of the substances interacting with the cell membrane]. AB - The surface activity and effect of histone, sodium deoxycholate and Triton X-100 on N+,K+-ATPase have been compared. It has been found that the coefficient of surface tightness on the borderline of suspension of ATPase containing membranes/air decreases with the increase in concentrations of these agents. The enzyme activity undergoes biphasic modification increasing with lower and decreasing with higher concentrations of the agents. The effectiveness of the agents depends on the charge of their molecules. The mechanism of interaction between Na+,K+-ATPase and the above modifiers is discussed. PMID- 3010519 TI - Intraductal carcinoma within a phyllodes tumor of the breast: a case report. AB - The first reported case in the world literature of a ductal carcinoma in situ located within a phyllodes tumor of the breast is presented. Previously reported cases of carcinoma in phyllodes tumors are reviewed, and the relationship between carcinoma arising in fibroadenomas and phyllodes tumors is discussed. PMID- 3010520 TI - [Properties of phosphoprotein phosphatase from the rat liver]. AB - Prosphoproteid phosphatase, an enzyme highly specific to lysyl-tRNA-synthetase and proteins of the high-molecular-multienzymic complex of aminoacyl-tRNA synthetases, was isolated from the rat liver. The data of electrophoresis in 4 30% PAAG with the presence of DS-Na have shown that phosphoproteid phosphatase is homogeneous and its molecular mass is 56 kDa. The isolated phosphoproteid phosphatase is activated by 2.5 mM Mg2+, Mn2+ and is inhibited by ions of univalent metals ions--200 mM Na+, 5 mM K+ as well as by 1 mM ATP, ADP, AMP. PMID- 3010521 TI - [Influence of effectors of hormone-sensitive adenylate cyclase on the activation system of photostimulated cyclic nucleotide phosphodiesterase from outer rod segments]. AB - The effect of preincubation of preparations of the outer segments of optic rods with the nonhydrolyzed analog GTP-guanilyl-5'-imidodiphosphate (Gpp(NH)p) and NaF, the combined effect of these agents as well as the action of (NH4)2SO4 (10 800 mM), MgSO4 (2-50 mM) and induction of peroxide oxidation of lipids are studied as applied to the catalytic activity of phosphodiesterase of cyclic nucleotides. Gpp(NH)p and NaF are shown to be tightly bound to GTP-binding proteins (G-proteins) of outer segments of optic rods, additional activation of phosphodiesterase in the presence of Gpp(NH)p being observed after preincubation with NaF and subsequent washing of the membrane. A problem on different binding sites of the ion F and Gpp(NH)p on G-proteins is discussed. It is found that (NH4)2SO4 does not affect the basal activity of phosphodiesterase but inhibits the activating effect of Gpp(NH)p and NaF on the enzyme. Induction of peroxide oxidation of lipids prevented by the addition of ionol (antioxidant) in a dose of 5.10(-4) M has the same effect. Changes in the concentration of Mg2+ in the medium influence insignificantly the basal activity of phosphodiesterase but are necessary for manifestation of the activating effect of Gpp(NH)p and NaF. PMID- 3010522 TI - [Enzymes of adenosine metabolism in lymphocytes and the functional state of the sympatho-adrenal system in tumor processes]. AB - A correlation between reactions of the sympathoadrenal system and the activity of adenosine transformation enzymes in lymphocytes is demonstrated in the dynamics of metastatic Lewis carcinoma development in C57Bl mice. In the period when metastases arise a decrease in the adenosine deaminase activity in lymphoid cells of the thymus and spleen is accompanied by drop in the content of DOPA, noradrenalin and adrenalin in adrenals. At the late stages of the tumour process a decrease in the amount of these compounds in adrenals is accompanied by the diminution of the adenosine deaminase activity and by an increase in the 5' nucleotidase activity in the thymus. Contrary changes are observed in spleen lymphocytes. The revealed disturbances may stimulate to a considerable extent the appearance and growth of metastases. PMID- 3010524 TI - [Syphilis profile among LAV/HTLV III antibody-positive and negative homosexual men]. PMID- 3010523 TI - [Occurrence of antibody to human T-cell leukemia/lymphovirus type III (LAV/HTLV III) in venereology clientele in Copenhagen]. PMID- 3010525 TI - [LAV/HTLV III antibody in patients with acute hepatitis B 1975-1984]. PMID- 3010526 TI - [The occurrence of LAV/HTLV III antibodies in patients with hemophilia in the County of Funen]. PMID- 3010527 TI - Anaplastic carcinoma of urinary bladder with oat cell features. AB - Anaplastic carcinoma of the urinary bladder, with oat cell-like components, is rare. We report such a tumor in a fifty-nine-year-old man, who died of rapidly disseminated carcinoma in one year despite postoperative radiotherapy and chemotherapy. PMID- 3010529 TI - [Effect of ultrasonics on internal ear receptors in chronic suppurative otitis media]. PMID- 3010528 TI - [Characteristics of morphological changes in nasal mucosa of workers exposed to chrysotile-asbestos dust]. PMID- 3010530 TI - Etiopathogenesis, clinical manifestations, and management of canine silica urolithiasis. AB - Silica uroliths were first recognized in dogs in the mid 1970s. Currently available data suggest that dietary factors may play a role in their pathogenesis. Diagnosis is facilitated by their typical jackstone appearance but must be verified by quantitative analysis. Surgery is the only feasible method of treatment. PMID- 3010531 TI - Canine uroliths. Analysis of data derived from 813 specimens. AB - This article contains an analysis of data compiled from 813 specimens of canine uroliths submitted to the Urinary Stone Analysis Laboratory at University of California School of Veterinary Medicine. PMID- 3010533 TI - [Comparison of the immunizing effectiveness of subunit vaccine and inactivated viral oil vaccine against infectious rhinotracheitis of cattle]. AB - Trials were conducted on rabbits and cattle to compare the immunizing effectiveness of the subunit vaccine against infectious bovine rhinotracheitis (IBR), representing antigens separated by the solubilization of the IBR virus infected cells by means of Triton X-100 with oil adjuvant, with the inactivated oil IBR vaccine. The rabbits inoculated and re-vaccinated with both vaccines in an interval of three weeks produced neutralizing antibodies in medium titres, the values of these antibodies were balanced in both groups. Cattle immunized with the subunit vaccine reacted to the inoculation and re-vaccination by producing serum antibodies of higher titres, as compared with the cattle inoculated with the virus vaccine; secretory antibodies were detected only after re-vaccination and had balanced values in both test groups. After intranasal infection with the virulent virus performed after 14 days from re-vaccination, the calves inoculated with the subunit and virus vaccines were protected against clinical disease whereas the non-inoculated control calves fell ill with symptoms characteristic of IBR. The immunized animals of both experimental groups had a smaller amount of virus p.i. in nasal secretions and for a shorter time than the control non inoculated calves. The intensity of multiplication and persistence of infectious virus excretion were the same in both experimental groups. PMID- 3010532 TI - [Experimental and natural infection with the enzootic leukosis virus of cattle]. AB - A trial was performed with heifers at the age of six to seven months. The animals were experimentally infected with the lymphocytes of a virus-productive donor. Infection was produced in all the nine cases, as demonstrated by means of the positive syncytial test. As indicated by the results of the trial, the antibodies to the enzootic bovine leucosis virus (BLV) were produced soon after experimental infection. A high sensitivity of the serum-neutralization test and the ELISA method was demonstrated in this connection: by these methods, the antibodies were identified already two to three weeks after experimental infection whereas by the immunodiffusion test they could be detected only after five weeks. Twenty-four animals were exposed to natural contact infection. Within 270 days of the trial, the disease after contact was recorded only in one heifer out of the four that were in close contact with the experimentally infected animals. In this case, as compared with experimental infection, the antibodies were produced much later- after 85 to 93 days. Leucosis was recorded in none of the remaining animals. The reasons why such a favourable result was obtained were the thorough disinfection of the stables after blood collections and the strict observance of the aseptic conditions. The results of experimental infection in three cows were identical with those obtained in young cattle. In the experimentally infected dairy cows, antibodies in milk were determined by the ELISA method. As found, in milk the antibodies to BLV appear two to three weeks later than they do in serum. The ELISA method of BLV antibody detection can be used for the identification of infected animals in herds where enzootic bovine leucosis occurs. PMID- 3010534 TI - [The incidence of herpesvirus hepatitis in parrots--Pacheco's disease--in Czechoslovakia]. AB - A highly fatal disease of parrots kept in zoological garden is described; it manifests itself as abrupt mass death of the birds. Deposits of necrotic changes with the occurrence of intranuclear inclusions of the Cowdry A type were found in the liver of dead parrots. The virus was isolated on chick embryos and tissue cultures of chick embryo cells. The bioassay was performed on Psittacus undulatus. The occurrence of herpesvirus nucleocapsids and complete virions in the infected tissue cultures was detected by means of electron microscopy. PMID- 3010535 TI - 'Kangaroo gait' in ewes: a peripheral neuropathy. AB - The clinical and pathological features of two lactating ewes with 'kangaroo gait', a locomotory disorder, are described, along with brief details of two further archival cases. Clinical neuropathological signs were consistent with a bilateral radial paresis and pathologically there was a polyneuropathy with preferential severe involvement of radial nerves. Flock incidence of the condition is low and previous experience suggests the clinical disorder is not progressive, recovery occurring at the end of lactation. The cause is unknown. PMID- 3010536 TI - Enteric virus-like particles associated with a stunting syndrome of chickens. PMID- 3010537 TI - Control of Aujeszky's disease. PMID- 3010538 TI - Persistent bovine virus diarrhoea virus infection in a bull. AB - Investigation of a sight defect in a pedigree bull, born as a result of artificial insemination and ovum transplantation, led to the finding that the animal was persistently infected with bovine virus diarrhoea virus. Virus was cultured from blood and from nasal and ocular swabs and was present in semen in high titre. At necropsy, virus was cultured from a wide range of tissues. The pathological findings are described and discussed as are the potential hazards of such infections. PMID- 3010539 TI - Serological surveillance for maedi-visna virus and caprine arthritis-encephalitis virus in Northern Ireland. AB - The results of serological testing for maedi-visna virus infection in indigenous and non-indigenous sheep in Northern Ireland, over a five year period (1980 to 1984) are presented. In tests carried out in 1980 and 1981 on pedigree breeds, 10 reactors were identified on seven farms and maedi-visna virus was isolated on three occasions from leucocyte cultures. None of the animals showed clinical signs of maedi-visna and all the reactors were subsequently destroyed. Nine of the reactors were imports from Scotland or the Republic of Ireland and the other was the progeny of an imported ewe. In subsequent tests (1982 to 1984) of exotic and indigenous flocks no further reactors were identified. A survey of goat herds for evidence of caprine arthritis-encephalitis virus infection was also negative. PMID- 3010540 TI - Hematologic values in calves infected with Sarcocystis cruzi. AB - Calves were inoculated with 2 X 10(5) Sarcocystis cruzi sporocysts. Red cell mass decreased dramatically between Days 21 and 35 post-infection and plasma volume increased concurrently, so that blood volume did not change significantly. Mild reticulocytosis and increased pyrimidine 5' nucleotidase activity in erythrocytes occurred between Days 35 and 42. Antiglobulin tests with anti-bovine IgG, IgM and C3 were negative, with the exception of a positive test for C3 in 1 of 6 infected calves. PMID- 3010541 TI - Detection and characterization of Leishmania species and strains from mammals and vectors by hybridization and restriction endonuclease digestion of kinetoplast DNA. AB - Leishmania parasites from animals, man or insect vectors were characterized by the gel electrophoresis of restriction endonuclease enzyme-produced mitochondrial (kinetoplast) DNA (kDNA) fragments and/or by DNA-DNA hybridization with 32P labelled cloned, or uncloned, kDNA fragment probes from type isolates. The electrophoretic separation of kDNA fragments is a sensitive method for detecting genetic similarities and differences among Leishmania. Parasites with similar kDNA restriction fragment patterns belong to the same schizodeme and schizodeme analysis is useful for studying Leishmania populations. Cloned, species-specific kDNA probes detected Leishmania in sandflies and in liver, spleen or blood preparations from infected animals. Cloned DNA probes also hybridized to immobilized kDNA from in vitro cultivated promastigotes and detected as few as 100 parasites in a species-specific manner. Sensitive DNA hybridization probes should be useful in research on the immunology, chemotherapy or epidemiology of animal and human leishmaniasis. PMID- 3010542 TI - Environmental impact of sewage sludge on livestock: a review. PMID- 3010543 TI - Toxic effect of the roasted and unroasted beans of Cassia occidentalis in goats. AB - The toxic effects of roasted and unroasted beans of the wild coffee, C occidentalis were compared. Both types of beans intoxicated goats in varying degrees, but roasting partially reduced the toxic effects of the beans. Histopathological, biochemical and enzyme histochemical studies showed that the toxin of C occidentalis damages the liver, vascular system, heart, and lungs. PMID- 3010544 TI - Infrequently used antidotes: indications and availability. PMID- 3010545 TI - The effect of parainfluenza virus type 3 on the phagocytic cell response of the ovine lung to Pasteurella haemolytica. AB - Groups of Caesarean-derived, colostrum-deprived lambs were inoculated by the intratracheal route with Pasteurella haemolytica 4 to 6 days after the inoculation of parainfluenza virus type 3 (PI3). Some were killed immediately (0 h) and others 24 h later. Control groups were inoculated with PI3 alone, P. haemolytica alone or media alone. Pulmonary phagocytic cells, P. haemolytica and PI3 were recovered by pulmonary lavage. The phagocytes were separated into alveolar macrophage (AM) and neutrophil fractions by density gradient centrifugation and examined biochemically and microbiologically. Twenty-four hours after the inoculation of P. haemolytica bacterial proliferation to greater than 0 h levels had occurred in four of six animals inoculated with P. haemolytica alone, two of eight inoculated with P. haemolytica 4 days after PI3 and all of eight inoculated with P. haemolytica 6 days after PI3. Mean bacterial numbers in animals inoculated with P. haemolytica 6 days after PI3 and killed at 24 h (10(9.1 +/- 1.9)) were significantly higher than they were in the other two groups killed at this time (PI3 4 days, P. haemolytica 24 h, mean = 10(5.3 +/- 1.7); P. haemolytica alone 24 h, mean = 10(4.5 +/- 2.9)). Pneumonic lesions were also more severe in the first group. This defect in pulmonary clearance and increase in the severity of pneumonia in animals inoculated with P. haemolytica 6 days after PI3 coincided with a 1000-fold decrease in virus titres in the lung between Day 6 and Day 7 after virus inoculation and the first detectable evidence of the host's immune response. The virus infection resulted in a significant increase in the number of AM that could be recovered from the lung and an increase in the number of AM with cytoplasmic vacuolation. However, there was no difference in the total number of AM or the number of vacuolated AM between animals that controlled the P. haemolytica infection and those in which proliferation of P. haemolytica occurred. The inoculation of P. haemolytica resulted in a 100-fold increase in the number of neutrophils in the lavage fluid, but there were no differences between virus-infected and uninfected animals, nor was there a difference between animals that controlled the P. haemolytica infection and those that did not.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3010546 TI - Peptide mapping of the group-specific antigens from the Australian bluetongue virus (BTV-20) and serotypes from southern Africa and North America. AB - The Australian bluetongue virus (BTV-20) was compared with six serotypes isolated in southern Africa and North America by peptide mapping of the virus proteins with group antigen properties. The p7 group antigens from each of the seven serotypes analysed did not have identical primary structures and a comparison of shared and unique tryptic peptides has been used as a means of estimating virus relationships. Whereas serological studies have suggested that BTV-20 is closely related to BTV serotypes 4 and 17, comparative peptide mapping of p7 indicates a different set of relationships with viruses from both southern Africa and North America. In contrast with cross-immune precipitation results, peptide mapping of p3 suggest that this protein is not a group specific antigen. PMID- 3010547 TI - Sequential isolation of rotavirus from individual calves. AB - An outbreak of neonatal calf diarrhea was studied on a breeding farm of Japanese indigenous beef cattle. During the breeding season of 1982, 43 calves were born over the period 27 February-28 April. All but one of the calves suffered from neonatal diarrhea and 5 died. Bovine rotavirus was isolated in cell cultures from fecal specimens of 39 (90.7%) of the 43 calves during the outbreak, strongly suggesting that this was the causative agent; the virus was readily isolated from 81 (83.5%) of 97 specimens of diarrhea. Rotavirus was subsequently isolated from the feces of 7 of the calves in early May, more than one month after the initial virus isolation in early March. Two of these calves were again rotavirus-positive in early June, 41 days after the second virus isolation. Diarrhea had ceased in all 7 calves in March. Some antigenic differences were shown by the neutralization test between the early and later isolates from one of these calves, suggesting either re-infection with a serologically different virus, or persistent infection with the original virus following antigenic modulation. PMID- 3010548 TI - Evaluation of a diagnostic antigen for the detection of Aujeszky's disease virus infected subunit-vaccinated pigs. AB - An early virus protein complex that is found in the maintenance medium of Aujeszky's disease (AD) virus-infected cells was evaluated as a subunit diagnostic antigen (SUDA) in the enzyme-linked immunosorbent assay (ELISA). This antigen was found in purer form and in larger quantities for up to 12 h post infection in the maintenance medium of AD virus-infected MDBK cell cultures than in the maintenance medium of virus-infected porcine Fallopian tube (PFT) and PK1a cell cultures. The SUDA was shown to be compatible with a lectin-derived subunit vaccine by the absence of positive ELISA reactions for antibody to this antigen in 25 AD virus-free subunit-vaccinated pigs. Following virus challenge, all fo 24 surviving vaccinated pigs seroconverted to SUDA within 10 days. Compatibility with the vaccine was further demonstrated by the absence of positive ELISA reactions for antibody to SUDA in 12 pigs that received five or six consecutive vaccine doses at 3-wk intervals. The sensitivity of the ELISA with SUDA was demonstrated by the detection of antibody in virus-infected vaccinated and non vaccinated pigs for at least 15 and 22 weeks, respectively, following exposure to virus. The SUDA was also economical: it was calculated that 8000-14 000 tests could be run with the antigen present in the maintenance medium of one 850 cm2 plastic tissue culture roller bottle of virus-infected MDBK cells. PMID- 3010549 TI - Development of specific antibodies mediating antibody-dependent cell-mediated cytotoxicity following experimental infection of cattle with bovine herpesvirus 1 and subsequent viral reactivation. AB - Evolution of sensitizing antibodies involved in antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-complement cell lysis and complement-facilitated ADCC was followed in bulls after primary infection by a wild strain of infectious bovine rhinotracheitis virus (bovine herpesvirus 1; BHV-1) and after experimental reactivation of the virus. These antibodies were detected between the 4th and the 7th day after primary infection, reached a maximum level after 2 weeks and rose slightly after reactivation of the virus following dexamethasone treatment. The presence of endogenous complement slightly enhanced the ADCC reaction. PMID- 3010550 TI - Unusual characteristics of a parvovirus isolated from a clinically ill steer. AB - A parvovirus was isolated from the feces of an 8- and 9-month-old steer that died acutely with hemorrhagic diarrhea and microscopic evidence of a coccidial infection. The concurrent intestinal parasitism in this steer appeared to play a role in the development of clinical disease. The viral isolate was identified as a bovine parvovirus (BPV) on the basis of its size (22 nm) and icosahedral morphology, the neutralization of viral cytopathology by antiserum to BPV, a strong immunofluorescent reaction with fluorescein-labeled antiserum to BPV, and the inhibition of viral hemagglutination of guinea-pig erythrocytes by antiserum to BPV. Cell cultures infected with this isolate showed a slight nuclear fluorescent reaction with fluorescein-labeled antiserum to canine parvovirus, suggesting an antigenic relationship to canine parvovirus. Patterns of hemagglutination for this isolate with human erythrocytes from 20 donors of various blood types differed from those obtained with the reference Abinanti strain of BPV. These results indicate that blood from multiple donors of a species may be necessary to confirm the presence or absence of viral hemagglutinating activity with clinical isolates of BPV. PMID- 3010552 TI - Two glycoproteins are produced from the rotavirus neutralization gene. AB - The major neutralization antigen of rotaviruses is an outer capsid glycoprotein, VP7, with an apparent molecular weight of 38,000 (38K). The simian rotavirus SA11 genome segment 9, which codes for VP7, contains two in-phase initiation codons, each of which is followed by a sequence that codes for a region of hydrophobic amino acids. We have determined that this gene is functionally bicistronic by analyzing the synthesis of VP7 in SA11-infected cells and in cell-free translation systems programmed with hybrid-selected, segment 9 specific mRNA and dog pancreatic microsomes. The translation of hybrid-selected gene 9 mRNA in wheat germ extracts yielded two distinct polypeptides of molecular weights 37K and 35.3K. In vitro translation in the presence of microsomes yielded one diffuse band of 38K that was converted into the 37K and 35.3K precursor bands by digestion with endoglycosidase H. Studies with a variant of SA11 that lacks the glycosylation site in VP7 confirmed these precursor-product relationships and extended them by indicating that the glycoprotein produced by translation from the first AUG contained a cleaved signal sequence whereas the glycoprotein produced by translation from the second AUG contained an uncleaved signal sequence. Immunoprecipitation with monospecific anti-VP7 serum and improved gel electrophoresis conditions allowed us to show that both VP7s were expressed at similar times in infected cells and both were found in purified virus particles of several different rotavirus strains. Whether these two VP7 glycoproteins are functionally distinct remains to be determined. PMID- 3010551 TI - Ross River virus mutant with a deletion in the E2 gene: properties of the virion, virus-specific macromolecule synthesis, and attenuation of virulence for mice. AB - A mutant of RRV T48 the prototype strain of Ross River virus has been isolated with a 21-nucleotide deletion in the gene coding for the envelope glycoprotein E2. Direct sequencing of the 26 S subgenomic RNA, together with HaeIII and TaqI restriction digest analysis of cDNA to RNAs from cells infected with the mutant virus (RRV dE2) and with RRV T48, were consistent with the deletion being the only major alteration in the mutant genome. The E2 protein of RRV dE2 virions had a higher electrophoretic mobility than that of RRV T48 E2 protein. Neither RRV dE2 nor RRV T48 virions contained more than trace amounts of E3, the small envelope glycoprotein found in Semliki Forest virus. RRV dE2 generated small plaques on Vero cell monolayers; plaque formation was not temperature-sensitive between 32 and 41 degrees. By comparison with RRV T48 the infectivity of RRV dE2 virions was thermolabile at 50 degrees. In BHK cells RRV dE2 grew with similar kinetics to RRV T48. Rates of synthesis of 26 S RNA and 49 S RNA were higher in cells infected with RRV dE2 than in cells infected with RRV T48. Virus-specific protein synthesis and shut-down of host protein synthesis occurred 2-3 hr earlier in RRV dE2-infected cells than in cells infected with RRV T48. Minor differences between the two viruses were observed in the profiles of virus-specific proteins generated in infected cells. In day-old mice RRV dE2 induced less severe symptoms of hind leg paralysis than did RRV T48. A small increase in LD50 and average survival time was observed in RRV dE2-infected mice by comparison with RRV T48 infected mice. Peak titers reached by RRV dE2 in the hind leg muscle, brain, and blood of day-old mice were 3-4 log units less than the titers reached during infection with RRV T48. In week-old mice the differences in virulence between the two strains were magnified: RRV dE2 induced no detectable symptoms even when injected at high doses (8 X 10(6) PFU) whereas the LD50 and average survival time for RRV T48 were unchanged from those in day-old mice. Peak RRV dE2 titers in hind leg muscle, brain, and blood, respectively, were 2, 5, and 5 log units less than the corresponding titers for RRV T48. Peak muscle titers reached by RRV dE2 were similar (approximately 10(8) PFU/g tissue) in day-old mice where lethality was high and in week-old mice where the virus was avirulent.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3010553 TI - In vivo and in vitro models of demyelinating diseases. XV. Differentiation influences the regulation of coronavirus infection in primary explants of mouse CNS. AB - Mouse oligodendrocytes and astrocytes, in primary cerebral explant cultures, were infected with JHMV and MHV3 coronaviruses. Contrary to previous findings with neural cells from the rat (S. Beushausen and S. Dales, 1985, Virology 141, 89 101), these agents show no discrimination in the tropism and have the ability to replicate in either type of murine glial cell. Effects of the differentiation inducer dbcAMP on levels of the myelinspecific enzyme 2':3'-cyclic nucleotide-3' phosphohydrolase (CNPase) activity and virus replication were determined. In the mouse system there was a gradual, continuous elevation of CNPase beyond 30 days whereas in comparable rat cell cultures maximum CNPase enhancement is elicited within 21 days (F. A. McMorris, 1983, J. Neurochem. 41, 506-515). After dbcAMP treatment replication of both coronaviruses was profoundly suppressed in murine oligodendrocytes, consistent with our findings on JHMV replication in treated rat oligodendrocytes. By contrast the replication of JHMV and MHV3 in dbcAMP-treated murine astrocytes was influenced only marginally. These findings provide further support for the hypothesis that susceptibility of rodents to CNS infection by coronaviruses is determined, in part, by the age-related maturation process of oligodendrocytes. PMID- 3010554 TI - Isolation of temperature-sensitive mutants of bacteriophage MB78 and correlation between the physical and genetic maps. AB - In order to study the biochemistry and genetics of the virulent virus MB78 of Salmonella typhimurium, 31 temperature-sensitive mutants of the phage were isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. These have been classified into six complementation groups (A through F). Linkage between different complementation groups has been mapped by using two factor crosses between representative members of each group. To correlate the physical and genetic maps of the phage, complementation between bacterial clones carrying plasmids with EcoRI fragments of the phage DNA as inserts and the ts mutants was studied. Good correlation between the physical and genetic maps has been obtained. Tentative locations of the ts mutations on the phage genome have thus been determined. PMID- 3010555 TI - Identification of a human cytomegalovirus virus DNA segment that complements an adenovirus 5 immediate early mutant. AB - Human cytomegalovirus (HCMV) complements adenovirus mutant dl312, which is completely defective for expression of the adenovirus immediate early E1a gene region, for lytic growth (Tevethia and Spector, 1984). This assay defines at least one HCMV function, activation of the transcription of adenovirus early genes in trans, that must be provided for dl312 replication in coinfected cells. We show here that trans-activation depended on the expression of one or more HCMV gene. Human embryonic lung cells were transfected with dl312 DNA-protein complex and either HCMV DNA or recombinant plasmids containing the adenovirus E1a gene region or HCMV (Towne strain) DNA fragments. Replication of dl312 occurred only in cells that received both DPC and either the E1a gene, HCMV DNA, or the XbaI-E HCMV DNA fragment (0.68 to 0.77 map units). In addition, we show that HCMV also complemented adenovirus mutant pm975 for growth in 0.2% serum. Since pm975 grows poorly in low concentrations of serum due to a defect in an E1a gene product, this assay identified a second HCMV E1a-like function. PMID- 3010556 TI - Cloning and sequencing of the genetic right end of bacteriophage T3 DNA. AB - The genetic right end of phage T3 DNA, from the beginning of gene 17, was cloned and sequenced. Genes 17, 18, and 19 were identified by comparing the sequence with the genetic map and by comparing the calculated and observed molecular weights of gene products. N-terminal amino acid sequence of the gene 17 product (gp17) predicted from the nucleotide sequence was consistent with the data from the analysis of purified gp17. Gene 17.5 was identified as the lysis gene on the basis of the presence of a nonsense codon within an open reading frame in the sequence of DNA from an amber mutant of lysis gene. In addition, five potential genes have been identified. Sequences corresponding to a promoter for phage T7 RNA polymerase (Rosa and Andrews, 1981) and to a class-III promoter for phage T3 RNA polymerase (Sarker et al., 1985) were found. The genomic organization and the nucleotide and deduced amino acid sequences of T3 were compared with those of T7. The genomic organizations of T3 and T7 were identical in this region. The sequence comparisons of T3 and T7 DNA point out the highly conserved sequences in all genes but also heavily varied regions in some genes. From these comparisons, possible implications with regard to structural and functional domains within several genes are discussed. PMID- 3010557 TI - Cloning and characterization of budgerigar fledgling disease virus, an avian polyomavirus. AB - The DNA of a virus isolated from fledgling budgerigars, designated BFDV, was cloned and analyzed with regard to its relationship to the polyomavirus subgroup of the papovavirus family. Under relaxed conditions, the DNA of BFDV cross hybridized with the DNAs of members of the polyomavirus subgroup, such as the mouse polyomavirus, the monkey viruses simian virus 40, stump-tailed macaque virus, and lymphotropic papovavirus, and the human viruses JCV and BKV. Under stringent conditions, however, no homologies could be detected. Furthermore, BFDV propagated in chicken embryo cells was antigenically related to the capsid antigen(s) of the other polyomaviruses. The virus was able to transform hamster embryo cells in vitro which is a typical feature of polyomaviruses. These data clearly indicate that BFDV is a new distinct member of the polyomavirus genus representing the first nonmammalian polyomavirus. PMID- 3010558 TI - The intracellular half-lives of nonreplicating nucleocapsids of DI particles of wild type and mutant strains of vesicular stomatitis virus. AB - We have used defective interfering (DI) particles purified free of all infectious virus to determine the intracellular biological stability of nonreplicating nucleocapsids of vesicular stomatitis virus (VSV). Following infection of BHK-21 cells or tc-7 cells with a low multiplicity of pure DI particles, we superinfected them at varying times afterward with a high multiplicity of infectious helper virus to allow replication of those DI particle nucleocapsids retaining biological activity. Careful quantitation of DI particles in the yields from each time point showed that the biological half-life of intracellular VSV Indiana wild type DI particle nucleocapsids was 6 hr in BHK-21 cells and 5.3 hr in tc-7 cells (not significantly different). However, a DI particle from a temperature-sensitive mutant (tsG31) of VSV exhibited a biological nucleocapsid half-life of 12.5 hr and a DI particle isolated following 5 years of persistent infection had a half-life of 18 hr. These findings have significance for the stability of DI particle activity in vivo during acute infections where virus and DI particles are not always present together in the same cells due to cycling interactions. The increased half-life of DI nucleocapsids after years of persistent infection contrasts with the decreased stability and debilitation generally observed in infectious virus from persistent infection. Finally, transcapsidation studies showed that the intracellular half-life differences between DI particles are due mainly to their RNA genomes rather than to the N protein which encapsidates them. PMID- 3010559 TI - Studies on herpes simplex virus type 1 glycoproteins using monoclonal antibodies. AB - Monoclonal antibodies against herpes simplex virus type 1 glycoproteins were isolated and utilized to study the synthesis and processing of glycoproteins B, C, and D (gB, gC, gD, respectively). Monoclonal antibodies against both gB and gD had higher virus-neutralizing activity when compared to that of gC. Differences among these glycoproteins were observed in their time of appearance in the virus infected cells. The presence of gD was detected at a very early stage of infection when compared to gB and gC. The localization of these glycoproteins during their synthesis and processing was studied. PMID- 3010560 TI - The spiroplasma virus 4 replicative form cloned in Escherichia coli transfects spiroplasmas. AB - The replicative form (RF) of spiroplasma virus 4 (SpV4) has been purified from infected cells of Spiroplasma melliferum strain G1 by alkaline lysis followed by low melting point agarose gel electrophoresis. A partial restriction map has been established. The circular RF was linearized by cutting at the unique ClaI restriction site and has been cloned in Escherichia coli HB101 using the plasmid pBR328 as a vector. The recombinant plasmid was purified by equilibrium centrifugation in ethidium bromide-cesium chloride gradient. After ClaI endonuclease digestion, the inserted SpV4 RF DNA was recovered by low melting point agarose gel electrophoresis and was recircularized by ligation. The cloned SpV4 RF DNA was demonstrated to be infectious by transfection. PMID- 3010561 TI - [History of influenza vaccine]. PMID- 3010562 TI - [Human rotavirus]. PMID- 3010563 TI - [Changes in the indicators of the immune system and cyclic nucleotides in neurocirculatory asthenia]. PMID- 3010564 TI - [Electroneuromyography in evaluating the efficacy of treatment with physical factors]. PMID- 3010565 TI - [Cholesterol-acceptor properties of high-density lipoproteins with respect to erythrocyte membranes in patients with ischemic heart disease]. AB - A capacity of high density lipoproteins (HDL), isolated from blood of patients with ischemic heart disease, to accept cholesterol of erythrocyte membranes was studied by means of monitoring, using spectra of electron paramagnetic resonance, a disease in the correlation period of rotation, in the patterns of regularity of stearic acid nitroxyl derivatives introduced into the membranes and by a decrease in the molar ratio "cholesterol/phospholipids" in erythrocytes. HDL2 and HDL3, isolated from blood plasma of the patients, were shown to bind cholesterol less effectively after incubation with erythrocytes as compared with the corresponding lipoproteins from blood plasma of healthy persons. Relationship between the phenomenon observed and the physico-chemical properties of HDL in ischemic heart disease are discussed. PMID- 3010567 TI - [Changes in the serotonin metabolism of rats with neurodystrophic process]. AB - Some mechanisms of alteration in serotonin content in tissues and an effect of the amine on development of visible trophic impairment in rats were studied under conditions of neurodystrophic impairments which occurred in the animals as a result of chronic stimulation of sciatic nerve. A long-term increase in serotonin content in tissues of rats with the neurodystrophic process was due to activation of its synthesis. Elevation in serotonin content in animals under conditions of chronic stimulation of peripheral nervous system might be of importance for development of neural and neurogenic dystrophies. PMID- 3010568 TI - [ATPase and acetylcholinesterase activity of the brain in hypothermia]. AB - Short-term hypothermia, caused by cooling of rats down to 20 degrees, decreased distinctly the Na+, K+-ATPase activity in brain homogenates incubated at 37 degrees and did not affect the enzyme activity in the homogenates incubated at 20 degrees. The longer hypothermia (2 hrs at 20 degrees) did not affect the Na+, K+ ATPase activity at 37 degrees (during incubation) and decreased the enzymatic activity in homogenates of middle brain and diencephalon at 20 degrees during the incubation. Contrary to Na+, K+-ATPase, the activity of acetylcholinesterase was markedly increased in brain tissues of rats with hypothermia (irrespective of the temperature of incubation) as compared with control animals. PMID- 3010566 TI - [Acid glycosidases in leukocytes of patients with chronic and acute myeloid leukemias]. AB - Activities of five acid glycosidases were examined in leukocytes of 30 healthy persons, 17 patients with chronic myeloid leukemia (CML) and 7 patients with acute myeloid leukemia (AML). In leukocytes of the patients with CML activities of alpha-D-mannosidase, N-acetyl-beta-D-hexosaminidase and beta-D-glucurodase were significantly higher (p less than 0.01) and those of alpha-D-galactosidase and alpha-D-glucosidase were somewhat higher (p less than 0.05) as compared with control leukocytes. Activities of all five glycosidases in leukocytes of patients with AML were within the normal limits. Glycosidase activities were also examined in leukocytes of three groups of patients with CML at different stages of the disease: chronic, progressive and blast crisis. All the enrymes exhibited the highest activity in the first group of patients; the activities of these enxymes in the second group were lower and those in the third group were close to normal. When CML leukocytes were fractionated using the Ficoll-verografin method, the activities of the enzymes in the interface fraction (unmatured cells) were higher than those in the bottom fraction (matured granulyocytes). These data suggest that the increased glycosidase activity in CML cells is due to the presence of the population of unmatured granulocytes which are distinct from blast cells. PMID- 3010569 TI - [Desensitization with acetylsalicylate and salicylate of fructose-1,6 diphosphatase from the rabbit liver and skeletal muscles to allosteric inhibition of AMP]. AB - Acetylsalicylate and salicylate partially desensitized fructose 1,6-biphosphatase from rabbit liver and skeletal muscle to allosteric AMP inhibition. The desensitization was accompanied by a decrease in cooperativity between allosteric sites especially distinct for the liver isoenzyme. The effect of salicylate on both isoenzymes was more pronounced than that of acetylsalicylate. No essential differences in the effect of the compounds studied on these isoenzymes were found. PMID- 3010571 TI - [Effect of Rous sarcoma virus on cytoplasmic membranes of sensitive and resistant cells]. AB - An increase in cholesterol and sialic acid levels in cytoplasmic membranes was registered in the course of Rous sarcoma-induced transformation of chick embryo cells. There was no such effect in virus-resistant human cells. PMID- 3010570 TI - [Effect of immunostimulants of steroidogenesis on the activity of various oxidative enzymes in the adrenal glands and other organs]. AB - Effect of IgG from rabbits, immunized with deoxyribonucleoprotein and with nuclear matrix of rat adrenal glands, on activities of cytochrome c oxidase, glucose-6-phosphate (G6PD) and lactate (LDH) dehydrogenases was studied in rat adrenal glands, liver and spleen tissues, myocardial and skeletal muscles. After administration of the antibodies and deoxynucleoprotein activation of the oxidative enzymes was tissue-dependent. Activities of cytochrome c oxidase and G6PD were distinctly increased in adrenal glands. In other tissues studied the enzymatic activity was not either increased (spleen, myocardium, skeletal muscle) or mediated through adrenal glands (liver tissue). The tissue-dependent activation of the oxidative enzymes, which accompanied the steroidogenic effect of the immunoglobulins studied, was considered as a result of interaction between the antibodies and tissue-specific antigen determinants in adrenal gland chromatin. PMID- 3010572 TI - [Thiamine metabolism in experimental hepatitis and the intake of Naftusia mineral water]. AB - The level and metabolism of vitamin B1 and its coenzymic form were studied in the tissues and subcellular structures of the internal organs of white rats with experimental toxic hepatitis, receiving mineral water naphtusya. It was found that naphtusya given per os stimulated the metabolism of thiamine pyrophosphate (TPP), enhanced its concentration in the hepatic tissue and intestinal mucosa, producing a stabilizing effect on the TPP activity. It is concluded that the mineral water naphtusya can be used for enteral correction of vitamin balance in hepatitis patients. PMID- 3010573 TI - [Compensatory hemodynamic mechanisms of patients with a history of myocardial infarct]. PMID- 3010574 TI - [Euphylline as a stimulant of gastric secretion (a review of the literature)]. PMID- 3010575 TI - [Embryonal cancer of the liver in an 18-year-old boy]. PMID- 3010576 TI - Leukocytes and tissue injury: the use of eicosapentaenoic acid in the control of white cell activation. AB - Eicosapentaenoic acid enriched diet may reduce the severity of acute inflammatory reactions by decreasing the synthesis of dienoic prostanoids and tetraenoic leukotrienes. The findings suggest that a nutritional approach to anti inflammatory therapy is an interesting possibility deserving further studies. PMID- 3010577 TI - [Leukotriene synthesis by gastrointestinal tissue and its pharmacologic modification]. AB - Tissues of the gastrointestinal tract synthesize leukotriene (LT) B4 and the sulfidopeptide-leukotrienes LTC4, LTD4 and LTE4 from endogenous substrate. Formation of leukotrienes was demonstrated using radioimmunoassay, high pressure liquid chromatography (HPLC) and bioassay. Under basal conditions the gastrointestinal tissues released minor amounts of leukotrienes only. Formation of lipoxygenase-derived products of arachidonic acid metabolism was, however, significantly increased in the presence of various stimuli. Thus, significant amounts of LTB4 and of sulfidopeptide-leukotrienes were released from colonic and gastric mucosa of guinea-pigs sensitized against ovalbumin when incubations were carried out in the presence of antigen. Antigen-induced leukotriene formation was not found in the muscularis propria and subserosal of ovalbumin-sensitized guinea pigs. Release of cyclooxygenase-derived metabolites of arachidonic acid, on the other hand, was most abundant in the subserosal layer of the guinea-pig colon and was not influenced by the immunological reaction. Inhibitors of cyclooxygenase, such as indomethacin, reduced gastrointestinal formation of prostaglandins, but not of leukotrienes. Inhibitors of 5-lipoxygenase, however, significantly decreased leukotriene formation. Synthesis of LTB4 and of sulfidopeptide leukotrienes was also found in human colonic mucosal tissue, using the divalent cation-ionophore A23187 as stimulating agent. HPLC analysis demonstrated that the sulfidopeptide-leukotrienes released were composed of a mixture of LTC4, LTD4 and LTE4. In addition, human colonic mucosal tissue contained high activities of enzymes that rapidly convert LTC4 to LTE4. As in most biological systems LTE4 is less active than LTC4 and LTD4 degrading enzymes might represent a local inactivating mechanism. Mucosal tissue of patients with Crohn's disease synthesized considerably more LTB4 and sulfidopeptide-leukotrienes than non inflamed mucosa.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010578 TI - Contribution of arachidonic acid metabolites to atherosclerosis. AB - Evidence is presented that arachidonic acid metabolites are involved to an important extent in platelet-arterial wall interaction. The perspectives opened up by these results are the possible prevention and therapy of atherosclerosis by a fish-diet containing eicosapentaenoic acid, the use of prostaglandins, releasers of endogenous PGI2 and thromboxane synthetase inhibitors. PMID- 3010579 TI - Regulation of prostacyclin production by vascular cells in in vitro and in vivo experimentals systems. AB - Prostacyclin (PGI2) is the major product of arachidonic acid (AA) metabolism in vascular cells. Its physiological role is linked to the cells' ability to produce PGI2 continuously in response to a variety of stimuli. Human endothelial cells or bovine smooth muscle cells in culture responded only once with PGI2 production to stimulation with AA, thrombin or ionophore A23187. Their refractoriness lasted about 6 hours. The same time was required by the cells to recover completely after inhibition of cyclooxygenase activity by aspirin. When the cells were repeatedly stimulated with AA in the presence of plasma no refractoriness was apparent. These results suggest that vascular cyclooxygenase is irreversibly self inactivated during oxygenation with AA. As shown in other experimental systems, deactivation could be attributed to the attack on the enzyme by oxidizing species formed during AA conversion and the plasmatic free radical-scavenger systems could protect the cells from inactivation. Thus, vascular cells in an artificial medium cannot be stimulated continuously to produce PGI2, but this process is regulated by negative feed-backmechanisms. When the cells are stimulated in the presence of plasma i.e. in more physiological conditions, they are much less sensitive to inactivation. PMID- 3010580 TI - The binding of fibrinogen to platelets in diabetes mellitus. AB - Platelets from diabetics are known to be more sensitive in vitro to a variety of aggregating agents, to produce more prostaglandin endoperoxides and thromboxane and to bind more 125I-fibrinogen than platelets from normal controls Fibrinogen binding to platelets is a pre-requisite for platelet aggregation. Previous studies suggested a role for prostaglandins and/or thromboxane A2 in the exposure of fibrinogen receptors on platelets. The present study compares fibrinogen receptors on platelets. The present study compares fibrinogen binding to hyperaggregable platelets from diabetic patients and to normal platelets when prostaglandin/thromboxane formation is suppressed by aspirin. It was found that pre-treatment with aspirin reduced collagen or thrombin-induced binding to platelets from non-retinopathic diabetics to the values seen in controls. By contrast, aspirin did not normalize binding to platelets obtained from retinopathic diabetics. The combination of aspirin with apyrase (an ADP scavenger) almost completely inhibited binding and aggregation of platelets from normal controls or non-retinopathic diabetics exposed to collagen or thrombin, whereas it only partially affected binding and aggregation of platelets from retinopathics. By using a monoclonal antibody (B59.2) to the platelet receptor for fibrinogen, we determined that this receptor was quantitatively and qualitatively the same on platelets from normal controls and diabetics. We conclude that increased fibrinogen binding and hyperaggregability of platelets from non-retinopathic diabetics is related to their capacity to form more prostaglandin endoperoxides/thromboxane than normal platelets. In contrast, hyperaggregability and increased binding of platelets from retinopathics appear at least partly related to a mechanism independent of ADP release and thromboxane synthesis. PMID- 3010581 TI - Alteration of platelet glucose transport system in atherosclerosis. AB - Functional and biochemical alterations of platelets in patients suffering from atherosclerosis were studied in our laboratory. One of the most striking alterations observed is in the platelet active glucose transport system. The Na+/K+ gradient dependent active transport system of glucose is found to be absent in the platelets of atherosclerotics. The platelet glucose transport kinetics in these subjects give unsaturable and linear kinetics. Furthermore, the specific glucose binding protein activity detected in the incubation fluid after cold osmotic shock to the platelets of normal subjects is found to be absent in the platelets of atherosclerotics. The platelet active glucose transport system is normal in juvenile onset diabetics, whereas it is impaired in maturity onset diabetics with clinical manifest atherosclerosis. The release inducers like ADP, adrenalin and collagen exert no effect on the platelet active glucose transport system. The specific glucose-binding protein is an unreleasable protein in the platelets of normal subjects. Hence, the absence of active glucose transport system in atherosclerotics is not due to the activated platelets in circulation. PMID- 3010582 TI - [Omega-3-fatty acids in fish: cell functions, health]. PMID- 3010583 TI - Hepatic resection for hepatocellular carcinoma associated with liver cirrhosis. PMID- 3010584 TI - Determination of the extent of feasible hepatic resection from hepatic blood flow. PMID- 3010585 TI - Liver resections in cirrhotic patients: a Western experience. PMID- 3010587 TI - [HTLV-III infection and the central nervous system]. PMID- 3010586 TI - Acquired immune deficiency syndrome. PMID- 3010588 TI - [Germinative testicular tumors. On the current status of diagnosis, therapy and prognosis]. AB - On the basis of results obtained in a multicentric study on testicular tumors (Bonn 1982) including our own group of patients, we give an outline of the present state of modern diagnosis, therapy, and prognosis concerning germinative tumors of the testes: Etiologically essential is an unknown dysontogenetic disturbance of both gonads. The only clinical predilection factor is cryptorchism. Among the new diagnostic methods, specific tumor markers and high resolution ultra-sonography have been proved especially useful. In non-seminomas, orchidectomy and lymphadenectomy is followed by chemotherapy instead of radiation therapy, today. Polychemotherapy has greatly improved the prognosis. Andrologically most interesting questions concerning sperm conservation, limited retroperitoneal lymphadenectomy, as well as late sequelae of chemotherapy cannot be definitely answered. Our findings rise new questions, which are fully discussed in concluding remarks. PMID- 3010589 TI - [Reaction of autogenous cortisol production in patients with extensive psoriasis vulgaris or atopic dermatitis following external administration of a 0.25% prednicarbate salve (W/O emulsion)]. AB - Prednicarbate is a new potent corticoid without halogenic groups which has been developed for topical application. During one week's application of 0.25% prednicarbate ointment (W/O Emulsion) two times daily in an ordinary dose, there could not be proved any significant suppression of the basal production of plasma cortisol in patients suffering from extensive dermatoses (such as psoriasis vulgaris or atopic dermatitis). After the end of treatment, investigation on the reservoir within the adrenal cortex by means of ACTH short test showed sufficient stimulation capacity of the adrenal gland in all patients examined. PMID- 3010590 TI - Alterations in the activities of rabbit erythrocyte membrane-bound enzymes induced by cholesterol enrichment and depletion procedures. AB - The effect of cholesterol enrichment and depletion of rabbit erythrocytes on the activities of membrane-bound enzymes, namely (Na+,K+)-stimulated ATPase, NAD+ase and acetylcholinesterase was examined. The cholesterol content of erythrocyte membranes has been modified by incubation of intact cells with sonicated egg lecithin/cholesterol vesicles (cholesterol/phospholipid molar ratio approx. 2) and with egg lecithin vesicles for time intervals up to 10 hours. The cholesterol/phospholipid molar ratio (CH/PL) of untreated rabbit red blood cell membranes was 0.92-0.94. Linear increase (up to CH/PL molar ratio 1.72-1.9) or decrease (up to CH/PL molar ratio 0.27-0.43) in cholesterol content of erythrocyte membranes was observed over the 10 hours of incubation with egg lecithin/cholesterol and egg lecithin liposomes respectively. Fusion of liposomes to the membrane or their attachment to the membrane surface was not a significant factor in the alteration of CH/PL ratio. (Na+,K+)-stimulated ATPase, NAD+ase and acetylcholinesterase activities were measured as a function of membrane cholesterol. The specific activities of all three enzymes were progressively decreased with increase in cholesterol content. Partial reversibility of the inhibitory effect of cholesterol was demonstrated by measurement on cells depleted again after cholesterol enrichment. This was confirmed by the fact that a lowering in cholesterol content evoked an analogous activation of enzymes. The possible implications of physicochemical modifications of bulk and annular lipids of membrane-bound enzymes in the inhibition mechanism are discussed. PMID- 3010592 TI - [Primates as laboratory models of viral persistence and the chronic course of viral encephalitis]. PMID- 3010591 TI - [Coronavirus infectivity of monkeys at the Sukhumi nursery]. PMID- 3010593 TI - [Paramagnetic centers of the blood and tissues in various dermatoses]. PMID- 3010594 TI - Sendai virus replication in Friend erythroleukemia cells. I. Acutely and persistently infected cells become resistant to virus-induced lysis. AB - Friend leukemia cells (FLC) are susceptible to infection by Sendai virus, a member of the paramyxovirus group. FLC constitute a most suitable model to study virus-host cell interactions, because they grow in suspension (thus avoiding the use of trypsin), and provide an easy way of deriving single-cell clones. When FLC are infected with Sendai virus at high m.o.i., a direct, extensive lysis of the cells ensues, whereas lower doses of virus result in a cytocidal infection whose lethality depends mainly on the virus used, standard or defective interfering egg grown Sendai virus (EGSV), and on the multiplicity of infection (m.o.i). At later times after infection, FLC become resistant to the Sendai induced lysis (SIL). The SIL resistance can be maintained in single-cell clones that had survived the first infection. The maintenance of the resistant phenotype of the clones requires the serial subcultivation of the cells in the presence of activated EGSV. The mechanisms that presumably regulate the appearance of SIL resistance in Sendai infected FLC are discussed. PMID- 3010595 TI - Coronavirus IBV: partial amino terminal sequencing of spike polypeptide S2 identifies the sequence Arg-Arg-Phe-Arg-Arg at the cleavage site of the spike precursor propolypeptide of IBV strains Beaudette and M41. AB - The spike protein of avian infectious bronchitis coronavirus comprises two glycopolypeptides S1 and S2 derived by cleavage of a proglycopolypeptide So, the nucleotide sequence of which has recently been determined for the Beaudette strain (Binns, M.M. et al., 1985, J. Gen. Virol. 66, 719-726). The order of the two glycopolypeptides within So is aminoterminus(N)-S1-S2-carboxyterminus(C). To locate the N-terminus of S2 we have performed partial amino acid sequencing on S2 from IBV-Beaudette labelled with [3H]serine and from the related strain labelled with [3H]valine, leucine and isoleucine. The residues identified and their positions relative to the N-terminus of S2 were: serine, 13; valine, 6, 12; leucine, none in the first 20 residues; isoleucine, 2, 19. These results identified the N-terminus of S2 of IBV-Beaudette as serine, 520 residues from the N-terminus of S1, excluding the signal sequence. Immediately to the N-terminal side of residue 520 So has the sequence Arg-Arg-Phe-Arg-Arg; similar basic connecting peptides are a feature of several other virus spike glycoproteins. It was deduced that for IBV-Beaudette S1 comprises 519 residues (Mr 57.0K) or 514 residues (56.2K) if the connecting peptide was to be removed by carboxypeptidase like activity in vivo while S2 has 625 residues (69.2K). Nucleotide sequencing of the cleavage region of the So gene of IBV-M41 revealed the same connecting peptide as IBV-Beaudette and that the first 20 N-terminal residues of S2 of IBV M41 were identical to those of the Beaudette strain. IBV-Beaudette grown in Vero cells had some uncleaved So; this was cleavable by 10 micrograms/ml of trypsin and of chymotrypsin. Partial N-terminal analysis of S1 from IBV-M41 identified leucine and valine residues at positions 2 and 9 respectively from the N terminus. This confirms the identification, made by Binns et al. (1985), of the N terminus of S1 and the end of the signal sequence of the IBV-Beaudette spike propolypeptide. N-terminal sequencing of [3H]leucine-labelled IBV-Beaudette membrane (M) polypeptide showed leucine residues at positions 8, 16 and 22 from the N-terminus; these results confirm the open reading frame identified by M.E.G. Boursnell et al. (1984, Virus Res. 1, 303-313) in the nucleotide sequence of M. The N-terminus of the nucleocapsid (N) polypeptide appeared to be blocked. PMID- 3010596 TI - Coronavirus IBV glycopolypeptides: locational studies using proteases and saponin, a membrane permeabilizer. AB - [35S]methionine-labelled avian infectious bronchitis virus (IBV) (strain 41) and its purified protein components and virions of IBV-Beaudette were incubated with 10 proteases. Several proteases hydrolysed all or some of the membrane glycopolypeptide (M; Mr 30K) and removed about 1.3K of peptide from the amino-(N )-terminus plus both glycans, as determined by SDS-polyacrylamide gel electrophoresis. N-terminal analysis of [3H]isoleucine-labelled M after hydrolysis by bromelain revealed that the first nine residues had been removed. After the virions had been permeabilised with saponin, a further 2.5K decrease in molecular weight was produced and this was shown to be from the carboxy-(C )terminus. When considered with the hydropathicity plot analysis of the amino acid sequence of M (Boursnell, M.E.G. et al., 1984, Virus Res. 1, 303-313) these results suggest that as few as 9-20 N-terminal amino acid residues may protrude at the outer membrane surface and that there is a highly protease sensitive sequence of an estimated 20-25 residues at the C-terminus of M exposed in the lumen of the virion. S2 but not S1 was cleaved to a major glycopolypeptide of approximately 71K by several proteases, and to 76K by trypsin. N-terminal sequencing of the 71K glycopolypeptide revealed that it had the same N-terminus as intact S2. After hydrolysis in the presence and absence of saponin it was concluded that S2 is very sensitive to hydrolysis near its carboxy terminus at residues close to the outer membrane surface. PMID- 3010597 TI - The expression and properties of polyoma virus middle-T antigen in simian cells. AB - SV40 late replacement vectors containing the polyoma middle-T coding sequences have been constructed. Mixed hybrid virus stocks have been obtained through complementation with a defective SV40 helper genome (dl 1055) following DNA transfection into CV-1 cells. Middle-T antigen is expressed in the infected simian cells at about 5-10 fold higher levels than in polyoma virus-infected mouse cells and has the pp60c-src-associated tyrosine-specific protein kinase activity in vitro. However, the 'specific activity' of the kinase in extracts of the infected CV-1 cells is lower than that observed in polyoma infected 3T6 cell extracts. The half-life of middle-T antigen in the CV-1 cells is about 4 h but the in vitro kinase activity associated with middle-T has a half-life of at least 8 h and hence appears to be stabilized. The in vivo phosphorylated species of middle-T has been shown by sucrose gradient analyses to be largely distinct from the middle-T with associated protein kinase activity in vitro. PMID- 3010599 TI - [Neuropharmacological analysis of the compensatory-recovery processes in deficiency of the generalization function in lobectomized cats]. AB - Compensatory-recovery abilities of the cat brain were studied in free behaviour under frontal deficiency by means of parenteral pharmacological influence on dopamin-, cholin- and GABA-ergic systems. Activation of dopaminergic structures in lobectomized cats restored complex forms of generalization, and GABA-ergic system stimulation improved simple forms of generalization. Excitation of cholinergic formations deepened decompensation of generalization function. PMID- 3010598 TI - Induction of thymidine kinase activity by viruses with group B DNA genomes: bovine cytomegalovirus (bovine herpesvirus 4). AB - The bovine herpesvirus type 4 (BHV-4) group has a slow replication cycle, a narrow host range, and cytopathogenic effects characteristic of cytomegaloviruses (CMV), but a Group B genome structure similar to that of lymphotropic Herpesvirus saimiri (HVS). Reference BHV-4 strain DN599 and BHV-4 strains N124 and FHV-2 induced in the cytosol fraction of thymidine kinase-negative (TK-) rabbit skin (RAB-BU) cell mutants a novel TK activity. The BHV-4-induced thymidine kinase (TK) differed from the principal cytosol TK of mock-infected cells in PAGE mobility (Rm) under non-denaturing conditions and in the capacity to efficiently substitute CTP for ATP as a phosphate donor. The BHV-4 thymidine phosphorylating activity could also be distinguished from many common herpesvirus-induced TKs because it lacked iododeoxycytidine phosphorylating activity. Iododeoxyuridine, trifluorothymidine and bromovinyldeoxyuridine inhibited [3H]thymidine (0.01 mM) phosphorylation by the BHV-4 enzyme in a dose-dependent manner, but arabinosylthymine and 2'-fluoro-5-methyl-arabinosyluracil (FMAU) were poor inhibitors of [3H]thymidine phosphorylation, and acyclovir and (dihydroxy-2 propoxymethyl)guanine (DHPG) did not inhibit at all at 60 and 40 times the concentrations of [3H]thymidine, respectively. PMID- 3010600 TI - [Malignancy grading of glial tumors. I. Astrocytomas]. AB - A total of 91 fibrillar, protoplasmic and gemistocytic astrocytomas, 56 pilocytic astrocytomas and 70 glioblastomas, surgically obtained from 1973 to 1979, were evaluated. A four-grade malignancy system was applied to the astrocytomas. The grading criteria for the astrocytomas included quantitative cell density, cellular and nuclear polymorphism, mitotic activity, proliferative changes in the vascular elements and degree of necrosis. Using these criteria it was possible to distinguish four grades. Astrocytoma grades 1 and 2 correspond to the rubric "low grade astrocytoma" in the English nomenclature, grades 3 and 4 to "high-grade astrocytoma". From the 91 fibrillar, protoplasmic and gemistocytic astrocytomas there were 12, 29, 31 and 19 tumors in grades 1 to 4 respectively. The corresponding distribution for the 56 pilocytic astrocytomas was 20, 29, 6, 1. Median survival for the fibrillar, protoplasmic and gemistocytic group for malignancy grades 1-4 were 43.8, 33.5, 8.4 and 5.6 months respectively. The corresponding figures for the pilocytic astrocytomas grades 1-3 were greater than 66, greater than 69, and 20.0 months. PMID- 3010601 TI - [Behavior of prolactin, estradiol and ACTH in maternal blood in delivery by cesarean section]. AB - The serum level of prolactin, estradiol (E2) and adrenocorticotropic hormones was determined pre-, intra- and postoperatively in 17 women with delivery by caesarean section. Three different anaesthetics were used before general anaesthesia and epidural anaesthesia. 9 women were able to nurse. Hormone levels of the nursing and non-nursing mothers were compared. An increased level of prolactin and significantly decreased of estradiol and adrenocorticotropic hormones was found in the nursing women postoperatively. The quotient of prolactin and adrenocorticotropic hormones is significant. It increased in nursing women in the childbed and remained almost constant with absent lactation. There were no differences according to methods of anaesthesia in hormones levels and secretion of milk. PMID- 3010602 TI - [Nourseothricin (streptothricin) inactivated by plasmid pIE 636-encoded acetyltransferase: detection of N-acetyl-beta-lysine in the inactivated product]. AB - Nourseothricin (streptothricin) can be inactivated by an acetyl transferase synthesized by E. coli strains containing plasmid pIE 636. Nourseothricin inactivated in the presence of 14C-acetyl-coenzyme A was purified and submitted to partial acidic hydrolysis. By electrophoresis of the hydrolysate a 14C containing substance moving only slowly towards the cathode could be isolated. This substance after complete hydrolysis yields only unlabelled beta-lysine. PMID- 3010603 TI - Laboratory procedures in adenoviruses. XII. Comparison of five ELISA methods for demonstration of hexon antigen. AB - To demonstrate adenovirus hexon, four different arrangements of a four-layer ELISA, to be performed with commercially available reagents, were compared with a three-layer ELISA. After evaluation of specific and control reagents and their dilutions, the guinea pig/rabbit or the rabbit/chicken method were equally practicable and sensitive as the three-layer method with rabbit serum and superior to two other methods. By testing about 100 stool samples and 74 throat swabs containing adenoviruses, it turned out that the sensitivity (1 ng/ml hexon) appears satisfactory for stool specimens in adenovirus enteritis of infants, but is much less sensitive than isolation in cell culture for adenoviruses in throat swabs. As confirmatory test, specimen were incubated with an adenovirus rabbit antiserum or normal serum and then tested. PMID- 3010604 TI - Phagocytosis as a defense mechanism against infection with leptospiras. AB - The role of macrophages in host defense was studied in vivo and in vitro. The intravenous administration of silica, an agent reported to selectively inactivate macrophages, increased the sensitivity to leptospiral infection and inhibited bacterial clearance. Active immunization with killed organisms or with leptospiral lipopolysaccharide (L-LPS), and passive immunization with a monoclonal antibody showed powerful protective effects against infection in mice. The effect of immunization decreased in silica-treated mice. These findings were supported by electron microscopic examination and observation of killing by macrophages in vitro. PMID- 3010605 TI - [Indices of collective immunity to the hepatitis A virus in different geographical regions]. AB - Antibodies to hepatitis A virus were determined in the sera of healthy population by enzyme immunoassay techniques. The distribution of antibodies in different age groups was compared, taking into account the proportion of persons with high, moderate and low titers. Among the surveyed regions, the highest proportion of nonimmune persons was detected in Moscow (45 %) and the lowest proportion, in Aralsk (7%) and Mongolia (3%). In some regions (Moscow, Vilnius) the characteristic feature of the immune structure of the population was the gradual increase of the occurrence of antibodies and their levels with age, while in other regions (Aralsk, Mongolia) the intensive acquisition of antibodies occurred at younger ages with a subsequent decline in antibody levels. Some of the possible mechanisms of the formation of population immunity to hepatitis A are discussed. PMID- 3010606 TI - [Immunological status of monkeys during acclimatization and its correction with levamisole]. AB - The work presents the results of investigations, carried out in different monkey species, on the physiological norms as regards the bactericidal factors of neutrophilic polymorphonuclear leukocytes in the blood, nonenzymatic lysosomal cationic proteins and myeloperoxidase, as well as on changes in these characteristics in monkeys at different periods of their acclimatization at the Institute of Experimental Pathology and Therapy, Sukhumi. The possibility of correcting the characteristics under study by means of the immunostimulating agent levamisole is shown. PMID- 3010607 TI - [Cyclic nucleotides in multiple sclerosis]. AB - The authors studied concentrations of cyclic nucleotides (cAMP and cGMP), as well as their ratio in the blood plasma and cerebrospinal fluid (CSF) of 83 patients with disseminated sclerosis of different forms and variants. An elevation in cAMP concentrations in the plasma and CSF detected in all the patients was more expressed in cases of a long standing and disseminated process. Blood levels of cAMP showed greater variability in the period of exacerbation versus remission of the disease. PMID- 3010608 TI - [Molecular mechanisms of substance dependence (regulatory-structural relations)]. AB - The article is devoted to a potentially possible molecular mechanism of the formation of narcomanic states: euphoria, tolerance, dependence, abstinence and polynarcotism. The regulatory-structural system including a structural component (enzyme, receptor, membrane, etc.), regulatory components (genome, the system of hormonal synthesis, etc.), and transport components (blood, etc.) may be considered a simple and sufficient model of "elementary toxicomanic unit" in the animal body. Proceeding from the above characteristics of functioning of "the toxicomanic unit", the cause of toxicomanogenesis should be sought in the regulatory components, in their inertia. Toxicomanogenesis in biological system is the consequence of four superimposed phenomena: "narcotic" interaction, functional limitedness, regulatory inertia and psychic ability to project, with the regulatory inertia playing the determinant role. PMID- 3010609 TI - [Papain in the treatment of spinal osteochondrosis]. PMID- 3010610 TI - Structure-activity relationships in distamycin A analogues: effect of alkyl groups on the pyrrole nitrogen at the non-amidine end of the molecule combined with methyl elimination in the following ring. AB - Distamycin A analogues 5a-f (R = CnH2n+1, n = 0-5) were synthesized using our previous strategy with some improved modifications and screened for their effects on herpes simplex virus (HSV-1). Virus yield assays show that 5a-5d were potent antiviral agents whereas 5e and 5f had lower activity. Considerable cellular toxicity was however observed for 5a-5c. Thus 5d combining significant antiviral activity with moderate cellular toxicity seems to be the most promising derivative in this series. PMID- 3010611 TI - [A collagen sponge impregnated with hydroxyapatite beads]. PMID- 3010612 TI - Arachidonic acid in psoriasis. Pathogenic role and pharmacological regulation. AB - AA and its derivatives are a family of potent mediators and regulators of inflammation. There are several pieces of evidence to suggest a pathophysiologic role of certain AA derivatives in psoriasis. The complexity of the pathways in the generation of LTs and other LO products suggests that carefully designed systems will be needed to establish the exact role of these compounds in the pathogenesis of diseases such as psoriasis, and to test the effects of pharmacological inhibitors. PMID- 3010613 TI - Cutaneous responses to lipoxygenase products of arachidonic acid. PMID- 3010615 TI - Altered phosphorylation as a possible basis for cyclic nucleotide abnormalities in atopic dermatitis. AB - Exposure of normal mononuclear leukocytes (MNL) to histamine causes heterologous desensitization accompanied by beta adrenergic receptor alterations and increased cyclic AMP-specific phosphodiesterase (PDE) activity. We have demonstrated similar abnormalities in MNL from patients with atopic dermatitis (AD). Considering that these dual changes might be due to altered phosphorylation, we have studied cyclic AMP-dependent protein kinase (PK-A) in untreated and histamine-treated normal MNL and in AD cells. We found that basal or endogenous phosphorylative activity was two-fold higher in preparations from patients with AD. The activity ratios of protein kinase increased significantly in normal MNL after histamine exposure. In contrast, cells from patients with AD failed to show this increase. These findings correlate with similarly increased PDE activity after histamine stimulation of normal, but not AD cells. The increased PK-A activity in both atopic and histamine-desensitized MNL correlates with membrane receptor changes and elevated PDE activity. This enchaned phosphorylation may account for the varied physiological and immunological abnormalities that have been described in AD. PMID- 3010614 TI - Functional changes of neutrophil responses in relapsing acne fulminans. AB - A patient suffering from an acute episode of acne conglobata demonstrated a transient decrease in several C5a induced polymorphonuclear cell functions, whereas the response to N-formylmethionyl peptides (FMLP) was normal. PMID- 3010616 TI - Balanced terminal chromosome translocations develop in EBV-derived, but non immortal cell lines from patients with mycosis fungoides. AB - Five lymphoblastoid cell lines established from patients with mycosis fungoides were followed during long-term culture. They all expressed the Epstein-Barr virus nuclear antigen. Although the established B-cell lines initially showed a normal karyotype, metaphases with balanced terminal translocations gradually occurred. These chromosome abnormalities occurred simultaneously with decreased cell proliferation leading in all cases to termination of the cell lines. This contrasts the usual finding that Epstein-Barr virus established B-lymphoblastoid cell lines are immortal. PMID- 3010617 TI - An epidemic of syphilis among homosexuals and bisexuals. AB - 22 cases of syphilis were diagnosed in the last part of 1982 in the Department of Dermatology and Venereology. Out of them 21 were male cases and 16 were homo- or bisexuals. Most cases were in the secondary stage and Herxheimer reaction only developed in these. Serological titers to both lipoidal and treponemal antigens were extremely high. All bisexual males were positive in herpes and CMV complement fixation test. Two of the homosexuals appeared with iridocyclitis and motile treponemes were isolated from the anterior chamber in one case. PMID- 3010618 TI - Herpes gestationis in association with neoplasma malignum generalisata. A case report. AB - A case of herpes gestationis (HG) and a hormone producing tumour in a woman is presented. The disease had a fulminant course and the patient deceased within months. Fifteen years ago a hydatidiform mole had been removed. Only rarely has HG been associated with hydatidiform mole and choriocarcinoma. We believe that our patient had a human chorionic gonadotrophin (HCG) producing germ cell tumour. Although extremely seldom encountered, the association of HG to neoplasms should be kept in mind. PMID- 3010619 TI - Acromegaly and insulin resistance: a case study. AB - Elevated levels of growth hormone (GH) alter both the glucose tolerance and the sensitivity of peripheral tissue to insulin. We have studied the relationship between impaired glucose metabolism and its variations with different plasma levels of endogenous GH in one patient with acromegaly. To do so, we studied the decline in blood glucose concentration, as induced by iv insulin infusion, from a given hyperglycaemic level. With high levels of GH (GH = 120 micrograms/l), the slope of the straight line representing the decrease in blood glucose after insulin infusion was -0.71, the time required to achieve normoglycaemic levels, 270 min, and the corrected area under the curve representing blood glucose 26 070 units2. After 10 months' bromocriptine treatment, GH plasma concentration fell to 8 micrograms/l, at which point the slope of the straight line was -1.40, the time required to achieve normoglycaemic levels 115 min, and the area under the curve 8956 units2. There was a greater total clearance of glucose when GH levels were lower (1.90 vs 1.00 ml/min/kg), as well as greater elimination of glucose from the extracellular glucose pool (4.02 vs 1.67 mg/min/kg). In conclusion, in this patient the elevated plasma levels of endogenous GH induced insulin resistance. Once GH levels were reduced by the administration of bromocriptine, glucose metabolism improved. PMID- 3010620 TI - Feedback effects of gonadal steroids and pituitary LH-releasing hormone receptors in the streptozotocin-induced diabetic rat. AB - The effects of streptozotocin (STZ)-induced diabetes mellitus on the positive feedback action of steroids on luteinizing hormone (LH) secretion have been investigated in the oestrogen-primed ovariectomized rat. Rats treated with 40 mg/kg STZ 2 weeks before experimentation showed an attenuated LH surge in response to progesterone, an effect only partially restored by insulin replacement. When the same dose of the drug was injected just 24 h before the progesterone treatment it had no effect on the LH surge, while a high dose of 80 mg/kg STZ completely abolished the positive feedback action of the steroid. Insulin treatment did not reverse this effect. In parallel the effects of 2-week and 24-h diabetes on pituitary LH-releasing hormone (LRH) receptors were studied. Pituitary binding of a long acting LRH analogue was reduced in the 2-week diabetic animals, although a more dramatic reduction was observed in the rats treated with the high dose of STZ 24 h before testing. The results suggest that diabetes impairs the positive feedback effects of gonadal steroids resulting in a reduced release of LRH. However, the impairment is unlikely to be caused simply by hyperglycaemia but by non-specific toxic side effects of STZ and/or other metabolic changes associated with diabetes. PMID- 3010621 TI - Stimulatory guanine nucleotide binding protein activity in the erythrocyte membrane of patients with pseudohypoparathyroidism type I and related disorders. AB - The activity of stimulatory guanine nucleotide regulatory protein (Ns) in the erythrocyte membrane was assayed by the reconstitution method using plasma membrane of cyc S49 mouse lymphoma cells in 18 patients with type I pseudohypoparathyroidism (PHP-I), 2 with pseudopseudohypoparathyroidism (PPHP) and 30 normal subjects, in parallel with other clinical parameters. The Ns activity as expressed by per cent of pooled standard (mean +/- SE) was 78.9 +/- 6.1 in PHP-I patients, which was significantly lower (P less than 0.01) than the value in normal subjects, 99.5 +/- 2.4. In PHP-I patients, the Ns activities (Y) were in significant correlation with three clinical parameters examined (X), i.e., with body height in standard deviation score from the mean of the normal population at the corresponding age, Y = 89.4 + 10.4X (r = 0.616, P less than 0.01); with urinary cAMP excretion in relation to creatinine [cAMP(nmol)/Cr(mg)], Y = 56.3 + 7.2X (r = 0.501, P less than 0.05); and with TSH levels in plasma (microU/ml), Y = 129 - 3X (r = 0.639, P less than 0.01). The Ns activities of PPHP were as low as 53.8 and 60.0. The decrease of Ns activity in the cell membrane may be implicated in the development of the clinical symptoms such as short stature, decrease in urinary excretion of cAMP and latent or manifest primary hypothyroidism in PHP-I and possibly in skeletal abnormality in PPHP. PMID- 3010622 TI - Cyclic AMP phosphodiesterase activity in mouse pancreatic islets. Effects of calmodulin and phospholipids. AB - The activity of cyclic AMP phosphodiesterase in mouse pancreatic islets was investigated. 85% of the total activity was found in a 27000 g supernatant fraction. The phosphodiesterase activity in the supernatant fraction, but not in the particulate fraction, was stimulated approximately 20% by Ca2+ (10(-5)M) and calmodulin (1 microM). The Km (cyclic AMP) of the unstimulated enzyme in the supernatant fraction was 20 microM, and the Vmax was 2 nmol/min X mg protein-1. The possible influence of a range of phospholipids was investigated. PI* and PS (150 micrograms/ml) inhibited the enzyme 20-30% both in the absence and presence of Ca2+/calmodulin, whereas PE, PC and PA did not affect the enzyme activity. ATP (1 mM) did not affect the particulate or supernatant fraction phosphodiesterase either in the absence or presence of Ca2+/calmodulin or Ca2+/phospholipid. It is concluded that, contrary to islet adenylate cyclase, islet cyclic AMP phosphodiesterase may be regulated by Ca2+/calmodulin. PMID- 3010623 TI - Characterization of immunoreactive corticotropin and corticotropin-releasing factor in human adrenal and ovarian tumours. AB - The distribution of immunoreactive ACTH (I-ACTH) and corticotropin-releasing factor (I-CRF) in the human adrenal was determined and these peptides were indentified in adrenocortical adenomas from patients with primary aldosteronism. Cushing's syndrome, and ovarian tumours and compared with the results in phaeochromocytomas. I-ACTH and I-CRF, mainly localized in the adrenal medulla from patients without endocrine disorders, showed a good correlation with the epinephrine concentrations. I-ACTH and I-CRF were present in all the above mentioned tumours. Gel filtration of I-ACTH and I-CRF from these tumours showed the presence of large molecular weight forms of these peptides as well as authentic peptides. The trypsinization study of I-CRF from phaeochromocytoma suggested that such large molecular weight forms were the precursors of authentic CRF. High performance liquid chromatography showed I-CRF in these tumours to be similar to hypothalamic CRF. Phaeochromocytoma tissue slices incorporated [3H]leucine into ACTH and CRF. These findings raised the possibility that I-ACTH and I-CRF are synthesized and processed in phaeochromocytoma. PMID- 3010624 TI - The role of phagosome formation in hydroxyl radical generation by human polymorphonuclear leukocytes: studies with normal and cytochalasin B-treated cell. PMID- 3010625 TI - Expression and regulation of transferrin receptors on human leukemic cells (HL 60) during cellular proliferation and differentiation. PMID- 3010626 TI - Comparison of midazolam (Ro 21-3981) and diazepam as complement of ketamine-air anesthesia in children. AB - A single-blind, randomized, prospective, comparative clinical trial was performed in 90 (30 X 3) pediatric patients, comparing a new water-soluble benzodiazepine, midazolam (Ro 21-3931) and ketamine-air (8 mg . kg-1) by intramuscular or intravenous routes at equipotent dose (0.2 mg . kg-1) with the standard anesthesia technique with diazepam (0.3 mg . kg-1) as an intravenous complement, during anesthesia for diagnostic urological procedures on an out-patient basis. Comparison between the three series of the mean values of maximal variation of hemodynamic parameters (systolic and diastolic blood pressures, and heart rate) during maintenance of anesthesia revealed no statistical significant difference in their values. Apnea was not observed in any patient. Local tolerance was excellent after i.v. administration, while good in the ketamine-midazolam i.m. series, 5 patients reported slight pain under pressure at the site of injection 24 hours after the end of anesthesia. No serious adverse reactions were observed in this clinical trial. We concluded that the efficacy of midazolam (Ro 21-3981) was excellent and comparable to that of the standard drug (diazepam), with the added advantage of better tolerance after both the intramuscular and intravenous administration, due to the rapid intramuscular absorption of midazolam, which starts simultaneously with that of ketamine. PMID- 3010627 TI - On-demand nalbuphine for post-operative pain relief. AB - Twenty-five patients with moderate to severe pain after major upper abdominal surgery chose to receive nalbuphine on demand from a Cardiff Palliator for pain relief. An initial i.v. injection of nalbuphine 20 mg was followed by 5 mg given over 90s in response to each successful demand. A maximum of 20 doses (100 mg) hr 1 of nalbuphine was available, plus additional bolus doses. Twelve patients obtained good pain relief and completed the 5 hour observation period. 13 patients withdrew from the study, 8 because of inadequate pain relief and 2 because of side-effects. At least half the patients who complained of inadequate pain relief at the time had no subsequent memory of the events. Despite high dosage in some cases (up to 200 mg in an hour) no clinically important cardiovascular or respiratory effects were observed. PMID- 3010628 TI - Retroviruses in Kaposi's sarcoma in acquired immune deficiency syndrome (AIDS). AB - The ultrastructure of Kaposi's sarcoma (KS) of the oral mucosa in patients with acquired immune deficiency syndrome (AIDS) was examined electron microscopically. The tumour consisted of pleomorphic vascular endothelial structures and spindle cell formations. The KS cells contained characteristically numerous multivesicular bodies, a large number of tubuloreticular structures and abundant Weibel-Palade bodies in their cytoplasm. Virus particles, 100-120 nm in diameter, were observed budding from the plasma membrane or as free particles already separated from the plasma membrane. Many mature virions manifested a dense cylindrical-shaped core. These virus particles and the human T-cell lymphotropic retroviruses subgroup HTLV-III are ultrastructurally identical. This report is based on recent immunological research. PMID- 3010629 TI - Vocal cord polyp with stromal atypia. A pseudosarcomatous lesion. AB - A large pedunculated vocal cord polyp containing numerous atypical stromal cells is described. The lesion was originally misinterpreted as malignant neoplasm. Follow-up data showed that the polyp was benign and adequately treated by local excision. PMID- 3010630 TI - [Nasopharyngeal fibroma. Apropos of 29 cases]. AB - This study is based on 29 juvenile angiofibroma, treated in 16 years. The average of age is 16.3 years but 24% of patients are over 20 years old. Scanner is the best mean to known the extension to the base of the skull and pterygoid fossa. Angiography is not yet necessary and may be dangerous. The surgical approach is transmaxillary in most cases, with a very low average of recurrence = 10% and 1 post operative death. PMID- 3010631 TI - An incidence peak of juvenile diabetes. Relation to Coxsackie B virus immune response. AB - All new cases of insulin dependent diabetes mellitus (IDDM) in children below 15 years of age were recorded prospectively during a 21-year period 1964-1984 in a defined uptake area with a relatively constant child population. The total number of children recorded was 222-111 boys and 111 girls. The number of new cases varied between 4 cases in 1968 and 20 in 1984; in 1983 seventeen new cases were recorded. Specific IgM antibodies against Coxsackie B virus (CBV), types 1-5 were measured by a reverse radioimmunoassay (RIA) technique in 24 consecutive patients collected during the period March 1982-January 1984, some of whom represented the recent period of a very high incidence of diabetes. Sixteen patients (67%) exhibited CBV IgM responses, strongly suggesting a current or recent CBV infection. The titres declined during the first few months of diabetes and seemed to be absent after the first half-year period. Among age-matched non-diabetic children scheduled for elective procedures during the same period, no cases with CBV-IgM antibodies were detected. Only three of the 16 IgM-RIA-positive patients showed a significant rise in the neutralising antibody titre against the same Coxsackie B type. It is concluded that CBV may play a pathogenetic role in induction of IDDM, and possibly more frequently so during periods with a high incidence of diabetes, at least in children below 15 years of age. PMID- 3010632 TI - Diet therapy for poorly controlled type 2 (non-insulin-dependent) diabetes mellitus. AB - Poorly controlled type 2 diabetes represents a major therapeutic problem. A classification of the disease, based on body weight and presence or absence of adequate insulin-secretion capacity, may be helpful in the choice of correct treatment. Calorie reduction is the most important therapeutic intervention in overweight patients. In the diabetic diet digestible carbohydrates should comprise at least 50 energy%, while the fat content should be reduced below 30 energy%. Controlled clinical studies show that the blood glucose control can be improved and the urinary glucose excretion be diminished by addition of dietary fibre. In obese type 2 diabetics supplemented fasting may be useful to achieve a rapid weight loss and an improved metabolic control. Although our knowledge with regard to the patho-physiology of type 2 diabetes and the effects of dietary treatment has increased during recent years, several important questions remain unanswered. Also there is a great need for education and training programmes to achieve improved compliance to the dietary advice given. PMID- 3010633 TI - Combined hepatocellular and mucinous carcinoma. AB - A rare autopsy case of combined liver cell and bile duct carcinoma (CLBC) occurring in a 51-year-old male with alcoholic liver cirrhosis is presented. Histologically, while the primary lesion was solely composed of well differentiated hepatocellular carcinoma (HCC), intrahepatic metastases consisted of a variable admixture of HCC and cholangiocarcinoma (CC) with excessive mucin production. Interestingly, the tumor cell cluster showing a trabecular growth pattern produced both bile and mucin, thus converting from HCC to mucinous CC. It is concluded that this liver malignancy is principally HCC with a marked tendency to transform into CC. The importance of the findings, especially the simultaneous production of bile and mucin within the same cell cluster, is emphasized in terms of the classification of CLBCs. PMID- 3010635 TI - Direct effects of ACTH on the ovary. AB - We have summarized the data of our former experiments which clearly demonstrate that ACTH increases ovarian blood flow in various mammals. Furthermore, when the ovary is primed with gonadotropic hormones it stimulates secretion of 17 beta oestradiol and progesterone. PMID- 3010634 TI - Structural and pharmacological differences between codfish and rat brain alpha 1 adrenergic receptors revealed by photoaffinity labeling with 125I-APDQ. AB - The photoaffinity probe 125I-APDQ has been used to characterize alpha 1-receptor peptides in the cod and rat brains. In the cod brain a major specific peptide of Mr = 68,000 could be covalently labeled by 125I-APDQ as revealed by SDS-PAGE. In the rat brain a specific peptide with Mr = 77,000 was instead labeled. When a number of adrenergic agonists and antagonists were tested for their ability to protect the labeling by 125I-APDQ their potencies were those expected for alpha 1 receptors in both species. The ligand binding peptide in the cod brain also distinguished between stereoisomers of epinephrine as expected for a physiological receptor. However, there was a distinct difference between the cod and rat alpha 1-receptor in that the beta-agonist 1-isoprenaline was equipotent to 1-norepinephrine in the cod whereas it was less potent in the rat. The protecting ability of the tested agents were also matched by their ability to displace the alpha 1-adrenergic ligand 3H-prazosin from alpha 1-receptor binding sites in brain membranes from both species. Thus, the codfish alpha 1-receptor seems to be different from mammalian alpha 1-receptors both structurally and pharmacologically. PMID- 3010636 TI - Haemodynamic effects of purinergic receptor stimulation in isolated fish gills. AB - The haemodynamic responses of the isolated, perfused gill of the tropical cichlid, Oreochromas (Sarotherodon) niloticus, to exogenous application of adenosine and adenosine triphosphate (ATP) showed that both drugs are potent vasoactive agents. However, while adenosine had vasoconstrictory effect, ATP induced vasodilation. ATP-induced vasodilation was reversibily inhibited in the presence of the enzyme, adenosine deaminase, and the P1 receptor antagonist, theophylline, but was unaffected by the P2 receptor antagonist, quinidine. This indicates that ATP acts specifically through P1 receptor stimulation. The significance of the inhibition of ATP by adenosine deaminase remains obscure since in purine metabolism the enzyme catalyzes the conversion of the haemodynamically active adenosine to inactive inosine. PMID- 3010638 TI - Cyclic AMP metabolism in the inner and outer layers of human myometrium near term. AB - The metabolism of cAMP which appears to be the intracellular mediator of various relaxing agents was studied in biopsies obtained during elective caesarean section from inner and outer myometrial layers outside the placental insertion. In the inner layer, L-epinephrine, PGE1, PGE2, PGF2 alpha and PGI2 stimulated the cAMP formation process while 6-keto PGF1 alpha was ineffective. The fact that some of these prostaglandins are well-known to promote contraction, confirms that the effects of drugs on uterine motility are not necessarily related to changes in the cAMP level. On the other hand, L-epinephrine and prostaglandins did not strongly influence the cAMP formation process in the outer layer. Kinetic analysis and purification assays of phosphodiesterase (PDE) which catalyzes the degradation of cAMP revealed the presence of multiple molecular forms of the enzyme in human pregnant myometrium. Qualitative and quantitative differences between the two layers appeared in the two forms separated from the soluble fraction by DEAE-cellulose chromatography. An unequal distribution of calmodulin was also observed in the inner and outer layers. Our results support the concept of the regulatory heterogeneity of the pregnant human uterus and suggest that the myometrial inner layer plays an important role in the regulation of uterine motility at the end of pregnancy. PMID- 3010637 TI - Can neonatal treatment with a related hormone adapt the receptor for itself? AB - Treatment of rats with endotoxin immediately after birth caused destruction of the cell membrane, resulting in depression of the thyroxin level and of the response to thyrotropin in adulthood. The thyroid gland of the rats treated neonatally with endotoxin failed to differentiate TSH from gonadotropin. Neonatal treatment (imprinting) with thyrotropin or gonadotropin after preexposure to the endotoxin improved the adult response to the exogenous hormone presented for imprinting after endotoxin. It appears that during the reconstruction stage following upon membrane perturbation in the critical period of receptor maturation, the adequate hormone or a related molecule can equally adapt the receptor for itself, but neither can fully compensate the perinatal membrane injury, nor the consequent diminution of receptor activity. PMID- 3010639 TI - Neuromodulation by adenine nucleotides, as indicated by experiments with inhibitors of nucleotide inactivation. AB - The adenine nucleotides AMP, ADP and ATP (3 X 10(-7) M and above) inhibited contractile responses to transmural nerve stimulation in guinea-pig ileum longitudinal muscle via a prejunctional action. Nucleotides assumed to inhibit the degradation of adenine nucleotides were employed to determine whether inhibition of contractile responses was elicited by adenine nucleotides per se, or required breakdown to adenosine. The IMP or 2'-deoxy AMP enhanced the prejunctional inhibitory effect elicited by AMP. A similar enhancement of the inhibitory effect of ADP and ATP was seen after administration of IDP and ITP, respectively. The inhibitory effect of adenosine was not enhanced by inosine, IMP or IDP. The 5'-nucleotidase inhibitor, TDP enhanced inhibition elicited by ADP. In contrast, alpha, beta-meADP did not influence the prejunctional inhibitory effect elicited by the adenine nucleotides. However, the combination of alpha, beta-meADP and IMP enhanced the inhibitory effect of ATP. The postjunctional contractile effect elicited by ADP and ATP was enhanced by pretreatment with inosine nucleotides, alpha, beta-meADP or TDP, indicating decreased inactivation of ADP and ATP during concurrent nucleotide administration. The fact that the prejunctional effect of adenine nucleotides can be enhanced by forms of pretreatment known to antagonize the breakdown of adenine nucleotides, constitutes strong evidence for prejunctional action per se by adenine nucleotides. PMID- 3010640 TI - Mechanisms underlying pre- and postjunctional effects of neuropeptide Y in sympathetic vascular control. AB - The effects of porcine neuropeptide Y (NPY) regarding sympathetic vascular control were studied in vitro on isolated rat blood vessels. The 10(-9)M NPY enhanced (about two-fold) the contractile responses to transmural nerve stimulation (TNS), noradrenaline (NA) and adrenaline (about two-fold) in the femoral artery. Higher concentrations of NPY (greater than 10(-8)M) caused an adrenoceptor-resistant contraction per se. The TNS-evoked [3H]NA efflux was significantly reduced by NPY in a concentration-dependent manner (threshold 10( 9)M). The calcium antagonist, nifedipine, abolished the contractile effects of NPY and the NPY-induced enhancement of NA contractions but did not influence the prejunctional inhibition of [3H]NA release. Receptor-binding studies showed that the ratio of alpha 1-to alpha 2-adrenoceptors in the femoral artery was 30:1. The NPY did not cause any detectable change in the number of alpha 1-or alpha 2 adrenoceptor binding sites or in the affinity of alpha 2-binding sites, as revealed by prazosin- and clonidine-binding, respectively. The NPY also inhibited the TNS-evoked [3H]NA release (by 42-86%) in the superior mesenteric and basilar arteries and in femoral and portal veins. The NPY still depressed TNS-evoked [3H]NA secretion from the portal vein in the presence of phentolamine. The NPY caused a clear-cut contraction in the basilar artery, increased the contractile force of spontaneous contractions in the portal vein, while only weak responses were observed in the superior mesenteric artery and femoral vein. The NA-induced contraction was markedly enhanced by NPY in the superior mesenteric artery, only slightly enhanced in the portal vein and uninfluenced in the femoral vein. In conclusion, in all blood vessels tested, NPY depresses the TNS-evoked [3H]NA secretion via a nifedipine-resistant action. Furthermore, NPY exerts a variable, Ca2+-dependent vasoconstrictor effect and enhancement of NA and TNS contractions. PMID- 3010641 TI - Possible involvement of the Ni-protein in the prejunctional inhibitory effect of a stable adenosine analogue (R-PIA) on noradrenaline release in the rat hippocampus. PMID- 3010643 TI - Reliability of record linkage in the Swedish Cancer-Environment Register. AB - The Swedish Cancer-Environment Register (CER) is intended to be used for studies on occupational cancer. CER was established by a computerized record linkage between the Swedish National Cancer Register (SCR) for 1961-1973 and the 1960 Population and Housing Census (PH 60) in order to obtain information on occupation, occupational status, economic activity (industry), place of work, domicile, etc. that might be useful for epidemiologic studies on cancer. Accurate inter-register linkage at individual level necessitates high quality of identification in both registers. The hit completeness in this respect was evaluated as 98.8 per cent, a figure regarded as acceptable for most studies based on the CER. Since a hit between SCR and PH 60 did not guarantee accuracy, the reliability of the record linkage was studied in random sample. With the aid of local and national population registries, 0.45 per cent (95% confidence interval 0.21-0.86%) false linkages were revealed in the sample. The CER was in 1982 supplemented with data notified to the SCR 1974-1979, and the hit completeness was 99.2 per cent for the total period. There are good grounds for assuming a lower rate of false linkages for the later period. PMID- 3010642 TI - Radiation biology of malignant melanoma. AB - The survival curves for melanoma cells exposed to single radiation doses in vitro and the specific growth delays for melanoma xenografts irradiated with single doses in vivo were found to differ considerably among individual cell lines and tumours. In fact, the differences could be almost as large as the largest differences observed among cell lines and xenografts from tumours of different histology with very different clinical radiocurability. Moreover, radiobiologic parameters that may have significant influence on tumour response to fractionated irradiation, e.g. growth rate, hypoxic fraction, reoxygenation ability, PLD repair capacity and contact repair capacity, were found to differ greatly in magnitude among individual melanomas. This review therefore concludes that malignant melanoma is a tumour type that is very heterogeneous in radioresponsiveness, i.e. malignant melanomas should no longer be considered to be radiation resistant in general. The values of the alpha/beta ratio derived from cell survival curves for melanoma cells irradiated in vitro and melanoma xenografts irradiated in vivo were found to cover a wide range relative to those for acutely and late responding normal tissues. Although these alpha/beta ratios are no more than estimates of the effective alpha/beta ratios in a clinical situation, they still indicated that hyperfractionation may be beneficial in the treatment of some melanomas, whereas others may be more efficiently treated by use of conventional fractionation regimes, either based on 2 Gy or higher doses per fraction. Consequently, optimum radiation therapy of malignant melanoma will probably require an individualized treatment strategy. In vitro assays for prediction of radiocurability and choice of treatment strategy for individual melanoma patients seem therefore highly warranted. PMID- 3010644 TI - Chest radiography in the follow-up of breast cancer. AB - The significance of routine chest radiography in the follow-up of breast cancer was evaluated. This evaluation also included correlation of symptoms with site of intrathoracic relapse. Ten per cent of the patients developed intrathoracic recurrence; 54 per cent thereof were asymptomatic. Eighty-six per cent of the intrathoracic relapses appeared within five years. The significance of prognostic factors was obvious: a higher stage or grade gave a higher risk of intrathoracic relapses. The presence of symptoms was dependent on the site of relapse. The disease-free survival and survival after recurrence were not significantly different in symptomatic and asymptomatic patients. The contribution of routine chest radiography to the detection of relapses in asymptomatic patients was poor: one positive finding among 263 chest radiographic examinations. Routine chest radiography should be limited to high risk groups (stage II, III and grade III), preferably within the first five years after the primary treatment. PMID- 3010645 TI - Energy and protein intake and nutritional status in non-surgically treated patients with small cell anaplastic carcinoma of the lung. AB - The spontaneous food intake and nutritional status was assessed in 23 patients with small cell anaplastic carcinoma of the lung before and two times during a treatment period of 6 weeks. Radiation therapy was given for 2 weeks followed by a course of chemotherapy and another 2 weeks of radiation therapy. The energy intake decreased during the treatment from 146 to 130 per cent of basal metabolic rate (p greater than 0.10). The protein intake remained unchanged (mean 0.9 g/kg body weight). There were insignificant and small losses of weight, body fat, free body mass and arm muscle circumference, and no changes were seen in serum albumin and serum transferrin. However, 6 patients suffered a weight loss of 5 per cent or more. No correlation existed between the nutritional parameters measured before treatment and the changes during treatment. Patients who suffered a loss of body weight could therefore not be singled out before the treatment. PMID- 3010646 TI - Radiation therapy of primary malignant lymphoma of the brain. AB - Primary malignant lymphoma of the brain (PMLB) is uncommon. Between 1975 and 1982, the authors observed 11 patients with histologically confirmed PMLB. Mean survival after radiation therapy was 7 months with 5 patients surviving for more than 2 years. Multifocal lesions were seen in 9 patients and spontaneous regression was seen at computed tomography in 2 patients. Radiation doses in excess of 30 Gy controlled the primary tumor, but intracranial recurrences occurred even after whole brain irradiation to 40 Gy. Only one patient had a relapse outside the central nervous system, and none had clinical evidence of seeding to the spinal canal. The authors postulate that PMLB usually is a multifocal intracranial disease, and that whole brain irradiation of at least 30 to 40 Gy should be given to all patients with this disease. PMID- 3010647 TI - Radiation treatment of testicular relapse in acute lymphoblastic leukemia. AB - Ten patients with testicular relapse among 128 cases of acute lymphoblastic leukemia are reported. At the time of the initial diagnosis of leukemia all patients with later testicular relapse showed one or more risk factors as predictive for leukemic infiltration of the testicles. All patients except one, who underwent orchiectomy and died 11 weeks after surgical intervention, received radiation therapy with doses ranging from 12 to 20 Gy and chemotherapy. The local control was excellent. Average survival time from testicular relapse to death was 68 weeks in 8 of 9 patients treated by irradiation and chemotherapy. One patient is still alive without signs of disease after 6 years. PMID- 3010649 TI - Interstitial 125I implantation in the retreatment of retroperitoneal soft tissue sarcoma. Report of a case. AB - Interstitial 125I was successfully used in the retreatment of a large recurrent malignant schwannoma in the retroperitoneum. After an average tumor dose of 160 Gy the tumor calcified and the patient is well, without disease 2 years later. PMID- 3010648 TI - Paternity after irradiation for testicular cancer. AB - According to the Medical Birth Registry (MBR) of Norway, 69 of about 430 patients irradiated for testicular cancer (stage I + II) during 1966-1978 fathered at least one child after radiation therapy (median observation time 136 months, range 36-191 months). A total of 95 children were born. Between 10 and 122 months elapsed between discontinuation of irradiation and the birth of the first child born after radiation therapy. Though the total doses to the abdominal irradiation field were higher in patients irradiated by a linear accelerator (1971-1978), than in those treated by a betatron (1966-1970), the gonadal doses were generally lower in the former group due to better gonadal shielding. In the children, the sex ratio and the median weight and length at birth were comparable to those values seen in a control group from the MBR. No increased frequency of malformations was observed. It is concluded that modern radiation therapy techniques allow post-irradiation fathership in a significant number of patients, without increased risk of neonatal problems or malformations in the children. PMID- 3010650 TI - Chordoma. Report on treatment results in eighteen cases. AB - Eighteen patients with a proven histologic diagnosis of chordoma were treated between 1949 and 1982. Four patients received only surgery, 4 patients only radiation therapy, and 10 patients received surgery and postoperative radiation therapy to a varying dose. The results suggest that a higher radiation dose gives longer recurrence-free survival, and that the best long term results can be achieved by combining surgery--as radically as possible--with radiation therapy to a dose level of 60 to 65 Gy. In view of the number of marginal recurrences (2 out of the 14 patients who received radiation therapy), the importance of choosing the right treatment volume is stressed. PMID- 3010651 TI - Early effects of preoperative irradiation upon the cell cycle composition in rectal adenocarcinomas. A flow-cytometric DNA investigation. AB - The DNA patterns were studied by means of flow cytometric analysis in 43 rectal adenocarcinomas. Ploidy level and cell cycle distribution were related to clinical stage and histopathology. The frequency of grossly aneuploid tumours and tumours with multiple aneuploid cell populations increased with more advanced clinical stages and with the degree of dedifferentiation. In 15 cases the DNA pattern was studied before and after preoperative irradiation. The ploidy level was not affected by irradiation. A pronounced increase in the proportion of G2 cells was found after irradiation. This G2 blockage was proportional to the amount of S-phase cells before irradiation. Since following irradiation the proportion of S-phase cells was low and the proportion of G1 cells unchanged, the existence of a high fraction of resting G1 tumour cells can be assumed. PMID- 3010652 TI - Cell proliferation and differentiation in the small intestine after irradiation with multiple fractions. AB - Qualitative and quantitative morphologic changes in rat small intestine were studied after abdominal exposure to multiple fractions of gamma radiation. One group of animals received 3 X 2 Gy with one fraction every 4 hours. Another group received two courses of this type with a 16 hour interval between the courses (total dose 6 X 2 Gy). A marked decrease in the number of crypt epithelial cells, and in mitotic and labelling indices, was observed up to 24 to 36 hours after the end of both regimens. Repair and recovery occurred within 72 hours after the end of the last exposure, and the epithelium regained normal morphology. At 1 and 4 hours after the end of the treatment the frequency of S-phase cells along the crypt was greatly reduced and at the following intervals labelled cells occupied the region where differentiation occurs in control animals. During recovery labelled cell distribution showed a gradual return to normal. No substantial differences between the effects of total doses of 6 and 12 Gy were shown except for a greater reduction in crypt epithelial cells at the early time intervals after the larger dose. PMID- 3010653 TI - Provoked repetitive healing of mature bone tissue following irradiation. A quantitative investigation. AB - A titanium implant, the bone harvest chamber (BHC), was used to investigate the regenerative capacity of mature bone after irradiation. One BHC was inserted in each proximal tibial metaphysis of a rabbit. One of these implant sites was irradiated (60Co single dose) to either 15 or 25 Gy while the other served as control. Newly formed bone grew through a canal that penetrated the implant. This newly formed bone was harvested from the implant every three weeks following irradiation and then quantified by microradiography and computer-assisted densitometry. In this way a ratio between bone formed on the irradiated side in comparison with the control could be established. An immediate depression in bone formation compared with the non-irradiated controls, was seen at both dose levels. A recovery in bone regenerative capacity was seen at 15 weeks after 15 Gy while the decrease in bone formation remained constant after 25 Gy during the 30 week follow-up period. PMID- 3010654 TI - Changes in energy metabolism following roentgen irradiation of in vivo growing Ehrlich ascites tumour cells studied by 31P magnetic resonance spectroscopy. AB - The energy metabolism in Ehrlich ascites tumour cells following in vivo irradiation of a dose of 5.0 Gy was studied in vitro in their ascites fluid up to 48 hours using 31P magnetic resonance spectroscopy measuring ATP, ADP and inorganic phosphate (Pi). The results are also related to radiation induced changes in cell cycle composition. ATP was reduced by more than 50 per cent 20 to 24 hours after irradiation but normalized at 48 hours. ADP was reduced to about half the normal level 24 to 48 hours after irradiation. When the ATP and ADP had reduced levels, the inorganic phosphate increased correspondingly. Addition of glucose to the ascites cell suspension at the time of minimum ATP level immediately raised the ATP:Pi ratio. Since the glucose concentrations in blood and in ascites fluid following irradiation were also reduced, lack of glucose for energy production might have been a major contributing factor for the reduced ATP production. PMID- 3010656 TI - Partial prevention of tritium induced uterine involution in mice by 2 mercaptopropionylglycine. AB - Pregnant Swiss albino mice were given on day 11.25 post conception a priming intraperitoneal (i.p.) injection of tritiated water at the activity levels 37, 74 or 185 kBq/ml body water, in the absence (control) or presence (experimental) of 2-mercaptopropionylglycine (MPG), 20 mg/kg body weight, given intraperitoneally 30 minutes before the tritium administration. The females were subsequently maintained on tritiated drinking water until term, at the above activity level, in the control series. The animals of the experimental series received in addition a daily i.p. injection of MPG at the same time of the day, until term. A third series received a daily injection of the drug, but no tritium, at the same dose rate. None of the females from the control series had parturition, and a gradual decline in their weight was recorded, exhibiting resorption. Treatment with MPG led to an obvious increase in embryonic survival in all groups, and even in the 185 kBq group two-thirds of the females had parturition. PMID- 3010655 TI - Effect of lactate on the recovery of CHO-KI cells from gamma radiation damage. AB - The effect of pre-exposure of CHO-KI cells to lactate on their capacity to recover from or repair radiation damage was examined. Prolonged pre-exposure to lactate and a number of inhibitors of glycolysis has previously been shown to reduce the cell survival after irradiation. Following a split dose of irradiation, cells pre-treated with lactate for 18 hours had a higher relative recovery factor than controls even though survival following a single dose was reduced. Where lactate was added just before irradiation it had a radiation protective effect on the single dose survival curve and no effect on recovery. To investigate a possible involvement of NADH generation via lactate dehydrogenase in the mechanism, oxamate, a non-metabolisable analogue of lactate, was investigated. It reduced the survival of irradiated CHO-KI cells following prolonged and short pre-exposure and did not significantly affect the recovery factor. It is suggested that since lactate and oxamate both inhibit glucose breakdown, their radiobiologic effects may be due to depletion of cellular energy substrates necessary for repair. In contrast, the increased recovery and short term radiation protective effect seen with lactate only may involve LDH activity. PMID- 3010658 TI - Microsurgery of gliomas. AB - The author describes his microsurgical operative technique used since 1980 for gliomatous tumours. Instead of extensive resection and lobectomy, a pergyral or intergyral persulcal approach with partial gyrectomy, interhemispheric, transsylvian and transventricular exposure of the tumour surface were used. The resection of the tumour begins from its centre. In the first phase 1980-1982 bipolar coagulation, micro-sucker and pincer were used, since 1983 tumour resections have been performed with the CO2 and Nd-Yag laser and CUSA. Tumours located in functionally important regions such as the speech area, thalamus, brain stem, etc. could be removed without additional morbidity and there was a rapid improvement in neurological deficits. The early prognosis of patients harbouring these tumours is improved thanks to minimized operative trauma. The quality of life during the recurrence free period is improved and surgery of recurrence is indicated more frequently than in the past. There is no evidence that these techniques influence the length of the total survival. The use of CT and MRI improved the early diagnosis of small tumours and intraparenchymal lesions. This requires exact intraoperative localization and identification of the lesion. The technical aspects of these procedures are described. Thanks to the improvement in operative technique some limitations of surgery such as location, nature of the tumour and the age of the patient have lost much of their importance. PMID- 3010657 TI - Late development of the medio-basal hypothalamic neuronal network around puberty. Some histochemical studies in the rat. PMID- 3010659 TI - Biophysics of left-handed Z-DNA. AB - Substantial new information in the past several years has been reported on different types of DNA structures and the possible biological consequences of these conformations. Detailed X-ray crystallographic analyses have provided a good understanding of several types of DNA structures. Biochemical studies demonstrated the concept of DNA microheterogeneity, i.e., the realization that more than one conformation can exist in a given DNA molecule. This mandates the presence of conformational junctions. Most cellular DNA is believed to exist in a right-handed structure. However, left-handed helices are stabilized by supercoiling. The energetics of the transition reveal a delicate balance between right-handed structures and left-handed helices. Several types of sequences adopt left-handed structures and several of these are known to serve as recombination hot spots. The Hha I restriction enzyme and methylase do not act on left-handed DNA, thus these agents may be used as probes for left-handed helices within suitable sequences. Furthermore, B-Z-DNA junctions contain few if any, non-paired bases at physiological superhelical densities. PMID- 3010660 TI - Sequence dependent DNA conformations: Raman spectroscopic studies and a model of action of restriction enzymes. AB - Raman spectra have been examined to clarify the polymorphic forms of DNA, A, B, and Z forms. From an analysis we found that the guanine ring breathing vibration is sensitive to its local conformation. Examination of nine crystals of guanosine residues in which the local conformations are well established revealed that a guanosine residue with a C3'endo-anti gives a strong line at 666 +/- 2 cm-1, O4'endo-anti at 682 cm-1, C1'exo-anti at 673 cm-1, C2'endo-anti at 677 cm-1 and syn-forms around 625 cm-1. Using this characteristic line, we were able to obtain the local conformations of guanosine moieties in poly(dG-dC). Such a sequence derived variation is suggested to be recognized by sequence specific proteins such as restriction enzymes. We found a correlation between sequence dependent DNA conformation and a mode of action of restriction enzymes. The cutting mode of restriction enzymes is classified into three groups. The classification of whether the products have blunt ends, two-base-long cohesive ends, or four-base long cohesive ends depends primarily on the substrate, not on the enzyme. It is suggested that sequence dependent DNA conformation causes such a classification by the use of the Calladine-Dickerson analysis. In the recognition of restriction enzymes, the methyl group in a certain sequence is considered to play an important role by changing the local conformation of DNA. PMID- 3010661 TI - Computer simulations of complex chemical systems. AB - In this paper, after a brief description on our approach to simulations of chemical systems, some of the results obtained are discussed. Four examples are reported: 1) liquid water simulation which takes in account a four-body potential; 2) hydration networks in a crystal; 3) water and ion structures in DNA; 4) proton tunneling in DNA base pairs. We include also a short description of a parallel system we have assembled. PMID- 3010662 TI - Agonist-induced inositol phospholipid metabolism and Ca++ flux in human platelet activation. PMID- 3010663 TI - Control and interrelation of aggregation and secretion; the roles of Ca2+, diacylglycerol and thromboxane with particular reference to ADP stimulation. PMID- 3010665 TI - Permeabilized platelets and exocytosis. PMID- 3010664 TI - Platelet receptors for thrombin. PMID- 3010666 TI - Energy requirements for stimulus-response coupling. PMID- 3010667 TI - Platelet protein phosphorylation. AB - As can be seen from this review, protein phosphorylation appears involved in both positive and negative regulation of platelets. To date, good evidence has been presented for the involvement of protein phosphorylation in the regulation of granule centralization (i.e. myosin light chain phosphorylation). It is probable that protein phosphorylation may also be involved in granule labilization, pseudopod formation and ATP synthesis. Protein phosphorylation in association with platelet activation appears mediated through calcium flux, in the case of myosin light chain phosphorylation, and through diglyceride or other substances in the case of 47P phosphorylation. A summary scheme is shown in Figure 1. PMID- 3010668 TI - Receptor-effector coupling in platelets: roles of guanine nucleotides. AB - Platelet-activating factor (PAF), which is thought to cause platelet aggregation and degranulation via a receptor-mediated activation of phospholipase C, had no direct action on PGE1-stimulated cyclic AMP formation in intact human platelets, although it caused a GTP and Na+-dependent inhibition of the adenylate cyclase activity of human platelet particulate fractions. Studies with PAF analogues indicated that the receptors mediating this inhibition of adenylate cyclase had structural specificity very similar or identical to that of the receptors mediating platelet aggregation. These results suggest that the PAF receptors linked to the activation of phospholipase C in intact platelets may, in membrane preparations, become coupled to the inhibition of adenylate cyclase via the guanine nucleotide-binding protein, Gi. Studies with permeabilized human platelets that secrete 5-HT on addition of low concentrations of Ca2+ showed that addition of either PAF or a guanine nucleotide decreased the Ca2+ required for secretion. When added together, PAF and GTP promoted secretion synergistically at low Ca2+ concentrations. Enhanced secretion of 5-HT was associated with increased formation of diacylglycerol. These results show that PAF can stimulate phospholipase C by both GTP-dependent and independent mechanisms. In intact human platelets, PAF receptors may interact preferentially with a guanine nucleotide binding protein that promotes phosphoinositide breakdown by phospholipase C, rather than with Gi. PMID- 3010670 TI - Characterisation of ADP receptors. PMID- 3010669 TI - Regulation of platelet phospholipid metabolism. PMID- 3010671 TI - Biological actions of prostacyclin and its pharmacological use in platelet studies. PMID- 3010672 TI - Fibrinogen and platelet function. PMID- 3010673 TI - Characterisation of factor VIII receptors. PMID- 3010674 TI - Characterization of thromboxane receptors in human platelets. PMID- 3010675 TI - Alterations of GABA-mediated synaptic transmission in human epilepsy. AB - Although animal models consistently indicate that gamma-amino-butyric acid (GABA) synaptic function (GABA levels, synthesis, uptake and/or receptors) is decreased in seizure states, there is little evidence to date in support of such a hypothesis for human epilepsy. This chapter presents the results of an in-depth study of the activity of the GABA-synthesizing enzyme L-glutamic acid decarboxylase (GAD) in brain tissue removed during neurosurgical resection for intractable epilepsy. The tissue studied is unique in that identified (by stereo EEG) foci were excised (rather than large blocks of tissue containing mixtures of foci and nonepileptic material) and compared with nonepileptic (stereo EEG and morphological definitions) tissue from the same patients. In patients in which there was no indication of a tumor, GAD activity in the foci was low in more than 50% of the patients examined. Furthermore, when the population distribution of GAD was compared in epileptic versus nonepileptic tissue fragments from all patients, the peak distribution of epileptic tissue fragments occurred at much lower GAD activities than for the nonepileptic fragments (0-20 versus 41-80 nmol CO2/mg protein X hr, respectively). A small subgroup of epileptic fragments occurred with a normal GAD distribution, indicating that the presence of an epileptic focus was not invariably associated with low GAD activity. When the low levels of GABA "A" binding sites in these epileptic tissue fragments are taken into consideration in combination with the low GAD levels, then it can be estimated that 60 to 70% of the present patient population had deficient GABAergic transmission in epileptic foci as compared to nonepileptic brain tissue from the same patients. It follows that the GABA hypothesis of human epilepsy is not an exclusive or unitary hypothesis, and some patients appear to have normally functioning GABA synapses (as assessed biochemically) in epileptogenic areas. Thus, other neurotransmitter and neurohumoral systems certainly play a role in the epileptic process. PMID- 3010676 TI - Role of the substantia nigra in GABA-mediated anticonvulsant actions. AB - The relationship between cerebral GABA content and susceptibility to seizures is addressed from the point of view of specific brain loci at which GABA synapses may control convulsive activity. The substantia nigra (SN) has been identified as a critical site at which GABA-agonist drugs act to reduce susceptibility to a number of types of experimentally induced generalized seizures. Moreover, the ability of GABA-elevating agents to protect against seizures in the maximal electroshock model is directly correlated with increases in GABA specifically in the nerve-terminal compartment of SN. Studies with 2-deoxyglucose indicate that a marked increase in metabolic activity in SN is a common feature of several types of generalized seizures; it is possible that some of this increased activity is associated with GABAergic nerve terminals that become activated in an attempt to suppress seizure spread. Because GABA has been shown to inhibit nigral efferents, it is likely that GABA terminals inhibit nigral projections that are permissive or facilitative to seizure propagation. In support of this, bilateral destruction of SN attenuated clonic and tonic chemoconvulsant and electroshock seizures. Other treatments capable of reducing nigral output, namely opiate agonists (morphine and D-Ala-Met-enkephalin), and substance P antagonist analogs, were also found to have anticonvulsant effects when applied bilaterally into SN. Thus, the seizure-facilitating nigral efferents may be subject to inhibition by both GABA and opiates and may normally be driven by substance P. Of the various outputs from SN, the GABAergic projections to thalamus, reticular formation and/or superior colliculus are most likely responsible for influencing seizure propagation. Experimental evidence does not indicate a significant role of pars compacta nigrostriatal dopamine neurons for controlling the various types of seizures subject to nigral influence. We propose that the inhibition of the GABAergic outputs from SN pars reticulata can suppress the progression of seizure discharge through circuits involving the target areas of these outputs. Because chemical or electrical stimulation of SN does not initiate convulsions, it appears that seizure activity generated elsewhere in the brain may be amplified or sustained by activity in these nigral outputs. PMID- 3010678 TI - GABA receptors, lipids, and gangliosides in cobalt epileptic focus. AB - The seizure state induced in the rat by cerebral cortical implantation of cobalt metal has been increasingly used to study a variety of neurochemical parameters. This experimental model of epilepsy affords the opportunity to study events prior to the development of seizures, during the period of intense seizure activity, and during the period when seizure activity has essentially terminated. The seizures that occur in this model are intermittent and paroxysmal and share many other similarities with human epilepsy. The crucial question with this model, and indeed with any experimental model of epilepsy, is whether the basic seizure producing mechanism(s) is similar. This question remains to be answered. There have been studies that show that changes in certain neurochemical parameters parallel the onset, intensity, and decline of seizure activity in cobalt epileptic animals. Although extremely interesting, by themselves they do not prove a cause-and-effect relationship. Such parallelism is more apparent in the case of GABA than in the cases of lipids and gangliosides. GABA and its synthetic enzyme, glutamic acid decarboxylase (GAD), are both at normal levels prior to the development of seizures, are significantly decreased during the period of seizures, and return toward control values at a time when seizures are no longer apparent. On the other hand, there is no change in postsynaptic GABA binding sites (Bmax) prior to seizures, a significant increase in Bmax during seizure activity, and a return toward normal by 21 days (when seizure activity has terminated). Studies that have been carried out on lipids and gangliosides in cobalt-induced epilepsy are not nearly as extensive nor are the results as positive as those that have been obtained in the case of GABA. They do, however, provide provocative findings that may well be related to the genesis of epilepsy. PMID- 3010677 TI - Benzodiazepine/barbiturate/GABA receptor-chloride ionophore complex in a genetic model for generalized epilepsy. AB - The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) acts through postsynaptic receptor sites which regulate membrane chloride ion channels. The GABA receptor-ionophore complex also contains modulatory receptor sites for two classes of centrally acting drugs, one for the benzodiazepines, and a second for both barbiturates and related depressants and for picrotoxin and related convulsants. The presence of these drug modulatory sites, directly on the GABA receptor protein, is consistent with other experimental observations; blocking GABA function can cause seizures, and augmenting GABA function can afford protection against seizures. This, and other circumstantial evidence, has suggested the possibility that a functional GABA deficit may be involved in some kinds of human epilepsy. Some neurochemical markers for GABA synapses have been reported to be altered in certain animal models as well as in human temporal lobe epilepsy. We have examined the postsynaptic GABA receptor complex using receptor binding assays for GABA, benzodiazepine (BZ), and barbiturate receptor sites in the seizure-susceptible gerbil, a genetic model of generalized epilepsy. A 30% deficit in BZ receptor binding was observed in the midbrain of seizure-sensitive animals relative to normal controls. This was shown by quantitative brain-slice binding autoradiography to involve a decrease in the number of binding sites in the substantia nigra (SN) and periaqueductal gray regions. A deficit in membrane receptors for BZs (which are linked to a subtype of postsynaptic GABA receptors) in a crucial region of brain might therefore contribute to seizure susceptibility in some kinds of epilepsy. PMID- 3010679 TI - Long-term potentiation and kindling: similar biochemical mechanisms? AB - For years, the hypotheses concerning the physiological mechanisms of epilepsy and spreading depression have implicated failures in inhibitory mechanisms and, in particular, GABA-mediated responses. More recent experiments have focused on the participation of excitatory neurotransmitters and especially on glutamate mediated responses in order to account for the long-lasting changes in the excitability of neurons found in epilepsy. Evidence supporting this view has been provided by the fact that two different types of manipulation resulting in long lasting changes in synaptic excitability, namely kindling and long-term potentiation (LTP) of synaptic transmission, result in modification of excitatory amino acid receptors. Kindling represents the progressive development of generalized seizures generated by repeated low levels of electrical stimulation of various limbic structures, and is generally accepted as a good model of epilepsy; it is associated with an increase in excitatory mechanisms and, in particular, with an increase in the number of glutamate binding sites that are presumed to represent a category of glutamate receptors. Similarly, LTP is elicited by brief bursts of electrical stimulation in monosynaptic excitatory pathways and is also associated with an increase in the number of the same type of glutamate binding sites. The present review compares the similarities between these two long-lasting forms of synaptic plasticity, and proposes that similar biochemical mechanism might underlie the changes in glutamate receptors. In addition, it describes a molecular mechanism that involves a calcium-dependent protease associated with postsynaptic membranes, the activation of which results in the unmasking of glutamate receptors. Moreover, since this mechanism has been recently implicated in the storage of some types of information in the mammalian telencephalon, these studies raise the possibility that epilepsy may represent a dangerous side-effect of an efficacious learning mechanism. PMID- 3010681 TI - Role of noradrenergic ascending system in extinction of epileptic phenomena. AB - This chapter reviews results which show that in electroshock-induced and chemically induced convulsions, audiogenic seizures of genetic epilepsy-prone rats and mice, in the kindling focus and the cobalt lesion seizure, susceptibility is modulated by a noradrenergic mechanism. In general, mechanisms that increase or decrease norepinephrine activities decrease or increase seizures, respectively, in these models. In the kindling phenomenon, since the seizure itself provokes an increase in norepinephrine (NE) turnover, with decreased beta-adrenoceptor binding and hyposensitivity to ionophoretic catecholamine application, down regulation could be the cause of hyperexcitability or a consequence of it. In the cobalt focus, supersensitivity to NE appeared when NE-containing terminal density decreased (denervation supersensitivity) and beta-receptor sites increased greater than 50%. Perfusion experiments with NE support the hypothesis that the cortical NE system inhibits the spread of chronic epileptogenic activities in the cobalt focus. In the quaking mouse and in the tottering mouse, noradrenergic dysfunction underlying epileptogenesis may be expressed as a hyperinnervation. PMID- 3010680 TI - A molecular approach to the calcium signal in brain: relationship to synaptic modulation and seizure discharge. AB - The synapse is a major regulatory site that has been implicated in modulating neuronal excitability and seizure discharge. Voltage-dependent calcium (Ca2+) entry at the synapse plays a major role in initiating neurotransmitter release and in regulating synaptic function. Thus, obtaining a molecular understanding of the effects of Ca2+ on synaptic modulation would provide important insights into the regulation of synaptic activity and, possibly, the biochemical basis for some forms of epilepsy. Calmodulin is a major Ca2+-binding protein in brain that has been implicated in mediating many of the second messenger effects of Ca2+ on neuronal function. The evidence implicating calmodulin in modulating synaptic excitability will be presented. Calmodulin was shown to be present at the synapse in association with synaptic vesicles and in the postsynaptic density. In addition, several calmodulin-regulated synaptic biochemical processes have been identified, including Ca2+- and calmodulin-regulated protein phosphorylation, vesicular neurotransmitter release, vesicle-membrane interactions, and neurotransmitter turnover. These results indicate that calmodulin may play an important role in synaptic modulation and provide a molecular approach to investigating the Ca2+ signal in brain. Several anticonvulsants have been shown to regulate some of calcium's effects on neuronal function. These anticonvulsants include phenytoin, carbamazepine, and the benzodiazepines. All of these compounds are effective against maximal electric shock (MES) seizure models in animals. Anticonvulsants were tested on several of the Ca2+-calmodulin-regulated synaptic biochemical systems. The results demonstrate that phenytoin, carbamazepine, and the benzodiazepines were effective in inhibiting calcium calmodulin protein kinase activity in membrane and purified kinase preparations, vesicle neurotransmitter release, vesicle-membrane interactions, and voltage-sensitive calcium uptake in intact synaptosomes. Phenobarbital, ethosuximide, trimethadione, valproic acid, and vinyl gamma-aminobutyric acid (GABA) were not effective in inhibiting these calcium-regulated processes. Thus, the effects of anticonvulsants on calcium-regulated processes were selective to a group of anticonvulsants that had been shown in several electrophysiological systems to antagonize some of the actions of calcium on neuronal excitability. These observations suggested the existence of specific membrane receptors that might mediate the effects of these anticonvulsants on neuronal function through the regulation of calcium-calmodulin-regulated processes.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3010682 TI - Looking for epilepsy genes: clinical and molecular genetic studies. AB - The complexity of the human genome creates special problems in understanding the genetic component of disease processes. An estimated 50,000 genes exist in the human genome, and it is reasonable to assume that mutation in any one of these genes may result in an inherited disorder. Because of the complex pattern of gene expression controlling the development and organization of the central nervous system (CNS), insights into the genetic component, if any, of diseases such as epilepsy are most accessible to analysis by genetic linkage studies. Advances in the manipulation of DNA have made possible more effective acquisition of genotypic information in humans by studying the inheritance of restriction fragment length polymorphisms (RFLPs) using cloned DNA probes. Two approaches exist to utilize this technology in studying inherited disorders. The first approach consists of genotypic determinations in affected families with cloned genes in which a mutation might result in the phenotype observed. Analysis of these data will show whether the inheritance of an allele of the candidate gene is linked to the disease. The second approach relies upon the construction with these probes of a linkage map for the human genome such that disease families can be screened in order to determine with which of these markers the phenotype is linked, indicating the map position of a gene associated with the inherited disorder. The use of these new approaches enables investigators to screen either specific biochemical defects in disease families or to identify the underlying genetic mechanisms in inherited disorders whose phenotype is expressed only in the intact human (84). The first step in localizing the chromosomal site of specific epilepsies is to define their pattern of inheritance. This determination is now being carried out for benign juvenile myoclonic epilepsy; 50 multigenerational families are being studied in three separate epilepsy programs in Los Angeles, Winston-Salem, North Carolina, and Berlin. Concurrent with these studies, investigators are combining the principles of classic linkage analysis, using 30 protein markers, with the use of RFLPs to determine the chromosomal location of juvenile myoclonic epilepsy. Two problems appear formidable, however. First, since the chromosomal location of specific epilepsies is unknown, the entire human genome must be screened.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3010683 TI - The accumulation of free arachidonic acid, diacylglycerols, prostaglandins, and lipoxygenase reaction products in the brain during experimental epilepsy. AB - There has been increasing biochemical evidence since 1970 that one of the targets for convulsion-induced changes is the cell membrane of neurons. This is partly based on the observation that following seizures, there are increased levels of diacylglycerols and free fatty acids, which are products of the degradation of the major component of cell membranes, phospholipids. In addition, the production of prostaglandins from the free fatty acid, arachidonic acid, is activated after convulsions. This implies that alterations in the metabolism of lipids in brain are a major effect of seizures, and that the further study of these biochemical pathways may reveal important information pertinent to defining the basic mechanism of seizures and seizure-related pathology and may help in the development of potentially effective treatments. The effects of seizures on brain lipid metabolism and some recent studies from our laboratory are described in this chapter. Our results demonstrate that in rat brain, dexamethasone--a phospholipase A2 inhibitor--attenuates bicuculline-induced free fatty acid accumulation in a dose-dependent manner; bicuculline-induced status epilepticus does not alter the activation (synthesis of arachidonoyl coenzyme A) or acylation of fatty acids as assayed in vitro, indicating that the availability of high energy cofactors (ATP) may be the critical factor responsible for decreased fatty acid acylation in vivo; bicuculline-induced fatty acid accumulation is localized mainly in the synaptosomal fraction of the rat brain; induction of seizures in the rat by bicuculline treatment produces a marked stimulation of lipoxygenase activity in synaptosomes that, in turn, results in a large increase in the synthesis of hydroxyeicosatetraenoic acids (HETEs). This effect is also observed following membrane depolarization with 45 mM K+, and bicuculline-induced status epilepticus stimulates the synthesis of prostaglandin D2. Possible mechanisms and consequences of alterations in specific lipids are described. Also, the possible involvement of a stimulated arachidonic acid cascade, particularly of hydroxylated products, in the release of neurotransmitters is discussed. Other aspects of the interaction between neurotransmission and the production of eicosanoids are reviewed. The metabolic pathways leading to the "lipid effect"- i.e., the production of free fatty acids, diacylglycerols, and arachidonic acid metabolites (eicosanoids)--are numerous and involve a wide variety of enzymes. The mechanism of this "lipid effect" may involve a seizure-induced overstimulation of normal lipid pathways that operate in neurotransmission.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3010684 TI - CT of adenoid cystic carcinoma of the trachea. AB - The CT features of six cases of adenoid cystic carcinoma of the trachea are presented and compared to the bronchoscopic, surgical, and pathologic findings. CT accurately demonstrated extratracheal extension of tumor, which occurred in all six cases and which was not visible on standard chest radiographs. This information was helpful in planning the surgical approach. However, CT consistently underestimated the longitudinal extension of the lesion because of partial volume averaging and the tendency of adenoid cystic carcinoma to grow submucosally. CT was also a poor predictor of mediastinal organ invasion. Because recent advances in tracheal resection and carinal reconstruction have made many of these lesions resectable, CT is frequently used in the evaluation of the operability of patients with adenoid cystic carcinoma. However, its usefulness in this regard is limited, and conventional techniques such as standard tomography continue to play a role in preoperative assessment. PMID- 3010685 TI - Cytomegalic inclusion virus encephalitis in patients with AIDS: CT, clinical, and pathologic correlation. AB - The computed tomographic (CT) scans of 10 patients with acquired immunodeficiency syndrome who had central nervous system (CNS) involvement by cytomegalovirus (CMV) were retrospectively reviewed and correlated with clinical data and pathologic findings. Diagnosis was established in all 10 patients by autopsy, which showed the pathognomonic "owl's eye" intracellular inclusions of CMV. In six patients CMV caused an initial CNS infection that was directly responsible for the patient's progressive encephalopathy and death. In four patients CMV caused a superimposed nondominant CNS infection that had no clinical expression in two. Cortical atrophy and mild hydrocephalus ex vacuo were seen on CT in all 10 patients. Positive findings on CT that could be attributed to infection with CMV were present in only three of the 10 patients, and in these three symptomatic cases autopsy correlation revealed that CT underestimated the degree of CNS involvement. In the other three symptomatic patients, CT showed no parenchymal abnormalities, while autopsy demonstrated diffuse cerebral involvement. In the four patients whose CNS was secondarily involved by CMV, CT showed changes proven at autopsy to be related only to the dominant infection with Toxoplasma gondii and to postoperative hematomas. CT did not demonstrate any abnormalities at the sites of CMV involvement, which were found at autopsy in this latter group. It was concluded that CT is not very sensitive for the detection of CMV encephalitis. PMID- 3010686 TI - Pituitary and adrenal CT of Cushing syndrome. AB - Determination of the site of excessive hormone production in Cushing syndrome is possible with biochemical tests in 80% of cases. High-resolution CT of both the pituitary and adrenal glands was used to evaluate eight patients with surgically verified ACTH-secreting pituitary microadenomas and one patient with ectopic Cushing syndrome. Three ACTH-secreting microadenomas were demonstrated by CT. Adrenal CT was normal in six of the eight patients with pituitary tumors. The patient with ectopic ACTH production had mild unilateral adrenal gland enlargement and a normal pituitary CT scan. Normal adrenal or pituitary CT scans do not exclude Cushing syndrome. PMID- 3010687 TI - Cranial CT findings in sclerosteosis. AB - Sclerosteosis or Van Buchem's disease is a rare genetic craniotubular hyperostosis that becomes evident in early childhood and is associated with progressive involvement of the skull. The pathologic changes in the cranium noted on CT are described in three cases. Although the disease is incurable, CT is useful to display the morbid anatomy of the cranium before palliative surgery. PMID- 3010688 TI - Evaluation of the thyroid nodule. AB - Evaluation of thyroid nodules challenges the most astute clinician. The history and the physical examination often identify those patients who require immediate surgical management. In other patients, time-honored thyroid function studies and thyroid scanning are helpful. Fine needle aspiration and computed tomography are also valuable in the diagnostic work-up. PMID- 3010689 TI - The Gorlin syndrome: a genetically determined disorder associated with cardiac tumor. PMID- 3010691 TI - Laboratory evaluation of silica gel sorbent tubes for sampling hydrogen fluoride. AB - The NIOSH sampling and analytical method for inorganic acids employs a silica gel sorbent tube for the collection of five common inorganic acids with simultaneous determination of these acids in a single sample by ion chromatography. When the method was extended to the determination of hydrogen fluoride (HF) the sampled HF reacted with the silica gel and glass fiber of the sampler, but the reaction products remained trapped on the sorbent. Silica gel samplers were evaluated for the collection of HF from laboratory generated atmospheres. Factors tested included capacity, storage stability, humidity, accuracy and precision. Based on comparison with impinger collection, the mean recovery of HF from silica gel tube samples was 100.7% with a precision of 0.144. PMID- 3010690 TI - The assessment of contractile reserve after thrombolytic therapy for acute myocardial infarction. AB - "Stunned" myocardium prevents the assessment of myocardial salvage after streptokinase. In order to unmask "stunning," we sought to evaluate left ventricular inotropic contractile reserve of patients after streptokinase. Radionuclide ventriculograms were obtained in 75 consecutive patients 2 weeks after myocardial infarction, at rest and during intravenous isoproterenol infusion. Resting and isoproterenol-stressed ejection fractions were compared in the patent and closed-infarct vessel groups. Although there was no difference in the resting ejection fractions between the patent group (0.48 +/- 0.02) and the closed group (0.48 +/- 0.02), isoproterenol increased the ejection fractions in the patent group (increase 0.14 +/- 0.01) significantly more than in the closed group (increase 0.06 +/- 0.01) (p less than 0.0001). Thus, despite identical resting ventricular function, the greater inotropic contractile reserve in the patent infarct vessel group suggests that restoration of blood flow in acute myocardial infarction salvages myocardium. PMID- 3010692 TI - In support of cardiac chronotropic beta 2 adrenoceptors. AB - The effects of atenolol (50 mg) and propranolol (40 mg) on exercise- and isoproterenol-induced heart rate increments were studied in 9 male volunteers. Propranolol reduced maximal heart rate from 187 +/- 4 to 146 +/- 7 beats/min and atenolol reduced it to 138 +/- 6 beats/min. There was no difference between the drugs at any point during exercise. Isoproterenol sensitivity was measured as the dose of isoproterenol required to increase resting heart rate by 25 beats/min (CD 25). Propranolol increased the CD-25 from 1.8 +/- 0.3 micrograms after placebo to 39 +/- 8 micrograms and atenolol increased the CD-25 to 8 +/- 2 micrograms. The increase by propranolol was significantly greater than that of atenolol. Intravenous atropine (0.04 mg/kg) did not alter the isoproterenol CD-25 during placebo or atenolol. The CD-25 with propranolol decreased after atropine (39 +/- 8 versus 25 +/- 5 micrograms) and was due to diminished plasma propranolol concentrations as the drug sensitivity (measured by Ka) was unchanged before (12 +/- 2 ml/ng) and after (10 +/- 3 ml/ng) atropine. These data support the hypothesis that moderate exercise is primarily a beta 1-mediated response and therefore equally antagonized by cardioselective and nonselective blockers, but that isoproterenol stimulates both beta 1 and beta 2 receptors. The greater ability of the nonselective agent to antagonize isoproterenol tachycardia with no significant change after atropine suggests the presence of cardiac beta 2 chronotropic receptors. The physiologic and pathologic importance of these receptors has yet to be determined. PMID- 3010693 TI - Epinephrine-induced hypokalemia: the role of beta adrenoceptors. AB - Epinephrine was infused intravenously in 9 normal volunteers to plasma concentrations similar to those found after acute myocardial infarction. This study was undertaken on 3 occasions after 5 days of treatment with placebo or the beta-adrenoceptor antagonist, atenolol, which is relatively beta 1 selective, or timolol, which blocks both beta 1 and beta 2 receptors. Epinephrine increased the systolic blood pressure (BP), decreased the diastolic BP and increased the heart rate modestly. These changes were prevented by atenolol. However, after timolol the diastolic BP rose by +19 mm Hg and heart rate fell by -8 beats/min. Epinephrine caused the corrected QT interval to lengthen (0.36 +/- 0.02 to 0.41 +/- 0.06 second). No significant changes were found in the corrected QT interval when subjects were pretreated with atenolol or timolol. The serum potassium decreased from 4.06 to 3.22 mmol/liter after epinephrine. Serum potassium decreased to a lesser extent to 3.67 mmol/liter after atenolol and actually increased to 4.25 mmol/liter after timolol. In a further study with a similar design another nonselective beta blocker propranolol also increased potassium after epinephrine. While atenolol also prevented hypokalemia in this study, it did not block the beta 2-receptor mediated decrease in diastolic BP. Epinephrine induced hypokalemia results from stimulation of a beta-adrenoceptor linked to membrane sodium/potassium adenosine triphosphatase causing potassium influx. This appears to be predominantly mediated by beta 2 receptors although beta 1 receptors may also play a part. PMID- 3010694 TI - Beta 2 receptors on myocardial cells in human ventricular myocardium. AB - Beta 2-adrenergic receptors comprise 40% of the total beta-receptor population in failing human right ventricles, as deduced from computer modeling of iodine-125 iodocyanopindolol-ICI 118,551 competition curves. A myocardial cell origin of at least some of the beta 2 subpopulation was demonstrated by documenting a beta 2 receptor mediated positive inotropic response to the selective beta 2 agonist zinterol. It is concluded that beta 2-adrenergic receptors are present on human ventricular myocardial cells. PMID- 3010695 TI - Postembedding immunogold labeling for electron microscopy using "LR White" resin. AB - A method is described for performing postembedding immunogold immunocytochemistry on sections of LR White-embedded tissues. Fixation of tissue in a combination of paraformaldehyde and glutaraldehyde, or with low concentrations of glutaraldehyde followed by partial dehydration, resulted in preservation of antigenicity for a variety of proteins in different tissue samples. Good structural preservation facilitated high-resolution immunolabeling when coupled with the use of purified monoclonal antibodies. The technique is straightforward and versatile, offering the potential for many immunocytochemical applications with minimal modifications. PMID- 3010697 TI - Phase II study of vincristine infusion in refractory small cell carcinoma of the lung. AB - Fifteen patients with extensive refractory small cell carcinoma of the lung received prolonged intravenous infusion of vincristine. All but one patient had previously been given vincristine by conventional bolus injection. Treatment consisted of a 0.5-mg bolus injection followed immediately by 0.25 mg/m2/day infusion which was continued for 5 days. Toxicity in general was minimal, but rapidly progressive disease precluded adequate assessment in the majority of patients. No objective responses were observed. Infusion of vincristine does not appear to be an efficacious salvage treatment for this disease. PMID- 3010696 TI - THC or Compazine for the cancer chemotherapy patient--the UCLA study. Part II: Patient drug preference. AB - Factors influencing preference for THC vs. Compazine (prochlorperazine) as an antiemetic agent during cancer chemotherapy were studied in 139 patients who received both medications in a double-blind randomized crossover design trial. Nausea reduction was the main determinant of preference. THC preference was associated with more, rather than fewer, drug-related side effects than Compazine, particularly sedation. Patients who reported being anxious or depressed did not experience accentuation of their mood states with either regime. Mood effects, nausea reduction, incidence of side effects, and drug preference were the same in patients under and over 50 years of age. Patients with a history of illicit drug use reported fewer side effects from THC, but reported no difference in drug preference or nausea reduction compared to those patients without a history of illicit use. PMID- 3010698 TI - Endocervical polyp with pseudosarcomatous pattern and cytoplasmic inclusions: an electron microscopic study. AB - A case of endocervical polyp with atypical stromal cells in a 32-year-old woman is reported. Light microscopy showed atypical stromal cells with eosinophilic cytoplasmic inclusions which, by electron microscopy, were nonmembrane-bound, compact fibrillary structures in close relationship with the cytoplasmic microfibrils. The ultrastructural features demonstrated a fibroblastic nature of bizarre cells. These inclusions are similar to those of infantile digital fibromatosis. These findings have not been reported previously. PMID- 3010699 TI - Enzyme histochemistry and thyroid neoplasia. AB - The authors have examined the enzyme histochemical staining of surgically removed human thyroid tissue in an attempt to identify markers that might be useful in the histopathologic diagnosis of thyroid neoplasms. Fresh thyroid glands and other tissues were fixed in cold (4 degrees C) 4% paraformaldehyde and embedded in glycol methacrylate. Forty-two specimens were studied in thin sections, which gave excellent histologic detail and enzyme preservation. Cytologic detail was similar to that in Papanicolaou-stained smears, with good definition of nuclear inclusions and grooves, particularly in cases of papillary carcinoma. The enzyme histochemical reactions studied were as follows: adenosine triphosphatase, alkaline and acid phosphatases, alpha-naphthyl acetate esterase, and 5' nucleotidase. Thyroid epithelial cells and the benign neoplasms derived from them were typically positive for 5'-nucleotidase, alpha-naphthyl acetate esterase, and acid phosphatase, and negative for adenosine triphosphatase and alkaline phosphatase. Staining for adenosine triphosphatase was present in papillary and follicular carcinomas and was seen in benign glands only under certain circumstances such as Graves' disease. The adenosine triphosphatase reaction therefore appears to be helpful in distinguishing between benign and malignant neoplasms derived from thyroid epithelium in humans and may be a useful adjunct to routine morphology. PMID- 3010700 TI - Predictive value of a screening test for antibodies to HTLV-III. AB - A test for the detection of antibodies to HTLV-III is available and will be widely used to screen donated blood to prevent transfusion-associated acquired immunodeficiency syndrome (AIDS). Based upon the sensitivity and specificity, the authors calculated the expected predictive values for different groups of asymptomatic individuals using Bayes' theorem. The prevalence of HTLV-III infection has great impact upon the positive and negative predictive values of the test. For a member of the general population there is a less than 3% chance that a positive test represents a true positive. High-risk patients, such as hemophiliacs, with a positive test will have a greater than 95% chance of actually having antibodies to HTLV-III, but the negative predictive value of the test in this group is less than ideal. The authors recommend that all positive tests for HTLV-III be confirmed by more specific methods when obtained in low risk people. Members of high-risk groups for AIDS should continue to refrain from donating blood, despite the availability of the screening test. PMID- 3010701 TI - Invasive partial mole. AB - A case of invasive partial hydatidiform mole requiring chemotherapy and hysterectomy in a 30-year-old white woman is presented. This is the first histologically and cytogenetically documented partial mole with persistent elevation of human chorionic gonadotropin (hCG) level and invasion of myometrium. There was no evidence of distant spread. PMID- 3010702 TI - Paucifascicular congenital sensory neuropathy in identical twins. AB - A male infant had sensory and autonomic dysfunction, and his identical twin had a similar clinical finding. One twin was extensively studied, utilizing sural nerve, skin, and conjunctival biopsy specimens, to evaluate the status of peripheral sensory axons. The results support an antenatal neurodevelopmental disturbance in axonal growth that affects sensory neurons and limits their distal extension. Neuropathologic studies of this patient closely resemble findings in hereditary sensory neuropathy type II; clinically, however, this patient resembles patients with congenital autonomic dysfunction and universal pain loss. Investigation of proximal and distal sural nerve, skin, and/or conjunctival biopsy specimens is recommended in patients with sensory and autonomic dysfunction to help differentiate these patients to assist in genetic counselling, treatment, and prognosis. It is possible that clinical overlap in such patients may result from a common neuropathic process, but with varying degrees of involvement. PMID- 3010703 TI - Four generations of probable person-to-person transmission of human monkeypox. AB - This paper examines an outbreak of five cases of human monkeypox which occurred in children belonging to two families living in the West Kasai region of Zaire during May-July 1983. Epidemiologic investigations suggest that the first case was infected from an animal source, possibly a monkey, and that each of the other four cases was infected from a previous human case. Three of these cases of presumed person-to-person transmission occurred in close household contacts. The other case infection occurred either by casual contact within the hospital compound, or possibly because of infection due to use of the same syringe for injections. Human monkeypox is the most important orthopoxvirus infection in the post-smallpox eradication period. The disease is a zoonosis and person-to-person transmission is rather difficult. Thus, this episode is a rare event and special analysis of the circumstances is discussed. However, it supports the necessity to carry out surveillance and research on this disease as recently reported by Arita et al. PMID- 3010704 TI - Fractionation of platelets according to size: functional and biochemical characteristics. AB - The functional and biochemical heterogeneity of platelets has been studied using graded differential centrifugation to fractionate human platelets according to size while maintaining their morphological and functional integrity as indicated by scanning electron microscopy and content of beta-thromboglobulin. Aggregation kinetics were studied by both optical and quenched-flow methods involving single particle counting. Large platelets were significantly more sensitive to ADP, but aggregated less rapidly than small platelets. Thrombin exerted a similar influence. Large platelets were also enriched in surface sialic acid and sulfhydryl groups and in internal glycogen, ATP, ADP, calcium, cyclic AMP, malonaldehyde, and succinate cytochrome c reductase when compared to small platelets, even when normalized per unit volume. ADP caused a more rapid breakdown of cyclic AMP in small platelets. Potential aging relationships were tested by isotope studies in rats. 75Se-selenomethionine was incorporated in vivo at a similar rate into all fractions. Large platelets labeled with 51Cr disappeared from circulation linearly and had a longer mean lifespan than small platelets, which disappeared exponentially. This behavior supports independent aging of platelet populations of differing size. The data suggest a distinct heterogeneity in platelet function and fate, which could derive from protection of large platelets against excessive activation by Ca2+-regulated events. PMID- 3010705 TI - Decreased adenosine deaminase (ADA) and 5'nucleotidase (5NT) activity in peripheral blood T cells in Hodgkin disease. AB - The purine metabolic enzymes adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and 5'nucleotidase (5NT) play an important role in normal lymphocyte differentiation. Abnormal levels of one or all of these enzymes have been associated with immunodeficiency diseases and lymphoproliferative disorders. ADA, PNP, and 5NT activity was measured in peripheral blood T cells from 24 patients with Hodgkin disease (HD) (12 in complete remission and 12 with active disease) to determine whether an association existed between enzyme abnormalities and the decreased cellular immune function previously described in this disorder. HD patients had a significantly decreased absolute lymphocyte count (1,618 +/- 1107/mm3; mean +/- SD) compared to controls (2,320 +/- 980; p less than .001). ADA, PNP, and NT activity was assessed in lymphocyte extracts by measuring the conversion of radiolabeled substrates to products over time. ADA activity expressed as mean +/- SEM nanomoles/10(6) lymphocytes/hr was significantly decreased in T cells from HD patients (84.6 +/- 7.5) compared to controls (128 +/ 12.3; p less than 0.025). Likewise, 5NT was significantly decreased in HD patients (12.7 +/- 1.3) compared to controls (24.0 +/- 3.6; p less than .005). There was not a significant difference in PNP activity between both groups. Low 5NT activity was present irrespective of whether patients had active disease (12.1 +/- 1.5) or were in unmaintained complete remission (14.5 +/- 2.4). These findings suggest that biochemical abnormalities may be responsible for or related to the persistent abnormalities in T-cell function noted throughout the clinical course of HD. PMID- 3010706 TI - Burkitt's cells can be triggered by teleocidin to secrete interferon-gamma. AB - The secretion of interferon (IFN)-gamma by T lymphocytes is mediated by the synthesis of interleukin 2 (IL-2) and the availability of IL-2 receptors. Since some Burkitt's lymphoma lines express Tac antigen and can be triggered to secrete IL-2 following activation with the new tumor promoter teleocidin, we addressed the question of whether the induction of IL-2 by B lymphocytes is accompanied by the induction of IFN-gamma. IFN-gamma has not been detected in any of the 25 cell lines studied, and following stimulation with teleocidin, we triggered the synthesis of IFN-gamma in JLP(C), a pre-Burkitt's cell line. The mechanism of IFN gamma secretion by B lymphocytes is not clear. Our findings demonstrate that the synthesis of IL-2 by B cells is not accompanied by IFN-gamma and suggest that the synthesis of IFN-gamma is not mediated by IL-2 or IL-1 or B-cell growth factor. Neutralization studies have shown that IFN-gamma secretion is not accompanied by the induction of IFN-alpha or IFN-beta. Our data imply that B cells can be triggered to secrete IFN-gamma under certain circumstances. Whether similar function occurs in vivo is not known. PMID- 3010707 TI - Humoral effects of long-term oral enalapril therapy. AB - Twenty-seven subjects with essential hypertension were prospectively followed for a minimum of 100 weeks, receiving either enalapril monotherapy or enalapril and hydrochlorothiazide combination therapy. Blood pressure and the renin-angiotensin aldosterone system were assessed following 4 weeks of placebo therapy, and 56 and 96 weeks of maintenance drug therapy. Blood pressure was well controlled with either form of therapy. Plasma renin activity remained stimulated following both long-term monotherapy and combination therapy. However, immunoreactive plasma angiotensin II concentration was not suppressed following either long-term monotherapy or combination therapy. Similarly, plasma aldosterone concentration was not suppressed following either form of therapy; indeed, combination therapy was associated with stimulation of plasma aldosterone concentration. We conclude that enalapril monotherapy or enalapril/hydrochlorothiazide therapy was effective in controlling blood pressure, but that long-term blood pressure control must be related to an angiotensin II independent antihypertensive mechanism. PMID- 3010709 TI - Sequences homologous to the human D1S1 locus present on human chromosome 3. AB - We have investigated the segregation, in somatic cell hybrids, of the human D1S1 locus, previously assigned to 1p36 by in situ hybridization. We have shown that the clone which defines this locus, lambda Ch4A-H3, originates from human chromosome 3, but contains a 1.7-kilobase (kb) PstI-HindIII repetitive element that is also present on chromosome 1, probably distal to PGD. The clone recognizes restriction fragment length polymorphisms within the single-copy sequence on chromosome 3 and one for the enzyme StuI in the repeated sequence on chromosome 1. These experiments thus expose a level of complexity in the D1S1 locus not revealed by earlier in situ hybridization studies. PMID- 3010708 TI - A DNA probe detecting multiple haplotypes of the human Y chromosome. AB - We have characterized a DNA probe (49f) that detects about 15 Y-specific TaqI bands corresponding to a low-copy number sequence. Five of these bands, each representing a single DNA fragment, can either be present, absent, or variable in length. Familial segregation studies have shown that the variations of these fragments are inherited in a Mendelian fashion and strictly Y-linked. A survey of 44 male individuals indicated that the five variable TaqI fragments detected by probe 49f can be considered as five independent allelic series. Each series represents the different and mutually exclusive allelic forms observed for a single DNA fragment. A total of 16 haplotypes, each defined by a different combination of the various forms of each of these five restriction fragment length polymorphisms, were observed among the 44 scored individuals. These TaqI restriction polymorphisms are not observed with other restriction digests and have therefore been attributed to point mutations. The five polymorphic fragments map to Yq11, a region that does not recombine with the X chromosome and are therefore not redistributed. This implies that an apparently independent reassortment of one of these series with respect to the others can be explained only on the basis of mutations that occurred several times (or reverted) during evolution of the Y chromosome. However, an examination of the different combinations of two or more allelic series suggests that some alleles are not randomly distributed and raises the possibility of establishing a genealogy of the human Y chromosome. PMID- 3010710 TI - The anonymous polymorphic DNA clone D1S1, previously mapped to human chromosome 1p36 by in situ hybridization, is from chromosome 3 and is duplicated on chromosome 1. AB - D1S1, a human anonymous DNA clone originally called lambda Ch4A-H3 or lambda H3, was mapped by two other laboratories to human chromosome 1p36 by in situ hybridization but its localization was not confirmed using a different mapping method. We used a panel of human-hamster somatic cell hybrids to show that there are copies of D1S1 on both chromosomes 1 and 3. The D1S1 clone itself is from chromosome 3, and part of it is duplicated at least twice on chromosome 1. A high frequency HindIII polymorphism detected by D1S1, believed to be at chromosome 1p36 on the basis of the in situ hybridization data, maps instead to chromosome 3. This finding demonstrates the importance of using two mapping methods to verify the localization of a gene or DNA segment, particularly a polymorphic one which itself may be used in mapping studies. It also raises the question of why in situ hybridization detected a duplicated portion of a clone but not the chromosomal origin of the clone itself. PMID- 3010711 TI - Four restriction fragment length polymorphisms revealed by probes from a single cosmid map to chromosome 19. AB - We have discovered and characterized a compound polymorphic locus on chromosome 19, defined by an arbitrary genomic DNA segment cloned into a cosmid vector. Four different restriction fragment length polymorphisms with minor allele frequencies equal to or greater than 10% are revealed by Southern hybridization of subclones of cosmid 1-13 with TaqI, MspI, BamHI, and HindIII digests of human DNAs. Seventy two percent of unrelated individuals are heterozygous at one or more loci, and seven of the 24 possible haplotypes occur with frequencies of 3%-38%. Using a somatic cell hybrid panel, we have mapped this locus to 19p13.2----19q13.3, whereas in situ hybridization suggests the probe is on 19p. Taken together, these results suggest localization to 19p13.2----19cen. The locus revealed by probes from cosmid 1-13 has been designated D19S11. PMID- 3010712 TI - The p97 antigen is mapped to the q24-qter region of chromosome 3; the same region as the transferrin receptor. AB - Since the p97 antigen, a membrane-associated iron-binding protein, has extensive amino acid sequence with homology with transferrin, is functionally related to the transferrin receptor, and has been previously mapped to chromosome 3, we have performed additional studies for regional mapping of the gene expressing p97 antigen. In these experiments, Chinese hamster-human cell lines were chosen that contained a large spectrum of autosomal human chromosomes, but mainly consisted of clones expressing all or a part of chromosome 3. These cell lines included a clone that previously allowed for mapping of human transferrin receptor to q22 qter region. Human p97 expression was assessed by specific binding of [125I]monoclonal antibody 96.5, and human transferrin receptor expression was tested by specific [125I]human transferrin binding and [125I]monoclonal antibody OKT-9 specific for human transferrin receptor. Based on these analyses, both human p97 antigenic expression and human transferrin receptor are mapped concordantly to the q24-qter region. These data and previous reports, therefore, suggest that the related iron-transport proteins are closely linked and may be under coordinate regulation. However, studies of several cell lines that exhibit up-regulation of human transferrin receptor expression with cellular proliferation, and down-regulation of receptor with increased transferrin-iron in the media, showed no change in expression of p97 antigen. p97 antigenic expression increased when melanocyte-stimulating hormone was added to a human melanoma cell line in tissue culture. These latter studies suggest that in mammalian cells the two proteins do not show coordinate regulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010713 TI - The heteromorphic marker on chromosome 18 using restriction endonuclease AluI. AB - The staining property of pericentromeric heterochromatin of chromosome 18 is compared by C-banding and restriction endonuclease AluI digestion methods. Only a small distal fraction of C-band of chromosome 18 is observed to be resistant to AluI treatment, which positively stained with subsequent Giemsa staining. The resistant fraction is characteristic and usually located toward the short arm. The extensive heterogeneity of constitutive heterochromatin revealed by AluI treatment is useful in demonstrating the heterozygosity of homologous chromosomes. This, in turn, may provide frequent markers to identify the chromosomes 18's. This present approach can be utilized in evaluation of the families to describe the origin of the extra chromosome 18 in Edward syndrome. As an example, one such family has been investigated where the additional chromosome 18 originated due to paternal nondisjunction at meiosis I. PMID- 3010714 TI - Exclusion of human chromosome 13q34 as the site of the cystic fibrosis mutation. AB - We have studied a family in which both cystic fibrosis (CF) and an unbalanced translocation between chromosomes 6 and 13 are found. As CF occurs in the child who is effectively monosomic for the translocated part of the long arm of chromosome 13, it was suggested that the locus of the gene mutation causing CF is on chromosome 13q34. The gene for human coagulation factor X is located at 13q34, and we have found a restriction fragment length polymorphism (RFLP) that is revealed by a cloned cDNA coding for this protein. Linkage analysis in eight CF families shows no evidence of cosegregation between CF and the gene for factor X, strongly suggesting that the locus for the defect causing cystic fibrosis is not at 13q34. PMID- 3010715 TI - Pathogenesis of osteoarthritis. AB - This article reviews the etiology and pathogenesis of osteoarthritis, particularly one of several current concepts concerning the possible central mechanisms regulating degradation of cartilage. According to this theory, degradation involves diffuse or focal exposure of the extracellular matrix to active neutral metalloproteinases, which then results in injury as well as initiation of repair processes. Diffuse matrix exposure is probably not a physiologic aberrancy but rather a pathologic result of either physical injury to local chondrocytes or inflammatory mediators. PMID- 3010716 TI - Norfloxacin versus vancomycin/polymyxin for prevention of infections in granulocytopenic patients. AB - Selective antimicrobial decontamination with norfloxacin was compared with vancomycin/polymyxin for prophylaxis of bacterial infections in granulocytopenic patients. In the group of patients receiving norfloxacin, there were a lower number of acquired gram-negative bacillary organisms per patient (0.88 versus 1.86, p = 0.002), fewer patients with documented infection (16 of 36 versus 20 of 30, p = 0.12), and fewer cases of gram-negative septicemia (0 of 36 versus five of 30, p = 0.02). Norfloxacin was better tolerated (30 of 36 versus 16 of 30 patients highly compliant, p = 0.02), and associated with fewer gastrointestinal side effects (eight of 36 versus 14 of 36 patients, p = 0.07). These results suggest that norfloxacin is a more tolerable and efficacious oral antimicrobial agent than vancomycin/polymyxin for the prevention of serious gram-negative bacillary infections in granulocytopenic patients. PMID- 3010717 TI - Changes in protoporphyrin distribution dynamics during liver failure and recovery in a patient with protoporphyria and Epstein-Barr viral hepatitis. AB - Acute liver failure with cholestasis, histologic and serologic evidence of Epstein-Barr viral infection, and associated autoimmune hemolytic anemia occurred in a patient with lifelong protoporphyria. Changes in previously established baseline protoporphyrin distribution dynamics in erythrocyte, plasma, and fecal excretion compartments were observed during the period of severe hepatic dysfunction and recovery. These changes were consistent with predictions of a previously described conceptual model for human protoporphyria. PMID- 3010720 TI - Electron microscopic detection of virus particles in the rhino mouse. AB - The rhino mouse, an experimental model for systemic lupus erythematosus, was found to have murine leukemia virus particles present in the skin. Immunoperoxidase studies with anti-type C virus antibody indicated viral antigen presence in the sebaceous cells and surrounding follicular cysts of the dermis. Electron microscope studies show virus particles, both type C extracellularly and type A intracellularly, around follicular cysts. Virus particle was labeled with specific ferritin-conjugated antitype C virus antibody. PMID- 3010719 TI - Acute and chronic complications of diabetes mellitus in older patients. AB - The chronic complications of diabetes are thought to be caused by an interaction between hyperglycemia or other metabolic consequences of insulin deficiency and other poorly defined independent genetic or environmental factors. Several important biochemical sequelae to hyperglycemia are discussed. Macrovascular disease appears to be primarily age-related in diabetic patients. The clinical course, manifestations, and management of diabetic complications are significantly altered when they appear against a background of the degenerative changes of aging, greatly complicating diagnosis and management. In elderly patients, the acute complications of diabetes--ketoacidosis and hyperosmolar dehydration--often occur in the context of chronic complications that greatly compound their management and increase their morbidity and mortality. PMID- 3010718 TI - Primary adrenocortical nodular dysplasia, a distinct subtype of Cushing's syndrome. Case report and review of the literature. AB - Non-iatrogenic Cushing's syndrome has been associated primarily with three entities: pituitary-dependent processes due to pituitary adenomas or microadenomas causing adrenal hyperplasia; pituitary-independent primary adrenal causes, predominantly unilateral adenomas, rarely multiple adenomas or adrenal carcinoma; ectopic sources of adrenocorticotropic hormone (ACTH) production. Although non-neoplastic bilateral adrenal disease generally has been ascribed to extra-adrenal stimulation, a rare cause of Cushing's syndrome that involves bilateral adrenal nodule formation independent of pituitary stimulation has been identified. Nodular adrenal diseases represent a confusion of terms in the literature, but one subgroup of Cushing's syndrome has most frequently--and, perhaps, most appropriately--been designated primary adrenocortical nodular dysplasia. A case of this unusual entity is presented, and previous case reports pertaining to this confusing area of adrenal hyperfunction are reviewed. The characteristic manifestations that separate this diagnosis from other types of nodular adrenal disease are also discussed. Recognition of this diagnosis, although rare, is important, as bilateral adrenalectomy in the treatment of choice. PMID- 3010721 TI - Hormonal effects of smoking--II: Effects on plasma cortisol, growth hormone, and prolactin. AB - Effect of smoking on the plasma levels of cortisol, growth hormone, and prolactin was evaluated in a group of smokers and nonsmokers. Plasma levels of these hormones were measured under basal conditions and following a short burst of smoking. In addition, to determine the mechanism of action of nicotine on the release of these hormones, rat renal cortical slices were incubated with nicotine and the generation of cyclic AMP was measured in vitro. Increasing concentrations of nicotine in the incubation medium resulted in increased generation of cyclic AMP. Basal levels of plasma cortisol were similar for both smokers and nonsmokers. After smoking, the cortisol levels increased significantly among smokers only and the levels achieved were significantly higher compared with nonsmokers. Mean prolactin curves were higher among nonsmokers compared with smokers, whereas growth hormone levels were similar in the two groups. These data suggest that the effects of smoking on pituitary/adrenal hormones differ among smokers and nonsmokers and that these effects may be mediated through increased generation of cyclic AMP induced by nicotine. PMID- 3010722 TI - The Perlman familial nephroblastomatosis syndrome. AB - In 1973, Perlman et al described a familial syndrome of bilateral renal hamartomas with or without nephroblastomatosis, macrosomia, islet cell hypertrophy, unusual facies, and early lethality. Two additional sibs were recently reported by Neri et al [1984]. We report on two sibs with polyhydramnios, fetal ascites, and abdominal muscular hypoplasia, visceromegaly, and subsequent development of Wilms tumor in one of them. Delineated features of this syndrome include visceromegaly, macrosomia, renal hamartomas, nephroblastomatosis, cryptorchidism in males, unusual facial appearance, polyhydramnios, fetal ascites, and Wilms tumor but do not include hemihypertrophy, omphalocele or umbilical abnormalities, aniridia, or other conditions known to be associated with Wilms tumor. This condition should be considered primarily in the differential diagnosis of fetal ascites without hydrops and possibly in the differential diagnosis of familial Wilms tumor, polyhydramnios, congenital hepatomegaly, or nephromegaly. PMID- 3010724 TI - Excretion of varicella-herpes zoster virus in breast milk. AB - Two cases involving breast-feeding and varicella-herpes zoster virus infection are presented. The possibility of viral excretion in the breast milk and its effects on continued breast-feeding are discussed. In neither case was the virus isolated from the breast milk. PMID- 3010723 TI - A crisis plan for pediatric code. PMID- 3010725 TI - Human papillomavirus infection of the cervix detected by cervicovaginal lavage and molecular hybridization: correlation with biopsy results and Papanicolaou smear. AB - Human papillomaviruses have previously been identified by molecular hybridization in the majority of dysplastic and cancerous lesions of the cervix. Since human papillomavirus types 16 and 18 have been strongly associated with cervical cancer, the identification of patients infected with these specific human papillomavirus types may provide useful prognostic information. We have developed a painless, noninvasive cervicovaginal lavage technique to collect exfoliated cervicovaginal cells, which can be reliably analyzed for the presence of human papillomavirus deoxyribonucleic acid by Southern blot analysis with the use of deoxyribonucleic acid cloned from human papillomaviruses 6, 11, 16, and 18. In a prospective study of 60 women referred to a colposcopy clinic for evaluation of abnormal Papanicolaou smears, we have detected human papillomavirus deoxyribonucleic acid in 16 of 17 (94%) women with a Class III (dysplasia) or IV (carcinoma in situ) Papanicolaou smear, five of 11 (45%) women with a Class II (atypical) Papanicolaou smear, and 10 of 34 (29%) women with a normal Papanicolaou smear. Detection of human papillomavirus deoxyribonucleic acid in cervicovaginal cells was indicative of a dysplastic cervical lesion in 19 of 20 (95%) patients irrespective of Papanicolaou smear results. We conclude that human papillomavirus deoxyribonucleic acid analysis in cervicovaginal cells is a sensitive method to detect dysplastic lesions of the cervix and may be useful in identifying patients with specific types of human papillomavirus infection, who are at risk to develop cervical cancer. PMID- 3010726 TI - Treatment of cytomegalovirus retinitis with dihydroxy propoxymethyl guanine. PMID- 3010728 TI - Renal transport and metabolism of nicotinic acid. AB - Renal metabolism and brush-border transport of nicotinic acid were studied in renal cortical slices and brush-border membrane vesicles exposed to a physiological concentration of vitamin (2.2-3.5 microM). Vesicle transport of [3H]nicotinic acid was found to be Na+ dependent and concentrative. The presence of a Na+ gradient resulted in a fivefold increase in the rate of nicotinic acid uptake over that observed with mannitol and caused a transient nicotinic acid accumulation two- to fourfold above the equilibrium value. The effects of membrane potential, pH, and elimination of Na+-H+ exchange were also studied. Cortical slices and isolated tubules exposed to 2.2 microM [14C]nicotinic acid took up vitamin and rapidly metabolized most of it to intermediates in the Preiss Handler (J. Biol. Chem. 233: 488-493, 1958) pathway for NAD biosynthesis; little free nicotinic acid was detectable intracellularly. The replacement of Na+ with Li+ in the bathing medium reduced total accumulation of 14C label primarily as a result of reduced nicotinic acid uptake. Cortical tissue concentrated free nicotinic acid only when the involved metabolic pathways were saturated by levels of nicotinic acid far in excess of what occurs in vivo. PMID- 3010727 TI - Intracellular diffusion gradients of O2 and ATP. AB - Endogenous enzymes with different subcellular localizations provide in situ probes to study O2 and ATP concentration at various sites within cells. Results from this approach indicate that substantial intracellular concentration gradients occur under some O2- and ATP-limited conditions. These studies, along with electron microscopic analyses and mathematical modeling, indicate that clustering and distribution of mitochondria are major factors in determining the magnitude and location of the concentration gradients. The mitochondria appear to be clustered in sites of high ATP demand to maximize ATP supply under conditions of limited production. The size of such clusters is limited by the magnitude of the O2 gradient needed to provide adequate O2 concentrations for mitochondrial function within the clusters. Thus microheterogeneity of metabolite concentrations can occur in cells without membranal compartmentation and may be important in determining the rates of various high-flux processes. PMID- 3010729 TI - Insulin-induced cytoplasmic alkalinization and glucose transport in muscle cells. AB - Insulin stimulates glucose uptake into muscle within minutes, preceding stimulation of glycolysis. Signals involved in stimulation of glycolysis include cytoplasmic alkalinization and specific intracellular proteolytic products. In contrast, the signals that mediate stimulation of glucose transport remain unknown. Here we explore whether the insulin-induced cytoplasmic alkalinization is an early event that precedes activation of sugar uptake, whether such alkalinization is causally related to stimulation of sugar uptake, and whether proteolytic activity mediates stimulation of hexose transport. Cytoplasmic pH (pHi) was measured in suspended skeletal muscle cells of the L6H9 line with the intracellularly trapped fluorescent pH indicator bis(carboxyethyl)carboxy fluorescein. At 37 degrees C, insulin (1 X 10(-7) M) produced an increase in pHi of 0.11 units in 10 min. This increase became apparent 2 min after addition of the hormone, and maximal elevation of pHi was observed after 10 min, remaining elevated for up to 60 min. Removal of the hormone with anti-insulin antiserum did not reverse pHi back to the resting level. The alkalinization was prevented by amiloride, by 5-(N,N'-disubstituted)amiloride analogues, and by isosmotic replacement of Na+ with N-methylglucamine+ or choline+. This suggests that insulin activates Na+-H+ exchange. In contrast, stimulation of 2-deoxy-D-glucose transport by insulin was not affected by replacement of external Na+ or by addition of amiloride. Monensin, an exogenous Na+-H+ exchanger, did not stimulate sugar transport even though it increased pHi. Proteinase inhibitors that block hormonal stimulation of glycolysis were ineffective in preventing stimulation of 2-deoxy-D-glucose transport by insulin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010730 TI - Polycations reduce vasopressin-induced water flow by endocytic removal of water channels. AB - Several polycations added to the luminal solution were found to inhibit the vasopressin (ADH)-induced water flow in toad urinary bladder but not the ADH induced increase in sodium transport or in urea permeability. Ultrastructural studies were conducted to evaluate the uptake of cationized ferritin. It was found that endocytosis of cationized ferritin by luminal cells was strikingly enhanced on exposure to ADH; this increased endocytosis was concomitant with inhibition of transepithelial ADH-induced water flow. Various maneuvers preventing endocytosis were also found to counteract the polycation-induced inhibition of the ADH effect. It is suggested that polycations are endocytosed in vesicles whose walls contain the water channels but not the urea or sodium channels. PMID- 3010731 TI - Photoinactivation of sodium-potassium-chloride cotransport in LLC-PK1/Cl 4 cells by bumetanide. AB - Rb+ uptake into LLC-PK1/Cl 4 cells can be subdivided into three components: 1) ouabain-sensitive uptake, 2) bumetanide-sensitive uptake, and 3) ouabain- and bumetanide-insensitive uptake. Exposure of cells to near-UV light in the presence of low concentrations of bumetanide produces a specific, irreversible inhibition of the bumetanide-sensitive uptake component, while not affecting the other two uptake components. Irreversible inhibition of bumetanide-sensitive transport is observed when measuring either cellular uptake or efflux and also when measuring 86Rb+ uptake into membrane vesicles. The irreversible inhibition is both concentration and time dependent and is blocked under conditions where the interaction of bumetanide with the Na+-K+-Cl- cotransporter is disturbed. We conclude that bumetanide, at low concentrations, can specifically and irreversibly inhibit the Na+-K+-Cl- cotransporter of LLC-PK1/Cl 4 cells. We suggest that this irreversible inhibition is the result of the photoactivation of an ether linkage in the bumetanide molecule, leading to a covalent binding of bumetanide to the Na+-K+-Cl- cotransporter. PMID- 3010732 TI - TRH and GRF stimulate release of growth hormone through different mechanisms. AB - Thyrotropin-releasing hormone (TRH) is an effective stimulator of growth hormone (GH) release from cultured adenohypophysial cells of chronically hypothyroid rats in vitro. The present study explored the question of cAMP and calcium mediation of the GH-stimulatory effect of TRH in this system. A maximally stimulatory concentration of TRH was added together with various concentrations of human GH releasing factor 40 (hGRF-40) whose action is cAMP mediated, or of dibutyryl cAMP (DBcAMP), to primary monolayer cultures of adenohypophysial cells from thyroidectomized rats. The GH responses to the combined addition of TRH with all doses of GRF or DBcAMP were fully additive, causing parallel elevations of the dose-response curves. Whereas the GH response to maximally effective concentrations of hGRF-40 and DBcAMP, added together, was not greater than that to either secretagogue alone, the inclusion of TRH increased the response to a new Emax. The calcium inhibitors, verapamil, EGTA, and CoCl2, markedly suppressed basal GH release and virtually completely blocked the GH response to TRH, suggesting calcium mediation. In chronically hypothyroid, urethan-anesthetized rats, the in vivo effect of the combined administration of maximally effective doses of TRH and GRF on plasma GH levels was also additive. These findings indicate that TRH stimulates GH release in adenohypophysial cells of hypothyroid rats by a cAMP-independent, calcium-dependent mechanism. PMID- 3010733 TI - H1 action of histamine on aldosterone and cortisol secretion by perfused dog adrenal. AB - The left adrenal of hypophysectomized-nephrectomized dogs was perfused in situ with artificial medium equilibrated with 95% O2-5% CO2. Repeated secretory responses of aldosterone to ACTH and histamine were almost reproducible during 2 h of perfusion, but those of cortisol and corticosterone gradually reduced. The secretion of aldosterone in response to histamine markedly increased at 1 microM and those of cortisol and corticosterone significantly increased at 1 and 10 microM, respectively. The secretory response of aldosterone to 10 microM histamine was comparable with that to 300 microU/ml ACTH, but the secretory responses of cortisol and corticosterone to 10 microM histamine were very much lower than those to 1 microU/ml ACTH. The secretory responses of these three steroids to 10 microM histamine were almost completely depressed by continuous administration of 10 microM pyrilamine maleate, but not abolished by that of 100 microM metiamide. The secretory responses of these three steroids to 10 microM histamine were slightly depressed by continuous infusion of 10 microM methysergide maleate. These results suggest that histamine at pathophysiological concentrations stimulates the secretion of aldosterone markedly, and those of cortisol and corticosterone slightly, via an H1 receptor of adrenocortical cells in the dog. PMID- 3010734 TI - Desensitization and redistribution of beta-adrenergic receptors on human mononuclear leukocytes. AB - We have used intact human mononuclear leukocytes (MNL) to examine desensitization of beta-adrenergic receptors in normal mammalian cells. MNL were prepared and radioligand binding experiments were performed at 4 degrees C. At this temperature the ligand [125I]iodocyanopindolol ([125I]ICYP) identified the same number of receptors as at 37 degrees C, and the agonist isoproterenol competed for this binding with high affinity (dissociation constant, Ki = 20 nM). At 37 degrees C, results were similar when the binding incubation was terminated after 1 min, but the apparent affinity of the receptors for isoproterenol was several 100-fold lower when the incubation was allowed to reach steady state. In desensitized MNL (prepared by incubating whole blood with 10 microM isoproterenol at 37 degrees C for 10 min, and then isolating and washing the MNL at 4 degrees C), isoproterenol-stimulated cAMP accumulation was reduced 63 +/- 4%. After desensitization, the total number of beta-receptors was unchanged, but isoproterenol and the hydrophilic antagonist CGP-12177 were able to compete with [125I]ICYP for binding to only 18 +/- 6% of these sites. Direct binding with [3H]CGP-12177 yielded similar results. These results demonstrate that isoproterenol promotes a rapid desensitization of beta-adrenergic receptors on MNL and a concomitant redistribution of receptors into a cellular compartment to which some ligands (including catecholamines) have restricted access. The findings demonstrate that redistribution of beta-receptors may be a mechanism mediating desensitization to catecholamines in normal mammalian cells. PMID- 3010735 TI - Ontogeny of gastric mucosal permeability responses to luminal H+ and bile salt in the rat. AB - Gastric mucosal responses to intraluminal instillation of acid (10, 50, and 150 mM HCl) and acidified solutions of sodium taurocholate (10 mM) were measured in rats between 5 and 60 days after birth. Net loss of H+ and gain of Na+, K+, and protein into the gastric lumen remained low and stable up to 25 days after birth. After that there were dose-dependent increases in H+ loss and Na+ and K+ appearance in the gastric lumen. Luminal protein appearance did not change with any concentration of acid tested in any of the ages examined. Gastric instillation of sodium taurocholate resulted in an increase in the rate of H+ loss as well as Na+ and K+ appearance in neonatal rats after 12-14 days. Protein appeared in the recovered gastric instillate and increased in parallel with Na+ and K+. In response to bile salt treatment, ulcers were only evident after 35 days of age. Injection of 8-day-old rats with corticosterone acetate (250 mg/kg) did not affect the ionic fluxes or protein output in response to acid alone or acidified bile salt solutions. These studies indicate that intragastric acid loads did not increase the ionic permeability of the gastric mucosa until 25 days after birth. The permeability changes occur earlier (day 12-14) if sodium taurocholate is added to the acid instillate. Prior to that time the mucosal permeability characteristics of the neonatal rat stomach are not altered by instillation of either acid loads alone or bile salt solutions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010736 TI - Effects of calcium antagonist TMB-8 on active Na and Cl transport in rabbit ileum. AB - The effects of 3,4,5-trimethoxybenzoate 8-(N,N-diethylamino)octyl ester (TMB-8), an agent that traps calcium within intracellular stores, were studied on active electrolyte transport in rabbit ileum under basal conditions and after altering transport by increasing the intracellular cAMP content or by exposure to two agonists that act by altering intracellular Ca2+ (carbachol and serotonin). TMB-8 decreased the ileal short-circuit current and increased active Na and Cl absorption by increasing the mucosal-to-serosal Na and Cl fluxes. These effects were reversed by increasing the bathing solution Ca2+ to 4 mM, a concentration that itself did not alter basal ileal transport. The maximum glucose- and amino acid (alanine)-induced increase in Na absorption in the ileum was not affected by TMB-8. The effects on basal transport of TMB-8 were not associated with a change in 45Ca2+ entry across the ileal serosal surface. TMB-8 did not alter cAMP induced secretion, as judged by its lack of effect on the increase in short circuit current caused by 8-bromo-cAMP (10(-4) M). TMB-8 totally prevented the transport effects of carbachol but did not inhibit the effects of serotonin. These data suggest a role for intracellular Ca2+ in regulation of basal ileal Na and Cl transport but not in cAMP-induced secretion. There appear to be several pools of intracellular Ca2+ involved in neurohumoral effects on active electrolyte transport. PMID- 3010737 TI - High Ca2+ inhibits AVP-dependent cAMP production in thick ascending limbs of Henle. AB - To further gain insights into the mechanisms underlying impaired urine concentration in hypercalcemia, effects of increasing Ca2+ concentrations in the incubation medium on cAMP production in response to 10(-8) M arginine vasopression (AVP) were examined in thick ascending limbs of Henle (MTAL) and collecting tubules (MCT) dissected from outer medulla of mouse kidney. Increasing Ca2+ in the incubation medium from 1.0 mM to either 2.0 mM or 5.0 mM inhibited AVP-dependent cAMP production in MTAL but not in MCT. This inhibition of AVP dependent cAMP production by 2.0 mM Ca2+ in MTAL was not reversed by verapamil or diltiazem. Also, Ca2+ ionophore A23187 did not inhibit AVP-dependent cAMP production in MTAL in the presence of 1.0 mM Ca2+. Increasing medium Ca2+ from 1.0 to 5.0 mM inhibited cAMP production in MTAL in response to both glucagon and forskolin by the magnitude comparable to that seen in response to AVP. These results show that high Ca2+ inhibits AVP-dependent cAMP production only in MTAL and not in MCT. In addition, the lack of effects of Ca2+ channel blockers and Ca2+ ionophore suggests that high ambient Ca2+ per se may inhibit AVP-dependent cAMP production in MTAL. The fact that high Ca2+ also suppressed cAMP production in response to glucagon or forskolin suggests that Ca2+ may inhibit AVP-dependent adenylate cyclase at postreceptor site(s), one of which is the catalytic unit of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010738 TI - Time-dependent attenuation of water flow in antidiuretic hormone-treated toad bladder. AB - Toad urinary bladders were subjected to sequential 30-min stimulation with antidiuretic hormone (ADH) followed by 30-min hormone washout over 4 h in the absence of a transmural osmotic gradient. Immediately thereafter, during test stimulation with hormone in the presence of a transmural gradient, transbladder water flow was profoundly inhibited, but intra(luminal)membrane particle aggregates, presumed markers of luminal membrane water permeability, were as numerous as in fully responsive controls. The protocol followed was designed to eliminate any distorting effect of prior water flow on cytoplasmic organization and to equalize, for both experimental and control tissues, the time of aggregate presence in the luminal membrane during final test stimulation. On the assumption that cytoplasmically stored and/or newly synthesized aggregates would be unaffected by the protocol followed, these observations appear to be consistent with the view that bladder refractoriness to prolonged ADH treatment may involve regulation of tissue water permeability at a resistance distal to the luminal membrane. PMID- 3010739 TI - Renal alpha-adrenoceptors and sodium excretion. PMID- 3010740 TI - Dobutamine-induced cardiac adaptations: comparison with exercise-trained and sedentary rats. AB - To investigate dobutamine-induced cardiac adaptations, we compared dobutamine treated with swim-trained and sedentary control rats. After 14 wk of treatment, heart rate was lower in the dobutamine-treated (279 +/- 6) and exercise-trained (287 +/- 4 beats/min) groups than in the control animals (305 +/- 3 beats/min; P less than 0.05). The exercised rats gained less weight (164 +/- 11 g; P less than 0.05) than the dobutamine-treated (238 +/- 16 g) and the control animals (231 +/- 12 g). Also, compared with the two other groups, the exercise group had higher relative heart weights (3.47 +/- 0.08 vs. 2.82 +/- 0.06 and 2.90 +/- 0.05 g/kg in the dobutamine and control groups, respectively; P less than 0.05) and lower epididymal fat pad weights (6.6 +/- 0.4 vs. 13.3 +/- 1.0 and 11.4 +/- 0.5 g/kg in the dobutamine and control groups, respectively; P less than 0.05). However, both the maximum heart rate produced by isoproterenol and the isoproterenol dose producing 50% of the peak heart rate response were similar among the three groups. Myocardial norepinephrine content, beta-adrenergic receptor number, and adenylate cyclase activation by isoproterenol, NaF, 5'-guanylyl imidodiphosphate, and forskolin also did not differ. Thus, although there were differences between the dobutamine-treated and the exercised rats, the two groups were similar in that they developed bradycardia that was not due to cardiac adrenergic desensitization. PMID- 3010742 TI - Antagonism of forskolin effects by adenosine in isolated hearts and ventricular myocytes. AB - Adenosine is known to antagonize the effects of catecholamine stimulation in atrial and ventricular tissue; however, its mechanism of action is unknown. Forskolin is an inotropic agent that causes an increase in cyclic AMP (cAMP) levels independent of receptor stimulation. We sought to test whether adenosine could attenuate the effects of forskolin in isolated perfused guinea pig hearts and isolated single ventricular myocytes. In isolated perfused hearts (n = 18), forskolin caused a concentration-dependent increase in left ventricular pressure and dP/dt. Adenosine (5 microM) antagonized the forskolin (0.35 microM)-induced increase in left ventricular pressure and dP/dt by 96 +/- 2 and 92 +/- 4% (means +/- SE), respectively. In contrast, in four hearts, adenosine was ineffective in attenuating the inotropic response to dibutyryl cAMP. In isolated ventricular myocytes (n = 10) 150 nM forskolin caused a significant increase in action potential duration and plateau. In voltage-clamp experiments (n = 8), 150 nM forskolin caused a 39 +/- 3% increase in the calcium current, which was antagonized by adenosine (50 microM) by 80%. Forskolin also caused an increase in contractility, as estimated by sarcomere shortening of the cell. Adenosine, and its analogue N6-R-phenylisopropyladenosine (L-PIA), antagonized the effects of 150 nM forskolin on the action potential and on sarcomere shortening. Dibutyryl cAMP had similar effects as forskolin, but they were not antagonized by adenosine. At higher concentrations of forskolin, above 300 nM, delayed after depolarizations and sustained spontaneous activity occurred that could be abolished by L-PIA. Forskolin caused a concentration-dependent increase in cAMP, measured in isolated ventricular myocytes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010741 TI - Vasoactive intestinal peptide: vasodilatation and cyclic AMP generation. AB - Vasoactive intestinal peptide (VIP) is a putative neurotransmitter that causes vasodilation when injected intravenously. To learn more about the vasodilator actions of VIP, studies were performed on the rat mesenteric arterial bed and on cultured smooth muscle cells derived from both the rat mesenteric artery and aorta. In isolated perfused rat mesenteric artery beds, VIP caused relaxation at the threshold concentration of less than 1 nM and a maximal fall in perfusion pressure similar to that obtained with isoproterenol. VIP stimulated adenylate cyclase in cultured vascular smooth muscle cells in a concentration range of 10( 10)-10(-6) M; maximum activity was increased 2.7 +/- 0.6- and 3.4 +/- 0.4-fold above basal in mesenteric and aortic smooth muscle cells, respectively. The threshold and the half-maximal concentrations of VIP for adenylate cyclase stimulation were more than 50-fold lower than those for both prostaglandin E1 and isoproterenol in cultured mesenteric and aortic cells. Similar effects on cyclic AMP generation in response to VIP were observed in isolated rat mesenteric artery rings. Immunocytochemical examination of the rat mesenteric artery bed in situ demonstrated dense innervation of small- and medium-size vessels with nerve fibers containing VIP-like immunoreactivity. Thus VIP is present in the rat mesenteric artery, a peripheral arterial bed, and is a potent vasodilator that can activate vascular smooth muscle adenylate cyclase and thus potentially contribute to the regulation of vascular tone. PMID- 3010743 TI - Effect of hypoxia and hypercapnia on ACE activity in the cerebral microcirculation of anesthetized dogs. AB - Angiotensin-converting enzyme (ACE) activity of the cerebral microcirculation of anesthetized dogs was measured from cerebral venous outflow curves after bolus injection of a synthetic ACE substrate, [3H]benzoyl-phenylalanyl-alanylproline ([3H]BPAP), into a common carotid artery. Cerebral BPAP metabolism was quantified by measuring the concentration of [3H]benzoyl-phenylalanine (the product of BPAP hydrolysis by ACE) in blood samples from the sagittal sinus after occlusion of the lateral sinuses with bone wax. Instantaneous BPAP metabolism in each sample increased as a function of time after injection, suggestive of perfusion heterogeneity, and averaged 59 +/- 4% (n = 8) over a single pass during normoxia and normocapnia. The ratio of Vmax (the maximal rate of cerebral BPAP metabolism) to Km (the concentration at Vmax/2), was calculated from instantaneous outflow curves using a model based on first-order kinetics. Increases in cerebral blood flow during either hypoxia or hypercapnia significantly reduced BPAP metabolism to 33 +/- 3 (n = 7) and 24 +/- 3% (n = 5), respectively; however, Vmax/Km of ACE activity (0.19 +/- 0.03 ml/s) was not affected by either condition. The lack of change in apparent kinetics of ACE activity (i.e., in Vmax/Km) during hypoxia or hypercapnia suggests that recruitment of cerebral capillaries was not a quantitatively significant factor in controlling BPAP metabolism with this degree of either hypoxia or hypercapnia. PMID- 3010745 TI - Reduced chronotropic responsiveness of the heart in experimental uremia. AB - Cardiac beta-adrenoceptor responsiveness was evaluated in experimental uremia by in vivo and in vitro techniques. Uremia was induced in rats by bilateral nephrectomy for 48 h. In rats with chronic intra-arterial and intravenous catheters, cardiovascular reflexes and the renin-angiotensin system were blocked with atropine, pentolinium, and a converting-enzyme inhibitor, respectively. Blood pressure (BP) and heart rate (HR) were continuously recorded. Cumulative doses of isoproterenol were injected intravenously. In uremic rats, the dose response curve for the HR response showed a lower maximal response (P less than 0.01) and no significant difference in 50% effective dose values compared with controls, whereas the BP decrease caused by isoproterenol was similar in control and uremic rats. When forskolin was injected intravenously to stimulate adenylate cyclase in a receptor-independent manner, the maximal HR increase was lower in uremic rats (P less than 0.01). beta-Adrenoceptor density and affinity, measured by 125I-cyanopindolol binding to sarcolemmal membranes, was not different between control and uremic rats. Also binding affinities for the agonist isoproterenol were not different between groups. Basal adenylate cyclase activity, as well as activity after maximal stimulation by isoproterenol and by forskolin were lower in uremic than in control rats (P less than 0.01). The results show that the chronotropic response of the heart is reduced in uremia. Such hyporesponsiveness may be due, at least in part, to a reduced activity of cardiac adenylate cyclase. PMID- 3010744 TI - Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. AB - Experiments were designed to determine the effects of oxygen-derived free radicals on the production and biological activity of endothelium-derived relaxing factor or factors released by acetylcholine. Rings of canine coronary arteries without endothelium (bioassay rings) were superfused with solution passing through a canine femoral artery with endothelium. Superoxide dismutase caused maximal relaxation of the bioassay ring when infused upstream, but not downstream, of the femoral artery; this effect of superoxide dismutase was inhibited by catalase. Infusion of acetylcholine relaxed the bioassay rings because it released a labile relaxing factor (or factors) from the endothelium. When infused below the femoral artery, superoxide dismutase and, to a lesser extent, catalase augmented the relaxations to acetylcholine. Superoxide dismutase, but not catalase, doubled the half-life of the endothelium-derived relaxing factor(s). This protective effect of the enzyme was augmented fivefold by lowering the oxygen content of the perfusate from 95 to 10%. These data demonstrate that: superoxide anions inactivate the relaxing factor(s) released by acetylcholine from endothelial cells and hyperoxia favors the inactivation of endothelium-derived relaxing factor(s). PMID- 3010746 TI - Sensitivity of cortisol-induced inhibition of ACTH and renin in fetal sheep. AB - Previous experiments demonstrated that increases in ovine fetal plasma cortisol concentration to maximal stress levels suppressed fetal plasma renin activity and completely inhibited fetal adrenocorticotropin (ACTH) responses to subsequent stress. This study was designed to quantitate the suppressive action of cortisol on both ACTH and renin. Fetal sheep between 117 and 131 days gestation were surgically prepared with chronically implanted catheters. At least 4 days after surgery, vehicle or cortisol (0.25, 0.5, 1, 2, or 3 micrograms/min) were infused for 5 h. One hour after the end of the vehicle or cortisol infusion, fetal ACTH and renin secretion were stimulated by intravenous infusion of sodium nitroprusside. Cortisol infusions suppressed basal plasma renin activity (caused by suppression of plasma renin concentration) to degrees that were related to the increases in fetal plasma cortisol concentration. After cortisol infusions, renin responses to hypotension were apparently suppressed to degrees not obviously related to the rate of cortisol infusion. Fetal plasma ACTH responses to hypotension were completely suppressed by increases in total and unbound fetal plasma cortisol concentration 1.6 and 1.7 ng/ml, respectively. These results demonstrate a high sensitivity of the fetal hypothalamopituitary unit and renin angiotensin system to cortisol. PMID- 3010747 TI - Effects of thyroid hormone replacement on beta-adrenergic responsiveness of food deprived rats. AB - Thyroid hormone levels and beta-adrenergic responsiveness after stimulation with the beta-adrenergic agonist isoproterenol were studied in four groups of male rats. The groups used were control rats, rats deprived of food for 72 h, rats administered 50 micrograms thyroxine (T4)/kg daily for 4 days, and rats deprived of food for 72 h and administered 50 micrograms T4/kg daily for 4 days. Food deprivation significantly decreased serum T4 and triiodothyronine (T3) levels, and administration of T4 significantly increased serum T4 and T3 levels in both fed and food-deprived rats. Administration of T4 led to lower body weights in both fed and food-deprived rats. Administration of isoproterenol led to increases in colonic and tail skin temperatures and heart rate. Food deprivation significantly attenuated the increased body temperatures and heart rate induced by isoproterenol, and administration of T4 to food-deprived rats returned these adrenergic responses to control levels. Administration of T4 to fed rats significantly increased the thermal and cardiac responses to isoproterenol above those of the control rats. Administration of isoproterenol also increased plasma glucose levels. Food deprivation significantly decreased both postsaline- and isoproterenol-stimulated glucose levels. However, administration of T4 was without effect on either the postsaline- or isoproterenol-stimulated glucose levels of either the fed or food-deprived rats. The decreases in T4 and T3 that accompany food deprivation may thus be responsible for some, but not all, of the reductions in beta-adrenergic responsiveness observed in food-deprived rats. PMID- 3010748 TI - Malignant fibrous histiocytoma tumor cells resemble fibroblasts. AB - The malignant fibrous histiocytomas (MFHs) are a histologically heterogeneous group of sarcomas that have been postulated to be derived from, or have the capacity to differentiate into, histiocytes. To determine whether MFH tumor cells actually express the features of histiocytes, i.e., bone marrow-derived cells of monocyte-macrophage lineage, we studied the antigenic and enzymatic phenotype of 13 MFHs in situ using frozen and plastic sections, respectively. Five pleomorphic three fibrous, two myxoid, two giant cell, and one histiocytic MFH were studied. While tumor cells in 12 of 13 cases were positive for HLA-A,B,C, tumor cells in all cases failed to express antigens present on bone marrow-derived macrophages, i.e., leukocyte common antigen (L3B12), HLA-DR, Leu-M3, and Leu-3a. Interestingly 8 of 13 cases were positive for CALLA. Although nonspecific, this may prove useful in differential diagnosis. Enzyme histochemistry demonstrated that tumor cells in 9 of 13 cases were positive for membrane 5' nucleotidase (5'N+). Four of these were also alkaline phosphatase positive (ALKP+). All cases were either negative or weakly positive for acid phosphatase (ACIDP) and alpha-naphthyl acetate esterase (ANAE). Tumor cells were unreactive for alpha-naphthyl butyrate esterase (ANBE) and adenosine triphosphatase (ATP). These findings indicate that MFH tumor cells do not express the enzymatic profile of cells of monocyte/macrophage lineage which are membrane 5'N-/ALKP- and ACIDP+/ANAE+/ANBE+/ membrane ATP+. In fact, these data suggest a similarity to fibroblasts which are membrane 5'N+, variably ALKP+, weakly ACIDP+/ANAE+, and ANBE-/membrane ATP-. Osteoclast-like giant cells present in two cases did express a histiocytic phenotype, suggesting that they are reactive elements not derived from admixed tumor cells. These results suggest that MFHs are primitive mesenchymal neoplasms, most likely sarcomas composed of poorly differentiated fibroblasts, and are unrelated to true histiocytic neoplasms. PMID- 3010749 TI - Pleomorphic xanthoastrocytoma. Immunohistochemical methods for differentiation from fibrous histiocytomas with similar morphology. AB - Three cases of primary intracranial tumours fulfilling the clinical and histological criteria of pleomorphic xanthoastrocytoma are presented. As is typical, they occurred in young people, aged 15, 17, and 22 years, and were composed of lipid-laden pleomorphic cells with frequent bizarre multinucleated forms and a prominent reticulin network. However, subsequent immunohistochemistry, further histological review, and clinical follow-up suggest that these tumours were different entities. In one case the tumour cells were negative for GFAP but positive with a panel of histiocytic markers. The lesion, which extended rapidly and caused death within 6 months, was assumed to be a true meningeal fibrous histiocytoma. The remaining two cases were positive for glial fibrillary acidic protein (GFAP) and S100 protein. One of these was mitotically active, contained areas of necrosis and vascular proliferation, and also led rapidly to death. This, we concluded, was a glioblastoma. The third case showed little mitotic activity and the patient remains well; this is probably a true pleomorphic xanthoastrocytoma. These results indicate that tumours with light microscopic appearance of pleomorphic xanthoastrocytoma require detailed immunohistochemical investigation. Only those lesions with low mitotic activity and undoubted evidence of glial origin should be accepted as true pleomorphic xanthoastrocytoma. Extensive necrosis in a tumour with GFAP-positive, lipid-rich cells indicates a lipidised glioblastoma, while positive histiocytic immunocytochemistry should suggest a fibrous histiocytoma. PMID- 3010750 TI - Electron microscopy of Plasmodium vivax exoerythrocytic schizonts grown in vitro in a hepatoma cell line. AB - Human hepatoma cells (HepG2-A16) have been shown to be useful for the growth of the exoerythrocytic stages of P. vivax. In order to determine whether the parasites grown in these cells are morphologically similar to those grown in vivo, we performed electron microscopy on the exoerythrocytic (EE) schizonts of P. vivax developed from sporozoites. This study showed for the first time that P. vivax EE schizonts within these cells closely resemble other plasmodial EE schizonts both in vivo and in vitro. PMID- 3010751 TI - Investigations of the vertebrate hosts of eastern equine encephalitis during an epizootic in Michigan, 1980. AB - A study was undertaken to investigate an increase in reported cases of clinical encephalitis due to eastern equine encephalitis (EEE) virus in horses and to determine the natural vertebrate hosts of that virus. Horses, birds, and small mammals were sampled at sites in a contiguous area in St. Joseph and Kalamazoo counties, Michigan, from 25 August to 5 September 1980. Serum samples from four horses acutely ill with encephalitis and 16 of 39 pasture mates of ill horses had neutralizing (N) antibody against EEE virus (46.5%); no viruses were isolated from these 43 sera. None of 24 draft horses from a site in St. Joseph County 12 km southeast of the affected sites had EEE antibody. A strain of Cache Valley virus was isolated from the blood of one of the 24 draft horses. No viruses were isolated, and no antibodies to EEE virus were detected in 28 blood samples from small mammals captured at sites where equine cases of encephalitis were occurring. Six strains of EEE virus, five of Highlands J virus, and one of Flanders virus were isolated from the blood of 401 wild birds belonging to 42 species captured at eight sites in both counties. A total of 29.9% of the wild birds had EEE antibody. Five species of domestic birds, mostly chickens and ring necked pheasants, were sampled in both counties. Six strains of EEE virus were isolated from 152 ring-necked pheasants; these included three isolates from the brains of dead birds. About 13% of 51 pheasants tested from two small flocks in backyard pens in Kalamazoo County and 9% of 103 pheasants tested from a large game farm in St. Joseph County had antibody to EEE virus. The source of the EEE virus and the factors responsible for this epizootic are unknown; however, the epizootic probably represented an explosive expansion of an enzootic level of virus transmission. PMID- 3010752 TI - Fatal outcome in Japanese encephalitis. AB - Forty-nine consecutive patients with laboratory-confirmed acute Japanese encephalitis were studied to identify risk factors present at hospital admission which were associated with a fatal outcome. Sixteen patients (33%) died. The following constellation of findings correlated with a fatal outcome: infectious virus in cerebrospinal fluid (CSF), low levels of Japanese encephalitis virus specific IgG and IgM in both CSF and serum, and a severely depressed sensorium. Age, sex, days ill before admission, distance from home to the hospital, past medical history, CSF protein content, and CSF leukocyte count were not significant risk factors. Among patients hospitalized for acute Japanese encephalitis, a vigorous virus-specific immunoglobulin response, both systemically and locally within the central nervous system, is a good marker for survival, and may be an inherently important factor in recovery from illness. PMID- 3010754 TI - Nonpalpable breast cancer: needle-localized biopsy for diagnosis and considerations for treatment. AB - Screening mammography as an adjunct to physical examination led to the discovery of 237 radiographically suspicious but nonpalpable breast lesions. Needle localization of the lesion preoperatively in the mammography suite followed by breast biopsy led to the diagnosis of 64 nonpalpable carcinomas, including 25 invasive, 16 minimally invasive, and 23 noninvasive cancers. Noninvasive and minimally invasive cancers were microscopic. Of the invasive lesions, 7 were 10 mm or less in diameter and 14 were 11 to 20 mm in diameter. Noninvasive and minimally invasive cancers tended to occur in younger women (average age 52 and 51 years, respectively), and almost uniformly appeared as clustered calcifications mammographically. Invasive cancers affected an older population (average age 65 years), and the mammographic appearance was that of a mass in the majority of cases. A variety of surgical procedures were carried out subsequent to biopsy to provide definite treatment of these nonpalpable breast cancers. A review of surgical specimens available from these procedures demonstrated a 27 percent incidence of residual disease at the biopsy site. In patients who underwent mastectomy, 34 percent had an unsuspected focus of cancer in another quadrant of the breast and an additional 14 percent had an unsuspected focus of epithelial atypia. No patient with either noninvasive or minimally invasive cancer was found to have axillary lymph node metastases. Twenty-nine percent of patients with invasive tumors demonstrated lymph node metastases in the axilla. Our results demonstrate the efficacy of preoperative needle localization to assist in the biopsy of nonpalpable breast lesions and the diagnosis of a significant number of early breast cancers. The treatment plan for patients with these cancers must address the high incidence of residual disease at the biopsy site, multicentricity, and the proved capacity for invasive lesions to metastasize to the axillary lymph nodes, regardless of the size of the primary tumor. PMID- 3010755 TI - [Interrelations between hormones and 3',5'-cyclic adenosine monophosphate in patients of reproductive age with uterine myoma]. PMID- 3010756 TI - [Effect of 17 beta-estradiol on 3',5'-cyclic AMP phosphodiesterase of uterine tissues in myoma]. PMID- 3010753 TI - Detection of eastern equine encephalomyelitis viral antigen in avian blood by enzyme immunoassay: a laboratory study. AB - An enzyme immunoassay (EIA) was evaluated for its efficacy at detecting eastern equine encephalomyelitis (EEE) virus in avian blood and brain specimens. Preliminary analysis of blood from experimentally infected house sparrows and naturally infected whooping cranes showed that EEE antigen could be detected with the EIA. Polyclonal mouse antibodies were selected for antigen capture, and rabbit antibodies were selected for antigen detection. Overnight antigen incubation increased sensitivity. The lower limit of EEE antigen detection was 10(3.5) TCID50/ml for a stock of virus. Sensitivity was 10% (2/20) for antigen detection in the blood of chicks inoculated with EEE virus less than 24 hr earlier. At 24 and 48 hr after infection, sensitivity was 100% (10/10). Sensitivity and specificity of antigen detection were excellent (100% for both) in house sparrows experimentally inoculated with EEE, Highlands J (HJ), western equine encephalomyelitis (WEE), or St. Louis encephalitis (SLE) virus and bled at 24 hr intervals. Cross-reactivity was observed, however, with high concentrations (10(5.5) TCID50/ml) of HJ virus. EEE antigen was detected in avian blood by the EIA after infectious virus had declined to undetectable levels. The EIA is a useful alternative to virus isolation in cell culture for diagnosis or detection of EEE virus infections in birds. The test has the advantages of being simple, rapid, and capable of detecting antigen in the absence of infectious virus. PMID- 3010757 TI - [Current concepts of fibrocystic mastopathy]. PMID- 3010758 TI - [Results of combined gamma-proton irradiation of patients with cervical cancer]. PMID- 3010759 TI - Primary adenoid cystic carcinoma of the skin. A clinical, histological, and immunocytochemical comparison with adenoid cystic carcinoma of salivary glands and adenoid basal cell carcinoma. AB - An adenoid cystic carcinoma of the skin was compared with three similar neoplasms of salivary glands and with an adenoid basal cell carcinoma, from unrelated cases. The histological and immunocytochemical details of these tumors were analyzed in an attempt to determine whether or not their differing clinical behaviors would be reflected in pathologic dissimilarities. Although the single adenoid cystic carcinoma of the skin did not recur or metastasize over a 10-year follow-up period, its morphologic and biochemical features were identical to those of biologically aggressive salivary gland tumors. All four adenoid cystic carcinomas contained carcinoembryonic antigen, epithelial membrane antigen, salivary-type amylase, and alpha-lactalbumin, and all bound peanut agglutinin. Three of four expressed positivity for S100 protein, and two contained low molecular-weight cytokeratin. In contrast, none was immunoreactive for beta-2 microglobulin, and only one displayed blood group isoantigen positivity. The adenoid basal cell carcinoma was negative for all immunological determinants, but it bound peanut agglutinin. Although these results should be regarded as preliminary, it appears that adenoid cystic carcinoma is a pathologically distinct and uniform entity, whether it occurs in the skin or in salivary glands. However, the clinical behavior of this tumor cannot be predicted on the basis of immunohistochemical or morphological studies. Finally, adenoid basal cell carcinoma is histopathologically and immunocytochemically separable from cutaneous adenoid cystic carcinoma. PMID- 3010760 TI - Congenital granular-cell neoplasms. An unusual case report with ultrastructural findings and a review of the literature. AB - Congenital granular-cell tumors are uncommon benign neoplasms of unknown cause that develop primarily in gingival tissue and usually do not recur after surgical excision. Untreated, these neoplasms either cease to grow or "spontaneously" regress after birth. In this report, we describe an unusual case of a child with several congenital granular-cell neoplasms that arose on the lips and continued to increase in size as the child grew. Histologic examination of the neoplasms revealed them to be composed of granular and mesenchymal cells associated with abundant collagen fibers and prominent vascular structures. Ultrastructural study disclosed typical granular cells and a preponderance of immature mesenchymal cells, some of which appeared to be transitional or early granular cells. We interpreted these findings to mean that primitive mesenchymal cells are the proliferative elements and precursors of granular cells in congenital granular cell neoplasms. The presence of numerous immature mesenchymal cells corroborates the clinical impression that the lesions were growing. Based upon the histological and ultrastructural findings reported here, and a review of the literature, we favor a mesenchymal or endothelial-cell origin for congenital granular cell neoplasms. PMID- 3010761 TI - Nucleic acid chromatography: substitute solid support material for RPC-5 columns. AB - Chromatography of tRNA and DNA fragments on columns of reverse-phase 5 (RPC-5) exchange material has been widely employed for analytical and preparative studies. The Plaskon bead that formed the solid support on which a quaternary amine was absorbed is no longer commercially available. A Voltalef bead is available and provides similar, though not identical, chromatograms for Asp-tRNA, Ser-tRNA, and certain DNA fragments. Procedures are described for preparation of the column packing and for long-term operation of the column. PMID- 3010762 TI - Phase separation of rat intestinal brush border membrane proteins using Triton X 114. AB - Rat intestinal microvillus membrane contains at least 24 polypeptides, of which 18 can be solubilized using Triton X-114 at 4 degrees C. Upon phase separation at 32 degrees C, 11 proteins separated nearly completely into the detergent-rich phase, while 9 proteins were found exclusively in the aqueous phase. Enzymes which were uniquely included in the detergent phase were alkaline phosphatase, leucine aminopeptidase, gamma-glutamyl transpeptidase, and Ca2+-Mg2+ ATPase. The proteins which were excluded from the detergent phase and found exclusively in the aqueous phase included the disaccharidases (glucoamylase, sucrase-isomaltase, trehalase, lactase) and the ileal receptor for the intrinsic factor-cobalamin complex. Integral membrane proteins can thus be separated during solubilization into two groups prior to further purification or characterization. PMID- 3010763 TI - A radiochemical study of irreversible adsorption of proteins on reversed-phase chromatographic packing materials. AB - A radiochemical study of the irreversible adsorption of proteins on commercial reversed-phase HPLC packing materials is reported. The conditions of study are similar to those used in HPLC separation of protein. The effects of the amount and contact time of two proteins, ovalbumin and cytochrome c, are reported. Additional results include the effect of column pretreatment with protein, silanophilic mobile-phase blocking agent, and type of packing material on the extent of irreversible adsorption. The loss process is shown to be at least biphasic and the mechanisms of loss distinct for different proteins. PMID- 3010764 TI - Sample preparation for inositol measurement: Sep-Pak C18 use in detergent removal. AB - The enzymatic-fluorometric method of myo-inositol quantitation by L.C. MacGregor and F.M. Matschinsky (1984, Anal. Biochem. 141, 382-389) has proved to be very useful in accurately measuring small amounts of myo-inositol. Although this procedure is quite satisfactory, relatively high concentrations of detergents, salts, NADH, and malate interfere. To extend the usefulness of the MacGregor and Matschinsky method we report here an extraction procedure which removed the interfering substances, yet allowed the recovery of close to 100% of the inositol. The procedure involved first passing the sample through a Sep-Pak C18 cartridge to remove detergent and then through a mixed-bed resin to remove the ionic constituents. The procedure with the Sep-Pak C18 cartridge is applicable to a wide variety of biological samples requiring detergent removal. PMID- 3010765 TI - The assay of ATP-sulfurylase. AB - An assay method for ATP-sulfurylase is described, based on the incorporation of 35SO4(2-) into adenosine-5'-phosphosulfate (and 3-phosphoadenosine-5' phosphosulfate) and the separation of these compounds by HPLC. Since the enzyme is easily inactivated at temperatures above 20 degrees C during the assay, the reaction time should not exceed 10 min. PMID- 3010767 TI - Parameter estimation for ligand binding systems kinetics applied to 1,25 dihydroxycholecalciferol. AB - A mathematical analysis of the kinetics of the hormone-receptor interaction was applied to the 1,25-dihydroxycholecalciferol-intestinal receptor system. The exact analytical solution and the numerical integration of the kinetic equation were installed in a Statistical Analysis System (SAS) computer program to estimate the rate constants of the reaction. Estimates of the parameters obtained by these two methods are similar, demonstrating that the numerical integration can be combined with the nonlinear regression procedure for least-squares parameter fitting using a simple SAS program. This enables estimation of kinetics rate constants when the kinetic equation cannot be solved analytically. The ratio of the rate constants (ka/kd) found by the nonlinear procedure is close to the independently determined equilibrium (Scatchard) constant in the nonlinear analysis. PMID- 3010766 TI - Use of 5-(4-dimethylaminobenzylidene)rhodanine in quantitating silver grains eluted from autoradiograms of biological material. AB - 5-(4-Dimethylaminobenzylidene)rhodamine, a silver-specific dye, was used in a colorimetric assay to quantitate the autoradiographic deposition of silver onto X ray film after exposure to sodium dodecyl sulfate-polyacrylamide gels of radiolabeled biological material. Silver grains were eluted from autoradiograms with 5 N potassium hydroxide, dissolved in nitric acid, and neutralized with 1 M Trizma Base. The concentration of silver was measured spectrophotometrically owing to the chelation properties of the dye. After corrections for background exposure were made, the silver contents of excised bands were then determined by comparison to a standard curve generated with silver nitrate. We have used this silver assay to quantitate the relative amount of each polypeptide band comprising the polyomavirus structural protein VP2 doublet. The method reported here has proven useful when densitometry is inconvenient (i.e., short distance between bands, irregular shape of bands, very faint bands) in addition to being inexpensive and simple to perform. PMID- 3010768 TI - Preparation of rat liver plasma membranes in a high yield. AB - Existing procedures for the preparation of rat liver plasma membranes are time consuming and generally produce low yields. A method is described in which a rat liver homogenate low-speed pellet is fractionated on a self-forming Percoll gradient. Plasma membranes can be removed from the gradient in a high yield along with much of the DNA in the liver homogenate. A second Percoll step performed in the presence of a low concentration of calcium ions separates the DNA from the plasma membranes. The final membrane fraction has high specific activities of marker enzymes with little contamination with microsomal, mitochondrial, Golgi, or lysosomal markers. PMID- 3010769 TI - A simplified procedure for the rapid identification of recombinant pAT153 plasmids in Escherichia coli HB101 cells. AB - Insertion of foreign DNA into the unique HindIII site of the high copy number plasmid pAT153 reduces but does not completely abolish the resistance of Escherichia coli HB101 cells to tetracycline. Recombinant DNA-containing colonies could then be phenotypically differentiated from non-recombinant ones by their smaller size on nutrient agar plates with ampicillin and tetracycline at a final concentration of 50 and 4 micrograms/ml, respectively. A wide variety of human cytomegalovirus DNA fragments have been found in pAT153 molecules propagated by the ampicillin-resistant tetracycline-sensitive bacteria selected. PMID- 3010770 TI - Isolation of DNA from fungal mycelia and sclerotia without use of density gradient ultracentrifugation. AB - A rapid method for extracting total DNA from Aspergillus flavus and Aspergillus parasiticus has been developed. The procedure can be completed in 2 h and yields 200 to 350 micrograms of DNA from 0.5 to 1.0 g wet wt of mycelia and 150 micrograms from 0.5 g of sclerotia. DNA samples had an OD260/OD280 of 1.6 to 1.8. Most of the DNA was at least 50 kb pairs in size and showed little degradation. DNA prepared by this method was used for restriction endonuclease digestion and Southern blotting. A DNA fragment containing the repeat unit of the ribosomal RNA genes of A. flavus has been identified. PMID- 3010771 TI - Use of specific collagenases for the isolation of rat liver cells with preserved lipase activities. AB - The heparin-releasable neutral lipase (EC 3.1.1.3) from rat liver is inactivated by the common preparations of collagenases (EC 3.4.24.3) used for the isolation of liver cells. We show that two collagenases purified from Clostridium histolyticum allow both the complete preservation of this lipolytic activity and a good viability of liver cells isolated by the usual perfusion protocol. PMID- 3010772 TI - The quantitative determination of 2'-deoxycytidine-5'-triphosphate in cell extracts by radioimmunoassay. AB - A radioimmunoassay (RIA) capable of quantitating dCTP in femtomolar amounts in cell extracts has been developed, and applied to human fibroblast cell lines and L5178Y mouse lymphoma lines. Cross reactivity of the antibody with CTP, though low (2.7%) has necessitated pre-RIA removal of CTP by either boronate affinity gel chromatography or sodium periodate oxidation. Fractions from the boronate gel column or aliquots of NaIO4-treated cell extract are quantitated directly by the RIA. Recovery of extracted dCTP standard taken through the entire procedure is quantitative and results are reproducible. Due to the high sensitivity of the quantitation step, dCTP can be accurately measured in relatively small numbers of cells--about 10(4) cells. PMID- 3010773 TI - Scanning molecular sieve chromatography of interacting protein systems: nonlinear least-squares analysis of small zone experiments. AB - A simple routine for nonlinear least-squares analysis is applied to small zone scanning data, where calibration of the gel column requires the use of fully characterized markers of known molecular size. Application of nonlinear least squares analysis eliminates the difficulty encountered because of scarcity of calibrating markers for gels whose porosities span certain size ranges by providing a sensitive measure of changes in molecular size of the interacting protein system, in order to determine the interaction parameters, size heterogeneity, and the centroid position for time difference chromatographic experiments. PMID- 3010774 TI - Solid-phase extraction and high-performance liquid chromatography analysis of lipoxygenase pathway products. AB - Liquid chromatography methods for quantitation of leukotrienes and HETEs (hydroxyeicosatetraenoic acids) in biological samples are described. Extraction is accomplished by acetonitrile precipitation of proteins followed by selective acetonitrile elution from a solid-phase C18 extraction cartridge. Isocratic elution of extracts from short reverse-phase columns with 3 microns C18-bonded silica results in elution of all components of interest in less than 10 min. The addition of the mobile-phase additives, trifluoroacetic acid and triethylamine, enable the peptido-leukotrienes to be eluted with excellent peak shape and unique elution times. As little as 1 pmol of each metabolite can be detected by uv spectrophotometry with a wavelength change from 280 to 235 nm midway through the chromatographic run. This method is demonstrated by the extraction and analysis of leukotriene and HETE standards from human serum. PMID- 3010775 TI - Tryptic peptide mapping of ubiquitin and derivatives using reverse-phase high performance liquid chromatography. AB - The conditions for tryptic digestion and subsequent peptide mapping of the ATP dependent proteolysis cofactor ubiquitin and its derivatives are described. In aqueous solution, the native ubiquitin which is composed of 76 amino acids undergoes only a single cleavage at arginine-74. Full digestion of ubiquitin was obtained in 6.5 M urea, although cleavages at lysine-33 and arginine-74 were slow. Peptide mapping was achieved by reverse-phase high-performance liquid chromatography with a C18 column using a trifluoroacetic acid/triethylamine buffer system and acetonitrile as eluants. The peptides, separated using a linear gradient, were identified by amino acid analysis. Derivatives analyzed by this method include oxidized, monoiodotyrosyl, and diiodotyrosyl ubiquitin. This technique will be useful in examining peptides of chemically modified ubiquitin with respect to extent and specificity of modification. In addition, this technique will be useful in comparing ubiquitin peptides of different organisms. PMID- 3010776 TI - Specific-primer-directed DNA sequencing. AB - A simple and rapid strategy for DNA sequence analysis based on the Sanger chain termination method is described. This procedure utilizes full-sized inserts of 1 to 4 kb of DNA cloned into M13 bacteriophage vectors. After the sequence of the first 600-650 bp of the insert DNA has been determined with the commercially available universal vector primer, a specific oligonucleotide is synthesized utilizing the sequence data obtained from the 3' end of the sequence and used as a primer to extend the sequence analysis for another 600-650 nucleotides. Additional primers are synthesized in a similar manner until the nucleotide sequence of the entire insert DNA has been determined. General guidelines for the selection of oligonucleotide length and composition and the use of unpurified primers are discussed. The use of the specific-primer-directed approach to dideoxynucleotide sequence analysis, in association with highly purified single stranded template DNA, reduces considerably the time required for the analysis of large segments of DNA. PMID- 3010777 TI - Effects of the hypolipidemic drug nafenopin on the zona glomerulosa of the rat adrenal cortex: morphological counterparts of functional alterations. AB - The effects of nafenopin, a hypolipidemic drug, on the zona glomerulosa of the rat adrenal cortex were investigated. Chronic nafenopin treatment significantly lowered serum cholesterol level, but did not alter blood aldosterone concentration, though the biosynthesis of adrenal cortico-steroid hormones seems to be largely dependent upon a continuous uptake of cholesterol from plasma lipoproteins. Stereology showed that the treatment provoked a notable lipid droplet depletion, coupled with a significant proliferation of smooth endoplasmic reticulum (SER) profiles. Since SER is known to be involved in the endogenous synthesis of cholesterol, the hypothesis is advanced that SER hypertrophy is a compensatory response enabling zona glomerulosa cells to maintain an adequate level of aldosterone output even in the absence of a normal supply of exogenous cholesterol. PMID- 3010778 TI - Ultrastructural and ultrahistochemical studies on the ventral aorta in larvae of a teleost, Poecilia reticulata. AB - The ultrastructure of the ventral aorta of Poecilia reticulata is described in larvae of variable age. In postnatal larvae this structure is located within the ventral pharyngeal wall. The tunica media consists of elongated smooth muscle cells, surrounded by a network of elastic fibrils (10 to 20 nm). The muscle cells are rich in free and membrane associated ribosomes, and contain a well developed Golgi apparatus surrounded by membrane-bound, electron dense bodies. During the first 21 d of postnatal life, the number of ribosomes declines gradually, whereas there is a corresponding increase in the amount of contractile material. In addition, the number of intercellular, elastic fibrils seems to increase with advancing age. No elastic lamina was seen in the present tissue, but in the 21 d old larvae the subendothelial elastic fibrils are sometimes aligned and may therefore make up an elastic lamina at a later stage of development. Probably, the elastic fibrils are composed of a central string (2-4 nm) of unknown chemical nature, covered by a layer of elastin. Thus, the elastic fibrillar network in young and adult bony-fish may be regarded as heavily reticulose elastin laminae, resembling those of the early fetal aorta in mammals. The present intercellular elastin is shown to be intensely stained with phosphotungstic acid at low pH and may therefore be rich in basic proteins. In 3 mm prenatal larvae the heart and the posterior part of the aorta are located at the outside of the body, whereas the anterior part occurs in the ventral pharyngeal wall. The aortic endothelium and tunica media consist of undifferentiated cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010779 TI - Glucose 6-phosphatase activity in pregnant and lactating mammary glands of the mouse. AB - Glucose 6-phosphatase activity was studied in the secretory epithelial cell and other cell types composing alveoli of the mammary gland (cytochemical study) and in the whole mammary gland (biochemical study) of pregnant and lactating mice. The reaction product for the enzyme activity was seen in the endoplasmic reticulum and nuclear envelope in secretory epithelial cells from all animals examined (days 7 and 14 of pregnancy, and days 0, 3, 10, and 20 of lactation. The amounts of the reaction product appeared scarce at day 7 of pregnancy, moderate at day 14 of pregnancy and day 0 of lactation, and abundant at days 3 and 10 of lactation. The reaction product, however, became generally scarce at day 20 of lactation. Biochemical activity was relatively low at days 7 and 14 of pregnancy and days 0 and 20 of lactation, while it was high at days 3 and 10 of lactation. The increased activity is probably related to functions of secretory epithelial cells in the lactating gland. PMID- 3010780 TI - CDC Guidelines for the Prevention and Control of Nosocomial Infections. Guidelines for prevention of surgical wound infections, 1985. PMID- 3010781 TI - The role of leukotriene B4 in the genesis of oxygen toxicity in the lung. AB - Leukotriene B4 (LTB4) is a metabolite of arachidonic acid that has potent chemotactic activity for polymorphonuclear leukocytes (PMN). Pulmonary oxygen toxicity is considered to be a good model of an acute inflammatory lung injury, and an increase in the number of PMN is found in the lungs acutely injured by hyperoxia. In order to estimate the role of LTB4 responsible for this influx of PMN, we measured the LTB4 by radioimmunoassay in lung lavages of rats exposed to hyperoxia for 60 h. We found that the level of LTB4 in lung lavages in rats exposed to hyperoxia for 60 h increased significantly compared with that in normoxic control rats. At the same time, the marked increase in the number of PMN in lung lavages and the decrease in the activity of NADPH-cytochrome c reductase in lung microsomes were also observed. The administration of AA861, a 5 lipoxygenase inhibitor, reduced not only the increase in LTB4 but also the increase in the number of PMN in lung lavages of rats exposed to hyperoxia for 60 h. Furthermore, treatment with AA861 also protected the decrease in the activity of NADPH-cytochrome c reductase. The effects of AA861 on these parameters were observed in a dose-dependent fashion. In addition, there is a good correlation between the level of LTB4 and the number of PMN in the lavage of rats exposed to hyperoxia for 60 h with or without AA861 administration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010782 TI - Recurrent genital herpes and suppressive oral acyclovir therapy. Relation between clinical outcome and in-vitro drug sensitivity. AB - To evaluate the association between in-vitro resistance of herpes simplex virus type 2 to acyclovir and breakthrough recurrences of herpes despite chronic suppressive therapy, we determined the in-vitro sensitivity of herpes simplex virus isolated before, during, and after therapy. One hundred eighty-three virus isolates from 107 patients were tested. Before therapy, the median amount of drug required to inhibit 50% of the virus in tissue culture (ID50) was 0.91 microgram/mL. The median ID50 after therapy was 0.99. Six isolates from patients with culture-positive breakthrough recurrences were evaluated. The median ID50 was 0.90 microgram/mL (range, 0.39 to 1.55). The development of breakthrough recurrences could not be correlated with infection with strains of herpes simplex virus type 2 that were resistant to acyclovir in vitro. Acyclovir-resistant strains are not commonly recovered from patients during acyclovir therapy, nor does there seem to be a high frequency of resistance after 4 months of chronic suppressive therapy. PMID- 3010783 TI - Recommendations for preventing transmission of infection with human T lymphotropic virus type III/lymphadenopathy-associated virus during invasive procedures. Centers for Disease Control, Department of Health and Human Services. AB - Previous recommendations on preventing the transmission of the human T lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) in the workplace have emphasized health care settings. The current recommendations complement those previously discussed by listing specific guidelines to be followed by health care workers who perform or assist in operative, obstetric, and dental invasive procedures. All such health care workers should be educated about the epidemiology, transmission, and prevention of HTLV-III/LAV infection. All workers should use gloves and follow the appropriate barrier precautions during invasive procedures. Workers should use extraordinary care to prevent injuries to the hands caused by needles, scapels, and other sharp instruments. No serologic testing of health care workers involved in invasive procedures is indicated because the risk of transmission in this setting is so low. Although there have been no reports of HTLV-III/LAV transmission during an operative procedure, this situation warrants attention to these additional precautions. PMID- 3010784 TI - Epstein-Barr virus infection and Hodgkin's disease. PMID- 3010786 TI - Biochemical and neuroendocrinological effects of electroconvulsive therapy. Discussion of Part II. PMID- 3010785 TI - Changes in gamma-aminobutyric acid biochemistry and seizure threshold. PMID- 3010788 TI - Neuroendocrine responses as a probe into the mechanisms of action of electroconvulsive therapy. PMID- 3010787 TI - Endogenous opioids and their receptors. Evidence for involvement in the postictal effects of electroconvulsive shock. PMID- 3010789 TI - The biochemistry of electroconvulsive therapy. Discussion of Part III. PMID- 3010790 TI - Neurochemical mechanisms of mood stabilization. Focus on electroconvulsive therapy. PMID- 3010792 TI - [Viral serology in ear diseases of presumably neuritic origin]. AB - A viral etiology is often advanced to explain the origin of Bell's palsy, sudden deafness and certain vertigos. Virology findings were compared in 72 patients with either chicken pox, herpes, zona or other "viral" infections, where the viral origin was not absolutely clear, and a population of healthy volunteers. Results confirmed the indefinite viral nature of these affections. However, recent studies have shown that definite viral disorders are not always associated with an increase in antibody levels, and the intervention of a virus should not be totally discounted in some of these affections, especially as several particularly demonstrative cases have provided confirmation of a virus origin, particularly the Epstein-Barr virus. PMID- 3010791 TI - Effects of electroconvulsive shock and serotonin axon lesions on beta-adrenergic and serotonin-2 receptors in rat brain. PMID- 3010793 TI - [Current strategy for the radiological exploration of the ear]. AB - CAT scan by thin slices offers the possibility of evaluation of the bone structures of the ear and their contents. The progressively increasing availability of the scanner should make access much more routine for study of the ear, with a concomitant reduction in tomographies, inadequate in a large number of circumstances. The 4 main indications are chronic otitis with cholesteatoma, trauma, acoustic neuromas and malformations. The respective performances of tomography and CAT scan in each of these situations are discussed and an investigation strategy is suggested. PMID- 3010794 TI - Multiple stenoses of the small intestine by metastatic spread from breast cancer. AB - We present a patient who had a left mastectomy for breast cancer and 3 years later developed intestinal obstruction due to metastatic spread to the small bowel. The small bowel follow through showed multiple stenoses of the small intestine leading to a variety of differential diagnostic considerations as metastatic spread from breast cancer to the small intestine has not been convincingly documented so far. PMID- 3010795 TI - Antibodies as diagnostic probes in the identification of anaplastic round cell malignancy. AB - The introduction of hybridoma technology has rapidly expanded the role of immunohistochemical analysis in identification of anaplastic malignancy. High affinity antibodies now allow the detection of a wide range of tissue specific antigens so that anaplastic tumours can be accurately classified without the need for costly and often unrewarding, time-consuming ancillary investigations such as electron microscopy, cell culture and chromosomal studies. This review examines the application of a selected panel of commercially available antibodies of proven specificity for the diagnosis of anaplastic round cell tumours. Details of specific examples are provided to illustrate the role of such tissue specific antibodies as diagnostic probes and the approach to anaplastic tumours in various sites is discussed. PMID- 3010796 TI - Steroidogenic expression of delta 5-3 beta-hydroxy pathway of androgen biosynthesis by human breast carcinoma. AB - In order to determine whether human mammary tumours could contribute to cholesterol and dehydroepiandrosterone biosynthesis in breast cancer patients, incubations of homogenates of infiltrating ductal carcinoma were carried out separately with 2-14C acetate (patient, n = 6; 21-80 years) and 7n-3H pregnenolone (patient, n = 5, 36-74 years) as substrates. With the aid of the reverse-isotope dilution technique, 14C cholesterol and 3H dehydroepiandrosterone were characterized; these two gave maximum yields of 2.38 X 10(-4)% and 9.42 X 10(-2)% respectively. These results indicate that there exists a potential for the synthesis of cholesterol and dehydroepiandrosterone in vivo. It is suggested that the biosynthetic pathway involved in the synthesis of the latter is; acetyl CoA----cholesterol----pregnenolone----17 alpha-hydroxypregnenolone--- dehydroepiandrosterone. PMID- 3010797 TI - Intratumour amyloidosis in Malaysians: an immunohistochemical study. AB - Congo red screening of tumour material examined at the Department of Pathology, University of Malaya revealed intratumour deposits of amyloid in 12% of nasopharyngeal carcinomas, 66% of basal cell carcinomas, 100% of medullary carcinomas of the thyroid, 56% of islet cell tumours of the pancreas, 1 out of 16 carcinoids and 1 out of 100 thyroid adenomas. All the deposits were permanganate resistant and did not contain AA protein, indicating that what was encountered was not secondary amyloid. The deposits showed variable staining for immunoglobulin light chains and amyloid P component with a standard peroxidase antiperoxidase method. The possibility that intratumour amyloid has a neoplastic origin is discussed. PMID- 3010798 TI - Sclerosing haemangioma in Singapore. AB - The Singapore experience with 11 cases of sclerosing haemangioma of lung seen from 1970-1984 is analysed. There is female preponderance (9/11) and the majority (8/11) were asymptomatic. Seven patients were treated by wedge resection and 4 had lobectomies. Microscopically, the lesion had variegated growth pattern with characteristic polygonal cells present in the stroma. All patients were well after surgery. The longest follow-up period is 7 years with no recurrence seen. This is an unusual, distinctive and benign entity which should be distinguished from other inflammatory conditions in the lung. PMID- 3010799 TI - Patterns of viral respiratory tract infections in Singapore. AB - A second retrospective study on viral respiratory tract infections, based on 12 years' laboratory reports for the influenza viruses, and 9 years' reports for the other respiratory viruses was made. Seasonal variation in incidence was found to occur with infections due to the influenza viruses, respiratory syncytial virus, and parainfluenzavirus type 1. Observations made previously on the age distribution of specific respiratory viral infections, and the association of the viruses with various respiratory syndromes were generally confirmed. PMID- 3010801 TI - In vitro or in vivo receptor binding: where does the truth lie? PMID- 3010800 TI - Cerebrospinal fluid polyamines: biochemical markers of malignant childhood brain tumors. AB - The clinical value of cerebrospinal fluid (CSF) polyamine determinations in childhood medulloblastoma has been suggested. We performed 72 CSF polyamine determinations in 35 children with primary brain tumors. Spermine values were normal and spermidine values were inconsistently elevated. CSF putrescine values, however, were consistently elevated in patients with histologically malignant brain tumors: medulloblastoma, ependymoma, pineal germ cell tumors, primitive neuroectodermal tumors, and brainstem gliomas. Children with supratentorial astrocytomas had normal CSF polyamine values. CSF putrescine values were closely correlated with clinical state, with the highest concentrations identified in patients with widely disseminated recurrent disease. We found CSF putrescine to be a sensitive indicator of active disease in childhood malignant brain tumors. Further investigation is warranted into the predictive value of CSF polyamines in determining tumor relapse before clinical or other diagnostic studies reveal recurrent disease. PMID- 3010802 TI - Giant axonal neuropathy: central abnormalities demonstrated by evoked potentials. AB - Previous studies of giant axonal neuropathy have reported clinical and pathological findings that indicate involvement of the central nervous system. We studied 3 boys with giant axonal neuropathy, who were 14 to 16 years of age, using auditory, visual, and somatosensory evoked potentials. Absence of waveforms and prolongation of peak and interwave latencies were found. Abnormalities were noted in all modalities. The auditory brainstem evoked response in particular indicated a significant increase in brainstem conduction time. These studies add clinical neurophysiological confirmation of the central nervous system involvement in this disorder and may also provide a means of quantitative evaluation of its progression. PMID- 3010803 TI - Assays of leukocyte chemotaxis. AB - A number of disorders of leukocyte motility and chemotaxis have been reported clinically. In this paper we present a rational approach for testing cell populations for a defect in leukocyte migration and for defining the cellular basis of any abnormality observed. The principles and difficulties of individual and population-type assays are discussed. PMID- 3010804 TI - Molecular mechanisms of platelet aggregation. AB - Recent studies suggest that platelet stimulation leads to the induction of specific fibrinogen receptors, which have been identified as the platelet membrane glycoprotein IIb-IIIa complex. The binding of fibrinogen initiates the reversible primary phase of aggregation. With a strong stimulus, thrombospondin is released from the platelet alpha-granules. By interacting with fibrinogen, thrombospondin serves to stabilize the platelet aggregates, which leads to a secondary irreversible phase of aggregation. PMID- 3010805 TI - Infections of the gastrointestinal tract in the immunocompromised patient. AB - Immunocompromised patients are susceptible to a variety of serious gastrointestinal infections. Among the most common or most severe are Mycobacterium avium-intracellulare, herpes simplex, candidiasis, cryptosporidiosis, and Salmonellosis. Some of these infections are difficult or impossible to treat successfully. The correct diagnosis often can only be established if it is first suspected and the appropriate studies are performed. PMID- 3010806 TI - Cellular mechanisms of cholestasis. AB - Concepts regarding the pathogenesis of cholestasis continue to evolve as investigational techniques improve and molecular mechanisms of bile formation are clarified. With the accumulation of information it is becoming increasingly evident that cholestasis results from not one but multiple disturbances in the sequence of events responsible for bile production. In addition to the inhibition of bile flow attributable to initiating events, cholestasis itself may inhibit bile flow by altering the intra- and extracellular environments of liver cells. Many questions about cholestasis remain unanswered, but future directions for research are suggested by the information currently available. PMID- 3010807 TI - Inherited complement component abnormalities. AB - Inherited deficiencies of early complement components are frequently associated with immune/rheumatic disorders and recurrent (Neisserial) infection with deficiency of late complement components. The genetic complexity of complement components is further demonstrated by electrophoretic allotypy and analysis of DNA restriction fragment length polymorphisms. PMID- 3010808 TI - DNA analysis in genetic disorders. AB - Recombinant DNA technology has applications in the analysis of an ever-increasing number of genetic disorders. This review discusses the basic genetic principles underlying recombinant DNA techniques, the general approach to analyzing genetic disorders using these methods, and finally the practical applications of DNA analysis in genetic disease. PMID- 3010809 TI - Cellular resistance to protozoal infection. AB - Several factors appear to determine cellular resistance to protozoal infection. As judged by the interaction at the cellular level between the oxygen-dependent and -independent antimicrobial mechanisms of the human mononuclear phagocyte and the intracellular protozoa, Toxoplasma gondii and Leishmania donovani, these determinants include the cell's state of maturation and activation, the magnitude of the phagocyte's oxidative burst capacity, the ability of the cell to respond specifically to parasite ingestion with the production of toxic oxygen intermediates, parasite susceptibility to H2O2, and the presence of and protozoan susceptibility to oxygen-independent mechanisms. PMID- 3010810 TI - Toxicity of parathyroid hormone in uremia. AB - The most significant complication of elevated parathyroid hormone (PTH) levels in uremia is the development of osteitis fibrosa cystica. The hormone also appears to play a role in soft-tissue and organ calcification, metabolic abnormalities (glucose, lipids), and electroencephalographic changes seen in uremic patients. Its role in the hematological abnormalities of uremia (anemia, bleeding) is controversial. A role for PTH in heart and skeletal muscle dysfunction in uremia has not been clearly established. Further studies are required to establish PTH as a "universal" toxin in uremia. PMID- 3010812 TI - Functional activities of hepatic lipoprotein receptors. PMID- 3010811 TI - Treatment of acid-peptic diseases by inhibition of gastric H+,K+-ATPase. AB - The substituted benzimidazoles are a new class of drugs with a unique antisecretory action. These agents have great potential for treatment of acid peptic disease because they produce substantial, long-lasting inhibition of gastric acid secretion by inhibiting gastric H+,K+-ATPase in the gastric parietal cell. Clinical trials of omeprazole, a substituted benzimidazole, indicate that it is safe and effective for short-term treatment of patients with duodenal or gastric ulcer, and it is highly effective for long-term treatment of patients with Zollinger-Ellison syndrome. PMID- 3010813 TI - Ca2+ and cyclic AMP in regulation of intestinal Na, K, and Cl transport. PMID- 3010814 TI - Functions of the endothelial cell surface. PMID- 3010816 TI - Intracellular pH regulation by vertebrate muscle. AB - Regulation of pHi in the face of acidosis resulting from contracture would appear to be of such fundamental importance to the physiology of the muscle cell that a process common to all muscle types seems a reasonable prediction. However, this has not been found to be the case. The transmembrane Na+ gradient clearly plays a major role and the process appears to be electroneutral in all three classes of muscle, but the transport mechanisms, even within the mammal, are different. It is an interesting observation that the ability of the muscle cell to regulate pHi in the presence of CO2, presumably governed by PHCO3, is related to PCl although there is little evidence for HCO3- permeation through Cl- channels. Virtually no recovery from CO2-induced acidosis is observed in normally polarized frog skeletal muscle, where PCl forms a large part of the resting conductance, whereas the same steady state pHi is recorded in the presence of various CO2 levels in mammalian smooth muscle, where PCl is very low. The study of pHi regulation in vertebrate muscle has provided important lessons for the subject as a whole. Experience in cardiac muscle has shown that if Na+-Ca2+ exchange is present, great care is required in interpretation of results where the transmembrane Na+ gradient is altered or where Ca2+ levels are changed. Interpretation may be even more complex, bearing in mind the recent reports that Ca2+ inhibits Na+-H+ exchange. "Indeed," it seems appropriate to conclude, "if a little knowledge is dangerous, where is the man who has so much as to be out of danger?" (Thomas Huxley). PMID- 3010815 TI - Endothelial cell metabolism of biogenic amines. PMID- 3010817 TI - Effects of growth factors on intracellular pH regulation. PMID- 3010818 TI - Intracellular pH regulation in epithelial cells. PMID- 3010819 TI - Mechanisms and consequences of pH-mediated cell regulation. PMID- 3010820 TI - ATP-driven H+ pumping into intracellular organelles. PMID- 3010821 TI - Neural grafting in the aged rat brain. PMID- 3010822 TI - Neuronal receptors. PMID- 3010823 TI - Mechanism of action of gonadotropin releasing hormone. AB - GnRH interacts with a plasma membrane receptor to provoke gonadotropin release, as well as regulate numbers of its own receptor and target cell responsiveness. Receptor numbers are altered in different physiological states of the animal. Microaggregation of the GnRH receptor mimics all known actions of the releasing hormone, and therefore is viewed as an early step in the molecular mechanism of hormone action. Internalized hormone is neither necessary nor sufficient for stimulation of known releasing hormone actions. Evidence summarized in the present work suggests that Ca2+ serves a role as a second messenger for GnRH stimulated gonadotropin release, and that it may be involved in receptor up regulation in response to the releasing hormone. In the former role, diacylglycerols by their action on protein kinase C, in a fashion independent of the Ca2+ -calmodulin system, may act as a signal amplifier. PMID- 3010825 TI - Mechanism of thyrotropin releasing hormone stimulation of pituitary hormone secretion. PMID- 3010824 TI - Inositol phospholipid metabolism in the kidney. AB - The unique features of renal phosphoinositide metabolism include an increase in tissue phosphoinositide levels induced by PTH. The significance of this finding remains unclear. Another unusual finding is the localization of phospholipase C activity in a BBMV preparation. As suggested in the review, the transducing mechanism involving cleavage of phosphoinositides by a phospholipase C would be expected to include a close association between phospholipase C and the plasma membrane. However, few attempts to localize phospholipase C activity in the plasma membrane have succeeded. The kidney also plays an unusual role in inositol metabolism in that it is the only organ that significantly catabolizes inositol. The kidneys also synthesize inositol. There is an enormous concentration of inositol in the outer medulla. This coexistence of significant inositol synthesis, breakdown, and the presence of extremely high amounts of free inositol is an intriguing but unexplained phenomenon. The substantial rate of endogenous renal inositol synthesis does not, however, preclude inositol deficiency states. There is a deficiency of inositol in diabetic peripheral nerve and in glomeruli isolated from diabetic rats. Such deficiencies may arise from a disturbance in the balance of synthesis, breakdown, and excretion of inositol, and particularly from the competition of glucose with the inositol transporter in the proximal tubule. Future studies of renal phosphoinositide metabolism need to address both basic cell biological questions and broader physiological or functional questions. The more basic issues include the question of which phosphoinositide is being attacked by agonist-stimulated phospholipase C. That is, are all the events explained by hydrolysis of PtdIns(4,5)P2, or are the other phosphoinositides hydrolyzed as well? Also, it would appear that stimulated phosphoinositide metabolism occurs quite early following receptor occupation, but there is still no way of selectively blocking stimulated phosphoinositide metabolism to see if it is a necessary first step in a cascade of events leading to cell response. Thus, the relationship of stimulated phosphoinositide metabolism to cell functions remains incompletely understood. At least two cellular functional or biochemical changes associated with stimulated phosphoinositide metabolism in the kidney have been identified, prostaglandin production and mesangial cell contraction. The regulation of prostaglandin production and its relationship to stimulated phosphoinositide metabolism are subjects of continuing study. The topic was recently reviewed by Hassid.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3010827 TI - Growth hormone releasing hormone. PMID- 3010826 TI - Endogenous opioid peptides and hypothalamo-pituitary function. PMID- 3010828 TI - Temperature receptors in the central nervous system. PMID- 3010829 TI - Oxy-radicals and related species: their formation, lifetimes, and reactions. PMID- 3010831 TI - Antioxidant defenses in the lung. PMID- 3010830 TI - Oxidant production and bactericidal activity of phagocytes. PMID- 3010832 TI - The relation of free radical production to hyperoxia. PMID- 3010834 TI - ATP and the regulation of renal cell function. PMID- 3010833 TI - Identification of cellular activation mechanisms associated with salivary secretion. AB - In recent years, our understanding of receptor-signalling mechanisms in the salivary glands has advanced considerably. Two receptor pathways exist, one involving cAMP, which primarily regulates enzyme secretion, and another involving the hydrolysis of PIP2, which regulates Ca2+ mobilization and, subsequently, monovalent ion fluxes probably important in ion and water secretion in the intact gland. Mobilization of Ca2+ results from both the release of internal Ca2+, and from Ca2+ entry from the extracellular space. The signal for Ca2+ release appears to be (1,4,5)IP3, one of the water soluble products of PIP2 hydrolysis. The mechanism controlling Ca entry is not understood, but speculation abounds. Hydrolysis of PIP2 also produces DG, which has a messenger role in activating a specific protein kinase, the C kinase. The C kinase interacts with Ca2+ mobilization in some as yet uncharacterized way in regulating enzyme secretion. PMID- 3010835 TI - Inhibition of murine cytomegalovirus lung infection and interstitial pneumonitis by acyclovir and 9-(1,3-dihydroxy-2-propoxymethyl)guanine. AB - We compared the effects of acyclovir (ACV) and 9-(1,3-dihydroxy-2 propoxymethyl)guanine (DHPG) on murine cytomegalovirus (MCMV) replication in lung and salivary gland tissues, the evolution of interstitial pneumonitis in vivo, and MCMV replication in mouse embryo cells in vitro. As measured by plaque reduction, ACV was more active than DHPG in vitro. In vivo, whether administered orally by gastric intubation or in the drinking water, or subcutaneously, DHPG was more effective than ACV in reducing MCMV titers in lung or salivary gland tissues. This was true in both normal and cyclophosphamide-treated mice. Neither drug was able to prevent MCMV interstitial pneumonitis, despite substantial reductions in virus titer, but both drugs reduced the severity of the pneumonitis. PMID- 3010838 TI - Use of an enzyme-linked immunosorbent assay performed directly on fixed infected cell monolayers for evaluating drugs against varicella-zoster virus. AB - An in situ enzyme-linked immunosorbent assay (ELISA), performed directly on fixed varicella-zoster virus-infected monolayers, was used to quantitate viral antigen, and by color reduction it was used to evaluate the activity of drugs against varicella-zoster virus. Color production in the ELISA (optical density) was directly related to the dose of input virus. Antigen representing 5 to 10 plaques could be detected 3 days after infection. The ELISA was specific and reproducible, as shown by absorption and repeat experiments, respectively. A color-reduction test by ELISA was compared with the conventional plaque-reduction assay for its ability to measure the antiviral activity of acyclovir, bromovinyldeoxyuridine, trifluorothymidine, and vidarabine against four strains of varicella-zoster virus. In all cases but one the 50% inhibitory doses were lower when measured by ELISA than by the plaque-reduction assay. This in situ ELISA color reduction method had the following advantages over the conventional plaque-reduction assay: the endpoint was an objective measurement; there was less well-to-well variation; the assay was sensitive to changes in plaque size as well as plaque number; it was less labor intensive. PMID- 3010837 TI - Membrane permeability changes associated with DNA gyrase inhibitors in Escherichia coli. AB - Inhibition of DNA synthesis in Escherichia coli B/r by a DNA gyrase inhibitor results in cell death after a 50-min lag period. Examination of the cells under phase-contrast and electron microscopes revealed that they appeared to undergo plasmolysis coincident with the onset of cell death. The inhibited cells were also found to become susceptible to low levels of detergent at this time. With a fluorescent membrane probe, the level of membrane permeability was assessed and found to increase concurrently with the decrease in culture viability. Analysis of the cell envelope constituents revealed that, other than a shift in the protein/lipid ratio, the compositions of the cell membranes were unperturbed. PMID- 3010836 TI - Plaque autoradiography assay for the detection and quantitation of thymidine kinase-deficient and thymidine kinase-altered mutants of herpes simplex virus in clinical isolates. AB - A plaque autoradiography assay to detect and quantitate thymidine kinase (TK) mutants of herpes simplex virus type 1 (HSV-1) and HSV-2 in clinical samples is described. This method utilizes the selective incorporation of [125I]iododeoxycytidine, a pyrimidine analog selectively phosphorylated by the HSV TK. Only cells infected with TK-competent virus will efficiently incorporate iododeoxycytidine and are the only cells detected by autoradiography. Furthermore, this assay discriminates between TK+ virus (TK competent) and TKA virus (TK altered or reduced). This ability to differentiate TK+ from TKA virus is enhanced when infected cells are labeled with [14C]thymidine in tandem with iododeoxycytidine labeling. Reconstruction experiments with mixtures of TK+ (HSV 1 Patton) virus and TK-deficient (TK-) (B2006) or TKA (IUDRr) mutants were performed to determine the limits of detection of this technique. Ten percent TK- or TKA virus was the lower limit for the detection of TK mutants in a mixed population, whereas 1 in 1,000 TK+ virus revertants could be detected in a TK- virus population. In reconstructed populations and 45 clinical samples, a good correlation existed between the increase in 50% inhibitory dose for acyclovir and the percent TK mutant virus present. Similarly, the results of this technique correlated well with the acyclovir phosphorylating activity of extracts from cells infected with isolates or reconstructed mixtures. Plaque autoradiography with [125I]iododeoxycytidine was able to distinguish mixed populations of TK+ and TK- virus and homogeneous populations of TKA virus. The tandem use of [125I]iododeoxycytidine and [14C]thymidine readily identified TKA virus, which appeared as TK+ virus when labeled with [14C]thymidine alone. This technique provides a sensitive screen for antiviral resistance due to alterations in the viral TK and can be used to analyze clinical samples. PMID- 3010839 TI - Genesis of a complex transposon encoding the OXA-1 (type II) beta-lactamase gene. AB - Plasmid R753-1-encoded resistance to ampicillin (by production of OXA-1 [type II] beta-lactamase), streptomycin, and sulfonamide was analyzed functionally and physically. The OXA-1 beta-lactamase gene on R753-1 could not transpose, whereas on some plasmids this gene was capable of transposition as part of transposon Tn2603. By using the nontransposable gene on R753-1 with Tn21 on a separate plasmid, we observed the genesis of a complex transposon with a structure similar to that of Tn2603. This finding confirms our previous hypothesis that Tn2603 has evolved directly from Tn21 through acquisition of the OXA-1 beta-lactamase gene by substitution of DNA segments. Furthermore, the mechanism of mobilization of pACYC184 derivatives by RecA-dependent homologous recombination was demonstrated. PMID- 3010840 TI - Activity of 9-(1,3-dihydroxy-2-propoxymethyl)guanine compared with that of acyclovir against human, monkey, and rodent cytomegaloviruses. AB - The activities of the purine acyclic nucleoside 9-(1,3-dihydroxy-2 propoxymethyl)guanine (DHPG) against two human and five animal strains of cytomegalovirus were compared with those of acyclovir. DHPG was significantly more active than acyclovir against all but one (mouse cytomegalovirus) of the strains tested, with 50% effective doses ranging from 5 to 13 microM, as determined by plaque reduction assays in human embryonic lung (MRC-5) and human embryonic tonsil cells. Both DHPG and acyclovir inhibited virus replication at concentrations considerably lower than those necessary to inhibit cell proliferation. In mode-of-action studies, the triphosphates of DHPG and acyclovir inhibited human cytomegalovirus DNA polymerase. DHPG phosphorylation to the active triphosphate was enhanced in infected cells; however, this enzymatic activity was unrelated to thymidine kinase. In animal studies, DHPG was slightly more effective than acyclovir in reducing mouse cytomegalovirus-induced mortality. PMID- 3010842 TI - Antiviral activity of 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodocytosine against human cytomegalovirus in human skin fibroblasts. AB - 2'-Deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodocytosine (FIAC) was shown to be a selective anti-human cytomegalovirus agent in vitro with a 50% antiviral effective dose of 0.6 microM (J. M. Colacino and C. Lopez, Antimicrob. Agents Chemother. 26:505-508, 1983) and a 50% cell growth inhibitory dose of 8 microM. Antiviral activity was more readily reversed with 10-fold excess thymidine, whereby the 50% effective dose was increased to 11.3 microM. FIAC-induced cytotoxicity was more readily reversed with 10-fold excess of deoxycytidine, whereby the 50% inhibitory dose was increased to greater than 100 microM. Thymidine was unable to reverse completely the antiviral activity of FIAC. Although, the extent of phosphorylation of thymidine, deoxycytidine, and deoxyuridine was 6-, 4-, and 4-fold greater, respectively, in human cytomegalovirus-infected cell lysates than in uninfected cell lysates, the extent of phosphorylation of FIAC was only 1.3-fold greater in human cytomegalovirus infected cell lysates than in uninfected cell lysates. By comparison, the extent of FIAC phosphorylation was 500 times greater in herpes simplex virus type 1 infected cells than in uninfected cell lysates. Methotrexate was 400 times more effective against human cytomegalovirus replication than it was against herpes simplex virus type 1 replication, indicating that thymidylate synthetase may be important for human cytomegalovirus replication. However, 10 microM FIAC did not inhibit thymidylate synthetase activity in uninfected or virus-infected cells as determined by their metabolism of [6-3H]deoxyuridine in the presence or absence of drug. FIAC at 1 microM suppresses and FIAC at 10 microM completely inhibits human cytomegalovirus DNA replication as indicated by Southern blot analysis. This inhibition was reversible. FIAC incorporation into the DNA of human cytomegalovirus strain AD169-infected cells was stimulated relative to that in nondividing, uninfected cells. PMID- 3010841 TI - Selective in vitro and in vivo activities of 5-(2-haloalkyl)pyrimidine nucleoside analogs, particularly 5-(2-chloroethyl)-2'-deoxyuridine, against herpes simplex virus. AB - 5-(2-Chloroethyl)-2'-deoxyuridine (CEDU), 5-(3-chloropropyl)-2'-deoxyuridine (CPDU), and 5-(2-chloroethyl)-2'-deoxycytidine (CEDC) were evaluated for activity against herpes simplex virus type 1 (HSV-1) and HSV-2 in vitro. Their MICs for HSV-1 in primary rabbit kidney cell cultures were 0.15, 0.20, and 0.60 micrograms/ml, respectively; their MICs for HSV-2 were about 10-fold higher. When tested in parallel, the reference compounds 5-ethyl-2'-deoxyuridine, 5-iodo-2' deoxyuridine, acyclovir, and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) gave MICs of 0.20, 0.18, 0.04, and 0.015 micrograms/ml, respectively. The antiviral indexes of CEDU, CPDU, and CEDC, as determined by the ratio of the minimum toxic dose for the normal host cell to the minimum inhibitory dose for HSV-1, were about 2,000, 100, and greater than or equal to 400, respectively. The three 5-(2 haloalkyl)pyrimidine derivatives were further evaluated for their antiviral effects in vivo. In hairless mice, CEDU suppressed the development of cutaneous HSV-1 lesions, and associated mortality, when applied topically at a concentration as low as 0.1%. For the treatment of systemic HSV-1 infection in Naval Medical Research Institute mice, a single oral dose per day of 5 mg of CEDU per kg achieved a significant reduction in the mortality rate. Against HSV-1 encephalitis, CEDU exerted a significant protective effect at a dosage of 50 mg/kg per day when administered intraperitoneally. CEDU was effective against systemic HSV-1 infection and HSV-1 encephalitis in mice at a 5- to 15-fold-lower dose than either BVDU or acyclovir. When given orally, CPDU and CEDC were considerably less active than CEDU against systemic HSV-1 infection. PMID- 3010843 TI - Plasmid-mediated aminoglycoside phosphotransferases in Haemophilus ducreyi. AB - Three clinical isolates of Haemophilus ducreyi, representing at least two subtypes, were shown to be resistant to streptomycin and kanamycin. They also produced a beta-lactamase and chloramphenicol acetyltransferase and were resistant to tetracycline. In the three strains the resistance to both aminoglycoside antibiotics was encoded by a plasmid of ca. 4.7 kilobases which apparently did not carry ampicillin, chloramphenicol, or tetracycline resistance genes, as determined after transfer to Escherichia coli by transformation. Resistance to streptomycin and kanamycin was due to the presence of two aminoglycoside phosphotransferases (APH). The enzyme modifying kanamycin was a 3',5"-APH of type I [APH(3',5")-I], as inferred from its substrate profile and immunological cross-reactivity with the APH(3',5")-I encoded by the transposable element Tn903. However, the APH(3',5")-I gene in H. ducreyi did not appear to be carried by Tn903. PMID- 3010844 TI - Mechanism of selective inhibition of human cytomegalovirus replication by 1-beta D-arabinofuranosyl-5-fluorouracil. AB - Four kinds of 1-beta-D-arabinofuranosyl-5-halogenouracil were examined for inhibition of human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV 1) and 2 (HSV-2) replication. 1-beta-D-Arabinofuranosyl-5-fluorouracil (ara-FU) was the most effective against HCMV, whereas 1-beta-D-arabinofuranosyl-5 bromouracil was the most effective against HSV-1 and HSV-2. The mechanism of action of ara-FU on HCMV replication was also studied. The dTTP pool size in human embryonic fibroblasts was increased 33-fold by HCMV infection. However, treatment with ara-FU decreased the size of the dTTP pool by approximately 50%. On the other hand, 1-beta-D-arabinofuranosyl-5-fluorouracil-5'-triphosphate inhibited HCMV DNA polymerase activity competitively with dTTP. These results suggest that ara-FU acts as a bifunctional inhibitor of HCMV replication. Ara-FU is phosphorylated by cellular thymidine kinase to 1-beta-D-arabinofuranosyl-5 fluorouracil-5'-monophosphate, which inhibits cellular thymidylate synthetase, which in turn decreases the dTTP pool size in infected cells. As the dTTP pool size is reduced, inhibition of viral DNA polymerase by 1-beta-D-arabinofuranosyl 5-fluorouracil-5'-triphosphate becomes more efficient. PMID- 3010846 TI - Comparison of itraconazole and fluconazole in treatment of cryptococcal meningitis and candida pyelonephritis in rabbits. AB - Itraconazole and fluconazole, two new triazoles, were examined for their antifungal activity in rabbits. Fluconazole easily crossed the blood cerebrospinal fluid barrier, and active drug was eliminated in high concentrations in the urine. On the other hand, itraconazole did not cross the blood-cerebrospinal fluid barrier in measurable amounts, and urine concentrations were variable. Despite differences in pharmacokinetics at the site of infection, both agents were equally effective in treating cryptococcal meningitis and candida pyelonephritis in animals. By using a ketoconazole-resistant strain of Candida albicans, we showed that there was cross-resistance in vivo between these two new triazole compounds. PMID- 3010845 TI - Broad geographical distribution of homologous erythromycin, kanamycin, and streptomycin resistance determinants among group D streptococci of human and animal origin. AB - The Emr, Kmr, and Smr determinants of the Streptococcus faecalis R plasmid pJH1 were cloned in Streptococcus sanguis with a streptococcal plasmid vector, pVA380 1. Each cloned determinant was used as a probe in hybridization reactions with dot blots containing plasmid-enriched DNA from 91 group D streptococcal isolates resistant to erythromycin, kanamycin, and streptomycin; the isolates were obtained from animal and human sources in a variety of geographical locations. Nearly 70% of the strains contained DNA that hybridized to each of the three resistance determinants from pJH1. Five plasmids mediating resistance to erythromycin, kanamycin, and streptomycin were examined in more detail. These plasmids varied in size between 26 and 105 kilobase pairs (kbp) and exhibited very different EcoRI restriction patterns. However, each plasmid contained the resistance determinants on a single 13- to 20-kbp EcoRI fragment. Southern blot hybridizations and additional restriction endonuclease digests revealed extensive DNA sequence homology and virtually indistinguishable restriction endonuclease maps within a 9- to 11-kbp region of each plasmid which included the resistance determinants. PMID- 3010847 TI - Prolonged herpes simplex virus latency in vitro after treatment of infected cells with acyclovir and human leukocyte interferon. AB - We previously demonstrated that herpes simplex virus type 1 (HSV-1) can be established in a latent form in vitro by the treatment of HSV-infected human cells with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) in combination with human leukocyte interferon (IFN-alpha). We now report that the substitution of BVDU with 9-[(2-hydoxyethoxy)methyl]guanine (acyclovir; ACV) during a combined treatment with IFN-alpha inhibited HSV-1 replication and established in vitro virus latency that could be maintained for a longer period after inhibitor removal and a continued incubation at 37 degrees C. By contrast, the treatment of HSV-1-infected cells with combined IFN-alpha and 9-(1,3-dihydroxy-2 propoxymethyl)guanine, a congener of ACV, failed to establish in vitro virus latency. Furthermore, none of these inhibitors used alone was sufficient to establish in vitro virus latency. The use of nucleoside analogs differing from BVDU in their modes of action has enabled us to initiate studies designed to extend in vitro virus latency. PMID- 3010848 TI - Inhibition of Micrococcus luteus DNA gyrase by norfloxacin and 10 other quinolone carboxylic acids. AB - The ability of norfloxacin, amifloxacin, cinoxacin, ciprofloxacin, flumequine, nalidixic acid, ofloxacin (OFL), oxolinic acid, perfloxacin, pipemidic acid, and rosoxacin to inhibit the in vitro supercoiling activity of Micrococcus luteus DNA gyrase was compared with the ability of each drug to inhibit the growth of the M. luteus strain from which the gyrase was purified. The potency of the quinolones as DNA gyrase inhibitors did not always correlate with antimicrobial potency. For example, OFL was a less potent inhibitor of gyrase than rosoxacin, yet the MIC of OFL was 16-fold lower than that of rosoxacin. Similarly, the MICs of norfloxacin and ciprofloxacin (the most potent of the antibiotics tested in these assays) were several hundredfold lower than the MIC of nalidixic acid (the least potent of these antibiotics), but the inhibition of purified gyrase by these two quinolones was only 8- to 16-fold lower than that of nalidixic acid. These results suggest that factors in addition to inhibition of gyrase supercoiling activity are important in determining the potency of these drugs. Further studies indicated that the uptake of norfloxacin, OFL, and amifloxacin by M. luteus cells may not account for the large differences in MICs observed for these drugs (MICs of 0.8, 2.0, and 128 micrograms/ml, respectively). PMID- 3010849 TI - Molecular evolution, species distribution, and clinical consequences of an endemic aminoglycoside resistance plasmid. AB - During the first 6 years after appearing in one hospital, a 92-kilobase conjugative plasmid, pBWH1, which encoded resistance to chloramphenicol and sulfonamides and determined TEM-1 beta-lactamase and 2''-aminoglycoside nucleotidyltransferase, underwent a variety of molecular changes. It was most prevalent initially in isolates of Klebsiella pneumoniae, then in isolates of Serratia marcescens, and finally, after nearly disappearing, in isolates of Enterobacter cloacae. Evolutionary changes in the plasmid did not account for its shifts in species distribution, since the original molecule was found in isolates of each species. The late resurgence of pBWH1 occurred after a copy of its original molecule entered a distinctive ornithine decarboxylase-negative strain of E. cloacae, new to the hospital. The resulting transconjugant strain, chromosomally resistant to topical silver salts and to cephalosporins, and with the addition of pBWH1-encoded aminoglycoside resistance, spread in the hospital by causing an outbreak of sepsis in the burn unit, where these were commonly used antibacterial agents. Thus, an endemic plasmid became prevalent in a new host species because one of its genes supplemented the fitness of an uncommon strain of the species for a particular clinical niche. PMID- 3010851 TI - Hospital distribution, persistence, and reintroduction of related gentamicin R plasmids. AB - A single plasmid clone was predominantly responsible for gentamicin resistance at the Minneapolis Veterans Administration Medical Center (MVAMC) for 9 years, although two unrelated R plasmids were found. Epidemiological data and restriction endonuclease analysis of 25 plasmid isolates suggested that the clonally derived plasmid population had persisted at the hospital. However, one case of reintroduction of the original epidemic plasmid from a hospital in another state was documented 4 years after the introduction of the original plasmid to the MVAMC. Resistance by the clonally derived plasmid population was not localized geographically within the MVAMC but rather was a hospital-wide problem. Furthermore, previously described classes of DNA rearrangement of the original plasmid were also widely disseminated in the hospital, implying that spread of strains bearing index-related plasmids was relatively unimpeded within the MVAMC despite extensive barrier isolation and cohorting measures. Potential environmental reservoirs of the plasmids were identified in hospital sinks and drains, but their relation to continued patient infection is not known. PMID- 3010850 TI - Genetic and biochemical characterization of norfloxacin resistance in Escherichia coli. AB - In Escherichia coli the frequency of spontaneous single-step mutation to high levels of resistance to the newer 4-quinolone agent norfloxacin was confirmed to be over 300-fold lower than that to the older agent nalidixic acid. Serial passage on incremental concentrations of drug was necessary to produce mutants highly resistant to norfloxacin. Genetic analysis of one such highly resistant strain identified two mutations conferring drug resistance. One mutation, nfxA, mapped around 48 min on the E. coli genetic map and was shown to be an allele of gyrA by studies demonstrating an increased drug resistance of DNA gyrase reconstituted with the gyrase A subunit isolated from the mutant strain. These findings also identified the DNA gyrase A subunit as a target of norfloxacin. The second mutation, nfxB, mapped between 20 and 22 min was associated with additional resistances to tetracycline, chloramphenicol, and cefoxitin and with decreases in outer membrane porin protein OmpF. The nfxA and nfxB mutations together accounted for most, but not all, of the norfloxacin resistance phenotype of this strain. PMID- 3010852 TI - Comparison of the in vitro and in vivo activity of the bis-triazole derivative UK 49,858 with that of amphotericin B against Histoplasma capsulatum. AB - The antifungal activity of UK 49,858, a difluorophenyl bis-triazole derivative, was evaluated in vitro against seven strains of Histoplasma capsulatum and in vivo in AKR and C57BL/6 murine models of histoplasmosis. UK 49,858 had a lower toxicity for AKR and C57BL/6 mice than amphotericin B did. The therapeutic index of UK 49,858 was 4.3 for AKR mice and 7.1 for C57BL/6; with amphotericin B it was 2 for both mouse strains. Given orally, UK 49,858 compared favorably with amphotericin B given intraperitoneally in either AKR or C57BL/6 mice infected with H. capsulatum. PMID- 3010853 TI - Effects of toluene permeabilization and cell deenergization on tetracycline resistance in Escherichia coli. AB - Resistance to tetracycline (Tcr) mediated by Tn10 and related Tcr determinants involves an inner membrane protein, TET (similar but not identical for different determinants), and a proton motive force-dependent efflux of tetracycline which keeps the drug away from its intracellular target, the ribosome (L. M. McMurry, R. E. Petrucci, Jr., and S. B. Levy, Proc. Natl. Acad. Sci. USA 77:3974-3977, 1980). However, the amount of tetracycline accumulated by bacteria does not always correlate with their resistance levels, suggesting that an additional resistance mechanism may be present. When we permeabilized susceptible and resistant Tn10-bearing cells with toluene, we found that protein synthesis in the two strains became equally sensitive to tetracycline. Therefore, the protein synthesis machinery was not a source of resistance, and an intact membrane was required for resistance. To determine whether resistance was entirely dependent on energy, we measured susceptibility to tetracycline after inhibition of proton motive force by starvation and specific inhibitors. An 80 to 90% loss of Tcr (measured by protein synthesis) resulted from partial deenergization of resistant cells. A remaining resistance (10- to 20-fold greater than that of susceptible cells) could not be eliminated by further deenergization. These findings indicated that, to a major extent, expression of Tn10 resistance required energy, presumably for tetracycline efflux. They also suggested the existence of a small component of Tcr having little or no energy dependence. Whether this component depends on tetracycline efflux or some other mechanism is not known, but presumably both high- and low-energy components of resistance reflect activity of TET protein. PMID- 3010855 TI - Influence of twenty potentially antiviral substances on in vitro multiplication of hepatitis A virus. AB - A multiwell tissue culture system was developed to study the influence of various substances on hepatitis A virus (HAV) propagation. A panel of 20 substances of different structure types, each with known effect against at least some viruses, was studied at a concentration of 100 microM. Three substances showed reproducible inhibition. The strongest inhibitor, arabinosylcytosine, also produced cytotoxic changes in cells down to a concentration of 1 microM, and its effect was considered as nonspecific. Amantadine and ribavirin showed a moderate effect at 100 microM. A stronger inhibition was seen at 250 and 500 microM, doses that are toxic and impractical for clinical use. Although no promising candidates for antiviral treatment of hepatitis A have emerged from the present study, the assay model described here would seem useful in the screening of substances with inhibitory effects on HAV. PMID- 3010854 TI - Incorporation of 1-beta-D-arabinofuranosylcytosine into DNA and mutagenesis of herpes simplex virus type 1. AB - To investigate the association between incorporation of 1-beta-D arabinofuranosylcytosine (ara-C) into herpes simplex virus type 1 (HSV-1) DNA and mutagenesis at selected genetic loci, we grew virus in the presence or absence of ara-C and compared the mutation frequencies. Both the forward mutation frequency of HSV-1 (KOS) to the tk- phenotype and the reversion frequency of a temperature sensitive mutant to the non-temperature-sensitive (ts+) phenotype were significantly increased following growth in ara-C. When the same viruses were grown in an inhibitor of the HSV-1 DNA polymerase that is not incorporated into DNA, no significant increase in the frequency of tk- or ts+ particles was observed. Furthermore, HSV-1 araAr, a mutant resistant to ara-C on the basis of reduced incorporation of the analog into viral DNA, did not demonstrate an increase in the number of tk- particles following growth in ara-C. These results suggest that mutagenesis of HSV-1 following growth in ara-C correlates with incorporation of the nucleoside analog into viral DNA. PMID- 3010856 TI - Protection of guinea pigs against local and systemic foot-and-mouth disease after administration of synthetic lipid amine (Avridine) liposomes. AB - Injection of the synthetic lipid amine, Avridine, in the form of liposomes, protected guinea pigs against the development of lesions from foot-and-mouth disease virus (FMDV) inoculated intradermally into the rear footpads. The animals were protected against the development of vesicles at the inoculation site as well as the systemic spread of virus. Maximal protection was obtained after intracardial injection of 5-10 mg doses of liposomal Avridine. Lower doses yielded decreased protection. Subcutaneous or intraperitoneal routes of liposomal Avridine injection were ineffective. Protection was maximal 0-24 h after injection of liposomes. Ethanol and emulsion formulations of Avridine could induce protection when injected intracardially but had toxic side effects. Guinea pigs protected against the first FMDV inoculation by liposomal and ethanol formulations of Avridine continued to be protected against lesions after a second inoculation 15-45 days later. FMDV protective antibody titers of these animals ranged from a low of less than 1:10 to greater than 1:1000. PMID- 3010857 TI - Production of herpes simplex virus type 1 thymidine kinase in the presence of thymidine analogues. AB - Treatment of herpes simplex virus type 1 (HSV-1) infected Vero, BHK, BHKtk- and LMtk- cells with 5-iodo-5'-amino-2',5'-dideoxyuridine (AIdUrd) caused increased synthesis of ICP36 and an increase in HSV-1 thymidine kinase (tk) activity at late times of infection. The overproduced ICP36 was identified as the HSV-1 encoded tk protein by immunoprecipitation. Whereas the thymidine analogue 5' amino-5'-deoxythymidine (AdThd) caused an increase in HSV-1 tk synthesis and activity in wild type Vero and BHK cells, 5-iodo-2'-deoxyuridine (IdUrd) caused a similar increase only in tk- cells (LMtk-, BHKtk-). In vivo and in vitro stabilization studies using a [35S]methionine pulse-chase experiment or heat inactivation studies with purified HSV-1 tk revealed that stabilization of tk by the analogues could not account for the extent of the observed increase. Since overproduction of tk is observed only at late times of infection, it is suggested that the presence of these thymidine analogues in either the viral DNA or the cellular nucleotide pools is responsible for the observed differential effects. PMID- 3010858 TI - Treatment of experimental herpes simplex virus type 1 encephalitis in mice with (E)-5-(2-bromovinyl)- and 5-vinyl-1-beta-D-arabinofuranosyluracil: comparison with bromovinyl-deoxyuridine and acyclovir. AB - The efficiency of (E)-5-(2-bromovinyl)- and 5-vinyl-1-beta-D arabinofuranosyluracil (BrVaraU, VaraU) as inhibitors of three herpes simplex virus type 1 (HSV-1) strains was assessed in comparison to (E)-5-(2-bromovinyl) 2'-deoxyuridine (BrVUdR), 9-(2-hydroxyethoxymethyl)guanine (ACV), and trisodium phosphonoformate (Na3PFA) using a plaque assay in human embryonic lung fibroblast (HELF) cell cultures. The following order of decreasing activity was found: BrVaraU greater than VaraU greater than BrVU-dR greater than ACV much greater than Na3PFA. In HELF cell cultures, the selectivity indexes of VaraU and BrVaraU were 10 times higher than those of BrVUdR and ACV. Protection of mice from encephalitis and death due to intracerebral (i.c.) infection with a clinical HSV 1 isolate was nearly complete if mice were treated intraperitoneally (i.p.) with two daily doses of VaraU and BrVaraU (100 or 200 mg/kg per day) over a period of 5 or 10 days. The efficacy was similar to ACV, but, using a treatment schedule of three daily i.p. doses over 10 days, with equimolar amounts of the nucleoside analogs, VaraU and BrVaraU (140 and 180 mg/kg per day) were superior to ACV (130 mg/kg per day) (P less than 0.05). PMID- 3010859 TI - Evaluation of the herpes simplex virus antiviral activity of pyrethrins. AB - Pyrethrins, complex esters extracted from Chrysanthemum cinerariaefolium, exhibit only minimal in vitro activity against herpes simplex virus (HSV). Employing a guinea pig model of HSV genital infection, no in vivo activity could be demonstrated. Although purported to be an effective remedy for the treatment of genital herpes, we were unable to demonstrate efficacy for either the oral administration of an alcoholic solution of pyrethrins or the topical application of pyrethrins in mineral oil. PMID- 3010862 TI - Energetic studies of lactose active transport in Escherichia coli membrane vesicles. AB - The energetics of D-lactate-driven active transport of lactose in right-side-out Escherichia coli membrane vesicles has been investigated with a microcalorimetric method. Changes of enthalpy (delta Hox), free energy (delta Gox), and entropy (delta Sox) during the D-lactate oxidation reaction in the presence of membrane vesicles are -39.9 kcal, -46.4 kcal, and 22 cal/deg per mole of D-lactate, respectively. The free energy released by this reaction is utilized to form a proton electrochemical potential (delta-microH+) across the membrane. The higher observed heat in the D-lactate oxidation reaction in the presence of carbonylcyanide m-chlorophenylhydrazone (a proton ionophore) supports the postulate that delta-microH+ is formed across the membrane vesicles. Thermodynamic quantities for the formation of delta-microH+ are delta Hm = 14.1 kcal, delta Gm = 0.6 kcal, and delta Sm = 45 cal/deg per mole of D-lactate. The efficiency in the free energy transfer from the oxidation reaction to the formation of delta-microH+ (defined by delta Gm/delta Gox) was 2%, as compared to that in the heat transfer (defined by delta Hm/delta Hox) of 35%. The energetics of the movement of lactose in symport with proton across the membrane as a consequence of the formation of delta-microH+ are delta H1 = -19 kcal, delta G1 = -0.5 kcal, and delta S1 = -62 cal/deg per mole of lactose. No heat of reaction is contributed by lactose movement across the membrane without symport with H+. PMID- 3010860 TI - Optimization of the BGM cell line culture and viral assay procedures for monitoring viruses in the environment. AB - An in-depth study of the continuous cell line designated BGM is described herein, and recommendations are made for standardizing cell culture and viral assay procedures. Based on data gathered from a survey of 58 laboratories using this cell line, a research plan was developed that included the study of growth media, sera, NaHCO3 levels, culture bottles, cell concentration, overlay media, agar, virus infection conditions, and cell-dissociating agents. Additionally, a comparative virus isolation study with BGM cells and nine other cell types was conducted with 37 sewage samples collected from nine different geographic areas. The results of the study indicated that the BGM cell line is superior for virus isolation when compared with the other cell types and that certain media and additives tend to increase BGM cell sensitivity to a specific group of viruses. A standardized procedure for cultivation of BGM cells is described which provides a more effective enterovirus assay system. PMID- 3010861 TI - Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. PMID- 3010863 TI - Intrinsic and artifactual pH buffering in chloroplast thylakoids. AB - After an increase in pH of the suspension medium, a pH gradient across the membrane of chloroplast thylakoids stored at pH greater than or equal to 6.5 is often maintained for several minutes. The intrinsic hydrogen ion buffering capacity of the thylakoid membranes between pH 6.5 and 8.5 is about 40 neq/mg chlorophyll, but can be artificially inflated by penetration of the external buffer into the thylakoid vesicle. A delta pH imposed across the thylakoid membrane by an acid/base transition cannot be estimated accurately by the fluorescent probe 9-aminoacridine, especially with osmotically shrunken thylakoids in which 9-aminoacridine appears to become bound or adsorbed to the membrane. This interaction may be related to the existence of the previously demonstrated special pool of slowly equilibrating, "sequestered" protons. PMID- 3010864 TI - Globoseries glycosphingolipids of human meconium. AB - Globoside and an extended globoseries glycosphingolipid with a blood group H determinant were isolated from pooled human meconia and structurally characterized by mass spectrometry, proton NMR spectroscopy, and degradational techniques using GC and GC-MS analyses. Both species contained mainly phytosphingosine and hydroxy fatty acids characteristic for human intestinal epithelial cells. With the same techniques also minor amounts of globoside with sphingosine and nonhydroxy fatty acids and a novel globoseries tetraglycosyl ceramide with a terminal N-acetylglucosamine were isolated and structurally characterized. PMID- 3010865 TI - The effect of pH on yields of hydroxyl radicals produced from superoxide by potential biological iron chelators. AB - The efficiency of conversion of superoxide to hydroxyl radicals was measured by determining the yields of fluorescent hydroxybenzoates. A variety of iron containing catalysts were tested. Citrate was the only organic salt which showed catalytic activity at neutral pH. Adenine nucleotides had little or no activity under similar conditions. Heme proteins were inactive and any catalytic activity measured with transferrin, lactoferrin, and conalbumin could be explained by free Fe3+ released by the former two at acid pH. Many of the potential catalysts tested showed maximum activity near pH 4.8, where the rate of dismutation of O2-. is highest. This suggests that in most systems the rate-controlling step in the superoxide-driven Fenton process was the formation of H2O2. It was concluded that, with the exception of citrate, none of the biological compounds tested were able to assist the conversion of O2-. to HO. with significant efficiency at neutral pH in homogeneous solutions. PMID- 3010866 TI - Functional constituents of the active site of human neutrophil collagenase. AB - A series of chemical modification reactions has been carried out to identify functional constituents of the active site of human neutrophil collagenase. The enzyme is reversibly inhibited by the transition metal chelating agent 1,10 phenanthroline, and inhibition is fully reversed by zinc. Removal of weakly bound metal ions by gel filtration inactivates collagenase, and activity is fully restored on immediate readdition of calcium. The enzyme is unaffected by reagents that modify serine, cysteine, and arginine residues. However, reaction with the carboxyl reagents cyclohexylmorpholinocarbodiimide and Woodward's Reagent K lowers the activity of the enzyme substantially. Acetylimidazole inactivates the enzyme, but activity is completely restored on addition of hydroxylamine. The enzyme is also inactivated by tetranitromethane, indicating that it contains an essential tyrosine residue. Acylation of collagenase with diethyl pyrocarbonate, diketene, acetic anhydride, or trinitrobenzenesulfonate inactivates the enzyme, and activity is not restored on addition of hydroxylamine, indicating the presence of an essential lysine residue. PMID- 3010867 TI - Sulfate-dependent iron oxidation by Thiobacillus ferrooxidans: characterization of a new EPR detectable electron transport component on the reducing side of rusticyanin. AB - Iron(II) oxidation by pH 2.5 HCl-washed cells of Thiobacillus ferrooxidans is known to be sulfate dependent. Sulfate dependence of the autooxidation of a novel component in the electron transport pathway is demonstrated. This component exhibits an electron paramagnetic resonance (EPR) signal in the oxidized state at g = 2.005 distinguishable from the g = 2.08 signal attributed to rusticyanin. The novel component is proposed to be a three-iron-sulfur cluster based upon the g value, lineshape, and temperature dependence. Oxyanion specificity for the EPR signal has the same dependence on sulfate as does iron(II) oxidation. By using azide to inhibit electron transfer to oxygen, sulfate was shown to be involved in electron transfer from the g = 2.005 component to the copper of rusticyanin. PMID- 3010868 TI - Glutaryl phosphatidylcholine effects on pH and composition dependent behavior of liposomes studied by spin-labeling method. AB - The influence of glutaryl phosphatidylcholine on the molecular organization of phosphatidylcholine liposomes was studied by spin-labeling technique. The ESR signals given by the 5-nitroxide stearic acid label showed that the presence of glutaryl lecithin (i) significantly increased the negative charge density of the polar liposome surface with increasing proton concentration depending on the bulk solution pH, and (ii) apparently decreased the packing (order) of the hydrophobic region close to the surface, essentially in the presence of saturated phospholipids. The spectral information--S (order parameter) and alpha N (isotropic nitrogen coupling constant)--resulted in the location of the probe near or in the polar zone of the membrane or in the hydrophobic region, depending on the protonation/deprotonation of the fatty acid carboxyl group of the probe. The microviscosity of the inner region of the membrane monitored by the 12- and 16-probes was not significantly altered by glutaryl lecithin. On the other hand, glutaryl lecithin has a lesser effect on liposomes containing anionic polar head groups, such as dipalmitoyl phosphatidylglycerol or phosphatidylinositol, the anionic charge of which already had the same effect on protonation of the polar surface. The temperature dependence of dipalmitoyl phosphatidylcholine liposome dynamic behavior indicates that the glutaryl lecithin effect is completely different above and below the gel-to-liquid crystalline phase transition point. PMID- 3010869 TI - Evidence for and partial characterization of three major and three minor chromatographic forms of human neutrophil myeloperoxidase. AB - Myeloperoxidase (MPO) is an enzyme found in the azurophil granules of neutrophils. Cation-exchange chromatography on carboxymethyl-cellulose previously has been used to demonstrate the heterogeneity of the peroxidase enzymes isolated from human neutrophils. In this study, fast protein liquid chromatography (FPLC) was used to separate and purify three major (I, II, and III) and three minor (IIa, IIIa, IIIb) forms of MPO from isolated neutrophil granules. Purity was confirmed by polyacrylamide gel electrophoresis in the presence of cetyltrimethylammonium bromide (CETAB-PAGE), by crossed immunoelectrophoresis, and by spectral characteristics. All three major forms were indistinguishable by immunodiffusion against rabbit antiserum, scanning spectrophotometry, and amino acid composition. They differed in their elution from a cation-exchange resin, inhibition by 3-amino-1,2,4-triazole, migration rate in CETAB-PAGE, and subunit molecular weight. Subunit molecular weight was examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All three major forms appeared to consist of heavy (H), intermediate (M), and light (L) peptides. The M peptide appeared to be derived from the H subunit. All L subunits exhibited a molecular weight of 14,500. The molecular weights for the H subunits varied, and were 60,000, 59,000, and 57,000 for MPO I, II, and III, respectively. The molecular weights for the M peptides were 44,100, 43,000, and 42,000 for MPO I, II, and III, respectively. The treatment of neutrophils, granules, and extracts with protease inhibitors and sodium azide did not block the appearance of three major forms of MPO. Thus, neither protease activity nor MPO autooxidation during extraction and purification procedures is responsible for the appearance of multiple chromatographic forms of MPO derived from human neutrophils. PMID- 3010870 TI - Induction of refractoriness of cyclic AMP responses to prostaglandin E1 and epinephrine by prior exposure of guinea pig macrophages to lipopolysaccharide. AB - Prior exposure of guinea pig macrophages to LPS (lipopolysaccharide) resulted in reduced cAMP-generating responses to prostaglandin E1 and epinephrine. LPS induced refractoriness was diminished when LPS treatment was carried out in the presence of an inhibitor of prostaglandin synthesis, hydrocortisone, or indomethacin, or an inhibitor of protein synthesis, cycloheximide. The release of arachidonic acid and its metabolites, especially prostaglandin E2 and thromboxane B2, increased during incubation of macrophages with LPS. These increases were efficiently antagonized by hydrocortisone, indomethacin, or cycloheximide. Preincubation of macrophages with prostaglandin E1 greatly reduced the subsequent responses of cAMP generation to prostaglandin E1 and unexpectedly also to epinephrine. Thus, increased production of prostaglandins during the LPS treatment is likely to be responsible for decreased cAMP responses to subsequent addition of prostaglandin E1 and epinephrine. PMID- 3010871 TI - The 3'-noncoding region of the chick myosin light-chain gene hybridizes to a family of repetitive sequences in the slime mold Dictyostelium discoideum. AB - During studies aimed at isolating myosin-specific genomic clones in Dictyostelium, we probed a lambda genomic library with a chicken myosin light chain sequence (pML10). Many lambda recombinant Dictyostelium clones hybridized to the pML10 cDNA insert, indicating that this sequence was reiterated in the Dictyostelium genome. It was found that the 3'-noncoding region (pML10-NC) alone was responsible for these results. Dictyostelium DNA contained approximately 65 copies of a sequence(s) similar but not identical to that of pML10-NC. Southern blot analysis showed that pML10-NC hybridized to many Dictyostelium genomic DNA fragments of varying sizes generated by digestion with EcoRI, HindIII, or AluI. In addition, each of the Dictyostelium clones was different in its size, restriction map, and flanking sequences. It seems likely, therefore, that the sequences which hybridized to pML10-NC are scattered throughout the Dictyostelium genome and similar but not identical to each other or to pML10-NC. Thus, probing with pML10-NC has allowed us to select a family of closely related but not identical sequences. These D. discoideum sequences are not found in other slime mold species. No RNA complementary to pML10-NC was found in vegetative cells, 18 h culmination stage, spores, or 1- and 2-h germinating spores. pML10-NC-related sequences were present in two other Dictyostelium species but were absent in the related genus Polysphondylium. PMID- 3010873 TI - Xanthine oxidase from human liver: purification and characterization. AB - Xanthine oxidase [EC 1.2.3.2] was purified 2000-fold from human liver. The last step of the procedure involved affinity chromatography. The resulting preparation showed two closely migrating bands of enzyme activity after gel electrophoresis under nondenaturing conditions. No other proteins were detected on these gels. The average particle mass of the enzyme was 300 kDa as determined by size exclusion chromatography. This together with results of gel electrophoresis under denaturing conditions suggested that the native enzyme was composed of two subunits of approximately 150 kDa each. The electrophoretic patterns also indicated that a portion of these subunits had undergone partial proteolysis. The substrate specificity of the purified human enzyme was studied using an assay in which phenazine ethosulfate coupled the transfer of electrons from the reduced enzyme to cytochrome c. Hypoxanthine, 2-hydroxypurine, xanthine, 2-aminopurine, and adenine were among the most efficient purine substrates studied. Most purine nucleosides tested were oxidized at detectable rates, but with relatively high Km values. The 2'-deoxyribonucleosides were more efficient substrates than were the corresponding ribonucleosides or arabinonucleosides. In a direct comparison with xanthine oxidase from bovine milk, the human enzyme showed a similar specificity toward purine substrates. However, considerable differences between the bovine and human enzymes were observed with nucleoside substrates. With xanthine as the substrate for the human enzyme, 20% of the total electron flow was univalently transferred to oxygen to produce superoxide radicals. PMID- 3010872 TI - Biological effects of the superoxide radical. AB - Can the superoxide radical exert deleterious effects independent of participating with H2O2 in the production of the hydroxyl radical? Examination of the superoxide-related literature reveals data suggesting an affirmative answer to this question. PMID- 3010874 TI - A calcium binding protein from Drosophila melanogaster which activates cAMP phosphodiesterase: comparison of this protein with porcine brain calmodulin. AB - A calcium binding protein from Drosophila melanogaster has been isolated and characterized. This protein shows several analogies with pig brain calmodulin in its molecular weight, isoelectric point, peptide maps, calcium binding properties, and ability to activate cyclic AMP phosphodiesterase. However, some differences were observed; the most remarkable one is the presence of tryptophan, an amino acid which is absent from all the calmodulins analyzed previously. PMID- 3010876 TI - Proton nuclear magnetic resonance studies of soybean lectin-monosaccharide interactions: computer analysis of complex binding behavior. AB - 1H NMR was used to quantify soybean lectin binding to monosaccharides, using presaturation of HOD plus a spin-echo sequence to observe sugar -NHCOCH3 and OCH3 to below 0.01 mM. Binding is in the very-slow-exchange limit; there is no broadening or shifting and only unbound sugar is observed for pH 5 to 8 and 25 to 75 degrees C. Preliminary results were consistent with those previously reported for methyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside (Me alpha-D-GalNAcp) (K = 3 X 10(4) liters mol-1 with four sites per tetramer). More detailed studies, however, gave concave Scatchard plots for methyl 2-acetamido-2-deoxy-alpha- and beta-D-galactopyranoside, (Me alpha- and Me beta-D-GalNAcp), best fitted using K1 values of (6-12) X 10(4) liters mol-1, K2 values less than or equal to 0.5 X 10(4) liters mol-1, and four sites of each type in D2O or 80% H2O at 25 degrees C and pH 7.2. Data for methyl alpha-D-galactopyranoside were fitted with K = 0.5 X 10(4) liters mol-1 and eight sites of the same K. Monosaccharides may be binding in the recently reported "hydrophobic sites" of soybean lectin. Both methyl 2 acetamido-2-deoxy-beta-D-galactofuranoside (Me beta-D-GalNAcf) and methyl 2 acetamido-2-deoxy-alpha-D-glucopyranoside (Me alpha-D-GlcNAcP) showed some binding by 1H NMR; K's were similar to those for the high-affinity sugars, but the occupancy was much lower. The soybean lectin in this study was saturated in Ca2+ (greater than or equal to 4 mol/tetramer), but low in Mn2+, with Mn2+ plus Mg2+ less than 4. We report new melting points for D-N-acetylgalactosamine, Me alpha-D-GlcNAcp, Me beta-D-GalNAcp, and Me beta-D-GalNAcf, and a fully listed program for fitting curved Scatchard plots using Apple IIc and IIe computers. PMID- 3010875 TI - Purification and inactivation-reactivation of phosphorylase phosphatase from the protein-glycogen complex. AB - Phosphorylase phosphatase purified from the protein-glycogen complex of rabbit muscle has a Mr of 34,000 by gel filtration and migrates as a single band of Mr 38,000 on sodium dodecyl sulfate gel electrophoresis, i.e., of the same size as the catalytic subunit of the sarcoplasmic complex of type 1 phosphatase (L. M. Ballou, D. L. Brautigan, and E. H. Fischer (1983) Biochemistry 22, 3393-3399). The enzyme, called PG-Ea, has a specific activity of 12,000 units/mg of protein and is essentially fully active, displaying at most a 20% further increase in activity on treatment with trypsin. As in the case of the catalytic subunit of the sarcoplasmic enzyme, tryptic attack decreases the size of PG-Ea to 33,000. PG Ea is completely inhibited by the modulator protein (inhibitor 2) after formation of a complex of Mr 70,000. On incubation of this complex at 30 degrees C, the catalytic subunit is converted (t 1/2 = 30 min) to an inactive form (Ei) that can be reactivated by the protein kinase FA (J. R. Vandenheede, S.-D. Yang, J. Goris, and W. Merlevede (1980) J. Biol. Chem. 255, 11,768-11,774) or to a lower extent by trypsin-Mn2+. Also the trypsinized PG-Ea is inhibited by inhibitor 2, it forms with this a Mr 70,000 complex and undergoes an even faster (t 1/2 = 10 min) conversion to an Ei form that can be reactivated by the kinase FA or to a lower extent by trypsin-Mn2+. Enzymological comparison of PG-Ea and trypsinized PG-Ea with the FA-activated, isolated catalytic subunit of the sarcoplasmic phosphatase (called EaFA, E. Villa-Moruzzi, L. M. Ballou, and E. H. Fischer (1984) J. Biol. Chem. 259, 5857-5863) and with its trypsinized form shows several similarities. The most relevant of these is the very specific interaction with inhibitor 2, that takes to inactivation and allows the following reactivation by FA or by trypsin-Mn2+. However, the inactivation-reactivation patterns show also some differences, namely (i) the t 1/2 of the conversion to Ei is longer for PG-Ea than for EaFA, and (ii) the reactivation of PG-Ea and trypsinized PG-Ea with trypsin-Mn2+ is only partial. Altogether the similarity but not identity of PG-Ea and EaFA would suggest that they are either two different conformations of the same molecule or two isozymes. PMID- 3010878 TI - Endogenous energy supply to the plasma membrane of dark aerobic cyanobacterium Anacystis nidulans: ATPase-independent efflux of H+ and Na+ from respiring cells. AB - The ejection of protons from oxygen-pulsed cells and the gradients of Na+ concentration (Na+o/Na+i at 150 mM external NaCl) and proton electrochemical potential (delta mu H+) across the plasma membrane of Anacystis nidulans were studied in response to dark endogenous energy supply. Saturating concentrations of the F0F1-ATPase inhibitors dicyclohexylcarbodiimide (F0) and 7-chloro-4 nitrobenz-2-oxa-1,3-diazole (F1) eliminated oxidative phosphorylation and lowered the ATP level from 2.6 +/- 0.15 to 0.7 +/- 0.1 nmol/mg dry wt while overall O2 uptake and delta mu H+ were much less affected. H+ efflux was inhibited only 60 to 75%. Aerobic Na+o/Na+i ratios (5.9 +/- 0.6) under these conditions remained 50% above the anaerobic level (2.1 +/- 0.2). Increasing concentrations of the electron transport inhibitors CO and KCN depressed H+ efflux and O2 uptake in parallel, with a pronounced discontinuity of the former at inhibitor concentrations, which reduced ATP levels from 2.6 to 0.8 nmol/mg dry wt, resulting in an abrupt shift of the apparent H+/O ratios from 4.0 +/- 0.3 to 1.9 +/- 0.2. Similarly, with KCN and CO the Na+o/Na+i ratios paralleled decreasing respiration rates more closely than decreasing ATP pool sizes. Ejection of protons also was observed when intact spheroplasts were pulsed with horse heart ferrocytochrome c or ferricyanide; the former reaction was inhibited, the latter was increased, by 1 mM KCN. Measurements of the proton motive force (delta mu H+) across the plasma membrane showed a strong correlation with respiration rates rather than ATP levels. It is concluded that the plasma membrane of intact A. nidulans can be directly energized by proton-translocating respiratory electron transport in the membrane and that part of this energy may be used by a Na+/H+ antiporter for the active exclusion of Na+ from the cell interior. PMID- 3010877 TI - The nucleotide sequence of two homogeneic Drosophila melanogaster tRNATyr isoacceptors: application of a rapid tRNA anticodon sequencing method using S-1 nuclease. AB - The nucleotide sequence of the two major Drosophila melanogaster tRNATyr isoacceptors was determined to be pC-C-U-U-C-G-A-U-A-m2G-C-U-C-A-G-D-D-G-G-acp3 U A-G-A-G-C-m2(2)G-G-psi-G-G-A-C-U-G/Q-psi-A-m1G-A-Um-C-C-A-U-A-G-m7 G-D-C-G-C-U-G G-U(T)-psi-C-A-m1A-A-U-C-C-G-G-C-U-C-G-A-A-G-G-A-A-C-C-AOH . The two isoacceptors differ by the presence of a G or a Q in the wobble position. Both contain a partial modification in position 54 (U/T). Thus, these tRNAs are transcribed from a single gene (or many genes with identical sequences). A fast and sensitive postlabeling method for sequencing tRNA anticodons is described. Nuclease S-1 treated tRNA is labeled with 5[32P]-pCp using T-4 RNA ligase. The tRNA fragments are then separated on 7 M urea/20% PAA gels. After autoradiography the RNA is eluted and digested with T-2 RNase. The nature of the labeled nucleotides is determined by two-dimensional thin-layer chromatography. The same method can be used to determine the 5' sequence of a tRNA by 3' labeling 5' tRNA halves with 5[32P]-pCp and subsequent chemical sequencing. PMID- 3010879 TI - Exogenous energy supply to the plasma membrane of dark anaerobic cyanobacterium Anacystis nidulans: thermodynamic and kinetic characterization of the ATP synthesis effected by an artificial proton motive force. AB - The net synthesis of ATP in dark anaerobic cells of Anacystis nidulans subjected to acid jumps and/or valinomycin pulses was characterized thermodynamically and kinetically. Maximum initial rates of 75 nmol ATP/min per mg dry weight at an applied proton motive force of -350 mV were obtained, the flow-force relationship (rate of ATP synthesis vs applied proton motive force) being linear between -240 and -320 mV irrespective of the source of the proton motive force. The pulse induced ATP synthesis was inhibited by uncouplers (H+ ionophores) and F0F1-ATPase inhibitors but not by KCN or CO. In order to obtain maximum rates of pulse induced ATP synthesis both a favorable stationary delta psi (-100 mV at pHo 9, preceding the acid jumps) and a favorable stationary delta pH (+2 units at pHo 4.1, preceding the valinomycin pulse) of the plasma membrane were obligatory, the effects of delta psi and delta pH being strictly additive. Moreover, the pulse induced ATP synthesis required a minimum total proton motive force of -200 to 250 mV across the plasma membrane; it also required low preexisting phosphorylation potentials corresponding to -400 mV in dark anaerobic, i.e., energy-depleted, cells. The results are discussed in terms of both a reversible H+-ATPase and a respiratory electron transport system occurring in the plasma membrane of intact Anacystis nidulans. PMID- 3010880 TI - Regulation of neutral cholesteryl esterase in arterial smooth muscle cells: stimulation by agonists of adenylate cyclase and cyclic AMP-dependent protein kinase. AB - Cultured arterial smooth muscle cells have been found to contain an activatable neutral cholesteryl esterase (EC 3.1.1.13). This enzyme is similar to that previously described in adipose tissue, adrenal cortex, and aortic homogenates. Although both the lysosomal (acid) and cytoplasmic (neutral) cholesteryl esterases were activated two- to threefold by the addition of 100 microM dibutyryl cyclic AMP, only neutral cholesteryl esterase was responsive to 100 microM dibutyryl cyclic AMP, 10 mM MgATP, and 50 micrograms/ml exogenous protein kinase when added together. Protein kinase inhibitor (10 micrograms/ml) reversed the action of cyclic AMP-dependent protein kinase; deactivation of neutral cholesteryl esterase was also shown to occur with 50 micrograms/ml phosphoprotein phosphatase. In addition, 0.2 microM prostacyclin, 50 microM forskolin, and an agonist of the beta-adrenergic receptor, 5 microM isoproterenol, significantly stimulated intracellular cyclic AMP accumulation and activated cholesteryl esterase in arterial smooth muscle cells. The data indicate that neutral cholesteryl esterase in arterial smooth muscle cells can be modulated by a phosphorylation-dephosphorylation system involving the cyclic AMP-dependent protein kinase-phosphoprotein phosphatase. Regulation of cholesteryl esterase by this mechanism may affect lipid accumulation in these arterial cells. PMID- 3010881 TI - Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: chromatographic, electrophoretic, and sedimentation behavior of active and inactive enzyme forms. AB - This paper describes the study of a highly purified pyrimidine nucleoside monophosphate kinase from rat bone marrow cells. Short-term storage (24 h at 4 degrees C) of the purified enzyme in the absence of dithiothreitol, a sulfhydryl reducing agent, led to considerable losses of enzyme activity. Most of the lost activity could be regained, however, by incubating the enzyme with 50 mM dithiothreitol. Enzyme stabilization by dithiothreitol and reactivation by dithiothreitol were enhanced in the presence of phosphate buffer. Severe enzyme inhibition was produced by micromolar concentrations of sulfhydryl group reagents. Chromatographic, electrofocusing, and sucrose gradient centrifugation experiments revealed that the enzyme has a molecular weight of about 26,000, an isoelectric point of 4.7, and a sedimentation coefficient of 2.5. These experiments were also carried out with enzyme preparations which had been almost completely inactivated by means of dialysis to remove dithiothreitol. Enzyme preparations of this type displayed at least one additional enzyme form. This form(s) was inactive but capable of being partially reactivated by dithiothreitol. The inactive form(s) exhibited the same apparent molecular weight as the native enzyme but possessed a higher isoelectric point (5.7). A working hypothesis was presented which states (1) that inactive enzyme forms arise because of disulfide bond formation, (2) that enzyme sulfhydryl groups are less susceptible to oxidation in the presence of phosphate buffer, and (3) that enzyme reactivation by dithiothreitol results from the regeneration of critical enzyme sulfhydryls. PMID- 3010882 TI - [AIDS--a further development]. AB - It has been recently established that AIDS is a virus infectious disease. HTLV-I essentially makes T4 lymphocytes malignant and induces their abnormal proliferation. However, on the way, when HTLV-I induces a decrease in the function of T4 lymphocytes, the symptoms of ARC or AIDS take place on occasion. HTLV-III/LAV possesses a central function to destroy T4 lymphocytes, but, when this function is blocked, it increases T4 lymphocyte proliferation. Accordingly, the concept of carriers of the AIDS virus and patients with AIDS-related complex or AIDS has been clarified. Simultaneously, it has been discovered that a multiplicity of amino acids exists on the envelope of isolated AIDS viruses, resulting in a variety of viral envelope antigenicities. This may support the presence of antigenic modulation, which might make the development of a vaccine more difficult. Therapy has been approached from the following points of view; blockers of reverse transcriptase, drugs for destroying the virus itself through the viral envelope, vaccination, replacement of patient's T4 lymphocytes with healthy bone marrow, and use of immunopotentiators. PMID- 3010883 TI - [New directions for the use of monoclonal antibodies in leukemia diagnosis and therapy]. AB - It has been just a decade since the advent of hybridoma technology, which has made it possible to obtain monoclonal antibodies using the method of Kohler and Milstein. The basic utilization and clinical application of monoclonal antibodies have been developed and generalized in leukemia and lymphoma. In addition, new exciting and specific approaches based on current knowledge of molecular biological techniques have been introduced, together with use of monoclonal antibodies. This paper introduces the following recent topics. Standardization of monoclonal antibodies detecting differentiation antigens. Chromosomal assignment of cell surface antigens. Immunoglobulin and T cell receptor genes as tumor markers. Autologous bone marrow transplantation with the use of antibodytoxin conjugates. Mouse-human chimeric monoclonal antibody. PMID- 3010884 TI - [Clinical evaluation of serum NSE and CEA in primary lung cancer patients]. AB - The clinical value of serum neuron specific enolase (NSE) as a tumor marker was evaluated in comparison with simultaneously measured serum CEA, using 74 cases of lung cancers, 13 cases of non-tumorous and non-neurological disease and 28 normal volunteers. The results obtained were as follows: Thirty-five out of the 74 lung cancer cases showed positive serum NSE levels. However, none of the cases of non tumorous, non-neurological disease and normal volunteers demonstrated more than 10ng/ml NSE which was significantly different from the results of lung cancer cases. We found a higher degree of NSE positiveness in cases of small cell carcinoma than in cases of other histological types. However, no obvious difference of CEA positiveness between any histological types was revealed. Both serum NSE and CEA showed an increase in positive rate in parallel with the progress of stages, including 80.4% of cases of stages III and IV disease which had positive serum levels of both or either of the two markers. However, we could not find any obvious correlation between serum NSE and CEA. The serum levels of both markers clearly decreased after surgery, serum CEA level being considered to be a better reflection of host tumor burden than NSE. In conclusion, the simultaneous testing of serum NSE and CEA is considered to be very useful for evaluating the clinical course or the effect of cancer therapy in lung cancer patients, with higher specificity and sensitivity. PMID- 3010885 TI - [A long-term survivor of primary hepatoma--a case suggesting the superiority of a multi-drug (OK-432, SPG, PSK) over mono-drug immunotherapy]. AB - The studied patient (70 years old, male) had primary hepatoma with rupture of the liver. We resected his left lobe partially and were able to save him. Since a residual tumor was found after surgery, we injected a total of 30 mg of MMC into the SCA 3 times. Under continuous administration of OK-432, the patient survived for 3 years and 6 months in remission. Because the tumor grew gradually, we started a multi-drug immunotherapy (OK-432 5 KE/2 W/intradermal, PSK/3.0 g/day/P.O., SPG/20 mg/2 W/I. M.) which we have advocated. In 6 months the tumor regressed to 1/3 of its original size. The patient is currently in good condition 4 years and 7 months after the operation. This case demonstrated the superiority of multi-drug immunotherapy over single-drug immunotherapy. PMID- 3010886 TI - [Randomized controlled study of high-dose metoclopramide and dexamethasone in the prevention of CDDP-induced emesis]. PMID- 3010887 TI - Pyoderma gangrenosum in a patient with HTLV-III antibody. PMID- 3010888 TI - Conservative treatment for breast cancer. Complications requiring reconstructive surgery. AB - Women who select conservative treatment for carcinoma of the breast (tumor excision followed by supervoltage radiation therapy) place a premium on breast preservation and aesthetics. When local control fails and they require a mastectomy, or when the aesthetic appearance is unacceptable, they may request breast reconstruction. The goal of this study is to evaluate a series of 10 patients who required reconstructive breast surgery after complications of conservative treatment. Patient classification: I. Breast or chest wall necrosis (3). II. Breast fibrosis and gross asymmetry (3). III. Local recurrence of breast cancer (5). IV. Positive margins after the initial lumpectomy (1). The mean age was 34 years. Radiation dosage average was 5252 rads with two patients receiving iridium-192 implant boosts. The reconstructive management was complex and usually required a major musculocutaneous flap because of the radiation effects. PMID- 3010889 TI - [Influence of a calcium overload on the genital system of the male rat]. AB - Treatment of "Sprague Dawley" immature rats with D3 vitamin subchronic doses during 25 days induces a sexual organs calcification, a delayed body development in general, and specially testis and seminal vesicles growth. The effect of this treatment on the development and secretions of the sex accessory organs is measured by the determination of fructose rate in the seminal liquid. PMID- 3010890 TI - [Characterization of adrenergic receptors in human cerebral arteries and the alteration after subarachnoid hemorrhage]. PMID- 3010891 TI - [Eicosanoids, vascular function and atherosclerosis]. AB - Icosanoides (prostaglandins, leukotrienes) seem to play an essential part in cardiovascular pathology. A range of experimental data obtained both in vitro and in vivo has resulted in a rapid progression of our understanding of their biochemical and functional properties and has opened up new fields of pharmacological research. However, a clear cut demonstration of their clinical relevance remains difficult. Improved methodology will no doubt provide more information about the importance of these compounds. For the present, we recommend reexamination of previously reported results. PMID- 3010892 TI - [Polyunsaturated fatty acids, atherosclerosis and thrombosis]. AB - This article is a short update of the role played by dietary polyunsaturated fatty acids in atherosclerosis and thrombosis, with special reference to the interactions between the platelets and endothelium. The n-3 family of fatty acids, especially eicosapentaenoic (20:5n-3) and docosahexaenoic acids (22:6n-3), seem to be beneficial in the prevention of these diseases. PMID- 3010893 TI - [Prospective study of 59 cases of viral meningitis in children. Clinical and virologic diagnosis. Epidemiology and physiopathology]. AB - In a prospective 2 year study of 59 cases of childhood meningitis, mumps was the most common etiological virus (39%), followed by enterovirus (27%). The analysis of the cases suggested that a diagnosis of the infectious agent may be arrived at using clinical data such as the degree of nuchal rigidity, the age of the patients, and the presence of associated parotiditis or macular rash. Pleiocytosis in the CSF was higher and included a larger percentage of lymphocytes during mumps meningitis than during enterovirus meningitis. Mumps or enterovirus were isolated in the CSF of 23% (mumps) and 27% (enterovirus) of the patients. Alpha interferon which was acid labile was detected in the CSF of 89% and 63% respectively of patients with mumps and enterovirus meningitis. PMID- 3010894 TI - Characterization of the DNA of the hamster papovavirus: IV. Transcription mapping of calf-thymus DNA polymerase II. AB - Nascent RNA, synthesized by calf thymus RNA polymerase II on restriction endonuclease BamHI linearized hamster papovavirus (HaPV) DNA, was rehybridized to the template strand under conditions allowing transcription R-loop formation. Hybrids, visualized by electron microscopy, were plotted and mapped according to the physical map of HaPV. Two predominant regions of transcription could be localized at 0.10--0.40 and 0.50--0.82 m.u., respectively. For the start sites of transcription at map positions 0.67 and 0.75, respectively, on the HaPV genome a transcription in opposite direction were estimated. This genome region harbours the putative origin of replication of HaPV DNA. These results suggest a distinct relatedness of HaPV to the polyomavirus group. PMID- 3010895 TI - [Viral carcinogenesis]. AB - A short overview of the history of tumor virology is given with respect to the special data of research in this field done by Arnold Graffi and his coworkers. The theory of viral etiology of tumors during the 75 years since its inauguration had to be proven by scientists fighting for the idea of viral agents being the cause of several types of cancer in animals and man. Today this theory is accepted; a series of human tumors is known as induced by viruses, too. Moreover, tumor viruses have increased our knowledge of mechanisms of malignant transformation and the discovery of one genes has opened new possibilities for analysis of the process of cancerogenesis. PMID- 3010897 TI - Angiomatoid malignant fibrous histiocytoma. PMID- 3010896 TI - UMPK polymorphism in the Polish population. AB - Studies on UMPK polymorphism were carried out in a sample of the Polish population numbering 462 subjects. The occurrence of three phenotypes was confirmed--UMPK 1, UMPK 2-1 and UMPK 2. From distribution of phenotypes, frequencies of determining genes were calculated with the following results: UMPK1-0.9762, UMPK2-0.0238, The usefulness of this system in paternity cases is 1.69%. PMID- 3010898 TI - Cystosarcoma phyllodes of the prostate. A pathologic and immunohistochemical study. AB - We treated two patients who had lesions in the prostate with histologic features similar to those of cystosarcoma phyllodes of the breast. In one case, the stroma progressed to a clearly sarcomatous appearance, whereas the other tumor had a cellular stroma that was mitotically inactive. This element was immunoreactive for vimentin and desmin in both cases but was negative for epithelial markers. In contrast, the epithelial component was immunoreactive for prostate-specific antigen and epithelial membrane antigen. Following surgical resection, both patients were well two and three years later, without local recurrence or distant metastasis. The histogenesis of these tumors is unknown. PMID- 3010900 TI - Prodrugs of 5-ethyl-2'-deoxyuridine, II. Syntheses and antiviral activities of 5' and 3'-ester derivatives. PMID- 3010899 TI - Primary lymphoma of the liver in the acquired immunodeficiency syndrome. AB - We describe a 78-year-old man with a diffuse large-cell lymphoma that was limited to the liver and was associated with micronodular cirrhosis and Kaposi's sarcoma that involved abdominal lymph nodes and gastric mucosa. The serum of the patient reacted positively to a test for human T-cell lymphotropic virus type III antibodies. We discuss the clinical and autopsy findings for this unusual patient, the criteria for the diagnosis of primary lymphoma of the liver, and its occurrence in patients with the acquired immunodeficiency syndrome. PMID- 3010901 TI - Segmental hepatic resection utilizing the ultrasonic dissector. AB - Hepatic resection continues to become a more widely accepted therapeutic modality, with increased use as improved imaging modalities more precisely define the nature and extent of various liver abnormalities. The surgical anatomy of the liver indicates that there are eight segments with single or multiple segmental resections able to be performed. The use of the ultrasonic dissector facilitates the performance of transparenchymatous segmental resection without obtaining vascular inflow or outflow control. This report describes the segmental anatomy of the liver and the use of the ultrasonic dissector. Thirteen patients have undergone segmental hepatic resection with the ultrasonic dissector. Five patients had cirrhosis. Mean +/- 1 SD operative time required for segmental resection was 128 +/- 57 minutes, and blood loss was 830 +/- 623 mL. Utilization of the ultrasonic dissector to perform segmental hepatic resection may increase our versatility in the management of various hepatic and biliary tract diseases. PMID- 3010902 TI - Efficacy of magnetic resonance imaging in 139 children with tumors. AB - One hundred thirty-nine children with neoplasms were studied using magnetic resonance imaging (MRI). This procedure was as accurate as computed tomography in predicting tumor histology, except that MRI was unable to detect small areas of tumor calcification. Magnetic resonance imaging could accurately identify the organ of origin of tumor masses and differentiate soft tissue from fat, fluid, or hemorrhage. In addition, MRI was helpful in planning surgery in many cases: It was better than computed tomography in defining the size and extent of soft tissue tumor masses. It was accurate in defining the extent of the spread of bone sarcomas in the bone marrow. Without requiring the injection of intravenous contrast agents, it accurately defined displacement, encasement, or invasion of major abdominal blood vessels by Wilms' tumors and neuroblastomas. As a means of evaluating pediatric neoplasms, MRI is noninvasive, painless, and well tolerated by children, and it uses no radiation. PMID- 3010903 TI - Second hepatoma developing 13 years after resection of first tumor. AB - A patient underwent a left-sided hepatic lobectomy for primary hepatocellular carcinoma 13 years ago and remained symptom free. He then presented with spontaneous rupture of a large tumor in the right lobe of the liver. Although this tumor proved to be primary hepatocellular carcinoma, there were significant histological differences between the two lesions, suggesting that this was a second primary liver tumor. Bleeding from the tumor was controlled by selectively ligating the branches supplying the area of hemorrhage. PMID- 3010904 TI - Interferon production in L929 cells under impaired translational conditions: comparison of rates of interferon, actin, Newcastle disease and encephalomyocarditis viruses mRNAs initiation of protein synthesis. AB - The pattern of Interferon (IFN) production in virus-infected cells has been compared with the rate of bulk cellular protein synthesis, on one hand, and with the synthesis of representative cell and virus proteins such as actin, the gamma and the NP proteins of encephalomyocarditis (EMC) and Newcastle Disease (NDV) viruses, on the other hand. This was investigated under conditions of impaired protein synthesis such as i) high osmolarity media, ii) a virus-induced shut-off, and iii) in cells exposed to relatively low doses of cycloheximide (CXM), which slow elongation of protein chain and thus favour the translation of low-affinity messenger RNAs (mRNAs). In each instance IFN production was compared with 35S methionine incorporation into TCA-precipitable materials and into SDS polyacrylamide gel-analysed proteins. Data obtained from each of the experimental approaches all indicate that IFN production and cellular protein synthesis are modified in a closely related fashion suggesting that their mRNAs share a similar degree of affinity for ribosomes. Conversely, two mRNAs coding for representative EMC and NDV virus proteins exhibit, respectively, the highest and the lowest affinity for ribosomes as compared to actin mRNA. PMID- 3010906 TI - Cleavage maps of human cytomegalovirus genome (strain Towne) determined by the use of cosmid cloning system. AB - Virion DNA of human cytomegalovirus strain Towne was partially digested with endonuclease Hind III and fragments larger than 29 kbp were ligated to cosmid pHC79. The whole viral DNA sequence has been cloned in large overlapping segments carried by 32 recombinant cosmid clones (a pIT series). A whole set of Hind III fragments has also been cloned (a pHI series). By using these, we have constructed corrected cleavage maps of strain Towne DNA for Hind III, Bam H I, EcoR I and Xba I. PMID- 3010905 TI - Neutralizing monoclonal antibodies directed to infectious bovine rhinotracheitis virus. AB - Infectious bovine rhinotracheitis virus (IBRV) has been shown in this report to have thirty-three polypeptides. Ten of the eleven polypeptides which can be labeled with (3H)-glucosamine are located on the surface of the virus since they can be surface labeled with sodium boro(3H)hydride. In order to define the immunologically important viral proteins, monoclonal antibodies were prepared against the virus and selected for their ability to neutralize infectivity. Four such hybridoma lines were obtained for characterization of the antigens that elicit neutralizing antibodies. The viral polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the specificity of each monoclonal antibody was determined by "Western" blot analysis and/or by immunoprecipitation of (35S)-methionine and (3H)-glucosamine labeled infected cell lysates by the monoclonal antibodies. One monoclonal antibody reacted with two glycoproteins, gp135 and gp78a, on the "Western" blot but immunoprecipitated three glycoproteins, gp135, gp78a, and gp54 from labeled infected cell lysates. The other three monoclonal antibodies immunoprecipitated a single glycoprotein, gp78b, from (3H)-glucosamine labeled infected cell lysates but not from (35S) methionine labeled infected cell lysates. PMID- 3010907 TI - Intracellular localization of rotaviral proteins. AB - The differential distribution of two SA 11 rotaviral capsid antigens in thin sections of infected cells was examined using antibody-coated colloidal gold electron-dense particles as specific post-embedding immunocyto-chemical labels. The treatment of thin sections of conventionally fixed and embedded tissue specimens with sodium metaperiodate allowed specific localization of the antigens in tunicamycin-treated, infected CV-1 cells. Both protein antigens were investigated with specific anti-rotavirus hyper-immune sera and with specific monoclonal antibodies. These studies showed that the major outer capsid glycoprotein, gp34, of SA11 rotavirus particles was mainly located within the cisternae and along the membranes of the rough endoplasmic reticulum. The antigen of the major inner capsid protein, p42, was identified attached to enveloped virus particles, and even more obviously, on laminar crystalline structures in the nucleus and cytoplasm of the infected cells. PMID- 3010909 TI - Further characterization of virus obtained from herpes simplex virus type 1 recurrences and primary infections. Influence of the temperature of incubation upon glycoprotein synthesis and virus release. Brief report. AB - The virus contained in clinical isolates of herpes simplex virus type 1 (HSV-1) which have not undergone previous in vitro passages (new isolates) differs from HSV-1 prototype strains with respect to infected cell glycoprotein pattern, and, most probably efficiency of virus egress at 37 degrees C. The differences can be abolished by lowering the temperature of incubation to 33 degrees C. A few tissue culture passages cause the conversion of the original virus to a virus undistinguishable from HSV-1 prototype strains with respect to the parameters mentioned above. PMID- 3010908 TI - Inoculation of guinea pigs with varicella-zoster virus via the respiratory route. AB - Guinea pigs were inoculated by the respiratory route with wild-type (Cyr) or vaccine (Oka) strain varicella zoster virus (VZV). Wild-type cell-free virus obtained by sonication produced neutralizing antibody responses in steroid treated animals when given via the intratracheal route, and induced neutralizing antibody as well as a pneumonitis in normal animals when given via the intrabronchial (i.b.) route. A humoral response also followed i.b. instillation of cell-associated wild-type or vaccine strain VZV. Prior i.b. administration of thioglycollate or exposure to hyperoxia altered the number and function of pulmonary macrophages, respectively, but viral susceptibility of the guinea pigs was not enhanced. Both strains of VZV could be isolated from bronchial washings up to 48 hours after i.b. instillation of cell-associated virus, but neither strain was isolated thereafter from cultures of bronchial washings or explanted lung tissues. PMID- 3010910 TI - Encephalomyocarditis virus can bind to and transfect non-permissive cells. AB - Mouse embryo fibroblasts and mouse adrenal tumor cells support the replication of encephalomyocarditis (EMC) virus, whereas rat glial and rat hepatoma cells are non-permissive. These differences in susceptibility were not due to the lack of virus attachment to rat cells. The findings that rat cells could be transfected with RNA derived from EMC virus indicates that the block in viral replication in these cells occurs at some point between attachment and uncoating of virus, probably at the level of uncoating. PMID- 3010911 TI - [Structure and functions of myoepithelium]. PMID- 3010912 TI - [Histogenesis of human embryonal cancer of the testis]. AB - Complex morphological study of 45 germ cell tumors of the testis revealed two types of histological structure of embryonal carcinoma. Tumour histostructure was related to the ultrastructural features of cells (I-IV types) forming them. The data obtained indicate that the process of differentiation of these tumours resembles initial stages of embryogenesis (morula, blastula, egg cylinder). Embryonal carcinoma cells appeared to be tumor stem cells in the development of teratoma, chorionepithelioma, yolk sac tumour. PMID- 3010913 TI - [Acrospiroma (clinico-morphological characteristics and histogenesis)]. AB - Clinico-morphological characteristics of 105 acrospiromas surgically removed within 10 last years is presented. In 6 patients the tumour was considered as a malignant acrospiroma which, according to the literature and the authors' material, is prone to recurrence. The features of a diagnostic significance are discussed in detail: the development of tumour in the cyst, growth of squamous cells and clear (due to the glycogen) cells, formation of structures resembling the sweat glands ducts. Electron-microscopic examination indicates the histogenetic link of acrospiroma with the elements of sweat gland ducts (intracytoplasmic ducts, periluminal cells, the lack of clear signs of secretion). All this relates also to the tumour described in the literature as a clear-cell hydradenoma. PMID- 3010914 TI - [Ultrahistochemistry of cardiomyocytes in acquired mitral lesions of the heart]. AB - A study of myocardium of patients with mitral stenosis was performed using ultrahistochemical methods at different stages of disease and in the course of valve replacement operation. A quantitative analysis of degenerative changes was introduced, the results of which correlated with the severity of heart failure. A pronounced heterogeneity in mitochondrial enzyme activity has been detected cytochemically. In some apparently "unaffected" cardiomyocytes an active alterative process was expressed in an increase of sarcolemma permeability for electron-microscopic tracers. An elevated fenestration of membrane barriers was noted at the stages of anoxic heart arrest. Using tannic acid it was possible to demonstrate a penetration of plasma protein-polysaccharide complexes into cell sarcoplasma. There were observed topographic variations in the distribution of acid phosphatase activity. PMID- 3010915 TI - [Peripheral cancer of the lung and tuberculosis]. AB - Morphological analysis of 90 observations with a clinical diagnosis of a small peripheral lung carcinoma is performed. The examination of an operation material confirmed diagnosis in 71 cases. Tuberculomas are found in 19 cases. The peripheral lung carcinoma was found to develop in 91.5% (65 cases) against the background of preexisting scars which in 73.8% (48 cases) had a post-tuberculosis and 26.2% (17) post-pneumonia origin. Scars were most frequently related to the healed forms of a focal secondary tuberculosis (30 cases) and sclerotic changes around tuberculomas (8). Post-tuberculosis scars provoking sclerosis and deformation of vessels, bronchi, bronchioles and alveoles with the development of an epithelial dysplasia, are one of the risk factors in the development of a peripheral lung carcinoma. The degradation and fibrinoid changes of a scar tissue infiltrated by a tumour and followed by destruction of scars are observed in peripheral lung carcinomas with a diameter more than 3 cm. PMID- 3010916 TI - Interaction between epidermal growth factor and triamcinolone acetonide in mouse palatal mesenchymal cells in vitro. AB - Palatal mesenchymal cells from mouse embryos (MEPM cells) had a high number of specific receptors for epidermal growth factor (EGF). At 37 degrees C, the number of high-affinity receptors (dissociation constant, Kd of 6.0 X 10(-10) M) was 2.0 X 10(5) per cell and of low-affinity receptors (Kd of 2.9 X 10(-9) M), 3.8 X 10(5) per cell. At 2 degrees C, a single class of receptors (Kd of 4.8 X 10(-9) M) was detected at 1.8 X 10(5) per cell. Triamcinolone acetonide, a potent cleft palate-inducing agent, slightly inhibited the recovery of 125I-labelled EGF binding capacity in MEPM cells after down regulation of EGF receptors; it did not affect the binding properties of 125I-labelled EGF in these cells. PMID- 3010917 TI - Peroxidase penetration in the crevicular epithelium of rat molar gingiva. AB - Rat molar gingiva was studied by tracer experiments employing horseradish peroxidase and by cytochemical demonstration of inorganic trimetaphosphatase. Though non-keratinized junctional epithelium appeared to have no effective diffusion barrier against the permeation of peroxidase, keratinized sulcular epithelium had an evident barrier for the tracer at the junction between the granular and cornified layers. The barrier to peroxidase permeation was closely associated with the presence of either numerous membrane-coating granules or a cornified layer. The basal cells of both junctional and sulcular epithelia frequently took up peroxidase by means of coated pits, vesicles and endocytotic vacuoles. Inorganic trimetaphosphatase activity was present in dense bodies, multivesicular bodies and vacuoles, but not necessarily related closely to peroxidase incorporation within the cytoplasm. PMID- 3010918 TI - Conjunctival cytology of adult chlamydial conjunctivitis. AB - We assessed the diagnostic value of ocular cytologic examination by reviewing Giemsa-stained smears of conjunctival scrapings. Of 387 patients with a clinical diagnosis of adult chlamydial conjunctivitis, intracytoplasmic inclusions were found in 30 (8%). Both polymorphonuclear leukocytes and lymphocytes were common; the predominant cell type was not useful to differentiate chlamydial from adenoviral conjunctivitis. More sensitive cytologic features included the presence of plasma cells, Leber cells, blastoid cells, and multinucleated cells. Giemsa-stained conjunctival cytologic examination can provide a useful method to support the clinical diagnosis and to direct further laboratory testing. PMID- 3010919 TI - Leukotrienes in the aqueous humor of patients with uveitis. AB - Utilizing chromatographic and radioimmunoassay techniques, we measured the concentration of leukotriene (LT) B4 and LTC4/D4/E4 in the aqueous humor of 14 patients with uveitis and seven patients undergoing penetrating keratoplasty or routine cataract extraction. Leukotriene B4 was detected in 11 of 14 patients with uveitis (mean, 0.96 pmole/mL), and LTC4/D4/E4 was found in 12 of 14 patients with uveitis (mean, 2.0 pmole/mL). In the control group, LTB4 was detected in six of seven patients (mean, 0.57 pmole/mL), and LTC4/D4/E4 was found in six of seven patients (mean, 1.9 pmole/mL). Leukotriene levels did not correlate with clinically assessed aqueous cell and flare. Mean levels of LT in patients with uveitis receiving corticosteroids were nearly double those found in non-steroid treated patients. Despite apparent differences in LT levels, none of the above differences reached statistical significance. PMID- 3010920 TI - Micropipette aspiration of human platelets after exposure to aggregating agents. AB - The present study examined the influence of activation on platelet deformability. Aspiration of cells after exposure to thrombin, adenosine 5' -diphosphate, or the calcium ionophore A23187 at concentrations producing shape change and stickiness revealed significant changes from control cells. At the lowest negative pressure, 4 X 10(-2) dynes/cm (-1 cm H2O), there were no differences in lengths of membrane segments aspirated from control and activated platelets. Each subsequent increase in negative pressure up to 35 X 10(-2) dynes/cm (-7.5 cm H2O) resulted in significantly longer aspirated segments on activated cells compared to control cells. Greater negative pressures did not cause further increases in lengths of membrane segments drawn into the pipette. Thus, activation, which results in constriction of the circumferential microtubule, makes more membrane available for aspiration as negative pressure is increased. In both control and activated platelets, the microtubule coils served as a barrier to further lengthening of aspirated membrane segments as negative pressure was increased beyond 35 X 10(-2) dynes (-7.5 cm H2O). PMID- 3010921 TI - Sexually transmissible diseases: an overview. PMID- 3010922 TI - Metabolism of glucose by preimplantation mouse embryos in the presence of glucagon, insulin, epinephrine, cAMP, theophylline and caffeine. AB - Neither insulin nor epinephrine influenced the incorporation of glucose into the acid-soluble or acid-insoluble glycogen pool of mouse embryos at the morula-early blastocyst stage during 5 h culture in the presence of radiolabelled glucose. During a 5 h chase culture of pulse-labelled embryos at this stage of development, acid-soluble glycogen labelled during the pulse was not utilized by the embryo but acid-insoluble glycogen was reduced. Addition of glucagon, insulin, epinephrine, cAMP, theophylline or caffeine during chase culture had no effect on the turnover of label in the glycogen pools of the embryo. These results indicate that the turnover of embryonic glycogen observed in vivo is not due to the direct effect of the hormones that regulate glycogen metabolism in the mother. Insulin was found to stimulate incorporation of glucose into non-glycogen macromolecules during both pulse and chase culture. Thus, whilst an effect of insulin on glycogen metabolism was absent, the anabolic effects of this hormone appear to have been expressed in the embryo at this stage of development. PMID- 3010923 TI - Autoradiographical localization of luteinizing hormone releasing hormone (LHRH) receptors on rat testicular intertubular cells fractionated on Percoll density gradients. AB - The specific binding of 125I-labelled [D-Ser(tBu)6,des-GlyNH2(10)] LHRH ethylamide (LHRH-A) to testicular intertubular cells fractionated on Percoll density gradients was investigated. The greatest binding per cell occurred in the density region which contained the largest proportion of Leydig cells (sp. gr. 1.0820-1.0585). Autoradiographs of the cells from this region confirmed that silver stains were predominantly located over the Leydig cell, significantly (P less than 0.01) more grains were observed over this cell type in the total binding fractions than in the non-specific binding fractions. However, 5.9% of cells other than Leydig cells (testicular macrophages and indeterminate connective tissue cells) from this region also displayed significant displaceable binding (P less than 0.01). The location of [125I]LHRH-A binding to cells in other density regions, which did not contain identifiable Leydig cells, could not be established by autoradiography. These results confirm that the Leydig cell possesses LHRH receptors, but also indicate that other testicular cells have specific, high-affinity binding sites for LHRH-A, and may either be responsive to direct stimulation by LHRH, or may partially mediate the effects of LHRH and its agonists on Leydig cell function. PMID- 3010924 TI - Paralysis in herpes zoster. AB - Herpes zoster is a relatively common disease which affects predominantly the middle-aged and elderly. The segmentally distributed cutaneous eruption, sensory changes, and pain make up the well known zoster syndrome. Motor loss is another aspect of this syndrome which is less well known but occurs in a significant number of cases, and is probably far more frequent than is recognised because the weakness is readily obscured by pain. Four cases of herpes zoster with motor involvement are described. Two cases had zoster paresis affecting the arm and hand, and one of these had, in limb, and one case had urinary retention owing to an atonic bladder. These cases serve to illustrate many of the clinical features of the zoster syndrome with motor involvement. The significant functional implications of unrecognised motor deficit, particularly in the elderly, are a prominent feature and highlight the importance of early accurate diagnosis and management. The pathogenesis and clinical features of this syndrome are discussed in the literature review. PMID- 3010926 TI - Metastatic astrocytoma. AB - Two women, treated with surgery and radiotherapy for cerebral astrocytoma, developed metastases. In one case a metastasis in a craniotomy scar was probably implanted at the time of surgery; at first this was sensitive to chemotherapy and radiotherapy, but it later recurred and became resistant to treatment, with resistant tumour spreading extensively through the scalp and the cervical lymphatic chain. In the other case tumour disseminated hematogenously to the bone marrow. Both patients died of metastatic disease with primary tumours apparently well controlled. The syndrome of metastatic astrocytoma may be under-diagnosed. PMID- 3010925 TI - Benign and malignant pleural effusions in former Wittenoom crocidolite millers and miners. AB - Serial plain chest radiographs taken between 1943 and 1982 for 280 claimants for compensation for asbestosis and 32 claimants for malignant pleural mesothelioma from the Wittenoom asbestos industry were reviewed by two observers to identify diffuse pleural thickening and pleural effusion. A pleural effusion which appeared and resolved within two years without radiographic or clinical evidence of underlying malignancy, infection or cardiac failure was seen in 15 cases by reader 1 and 24 cases by reader 2. Eighteen cases of effusion, determined from clinical records to be caused by malignant pleural mesothelioma, were seen by reader 1 and 20 by reader 2. The latent periods between commencing work and the first radiograph showing effusion were much shorter for subjects with benign asbestos pleural effusion than for subjects with effusion due to malignant pleural mesothelioma, although there was considerable overlap in the range. The longest latent period for benign asbestos pleural effusion was 22 years and the shortest period for effusion due to malignant pleural mesothelioma was 12 years. The latent period for benign asbestos pleural effusion was inversely related to total cumulative exposure, whereas that for effusion due to malignant pleural mesothelioma was significantly shorter for subjects who had worked in the mill than for those who had worked in the mine. A long latent period and a history of working in the mill were significant discriminators for a malignant as opposed to a benign basis for an effusion. The appearance of a benign asbestos pleural effusion appeared to be a risk factor for the severity of subsequent diffuse pleural thickening.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3010927 TI - Recurrent cholangitis caused by hepatocellular carcinoma. AB - A patient is described with recurrent cholangitis and longstanding alcoholic cirrhosis. The cause of the cholangitis was found to be intraductal biliary tract invasion by hepatocellular carcinoma. A review of the literature suggests that this is an unusual manifestation of a common malignancy. PMID- 3010928 TI - Arbovirus infection in a Murray Valley community. AB - Serum antibodies to Ross River virus and Murray Valley encephalitis virus were measured during 1974-1975 in residents of Echuca, an urban Murray Valley community. A representative group of volunteers was obtained by random selection of households. The prevalence of antibodies to both viruses increased progressively with age. Prevalence was equal in both sexes for both viruses in all age groups, indicating that the risk of infection was mainly determined by geography rather than by personal activities. Antibody levels remained unchanged in the following year when there was no disease activity in the area. The stability of antibody levels permitted retrospective estimates of mean rates of infection. These were approximately 0.4% per annum for both viruses when age was used as the index for years of exposure. With allowance for other factors, the best estimate for both virus infections is probably closer to 1%. The morbidity rate for Ross River virus infection appeared to be low. It is concluded that infection with Ross River virus and with Murray Valley encephalitis-related viruses is endemic in the Murray Valley. PMID- 3010930 TI - Foot-and-mouth disease in cattle, buffalo and sheep in Java. PMID- 3010929 TI - Combined spironolactone and converting-enzyme inhibitor therapy for refractory heart failure. AB - Three cases are described of severe congestive heart failure which failed to respond to digitalis, high doses of loop diuretics, and converting-enzyme inhibitors. The addition of spironolactone produced a substantial diuresis associated with clinical and radiological improvement in each case. Although a reversible deterioration in renal function was observed, this drug combination represents a therapeutic option in end-stage heart failure. PMID- 3010931 TI - Neonatal treatment with monosodium glutamate does not alter grooming behavior induced by novelty or adrenocorticotropic hormone. AB - The increased grooming behavior observed in a novel environment has been attributed to release of peptides derived from proopiomelanocortin (POMC), such as ACTH, alpha-melanocyte-stimulating hormone (alpha-MSH), or beta-endorphin, which themselves can elicit grooming. This is because novelty-induced grooming is attenuated both by hypophysectomy and by antiserum to ACTH injected into the cerebral ventricles. Administration of monosodium glutamate (MSG) to neonatal rats destroys neurons in the arcuate nucleus of the hypothalamus, depleting the brain of POMC peptides, and also hypothalamic dopamine and choline acetyl transferase activity. Neonatal MSG treatment did not significantly alter the grooming scores of adult rats in either home or novel environments compared to saline-treated animals. There were also no differences between MSG-and saline treated rats in the grooming scores observed following graded doses of ACTH1-24 (0.2-1.0 micrograms) administered intracerebroventricularly. Thus if increased grooming in the novel environment is due to release into the ventricles of ACTH, alpha-MSH, beta-endorphin, these peptides more likely derive from the pituitary rather than from brain cells, although the failure of the MSG treatment to produce quantitative depletions of cerebral POMC peptides, especially in the brain stem, leaves open the latter possibility. PMID- 3010932 TI - History of genetic engineering of laboratory and farm animals. PMID- 3010933 TI - Expression of the bovine growth hormone gene in cultured rodent cells. PMID- 3010934 TI - Application of bioengineering to disease diagnosis. AB - Traditional approaches to diagnosing disease include clinical observations, pathological changes in tissues, and searches for the etiology, by isolation, or identification of microorganisms, or by serological methods. Development of techniques for studying the molecular biology of microorganisms, manipulation of cellular systems, and improved immunoassays have contributed to better diagnostic technology. Recombinant DNA technology has made it possible to apply highly specific probes consisting of nucleotide sequences that hybridize with complementary sequences of microorganisms. The specific techniques utilized include Southern, northern, and dot blot hybridization and in situ hybridization. Identification of proteins of microorganisms is done by western blot, dot blot, and in situ peroxidase-antiperoxidase techniques. These techniques have the promise of being highly specific and rapid methods for diagnosis of disease. PMID- 3010935 TI - Recombinant DNA approaches to feline leukemia virus immunization. AB - We have utilized 2 recombinant DNA strategies for immunization against FeLV in cats: (a) modified live virus was attenuated by mutation and recombination, and (b) an immunogen, consisting of subunit envelope protein, was prepared in genetically engineered yeast. Results indicated that the genetically manipulated live virus preparations were not protective against FeLV challenge because they were either not attenuated in virulence or were not sufficiently antigenic. Immunization with yeast-synthesized FeLV envelope protein followed by the modified live virus gave protective immunity in cats under experimental conditions. Future immunization attempts will concentrate on enhancing the immunogenic potency of the yeast- synthesized FeLV envelope protein. PMID- 3010936 TI - Gene transfer into animals by retroviral vectors. PMID- 3010937 TI - Casein genes and genetic engineering of the caseins. PMID- 3010938 TI - [Polioencephalomyelitis induced by porcine enterovirus serotype 2 in a pig fattening plant]. PMID- 3010939 TI - Activity of recombinant human alpha interferon against influenza virus infection in mice. AB - The in vivo antiviral activity of recombinant human leukocyte hybrid interferon, HuIFN-alpha AD, was examined. Results showed that this material in highly purified form did not protect mice against a lethal dose of influenza virus, although administration of natural MuIFN-alpha/beta to mice infected with a lethal dose of influenza virus had a marked protective effect. The effect of alveolar macrophages treated with IFN on influenza virus replication was examined in vitro. The antiviral activity of alveolar macrophages treated with HuIFN-alpha AD was lower than that of MuIFN-alpha/beta. It is concluded that HuIFN-alpha AD is effective in direct inhibition of influenza virus, but not in indirect inhibition mediated by alveolar macrophages or in protection of mice from influenza virus infection. PMID- 3010940 TI - Nature and properties of human platelet vasopressin receptors. AB - The binding of 3H-labelled [8-arginine]vasopressin to human platelets or crude platelet membranes was examined. Both preparations specifically bound [8 arginine]vasopressin. The binding increased linearly with protein concentration, it was temperature- and time-dependent, saturable and could be reversed to a large extent by EDTA (10 mM). In this latter case, addition of an excess of MgCl2 (20 mM) restored the initial level of binding. Intact platelets and membranes derived from these platelets presented a single population of binding sites with a dissociation constant (Kd) of 1.3 +/- 0.2 and 1.8 +/- 0.3 nM and a maximal binding capacity of 142 +/- 48 and 270 +/- 17 fmol/mg of protein, respectively. The Kd values of various analogues correlated well with those determined on rat liver membrane V1 vasopressin receptors but not with those determined on rat kidney membrane V2 receptors. PMID- 3010941 TI - Activation of rat liver adenylate cyclase by guanosine 5'-[beta,gamma imido]triphosphate and glucagon. Existence of reversibly and irreversibly activated states of the stimulatory GTP-binding protein. AB - The effects of guanosine 5'-[beta-thio]diphosphate (GDP[S]) on the kinetics of activation of rat liver membrane adenylate cyclase by guanosine 5'-[beta,gamma imido]triphosphate (p[NH]ppG) were examined. GDP[S] caused immediate inhibition of the activation by p[NH]ppG at all time points tested. Substantial inhibition by GDP[S] was observed even after the time required for the enzyme to reach its steady-state activity, but the extent of inhibition became progressively smaller as the preincubation time with p[NH]ppG increased. The rate at which adenylate cyclase became quasi-irreversibly activated was a strictly first-order process. In the presence of glucagon, the formation of the irreversibly activated state was much slower. A combination of GDP[S] and glucagon could partially reverse the quasi-irreversible activation by p[NH]ppG. Glucagon decreased the lag time required for p[NH]ppG to activate adenylate cyclase and increased the extent of activation by p[NH]ppG. This stimulatory effect of the hormone on top of guanine nucleotide decreased on preincubation with p[NH]ppG, but not with GTP. Our results suggest that the activation of adenylate cyclase by non-hydrolysable GTP analogues is a two-stage process: the formation of a reversibly activated form (G rev) is a rapid process, followed by a much slower formation of the quasi irreversibly activated form (G irr). Glucagon can stimulate G rev but not G irr, and can partially facilitate the formation of the G rev from the G irr state. PMID- 3010943 TI - Immunochemical characterization of phosphatidylinositol 4-phosphate kinase from rat brain. AB - Affinity-purified antibodies were used to identify a protein of molecular mass 45 kDa (45 kDa protein) in rat brain cytosol as phosphatidylinositol 4-phosphate (PtdIns4P) kinase. Antibodies were raised in rabbits by immunization with the purified 45 kDa protein. Anti-(45 kDa protein) immunoglobulins were isolated by affinity chromatography of the antiserum on a solid immunosorbent, which was prepared by coupling a soluble rat brain fraction, the DEAE-cellulose pool containing 10-15% 45 kDa protein, to CNBr-activated Sepharose 4B. The purified IgGs were specific for the 45 kDa protein as judged by immunoblot and by immunoprecipitation. The purified anti-(45 kDa protein) IgGs inhibited the enzyme activity of partially purified PtdIns4P kinase, whereas preimmune IgGs were ineffective. Immunoprecipitation of the 45 kDa protein from the partially purified enzyme preparation with the purified IgGs resulted in a concomitant decrease in the amount of 45 kDa protein and in PtdIns4P kinase activity. The amount of 45 kDa protein remaining in the supernatant and the activity of PtdIns4P kinase correlated with a coefficient of r = 0.87. The evidence presented lends further support for the notion that the catalytic activity of PtdIns4P kinase in rat brain cytosol resides in a 45 kDa protein. PMID- 3010942 TI - Interaction between calmodulin and five different spin-labelled chlorophenothiazines. AB - The binding to purified calmodulin of five spin-labelled derivatives of chlorophenothiazine was investigated by e.s.r. spectrometry and by the antagonizing potency on the calmodulin-dependent activation of myosin light chain kinase. The results of a comparative study and the influence of pH and ionic strength on the binding support the occurrence of an electrostatic binding involving the terminal amino group of the side-chain of the chlorophenothiazine. These results are discussed in relation to the specificity of the interaction that holds the antipsychotic drug-calmodulin complex together. PMID- 3010944 TI - Glucose-stimulated sequestration of Ca2+ in clonal insulin-releasing cells. Evidence for an opposing effect of muscarinic-receptor activation. AB - Net fluxes of Ca2+ and acid production were studied in clonal insulin-releasing cells (RINm5F) by using colour indicators and dual-wavelength spectrophotometry. After equilibration with a medium containing 10-20 microM-Ca2+, only minimal amounts of Ca2+ (0.08 mmol/kg of protein) were released from the cells by subsequent additions of the respiratory blocker antimycin A and the Ca2+ ionophore A23187. The presence of 20 mM-glucose resulted in an almost 5-fold increase of the acid production and in a stimulated net uptake of Ca2+. The latter process was independent of the extracellular Ca2+ concentration and reached saturation after 20 +/- 1 min, when it corresponded to 1.18 +/- 0.07 mmol of calcium/kg of protein. Whereas the thiol reagent iodoacetamide suppressed the acid production, interference with mitochondrial function by using antimycin A or the uncoupler carbonyl cyanide m-chlorophenylhydrazone had the opposite effect. The latter two drugs induced a selective release of Ca2+ from a pool containing 35% of that taken up during glucose exposure. Most of the remaining Ca2+ was liberated by A23187 or iodoacetamide. Carbamoylcholine was also selective in mobilizing glucose-stimulated calcium, but this calcium (17%) appeared to originate from the pool insensitive to mitochondrial poisons. The action of carbamoylcholine was blocked by atropine and did not depend on the presence of extracellular Na+. The opposite effects of glucose and muscarinic-receptor activation on a non-mitochondrial calcium pool are consistent with participation of the endoplasmic reticulum in the glucose-induced sequestration of Ca2+ in pancreatic beta-cells. PMID- 3010946 TI - An investigation of the ligand-binding properties of Pseudomonas aeruginosa nitrite reductase. AB - The low-temperature e.p.r. and m.c.d. (magnetic-circular-dichroism) spectra of Pseudomonas aeruginosa nitrite reductase, together with those of its partially and fully cyanide-bound derivatives, were investigated. The m.c.d. spectra in the range 600-2000 nm indicate that the native axial ligands to haem c are histidine and methionine, and furthermore that it is the methionine ligand that must be displaced before cyanide binding at this haem. The m.c.d. spectra in the range 1000-2000 nm contain no charge-transfer bands arising from low-spin ferric haem d1, a chlorin. New optical transitions in the region 700-850 nm were found for the cyanide adduct of haem d1. The g-values of haem d1 in the native enzyme are 2.51, 2.43 and 1.71, suggesting co-ordination by two histidine ligands in the oxidized state. There is clear evidence in the e.p.r. data of an interaction between the c and d1 haem groups. This is not apparent in the optical spectra. The results are interpreted in terms of haem groups that are remote from each other, their interaction being mediated through protein conformational changes. The possible implications of this in relation to reduction processes catalysed by the enzyme are considered. PMID- 3010945 TI - Salivary apyrase of Rhodnius prolixus. Kinetics and purification. AB - The salivary apyrase activity of the blood-sucking bug Rhodnius prolixus was found to reside in a true apyrase (ATP diphosphohydrolase, EC 3.6.1.5) enzyme. The crude saliva was devoid of 5'-nucleotidase, inorganic pyrophosphatase, phosphatase and adenylate kinase activities. ATP hydrolysis proceeded directly to AMP and Pi without significant accumulation of ADP. Km values for ATP and ADP hydrolysis were 229 and 291 microM respectively. Ki values for ATP and ADP inhibition of ADP and ATP hydrolysis were not different from the Km values, and these experiments indicated competitive inhibition. Activities were purified 126 fold by combined gel filtration and ion-exchange chromatography procedures with a yield of 63%. The purified enzyme displayed specific activities of 580 and 335 mumol of Pi released/min per mg of protein for ATP and ADP hydrolysis respectively. The action of the purified enzyme on several phosphate esters indicates that Rhodnius apyrase is a non-specific nucleosidetriphosphate diphosphohydrolase. PMID- 3010947 TI - Removal of haem from lipids extracted from intact erythrocytes with particular reference to polyphosphoinositides. AB - With the use of a modified acid version of a current lipid-extraction technique [Bligh & Dyer (1959) Can. J. Biochem. Physiol. 37, 911-917], 92% of phosphatidylinositol 4,5-bisphosphate obtained by means of three sequential extractions from intact human erythrocytes was obtained during the first one. Some 95% of the haem co-extracted with the lipids could then be removed, with a maximal loss of polyphosphoinositides of less than 3%. About 58 nmol of phosphatidylinositol 4,5-bisphosphate was found per ml of erythrocytes. PMID- 3010949 TI - Insertion of transposon Tn7 into the Escherichia coli glmS transcriptional terminator. AB - The transposon Tn7 is unusual as it transposes at high frequencies from episomal elements to a unique site in the Escherichia coli chromosome. This unique site is within a region of dyad symmetry that we have demonstrated to be the transcriptional terminator of the glmS gene which encodes the glutamine amidotransferase, glucosamine synthetase. Transposition of Tn7 abolishes termination of glmS transcription at this site; the transcripts now extend into the left end of Tn7 and terminate at a new site, tm, 127 base pairs from the left end of Tn7. This region of the transposon contains a long open reading frame which encodes a protein sequence that is significantly related to the transposase proteins of the transposable elements IS1 and Tn3. A weak transcript has been identified that emanates from a promoter on the 5' side of this reading frame. This promoter is over-run by glmS transcripts and so it appears that expression of the Tn7 transposase may be regulated by promoter occlusion. PMID- 3010950 TI - Phosphoinositide reorganization in human erythrocyte membrane upon cholesterol depletion. AB - The effect of cholesterol depletion on the activity of phosphatidylinositol/phosphatidylinositol 4-phosphate and diacylglycerol kinases and polyphosphoinositide phosphodiesterase has been studied in isolated membranes of human normal and cholesterol-depleted erythrocytes. Polyphosphoinositide synthesis (phosphatidylinositol/phosphatidylinositol 4-phosphate kinase activities) were found to depend on the permeability and sidedness characteristics of the membrane vesicles, which could limit the accessibility of ATP for the enzymes. When measured under proper conditions, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate synthesis were decreased in cholesterol-depleted membranes as compared with control membranes. The same level of synthesis could be obtained in both membranes by the addition of phosphatidylinositol (and Triton X-100) or of phosphatidylinositol 4-phosphate. Phosphatidic acid synthesis (diacylglycerol kinase activity) was also decreased in cholesterol-depleted membranes as compared with control membranes when measured in the presence of Ca2+. Addition of diolein (and Triton X-100) caused a large increase in phosphatidic acid synthesis which reached approximately the same level in both membranes. This showed that the apparent inhibition of polyphosphoinositide and phosphatidic acid synthesis was not due to a loss or to an inactivation of the kinases. Ca2+-activated polyphosphoinositide phosphodiesterase promoted the hydrolysis of 65-70% of the polyphosphoinositides in control and of only 45-55% in cholesterol-depleted membranes without changing the Ca2+ concentration for half-maximum hydrolysis (1 microM). Upon addition of sodium oleate, the extent of polyphosphoinositide hydrolysis became identical in both membranes, indicating again that there was no loss nor inactivation of the polyphosphoinositide phosphodiesterase in the cholesterol-depleted membranes. Since the concentration of the polyphosphoinositides was not changed by cholesterol depletion [Giraud, M'Zali, Chailley & Mazet (1984) Biochim. Biophys. Acta 778, 191-200], the reduction in both their synthesis and degradation observed here could be attributed to a reorganization of the phosphoinositides in membrane domains where they were not accessible to the kinases and phosphodiesterase. The reduction in phosphatidic acid synthesis was likely caused by a reduction in the total amount of the substrate diacylglycerol in cholesterol depleted membranes as already shown [Giraud, M'Zali, Chailley & Mazet (1984) Biochim. Biophys. Acta 778, 191-200]. PMID- 3010948 TI - Structure and function of repetitive DNA in eukaryotes. PMID- 3010951 TI - Catalytic unit-independent phosphorylation and dephosphorylation of type II regulatory subunit of cyclic AMP-dependent protein kinase in rat liver plasma membranes. AB - Rat liver plasma membranes contain a 55 kDa protein which proved to be identical with type II regulatory subunit (RII) of the cyclic AMP-dependent protein kinase (kinase A) by several criteria (gel electrophoretic behaviour, peptide map, position of the autophosphorylated site). Analysis of phosphopeptide maps revealed that the membrane-bound RII was phosphorylated by a kinase which is unrelated to the catalytic unit (C) of kinase A. Dephosphorylation of the membrane-bound RII by an endogenous phosphatase was stimulated by both cyclic AMP and fluoride. Addition of C did not stimulate dephosphorylation even in the presence of ADP; moreover, protein inhibitor of C did not modify the effects of cyclic AMP or fluoride. The effects of both cyclic AMP and fluoride were, however, inhibited by C. Results indicate that rat liver plasma membranes contain a phosphorylation-dephosphorylation system for which RII is a relatively specific substrate. PMID- 3010952 TI - Modulation of cyclic AMP action in the dog thyroid by its agonist and antagonist Sp- and Rp-adenosine 3',5'-monophosphorothioate. AB - The diastereoisomeric forms of adenosine 3',5'-monophosphorothioate, Sp-cAMPS and Rp-cAMPS, have been shown to mimic and to inhibit activation of protein kinase type I and type II by cyclic AMP. In the present work, Sp-cAMPS mimicked thyrotropin (TSH) action on thyroid hormone secretion and protein iodination in dog thyroid slices, whereas Rp-cAMPS antagonized those effects. The phosphorothioates have been tested as inhibitors or activators of the three major phosphodiesterases: the Ca2+/calmodulin-sensitive form, the cyclic GMP-stimulated form and the cyclic AMP-specific enzyme. At 100 microM Sp-cAMPS inhibited the three enzyme activities. In contrast, Rp-cAMPS failed to stimulate activity of the three enzymes. From a comparison of the biological properties of Sp- and Rp cAMPS and 3-isobutyl-1-methyl xanthine, it is suggested that one site of action of the phosphorothioates is on the cyclic AMP-dependent protein kinases, i.e. the effects of Sp-cAMPS and Rp-cAMPS observed in intact cell can be ascribed to the agonistic and antagonistic effects on the cyclic AMP-dependent protein kinases. However, partial inhibition of phosphodiesterase activities by the phosphorothioates cannot be excluded. PMID- 3010954 TI - ATP binding to bovine heart cytochrome c oxidase. A photoaffinity labelling study. AB - ATP influences the kinetic properties of cytochrome c oxidase. A photoactivatable radioactive ATP analogue was used to localize the nucleotide-binding site on the bovine heart enzyme. Subunits IV and VIII were specifically labelled, suggesting that these two nuclear-coded polypeptides may play a regulatory role on the oxidase functions. PMID- 3010953 TI - Copper + zinc and manganese superoxide dismutases inhibit deoxyribose degradation by the superoxide-driven Fenton reaction at two different stages. Implications for the redox states of copper and manganese. AB - When OH. radicals are formed in a superoxide-driven Fenton reaction, in which O2. is generated enzymically, deoxyribose degradation is effectively inhibited by CuZn- and Mn-superoxide dismutases. The products of this reaction are H2O2 and a Fe3+-EDTA chelate. The mixing of H2O2 and a Fe3+-EDTA chelate also generates OH. radicals able to degrade deoxyribose with the release of thiobarbituric acid reactive material. This reaction too is inhibited by CuZn- and Mn-superoxide dismutases, suggesting that most of the OH. is formed by a non-enzymic O2.- dependent reduction of the Fe3+-EDTA chelate. Since the reaction between the Fe3+ EDTA chelate and H2O2 leads to a superoxide dismutase-inhibitable formation of OH. radicals, it could suggest a much wider protective role for the superoxide dismutase enzymes in biological systems. Urate produced during the reaction of xanthine oxidase and hypoxanthine limits deoxyribose degradation as well as the effectiveness of the superoxide dismutase enzymes to inhibit damage to deoxyribose by H2O2 and the Fe3+-EDTA chelate. Some of this damage may result from an O2.--independent pathway to OH. formation in which urate reduces the ferric complex. PMID- 3010955 TI - Interaction of 4-hydroxynonenal-modified low-density lipoproteins with the fibroblast apolipoprotein B/E receptor. AB - The incorporation of the lipid peroxidation product 4-hydroxynonenal into low density lipoprotein (LDL) increases the negative charge of the particle, and decreases its affinity for the fibroblast LDL receptor. It is suggested that this modification may occur in vivo, and might promote atherogenesis. PMID- 3010956 TI - Desensitization and antagonism of vasopressin-induced phosphoinositide metabolism and elevation of cytosolic free calcium concentration in human platelets. AB - The receptor mechanisms underlying vasopressin-induced human platelet activation were investigated with respect to stimulation of phosphoinositide metabolism and changes in the cytosolic free Ca2+ concentration ([Ca2+]i). Vasopressin stimulated phosphoinositide metabolism, as indicated by the early formation of [32P]phosphatidic acid ([32P]PtdA) and later accumulation of [32P]phosphatidylinositol ([32P]PtdIns). In addition, vasopressin elicited a transient depletion of [glycerol-3H]PtdIns and accumulation of [glycerol-3H]PtdA. The effects of vasopressin on phosphoinositide metabolism were concentration dependent, with half maximal [32P]PtdA formation occurring at 30 +/- 15 nM vasopressin. In the presence of 1 mM extracellular free Ca2+, vasopressin induced a rapid, concentration-dependent elevation of [Ca2+]i in quin2-loaded platelets: half-maximal stimulation was observed at 53 +/- 20 nM-vasopressin. The V1 receptor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]-vasopressin selectively inhibited vasopressin (100 nM)-induced [32P]PtdA formation [I50 (concn. giving 50% inhibition) = 5.7 +/- 2.4 nM] and elevation of [Ca2+]i (I50 = 3 +/- 1.5 nM). Prior exposure of platelets to vasopressin rendered them unresponsive, in terms of [32P]PtdA formation and elevation of [Ca2+]i, to a subsequent challenge with vasopressin, but responsive to a subsequent challenge with U44069, a thromboxane A2 mimetic. These results indicate that vasopressin-induced human platelet activation is initiated by combination with specific V1 receptors on the platelet, and that the sequelae of receptor occupancy (stimulation of phosphoinositide metabolism and elevation of [Ca2+]i) are equally susceptible to inhibition by receptor antagonists and by receptor desensitization. PMID- 3010958 TI - Characterization of membrane-associated growth inhibitor activity for Ehrlich ascites mammary carcinoma cells. AB - Purified plasma membranes obtained from bovine milk or from bovine mammary gland inhibited in vitro growth of Ehrlich ascites mammary carcinoma cells. Plasma membranes derived from tumor cells or from tumor tissue were inactive. Growth inhibition by milk fat globule membranes was shown to be due to release of membrane-associated inhibitory factors into the medium. Gentle removal of proteins from the surface of milk fat globule membranes succeeded in recovery of a fraction inhibiting cell growth specifically. The same fraction contained the antigen cross-reacting with an antiserum raised against the previously purified 13 kD growth inhibitor from bovine mammary gland. PMID- 3010957 TI - Changes in permeability to protons and other cations at high proton motive force in rat liver mitochondria. AB - We have confirmed that the respiration rate of rat liver mitochondria can be substantially inhibited with only a small drop in proton motive force. We have directly measured the passive proton permeability as a function of delta psi by using K+ diffusion potentials and have shown that there is a large increase in proton permeability at high delta psi. This can quantitatively account for the inhibitor titrations of respiration. delta psi and delta pH were shown to have roughly equal effects on the relatively high respiration rate in static head. The permeabilities to K+, tetramethylammonium+ and choline+ were shown to increase greatly at high delta psi, in a similar way to proton permeability, indicating a similar mechanism of entry. PMID- 3010959 TI - Development of glutamate binding sites in the visual structures of the rat brain. Effect of visual pattern deprivation. AB - The postnatal development of the Na-independent 3H-glutamate binding sites has been studied in the retina, lateral geniculate nucleus, superior colliculus, frontal and visual cortex of the rat. In the visual cortex, lateral geniculate nucleus and superior colliculus the highest binding was found at postnatal day 15. Until day 25 glutamate binding decreases drastically reaching the adult values. In contrast, in the retina and in the frontal cortex binding exhibits a maximum already at postnatal day 10 and then decreases to reach the adult value at day 25. Comparing glutamate binding within the visual areas and frontal cortex, highest binding was found in the lateral geniculate nucleus at all stages of age studied. Unilateral eyelid closure from day 11 postnatally resulted in a decreased binding level in the lateral geniculate nucleus ipsilateral to the sutured eye of both 25- and 90-day-old monocularly deprived rats in comparison to controls. A decreased glutamate binding was also observed in the retina of both eyes of 90-day-old monocularly deprived animals, which was not detectable in rats monocularly deprived only until the age of 25 days. The other regions studied were not affected by monocular deprivation. In contrast to that, monocular deprivation until postnatal day 90 failed to affect glutamate high-affinity uptake in both retinas. Binocular deprivation had no effect on glutamate binding in the retina of adult rats. Since monocularly deprived rats use their open eyes for longer periods of time than animals with both eyes open [1], the decreased glutamate binding in the lateral geniculate nucleus might be the consequence of a down-regulation of the increased functional activity of the cortico-geniculate pathway of the non-deprived (open) eye. The decreased glutamate binding in the retina of both eyes of monocularly deprived animals suggests a physiological coupling between both retinas and/or central nervous control of retinal glutamatergic mechanism. PMID- 3010961 TI - Demonstration of corticotropin-releasing factor binding sites on human and rat erythrocyte membranes and their modulation by chronic ethanol treatment in rats. AB - In a previous study we reported the presence of specific corticotropin-releasing factor (CRF) binding sites in peripheral tissues of the rat (Endocrinology, 116, 2152, 1985). Using 125I-labeled rat or human CRF, specific CRF binding sites were identified on rat and human erythrocytes, but not on lymphocytes or platelets. Furthermore, identical CRF binding was observed in the presence of intact erythrocytes or lysed erythrocyte membranes. Maximal binding of 125I-CRF occurred within 25 min at 4 degrees C and was saturable. Scatchard analysis of CRF binding to erythrocyte membranes revealed the existence of a single class of binding site. Chronic exposure of rats to ethanol vapor, known to lower specific CRF binding to pituitary tissue by 35%, also decreased 125I-rat CRF binding to erythrocyte membranes by approximately 45%, which was due to a decrease in the number of CRF binding sites. The parallel decrease of CRF binding to rat erythrocyte and pituitary membranes following chronic ethanol treatment suggests that CRF binding to erythrocyte and pituitary membranes is modulated in a similar direction, which further suggests that the determination of CRF binding to erythrocytes may provide an important clinical tool to indirectly assess CRF receptor levels in the pituitary gland and thereby enhance our understanding of ethanol-induced disorders of the hypothalamic-pituitary-adrenal axis in patients. PMID- 3010962 TI - The ATP, Mg-dependent phosphatase: role of Mg ions in the expression of the phosphorylase phosphatase activity. AB - The activation of the ATP, Mg-dependent phosphatase [FCM] by kinase FA has been shown to involve the phosphorylation or thiophosphorylation of the modulator subunit [M] and the consequent isomerization of the catalytic subunit [FC] into the active conformation. The inactive catalytic subunit [free FC] exhibits substantial activity in the presence of non-physiological concentrations of Mn ions whereas the Mn2+-activation of the intact FCM-enzyme requires the proteolytic destruction of the modulator subunit. The present study points to the importance of Mg2+ in the activation of the phosphatase. The inactive catalytic unit can be activated by millimolar concentrations of Mg2+ and the thiophosphorylated FCM-enzyme only expresses its phosphorylase phosphatase activity after a subsequent trypsin treatment in the presence of Mg ions. PMID- 3010960 TI - Degradation of DNA by metalloanthracyclines: requirement for metal ions. AB - Metallodaunomycin has been shown to cleave DNA only in the presence of oxygen, a reducing agent and a metal ion under reaction conditions similar to those used for the cuprous-phenanthroline complex. The intermediacy of 02-. and H2O2 has been substantiated by experiments with superoxide dismutase and catalase, respectively. Only partial inhibition by OH. scavengers was observed. An important feature of the reaction is that no specificity for Cu(II) was observed. This observation has led us to propose a reaction mechanism different from that proposed for the cuprous-phenanthroline complex. The mechanism proposed includes a catalytic role for metal ions other than Cu(II) as well as the direct participation of products of metal-catalyzed redox reactions such as semiquinone and/or hydroquinone of daunomycin. PMID- 3010963 TI - Difference in molecular size of receptors for alpha-rat atrial natriuretic polypeptide among the kidney, aorta, and adrenal gland as identified by direct UV photoaffinity labeling. AB - In order to identify the molecular size of receptors for alpha-rat atrial natriuretic polypeptide (alpha-rANP), we utilized the direct UV irradiation method for photoaffinity labeling with the biologically active [125I] alpha-rANP. In the preparation of isolated glomerulus and the inner medullary collecting duct (IMCT)-rich fraction, the autoradiograms of the electrophoresed sodium dodecyl sulfate (SDS)-polyacrylamide gels showed a single radioactive band which is displaceable with unlabeled alpha-rANP. The dose-dependent displacement fit very well with a binding-inhibition curve representing the binding affinity of 6.5 X 10(-10) M. The molecular size of the ligand-receptor complex was about 65,000 daltons for both glomerulus and IMCT-rich fraction. In contrast, in homogenate of the aorta and adrenal gland, the ligand-receptor complex was 140,000 daltons. PMID- 3010965 TI - Stimulation of superoxide release in neutrophils by 1-oleoyl-2-acetylglycerol incorporated into pH-sensitive liposomes. AB - Incorporation of 1-oleoyl-2-acetylglycerol (OAG) into multilamellar liposomes composed of egg phosphatidylethanolamine (PE) and arachidonic acid (AA) resulted in a significant enhancement of superoxide release by guinea pig neutrophils when compared to free OAG. OAG incorporated into liposomes containing phosphatidylcholine and arachidonic acid were generally less effective than free OAG. The potency of the liposomes correlates well with the ability of the liposomes to undergo lipid mixing at acidic pH. The enhanced effect of liposome associated OAG could be related to exposure to an acidic environment in the endosomes/lysosomes once liposomes are endocytosed by neutrophils. PMID- 3010964 TI - Immunological identification of aa3-type cytochrome oxidase in membrane preparations of the cyanobacterium Anacystis nidulans. AB - Membranes were isolated from the cyanobacterium Anacystis nidulans by French press extrusion of lysozyme-treated cells. The membranes were solubilized with sodium dodecylsulfate and subjected to denaturing polyacrylamide gel electrophoresis. Separated polypeptides were transferred to nitrocellulose by Western blotting, and incubated with antibodies against aa3-type cytochrome oxidase of Paracoccus denitrificans; antibodies against subunits I and II, and against the holoenzyme, were used and gave pronounced complementary cross reaction with two of the Anacystis membrane polypeptides corresponding to molecular weights of approximately 55,000 and 32,000, respectively. From this we conclude that an aa3-type cytochrome oxidase is present in Anacystis nidulans as was previously suggested from spectral evidence (G.A.Peschek, Biochim.Biophys.Acta 635 (1981) 470-475), and that this enzyme is composed of at least two subunits with apparent homology to subunits I and II of the corresponding Paracoccus cytochrome oxidase. PMID- 3010966 TI - Phosphorylation of erythrocyte membrane liberates calcium. AB - Washed and permeabilized human erythrocyte ghosts were found to discharge calcium on treatment with ATP. Concomitantly, there was a decrease in phosphatidylinositol (PI) and an increase in phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2). These results support the hypothesis that an inositide shuttle, PI in equilibrium PIP in equilibrium PIP2, operates to maintain intracellular Ca2+ levels. The cation is thought to be sequestered in a cage formed by the head groups of two acidic phospholipid molecules, e.g., phosphatidylserine and phosphatidylinositol, with participation of both PO and fatty acid ester CO groups. These cages are stabilized by inter headgroup hydrogen bonding. When the inositol group is phosphorylated in positions 4 and 5, inter-lipid hydrogen bonding is disrupted and the cage opens to release its Ca2+. PMID- 3010967 TI - Human breast cancer cells synthesize and secrete an EGF-like immunoreactive factor in culture. AB - A human breast cancer cell line, strain MCF-7, in culture synthesized and secreted a large amount of a polypeptide (designated as MCF-7 EGF) immunologically related to human epidermal growth factor (hEGF). The molecular weight of MCF-7 EGF estimated by gel filtration on Sephadex G-75 was similar to that of hEGF from human urine. On isoelectric focusing analysis, MCF-7 EGF gave a major peak at pH 4.6 and a minor peak at pH 5.0. In our enzyme immunoassay system, however, the dose-response curve of MCF-7 EGF did not show good parallelism with that of standard hEGF. From these results, it is suggested that MCF-7 cells synthesize and secrete a polypeptide immunologically related to hEGF into the culture medium. PMID- 3010968 TI - A possible requirement for arachidonic acid lipoxygenation in the mechanism of phagocytic degranulation by human neutrophils stimulated with aggregated immunoglobulin G. AB - Aggregated immunoglobulin G (AggIgG) caused a concentration-dependent extracellular release of granule-associated lysozyme and myeloperoxidase (MPO) from human neutrophils. Generation of the 5-lipoxygenase product of arachidonic acid (AA) metabolism, 5(S),12(R)-dihydroxy-6,14-cis,8,10-trans-eicosatetraenoic acid [leukotriene B4 (LTB4)], by neutrophils is exposed to AggIgG occurred in the presence but not absence of exogenous AA. U-60,257B (piriprost potassium), an inhibitor of leukotriene synthesis, caused a dose-related suppression of LTB4 production and granule exocytosis by AggIgG-treated cells. These data suggest that a lipoxygenase product of AA metabolism may mediate AggIgG-induced phagocytic release of granule constituents from neutrophils. PMID- 3010969 TI - Inhibition of SV40 DNA replication in vitro by 1-N-acyl-3"-N-(trifluoroacetyl) kanamycin. AB - A new antiviral aminoglycoside antibiotic, 1-N-eicosanoyl-3''-N-(trifluoroacetyl) kanamycin, was found to inhibit SV40 DNA replication in vitro. Several other aminoglycoside antibiotics examined did not exhibit a significant inhibition of SV40 DNA replication. The elongation of the SV40 DNA strand was profoundly affected by this agent. The degree of inhibition was decreased by increasing the amount of DNA template, but not by increasing the amount of enzyme. The inhibition of SV40 DNA replication is attributed to the interaction between the agent and the DNA template. PMID- 3010970 TI - Wheat germ phosphoglycerate mutase: evidence for a metalloenzyme. AB - Wheat germ phosphoglycerate mutase, exposed to 3.4 M guanidinium chloride at 22 degrees C and pH 7.8, slowly undergoes time-dependent inactivation which can be fully reversed by adding excess Co2+ or Mn2+ to a 50-fold dilution of the denaturing medium. Titration of the denatured enzyme with either Co2+ or Mn2+ shows that wheat germ mutase preferentially binds Co2+. Assuming 1:1 complexation between metal atom and protein, the apparent dissociation constants (Kd) for E Co2+ and E Mn2+ at 22 degrees C and pH 8.7 are approximately 1.06 and 1.84, respectively. Other metal atoms (e.g., Cr2+, Cu2+, Fe2+, Fe3+, Mg2+, and Ni2+) have no effect in restoring the apoenzyme's catalytic activity. At low concentrations (0.11-0.23 mM) Zn2+ partially restores activity, but promotes protein precipitation at elevated concentrations. Evidence suggests that all bisphosphoglycerate-independent phosphoglycerate mutases require either an intra- or an extramolecular metal atom in order to function. Attempts to characterize wheat germ mutase as a glycoprotein have yielded negative results. PMID- 3010971 TI - A guanine nucleotide-dependent regulatory protein couples substance P receptors to phospholipase C in rat parotid gland. AB - Electrically permeabilized cells of rat parotid gland, prelabelled with [3H] inositol, synthesized [3H]-inositol phosphates (IP3 and IP2) when stimulated with alpha 1-adrenergic, muscarinic-cholinergic, and substance P receptor-agonists. Non-hydrolyzable analogues of GTP (GTP gamma S and GppNHp) also stimulated [3H] IP3 formation by permeabilized cells and they potentiated the stimulation by receptor-agonists. These effects of guanine nucleotides occurred only with GTP analogues and only in permeabilized cells indicating an intracellular site of action. NaF stimulated [3H]-IP3 accumulation, an effect that was not entirely attributable to the ability of F- to inhibit (1,4,5)IP3 degradation. These results suggest that a guanine nucleotide-dependent regulatory protein couples Ca2+-mobilizing receptors to phospholipase C in parotid gland. PMID- 3010972 TI - Interaction of anti-iron-sulfur protein and anti-ubiquinone binding protein antibodies with complex III of beef heart mitochondria. AB - Ubiquinol-cytochrome c reductase activity of Complex III was substantially inhibited by anti-iron-sulfur protein antibody, whereas it was not affected by anti-ubiquinone binding protein antibody. Enzyme-linked immunosorbent assay indicated that anti-ubiquinone binding protein antibody do not bind to the complex, but that it binds to Complex III of which iron-sulfur protein and phospholipids have been depleted. These results indicate that some of the antigenic sites of the iron-sulfur protein are located on the surface of Complex III, while the antigenic sites of the ubiquinone binding protein are inaccessible to antibody owing to the interaction with iron-sulfur protein and/or phospholipids in the complex. PMID- 3010973 TI - Peptide hydroxamic acids inhibit skin collagenase. AB - A number of peptide hydroxamic acids have been synthesized and have been shown to be inhibitors of human skin collagenase. One of these, Z-Pro-Leu-Gly-NHOH, has an IC50 value of 4 X 10(-5)M. Corresponding peptides with different C-terminal functional groups, such as amide, carboxylate and aldehyde, showed little or no inhibition, indicating the importance of the hydroxamate functional group. In addition, the peptide sequence of this effective inhibitor corresponds closely to that of the cleavage site of native collagen, the substrate for the enzyme. Thus, substrate analogs incorporating a suitable metal coordinating group serve as potential inhibitors of human collagenase. PMID- 3010974 TI - Copper induces luteinizing hormone release and desensitization of pituitary gonadotropes. AB - Copper stimulated LH release from cultured rat pituitary cells in a dose-and time dependent manner. After 4 h of incubation with 10 mu M Cu2+, LH release was stimulated by 3-fold. The release of LH stimulated by Cu2+ was Ca2+ dependent, thus excluding the possibility that the releasing activity of this divalent cation was due to a toxic effect on pituitary cells. The stimulatory action of Cu2+ is substantially mediated via the GnRH-receptors since Cu2+ inhibited 125I Buserelin binding and since GnRH-antagonist blocked most of the Cu2+-stimulated LH release (80%). Both GnRH (1 microM) and Cu2+ (10 microM) induced desensitization of pituitary cells to a subsequent stimulation of either GnRH (0.5 nM) or Cu2+ (10 microM). However, in contrast to GnRH, Cu2+ did not induce down regulation of GnRH receptors. These findings suggest that the Cu2+ effects are mainly mediated through the GnRH receptors. PMID- 3010975 TI - Enhanced superoxide dismutase activity of pulsed cytochrome oxidase. AB - The superoxide dismutase (SOD) activity of beef heart cytochrome oxidase, both in the resting (as isolated) and pulsed (reduced and reoxidized) states, has been investigated using their ability to inhibit the autoxidation rate of pyrogallol and epinephrine. Resting oxidase showed variable SOD activity, while in the pulsed state the SOD activity of cytochrome oxidase (CcO) increased by an order of magnitude. These results are discussed in terms of a physiological role for the pulsed oxidase. PMID- 3010976 TI - The ligand binding subunit of the insulin-like growth factor 1 receptor has properties of a peripheral membrane protein. AB - 125I-insulin-like growth factor 1 was cross-linked to its receptor in human placenta microsomal membranes. The microsomes were treated with urea, with dithiothreitol or with both reagents prior to centrifugation at 100,000 X g. We found that greater than 80% of the label was membrane-associated following separate treatment with urea or dithiothreitol, but greater than 80% of the radioactivity remained in the supernatant after simultaneous exposure to both reagents. In identical experiments employing 125I-epidermal growth factor, no condition led to the release of greater than 10% of label from the membrane. We conclude that the ligand binding subunit of the insulin-like growth factor 1 receptor, like peripheral membrane proteins, lacks a membrane anchoring domain. PMID- 3010978 TI - Sequential binding of actin monomers to plasma gelsolin and its inhibition by vitamin D-binding protein. AB - Functional studies that distinguish free from actin-bound gelsolin based on the ability of the former to sever actin filaments reveal that the binding of actin monomers to gelsolin is highly cooperative and can be prevented by prior incubation of actin with vitamin D-binding protein (DBP), even though the apparent affinity of gelsolin for actin is 50-fold greater than that of DBP. Measurements of actin binding by immunoprecipitation and pyrene-actin fluorescence establish that DBP-actin complexes do not bind to gelsolin and that DBP removes one of the actin monomers in a 2:1 actin-gelsolin complex. These studies may explain why DBP-actin complexes exist in blood plasma in vivo in the presence of free gelsolin and suggest that the interaction of gelsolin with actin in cells and plasma may be regulated in part by actin monomer binding proteins. PMID- 3010977 TI - Aurintricarboxylic acid and Evans Blue represent two different classes of anionic compounds which selectively inhibit the cytopathogenicity of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus. AB - Aurintricarboxylic acid, an anionic triphenylmethane dye, and Evans Blue, an anionic compound structurally related to suramin, are, like suramin itself, inhibitors of human T-cell lymphotropic virus type III (HTLV-III)/ lymphadenopathy-associated virus (LAV) in vitro. These compounds may be targeted, at least in part, at the HTLV-III/LAV reverse transcriptase. The lack of any appreciable cytostatic action of aurintricarboxylic acid, Evans Blue and suramin against several murine and human cell lines, their inability to inhibit cellular DNA, RNA and protein synthesis, and their high lethal dose-50 (greater than or equal to 0.340 g/kg) for NMRI mice point to the selectivity of the compounds as inhibitors of HTLV-III/LAV. PMID- 3010979 TI - Dexamethasone and estrogen regulate Xenopus laevis albumin mRNA levels. AB - The regulation of albumin mRNA levels by steroid hormones was examined in a primary Xenopus liver culture system. In the absence of exogenous steroid hormone, albumin mRNA levels declined rapidly in culture. Dexamethasone is required for preservation of albumin mRNA levels in culture and effects a greater than 10 fold induction of albumin mRNA in cultures maintained in steroid hormone free medium. Estrogen can override the effect of dexamethasone and elicits a decline of greater than 80% in albumin mRNA levels. Our cDNA clones of the mRNAs encoding the two 74,000 dalton and one 72,000 dalton cellular albumins allowed us to show that all three albumin mRNAs were down regulated by estrogen. PMID- 3010980 TI - The interaction of phosphate with the purple acid phosphatase from beef spleen: evidence that phosphate binding is accompanied by oxidation of the iron chromophore. AB - Reaction of the reduced (pink) form of the purple acid phosphatase from beef spleen with excess phosphate at pH 5.0, monitored by optical and low temperature EPR spectroscopy and by measurement of enzymatic activity, results in parallel loss of activity and oxidation of the iron chromophore. Colorimetric and radiochemical (32P) experiments indicate the presence of one mole of tightly bound phosphate in the oxidized (purple) form of the enzyme; this phosphate is released upon reduction. Acid hydrolysis of 32P-phosphate-containing enzyme, followed by high voltage paper electrophoresis, gave no evidence for significant amounts of acid-stable phosphoamino acids. PMID- 3010981 TI - Effects of diabetes on fructose 2, 6-P2, glucose 1, 6-P2 and 6-phosphofructo 2 kinase in rat liver. AB - The levels of fructose 2,6-P2 and 6-phosphofructo 2-kinase have been found to be decreased in the liver of both ketotic and non-ketotic diabetic rats, a good correlation between fall of hepatic fructose 2,6-P2, ketonemia and glycemia being observed. The "total" 6-phosphofructo 2-kinase activity and the "active" (non phosphorylated) from of the enzyme were decreased to a different extent, resulting in a fall of the "active"/"total" activity ratio. Hepatic levels of glucose 1,6-P2 were lowered only in ketotic diabetes. Insulin treatment normalized all the values studied. Insulin administration to control rats decreased the hepatic levels of fructose 2,6-P2 and did not affect glucose 1,6-P2 levels. It also decreased the "active" form of 6-phosphofructo 2-kinase, without significantly altering the "total" activity. PMID- 3010982 TI - Mitoxantrone affects topoisomerase activities in human breast cancer cells. AB - The effects of mitoxantrone, an antineoplastic DNA intercalator, on topoisomerase I and II were studied in two human breast cancer cell lines. A large increase of topoisomerase I activity was found when cells were exposed to various doses of mitoxantrone. Maximal effect was achieved with 20 and 40 ng/mL in T47D and MCF-7 cells respectively. The enhancement on topoisomerase I activity seemed to be reversible, to be dependent on time of exposure to the drug and to require cellular integrity. Type II topoisomerase was inhibited in T47D cells after treatment for one hour with 10 ng/mL of mitoxantrone and enzyme activity was undetectable at higher doses (40 ng/mL). This inhibitory effect did not take place in vitro unless the concentration of the intercalator was increased to 400 500 ng/mL. PMID- 3010983 TI - The kanamycin resistance gene expression in Escherichia coli as affected by specific yeast sequences. AB - It was shown that the transcription initiation of the kanamycin resistance gene (the Km gene) from transposon Tn5 in E.coli HB101 strain can be provided by specific yeast sequences. To localize a region of the yeast DNA involved in transcription initiation, a number of hybrid plasmids containing promoterless part of the Km gene fused with DNA sequences from chromosome III of the yeast Saccharomyces cerevisiae was constructed. Comparison of nucleotide sequences lying in the LTR of the yeast transposon Tyl-17 with the consensus bacterial promoter revealed strong similarities. PMID- 3010984 TI - Effects of acidotropic compounds on the secretory pathway: inhibition of secretion and processing of the third and fourth components of complement. AB - Acidotropic compounds (also termed lysosomotropic) such as chloroquine and amantadine interfered with processing of the single-chain precursors to the third and fourth components of complement (C3 and C4) by the human hepatoma-derived cell line HepG2. When these compounds were added to culture medium, the precursors of C3 and C4 became the major secretory forms in contrast to the normal secretion of C3 and C4 as their mature forms. In addition, secretion of C3, C4, and total protein was inhibited by these compounds. Our results indicate that lysosomotropic agents, in addition to their well recognized effects on lysosomes and endosomes, inhibit functions of the secretory pathway. PMID- 3010985 TI - Spectroscopic characterization of plastocyanin from hornwort. AB - Plastocyanin isolated from an aquatic higher plant, Ceratophyllum demersum L. (hornwort), has been characterized by electronic absorption, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopies. The visible absorption, CD, and EPR spectra of hornwort plastocyanin indicated a complete similarity of blue copper center to those of terrestrial higher plants and algae. However, the ultraviolet absorption spectrum of hornwort plastocyanin exhibited a lower tyrosine (Tyr) and a higher phenylalanine (Phe) content of the protein comparing with other plastocyanins. The ratio of Phe/Tyr residues was estimated to be 9 by amino acid analysis. The hornwort plastocyanin resembles in amino acid composition terrestrial higher plant plastocyanins rather than alga plastocyanins but is peculiar in the content of Phe and Tyr residues. PMID- 3010987 TI - Carbachol-induced accumulation of inositol-1-phosphate in neurohybridoma NCB-20 cells: effects of lithium and phorbol esters. AB - Clonal neurohybridoma NCB-20 cells expressed muscarinic cholinergic receptors coupled to phospholipase C. Addition of carbachol in the presence of Li+ to cells prelabeled with 3H-inositol increased 3H-inositol-l-phosphate (3H-IP1) accumulation by more than 4-fold with an EC50 of about 50 microM. This carbachol induced response was blocked by atropine and pirenzepine with a Ki of 0.5 and 25 nM, respectively. The EC50 of Li+ for the carbachol-induced phosphoinositide turnover was 17 +/- 1.2 mM compared with a value of 1.8 +/- 0.2 mM in brain slices, suggesting the presence of an unusual type of inositol-l-phosphatase in NCB-20 cells. Carbachol-induced IP1 accumulation in these cells was potently and noncompetitively inhibited by the biologically active phorbol esters, phorbol dibutyrate (PDB) and phorbol myristate diacetate (PMA), while the biologically inactive phorbol, 4 beta-phorbol, failed to affect this phosphoinositide breakdown. The basal IP1 accumulation was also significantly attenuated by PDB and PMA but not by 4 beta-phorbol. PMID- 3010986 TI - The EPR spectra of the cytochrome b-c1 complex of Rhodopseudomonas sphaeroides. AB - The purified cytochrome b-c1 complex of Rhodopseudomonas sphaeroides has two b cytochromes distinguishable by optical, thermodynamic and electron paramagnetic resonance criteria (gz values are approximately equal to 3.75 and approximately equal to 3.4). EPR features typical of a Rieske iron sulfur cluster (g values of 2.03 1.90 and 1.81) and a c1 type cytochrome (g approximately equal to 3.4) were also observed. The b and c1 cytochromes were individually purified from the complex. The cytochrome c1 retained its native EPR spectrum. The b cytochrome lost over 90% of the intensity from the 'b566 type' heme site (g approximately equal to 3.75), while the 'b561 type' heme site (g approximately equal to 3.4) retained its native EPR spectrum. PMID- 3010988 TI - DNA strand scission by hemin-thiolate complexes as models of cytochrome P-450. AB - Hemin-thiolate complexes, as chemical models for cytochrome P-450 monooxygenases, have been shown to cause strand scission of DNA. Circular super-coiled DNA was degraded to open-circular and linear forms by these complexes in 30 min at pH 7.8 under aerobic conditions, the degradation depending on the structure of the thiol ligand and the ratio of thiol ligand to hemin concentration. The relationship between the structure of the thiol ligand and DNA strand cleaving activity was examined. Complete cleavage of DNA was observed by complexes containing TGE and ME at 400-600 moles excess of thiol ligand to hemin, those containing Cys, CysMe, and CysEt at 50-200 moles excess, and those containing MPG, GSH, penta- and nona peptides at 5-20 moles excess. Complexes containing NACys and MEA caused no cleavage of DNA. Inhibition experiments suggested the involvement of active oxygen species in the cleavage. PMID- 3010989 TI - Comparison of the regulatory and catalytic subunits of cAMP dependent protein kinase from Dictyostelium discoideum and bovine heart using polyclonal antibodies. AB - The purified regulatory (R) and catalytic (C) subunits of cAMP dependent protein kinase (cAK) from the primitive eukaryote Dictyostelium discoideum have been compared with the homologous proteins from bovine heart by SDS-PAGE followed by Western blotting using polyclonal antibodies. No cross-reaction could be demonstrated by this technique although the slime mold subunits share several functional properties with their mammalian counterparts and are able to form functional hybrid holoenzymes. PMID- 3010990 TI - Spin-trapping of superoxide ion by a water-soluble, nitroso-aromatic spin-trap. AB - Spin-trapping of superoxide ion, O2-, which is produced from two different sources (OH(-)-DMSO and xanthine-xanthine oxidase systems), was investigated by use of a water-soluble, notroso-aromatic spin trap, sodium 3,5-dibromo-4 nitrosobenzene-sulfonate (DBNBS). It was found that O2- from all sources was easily trapped by DBNBS to yield the stable O2- adduct showing the ESR spectrum consisting of a triplet of a triplet [aN (1) = 12.63 G and aH (2) = 0.71 G]. Hydroperoxy radical (HO2.), which can be generated from the oxidation of hydrogen peroxide with Ce4+ ion, was not trapped by DBNBS. These results indicate that the trapped radical is O2-, but not HO2.. PMID- 3010991 TI - Sites of phosphorylation in recombinant human interferon-gamma. AB - Recombinant human interferon-gamma was phosphorylated with ATP and c-AMP dependent protein kinase. After phosphorylation, interferon-gamma was separated from the adenosine phosphates and the kinase and analyzed by SDS-PAGE, reverse phase HPLC, and HPLC peptide mapping. Comparison of the S. aureus V8 protease maps of intact and phosphorylated interferon-gamma showed that the maps were identical except that one peptide fragment elutes earlier in the map of the phosphorylated sample. This peptide was identified as the C-terminal fragment containing two serinyl phosphorylation sites at positions 132 and 142. This phosphorylated interferon-gamma exhibited a slightly higher specific antiviral activity than the intact protein. PMID- 3010992 TI - Novel pathway for the metabolism of 6-trans-leukotriene B4 by human polymorphonuclear leukocytes. AB - Polymorphonuclear leukocytes convert arachidonic acid to leukotriene B4 as well as to two 6-trans isomers of this substance. Both leukotriene B4 and 6-trans leukotriene B4 are metabolized by a hydroxylase in human polymorphonuclear leukocytes to 20-hydroxy metabolites. We have now found a second, previously unknown, metabolic pathway for 6-trans-leukotriene B4 involving reduction of either the 6- or the 10- double bond. One of the two major metabolites of 6-trans leukotriene B4 in human polymorphonuclear leukocytes is formed by the action of this reductase, followed by hydroxylation by leukotriene B4 20-hydroxylase. On the basis of ultraviolet (maximum absorbance at 232 nm) and mass spectral evidence, this product is either 5,12,20-trihydroxy-6,8,14-eicosatrienoic acid or 5,12,20-trihydroxy-8,10,14-eicosatrienoic acid. PMID- 3010993 TI - Partial purification of phosphoinositide phospholipase C from human platelet cytosol; characterization of its three forms. AB - Three forms (I, IIa and IIb) of phospholipase C, hydrolyzing specifically inositol phospholipids, were resolved from human platelet cytosol and partially purified by DEAE-cellulose, phenyl-Sepharose, Ultrogel ACA-44 and hydroxylapatite column chromatographies. All three forms exhibited pH optimum at 6.5 - 7.0 in the presence of deoxycholate and their molecular weights were 67,000 (form I), 120,000 (IIa) and 70,000 (IIb). These enzymes hydrolyzed both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate in Ca2+-dependent manner; their maximal activities for phosphatidylinositol hydrolysis were obtained at 10(-4) to 10(-3) M Ca2+, whereas phosphatidylinositol 4,5 bisphosphate was preferentially hydrolyzed at lower concentration of Ca2+. PMID- 3010995 TI - In vitro activity profiles of cyclic and linear enkephalin pseudopeptide analogs. AB - The peptide bond in the 4-5 position of the cyclic and linear enkephalin analogs H-Tyr-cyclo[-D-Lys-Gly-Phe-L(or D)-Leu-] and H-Tyr-D-Ala-Gly-Phe-L(or D)-Leu-OH was replaced by a thiomethylene ether linkage. Each of the configurational isomers of the cyclic pseudopeptide H-Tyr-cyclo[-D-Lys-Gly-Phe psi [CH2S]L(or D) Leu-] showed high potency in both the guinea pig ileum and the mouse vas deferens assay and, therefore, had no preference for either mu- or delta-opioid receptors, in contrast to the cyclic parent peptides H-Tyr-cyclo[-D-Lys-Gly-Phe-L(or D)-Leu ] which are mu-receptor selective. The loss of selectivity observed with the cyclic pseudopeptides may be due to the greater flexibility of their 18-membered ring structures as a consequence of the peptide bond substitution. The linear pseudopeptide analogs were both less potent and less delta-receptor selective than their parent compounds. These results indicate that thiomethylene ether peptide bond replacements can have a pronounced effect on the activity profile of peptide hormones and neurotransmitters. PMID- 3010994 TI - Restriction maps and restriction fragment length polymorphisms of the human 21 hydroxylase genes. AB - Restriction maps were constructed for the two human 21-hydroxylase genes (21-OHA and 21-OHB) by using DNA from subjects homozygous for a deletion of each gene. Comparing the patterns of these two genes, a KpnI restriction site occurred in the 21-OHA gene in place of a TaqI site in the 21-OHB gene about 1-kb from the 5' end of the gene, and an extra EcoRI site was located 500 bp 5' to the common EcoRI site. The DNA of fourteen unrelated normal subjects was digested with nine restriction endonucleases (AccI, BamHI, BgIII, EcoRI, HindIII, KpnI, MspI, SacI and TaqI). Restriction fragment length polymorphisms were found with EcoRI, HindIII and AccI that resulted from polymorphic endonuclease sites outside the genes. PMID- 3010996 TI - Interferon alpha/beta selectively antagonises down-regulation of mannosyl-fucosyl receptors on activated macrophages by interferon gamma. AB - Previous reports have described synergism of various interferon preparations in anticellular and antiviral activity. We report that recombinant interferon (rIFN gamma) and IFN alpha/beta mediate distinct, antagonistic effects on expression of a lectin-like receptor for mannose and fucose (MFR) on mouse peritoneal macrophages (M phi). IFN gamma down-regulates MFR activity, a highly reproducible change in mouse M phi activated to secrete enhanced levels of o-2/H2o2. IFN alpha/beta enhances MFR activity and prevents the action of IFN gamma when added in combination. Antagonism is selective for this M phi activation marker and requires a minimum 4 h exposure period to rIFN gamma, during which IFN alpha/beta can prevent its action. PMID- 3010997 TI - Identification of two new calcium dependent hydrolases in the human heart. AB - We have identified and isolated two new calcium-activated neutral hydrolases from human ventricular muscles. The one is an esterase, of which molecular weight was 300,000, required millimolar concentration of Ca2+, hydrolyzed Ac-Tyr-OEt X H2O, optiaml pH at 7.0. The other is an amidase, of which molecular weight was 70,000, also required millimolar concentration of Ca2+, hydrolyzed a synthetic substrate for chymotrypsin, Suc-Leu-Leu-Val-Tyr-MCA, with optimal pH at 7.2. Both enzymes did not degrade casein or contractile proteins (myosin, actin, troponin and tropomyosin). Their activities were not inhibited by exogenous protease inhibitors, leupeptin, antipain, monoiodoacetic acid and chymostatin, while the amidase activity was blocked by the endogenous inhibitor against calcium activated neutral protease (CANP). Thus, their characters are different from chymotrypsin or CANP and they seems to be new hydrolases in the human heart. PMID- 3010998 TI - Epoxidation of alkenes by chloroperoxidase catalysis. AB - Chloroperoxidase from Caldariomyces fumago catalyzes the peroxidation of alkenes to epoxides. This enzyme is the only haloperoxidase of four tested capable of carrying out the reaction. These results further establish chloroperoxidase as a unique haloperoxidase, and adds this enzyme to the short list of other enzymes (e.g., cytochrome P-450) known to epoxidize alkenes. PMID- 3010999 TI - A multisubstrate Ca2+ and cyclic nucleotide independent kinase from vascular smooth muscle: modulation of activity by polycations. AB - A multisubstrate Ca2+ and cyclic nucleotide independent kinase (Mr = 47,000) was purified from bovine aortic smooth muscle. Phosphorylation of glycogen synthase by this enzyme was polycation modulable. Low concentrations of polylysine (0.04 0.16 microM) stimulated phosphorylation 2-7 fold, whereas higher concentrations suppressed phosphorylation. Glycogen synthase converted to its glucose 6-PO4 dependent form following phosphorylation in either the presence (7 mol 32P/mol synthase) or absence (4 mol 32P/mol synthase) of polylysine: extent of conversion correlated to extent of phosphorylation. Seven of 14 potential substrates tested were phosphorylated: kinase activity was greatest for phosvitin followed by casein, the receptor protein from type 2 cAMP-kinase, histone H2b, phosphorylase kinase, glycogen synthase, and myocardial myosin light chains. Phosphorylation of phosvitin or synthase was inhibited by heparin (1/2 maximally by 0.5 microgram/ml without salt and 37 micrograms/ml with 150 mM NaCl). The results suggest that the enzyme may participate in regulating arterial glycogen metabolism and that such regulation may be modulated by polycationic and polyanionic effectors. PMID- 3011000 TI - Enhanced inositide turnover in brain during bicuculline-induced status epilepticus. AB - Because brain inositides are enriched in the 1-stearoyl-2-arachidonoyl species, they form a likely source for the tetraenoic free fatty acids (FFA) and diacylglycerols (DG) that are accumulated during seizures. To study inositide turnover during bicuculline-induced seizures, rats were injected intraventricularly and bilaterally with 10-20 microCi 32P, mechanically ventilated and sacrificed by 6.5 KW head-focused microwave irradiation. Seizure activity was recorded by electroencephalography. Bicuculline-induced seizure activity resulted in: a) almost 50% increase in 32P labeling of phosphatidic acid (PA); phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2) also increased (24% and 36%, respectively); b) no change in other lipids; and c) water-soluble phosphodiesteratic degradation products, analyzed by high voltage paper electrophoresis, increased 24% in the amount of radiotracer recovered as inositol 1,4-bisphosphate (IP2) and by 44% in the amount recovered as inositol 1,4,5-trisphosphate (IP3). These data indicate that during experimental status epilepticus the cerebral inositide cycle is accelerated: PIP2----(IP3----IP2--- IP----I) + DG----PA----PI----PIP----PIP2. PMID- 3011001 TI - Inhibitory effects of the analgesic neuropeptides kyotorphin and neo-kyotorphin on enkephalin-degrading enzymes from monkey brain. AB - The inhibitory effects of the analgesic neuropeptides kyotorphin (Tyr-Arg) and neo-kyotorphin (Thr-Ser-Lys-Tyr-Arg) on enkephalin-degrading enzymes were studied. The enzyme used were aminopeptidase (AP), dipeptidyl aminopeptidase (DPP), enkephalinase-A (ENK-A), and angiotensin-converting enzyme (ACE), which were prepared from the monkey brain membrane fraction. Kyotorphin inhibited only DPP (IC50, 18 microM), and the mode of inhibition was non-competitive (Ki, 6 microM). Neo-kyotorphin inhibited AP, DPP, and ACE with IC50 values of 131, 306, and 200 microM, respectively, but the inhibition of the enzyme activities were not effective. The selective inhibition by kyotorphin suggested that kyotorphin might protect the released Met-enkephalin against enzymatic degradation by DPP. Thus, kyotorphin may not only induce the release of Met-enkephalin but also stabilize the released neuropeptide. PMID- 3011002 TI - The complete amino acid sequence of the cellular retinoic acid-binding protein from bovine retina. AB - The complete amino acid sequence of cellular retinoic acid-binding protein from bovine retina was obtained by Edman degradation of peptides obtained from enzymatic and CNBr digests. The 136 residue sequence is identical to that reported recently for the protein from bovine adrenal tissue indicating that the same gene is expressed in both tissues. PMID- 3011003 TI - A new pathway of autotrophic growth utilizing carbon monoxide or carbon dioxide and hydrogen. AB - The discovery of a pathway of autotrophic growth is described in which carbon monoxide or carbon dioxide and hydrogen is used as the source of carbon and energy. Carbon monoxide dehydrogenase is the central enzyme catalyzing the final steps in the synthesis of acetyl-CoA which is utilized in the anabolic reactions. PMID- 3011004 TI - Effect of cAMP on cytosol protein phosphorylation in placental tissue of different gestational age. AB - Phosphorylation of cytosol proteins in placental tissue of different gestational age has been studied. Cytosol protein phosphorylation was stimulated by exogenous cAMP only in term placentae and remained unchanged in first and second trimester placentae. Exogenous proteins, histone and casein, were intensively phosphorylated by cytosol kinases with maximal activities in first trimester cytosol preparations. Exogenous cAMP stimulated histone phosphorylation, but it had no effect on casein phosphorylation. On the basis of the obtained results it can be concluded that endogenous protein phosphorylation in first and second trimester placental cytosol is cAMP independent. PMID- 3011005 TI - Periplasmic cytochrome C552 of Escherichia coli K12. Oligomeric structure and its role in nitrite reduction. AB - Perisplasmic cytochrome C552 was purified from a cold shock fluid. We demonstrated that this protein could reduce nitrite into ammonium. The reducing equivalents could be donated by formate or NADH through some segment of the membrane respiratory chain. FAD addition was necessary when pure cytochrome was used to reduce nitrite. The predominant form was found to have a molecular weight of about 65 to 70 K daltons, composed of three 21,000 d. subunits. Hemes may or may not be present on the subunit. PMID- 3011006 TI - Induction of DNA breakage and suppression of DNA synthesis by the OH radical generated in a Fenton-like reaction. AB - The effect of the OH radical, generated in a Fenton-like reaction, on DNA structure and function was studied in a monkey kidney cell line (Vero). DNA single strand breaks were detected following exposure to 10- 100 microM concentrations of H2O2 on ice. These breaks were repaired very rapidly, and addition of the poly (ADP-ribose) transferase inhibitor, 3-aminobenzamide, resulted in an accumulation of breaks. DNA synthesis was inhibited at concentrations as high as 1- 30 mM, this effect also being reversible in approximately 60 min. 3-aminobenzamide did not affect the rate of DNA synthesis inhibition by H2O2. PMID- 3011007 TI - Neurochemical correlates of opiate receptor regulation. PMID- 3011008 TI - Perturbations of rat intestinal brush border membranes induced by Ca2+ and vitamin D3 are detected using steady-state fluorescence polarization and alkaline phosphatase as membrane probes. AB - Rat intestinal brush border membranes (BBM) were prepared by discontinuous sucrose gradient centrifugation. This specific fraction contained alkaline phosphatase activity. A dramatic decrease in the specific activity of the BBM bound alkaline phosphatase was observed at different concentrations of Ca2+ and vitamin D3. Studies of the temperature dependence (5-40 degrees) of alkaline phosphatase reveal a change in energy of activation (slope of the Arrhenius plot) at 23.0 +/- 1.1 degrees which was elevated to 27.8 +/- 1.3 degrees in BBM treated with Ca2+, while it was depressed to 17.2 +/- 1.2 degrees in BBM treated with vitamin D3. Membrane lipid fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), was significantly greater in BBM treated with vitamin D3 and significantly lower in Ca2+ treated BBM. A lipid thermotropic transition temperature was observed at 22.2 +/- 1.2 degrees which rose to 28.3 +/ 1.4 degrees in Ca2+ treated BBM and reduced to 17.0 +/- 1.2 degrees in vitamin D3 treated BBM. The biological significance of these results is discussed in terms of modifications in the lipid-protein interactions in BBM, induced by Ca2+ and vitamin D3, and the implications in the physiological intestinal transport processes of calcium. PMID- 3011009 TI - Differences in Ca2+ entry blockers revealed by effects on adenosine- and adrenergic- receptors and cyclic AMP levels of [2-3H]adenine-prelabeled vesicles prepared from guinea pig brain. AB - Three major classes of Ca2+ entry blockers, classified according to effects on cardiac and vascular smooth muscle, were tested. Vesicles prepared from cerebral cortex and stimulated by adenosine and epinephrine constituted adenosine and alpha-adrenergic receptor systems respectively. Vesicles prepared from cerebellum and stimulated by epinephrine constituted the beta-adrenergic receptor system. Experiments with adenosine were also performed with vesicles formed or incubated in the absence of exogenous Ca2+. The results indicate that Ca2+ entry blockers had a variety of effects, even within classes of drugs. Vascular-selective group A Ca2+ entry blockers such as nifedipine and nisoldipine antagonized adenosine, but the structurally-related drug nitrendipine was inactive. Inhibition was competitive with adenosine and independent of exogenous Ca2+. In contrast to receptor-binding studies requiring high ratios of the drugs to adenosine receptor radioligands, nifedipine and nisoldipine were inhibitory at equimolar concentrations with adenosine. Non-selective group A Ca2+ entry blockers such as diltiazem and verapamil were inactive against adenosine. Group B Ca2+ entry blockers, prenylamine and perhexilene, increased cyclic AMP (cAMP) levels of vesicles stimulated by adenosine but not by epinephrine or under basal conditions. This effect was observed only in vesicles that had been formed in the presence of Ca2+. Ca2+ entry blockers also exhibited effects on adrenergic receptors unrelated to effects on adenosine. Verapamil and prenylamine acted as alpha-adrenergic antagonists and only prenylamine acted as a beta-adrenergic antagonist. However, the vesicle system also revealed indirect blocking actions of nifedipine on adrenergic receptor systems. The actions of the Ca2+ entry blockers are discussed in relation to the special usefulness of nifedipine in the treatment of patients with defective atrioventricular conduction and also in relation to the unique ability of group B Ca2+ entry blockers to selectively inhibit Ca2+ and calmodulin activated phosphodiesterase. However, some caution must be applied in drawing conclusions relating to the cardiovascular actions of these drugs from data generated in a neuronally-derived model. PMID- 3011010 TI - Deoxyribosyl exchange reactions leading to the in vivo generation and regeneration of the antiviral agents (E)-5-(2-bromovinyl)-2'-deoxyuridine, 5 ethyl-2'-deoxyuridine and 5-(2-chloroethyl)-2'-deoxyuridine. AB - In the rat, the highly potent anti-herpes drug (E)-5-(2-bromovinyl)-2' deoxyuridine (BVdUrd) is rapidly converted to its base (E)-5-(2-bromovinyl)uracil (BVUra) through the action of pyrimidine nucleoside phosphorylases. However, BVdUrd can be regenerated or even generated de novo from BVUra by a pentosyl transfer reaction upon the administration of 2'-deoxythymidine (dThd), 2' deoxyuridine (dUrd) or 5-ethyl-2'-deoxyuridine (EtdUrd). The antiherpetic drugs EtdUrd and 5-(2-chloroethyl)-2'-deoxyuridine (ClEtdUrd) can also be regenerated or generated de novo from their respective bases 5-ethyluracil (EtUra) and 5-(2 chloroethyl)uracil (ClEtUra), by a pentosyl transfer mediated by the administration of dThd or dUrd as deoxyribosyl donor. The generation or regeneration of BVdUrd, EtdUrd and ClEtdUrd from their bases (BVUra, EtUra and ClEtUra, respectively) is readily achieved because the latter have long half lifes. Thus, the active anti-herpes drugs can be (re)generated repeatedly after a single administration of these nucleosides or their bases, followed by repeated administrations of dUrd. PMID- 3011011 TI - Affinity labeling of the DDT1 MF-2 cell alpha 1-adrenergic receptor with [3H]phenoxybenzamine. AB - In this study, we used phenoxybenzamine to label the alpha 1-adrenergic receptor of a smooth muscle cell line. Our results demonstrate a dose-dependent occupancy of alpha 1-adrenergic receptors by phenoxybenzamine determined by competition for the [3H]prazosin binding site. Following incorporation of [3H]phenoxybenzamine, partially purified membranes were solubilized and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Despite numerous Coomassie blue-stained bands, only three bands, Mr = 80,000 +/- 500, Mr = 33,000 +/- 2,000, and Mr = 21,000 +/- 400 (N = 4), were labeled with [3H]phenoxybenzamine as determined by autofluorography. Incorporation of [3H]phenoxybenzamine into the Mr = 80,000 band, but not the Mr = 33,000 and Mr = 21,000 bands, was affected by adrenergic agonists and antagonists in a manner consistent with an alpha 1-adrenergic interaction. Labeling of the Mr = 33,000 and Mr = 21,000 bands was partially blocked by phenoxybenzamine. We conclude that [3H]phenoxybenzamine can be used as an affinity probe for the alpha 1-adrenergic receptor and that the ligand binding site of the alpha 1-adrenergic receptor resides in a Mr = 80,000 protein. PMID- 3011012 TI - Leukocyte recruitment in the subcutaneous sponge implant model of acute inflammation in the rat is not mediated by leukotriene B1. AB - The subcutaneous sponge implant model of acute inflammation in the rat has been evaluated as a suitable test system for evaluating the potential anti inflammatory efficacy of 5-lipoxygenase inhibitors. The inflammatory parameters measured were exudate volume and leukocyte recruitment. Specific radioimmunoassays were used to measure (1) 5-lipoxygenase (LPO) and cyclo oxygenase (CO) activity in exudate leukocytes stimulated ex vivo with A23187, and (2) the LTB4 and PGE2 content of inflammatory exudate. The NSAIDs flurbiprofen and indomethacin inhibited cell recruitment, exudate volume and CO activity with ED50S of approximately 1 mg per kg p.o. but failed to inhibit LPO activity at 10 mg per kg p.o. Nafazatrom (Bayer 6575), quercetin and NDGA, which inhibit LPO activity in vitro, were inactive against all parameters when dosed at 100 mg per kg p.o. The "mixed inhibitors" BW755C and phenidone were approximately equipotent inhibitors of LPO activity but BW755C was 10 times more potent than phenidone against CO activity. BW755C was also greater than 10 times more potent at inhibiting cell recruitment and exudate volume than phenidone suggesting that the anti-inflammatory efficacy of the mixed inhibitors reflect their potency against CO rather than LPO activity. Time course studies demonstrated that the inhibitor effects of BW755C and phenidone on leukocyte recruitment reflected a reduction in the PGE2 but not the LTB4 content of the inflammatory exudate. Polyester sponges soaked in high concentrations of LTB4 caused only a modest (2-fold) increase in leukocyte recruitment whilst physiological levels were inactive. The results taken together suggest that CO products make a major contribution to leukocyte recruitment in this model whilst the LPO product LTB4 has little role. This model therefore is of little value for evaluating the anti-inflammatory efficacy of 5 lipoxygenase inhibitors. Moreover, the rat would appear to be unsuitable for evaluating the role of LTB4 in acute inflammation. PMID- 3011013 TI - Alloxan toxicity in human and canine spermatozoa. Possible biochemical basis for a species difference in sensitivity. AB - In view of the well known species differences in the sensitivity of pancreatic B cells to the toxic glucose analogue alloxan, it was tested whether spermatozoa from two species with a different diabetogenic effect of alloxan displayed a similar difference in their sensitivity to this drug. In canine spermatozoa, less than 2 mM alloxan profoundly reduced the rate of glucose oxidation and cellular motility whereas more than 5 mM was required to significantly alter these parameters in human spermatozoa. Such species difference was not observed in spermatozoal sensitivity towards the inhibitory effects of tert-butyl hydroperoxide. The phenomenon is not attributable to a different rate of alloxan uptake since the drug is not incorporated by dog or human spermatozoa. The alloxan toxicity was counteracted by D-glucose and its 3-O-methyl analogue in both species, and was potentiated by ascorbic acid; however, only in man. The protective effect of D-glucose was much less marked in tert-butyl hydroperoxide cytotoxicity. It is concluded that the observed species difference in spermatozoal alloxan sensitivity is not related to differences in alloxan uptake or in sensitivity to organic peroxides; differences in cellular scavenging of superoxide anion radicals and/or ascorbic acid metabolism may explain the lower sensitivity of human spermatozoa for alloxan. PMID- 3011014 TI - Purine effects on (3H)-clonidine binding to rat brain. PMID- 3011015 TI - Inhibition of phorbol ester-stimulated chemiluminescence and superoxide production in human neutrophils by fructose 1,6-diphosphate. PMID- 3011016 TI - The pharmacological mediation of epithelial cell proliferation. AB - In the past, it has been necessary for pharmacological intervention of epithelial proliferation to mostly be limited to non-specifically arresting or killing actively proliferating cells. As our understanding of the mechanisms involved in mediating the processes of growth and differentiation increases, we can hope to see the development of a new pharmacology, in which proliferation of individual systems may be regulated in a less drastic manner. PMID- 3011017 TI - Oxidation of 1,4-dihydropyridines by prostaglandin synthase and the peroxidic function of cytochrome P-450. Demonstration of a free radical intermediate. AB - Oxidation of 1,4-dihydropyridines by the hydroperoxidic function of cytochrome P 450 and prostaglandin synthase was investigated using felodipine as a model substance. Nifedipine and the 2,6-dichlorophenyl analogue of felodipine were used in some experiments with similar results. Felodipine was metabolized to a pyridine metabolite in vitro when incubated with liver microsomes and cumene hydroperoxide, as well as with ram seminal vesicle microsomes and arachidonic acid. The oxidation of 1,4-dihydropyridines is proposed to proceed via formation of a free radical intermediate, judging from EPR analysis with the spin trap POBN. When reduced glutathione was added the EPR signal was decreased as well as the formation of the pyridine metabolite while an oxidation of glutathione was observed. This effect was due to a reduction of the radical intermediate back to felodipine by glutathione. Felodipine interacts with the hydroperoxidase activity of prostaglandin synthase, since the pyridine metabolite was formed also when 15 hydroperoxyeicosatetraenoic acid was used as a substitute for arachidonic acid. Indomethacin could only inhibit the metabolism of felodipine when arachidonic acid was used as substrate. The cooxidation of felodipine by prostaglandin synthase is associated with an increased metabolism of arachidonic acid. This was further supported by a stimulated oxygen consumption and an increased formation of prostaglandin E2. PMID- 3011018 TI - Hydrolysis of S-2-(3-aminopropylamino)ethylphosphorothioate (WR-2721). AB - The hydrolysis reaction of S-2-(3-aminopropylamino)ethylphosphorothioate (WR 2721), a radioprotective agent currently undergoing clinical trials, was studied under a variety of experimental conditions in order to provide more complete data and to reconcile significant differences found between two previous studies. 31P NMR spectroscopy was primarily used to follow the reaction, but comparable results were also obtained in parallel studies using a spectrophotometric technique and a technique involving liquid chromatography with electrochemical detection, in which the free sulfhydryl product, 2-(3 aminopropylamino)ethanethiol (WR-1065), was measured. Upon hydrolysis, inorganic phosphate and the free sulfhydryl group were formed by cleavage of the P-S bond. The reaction rate versus pH profile at 30 degrees in 42.5 mM buffer, mu = 127.5 mM, showed primarily hydrolysis of the monoanion, with an acid-catalyzed reaction below pH 1.5 to 2.0 involving the neutral species of the ester. The energy of activation at pH 4.0 in 42.5 mM acetate buffer was 25.7 kcal/mole (23.1 kcal/mole by liquid chromatography with electrochemical detection). The entropy of activation at pH 4.0, 36 degrees was positive, and there was a deuterium isotope effect on the reaction. A small buffer effect on the rate of the reaction at pH 4.0 and pH 5.0 was found to include contributions from both general acid and general base catalysis. These data are consistent with a mechanism for hydrolysis of the monoanion involving a partially rate-determining proton transfer to the sulfur atom and the formation of metaphosphate ion, which is rapidly hydrolyzed to inorganic phosphate. PMID- 3011019 TI - Selective inhibition of herpes simplex virus ribonucleoside diphosphate reductase by derivatives of 2-acetylpyridine thiosemicarbazone. AB - The effects of thiosemicarbazone derivatives of 2-acetylpyridine on mammalian and viral ribonucleoside diphosphate reductases were investigated. The enzymes were partially purified from uninfected and herpes simplex virus type-1 (HSV-1) infected KB cells by sequential salt fractionation with streptomycin sulfate and ammonium sulfate and by affinity chromatography on ATP-agarose. The five thiosemicarbazone derivatives investigated were all potent inhibitors of the virus-induced reductase. Fifty percent inhibitory concentrations (IC50 values) range from 2 to 13 microM. Four of the five derivatives also were inhibitors of the host cell reductase (IC50 values = 7-34 microM). A semicarbazone was inactive against the cellular enzyme and relatively weak as an inhibitor of the viral enzyme (IC50 = 340 microM). Four of six compounds were preferential inhibitors of the viral reductase based on a comparison of IC50 values (5- to greater than 85 fold difference). Kinetic experiments revealed that inhibition of the HSV-1 reductase by the thiosemicarbazones was noncompetitive with respect to CDP and dithiothreitol. A comparison of the inhibitory effects of 2-acetylpyridine thiosemicarbazone itself on viral reductase and on virus replication in vitro demonstrated a similarity in the dose-response relationships for the two parameters. This observation supports the hypothesis that the HSV-induced ribonucleoside diphosphate reductase is an important target for the design of antiviral drugs. PMID- 3011021 TI - Human parvovirus arthropathy. PMID- 3011020 TI - The effect of organophosphorous insecticide Wofatox 50 EC on the adenylate cyclase activity of chicken embryo muscle. PMID- 3011022 TI - Comparative alpha- and beta-adrenoceptor activity of 2- and 6-ring-chlorinated noradrenaline analogues. AB - The alpha- and beta-adrenoceptor activity of the 2- and 6-ring-chlorinated analogues of noradrenaline (norepinephrine) were evaluated in vitro. The 2-chloro substituted analogue exhibits a far greater affinity for beta 1-chronotropic receptors than the 6-chloro-substituted analogue, whereas no significant differences are apparent for their alpha-adrenergic affinities. PMID- 3011024 TI - Binding surface analysis: an intuitive yet quantitative method for the design and analysis of ligand binding studies. AB - Ligand binding techniques are widely used to measure drug, hormone, and neurotransmitter receptors. Despite its wide application, quantitative methods of data analysis have not been widely adopted. This paper reviews some of the problems associated with semiquantitative methods of data analysis, and describes a mathematically based approach to the design and analysis of ligand binding experiments. Widely spaced concentrations of radiolabeled ligand are displaced by concentrations of cold drug. Analysis of these curves, which describe a "binding surface," with computer curve-fitting programs significantly increases the power of the ligand-binding technique. PMID- 3011023 TI - Long-term effects of guar gum on blood lipids. AB - While guar gum has been shown to lower total cholesterol and low density lipoprotein cholesterol (LDL-C) in diabetic patients over the short-term, the long-term effects are less well studied and may be unpredictable. Granola bars with and without 6.6 g guar gum were developed and fed to 16 adult volunteers with Type II diabetes mellitus who had been randomized in a double-blind fashion into guar and placebo groups of equal size. Four to six bars were consumed daily with an ad lib diet over a 6-month period. Total cholesterol, total high density lipoprotein cholesterol (HDL-C), subfractions HDL2-C and HDL3-C, LDL-C, and beta apoprotein were measured at 0 and 6 months. Although LDL-C was lower and triglycerides higher at 6 months than at baseline, these changes were of equal magnitude and direction in both guar and placebo groups. Using each subject as his own control, only the change in triglycerides was statistically significant (P less than 0.025). When male subjects alone were analyzed, the guar group showed a statistically significant decrease in LDL, while the placebo group did not. Other lipid parameters were not significantly changed during the study, despite a positive effect on carbohydrate metabolism from the guar bars. The data suggest either that the hypolipemic effects of guar gum in patients with Type II diabetes mellitus are not sustained for 6 months, or the effects occur only in men. PMID- 3011025 TI - Place-conditioning properties of mu, kappa, and sigma opioid agonists. AB - The place-conditioning capacities of morphine, ketocyclazocine, ethylketocyclazocine, bremazocine, U 50488, d,1-N-allylnormetazocine (NAN), d NAN, 1-NAN, and the effects of the opioid antagonists naloxone, WIN 44,441-3, and MR 2266BS were assessed in adult male rats using a three-chambered place conditioning apparatus. Morphine, ketocyclazocine, ethylketocyclazocine, d,1-NAN, and 1-NAN at doses ranging from 0.25 to 4 mg/kg induced place-preferences for the compartment that was paired with drug administration during the conditioning process. One, 2, or 4 mg/kg of d-NAN had little effect. Naloxone, at doses of 1, 2, 4, and 10 mg/kg, conditioned strong place-aversions in rats; that is, on test day, rats spent significantly more time in the compartment that was not paired with drug-treatment. Bremazocine (0.05 to 4 mg/kg) and U 50488 (0.4 to 4 mg/kg) also conditioned significant, dose-related place-aversions. The results of the putative kappa opioid antagonists were mixed; MR 2266BS caused a dose-related place-aversion while the WIN 44,441-3 produced place-preference. Two mg/kg, but not 0.02 or 0.2 mg/kg, of naloxone administered prior to conditioning with 4 mg/kg of morphine, ethylketocyclazocine, and d,1-NAN, or 2 mg/kg of ketocyclazocine, resulted in place-aversions similar in magnitude to those found after naloxone-conditioning alone. Thus, the mu agonist morphine, the kappa agonists ketocyclazocine and ethylketocyclazocine, the sigma-agonist d,1-NAN, and the levo isomer of NAN all induce place-preferences in the rat according to the place-conditioning paradigm. In contrast, the kappa agonists bremazocine and U 50488, and the kappa antagonist MR 2266BS, induced place-avoiding responses. Since the development of place-preference after morphine, ketocyclazocine, ethylketocyclazocine, and d,1-NAN was antagonized by a kappa-receptor-antagonist dose of naloxone (2 mg/kg) and not by mu-receptor-antagonist doses, these data provide evidence that these particular mu, kappa, and sigma opioids may share a common underlying pharmacologic action. PMID- 3011027 TI - Evaluation of muscle damage in electrical burns. 99mTc-pyrophosphate. PMID- 3011026 TI - Plasma delta-9-tetrahydrocannabinol in pregnant sheep and fetus after inhalation of smoke from a marijuana cigarette. AB - Seven pregnant ewes were prepared with open-ended tracheal T tubes and with catheters in maternal femoral artery and in central circulation of fetus. Several days postoperatively, at 129-132 days gestation, ewes inhaled smoke from one marijuana cigarette containing 3.19% delta-9-tetrahydrocannabinol (delta-9-THC). Smoke was produced continuously in a hand-held chamber and delivered to the protruding arm of the tracheal tube. Samples of maternal and fetal blood were taken during the 8-10 minute smoking period and at intervals up to 24 hours. Delta-9-THC was detected in maternal plasma at 3 minutes and peaked at 10 minutes. Fetal plasma delta-9-THC reached detectable levels in 5 animals by 10 minutes. Maximum mean level was reached at 90 minutes and remained nearly constant until the fourth hour. Fetal delta-9-THC levels remained lower than maternal levels at all times. The terminal half-life of delta-9-THC in fetal and maternal plasma exceeded 10 hours. PMID- 3011028 TI - Health effects of omega-3 fatty acids. PMID- 3011030 TI - [Current aspects of the problem of arterial hypertension]. PMID- 3011029 TI - [Derivatives of arachidonic acid. I. Structure, biosynthesis and biological role]. PMID- 3011031 TI - Selection of a suitable internal standard in head space gas chromatographic breath ethanol analysis after adsorption on silica gel. PMID- 3011033 TI - The human interleukin-2 receptor. PMID- 3011032 TI - The receptor with high affinity for immunoglobulin E. PMID- 3011035 TI - Characterization of microsomal ATPases from developing human placenta. AB - Activities and some properties of microsomal ATPases have been studied in developing human placenta. The enzyme activities (Na+ + K+ + Mg2+, Mg2+, and Ca2+ dependent) in the placenta increase steadily with gestational age until the 18th to 21st week, and decrease in the second half of pregnancy. Mg2+-dependent and Na+ + K+ + Mg2+-dependent ATPases possess nearly the same Km (apparent) for ATP, while the Ca2+-dependent enzyme shows a different one. Mg2+-dependent ATPase shows higher substrate affinity than Ca2+-dependent ATPase, although the Vmax of the Mg2+-dependent enzyme is lower than that of the latter. However, for each enzyme, the Km remains almost constant and Vmax varies during ontogenic development. Vmax of the enzymes decline at term. The enzymes are heat-labile, unaffected by amino acids, namely, L-phenylalanine, L-leucine, and L-tryptophan, and deoxycholate inhibits the enzyme activities by about 50%. PMID- 3011034 TI - Lymphadenopathy-associated-virus infection and acquired immunodeficiency syndrome. PMID- 3011036 TI - Hormonal effects on the activities of glycosidases in the endometrium of rabbit uterus. AB - The activities of several glycosidases in the lysosomal fraction of the uterine endometrium of rabbit were measured using 4-MU-glycosides as substrates. The specific activity of beta-N-acetylglucosaminidase was the highest, which was followed by beta-galactosidase, beta-glucuronidase, and alpha-galactosidase in this order. beta-Glucosidase had the lowest activity among the glycosidases examined. In order to examine the hormonal effects on these glycosidases, the lysosomal fractions were prepared from the uterine endometrium of the control, estrogen-treated, and progesterone-treated rabbits. In all glycosidases examined, except for beta-glucosidase, the specific activity was highest in the lysosome obtained from estrogen-treated rabbit. The specific activity in the lysosome from the progesterone-treated rabbit was between that from the estrogen-treated rabbit and that from control. Hormonal treatments, however, affected neither pH optimum curves nor isozyme patterns of these glycosidases. PMID- 3011037 TI - Tourniquet shock in rats: effects of allopurinol on biochemical changes of the gastrocnemius muscle subjected to ischemia followed by reperfusion. AB - Recent data suggest that oxygen free radicals are implicated in the pathogenesis of ischemic injury. This study evaluates the effects of allopurinol, a xanthine oxidase (XO) inhibitor, on malonaldehyde generation, free sulfhydryl levels, oxygen consumption, and water contents of rat gastrocnemius muscles of female Sprague-Dawley rats subjected to tourniquet shock and after hind-limb reperfusion. Serum lactic dehydrogenase isozyme patterns after ligature release were also examined. Our results show that the four muscle parameters were not altered during 5 hr of ischemia, but that on hind-limb reperfusion, malonaldehyde production, SH levels, O2 consumption, and water contents were significantly altered in the control animals, but not in those pretreated with allopurinol. LDH serum patterns of the untreated animals showed the presence of all five isoforms; these were much less evident in the drug-protected rats. Our data suggest that following ischemia, the affected muscles are unable to recover their normal function when reperfusion is resumed. The subsequent damage is probably due to the generation of cytotoxic superoxide radicals formed during the XO-catalyzed transformation of hypoxanthine to uric acid on tissue reoxygenation. The severity of tissue damage is related to the duration of the ischemic episode possibly due to hypoxanthine accumulation during ischemia. PMID- 3011038 TI - Myokinase and contractile function of glycerinated muscle fibers. AB - Glycerinated rabbit psoas muscle fibers containing native CPK, ATPase, and myokinase activities were used and isometric contraction and relaxation responses to either ADP or ATP + CP or to ATP alone in the presence and absence of P1, P5 di(adenosine-5'-pentaphosphate), a myokinase inhibitor, were compared. In previous (14) work it was shown that CP generated more efficient and faster contraction and relaxation of glycerinated muscle fibers than ATP. The present work deals with the role of myokinase in the differential response of fibers to CP and ATP. Inhibition of the myokinase activity of these fibers caused slight diminution of the rate of contraction at physiological concentrations of ATP. Uninhibited fibers were not able to reach maximum contraction, because the tension began to drop gradually even in the presence of Ca2+. Addition of Ap5A permitted maximum contraction and the ability to stay at the contracted state. In the case of CP + adenosine nucleotides (ATP or ADP), myokinase activity decreased the rate of tension development which was statistically significant after 5-7 sec of contraction. Thus, a higher tension was obtainable when myokinase was inhibited. At high concentration of adenine nucleotides (greater than 2 mM) and in the absence of Ap5A, not only the maximum tension never was reached, but a spontaneous drop in tension was observed before addition of EGTA, as was seen with ATP alone. Relaxation was faster and more complete in the presence of uninhibited myokinase activity except that the ADP was low (125 mM). These observations provide further evidence for a close functional interaction of these three enzymes in the mechanism of contraction and relaxation, giving further support to the notion of the creatine-phosphocreatine energy shuttle. PMID- 3011040 TI - [CT findings of growth hormone secreting pituitary adenomas]. AB - The value of high-resolution computed tomography (CT) in the diagnosis of pituitary adenoma has recently been stressed, especially of the coronal view with contrast enhancement. Analysis of the CT scans of 33 growth hormone (GH) secreting pituitary adenomas was done (11 cases of microadenomas, 7 cases of intrasellar adenomas and 15 cases of macroadenomas with suprasellar extension). In macroadenomas, the density was high in five cases, high with isodense portion in two cases, mixed in four cases, isodense in three cases, and isodense and low dense in one case. Six adenomas showed homogeneous density and nine were heterogeneous. After contrast enhancement, two cases showed marked enhancement, ten cases mild and three cases ring enhancement. Margin of adenoma was smooth in nine cases and irregular in six. Among seven cases of intrasellar adenoma one accompanied primary empty sella. In microadenomas ten of eleven cases had hypodense mass inside the normally enhanced pituitary gland. The margin was ill defined in seven cases and well-defined in three. Eight cases had pituitary height 7 mm or more. Upper surface of the pituitary gland was convex upward in five cases, flat in four and concave in two. Deviation of pituitary stalk was found in seven cases. Bony changes of sellar floor were recognized in three cases. There was a tendency that serum GH level increased with the increment of the size of adenoma. Serum GH levels in adenomas with ring enhancement were lower than those in the homogeneously enhanced adenomas of similar size. One case with marked enhancement showed the highest GH level among all adenomas of the presented series.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011039 TI - [Aicardi syndrome: an unusual association of neoplastic or cystic lesions]. PMID- 3011041 TI - Dental treatment of hepatitis B carriers and HTLV III-antibody-positive patients. PMID- 3011044 TI - Pseudocholinesterase activity and atracurium v. suxamethonium block. AB - Serum pseudocholinesterase activity in pregnant women was assayed spectrophotometrically using either propionylthiocholine or suxamethonium as substrate. All patients had a normal phenotype as indicated by normal dibucaine and fluoride numbers. However, the mean cholinesterase activity was lower than in the general population. There was a negative correlation between cholinesterase activity and the duration of neuromuscular blockade following suxamethonium, but no correlation was observed between the cholinesterase activity and the duration of block following atracurium. PMID- 3011042 TI - Altered platelet alpha 2 adrenoreceptor in acute myocardial infarction and its relation to plasma catecholamine concentrations. AB - Changes in platelet alpha 2 adrenoreceptors and their relation to plasma catecholamine concentrations were studied in 11 patients with acute transmural myocardial infarction. A radiolabelled alpha 2 adrenoreceptor antagonist, [3H] yohimbine, was used to assay alpha 2 adrenoreceptors on platelet membranes, and plasma catecholamine concentrations were measured by high performance liquid chromatography. The number of platelet alpha 2 adrenoreceptors, the dissociation constant, and plasma noradrenaline and adrenaline concentrations were studied 6.6 (3.3) (mean (SD)) hours after the onset of acute myocardial infarction and one month later. The mean (SD) number of adrenoreceptors increased significantly from 94.5 (50.5) fmol/mg protein immediately after infarction to 157.0 (65.7) fmol/mg protein one month later. The dissociation constant, however, did not change significantly (4.33 (1.40) nmol/l vs 4.37 (1.22) nmol/l). Raised noradrenaline (5.60 (4.37) nmol/l) and adrenaline (0.28 (0.14) nmol/l) concentrations had fallen significantly to normal values (1.21 (0.67) and 0.09 (0.05) nmol/l respectively) a month after infarction. The decrease in the number of alpha 2 adrenoreceptors soon after infarction may be beneficial because such a change will reduce the strength of various reactions to catecholamines, such as vasoconstriction. PMID- 3011043 TI - Cinchocaine and amethocaine inhibit activation and activity of superoxide production in human neutrophils. AB - Local anaesthetics, cinchocaine and amethocaine, inhibited superoxide production in human neutrophils stimulated by phorbol ester, with IC50 (concentration of a drug giving 50% activity of control) values of 0.2 and 0.6 mmol litre-1, respectively. These anaesthetics inhibited protein (de)phosphorylation and mobilization of membrane-associated calcium, both of which are probable signal transmission mechanisms for the induction of superoxide production. Local anaesthetics also inhibited NADPH oxidase, responsible for superoxide production, with IC50 = 0.3 and 0.9 mmol litre-1, respectively. The results indicate that cinchocaine and amethocaine inhibit superoxide production in human neutrophils by affecting not only the catalytic activity, but also the activation, of NADPH oxidase. PMID- 3011045 TI - Atracurium infusion in liver transplantation. AB - A continuous infusion of atracurium was used to provide neuromuscular blockade in 25 adult patients undergoing liver transplantation following atracurium, suxamethonium or vecuronium for intubation. Blockade was monitored by recording evoked electromyographic response and maintained with a mean infusion rate of 0.38 +/- 0.14 mg kg-1 h-1 during 306 +/- 80 min of operation. Atracurium requirements appeared to be less during the anhepatic period and greater after removal of the vascular clamps on the new liver. No instances of arterial hypotension or anaphylactoid reactions attributable to atracurium were reported. It can be concluded that a continuous infusion of atracurium is a safe, effective and convenient technique of ensuring neuromuscular blockade during liver transplantation, at a rate of infusion no different from that needed in patients with normal hepatic function. PMID- 3011046 TI - Age and the pharmacokinetics of angiotensin converting enzyme inhibitors enalapril and enalaprilat. AB - The pharmacokinetics of angiotension converting enzyme (ACE) inhibitors enalapril (10 mg orally) and its active metabolite, enalaprilat (10 mg intravenously) were studied in nine young healthy volunteers aged 22-30 years and nine sex matched elderly subjects aged 65-73 years. After both drugs, a biexponential curve was fitted to the decline in plasma enalaprilat concentration. Area under the plasma concentration-time curve (AUC) was greater in the elderly for both drugs. Clearance (CL) and clearance/bioavailability (CL/F) were less in the elderly for enalaprilat and enalapril, respectively. There was no difference in F between young (0.62 +/- 0.16) and elderly subjects (0.61 +/- 0.15). Enalaprilat CL and enalapril CL/F were significantly and positively correlated to endogenous creatinine clearance. There was a significant difference in the weight corrected volume of distribution at steady state after enalaprilat between the young and elderly (P less than 0.02). The relationship between plasma enalaprilat concentrations and percentage ACE inhibition, using the Hill equation, showed no difference in the sensitivity to ACE inhibition between the young and the elderly group. The pharmacokinetic differences observed are likely to be related to an age dependent decline in renal function as well as changes in body composition. Kinetic differences partly explain the greater pharmacodynamic response in the elderly. PMID- 3011048 TI - Racial differences in drug responses--a comparative study of trimazosin and alpha 1-adrenoceptor responses in normotensive Caucasians and West Africans. AB - The possible racial differences in alpha 1-adrenoceptor responsiveness and the blood pressure and heart rate responses following alpha 1-adrenoceptor antagonism with trimazosin have been investigated in matched groups of six Caucasians and six Nigerians. There were no significant differences between the racial groups in the blood pressure and heart rate responses to oral (200 mg) and intravenous (100 mg) trimazosin. alpha 1-adrenoceptor responsiveness was similar in both groups after placebo and following both active treatments. There were only minor pharmacokinetic differences with the Caucasians having a larger volume of distribution, and a longer terminal elimination half-life for the metabolite, 1 hydroxy-trimazosin. These results suggest a similarity in peripheral vascular alpha 1-adrenoceptor mechanisms and show no major significant racial differences in the pharmacokinetics and pharmacodynamics of trimazosin. PMID- 3011047 TI - Age and the pharmacodynamics of angiotensin converting enzyme inhibitors enalapril and enalaprilat. AB - The effect of age on the pharmacodynamic responses to converting enzyme inhibitors, enalapril and enalaprilat was investigated in nine young (22-30 years) and nine sex-matched elderly (65-73 years), healthy volunteers. The groups differed in baseline blood pressure, young 121/64 mmHg, elderly 142/75 mmHg (P less than 0.01), but not in sodium intake or body weight. Both enalapril and enalprilat produced significant falls in blood pressure in both groups but no increase in heart rate in the supine or erect posture. The blood pressure fall was significantly greater in the elderly on both treatments and in both erect and supine posture. The greater fall in blood pressure in the elderly was associated with a more prolonged inhibition of plasma angiotensin converting enzyme activity. The apparent age-related difference in response appears to be due to the difference in baseline blood pressures between the groups. Further study of the usefulness of chronic dosing with angiotensin converting enzyme inhibitors in hypertension in the elderly is indicated. PMID- 3011049 TI - Genetic predisposition to human lung cancer. AB - The influence of polymorphic variants of the human c-Ha-ras gene on predisposition to lung cancer has been investigated. The human c-Ha-ras gene has been shown to reside on a polymorphic BamH1 restriction fragment. This restriction fragment length polymorphism (RFLP) results from variation in the size of a region of repetitive DNA 3' to the gene. An attempt has been made to characterise and compare the c-Ha-ras RFLP's in a normal population and in a group of cancer patients. DNA was extracted from the white blood cells of 101 normal donors and four common Ha-ras alleles identified, with occasional rare alleles of various sizes. The allele frequencies were examined in 132 lung cancer patients, comprising 66 individuals with small cell carcinoma of the lung (SCCL) and 66 with non-small cell carcinoma of the lung (non-SCCL). An abnormal allele distribution was found in individuals with non-SCCL compared to both control and SCCL values suggesting a degree of genetic pre-position to non-SCCL. In addition, analysis of the Ha-ras RFLP's in solid samples inferred a deletion of material from the short arm of chromosome 11 in two of 16 informative samples. PMID- 3011050 TI - Effect of transformation by Rous sarcoma virus on the character and distribution of actin in Rat-1 fibroblasts: a biochemical and microscopical study. AB - Actin has been measured in subcellular fractions from Rat-1 fibroblasts and in Rous sarcoma virus-transformed Rat-1 cells (VIT), using the DNase 1 inhibition assay. The transformed cells showed a significant shift in the actin monomer (G)in equilibrium with polymer (F) equilibrium within the cell cytosol, and a significant increase in actin in the Triton-insoluble cytoskeletal core in comparison with untransformed cells. This incorporation of actin into the cytoskeletal core fraction is associated with a change in filamentous actin assemblies from 'stress fibre' patterns to punctate filament aggregates. These differences have been correlated with changes in morphology, in actin, vinculin and alpha-actinin distribution, in adhesion plaque formation and with the production of pp60v-src-associated protein kinase activity in the transformed cells. Changes in actin distribution and its polymerization in response to src gene expression may play an important role in the determination of the transformed cell characteristics. PMID- 3011051 TI - Faecal pH, dietary fibre intake, and proneness to colon cancer in four South African populations. AB - In a series of South African populations, mean faecal pH values were found to be: rural and urban blacks, 6.12 and 6.15; Indians 6.21; coloureds (Eur-African Malay), 6.29; these are significantly lower (p less than 0.01) than that of whites, 6.88. Apart from that of the coloureds, mean values for series of children and adults did not differ significantly. In the populations mentioned, corresponding mean dietary fibre intakes of children's mothers (or associates of mothers) were all relatively low, namely, roughly 25 g, 18 g, 20 g, 21 g, 23 g, respectively. Frequency of colon cancer (also other non-infective bowel diseases, e.g. appendicitis) is very low in rural and urban blacks, is low in Indians and coloureds, yet much higher in whites. Thus, in these different ethnic populations, rarity or low frequency of colon cancer is associated more with low faecal pH than with level of dietary fibre intake, suggesting that components additional to fibre have a role in determining the milieu interieur of the bowel and its proneness to disease. PMID- 3011052 TI - Further experience with Kaposi's sarcoma in Uganda. AB - Four Ugandan patients (1 women, 3 men) with generalized Kaposi's sarcoma (KS) were seen in the Uganda Cancer Institute between October 1983 and December 1984. They presented with generalized lymphadenopathy, plaques/nodules on the body, general swelling of the head, oral and visceral involvement and respiratory distress. Initial responses to adriamycin as a single or a combination chemotherapy of actinomycin D, vincristine, adriamycin and imidazole carboxamide appeared to be favourable but no sustained response was obtained. Serological tests for human T-lymphotropic virus (HTLV-II) antibodies were positive in all 4 cases. PMID- 3011054 TI - Derivation and preliminary characterisation of adriamycin resistant lines of human lung cancer cells. AB - We have produced adriamycin (ADM)-resistant variants of the human lung cancer cell lines NCI-H69 (small cell), MOR (adenocarcinoma) and COR-L23 (large cell) but have failed to produce resistant variants of two other small cell lines. In each case, the derivation protocol took 7-9 months and included a period of drug free growth. All three resistant lines show reduced cellular content of ADM after 1 h exposure when compared with their controls. During prolonged incubation of control and resistant NCI-H69 cells in 0.4 microgram ml-1 ADM, the ADM content of resistant cells was 6-7 times lower than that of control cells. The ratio of ADM doses to suppress growth of the two lines, however, was in the range of 40-200X. The ADM-resistant variant of NCI-H69 was also resistant to vincristine, colchicine, VP16, mitozantrone, 4' epiadriamycin and 4' deoxyadriamycin, somewhat resistant to melphalan but not resistant to aclacinomycin A, bleomycin of CCNU. The resistance to ADM could be partially overcome by the use of verapamil, an inhibitor of calcium transport. PMID- 3011053 TI - Neuron specific enolase expression in carcinoma of the lung. AB - The value of neuron specific enolase (NSE) immunoreactivity as a marker for small cell lung cancer (SLC) has been assessed using a monoclonal antibody (MCAB) against NSE, MCAB specificity was confirmed using purified enolase isoenzymes, sections of human brain, a panel of lung tumours, neuroendocrine and non neuroendocrine tumours and normal tissues. Using this MCAB in radioimmunoassay and immunohistochemistry, NSE immunoreactivity was detected in all SCLC material examined. However, considerable reactivity was also observed in a number of non small cell lung cancer cell lines and tumour biopsy specimens. Furthermore, intratumoral heterogeneity with respect to NSE immunostaining was observed in several cases. Factors which may underlie such intratumoral phenotypic diversity were assessed using flow cytometry together with MCABs directed against both NSE and non-neuronal enolase. Such studies revealed that enolase expression in cells which were no longer actively proliferating differed markedly from that of cells in exponential growth. Furthermore, cells grown under conditions of increasing hypoxia exhibited increased enolase expression relative to those grown under oxygenated conditions. It is concluded from these studies that NSE immunoreactivity per se is an unreliable marker for the SCLC phenotype. PMID- 3011055 TI - 201T1 to 67Ga uptake ratio as an indicator for predicting tumour doubling time in human pulmonary neoplasms. PMID- 3011056 TI - Macrophage transmission of suppressor signal for suppression of delayed hypersensitivity and humoral response in JEV-infected mice. AB - Japanese encephalitis virus (JEV) infection induces suppressor T-cells (Ts1) which suppress both the humoral (Ts-PFC) and cell mediated (Ts-DTH) immune response by producing soluble suppressor factors. This study shows that in the JEV model, both TS-PFC and Ts-DTH mediate suppression by recruiting a second subpopulation of suppressor T-cells, the Ts2-PFC and Ts2-DTH. The signal between Ts1 and Ts2 is transmitted by macrophages (M phi). The suppressor factors are adsorbed by peritoneal or splenic M phi. Both heat-killed and live M phi are capable of adsorbing suppressor factors but only live M phi are capable of presenting the signal to T-cells. Thus these are at least two generations of suppressor T-cells in the JEV-specific suppressor pathway and the presence of M phi is obligatory for transmission of the signal. PMID- 3011057 TI - Detection of hepatitis B virus DNA in hepatocellular carcinoma. AB - DNA-DNA hybridization was used to examine tissue from 31 patients with hepatocellular carcinoma (HCC) in Taiwan for the presence of the hepatitis B virus genome. Twenty-four of the DNA samples had discrete high molecular weight bands when cut with HindIII and hybridized with a radiolabelled HBV DNA probe, and similar results were obtained with 25 of the samples when cut with EcoRI. Thus integrated HBV DNA was present in almost 80% of the specimens. None of the samples had a similar pattern and most contained HBV DNA integrated at multiple sites. Seventeen out of 19 HBsAg carriers and one out of two patients with anti HBs were found to have integrated HBV DNA in the tumour tissue, providing further evidence of the strong association between HBV infection and development of hepatocellular carcinoma. PMID- 3011058 TI - The in vitro activities of a highly carcinogenic mineral fibre--potassium octatitanate. AB - The in vitro activities of a highly carcinogenic potassium octatitanate fibre (Fybex) have been investigated. This material caused a low level of in vitro transformation in C3H10T1/2 cells and is thus more active in this assay than the UICC amphibole asbestos samples but less active than fibrous erionite. This ranking is in accord with carcinogenicity in vivo. However Fybex had no detectable activity in an assay for DNA damage based on the S1-nuclease sensitivity of the DNA from exposed cells. In that assay crocidolite asbestos was more active than either of the more carcinogenic dusts. It is suggested that the activities of amphibole asbestos in assays for genetic toxicity may depend on the production of free radicals via a catalytic reaction requiring the presence of transition elements which are either not present or only present at low concentrations in erionite or Fybex. This type of reaction may not be related to in vivo carcinogenicity. The action of fibrous dusts against macrophage-like cells, measured in this instance by the release of arachidonic acid, remains one of the best measures of biological activity. PMID- 3011059 TI - Purification of haemopoietic progenitor cells from patients with chronic granulocytic leukaemia using percoll density gradients and elutriation. AB - Purification of haemopoietic progenitor cells from chronic granulocytic leukaemia buffy coat preparations requires a multistep approach using complementary cell separation techniques. In this study Percoll density gradient centrifugation and centrifugal elutriation were used to isolate large numbers of immature progenitor cells. Percoll density gradients were valuable as a first separation step: CFU-GM and CFU-GEMM could be enriched 75-fold in a light density fraction of d less than 1.056 g/ml and the technique could be adapted to cope with more than 10(10) buffy coat leucocytes. Progenitors cells were concentrated 3-fold by elutriation used as single method to separate buffy coat cells or when used to purify further light density Percoll fractions. When Percoll gradients and elutriation were used sequentially, undifferentiated mononuclear cells were enriched to more than 90% purity and between 5% and 40% of these cells formed CFU-GM or BFU-E colonies consisting of more than 40 cells. The enriched fractions were further characterized with monoclonal antibodies. The density and elutriation profiles of these colony forming cells resembled corresponding profiles of cells that reacted with the monoclonal antibody BI-3C5, which recognizes an antigen on primitive haemopoietic progenitor cells. Physical separation methods are a valuable first stage in the attempt to procure relatively pure myeloid progenitor cell populations, whose characteristics can then be further studied at a cellular or molecular level. PMID- 3011060 TI - Repair of episiotomies and perineal tears. PMID- 3011061 TI - Episiotomy repair--immediate and long-term sequelae. A prospective randomized study of three different methods of repair. AB - Three methods of episiotomy repair were randomly assigned after 900 consecutive deliveries. The three procedures were: (1) continuous No. 00-plain catgut in the vagina; No. 00-plain catgut interrupted stitches in the perineal muscles and fascia, and No. 00-nylon interrupted stitches in the skin. (2) The same technique as in (1), but with No. 0-polyglycolic acid (Dexon) in all layers. (3) The suture material as in (2), but used with a subcuticular technique. The women treated with method 3 reported statistically significant less pain and disabilities in the early puerperium. Three months after delivery 262 women (33%) still had perineal complaints which could be directly related to the episiotomy in 25% (8% of total number). The group treated with method 3 had the best long-term results and we conclude that the subcuticular technique using polyglycolic acid should be the method of choice. PMID- 3011062 TI - Purification and characterization of Plasmodium berghei DNA topoisomerases I and II: drug action, inhibition of decatenation and relaxation, and stimulation of DNA cleavage. AB - It has recently been suggested that topoisomerases could be important targets for drugs used in several diseases. This prompted us to purify and characterize the topoisomerases I and II present in the erythrocytes of protozoan parasites of the genus Plasmodium, the causative agent of malaria, in order to later use these enzymatic systems in antimalarial drug assays. The topoisomerases were purified from Plasmodium berghei, a parasite of mouse red cells. The Plasmodium topoisomerase II consists of two subunits with a molecular weight of about 160K. The enzyme is ATP- and Mg2+-dependent. The conditions for the reactions of relaxation, unknotting, decatenation, and catenation were found to be similar to those observed with enzymes from other eukaryotic cells. The Plasmodium topoisomerase I is a monomeric enzyme with a Mr of 70K-100K. It is ATP independent and K+- or Na-dependent. Mg2+ is not required for relaxation but stimulates the reaction. Topoisomerase II was more sensitive to drug action than topoisomerase I. The most active drugs were the ellipticine derivatives. The antimalarial drugs, currently used in human clinical therapy, were poor inhibitors. Some antitumoral drugs stimulated the double-stranded DNA cleavage activity of Plasmodium topoisomerase II, like that of mammalian topoisomerases II. Antimalarial drugs had no stimulating activity. It is therefore suggested that Plasmodium topoisomerases are not good targets for antimalarial drugs. PMID- 3011063 TI - Characterization of a cDNA coding for human factor XII (Hageman factor). AB - Affinity-purified antibody against human factor XII (Hageman factor) has been radiolabeled with 125I and employed as a probe to screen a human liver cDNA expression library prepared in lambda gt11. Approximately 3.5 X 10(6) recombinant phages were screened for factor XII, and two positive clones were identified and plaque purified. The largest cDNA coding for factor XII was 1571 base pairs in length and coded for amino acid residues 127-596 in the mature protein, a termination codon of TGA, a 3' noncoding sequence of 147 nucleotides, and a poly(A) tail of 11 nucleotides. The second clone contained an insert of 1334 base pairs and coded for amino acid residues 200-596. The amino acid sequence predicted by the cDNAs was in excellent agreement with that previously determined by amino acid sequence analysis. The amino acid and DNA sequences in human factor XII showed considerable homology with the corresponding domains in other serine proteases, including prothrombin, plasminogen, tissue plasminogen activator, and urokinase. PMID- 3011065 TI - Reconstitution of membrane proteins: catalysis by cholesterol of insertion of integral membrane proteins into preformed lipid bilayers. AB - The presence of cholesterol in small unilamellar vesicles (ULV) of dimyristoylphosphatidylcholine (DMPC) catalyzes fusion of the vesicles at temperatures below the upper limit for the gel to liquid-crystalline phase transition of the DMPC. The extent to which ULV grow depends on the concentration of cholesterol in the vesicles and on temperature. Maximum growth occurs at 21 degrees C. It decreases as the temperature is lowered below 21 degrees C. Growth does not occur at temperatures above the phase transition. In addition, the presence of cholesterol in ULV of DMPC catalyzes the insertion of integral membrane proteins into the vesicles. Thus, bacteriorhodopsin from Halobacterium halobrium, UDPglucuronosyltransferase (EC 2.4.1.17) from pig liver microsomes, and cytochrome oxidase from beef heart mitochondria formed stable lipid-protein complexes spontaneously when added to ULV containing cholesterol at temperatures under which these vesicles would fuse. Incorporation of these proteins into the ULV of DMPC did not occur in the absence of cholesterol or in the presence of cholesterol when the temperature of the system was above that for the phase transition. It appears that cholesterol lowers the energy barrier for fusion of ULV of DMPC and for insertion of integral membrane proteins into these bilayers. Studies with bacteriorhodopsin suggest that the energy barrier for insertion of proteins into ULV containing cholesterol is smaller than the energy barrier for fusion of the ULV with each other. PMID- 3011064 TI - p-(Chloromercuri)benzenesulfonate binding by membrane proteins and the inhibition of water transport in human erythrocytes. AB - The binding of [203Hg]-p-(chloromercuri)benzenesulfonate to the membrane proteins of human erythrocytes and erythrocyte ghosts was examined under conditions where binding to the bulk of membrane sulfhydryl groups was blocked by N ethylmaleimide. Binding was essentially complete within 90 min when approximately 40 nmol was bound per milligram of membrane protein. This binding was correlated with the inhibition of water transport measured by an NMR technique. Maximal inhibition was observed with the binding of approximately 10 nmol of p (chloromercuri)benzenesulfonate/mg of membrane protein. Under these conditions, both band 3 and band 4.5 bound 1 mol of inhibitor/mol of protein. In contrast to previous experiments, these results indicate that band 4.5 proteins as well as band 3 have to be considered as playing a role in water transport. PMID- 3011066 TI - Interaction of enkephalins and des-tyrosyl-enkephalins with synaptosomal plasma membrane vesicles: enkephalin binding and inhibition of proline transport. AB - Leucine- and methionine-enkephalins inhibit the Na+-dependent transport of proline into plasma membrane vesicles derived from synaptosomes. Glycine transport is weakly inhibited by enkephalins whereas there is no inhibition of transport of glutamic acid, aspartic acid, or gamma-aminobutyric acid. The inhibition of proline uptake is observed with des-tyrosyl-enkephalins but not with morphine, dynorphin(1-13), or beta-endorphins. Furthermore, enkephalin induced inhibition of proline transport is not antagonized by naloxone. [Leu]enkephalinamide and modified [Leu]enkephalins with greater selectivity for the delta-subclass of enkephalin binding sites are less effective than [Leu]enkephalin in the inhibition of proline transport. Specific binding of [3H]Leu-enkephalin to the plasma membrane vesicles is demonstrated, and des-Tyr [Leu]enkephalin competes with Leu-enkephalin for [Leu]enkephalin binding sites. The similarity in the concentrations of des-Tyr-[Leu]enkephalin required to compete for specific [Leu]enkephalin binding and to inhibit proline transport suggests that a specific subclass of enkephalin binding sites, distinguished by their recognition of both the enkephalins and their des-tyrosyl derivatives, may be associated with the synaptic proline transport system. PMID- 3011068 TI - Isolation of pyrophosphohistidine from pyrophosphorylated pyruvate, phosphate dikinase. AB - The pyrophosphoryl form of pyruvate, phosphate dikinase was prepared by incubation with adenosine 5'-[gamma-32P]triphosphate and isolated by gel chromatography. Previously a phosphorylated moiety had been isolated from the enzyme and was shown to be bound through a phosphoramidate linkage to the 3' nitrogen of a histidine residue [Spronk, A. M., Yoshida, H., & Wood, H. G. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4415]. This histidine residue has been considered to be the pyrophosphoryl and phosphoryl carrier between the three subsites of this enzyme. Previous attempts to isolate the putative [32P]pyrophosphohistidine have been unsuccessful due to the lability of the [32P]pyrophosphoryl-enzyme. By stabilization of the [32P]pyrophosphoryl-enzyme with diazomethane, it has been possible to isolate a [32P]-pyrophosphohistidine from the hydrolysates. To our knowledge this work constitutes the first direct demonstration of a pyrophosphorylated histidyl residue in an enzyme. PMID- 3011067 TI - Influence of anions and pH on the conformational change of horse liver alcohol dehydrogenase induced by binding of oxidized nicotinamide adenine dinucleotide: binding of chloride to the catalytic metal ion. AB - The conformational change of horse liver alcohol dehydrogenase induced by binding of NAD+ was studied by electronic absorption spectroscopy using cobalt as a spectroscopic probe in the active site. The complex of the enzyme with NAD+ exists in an acidic and an alkaline form. The transition between the two forms proceeds through several intermediates and is controlled by an apparent pKa of 6.9. Only at pH values below this pKa can a complex between enzyme, NAD+, and Cl- be formed. The spectral changes indicate that chloride displaces the cobalt-bound water molecule in a tetracoordinate structure. We conclude that a negative charge at the active site is necessary to stabilize the closed conformation of the enzyme in the presence of NAD+. Spectral correlations are given which strongly support the postulation of a metal-bound alkoxide in the closed structure of the enzyme as an essential feature of the catalytic mechanism of horse liver alcohol dehydrogenase. PMID- 3011069 TI - Receptor binding and adenylate cyclase activities of glucagon analogues modified in the N-terminal region. AB - In this study, we determined the ability of four N-terminally modified derivatives of glucagon, [3-Me-His1,Arg12]-, [Phe1,Arg12]-, [D-Ala4,Arg12]-, and [D-Phe4]glucagon, to compete with 125I-glucagon for binding sites specific for glucagon in hepatic plasma membranes and to activate the hepatic adenylate cyclase system, the second step involved in producing many of the physiological effects of glucagon. Relative to the native hormone, [3-Me-His1,Arg12]glucagon binds approximately twofold greater to hepatic plasma membranes but is fivefold less potent in the adenylate cyclase assay. [Phe1,Arg12]glucagon binds threefold weaker and is also approximately fivefold less potent in adenylate cyclase activity. In addition, both analogues are partial agonists with respect to adenylate cyclase. These results support the critical role of the N-terminal histidine residue in eliciting maximal transduction of the hormonal message. [D Ala4,Arg12]glucagon and [D-Phe4]glucagon, analogues designed to examine the possible importance of a beta-bend conformation in the N-terminal region of glucagon for binding and biological activities, have binding potencies relative to glucagon of 31% and 69%, respectively. [D-Ala4,Arg12]glucagon is a partial agonist in the adenylate cyclase assay system having a fourfold reduction in potency, while the [D-Phe4] derivative is a full agonist essentially equipotent with the native hormone. These results do not necessarily support the role of an N-terminal beta-bend in glucagon receptor recognition. With respect to in vivo glycogenolysis activities, all of the analogues have previously been reported to be full agonists.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011070 TI - Control of the redox potential of cytochrome c and microscopic dielectric effects in proteins. AB - X-ray structural information provides the opportunity to explore quantitatively the relation between the microenvironments of heme proteins and their redox potentials. This can be done by considering the protein as a "solvent" for its redox center and calculating the difference between the electrostatic energy of the reduced and oxidized heme. Such calculations are presented here, applying the protein dipoles-Langevin dipoles (PDLD) model to cytochrome c. The calculations focus on an evaluation of the difference between the redox potentials of cytochrome c and the octapeptide-methionine complex formed by hydrolysis of cytochrome c. The corresponding difference (approximately 7 kcal/mol) is accounted for by the PDLD calculations. It is found that the protein provides basically a low dielectric environment for the heme, which destabilizes the oxidized heme (relative to its energy in water). The effect of the charged propionic acids on the heme is examined in a preliminary way. It is found that the negative charges of these groups are in a hydrophilic rather than a hydrophobic environment and that the protein-water system provides an effective high dielectric constant for their interaction with the heme. The dual nature of the dielectric effect of the cytochrome (a low dielectric constant for the self energy of the heme and a high dielectric constant for charge-charge interactions) is discussed. The findings of this work are consistent with the difference between the folding energies of the reduced and oxidized cytochrome c. PMID- 3011071 TI - Guanosine 3',5'-cyclic monophosphate stimulates release of actively accumulated calcium in purified disks from rod outer segments of bovine retina. AB - Parallel lines of evidence have suggested that light initiates changes in both cGMP metabolism and calcium levels in rod outer segments (ROS). We report that cGMP stimulates release of a pool of Ca2+ actively accumulated within purified ROS disks. Disks were purified and actively loaded with 45Ca2+ by an associated ATP-dependent calcium uptake activity as previously described [Puckett, K.L., Aronson, E.T., & Goldin, S.M. (1985) Biochemistry 24, 390-400]. Spikes of 45Ca2+ released from disks were observed in a rapid superfusion system. The Ca2+ release was specifically stimulated by physiological levels of cGMP (Kapp approximately 20 microM; Hill coefficient = 1.7). 8-Bromo-cGMP could also activate the release mechanism, but cAMP was ineffective. At cGMP levels of greater than or equal to 100 microM, approximately 20% of the loaded Ca2+ was released. The Ca2+ release rate at saturating cGMP levels reached a maximum within the 10-s time resolution of the assay system. In contrast to other recent reports of cGMP activation of ROS ion conductances, the majority of the release activity terminated in a spontaneous manner, suggestive of an intrinsic inactivation process. The amount of Ca2+ released and the release kinetics were similar to the presence or absence of an unbleached pool of rhodopsin. Cyclic nucleotides did not stimulate release from disks passively equilibrated with 45Ca2+, i.e., in the absence of ATP but otherwise under identical conditions. Preincubation of the disks with cGMP also reduced the level of ATP-dependent Ca2+ uptake (approximately 30%); this apparent inhibition may be due to activation of the release mechanism, rather than direct modulation of the uptake activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011073 TI - Natural variation of tyrosyl-tRNA synthetase and comparison with engineered mutants. AB - We report the cloning and sequence analysis of the gene for the tyrosyl-tRNA synthetase from Bacillus caldotenax and properties of the gene product. The amino acid sequence of the tyrosyl-tRNA synthetase was found to be 99% homologous with the corresponding enzyme from B. stearothermophilus, with only four amino acid differences. Two of these natural variations were found to involve active site residues of the enzyme and correspond to mutations that have been engineered previously in vitro. One, Thr-51----Ala-51, produced a more active enzyme, possessing a higher value of kcat/KM for ATP. Position 51 is a "hot spot" in the tyrosyl-tRNA synthetase, differing in enzymes derived from Escherichia coli, B. stearothermophilus, and B. caldotenax. The other, His-48----Asn-48, is found to be a neutral mutation but is in one of the rare regions that are conserved with other aminoacyl-tRNA synthetases. The equivalence of histidine and asparagine at position 48 extends the homology in this region to more enzymes. These residues, His-Ile-Gly-His, and now His-Ile-Gly-Asn, form part of the binding site for ATP in the transition state of the reaction. Although B. caldotenax is an obligate thermophile with an optimal growth temperature of 80 degrees C, as much as 20 degrees C above the growth optima of strains of Bacillus stearothermophilus, its tyrosyl-tRNA synthetase has an identical thermal stability in vitro to that from B. stearothermophilus. PMID- 3011072 TI - Differential and analytical subfractionation of rat liver components internalizing insulin and prolactin. AB - Receptor-mediated endocytosis of 125I-insulin and 125I-prolactin into liver parenchymal cells has been studied by quantitative subcellular fractionation. Differential centrifugation yielded three particulate fractions, N (nuclear), ML (large granule), and P (microsomes), and a final supernatant (S). Quantitative differences in the extent and rates of accumulation of 125I-insulin and 125I prolactin into the fractions were observed. The acidotropic agent chloroquine and the microtubule disrupting agent colchicine were administered separately to rats. The agents increased significantly the T 1/2 of hormone clearance from the liver and augmented the accumulation of both ligands in the low-speed ML fraction. However, differences in the rates of accumulation of insulin and prolactin into all cell fractions were still maintained. Analytical centrifugation of each of the particulate fractions was carried out in order to determine if different endocytic components were specific to insulin or prolactin internalization. This was not the case. An "early" endosomal component of density 1.11 was identified in microsomes. A "late" endosome of density 1.10 was identified in the large granule (ML) fraction. Both endosomal components appeared to accumulate insulin and prolactin but at different rates. Marker enzyme analysis identified the presumed plasma membrane component in microsomes (density approximately 1.155). This component showed a significant difference in the rate of loss of 125I insulin (T 1/2 approximately 4.1 min) as compared to that of 125I-prolactin (T 1/2 approximately 12.7 min). A further difference in the handling of the ligands was observed in early endosomes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011075 TI - Chicken U2 and U1 RNA genes are found in very different genomic environments but have similar promoter structures. AB - We have cloned and analyzed a gene that codes for chicken U2 small nuclear RNA (snRNA). In the haploid chicken genome, there are approximately 35-40 copies of the U2 RNA gene arranged in tandemly repeated units 5.35 kilobase pairs in length. This U2 gene organization contrasts with that of chicken U1 RNA genes, which are found in heterogeneous genomic environments. Although U snRNA genes are transcribed by RNA polymerase II, they lack the usual TATA and CAAT homologies found in the 5' control regions of most RNA polymerase II transcription units. Nevertheless, a comparison of chicken U2 and U1 RNA gene 5'-flanking DNA sequences reveals two upstream blocks of homology which are also evolutionarily conserved in U2 and U1 RNA genes of other vertebrate species. The first block of conserved sequence is centered around position -55 relative to the RNA cap site, and the other is located near position -200. Interestingly, stretches of sequence with the potential to form Z DNA are located either within or immediately adjacent to both of these two conserved upstream sequence elements, suggesting a possible role for Z DNA in U1/U2 gene expression. Moreover, the chicken U2 and U1 gene promoter regions also contain specific short sequences (i.e., the hexamer GGGCGG and the octamer ATGCAAAT) that have been shown to be required for the expression of a number of mRNA-encoding genes. These findings suggest that the transcription of snRNA genes is controlled by a complex set of factors, some shared with other RNA polymerase II transcription units and others which may be unique to the snRNA genes. PMID- 3011074 TI - New pepsin-solubilized low molecular weight collagenous component possibly unique to periodontal ligament. AB - Limited pepsin digestion of bovine periodontal ligament releases genetic types I, III, and V collagen and a high cystine containing low molecular weight collagenous component. Salt fractionation and molecular sieve chromatography allowed the isolation of the latter as an apparently pure homogeneous moiety which had an approximate molecular mass of 30 000 daltons. Reduction with mercaptoethanol yielded a single 10 000-dalton band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This led us to conclude that the newly isolated low molecular weight collagen fragment consists of three similar molecular weight chains. Unreduced collagen-like glycoprotein (CGP) [Jander, R., Troyer, D., & Rauterberg, J. (1984) Biochemistry 23, 3675-3681] after extraction from tissues with collagen denaturing solvents yields the GP140 glycoprotein upon reduction and does not release any collagen fragment below 90 000 daltons upon mild or vigorous pepsin digestion. The GP140 glycoprotein [Heller-Harrison, R. A., & Carter, W. G. (1984) J. Biol. Chem. 259, 6858-6864] isolated by extraction under reducing and collagen denaturing solvent conditions did not yield a collagen fragment below 40 000 daltons after pepsin treatment. It was clearly shown that both CGP and GP140 yield type VI collagen fragments in the above-cited reports. Since this report demonstrates that the Mr 30 000 collagen fragment is only released by pepsin treatment of nondenaturing solvent treated periodontal ligament and that only very small peptides are found in denaturing solvent treated tissue after pepsin digestion, it is concluded that the newly isolated Mr 30 000 collagen fragment reported here is not derived from type VI collagen.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011077 TI - Solubilization and reassembly of the mitochondrial benzodiazepine receptor. AB - We have solubilized and reassembled the peripheral-type benzodiazepine receptor, a component of the mitochondrial outer membrane, from rat adrenal gland mitochondria. The ligand binding site of this receptor undergoes denaturation during solubilization in digitonin, Triton X-100, or 3-[(3 cholamidopropyl)dimethylammonio]-1-propanesulfonate at detergent concentrations above 0.1%, which is evident from the loss of high-affinity binding of [3H]PK11195, a ligand selective for the mitochondrial benzodiazepine receptor. The conformation of the binding site for PK11195 can be stabilized during solubilization in sodium cholate by relatively low concentrations of supplementary soybean lipid. Drug displacement studies demonstrate that the pharmacological properties of the receptor are preserved under these conditions. Electron micrographs of the solubilized preparation show a heterogeneous population of many small particles (less than 100 A) and some larger membranous aggregates (up to 500 A). Sucrose gradient centrifugation indicates that these lipoprotein complexes are of high buoyant density. They can be incorporated in liposomes via cholate dialysis in the presence of additional supplementary lipid. The behavior of the mitochondrial benzodiazepine receptor during solubilization and reassembly suggests that it is an integral protein of the outer membrane. PMID- 3011076 TI - Active nonaromatic intermediates in the conversion of steroidal estrogens into catechol estrogens. AB - A mechanism is proposed for mixed-function oxidase-catalyzed formation of the catechol estrogens 2-hydroxy- and 4-hydroxyestradiol from estradiol. This mechanism involves nonaromatic epoxyenones as intermediates. The isomeric 1 alpha,2 alpha-epoxy-17 beta-hydroxyestr-4-en-3-one and 1 beta,2 beta-epoxy-17 beta-hydroxyestr-4-en-3-one (the latter as its 17-acetate) were synthesized from 17 beta-hydroxy-5 alpha-estran-3-one. The isomeric 4 alpha,5 alpha-epoxy-17 beta hydroxyestr-1-en-3-one and 4 beta,5 beta-epoxy-17 beta-hydroxyestr-1-en-3-one were prepared from 19-nortestosterone. From incubations of [6,7-3H]estradiol with microsomes from MCF-7 human breast cancer cells, which principally catalyze the formation of 2-hydroxyestradiol from estradiol, we were able to isolate a 3H labeled product with the chromatographic properties of 1 beta, 2 beta-epoxy-17 beta-hydroxyestr-4-en-3-one (as its 17-acetate). The soluble protein fraction of homogenates of rat liver, which is devoid of estrogen 2-/4-hydroxylase activity, has been shown to catalyze the formation of 2- and 4-hydroxyestradiol from the 1 alpha,2 alpha-epoxide and from the 4 alpha,5 alpha- and 4 beta,5 beta-epoxides, respectively. We suggest that these results taken together strongly support a role for epoxyenones as intermediates in the formation of catechol estrogens. PMID- 3011078 TI - Interaction of intestinal brush border membrane vesicles with small unilamellar phospholipid vesicles. Exchange of lipids between membranes is mediated by collisional contact. AB - The kinetics of lipid transfer from small unilamellar vesicles as the donor to brush border vesicles as the acceptor have been investigated by following the transfer of radiolabeled or spin-labeled lipid molecules in the absence of exchange protein. The labeled lipid molecules studied were various radiolabeled and spin-labeled phosphatidylcholines, radiolabeled cholesteryl oleate, and a spin-labeled cholestane. At a given temperature and brush border vesicle concentration similar pseudo-first-order rate constants (half-lifetimes) were observed for different lipid labels used. The lipid transfer is shown to be an exchange reaction leading to an equal distribution of label in donor and acceptor vesicles at equilibrium (time t----infinity). The lipid exchange is a second order reaction with rate constants being directly proportional to the brush border vesicle concentration. The results are only consistent with a collision induced exchange of lipid molecules between small unilamellar phospholipid vesicles and brush border vesicles. Other mechanisms such as collision-induced fusion or diffusion of lipid monomers through the aqueous phase are negligible at least under our experimental conditions. PMID- 3011079 TI - Kinetics and extent of fusion between Sendai virus and erythrocyte ghosts: application of a mass action kinetic model. AB - The kinetics and extent of fusion between Sendai virus and erythrocyte ghosts were investigated with an assay for lipid mixing based on the relief of self quenching of fluorescence. The results were analyzed in terms of a mass action kinetic model, which views the overall fusion reaction as a sequence of a second order process of virus-cell adhesion followed by the first-order fusion reaction itself. The fluorescence development during the course of the fusion process was calculated by numerical integration, employing separate rate constants for the adhesion step and for the subsequent fusion reaction. Dissociation of virus particles from the cells was found to be of minor importance when fusion was initiated by mixing the particles at 37 degrees C. However, besides the initiation of fusion, extensive dissociation does occur after a preincubation of a concentrated suspension of particles at 4 degrees C followed by a transfer of the sample to 37 degrees C. The conclusion drawn from the levels of fluorescence increase obtained after 20 h of incubation is that in principle most virus particles can fuse with the ghosts at 37 degrees C and pH 7.4. However, the number of Sendai virus particles that actually fuse with a single ghost is limited to 100-200, despite the fact more than 1000 particles can bind to one cell. This finding may imply that 100-200 specific fusion sites for Sendai virus exist on the erythrocyte membrane. A simple equation can yield predictions for the final levels of fluorescence for a wide range of ratios of virus particles to ghosts.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011080 TI - Restriction endonuclease EcoRI alters the enantiomeric preference of chiral metallointercalators for DNA: an illustration of a protein-induced DNA conformational change. AB - A conformational change in the DNA plasmid ColE1 appears to occur upon specific binding of the restriction endonuclease EcoRI. Enzyme association alters the chiral discrimination found in binding metallointercalators to DNA sites. The complexes tris(1,10-phenanthroline)ruthenium(II), Ru(phen)3(2+), tris(4,7 diphenyl-1,10-phenanthroline)ruthenium(II), Ru(DIP)3(2+), and tris(4,7-diphenyl 1,10-phenanthroline)cobalt(III), Co(DIP)3(3+), in general, bind stereoselectively to DNA helices, with enantiomers possessing the delta configuration bound preferentially by right-handed B-DNA. In the presence of EcoRI, however, this enantioselectivity is altered. The chiral intercalators, at micromolar concentrations, inhibit the reaction of EcoRI, but for each enantiomeric pair it is the lambda enantiomer, which binds only poorly to a B-DNA helix, that inhibits EcoRI preferentially. Kinetic studies in the presence of lambda-Ru(DIP)3(2+) indicate that the enzyme inhibition occurs as a result of the lambda enantiomer binding to the enzyme-DNA complex as well as to the free enzyme. Furthermore, photolytic strand cleavage experiments using Co(DIP)3(3+) indicate that the metal complex interacts directly at the protein-bound DNA site. Increasing concentrations of bound EcoRI stimulate photoactivated cleavage of the DNA helix by lambda-Co(DIP)3(3+), until a protein concentration is reached where specific DNA recognition sites are saturated with enzyme. Thus, although lambda Co(DIP)3(3+) does not bind closely to the DNA in the absence of enzyme, specific binding of EcoRI appears to alter the DNA structure so as to permit the close association of the lambda isomer to the DNA helix. Mapping experiments demonstrate that this association leads to photocleavage of DNA by the cobalt complex at or very close to the EcoRI recognition site.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011081 TI - Amino acid sequence of human histidine-rich glycoprotein derived from the nucleotide sequence of its cDNA. AB - A lambda gt 11 library containing cDNA inserts prepared from human liver mRNA has been screened with an affinity-purified antibody to human histidine-rich glycoprotein (HRG) and then with a restriction fragment isolated from the 5' end of the largest cDNA insert obtained by antibody screening. A number of positive clones were identified and shown to code for HRG by DNA sequence analysis. A total of 2067 nucleotides were determined by sequencing 3 overlapping cDNA clones, which included 121 nucleotides of 5'-noncoding sequence, 54 nucleotides coding for a leader sequence of 18 amino acids, 1521 nucleotides coding for the mature protein of 507 amino acids, a stop codon of TAA, and 352 nucleotides of 3' noncoding sequence followed by a poly(A) tail of 16 nucleotides. The length of the noncoding sequence of the 3' end differed in several clones, but each contained a polyadenylylation or processing sequence of AATAAA followed by a poly(A) tail. More than half of the amino acid sequence of HRG consisted of five different types of internal repeats. Within the last 3 internal repeats (type V), there were 12 tandem repetitions of a 5 amino acid segment with a consensus sequence of Gly-His-His-Pro-His. This repeated portion, referred to as a "histidine-rich region", contained 53% histidine and showed a high degree of similarity to a histidine-rich region of high molecular weight kininogen. PMID- 3011082 TI - Proliferation dependence of topoisomerase II mediated drug action. AB - Topoisomerase II mediated DNA scission induced by both a nonintercalating agent [4'-demethylepipodophyllotoxin 4-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP 16)] and an intercalator [4'-(9-acridinylamino) methanesulfon-m-anisidide (m AMSA)] was studied as a function of proliferation in Chinese hamster ovary (CHO), HeLa, and mouse leukemia L1210 cell lines. Log-phase CHO cells exhibited dose dependent drug-induced DNA breaks, while plateau cells were found to be resistant to the effects of VP-16 and m-AMSA. Neither decreased viability nor altered drug uptake accounted for the drug resistance of these confluent cells. In contrast to CHO cells, plateau-phase HeLa and L1210 cells remained sensitive to VP-16 and m AMSA. Recovery of drug sensitivity by plateau-phase CHO cells was found to reach a maximum approximately 18 h after these cells regained exponential growth and was independent of DNA synthesis. DNA strand break frequency correlated with cytotoxicity in CHO cells; log cells demonstrated an inverse log linear relationship between drug dose (or DNA damage) and colony survival, whereas plateau-derived colony survival was virtually unaffected by increasing drug dose. Topoisomerase II activity, whether determined by decatenation of kinetoplast DNA, by cleavage of pBR322 DNA, or by precipitation of the DNA-topoisomerase II complex, was uniformly severalfold greater in log-phase CHO cells compared to plateau-phase cells. PMID- 3011084 TI - Detection, characterization, and quenching of the intrinsic fluorescence of bovine heart cytochrome c oxidase. AB - The intrinsic fluorescence of lauryl maltoside solubilized bovine heart cytochrome c oxidase has been determined to arise from tryptophan residues of the oxidase complex. The magnitude of the fluorescence is approximately 34% of that from n-acetyltryptophanamide (NATA). This level of fluorescence is consistent with an average heme to tryptophan distance of 30 A. The majority of the fluorescent tryptophan residues are in a hydrophobic environment as indicated by the fluorescence emission maximum at 328 nm and the differing effectiveness of the quenching agents: Cs+, I-, and acrylamide. Cesium was ineffective up to a concentration of 0.7 M, whereas quenching by the other surface quenching agent, iodide, was complex. Below 0.2 M, KI was ineffective whereas between 0.2 and 0.7 M 15% of the tryptophan fluorescence was found to be accessible to iodide. This pattern indicates that protein structural changes were induced by iodide and may be related to the chaotropic character of KI. Acrylamide was moderately effective as a quenching agent of the oxidase fluorescence with a Stern-Volmer constant of 2 M-1 compared with acrylamide quenching of NATA and the water-soluble enzyme aldolase having Stern-Volmer constants of 12 M-1 and 0.3 M-1, respectively. There was no effect of cytochrome c on the tryptophan emission intensity from cytochrome c oxidase under conditions where the two proteins form a tight, 1:1 complex, implying that the tryptophan residues near the cytochrome c binding site are already quenched by energy transfer to the homes of the oxidase. The lauryl maltoside concentration used to solubilize the enzyme did not affect the fluorescence of NATA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011083 TI - Iron-depleted reaction centers from Rhodopseudomonas sphaeroides R-26.1: characterization and reconstitution with Fe2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+. AB - Reaction centers (RCs) from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26.1 were depleted of Fe by a simple procedure involving reversible dissociation of the H subunit. The resulting intact Fe-depleted RCs contained 0.1 0.2 Fe per RC as determined from atomic absorption and electron paramagnetic resonance (EPR) spectroscopy. Fe-depleted RCs that have no metal ion occupying the Fe site differed from native RCs in the following respects: (1) the rate of electron transfer from QA- to QB exhibited nonexponential kinetics with the majority of RCs having a rate constant slower by only a factor of approximately 2, (2) the efficiency of light-induced charge separation (DQA----D+QA-) produced by a saturating flash decreased to 63%, and (3) QA appeared readily reducible to QA2-. Various divalent metal ions were subsequently incorporated into the Fe site. The electron transfer characteristics of Fe-depleted RCs reconstituted with Fe2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+ were essentially the same as those of native RCs. These results demonstrate that neither Fe2+ nor any divalent metal ion is required for rapid electron transfer from QA- to QB. However, the presence of a metal ion in the Fe site is necessary to establish the characteristic, native, electron-transfer properties of QA. The lack of a dominant role of Fe2+ or other divalent metals in the observed rate of electron transfer from QA- to QB suggests that a rate-limiting step (for example, a protonation event or a light induced structural change) precedes electron transfer. PMID- 3011085 TI - Human papillomaviruses and cancer. PMID- 3011086 TI - Cellular and viral fos genes: structure, regulation of expression and biological properties of their encoded products. PMID- 3011087 TI - Immunochemical evidence for an inactive form of cytochrome oxidase in mitochondrial membranes of ethanol-fed rats. AB - Previous studies have established that rats fed ethanol chronically exhibit a 50% decrease in hepatic mitochondrial cytochrome oxidase compared to pair-fed controls, based on both heme aa3 content and specific activity. To determine whether the 'missing' 50% of cytochrome oxidase is present in the membrane but catalytically inactive, or entirely absent, we used immunochemical techniques to determine the content of cytochrome oxidase protein in hepatic submitochondrial particles. Rabbit antiserum against purified rat liver cytochrome oxidase precipitated cytochrome oxidase from detergent-solubilized submitochondrial particles. Immunoinhibition titrations of a fixed amount of anti-oxidase serum with increasing amounts of submitochondrial particle protein showed that similar percentages of added oxidase activity were recovered in supernatants after immunoprecipitation with preparations from both alcoholic and control rats. Similarly, titrations of a fixed amount of submitochondrial particle protein with increasing amounts of antiserum showed comparable decreases in oxidase activity. Equivalent amounts of protein were obtained in immunoprecipitates from both preparations. Immunoprecipitates demonstrated comparable oxidase subunit profiles by electrophoresis, except that one additional band, migrating in the region of oxidase subunit IV, was present in samples from alcoholic rats. The data indicate that cytochrome oxidase immunologic reactivity is quantitatively similar in both types of membranes. The results suggest that the 'missing' cytochrome oxidase is actually present within the membranes of alcoholic animals in an inactive form, apparently devoid of heme aa3. PMID- 3011088 TI - The effects of pH and ionic strength on cytochrome c oxidase steady-state kinetics reveal a catalytic and a non-catalytic interaction domain for cytochrome c. AB - The influence of pH and ionic strength on the steady-state kinetics of purified bovine cytochrome c oxidase was studied by spectrophotometry. At low ionic strength, increasing the pH in the range between 5.4 and 8.6 resulted in a slight decrease in maximal turnover numbers of the high-affinity and the low-affinity reactions. The high-affinity Km was also found to decrease with increasing pH. The ionic-strength dependence of the steady-state kinetics of positively charged cytochrome c oxidase at pH 6.2 and that of negatively charged cytochrome c oxidase at pH 7.8 were similar; in both cases, high-affinity Km values and high affinity and low-affinity TNmax values increased with ionic strength. The low affinity Km was independent of both pH and ionic strength. Above I = 100 mM, no low-affinity reaction could be observed. A description of the electrostatic interactions between cytochrome c and cytochrome c oxidase, based on the overall monopoles and overall dipoles of the two proteins, could not explain our data. We propose that at I greater than or equal to 25 mM such an approximation cannot be used for electrostatic interactions between large proteins, since the assumption that all charges on the surfaces of the reacting proteins would contribute equally to the electrostatic interaction is not valid. A qualitative description of electrostatic interactions between the two cytochromes based on limited electrostatic interaction domains on the cytochrome c oxidase surface was found to be in good agreement with all our data and supports the model of Speck et al. (Speck, S.H., Dye, D. and Margoliash, E. (1984) Proc. Natl. Acad. Sci. USA 81, 347-351), who proposed one catalytic and one non-catalytic cytochrome c binding site. It is proposed that the allosteric effect of the cytochrome c at the non catalytic site is of an electrostatic nature. At high ionic strength (occurring in vivo), this cytochrome c molecule would then no longer affect the catalytic site, resulting in the absence of the low-affinity reaction. PMID- 3011089 TI - Iron-histidine stretching Raman line and enzymic activities of bovine and bacterial cytochrome c oxidases. AB - Resonance Raman spectra of the reduced form of cytochrome c oxidase isolated from bovine heart and the thermophilic bacterium PS3 were investigated in relation to their H+-pumping- and cytochrome-c-oxidizing activities, which were varied by incubating the enzyme at raised temperatures or at alkaline pH at room temperature. For both the bovine and PS3 enzymes, the intensity of the iron histidine stretching Raman line of the ferrous a3 heme (214 cm-1) exhibited an incubation-temperature-dependent change, which fell between the similar curves of the H+-pumping and cytochrome-c-oxidizing activities. The intensities of the formyl CH=O stretching Raman line of the ferrous a3 heme (1665 cm-1) as well as of other lines were insensitive to the heat treatment. The iron-histidine stretching Raman line of both enzymes showed pH-dependent intensity change which was nearly parallel with the pH dependence of cytochrome-c-oxidizing activity. Therefore, deprotonation affecting the 214 cm-1 Raman line is responsible for the decrease of activity. This limited alkaline treatment to the PS3 enzyme was reversible and the recovered enzyme exhibited Raman intensities and enzymic activities similar to the native one. However, the neutralized, bovine enzyme with a similar intensity of the 214 cm-1 line showed increased cytochrome-c oxidizing activity and null H+-pumping activity. PMID- 3011090 TI - The interaction of saccharides with lipid bilayer vesicles: stabilization during freeze-thawing and freeze-drying. AB - The fusion of small unilamellar vesicles of phosphatidylcholines during freeze thawing and freeze-drying/rehydration, and the suppression of fusion under these conditions by various saccharides, was investigated by gel filtration on Sepharose 4B, quasielastic light scattering, high-resolution 1H-NMR, ESR spin labeling, and differential scanning calorimetry. Freeze-thawing and freeze-drying of aqueous small unilamellar vesicle suspensions in the presence of sufficient sucrose had no significant effect on the average size and size distribution of small unilamellar vesicles. In the presence of sucrose the structural integrity and the permeability properties of the phosphatidylcholine bilayers were retained during freeze-thawing and freeze-drying. A comparison of the stabilizing effect of sucrose with that of trehalose and glucose showed that the stabilization is not sugar-specific but is a general property of saccharides. The fraction of small unilamellar vesicles recovered after freeze-thawing depended on the saccharide/phosphatidylcholine molar ratio. The mechanism of the cryoprotective effect involves binding of the sugar to the phospholipid polar group, probably through hydrogen bonding. PMID- 3011091 TI - Inside-out basolateral plasma membrane vesicles from rat kidney proximal tubular cells. AB - A method for preparation of highly purified basolateral plasma membranes from rat kidney proximal tubular cells is reported. These membranes were assayed for the presence of vesicles as well as for their orientation. (Na+ + K+)-ATPase activity and [3H]ouabain binding studies with membranes treated with or without SDS revealed that the preparation consisted of almost 100% vesicles. The percentage of inside-out vesicles was found to be approx. 70%. This percentage was determined measuring the (Na+ + K+)-ATPase activity in K+-loaded vesicles and in membranes treated with or without trypsin and SDS. These membranes represent a very efficient tool to assay the correlation between active transport and ATPase activities in basolateral plasma membranes from rat kidney proximal tubular cells. PMID- 3011092 TI - Na+-ATPase is a different entity from the (Na+ + K+)-ATPase in rat kidney basolateral plasma membranes. AB - In this work, we present evidence in agreement with the hypothesis that there exist two Na+-stimulated ATPase activities in basolateral plasma membranes from rat kidney proximal tubular cells: (1) (Na+ + K+)-ATPase activity, which is inhibited by ouabain and by treating the membranes with trypsin, is insensitive to furosemide and reaches maximal activity upon treatment with SDS at an SDS/protein ratio of 1.6; (2) the Na+-ATPase activity, which is insensitive to ouabain and to trypsin treatment, is inhibited by furosemide and reaches maximal activity upon treatment with SDS at an SDS/protein ratio of 0.4. PMID- 3011093 TI - The activation of phosphatase activity of the Ca2+-ATPase from human red cell membranes by calmodulin, ATP and partial proteolysis. AB - Depending on the assay conditions, the ability of the Ca2+-ATPase from intact human red cell membranes to catalyze the hydrolysis of p-nitrophenylphosphate is elicited by either calmodulin or ATP. The response of the phosphatase activity to p-nitrophenylphosphate, ATP, Mg2+ and K+ is the same for the activities elicited by ATP or by calmodulin, suggesting that a single process is responsible for both activities. In media with calmodulin, high-affinity activation is followed by high-affinity inhibition of the phosphatase by Ca2+ so that the activity becomes negligible above 30 microM Ca2+. Under these conditions, addition of ATP leads to a large decrease in the apparent affinity for inhibition by Ca2+. In membranes submitted to partial proteolysis with trypsin, neither calmodulin nor Ca2+ are needed and phosphatase activity is maximal in media without Ca2+. This is the first report of an activity sustained by the Ca2+-ATPase of red cell membranes in the absence of Ca2+. Under these conditions, however, ATP still protects against high-affinity inhibition by Ca2+. These results strongly suggest that during activation by calmodulin, Ca2+ is needed only to form the calmodulin-Ca2+ complex which is the effective cofactor. Protection by ATP of the inhibitory effects of Ca2+ and the induction of phosphatase activity by ATP + Ca2+ suggests that activation of the phosphatase by Ca2+ in media with ATP requires the combination of the cation at sites in the ATPase. Results can be rationalized assuming that E2, the conformer of the Ca2+-ATPase, is endowed with phosphatase activity. Under this assumption, either the calmodulin-Ca2+ complex or partial proteolysis would elicit phosphatase activity by displacing the equilibrium between E1 and E2 towards E2. On the other hand, ATP + Ca2+ would elicit the activity by establishing through a phosphorylation-dephosphorylation cycle a steady-state in which E2 predominates over other conformers of the ATPase. PMID- 3011094 TI - Preferential association of apocytochrome c with negatively charged phospholipids in mixed model membranes. AB - The mitochondrial precursor protein, apocytochrome c, binds to model membranes containing negatively charged phospholipids (Rietveld, A., Sijens, R., Verkleij, A.J. and Kruijff, B. (1983) EMBO J. 2, 907-913). In the present paper the effect of apocytochrome c on the lipid distribution in model membranes, consisting of neutral and acidic phospholipids, is examined. Both ESR and fluorescence energy transfer experiments show that the protein preferentially interacts with the negatively charged phospholipid in the mixed model membranes. Semi-quantitative analysis of the fluorescence energy transfer from the single tryptophan in apocytochrome c to the parinaric acid in phosphatidylserine or phosphatidylcholine in mixed bovine brain phosphatidylserine/egg phosphatidylcholine vesicles reveals and average donor-acceptor distance of 22-26 A and 26-30 A for phosphatidylserine and phosphatidylcholine, respectively. In addition, these experiments demonstrate that this preferential interaction does not induce the separation of large domains enriched in complexes of apocytochrome c with negatively charged phospholipids and domains enriched in neutral lipids. PMID- 3011095 TI - Relationship between DNA acid-solubility and frequency of single-strand breaks near apurinic sites. AB - Using [32P]DNA alkylated with [3H]methyl methanesulfonate, depurinated by heating at 50 degrees C for various periods, then treated with sodium hydroxide, a table was constructed giving the DNA fraction soluble in 5% perchloric acid at 0 degree C as a function of the frequency of strand breaks. The alkaline treatment placed a break near each apurinic site; the apurinic sites were counted in two ways which gave consonant results: by the loss of [3H]methyl groups and by reaction with [14C]methoxyamine. The 32P label of DNA was used to measure the acid solubility. PMID- 3011097 TI - Perturbation of Pseudomonas cytochrome oxidase by guanidine hydrochloride to detect differential stabilization of the heme d1 and heme c moieties. AB - The optical properties of Pseudomonas cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.2) were monitored as a function of guanidine hydrochloride (Gdn X HCl) concentration to probe for differential stabilization of its prosthetic groups, heme d1 and heme c. The protein fluorescence intensity increased with the Gdn X HCl concentration, revealing two transitions, a sharp one between 1.3 and 1.5 M Gdn X HCl, and a second less well defined extending from 2.5 to 4.5 M. Only the transition at the lower Gdn X HCl concentrations was present in titrations followed using the emission maxima. The spectral maximum for native Pseudomonas cytochrome oxidase was at approx. 335 nm and shifted to approx. 350 nm above 2 M Gdn X HCl. The heme d1 absorbance at 638 nm decreased with increasing [Gdn X HCl], giving a transition at 1.3-1.5 M, and no transition up to 4 M Gdn X HCl when the heme c was monitored at 525 nm. Along with the decrease at 638 nm, an absorption band appeared at 681 nm, suggesting heme d1 release into solution. Fluorescence titration of heme d1-depleted enzyme, prepared by gel filtration, showed a single transition similar to the transition occurring in the intact enzyme at high Gdn X HCl concentrations. Circular dichroism spectra revealed clearly distinguishable transitions for the heme d1 and heme c near 1.5 and 3.0 M Gdn X HCl, respectively. These results suggest that the two hemes are in regions of the protein with different stabilities which may represent distinct structural domains. PMID- 3011098 TI - Reversible inactivation of ribonucleases by ADPribosylation. AB - The activity of purified bovine seminal RNAase and pancreatic RNAase A (EC 3.1.27.5) has been investigated following in vitro ADPribosylation in the presence of nuclear ADPribosyltransferase (EC 2.4.2.30) and NAD+ X ADPribosylation of these enzymes was correlated with a significant decrease in their activities. Approximately three residues of ADPribose were present per mol of enzyme. Removal of the bound ADPribose restored enzyme activity to near normal levels. Similar results were obtained with nuclei isolated from bull seminal vesicles as an endogenous source of seminal RNAase and nuclear ADPribosyltransferase. The findings suggest that in vitro ADPribosylation has a reversible inactivating effect on ribonucleases. PMID- 3011096 TI - The effect of the trp repressor from Escherichia coli on the structure and dynamics of dA20dT20. AB - We have determined the effect of the tryptophan (trp) repressor from Escherichia coli on the structure and dynamics of dA20dT20. The structure was determined using time-dependent nuclear Overhauser effects and spin-lattice relaxation times. The deoxyribose conformation is near C3' endo for the thymine residues, and a mixture of about 30% C3' endo and 70% C2' endo for the adenine residues. The glycosidic torsion angles are -50 degrees for T and -60 degrees for A. The roll is 20 degrees and the propellor twist is about 29 degrees. The conformation is consistent with recent calculations (Rao, K. and Kollman, P.A. (1985) J. Am. Chem. Soc. 107, 1507-1511). The rate constant for exchange of the imino protons is similar to that usually found for AT base-pairs, with an activation energy of 20 +/- 2 kcal/mol, and an activation entropy of 17 +/- 7 cal/mol per K. The repressor greatly retards the exchange of imino protons, and the activation energy increases to 38 kcal/mol. There are small changes in the structure of the DNA on forming the complex, with the adenine and thymidine residues becoming more similar in conformation. PMID- 3011099 TI - Hydrolysis of cyclic nucleotides by a purified cGMP-stimulated phosphodiesterase: structural requirements for hydrolysis. AB - Hydrolysis of cyclic AMP and cyclic GMP analogues by a purified cGMP-stimulated phosphodiesterase from bovine adrenal tissue was investigated by reversed-phase HPLC. The results indicate that both a negative charge and an equatorial oxygen atom located at the cyclic phosphate residue are absolute requirements for the process of hydrolysis. Other substituents only gradually decreased the apparent hydrolytic activity. C-8-substituted derivatives were generally poor substrates due to the limited ability of these compounds to rotate freely around the glycosidic bond. While C-6- and 0-2'-substituted analogues carrying bulky substituents were also poorly hydrolysed, all other derivatives, including different C-2-, C-6-, 0-3'- and 0-5'-modified cyclic nucleotides, were good substrates. We consistently observed that cyclic GMP and cyclic GMP analogues were better hydrolysed than the corresponding cyclic AMP analogues. Hydrolysis was correlated with neither the hydrogen bond donor/acceptor abilities nor the hydrophobicity of selected cyclic nucleotide analogues. Based on quantum-chemical calculations of the size and direction of the dipole moments of different purine bases, we propose that the polarization of inducible amino acid side-chains within the binding site is involved in the differential binding of adenine derived and guanine-derived nucleotides. However, the size of the dipole moment alone is not sufficient to explain the observed cGMP-preference. Rather, the direction of the polarization power relative to the other molecular structures involved in binding and hydrolysis seems to be the molecular mechanism by which the enzyme is able to discriminate between cAMP- and cGMP-like structures. PMID- 3011100 TI - Proton and nitrogen-15 NMR studies of ferricytochrome c cyanide complexes: remarkable conservation of the heme environment among organisms of diverse origin. AB - The native ferric and cyanide-bound ferric forms of nine vertebrate and two yeast cytochromes c have been investigated by high-resolution proton nuclear magnetic resonance spectroscopy. Spectral comparisons have been made among the cytochromes with emphasis on the signal positions for heme and amino acid ligand protons. Consistent with earlier more limited studies of native ferric cytochromes c, the paramagnetically shifted proton NMR signals show little variation among species with up to 50% substitution of amino acids. Proton NMR spectra for the cyanide complexes also show little variation among species. The nitrogen-15 signal for the coordinated cyanide ion is known to be highly variable among other hemoproteins, but the signal covers a range of only 855 to 865 ppm (nitrate ion reference) for vertebrate cytochromes c and 884 to 886 ppm for yeast cytochromes c. The cyanide ligand probe thus reports an amazing conservation of the heme and proximal ligand environment among the cytochromes. Comparative proton and nitrogen-15 chemical shift values are consistent with a slightly stronger proximal histidine imidazole hydrogen bond to an amino acid carbonyl function than is the case for hemoglobin and myoglobin. PMID- 3011101 TI - A comparison of the reversibility of phosphoethanolamine transferase and phosphocholine transferase in rat brain microsomes. AB - The reversibility of phosphoethanolamine transferase (EC 2.7.8.1) in rat brain is demonstrated in this paper. Microsomal ethanolamine glycerophospholipids were prelabeled with an intracerebral injection of [3H]ethanolamine 4 h before killing young rats. Labeled CDPethanolamine was produced by incubation of the microsomes with CMP, although to a lesser extent than for the previously observed release of CDPcholine. Ethanolamine and choline glycerophospholipids were labeled with [2 3H]glycerol by incubation with primary cultures of rat brain. Microsomes from rat brains, with diisopropyl phosphofluoridate for inhibition of lipases, were incubated with the labeled glycerophospholipids separately, and labeled diacylglycerols were produced. The kinetic parameters of phosphoethanolamine transferase and phosphocholine transferase (EC 2.7.8.2) were compared by incubating rat brain microsomes with [3H]CMP. Inclusion of AMP in the reaction mixture was necessary in order to inhibit the hydrolysis of CMP by an enzyme with the properties of 5'-nucleotidase (EC 3.1.3.5). For phosphoethanolamine transferase and phosphocholine transferase respectively, the Km values for CMP were 40 and 125 microM and the V values were 2.3 and 21.6 nmol/h per mg protein. The reversibility of both enzymes permits the interconversion of the diacylglycerol moieties of choline and ethanolamine glycerophospholipids. During brain ischemia, a principal pathway for degradation of ethanolamine glycerophospholipids may be by reversal of phosphoethanolamine transferase followed by hydrolysis of diacylglycerols by the lipase. PMID- 3011102 TI - Phospholipid-transfer proteins in human liver and primary liver carcinoma. AB - Investigations have been carried out on phospholipid-transfer activity of the cytosol and the phospholipid composition of subcellular membranes from human liver and primary liver carcinoma. In both human liver and primary liver carcinoma cytosolic fractions, the transfer activity for phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin has been observed for the first time. The transfer rate of PC and PE in normal human liver was almost equal, whereas sphingomyelin-transfer activity was much slower. In carcinoma cells, the transfer activity for PE and PC was significantly enhanced, while sphingomyelin transfer remained unchanged. Comparative investigations with HepG2 cultured cells have revealed a high PE-transfer activity in this cell line. Parallel with the phospholipid-transfer activity modifications in neoplasic cells, changes in the phospholipid composition of microsomes and mitochondria have been observed. The content of PC and PE in hepatocarcinoma cells was decreased in microsomes, while in the mitochondria it was increased. The possible role of the phospholipid-transfer proteins in the maintenance of membrane composition and structure is discussed. PMID- 3011103 TI - Regulation of lipid peroxidation by ATP synthetase substrates in rat liver mitochondria. AB - The addition of cumene hydroperoxide to succinate-energized mitochondria has been shown to result only in an insignificant acceleration of lipid peroxidation. Phosphate accelerates this process, while ADP reverses the phosphate effect. The phosphate and ADP effects are revealed in the mitochondrial matrix. N Ethylmaleimide, the phosphate transport inhibitor, taken at low concentrations, prevents the phosphate effects; accordingly, carboxyatractyloside, the nucleotide transport inhibitor, prevents the ADP effects. The addition of an uncoupler to the energized mitochondria has no effect on the induction of lipid peroxidation by cumene hydroperoxide in the presence of phosphate and does not reverse the ADP effect. PMID- 3011104 TI - Relationship between the displacement of phosphatidate phosphohydrolase from the membrane-associated compartment by chlorpromazine and the inhibition of the synthesis of triacylglycerol and phosphatidylcholine in rat hepatocytes. AB - Glycerolipid synthesis was studied in isolated hepatocytes by using 177 microM [14C]oleate and 1 mM [3H]glycerol. Chlorpromazine (25-400 microM) inhibited the synthesis of phosphatidylcholine and triacylglycerol. This was accompanied by an average increase of 12-fold in the accumulation of the labelled precursors in phosphatidate at 200 microM chlorpromazine and a decrease in the conversion of phosphatidate to diacylglycerol of 76%. These results indicate that part of the inhibition of the synthesis of phosphatidylcholine and triacylglycerol occurs at the level of phosphatidate phosphohydrolase. The relative rate of triacylglycerol synthesis at different concentrations of chlorpromazine was approximately proportional to the rate of conversion of phosphatidate to diacylglycerol. Phosphatidylcholine synthesis increased at higher rates of conversion of phosphatidate to diacylglycerol, but it was relatively independent of the latter rate when this was inhibited by more than about 30% with chlorpromazine. The addition of oleate to the hepatocytes caused a translocation of phosphatidate phosphohydrolase from the cytosol to the membrane-associated compartment. Chlorpromazine had the opposite effect and displaced the phosphohydrolase from the membranes in the presence or absence of oleate. There was a highly significant correlation between the activity of phosphatidate phosphohydrolase that was associated with the membranes of the hepatocytes and the calculated conversion of [3H]phosphatidate to diacylglycerol. Chlorpromazine also antagonized the association of the phosphohydrolase with microsomal membranes when cell-free preparations were incubated with combinations of oleate and spermine. Furthermore, it inhibited the transfer of the soluble phosphohydrolase to microsomal membranes that were labelled with [14C]phosphatidate and thereby decreased diacylglycerol production. It is concluded that part of the action of chlorpromazine in inhibiting the synthesis of triacylglycerol and phosphatidylcholine occurs because it prevents the interaction of the soluble phosphatidate phosphohydrolase with the membranes on which glycerolipid synthesis occurs. This in turn prevents the conversion of phosphatidate to diacylglycerol. PMID- 3011105 TI - Incorporation of [1-14C]arachidonic and [1-14C]eicosapentaenoic acids into the phospholipids of peripheral blood neutrophils from the plaice, Pleuronectes platessa L. AB - The incorporation of [1-14C]arachidonic acid and [1-14C]eicosapentaenoic acids into phospholipids was studied in peripheral blood neutrophils from plaice, a marine fish whose lipids are rich in (n-3) polyunsaturated fatty acids. The incorporation of both labelled fatty acids into phospholipids was approximately equal when presented individually, and at equal concentration, to the cells. Arachidonic acid was relatively preferred when both acids were present in the incubations. When incorporation was expressed per unit mass of phospholipid class, the order of incorporation was PI greater than PC greater than PE greater than PS greater than sphingomyelin for both fatty acids. However, the specificity for incorporation into PI was significantly greater with arachidonic acid. Eicosapentaenoic acid was incorporated into PC to a greater extent than arachidonic acid. The results are discussed in relation to the possible roles of arachidonic acid and eicosapentaenoic acid in polyunsaturated fatty acid metabolism in plaice neutrophils. PMID- 3011106 TI - Pertussis toxin effects on adenylate cyclase activity, cyclic AMP accumulation and lipolysis in adipocytes from hypothyroid, euthyroid and hyperthyroid rats. AB - Adipocytes from hypothyroid rats have a decreased responsiveness to agents that activate adenylate cyclase, whereas cells from hyperthyroid rats have an increased responsiveness as compared to the controls. This is reflected in cyclic AMP accumulation as well as lipolysis. Administration of pertussis toxin to rats or its in vitro addition to adipocytes increased basal lipolysis and cyclic AMP accumulation as well as the response to norepinephrine or forskolin. The effects of thyroid status was not abolished by toxin treatment. Pertussis toxin-catalyzed ADP ribosylation of Ni was increased in adipocyte membranes from hypothyroid rats as compared to those from euthyroid rats. However, no change in sensitivity to N6 (phenylisopropyl)adenosine was observed. The data suggest that the amount of Ni might not be rate-limiting for the inhibitory action of adenosine. A consistent decrease in maximal lipolysis was observed in freshly isolated adipocytes from hypothyroid animals as compared to those from the controls. Such defective maximal lipolysis was not corrected by adenosine deaminase or in vivo administration of pertussis toxin. The relationship between cyclic AMP levels and lipolysis suggests that in fat cells from hypothyroid rats either the cyclic AMP dependent protein kinase or the lipase activity itself may limit maximal lipolysis. There appears to be multiple effects of thyroid status on lipolysis involving factors other than those affecting adenylate cyclase activation. PMID- 3011107 TI - The C-terminus of type I collagen is a major binding site for heparin. AB - The binding of collagens and fragments of type I collagen to heparin was studied by gel electrophoresis and affinity chromatography. Samples bound in 150 mM NaCl/10 mM Hepes (pH 6.5) were eluted with 2 M NaCl, 6 M urea, or a linear gradient of 0.15-1.0 M NaCl. The triple-helical conformation was shown to be essential for binding. The vertebrate collagenase-generated C-terminal fragment, TCB, was shown to have greater binding affinity for heparin than the N-terminal TCA fragment. Both type II collagen and the NC1 domain of type IV collagen bound to heparin, whereas pepsin-solubilized tetrameric type IV failed to bind. PMID- 3011108 TI - Ca2+ and phorbol ester activation of protein kinase C at intracellular Ca2+ concentrations and the effect of TMB-8. AB - A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from human polymorphonuclear leukocytes and shown to be identical to bovine protein kinase C. The Ca2+ activation of the enzyme was studied and the Ca2+ concentrations required to activate the enzyme were compared to free cytosolic Ca2+ concentrations in resting and activated polymorphonuclear leukocytes. The free calcium concentrations in the cytosol and in the enzyme assay mixture were determined using the calcium indicator quin 2. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range, but was not activated by phosphatidylserine at Ca2+ concentrations corresponding to the intracellular free Ca2+ concentration under resting conditions. However, at similar Ca2+ concentrations (less than 2.5 X 10(-7) M) the enzyme was highly activated by phorbol 12-myristate 13-acetate (PMA) or diolein in the presence of phosphatidylserine. It was demonstrated that PMA stimulation of human polymorphonuclear leukocytes did not induce any increase in the level of the intracellular free calcium concentration. It was concluded that PMA activation of protein kinase C occurred independently of a rise in the intracellular Ca2+ concentration. K0.5 (half-maximal activation) for the PMA activation of purified protein kinase C was shown to be equivalent to the K0.5 for PMA stimulation of superoxide (O-2) production in human polymorphonuclear leukocytes, suggesting that protein kinase C is involved in activation of the NADPH oxidase. The presumed intracellular Ca2+ antagonist TMB-8 inhibited the PMA-induced superoxide production, but neither by an intracellular Ca2+ antagonism nor by a direct inhibition of protein kinase C activity. PMID- 3011109 TI - Interaction of heme nonapeptide derived from cytochrome c with microsomal reductases. AB - The interaction of heme nonapeptide (a proteolytic product of cytochrome c) with purified NADH:cytochrome b5 (EC 1.6.2.2) and NADPH:cytochrome P-450 (EC 1.6.2.4) reductases was investigated. In the presence of heme nonapeptide, NADH or NADPH were enzymatically oxidized to NAD+ and NADP+, respectively. NAD(P)H consumption was coupled to oxygen uptake in both enzyme reactions. In the presence of carbon monoxide the spectrum of a carboxyheme complex was observed during NAD(P)H oxidation, indicating the existence of a transient ferroheme peptide. NAD(P)H oxidation could be partially inhibited by cyanide, superoxide dismutase and catalase. Superoxide and peroxide ions (generated by enzymic xanthine oxidation) only oxidized NAD(P)H in the presence of heme nonapeptide. Oxidation of NAD(P)H was more rapid with O2- than O2-2. We suggest that a ferroheme-O2 and various heme-oxy radical complexes (mainly ferroheme-O-2 complex) play a crucial role in NAD(P)H oxidation. PMID- 3011110 TI - Hypertonic glycerol induces a respiratory burst in leukocytes. AB - Exposure to hypertonic glycerol induced cyanide-insensitive oxygen consumption and formation of superoxide anion (O-2) in leukocytes such as porcine blood polymorphonuclear leukocytes, guinea pig peritoneal leukocytes and guinea pig alveolar macrophages. Generation of O-2 occurred after a short lag time, remained maximal for a certain time and then stopped. Its termination was not due to cell damage, since cells exposed to glycerol did not release cytosolic enzymes such as lactate dehydrogenase and exhibited a subsequent respiratory burst upon addition of other stimulators such as myristic acid and phorbol myristate acetate. The period of O-2 generation increased linearly as a function of the glycerol concentration; cells exposed to 20% (v/v) glycerol produced O-2 for 10 min. The maximal velocity of O-2 generation also increased with the concentration of glycerol, reaching a plateau at 10% glycerol. Membrane vesicles isolated from the cells exposed to 20% glycerol showed high activity of NADPH-dependent O-2 generation as compared to those of unexposed cells. Activation of leukocytes by glycerol was not accompanied by degranulation, unlike stimulation by phagocytosis. A marked change in shape of the cell membrane of glycerol-treated cells was observed by light and scanning electron microscopies. PMID- 3011111 TI - Reaction of superoxide anions with melanins: electron spin resonance and spin trapping studies. AB - Scavenging of superoxide radicals by melanin is a possible factor in the photoprotection afforded by melanin pigments. The reaction between superoxide anions and melanins has been studied by electron spin resonance and spin trapping methods. It was found that superoxide anions react to produce melanin free radicals in a reaction inhibited by superoxide dismutase but not by catalase. The rate of radical formation depends on the concentration of melanin and superoxide, the pH of the medium and the presence of diamagnetic metal ions. The melanin pigment competes with the enzyme superoxide dismutase for removal of superoxide radicals. It was found that the xanthine-xanthine oxidase system is not suitable for studying the reaction of superoxide with melanin, as the enzymatic activity of xanthine oxidase is considerably inhibited by melanin. PMID- 3011113 TI - Sequential changes in brown adipose tissue composition, cytochrome oxidase activity and GDP binding throughout pregnancy and lactation in the rat. AB - The sequential appearance of changes in interscapular brown adipose tissue composition, cytochrome oxidase activity and GDP binding was studied throughout pregnancy and lactation in the rat. Brown adipose tissue was hypertrophied during pregnancy because of progressive lipid accumulation, whereas its mitochondrial component and GDP binding to brown fat mitochondria were unchanged. In early lactation (day 5) there was a decrease in the overall GDP binding to brown fat only because of the lower mitochondrial protein content. In late stages of lactation (days 10 and 15), the amount of tissue and its mitochondrial protein content were minimal and the GDP binding per mitochondrial protein decreased substantially. Scatchard analysis in day-15-lactating rats indicated a large decrease in GDP binding sites without any changes in affinity. It is concluded that the diminished thermogenic activity of brown fat in lactation is attained through changes at different structural levels of the tissue occurring in a characteristic sequential trend; first a reduction in its mitochondrial component, and only later, at mid-lactation, a decrease in the specific mitochondrial proton conductance pathway activity. PMID- 3011114 TI - Differential composition of cytosol 5'-nucleotidases between T and B lymphoblasts. AB - WI-L2 cells (a B-lymphoblastoid cell line) were more resistant than CEM cells (a T-lymphoblastoid cell line) to deoxyadenosine, ara-A (9-beta-D arabinofuranosyladenine), or ara-C (1-beta-D-arabinofuranosylcytosine) inhibition. This was caused by a difference in the composition of cytosol 5' nucleotidases between WI-L2 and CEM cells. In intact cells, the endogenous production of deoxyadenosine from WI-L2 cells deficient in adenosine kinase (EC 2.7.1.20) and deoxycytidine kinase (EC 2.7.1.74) was consistently high, despite changes in endogenous adenosine production. Endogenous production of deoxyadenosine from CEM cells deficient in adenosine kinase and deoxycytidine kinase was, however, coordinated with endogenous adenosine production. In broken cells, cytosol dAMPase (2'-deoxyadenosine 5'-monophosphate 5'-nucleotidase) activity of WI-L2 cells was 3-5-fold higher than that of CEM cells. dAMPase activity could be separated from ATP-activated IMPase (inosine 5'-monophosphate 5'-nucleotidase) by gel filtration (molecular weight: dAMPase; 39,000-46,000; ATP activated IMPase, greater than 150,000). Cytosol ATP-activated IMPase and dAMPase were isolated by phosphocellulose or DEAE-Bio-Gel A chromatography from non specific phosphatases. The ATP-activated IMPase showed only marginal activity towards dAMP (2'-deoxyadenosine 5'-monophosphate), ara-AMP (9-beta-D arabinofuranosyladenine 5'-monophosphate), or ara-CMP (cytosine-beta-D arabinofuranoside 5'-monophosphate), even in the presence of ATP. The activity of ATP-activated IMPase was similar in WI-L2 and CEM cells. dAMPase was separated into two peaks by DEAE-Bio-Gel A chromatography; one of these peaks degraded ara AMP and ara-CMP. The activities of both peaks from WI-L2 cells were higher than those from CEM cells. These results show that the degradation of dAMP, ara-AMP or ara-CMP was more specific and rapid in WI-L2 than in CEM cells. PMID- 3011112 TI - Combined use of 1H-NMR and GC-MS for metabolite monitoring and in vivo 1H-NMR assignments. AB - Thirty-three metabolites were observed in perchloric acid extracts of four different tissues by in vitro 1H-NMR, GC-MS and alcohol dehydrogenase assay, and the information was used to interpret an in vivo two-dimensional nuclear Overhauser effect 1H-NMR spectrum. The metabolite profiles of the different tissues indicate a number of potential tissue-specific markers: N-acetylaspartate and gamma-aminobutyric acid for rat brain, glutamine/glutamic acid ratio for dog heart, arginine and sucrose for carrot, and t-aconitate, sucrose, asparagine/aspartic acid concentration ratios for corn roots. gamma-Aminobutyric acid and malate can be regarded as metabolic indicators for stressed corn roots. Concentrations of threonine and valine in corn roots were constant under hypoxic and salt stress, and can serve as internal standards for both in vivo and in vitro NMR studies. The in vitro information was further used to identify 12 compounds from the in vivo 1H-NMR spectra (including the two-dimensional nuclear Overhauser effect spectrum) of a carrot cylinder by correlating the chemical shift and nuclear Overhauser effect information. Thus, our choice of methods with a capability for structural determination allows the characterization of complex tissue extracts with minimum sample preparation, and supports, as well as complements, in vivo 1H-NMR investigations of metabolism. PMID- 3011115 TI - Modification of transmembrane electron transport activity in plasma membranes of simian virus 40 transformed pineal cells. AB - Changes have been found in the plasma membrane enzyme system which carries out transmembrane electron transport and associated proton transport in Simian virus 40 (SV40) temperature-sensitive A (tsA) mutant-transformed rat pineal cell line, RPN209-1. This cell line was temperature-sensitive for the maintenance of transformation. RPN209-1 cells expressed the transformed phenotype (rapid growth, high cell density, and cloning in soft agar) at the permissive temperature (33 degrees C) and the nontransformed phenotype (slower growth, lower saturation density, and lower cloning efficiency in soft agar) at the nonpermissive temperature (40 degrees C). The reduction of external ferricyanide, hexaammine ruthenium and diferric transferrin was used to measure the transmembrane redox activity. The transformed RPN209-1 cells expressed a lower transmembrane redox activity, which is more sensitive to the antitumor drug adriamycin, when compared to the cells with a nontransformed phenotype. The lower transmembrane redox activity is associated with a decrease in the affinity for ferricyanide and a change in Vmax of the enzyme. Since the transformed cells have 25% lower concentration of NADH, the decrease in Vmax may be partly based on substrate limitation. Ionic strength variation in the assay media shows that the change in activity with transformation is not based on change in cell-surface change. Treatment with neuraminidase, however, indicates that sialic acid is important for enzyme activity, consistent with previous proposals that the transmembrane enzyme is a glycoprotein. The proton extrusion associated with transplasma membrane electron transport is increased in transformed cells relative to the rate of ferricyanide reduction. A relation between proton pumping transplasma membrane electron transport and growth stimulation by external oxidants is discussed. PMID- 3011116 TI - Cyclosporin A depolarizes cytoplasmic membrane potential and interacts with Ca2+ ionophores. AB - Cytoplasmic membrane potential of mouse lymphocytes was determined with flow cytometry and fluorescence spectroscopy using 3,3'-dihexylcarbocyanine iodide (DiOC6(3)). The amount of this lipophilic cation incorporated into the cytoplasmic membrane is dependent upon the transmembrane potential, so the dye is suitable for continuous monitoring of this parameter, under controlled conditions. Membrane potential of the cells was decreased in the presence of cyclosporin A and cyclosporin G in a dose-dependent manner. However, the depolarization caused by Ca2+ ionophores, ionomycin and A23187, was reduced in the presence of cyclosporin A. Electron spin resonance spectroscopy with 5 doxylstearic acid as a probe indicated that cyclosporin A decreased the apparent motional freedom of membrane lipids. These data suggest incorporation of cyclosporin A into the cytoplasmic membrane, causing changes in ion fluxes. The membrane potential change induced by cyclosporin A may have selective biological consequences in certain subpopulations of lymphocytes. PMID- 3011117 TI - Metabolism of S-adenosyl-L-methionine in intact human erythrocytes. AB - Freshly isolated human erythrocytes contain S-adenosyl-L-methionine (AdoMet) at a concentration of about 3.5 mumol/l cells. When such cells are incubated in a medium containing 30 microM L-methionine, 18 mM D-glucose and 118 mM sodium phosphate (pH 7.4), intracellular AdoMet levels continuously decrease to a value of about 0.1 microM after 24 h. This occurs in spite of the fact that the cellular concentrations of the substrates for the AdoMet synthetase reaction, ATP and L-methionine, remain relatively constant. In a search for incubation conditions that lead to stable levels of AdoMet in incubated cells, we have developed a sodium-Hepes-buffered medium which includes 1 mM adenine and a stoichiometric excess of MgCl2 over its ligand, phosphate. The inclusion of magnesium ion (and a reduction in phosphate) appears to increase intracellular free Mg2+, which is required for full activity of the erythrocyte AdoMet synthetase. Even in the presence of MgCl2, however, the AdoMet pool level can drop 4-6-fold within the first 2 h of incubation. We present evidence that suggests that this initial fall in the cellular AdoMet level may be due to the activation of AdoMet-dependent protein carboxyl methyltransferase, an enzyme which accounts for a large fraction of the total cellular AdoMet utilization. Adenine, or related compounds in the medium may prevent this activation, although the mechanism of this action is not clear at present. PMID- 3011118 TI - The influence of pH and membrane potential on passive Na+ and K+ fluxes in human red blood cells. AB - Passive (ouabain-insensitive) Na+ and K+ effluxes from human red blood cells were measured over the range pHo 6.2-8.5. On raising pHo, Na+ efflux increased and this was mainly attributable to the piretanide-sensitive component: K+ efflux likewise but attributable to both piretanide-sensitive and piretanide-insensitive components. On replacing Cl- with non-penetrating anions (mainly gluconate), Na+ and K+ effluxes increased, mostly attributable to the piretanide-insensitive components. On restoring pHi either by reducing pHo or by applying DIDS, the influence of pHo on Na+ and K+ effluxes was diminished. These results suggest that pHi rather than Em is the dominant influence. Passive Na+ and K+ effluxes and influxes in the presence of bumetanide were tested fro conformity to the Ussing independence relationship. For K+, the calculated and observed ratios agreed, indicating that the sodium pump, 'cotransport' and leak wholly account for K+ fluxes in human red blood cells. For Na+, the ratios did not agree and a 1:1 Na+/Na+ exchange did not account for the discrepancy. Pathways for Na+ appear to be more numerous than for K+. PMID- 3011119 TI - Cyclic AMP inhibition of phosphoinositide turnover in human neutrophils. AB - The effect of increased intracellular levels of cyclic AMP on phosphoinositide metabolism was studied in human neutrophils stimulated with fMet-Leu-Phe. Intracellular cyclic AMP was raised by preincubation either with dibutyryl cyclic AMP and theophylline or with prostaglandin E1. Concentrations of dibutyryl cyclic AMP and theophylline fully inhibitory for the metabolic responses inhibited phosphoinositide breakdown and phosphatidic acid formation to a large extent. The accumulation of the water-soluble inositol phosphates was also measured. In agreement with the data obtained on the phospholipids, inositol phosphate generation was found to be severely, though not completely, reduced. Treatment with dibutyryl cyclic AMP and theophylline also inhibited resynthesis of membrane inositol lipids. Treatment with prostaglandin E1 had a similar, though less, marked effect on inositol lipid turnover, which was parallel with a smaller inhibition of metabolic responses. We therefore suggest that the elevation of intracellular cyclic AMP mainly affects neutrophil responses by inhibiting the phosphoinositide cycle. PMID- 3011120 TI - Induction of corticosteroid biosynthetic pathway by ACTH and high-density lipoprotein in newborn rat adrenocortical cells cultured in serum-free medium. AB - In order to investigate the role of rat high-density lipoprotein (HDL) on adrenal cholesterol accumulation and steroidogenic pathways (corticosteroid, i.e., 21 hydroxysteroid biosynthesis and reductive metabolism of progesterone), newborn rat adrenal cells cultured in serum-free medium were used. Incubation of [4 14C]cholesterol-HDL in serum-free medium compared to those in medium with lipoprotein-deficient serum, in serum-free medium with ACTH compared to those without ACTH, both showed an increase of labelled cholesterol in cells and of labelled 21-hydroxysteroids excreted in medium. Substitution of serum supplemented medium by serum-free and cholesterol-free medium led to a deep decrease of ACTH-induced steroid biosynthesis with a predominance of 20 alpha reduced steroids; addition of HDL restored the corticosteroid biosynthesis and decreased the reductive metabolism. Addition of increased concentrations of HDL (7-150 micrograms cholesterol/ml) enhanced, in a saturable fashion, the total cholesterol uptake and the corticosteroid biosynthesis. The total cholesterol accumulation in cells exceeded by 4-fold the steroid production at saturation. The ratio between the two steroidogenic pathways increased up to 40 at saturation in favor of corticosteroids. These results suggest that HDL is at least partly internalized and that probably its constituents contribute greatly to the control of the two different steroidogenic pathways. PMID- 3011121 TI - (Thr-59)-insulin-like growth factor I stimulates 2-deoxyglucose transport in BC3H1 myocytes through the insulin-like growth factor receptor, not the insulin receptor. AB - The murine non-fusing muscle cell line contains distinct receptors for insulin and insulin-like growth factors. Pretreatment of myocytes with insulin for 20 h at 37 degrees C inhibits the binding of [125I]iodoinsulin by 60% without affecting the binding of [125I]iodoinsulin-like growth factor I. The ED50 values for down-regulation of the insulin and insulin-like growth factor receptor by their respective ligands are 1 nM and 3 nM, respectively. Insulin, (Thr-59) insulin-like growth factor I and multiplication-stimulating activity stimulate 2 [3H]deoxyglucose transport in myocytes with ED50 values of 5 nM, 5.6 nM and 33 nM, respectively. In order to determine whether (Thr-59)-insulin-like growth factor I stimulates 2-[3H]deoxyglucose transport in myocytes via its own receptor or the insulin receptor, we determined the activity of these peptides after down regulation of the insulin receptor. The rate of 2-[3H]deoxyglucose transport in myocytes pretreated with insulin (5 nM) is elevated but returns to control levels by 1 h after the washout of insulin. The dose-response curve for insulin stimulated 2-[3H]deoxyglucose transport is shifted to the right (ED50 greater than 100 nM) immediately after insulin washout but is normal by 1 h after insulin washout. In contrast, the dose-response curve for (Thr-59)-insulin-like growth factor I is unchanged in insulin-pretreated cells immediately after insulin washout. These data show that (Thr-59)-insulin-like growth factor I stimulates 2 [3H]deoxyglucose transport in myocytes by acting through an insulin-like growth factor receptor and not through the insulin receptor. Since multiplication stimulating activity is 6-fold less active than (Thr-59)-insulin-like growth factor, they both may be acting through a type 1 insulin-like growth factor receptor. PMID- 3011122 TI - Increased protein ADPribosylation in HeLa cells exposed to the anti-cancer drug methotrexate. AB - DNA single-strand breaks (about 200-300 per genome) were transiently detected during the first hour when HeLa cells were incubated for up to 24 h with 100 microM methotrexate. There was an expected increase in ADPribosyltransferase activity, which reached a maximum 2-3-fold stimulation at 3 h but which was still greater than in control cells after 24 h. When hypoxanthine (25 microM) was present in the incubations together with the methotrexate the transferase was no longer activated, although basal, control levels of activity were still present. DNA strand breaks were reduced in number but were still just detectable under these conditions. Cellular NAD+ levels were mostly unaffected by the various drug treatments, except for a small transient decrease after 1 h, possibly as a result of the transferase activation. Methotrexate did not cause an increase in the rate of ADPribose degradation. Degradation of ADPribose residues labelled in a preincubation period in permeabilized cells was more extensive at pH 6.0 was a 50% loss of acid-insoluble radioactivity in 30 min at 26 degrees C. At pH 8.0 the loss did not exceed 30-35% even after 90 min incubation. The activation of the transferase is reflected in a general increase in protein ADPribosylation detected by autoradiography of 32P-labelled proteins in 6.25-18.25%T gradient acrylamide gels. There were three major acceptors with molecular masses of 17, 100 and over 100 kDa, which could be respectively a histone, a transferase derived peptide fragment and the transferase itself. When ADPribosyltransferase was inhibited with 3-amino-benzamide DNA single-strand breaks were no longer detected. However, this had no observably signficant effect on the kinetics of loss of cell viability (from Trypan blue uptake), cell number or colony-forming ability. Similar results are observed in most cases when the activation of the transferase, resulting from the incubation of cells with methotrexate, is inhibited by hypoxanthine. We conclude from such observations that the enhanced protein ADPribosylation seen in the cells exposed to methotrexate is a direct consequence of drug-exposure, but does not have any significant influence over the course of events leading ultimately to cell death. PMID- 3011123 TI - Phorbol esters, vasopressin and angiotensin II block alpha 1-adrenergic action in rat hepatocytes. Possible role of protein kinase C. AB - The effect of vasopressin, angiotensin II and phorbol myristate acetate on the alpha 1-adrenergic action (induced by epinephrine + propranolol), was studied. We selected three conditions: (a) ureagenesis in medium without added calcium and containing 25 microM EGTA; (b) ureagenesis using cells from hypothyroid animals, and (c) gluconeogenesis from dihydroxyacetone. Under these conditions epinephrine + propranolol produces clear metabolic effects, whereas the vasopressor peptides do not (although they stimulate phosphoinositide turnover). It was observed that the vasopressor peptides and the active phorbol ester inhibited in a concentration-dependent fashion the effect of epinephrine + propranolol. It is suggested that activation of protein kinase C by phorbol esters or physiological stimuli (hormones that activate phosphoinositide turnover, such as vasopressin or angiotensin II) modulate the hepatocyte alpha 1-adrenergic responsiveness. PMID- 3011125 TI - [Biosynthesis of fibronectin by human embryonal fibroblasts transformed by SV-40 virus]. AB - Fibronectin biosynthesis by human embryonic fibroblasts transformed with virus SV 40 was studied in intact cells and in a cell-free protein synthesizing system on free and membrane-bound polyribosomes isolated from these cells. It was found that fibronectin release from transformed fibroblasts into the culturing medium was decreased 4.5-fold, while its per cent content--2-fold. The amount of fibronectin precipitated by antibodies in the course of an immunoprecipitation reaction in transformed cells appeared to be somewhat higher than in normal cells, although when expressed on a per cent basis this content was decreased only 1.5-fold. However, the content of fibronectin monomer with Mr = 220 kD exceeded that in normal fibroblast cell material 1.6 times. Study on fibronectin biosynthesis in a cell-free system revealed that in transformed cells 45% of fibronectin is synthesized on free polyribosomes as compared to 13% in normal fibroblasts. It is assumed that the decreased fibronectin biosynthesis in human fibroblasts transformed with virus SV-40 results in spatial uncoupling of polyribosomes and membrane structures responsible for protein transport from the cell, as a result of which a significant part of fibronectin synthesized by transformed fibroblasts undergoes intracellular degradation. PMID- 3011124 TI - Homologous and heterologous desensitization of one of the pathways of the alpha 1 adrenergic action. Effects of epinephrine, vasopressin, angiotensin II and phorbol 12-myristate 13-acetate. AB - Activation of protein kinase C blocks the alpha 1-adrenergic action in hepatocytes. Preincubation of hepatocytes (in buffer with or without calcium) with vasopressin, angiotensin II, phorbol myristate acetate (PMA) or epinephrine + propranolol markedly diminished the alpha 1-adrenergic responsiveness of the cells (stimulation of ureagenesis) assayed in buffer without calcium. On the contrary, when the alpha 1-adrenergic responsiveness was assayed in buffer containing calcium no effect of the preincubation with vasopressin, angiotensin II or PMA was observed. Preincubation with epinephrine diminished the alpha 1 adrenergic responsiveness of the cells. In hepatocytes from hypothyroid rats the preincubation with the activators of protein kinase C (vasopressin, angiotensin II, phorbol 12-myristate 13-acetate and epinephrine) reduced markedly the alpha 1 adrenergic responsiveness of the cells, whereas in identical experiments using cells from adrenalectomized rats only the preincubation with epinephrine diminished the responsiveness. It is concluded that activation of protein kinase C induces desensitization of the alpha 1-adrenergic action in hepatocytes and that the calcium-independent pathway of the alpha 1-adrenergic action (predominant in cells from hypothyroid animals) resensitizes more slowly than the calcium-dependent pathway (predominant in cells from adrenalectomized rats). Epinephrine in addition to inducing this type of desensitization (through protein kinase C) leads to a further refractoriness of the cells towards alpha 1 adrenergic agonists. PMID- 3011126 TI - [The role of calmodulin (delta-subunit) in the activation of phosphorylase kinase from rabbit skeletal muscles]. AB - A comparative study on the structure of nonactivated and activated forms of phosphorylase kinase was carried out. The enzyme was activated by incubation in alkaline medium (pH 8.5), by phosphorylation with cAMP-dependent protein kinase and by limited proteolysis. The comparative analysis was based on the use of hydrophobic chromatography on phenyl-sepharose and electrophoresis in polyacrylamide gel density gradient. Activation of the enzyme was accompanied by separation of a low molecular weight component (Mr about 17 000). Using chromatography on phenyl-sepharose, this low molecular weight protein was obtained in a homogeneous state. It was found that the properties of the protein are close to those of calmodulin. The presence of calmodulin in phosphorylase kinase preparations was judged upon by the activation of the calmodulin-dependent form of phosphodiesterase. The boiled and subtilisin-treated kinase activates phosphodiesterase in the same way as does bovine brain calmodulin. The experimental results suggest that the delta-subunit is a protein inhibitor of the enzyme. PMID- 3011127 TI - [Possible mechanism of the protective effect of phosphocreatine on the ischemic myocardium]. AB - The uptake of 32P-phosphocreatine by control and ischemic isolated perfused rat hearts has been studied. The rate of phosphocreatine (PCr) uptake by the hearts after 35 minutes of ischemia was two times that in control hearts at 0.5-10 mM PCr in the perfusate. At 10 mM PCr in the perfusate, this rate was 182 nmoles/min/g dry weight. The 5'-nucleotidase and phosphatase activities were found in the crude plasma membrane fraction of rat heart. The pH-dependence of these enzymes was examined. The 5'-nucleotidase activity decreased with a drop in pH from 8.0 to 6.0. The phosphatase activity in the crude plasma membrane fraction of rat heart was increased 2-fold with a decrease in pH from 8.0 to 6.0. The 5'-nucleotidase activity was inhibited by 10 mM PCr in the presence of 5 mM Mg2+. This inhibition was pH-dependent with a maximum (58%) at pH 6.0. The inhibition of phosphatase activity by PCr was independent of pH and reached 20% in the presence of 10 mM PCr. Some feasible mechanisms of the protective effect of PCr on ischemic myocardium are discussed. PMID- 3011128 TI - [Role of temperate phage in bacterial dissociation]. AB - The analysis of literary and own data testifies that the dissociants may appear in bacteria population from spontaneous mutations and transfer of genetic material (conjugation, transformation, transduction). The phage conversion and different DNA reorganizations within a cell where prophage plays an active role, probably introduce the largest contribution into the dissociative transitions of variants which occur with high frequency (about 10(-2)-10(-4). The dissociation of various bacteria has been studied with different degree. The role of temperate phage has been shown in splitting of bacteria into variants in the genera Mycobacterium, Corynebacterium, some Bacillus, Clostridium, Staphylococcus, some enterobacteria, Yersinia, Vibrio Pseudomonas, Rhizobium, Nostoc; the participation of prophage in dissociation of bacteria of the genera Xanthomonas, Erwinia, Bacteroides is proposed. A method for obtaining the nondissociating S variants for stability of biologically active substances synthesized by cells has been suggested. PMID- 3011129 TI - Human corticotropin-releasing hormone in depression--correlation with thyrotropin secretion following thyrotropin-releasing hormone. AB - Twenty-two subjects (11 patients with major endogenous depression and 11 controls) received an intravenous test dose of 100 micrograms human corticotropin releasing hormone (h-CRH). Corticotropin (ACTH), but not cortisol, responses were blunted in depressives. Basal cortisol secretion was higher in depressives than in controls and was negatively correlated to the corticotropin response following h-CRH. This finding indicates the integrity of the glucocorticoid-dependent negative feedback regulation in depression and supports the view that hypercortisolism in depression is primarily due to a suprapituitary disturbance. Comparison of ACTH responses after h-CRH with thyrotropin (TSH) output following thyrotropin-releasing hormone (TRH) revealed a positive correlation (r = 0.65, p less than 0.001). The concordance between ACTH and TSH responses after specific challenges suggests that regulation of both systems is at least in part under a common control. PMID- 3011130 TI - The benzodiazepine antagonist Ro 15-1788 does not antagonize the ethanol withdrawal syndrome. AB - It has been suggested that the signs and symptoms of the ethanol withdrawal syndrome may be due to the increased production of an "inverse agonist" that binds to the central benzodiazepine (BZ) recognition site in the brain. Ro 15 1788 (a potent antagonist at the central BZ recognition site), diazepam, and Ro 15-1788 plus diazepam were administered to groups of rats undergoing overt ethanol withdrawal. Ro 15-1788 did not alter the severity of the ethanol withdrawal reactions, but antagonized the ameliorative effect of diazepam. The results of our studies suggest that (1) the ethanol withdrawal syndrome is not produced by an endogenous ligand acting on the central BZ recognition site, and (2) diazepam decreases the severity of the ethanol withdrawal syndrome, at least in part, by its action at the central BZ recognition site. PMID- 3011131 TI - Effects of intermittent and continuous haloperidol administration on the dopaminergic system in the rat brain. AB - The after-effect of intermittent and of continuous treatment with haloperidol on the dopaminergic system of the rat brain was studied. Each rat was treated for 14 days with either a single daily intraperitoneal injection of haloperidol (intermittent haloperidol group) or with a subcutaneously implanted pump that released haloperidol for 14 days (continuous haloperidol group). The total amount of haloperidol administered was 28 mg/kg in each animal of both groups. On the seventh day after cessation of injections or removal of pumps, the changes in dopamine (DA) metabolism after a challenge dose of haloperidol (1 mg/kg, intraperitoneally) were noted, and the number of [3H]spiperone binding sites in the striatum were recorded. The continuous haloperidol group showed a greater tolerance response to the influence of haloperidol on stimulation of DA turnover and also showed a larger increase in the number of [3H]spiperone binding sites than the intermittent haloperidol group. It is concluded that continuously administered haloperidol exerts a stronger effect on DA transmission, which in turn produces a greater tolerance to an acute dose of haloperidol than intermittent haloperidol administration. PMID- 3011132 TI - Behavioral manifestations of paraneoplastic encephalopathy. PMID- 3011133 TI - Changes in the 31P-NMR spectra of cats receiving lithium chloride systemically. PMID- 3011134 TI - Cyclic AMP-dependent protein kinase isozymes of bovine epididymal spermatozoa: evidence against the existence of an ectokinase. AB - By using ethidium bromide fluorescence to measure cellular permeability and the photoaffinity probe, 8-azido-[32P] cyclic adenosine monophosphate (cAMP), to label cAMP-dependent protein kinases, washed bovine epididymal spermatozoa were examined for the presence of "ectokinases" on the sperm surface. In washed, intact spermatozoa, three proteins of Mr 49,000, 54,000, and 56,000 specifically bound 8-azido-[32P] cAMP. The Mr 49,000 protein corresponded to the type I regulatory subunit while the Mr 56,000 and 54,000 proteins comigrated with phosphorylated and dephosphorylated forms, respectively, of type IIA regulatory subunit of bovine heart. The addition of Nonidet P-40 (0.1%) increased the radioactive labeling of all three proteins and caused the appearance of a cAMP binding protein of Mr 40,000, which was likely a proteolytic fragment of the regulatory subunit. Although these data could support the concept of a surface location for regulatory subunits in spermatozoa, it was necessary to determine if the appearance of cAMP binding sites was correlated with the loss of membrane integrity. A population of washed epididymal spermatozoa appeared to contain 10 20% damaged cells based on ethidium bromide fluorescence. The same population of cells also had 10-20% of the regulatory subunits of the cAMP-dependent protein kinase accessible to labeling with the cyclic AMP photoaffinity probe. When spermatozoa were sonicated for increasing lengths of time, ethidium bromide fluorescence was found to be related directly to the relative amount of regulatory subunit labeling by the probe. It is suggested that the major apparent cAMP-dependent "ectokinases" in sperm represent artifacts resulting from cellular damage. PMID- 3011135 TI - Micropuncture studies of receptor-mediated endocytosis of transferrin in the rat epididymis. AB - The concentration of transferrin in fluids collected by micropuncture techniques from the rete testis and zones 1A, 2, 5A, and 6B of the rat ductus epididymidis was 44, 527, 113, and 49 ng/microliter, respectively. When changes in fluid volume were taken into account, it was found that transferrin was concentrated by the efferent ducts, whereas the amount of endogenous luminal transferrin declined from the caput to the cauda epididymidis. Using transferrin-gold as an electron dense marker, we showed that the decline in the concentration of transferrin along the epididymis could be attributed to its cumulative receptor-mediated endocytosis by the lining principal cells. No significant difference in the net receptor-mediated endocytosis of transferrin-gold was found between the proximal caput and corpus epididymidis in vivo. PMID- 3011136 TI - Identification of calpain II in porcine sperm. AB - The role that proteolytic enzymes may play in membrane-associated phenomena of sperm has been the subject of extensive investigation. In the present study, we have examined the possibility that a Ca2+-activated, neutral protease, calpain II, may be associated with sperm membranes. Using indirect immunofluorescence with primary antibodies, which are polyclonal and monoclonal antibodies directed against the 80 kDa subunit of calpain II, we have established the presence of this antigen in porcine sperm. Staining by anticalpain II (80 kDa subunit) of the apical segment of the acrosomal cap and basal body (centriolar) region was seen consistently. Variable staining of the sperm tail was also observed. These observations, combined with our positive identification of a 80 kDa protein in acrosomal membranes (via immunoblot), document the association of this protease with sperm membranes. The proximity of calpain II to the acrosome suggests a potential role for the protease in the Ca2+-mediation of the acrosome reaction. PMID- 3011137 TI - Tissue reactions to calcined bone graft. AB - Calcined bone blocks made by heating raw bovine bone at 1200 degrees C were implanted into defects in canine femoral condyles. X-ray diffraction patterns showed that the calcined bone was well-crystallized hydroxyapatite. Rapid, good new bone growth was observed around the calcined bone which was gradually absorbed along its surface and also by the Haversian canals. PMID- 3011139 TI - Structural analysis of hydroxyapatite coatings on titanium. AB - Hydroxyapatite from two sources was electrophoretically deposited onto flat titanium plate material. Depending upon the deposition conditions various changes in the structure of the ceramic were identified. A well-adhering Ti-P compound was present at the interface. Hydroxyapatite oxygenated to various degrees and tetracalcium phosphate were reproducibly formed in the coating. PMID- 3011138 TI - Macropore tissue ingrowth: a quantitative and qualitative study on hydroxyapatite ceramic. AB - The aim of this study was to obtain more information about macropore tissue ingrowth into the pores of sintered hydroxyapatite implanted in the rat middle ear, for the assessment of the usefulness of this material in reconstructive middle-ear surgery. The exudate filing the pores during the early post-operative period was gradually replaced by equal amounts of fibrous tissue and bone. The percentage of the macropore area occupied by bone was directly correlated with the macropore size. Bone was deposited not only from the pore wall towards the pore centre, but also in the opposite direction. Bonding osteogenesis was demonstrated. At sites of mechanical irritation, the presence of multinucleated cells and proliferatively active mononuclear phagocytes persisted for as long as a year. Under appropriate conditions hydroxyapatite seems to be a promising material for bone substitution in reconstructive middle-ear surgery. PMID- 3011140 TI - Sequence-dependent recognition of DNA duplexes: netropsin complexation to the TATA site of the d(G-G-T-A-T-A-C-C) duplex in aqueous solution. PMID- 3011141 TI - Papillomaviruses and their involvement in oncogenesis. AB - Papillomaviruses have been identified as the causative agents of benign epithelial proliferation in many animals including man. Recent evidence has shown that each viral type will only infect a specific species and tissue. Furthermore, certain papillomavirus types have been found associated with lesions capable of malignant conversion, particularly in response to secondary physical or chemical factors. Many advances have been made in identifying particular papillomavirus types inducing papillomas capable of progression and in the identification of the virally encoded functions involved in viral replication and cellular transformation. PMID- 3011142 TI - Management of irritable bowel syndrome. AB - Few drugs are of proven efficacy in irritable bowel syndrome. Bulking agents probably relieve constipation, spasmolytics may alleviate pain and antidiarrhoeals help control urgency and diarrhoea. With a combination of reassurance and therapeutic intervention up to 75% of patients can be expected to improve. For the 25% who do not, alternative therapies such as stress management, psychotherapy or hypnotherapy may prove effective. PMID- 3011143 TI - [The role of ACTH(5-8) and beta-MSH(5-8) fragments in the organization of the self-stimulation reaction]. AB - The experiments on rats have shown that intraperitoneal administration of ACTH5-8 fragments in a dose of 40 ng per kg altered considerably the character of self stimulation reaction and the behaviour of rats. Searching activity and self stimulation reaction were intensified, with the latter characterized by the onset of aversive components, that disappeared 24 hours later. Activation depended on the site of stimulation. Two phases of activity were noted (the first 0.5-1 h and the second 4.5-6 h after ACTH5-8 injection). beta-MSH5-8 fragment, when injected intraperitoneally in a dose of 20 ng per kg, had no effect on self-stimulation reaction and the behaviour of animals. PMID- 3011145 TI - [Arterio-venous differences in the vascular and thrombocytic indices of the hemostasis system]. AB - Arterio-venous difference in the functional activity of platelets and vascular wall was studied in Wistar rats. It was found that platelets, unlike leukocytes, were functionally more active in arterial than in venous blood. Athrombogenic activity of arteries was higher than that of veins. PMID- 3011144 TI - [The role of the cardiac-pulmonary blood volume in the changes in left ventricle output during stimulation of the afferent fibers of somatic nerves]. AB - Stimulation of tibial nerve afferent fibers has revealed heterogeneous shifts of left ventricular output, as well as pulmonary artery and posterior vena cava blood flow in anesthetized cats. Uniform changes in left ventricular output and pulmonary artery blood flow were noted in the majority of cases, with venous return most often exceeding pulmonary artery blood flow. beta-adrenoreceptor blockade failed to influence changes in pulmonary artery blood flow. It is concluded that the increase in pulmonary artery blood flow depends on the rise in venous return, but not on neurogenic influence upon the right ventricle. The reduction in left ventricular output is the result of decreased right ventricular outflow due to its overload caused by pulmonary vasoconstriction. PMID- 3011146 TI - [Mutagenesis and cloning of hly determinants of the paP20 plasmid]. AB - The data obtained indicate that pAP20 plasmid determinants for the synthesis and excretion of Escherichia coli haemolysin are located on the plasmid SalI-fragment f3. The size of f3 fragment is 12 X 10(6) dalton. PMID- 3011148 TI - Clinical and phenotypic diversity of T cell lymphomas. AB - Forty-one cases of T cell lymphoma were identified on the basis of morphology and the expression of two or more T cell antigens with an absence of B cell markers. Mycosis fungoides and lymphoblastic lymphoma were excluded. Marked clinical, morphological, and immunologic diversity was observed. Cutaneous lymphoma was found in approximately 50% of the patient group, and 27% had a prior history of dermatologic or immunologic disease. No correlations among immunologic and morphologic phenotypes and clinical course were apparent. Survival data was comparable to that of a concurrent group of non-T cell lymphoma patients studied at this institution, suggesting that, contrary to previous reports, T cell lymphoma may not necessarily confer a more unfavorable prognosis. Prospective studies using uniform treatments are necessary to address the clinical significance of the T cell phenotype definitively, independent of established histologic and clinical features. PMID- 3011147 TI - Defective platelet-fibrinogen interaction in hereditary canine thrombopathia. AB - A unique, intrinsic, hereditary canine platelet disorder attributable to abnormal fibrinogen receptor availability is described. Thrombopathic platelets from 13 severely affected basset hounds failed to aggregate in response to all agonists tested except thrombin. Normal platelet interaction with the various stimuli was inferred on the basis of their ability to elicit unimpaired shape change in thrombopathic platelets. No quantitative differences in major platelet membrane glycoproteins, intraplatelet fibrinogen, adenine nucleotides, or serotonin uptake were detected. Dense granule secretion was impaired. The ultrastructural appearance of thrombopathic platelets was normal. Fibrinogen-platelet interaction was evaluated by reacting platelet-rich plasma (PRP) with fibrinogen coupled to polymeric acrylonitrile beads and scoring the extent of stimulus-induced agglutination. The aggregatory responses of normal and thrombopathic platelets were closely correlated with fibrinogen receptor availability. In contrast to human platelets, epinephrine-stimulated canine platelets did not interact with immobilized fibrinogen, and arachidonate generally induced only weak agglutination. Thrombopathic platelets agglutinated fibrinogen beads at reduced rates when stimulated with physiologic doses of thrombin and high-dose calcium ionophore, A23187. Our data suggest that thrombin-mediated induction of canine platelet fibrinogen receptors may proceed by pathway(s) alternate to those shared by other platelet agonists, and/or that secreted granule constituents may act synergistically with thrombin to overcome inhibition of signal-response-coupled reactions mediating the interaction of fibrinogen with its receptor. This congenital platelet defect provides further evidence, in a species other than human, for the pivotal role of fibrinogen receptor induction in platelet aggregation. PMID- 3011149 TI - T cell-derived migration-inhibitory factor and colony-stimulating factor share common structural elements. AB - Migration-inhibitory factor (MIF) is a lymphokine that acts to localize mononuclear phagocytes (monocytes and macrophages) and perhaps to activate them. Mo cells are a human T cell leukemia virus II-infected T cell line previously shown to secrete large quantities of MIF upon stimulation with phytohemagglutinin and phorbol myristate acetate. MIF was purified from Mo cell-conditioned medium by gel filtration, phenyl-Sepharose affinity chromatography, isoelectrofocusing, and reverse-phase high-performance liquid chromatography (RP-HPLC). Overall purification was 6,000-fold. The purified MIF fraction was found to display potent colony-stimulating factor (CSF) activity when assayed on human bone marrow cells. The double peak of MIF activity as shown by C 18-RP-HPLC coincided with the double peak of CSF activity. A monoclonal antibody selected for its anti-MIF activity absorbed both the CSF and the MIF activity. These findings indicate that MIF and CSF are either identical molecules or closely related molecules with common structural elements. PMID- 3011151 TI - Absence of oncogene amplifications and occasional activation of N-ras in lymphoblastic leukemia of childhood. AB - To examine whether determination of (1) the copynumber or restriction pattern of certain oncogenes or (2) the mutational activation of the N-ras gene might contribute to the risk classification of acute lymphoblastic leukemia of childhood (ALL), we investigated DNA isolated from lymphoblasts of untreated patients. Restriction enzyme analysis of cellular oncogenes was performed on DNA of 25 patients. No rearrangements could be demonstrated within or near the genes c-myc, c-myb, c-abl, bcr, c-Ki-ras, and N-ras. No amplifications of these genes nor of N-myc or c-Ha-ras were present. Eight of 21 patients were heterozygote for "rare" Ha-ras allelic restriction fragments that have been associated with an increased risk of developing a malignancy. These patients were clinically indistinguishable from patients lacking these fragments. The breakpoint cluster region (bcr) that is rearranged in all patients with Philadelphia chromosome positive chronic myeloid leukemia, was normal in all cases, including at least one patient with Philadelphia chromosome positive ALL. A 2.8 kb HindIII fragment of a hitherto unknown gene or pseudogene related to v-myb probably derives from the Y chromosome. Nineteen patients were examined for point mutations in the N ras gene, using a novel synthetic oligonucleotide hybridization assay. In two patients activating point mutations were present, both in positions 1 of the 12th codon. Both patients were somewhat older than the others (16 and 11 years), had L2 morphology, and were shown to have high growth fractions of tumor in their bone marrow. PMID- 3011150 TI - Selective inhibition of the growth of human erythroid bursts by monoclonal antibodies against transferrin or the transferrin receptor. AB - The relative requirements of colonies derived from erythroid (BFU-E) and myeloid (CFU-c) progenitors for transferrin were examined using monoclonal antibodies directed against the transferrin molecule (TF-6) or its cell surface receptor (TFR-A12, TFR1-2B). Growth of erythroid bursts was profoundly reduced at concentrations of all three antibodies that had no effect on CFU-c-derived colonies. When TFR1-2B was layered over cultures established one to seven days previously, further burst development was inhibited, and degeneration of early erythroid colonies was observed. Addition of erythropoietin augmented transferrin receptor expression on cells harvested after 1 to 2 weeks in culture and analyzed by flow cytometry. Recombinant human erythropoietin gave results comparable to those obtained in experiments using human urinary erythropoietin. Analysis of erythroblasts plucked directly from culture plates confirmed the presence of transferrin receptors on BFU-E-derived colonies. Thymidine incorporation was maximal early in the second week of culture and coincided with high transferrin receptor expression. These data demonstrate that transferrin must be available into the second week of culture to support the growth and differentiation of BFU E-derived erythroid bursts, that the generation of erythroid colonies from BFU-E is more dependent on transferrin than myeloid colony formation from CFU-c, and that erythropoietin modulates the expression of transferrin receptors on growing bursts. PMID- 3011153 TI - HTLV-III antibody status in household contacts of seropositive hemophiliacs. PMID- 3011154 TI - Calmodulin stimulation of plasmalemmal Ca2+-pump of canine aortic smooth muscle. AB - Plasma-membrane-enriched fractions of canine aortic smooth muscle possess an ATP supported Ca2+ accumulation which has an absolute requirement for Mg2+ and a high affinity for Ca2+ (Km approximately 0.5 microM). The rate of ATP-supported Ca2+ transport is not affected by several calmodulin antagonists, but is stimulated by exogenously added calmodulin. The maximal effect of calmodulin on the rate of ATP dependent Ca2+ transport (at 5.0 microM Ca2+) occurs at 10 micrograms/ml calmodulin and represents an approximately 3-fold stimulation. This calmodulin stimulation of Ca2+ transport does not require pretreatment of the membranes by EGTA and is an intrinsic property of the plasma membranes. A high-affinity Ca2+ ATPase (Km for Ca2+ approximately 0.5 microM) is also present in the aortic smooth muscle plasma membrane. This high-affinity Ca2+-ATPase does not require Mg2+ for catalytic activity, but is in fact inhibited by increasing Mg2+ concentrations. Calmodulin at concentrations effective for the stimulation of the ATP-dependent Ca2+ transport has no effect on the high-affinity Ca2+-ATPase activity or on the basal ATPase activity stimulated by 5 mM Mg2+ or Ca2+. Our results indicate that isolated plasma membranes of canine aortic smooth muscle contain no endogenous calmodulin. The ability of exogenously added calmodulin to stimulate the rate of ATP-dependent Ca2+ transport by vascular smooth muscle plasma membranes suggests that calmodulin may play a role in lowering the cytoplasmic concentration of ionized calcium during vasodilatation. An Mg2+ independent, but not an Mg2+-dependent high-affinity Ca2+-ATPase, was identified in the plasma membranes. This may be separate from the plasmalemmal Ca2+-pump. PMID- 3011152 TI - Induction of Tac antigen and proliferation of myeloid leukemic cells by ATL derived factor: comparison with other agents that promote differentiation of human myeloid or monocytic leukemic cells. AB - We studied seven cases of myeloid leukemia at various differentiation stages to investigate the response of leukemic cells to phorbol 12-myristate 13-acetate (PMA) and various biological factors. gamma-Interferon (gamma-IFN)-treated cells expressed higher amounts of Fc receptors on leukemic cells in five out of seven cases. Expression of HLA-DR antigen of gamma-IFN-treated leukemic cells was significantly enhanced in three cases. PMA did not induce Fc receptors or HLA-DR antigen on these cells. Induction of Tac antigen, a putative interleukin 2 (IL 2) receptor, was observed in two cases after cultivation with PMA or with a novel lymphokine, adult T cell leukemia-derived factor (ADF). Cells from one of these patients expressed Tac antigen immediately after cell separation, and expression of Tac antigen was augmented by PMA and ADF. Interleukin 1 (IL 1) or IL 2 did not induce Tac antigen. Leukemic cells from this patient also proliferated vigorously in the presence of ADF but not PMA, IL 1, or IL 2. PMID- 3011155 TI - Serum enzyme concentrations in untreated acute myeloid leukaemia. AB - Serum concentrations of lactate dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (HBD), phosphohexose isomerase (PHI), leucine aminopeptidase (LAP), beta-glucuronidase (beta-gluc) and angiotensin-converting enzyme (ACE), were measured in 107 cases of untreated acute myeloid leukaemia which were classified by morphological, immunological and cytochemical criteria. The results show that serum LDH was increased in most cases, irrespective of leukaemic subtype, serum PHI and LAP were significantly higher in monocytic variants and serum beta-gluc and ACE levels were generally within normal limits. In addition, significant (p less than 0.05) relationships were found between serum LDH, PHI, LAP and beta gluc concentrations and the numbers of circulating leucocytes. PMID- 3011156 TI - Cournand lecture. Expiratory partial pressure curves in the diagnosis of emphysema. AB - The deformation of expiratory partial pressure curves of respiratory and inert gases (O2, CO2, He, Ar, SF6), as measured by means of mass spectrometry, was analysed in healthy subjects and in patients with asthma, chronic bronchitis and/or pulmonary emphysema. Patients with emphysema could best be distinguished from the other groups when the comparison was based on the shape of expirogram phase II. Phase II was characterized by the volume V25-50 which was expired between 25% and 50% of the inspiratory/end-tidal partial pressure difference. Comparative measurements of V25-50 of O2, CO2, He and SF6 in 10 healthy subjects (mean age: 30.8 yr) and in 10 patients with advanced emphysema (mean age: 60.8 yr) showed a linear increase of V25-50 with increasing inspired volume (VI) being 3-5 times higher in the patients than in normals. V25-50 increased with the sequence He less than SF6 approximately equal to CO2 less than O2 in both groups. The separation between phase II volumes of He and SF6 was more pronounced in the patients with emphysema than in the healthy subjects. Variations of the breathing pattern in eight healthy subjects revealed a linear increase of V25-50 with increasing VI and, to a lower extent, with increasing respiratory frequency, while a decrease of V25-50 with increasing breath-holding time and only very small changes with variations of inspiratory as well as expiratory duration were observed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011157 TI - From stimulus-secretion coupling to stimulus-hydrosmotic response. PMID- 3011158 TI - Hydrosmotic response to vasopressin in frog skin. Control by endogenous factors. AB - The A23187 calcium ionophore strongly inhibits the hydrosmotic response to vasopressin. On the contrary neither exogenous cAMP nor theophylline-induced hydrosmotic response are affected by Ca++-ionophore. The effects obtained with A23187 suggest that calcium ions modulate vasopressin-induced osmotic water flow by interfering with a pre-cAMP step. The increase in intracellular calcium concentration induced by A23187, in fact, may inhibit the rate of cAMP formation by interfering with the vasopressin-stimulated adenylate cyclase system. This inhibition seems to be restricted to a locus proximal to the catalytic subunit of adenylate cyclase, whereas the hydrosmotic response to forskolin, a non-hormonal activator, is not affected by calcium ionophore addition. In order to clarify whether calcium ions act directly on the adenylate cyclase system or activate a more complex pathway, we studied the interaction between calcium and prostaglandins in modulating the hydrosmotic response to vasopressin. Direct measurements by radioimmunoassay of PGE2 release in the serosal medium show that A23187 notably increases PGE2 release. Furthermore, the inhibition of prostaglandin biosynthesis by hydrocortisone (a phospholipase inhibitor) or by indomethacin and naproxen (agents that inhibit arachidonic acid oxygenase) results in augmented vasopressin-stimulated water flow and prevents the inhibitory effect of the ionophore. Collectively these results strongly suggest that the effects obtained with A23187 on hydrosmotic response to ADH, are closely linked to calcium-stimulated PGE2 biosynthesis. PMID- 3011159 TI - Regulation of ADH-stimulated water flow at a post-luminal barrier in toad bladder. AB - Recent studies show that ADH-stimulated water flow across toad bladder may be regulated at a site other than the luminal membrane. In these studies luminal membrane particle aggregate frequency has been used as a measure of luminal membrane water permeability. In fully stretched bladders the relationship between total tissue permeability and aggregate frequency is curvilinear, rather than linear. This implies a resistance in series with the luminal membrane that can become rate-limiting for water flow during ADH stimulation. The possibility that transtissue water movement is actually regulated at such a post-luminal membrane resistance is suggested by the finding that within 30 min following exposure to hormone, water flow becomes attenuated without any change in aggregate frequency. Supporting this possibility, recent data from follow-up studies suggest that the apparent water permeability per luminal membrane aggregate is not reduced with time. Finally, for bladders in which prostaglandin synthesis is inhibited (by naproxen), increases in both base-line water flow and water flow consequent to treatment with a submaximal dose of ADH (0.125 mU/ml), are much less than expected from simultaneously observed changes in luminal membrane aggregate frequency. In parallel experiments to these, moreover, direct measurements of luminal membrane water permeability from the rate of change of cell volume consequent to a transluminal membrane osmotic challenge, confirm that luminal membrane water permeability increases to the extent expected from changes in aggregate frequency. All of the data taken together argue for a post-luminal membrane barrier in toad bladder which regulates tissue permeability during ADH stimulation. PMID- 3011160 TI - Variations of tetrahydrocannabinol content in cannabis plants to distinguish the fibre-type from drug-type plants. AB - There are many different species of cannabis plants, but their psychoactive properties mainly depend on the concentration of tetrahydrocannabinol (THC), which may vary according to genetic factors and environmental influences. On the basis of the THC content all cannabis plants are divided into fibre-type and drug type plants. The fibre-type plant does not exceed 0.4 per cent of THC while the drug-type plant usually contains up to 5 per cent of THC, though higher percentages (up to 10 per cent) have been reported. A study of the characteristics of cannabis seeds and the influence of environmental conditions on the content of THC in cannabis plants grown in northern, southern and insular Italy has shown that the fibre-type plants contain mean values of THC in a range from 0.058 to 0.299 per cent. The content of THC in the drug-type plants grown in Sicily and Tuscany ranged from 0.82 to 1.31 per cent +/- 0.49 per cent. In 1984, the Commission of the European Communities prepared a regulation to prevent diffusion of the drug-type cannabis, providing that raw material could not be imported if its THC content exceeded 0.5 per cent from 1984 to 1987 and after that period the maximum limit would be set up to 0.3 per cent. PMID- 3011161 TI - Experimental cultivation of cannabis plants in the Mediterranean area. AB - In research carried out in 1982, which included the cultivation of cannabis plants with low, medium and high levels of delta 9-tetrahydrocannabinol (THC), the authors have determined the parameters for individualization and classification of cannabis plants according to their intoxicant potential. This can help to provide courts of law with valid supportive expertise on cannabis trafficking cases. The parameters are the percentages of THC in cannabinoids and in the dried substance of a plant, as well as the percentage of cannabinoids in the dried substance. On the basis of these parameters, the authors have found that a cannabis plant in which the percentage of THC exceeds 50 per cent of the total amount of cannabinoids of the extractable resin and 0.3 per cent of the total amount of dried substance, and in which the amounts of resin and cannabinoids are substantial, has a considerable intoxicant potential and is liable to be used for illicit production of cannabis for abuse. On the contrary, a plant with a THC level below 50 per cent of the cannabinoids and 0.3 per cent of the dried substance, in addition to a low level of total cannabinoids, has low intoxicant potential and can be used in industry for the production of oil and rope. On the basis of these parameters it is also possible to predict the intoxicant potential of a young cannabis plant harvested at a relatively early stage of its development. PMID- 3011162 TI - The physical and chemical features of Cannabis plants grown in the United Kingdom of Great Britain and Northern Ireland from seeds of known origin--Part III: Third and fourth generation studies. AB - Two further generations of Cannabis plants have been grown from seeds produced by earlier generation plants which were raised in the United Kingdom of Great Britain and Northern Ireland in 1980 and 1981. The original seedstocks were from known countries of origin. Although there are exceptions, both the physical and chemical characteristics of the third and fourth generation plants generally resemble those of their parents. The yields of cannabis vary substantially from year to year. The total tetrahydrocannabinol contents also vary, but are comparable with the levels in the original plants. Tetrahydrocannabinolic acid continues to predominate over free tetrahydrocannabinol and is much higher than in the original plants. PMID- 3011163 TI - Use of descending thin layer chromatography for identification of cannabinoids. AB - The article describes a technique that uses descending thin layer chromatography (TLC) for identification of cannabinoids. The technique employs a partition system of two-dimensional descending TLC, in which toluene is used as the eluting solvent. The quantity of cannabinoids obtained by TLC has been confirmed by gas chromatography (GC). The technique presented in the article has proved useful for the analysis of cannabinoids. PMID- 3011164 TI - Cannabinoid content of cannabis grown on the Danish island of Bornholm. AB - The analysis in 1983 of representative samples taken from fruiting tops of seized cannabis plants illicitly grown in 19 localities on the Danish island of Bornholm showed that the average (mean) content of the total tetrahydrocannabinol (delta 9 tetrahydrocannabinol (THC) + delta 9-tetrahydrocannabinolic acid) was approximately 1.55 per cent, ranging from 0.1 to 4.2 per cent. There was no significant difference in the total THC content of cannabis plants from the Danish island of Bornholm and the content found by other authors in cannabis plants grown during the period from 1968 to 1972 in Afghanistan, India, Mexico, South Africa and Thailand. However, studies carried out on fresh cannabis seized on entry into the United Kingdom of Great Britain and Northern Ireland during the period 1975-1981 showed a larger content of THC (ranging from 2.3 to 4.9 per cent) than in cannabis plants from Bornholm. PMID- 3011165 TI - Stereospecific antiarrhythmic effect of opioid receptor antagonists in myocardial ischaemia. AB - The effects of the stereoisomers of two different antagonists at opioid receptors were examined on the ventricular arrhythmias that result from acute coronary artery ligation in anaesthetized male rats. (-)-Mr 1452(but not the (+)-isomer, Mr 1453) reduced, in a dose-dependent manner, the number of ventricular ectopic beats and the incidence or duration of both ventricular tachycardia and fibrillation. (-)-WIN 44,441-3 (but not its (+)-isomer, WIN 44,441-2) had a similar protective effect in ischaemia. These results suggest that antagonism of the effects of endogenous opioid peptides at specific receptors results in reduced severity of arrhythmias in myocardial ischaemia. PMID- 3011166 TI - Treatment of acute myocardial ischaemia with a selective antagonist of thromboxane receptors (BM 13.177). AB - In order to elucidate the role of endogenous thromboxane A2 in myocardial ischaemia, cats were subjected to 5 h of permanent occlusion of the left anterior descending coronary artery (LAD) and treated with the thromboxane receptor antagonist BM 13.177 (5 mg kg-1 h-1, i.v.). In comparison with vehicle-treated LAD-occluded cats, BM 13.177 significantly attenuated the loss of creatine phosphokinase-specific activity from the ischaemic myocardium and antagonized the ischaemia-induced rise in the ST-segment of the electrocardiogram. BM 13.177 at the dose used did not reduce plasma thromboxane levels or ischaemia-induced platelet aggregate formation but considerably antagonized thromboxane-dependent platelet secretion ex vivo. The study demonstrates some beneficial effects of selective blockade of thromboxane receptors on biochemical and electrophysiological parameters of acute myocardial ischaemia. PMID- 3011167 TI - Effects of relaxants on electrical and mechanical activities in the guinea-pig tracheal muscle. AB - In isolated tracheal muscles of the guinea-pig, effects of several relaxants were studied by simultaneously recording the membrane potential and mechanical response. Intracellular recordings showed regular slow waves in most preparations. There was a close correlation between membrane potential and slow wave amplitude. The linear regression line of the slow wave amplitude (Y mV) on the membrane potential (X mV) could be expressed by Y = -0.35X-5.9. The mean values of resting potential and slow wave amplitude were -50.6 +/- 0.6 mV and 11.9 +/- 0.5 mV, respectively. Relaxant drugs used (isoprenaline, terbutaline, adrenaline, noradrenaline, theophylline, forskolin and dibutyryl cyclic AMP) all produced hyperpolarization of the membrane and abolished the slow wave. The degree of relaxation was closely related to these electrical responses, although the recovery of electrical responses was faster than the mechanical response. It was concluded that the relaxation caused by the agents, which are known to increase the intracellular cyclic AMP level, was accompanied by a clear hyperpolarization and suppression of slow waves. PMID- 3011168 TI - Quantitative evaluation of the potencies of GABA-receptor agonists and antagonists using the rat hippocampal slice preparation. AB - CA1 population spikes recorded in the rat hippocampal slice were used to assess quantitatively the potencies of GABA-receptor agonists and antagonists on mammalian CNS neurones. Apart from GABA itself, GABA A-receptor agonists inhibited the CA1 population spikes with potencies that correlated closely (r = 0.96) with their ability to displace [3H]-GABA from GABAA-binding sites. The low potency of GABA in this preparation was attributed to the action of uptake processes as the GABA uptake inhibitor, cis-4-hydroxynipecotic acid (2 X 10(-4) M), produced an approximate 6 fold increase in the potency of GABA whilst having no effect on the potency of 4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridin-3-ol (THIP), a GABAA-receptor agonist which is not a substrate for the GABA uptake system. The inhibitory effects of the selective GABAA-receptor agonists isoguvacine and muscimol were antagonized by bicuculline methochloride, which shifted the dose-response curves to the right in a parallel manner. The Schild plots for bicuculline methochloride against isoguvacine and muscimol had slopes of 1 and gave pA2 values of 6.24 and 6.10, respectively. Picrotoxin also antagonized the inhibitory effects of isoguvacine and produced parallel shifts to the right of the dose-response curve. However, the Schild plot for picrotoxin had a slope significantly less than unity (0.82) and gave a pA2 value of 6.89. The novel GABAA-receptor antagonist, pitrazepin, antagonized the inhibitory effects of isoguvacine in an apparently competitive manner. The Schild plot had a slope of 1 and gave a pA2 of 6.69. 6 The inhibitory effects of baclofen, GABA and kojic amine were not antagonized by GABAAreceptor antagonists and were presumed to be mediated by actions at GABA5-receptors. 7 The inhibitory effects of THIP and isoguvacine were antagonized with the same potency by bicuculline methobromide. These results do not support the suggestion that THIP acts preferentially at a 'synaptic' bicuculline-sensitive, GABA receptor. 8 It is concluded that the CAI population spike in the rat hippocampal slice is a useful test system for the quantitative analysis of both GABAA- and GABAB-receptor agonists and antagonists. PMID- 3011170 TI - An oxytocin receptor in anococcygeus muscles isolated from male mice. AB - The nature of the neurohypophyseal peptide receptor in the anococcygeus muscles from male mice was investigated. The rank order of potency of naturally occurring peptides was oxytocin greater than Arg-vasotocin greater than Arg-vasopressin greater than Lys-vasopressin, which is similar to that found in the uterus and mammary gland. Selective agonists on the oxytocin (OT) receptors of the uterus and mammary gland (Thr4-OT; Gly7-OT; Thr4-Gly7-OT) were also potent agonists in the mouse anococcygeus. Competitive antagonists of uterine responses to oxytocin (dP-TyrMe-Thr4-OT; dP-TyrMe-OT; dP-Thr4-OT; dp-Orn8-OT) were also competitive antagonists of oxytocin-induced contractions of the mouse anococcygeus. It is concluded that the neurohypophyseal peptide receptor of the male mouse anococcygeus is of the oxytocin type; antagonist pA2 values suggest that this receptor resembles, but may not be identical to, the uterine oxytocin receptor. Possible physiological and pharmacological implications of these observations are discussed. PMID- 3011171 TI - Mechanism of gastric antisecretory effect of SCH 28080. AB - The mechanism of the gastric antisecretory action of SCH 28080 has been studied utilizing two different in vitro test systems, isolated and enriched parietal cells from the guinea-pig and guinea-pig gastric membranes purified and enriched with K+/H+-ATPase. In guinea-pig isolated and enriched parietal cells SCH 28080 inhibited the acid response to histamine and high K+ concentrations with IC50 values not significantly different from each other. SCH 28080 inhibited the purified K+/H+-ATPase measured in the presence of 5 mM KCl with an IC50 value of 1.3 microM. Kinetic studies indicated a competitive inhibition of ATPase by SCH 28080 with respect to K+. Studies on Na+/K+-ATPase showed that this enzyme was only slightly depressed by SCH 28080. It is concluded that SCH 28080 acts with high selectivity on the parietal cell K%/H+-ATPase, establishing its antisecretory effect by a competitive interaction with the high affinity K+-site of the gastric ATPase. PMID- 3011169 TI - Adenosine inhibits epileptiform activity arising in hippocampal area CA3. AB - The ability of adenosine and structurally-related compounds to inhibit epileptiform activity induced by bicuculline in the CA3 region of the hippocampal slice of the rat was examined. Bath application of all purinoceptor agonists tested reduced the frequency of generation of burst potentials. Analysis of dose response curves yielded the following IC50 values: adenosine, 1.5 microM; 2 chloroadenosine, 0.144 microM; 5'-(N-ethyl)carboxamidoadenosine, 30.2 nM; L phenylisopropyladenosine, 12.1 nM; cyclohexyladenosine, 7.9 nM. Theophylline (30 microM) increased the rate of bursting and antagonized the effect of exogenous adenosine. Dipyridamole (0.03-1 microM) reduced the occurrence of burst firing. In slices untreated with bicuculline, theophylline (30 microM) and adenosine deaminase (10 micrograms ml-1) induced bursting activity. These results demonstrate that purinoceptor agonists can suppress epileptiform activity in the hippocampus and suggest that adenosine may act as an endogenous anticonvulsant. PMID- 3011172 TI - Adenosine-modulation of cholinergic and non-adrenergic non-cholinergic neurotransmission in the rabbit iris sphincter. AB - The characteristics of smooth muscle responses to transmural nerve stimulation in the rabbit iris sphincter were examined. Transmural stimulation elicited a composite contractile response that could be divided in two phases. Atropine abolished the phase I contraction and inhibited the phase II contraction. The atropine-resistant component of the phase II contraction which was unaltered by sympathetic denervation, was mimicked by substance P and abolished by capsaicin. Adenosine inhibited the phase I contraction. The adenosine analogue L-N6 phenylisopropyladenosine (L-PIA) was more potent than 5'-N ethylcarboxamideadenosine (NECA) in mimicking this adenosine effect. By contrast, adenosine enhanced the phase II contraction in non-pretreated preparations, as well as the atropine-resistant capsaicin-sensitive part of this contraction. Here, NECA was more potent than L-PIA. Adenosine, NECA, L-PIA and D-PIA also enhanced the atropine-sensitive component of the phase II contraction, as well as the contractile response to exogenous acetylcholine or carbachol, but not to exogenous substance P. In this respect, L-PIA was the most powerful adenosine analogue with at least 10 fold higher potency than D-PIA. The adenosine antagonist 8-p-sulphophenyltheophylline enhanced the phase I contraction and decreased the capsaicin-sensitive non-adrenergic non-cholinergic component of the phase II contraction. We conclude that adenosine inhibited the nerve-induced cholinergic twitch (phase I) responses by action at prejunctional A1-receptors. Furthermore, adenosine enhanced the phase II contractile responses via postjunctional enhancement of the cholinergic transmission by action at A1 receptors, and via enhancement of the non-adrenergic non-cholinergic transmission by action at presumably prejunctional A2 receptors. PMID- 3011173 TI - The effect of alpha, beta-methylene ATP on the depolarization evoked by noradrenaline (gamma-adrenoceptor response) and ATP in the immature rat basilar artery. AB - Depolarizations evoked by noradrenaline that were resistant to alpha- and beta adrenoceptor antagonists were recorded in the rat basilar artery. These gamma adrenoceptor-mediated responses and the depolarizations to adenosine triphosphate (ATP) were blocked by pretreatment of the tissue with alpha, beta-methylene ATP. These data are discussed with respect to the selectivity of alpha, beta-methylene ATP. PMID- 3011174 TI - A controlled trial of continuous lumbar traction in the treatment of back pain and sciatica. AB - A controlled trial of continuous lumbar traction in the treatment of back pain and sciatica showed similar improvements in both the treated group (weighted traction) and the control group (simulated traction). The findings of this study question the justification of admitting patients with back pain into hospitals for purposes of traction alone. PMID- 3011175 TI - A phase I study of mixed (neutron and photon) irradiation using two fractions per day in the treatment of high-grade astrocytomas. AB - A Phase I study of the treatment of 50 patients with high-grade astrocytomas by mixed schedule (neutron and photon) irradiation given in 12 fractions over 4 weeks is reported. The neutron and photon fractions were separated each day by 2 3 h. A total neutron dose of 6.36 Gy (8% gamma) and 20.40 Gy of photons was prescribed. Treatment was well tolerated and there was no clinical evidence of radiation-related morbidity in the brain. The median survival was 6.9 months, similar to that expected after photon irradiation alone. A multivariate analysis of prognostic variables in presented. PMID- 3011176 TI - Prominent periportal echogenicity: its sonographic evaluation and its significance. AB - Prominent periportal echogenicity was detected during sonographic examination of patients suffering from recurrent pyogenic cholangitis, hepatocellular carcinoma and acute cholecystitis. To document the finding, 140 normal individuals were studied to establish a norm for the evaluation of the periportal echogenicity. The significance of this sonographic finding and its possible aetiology are discussed. PMID- 3011177 TI - Menetrier's disease in childhood associated with cytomegalovirus infection: a case report and review of the literature. PMID- 3011178 TI - Protection of mouse chromosomes against whole-body gamma irradiation by SH compounds. PMID- 3011179 TI - An unusual hepatic cancer. PMID- 3011180 TI - Treatment of first episodes of acute anal fissure: prospective randomised study of lignocaine ointment versus hydrocortisone ointment or warm sitz baths plus bran. AB - One hundred and three patients with an acute first episode of posterior anal fissure were randomised to receive a three week trial of lignocaine ointment (n = 33) versus hydrocortisone ointment (n = 35) or warm sitz baths combined with an intake of unprocessed bran (n =35). Seven patients were withdrawn owing to failure to adhere to the trial protocol. After one and two weeks of treatment symptomatic relief was significantly better among patients treated with sitz baths and bran than among patients treated with lignocaine ointment or hydrocortisone ointment. After three weeks there was no difference in symptomatic relief among the three groups. Patients treated with lignocaine, however, had significantly fewer healed fissures (60%) than patients treated with hydrocortisone (82.4%) or warm sitz baths and bran (87%). In this study warm sitz baths plus an intake of unprocessed bran came out as the treatment of choice for an acute first episode of posterior anal fissure. This treatment is cheap, has no potential serious side effects, and brings the best and quickest relief of symptoms. PMID- 3011181 TI - Spontaneous remission and relapse in adult T cell lymphoma/leukaemia associated with HTLV-I. PMID- 3011182 TI - HTLV-III: should testing ever be routine? PMID- 3011183 TI - Bacterial contamination of the small intestine of infants with enteropathogenic Escherichia coli and other enteric infections: a factor in the aetiology of persistent diarrhoea? AB - The duodenal microflora was studied during the first week of diarrhoea in 40 infants with acute infectious diarrhoea of different aetiologies and compared with that in a convalescent group and a group in whom diarrhoea of known aetiology had persisted for more than 14 days after an acute onset. In the acute phase 16 of the 40 infants had more than 10(4) colony forming bacteria/ml, predominantly upper respiratory commensals. In over half of the infants infected with enteropathogenic Escherichia coli a faecal type flora was found in the duodenum. This flora included the enteropathogenic E coli serotype isolated from the stool in three quarters of cases. Infants with persisting diarrhoea had significantly more faecal type bacteria in the duodenum than either those with acute diarrhoea or the convalescent group. In addition, there was a significant further increase in Enterobacteriaceae in infants whose persistent diarrhoea occurred after infection with enteropathogenic E coli. Infections with enteropathogenic E coli may have a predilection for disturbing the duodenal microflora, which may contribute to the development of persistent diarrhoea. PMID- 3011184 TI - Intrathecal synthesis of antibodies to HTLV-III in patients without AIDS or AIDS related complex. AB - De novo synthesis in the central nervous system of IgG antibodies to human T cell lymphotropic virus type III (HTLV-III) (lymphadenopathy associated virus) was shown in seven of 10 seropositive men who had syphilis but not the acquired immune deficiency syndrome (AIDS) or AIDS related complex. None of these men showed neurological symptoms when the serum and cerebrospinal fluid were collected. Pleocytosis was present in all 10. Of the seven men who showed evidence of intrathecal synthesis of antibodies, five had increased total concentrations of IgG and four had oligoclonal IgG bands in their cerebrospinal fluid. Oligoclonal bands were also present in one man who did not have any antibodies. Longitudinal study of one man showed that seroconversion preceded intrathecal synthesis of antibody specific to HTLV-III. The appearance of antibody in the cerebrospinal fluid was accompanied by a transient rise in mononuclear cell count and the appearance of oligoclonal bands. The presence of clones of B cells specific to HTLV-III in the central nervous system of these patients without persisting neurological symptoms suggests that HTLV-III enters the central nervous system in the early stages of infection. PMID- 3011186 TI - Cancer of the liver and the use of oral contraceptives. AB - A case-control study of the use of oral contraceptives was conducted among women certified as having died from cancer of the liver in the period 1979-82 and in the age range 20-44 years. An age matched group of women who died from other causes, not related to use of oral contraceptives, in the same period were used as controls. Information about use of oral contraceptives was obtained from the general practitioners' notes for both cases and controls. Information was obtained for 30 women with histologically confirmed liver cancer, 19 with hepatocellular carcinoma and 11 with cholangiocarcinoma, and for 147 controls. The results were analysed after adjusting for age at diagnosis and year of birth and showed that use of oral contraceptives was associated with a significantly (p less than 0.05) raised relative risk for hepatocellular carcinoma of 3.8 (95% confidence interval 1.0 to 14.6) and use for eight years or more was associated with a significantly (p less than 0.01) increased relative risk of 20.1 (2.3 to 175.7). There were no apparent increases in risk for cholangiocarcinoma. Despite the small number of cases in this study and the methodological problems in assessing use of oral contraceptives from general practitioners' notes, the results were consistent with other similar studies. Although in the United Kingdom primary liver cancer remains an exceptionally rare disease, especially in young women, further research on the role of oral contraceptives is needed in those countries where it is a much more common disease. PMID- 3011187 TI - Oral contraceptives and hepatocellular carcinoma. PMID- 3011185 TI - Oral contraceptives and hepatocellular carcinoma. AB - A series of 26 white women aged under 50 who developed hepatocellular carcinoma in a non-cirrhotic liver were studied for the possible role of oral contraceptives. Eighteen of the women had used the "pill" for a median of eight years. Over 1300 women whose use of the pill had been determined in another study served as controls. Patients and controls were divided into five age and four calendar groups and the relative risks associated with oral contraceptives calculated by multivariate analysis. Short term use of the pill was not associated with an increased risk of tumour development; nevertheless, use for eight years or more was associated with a 4.4-fold increased risk (p less than 0.01). When patients with markers of hepatitis B virus infection were excluded the relative risk was 7.2 (p less than 0.01). In both instances the absolute risk for developing hepatoma remained low. PMID- 3011188 TI - Peptidergic modulation of a neuromuscular junction in Aplysia: bioactivity and immunocytochemistry. AB - Three endogenous peptides were assayed for bioactivity at an Aplysia neuromuscular junction. Evoked contractions were enhanced by Phe-Met-Arg-Phe-NH2 (FMRFamide) and suppressed by arginine vasotocin; small cardioactive peptide B (SCPB) also enhanced contractions at low concentrations, but caused suppression at higher doses. In accordance with their putative roles as neuromodulators, immunocytochemistry revealed FMRFamide-like and SCPB-like fibers on the muscle surface. PMID- 3011189 TI - Inhibitory potentials in neurons of the deep layers of the in vitro neocortical slice. AB - Neocortical neurons in slices of the rat sensorimotor region maintained in vitro generate postsynaptic potentials (PSPs) in response to focal extracellular stimulation. These PSPs are mainly depolarizing at the resting membrane potential (Vm) but a sequence of depolarizing-hyperpolarizing potentials is often disclosed by depolarizing the Vm. The stimulus-induced hyperpolarization can last up to 1000 ms and show two components: the early one (peak latency 10-20 ms), is inverted by diffusion of Cl- into the cell; the late one is diminished by augmenting [K+]o. The membrane conductance is increased throughout the stimulus induced hyperpolarization, mainly during the first 10-60 ms. A decrease in excitability results from both the hyperpolarizing trend and the conductance increase. The latter is more effective in decreasing depolarizing than hyperpolarizing pulses of current injected intracellularly. PMID- 3011190 TI - Selective solubilization of physalaemin-type substance P binding sites from rat brain membranes by glycodeoxycholate and NaCl. AB - Active substance P binding sites were solubilized from rat brain membranes by treatment with 0.125% sodium glycodeoxycholate and 1 M NaCl. About 50% of the binding activity in membrane-bound binding sites was recovered in the solubilized fraction after centrifugation at 105,000 g for 1 h. [3H]Substance P absorbed extensively to glass tubes and glass filters, but the absorption was greatly reduced by siliconizing glass tubes and preincubating glass filters in a solution containing poly-D-lysine and bovine serum albumin. [3H]Substance P was found to bind the solubilized receptors in a saturable fashion with a Bmax of 145 fmol/mg protein and a Kd of 4.6 nM, and these bindings were completely replaced by low concentrations of unlabeled substance P and physalaemin. PMID- 3011191 TI - The response to viscero-peritoneal nociceptive stimuli is reduced in experimental arthritic rats. AB - Reciprocal inhibitory influences of heterotopic nociceptive stimuli have been known for centuries (counter-irritation phenomena). To assess whether such phenomena could be measured objectively in a chronic pain model, we have investigated the writhing behaviour induced by a viscero-peritoneal nociceptive stimulus (i.p. acetic acid) in arthritic rats. The latter was induced by s.c. injection of mycobacterium butyricum suspended in oil into the base of the tail and experiments carried out at various (1-9 weeks) post-inoculation periods. To assess the arthritis-induced hyperaesthesia, the threshold for struggle triggered by a calibrated pressure applied on an inflamed hindpaw (Randall-Selitto test) was measured before each experiment. A clear relationship was observed between the two parameters during the evolution of the disease. In the most severe phase (2nd to 5th week post-inoculation), when arthritis-induced hyperaesthesia was most pronounced (50% reduction of the threshold for struggle), writhing behaviour was strongly reduced (80%). At that time, writhing behaviour was enhanced by naloxone (0.4 mg/kg i.v.). In the following period (5-9 weeks), the progressive decrease of hyperaesthesia was correlated with a recovery of the writhing behaviour. We conclude that in this chronic pain model, the behavioral reaction to a viscero-peritoneal nociceptive stimulus is impaired. Endogenous opioid systems could be involved in this phenomenon. Such heterotopic inhibitory processes could be relevant to some paradoxical clinical observations such as the masking of a pain by the experience of pain at another locus. PMID- 3011192 TI - Dendritic morphology of dopaminergic cells revealed by intracellular injection of Lucifer yellow in fixed carp retina. AB - The dendritic morphology of dopaminergic cells in carp retinas was investigated by identifying their fluorescent cell bodies in isolated, aldehyde-fixed preparations and injecting them iontophoretically with Lucifer yellow CH (LY) under microscopic control. The LY-injected cells were examined in flatmount and in radial cryosections. The cell bodies were located at the inner margin of the inner nuclear layer (the amacrine cell sublayer), and gave rise to 3-5 primary dendrites that branched repeatedly within the inner plexiform layer to form a narrow-field, diffusely branched dendritic tree. In some cases, a distal process was found to extent to the outer plexiform layer, representing the interplexiform type of cells. The 59 filled cells had roughly round or oval dendritic fields covering an area of 0.102 +/- 0.003 mm2 (361 +/- 57 micron in diameter) in the intermediate retinal region. The density of dopaminergic cells in this region was 31 cells/mm2 and, therefore, the dendritic field coverage of these cells approximately 3.0. PMID- 3011194 TI - Diurnal rhythms in neurotransmitter receptor binding and choline acetyltransferase activity: different patterns in two rat lines of Wistar origin. AB - Diurnal cycles for 8 ligand receptor pairs and choline acetyltransferase (ChAT) activity in 3 brain regions differed markedly in two rat lines, both of Wistar origin. Statistically significant differences between diurnal cycles in the two rat lines were found in the following parameters: 24 h means in 6 of 11 measurements, magnitude of cycle amplitudes, and phase position in 6 of 11 measurements, up to complete reversal of the acrophases in the case of ChAT activity in hippocampus. The importance of these findings--such major differences in two closely related rat lines--is obvious in any attempt to compare receptor binding studies per se between laboratories using the same strain but not line, in studies of receptor rhythm characteristics, and in particular, for analysing the effects of brain-reactive drugs. While there are some reports on strain dependency of cyclic functions, we are not aware that line-dependency has previously been described. PMID- 3011193 TI - Effects of tissue preincubation and hypoxia on CA3 hippocampal neurons in the in vitro slice preparation. AB - Using the in vitro hippocampal slice preparation, we studied the electrophysiological properties of pyramidal cells in tissue that was 'preincubated' (2-6 h in a large, static volume of oxygenated bathing medium) before being placed in an interface chamber for study. Striking differences were found in 'preincubated' vs 'non-preincubated' CA3 cells. The preincubated cells had more negative resting potentials, higher input resistance, lower threshold for stimulus-evoked burst discharge and larger hyperpolarizing afterpotentials. Cells in the preincubated CA3 region were also more likely to show spontaneous synchronized burst discharge, but were relatively resistant to hypoxia-induced spreading depression. CA1 cells were less dramatically affected by preincubation, showing little difference from their non-preincubated counterparts. Possible mechanisms involved in the CA3 preincubation effect, including glial buffering alterations and changes in Na+, K+-ATPase activity, are discussed. PMID- 3011195 TI - Cyclic AMP or forskolin rapidly attenuates the depressant effects of opioids on sensory-evoked dorsal-horn responses in mouse spinal cord-ganglion explants. AB - Exposure of fetal mouse spinal cord-ganglion explants to morphine (greater than 0.1 microM) results in naloxone-reversible, dose-dependent depression of sensory evoked dorsal-horn synaptic-network responses within a few minutes. After chronic opiate exposure (1 microM) for 2-3 days, these dorsal cord responses recover and can then occur even in greater than 10 microM morphine. In the present study, when naive explants were treated with forskolin (10-50 microM)--a selective activate activator of cyclase (AC)--for 10-30 min prior to and during exposure to morphine (0.1-0.3 microM) or D-Ala2-D-Leu5-enkephalin (0.03-0.1 microM), the usual opioid depressant effects on dorsal-horn responses generally failed to occur (10-30 min tests). Dibutyryl cyclic AMP (10 microM) or the more lipid soluble analog, dioctanoyl cyclic AMP (0.1 mM), produced a similar degree of subsensitivity to opiates as 10 microM forskolin. With high levels of forskolin (50 microM), even concentrations of morphine up to 1-10 microM were far less effective in depressing cord responses. These effects of exogenous cAMP analogs and forskolin on cord-ganglion explants are probably both mediated by increases in intracellular cAMP. The marked decrease in opioid sensitivity of cAMP or forskolin-treated cord-ganglion explants provides significant electrophysiologic data compatible with the hypothesis that neurons may develop tolerance and/or dependence during chronic opioid exposure by a compensatory enhancement of their AC/cAMP system following initial opioid depression of AC activity. Previous evidence relied primarily on behavioral tests and biochemical analyses of cell cultures. It will be of interest to determine if dorsal-horn tissues of cord ganglion explants do, in fact, develop increased AC/cAMP levels as they express physiologic signs of tolerance during chronic exposure to opioids. PMID- 3011196 TI - Dopaminergic control of male sex behavior in rats: effects of an intracerebrally infused agonist. AB - Systemically-administered dopaminergic drugs have been found to facilitate sexual behavior of men and male rats. The present experiments investigated the localization within the brain of dopaminergic effects on copulation of male rats. Apomorphine, a dopamine agonist, was microinfused into the medial preoptic area, caudate-putamen, nucleus accumbens, lateral septum and lateral ventricle. The lowest dose of apomorphine (0.2 microgram) infused into the ventricle reduced the number of ejaculations, slowed the rate of intromitting and decreased the percentage of mounts on which the male gained vaginal intromission. The higher two doses (0.5 and 2.0 micrograms) infused into the medial preoptic area and, in some cases, the ventricle, increased the number of ejaculations and the percentage of mounts with vaginal intromission, increased the rate of intromitting and decreased the latency to ejaculate and the postejaculatory interval before resuming copulation. Infusions into the caudate-putamen and lateral septum were without effect. Those into nucleus accumbens produced only a slight dose-related decrease in latency to begin copulating. The copulatory impairments associated with infusions of the lowest dose into the ventricle may have resulted from stimulation of autoreceptors, or from preferential stimulation by low doses of an undetermined area. The facilitative effects of the two higher doses into the medial preoptic area and lateral ventricle may have been due to stimulation of dopaminergic postsynaptic receptors. PMID- 3011197 TI - Differentiation of a cell line from olfactory epithelium is induced by dibutyryladenosine 3',5'-cyclic monophosphate and 12-O-tetradecanoylphorbol-13 acetate. AB - Olfactory receptor neurons undergo continual replacement with new cells differentiating from a population of basal cells located in the olfactory epithelium. We have previously described the isolation of a cell line from murine olfactory epithelium (MOE CL1) which is now shown to be sensitive to two differentiation promoting agents, dibutyryladenosine 3',5'-cyclic monophosphate (db-cAMP) and 12-O-tetradecanoylphorbol-13-acetate (TPA). Cells grown in the presence of db-cAMP or TPA, after a lag phase of 72-96 h, became bipolar, with long neurite-like processes. Cells conditioned for at least one passage in db cAMP or TPA, or cells exposed to a mixture of db-cAMP and TPA, attained a bipolar morphology within 24 h. Cultures grown with db-cAMP, TPA or db-cAMP + TPA showed significant increases in the percentage of process-bearing cells. An increase in population doubling time and decrease in saturation density were also observed in cultures exposed to db-cAMP. Cells exposed to TPA continued to divide at the same rate as control cells, while those treated with db-cAMP + TPA underwent terminal differentiation. Peripheral-type benzodiazepine binding sites, which within the olfactory epithelium are localized on the receptor neurons, were present in homogenate membranes of undifferentiated cells. Cells differentiated by exposure to db-cAMP + TPA for 7 days showed a 65% increase in the density (Bmax) of peripheral-type benzodiazepine binding sites. Undifferentiated cells in control cultures showed no spontaneous changes in membrane potential or regenerative responses during current injection. Small, bipolar cells in morphologically differentiated cultures, however, were capable of generating several types of spontaneous changes in membrane potential including slow, regular oscillations; irregular, slow and fast depolarizations; and trains of action potentials. PMID- 3011198 TI - Sodium increases angiotensin II receptors in neuronal cultures from brains of normotensive and hypertensive rats. AB - Neuronal cultures from one-day-old brains of Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats were used to determine the effect of sodium ions on angiotensin II (Ang II) receptors in order to understand the mechanism of action of sodium at the cellular level. Incubation with sodium chloride of neuronal cultures from WKY rats caused a rapid and dose-dependent increase in the specific binding of 125I-Ang II to its receptors. A 260% increase in the binding was observed with 150 mM NaCl. Neuronal cultures from SH rat brains showed a similar sodium-stimulated increase in Ang II binding; however this increase was 150% greater than that observed in neuronal cultures from WKY rat brain. Kinetic studies of the effects of sodium ions on both WKY and SH neuronal cultures showed an increase in the dissociation of 125I-Ang II from its receptors in the presence of sodium ions. In addition, Scatchard analysis revealed that the increase in binding caused by sodium was due to an increase in the number of Ang II receptors. These observations indicate that sodium ions increase the number of Ang II specific receptors in intact neuronal cells and that this stimulation was more pronounced in neuronal cells from SH rat brains compared with WKY controls. PMID- 3011199 TI - Diethyldithiocarbamate increases distribution of cadmium to brain but prevents cadmium-induced neurotoxicity. AB - Dithiocarbamates exhibit potent metal-binding properties which have been exploited in a variety of applications, one of which is chelation therapy for heavy metal toxicity. Such therapy, however, promotes the accumulation of metals in the brain, a side effect which may result in neurotoxicity. To examine this possibility we used morphological and biochemical indices to assess the effects of diethyldithiocarbamate (DDC) on cadmium-induced neurotoxicity in the newborn rat. Co-administration of DDC prevented the neurotoxic effects of cadmium while causing a persistent increase in the distribution of cadmium to brain. PMID- 3011200 TI - Electrical stimulation reveals relatively ineffective sural nerve projections to dorsal horn neurons in the cat. AB - Electrical stimulation of the sural nerve (SN) revealed input from sural nerve afferents to L6 and L7 dorsal horn neurons that were not apparent using natural mechanical stimuli, especially in cells with variable latency responses to SN stimulation. Nearly all (31/32) cells that had reliable, fixed latency responses to SN stimulation also had an excitatory receptive field (RF) in the region of skin innervated by the sural nerve (SN region). About one-third (20/57) of the cells with variable latency responses to SN stimulation, however, had an RF outside the SN region. Most (130/146) cells with no response to SN stimulation had RFs outside the SN region. There were no obvious differences between variable latency cells with RFs in the SN region vs those with RFs outside it in latency of response to SN stimulation, recording depth, RF sizes or modality properties. In a subsample of 31 postsynaptic dorsal column neurons all cells responding to SN stimulation also had an RF in the SN region. Strengthening of relatively ineffective projections from the sural nerve by lesions might be expected to lead to an increase in the proportion of cells responding with impulses to natural stimulation of the skin innervated by the sural nerve, and, hence, to an increase in average RF size. PMID- 3011201 TI - Antagonism of footshock stress-induced inhibition of intracranial self stimulation by naloxone or methamphetamine. AB - Rats were trained to lever-press for intracranial self-stimulation (ICSS) with electrodes implanted in the ventral tegmental area (VTA). The effect of inescapable footshock on response rates to ICSS was examined in the present study. Markedly decreased response rates to ICSS were observed 15 min to 24 h following inescapable footshock. Naloxone (10.0 mg/kg) itself was without effect on response rates to ICSS, but completely antagonized the decreased response rates by the stressor treatment. A relatively low dose of methamphetamine (0.5 mg/kg), which showed no effect on ICSS rates in naive rats, also antagonized the decreased response rates to ICSS. The present results suggest that inescapable footshock may release endorphin in the mesolimbic or mesocortical area; the released endorphin may act on dopaminergic nerve endings and interrupt dopaminergic transmission. The decreased activity of dopaminergic neurons may cause the decreased response rates to ICSS. PMID- 3011202 TI - Analgesia produced by low doses of the opiate antagonist naloxone in arthritic rats is reduced in morphine-tolerant animals. AB - In a model of experimental chronic pain (adjuvant-induced arthritic rats), low doses of the opiate antagonist naloxone produced a profound analgesia. Maximum analgesia was seen with 3 micrograms/kg (i.v.). In contrast, hyperalgesia was obtained with much higher doses (1-3 mg/kg, i.v.). The hyperalgesic effects were not affected in arthritic animals rendered tolerant to morphine, but the paradoxical analgesic effects were significantly reduced. This decrease suggests that naloxone analgesia involves interaction with opiate receptors and that the operation of endorphinergic systems differs in normal animals and animals which experience persistent pain. PMID- 3011203 TI - Electrophysiology of rat thalamo-cortical relay neurons: an in vivo intracellular recording and labeling study. AB - Membrane properties and responses to frontal cortical stimulation were studied on electrophysiologically and morphologically identified thalamo-cortical neurons in anesthetized rats. Those neurons generated membrane responses resembling the low threshold Ca-spike, gK(Ca) and IA that have been previously demonstrated in in vitro studies of thalamic neurons. Stimulation of the frontal cortex evoked a sequence of responses; antidromic spike, initial depolarization, long duration hyperpolarization and a short period of depolarization. The initial depolarization was considered to be a monosynaptic excitatory postsynaptic potential (EPSP) which overlapped with an inhibitory postsynaptic potential (IPSP). Major constituents of the long duration hyperpolarization were considered to be short duration IPSPs and long duration disfacilitations of cortical inputs. PMID- 3011204 TI - A quantitative estimate of the contribution made by various receptor categories to the depolarizations evoked by some excitatory amino acids in the olfactory cortex. AB - A study has been undertaken to assess the percentage contributions made by N methyl-D-aspartate (NMDA), kainate and quisqualate receptors to the composite depolarizations evoked by L-cysteate, L-cysteinesulphinate, L-homocysteate and S sulpho-L-cysteine in the rat olfactory cortex slice. The percentage contribution made by NMDA receptors, which was quantified by measuring the reduction in agonist responses in the presence of the highly selective NMDA receptor antagonist 2-amino-5-phosphonopentanoate (0.1 mM), was: L-homocysteate, 73%; S sulpho-L-cysteine, 65%; L-cysteate, 42% and L-cysteinesulphinate, 30%. Responses mediated by NMDA, kainate and quisqualate receptors were abolished by a 'desensitization' procedure involving repeated application of a mixture containing high concentrations of the selective agonists followed by perfusion of the non-selective receptor antagonist cis-2,3-piperidine dicarboxylate (5 mM). Following this procedure, responses to L-homocysteate and S-sulpho-L-cysteine were almost abolished and simple calculation gave the contribution of kainate plus quisqualate receptors to the agonist responses as: L-cysteinesulphinate, 46%; L-cysteate, 34%; S-sulpho-L-cysteine, 28% and L-homocysteate, 23%. However, approximately 24% of the composite depolarizations evoked by L-cysteate and L cysteinesulphinate was mediated by a mechanism not involving NMDA, kainate or quisqualate receptors, neither did it reflect possible electrogenic uptake of the amino acids nor an interaction with 2-amino-4-phosphonobutyrate receptors. It is suggested that this fraction of the depolarizations evoked by L-cysteate and L cysteinesulphinate might be due to a non-receptor-mediated release of K+ or, perhaps, to activation of an as yet unidentified receptor category. PMID- 3011205 TI - A rapid method for isolation of synaptosomes on Percoll gradients. AB - A new rapid method for fractionation of crude synaptosomes (postmitochondrial pellet, P2) on a discontinuous 4-step Percoll gradient is described. The homogeneity and integrity of the 5 major subcellular fractions were determined by analysis of the distribution of protein, lactate dehydrogenase, cytochrome oxidase, pyruvate dehydrogenase, synapsin I (a synaptic vesicle marker) and the myelin basic proteins. The biochemical results were substantiated by quantitative electron microscopy. Fractions 3, 4 and 5 were enriched in synaptosomes and contained 19.7, 40.6 and 19.5% of the intact, identifiable synaptosomes in P2, respectively. Fraction 1 was enriched in membranous material, fraction 2 in myelin and fraction 5 in extrasynaptosomal mitochondria. The synaptosomes in fractions 3, 4 and 5 differed in their size, and their content of mitochondria, synapsin I and neurotransmitters. These results suggest that partial separation of different pools of synaptosomes has been achieved. The synaptosomes in fractions 3, 4 and 5 are viable, as they take up calcium, phosphate and noradrenaline; they are metabolically normal as judged by their ability to perform protein phosphorylation and they respond normally to depolarization by increasing calcium uptake, protein phosphorylation and neurotransmitter release. The synaptosomes in fraction 4 are relatively homogeneous and appear to be free of contamination from lysed synaptosomes and synaptic plasma membranes. This constitutes a major advantage of the Percoll method over traditional procedures which involve centrifugation to equilibrium. We have therefore confirmed (J. Neurochem., 43 (1984) 1114-1123) the advantages of Percoll use over traditional procedures, while further reducing the time taken, and extended our analysis to show that the present procedure provides a fractionation of synaptosomes into different pools of viable synaptosomes. PMID- 3011206 TI - Pipecolic acid antagonizes barbiturate-enhanced GABA binding to bovine brain membranes. AB - L-Pipecolic acid, a brain L-lysine metabolite, is found to inhibit GABA binding to bovine synaptic membranes strongly in the presence of hexobarbital (IC50 = 2 X 10(-10)M) or pentobarbital (IC50 = 2 X 10(-9)M), but only slightly (less than 10%) by itself. Longer dialysis increases this binding inhibition. Hill plots indicate heterogeneity of L-pipecolic acid displaceable GABA binding sites. L Pipecolic acid may be an endogenous ligand acting as a neuromodulator on the GABA receptor ionophore complex, or it may act on its own membrane binding sites exerting an allosteric effect on the GABA receptor complex. This discovery may be useful for further defining pharmacological and biochemical differences between the GABA receptors in the brain. PMID- 3011207 TI - Reinnervation of the nucleus accumbens and frontal cortex of the rat by dopaminergic grafts and effects on hoarding behavior. AB - Embryonic dopaminergic neurons were implanted in the form of a cellular suspension in the nucleus accumbens previously deprived of its dopaminergic innervation by a local injection of 6-hydroxydopamine. The graft provided a dopaminergic reinnervation to the nucleus accumbens, the anteromedial striatum, the anteromedial frontal cortex and also, in some cases, of the septum. The pattern of reinnervation was specific for each structure and similar to the innervation provided by mesocorticolimbic dopaminergic neurons to these same structures in the normal animal. The graft restored the locomotor stimulatory action of amphetamine which was abolished in the lesioned controls. Hoarding behavior, which was disrupted following the lesion, was not reinstated by the graft alone. However, if the grafted neurons were stimulated by a small dose (0.2 mg/kg, i.p.) of (+)-amphetamine, hoarding reappeared in the grafted animals, while the same dose of amphetamine had no effect in the lesioned controls. PMID- 3011208 TI - Morphine analgesia is potentiated in adult rats prenatally exposed to ethanol. AB - Exposure to inescapable, intermittent footshock elicits an opioid-mediated stress induced analgesia in rats. We have previously shown that this response is markedly potentiated in adult rats, prenatally exposed to ethanol. To further investigate our hypothesis that endogenous opioid pain-inhibitory systems are modified by prenatal ethanol exposure, we have measured the analgesic response to morphine, in vitro brain opiate receptor binding characteristics, and occupation of brain opiate receptors following systemic administration of morphine. Compared to controls, rats prenatally exposed to ethanol had significantly enhanced morphine analgesia. This enhancement, however, does not appear attributable to changes in number or affinity of mu or delta opiate receptors, or to altered occupation of receptors by morphine. PMID- 3011209 TI - Clorgyline and desipramine alter the sensitivity of [3H]noradrenaline release to calcium but not to clonidine. AB - Synaptosomes (P2) were prepared from cerebral cortices of control rats and from those which had received clorgyline (1 mg/kg/day for 21-28 days) or desipramine (10 mg/kg/day for 21-28 days). Following incubation with [3H]noradrenaline (500 nM/15 min, 37 degrees C), aliquots of the synaptosomes were gently filtered onto Whatman GF/A filters and superfused with Krebs buffer (pH 7.5, 37 degrees C) for a maximum period of 2 h. During this time, the basal efflux of tritiated materials (approximately 75% noradrenaline) together with K+-evoked release of the amine and metabolites, were measured. Chronic antidepressant drug regimens increased the K+-stimulated release, but its attenuation by clonidine was not altered. Thus, chronic antidepressant drug regimens do not apparently alter presynaptic alpha 2-adrenoceptors. These results suggest that the reported antidepressant drug induced decreases in [3H]clonidine binding, occur on sites which are postsynaptic to noradrenergic neurones. Following the chronic antidepressant drug regimens, the sensitivity of the [3H]noradrenaline release process to Ca2+ is significantly increased. This change may explain the enhanced K+-evoked release which follows the antidepressant drug regimens. It is proposed that this increased sensitivity of the [3H]noradrenaline release process may be an adaptation to the decrease in neuronal firing which have been reported following antidepressant drug treatments. PMID- 3011210 TI - Dopaminergic amacrine cells in the retina of Japanese dace. AB - The morphological and physiological identification of the cells in the inner retina of Japanese dace was made by means of intracellular single cell recording and dye injection techniques. Our flat mounts and physiological classification revealed that 97 out of 102 sample cells were amacrine cells except for 3 transient neurons possibly being ganglion cells. Only two cells were identified as interplexiform cells. The double-labeling histochemical technique showed that 17 cells including the two interplexiform cells are dopaminergic neurons. Therefore, 15 of 97 amacrine cells in dace retina were dopaminergic cells, a finding which is different from the previously published data. PMID- 3011211 TI - Effects of footshock stress and morphine on natural killer lymphocytes in rats: studies of tolerance and cross-tolerance. AB - Exposure to a form of footshock stress known to cause opioid-mediated analgesia suppresses the cytotoxic activity of natural killer (NK) cells in rats. This suppression is blocked by the opioid antagonist, naltrexone and is mimicked by morphine administration, suggesting mediation by opioid receptors. Supporting this hypothesis, we now report that the morphine-induced suppression of NK activity shows tolerance after 14 daily injections. The NK-suppressive effect of stress, however, shows neither tolerance with repetition nor cross-tolerance in morphine-tolerant rats. PMID- 3011212 TI - Effects of diazepam on circadian phase advances and delays. AB - Phase advances of hamster locomotor rhythms, which normally can be induced by light pulses in the late subjective night, were blocked in a dose-dependent manner by the benzodiazepine, diazepam. Light-induced phase delays were unaffected at doses that significantly blocked phase advances. Diazepam caused small phase delays of the free-running rhythm when given without a light pulse at either phase advance or phase delay time points. These results are discussed with regard to the possibility that different neurochemical mechanisms are required to process light-induced phase advances and delays and that GABA neurotransmission may be involved in the modulation of light input to the clock. PMID- 3011214 TI - The ontogeny of the laminar distribution of beta-adrenergic receptors in the visual cortex of cats, normally reared and dark-reared. AB - Patterns of distribution of beta 1 and beta 2 adrenergic receptors were examined autoradiographically in slide-mounted sections from the visual cortical areas of 22 developing cat brains, using [125I]iodopindolol as the ligand in combination with displacers specific for beta 1 and beta 2 subtypes of adrenergic receptors. Within visual cortical areas 17 and 18 of adult brains, the density of beta 1 and beta 2 adrenergic receptors was highest in laminae I-III, lowest in lamina IV, and intermediate in laminae V-VI. For beta 1 adrenergic receptors, this laminar distribution was also seen in visual area 19 as well as in the non-visual area 7 that is lateral to area 19. By contrast, the distribution of beta 2 adrenergic receptors varied across cortical areas, such that its density was more homogeneous across the laminae in area 19, and decreased in all laminae in area 7. This pattern of distribution in adult brains was already formed at the beginning of the critical period and was not disturbed by dark-rearing. PMID- 3011213 TI - The development of a patchy organization of the rat striatum. AB - The rat striatum can be divided into patch and matrix compartments. Patches, as marked by high opiate receptor binding, first emerge perinatally from a dense, diffuse field of striatal opiate binding. Our quantitative analysis revealed that the patch compartment formed its peak proportion of the total striatal area at postnatal day 7. After this time, patches occupied a smaller proportion of the striatum, reflecting the fact that the number of patches and mean area per patch reached near adult levels during the first postnatal week, yet the volume of the striatum as a whole continued to increase for several weeks postnatally. Results from transplant and early postnatal lesion experiments suggested that connections between the striatum and other brain areas are important for the formation and/or maintenance of the patch and matrix compartments. Transplants of embryonic striatum to cavities in the cortex of young adult hosts developed diffuse opiate receptor binding but not dopamine receptor binding. Significantly, the opiate receptor binding seen in the transplants was never organized into the dense patches normally seen in the adult striatum. In a few transplants areas of relatively higher opiate receptor binding occurred in areas of relatively low neuronal cell density, as is seen in early normal development, but the dense adult patches never developed. Coronal diencephalic hemisections, but not decortications, in the early postnatal period produced drastic shrinkage of the striatum and, more importantly, a large decrease in opiate receptor patches when expressed as a proportion of total striatal area. Neuronal connections with more caudal brain structures may play a role in the final differentiation and maintenance of the striatal compartments. PMID- 3011215 TI - Immunocytochemically defined astroglia from fetal, newborn and young adult rats express beta-adrenergic receptors in vitro. AB - Autoradiography of radioligand binding was used to assess the expression of beta adrenergic receptors (beta-AR) by immunocytochemically identified astroglia cultured from the cerebral cortices of rats 16 days in gestation through 28 days postnatal (DPN). Polygonal astroglia isolated from animals at each age examined were found to exhibit large numbers of beta-AR. In contrast, only low levels of beta-AR could be detected on process-bearing astroglia and fibroblasts. Quantitative analysis showed that there was an increase in the density of beta-AR on polygonal astroglia between 16 days in gestation and 1 DPN. This increase in beta-AR receptor density was present whether the cells were grown for long periods of time in culture (8-22 days) or for short periods of time in culture (1 5 days). The results also suggest that differences in the level of receptor expression between cells grown in short-term and long-term culture may be due in part to culture methodology. PMID- 3011216 TI - Late development of intrinsic excitation in the rat neostriatum: an in vitro study. AB - Functional maturation of intrinsic circuitry in the neostriatum was studied by intracellular recording and intracellular staining with Lucifer yellow in slices obtained from rat pups at postnatal days (P)1-20 and from adult rats. The most striking observation was that intrastriatal stimulation elicited predominantly inhibitory responses in slices obtained from animals of P1-6. In contrast, intrastriatally evoked responses in slices obtained after P10 were predominantly excitatory. The inhibitory postsynaptic potentials (IPSPs) recorded in slices obtained from pups were blocked by bicuculline (50 microM) and exhibited a reversal potential at about -60 mV which shifted in depolarizing direction when intracellular Cl- activity increased. Thus, these IPSPs correspond to IPSPs observed in adult animals. It is concluded that maturation of excitatory synapses is the main change during postnatal development. The changes of postsynaptic potentials were paralleled by the appearance of spines on dendrites around P7 as revealed by intracellular staining. The apparent input resistances and time constants of young neurons were very high and responses to large current injections were often distorted by humps which could not be observed in adult neurons. Only young neurons responded with bursts to synaptic activation in the presence of bicuculline (50 microM). It appears that dendritic conductances have a stronger influence on somatic discharge in the electrotonically compact young neurons than in adult neurons. PMID- 3011217 TI - Central nervous system mediation of positive and negative reinforcement in neonatal albino rats. AB - Two classes of affective behaviors served as assays for central opioid mediation of affect during development. On the positive side, we found that 5-day-old rats came to prefer a novel odor that predicted the delivery of morphine intracerebroventricularly, thereby replicating previous findings with peripheral morphine. Moreover, central naltrexone blocked the odor preference formation associated with peripheral morphine. In examining responses to pain and stress, we found that central morphine decreased sensitivity to pain, as measured by paw withdrawal to heat and decreased ultrasonic vocalizations during isolation. Conversely, naltrexone delivered intracerebrally increased sensitivity to a painful stimulus and markedly increased distress vocalizations relative to untreated isolates. Thus, the present results imply that central opioid systems are functional behaviorally in neonatal rats mediating affective responses. PMID- 3011218 TI - Maturation of the adrenocortical stress response: neuroendocrine control mechanisms and the stress hyporesponsive period. AB - During the first two weeks of life rat pups show a markedly reduced adrenocortical response to stress, and this period of adrenocortical quiescence has been termed the 'stress non-responsive period' (SNRP). The adaptive value of the SNRP can be understood in terms of the effects of glucocorticoids on CNS development: excessively high or low corticoid levels are associated with abnormal neural and behavioral development. We have attempted to explain adrenocortical activity during this period in terms of the unique pattern of glucocorticoid-receptor concentrations that exist in the brain and pituitary of the neonatal rat. This pattern of receptor concentrations results in a negative feedback condition at the level of the brain and pituitary that ensures the low, stable corticoid levels that appear to be optimal for neuronal development in glucocorticoid-sensitive brain regions. PMID- 3011219 TI - Kappa opioid receptors in human lumbo-sacral spinal cord. AB - [3H]Etorphine and [3H]ethylketocyclazocine bind with high affinity (Kd between 0.25-2.0 nM) to a single class of sites in human lumbo-sacral spinal cord. Other ligands such as [3H]morphine, [3H]dihydromorphine and [3H]D-Ala2, D-Leu5 enkephalin (DADLE) did not bind to significant number of sites under our incubation conditions. Ligand selectivity pattern strongly suggests that [3H]etorphine labels kappa opioid binding sites in the human lumbo-sacral spinal cord since benzomorphans and oripavines are much more potent than mu and delta agonists. Furthermore, [3H]etorphine and [3H]ethylketocyclazocine binding is sensitive to high concentrations of DADLE suggesting that these sites are of the kappa 2 sub-type. Finally, the visualization of these sites by receptor autoradiography demonstrates that they are mainly concentrated in lamina II and III of the dorsal horn. Moderate densities of sites are present around the central canal. Thus, it is possible that kappa opioid binding sites could be involved in the control of sensory and autonomic functions in the human lumbo sacral spinal cord. PMID- 3011220 TI - Subpallidal projections to the mesencephalic locomotor region investigated with a combination of behavioral and electrophysiological recording techniques. AB - Locomotor activity was increased as a function of current intensity in a series of rats with chronic stimulating electrodes in the tegmental pedunculopontine nucleus. Single pulse stimulation of these same mesencephalic locomotor region (MLR) sites antidromically activated more than one-quarter of the neurons sampled in the subpallidal area. In a second series of rats locomotor activity was increased by chronic stimulation of the zona incerta as well as by chronic stimulation of MLR. Approximately one-quarter of subpallidal neurons sampled were antidromically activated by single pulse stimulation of zona incerta and/or MLR. Thirty-one of the subpallidal neurons were antidromically activated by stimulation of both zona incerta and MLR and, according to the results of the reciprocal collision test, about half this number had branching projections to the zona incerta and MLR. These observations provide additional evidence that subpallidal-MLR neurons are associated with locomotion. PMID- 3011221 TI - Non-overlapping thalamocortical projections for separate forepaw digits before and after cortical reorganization in the raccoon. AB - The fluorescent dye retrograde tracing technique was used to examine the projection from ventroposterior lateral thalamus to primary somatosensory cortex in the raccoon. Results are presented from fifteen experiments using Fast Blue in combination with either Nuclear Yellow or Diamidino Yellow Dihydrochloride. Injections of two dyes into adjacent but separate digit regions of cortex produced single-labeling of thalamic neurons, suggesting that projections for each digit are separate and independent with no branching to adjacent parts of the somatotopic map. Double-labeling was only seen when the cortical injection sites were overlapping. Similar results were seen in six cases in which amputation of the 5th digit of the contralateral forepaw was carried out four months earlier. These results suggest that neither the strengthening of existing collaterals nor growth of new collaterals in the thalamocortical projection are likely to be responsible for the physiological reorganization that is seen in raccoon cortex after peripheral deafferentation. PMID- 3011223 TI - [Molecular models of the interaction between T4 antigen and HLA class II antigens or the LAV virus]. AB - HLA class II beta chains contain in their aminoterminal polymorphic domain a highly conserved tetrapeptide (RFDS) also present in protein F encoded by the LAV virus. Homology between this tetrapeptide and the fibronectin cell-attachment site (RGDS) has suggested a role in cell adhesion processes. I propose here that such a structure, that I call adhesiotope, would allow stabilization of intermolecular contacts between molecules present at the surface of interacting cells or viruses. Analysis of a three dimensional model of the T4 antigen suggests that the tetrapeptide RADS is located at the surface of the aminoterminal, immunoglobulin-like domain. A model is proposed in which interaction between the adhesiotopes present in class II antigens (RFDS) and T4 (RADS) is the molecular basis of conjugate formation between antigen presenting cells and T helper lymphocytes. The LAV virus, having the RFDS adhesiotope on its surface, would mimic class II antigens in their interaction with T4 and infect selectively T4 positive cells, resulting in the acquired immune deficiency syndrome. PMID- 3011222 TI - [A DNA topoisomerase from trypanosomes able to catenate, decatenate and unknot DNA without ATP]. AB - A novel DNA-topoisomerase from Trypanosoma cruzi was partially purified. The enzyme, without ATP addition, catalyzes decatenation of kinetoplast DNA, catenation of circular supercoiled DNAs and unknotting of P4 phage DNA. The presence of Mg++ is required as well as a suitable concentration of KCl. In stoichiometric conditions the trypanosome enzyme induces double-strand DNA cleavage. The reaction is highly stimulated by some chemicals. Such characteristics allow to include this enzyme into the type II class of DNA topoisomerases. PMID- 3011224 TI - Dermatologic manifestations of internal cancer. AB - The cutaneous manifestations of internal cancer can develop either before or after the presence of an underlying tumor has been established. These signs may result either from the physical presence of tumor cells in the skin or from presumed metabolic effects of tumor cells located at visceral sites. Occasionally, skin involvement in cancer patients is biologically unrelated to the tumor but is instead part of a well-defined inherited syndrome featuring an increased incidence of internal cancer. Whatever the association, inspection of the skin remains an essential part of the complete physical examination. PMID- 3011225 TI - Ovarian cancer. AB - Early diagnosis is the most effective means of reducing the currently high mortality rate associated with ovarian cancer. The palpation of what appears to be a normal size ovary in a premenopausal woman suggests an ovarian tumor in a postmenopausal woman. Ovarian cancer should be ruled out in any woman 40 years of age or older who has persistent, unexplained GI symptoms. Ninety percent of all ovarian tumors are of epithelial origin. Treatment consists of total hysterectomy, bilateral salpingo-oophorectomy, omentectomy, and appendectomy. Instillation of P32 is optional. In stages IIb, III, and IV tumors, chemotherapy is advised; in stages I and IIa, the use of prophylactic chemotherapy must be judged on an individual basis. In children, ovarian cancer that is beyond the localized stage is one of the most frustrating of all gynecologic diseases. Total surgical extirpation of disease is the only hope for cure; for now, early diagnosis is more chance than scientific method. Thanks to better public and professional education, ovarian cancer is now being diagnosed at an earlier stage. The earlier the diagnosis, the greater the chance for cure. It is becoming obvious that ovarian cancer is a disease of the GI tract, and physicians treating ovarian cancer should be prepared to deal with bowel-associated problems. The practice of tapping women with ascites for diagnosis as well as doing an exploration merely to obtain a biopsy should be discouraged. Unless the physician is prepared to carry out the optimal surgical approach for the patient, it is crucial that the patient be referred to either a center or to a physician who is actively engaged in the day-to-day care of cancer patients. With the combined use of all the available treatment methods, patients with ovarian cancer are now living longer and more comfortably. There is also indication that their long-term survival will be increased. The one message that is important for both patients and physicians is that the gloom and doom of the 1960s and 1970s can now be replaced by a spirit of optimism. PMID- 3011226 TI - The metabolic cancer therapy of Harold W. Manner, Ph.D. PMID- 3011227 TI - [Platelet adhesion, aggregation and liberation and cyclic nucleotide level in patients with leukemia]. PMID- 3011228 TI - [General status and prospect of the use of monoclonal antibodies in endocrinology]. PMID- 3011229 TI - Type I collagen shows a specific binding affinity for bovine dentin phosphophoryn. AB - Bovine dentin phosphophoryn was iodinated with 125I, then tested for binding to native monomeric collagen, to collagen fibrils, and to gelatin. The phosphophoryn was found to bind reversibly, but specifically, to both collagen monomers and fibrils, but not to denatured collagen (gelatin). Competitive binding studies showed that bovine serum albumin, fibronectin, and bovine bone 34K glycoprotein (osteonectin) did not compete with phosphophoryn and did not inhibit its binding to collagen fibrils. Phosvitin, a phosphoserine-rich protein, did compete, but sixfold higher concentrations of phosvitin than of unlabeled phosphophoryn were required to reduce iodinated phosphophoryn binding to the same extent. Quantitative analyses of the binding showed binding to be limited to the fibril surfaces. Bound phosphophoryn enhanced the uptake of 45Ca onto collagen fiber surfaces. These data support the hypothesis that, in dentin, the phosphophoryn plays an important role in localizing the calcium binding leading to the growth of collagen-oriented calcium hydroxyapatite crystals. PMID- 3011231 TI - Adrenocortical responses to corticotropin-releasing factor in the rat. AB - The time course of plasma corticosterone was measured in male Sprague-Dawley rats whose endogenous release of ACTH had been blocked following rapid i.v. injections of doses ranging from 0.003 to 10 micrograms corticotropin-releasing factor (CRF) per rat and during i.v. infusions at rates ranging from 0.001 to 20 ng CRF X min 1 X 100 g body weight-1. The range of the dose-response curve, following rapid injection, extends from 0.01 to 0.37 micrograms CRF, whereas it extends over a 20 000-fold range from 0.001 to 20 ng CRF X min-1 X 100 g body weight-1 during a continuous infusion. The delayed response to a small rate of CRF could be ascribed to a relatively long time of residence of CRF in the plasma which implies that a relatively long period of time is required until a minimal plasma CRF concentration is reached after the onset of a continuous infusion of CRF at a small rate. When presented with a prolonged infusion of CRF at a large rate, the pituitary secretion of ACTH is rapidly turned on at a rate which exhibits the characteristics of a prolonged secretion at a constant large magnitude. PMID- 3011232 TI - Choline analogues: their use in studies of acetylcholine synthesis, storage, and release. AB - The objective of this article is to illustrate how choline analogues might provide insight into mechanisms that regulate the synthesis, storage, and release of acetylcholine (ACh). Studies with false neurotransmitters provide information about the origin of releasable transmitter. Thus, false esters that distribute like ACh to vesicle-bound stores are as releasable as is ACh, but esters that poorly localize to synaptic vesicles are poorly releasable. Studies of choline analogue uptake provide information about the structural specificity of that transport process and, also, show that choline uptake is regulated in response to activity. Thus, stimuli that normally release transmitter increase the rate of choline transport, presumably to provide more precursor for ACh synthesis. However, the relationship between precursor delivery and product formed can be dissociated, suggesting that some factor in addition to choline delivery is involved in ACh synthesis regulation. Studies with a compound (AH5183), which inhibits ACh uptake by synaptic vesicles, provide information about the relationship of ACh stores and releasable transmitter. In the presence of AH5183 some 15% of nerve terminal ACh is released in response to nerve impulses, suggesting the existence of a small population of vesicles that contain readily releasable ACh. In presence of AH5183, ACh synthesis is activated even when ACh release is depressed, showing that transmitter synthesis can be regulated by some factor other than nerve terminal ACh levels. PMID- 3011230 TI - Changes in heated and in laser-irradiated human tooth enamel and their probable effects on solubility. AB - Enamel of intact human teeth laser irradiated in vitro under certain conditions is known to have less subsurface demineralization than unirradiated enamel on exposure to acid; consequently, the potential use of laser irradiance to reduce caries is apparent. The laser-induced physical and/or chemical changes that cause this reduced subsurface demineralization are not known. A laser-irradiated tooth enamel surface will have a temperature gradient that decreases towards the dentin junction. Dependent on irradiant conditions, the temperature may range from greater than 1400 degrees C at the surface to near normal at the dentin-pulp junction. Along this steep temperature gradient, different compositional, structural, and phase changes in the tooth enamel are to be expected. Identification of changes occurring along this gradient has bearing on understanding the dissolution reduction mechanism and, in turn, optimizing its effect. Changes in laser-irradiated material from the highest temperature region have been characterized, but those occurring in sequential layers of decreasing temperatures have not. Since the laser-induced changes are expected to primarily arise from localized heating, previously reported thermally induced changes in tooth enamel on heating in conventional furnaces were utilized to infer corollary changes along the gradient in laser-irradiated tooth enamel. These thermally inferred changes which resulted in modifications in the tooth enamel apatite and/or newly formed phases were correlated with their probable effects on altering solubility. A temperature gradient range from 100-1600 degrees C was considered with subdivisions as follows: I, 100-650 degrees C; II, 650-1100 degrees C; and III, greater than 1100 degrees C. Two of the products formed in range III, alpha-Ca3(PO4)2 and Ca4(PO4)2O, and also identified in the fused melted material from laser-irradiated tooth enamel, are expected to markedly increase solubility in those regions that contain considerable amounts of these compounds. Products and changes occurring in range II, separate phases of alpha- and/or beta-Ca3(PO4)2 and a modified phase of apatite, may increase or decrease the solubility depending on the Ca/P ratio and the resultant amounts of alpha-, beta-Ca3(PO4)2 formed. Modifications in tooth enamel apatite effected in range I are expected to decrease its solubility; the formation of pyrophosphate in this range may have a substantial effect on reducing the solubility rate.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3011233 TI - Effect of various growth conditions on spore formation and bacillomycin L production in Bacillus subtilis. AB - Bacillomycin L is produced by Bacillus subtilis NCIB 8872 in the stationary phase; it is excreted into the culture medium, without prior accumulation in the bacterial cells. The production of bacillomycin L is largely dependent on the composition of the culture medium. The action of specific inhibitors of sporulation, netropsin and diethyl malonate, on antibiotic synthesis is dependent on the composition of the culture medium. Although they occurred at the same time, there appears to be no direct correlation between sporulation and antibiotic synthesis. PMID- 3011234 TI - Surgical treatment of lung cancer: promise and problems of early diagnosis. AB - Recent studies have shown that the survival of patients with lung cancer is improved if the tumour is resected before it becomes larger than 3 cm in diameter and before it spreads to lymph nodes. While this suggests a positive benefit from early detection, recent mass-screening studies have claimed that the benefit obtained from this procedure is illusory because it relates to a lead-time bias. This study reports the results of surgical resection of 143 primary lung cancers. The data confirmed that the predicted 5-year survival was greatest (74%) following resection of lesions that were less than 3 cm in diameter without node involvement. Analysis showed that the age of these patients was 63 +/- 8 years, the same as in patients with larger tumours and more extensive node involvement. This suggests that tumours progress rapidly from a stage at which resection is beneficial to stages at which it is not. Although it is desirable that tests predict the presence of small tumours, the high requirements for sensitivity and specificity at current prevalence rates for lung cancer make this goal impractical. PMID- 3011235 TI - The experience and effectiveness of the Nova Scotia Rh program, 1964-84. AB - A program to reduce the incidence of erythroblastosis fetalis was started in Nova Scotia in 1964. Up to the end of 1984, 120 fetuses received 247 intrauterine transfusions. The survival rate was 45.6% in the first 10 years of the program and 66.7% in the next 11 years. For fetuses at or over 26 weeks' gestation the figures were 51.5% and 73.7% respectively. Postpartum prevention was started in 1968, with administration of Rh immune globulin (RhIG) to Rh-negative unimmunized women within 72 hours after the birth of an Rh-positive infant. Antepartum prevention, started in 1979, consisted of administration of RhIG at 28 weeks' gestation to Rh-negative unimmunized women. The effectiveness of the prevention program was evaluated by enumerating the known cases of Rh(D) alloimmunization in the province from 1982 to 1984: 55 cases were identified, a rate of 1.5 per 1000 births instead of the expected rate of about 10 per 1000. PMID- 3011236 TI - Serum deoxythymidine kinase in small cell carcinoma of the lung. Relation to clinical features, prognosis, and other biochemical markers. AB - Thymidine kinase (s-TK), lactate dehydrogenase (LDH), and carcinoembryonic antigen (CEA) were determined in pretreatment serum from 125 patients with small cell carcinoma of the lung. The distribution of marker levels into three ranges, when including all patients were as follows: s-TK less than 5 units 49%, 5-less than 10 units 25%, greater than or equal to 10 units 26%; LDH less than 6.7 mukat 31%, 6.7-less than 13.4 mukat 48%, greater than or equal to 13.4 mukat 21%; CEA less than 7.5 micrograms/l 51%, 7.5-less than 15 micrograms/l 25%, greater than or equal to 15 micrograms/l 24%. The percentages of patients with limited and with extensive disease within each range were s-TK less than 5 82/18, 5-less than 10 29/71, greater than or equal to 10 9/91; LDH less than 6.7 76/24, 6.7-less than 13.4 51/49, greater than or equal to 13.4 21/79; CEA less than 7.5 70/30, 7.5-less than 15 39/61, greater than or equal to 15 23/77. Analyses in relation to metastases present showed that patients with skeletal and bone marrow metastases had significantly higher s-TK and LDH than those without, while this was not the case for CEA. A strong correlation between s-TK and LDH level, a weaker correlation between CEA and s-TK, and no correlation between CEA and LDH level, was found. Both the level of s-TK and LDH correlated to the patients' performance, as defined by the Karnofsky index. These correlations were mainly confined to the patients with extensive disease. Analyses of the prognostic capacity of variables showed that s-TK, stage, and Karnofsky index could divide the patients into groups with highly significant difference in survival time, while LDH and CEA were of less value. Longitudinal studies showed that the serum markers mirrored the disease activity, with the exception that highly increased s TK was found during remission induction for some patients. It was concluded that the expression of pathologic levels for the serum markers were dependent on different biological parameters. Of the serum markers, only s-TK was judged useful for estimation of disease spread and prognosis of the individual patient. PMID- 3011237 TI - Primary oral etoposide therapy in gestational trophoblastic disease. An update. AB - Sixty patients who developed persistent or metastatic gestational trophoblastic disease (GTD) received primary oral etoposide therapy (VP 16-213). Twelve patients had metastatic GTD. Fifty-nine patients achieved biochemical remission. One patient had marked nausea and vomiting and the therapy was switched to a methotrexate/folinic acid regimen. Three patients developed relapse of GTD, giving a relapse rate of 5.1%. Etoposide is an active drug against choriocarcinoma. Its use should not be restricted to drug-resistant GTD. PMID- 3011238 TI - Histogenesis of adenoid cystic carcinoma of the salivary glands. Light and electronmicroscopic study. AB - This ultrastructural study, based on 12 cases of adenoid cystic carcinoma of the major and minor salivary glands, was conducted to determine the role and extent of participation of myoepithelial cells in their histogenesis. The tumors were composed of four major cell types; intercalated duct, myoepithelial, secretory, and pluripotential reserve/stem cells. The cellular composition of adenoid cystic carcinoma is similar to that in the "terminal tubule" complex stage of a developing salivary gland except that in the tumor the pluripotential reserve/stem cells differentiate predominantly along the intercalated duct cell line rather than secretory cells as in the acinic cell carcinoma. Furthermore, adenoid cystic carcinoma appears to contain a far greater number of myoepithelial cells than acinic cell carcinomas. PMID- 3011240 TI - Chromosomal evolution in malignant human gliomas starts with specific and usually numerical deviations. AB - Our previous karyotypic studies of malignant human gliomas have demonstrated that their most consistent early or primary gross changes include gains of #7, losses of #10, #22, and the gonosomes, and the presence of double minutes. Karyotypes of 15 additional malignant human gliomas reported here have confirmed these observations and, by enlarging our series, we can now show that in addition to double minutes, certain other gross structural abnormalities also are clearly associated with the early evolution of this type of tumor. The most prevalent deviations are deletions and translocations involving 9p. Other chromosomes commonly involved in rearrangements are #1, #6, and #13, and less frequently #7, #11, and #16. PMID- 3011239 TI - Enzymes of purine metabolism in hairy cell leukemia. AB - Differences in activities of the purine degradative enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and 5'-nucleotidase (5'NT), have been observed among different classes of lymphoid malignancies. Recent studies have shown that hairy cell leukemia (HCL) may respond to treatment with the ADA inhibitor, 2-deoxycoformycin. This study demonstrates that the cells of HCL have significantly lower levels of ADA and 5'NT (P always less than 0.01) when compared to levels in normal B- or T-lymphocytes, but have higher levels of PNP (P less than 0.001 for both comparisons). Recent studies have shown that when treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), cells of B-cell chronic lymphatic leukemia (B-CLL) acquire phenotypic characters of HCL. The authors have therefore also investigated the changes in enzyme pattern of B-CLL after incubation with TPA B-CLL cells are characterized by low levels of ADA, PNP, and 5'-NT, but TPA caused a marked increase in PNP activity (P less than 0.001, t test for paired samples), a pattern similar to HCL. The results from biochemical studies are thus in accordance with the hypothesis that HCL cells are more mature than B-CLL cells. The special enzyme profile of HCL suggests that a PNP inhibitor might also be effective in the treatment of this disease. PMID- 3011241 TI - Karyotyping and DNA flow cytometry of mature residual teratoma after intensive chemotherapy of disseminated nonseminomatous germ cell tumor of the testis: a report of two cases. AB - Karyotyping and DNA flow cytometry was performed on mature residual teratoma cells following intensive chemotherapy of disseminated nonseminomatous germ cell tumor of the testis to study its biology. We report herein a successful method for short-term tissue culture and karyotyping of retroperitoneal residual mature teratoma in two cases. In vitro morphology confirmed that the cultured cells were nonembryonal carcinoma cells. Both mature residual teratomas were highly aneuploid and possessed the i(12p) marker characteristic of testicular germ cell tumors. A clone in the retroperitoneal residual lesion of one of the patients showed a DNA-index different from the primary tumor and might represent a clone unmasked by chemotherapy. In view of these data, which are in agreement with recent reports on secondary non-germ cell malignancies arising in mature residual teratoma, aggressive surgery of mature residual lesions seems justified. PMID- 3011242 TI - Conditioned tumorigenicity of activated oncogenes. AB - Virally transduced oncogenes (v-onc) have a restricted target cell spectrum. They transform only a small part of the cell types in which they are expressed. Temperature-sensitive (ts) mutant studies have shown that some of them may act by blocking specific steps of maturation. If the cell can bypass the block, e.g., by a temporary switch off of the temperature-sensitive transforming protein, reexpression of the oncogene product at the permissive temperature may be unable to restore the transformed phenotype. Consideration of these facts, together with evidence concerning the reversion of the transformed phenotype and the suppression of tumorigenicity in hybrids derived from the fusion of normal and malignant cells, leads to the concept of "conditioned tumorigenicity." It states that the transforming and/or tumorigenic effect of a given oncogene, activated by structural or by regulatory changes, is restricted to specific and often quite narrow differentiation or maturation windows within each susceptible lineage. A similar restriction seems to apply to oncogenes activated by chromosomal translocation. The regular juxtaposition of the c-myc gene to one of the three immunoglobulin loci in Burkitt's lymphoma, mouse plasmacytoma, and rat immunocytoma is a case in point. The myc-carrying chromosome can break at many different places, within, upstream, or downstream of the gene, but not within its coding exons. This suggests that the break occurs at random and the myc protein plays an essential role in the selective, i.e., tumorigenic process. If so, other oncogenes should be equally transposable to the "Ig hot spots" during the long series of cell divisions in the preneoplastic target cell population that characterizes the prehistory of both BL and MPC. In other human B-cell leukemias and lymphomas, other (e.g., 11;14 and 14;18) translocations have been found, confirming that this can actually occur, but only in histologically different neoplasms. The exclusive involvement of myc in BL and MPC must be relatable to the specific functional features of the precursor cells and to the normal role of the myc protein. Recent evidence indicates that the myc gene is regularly turned off before or at the time when the cell enters a pathway that is programmed to lead it towards a resting Go state. Clonally expanded B-cells are believed to turn into resting memory cells upon waning of the antigenic stimulus. The normal, nontranslocated myc allele is regularly switched off in both BL and MPC, indicating that the cell has already obeyed a program involving the down regulation of myc.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3011243 TI - Antibody-Pseudomonas exotoxin A conjugates cytotoxic to human breast cancer cells in vitro. AB - Breast tumor selective antibodies (MAB) conjugated to Pseudomonas exotoxin A (PE) formed immunotoxins with potent cytotoxicities for human breast tumor cell lines. The most effective of the MAB-PE conjugates were about 500-fold more toxic to the breast tumor target cell lines than to the nontarget human fibroblast cell line. Specificity of cytotoxicity by MAB-PE conjugates was demonstrated by protection of target cells in the presence of excess unconjugated homologous antibody. A MAB PE conjugate inhibited protein synthesis more rapidly than the corresponding MAB ricin toxin A chain (RTA) conjugate. Generally, there was a direct correlation between the cytotoxicity of RTA and PE when conjugated to the same MAB: MABs that made highly cytotoxic RTA conjugates made effective PE conjugates and MABs that made poorly cytotoxic RTA conjugates made PE conjugates of low cytotoxicity; however, one MAB-PE immunotoxin was cytotoxic to the human breast tumor cell lines, whereas the corresponding MAB-RTA immunotoxin was noncytotoxic at the highest dose tested. In contrast to MAB-RTA conjugates, which require a cleavable (disulfide) linkage for maximal in vitro cytotoxicity, MAB-PE conjugates were about equally cytotoxic when linked by either a cleavable or noncleavable (thioether) bond. PMID- 3011245 TI - Arrest of the proliferation of renal and prostate carcinomas of human origin by inhibition of mitochondrial protein synthesis. AB - The results described in this paper demonstrate that proliferation arrest by low concentrations of tetracyclines, which has previously been shown in experiments with animal tumor systems, can also be achieved in tumor systems of human origin. Tetracyclines specifically inhibit mitochondrial protein synthesis. Prolonged and continuous impairment of protein synthesis inside the mitochondria leads to reduction of the cellular concentration of the polypeptide products which are coded and synthesized within mitochondria. These products are part of the oxidative phosphorylative system of the cell. Long-term tetracycline treatment leads to a decrease of oxidative ATP-generating capacity as monitored by cytochrome c oxidase activity. This may cause severe energetic or metabolic disturbances which explain the proliferation arrest observed. Proliferation arrest, provided that mitochondrial protein synthesis is blocked effectively, is found in vitro as well as in vivo. It is shown that the effect of doxycycline is not limited to cytostasis; prolonged doxycycline treatment is clearly cytotoxic for the tumor cells. PMID- 3011244 TI - In vivo enhancement of antitumor immunity by interleukin 2-rich lymphokines. AB - The ability of interleukin 2 (IL-2) to enhance in vivo antitumor immunity has been evaluated in the line 1 alveolar cell carcinoma (L1) model of BALB/c mice. A crude supernatant from phorbol myristate acetate exposed EL-4 cells rich in IL-2 plus other lymphokines (EL-4 IL-2), a concanavalin A-induced supernatant from murine splenocytes (Con A IL-2), and recombinant IL-2 (rIL-2) provided by Biogen were tested. Mice were immunized with a cloned population of L1 cells (10(6) irradiated L1 cells given s.c. in the left inguinal region) followed by s.c. injections of EL-4 IL-2, Con A IL-2, or rIL-2 given to the same site. Two immunizations of L1 cells each followed by IL-2 administration were given prior to challenge with live L1 cells s.c. on the right chest wall. Mice receiving EL-4 IL-2 survived significantly longer than those receiving L1 cells only. Daily administration of EL-4 IL-2 for 7 days after the last L1 immunization was significantly better than 3 days (P less than 0.01) which in turn was significantly better than 1 day (P less than 0.05). Among the doses tested (normalized in vitro to the Biologic Response Modifiers Program IL-2 standard) 404 units of IL-2/injection was optimal. The EL-4 IL-2 had to be injected adjacent to the site of L1 cells; s.c. injection at a distant site or i.p. was not effective. When rIL-2 or Con A IL-2 was substituted for EL-4 IL-2, survival was not prolonged; however, if Con A IL-2 (low IL-2 levels) was supplemented with rIL-2 to 404 units of IL-2, it augmented immunity as well as 404 units of EL-4 IL 2. The data suggest that IL-2 is not the only lymphokine active in augmenting antitumor immunity induced by L1 cells. Some preliminary experiments indicate that a multilymphokine approach may have potential clinical relevance. PMID- 3011247 TI - Expression of potentially lethal damage in Chinese hamster cells exposed to hematoporphyrin derivative photodynamic therapy. AB - Experiments were performed to determine whether the expression and/or repair of potentially lethal damage could be observed in mammalian cells exposed to hemataporphyrin derivative (HPD) photodynamic therapy (PDT). Photodynamic therapy was combined with posttreatment protocols known to inhibit the repair of potentially lethal damage in cells treated with X-rays, ultraviolet radiation, or alkylating agents. Potentiation of lethal damage from photodynamic therapy was induced by hypothermia (4 degrees C) following short (1 h) or extended (16 h) HPD incubation conditions. Caffeine potentiated the lethal effects of PDT only when cells were incubated with HPD for extended time periods. However, 3 aminobenzamide had no effect on the cytotoxic actions of PDT following either short or extended HPD incubations. Recovery from potentially lethal damage expressed by posttreatment hypothermia was complete within 1 h, while recovery from potentially lethal damage expressed by posttreatment caffeine required time periods of up to 24 h. The lack of effect of 3-aminobenzamide on expression of potentially lethal damage following photodynamic therapy may be related to direct inhibition of adenosine diphosphoribose transferase by photodynamic therapy. These results indicate that the expression and repair of potentially lethal damage can be observed in cells treated with PDT and will vary as a function of porphyrin incubation conditions. PMID- 3011246 TI - Selective killing of human T-lymphotropic virus-I infected leukemic T-cells by monoclonal anti-interleukin 2 receptor antibody-ricin A chain conjugates: potentiation by ammonium chloride and monensin. AB - Adult T-cell leukemia is a progressive disease produced by infection of mature T cells with the human T-lymphotropic virus-I (HTLV-I). These retrovirus infected T cells express large numbers of receptors for interleukin 2 (or T-cell growth factor). Due to the presence of these receptors, these leukemic T-cells can be selectively killed in vitro by monoclonal anti-interleukin 2 receptor antibody covalently linked to the A chain of the plant toxin, ricin (anti-TAC-A), suggesting that such immunotoxins may be useful in the therapy of this disease. In this report we demonstrate that the lysosomotropic agent ammonium chloride and the carboxylic ionophore monensin substantially potentiate the cytotoxicity of anti-TAC-A on HUT 102 cells, a long-term cultured HTLV-I infected T-cell line. Anti-TAC-A alone produces half-maximal inhibition of protein synthesis in HUT 102 cells at a concentration of 2.2 X 10(-10) M (the 50% inhibitor, concentration). Addition of ammonium chloride or monensin augments the potency of anti-TAC-A killing 100-fold (50% inhibitory concentration = 2.5 X 10(-12) M) and 400-fold (50% inhibitory concentration = 8 X 10(-13) M), respectively; furthermore, these agents accelerate rate of anti-TAC-A intoxication and increase the specific killing of interleukin 2 receptor-bearing leukemic cells. At concentrations which cause only minor harm to colony forming hematopoietic progenitor cells (granulocyte-erythroid, monocytic, megakaryocytic colony forming unit, granulocyte-macrophage colony forming unit, macrophage colony forming unit, and granulocyte colony forming unit), anti-TAC-A alone is able to eliminate greater than 99.99% of an HTLV-I infected T-cell population. In the presence of ammonium chloride or monensin, respectively, a 3.5- and 20-fold greater cytoreduction of HTLV-I infected T-cells is achieved. Combined treatment with anti-TAC-A and monensin may offer an efficient and highly selective means of purging bone marrow of patients with adult T-cell leukemia, which may then be used for autologous marrow transplantation. PMID- 3011248 TI - Inhibition of chemotactic peptide-induced phosphoinositide hydrolysis by phorbol esters through the activation of protein kinase C in differentiated human leukemia (HL-60) cells. AB - Phorbol-12,13-dibutyrate (PDBU), a tumor-promoting and protein kinase C activating phorbol ester, inhibited formylmethionylleucyl-phenylalanine-induced generation of inositol mono-, bis-, and tris-phosphates from the hydrolysis of phosphoinositides in human leukemia (HL-60) cells, which had been differentiated to polymorphonuclear leukocyte-like cells by pretreatment with dibutyryl cyclic adenosine 3':5'-monophosphate. PDBU did not alter the binding of formylmethionylleucyl-phenylalanine to the cells. Other protein kinase C activating substances such as 12-O-tetradecanoylphorbol-13-acetate and 1-oleoyl-2 acetyl-glycerol could substitute for PDBU, but 4 alpha-phorbol-12,13-didecanoate, which is inactive for both tumor promotion and protein kinase C activation, was ineffective in this capacity. Prolonged treatment of the cells with PDBU resulted in the down-regulation and decrease of protein kinase C activity to the level of 30-40% of that in the control cells. In the down-regulated cells, formylmethionylleucylphenylalanine still induced generation of the phosphorylated inositols to the same extent as that in the control cells, but the inhibition of this reaction by PDBU was reduced to 30-50% as compared with that in the control cells. These results strongly suggest that tumor-promoting phorbol esters inhibit the agonist-induced phosphoinositide hydrolysis through the activation of protein kinase C in the differentiated HL-60 cells. PMID- 3011249 TI - Metabolic activation and cytotoxicity of 4-ipomeanol in human non-small cell lung cancer lines. AB - In the normal lungs of many animal species, 4-ipomeanol is transformed to a highly reactive metabolite preferentially in pulmonary bronchiolar Clara cells and to a lesser extent in alveolar type II cells, potentially leading to damage or destruction of these cell types. Since Clara cells and type II cells are suspected sites of origin of certain "non-small cell" lung cancers, the metabolic activation of 4-ipomeanol (measured by the metabolism-dependent covalent binding of 4-ipomeanol to cellular macromolecules) was compared in two human non-small cell carcinoma derived cell lines (NCI-H322 and NCI-H358) and two human small cell carcinoma derived cell lines (NCI-H128 and NCI-H69). Metabolic activation of 4-ipomeanol was evident in the non-small cell lines; the production of covalently bound metabolite was somewhat greater in NCI-H322 (morphology related to Clara cells) compared to NCI-H358 (morphology related to alveolar type II cells), but was entirely undetectable in the small cell lines. The activation pathway was concentration (4-ipomeanol) and time dependent and followed Michaelis-Menten kinetics. Metabolism to the reactive intermediate required oxygen and was strongly inhibited by carbon monoxide. Covalent binding was enhanced in the non small cell lines by prior incubation with beta-naphthoflavone and by supplementation of the incubate with exogenous reduced nicotinamide adenine dinucleotide phosphate. 4-Ipomeanol was more cytotoxic to the non-small cell lines than to the small cell lines under the in vitro growth conditions used. These studies indicate that certain human non-small cell lung cancers have metabolic characteristics of normal bronchiolar Clara cells and alveolar type II cells; these results would therefore be consistent with an origin of these tumors from Clara cells or type II cells, respectively. The present studies indicate that the further preclinical testing and development of 4-ipomeanol is warranted, with a view toward possible clinical evaluation against human lung cancers. PMID- 3011250 TI - Generation of reactive oxygen radicals through bioactivation of mitomycin antibiotics. AB - Mitomycin C (MC) is a naturally occurring anticancer agent which has been shown to be more cytotoxic to hypoxic tumor cells than to their aerobic counterparts. The mechanism of action of this agent is thought to involve biological reductive activation, to a species that alkylates DNA. A comparison of the cytotoxicity of MC to EMT6 tumor cells with that of the structural analogues porfiromycin (PM), N (N',N'-dimethylaminomethylene)amine analogue of mitomycin C (BMY-25282), and N (N',N'-dimethylaminomethylene)amine analogue of porfiromycin (BL-6783) has demonstrated that PM is considerably less cytotoxic to aerobic EMT6 cells than MC, whereas BMY-25282 and BL-6783 are significantly more toxic. The relative abilities of each of these compounds to generate oxygen free radicals following biological activation were measured. Tumor cell sonicates, reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase, xanthine oxidase, and mitochondria were used as the biological reducing systems. All four mitomycin antibiotics produced oxygen radicals following biological reduction, a process that may account for the aerobic cytotoxicity of agents of this class. The generation of relative amounts of superoxide and hydroxyl radical were also measured in EMT6 cell sonicates. BMY-25282 and BL-6783 produced significantly greater quantities of oxygen free radicals with the EMT6 cell sonicate, reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase, and mitochondria than did MC and PM. In contrast, BMY-25282 and BL-6783 did not generate detectable levels of free radicals in the presence of xanthine oxidase, whereas this enzyme was capable of generating free radicals with MC and PM as substrates. MC consistently produced greater amounts of free radicals than PM with all of the reducing systems. BMY-25282, BL-6783, and MC all generated hydroxyl radicals, while PM did not appear to form these radicals. The findings indicate that a correlation exists between the ability of the mitomycin antibiotics to generate oxygen radicals and their cytotoxicity to aerobic EMT6 tumor cells. PMID- 3011251 TI - Selective killing of simian virus 40-transformed human fibroblasts by parvovirus H-1. AB - A normal strain of human foreskin fibroblasts, two SV40-transformed derivatives with finite and infinite life spans, and an established line of SV40-transformed newborn human kidney cells are compared for their susceptibility to infection with parvovirus H-1. H-1 inocula, which do not detectably alter the growth of normal cells, cause a progressive degeneration of all three SV40-transformed cultures. The resistance of normal cells is not a membrane phenomenon since they adsorb and take up H-1 as efficiently as the transformants. Moreover, the fraction of infected cells supporting the synthesis and nuclear migration of H-1 proteins is similar in normal and SV40-transformed cultures. On the other hand, the enhanced H-1 sensitivity of transformed cells correlates with a 5- to 30-fold increase in their accumulation of newly synthesized parvoviral DNA, as compared with normal cultures. This stimulation of H-1 DNA replication is most pronounced for the amplification of duplex replicative forms, although the conversion of parental single-stranded DNA to replicative forms is also enhanced to a smaller extent. In addition, SV40-transformed cells support productive H-1 infection and release a burst of infectious virus, whereas no H-1 production can be detected in the normal cell strain. The latter difference was confirmed for another series of 7 normal and 16 SV40-transformed strains of human skin fibroblasts. Altogether, these results indicate that intracellular limitations on H-1 DNA replication are associated with the abortive nature of the parvoviral life cycle in normal human fibroblasts and are overcome after SV40 transformation, resulting in the selective killing of the transformants. This observation raises the possibility that oncolysis might contribute to the oncosuppressive activity displayed by parvoviruses in vivo. PMID- 3011252 TI - Stable integration of woodchuck hepatitis virus DNA in transplanted tumors and established tissue culture cells derived from a woodchuck primary hepatocellular carcinoma. AB - The fate of integrated woodchuck hepatitis viral (WHV) DNA was systematically investigated in DNA samples from primary hepatocellular carcinoma (HCC) of woodchucks, solid tumors transplanted in athymic mice derived from a primary HCC of woodchuck, and an established cell line of tissue culture originating from the transplanted tumor. In four of five woodchuck primary HCCs, WHV DNA integration was demonstrated in addition to various amounts of extrachromosomal replicative intermediate WHV DNA. The integration pattern of the primary HCCs does not indicate a common integration site on the host chromosome. The integration pattern in the established cells is identical to that in the transplanted tumor and similar but slightly different from that of the primary HCC. No extrachromosomal or replicative intermediates of WHV DNA were detected in the transplanted tumors or in the established cells of tissue culture. There are three integration sites on the chromosomes of the established cells. Results of Southern hybridization and restriction maps of cloned fragments suggest that all of these integrated WHV DNA sequences are not a complete genome but a part of the genome. A small portion corresponding to the cohesive region of the genome was not detected in all of these integrated WHV DNA. A positive role of WHV DNA integration on the generation of HCC is strongly suggested by the high incidence of WHV DNA integration in woodchuck primary HCCs and the stable maintenance of a certain mode of WHV DNA integration in the hepatoma-derived cell populations during passages of transplantation or serial growth of tissue culture. PMID- 3011253 TI - Induction of exocrine pancreatic, bile duct, and thyroid gland tumors in offspring of Syrian hamsters treated with N-nitrosobis(2-oxopropyl)amine during pregnancy. AB - We examined the effect on the Syrian hamster fetal pancrease of N-nitrosobis(2 oxopropyl)amine (BOP), a potent pancreatic carcinogen in adult hamsters. Pregnant Syrian hamsters (F0 generation) were treated with BOP (10 mg/kg body weight) at the 8th, 10th, 12th, and 14th days of gestation (for a total dose of 40 mg/kg body weight). Treatment was well tolerated and all hamsters delivered, at term, pups (F1 generation = 24 females and 27 males) with no abnormalities in number per mother or in physical and behavioral conditions, when compared to matched F1 controls (20 females and 17 males). The experiment was terminated when hamsters in each group (F0 and F1) were 46 weeks old. Pancreatic tumors were found in 89% of the BOP-treated F0 generation and in 5 (50%) of their male litters, but none was seen in their female progeny or in any hamsters from the F1 control group. Tumors in the BOP-treated F0 generation hamsters were ductular adenomas (78%), ductular carcinomas in situ (11%), and ductal/ductular carcinomas (33%). Tumors in their litters were ductular adenomas (20%), ductular carcinomas in situ (10%), and poorly differentiated tumors (20%) that resembled human pancreatoblastomas. The incidence of common duct polyps (44%), gallbladder polyps (44%), and cholangiomas (44%) was significantly higher in the BOP-treated F0 generation than in their litters (which had incidences of 10, 0, and 40%, respectively). Pulmonary and renal neoplasms occurred only in the F0 generation, whereas ovarian and thyroid gland neoplasms were found only in the F1 generation. Results indicate a differing susceptibility of fetal and maternal tissues to BOP. PMID- 3011254 TI - Ah receptor mediating induction of aryl hydrocarbon hydroxylase: detection in human lung by binding of 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin. AB - In laboratory animals and in mouse hepatoma cells in culture the Ah receptor previously has been shown to mediate induction of aryl hydrocarbon hydroxylase (cytochrome P1-450) by 3-methylcholanthrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. We examined human lung cytosols to determine whether the Ah receptor was present in human tissues. Cytosol was prepared from grossly normal lung tissue obtained at pulmonary lobectomy for presumed lung cancer from 53 consecutive adult patients including 32 males (42-77 years old) and 21 females (18-81 years old). Ah receptor in the cytosols was identified and quantitated by specific binding of [3H]TCDD after separation by ultracentrifugation on sucrose gradients. Specific binding of [3H]TCDD to a component which met the criteria for Ah receptor was detected in 10 of the 53 specimens. As previously established in tissues from laboratory animals, the specific [3H]TCDD-binding component sedimented approximately 9S. Binding of [3H]TCDD to the 9S component was competitively inhibited by incubation in the presence of 2,3,7,8-tetrachlorodibenzofuran, dibenz(a,h)anthracene, and nonradioactive TCDD, all known to be potent agonists for Ah-receptor-mediated induction of aryl hydrocarbon hydroxylase. Specific Ah receptor also was detected in some specimens by direct binding of [3H]-3-methylcholanthrene. The human population studied exhibited striking heterogeneity in Ah receptor concentrations. Only 10 of the 53 individuals studied had detectable Ah receptor. In specimens with detectable specific binding, the mean concentration of binding sites was 6.9 +/- 1.2 (SE) fmol/mg cytosolic protein. These concentrations are approximately 10-30% of the concentrations of Ah receptor found in lung cytosols from laboratory animals. Our experiments indicate that the Ah receptor can be detected in lung cytosol from some humans and suggest that the regulatory mechanism mediating human cytochrome P1-450 induction may be similar to that in the murine model. Aryl hydrocarbon hydroxylase, the major enzyme induced under control of the Ah receptor, plays an important role in the metabolism of several carcinogens including polycyclic aromatic hydrocarbons such as benzo(a)pyrene. It is possible that differences in the Ah receptor content within the human population may be genetically based and that variation at the Ah receptor level may be an important determinant of individual susceptibility to certain chemically induced cancers. PMID- 3011255 TI - Myoclonus and parkinsonism. AB - We have observed three patients in whom the onset of parkinsonian signs was clinically associated with myoclonic movements. In each case, the parkinsonian signs were relatively mild and myoclonus was the major abnormality. The presentation of these three unusual cases within 1 year may be coincidental or may reflect a common pathogenetic mechanism. Adrenocorticotropic hormone was given to all three patients. It ameliorated the myoclonus in two patients but had no effect on the third. PMID- 3011256 TI - Neurotoxicity and efficacy of combined vinca alkaloids in breast cancer. AB - We treated 17 patients with metastatic breast cancer with the four-drug combination of vincristine (0.5-1.0 mg), vinblastine (4 mg/m2), doxorubicin (40 mg/m2), and cyclophosphamide (400 mg/m2). The two vinca alkaloids appeared to be synergistic in causing acute, reversible neurotoxicity, manifested primarily as diffuse pains involving one or more of jaws, limbs (particularly joints), chest, and abdomen. The response rate of 70% was no higher than that noted previously with a regimen identical to this one except for the exclusion of vincristine. Further studies of combined vinca alkaloids are not recommended in the treatment of breast cancer. PMID- 3011258 TI - Neurotransmitter receptor binding studies predict antiemetic efficacy and side effects. AB - Radioligand binding studies were used to analyze the interaction of antiemetics with central dopamine D2 and alpha-adrenergic1 receptors. The affinity of antiemetics for dopamine D2 receptors labeled by 3H-spiperone significantly correlated with clinically effective drug doses (r = 0.92; P less than 0.01). Furthermore, the relative inhibition of alpha-adrenergic1 receptors as measured by 3H-WB 4101 binding predicts the clinical side effects of sedation and orthostatic hypotension. Neurotransmitter receptor binding analysis provides a rapid and sensitive technique for measuring antiemetic potency as well as associated side effects. PMID- 3011257 TI - Randomized dose-response evaluation of etoposide in small cell carcinoma of the lung: a Southeastern Cancer Study Group Trial. AB - To evaluate postulated dose-response relationships of etoposide (VP-16) for patients with recurrent small cell carcinoma of the lung, a prospectively randomized study was undertaken. VP-16 was administered iv at three dose levels: 300, 600, and 900 mg/m2. Based on historical information, a 20% response rate was anticipated in the standard-dose level and the study was designed to be able to detect a response rate of 40% in either of the high-dose levels. The planned number of patients required in each arm was 45, with an alpha-level of 0.1 and a beta-level of 0.2. The total number of patients actually entered was less than the planned number due to a low response rate. Seventy-seven of 79 treated patients were eligible, and 26, 27, and 26 patients were treated at each dose level, respectively. Toxicity was predominantly hematologic, with the higher dose levels substantially more toxic. Response to therapy was infrequent, with only four partial responses achieved and distributed between all dose levels. In this study, using previously treated patients, VP-16 at standard-dose or at moderate dose increments had minimal activity. PMID- 3011259 TI - Vindesine and mitomycin in the treatment of advanced non-small cell lung cancer: a Southeastern Cancer Study Group Trial. PMID- 3011260 TI - Possible mitoxantrone-induced amenorrhea. AB - A patient with adenoid cystic carcinoma was treated with mitoxantrone at a dose of 12 mg/m2 every 3 weeks. After the fifth dose, she developed amenorrhea accompanied by vasomotor instability (hot flashes). Subsequent serum gonadotropin and estradiol levels confirmed the postmenopausal state. The mechanism of mitoxantrone-associated amenorrhea is most likely due to direct toxic effects on the ovary, as is seen with other forms of chemotherapy. Ovarian dysfunction with mitoxantrone has not been previously reported. The important consequences of chemotherapy-induced ovarian failure are described. This toxic effect may be more frequently described as the drug becomes more widely available. PMID- 3011261 TI - Phase II study of mitomycin in small cell lung carcinoma. PMID- 3011262 TI - Distribution of intrapleural and intravenous Corynebacterium parvum in humans; 99mTc-, and 131I-labeled bacteria. AB - The distribution of Corynebacterium parvum labeled with 131iodine or 99mtechnetium was studied in 17 patients with bronchogenic carcinoma. The labeled bacteria were given intravenously or intrapleurally and monitored by whole-body gamma tracking and samples of blood and urine. Even though the rate of physical decay is quite different for 131iodine and 99mtechnetium, the tracking time of labeled bacteria was limited to 24 h after injection for both radioactive isotopes. Technetium labeling was preferred because of greater imaging resolution and less radiation dose to the patient. Following intravenous administration, labeled C. parvum was found predominantly in the liver and spleen, and in a lesser amount in the lung. Radioactivity was confined to the pleural cavity after intrapleural injection. These results suggest the combined intravenous and intrapleural route of adjuvant immunosupportive agents such as C. parvum for operable lung cancer patients. PMID- 3011263 TI - Immunomodulating activity in supernatants from EBV immortalized lymphocytes. AB - Our earlier work has demonstrated that EBV immortalized B lymphocytes are involved in a factor dependent autostimulatory cycle. Soluble growth stimulating activity was released into culture supernatants by these growing B cells. Growth enhancing (GE) media from B lymphocyte lines, immortalized by EBV infection, contained soluble factor(s) which modulated the Con A response of normal human mononuclear cells. Conditioned media from these lines affected the Con A response in a biphasic manner, stimulating the blastogenic response at lower concentrations, while inhibiting at higher concentrations. At stimulatory concentrations, the blastogenic response to Con A began earlier than in controls and was markedly enhanced by day 2. GE media reduced the initial response of purified B cells to pokeweed mitogen. GE media did not support growth of IL-2 dependent cells. GE media from some EBV-carrying B cell lines had measurable IL-1 activity in the mouse thymocyte PHA response. GE media from LPS stimulated B lymphocyte lines produced significant IL-1-like effects on stimulated mouse thymocytes. These results suggested that these B cell lines may produce IL-1-like factors that cooperate in T cell responses. The possibility that such factors may play a role in B lymphocyte transformation by EBV is discussed. PMID- 3011264 TI - [Changes in the activity of plasma renin and blood aldosterone and in that of the enzyme converting angiotensin I to angiotensin II during the dialytic treatment of patients with chronic renal insufficiency]. PMID- 3011265 TI - [Effects of antihypertensive therapy with enalapril maleate on morphological and functional parameters of the left ventricle in hypertensive subjects]. PMID- 3011267 TI - Cecal angiodysplasia localized by 99mtechnetium blood-pool scintigraphy and specimen venography. AB - Angiodysplasia of the right colon may be difficult to diagnose. The usual methods of choice, selective abdominal angiography and colonoscopy, may be impracticable or fail. When bleeding occurs, 99mtechnetium blood-pool scintigraphy is a simple and reliable method of localizing vessel leakage. We present a case of an 84-year old woman with severely bleeding angiodysplasia, where the source of bleeding was localized by means of scintigraphy using 99mTc in vivo labeled red blood cells. After right hemicolectomy, the angiodysplastic lesion was confirmed by specimen venography utilizing a barium gelatin mixture. PMID- 3011266 TI - Pathophysiology of hypertension in the elderly. AB - Combined systolic and diastolic arterial hypertension and isolated systolic hypertension in the elderly are proven risk factors for stroke, sudden death, coronary artery disease, and congestive heart failure. Because hemodynamics, vascular and cardiac adaptations, fluid volume, and endocrine functions are distinctly altered in the elderly hypertensive patient compared with a younger patient, antihypertensive treatment should be individualized, and an unsophisticated regimen, such as a stepped-care approach, is too rigid to be as beneficial for elderly hypertensive patients as for young hypertensive patients. PMID- 3011268 TI - Correlation between effects of hydralazine on force and on the adenyl cyclase system of ventricular myocardium in dogs and cats. AB - Hydralazine is a potent arteriolar dilator, which increases cardiac output in patients with heart failure. Previous studies suggested that these beneficial effects might be due in part to a positive inotropic effect. The present study further investigated the effect of hydralazine on myocardial contractility and adenyl cyclase activity. In isolated cat papillary muscles, bath concentrations of hydralazine up to 10(-4) mol X litre-1 did not alter force development, whereas 10(-3) mol X litre-1 hydralazine increased isometric force by 31%. This effect was blocked by 10(-6) mol X litre-1 propranolol and was absent after catecholamine depletion produced by previous reserpine treatment. In canine ventricular myocardium hydralazine in all concentrations used (10(-7) to 10(-3) mol X litre-1) increased control adenyl cyclase activity. This increase was statistically significant in 10(-6) to 10(-3) mol X litre-1 concentrations, reaching a maximum of 69.5% at 10(-4) mol X litre-1. In cat ventricular myocardium 10(-6) to 10(-3) mol X litre-1 hydralazine increased the cyclic AMP production, although to a lesser magnitude than that in canine tissue. Hydralazine 10(-5) mol X litre-1 produced a 37.8% increase, and the maximum effect of 45.2% occurred at 10(-3) mol X litre-1. The positive effects of hydralazine were completely abolished by the addition of propranolol in dogs as well as in cats. Thus the adenyl cyclase stimulation induced by hydralazine is mediated through the beta receptor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011269 TI - Mechanisms and time course of beta 1 adrenoceptor desensitisation in mammalian cardiac myocytes. AB - To study the mechanisms and time course of beta1 adrenoceptor desensitisation in mammalian heart tissue neonatal rat cardiac myocytes (greater than 90% pure) were cultured in serum free medium. Cells were exposed to 1 mumol X litre-1 (-) isoprenaline for 30 min, 4 h, and 16 h. In myocyte membranes mean(SEM)) 125I iodocyanopindolol binding was 167(46) pmol X litre-1 (n = 5) and did not differ at 30 min, 4 h, or 16 h in control compared with (-)-isoprenaline treated cells. The maximum number of binding sites was 84(32) fmol X mg protein-1 and was unchanged at 30 min, but (-)-isoprenaline stimulated adenylate cyclase activity significantly decreased from 221(62) to 103(37) pmol X mg protein-1 30 min-1. (-) Isoprenaline competition curves at 30 min showed a significant increase in the proportion of low affinity binding sites from 46% to 62% (n = 5). By 4 h the maximum number of binding sites was significantly decreased by 54%, adenylate cyclase activity remained depressed, and agonist affinity decreased threefold in the (-)-isoprenaline treated cells. At 16 h (-)-isoprenaline treated cells showed alterations similar to the 4 h values in the maximum number of binding sites, adenylate cyclase activity, and affinity for (-)-isoprenaline. (-)-Isoprenaline stimulated adenylate cyclase activity took 72 h to recover after desensitisation. Overnight ultracentrifugation of the cytosol showed a significant 40% increase in beta adrenoceptor density in cells exposed to (-)-isoprenaline for 4 h (n = 5), suggesting receptor internalisation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011270 TI - [Comprehensive diagnostic approaches in tumor metastases in the skeleton. Results of radionuclide examination]. PMID- 3011271 TI - Consequences of widespread deregulation of the c-myc gene in transgenic mice: multiple neoplasms and normal development. AB - We have constructed a transgenic mouse strain in which a mammary tumor virus LTR/c-myc fusion gene is anomalously expressed in a wide variety of tissues. The deregulated c-myc transgene, now glucocorticoid inducible, contributes to an increased incidence of a variety of tumors, including those of testicular, breast, lymphocytic (B cell and T cell), and mast cell origin. The deregulated gene does not, however, otherwise disturb cell proliferation, nor does it interfere with normal development in these animals. Moreover, since not all tissues that express the transgene develop neoplasms, these results begin to define the transforming spectrum of the c-myc oncogene. They also extend to several organ systems the notion that elements in addition to an activated c-myc gene are required to induce malignancy in the living organism. PMID- 3011272 TI - Chromatin assembly during SV40 DNA replication in vitro. AB - A cytosol extract from human 293 cells supports efficient replication of SV40 origin-containing plasmid DNA in the presence of the SV40 T antigen. Addition of a nuclear extract from the same cells promotes negative supercoiling of the replicated DNA but not the bulk of the unreplicated DNA. The level of superhelicity is affected by the concentrations of T antigen and nuclear extract factors and by the time of addition of the nuclear extract. The replicated DNA in isolated DNA-protein complexes resists relaxation by purified HeLa cell topoisomerase I. Micrococcal nuclease digestion, sucrose gradient sedimentation, and electron microscopy demonstrate that the negative supercoils result from assembly of the replicating DNA into a chromatin structure. These results suggest that, during DNA replication, the core histones can be assembled on both sides of the replication fork by an active, replication-linked mechanism that does not require a template of preexisting nucleosomes. PMID- 3011273 TI - The use of psoralen-modified DNA to probe the mechanism of enhancer action. AB - Psoralen-modified DNA was used to study the SV40 enhancer-dependent transcription of the human beta-globin gene. When the enhancer is separated from the beta globin gene by psoralen adducts on one side and plasmid vector sequences on the other, expression of the gene is strongly inhibited. When placed on the same side of the enhancer as the vector sequences, psoralen adducts have little effect on transcription unless they are located near the transcriptional start site. These results suggest that the inhibition of the transcription of a gene linked to an enhancer in a circular DNA requires the presence of blocking agents on both sides of the gene and that the vector sequences are already blocking enhancer action on one side. Psoralen monoadducts are sufficient to inhibit transcription; the formation of interstrand psoralen cross-links is unnecessary. We assess models for the enhancer mechanism in light of these results. PMID- 3011274 TI - Transcription of the human beta-globin gene is stimulated by an SV40 enhancer to which it is physically linked but topologically uncoupled. AB - One postulated mechanism for how the SV40 enhancer stimulates transcription of linked genes involves the enhancer as a binding site for a sequence-specific "gyrase" activity. We sought to test this hypothesis directly by constructing a novel heteroduplex circle, termed a tailed-circle, in which one of the strands contains an extra palindromic sequence base-paired into a hairpin structure. The human beta-globin gene is placed in the circle and the SV40 enhancer on the hairpin tail, where a bound topoisomerase cannot supercoil the circle. Upon transfection of this DNA into HeLa cells the SV40 enhancer on the hairpin arm is still able to stimulate transcription of the beta-globin gene. Southern blot analysis of the DNA after transfection does not demonstrate any repair or replication of the tailed-circle in vivo. These results argue against the sequence-specific gyrase model for SV40 enhancer action. PMID- 3011275 TI - The enfolding arms of EcoRI endonuclease: role in DNA binding and cleavage. AB - The N-terminal segments of the EcoRI endonuclease dimer form part of mobile "arms" that encircle DNA in the recognition complex. By treating endonuclease TCGCGAATTCGCG complexes with proteases, we have prepared a series of deletion derivatives lacking defined segments of the N-terminal region. The 5-12 segment is essential for DNA cleavage and forms one electrostatic interaction (per subunit) with DNA phosphate. These ionic contacts are directly across the double helix from the scissile phosphodiester bonds; they thus may permit the enfolding arms to immobilize DNA in apposition to the catalytic cleft and/or contribute to the unusual "kinked" conformation of DNA in the complex. Sequence specificity is fully retained when 28 residues are deleted from the N-terminus, but the complexes dissociate more rapidly. PMID- 3011276 TI - Autocrine saturation of pro-urokinase receptors on human A431 cells. AB - Single-chain pro-urokinase (pro-uPA) is present both in the medium and lysate of the A431 epidermoid carcinoma cell line. Most of the cell-associated pro-uPA is on the cell surface, as shown by indirect immunofluorescence and by surface iodination. Pro-uPA is not an integral membrane protein but is bound to a specific surface receptor that is completely saturated. A mild acid treatment uncovers the surface receptors by dissociating pro-uPA. Resaturation of uncovered receptors has been studied by reincubating cells in normal medium; within 40 min, 50% of the free sites are reoccupied. Excess uPA-specific antibodies prevent rebinding of ligand to the receptors. Thus, A431 cells first secrete uPA, which then binds to the surface receptor. We propose that the synthesis of uPA and uPA receptor by the same cell may provide a pathway for the activation of the metastatic potential of malignant cells. PMID- 3011277 TI - Structure and function of the S1 nuclease-sensitive site in the adenovirus late promoter. AB - We analyzed an S1 nuclease-sensitive site present in supercoiled, but not linear, recombinant plasmids containing the adenovirus late promoter. S1 nicking was detected on both strands, primarily in the TATA box. Analysis of deletion mutants showed that sequences upstream of -47 and downstream of -12 are not required for S1 cutting. However, a number of different base substitution mutations in stretches of G residues upstream and/or downstream of the TATA box were sufficient to eliminate S1 cutting. When the transcriptional activities of these mutant promoters were assayed in vivo, six of seven mutants lacking the ability to form the S1-sensitive structure showed no reduction in transcriptional potential. In fact, several showed increased promoter activities. These data show that the S1 nuclease cutting site in the adenovirus late promoter has precise nucleotide sequence requirements for its formation. However, the ability of recombinant plasmids to adapt this conformation in vitro is not necessary for such plasmids to serve as templates for transcription in vivo. PMID- 3011278 TI - A second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein. AB - The poliovirus polyprotein is cleaved at three different amino acid pairs. Viral polypeptide 3C is responsible for processing at the most common pair (glutamineglycine). We have found that a cDNA fragment encoding parts of the capsid protein region (P1) and the nonstructural protein region (P2), and including the P1-P2 processing site (tyrosine-glycine), can be expressed in E. coli. The translation product was correctly processed. Disruption of the coding sequence of 2A, a nonstructural polypeptide mapping carboxy-terminal to the tyrosine-glycine cleavage site, by linker mutagenesis or deletion, prevented processing. Deletion of the adjacent polypeptide 2B had no such effect. Antibodies against 2A specifically inhibited processing at the 3C'-3D' processing site (tyrosine-glycine) in vitro. We conclude that poliovirus encodes the second proteinase 2A, which processes the polyprotein at tyrosine-glycine cleavage sites. PMID- 3011279 TI - Role of DNA topology in Mu transposition: mechanism of sensing the relative orientation of two DNA segments. AB - DNA strand transfer at the initiation of Mu transposition normally requires a negatively supercoiled transposon donor molecule, containing both ends of Mu in inverted repeat orientation. We propose that the specific relative orientation of the Mu ends is needed only to energetically favor a particular configuration that the ends must adopt in a synaptic complex. The model was tested by constructing special donor DNA substrates that, because of their catenation or knotting, energetically favor this same configuration of the Mu ends, even though they are on separate molecules or in direct repeat orientation. These structures are efficient substrates for the strand transfer reaction, whereas appropriate control structures are not. The result eliminates tracking or protein scaffold models for orientation preference. Several other systems in which the relative orientation of two DNA segments is sensed may utilize the same mechanism. PMID- 3011280 TI - Genetic evidence that Tn10 transposes by a nonreplicative mechanism. AB - We present genetic evidence that the tetracycline resistance element Tn10 transposes by a nonreplicative mechanism. Heteroduplex Tn10 elements containing three single base pair mismatches were constructed on lambda phage genomes and allowed to transpose from lambda into the bacterial chromosome. Analysis of TetR colonies resulting from such transpositions suggests that information from both strands of the transposing Tn10 element is transmitted faithfully to its transposition product. The simplest interpretation of these results is that the transposing element is excised from the donor molecule and inserted into the target molecule without being replicated. A mismatch 70 base pairs from one end of the transposon is preserved, suggesting that there is little or no replication, even at the termini of the element, during transposition in vivo. PMID- 3011282 TI - An anticatalytic monoclonal antibody to avian plasminogen activator: its effect on behavior of RSV-transformed chick fibroblasts. AB - A monoclonal antibody has been raised against the serine protease, plasminogen activator (PA) produced by Rous sarcoma virus-transformed chick embryo fibroblasts (RSVCEF), and selected for its ability to inhibit the catalytic activity of PA. The high specificity and anticatalytic nature of the antibody has allowed probing of the direct role of PA in cellular behavior. Microgram quantities of monoclonal IgG inhibit the overgrowth and the morphological changes associated with RSVCEF transformation and the degradation of extracellular matrix mediated by RSVCEF, indicating a catalytic role for PA in these cellular processes. Specific cleavage of extracellular matrix proteins by immunoaffinity purified PA in the complete absence of plasminogen demonstrates a direct catalytic involvement of PA in matrix degradation. PMID- 3011281 TI - DNA primase of human mitochondria is associated with structural RNA that is essential for enzymatic activity. AB - DNA primase isolated from human mitochondria sediments in glycerol density gradients at 30S and 70S. These unusually high sedimentation coefficients are a result of association of the primase activity with RNA. Treatment of primase with nuclease not only affects its sedimentation behavior, but also inactivates the primase activity. The major RNA species that cofractionates with primase activity is shown by direct sequence analysis to be cytosolic 5.8S ribosomal RNA (rRNA). Specific degradation of endogenous 5.8S rRNA using ribonuclease H and oligonucleotides complementary to 5.8S rRNA results in reduction of primase activity. Other small RNAs may play a structural role in the formation of an active DNA primase complex. PMID- 3011283 TI - The treatment of herpesvirus infections. PMID- 3011284 TI - The use of submicroscopic gold particles combined with video contrast enhancement as a simple molecular probe for the living cell. AB - We describe a new approach to probe the molecular biology of the living cell that uses small colloidal gold particles coupled to specific ligands. They are visualized in cells by bright-field, video enhanced contrast microscopy. We describe the basic aspects of the technique and provide examples of applications to intracellular motility, cell membrane dynamics, receptor translocation, internalization, and intracellular routing. We also provide examples of the use of this approach in immunospecific labelling of cells and tissue sections. PMID- 3011286 TI - [Beta-thalassemia in the Chinese: polymorphic restriction site haplotype analysis of the beta-globin gene cluster]. PMID- 3011287 TI - [Studies on the effect of lipoproteins and apoproteins on lipoprotein receptors. IV. Comparison of the physico-chemical properties of LDL between normal and hyperlipidemic rabbit serum]. PMID- 3011285 TI - Regulation of sperm flagellar movement by protein phosphorylation and dephosphorylation. AB - Flagellar motility of Triton models of sea urchin spermatozoa was reactivated by cyclic AMP-dependent protein kinase and a protein factor, termed motility activator, both of which were prepared from the detergent-extract of sea urchin spermatozoa. It was shown that phosphorylation of the motility activator by the protein kinase is necessary for the reactivation of flagellar motility [Ishiguro et al, J. Cell Biol. 92:777-782, 1982; Murofushi et al, in "Biological Functions of Microtubules and Related Structures," Academic Press, 1982]. Reactivating factor was also detected in a KCl-extract of the axoneme fraction devoid of the detergent-extractable materials. The activity of this factor was also cyclic AMP- and protein kinase-dependent. Furthermore, when freshly prepared Triton models were treated with phosphoprotein phosphatase prepared from bovine cardiac muscle, the flagellar motility was drastically suppressed. This inhibition of the motility was partially recovered by the addition of cyclic AMP and protein kinase to the phosphatase-treated models. PMID- 3011288 TI - [Isolation of a new strain of EB virus with lytic and transforming properties]. PMID- 3011289 TI - [Discussion on the problems in making a physical map of DNA]. PMID- 3011290 TI - [Analysis of genotypes and haplotypes determined by the polymorphic restriction endonuclease site on the beta-globin gene in the Chinese]. PMID- 3011291 TI - [Affinity of different cell lines for HpD in vitro]. PMID- 3011292 TI - [Studies on the content of trace elements in the hair of patients with Wilson's disease and healthy individuals]. PMID- 3011293 TI - [Studies on polymorphism in the beta-globin gene cluster in the Chinese I. Polymorphic restriction endonuclease hinc II site 5' to epsilon-globin gene]. PMID- 3011295 TI - [Antigenicity determination of inactivated poliovirus--the micro-neutralization inhibition test]. PMID- 3011297 TI - [Preparation and assay of calmodulin]. PMID- 3011294 TI - [Influence of gossypol on the potassium metabolism of human erythrocytes]. PMID- 3011296 TI - [Studies on platelet aggregation and 14C-serotonin release in patients with uremia]. PMID- 3011298 TI - [Determination of aqua armeniacae in syrupus bulbus Fritillariae cirrhosae]. PMID- 3011299 TI - [Effect of "bai-jin-jiang-zhi-wan" on blood lipids, blood rheology and cyclic nucleotides in rats with experimental hyperlipidemia]. PMID- 3011300 TI - [Benign pathology of the breast. Diagnostic protocol and therapeutic indications]. AB - The frequency of benign mammary disease and the need to differentiate between benign and malignant tumors without the indiscriminate use of instrumental examinations and biopsies have prompted the Cooperative Interdisciplinary Group on Mammary Neoplasms to adopt the diagnostic protocol and propose a number of therapeutic indications. Though involving a progressive reduction in biopsies, the use of the protocol has not led to failure to recognize carcinomas, the number of identified malignancies being maintained within the anticipated limits. PMID- 3011301 TI - Human tanapox in Zaire: clinical and epidemiological observations on cases confirmed by laboratory studies. AB - Human tanapox, a mild disease characterized by a short febrile illness associated with one or more skin lesions, is important because of its possible confusion with smallpox. The article describes clinical and epidemiological features of 264 laboratory-confirmed tanapox cases observed in a geographically limited area in northern Zaire over the period 1979-83. PMID- 3011302 TI - Improved surveillance of Japanese encephalitis by detection of virus-specific IgM in desiccated blood specimens. AB - An IgM antibody-capture type enzyme-linked immunoassay (MAC ELISA) was compared with the haemagglutination inhibition method (HI) for establishing a laboratory diagnosis of acute Japanese encephalitis (JE) virus infection using specimens of dried blood eluted from filter paper strips. Paired samples from 243 encephalitis patients, which had been obtained by mail through a national surveillance programme in Thailand, were tested. During the peak of the 1983 encephalitis epidemic, 72% of cases were diagnosed as Japanese encephalitis by MAC ELISA, compared with only 38% by HI. During non-epidemic periods, the proportions diagnosed as Japanese encephalitis by MAC ELISA or HI were 26% and 33%, respectively. Detection of IgM anti-JE activity by the antibody-capture immunoassay is superior to the HI method for establishing a diagnosis of acute Japanese encephalitis using dried blood specimens. PMID- 3011304 TI - Carcinogen induced asynchronous replication of polyoma DNA is mediated by a trans acting factor. AB - We have demonstrated that carcinogen damage to DNA induces the production of cellular factors that act in trans to enhance the asynchronous replication of polyoma viral DNA. Exposure of a polyoma virus-transformed rat cell line to benzo[a]pyrene-7,8-diol-9,10-oxide (BPDE), the ultimate carcinogenic metabolite of benzo[a]pyrene, led to the accumulation of heterogeneously sized free viral DNA molecules which contain polyoma origin sequences as well as cellular sequences that flank the integrated viral DNA. When the sequence gpt was linked to the polyoma early region and transfected into rat cells, it underwent asynchronous replication in response either to direct treatment of the transfected cells with BPDE, or to fusion of untreated transfected cells with normal cells previously exposed to BPDE. Transient arrest of the cell cycle by hydroxyurea, isoleucine deprivation or methotrexate caused a slight enhancement of viral DNA replication when compared with BPDE. Both aphidicolin, an inhibitor of DNA polymerase alpha, and 3-aminobenzamide, an inhibitor of poly[ADP]ribosyl transferase, caused marked inhibition of BPDE-induced viral DNA synthesis. The induction of a trans-acting factor in response to damage of cellular DNA may be relevant to synergistic interactions between environmental chemicals and DNA viruses in cell transformation and to the general phenomenon of gene amplification. PMID- 3011303 TI - The use of the single radial haemolysis technique in the serological diagnosis of dengue and Japanese encephalitis virus infections. AB - The single radial haemolysis test for the serological diagnosis of suspected dengue and Japanese encephalitis virus infections uses crude virus antigens with a haemagglutinin titre of 1:320 or 1:640. The results, which may be read 3 hours after the addition of a patient's serum, showed a general agreement between this test and haemagglutination-inhibition tests in the number of case diagnoses that were confirmed. The antibody responses of individual patients shown by the two tests, however, were different, which suggests that the two tests may not be measuring the same antibody. The single radial haemolysis test can distinguish between dengue and Japanese encephalitis viruses using specific mouse hyperimmune sera. Tests on a limited number of sera from Japanese encephalitis patients also showed no cross-reactions with dengue virus antigens in those cases having a low titred but significant fourfold antibody rise to Japanese encephalitis antigen. PMID- 3011305 TI - Effects of anti-promoters and strain of mouse on tumor promoter-induced oxidants in murine epidermal cells. AB - The induction of oxidant production in mouse epidermal cells by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) can be suppressed by many, but not all, known inhibitors of mouse skin tumor promotion. Members of the anti oxidant category that were tested were ranked in the following order, 7,8 benzoflavone greater than butylated hydroxyanisole greater than ascorbic acid. In the retinoid category, retinoic acid was only moderately effective, while the trimethoxymethylphenyl analog had at least twice the inhibitory activity. Among the six protease inhibitors examined, tosylphenylalanine chloromethylketone and tosyllysine chloromethylketone were effective in diminishing the response while tosylarginine methylester, antipain, leupeptin and soybean trypsin inhibitor were ineffective, suggesting that proteases are probably not involved in the oxidant response. Several agents, trifluoperazine, trisialoganglioside and diolein, that have been shown to suppress TPA activity in other cell systems were also found to suppress the oxidant response. Finally, the extent of the oxidant response was found to correlate with sensitivity to TPA tumor promotion among the three strains of mice tested: SSIn greater than SENCAR greater than C57BL/6J. PMID- 3011306 TI - Biochemical significance of enhanced activity of fluorinated 1,25 dihydroxyvitamin D3 in human cultured cell lines. AB - Several human cancer cells possess receptors for 1,25-dihydroxyvitamin D3[1,25 (OH)2D3]. In these cells 1,25-(OH)2D3 has a biphasic concentration-dependent regulatory effect on cell replication and specifically induces its own metabolism. We have studied the effects on these parameters of the native hormone together with those of two analogues fluorinated at the 24-carbon and of 1,24R,25 trihydroxyvitamin D3[1,24R,25-(OH)3D3]. The difluorinated analogue 24,24-difluoro 1,25-(OH)2D3[24,24-F2-1,25-(OH)2D3] is an approximately fivefold more potent inhibitor of cellular replication than the native hormone, while 1,24R,25-(OH)3D3 is about fivefold less potent. This enhanced potency of the fluorinated analogue parallels its enhanced potency in in vivo studies of its effects on calcium and mineral metabolism. However, although the analogue retains replication stimulatory activity, it is clearly no more potent than the native hormone in this activity: 1,24R,25-(OH)3D3 has no significant stimulatory activity. Exposure of the cells to 1,25-(OH)2D3 at 0.05 nM for 6 h increases the subsequent conversion of labelled hormone to aqueous phase soluble compounds by 6.7-fold. None of the other compounds had a similar effect at this concentration. At 10 nM all 1-hydroxylated compounds increased aqueous phase radioactivity about equally (13 to 17-fold); this effect is still specific since 25-OH D3 had no such effect even at 10 nM. Studies on the effects of the fluorinated analogues upon receptor binding of hormone in cell cytosols and uptake of hormone by intact cells clearly demonstrate that the enhanced activity of these analogues is not due to higher receptor affinity or more rapid access to intracellular receptor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011308 TI - Free radicals and myocardial injury: pharmacologic implications. PMID- 3011307 TI - George E. Brown memorial lecture. Role of atrial peptides in body fluid homeostasis. AB - Extracts of mammalian atria, but not ventricles, induce marked diuresis, natriuresis, and reduction in blood pressure when infused systemically in rats and dogs. These extracts also inhibit aldosterone biosynthesis and renal renin release. Natriuretic peptides, 21 amino acids and longer, have been isolated from atria of rodents and man, and share a nearly homologous amino acid sequence at the carboxyterminus. Natriuretic activity resides in a 17-amino acid ring formed by a disulfide bridge, and the C-terminal Phe-Arg appears necessary for full biological potency. The deoxyribonucleic acid-encoding atrial natriuretic peptides have been cloned and the gene structure elucidated. Reduction of the diuretic and natriuretic responses to an acute volume load by right atrial appendectomy first suggested a role for atrial peptides in the physiological response to plasma volume expansion. Subsequently, release of peptides with natriuretic and spasmolytic properties from isolated heart preparations in response to right atrial distension was demonstrated by bioassay and radioimmunoassay. The presence of these peptides in normal rat and human plasma in concentrations of 20-100 pM, and the findings of increased levels in response to acute and chronic plasma volume expansion, rapid atrial tachyarrhythmias, systemic hypertension, congestive heart failure, and renal insufficiency imply that they play an important role in body fluid homeostasis. The mechanisms by which atrial peptides increase renal salt and water excretion are as yet unclear. Renal vascular effects have been consistently demonstrated, and limited evidence for direct actions on tubule ion transport has also been reported recently. In vitro, these peptides cause precontracted vascular and nonvascular smooth muscle to relax, mediated by a direct action on smooth muscle cells. Specific receptors for these peptides have been characterized in crude membranes prepared from whole kidney homogenates and adrenal glomerulosa cells, in intact glomeruli and cultured glomerular mesangial cells, and in intact bovine aortic smooth muscle and endothelial cells. Natriuretic peptides stimulate cyclic guanosine monophosphate accumulation in target tissues, and augment particulate guanylate cyclase activity in membrane fractions, suggesting that cyclic guanosine monophosphate is the second messenger mediating their cellular action. PMID- 3011309 TI - AIDS and the practice of ophthalmology. PMID- 3011310 TI - Effect of nedocromil sodium on the immediate response to antigen challenge in asthmatic patients. AB - In this double-blind placebo controlled crossover study the protective effect of nedocromil sodium against bronchial allergen challenge was evaluated in ten asthmatic patients. Single doses of 0.5, 1.0 and 2.0 mg nedocromil sodium, administered as a pressurized aerosol 3 hr prior to challenge, were each significantly more effective than placebo in inhibiting allergen-induced bronchoconstriction. The 1.0 and 2.0 mg doses of the active drug were significantly more effective than the 0.5 mg dose. No significant difference was demonstrated between the 1.0 and 2.0 mg doses. Duration of protection beyond 3 hr was not tested. The results suggest that nedocromil sodium may be a potentially useful therapeutic agent and is worthy of further evaluation for the treatment of reversible obstructive airways disease. PMID- 3011311 TI - Prospects for modifying the allergic response by fish oil diets. PMID- 3011312 TI - Perchloric acid interference in enzymatic-fluorimetric-continuous-flow assay methods for measuring glucose, lactate, pyruvate, alanine, glycerol, and 3 hydroxybutyrate in blood. AB - Perchloric acid is commonly used to denature and precipitate proteins in samples before various metabolites are measured in tissue, blood, and other body fluids. However, perchloric acid can interfere in the analytical process, possibly by inhibiting the enzymes used. We have determined the effects of perchloric acid on measurements of glucose, lactate, pyruvate, alanine, glycerol, and 3 hydroxybutyrate in blood by enzymatic-fluorimetric-continuous-flow assays. There was a net increase or decrease in the apparent concentration of some of these metabolites when the perchloric acid concentration in the samples differed from that of the reference standards-some of these differences were due to the concentration of perchlorate ion and some to the pH of the acid extracts. The results show the need either to add a fixed amount of blood to perchloric acid or to neutralize and remove the perchlorate. PMID- 3011313 TI - Increased lactate dehydrogenase isoenzyme 1 in serum and tumor tissue of a patient with small-cell carcinoma. AB - Histological examination of supraclavicular lymph node tissue obtained at biopsy from a 63-year-old man disclosed metastatic small-cell carcinoma. On admission and for four days subsequently, total lactate dehydrogenase (LD; EC 1.1.1.27) activity in serum was 6.5 times normal; studies of LD isoenzyme showed persistently increased LD-1, with LD-1 greater than LD-2. Isoenzyme electrophoresis of tissue homogenates prepared from the patient's tumor also showed the LD-1 greater than LD-2 pattern. Isoenzyme studies for supraclavicular lymph node tissue from five control subjects showed contrasting isoenzyme patterns as compared with the patients in whom LD-2, LD-3, and LD-4 predominated. Because these abnormalities were persistent, they differ from the temporal sequence for LD usually seen in myocardial infarction. This emphasizes the importance of repetitive sampling for clinical interpretation of data on this enzyme. PMID- 3011314 TI - Peroxidase activity and estradiol receptors in human breast cancer. AB - In order to establish the relation between peroxidase activity in human breast carcinoma and its estrogen-dependency, estradiol receptors and their peroxidase activities were determined by a very sensitive fluorimetric method in 61 carcinomas. The geometrical mean of peroxidase activity in the 34 estradiol receptor negative carcinomas, 12.8 mU/g tissue, was significantly higher (p less than 0.05) than that of the 27 estradiol receptor positive cases, with a mean of 10.57 mU/g tissue. Necrosis and/or inflammation were found more frequently in the estradiol receptor negative carcinomas (p less than 0.01) than in the estradiol receptor positive. It is concluded that peroxidase activity is linked more to the typical biological behavior of the tumor than to tumoral estrogen-dependency. Thus, the estradiol receptor negative tumors, which showed a more aggressive behavior associated with frequent necrosis and intratumoral inflammatory signs, had a greater peroxidase activity. PMID- 3011317 TI - Determination of volatile fatty acids by gas chromatography on a capillary and a megabore column. PMID- 3011316 TI - Estimation of NADH oxidation in human skeletal muscle mitochondria. AB - Assay procedures are described for the detection of defects in the process of NADH oxidation by the respiratory chain in human skeletal muscle biopsy specimens. The procedures allow determination of rotenone-sensitive NADH: O2 oxidoreductase and NADH: ubiquinone-1 oxidoreductase activity not only in isolated mitochondria but also in post-nuclear supernatants. The use of ferricyanide as electron acceptor for estimation of NADH dehydrogenase activity is inadequate when only applied on a disrupted mitochondrial preparation. PMID- 3011318 TI - IgG thyrotrophin receptor antibody activity in Graves' disease; a study of TSH agonist and antagonist activities by isoelectric focusing. AB - The distribution of TSH receptor antibody activity in the 7S and 19S fractions of Graves' sera has been re-evaluated. Serum fractions were obtained by gel filtration from 12 Graves' sera and assayed for TSH receptor binding activity in a radioreceptor assay. Thyroid stimulating activity was determined in a cultured porcine thyroid cell bioassay. In apparent contrast to the findings of Baker et al. (1983) TSH receptor binding activity was confined to the 7S gel filtration fraction, containing IgG, and was not detected in the 19S fraction, containing IgM. Similarly thyroid stimulating activity was detected only in the 7S fraction. 7S fractions from seven Graves' sera were fractionated by isoelectric focusing and the fractions analysed for TSH receptor binding activity and TSH agonist and antagonist activities. Five of the IgGs showed TSH agonist activity and in all five, the peak thyroid stimulating activity (measured by stimulation of cyclic AMP release from isolated porcine thyroid cells) was in fractions with a pI of between 8.0 and 9.5. In four of these five IgGs, TSH receptor binding activity showed similar isoelectric distribution to the thyroid stimulating activities. High levels of TSH receptor binding activity without associated TSH agonist or antagonist activity were however observed in some isoelectric fractions of the fifth stimulating Graves' IgG studied. All the isoelectric fractions from the fifth IgG with thyroid stimulating activities contained TSH receptor binding activity. Two of the Graves' IgGs showed TSH antagonist activity and both the TSH receptor binding and TSH antagonist activities of these IgGs showed similar isoelectric distribution with the peak activities at a pI of around 9.0. Consequently, it was not possible to separate TSH agonist or TSH antagonist activities from TSH receptor binding activity in seven Graves' sera by isoelectric focusing although in one IgG several isoelectric fractions contained isolated receptor binding activity. These findings are in keeping with the hypothesis that the biological activities of Graves' IgGs are intimately related to their ability to bind to the TSH receptor. PMID- 3011315 TI - Rapid diagnosis of vitamin D-dependent rickets type II by use of phytohemagglutinin-stimulated lymphocytes. AB - The interactions of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] with phytohemagglutinin (PHA)-stimulated lymphocytes from normal subjects and three patients with vitamin D-dependent rickets (DDR) type II were investigated. Impaired nuclear uptake and normal cytosol binding of [3H]1,25-(OH)2D3 were observed with PHA-stimulated lymphocytes of these patients as with their cultured skin fibroblasts. Furthermore, the incorporation of [14C]thymidine into PHA stimulated lymphocytes of the patients was not reduced by 1,25-(OH)2D3, which is known to inhibit proliferation of various cells. These findings suggest that 1,25 (OH)2D3 receptors are reduced or absent in patients with DDR type II. Thus, the capacities of cytosol binding and nuclear uptake of 1,25-(OH)2D3 in PHA stimulated lymphocytes seem to reflect those of endo-organs such as the intestine and bone. These findings show that a test of the effect of 1,25-(OH)2D3 on thymidine incorporation into PHA-stimulated lymphocytes is useful for rapid diagnosis of DDR type II. PMID- 3011319 TI - TSH receptor antibody synthesis by thyroid lymphocytes. AB - Several indirect observations have indicated that lymphocyte in the thyroid may be an important site of TSH receptor antibody synthesis in Graves' disease and we now describe an investigation of this possibility using improved lymphocyte isolation and TSH receptor antibody assay procedures. Our studies demonstrate that thyroid lymphocytes spontaneously produce TSH receptor antibody in culture. Furthermore, experiments with mitogen tend to suggest that these cells, in contrast to lymphocytes from lymph nodes draining the thyroid, are part of an active immune response to the TSH receptor. PMID- 3011321 TI - Alpha-human atrial natriuretic polypeptide-induced rise of plasma and urinary cyclic 3'5'-guanosine monophosphate concentration in human subjects. AB - Synthetic alpha-hANP was infused into 8 normotensive, euvolemic volunteers to examine the effects on plasma and urinary cyclic-GMP. The dose given and duration of alpha-hANP (0.1 micrograms/kg/min, 20 min) led to the significant diuresis, saliuresis and hypotensive effects in each subject. The mean plasma cyclic-GMP concentration was clearly elevated at 5 min after infusion and attained 8 fold of the mean basal concentrations by the end of infusion. The mean urinary cyclic-GMP excretion rate also showed 3.5 fold increases of the basal excretion rate with synthetic alpha-hANP administration (before loading: 0.69 +/- 0.22 p moles/g creat., after loading: 2.85 +/- 0.28 p moles/g creat.). However, there was no apparent change on plasma or urinary cyclic-AMP during the infusion. The result clearly demonstrated that synthetic alpha-hANP selectively induces the cyclic-GMP in concomitant with the marked diuretic, natriuretic and hypotensive effects in human subjects. PMID- 3011322 TI - Aging and vasodilation to atrial peptides. AB - Alterations in the levels or activity of atrial peptides may be associated with cardiovascular pathologies such as hypertension and congestive heart failure that are more common in the elderly. In the present study, we examined the possibility that vascular relaxation to atrial peptides may be affected by animal age. In vitro evaluation of the relaxation produced by atrial peptide-25 (AP-25) in serotonin-contracted rat aorta, carotid artery and mesenteric artery indicated that relaxation was greatest in tissues from rats 1-2 months of age. Nevertheless, roughly 80% of the maximal possible relaxation to AP-25 occurred in arteries from older rats (up to 18-19 months of age). A similar profile of relaxant responsiveness occurred with AP-21 (atriopeptin I). Unlike the arterial preparations examined, portal veins from rats of all ages relaxed similarly to AP 25, consistent with the lack of age-related changes in relaxation to beta agonists in the rat portal vein. In vivo, AP-25 given intravenously lowered blood pressure to a similar extent in rats of all ages. Thus, the greater in vitro sensitivity of arteries from rats 1-2 months of age did not result in a greater reduction in blood pressure in these young rats. This latter observation is consistent with the possibility that the effect of atrial peptides on blood pressure may not be associated with an arterial event, but rather with an alteration in venous return and/or cardiac output as previously proposed. Since arteries from older rats were able to respond to atrial peptides, our studies add further support to the impetus to develop clinical agents that might either enhance atrial peptide levels or mimic the action of atrial peptides since no major decline in receptor function of atrial peptides occurred with advanced age. PMID- 3011320 TI - Measurement of insulin-like growth factor-II by radioreceptor assay using ovine placental membranes. AB - A new radioreceptor assay for insulin-like growth factor-II (IGF-II), using receptors on ovine placental membranes, is described. Half-maximal displacement of specifically bound radioiodinated human IGF-II tracer was seen at 1.0 ng/tube of unlabelled IGF-II. The cross-reactivity of IGF-I was 1%, and insulin was entirely without effect. Measured on serum samples from 100 healthy adults, the mean IGF-II concentration (+/- SD) was 576 +/- 160 ng/ml. Identical mean values were seen for all adult age groups up to 65 years. The mean value for 10 acromegalic adults was 583 +/- 155 ng/ml, and for 9 GH-deficient subjects, 161 +/ 26 ng/ml (P less than 0.001 compared to normals). Of eight patients with chronic renal failure, none had an IGF-II level less than 2SD above the normal mean. No significant effect of renal dialysis was seen. In groups of patients with gastric, breast, lung, testicular, oat cell, ovarian, colonic and prostatic carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, sarcoma and teratoma (5-12 patients per group), mean IGF-II levels were in the lower part of the normal range. Thus this study does not provide evidence supporting a role for excessive IGF-II production in the growth of any of these tumour types. PMID- 3011323 TI - Fine needle aspiration biopsy in salivary gland tumours. AB - Of 105 tumours of the major salivary glands, 90 were benign and 15 malignant. In benign tumours a correct preoperative diagnosis was made by fine needle aspiration biopsy in 84%, and none were falsely classed as malignant. In the malignant tumours, only 8 out of 15 (53%) were correctly diagnosed as malignant while 7 were misdiagnosed as benign. It is concluded that in benign salivary gland tumours there is good accordance between fine needle aspiration biopsy and the final histological report, in contrast to the malignant tumours where this is less convincing. Fine needle aspiration biopsy is a valuable diagnostic tool, but the result should be carefully evaluated, regarded as only part of the clinical picture and not solely relied on. PMID- 3011324 TI - Not all potently neutralizing, vaccine-induced antibodies to Epstein-Barr virus ensure protection of susceptible experimental animals. AB - Cottontop tamarins have been immunized with a high molecular weight, Epstein-Barr (EB) virus membrane antigen (MA) glycoprotein (gp340) separated by monoclonal antibody immunoaffinity chromatography (MCABgp340). Specific antibody production was monitored by immunofluorescence, a highly sensitive enzyme-linked immunosorbent assay (ELISA), and virus neutralization tests, and was found to reach high titre after 4/5 inoculations. The animals were challenged with a 100% lymphomagenic dose of EB virus but despite possessing powerful neutralizing antibodies were not protected against tumour causation by the virus. This result contrasts with that of earlier experiments in which tamarins with neutralizing antibodies induced by gp340 prepared by a molecular weight-based method (MWgp340) were protected. The reasons for this difference in protection associated with vaccine molecules prepared in different ways are discussed together with the need for parameters other than neutralizing antibody for use in the assessment of subunit immunogens against EB virus. PMID- 3011325 TI - Secretory IgA and IgM antibodies to E. coli O and poliovirus type I antigens occur in amniotic fluid, meconium and saliva from newborns. A neonatal immune response without antigenic exposure: a result of anti-idiotypic induction? AB - Antibodies to E. coli O-antigens and poliovirus type I antigen as well as total SIgA were analysed using enzyme-linked immunosorbent assay (ELISA), in amniotic fluid and in meconium, urine and saliva from neonates. Secretory IgA and IgM antibodies to E. coli and poliovirus antigens were found in saliva as well as in most meconium samples taken during the first day of life. SIgA could be quantified in all types of samples including amniotic fluid. The finding of secretory IgA and IgM antibodies to E. coli and poliovirus type I antigens in early samples from an infant of a hypogammaglobulinaemic mother, given regular intravenous (i.v.) immunoglobulin prophylaxis, but still lacking IgA and IgM antibodies, supports a fetal origin for at least part of the secretory antibodies detected in the different samples. Since it is unlikely that the fetus has been exposed to poliovirus, which is rare in Sweden, it is hypothesized that the stimulus inducing the SIgA and IgM antibodies found in the neonate could be anti idiotypic antibodies from the mother. PMID- 3011327 TI - The effect of aging on alpha-adrenoceptors and their responses in rabbits. AB - The effects of age on alpha-adrenoceptor responses, sensitivity and number were studied in rabbits aged from 1 to 36 months. Three types of investigation were carried out: conscious animal studies, isolated tissue studies and radioligand binding studies. Specific [3H]-prazosin binding decreased with age in both spleen and heart suggesting that the number of alpha 1-receptors declined at least in the tissues studied. The specific binding of [3H]-clonidine to spleen membranes and [3H]-yohimbine to platelets was not affected by age. In vitro responsiveness to alpha-adrenoceptor agonists decreased with age in abdominal aorta and renal artery, while the affinity of adrenoceptors for prazosin (pA2) was not altered. The decrease may be non-specific as responses to potassium were also altered. No change in alpha 2-adrenoceptor mediated platelet aggregation was observed. No change in pressor or depressor responses to full adrenoceptor agonists or to antagonists was observed in vivo. However, responses to clonidine, which is a partial agonist at alpha 1-adrenoceptors, were decreased. While aging influenced alpha-adrenoceptor subtypes differently, there was no direct relation between functional changes and number of receptors. PMID- 3011326 TI - T lymphocyte regeneration after transplantation of T cell depleted allogeneic bone marrow. AB - The regeneration of T cell subsets was studied with double immunofluorescence marker methods in 37 patients who received HLA matched T lymphocyte depleted bone marrow transplants (BMT) as part of the treatment for their haematological disease. A cocktail of anti-pan-T (CD6: MBG6) and anti-suppressor/cytotoxic-T cell (CD8: RFT8) monoclonal antibodies was used with rabbit serum as a source of cytolytic complement to achieve selective T cell lysis. The T8+ cells reached low normal values around 60 days post-transplant and remained within the normal range throughout the study (greater than 150 days). This observation is in contrast to our previously published results in patients who, after receiving BMT without efficient T cell depletion, had increased numbers of circulation T8+ cells from 60 days post-transplant. In the present study Leu-7+, RFT8- cells reached normal values rapidly but the reconstitution of T4+ lymphocytes was slow: low normal levels were reached only around day 150 following BMT. The degree of graft-versus host disease (GVHD) seemed to be related to the number of residual T cells infused: two of the three patients who received the highest numbers of T cells developed Grade II III; otherwise GVHD was minimal. Among the clinical parameters studied cytomegalovirus (CMV) immune status moderately influenced reconstitution: at 55-90 days post-transplant T8+ cells were present at the upper normal levels in seven out of 15 patients receiving BMT from CMV seronegative donors, but in none of the 16 individuals receiving BMT from seropositive donors. CMV related complications were relatively uncommon. Thus the most significant factor in preventing 'T8+ cell overshoot' and T cell imbalance during regeneration appears to be the depletion of T (including T8+) lymphocytes from marrow. The differences of T8+ cell reconstitution in this and previous studies may reflect a different regeneration pattern from T cell precursors as opposed to inoculated mature T cells. PMID- 3011328 TI - The effect of the 'hypertensinogenic' steroid, 9 alpha-fluorocortisol on blood pressure in sheep with ACTH-induced hypertension. AB - The effect of treatment with 9 alpha-fluorocortisol (9 alpha FF), a steroid which causes hypertension in sheep, was examined in sheep with ACTH-induced hypertension. ACTH treatment alone increased mean arterial pressure (MAP), plasma Na concentration, water intake and urine volume and decreased plasma K concentration. 9 alpha FF treatment, for 3 days during continuing ACTH administration, did not change blood pressure but increased heart rate, water intake and urine volume and decreased urinary K excretion. As 9 alpha FF did not cause a further increment in blood pressure in sheep with ACTH-induced hypertension it is possible that both ACTH and 9 alpha FF may produce hypertension by similar mechanisms. PMID- 3011329 TI - Time course of changes in blood pressure, aldosterone and body fluids during enalapril treatment: a double-blind randomized study vs hydrochlorothiazide plus propranolol in essential hypertension. AB - Aldosterone suppression is said to play a major role in the long term hypotensive efficacy of angiotensin converting enzyme inhibitors. However, in previous reports from other laboratories, plasma volume has been found mostly increased and sodium balance sometimes positive. The effects of the angiotensin converting enzyme inhibitor enalapril (10-40 mg/day, p.o., for 6 weeks) on blood pressure, body fluid volumes, renal function and plasma aldosterone were compared to those of hydrochlorothiazide (50 mg/day, p.o.) alone for 2 weeks and in association with propranolol (80-160 mg/day, p.o.) for 4 more weeks during a randomized double-blind parallel study in 14 essential hypertensives. Hydrochlorothiazide alone and in combination with propranolol induced slight and not significant change in either blood pressure and body fluids. The maximum hypotensive response to enalapril was achieved only after 2 weeks of continuous treatment possibly because after 1 week the hypotensive efficacy was lessened by a significant (P less than 0.05) fluid retention secondary to a transient and not significant fall in renal perfusion. At this time aldosterone was not significantly changed compared to pretreatment values. After 6 weeks on enalapril, blood pressure was significantly reduced, plasma aldosterone further but not significantly decreased and extracellular fluid volume was normal. These findings indicate that aldosterone suppression contributes to the blood pressure lowering effect of enalapril by offsetting the salt and water retention observed on starting treatment and due to direct vasodilation. PMID- 3011330 TI - The relationship between tissue levels of cyclic GMP and tracheal smooth muscle relaxation in the guinea-pig. AB - The effect of cyclic GMP was investigated using guinea-pig tracheal smooth muscle. 8-Bromo-cyclic GMP showed a dose-dependent relaxation of spontaneous tension of tracheal smooth muscle. Administration of sodium nitroprusside induced dose-dependent relaxation of tracheal smooth muscle as well as an increase in tissue levels of cyclic GMP. Nicorandil, N-(2-hydroxyethyl) nicotinamide nitrate showed dose-dependent relaxation of tracheal smooth muscle and an increase in cyclic GMP levels in the tissue. N-(2-aminoethyl) nicotinamide dihydrochloride, which is a nicorandil derivative and differs minimally in its molecular structure (-NH2 vs -NO2), had neither a relaxant effect on tracheal smooth muscle nor did it increase the level of cyclic GMP in the tissue. The rise cyclic GMP levels preceded the relaxation of tracheal smooth muscle induced by sodium nitroprusside. These results suggest that cyclic GMP is one of the relaxant factors and that nitro-derivatives exhibit their relaxant effect on the smooth muscle mediated by an increase in cyclic GMP level. PMID- 3011331 TI - Characterization of thromboxane A2 receptors in the human fetal placental vessels and umbilical vein. AB - An in vitro examination has been made of the thromboxane A2 receptors in human fetal placental villous vessels and umbilical veins utilizing the TxA2 agonist U46619 and its competitive antagonist AH22921. U46619 was a potent constrictor of both preparations. The EC50 were 25.3 nmol/l (s.e.m. = 2.5, n = 8) for causing constriction of perfused villous vessels and 22 nmol/l (s.e.m. = 5, n = 17) for contraction of the venous longitudinal strip. AH22921 competitively antagonized responses to U46619 in both preparations. The pA2 values were not significantly different and their 95% confidence limits, obtained for its ability to antagonize responses to U46619 in villous vessels and the umbilical vein, were 8.0 (7.3-8.8) and 7.1 (6.3-7.9) respectively. It is concluded that TxA2 receptors in both the human fetal placental villous vessels and umbilical vein may be similar. They also may be similar to those in human platelets and pulmonary blood vessels. PMID- 3011332 TI - The mediators of allergic contraction of human airway smooth muscle: a comparison of bronchial and lung parenchymal strip preparations. AB - Antigen-induced contractions of passively sensitized preparations of human bronchi and lung parenchymal strips were studied. Mepyramine did not affect the magnitude or time course of the contractile responses of either preparation. Indomethacin potentiated the responses of lung parenchymal strips, but inhibited those of bronchial preparations. Responses of both tissues were inhibited by the leukotriene antagonist FPL 55712, by the lipoxygenase inhibitor BW 755C, and by the anti-allergic compound disodium cromoglycate Disodium cromoglycate and BW 755C inhibited histamine release from lung parenchymal strips. The results indicate an important role for leukotrienes in allergic contraction of both large and small human airways, while histamine is of little importance. Bronchoconstrictor prostaglandins may also contribute to the response of large airways. PMID- 3011333 TI - Neuromuscular blocking activity of a crude aqueous extract of Ipomoea fistulosa. AB - The rat phrenic nerve hemidiaphragm and chick biventer-cervicis preparations were used to investigate the effect of a crude aqueous extract of Ipomoea fistulosa. Electrically-induced skeletal muscle twitches of the hemidiaphragm were blocked by the extract and by tubocurarine. The anticholinesterase, physostigmine, intensified the blockade by the extract. The extract produced contracture of the chick biventer-cervicis preparation. It is suggested that the extract possesses neuromuscular blocking activity of a depolarizing nature. PMID- 3011334 TI - [What is the effect of hydroxylapatite ceramics on bone formation?]. PMID- 3011335 TI - [Pleomorphic adenoma as a risk factor. A case report]. PMID- 3011336 TI - Morphological and biochemical observations in rat nephron epithelia following cyclosporine A (CsA) treatment. AB - Morphology, urinary enzyme excretion and mitochondrial function was studied in rats fed over a period of 30 days with 20 and 40 mg CsA/kg body weight. Already on day 8 vacuolisation and augmentation of autophagic vacuoles, lipid droplets and a loss in microvilli can be observed in the S-3 segment of the proximal nephron using the lower CsA dose. These effects are enhanced during the treatment period. The overall effect, however, is a subtle one. The dose of 40 mg/kg produces more pronounced cellular alterations, a more severe vacuolisation, but also focal prenecrotic damage of proximal tubular S-2 and S-3 cells. The cells altered in that manner amount to roughly 5% of the total proximal tubular epithelium. Enhanced urinary excretion of the proximal cytosolic enzymes, fructose-1,6-bisphosphatase, glutathione S-transferase, pyruvate kinase and the lysosomal N-acetyl-beta-D-glucosaminidase appear to parallel the morphologic changes, whereby only the excretion of pyruvate kinase is significantly elevated on day 30 using 40 mg/kg. Decrease in oxidative phosphorilation capacity (ADP:O ratio) was found with both CsA doses, however, this result seems to be dissociated from changes in morphology and enzyme excretion. Studies on isolated tubular fragments in vitro, exposed to CsA exhibited an inhibition of the cytosolic malate dehydrogenase isoenzyme, which could be interpreted as a possible source of the CsA induced tubular alteration. PMID- 3011337 TI - Cyclosporine nephrotoxicity: comparative cytochemical study of rat kidney and human allograft biopsies. AB - The aim of this study was to detect early renal changes in the rat. Female Wistar rats received oral doses of cyclosporine (12.5, 25 or 50 mg/kg daily). The duration of the experiment was 1, 2, and 3 weeks. Controls received the vehicle only (olive oil). The following alterations were seen by light microscopy: Hypertrophy of the juxtaglomerular apparatus (PAS stain). Cytoplasmic droplets of neutral fat (Oil Red 0) in clusters of cortical tubules, probably belonging to the same nephron. Both the above phenomena increased with dosage and duration of treatment and were absent in controls. In the fat containing tubulus (FCT) brush border staining (alkaline phosphatase) was decreased or absent. Since after PAS the brush border was visualized in many FCT, it is concluded that many FCT were proximal tubulus (PT) of which the brush border has been damaged. In FCT mitochondrial staining (Cytochrome oxidase activity) was strongly decreased or absent. Mean lysosomal volume (acid phosphatase and dipeptidase II) is increased in the PT; in some cyclosporine animals, lysosomes were enlarged, while in others they were comparable to controls. Electron microscopy showed in some PT cells an increased number of empty vacuoles and focal alteration of mitochondria. Normal mitochondria were present next to grossly altered mitochondria. Autophagocytosis of mitochondria was clearly present. The lysosomes appeared swollen and contained electron dense material, not organised in the typical 50 A pattern of myeloid figures. These morphological changes suggest a defect of mitochondrial metabolism, leading to lipid deposition in PT. The mitochondrial metabolism can be disturbed by a direct toxic effect of cyclosporine or indirectly via ischemia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011338 TI - Alterations in molecular structure of renal mitochondria associated with cyclosporine A treatment. AB - We examined whether ultrastructural changes in renal mitochondria associated with Cyclosporine A treatment might reflect underlying alterations in mitochondrial molecular structure. A nonrejected renal transplant removed from a patient treated with Cyclosporine A showed a decreased level of beta subunit antigen of mitochondrial F1-ATPase compared to controls. This decrease was more marked in the medulla than in the cortex (56% vs. 70% of pooled controls). Spontaneously hypertensive rats treated with a high dose of Cyclosporine A showed a similar decrease of beta-subunit antigen in the renal medulla and decreased levels of two subunit antigens of cytochrome c oxidase, but had increased medullary levels of the alpha subunit antigen of the F1-ATPase. Such changes were not detected in a normotensive strain of rats. Our data indicate that Cyclosporine A administration is associated with structural alterations in renal mitochondria at the molecular level, predominantly in the medulla, and that additional renal damage due to ischemia or hypertension may predispose to this cyclosporine effect. PMID- 3011339 TI - Effect of short-term cyclosporine A administration on urinary acidification. AB - This study was designed to investigate the short-term effect of cyclosporine A (CyA) at a dose of 25 mg/kg body weight, on urinary acidification and renal potassium handling. Rats treated with CyA for 8 days developed metabolic acidosis (Blood pH 7.34 +/- 0.01, Blood HCO3 20 +/- 0.9 mEq/l) while those treated for 3 days did not (Blood pH 7.39 +/- 0.01, HCO3 24 +/- 1.0 mEq/l). Fractional HCO3 excretion was low in both groups indicating that bicarbonate reabsorption in the proximal nephron was unimpaired. Distal hydrogen ion secretion evaluated by the ability to increase urinary pCO2 in a highly alkaline urine was impaired in both groups (urinary pCO2 61 +/- 2.3 mmHg and 50 +/- 2.5 mmHg in rats treated with CyA for 3 and 8 days, respectively as compared to controls 72 +/- 3.0 mmHg, p less than 0.01). Under basal conditions, renal potassium excretion was lower in CyA treated rats than in controls. This was observed in association with a decrease in GFR in rats treated with CyA for 8 days (GFR 1.3 +/- 0.3 ml/min) but not in those treated for 3 days (GFR 2.2 +/- 0.4 ml/min). Rats treated with CyA for 3 days were able to increase potassium excretion normally in response to both sodium sulfate infusion and to an acute potassium infusion. In rats treated with CyA for 8 days, acute potassium loading failed to elicit an increase in fractional potassium excretion (from 32 +/- 5.3 to 28 +/- 2.3%) despite an increase in plasma K (from 3.0 +/- 0.2 to 8.4 +/- 0.3 mEq/l) and urine flow (from 11 to 36 mu ml/min).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011340 TI - Treatment of septic olecranon and prepatellar bursitis with percutaneous placement of a suction-irrigation system. A report of 12 cases. AB - Ten cases of septic olecranon bursitis and two cases of septic prepatellar bursitis were treated in the period from 1975 to 1980 with antibiotics and percutaneous tube placement for suction-drainage and local antibiotic irrigation. All patients had positive bacterial cultures: Staphylococcus aureus in nine, beta hemolytic Streptococcus in two, and Staphylococcus epidermidis in one. Intravenous antibiotics, local suction-drainage, and irrigation with a solution of 1% kanamycin and 0.1% polymyxin controlled the infection in each case. The antibiotic treatment averaged 19 days, compared with 24 days in a series in which suction-irrigation was not used. In contrast with studies in which aspiration or incision and drainage were performed, there were no complications or recurrences. Percutaneous suction-irrigation appears to be a safe, effective method of treatment that is particularly beneficial in severe cases of septic bursitis in which continuous drainage is desirable. PMID- 3011341 TI - [Study on adrenocorticotrophin immunoactivity in adrenoleukomyeloneuropathy]. PMID- 3011342 TI - [A case of mamushi envenomation resembling myasthenic syndrome with a waxing phenomenon]. PMID- 3011344 TI - Heterogeneity of anterior pituitary cell antibodies detected in insulin-dependent diabetes mellitus and adrenocorticotropic hormone deficiency. AB - A sensitive assay method for pituitary cell antibodies (PitCA) was established by a biotin/avidin system using rat pituitary. Results in 24 cases out of 81 insulin dependent diabetic patients and 10 cases of 21 adrenocorticotropic hormone (ACTH) deficient patients were positive for autoantibodies to anterior pituitary cell cytoplasm. PitCA observed in the sera of insulin-dependent diabetic patients were suspected of being pituitary specific and independent with islet cell antibodies (ICA) and islet cell surface antibodies (ICSA). In the sera of ACTH deficient patients, PitCA were frequently absorbed with liver acetone powder. Populations of insulin-dependent diabetes mellitus (IDDM) are almost equal in males and females. A total of 16 cases out of 21 ACTH deficient patients were female. These results suggest that heterogenous PitCA are involved in the sera of those patients with IDDM and ACTH deficiency. PMID- 3011343 TI - Intracellular sodium concentration and transport in red cells in essential hypertension, hyperthyroidism, pregnancy and hypokalemia. AB - Intracellular sodium content ([Nai]), ouabain-sensitive ('Na-K ATPase') and ouabain-insensitive ('passive permeability') sodium efflux, Na-K cotransport and Na-Li ('Na-Na') countertransport were estimated in erythrocytes in 39 control subjects, 20 patients with essential hypertension, 14 patients with hypokalemia of renal or unknown etiology, 13 hyperthyroid patients and 19 pregnant women. In normokalemic essential hypertension there was only a moderate, but significant elevation of the activity of the Na-Li countertransport system. In the group of patients with hypokalemia, there was a significant increase of [Nai], ouabain insensitive sodium efflux and Na-Li countertransport. In hyperthyroidism, a marked decrease of Na-Li countertransport was associated with a marked elevation of [Nai], in pregnancy an elevation of the Na-Li countertransport with a [Nai] 43% lower than the control values. The ouabain-sensitive sodium efflux was elevated in hyperthyroidism and hypokalemia, in which [Nai] was increased. In the control subjects there was a positive linear correlation between ouabain sensitive sodium efflux and [Nai]. The sodium component of the Na-K cotransport was decreased to about one third of the unchanged furosemide-sensitive potassium component during pregnancy. CONCLUSIONS: The changes of cellular sodium metabolism in essential hypertension are of minor degree as compared to those in the other conditions studied. Cellular sodium metabolism in blood cells is influenced by thyroid hormones and metabolic disorders. Na-Li countertransport, i.e. Na-Na countertransport, seems to be involved in the regulation of [Nai]: an increase of its activity diminishes [Nai] (pregnancy); a decrease elevates [Nai] (hyperthyroidism). Ouabain-sensitive sodium efflux, i.e. 'Na-K ATPase', is mainly regulated by its substrate, [Nai]. PMID- 3011345 TI - Proton chemical shift imaging. AB - Proton resonance spectra contain two broad classes of hydrogen-containing compounds: those that are fat-like and those that are water-like. Proton chemical shift imaging is a means to produce water or fat proton images. Three methods of proton chemical shift imaging are discussed. PMID- 3011346 TI - Some aspects of the use of contrast agents in magnetic resonance imaging. AB - The parameters for signal intensity in magnetic resonance imaging (MRI) are classified and shortly described. Proton density, T1 and T2 are discussed as tissue parameters to be influenced in different ways by different types of contrast agents. Conclusions for the development of contrast agents for MRI are drawn. The influence of the concentration of paramagnetic substances on signal intensity is shortly described. The good tolerance of Gd-DTPA as a contrast agent for MRI is reported. Its favorable use is shown in three examples of brain tumors. PMID- 3011347 TI - In vivo tissue analysis by NMR spectroscopy. AB - Nuclear magnetic resonance (NMR) spectroscopy can be applied to study metabolism and physiology in living tissues and organisms. It is based upon the ability to identify a number of important metabolites in the NMR spectra. Limiting factors are sensitivity and resolution. Concentrations of small metabolites are 4 orders of magnitude lower than those of tissue water and lipids. The consequent reduction in signal intensity leads to the need of signal averaging, the use of surface coils, and large volumes of interest. High magnetic fields are necessary, both to improve spectral resolution and sensitivity. 31P NMR allows one to measure various phosphate metabolites, such as ATP, phosphocreatine (PCr), and inorganic phosphate (Pi). From the chemical shift of the P1 resonance it is possible to determine the intracellular pH value. 31P NMR is therefore particularly suited to follow energy metabolism. 1H NMR spectroscopy can also be used to measure small metabolites. To do this it is necessary to implement techniques for suppression of the intense water and lipid signals. It has been possible to measure various metabolites, such as lactate, N-acetylaspartate, and amino acids in the brains of laboratory animals. 13C NMR spectroscopy can be used to measure and characterize high-concentration components such as lipids and glycogen. The introduction of 13C-labeled substrates allows one to follow metabolism by the 13C NMR method. PMID- 3011348 TI - Abnormal B-cell response to T-cell-independent polyclonal B-cell activators in homosexuals presenting persistent generalized lymph node enlargement and HTLV-III antibodies. AB - B-cell functions were investigated in a well-defined high-risk group for the development of AIDS/AIDS-related complex (ARC). Stimulation of mononuclear cells (MNC) with T-cell-independent polyclonal B-cell activators failed to increase high spontaneous IgG levels observed in vivo and in vitro. The secretion of IgM following stimulation with Klebsiella M (Klebs M) or Salmonella (Salm) membrane preparation increased by a factor of 4 to 6 and thus ranged between the results of the control group and those of AIDS/ARC patients; the response to a T-cell independent B-cell mitogen, Staphylococcus aureus Cowan I (SAC), showed profound abnormalities as well in this group. This indicates that functional B-cell abnormalities can be seen in addition to T-cell dysfunctions in patients at increased risk for the development of AIDS/ARC. PMID- 3011349 TI - T-CLL and allied diseases: new insights into classification and pathogenesis. PMID- 3011350 TI - The role of magnetic resonance imaging techniques in the evaluation of orbital and ocular disease. AB - Technical developments have resulted in a great improvement in the quality of magnetic resonance imaging (MRI) of the orbit. With surface coil data acquisition spatial resolution of less than a millimetre can now be achieved, and contrast discrimination is such that the cortex and nucleus of the lens can be distinguished. The application of MRI to the diagnosis of orbital and ocular pathology was studied in a group of 51 patients with a wide range of pathology. Advantages of MRI over computed tomography (CT) included the avoidance of ionising radiation, the direct multiplanar facility and the use of flow-dependent sequences to identify pathological vessels without the need for contrast medium; although CT was superior in showing bone detail. Only a limited discrimination between different tumour types is possible by assessment of their MRI characteristics. Research is currently being directed towards achieving thinner slices, shorter data acquisition times and removal of the high signal from retrobulbar fat. PMID- 3011351 TI - Alterations of intestinal membrane-bound enzymes in three types of hypertensive rats. AB - Alkaline phosphatase, sucrase, Na+,K+-ATPase and Mg2+-ATPase specific activities of crude membrane fractions, prepared from duodenal, jejunal, ileal and colonic mucosa, have been estimated in three types of hypertensive rats: the spontaneously hypertensive rat (SHR), the DOCA-saline treated rat and the renovascular rat (Goldblatt one-kidney, one-clip rat; 1K-1C). Alkaline phosphatase and sucrase specific activities have been measured in purified jejunal brush-border membranes. When compared with its normotensive age-matched control (WKY rat), the SHR has a lower activity of alkaline phosphatase in duodenal and jejunal crude membrane fractions, whereas a higher activity in colonic Na+,K+-ATPase is recorded. In purified jejunal brush-border membranes, lower alkaline phosphatase activity and higher sucrase activity were found. These differences occur in the young prehypertensive SHR as well as in the adult animal. In the DOCA-treated rat, the only significant alteration in crude membrane fractions is a decreased Mg2+-ATPase activity at all regions of intestinal mucosa. In purified jejunal brush-border membranes both alkaline phosphatase and sucrase activities are increased at 4 or 7 weeks but especially at 13 weeks of hypertension. In the 1K-1C rat, no significant modification appears in crude membrane fractions or in purified jejunal brush-border membranes, but a decrease in alkaline phosphatase and in sucrase activities is probable after 13 weeks of hypertension. Since alterations of the intestinal enzymes are different in the three types of hypertensive rats it is concluded that the changes are not secondary to the hypertension condition. In the SHR, these alterations are present in the young prehypertensive animal.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011352 TI - The effects of alcohol on local, neural and humoral cardiovascular regulation. PMID- 3011353 TI - An eight-year study of the viral agents of acute gastroenteritis in humans: ultrastructural observations and seasonal distribution with a major emphasis on coronavirus-like particles. AB - During an 8-yr period, 862 stool specimens from patients with gastroenteritis were examined by electron microscopy after negative staining with 2% phosphotungstic acid (pH 6.5). Forty-one percent of the specimens submitted over an 8-yr period were determined to be positive for virus or viruslike particles belonging to one or more of seven morphologically distinct viral groups. Coronavirus-like particles (CVLPs) were present in 69.8% of the positive stool specimens. Membranous profiles containing "complement-type" holes (10 nm in diameter) were identified in some preparations containing CVLPs. The second most prevalent viral agent found in stool specimens was the rotavirus (17% of all positive stools). The incidence of other viruses identified in the survey were as follows: adenovirus 4.5%, picorna/parvovirus agents 2.9%, Norwalk-like agent 2.9%, astrovirus 1.9%, and calicivirus 0.5%. Unclassified small round viruses (approximately 25-30 nm in diameter) represented 0.5%. It was also determined that there was a seasonal distribution in excretion of all viruses except for CVLPs. A greater number of viruses were identified in the cooler, drier months of the year. PMID- 3011354 TI - CT evidence of calcification within a small cell carcinoma of the lung. AB - Calcification in bronchogenic carcinoma is rare. When found it is usually focal in nature and felt by many to be coincidental granulomatous disease. To our knowledge this case report is the first computerized tomography (CT) description of diffuse, amorphous calcification in bronchogenic carcinoma, specifically, small cell. PMID- 3011355 TI - Hemorrhagic primary intracranial neoplasms: clinical-computed tomographic correlations. AB - Ten patients with symptomatic peritumoral hemorrhage were analyzed. The neoplasms included glioblastoma multiforme (9 cases) and oligodendroglioma (1 case). Two presented with stroke syndromes and initial CT showed an intracerebral nonenhancing hematoma. There was clinical improvement with CT evidence of resolution of the hematoma; however clinical deterioration occurred within 4-6 weeks and CT findings were consistent with an enhancing neoplasm. Eight patients had the sudden onset of neurological symptoms. The neurological deficit subsequently stabilized (2 cases) or worsened (6 cases). Initial CT showed evidence of an intracerebral hemorrhage on the noncontrast scan with evidence of heterogeneous ring enhancement on the post-contrast CT scan. PMID- 3011356 TI - Magnetic resonance study of the structure and functions of cytochrome P450. AB - Cytochrome P450 is a membrane-bound enzyme providing oxidation of numerous organic compounds in organisms. The objective of this review is to show the wide possibilities that are provided by Electron Spin Resonance (ESR) and Nuclear Magnetic Resonance (NMR) techniques to the study of the structure and functions of this unique enzyme. High sensitivity of ESR spectra of cytochrome P450 to its functional state and interaction with substrates and inhibitors is illustrated. NMR and proton relaxation make it possible to obtain unique information about the structure of the active center of cytochrome P450 under physiological conditions. ESR and NMR methods allow one to obtain structural data on location of substrates, inhibitors, and their spin-labeled analogs with respect to Fe3+ ions in the enzyme-active center. Of special interest seems to be coupling of ESR with the affinity modification method. For this purpose, the spin-labeled analogs of cytochrome P450 substrates containing alkylating groups were used. As a result, an important datum has been obtained on the structure of active centers of cytochrome P450 in microsomes and in a highly purified state. In conclusion, the problems of the structure and functions of cytochrome P450, which can be most efficiently resolved with the use of magnetic resonance methods, are discussed. PMID- 3011357 TI - Food applications and the toxicological and nutritional implications of amorphous silicon dioxide. AB - The chemical and physical characteristics of the different types of amorphous silicon dioxide contribute to the versatility of these compounds in a variety of commercial applications. Traditionally, silicas have had a broad spectra of product usage including such areas as viscosity control agents in inks, paints, corrosion-resistant coatings, etc. and as excipients in pharmaceuticals and cosmetics. In the food industry, the most important application has been as an anticaking agent in powdered mixes, seasonings, and coffee whiteners. However, amorphous silica has multifunctional properties that would allow it to act as a viscosity control agent, emulsion stabilizer, suspension and dispersion agent, desiccant, etc. The utilization of silicas in these potential applications, however, has not been undertaken, partially because of the limited knowledge of their physiochemical interactions with other food components and partially due to their controversial status from a toxicological point of view. The main goal of this review is to compile current information on the incorporation of amorphous silicon dioxide as a highly functional and viable additive in the food processing industry as well as to discuss the most recent toxicological investigations of silica in an attempt to present some of the potential food applications and their concomitant toxicological implications. Some of the more significant differences between various silicas and their surface chemistries are presented to elucidate some of their mechanisms of interaction with food components and other biological systems and to aid in the prediction of their rheological or toxicological behavior. PMID- 3011358 TI - Sodium-potassium-dependent-ATPase activity in Emory mouse lens. AB - Previous morphological and biochemical studies indicate that a late appearing hereditary Emory mouse cataract may be a good model for certain human senile cataracts. The development of lenticular opacity in the Emory mouse is a slow process which provides an opportunity to conduct analysis of the progression of alterations that lead to cataract development. Biochemical investigations have not yet demonstrated any specific correlation between alterations in the lens and the extent of opacity. We have conducted studies to determine the role of Na+K+ ATPase in the development of cataract in the Emory mouse. In this report we present results obtained on the site and level of activity of Na+K+-ATPase in six and twelve-month-old Emory mouse lenses in which visible cataractous changes are beginning to appear. CFW mice (the parent strain) were used for controls in this study. Ultrastructural cytochemistry for the localization of Na+-K+-ATPase exhibited the enzyme reaction product for this enzyme to be present mainly between the lateral epithelial cell membranes and between the apical epithelial cell membranes and superficial cortical fiber membranes. In cortical fibers the reaction product was localized between fiber membranes. Although there was very little or no significant differences in the extent of reaction product in epithelial cells, the reaction product in the cortical fibers of six-month-old Emory mouse was less extensively distributed as compared to lenses from control CFW mice of the same age.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011359 TI - Localization and characterization of specific receptors for atrial natriuretic factor in the ciliary processes of the eye. AB - By light and electron microscope radioautography in vivo, competitive binding sites for 125I-Arg 101-Tyr 126 atrial natriuretic factor were localized mostly on the "pigmented" epithelium of the rat ciliary process. Further investigation using isolated ciliary processes from rabbits demonstrated the presence of specific receptors for 125I-atrial natriuretic factor. In addition, synthetic atrial natriuretic factor inhibited basal and stimulated adenylate cyclase activity. These results demonstrate for the first time the presence of specific receptors for atrial natriuretic factor in the ciliary processes which are negatively coupled to adenylate cyclase. The possible role of this peptide in the control of intraocular pressure is suggested. PMID- 3011360 TI - Yellow nail syndrome. AB - The case of a patient with yellow nail syndrome (YNS), an infiltrating duct carcinoma of the breast, and giant cell interstitial pneumonitis (GIP) is presented. YNS has not been previously described in association with GIP. There was improvement of the yellow fingernails following surgery and chemotherapy for the breast cancer. The possible pathogenesis of the yellow nails in this case and its management are presented. A wide variety of conditions are associated with YNS, which has been reported to respond to various treatment modalities. The most likely cause in our case is impaired lymphatic drainage. However, treatments that do not affect lymphatic drainage also have been reported to be successful. PMID- 3011362 TI - Two subsets of human alphoid repetitive DNA show distinct preferential localization in the pericentric regions of chromosomes 13, 18, and 21. AB - We have isolated and characterized two human middle repetitive alphoid DNA fragments, L1.26 and L1.84, which localize to two different sets of chromosomes. In situ hybridization revealed both repeats to have major and minor binding sites on the pericentric regions of several chromosomes. Probe L1.26 maps predominantly to chromosomes 13 and 21. Probe L1.84 locates to chromosome 18. Minor hybridization sites for both probes include chromosomes 2, 8, 9, and 20; in addition, L1.26 revealed minor sites on chromosomes 18 and 22. The binding to these sites strongly depends on hybridization conditions. In Southern blot hybridizations to total human DNA, both L1.26 and L1.84 give the same ladder pattern, with a step size of 170 bp, indicating their presence as tandem repeats, but with different band intensities for each probe. The chromosome-specific nature of particular multimers was confirmed by Southern blot analyses of a human rodent hybrid cell panel. We conclude that L1.26 and L1.84, with their related sequences, constitute subfamilies of alphoid DNA that are specific for subsets of chromosomes and, in some cases, possibly even for single chromosomes. PMID- 3011361 TI - Effect of cannabinoids on spermatogenesis in vivo: a cytological study. AB - The cytogenetic effects of delta 9-tetrahydrocannabinol (THC) and cannabinol (CBN) (10 mg/kg) were investigated in hybrid mice of genotype (C57BL x C3H)F1. Mice were treated for 5 consecutive days with the specific cannabinoid; 16 days after the last treatment the meiotic cells were evaluated. Analysis of the spermatocyte bivalents at the first meiotic metaphase failed to reveal any numerical or structural abnormality. Contrary to previous reports we failed to find any major meiotic abnormalities associated with THC and CBN treatments. There was no evidence of ring or chain figures. The centromeric banding procedure (C banding) was used to support observations of Giemsa stained cells. It has previously been reported that cannabinoids suppress the incorporation of 3H uridine into pachytene spermatocytes and round spermatids; the absence of chromosome aberrations in the present study suggests that an adverse effect appears to be present, but may be at a level other than cytogenetic. PMID- 3011364 TI - DNA polymorphisms indicate loss of heterozygosity for chromosome 11 of D98AH2 cells. AB - Studies with cell hybrids of normal diploid cells fused with tumorigenic D98AH2 (D98) cells had implicated human chromosome 11 of a normal cell as carrying tumorigenicity suppressing information. The cervical carcinoma-derived D98 (HeLa) cells contain two copies of chromosome 11. In this study, analysis of restriction fragment length polymorphism of DNA from D98 cells digested with one of nine restriction endonucleases and hybridized with five DNA probes for highly polymorphic regions on the short arm of chromosome 11 detected no heterozygosity at the insulin (INS), Harvey murine sarcoma virus 1 (HRAS1), and the beta-globin cluster (HBBC) regions. The low probability of an individual being homozygous at all these loci suggests that the Nos. 11 of the D98 cells are both copies of only one of the original homologs, or at least of the short arm segment examined. This indicates that the D98 cells could express altered or lost genes associated with tumorigenicity, even if such changes were recessive. In tumorigenically suppressed hybrids the Nos. 11 of the normal cell could then be complementing this genetic defect of the D98 cells. PMID- 3011363 TI - Linkage of cystic fibrosis to the pro alpha 2(I) collagen gene, COL1A2, on chromosome 7. AB - A linkage has been detected between the locus for cystic fibrosis (CF) and the pro alpha 2(I) collagen gene (COL1A2) which is located in the region q21.3--- q22.1 of chromosome 7. Based on the combined linkage data derived from 50 informative two-generation nuclear families collected in Canada and Denmark, the distance between COL1A2 and CF is estimated to be 19 centiMorgans. Close linkage has also been detected between COL1A2 and the DNA marker D7S15 (formerly D0CRI 917) and the serum enzyme activity marker paraoxonase (PON), both of which have previously been found linked to CF. The results of the two-point and three-point linkage analyses indicate that the most probable order of these four genetic loci is COL1A2-D7S15 - PON - CF. PMID- 3011366 TI - [Classification and malignancy grading of bone tumors]. PMID- 3011365 TI - Evaluation of the immunostimulating activity of erythromycin in man. AB - The effects of erythromycin on the immune system have been studied in healthy volunteers and patients suffering from chronic bronchopneumonial diseases, by means of the following assays: phagocytosis, natural killer activity and superoxide anion production. The tests were performed before and after oral administration of 1 g of erythromycin. The findings suggest that erythromycin enhances phagocytosis by means of increasing ingestion of microorganisms, superoxide anion (O2-) production as well as natural killer activity. Under the experimental conditions described these effects appear 4-6 h after drug intake and reach their maximum around the 8th hour. PMID- 3011368 TI - [Promoting effect of croton oil on the induction of lip cancer with herpes simplex virus in mice]. PMID- 3011367 TI - [Chemotherapy of bone tumors]. PMID- 3011369 TI - [Ultrastructural changes and histogenesis of large cell cancer of the lung]. PMID- 3011370 TI - Chemosensitivity of human small cell carcinoma of the lung detected by flow cytometric DNA analysis of drug-induced cell cycle perturbations in vitro. AB - A method based on detection of drug-induced cell cycle perturbation by flow cytometric DNA analysis has previously been described in Ehrlich ascites tumors as a way to estimate chemosensitivity. The method is extended to test human small cell carcinoma of the lung. Three tumors with different sensitivities to melphalan in nude mice were used. Tumors were disaggregated by a combined mechanical and enzymatic method and thereafter have incubated with different doses of melphalan. After incubation the cells were plated in vitro on agar, and drug induced cell cycle changes were monitored by flow cytometric DNA analysis. Melphalan produced a dose-related S phase accumulation in the two sensitive tumors, whereas no changes in the cell cycle distribution were found in the resistant tumor. The size of S phase accumulation correlated to the chemosensitivity in vivo. For low concentrations of melphalan, the S phase accumulation was accompanied by G2 + M accumulation. The results indicate that the method may be extended to sensitivity testing of human solid tumors, including screening for new agents. PMID- 3011372 TI - Common peroneal nerve palsy associated with pelvic surgery for cancer. An analysis of 11 cases. AB - Eleven occurrences of common peroneal palsies following pelvic surgery for malignant conditions are reported. The patients' clinical course and possible mechanisms of nerve injury were reviewed. It was concluded that the current belief that all peripheral neuropathies occurring under general anesthesia are preventable may not be applicable to patients with pelvic cancer who must undergo tedious, lengthy, meticulous, extirpative surgery in the dorsal lithotomy position. In patients with tumors that are seemingly isolated to the pelvis, amenable for surgical resection, the possible risk of a peripheral nerve injury is superseded if beneficial effects are obtained by controlling the local manifestations of the tumor. PMID- 3011371 TI - Measurement of myeloid maturation by flow cytochemistry in HL-60 leukemia: esterase is inducible, myeloperoxidase is not. AB - The phenomenon of leukemic cell maturation requires a measurement of myeloid maturation to understand the process and to exploit it as a means of therapy for leukemia. The HL-60 leukemic cell line was used as a model of induced leukemic cell maturation in order to develop a method of quantitating granulocytic and monocytic maturation in response to drug therapy. An automated flow cytochemistry system (Hemalog-D) was employed to measure mean cell volume, myeloperoxidase (MPO), and nonspecific esterase (NSE). For granulocytic maturation induced by vitamin A or DMSO, MPO and cell volume decreased by 50%, maintaining a constant mean cellular MPO concentration throughout maturation from promyelocyte to neutrophil-like forms. For monocytic maturation induced by low-dose ARA-c, the mean NSE increased substantially, while cell volume remained constant. Unlike MPO concentration, NSE was truly inducible and thus a useful quantitative measure of maturation caused by low-dose ARA-c. Flow cytochemistry and cytofluorometry may be developed to allow for quantitative monitoring of therapeutic trials of induced maturation in human leukemias. However, this will require adapting these techniques to the complexity of human leukemias in vivo, and the necessity of handling heterogeneous populations encountered in bone marrow samples. PMID- 3011374 TI - [Effect of selenomethionine on proton magnetic relaxation of liver tissue in x irradiated animals]. PMID- 3011375 TI - [Nucleotide sequence of cDNA and primary structure of the NA+,K+-ATPase beta subunit from the swine kidney]. PMID- 3011376 TI - [Cyclic GMP-phosphodiesterase from the cattle retina. Amino acid sequence of the gamma subunit]. PMID- 3011373 TI - Human T-cell lymphotropic virus type III (HTLV-III) core antigens: synthesis in Escherichia coli and immunoreactivity with human sera. AB - Fragments of human T-cell lymphotropic virus type III (HTLV-III) proviral DNA carrying the gene for the core antigen (gag) was cloned in the plasmid REV. Several of the recombinants direct high levels of synthesis of the antigens. One clone, pG1, produced a hybrid protein containing 13 amino acid residues of the carboxyl terminus of the 17 kD virion protein, the entire p24, the major core protein of HTLV-III, and 74 amino acid residues of the amino terminal of the 15 kD core ribonucleoprotein. A second clone, pG2, was similar to pG1 except that it contained no p17 sequences and was missing the amino-terminal 77 amino acid residues of the p24. A third clone, pG3, was similar to pG2, except that all but 56 amino acids of the carboxyl terminus of p24 were removed. All three proteins were found to be strongly immunoreactive with anti-HTLV-III antibodies present in sera from patients with acquired immune deficiency syndrome (AIDS) or AIDS related complex (ARC). In addition, pG1 and pG2, but not pG3, reacted with a monoclonal antibody (M26) specific for the p24 virion core protein. Whereas all three reacted with an anti-p15 monoclonal antibody, none of the clones reacted with an anti-p17 monoclonal antibody. These results provide direct evidence to support the predicted assignment of the coding region of the gag gene of HTLV III. The product from pG2 was purified and was found to be potentially useful for the detection of anti-p24 antibodies in sera from patients with AIDS or ARC and from individuals at risk from AIDS. PMID- 3011377 TI - [Bacterial degradation of alkylbenzosulfonates]. PMID- 3011378 TI - [Double infection with early summer meningoencephalitis virus and Borrelia burgdorferi]. AB - A 68-year-old woman developed a meningoencephalitis 18 days after a tick bite. IgG and IgM antibodies against tick-encephalitis virus were demonstrated, by enzyme-linked immunoabsorbent assay, in both serum and cerebrospinal fluid (csf). Lymphoplasmocytic pleocytosis was present in csf for over six weeks, as was an increased IgM level. Three weeks after the onset of neurological symptoms and clearing of the encephalitis there occurred multiple peripheral pareses in the left leg which were slow to regress. Retrospectively, IgM and IgG antibodies against Borrelia burgdorferi were demonstrated in deep-frozen serum and csf. Since IgG antibodies against Borrelia burgdorferi, locally synthesised in csf, could also be demonstrated, it must be assumed that the patient had a double infection. It is suggested that in confirmed cases of tick-encephalitis with an atypical course an additional infection with Borrelia should be considered, because if present the latter can be successfully treated with high doses of penicillin. PMID- 3011379 TI - [LAV/HTLV-III and respirators]. PMID- 3011380 TI - [Fractionated afterloading: an advance in the interstitial irradiation of inoperable malignant brain tumors]. PMID- 3011382 TI - [Catechol estrogens and their physiologic relevance]. PMID- 3011381 TI - [Diphenylhydantoin in the prevention and therapy of AIDS?]. PMID- 3011383 TI - [Concentration of specific antibodies to enzootic bovine leukosis virus in individual and collective milk samples]. PMID- 3011384 TI - [Detection of IBR/IPV, leukosis and brucellosis antibodies in samples of bulk milk with ELISA using a simple concentrating method]. PMID- 3011385 TI - [Comparative studies of the detection of Aujeszky virus from filtered and unfiltered study samples]. PMID- 3011387 TI - Colorectal cancer under the age of twenty years. PMID- 3011388 TI - Adsorption thermodynamics of carbofuran on antimony (V) silicate cation exchanger. AB - The adsorption thermodynamics of carbofuran has been studied on antimony (V) silicate cation exchanger at 30 and 50 degrees C. The adsorption isotherms of carbofuran have been found to follow the Freundlich adsorption model and yield "S" class isotherms. The order of adsorption of carbofuran is in accordance with the partial molal free-energy changes in the exchanger. The thermodynamic equilibrium constant (K0), standard free energy (delta G degrees), enthalpy (delta H degrees), and entropy (delta S degrees) changes have also been calculated for predicting the nature of adsorption. PMID- 3011389 TI - Effect of different salt leachates on the movement of some phosphorus containing pesticides in soils using thin layer chromatography. AB - The influence of pH, leachates of alkaline and saline salts, inorganic fertilizers, and surfactants on the movement of eight organophosphorus pesticides, viz., DDVP, diazinon, Ekatin, Folithion, malathion, metasystox, parathion methyl, and Rogor has been studied using soil thin layer chromatographic techniques. The variation in the movement of pesticides under different solvent amendments are expressed in terms of Rf, RB, and RM values and are explained on the basis of adsorption and leachability. PMID- 3011390 TI - 6-Methylmercaptopurine riboside resistance in human lymphocytes in the in vivo somatic cell mutation test. AB - Additional drug-resistance markers are being investigated to broaden the in vivo somatic cell mutation test in human lymphocytes (PBL). The adenosine kinase (AK) locus was chosen for study because Gupta and Singh [Gupta RS, Singh B: Mutat Res 113:441-454, 1983] have demonstrated that in Chinese hamster ovary cells, mutants affected at this locus are obtained at a very high spontaneous frequency and that the response of this locus to different types of mutagens was comparable to that of the hypoxanthine-guanine phosphoribosyl transferase locus. The adenosine analog 6-methylmercaptopurine riboside (MeMPR) was used as the selective agent for obtaining AK-deficient mutants. Cultures of mitogen-stimulated PBL were set up in the presence (test) and absence (control) of the selective agent. Resistant cells capable of synthesizing DNA in the presence of MeMPR were labeled with tritiated thymidine and enumerated autoradiographically. The variant frequency (Vf) was calculated as the ratio of the number of labeled nuclei in the test relative to that in the control. Human PBL were found to be sensitive to MeMPR inhibition of DNA synthesis and exhibited a dose-dependent decrease in Vf with increasing concentrations of MeMPR. However, no leveling off of the dose-response curve was observed. Thus the background level of Vf was probably lower than the practical detection limit of the test (4.0 X 10(-7) with a 50-ml blood sample). It was concluded that, because of the autosomal recessive nature of the AK gene, the background Vf in human PBL is too low to allow a useful baseline to be established for the in vivo somatic mutation test. PMID- 3011391 TI - Primary structure and spectroscopic studies of Neurospora copper metallothionein. AB - When Neurospora crassa is grown in the presence of Cu(II) ions, it accumulates the metal with the concomitant synthesis of a low molecular weight copper-binding protein. The molecule binds 6 g-atom of copper per mole protein (Mr = 2200) and shows a striking sequence homology to the zinc- and cadmium-binding vertebrate metallothioneins. Absorption, circular dichroism, and electron paramagnetic resonance spectroscopy of Neurospora metallothionein indicate the copper to be bound to cysteinyl residues as a Cu(I)-thiolate complex of the polymeric mu thiolate structure [Cu(I)6RS7]-. This metal-binding mode is also in agreement with the unusual luminescence of the protein. Spectral perturbation studies with HgCl2 and p-(chloromercuri)benzoate suggest that the 6 Cu(I)ions are coordinated to the seven cysteinyl residues in the form of a single metal cluster. Neurospora apometallothionein is also capable of binding in vivo group IIB metal ions [Zn(II), Cd(II), and Hg(II)] as well as paramagnetic Co(II) ions with an overall metal-to-protein stoichiometry of 3. The spectroscopic properties of the fully substituted forms are indicative of a distorted tetrahedral coordination. However, metal titration of the apoprotein shows the third metal ion to be differently coordinated than the other two metal ions. This difference can be explained by the presence of only seven cysteine residues in Neurospora metallothionein as opposed to nine cysteine residues in the three-metal cluster of the mammalian metallothioneins. PMID- 3011386 TI - Enalapril. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in hypertension and congestive heart failure. AB - Enalapril maleate is an orally active angiotensin-converting enzyme inhibitor. It lowers peripheral vascular resistance without causing an increase in heart rate. Enalapril 10 to 40 mg/day administered either once or twice daily is effective in lowering blood pressure in all grades of essential and renovascular hypertension, and shows similar efficacy to usual therapeutic dosages of hydrochlorothiazide, beta-blockers (propranolol, atenolol and metoprolol) and captopril. Most patients achieve adequate blood pressure control on enalapril alone or with hydrochlorothiazide. In patients with severe congestive heart failure resistant to conventional therapy, enalapril improves cardiac performance by a reduction in both preload and afterload, and improves clinical status long term. Enalapril appears to be well tolerated, with few serious adverse effects being reported. It does not induce the bradycardia associated with beta-blockers or the adverse effects of diuretics on some laboratory values. In fact, the hypokalaemic effect of hydrochlorothiazide is attenuated by the addition of enalapril. The incidence of the main (but rare) side effects of hypotension in hypovolaemic patients and reduced renal function in certain patients with renovascular hypertension, which are also seen with captopril, might be reduced by careful dosage titration, discontinuation of diuretics, and monitoring of at-risk patients. Thus, enalapril is a particularly worthwhile addition to the antihypertensive armamentarium, as an alternative for treatment of all grades of essential and renovascular hypertension. It also shows promise in the treatment of congestive heart failure. PMID- 3011393 TI - Enhancement of influenza virus infections by secalonic acid D. AB - Secalonic acid D (SAD), a hepatotoxic, teratogenic, and slightly mutagenic metabolite of Penicillium oxalicum has been identified as a natural contaminant of grain dust. Secalonic acid D was administered intraperitoneally to male ICR mice that were exposed to influenza virus aerosols 5 days earlier. The mortality rate was significantly higher (p less than 0.001) in mice subjected to both influenza and SAD than those subjected to influenza alone. Virus titers in lung tissue samples at selected time intervals appeared similar for both influenza and influenza-SAD treated groups of mice for 9 days after exposure to the virus. After 9 days, influenza-SAD treated mice appeared to have higher virus titers. No difference in the pathological progression of pneumonia was discernible between these two groups of mice. The influenza-SAD group, in addition to pneumonia, exhibited severe hepatic necrosis characteristic of SAD administration. Mice infected with influenza virus followed by administration of SAD responded with significantly lower (p less than 0.05) antibody titers to influenza virus than mice exposed to influenza virus alone. PMID- 3011392 TI - Characterization of a highly negative and labile binding protein induced in Euglena gracilis by cadmium. AB - The physiochemical properties and physiological significance of the cadmium binding protein (CdBP) of the algae Euglena gracilis have been studied. Following in vivo exposure of cells to 0.4 or 1.3 micrograms/mL of Cd2+, all the cytosolic Cd is bound to high molecular weight species. At 4.7 micrograms/mL, appreciable CdBP has formed in cells grown under illumination or in the dark. An analogous ZnBP could not be isolated from control or Zn-exposed (20 micrograms/mL) cells, but zinc and a trace of copper were bound to the CdBP when 2-mercaptoethanol (2 ME) is added to the homogenates of Cd-treated cells and the buffers used during isolation. The large pool of very low molecular weight zinc species previously reported is increased when cells are exposed to high cadmium levels. Two distinct species, BP-1 and BP-2 are resolved by ion-exchange chromatography on DEAE Sephadex. Unusually high conductivities (25 and 40 mSiemen) are required to displace them, indicating that they are very negatively charged proteins at pH 8.6. The pH for half-titration of bound Cd2+ is between 5 and 6. EDTA (0.4 M) and the CdBP isolated by gel-exclusion chromatography react biphasically with pseudo first-order rate constants of 4 +/- 3 X 10(-4) sec-1 and 7 +/- 2 X 10(-5) sec-1. Neither form of the CdBP cross-reacts with antibodies to rat liver metallothionein (MT) antibodies. The structural, chemical, and functional differences between the Euglena CdBPs and mammalian MTs are discussed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011394 TI - An overview of species differences in the effects of a water extract of cotton bract on isolated airway smooth muscle, and effects of E. coli lipopolysaccharide. AB - Our laboratory has been comparing the activity of a water extract of cotton bract (CBE) with the isolated trachealis smooth muscle of the dog, guinea pig, and cat. CBE induced contractions that were not mediated by 5-hydroxytryptamine (5-HT), histamine, or muscarinic receptors. The active agent(s) in CBE was dialyzable (less than 14,000 molecular weight), and substantial activity was retained after low-temperature ashing. CBE potentiated contractions of dog trachealis to histamine and 5-HT and relaxation responses to isoproterenol, whereas it had no effect on responses to methacholine and KCl. In the guinea pig trachealis, CBE reduced responsiveness to KCl, potentiated relaxations to adenosine and ATP, and did not alter the responses to the remaining agents. Responses of cat trachealis to KCl and isoproterenol were potentiated by CBE, while those to 5-HT were unaffected. Neurogenic cholinergic contractile responses were potentiated by CBE in the trachealis of the dog, but not of the guinea pig, while neurogenic relaxations were potentiated by CBE in guinea pig trachealis but not in the dog trachealis. There are thus marked species differences in the acute effects of CBE on airway smooth muscle. Due to recent interest in the possible involvement of bacterial endotoxins in the etiology of byssinosis, we examined the effects of E. coli lipopolysaccharide (LPS) in guinea pig trachealis. An initial examination revealed that LPS potentiated responses to histamine, but not those to methacholine and isoproterenol. This effect vanished upon a second appraisal with a different batch of LPS. The effect of LPS in airway smooth muscle is thus, at present, equivocal. PMID- 3011395 TI - Biologic activity of purified cotton bract extracts in man and guinea pig. AB - Purified aqueous extracts of cotton bract induce acute airway constriction in healthy volunteers never before exposed to cotton bract. The response is similar to that of textile workers who inhale cotton dust. Approximately 60% of volunteers respond to bract extract with significant decreases in lung function, and these volunteers show an increased number of lymphocytes present in their lungs. Following inhalation of bract, the percent of polymorphonuclear leukocytes increases. Macrophages obtained by bronchoalveolar lavage from volunteers pre challenged with bract extract release increased amounts of chemotactic factor and superoxide anion. Efforts to detect release of histamine and leukotrienes in volunteers following challenge with bract show no increase in urinary histamine and no significant release of leukotrienes in lung lavage fluid. Purified extracts exhibit chemotactic activity in vitro. They also contract guinea pig ileal longitudinal muscle in vitro. This preparation contains mast cells but no basophils, and the H-1 blocker, mepyramine blocks the contraction. Purified bract extracts contain no histamine or endotoxin but other contractors of smooth muscle may be present. The purified extract exhibits spectral, fluorescent, and radioimmune assay properties similar to a leukotriene B-like component. Cotton bract appears to have direct as well as cell-mediated activities. PMID- 3011396 TI - Pulmonary response and intrapulmonary lipids in rats exposed to bismuth orthovanadate dust by inhalation. AB - Rats were exposed to 0, 1.2, or 0.11 mg/liter of uncoated bismuth orthovanadate (BOV), 1.3 or 0.15 mg/liter of silica-coated BOV, or 1.9 mg/liter of silica coated TiO2 for 2 weeks. Rats were killed 0, 1, 3, 6, and 12 months postexposure (PE). After the rats were exposed for 2 weeks their pulmonary response to silica coated TiO2 was characterized by dust-laden macrophage (dust cell) response with hyperplasia of type II pneumocytes. The lung reaction to uncoated BOV or silica coated BOV was both similar to that of silica-coated TiO2 and dose-related. After the rats were exposed for 3 months, foamy macrophage infiltration in silica coated TiO2 was evident. Alveolar proteinosis, foamy macrophages with cholesterol clefts, and hyperplastic type II pneumocytes were observed in rats exposed to uncoated BOV or silica-coated BOV. By 6 months PE, the lung exposed to silica coated TiO2 was restored to essentially normal architecture with removal of most dust cells. In the silica-coated BOV or uncoated BOV exposure groups, alveolar proteinosis and cholesterol granulomas became obvious with degenerative foamy macrophages. After 1 year PE, the lungs exposed to silica-coated TiO2 were almost normal with only a few dust cell aggregates remaining. The pulmonary lesions of silica-coated or uncoated BOV were reduced but alveolar proteinosis and cholesterol granulomas still persisted. Electron microscopy revealed massive accumulation of intraalveolar phospholipid material with hyperplastic type II pneumocytes showing overloaded myelin figures after 2 weeks exposure. The increase in phospholipid at 1 month PE and sterol content at 3 months PE in the lungs correlated with the accumulation of intraalveolar myelin figures and lamellar structure, foamy macrophage infiltration, and occurrence of cholesterol clefts. PMID- 3011397 TI - Ice nucleation activity of Pseudomonas fluorescens: mutagenesis, complementation analysis and identification of a gene product. AB - A DNA fragment of 7.5 kb from Pseudomonas fluorescens MS1650 confers an ice nucleation phenotype when cloned in Escherichia coli. This DNA encodes a protein with an apparent mol. wt of 180 kd, which is found in both inner and outer membrane fractions of transformed E. coli cells. Insertion mutations throughout a 3.9-kb region cause deficiency in ice nucleation, and eliminate the 180-kd protein. Complementation is not observed between any pair of mutations, suggesting that the nucleating phenotype is encoded by a single transcriptional unit. Mutations in most parts of the 3.9-kb region are not completely deficient in phenotype: they still generate ice nuclei at low frequency. One insertion mutation was found to generate pseudowild revertants, which had undergone deletions of the entire insertion and some of the adjacent sequence; these could account for the incomplete deficiency. These deletions displayed depressed nucleation temperatures, but their nucleation frequencies were close to that of the wild-type gene. PMID- 3011398 TI - Surface expression of viral glycoproteins is polarized in epithelial cells infected with recombinant vaccinia viral vectors. AB - In polarized epithelial cells, maturation sites of enveloped viruses that form by budding at cell surfaces are restricted to particular membrane domains. Recombinant vaccinia viruses were used to investigate the sites of surface expression in the Madin-Darby canine kidney (MDCK) cell line of the hemagglutinin (HA) of influenza virus, the G glycoprotein of vesicular stomatitis virus (VSV), and gp70/p15E of Friend murine leukemia virus (MuLV). These glycoproteins could be demonstrated by immunofluorescence on the surfaces of MDCK cells as early as 4 h post-infection. In intact MDCK monolayers, vaccinia recombinants expressing HA produced a pattern of surface fluorescence typical of an apically expressed glycoprotein. In contrast, cells infected with vaccinia recombinants expressing VSV-G or MuLV gp70/p15E exhibited surface fluorescence only when monolayers were treated with EGTA to disrupt tight junctions, as expected of glycoproteins expressed on basolateral surfaces. Immunoferritin labeling in conjunction with electron microscopy confirmed that MDCK cells infected with the HA recombinant exhibited specific labeling of the apical surfaces whereas the VSV-G and MuLV recombinants exhibited the respective antigens predominantly on the basolateral membranes. Quantitation of surface expression by [125I]protein A binding assays on intact and EGTA-treated monolayers confirmed the apical localization of the vaccinia-expressed HA and demonstrated that 95% of the VSV-G and 97% of the MuLV gp70/p15E glycoproteins were localized on the basolateral surfaces. These results demonstrate that glycoproteins of viruses that normally mature at basolateral surfaces of polarized epithelial cells contain all of the structural information required for their directional transport to basolateral plasma membranes. PMID- 3011399 TI - Microaggregation of hormone-occupied epidermal growth factor receptors on plasma membrane preparations. AB - The rotational diffusion of the complexes of epidermal growth factor (EGF) with its specific receptor on plasma membrane vesicles prepared from human epidermoid carcinoma A431 cells was studied using the time-resolved polarization of phosphorescence of erythrosin-labeled hormone. The measured rotational correlation times of 16-20 microseconds at 4 degrees C are consistent with monomeric freely diffusing EGF receptor. Upon increasing the temperature to 37 degrees C, the rate of rotational diffusion slows down as evidenced by an increase in the correlation time to 75 microseconds. This finding suggests that small clusters of the occupied EGF receptor (microaggregation) form at the higher temperature, a property we have reported previously for occupied receptors on living A431 cells. Subsequent cooling of the membranes leads to a partial reversal of the microaggregation. We conclude that clustering of occupied EGF receptors can proceed at 37 degrees C in the absence of metabolic energy and external interactions, e.g. with components of the cytoskeleton, and thus reflects inherent properties of the receptor protein in its natural environment. A lag phase in the time course of microaggregation observed with the isolated membrane preparations may reflect cooperativity in the process of receptor association. PMID- 3011400 TI - Digestion of the chicken beta-globin gene chromatin with micrococcal nuclease reveals the presence of an altered nucleosomal array characterized by an atypical ladder of DNA fragments. AB - The structure of the chicken adult beta-globin gene chromatin in immature and mature erythrocyte nuclei has been analysed using micrococcal nuclease digestion. The resulting DNA fragments were blotted onto DBM-papers and probed with labelled DNA fragments spanning the adult beta-globin gene and its 5'- and 3'-flanking regions. The structure of the nucleosomes within and in the regions flanking the adult beta-globin gene appears to be altered in at least two ways in erythrocyte chromatin, when compared with either bulk or inactive ovalbumin gene chromatin. First, oligomeric DNA fragments containing the beta-globin gene are released faster than those of either bulk or ovalbumin gene chromatin. Second, although the difference in size of the liberated oligomeric DNA fragments is similar to the nucleosomal repeat length of bulk and ovalbumin gene chromatin, the individual oligomers are approximately 100 bp shorter than their bulk or ovalbumin gene counterparts, most noticeably when the nuclease digestion is performed at 37 degrees C. This results in an atypical ladder of approximately 300, 500, 700, 900 bp instead of the canonical chicken erythrocyte ladder which is an integral multiple of 207 bp. The same ladder was obtained from immature erythrocytes, in which the beta-globin gene is actively transcribed, and from mature erythrocytes, in which it is considered to be inactive with RNA polymerase molecules clustered in the 5' moiety of the gene. This indicates that the alteration of the nucleosomal structure is not due to transcription per se.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011402 TI - Mutations around the NG59 lesion indicate an active association of polyoma virus middle-T antigen with pp60c-src is required for cell transformation. AB - The transforming activity of polyoma virus middle-T antigen is believed to be dependent on its ability to form a complex with the cellular tyrosine protein kinase, pp60c-src. This hypothesis is based on observations of mutants of middle T which demonstrated a correlation between these two activities. To investigate further the significance of pp60c-src association in transformation by middle-T, a series of deletion and point mutants were constructed around the NG59 lesion since this region has been implicated in pp60c-src binding. Analysis of the middle-T variants revealed a complete correlation between the presence of associated activated pp60c-src and the ability to transform. Further, this ability of pp60c-src to associate with middle-T may depend on the presence of a beta-turn between amino acids 177 and 180. The results indicate the NG59 phenotype results from the introduction of an isoleucine residue between amino acids 177 and 178 rather than the transition mutation at 179. The mutant MG1 is a single point mutation (at residue 180) and represents the smallest change in the middle-T which abolishes both the transformating and kinase activity of middle-T. Taken together, the data suggest the region surrounding the NG59 lesion is involved in the formation of an active complex between middle-T and pp60c-src and strongly suggest that this association is an absolute requirement for polyoma virus-induced transformation. PMID- 3011401 TI - Identification of a provirally activated c-Ha-ras oncogene in an avian nephroblastoma via a novel procedure: cDNA cloning of a chimaeric viral-host transcript. AB - Retrovirus without oncogenes often exert their neoplastic potential as insertional mutagens of cellular proto-oncogenes. This may be associated with the production of chimaeric viral-host transcripts; in these cases; activated cellular genes can be identified by obtaining cDNA clones of bipartite RNAs. This approach was used in the analysis of chicken nephroblastomas induced by myeloblastosis-associated virus (MAV). One tumor contained a novel mRNA species initiated within a MAV LTR. cDNA cloning revealed that this mRNA encodes a protein of 189 amino acids, identical to that of normal human Ha-ras-1 at 185 positions, including positions implicated in oncogenic activation of ras proto oncogenes; there are no differences between the coding sequences of presumably normal Ha-ras cDNA clones from chicken lymphoma RNA and the tumor-derived cDNAs. The chimaeric mRNA in the nephroblastoma is at least 25-fold more abundant than c Ha-ras mRNA in normal kidney tissue, and a 21-kd ras-related protein is present in relatively large amounts in the tumor. We conclude that a quantitative change in c-Ha-ras gene expression results from an upstream insertion mutation and presumably contributes to tumorigenesis in this single case. Little or no increase in c-Ha-ras RNA or protein was observed in other nephroblastomas. PMID- 3011403 TI - Adenovirus E1A-mediated regulation of class I MHC expression. AB - Expression of class I MHC transplantation antigens has been shown to be reduced in baby rat kidney (BRK) cells transformed by highly oncogenic adenovirus type 12 (Ad12), as compared with untransformed cells and cells transformed by non oncogenic Ad5. Here we show that this reduction of class I expression also occurs in a variety of other primary cell cultures transformed by Ad12, and that reduction of class I gene expression occurs for all class I loci. Transfection of Ad5E1 into class I-negative Ad12-transformed BRK cells leads to complete restoration of class I expression. Introduction of Ad12E1 into most class I positive established cell lines does not result in suppression of class I expression. However, transfection of the Ad12E1A region into a class I-positive cell line which was immortalized by a mutant Ad12E1A region resulted in suppression of class I gene expression, implying that the suppression of class I activity in Ad12-transformed cells is due to an active switching-off process. PMID- 3011404 TI - Identification of the human papilloma virus-1a E4 gene products. AB - Antibodies prepared against a human papilloma virus-1 (HPV-1) E4/beta galactosidase fusion protein identified several polypeptides in HPV-1, but not HPV-2 or 4, induced papillomas. The major E4 protein, that represented up to 30% of total cellular protein, was a 16/17-K doublet which was purified by column chromatography and analysed for amino acid content. A peptide derived by chymotryptic digestion was purified by h.p.l.c. and subjected to amino acid sequencing. The unique sequence obtained, Gly-His-Pro-Asp-Leu-Ser-Leu, identified the 16/17-K doublet as a product of the HPV-1 E4 gene region. Antibodies to both the E4/beta-galactosidase fusion protein and the 16/17-K doublet identified two smaller polypeptides (10/11-K) which may represent spliced products of E4. We propose that the products of the HPV-1 E4 gene region are not classical DNA tumor virus early proteins and suggest that they play a role in virus maturation. PMID- 3011405 TI - Characterization, cloning and sequence analysis of the CDC25 gene which controls the cyclic AMP level of Saccharomyces cerevisiae. AB - The cell division cycle of the yeast Saccharomyces cerevisiae is triggered at the stage called 'START'. Many results strongly suggest that adenylate cyclase is an essential element of the control of START. We report here results arguing for a positive control of the cAMP level by the CDC25 gene, another gene of START. Firstly, cdc25 cells can be rescued by extracellular cAMP. Secondly, the cellular cAMP content drops when thermosensitive cdc25 mutant cells are shifted to restrictive temperature. We report the molecular cloning of the CDC25 gene by complementation of cdc25 mutant cells. The identity of the cloned gene was confirmed by site-specific gene re-integration experiments and segregation analysis: the isolated fragment is shown to integrate into the cdc25 gene locus. When transferred in cdc25 mutant cells this DNA prevents the drop of the cAMP level at restrictive temperature. This gene is transcribed in a 5200-nucleotides mRNA. We have determined the nucleotide sequence of a 5548-bp DNA fragment which shows an uninterrupted open reading frame (ORF) coding for a 1587-amino acid polypeptide chain. Only the C-terminal part of the ORF appears to be essential for the complementation of the cdc25-5 allele, suggesting a multidomain protein. PMID- 3011406 TI - Multiple sequence motifs are involved in SV40 enhancer function. AB - A systematic mutagenesis of the SV40 enhancer indicates that it spans approximately 100 bp and is composed of at least two distinct DNA domains which exhibit very little enhancing activity on their own. Their association results in a 400-fold enhancement of transcription, virtually irrespective of their relative orientation and, to some extent, of the distance between them. Enhancer activity can also be generated by duplication of either domain. We show also that the activity of each domain is due to the presence of several specific sequence motifs. These motifs are found assorted in different combinations in other viral and cellular enhancers. PMID- 3011407 TI - The integrase family of site-specific recombinases: regional similarities and global diversity. AB - A combination of two methods for detecting distant relationships in protein primary sequences was used to compare the site-specific recombination proteins encoded by bacteriophage lambda, phi 80, P22, P2, 186, P4 and P1. This group of proteins exhibits an unexpectedly large diversity of sequences. Despite this diversity, all of the recombinases can be aligned in their C-terminal halves. A 40-residue region near the C terminus is particularly well conserved in all the proteins and is homologous to a region near the C terminus of the yeast 2 mu plasmid Flp protein. This family of recombinases does not appear to be related to any other site-specific recombinases. Three positions are perfectly conserved within this family: histidine, arginine and tyrosine are found at respective alignment positions 396, 399 and 433 within the well-conserved C-terminal region. We speculate that these residues contribute to the active site of this family of recombinases, and suggest that tyrosine-433 forms a transient covalent linkage to DNA during strand cleavage and rejoining. PMID- 3011408 TI - Sequence and domain relationships of ntrC and nifA from Klebsiella pneumoniae: homologies to other regulatory proteins. AB - We have determined the nucleotide sequences of two genes from Klebsiella pneumoniae, nifA, the nif-specific activator of transcription and ntrC, the bifunctional regulatory protein involved in 'nitrogen control'. These sequences differ significantly from those previously published. In particular, nifA extends 40 codons beyond the stop codon reported earlier. This extension encodes a putative DNA-binding domain strongly homologous to the Rhizobium meliloti nifA protein and to some extent to the ntrC protein. In all three proteins this domain is linked by a segment of variable length to a strongly conserved central domain of 240 residues. A short segment having the properties of an interdomain linker joins the central region to an N-terminal domain, which is weakly related in the case of the two nifA proteins. This homology is not shared by the N-terminal domain of ntrC, which is clearly but unexpectedly related to the N-terminal domains of a diverse set of procaryotic pleiotropic control proteins, including ompR, dye and nusA from Escherichia coli and spoOA and spoOF from Bacillus subtilis. PMID- 3011410 TI - Cryo-electron microscopy of vitrified SV40 minichromosomes: the liquid drop model. AB - The structure of SV40 minichromosomes has been studied by cryo-electron microscopy of vitrified thin layers of solution. In high-salt buffer (130 mM NaCl), freshly prepared minichromosomes are condensed into globules 30 nm or more in diameter. On the micrograph, they appear to be formed by the close packing of 10 nm granules which give rise to a 10 nm reflection in the optical diffractogram. The globules can adopt many different conformations. At high concentration, they fuse into a homogeneous 'sea' of closely packed 10 nm granules. In low-salt buffer (less than 10 mM NaCl), the globules open, first into 10 nm filaments, and then into nucleosome-strings. The 'liquid drop' model is proposed to explain the condensed structure of the minichromosome in high-salt buffer: nucleosomes stack specifically on top of one another, thus forming the 10 nm filaments. 10 nm filaments in turn, tend to aggregate laterally. Optimizing both these interactions results in the condensation of 10 nm filaments or portions thereof into a structure similar to that of a liquid. Some implications of this model for the structure of cellular chromatin are discussed. PMID- 3011409 TI - Subcellular localisation of the middle and large T-antigens of polyoma virus. AB - The distribution of two of the polyoma virus early proteins (the large and middle T-antigens) in lytically infected mouse cells and transformed rat cells has been investigated by indirect immunofluorescence and immuno-electron microscopy using well-characterised monoclonal antibodies. By these techniques, the viral large T antigen was found almost exclusively in the nucleus, sometimes in association with nuclear pores, but never in the nucleolus. In lytically infected, but not transformed cells, fluorescence was detected in discrete areas ('hot spots') within the nucleus and, in a minor population of lytically infected cells, cytoplasmic immunoreactive material was observed. The viral middle T-antigen was found in association with most cytoplasmic membranes and in the majority of cells mainly in the endoplasmic reticulum. Only a fraction of the staining was observed in the plasma membrane and no staining in the nucleoplasm was observed. The data suggest that the site of action of the major transforming activity of polyoma virus need not be at the plasma membrane. Functions associated with the viral antigens are discussed in terms of their subcellular distributions within cells. PMID- 3011411 TI - Gene conversion-like mechanisms may generate polymorphism in human class I genes. AB - The nucleotide sequences of the human class I major histocompatibility complex genes HLA-B27k and HLA-B27w have been determined. They differ by only four nucleotides over a stretch of 14 bp in exon 2, resulting in three amino acid exchanges at positions 77 (Asp to Asn), 80 (Thr to Ile) and 81 (Leu to Ala). The distribution of these nucleotide substitutions suggests a gene conversion-like event responsible for the generation of these HLA-B27 subtypes. The mechanisms underlying the generation of new polymorphic variants in man are therefore probably identical to the gene conversion-like events postulated in the generation of H-2Kbm class I mutants in the mouse. PMID- 3011412 TI - The immunoglobulin heavy-chain B-lymphocyte enhancer efficiently stimulates transcription in non-lymphoid cells. AB - The mouse immunoglobulin heavy-chain (IgH) B-lymphocyte enhancer stimulates transcription from heterologous promoters 20- to 40-fold when transfected into several non-lymphoid cell lines. Stimulation in B-lymphocyte melanoma cell-lines is only about 5--10 times better. A central sequence is equally active in both cell types, whilst flanking sequences, on either side of the common enhancer sequences, specifically stimulate transcription in myeloma cells. These results suggest that there are factors in non-lymphoid cells that can interact with the IgH enhancer to stimulate transcription. PMID- 3011413 TI - Direct evidence that p40x of human T-cell leukemia virus type I is a trans-acting transcriptional activator. AB - Human T-cell leukemia virus type I has a unique sequence, pX, between the env gene and the 3'LTR (long terminal repeat). This sequence codes for p40x, which was proposed to trans-activate transcription from the LTR. Recently, we identified novel pX proteins coded by frame III, which mostly overlaps frame IV (x-lor, coding for p40x), in a region also overlapped by frame II. To determine which product is responsible for the trans-acting function, we constructed an active provirus clone, pMTPX, that contained a genomic fragment of the env, pX and 3'LTR, and introduced site-directed mutations into the active site. The effects of various deletions and point mutations that distinguished each of the overlapping open reading frames (ORFs), II, III and IV, on trans-activation of pLTR-CAT were treated by co-transfection assays. The results showed that only mutations which affected p40x expression resulted in loss of activity for transcriptional activation. These findings clearly indicate that p40x coded by frame IV is responsible for the transcriptional activation of the LTR. This conclusion was confirmed by studies on expression of cDNA of pX mRNA. PMID- 3011415 TI - A GAL family of upstream activating sequences in yeast: roles in both induction and repression of transcription. AB - Binding sites for the GAL4-positive regulatory protein have been identified upstream of six galactose-inducible genes of Saccharomyces cerevisiae on the basis of (i) protection in DNAse I footprints, (ii) loss of protection when excess GAL4-binding oligonucleotide is added and (iii) homology with a 23-bp dyad symmetric consensus sequence. Many of the binding sites have been shown to function as upstream activating sequences. The number of binding sites upstream of the various genes ranges from one to four, but a feature is conserved: in cases of multiple sites there is a pair with highest binding affinity located at dyad--dyad distances of 82--87 bp. We suggest that a pair of sites facilitates repression by the GAL80-negative regulatory protein, on the basis of (i) a correlation of a pair of sites (or only one) with full (or only partial) repression and (ii) the introduction of a second site abolishing transcription occurring with one. PMID- 3011414 TI - Hormonally induced alterations of chromatin structure in the polyadenylation and transcription termination regions of the chicken ovalbumin gene. AB - We have studied the chromatin structure of a 16-kb region of the chicken genome containing the 3'-terminal 2 kb of the ovalbumin pre-mRNA coding sequence and the 14-kb segment located immediately downstream from the main mRNA polyadenylation site. Using the indirect end-labelling technique, four major and two minor DNase I-hypersensitive regions were found in the oviduct chromatin, whereas they were not present in liver, kidney or erythrocyte chromatin. The first hypersensitive region (region A) was present in chromatin of oviducts from laying hen and estrogen- or progesterone-stimulated immature chicks, in which the ovalbumin gene is expressed, but not in the chromatin of 'acute withdrawn' chicks where the gene is no longer transcribed. Region A spans 1.3 kb, from 7.2 to 8.5 kb downstream from the ovalbumin gene capsite (position +1), and encompasses the 3' moiety of the last exon including the major polyadenylation signal and polyadenylation site located at +7546 and +7564, respectively. Region A also contains a minor polyadenylation signal present at +7294 and the corresponding polyadenylation site at +7368. Two putative termination sequences at +8445 and +8483 are also found at the 3' extremity of region A in a 170-bp DNA segment within which 90% of the ovalbumin primary transcripts apparently terminate. Two minor hormone independent DNase I-hypersensitive regions (a1 and a2) located at +8.6 and +8.8 kb are also specific to oviduct chromatin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011416 TI - Overproduction of human Cu/Zn-superoxide dismutase in transfected cells: extenuation of paraquat-mediated cytotoxicity and enhancement of lipid peroxidation. AB - The 'housekeeping' enzyme Cu/Zn-superoxide dismutase (SOD-1) is encoded by a gene residing on human chromosome 21, at the region 21q22 known to be involved in Down's syndrome. The SOD-1 gene and the SOD-1 cDNA were introduced into mouse L cells and human HeLa cells, respectively as part of recombinant plasmids containing the neoR selectable marker. Human and mouse transformants were obtained that expressed elevated levels (up to 6-fold) of authentic, enzymatically active human SOD-1. This enabled us to examine the consequences of hSOD-1 gene dosage, apart from gene dosage effects contributed by other genes residing on chromosome 21. Human and mouse cell clones that overproduce the hSOD 1 had altered properties; they were more resistant to paraquat than the parental cells and showed an increase in lipid peroxidation. The data are consistent with the possibility that gene dosage of hSOD-1 contributes to some of the clinical symptoms associated with Down's syndrome. PMID- 3011417 TI - Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life? AB - Mu transposons carrying the chloramphenicol resistance marker have been inserted into the cloned Escherichia coli genes sodA and sodB coding for manganese superoxide dismutase (MnSOD) and iron superoxide dismutase (FeSOD) respectively, creating mutations and gene fusions. The mutated sodA or sodB genes were introduced into the bacterial chromosome by allelic exchange. The resulting mutants were shown to lack the corresponding SOD by activity measurements and immunoblot analysis. Aerobically, in rich medium, the absence of FeSOD or MnSOD had no major effect on growth or sensitivity to the superoxide generator, paraquat. In minimal medium aerobic growth was not affected, but the sensitivity to paraquat was increased, especially in the sodA mutant. A sodA sodB double mutant completely devoid of SOD was also obtained. It was able to grow aerobically in rich medium, its catalase level was unaffected and it was highly sensitive to paraquat and hydrogen peroxide; the double mutant was unable to grow aerobically on minimal glucose medium. Growth could be restored by removing oxygen, by providing an SOD-overproducing plasmid or by supplementing the medium with the 20 amino acids. It is concluded that the total absence of SOD in E. coli creates a conditional sensitivity to oxygen. PMID- 3011418 TI - Are single-stranded circles intermediates in plasmid DNA replication? AB - Plasmid pC194 exists as circular double-stranded and single-stranded DNA in Bacillus subtilis and Staphylococcus aureus. We report here that the plasmid pHV33, composed of pBR322 and pC194, exists as double- and single-stranded DNA in Escherichia coli, provided that the replication functions of pC194 are intact. Single-stranded pHV33 DNA is converted to double-stranded DNA by complementary strand synthesis probably initiated at rriB, a primosome assembly site present on pBR322. The efficiency of complementary strand synthesis affects the double stranded copy number, which suggests that single-stranded DNA is a plasmid replication intermediate. PMID- 3011419 TI - Regulation of transferrin receptor expression at the cell surface by insulin-like growth factors, epidermal growth factor and platelet-derived growth factor. AB - Addition of platelet-derived growth factor (PDGF), recombinant insulin-like growth factor I (rIGF-I) or epidermal growth factor (EGF) to BALB/c 3T3 fibroblasts causes a marked increase in the binding of [125I]diferric transferrin to cell surface receptors. This effect is very rapid and is complete within 5 min. The effect of EGF is transient, with [125I]diferric transferrin binding returning to control values within 25 min. In contrast, PDGF and rIGF-I cause a prolonged stimulation of [125I]diferric transferrin binding that could be observed for up to 2 h. The increase in the binding of [125I]diferric transferrin caused by growth factors was investigated by analysis of the binding isotherm. Epidermal growth factor, PDGF and rIGF-I were found to increase the cell surface expression of transferrin receptors rather than to alter the affinity of the transferrin receptors. This result was confirmed in human fibroblasts by the demonstration that EGF, PDGF and rIGF-I could stimulate the binding of a monoclonal antibody directed against the transferrin receptor (OKT9) to the cell surface. Furthermore, PDGF and rIGF-I stimulated the sustained uptake of [59Fe]diferric transferrin by BALB/c 3T3 fibroblasts, while EGF transiently increased uptake. Thus the effect of these growth factors to increase the cell surface expression of the transferrin receptor appears to have an important physiological consequence. PMID- 3011420 TI - Deletion mutants of Harvey ras p21 protein reveal the absolute requirement of at least two distant regions for GTP-binding and transforming activities. AB - Deletions of small sequences from the viral Harvey ras gene have been generated, and resulting ras p21 mutants have been expressed in Escherichia coli. Purification of each deleted protein allowed the in vitro characterization of GTP binding, GTPase and autokinase activity of the proteins. Microinjection of the highly purified proteins into quiescent NIH/3T3 cells, as well as transfection experiments utilizing a long terminal repeat (LTR)-containing vector, were utilized to analyze the biological activity of the deleted proteins. Two small regions located at 6-23 and 152-165 residues are shown to be absolutely required for in vitro and in vivo activities of the ras product. By contrast, the variable region comprising amino acids 165-184 was shown not to be necessary for either in vitro or in vivo activities. Thus, we demonstrate that: (i) amino acid sequences at positions 5-23 and 152-165 of ras p21 protein are probably directly involved in the GTP-binding activity; (ii) GTP-binding is required for the transforming activity of ras p21 and by extension for the normal function of the proto oncogene product; and (iii) the variable region at the C-terminal end of the ras p21 molecule from amino acids 165 to 184 is not required for transformation. PMID- 3011421 TI - Activation of the pp60c-src kinase during differentiation of monomyelocytic cells in vitro. AB - The proto-oncogene c-src, the cellular homolog of the Rous sarcoma virus (RSV) transforming gene v-src, is expressed in a tissue-specific and age-dependent manner. Its physiological function, although still unknown, appears to be more closely related to differentiation processes than to proliferation processes. To obtain more information about the physiological role of the c-src gene in cells, we have studied differentiation-dependent alterations using the human HL-60 leukaemia cell line as a model system. Induction of monocytic and granulocytic differentiation of HL-60 cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) and dimethylsulfoxide (DMSO) is associated with an activation of the pp60c-src tyrosine kinase, but not with increased c-src gene expression. Control experiments exclude an interaction of TPA and DMSO themselves with the pp60c-src kinase. PMID- 3011422 TI - Expression of integrated Rous sarcoma viruses: DNA rearrangements 5' to the provirus are common in transformed rat cells but not seen in infected but untransformed cells. AB - The study of Rous sarcoma virus (RSV)-infected rat cell clones offers a novel approach to unravelling the mechanisms controlling eukaryotic gene expression. RSV-transformed rat cell clones frequently contain duplicated proviral sequences immediately upstream of an intact provirus. This category of proviral rearrangement is not seen in cells that remain untransformed after RSV infection nor in subsequently segregating transformants. These results suggest that such rearrangements occur during or soon after proviral integration, and that they may favour early proviral expression. PMID- 3011424 TI - Several hundred base pairs upstream of Drosophila hsp23 and 26 genes are required for their heat induction in transformed flies. AB - We have used the P-element-mediated transformation of Drosophila germ line to study the 5' DNA sequences involved in the thermal inducibility of the genes for heat shock proteins hsp23 and 26. The results are strikingly different from those previously obtained in heterologous systems. For hsp23, each successive shortening of the promoter region from 618 to 402, 321 and 263 bp clearly decreased the expression. A construct with only 149 bp was not inducible at all. For hsp26, all the regulatory elements appear to be clustered in the first 350 bp upstream from the cap site. Clones with 171 bp showed a 4- to 10-fold decrease in induction depending on the transformed line, and those with only 52 bp were not expressed. The results suggest that at least three Pelham consensus sequences are required for the full expression of these two genes. The direct involvement of one of these consensus sequences has been assessed: a 6-bp deletion within the proximal element of the hsp26 gene strongly reduced its inducibility. Our results also indicate that X-linked hsp genes exhibit either partial dosage compensation or none at all. PMID- 3011423 TI - A transcriptional enhancer sequence of HTLV-I is responsible for trans-activation mediated by p40 chi HTLV-I. AB - Human T-cell leukemia virus type I (HTLV-I) contains a unique sequence pX that is located between env and the 3' long terminal repeat (LTR) and codes for three pX proteins, p40 chi, pp27 chi-III and pp21 chi-III. One of these proteins, p40 chi, was previously shown to activate transcription from the LTR in a trans-acting manner, which suggested that it activated some cellular genes involved in leukemogenesis. In this study, the sequences in the LTR responsible for this trans-activation were analyzed. Construction of deletion mutants of the LTR in pLTR-CAT and measurement of their activities in trans-activated expression of the CAT gene showed that sequences upstream of the TATA box were responsible for the trans-activation mediated by p40 chi. The active unit was identified as an enhancer sequence containing direct repeats by inserting it into an enhancer minus SV40 promoter. Thus, it was concluded that an enhancer sequence in HTLV-I LTR is responsible, at least in part, for transcriptional trans-activation mediated by the viral product p40 chi. PMID- 3011426 TI - The E35 stopper mutant of Neurospora crassa: precise localization of deletion endpoints in mitochondrial DNA and evidence that the deleted DNA codes for a subunit of NADH dehydrogenase. AB - Two types of defective mitochondrial DNA molecules with large deletions (5 kbp and 40 kbp) have previously been identified in the stopper mutant, E35, of Neurospora crassa. The junction fragments spanning the deletion endpoints have now been cloned and sequenced, and their sequences compared with those of the corresponding wild-type fragments. We show that both types of defective mitochondrial DNAs result from deletions of sequences flanked by short direct repeats, which are themselves parts of larger inverted repeat sequences. In every case, the short direct repeat sequences consist of a run of pyrimidines in one strand and purines in the other. We also report the sequence of a 2151-bp HindIII fragment, which is deleted in both of the defective mitochondrial DNAs. Besides the previously identified gene for a methionine tRNA, the 2151-bp DNA sequence contains an open reading frame with the potential to code for a hydrophobic protein 583 amino acids long. This hydrophobic protein has three blocks of significant homology with proteins coded by URF2 found in other mitochondrial genomes. Since the mammalian mitochondrial URF2 has recently been shown to code for a subunit of NADH dehydrogenase, part of the DNA sequence missing in the E35 stopper mutant of N. crassa may also code for a subunit of NADH dehydrogenase. PMID- 3011425 TI - Structure of RNA polymerase II promoters. Conformational alterations and template properties of circularized Saccharomyces cerevisiae GAL1-GAL10 divergent promoters. AB - A DNA fragment encompassing the Saccharomyces cerevisiae GAL1--GAL10 divergent promoters (914 bp) has been circularized in vitro with T4 DNA ligase. We have defined a set of conditions that allows the production of a series of nine topoisomers covering a range from relaxed to highly negatively supercoiled DNA. Topoisomers were recovered in pure form from agarose gels and were analysed singly for the presence of sites sensitive to the single strand-specific endonuclease Pl. In this way, the occurrence of conformational alterations as a function of the linking deficiency of the closed DNA domain has been determined. Interestingly, sites of Pl hypersensitivity localize on the three sequences identified as relevant for the in vitro transcription of the GAL1 moiety of the divergent promoter: the upstream activator sequence (UAS), the TATA sequence, and the RNA initiation site (RIS). In vitro transcription with purified S. cerevisiae RNA polymerase II shows that activation of transcription parallels the appearance of conformational alterations on the UAS, the TATA and the RIS sequences. PMID- 3011427 TI - Variations of intramolecular ligation rates allow the detection of protein induced bends in DNA. AB - A method requiring minute amounts of DNA and protein is proposed for the detection of DNA bending in solution. Local bending, induced by the binding of a protein at its stereospecific site, must increase the probability of circularization of short DNA fragments and hence the rate of formation of the corresponding minicircles. This effect should be highly sensitive to the location of the protein-binding site on the DNA fragment. Simple controls allow other possible interpretations to be ruled out. The deformation due to the cyclic AMP receptor protein when it interacts with its stereospecific site at the lactose control region is studied by this method. It is shown in this case that untwisting is negligible and bending significant. Further measurements are required to assess the actual value of the radius of curvature of the DNA double helix in this complex. PMID- 3011428 TI - Antibodies against the benzylpenicilloyl moiety as a probe for penicillin-binding proteins. AB - Antibodies against the benzylpenicilloyl determinant were used to identify complexes of benzylpenicilloyl and penicillin binding protein (PBP) of several bacterial species on immunoblots. Since radioactive penicillin was not needed, this technique readily allowed in vivo labeling studies even in Escherichia coli, where the saturating concentration was around 0.6 mg/ml. The antibodies showed no substantial cross-reactivity to other beta-lactam-PBP complexes with the exception of 6-aminopenicillanic acid. Surprisingly, some penicilloyl-PBP were hardly recognized by the antiserum, whereas the others could be stained according to the amount of penicillin bound. PMID- 3011429 TI - The pterin (bactopterin) of carbon monoxide dehydrogenase from Pseudomonas carboxydoflava. AB - Radioactively labeled carbon monoxide (CO) dehydrogenase has been obtained in good yield and purity from Pseudomonas carboxydoflava grown in the presence of [32P]phosphate. One enzyme molecule contained an average of 8.32 molecules of phosphate. The entire phosphate content was confined to 2 molecules of FAD and 2 molecules of a pterin. These were noncovalently bound. Molybdoenzyme cofactors could be extracted into N-methyl formamide; pterins were isolated by thin-layer chromatography. CO dehydrogenase contained a novel pterin, different from molybdopterin, which was also resolved in other bacterial molybdoenzymes. Therefore, it was tentatively named bactopterin. The characteristic features of bactopterin were as follows. A relative molecular mass, Mr, of 730 which was much greater than that of molybdopterin (330) (Mr values refer to molybdenum-free forms of the cofactors; presumably, the latter were also devoid of the sulfhydryl groups contained in the native compounds). A content of 2 molecules of phosphate/molecule compared to only 1 phosphate in molybdopterin. Bactopterin was three times less susceptible to air oxidation than molybdopterin. Native bactopterin was cleaved by perchloric acid into two phosphorous-containing fragments with Mr of 330 and 420. The smaller one is believed to be very similar to molybdopterin, the larger one was not a pterin but probably contained an aromatic structure. PMID- 3011430 TI - Sequence divergence and selection of cap sites in the rat gamma-crystallin gene family. AB - The transcription initiation sites of the six rat gamma-crystallin genes were mapped by combining the results of primer extension and S1 nuclease mapping experiments. To obtain more accurate results from the S1 nuclease mapping experiments, intron-deleted clones were constructed by a novel and efficient modification of existing methods involving the use of primer extension products to seal the exons. Four of the six gamma-crystallin genes have multiple transcription start sites. The major and most of the minor transcripts start with an adenosine. Analysis of the 5' flanking sequences of the gamma-crystallin genes shows that the sequence determining the position of the cap site is merely -CA- and that its optimal distance from the first T of the TATA box is 32 base pairs. Our data further suggest that an A to G transition in the first two base pairs of the Goldberg/Hogness box of one the genes does not affect the position of its major cap site. This, together with the fact that most minor transcription start sites are located upstream from the major cap sites, suggests that in the long TATA boxes of the rat gamma-crystallin genes the major RNA polymerase 'trap site' is not directly at the beginning of the TATA sequence. PMID- 3011431 TI - Stoichiometry of mitochondrial cytochromes P-450, adrenodoxin and adrenodoxin reductase in adrenal cortex and corpus luteum. Implications for membrane organization and gene regulation. AB - We have estimated the concentrations of cytochromes P-450scc and P-45011 beta and the electron-transfer proteins adrenodoxin reductase and adrenodoxin in the adrenal cortex and corpus luteum using specific antibodies against these enzymes. While in the adrenal cortex the concentrations of these enzymes are relatively constant in different animals and show no significant sex differences, in corpora lutea they vary considerably and can increase at least up to fifty-fold over the levels found in the ovary. The average relative concentrations of adrenodoxin reductase, adrenodoxin and P-450 are 1:3:8 in the adrenal cortex (which has two cytochromes P-450, P-450scc and P-450(11) beta, in equal concentrations) and 1:2.5:3 in the corpus luteum (which has only P-450scc). We further present evidence that the levels of cytochrome c oxidase also show a degree of correlation with the levels of the mitochondrial steroidogenic enzymes. PMID- 3011432 TI - Structure of mouse type IV collagen. Amino-acid sequence of the C-terminal 511 residue-long triple-helical segment of the alpha 2(IV) chain and its comparison with the alpha 1(IV) chain. AB - The sequence of 511 residues from the C-terminal portion of the triple helix of mouse alpha 2(IV) chain was determined by using the pepsin fragment P2 of collagen IV and two cDNA clones selected from an Engelbreth-Holm-Swarm (EHS) tumor library. The sequence contains nine interruptions of the triplet repeat Gly Xaa-Yaa ranging in size from single insertions or deletions up to stretches of eleven amino acid residues. Five of these interruptions match those present in the homologous segment of the alpha 1(IV) chain but are otherwise different in length and/or sequence. A low homology was found for the triplet regions of the alpha 1(IV) and alpha 2(IV) chain which constitute more than 90% of the sequence. The data indicate a remote evolutionary relationship of the triple-helical sequences of the two constituent chains of basement membrane collagen. PMID- 3011433 TI - Selective dinucleotide-primed in vitro transcription of a cloned fragment of cauliflower mosaic virus DNA is dependent on a limited region of the viral genome. AB - We have previously shown that plant RNA polymerase II preferentially forms ternary transcription complexes on a cloned fragment of the cauliflower mosaic virus genome in the presence of a particular dinucleotide/purine NTP combination (ApG + ATP). This preferential interaction is observed when the viral sequences are present on a discrete circular molecule. Deletion of a 205-bases-pair region abolishes this selectivity. The deleted region contains a considerable number of symmetrical or repeating elements. The use of nuclease S1 as a probe shows that this region contains a homopurine-homopyrimidine sequence which is extremely sensitive to this enzyme, indicating its capacity to adopt a non-B DNA conformation. A possible alternative structure of these sequences, which may explain the preferential interaction with the RNA polymerase, is presented. PMID- 3011434 TI - High-performance liquid chromatography using spherical aggregates of hydroxyapatite micro-crystals as adsorbent. AB - High-performance liquid chromatography (HPLC) using spherical aggregates of hydroxyapatite (HA) microcrystals as adsorbent has been developed; preliminary performance tests were carried out by using several types of protein. In comparison with previously developed plate-like HA packed columns for HPLC, spherical HA packed columns show considerably high chromatographic resolutions in spite of extremely reduced column lengths of 0.5-3 cm. The pressure generated by the latter columns is much higher than that generated by the former, however. PMID- 3011435 TI - A comparative study on iron sources for mitochondrial haem synthesis including ferritin and models of transit pool species. AB - The rates of reaction of various exogenic iron(III) complexes with deuteroporphyrin IX in isolated mitochondria to form deuterohaem were measured. Ferritin was shown to supply iron readily for haem synthesis if the ferritin iron was reductively mobilised by the mitochondrial respiratory chain with succinate as substrate and FMN as mediator. In contrast, polynuclear complexes of iron(III) were able to form deuterohaem without added FMN. Rates of haem formation are about five times higher for the lowest polynuclear units than for ferritin. Sorbitol, gluconate, and bovine serum albumin were used as scavengers for polynuclear complexes with restricted size. Strong chelators of iron(II) compete favourably for deuterohaem formation, which supports the multistep mechanism for haem formation suggested by a priori arguments. Rates of deuterohaem formation were measured in homologous and heterologous systems of ferritins and mitochondria. Slightly differing rates of haem formation were shown to originate in different rates of iron mobilisation from the ferritins. The lack of species specificity in the interaction of ferritin with mitochondria also shows up in the linear dependence of ferritin binding on its bulk concentration as measured using 3H-labeled ferritin. Rates of haem formation are virtually the same in mitoplasts and mitochondria which indicates insignificant influences of the outer membrane. The hypothesis of low polynuclears as major components of the intracellular transit iron pool implies that both ferritin and transit iron pool species are largely equivalent sources of iron for mitochondrial haem synthesis. PMID- 3011437 TI - Enzymatic wound cleaning and absorbable sutures. An experimental study on Varidase and Dexon sutures. AB - The effect of an enzymatic preparation for wound cleaning (Varidase) on the mechanical properties of absorbable sutures (Dexon) was studied in vitro and in vivo in a rat model. In vitro the sutures demonstrated a significant decrease in strength (breaking strength and energy absorption) and extensibility after 12 days of incubation in saline. Incubation in Varidase, however, further decreased the mechanical properties significantly. The stiffness of the sutures was independent of treatment and time. In vivo changes of mechanical properties of the sutures resemble those of the in vitro study, except for a decrease in stiffness of the sutures. The sutures in a primary closed wound had the same strength (energy absorption) as the sutures of an open wound treated by saline, while the sutures of an open wound treated by Varidase tended to have a decreased strength (p = 0.08). This study supports the hypothesis that an enzymatic process may be involved in the degradation of Dexon. The continuous use of Varidase in Dexon-sutured wounds for a period longer than a few days is, therefore, questioned. PMID- 3011436 TI - Diagnosis of exercise-induced left bundle branch block at rest by scintigraphic phase analysis. AB - Accurate diagnosis of diseases of the ventricular conducting system is essential for their appropriate therapy. Some conduction abnormalities, such as exercise induced left bundle branch block (EX-LBBB), are not apparent on resting electrocardiograms. Phase analysis of rest and exercise radionuclide ventriculograms (RVG's) was used to compare four EX-LBBB patients with six normal controls. All patients had normal resting electrocardiograms, ejection fractions, and visually normal wall motion. First harmonic phase images were generated reflecting the timing of ventricular contraction. Dynamic phase displays were reviewed and graded in a blinded fashion by three independent experienced observers. Phase angle histograms of the right and left ventricle were determined for both resting and exercise images. The mean phase angle and standard deviation were also calculated for each ventricle. Visual grading of the resting phase images failed to show a significant difference between normal patients and patients with EX-LBBB. Quantitative analysis, however, revealed a significant difference in mean phase angle differences (LV-RV) in resting studies: 0.8 degrees (+/- 1.9 degrees SEM) in normals versus 9.3 degrees (+/- 2.3 degrees SEM) in EX-LBBB patients (P less than 0.03). Exercise accentuated the phase angle differences: 1.8 degrees in normals vs. 31.2 degrees in EX-LBBB patients (P less than 0.001). Quantitative phase analysis of resting RVG's permits the diagnosis of cardiac conduction disease that is not apparent on the resting EKG and may result in better monitoring and treatment. PMID- 3011438 TI - Epidemiology, diet and colorectal cancer. PMID- 3011439 TI - Augmentation of human blood monocyte microbicidal activity by RU 41740, a glycoprotein extract from Klebsiella pneumoniae. AB - Non-specific activation of host defences may have a significant impact on the outcome of infections in the immunocompromized patient. RU 41740, a glycoprotein extract from Klebsiella pneumoniae, effective in increasing resistance to experimental infections in animals, has been examined in vitro for its effect on human blood monocyte locomotion, phagocytosis, killing of Candida albicans, and release of superoxide anion. RU 41740 had no chemo-attractant activity nor any effect on monocyte chemotactic and phagocytic function. Candidacidal capacity and superoxide anion production by monocytes were significantly enhanced after preincubation with RU 41740 greater than 1.0 microgram/ml. The effect was dose- and time-dependent and was not influenced by the presence of lymphocytes or their culture supernatants. This suggests a direct interaction with monocytes as the mechanism of action of the extract. PMID- 3011440 TI - Effects of angiotensin converting enzyme inhibitor, perindopril, on autonomic reflexes. AB - The effect of the angiotensin converting enzyme inhibitor, perindopril, on autonomic function was assessed in a double blind, placebo controlled, crossover study in 10 normotensive males. Eight milligram of perindopril given orally lowered blood pressure without a change in heart rate. Perindopril enhanced the vagally mediated heart rate variation with deep breathing. There was no impairment of the responses to either bicycle exercise at 175 W for 5 min or isometric handgrip. The pressor response to cold was not changed and the response to the Valsalva manoeuvre was unaltered. These results suggest that the absence of tachycardia after perindopril may be in part related, as has been reported with other converting enzyme inhibitors, to enhanced cardiac parasympathetic tone. Vagomimetic action may be a property of converting enzyme inhibitors in general. PMID- 3011441 TI - Inhibition of human neutrophil degranulation by forskolin in the presence of phosphodiesterase inhibitors. AB - The secretory response of cytochalasin B-treated human polymorphonuclear neutrophils to the peptide chemoattractant f-Met-Leu-Phe (FMLP), the calcium ionophore A23187 and other secretagogues was measured by assaying neutrophil supernatants for the granular enzymes beta-glucuronidase and lysozyme. The dose dependent enzyme secretion in response to 10(-8)-10(-4) M FMLP and A23187 was unaffected by pretreatment with 10-75 microM forskolin (an activator of adenylate cyclase), but inhibited by high concentrations of prostaglandins E1 and E2. The phosphodiesterase inhibitors isobutyl-methyl-xanthine (IBMX), papaverine and Ro 20-1724 dose dependently inhibited enzyme secretion from FMLP- or A23187-treated cells, and this effect was augmented in the presence of 50-75 microM forskolin. Similar results for PGE1, forskolin and forskolin/IBMX combinations were also obtained using leukotriene B4, platelet activating factor and C5a des-Arg as secretagogues. We conclude that the adenylate cyclase system of human neutrophils is activatable by forskolin, but that the regulatory effects of adenylate cyclase stimulants in these cells are greatly attenuated unless cyclic AMP phosphodiesterases are inhibited. Thus the phosphodiesterase activity of neutrophils may be of functional importance and is relevant to the modulation of neutrophil activity in inflammation. PMID- 3011442 TI - Anti-ischemic actions of a new thromboxane receptor antagonist, SQ-29,548, in acute myocardial ischemia. AB - Thromboxane A2 (TxA2) has been implicated as a mediator of ischemic damage to the myocardium. A new, selective thromboxane receptor antagonist, SQ-29,548 (2 mg/kg bolus + 2 mg/kg per h infusion) was studied for its effects on the extension of ischemic damage following acute myocardial ischemia (MI) in the rat. Administration of SQ-29,548 to sham MI rats had no significant effect on mean arterial blood pressure or heart rate over the 6 h experimental protocol. Ischemic damage was assessed by measurement of the depletion of creatine kinase (CK) activity and amino-nitrogen concentration from the myocardium. Six hours following ligation of the left main coronary artery, there was a significant loss of both CK (P less than 0.001) and amino-nitrogen (P less than 0.001) from the left ventricular free wall (LVFW). Administration of SQ-29,548 significantly blunted this loss of CK activity (P less than 0.01) and amino-nitrogen concentration (P less than 0.001) from the ischemic myocardium. Furthermore, the survival rate at 6 h following acute coronary artery ligation was 100% (7/7) for rats given SQ-29,548 and 58% (11/19) for rats given only the vehicle (P less than 0.05). These data indicate that SQ-29,548 significantly prevents the extension of ischemic damage in the myocardium and improves survival following acute coronary artery ligation, suggesting an important role for TxA2 in the pathophysiology of acute myocardial ischemia. PMID- 3011443 TI - Regulation of beta-adrenoceptor density and function in rat vas deferens. AB - beta-Adrenoceptor density and responsiveness were examined in rat vas deferens following surgical and pharmacological treatments. Receptor density was measured by Scatchard analysis of saturation isotherms of specific [125I]pindolol ([125I]PIN) binding in membrane homogenates. Functional responsiveness was measured by isoprenaline-induced inhibition of field stimulated (60 V, 1 ms, 0.1 Hz) or 40 mM K+-induced contractions. Four days following surgical denervation of vas deferens there was no change in the density of [125I]PIN binding sites, suggesting that these sites are not located on prejunctional neurons. Neither 7 day bilateral adrenalectomy, 21 day denervation, nor 7 days treatment with 10 mg/kg per day desmethylimipramine caused changes in either the potency of isoprenaline in inhibiting contraction or the density of [125I]PIN binding sites compared to controls. Infusion of 3 mg/kg per day isoprenaline for 8 days significantly reduced the potency of isoprenaline in inhibiting field stimulated contractions, reduced the maximum degree of inhibition, and reduced the density of [125I]PIN binding sites. These results suggest that beta-adrenoceptor density and responsiveness in rat vas deferens are not affected by removal of adrenal hormones or neuronal stimulation, but that receptor density and responsiveness can be decreased by increasing the concentration of beta-adrenoceptor agonists at the receptor. Therefore, beta-adrenoceptors in rat vas deferens probably receive little tonic stimulation under normal circumstances. PMID- 3011444 TI - Leukotriene C4 binding sites in the rat central nervous system. AB - Binding sites for [3H]LTC4 were observed in crude membrane preparations of rat central nervous system tissue. Equilibrium binding studies indicated one high affinity [3H]LTC4 binding site with a KD of 31.4 +/- 3.4 nM for whole brain preparations. The binding was highly specific for [3H]LTC4 and could be inhibited by the SRS-A antagonist FPL 55172. Specific binding was increased with both mono- and di-valent ions. Regional distribution studies revealed a three-fold difference in binding capacity within different regions of the brain with the highest binding capacity in the brainstem (94.1 +/- 6.9 fmol/mg of protein) and the lowest in the hypothalamus (29.6 +/- 12.8 fmol/mg of protein). In addition, weak low capacity binding was observed for [3H]LTB4 and [3H]LTE4, while no saturable binding was observed for [3H]LTD4. The order of selectivity in inhibiting [3H]LTC4 binding was LTC4 much greater than LTD4 = LTE4 greater than LTB4. PMID- 3011445 TI - Effect of BM-5, a presynaptic antagonist-postsynaptic agonist, on cortical acetylcholine release. AB - The effect of N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl) acetamide (BM-5) on acetylcholine release from the cerebral cortex was investigated in unanaesthetized and urethane-anaesthetized rats. BM-5 at doses ranging from 0.3 to 5 mg/kg i.p. enhanced acetylcholine output in both groups of rats. The maximum increase occurred with 0.5 mg/kg in the unanaesthetized and 2 mg/kg in the anaesthetized rats. The effect lasted approximately 60 min. At the largest doses peripheral muscarinic effects including salivation, chromodachryorrhea and rhinorrhea were also seen. These results demonstrate that BM-5 exerts presynaptic antagonistic and postsynaptic agonistic effects on muscarine receptors in vivo also. PMID- 3011446 TI - Decreased Na+-K+-ATPase activity and [3H]ouabain binding sites in various tissues of spontaneously hypertensive rats. AB - Na+-K+-ATPase activity and [3H]ouabain binding were studied in cerebral cortex, kidney and heart isolated from spontaneously hypertensive rats (SHR) in both the prehypertensive (6 week old) and the hypertensive stages (14 week old). Na+-K+ ATPase activity of heart and kidney was found to be decreased by about 38 and 16% in the prehypertensive and hypertensive stages of SHR respectively; that of cerebral cortex decreased by 23.5% only in the hypertensive stages. Similar results were obtained by pretreatment of membranes with either 0.001% Triton X 100 or by increasing the K concentration from 4.7 to 12.7 mM in the Krebs solution. No significant differences in microsomal protein yield were noted between prehypertensive or hypertensive SHR and the age-matched WKY rats. The study of binding of [3H]ouabain to cerebral cortex, kidney and heart showed that the decreased Na+-K+-ATPase in hypertensive SHR was due to a 31.6, 21.8 and 41.3% reduction in the number of high affinity binding sites respectively, while the affinity constants (Kd) of ouabain binding sites on this enzyme in cerebral cortex, kidney and heart of the normotensive WKY rats were 26.5, 455.9 and 74.7 nM respectively and those from the hypertensive SHR were not altered. The plasma K concentration of the SHR in the prehypertensive and hypertensive stages was 4.07 and 4.13 mM, respectively, significantly less than that of the age-matched WKY rats. It appears that the decrease of plasma K and Na+-K+-ATPase activity in heart and kidney in SHR is derived from a genetic defect and may be related to the abnormal Na handling in this genetically hypertensive strain. PMID- 3011447 TI - Structure-activity studies on the activity of a series of cyclopentane GABA analogues on GABAA receptors and GABA uptake. AB - A series of analogues of gamma-aminobutyric acid (GABA) has been investigated for GABA mimetic activity on the isolated guinea-pig ileum, facilitation of [3H]diazepam binding in rat brain membranes and inhibition of [3H]GABA uptake from rat brain cortical slices. The derivatives tested include six racemic amino acids, all of which contain a 'GABA backbone' with the conformation restricted by a cyclopentane or cyclopentene ring system. From the more potent analogues, five optically pure compounds, including (+)-(4S)-4-aminocyclopent-1-ene-1-carboxylic acid and its (-)-4R enantiomer, have also been assessed as GABA agonists. Of the racemic analogues, 4-aminocyclopent-1-ene-1-carboxylic acid and trans-3 aminocyclopentane-1-carboxylic acid were the most potent at GABAA receptors, while most of the analogues had considerable activity on GABA uptake. The individual resolved isomers of 4-aminocyclopent-1-ene-carboxylic acid and of trans-3-aminocyclopentane-1-carboxylic acid displayed great specificity for GABA receptor and GABA uptake sites. For example (+)-(4S)-4-aminocyclopent-1-ene-1 carboxylic acid was approximately twice as potent as GABA and about 600 times more active than the (-)-4R isomer on the guinea-pig ileum, while it was not significantly active as a GABA uptake inhibitor at 500 microM. On the other hand, its (-)-4R isomer was selective for inhibiting GABA uptake with an IC50 equal to that of racemic nipecotic acid. PMID- 3011448 TI - Bradykinin-induced endothelium-dependent relaxation of bovine intrapulmonary artery and vein. AB - Bradykinin induced relaxation and cyclic GMP accumulation in both bovine intrapulmonary artery and vein. Both the relaxant responses and the accompanying cyclic GMP accumulations were abolished or markedly reduced by intimal rubbing or pretreatment with the guanylate cyclase inhibitor, methylene blue. These findings indicate that both bovine intrapulmonary artery and vein exhibit endothelium dependent relaxation in response to bradykinin, and that the relaxant responses in both vessels are associated with cyclic GMP accumulation. PMID- 3011449 TI - Molecular size of alpha 1- and beta-adrenoceptors in rat brain cortex as determined by a radiation inactivation method. AB - Frozen whole rat cerebral cortex was exposed to 10 MeV electrons from a linear accelerator. Based on the theory of target size analysis, the in situ molecular weight of alpha 1-adrenoceptors (labelled by [3H]prazosin) and beta-adrenoceptors (labelled by [3H]dihydroalprenolol) was 57 800 daltons and 42 600 daltons, respectively. PMID- 3011450 TI - Imaging benzodiazepine receptors in man with [11C]suriclone by positron emission tomography. PMID- 3011451 TI - Enhanced phosphorylation of arterial particulate proteins by cyclic nucleotides and human atrial natriuretic factor. PMID- 3011452 TI - Adrenal-independent, anti-shock effect of ACTH-(1-24) in rats. PMID- 3011453 TI - Adenosine modulation of adrenergic neurotransmission in the human fallopian tube. AB - The effects of adenosine and adenosine analogues on nerve-induced contractile responses and [3H]noradrenaline ([3H]NA) release, were studied in the isthmic part of human oviducts. Adenosine and L-N6-phenylisopropyladenosine (L-PIA) could enhance neurogenic contractile responses in preparations obtained mainly in the proliferative phase. At higher concentrations, adenosine derivatives inhibited contractile responses to nerve stimulation in both proliferative and secretory phase, with the potency order: 5'-N-ethylcarboxamideadenosine (NECA) greater than or equal to L-PIA much greater than D-PIA. This indicated actions at both stimulatory A1- and inhibitory A2-receptors. Adenosine, L-PIA and NECA but not D PIA inhibited [3H]NA release during nerve stimulation. The relative potency order for the prejunctional inhibition was compatible with an action at A1-receptors. Furthermore, adenosine was found to modulate nerve-induced contractions via postjunctional stimulatory A1- and inhibitory A2-like receptors. The postjunctional effects may be influenced by cyclic hormonal changes. The adenosine antagonist 8-p-sulfophenyltheophylline (PSoT) reversibly antagonized the stimulatory and inhibitory effects by adenosine and analogues. PMID- 3011454 TI - High correlation between the localization of [3H]TCP binding and NMDA receptors. PMID- 3011455 TI - A new photoaffinity probe, 4-amino-2-[4-(4-azidocinnamoyl)piperazino]-6,7 dimethoxyqu inazoline, for alpha 1-adrenoceptors. AB - A photoaffinity probe for alpha 1-adrenoceptors was synthesized and its properties examined on rat brain membrane preparations. The binding of 4-amino-2 [4-(4-azidocinnamoyl)piperazino]-6,7-dimethoxyquin azoline (ACP) to these receptors was of high affinity (KD = 1.05 nM) and reversible in the dark. A dose dependent decrease in the concentration of [3H]prazosin binding sites without a change in KD was observed when membranes were preincubated with ACP, photolyzed, and then extensively washed prior to assay. This reduction in receptor concentration was prevented by alpha 1-adrenergic ligands. The specificity of ACP for alpha 1-receptors was further demonstrated by its inability to compete with [3H]dihydroalprenolol and [3H]yohimbine binding in these same membranes. Also, the concentrations and affinity constants of beta-adrenoceptors and alpha 2 adrenoceptors were unaffected in membranes which had been photolyzed after preincubation with ACP. No reduction in concentration of alpha 1-adrenoceptors was detected if ACP was photolyzed prior to incubation with receptors or if ACP was maintained in darkness throughout the experiment. The results suggest that ACP is a specific and sensitive photoprobe that may be useful for further studies on alpha 1-adrenoceptor coupled systems and that may be particularly suited for use in cell culture work. PMID- 3011456 TI - Leukotriene effects upon the transgastric potential difference and pepsin secretion. AB - The effects of leukotrienes on gastric mucosal function in vivo, and on acid and pepsin secretion in vitro were investigated. In cats, treatment with leukotrienes C4 (LTC4), D4 (LTD4), and E4 (LTE4) caused significant decreases in the transgastric electrical potential difference (P.D.) and significant increases in pepsin secretion, which returned toward control levels over 30-90 min. No detectable changes were observed in either acid concentration or gastric secretion volume over the entire 210 min experimental period. Leukotriene B4 (LTB4) had no effect upon any of these parameters. Treatment with LTD4 in isolated rabbit gastric glands resulted in significant increases in pepsin secretion, with no changes observed in aminopyrine accumulation (acid secretion). These results indicate that exogeneous LTC4, LTD4 and LTE4 can affect certain gastric mucosal functions. PMID- 3011457 TI - [3H]imipramine displacement and 5HT uptake inhibition by tryptoline derivatives: in rat brain 5-methoxytryptoline is not the autacoid for [3H]imipramine recognition sites. AB - A putative endacoid capable of displacing [3H]imipramine from its high affinity binding site and of inhibiting [3H]serotonin (5HT) uptake has been partially purified from rat brain tissue. It appears to be unevenly distributed in various rat brain structures following a pattern that only partially matches the extent of the serotonergic innervation in the rat brain structures investigated. The highest amounts have been recovered in striatum followed by hippocampus, cerebral cortex, brain stem and less in diencephalon, cerebellum, hypothalamus, olfactory bulb. Virtually no inhibitory activity on [3H]imipramine binding or on [3H]5HT uptake in addition to 5HT has been found in rat pineal extracts. Its absence in the pineal and various chemicophysical properties discussed in this report suggest that the rat brain endacoid for the imipramine binding site is not 5 methoxytryptoline, a compound previous proposed as the candidate for the role of endogenous ligand of [3H]imipramine recognition site. Moreover, the study of a series of tryptoline derivatives indirectly supports these conclusions. PMID- 3011459 TI - Inhibitory effects of clonidine on bronchospasm induced in guinea-pigs by vagal stimulation or antigen challenge. AB - The effects of clonidine on the bronchospastic responses induced by vagal stimulation or antigen challenge were studied in anaesthetized guinea-pigs. Electrical stimulation of the vagus nerves by 2-4 Hz induced a vigorous, mainly atropine-sensitive bronchoconstriction, which was strongly inhibited by clonidine (0.05 mg/kg i.v.). The inhibitory effect of clonidine was significantly reduced by the alpha 2-adrenoceptor antagonist yohimbine (1 mg/kg i.v.). Another series of experiments was done with ovalbumin-sensitized guinea-pigs. Respiratory anaphylaxis was induced by antigen inhalation resulting in an increase of pulmonary resistance from 100% (baseline) to about 190% in the control group. Animals pretreated with a clonidine aerosol (0.03%) showed a marked inhibition of the bronchospastic response. It is suggested that the inhibition of the bronchospastic responses induced by clonidine may be mediated by a stimulation of alpha 2-adrenoceptors, which exerts an inhibitory control of the excitatory vagal activity in the guinea-pig airways. PMID- 3011458 TI - Differential electrographic patterns for specific mu- and delta-opioid peptides in rats. AB - Cortical electroencephalographic (EEG) recordings were performed on rats after i.v. administration of morphine and specific mu- and delta-opioid peptides. DAGO (Tyr-D X Ala-Gly-N X Me X Phe-Gly-ol), the mu-selective peptide, produced repetitive paroxysmal discharges organized in a pattern analogous to that seen in tonic clonic seizures at doses which produced analgesia while DTLET (Tyr-D X Thr Gly-Phe-Leu-Thr), the delta-selective peptide, produced 'petit-mal'-like seizures at doses which caused neither analgesia nor catatonia. It is suggested that the delta receptor is preferentially implicated in the epileptogenic spectrum of opioids. PMID- 3011460 TI - Phosphoinositide hydrolysis mediated by histamine H1-receptors in rat brain cortex. AB - Histamine stimulated the accumulation of [3H]inositol 1-phosphate in the presence of lithium in [3H]inositol-prelabelled slices from rat brain cortex in a concentration-dependent manner, with an EC50 value of 94.7 microM. High concentrations of antagonists of histamine H2 receptors, muscarinic receptors, alpha 1-adrenoceptors and serotonin receptors did not inhibit the effect. The histamine H1-receptor antagonists mepyramine, triprolidine, promethazine, d chlorpheniramine and the tricyclic antidepressant doxepin inhibited the response with Ki values corresponding to an interaction with histamine H1-receptors. The EC50 for the response was about three times lower than the Ki value (approximately 300 microM) for the inhibition by histamine of [3H]mepyramine binding to membranes from rat brain cortex. Partial inactivation of H1-receptors with the alkylating antagonist phenoxybenzamine resulted in similar reductions in [3H]mepyramine binding sites and in the maximal histamine-induced [3H]inositol 1 phosphate accumulation, without affecting the KD for the radioligand or the EC50 for the response. The apparent dissociation constant for histamine calculated from these experiments (KA = 92.2 microM) was not different from the EC50 value. The present results indicate that histamine-stimulated phosphoinositide hydrolysis in rat brain cortex is mediated by H1-receptors and that no receptor reserve is present. PMID- 3011461 TI - Antagonistic effects of bisoprolol on several beta-adrenoceptor-mediated actions in anaesthetized cats. AB - The beta-adrenoceptor antagonistic activity of i.v. administered bisoprolol ((+/ )-1-[4-(2-isopropoxyethoxymethyl)-phenoxy] -3-isopropylamino-2-propanol, hemifumarate) was studied under two different sets of experimental conditions in anaesthetized cats and compared to the activity of atenolol and propranolol. The responses of several target organs to beta 2-adrenoceptor stimulation were used: inhibition of isoprenaline effects on diastolic blood pressure, hindlimb perfusion pressure, soleus muscle contractility and histamine aerosol-induced bronchoconstriction. The inhibition of isoprenaline-induced tachycardia served as indicator of beta 1-antagonism. The slopes of agonist dose ratio vs. antagonist dose effect were close to unity for propranolol but deviated from unity for atenolol and even more so for bisoprolol. In spite of the ensuing difficulty of comparisons, bisoprolol showed the most pronounced selectivity indices (10-20), followed by atenolol (1-7.5) and propranolol (0.3-1.6). Thus, bisoprolol exhibited a higher degree of beta 1-selectivity in the cat than did atenolol, regardless of the parameter used for measurement of beta 2-antagonism. Propranolol proved to be non-selective or even had a somewhat higher affinity for beta 2- than for beta 1-adrenoceptors. PMID- 3011462 TI - [3H]clonidine and [3H]yohimbine binding to solubilized alpha 2-adrenoceptors from rat cerebral cortex. AB - Alpha 2-adrenoceptors were solubilized from rat cerebral cortex using the zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). The CHAPS extract retained binding activity for [3H]clonidine and [3H]yohimbine. Treatment of membranes with 10 mM CHAPS solubilized about 30% of the [3H]clonidine binding sites in the starting membranes. A Scatchard plot of [3H]clonidine binding to the CHAPS extract showed a non-linear curve, indicating the existence of the two distinct binding components. The effects of GTP and cations on alpha 2-agonist and antagonist binding to the CHAPS extract were similar to the effects in membrane preparations. Sepharose CL-4B column chromatography showed the alpha 2-agonist binding complex to be a larger molecule, with a Stokes radius of 85 A, than the alpha 2-antagonist binding complex with a radius of 71 A. These results indicate that the complexes between the alpha 2-adrenoceptors and GTP binding regulatory proteins remain intact throughout the CHAPS solubilization procedure. PMID- 3011463 TI - Alpha-adrenoceptor antagonistic and calcium antagonistic effects of nicergoline in the rat isolated aorta. AB - The activity of the alpha-adrenoceptor antagonist nicergoline, a molecule composed of two constituent parts, ergoline and bromonicotinic acid, was investigated in the rat isolated aorta. Nicergoline (10 nM-0.1 microM) displaced concentration-effect curves elicited by noradrenaline and phenylephrine to the right and inhibited maximal responses elicited by both alpha-adrenoceptor agonists without significantly affecting prostaglandin F2 alpha-induced contractions. Higher concentrations of nicergoline (1 microM-50 microM) displaced to the right the concentration-effect curves elicited by calcium in a depolarizing medium. This calcium antagonist activity was not shared by either of the constituent parts. Nicergoline 100 microM abolished the 45Ca influx induced into rat aorta by 100 mM K+-containing physiological solution. The selectivity of nicergoline for alpha 1-adrenoceptors seen in binding experiments also depends on the presence of the bromonicotinic moiety of the molecule. It is concluded that nicergoline, but not its substituent parts, displays both alpha 1-adrenoceptor and calcium antagonism. The latter property may account for some of the observed effects of this compound. PMID- 3011464 TI - Effects of nitroprusside on pancreatic exocrine secretion and cyclic nucleotide concentration in the dog pancreas. AB - The effects of intravenous injection of nitroprusside on pancreatic exocrine secretion and on pancreatic cyclic AMP and cyclic GMP concentrations of mongrel dogs were investigated. Nitroprusside (10-100 micrograms/kg) increased the exocrine secretion together with the increase in cyclic GMP concentration dose dependently but did not affect the cyclic AMP concentration. These results suggest that nitroprusside causes exocrine secretion from the dog pancreas mediated through an increase of intracellular cyclic GMP concentration. PMID- 3011465 TI - Species differences in the angiotensin converting enzyme activity of mammalian serum. AB - Angiotensin converting enzyme (ACE; EC 3. 4. 15. 1) activities were compared in the serum of various mammals. Cattle, human, monkey, and swine serum showed enzyme levels from 3.7 to 67 mU/ml. Relatively high enzyme activity was observed in rodents, the rat (Wistar) and mouse (BALB/c) showing levels of 93 +/- 7 and 1052 +/- 165 mU/ml, respectively. Among the mammalian sera examined, that of the guinea pig contained the highest ACE level, 2262 +/- 574 mU/ml. No age-related difference in enzyme activity was observed in 10-day-old to 1-year-old guinea pigs. PMID- 3011466 TI - Herpes simplex virus complement fixing antibody and herpes B virus serum neutralizing antibody in sera of wild and laboratory-bred cynomolgus monkeys. AB - The result of the complement fixation (CF) test for the antibody to herpes simplex virus (HSV) in sera of the cynomolgus monkeys was compared with that of the neutralization test (NS) for the antibody to herpes B virus (HBV) in the same sera. Fifty-seven (74%) of 77 wild-originated monkeys were positive for HSV-CF, while 65 (84%) of the 77 animals were positive for HBV-SN. All of the 57 CF positive cases were also positive for HBV-SN. On the other hand, 30 (75%) of 40 laboratory-bred monkeys had neither HSV-CF antibody nor HBV-SN antibody. Remaining 10 of the 40 laboratory-bred animals were positive for HSV-CF. However, no HBV-SN antibody was detected in nine of the 10 HSV-CF positive animals. These results suggest that the HSV-CF test may be as satisfactory as the HSV-SN test as a practical measure for rough screening of HBV infection in the cynomolgus monkey in laboratories having no containment unit for handling HBV. PMID- 3011468 TI - Intracellular pH controls growth factor-induced ribosomal protein S6 phosphorylation and protein synthesis in the G0----G1 transition of fibroblasts. AB - Mitogen-induced intracellular alkalinization mediated by activation of a Na+/H+ antiporter is a common feature of eukaryotic cells stimulated to divide. A Chinese hamster fibroblast mutant (PS120) lacking Na+/H+ antiport activity (Pouyssegur et al., Proc natl acad sci US 81 (1984) 4833) [42] possesses an intracellular pH (pHi) 0.2-0.3 units lower than the wild type (CCL39) and requires a more alkaline pHout (pHo) for growth. Here, we show that serum stimulated ribosomal protein S6 phosphorylation, protein synthesis activation and DNA synthesis re-initiation are pH-regulated events that display a similar threshold pHo value (6.60) in CCL39 cells. pH-Dependencies for initiation of all three events are shifted toward higher pHo values in the mutant PS120, indicating that growth factor-induced alkalinization has a permissive effect on the pleiotypic response. However, cytoplasmic alkalinization per se is insufficient to trigger S6 phosphorylation, polysome formation, and subsequent DNA synthesis. Transient exposure to a non-permissive pHo (6.5) inhibits both the rate of leucine incorporation into proteins and the progression through the G1 phase of the cell cycle. In contrast, cells committed to DNA synthesis are unaltered by the acidic pHo. These observations suggest that pHi by controlling the rate of protein synthesis play a determinant role in the control of cell division. PMID- 3011467 TI - Two cases of spontaneous malignant fibrous histiocytoma in Wistar rats. AB - Spontaneous malignant fibrous histiocytoma (MFH) of subcutaneous tissues was found in 2 Wistar rats of a 18-month-old male and a 21-month-old female. These tumors consisted of spindle, oval, myxoid and giant cells. In the female rat, a similar histological pattern was found in the metastatic tumor nodules in the lung and liver. In the electron microscopy, four differentiated cells of fibroblast-like, histiocyte-like, giant and undifferentiated cells were identified in these tumors. Spontaneous MFH of Wistar rats in the present study was quite similar to those reported in rats of other strains. PMID- 3011469 TI - Fusion of fluorescently labeled Sendai virus envelopes with living cultured cells as monitored by fluorescence dequenching. AB - Fluorescently labeled (bearing N-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine (N-NBD-PE)) reconstituted Sendai virus envelopes (RSVE) were used to study fusion between the viral envelopes and cultured living cells such as lymphoma, Friend erythroleukemia cells (FELC) and L cells. Incubation of fusogenic viruses with the above cell lines resulted in a relatively high degree (40-45%) of fluorescence dequenching. On the other hand, incubation of unfusogenic (trypsin or phenylmethylsulfonylfluoride (PMSF)-treated) RSVE with these cells led to very little (6-9%) fluorescence dequenching. The degree of fluorescence dequenching was linearly correlated to the surface density of the virus-inserted N-NBD-PE molecules. Fluorescence photobleaching recovery experiments showed that fusion of fluorescent RSVE with FELC resulted in an infinite dilution of the fluorescent molecules in the recipient cell membranes. The fluorescent probe 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (N-NBD-Cl) was covalently attached to envelopes of intact Sendai virions without significantly impairing their biological activity. Incubation of fluorescently labeled, intact Sendai virions with cultured cells resulted in about 20% fluorescence dequenching. The present data clearly indicate that fluorescently labeled Sendai virions can be used for a quantitative estimation of the degree of virus-membrane fusion. PMID- 3011470 TI - Endonuclease banding of isolated mammalian metaphase chromosomes. AB - Evidence is presented that endonuclease digestion of isolated, unfixed chromosomes results in the production of banding patterns similar to those produced by digestion of fixed, air-dried chromosomes. Mouse L cell chromosomes were isolated under acidic or relatively neutral pH conditions, exposed in situ (as wet mounts on glass slides) or in vitro (in suspension) to micrococcal nuclease, Alu I or Eco RI, treated with a buffered salt solution, and stained with Giemsa. After any of these endonuclease treatments in situ, the centromeric regions of the chromosomes were intensely stained, characteristic of the C banding observed in fixed chromosomes exposed to the same treatments. Although the fixed chromosomes were morphologically well-preserved after endonuclease digestion, the morphology of chromosomes digested in situ was variable, ranging from normal to swollen to highly distorted chromosomes. In the latter, the endonucleases induced dispersion of non-C-band chromatin; however, C-bands were still apparent as condensed, differentially-stained regions. Exposure of isolated chromosomes to Alu I in vitro also resulted in well-defined C-banding and led to the extraction of about 70% of the chromosomal DNA. From these results, the mechanism of endonuclease-induced C-banding appears to involve the dispersion and extraction of digested chromatin. PMID- 3011471 TI - The effect of dexamethasone on epidermal growth factor binding and stimulation of proliferation in young and senescent WI38 cells. AB - Addition of 0.14 microM dexamethasone (DEX) to young log-phase WI38 cultures seeded at various densities in serum-free medium containing 4.1 nM epidermal growth factor (EGF) resulted in a synergistic increase in proliferation and final cell density. The action of DEX plus EGF was stimulatory but not synergistic in young confluent cultures. DEX plus EGF had no synergistic effect on senescent cells either during log phase or at confluence. Analysis of the effect of DEX on [125I]EGF binding revealed no statistically significant changes in either the number of binding sites or the apparent dissociation constant of the EGF-receptor complex. PMID- 3011472 TI - A simple quantitative method for the estimation of free ecto-sulfhydryl groups of spermatozoa. AB - A simple rapid quantitative method has been developed for the estimation of sperm ecto-SH groups on the basis of their high affinity binding to the mercurial: [203Hg]p-chloromercuriphenylsulfonic acid (PCMPS) used as a surface probe. The thiol reagent did not penetrate the sperm plasma membrane, as evidenced by the extremely rapid time course of the binding reaction and undetectable uptake of [203Hg]PCMPS by intact goat spermatozoa. The binding reaction was not due to contaminating broken or damaged cells, if any. The method consists of incubating of highly motile goat spermatozoa with PCMPS in a modified Ringer solution at 37 degrees C for 5 min, agglutination of the labelled cells with polyethyleneimine (100 micrograms/ml) and filtration and washing of the cell suspension through Whatman No. 1 filter discs under mild vacuum. The binding interaction is proportional to cell concentration, specific and saturable at 50 microM PCMPS. The method is capable of estimating free ecto-SH as low as 25 pmoles. Spermatozoa possess 286 +/- 61 pmoles of free ecto-SH groups/10(6) cells. Scatchard analysis showed the presence in goat spermatozoa of multiple classes of ecto-SH groups differing in their affinity for PCMPS. PMID- 3011473 TI - The effect of retinoic acid on protein phosphorylation in mouse melanoma cells. AB - Vitamin A inhibits growth and increases the activity of cAMP-dependent protein kinase in B16 mouse melanoma cells. In this report we show that retinoic acid (RA) treatment of intact cells alters their subsequent in vitro protein phosphorylation, but we could not demonstrate any changes in in vivo protein phosphorylation. A 48-h treatment with RA results in a concentration-dependent decrease of protein phosphorylation of a 95K molecular weight (MW) protein in both supernatant and particulate fractions. The phosphorylation of this protein does not appear to be regulated by cAMP. Proteins at 92K and 82K MW in the supernatant fraction are increased in phosphorylation. The former (but not the latter) is regulated by cAMP. In the particulate fraction a variety of proteins 12K-68K MW are increased in phosphorylation, as the cells are treated with increasing amounts of RA. The phosphorylation of most of these proteins is regulated by cAMP. Another inhibitor of B16 cell growth, melanocyte-stimulating hormone (MSH) also alters protein phosphorylation. At short incubation periods (1 h), this hormone stimulates phosphorylation of a number of proteins (17-40K MW), while in longer incubation periods (48 h) phosphorylation is inhibited. All of these phosphorylations appear to be regulated by cAMP. We attempted to repeat these observations using intact-cell phosphorylation with 32PO4. In two experiments we saw small changes in the phosphorylation of proteins. In most experiments, however, we could find no change in the phosphoproteins. Further experiments have led us to question the in vivo phosphorylation, since treatment of the cells with MSH, cholera toxin, or db-cAMP also did not affect intact-cell protein phosphorylation. We have previously documented that under these latter conditions cAMP levels are greatly elevated and cAMP-dependent protein kinase is activated. The in vitro phosphorylation results suggests that in RA-treated cells, kinase activities and/or protein substrate levels are changing. However, the physiological significance of the particular MW phosphoproteins changes we have described must await resolution of the in vivo phosphorylation data. PMID- 3011474 TI - Plasma membranes purified from myeloid leukemia cells before and after differentiation. I. Characterization of spectrin-like proteins and increased association of actin. AB - Two-step sucrose density gradient centrifugation was used to isolate the plasma membrane of a myeloid leukemia cell line (Ml). Calspectin (or fodrin) was identified in the Triton-insoluble fraction from the plasma membrane, and the molecular size and actin- and calmodulin-binding activity were studied. During differentiation of this cell line, which accompanied the induction of cell motility and phagocytic activity, the membrane-bound actin increased dramatically, whereas calspectin increased only slightly. Therefore, calspectin does not appear to have a major function in the increased binding of actin filaments to the plasma membrane, a requirement for the induction of cell motility. PMID- 3011476 TI - Preparation of cytoplasmic bodies (cytospheres) from isolated hepatocytes and their biochemical properties. AB - The controlled centrifugation of isolated rat hepatocytes at 260 000 g results in the formation of membrane-bounded cell fragments that we have termed 'cytospheres'. A method is described for the isolation of these cytospheres. Cytospheres are spherical, have a mean diameter of 9.2 +/- 3.2 microns (SD) and a protein content of 225 +/- 12 mg/g wet wt. About 3% of the protein from the original isolated hepatocyte suspension is recoverable. Transmission electron microscopy (TEM) shows cytospheres to possess a trilaminar membrane, and a finely granular hyaloplasm generally devoid of organelles, filaments and microtubules. Freeze-fracture studies reveal a membrane structure typical of a plasma membrane. Ouabain and wheat germ agglutinin (WGA)-binding studies indicate that the original orientation of the plasma membrane is maintained throughout the formation of the cytospheres. The cytospheres have also been characterized biochemically. Cytospheres are enriched in the enzymes normally associated with the hyaloplasm, whereas the activities of enzymes localized in organelles are greatly diminished. Lipid analysis of the cytosphere membrane indicates that it is derived from the plasma membrane of the hepatocyte. Cytospheres are sensitive to changes in the osmolarity and ionic composition of their environment. Cytospheres should therefore prove a useful preparation for the study of hyaloplasm metabolism and of plasma membrane receptor and permeability properties. PMID- 3011475 TI - Human histone gene organization. Identification of a histone gene polymorphism prevalent in a black population. AB - Analysis of the restriction enzyme digests of total genomic DNAs from a broad spectrum of human cell lines and from individuals with different genetic backgrounds, by hybridization with a series of cloned human histone sequences, indicated restriction site polymorphisms (RSPs) for two adjacent human histone genes which reside on chromosome 1. In most cell lines and individuals examined we observed a single 2.05 kb H4 histone HindIII fragment and a 7.0 kb H3 histone HindIII fragment. In contrast, the polymorphisms were manifested as a 2.15 kb H4 HindIII fragment and a 9.1 kb H3 HindIII fragment. From population studies, we were able to show that there is no linkage disequilibrium between these two polymorphic restriction sites. Nor was there any apparent correlation between the presence of the H3/H4 histone polymorphisms and maintenance of the transformed karyotype, passage in culture, transformation or tumor progression. These chromosome 1 H3 and H4 histone gene polymorphisms are common in the American Black population and, in our survey of individuals, were not found in the American Caucasian population. Among the American Blacks studied, the frequency of the H3 HindIII(-) allele is 43% and of the H4 HindIII(-) allele 30%. In limited family studies, we were unable to detect recombination between these two physically linked alleles. PMID- 3011477 TI - Elevated level of beta-adrenergic receptors in hepatocytes from regenerating rat liver. Time study of [125I]iodocyanopindolol binding following partial hepatectomy and its relationship to catecholamine-sensitive adenylate cyclase. AB - Hepatocytes from regenerating rat liver show an enhanced epinephrine-sensitive adenylate cyclase activity and cAMP response, which may be involved in triggering of the cell proliferation. We have determined adrenergic receptors and adenylate cyclase activity in hepatocytes isolated at various time points after partial hepatectomy. The number of beta-adrenergic receptors, measured by binding of [125I]iodocyanopindolol ([125I]CYP) to a particulate fraction prepared from isolated hepatocytes, increased rapidly after partial hepatectomy as compared with sham-operated or untreated controls. The maximal increase, which was observed at 48 h, was between 5- and 6-fold (from approximately 1 800 to approximately 10 500 sites per cell). Thereafter, the number of beta-adrenergic receptors decreased gradually. Competition experiments indicated beta 2-type receptors. Parallelism was found between the change in the number of beta 2 adrenergic receptors and the isoproterenol-responsive adenylate cyclase activity. The number of alpha 1-adrenergic receptors, determined by binding of [3H]prazosin, was transiently lowered by about 35% at 18-24 h, with no significant change in Kd. Although the results of this study do not exclude the possibility of post-receptor events, they suggest that the increased number of beta 2-adrenergic receptors is a major factor responsible for the enhanced catecholamine-responsive adenylate cyclase activity in regenerating liver. PMID- 3011478 TI - The use of Rous sarcoma virus transformation mutants with differing tyrosine kinase activities to study the relationships between vinculin phosphorylation, pp60v-src location and adhesion plaque integrity. AB - Tyrosine-specific phosphorylation of cellular proteins has been implicated in the neoplastic transformation of cells by Rous sarcoma virus (RSV). One of the putative substrates for the src gene product (pp60v-src) of RSV is the cytoskeletal protein vinculin, giving rise to the hypothesis that tyrosine specific phosphorylation of vinculin disrupts adhesion plaque integrity, leading to the characteristic rounded morphology of RSV-transformed cells. We have investigated this hypothesis by analysing the properties of fibroblasts transformed by conditional and non-conditional mutants of RSV which confer different morphologies on infected cells, with respect to formation of microfilament bundles, formation of vinculin-containing adhesion plaques, the deposition of a fibronectin-containing extracellular matrix, the localization of pp60v-src and the tyrosine-specific phosphorylation of vinculin. Cells transformed by the temperature-sensitive (ts) RSV mutant LA32 cultured at 41 degrees C were morphologically normal, and contained prominent microfilament bundles and well-developed adhesion plaques. However, these cells had a fully active pp60v-src kinase, had pp60v-src concentrated in their adhesion plaques and contained vinculin which was heavily phosphorylated on tyrosine residues. Cells transformed by a recovered avian sarcoma virus, rASV 2234.3 exhibited a markedly fusiform morphology with pp60v-src concentrated in well-developed adhesion plaques and an elevation of the phosphotyrosine content of vinculin. Cells transformed by LA32 at restrictive temperature comprise morphologically normal cells, indistinguishable from untransformed CEF, yet which contain tyrosine phosphorylated vinculin and suggest that neither tyrosine-specific phosphorylation of vinculin nor pp60v-src concentration in adhesion plaques is sufficient for the rounded morphology of RSV-transformed cells. PMID- 3011479 TI - Establishment of a differentiated mesodermal line from P19 EC cells expressing functional PDGF and EGF receptors. AB - Aggregation of pluripotent P19 embryonal carcinoma (EC) cells in the presence of DMSO induces differentiation to various mesodermal cell types, including spontaneously contracting muscle. We have established clonal cell lines from these cultures and characterized one (MES-1) in particular for its response to growth factors. In contrast to the undifferentiated stem cells, but as a number of myoblast and muscle cell lines, MES-1 cells respond to both carbachol and bradykinin by the rapid release of Ca2+ from intracellular stores. In addition, MES-1 express receptors for and respond mitogenically to epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Isolated membranes from these cells retain the capacity to bind both ligands; addition of EGF to membranes induces endogenous phosphorylation of several proteins, including the EGF receptor itself and a 38 kD protein, while addition of PDGF specifically induces phosphorylation of the PDGF receptor. By contrast, other derivatives of P19, isolated from retinoic acid (RA)-treated aggregates and resembling neuroectodermal or endodermal cell types respond only to EGF; PDGF neither binds nor induces phosphorylation and a mitogenic response in these cells. During differentiation from EC cells therefore MES-1 cells developed a combination of growth factor receptor characteristics typical of somatic mesodermal cells and indicate that such receptors on EC-derived mesodermal cells are also functional. PMID- 3011480 TI - Isolation of an MSP1-derived transcriptionally active nucleolar particle. AB - A discrete macromolecular structure with inherent transcription activity has been isolated from Novikoff nucleoli. The particle (350 A diameter) was released from the intact nucleoli by digestion with restriction endonuclease (Msp1) and is enriched in repetitive DNA sequences both 5' and 3' to the ribosomal genes. In vitro transcription analyses suggest that the particle contains the rDNA polymerase template, and that its 12-15 major proteins are responsible for transcriptional activity without added soluble protein factors. PMID- 3011481 TI - Purification of calpain II from rat lens and determination of endogenous substrates. AB - Calpain II (EC 3.4.22.17), a calcium-dependent neutral protease, was purified approximately 7000-fold from the soluble of rat lens. The estimated molecular weight of rat lens calpain II was 120,000, and the enzyme was composed of 80,000 and 28,000 MW subunits. Calpain II required 400 microM calcium, a reducing agent, and pH = 7.5 for maximal activity. The enzyme could not be activated by magnesium, and was inhibited by leupeptin and iodoacetate, but not by phenylmethylsulfonyl fluoride. Purified calpain II degraded rat alpha, beta H-, and beta L-crystallins, insoluble proteins, and intrinsic membrane proteins, gamma-Crystallin was not degraded. The proteolysis caused by purified calpain II was similar to proteolysis occurring during the formation of several experimental cataracts in rodents; this suggested that the enzyme may play a role in cataract formation. PMID- 3011482 TI - ATP hydrolysis kinetics of Na,K-ATPase in cataract. AB - The steady-state kinetics of hydrolysis of Mg2+ ATP by the epithelial Na,K-ATPase of individual human lenses were determined. Among the cataract lens population, four distinct kinetic types were observed: negative kinetic co-operativity. Michaelis-Menten kinetics, positive kinetic co-operativity, and substrate inhibition kinetics. Negative kinetic co-operativity and Michaelis-Menten kinetics were also observed in a group of presumably clear lenses from non diabetic individuals ages 16-42 years. Substrate inhibition kinetics were found to be prevalent in individuals with mature onset diabetes. Substrate inhibition kinetics were also observed for Na,K-ATPase isolated from lenses which had been incubated in high glucose. It would appear that this modification leads to an inhibition of Na,K-ATPase-dependent K+ influx into these cultured lenses. PMID- 3011483 TI - Effect of a monoclonal antibody against GPs IIb-IIIa on platelet aggregation and ATP secretion. AB - A murine monoclonal antibody against glycoproteins IIb-IIIa of platelet membrane completely abolished platelet aggregation induced by epinephrine, arachidonic acid, and low concentration of collagen and thrombin, but it had only minor inhibitory effects on aggregation induced by ADP, higher amounts of collagen and thrombin, and these agents combined in pairs. The simultaneous studies of aggregation and ATP secretion demonstrated that aggregation plays an essential role in stimulating the platelet-release reaction, except for the thrombin induced ATP secretion that seems to be largely dependent on fibrinogen binding to platelet surface. PMID- 3011484 TI - Spatial relationship of histochemically demonstrable patches in the mouse superior colliculus. AB - Patches of high phosphorylase activity are found in the intermediate and dorsal deep grey layers of the mouse superior colliculus when either coronal or sagittal sections are cut. These patches indicate that the phosphorylase a activity is arranged in a continuous lattice composed of bands of high phosphorylase a activity with a width of 100-200 microns that surround pale islands of low activity. This lattice was demonstrated by cutting surface parallel sections through the partially flattened superior colliculus. An almost identical lattice is observed in sections incubated to demonstrate total phosphorylase or cytochrome oxidase (CYO) activity. This phosphorylase/CYO lattice extends over the entire area of the superior colliculus. A discontinuous staining pattern is also observed in the intermediate and deep grey layers of both sagittal and coronal sections incubated for acetylcholinesterase (AChE) activity. The staining is arranged in two discontinuous sheets of intense activity that are joined together by vertical streamers. In surface parallel sections the AChE activity is found to form a network pattern which extends over the entire extent of the superior colliculus but which becomes fainter at the anterior pole. The phosphorylase/CYO lattice is not in register with the AChE lattice and the two seem to be organized independently of each other despite occurring at the same depth. PMID- 3011486 TI - Electrophysiologic evidence for connections between the supraoptic and the arcuate/ventromedial hypothalamic nuclei in the rat. AB - Extracellular action potentials were recorded from 48 single units located in the hypothalamic arcuate and ventromedial nuclei. Fifteen percent of the cells were identified as projecting to the median eminence and some of these cells may have belonged to the tuberoinfundibular dopaminergic systems. Responses of all cells to stimulation of the ipsilateral supraoptic nucleus were recorded; 17% of ventromedial nucleus neurons were antidromically identified as projecting to the supraoptic nucleus. None of the latter cells was also identified as projecting to the median eminence. Three of six identified tuberoinfundibular and eight unidentified ventromedial nucleus cells were found to be excited by stimulation of the supraoptic nucleus. One arcuate cell identified as projecting to the median eminence was nonresponsive to supraoptic stimulation. Orthodromic inhibitory responses were recorded from 17% of all cells recorded but no inhibitory responses were recorded from cells identified as projecting to the median eminence. We suggest that these results may provide some neurophysiologic explanations for the observed interrelationships between oxytocin and prolactin secretion, and between vasopressin and growth hormone secretion. PMID- 3011487 TI - Convulsions may alter the specificity of kappa-opiate receptors. AB - Morphine, a mu-opiate agonist, and ethylketazocine, a kappa-opiate agonist, produce distinct behavioral, pharmacologic, and biochemical effects. In the mouse, large doses of morphine produce convulsions that are usually lethal and that cannot be blocked by naltrexone, whereas ethylketazocine produces nonlethal clonic convulsions that can be blocked by naltrexone. Moreover, mice made tolerant to morphine failed to show cross-tolerance to ethylketazocine, suggesting that the convulsions induced by these drugs are not mediated via a common opioid mechanism. Following a series of electroconvulsive shocks, both morphine and ethylketazocine produced clonic convulsions that were not lethal and that could be blocked by naltrexone. Furthermore, electroconvulsive shock-treated animals made tolerant to morphine-induced convulsions showed cross-tolerance to ethylketazocine. These data suggest that electroconvulsive shock may alter kappa opioid systems in such a way as to allow mu-agonists to be functional at these sites. PMID- 3011485 TI - Collateral specific long term potentiation of the output of field CA3 of the hippocampus of the rat. AB - Long term potentiation (LTP) in response to brief high frequency trains has been reported for many pathways in the hippocampus. The mechanisms involved are still unclear. The present experiments set out to confirm reports in the literature that LTP of output from CA3 neurons can be specific to particular collaterals. Single pulses delivered to area CA3 produced field responses nearly simultaneously in area CA1 and in the lateral septum (LS). High frequency stimulation of CA3 produced long term potentiation of CA1 but not LS responses. The CA1 response to stimulation of the contralateral hippocampus did not potentiate when the CA1 response to CA3 stimulation showed long term potentiation. The CA1 and LS responses to CA3 stimulation showed similar strength duration, strength-amplitude and frequency following characteristics. Their latencies were comparable to the latencies of antidromic activation of CA3 cells from CA1 and LS. Movement of stimulating electrodes to the region of the Schaffer collaterals increased the latency of the LS response and decreased the latency of the CA1 response but left the sum of these latencies unchanged. It was concluded that the CA3 and Schaffer stimulation were activating LS and CA1 collaterals of the same CA3 neurons. CA1 and LS responses to CA3 stimulation showed somewhat different paired pulse and frequency potentiation characteristics. These data confirm reports in the literature that long term potentiation is both input specific and collateral-specific.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011489 TI - Caenorhabditis elegans: comparisons of chemotactic behavior from monoxenic and axenic culture. AB - Significant differences in chemotactic response of Caenorhabditis elegans were demonstrated for nematodes from monoxenic culture as compared to nematodes from axenic culture. These results support those of a previous study in which large differences in growth, development, behavior, and longevity were shown for C. elegans in comparative assays of the monoxenic and axenic regimes. PMID- 3011488 TI - Plasma ACTH and corticosterone responses to limbic kindling in the rat. AB - Changes in plasma ACTH and corticosterone concentrations were measured in individual cannulated rats at stages 1 and 5 of limbic kindling induced by electrical stimulation of the basolateral amygdala or the dorsal hippocampus. At both stages, a stimulation of either structure produced swift surges, first of ACTH and then of corticosterone. At stage 5 of hippocampal stimulation, ACTH baseline concentrations were four times higher than in the controls. The results are discussed in relation to the central control of the adrenocorticotropic system and to the neuroendocrine correlates of the kindling process. PMID- 3011490 TI - Glucose-6-phosphatase activity in normal and denervated developing chick gastrocnemii: reappraisal of glycogenolytic and glycolytic metabolism in skeletal muscle. AB - Biochemical changes in glycogen content and activity levels of phosphorylase (EC 2.4.1.1), glucose-6-phosphatase (EC 3.1.3.9), phosphohexose isomerase (EC 5.3.1.9) and aldolase (EC 4.1.2.13) have been studied in normal and denervated whole gastrocnemius muscle and its three fasciculi, viz., pars externus, medius and internus up to 9 weeks in chicks. Glycogen content as well as phosphorylase, phosphohexose isomerase and aldolase decrease in normal muscle with advancement of postembryonic growth whereas transiently increased glucose-6-phosphatase reveals an inverse relationship with these parameters. Denervated muscles demonstrate loss of glycogen and related enzymes owing to ablation of neural supply during the initial 4 weeks. Denervation results in a delayed stimulation of glycogenolysis and glycolysis which seems to be governed by decreasing activity of glucose-6-phosphatase. The significance of glucose-6-phosphatase in the regulation of glycogenolysis and glycolytic metabolism of normal and denervated skeletal muscle is discussed. PMID- 3011491 TI - The action of various vitamin D3 metabolites on calcium and phosphorus metabolism in chick embryo calvariae. AB - Chick embryos from vitamin D-deficient hens given physiological doses of 1,25 dihydroxyvitamin D3 or 24,25-dihydroxyvitamin D3 or both become severely hypocalcemic, hyperphosphatemic and fail to hatch as compared to those derived from hens given 25-hydroxyvitamin D3 or 24,25-difluoro-25-hydroxyvitamin D3. Calvariae from the former contain less mineral and on incubation in vitro produce significantly lower calcium and higher phosphate concentration in the medium than do the calvariae derived from the embryos of hens supported on 25-hydroxyvitamin D3 or 24,24-difluoro-25-hydroxyvitamin D3. PMID- 3011492 TI - Hydroxyl radicals are not involved in NADPH dependent microsomal lipid peroxidation. AB - NADPH dependent H2O2 formation in microsomes in the presence of chelated iron leads to formation of hydroxyl radicals. Enhancement of hydroxyl radical generation (via ferric-EDTA or sodium azide) did not result in a concomitant increase in lipid peroxidation; rather, a decrease was observed. Moreover, the hydroxyl radical scavenger DMSO did not inhibit lipid peroxidation. This comparison of hydroxyl radical formation with lipid peroxidation suggests that hydroxyl radicals do not play a part in NADPH-dependent lipid peroxidation. PMID- 3011495 TI - Autonomic effects of centrally administered dermorphin in conscious rabbits. AB - The administration of 1 microgram dermorphin into the mesencephalic ventricle of conscious rabbits modifies some autonomic functions, heart rate, respiratory rate and temperature decreasing in a statistically significant way. Naloxone completely reverses such effects. Pretreatment with drugs which inhibit serotoninergic, catecholaminergic, Gabaergic and cholinergic systems does not affect these central dermorphin actions. PMID- 3011494 TI - [Monoclonal antibodies in pharmacology]. AB - The paper outlines technology of monoclonal antibody manufacture. Pharmacological applications of monoclonal antibodies are considered. The list is given of produced by the late 1984 stable hybridomas to various receptors and some proteins being of interest to pharmacology. The possibility of using monoclonal antibodies as carriers for drug transport is discussed. PMID- 3011493 TI - [Action of substances altering the intracellular cAMP level on the enzymes of the oxidative metabolism of xenobiotics]. AB - The substances decreasing (insulin subcutaneously 30 U/kg single dose) and increasing (isadrin, theophylline orally 30 mg/kg five times with 12-hour intervals) the intracellular level of cAMP exert varying effects of the activities of mono-oxygenases of the rat liver endoplasmic reticulum. Insulin decreases aniline binding with cytochrome P-450 and the rate of its p hydroxylation (after 6, 12 hours), the content of cytochrome P-450 and the rate of NADP.H oxidation (after 12 hours), the rate of NAD.H oxidation (after 24 hours). The activity of NADP.H-nitrotetrazolium reductase, content of cytochrome B5, the rate of aniline p-hydroxylation are increased by theophylline, and the rate of NADP.H and NAD.H oxidation and the content of cytochrome P-450 are increased by theophylline and isadrin. PMID- 3011497 TI - Physico-chemical study of complex formation of DNA with wild-type and mutant E. coli RNA polymerases. Recognition properties of beta-subunit. AB - Complex formation of T7 DNA with RNA polymerase from E. coli B/r WU-36-10-11-12 (E. coli W12) and its rifampicin-resistant mutant rpoB409 was studied. The rpoB409 mutant possesses a highly pleiotropic effect due to alteration in the RNA polymerase beta-subunit structure. The two RNA polymerases have been previously shown to differ in gene selection during RNA synthesis on T7 DNA. In this study it was found that the change in selective properties of the mutant RNA polymerase occurs during its interaction with DNA, the general ability of the enzyme to melt DNA being unaffected. PMID- 3011496 TI - Modulation of cytosolic protein kinase C activity by ferricyanide: priming event seems transmembrane redox signalling. A study on transformed C3H/10T1/2 cells in culture. AB - Transformed 3T3/10T1/2 cultured cells incubated with ferricyanide caused a decrease of 2 mM EDTA extractable cytosolic protein kinase C activity in 2 min, whereas 5 or 20 min ferricyanide treatment reverted the enzyme activity to that observed without ferricyanide. The ferricyanide effect in 2 min was abolished by amiloride and sustained by ouabain. Thus, deactivation-activation of cytosolic protein kinase C is attributed to an unknown signal generation during H+ accumulation coupled with the Na+/H+ exchange phase. In this mechanism the priming event concerns the transmembrane redox process shedding H+ into the cell interior while impermeant ferricyanide acts as a unique electron acceptor. PMID- 3011498 TI - Activation of chicken liver fructose- 1,6-bisphosphatase by oxidized glutathione. AB - Treatment of chicken liver fructose- 1,6-bisphosphatase with oxidized glutathione (GSSG) leads to an increase in activity. This activation is markedly enhanced if treatment is performed in the presence of AMP or Mn2+. The effects of AMP and Mn2+ appear to be synergistic. The maximal activation is over 13-fold and is accompanied by the disappearance of 4 sulfhydryl groups per molecule of enzyme. Both fructose 1,6-bisphosphate and fructose 2,6-bisphosphate can largely prevent this activation. Activation can be reversed by dithiothreitol or cysteine. It appears that GSSG activates this enzyme by thiol/disulfide exchanges with the enzyme's specific sulfhydryl groups. PMID- 3011499 TI - Identification of creatine as a cofactor of thiamin-diphosphate kinase. AB - Thiamin-diphosphate (TDP) kinase which catalyzes thiamin triphosphate formation from TDP requires a low-molecular-mass cofactor in addition to ATP and Mg2+. The cofactor was isolated in a crystalline form from pig skeletal muscle and identified as creatine by proton NMR, mass spectrometry, infrared spectrometry and elemental analysis. The isolated cofactor and authentic creatine supported the same activity of partially purified TDP kinase at identical molar concentrations. Neither creatine phosphate nor creatinine showed activity as a cofactor. This is the first report showing evidence of the existence of a creatine-dependent enzyme. PMID- 3011500 TI - Characterization of new gangliosides of the lactotetraose series in murine xenografts of a human glioma cell line. AB - The major mono- and disialogangliosides of the extensively characterized established human glioma line D54MG were isolated and purified from subcutaneous solid xenografts grown in athymic (nu/nu) mice. Structural determination showed that they belonged to the lactotetraosylceramide series. The sialyllactotetraosylceramide contained 90% N-glycolyl- and 10% N-acetylneuraminic acid linked in an alpha 2-3 linkage (IV3NeuGc-LcOse4Cer, IV3NeuAc-LcOse4Cer). The disialogangliosides had a previously undescribed type of structure with sialic acids linked to the terminal galactose in an alpha 2-3 linkage and to N acetylglucosamine in an alpha 2-6 linkage. Not only did species with NeuAc or NeuGc occur, but also species with mixtures of the two sialic acids, e.g. NeuAc and NeuGc. The schematic structures of the new disialogangliosides are (Formula:see text). PMID- 3011501 TI - Predicted sequence of the host-protective immunogen of infectious bursal disease virus. AB - The genome of Australian strain 002-73 of infectious bursal disease virus (IBDV) has been cloned as cDNA fragments into an expression library based on pUR plasmid vectors. Recombinant colonies were selected with a monoclonal antibody specific for the 32-kDa host-protective immunogen of IBDV and fully characterized by nucleotide sequence analysis. The amino acid sequence of several tryptic peptides derived from the native 32-kDa structural protein has confirmed the coding region assignment and nucleotide sequence data. We believe this to be the first published sequence of a birnavirus-encoded protein and these data may provide the basis for an effective subunit vaccine against IBDV. PMID- 3011502 TI - Leukotriene B4 stimulation of phosphatidylinositol turnover in macrophages and inhibition by pertussis toxin. AB - The influence of leukotriene B4 (LTB4) on phosphatidylinositol (PI) cycle activity was investigated in the guinea pig alveolar macrophage. In contrast to the observation reported in leukocytes [(1984) Proc. Natl. Acad. Sci. USA 81, 5966-5969], LTB4 was found to stimulate PI cycle activity in the macrophage (transient decrease in phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate, transient elevation of 1,2-diacylglycerol and elevation of phosphatidic acid) similar to the stimulation of the PI cycle by the chemotactic formyl peptides. Preincubation of macrophages with 1 microgram/ml of pertussis toxin, for 1 h at 37 degrees C, blocked LTB4 and formyl peptide stimulated O-2 production and PI cycle activity. Therefore, both stimulants are proposed to act through the same coupling protein to activate phospholipase C in the early stages of macrophage activation. PMID- 3011503 TI - EPR of a novel high-spin component in activated hydrogenase from Desulfovibrio vulgaris (Hildenborough). AB - The EPR of reoxidized hydrogenase from Desulfovibrio vulgaris (H.) has been reinvestigated. In contrast to other workers [(1984) Proc. Natl. Acad. Sci. USA 81, 3728-3732] we find the axial signal with g = 2.06; 2.01 to be only a minor component of concentration 0.03 spin/mol. In the spectrum of fully active reoxidized enzyme this signal is overshadowed by a rhombic signal (0.1 spin/mol) with g = 2.11; 2.05; 2.00 reminiscent of the only signal found for other oxidized bidirectional hydrogenases. In addition, a novel signal has been detected with geff = 5.0 which, under the assumptions that S = 2 and [delta ms] = 2, quantitates to roughly one spin/mol. Ethylene glycol affects the relative intensity of the different signals. It is suggested that O2 sensitization parallels a spin-state transition of an iron-sulfur cluster. PMID- 3011504 TI - Binding inhibition by monoclonal antibodies of ovine placental lactogen to growth hormone receptors in human liver. AB - In the radioreceptor assay for growth hormone (RRA-GH) using [125I]iodo-hGH, hGH and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, ovine placental lactogen (oPL) is capable of inhibiting the binding of [125I]iodo-hGH in a parallel manner with hGH and in equipotency. Similarly, in the RRA-GH by employing [125I]iodo-oPL, oPL and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, hGH is also equipotent as oPL in inhibiting the binding of [125I]iodo-oPL in a parallel fashion. The addition of monoclonal antibodies against oPL in the assay was effective in inhibiting the binding of [125I] iodo oPL to human liver, but could not, however, inhibit the binding of [125I]iodo-hGH to human liver. Furthermore, the addition of the monoclonal antibodies in the RRA GH did not affect the parallelism of the oPL standard but lowered the total binding of oPL. Our studies indicate that the structure of the binding sequence in oPL which binds to the GH receptor of human liver is not identical to the equivalent sequence of hGH and that the monoclonal antibodies compete with GH receptors in human liver for the binding of oPL. PMID- 3011505 TI - Activation of phosphatidylinositol turnover by neurotensin receptors in the human colonic adenocarcinoma cell line HT29. AB - Association of neurotensin to its receptor in HT29 cells increases the intracellular concentration of inositol phosphates. A rapid (20-30 s), transient stimulation of inositol trisphosphate (275% of the basal level) and inositol bisphosphate (420%) is first observed, followed by a slower, stable increase in inositol monophosphate (170%). Half-maximal stimulation of the three inositol phosphates was obtained with 50-100 nM neurotensin. These results indicate that neurotensin is able to regulate intracellular Ca2+ levels in HT29 cells by using inositol trisphosphate as a second messenger. PMID- 3011506 TI - Solubilization of trehalase from rabbit renal and intestinal brush-border membranes by a phosphatidylinositol-specific phospholipase C. AB - Trehalase (EC 3.2.1.28) associated with renal and intestinal brush-border membranes was solubilized by highly purified phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) from Bacillus thuringiensis, but not by phosphatidylcholine-hydrolyzing phospholipase C (EC 3.1.4.3) from Clostridium welchii or phospholipase D (EC 3.1.4.4) from cabbage. The solubilized trehalase was not adsorbed on phenyl-Sepharose, indicating that it was hydrophilic. Phosphatidylinositol-specific phospholipase C also converted Triton X-100 solubilized amphipathic trehalase into a hydrophilic form. These results suggest that trehalase is bound to the membrane through a direct and specific interaction with phosphatidylinositol. PMID- 3011507 TI - Carbachol and oxytocin stimulate the generation of inositol phosphates in the guinea pig myometrium. AB - In the guinea pig myometrium prelabelled with myo-[2-3H]inositol, carbachol and oxytocin enhanced a concentration-dependent and rapid release of IP3 which preceded that of IP2 and IP1. The specific receptor-mediated phospholipase C activation degrading PIP2 to IP3 did not require the presence of extracellular Ca2+. The ionophore A23187 as well as K+ depolarization failed to increase inositol phosphate accumulation. It is proposed that IP3 could have a role in the contraction of uterine smooth muscle elicited by the activation of muscarinic as well as of oxytocin receptors. PMID- 3011508 TI - Phosphorylation of pig brain diacylglycerol kinase by endogenous protein kinase. AB - Pig brain diacylglycerol kinase did not catalyze autophosphorylation. However, the kinase was phosphorylated on serine, when immunoprecipitated from the partially purified enzyme preparation preincubated with Mg2+ and [gamma-32P]ATP. The action of the endogenous protein kinase phosphorylating diacylglycerol kinase was independent of cyclic nucleotides and Ca2+, and became maximum at pH 5.5. Although the extent of enzyme phosphorylation was limited (maximally about 0.25 mol Pi incorporated per mol kinase), the results show that diacylglycerol kinase can be a phosphoprotein. PMID- 3011509 TI - Mechanism of glucagon stimulation of fructose-1,6-bisphosphatase in rat hepatocytes. Involvement of a low-Mr activator. AB - Isolated rat hepatocytes were incubated in the absence or presence of glucagon and the activity of fructose-1,6-bisphosphatase was measured in cell extracts. After glucagon treatment the Vmax was increased (20-50%) whereas the Km remained unchanged. The stimulation was complete at 5 min after addition of glucagon. The glucagon concentration needed for maximal stimulation was 10(-9) M. After gel filtration the fructose-1,6-bisphosphatase activity in extracts of glucagon treated cells was lowered to the control level. The effect of glucagon could not be completely mimicked by dibutyryl cAMP. The data indicate that in addition to the possible regulatory role of enzyme phosphorylation, a positive effector is involved in the stimulation of fructose-1,6-bisphosphatase activity by glucagon. PMID- 3011510 TI - Determination of the stoichiometry of redox-linked proton translocation from the kinetics of pulse experiments. A simulation study. AB - We have previously published a simple kinetic model to analyse possible pitfalls in kinetic measurements of H+/O ratios in mitochondria [(1984) FEBS Lett. 178, 187-192]. While this model demonstrated how relative electrode response times may affect the results, it did not adequately describe the kinetics of proton back diffusion across the membrane. Here this model is further developed and improved, and shown to give a good quantitative description of both oxygen-pulse type experiments as well as of experiments where the reaction is started by photolysis of the cytochrome c oxidase-CO complex. Simulations based on this model reveal that the extrapolation procedure used by Lehninger et al. [e.g. (1984) J. Biol. Chem. 259, 4802-4811] to estimate the H+/O ratio will tend to yield overestimated values. This is mainly due to the back-diffusion of protons into the mitochondria, which is not correctly accounted for by this extrapolation. PMID- 3011512 TI - The regulation of phosphofructokinase in epimastigote Trypanosoma cruzi. AB - Glycosomal (microbody)-enriched fractions prepared from epimastigote Trypanosoma cruzi were used as a partially purified source of phosphofructokinase. D-Fructose 6-phosphate showed sigmoidal kinetics at pH 7.0, but hyperbolic kinetics at pH 8.0. Various adenosine nucleotides were positive effectors; 5'-AMP was the most powerful. ATP showed hyperbolic kinetics under all conditions tested. Several described inhibitors and activators of mammalian phosphofructokinase were without significant effect on the trypanosomal enzyme; the absence of effect of D fructose 2,6-bisphosphate is of particular note. PMID- 3011511 TI - Regulation of cholesterol ester hydrolase by cyclic AMP-dependent protein kinase. AB - Phosphorylation of cholesterol ester hydrolase by cyclic AMP-dependent protein kinase results in activation of both cholesterol ester and triacylglycerol hydrolase activities. Activation against both substrates correlates closely with phosphorylation in time course experiments. Proteolytic digestion of phosphorylated cholesterol ester hydrolase, followed by peptide mapping, indicates the presence of a single phosphorylation site on the enzyme. Phosphoserine is the only phosphoamino acid detected following partial acid hydrolysis of the phosphorylated enzyme. PMID- 3011514 TI - Semiquinone anion radicals formed by the reaction of quinones with glutathione or amino acids. AB - Using ESR spectroscopy, we show that benzoquinone, 1,4-naphthoquinone and 5 hydroxy-1,4-naphthoquinone react readily with thiol containing compounds, such as glutathione, to form their respective semiquinone anion radicals. These quinones react similarly, but less readily, with the amino group of amino acids. The therapeutic or toxicological significance of the formation of semiquinone anion radicals from the reaction of quinones with nucleophiles, such as thiols and amines, remains to be assessed. PMID- 3011513 TI - Abolishment of inhibitory effects of 3'-deazaadenosine on superoxide generation of guinea pig phagocytes by pre-exposure to phorbol myristate acetate. AB - Superoxide generation by guinea pig macrophages and polymorphonuclear leukocytes induced by wheat germ agglutinin, immune complexes or formyl-methionyl-leucyl phenylalanine was inhibited considerably by 3'-deazaadenosine. The pre-exposure of the 3'-dezazaadenosine-treated cells to a small amount of phorbol myristate acetate abolished the inhibitory effect of 3'-deazaadenosine on the generation of superoxide. PMID- 3011515 TI - Chemical crosslinking of different forms of the simian virus 40 large T antigen using bifunctional reagents. AB - Chemical crosslinking was used for a direct analysis of the different forms of large tumor (T) antigen, the simian virus 40 A gene product. The first subclass, sedimenting at 14-16S, is composed of monomeric to tetrameric units, whereas the second, sedimenting at 5-6S, only contains dimers and monomers of T. The occurrence of oligomeric structures of T in solution which are higher than dimers suggests the possibility of direct binding of such trimers or tetramers to the origin of replication of the viral DNA as an alternative to the formation of these structures by aggregation of bound dimers or monomers after their sliding along the DNA. PMID- 3011516 TI - Fine needle aspiration cytology and exfoliative biliary cytology in the diagnosis of hilar cholangiocarcinoma. AB - Nineteen patients with suspected malignant obstruction at the confluence of the bile ducts had exfoliative biliary cytology and fine needle aspiration cytology performed. Of these patients 14 cases were histologically proven to be cholangiocarcinoma, and 3 others followed a clinical course which was clearly malignant. Fine needle aspiration cytology gave a true positive result in 14 patients (87.5%), whereas exfoliative cytology was positive in 11 (73%). There were no false positive results and no complications from either procedure. Both the cytological procedures are rapid and safe and are useful for preoperative planning of surgical and intraoperative diagnosis. PMID- 3011517 TI - Parathyroid carcinoma: a case with recurrence treated with extensive vascular surgery to the neck. AB - Parathyroid carcinoma is a slow growing tumor, and the patients most often die from complications to the hypercalcemia. Therefore, any attempt should be made to remove local recurrence and metastasis surgically, as medical treatment is disappointing. A case treated with extensive vascular surgery to the neck is reported. PMID- 3011518 TI - Transport of ions across the blood-brain barrier. AB - Capillaries in the brain are formed by a uniquely specialized endothelial cell that regulates the movement of substances between blood and brain. Although they provide an impermeable barrier to some solutes, brain capillary endothelial cells facilitate the transcapillary exchange of others. In addition, they contain specific enzymes that contribute to a metabolic blood-brain barrier by limiting the movement of compounds such as neurotransmitters across the capillary wall. Studies of sodium and potassium transport by brain capillaries indicate that the endothelial cell contains distinct types of ion transport systems on the two sides of the capillary wall, i.e., the luminal and antiluminal membranes of the endothelial cell. As a result, specific solutes can be pumped across the capillary against an electrochemical gradient. These transport systems are likely to play a role in the active secretion of fluid from blood to brain and in maintaining a constant concentration of ions in the brain's interstitial fluid. In this way, the brain capillary endothelium is structurally and functionally related to an epithelium. PMID- 3011519 TI - Atriopeptin biochemical pharmacology. AB - Several low-molecular-weight peptides that possess potent natriuretic, diuretic, and vascular smooth muscle relaxant activity have been isolated from atrial extracts. Elucidation of their structure indicates that they consist of a 17 membered ring of amino acids formed by a cystine disulfide bond and that they differ only in the composition of the amino and carboxy termini. The 24-amino acid peptide atriopeptin (AP) III was selected as the reference compound for structure-activity studies. Amino-terminal amino acid extensions on APIII markedly increase the natriuretic-diuretic but not the renal vasodilatory response in anesthetized dogs, which suggests a heterogeneity of AP receptors in renal tubular and vascular tissues. Radioligand (125I-labeled APIII) binding studies with fresh rat kidney slices indicate that the primary renal sites of specific AP binding are in the glomerulus and in the papillary segment of the medulla, thus implicating these structures in the natriuretic-diuretic effect. Data obtained from radioimmunoassay, chromatographic migration, vasorelaxant biological activity, and peptide sequence analysis indicate that Ser-Leu-Arg-Arg APIII is the major circulating form of low-molecular-weight atrial peptide present in rat plasma. Circulating APs fulfill many of the criteria for involvement in the endocrine regulation of fluid and electrolyte homeostasis. PMID- 3011520 TI - Mechanisms of release and renal tubular action of atrial natriuretic factor. AB - Inasmuch as atrial natriuretic factor (ANF) is apparently involved causally in the renal response to acute hypervolemia, it became of interest to study cellular mechanisms of release and renal tubular action. To study release mechanisms, freshly excised rat heart atria were incubated in vitro. Activation of the cellular adenylate cyclase system by either beta-adrenergic stimulation or the vasopressin analog deamino-8-D-arginine vasopressin did not result in ANF release. By contrast, activation of the polyphosphoinositide system by alpha adrenergic stimulation or stimulation of the V1-type vasopressin receptors, and by a calcium ionophore or active phorbol ester, significantly increased natriuretic activity in the medium and reduced it in tissue. It is concluded, therefore, that activation of this latter system is the mechanism for ANF secretion from atrial myocytes. To test the effect of ANF on tubular transport in the medullary collecting duct, microcatheterization was used in rats before and during i.v. infusion of synthetic atrial peptide (23 amino acids). It was found that tubular delivery of salt to this part of the nephron was increased, and that reabsorption in the duct itself was reduced. In control experiments, increased delivery was associated with proportionately increased reabsorption, which demonstrated glomerulotubular balance in the nephron segment under normal conditions. The natriuretic effect of ANF, therefore, was not caused solely by enhanced tubular load, but included specific inhibition of duct sodium reabsorption as an essential feature of the renal response. PMID- 3011521 TI - Renal hemodynamic and natriuretic effects of atrial natriuretic factor. AB - In this article we review the renal hemodynamic and excretory actions of atrial natriuretic factor (ANF) and consider some of the mechanisms of its vascular and natriuretic effects. ANF leads to a marked, sustained, and parallel increase in whole-organ and superficial single-nephron glomerular filtration rate (GFR) while mean blood pressure is decreased and renal blood flow (RBF) is unchanged or even decreased. The increase in GFR is caused by an efferent arteriolar vasoconstriction or by a combination of afferent vasodilation and efferent vasoconstriction. ANF also leads to a decrease in the hypertonicity of the innermedullary interstitium. Together with the increase in GFR, this phenomenon accounts wholly or in great part for the ANF-induced natriuresis. The overall renal vascular effects of ANF are complex and may tentatively be conceptualized as a behavior of a functional partial agonist: slight vasoconstriction in vasodilated kidneys, no sustained effects on the vascular resistance in normal kidneys, and vasodilation in vasoconstricted kidneys. The vasoconstrictor effect of ANF may be direct or indirect and depends on extracellular calcium whereas the antagonist effect likely results from alterations in intracellular calcium homeostasis. The data raise the perspective that ANF is not only a powerful natriuretic substance but has the potential of being an important modulator of GFR and RBF in intact animals. PMID- 3011522 TI - Leukocyte stimulation: receptor, membrane, and metabolic events. Introduction and summary. PMID- 3011524 TI - Dual role for guanine nucleotides in stimulus-secretion coupling. AB - We have made mast cells and neutrophils permeable to gain access to the cytosol and thus to manipulate the composition of the cytosol. Secretion from both cell types can be triggered by elevation of cytosol Ca2+ to concentrations approaching 10-6 M; alternatively, secretion from mast cells, and of beta-glucuronidase (but not lysozyme) from neutrophils, can be triggered in the absence of Ca2+ by introducing stable analogs of GTP. We propose that GTP acts at two intracellular guanine nucleotide regulatory proteins (N proteins) in the stimulus-secretion sequence. By interaction with Np located on the inner face of the plasma membrane, it activates polyphosphoinositide phosphodiesterase to yield inositol phosphates and diacylglycerol. By interaction with Ne, situated distal to the site of action of Ca2+ and protein kinase C, it directly activates the exocytotic process without intervention of the products of polyphosphoinositide hydrolysis. PMID- 3011525 TI - Characterization and assay of the nuclear and cytosolic progesterone (P) receptors in Premarin-primed human endometrium and myometrium after single-dose P administration. AB - Progesterone receptors (RP) were characterized and measured in the 0.4 M KCl nuclear extract and in the cytosol of 7 to 11 days Premarin (Premarin, Ayerst Laboratories, New York, NY) primed human endometrium and myometrium with [3H]R5020 as the ligand. The test group comprised 12 women who received progesterone injection 1 to 3 hours before tissue collection; the control group numbered 10 women. Receptor characteristics in both the endometrium and the myometrium, in the nuclear extract and cytosol in the test group and in the cytosol in the control group, were similar and were demonstrated by (1) high specificity, (2) sedimentation constant of 4.7 to 6.5S in the cytosols and 2.8 to 3.9S in the nuclear extract, (3) saturable affinity with Kd of 2.2 to 3.5 nM and 1.9 to 3.3 nM, respectively. Total RP levels (nuclear extract and cytosol) ranged from 1030 to 13,270 and 1280 to 17,300 fmol/mg DNA, respectively, in the endometria of women in the test and control groups. In 13 of 14 women myometrial total RP levels were higher than in the endometrium. There was a significant difference in the distribution of the RP between the test and the control groups: 50% and 48% of the receptor were measured in the nuclear extract in the endometrium and myometrium, respectively, in the test group, compared with 25% and 21% in the control group (P less than 0.001). PMID- 3011523 TI - Pertussis toxin as a probe of neutrophil activation. AB - In reviewing our own and other work, it is clear that pertussis toxin treatment of neutrophils causes a time- and concentration-dependent inhibition of granule enzyme secretion induced by formylmethionylleucylphenylalanine (fMet-Leu-Phe), C5a, leukotriene (LT) B4 and platelet-activating factor (PAF). Chemotaxis, O2- generation, aggregation, and arachidonic acid production induced by fMet-Leu-Phe are also inhibited by pertussis toxin. Granule enzyme release caused by A23187 or phorbol 12-myristate 13-acetate is not inhibited. The inhibition of neutrophil function correlates closely with the NAD-ribosylation of a 41,000-dalton protein in the neutrophil plasma membrane, presumably the GTP-binding regulatory protein Ni. Pertussis toxin treatment prevents or obtunds the increased influx of Ca2+ induced by fMet-Leu-phe and LTB4, but not that caused by stimulation of neutrophils with PAF. Pertussis toxin prevents the receptor-induced breakdown of polyphosphoinositides in intact neutrophils and isolated membrane and prevents or decreases the production of inositol 1,4,5-trisphosphate (IP3) and 1,2 diacylglycerol. The hypothesis advanced by us and others is that pertussis toxin interacts with a GTP-binding regulatory protein identical or similar to Ni, which couples receptor-chemotactic factor interaction to phospholipase C activation. Inhibition of the activation prevents the production of IP3 and the resulting release of Ca2+ from intracellular stores and of 1,2-diacylglycerol and thus, the activation of protein kinase C. The lack of these two mediators is the immediate cause of the depression of neutrophil activation resulting from pertussis toxin. Some of the limitations and uncertainties of our present knowledge with respect to this hypothesis are discussed. PMID- 3011526 TI - [Synaptic potentials of smooth muscles of the human small intestine]. PMID- 3011527 TI - [Characteristics of receptors of human placental blood vessels]. PMID- 3011528 TI - [Interaction of adrenergic and serotoninergic mechanisms in modulation of seizure activity of the brain]. AB - Electrical stimulation of the raphe nuclei and locus coeruleus induces inhibition of the penicillin-induced seizure activity. Adrenoblocking agents facilitate the effects of raphe nuclei on the seizure activity. PMID- 3011529 TI - Analysis of residual vulcanization accelerators in baby bottle rubber teats. AB - An analytical method was established for the determination of dialkyldithiocarbamates (DTCs) in chloroform-acetone extracts from rubber teats for baby bottles. DTCs in the extracts were derivatized into ethyl esters and analysed by gas chromatography employing nitrogen-phosphorus detection. Dimethyldithiocarbamate and diethyldithiocarbamate were detected at levels up to 3.2 micrograms/g rubber and up to 4.6 micrograms/g rubber (as dithiocarbamic acid), respectively, in the extracts from commercially available isoprene rubber tests. DTCs can form secondary amines by acid hydrolysis, although the levels of DTCs in the extracts only made a minor contribution to the total level of measured secondary amine precursors. PMID- 3011530 TI - Plasma ACTH levels and beta-endorphin-like immunoreactivity following administration of luteinizing hormone-releasing hormone in Nelson's syndrome. AB - The effect of LH-RH (25 micrograms as a single i.v. bolus) on plasma corticotropin (ACTH) levels and beta-endorphin-like immunoreactivity was studied at 30 and 60 min in eight women with Nelson's syndrome. Plasma ACTH concentrations increased in three of them, while beta-endorphin-like immunoreactivity, measured in six cases, rose significantly at 30 min in all the patients under investigation. In the control group containing seven women with Nelson's syndrome, placebo (0.9% sodium chloride) administration did not induce any significant changes in ACTH concentrations or in beta-endorphin-like immunoreactivity. Our results suggest that a paradoxical stimulatory influence of LH-RH on pituitary Nelson's adenomas may play an important role in the adenoma hormonal activity and, perhaps, growth. Such an effect could be responsible for a rapid development of some pituitary neoplasms during pregnancy. PMID- 3011532 TI - Cyclic AMP and cell differentiation in amphibian embryonic explants. AB - Conflicting results have been published concerning the effects of cyclic nucleotides on amphibian cell differentiation. Here we report the effects of cyclic adenosine monophosphate (cAMP) and dibutyryl-cyclic adenosine monophosphate (db-cAMP) on isolated explants from late blastulae of Ambystoma mexicanum and Xenopus laevis. Both cAMP and db-cAMP (10(-4)-10(-9) M) promote 'neuralizing' differentiation in Ambystoma explants. Xenopus explants treated with the nucleotides (10(-4), 10(-6), 10(-8) M) LiCl or heparan sulphate only give rise to ciliated aggregates or dissociation. The results confirm observations that different amphibian species react in different ways to activating chemicals. PMID- 3011531 TI - Serum copper and zinc concentrations in intrahepatic cholestasis of pregnancy: a controlled study. AB - A prospective, controlled study was undertaken in parturients with normal pregnancy and in parturients with intrahepatic cholestasis of pregnancy (IHC) to assess changes in maternal serum copper and zinc concentrations. Cord serum levels of these trace metals were measured, as well. Copper and zinc were measured by particle-induced X-ray emission (PIXE) analysis on two occasions during pregnancy; and immediately, 5 days and 5 weeks after delivery. Maternal serum copper was significantly higher in the IHC group as compared to the control group at 36-40 weeks of pregnancy and immediately and 5 days after delivery. There were no statistically significant differences between the control and study groups for maternal and cord serum zinc and cord copper concentrations. A positive correlation between maternal serum copper and serum aspartate aminotransferase was observed. PMID- 3011533 TI - Loss and reappearance of transferrin receptors in human leukemic cell lines. AB - Serum transferrin (the iron binding protein) exerts its iron carrier function at the cell surface after binding to the appropriate receptor (TrR). In this work it is demonstrated that differentiating agents induce loss of TrR from the surface of three leukemic cell lines (Molt-3, HL-60 and K-562). Loss of TrR correlates with change in morphology and induction of phenotypic markers of the differentiated cells. Removal of the differentiating agent from the culture is followed by reexpression of TrR on the cell surface. The data presented in this paper suggest that TrR may play a regulatory role in cell differentiation and malignant transformation. PMID- 3011535 TI - Calcium alters the properties of [3H]diazepam binding sites in rat brain membranes. AB - [3H]diazepam [( 3H]DZ) was used as a ligand to study the effects of Ca2+ on benzodiazepine binding to rat brain membranes. [3H]DZ bound at a single class of binding sites showing KD and Bmax values of 5.4 nM and 852 fmol/mg protein respectively. These values are consistent with previous reports. Amongst the various divalent cations tested Mg2+, Fe2+, Cd2+ and Sr2+ had no significant effect on [3H]DZ binding. Mn2+, Ba2+, Co2+, Ni2+ and La3+ enhanced radioligand binding, whereas Ca2+, Zn2+ and Pb2+ inhibited of [3H]DZ binding by Ca2+ was concentration-dependent. 50% inhibition occurred at a Ca2+ concentration of 5.6 mM. The Hill coefficient for the inhibition was 1.03, displaying noncooperativity. The effect of Ca2+ on [3H]DZ binding could be prevented by La3+ but was not reversed by EGTA. A kinetic analysis of Ca2+ inhibition of [3H]DZ binding indicates that Ca2+ inhibited competitively. Analysis of binding isotherms indicates that both KD and Bmax were altered at the [3H]DZ binding sites. The marked increase in KD value in the presence of Ca2+ (5 mM) can be explained by a drastic increase in the dissociation rate constant. It was suggested that Ca2+ may induce a conformational change in the diazepam binding sites on rat brain membranes. The unchanged Hill coefficient in the presence or absence of Ca2+ indicates that a single population of binding sites was labeled by [3H]DZ. The calmodulin antagonists, trifluoperazine and W-7 were weak inhibitors of [3H]DZ binding. PMID- 3011534 TI - Analytical study on Na-K-ATPase (and cysteine insensitive p nitrophenylphosphatase) in rat kidney-cortex microsomes subfractioned by zonal centrifugation. AB - The common use of Na-K-ATPase as a marker enzyme for basolateral membranes in the kidney is based on the microscopic localization of the enzyme by the cytochemical assay of Na-K-ATPase as cysteine insensitive p-nitrophenylphosphatase (Ernst S.A., J. Cell Biol. 66, 586-606, 1975). Rat kidney cortex plasma membranes were therefore fractionated by differential pelleting in isotonic sucrose, followed by equilibrium banding in linear sucrose gradients, to compare the distribution of "biochemical" and "cytochemical" assayed Na-K-ATPase. In all fractions, the distribution of Na-K-stimulated Mg-dependent ATPase differed from the distribution of cysteine insensitive p-nitrophenylphosphatase (alkaline phosphatase). Evidence is presented that this difference is not only due to the separation of plasma membranes from different cell types, but simply reflects different membrane location of the enzymic activities. PMID- 3011536 TI - Investigations of primary and secondary structure of porcine ubiquitin. Its N epsilon-acetylated lysine derivative. AB - N epsilon-acetylation in vitro of internal lysyl residues of Ub by p-nitro-phenyl acetate at pH 8.0 was performed. The position of acetylation sites are determined. (e.g. Fully acetylated: Lys-6, Lys-11 and Lys-33; partially free internal lysines: Lys-27, Lys-29; Lys-48 and probably Lys-63.) 55 cycles Edman degradation were performed and the first 53 N-terminal residues were identified. Secondary structural studies of ubiquitin have been carried out using the circular dichroism (CD) technique. No changes are noted upon heating to 100 degrees C at neutral pH even in the presence of 8 M urea but in 6 M guanidine-HCl extensive modification results. Ubiquitin with an average of 4.4 of its 7 lysines in the N epsilon-acetyl form shows little deviation from native protein. After reduction with dithiothreitol and subsequent removal of the mercaptan, significant changes in the secondary structure are noted. Circular dichroic measurements of ubiquitin indicated an alpha-helical content of about 10% whereas the secondary structural predictions of Chou and Fasman suggest a level of about 45%. PMID- 3011537 TI - Cyclic AMP-dependent phosphorylation, Ca2+ and calmodulin: possible influences on acyltransferases and pyruvate dehydrogenase activities of rat mammary gland. AB - Specific activities of diacylglycerol acyltransferase, glycerol 3-phosphate acyltransferase and pyruvate dehydrogenase were studied in virgin, pregnant, lactating and involuting rat mammary glands. An inverse relationship was evident between cAMP binding to protein kinase(s) and the activities of the above enzymes in lactating rat mammary glands. Results suggested that free Ca2+ concentration may also contribute to control of the activity of pyruvate dehydrogenase in these glands. However, no consistent change was observed between the activities of these enzymes and cAMP binding in young, pregnant and involuting rat mammary glands. Calmodulin levels paralleled bound Ca2+ except in lactating rats. Almost all parameters studied peaked on day 8 of lactation. PMID- 3011539 TI - Molecular basis for substrate specificity of protein kinases and phosphatases. AB - Regulation of various metabolic processes occurs by the phosphorylation/dephosphorylation of enzymes. Both the protein kinases that catalyze the phosphorylations and the protein phosphatases that catalyze the dephosphorylations display relatively broad specificity, reacting with a number of distinct sites in target enzymes. In this way changes in the activity of a particular kinase or phosphatase can cause coordinated and pleiotropic responses. However, the kinases and phosphatases do not exhibit a one-to-one correspondence in their reactions. Residues at different positions may be phosphorylated by a single kinase, yet dephosphorylated by different individual phosphatases. Conversely, sites which are substrates for different individual kinases may be dephosphorylated by a single phosphatase. In exploring the molecular basis for these differences this article shows that whereas kinases react with specific primary structures that often times appear as beta bends, the phosphatases recognize higher order structure, less strictly ruled by amino acid sequence surrounding the phosphorylated site. The differences, seen in the ability of these enzymes to utilize synthetic peptide substrates, might be rationalized in terms of function. Kinases need protruding segments of structure that can be enwrapped to exclude water, thereby minimizing ATP hydrolysis and enhancing phosphotransferase activity. On the other hand phosphatases are hydrolytic enzymes that may operate especially well on protein interfaces. Hydrolytic action often measured with p-nitrophenylphosphate is not necessarily indicative of a protein phosphatase and consideration of the mechanism reveals why this substrate can be misleading.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011538 TI - Characterization and cAMP inhibition of a lysyl-(N-epsilon-5'-phospho) adenosyl phosphoamidase in Dictyostelium discoideum. AB - A lysyl-(N-epsilon-5'-phospho) adenosyl phosphoamidase activity has been identified in Dictyostelium discoideum. Conjugates, formed by coupling AMP via a phosphoamide bond to the epsilon amino group of lysine in avidin and tuftsin, served as substrate. Lysyl-N-epsilon-5'-phosphoadenosine and adenosine phosphoramidate (AMPNH) were substrates as well. The phosphoamidase liberated AMP from all four compounds but did not degrade cAMP. Approximately 90% of the phosphoamidase activity was inhibited competitively by 100 microM cAMP with an apparent Ki of 35 microM for all substrates. PMID- 3011540 TI - The function of Mg-ATP in interactions between the regulatory and catalytic subunits of type I cAMP-dependent protein kinase from rabbit skeletal muscle. AB - The regulatory subunit of Type I cAMP-dependent protein kinase from rabbit skeletal muscle can bind [3H]cAMP to form the R-[3H]cAMP complex, and the slow phase of the enhanced exchange of free cAMP with [3H]cAMP from the R-[3H]cAMP complexes was studied under various conditions using the equilibrium isotope exchange technique. Results indicate that Mg-ATP and the catalytic subunit are absolutely required for the enhanced exchange reaction to occur, but phosphorylation of the regulatory subunit by Mg-ATP does not play a determining role in the slow rate of the dissociation/association of the Type I protein kinase in the presence of cAMP and the catalytic subunit. We interpret the role of Mg-ATP as being one in which it may provide the structural attributes required for formation of a stabilized transient state of the cAMP-regulatory subunit catalytic subunit ternary complex, an obligatory intermediate involved in the dissociation/association of Type I cAMP-dependent protein kinase. PMID- 3011542 TI - Mechanism of activation of transcription by the complex formed between cyclic AMP and its receptor in Escherichia coli. PMID- 3011541 TI - Insulin, glucagon and the receptor-mediated control of cyclic AMP concentrations in liver. Twenty-second Colworth medal lecture. PMID- 3011543 TI - Binding sites of restriction endonucleases. PMID- 3011544 TI - Action of restriction endonucleases on phosphorothioate DNA. PMID- 3011545 TI - Structural and catalytic properties of specific complexes between Tn3 resolvase and the recombination site res. PMID- 3011546 TI - DNA sequence specificity of Tn21 resolvase. PMID- 3011547 TI - Sympathetic and hormonal regulation of brown adipose tissue thermogenesis. PMID- 3011548 TI - Brown adipose tissue thermogenesis in neonatal and cold-adapted animals. PMID- 3011549 TI - Stimulated inositol phospholipid breakdown and myoblast fusion. PMID- 3011550 TI - Lipoidal anti-inflammatory prodrugs as local targeting agents. PMID- 3011551 TI - Strategies for the inhibition of neuropeptide-metabolizing enzymes. PMID- 3011552 TI - DNA supercoiling. PMID- 3011553 TI - DNA breakage by Escherichia coli DNA gyrase in vitro and in vivo. PMID- 3011554 TI - Inhibitors of trypanosome topoisomerases. PMID- 3011555 TI - Gyrase inhibitors and intracellular DNA supercoiling. PMID- 3011556 TI - Arachidonic acid inhibition of insulin action and phosphoinositide turnover in fat cells. AB - Incubation of isolated rat adipocytes with 1 microM arachidonic acid (20:4) coupled to equimolar amounts of bovine serum albumin (BSA) results in the cellular uptake of the fatty acid and a subsequent inhibition of insulin stimulated antilipolysis and lipogenesis without altering glucose transport. These effects are apparently not mediated at the insulin receptor level since insulin binding is not altered in arachidonate-enriched fat cells. In addition, effects on antilipolytic and lipogenic are not specific for arachidonic acid. Oleic or palmitic acid can mimic these effects in both insulin-stimulated and PGE2-stimulated cells. Adipocyte enrichment with 20:4, however, specifically inhibits the insulin-stimulated turnover of phosphoinositides. The latter can be specifically prevented by preincubation with ibuprofen. These results suggest that the level of intracellular arachidonate may play a major role in modulating insulin-stimulated phosphoinositide turnover and thereby indirectly regulate certain aspects of insulin action which involve lipid metabolism. PMID- 3011558 TI - Hormonal regulation of extracellular plasminogen activators and Mr approximately 54,000 plasminogen activator inhibitor in human neoplastic cell lines, studied with monoclonal antibodies. AB - We have studied the regulation by glucocorticoids and dibutyryl cAMP of the amounts of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA) and a Mr approximately 54000 plasminogen activator inhibitor accumulated in serum-free conditioned culture fluid by a human fibrosarcoma, a human glioblastoma and a human melanoma cell line (HT-1080, UCT/gl-1 and Bowes). For the quantitation of u-PA and t-PA, we used sandwich-type ELISA with a combination of polyclonal and monoclonal antibodies. For an estimation of variations in the amount of the inhibitor, we used sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by Coomassie blue staining of conditioned culture fluid proteins, the inhibitor protein band being identified by its selective removal by passage of the conditioned culture fluids through a column with monoclonal antibodies against the inhibitor. The modulation of the 3 proteins by the hormonal agents varied greatly between the cell lines. The proteins were independently regulated, in the sense that the hormonal agents did not concomitantly change their levels in the direction expected either to increase or decrease total extracellular plasminogen activator activity. In conditioned culture fluids containing both t-PA and inhibitor, the two were present in the medium as a Mr approximately 120 000 complex. In contrast, no u-PA inhibitor complexes were found in conditioned culture fluid from any of the cell lines; this is likely to be due to the occurrence of u-PA in the culture fluid in the one-chain proenzyme form, which, unlike active u-PA, does not react with the inhibitor. These findings illustrate the complexity of the regulation of extracellular plasminogen activator activity, and imply that the presumed functional diversity of u-PA and t-PA may be related to their independent regulation. PMID- 3011557 TI - Role of cytosolic calcium in the control of cAMP content by calcium in bovine parathyroid cells. AB - In the present studies we used the calcium (Ca2+)-sensitive dye Quin-2 to determine whether the cytosolic (Cyt) Ca2+ mediates the effects of extracellular (EC) Ca2+ on cAMP accumulation through changes in adenylate cyclase and phosphodiesterase activity in bovine parathyroid cells (bPTC). In dispersed (d) bPTC, increasing the EC Ca2+ from 0.5 to 2 mM produces a rise in the Cyt Ca2+ from 179 to 646 nM which is associated with a 52% inhibition of agonist stimulated cAMP accumulation. Over this range of free Ca2+ adenylate cyclase activity decreased by approx. half (57%) and phosphodiesterase activity increases 2-fold (101%) suggesting that changes in the Cyt Ca2+ can account for the effects of EC Ca2+ on cAMP through changes in these enzymes. Unlike dbPTC, 4-day-old cultured bPTC show only a 23% suppression of cAMP by high EC Ca2+ and a reduced rise in the Cyt Ca2+ from 0.5 to 3 mM EC Ca2+. Although there is no reduction in the Ca2+ sensitivity of adenylate cyclase, phosphodiesterase activity shows no change at varied free Ca2+. Thus, this diminished Ca2+ sensitivity of phosphodiesterase activity, and the decreased rise in Cyt Ca2+ relative to EC Ca2+ may both contribute to the resistance to the effects of EC Ca2+ on cAMP content in cultured cells. Because in addition to Cyt Ca2+, protein kinase C may also mediate the effects of EC Ca2+ on PTH release, we studied the effects of TPA (12-alpha-tetradecanoylphorbol 13-acetate) on agonist-stimulated cAMP in dbPTC.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011559 TI - Sex steroid and prostaglandin interactions upon the purified rat myometrial plasma membranes. AB - [14C]Estradiol, [14C]estrone, [14C]progesterone and [3H]prostaglandins (PGs) E2 and F2 alpha, when incubated with myometrial plasma membranes (MPM) at a concentration of 1 X 10(-6) M for 1 h at 37 degrees C, bind into MPM at pmolar concentrations. Unlabeled steroids inhibited [3H]PGE2 and [3H]PGF2 alpha binding to MPM in a dose-dependent manner. Membrane-bound and free steroids or PGs were found to be essentially unchanged under the present incubation conditions. Ca2+ ions up to 10 mM increased steroid binding into MPM. Molecular interactions between steroids and MPM were assessed by measuring the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), and by estimating the changes in the allosteric properties of MPM-bound (Na+ + K+)ATPase by fluoride (F ). Steroids appear to increase the MPM fluidity, evaluated through changes in the Hill coefficient for MPM-bound (Na+ + K+)ATPase by F- and by the fluorescence polarization method. Binding of sex steroids to MPM increased the membrane fluidity and decreased the binding of the uterus stimulatory PGs by membrane receptors. These studies provide a basis for postulating that a 'non-genomic' mechanism of sex steroids induces reduction of uterine contractions. PMID- 3011560 TI - Changes in endonuclease activity during growth and regression of the Shionogi mammary carcinoma. AB - Autodigestion of nuclei isolated from the androgen-dependent Shionogi mouse mammary carcinoma revealed the presence of endogenous endonuclease activity which was stimulated by Ca2+ and Mg2+ and generated a repeating pattern of DNA fragments with an average monomeric size of 179 base pairs. The nuclear concentration of endonuclease activity declined rapidly after castration to a level 45% of that measured in tumours from non-castrated hosts. Most of the endonuclease activity (80%) could be extracted in a soluble form after sonication of the nuclei and subsequent centrifugation. The reduced concentration of endonuclease activity in regressing tumours following castration was not due to changes in chromatin template conformation and could be restored to normal levels within 1 day of androgen treatment. Examination of DNA synthetic activity and the concentration of nuclear androgen receptors during tumour regression indicated that the decrease in endonuclease activity paralleled the declining rate of DNA synthesis but was preceded by a loss of nuclear androgen receptors. Together these results suggest that the endonuclease activity of the Shionogi carcinoma is more likely involved with androgen-stimulated cell proliferation rather than with the autophagic mechanism responsible for tumour regression and cell death. PMID- 3011562 TI - Iodination of thyroglobulin molecules depends on their diffusion velocity in follicular colloid. AB - We have studied in vitro the effects of altered physicochemical properties of thyroglobulin molecules in solution and of the solution itself on iodination kinetics and hormone synthesis. Any change in hydrodynamic properties had a far greater effect in compartmentalized systems, obtained by coating the test tubes with peroxidase, than in conventional homogeneously mixed systems. Increasing thyroglobulin concentration in a range still far below that existing in vivo greatly retarded iodination and hormone synthesis. In contrast, a number of physiological and non-physiological changes of thyroglobulin structure, such as desialylation, preiodination, oxidation and denaturation, strikingly accelerated iodination. The highly variable physical-chemical state of thyroglobulin molecules appears to be a main determinant of protein diffusion within the colloid and, thereby, of iodination kinetics and rate of hormone synthesis. Moreover, alterations of the physicochemical state of thyroglobulin molecules may explain some hitherto ill-understood diffusion phenomena in live follicles. PMID- 3011561 TI - Tunicamycin sensitivity of prolactin, insulin and epidermal growth factor receptors in rat liver plasmalemma. AB - We have used the glycosylation inhibitor tunicamycin to assess the stability of the receptors for prolactin, insulin and epidermal growth factor (EGF) in rat liver cell membrane. Direct binding studies on liver plasmalemma fractions which were isolated from tunicamycin-treated rats revealed a rapid loss of prolactin receptors (t1/2 approximately 35 min) with a more prolonged half-life for insulin (10 h) and EGF receptors (8 h). The rates of receptor loss were similar to the respective half-lives of the receptors as documented by others using cultured cells. The respective ligands for each receptor were lost more rapidly from liver, i.e. prolactin, t1/2 approximately 10 min, insulin, t1/2 approximately 5 min and EGF, t1/2 approximately 17 min. Previous studies have shown ligand loss in vivo to be receptor mediated. Thus, receptors and their ligands do not turn over synchronously in vivo. These studies also point to a major role for N-linked oligosaccharide side chains in the functional insertion of prolactin, insulin and EGF receptors into the hepatocyte cell surface in vivo. PMID- 3011563 TI - Affinity labelling of steroid hormone receptors. AB - Affinity labelling techniques have proved indispensable for the study of reversible biological recognition systems, since they conserve ligand-receptor interaction by covalent linkage. Using photo- and electrophilic labelling, it has become possible to unequivocally identify steroid hormone receptors and their proteolytic degradation products and it is simple to establish receptor peptide maps even in crude receptor preparations. The isolation of receptor proteins has been greatly simplified, as their integrity can be analyzed at any step of a purification protocol by SDS-PAGE analysis after crosslinking. Moreover, affinity labelled receptors can be purified under denaturing conditions, e.g., in high resolving preparative SDS-PAGE, and the material obtained can be efficiently used to generate anti-receptor antibodies. Peptide mapping after crosslinking of related receptors has been used to assess the degree of structural homology between different forms of steroid hormone receptors and receptors of different species. Peptide sequence analysis of purified crosslinked receptor protein and anti-receptor antibodies have provided the basis for cloning corresponding genes. Techniques have been established to demonstrate--via crosslinking--that the cloned DNA sequences correspond to the receptor gene binding the correct ligand. The analytical and preparative crosslinking methods developed for steroid receptors are potentially important for the study of any system in which signal transduction proceeds via the reversible interaction between biological macromolecules. PMID- 3011564 TI - Effect of oxidizing agents on rabbit mammary prolactin receptors. AB - Treatment of rabbit mammary membrane-bound and solubilized prolactin receptors with the oxidizing agents N-chlorobenzene sulfonamide and N-bromosuccinimide resulted in total inactivation of the ability of the receptor to bind prolactin. A similar inactivation was obtained with 2-hydroxy-5-nitrobenzylbromide. The results suggest the possible importance of tryptophan in maintaining the activity of the prolactin receptor. PMID- 3011565 TI - Homologous up-regulation of the prolactin receptor in rat prostatic explants. AB - Exposure of explants of rat ventral prostates and a rat Leydig cell tumour to ovine prolactin for 20 h caused alterations of the subsequent membrane binding of 125I-human prolactin to an extent and in a direction dependent on the dose of hormone used. Low prolactin concentrations (1-10 micrograms/2 ml) were associated with an increase in binding (up-regulation) which was 75% in the case of the prostatic tissue and 500% in the case of the tumour tissue above control levels. Higher concentrations caused a dose-dependent decrease in binding to below control levels (down-regulation), alterations which could not be explained by receptor occupancy. Time studies with an up-regulatory dose of hormone (3 micrograms/2 ml) indicated that the effects of prolactin on its receptor did not begin to become manifest until after 6-12 h of culture. The results suggest that homologous up-regulation of prolactin binding may be a general feature of prolactin target organs and that explant cultures of prostatic tissue may provide a convenient model for exploring its mechanisms. PMID- 3011566 TI - 3B55: a repetitious sequence family which is transcribed and proportionately replicated in germ-line polyploid nuclei of Calliphora erythrocephala. AB - The chromosomes of dipteran polyploid nurse cell nuclei are functionally analogous to the oocyte lampbrush chromosomes of the Amphibia. In investigating the transcriptional and replicative activity of these nuclei in Calliphora erythrocephala we have identified a cloned highly repetitious DNA fragment which shows enhanced transcriptional activity in these cells and different replicative behavior in these germ-line polyploid nuclei as opposed to somatic polytene nuclei. The clone, 3B55, contains a 6.8-kb insert which consists primarily of tandemly repeated 200-bp sequences defined by RsaI sites. From Southern hybridizations to diploid (embryonic) genomic DNA, 3B55-related DNA was calculated to represent a significant fraction of the haploid genome (0.8% or 5000 kb). In situ hybridizations established that these sequences are present in the pericentric regions of four of the six chromosomes. Thus the 3B55 sequences have the properties of a satellite-type DNA family. Quantitation of the 3B55 200 bp monomer (which represents approximately 60% of the genomic 3B55 DNA sequences in all tissues examined) revealed that in somatic polytene salivary gland nuclei, 3B55 DNA is highly under-replicated to give a monomer representation of only 48 +/- 11 kb per haploid genome. However, in germ-line nurse cell nuclei, 3B55 DNA is proportionately replicated to give a monomer genomic representation (3560 +/- 344 kb) equivalent to that of diploid DNA (3011 +/- 202 kb). Transcripts complementary to 3B55 sequences are at least 25 times more abundant in total nurse cell nuclear RNA than in total embryonic nuclear RNA. These findings suggest an association of the 3B55 sequence family with some germ-line specific function. PMID- 3011567 TI - Characterization of nerve growth factor binding to cultured neural crest cells: evidence of an early developmental form of the NGF receptor. AB - Cultured neural crest cells undergoing differentiation have been shown to contain a subpopulation of cells with specific receptors for nerve growth factor (NGF). These cells are the potential targets of NGF during differentiation and development. This study was done to pharmacologically characterize the binding of NGF to long-term (1- to 3-week) cultures of quail neural crest cells. The data indicate that 125I-NGF binding was specific and saturable, with less than 20% nonspecific binding. Scatchard analysis revealed the presence of one type (class) of receptors with a binding constant (Kd) similar to that of the low-affinity binding site described for embryonic dorsal root and sympathetic ganglia (approximately 3.2 nM). This was corroborated by displacement experiments (Kd of 1.3 nM), in which 125I-NGF binding was measured in the presence of increasing concentrations of nonradioactive NGF. In addition, affinity labeling revealed that the 125I-NGF-receptor complex had a molecular weight of about 93K, characteristic of the low-affinity NGF receptor of PC12 cells. The NGF receptor of cultured neural crest cells was trypsin-sensitive, as is typical of the low affinity NGF binding sites. These findings indicate that differentiating neural crest cells lack high-affinity 125I-NGF binding sites. In contrast, embryonic dorsal root and sympathetic ganglia cells, known NGF targets, have both high- and low-affinity receptors. Measurements of the differential release of surface-bound 125I-NGF indicated that a relatively small amount (about 14%) of NGF is internalized over a 1-hr period. Cultured neural crest cells which bear NGF receptors were also shown by light microscopic radioautographic techniques to incorporate [3H]thymidine. I suggest, therefore, that cultured neural crest cells which have not terminally differentiated, as judged by morphological criteria and continued proliferation, may express an early developmental form of the NGF receptor. PMID- 3011568 TI - Poly(A) shortening of coregulated transcripts in Drosophila. AB - The 71E ecdysterone-regulated puff of Drosophila melanogaster contains a cluster of six coregulated "late" genes which are expressed in the prepupal salivary gland. The resulting transcripts exhibit a decrease in their length during the 12 hr period in which they accumulate. Using the enzyme ribonuclease H, we show that this size decrease is a result of a progressively shorter poly(A) tract and suggest that these transcripts undergo an active sequential shortening of their poly(A) tracts in prepupal salivary glands. It is interesting to note that this shortening precedes the complete loss of these transcripts from the RNA population. PMID- 3011569 TI - Bordetella heat-labile toxin: further purification, characterization and mode of action. AB - The heat-labile toxin (HTL) was purified from sonic extracts of Bordetella bronchiseptica and Bordetella pertussis cells by a series of hydrophobic interactions, density gradient centrifugation, gel filtration, isoelectric precipitation, and isoelectric focusing. A 114-fold purification was regularly obtained with a yield of 31%. A dose of 0.75 ng was dermonecrotizing in guinea pigs. HLT is a simple protein (pI 6.9) with a molecular weight by gel filtration of 102,000 which consists of two polypeptides of 30,000 and 24,000 molecular weight. Amino acid analysis showed 15 common amino acids and the absence of methionine. The dermonecrotizing activity was inactivated at 56 degrees C, and at above pH 10 or below pH 5. Effects of various ions, detergents, enzymes and other chemical agents on HLT were determined. When instilled on the surgically exposed peripheral blood vessels of guinea pigs or suckling mice, HLT induced vasoconstriction within 15 min resulting in the decrease of blood flow, followed by manifestations of ischemia, diapedesis and petechial hemorrhage during the following 5 h. HLT activity on arterioles was unaffected by adrenergic or cholinergic blockades. Biochemically, HLT significantly inhibited in vitro the activity of Na+ - K+ ATPase prepared from rat kidney. A possible mechanism by which HLT induces dermohemorrhagic necrosis and splenic atrophy, is discussed. PMID- 3011570 TI - Conditioning of aversion to alcohol orosensory cues in 5- and 10-day rats: subsequent reduction in alcohol ingestion. AB - Two experiments examined in 5- and 10-day-old rat pups the impact of alcohol olfactory aversions upon subsequent alcohol intake. In Experiment 1 it was observed that at both ages, animals given a single pairing of alcohol odor and LiCl subsequently consumed less alcohol than those in any of 4 control conditions (alcohol odor unpaired with LiCl, alcohol odor exposure, lemon odor paired with LiCl, and untreated animals). In Experiment 2, pups of both age groups were given LiCl following exposure to alcohol odor, the infusion of an alcohol solution or both stimuli simultaneously. Rats given explicitly unpaired presentations of the different conditioned stimuli and internal malaise served as controls. It was observed that equivalent aversions to alcohol ingestion were expressed whether the infants had experienced the alcohol odor, the alcohol infusion, or both, paired with toxicosis. These experiments extend the ontogenetic spectrum of circumstances in which olfactory experiences affect subsequent ethanol ingestion and also may indicate an early nondifferentiated processing of odorant and gustatory cues arising from an ethanol stimulus. PMID- 3011571 TI - Long-term plasma glucose normalization in experimental diabetic rats with macroencapsulated implants of benign human insulinomas. AB - Permselective tubular membranes (1 mm i.d.) were filled with fragments of nine freshly resected human insulinomas, closed at both ends, and implanted in the peritoneal cavity of 30 streptozocin-induced diabetic rats. In 14 animals, nonfasting plasma glucose (PG) and insulin levels were normalized by these immunoprotected transplants for up to 1 yr (PG from 520 +/- 12 to 142 +/- 3 mg/100 ml; insulin from 6 +/- 0.5 to 44 +/- 3 microU/ml). These animals showed the same weight gain after 12 mo of observation as 20 controls. The remaining 16 animals showed an incomplete or transient correction of their diabetes and survived 4-6 mo, versus less than 8 wk in untreated animals. Removal of the membrane-encapsulated insulin-secreting tissue from 8 successfully treated rats led to hyperglycemia and death within 10 days. Histology and electron microscopy of insulinoma tissue retrieved after long-term implantation showed functionally active endocrine cells and no evidence of graft rejection. In vitro perifusion gave similar results for encapsulated and nonencapsulated insulinoma tissue. The amount of insulin secreted was quite variable, and responsiveness of the insulinoma to changes in glucose concentration of the surrounding medium was observed in three out of the five tumors studied. These observations establish the effectiveness of immunoseparation by a synthetic membrane in a pancreatic xenograft model. PMID- 3011572 TI - Binding and internalization of insulin and insulin-like growth factors by isolated brain microvessels. AB - Isolated brain capillaries were used as a model system to test for binding and internalization of insulin and insulin-like growth factors (IGF) I and II. At 37 degrees C, the maximum specific binding of the 125I-labeled peptides was 48.0 +/- 0.8%/mg capillary protein for IGF I, 40.6 +/- 1.4% for IGF II, and 15.1 +/- 0.6% for insulin. The concentration of unlabeled peptide needed to cause a 50% decrease in the maximum binding (ID50) was 22 ng/ml (2.9 nM), 25 ng/ml (3.3 nM), and 7 ng/ml (1.2 nM) for IGF I, IGF II, and insulin, respectively. Unlabeled insulin competed poorly for the IGF I receptor, requiring 5000 ng/ml (667 nM) to cause a 50% reduction in binding, and did not compete at all for the IGF II receptor at concentrations up to 10(5) ng/ml (17.8 microM). The IGF I receptor was further characterized by reduced polyacrylamide gel electrophoresis of the disuccinimidyl suberate cross-linked 125I-labeled IGF I receptor. The gel showed a distinct band at 133,000 Mr that was abolished by 0.6 microgram/ml (80 nM) unlabeled IGF I but not by 10.0 micrograms/ml (1780 nM) unlabeled insulin. Peptide internalization was monitored by the acidwash technique. Only 22% of the bound IGF I was internalized, but 50% of the insulin and 43% of the IGF II were acid resistant. Capillaries prelabeled with internalized 125I-insulin could then export radioactivity into fresh, insulin-free media in a time- and temperature dependent manner. However, high-performance liquid chromatography (HPLC) and trichloroacetic acid (TCA) analysis of the released material showed that it consisted mostly of degraded peptide.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011573 TI - Purification of putative insulin-sensitive cAMP phosphodiesterase or its catalytic domain from rat adipocytes. AB - Insulin acutely inhibits the catecholamine-stimulated rise in cAMP levels in fat, liver, and muscle primarily through stimulation of the enzyme cAMP phosphodiesterase (PDE). Adipocytes from rat epidydimal fat pads were exposed to insulin and fractionated by centrifugation. Whereas the cytosolic fraction contained a low-affinity cAMP PDE that was unaffected by insulin, the activity of a high-affinity enzyme residing in a particulate fraction was increased by insulin. This enzyme activity could be solubilized with nonionic detergent and chromatographed on ion exchange followed by chromatofocusing. The resulting enzyme preparation was subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Silver staining revealed a single band with a molecular weight of 60,000. This apparent molecular weight was verified by calculation of the hydrodynamic properties of the enzyme. Evaluation of its kinetic properties indicated that the enzyme activity residing in this solubilized 60,000-Mr protein exhibited lower affinity than the membrane-bound enzyme but was still specific for cAMP. Activation of this enzyme may be one of the primary mechanisms by which insulin counteracts the effects of adenylate cyclase-stimulating hormones. PMID- 3011574 TI - Coxsackie virus B4 produces transient diabetes in nonhuman primates. AB - Cynomolgus, rhesus, and Cebus monkeys failed to show glucose tolerance or insulin secretion abnormalities after infection with encephalomyocarditis virus or Coxsackie virus B4. Patas monkeys also showed no abnormalities after infection with encephalomyocarditis virus. However, patas monkeys infected with Coxsackie virus B4 or treated first with a subdiabetogenic dose of streptozocin and then infected sequentially with Coxsackie viruses B4 and B3 showed transient elevation of glucose tolerance tests, depressed insulin secretion, and glucose in the urine. Our experiments in nonhuman primates support earlier studies in mice and humans that under certain circumstances, Coxsackie viruses can cause abnormalities in glucose homeostasis. PMID- 3011575 TI - Superoxide production of polymorphonuclear leukocytes in surgical patients with gastric cancer. AB - Superoxide production (SOP) by polymorphonuclear leukocytes (PMNs) in 65 gastric cancer patients, who were in preoperative state and had received no medical therapy, was assayed in order to evaluate the bactericidal activity of PMNs in cancer patients, as well as to determine the correlation of SOP by PMNs with postoperative prognoses and several factors by which extent of disease and the clinical or histological character of gastric carcinoma were defined. Patients with stage II disease had a tendency to have an increased SOP by PMNs, and furthermore, as the disease progressed, the SOP by PMNs decreased with significant depression being noted in stage III and IV cases compared to healthy controls. Significantly reduced SOP by PMNs was observed in n2 and n3 cases and with se, si, sei and/or ps(+) pathological invasion. SOP by PMNs in patients with Borrmann II type and/or poorly differentiated adenocarcinoma was significantly depressed. Patients who suffered from septic complications showed a significant depression in SOP by PMNs compared with the controls and no complication group. These results suggest that advanced gastric cancer patients may have defective oxidative PMN metabolism, and that a decrease of SOP is a contributory cause of high susceptibility to postoperative infection in cancer patients. PMID- 3011576 TI - Effects of cyclic nucleotides, betazole hydrochloride and acetylcholine on pepsinogen secretion from isolated rabbit gastric mucosa. AB - The effects of dibutyryl cyclic AMP (db-cAMP), dibutyryl cyclic GMP (db-cGMP), betazole hydrochloride (betazole) and acetylcholine (ach.) on pepsinogen secretion from isolated rabbit gastric mucosa were studied using an organ culture system. The 10(-3) M db-cAMP, but not db-cGMP of any concentration, produced a significant enhancement of pepsinogen secretion into the culture medium. In the presence of aminophylline (phosphodiesterase inhibitor), betazole stimulated pepsinogen secretion at concentrations of 10(-8), 10(-6) and 10(-4) M. The 10(-4) M betazole stimulated pepsinogen secretion most strongly (208% of control) and 10(-6) M betazole induced submaximal secretion (137% of control). Ach. stimulated pepsinogen secretion at 10(-8), 10(-6), 10(-4) and 10(-2) M. The peak secretion occurred at 10(-4) M ach. (303% of control). Betazole (with aminophylline) against a background of 10(-6) M ach. (submaximal stimulation dose), produced an intense stimulation of pepsinogen secretion at the concentrations of 10(-8), 10( 6) and 10(-4) M, and the secretion rate expressed as percent of control were 277, 350 and 329%, respectively. These responses were not considered the additive action by betazole and ach. but the potential interaction between the two agents in pepsinogen secretion. From these findings, we conclude that betazole-ach. interdependency exists in in vitro pepsinogen secretion. PMID- 3011578 TI - The Japanese Society of Gastroenterology. Proceedings of the 71st general meeting. Sapporo, Japan, May 22-24, 1985. PMID- 3011577 TI - Superoxide anion generating capacity and lysosomal enzyme activities of Kupffer cells in galactosamine induced hepatitis. AB - To elucidate the function of the reticuloendothelial system of liver in hepatic injury, we investigated the effect of endotoxins on superoxide anion (O-2) generating capacity and lysosomal enzyme activities of Kupffer cells isolated from rats treated with galactosamine (Gal N), with Gal N supplemented with polymyxin B (Polymyxin B-Gal N), with lipopolysaccharide (LPS) and from control rats. After collagenase digestion of the liver and centrifugation over metrizamide gradient, Kupffer cells were prepared by the dish adherence procedure. O-2 production by the cells was examined as chemiluminescence during phagocytosis of latex particles and beta-glucuronidase activities were analyzed. High titers of endotoxemia were detected in LPS and Gal N rats by limulus test, while a low endotoxemia titer was found in Polymyxin B-Gal N rats. Hepatocyte damage was found in Gal N rats, but little was recognized in LPS and Polymyxin B Gal N rats. In the latter groups, Kupffer cells, activated by endotoxins, showed the enhancement of chemiluminescence and a release of lysosomal enzyme. Though lysosomal enzyme was released from Kupffer cells in Gal N rats, chemiluminescence was slightly suppressed in spite of the high titer of endotoxemia. These results appear to be related to the consumption of O-2 during liver injury. The functional state of Kupffer cells was thus changed by the grade of endotoxemia and hepatic injury. PMID- 3011579 TI - Evidence for abnormal cholinergic neuromuscular transmission in diabetic rat small intestine. AB - Proximal and distal rat small intestine from control, diabetic, and insulin treated diabetic rats was cut into strips measuring 6.0 X 10.0 mm. Strips cut along the oral-caudal axis were called longitudinal strips, while those cut 90 degrees to that axis were called circular strips. The strips were stretched to their optimum lengths and subjected to electrical field stimulation (0.1-1.0-ms pulse duration, 30-270 mA, 1-26 Hz) in the presence of Krebs' solution and Krebs' solution plus 10(-6) M atropine. Field stimulation produced atropine-sensitive and atropine-resistant contractions in all strips. Significant differences among the three groups were found in the amplitudes of atropine-sensitive contractions in strips from distal longitudinal muscle. Controls showed the highest amplitude contractions and diabetics the lowest, whereas the insulin-treated diabetics showed contractions intermediate in amplitude. No significant differences were noted among the atropine-resistant contractions. Field stimulation delivered at pulse durations of 5.0 and 50.0 ms in the presence of neural blockade with tetrodotoxin (5 X 10(-6) M) produced similar contraction amplitudes among the three groups. These results suggest that streptozotocin-induced diabetes mellitus is associated with defective cholinergic neuromuscular transmission in the myenteric plexus of the distal small intestine. Insulin therapy seems to improve the abnormality. PMID- 3011580 TI - Corynebacterium parvum-elicited hepatic macrophages demonstrate enhanced respiratory burst activity compared with resident Kupffer cells in the rat. AB - We have recently demonstrated that release of oxygen-derived free radicals by activated hepatic macrophages may be involved in the pathogenesis of a rat model of liver injury induced by Corynebacterium parvum and endotoxin. In the present study we have compared the respiratory burst activity of isolated normal rat Kupffer cells with that of hepatic macrophages elicited by C. parvum. Superoxide production (O2-.) and glucose oxidation via the hexose monophosphate shunt (HMPS) were low in normal Kupffer cells, but were significantly increased (O2-. 2.1 fold, HMPS 1.7-fold) by phorbol myristate acetate, a stimulant of the respiratory burst. Corynebacterium parvum-elicited hepatic macrophages demonstrated significantly enhanced superoxide production and HMPS activity compared with normal Kupffer cells, both in the absence of specific stimuli (O2-. 3.3-fold, HMPS 5.3-fold) and after exposure to phorbol myristate acetate (O2-. 4.5-fold, HMPS 5.3-fold). These results demonstrate that normal Kupffer cells are capable of exhibiting respiratory burst activity, but this is markedly increased for hepatic macrophages elicited by an inflammatory stimulus. PMID- 3011581 TI - Neurologic syndrome associated with low levels of vitamin E in primary biliary cirrhosis. AB - A 41-yr-old woman with primary biliary cirrhosis developed weakness and wasting in proximal muscles, areflexia, and decreased proprioceptive and vibratory sensation. Investigations revealed law serum levels of vitamin E and electromyographic and muscle biopsy changes consistent with a neuropathy. Sural nerve histology demonstrated axonal dystrophy with patchy demyelination. These features closely resemble a neurologic syndrome associated with chronic cholestatic liver disease and vitamin E deficiency in children. Adults with chronic cholestasis may also be susceptible to neurologic damage from prolonged malabsorption of vitamin E. PMID- 3011583 TI - Healing of benign gastric ulcer with low-dose antacids and fiber diet. AB - A randomized, double-blind, placebo-controlled trial was conducted to determine the efficacy of a low-dose aluminum-magnesium antacid regimen (Link one tablet q.i.d.) (total neutralizing capacity 120 mmol HCl/day) in combination with a high or a low-fiber diet in ulcer healing and relief of symptoms in patients with benign gastric ulcer. After 6 wk, the ulcer healed in 28 (67%) of the 42 patients treated with antacids compared with 11 (25%) of the 44 patients treated with placebo (p less than 0.001). Antacids were also significantly more effective than placebo in the relief of symptoms. The dietary treatment did not significantly influence ulcer healing or ulcer symptoms. Constipation was more frequently seen with the low- than with the high-fiber diet (p less than 0.01). No significant side effects from antacids were recorded. PMID- 3011582 TI - Patients with a high jejunostomy do not need a special diet. AB - Absorption from a chemically defined liquid feed consisting of small peptides, oligosaccharides, and little fat (half medium-chain triglycerides) was compared with that from a feed of whole protein, polysaccharides, and long-chain triglycerides in 7 patients with less than 150 cm of jejunum ending in a stoma. Comparisons of absorption from three solid food diets varying in their fiber and fat content but containing equal amounts of nitrogen and minerals were also made in 4 of the patients. There were no consistent differences between the two liquid or three solid-food diets in percentage of calorie, nitrogen, or fat absorption. The absolute loss of fat depended on the fat intake, but larger losses did not appear detrimental. A liquid diet consisting of peptides, oligosaccharides, and medium-chain triglycerides is not more beneficial than a polymeric diet in patients with a high jejunostomy. A liberal attitude is appropriate toward the fat and fiber content of the diet. Electrolyte supplements, especially sodium and magnesium, are often needed. PMID- 3011584 TI - Primary and metastatic tumors of the liver associated with cirrhosis. A study based on laparoscopy and autopsy. AB - In a series of 2,538 cases of cirrhosis seen at laparoscopy there were 140 primary liver carcinomas and 19 cases of metastases to a cirrhotic liver out of a total of 167 extrahepatic neoplasms associated with cirrhosis. In an autopsy series of 1,073 cases of cirrhosis there were 190 primary liver carcinomas and 22 cases of metastases to a cirrhotic liver out of a total of 98 extrahepatic neoplasms. In another autopsy series of 498 cases of cirrhosis there were 71 primary liver carcinomas and 18 cases of metastases to a cirrhotic liver out of a total of 58 extrahepatic neoplasms. The laparoscopy series showed a predominance (31.8%) of esophageal tumors associated with cirrhosis, but these tumors rarely gave rise to liver metastases (3.7%); in the autopsy series there was a predominance (35.3%) of tumors of the portal territory, giving rise to metastases in cirrhotic livers in 35.2% of cases. PMID- 3011585 TI - Endoscopic and pathologic features of esophageal lymphoma: a report of four cases in patients with acquired immune deficiency syndrome. PMID- 3011586 TI - [The Aids problem in pregnant women--a challenge to the obstetrician]. AB - Nine pregnant women with the AIDS problem were under treatment and care at the Department of Gynaecology of the University of Berlin at Charlottenburg during 1985. Of these, seven had HTLV-III antibodies only, whereas one woman showed additional clinical signs such as lymphoadenopathy and opportunistic infections; another woman developed exanthemas during pregnancy that pointed to a possible manifestation of AIDS. The increasing incidence in our clinic of pregnant women infected with or actually suffering from AIDS is due to the fact that for several years we have been conducting heroin withdrawal programmes in pregnant women. It must be born in mind, however, that generally more and more women outside the high-risk groups covered by anamnesis are being infected with HTLV-III. Screening tests for HTLV-III can help to clarify matters and make it possible to care adequately for the patient and to guarantee the obstetric team an appropriate management of the risk of infection. However, it must be considered as a minimum demand to be made on the obstetrician to conduct tests for HTLV-III antibodies in women of the high-risk groups (heroin addicts, prostitutes, blood transfusion recipients, fathers at risk). Based on update knowledge, the authors supply pointers to a more realistic information of pregnant women. Suggestions are advanced in respect of care during pregnancy, childbirth and puerperium. PMID- 3011587 TI - [Endocrine fertility-restricting factors in clomiphene therapy]. AB - A marked discrepancy between a high ovulation rate (70-80%) and a low pregnancy rate (30-35%) is generally observed during clomiphene therapy in female infertility. Disturbances in follicular maturation, inadequate secretory transformation of the endometrium and influence on the quality of cervical mucus are discussed as cause for this phenomenon. The purpose of this study was to investigate whether changes in endocrine patterns might lead to these changes thus limiting the success of clomiphene therapy. In 7 patients gonadotropins, prolactin, ovarian and adrenal androgens, sexual hormone binding globuline (SHBG) and plasma levels of clomiphene were measured at short intervals during a stimulated cycle. The increase of testosterone and androstendione promoted by clomiphene during the follicular phase is in accordance with previous observations. The decline in SHBG due to an antioestrogenic effect on its hepatic production leads to an additional increase in free, biologically active androgens. These androgens interfere with follicular maturation, causing atresia and premature luteinisation. As a direct effect of clomiphene on the adrenal gland, we observed an elevation of DHEAS. The significance of this finding remains unclear. During the follicular phase prolactin remained suppressed inspite of supraphysiological oestrogen levels. In the late luteal phase the diminished antioestrogenic influence on lactotrophic cells causes a 80% rise in prolactin that in some cases may impair luteal function. The average plasma half life of clomiphene was found to be 12 days. This is in accordance with the half life of the closely related compound tamoxifen. Clomiphene, also present in the luteal phase of the cycle, may cause inadequate secretory transformation of the endometrium by blocking endometrial oestrogen receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011588 TI - Population genetics of an expanding family of mobile genetic elements. AB - A model of an expanding family of dispersed repetitive DNA was studied. Based on the previous result of the model of duplicative transposition, an approximate solution to give allelism and identify coefficients as functions of time was obtained, and theoretical predictions were verified by Monte Carlo experiments. The results show that, even if the copy number per genome increases very rapidly, allelism and identity coefficients may take a long time to reach equilibrium. The changes of allelism and allelic identity are similar to that of homozygosity at an ordinary single locus, whereas that of nonallelic identity can be much slower, particularly when the copy number per genome is large. Thus, many existing families of highly repetitive sequences may represent nonequilibrium states for nonallelic identity. The present model may be extended to include other evolutionary forces such as gene conversion or the recurrent insertion from normal gene copies. PMID- 3011590 TI - Structural genes for phosphatases in Aspergillus nidulans. PMID- 3011589 TI - Evolutionary change of restriction cleavage sites and phylogenetic inference. AB - Mathematical formulas are developed for the evolutionary change of restriction cleavage sites in a DNA sequence, allowing unequal rates between transitional and transversional types of nucleotide substitution. Formulas are also developed for the probability of having a particular pattern of site changes among evolutionary lineages, such as parallel gains or losses of sites, and for inferring the presence or absence of a restriction site in an ancestral sequence from data on the present-day sequences. The unordered compatibility method is proposed for inferring the phylogenetic relationships among relatively closely related organisms, treating restriction sites as cladistic characters. Formulas are derived for the probability (P+) of obtaining the correct network for a given number (N) of informative sites for the cases of four and five species. These formulas are applied to evaluate the performance of the method and to estimate the N value required for P+ to be 95% or larger. The method performs well when the branches between ancestral nodes and the branches leading to the two most recent species are more or less equal in length, but performs poorly when the latter two branches are considerably longer than the former. PMID- 3011591 TI - Phosphatase regulation in Aspergillus nidulans: responses to nutritional starvation. PMID- 3011592 TI - Tn1721 derivatives for transposon mutagenesis, restriction mapping and nucleotide sequence analysis. AB - New derivatives of the tetracycline-resistance transposon Tn1721 that carry resistances to chloramphenicol, tetracycline, kanamycin and streptomycin are described. These elements are provided on various plasmid vehicles and as chromosomal insertions to extend the range of targets for Tn mutagenesis. Single EcoRI sites at the ends of these transposons proved most useful for physical mapping, for the generation of new EcoRI sites in cloning experiments, for end labelling and for sequencing of DNA adjacent to an insertion. PMID- 3011593 TI - Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids. AB - A hybrid plasmid, pJS37, was made by combining pLS1, which confers tetracycline (Tc) resistance, and pC194, which confers chloramphenicol (Cm) resistance. Both pJS37 (7.3 kb) and its derivative pJS140 (6.0 kb), from which pC194 replication genes were removed, were structurally and segregationally stable when introduced into Streptococcus pneumoniae and grown either in the presence of Tc or in the absence of drug. However, both hybrid plasmids underwent systematic deletion when grown in the presence of Cm. One of the deleted forms, pJS4 (3.4 kb), could not be maintained in the absence of a helper plasmid; two others, pJS3 (4.1 kb) and pJS5 (3.8 kb), lost the tet gene but retained the replication functions of pLS1. They both expressed very high levels of Cm acetyltransferase (CAT), which, in the case of pJS5, were constitutive. Nucleotide sequence determination of the deletion junctions in pJS3 and pJS5 indicated that the deletions occurred, presumably by recombination, between short direct repeats of 6 and 9 bp, respectively. In both cases the tet promoter was juxtaposed to the cat gene. In the case of pJS5, the deletion removed a sequence that sequestered the ribosome binding site (RBS) for cat, thereby rendering constitutive the production of CAT. The increased resistance to Cm afforded by the hyperexpression of the cat gene apparently provided a positive selective advantage for the accumulation of the deleted forms in the plasmid pool. PMID- 3011594 TI - Ribosomal proteins are encoded by single copy genes in Dictyostelium discoideum. AB - Five recombinant plasmids which encode ribosomal proteins (r-proteins) from Dictyostelium discoideum have been isolated. Poly(A) + RNA was size-fractionated by preparative agarose gel electrophoresis and a fraction encoding proteins of less than 35 kDa was used to construct a cDNA library in the plasmid vector pBR322. Individual clones from the library were screened by hybrid-selected translation and those encoding r-proteins were identified by co-migration of the translation products in two-dimensional gel electrophoresis with marker proteins purified from Dictyostelium ribosomes. Initial characterization using the five cDNA plasmids indicates that these r-proteins are encoded by single copy genes and that they are not tightly clustered in the genome. PMID- 3011595 TI - Relaxed specificity of the EcoRV restriction endonuclease. AB - The EcoRV restriction endonuclease normally shows a high specificity for its recognition site on DNA, GATATC. In standard reactions, it cleaves DNA at this site several orders of magnitude more readily than at any alternative sequence. But in the presence of dimethyl sulphoxide and at high pH, the EcoRV enzyme cleaves DNA at several sites that differ from its recognition site by one nucleotide. Of the 18 (3 X 6) possible sequences that differ from GATATC by one base, all were cleaved readily except for the following 4 sites: TATATC, CATATC, GATATA and GATATG. However, two of the sites that could be cleaved by EcoRV in the presence of dimethyl sulphoxide, GAGATC and GATCTC, were only cleaved on DNA that lacked dam methylation: both contain the sequence GATC, the recognition site for the dam methylase of Escherichia coli. PMID- 3011596 TI - Cloning and expression of the bacteriophage T3 RNA polymerase gene. AB - The gene that encodes the RNA polymerase of bacteriophage T3 (gene 1) has been cloned into a pBR322 derivative under the control of an inducible lacUV5 promoter. Large quantities of the protein are synthesized after induction of cells that carry this plasmid. RNA polymerase purified from these overproducing cells selectively initiates transcription from T3 promoter sequences as demonstrated by transcription of a dual promoter plasmid that carries both T3 and T7 promoters. Cells that carry the T3 RNA polymerase gene can complement amber mutants of T3 that are defective in gene 1 but not gene 1 amber mutants of T7, and vice versa; this experiment demonstrates the specificity of these enzymes in vivo. PMID- 3011597 TI - Androgen induction of alcohol dehydrogenase in mouse kidney. Studies with a cDNA probe confirmed by nucleotide sequence analysis. AB - A cDNA clone for the beta-chain of human alcohol dehydrogenase (ADH) was used to isolate several cross-hybridizing clones from a mouse liver cDNA library. Clones pADHm9 and a portion of pADHm12 were sequenced. pADHm9 coded for a sequence of 151 C-terminal amino acids and some untranslated sequences from the 3' end of its corresponding mRNA. This clone was identified as an Adh-1 cDNA clone. Consistent with the known expression of Adh-1, this gene was expressed constitutively in liver, whereas the Adh-3 gene product was found only in stomach, lung and reproductive tissues. Furthermore, the translated region of the cDNA shared 91% amino acid sequence homology with rat liver ADH. [32P]pADHm9 was used as a hybridization probe to study the mechanism of androgen induction of kidney ADH activity. Induction of A/J female mice by androgen resulted in a dramatic increase in the steady-state level of Adh-1 mRNA content which correlated with the level of enzyme induction. The size of the mRNA obtained from control or induced kidney and liver tissues was indistinguishable by Northern analysis. [32P]pADHm9 was also used to probe restriction fragments of genomic DNA obtained from several inbred mouse strains. The hybridization patterns, considered with the genetic evidence, suggested that pADHm9 recognized sequences which may be present as only a single copy in the genome. No restriction fragment length polymorphisms were observed among the several inbred mouse strains examined. PMID- 3011598 TI - Genes from the cyanobacterium Agmenellum quadruplicatum isolated by complementation: characterization and production of merodiploids. AB - The isolation of several biosynthetic genes from a cyanobacterium, Agmenellum quadruplicatum, by complementation of auxotrophic mutations in Escherichia coli, and their partial characterization, is described. Although our search for such genes has not been exhaustive, it appears that complementation of E. coli mutations may be of limited utility for the identification and/or isolation of cyanobacterial genes. Despite some overlap in the complementation abilities of these isolated cyanobacterial DNA fragments, the genes that we have studied in some detail are not located in operons. We have used mutagenized versions of these cyanobacterial DNA fragments to produce mutant phenotypes in the cyanobacterium, but clean auxotrophs were not obtained. Complementation of these mutant phenotypes can be obtained when the appropriate wild-type DNA fragment is introduced into the cyanobacterium on a shuttle vector. Recombination between two copies of a cyanobacterial gene occurs at high frequency in the cyanobacterium. PMID- 3011599 TI - Cloning and expression of Bacillus subtilis phage DNA methyltransferase genes in Escherichia coli and B. subtilis. AB - The DNA methyltransferase (Mtase) genes of the temperate Bacillus subtilis phages SPR (wild type and various mutants), phi 3T, rho 11 and SP beta have been cloned and expressed in Escherichia coli and B. subtilis host-plasmid vector systems. Mtase activity has been quantitated in these clones by performing in vitro methylation assays of cell-free extracts. The four-phage Mtase genes differ in the amount of Mtase synthesized when transcribed from their genuine promoters. In B. subtilis as well as in E. coli the SPR Mtase is always produced in smaller amounts than the other phage Mtases. Expression levels of the SPR Mtase are dependent on the strength of the upstream vector promoter sequences. Overproduction of the SPR wild-type and mutant enzymes was achieved in E. coli (inducible expression) by fusions to the lambda pL or the tac promoter and in B. subtilis (constitutive expression) by means of the phage SP02 promoter. PMID- 3011600 TI - Isolation of the positive-acting regulatory gene PHO4 from Saccharomyces cerevisiae. AB - We have isolated a 10.2-kb fragment of yeast DNA from a genomic library of recombinant centromeric YCp50 plasmids, which complements a mutation in the PHO4 gene of Saccharomyces cerevisiae. The identity of the PHO4 gene on this plasmid was established by integration of a subfragment into the PHO4 region of the yeast chromosome. Analysis of a series of plasmid subclones covering different regions of the original yeast DNA insert localized the PHO4 gene within a 2.25-kb sequence. Southern hybridization of total genomic DNA prepared from wild-type strains and from integrative transformants show that the PHO4 gene consists of unique yeast DNA sequences and is present at a single copy in the S. cerevisiae genome. RNA blot hybridization mapping of transcripts within this genomic region identify the PHO4 transcript as a 1.7-kb, low-abundancy, constitutively expressed and polyadenylated RNA. PMID- 3011601 TI - Use of bacterial DHFR-II fusion proteins to elicit specific antibodies. AB - Plasmids containing the coding region of the type II dihydrofolate reductase (DHFR) specified by R388 have been used to alter the amino acid (aa) sequence at the C-terminus of this protein. These plasmids have a unique cloning site in the C-terminal portion of the 78-aa coding region. Insertions of DNA fragments into this site produced plasmids that code for proteins with 6- to 80-aa extensions. The vectors were constructed to terminate translation in all three phases beyond the position of insertion of foreign DNA. Random DNA fragments from the major sperm protein (MSP) gene of Caenorhabditis elegans produced by DNase I cleavage were inserted into these vectors. Cell extracts from colonies containing MSP sequences were examined by gel electrophoresis and immunoblotting. One of the hybrid DHFR-MSP proteins was isolated and antibody was prepared to it. This antibody preparation reacted with MSP in immunoblots of purified MSP and whole cell extracts of the worm. A rapid purification procedure for the DHFR is presented. PMID- 3011603 TI - Isolation and characterization of human blood-coagulation factor X cDNA. AB - Using synthetic oligodeoxynucleotides as probes, we have isolated factor X cDNA from human liver cDNA library. We sequenced the 1430-bp cDNA which spans the coding region of the mature factor X and contains the polyadenylation signal and poly(A) tail. The amino acid (aa) sequence is in agreement with the published aa sequence. The nucleotide (nt) sequence of cDNA confirmed that factor X is synthesized and secreted as a single-chain precursor, and then converted into dimeric form by proteolytic cleavage of an internal tripeptide. From the nt sequence, it was also predicted that like other secretory proteins, human factor X is synthesized with a leader sequence (prepro-protein). The 5'-coding region of factor X cDNA is 60 and 40% homologous to the corresponding regions of factor IX and prothrombin genes, respectively. This supports the hypothesis of gene evolution by gene duplication followed by divergence. PMID- 3011602 TI - Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens. AB - Two cDNA clones encoding two major human trypsinogen isozymes were isolated from a human pancreatic cDNA library. The deduced amino acid (aa) sequences of the two trypsinogen precursors are found to have 89% sequence homology, and have the same number of aa (247), including 15 aa for a signal peptide and 8 aa for an activation peptide. Southern blot analysis of human genomic DNA using the cloned cDNA as a probe, revealed that the human trypsinogen genes constitute a multigene family of more than ten genes. PMID- 3011604 TI - Orientation and sequence analysis of right ends and target sites of bacteriophage mu and D108 insertions in the plasmid pSC101. AB - We have isolated four independent insertions of the entire 37-kb D108cts 10 genome in the low-copy-number plasmid pSC101 in vivo. They were all formed by replicative transposition during the D108 lytic cycle. The orientation of these four insertions was found to be the same, with the left ends facing towards pSC101 replication, and the right end facing in the direction of all pSC101 transcription, as was previously found for a Mucts62 insertion in pSC101, pMC321. The exact sites of insertion of two of the D108 prophages, as well as the Mu prophage, have been determined by sequence analysis. All three insertions caused a 5-bp duplication of pSC101 sequences at the target site, as has been found for insertions formed by conservative integration upon lysogeny. Moreover, we have determined the nucleotide sequence of the first 75 bp of the right end of D108 and, though this end is interchangeable with the right end of Mu as a substrate for either phage's transposition functions, there are a number of nucleotide differences between them. PMID- 3011605 TI - Cloning and characterization of the Schizosaccharomyces pombe DNA ligase gene CDC17. AB - The Schizosaccharomyces pombe CDC17 gene has been cloned by complementation of the cdc17 mutant coding for temperature-sensitive DNA ligase. An allele-specific suppressor active only in the presence of a high osmotic pressure was also isolated. The cloned CDC17 gene failed to complement the analogous DNA ligase mutation, cdc9, in Saccharomyces cerevisiae, although the reverse complementation was successful [Barker and Johnston, Eur. J. Biochem. 134 (1983) 315-319]. The CDC17 gene specifies a 2.8-kb transcript. PMID- 3011606 TI - A Bacillus subtilis plasmid that can be packaged as single-stranded DNA in Escherichia coli: use for oligodeoxynucleotide-directed mutagenesis. AB - A Bacillus subtilis/Escherichia coli shuttle vector was modified to contain the origin of DNA replication of the E. coli filamentous phage f1, in both orientations. Upon superinfection with and f1-related phage of an E. coli strain containing either of the modified vectors, the single-stranded (ss) form of the plasmid was packaged in virions and released to the culture medium. Each of these ss DNAs has been purified from the virions and used as a template for oligodeoxynucleotide-directed mutagenesis. The resulting mutations were demonstrated by DNA sequencing. The capacity of these vectors to be isolated as phage ss DNA from E. coli and to replicate as plasmids in B. subtilis makes them convenient substrates for the production of oligodeoxynucleotide-directed mutations for studies in B. subtilis. PMID- 3011607 TI - Kanamycin-resistant vectors that are analogues of plasmids pUC8, pUC9, pEMBL8 and pEMBL9. AB - Analogues of the cloning vectors pUC8, pUC9, pEMBL8 +/- and pEMBL9 +/- that have kanamycin resistance (KmR) instead of ampicillin resistance (ApR) as the selectable marker have been developed. HindIII and SmaI sites within the KmR gene have been removed so that all of the cloning sites in the multi-linker region of these plasmids may be used except the AccI site. PMID- 3011609 TI - Nucleotide sequence of the R26 chloramphenicol resistance determinant and identification of its gene product. AB - The cml gene of plasmid R26 is carried on a 1.9-kb HindIII fragment and specifies low-level, inducible resistance to chloramphenicol (Cm). In this paper we report the identification of its product as an approx. 31 kDa protein in minicell experiments, and the determination of the nucleotide sequence of cml, which indicates that the gene product is a relatively hydrophobic protein of Mr 33,800. The protein has no detectable homology to other characterised chloramphenicol resistance (CmR) proteins, nor any to the membrane-associated tetracycline resistance (TcR) proteins. The presumptive ribosome-binding site (RBS) of cml mRNA is within a region showing potential for secondary structure. PMID- 3011608 TI - The methionine initiator tRNA genes of yeast. AB - We have isolated three distinct tRNAimet genes from a yeast DNA clone bank. The complete sequence of two shows that these genes are colinear with the mature tRNAimet and supports the RNA sequence of tRNAimet. Southern analysis of yeast genomic DNA indicates the presence of four copies of tRNAimet gene per haploid genome. PMID- 3011611 TI - [Hygienic characteristics of the working conditions for workers in the large tonnage manufacture of carmabide]. PMID- 3011612 TI - [Hygienic significance of the combined effects of silica condensation aerosol and ozone]. PMID- 3011610 TI - Herpes simplex virus immediate-early protein ICP4 in murine models of latency. AB - Herpes simplex virus-1 (HSV) infection of trigeminal and dorsal root ganglia was established in mice via corneal scarification and footpad injection, respectively. Trigeminal and dorsal root ganglia were removed during the acute and latent stages of infection and processed for the immediate-early HSV polypeptide ICP4 (VP175) using both a monoclonal reagent and polyclonal antiserum and the avidin biotin complex immunoperoxidase method. ICP4 (VP175) antigen was readily detected during the acute infection of both dorsal and trigeminal ganglia, but not during latency. This antigen could again be detected by reactivation of the latent virus by explanation and organ culture. The detection of ICP4 (VP175) during latency in a rabbit model but not in a murine model may correlate with biologic differences in each system (e.g., rabbits spontaneously reactivate). Alternatively, the discrepancy could reflect viral strain characteristics or may imply that ICP4 (VP175) need not be constitutively expressed (at detectable levels) in all models of latent infection. PMID- 3011613 TI - Phosphorylase phosphatase and phosphatase activating-kinase FA in growing rat muscles. AB - The activities of phosphorylase phosphatase and of FA, the kinase that activates phosphatase, were measured in rat skeletal muscle from birth to 200 g body weight. Throughout this period part of phosphatase was always spontaneously active. The activity could be further increased by trypsin, but did not additionally increase when Mn2+ was present. During the first 15-20 days of life most of the phosphatase was cytosolic. Then it decreased in this fraction and more phosphatase was found in glycogen particles, to reach the adult level at about 50 g body weight. Also the activity of the kinase FA was lower for the first 10 days, then it increased attaining the adult level again at about 50 g. These results are compared to those on phosphorylase activity and glycogen level in muscle and on serum insulin during growth. PMID- 3011615 TI - Reactivation of cytomegalovirus in endometrial cells by estradiol. AB - The present study uses an in vitro model to test the influence of hormones on the replication and reactivation of cytomegalovirus (CMV) in endometrial tissue culture. Infection of epithelial cells of the endometrium with CMV in the presence of a high concentration of estradiol resulted in only a slightly higher yield of infectious virus. Progesterone did not cause any effect on viral replication. Arrest of CMV replication was achieved by reduction of the pH of the incubation medium. No infectious virus was detectable after incubation of infected cultures at pH 5.8. In the above-described conditions following the arrest of CMV replication by low pH, estradiol treatment was capable of reactivating the virus. The possibility that the increase in CMV infection observed in pregnancy is caused by reactivation of latent virus provoked by hormonal factors will be discussed. PMID- 3011614 TI - Gestational trophoblastic disease: the significance of vaginal metastases. AB - Five patients with gestational trophoblastic disease whose presenting symptom was hemorrhage from vaginal metastases have been added to our previous report. The clinical features, management, and responses to treatment are outlined. All the patients required suturing of the bleeding lesions under general anesthetic to arrest the hemorrhage. In addition one patient needed selective arterial embolization. This did not compromise the response to chemotherapy. We confirm our previous view that the presence of vaginal metastases should be classified as a high-risk factor and that these patients be treated with multiple agent chemotherapy from the outset. PMID- 3011616 TI - Beneficial effects of defibrotide against myocardial ischemia and decline of beta adrenoceptor function in the rabbit. AB - Defibrotide is a polydeoxyribonucleotide of mammalian origin which possesses profibrinolytic effect and PGI2-releasing capacity. Because of these properties, defibrotide has antithrombotic effects which are demonstrated in various experimental models of venous and arterial thrombosis. The present study indicates that defibrotide clearly protects against myocardial damage in the rabbit 3 days after total coronary artery occlusion. Furthermore, defibrotide prevents the decline of beta-adrenergic receptor function, a phenomenon related to excessive circulating catecholamines that occurs during the myocardial infarct. Defibrotide prevents the dramatic fall of creatine phosphokinase activity in the ischemic ventricle: metabolic changes which reflect changes in the cells affected by prolonged ischemia. Assumptions about the mode of action of defibrotide are given particularly in consideration of the interaction between the fibrinolytic activity and the PGI2-releasing capacity of this substance. PMID- 3011618 TI - [Transient neonatal metabolic acidosis]. PMID- 3011617 TI - In vivo effects of defibrotide on platelet c-AMP and blood prostanoid levels. AB - Patients were treated with defibrotide (3 X 200 mg/day) for 7 days. Plasma PGI2 and TXB2 levels were measured by RIA using EDTA-aspisol as anticoagulant and cyclooxygenase inhibitor. Isolated platelet c-AMP levels were also determined by RIA, using EDTA-dipyridamole as anticoagulant and phosphodiesterase inhibitor. Blood PGI2 levels were found to increase significantly upon treatment while the increase in TXB2 levels was not significant. Blood PGI2/TXB2 ratio increased 51%, 30 min after intravenous injection and it remained 28% higher during therapy than the predrug blood level. Significantly higher platelet c-AMP levels were also obtained after the injection of drug (0.02 less than p less than 0.05). PMID- 3011620 TI - [Effects of misoprostol, (+/-)-methyl (11 alpha, 13E)-11, 16-dihydroxy-16-methyl 9-oxoprost-13-en-l-oate, on various gastric and duodenal lesions in rats]. AB - Male Sprague-Dawley rats (230-280 g), either fasted for 15-24 hr or non-fasted prior to experiments, were used. Misoprostol (3-100 micrograms/kg, p.o.) dose dependently inhibited the development of 150 mM HCl X aspirin (100 mg/kg)-, 150 mM HCl X 60% ethanol-, and aspirin (150 mg/kg)-induced gastric lesions. Misoprostol (30, 100 micrograms/kg, p.o.), given twice daily for 4 days, significantly inhibited prednisolone (50 mg/kg given once daily for 4 days) induced gastric lesions. Misoprostol (30 or 2 X 300 micrograms/kg, p.o.) also significantly inhibited water-immersion stress (21 degrees C, 10 hr)-induced gastric lesions or mepirizole (200 mg/kg)-induced duodenal lesions, respectively. In contrast, misoprostol (30-300 micrograms/kg, p.o.) had no effects on indomethacin (25 mg/kg)- and mepirizole (200 mg/kg)-induced gastric lesions. Misoprostol (30 micrograms/kg, p.o.) had no effect on gastric secretion in pylorus-ligated preparations (4 hr), but it (100 or 300 micrograms/kg, p.o.) significantly increased the volume and pepsin output. Gastric motility, either normal or enhanced with indomethacin (25 mg/kg), was inhibited by misoprostol (30 or 300 micrograms/kg, p.o.). Misoprostol (30 micrograms/kg, i.d.) significantly stimulated duodenal HCO3- secretion. Mechanisms by which misoprostol inhibits various gastric lesions remain unknown. However, the stimulatory activity on duodenal HCO3- secretion appears to be involved in the preventive effect of misoprostol on the development of duodenal lesions. The effects of cimetidine and 16,16-dimethyl PGE2 were also studied and compared with those of misoprostol. PMID- 3011619 TI - [Vasoconstrictor responses of isolated pig coronary arteries]. AB - In helical strips of pig coronary arteries, histamine, serotonin, acetylcholine and a stable analogue of thromboxane A2 (9, 11-epithio-11, 12-methano TXA2: s TXA2) produced a dose-dependent contraction. The histamine-induced contraction was suppressed by treatment with chlorpheniramine, suggesting an involvement of H1 receptors. Contractile responses to serotonin were attenuated by not only ketanserin, an S2 antagonist, but also by cinanserin and methysergide. Relaxation induced by serotonin in preparations treated with high concentrations of ketanserin were inhibited by cinanserin and methysergide. Norepinephrine contracted coronary arteries treated with propranolol. Contractile responses to norepinephrine were reversed to relaxations by prazosin, which were abolished by treatment with yohimbine. Contractile responses to histamine were potentiated by treatment with low concentrations of serotonin or s-TXA2. Contractile responses to serotonin were also potentiated by low concentrations of histamine or s-TXA2. Removal of the endothelium from pig coronary arterial strips potentiated contractions induced by serotonin, histamine and norepinephrine. These results suggest that, in addition to damaged endothelium, integrating action of endogenous vasoconstrictors, including histamine, serotonin, TXA2 and norepinephrine, may play an important role in producing coronary vasospasm. PMID- 3011623 TI - [After care following heart valve replacement in general practice and a heart support group. 1: Diagnostic measures and drug therapy]. PMID- 3011622 TI - [Adenocarcinoma of the urachus. A case report]. PMID- 3011621 TI - [Diagnosis and therapy of the most frequent APUDomas. Insulinoma, gastrinoma and C cell carcinoma of the thyroid gland]. PMID- 3011624 TI - High concentration of locally administered human fibroblast interferon in a glioblastoma multiforme. Discrepancy between clinical response and sensitivity of the tumor cells to the interferon in vitro. PMID- 3011625 TI - Physiologic implications of the autonomic aberrations in cystic fibrosis. AB - Cystic fibrosis patients and their parents have increased alpha-adrenergic sensitivity, increased cholinergic sensitivity, and reduced beta-adrenergic sensitivity. This combination of autonomic aberrations has been associated with increased airway reactivity in other disease populations. Although studies of airway reactivity are difficult to interpret in the cystic fibrosis patients themselves, the parents have no apparent pulmonary infection or inflammation, and one-third of these people have increased airway reactivity. Moreover, parents of children with cystic fibrosis have increased prevalence of wheezing and lung disease in childhood. Airway reactivity has been associated in other populations, with increased risk of obstructive pulmonary disease. Further studies are required to test the hypothesis that heterozygosity for CF is a risk factor for development and progression of obstructive pulmonary disease. PMID- 3011626 TI - Morphine induced thyroxine release from rat thyroid gland in vitro. AB - Male rat thyroid glands were incubated for two hours in Krebs-Ringer bicarbonate buffer with different amounts of morphine and/or naloxone. Five micrograms/ml morphine produced a significant increase in the T4 concentration of incubation medium, and resulted in an accumulation of cAMP in the tissue. Naloxone did not change the T4 release but its incubation with morphine prevented the morphine induced changes. Similarly, naloxone inhibited the morphine-induced accumulation of cAMP in the thyroid tissue. PMID- 3011627 TI - Calcitonin induced increase in ACTH, beta-endorphin and cortisol secretion. AB - The response of ACTH, beta-endorphin and cortisol to calcitonin administration was investigated in 8 subjects with recent fractures of the vertebrae due to postmenopausal or senile osteoporosis (Ost) and in seven normal healthy controls (NC). A significant increase of the three hormones was observed in 13 subjects. The maximum increase was observed between 15 and 60 min.: the cortisol level (microgram/100 ml) rose from 14.3 +/- 1.9 to 24.8 +/- 3.2 (P less than 0.05) in Ost and from 7.7 +/- 0.6 to 21.7 +/- 1.7 (P less than 0.001) in NC, the beta endorphin (pmol/l) from 5.8 +/- 0.6 and to 21.2 +/- 1.3 in OST (P less than 0.001) and from 5.9 +/- 0.4 to 21.9 +/- 4.5 (P less than 0.01) in NC and the ACTH levels (pg/ml) from 21.3 +/- 5.7 to 61.7 +/- 3.6 (P less than 0.001) in OST and from 30.0 +/- 6.2 to 58.8 +/- 7.5 (P less than 0.05) in NC. The results indicate a possible role of calcitonin in modulating the anterior pituitary function. It also suggests that the analgesic effect of calcitonin might be mediated by the increase of beta-endorphin. The possibility that this analgesic effect of calcitonin is due to its direct binding to the opiate receptors was excluded in the present study by in vitro binding assay. PMID- 3011628 TI - Effect of thiamin deficiency on adrenocorticotrophin and vasopressin in the rat hypothalamus. PMID- 3011629 TI - The possible involvement of cytochrome P-450 monooxygenase in AVP-induced ACTH secretion. AB - AVP (10(-7) M) induced ACTH as well as PGE2 release from rat anterior pituitary quarters. Inhibitors of P-450 monooxygenase, metyrapone (10 mM) and piperonyl butoxide (1 mM and 10 mM) attenuated the ACTH and PGE2 response to AVP. 7,8 benzoflavon (10 mM) which inhibits 3-methylchloranthrene inducible form of P-450 isoenzymes showed no inhibition of AVP-induced ACTH secretion. The decrease in ACTH response to AVP was still observed following the inhibition of prostaglandin synthesis by indomethacin. These results suggest that cytochrome P-450 monoocygenase systems are involved in the process of AVP-induced ACTH secretion, 3-methylchloranthrene inducible form of P-450 isoenzymes do not seem to be involved in this process. PMID- 3011630 TI - Frequency and significance of tumor thrombi in esophageal varices in hepatocellular carcinoma associated with cirrhosis. AB - Histological examination of the wall of the stomach and esophagus in patients with hepatocellular carcinoma associated with cirrhosis demonstrated intravariceal tumor thrombi in 13 (23.6%) of 55 cases studied. There were distant hematogenous metastases in 31 of them, of whom 12 (38.7%) had variceal tumor thrombi. Tumor thrombi were of varying sizes, and tumor cells appeared either intact, degenerated or necrotic. In seven cases, there was a firm adhesion of thrombi onto the vascular wall suggesting possible mural infiltration, but no extravascular metastases were noted grossly. These findings suggest a possibility of metastasis of hepatocellular carcinoma to the stomach and esophagus via the portal vein. It is also suggested that the degree of varices is not increased by tumor thrombus formation per se, and that both varices and tumor thrombi are due to extensive hepatofugal collateral circulation. Considering that 12 of 13 cases of intravariceal tumor thrombi had lung metastases, a portal vein-varices-lung route is possible for lung metastasis beside the established route through the hepatic vein in hepatocellular carcinoma. PMID- 3011631 TI - Androgen and estrogen receptors in hepatocellular carcinoma and in the surrounding noncancerous liver tissue. AB - Both androgen and estrogen receptors were studied in human hepatocellular carcinoma and noncancerous liver tissue surrounding it. Androgen receptor was detected in the cytosol and/or nucleosol of 4 of 8 cancerous tissues and 1 of 6 noncancerous tissues. The levels of androgen receptor in hepatocellular carcinomas ranged from 3.4 to 37.6 fmoles per mg protein with dissociation constants (Kd) of 0.226 - 51.3 X 10(-9) M. That in the surrounding noncancerous tissue was 2.1 fmoles per mg protein with Kd of 0.941 X 10(-9) M. Estrogen receptor was detected in the cytosol of 1 of 7 cancerous tissues, while it was detected in the cytosol and/or nucleosol of 3 of 7 noncancerous tissues. The level of estrogen receptor in hepatocellular carcinoma was 4.9 fmoles per mg protein with Kd of 1.20 X 10(-9) M, and those in the surrounding noncancerous tissues ranged from 2.6 to 1,073 fmoles per mg protein with Kd of 0.223 - 3.15 X 10(-9) M. The results suggest that the expression of androgen receptor may be augmented in association with malignant transformation of hepatocytes while the expression of estrogen receptor may be rather suppressed and that some of hepatocellular carcinomas may be androgen-dependent. PMID- 3011632 TI - Relationships between 34 HLA-A, HLA-B and HLA-DR antigens and three serological markers of viral infections in alcoholic cirrhosis. AB - In order to study the genetic risk of alcoholic cirrhosis, the frequency of 26 HLA-A and -B antigens was compared in 184 normal controls, 175 alcoholic cirrhotic patients and 83 alcoholic patients with hepatic steatosis of carefully selected ethnic origin. Eight HLA-DR antigens were also determined in 95 subjects of the normal control group and 63 patients of the alcoholic cirrhosis group. The incidence of hepatitis B virus antibodies (anti-HBc and anti-HBs) was defined in 74 patients of the alcoholic steatosis group, 170 patients of the alcoholic cirrhosis group and 111 normal controls different from the previously mentioned normal control group. The incidence and the titers of cytomegalovirus and rubella antibodies were also determined in 93 patients of the alcoholic cirrhosis group and the 111 normal controls. Serum immunoglobulin concentrations were measured in the same 93 cirrhotic patients. Compared with the controls, the alcoholic cirrhosis group revealed a significantly higher frequency of HLA-B15 (21.7 vs. 9.8%, p less than 0.00025, corrected p less than 0.050) and HLA-DR4 (38.1 vs. 17.9%, p less than 0.005, corrected p less than 0.050) and a significantly lower frequency of HLA-B13 (2.9 vs. 11.4%, p less than 0.025, corrected p less than 0.050). As for the frequency of all other HLA antigens, there was no significant difference between the three groups (normal controls, alcoholic cirrhosis and alcoholic steatosis).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011633 TI - Anatomy of the renin-angiotensin system in the normal and pathological kidney. AB - In this review we describe the contributions made by immunocytochemistry to our knowledge of the renin-angiotensin system in the normal and the pathological kidney. Most of the renin-secreting cells appear to be on the outer aspect of the vessel wall, supporting the view that renin is secreted mainly into the interstitium of the kidney rather than into the lumen of the vessel. Angiotensin II immunoreactivity is present within renin-secreting cells. The angiotensin II appears to be present in high concentration in the renin storage granules and is therefore presumably secreted from the cell with renin. The pathways by which renin is secreted from the cell have also been clarified. In pathological kidneys, the reactions of renin-secreting cells to variation in functional demand have been confirmed. Renin-containing cells have also been found in most types of renal tumours and occasional cases probably secrete renin or prorenin into the blood. In renal tumours and in the developing kidney (in all species studied) the renin-containing cells are also intimately associated with blood vessels. PMID- 3011634 TI - The ultrastructure of medullary, atypical medullary and non-medullary carcinomas of the breast. AB - Three medullary, eight atypical medullary and four non-medullary carcinomas of the breast were studied by transmission electron microscopy. Detailed comparison of a number of structural, cytoplasmic and nuclear features failed to confirm previous suggestions that medullary carcinoma cells have a distinctive ultrastructure. Electron microscopy is thus unlikely to be useful in the differential diagnosis of the tumours, nor does it suggest a basis for the good prognosis of medullary carcinoma. PMID- 3011635 TI - Adenoid cystic carcinoma of the breast: a case with axillary lymph node metastasis. AB - A breast tumour with proven lymph node metastasis is conclusively characterized as an adenoid cystic carcinoma using immunocytochemistry and electron microscopy. The majority of tumour cells showed certain of the characteristic features of myoepithelial cells while the pseudocystic spaces contained large amounts of reduplicated basal lamina. A small proportion of tumour cells, however, showed epithelial differentiation with the formation of true lumina. PMID- 3011636 TI - Adenoid cystic (cylindromatous) carcinoma of the trachea: an ultrastructural study. AB - Adenoid cystic (cylindromatous) carcinoma of the trachea is a rare tumour. The histological features in previously reported cases have been widely variable; only one previous ultrastructural study has been undertaken. Three cases are reported in the present study and the ultrastructural findings are reviewed. The tumours were composed of two types of cells, mature ductal and immature myoepithelial. The cylindromatous stroma appeared as a fibrillar substance which was also found between the undifferentiated myoepithelial cells and appeared to be a product of these cells. PMID- 3011637 TI - Calcium pyrophosphate crystal arthropathy: a biomineralization disorder. PMID- 3011638 TI - The role of human papillomaviruses in the pathogenesis and histologic classification of precancerous lesions of the cervix. AB - It is clear that the relation between HPV infection and cervical neoplasia is more complex than initially realized. Preliminary molecular virologic data suggest preferential distributions of low- and high-risk HPV types in CIN that tend to correlate with the morphologic appearance. Thus, mild and moderate dysplasias (CIN I and II) contain a diverse distribution of HPV types, including a minority that have a high risk of malignant potential. HPV, therefore, appears to play a major role as a promoter. Neoplastic transformation is probably determined by specific HPV types but, in addition, requires initiation by some other carcinogenic stimulus, e.g., HSV II, cigarette smoking. Despite numerous studies, performed during the past 30 years, the long-term behavior of dysplasia remains uncertain. The natural history of HPV-associated lesions is unknown. Until this information is available, it is recommended that the conventional dysplasia--CIS or CIN nomenclature be used. The presence of associated viral changes can be considered and added to the diagnosis, e.g., "moderate dysplasia (CIN II) with evidence of papillomavirus infection." Treatment should be the same for all intraepithelial lesions, regardless of the presence of morphologic evidence of HPV. In the future, it may be necessary to modify the classification of precancerous lesions of the cervix if it is shown that a specific HPV type induces a characteristic morphologic alteration or that the HPV type, in and of itself, has greater prognostic significance. Until then, confusion will be minimized and management optimized if the conventional dysplasia--CIS or CIN nomenclature is employed. PMID- 3011639 TI - The phenotypic diversity of peripheral T-cell lymphomas: the Southeastern Cancer Study Group experience. AB - Four member institutions of the Southeastern Cancer Study Group (SECSG) investigated 27 cases of malignant lymphoma proved to be of T-cell origin by a frozen section immunoperoxidase technique. The specimens were sent to one central laboratory in Michel's transport medium, where phenotyping studies were performed with a large number of monoclonal antibodies. The phenotypes encountered differed as a group from that reported for lymphoblastic lymphoma, but there was significant diversity within the peripheral T-cell lymphomas. Most tumors were of a mature helper/inducer phenotype (Leu-3+, Leu-2-), but nine of the 27 lymphomas expressed Leu-3 and Leu-2 in other combinations. Half of the lymphomas expressed abnormal T-cell phenotypes in that one or more pan-T-cell markers usually present in nonneoplastic T-cell proliferations were absent. Antibody 3A1 was the pan-T marker that was most frequently lacking in the peripheral T-cell lymphomas. The tumors were also studied for their expression of three markers associated with T cell activation--HLA-DR, transferrin receptor, and interleukin 2 receptor. The majority of the lymphomas expressed one or more activation markers. However, these three markers appear to be expressed independently. In general, there was no simple correlation between the phenotype of the tumor and the histologic appearance, although neoplasms of morphologically higher grades were somewhat more likely to express T-cell activation markers. PMID- 3011640 TI - Subtypes of small cell carcinoma of the lung: morphometric, ultrastructural, and immunohistochemical analyses. AB - Fifty-three surgically resected small cell carcinomas of the lung were studied morphometrically, electron microscopically, immunohistochemically, and in terms of possible site of origin. Four subtypes of small cell carcinomas were identified: oat cell carcinoma (OAT), small cell carcinoma of the intermediate cell type (INT), combined oat cell carcinoma, and undifferentiated carcinoma of the small cell type (UCS). The latter type is presumed to be non-neuroendocrine. Morphometric analysis showed considerable overlap among OAT, INT, and UCS with respect to nuclear area, cell area, and nuclear/cytoplasmic ratio. Ultrastructurally, significantly more carcinomas categorized as OAT and INT contained neurosecretory granules than did those in the UCS category (P less than 0.01 and 0.05, respectively). Cells with tonofibrils were more frequent in UCSs than in OATs and INTs. Immunohistochemically, fewer UCSs than OATs contained cells with gastrin-releasing peptide, neuron-specific enolase, and Leu-7 (P = 0.5, P less than 0.05, and P less than 0.05, respectively). UCSs were located more frequently at the periphery of the lung than were OATs (P less than 0.01) and INTs (P = 0.06). These findings suggest that UCS may be a pathologic entity distinct from the typical neuroendocrine-type small cell carcinoma and that this subtype probably corresponds to small cell carcinoma with a "large cell component," and to very poorly differentiated adenocarcinoma and squamous cell carcinoma of the small cell type. PMID- 3011641 TI - Diffuse intestinal ulceration after marrow transplantation: a clinicopathologic study of 13 patients. AB - The cases of 13 allogeneic marrow transplant recipients who had undergone laparotomy for manifestations of severe enteritis were reviewed to determine the causes of the severe intestinal disease and to assess the relation between clinical, histologic, and microbiologic findings. Laparotomies were performed a median of 63 days (range, 11 to 273 days) after transplantation for suspected peritonitis, intestinal obstruction, or bleeding. Intestinal tissue was available from small bowel resections in nine patients, intraoperative biopsies in one, and from autopsies in three patients who died shortly after laparotomy. Widespread small bowel ulceration was present in all 13 cases. Four causes of ulceration were identified: chemoradiation toxicity (n = 2), acute graft-versus-host disease (GVHD) (n = 5), opportunistic infections superimposed on either GVHD or toxicity from chemotherapy (n = 4), and Epstein-Barr virus-associated lymphoproliferative disorder (n = 2). Intestinal infections, unrecognized before laparotomy, were due to cytomegalovirus (CMV), herpes simplex virus (HSV), adenovirus, and Torulopsis glabrata. CMV- and HSV-infected cells, often lacking diagnostic inclusions, were identified in the intestine by in situ hybridization with biotinylated DNA probes. Eleven patients died in the perioperative period, and two died 452 and 558 days after surgery of complications of chronic GVHD. Poor outcomes were related to extensive intestinal involvement, which was commonly underestimated before surgery, failure to diagnose intestinal infections early, poor marrow function, impaired immunity, and refractoriness of severe GVHD. PMID- 3011643 TI - Assignment of the human gamma-crystallin gene cluster (CRYG) to the long arm of chromosome 2, region q33-36. AB - The gamma-crystallins of the human eye lens are encoded by a multigene family of which at least six genes have recently been assigned to chromosome 2. We have now localized these genes to the distal region of the long arm of chromosome 2 (region q33-36, most probably q34-35) using somatic cell hybrids containing different parts of this chromosome and by in situ hybridization. The gamma crystallin genes map to the same chromosomal region as IDH-1. Similar linkage exists between the loci Len-1 and Idh-1 on mouse chromosome 1. PMID- 3011645 TI - Beta-globin gene polymorphism in the Saudi Arab population. AB - DNA mapping with the restriction endonucleases, Hpa I and Mst II, has been used to investigate beta-globin gene polymorphism in the Saudi Arab population. Using Hpa I digestion, 13.0 kb and 7.6 kb fragments were found in association with the beta A and beta S genes. The frequency of the polymorphic forms in two regions investigated vary significantly. In Al-Hafouf and the surrounding villages, situated in the Eastern Province of Saudi Arabia, the frequencies of association of the beta A gene with the Hpa I 7.6 kb, 7.0 kb, and 13.0 kb fragments were 0.866, 0.043, and 0.071, respectively. The frequency of association of beta S with the 7.6 kb and 13.0 kb fragments resulting from the Hpa I digestion were 0.875 and 0.125. In Khaiber, Tehamat-Aseer, and surrounding villages, in the Western Province, the frequency of association of beta A with 7.6 kb, 7.0 kb, and 13.0 kb fragments were 0.836, 0.027, and 0.0136, respectively, while that of beta S was 0.250 and 0.750 with 7.0 kb and 13.0 kb Hpa I fragments, respectively. Using Mst II digestion, beta A was found to be linked to a 1.15 kb fragment, while beta S was linked to a 1.35 kb fragment. The normal (Hb AA), heterozygotes (Hb AS), and homozygotes (Hb SS) gave 1.15, 1.15/1.35, and 1.35 kb fragments, respectively. The results of this study show extensive polymorphism at the Hpa I restriction site of the beta A and beta S globin genes with the different polymorphic forms existing at a variable frequency in different regions of Saudi Arabia. PMID- 3011644 TI - Characteristics and distribution of beta thalassemia haplotypes in South China. AB - Eleven restriction site polymorphisms in the beta-globin gene cluster were determined in 48 Chinese with homozygous beta-thalassemia and their parents. Seven haplotypes were identified as associated with the beta thal chromosome and 25 with the beta A chromosome. The distribution of the various beta thal haplotypes in different regions of South China was mapped and discussed in relation to prenatal diagnosis and migration of the Chinese people. PMID- 3011647 TI - The pro alpha 2(V) collagen gene (COL5A2) maps to 2q14----2q32, syntenic to the pro alpha 1 (III) collagen locus (COL3A1). AB - A recombinant probe specific for the pro alpha 2 chain of human Type V collagen has been used for the localization of the corresponding gene (COL5A2) to chromosome 2. Regional mapping by in situ hybridization and analysis of DNA from human X rodent cell lines indicated that COL5A2 is confined within the segment 2q14----2q32, thus syntenic to the pro alpha 1 (III) collagen gene (COL3A1). PMID- 3011646 TI - Heterozygosity for phosphodiester glycosidase deficiency: a novel human mutation of lysosomal enzyme processing. AB - We have carried out studies on the fibroblasts of III-3, a clinically normal Lebanese individual previously reported to have abnormally high plasma lysosomal enzyme levels. Mannose-6-phosphate (man-6-P) receptors in III-3 fibroblasts were found to be functioning normally, but the cells had only half normal levels of phosphodiester glycosidase activity. Pinocytosis of III-3 fibroblast secreted beta-hexosaminidase B (hex B) into Sandhoff disease fibroblasts was 18% of control, and the apparent KD for binding of III-3 hex B to man-6-P receptors was 3.7 X 10(-9) M compared to 1.25 X 10(-9) M for control enzyme. Hex B secreted by III-3 fibroblasts included an enzyme pool less electro-negative than control enzyme which had a very low affinity for man-6-P receptors and which did not bind to DEAE-Sephadex. Treatment of this abnormal hex B with exogenous placental phosphodiester glycosidase increased its binding to man-6-P receptors three-fold. Secretion rates of seven lysosomal enzymes from III-3 fibroblasts were, on average, twice as great as rates measured for two I-cell disease heterozygote fibroblast lines. The results suggest that III-3 fibroblasts are heterozygous for phosphodiester glycosidase deficiency. The possibility that an individual homozygous for this enzyme deficiency would develop I-cell disease is discussed. PMID- 3011642 TI - Diagnosis of genetic disease using recombinant DNA. AB - Recombinant DNA technology promises to make an important contribution to the analysis and diagnosis of inherited human disease. Direct detection and analysis of various genetic defects at the DNA level are now possible using cloned gene or oligonucleotide probes. In addition, the use of restriction fragment length polymorphisms associated with linked DNA segments should permit not only the diagnosis of hitherto undetectable disease states but also the chromosomal localization of the loci responsible. The eventual isolation of disease loci should lead to a better understanding of the molecular basis of inherited disease. PMID- 3011648 TI - The genes for human gastrin and cholecystokinin are located on different chromosomes. AB - The polypeptide hormones gastrin and cholecystokinin are structurally related, having the identical pentapeptide GWMDF located at their C-terminus. The precursors to these two hormones also show amino acid homology, suggesting that they may have a common ancestral origin. Recombinant DNA clones corresponding to gene fragments encoding human gastrin and cholecystokinin were used to determine their respective chromosomal localization by analyzing human-rodent cell lines. We have assigned the cholecystokinin gene to human chromosome 3q12-3pter and the gastrin gene to chromosome 17q. PMID- 3011649 TI - Length polymorphism in the pro alpha 2(I) collagen gene: an alternative explanation in a case of Marfan syndrome. AB - A 38 base pair (bp) insertion in the pro alpha 2(I) collagen gene (COL1A2) of a patient with Marfan syndrome has been proposed to be the possible cause of the disease (Henke et al. 1985). However, analysis of this insertion in DNA from the patient in question and from random normal individuals reveals it to be a common polymorphism. We suggest that the 38 bp insertion is not related to the primary defect in this case of Marfan syndrome. PMID- 3011650 TI - Correlation between alimentary mycotoxin contamination and specific diseases. PMID- 3011651 TI - The functional inhibition of activated C1 inhibitor in normal human serum causes spontaneous consumption of the complement components C2, C3, C4, and factor B. AB - The human complement components C1r, C1s, C4, C3, factor B, and/or activated C1INH were functionally blocked in normal human serum (NHS) and EGTA- or EDTA treated NHS by polyclonal monospecific Fab'-fragments to the individual components. The results of inhibition experiments are compatible with the formation of a classical pathway fluid-phase C3 convertase (C4b2a) spontaneously generated by the inhibition of activated C1INH. This process in both NHS and EGTA NHS was accompanied by the consumption of C2, C4, C3, and factor B but only by poor enhancement of C5 conversion. Blocking subcomponent C1r, completely inhibited spontaneous activation of the complement components, indicating that the control of C1r hydrolysis is the essential role of activated C1INH as a regulator of C1 activation in NHS. Non-complement serum proteases were inactive during the initiation of the activation process. The presence of blood cells during functional inhibition of activated C1INH in NHS slightly decreased the consumption of C3 but not of C2 and C4. PMID- 3011652 TI - Comparative evaluation of the effect of pharmacological agents on endocytosis and coendocytosis of IgE by rat basophilic leukaemia cells. AB - The aggregation of IgE bound to rat basophilic leukaemia (RBL) cells leads to the exocytosis of mediators, the endocytosis of the antigen-aggregated mouse IgE anti DNP, as well as the coendocytosis of some unaggregated monomeric rat IgE (IR162) and/or unbound receptors. We describe here the relative effect on endocytosis and coendocytosis of various pharmacological agents that block or enhance exocytosis. We have previously shown that, unlike exocytosis, endocytosis by RBL and normal rat mast cells was independent of extracellular calcium. We show here that the presence of calcium chelators or antagonists also had no effect on endocytosis of cross-linked IgE. However, coendocytosis of non-cross-linked IgE was partially inhibited by the elimination of extracellular calcium and the addition of calcium chelators such as EDTA or EGTA-Mg2+. Moreover, the addition of calcium antagonists such as Ni2+ and Co2+ (5 mM) to an incubation mixture containing Ca2+ (1 mM) resulted in the complete inhibition of coendocytosis without affecting endocytosis. Other inhibitors of exocytosis such as sodium azide (10-2M), quercetin (10-4M) and dibutyryl cyclic AMP (10-2 M) blocked coendocytosis completely but had no effect on endocytosis. Sodium azide (10 mM) in combination with 2-deoxyglucose (10 mM) effectively inhibited (90%) endocytosis. Cytochalasin B (10-4 M), which was shown to enhance serotonin release, had no effect on the extent of endocytosis or coendocytosis observed 20 min after the initiation of aggregation. Thus, in RBL cells, endocytosis, coendocytosis and exocytosis exhibit distinguishable sensitivities to some pharmacological drugs. PMID- 3011653 TI - Production of human monoclonal IgG and IgM antibodies with anti-D (rhesus) specificity using heterohybridomas. AB - Heterohybridomas secreting human IgM and IgG anti-D antibodies of the rhesus blood group system have been established by fusion of EBV-transformed anti-D secreting cells with the mouse myeloma cells X63-Ag8.653. Both classes of antibody reacted with all Rh-positive cells, some Du cells but not with Rh negative or DB cells. Concentrations of both antibodies reached between 25 micrograms/ml and 50 micrograms/ml in the culture supernatants. The cell lines have been maintained in culture for 14 months and have been shown to be suitable for large-scale production of antibody. PMID- 3011654 TI - Hydroxyl radical scavengers inhibit human lectin-dependent cellular cytotoxicity. AB - The role of oxygen-derived free radicals (ODFR) in lectin-dependent cellular cytotoxicity (LDCC) in humans was investigated. The hydroxyl radical traps thiourea, methanol, ethanol and phenol were effective in inhibiting LDCC, as was DABCO, a singlet oxygen quencher. The proposed pathway of hydroxyl radical production in living cells is either an iron catalysed Haber-Weiss reaction or a Fenton reaction. The effect of inhibitors of these pathways was investigated. The superoxide anion scavengers superoxide dismutase, ferricytochrome c and Tiron were without effect. It was shown that Tiron inhibits the lucigenin-amplified chemiluminescence produced by the action of xanthine oxidase, and also the lucigenin-amplified chemiluminescence produced by activated PMN, suggesting that this agent (Tiron) scavenges intracellular superoxide anion. Catalase gave slight inhibition of LDCC only. The ferric iron chelator desferrioxamine gave no protection of the target cells, while the ferrous chelator, 1,10-phenanthroline, inhibited LDCC and partially prevented the detection of hydroxyl radicals generated by the Fe2+-H2O2 system. Cibacron blue, an agent that inhibits NAD(P)H linked enzymes, also inhibited LDCC. The cyclo-oxygenase inhibitors indomethacin and salicylate were without effect, while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) inhibited cytolysis. None of the LDCC inhibitors was cytotoxic to the effector cells or to the target cells, neither did they inhibit lymphocyte-target binding. The findings would suggest that hydroxyl radicals have a role to play in human T-cell mediated cytolysis, either as the active lytic agent or as an epiphenomenon. PMID- 3011655 TI - Interaction between herpes simplex type 1-induced Fc receptor and human and rabbit immunoglobulin G (IgG) domains. AB - Cells infected with herpes simplex virus type 1 (HSV-1) express a cell surface receptor able to bind to the Fc region of immunoglobulin G (IgG). The ability of HSV-1-infected cells to bind 125I-labelled human and rabbit IgG and IgG fragments was studied to localize the site of interaction to the C gamma 2 or C gamma 3 domains of IgG. 125I-labelled IgG and IgG Fc fragments consisting of C gamma 2 and C gamma 3 domains bound strongly to HSV-infected cells and did not bind to uninfected cells. In contrast, 125I-labelled F(ab')2, Facb [consisting of F(ab')2 and C gamma 2 domains] and pFc' (consisting of C gamma 3 domains) fragments did not bind to any of these cells. Unlabelled IgG and IgG Fc fragments inhibited the interaction between 125I-labelled rabbit IgG Fc and the HSV Fc receptor, whereas F(ab')2, Facb and pFc' fragments failed to inhibit this interaction. These data indicate that the HSV Fc receptor requires both the C gamma 2 and C gamma 3 domains for interaction with the IgG molecule analogous to the known interaction of protein A of Staphylococcus aureus, the Fc binding proteins of Group A, C and G streptococci, and certain human rheumatoid factors. PMID- 3011657 TI - [Histologic considerations on fibrous papules of the nose]. PMID- 3011656 TI - Cell surface receptors for sulphated polysaccharides: a potential marker for macrophage subsets. AB - The expression of a diverse array of receptors for sulphated polysaccharides on lymphocytes has been demonstrated by Parish & Snowden (1985). This paper presents evidence to suggest that other cell types, namely macrophages, polymorphonuclear leucocytes, mast cells and fibroblasts, can bind similar polysaccharides. Using a rosetting assay and eleven structurally unique polysaccharides, each cell type was observed to bind a characteristic array of these polysaccharides. Analysis of the polysaccharide reactivity of macrophages revealed that BCG-activated and thioglycollate-elicited macrophages express an expanded repertoire of reactivity compared to resident peritoneal macrophages. For example, only thioglycollate elicited macrophages, but not resident and BCG-activated peritoneal macrophages, reacted with the glycosaminoglycans, chondroitin-4-sulphate, chondroitin-6 sulphate and dermatan sulphate, while both BCG- and thioglycolate-activated, but not resident peritoneal macrophages, bound pentosan polysulphate-coupled sheep erythrocytes. The expression of the receptors for chondroitin-4 and -6-sulphate was observed to be cyclic and peaked at 2 and 5-6 days after thioglycollate treatment. Preliminary analyses of the functional significance of the observed binding of polysaccharides to macrophages revealed that heparin, fucoidan and kappa-carrageenen were specifically endocytosed. However, endocytosis of all other test polysaccharides was not observed. Finally, polysaccharide-coupled sheep erythrocytes were not phagocytosed, even though they interacted strongly with the macrophage surface. The possible relevance of these observations to an inflammatory response and as a means of identifying cellular subsets is discussed. PMID- 3011658 TI - [Evaluation of the risk of AIDS in homo- and heterosexual males at the Milan Antivenereal Center]. PMID- 3011659 TI - Dissection of HLA class II haplotypes in HLA-DR4 homozygous individuals. AB - In order to identify better markers for HLA-DR4-associated autoimmune disorders, we have studied the complexity of the HLA class II region in DR4-positive cells at the DNA level and compared the DNA polymorphism with that defined by serology, mixed lymphocyte culture (MLC) reactivity, and protein chemistry. At the DNA level, HLA-DR4 can be characterized by a homogeneous pattern of bands hybridizing to HLA class II cDNA probes. Besides, subtypes can be defined within DR4 using HLA-DR beta, -DQ alpha, and -DQ beta cDNA probes in Southern blot analysis. Three subtypes are found using the DR beta cDNA probe. One of these subtypes correlates with the cellularly defined Dw15 specificity, another with the serologically defined LB4 and LB14 specificities. None of the restriction fragment length polymorphism (RFLP) patterns coincide with the MLC-defined DR4 subtypes Dw4, Dw10, Dw13, and Dw14 separately. Variation of two fragments hybridizing to the DQ alpha cDNA probe obtained after either Pvu II or Taq I digestion yields three subtypes. Pvu II- and Eco RI-digested DR4 DNA give rise to three DQ beta detectable subtypes. Correlation between these subtypes, isoelectric point variation of DQ molecules, and the DQ-related allelic system TA10/2B3 are demonstrated. Some of the patterns obtained with DQ alpha and DQ beta cDNA probes display heterozygosity in the DQ region, as demonstrated by family segregation. No correlation was observed between DQ and the cellularly defined Dw determinants. A new polymorphism has been obtained with the DQ alpha probe, probably due to DX polymorphism. DR beta RFLP divides the LB14 supertypic specificity into two new subtypes. A combination of the four different techniques applied to a panel of 16 DR4 homozygous cell lines reveals at least nine different haplotypes in DR4. These newly defined haplotypes may be of help in further studies concerning the relationship of micropolymorphism with several diseases. PMID- 3011661 TI - A mathematical insight to poliovaccine failures. PMID- 3011662 TI - Effect of incubation temperatures on the replication of herpes simplex virus in chicken embryos--a virological and histopathological study. PMID- 3011660 TI - Melanotic prognoma--report of a case and literature review. PMID- 3011663 TI - Osteoclast-like giant cells in breast carcinoma. PMID- 3011664 TI - Specific measurement of angiotensin metabolites and in vitro generated angiotensin II in plasma. AB - Combining high-performance liquid chromatography with radioimmunoassay enabled the precise measurement of different angiotensins and their metabolites in plasma. Peptides were extracted from 2 ml of plasma by reversible adsorption to phenylsilyl-silica, separated by isocratic high-performance liquid chromatography, and quantitated by radioimmunoassay using a sensitive but suitably cross-reacting angiotensin II antiserum. For the C-terminal angiotensin II metabolites (2-8)heptapeptide, (3-8)hexapeptide, and (4-8)pentapeptide, overall recoveries of 10 fmol peptide added to 1 ml of plasma were (mean +/- SD), 74 +/- 6, 68 +/- 8, and 67 +/- 11%, respectively. The detection limit for these peptides in plasma was 0.2 fmol/ml. Blanks were below the detection limits. In eight seated normal subjects treated for 4 days with enalapril, 20 mg p.o., q.d., angiotensin II metabolites tended to decrease during the 4 postdrug hours. However, their cumulated concentration in relation to octapeptide increased from 54 to 163% on Day 1 and from 62 to 103% on Day 4. After 4 hours of converting enzyme inhibition with enalapril there was still a close correlation between plasma renin activity and angiotensin-(1-8)octapeptide level (r = 0.83, p less than 0.05) and between blood angiotensin I and angiotensin-(1-8)octapeptide levels (r = 0.86, p less than 0.01). Adding angiotensin I in vitro raised the angiotensin-(1-8)octapeptide levels after incubation at 4 degrees C for 4 hours. Thus, immunoreactive "angiotensin II" does not disappear after converting enzyme inhibition largely because of the cumulated contribution of cross-reacting metabolites and partly because of in vitro generation of true angiotensin II. PMID- 3011665 TI - Effects of calcium infusion on blood pressure in hypertensive and normotensive humans. AB - Disorders of calcium and parathyroid hormone homeostasis have been reported in subjects with essential hypertension. In many of these studies, dietary intakes of sodium and calcium were not carefully controlled. The present study was designed to compare calcium and parathyroid hormone homeostasis in normal and hypertensive subjects on controlled dietary sodium and calcium intakes and to examine the impact of dietary sodium loading on hemodynamic and metabolic responses to infused calcium. Seven subjects with essential hypertension and seven age-matched and sex-matched controls were studied while consuming a standard diet containing 600 mg of elemental calcium. Each subject was studied while consuming 10, 160, and 510 mEq of sodium per day, before, during, and after a 3-hour calcium infusion (3.75 mg/kg/hr). Before calcium infusion, hypertensive subjects had increased urinary cyclic adenosine 3',5'-monophosphate excretion independent of sodium intake (p less than 0.05). Urinary potassium excretion was greater in normotensive than in hypertensive subjects (p = 0.002). At baseline, dietary sodium intake had no effect on systolic, diastolic, or mean arterial pressure. During calcium infusion, systolic pressure increased in both groups, whereas diastolic pressure increased only when dietary sodium content was high and mean arterial pressure increased only in hypertensive subjects (p = 0.007). Together, these data provide evidence for interactions between dietary sodium intake and the cardiovascular response to calcium. They confirm that hypertensive subjects exhibit enhanced parathyroid gland function even when dietary factors are controlled, and they suggest that these subjects are more sensitive to the cardiovascular effects of short-term calcium infusion. PMID- 3011667 TI - Characteristics of aggregated immunoglobulin G as an immunologic phagocytic stimulus for granule enzyme release from human neutrophils. AB - Aggregated immunoglobulin G (AggIgG) induced a time- and concentration-dependent phagocytic release of granule-associated lysozyme and myeloperoxidase (MPO) from human neutrophils. Degranulation was significantly enhanced in the presence of calcium or magnesium, and maximum granule exocytosis was observed when both divalent cations were present. AggIgG-stimulated enzyme release was inhibited with the intracellular calcium antagonist, TMB-8[8-(N,N-diethylamino)-octyl (3,4,5-trimethoxy)benzoate] in the absence of extracellular calcium. DIDS (4,4' diisothiocyano-2,2'-disulfonic acid stilbene), a permeant anion channel blocker, also suppressed AggIgG-induced degranulation. Cycloheximide, an inhibitor of protein synthesis, enhanced granule exocytosis from AggIgG-treated neutrophils. Two inhibitors of transmethylation reactions, 3-deazaadenosine (3-DZA) and homocysteine thiolactone (HCTL) in combination, suppressed AggIgG-elicited granule enzyme release. These data indicate that AggIgG is a useful probe for investigating the requirements for phagocytic enzyme release from human neutrophils. PMID- 3011666 TI - Oxygen radical production by neutrophils from patients with bacterial infection and rheumatoid arthritis. Measurement of hydrogen peroxide may most accurately represent enhancement of oxygen radical production during infection. AB - The production of three kinds of oxygen radicals (superoxide, hydrogen peroxide, and hydroxyl radicals) by neutrophils from patients with bacterial infection or rheumatoid arthritis was measured. The stimulators used in this study were opsonized zymosan (1 mg/ml), phorbol myristate acetate (20 ng/ml), A23187 (1 microM), and platelet activating factor (1 microM). Oxygen radical production by neutrophils from patients with rheumatoid arthritis was not significantly different from that of the control group. Hydrogen peroxide production by the neutrophils from patients with bacterial infection was significantly enhanced by only opsonized zymosan, but the production of the other kinds of oxygen radicals was not. Cytochalasin B reduced the production of hydrogen peroxide induced by opsonized zymosan more markedly than that of any other kind of oxygen radical. The measurement of hydrogen peroxide is suggested to be the most accurate indicator of the enhancement of intracellular production of oxygen radicals by neutrophils during infection. PMID- 3011668 TI - Calcium ionophore and chemotactic peptide stimulation of peptidoleukotriene synthesis in DMSO-differentiated HL60 cells. AB - The human promyelocytic leukemia cell line HL60 can be differentiated to mature granulocytes upon exposure to DMSO (1.3%, 6 days). The ability of these cells to metabolize arachidonic acid via the 5-lipoxygenase pathway to form 5-HETE, LTB4, and 5,12-diHETEs, has been previously documented. However, the production of peptidoleukotrienes by DMSO-differentiated HL60 cells has not been previously reported. Arachidonic acid metabolites produced via 5-lipoxygenase were identified by reverse-phase, high-performance liquid chromatography, immunoreactivity specific for peptidoleukotriene, glutamyl transpeptidase transformation, characteristic UV spectra, and GC mass spectra. Leukotriene synthesis in the DMSO-differentiated HL60 cell is maximal at 5 min when stimulated with the calcium ioniphore, A23187 (1 microM), in the presence of calcium. These cells produce 12.94 +/- 1.8 ng/10(6) cells of LTC4 and 3.8 +/- 0.4 ng/10(6) cells of LTB4. LTC4 and LTB4 are also synthesized in the undifferentiated cell when stimulated with 1 microM A23187 and 1 mM Ca2+, but in much smaller quantities, i.e., 1.91 +/- 0.42 ng/10(6) cells of LTC4 and 0.41 ng +/- 0.06/10(6) cells of LTB4. The synthetic chemotactic peptide, f-Met-Leu-Phe, also elicits formation of LTC4 and LTB4 in a dose-dependent manner in the presence of exogenously added calcium. Maximal stimulation of DMSO-differentiated cells with f-Met-Leu-Phe produces 2.5 +/- 0.2 ng of LTC4 and 1.45 +/- 0.2 ng of LTB4 per 10(6) cells. The observation that DMSO-differentiated HL60 cells produce LTC4, as well as other 5-lipoxygenase products, increases the utility of this cell line for unraveling the regulation of leukotriene biosynthesis by granulocytes. PMID- 3011669 TI - Arachidonic acid metabolism in bovine alveolar macrophages. Effect of calcium ionophore on lipoxygenase products. AB - The production of lipoxygenase metabolites of arachidonic acid was studied in bovine alveolar macrophages (BAM). Unstimulated macrophages produced small amounts of LTB4 (0.2 +/- 0.2 ng/10(6) BAM) but monohydroxyeicosatetraenoic acids (5-, 12-, and 15-HETE) usually were not detectable. Both exogenous arachidonic acid and the calcium ionophore A23187 induced production of LTB4, 5-, 12-, and 15 HETE, of which 60-80% was 5-HETE. Combined challenge of BAM with both exogenous arachidonic acid and A23187 was more effective in the production of these metabolites than with either stimulus alone. The generation of the peptidoleukotrienes LTC4, LTD4, and LTE4 by BAM could not be detected under these in vitro conditions. Our results demonstrate that bovine alveolar macrophages produce similar lipoxygenase metabolites of arachidonic acid in response to A23187, as do human alveolar macrophages stimulated with the same agonist. PMID- 3011670 TI - Bee venom melittin blocks neutrophil O2- production. AB - Bee venom (BV) is used in folk medicine to treat arthritis. It has antiinflammatory effects in animal models of rheumatic disease. We have studied the effects of BV on human neutrophil production of superoxide (O2-) and hydrogen peroxide, finding potent, nontoxic, dose-dependent production inhibition. Melittin, the major fraction of BV (50-70%) shows high-affinity calmodulin binding (Kd 3 nM). Drugs which bind calmodulin, such as trifluoperazine, inhibit O2- production by human neutrophils. For these reasons we have investigated the effect of melittin and other BV peptides on O2- production by human peripheral blood leukocytes. We show that melittin inhibited O2- production both pre- and poststimulation in contrast to other BV fractions which were without effect. Oxygen radicals and their derivatives from inflammatory cells are implicated in the tissue damage occurring during inflammation. The inhibition is due to a direct effect on cells, and not indicator medium, dismutation, toxic or scavenging effects. We propose that melittin may serve as a prototype small (mol wt 1280), cationic, amphipathic, calmodulin-binding, membrane-active, superoxide production-inhibiting peptide, providing a model for peptides which could have a role in in vivo regulation of radical production. PMID- 3011671 TI - Effect of sublethal gamma radiation on host defenses in experimental scrub typhus. AB - The effect of sublethal gamma radiation on inbred mice chronically infected with scrub typhus rickettsiae was examined. Inbred mice which were inoculated with the Gilliam or Karp strain of Rickettsia tsutsugamushi by the subcutaneous route harbored the infection for at least 1 year. Irradiation of these animals at 12 or 52 weeks postinoculation with normally sublethal levels induced a significantly higher percentage of rickettsemic mice (recrudescence) than was seen in the unirradiated, similarly infected control animals. In addition, sublethal irradiation at 12 weeks induced a quantitative increase in total rickettsiae. Homologous antibody titers to the rickettsiae were examined for 5 weeks after irradiation to determine the role of the humoral response in radiation-induced recrudescence. Unirradiated, infected mice showed consistent titers of about 320 throughout the 5-week observation period, and the titer was not affected by exposure of up to 500 rads of gamma radiation. Drug dose-dependent radioprotection and modification of recrudescence was noted in infected, irradiated mice treated with the antiradiation compound S-2-(3 aminopropylamino)ethyl phosphorothioic acid. The results of this investigation supported the conclusion that the recrudescence of a chronic rickettsial infection in the appropriate host after immunological impairment due to gamma radiation can result in an acute, possibly lethal rickettsemia. PMID- 3011672 TI - Effect of polymyxin B and colimycin on induction of plasminogen antiactivator by lipopolysaccharide in human endothelial cell culture. AB - The effect of lipopolysaccharide (LPS) on the production of fibrinolytic inhibitor by human endothelial cells was determined because results of previous experiments have shown us that it is possible to stimulate this synthesis with muramyl dipeptide. Treatment of these cells with LPS resulted in a marked enhancement of fibrinolytic inhibitor, as estimated in a urokinase-induced fibrinolysis assay. A dose-response curve was obtained for LPS concentrations ranging from 10 to 1,000 ng/ml, thus demonstrating the great sensitivity of these cells. This inhibitor did not reduce plasmin activity and formed complexes with high- and low-molecular-weight urokinase as visualized by fibrin enzymography on sodium dodecyl sulfate-polyacrylamide electrophoretic gels. The molecular weight of this inhibitor was estimated to be 54 to 58 kilodaltons. These findings led us to conclude that LPS stimulates formation of a plasminogen antiactivator. This LPS effect could be suppressed by polymyxin B and colimycin. The stimulatory effect of muramyl dipeptide required doses which were at least 1,000 times greater than those of LPS and was not decreased by polymyxin B. These results show the possibility of independent modulation of plasminogen antiactivator production at the endothelial level, which could be important in endotoxemia. Under these conditions colimycin might have an additional advantage for clinical use because of its ability to prevent fibrinolytic inhibition. PMID- 3011673 TI - Enhanced resistance against Listeria monocytogenes at an early phase of primary infection in pregnant mice: activation of macrophages during pregnancy. AB - We investigated the pregnancy-induced changes in macrophage activity which are important in the expression of host defense against infections. Several macrophage functions were examined by using Listeria monocytogenes. In pregnant mice, prolonged survival and enhanced in vivo elimination of bacteria were observed in the early phase of primary infection. Functions of peritoneal macrophages, including in vitro phagocytosis intracellular killing, glucose consumption, generation of superoxide anion, and intracellular beta-glucuronidase activity were shown to be enhanced in pregnant mice. These findings indicate that pregnancy enhances macrophage functions qualitatively. Possible mechanisms for this enhancement and the significance of macrophage activation for pregnant hosts are discussed. PMID- 3011674 TI - Differential contribution of chemotaxins and opsonins to neutrophil-mediated killing of Schistosoma mansoni larvae. AB - In neutrophil-mediated killing of schistosomula, antibody and serum complement serve two functions: (i) opsonization of the target pathogen and (ii) generation of cell-activating, chemotactic peptides. We studied neutrophil-mediated schistosomulum killing in experiments designed to isolate these functions of antibody and complement in the cell activation response of neutrophils to external pathogens. Schistosomula opsonized by antibody or antibody plus complement induced neutrophil-mediated killing, while complement-opsonized larvae did not. Antibody enhancement of cell-mediated killing correlated with increased cell-to-parasite adherence, while cell-mediated killing in the presence of fresh normal human serum (providing chemotactic factor stimulation and complement opsonization of larvae) demonstrated a dissociation between neutrophil adhesive and killing responses. Exogenous chemotactic stimuli were found to enhance significantly neutrophil-mediated killing of both opsonized and unopsonized larvae. Experiments involving parabiotic chambers confirmed that chemotactic factor-stimulated neutrophils may cause significant parasite mortality without direct cell-parasite contact. In further analysis of the neutrophil response to surface and fluid-phase stimulation, release of neutrophil cytotoxic mediators was found to vary according to the type of stimulus provided: cell exposure to either larvae opsonized by antibody plus complement or to chemotactic factors provoked low-to-moderate cell release of superoxide anion, granule enzymes, and arginase; when opsonic and chemotactic stimuli were combined, a greater-than additive secretory response was noted. Under such maximal stimulation, oxidative and nonoxidative mediators were synergistic in effecting neutrophil-mediated parasite killing. The primary function of complement in cell-mediated parasite killing appears to be to promote chemotactic-factor-mediated cellular release of toxic agents and not cell-target linkage, whereas antibody-mediated adherence is associated with concurrent cellular activation. Both neutrophils adherent to the antibody-opsonized schistosomulum and chemotactic-factor-stimulated cells (adherent and not adherent to the parasite) appear to contribute significantly to its demise. PMID- 3011675 TI - A preclinical evaluation of aminopyridines as putative therapeutic agents in the treatment of botulism. AB - 4-Aminopyridine and 3,4-diaminopyridine were evaluated for their abilities to delay the onset of paralysis due to botulinum neurotoxin types A, B, and E. Experiments were done on phrenic nerve-hemidiaphragm preparations excised from mice. At a concentration that produced an enhancement in muscle twitch amplitude, 4-aminopyridine and 3,4-diaminopyridine delayed the onset of paralysis due to botulinum toxin type A. Under the same conditions, the drugs did little to protect tissues against botulinum toxin types B and E. 3,4-Diaminopyridine was also evaluated for its ability to reverse the paralysis due to botulinum toxin. Experiments were done on rat phrenic nerve-hemidiaphragm preparations that had previously been poisoned in vivo. The drug produced transient increases in neuromuscular transmission, with the effect being greater for botulinum neurotoxin type A than for botulinum neurotoxin types B and E. Equivalent types of experiments were done with tetanus toxin. The results with 3,4-diaminopyridine showed that tetanus toxin resembled botulinum toxin types B and E. The data help to clarify the role of aminopyridines as therapeutic agents in the treatment of botulism. They also provide insights into the mechanism of action of clostridial neurotoxins. PMID- 3011677 TI - Western blot technique in the serological evaluation of three LAV/HTLV III infected Italian families. AB - In order to confirm suspected LAV/HTLV III infection, serological evaluation of patients is of utmost importance. ELISA is currently being employed on a large scale for screening, but like the immunofluorescence assay, it has a variable rate of possible non-specific positivity. On the other hand, the Western Blot (WB) technique can detect antibodies to different viral proteins. In this paper we are reporting the serological patterns of three LAV/HTLV III-infected families. In particular, their viral protein-specific antibody patterns are described. With the exception of one child, all the patients tested showed seropositivity in both ELISA and WB. In the one child mentioned above, ELISA and immunofluorescence positivity were due to non-specific binding. Two out of three children tested showed a close correlation between a severe clinical course and the absence of p25-specific IgM. In contrast, one child showing a switch from IgM to p25-specific IgG antibodies had a favorable clinical course. We observed a family in which vertical transmission of LAV/HTLV III from the mother to her neonate seems not to have happened; the child was seronegative and healthy at the age of one. At birth, this neonate had LAV/HTLV III-specific IgG corresponding to the mother's pattern, but it lacked viral-specific IgM. Its mother had transmitted the viral infection to her first child, who died of AIDS. Preliminary suggestions are made about the detection of different specific antibodies and clinical features; the utility of WB is emphasized. PMID- 3011678 TI - Comparison of the binding of gram-negative bacterial endotoxin by polymyxin B sulphate, colistin sulphate and colistin sulphomethate sodium. AB - Polymyxins are cyclic polypeptide antibiotics. In addition to their bactericidal activity they bind lipid A and neutralize the biological effects of bacterial endotoxin. We have studied the three available polymyxin preparations: polymyxin B sulphate (PB), colistin sulphate (CS) and colistin sulphomethate sodium (CMS), and compared their endotoxin binding capacity at equivalent therapeutic dosage. Each polymyxin was bound to a column of Sepharose 4B and challenged with 5 micrograms of endotoxin from Escherichia coli O127:B8. Recovery of endotoxin in the eluate was measured by a quantitative Limulus lysate microassay. PB and CS bound 94% of the challenge dose, CMS 89% and the control column (Sepharose alone) 24%. These results suggest that parenteral CMS (the least toxic polymyxin) retains useful anti-endotoxin capacity, and that in neutropenic patients, oral polymyxin may exert both anti-endotoxin and antimicrobial effects. PMID- 3011676 TI - Low endotoxic activities of synthetic Salmonella-type lipid A with an additional acyloxyacyl group on the 2-amino group of beta (1-6) glucosamine disaccharide 1,4'-bisphosphate. AB - A synthetic lipid A (Salmonella type, compound 516), beta (1-6)-linked D glucosamine disaccharide 1,4'-bisphosphate, with three acyloxyacyl groups and one hydroxyacyl group, i.e., (R)-3-hexadecanoyloxytetradecanoyl, (R)-3 hydroxytetradecanoyl, (R)-3-dodecanoyloxytetradecanoyl, and (R)-3 tetradecanoyloxytetradecanoyl groups at the 2-amino, 3-hydroxyl, 2'-amino, and 3' hydroxyl groups, respectively, was less biologically active than the synthetic Escherichia coli-type lipid A (compound 506), which has only two acyloxyacyl groups at the 2' and 3' positions and is substituted with a (R)-3 hydroxytetradecanoyl group at the 2-amino group. Compound 516 exhibited considerably weaker pyrogenic and leukopenic activity than compound 506, and it scarcely prepared rabbit skin for the Shwartzman reaction and lacked lethal toxicity on chicken embryos, although its lethal toxicity in galactosamine-loaded mice was as strong as that of compound 506. Compound 516 was also less active than compound 506 or natural E. coli lipid A (from Restrain F515) in other biological test systems, such as the Limulus test, stimulation of macrophages and lymphocytes, and interferon-inducing activity but not for interleukin-1 induction or complement activation. This observation suggests that there is an optimal number of acyloxyacyl groups on the glucosamine backbone for producing the biological activities of lipid A, especially the endotoxic activities. The 4' monophosphate analog (compound 514) of compound 516 in general had significantly weaker activity than compound 516 in the above assays, most probably because of its greater hydrophobicity and consequently lower solubility in assay systems. Bacterial R595 lipid A derived from S. minnesota Re-mutant, which is a mixture of compounds 516 and 506, their 4'-monophosphate analogs and other compounds, exerted intermediate degrees of activity between compounds 506 and 516 in the various test systems employed. PMID- 3011679 TI - Current views on the role of opioid receptors and endorphins in anesthesiology. PMID- 3011680 TI - Evaluation of the mechanisms involved in sodium diethyl dithiocarbamate-induced immunomodulation using the hydrophilic analog, sodium N-methyl-D-glucamine dithiocarbamate. AB - Sodium diethyl dithiocarbamate (DE-DTC) is a lipophilic low molecular weight sulphur compound previously demonstrated to be a potent immunomodulator but cytotoxic in vitro. In this work, we studied the effects on a hydrophilic analog of DE-DTC, sodium N-Methyl-D-glucamine dithiocarbamate (NMG-DTC) on immune responses to a hapten carrier conjugate and on mitogen-induced lymphoproliferation. NMG-DTC, in contrast to DE-DTC, did not modify the responses to a hapten-carrier conjugate. The immunomodulatory activity of DE-DTC appeared to be linked with its lipophilicity. NMG-DTC had a slight inhibitory effect on thymidine incorporation by lymphocytes stimulated by mitogens as compared to that of DE-DTC. DE-DTC was cytotoxic possibly at the cell membrane level; cytotoxicity was not related to the chelating properties of DE-DTC in the culture medium. On the other hand, NMG-DTC appeared to be a less cytotoxic molecule. Therefore it could be useful to study the effects of the dithiocarbamate moiety at the cellular level. PMID- 3011681 TI - Superoxide anion generation during airway anaphylaxis. AB - Extracellular release of superoxide anion (O-2) during in vitro anaphylaxis in guinea pig trachealis was found to exhibit both a time- and a concentration dependent generation of O-2. Trachealis O-2 release was unaffected by treatment with diphenhydramine HCl (10(-5) M, 10(-6) M). While indomethacin (10(-5) M) augmented anaphylaxis tension it did not enhance O-2 release. However, the semiselective SRS-A antagonist FPL 55712 (10(-5) M) resulted in essentially complete inhibition of O-2 generation. These data indicate that O-2 generation occurring during acute airway anaphylaxis is associated with the activation of SRS-A products. PMID- 3011682 TI - The importance of the Leydig cells in the vascular response to hCG in the rat testis. AB - The increase in permeability of the testicular blood vessels following an injection of hCG into rats is abolished completely if the animals are treated 3 days earlier with ethane dimethane sulphonate (EDS), a compound that effectively eliminates Leydig cells from the testes. As there is other evidence that androgens or prostaglandins are not involved in this vascular response, further studies will be necessary to determine whether these data mean that another vasoactive substance is secreted by the Leydig cells or whether the EDS also eliminates other cells besides the Leydig cells, for example the mast cells found in the vicinity of the testicular artery. PMID- 3011683 TI - Dose and time related responses of the irradiated prepubertal rat testis. I. Leydig cell function. AB - The testes of prepubertal rats were locally irradiated with 300 kVp x-rays to doses of between 1 and 15 Gy. Leydig cell function was assessed between 2 and 36 weeks post-irradiation. Dysfunction was observed at two weeks as evidenced by a reduction in serum levels of testosterone to between 40 and 70% of control, with a threshold dose of about 5 Gy. Endocrine deficiencies in the Leydig cell population were indicated at later times by increased serum levels of LH, although serum testosterone concentrations recovered to control levels. The elevations in serum LH increased with time suggesting progressive Leydig cell failure. PMID- 3011684 TI - New abnormal isoenzyme of 5'-nucleotide phosphodiesterase in the serum of human hepatoma. AB - 5'-nucleotide phosphodiesterase (5'-NP) isoenzymes were separated from the serum of human primary liver cancer (PLC) patients by 4-20% tubular polyacrylamide gel gradient electrophoresis. Electrophoresis showed 11 isoenzymes in which bands I, V and VI (from anode to cathode) were all absent in 125 normal control sera; bands V and VI were detectable in 31(73.8%) of 42 patients with PLC, but absent in 70 cases of other diseases. Bands V and VI may represent specific isoenzyme bands of PLC, comprising a new addition to the markers used in the diagnosis of this disease. By the same method, it was also found that the abnormal serum isoenzyme bands of PLC correspond to those found in the cord blood sera of the newborn and fetus. These abnormal isoenzymes diminish gradually after birth and all disappear when children reach the age of 10. PMID- 3011685 TI - Human papillomavirus DNA sequences in penile carcinomas in Brazil. AB - To detect the presence of human papillomavirus (HPV) DNA in penile cancers, 18 squamous-cell carcinomas were studied by Southern blot analysis. All specimens were from Brazilian patients ranging in age from 31 to 78 years. Of these, 7 harbored DNA sequence homology. Most positive specimens contained more than 100 copies of viral DNA. No HPV 16 was detected in our samples. Presence of HPV DNA sequences in the samples studied was not correlated with the patients' age or race, nor with extent or progress of disease. PMID- 3011686 TI - Inhibitors of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced multinucleated cell formation and HTLV-I p19 antigen expression in HTLV-I-infected T-cell line KH-2Lo. AB - To elucidate the biological mechanisms of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phenotypic changes in HTLV-I virus-infected human T-cell line KH 2Lo cells, inhibitors of TPA-induced ornithine decarboxylase (ODC), protein kinases and calmodulin were examined for their effects on TPA-induced multinucleated cell formation and HTLV-I p19 antigen expression. Among the inhibitors of TPA-induced ODC activity, alpha-difluoromethyl ornithine (DFMO), 1,25(OH)2D3 and its analogues, and retinoic acid were tested. As inhibitors of protein kinases, 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), N-[2 (methylamino)ethyl]-5-isoquinoline-sulfonamide (H-8) and 1,(5 isoquinolynylsulfonyl)-2,3-dimethylpiperazine (H-5) were used. In addition, a calmodulin inhibitor, N-(4-aminobutyl)-5-chlor-2-naphthalenesulfonamide (W13) and its inactive analogue, N-(4-aminobutyl)-2-naphthalenesulfonamide (W12) were also tested. 1,25(OH)2D3 and its active analogues inhibited both TPA-induced HTLV-I p19 antigen expression and multinucleated cell formation after 4 days of culture with TPA. On the other hand, an inhibitor of ODC, DFMO, the protein kinase inhibitors, the calmodulin inhibitor and retinoic acid suppressed TPA-induced HTLV-I p19 expression but did not suppress multinucleated cell formation. PMID- 3011687 TI - The effect of carbaryl on the arachidonic acid metabolism and superoxide production by mouse resident peritoneal macrophages challenged by zymosan. AB - Carbaryl, a broad spectrum insecticide with anticholinesterase activity, was tested for its ability to disturb resident peritoneal macrophages stimulated by opsonized zymosan. The effect of carbaryl on superoxide production and on the release of [1-14C] arachidonic acid and 14C-labelled prostaglandins was dose dependent. For 2.5 X 10(-6) M of carbaryl, superoxide production and prostaglandin release were not significantly inhibited. At 12.5 X 10(-6) M, the inhibitory effect was apparent for superoxide production (33%) and for the release of 6 KPGF1 alpha (60%), PGE2 (42%), PGF2 alpha (38%), PGD2 (33%). Carbaryl had no effect on the level of free arachidonic acid. Insecticide at 12.5 X 10(-6) M significantly decreased the deacylation of the phosphatidylcholine (20%). Incubation of resident peritoneal macrophages with indomethacin studied conjointly decreased only the prostaglandin release. These results suggest that carbaryl decreases the sequence of events following the binding of a particulate agent to its receptor and leading to the induction of phospholipase activity and the subsequent release of 20:4 and the oxidative burst in the cells. The effect of this pesticide on phospholipid metabolism and its consequences on macrophage stimulation are discussed. Ecto-serine esterase inhibition in the effect mechanism of the pesticide was suggested. PMID- 3011688 TI - Evidence against functional adenosine receptors on murine lymphocytes. AB - The effects of adenosine, 2-chloroadenosine (2Cl Ado) and N6 phenylisopropyladenosine (PIA) were examined on peripheral blood and splenic lymphocytes from mice. Lectin-stimulated DNA synthesis was antagonized by adenosine and 2Cl Ado at high concentrations. Lower concentrations of all three nucleosides produced an enhancement of lectin-stimulated thymidine uptake in splenic lymphocytes. Peripheral blood lymphocytes were found to exhibit only inhibitions of mitogenic stimulation suggesting a difference in response to nucleoside exposure between spleen and peripherally circulating cells. The synthesis of antibody to sheep red blood cells was inhibited in a non-cytotoxic manner by 2Cl Ado and PIA while adenosine was without effects. The receptor antagonist 8-phenyltheophylline was found to block nucleoside increases in thymidine uptake and low concentrations of 2Cl Ado with regard to antibody production. The effects of high concentrations of 2Cl Ado or PIA on humoral responses were not antagonized by receptor blockade. The data suggest that functional alterations of lymphocyte responses to nucleoside exposure are not a consequence of surface receptors for adenosine nucleosides in murine cells. PMID- 3011690 TI - Synthesis of pseudo-peptide analogues of the C-terminal tetrapeptide of gastrin and evaluation of their biological activity on acid secretion. AB - The syntheses of pseudo-tetrapeptides Boc-Trp-psi (CH2-NH)-Met-Asp-Phe-NH2 21 and Boc-Trp-Met-psi (CH2-NH)-Asp-Phe-NH2 20, representing the C-terminal tetrapeptide sequence of gastrin, in which amide bonds were replaced by CH2-NH bond, are described, as well as the syntheses of pseudo-peptide analogues Boc-Trp-psi (CH2 NH)-Nle-Asp-Phe-NH2 16, Boc-Trp-Nle-psi (CH2-NH)-Asp-Phe-NH2 11, and Boc-Trp-Nle Asp-psi (CH2-NH)-Phe-NH2 5, in which the methionyl residue was replaced by a norleucyl residue. Pseudo-peptides 16 and 21, in which the amide bond between Trp and Met (or Nle) was substituted by a CH2-NH bond, stimulated gastric acid secretion in the rat in vivo. Pseudo-peptides 11 and 20, where the amide bond between Met (or Nle) and Asp was replaced by a CH2-NH bond, did not exhibit any activity on acid secretion in the rat in vivo but were potent inhibitors of pentagastrin-induced acid secretion. Peptides 11, 16, 20 and 21 all recognize the gastrin receptor on a mucosal cell preparation. Pseudo-peptide 5, in which the amide bond between Asp and Phe was replaced by a CH2-NH bond, was a less potent inhibitor of pentagastin-induced acid secretion and had a weaker affinity than the other pseudo-peptides. PMID- 3011689 TI - Effect of a high carbohydrate, low sodium and low fat diet in type 2 diabetics with moderate hypertension. AB - The effect of an intended diet, high in cereal fibre, low in fat and sodium was assessed over a 3-month period in 13 type 2 diabetic patients with moderate hypertension (diastolic blood pressure greater than 105 and less than 115 mmHg without antihypertension drug therapy). Eleven patients completed the study and two patients were withdrawn owing to an increase of blood pressure above initial values after 1 month. Using a compliance scoring system by an observer unaware of blood pressure response, patients were divided into those compliant to the dietary regimen (n = 7; group A) and those who were not, and therefore were considered controls (n = 4; group B). Group A demonstrated significant reductions in systolic (190.4 +/- 18 to 166.6 +/- 22.4 mmHg; P less than 0.02) and diastolic blood pressure (113.1 +/- 3.7 to 103.3 +/- 9.1 mmHg; P less than 0.01), weight (78.5 +/- 5.6 to 74.3 +/- 6.8 kg; P less than 0.02), daily urinary sodium excretion (210.3 +/- 79.9 to 120.3 +/- 56.1 mmol; P less than 0.02) and serum LDL levels (P less than 0.02). A reduction in glycosylated haemoglobin of 2.2 per cent was also noted. Three patients achieved a diastolic blood pressure level below 100 mmHg. In contrast, no significant changes occurred in group B. In particular, systolic and diastolic blood pressure (111.0 +/- 2.2 to 110.3 +/- 8.9 mmHg) remained unchanged. We conclude that the modified diet may have a hypotensive effect in diabetic subjects with moderate hypertension. However, the degree of blood pressure reduction suggests that this diet could, at best, only be considered an adjunct to conventional antihypertensive drug therapy. PMID- 3011691 TI - Chemical properties of water-soluble porphyrins. 4. The reaction of a 'picket fence-like' iron (III) complex with the superoxide oxygen couple. AB - Solution properties of the iron-(III) 'picket-fence-like' porphyrin, Fe(III) alpha,alpha,alpha, beta-tetra-ortho (N-methyl-isonicotinamidophenyl) porphyrin, (Fe(III)PFP) were investigated. These were acid/base properties of the aquo complex with pKa of 3.9 and its aggregation (formation of dimer with K = 1 X 10( 10) dm3 mol-1), complex formation with cyanide ions and 1-methyl imidazole (1 MeIm), spectral properties of the three iron complexes in their ferric and ferrous form and the one-electron reduction potential of these complexes. Knowing these properties, the reaction of the ferric complexes, aquo, dicyano and bis (1 MeIm), with the superoxide radical and other reducing radicals were studied using the pulse radiolysis technique. The second-order reaction rate constant of O2- with the iron (III) aquo complex which governs the catalytic efficiency of the metalloporphyrin upon the disproportionation of the superoxide radical was 7.6 X 10(7) dm3 mol-1 s-1, two orders of magnitude faster when compared to the reaction of each of the other complexes. The reduction by other radicals with all iron (III) complexes had similar second-order rate constants (10(9) to 10(10) dm3 mol 1 s-1). The reduction reaction in all cases produced Fe(II)PEP and no intermediate was found. The oxidation reaction of Fe(II)PEP by O2- was one order of magnitude faster when compared to the reduction of Fe(III)PFP by the same radical. Since the reactivity of O2- toward the three iron (III) porphyrin complexes follows their reduction potentials, it is suggesting the formation of a peroxo Fe(II) porphyrin as an intermediate. The reactions of the Fe(II)PFP complexes with dioxygen were also studied. The aquo complex was found to be first order in O2 and second order in Fe(II)PFP, suggesting the formation of a peroxo Fe(II) porphyrin as an intermediate. The intermediate formation was corroborated by evidence of the rapid CO binding reaction to the aquo complex of Fe(II)PFP. The two other complexes reacted very slowly with O2 as well as with CO. PMID- 3011693 TI - Hospital experience with varicella-zoster virus. AB - Varicella-zoster virus (VZV), one of the most common highly communicable agents of disease, stimulates aggressive infection control measures. In a 1-year period, at one hospital, at least 93 inpatients (82 adult patients, 11 pediatric patients) and 2 hospital staff with active varicella-zoster infections served as potential sources of nosocomial infection. Six incidents of exposure to the virus that occurred without the protection of standard infection control precautions were investigated by the infection control surveillance team. One hundred fifty six patients and 353 hospital staff were exposed. Fifty-one patients had no history of varicella-zoster infection, but only five were susceptible by serologic testing. One hundred one staff members had no history of varicella zoster, but only 11 were susceptible by serologic testing. These exposures resulted in three secondary varicella-zoster infections, six courses of varicella zoster immune globulin prophylaxis and furlough of 13 staff members. Epidemiologic investigation consumed approximately 356 hours of staff time, and management of exposed persons cost approximately $41,500. Prospective knowledge of the immune status of health care workers would vastly decrease the time and effort required to control hospital VZV exposures. PMID- 3011694 TI - Management of choroidal melanomas. PMID- 3011692 TI - 99mTc bone scanning agents--II. Adsorption of 99mTc(Sn)pyrophosphate complexes on the mineral phase of bone. AB - The adsorption of pyrophosphate, tin-pyrophosphate and 99mTc(Sn)pyrophosphate on Ca3(PO4)2 was investigated at pH 4.0 and pH 7.4. All components were radioactively labeled. Tin and reduced technetium were in most cases almost completely bound. The adsorption of pyrophosphate, tin(II) and technetium-99m at pH 4.0 was higher than at pH 7.4. The presence of tin gave rise to an increase of the pyrophosphate adsorption that was much larger than can be accounted for by a stoichiometric adsorption of tin-pyrophosphate. It is concluded that tin and technetium are bound as negatively charged complexes with pyrophosphate. Finally it is argued that the fraction of the bone scanning agent that reaches the bone surface is adsorbed completely by the mineral phase. PMID- 3011696 TI - The surgical management of parasternal recurrence of breast cancer. AB - Although parasternal recurrence of breast cancer can be adequately controlled by radiation therapy, at times, surgical excision is more effective. In this paper, a new technique for the radical resection of the parasternal portion of the chest wall, including dissection of the internal mammary and supraclavicular lymph nodes, is proposed. Between 1977 and 1984, eleven patients with parasternal recurrence were treated in our department. Five of these cases underwent this kind of operation and four of them are still alive without further recurrence; the remaining patient developed skin metastasis postoperatively. One patient refused radiation therapy but, in any case, radiation therapy failed to control the parasternal recurrences in the other five patients. This new operation is a rational and effective mode of treatment for parasternal recurrence. PMID- 3011695 TI - Acquired immunodeficiency syndrome (AIDS): the disease and its ocular manifestations. PMID- 3011697 TI - Small cell carcinoma of the lung: is there a place for surgery? AB - In many cases, surgery is not considered for anaplastic small cell carcinoma even in localized lesions. A review of the literature and the results obtained in our series of 30 patients prompt us to recommend surgery in stage I disease. PMID- 3011698 TI - Functioning insulinoma with a twenty-year history. AB - A case of functioning beta islet cell tumor (insulinoma) producing incapacitating hypoglycemia for 20 years before being diagnosed by means of pancreatic angiography is presented. Laparotomy with local tumor excision was curative. PMID- 3011699 TI - Enucleable liver tumors in cirrhotic patients. PMID- 3011700 TI - Diagnostic value of urinary cyclic AMP measurement in patients with calcium nephrolithiasis. AB - One hundred patients with recurrent calcium nephrolithiasis were submitted to the Pak test. At fasting state hypercalciuria was found in 27 cases, while a group of 16 further patients became hypercalciuric after oral calcium load. Only measurement of urinary cAMP excretion in both conditions made it possible to diagnose renal hypercalciuria in 9 out of 27 patients in the former group; according to test results 4 patients were expected to have primary hyperparathyroidism, but afterwards the disease was identified in only one case. PMID- 3011701 TI - In vitro study of local solubility of uric acid stones with lithium carbonate. AB - The possibilities of local dissolution of uric acid calculi with Na hydrocarbonate, K carbonate and lithium carbonate were examined in vitro. The most promising results were obtained with lithium carbonate. The necessity for studies of the compound in laboratory animals before its use in humans is emphasized. PMID- 3011702 TI - Leukotriene modulation of chloride transport in frog cornea. AB - The present study has identified the metabolites of arachidonic acid (AA) formed by the isolated frog cornea and has shown that this tissue is capable of the biosynthesis of both lipoxygenase and cyclo-oxygenase pathway metabolites. The cyclo-oxygenase (CO) metabolites found in greatest abundance were the prostaglandins E2 and F2a (PGE2 and PGF2a). The major lipoxygenase (LO) pathway metabolite found by thin-layer chromatography (TLC) was leukotriene B4 (LTB4), whereas leukotriene C4 (LTC4) biosynthesis was detected by radioimmunoassay. These leukotrienes (LTs) are highly potent modulators of active chloride transport, since incubation with LTC4 (10(-7)-10(-9) M) resulted in a dose dependent stimulation of both the Cl- originated short-circuit current (SCC) and potential difference (PD). In contrast, LTB4 (10(-7)-10(-9) M) inhibited both of these parameters. Both LTC4 and LTB4 exerted these effects only when applied to the endothelial side. Preincubation with the leukotriene receptor antagonist, FPL 55712 completely blocked the response to LTC4, indicating that the action of LTC4 in the cornea is receptor-mediated. In this report the authors show that LTB4 and LTC4 are formed by the cornea and that they are potent modulators of the SCC and PD in chamber experiments. The possibility exists that LTB4 and LTC4 may function as endogenous regulators of net Cl- transport in the cornea, since the addition of these metabolites resulted in a dose-dependent stimulation (with LTC4), and inhibition (with LTB4), of both the short-circuit current (SCC) and potential difference (PD). PMID- 3011703 TI - Comparative replication of HSV-1 in BALB/c and C57BL/6 mouse embryo fibroblasts in vitro. AB - Herpes simplex virus type 1 (HSV)-host cell interactions were studied in fibroblasts from inbred mice by measuring virus replication, virus adsorption, infectious center formation, and single-cell virus production. BALB/c mouse embryo fibroblasts (MEF) produced more intracellular and extracellular virus than C57BL/6 MEF, with differences in virus production first appearing at 16 hr after inoculation. Virus yield in C57BL/6 cells peaked earlier (16 hr) and at a lower level than in BALB/c cells (20 hr). These results were explained by a difference in single-cell virus replication, rather than less efficient adsorption or the presence of cells that could not be infected. Host-related variation in the ability of infected cells to support HSV replication may account, in part, for differences in the severity of HSV ocular disease. PMID- 3011705 TI - Aids on AIDS revisited. PMID- 3011704 TI - Pediatric radiation oncology. PMID- 3011707 TI - A possible association between lung cancer and a geological outcrop. PMID- 3011706 TI - The complete amino acid sequence of bovine skeletal muscle acylphosphatase. AB - The primary structure of bovine skeletal muscle acylphosphatase was determined by performing the sequence analyses of the complete series of tryptic peptides. The amino acid composition of the entire series of peptic peptides was used to reconstruct the sequence by the overlapping method. The proposed structure is further confirmed by analogy with known amino acid sequences of acylphosphatase from skeletal muscle of other vertebrate species. The length of the polypeptide chain is 98 residues, identical to the length of the enzymes from other known mammalian species, but different from that found in turkey. The enzyme is NH2 acetylated and a comparison with the analogous molecular forms from other vertebrate species indicates that there are several long polypeptide stretches strictly conserved (93-97% identical position among mammals, and about 80% between calf and turkey enzymes). PMID- 3011708 TI - Phenoxymethyl penicillin acylase: sources and study--a sum up. PMID- 3011709 TI - Electron microscopic localization of 5'-nucleotidase in rat salivary glands. Comparative enzyme- and immunohistochemical studies. AB - The localization of 5'-nucleotidase in rat parotid and submandibular glands was investigated at the electron microscope level by an immunohistochemical technique using a highly specific antibody, and the results were compared with those obtained using the newley developed cerium method for enzyme histochemistry. Both methods demonstrated that 5'-nucleotidase is located on the external surface of the luminal plasma membranes of acinar cells as well as on intercalated and striated ductal cells. In the basolateral membranes of these cells, the portions adjacent to myoepithelial cells exhibited intense reaction products, but the other areas of plasma membranes contained only trace amounts of the reaction products. Both cerium-based enzyme histochemistry and immunohistochemistry showed that myoepithelial cells retain the enzyme on their plasma membranes. Neither method produced reaction products in the intracytoplasmic structure of constitutive cells of the salivary glands. We discuss the usefulness of the cerium-ion method for the demonstration of 5'-nucleotidase activity and compare it with the traditional lead-ion method. PMID- 3011710 TI - Localization of an aminoglycoside (streptomycin) in the inner ear after its systemic administration. A histochemical study using fluorescence microscopy. AB - We used the simple method of direct cytofluorescence to detect the presence of the aminoglycoside, streptomycin, in the inner ear after its systemic administration. In the cochlea, fluorescence was observed in the organ of Corti, the spiral ganglion, the nerve fibres, the vascular stria and Reissner's membrane; in the vestibulum, fluorescence was seen in the crista ampullaris and the planum semilunatum. The localization of the drug was related to the distribution of its specific receptor, triphosphoinositide (TPI); therefore, it is reasonable to assume that aminoglycosides exert their toxic effects by binding to TPI. PMID- 3011711 TI - Cf-252 neutron brachytherapy of short duration for bulky neck tumors. AB - Cf-252 (Cf) neutron brachytherapy (NT) was tested for treatment of bulky neck tumor masses in 13 patients. Complete regression (CR) or partial regression (PR) was obtained in 100% and local control in 85%. Tumors cleared rapidly and completely after an implant therapy of 4-7 hrs. Short duration Cf-NT can produce rapid and complete clearance of bulky tumors. Efficacy was independent of the implant time duration and without unusual adverse side effects or complications. The median survival time (MST) of patients dying of their disease was 6.8 months and 48% of treated patients survived greater than 18 months despite their advanced diseased status. PMID- 3011712 TI - Prognostic influence of TNM staging and LDH levels in small cell carcinoma of the lung (SCCL). AB - To better define the prognostic factors influencing the response to therapy and survival in small cell carcinoma of the lung (SCCL), an expanded "TNM" type staging system was developed and investigated in a series of 73 protocol treated patients. Because serum LDH levels at disease presentation have been correlated to disease extent, response to therapy, and treatment outcome in a number of malignancies, including SCCL, these interrelationships were also analyzed in the protocol patients. The TNM system was found to be a more descriptive and specific "shorthand" for denoting sites of involvement and for indicating the body burden of tumor than the traditional limited-extensive disease (LD-ED) system. A clear statistical advantage could not be shown over the LD-ED system for predicting chemotherapy response or survival, although there were trends suggesting the TNM system could divide patients into three prognostic subgroups. Serum LDH proved to be a useful index of disease extent and therapy outcome. LDH levels at presentation were proportionately higher with more extensive tumor, measured by either the LD-ED or TNM staging. High LDH predicted poorer responses to chemotherapy and lower survival within similar stage subgroups compared to patients with normal LDH levels. The negative effect of elevated LDH was independent of hepatic involvement and did not predict subsequent hepatic failure in any consistent way. The SCCL TNM staging system proposed needs further refinement and should be tested with larger patient numbers. LDH, along with other tumor markers recently identified, need to be integrated into the staging system to form an overall prognostic index. PMID- 3011714 TI - Noninfectivity of semen from bulls infected with bovine leukosis virus. AB - An opportunity for study of the potential role of semen in the transmission of bovine leukosis virus (BLV) was provided when a Jersey herd was found to be BLV seronegative. This was a closed herd; new genetic material had been introduced by artificial insemination (AI), using semen collected and processed at 7 AI centers in the United States. Of 66 donor bulls from which semen had been collected for AI use in the herd during the 5 years the herd remained seronegative, 24 were consistently BLV-seropositive and 2 became seropositive for BLV during the study. Semen collected from the BLV-seropositive bulls accounted for 1,019 semen units, representing 48.3% of the semen purchased. The maintenance of BLV seronegativity in this herd for 5 years, when semen from BLV-seropositive bulls was used for AI, provided evidence for the lack of infectivity of BLV in bovine semen. PMID- 3011713 TI - Protection of mouse jejunal crypt cells by WR-2721 after small doses of radiation. AB - The ability of WR-2721 to protect jejunal crypt cells after single doses and multifractionated doses of radiation was studied. Effective dose survival curves for jejunal crypt cells were constructed over the dose range of 230 to 1600 cGy. WR-2721 was given 30 minutes before each fraction, in a regimen consisting of 200 mg/kg before the first radiation fraction, followed at 3 hr intervals by 100 mg/kg for a total of 12 drug doses for the largest number of fractions. Fractionation protocols were designed with common dose fractions in regimens with different fraction numbers, allowing a test of the hypothesis of equal effect per fraction and an estimate of the initial number of clonogens per crypt in both the drug treated and non-drug treated mice. The hypothesis of equal effect per fraction could not be rejected in either the drug or non-drug treated mice. An average number of 137 clonogens per crypt was estimated for the non-drug treated mice and 81 clonogens per crypt in the drug treated mice; the difference between these two values was not significant. The protection factor decreased with decreasing dose ranging from a high of 1.47 (95% C.L. = 1.44 to 1.50) after a single dose of 2000 cGy to a low of 1.21 (95% C.L. = 1.08 to 1.37) after 200 cGy. Analysis of the data using either the linear quadratic (LQ) or two-component (TC) model of cell survival showed that WR-2721 was not dose-modifying over the dose range tested. Analysis using the LQ model showed that both beta and alpha were modified by WR-2721, by 50% and 20% respectively. These data indicate that protection by WR-2721 can be expected to decrease with dose although there is some protection after clinically relevant doses. PMID- 3011715 TI - Embryo transfer and transmission of bovine leukosis virus in a dairy herd. AB - Transmission of bovine leukosis virus (BLV) by embryo transfer was investigated in a large commercial Holstein herd. One hundred and sixteen calves, transplanted as embryos from BLV-positive cows into BLV-negative heifers, were serologically negative, as were recipients, whereas 5 of 29 (17%) calves transplanted as embryos into BLV-positive recipients were infected with BLV, as evidenced by antibodies in the agar gel immunodiffusion test. PMID- 3011716 TI - Eradication of ovine progressive pneumonia from sheep flocks. AB - Two management methods for controlling ovine progressive pneumonia in sheep were evaluated. By a test and cull method, wherein seropositive sheep were culled from the flock, the causative virus was eradicated from a closed flock and controlled in an open flock. By an isolate-and-test method, wherein lambs were removed from infected ewes before nursing and reared in isolation, the virus was eradicated in 1 of 2 attempts to establish virus-free flocks. It was concluded that either method should be effective in controlling ovine progressive pneumonia. Results indicated that annual serologic monitoring of a virus-free flock would be necessary to ensure that the virus-free status is maintained. PMID- 3011717 TI - Use of tears for diagnosis of feline leukemia virus infection. AB - A comparison was made of the use of serum, tears, and saliva for the detection of feline leukemia virus (FeLV) infection in cats. Cotton swabs were used to collect saliva, and tear-test strips were used to collect tears. Specimens were analyzed by a commercially available ELISA. Using a 10- to 15-minute specimen incubation period, FeLV was detected in 70% of the saliva specimens and in 73% of the tear specimens from viremic (serum-positive) cats. Feline leukemia virus antigen was not detected in saliva and tear specimens from serum-negative cats. The sensitivity of the tear assay was improved by increasing the incubation time to 24 hours. Tear strips could be air-dried and stored at room temperature for up to 7 days without any appreciable loss of activity. Client-owned and experimentally infected laboratory cats were tested for FeLV, using air-dried tear-test strips and a 24-hour incubation period. Tears were positive (contained FeLV antigen) in 65 of 72 (90%) serum-positive cats and did not contain antigen in 46 of 46 (100%) serum-negative cats. Results of ELISA obtained from serum and tears also were compared with results obtained from indirect fluorescent antibody testing of blood smears. Results of indirect fluorescent antibody and ELISA compared favorably with each other and with the results of tear testing. PMID- 3011718 TI - Hyperprogesteronemia associated with Sertoli cell tumor and alopecia in a dog. AB - Hyperprogesteronemia was found in a dog with alopecia and Sertoli cell tumor. Alopecia began in the lumber areas; the entire coat was dull and dry, and epilated easily. The only laboratory abnormalities were high serum progesterone concentration and incomplete suppression of cortisol after low-dose dexamethasone administration. The hair regrew after castration, and the progesterone concentration decreased toward normal. PMID- 3011719 TI - Opposing viewpoints on endocrine tests persist. PMID- 3011720 TI - It's time to unamend the insulin-glucose ratio. PMID- 3011721 TI - Effect of forphenicinol on gamma-interferon production in mice sensitized with BCG. AB - Forphenicinol stimulated the production of BCG-induced gamma-interferon in BCG sensitized mice as much as 11-fold whereas it did not induce interferon production in unsensitized mice. This stimulatory effect was also observed in mice immuno-suppressed by cyclophosphamide. Furthermore, BCG-induced gamma interferon production was reduced in mice by treatment with an anti-macrophage agent (silica gel), and forphenicinol administration into such mice could augment their interferon production. Transplantation of macrophages from forphenicinol treated mice into mice injected with silica gel remarkably enhanced the BCG induced interferon production in the recipients. These results suggest that the stimulatory effect of forphenicinol on BCG-induced gamma-interferon production in mice was due to the activation of macrophages. PMID- 3011722 TI - Effect of FSH treatment on LH and FSH receptors in chronic cystic-ovarian diseased dairy cows. AB - This experiment was conducted to 1) determine whether chronic cystic-ovarian diseased (CCOD) cows fail to respond to luteinizing hormone (LH) treatment because of a lack of adequate ovarian LH receptors and 2) determine the effect of follicle stimulating hormone (FSH) treatment on ovarian LH and FSH receptors in ovaries of CCOD cows. The CCOD cows were those that did not resume cyclic ovarian activity after repeated treatment with human chorionic gonadotropin (hCG) and(or) LH-releasing hormone (LHRH) and were considered chronic by veterinarians. Thirteen CCOD cows were purchased from producers; six of them were injected with 5 mg FSH twice daily for 3 or 5 d (TCCOD) and the remaining seven remained untreated. Seven control (noncystic) cows in the luteal phase of the estrous cycle were injected with Lutalyse approximately 48 to 50 h before slaughter so they would be in the follicular phase (FP) of the cycle at the time of slaughter. Analysis of serum and pituitaries showed no differences (P greater than .05) in mean concentrations of serum or pituitary LH and FSH or pituitary LHRH receptor concentration and affinity among FP, CCOD and TCCOD cows. Ovarian follicle wall concentrations of receptors for LH (3.2 +/- .6; 13.0 +/- 2.5; 22.4 +/- 5.1 fmol/mg protein) and FSH (10 +/- 2.6; 43 +/- 7.2; 29 +/- 6.7 fmol/mg protein) were lower (P less than .05) in CCOD cows compared with FP and TCCOD cows, respectively. The same pattern was observed for concentrations of granulosa cell LH and FSH receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011723 TI - Current leads in antiviral chemotherapy. PMID- 3011724 TI - Variation in the potentiation of beta-lactam antibiotic activity by clavulanic acid and sulbactam against multiply antibiotic-resistant bacteria. AB - Using a broth microdilution method, we studied 120 strains of beta-lactamase producing clinical isolates to determine the potentiating effect of clavulanic acid and sulbactam (both beta-lactamase inhibitors) on the antibacterial activity of a variety of beta-lactam antibiotics. All antibiotics were tested alone and in combination with each inhibitor simultaneously using low (10(5) cfu/ml) and high (10(7) cfu/ml) inocula. Against the strains of Escherichia coli and Haemophilus influenzae the addition of both inhibitors significantly potentiated the activity of most antibiotics at the low inoculum. At the high inoculum, however, this effect was abrogated against E. coli and most strains of H. influenzae. Antibiotic activity was significantly enhanced by the inhibitors at both inoculum sizes with Bacteroides fragilis. Antibiotic resistance of Pseudomonas aeruginosa was little affected by the inhibitors. Among methicillin-resistant staphylococci, antibiotic potentiation by the inhibitors was more significant for Staphylococcus aureus than the coagulase-negative staphylococci. PMID- 3011725 TI - Sultamicillin--a new antibiotic in the treatment of persistent lower respiratory tract infections caused by Haemophilus influenzae. AB - Haemophilus influenzae is a frequent cause of recurrent or chronic lower respiratory tract infections in patients suffering from cystic fibrosis (CF) and other chronic obstructive pulmonary disease (COPD). Ampicillin and its derivatives are routinely used in treatment, but resistant strains producing beta lactamase frequently necessitate the use of other antibiotics. Sultamicillin is a compound agent for oral use in which ampicillin and the beta-lactamase inhibitor sulbactam are linked as a double ester. This combination is active in vitro against many beta-lactamase producing bacteria including ampicillin-resistant H. influenzae. Eight CF children and ten children with other COPD suffering from chronic or recurrent H. influenzae infection of the lower respiratory tract were treated with sultamicillin orally, 25 mg/kg, 12-hourly, for two weeks. Nine infections were caused by ampicillin-resistant strains. At the end of the treatment 65% of the patients were free of H. influenzae. The only adverse reaction was diarrhoea which occurred in 14 patients, and necessitated withdrawal of one patient from the study. PMID- 3011727 TI - Cytochemical localization of Na+-K+-ATPase in rat type II pneumocytes. AB - The distribution of sodium-potassium-activated adenosinetriphosphatase (Na+-K+ ATPase) in the alveolar portion of rat lungs was examined by indirect immunofluorescence with the use of a mouse monoclonal anti-rat Na+-K+-ATPase and by ultrastructural cytochemistry using p-nitrophenylphosphate as substrate. The reaction was inhibitable by 10 mM ouabain or by the omission of K+ from the reaction mixture. Cysteine or levamisole was used to inhibit alkaline phosphatase activity. By immunofluorescence, staining was confined to cuboidal cells in alveolar spaces. These were tentatively identified as type II pneumocytes. By ultrastructural cytochemistry reaction product was present on the cytoplasmic side of the basolateral membranes of type II pneumocytes. No reaction product was observed in type I pneumocytes or in endothelium. These results indicate that type II pneumocytes contain more Na+-K+-ATPase, an enzyme important in vectorial electrolyte transport, than type I pneumocytes or endothelial cells. More sensitive methods, however, are required to determine the amounts and distribution of this enzyme in type I pneumocytes and pulmonary vascular endothelial cells. PMID- 3011726 TI - The new quinolones and their combinations with other agents for therapy of severe infections. AB - New quinolones are very potent compounds against Gram-negative bacteria, including Pseudomonas, and staphylococci. They are particularly interesting because of their oral availability and their pharmacokinetic properties. When combined with other common antibiotics they appear to have mostly indifferent effects in vitro. Clinical results have shown their efficacy when used alone for the treatment of osteomyelitis, complicated urinary tract infections and prostatitis. Nevertheless, in 6% of cases resistant strains were selected (Klebsiella, Enterobacter, Serratia, P. aeruginosa and staphylococci). The use of combinations of quinolones with other antibiotics was successful in the treatment of osteomyelitis and severe nosocomial infections including bacteraemia, and seems to prevent emergence of resistant strains. PMID- 3011728 TI - Lung beta-adrenoreceptor blockade affects perinatal surfactant release but not lung water. AB - We induced beta-adrenergic receptor blockade at 28 days gestation in the fetal rabbit with an irreversible beta-antagonist, bromace-tylalprenolomenthane (BrAlp). There was a marked decrease in concentration of available receptors in lung with increasing doses of BrAlp. BrAlp treatment decreased isoproterenol, but not prostaglandin, stimulated adenosine 3',5'-cyclic monophosphate (cAMP) generation in lung minces, and had no effect on activation of adenylate cyclase through non-beta-receptor-mediated components of the cyclase system in particulate preparations. Phospholipid recovery via lung lavage was significantly less from treated fetuses than from controls in groups delivered by cesarean section at 30 days (-31%) or vaginally at 31 days (-34%) and not allowed to air breathe. However, if fetuses from either group were allowed to air breathe, the difference was abolished. BrAlp treatment did not affect the phospholipid composition in lavage fluid, the rate of phosphatidylcholine synthesis, or tissue content of total or saturated phosphatidylcholine. Beta-adrenergic receptor blockade did not produce a significant change in lung water content either at or after birth regardless of the route of delivery. These data indicate that endogenous catecholamines play a role in surfactant secretion in both the fetal and newborn rabbit. We found no effects of BrAlp treatment on lung water, suggesting perhaps a less important role of endogenous catecholamines or that fewer receptors are required for this response than remained after treatment. PMID- 3011729 TI - Linear programming analysis of VA/Q distributions: limits on central moments. AB - Linear programming examines the boundaries of infinite sets. We used this method with the multiple-inert gas-elimination technique to examine the central moments and arterial blood gases of the infinite family of ventilation perfusion (VA/Q) distributions that are compatible with a measured inert gas-retention set. A linear program was applied with Monte-Carlo error simulation to theoretical retention data, and 95% confidence intervals were constructed for the first three moments (mean, dispersion, and skew) and the arterial PO2 and PCO2 of all compatible blood flow distributions. Six typical cases were studied. Results demonstrate narrow confidence intervals for both the lower moments and predicted arterial blood gases of all test cases, which widen as moment number or error increase. We conclude that the blood gas composition and basic structure of all compatible VA/Q distributions are tightly constrained and that even subtle changes in this structure, as may occur experimentally, can be identified. PMID- 3011731 TI - Characterization of bacteriophages infecting Streptomyces erythreus and properties of phage-resistant mutants. AB - Three bacteriophages infecting Streptomyces erythreus, called G3, G4 and G5, were isolated and characterized. They contain double-stranded linear DNA molecules with cohesive ends. The restriction map of G3 DNA (48 kilobases long) for four restriction endonucleases and that of G4 DNA (43 kilobases long) for seven restriction endonucleases are reported. Restriction analysis and hybridization experiments showed that G3 and G4 share little DNA homology, while G4 and G5 are apparently identical except for an additional EcoRI site present in G5. The region containing this EcoRI site has been mapped on G4 DNA. Microbiological and serological data showed that G5 is very similar to G4. G3- and G4-resistant mutants of S. erythreus PS1 were isolated. The screening of phage-resistant mutants showed a high frequency of strains with increased erythromycin production. The mechanism of phage resistance of strain PS3 (G3 resistant) and of strain PS16 (G4 resistant) was examined. The DNA of the resistant strains contains no phage DNA, ruling out lysogeny as a cause of phage resistance. Transfection of strains PS1, PS3, and PS16 with DNA of the three phages showed the same efficiency, indicating that resistance is at the level of the bacterial wall. PMID- 3011730 TI - Kidney glomerular explants in serum-free media: demonstration of intracellular antioxidant enzymes and active oxygen metabolites. AB - Guinea pig glomeruli were grown for 22 d in a serum-free medium composed of Waymouth's MB 752/l supplemented with sodium pyruvate, nonessential amino acids, and antibiotics (the basic medium). Intracellular cellular activity of the antioxidant enzymes superoxide dismutase (SOD; both copper-zinc [Cu,Zn] and manganese [Mn] forms) and catalase, and intracellular active oxygen metabolites (hydrogen peroxide [H2O2] and superoxide [O2-.]) were measured with time in culture. The results were compared to results obtained from glomeruli grown in different serum-free media, including the basic medium plus fibronectin (FN), the basic medium plus transferrin and FN, and a complex medium containing insulin, transferrin, selenium (Se), triiodothyronine, and FN (complete medium). In general, although the intracellular activity of antioxidant enzymes and active oxygen metabolites varied over time in culture in all media, there were only a few statistically significant differences among different media. Both CuZn SOD and Mn SOD activity were demonstrated in isolated glomeruli. The CuZn SOD activity per DNA ratio decreased slightly with time in culture in all media tested except the complete medium, in which CuZn SOD activity per DNA ratio remained more constant. The Mn SOD activity per DNA ratio did not vary significantly over time in culture. Catalaselike activity was very low in isolated glomeruli and declined sharply with time in culture in all media except the complete medium. Both H2O2 and O2-. were detected intracellularly in glomerular culture. Our results indicate that intracellular antioxidant enzymes and active oxygen metabolites in glomeruli vary with time in culture and, in some instances, with culture conditions. PMID- 3011732 TI - Cloning and expression of the phospho-beta-galactosidase gene of Staphylococcus aureus in Escherichia coli. AB - The phospho-beta-galactosidase gene of Staphylococcus aureus was cloned in Escherichia coli. This was done by first isolating a staphylococcal transposon Tn551-induced mutant which rendered phospho-beta-galactosidase synthesis partially constitutive because of an insertion nearby this lac structural gene. This allowed selection in E. coli of chimeric plasmids which expressed the erythromycin resistance determinant of Tn551. A 26-kilobase (kb) BamHI insert in plasmid pBR322 was isolated which encoded phospho-beta-galactosidase, as determined by phospho-beta-galactosidase activity measurements. Maxicell experiments showed the presence of 56-, 13.5-, and 31-kilodalton proteins encoded by the staphylococcal DNA. The presence of the 56-kilodalton protein correlated with phospho-beta-galactosidase activity and corresponded in molecular weight to the reported value for the purified enzyme. The nature of the other proteins is unknown. Phospho-beta-galactosidase was apparently expressed in E. coli by a promoter contained within a 2.1-kb EcoRI chromosomal DNA fragment. This fragment, when inserted into a chloramphenicol acetyl transferase promoter detection plasmid, was transcriptionally active in both E. coli and Bacillus subtilis but was much more active in the latter host. PMID- 3011733 TI - camR, a negative regulator locus of the cytochrome P-450cam hydroxylase operon. AB - A 4.27-kilobase insert from a HindIII DNA library of Pseudomonas putida carrying the CAM plasmid allowed coordinate expression of genes camD and camC under control of camR, an upstream regulator. The camC gene specifies cytochrome P 450cam, and camD specifies the 5-exo-alcohol dehydrogenase. A 1.38-kilobase deletion from the insert results in the constitutive expression of genes camC and camD; transformation in trans restores the substrate control, indicating that camR is a negative regulator. PMID- 3011735 TI - Molecular cloning, DNA nucleotide sequencing, and expression in Bacillus subtilis cells of the Bacillus macerans cyclodextrin glucanotransferase gene. AB - The gene for cyclodextrin glucanotransferase from Bacillus macerans was cloned in an Escherichia coli bacteriophage, lambda D69, and was recloned in a Bacillus subtilis plasmid, pUB110. Starting from an ATG initiation codon, a unique reading frame was shown to extend for 2,142 base pairs (714 amino acids). The nucleotide sequence revealed that the enzyme is composed of two identical subunits. PMID- 3011734 TI - Identification and cloning of genes involved in phaseolotoxin production by Pseudomonas syringae pv. "phaseolicola". AB - Genes involved in the production of phaseolotoxin by Pseudomonas syringae pv. "phaseolicola" NPS3121 were identified by Tn5 mutagenesis and cosmid cloning. A total of 5,180 kanamycin-resistant colonies were screened for the loss of phaseolotoxin production by a microbiological assay. Six independent, prototrophic, Tox- mutants were isolated that had Tn5 insertions in five different EcoRI fragments. All six mutants had Tn5 inserted in the same KpnI fragment, which had a length of ca. 28 kilobases including Tn5. The mutants produced residual toxin in vitro. An EcoRI fragment containing Tn5 and flanking sequences from mutant NPS4336 was cloned and used to probe a wild-type genomic library by colony hybridization. Seven recombinant plasmids showing homology to this probe were identified. Each Tox- mutant was restored in OCTase-specific toxin production by two or more of the recombinant plasmids. The data suggest that at least some of the genes involved in phaseolotoxin production were clustered in a large KpnI fragment. No homology was detected between the Tn5 target fragment cloned from mutant NPS4336 and the total genomic DNA from closely or distantly related bacteria that do not produce phaseolotoxin. PMID- 3011736 TI - IS222, a new insertion element associated with the genome of Pseudomonas aeruginosa. AB - A new insertion element, IS222, was identified to be associated with the DNA of a mutant strain of the converting Pseudomonas aeruginosa bacteriophage D3. The insertion sequence was 1,350 base pairs in size and possessed terminal inverted repeats. The nucleotide sequence contained single cleavage sites for EcoRI and PvuI but none for BamHI, PstI, HindIII, SmaI, or SalI. By Southern hybridization analysis, no homology was found with genomic DNA from P. aeruginosa PAT or Escherichia coli. Genomic DNA from the phage host, P. aeruginosa PAO, contained two sequences homologous to IS222. PMID- 3011737 TI - Contrasting mechanisms of envZ control of mal and pho regulon genes in Escherichia coli. AB - The envZ11 missense mutation in the regulatory gene envZ pleiotropically repressed synthesis of OmpF, alkaline phosphatase, and several proteins of the maltose regulon. Procaine treatment of wild-type cells resulted in the same phenotype through an envZ+-mediated mechanism. Here we show that envZ11-procaine act differently on the mal and pho regulons. In the mal system, the expression of the positive regulator gene malT, measured as beta-galactosidase activity of a malT-lac+ operon fusion, was drastically reduced by procaine treatment or by the envZ11 mutation. In contrast, expression of the positive regulator of the pho regulon phoB was not reduced by procaine treatment. The products of the regulatory genes phoM, phoR, and phoU were also not required for procaine action. Procaine and envZ11 inhibited expression of only two products of the pho regulon, alkaline phosphatase and the PhoE porin. The conclusion that envZ11-procaine act differently on the mal and the pho regulons is supported by our ability to isolate second-site mutations with a Mal+ PhoA- phenotype in an envZ11 strain. PMID- 3011738 TI - Nucleotide sequence of the surface exclusion genes traS and traT from the IncF0 lac plasmid pED208. AB - pED208 is a 90-kilobase conjugative plasmid belonging to the incompatibility group IncF0 lac. The surface exclusion system from this plasmid was cloned and sequenced, and two genes demonstrated exclusion ability. traS encoded a 186-amino acid hydrophobic protein which, when transcribed from a vector promoter, caused exclusion of pED208. The product of traT (TraTp) was a 245-residue protein which was highly expressed independently of a vector promoter in Escherichia coli minicells. The TraTp from pED208 was homologous with traT products from the IncF plasmids R-100 and F (80% homology), but recombinants containing the pED208 surface exclusion system excluded F poorly. PMID- 3011739 TI - Excretion of the penicillinase of an alkalophilic Bacillus sp. through the Escherichia coli outer membrane is caused by insertional activation of the kil gene in plasmid pMB9. AB - Most of the cloned penicillinase from alkalophilic Bacillus sp. strain 170 and alkaline phosphatase were released into the culture medium by Escherichia coli strains bearing plasmid pEAP1 or pEAP2 (T. Kudo, C. Kato, and K. Horikoshi, J. Bacteriol. 156:949-951, 1983). We analyzed the basis for excretion of periplasmic enzymes in the cells bearing these plasmids. Several experiments such as subcloning, insertion of a chloramphenicol acetyltransferase cartridge, and DNA sequencing were done. A dormant kil gene in plasmid pMB9 was expressed by a promoter of the inserted DNA fragment of alkalophilic Bacillus sp. strain 170, and as a result, the outer membrane of E. coli became permeable, allowing the proteins to be excreted without cell lysis. PMID- 3011740 TI - Reversibility of SOS-associated division inhibition in Escherichia coli. AB - In Escherichia coli the SOS response, induced by DNA-damaging treatments, includes two systems of cell division inhibition, SfiA and SfiC, which are thought to prevent cell division by interacting with the division protein FtsZ. It is shown here that SfiA-mediated division inhibition is readily reversible, even in the absence of de novo protein synthesis, suggesting that functional FtsZ molecules can be recovered from SfiA-FtsZ complexes. The action of SfiC, on the other hand, is essentially irreversible; induction by expression of the recA (Tif) mutation for 60 min results in division inhibition that continues for at least 180 min after the end of the induction period. An excess of the presumed target molecule FtsZ, furnished by a multicopy plasmid, suppresses the action of SfiA but not SfiC. Simultaneous induction of SfiA and SfiC results in irreversible division inhibition, showing that SfiC is epistatic to SfiA. The irreversibility of SfiC action is most readily accounted for by assuming that the SfiC product, unlike SfiA, is stable. The reversibility of SfiA action is slower in a lon mutant, in which the SfiA protein is partially stabilized. From the kinetics of division resumption in the absence of protein synthesis, we estimated the in vivo half-life of the SfiA protein to be 10 min in a lon+ strain and 170 min in a lon mutant. PMID- 3011742 TI - Cloning and characterization of the dcm locus of Escherichia coli K-12. AB - The dcm locus of Escherichia coli K-12 has been shown to code for a methylase that methylates the second cytosine within the sequence 5'-CC(A/T)GG-3'. This sequence is also recognized by the EcoRII restriction-modification system coded by the E. coli plasmid N3. The methylase within the EcoRII system methylates the same cytosine as the dcm protein. We have isolated, from a library of E. coli K 12 DNA, two overlapping clones that carry the dcm locus. We show that the two clones carry overlapping sequences that are present in a dcm+ strain, but are absent in a delta dcm strain. We also show that the cloned gene codes for a methylase, that it complements mutations in the EcoRII methylase, and that it protects EcoRII recognition sites from cleavage by the EcoRII endonuclease. We found no phage restriction activity associated with the dcm clones. PMID- 3011741 TI - Nucleotide sequence of a DNA segment promoting transcription in Pseudomonas putida. AB - A DNA segment that promotes gene expression in Pseudomonas putida was identified in pTN8, a mutant plasmid of an RP4-TOL recombinant. A promoter on the segment was cloned with a promoter-probe vector containing the xylE gene of the TOL plasmid. The xylE gene was expressed under the control of the promoter, and the gene product catechol 2,3-dioxygenase was constitutively synthesized. As analyzed by an S1 nuclease protection assay, the amount of mRNA produced in P. putida was more than that in Escherichia coli. Fine S1 nuclease mapping and reverse transcriptase mapping revealed three tandem transcription start sites in both P. putida and E. coli. The nucleotide sequence preceding the transcription start sites was determined; a part of this sequence contained a sequence homologous to E. coli promoter sequences. A tentative consensus sequence for P. putida constitutive promoters is proposed. PMID- 3011743 TI - Overproduction of FtsZ suppresses sensitivity of lon mutants to division inhibition. AB - Escherichia coli lon mutants are sensitive to UV light and other DNA-damaging agents. This sensitivity is due to the loss of the lon-encoded ATP-dependent proteolytic activity which results in increased stability of the cell division inhibitor SulA. Introduction of the multicopy plasmid pZAQ containing the ftsZ gene, which is known to increase the level of FtsZ, suppressed the sensitivity of lon mutants to the DNA-damaging agents UV and nitrofurantoin. Alterations of pZAQ which reduced the expression of ftsZ reduced the ability of this plasmid to suppress the UV sensitivity. Examination of the kinetics of cell division revealed that pZAQ did not suppress the transient filamentation seen after exposure to UV, but did suppress the long-term inhibition that is normally observed. lon strains carrying pZAQ could stably maintain a multicopy plasmid carrying sulA (pBS2), which cannot otherwise be introduced into lon mutants. In addition, the increased temperature sensitivity of lexA(Ts) strains containing pBS2 was suppressed by pZAQ. These results suggest that SulA inhibits cell division by inhibiting FtsZ and that this interaction is stoichiometric. PMID- 3011745 TI - Essential structure in the cloned transforming DNA that induces gene amplification of the Bacillus subtilis amyE-tmrB region. AB - Bacillus subtilis B7, a mutant which acquired gene amplification of the amyE-tmrB region, showed, as a result, hyperproductivity (about a 5- to 10-fold increase) of alpha-amylase and tunicamycin resistance. The mutational character was transferred to recipient cells by competence transformation. A 14-kilobase (kb) EcoRI chromosomal DNA fragment of strain B7 was found to have the transforming activity. We cloned a 6.4-kb EcoRI fragment on a phage vector lambda Charon 4A through a spontaneous deletion of 7.6 kb from the 14-kb fragment and subcloned a 1.6-kb HindIII fragment on pGR71. The cloned 6.4-kb EcoRI and 1.6-kb HindIII fragments retained the transforming activity of inducing gene amplification of the amyE-tmrB region. At the junction point (J) of the repeating units (16 kb), the tmrB gene was linked to a DNA region (M) located 4 kb upstream of amyE. The essential structure of the cloned, transforming (gene amplification-inducing) DNA was deduced to be that around J. The subcloned 1.6-kb HindIII fragment that retained the transforming activity was shown to be almost solely composed of the tmrB-J-M region. In addition, the DNA sequence around J was determined. PMID- 3011746 TI - Structural similarity between the lepidoptera- and diptera-specific insecticidal endotoxin genes of Bacillus thuringiensis subsp. "kurstaki" and "israelensis". AB - A gene from Bacillus thuringiensis subsp. "israelensis" was cloned from the large plasmids of this subspecies and was shown to code for a mosquitocidal polypeptide. The gene could be expressed in either Escherichia coli, Bacillus subtilis, or B. thuringiensis subsp. "israelensis" to produce the larvicidal activity. Similarly, a Lepidoptera-specific toxin gene from B. thuringiensis subsp. "kurstaki" was also cloned and expressed in E. coli and B. subtilis. Both cloned genes were sequenced and subjected to computer analysis. A long open translational reading frame coded for the B. thuringiensis subsp. "kurstaki" gene product. However, the B. thuringiensis subsp. "israelensis" clone was composed of two adjacent open reading frames oriented as if they were in a transcriptional operon. The products of the cloned genes retained their specificity for either Lepidoptera or Diptera. The control regions immediately preceding the toxin genes of both B. thuringiensis subspecies showed considerable DNA homology, most likely because both toxins are expressed only during sporulation. In addition, the deduced amino acid sequences from the two contiguous B. thuringiensis subsp. "israelensis" genes bore a striking resemblance to the deduced amino acid sequence from the single larger B. thuringiensis subsp. "kurstaki" gene, as if these two arrangements were evolutionarily related. PMID- 3011744 TI - Metabolism of the phospholipid precursor inositol and its relationship to growth and viability in the natural auxotroph Schizosaccharomyces pombe. AB - Phospholipid metabolism in the fission yeast Schizosaccharomyces pombe was examined. Three enzymes of phospholipid biosynthesis, cytidine diphosphate diacylglycerol synthase (CDP-DG), phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase, were characterized in extracts of S. pombe cells. Contrary to an earlier report, we were able to demonstrate that CDP-DG served as a precursor for PI and PS biosynthesis in S. pombe. S. pombe is naturally auxotrophic for the phospholipid precursor inositol. We found that S. pombe was much more resistant to loss of viability during inositol starvation than artificially generated inositol auxotrophs of Saccharomyces cerevisiae. The phospholipid composition of S. pombe cells grown in inositol-rich medium (50 microM) was similar to that of S. cerevisiae cells grown under similar conditions. However, growth of S. pombe at low inositol concentrations (below 30 microM) affected the ratio of the anionic phospholipids PI and PS, while the relative proportions of other glycerophospholipids remained unchanged. During inositol starvation, the rate of PI synthesis decreased rapidly, and there was a concomitant increase in the rate of PS synthesis. Phosphatidic acid and CDP-DG, which are precursors to these phospholipids, also increased when PI synthesis was blocked by lack of exogenous inositol. The major product of turnover of inositol containing phospholipids in S. pombe was found to be free inositol, which accumulated in the medium and could be reused by the cell. PMID- 3011748 TI - Role of cell cohesion in Myxococcus xanthus fruiting body formation. AB - Dsp mutants of Myxococcus xanthus have a complex phenotype with abnormal cell cohesion, social motility, and development. All three defects are the result of a single mutation in the dsp locus, a region of DNA about 14 kilobases long. Cohesion appears to play a central role in social motility, since nonsocial mutants exhibit weak agglutination or, in the case of Dsp cells, no agglutination (L. J. Shimkets, J. Bacteriol. 166:837-841, 1986). However, Dsp cells can be agglutinated by cohesive strains of M. xanthus. This provided the opportunity to examine the role of cohesion during development by comparing the developmental phenotype of Dsp cells with that of Dsp cells mixed with cohesive strains. Dsp mutants were unable to complete any of the developmental behaviors: aggregation, fruiting body formation, developmental autolysis, and sporulation. Contact with cohesive strains seemed to restore some developmental characteristics to the Dsp cells. When allowed to develop with wild-type cells, Dsp cells accumulated in fruiting bodies and underwent developmental autolysis, but did not form a significant portion of the spore population. Igl mutants, which may be similar to the previously described frizzy mutants, are cohesive strains that are unable to form fruiting bodies. Mixing Igl cells with Dsp cells under developmental conditions resulted in fruiting body formation, although the Dsp cells were unable to form significant levels of myxospores. In spite of their inability to sporulate under developmental conditions, Dsp mutants did not appear to be defective in the sporulation process. In fact, they formed normal levels of myxospores in response to the chemical inducer glycerol. PMID- 3011747 TI - Stimulation of dihydroxyacetone and glycerol kinase activity in Streptococcus faecalis by phosphoenolpyruvate-dependent phosphorylation catalyzed by enzyme I and HPr of the phosphotransferase system. AB - Recently we reported the phosphoenolpyruvate (PEP)-dependent phosphorylation of a 55-kilodalton protein of Streptococcus faecalis catalyzed by enzyme I and histidine-containing protein (HPr) of the phosphotransferase system (J. Deutscher, FEMS Microbiol. Lett. 29:237-243, 1985). The purified 55-kilodalton protein was found to exhibit dihydroxyacetone kinase activity. Glycerol was six times more slowly phosphorylated than dihydroxyacetone. The Kms were found to be 0.7 mM for ATP, 0.45 mM for dihydroxyacetone, and 0.9 mM for glycerol. PEP dependent phosphorylation of dihydroxyacetone kinase stimulated phosphorylation of both substrates about 10-fold. Fructose 1,6-diphosphate at concentrations higher than 2 mM inhibited the activity of phosphorylated and unphosphorylated dihydroxyacetone kinase in a noncompetitive manner. The rate of PEP-dependent phosphorylation of dihydroxyacetone kinase was about 200-fold slower than the phosphorylation rate of III proteins (also called enzyme III or factor III), which so far have been considered the only phosphoryl acceptors of histidyl phosphorylated HPr. P-Dihydroxyacetone kinase was found to be able to transfer its phosphoryl group in a backward reaction to HPr. Following [32P]PEP-dependent phosphorylation and tryptic digestion of dihydroxyacetone kinase, we isolated a labeled peptide composed of 37 amino acids, as determined by amino acid analysis. The single histidyl residue of this peptide most likely carries the phosphoryl group in phosphorylated dihydroxyacetone kinase. PMID- 3011749 TI - Suppressors of the secY24 mutation: identification and characterization of additional ssy genes in Escherichia coli. AB - We previously reported (Shiba et al., J. Bacteriol. 160:696-701, 1984) the isolation and characterization of the mutation (ssy) that suppresses the protein export defect due to the secY24(Ts) mutation and causes cold-sensitive growth of Escherichia coli. This report describes more systematic isolation of ssy mutations. Among temperature-resistant revertants of the secY24 mutant, 65 mutants were found to be cold sensitive. These cold-sensitive mutations have been classified by genetic mapping. Twenty-two mutations fell into the ssyA class previously described. The remaining mutations were located at five new loci: ssyB at 9.5 min between tsx and lon; ssyD around 3 min; ssyE at 72.5 min near secY; ssyF at 20.5 min within rpsA; and ssyG at 69.0 min near argG. Two predominant classes, ssyA and ssyB, are probably affected in protein synthesis at the elongation step, whereas the ssyF mutant contained an altered form of ribosomal protein S1 (the gene product of rpsA). These cold-sensitive ssy mutations which suppress secY24 may define genes whose function is somehow involved in the secY dependent protein secretion mechanism. However, the existence of multiple suppressor loci makes it unlikely that all of these genes specify additional components of the export machinery. A delicate balance may exist between the systems for synthesizing and exporting proteins. PMID- 3011750 TI - Mutations in the araC regulatory gene of Escherichia coli B/r that affect repressor and activator functions of AraC protein. AB - Mutations in the araC gene of Escherichia coli B/r were isolated which alter both activation of the araBAD operon expression and autoregulation. The mutations were isolated on an araC-containing plasmid by hydroxylamine mutagenesis of plasmid DNA. The mutant phenotype selected was the inability to autoregulate. The DNA sequence of 16 mutants was determined and found to consist of seven different missense mutations located within the distal third of the araC gene. Enzyme activities revealed that each araC mutation had altered both autoregulatory and activator functions of AraC protein. The mutational analysis presented in this paper suggests that both autoregulatory and activator functions are localized to the same determinants of the AraC protein and that the amino acid sequence within the carboxy-terminal region of AraC protein is important for site-specific DNA binding. PMID- 3011751 TI - Role of small subunit (IlvN polypeptide) of acetohydroxyacid synthase I from Escherichia coli K-12 in sensitivity of the enzyme to valine inhibition. AB - Most of the coding sequence for the IlvN polypeptide subunit of acetohydroxyacid synthase I was deleted from the ilvB+ ilvN+ plasmid pTCN12 by in vitro methods. Several ilvB+ delta ilvN derivatives of pTCN12 were identified among transformants of a strain otherwise lacking any acetohydroxyacid synthase. Deletion derivatives produced an enzymatically active IlvB polypeptide, as shown by the Ilv+ phenotype of transformed cells and by immunologic and enzymatic assays. However, whereas the growth of pTCN12 transformants was sensitive to valine inhibition, growth of the ilvB+ delta ilvN transformants was relatively resistant. Moreover, in vitro analyses confirmed that both acetolactate and acetohydroxybutyrate synthesis in extracts of the ilvB+ delta ilvN transformants was resistant to valine inhibition, in comparison with that in extracts of pTCN12 transformants or with that catalyzed by purified acetohydroxyacid synthase I. The IlvN polypeptide had a minimal effect, if any, on IlvB polypeptide accumulation as measured by immunoprecipitation, but its absence resulted in a greater than 10 fold reduction in enzyme specific activity. PMID- 3011753 TI - Escherichia coli K-12 envelope proteins specifically required for ferrienterobactin uptake. AB - Escherichia coli genes specifically required for transport of iron by the siderophore enterobactin are designated fep. The studies reported here were initiated to identify and localize the fepB product. The plasmid pCP111, which consisted of an 11-kilobase E. coli DNA fragment containing fepB ligated to pACYC184, was constructed. The fepB gene was subcloned; in the process, complementation tests and Tn5 mutagenesis results provided evidence for the existence of a new fep gene, fepC. The order of the transport genes in the ent gene cluster is as follows: fepA fes entF fepC fepB entE. Minicell, maxicell, and in vitro DNA-directed protein synthesizing systems were used to identify the fepB and fepC products. The fepC polypeptide was 30,500 daltons in standard sodium dodecyl sulfate-polyacrylamide gels. The fepB gene was responsible for the appearance of three or four bands (their apparent molecular weights ranged from 31,500 to 36,500) in sodium dodecyl sulfate-polyacrylamide gels, depending on the gel system employed. The largest of these was tentatively designated proFepB, since it apparently had a leader sequence. Localization experiments showed that FepC was a membrane constituent and that mature FepB was present in the periplasm. An additional polypeptide (X) was also encoded by the bacterial DNA of pCP111, but its relationship to iron transport is unknown. The results indicated that ferrienterobactin uptake is mediated by a periplasmic transport system and that genes coding for outer membrane (fepA), periplasmic (fepB), and cytoplasmic membrane (fepC) components have now been identified. PMID- 3011752 TI - Nucleotide sequence, transcript mapping, and regulation of the RAD2 gene of Saccharomyces cerevisiae. AB - We determined the nucleotide sequence, mapped the 5' and 3' mRNA termini, and examined the regulation of the RAD2 gene of Saccharomyces cerevisiae. A long open reading frame within the RAD2 transcribed region encodes a protein of 1,031 amino acids with a calculated molecular weight of 117,847. A disruption of the RAD2 gene that deletes the 78 carboxyl terminal codons results in loss of RAD2 function. The 5' ends of RAD2 mRNA show considerable heterogeneity, mapping 5 to 62 nucleotides upstream of the first ATG codon of the long RAD2 open reading frame. The longest RAD2 transcripts also contain a short open reading frame of 37 codons that precedes and overlaps the 5' end of the long RAD2 open reading frame. The RAD2 3' mRNA end maps 171 nucleotides downstream of the TAA termination codon and 20 nucleotides downstream from a 12-base-pair inverted repeat that might function in transcript termination. Northern blot analysis showed a ninefold increase in steady-state levels of RAD2 mRNA after treatment of yeast cells with UV light. The 5' flanking region of the RAD2 gene contains several direct and inverted repeats and a 44-nucleotide-long purine-rich tract. The sequence T G G A G G C A T T A A found at position -167 to -156 in the RAD2 gene is similar to a sequence present in the 5' flanking regions of the RAD7 and RAD10 genes. PMID- 3011754 TI - Specific excretion of Serratia marcescens protease through the outer membrane of Escherichia coli. AB - A DNA fragment of Serratia marcescens directing an extracellular serine protease (Mr, 41,000) was cloned in Escherichia coli. The cloned fragment caused specific excretion of the protease into the extracellular medium through the outer membrane of E. coli host cells in parallel with their growth. No excretion of the periplasmic enzymes of host cells occurred. The cloned fragment contained a single open reading frame of 3,135 base pairs coding a protein of 1,045 amino acids (Mr 112,000). Comparison of the 5' nucleotide sequence with the N-terminal amino acid sequence of the protease indicated the presence of a typical signal sequence. The C-terminal amino acid of the enzyme was found at position 408, as deduced from the nucleotide sequence. Artificial frameshift mutations introduced into the coding sequence for the assumed distal polypeptide after the C terminus of the protease caused complete loss of the enzyme production. It was concluded that the Serratia serine protease is produced as a 112-kilodalton proenzyme and that its N-terminal signal peptide and a large C-terminal part are processed to cause excretion of the mature protease through the outer membrane of E. coli cells. PMID- 3011755 TI - Use of targeted insertional mutagenesis to determine whether chondroitin lyase II is essential for chondroitin sulfate utilization by Bacteroides thetaiotaomicron. AB - Bacteroides thetaiotaomicron produces two inducible chondroitin lyases (I and II) when it is grown on chondroitin sulfate. Both enzymes have very similar biochemical properties. To determine whether both enzymes are required for growth on chondroitin sulfate, we constructed a Bacteroides suicide vector, pE3-1, and used it to create an insertional mutation that interrupts the chondroitin lyase II gene of Bacteroides thetaiotaomicron. pE3-1 contains a 4.4-kilobase cryptic B. eggerthii plasmid (pB8-51), the Escherichia coli cloning vector pBR328, and the EcoRI D fragment from the conjugative B. fragilis plasmid pBF4. A 0.8-kilobase fragment from the center of the B. thetaiotaomicron chondroitin lyase II gene was inserted in pE3-1 to create pEG817. Although, pEG817 is stably maintained in E. coli and can be mobilized into B. thetaiotaomicron by the IncP plasmid R751, pEG817 is not maintained as a plasmid in Bacteroides spp. When pEG817 was mobilized into B. thetaiotaomicron, with selection for a drug marker on pEG817, transconjugants were obtained which had pEG817 inserted into the chondroitin lyase II gene. Western blot analysis was used to confirm that intact chondroitin lyase II is not produced in the mutant. The mutant was able to utilize chondroitin sulfate as a sole source of carbon, although no active chondroitin lyase II was produced. Thus chondroitin lyase I alone appears to be sufficient for growth on chondroitin sulfate. The mutant also had some minor changes in its outer membrane protein profile. However, there was no evidence that any of the major chondroitin sulfate-associated polypeptides in the outer membrane were affected by the insertion in the chondroitin lyase II gene. PMID- 3011756 TI - Cloning and physical characterization of chromosomal conjugative elements in streptococci. AB - We used a directed insertion method to introduce a nonreplicating vector plasmid into the large conjugative cat-tet element found in the chromosome of Streptococcus pneumoniae BM6001 and derivatives. To direct insertion preferentially to the conjugative element, we transferred it by conjugation to Streptococcus faecalis and then used DNA from this strain as a source of restriction nuclease fragments for ligation to digests of the vector pVA891, which can replicate in Escherichia coli but not in streptococci. This ligation mix was used to transform pneumococcal cells carrying the cat-tet element, with selection for the erythromycin resistance carried by pVA891. Eight such isolates were found, and transformation and conjugation tests showed that in each case the vector had inserted into the conjugative element, as expected. DNA from these pneumococcal strains generated a variety of E. coli plasmids which provide tools for obtaining a detailed restriction map and for defining other structural features of the streptococcal conjugative element. PMID- 3011757 TI - Structure of a conjugative element in Streptococcus pneumoniae. AB - We have cloned and mapped a 69-kilobase (kb) region of the chromosome of Streptococcus pneumoniae DP1322, which carries the conjugative omega (cat-tet) insertion from S. pneumoniae BM6001. This element proved to be 65.5 kb in size. Location of the junctions was facilitated by cloning a preferred target region from the wild-type strain Rx1 recipient genome. This target site was preferred by both the BM6001 element and the cat-erm-tet element from Streptococcus agalactiae B109. Within the BM6001 element cat and tet were separated by 30 kb, and cat was flanked by two copies of a sequence that was also present in the recipient strain Rx1 DNA. Another sequence at least 2.4 kb in size was found inside the BM6001 element and at two places in the Rx1 genome. Its role is unknown. The ends of the BM6001 element appear to be the same as those of the B109 element, both as seen after transfer to S. pneumoniae and as mapped by others in pDP5 after transposition in Streptococcus faecalis. We see no homology between the ends of the BM6001 element and find no evidence suggesting that it ever circularizes. PMID- 3011759 TI - Structural analysis of plasmid and chromosomal loci involved in site-specific excision and integration of the SLP1 element of Streptomyces coelicolor. AB - SLP1int (integrated [int] form of Streptomyces lividans plasmid 1 [SLP1]) is a Streptomyces coelicolor A3(2) transmissible sequence capable of autonomous replication as well as site-specific integration into and excision from the S. coelicolor chromosome. We report here that the plasmid and chromosomal loci involved in the integration of SLP1 and the two loci at which the recombination occurs during excision all share at least 111 base pairs of a 112-base-pair DNA sequence. Recombinational cross-over during integration or excision occurred nonrandomly within the common att sequence at or near a 25-base-pair inverted repeat. We suggest that chromosomally integrated plasmidogenic segments such as SLP1int may be involved in the acquisition and structural organization of genes encoding the diverse metabolic capabilities observed in different streptomycetes. PMID- 3011758 TI - Interaction of FtsA and PBP3 proteins in the Escherichia coli septum. AB - Mutations in the ftsA gene of Escherichia coli conferred a higher resistance to lysis induced by penicillin or by a combination of cefsulodin and furazlocillin. The ftsA2 allele codes for an FtsA protein which is inactive at 42 degrees C but is able to regain its activity once it is transferred back to 30 degrees C; ftsA2 filaments formed at 42 degrees C in the presence of penicillin divided once the penicillin was removed and the temperature was lowered to 30 degrees C. Potential septation sites in the filaments of wild-type cells treated in the same way remained inactive. The binding of a radioactively labeled derivative of ampicillin to penicillin-binding protein 3 (PBP3) was significantly decreased in strain D-3, containing the mutant allele ftsA3, when the binding assay was performed at the restrictive temperature. A molecular species able to cross-react with an anti-PBP3 serum was nevertheless found to be present in the envelope of D 3 cells. These observations suggested that the FtsA protein, a protein with a structural and regulatory role in septation, and PBP3, a protein enzymatically active in the synthesis of murein for septation, interact with each other. PMID- 3011760 TI - Central dopaminergic and noradrenergic receptor blockade in a patient with neuroleptic malignant syndrome. AB - During treatment with clomipramine and haloperidol, a 54-year-old depressed woman exhibited a typical neuroleptic malignant syndrome (NMS). Among results of biologic tests performed at least 2 weeks after discontinuation of all psychotropic treatment, the absence of normal growth hormone response after both apomorphine and clonidine challenge tests and increased levels of cerebrospinal fluid homovanillic acid and urinary 3-methoxy-4-hydroxyphenylglycol suggest that NMS may be related to central dopaminergic and possible alpha-noradrenergic receptor blockade. PMID- 3011762 TI - Crystallization and properties of myeloperoxidase from normal human leukocytes. AB - Myeloperoxidase was purified from normal human leukocytes in a crystalline state. Two types of crystals were obtained by the batchwise and dialysis crystallization methods, one of which had a bipyramidal shape belonging to the orthorhombic system. Three multiple forms of human myeloperoxidase were separated from the crystalline enzyme by CM-Sepharose chromatography with sodium chloride gradient elution. These three multiple forms were found to have very similar enzymatic, spectroscopic, and chemical properties. However, slight differences were observed in their amino acid compositions and the molecular weights of their large subunits determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Hemi-myeloperoxidase was prepared from holo-myeloperoxidase by reduction with dithiothreitol and modification with iodoacetamide, and the molecular shapes of the holo- and hemi-enzymes were determined by analytical ultracentrifugation. The axial ratios were calculated to be 2.4-3.5 for the holo enzyme and 2.9-3.1 for the hemi-enzyme. These results suggest that the shapes of the two enzymes are more spherical in solution than the proposed structural model previously reported. PMID- 3011761 TI - Alkaline phosphodiesterase I release from eucaryotic plasma membranes by phosphatidylinositol-specific phospholipase C. I. The release from rat organs. AB - From various rat organs, alkaline phosphodiesterase I was liberated by the action of phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis. Especially, a large amount of alkaline phosphodiesterase I was released from slices of small intestine, testis, lung, and kidney, but not from pancreas and liver. The release of the enzyme induced by phospholipase C was dependent on, or proportional to, the reaction time and the concentrations of the phospholipase C and the weight of the slices of small intestine or testis. Furthermore, little enzyme was released from the homogenate of pancreas. These results suggest an important role of phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membranes of rat small intestine and pancreas. The alkaline phosphodiesterase I released from slices of rat small intestine and testis had a molecular weight of about 240,000, and was activated by Mg2+ and Ca2+ but inhibited by EDTA. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9, having the Km values of 0.36 mM (small intestine) and 0.25 mM (testis). The intestinal enzyme differed from the testis enzyme in pI values, thermostability, and Arrhenius plot having a single breakpoint. PMID- 3011763 TI - Is cytochrome b558 translocated into the plasma membrane from granules during the activation of neutrophils? AB - Two laboratories (Borregaard et al. (1984) J. Biol. Chem. 259, 47; Ohno et al. (1985) J. Biol. Chem. 260, 2409) have reported that a b-type cytochrome (b558) was translocated into plasma membranes from specific granules in activated neutrophils. In an attempt to examine the cytochrome b translocation, porcine neutrophils were activated by treatment with surface-active agents such as myristate (MA) and phorbol myristate acetate (PMA), and then the postnuclear supernatants of both activated and unactivated cells were fractionated by Percoll density gradient centrifugation with a Zonal rotor. In activated neutrophils, high O2- generating activity was found in the plasma membrane fraction, which showed a peak of Na, K-ATPase activity as a marker enzyme. Cytochrome b558 was recovered 74 to 78% in the plasma membrane fraction and 14 to 16% in granules in either activated or unactivated cells. No change in specific content of cytochrome b558 was observed in plasma membranes before and after activation of cells. Furthermore, in both activated and unactivated cells, vitamin B12-binding protein, a specific granule marker, was mainly found in the bottom fractions and scarcely at all in plasma membranes. These results suggest that no translocation of cytochrome b558 occurs during activation of neutrophils. PMID- 3011764 TI - Molecular cloning of herpes simplex virus type 2 DNA. AB - Restriction enzyme HindIII digestion of the whole genome of herpes simplex virus type 2 strain 186 yielded 10 DNA fragments with molecular weights ranging from approximately 22 X 10(6) to 1.2 X 10(6), which were cloned into the HindIII site of bacterial plasmid pACYC 184. The cloned fragments were identified by hybridization to HSV-2 virus DNA and by double digestion with restriction endonucleases. The recombinant plasmids, even if they carried DNA sequences with molecular weights of more than 10(7), were efficiently replicated in E. coli HB101. PMID- 3011765 TI - Purification and characterization of a phosphoprotein phosphatase that dephosphorylates myosin and the isolated light chain from chicken gizzard smooth muscle. AB - A phosphatase that dephosphorylates myosin and the isolated light chain has been purified to near homogeneity from chicken gizzard smooth muscle. The molecular weight of the enzyme was estimated to be 100,000 and 35,000 under native and denatured conditions, respectively. It requires Mg2+ or Mn2+. The activity was measured quantitatively with a coupled enzyme system with the aid of myosin light chain kinase. The Vm and Km were determined to be 23.4 mumol/mg/min and 4.2 microM, respectively, with the isolated light chain as substrate under the optimal conditions (5 mM Mg2+ at pH 8.45). The specific activity with myosin as substrate at a concentration of 0.9 microM was found to be 1.25 mumol/mg/min, which was about one-fifth of the activity for the isolated light chain under the same conditions. The phosphatase seems to be specific to gizzard myosin. It may play an important role in the regulation of the myosin-actin interaction in smooth muscle. PMID- 3011766 TI - Glutamate synthase from Bacillus subtilis PCI 219. AB - Reduced pyridine nucleotide dependent glutamate synthase [L-glutamate: NADP+ oxidoreductase (transaminating); EC 1.4.1.13] was purified to homogeneity from Bacillus subtilis PCI 219. The molecular weight of the enzyme was 210,000, and the enzyme was composed of two nonidentical subunits with molecular weights of 160,000 and 56,000. The absorption and CD spectra of the enzyme indicated that the enzyme is an iron-sulfur flavoprotein. The enzyme was found to contain 1:1:7.4:8.7 mol of FMN, FAD, iron atoms, and acid-labile sulfur atoms per mol (MW 210,000). EPR measurements of the NADPH-reduced enzyme at 77K revealed the formation of a stable flavin semiquinone intermediate; however, none of the signals originating from the iron-sulfur cluster was observed. Still at 4.2K the EPR signals in the region of g = 2, which may originate from the paramagnetic iron-sulfur cluster, were clearly observed for both the isolated and dithionite reduced states of the enzyme. The enzyme exhibited a wide coenzyme specificity, and either NADPH or NADH could be used as electron donor, although the latter was less effective. The enzyme activity was also expressed when ammonium chloride was substituted for L-glutamine. The optimum pHs for NADPH-Gln-, NADH-Gln-, and NADPH NH3-dependent reactions were 7.8, 6.9, and 9.4, respectively. The apoenzyme exhibited substantial inactivation of the Gln-dependent activities but still retained the NH3-dependent activities. Enzyme reduction-oxidation experiments, initial velocity experiments, and product inhibition patterns revealed that both the NADPH-Gln- and NADH-Gln-dependent reactions coincided with the two-site ping pong uni-uni bi-bi kinetic mechanism, while the NADPH-NH3-dependent reaction deviated from Michaelis-Menten kinetics. The Gln-dependent activities were inhibited by several TCA cycle members, especially L-malate and fumarate, as well as L-methionine-SR-sulfoximine, pyridoxal-5'-phosphate, and pCMB. The regulation of the glutamate synthase, glutamine synthetase [EC 6.3.1.2], and glutamate dehydrogenase [EC 1.4.1.3] activities was examined with cultures of cells grown with various nitrogen and carbon sources. PMID- 3011767 TI - Purification and characterization of a trypsin-like protease in the submandibular gland of rats. AB - A trypsin-like protease (named RSP-V) was purified to homogeneity from rat submandibular glands by isoelectric focusing and high-performance liquid chromatography. The purified enzyme had an isoelectric point of 5.3 and an apparent molecular weight of 25,000, and consisted of two subunits with molecular weights of 19,500 and 6,000. RSP-V hydrolyzed BAEE, BAPA, and TAME, but not ATEE or BTPA. It had an optimum pH at around 10.0. RSP-V was strongly inhibited by soybean trypsin inhibitor, aprotinin, leupeptin, antipain, and benzamidine, but not by ovomucoid trypsin inhibitor, p-CMB, or iodoacetic acid. This enzyme partly resembled, but was not identical with, tonin. It was also different from kallikrein, salivain, and glandulain in rat submandibular gland. Although the physiological role of RSP-V has not yet been clarified, this enzyme inactivated dopa decarboxylase alone among catecholamine-synthesizing enzymes. PMID- 3011768 TI - Purification and properties of a cytochrome b560-d complex, a terminal oxidase of the aerobic respiratory chain of Photobacterium phosphoreum. AB - A cytochrome b560-d complex, a terminal oxidase in the respiratory chain of Photobacterium phosphoreum grown under aerobic conditions, was purified to near homogeneity. The purified oxidase complex is composed of equimolar amounts of two polypeptides with molecular weights of 41,000 and 54,000, as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. It contains 10.2 nmol of protoheme and 22.5 nmol of iron/mg of protein. The enzyme is a "cytochrome bd type oxidase," showing absorption peaks at 560 and 625 nm in its reduced minus oxidized difference spectrum at 77K. This oxidase combined with CO, and its CO difference spectrum at room temperature in the Soret region showed a peak at 418 nm and a trough at 434 nm. In addition, a trough at 560 nm (cytochrome b), and a trough at 620 nm and a peak at 639 nm (cytochrome d) were observed in the CO binding spectrum. This cytochrome b560-d complex catalyzed the oxidation of ubiquinol-1 and ascorbate in the presence of N,N,N',N'-tetramethyl-p phenylenediamine dihydrochloride or phenazine methosulfate. The oxidase activity required phospholipids and was inhibited by the respiratory inhibitors, KCN and NaN3, and the divalent cation, ZnSO4. Formation of a membrane potential by the cytochrome b560-d complex reconstituted into liposomes was observed with the fluorescent dye, 3,3'-dipropylthiodicarbocyanine iodide, on the addition of ubiquinol-1, showing that the enzyme provided a coupling site for oxidative phosphorylation. PMID- 3011769 TI - Dissociation of Ca2+ mobilization from breakdown of phosphatidylinositol 4,5 bisphosphate in activated human platelets. AB - The early breakdown of phosphatidylinositol 4,5-bisphosphate in human platelets stimulated by a threshold concentration of either collagen or thrombin was inhibited by 5 mM NaF through its inhibition of phospholipase C activity. However, 5 mM NaF did not inhibit Ca2+ mobilization due to the stimuli from internal stores, but it did inhibit the influx of extracellular Ca2+ through its suppression of thromboxane A2 formation. PMID- 3011770 TI - The amino-terminal hydrophobic region of the small subunit of calcium-activated neutral protease (CANP) is essential for its activation by phosphatidylinositol. AB - Ca2+-Activated neutral protease (CANP), that consists of 80K and 30K subunits, is converted to a low-Ca2+-requiring form by autolysis in the presence of Ca2+. Phosphatidylinositol greatly reduces the Ca2+-requirement for the autolysis of native CANP. However, this effect was not observed for CANP with a trimmed 30K subunit lacking the NH2-terminal hydrophobic and glycine-rich region. This suggests that the NH2-terminal hydrophobic region of the 30K subunit is important for the interaction of CANP with the cell membrane and that the calcium sensitivity of CANP is increased at the cell membrane through the effect of phosphatidylinositol. PMID- 3011772 TI - Membrane phospholipid synthesis in Escherichia coli: alteration by glycerol and physiological consequences in a pss mutant. AB - Escherichia coli mutants harboring the pss-1 allele (coding for a temperature sensitive phosphatidylserine synthase) are temperature sensitive for growth and synthesize less phosphatidylethanolamine at higher temperatures, giving rise to abnormal membrane phospholipid compositions. To obtain information concerning the determinant for the phospholipid polar headgroup composition and the lethal factor in the defective membranes, we have examined the effect of increased supply of sn-glycerol 3-phosphate on the phospholipid synthesis and the growth ability of a pss-1 mutant. For this purpose, a pair of E. coli K-12 derivatives isogenic except for the pss-1 allele was constructed from strain BB26-36 to harbor the mutations related to glycerol metabolism (glpD3, glpR2, glpKi, and phoA8). Pulse- and uniform-labeling of phospholipids with 32P at 42 degrees C in a synthetic medium with (0.2%) or without glycerol showed that glycerol further lowered the temperature-sensitive formation of phosphatidylethanolamine, removed the phosphatidate and CDP-diacylglycerol accumulated in the absence of glycerol, and resulted in an increase in cardiolipin content in the pss-1 mutant. The phospholipid synthesis and contents in the pss+ strain were not significantly affected by glycerol. Glycerol in the medium markedly enhanced the growth defect of the pss-1 mutant, which was remediable by sucrose. The results indicate that the intracellular pool of sn-glycerol 3-phosphate is the limiting factor for acidic phospholipid synthesis in the pss-1 mutant, and cardiolipin unusually accumulated is injurious to the functional E. coli membranes. Possible determinants for the phospholipid composition of the wild-type E. coli cells are also discussed on the basis of the present observations. PMID- 3011771 TI - Calmodulin-binding protein (55K + 17K) of sea urchin eggs has a Ca2+- and calmodulin-dependent phosphoprotein phosphatase activity. AB - A calmodulin-binding protein from sea urchin eggs consisting of two subunits (55 and 17K-daltons) was identified as a Ca2+-dependent phosphoprotein phosphatase similar to calcineurin in mammalian brain and to phosphatase 2B in skeletal muscle. Peptide mappings showed that the 55K subunit was different from 61K subunit of calcineurin, whereas the 17K subunit was similar to 19K subunit of calcineurin but different from calmodulin. The 55K + 17K protein of sea urchin eggs dephosphorylated 32P-inhibitor-1 in a Ca2+- and calmodulin-dependent manner. Vmax and Km for inhibitor-1 in the presence of Ca2+ and calmodulin were 2,100 pmol Pi/min/mg and 2.7 microM. Ca2+-dependent phosphatase activity for inhibitor 1 was detected in homogenates of both unfertilized and fertilized eggs, but was not detected in isolated cortices and mitotic apparatus. PMID- 3011774 TI - Purification and properties of an auxin-binding protein from maize shoot membranes. AB - An auxin-binding protein was purified from membranes of maize shoots including the coleoptiles, leaf rolls and mesocotyls. The method of Ca2+-promoted sedimentation of membrane particles was adopted for large-scale preparation. The auxin-binding protein was solubilized from the acetone-washed membranes, and purified by successive chromatographies on DEAE-Sephacel, 1-naphthylacetic acid linked AH-Sepharose 4B, and Sephadex G-100 columns. The yield of the purified protein was about 0.2 mg from 1 kg of shoots. The binding protein exists as a dimer with a subunit molecular weight of 21,000, and possesses one auxin-binding site per dimer. The preparation also contains a minor molecular form with a subunit molecular weight of 20,000. The auxin-binding protein is not a hydrophobic protein, as judged from its amino acid composition and solubility. The circular dichroic (CD) spectrum of the binding protein resembles the spectrum anticipated from the beta-structures, and shows no alpha-helix characteristic in the secondary structure. The CD spectral changes induced on the binding of auxin and its antagonist seem to be related to the receptor function. The affinity of the binding protein for auxin is dependent on pH, with an optimum at pH 5.0, while the binding protein is unstable below pH 6. We discuss here the intracellular localization of the auxin-binding protein from the view point of the controversial pH-dependence of the binding affinity and stability. PMID- 3011773 TI - Structural studies on glycolipids of shellfish. V. Gala-6 series glycosphingolipids of the marine snail, Chlorostoma argyrostoma turbinatum. AB - A series of glycosphingolipids and phosphonoglycosphingolipid containing only galactose as the sugar component were isolated from the marine snail, Chlorostoma argyrostoma turbinatum. The structures of these lipids were studied by methylation analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy and fast atom bombardment mass spectrometry, and characterized as follows: the glycosphingolipids galactosyl beta(1-1)ceramide, galactosyl beta(1 6)galactosyl beta(1-1)ceramide, galactosyl beta(1-6)galactosyl beta(1 6)galactosyl beta(1-1)ceramide and galactosyl beta(1-6)galactosyl beta(1 6)galactosyl beta(1-6)galactosyl beta(1-1)ceramide, and phosphonoglycosphingolipid N-methylaminoethylphosphonyl galactosyl(1-1)ceramide. The main molecular species of the ceramide moiety were hexadecanoyl octadecasphingenine and hydroxyhexadecanoyl-octadecasphingadienine in all of these sphingolipids. PMID- 3011775 TI - The small subunits of calcium dependent proteases with different calcium sensitivities are identical. AB - The small subunits of two calcium dependent proteases from rabbit with different calcium sensitivities were isolated by high performance liquid chromatography (HPLC) and their properties were compared. The isolated subunits were indistinguishable on polyacrylamide gel electrophoresis and HPLC. Their amino acid compositions were identical, and the peptides obtained on their digestion with lysyl-endopeptidase showed identical peptide maps on HPLC. Furthermore, the amino acid compositions and partial amino acid sequences of the corresponding peptides purified by HPLC were the same. These results indicate that the two calcium protease isozymes possess the same small subunit. PMID- 3011776 TI - Conformations of fibroblast and E. coli-derived recombinant human interferon-beta s as studied by nuclear magnetic resonance and circular dichroism. AB - The conformations of fibroblast and E. coli-derived recombinant human interferon beta s were studied by circular dichroism and nuclear magnetic resonance spectroscopy in the acidic pH region of 4.6 to 1.6. Both interferons have very similar conformations with high alpha-helix contents (approximately 70%). These results suggest that glycosylation does not appreciably change the conformation of human interferon-beta. Moreover, a slow conformational change is observed below pH 2.0, which induces the disruption of beta-sheets. PMID- 3011777 TI - Interaction of the 56,000-dalton phosphoprotein phosphatase from reticulocytes with regulin and inhibitor 2. AB - The interaction of divalent metal ions with a homogeneous 56,000-dalton phosphoprotein phosphatase isolated from rabbit reticulocytes was studied. The effects of the ions on enzymatic activity and on fluorescence from a 3-(4 maleimidylphenyl)-4-methyl-7-(diethylamino)coumarin derivative of the protein were compared. Enzymatic activity is dependent on Mn2+. The apparent association constant for Mn2+ is about 0.5 mM-1 as judged from enzymatic activity and from changes in fluorescence caused by binding of the metal ion; Ca2+ and Mg2+ do not affect enzymatic activity and appear not to bind tightly to the enzyme; however, Co2+, Fe2+, and Zn2+ bind to the protein and inhibit the Mn2+-activated enzyme. The 56,000-dalton phosphoprotein phosphatase was found to interact with regulin, a spectrin-associated protein also isolated from reticulocytes, and with skeletal muscle phosphatase inhibitor 2. The interaction was followed by changes in the enzymatic activity and by quenching of fluorescence from the coumarin derivative of the phosphatase. Homogeneous regulin (Mr approximately 230,000) increases the activity of the enzyme severalfold; this stimulation is Mn2+-dependent. Inhibitor 2 decreases enzyme activity but only if the two proteins are preincubated in the absence of Mn2+. Comparable differences in the effect of Mn2+ were also observed in parallel experiments in which changes in fluorescence from the coumarin labeled 56,000-dalton phosphatase were measured. In these experiments, it was shown that Mn2+ enhances the interaction between regulin and the 56,000-dalton phosphatase, but inhibits the interaction between the phosphatase and inhibitor 2. PMID- 3011778 TI - Different conformations of the 3' termini of initiator and elongator transfer ribonucleic acids. An EPR study. AB - The 3' ends of transfer ribonucleic acids were covalently labeled with a nitroxide spin label. The 3' end of initiator tRNA (tRNAMetf) from Escherichia coli shows different motional behavior than the 3' terminus of elongator tRNAs as monitored by EPR. The line shapes of the EPR spectra are quite sensitive to the buffer conditions, as shown by measurements in 4 different buffers. The data are consistent with a constrained or folded back 3' terminus in the initiator tRNA as opposed to the freely rotating elongator 3' terminus. The EPR spectra are also sensitive to aggregation of the tRNA. PMID- 3011779 TI - Inhibition of monoterpene cyclases by sulfonium analogs of presumptive carbocationic intermediates of the cyclization reaction. AB - The enzymatic cyclization of geranyl pyrophosphate to monoterpenes is thought to proceed through a series of carbocation-pyrophosphate anion paired intermediates. Sulfonium analogs of two putative carbocationic intermediates of the cyclization sequence were shown to be inhibitors of the conversion of the acyclic precursor to the bicyclic monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate by partially purified cyclase preparations from sage (Salvia officinalis). The sulfonium analog of the tertiary allylic, linalyl, intermediate (i.e. methyl-(4 methylpent-3-en-1-yl)vinyl-sulfonium perchlorate) provided respective Ki values of 2.5 microM and 3.0 microM against the cyclization to alpha-pinene and bornyl pyrophosphate at a substrate concentration of 5 microM, whereas the sulfonium analog of the monocyclic, alpha-terpinyl, intermediate (i.e. dimethyl-(4 methylcyclohex-3-en-1-yl) sulfonium iodide) exhibited respective Ki values of 3.4 microM and 3.9 microM against the same two cyclizations. The potency of inhibition in all cases increased with increasing substrate concentration, indicating that the affinity of the enzymes for the sulfonium analogs was increased by the presence of the pyrophosphate ester. Inorganic pyrophosphate at a concentration of 50 microM, which alone had little influence on the cyclizations, increased the effectiveness of inhibition of the sulfonium analogs severalfold, and the apparent Ki for inorganic pyrophosphate was reduced manyfold by the presence of either analog at 5 microM. That the combination of sulfonium analog and pyrophosphate provided synergistic inhibition of the electrophilic cyclizations indicated that the cyclases bind the paired species more tightly than either partner alone. Specificity studies suggested that inhibition by the above sulfonium ion:pyrophosphate pairs was due to both electronic and structural resemblance to intermediates of the reaction. PMID- 3011780 TI - The effects of base analogue substitutions on the cleavage by the EcoRI restriction endonuclease of octadeoxyribonucleotides containing modified EcoRI recognition sequences. AB - It has been proposed that recognition of specific DNA sequences by proteins is accomplished by hydrogen bond formation between the protein and particular groups that are accessible in the major and minor grooves of the DNA. We have examined the DNA-protein interactions involved in the recognition of the hexameric DNA sequence, GAATTC, by the EcoRI restriction endonuclease by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues. The base analogues hypoxanthine, 2-aminopurine, 2,6-diaminopurine, N6-methyladenine, 5 bromouracil, uracil, 5-bromocytosine, and 5-methylcytosine were incorporated as single substitutions into the octadeoxyribonucleotide d(pG-G-A-A-T-T-C-C). The effects of the substitutions on the interactions between the EcoRI endonuclease and its recognition sequence were monitored by determining the steady state kinetic values of the hydrolysis reaction. The substitutions resulted in effects that varied from complete inactivity to enhanced reactivity. The enzyme exhibited Michaelis-Menten kinetics with those substrates that were reactive, whereas octanucleotide analogues containing N6-methyladenine at either adenine position, uracil at the second thymine position, or 5-bromocytosine or 5-methylcytosine at the cytosine position were unreactive. The results are discussed in terms of possible effects on interactions between the enzyme and its recognition site during the reaction. An accompanying paper presents the results of a similar study using these oligonucleotides with the EcoRI modification methylase. PMID- 3011781 TI - The effects of base analogue substitutions on the methylation by the EcoRI modification methylase of octadeoxyribonucleotides containing modified EcoRI recognition sequences. AB - We have examined the DNA-protein interactions involved in the recognition of a specific hexameric sequence, GAATTC, by the EcoRI modification methylase by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues. The base analogues 2-aminopurine, 5-bromocytosine, 5-bromouracil, 2,6 diaminopurine, hypoxanthine, 5-methylcytosine, N6-methyladenine, and uracil were incorporated as single substitutions into the octadeoxyribonucleotide d(pG-G-A-A T-T-C-C). The effects of the substitutions on the ability of the enzyme to methylate the modified substrates were monitored by determining the steady state kinetic values of the reaction in the presence of the cosubstrate, S adenosylmethionine. The substitutions resulted in effects ranging from complete inactivity to enhanced reactivity. The enzyme exhibited Michaelis-Menten kinetics with those analogues that were active, whereas the octanucleotides containing hypoxanthine at the guanine site, N6-methyladenine at the first or 2-aminopurine at the second adenine site, or uracil at the second thymine site were completely inactive. The results are discussed in terms of the possible interactions between the methylase and its recognition sequence. In addition, the interactions are compared to those of the EcoRI restriction endonuclease, which has been similarly tested with the same analogue oligonucleotides. The results of that study are reported in an accompanying paper. Although both enzymes recognize the same sequence, they do so in different ways. PMID- 3011782 TI - ACTH induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase, cholesterol biosynthesis, and steroidogenesis in primary cultures of bovine adrenocortical cells. AB - The current studies demonstrate that corticosteroidogenesis can be maintained by primary cultures of bovine adrenocortical cells under lipoprotein-depleted conditions. The cholesterol necessary as substrate for steroid synthesis was found to arise from de novo synthesis within these cells. Adrenocorticotropin (ACTH) increased 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity 5-fold within 12 h after addition to the medium. The increase in activity apparently represented accumulation of enzyme as determined by protein blotting and immunodetection. The predominant immunodetectable species of HMG-CoA reductase from bovine adrenal cells was 97,000 daltons; no higher molecular mass species was detectable. The ACTH induction of HMG-CoA reductase activity could be prevented after inhibition of cholesterol conversion to pregnenolone with clotrimazole. These results are suggestive that ACTH increases adrenocortical cholesterol biosynthesis and HMG-CoA reductase activity after conversion of a cellular pool of cholesterol and/or oxysterol into steroid. The increased rate of cholesterol biosynthesis is then capable of maintaining ACTH-promoted steroid production. This is the first study, in vitro, to demonstrate an ACTH-promoted accumulation of HMG-CoA reductase of adrenocortical cells. PMID- 3011783 TI - The binding of fucose-containing glycoproteins by hepatic lectins. Re-examination of the clearance from blood and the binding to membrane receptors and pure lectins. AB - The nature of the hepatic receptors that bind glycoproteins through fucose at the non-reducing termini of oligosaccharides in glycoproteins has been examined by three different approaches. First, the clearance from blood of intravenously injected glycoproteins was examined in mice with the aid of neoglycoproteins of bovine serum albumin (BSA). The clearance of fucosyl-BSA was rapid and was not strongly inhibited by glycoproteins that inhibit clearance mediated by the galactose or the mannose/N-acetylglucosamine receptors of liver. The clearance of Fuc alpha 1,3(Gal beta 1,4)GlcNAc-BSA (where Fuc is fucose) was inhibited weakly by either Fuc-BSA or Gal beta 1,4GlcNAc-BSA but strongly by a mixture of the two neoglycoproteins, suggesting that its clearance was mediated by hepatic galactose receptors as well as by a fucose-binding receptor. Second, the binding of neoglycoproteins to a membrane fraction of mouse liver was examined. Fuc-BSA binding to membranes was Ca2+ dependent but was not inhibited by glycoproteins that would inhibit the galactose or the mannose/N-acetylglucosamine receptors. In addition, the binding of Fuc-BSA and Gal beta 1,4GlcNAc-BSA differed as a function of pH, in accord with binding of Fuc-BSA through fucose-specific hepatic receptors. Finally, the binding of neoglycoproteins to the pure galactose lectin from rat liver was examined. Neither Fuc-BSA nor Fuc alpha 1,2Gal beta 1,4GlcNAc BSA bound the galactose lectin, although Fuc alpha 1,3(Gal beta-1,4) GlcNAc-BSA bound avidly. Taken together, these studies suggest that a fucose-binding receptor that differs from the galactose and the mannose/N-acetylglucosamine receptors may exist in rat and mouse liver. PMID- 3011784 TI - Separation of two populations of endocytic vesicles involved in receptor-ligand sorting in rat hepatocytes. AB - Rat hepatocytes incubated in high K+ buffer (all Na+ is replaced by K+) internalize glycoproteins bearing terminal galactose moieties but are not able to deliver them to lysosomes (Baenziger, J. U., and Fiete, D. (1982) J. Biol. Chem. 257, 6007-6009). Instead, internalized ligand accumulates in a prelysosomal compartment(s) with a density similar to that of plasma membrane. We have separated two populations of prelysosomal endocytic vesicles from hepatocytes incubated in high K+ buffer. The vesicle population VR.L has a mean density of 1.14 by sucrose gradient centrifugation and contains functionally active Gal/GalNAc-specific receptor which is able to bind intravesicular ligand. The vesicle population VL has a mean density of 1.19. It contains ligand, but is deficient in Gal/GalNAc-specific receptor when compared to VR.L. These two vesicle populations appear to arise from intracellular organelles which participate in receptor-ligand segregation in rat hepatocytes. Pulse-chase experiments indicate that ligand passes from VR.L to VL. VR.L and VL are also detected in hepatocytes incubated in buffers containing physiologic amounts of Na+; however, the proportion of ligand found in VL is less than in cells incubated in K+-containing buffer. The primary effect of high K+ buffer is to prevent exit of ligand from VL whereas the accumulation of ligand in VR.L is likely secondary to the effect on VL. Membrane protein constituents of VR.L and VL were identified by vectorial lactoperoxidase labeling using a galactosyl conjugate of lactoperoxidase. Vesicles containing Gal-lactoperoxidase were isolated and labeling initiated by addition of 125I, glucose, and glucose oxidase. The labeling patterns for VR.L and VL by sodium dodecyl sulfate polyacrylamide gel electrophoresis were distinct from the more complex labeling pattern obtained at the cell surface. Analysis by two-dimensional electrophoresis demonstrated a highly selective labeling pattern with only a small number of differences between VR.L and VL. This suggests that the major membrane components of the compartments prior to and following receptor-ligand segregation are the same. Thus, receptors may be selectively removed from these membranes during the process of receptor-ligand segregation. PMID- 3011785 TI - Disulfides of the lutropin receptor. AB - Affinity cross-linking of the lutropin receptor with 125I-human choriogonadotropin (hCG) on porcine granulosa cells produced four distinct homone receptor complexes under reducing conditions. They contain 18-, 24-, 28-, and 34 kDa components (Ji, I., Bock, J. H., and Ji, T. H. (1985) J. Biol. Chem. 260, 12815-12821). Photoaffinity labeling and cross-linking produced 136-, 102-, and 74-kDa hCG-receptor complexes under reducing conditions and the 136-kDa complex under nonreducing conditions. In addition, the unreduced 102-kDa complex was seen in photoaffinity labeling but not in cross-linking. When the unreduced 136-kDa complex was reduced, the 102- and 74-kDa complexes were generated, indicating release of the 34- and the 28-kDa components in two steps. When the unreduced 102 kDa complex was reduced, the 74-kDa complex was produced, indicating the release of a 28-kDa component. The 74-kDa complex could not be reduced but was cleaved by alkaline treatment to produce the hCG alpha beta dimer. The results indicate that the 24-kDa component is released from the 74-kDa complex, since the apparent mass of the hCG alpha beta dimer on gels is 50 kDa. The 24-kDa component appears to be the initial site for photoaffinity labeling or cross-linking and to be disulfide linked to the 28-kDa component which is in turn disulfide linked to the 34-kDa component. These intercomponent disulfides exist in some receptors but not all. Formation of the disulfide-linked 136-kDa band required the presence of a sulfhydryl-blocking agent, N-ethylmaleimide. In particular, the 34-kDa component was vulnerable to reduction. There was no significant evidence of disulfides between the hormone and any of the receptor components. PMID- 3011786 TI - Phosphorylation and transformation sensitivity of a major collagen-binding protein of fibroblasts. AB - Using affinity chromatography with immobilized gelatin and native type I collagen, we have identified the major collagen-binding proteins in Nonidet P-40 extracts of chick embryo fibroblasts labeled with [35S] methionine. After washing the gelatin- or collagen-Sepharose beads with high ionic strength buffer, a 47,000-dalton protein was the only major protein besides fibronectin found to bind to these affinity beads. The isoelectric point of this protein was approximately 9.0, with a closely spaced minor spot. The total amount and the synthesis of this collagen-binding protein were both decreased in Rous sarcoma virus-transformed cells. This collagen-binding protein was found to be phosphorylated by incubating intact cells with [32P]orthophosphate. Phosphoamino acid analysis revealed that serine and threonine residues were phosphorylated, but tyrosine was not. Although quantities of the 47,000-dalton protein labeled with [35S]methionine were decreased by a factor 2.5 after transformation, the incorporation of [32P]orthophosphate/unit of protein was 5-7-fold higher in transformed cells. In temperature-sensitive mutant virus-infected cells, the amount of the 47,000-dalton protein was also decreased at the temperature permissive for transformation, and the incorporation of [32P]orthophosphate/protein was also increased. These studies establish that a major membrane-associated collagen-binding protein of fibroblasts is phosphorylated and that it is altered in both total quantity and degree of phosphorylation after malignant transformation. PMID- 3011787 TI - Characterization of the DNA binding domain of bacteriophage lambda O protein. AB - The lambda O and P gene products are required for the initiation of lambda DNA replication. In order to study the biochemistry of this process, we have constructed plasmids that carry the lambda O gene, P gene, and half of the O gene coding for the amino-terminal half of the O protein. Each is under the control of the inducible lambda promoter, PL. We have purified these three proteins from induced cells carrying the plasmids. Our results show that the amino-terminal portion of the O protein binds to the lambda origin of replication in a manner similar to the intact lambda O protein, demonstrating that the amino-terminal portion of O protein contains the DNA binding domain. Using chromatographic procedures, we have isolated a complex of lambda O and P proteins with lambda dv DNA. The amino-terminal portion of the O protein does not complex with P protein under the same conditions. This suggests that the specificity of the lambda O protein for P protein resides in the carboxyl-terminal half of the lambda O protein. Our results also show that, while the intact O protein is active in in vitro replication of lambda dv plasmid DNA, the amino-terminal portion of the O protein is inactive and is a competitive inhibitor of the lambda O protein in this reaction. These results confirm previous genetic observations that were interpreted as indicating a bifunctional structure for the lambda O protein with the amino-terminal domain recognizing the lambda origin of replication and the carboxyl-terminal domain interacting with the lambda P protein. PMID- 3011788 TI - The 5' flanking region of the ornithine transcarbamylase gene contains DNA sequences regulating tissue-specific expression. AB - Ornithine transcarbamylase (OTCase) is a mitochondrial matrix enzyme that catalyzes the 2nd step in the mammalian urea cycle. The gene encoding OTCase is located on the X chromosome and expression of OTCase is limited almost exclusively to hepatocytes. We have characterized a lambda phage recombinant, isolated from a mouse genomic library, that spans the first two exons of the mouse OTCase gene. Nuclease S1 mapping and primer extension analysis of this clone allowed us to determine that the transcription start site is 136 base pairs (bp) upstream from the translation initiation codon. Two TATA-like sequences were found 25 and 153 bp from the transcription initiation point. An 800-bp fragment containing the 5' flanking region of the OTCase gene was fused upstream to the coding sequence of the chloramphenicol acetyltransferase gene to assay promoter activity. This plasmid was introduced into mouse fibroblast NIH 3T3 cells and human hepatoma Hep G2 cells by the calcium phosphate co-precipitation method. After DNA transfection chloramphenicol acetyltransferase activity was observed only in Hep G2 cells. We conclude that this 800-bp fragment contains sufficient information to control OTCase gene expression in a tissue-specific manner, probably by interacting with trans-acting factor(s) which are not present in the other cell line. PMID- 3011789 TI - Platelet-activating factor and leukotriene biosynthesis is inhibited in polymorphonuclear leukocytes depleted of arachidonic acid. AB - Rat peripheral or elicited polymorphonuclear leukocytes 90% deficient in arachidonic acid incorporate, after stimulation with the calcium ionophore A23187, 86% less acetate into platelet-activating factor than control. The total amount of platelet-activating factor in the ionophore stimulated elicited polymorphonuclear leukocytes deficient in arachidonate, measured by gas chromatography-negative ion chemical ionization mass spectrometry, was 84% less than that of control. The mass spectrometry also revealed the presence of various molecular species of platelet-activating factor ranging from 1-O-tetradecyl to 1 O-nonadecyl forms in both the deficient and control cells. However, the 1-O hexadecyl was the predominate molecular species representing 79 and 96% of the total platelet-activating factor in the respective deficient and control cells. Other molecular species were less than 1.5 and 8.5% of the total for control and deficient polymorphonuclear leukocytes, respectively. Leukotriene B4 formation was also inhibited by 90% in the deficient cells. Both platelet-activating factor and leukotriene B4 biosynthesis could be partially restored in arachidonic acid deficient cells by prelabeling the cells with arachidonate. This represents the first dietary link with platelet-activating factor biosynthesis. PMID- 3011790 TI - Herpesvirus infection prevents activation of cytoplasmic cholesteryl esterase in arterial smooth muscle cells. AB - Herpesvirus infection has been shown to alter the cholesteryl ester cycle in avian arterial smooth muscle cells, resulting in cytoplasmic cholesteryl ester accumulation (Hajjar, D. P., Falcone, D. J., Fabricant, C. G., and Fabricant, J. (1985) J. Biol. Chem. 260, 6124-6128). In this study, we attempted to define some of the regulatory mechanisms associated with the control of cytoplasmic cholesteryl esterase in Marek's disease herpesvirus (MDV)-infected cells. We found that cholesteryl esterase activity in MDV-infected cells could not be activated by dibutyryl cyclic AMP, dibutyryl cyclic AMP added together with protein kinase, or agonists of adenylate cyclase. Activation of cytoplasmic cholesteryl esterase activity occurred in uninfected cells and in cells infected with a control virus, turkey herpesvirus. Furthermore, the rate of cholesterol efflux from arterial smooth muscle cells challenged with dibutyryl cyclic AMP was unchanged in MDV-infected cells as compared to uninfected or turkey herpesvirus infected cells in which efflux was increased. We propose that the reduced cytoplasmic cholesteryl esterase activity in lipid-laden, herpesvirus-infected cells is due partly to its inability to be activated by the cyclic AMP-protein kinase mechanism. This may contribute to the pathologic changes seen in MDV infected arterial cells, including accumulation of intracellular cholesteryl esters. PMID- 3011791 TI - Transcriptional and post-transcriptional regulation of L-type pyruvate kinase gene expression in rat liver. AB - The effects of starvation, refeeding a diet high in carbohydrate, administration of glucagon and cyclic AMP, thyroidectomy, and adrenalectomy on transcription of the gene for liver L-type pyruvate kinase and on the accumulation of cytoplasmic mRNA for L-type pyruvate kinase were investigated in rat. Transcription of the gene was undetectable in either fasted or protein-fed rats. Refeeding fasted rats a carbohydrate-rich diet stimulated an increase in L-type pyruvate kinase mRNA, preceded by an increase in the gene transcription. Transcription was maximal at 12 h of refeeding, decreasing to 10% of maximum at 72 h. The level of L-type pyruvate kinase mRNA remained constant at 50% of maximum for at least 120 h. Neither thyroidectomy nor adrenalectomy affected gene transcription in fasted rats refed the carbohydrate-rich diet, despite a decrease in mRNA abundance to 40 and 20%, respectively, of controls fed a normal diet. Glucagon or cyclic AMP totally blocked the increase in transcription of the L-type pyruvate kinase gene caused by feeding a carbohydrate-rich diet to previously fasted rats. Nevertheless, the level of L-type pyruvate kinase mRNA remained high for 3 h after glucagon administration. After 3 h, the mRNA decreased rapidly with a half life less than 1 h. Thus, expression of the gene for L-type pyruvate kinase is regulated at both transcriptional and post-transcriptional levels. The transcription is regulated by two major effectors, one positive, namely carbohydrates, and one negative, namely glucagon (via cyclic AMP). Both agents probably act at the level of the mRNA stability as well. Glucocorticoids and thyroid hormones do not regulate transcription of the gene for L-type pyruvate kinase but do appear to be required for a normal accumulation of the transcripts in the cytoplasm. PMID- 3011792 TI - Dephosphorylation of phosphoproteins of human liver plasma membranes by endogenous and purified liver alkaline phosphatases. AB - Purified alkaline phosphatase and plasma membranes from human liver were shown to dephosphorylate phosphohistones and plasma membrane phosphoproteins. The protein phosphatase activity of the liver plasma membranes was inhibited by levamisole, a specific inhibitor of alkaline phosphatase, and by phenyl phosphonate and orthovanadate, but was relatively insensitive to fluoride (50 mM). Endogenous membrane protein phosphatase activity was optimal at pH 8.0, compared to pH 7.8 for purified liver alkaline phosphatase. Plasma membranes also exhibited protein kinase activity using exogenous histone or endogenous membrane proteins (autophosphorylation) as substrates; this activity was cAMP-dependent. Autophosphorylation of plasma membrane proteins was apparently enhanced by phenyl phosphonate, levamisole, or orthovanadate. The dephosphorylation of phosphohistones by protein phosphatase 1 was not inhibited by levamisole but was inhibited by fluoride. Inhibition of endogenous protein phosphatase activity by orthovanadate during autophosphorylation of plasma membranes could be reversed by complexation of the inhibitor with (R)-(-)-epinephrine, and the dephosphorylation that followed was levamisole-sensitive. Neither plasma membranes nor purified liver alkaline phosphatase dephosphorylated glycogen phosphorylase a. These results suggest that the increased [32P]phosphate incorporation by endogenous protein kinases into the membrane proteins is due to inhibition of alkaline phosphatase and that the major protein phosphatase of these plasma membranes is alkaline phosphatase. PMID- 3011793 TI - The nucleotide sequences of the rbsD, rbsA, and rbsC genes of Escherichia coli K12. AB - The nucleotide sequences of rbsD, rbsA, and rbsC have been determined. These genes encode components of the high affinity ribose transport system in Escherichia coli, and together with the sequences of rbsB (Groarke, J.M., Mahoney, W.C., Hope, J.N., Furlong, C.E., Robb, F.T., Zalkin, H., and Hermodson, M.A. (1983) J. Biol. Chem. 258, 12952-12956) and rbsK (Hope, J.N., Bell, A.W., Hermodson, M.A., and Groarke, J.M. (1986) J. Biol. Chem. 261, 7663-7668), they complete the nucleotide sequence of the first five genes of the rbs operon. Nuclease S1 mapping places the transcriptional start site for the operon 29 base pairs upstream from the most likely translational start site for rbsD. The open reading frames of rbsD, rbsA, and rbsC encode proteins of 139, 501, and 321 amino acid residues, respectively. The character of the proteins varies widely, from very hydrophilic for the rbsA product to exceedingly hydrophobic for the rbsC product. The intercistronic spaces between the three genes are very short, with the stop codons of the upstream genes overlapping the ribosome-binding sites of the downstream genes. This may imply translational control of expression of these genes, the products of which presumably form a membrane-bound transport complex. PMID- 3011794 TI - Ribokinase from Escherichia coli K12. Nucleotide sequence and overexpression of the rbsK gene and purification of ribokinase. AB - The nucleotide sequence of a 1455-base pair TaqI-HinfI fragment of the rbs operon of Escherichia coli K12 has been determined. It includes the 3' terminus of rbsB (the gene for ribose-binding protein) and the entire rbsK gene, encoding ribokinase. Potential consensus promoter sequences and a stable stem-loop structure are present in the rbsB-rbsK intercistronic region. The regulatory significance of these sequence features is discussed with respect to the rbs operon. rbsK has been cloned downstream from the Serratia marcescens trp promoter on a multicopy plasmid. Cells harboring this plasmid, when grown on minimal ribose plus ampicillin, express ribokinase at the level of 2% of the soluble protein, and induction with indoleacrylic acid raises ribokinase levels another 8 fold. Ribokinase has been purified to homogeneity (216 mumol/min/mg) from a strain harboring this plasmid. Protein sequence analyses of peptides generated by cyanogen bromide cleavage and o-iodosobenzoic acid cleavage confirmed the translation initiation site and the reading frame of the DNA sequence. Amino acid compositions of native ribokinase and the C-terminal dodecapeptide agree with the predicted amino acid compositions, confirming the accuracy of the DNA sequence and the translation termination site. PMID- 3011795 TI - Purification of insulin-like growth factor I receptor from human placental membranes. AB - Insulin-like growth factor (IGF) I receptor was purified from Triton X-100 solubilized human placental membranes by wheat germ agglutinin-Sepharose chromatography followed by immunoaffinity chromatography using alpha IR-3, a monoclonal antibody directed against the IGF-I receptor. Purification of 3200 fold and 2800-fold was achieved from wheat germ agglutinin-Sepharose eluates with regard to IGF-I binding and kinase activities. Sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions revealed two major protein bands corresponding to the alpha and beta subunits of the receptor, which accounted for at least 90% of the protein content. The purified receptor bound 10 20 micrograms of IGF-I/mg of protein and was more than 95% free of contamination by insulin receptor. It sedimented in glycerol gradients as a single species with a sedimentation coefficient of 13.7 S and gave three protein bands with Mr = approximately 300,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, indicating that alpha 2 beta 2 is an intact form of the IGF-I receptor. The purified receptor, when incubated with [gamma-32P] ATP, became phosphorylated at tyrosine residues of its beta subunit. This was stimulated 3-fold by IGF-I. It also had IGF-I-stimulated tyrosine kinase activity (5264 pmol of 32P incorporated/min/mg of protein) toward a synthetic peptide corresponding to the autophosphorylation site of pp60src. These data strongly suggest that it is a tyrosine-specific protein kinase. PMID- 3011796 TI - Mammalian alpha 1-adrenergic receptor. Purification and characterization of the native receptor ligand binding subunit. AB - alpha 1-Adrenergic receptors from a cultured smooth muscle cell line (DDT1 MF-2) have been solubilized with digitonin and purified to apparent homogeneity by sequential chromatography on a biospecific affinity support (Sepharose-A55453 (4 amino-6,7-dimethoxy-2-[4-[5-(4-amino-3-phenyl) pentanoyl]-1-piperazinyl] quinazoline), an alpha 1 receptor-selective antagonist), a wheat germ agglutinin agarose gel, and a high performance steric exclusion liquid chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveals a peptide with an apparent Mr = 80,000 that co-migrates with the peptide labeled by the specific alpha 1-adrenergic receptor photoaffinity probe 4-amino-6,7-dimethoxy-2 [4-[5-(4-azido-3-[125I]iodophenyl)pentanoyl] -1-piperazinyl] quinazoline. The specific activity (approximately 13,600 pmol of ligand binding/mg of protein) of purified receptor preparations is consistent with that expected for a pure peptide of Mr = 80,000 containing a single ligand binding site. Overall yields approximate 14% of initial crude particulate binding. The purified receptor preparations bind agonist and antagonist ligands with appropriate alpha 1 adrenergic specificity, stereoselectivity, and affinity. Peptide maps of the pure alpha 1-adrenergic receptor and the pure human platelet alpha 2-adrenergic receptor (Regan, J.W., Nakata, H., DeMarinis, R.M., Caron, M.G., and Lefkowitz, R.J. (1986) J. Biol. Chem. 261, 3894-3900) using several different proteases suggest that these two receptors show little if any structural homology. PMID- 3011797 TI - Absence of reactive intermediates in the formation of catechol estrogens by rat liver microsomes. AB - Release of 3H2O from regiospecifically labeled estradiol was measured during 2 hydroxylation of this estrogen by rat liver microsomes. The amount of tritium remaining in the isolated catechol estrogen was also determined. Virtually all the tritium was removed from C-2 during the reaction confirming the absence of an NIH shift. About 20% of the tritium at C-1 was also lost without any such change occurring at C-4 or C-6,7 of the steroid molecule. These findings provide evidence for the formation of an arene oxide or o-semiquinone intermediate during the conversion of estradiol to 2-hydroxyestradiol. No indication of adduct formation at either C-1 or C-4 during this biotransformation was obtained although the 2-hydroxylated product was able to react with a nucleophile such as glutathione. The different regiospecificity of tritium loss in the generation of catechol estrogens and in their subsequent reaction leads to the important conclusion that the reactive intermediates in the two processes must be different. The possible role of catechol estrogens in neoplastic transformation is discussed. PMID- 3011798 TI - A brain phosphatase with specificity for microtubule-associated protein-2. AB - A protein phosphatase has been isolated from brain using, as assay substrate throughout the purification, microtubule-associated protein-2 which had been phospho-labeled by its associated kinase. In contrast to other protein phosphatases, this phosphatase can effectively release phosphate from both the microtubule-binding and projection domains of microtubule-associated-protein-2. This enzyme appears to be a distinct, specific phosphatase that does not readily fit into previous classification schemes and is possibly the enzyme responsible for dephosphorylating microtubule-associated protein-2 in vivo. PMID- 3011799 TI - Coupling between the sodium and proton gradients in respiring Escherichia coli cells measured by 23Na and 31P nuclear magnetic resonance. AB - The relationship between the steady-state sodium gradient (delta pNa) and the protonmotive force developed by endogenously respiring Escherichia coli cells has been studied quantitatively, using 23Na NMR for measurement of intracellular and extracellular sodium concentrations, 31P NMR for measurement of intracellular and extracellular pH, and tetraphenylphosphonium distribution for measurement of membrane potential. At constant protonmotive force, the sodium concentration gradient was independent of extracellular concentrations over the measured range of 4-285 mM, indicating that intracellular sodium concentration is not regulated. The magnitude of delta pNa was measured as a function of the composition and magnitude of the protonmotive force. At external pH values below 7.2, delta pNa was parallel to delta pH but showed no simple relationship to the membrane potential; above pH 7.2 the parallel relationship began to diverge, with delta pH continuing to decrease but delta pNa starting to level off or increase. Although plots of delta pNa versus delta pH had slopes of close to 1, the value of delta pNa consistently exceeded that of delta pH by approximately 0.4 units, indicating a partially electrogenic character to the putative H+/Na+ antiport. The apparent stoichiometry was 1.13 +/- 0.01 at external pH below 7.2. The possible significance of this nonintegral stoichiometry is discussed according to a model in which two distinct integral stoichiometries (possibly 1H+/1Na+ and 2H+/1Na+) are available with some relative probability; the model predicts futile cycling of sodium ions and a dissipative proton current. In the course of this study, we discovered that the magnitude of the pH gradient developed by the cells was osmolarity-dependent, yielding steady-state intracellular pH values that varied from 7.1 at 100 mosm to 7.7 at 800 mosm. PMID- 3011800 TI - Nitrobenzyl radical metabolites from microsomal reduction of nitrobenzyl chlorides. AB - The o-, m-, and p-nitrobenzyl chlorides are reduced aerobically and anaerobically by NADPH and rat hepatic microsomes. Under aerobic conditions, these nitro anion radicals reduce oxygen to superoxide as demonstrated by oxygen consumption and spin trapping of superoxide with 5,5-dimethyl-1-pyrroline N-oxide. At low oxygen concentration, the p- and o-nitro anion radicals undergo intramolecular electron transfer and decompose to carbon-centered nitrobenzyl radicals, which can be spin trapped with t-nitrosobutane. The p-nitrobenzyl (o-nitrobenzyl) radical adduct was characterized by a nitrogen hyperfine splitting of 16.5 G (17.1 G) and two equivalent beta-hydrogen hyperfine splittings of 10.6 G (14.4 G). The spin trap 5,5-dimethyl-1-pyrroline N-oxide also yields adducts characteristic of carbon centered free radicals. This unimolecular decomposition is much faster than the disproportionation decay, which is characteristic of most nitro anion radicals, and the primary o- and p-nitrobenzyl chloride anion radicals never achieve detectable concentrations. The nitrobenzyl radical trapping is not inhibited by metyrapone or CO. In contrast, the m-nitrobenzyl anion radical does achieve a detectable steady-state concentration, which is increased 20% by either metyrapone or a CO atmosphere. PMID- 3011801 TI - Partial identity of the 2-oxoglutarate and ascorbate binding sites of prolyl 4 hydroxylase. AB - Various hydroxybenzenes, hydroxybenzoic acids, and related compounds resemble structurally both 2-oxoglutarate and ascorbate, two reactants needed in the reaction of prolyl 4-hydroxylase. These substances were found to inhibit prolyl 4 hydroxylase competitively with respect to both cosubstrates. Ortho-dihydroxy derivatives, which are capable of chelating the enzyme-bound iron, were the most effective inhibitors, with Ki values of about 5 microM. In contrast, pyridine 2 carboxylates, which have previously been reported to inhibit the enzyme competitively with respect to 2-oxoglutarate, were found to inhibit it uncompetitively with respect to ascorbate. In a separate set of experiments the side chain of the ascorbate molecule was shown to make no significant contribution to the binding of the reductant to the enzyme, as D(-)-isoascorbate and 5,6-O-isopropylidene ascorbate gave essentially the same Vmax and Km values as ascorbate. On the other hand, structural modifications of the ring atoms that abolished the chelating capacity destroyed both the cosubstrate and inhibitory activity, as in L-galactono gamma-lactone. The ascorbate binding site therefore appears to consist of two cis-positioned coordination sites of the enzyme-bound iron and is thus partially identical to the binding site of 2-oxoglutarate. This mode of interaction suggests that ascorbate reduces the enzyme-bound iron through an "inner-sphere" mechanism. The inhibitors studied appear to react at different phases of the catalytic cycle, determined by the oxidation state of the enzyme bound iron atom. PMID- 3011802 TI - Characterization of phorbol ester- and diacylglycerol-stimulated secretion in permeable GH3 pituitary cells. Interaction with Ca2+. AB - Prolactin (PRL) release in permeable GH3 pituitary cells was stimulated by the protein kinase C activators 12-O-tetradecanoylphorbol 13-acetate (TPA) and 1 oleoyl-2-acetyl-sn-glycerol (OAG). Both agents stimulated secretion at 10 nM Ca2+, but higher [Ca2+] (greater than 0.1 microM) potentiated TPA and OAG action. Maximal potentiation occurred at 1 microM calculated free Ca2+, and a similar value was obtained when the cytoplasmic [Ca2+] was measured with the Ca2+ sensitive dye Quin 2. Release of a secretory sulfated proteoglycan was also stimulated by TPA and OAG in permeable GH3 cells, with characteristics similar to those for PRL release. Trifluoroperazine, polymyxin B, neomycin, and 8 (diethylamino)octyl-3,4,5-trimethoxybenzoate all inhibited both TPA- and Ca2+ stimulated PRL release, but in each case the half-maximal inhibitory concentrations were approximately 2-fold higher for TPA-stimulated release compared to Ca2+-stimulated release. Thyrotropin-releasing hormone (TRH) and guanosine 5'-Q-thiotriphosphate, which stimulate polyphosphoinositide breakdown in permeable cells, were found to be only weak stimulators of PRL release, compared to TPA and exogenous diacylglycerol. However, a much stronger effect of TRH was seen if cells were briefly treated with TRH prior to permeabilization. PRL release from TRH-pretreated permeable cells resembled TPA- and OAG-stimulated secretion, with [Ca2+] greater than 0.1 microM potentiating the effect of TRH pretreatment. These studies support the hypothesis that PRL release in GH3 cells can be stimulated directly by a diacylglycerol-activated secretory mechanism whose activity is modulated by [Ca2+]. PMID- 3011803 TI - Rat liver glutathione S-transferases. DNA sequence analysis of a Yb2 cDNA clone and regulation of the Yb1 and Yb2 mRNAs by phenobarbital. AB - We have constructed a cDNA clone, pGTA/C48, which is complementary to the rat liver glutathione S-transferase Yb2 mRNA. Recombinant clone pGTA/C48 contains a cDNA insert of 845 base pairs which overlaps nucleotides 108-952 of the Yb1 cDNA clone, pGTA/C44, described previously by our laboratory (Ding, G. J.-F., Lu, A. Y. H., and Pickett, C. B. (1985) J. Biol. Chem. 260, 13268-13271). Over the protein coding region of the Yb1 and Yb2 cDNA clones there is an 84% nucleotide sequence homology, whereas the 3' untranslated regions are only 32% homologous. The complete amino acid sequence of the Yb2 subunit has been determined from a combination of DNA sequence analysis of pGTA/C48 and conventional protein sequence analysis of the glutathione S-transferase Yb1 Yb2 heterodimer. The Yb2 subunit is comprised of 218 amino acids with a molecular weight of 25,705 and has an amino acid sequence which is 79% homologous to the sequence of the Yb1 subunit. We have utilized the divergent 3' untranslated regions of three rat liver glutathione S-transferase cDNA clones as specific probes to determine the effect of phenobarbital on the level of Yb1, Yb2, and Yc mRNAs. Our results clearly show that the Yb1 and Yb2 mRNAs are elevated approximately 5-6-fold by phenobarbital administration; whereas the Yc mRNA is only modestly elevated by this xenobiotic. Finally, our data suggest that the Yb2 subunit is encoded by a gene(s) which is distinct from the Yb1 gene(s) and provides direct evidence for the existence of multiple glutathione S-transferase Yb genes in the rat. PMID- 3011804 TI - Protein phosphorylation during activation of Na+/H+ exchange by phorbol esters and by osmotic shrinking. Possible relation to cell pH and volume regulation. AB - In lymphocytes, the Na+/H+ antiport can be stimulated by 12-O tetradecanoylphorbol 13-acetate (TPA) and by osmotic shrinking. Since TPA acts by stimulating protein kinase C, we undertook experiments to determine if protein phosphorylation also underlies the osmotic stimulation of the antiport. We found that at least one of the membrane polypeptides labeled in cells treated with TPA is also phosphorylated by hypertonic shrinking. In both instances phosphorylation is alkali labile and associated with serine and threonine residues. We tested the possibility that shrinking activates phospholipase C, thereby stimulating protein kinase C through release of diacylglycerol. No decrease in phosphatidylinositol 4,5-bisphosphate levels was detected in hypertonically treated cells. Moreover, the concentrations of inositol phosphates, including inositol trisphosphate, were not altered in shrunken cells. Thus, shrinking does not appear to activate phospholipase C. Whereas TPA induced intracellular redistribution of soluble protein kinase C, no such effect was detected in osmotically activated cells. It was concluded that osmotic stimulation of the Na+/H+ antiport is associated with activation of protein phosphorylation by a kinase that is similar, but not identical to protein kinase C. Experiments in Na+-free or amiloride-containing media indicate that phosphorylation is not a consequence of activation of the antiport. PMID- 3011805 TI - Cyclic AMP and phorbol esters separately induce growth inhibition, calcitonin secretion, and calcitonin gene transcription in cultured human medullary thyroid carcinoma. AB - We have previously reported that the phorbol ester, 12-O-tetradecanoyl phorbol 13 acetate (TPA) induces, in the TT cell line of human medullary thyroid carcinoma, decreased cellular proliferation, increased calcitonin secretion, and enhanced calcitonin gene transcription (deBustros, A., Baylin, S. B., Berger, C. L., Roos, B. A., Leong, S. S., and Nelkin, B. D. (1985) J. Biol. Chem. 260, 98-104). The cellular responses evoked by TPA are thought to be mediated by protein kinase C. In the present study, we have investigated whether protein kinase A, another key mediator of extracellular signal transduction, may also alter the differentiation status of the TT cells. We find that the effects of cAMP, an activator of protein kinase A, on cellular growth, calcitonin secretion, and calcitonin gene transcription almost parallel those of TPA. We also show that both TPA and cAMP lead to similar increases of both major mRNA species encoded within the calcitonin gene, calcitonin itself and the neuropeptide calcitonin gene-related peptide. In addition, cAMP increases nuclear calcitonin and calcitonin gene related peptide mRNA precursors to a greater extent (8-10-fold) than it does the mature cytoplasmic mRNA species (2-4-fold). The effects of TPA and cAMP on the TT cells are additive rather than synergistic. Furthermore, TPA evokes no increase in intracellular cAMP. We thus conclude that TPA and cAMP can trigger, independently, in the TT cells, a similarly programmed set of events resulting in a more differentiated phenotype. These cells provide a model system to explore how these two pathways of signal transduction converge to regulate molecular events such as the transcription of the calcitonin gene. PMID- 3011806 TI - Multiple forms and cellular localization of Drosophila DNA topoisomerase II. AB - Purified type II topoisomerase from Drosophila melanogaster embryos was reported earlier to contain a major polypeptide of 166,000 daltons and several smaller peptides between 132,000 and 145,000 daltons (Shelton, E. R., Osheroff, N. and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9530-9535). Using purified topoisomerase II we have raised antibodies against the 132,000-166,000-dalton cluster of polypeptides. In this paper we demonstrate that at least three of these polypeptides are also present in embryos immediately upon lysis. Using antigen-affinity purified antibody from the cluster of purified topoisomerase II antigens, we have also discovered several smaller polypeptides in the molecular size range of 30,000-40,000 daltons in embryo extracts. These observations suggest the presence of multiple forms of DNA topoisomerases in the cell. In addition, we demonstrate that purified Drosophila topoisomerase II antibody recognizes yeast topoisomerase II antigens expressed by lambda gt 11-yeast topoisomerase II recombinants (Goto, T. and Wang, J. C. (1984) Cell 36, 1073 1080) establishing a structural homology between yeast and Drosophila enzymes. Antibody preparations were also used to localize the distribution of topoisomerase II in polytene nuclei. In contrast with the distribution of topoisomerase I which is located primarily at puffs, the Drosophila topoisomerase II is distributed generally along the chromosomes paralleling the distribution of DNA itself. PMID- 3011807 TI - Molecular and mechanical property changes during aging of bone cement in vitro and in vivo. AB - The changes in mechanical properties and free radical concentration of curing Simplex P Radiopaque Bone Cement in vivo and in vitro conditions were studied. Samples were prepared so that each in vivo sample that cured and aged in the canine femoral intramedullary cavities had an in vitro counterpart that was cured and aged in a constant-temperature saline bath at 37 degrees C. An electron paramagnetic resonance (EPR) spectrometer was used to measure the growth and decay (curing) of polymerization radicals. The results of EPR measurements showed that the curing (disappearance of free radicals) of in vivo samples takes a much longer time (more than 4 weeks) than in vitro curing (less than 2 weeks). The mechanical tests indicate that, whether aged in vivo or in vitro, the strength increased rapidly for the first 1-2 weeks and then slight increases were seen for up to 6 months. PMID- 3011808 TI - The effect of annealing treatments on the tensile properties and hydrolytic degradative properties of polyglycolic acid sutures. AB - The purpose of this research was to examine the effect of annealing treatments on the mechanical properties of polyglycolic acid sutures, and their subsequent influence on PGA degradation properties. An attempt was made to develop a better understanding of the degradation mechanism of synthetic absorbable sutures, including the relationship of their structure, morphology, and mechanical properties. PGA sutures were annealed under selected axial strain (freely hung, 0%, 1%, 10%), at four temperatures (150 degrees C, 170 degrees C, 180 degrees C, 190 degrees C), and two times (5 and 20 min). The annealed PGA specimens were then subjected to hydrolysis in phosphate-buffer solutions (pH = 7.4) for up to 28 days at 37 degrees C. Tensile properties were used to evaluate the effect of annealing treatments. The data were subjected to statistical analysis using the SAS system. All of the one-, and many of the two- and three-factor interactions were found to be statistically significant. Annealing treatments did alter the mechanical properties of PGA sutures, as well as their degradation properties. Except for the increase in tenacity of samples with increasing percent extension, all other respects of the annealing treatments resulted in lower tenacity and breaking elongation when compared with the control samples. Sutures that have been exposed to any level of axial tension during annealing, however, exhibited a lower rate of hydrolytic degradation than the freely hung suture samples. The reduction of the characteristics of fiber structure due to the tendency of the tie-chain molecules to acquire the less constrained conformations and thus to bring the crystal blocks they connect back to the original arrangement before drawing is believed to be responsible for the freely hung specimens to behave quite differently from the clamped and stressed PGA samples. PMID- 3011809 TI - Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane. AB - Oligonucleotide-directed mutagenesis was used to construct chimeric cDNAs that encode the extracellular and transmembrane domains of the vesicular stomatitis virus glycoprotein (G) linked to the cytoplasmic domain of either the immunoglobulin mu membrane heavy chain, the hemagglutinin glycoprotein of influenza virus, or the small glycoprotein (p23) of infectious bronchitis virus. Biochemical analyses and immunofluorescence microscopy demonstrated that these hybrid genes were correctly expressed in eukaryotic cells and that the hybrid proteins were transported to the plasma membrane. The rate of transport to the Golgi complex of G protein with an immunoglobulin mu membrane cytoplasmic domain was approximately sixfold slower than G protein with its normal cytoplasmic domain. However, this rate was virtually identical to the rate of transport of micron heavy chain molecules measured in the B cell line WEHI 231. The rate of transport of G protein with a hemagglutinin cytoplasmic domain was threefold slower than wild type G protein and G protein with a p23 cytoplasmic domain, which were transported at similar rates. The combined results underscore the importance of the amino acid sequence in the cytoplasmic domain for efficient transport of G protein to the cell surface. Also, normal cytoplasmic domains from other transmembrane glycoproteins can substitute for the G protein cytoplasmic domain in transport of G protein to the plasma membrane. The method of constructing precise hybrid proteins described here will be useful in defining functions of specific domains of viral and cellular integral membrane proteins. PMID- 3011810 TI - Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils. AB - Cytosolic free calcium concentration, [Ca2+]i, and exocytosis of azurophil granules (beta-glucuronidase), specific granules (vitamin B12-binding protein), and secretory vesicles (gelatinase) were measured concomitantly in intact human neutrophils under steady state [Ca2+]i. The cells were loaded with the fluorescent calcium indicator quin2 in the presence or absence of extracellular Ca2+, and steady state [Ca2+]i levels ranging from 20 to greater than 2,000 nM were obtained by adding the Ca2+ ionophore ionomycin at various concentrations of extracellular calcium. The extent of exocytosis from the three granule populations was found to be a function of [Ca2+]i. The minimal [Ca2+]i that caused significant release (threshold [Ca2+]i) was approximately 200-300 nM and was similar for all three compartments. Marked differences, however, were found when the [Ca2+]i for half-maximal exocytosis (EC50) was determined. In the absence of cytochalasin B the EC50 was 1,100 +/- 220 nM and 1,600 +/- 510 nM for specific granules and secretory vesicles, respectively, and approximately 6,000 nM for azurophil granules. Cytochalasin B did not affect the threshold [Ca2+]i but decreased the EC50 and enhanced the rate of exocytosis. In the presence of cytochalasin B the EC50 was approximately 600 nM both for secretory vesicles and specific granules, and approximately 2,600 nM for azurophil granules. The addition of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine dramatically changed the [Ca2+]i dependency of granule secretion: It decreased the threshold [Ca2+]i to less than 20 and less than 50 nM, and the EC50 to 50 and 200 nM for specific and azurophil granules, respectively, and it significantly increased the rate of exocytosis. Thus, the additional signal(s) provided by receptor activation markedly lower(s) the Ca2+ requirement of the exocytotic process. Furthermore, these results indicate that the secretion from three different granule populations within the same cell type are differently modulated by [Ca2+]i. PMID- 3011812 TI - Differential localization of mRNAs of collagen types I and II in chick fibroblasts, chondrocytes, and corneal cells by in situ hybridization using cDNA probes. AB - We have employed a highly specific in situ hybridization protocol that allows differential detection of mRNAs of collagen types I and II in paraffin sections from chick embryo tissues. All probes were cDNA restriction fragments encoding portions of the C-propeptide region of the pro alpha-chain, and some of the fragments also encoded the 3'-untranslated region of mRNAs of either type I or type II collagen. Smears of tendon fibroblasts and those of sternal chondrocytes from 17-d-old chick embryos as well as paraffin sections of 10-d-old whole embryos and of the cornea of 6.5-d-old embryos were hybridized with 3H-labeled probes for either type I or type II collagen mRNA. Autoradiographs revealed that the labeling was prominent in tendon fibroblasts with the type I collagen probe and in sternal chondrocytes with the type II collagen probe; that in the cartilage of sclera and limbs from 10-d-old embryos, the type I probe showed strong labeling of fibroblast sheets surrounding the cartilage and of a few chondrocytes in the cartilage, whereas the type II probe labeled chondrocytes intensely and only a few fibroblasts; and that in the cornea of 6.5-d-old embryos, the type I probe labeled the epithelial cells and fibroblasts in the stroma heavily, and the endothelial cells slightly, whereas the type II probe labeled almost exclusively the epithelial cells except for a slight labeling in the endothelial cells. These data indicate that embryonic tissues express these two collagen genes separately and/or simultaneously and offer new approaches to the study of the cellular regulation of extracellular matrix components. PMID- 3011813 TI - Regulation of the expression of the SV40 T-antigen coding gene under the control of an rDNA promoter. AB - We have constructed a hybrid gene in which the SV40 T-antigen coding gene is driven by a mouse rDNA promoter and we have compared its expression to that of an SV40 T-antigen coding gene under the control of its own promoter. The comparison has been carried out in microinjected cells, in transfected cells, and in stable cell lines carrying the respective T-antigen coding genes in an integrated form. These cell lines were derived from ts AF8 cells, a mutant which is temperature sensitive for RNA polymerase II activity. The hybrid gene clearly expresses T antigen, albeit less efficiently than when the T antigen coding gene is under the control of the SV40-promoter. We also show that the expression of T-antigen by the hybrid gene is 50% inhibited by an antibody against RNA polymerase I. In tsAF8 cells carrying the hybrid gene, T-antigen is still expressed at the restrictive temperature (where RNA polymerase II is inactive) at a level again about 50% of controls. However, our findings also confirm those of Smale and Tjian (Mol. Cell. Biol. 5:352, 1985) that such hybrid genes are in part transcribed by RNA polymerase II and generate abnormal transcripts. PMID- 3011814 TI - Collagen synthesis by murine bone marrow cell culture. AB - Collagen types synthesized by murine bone marrow cells were studied and the effect of lithium chloride on collagen biosynthesis in vitro was investigated. In the liquid culture system used, an adherent, mixed cell population supports hemopoiesis. Radioactive labeling of cell cultures and subsequent fractionation with ammonium sulfate, enzyme digestion, immune precipitation, and gel electrophoresis indicated that the bone marrow cells synthesized precursors to collagen types I, III, and IV, and fibronectin. A previously undescribed molecule or fragment with an apparent molecular weight of 17,000 daltons that was susceptible to bacterial collagenase and containing no interchain disulfide bonds was also identified in the culture media of both control and lithium-treated cells. Lithium treatment did not affect the types of collagen synthesized, although the relative proportions of collagen types may differ from controls. However, lithium does have an effect on the appearance of some, as yet unidentified, non-collagenous components in the cell culture media. PMID- 3011816 TI - Temporal order of replication of genes responsible for hypoxanthine phosphoribosyl transferase and Na+/K+ ATPase in chemically transformed human fibroblasts. AB - The cytotoxic and mutagenic effects of a direct perturbation of DNA during various portions of the DNA synthetic period (S phase) of a chemically induced, transformed line (Hut-11A cells) derived from diploid human skin fibroblasts were examined. The cells were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method resulted in over 90% synchrony, although approximately 20% of the cells were noncycling. Synchronized cells were treated for each of four 2-h periods during the S phase with 5-bromodeoxyuridine (BrdU) followed by irradiation with near ultraviolet (UV). The BrdU-plus-irradiation treatment was cytotoxic and mutagenic, while treatment with BrdU alone or irradiation alone was neither cytotoxic nor mutagenic. The cytotoxicity was dependent upon the periods of S phase during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, particularly in the first 2 h of S phase, whereas scare lethality was observed in late S phase. The BrdU-plus irradiation treatment induced ouabain- and 6-thioguanine-resistant mutants, while BrdU alone or irradiation alone was not mutagenic. Ouabain-resistant mutants were induced during early S phase by the BrdU-plus-irradiation treatment. 6 Thioguanine-resistant mutants, however, were induced during middle to late S phase. These results suggest that a certain region or regions in the DNA of Hut 11A cells, as designated by their specific temporal relationship in the S phase, may be more sensitive to the DNA perturbation by BrdU treatment plus near-UV irradiation for cell survival and that gene(s) responsible for Na+/K+ ATPase is replicated during early S phase and gene(s) for hypoxanthine phosphoribosyl transferase is replicated during middle to late S phase. PMID- 3011811 TI - Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I. Activation of protein kinase C and inhibition of epidermal growth factor binding. AB - Addition of bombesin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an Mr 80,000 cellular protein (designated 80k). The effect was both concentration and time dependent; enhancement in 80k phosphorylation could be detected as early as 10 s after the addition of peptide. Recently, a rapid increase in the phosphorylation of an 80k cellular protein after treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact fibroblasts (Rozengurt, E., A. Rodriguez-Pena, and K. A. Smith, 1983, Proc. Natl. Acad. Sci. USA., 80:7244-7248; Rozengurt, E., A. Rodriguez-Pena, M. Coombs, and J. Sinnett-Smith, 1984, Proc. Natl. Acad. Sci. USA., 81:5748-5752). The 80k phosphoproteins generated in response to bombesin and to phorbol 12,13-dibutyrate were identical as judged by one- and two-dimensional PAGE and by peptide mapping after partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with phorbol 12,13-dibutyrate, which leads to the disappearance of protein kinase C activity, blocked the ability of bombesin to stimulate 80k. Bombesin also caused a rapid (1 min) inhibition of 125I-labeled epidermal growth factor (125I-EGF) binding to Swiss 3T3 cells. The inhibition was both concentration and temperature dependent and resulted from a marked decrease in the affinity of the EGF receptor for its ligand. Peptides structurally related to bombesin, including gastrin-releasing peptide, also stimulated 80k phosphorylation and inhibited 125I-EGF binding; both effects were selectively blocked by a novel bombesin antagonist. These results strongly suggest that these responses are mediated by specific high-affinity receptors that recognize the peptides of the bombesin family in Swiss 3T3 cells. While an increase in cytosolic Ca2+ concentration does not mediate the bombesin inhibition of 125I-EGF binding, the activation of protein kinase C in intact Swiss 3T3 cells by peptides of the bombesin family may lead to rapid inhibition of the binding of 125I-EGF to its cellular receptor. PMID- 3011815 TI - Characterization of water in unfertilized and fertilized sea urchin eggs. AB - The water in unfertilized and fertilized sea urchin eggs was characterized with a proton nuclear magnetic resonance (NMR) titration method assuming fast proton diffusion (FPD) between water compartments. This method involves stepwise dehydration with sequential T1 relaxation time and water content determinations. The results analyzed by the FPD model give evidence of intracellular water compartments with three different correlation times: 6 X 10(-12) sec (bulk water), 1 X 10(-10) sec (structured water) and about 2 X 10(-9) sec (bound water). Fertilization is accompanied by a substantial increase in bulk water (from 111 to 414 g H2O per 100 g dry mass) and by a decrease in the water of hydration (from 128 g to 56 g per 100 g dry mass). This study shows that 54% of the water in the unfertilized sea urchin egg has motional properties different from bulk water and that this percentage decreases dramatically shortly after fertilization. Most of the change in T1 relaxation rate observed at fertilization can be accounted for by uptake of bulk water associated with elevation of the fertilization membrane. PMID- 3011817 TI - Human restriction fragment length polymorphisms and cancer risk assessment. AB - The polymorphic restriction fragments of the human Ha-ras locus, produced by the variable tandem repetition (VTR) of a short consensus sequence, fall into three classes based on allelic frequencies. Alleles of the "rare" class (individual frequencies less than 0.5%) have been detected only in white blood cell and tumor DNA of cancer patients. This phenomenon is independent of ethnic origin. No significant association of rare alleles with cancer patients has been demonstrated at an independent tandem repeat locus, VTR4.1. The results suggest that the Ha-ras restriction fragment length polymorphism is useful in cancer risk assessment. PMID- 3011818 TI - Epidermal growth factor: biology and receptor metabolism. AB - Epidermal growth factor (EGF) is a small (Mr 6045) protein that stimulates cell proliferation in cell culture systems and in intact animals. This growth factor has been isolated from rodents and human material and probably exists in nearly all animal species. In humans EGF has been detected in many body fluids and receptors for the growth factor are also ubiquitous. While the mitogenic activity of EGF has been most frequently reported, it clearly has other functions, such as the inhibition of gastric acid secretion, that are unrelated to mitogenic responses. Correspondingly, receptors for EGF have been localized on cells that are rapidly proliferating and cells that are essentially non-proliferating. Nevertheless, it has not been possible to define experimentally the biological function(s) of the endogenous EGF present in the intact animal. Studies of the mechanism of action of EGF have concentrated, to date, on the plasma membrane receptor that specifically binds this ligand. The receptor is undoubtedly the first cellular component that mediates the eventual biological response(s) of the cell to this extracellular signal. Studies of the EGF receptor have shown that this molecule, which has no subunit structure, functions not only in ligand recognition, but also may produce an intracellular 'second message'. The receptor contains a protein kinase activity that is activated by the binding of EGF and it is this enzymic function that may yield the critical 'second messenger', by phosphorylation of an intracellular protein. Although intracellular targets of this EGF-sensitive protein kinase have been identified, it has not been possible to demonstrate their relevance as regulatory mediators of EGF activity. PMID- 3011819 TI - Molecular dissection of the human transferrin receptor. AB - Transferrin is the major iron carrier protein in vertebrates and is required for maintenance of cell viability. To deliver iron, transferrin binds to its receptor, the complex is internalized and directed into acidic vacuoles where iron is dissociated and the ligand-receptor complex is recycled back to the plasma membrane. The transferrin receptor is a transmembrane glycoprotein, composed of two disulphide-bonded subunits (each of apparent Mr 90 000). It contains three N-linked glycan units and is post-translationally modified with both phosphate and fatty-acyl groups. The primary structure of the receptor consists of 760 amino acids divided into three domains. Starting from the N terminal residue the cytoplasmic domain consists of 62 amino acids, followed by 26 predominantly non-polar residues, which constitute the transmembrane domain, and 672 residues form the C-terminal extracellular domain. It does not contain an N-terminal cleavable signal sequence. PMID- 3011821 TI - Receptor-mediated endocytosis of transferrin and epidermal growth factor receptors: a comparison of constitutive and ligand-induced uptake. AB - The distribution of cell surface receptors for transferrin-iron and epidermal growth factor (EGF) on the surface of cultured epithelioid (A431) cells has been identified by immunocytochemical electron microscopy. The patterns of movement displayed by these two receptor populations as they transfer to their sites of internalization on the cell surface are different. The movement of recycling transferrin receptors over the surface is ligand-independent whereas EGF receptors are more stable residents and remain monodisperse until they bind ligand. Prior to uptake transferrin receptors cluster, predominantly within existing clathrin-coated pits while the aggregates formed by EGF ligand-receptor complexes induce new membrane invaginations. These results are discussed in relation to receptor populations concerned with constitutive, high capacity uptake processes and receptors involved in signal transduction. PMID- 3011820 TI - Amplification and overexpression of the EGF receptor gene in primary human glioblastomas. AB - The expression of epidermal growth factor (EGF) receptor in brain tumours of glial origin was studied at the protein, mRNA and genomic levels. Four out of 10 glioblastomas that overexpress EGF receptor also have gene amplification. The amplified genes appear to be rearranged, generating an aberrant mRNA in at least one of these tumours. Such receptor defects may be relevant to tumorigenesis of human glioblastomas. PMID- 3011822 TI - Phosphoinositides and cell proliferation. AB - Certain growth factors act by stimulating the hydrolysis of inositol lipids to yield putative second messengers such as diacylglycerol (DG) and inositol trisphosphate (IP3). One function of the former is to stimulate C-kinase, which may act by switching on a sodium/hydrogen exchanger to induce the increase in pH that appears to have a permissive effect on DNA synthesis. Studies on Swiss 3T3 cells have revealed that growth factors stimulate an increase in two separate isomers of IP3. In addition to inositol 1,4,5-trisphosphate there was a large increase in inositol 1,3,4-trisphosphate. While the former functions to elevate intracellular calcium, which has been implicated in the control of growth of many different cell types, the function of the latter is unknown. Since the 1,3,4 isomer turns over very slowly, it may control long-term events and thus could play a role in cell growth. There are other growth factors such as insulin and epidermal growth factor (EGF), which apparently do not work through the inositol lipids but they may initiate ionic events similar to those just described for calcium-mobilizing receptors. The bifurcating signal pathway based on IP3/Ca2+ and DG/C-kinase provides an interesting framework within which to consider the mode of action of oncogenes. PMID- 3011823 TI - The structure and biosynthesis of epidermal growth factor precursor. AB - The structure of mouse submaxillary gland epidermal growth factor (EGF) precursor has been deduced from complementary DNAs. The mRNA is approximately 4800 bases and predicts prepro EGF to be a protein of 1217 amino acid residues (133 X 10 Mr). EGF (53 amino acid residues) is flanked by polypeptides of 188 and 976 residues at its carboxy and amino termini, respectively. The amino terminus of the precursor contains seven cysteine-rich peptides that resemble EGF. Towards the carboxy terminus is a 20-residue hydrophobic membrane spanning domain. The mild portion of the EGF precursor shares a 33% homology with the low density lipoprotein receptor, which extends over 400 amino acid residues. These features suggest that EGF precursor could function as a membrane-bound receptor. RNA dot blot analysis and in situ hybridization show EGF mRNA to be abundant in the submaxillary gland, kidney and incisor tooth buds. Lower EGF mRNA levels were found in the lactating breast, pancreas, small intestine, ovary, spleen, lung, pituitary and liver. In the kidney EGF mRNA was most abundant in the distal convoluted tubules. Analysis of EGF precursor biosynthesis in organ culture of the submaxillary gland and kidney showed differential processing of the precursor in the two tissues. In the submaxillary gland immunoreactive low molecular weight EGF was produced, but in the kidney the high molecular weight precursor was not processed. In the distal convoluted tubule of the kidney EGF precursor may act as a receptor that is involved in ion transport. PMID- 3011825 TI - Insulin-like growth factor receptors. AB - There are two types of insulin-like growth factor (IGF) receptors. The type I receptor generally binds IGF-I more tightly than IGF-II and also interacts weakly with insulin. The type II receptor prefers IGF-II over IGF-I and does not recognize insulin. The type I receptor is made up of an alpha binding subunit (Mr 130 000) and a beta subunit (Mr 95 000) probably organized as a heterotetramer (alpha 2 beta 2). The type II receptor consists of a single binding unit (Mr 250 000). IGF stimulates phosphorylation of the beta subunit of the type I receptor in whole cells and solubilized receptor preparations. Tyrosine kinase activity is associated with the type I receptor, resulting in autophosphorylation of the beta subunit and phosphorylation of exogenous substrates. In contrast, phosphorylation of the type II receptor in whole cells is less IGF-dependent, solubilized receptor preparations are not phosphorylated, and purified type II receptors do not exhibit tyrosine kinase activity toward the artificial substrate poly(Glu, Tyr)4:1. There are many similarities between the type I IGF receptor and the insulin receptor; however, different ligand-binding properties, subtle differences in the size of alpha and beta subunits, and immunoreactivity toward anti-receptor antibodies allow us to distinguish between these two receptors. The presence of both IGF receptors as well as insulin receptors on most cells and cross-reactivity of ligands for binding to these receptors present difficulties in assigning a particular biological response to a specific receptor. The type I receptor is down-regulated by ligand while in several cell types the type II receptor is rapidly up-regulated by insulin; the mechanism of up-regulation appears to be a translocation of type II receptors to the cell surface. There are two classes of serum binding proteins for IGF, a Mr 150 000 species found in adult blood and a Mr 40 000 species, which predominates in foetal blood. Like the type II receptor, IGF binding proteins do not bind insulin. The binding site on the type II receptor can be distinguished from the binding protein sites by a hybrid molecule Ainsulin-BIGF-I, which recognizes the binding protein but not the type II receptor. Binding proteins produced by cells in culture may cause confusion in the interpretation of experiments that are designed to study the binding of radiolabelled IGF to cell surface receptors in monolayer culture. PMID- 3011824 TI - Synergistic signals in mitogenesis: role of ion fluxes, cyclic nucleotides and protein kinase C in Swiss 3T3 cells. AB - A fundamental feature in the action of most mitogenic agents when added to quiescent cells in serum-free medium is that they exhibit striking synergistic effects when applied in specific combinations. A tenable hypothesis of growth control must provide a cogent explanation for the molecular mechanisms underlying this complex pattern of synergistic effects. To gain an understanding of the mechanisms by which these synergistic effects arise, we studied the initial cellular responses associated with the interaction of mitogenic factors and hormones with the cell, including changes in cation fluxes, cyclic nucleotides and cellular phosphoproteins. In this paper, some of our recent results on the early signals and responses elicited by multiple growth-promoting agents in quiescent cultures of Swiss 3T3 cells will be summarized. On the basis of the emerging information, we propose a framework that integrates early events and synergistic effects in a unified hypothesis of growth control. PMID- 3011826 TI - Platelet-derived growth factor: mechanism of action and relation to oncogenes. AB - Recent studies of platelet-derived growth factor (PDGF) have revealed several structural and functional similarities between this growth factor or components linked to its mechanism of action and certain oncogene products: PDGF itself has a structural homology with the transforming protein of simian sarcoma virus, the PDGF receptor has a functional homology (tyrosine kinase activity) with a family of oncogene products, and PDGF induces the expression of the cellular counterparts of myc and fos. In addition, several tumour cell lines have been found to produce PDGF-like growth factors, which may cause autocrine stimulation of growth. We interpret these findings as indicating that regulatory components along the PDGF-dependent mitogenic pathway may have oncogenic properties if they are inappropriately expressed or activated. PMID- 3011827 TI - Glycogen synthesis and immunocytochemical study of fructose-1,6-biphosphatase in methionine sulfoximine epileptogenic rodent brain. AB - The effects of the convulsant methionine sulfoximine (MSO) on the glucose pathway have been investigated in mouse and rat brain. The key gluconeogenic enzyme fructose-1,6-biphosphatase (FBPase) (EC 3.1.3.11) was immunostained by rat anti FBPase antibody. The rat cortex slices were very lightly stained, almost unstained in controls. After MSO injection, there was a marked staining only in astrocytes (perikarya, processes, and end feet). The activity of this enzyme also increased. MSO induced an increase of 63% in the stability at heating (47 degrees C) and of 36% in the stability at proteolysis (trypsin, 10 micrograms/ml) of FBPase. The convulsant had no effect on the concentrations of the metabolites related to the FBPase-phosphofructokinase step, i.e., fructose-1,6-biphosphate, glyceraldehyde-3-phosphate, and dihydroxyacetone phosphate, before, during, or after the convulsions. These results show that the cellular site of glucose pathway impairment induced by MSO in rodent brain is presumably the astroglial cells and that one mechanism of glycogenesis could be the reinforcement of the molecules of FBPase, which enhances gluconeogenesis. A hypothetical diagram of glucose metabolism under the effect of MSO has been proposed. PMID- 3011828 TI - Effects of clonidine on cerebral blood flow and the response to arterial CO2. AB - CBF, as measured by the clearance of 133Xe or 85Kr in the pentobarbital anesthetized cat, displays a monotonic increase as the PaCO2 is elevated over a range of 20-60 mm Hg (slope Xe, 1.65 +/- 0.14 ml/100g/min/mm Hg; slope Kr, 1.40 +/- 0.11 ml/100 g/min/mm Hg). Clonidine (20 micrograms/kg i.v.), a centrally acting, alpha 2-preferring agonist, reduced the slope of the PaCO2-CBF response functions for Xe and Kr by 70 and 64%, respectively. Clonidine reduced normocarbic CBF-Xe by 36%, but had no effect on normocarbic CBF-Kr. ST-91, a polar structural analog of clonidine that does not cross the blood-brain barrier, did not reproduce the effects of clonidine when administered at an equivalent dose. This indicates that the effects of clonidine observed were secondary to its action on central rather than peripheral sites. In addition to the effects on the clearance of CBF markers, clonidine reduced the increased MABP otherwise evoked by elevated PaCO2. Reduction in the MABP response to PaCO2 did not account for the lowering of CBF during hypercarbia. In separate experiments where MABP was elevated to correspond with the PaCO2-MABP response observed in the absence of clonidine, a comparable reduction in the slope of the PaCO2 response was also observed. In addition, the pressure autoregulatory response was unaltered after clonidine treatment. These observations suggest that the central action of alpha 2-receptors on the CBF-CO2 response cannot be attributed to an altered perfusion pressure. PMID- 3011829 TI - AIDS: what is now known. II. Epidemiology. PMID- 3011830 TI - The use of opioids in the management of pain. PMID- 3011831 TI - Sensitive method for bromide determination in atmospheric aerosols. PMID- 3011832 TI - Quantitative determination of acivicin in bulk drug and pharmaceutical formulations by ion-pair high-performance liquid chromatography. AB - An ion-pair reversed-phase high-performance liquid chromatographic method for the determination of the purity of acivicin and the amount of drug in a sterile powder and two sterile solution formulations is described. The method displays good recoveries (98.4-100.4%) for all formulations and a linear range of 0.002-20 micrograms of drug injected. Estimates of assay precision were 1.3% for the bulk drug, 0.6% for sterile solution formulations and 1.6% for the sterile powder formulations. PMID- 3011833 TI - Profiling of organic acids and polyols in nerves of uraemic and non-uraemic patients. AB - Organic acids, polyols and lipid-bound polyols in the cauda equina nerves of uraemic patients and non-uraemic patients were analysed with high-resolution gas chromatography-mass spectrometry. In the uraemic nervous tissue, the concentrations of myoinositol and 4-hydroxyphenylacetic acid were increased. Levulinic acid was first detected in the nervous tissue as a normal component. 1 Deoxyglucose and free and lipid phosphatide scylloinositol were detected in the nervous tissue as normal components. PMID- 3011834 TI - Thyroxine therapy in patients with severe nonthyroidal illnesses and low serum thyroxine concentration. AB - A randomized prospective study was done to assess the response of hypothyroxinemic patients with severe nonthyroidal illnesses to T4 therapy. Patients admitted to a medical intensive care unit who had a total serum T4 concentration less than 5 micrograms/dl were randomly assigned to a control (12 patients) or a T4 treatment group (11 patients). L-T4 in a dose of 1.5 micrograms/kg was given iv each day for 2 weeks. In the treatment group, serum T4 and free T4 concentrations significantly increased by day 3 and were normal on day 5. Serum TSH levels decreased significantly in the T4 treatment group, as did the TSH response to TRH. A significant rise in serum T3 occurred in the control group on day 7, but was delayed until day 10 in the treatment group. Mortality was equivalent in the 2 groups (75% control vs. 73% treatment). Regardless of group assignment, survivors and nonsurvivors were completely separable based on baseline T3 to T4 ratios [17.0 +/- 1.8 (+/- SE) ng/micrograms in survivors vs. 7.0 +/- 0.7 in nonsurvivors; P less than 0.001]. Angiotensin-converting enzyme was significantly reduced in the T4 treatment group, but did not rise significantly in response to treatment. T4 therapy was not beneficial in this population of intensive care unit patients, and by inhibiting TSH secretion, it may suppress an important mechanism for normalization of thyroid function during recovery. PMID- 3011835 TI - Treatment of hyperthyroidism with a small single daily dose of methimazole. AB - The duration of action of methimazole (MMI) was studied in patients with hyperthyroidism due to Graves' disease. Perchlorate discharge tests performed 24 h after MMI administration revealed greater than 10% discharge in 77% of 53 patients who received a single dose of 15 mg MMI and in 74% of 23 patients who received 30 mg. The mean percent discharges were 41.5 +/- 26.4% (+/- SD) and 35.4 +/- 28.0, respectively. Based on these results, hyperthyroidism was treated with a single daily dose (SDD) of 15 mg in 43 patients and with 30 mg in 32 patients, and the results were compared with retrospective analysis of 50 patients who were treated with divided doses of MMI (10 mg, 3 times daily). Within 12 weeks, 93% of the patients treated with 15 mg SDD, 91% treated with 30 mg SDD, and 86% treated with divided doses were euthyroid. The mean times to achieve euthyroidism in these patients were 5.3 +/- 3.6 (+/- SD), 5.3 +/- 3.1, and 5.6 +/- 3.0 weeks, respectively. Side-effects occurred in 2 patients treated with 15 mg SDD and in 6 treated with 30 mg SDD. We conclude that a single daily dose of 15 mg MMI is not only effective in most patients with Graves' hyperthyroidism, but also less frequently causes adverse effects. PMID- 3011837 TI - Hypocalcemia and hypercalcemia in patients with rhabdomyolysis with and without acute renal failure. AB - Patients with rhabdomyolysis (RBD) and acute renal failure (ARF) are hypocalcemic during the oliguric phase of ARF and over 30% develop hypercalcemia during the diuretic phase. The present study examined the factors underlying these derangements in calcium metabolism in 15 patients: 7 with RBD and ARF, 4 with RBD only, and 4 with ARF only. All patients had hypocalcemia on admission and the hypocalcemia was more pronounced in those with RBD and ARF. All patients with RBD independent of the presence or absence of ARF had calcium deposition in soft tissues as documented by technetium-99 scan. In 4 patients with RBD and ARF, hypercalcemia developed during the diuretic phase at a time when Serum PTH levels were undetectable. Only patients with RBD and ARF had a significant increase in serum levels of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D [1,25(OH)2D] during the diuretic phase and both the increments in and the levels of 1,25(OH)2D were significantly greater in those who were hypercalcemic. The data indicate that 1) hypocalcemia occurs in RBD independent of ARF and is most likely related to calcium deposition in injured tissues, and 2) elevation in serum levels of 1,25(OH)2D plays an important role in the genesis of hypercalcemia during the diuretic phase of patients with RBD and ARF. Our observations suggest that extrarenal production of 1,25(OH)2D may occur in these patients, and/or that the renal production of 1,25(OH)2D may not be so tightly controlled as it is in normal subjects. PMID- 3011836 TI - Increased cortisol production in women runners. AB - Because we previously found increased basal serum cortisol levels in women runners, we examined adrenocortical function in amenorrheic running women (AR), eumenorrheic running women (R), and normal nonexercising women (NC) in further detail. Mean 24-h urinary cortisol levels were significantly elevated (P less than 0.001) in six AR [45.1 +/- 7.2 (+/- SEM) micrograms/24 h] and eight R (38.5 +/- 6.9 micrograms/24 h) compared to four NC (13.9 +/- 2.8 micrograms/24 h). After adrenal suppression with 2 mg dexamethasone, integrated responses and absolute maximal elevations in serum cortisol levels in response to 10 micrograms/m2 exogenous ACTH (1-24) administered as an iv bolus dose, were not significantly different among six AR, six R, and six NC. This dose of ACTH results in maximal steroid release. The disappearance rates of cortisol (5 mg, iv) after dexamethasone suppression were similar in four AR, five R, and four NC and corresponded to a two-compartment model with mean half-lives of 4.9 and 93.8 min, respectively. Cortisol-binding globulin levels were also similar among the groups. These data document higher cortisol secretion and suggest increased ACTH secretion in running women. PMID- 3011838 TI - Lack of inhibitory effect of alpha-human atrial natriuretic polypeptide on aldosteronogenesis in aldosterone-producing adenoma. AB - The effects of synthetic alpha-human atrial natriuretic polypeptide (alpha hANP) on aldosteronogenesis in normal and aldosterone-producing adenoma cells (APA cells) in primary monolayer cultures were studied. alpha hANP significantly inhibited aldosterone secretion from normal adrenal cells in culture, but had no inhibitory effect on aldosterone secretion from APA cells in the presence or absence of 10(-8) M ACTH. alpha hANP enhanced the accumulation of intracellular cGMP in normal adrenal cells in culture, but not in APA cells. Visualization of [125I]iodo-alpha hANP-specific binding sites in APA and adjacent normal adrenal tissues by an in vitro receptor autoradiographic technique showed that these sites were localized only in normal adrenal tissue, but not in APA tissue. These results suggest that the lack of an inhibitory effect of alpha hANP on aldosteronogenesis in APA cells may be due to the absence of alpha hANP-specific receptor sites in APA cells. PMID- 3011840 TI - Presence of renin, angiotensinogen, and converting enzyme in human pituitary lactotroph cells and prolactin adenomas. AB - Renin, angiotensinogen, and converting enzyme were detected in 10 normal human pituitary glands by immunohistochemical techniques. Renin was stained by polyclonal and monoclonal antibodies directed against human renin, and an antibody directed against the renin prosegment revealed the presence of prorenin. Immunoreactive angiotensinogen and angiotensin I-converting enzyme were found in the same cells as renin. Using serial sections and double immunohistochemical labeling with a PRL antiserum, all of the proteins of the renin-angiotensin system appeared to be localized within the lactotroph cells, and no component of the renin system was detected in any of the other pituitary cells. Renin, angiotensinogen, and angiotensin I-converting enzyme also were found in 6 PRL secreting adenomas as well as in a mixed PRL/GH-secreting adenoma. The renin content of a PRL adenoma was about 1/100th that of a normal kidney. Renin activity could be blocked by an anticatalytic human renin antibody. No renin, angioten-sinogen, or angiotensin I-converting enzyme was found in 6 GH-secreting adenomas, 1 corticotroph adenoma, or 10 nonsecreting pituitary adenomas. The colocalization of proteins of the renin-angiotensin system suggests production of angiotensin II within the lactotroph cells and favors the hypothesis of a paracrine action of this peptide. PMID- 3011839 TI - Hormonal regulation of P450scc (20,22-desmolase) and P450c17 (17 alpha hydroxylase/17,20-lyase) in cultured human granulosa cells. AB - Conversion of cholesterol to pregnenolone in man is mediated by a single enzyme, P450scc. To study possible regulation of the single P450scc gene in ovarian steroid synthesis, we incubated human granulosa cells with potential hormonal stimulators, measured P450scc mRNA accumulation by hybridization to 32P-labeled human P450scc cDNA, and compared the results to secretion of progesterone into the culture medium. Primary cultures of human granulosa cells were optimally responsive after 8-14 days of culture. Incubation with hCG (1.0-100 ng/ml), FSH (1.0-50 ng/ml), and (Bu)2cAMP (0.02-2.0 mM) increased P450scc mRNA accumulation and progesterone secretion in dose-dependent fashions. Maximal stimulation increased P450scc mRNA accumulation and progesterone secretion to 490% and 240% of control values, respectively, with hCG, to 166% and 168% with FSH, and to 495% and 380% with (Bu)2cAMP. PRL (to 100 ng/ml), ACTH (10(-6) M), and butyric acid (2 mM) had no significant effect on progesterone secretion or P450scc mRNA accumulation. These data indicate gonadotropin-specific stimulation of cAMP mediated regulation of P450scc mRNA accumulation in human granulosa cells, presumably mediated by increased P450scc gene transcription. Ovarian estrogen synthesis may require both thecal and granulosa cells, although this two-cell theory of estrogen synthesis is unproven in man. To examine this theory, we probed the same blots used in the experiments described above with 32P-labeled human P450c17 cDNA (P450c17 is the single enzyme mediating both 17 alpha hydroxylase and 17,20-lyase activities). Only miniscule amounts of P450c17 mRNA were found in the human granulosa cells, and the amounts did not increase in response to any of the above stimuli. These data strongly support the two-cell theory of human ovarian estrogen synthesis. PMID- 3011841 TI - Severely deficient binding of 1,25-dihydroxyvitamin D to its receptors in a patient responsive to high doses of this hormone. AB - A 1.6-yr-old Hispanic boy with sparse hair, muscle weakness, severe growth retardation, and rickets was found to have hypocalcemia, secondary hyperparathyroidism, and high circulating 1,25-dihydroxyvitamin D [1,25-(OH)2D] levels. One year of treatment with a high dose of 1,25-(OH)2D3 (20 micrograms, orally, daily) resulted in marked subjective and radiological improvement; there was a parallel improvement in serum calcium, phosphorus, PTH, alkaline phosphatase, and osteocalcin concentrations. Soluble extracts from cultured skin fibroblasts did not bind [3H]1,25-(OH)2D3 using standard methods. To evaluate the possibility of receptors with abnormally low affinity, we tested for binding with [3H]1,25-(OH)2D3 concentrations higher than those usually used. In one experiment, there was a suggestion of low affinity binding. Hormone receptors with abnormally low affinity for 1,25-(OH)2D may explain this patients resistance to 1,25-(OH)2D. Therapy to maintain extremely high serum 1,25-(OH)2D3 concentrations markedly improved mineral homoeostasis, but did not affect the hair disorder. PMID- 3011842 TI - Decreased beta-adrenergic sensitivity in insulin-dependent diabetic subjects. AB - To study beta-adrenergic sensitivity in diabetes mellitus, we performed isoproterenol sensitivity tests in 34 insulin-dependent diabetic patients and 10 age-matched normal subjects. beta-adrenergic sensitivity [defined as the dose of isoproterenol required to increase the resting heart rate by 25 beat/min, (I25)] was significantly higher in the diabetic group (4.07 +/- 1.4 micrograms, mean +/- SD) than in the normal group (2.02 +/- 1.49 micrograms). A comparison of I25 of normal subjects and diabetic patients as a function of age showed that the latter were significantly less sensitive to beta-adrenergic stimulation at all ages (P less than 0.01). In diabetic patients, beta-adrenergic sensitivity also increased with the duration of diabetes (r = 0.64, P less than 0.0005), but the correlation was stronger when the age of the patients and the duration of the diabetes were both taken into consideration (r = 0.72, P less than 0.0005). We conclude that beta-adrenergic sensitivity is diminished in patients with type I diabetes mellitus of all ages. PMID- 3011844 TI - Adenylate cyclase activity in membrane fractions of adrenal tissue of human anencephalic fetuses. AB - There is greater basal and ACTH-stimulated adenylate cyclase activity in membrane fractions prepared from the neocortex of human fetal adrenal (HFA) tissue than in similar preparations from the fetal zone. In this study, the specific activity of adenylate cyclase was determined in membrane preparations of adrenal tissue obtained from anencephalic fetuses (n = 5) varying in gestational age from 17-43 weeks. The basal adenylate cyclase activity in membrane fractions of adrenals of anencephalics was 2.9 +/- 2.1 (mean +/- SEM) pmol mg protein-1 min-1, 3-5% of the average specific activity in membrane preparations of fetal zone or neocortex of normal fetuses. ACTH (10(-10)-10(-4) M) in the incubation mixture stimulated adenylate cyclase activity 2- to 5-fold in whole HFA membrane fractions. In contrast, ACTH, when added to adrenal membrane preparations of the anencephalics, did not stimulate adenylate cyclase activity. Furthermore, sodium fluoride or forskolin stimulated adenylate cyclase activity markedly in HFA membrane preparations of normal fetuses, but did not affect enzyme activity in adrenal membrane preparations of the anencephalics. In conclusion, the basal activity of adenylate cyclase in adrenal membrane preparations of anencephalics was low and unresponsive to brief exposure to ACTH, sodium fluoride, or forskolin. These findings as well as those of our previous investigations suggest that the expression of HFA adenylate cyclase may be regulated in part by ACTH. PMID- 3011843 TI - Requirement of mineralocorticoid in congenital adrenal hyperplasia due to 11 beta hydroxylase deficiency. AB - Marginal salt loss occurs in patients with congenital adrenal hyperplasia due to 11 beta-hydroxylase (11-OHase) deficiency treated with dexamethasone and is accompanied by increased PRA. The present study was undertaken to evaluate the effect of the stimulated renin-angiotensin system on pituitary-adrenal suppression. Seven patients with 11-OHase deficiency were subjected to a series of treatments with dexamethasone, cortisol, and combined cortisol and 9 alpha fluorohydrocortisone. The latter combination suppressed PRA and sodium excretion, and produced better control of the pituitary-adrenal axis, as measured by plasma ACTA and serum 11-deoxycortisol. We conclude that in children with 11-OHase deficiency, PRA needs to be monitored, and when it is elevated, mineralocorticoid replacement is indicated. PMID- 3011845 TI - A study of 25 patients with chronic granulomatous disease: a new classification by correlating respiratory burst, cytochrome b, and flavoprotein. AB - Twenty-five patients suffering from chronic granulomatous disease (CGD) and their families were investigated. Defects in the superoxide generating system were characterized at the level of the heme-containing cytochrome b and of the FAD containing flavoprotein, both localized in the plasma membrane of granulocytes. It was confirmed that in most of the typical cases (18 of 22), the complete inability of superoxide generation was associated with the absence of detectable cytochrome b. Mothers but not fathers of such male patients were characterized by a diminished content of cytochrome b, confirming that the affected gene is localized on the X chromosome. In contrast, the granulocytes of four other typical patients (two female and two male) contained normal amounts of cytochrome b, whereas oxidative activity was absent. Since no abnormality of oxidative activity as well as of cytochrome b was found in granulocytes of the mothers and fathers of these patients, an autosomal recessive mode of inheritance of the disease is probable. The flavoprotein deficiency found in the granulocytes of four male patients was always associated with an absence of detectable cytochrome b. This could indicate a structural relationship between flavoprotein and cytochrome b (e.g., a flavocytochrome). Three further patients with mild X-linked CGD contrasted with the patients with severe or classic X-linked disease; the oxidative activity of their phagocytes was diminished but not absent, and the cytochrome b present, albeit in small amounts. PMID- 3011846 TI - [Studies on tissue corticotropin-releasing factor]. PMID- 3011848 TI - Development and application of monoclonal antibodies for specific detection of human enteric adenoviruses. AB - Monoclonal antibodies were prepared against enteric adenovirus by fusing P3-NS1/ Ag4-1 mouse myeloma cells with lymphocytes from BALB/c mice immunized with enteric adenovirus 40 (Ad40) G2297. Of the several putative clones secreting antibodies to adenovirus, five were found to react specifically to the enteric adenovirus. The specificity of two of these monoclones which recognize a single antigen of a molecular size of 17 kilodaltons was evaluated against 78 clinical isolates. One monoclone (5D8/2C2) reacted with both Ad40 and Ad41, and the other monoclone (2H6/C11) recognized Ad40 only in an enzyme-linked immunosorbent assay (ELISA). These ELISA results correlated well with those of the specific neutralization test or DNA restriction endonuclease analysis or both. The use of this rapid ELISA with these monoclones will find applications in the diagnosis of enteric adenovirus and should facilitate the epidemiologic studies of enteric adenovirus gastroenteritis. PMID- 3011847 TI - The role of beta-lactamase in staphylococcal resistance to penicillinase resistant penicillins and cephalosporins. AB - We showed that most Staphylococcus aureus strains that have borderline or intermediate susceptibility to the penicillinase-resistant penicillins (PRPs) react this way because of the activity of their beta-lactamase on these antimicrobial agents. These strains produced large amounts of staphylococcal beta lactamase that rapidly hydrolyzed penicillin and partially hydrolyzed the PRPs. Susceptibility to hydrolysis was penicillin greater than oxacillin greater than cephalothin greater than methicillin. The borderline results and the hydrolysis could be prevented by the beta-lactamase inhibitors clavulanic acid and sulbactam. For intrinsically methicillin-resistant (heteroresistant) S. aureus, the inhibitors reduced the penicillin MICs, but the strains remained resistant to all the beta-lactam antimicrobial agents, including penicillin. We conclude that the borderline in vitro susceptibility or resistance to PRPs in most of these S. aureus strains is mediated by beta-lactamase and they are not heteroresistant or intrinsically resistant. We do not know whether this in vitro resistance is expressed clinically. PMID- 3011850 TI - Phosphatase activity as a criterion for differentiation of oral mycoplasmas. AB - Phosphatase activity was found to be applicable as a criterion for the differentiation of Mycoplasma salivarium and Mycoplasma orale, predominant constituents of oral mycoplasmal flora. Therefore, a simple procedure for the phosphatase activity assay was established as a screening test for the differentiation of oral mycoplasmas. PMID- 3011849 TI - Immune-specific gamma interferon production correlates with lymphocyte blastogenesis. AB - Gamma interferon (IFN-gamma) production, measured by a new commercially available radioimmunoassay, and lymphocyte blastogenesis were investigated in human peripheral lymphocyte cultures from healthy adults stimulated by crude cytomegalovirus (CMV) antigen. Mononuclear cells were obtained from 32 healthy adults (18 CMV seropositive [S+] and 14 CMV seronegative [S-]) by Ficoll-Hypaque gradients and cultured in microtiter plates containing CMV antigen. Lymphocyte blastogenesis ([3H]thymidine uptake) and IFN-gamma were determined on day 6. The mean stimulation index for S+ individuals was significantly greater than that for S- individuals (P less than 0.001). Similarly, the IFN-gamma stimulation index was greater for S+ individuals than for S- individuals (P less than 0.005). A significant increase in the concentration of IFN-gamma (10 NIH units/ml) was observed for S+ individuals at 24 h of antigen stimulation, with peak levels at 4 days. The radioimmunoassay for IFN-gamma production by antigen-stimulated lymphocytes in vitro (IMRX; Centocor Inc., Malvern, Pa.) is a rapid and sensitive measure of cell-mediated immunity. PMID- 3011851 TI - Sensitivity of different assay systems for immunoglobulin M responses to varicella-zoster virus in reactivated infections (zoster). AB - An immunoglobulin M response to varicella-zoster virus was detected in 70% of zoster patients by solid-phase radioimmunoassay, in 52% by indirect immunofluorescence, in 48% by neutralization on sucrose density gradient fractions, and in 27% by an antibody class capture enzyme immunoassay. The patients showed marked variations in their varicella-zoster virus immunoglobulin M responses detectable in the different assays. PMID- 3011852 TI - Percoll-purified Treponema pallidum, an improved fluorescent treponemal antibody absorbed antigen. AB - Percoll-purified Treponema pallidum was evaluated as a fluorescent treponemal antibody-absorbed antigen. Borderline and false-positive reactions were essentially eliminated, resulting in sensitivity and specificity of 100 and 95.5%, respectively. The lack of background debris improved the ease and speed of reading the test. PMID- 3011853 TI - Rapid detection of cytomegalovirus in bronchoalveolar lavage specimens by a monoclonal antibody method. AB - Cytomegalovirus (CMV), a common cause of pneumonia in immunocompromised subjects, is conventionally diagnosed in the laboratory by tube cell culture assays or by detection of characteristic inclusions in histologic sections. Of 160 immunocompromised patients, CMV infection was diagnosed in 19 subjects by bronchoalveolar lavage (BAL), using a monoclonal antibody directed against an early nuclear antigen of the virus. Cytospin preparations from BAL and MRC-5 cell cultures inoculated with the BAL specimens yielded positive results for 6 (31.6%) and 18 (94%) of the 19 subjects, respectively, within hours of the bronchoscopic procedure, whereas conventional tube cell cultures were positive for 11 of the 19 subjects (57.9%) only after an average of 9.3 days. The monoclonal antibody method permitted easy and rapid detection of CMV in BAL specimens. PMID- 3011854 TI - Comparison of detection of antibody to the acquired immune deficiency syndrome virus by enzyme immunoassay, immunofluorescence, and Western blot methods. AB - There was 100% agreement between enzyme immunoassay (EIA) (Abbott Laboratories), Western blot, and indirect immunofluorescence (IF) when these three methods were used to measure antibody to the acquired immune deficiency syndrome (AIDS) virus in sera from 142 high-risk individuals, indicating that IF was a sensitive alternative method for detecting antibody to this agent. Thirty-two (64%) of 50 EIA-positive plasma specimens from a blood bank and 6 (21%) of 28 EIA-positive sera from alternative testing sites were negative by IF. In addition, two EIA negative sera from the latter group were positive by IF. Western blotting agreed with IF on those 40 specimens which gave discrepant results by EIA and IF. The IF method was determined to be equal to Western blotting in sensitivity and specificity for detection of AIDS antibody, and it was found to be useful for confirming positive EIA results, especially in specimens from individuals in low risk groups. PMID- 3011855 TI - Surveillance of mice for antibodies to murine cytomegalovirus. AB - The sera of 256 mice from nine commercial sources were screened for antibodies to murine cytomegalovirus (MCMV) because a surveillance of this virus has not been reported in the literature for over a decade. Although no evidence of antibodies to MCMV were detected by complement fixation or nuclear anticomplement immunofluorescence, 54.7% of these sera did have antibodies that were detected by enzyme-linked immunosorbent assay. These data emphasize the need for proper containment of laboratory mice to prevent the potential outbreak of acute MCMV infection. Including MCMV antibody surveillance by enzyme-linked immunosorbent assay in routine health monitoring of mice and imparting these findings in an analysis of the role of MCMV on interpretation of experimental results is advised. PMID- 3011856 TI - Actin-attached and detached crossbridges in myofibrils: segregation into two populations according to their sensitivity to proteolytic digestion of myosin heavy chain. AB - Tryptic digestion of myofibrils was used to assess the interaction of crossbridges with thin filaments in the presence of ATP analogues. The relative amounts of 200 kDa fragment produced by trypsin from myosin heavy chain when the crossbridge is attached to actin, and of 160 kDa fragment produced when the crossbridge is detached from actin, served as a measure of crossbridge-actin interaction. In rigor only the 200 kDa fragment was produced suggesting that a great majority of the crossbridges were strongly attached to actin; in the presence of MgPPi at 0 degrees C only the 160 kDa fragment was finally produced suggesting that eventually all crossbridges detached from actin. In the presence of MgPPi or MgAMPPNP at 25 degrees C both 200 and 160 kDa fragments were present for several minutes after myosin heavy chain had been completely digested, suggesting that two populations of crossbridges (attached and detached) co existed at the same time within the myofibril. It is concluded that the addition of ATP analogues to muscle does not simply affect the chemical equilibrium of binding of myosin heads to actin but that it causes rapid dissociation of one crossbridge population without significant effect on binding to actin of the remaining crossbridge population. PMID- 3011858 TI - Glycosaminoglycan antigens in peripheral nerve. Studies with antibodies from a patient with neuropathy and monoclonal gammopathy. AB - An immunoblot staining procedure was developed for the detection of antibody binding to glycosaminoglycans (GAGs). The method was used to study the binding of a human monoclonal antibody (M-protein) from a patient with peripheral neuropathy, previously found to react with proteoglycans (PGs). GAGs prepared from human peripheral nerve were separated by electrophoresis on cellulose acetate membranes, transferred onto nitrocellulose sheets, and immunostained with the M-protein. The M-protein bound to a single GAG band with intermediate mobility which eluted with 1.25 N NaCl on ion-exchange chromatography and was chondroitinase sensitive. The M-protein appeared to bind to chondroitin sulfate containing proteoglycans in peripheral nerve. PMID- 3011857 TI - Role of the blood-brain barrier in the establishment of the immune response against polyoma virus-induced cerebral tumours in hamsters. AB - The existence of an immunological blood-brain barrier (BBB) is well established but its role in cerebral tumour immunology is less well defined. Attempting to clarify this problem we tested the graft rejection of polyoma virus-induced central nervous (CNS) tumours in hamsters after systemic or intracerebral immunization with polyoma virus. Animals were immunized by intracerebral or subcutaneous inoculations of polyoma virus before tumours were induced by intracerebral or intramuscular graft of polyoma-transformed hamster neuroglial cells. The growth of cerebral and muscular tumours was significantly inhibited in animals immunized subcutaneously. In animals immunized intracerebrally the inhibition of growth was highly significant for cerebral tumours and only very slight for intramuscular tumours. These results suggest that the blood-brain barrier allowed immunocompetent effector cells to penetrate inside the CNS but prevented the locally elicited cell-mediated immune response from diffusing outside the CNS. The ability of the brain to develop a local immune response and the partial lack of circulation of immunocompetent cells to cross the BBB could be mainly responsible for the special immune status of the CNS and may greatly interfere with the establishment of an efficient immune response toward brain tumours. PMID- 3011859 TI - Expression of the human c-fms proto-oncogene product (colony-stimulating factor-1 receptor) on peripheral blood mononuclear cells and choriocarcinoma cell lines. AB - The c-fms gene product is related, and possibly identical, to the receptor for the mononuclear phagocyte colony stimulating factor, CSF-1. Using antisera to a recombinant v-fms--coded polypeptide, glycoproteins encoded by the human c-fms locus were detected in mononuclear cells from normal peripheral blood and in promyelocytic HL-60 cells 24 h after induction of monocytic differentiation with phorbol ester. The 150-kD human c-fms--coded glycoprotein was expressed at the cell surface, was active as a tyrosine-specific protein kinase in vitro, and shared primary structural features with the product of the feline retroviral v fms oncogene. A biochemically indistinguishable glycoprotein was detected in human choriocarcinoma cell lines. Like peripheral blood mononuclear cells and phorbol ester-treated HL-60 cells, the choriocarcinoma cells expressed high affinity binding sites for human CSF-1. In addition to serving as a lineage specific growth factor in hematopoiesis, CSF-1 may play a role in normal trophoblast development. PMID- 3011860 TI - Anti-F(ab')2 antibodies in thrombocytopenic patients at risk for acquired immunodeficiency syndrome. AB - 22 homosexual or narcotic addict patients at risk for acquired immunodeficiency syndrome (AIDS) or with AIDS, were studied for the presence of antiimmunoglobulin antibodies and circulating immune complexes (20 were thrombocytopenic, 6 had AIDS). Circulating immune complex levels were 10-fold higher than levels in normal subjects. IgG anti-F(ab')2 antibodies were noted in homosexual as well as narcotic addict patients. Of 16 homosexual patients, 7 had IgG anti-F(ab')2 antibody of moderate to marked titer with broad reactivity against autologous, homologous, and control F(ab')2 fragments. Three others demonstrated limited reactivity against one or two F(ab')2 fragments. The remaining six patients were negative. Six of six narcotic addict patients had IgG anti-F(ab')2 antibody, five with limited reactivity, one with broad reactivity. In contrast, neither elevated circulating immune complexes nor anti-F(ab')2 antibodies were detectable in six autoimmune thrombocytopenic patients. Anti-F(ab')2 antibody could be affinity purified from serum or circulating immune complexes. Anti-F(ab')2 reactivity correlated with circulating immune complex levels, r = 0.83, P less than 0.01. PMID- 3011861 TI - Treatment of verapamil toxicity in intact dogs. AB - The treatment of verapamil toxicity was examined in lightly sedated dogs. Verapamil, administered as a bolus (0.72 mg/kg) followed by a continuous infusion (0.11 mg/kg per min), decreased cardiac output (CO) from 3.1 +/- 0.1 to 1.7 +/- 0.1 liter/min (P less than 0.001), heart rate (HR) from 85 +/- 4 to 57 +/- 3 beats/min (P less than 0.001), left ventricular derivative of pressure with respect to time (LV dP/dt) from 2,085 +/- 828 to 783 +/- 78 mm Hg/s (P less than 0.001), mean aortic pressure (AO) from 77 +/- 4 to 38 +/- 2 mm Hg (P less than 0.001) and stroke volume from 39 +/- 3 to 28 +/- 2 ml/beat (P less than 0.01). In verapamil-toxic animals isoproterenol increased HR, CO, LV dP/dt, and AO; calcium chloride increased LV dP/dt and AO; norepinephrine, epinephrine, and dopamine increased CO, AO, and LV dP/dt, atropine increased HR, CO, and AO. Phenylephrine (13-55 micrograms/kg per min) produced no changes except a small increase in AO while very high dose phenylephrine (300 micrograms/kg per min) increased AO, CO, and LV dP/dt. 4-Aminopyridine (4-AP) increased HR, CO, LV dP/dt, and AO. When administered prior to verapamil, 4-AP prevented the development of verapamil toxicity as shown by the significantly higher AO (P less than 0.001), CO (P less than 0.01), and LV dP/dt (P less than 0.01) when 4-AP followed by verapamil was compared to verapamil alone. In conclusion, there does not appear to be a single specific therapy for verapamil toxicity, however it can be partially corrected by presently available pharmacologic therapy and 4-AP. PMID- 3011862 TI - Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension. AB - Two groups of adult male Munich-Wistar rats and a third group of nondiabetic age matched and weight-matched normal control rats underwent micropuncture study 1 mo, and morphologic studies 14 mo, after induction of streptozotocin diabetes or sham treatment. All animals were fed standard rat chow. Diabetic rats received daily ultralente insulin to maintain stable moderate hyperglycemia (approximately 350 mg/dl). In addition, one group of diabetic rats was treated with the angiotensin I converting enzyme inhibitor, enalapril, 15 mg/liter of drinking water. Average kidney weight, whole kidney and single-nephron glomerular filtration rate, and glomerular plasma flow rate were elevated to similar values in both groups of diabetic rats, relative to normal control rats. Non-enalapril treated diabetic rats exhibited significant elevations in mean glomerular capillary hydraulic pressure and transcapillary hydraulic pressure gradient, compared with the other groups studied, and only this group eventually developed marked and progressive albuminuria. Likewise, histological examination of the kidneys at 14 mo disclosed a high incidence of glomerular structural abnormalities only in non-enalapril-treated diabetic rats. These findings indicate that prevention of glomerular capillary hypertension in rats with diabetes mellitus effectively protects against the subsequent development of glomerular structural injury and proteinuria. This protection is afforded despite pronounced hyperglycemia and elevated levels of glucosylated hemoglobin, further supporting our view that hemodynamic rather than metabolic factors predominate in the pathogenesis of diabetic glomerulopathy. PMID- 3011864 TI - Subepidermal vesicular dermatosis and sensory peripheral neuropathy caused by pyridoxine abuse. AB - A woman who had ingested 2 gm of pyridoxine (vitamin B6) daily for 2 years for menstrual water retention developed a subepidermal vesicular eruption on the dorsa of the hands and toes, as well as a sensory peripheral neuropathy. The cutaneous and neurologic manifestations subsided about 2 months after discontinuation of the pyridoxine. The possible relationship of subepidermal vesicular eruptions caused by pyridoxine abuse to epidermolysis bullosa acquisita is discussed. PMID- 3011863 TI - Therapeutic advantage of converting enzyme inhibitors in arresting progressive renal disease associated with systemic hypertension in the rat. AB - Micropuncture and morphologic studies were performed in six groups of male Munich Wistar rats after removal of the right kidney and segmental infarction of two thirds of the left kidney. Groups 1 and 4 received no specific therapy. Groups 2 and 5 were treated with the angiotensin I-converting enzyme inhibitor, enalapril, 50 mg/liter, in the drinking water. Groups 3 and 6 were treated with reserpine (5 mg/liter), hydralazine (80 mg/liter), and hydrochlorothiazide (25 mg/liter). All rats were fed standard chow. Groups 1-3 underwent micropuncture study 4 wk after renal ablation. Untreated group 1 rats exhibited systemic hypertension and elevation of the single nephron glomerular filtration rate (SNGFR) due to high average values for the mean glomerular transcapillary hydraulic pressure gradient (delta P) and glomerular plasma flow rate (QA). In group 2 rats, treatment with enalapril prevented systemic hypertension and maintained delta P at near-normal levels without significant reduction in SNGFR and QA. In contrast, triple drug therapy normalized systemic hypertension, but failed to lower delta P in group 3 rats. Groups 4-6 were followed for 12 wk after renal ablation. Untreated group 4 rats demonstrated continuous systemic hypertension, progressive proteinuria, and glomerular structural lesions, including mesangial expansion and frequent areas of segmental sclerosis. In group 5 rats, treatment with enalapril maintained systemic blood pressure at normal levels over the 12-wk period and dramatically limited the development of proteinuria and glomerular lesions. Despite equivalent systemic blood pressure control in group 6 rats, failure of triple drug therapy to control glomerular hypertension was associated with progressive proteinuria and glomerular lesions comparable to those seen in untreated group 4 rats. Thus, unless glomerular capillary hypertension is corrected, control of systemic blood pressure is insufficient to prevent progressive renal injury in rats with reduced renal mass. PMID- 3011866 TI - Hereditary hypophosphatemia rickets: an important awareness for dentists. AB - Because X-linked hypophosphatemia VDRR is reported to be the most common form of rickets in the United States today, it is important for dentists to be aware of this condition. Characteristic dental findings are often the first clinically noticeable signs of the disease. The confirmation of hypophosphatemia with dental findings will permit early diagnosis and prevent crippling rachitic deformities, otherwise certain to follow. Reports in the literature confirm the validity of conservative, prophylactic full coverage restorations in patients with VDRR. PMID- 3011865 TI - Follicular development after administration of adrenocorticotropin to nonlactating Holstein cows. AB - The effect of chronic administration of adrenocorticotropin on ovarian follicular development was studied. Twelve nonlactating Holstein cows received either 100 IU adrenocorticotropin (n = 6) or saline (n = 6) at 12-h intervals, commencing d 16 and continuing until d 23 of an induced estrous cycle (estrus = d 0). Cows were slaughtered on d 24, ovaries collected, and number of visible antral follicles recorded. Estradiol-17 beta, androstenedione, and testosterone in follicular fluid, and luteinizing hormone and follicle-stimulating hormone receptors in follicular tissue of the largest follicles were determined. Largest follicles were classified as ovulatory or nonovulatory based on the estrogen to androgen ratio. One cow treated with adrenocorticotropin, but none treated with saline, had ovulated by slaughter. The numbers of small, medium, and large antral follicles were 0, 1, and 5 for cows treated with adrenocorticotropin and 0, 1, and 6 for cows treated with saline. Follicular diameter (15.0 +/- 1.0 versus 14.0 +/- 2.0 mm) and follicular fluid volume (2.9 +/- .8 versus 2.2 +/- .5 ml) of the largest follicle in cows treated with adrenocorticotropin or saline were not different. No differences were found in the number of luteinizing hormone and follicle stimulating hormone receptors nor in the proportion of ovulatory versus nonovulatory follicles between treatments. We conclude that adrenocorticotropin administered at 100 IU every 12 h during the follicular phase does not significantly alter follicular development in the nonlactating dairy cow. PMID- 3011867 TI - Light scattering and gloss of an experimental quartz-filled composite. AB - For samples of polymethylmethacrylate with and without quartz filler, the inverse of the contrast-gloss ratio is shown to be related to surface roughness and to the optical scattering coefficient. This finding adds to the importance of optical scattering, which has been widely studied because of its relation to color and translucency of materials. Furthermore, optical scattering by composite fillers is shown to be linearly related to the concentration of the filler material within the range of concentrations studied. Quartz fillers were incorporated at concentrations from 5 to 20 weight percent and were short fibers or granular powder, with the granular particles ranging in median equivalent spherical diameter from 15 to 3.3 micron. The efficiency of optical scattering for the granular quartz filler increased as the size of the filler decreased. PMID- 3011868 TI - The influence of chemically-induced modifications of root surfaces on cell migration, attachment, and orientation. AB - Surface demineralization of tooth root surfaces has been shown to improve re attachment of cells and to promote tissue reconstruction following periodontal surgery. Exposure of collagen fibers has been thought to facilitate migration, attachment, and orientation of fibroblasts on the root surface. However, using an in vitro assay, we have found that both attachment and orientation of human gingival fibroblasts on demineralized dentin surfaces are further improved following digestion of the exposed collagen with bacterial collagenase. In contrast, pronase and trypsin digestion of the surface collagen had no significant effect, whereas heat denaturation had an inhibitory effect. Dissociative extraction of the demineralized dentin slices with 4 mol/L guanidine hydrochloride (GuHCl) also improved attachment and orientation, and when undemineralized dentin was subjected to dissociative extraction, cell attachment was comparable and orientation superior to that on demineralized surfaces. These studies indicate that demineralization is not a prerequisite for facilitating attachment, and that enhanced attachment and orientation of cells are not dependent upon a collagenous substratum. PMID- 3011869 TI - The vulnerability of unexposed human dental roots to demineralization. AB - Crowns and roots of human molars, the roots from which had not been exposed to the oral environment, were exposed for 0, 3.5, 7, and 14 days to buffer solutions which were undersaturated or supersaturated with respect to hydroxyapatite. Densitometric measurements on contact-microradiograms of transverse sections of the crowns and of the cervical parts of the roots yielded plots of the mineral content as a function of the distance to the outer surface. From these plots, the rate of demineralization was calculated. It was found that the mineral of the roots dissolved even in buffer solutions which were supersaturated with respect to hydroxyapatite. Comparison of the results obtained from the crowns with those from the roots showed that the root hard tissues were more vulnerable to demineralization than was the dental enamel. PMID- 3011870 TI - Complement and experimental respiratory failure. AB - Activation of the complement system within the lung can lead to acute pulmonary damage and dysfunction. Based on a variety of experimental models it is now apparent that lung injury is related to complement-induced generation of oxygen derived free radicals from neutrophils and from macrophages. In addition to the oxygen radicals, it is also possible that the conversion of hydrogen peroxide by myeloperoxidase to hypochlorous acid also contributes to the injury. Exposure of the pulmonary microvasculature to oxygen radicals generated from complement activated neutrophils causes focal damage and necrosis of endothelial cells. IgG immune complex-induced injury of lung is also complement and neutrophil dependent and oxygen radical mediated. In contrast, lung injury produced by IgA immune complexes is neutrophil independent, complement dependent and oxygen radical mediated. There is now increasing evidence that oxygen radicals are not only directly tissue-toxic but also able to potentiate the activity of leukocytic proteases. In all of these models the lung can be protected from injury by pretreatment of the animals with either scavengers of hydroxyl radical or with agents that prevent its formation (e.g. catalase, iron chelators). Data from these models may have direct clinical relevance to conditions such as adult respiratory distress syndrome where lung injury is probably oxygen radical mediated. PMID- 3011871 TI - Oral prodromal signs of a central nervous system malignant neoplasm--glioblastoma multiforme: report of case. AB - A fatal case of glioblastoma multiforme, initially appearing as orofacial pain of unknown cause, is reported. The report of case illustrates the multidisciplinary approach that, at times, is required to diagnose elusive orofacial pain. PMID- 3011872 TI - Radionuclide assessment of left ventricular diastolic filling in diabetes mellitus with and without cardiac autonomic neuropathy. AB - Indexes of left ventricular diastolic filling were measured by radionuclide ventriculography in 28 patients with insulin-dependent diabetes mellitus without evidence of ischemic heart disease. Six patients (21%) had abnormal diastolic filling and differed from diabetic patients with normal filling in their greater severity of cardiac autonomic neuropathy, assessed by noninvasive means, and their lower plasma norepinephrine levels in the supine (131.1 +/- 24.7 versus 356.2 +/- 58.4 pg/ml, p less than 0.01) and upright (224.9 +/- 47.8 versus 673.3 +/- 122.3 pg/ml, p less than 0.005) positions. The diabetic patients determined as having cardiac autonomic neuropathy (n = 15) had depressed left ventricular diastolic filling compared with subjects free of autonomic neuropathy, whether measured as the time to peak filling rate (154.2 +/- 12.0 versus 119.1 +/- 10.6 ms, p less than 0.05) or the time to peak filling rate normalized to the cardiac cycle length (24.3 +/- 2.2 versus 16.2 +/- 1.5%, p less than 0.01). Of the various tests of autonomic nervous system function, the strongest correlate of impaired diastolic filling was orthostasis, measured as the decrease in systolic blood pressure with standing (r = 0.584, p less than 0.001). Thus, in patients with diabetes mellitus, alterations in sympathetic nervous system activity are associated with abnormalities of left ventricular diastolic filling. PMID- 3011874 TI - Beta-adrenergic responses in drug-free subjects with asthma. AB - Young atopic subjects with asthma who had taken no medication for 30 days and had normal pulmonary function were compared to atopic and nonatopic control subjects in several measures of beta-adrenergic response. Subjects with asthma required a larger dose of infused isoproterenol (14.3 +/- 3.9 ng/kg/min) to increase pulse pressure by a target greater than 22 mm Hg than nonatopic control subjects (8.7 +/- 3.3 ng/kg/min). Asymptomatic atopic control subjects had intermediate sensitivity (12.0 +/- 6.0 ng/kg/min) (F = 3.67; p less than 0.05). At the dose of infused isoproterenol producing the target pulse pressure response, the increase in low-frequency (1 to 8 cycles per minute) heart rate variability was less in subjects with asthma (107 +/- 34% increase in normal subjects versus 9 +/- 21% increase in subjects with asthma; p less than 0.05). In addition, subjects with the least beta-adrenergic responsiveness had the most reactive airways. Airway reactivity (assessed by the provocative concentration of methacholine causing a 20% fall in FEV1) correlated with both the dose of isoproterenol required for the target pulse pressure response (Rs = -0.46; p less than 0.05) and the isoproterenol-induced increase in low-frequency heart-rate variability (Rs = 0.47; p less than 0.05). In contrast, in lymphocytes and granulocytes from these same subjects, the cAMP response to isoproterenol and beta-adrenergic receptor number were identical in normal subjects and subjects with asthma and unrelated to airway reactivity or to cardiovascular beta-adrenergic responses. Thus, different results for beta-adrenergic responsiveness in subjects with asthma are obtained in different tests in the same subjects. PMID- 3011873 TI - Toluene diisocyanate-induced airway hyperreactivity and pathology in the guinea pig. AB - We assessed the nature and progression of airway mucosal disease and histaminic reactivity in English short-haired guinea pigs at 2, 24, 72, 168, and 504 hours after toluene diisocyanate (TDI) exposure (4 hours of 3 ppm of TDI for 5 consecutive days). To also determine whether TDI-specific, IgE-like antibodies developed in TDI-exposed animals, passive cutaneous anaphylaxis testing was done 28 days after TDI. Bronchial reactivity was determined serially by measuring specific airway conductance as a function of increasing doses of aerosolized histamine in six exposed and three control animals studied intact and unanesthetized. The remaining 10 exposed and 10 control guinea pigs were sacrificed in groups of two at each time point to obtain airway tissue for light microscopic examination. We found that airway hyperreactivity to histamine occurred after TDI in all animals tested. It was maximal 2 hours after the 5-day exposure and remitted by 72 hours. In addition, marked airway obstruction occurred after TDI that persisted for at least 168 hours. There were dramatic signs of airway mucosal damage associated with the bronchial hyperreactivity that included substantial decreases in epithelial cilia, mucin content, and mast cells, as well as squamous metaplasia, numerous mitotic figures, and a prominent polymorphonuclear leukocytic infiltrate. Passive cutaneous anaphylaxis tests in exposed animals were negative. Our results suggest that TDI-induced bronchial hyperreactivity may be related to airway mucosal injury and inflammation. PMID- 3011875 TI - A critical review of food fiber analysis and data. AB - Epidemiologists, research scientists, and dietitians need data on the dietary fiber content of foods. This article provides a provisional table on dietary fiber, compiled after a thorough search of the literature and a critical evaluation of the analytical methodology employed. To make fully understandable the limitations and problems associated with the current dietary fiber data base, a short review of what is meant by the term dietary fiber and the complex chemical structures of the major dietary fibers--cellulose, hemicellulose, pectin, and lignin--are presented. A short description of the numerous analytical methods for quantifying dietary fiber, including the neutral detergent fiber procedure, the various enzymatic gravimetric procedures, and the analytic schemes for measuring the major dietary fiber fractions is also given, along with the strengths and weakness of the various procedures. The table on foods commonly eaten in the United States is meant as an interim guide for menu planning and dietary evaluation until newer data become available. Data are most limited on legumes and the numerous specialty baked products and breads available in this country. PMID- 3011876 TI - Serum lipid response to oat product intake with a fat-modified diet. AB - Healthy adult volunteers (no. = 208), men and women aged 30 to 65 years, participated in a 12-week study on dietary fat modification plus oat product ingestion (60 gm/day) to test whether moderate daily intake of oat bran and oatmeal enhanced serum lipid response. During weeks 0 to 6, all participants followed the American Heart Association fat-modified eating style. At 6 weeks, participants were randomly assigned to one of three groups. All participants continued to follow the fat-modified eating pattern; groups 1 and 2 were asked during weeks 7 to 12 to consume two servings of either oat bran or oatmeal per day, for a total of 60 gm/day isocalorically substituted for other carbohydrates. Group 3 ingested no oat products. At baseline, the group mean cholesterol level was 208.4 mg/dl. After 6 weeks of dietary fat intervention, the level was 197.6- a fall of 10.8 mg/dl (5.2%). At 12 weeks, the mean serum cholesterol level fell further, by 5.6, 6.5, and 1.2 mg/dl for groups 1, 2, and 3, respectively. Group mean weight loss was small--1.9 lb during the first 6 weeks and 0.6 to 0.8 for the three groups during weeks 7 to 12. Reported oat product ingestion was 39 and 35 gm per person per day, respectively, for groups 1 and 2 (2.2 and 1.4 servings per person per day, respectively). Dietary fat composition remained similar among the three groups during weeks 7 to 12. Pooled results indicated that the addition of oat products at a moderate and practical level enhanced serum lipid response (p less than .05) to a fat-modified eating pattern among free-living adults. PMID- 3011877 TI - Variability in the dietary fiber content of wheat and mixed-grain commercial breads. PMID- 3011878 TI - Sensitive quantitation of endonuclease kinetics. AB - Described here is an assay permitting exclusive quantitation of the kinetics of endonucleolysis by a mixed-function nuclease at substrate DNA concentrations much lower than those necessary with other assay methods. It makes possible determination of Michaelis parameters Km and kcat for endonuclease activity of such enzymes. The sensitivity of the assay method to very low DNA concentrations is obtained through use of 32P-labeled DNA as substrate. Digested DNA is electrophoresed, and computerized analysis of an autoradiogram made from the gel gives the extent of digestion. The analysis produces a profile of weight fraction vs. DNA fragment size for each sample taken from a nuclease-DNA reaction mixture. Each experimental profile is compared to a family of theoretical profiles generated by computer using theory of polymer statistics and assuming random cleavage. Theoretical profiles are found whose shapes most closely match those of the experimental profiles. Associated with each best-fitting theoretical profile is a value of the number of cuts made in the DNA sample. These values, plotted against time, give initial reaction velocities. Initial velocities from experiments at different DNA concentrations have been used to obtain Km and kcat for micrococcal nuclease under specific reaction conditions. PMID- 3011880 TI - Displacement analysis of binding inhomogeneities in crude extracts of receptors. AB - Guidelines are given to distinguish different kinds of binding inhomogeneities of non-radiolabeled ligands in crude extracts of receptors if an appropriate 'binding analogue' of the displacers is available in radiolabeled form. Three minimal models for the simplest types of binding inhomogeneities are analysed theoretically. These models include a cooperative system (with two interacting sites on the same receptor molecule) and two non-cooperative systems (one of them with a single-site receptor having two conformational states in equilibrium and the other with two single-site receptors independent of each other). In certain cases one can distinguish these systems experimentally. Furthermore, if a group of displacers is already classified according to the above models, then dissociation constants can be determined. The quantitative comparison of these displacers on the basis of their dissociation constants is more appropriate (e.g. in Quantitative Structure Activity Relationship studies) than on the basis of their ID50 and Ki values or Hill coefficients, which is often done. PMID- 3011879 TI - An economic water-jacketed vacuum-enforced mini-column system for quick separation of double and single stranded DNA. AB - A jacketed column system that can be applied in separation of double and single stranded DNA by hydroxylapatite chromatography is described. The construction of the column allows high flow rates and a short manipulation time. The system is simple, inexpensive, and easy to construct. PMID- 3011882 TI - Absorption, deposition and distribution of dietary aluminium in immature rats: effects of dietary vitamin D3 and food-borne chelating agent. AB - We report the levels of aluminium, calcium and potassium in selected tissues of growing rats administered dietary or subcutaneous aluminium, and also the effect of dietary aluminium in combination with cholecalciferol, or with lactose plus a dietary chelating agent. Dietary aluminium decreased the growth rate of normal rats and increased the deposition of aluminium in the tissues. Animals given lactose with a dietary chelator showed a 17 - 100% increase in brain, heart, and muscle aluminium concentration in comparison with those fed aluminium alone. Animals fed both aluminium with cholecalciferol also showed increased levels (12 39%) of aluminium, chiefly in muscle and heart in comparison with those fed aluminium alone. Aluminium deposition was correlated positively with Ca2+ and K+ levels among each of these tissues. We conclude that in normal growing rats aluminium deposition is increased in heart and muscle in the presence of vitamin D3 and in brain, heart and muscle in the presence of lactose and a dietary chelating agent. PMID- 3011881 TI - Regulation of aflatoxin biosynthesis: effect of adenine nucleotides, cyclic AMP and N6-O2' -dibutyryl cyclic AMP on the incorporation of (1-14C)-acetate into aflatoxins by Aspergillus parasiticus NRRL-3240. AB - Adenosine triphosphate (ATP) was found to inhibit the incorporation of labelled acetate into aflatoxins while adenosine diphosphate (ADP) and adenosine monophosphate (AMP) stimulated the process. Exogenous cyclic AMP and its derivative, N6-O2' -dibutyryl cyclic AMP produced efficient (1-14C)-acetate incorporation into aflatoxins at all the concentrations tried (0.1, 0.5 and 1 mM). The stimulation of incorporation of labelled acetate into aflatoxins was significant at 0.1 and 1 mM concentration of the cyclic nucleotide but was found to be maximum at 0.5 mM concentration. PMID- 3011883 TI - Synovial sarcoma in the foot. AB - Synovial sarcoma is a rare malignancy that occurs most frequently in the extremities. Its onset and associated symptoms are often insidious and therefore can be misleading. The authors review this primary malignancy and offer a case report to illustrate the importance of including such entities in the differential diagnosis and subsequent treatment of soft tissue masses found in the foot. PMID- 3011884 TI - Effects of danazol on steroidogenesis and gonadotropic responsiveness in isolated human preovulatory follicular cells. AB - In order to investigate the influence of danazol on steroidogenesis and gonadotropic responsiveness of human follicular cells, granulosa and thecal cells of preovulatory follicles were isolated and separately incubated for short term periods. Human chorionic gonadotropin (hCG) (100 IU/ml), FSH (0.5 IU/ml) and danazol (10 micrograms/ml) alone or in combination were added to the incubation medium. Following incubation the cellular cyclic adenosine 3'5' monophosphate (cAMP) levels and the medium content of progesterone (P), androstenedione (A) and 17 beta-estradiol (E2) were determined. All follicles included in the study were classified as nonatretic and well developed, i.e. less than 3 days before ovulation. Human chorionic gonadotropin caused an increase in cAMP formation in both cell-types and this effect was significantly counteracted by danazol in vitro. In granulosa cells danazol tended to counteract a stimulatory effect of FSH on cAMP formation. No significant influence of danazol was found on the basal steroid formation of both cell types during short term incubation. On the other hand, danazol significantly counteracted the FSH stimulated P formation of the granulosa cells and the hCG stimulated A and E2 formation of the thecal cells. It is concluded that danazol inhibits gonadotropin-stimulated steroidogenesis locally in the human follicular cells and that this effect of danazol is mediated via the cyclic AMP system. PMID- 3011885 TI - ACTH 1-17 stimulates GH pituitary secretion in humans. AB - Nineteen normal subjects (12 men and 7 women) were injected with 100 micrograms of ACTH 1-17. Additionally 6 male subjects were studied twice at 3-day intervals with random infusions of ACTH 1-17 and saline. A clear GH response to ACTH 1-17 infusions (GH peak higher than 5 ng/ml) was documented in 10 out of 12 males and in 6 out of 7 women. In the 6 male subjects studied twice, clear-cut GH increments were observed only after peptide administration. PRL levels decreased throughout the study period both in male and female subjects; however, when the PRL percentage decline was evaluated in the same group of subjects after saline and ACTH 1-17, the more obvious decrease of PRL levels after the peptide infusion was not statistically significant. No variation of LH, FSH and TSH levels was documented. With the exception of the specific increase of cortisol levels, no significant change in peripheral steroid pattern (Te, E2, DHEA-S) was observed. In this experiment the effect on GH secretion was quite evident in both sexes. This effect was obtained using the lowest dosage of ACTH preparation documented in the literature. PMID- 3011886 TI - Steroidogenesis by the immature rhesus monkey ovary in vitro. AB - Primates are believed to have a low level of ovarian steroidogenic activity during prepubertal development. In order to study the rate limiting factors associated with the low level of steroidogenesis, ovaries from prepubertal rhesus monkeys were quartered and incubated for 48 h at 37 C in minimum essential medium. These ovaries secreted 687 +/- 347 pg estradiol/mg ovary and 299 +/- 35 pg progesterone/mg ovary during 48 h of incubation. The addition of 100 ng luteinizing hormone (LH) or 1 mM dibutyryl (Bu)2 cAMP failed to increase significantly estradiol or progesterone secretion. Furthermore, the addition of either progesterone or androstenedione failed to augment estradiol secretion. The presence of either LH or (Bu)2 cAMP with the steroidal substrates also failed to augment estradiol secretion. In contrast, the addition of (Bu)2 cAMP with lipoprotein-derived cholesterol significantly stimulated a two-fold increase in progesterone secretion. The presence of LH in the lipoprotein-supplemented medium failed to augment progesterone secretion. These results suggest that prepubertal monkey ovaries lack the ability to respond to LH, probably due to a lack of gonadotropin receptors or failure of the receptor to stimulate cAMP synthesis. Furthermore, the failure of progesterone and androstenedione to augment estradiol secretion suggests that some cellular components needed to induce aromatase activity are not functional in the prepubertal primate ovary. PMID- 3011888 TI - A rapid method for raising monospecific antisera against polio viruses in monkeys. PMID- 3011887 TI - Metabolic and ultrastructural aspects of experimental diabetic glomerulopathy. AB - The effects of alloxan induced diabetes and insulin treatment on rat kidney glomerular ion transport and the oxidative phosphorylation were investigated in order to correlate metabolic and ultrastructural alterations. In alloxan diabetic rats an early decrease of Na+K+ ATPase activity in isolated glomeruli preparations and the subsequent uncoupling of oxidative phosphorylation were observed. These metabolic alterations are associated with structural changes such as the thickening of the basement membrane and the disorganization of both endothelial and mesangial cells. Insulin treatment induces a slow and only partial recovery of metabolic changes. Ultrastructural features of the kidney cortex were almost completely normalized after only two months of insulin treatment. PMID- 3011889 TI - Increased sympatho-adrenal tone and adrenal medulla reactivity in DOCA-salt hypertensive rats. AB - Sympatho-adrenal tone and reactivity were evaluated in anaesthetized normotensive and DOCA-salt hypertensive rats, by measuring arterial plasma concentrations of norepinephrine and epinephrine under basal conditions and following bilateral carotid occlusion. Baseline norepinephrine levels were significantly higher in DOCA-salt hypertensive animals than in their respective normotensive controls, whether they were studied with intact vagi or following bilateral vagotomy. The possibility of a relationship between the increased basal sympathetic fibres and the maintenance of DOCA-salt hypertension is strongly suggested by the finding of a significant correlation between mean arterial pressure (MAP) and basal circulating norepinephrine values in those animals. Furthermore, the epinephrine increase following carotid occlusion was found to be markedly potentiated in hypertensive animals (intact or vagotomized), suggesting adrenal medullary hyperreactivity to baroreflex activation in this model of hypertension. In normotensive rats the epinephrine increase induced by the carotid occlusion was greatly potentiated by the administration of an alpha 2-antagonist (yohimbine), and completely abolished by administration of an alpha 2-agonist (clonidine). In contrast, the epinephrine response to carotid occlusion, which is already enhanced in hypertensive animals, was unaffected by the same treatments. These results therefore suggest that adrenal medullary hyperreactivity observed in DOCA salt hypertensive rats may be due to a dysfunction of an alpha 2-adrenergic mechanism modulating adrenal medullary secretion. PMID- 3011890 TI - Influence of converting enzyme inhibition on the hormonal and renal adaptation to hyper- and hyponatraemic dehydration. AB - The present study was designed to investigate in rats the influence of converting enzyme inhibition with captopril on blood pressure, plasma urea, plasma renin concentration (PRC), plasma aldosterone and plasma vasopressin, and to define the interrelationships between PRC and these variables during equal degrees of either hyponatraemic (furosemide, 40 mg/kg for 2 days) or hypernatraemic (48-h water deprivation) dehydration. Chronic treatment with captopril (40 mg/kg daily) decreased blood pressure by 19% in normally hydrated treated rats, by 27% in water-deprived treated rats and by 40% in furosemide-treated rats. Plasma renin concentration, plasma aldosterone and plasma vasopressin were increased during both hypo- and hypernatraemic dehydration. Captopril decreased plasma aldosterone in water-deprived and furosemide-treated rats, whereas plasma vasopressin was unchanged. The significant correlation observed between plasma aldosterone and PRC in non-treated rats persisted in treated rats, the same level of plasma aldosterone being observed at values of PRC 10 times higher. On the other hand, the correlation between plasma vasopressin and PRC did not persist in captopril treated rats. An increase in plasma urea was observed in both water-deprived treated rats and furosemide-treated rats. These data indicate that during hypo- and hypernatraemic dehydration, the renin-angiotensin system plays a role in regulating blood pressure, urea elimination and plasma aldosterone, but vasopressin regulation is not modified by its inhibition. PMID- 3011892 TI - Two-kidney, one clip renal hypertension in the marmoset. AB - The purpose of this study was to assess whether two-kidney, one clip (2K1C) renal hypertension can be induced in the marmoset. During the first 3-5 weeks after renal arterial clipping, blood pressure (BP) and plasma renin activity (PRA) increased in approximately one-third of the operated marmosets. However, within 10 weeks after clipping, BP and PRA had returned to control values. There was a significant positive correlation between BP and log PRA 3 and 5 weeks after the operation, but no correlation was observed at 10 weeks. In a selected group of marmosets with the highest values of BP (greater than 140 mmHg; n = 4), the converting enzyme inhibitor, enalapril (2 mg/kg s.c.) lowered BP by 58 +/- 7 (s.e.m.) mmHg when given 3 weeks after clipping. At 18 weeks the response to enalapril was only -17 +/- 6 mmHg. These results demonstrate that unilateral renal arterial clipping in marmosets results in a transient renin-dependent hypertension. Marmosets in this initial hypertensive phase could be useful for investigating the antihypertensive effects of inhibitors of human renin. PMID- 3011891 TI - Nutrient intake, blood pressure, serum and urinary prostaglandins and serum thromboxane B2 in a controlled trial with a lacto-ovo-vegetarian diet. AB - Fifty-nine healthy omnivores volunteered for a randomized crossover trial with a lacto-ovo-vegetarian (L-O-V) diet. Twenty-one 1-day diet records were kept throughout the project as a means of assessing food and nutrient intakes, and samples of serum and urine were assayed to evaluate change in prostanoid metabolism. While on the L-O-V diet subjects ate more vegetable protein, wholegrain cereals, polyunsaturated oils, fruits and vegetables, and avoided eating meat, fish or poultry. The L-O-V diet contained significantly more polyunsaturated fatty acids, fibre, vitamin C, vitamin E, magnesium, calcium and potassium, and less total protein, saturated fat, monounsaturated fat and vitamin B12 than the control omnivore diet. Changes in nutrient intakes were subjected to principal components analysis to identify dimensions of change in nutrient intakes. Three Factors accounted for 83% of the total variation in dietary intake. Blood pressure changes were significantly and negatively (F = 17.4, P less than 0.001 for systolic; F = 6.09, P = 0.02 for diastolic pressure) related to individual scores for only one Factor--that representing an increase in intake of polyunsaturated fat, fibre, vitamin C, vitamin E, calcium and magnesium, and a fall in intake of protein and vitamin B12. Blood pressure changes were unrelated to change in body weight or sodium intake. Serum and urinary prostanoids were not affected by eating the L-O-V diet. PMID- 3011893 TI - Stress levels of adrenaline amplify the blood pressure response to sympathetic stimulation. AB - The possibility that sympathetic pressor responses are modulated by adrenaline mediated facilitation of neuronal noradrenaline release was explored in 17 subjects with borderline hypertension. Infusion of adrenaline, which raised plasma adrenaline by a factor of 8 to 9, augmented the rise in systolic and diastolic arterial pressure induced by standardized cold pressor and isometric exercise tests. The heart rate response to these tests was not affected. When a low dose of propranolol was given on top of the adrenaline infusion before the cold pressor test, the blood pressure response to cold exposure was not different from the response observed when the test was performed during saline infusion. Plasma noradrenaline was higher during adrenaline infusion then during saline infusion, both before and after the cold pressor and isometric exercise tests, and the effect of adrenaline on plasma noradrenaline was antagonized by propranolol. These observations are consistent with the hypothesis that stress levels of circulating adrenaline may amplify sympathetic pressor responses by facilitation of the release of transmitter noradrenaline. PMID- 3011894 TI - Production of nephritic factor of the alternative complement pathway by Epstein Barr virus-transformed B cell lines derived from a patient with membranoproliferative glomerulonephritis. AB - Nephritic factor of the alternative complement pathway (C3NeF) is an IgG autoantibody which binds to and stabilizes the C3 convertase (C3bBb) enzyme, and which has been detected mainly in sera from patients with membranoproliferative glomerulonephritis (MPGN) and partial lipodystrophy. To study the production of C3NeF, mononuclear cells isolated from the peripheral blood of patients with MPGN and C3NeF activity in their sera were infected with Epstein Barr virus (EBV) to establish active B lymphocyte cell lines. By using a modified C3NeF screening assay, we detected C3NeF activity in the supernatant of a B cell line derived from a patient with MPGN Type II, but in none of the supernatants of B cell lines derived from normal individuals. C3NeF-positive supernatants were investigated for their ability to conserve classical or alternative pathway C3 convertase activity by using EAC3bBb and EAC4b2a stabilization assays. C3NeF-positive supernatants stabilized the C3bBb convertase activity, but not the C4b2a convertase activity. Studies of the supernatants, using anti-human IgG affinity columns, showed that the C3NeF activity was in the IgG fraction; furthermore, C3NeF antibody agglutinated sheep erythrocytes coated with C3bBb, but not with C3b alone. On gel electrophoresis, both heavy and light chains of the C3NeF were comparable in size to that of normal human IgG molecules. We conclude that C3NeF, produced in vitro by EBV-transformed B cell lines derived from a patient with MPGN Type II, is functionally identical to the conventional C3NeF in serum. In vitro preparation of homogeneous NeF(s) should greatly facilitate the studies of the role of these autoantibodies in complement dysmetabolism. PMID- 3011895 TI - Properties of a specific interleukin 1 (IL 1) receptor on human Epstein Barr virus-transformed B lymphocytes: identity of the receptor for IL 1-alpha and IL 1 beta. AB - The properties of specific human interleukin 1 (IL 1) receptors on human Epstein Barr virus-transformed B lymphocytes (EBV-B) were studied. Purified human IL 1 beta from a myelomonocytic cell line (THP-1) was labeled with 125I by the Bolton Hunter method without detectable loss of biological activity. Among four EBV-B cell lines tested, a pre-B cell type (VDS-O) specifically bound the highest amount of 125I-IL 1-beta. Maximal binding was reached within 20 min at 4 degrees C. Scatchard plot analysis of the binding of 125I-IL 1-beta to VDS-O cells yielded a Kd (dissociation constant) of 2.4 to 5.9 X 10(-10) M with 110 to 220 binding (receptor) sites/cell. The binding of 125I-IL 1-beta to VDS-O cells was also inhibited by F(ab)'2 fragments of anti-human IL 1 and recombinant human IL 1 alpha, as well as by unlabeled human IL 1-beta but not by recombinant lymphotoxin, recombinant tumor necrosis factor, or phorbol myristic acid, suggesting that IL 1-alpha and IL 1-beta bind specifically to the same receptor. The m.w. of IL 1 receptor on human EBV-B cells was estimated to be 60,000 by both the chemical cross-linking method and high pressure liquid chromatography (HPLC) gel filtration analysis of receptor extracted from membrane enriched fraction by a non-ionic detergent (CHAPS). The isoelectric point of solubilized human IL 1 receptor was 7.3 on HPLC chromatofocusing. The evidence of existence of IL 1 receptor on human EBV-B cells additionally supports the hypothesis that IL 1 may be an autocrine signal for these cells. PMID- 3011897 TI - Oxidative inactivation of pneumolysin by the myeloperoxidase system and stimulated human neutrophils. AB - Pneumolysin, a hemolytic toxin from Streptococcus pneumoniae, is a member of the group of thiol-activated, oxygen-labile cytolysins produced by various Gram positive bacteria. The toxin activity of pneumolysin, as determined by lysis of 51Cr-labeled human erythrocytes, was destroyed on exposure to the neutrophil enzyme myeloperoxidase, hydrogen peroxide, and a halide (chloride or iodide). Detoxification required each component of the myeloperoxidase system and was prevented by the addition of agents that inhibit heme enzymes (azide, cyanide) or degrade H2O2 (catalase). Reagent H2O2 could be replaced by the peroxide generating enzyme system glucose oxidase plus glucose. The entire myeloperoxidase system could be replaced by sodium hypochlorite at micromolar concentrations. Toxin inactivation was a function of time of exposure to the myeloperoxidase system (less than 1 min), the rate of formation of H2O2 (0.05 nmol/min), and the concentration of toxin employed. Toxin that had been inactivated by the myeloperoxidase system was reactivated on incubation with the reducing agent dithiothreitol. Pneumolysin was also inactivated when incubated with human neutrophils (10(5)) in the presence of a halide and phorbol myristate acetate, an activator of neutrophil secretion and oxygen metabolism. Toxin inactivation by stimulated neutrophils was blocked by azide, cyanide, or catalase, but not by superoxide dismutase. Neutrophils from patients with impaired oxygen metabolism (chronic granulomatous disease) or absent myeloperoxidase (hereditary deficiency) failed to inactivate the toxin unless they were supplied with an exogenous source of H2O2 or purified myeloperoxidase, respectively. Thus, inactivation of pneumolysin involved the secretion of myeloperoxidase and H2O2, which combined with extracellular halides to form agents (e.g., hypochlorite) capable of oxidizing the toxin. This example of oxidative inactivation of a cytolytic agent may serve as a model for phagocyte-mediated detoxification of microbial products. PMID- 3011896 TI - Differential stimulation of the respiratory burst and lysosomal enzyme secretion in human polymorphonuclear leukocytes by synthetic diacylglycerols. AB - Binding of chemoattractants to receptors on human polymorphonuclear leukocytes (PMN) stimulates the phosphodiesteric cleavage of phosphatidylinositol 4,5 bisphosphate to produce inositol 1,4,5-trisphosphate and 1,2-diacylglycerols. To investigate the possible second messenger function of diacylglycerols in PMN activation, we tested the ability of a series of synthetic sn 1,2 diacylglycerols, known to stimulate protein kinase C in other systems, to promote superoxide anion release, oxygen consumption, lysosomal enzyme secretion, and chemotaxis. None of the diacylglycerols initiated the chemotactic migration of PMN. Several of the diacylglycerols however, were, active in stimulating superoxide anion release and lysozyme secretion, with dioctanoylglycerol (diC8) being the most potent. Unexpectedly, didecanoylglycerol (diC10) induced lysosomal enzyme secretion, but failed to stimulate superoxide production or oxygen consumption. All other biologically active diacylglycerols tested displayed similar EC50 for stimulating lysozyme secretion and superoxide production. The ability of the diacylglycerols to compete for phorbol dibutyrate (PDBu) binding in intact PMN suggested a mechanism for the divergent biological activity of diC10. Although the compounds that stimulated both superoxide production and lysosomal enzyme secretion competed for essentially all [3H]PDBu binding from its receptor, diC10, which only stimulated secretion, competed for 45% of the bound [3H]PDBu. Thus diacylglycerols can selectively activate certain functions of leukocyte chemoattractant receptor. The data suggest that a discrete pool of protein kinase C may mediate activation of the respiratory burst in PMN. PMID- 3011898 TI - Effects of tunicamycin on the expression and function of formyl peptide chemotactic receptors of differentiated HL-60 cells. AB - We previously showed that formyl peptide chemotactic receptors (FPCR) of human phagocytic cells contain at least two asparagine-linked oligosaccharide chains located at the distal end of the receptor. The requirement of these N-linked oligosaccharide chains for expression and function of FPCR was investigated in HL 60 cells induced to differentiate by N6,O2-dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) in the presence or absence of 5 micrograms/ml tunicamycin. Tunicamycin did not prevent the changes in morphology associated with Bt2cAMP-induced differentiation of HL-60 cells. Autoradiographic analysis after SDS-PAGE of FPCR affinity labeled with N-formyl-Nle-Leu-Phe-Nle-[125I]iodo-Tyr-Lys (formyl 125I hexapeptide) and ethylene glycol bis(succinimidyl succinate) demonstrated that greater than 95% of FPCR expressed by tunicamycin-treated cells completely lacked N-linked oligosaccharide (Mr 32,000), and no fully glycosylated FPCR (Mr 62,000 to 85,000) was detectable. Scatchard analysis of formyl 125I-hexapeptide binding indicated the presence of two classes of binding sites for both control and tunicamycin-treated cells (control cells, 82,000 +/- 32,000 sites/cell with Kd 10.0 +/- 4.3 nM and 520,000 +/- 40,000 sites/cell with Kd 250 +/- 80 nM; tunicamycin-treated cells, 11,000 +/- 5000 sites/cell with Kd 3.0 +/- 1.9 nM and 470,000 +/- 70,000 sites/cell with Kd of 500 +/- 140 nM). Both control and tunicamycin-treated cells augmented superoxide anion release, exhibited a migratory response, and showed a transient rise in intracellular free Ca2+ upon stimulation with N-formyl-Nle-Leu-Phe. However, the responses of the tunicamycin treated cells were less than that of the control cells. The present studies demonstrate that N-glycosylation of FPCR is not essential for cell surface expression or for several FPCR-mediated cell responses. PMID- 3011899 TI - Development of receptors for leukotriene B4 on HL-60 cells induced to differentiate by 1 alpha,25-dihydroxyvitamin D3. AB - The incubation of HL-60 human promyelocytic leukemia cells for 7 days with 100 nM 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced differentiation into monocyte-like cells, as assessed by morphologic and biochemical characteristics. Stereospecific receptors for leukotriene B4 (LTB4) developed on the surface of the HL-60 cell-derived monocytes that had the capacity to transduce LTB4 stimulation of a transient increase in the cytosolic concentration of calcium ([Ca+2]in). HL-60 cell-derived monocytes, but not undifferentiated HL-60 cells, expressed a high affinity subset of 6400 +/- 3700 receptors per cell with a dissociation constant (Kd) of 2.3 +/- 1 nM (mean +/- SD, n = 3) and a low affinity subset of approximately 2.2 X 10(6) receptors per cell with an apparent Kd of 680 +/- 410 nM. Derivatives of LTB4 inhibited the binding of [3H]LTB4 to HL 60 cell-derived monocytes with a rank order of potency of LTB4 greater than 20-OH LTB4 greater than 3-aminopropyl amide-LTB4, which is similar to the order for LTB4 receptors of human blood PMNL. In contrast, leukotrienes C4 and D4 and formyl-methionyl chemotactic peptides did not inhibit the binding of [3H] LTB4, which demonstrates the specificity of these receptors for isomers of 5,12 dihydroxy-eicosatetraenoic acid. LTB4 stimulated an increase in [Ca+2]in in HL-60 cell-derived monocytes which reached 50% of the maximal level at an LTB4 concentration of 0.5 nM (EC50). Preincubation of HL-60 cell-derived monocytes with 10 nM LTB4 resulted in a selective loss of high affinity receptors, as assessed by binding of [3H]LTB4, and a 200-fold increase in the EC50 for stimulation by LTB4 of increases in [Ca+2]in, without alterations in either the low affinity receptors for LTB4 or the responsiveness of [Ca+2]in to formyl methionyl chemotactic peptides. HL-60 cells that are induced to differentiate into monocytes thus develop stereospecific receptors for LTB4 with binding and transductional characteristics similar to those of human blood PMNL. PMID- 3011900 TI - Platelet-activating factor (PAF) mediation of rat anaphylactic responses to soluble immune complexes. Studies with PAF receptor antagonist L-652,731. AB - A new synthetic compound, L-652,731 (trans-2,5-(3,4,5-trimethoxyphenyl) tetrahydrofuran), which has been demonstrated by Hwang et al. to be a potent and specific platelet-activating factor (PAF) receptor antagonist causes 100% inhibition of 1 microM PAF-induced neutrophil degranulation at 50 microM, but has no effect on neutrophil degranulation induced by precipitating immune complexes (323 micrograms/ml), fMet-Leu-Phe (10(-7) M), or the calcium ionophore A23187 (10(-5) M). Intravenous infusion of 1 mumol L-652,731 results in almost 100% inhibition of hypotension induced by PAF but not that induced by isoproterenol, histamine, bradykinin, or acetylcholine. With the use of this novel PAF receptor antagonist, the in vivo mediator role of PAF in the soluble immune complex induced hypotension, extravasation, vascular lysosomal hydrolase secretion, and neutropenia in rats was determined. The hypotension, extravasation, and lysosomal hydrolase release induced by immune complex infusion take 2 to 10 min longer to occur than the same responses elicited by PAF infusion. The neutropenia response is immediate with both stimuli. L-652,731 when orally administered to rats (20 mg/kg, 1.5 hr before PAF infusion) inhibited PAF-induced hypotension (69%), extravasation (76%), vascular lysosomal hydrolase release (79%), and neutropenia (73%). The same L-652,731-dosing regimen inhibited immune complex-stimulated hypotension (87%), extravasation (77%), and vascular lysosomal hydrolase release (31%). The initial and complete neutropenia induced by immune complex infusion was not inhibited in L-652,731-pretreated rats, but the rate of return of neutrophils to the blood was faster in the latter rats. Rats with blocked circulation to the liver still exhibited extensive extravasation and vascular lysosomal hydrolase release in response to PAF, but there was no extravasation and greatly reduced hydrolase release in response to immune complexes. Thus PAF is indicated to be a major mediator of soluble immune complex-induced hypotension and vascular permeability and a minor mediator of immune complex-induced lysosomal hydrolase release in rats. PAF probably does not mediate the initial and complete neutropenia stimulated by immune complexes. The liver is probably the major site for PAF production in response to circulating immune complexes. PMID- 3011901 TI - Expression of transfected human interferon-gamma DNA: evidence for cell-specific regulation. AB - The production of interferon-gamma (IFN-gamma) has been limited to two specific cell types of the immune system, T cells and large granular lymphocytes. Interleukin 2 (IL 2) appears to be the primary physiologic stimulus for IFN-gamma production in vitro, but other agents, such as antigens, phorbol myristic acetate, concanavalin A, or other plant lectins, may also act as effective inducing agents for IFN-gamma production. Little is known, however, as to the role, if any, that genetic factors may play in the induction process. We now report that, on stable transfection of the genomic human IFN-gamma 8.6 Kb BamH DNA fragment into a mouse T lymphoblast cell line, both mRNA expression and synthesis of human IFN-gamma were stimulated by both the physiologic ligand IL 2 and phorbol ester. In contrast, we have been unable to induce with extracellular stimulants IFN-gamma production or cytoplasmic mRNA after introduction of this gene into NIH 3T3 fibroblasts, thus suggesting that the extracellular regulation of the expression of IFN-gamma may be controlled by a developmental mechanism(s) intrinsic for cells of lymphoid lineage. PMID- 3011902 TI - The low affinity 40,000 Fc gamma receptor and the transferrin receptor can be alternative or simultaneous target structures on cells sensitive for natural killing. AB - The role of the low avidity 40,000 dalton receptor for IgG (Fc gamma R) present on K562 and U937 cells in sensitivity to natural killing (NK) was studied by using a murine monoclonal antibody (mAb) specific for the 40,000 dalton Fc gamma R (alpha Fc gamma R mAb). Pretreatment of K562 target cells with intact alpha Fc gamma R mAb or its Fab fragment or anti-transferrin receptor (alpha TFR) mAb partially blocked in a dose-dependent manner, NK activity to K562 cells. However, combined pretreatment with alpha Fc gamma R and alpha TFR mAb completely blocked NK activity against K562 targets. As compared with K562 cells, lower levels of NK were elicited against Molt-4, U937, HL-60, and Daudi targets. Although NK activity to Molt-4 targets was not affected by alpha Fc gamma R mAb, it was fully prevented by pretreatment with alpha TFR mAb. In contrast, NK to U937 cells was not influenced by alpha TFR mAb, but it was strongly inhibited by alpha Fc gamma R mAb. The resistance of 3H-TdR-prelabeled adherent HEp-2 cells to natural cell mediated cytotoxicity was not affected by either mAb. Lectin-dependent cell mediated cytotoxicity (LDCC) against HEp-2 cells due to the presence of concanavalin A, and was completely abrogated by pretreatment of the targets with alpha TFR mAb, but was unaffected by alpha Fc gamma R mAb. By use of the flow cytometer, a significant correlation was detected between the relative expression of 40,000 dalton Fc gamma R and the susceptibility to NK, whereas the expression of TFR was discordant from NK sensitivity. As determined in the single cell cytotoxicity assay alpha Fc gamma R mAb reduced the frequency of target binding effector cells without affecting the number of dead bound targets. This pattern of inhibition was found against both K562 and U937 targets. Alternatively, alpha TFR mAb inhibited both binding and killing of K562 and Molt-4 targets. Because pretreatment of HEp-2 cells with alpha TFR mAb did not influence conjugate formation, the blocking of LDCC to HEp-2 cells by alpha TFR mAb can be related to post-binding events. These data show that although both the 40,000 dalton Fc gamma R and the TFR can be target structures for NK cell recognition, the TFR may also play an important role in the post-binding events. PMID- 3011903 TI - Anti-tumor properties of lymphocytes activated by the oxidizing mitogens. AB - Human peripheral blood mononuclear cells when activated with the oxidizing mitogens, neuraminidase/galactose oxidase or sodium periodate, express cytolytic activity for freshly isolated tumor cells and for a variety of cell lines, including NK-resistant solid tumor lines. Normal lymphoid cells are not targets for cytotoxicity and do not inhibit lysis of susceptible targets mediated by the oxidizing mitogen-activated mononuclear cells. The cytotoxic response is rapidly generated and reaches peak levels at 48 hr. The oxidizing mitogens induce expression of IL 2 receptors on peripheral blood mononuclear cells. Combined treatment of cells with IL 2 and the oxidizing mitogens results in a marked enhancement of cytotoxicity. Enhancement is achieved at levels of IL 2 that alone result in minimal generation of cytotoxic cells. Growth of a human renal cancer cell line in nude mice was inhibited when the renal cancer cells were injected together with oxidizing mitogen-activated human mononuclear cells. These studies indicate that oxidizing mitogen-activated cells provide a potentially valuable source of material for the adoptive immunotherapy of tumors. PMID- 3011904 TI - High and low affinity receptors for interleukin 2: implications of pronase, phorbol ester, and cell membrane studies upon the basis for differential ligand affinities. AB - Cellular binding sites for IL 2 exist in two forms which differ with respect to their apparent affinity for the factor. The present studies were designed to evaluate various models for the difference. Receptor-mediated internalization and covalent receptor-ligand coupling were discounted as explanations on the basis of ligand binding and elution studies on permeabilized cells and cell membranes. Phosphorylation of the receptor during activation of protein kinase C failed to modulate the ratio of high and low affinity sites, demonstrating that it also did not provide a potential mechanism. Selective destruction of low affinity receptors with pronase, on the other hand, indicated that the two forms of binding sites differed significantly in their cell surface structure. Either the two types of receptor consist of distinct molecules or the conformation of the high affinity binding sites renders them more resistant to proteolysis. Antibody inhibition studies revealed that the high affinity receptors remaining after protease treatment and their low affinity counterparts both utilized the same ligand-binding component. Thus, this result ruled out the possibility of two totally distinct receptor structures. Together, the findings support the hypothesis that other membrane components modify the conformation of the ligand binding polypeptide to confer a high affinity protease-resistant configuration. PMID- 3011905 TI - Effect of cyclosporin A and dexamethasone on interleukin 2 receptor gene expression. AB - Here we demonstrate that CsA and DEX, at concentrations that markedly inhibited PHA-induced proliferation and IL 2 mRNA accumulation, partially diminished the expression of receptors for IL 2 on PBMC. This inhibition of IL 2 receptor expression occurred at a pretranslational level and involved a reduction in both high affinity and low affinity forms of the receptor. Although both CsA and DEX inhibited IL 2 receptor expression by about 50%, only CsA blocked the PHA mediated induction of IL 2 responsivity in PBMC cultures. These data provide evidence that 1) CsA and DEX suppress the proliferation of T lymphocytes through distinct (though perhaps overlapping) mechanisms, 2) CsA (but not DEX) blocks the PHA-mediated induction of signals necessary for T cells to become capable of proliferating in response to IL 2, and 3) T cells regulate the expression of their genes for IL 2 and IL 2 receptors, at least in part, through independent mechanisms. PMID- 3011906 TI - A guanine nucleotide regulatory protein controls polyphosphoinositide metabolism, Ca2+ mobilization, and cellular responses to chemoattractants in human monocytes. AB - Previous studies demonstrated that oligopeptide chemoattractant receptors on PMN and macrophages exist in high and low affinity states which are interconvertible by guanosine di- and triphosphates. These observations suggest that guanine nucleotide regulatory (N) proteins play a role in phagocyte activation by chemotactic factors. The data presented here indicate that chemotactic factor receptors on monocytes utilize an N protein to activate phospholipase C and subsequent biologic responses by the cells. This conclusion is based on the findings that inactivation of an N protein of 41,000 m.w. by Bordetella pertussis toxin (PT) treatment abolishes monocyte responsiveness to chemoattractants but not to lectins, PMA, or the Ca2+ ionophore A23187. Treatment with PT inhibited IP3 production, Ca2+ mobilization, and cellular activation as assessed by chemotaxis and changes in forward light scattering in response to the chemoattractants by at least 80%. Therefore, a PT-sensitive N protein plays an important role in the activation of monocytes by chemoattractants. PMID- 3011907 TI - Enteral and systemic release of leukotrienes during anaphylaxis of Nippostrongylus brasiliensis-primed rats. AB - Rats with acquired immunity to the intestinal nematode Nippostrongylus brasiliensis develop anaphylaxis after i.v. challenge with an extract of worm antigen, with the small intestine being the primary shock organ. In the present study we have shown that these events were associated with significant elevations in intestinal and plasma concentrations of leukotrienes LTB4 and LTC4. The changes were observed in immune rats over 10-, 30-, and 60-min intervals after antigen challenge but were absent in control animals. These lipid mediators were identified both in the perfusate of the gut lumen, which contained large quantities of mucus, and in homogenates of intestinal tissue. In addition, significant elevations in the concentrations of plasma LTB4 and LTC4 were detected in immune challenged rats but not in controls. Leukotrienes were identified by radioimmunoassay and validated by reverse-phase high-performance liquid chromatography (RP-HPLC). RP-HPLC analysis of SRS-A leukotrienes in immune challenged rats indicated that LTC4 was the predominant sulfidopeptide leukotriene at 10 min, with almost complete biodegradation to LTD4 and LTE4 within 30 min. Infected rats also had significant increases in the numbers of intestinal mucosal mast cells (MMC) and eosinophils. Evidence of MMC activation during anaphylaxis was obtained by showing significant elevations of intestinal and systemic concentrations of their exclusive serine enzyme, rat mast cell proteinase II (RMCPII). Thus, the release of substantial amounts of leukotrienes in the gut and plasma of N. brasiliensis-primed rats after interaction with worm antigens suggests that these potent mediators may play an important role in allergic-type hypersensitivity known to occur during immune reactions against parasitic helminths. PMID- 3011908 TI - The effects of derivatives of histamine on natural suppressor cells. AB - Histamine is an impressive modulator of immune functions at least via its effects on lymphoid cells. Its in vivo effects will not be used practically as long as they produce the profound cardiovascular and pulmonary effects for which the drug is known. A series of 13 congener derivatives and conjugates of histamine was constructed and was tested to investigate whether chemical alterations would result in pharmacologic actions on leukocytes that were more potent and effect specific than histamine. The new compounds, which contained spacer groups of varying lengths between ligand and carrier and with various aromatic modifying groups, showed potencies widely different from histamine when tested in natural suppressor cells. Some compounds showed selective effects on natural suppressor cells in that they were inactive on myocardial tissue, whereas other compounds were selectively active on the myocardium. Some compounds augmented the suppressive capacity of natural suppressor cells in mixed leukocyte reactions via H1 receptors. Our scheme might be more widely extrapolated to other low m.w. immune modulators in an attempt to make them lymphocyte specific. The data also encourage the in vivo testing of selected histamine analogues as selective modulators of immunity. Some of these modulators might be experimentally useful in vivo because they may lack actions in other tissues. PMID- 3011909 TI - In vitro infection of human monocytes with human T lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV). AB - We explored the possibility that normal human monocytes can be infected with the retrovirus human T lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV). The T4 antigen, believed to be the receptor for HTLV-III/LAV binding to CD4 cells, is found on monocytes at low levels. Anti-T4A, which recognizes an epitope on the T4 molecule, inhibits viral binding to monocytes, and virus inhibits anti-T4A binding, although inhibition in both cases is not total. Virus particles were detected in HTLV-III/LAV-pulsed monocytes by electron microscopy as early as 10 min and for up to 3 days after inoculation, although budding virus was not observed. Monocytes were exposed to virus, were washed, and were cultured. Monocyte cultures were monitored by conventional assays for virus replication: immunofluorescence detection of cytoplasmic virus, supernatant reverse transcriptase activity, and supernatant virus antigen. These assays were either negative or at the lower limits of positivity. However, the amount of infectious virus was shown to increase over time in monocyte cultures by harvesting monocytes or their culture supernatants and titrating them into assay cultures containing stimulated T cells. Virus recovery from monocytes and virus recovery from T cells differed both quantitatively and qualitatively. Recovery from T cells and T cell supernatants peaked at 3 to 6 days and declined thereafter. Recovery from monocytes and monocyte supernatants increased over time in culture and never attained the levels of T cell cultures. Taken together, these studies indicate that HTLV-III/LAV binds to monocytes via the T4 molecule and enters the cells. Infectious virus is retained and increases with time in infected monocyte cultures. Both viral binding and infection are at low levels compared with levels in T cells. Unlike the usual infection of T cells characterized by high level virus replication with cell depletion, the infection appears to be persistent in monocytes. PMID- 3011910 TI - Germ-line sequence of the DH segment employed in Ars-A antibodies: implications for the generation of junctional diversity. AB - The majority of antibodies produced by A/J mice in response to p azophenylarsonate belong to the Ars-A family. These antibodies have the conserved sequence cys-ala-arg-ser-x-tyr-tyr (in which x is variable) spanning the V-D junction of the heavy chain. The cys-ala-arg residues are accounted for in the sequence of the A/J VH gene; the tyr-tyr are believed to be specified by the A/J DH segment, although this assumption is based on the DFL16.1 sequence derived from BALB/c mice. This implies that the ser-x is generated by joining imprecision and/or N segment addition. More recent data have revealed that the codon specifying the junctional serine residue is highly conserved (TCN, where N is usually G), suggesting a germline origin. Because there is no obvious way to generate this codon from the A/J Ars-A VH gene, we examined the involvement of the A/J DH segment in the generation of this junctional residue by cloning and sequencing the A/J equivalent to DFL16.1. We have established that this DH segment is polymorphic among BALB/c and A/J at the nucleic acid sequence level, and that it does not encode the junctional serine. This implies that a mechanism other than joining imprecision or random N segment addition operates at V-D junctions of Ars-A heavy chains. PMID- 3011911 TI - Molecular analysis of the rat MHC. I. Delineation of the major regions in the MHC and in the grc. AB - Southern blot analysis with liver DNA from a unique series of recombinant (R10, R11, R16, R18, R21, and R22), congenic (Y0.1U.grc+, Y0.1U.grc+/Y0.1L.grc, and Y0.1L.grc) and inbred rats has been performed to examine the restriction fragment length polymorphisms of class I genes. After digestion with Xba I or Eco RI, the genomic DNA was resolved on agarose gels, was transferred to nitrocellulose membranes, and was hybridized with murine H-2 cDNA probes. Eighteen to 25 bands of varying intensities could be clearly resolved in any given strain. Analysis of these hybridization patterns detected restriction fragment length polymorphisms that permitted the assignment of 17 specific fragments to regions within the major histocompatibility complex: RT1.A, RT1.B/D, and the RT1.E-grc-T1 alpha region. Fragments have been identified that are specific for grc, grc+, and RT1.E, and mark the junction sites between these loci. In addition, several markers identify the region around the sites of recombination in some strains. The hybridization pattern of the R18 recombinant had a unique band that specified a point of recombination within the grc. The recombinant R11 presented a unique restriction pattern unrelated to either of the parental strains or other related strains. This result suggests that R11 arose from a recombination event(s) undetected by conventional serologic methods. PMID- 3011912 TI - Mononuclear phagocyte function in SLE. I. Bipartite Fc- and complement-dependent dysfunction. AB - To determine the relative contributions of Fc- and complement-mediated immune clearance to the overall mononuclear phagocyte system (MPS) dysfunction in SLE, we performed a kinetic analysis of clearance rate data from 32 patients and 49 normal controls. Three rate constants regulating complement-mediated MPS clearance and one rate constant regulating Fc-mediated MPS clearance were evaluated. Mean values for rate constants regulating complement-mediated phagocytosis (k4) and Fc-mediated clearance (k3) were significantly lower for the patient population as a whole when compared with normal controls (p less than 0.01 and p less than 0.001, respectively). Further analysis by clinical subgroups revealed that mean k3 values were significantly low for all but the inactive nonrenal subset of patients, whereas mean k4 values were significantly low for both the active and inactive renal patients but not for nonrenal patients. Rate constant values for Fc-mediated clearance correlated significantly with disease activity scores for the entire SLE group (p less than 0.001) as well as for the subset of patients with active and inactive renal disease (p less than 0.001). Neither k3 nor k4 correlated significantly with anti-DNA antibody, titers, total hemolytic complement levels, or circulating immune complexes. These data indicate that both Fc- and complement-mediated clearance defects occur in SLE. Nonrenal patients have at least one clearance mechanism intact, whereas immune complex glomerulonephritis is associated with dysfunctions in both of these clearance mechanisms. PMID- 3011913 TI - An enzyme-linked immunosorbent assay for the detection of autoantibodies to albumin. AB - The development of an enzyme-linked immunosorbent assay (ELISA) for anti-albumin autoantibodies (AAA), using immobilized monomeric or glutaraldehyde-polymerized human, bovine or egg albumin, is described. Major problems in detection by the ELISA of AA against human albumin (HSA) were due to high 'non-specific' binding with the commercial anti-human immunoglobulin antisera used and to interference by IgM/HBs circulating complexes. However, it was found that AAA are not species specific and that these problems may be overcome using immobilized bovine (BSA) or egg (EggA) albumin. AAA were found to have a similar affinity for BSA as for HSA but slightly lower for EggA, while AAA affinities for the monomeric forms were lower than for the corresponding polymeric albumins. All sera from the 28 normal subjects tested were found to contain both IgM- and IgG-AAA. Patients with acute hepatitis B (n = 23) had significantly lower titres of IgM-AAA than normal subjects, as did chronic HBV carriers with (n = 33) or without (= 17) underlying liver disease, while IgG-AAA titres were reduced only in the acute hepatitis patients. These findings support the concept that AAA have a normal physiological function (probably for removal of effete albumin molecules) and that, in HBV infection, there is a decrement in titres that may be related to the clearance of the virus. PMID- 3011914 TI - Single cell cloning of Epstein-Barr virus transformed cells in 20-microliter hanging drops. AB - A simple method for cloning EBV transformants from single cells in 20-microliter hanging drops is described. The hanging-drop method allows direct verification prior to the addition of irradiated feeder cells that clones will be established from single cells. The average efficiency of cloning from wells containing a single transformed cell was 74%, ranging from 60 to 95% in eight separate experiments. PMID- 3011915 TI - Complement receptors (CR) and cytotoxic responses: monoclonal antibodies directed against CR1 and CR3 inhibit the generation of human allospecific and virus specific cytotoxic cells in vitro. AB - A variety of cellular immune responses involve complement factors which bind to specific receptors, and modulate or effect a specific reaction. Monoclonal antibodies (MAb) have been generated against complement receptors (CR) 1 and 3, which were utilized to investigate human allogeneic and Epstein-Barr virus specific cytotoxic cells in vitro. MAb OKM1, which binds to the C3bi CR (CR3), and MAb M710, which binds to the C3b/4b CR (CR1), inhibited the generation of both allogeneic and virus specific cytotoxic responses in vitro in a dose dependent way; doses of 1 microgram/ml (or greater) completely abrogated the cytotoxic responses. Inhibition of these responses was observed when the MAb was added to the cultures at any time point except the last two days. In addition, treatment of the responder (but not the stimulator cells) with either MAb resulted in complete inhibition of cytotoxic responses. These experiment indicate that complement receptors participate in the generation of human cytotoxic responses in vitro. PMID- 3011916 TI - DNA alterations photosensitized by tetracycline and some of its derivatives. AB - Bacteriophage M13 mp10 DNA were irradiated with near-UV light in the presence of tetracycline derivatives and primed with synthetic oligonucleotide to be used for DNA synthesis using Escherichia coli DNA polymerase. Chain terminations were observed by denaturing polyacrylamide gel electrophoresis and mapped precisely. All the synthesis stops occurred before or at the level of guanine residues, showing that the photoreaction mediated by tetracycline derivatives led to a preferential alteration of guanine residues. These lesions were demonstrated to be induced in DNA through a pathway involving singlet oxygen. Tetracycline derivatives also photoinduced the breakage of the DNA sugar-phosphate backbone monitored by the conversion of supercoiled phi X174 DNA to a relaxed form. This lesion was shown to be initiated by hydroxyl radicals. The production of this free radical has been confirmed by electron paramagnetic resonance (EPR) spin trapping experiments using 5,5-dimethyl-1-pyrroline-N-oxide as spin trap. In addition to the EPR signal due to OH radicals trapping another unassigned signal has been detected. PMID- 3011917 TI - Intranasal interferon-alpha 2b for seasonal prophylaxis of respiratory infection. AB - Efficacy of intranasal recombinant alpha interferon (IFN-alpha 2b) was evaluated over a four-week period. The first 400 participants received either 1,500,000 IU of IFN-alpha 2b or placebo twice daily. Rhinovirus infections were prevented (protective efficacy, 76%). Parainfluenza infections were not prevented, but symptoms in associated episodes of disease were significantly reduced. The medication was generally well tolerated, but side effects were often observed. The most commonly reported symptom was blood-tinged mucus. A pilot study of IFN alpha 2b or placebo administered on a once-daily dose schedule was also carried out in 150 participants. There was a suggestion of continued efficacy with reduced side effects. Overall, these findings would limit the use of IFN-alpha 2b on the twice-daily schedule to shorter time periods or to special situations in which the efficacy clearly outweighs side effects, and they encourage further examination of other dosage schedules. PMID- 3011918 TI - Acute genital infection in guinea pigs: effect of recombinant interleukin-2 on herpes simplex virus type 2. AB - Human recombinant interleukin-2 (rIL-2) modifies infection with herpes simplex virus type 2 (HSV-2) in normal guinea pigs. Animals were injected sc with rIL-2 twice a day, beginning 24 hr before infection and continuing for three subsequent days. Guinea pigs were assigned to five different regimens in which rIL-2 was administered daily at dosages from 8 X 10(3) U/kg to 8 X 10(5) U/kg. After intravaginal inoculation with HSV-2, 83% of 24 control animals developed apparent genital herpes, and 5% had asymptomatic viral shedding. Animals receiving 4 X 10(4) U or 2 X 10(5) U or rIL-2/kg showed a significantly lower rate of infection (P less than .001 by chi 2) and a lower number of lesions and more rapid healing than did the infected animals in the control (untreated) group. The mortality in the untreated group (28%) was higher than in the animals in the 2 X 10(5) U/kg treatment group (7%). No difference was observed between the control, the 8 X 10(3) U/kg, and the 8 X 10(5) U/kg treatment groups with respect to severity of infection or mortality. Animals examined two to four weeks after inoculation had lymphocyte stimulation indices (induced with HSV-2 antigen) greater than 3.5 and titers of antibody between 2.5 log10 and 4.5 log10 by radioimmunoassay. In the disease-free animals, stimulation indexes were less than 2 and antibodies were undetectable. The mean stimulation index was 54 +/- 16 in the diseased control animals and 12 +/- 7 (P less than .05) in the 2 X 10(5) U/kg treatment group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011920 TI - Detection of cytomegalovirus in urine by nonisotopic DNA hybridization. PMID- 3011919 TI - Cloned, random chromosomal sequences as probes to identify Salmonella species. AB - We have developed and evaluated a new technique--chromosomal probe fingerprinting -to differentiate between strains of Salmonella species by using sequences of cloned chromosomal DNA as probes to highlight restriction site heterogeneity. Chromosomal probe fingerprint patterns were compared with other strain-typing methods and epidemiological data. Seventeen isolates of Salmonella typhimurium recovered from 11 outbreaks had six unique chromosomal probe fingerprint patterns. Most strains of Salmonella dublin, including some that had identical plasmid profiles and restriction endonuclease analysis patterns, could be distinguished by this method. On the other hand, eight of nine isolates of Salmonella enteritidis had a common chromosomal probe fingerprint pattern, although there were differences in plasmid profiles and the isolates had been collected over a lengthy time interval from widely disparate geographic locations. These results suggest clonal dissemination of some Salmonella serovars. PMID- 3011921 TI - Use of restriction endonuclease digestion to analyze strains of human cytomegalovirus isolated concurrently from an immunocompetent heterosexual man. PMID- 3011922 TI - The risk of transmitting cytomegalovirus to patients receiving blood transfusions. PMID- 3011923 TI - HTLV-III/LAV infection in nine children infected by a single plasma donor: clinical outcome and recognition patterns of viral proteins. PMID- 3011924 TI - Herpes zoster and the classification of HTLV-III/LAV-related diseases. PMID- 3011925 TI - [Transforming activity and protein kinase activity of p60c-src]. PMID- 3011926 TI - [Lipoxygenase reaction and leukotrienes]. PMID- 3011927 TI - Dependence of proliferative activity of lymphocytes on basal levels of lymphocytic cyclic nucleotides in lepromatous leprosy patients. AB - The correlation between the lymphocyte blast transformation test and the basal levels of cyclic nucleotides (cAMP and cGMP) in lymphocytes was studied in 30 patients with lepromatous leprosy and 14 healthy persons. Cells in cultures of whole blood were stimulated with PPD and PHA. As compared to healthy persons, leprosy patients showed inversion of the role of cGMP basal levels in proliferative activity of lymphocytes in a response to PPD. The data obtained give rise to a hypothesis which might explain the low effectiveness of immune stimulators in leprosy. PMID- 3011928 TI - [Clinical study of blood cell differentiation]. PMID- 3011929 TI - [Sequential salivary scintigraphy with technetium-99m pertechnetate in healthy adults]. PMID- 3011930 TI - Presence of a plasma transthyretin (prealbumin) variant in familial amyloidotic polyneuropathy in a kindred of Greek origin. AB - Human plasma transthyretin (TTR, a protein formerly called prealbumin) is known to be associated with familial amyloidotic polyneuropathy (FAP) of autosomal inheritance. A variant TTR with a methionine-for-valine substitution at position 30 has been described as the major protein component of amyloid in Portuguese and Japanese patients with FAP and in patients of Swedish ancestry with FAP. In these patients, TTR(Met30) also circulates in relatively low concentration in the plasma. TTR variants having substitutions in other positions have also been reported in a patient of Jewish origin with FAP. We now report studies on TTR from an FAP kindred of Greek ancestry. By peptide mapping analysis, plasma TTR from the propositus was compared with TTR from a Portuguese patient with FAP. TTR(Met30) was found to circulate in the blood plasma of the Greek propositus. By use of a recently developed immunoblotting technique, this variant TTR was also detected in some of the relatives of the propositus. Future studies of this mutant gene among ethnically different FAP populations might contribute to an understanding of selection and persistence of the mutation. PMID- 3011931 TI - Effects of pH on adrenal angiotensin receptors and responses. AB - Ambient hydrogen ion concentration modulates the effects of angiotensin II (AII) on adrenal aldosterone secretion, but the mechanism of this modulation is unknown. We examined the influence of pH on AII receptors and responses in bovine adrenal glomerulosa cells. Lowering pH from 7.4 to 6.8 increased AII binding 20.5% and increased maximal All-stimulated aldosterone secretion 43%. By contrast, at pH 8.0, All binding and stimulation of aldosteronogenesis fell by 56.6% and 39%, respectively. Effects on All binding intermediate to these changes were observed at pH 7.1 and 7.7. Similar effects of altered pH were observed on All binding to a crude membrane fraction of bovine glomerulosa cells. Analysis indicated that pH primarily affected receptor number rather than affinity. In studies of proposed postreceptor mediators of All actions, pH had no effect on All stimulation of phosphatidylinositol turnover or All inhibition of calcium influx. The results show that pH affects All interaction with its receptors. The larger magnitude of the change in aldosterone compared with receptor binding suggests that a postreceptor step is also altered by pH; however, this postreceptor step is not reflected in calcium influx or phospholipid turnover. PMID- 3011932 TI - Self-limitation of the oxidative burst of rat polymorphonuclear leukocytes. AB - The oxidative response of rat polymorphonuclear leukocytes stimulated by phorbol myristate acetate and N-formyl-methionyl-leucyl-phenylalanine was studied. Ferricytochrome reduction and peroxidase-catalyzed decrease of scopoletin fluorescence were used to monitor O-2 and H2O2 release in the extracellular medium. Oxygen consumption was also measured in some experiments. Decrease of chlortetracyclin fluorescence after stimulation of dye-loaded cells was used to study an early step of cell stimulation. Finally, a possible relationship between cell responses and the medium redox potential was explored. Three major conclusions were obtained: Ferricytochrome reduction is dependent on the total cytochrome concentration, and a simple mathematical model allows a tentative estimate of total superoxide anion production by stimulated cells. Increasing cell concentration results in a decrease of individual cell response, and this may be accounted for by a direct inhibition of cell-released hydrogen peroxide on the reactivity of leukocytes. Further, H2O2 may be shown to inhibit an early step of cell response. The solution redox potential does not influence cell reactivity, since it may be dramatically decreased without inhibiting cell response. PMID- 3011933 TI - Increased leukocyte diversity and responsiveness to B-cell and T-cell mitogens in cell suspensions prepared by enzymatically dissociating murine lymph nodes. AB - The isolation of viable follicular dendritic cells (FDCs) from murine lymph nodes requires that the nodes be enzymatically dissociated with collagenase and the protease dispase. The present study was undertaken to compare leukocyte populations derived from enzymatically dissociated and mechanically disrupted lymph nodes. We examined cell viability, cell number, cell types, and the proliferative response to the mitogens LPS, PHA, and Con A. Cells were prepared by taking the inguinal, brachial, axillary, and popliteal lymph nodes from one side of the animals and mechanically disrupting them. The corresponding lymph nodes from the other side of the same animals were digested with enzymes. The enzyme-treated lymph nodes yielded substantially more cells and had a higher percentage of viable cells. Over 40% more viable cells were available for cell culture using the enzyme dissociation method. The numbers of FDCs, macrophages, plasma cells, and fibroblasts were clearly increased. The cells dispersed by enzymatic means responded better than the mechanically dispersed cells to both B cell and T-cell mitogens. This was especially striking in cultures at lower cell densities. We conclude that the method of cell dissociation has a marked effect on the types of viable cells released and available for culture, as well as on the ability of the cells to respond. We believe the cell types released by enzymatic dissociation and their response in culture more accurately reflect conditions in vivo. PMID- 3011934 TI - Chemical modification of human neutrophil membrane proteins: effect on fMet-Leu Phe binding and function. AB - [3H]fMet-Leu-Phe binding to human neutrophil membrane proteins was shown to be inhibited by pretreatment of membranes with the histidine-preferring reagent diethylpyrocarbonate in a concentration- and time-dependent fashion. The inhibition was partially reversed by hydroxylamine and was affected by pH. The pH profile for inhibition and the partial reversibility of the inhibition by hydroxylamine are consistent with a modification of the histidine residue by diethylpyrocarbonate. The addition of unlabeled fMet-Leu-Phe to the membrane preparation prior to diethylpyrocarbonate treatment provided protection from the binding inhibition following washout of unlabeled fMet-Leu-Phe and unreacted reagent. Cells treated with diethylpyrocarbonate were inhibited in their ability to produce superoxide anions in response to fMet-Leu-Phe, but the concentration of the chemotactic factor required to obtain 50% of the response was alike for treated or untreated cells. These results suggest that a histidine residue at or near the receptor binding site for fMet-Leu-Phe is required for binding and cell activation. Neither N-acetylimidazole, an agent that preferentially reacts with tyrosine, nor acetic anhydride, which reacts with lysyl groups, affected [3H] fMet-Leu-Phe binding to plasma membrane proteins or superoxide production by intact cells. Scatchard analysis of the binding inhibition owing to diethylpyrocarbonate was consistent with a loss of receptor number rather than a change in affinity. PMID- 3011935 TI - Tetrahydrocannabinol-induced suppression of macrophage spreading and phagocytic activity in vitro. AB - The effects of tetrahydrocannabinol (THC) on several parameters of macrophage function in vitro were assessed. Delta 9 THC added to cultures of normal mouse peritoneal cells in vitro affected the ability of the cells to spread on glass surfaces and also had some effect on their ability to phagocytize yeast. These effects were dose related. A concentration of 20 micrograms of THC almost completely inhibited macrophage spreading, but it also decreased viability and the total number of these cells. Doses of 10 or 5 micrograms of THC also inhibited spreading but had little effect on cell viability or number. A dose of 1.0 microgram of THC had some inhibitory effect on spreading and the lowest dose affecting spreading appeared to be about 0.05 micrograms per culture. Higher doses of THC were necessary to inhibit phagocytosis of yeast particles as determined by direct microscopic examination or use of radiolabeled yeast as the test particles. These results indicate that several readily measured functions of macrophages may be suppressed by THC. PMID- 3011936 TI - Accumulation of activated mononuclear phagocytes in the liver following lipopolysaccharide treatment of rats. AB - Lipopolysaccharide (LPS) is a toxic bacterial cell wall component that is rapidly cleared from the portal circulation by Kupffer cells. To determine if interaction with LPS causes the accumulation and activation of mononuclear phagocytes (MNP) in the liver, we compared the morphological and functional characteristics of MNP obtained from livers of rats treated with LPS (5 mg/kg, intravenously [IV]) with normal resident Kupffer cells. MNP were isolated from rat livers by combined collagenase/pronase perfusion, selective digestion, and differential centrifugation on a metrizamide gradient. MNP obtained from livers of LPS-treated rats were found to display morphologic and functional characteristics of activated macrophages. These cells were generally larger than resident cells, were highly vacuolated, and adhered to culture dishes more rapidly. Both cell types phagocytized sheep red blood cells (sRBC) in a time-dependent manner, reaching a maximum after 60-75 min incubation with sRBC. However, MNP from livers of LPS-treated rats phagocytized 10-15 times more sRBC than resident Kupffer cells from untreated animals. Employing the Boyden chamber technique, both cell types were also found to be chemotactic to a number of stimuli including the complement fragment, C5a, the tumor promoter, 12-0-tetradecanoyl-phorbol-13 acetate (TPA), and collagenous peptides related to tissue breakdown products. MNP from livers of LPS-treated rats were generally 10-15 times more responsive to the chemoattractants than resident Kupffer cells. In addition, both resident Kupffer cells and MNP from LPS-treated rats were found to release superoxide anion in response to stimulation by C5a and TPA. Taken together these results suggest that LPS treatment of rats leads to the recruitment and activation of MNP in the liver. PMID- 3011938 TI - Immunotherapy of primary liver cancer. A control study of 51 patients. PMID- 3011937 TI - Cell-surface carbohydrates in cell recognition and response. AB - Complex carbohydrates coat the surfaces of cells and have the potential to carry the information necessary for cell-cell recognition. Sugar-specific receptors (lectins) are also present on cells, and can interact with sugars on apposing cells. This may result in the adhesion of the two cells via carbohydrates and specific cell-surface receptors. Such carbohydrate-directed cell adhesion appears to be important in many intercellular activities including infection by bacteria and viruses, communication among cells of lower eukaryotes, specific binding of sperm to egg; and recirculation of lymphocytes, among others. New approaches involving synthesis of chemically defined cell-surface analogs, in conjunction with inhibition experiments, are beginning to reveal the mechanics of a potential carbohydrate "language" involved in intercellular interactions. PMID- 3011939 TI - Tamoxifen in the treatment of advanced breast cancer. Clinical experience at Siriraj Hospital. PMID- 3011940 TI - Lactogenic hormone receptors in mammary membrane preparations from prepartum and 60 and 180 day post-partum Holstein cattle. AB - Mammary tissue from nine Holstein cows was collected within 1 week of parturition and at 60 and at 180 days post partum. Blood samples were collected by puncture of the coccygeal vein or artery at 6-h intervals from 2 days before to 2 days after surgery. A membrane-enriched fraction of tissue homogenates was prepared by differential centrifugation. A microcomputer program (LIGAND) provided estimates of the dissociation constant (Kd) and receptor concentrations using Scatchard analysis of competition of radioiodinated human GH by bovine prolactin (NIH-bPRL 6), ovine prolactin (NIH-oPRL-15) and unlabelled human GH (NIH-hGH-I-1). Split plot analysis of variance of hormone data indicated that mean prolactin concentrations during the periparturient period were greater than those at 60 or 180 days post partum. However, no differences were evident in prolactin content of blood samples collected immediately before biopsy. Analysis of variance of Scatchard data indicated that the Kd of the lactogenic hormone receptor did not differ at different stages of lactation, and averaged 89.7 nmol/l. Receptor concentrations were lower during the prepartum period than at 60 and 180 days post partum (0.65 vs 1.2 and 1.5 fmol/mg membrane protein respectively). The Kd of the lactogenic hormone receptor was similar when estimated with NIH-bPRL-6 or NIH-oPRL-15 competition, but 100-fold greater when estimated with NIH-hGH-I-1. It is concluded that lactogenic hormone receptor concentrations in bovine mammary tissue increase with the onset of lactation, with a pattern similar to that observed in non-ruminants. PMID- 3011941 TI - Five new insulin-producing cell lines with differing secretory properties. AB - Five cell lines have been derived from a rat transplantable islet cell tumour using two different methods. The lines differ in morphology and contain and release different amounts of insulin and glucagon (insulin content, 1-90 pmol/10(6) cells; insulin release, 6-250 pmol/10(6) cells per 24 h; glucagon content, less than 0.005-35 pmol/10(6) cells; glucagon release, less than 0.05-10 pmol/10(6) cells per 24 h). All the lines responded to the presence of the secretagogues leucine (20 mmol/l) plus theophylline (5 mmol/l) by increasing the rate of release of insulin approximately twofold. A high extracellular concentration of potassium (40 mmol/l) caused a three- to tenfold calcium dependent increase in release of insulin and a parallel release of glucagon. Increasing the concentration of glucose from 2.8 to 16.7 mmol/l did not alter the rate of insulin release by any of the cell lines. PMID- 3011942 TI - Dexamethasone and 8-bromo-cyclic AMP depress the incorporation of [3H]thymidine into mouse condylar cartilage by different pathways. AB - We examined whether cyclic AMP (cAMP) affects the incorporation of [3H]thymidine into cartilage cells and, if so, whether this action could be related to the inhibitory effect of glucocorticoid hormones on the growth of ossifying cartilage. Incorporation of [3H]thymidine into trichloroacetic acid-precipitable material by mouse cartilage was measured concomitantly with the concentration of cAMP. Dexamethasone (1 mumol/l) significantly (P less than 0.05) depressed the incorporation of [3H]thymidine. The cAMP analogue 8-bromo-cAMP (0.01-1 mmol/l) also depressed the incorporation of the radionucleotide in a dose-dependent fashion. When various concentrations of 8-bromo-cAMP were added with dexamethasone (1 mumol/l), no apparent changes took place compared with the effect of dexamethasone alone. The phosphodiesterase inhibitor 3-isobutyl-1 methylxanthine (0.2-1 mmol/l) elicited an inhibitory effect on [3H]thymidine incorporation and a stimulatory influence on cartilage cAMP concentrations. Dexamethasone, at doses (0.01-1 mumol/l) causing significant inhibition of [3H]thymidine incorporation, failed to increase cartilage levels of cAMP. It seems, therefore, that the depressive effect of dexamethasone on [3H]thymidine incorporation in condylar cartilage is not mediated through an increase of cAMP in the tissue. PMID- 3011943 TI - The actions of N-terminal fragments of corticotrophin on steroidogenesis in dispersed rat adrenal cells in vitro. AB - The finding that the rat adrenal zona glomerulosa cell shows specific sensitivity to stimulation by alpha-MSH and related peptides has been confirmed both in vivo and in vitro, raising the possibility that alpha-MSH may have a physiological role in the control of glomerulosa function and aldosterone secretion. To define more closely the structural features which confer teh specificity of the glomerulosa response, other ACTH derived peptides have been tested for their specificity of actions on rat adrenal cells in vitro. The peptides tested were ACTH(5-24), ACTH(1-12), ACTH(1-14), ACTH(1-15), ACTH(1-16) and ACTH(1-17). Their actions were compared with those of alpha-MSH and ACTH(1-24). All of the ACTH derived peptides stimulated glomerulosa corticosterone production with sensitivities similar to that of alpha-MSH; minimum effective concentration was 10 nmol/l. Also, like alpha-MSH, the shorter ACTH peptides stimulated aldosterone production only relatively weakly in these cells from animals on normal sodium intake. Only ACTH(5-24), ACTH(1-16) and ACTH(1-17) stimulated fasciculata/reticularis cells at concentrations up to 1 mumol/1. The actions of all of the shorter peptides were thus unlike those of ACTH(1-24) which stimulates both cell types with approximately equal sensitivity, and which furthermore strongly stimulates aldosterone production. The data suggest that the 18-24 region of the ACTH molecule contains the signal for a fasciculata/reticularis response, and the region 1-13 that for glomerulosa specificity. They confirm the view that, in the rat, alpha-MSH itself may be the specific pituitary glomerulosa stimulating agent which much experimental work has predicted. They also indicate that synthetic ACTH(1-17) analogues should be used with caution. PMID- 3011944 TI - Effects of stimulation on steroid output and perfusion medium flow rate in the isolated perfused rat adrenal gland in situ. AB - Using the in-situ, isolated, perfused rat adrenal system, the actions of adrenal stimulants on steroidogenesis and perfusion medium flow rates (under constant perfusion pump conditions) have been studied. In a series of 100 experiments, initial rates of corticosterone output and flow rates were found to be positively correlated, although there was no such relationship between initial rates of aldosterone output and flow rates. Furthermore, in stable perfusion conditions, bolus injections of ACTH increased both flow rate and steroid output in a dose related manner. In individual experiments there was a clear correlation between corticosterone and flow, but the association between aldosterone secretion rate and flow was less evident. It is possible that this discrepancy arises because of temporal differences in the responses of these two steroids. Flow was also stimulated by dibutyryl cyclic AMP (dbcAMP), with correlations with steroid output similar to ACTH, but the specific zona glomerulosa stimulants angiotensin II amide and potassium ions had, if anything, inhibitory effects on flow, but only at high concentrations. The data suggest that ACTH and dbcAMP evoke specific responses in the adrenal vasculature, resulting in relatively decreased intraglandular vascular resistance. They furthermore suggest that the secretory functions of the inner adrenocortical zones are subject to the additional control of vascular elements in the intact gland. PMID- 3011945 TI - Prevalence of goitre and hypothyroidism in Southern Tanzania: effect of iodised oil on thyroid hormone deficiency. AB - In the Southern Highlands of Tanzania the prevalence of endemic goitre due to iodine deficiency is in the range of 90% and hypothyroidism in the range of 50% of schoolchildren. The present study confirms these data and documents the beneficial effect of Lipiodol injections on thyroid function in children around the age of puberty compared with untreated children from the same villages. On the other hand, a decrease in the prevalence of goitre could not be shown. A beneficial effect is shown for infants of mothers who received iodine during pregnancy. It seems that this form of supplementation is sufficient for breast fed children for more than three years, even when a second child has been delivered in the meantime. In contrast, older siblings of these babies may become hypothyroid when breast feeding is stopped. The determination of thyroid autoantibodies in iodine treated and untreated children and in young adults showed no increasing prevalence of positive findings thus excluding iodine induced chronic thyroiditis at least in the young target population. PMID- 3011946 TI - Tumor necrosis factor/cachectin interacts with endothelial cell receptors to induce release of interleukin 1. AB - Tumor necrosis factor/cachectin (TNF) has been implicated as a mediator of the host response in sepsis and neoplasia. Recent work has shown that TNF can modulate endothelial cell hemostatic properties, suggesting that endothelium is a target tissue for TNF. This led us to examine whether endothelial cells have specific binding sites for TNF and augment the biological response to TNF by elaborating the inflammatory mediator, IL-1. Incubation of 125I-recombinant human TNF with confluent, cultured human umbilical vein endothelial cells resulted in time-dependent, reversible, and saturable binding. Binding was half-maximal at a TNF concentration of 105 +/- 40 pM, and at saturation 1,500 molecules were bound per cell. Heat-treated TNF, which is biologically inactive, did not bind to endothelium. In addition to surface binding, TNF induced the elaboration of IL-1 activity by endothelial cells in a time-dependent manner. Generation of IL-1 activity required protein synthesis and was half-maximal at a TNF concentration of 50 +/- 20 pM. IL-1 activity from TNF-treated endothelium could be adsorbed by an immobilized antibody to IL-1. Heat-treated TNF was ineffective in eliciting endothelial cell IL-1. These data indicate that TNF can bind specifically to endothelium and initiate a cascade of inflammatory and coagulant events on the vessel surface potentially central to the host response to neoplasia and sepsis. PMID- 3011948 TI - Mapping of a second recombination hot spot within the I-E region of the mouse H-2 gene complex. AB - The crossover points of nine intra-I region recombinant mouse strains were determined by restriction fragment analysis. The recombinants were examined for the presence of k and p haplotype specific DNA restriction endonuclease sites. These restriction sites were a Sac I site between the E beta and E beta 2 genes, a Hpa I site within the E beta 2 gene, and a Rsa I site approximately 1 kb to the right of the E alpha gene. Seven of the recombinants were found to have crossovers between the Hpa I and the Rsa I site. This analysis suggests that a recombination hot spot exists within this segment. This segment is approximately 12-14 kb, and contains the E alpha gene and the intervening sequence between the E beta 2 and E alpha genes. PMID- 3011947 TI - Cyclooxygenase blockade elevates leukotriene E4 production during acute anaphylaxis in sheep. AB - We examined changes in the levels of eicosanoids in blood and pulmonary lymph of anesthetized sheep undergoing acute anaphylaxis. Within 1-3 min of intravenous antigenic challenge of previously sensitized sheep, there were approximately 7-30 fold elevations in mean arterial plasma levels of thromboxane B2 and 6 ketoprostaglandin F1 alpha, respectively, as measured by RIA. Negligible changes in levels of these cyclooxygenase products were found in both nonsensitized sheep and in sensitized sheep treated with indomethacin before antigenic challenge. In contrast, no changes in levels of sulfidopeptide leukotrienes (SPLT) in pulmonary lymph were detectable by RIA during anaphylaxis in sensitized or nonsensitized sheep, but levels of SPLT in indomethacin-treated sensitized sheep increased more than fivefold above levels in lymph from both other groups of animals. The immunoreactive SPLT in lymph from indomethacin-treated sheep was accounted for as LTE4, as demonstrated by mobility on HPLC and absorbance at 280 nm. These results support the possibility that certain undesirable effects of nonsteroidal antiinflammatory drugs, such as cardiopulmonary reactions in aspirin-sensitive individuals, and impaired renal and cardiac function during therapy with these drugs, may be related in part to augmented synthesis of the 5-lipoxygenase pathway products, especially those of the sulfidopeptide class. Increased LT production could also limit the antiinflammatory effectiveness of these drugs in many disease states. PMID- 3011950 TI - Simultaneous spectrophotometric calibration of wavelength and absorbance in an interlaboratory survey using holmium oxide (Ho2O3) in perchloric acid as reference, compared with p-nitrophenol and cobaltous sulphate solutions (1978 1984). AB - The wavelength accuracy of ten different types of spectrophotometer was tested with holmium perchlorate solutions. It was found to be good, with mean deviations from the literature values of maximally 0.3 nm. Standard deviations over the entire spectral range were within 0.75 nm. The absorbance accuracy for different types of instruments was generally within 5%, except in the 287 nm range where higher deviations were found. The sharpness of the holmium peaks, in combination with band width and sensitivity of the instruments, troubled the majority of the participants. 150 spectrophotometers were involved in the surveys. Linearity of the spectrophotometers was tested with p-nitrophenol and cobaltous sulphate and found to be satisfactory. PMID- 3011949 TI - Recognition of cloned vesicular stomatitis virus internal and external gene products by cytotoxic T lymphocytes. AB - It has generally been assumed that most if not all CTL specific for vesicular stomatitis virus (VSV)-infected cells recognize the viral glycoprotein (G), an integral membrane protein abundantly expressed on infected cell surfaces. Using recombinant vaccinia viruses containing copies of cloned VSV genes to examine CTL recognition of VSV, we have confirmed that G is recognized by VSV-specific CTL. More interestingly, however, we have also found that nucleocapsid protein (N), an internal virion protein, can be detected on infected cell surfaces using mAb, and serves as a major target antigen for VSV-specific CTL. In contrast to the highly serotype-specific recognition of G, N is recognized by a major population of CTL able to lyse cells infected with either the Indiana or New Jersey VSV serotypes. Using target cells expressing a cloned MHC class I gene, we could directly show that CTL recognition of N occurs in the context of the MHC Ld molecule. PMID- 3011951 TI - Attenuated cardiovascular effects of prostaglandin I2 and prostaglandin F2 alpha in cold acclimated American bullfrogs, Rana catesbeiana. AB - American bullfrogs, Rana catesbeiana respond to prostaglandins with changes in heart rate and blood pressure. These studies compare responses of warm (22 degrees C) and cold acclimated (5 degrees C) bullfrogs to prostaglandins. Gas chromatographic analysis determined equivalent fatty acid profiles in total lipids of heart and artery tissue from warm and cold acclimated animals. Arachidonic acid was the fatty acid precursor found in greatest abundance in both groups. For cardiovascular experiments, bullfrogs were cannulated by using a T cannula implanted in the right sciatic artery. In warm acclimated bullfrogs, preinfusion systemic arterial pressure (SAP) was 14.7 +/- 0.5 mm Hg, and heart rate was 33.0 +/- 1.7 beats/min. Cold acclimated bullfrogs had SAP values of 8.0 +/- 0.8 mm Hg, and heart rate was 6.9 +/- 0.3 beats/min. Arachidonic and eicosapentaenoic acid infusions (2,000 micrograms/kg body weight [bw]) were hypertensive in cold acclimated and hypotensive in warm acclimated animals. These effects were blocked by indomethacin (4 mg/kg bw). In both warm and cold acclimated bullfrogs, prostaglandin F2 alpha (3-100 micrograms/kg bw) was hypertensive, while prostaglandin I2 (0.03-3 micrograms/kg bw) was hypotensive, with both prostaglandins stimulating a greater absolute response in warm acclimated animals. In addition, both prostaglandins increased heart rate in warm but not in cold acclimated bullfrogs. The results suggest diminished cardiovascular sensitivity to prostaglandins at low environmental temperatures. PMID- 3011952 TI - CNPase activity in the vertebrate retina, retinal pigmented epithelium, and choroid. AB - The activity of the enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase, E.C.3.1.4.37) has been studied in the retina of three vertebrate species. Activity was highest in the goldfish, followed by Xenopus laevis and Rana pipiens. Also, high activity levels were found in goldfish retinal pigment epithelium and choroid, but not in the other two species. When added to in vitro culture systems, 2',3'-cyclic nucleotides were found to have no effect on goldfish cone retinomotor movement, but caused a marked inhibition of Rana pipiens rod outer segment disc membrane shedding. It is suggested that CNPase may play a role in cellular processes requiring membrane structural reorganization. PMID- 3011953 TI - Effects of the lethal yellow (Ay) and recessive yellow (e) genes on the population of epidermal melanocytes in newborn mice. AB - Very few melanocytes can be detected by the DOPA reaction in the dorsal epidermis of newborn lethal yellow mice (Ay/a). Nevertheless, the epidermis contains a considerable number of melanoblasts (cells positive for the combined DOPA premelanin reaction). On the other hand, numerous melanocytes as well as melanoblasts are found in the dorsal epidermis of black mice (a/a). The number of epidermal melanoblasts is smaller in (Ay/a than in a/a mice even though the same number of melanocytes is found in the dermis of these animals. It seems probable that the product of the A y gene suppresses either the differentiation or the proliferation of epidermal melanoblasts. The number of melanoblasts plus melanocytes in day-17 embryos from a cross between Ay/a and a/a mice shows a bimodal distribution. It seems possible that half of the embryos were Ay/a and possessed a reduced number of melanoblasts and melanocytes. This result seems to suggest that the Ay gene is active at this embryonic stage. In contrast to the case for the epidermis from Ay/a mice, numerous DOPA-positive melanocytes were detected in the epidermis from e/e mice. However, the total number of melanoblasts plus melanocytes in e/e epidermis did not differ from that in Ay/a epidermis, suggesting that the mode of action of the e gene in the epidermis is different from that of the Ay gene. PMID- 3011954 TI - Analysis of restriction fragment length polymorphisms in deoxyribonucleic acid (DNA) recovered from dried bloodstains. AB - Deoxyribonucleic acid (DNA) was recovered from dried bloodstains aged up to three years and shown to be of high molecular weight. DNA was digested with restriction endonucleases and fractionated by agarose gel electrophoresis. Following transfer to a filter, DNA was hybridized with two different radioactively labeled recombinant probes which recognize highly polymorphic regions in human DNA. The autoradiographic pattern observed was not altered by sample age, and the size of the alleles was consistent with those observed in the general population. Therefore, DNA of high molecular weight prepared from dried blood samples can be used for identification. PMID- 3011955 TI - Application of deoxyribonucleic acid (DNA) polymorphisms to the analysis of DNA recovered from sperm. AB - Sperms, collected following sexual activity of volunteers, were processed to isolate high-molecular weight deoxyribonucleic acid (DNA). These DNA samples were digested with particular restriction endonucleases and analyzed with probes that recognize polymorphic DNA regions within the human genome. The pattern of restriction fragment length polymorphisms (RFLP) detected by this test is identical to that observed with DNA prepared from blood of the male sexual partner. Therefore, RFLP analysis can be used to exclude or to determine the probable identity of an assailant in rape cases. PMID- 3011956 TI - Confirmation of Syva enzyme multiple immunoassay technique (EMIT) d.a.u. and Roche Abuscreen radioimmunoassay (RIA) (125I) urine cannabinoid immunoassays by gas chromatographic/mass spectrometric (GC/MS) and bonded-phase adsorption/thin layer chromatographic (BPA-TLC) methods. AB - Thirty human urines screened positive by the Syva enzyme multiple immunoassay technique (EMIT) d.a.u. urine cannabinoid assay were also positive for the major marijuana urinary metabolite 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) when assayed by gas chromatographic/mass spectrometric (GC/MS) and a noninstrumental qualitative bonded-phase adsorption/thin-layer chromatographic (BPA-TLC) technique. The noninstrumental BPA-TLC procedure was the simpler of the two techniques to perform and interpret. Assay of these same samples by the Roche Abuscreen radioimmunoassay (RIA) for cannabinoids (125I) revealed that reliance on the 100-ng/mL equivalent positive calibrator yielded a high incidence of false negative results (10 out of 30). The performance of these same 4 assays on 30 true negatives also was evaluated. All samples were negative for cannabinoids by EMIT and RIA, and for THC-COOH by BPA-TLC. GC/MS assay, however, detected spurious low levels of approximately 5-ng/mL THC-COOH in two instances. Because of this, a reliability level of 10 ng/mL was set for the routine quantitative confirmation of THC-COOH by the GC/MS method. PMID- 3011957 TI - Profiles of delta 9-tetrahydrocannabinol metabolites in urine of marijuana users: preliminary observations by high performance liquid chromatography radioimmunoassay. AB - Metabolic profiles of 11-nor-9-carboxylic acid-delta 9-tetrahydrocannabinol (COOH THC) and other THC metabolites were determined in an infrequent and a frequent marijuana user by high performance liquid chromatography-radioimmunoassay (HPLC RIA). In the infrequent user, no unconjugated COOH-THC was detected in urine samples for the first 8 h following smoking, whereas this metabolite was detected in the urine samples from a frequent user. A metabolite was also detected in the frequent user, which was not present in the urine sample from the infrequent user. PMID- 3011958 TI - Cloning and expression of the Erwinia chrysanthemi asparaginase gene in Escherichia coli and Erwinia carotovora. AB - A genomic library of Erwinia chrysanthemi DNA was constructed in bacteriophage lambda 1059 and recombinants expressing Er. chrysanthemi asparaginase detected using purified anti-asparaginase IgG. The gene was subcloned on a 4.7 kb EcoRI DNA restriction fragment into pUC9 to generate the recombinant plasmid pASN30. The position and orientation of the asparaginase structural gene was determined by subcloning. The enzyme was produced at high levels in Escherichia coli (5% of soluble protein) and was shown to be exported to the periplasmic space. Purified asparaginase from E. coli cells carrying pASN30 was indistinguishable from the Erwinia enzyme on the basis of specific activity [660-700 units (mg protein)-1], pI value (8.5), and subunit molecular weight (32 X 10(3]. Expression of the cloned gene was subject to glucose repression in E. coli but was not significantly repressed by glycerol. Recombinant plasmids, containing the asparaginase gene, when introduced into Erwinia carotovora, caused increased synthesis of the enzyme (2-4 fold higher than the current production strain). PMID- 3011959 TI - The characterization and cloning of a gluconate (gnt) operon of Bacillus subtilis. AB - The enzymes involved in gluconate utilization in Bacillus subtilis seemed to be gluconate permease and gluconate kinase. Several mutants unable to grow on gluconate were isolated. The mutations they harboured (gnt) were clustered between iol-6 and fdp-74 on the B. subtilis chromosome (a tentative map order of gnt-10, gnt-4, gnt-26, gnt-23 and gnt-9 was obtained). The gnt-10 mutation seemed to be located within the structural gene of the kinase, and the gnt-23 and gnt-26 mutations seemed to be within that of the permease. An EcoRI fragment (4.5 MDal) containing an intact gluconate (gnt) operon consisting of these two structural genes was cloned in phage phi 105 by prophage transformation and was mapped physically. The physical location of the mutations coincided with their order on the genetic map. The HindIII-A fragment (2.4 MDal), which corrects all the gnt mutations, was subcloned in plasmid pC194. The fragment contained the structural genes for the gluconate permease and kinase, but not the regulatory region of the gluconate operon. PMID- 3011960 TI - Characterization of the ColE9-J plasmid and analysis of its genetic organization. AB - We have determined the restriction and functional map of the ColE9-J plasmid. By sub-cloning and transposon mutagenesis we have shown that the ColE9imm gene and the ColE5imm gene present on the ColE9-J plasmid are located on separate EcoRI fragments. Using an expression vector we have demonstrated the presence of two lys genes on the ColE9-J plasmid, both of which are dependent upon the colicin E9 structural gene promoter. Promoter mapping studies imply that the colicin E9 structural gene and the ColE5imm gene are transcribed in the same direction, but that the ColE9imm gene is transcribed in the opposite orientation. PMID- 3011961 TI - Decreased particulate NADH oxidase activity in Bacillus subtilis spores after polymyxin B treatment. AB - The activities of several enzymes of polymyxin B-treated dormant and germinated spores of Bacillus subtilis were examined. The particulate NADH oxidase of the antibiotic-treated spores showed considerably lower specific and total activities compared with those of untreated ones. The specific and total NADH oxidase activities of untreated spores increased 12- and 15-fold respectively during germination, whereas increases during germination of polymyxin B-treated spores were inhibited. The specific and total activities of particulate NADH cytochrome c reductase of dormant spores were decreased by polymyxin B treatment in almost the same proportion as those of the particulate NADH oxidase. The specific activity of NADH dehydrogenase of dormant spores remained unchanged after antibiotic treatment but the total activity fell considerably. The activities of other enzymes examined were similar for untreated dormant and germinated spores and antibiotic-treated spores. The respiration of polymyxin B-treated dormant spores was inhibited at the same time as the start of germination. Morphologically, polymyxin B-treated dormant spores lost a laminar structure of the cortex and details of the spore protoplast. The inhibitory mechanism of particulate NADH oxidase activity of polymyxin B-treated dormant spores is discussed. PMID- 3011963 TI - Genetic heterogeneity among isolates of Coxiella burnetii. AB - Chromosomal and plasmid DNA have been extracted from six isolates of Coxiella burnetii, the aetiological agent of Q fever. Restriction fragment length polymorphisms detected after HaeIII digestions of chromosomal DNA revealed four different patterns that distinguished the American from the European isolates, and the Nine Mile phase I prototype strain from a spontaneously derived, isogenic phase II nonrevertant variant. At least one of the HaeIII fragments visible in the pattern from Nine Mile phase I and not in that from Nine Mile phase II could not be detected by DNA-DNA hybridization, and thus may have been deleted during the phase transition. Comparison of Nine Mile phase II, which does not survive animal passage, with Grita M44 phase II, which does, indicated that the HaeIII fragment was present in the Grita strain. These results suggest that this HaeIII fragment may be concerned with functions necessary to survive the cellular immune response in vivo. Isolates from two human endocarditis cases showed the greatest divergence from all the other isolates, having at least five fragments of unique mobility in the HaeIII digestion pattern of their chromosomal DNA. Also, a plasmid obtained from these two isolates was 2 to 3 kb larger than the plasmid present in the other five isolates, and its restriction pattern could be distinguished from that of the other plasmids by several endonucleases. Detection of chromosomal and plasmid restriction fragment length polymorphisms among strains of phase I or phase II C. burnetii from various geographical locations and environmental sources will facilitate Q fever diagnosis and strain identification. PMID- 3011962 TI - spoIID operon of Bacillus subtilis: cloning and sequence. AB - The locus spoIID, involved in the sporulation of Bacillus subtilis, was cloned into derivatives of the temperate phage phi 105. Two recombinant phages were obtained which contain chromosomal DNA covering 1.6 kbp. They are both able to complement mutations spo-68 and spo-298. These mutations, which were believed to be in different loci, spoIID and spoIIC respectively, were shown to be closely linked, and both map at the position assigned to spoIID on the genetic map of B. subtilis. The sequence of 1656 bp carrying the spoIID locus was determined. Only one open reading frame was found; this codes for a protein of 343 amino acids. It is preceded by a ribosome binding site and possible recognition sequences for sigma 32- and sigma 29-RNA polymerases. Studies of the locus by means of integrational plasmid vectors defined the outer limits of the transcriptional unit. These results are completely compatible with the sequence data. The combination of sequence and mapping and the information obtained by the use of integrational plasmids confirm that the spoIID locus functions as a monocistronic operon. PMID- 3011965 TI - Isolation and restriction endonuclease analysis of mycobacterial DNA. AB - A method for the isolation of DNA from mycobacteria propagated in vitro is described that utilizes organic solvents to extract lipoidal components from the outer membrane, and digestion with a protease (nagarse) and lysozyme to penetrate the cell wall. The mycobacterial cells were lysed by the addition of detergent and the DNA was purified by digestion with pronase, sequential phenol and chloroform extractions, and digestion with RNAase A. The isolated DNA, which was obtained in good yields, was of a relatively high Mr and could be readily digested by restriction endonucleases. By this method, the genomes of Mycobacterium avium, M. intracellulare, M. lepraemurium, 'M. lufu', M. marinum, M. phlei, M. scrofulaceum, M. smegmatis and M. tuberculosis were isolated and the restriction endonuclease digestion patterns analysed. Each species could be distinguished by the digestion patterns, indicating that this approach can be used for identifying mycobacterial species. This approach is also sufficiently sensitive to differentiate strains since we were able to distinguish two independently isolated strains of M. tuberculosis, H37 and H4. In addition, no evidence was obtained for the presence of methylcytosine residues in the sequences 5'.CCGG.3',5'.CCCGGG.3',5'.CC(A/T) GG.3' or for methyladenine at 5'.GATC.3' in the DNA of the nine mycobacterial species examined using pairs of restriction enzymes that recognize and cleave at the same nucleotide sequence but differ in their sensitivity to 5-methylcytosine or 6N-methyladenine. PMID- 3011964 TI - Transcriptional regulation of the mercury-resistance genes of transposon Tn501. AB - Expression of the mercury-resistance (mer) genes of the transposon Tn501 is positively and negatively controlled by the product of the merR gene. DNA sequence analysis has identified three open reading frames as potential candidates for this gene, one of which is oriented divergently with respect to the mercury-resistance genes. We have demonstrated that although RNA polymerase will bind to fragments containing the potential control regions for all three reading frames, only the control region for this divergent reading frame shows detectable promoter activity in vivo. Transcription of this reading frame is required for repression and induction of mer transcription. We have also shown that the Tn501 merR gene product negatively regulates its own synthesis, and have identified the start point of the transcript for this reading frame and for the mercury-inducible transcript of the mercury-resistance genes. PMID- 3011966 TI - Adenylate kinase activity in Mycobacterium leprae. AB - Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) was detected in partially purified preparations of cell-free extracts of Mycobacterium leprae. The apparent Km values of M. leprae adenylate kinase for ADP and Mg2+ were 1 X 10(-4) M, respectively. The enzyme was heat-labile: loss of activity by 80% at 45 degrees C and over 90% at 60 degrees C occurred within 5 min. M. leprae adenylate kinase was distinct from armadillo adenylate kinase in respect of affinity for substrate and heat-sensitivity. PMID- 3011967 TI - Suppression and enhancement of humoral antibody formation by herpes simplex virus types 1 and 2. AB - Intraperitoneal infection of mice and rats by herpes simplex virus type 2 (HSV-2) but not type 1 (HSV-1) resulted in suppression of antibody formation on subsequent challenge with HSV-1 or HSV-2. Application of silica considerably enhanced antibody formation after primary HSV-1 infection, but only slightly after primary HSV-2 infection. Suppression induced by HSV-2 was, however, reduced significantly by injection of silica 21 days later, on the day of the second injection of HSV-2. Suppression could be detected soon after infection by HSV-2. The degree of this suppression depended on the dose of the injected virus and was abolished by u.v. irradiation of the virus prior to inoculation. Likewise the weak antibody response induced by HSV-2 was abolished for both neutralizing and ELISA antibodies. Infections with HSV-1 evoked considerable numbers of HSV specific antibody-producing B cells, when assessed by an enzyme-linked immunospot assay. The B cell response to HSV-2, however, was very weak. Silica considerably enhanced the number of specific antibody-producing B cells only during primary HSV-1 infections. The present results in combination with earlier data demonstrate the central role of macrophages, which seem to be the primary target affected by silica, for enhancement and suppression of HSV-induced antibody generation. PMID- 3011968 TI - X-linkage of the early in vitro alpha/beta interferon response of mouse peritoneal macrophages to herpes simplex virus type 2. AB - The genetics of the early interferon response of mouse peritoneal cells to infection with herpes simplex virus type 2 (HSV-2) was studied in susceptible BALB/c and more resistant C57BL/6 mice and in reciprocal crosses between these mice. Wash-outs of the peritoneal cavity of normal C57BL/6 mice contained significantly more cells than wash-outs from BALB/c mice. Therefore, interferon induction with HSV-2 was studied under standardized conditions in vitro. Peritoneal cells reacted to HSV-2 infection by interferon production in a virus dose-dependent manner. Interferon was detected first after 2 h and peaked after 24 h. Cells from C57BL/6 mice of each sex produced significantly more early interferon than cells from BALB/c mice, and cells from female BALB/c mice produced more interferon than cells from males. This difference was not seen with C57BL/6 mice. Cultures of highly purified adherent cells yielded approximately 10 times as much interferon as cultures of non-adherent cells. Since treatment of cells with carbonyl iron and silica significantly reduced the amount of interferon produced, whereas 2000 rad of irradiation had no obvious effect, it is concluded that the main interferon-producing cell in the peritoneal cavity of mice in response to HSV-2 is of the monocyte/macrophage lineage. Interferon production in peritoneal cells was found to be quantitatively influenced by X linked loci in that cells from male (BALB/c female X C57 male) F1 mice, which inherit the X chromosome from the low-responding BALB/c females, produced significantly lower amounts of interferon than cells from the other three F1 generation genotypes. All interferons were characterized as alpha/beta interferon. It is suggested that the early production of alpha/beta interferon in response to HSV-2 is influenced by X-linked loci, which might be involved in sex linked differences in resistance to human herpesviruses. PMID- 3011969 TI - Conversion of a fraction of the unique sequence to part of the inverted repeats in the S component of the herpes simplex virus type 1 genome. AB - A novel genome variant of herpes simplex virus type 1 (HSV-1) was isolated, in the S component of which a fraction of the unique sequence (map units 0.865 to 0.880) was converted to part of the diploid inverted repeats and another fraction of the unique sequence (map units 0.937 to 0.955) had been deleted. The S component of the variant consisted of a shortened unique sequence (map units 0.880 to 0.937) and a pair of elongated inverted repeats (map units 0.820 to 0.880, and 0.937 to 1.000). The conversion occurred as a result of a recombination event between two points (map units 0.880 and 0.937) having a 5 base pair stretch (5'-CCCCG-3') of homology, in an inverted direction in the unique sequence. An involvement of the mechanism causing L-S inversion was inferred to account for the generation of the variant, as there are multiple copies of the 5'-CCCCG-3' stretch in the 'a' sequences. The occurrence of the variant indicates that the products of HSV-1 genes US9, US10, US11 and US12 are unnecessary for an HSV-1 productive infection in tissue culture cells, and also suggests the presence of a mechanism whereby expansion of the inverted repeats could occur. PMID- 3011970 TI - Characteristics of the induction of a new protein kinase in cells infected with herpesviruses. AB - The appearance of a recently described protein kinase activity (virus-induced protein kinase, ViPK) has been studied during infection of hamster fibroblasts with pseudorabies virus or with herpes simplex virus type 1 (HSV-1). An enzyme activity with comparable catalytic properties was induced in both cases, and had broadly similar kinetics of appearance to that of the viral DNA polymerase. The amount of active ViPK detected depended on the multiplicity of infection, and no ViPK was induced after the viruses had been subjected to irradiation with u.v. light. When cells were infected with the tsK mutant of HSV-1, ViPK was induced at the permissive but not at the restrictive temperature. The ViPK preparations obtained from cells infected with each virus differed in chromatographic properties on anion-exchange and gel-permeation resins. These results indicate that expression of the viral genome is required for induction of ViPK. They suggest that the enzyme may be encoded by the viral genome, but do not provide proof of this. PMID- 3011971 TI - Location and nucleotide sequence of the Orgyia pseudotsugata single nucleocapsid nuclear polyhedrosis virus polyhedrin gene. AB - A restriction endonuclease map was determined for the Orgyia pseudotsugata single nucleocapsid nuclear polyhedrosis virus (SNPV) genome. The order of the fragments generated by the enzymes BglII, BamHI and XbaI was analysed using double digestion of the total genome and digestion of isolated restriction fragments. The location of the polyhedrin gene was then determined using a cloned polyhedrin gene from the O. pseudotsugata multiple nucleocapsid NPV (MNPV) as a hybridization probe. A fragment containing this gene was cloned, mapped, subcloned and the nucleotide sequence of a 1.3 kb fragment was determined which contained the entire polyhedrin reading frame and some flanking sequences. This gene demonstrated 76% nucleotide sequence homology and 87% amino acid sequence homology to the Autographa californica MNPV polyhedrin sequence. A probable regulatory element was identified which is common to the 5' flanking region of all hypertranscribed late genes (polyhedrin and 10K proteins) which have been examined in baculoviruses. PMID- 3011973 TI - The herpes simplex virus type 2 alkaline DNase activity is essential for replication and growth. AB - A mutant of herpes simplex virus type 2 (HSV-2), which is temperature-sensitive (ts) for the induction of an alkaline DNase activity, was examined at a number of different temperatures. Induction of DNase activity by this mutant resembled that of wild-type (wt) virus at 31 degrees C but was greatly reduced at 38.5 degrees C and barely detectable at 39.2 degrees C. Virus DNA synthesis showed similar patterns, exhibiting wt levels at 31 degrees C, reduced levels at 38.5 degrees C and very little incorporation at 39.2 degrees C. Similarly, virus growth in cells infected with this mutant was equal to that of wt at 31 degrees C, slightly reduced at 38.5 degrees C but considerably reduced at 39.2 degrees C. Marker rescue of the ts DNase lesion restored wt levels of virus DNase activity, of virus DNA synthesis and of virus growth, thus providing direct evidence that HSV DNase activity is essential for virus replication. PMID- 3011972 TI - DNA inversion in bacteriophage Mu: characterization of the inversion site. AB - Gin-mediated site-specific recombination promotes inversion of the G segment of phage Mu. The crossover takes place between two 34 bp-long inverted repeat sequences flanking the G segment. We have characterized the inversion site, the target for the site-specific recombination mechanism. An artificial invertible segment was constructed which consists of parts of the invertible segments of Mu and phage P1, which in this respect are largely homologous. Upon inversion of this hybrid segment the crossover site could be located, by DNA sequencing, in the ACCT sequence of the centre of symmetry in the inverted repeat in Mu. The hybrid Mu-P1 segment inverts at a lower frequency than its parental invertible segments probably because of the mismatches between the inverted repeats of Mu and P1. This suggests that base pairing between the inverted repeats is an intermediate step in recombination. Plasmids with subcloned G segments lacking the adjacent beta region of Mu or the corresponding region in P7, a relative of P1, are deficient in inversion. By analysis through site-specific mutagenesis of Mu DNA, an enhancer element with multiple recognition sites was identified which is necessary for efficient inversion. This component of the inversion site was located in a 170 bp segment within the Mu beta region, 30 bp to the right of the inverted repeat sequence, but can be separated from the crossover site by a 1200 bp insertion without losing its effect. PMID- 3011974 TI - Differentiation of serum antibodies from pigs vaccinated or infected with Aujeszky's disease virus by a competitive enzyme immunoassay. AB - A competitive enzyme immunoassay was developed to detect antibodies to a glycoprotein (gI) of Aujeszky's disease virus. Infected cell monolayers were used as antigen and a monoclonal antibody directed against an epitope of gI as indicator antibody. It was demonstrated that pigs vaccinated with the Bartha, BUK or NIA-4 strains did not produce antibody to the epitope of gI, whereas all wild type viruses tested did induce this antibody. The antibody to the gI epitope persisted for at least 15 weeks. The present test, which enables us to distinguish pigs vaccinated with certain attenuated strains from pigs infected with wild-type Aujeszky's disease virus, may be of great value in future combined vaccination-eradication programmes for Aujeszky's disease. PMID- 3011975 TI - Analysis of structural properties which possibly are characteristic for the 3' terminal sequence of the genome RNA of flaviviruses. AB - Recently we have shown that an open reading frame comprising 10290 nucleotides is present on the infectious, single-stranded genome RNA of the West Nile flavivirus. We have now isolated cloned cDNA representing the 3'-terminal untranslated region of this molecule. The sequence of this region which comprises 571 nucleotides is given in this report. Recently, the nucleotide sequence of the genome RNA of the yellow fever flavivirus has been described. A comparative analysis of the 3'-terminal untranslated nucleotide sequences present in each genome suggests that in flaviviruses this region probably has the following properties. It has a heteropolymeric sequence at the 3' terminus. It contains one or more oligonucleotide sequences that are repeated. An extensive stem and loop structure can be folded from the nucleotide sequences present at the 3' terminus. The stem of this structure contains a conserved region introducing a defined mismatch into the stem. The loop of this structure probably contains short conserved oligonucleotide sequences in analogous positions. In both viruses the oligonucleotide CAUAUUGAC (AG)CC(UA) GGGA(UA) AGAC lies closely in front of the sequence which can be folded into the stem of the 3'-terminal stem and loop structure and the oligonucleotide CUAGAGGUUAGAGGAGACCC is strictly conserved between both viruses. The analyses indicate that the 3'-terminal untranslated region of the genome of flaviviruses probably has rather unique characteristics of primary and secondary structure. Possible implications of these findings are discussed. PMID- 3011976 TI - Persistence of virulent Semliki Forest virus in mouse brain following co inoculation with defective interfering particles. AB - Semliki Forest virus (SFV) normally causes an acute lethal encephalitis in mice following intranasal inoculation. However, animals co-administered with 10 LD50 SFV and defective interfering (DI) SFV survive the infection without clinical signs of disease. In this report we demonstrate the isolation of infectious virus from the brains of 12/169 protected mice up to 6.5 months post-infection. Although, with one exception, mice were clinically normal, five of 12 of the SFV isolates were identical to the original virus as judged by plaque morphology, maximum temperature for growth, virulence in mice and pathology. Others were less virulent (although not any were plaque or temperature-sensitive mutants) and on re-inoculation into fresh mice caused a demyelinating pathology which was not an attribute of the original inoculum. How the virulent virus can persist in brain, sometimes in amounts in excess of 100 LD50, without causing disease remains to be determined. PMID- 3011977 TI - Very rapid generation/amplification of defective interfering particles by vesicular stomatitis virus variants isolated from persistent infection. AB - Multiply cloned variants of vesicular stomatitis virus (VSV) were found to generate/amplify defective interfering (DI) particles at a rate greatly exceeding the rates normally observed for wild-type VSV (or for other mutants of VSV). A single undiluted passage of the first clonal pool of this variant virus produced concentrated visible bands of DI particles on sucrose gradients whereas wild-type and other mutant strains of VSV required from three to six or more serial undiluted passages. Since DI particle amplification by wild-type VSV at each undiluted passage can exceed 10,000-fold enrichment, these variant virus clones were generating/amplifying DI particles many millions of times more rapidly than were wild-type and other mutant strains of VSV. This rate of generation/amplification is so high that it was not feasible to obtain accurate estimates of the rates of generation (or amplification) of these DI particles. PMID- 3011978 TI - Is rodent virus contamination of monoclonal antibody preparations for use in human therapy a hazard? PMID- 3011979 TI - Involvement of infants, children, and adults in a rotavirus gastroenteritis outbreak in a kibbutz in southern Israel. AB - An outbreak of acute gastroenteritis in a kibbutz in southern Israel, characterized by diarrhea, fever, vomiting, and abdominal pain, involved 32 kibbutz members of all ages. Nineteen percent of the children and 3.5% of the adults were ill. Transmission of the illness occurred in direct proportion to the degree of close contact, involving first infants, then mothers and nursery staff, and only later youngsters, adolescents, and fathers. Stool samples obtained from 32 kibbutz members with clinical illness and from 44 asymptomatic close contacts were examined for the presence of rotavirus antigen. Fifty-six percent of symptomatic members were positive for rotavirus antigen as compared with 4.5% of asymptomatic close contacts. Positivity of stool samples correlated inversely with the number of days elapsed after onset of illness until the sample was obtained. Serologic studies carried out on acute and convalescent sera of symptomatic and asymptomatic subjects further supported a rotavirus etiology for the outbreak. RNA profiles of stool sample extracts obtained by polyacrylamide gel electrophoresis and silver staining indicate that one electropherotype may have been responsible for the outbreak. PMID- 3011980 TI - Prevalence of delta infection in the western Pacific region. AB - The prevalence of coinfection and superinfection with the delta agent was studied in 2,645 hepatitis B surface antigen (HBsAg)-positive subjects from six countries and nine islands in the Western Pacific region. The study group comprised 262 patients with acute hepatitis B and 2,383 chronic carriers of HBsAg, of whom 278 were suffering from chronic liver disease or primary hepatocellular carcinoma. While major foci of infection were observed in Nauru, Niue, and Western Samoa, delta infection appears to be uncommon in other parts of the region. PMID- 3011981 TI - Epstein-Barr virus serology in bone marrow transplantations: a one-year retrospective study with detection of EBV IgM-VCA-specific antibodies. AB - The specific antibody response to Epstein-Barr virus (EBV) antigens of 41 bone marrow transplant recipients with leukemia or aplastic anemia was examined retrospectively by immunofluorescence test (IF) over 1 year. We observed high titers (greater than 640) of IgG-viral capsid antigen (VCA) with emergence of IgG early antigen (EA) and frequent absence or low levels of Epstein-Barr nuclear antigen (EBNA) antibodies. After absorption to remove rheumatoid factor (RF), five of the 41 recipients had IgM-VCA antibody to EBV, which appeared between weeks 26 and 48 after BMT and persisted for 1-4 months. No heterophil antibodies were detected in these sera, and none of the five recipients had a history of infectious mononucleosis. PMID- 3011982 TI - Establishment and characterization of a chronic infectious mononucleosislike syndrome in common marmosets. AB - Epstein-Barr virus (EBV) was inoculated into two species of marmosets. Successful infection was established in the majority of the animals of one species, Callithrix jacchus, as evidenced by the development of high, persistent levels of antibody against virus-specific capsid and early nonstructural proteins. Antibodies also were produced against the major membrane antigen and, in some animals, against EBV nuclear antigen (EBNA) 2 but not against EBNA 1. This is the antibody profile normally noted in individuals with chronic infectious mononucleosis (IM). EBV-induced lymphoproliferation was not seen, and EBV specific proteins were not detected in the peripheral blood lymphocytes of infected animals. Hence, EBV infection in C. jacchus apparently does not generally include extensive B-cell involvement. However, the marmosets clearly are useful as a model for EBV primary infection and also possibly for chronic IM. PMID- 3011983 TI - The interaction of 1-alkyl-4,4-diphenylpiperidines with the 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine receptor binding site. AB - 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a selective neurotoxin which produces degeneration of the nigrostriatal bundles in the central nervous system of man and animals. In these areas of the brain are concentrated the receptor binding sites for [3H]MPTP. 1-Alkyl-4, 4-diphenylpiperidines displace [3H]MPTP from these binding sites with Ki values in the micromolar range. The t butyl analogue in this class of substances, budipine, is a novel therapeutic agent for Parkinsonism whose mechanism has not yet been fully clarified. The affinity of budipine for the MPTP receptor binding site was determined as a Ki value of 2.2 microM. Other 4, 4-diphenylpiperidine derivatives such as 1-methyl 4, 4-diphenylpiperidine and 1-i-propyl-4, 4-diphenylpiperidine have substantially lower affinities. Monoamine oxidase inhibitors such as deprenyl, pargyline and harmaline have affinities to the MPTP receptors which parallel their affinity for the B type of monoamine oxidase (MAO B). This supports the theory that the MPTP receptor binding sites is identical with membrane bound MAO B. PMID- 3011984 TI - The inhibition of the cage-leaving response--a model for studies of the serotonergic neurotransmission in the rat. AB - It was observed that rats that had been given drugs that enhance serotonergic neurotransmission, e.g. the serotonin releasing compounds p-chloroamphetamine (PCA) and fenfluramine, the MAO-A inhibitors and serotonin releasing agents amiflamine and alpha-ethyltryptamine and the serotonin agonists 5-methoxy-N, N dimethyltryptamine (5-MeODMT), 8-hydroxy-2-(di-n-propylamino) tetraline (8-OH DPAT), m-chlorophenyl piperazine (m-CPP) and 5-methoxy-3 (1,2,3,6 tetrahydropyridin-4-yl)1H-indole (RU 24969), did not leave their home-cages when the grid-covers were removed in contrast to normal rats who almost immediately left the cages. The association between the serotonin neurotransmission and the inhibitory effect of PCA on the cage-leaving response was indicated by the findings that 1. Serotonin uptake inhibitors (alaproclate and citalopram) antagonized the effect of PCA. 2. High, neurotoxic doses of PCA antagonized the effect of PCA when tested one week after the former administration. The serotonin uptake inhibitor zimeldine counteracted the effect of neurotoxic PCA. 3. Depletion of brain serotonin with p-chlorophenylalanine counteracted the effect of acute PCA. 4. Repeated treatment of rats for 7 days with zimeldine, amiflamine, alpha-ethyltryptamine or clorgyline plus a low dose of PCA counteracted the effect of acute PCA probably due to a functional down-regulation at postsynaptic receptors. Clorgyline or a low dose of PCA by themselves had no effect. 5. Compounds interacting with dopamine or noradrenaline mechanisms, e.g. alpha-methyltyrosine, N-2-chloroethyl-N-ethyl-2-bromobenzylamine (DSP 4), pimozide, remoxipride and prazosin did not antagonize the effect of PCA nor did (+)-amphetamine inhibit the cage-leaving response. None of the serotonin receptor antagonists (cinanserin, ketanserin, metergoline, methysergide, metitepine, mianserin, pirenperone) blocked the inhibition of the cage-leaving response produced by PCA, indicating that the receptors involved may not be of the S1- and S2- types. Observation of the cage-leaving response may be a valuable technique in studies of drugs that enhance the serotonin neurotransmission in the rat brain. PMID- 3011985 TI - Studies on rat brain catecholamine synthesis and beta-adrenoceptor number following administration of electroconvulsive shock, desipramine and clenbuterol. AB - The effects of administration to rats of repeated electroconvulsive shock (ECS), clenbuterol and desipramine (DMI) on beta-adrenoceptor number in cortex, and noradrenaline (NA) and dopamine (DA) turnover in whole brain has been investigated by examining the rate of decline of NA concentration (kNA) following injection of alpha-methyl-p-tyrosine. A single injection of clenbuterol (5 mg/kg) raised brain NA content and decreased the rate constant (kNA), leaving the turnover rate unaltered. Acute DMI injection decreased kNA and turnover rate, while a single ECS did not change NA metabolic rate. Repeated treatment with either ECS (5 seizures over 10 days), clenbuterol (5 mg/kg for 14 days) or DMI (5 mg/kg twice daily for 14 days) decreased beta-adrenoceptor density in cortex. No change in NA content, rate constant or turnover rate was observed after repeated ECS or clenbuterol administration. Ninety min after the last dose of DMI brain NA content was significantly decreased but kNA was unchanged compared with control animals, possibly because of the presence of subsensitive presynaptic alpha 2 adrenoceptors. At 18 hours after the last dose brain NA content was still lower than control animals but kNA was enhanced. This presumably a "withdrawal" effect, the uptake inhibitory effect of the drug now being decreased. The treatments had little effect on DA turnover apart from DMI decreasing synthesis rate. Clearly there is no obvious relationship between the ability of antidepressant treatments to alter NA turnover and decrease beta-adrenoceptor number. PMID- 3011987 TI - Imidazolate bridged heterobinuclear complexes of zinc(II)tetraphenylporphyrin. Modeling cytochrome c oxidase. AB - The reaction of a copper(II) or nickel(II) imidazolate complex (M[CBP-PHEN-4-CHO Im]) with zinc(II)tetraphenylporphyrin (TPP) in toluene results in the formation of an imidazolate bridged heterobinuclear axial adduct. Conversion of the four coordinated Zn(TPP) to the five-coordinated species is followed in the visible region between 700 and 500 nm. Isosbestic behavior is exhibited at 523, 556, 588, and 638 nm by solutions of Zn(TPP) to which varying amounts of the metal imidazolate complex are added, indicating the existence of an equilibrium between Zn(TPP) and its axial adduct. The products exhibit maxima beta and alpha bands at 566 and 606 nm, respectively, which are red-shifted from 548 and 588 nm for Zn(TPP) and yield epsilon alpha/epsilon beta ratios of 0.57 and 0.55 for the Ni(II) and Cu(II) adducts, respectively. The binding of the metal imidazolate complexes is thought to closely resemble that of N-methylimidazole, N-CH3Im, rather than imidazolate, owing to the close spectral similarities with the adduct of the former and significant differences from the latter. Formation constants were determined using the 548-nm beta band of Zn(TPP) in the 293-308 K range by the method of Rose and Drago. At 25 degrees C, K = 152,000 M-1 and 110,000 M-1 for the copper and nickel adducts, respectively. Comparison of these values to that of 54,100 M-1 for N-CH3Im indicates that the metal-imidazolate complexes are considerably more reactive. Van't Hoff plots for the two series are very similar with enthalpies of -41.9 and -43.3 kJ/mole respectively. The structural core of these complexes is similar to the imidazolate bridged model of cytochrome c oxidase in that they contain a metal imidazolate axially adducted to a metalloporphyrin. PMID- 3011986 TI - An electron paramagnetic resonance study of bovine alpha-lactalbumin-metal ion complexes. AB - alpha-lactalbumin has at least three distinct cation binding regions: a Ca(II) Gd(III) site, a Cu(II)-Zn(II) site and a VO2+ site as observed from electron paramagnetic resonance (EPR) studies of complexes with the bovine protein. Gadolinium, which bound to the calcium site of the protein with a subnanomolar dissociation constant, yielded EPR spectra at 9.5 GHz (X-band) that exhibited features from g = 8 to g = 2. At 35 GHz (Q-band) the central fine structure transition (Ms = 1/2----Ms = -1/2) gave a well-defined powder pattern. The zero field splitting was large, as reflected in the second-order splitting of the central fine structure transition of about 1 kG. There was also evidence for additional, low affinity binding site(s) for Gd(III). Addition of either Zn(II) or Al(III) did not affect the amplitudes or positions of the bound Gd(III) EPR spectrum. The Cu(II)-alpha-lactalbumin complex gave a typical axially symmetric spectrum (g parallel = 2.260, g perpendicular = 2.056, A parallel = 171 G) with a partially resolved superhyperfine interaction attributable to at least one directly coordinated nitrogen ligand. Addition of Cu(II) to Gd(III)-alpha lactalbumin gave an EPR spectrum that was a superposition of signals from the individual Gd(III)- and Cu(II)-alpha-LA spectra. The absence of any magnetic interactions in the Gd(III)-Cu(II)-alpha-lactalbumin species indicated that the two cation sites were more than 10 A apart. On the other hand, addition of Zn(II) to Cu(II)-alpha-lactalbumin gave a set of EPR lines due to free or loosely bound Cu(II), confirming that the Cu(II) was displaced by zinc.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3011989 TI - Catalytic activity of a copper(II)-oxidized glutathione complex on aqueous superoxide ion dismutation. AB - The study of the catalytic activity of a Cu(II)-oxidized glutathione system upon the disproportionation of superoxide radicals shows that the mononuclear complex MA catalyzes dismutation in the pH range 7-9. The corresponding first-order rate constant of value kcat congruent to 6 X 10(6) M-1 sec-1 is pH independent, whereas the second-order rate constant ks for the reference solutions is pH dependent. The kcat constant is about 10-, 100-, and 300-fold higher than the ks constant at pH 7, 8, and 9, respectively. The measured effect is explained in terms of free axial sites in the square-planar arrangement around the copper ion. PMID- 3011988 TI - PDN-1: a spin-labeled analog of cis-diamminedichloroplatinum(II). Biophysical studies in aqueous, lipophilic, and biological media. AB - Biophysical and biochemical studies on a biradical-labeled analog of Cisplatin, PDN-1, in aqueous, biological, and lipophilic media are presented. The ESR spectra from PDN-1 in aqueous meida show five-line spectra typical of biradicals in which intramolecular spin exchange occurs. The spectrum of PDN-1 in 1-octanol is characterized by three broad lines overlying a fourth line that is approximately three times as wide as the others. Although the characteristics of the aqueous spectra differ substantially from those of the octanol spectra, both the midfield peak height and the integral of the absorption spectrum can be used as a linear measure of PDN-1 concentration in either media. The smallest concentration used in these linear calibrations was equivalent to the detection of 60 femptomoles of PDN-1 at a signal-to-noise ratio of 30. PDN-1 is shown to "decay" in tissue culture medium which involves either the reduction of a nitroxyl moiety, the labilization of an amino bond, or a combination of both. Spectra of intracellular PDN-1 show that both a mobile ("free") fraction and slowly tumbling ("bound") fraction of the compound can be detected within CHO cells. The spin-labeled cisplatin method is at least as sensitive as conventional chromatographic and spectroscopic methods, yet has the added advantage of offering information about the molecular environment of the complex. PMID- 3011990 TI - Reactions of triethylphosphine gold(I) complexes with heme proteins: novel spin state changes in cytochrome b562, myoglobin, and hemoglobin. AB - Reactions of bacterial Fe(III) cyt b562, HbO2, met Hb and met Mb with Et3PAuCl and Et3PAuNO3 (and some related complexes) have been investigated by electronic absorption and EPR and NMR spectroscopy. Except for met Hb, which denatured, the products were novel high-spin Fe(III) heme proteins. The reactions of cyt b562 and Mb were reversible. Two distinct kinetic steps were observed in the autoxidation of HbO2 and MbO2. These may involve the liberation of superoxide. Autoxidation of HbO2 occurred more rapidly than that of MbO2. The kinetics of the spin-state change of cyt b562 were too fast to measure by conventional (spectrophotometric) methods. The reaction of Et3PAuCl with HbO2 was not blocked by N-ethylmaleimide. The reactions are discussed in terms of attack by Et3PAu+ on histidine residues in the hydrophobic haem pockets of the proteins. PMID- 3011992 TI - Biochemical changes in central nervous system membranes in experimental allergic encephalomyelitis. AB - Biochemical and morphological studies of myelin subfractions were undertaken on Lewis rats during the early stage of the development of experimental allergic encephalomyelitis (EAE). Myelin subfractions, obtained by sucrose density gradient centrifugation at 10 days post-induction, were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assayed for 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity. Aliquots were processed for electron microscopic analysis. When comparing the myelin subfractions of EAE affected animals with those of controls, differences were observed only in the light fractions, i.e., a decrease in the specific activity of CNPase and in the percentage of basic proteins relative to the total proteins of the fraction. This decrease was also evident in the basic protein/proteolipid protein ratio which is frequently used in the literature. In addition, electron microscopic observations demonstrated strong differences in the morphology of the same fraction. These findings suggest that the light fraction is the most sensitive in the early stages of the disease and must play a key role in demyelinating processes. PMID- 3011991 TI - Polyphosphoinositides as a probable source of brain free fatty acids accumulated at the onset of ischemia. AB - The quantitative relationship between phosphoinositides and free fatty acids (FFAs) in brain ischemia was studied by measuring contents of individual fatty acids in phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4 phosphate (PIP), phosphatidylinositol (PI), phosphatidic acid (PA), diacylglycerol (DAG), and the FFA pool. Various periods of complete ischemia (1, 3, 10, and 30 min) were produced by decapitation. Ischemia of 1-3 min caused rapid decreases in PIP2 and PIP content together with preferential production of stearic and arachidonic acids in the DAG and FFA pools. The decrement in levels of these fatty acid residues in polyphosphoinositides was sufficient to account for their increment in levels in the enlarged DAG and FFA pools. After 10 min of ischemia, levels of PIP2, PIP, and DAG approached plateau values, but levels of all FFAs continued to increase. The increases in content of DAG and FFAs at later ischemic periods could not be accounted for by the decreases in content of PIP2 and PIP, PI and PA levels showed only transient and subtle changes. These results indicate that, at the onset of ischemia, phosphodiesteric cleavage of PIP2 and PIP and subsequent deacylation by lipases are primarily responsible for the preferential increase in levels of free stearic and arachidonic acids and that, later, hydrolysis of other phospholipids plays a major role in the continuous accumulation of FFAs. PMID- 3011993 TI - Studies of the regulation of basal adenylate cyclase activity by membrane polyunsaturated fatty acids in cultured neuroblastoma. AB - The role of membrane polyunsaturated fatty acids (PUFAs) in the regulation of basal adenylate cyclase activity was examined in intact N1E-115 neuroblastoma cells. Addition of linoleic acid (50 microM) to the culture medium for 48 h resulted in a significant increase in phospholipid PUFA content and in a two- to fivefold increase in basal accumulation of cyclic AMP (cAMP). Both phenomena were reversed on removal of linoleate from the medium. PUFA enrichment stimulated cell proliferation by approximately 20% without altering the relative proportion of cellular protein. The supplemented cells synthesized significantly larger amounts of prostaglandin (PG) E and D than did the controls; however, blockade of PG synthesis by indomethacin or ibuprofen did not alter cAMP formation. Supplemented cells contained higher levels of malondialdehyde (MDA) than did controls, and MDA formation was reduced by coculture with alpha-tocopherol; however, its inclusion in the medium did not affect cAMP accumulation. Linoleate-supplemented cells responded to cyclase-activating agonists to the same extent as did control cells. Responses to inhibitory agonists (e.g., isoproterenol and carbamylcholine) were altered, but not to a sufficient extent to account for the PUFA-dependent increases in basal adenylate cyclase activity. PMID- 3011994 TI - Effect of chronic desipramine treatment on dihydroalprenolol, imipramine, and desipramine binding sites: a quantitative autoradiographic study in the rat brain. AB - Desmethylimipramine (DMI) administered once daily for 10 days caused a significant decrease in beta-adrenergic receptor binding, as measured by quantitative autoradiography in discrete brain regions. The decrease was observed 72 h after the last injection throughout the cortex and in hippocampus but not in other regions, much richer in beta-receptors, such as the caudate, olfactory tubercle, superior colliculus, dorsomedial thalamus, substantia nigra, or pineal. The same paradigm did not affect imipramine (IMI) binding in the cortex or in regions with high concentrations of IMI binding sites. DMI binding was not decreased, either. Significant increases in DMI binding were observed in frontal cortex and in the ventral aspect of the bed nucleus of the stria terminalis. We conclude that a reduction in tricyclic binding is not a general phenomenon following chronic treatment with tricyclic antidepressants, and changes in binding, when they do occur, are not correlated with areas of high binding site density. PMID- 3011995 TI - Analysis of cyclic AMP-dependent changes in intermediate filament protein phosphorylation and cell morphology in cultured astroglia. AB - Receptor agonists that increase cyclic AMP levels in cultured astroglia have been shown to increase 32P-labeling of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin in these cells. Experiments were designed to determine if the increase in 32P-labeling resulted from either an increase in the turnover or net number of phosphates associated with the intermediate filament proteins and if the phosphorylation of these proteins causally affected astroglial morphology. Time course experiments indicated that 6 8 h were required to reach steady-state 32P-labeling of both GFAP and vimentin. Treatment with forskolin (10 microM) after steady-state 32P-labeling increased GFAP and vimentin phosphorylation fourfold and twofold, respectively, and also induced a morphological change from polygonal to process-bearing cells within 20 30 min of drug addition. Cells incubated in media containing brain extract (30%) for 24 h at 37 degrees C and then 3 h at 23 degrees C underwent changes from polygonal to process-bearing cells with no apparent increase in the phosphorylation of either GFAP or vimentin. Treatment of process-bearing cells (induced by brain extract) or polygonal cells with 10 microM forskolin at 23 degrees C resulted in a three- to fourfold increase in GFAP phosphorylation without significant morphological changes. These results suggest that forskolin stimulation of GFAP and vimentin increases net number of phosphates associated with these intermediate filament proteins and that the resulting increase in phosphorylation can be dissociated from morphological changes. PMID- 3011996 TI - Critically ill polyneuropathy: electrophysiological studies and differentiation from Guillain-Barre syndrome. AB - A polyneuropathy of varying severity has been observed in association with sepsis and critical illness in 15 patients. Since clinical evaluation is often difficult, electrophysiological studies provided definitive evidence for polyneuropathy. These revealed reductions in the amplitudes of compound muscle and sensory nerve action potentials, the most marked abnormality. Near-nerve recordings confirmed such reductions for sensory fibres. Needle electromyography revealed signs of denervation of limb muscles. Phrenic nerve conduction and needle electromyographic studies of chest wall muscles suggested that the polyneuropathy partially explained difficulties in weaning patients from the ventilator, an early clinical sign. No defect in neuromuscular transmission was demonstrated, despite the use of aminoglycoside antibiotics in some patients. In those who survived the critical illness, clinical and electrophysiological improvement occurred. The 15 critically ill polyneuropathy patients were compared with 16 Guillain-Barre syndrome patients observed during the same period. The analysis showed that the two polyneuropathies are likely to be separate entities that can be distinguished in most instances by the predisposing illness, electrophysiological features and cerebrospinal fluid results. PMID- 3011997 TI - Herpes simplex virus infection in capsaicin-treated mice. AB - Following inoculation into the snout herpes simplex virus (HSV) spread to neurons in mouse trigeminal ganglion and subsequently to the brain. Capsaicin treatment of neonatal mice, which causes a loss of unmyelinated sensory neurons, some of which contain substance P, reduced the mortality rate of HSV-infected mice. Moreover, a lower percentage of mice survived the infection with reactivatable virus. There was also an extensive infection of glial cells proximal to the transitional zone in the trigeminal root between the peripheral and central nervous system. Distal to this zone there was an accumulation of substance P immunoreactivity in centrally directed fibres. This amplified degenerative effect on central branches of the substance P containing sensory nerves by glial infection may contribute to the deafferentiation pain syndrome following HSV infection. PMID- 3011998 TI - Isolation of JC virus capsomer-like structures from progressive multifocal leukoencephalopathy brain. AB - Brain tissue from a patient with progressive multifocal leukoencephalopathy (PML) was analyzed by molecular biological and electron-microscopic techniques. Viral DNA was isolated directly from brain tissue, cloned into a plasmid vector, and subjected to restriction endonuclease analysis. The pattern of restriction fragments identified by gel electrophoresis was almost indistinguishable from that of prototype JC virus. By this procedure the etiologic agent of PML in this patient was identified without the isolation of infectious virus. After centrifugal clarification of brain homogenates, high speed centrifugal pellets were studied by electron microscopy. Large numbers of 9-nm polygonal particles, sometimes in paracrystalline arrays, were observed. It was thought likely that these particles were capsomer subunits of 41-43 nm JC virus virions. That the particles were capsomers was supported by negative stain electron microscopy, including reconstruction studies with simian virus 40. PMID- 3012000 TI - The production of focal herpes encephalitis in mice by stereotaxic inoculation of virus. Anatomical and behavioral effects. AB - A low virulence strain of herpes simplex type 1 was microinjected into the hippocampus of BALB/c mice. Intense replication of virus at the inoculum site was followed by spread of viral antigen to the afferent connections of the hippocampus. Surviving animals showed focal damage of limbic structures and specific behavioral abnormalities generally consistent with hippocampal damage. This procedure thus produces an animal model which more closely resembles human herpes encephalitis than those previously reported. PMID- 3011999 TI - The primary role of the Erb's point--axilla segment in median and ulnar motor nerve conduction determinations in alcoholic neuropathy. AB - Motor nerve conduction velocity (MNCV) was explored in the Erb's point-axilla (N A) nerve segment of median and ulnar nerves, bilaterally, in 10 patients with a history of prolonged heavy drinking but in whom no other predisposing factors to peripheral neuropathy were found. For comparison, MNCV was determined also in the axilla-elbow (A-E), elbow-wrist (E-W) nerve segments, as well as the motor terminal latency (MTL) of the same nerves. A total of 140 nerve segments were tested tested but only 133 results were obtained. Abnormal MNCV or MTL was found in 36 or 27% of all tested nerve segments. From the latter, 38.9% were in the N-A nerve segments. Of N-A nerve segments tested, reduction in MNCV was found in 46.7%. Our results are statistically significant. PMID- 3012001 TI - Progressive axonopathy: an inherited neuropathy of boxer dogs. 3. The peripheral axon lesion with special reference to the nerve roots. AB - Progressive axonopathy is an autosomal recessive inherited neuropathy of Boxer dogs with lesions in the CNS and PNS. This paper describes the axonal changes in the lumbar and cervical nerve roots and tibial nerve. By 2 months of age the proximal paranodal areas of many larger diameter fibres show small axonal swellings, sometimes with attenuation or loss of the associated myelin sheath. Axoplasmic changes within swollen and non-swollen fibres include disorganization of the peripheral neurofilaments and small accumulations of vesicles and vesiculo tubular profiles, particularly in the sub-axolemmal area. Occasional fibres, more often in the cervical roots, are massively distended with disorganized neurofilaments. The frequency of the membranous accumulations decreases with progression of the disease. Many axons show a markedly irregular or corrugated outline and are surrounded by an attenuated sheath. The peripheral axonal cytoskeleton is disorganized and misaligned, whereas the central structures maintain a more normal arrangement. Regenerating axonal clusters are common in the cervical ventral roots but occur infrequently in the lumbar roots. Similar axonal changes occur in the peripheral nerves but at a much lower frequency. Any membranous accumulations or cytoskeletal disorganization are more probable in the proximal tibial nerves, while the frequency of axonal degeneration and regeneration increases distally. The morphological appearances indicate gross disturbances in axon-sheath cell relationships and suggest that abnormalities in the transport of various axoplasmic organelles may be involved in the pathogenesis of the axonal lesion. PMID- 3012002 TI - Reorganization of synaptic ultrastructure at facilitated lobster neuromuscular terminals. AB - Prolonged stimulation of the single excitor axon to the lobster distal accessory flexor muscle in the presence of ouabain caused long-term facilitation at its neuromuscular synapses. Hence the extracellularly recorded synaptic potentials failed less frequently and increased their mean amplitude, compared to the non facilitated (control) potentials from homologous sites in the contralateral muscle. The fine structure of synaptic terminals between matched pairs of facilitated and control preparations was compared with the aid of serial section electron microscopy. Differences between facilitated and control preparations were similar both when the latter were bathed in normal saline or ouabain containing saline, suggesting that the changes were related to the electrical stimulation rather than to the presence of ouabain. First, the facilitated terminals were smaller in surface area than the control. Second, the number and size of synaptic contacts in the facilitated terminals resembled those in the control. Third, presynaptic dense bodies or active sites increased in number although their sizes remained unaltered in the facilitated terminal. This increase is attributed to the addition of dense bodies at existing synaptic contacts since synaptic contacts remained constant in number between facilitated and control preparations. Fourth, the number and size of synaptic vesicles were unaffected by prolonged stimulation although there was a redistribution of vesicles such that they appeared to be channelled in distinct streams to synaptic contacts. Fifth, mitochondria increased in number and were situated closer to the dense bodies at facilitated nerve terminals than at control terminals. Overall, these changes denote considerable reorganization of the synaptic terminals associated with elevated transmitter release. PMID- 3012003 TI - Scanning electron microscopic studies of bullfrog sympathetic neurons exposed by enzymatic removal of connective tissue elements and satellite cells. AB - The ninth and tenth abdominal sympathetic ganglia of bullfrogs were studied by light microscopy and transmission and scanning electron microscopy after the removal of the connective tissue elements overlying the neurons. Digestion of tissues with trypsin and subsequent acid hydrolysis exposed the unipolar neurons, which remained covered by their satellite cells. The preganglionic innervation was visible on the proximal segment and axon hillock region of the postganglionic neurite. Clusters of small cells seen at the periphery of ganglia probably corresponded to groups of cells with abundant catecholamine-containing granules (SIF cells). Digestion with collagenase and protease removed some or all of the satellite cells in addition to the connective tissue. The true neuronal surfaces had short finger-like processes, whereas the external surfaces of satellite cells were smooth. Preganglionic nerve varicosities were clearly visible on the proximal segment of the postganglionic neurite, on the axon hillock and on the cell body of neurons. A few axonal varicosities were fractured to reveal the synaptic vesicles within. The possible effects of the distribution and glial ensheathment of nerve varicosities on their function are discussed. PMID- 3012004 TI - Effects of enalaprilic acid on sodium excretion and renal hemodynamics in essential hypertension. AB - The effects of MK 422 (enalaprilic acid) on renal function and electrolyte excretion were assessed in 14 patients with essential hypertension on a sodium intake of 100 mmol/day. Injection of MK 422 led to a prompt fall in blood pressure (p less than 0.01). Effective renal plasma flow increased by 9 +/- 4% (p less than 0.01) within 1 hour, an increase that persisted for a least 5 hours. Glomerular filtration rate did not change, so filtration fraction decreased by 6 +/- 2% (p less than 0.01). Sodium excretion increased with a maximum of 61 +/- 17% (p less than 0.01) after 5 hours, and potassium excretion fell (p less than 0.01). The log of the initial plasma renin activity correlated with the changes in blood pressure (r = 0.59, p less than 0.05) in effective renal plasma flow (r = 0.59, p less than 0.05) and in sodium excretion (r = 0.65, p less than 0.01). All the renal effects of MK 422 could be reversed by infusion with angiotensin II. PMID- 3012005 TI - Erythrocyte sodium-potassium activities, plasma natriuretic activity, and peripheral vascular resistances during hemodialysis or hemofiltration. AB - The effect of hemodialysis (acetate buffer) or hemofiltration on blood pressure, heart rate, peripheral vascular resistances, red blood cells ionic fluxes, and plasma natriuretic activity has been studied in six male patients treated for end stage renal disease. The hemodynamic response to these two modes of treatment markedly differs. Whereas, peripheral resistances increase and heart rate is not affected during hemofiltration, a decrease in blood pressure, tachycardia, and vasodilation is observed during hemodialysis. However, in both therapeutic approaches, red blood cell ouabain-sensitive sodium-potassium pump activity increases in a similar way, and the plasmatic natriuretic activity decreases, whereas the vascular response to norepinephrine is reduced. All of these changes were strongly correlated to the amount of fluid removed. The natriuretic activity may thus play a role in the regulation of blood pressure and hemodynamic adjustments to fluid removal in chronic renal failure between two dialyses, but its action is not predominant during the dialysis session itself. PMID- 3012006 TI - The therapeutic effect of a new angiotensin-converting enzyme inhibitor, enalapril maleate, in idiopathic hyperaldosteronism. AB - Patients with idiopathic hyperaldosteronism (IHA) manifest hypertension, hypokalemia, and renin suppression. IHA is thought to have one of three possible etiologies: zona glomerulosa autonomy, an aldosterone secretory factor, or angiotensin-II (A-II) adrenal hypersensitivity. To determine the contribution of A-II adrenal hypersensitivity in IHA, four patients with IHA were treated with a new angiotensin-converting enzyme inhibitor, enalapril, on a controlled diet (sodium [128 mEq/day] and potassium [80 mEq/day]) in a metabolic unit. The results of this study demonstrate that enalapril therapy in three of four patients normalized blood pressure, improved potassium balance, elevated PRA, reversed the postural increment in plasma aldosterone concentration (PAC), and reduced aldosterone secretion to normal. The fourth patient with bilateral macronodular disease, on the other hand, had no improvement in any of the above indices, despite maximal doses of enalapril (80 mg/day). This patient, however, may have had bilateral adrenal adenomas, based on extremely elevated 18-OH corticosterone levels (greater than 100 ng/dl), and because of a lack of adrenal A-II hypersensitivity, demonstrated by a fall in pre-enalapril, postural-, and lasix-induced PAC. In conclusion, enalapril improved the hypertension, hypokalemia, renin suppression, and hyperaldosteronism in three patients with IHA over 28 days of therapy. The results of this study suggest an etiologic role of A II adrenal hypersensitivity in IHA. PMID- 3012007 TI - Hyperdiploidy and chromosomal rearrangements define the anaplastic variant of Wilms' tumor. AB - Flow cytometric measurement of the DNA content of Wilms' tumor cells revealed a striking correspondence with the histologic subtype and treatment outcome. In the 48 cases studied, a hyperdiploid DNA content ranging from 1.7 to 3.2 times the result for normal diploid cells distinguished all but one of the ten anaplastic tumors. Lower values, from 1.0 to 1.4 times the diploid DNA content, characterized the nonanaplastic specimens. By Kaplan-Meier analysis, the probability of achieving 3 years of relapse-free survival was significantly lower in the group with higher DNA content (0.42 v 0.87, P less than .01). Analysis of banded chromosomes for a subset of 22 patients contributed important information beyond the flow cytometric study. Cases of anaplasia associated with poorer responses to therapy showed numerous complex translocations, whereas all others lacked such changes. By combining flow cytometric techniques and conventional methods of chromosome analysis, it should be possible to identify those patients with Wilms' tumor who are most likely to fail therapy. The biologic implication of these findings is that the development of clinical drug resistance in Wilms' tumor is a result of the genetic instability of the malignant clone. PMID- 3012008 TI - Etoposide (VP-16) and cisplatin in previously treated small-cell lung cancer: clinical trial and in vitro correlates. AB - Treatment of patients with relapsed small-cell lung cancer (SCLC) has been uniformly unsuccessful. Recently, the combination of etoposide (VP-16) and cisplatin in this setting has been reported to result in up to 50% response rates. We treated 29 patients with relapsed SCLC with this combination and found only a 12% response rate. The discrepancy between our results and those of others is most likely due to differences in prior treatment of the patients, although the effect of dose and schedule modifications are considered. Our patients had received a six-drug regimen over a median of 7 months and had a median drug-free interval before this treatment of only 3 weeks. Evidence is presented that suggests that this aggressive initial therapy affected both host tolerance to further treatment and the development of tumor resistance at the cellular level. PMID- 3012009 TI - Frequency dependence of synaptic transmission in nucleus of the solitary tract in vitro. AB - Afferent fibers from visceral sensory receptors enter the medulla oblongata, form the solitary tract, and synapse with neurons in the nucleus of the solitary tract. In the present study longitudinal slices were prepared from guinea pig medulla in order to examine the properties of transmission at these synapses in vitro. Synaptic responses to selective stimulation of solitary tract fibers were recorded intracellularly from neurons in an area, close to the obex and immediately medial and lateral to the tract, where arterial baroreceptor fibers are known to terminate. The amplitude of maximally evoked postsynaptic potentials (PSPs) in solitary tract neurons was strongly dependent on stimulus frequency. On increasing frequency from 0.5 to 20 Hz, a PSP depression of 80% was reached in 4 8 s. The mean depression was 35% at 5 Hz and 60% at 10 Hz. Sufficient local connections were retained in vitro that solitary tract stimulation evoked disynaptic inhibitory potentials and long latency, possibly polysynaptic, excitatory potentials in some neurons. The possibility that frequency-dependent changes in the efficacy of these local synaptic circuits contributed to PSP depression was examined. The role of postsynaptic inhibition in synaptic depression was tested by examining the frequency dependence of PSPs at membrane potentials close to the reversal of their excitatory component. The resulting hyperpolarizing PSPs were also depressed suggesting that a facilitation of postsynaptic inhibition at high frequency does not underlie the depression. The contribution of depression in multisynaptic excitatory pathways to PSP depression was assessed by exclusion. At low stimulus intensities, excitatory synaptic events with no long latency components were evoked. These events exhibited a similar frequency dependence to that of maximal PSPs. These results suggest that mechanisms operating at synapses made by solitary tract fibers are responsible for the frequency dependence of PSPs recorded in solitary tract neurons. Such mechanisms might contribute to the adaptation of some cardiovascular reflexes initiated by baroreceptors. PMID- 3012010 TI - Opioid peptide immunoreactivity in spinal and trigeminal dorsal horn neurons projecting to the parabrachial nucleus in the rat. AB - The parabrachial nucleus (PB) is the major relay for ascending visceral afferent information from the nucleus of the solitary tract to the forebrain. We have recently found that PB in the rat also receives a substantial afferent projection from neurons in the marginal zone of the entire length of the spinal and trigeminal dorsal horn. Immunoreactive perikarya stained with antisera against several neuropeptides--including dynorphin, enkephalins, and substance P--have been identified in the marginal zone. We therefore investigated the chemical specificity of the spinoparabrachial projection by combining fluorescent retrograde tracing with immunofluorescence for substance P, dynorphin A1-17, met enkephalin, and two enkephalin precursor fragments (proenkephalin 192-203 and peptide E). Following PB injections of fluorescent dyes, about half of the retrogradely labeled neurons in the marginal zone stained with antisera against either dynorphin or enkephalin series peptides. Elution-restaining experiments indicated that the dynorphin- and enkephalin-immunoreactivities were contained within separate populations of marginal zone neurons. We could not identify any substance P-immunoreactive perikarya in the marginal zone, but substance P immunoreactive fibers were seen in close apposition to retrogradely labeled, opioid-immunoreactive cell bodies and dendrites. These results indicate that the dynorphin- and enkephalin-immunoreactive perikarya in the marginal zone of the dorsal horn represent independent neuronal populations. These opioid immunoreactive neurons, which are believed to have extensive local collateral connections, are the main source of a long ascending projection to the parabrachial nucleus in the rat. Furthermore, opioid neurons in the marginal zone may receive substance P-immunoreactive primary sensory afferents. PMID- 3012011 TI - Multiple calcium-activated neutral proteinases (CANP) in mouse retinal ganglion cell neurons: specificities for endogenous neuronal substrates and comparison to purified brain CANP. AB - Calcium-activated neutral proteinases (CANPs) and their specificities for axonally transported proteins were studied within intact axons of mouse retinal ganglion cell (RGC) neurons in vitro. Two CANP activities with markedly different properties were identified. CANP B, at endogenous calcium levels, selectively cleaved the 145,000 Da (145 kDa) neurofilament protein subunit to yield 143 and 140 kDa neurofilament proteins that are also major constituents of the axonal cytoskeleton. This process represents a posttranslational modification of the neurofilament protein subunit rather than the initial step in its degradation (Nixon et al., 1982, 1983). A second calcium-activated neutral proteinase activity, CANP A, appeared only when calcium levels in the incubating medium were 100 microM or higher. CANP A degraded most proteins in RGC axons but acted considerably more rapidly on high-molecular-weight species. In particular, a 290 320 kDa protein in the Group IV (SCb) phase of axoplasmic transport was degraded 3 X faster than other major axonal proteins, including neurofilament proteins and fodrin. When maximally expressed, CANP A activity represented an enormous proteolytic potential in RGC axons--more than 50% of the total axonal content of proteins larger than 60 kDa could be hydrolyzed within 5 min. The calcium requirements, inhibitor profile, and substrate specificity of CANP A were similar to those of mCANP, the major CANP of mouse brain purified to homogeneity, suggesting that these enzymes may be the same or highly related proteins. The existence in a single neuron type of two CANP activities with markedly different substrate specificities and enzymatic properties emphasizes the possible functional diversity of calcium-activated neutral proteinases in neurons. These functions include the posttranslational modification, as well as degradation of neuronal proteins. PMID- 3012012 TI - Fodrin degradation by calcium-activated neutral proteinase (CANP) in retinal ganglion cell neurons and optic glia: preferential localization of CANP activities in neurons. AB - The activity of calcium-activated neutral proteinases (CANPs) toward endogenous substrates was measured in axons of retinal ganglion cell (RGC) neurons and separately in adjacent optic glia under in vitro conditions that preserved the ultrastructure and anatomic relationships between these cellular elements. RGC neurons and optic glia both expressed CANP activity. In contrast to RGC axons, which contained at least two CANP activities with calcium requirements in the millimolar (CANP A) and micromolar (CANP B) range (Nixon et al., 1985), CANP activity in optic glia was detectable only at millimolar calcium concentrations. When maximally activated, CANP(s) in optic glia exhibited a broad specificity for endogenous proteins but degraded larger proteins at a faster rate. The cytoskeletal protein fodrin (brain spectrin) was among the most susceptible endogenous substrates in RGC axons or glia. The similar properties of fodrin in neurons and glia, including its susceptibility to a purified millimolar calcium sensitive brain CANP (mCANP), provided the basis for using this protein as a substrate to compare the relative activity of neuronal and glial CANPs in situ. Fodrin degradation mediated by CANPs proceeded at least 6 X more rapidly in intact RGC axons than in optic glia. Comparable differences in the relative degradation rates of the total neuronal and glial protein pools were also observed. These results indicate that the potential activity of CANPs is substantially greater in RGC neurons than in glia. The enormous potential activity and preferential localization of multiple CANP activities in RGC neurons support previously hypothesized roles for CANPs in the processing of axonally transported proteins and in the regulation of neuronal cytoskeletal dynamics and geometry.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012013 TI - Immunofluorescent localization of two different Na,K-ATPases in the rat retina and in identified dissociated retinal cells. AB - Two isozymes of the Na,K-ATPase are made in the brain and retina. Antisera have been produced that contain antibodies specific for each of the catalytic subunits of the two isozymes, alpha and alpha(+) (Sweadner & Gilkeson, 1985a). To determine which classes of cells express which form of the Na,K-ATPase, and whether cells can express both forms, the antisera were used to stain sections of the rat retina and dissociated retinal cells by indirect immunofluorescence. In sections of the retina, bright stain specific for both isozymes was seen in the inner segments of the photoreceptor cells, the outer and inner plexiform layers, and in the ganglion cell axons. Cell bodies were outlined in the inner nuclear layer. Stain specific for the alpha isozyme was seen in adhering pigment epithelium and at the position of the apical processes of the Muller glial cells. In sections of the optic nerve, the nerve itself stained for both isozymes, although the stain for the alpha(+) isozyme was stronger. The sheath, in contrast, stained selectively for the alpha isozyme. Cells were dissociated from the retina, and they were identified by their morphology and by cell class specific monoclonal antibodies. Muller glial cells were found to stain brightly for only the alpha isozyme, while horizontal and ganglion cells stained brightly for both isozymes. Bipolar cells stained faintly for only the alpha(+) isozyme, while amacrine cells stained faintly for both isozymes. The evidence indicates that different kinds of neurons can express one or both isozymes of the Na,K ATPase, and at quantitatively different levels. PMID- 3012014 TI - The glycine receptor deficiency of the mutant mouse spastic: evidence for normal glycine receptor structure and localization. AB - Homozygotes of the mutant mouse spastic exhibit reduced binding of 3H-strychnine to homogenates from various regions of the CNS compared with unaffected littermates (White and Heller, 1982). Here we report evidence that the spastic mutation coincides with a reduced concentration and an unaltered structure of the glycine receptor in spinal cord. Scatchard analysis of 3H-strychnine binding revealed a single binding site with a Bmax of 267 +/- 62 fmol/mg protein for spastic and of 864 +/- 220 fmol/mg protein for control mice; no difference was found for the corresponding KD values. Also Ki values of glycine for 3H strychnine binding and displacement of 3H-strychnine by beta-alanine and taurine were indistinguishable for both preparations. Photoaffinity labeling of synaptic membranes with 3H-strychnine identified an Mr = 48,000 polypeptide in both control and spastic mouse membranes. Tryptic digestion of these membranes produced radiolabeled peptide fragments of identical molecular weights, suggesting that the proteolytic cleavage sites around the antagonist binding site are conserved in the mutant glycine receptor protein. Glycine receptors from both control and mutant mice were purified by affinity chromatography on aminostrychnine agarose. SDS/PAGE revealed three polypeptides of Mr = 48,000, 58,000, and 93,000 in both receptor preparations. Monoclonal antibodies directed against different subunits of the glycine receptor were applied to an enzyme linked immunosorbent assay. The same pattern of immunoreactivity was obtained for glycine receptor from spinal cord of spastic homozygotes, control mice, and rats, suggesting conservation of the antigenic epitopes in the mutant receptor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012015 TI - Interchange of callosal and association projections in the developing visual cortex. AB - Neurons projecting transitorily into the corpus callosum from area 17 of the cat were retrogradely labeled by the fluorescent tracer Fast Blue (FB) injected into contralateral areas 17 and 18 on postnatal days 1-5. During the second postnatal month these neurons were still labeled by the early injection, although they had eliminated their callosal axon. At this time, 15-20% of these neurons could be retrogradely relabeled by injections of Diamidino Yellow (DY) into ipsilateral areas 17 and 18, but few or none by similar injections in the other areas that receive from area 17 (19, 21a, PMLS, 20a, 20b, DLS). Similarly, area 17 neurons projecting transitorily to contralateral area PMLS during the first postnatal week could be relabeled by DY injections in ipsilateral areas 17 and 18 but not in PMLS. Already around birth, many transitorily callosal neurons in area 17 send bifurcating axons both to contralateral areas 17 and 18 and ipsilateral area 18. It is probable that during postnatal development some of these neurons selectively eliminate their callosal axon collaterals and maintain the projection to ipsilateral area 18. In fact, some transitorily callosal neurons in area 17 can be double-labeled by simultaneous perinatal injections of FB in contralateral areas 17 and 18 and of a new long-lasting retrograde tracer, rhodamine-conjugated latex microspheres, in ipsilateral area 18. The same neurons can then be relabeled by reinjecting ipsilateral area 18 with DY during the second postnatal month. This finding, however, does not exclude the possibility that some transitorily callosal neurons send an axon to ipsilateral area 18 after eliminating their callosal axon. In conclusion, area 17 neurons that project transitorily through the corpus callosum later participate, probably permanently, in ipsilateral corticocortical projections but selectively to areas 17-18. The mechanism responsible for this selectivity is unknown, but it may be related to the differential radial distribution (i.e., to birth date) of area 17 neurons engaged in the various corticocortical projections. The problems raised by the use of long-lasting retrograde fluorescent tracers in neurodevelopmental studies and by the quantification of results of double- and triple-labeling paradigms are also discussed. PMID- 3012016 TI - Noxiustoxin, a short-chain toxin from the Mexican scorpion Centruroides noxius, induces transmitter release by blocking K+ permeability. AB - Noxiustoxin (NTX), a 39 amino acid peptide purified from the venom of the Mexican scorpion Centruroides noxius, has been shown to block voltage-dependent K+ currents in the squid giant axon (Possani et al., 1982; Carbone et al., 1982). Although several other drugs known as K+ channel blockers in the squid axon also act on isolated nerve terminals to produce an increase in transmitter release, these releasing effects have not been shown to be related to a decrease of K+ permeability in synaptosomes (Vizi et al., 1977; Tapia and Sitges, 1982). In this work we show that NTX increases 3H-GABA release from perfused mouse brain synaptosomes. This effect was not blocked by TTX. Ca2+ channel blockers (verapamil or Co2+) or the absence of external Ca2+ prevents the releasing effect of this toxin. NTX does not seem to induce transmitter release by directly increasing Ca2+ permeability: The K+ ionophore valinomycin completely inhibits the release induced by NTX, as well as that evoked by the K+ channel blocker 4 aminopyridine; in contrast, the release evoked by a Ca2+ ionophore is not blocked by valinomycin. These findings strongly suggest that the releasing effect of NTX is mediated by a decrease in K+ permeability. External Ca2+ is needed only in order to couple this stimulus with the release process. Consistent with this hypothesis, we present evidence that NTX blocks the efflux of 86Rb+ from synaptosomes. An extended comparison of the effect of 4-aminopyridine with that of NTX is also reported. PMID- 3012018 TI - A new method for analyzing the distribution of fluoride in human enamel. PMID- 3012019 TI - Tomographic and CT features of the petrous bone in Lobstein's disease. One case and a review of the literature. PMID- 3012017 TI - Autoradiographic localization of sigma receptor binding sites in guinea pig and rat central nervous system with (+)3H-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine. AB - (+)3H-3-PPP [(+)3H-3-(3-Hydroxyphenyl)-N-(1-propyl)-piperidine] binds with high affinity to brain membranes with a pharmacological profile consistent with that of sigma receptors. The distribution of (+)3H-3-PPP binding sites in brain and spinal cord of both guinea pig and rat has been determined by in vitro autoradiography with binding densities quantitated by computer-assisted densitometry. (+)3H-3-PPP binding to slide-mounted brain sections is saturable and displays high affinity and a pharmacological specificity very similar to sites labeled in homogenates. (+)3H-3-PPP binding sites are heterogeneously distributed. Highest concentrations of binding sites occur in spinal cord, particularly the ventral horn and dorsal root ganglia; the pons-medulla, associated with the cranial nerve and pontine nuclei and throughout the brain stem reticular formation; the cerebellum, over the Purkinje cell layer; the midbrain, particularly the central gray and red nucleus; and hippocampus, over the pyramidal cell layer. Lowest levels are seen in the basal ganglia and parts of the thalamus, while all other areas, including hypothalamus and cerebral cortex, exhibit moderate grain densities. Quinolinic acid-induced lesions of the hippocampus indicate that (+)3H-3-PPP labels hippocampal pyramidal cells and granule cells in the dentate gyrus. Intrastriatal injection of ibotenic acid dramatically reduces (+)3H-3-PPP binding in this area, while injection of 6 hydroxydopamine produces a relatively slight decrease. The distribution of (+)3H 3-PPP binding sites does not correlate with the receptor distribution of any recognized neurotransmitter or neuropeptide, including dopamine. However, there is a notable similarity between the distribution of (+)3H-3-PPP sites and high affinity binding sites for psychotomimetic opioids, such as the benzomorphan (+)SKF 10,047. PMID- 3012020 TI - Simplified detection system for neuroreceptor studies in the human brain. AB - A simple, inexpensive dual-detector system has been developed for measurement of positronemitting receptor-binding drugs in the human brain. This high efficiency coincidence counting system requires that only a few hundred microcuries of labeled drug be administered to the subject, thereby allowing for multiple studies without an excessive radiation dose. Measurement of the binding of [11C]carfentanil, a high affinity synthetic opiate, to opiate receptors in the presence and in the absence of a competitive opiate antagonist indicates the potential utility of this system for estimating different degrees of receptor occupation in the human brain. PMID- 3012021 TI - Nuclear magnetic resonance proton imaging of bone pathology. AB - Thirty-two patients with diversified pathology were examined with a supraconductive NMR imager using spin echo with different TR and TE to obtain T1 and T2 weighted images. They included 20 tumors (12 primary, eight metastasis), six osteomyelitis, three fractures, two osteonecrosis, and one diffuse metabolic (Gaucher) disease. In all cases except for the stress fractures, the bone pathology was clearly visualized in spite of the normal lack of signal from the compact cortical bone. Nuclear magnetic resonance (NMR) imaging proved to be at least as sensitive as radionuclide scintigraphy but much more accurate than all other imaging procedures including computed tomography (CT) and angiography to assess the extension of the lesions, especially in tumors extended to soft tissue. This is due both to easy acquisition of sagittal and coronal sections and to different patterns of pathologic modifications of T1 and T2 which are beginning to be defined. It is hoped that more experience in clinical use of these patterns will help to discriminate between tumor extension and soft-tissue edema. We conclude that while radionuclide scintigraphy will probably remain the most sensitive and easy to perform screening test for bone pathology, NMR imaging, among noninvasive diagnostic procedures, appears to be at least as specific as CT. In addition, where the extension of the lesions is concerned, NMR imaging is much more informative than CT. In pathology of the spine, the easy visualization of the spinal cord should decrease the need for myelography. PMID- 3012022 TI - Resolution of massive technetium-99m methylene diphosphonate uptake in the stomach in vitamin D intoxication. AB - Vitamin D intoxication, which may result from zealous intake of health food supplements, may cause metastatic calcification. This is the first reported case of a patient with vitamin D intoxication who had massive gastric uptake of [99mTc]MDP, but no lung uptake, with histologic documentation of the metastatic calcification by gastric biopsy. It is probable that the metastatic calcification was a highly metabolic process in this patient since the gastric uptake resolved within 3 wk when serum calcium and phosphate had returned to normal. PMID- 3012023 TI - Joint scintigraphy using technetium-99m pyrophosphate in experimental hemarthrosis. AB - To determine the validity of a method for induction of experimental hemarthrosis in dogs and for the nuclear imaging of hemarthrosis, serial technetium-99m pyrophosphate [( 99mTc]PYP) flow and blood-pool scans were performed monthly in eight dogs who received bi-weekly injections of autologous blood into their femoro-tibial joints (also called stifle joint). In four control dogs, one joint was injected with saline while the other joint received only a sham injection. In addition, two dogs received intra-articular injections of autologous blood into their right stifle joint and saline into their left stifle joint. These dogs were studied with 99mTcO4 joint scintigraphy at monthly intervals. The dogs were periodically taken out of the study and explored surgically. Pathologic examination of synovial tissue was performed. Serial radiographs were also obtained and correlated with the scan and surgical findings. There was a striking abnormal increase in blood-pool activity of [99mTc]PYP in the treated stifle joints, commencing at the first examination after 1 mo of blood injections and continuing for the length of the study. All radiographs showed only minimal joint space widening and some soft-tissue swelling. On pathologic examination, both grossly and microscopically, there was profuse pannus formation, with intense inflammatory infiltrate replacing much of the subsynovial fat. The scintigraphic findings correlated well with these pathologic findings. This study not only validates this method for simulating hemophilic hemarthrosis but also suggests that [99mTc]PYP joint scintigraphy is a simple, and noninvasive method for monitoring the early changes in hemophilic arthropathy and is superior to pertechnetate imaging for this disease process. Instead of the previously recommended delayed bone images, we recommend, in addition, flow studies to assess joint hypervascularity and immediate static images to visualize the synovium and joint capsule. PMID- 3012024 TI - Stimulated salivary clearance of technetium-99m pertechnetate. PMID- 3012025 TI - Failure to visualize acutely injured kidneys with technetium-99m DMSA does not preclude recoverable function. AB - A 35-yr-old patient developed severe acute tubular necrosis requiring hemodialysis. A [99mTc]dimercaptosuccinic acid scan of the kidneys showed no renal uptake at 4 or 24 hr, but the patient subsequently recovered normal renal function as judged by a normal serum creatinine. Based on this case report and a review of the literature, one cannot assume irreversible loss of function in patients with acute renal failure, based on the absence of radiopharmaceutical uptake by the kidneys. PMID- 3012027 TI - Impact of radiocontaminants in commercially available iodine-123: dosimetric evaluation. AB - Iodine-123 (123I) is considered by some to be the radionuclide of choice for thyroid scintigraphy because of its ideal physical and biological characteristics and low radiation absorbed dose to the thyroid. However, commercially available 123I (p,2n) and (p,5n) have radiocontaminants. The MIRD formalism was used to estimate the absorbed dose to the thyroid for various age groups receiving recommended administered activities at the time of delivery and at two half-lives assuming radiocontamination levels specified by the suppliers. The calculations demonstrate that an 131I uptake with a technetium-99m scan at the time of delivery results in less absorbed dose to the thyroid than an 123I (p,2n) scan and uptake. At two half-lives the absorbed dose triples and becomes equivalent to the dose from an 131I scan. The absorbed dose from an 123I (p,5n) scan at two half-lives is higher than that of an 123I (p,2n) scan at the time of delivery. Iodine-123 capsules should not be decayed down in order to obtain a recommended pediatric administered activity. There appears to be no dosimetric advantage of commercially available 123I for thyroid scintigraphy for adults or most children. PMID- 3012026 TI - Enhancement of hepatic gallium-67 uptake by asialo-transferrin. AB - A significant fraction of intravenously injected 67Ga localizes in the liver. The mechanism of 67Ga uptake by the liver is not clear. Hepatocyte membranes contain galactose-specific receptors which selectively remove asialo-glycoproteins from the circulation by way of endocytosis. In this investigation, we examined whether endocytic uptake of asialo-transferrin would involve 67Ga transport into hepatocytes. We demonstrated that asialo-transferrin bound 67Ga as effectively as apotransferrin. Human asialo-transferrin markedly enhanced 67Ga uptake by isolated, perfused rat livers. Human asialo-orosomucoid, but not orosomucoid competitively inhibited hepatic uptake of the 67Ga asialo-transferrin complex, indicating that hepatic 67Ga uptake in the presence of asialo-transferrin occurred by way of galactose-specific receptors. Our results point to a novel pathway for metal ion transport into hepatocytes by way of galactose receptor mediated endocytosis. PMID- 3012028 TI - Detection of diffuse glomerular lesions in rats: I. Comparisons of conventional radioactive agents. AB - Conventional renal diagnostic agents, [131I]hippuran, [99mTc]glucoheptonate (GHA), and [99mTc] dimercaptosuccinate (DMS) were compared with [99mTc] or [111In] diethylenetriaminepentaacetic (DTPA) for the detection of glomerular damage in rats compared with controls. The glomerular lesions were induced by the i.v. injection of puromycin aminonucleoside (PA) 9 days before the radionuclide studies, a model of spontaneous "minimal change" glomerulonephritis in humans. Computer-generated early renal uptake of [99mTc]DTPA or GHA correlated with the glomerular filtration rate (GFR) quantitated by biexponential plasma clearance of DTPA administered by single i.v. injection. The early renal uptake of hippuran and DMS correlated poorly with GFR as assessed by DTPA clearance. However, the 2 hr renal retention of DMS correlated well with the DTPA clearance. None of the parameters measured with [131I]hippuran correlated well with DTPA clearance, probably because of decreased protein plasma binding of hippuran secondary to hypoproteinemia in this experimental model. It was concluded that none of these agents was superior to labeled DTPA for the detection of glomerular damage in this experimental model. PMID- 3012029 TI - Quantitative relationship between global left ventricular thallium uptake and blood flow: effects of propranolol, ouabain, dipyridamole, and coronary artery occlusion. AB - The quantitative relationship between fractional myocardial thallium uptake and radioactive microsphere-determined flow was studied in 33 open chest dogs under baseline conditions during increased coronary flow (dipyridamole), decreased coronary flow (propranolol and coronary artery stenosis), inhibition of Na-K ATPase (ouabain), and regional infarction. Myocardial contents of thallium and microspheres were compared in left ventricular (LV) biopsies taken 5, 10, 15, 30, and 60 min after thallium injection, expressed as fractions of injected dose. Maximal LV thallium uptake occurred 10 min after injection and the 10-min values were therefore used for subsequent comparisons. Combining all dogs, fractional LV thallium content (% injected dose) correlated well with fractional LV blood flow (% cardiac output) (r = 0.95). However, for fractional LV flows in the low, normal, or moderately elevated range (LV flow/cardiac output less than 9%), thallium content consistently exceeded flow by about 15%. This relationship was not altered by ouabain or regional ischemia or infarction. For greatly elevated fractional LV flows (greater than 9%), thallium content was not significantly different from flow. To explain these differences, myocardial and systemic extraction fractions for thallium were determined in eight dogs with a dual tracer method. At baseline, myocardial extraction fraction was significantly greater than systemic (88 +/- 0.4% compared with 75 +/- 1%, p less than 0.001). During dipyridamole, myocardial extraction fraction decreased and myocardial and systemic values were no longer significantly different (82 +/- 1% compared with 79 +/- 1%). These results show that the fraction of injected thallium dose taken up by the LV myocardium exceeds the delivered fraction of cardiac output over a wide range of LV flows, and is not altered by ouabain-induced inhibition of sodium-potassium ATPase or regional myocardial infarction. This difference is explained by a greater myocardial than systemic extraction fraction for thallium. During high LV flows produced by dipyridamole, fractional LV thallium uptake and flow become similar as myocardial and systemic extraction fractions equalize. PMID- 3012030 TI - Characterization of beta-adrenoreceptors in vivo with iodine-131 pindolol and gamma scintigraphy. AB - The aim of this study to was to assess the feasibility of using iodopindolol to delineate myocardial beta-adrenoreceptors in vivo. Preliminary biodistribution studies indicated that binding of 131I-d,I-pindolol in the heart was stereospecific, saturable, and displaceable by I-propranolol but not by phenoxybenzamine. However, considerable nonspecific binding was encountered. Subsequently, the stereoisomer, 131I-I-pindolol, was shown to be a high affinity beta-adrenoreceptor antagonist (Kd approximately 0.37 nM) as assessed by Scatchard analysis, and one exhibiting marked specific uptake in lung and heart in rabbits. In contrast, 131I-d-pindolol exhibited no specific binding in rabbit left ventricular membrane preparations nor specific organ uptake. Gamma camera scintigraphy with both isomers demonstrated that the I-isomer accumulated in lung and heart, and that its accumulation was blocked by I-propranolol. In contrast, d isomer uptake was nonspecific and diffuse. The results indicate that it should be possible to externally visualize receptors by differentiating specific and nonspecific binding components of a ligand in vivo with the use of radiolabeled stereoisomers. PMID- 3012032 TI - An historical review of positions in baccalaureate education in nursing as basic preparation for professional nursing practice 1960-1984. AB - "The call for the baccalaureate degree in nursing as the educational basis for the profession has a lengthy history. Nursing leaders have suggested the need for the baccalaureate for more than a half century" (Education for, 1983, p. 3). The article culls comments concerning baccalaureate education as entry into professional nursing practice from published statements of selected organizations and groups, and places them in a historical perspective from 1960-1984. Materials from the American Nurses' Association (ANA), National League for Nursing (NLN), American Association of Colleges of Nursing (AACN), American Society for Nursing Service Administrators (ASNSA), Institute of Medicine (IOM), and the National Commission on Nursing are included. PMID- 3012031 TI - Digoxin-like immunoreactivity in human body fluids. PMID- 3012033 TI - Predicting the success of minority students in a baccalaureate nursing program. AB - Entering grade point average (ENTGPA), American College Test Assessment (ACT), high school rank (HSRANK), high school GPA (HSGPA), number of college credit hours prior to program admission (HRSPTA), age at admission, and an index of applicant motivation and related experience (MEP) were investigated to determine the best predictive combination of variables for success among minorities in a baccalaureate nursing program. Final GPA, program completion, and State Board Examination (SBTPE) performance were used as indicators of success. Minority students (N = 145) admitted between 1971-1981 were identified by record review. Two minority subgroups, blacks (n = 111) and nonblack minorities (n = 34) were compared using multiple regression and discriminant analysis procedures. ACT was the strongest, most consistent predictor of SBTPE performance and final GPA for all minorities. ENTGPA and ACT provided substantial predictive power for both subgroups, but explained markedly less variance for blacks. HSGPA, HRSPTA, and HSRANK explained some variance differently by subgroup. ENTGPA provided the only discrimination between graduates and dropouts. Cognitive attributes are critical to academic success among minorities, although predictors may vary in explanatory power by minority group. Variables interfering with program completion need to be explored. PMID- 3012034 TI - The effect of nonspecific imaging practice on the mental imagery ability of nursing students. AB - Mental imagery practice has assisted in the acquisition of psychomotor skills in several fields. Nursing education programs include many psychomotor skills which students need to learn quickly and efficiently. Examining imagery ability might be the first step in improving methods of teaching psychomotor nursing skills. This study hypothesized that subjects with low imagery who were exposed to non specific imaging practice could have their imagery ability enhanced through the use of imaging exercises. A within subjects experimental design was used. Sixty sophomore nursing students were divided into high and low imagers based on scores on the shortened form of the Betts' QMI Vividness of Imagery Scale. Each group received one of two treatments--nonspecific imaging practice sessions or relaxation practice sessions; all subjects were posttested on the Betts'. A one way analysis of variance with followup planned comparison tests using power analysis showed a highly significant main effect for low imagers. PMID- 3012035 TI - A taxonomy of games and simulations for nursing education. AB - The purposes of this study were to locate games and simulations available for nursing education, to categorize these materials to make them more accessible for nurse educators, and to determine how nursing's use of instructional games might be enhanced. Twenty-nine games and simulations were identified in the nursing literature. The taxonomy developed for this study described three types of process games: clinical, pathophysiologic and psychosocial. These games were further analyzed using a modified version of Ruben's (1977) classification, which compared and contrasted games by structural components and control parameters. The taxonomy is intended to guide nurse educators in conveniently locating games and simulations which may fit or be tailored to fit individual learning objectives. Recommendations include instituting standards for describing games and simulations, establishing a clearinghouse for disseminating information on nursing games and initiating research on the effectiveness of games and simulations as instructional strategies. PMID- 3012036 TI - Correlates of university nurse faculty publication productivity. AB - The purpose of this study was to identify factors which may be important in the publication productivity of university nurse faculty. Two central research questions were addressed: 1) What relationship exists between selected professional, educational, and career variables and the publication productivity of university nurse faculty members? 2) What is the typical publication productivity profile of university nurse faculty? The population consisted of 422 full-time tenure tract nurse faculty teaching in seven nursing schools that offered baccalaureate, master's and doctoral programs and were located in public Research Universities I. All data were obtained through the use of a questionnaire. Completed questionnaires were received from 80 percent of the respondents. Faculty not meeting the criteria for the study and all instructors were eliminated from analysis. Data were ultimately analyzed for 261 subjects. Thirty-two variables were found to have a significant relationship to faculty publication productivity. Eleven of these variables (highest degree, years since first master's, age, rank, teaching responsibilities, time spent teaching, time spent in research, hours of clinical instruction, teaching and research preferences, journals received, beliefs about the desirable relationship between publication and promotion and tenure) and five motivational variables were included in a regression analysis. These 16 variables grouped into three clusters, accounted for .4845 percent of the total variation in university nurse faculty publication productivity. Current job socialization factors and motivational factors accounted for a significant amount of variation in faculty publication productivity even when highest degree, years since first master's, age, and rank were controlled. PMID- 3012037 TI - Journal writing: a key to promoting critical thinking in nursing students. PMID- 3012038 TI - 4MAT: a method of stimulating holistic learning. PMID- 3012039 TI - Home-bound hospice as a learning environment for mental health nursing students- a model approach. PMID- 3012040 TI - Development and evaluation of a summative clinical grading tool. PMID- 3012041 TI - Facilitating student involvement in large classroom settings. AB - Diminishing resources and increasing student enrollment in the basic and post basic programs at the Faculty of Nursing, University of Alberta, have led to large teacher-student ratios in the area of classroom lectures. This article is a discussion about the issue of student involvement in the learning process where there may be 100 or more students in one class. The meaning of student involvement, factors which inhibit student involvement, and strategies to increase student involvement in a large classroom situation are addressed. PMID- 3012042 TI - Pulmonary nurse specialists: preparation for the advanced practice role. AB - A survey was done to determine the factors that influence nurses' decisions to prepare as specialists in pulmonary nursing. A questionnaire that included items concerned with biographic variables and perceptions toward pulmonary nursing was distributed on selected adult units of two medical centers and one community hospital in south central Connecticut. Data were collected from 113 medical surgical nurses. Subjects were primarily women (96.5%) and in the 20-30 age range (mean = 30, SD = 7.06, median = 27.6). On the average, subjects had been employed for 7.5 years (7.03, median = 5.37). Thirty percent of the sample expressed a desire to work with pulmonary patients. This group had a positive score on the semantic differential (p = 0.0001). Nurses (N = 11) who had worked with a pulmonary clinical specialist rated their impression of caring for pulmonary patients more positively than those who had not (p = 0.044). Although only 13 subjects expressed an interest in preparing as a pulmonary nurse specialist, they had a positive score on the semantic differential (p = 0.0001). Limitations, recommendations for further study, and implications for nursing and nursing education are addressed. PMID- 3012043 TI - Hours of contact and their relationship to students' evaluations of teaching effectiveness. AB - This study was designed to determine if hours of contact with a teacher influenced a student's perceptions of teaching effectiveness. Three hundred forty one nursing students used a researcher developed instrument to rate five teachers of nursing on classroom teaching effectiveness, and one of the five teachers on clinical teaching effectiveness. Results indicated that a positive relationship existed between students' ratings of their clinical teacher's teaching effectiveness in the classroom and clinical area. The significant Pearson correlations for the five teachers ranged from .61 to .91 (p = .000). The hypothesis, that increased contact hours with a teacher would have a positive influence on students' evaluations of a teacher, received support. Three of five clinical groups of students gave their clinical instructors significantly higher scores for classroom teaching effectiveness than did other students. PMID- 3012044 TI - Invitational teaching behaviors in the associate degree clinical setting. AB - Teaching in the clinical area provides some unique problems for the nurse educator. An effective clinical instructor is one whose teaching behaviors promote learning. This study investigated the relationship between the attitudes associate degree nursing students have toward their clinical learning experiences and their perceptions of the Invitational Teaching behaviors of the clinical instructor. The Invitational Teaching Survey and the Student Affective Outcome Measure (Amos, Purkey, & Tobias, 1984) were adapted to describe teaching behaviors in the clinical setting. Reliability studies were conducted on the revised instruments. The total scores and subscores yielded 15 relationships which were compared using Pearson Product Moment Coefficients. The correlations coefficients ranged from .39 to .69 and all were statistically significant at p less than .01. The results of this study indicated a strong relationship between students attitudes toward their clinical experience and their perceptions of the inviting teaching behaviors of their clinical instructor. The study yielded a reliable tool which can be used by instructors to identify the students' perceptions of their Invitational Teaching behaviors in the clinical setting. PMID- 3012045 TI - Using computers to teach basic facts in the nursing curriculum. AB - This article reports on the field testing of a set of flexible computer assisted instruction (CAI) programs to supplement more traditional approaches to a unit of instruction on respiratory assessment for undergraduate nursing students. The results indicate that the students reacted favorably to the CAI materials and that the performance of students improved significantly when they used the experimental programs. This field test offers useful insights into how microcomputers can be integrated into the nursing curriculum. PMID- 3012046 TI - Quality circles in nursing education. PMID- 3012047 TI - A peer evaluation for measuring team teaching effectiveness. AB - The peer evaluation tool for team teaching was developed to supply data for the team teaching component of the comprehensive evaluation procedure. The tool is used most effectively when the evaluators are familiar with the criteria, and have had an opportunity to work with and observe a colleague's performance for an extended period of time. It lends itself for use in yearly evaluation of non tenured faculty and faculty applying for promotion of tenure. The criteria written as performance objectives can also provide direction for one's self evaluation of teaching performance and serve as a standard for professional growth. PMID- 3012048 TI - Changing needs of society and the response of nursing education. PMID- 3012049 TI - Health care and cost containment for the homeless: curricular implications. PMID- 3012050 TI - Tissue distribution of 7-dehydrocholesterol, vitamin D3 and 25-hydroxyvitamin D3 in several species of fishes. AB - A high-performance liquid chromatographic (HPLC) method for simultaneous determination of 7-dehydrocholesterol (7-DHC), vitamin D3 and 25-hydroxyvitamin D3 (25-OH-D3) in tissues of fishes was established, and using this method the tissue distribution of the sterols in lamprey (Entosphenus japonicus), great blue shark (Prionace glauca), skipjack (Katsuwonus pelamis) and albacore (Thunnus alalunga) was investigated. The results are summarized in the following: Although the alimentary canal, gall bladder and roe of lamprey and the alimentary canal of great blue shark contained comparatively high levels of 7-DHC (higher than 2,000 ng/wet tissue g), the other tissues of lamprey and great blue shark and all tissues of skipjack and albacore contained only low levels of 7-DHC (lower than 1,000 ng/g). There was no significant correlation between the levels of 7-DHC and vitamin D3. The contents of 7-DHC in the skin of skipjack and albacore were only 1/1,000 of those in the skin of rats. Although the contents of vitamin D3 in the liver of skipjack and albacore were extremely high (41,240 and 21,000 ng/g, respectively), those in the skin were very low (454 and 257 ng/g, respectively). 25-OH-D3 was detected in the viscera of skipjack, but the levels were not very high (lower than 150 ng/g). These levels were not significantly correlated with those of vitamin D3. The results suggest that large quantities of vitamin D3 in the liver of skipjack and albacore are supplied by other biosynthetic routes or by intake of vitamin D3 rather than by photochemical biosynthesis. PMID- 3012051 TI - Effects of vitamin E deficiency and non-biological antioxidant (DPPD) on the function of the pituitary-gonadal axis of the rat. AB - The effects of vitamin E deficiency on pituitary-gonadal function in rats and the preventive effects of N,N'-diphenyl-p-phenylene diamine (DPPD) administration were examined by measuring levels of pituitary and plasma follicle-stimulating hormone (FSH) and luteinizing hormone (LH), serum and testicular levels of testosterone, and affinity and receptor sites of FSH and LH in the testis by radioimmunoassay, at 180 days after feeding of a vitamin E-deficient diet and DPPD-administered diet. Light and electron microscopic examinations were also performed on the pituitary gland and testis. In the vitamin E-deficient rats, serum and liver alpha-tocopherol concentrations decreased significantly and erythrocyte hemolytic rate and serum and tissue malondialdehyde levels increased significantly. However, the increase of hemolytic rate and malondialdehyde concentration in the vitamin E-deficient rats was somewhat lessened by the administration of DPPD. In the vitamin E-deficient rats, the gonadotropic cells in the pituitary gland manifested accelerated secretory function indicated by enlargement of cells, development of Golgi apparatus and accumulation of secretory granules, while FSH and LH concentrations in the pituitary and serum were not affected by vitamin E deficiency. However, the testosterone concentrations in the plasma and testis were significantly lower in the vitamin E deficient rats. The decrease of testosterone in plasma and tissue was prevented by the administration of DPPD, while the degeneration of seminiferous tubules was not completely restored by DPPD. It is concluded that DPPD can compensate to some degree for the lack of antioxidative activity due to vitamin E deficiency. PMID- 3012052 TI - [Physiological characteristics of first-order horizontal canal neurons in guinea pigs. 2. Response to angular acceleration]. PMID- 3012053 TI - Transplacental passage of acyclovir. PMID- 3012054 TI - Use of digoxin in infants and children, with specific emphasis on dosage. AB - The foregoing discussion leads to several general conclusions regarding the use of digoxin in the pediatric patient. First, pharmacokinetic studies indicate that somewhat higher doses are required in the infant to attain the same serum levels as in the adult. Important sources for this difference appear to be more rapid body clearance of digoxin and larger volume of distribution in the infant. Second, higher serum digoxin levels are not indicated on the basis of decreased myocardial uptake of digoxin in the infant. Tissue uptake of digoxin, as indicated by myocardium/serum digoxin ratios, is higher in infants and children than in adults. Third, according to results of animal studies, the inotropic sensitivity to digoxin in the young is probably greater--certainly not less--than in the adult. This is opposite to a commonly held view that the immature heart is less sensitive to cardiac glycosides and therefore requires higher serum levels for a therapeutic effect. Rather, the infant has decreased sensitivity of the conduction system to digitalis toxicity, and healthy myocardium less prone to arrhythmia than the adult. Therefore the infant may tolerate, but does not require, higher serum levels of digoxin. Fourth, high levels of serum digoxin (greater than 2 ng/ml) are not associated with greater inotropic effects in the pediatric patient. The higher dosages of digoxin are, instead, associated with greater frequency of toxic effects, especially in infants receiving concomitant diuretic therapy. Therefore, a digoxin dosage recommendation is presented, that will result in mean serum digoxin levels of 1.1 to 1.7 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012055 TI - Neurologic complications in oral polio vaccine recipients. AB - Between April 1982 and June 1983 four children 3 to 24 months of age were referred for evaluation of neurologic abnormalities found to be compatible with vaccine-related poliovirus infection, which had not been suspected by referring physicians. Patients were epidemiologically unrelated residents of Indiana, and none had prior symptoms suggestive of immunodeficiency. All had received poliovirus vaccine orally (first dose in three, fourth dose in one) and a diphtheria-tetanus-pertussis injection in the left anterior thigh within 30 days of symptoms. A vaccine-like strain of poliovirus was isolated from each patient, and each had symptoms (left leg paralysis in three; developmental regression, spasticity, and progressive fatal cerebral atrophy in one) persisting for at least 6 months. Immune function was normal in two with poliovirus type 3 infection, and abnormal (hypogammaglobulinemia, combined immunodeficiency) in two with type 1 and type 2 infection, respectively. The incidence of observed vaccine related poliovirus infection in Indiana recipients of orally administered poliovirus vaccine was 0.058 per 100,000 per year, significantly greater (P less than 0.001) than predicted. PMID- 3012056 TI - Changes in cerebral blood volume and cytochrome aa3 during hypertensive peaks in preterm infants. AB - Relative changes in cerebral blood volume and in the oxidation/reduction state of cytochrome aa3, the terminal member of the electron transport chain in oxidative metabolism, can be simultaneously observed with near infrared spectroscopy. Using this technique, we studied movement-associated blood pressure elevations in three nonparalyzed very low birth weight infants receiving mechanical ventilation. We defined hypertensive peaks as increases in systolic and diastolic blood pressures greater than or equal to 30% over baseline and lasting at least 2 seconds. Ninety percent of monitored time, an increase in tissue blood volume (tBV) immediately followed each blood pressure elevation, with deoxygenated hemoglobin providing the sole or predominant increase in tBV. A simultaneous shift of cytochrome aa3 to a more reduced state usually accompanied the rise in tBV, probably indicating a transient imbalance between oxygen delivery and cellular oxygen utilization and a failure of mechanisms that normally regulate cerebral oxygenation. The consistent association of hypertensive peaks with body movement, coughing, and breath holding, and the predominant increase in deoxygenated hemoglobin suggest that increased intrathoracic pressure transiently impedes cerebral venous return. The repeated fluctuations in intracerebral blood volume and associated shifts to greater cytochrome aa3 reduction with hypertensive peaks provide a possible explanation for the association of fluctuating blood pressure patterns and increased risk for intraventricular hemorrhage. PMID- 3012057 TI - Wilms' tumor in the neonate: a report from the National Wilms' Tumor Study. AB - Renal neoplasms in the neonate are quite uncommon. Twenty-seven of the 3,340 patients (0.8%) registered on the National Wilms' Tumor Studies from 1969 through April 1984, were 30 days old or less. Of these 27 patients, 18 had mesoblastic nephroma, 1 had a malignant rhabdoid tumor of the kidney, and 4 others had nonneoplastic lesions. The remaining four infants were reviewed in detail. All had favorable histology Wilms' tumors; none had distant metastasis at diagnosis. Treatment ranged from surgery alone to excision plus three-drug therapy for 15 months. All fared well. The patient with Stage I rhabdoid tumor died at eight weeks of age in spite of aggressive four-drug therapy. This review supports the view that Wilms' tumor in the neonate is extremely rare. PMID- 3012058 TI - Effects of two cannabinoids on hepatic microsomal cytochrome P-450. AB - The effects of cannabidiol (CBD) and delta 9-tetrahydrocannabinol (delta 9-THC) on the synthesis and degradation of hepatic microsomal cytochrome P-450 were studied in mice. Cannabinoids used (10, 50 and 100 mg/kg, i.p.) did not affect delta-aminolevulinic acid synthetase activity in the liver. delta 9-THC-treatment (10, 50 and 100 mg/kg, i.p.) markedly stimulated heme oxygenase activity in hepatic 18000 X g supernatant fractions in a dose-dependent manner, whereas CBD treatment was without effect. In vitro experiments, CBD and delta 9-THC (40 to 160 microM) markedly inhibited nicotinamide adenine dinucleotide phosphate (NADPH)-induced lipid peroxidation in hepatic microsomes. When CBD was incubated with the hepatic microsomes in the presence of an NADPH-generating system, cytochrome P-450 content decreased significantly. However, delta 9-THC showed no effects in similar experiments. The rate of decrease in the cytochrome P-450 content using CBD (160 microM) was 0.212 nmol/mg protein/20 min in microsomes from control mice. This value increased significantly in microsomes from phenobarbital-treated mice (0.792 nmol/mg protein/20 min) but not in those from 3 methylcholanthrene-treated mice (0.190 nmol/mg protein/20 min). The metabolic rate (per nmol cytochrome P-450) of CBD was also increased significantly by phenobarbital-treatment but not by 3-methylcholanthrene-treatment. These results suggest that CBD metabolites rather than CBD itself, play some role in the decreasing effect on cytochrome P-450 content in the hepatic microsomes in vitro, and that the microsomal formation of reactive metabolite of CBD is increased by phenobarbital-treatment. PMID- 3012059 TI - Conditioned suppression and opioid kappa receptor in mice. AB - Mice exhibited a marked suppression of motility (conditioned suppression) when placed in the same environment in which they had previously received an electric footshock. Furthermore, chronic morphine (mu agonist)-pretreated mice, as well as chronic vehicle-pretreated mice, exhibited the conditioned suppression but chronic ethylketocyclazocine (kappa agonist)- and pentazocine (kappa agonist) pretreated mice did not. On the other hand, in the synaptic membranes of the chronic vehicle- and morphine-pretreated mice showing conditioned suppression, the specific binding of [3H]phencyclidine (sigma agonist) and [3H]naloxone (mu antagonist) significantly increased, while the specific binding of [3H]ethylketocyclazocine did not change compared to those of the corresponding control groups. However, in the chronic pentazocine- and ethylketocyclazocine pretreated groups showing non-conditioned suppression, the specific binding of [3H]phencyclidine and [3H]naloxone were not altered. Moreover, a decrease of [3H]ethylketocyclazocine binding was observed in the chronic pentazocine pretreated group. These results suggest that the binding function of different opioid receptor subtypes may be altered differently by stress, and that the kappa receptor may be important for the conditioned suppression of motility in mice. PMID- 3012060 TI - Potent platelet antiaggregant action of an antiarrhythmic peptide (AAP). AB - A potent platelet antiaggregant action of an antiarrhythmic peptide (AAP) was demonstrated to be a cause of the antithrombotic effect. AAP (10, 20 or 40 mg/kg, i.v.) inhibited ex vivo platelet aggregation induced by collagen in a dose dependent manner. AAP also inhibited the platelet aggregation of platelet-rich plasma (PRP) induced by collagen, Ca-ionophore A-23187, adenosine diphosphate (ADP), thrombin or arachidonic acid in vitro. The IC50 was 2.5 mM for collagen, 1.7 mM for A-23187, 5 mM for ADP, 0.4 mM for thrombin and 0.15 mM for arachidonic acid. The aggregation inhibitory activity of the peptide on washed platelet (WP), in a Ca2+-free medium, was stronger than on PRP. The IC50 was 1 mM for collagen and 20 microM for A-23187. No significant difference was found between antiaggregant activities of platelet-free plasma (PFP) from AAP-treated rats and PFP from normal rats supplemented with AAP. The direct action of AAP on platelets was also supported by the incorporation of AAP into platelet cytoplasma which caused a decrease of Ca2+-dependent 3':5'-cyclic nucleotide phosphodiesterase (Ca PDE) activity. It was considered that AAP showed its platelet aggregation inhibitory activity by decreasing intracellular Ca2+ concentration through the inhibition of Ca-PDE activity. PMID- 3012061 TI - Dietary fiber. PMID- 3012062 TI - Effect of dietary fats on membrane lipids and functions. PMID- 3012063 TI - Biologic interaction of prostaglandin, thromboxane and prostacyclin: potential clinical applications. AB - Prostaglandins are vasoactive agents which have potent and varied effects depending on the species, conditions and organs tested. The clinician wishing to gain a significant overview of the field from current research literature has a demanding task for himself. A review of biologic interactions is exactly what is needed in a consideration of possible clinical applications of prostaglandins. Thus, it is necessary first to recount the last five years' advances in prostaglandin research. Only then will the listing and discussion of some diseases soon to benefit from the application of research be meaningful. PMID- 3012064 TI - The role of dietary fiber in health and disease. AB - Dietary fiber has been, for several years, the glamour ingredient in popular nutrition. Based on epidemiological evidence, lack of fiber in the diet has been impugned as a major risk factor for development of colon cancer, heart disease, diabetes and a variety of lesser ills. Animal experiments suggest that some components of the complex mixture of substances called fiber will reduce cholesterol levels to a modest extent and will inhibit atherosclerosis induced by diet. In man the data center on the effects of fiber on plasma cholesterol levels and some fibers such as pectin or guar exert significant hypocholesterolemic effects whereas others, such as bran, do not. The situation is similar with regard to colon cancer. Some types of fiber, bran and cellulose for instance, inhibit experimentally induced colon cancer. There are a number of ways of establishing experimental colon cancer; feeding the carcinogenic agent, injecting it, or instilling it intrarectally. There also exists a variety of carcinogenic agents. The effect of fiber is the sum of the type of fiber and carcinogen used and the mode of establishing the cancer. Different combinations give different results in animal studies. In man the data bearing on this subject are wholly epidemiological. A few case-control studies have provided suggestions that low fiber diets may predispose to colon cancer but these studies point to a dietary life-style in which many components other than fiber vary. The most notable success in wedding practice to hypothesis has been in the area of diabetes. Here it has been shown clearly that increasing dietary fiber results in reductions in lipemia, glycemia and insulin requirement. What remains? More work in the cancer and heart disease fields but mainly a greater effort to identify the specific structure of those fibers which exert a beneficial effect. This will have the two fold benefit of identification of specifically useful structural types of fiber and of possibly providing clues to mechanism of action or of carcinogenicity. Most experts agree that a modest increase in intake of fiber will have a generally beneficial effect but they can only support these statements with epidemiological proof. Future research must include studies designed to confirm the epidemiological findings and to identify the specific components responsible for them. PMID- 3012065 TI - Effect of repeated restraint stress, desmethylimipramine or adrenocorticotropin on the alpha and beta adrenergic components of the cyclic AMP response to norepinephrine in rat brain slices. AB - The cyclic AMP response to catecholamines in rat cortical slices is mediated by a beta adrenergic receptor which is coupled to adenylate cyclase and an alpha adrenergic receptor which potentiates the response to beta receptor stimulation. The present studies examined the effects of repeated restraint stress, adrenocorticotropin or desmethylimipramine administration on the beta and alpha adrenergic components of this response. Restraint was found to produce a small nonsignificant decrease of the beta receptor response accompanied by a significant reduction of the alpha receptor-induced potentiation of the beta response. Desmethylimipramine was found to lower the cyclic AMP response to beta receptor stimulation but not to alter the alpha-induced potentiation of the beta response. Adrenocorticotropin, like restraint stress, was found to reduce only the alpha-induced potentiation of the beta response. Experiments with adenosine and histamine showed that restraint stress lowered the alpha-induced potentiation of cyclic AMP responses to these neurohormones also. It is concluded that restraint stress acts primarily to reduce the response to stimulation of central alpha adrenergic receptors whereas desmethylimipramine acts primarily to reduce the response to stimulation of beta adrenergic receptors. Adrenocorticotropin has the same effect as restraint stress suggesting that pituitary adrenal hormones mediate the stress effect. PMID- 3012066 TI - Effect of PGD2 on cardiac contractility: a negative inotropism secondary to coronary vasoconstriction conceals a primary positive inotropic action. AB - The purpose of this study was to characterize by pharmacological means the inotropic action of prostaglandin D2 (PGD2) in the guinea-pig heart. In the whole heart perfused at constant pressure, PGD2 (0.01-10 micrograms) reduced coronary flow rate and decreased left ventricular contractile force in a dose-dependent manner. When the coronary vasoconstricting effect of PGD2 was antagonized by the PG antagonist sodium p-benzyl-4[1-oxo-2-(4-chlorobenzyl)-3-phenyl propyl]phenyl phosphonate (N-0164), by the cyclooxygenase inhibitor indomethacin or by the thromboxane synthetase inhibitor sodium (E)-3-[4-(1-imidazolyl-methyl)phenyl]-2 propanoate (OKY 046), the negative inotropic response to PGD2 was attenuated or completely abolished and a positive inotropic effect was unmasked. In the isolated left atrium or right ventricular papillary muscle preparations, PGD2 induced only a positive inotropic response. The atrial response to PGD2 was unaffected by N-0164, indomethacin or propranolol but was markedly decreased by carbachol or adenosine. Conversely, the response of the papillary muscle to PGD2 was potentiated by papaverine. Thus, these data indicate that PGD2 has a primary positive inotropic effect, which may involve cyclic AMP metabolism. On the other hand, because PGD2 is also a potent coronary vasoconstrictor, the secondary negative inotropic effect of PGD2 predominates. PMID- 3012069 TI - Acute, stress-induced changes in the benzodiazepine/gamma-aminobutyric acid receptor complex are confined to the chloride ionophore. AB - Rapid and robust changes in the chloride ionophore component of the benzodiazepine/gamma-aminobutyric acid receptor complex ("supramolecular complex") were observed in the central nervous system of rats exposed to a brief, ambient temperature swim stress. Stress-induced modification of the chloride ionophore was manifest as an increase in the efficacy of halides (Cl-, Br- and I ) to enhance [3H]flunitrazepam binding. In contrast, neither gamma-aminobutyric acid-enhanced [3H]flunitrazepam binding nor [3H] flunitrazepam binding assayed in the absence of halide ions was altered by stress. Furthermore, the number of [35S]t-butylbicyclophosphorothionate binding sites and the apparent affinity of this radioligand were increased as a result of stress, as was the ability of Cl- and low concentrations of muscimol to increase the binding of this radioligand. These changes could represent the physiological attempt of an organism to compensate for stressful changes in the environment. PMID- 3012068 TI - Relative presynaptic and postsynaptic effects of urapidil on adrenoceptors in the rabbit pulmonary artery. AB - Urapidil is a new antihypertensive drug which is thought to possess unique antiadrenergic properties. It is definitely a post-synaptic alpha adrenergic antagonist, but it also claimed to be a presynaptic alpha adrenergic agonist resulting in inhibition of norepinephrine release. To test the relative presynaptic and postsynaptic activity and selectivity in an isolated rabbit pulmonary arterial preparation, two series of studies were performed. The first evaluated the effects of urapidil on electrically evoked 3H overflow in the superfused vascular strip preincubated with [3H]norepinephrine. No significant inhibition of 3H overflow was observed. Rather, urapidil appeared to possess relatively weak presynaptic alpha-2 adrenergic antagonistic properties which were reversed by clonidine. The concentration producing a 30% increase in 3H overflow (EC30pre) was 7070 nM. The second series of studies evaluated the effects of urapidil on the concentration-contractile response curve to norepinephrine. Schild plot analysis yielded a pA2 value of 6.24 and a KBpost of 575 nM. One measure of the relative pre- and postsynaptic antagonistic potency is the EC30pre/KBpost ratio. For urapidil, this was 12.3, suggesting that the drug is a partially selective postsynaptic alpha adrenergic antagonist, with approximately 4.5 times the postsynaptic selectivity of phentolamine, but with 1/18 the potency of phentolamine to block postsynaptic alpha receptors. PMID- 3012067 TI - (-)-[3H] desmethoxyverapamil labels multiple calcium channel modulator receptors in brain and skeletal muscle membranes: differentiation by temperature and dihydropyridines. AB - The verapamil-like calcium channel modulator, (-)-[3H]desmethoxyverapamil binds to multiple sites in microsomal membrane preparations from brain and skeletal muscle. In brain the Kd values of the sites are 0.55 +/- 0.1 and 61.8 +/- 18 nM for the high and low affinity sites and the maximum binding values are 0.22 +/- 0.04 and 4.6 +/- 1.0 pmol/mg of protein, respectively. Equilibrium analysis of saturation data in skeletal muscle membranes shows only one site with an affinity of 7.2 +/- 0.8 nM and a maximum binding of 2.96 +/- 0.32 pmol/mg of protein. However, a low affinity site with an estimated Kd of 152 nM is indicated in dissociation kinetic studies. Dihydropyridine calcium channel modulators regulate the binding of desmethoxyverapamil in a temperature-dependent fashion with (+)-PN 200110 decreasing (-)-[3H]desmethoxyverapamil binding more at 0 degrees C than at higher temperatures and, at 37 degrees C, enhancing binding in skeletal muscle. The influence of (+)-desmethoxyverapamil on (+)-[3H]PN 200110 binding is unchanged by temperature variations, whereas interactions of the (-)-enantiomer are altered markedly with more inhibition at 0 degrees C than at higher temperatures and, in skeletal muscle, stimulation of binding at 37 degrees C. Dissociation studies indicate that the two sites labeled by (-)-[3H] desmethoxyverapamil in skeletal muscle interact in a negative heterotropic cooperative fashion. Dihydropyridines appear to slow the dissociation of ligand from the low affinity site, whereas diltiazem accelerates the dissociation of (-) [3H]desmethoxyverapamil from the high affinity site. These results suggest that the high and low affinity sites labeled by (-)-[3H]desmethoxyverapamil, respectively, represent the verapamil and diltiazem receptors in brain and skeletal muscle. PMID- 3012070 TI - Malotilate (diisopropyl 1,3-dithiol-2-ylidenemalonate) increases liver de novo purine synthesis and amidophosphoribosyltransferase activity. AB - The effect of malotilate (diisopropyl 1,3-dithiol-2-ylidenemalonate) on de novo purine synthesis was studied in rat liver in vivo and in primary cultured rat hepatocytes in vitro. In the in vivo system, malotilate increased [14C]glycine incorporation by 1.4- to 1.7-fold into acid soluble hepatic purines 4 to 18 hr and the rapidly miscible glycine pool size at least by 2.0-fold 12 hr after administration. Malotilate did not change 5-phosphoribosyl-1-pyrophosphate concentration but increased significantly amidophosphoribosyltransferase (E.C. 2.1.2.14) activity per unit of DNA or per unit of liver protein. Malotilate also increased significantly the hepatic cyclic AMP concentration 2 to 12 hr after administration. In the in vitro system, malotilate increased [14C] formate incorporation into acid soluble purines with accompanying increases in 5 phosphoribosyl-1-pyrophosphate availability and the specific activity of amidophosphoribosyltransferase. These results suggest that malotilate increases the rate of de novo purine synthesis in the liver by its direct action on hepatocytes, the mechanism of this increase is due to the combined increase of amidophosphoribosyltransferase activity and 5-phosphoribosyl-1-pyrophosphate supply and cyclic AMP may play a role for this response. PMID- 3012071 TI - Inhibition of Na+ flux in Chinese hamster ovary cells and Swiss 3T3 cells by a potent new derivative of amiloride, methylisopropyl-amiloride. AB - Amiloride inhibits the mitogen-stimulated Na+ influx of cultured fibroblasts with very low affinity. We have therefore analyzed eight new derivatives of amiloride for their efficacies of inhibiting the Na+ flux in Chinese hamster ovary cells (CHO-K1). Four of these analogs demonstrate markedly enhanced potencies relative to amiloride. One of the derivatives, methylisopropyl-amiloride (MIA), is approximately 900-fold more potent (ID50 = 42 nM) than amiloride in inhibiting Na+ uptake of these cells. Inasmuch as external Na+ ions antagonize amiloride inhibition of Na+ influx competitively, we investigated the ability of MIA to inhibit Na+ flux in the absence of external Na+ ions. The ID50 value determined for MIA inhibition of CHO-K1 cell Na+ efflux from Na+-loaded cells into Na+-free media is 15 nM. We also examined the efficacy of MIA for Na+ efflux inhibition in a nontransformed fibroblast cell line, Swiss 3T3. The ID50 value for inhibition of Na+ efflux from 3T3 cells is comparable to that for CHO-K1 cells. Thus, we have identified a highly potent derivative of amiloride, MIA, that inhibits Na+ flux at nanomolar concentrations in both a transformed and a nontransformed cell line. It is possible that MIA may serve as a useful tool for biochemical characterization of the mitogen-stimulated Na+ transporter and for assessment of the role of this transporter in mitogenesis. PMID- 3012072 TI - Marihuana smoking suppresses luteinizing hormone in women. AB - Smoking a single 1-g marihuana cigarette containing 1.8% delta 9 tetrahydrocannabinol induced a 30% suppression of plasma luteinizing hormone levels (P less than .02) in women during the luteal phase of the menstrual cycle. After marihuana placebo cigarette smoking, no luteinizing hormone suppression was observed in the same women under double-blind conditions. Marihuana may have adverse effects upon reproductive function during the luteal phase of the menstrual cycle as a consequence of gonadotropin inhibition. PMID- 3012073 TI - Baclofen suppresses hippocampal epileptiform activity at low concentrations without suppressing synaptic transmission. AB - Baclofen is used clinically to treat spasticity, but has received little attention as a potential antiepileptic agent. To explore the antiepileptic potential of baclofen further, we tested its effect on stimulus train-induced bursting, an in vitro model of hippocampal epileptiform activity. In hippocampal slices prepared from male rats, extracellular field potentials were recorded in stratum pyramidale of CA3, and electrical stimuli were delivered to s. radiatum of CA3. After stable responses to single stimuli were established, stimulus trains were delivered every 5 min until stable triggered and spontaneous population bursting were elicited. (+/-)-Baclofen was bath-applied to the slices at varying concentrations to study its ability to suppress synaptic transmission and epileptiform activity. EC50 values for suppression of orthodromic population spike amplitude, of triggered burst duration and of spontaneous burst frequency were 2300, 355 and 26.9 nM, respectively; all statistically significantly different. These findings suggest that baclofen suppresses epileptiform electrical activity in the hippocampus at concentrations well below those which suppress normal synaptic transmission, and support renewed consideration of baclofen as an antiepileptic agent. PMID- 3012074 TI - Leukotriene B4 and 20-hydroxyleukotriene B4 contract guinea-pig trachea strips in vitro. AB - In contrast to its established myotropic effect on guinea-pig lung parenchyma, the myotropic action of leukotriene B4 on the trachea is uncertain. Our characterization of its effects on the latter organ indicates that leukotriene B4 contracts guinea-pig trachea zig-zag strips in a concentration-dependent manner from 5 X 10(-9) to 5 X 10(-7) M. Leukotriene B4 was at least 10 times more potent than histamine, but 10 times less potent than leukotriene C4. Similar effects were evident with 20-hydroxyleukotriene B4; however, this metabolite contracted the trachea less forcefully. Tracheas developed tachyphylaxis after cumulative administration of leukotriene B4, but not 20-hydroxyleukotriene B4. The myotropic effect of leukotriene B4 was attributable to an indirect mechanism involving formation of cyclooxygenase metabolites of arachidonic acid. For example, the levels of prostaglandin E2 and prostaglandin F2 alpha released into the incubation medium correlated with the contractile response, and suppression of their biosynthesis with cyclooxygenase inhibitors eliminated that response. We conclude that myotropic effects of leukotriene B4 occur in central airways in addition to peripheral airways. The contribution of leukotriene B4 to tracheal bronchospasm is not necessarily negligible. PMID- 3012075 TI - Inhibition of gastrointestinal transit by morphine in rats results primarily from direct drug action on gut opioid sites. AB - The local intestinal component of the constipating action of morphine was assessed through several integrated approaches in an in vivo animal model. The doses of systemically administered morphine reducing to 50% of drug-free controls (ID50) the small intestinal transit of a charcoal meal fed by gavage to overnight fasted rats were 0.04 and 3.8 mg/kg i.p. and p.o. and 0.5 mg/kg either s.c. or i.v., respectively. Transit inhibition with any of these morphine doses occurred within 10 min and was still measurable 20, 30, 45 and 240 min after i.p., i.v., s.c. and p.o. administration, respectively. Morphine given by any of these systemic routes did not reduce significantly transit in rats receiving the putative peripheral antagonist quaternary naloxone (1 mg/kg i.p., 5 min before morphine) that, unlike naloxone, failed to reverse transit inhibition (to about 50% of drug-free controls) induced by 0.08 mg/kg i.c.v. of morphine. Radioassay of thin-layer chromatograms of extracts of central nervous system, plasma, small intestine and small intestinal longitudinal muscle of rats given tritium-labeled morphine and also tested for gastrointestinal transit, showed that morphine concentrations in the longitudinal muscle (with attached myenteric plexus) after i.v., i.p. and s.c. injection fell within a relatively narrow range and were consistent with the appropriate transit scores. Morphine levels in the central nervous system of the same rats were lower than in any other tissue assayed, presented considerable differences depending on administration routes and did not correlate at all with the corresponding intestinal effects. Morphine administered directly into the rat cerebral ventricles effectively inhibits gastrointestinal transit through an opioid-sensitive central nervous system-located action site.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012076 TI - Responses mediated via beta-1 adrenoceptors but not beta-2 adrenoceptors exhibit supersensitivity after chronic reserpine pretreatment. AB - The purpose of this study was to examine whether reserpine pretreatment induces supersensitivity of both beta-1 and beta-2 adrenoceptor-mediated responses. Guinea pigs received reserpine (0.5 mg kg-1 s.c. or i.p.) daily for 7 days. Isolated tissues were set up in the presence of phentolamine (5 microM) and metanephrine (10 microM) and the sensitivity to isoproterenol and, where possible, a partial agonist (ritodrine, salbutamol or prenalterol) was determined. The beta adrenoceptor-mediated responses were recorded as the increase in rate and tension of right and left atria, inhibition of carbachol induced contractions of ileum, relaxation of aortic spirals contracted with histamine, inhibition of transmurally stimulated vas deferens and relaxation of tracheal spirals and lung strips with intrinsic tone. The atria exhibited supersensitivity after reserpine pretreatment (s.c. and i.p.) as a leftwards shift of the isoproterenol concentration-response curve and elevation of the prenalterol maximum response. The ileum was also supersensitive, but only when tissues from animals receiving i.p. reserpine were compared with shams, which themselves were subsensitive or when reserpine was administered s.c. The trachea was also supersensitive, but not the aorta, lung and vas deferens, the responses of which are mediated via beta-2 adrenoceptors. In contrast, beta-1 adrenoceptors are involved in the atrial, ileal and tracheal responses. Therefore, only responses mediated via beta-1 adrenoceptors exhibited reserpine-induced supersensitivity which supports the hypothesis that beta-1 but not beta-2 adrenoceptors receive a sympathetic innervation. PMID- 3012077 TI - Ontogeny of renal alpha-1 and alpha-2 adrenoceptors in the spontaneously hypertensive rat. AB - Renal alpha-1 and alpha-2 adrenoceptors were characterized during the development of the spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto rats through Scatchard analysis of [3H]prazosin and [3H]yohimbine binding to kidney membrane preparations in an attempt to correlate biochemical changes with the reported functional changes occurring in hypertension development in the SHR. Renal alpha-1 and alpha-2 receptor density was higher in SHR than in Wistar-Kyoto rats at all ages tested. In contrast to Wistar-Kyoto rats, in which the number of alpha-1 and alpha-2 receptors remained relatively constant with age, the number of renal alpha-1 adrenoceptors in the SHR was lowest at the 4th week of age (61 fmol/mg) increasing transiently at 5 weeks and then again at 8 week reaching a plateau at that time. The maximum number of binding sites for [3H]yohimbine binding in the SHR was also age dependent. The number of renal alpha-2 adrenoceptors in the SHR was lowest at 4 weeks of age (125 fmol/mg) increasing to 220 fmol/mg at 5 weeks of age and to 260 fmol/mg at 8 weeks of age. Adult levels (308 fmol/mg) were reached by 18 weeks of age. Unlike receptor densities, affinity constants were not significantly altered during postnatal development. The changes in alpha-1 and alpha-2 adrenoceptors in the kidneys of SHR may suggest an important developmental role which is not yet understood. PMID- 3012078 TI - Effect of cholera toxin and pertussis toxin on opioid tolerance and dependence in the guinea-pig myenteric plexus. AB - Opioid tolerance and dependence are characterized in terms of inhibitory and excitatory neuronal mechanisms. Bacterial toxins were used to investigate these phenomena in the isolated guinea-pig ileum (GPI) and mouse vas deferens (MVD). Cholera toxin (CT) and pertussis toxin (islet activating protein; IAP) have been demonstrated to selectively impair the function of either the stimulatory or inhibitory nucleotide regulatory protein, mediating the signal transmission to the adenylate cyclase. It has been found that CT failed to affect electrically evoked twitch tension in naive and tolerant GPI and MVD. Twitch tension evoked by excitatory drugs, e.g. neurotensin, was attenuated in the GPI, but not in the MVD. CT did not interfere with the actions of opioids or nonopioids in either the GPI or the MVD. Similarly, IAP failed to affect electrically evoked twitch tension. It virtually lacked an effect on the inhibitory action of opioids and nonopioids on electrically stimulated naive and tolerant GPI and MVD. On the other hand, both CT and IAP dose-dependently attenuated the naloxone precipitated withdrawal contracture in the GPI rendered opioid-dependent. These findings may suggest that opioid receptors at nerve terminals of the GPI and the MVD are not linked to adenylate cyclase, and those receptors relate to the state of tolerance. In contrast, opioid receptors located at nerve somata may be linked to adenylate cyclase. Those receptors may be associated with the development of dependence. The experiments with CT suggest a synaptic excitatory input of neighboring nerve elements necessary for the generation of a withdrawal contracture. PMID- 3012079 TI - [Radiologic surveillance of irradiated glomal tumors]. AB - A favorable clinical result was obtained in 10 patients with jugular glomal tumors treated by external radiotherapy (approximately 50 Gy), alone or combined with surgical treatment, and preceded in some cases by embolization. Radiologic review examinations showed complete stability of lesions without bone reconstruction phenomena, in spite of a decrease in tympanic mass and regression of functional symptoms. PMID- 3012080 TI - Differential regulation of two molecular forms of a mu-opioid receptor type by sodium ions, manganese ions and by guanyl-5'-yl imidodiphosphate. AB - The rabbit cerebellum contains a very high proportion (up to 80%) of mu-opioid receptor sites (Meunier, J.C., Kouakou, Y., Puget, A. and Moisand, C., Mol. Pharmacol. 24, 23-29, 1983). A membrane fraction derived therefrom is labeled either with the opioid agonist, 3H-etorphine or with the opioid antagonist, 3H diprenorphine, and solubilized with digitonin. Centrifugation of the soluble extracts in linear sucrose gradients reveals that bound 3H-etorphine sediments faster than does bound 3H-diprenorphine: 12S vs 10S. Pre-incubation of membranes and radioligand in the presence of 120 mM NaCl results in considerably decreased recovery of the 3H-agonist in 12S form while recovery of the 3H-antagonist in 10S form is substantially increased. The opposite situation is observed when the membranes have been prelabeled with radioligand in the presence of 1 mM MnCl2. Guanyl-5'-yl imidodiphosphate, a metabolically stable structural analog of GTP is found to selectively reduce recovery of labeled 12S receptors while it does not affect that of labeled 10S receptors. These data indicate that the mu-opioid receptor from rabbit cerebellum is capable of existing in two forms which differ in apparent molecular size: an "antagonist" (10S) form of apparent Mr approximately 230,000 which is stabilized in the presence of sodium ions and an "agonist" (12S) form of apparent Mr approximately 300,000 which, unlike the antagonist one, is sensitive to guanyl-5'-yl imidodiphosphate. It is thought that the form of larger apparent size represents the mu-opioid receptor associated with a guanine nucleotide regulatory protein. PMID- 3012081 TI - Is there a common, high-affinity opioid binding site in rat brain? AB - At low concentrations, 3H-naloxone apparently bound to two sites, of high (KD 0.50 nM) and low (KD 2.0 nM) affinity. Binding to the high affinity site was preferentially blocked by naloxonazine. This is consistent with the high and low affinity sites representing the mu 1 and mu 2 sites respectively. Binding of 3H naloxone to the mu 1 and mu 2 sites was differentially inhibited by opioids. Compared to mu 2 binding, DADLE and DAGO preferentially inhibited mu 1 binding. DADLE inhibited the binding of 3H-DAGO potently and in a competitive manner. DAGO inhibited the binding of 3H-DADLE from two sites for which DAGO had high and low affinities. Scatchard analysis indicated that both 3H-DAGO and 3H-DADLE bound to one class of sites, with 3H-DADLE having a 2-3 fold greater Bmax. It is concluded that 3H-opioids bind to at least three sites--mu 1, mu 2 and delta. The mu 1 site represents a high affinity binding site for both opioid peptides and opioid alkaloids. DAGO is a selective ligand for the mu 1 site, whilst DADLE interacts with mu 1 and delta sites with similar affinities. PMID- 3012082 TI - Prenatal diagnosis of a congenital mesoblastic nephroma. A case report. AB - Fetal urinary tract malformations are diagnosed frequently with ultrasonography. There are many published papers on in utero detection of fetal cystic abnormalities. In this case the prenatal diagnosis of a solid kidney mass was made, and histopathologic examination after surgical resection revealed congenital mesoblastic nephroma. PMID- 3012083 TI - Documentation of subclinical trophoblastic embolization with invasive cardiac monitoring in a woman with a molar pregnancy. A case report. AB - Patients presenting with hydatidiform mole are at risk of developing trophoblastic deportation. This clinical syndrome is seen more frequently in patients with uteri greater than 16 weeks in size and/or larger than expected for the gestational age. A pulmonary artery catheter was inserted in a patient, demonstrating the recovery of trophoblast from the pulmonary artery. This invasive technique can aid in the differential diagnosis and precise administration of intravenous fluid in this clinical situation. PMID- 3012084 TI - Indole-phenol bioisosterism. Synthesis and antihypertensive activity of a pyrrolo analogue of labetalol. AB - The synthesis of 5-[hydroxy-2-[(1-methyl-3-phenylpropyl)amino]ethyl]-1H-indole-7- carboxamide, 5, a pyrrolo analogue of labetalol, is described. Compound 5 was found to reduce blood pressure in spontaneously hypertensive rats with an ED50 of 5 mg/kg po, without causing any decrease in heart rate. Isolated tissue studies with 5 shows that it is a nonselective beta-adrenoceptor antagonist and that it is a weaker alpha-adrenoceptor antagonist with a relative selectivity for alpha 1 receptors. Additionally, the compound displayed significant beta-adrenoceptor intrinsic sympathomimetic activity. Evidence is presented that the beta adrenoceptor antagonist and agonist properties of 5 are mediated via hydrogen bond formation with the receptor. PMID- 3012085 TI - Probes for narcotic receptor mediated phenomena. 12. cis-(+)-3-Methylfentanyl isothiocyanate, a potent site-directed acylating agent for delta opioid receptors. Synthesis, absolute configuration, and receptor enantioselectivity. AB - The first enantiomeric pair of irreversible opioid ligands [(+)- and (-)-4] were synthesized in greater than 99.6% optical purity as determined by HPLC analysis of diastereoisomeric derivatives of the intermediate 3-methyl-N-phenyl-4 piperidinamine enantiomers. Single-crystal X-ray analysis of the (R,R)-L-(+) tartaric acid salt of (-)-9 revealed the absolute configuration to be 3S,4R. The absolute configuration of (-)-3 [cis-(-)-3-methylfentanyl] and (-)-4 derived from (-)-9 is thus 3S,4R and that of (+)-3 and (+)-4 is 3R,4S. The (+) enantiomer of 4 (SUPERFIT) was shown to be highly potent and specific for acylation of delta opioid receptors (to the exclusion of mu) in rat brain membranes like its achiral prototype FIT and was about 10 times as potent as the latter in this assay. The (+)-4 was about 5 times as potent as FIT in acylation of delta receptors in NG108 15 neuroblastoma X glioma hybrid cells and about 50 times as potent as its enantiomer. Both FIT and (+)-4 behaved as partial agonists in inhibition of delta receptor coupled adenylate cyclase in NG108-15 membranes and (+)-4 was 5-10 times more potent than FIT and about 100 times more potent than its enantiomer in this assay. Dibromination of amine 12, catalytic exchange of bromine with tritium gas, and reaction of the labeled amine with thiophosgene afforded [3H]-(+)-4 with a specific activity of 13 Ci/mmol. Previous experiments indicated (+)-4 acylates the same 58 000-dalton glycoprotein previously shown to be acylated by FIT but with less nonspecific labeling. In view of the high potency and specificity of (+)-4 and the availability of its enantiomer, it seems likely that these compounds will prove to be valuable tools for study of the opioid receptor complex. PMID- 3012086 TI - Dog coronary artery adenosine receptor: structure of the N6-aryl subregion. AB - Previous structure-coronary vasoactivity correlations of the N6-alkyladenosine analogues of N6-[(R)-1-phenyl-2-propyl]adenosine, 1, support the hypothesis that the coronary artery A2 adenosine receptor contains an N6 region of specialized structure. The part of this receptor region that binds the 2-propyl moiety of 1 determines stereoselectivity and contributes to coronary vasoactivity. The present study uses 92 adenosine analogues containing an aryl group in the N6 substituent to test the hypothesis that the N6 receptor region contains an aryl subregion that binds the phenyl moiety of 1 and thereby contributes to its coronary vasoactivity. N6-Aralkyladenosines are often more potent than their alkyl congeners. Two methylene residues seem to provide optimum separation of the aryl group from N6. Among adenosines with semirigid N6 substituents, N6-[(1R,2S) trans-2-phenylcyclohexyl]adenosine was uniquely active, evidence that when 1 occupies the receptor, the axis of the propyl C-1 to phenyl C-1 bond is nearly in the plane described by N6 and propyl C-1 and C-2. The torsion angle around this bond is unknown. Replacing the phenyl group of N6-2-phenethyladenosine with a thienyl or a 3-pyridyl group raises activity. The structure-activity relationships of the N6-(arylethyl)-, the N6-(arylmethyl)-, and the N6 phenyladenosines differ strinkingly from each other. Taken together, such results support the idea that the N6 region of the dog coronary artery A2 adenosine receptor includes an aryl subregion. PMID- 3012087 TI - Cardiac glycosides. 6. Gitoxigenin C16 acetates, formates, methoxycarbonates, and digitoxosides. Synthesis and Na+,K+-ATPase inhibitory activities. AB - A series of 17 gitoxigenin 16 beta-formates, acetates, and methoxycarbonates was synthesized, including their 3 beta-acetates, formates, and digitoxosides. A 16 beta-formate group was generally found to increase activity 30 times, a 16 beta acetate group 9-12 times, while a 16 beta-methoxycarbonate decreased activity by two-thirds. 3 beta-Formates and acetates had little effect on activity by themselves, but sometimes reduced the activity-increasing properties of 16 beta formates and acetates. A 3 beta-digitoxoside increases the activity of gitoxigenin by 15 times, but the effect is less if the 16 beta-group is esterified. And finally, a 16-one decreases activity dramatically. These data suggest an important role for C16 esters and possibly the presence of a separate binding site on Na+,K+-ATPase corresponding to the cardenolide C16 position. PMID- 3012088 TI - A new epsilon globin HincII variant fragment length in a South African Negroid family. AB - A new HincII epsilon globin variant is reported in a South African Negroid family. The usual HincII epsilon globin fragment lengths are 8.0 and 3.7 kb and the variant described here is 14.0 kb in length. The 14.0 kb fragment was generated by a site alteration removing the 3' HincII site on a chromosome that already lacked the 5' HincII site. It appears that there are differences among races with regard to the frequencies of the 8.0 and 3.7 kb alleles. The 3.7 kb allele is the more common form in Caucasoids whereas the 8.0 kb allele occurs in the majority of Negroid and Khoisan subjects. PMID- 3012089 TI - A highly reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Bombyx mori. AB - A library of low Cot DNA (Cot is the molar concentration of DNA times the incubation time in seconds) from Bombyx mori was used to isolate five independent clones of highly reiterated sequences from the genome of this organism. Sequence analysis revealed that all five clones belong to a single family of repetitive DNA elements, which we have named Bm1, and whose reiteration frequency is approximately 2.3 X 10(4) copies per haploid genome. Probing of a Bombyx genomic library (in lambda phage) with a Bm1 clone reveals that this repetitive sequence is dispersed throughout the genome. The pattern of interspersion was confirmed by Southern blot mapping of a large (270 X 10(3) base-pairs) domain of the chorion locus of Bombyx, where at least 13 independent regions were found to hybridize to Bm1. Four additional Bm1 elements have been sequenced from a 4.8 X 10(3) base pair genomic fragment containing an early chorion gene. Two of these four elements are bounded by short (4 to 12 base-pairs) direct repeats. The nine Bm1 elements which have been sequenced are greater than 88% homologous to each other, and tend to fall in at least two size classes (253 base-pairs and 450 base pairs). Seven of the nine Bm1 elements have a short 6 to 10 base-pair oligo(A) sequence at the 3' end. A sequence of about 29 base-pairs at the 3' end, including the oligo(A), shows 86% homology to the equivalent 3'-terminal domain of human Alu family repetitive elements. A 129 base-pair domain at the 5' end of Bm1 shows 66% homology to a Drosophila valine transfer RNA gene; thus the 5' end of Bm1 may contain the split internal RNA polymerase III promoter that is characteristic of most transcribed tRNA-like retroposons. Dot-blot analysis of Bombyx RNA shows that Bm1 DNA is indeed transcribed, and that the transcripts are well-represented in the total RNA of an ovarian-derived permanent cell line and posterior silk glands early in the fifth instar, but are less abundant in the RNA of pupae or silk glands late in the fifth instar. PMID- 3012090 TI - Organization and evolution of a higher plant alphoid-like satellite DNA sequence. AB - Two recombinant plasmids containing, respectively, three and eight tandem repeats of a 177 base-pair (bp) element from radish nuclear DNA have been isolated. These plasmids were used as probes to investigate the organization and the copy number of this element within the genome. This sequence is present in congruent to 0.6 million copies. Restriction analysis provides evidence for sequence heterogeneity and reveals the occurrence of non-overlapping subfamilies. Nine units were sequenced and found to be remarkably conserved. However, sequences in the two clones clearly belong to two distinct subgroups. Our data suggest that these sequences evolved in a concerted manner and that homogenization mechanisms such as gene conversions certainly took place. The 177 bp sequence is made from three 60 bp blocks that are derived from a common ancestor. Exchanges between the three blocks probably occurred before they became fixed as a patchwork of short sequences, the 177 bp element. This unit of 177 bp was then amplified in several steps. The presence of such a repeated sequence can be detected in other Cruciferae when hybridizations are carried out under low stringency conditions. Direct comparison with a previously published mustard satellite DNA sequence indicates a similar organization and a 75% homology. Homology was also found with shorter regions (congruent to 60 bp) of broad bean and corn satellite DNA. Finally, homology was also found with several animal alphoid sequences, suggesting that this family also occurs in the plant genomes. PMID- 3012091 TI - Complete nucleotide sequences of the nuclear pseudogenes for cytochrome oxidase subunit I and the large mitochondrial ribosomal RNA in the sea urchin Strongylocentrotus purpuratus. AB - Nucleotide sequencing of the sea urchin nuclear genomic homologues of two mitochondrial genes, cytochrome oxidase subunit I (COI) and 16 S ribosomal RNA, shows clearly that they are both pseudogenes. The COI homologue has accumulated numerous single-base changes causing non-conservative amino acid substitutions, as well as many small insertions and deletions, most of which result in frameshifts. There is no continuous open reading frame and eight unmutated TGA codons persist. A genomic repetitive element is found between the break points of two rearrangements that have occurred in the region. By solution hybridization in RNA excess, we were unable to detect transcripts colinear with the complete nuclear COI sequence, using Strongylocentrotus purpuratus gastrula RNA, at a detection limit of 10(-6) of total RNA. Transcripts restricted to the 3' end of the COI pseudogene may be present, however, but at an extremely low level. Comparison of the 16 S/COI junctions in nuclear and mitochondrial DNA suggests a possible complementary DNA-mediated conversion of the 16 S pseudogene subsequent to its original transposition into nuclear DNA. We have estimated the likely age of the nuclear sequence element from the divergence between nuclear and mitochondrial sequences and from cross-hybridization with the genomes of other sea urchin species. With both methods, an age of more than 30 million years is suggested. PMID- 3012092 TI - Dosage compensation at the sgs4 locus of Drosophila melanogaster. AB - The X-linked sgs4 gene of Drosophila melanogaster encodes a salivary glue protein. Non-dosage-compensated alleles of this gene have been described by G. Korge, in which males accumulate only about half of the Sgs4 polypeptide as do females. We show that the Sgs4 mRNA levels in dosage-compensated and non-dosage compensated strains parallel the levels of Sgs4 polypeptide. Korge's genetic analysis of one of the non-dosage compensated alleles suggests that dosage compensation is controlled by sequences that lie 5' to the coding region. Sequences important for regulating the absolute levels of transcription of sgs4 have been identified as residing within a region of 600 base-pairs 5' to the site of transcription initiation. We have sequenced this region from two non-dosage compensated strains and find no significant deviation from the sequence of dosage compensated alleles. We therefore conclude that sequences that act to mediate the dosage compensation of sgs4 must lie outside this 600 base-pair region. PMID- 3012094 TI - Physical state, expression and regulation of two glucocorticoid-controlled genes on bovine papilloma virus vectors. AB - We have studied the extrachromosomal maintenance and the transcription regulation of two glucocorticoid-inducible genes on bovine papilloma virus (BPV) vectors in c127 mouse fibroblasts. These genetic elements were the rat tryptophan oxygenase (TOase) gene promoter, which is active in vivo only in hepatocytes, and the long terminal repeat of the mouse mammary tumor virus (MMTV-LTR). From both genes, fusions of the 5'-flanking region of the transcription unit to the bacterial gene for chloramphenicol acetyltransferase (CATase) were constructed. These fusion genes were inserted either into pCGBPV9, a BPV vector encoding G418 resistance, into pBPV-BV1, a vector containing "stabilizing" segments of the human beta globin gene, or into a BPV construct, whose bacterial plasmid sequences could be removed before transfection. Five constructs of the two latter groups, selectable in c127 cells only as foci, were normally maintained in the extrachromosomal state. In contrast, three out of five constructs based on pCGBPV9 and selectable for resistance against G418 were maintained in a high molecular weight form, most probably of intrachromosomal concatemeric nature, while the remaining two G418 resistant constructs appeared alternatively in this or the extrachromosomal monomeric form. In contrast to its absence of expression in fibroblasts in vivo, the TOase gene element present on BPV vectors was found to be active in fibroblasts in these transfection experiments. As judged by CATase activities and for TOase also by mapping of the transcription start sites, transcription of both genes was under hormonal regulation. All BPV vectors proved to be useful tools in the study of these regulated genes, and in only one out of ten constructs was regulation atypical, possibly due to effects from flanking vector sequences. PMID- 3012093 TI - High frequency DNA rearrangements associated with mouse centromeric satellite DNA. AB - A DNA transformed mouse cell line, generated by the microinjection of a pBR322 plasmid containing the herpes thymidine kinase (tk) gene, was observed to exhibit a high frequency of DNA rearrangement at the site of exogenous DNA integration. The instability in this cell line does not appear to be mediated by the tk inserts or the immediately adjacent mouse DNA, but instead may be a consequence of the larger host environment at the chromosomal site of tk insertion. Results obtained from restriction analysis, in situ chromosome hybridizations, and cesium chloride density-gradient fractionations indicate that the tk inserts are organized as a single cluster of direct and inverted repeats embedded within pericentromeric satellite DNA. To determine the molecular identity of the flanking host sequences, one of the mouse-tk junction fragments was cloned, and subsequent restriction and sequence analyses revealed that this DNA fragment consists almost entirely of classical mouse satellite DNA. On the basis of these observations, we suggest that the instability in this cell line may reflect the endogenous instability or fluidity of satellite DNA. PMID- 3012095 TI - Rate of treadmilling of actin filaments in vitro. AB - Actin filaments capped at the barbed ends were formed by polymerizing monomeric actin onto a gelsolin-actin complex. The rate of depolymerization and polymerization of the pointed ends was determined by diluting gelsolin-capped actin filaments into various concentrations of monomeric actin. Under the conditions of the experiments (100 mM-KCl, 2 mM-MgCl2 at 37 degrees C) the rate constant of dissociation of subunits both from a shortening and a lengthening filament was found to be 0.21 s-1. As the rate of dissociation of subunits from the slow pointed end determines the rate of treadmilling, it is concluded that actin filaments treadmill with a rate of about 2 micron/h. PMID- 3012097 TI - Template-directed synthesis on the oligonucleotide d(C7-G-C7). AB - When the deoxynucleotide template d(C7-G-C7) is incubated with the activated nucleotides 2-MeImpG and 2-MeImpC, a series of oligomers of G up to the sevenmer and a series of copolymers of composition GnC with n = 3 to 13 are formed. Oligomers GnC with n greater than 7 are completely degraded by pancreatic ribonuclease, establishing that they contain a 3' to 5' internucleotide bond between 5'-C and 3'-G within a sequence of the form (pG)ipC(pG)j. As expected, (pG)7-Cp and (pG)6-Cp are major hydrolysis products. Detailed analysis of the product distribution shows that a substantial fraction of the oligomeric products are of the type (pG)ipC(pG)j with i less than 7. This shows that product synthesis does not necessarily begin at the 3' terminus of the template. The significance of this finding in terms of the origin of molecular replication is discussed. PMID- 3012096 TI - Structure of bovine papillomavirus type 1 DNA in a transformed mouse cell line. AB - Linearized bovine papillomavirus type 1 (BPV-1) DNA was introduced into mouse C127 cells, where it recircularized and replicated as an intact monomeric, extrachromosomal circular form in the resulting transformants. These cells contained a mixture of complex high molecular weight forms that were converted to a linear form of approximately BPV-1 size upon digestion with an enzyme that cuts once within the BPV-1 genome. Further analysis of one of these cell lines revealed that these high molecular weight forms consisted of two components. One was detected on agarose gels as a diffuse smear of slow-migrating material representing linear forms that were tightly associated with host chromosomes, probably by integration. The second component was composed of discrete-sized oligomeric open and supercoiled extrachromosomal circular forms of up to approximately 48 X 10(3) base-pairs (6 tandemly linked BPV-1 genomes) in size. No catenated (interlocked) forms could be detected. PMID- 3012098 TI - Dihydrofolate reductase. 1H resonance assignments and coenzyme-induced conformational changes. AB - Lactobacillus casei dihydrofolate reductase has been studied in solution by one and two-dimensional 1H nuclear magnetic resonance (n.m.r.) spectroscopy at 500 MHz. By using a combination of n.m.r. methods in conjunction with the crystal structure of the enzyme-methotrexate-NADPH complex, resonances have been assigned for 32 of the 162 residues of the enzyme. These are widely distributed throughout the structure of the protein, and include all the histidine and tyrosine residues, as well as several valine, leucine, isoleucine and phenylalanine residues. The assignments have been made for the enzyme-methotrexate and enzyme methotrexate-NADP+ complexes as well as the enzyme-methotrexate-NADPH complex. Comparison of assigned resonances in the spectra of the three complexes has permitted a preliminary assessment of structural differences between them. The beta-sheet "core" of the protein is unaffected by coenzyme binding, but two regions of the structure that undergo coenzyme-induced conformation changes have been identified. These are the loop comprising residues 13 to 23, and alpha-helix C (residues 42 to 49). PMID- 3012099 TI - Prevention of ventricular fibrillation by metoprolol in a pig model of acute myocardial ischaemia: absence of a major arrhythmogenic role for cyclic AMP. AB - Absence of a Major Arrhythmogenic Role for Cyclic AMP. Journal of Molecular and Cellular Cardiology (1986) 18, 375-387. We examined the mechanism whereby beta adrenoceptor antagonism exerts an antiarrhythmic effect in early myocardial ischaemia. Ligation of the anterior descending coronary artery in the anaesthetized open-chest pig resulted in severe transmural anteroseptal ischaemia. Blood flow in the mid-ischaemic zone 20 min after ligation was decreased to 5.7 +/- 0.7% of the preligation control value. Epicardial ST-segment deflections of 6.7 +/- 0.4 mV were recorded over this zone. A distinct phase of ventricular arrhythmias was evident about 10 to 30 min after ligation. A high incidence of ventricular fibrillation (14/16 pigs) was associated with a circumstantial increase in levels of cyclic AMP in ischaemic tissue. Twenty minute values were: 1.10 +/- 0.06, P less than 0.05 v. the non-ischaemic tissue level of 0.86 +/- 0.05 nmol/g. Propranolol 3 mg/kg IV, metoprolol 20 mg/kg IV or sotalol 10 mg/kg IV were given between 30 min prior to and 10 min after ligation. Adequate beta-adrenoceptor antagonism by each agent could be proven. Metoprolol decreased the incidence of ventricular fibrillation (2/13, P less than 0.0005 v. control group), while propranolol or sotalol did not. All three beta-antagonists decreased tissue levels of cyclic AMP prior to ligation. However, the temporary increase in ischaemic tissue after ligation could not be prevented. Furthermore, cyclic AMP in ischaemic tissue 20 min after ligation was higher in the metoprolol group than in the propranolol or sotalol group (0.94 +/- 0.04 v. 0.81 +/- 0.02 P less than 0.05, and 0.79 +/- 0.03 nmol/g P less than 0.01, respectively). Blood flow in the mid-ischaemic zone of the metoprolol group was increased to 8.6 +/- 0.6% of preligation control value (P less than 0.0001 v. control group). In contrast, blood flow in the mid-ischaemic zone of the propranolol or sotalol group was decreased. Metoprolol also reduced epicardial ST-segment deflections over the mid-ischaemic zone to 3.5 +/- 0.2 mV (P less than 0.0001 v. control group). ST-segment deflections in the propranolol group were increased. The mechanism whereby metoprolol prevented ventricular fibrillation may be explained by a decrease in the severity of ischaemia but not in terms of changes of tissue levels of cyclic AMP. PMID- 3012100 TI - Cure rates in small cell and non-small cell carcinoma of the lung utilizing high dose radiotherapy and chemotherapy. AB - From 1967 to 1977, 72 patients with small cell carcinoma of the lung were seen. Thirty-five of these patients had unilaterally localized lesions (limited disease) and were treated with cobalt 60 radiation therapy (6,000 rad in six weeks) followed by chemotherapy consisting of cyclophosphamide (Cytoxan), vincristine, methotrexate and lomustine (CCNU) (Group A). The remaining 37 patients with extensive disease were treated with similar chemotherapy alone, or in combination with local palliative radiotherapy to the symptomatic area (Group B). For Group A the five-year survival rate was 20 percent, while for both groups combined, it was only 5 percent.During this same period 560 patients with non small cell carcinomas were treated. The five-year survival rate for those patients with operable, resectable lesions was 33 percent, while for those with unilateral, inoperable, unresectable lesions, it was 10 percent. Thus, it would appear that the results in limited small cell and non-small cell carcinomas of the lung utilizing high-dose radiotherapy followed by chemotherapy are comparable, and that limited small cell carcinoma of the lung patients with high dose radiotherapy followed by chemotherapy can survive longer than those patients with stage III, non-small cell lung carcinoma.While the two- to five-year survival rates in small cell carcinoma demonstrate no appreciable differences, in non-small cell carcinomas there are significant two- to five-year survival differences. These improved results probably are due to the increased sensitivity of small cell carcinoma to high-dose local radiotherapy and to the chemotherapeutic vulnerability of circulating and microscopic metastatic cancer cells. PMID- 3012101 TI - Bipyridylium herbicide toxicity in vitro: comparative study of the cytotoxicity of paraquat and diquat toward the pulmonary alveolar macrophage. AB - In vitro exposure of adult rat alveolar macrophages to either paraquat or diquat resulted in concentration dependent cytotoxicity (cell death). The herbicide paraquat, however was statistically significantly more potent toward these cells than was diquat. The LC50 value for paraquat (8-h exposure, 37 degrees C) was determined to be 0.94 mM [95% confidence interval (C.I.) 0.79-1.12 mM], whereas the corresponding LC50 value for diquat was 1.97 mM (C.I. 1.58-2.51 mM). Interestingly, diquat was shown to enter these cells to a much greater extent than was paraquat. The latter data, while seemingly contradictory to the above findings, is consistent with other reported findings in this study that show that cell respiration, as measured by loss of oxygen consumption, was more sensitive to diquat than it was to paraquat. Also, only paraquat cytotoxicity was found to be dependent on oxygen tension and could be altered by the presence of antioxidant enzymes in the culture medium. Both compounds, however, were found to be equipotent toward purified mitochondria. Both paraquat and diquat were able to uncouple oxidative phosphorylation and induce active oxygen species (superoxide anions and hydrogen peroxide) from this organelle. It is concluded that free radical pathology is the most likely mechanism of action by which paraquat is cytotoxic toward these cells, but that diquat poisoning probably originates from some other mode of action. PMID- 3012102 TI - Pulmonary deposition and effects of inhaled silica particles after short-term exposures in the rat. AB - The present study was designed to evaluate the pulmonary deposition and the effects of inhaled silica particles in the rat model. Wistar (W/M strain) rats were exposed to silica aerosols generated from a fluidized bed dust generator for 1 hr a day, intermittently for 6 days, using a "nose-only" inhalation chamber. After the cessation of the exposures, analysis of lavaged bronchoalveolar cells (BAC) and histological examinations of lungs and tracheobronchial lymph nodes (TBLN) were performed during a period of 6 months. Total cell yields and the proportions of alveolar macrophages (AM) in BAC were not altered, whereas the proportions of lymphocytes in BAC were significantly increased in the exposed animals. Although the proportions of silica-laden AM in BAC were gradually decreased during the 6 months, particle-laden AM were predominantly and persistently observed in the alveoli under light microscopy. Silica particles were also identified in macrophages of granulomatous nodules in pulmonary peribronchial lymphoid tissues(PBLT) and TBLN, indicating the translocation of particles via the lymph. Associated with pulmonary particle deposition, some characteristic histopathological features were evident, including thickening of alveolar duct bifurcations and lymphocyte infiltrations both in the alveolar sacs and around the interstitial blood vessels. At later months after the exposures, the alveolar interstitium was thickened with the increase of fibroblasts and collagen. These results implicate that short-term exposures of silica particles in the rat can evoke histopathologic changes in the lungs and lymphatic tissues, associated with their deposition and translocation. PMID- 3012103 TI - Confronting cisternae in human hepatocellular carcinoma. AB - The Authors report the presence of confronting cisternae (CC) in two cases of poorly differentiated human hepatocellular carcinoma. They are interpreted as a peculiar phenotypical alteration of the tumor cells, probably related to a disturbance of the morphogenesis of nuclear and endoplasmic membranes. PMID- 3012105 TI - Mitochondrial abnormalities in hepatocytes adjacent to fibrolamellar hepatocellular carcinoma. PMID- 3012104 TI - Effects of fenvalerate on enzymes of rat liver cell membranes and microsomes. PMID- 3012106 TI - [Response characteristics in the cortical taste cells as elicited by taste stimuli]. PMID- 3012107 TI - [The role of endogenous amines in anaphylactic shock in rats]. PMID- 3012108 TI - Immune status to congenital infections by TORCH agents in pregnant Saudi women. PMID- 3012109 TI - Novel crystalline sheets of Na,K-ATPase induced by phospholipase A2. AB - Treatment of purified preparations of Na,K-ATPase by phospholipase A2 has led to the formation of two-dimensional crystals of the protein. Control tests with another phospholipase and two detergents have shown that crystallization occurs as the result of hydrolysis and/or solubilization of the phospholipids in the enzyme vesicles. Experimentation with various buffer systems has indicated that reduction in the amount of phospholipids alone is sufficient for inducing the formation of crystalline sheets. Inclusion of crystal inducing ions in the buffer facilitates the crystallization process, resulting in more extensive arrays. The new crystalline sheets are exclusively dimeric with average unit cell dimensions: a = 15.8 +/- 0.4 nm, b = 4.9 +/- 0.2 nm, and gamma = 64 +/- 3 degrees. Examination of the micrographs shows that the initial intermolecular interaction leading to the formation of sheets is between the alpha subunits. Results from this study suggest that removal and/or modification of phospholipids by phospholipases could prove successful in crystallizing those membrane proteins in which excess lipid is the main barrier to the formation of two-dimensional arrays. PMID- 3012110 TI - Localization of the v-rel protein in reticuloendotheliosis virus strain T transformed lymphoid cells. AB - The protein (p59rel) encoded by the transforming gene of reticuloendotheliosis virus strain T (REV-T) has been identified in REV-T-transformed avian lymphoid cells by using antisera raised against synthetic peptides whose sequences were derived from three nonoverlapping regions of v-rel (N. R. Rice, T. D. Copeland, S. Simek, S. Oroszlan, and R. V. Gilden, Virology 149:217-229, 1986). To obtain polyclonal antibodies directed against a larger number of p59rel epitopes, a 262 amino acid segment was expressed in bacteria. Antisera raised against this fusion protein (v-delta-rel) precipitated p59rel from lysates of [35S]methionine-labeled REV-T-transformed cells, thus confirming previous results obtained with the peptide antisera. We used this new antiserum to localize p59rel in REV-T transformed cells by subcellular fractionation using differential centrifugation and by indirect immune fluorescent staining. After fractionation and immune precipitation, the majority of p59rel was found in the cytosolic fraction. Indirect immunofluorescence experiments also gave results consistent with the cytoplasmic localization of the v-rel protein in transformed lymphoid cells. In previous studies (Rice et al., Virology 149:217-229, 1986) it was shown that immune precipitates formed with one of the three p59rel peptide antisera possessed in vitro protein kinase activity. Immune precipitates formed with the fusion protein antiserum also showed kinase activity in the in vitro assay. Most of this activity was found in the soluble cytoplasmic fraction, indicating that the kinase may be p59rel or a protein closely associated with it. PMID- 3012111 TI - Recent clinical isolates of cytomegalovirus suppress human cytomegalovirus specific human leukocyte antigen-restricted cytotoxic T-lymphocyte activity. AB - Peripheral blood mononuclear cells harvested from healthy adults seropositive for human cytomegalovirus (HCMV) and cultured with laboratory strain AD-169 demonstrated human leukocyte antigen-restricted and HCMV-specific killing on target cells infected with either HCMV laboratory strain AD-169 or recent low passage HCMV isolates. These results indicated that the determinants recognized by cytotoxic T lymphocytes (CTLs) are shared among different strains of HCMV. However, when low-passage isolates, rather than high-passage AD-169 virions, were used to stimulate CTL activity, the lytic response was significantly lower against all targets. Mixing of AD-169 and low-passage HCMV isolates induced low CTL activity. Collectively, the findings suggest that low-passage HCMV isolates have dual effects--antigenic stimulation and immunosuppression--whereas laboratory strain AD-169 is primarily immunogenic. The study of several recent isolates indicated that they varied in their ratio of immunostimulation to suppression, that infectious virus was necessary to produce suppression, and that suppressive isolates did not have to be present at the initiation of culture to exert their suppressive effects. PMID- 3012112 TI - Identification with monoclonal antibodies of virus-specific DNA-binding proteins in the nuclei of cells infected with three serotypes of Marek's disease virus related viruses. AB - Two groups of virus-specific polypeptides were identified in the nuclei of infected cells by cross-reacting monoclonal antibodies with three serotypes of Marek's disease virus. Of these, a 135,000-molecular-weight polypeptide common to all three serotypes was found to bind to both double-stranded and single-stranded DNAs. PMID- 3012113 TI - Direct detection of exogenous mouse mammary tumor virus sequences in lymphoid cells of BALB/cfC3H female mice. AB - The presence of exogenous mouse mammary tumor virus (MMTV) (C3H) DNA sequences in lymphoid tissue (spleen, bone marrow, and thymus) and nonlymphoid tissue (liver and kidney) of BALB/cfC3H female mice was directly assessed by DNA hybridization methods. Lymphoid tissues were found positive for integrated MMTV(C3H) sequences in females as young as 4 weeks. In most samples, the level of splenic MMTV(C3H) infection was low (2 to 5%). Infection remained throughout the life of the animal. The percentage of spleen samples found positive for exogenous viral infection was significantly higher in females bearing mammary tumors, whether virgin or multiparous. Liver and kidney DNAs were negative for exogenous MMTV sequences, suggesting tissue type selectivity in MMTV infection. PMID- 3012115 TI - Protective effect of monoclonal antibodies on lethal mouse hepatitis virus infection in mice. AB - Neutralizing and nonneutralizing monoclonal antibodies to the peplomer glycoprotein and nucleocapsid protein of a mouse hepatitis virus (MHV), MHV-NuU, protected mice against lethal MHV-2 challenge. Histopathologically, livers of mice receiving protective antibodies showed some focal necrotic lesions with remarkable cellular infiltration instead of fulminant hepatitis caused by MHV-2. PMID- 3012114 TI - A conserved cis-acting sequence in the 5' leader of avian sarcoma virus RNA is required for packaging. AB - The deletion of a conserved sequence of ca. 30 nucleotides in the 5' noncoding leader region of an avian sarcoma virus DNA clone resulted in a loss of infectivity after transfection of chicken embryo fibroblasts. Genetic and biochemical analysis of a representative mutant demonstrated that the env gene was expressed normally. Thus, viral RNA transcription, splicing, and translation were not impaired. The amount of mutant viral RNA encapsidated into virions, however, was severely reduced despite the presence of helper-virus. We conclude that the deleted sequence is an essential cis-acting packaging signal. PMID- 3012116 TI - Dispersed chromosomal localization of the proto-oncogenes transduced into the genome of Mill Hill 2 or E26 leukemia virus. AB - Both Mill Hill 2 and E26 retroviruses have transduced two cellular genes--c-myc and c-mil/mht (Mill Hill 2) and c-myb and c-ets (E26). We localized the genes transduced by these viruses to different chromosomes: c-myc and c-myb to relatively large chromosomes and c-mil/mht and c-ets to microchromosomes. Thus, like avian erythroblastosis virus, each of these retroviruses has transduced two cellular genes unlinked in the chicken genome. PMID- 3012118 TI - AKR ecotropic murine leukemia virus SL3-3 forms envelope gene recombinants in vivo. AB - The ecotropic AKR virus SL3-3 was injected into neonatal mice of the high leukemia strains HRS/J and CWD/J and the low-leukemia strains CBA/J, SEA/J, and NIH Swiss. SL3-3 was highly leukemogenic in each strain, and 90% of the inoculated animals died by 6 months of age. T1 oligonucleotide fingerprint analysis of the genomic RNAs of viruses recovered from 9 of 13 leukemic animals revealed the presence of the SL3-3 virus and recombinant viruses with polytropic virus-related envelope gene sequences. Recombinant proviruses were detected by the Southern blot technique in the DNAs of 17 of 18 tumors. The pattern of substitutions within the envelope genes of the SL3-3 recombinant viruses appeared to be dependent on the strain of the animal. These observations indicate that the SL3-3 virus formed envelope gene recombinants in vivo in each of the strains that were studied. However, the role of these recombinants during leukemogenesis remains to be defined. PMID- 3012117 TI - Human cytomegalovirus virion-associated protein with kinase activity. AB - Protein kinase activity was detected in immunoprecipitates of human cytomegalovirus virions and infected cells by using a monoclonal antibody directed against an abundant 68,000-dalton virion structural protein. Purification of this protein by electrophoresis confirmed that the kinase activity was associated with this protein. The kinase activity was dependent on divalent cations (Mg2+, Mn2+) and cyclic nucleotide independent and exhibited optimal activity at pH 7 to 8. The kinase phosphorylated threonine and serine but not tyrosine. PMID- 3012119 TI - In vitro characterization of a thermolabile herpes simplex virus DNA-binding protein. AB - The major herpes simplex virus DNA-binding protein, ICP8, was purified from cells infected with the herpes simplex virus type 1 temperature-sensitive strain tsHA1. tsHA1 ICP8 bound single-stranded DNA in filter binding assays carried out at room temperature and exhibited nonrandom binding to single-stranded bacteriophage fd DNA circles as determined by electron microscopy. The filter binding assay results and the apparent nucleotide spacing of the DNA complexed with protein were identical, within experimental error, to those observed with wild-type ICP8. Thermal inactivation assays, however, showed that the DNA-binding activity of tsHA1 ICP8 was 50% inactivated at approximately 39 degrees C as compared with 45 degrees C for the wild-type protein. Both wild-type and tsHA1 ICP8 were capable of stimulating viral DNA polymerase activity at permissive temperatures. The stimulatory effect of both proteins was lost at 39 degrees C. PMID- 3012120 TI - Neutralizing antibodies to visna lentivirus: mechanism of action and possible role in virus persistence. AB - Lentiviruses are nononcogenic retroviruses that cause persistent infections and slowly progressive diseases. Visna virus, a lentivirus of sheep, persists in cells of the macrophage lineage despite the presence of neutralizing antibodies in the animal. These antibodies are measured by prevention of virus replication in sheep fibroblast cell cultures. In this study we have compared the antiviral properties of the antibodies in sheep fibroblast and macrophage cell cultures, the latter being more relevant to infection in the animal. Using infectivity assays, binding of radiolabeled virus to cell membranes, cellular processing of labeled virus into acid-precipitable and acid-soluble components, and in situ hybridization of viral nucleic acid, we show that the antibodies prevented virus replication in both fibroblasts and macrophages. However, the site of neutralization differed between the two cell types. In fibroblasts, the site of virus neutralization was at the cell membrane, when the antibodies prevented virus attachment. In macrophages, virus incubated with the antibodies was phagocytized rapidly, followed by uncoating of the virions. However, virus RNA was not transcribed. Despite this ability of the antibodies to abort virus replication in macrophages, the kinetics of binding of the antibodies to the virus was much slower than the binding of virus to the macrophages. Therefore, persistent virus replication in immune sheep may be the result of virus spreading from macrophage to macrophage before the agent can be neutralized by antibodies in the plasma. PMID- 3012123 TI - Granular cell tumor of the glans penis. AB - We report a rare case of granular cell tumor of the glans penis. The surgical management and clinical implications are described. PMID- 3012121 TI - Localization of the feline sarcoma virus fgr gene product (P70gag-actin-fgr): association with the plasma membrane and detergent-insoluble matrix. AB - The v-fgr oncogene codes for a unique transforming protein (P70gag-actin-fgr) that contains virus-specific determinants and cell-derived sequences for both a tyrosine-specific kinase domain and an actin domain. We examined the subcellular distribution of the v-fgr protein by immunofluorescence microscopy and various cell fractionation techniques. By immunofluorescence, the v-fgr protein was localized in a diffuse cytoplasmic pattern within transformed cells. The v-fgr protein was not detectable at substratum adhesion sites. Crude membrane preparations (P100) obtained from fgr-transformed cells contained elevated levels of P70gag-actin-fgr. Further analysis of membranes on discontinous sucrose gradients revealed that P70gag-actin-fgr cofractionated with plasma membranes. Using an alternate method of fractionation, we found that the majority of the v fgr protein remained with the insoluble matrix obtained by treating cells with a buffer containing Triton X-100. When membranes were similarly treated with detergent, nearly all of v-fgr protein remained with the residual insoluble matrix. These results suggest that the transforming activity of P70gag-actin-fgr may be directed to subcellular cytoskeletal targets at or near the cytoplasmic face of the plasma membrane. PMID- 3012125 TI - Tumor of the testicle: a case of melanotic neuroectodermal tumor of infancy. AB - We describe an 8-month-old boy with a tumor involving the right testicle and epididymis. Anatomical and pathological examination of this tumor revealed embryological tissue and melanin granules derived from the neural crest. This is an uncommon neoplasm, which is especially rare in the testicles. The patient was free of disease after 30 months. PMID- 3012122 TI - Genetic basis for altered pathogenesis of an immune-selected antigenic variant of reovirus type 3 (Dearing). AB - In this paper we provide a step by step comparison of the pathogenesis of murine infection caused by reovirus type 3 (Dearing) and an antigenic variant (K) selected by its resistance to neutralization with a monoclonal antibody (G5) directed against the T3 hemagglutinin. To show that specific changes in the biologic properties of variant K were due to mutation in the S1 double-stranded RNA segment (gene), which encodes the viral hemagglutinin, we generated a reassortant virus ("1 HA K") containing the variant K S1 gene and compared its properties to variant K and to a reassortant ("1 HA 3") containing the T3 (Dearing) S1 gene. These studies, in conjunction with our previous nucleotide sequence analysis of the S1 genes of variant K and T3 (Dearing) [R. Bassel-Duby, A. Jayasuriya, D. Chatterjee, N. Sonenberg, J. V. Maizel, Jr., and B. N. Fields, Nature (London) 315:421-423, 1985; R. Bassel-Duby, D. R. Spriggs, K. L. Tyler, and B. N. Fields, submitted for publication], indicate that a single amino acid change in the T3 hemagglutinin can alter viral growth and tropism within the central nervous system without affecting either its primary replication in the intestine or its pattern of spread to or within the central nervous system. PMID- 3012124 TI - Relaxant effect of forskolin in rabbit detrusor smooth muscle: role of cyclic AMP. AB - Forskolin caused a concentration dependent relaxation of rabbit detrusor muscle strips. The relaxant effect of forskolin was potentiated by the cyclic AMP sensitive phosphodiesterase inhibitor, Ro20-1274. Pretreatment with the beta adrenergic antagonist, propranolol, did not inhibit the relaxation of rabbit detrusor induced by forskolin, whereas the relaxation response to forskolin was inhibited in part by the adenylate cyclase inhibitor, SQ22536. Forskolin also increased cyclic AMP levels significantly in rabbit detrusor muscle. These data suggest that detrusor muscle relaxation by forskolin may be mediated by cyclic AMP and that forskolin may activate adenylate cyclase without stimulating beta adrenergic receptors in detrusor muscle. PMID- 3012127 TI - Isolation of a retrovirus and a herpesvirus from a captive California sea lion. AB - A non-oncogenic retrovirus was isolated from an explanted skin biopsy from a captive California sea lion (Zalophus californianus) with a history of recurring skin lesions. The morphology of the viral particles in electron photomicrographs was characteristic of a foamy virus, a retrovirus in the subfamily Spumavirinae. Viral cytopathic effects consistent with foamy virus infection were observed in subsequent explants of skin and lymph nodes and co-cultivated peripheral blood leukocytes. The sea lion with the persistent foamy virus infection later died from pericarditis caused by Pasteurella multocida. A herpesvirus was isolated from explants of lung. PMID- 3012128 TI - Antibodies to vesicular stomatitis New Jersey type virus in a population of white tailed deer. PMID- 3012129 TI - Clarifying electron micrograph labeling. PMID- 3012126 TI - Modifiers of calcium oxalate crystallization found in urine. II. Studies on their mode of action in an artificial urine. AB - The relative potencies of various modifiers of the crystallization of calcium oxalate (CaOx) were determined under "whole urine equivalent" conditions using a batch crystallizer. The system was used to measure changes in the degree of agglomeration of CaOx crystals produced spontaneously at a level of supersaturation within the range found in the urines of recurrent CaOx stone formers. The modifiers tested included RNA, heparin, chondroitin-4-sulphate, pyrophosphate (at pH 5, 6 and 7), Tamm-Horsfall mucoprotein and a greater than 10,000 dalton fraction of macromolecules obtained from pooled normal urine by dialysis. The effects of these urinary constituents were also measured on the zeta potential produced at the surface of CaOx crystals. In the case of pyrophosphate, there was a clear correlation between the degree of inhibition of agglomeration and the zeta potential observed on the crystal surface indicating that, for this ion, repulsive electrostatic forces dominate the tendency for CaOx crystals to agglomerate. For the other modifiers tested, the tendency to agglomerate appeared to be dependent on the balance between the positive viscous binding ("sticky") forces and the negative electrostatic forces produced by these macromolecules on the CaOx crystal surface. PMID- 3012130 TI - The presentation by the CDC of data on AIDS. PMID- 3012132 TI - Human T-cell lymphotropic viruses: syncytia formation. PMID- 3012131 TI - Surveillance for AIDS in a central African city. Kinshasa, Zaire. AB - Surveillance for acquired immunodeficiency syndrome (AIDS) in Kinshasa, Zaire, was initiated in July 1984, using a modified version of the case definition developed by the Centers for Disease Control. During the first eight months, 332 patients met all clinical and laboratory criteria; surveillance information was available for 295 (89%) of these patients. Of the sera tested from these patients, 99% had antibodies to human T-cell lymphotropic virus type III/lymphadenopathy-associated virus by both enzyme-linked immunosorbent assay and Western blot procedures. The male-female case ratio was 1:1.1; the mean age of patients was 33.6 years (median, 32 years; range, 1.5 to 64 years); and men were significantly older than women (mean, 37.4 vs 30.0 years). The estimated incidence rate for adults in Kinshasa is 380 cases per 1 million people per year. Peak age-specific incidence rates for men and women occurred among the 30- to 39 year age group, although the rate for men in this age group was 24% higher than the rate for women (786 vs 601 per 1 million). A reasonable estimate of the current annual incidence of AIDS is 550 to 1,000 cases per 1 million people. Surveillance of AIDS in Zaire provides important information on transmission patterns and rates in Africa. PMID- 3012133 TI - Leads from the MMWR. Classification system for human T-lymphotropic virus type III/lymphadenopathy-associated virus infections. PMID- 3012134 TI - High-altitude pulmonary edema. Characteristics of lung lavage fluid. AB - To evaluate the cellular and biochemical composition of bronchoalveolar fluid in high-altitude pulmonary edema (HAPE), we performed bronchoalveolar lavage in three climbers with HAPE in a research facility at 4400 m on Mount McKinley. Three healthy climbers were used as controls. The HAPE fluids contained marked increases in high-molecular-weight proteins, erythrocytes, and leukocytes, most of which were alveolar macrophages. The HAPE fluids also contained detectable amounts of leukotriene B4 and other lipoxygenase products of arachidonic acid metabolism, complement fragments (C5a), inhibitors of neutrophil chemotaxis, and acid proteases but not hydroxyproline, a constituent of collagen. The data from this study indicate that HAPE involves a transient "large pore" leak in the pulmonary circulation. Despite the presence of two potent mediators of inflammation, leukotriene B4 and C5a, HAPE is not characterized by the intense neutrophil accumulation that is typical of other forms of acute lung injury. PMID- 3012135 TI - [Effects of fentanyl on pulmonary airway dynamics in the rabbit]. PMID- 3012136 TI - [Effect of trimethaphan-induced hypotension on pituitary-adrenocortical function]. PMID- 3012138 TI - [Primary diffused infiltrating (linitis plastica) type of colonic cancer--case report]. AB - The primary diffused infiltrating type of colonic cancer is characteristically rare, and has a high rate of malignancy. A 54-year-old Japanese man was admitted to our hospital complaining of abdominal pain and decreased stools. Sigmoid colonic cancer was suspected as a result of the Ba-enema and colonic fiberscopy examination, but the biopsy results were negative. The sigmoid colon was resected extensively. Histopathologically, the cancer was diagnosed as being a signet ring cell carcinoma of the sigmoid colon. Although the patient received postoperative chemotherapy, he died of carcinomata peritonitis 175 days after surgery. PMID- 3012137 TI - [Nuclear DNA content and focal relapse of patients with small-cell carcinoma of the lung]. AB - The relationship between the nuclear DNA content and the focal relapse of 17 patients with small-cell carcinoma completely responsive to treatment was studied. The nuclear DNA content was measured at 550 nm using a microspectrophotometer. The nuclear DNA histogram pattern was classified as either "Type A" (near-diploid) or "Type B" (hyperdiploid). Of the nine patients with type A, only one was found to have focal relapse of the intrathoracic lesion. However, of the eight patients with type B, focal relapse was observed in six (75%). No significant difference was discerned between the metastasis to other organs and the nuclear DNA histogram patterns. The nuclear DNA histogram patterns in small-cell carcinoma of the lung are correlated to the outcome of the patients. PMID- 3012139 TI - [Retroperitoneal malignant fibrous histiocytoma--report of a case]. AB - A 52-year-old man with a three-month history of left epigastralgia and body weight loss was referred to us for a possible abdominal tumor in March 1984. Retroperitoneal malignant tumor was suspected by ERCP, angiography, US, and CT. At laparotomy, a child's-head-sized retroperitoneal tumor, which weighed 1,800 g, was resected. Pathological examination revealed the diagnosis of retroperitoneal malignant fibrous histiocytoma (MFH). He died because of recurrence in the retroperitoneum six months after the operation. We selected 26 Japanese cases of retroperitoneal MFH and discussed their clinical findings and prognosis. PMID- 3012140 TI - [Pathomorphological study of thorotrast-related intrahepatic cholangiocarcinoma- a comparison with non-thorotrast cases]. AB - Thirty-five autopsy cases of Thorotrast (TH)-related intrahepatic cholangiocarcinoma were morphologically studied with a comparison to 45 non-TH cases. Latent periods ranged from 25 to 48 years, with a mean of 34.1 +/- 6.6 years. As to tumor location, the peripheral-middle type, in which the main tumor was located in the periphery to middle portion of the liver, was the most common (89.2%) in the TH-related cases, and the hilar type, in which the main tumor was located in the hepatic hilum, was the most common (78.8%) in the non-TH cases. However, there was no close relationship between the distribution of TH deposits and tumor location by soft-X ray examination of the liver slices. Grossly, the massive type with an infiltrative growth was the most common both in the TH- and non-TH cases. Histologically, there were no remarkable differences between the two groups, and tubular adenocarcinoma with varying degrees of fibrous stroma was the most common. In noncancerous areas, proliferation of the bile ducts with slight to moderate atypism and ductular proliferation around Glisson's capsule were found in 30%, and 10%, respectively, of the TH-related cases. However, such changes were also found in the non-TH cases at almost the same incidence. PMID- 3012141 TI - [The effects of neocarzinostatin on superoxide production by monocyte-derived macrophages]. AB - Monocyte-derived macrophages (M phi) from cancer patients injected with low doses of Neocarzinostatin (NCS, 500 units/day, three times a week) 12 times produced significantly more superoxide (O2-) than controls. Lymphocyte functions, such as PHA response, surface marker and serum IAP, before and after NCS injections were the same. M phi from normal persons cultured with NCS (0.4 microgram/ml) for three days produced more O2- than controls, but those cultured with rINF gamma did not. These results suggest that the increased O2- production of M phi from patients taking low doses of NCS may be due to the direct action of NCS on the M phi. PMID- 3012142 TI - [Resected hepatocellular carcinoma with macroglobulinemia--a case report]. AB - A 54-year-old man had been diagnosed as having macroglobulinemia 10 years earlier; Ultrasonography and liver scintigraphy showed a space-occupying lesion in the right lobe of the liver; CT scan showed abnormal density in the same region. IgG was more than 7,000 mg/dl. The tumor was isolated, and liver function was good. On laparotomy, biopsy of the tumor and one of the enlarged lymph nodes in the hepatoduodenal ligament was undertaken. The diagnosis the from frozen tumor section was hepatocellular carcinoma; malignancy was not found in the lymph node. Right lobectomy was done. The patient left the hospital two months later after an uneventful recovery. PMID- 3012143 TI - [A case of bilateral metastatic breast carcinoma from gastric carcinoma]. AB - The patient was a 48-year-old woman who had undergone radical surgery for gastric carcinoma in 1981. Seventeen months after the operation, recurrence of gastric carcinoma at the anastomotic stoma was found, and total gastrectomy was performed. Fourteen months after the second operation, she was revealed to have bilateral breast tumors and underwent exploratory excision. Histological examination suggested primary malignant tumor of the breast. Bilateral standard radical mastectomy was done seven days after the excision. Final histological examination revealed metastatic breast cancer originating from the stomach. She died from general metastasis seven months after the radical mastectomy in spite of adjuvant immunochemotherapy. PMID- 3012144 TI - [A case of cholangiocarcinoma and dysgerminoma associated with Turner's syndrome]. AB - A rare case of cholangiocarcinoma and dysgerminoma synchronously associated with Turner's syndrome was reported. A 53-year-old woman was admitted to our hospital on July 12, 1984 due to intrahepatic and left inguinal tumors. Physical examination revealed the typical characteristics of Turner's syndrome. The karyotype from myelocyte and fibrocyte culture was interpreted as 45, X. The resected intrahepatic tumor was cholangiocarcinoma that had invaded to the transverse mesocolon and the duodenum, while the inguinal lesion was dysgerminoma derived from a dysgenetic gonad. As far as we have been able to ascertain through our investigations of the literature on malignant disease in Turner's syndrome, the association of cholangiocarcinoma with Turner's syndrome has not been reported previously. PMID- 3012145 TI - [A case of primary malignant fibrous histiocytoma of the vagina]. AB - A 52-year-old woman visited us complaining of a recurrent vaginal tumor, which was diagnosed histologically as malignant fibrous histiocytoma of the vagina. Computed tomography revealed multiple metastatic lesions in the liver, lymph nodes and vertebrae. The patient was treated with combination chemotherapy consisting of cyclophosphamide, adriamycin and cis-platinum, but the tumor did not yield readily to the treatment. We believe that this is the first reported case of this tumor in this location in Japan. PMID- 3012146 TI - [Lymphocytapheresis therapy. 2. Adult T-cell leukemia]. PMID- 3012148 TI - [In vivo evaluation of the development of biocompatible materials]. PMID- 3012147 TI - [A case report of acute myelogenous leukemia treated by bone marrow transplantation from a three-year-old sister and managed in a bed isolator]. PMID- 3012149 TI - [Immobilization of physiological substances and slow-releasing medical materials]. PMID- 3012150 TI - [Mechanism of secretion of saliva]. PMID- 3012151 TI - [Histological classification of lung cancer cells]. PMID- 3012153 TI - [Cytodiagnosis of lung cancer. Electron microscopic observation of lung cancer]. PMID- 3012152 TI - [Cytodiagnostic criteria in lung cancer]. PMID- 3012155 TI - [Cytodiagnosis of lung cancer. B. The general type. b. Large cell carcinoma]. PMID- 3012154 TI - [Cytodiagnosis of lung cancer. A. The central (hilar) type. b. Small cell carcinoma]. PMID- 3012156 TI - [Cytodiagnosis of lung cancer. C. Special types]. PMID- 3012158 TI - [Nucleoside mono- (di-)phosphate kinase activities in guinea pig epidermis]. PMID- 3012157 TI - [Large amount of intra-arterial injection of iodized oil (Lipiodol) in the treatment of unresectable hepatoma]. PMID- 3012159 TI - Detection of antibodies to human T-lymphotropic virus type III in various non human primates. PMID- 3012160 TI - Adult adrenoleucodystrophy with hypercortisolemia. AB - A 39-year-old male showed rapid progression of cerebral white matter involvement after 8 years' duration of adrenomyeloneuropathy. Increased ratio of C:26 to C:22 in the fatty acid composition of sphyngomyelin in the blood and needle-like cytoplasmic inclusions in the biopsied peripheral nerve were indicative of adrenoleucodystrophy or adrenoleucomyeloneuropathy. Plasma ACTH levels were extremely elevated, but most of ACTH immunoactivity was eluted at the molecular weight of 55000. Plasma cortisol levels were initially within normal range and were elevated in the final stage. Possible mechanism of elevated ACTH in this disorder was briefly discussed. PMID- 3012161 TI - Localization of renal kallikrein-kinin system components in the kidney. AB - In a study using a stop-flow technique in dog kidney, the existence of kallikrein and kinin was recognized in distal tubules. The presence of kininase I was seen in both distal and proximal tubules, and also partly in the distal tubules. The presence of kininase II in the distal tubules was again confirmed by pretreatment with SQ14225. No evidence of kinin formation, however, was obtained in the proximal nephrons in stop-flow method. From these results, it was suggested that kininase I and II localized in proximal tubules may destroy the kinin filtered from glomeruli at the proximal level, while kallikrein and kininogen and also kininase I and II in the distal tubules may regulate the activity of the renal kallikrein-kinin system in the distal nephrons. PMID- 3012162 TI - Influence of some dopaminoceptor agents on nitrazepam-induced sleep in the domestic fowl (Gallus domesticus) and rats. AB - The influence of apomorphine, levodopa and haloperidol was studied on nitrazepam sleep using young chicks and rats. In addition, the influence of dopamine and ADTN was studied in young chicks. Nitrazepam dose-dependently (0.4-51.2 mg/kg, i.p.) induced behavioural sleep in chicks. However, higher doses of nitrazepam (12.8-51.2 mg/kg, i.p.) were required to induce behavioural sleep in rats. Dopamine (12.5-100 mg/kg, i.p.) and ADTN (2.5-80 mg/kg, i.p.) delayed the onset but prolonged nitrazepam sleep in chicks: these effects were statistically significant. Levodopa (12.5-100 mg/kg, s.c.) and apomorphine (0.2-0.8 mg/kg, s.c.) profoundly delayed the onset and shortened the duration of nitrazepam sleep in both chicks and rats. Noradrenaline (20-80 mg/kg, i.p.) shortened the onset and prolonged nitrazepam sleep in chicks. Pimozide (1-8 mg/kg, i.p.) potentiated nitrazepam sleep and antagonized the effects of dopamine, levodopa and ADTN on nitrazepam sleep in chicks. Similarly, haloperidol (0.5-1.0 mg/kg, i.p.) potentiated nitrazepam sleep and antagonized the effects of levodopa and apomorphine on nitrazepam sleep in rats. The EEG synchronization and decreased EMG induced by nitrazepam (1.6 mg/kg, i.p., and 12.8 mg/kg, i.p., for chicks and rats, respectively) were antagonized by levodopa (12.5 mg/kg, s.c.). The behavioural and electroencephalographical results suggest that enhancement of dopaminergic neurotransmission may be involved in the mechanisms of wakefulness in both chicks and rats. PMID- 3012163 TI - Species difference of (2R,4R)-2-(o-hydroxyphenyl)-3-(3-mercaptopropionyl)-4 thiazolidinec arb oxylic acid (SA446) in inhibition of angiotensin converting enzyme. AB - A marked species difference was observed both in vitro and in vivo in the activity of SA446, an orally active inhibitor of angiotensin converting enzyme (ACE), as compared with that of captopril in five different animal species. The activity of SA446 in vitro in inhibiting plasma ACE correlated with the activity in vivo as determined by inhibition of the pressor response to angiotensin I (AI). SA446 was more potent as an inhibitor of AI response in dogs, cats and rabbits than in guinea pigs and rats. Furthermore, ACE activity in whole blood in vivo was inhibited by SA446, and the activity of SA446 was also more potent in dogs than in rats. The concentration of SA446 in the ultrafiltrate of blood (free form) was significantly higher in dogs than in rats, while no difference was observed in level of SA446 in the whole blood (free and protein-bound form) between these two species after intravenous injection. The binding rate of SA446 to plasma protein of rats in vitro was more than twice as high as that of dogs. These results suggest that the difference in the protein binding rate of SA446 is reflected in ultrafiltrate level and is one of the important components in defining the species difference in SA446 action. PMID- 3012164 TI - Comparison of acute hemodynamic effects of MC-838, a new angiotensin-converting enzyme inhibitor, with captopril in anesthetized dogs. AB - Effects of a new angiotensin-converting enzyme inhibitor, N-[3-(N cyclohexanecarbonyl-D-alanylthio)-2-methylpropanoyl] -L-proline calcium (MC-838), on the systemic and coronary circulation were evaluated in anesthetized dogs, and the effects were compared with those of captopril. Administration of MC-838 (0.1, 0.3, 1.0 and 3.0 mg/kg, i.v.) produced a gradual and dose-dependent decline in aortic pressure associated with no marked changes in coronary blood flow, heart rate and LVdP/dt. Captopril (0.01, 0.03, 0.1 and 0.3 mg/kg, i.v.) also caused a dose-related decrease in aortic pressure, but the significant hypotension appeared more rapidly than that of MC-838. Both MC-838 and captopril inhibited selectively the pressor response to angiotensin I in a dose-related manner. The doses of MC-838 and captopril to lower mean aortic pressure by 10 mmHg from the pre-drug value were 2.8 mg/kg and 0.03 mg/kg, respectively; those of these drugs to cause 50% inhibition of angiotensin I-pressor response were 1.0 mg/kg and 0.04 mg/kg, respectively. When administration of MC-838 (3.0 mg/kg) was repeated three times at a 30 min-interval, the second and third injections caused no additional hypotension, while each of the repeated injections of captopril (0.3 mg/kg) produced significant hypotension. These results indicate that MC-838 inhibits angiotension I-conversion and decreases systemic blood pressure more slowly and persistently than captopril in anesthetized dogs. PMID- 3012166 TI - [A case of hyponatremic bronchorrhea]. PMID- 3012165 TI - Cardiotonic and coronary vasodilatatory effects of amrinone in the canine heart lung preparation with a support dog as compared with those of dobutamine. AB - Analysis of the relative magnitude of the positive inotropic and chronotropic effects and the coronary vasodilating effects of amrinone conducted in the canine heart-lung preparation with a support dog in comparison with those of dobutamine demonstrated that amrinone was a preferential coronary vasodilator rather than a selective positive inotropic agent. An in vitro experiment in the canine papillary muscle suggested the initiation of the slow response action potential as a mechanism of the positive inotropic effect of amrinone. PMID- 3012167 TI - [Lung cancer due to exposure to bis (chloromethyl) ether]. PMID- 3012168 TI - [The value of 99mTc-DMSA uptake in renal function test]. PMID- 3012169 TI - [Host defense mechanism in pyelonephritis--activation of peritoneal macrophage and its protective effect in the rat with retrograde Proteus mirabilis pyelonephritis]. PMID- 3012170 TI - [An inhibitory effect of 1 alpha, 25-dihydroxyvitamin D3 on the growth of the receptor-containing human renal carcinoma cell lines]. PMID- 3012172 TI - Electronmicroscopy of sciatic nerves in aging rats with spontaneous radiculoneuropathy. PMID- 3012171 TI - [Malignant fibrous histiocytoma of bladder combined with inverted papilloma. Report of a case]. PMID- 3012173 TI - Establishment of the attenuated strain of porcine parvovirus for the live vaccine and its biological-immunological characteristics. PMID- 3012174 TI - A survey of feline respiratory infections. PMID- 3012175 TI - Heterogeneity of Epstein-Barr virus derived from a nasopharyngeal carcinoma that has transforming and lytic properties. AB - The biological activities of Epstein-Barr virus (EBV) from a human epithelial hybrid cell line derived from a nasopharyngeal carcinoma (NPC-KT) were studied. Low concentrations of virus from the NPC-KT cell line stimulated DNA synthesis of human cord blood lymphocytes (CBL) and induced CBL to form EBV nuclear antigen positive continuous cell lines. The CBL exposed to higher doses of virus showed stimulated DNA synthesis 2 days after infection, followed by cell lysis. Virus from the NPC-KT cell line induced EBV-specific antigens in nonproducer Raji cells and reduced the colony-forming ability of the super-infected cells. The ratio of transforming activity to early antigen-inducing ability was not constant during cell passage. The data obtained suggest that NPC-KT clone 1 cells are producing virus with cytotoxic and transforming properties. PMID- 3012177 TI - Determining compliance with a dietary fiber supplement. AB - The purpose of this pilot study was to evaluate possible ways to determine compliance with a dietary fiber supplement. A wheat bran supplement (30 g daily) significantly increased mean daily wet and dry stool weights (SW) in 7 adults, when compared to SW during an ad libitum low-fiber diet (paired t-test, P less than .01). Because unpaired data would be used during a clinical trial, distribution of the 7 ad libitum low-fiber mean SW observations was used to establish a reference distribution and an upper confidence limit against which the bran supplement SW could be compared. Only one of the seven bran supplement mean SW was above the confidence limit of the low-fiber period, independent of the number of days of collection (2-10) used to calculate the individual mean daily SW. Total fecal output over varying periods of time (2-10 days) suffered the same intersubject and intrasubject variability. Most (5-6) of the bran mean daily SW were above the group mean SW of the low-fiber period. However, this dose of bran was large enough to significantly decrease calcium absorption, and differences in SW produced by lower doses of wheat bran would probably not be as great. The bulk (greater than or equal to 80%) of a single dose of a fecal marker, chromium sesquioxide, which could be incorporated into a specific day's fiber supplement, was recovered in 5 days of excretion during the control period and in 4 days during the bran period. However, the blue color of the chromium before ingestion is clearly a negative feature. Another marker, polyethylene glycol, could not be recovered in excreta when transient time was 4 days or more. In a separate study, demonstration of very little overlap in the concentration of fecal neutral detergent fiber between the control and bran periods suggests that fecal fiber may be a marker of compliance with a fiber supplement. PMID- 3012176 TI - Attachment of antinuclear antibodies to nasopharyngeal carcinoma or other cells during preparation of biopsy imprints. AB - The presence of Epstein-Barr virus (EBV) genomes in nasopharyngeal and other carcinomas or Burkitt's and other B-cell lymphomas can be established by the demonstration of viral nucleic acid sequences in DNA extracts from biopsy specimens, the detection of EBV-associated nuclear antigen (EBNA) in biopsy imprints, and the inhibition of leukocyte migration by tumor extracts. Of these techniques, the detection of EBNA-positive tumor cells can be performed most readily in the laboratory. This report shows that a patient's antibodies to nuclear antigens can gain access to cell nuclei during the preparation of imprints. If the antibodies are directed against EBNA, nuclear immunofluorescence is elicited solely in the tumor cells when only complement (C') and fluorescein labeled antibodies to C' are applied to the imprints without prior exposure to anti-EBNA-positive sera. If nonspecific antinuclear antibodies (ANA) are involved, the nuclear immunofluorescence seen in the EBNA-specific and control assays is not limited to the tumor cells but extends to any normal cells that may be present in the imprints. Furthermore, nuclear fluorescence is elicited when solely an anti-human IgG conjugate is applied because ANA is measurable by indirect immunofluorescence, whereas detection of EBNA requires augmentation of the antigen-antibody complexes by C', which differentiates further between EBNA specific and nonspecific staining. Attachment of antibodies to nuclei can be avoided by minimizing the deposit of blood during imprint preparation and by rapid drying of the imprints. Similar results are obtained experimentally when smears of lymphoblasts are made in the presence of anti-EBNA or ANA. PMID- 3012178 TI - Transfer of blood lymphocytes and macrophages between histocompatible progressor and regressor chickens infected with Rous sarcoma virus. AB - The development of histocompatible White Leghorn (progressor) and Arkansas Regression (regressor) chicken lines was described. When challenged with Rous sarcoma virus, progressor chickens developed fatal tumors while the regressor chickens eliminated the sarcoma. When sensitized histocompatible peritoneal macrophages and blood lymphocytes were transferred from regressor donors to progressor recipients, they both eradicated growing tumors. Histoincompatible cells were ineffective in inducing tumor remission. Within the two age groups tested, the sensitized blood lymphocytes and macrophages were only effective when transferred between age-matched donor and recipient chickens. PMID- 3012179 TI - N-nitroso-N-methylurea-induced mammary carcinogenesis: effect of prolactin on expression of Ia antigen by tumor cells. AB - Prolactin (PRL) increases of Ia antigen (Ia Ag) expression in female Sprague Dawley rats with N-nitroso-N-methylurea [(NMU) CAS: 684-93-5]-induced mammary tumors were studied. The effectiveness of PRL was examined when cancers appeared about 2-3 months after the first NMU administration. Rats with NMU-induced mammary tumors were divided into 3 groups: Group 1 was treated with 30 micrograms ovine PRL (o-PRL) in daily sc injections for 5 days. Group 2 received 0.5 mg 2 alpha-bromoergocryptine (CB-154), a known inhibitor of pituitary gland secretion, daily in sc injections for 6 days. Group 3 was the control group. Ia Ags expressed by NMU-induced mammary tumor cells were then quantified successively by double labeling [protein membrane cells with iodine-131 and anti-Ia monoclonal antibody (MoAb) with iodine-125]; then isolation and quantification of the doubly labeled immune complex were performed by affinity chromatography and chromatofocusing successively. When the specific activity of glycoproteins is known, the amount of glycoproteins that bind specifically to the anti-Ia MoAb can be deduced. In NMU-induced rat mammary tumor controls, about 5% of the purified glycoproteins bound specifically to the MoAb, and the amount increased to 8% for NMU-induced rat mammary tumors treated with 30 micrograms o-PRL daily for 5 days and decreased to 2.5% in NMU-induced rat mammary tumors treated with 0.5 mg CB 154 daily for 6 days. Total PRL receptor levels were measured in all tumors tested. For control NMU-induced rat mammary tumors, total PRL receptor levels were 6.35 +/- 1.40 fmol/mg protein, 7.20 +/- 2.40 fmol/mg protein for NMU-induced rat mammary tumors treated with o-PRL, and 6.81 +/- 2.34 fmol/mg protein for NMU induced rat mammary tumors treated with CB-154. Our results demonstrated that treatment of NMU-induced rat mammary tumors with PRL increased the amount of Ia Ag expression by tumor cells and should prove very useful to the understanding of the biology of PRL in the tumorogenesis of the mammary gland. PMID- 3012180 TI - Internal malignancy in genetic hemochromatosis. PMID- 3012181 TI - Bone marrow transplantation: current perspectives and future directions. PMID- 3012182 TI - Carcinoma of the nipple. PMID- 3012183 TI - [Complex radionuclide diagnosis of necrotic and ischemic lesions of the myocardium]. PMID- 3012184 TI - [Various humoral factors regulating blood pressure in patients with hypertension during treatment by an impulse magnetic field]. AB - Renin activity, aldosterone, prostaglandin (PGF2 alpha and PGB) and cyclic nucleotide levels and catecholamine excretion were measured in 165 essentially hypertensive patients exposed to therapeutic effects of "running" impulse magnetic field (RIMF). The correction of arterial blood pressure in RIMF-treated patients is shown to be mediated by BP-controlling humoral factors, the magnitude and direction of changes in levels and activity of biologically-active substances and hormones being determined by their respective baselines. A decrease of hyperfunction, as reflected in elevated hormonal production, and an increase of hypofunction were the most common therapeutic effect of RIMF exposure. PMID- 3012186 TI - Inhibitory effect of 1 alpha,25-dihydroxyvitamin D3 on the growth of the renal carcinoma cell line. AB - We studied the effect of vitamin D compounds on the growth of the human renal carcinoma cell line (KU-2) and discovered a receptor protein specific for the active form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3. The KU-2 cell line was established from a pulmonary metastasis of renal cell carcinoma in a patient with hyperhemoglobinemia. The cells were tumorigenic in nude mice and clonogenic in a soft agar culture. Vitamin D3 derivatives suppressed proliferation of KU-2 cells in a monolayer culture and also clonogenicity in a soft agar culture dose dependently. Of the vitamin D3 derivatives tested, 1 alpha,25-dihydroxyvitamin D3 was the most potent in inhibiting cell growth, followed successively by 1 alpha,24R,25-trihydroxyvitamin D3, 25-hydroxyvitamin D3, 1 alpha-hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 in that order. Analysis of the cell cycle phase of treated and non-treated KU-2 cells revealed that the action of 1 alpha,25 dihydroxyvitamin D3 was not phase-specific but simply extended the doubling time of the cells. Radioreceptor assay and sucrose density gradient analysis of the cytosol showed that KU-2 cells contained a 3.2S receptor protein to which 1 alpha,25-dihydroxyvitamin D3 was specifically bound (Kd = 20.8 +/- 4.8 pM, Nmax = 87 +/- 24 fmole/mg protein, 4000 molecules/cell). On the other hand, the equilibrium dissociation constant of internalization of 1 alpha,25 dihydroxyvitamin D3 (Kint) by intact KU-2 cells was 1.2 nM and the internalizing capacity was 33 fmole/8 X 10(6) cells (2500 molecules/cell) in the 10% serum medium, which was the same as that used in the growth study.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012185 TI - Hypernatremia inhibits NaHCO3 reabsorption and associated NaCl reabsorption in dogs. AB - To examine the effect of selective rise of plasma NaCl concentration (hypernatremia) on NaHCO3 reabsorption and associated NaCl reabsorption remaining during continuous ethacrynic acid infusion, hypertonic NaCl solution was infused in three groups of anesthetized volume-expanded dogs. In six dogs examined at constant hematocrit and plasma pH, bicarbonate and water reabsorptions were inversely related to PNa and reduced by 37% and 39% respectively by raising PNa from 140 to 200 mM. Chloride reabsorption remained essentially constant until PNa exceeded 170 to 180 mM. At PNa 200 mM, sodium reabsorption was reduced by 22 +/- 6%. In six other dogs, mechanical variations of GFR showed that the inhibitory effects of hypernatremia (PNa 199 +/- 3 mM) were less pronounced at low GFR. After subsequent administration of acetazolamide (30 mg/kg body wt), only 20% of control bicarbonate reabsorption remained and glomerulo-tubular balance was completely abolished. Both hypernatremia and acetazolamide inhibited NaHCO3 and NaCl reabsorption in a molar ratio of about 1:2, as in normonatremic dogs. Finally, experiments in six dogs showed that the inhibitory effects of hypernatremia (PNa 213 +/- 4 mM) were not altered by varying PCO2 and plasma pH. We conclude that hypernatremia inhibits paracellular water and NaCl reabsorption in the proximal tubules by reducing the osmotic force caused by transcellular NaHCO3 reabsorption. A rise in PNa does not stimulate transcellular NaCl reabsorption during distal inhibition by ethacrynic acid. PMID- 3012187 TI - Chronic progressive renal lesions induced by lithium. AB - New Zealand white rabbits, eight fed lithium (Li) (50 to 250 mmole LiCl/kg food) and seven controls (C) had sequential open renal biopsies at zero, one, three, six, and 12 months. A distinctive histological lesion, consisting of cytoplasmic vacuolation and accumulation of glycogen in cells lining distal convoluted tubules and collecting ducts, was present in Li (one, three, six, and 12 months), but was absent in Li prior to lithium (zero months) and in C (zero, one, three, six, and 12 months). Histological changes of chronic focal interstitial nephropathy namely, interstitial fibrosis, (quantitated by point counting), tubular atrophy and cast formation (quantitated by digitization), and glomerular sclerosis (determined as the percent of sclerosed glomeruli) showed significant differences between Li and C from as early as one month (interstitial fibrosis, P less than 0.02; tubular atrophy, P less than 0.05; casts, P less than 0.05), and up to 12 months (glomerular sclerosis, P less than 0.05). Distal tubular dilatation and microcyst formation, (quantitated by digitization) was also marked in Li compared with C from one month (P less than 0.05). The degree of distal tubular dilatation and other changes of interstitial nephropathy tended to progress with duration of lithium exposure. Macroscopically, Li kidneys (12 months) were pale, granular, and exhibited microcysts. Raised blood urea (P less than 0.02) and serum creatinine levels (P less than 0.05) were also late features (12 months) of lithium-induced nephropathy. The data support the view that lithium induces chronic renal lesions. The precise relationship between the distinctive distal tubular lesion, distal tubular dilatation and focal interstitial nephropathy remains speculative. PMID- 3012188 TI - [Cancer of the pancreas (clinico-anatomical analysis)]. PMID- 3012189 TI - [Significance of leukotrienes in chronic respiratory tract diseases in childhood]. AB - The role of lipoxygenase products was studied in children suffering from chronic diseases of the lung. Leukotrienes C4, D4, E4 and B4 were measured by high performance liquid chromatography (HPLC) and a specific radioimmunoassay (RIA) for C4. Elevated levels (up to 40 ng/ml), especially for leukotriene E4, were found in plasma of asthmatic and bronchitic patients (leukotriene C4 concentrations varied between 0.05 and 40 ng/ml, mean 4.9 +/- 7.8 ng/ml). In healthy donors the concentrations were below the detection limits of HPLC, leukotriene C4 ranging between 5 +/- 4 ng/ml (RIA data). The conversion of leukotriene C4 to D4 and E4 was observed by incubating the samples with synthetic leukotriene C4. The half-life of leukotriene C4 in plasma varied greatly, ranging from less than 12 min to 72 min (mean 39 +/- 16 min). Bronchial lavages yielded leukotriene C4 concentrations of 0.2 to 7 ng. Leukotriene E4 was detected in 10 of 41 cases. Conversion of leukotriene C4 did not occur in 50% of all cases, but was regularly observed in putrid lavages. These data suggest that leukotrienes play an important role in allergic and infectious lung diseases. PMID- 3012190 TI - Combined pituitary function-test with four hypothalamic releasing hormones. AB - Anterior pituitary function was investigated in ten healthy subjects by administering a combination of 200 micrograms thyrotropin releasing hormone (TRH), 100 micrograms gonadotropin releasing hormone (GnRH), 100 micrograms growth hormone releasing factor (GRF1-44), and 100 micrograms human corticotropin releasing factor (CRF). The same test protocol was performed in all subjects after pretreatment with 0.25 mg terguride. Five subjects were tested only with TRH and GnRH, five only with CRF, and six only with GRF. There was a prompt increase in all hormones after the administration of the four releasing hormones (RH). Pretreatment with terguride lowered the prolactin (PRL) increase (p less than 0.01) as well as the thyrotropin (TSH) peak (p less than 0.05) compared with the test without dopamine agonist pretreatment. The PRL levels after combined RH administration were significantly higher than after TRH and GnRH alone. Although four of the five subjects had higher TSH levels after combined RH administration than after TRH and GnRH alone, the difference was not significant. Other hormones were not significantly influenced by the combined RH administration or dopamine agonist pretreatment. Despite the fact that the interaction of the different releasing hormones and dopamine agonists influences the pituitary hormone response, combined RH administration seems to be a useful test for evaluating pituitary function also in patients receiving dopamine agonist therapy. PMID- 3012192 TI - [New trends in the treatment of diabetes mellitus]. PMID- 3012193 TI - Change of membrane fluidity in HeLa cells at an early stage of a poliovirus infection. PMID- 3012194 TI - A case of malignant fibrous histiocytoma of the pleura. PMID- 3012191 TI - Neopterin in AIDs, other immunodeficiencies, and bacterial and viral infections. AB - An increase in total urinary neopterin was observed in 12 of 13 patients with acquired immunodeficiency syndrome (AIDS), seven of 13 patients with lymphadenopathy, one of six healthy homosexual males, seven of ten adult patients with staphylococcal pneumonia, 11 of 12 children with viral infections, four of seven children with bacterial infections, and 12 of 13 children with various immune defects. Extremely high values of total urinary neopterin and monapterin were observed in severely ill patients with AIDS and those with familial hemophagocytic lymphohistiocytosis. Neopterin excretion was normal in two AIDS patients with Kaposi's sarcoma, but without opportunistic infections at that time. On reexamination of one of these patients later on, elevated neopterin values were noted. Parallel increases in neopterin and monapterin were found, whereas biopterin was usually normal. The increase in total neopterin was mainly due to 7,8-dihydroneopterin and was accompanied by an increase in 3' hydroxysepiapterin. Increased neopterin in urine is assumed to reflect the increase in GTP pool and GTP cyclohydrolase I activity as observed in stimulated monocytes. Thus, neopterin, as a measure of the activation of the nonspecific cellular immune system, may be used diagnostically to detect allograft rejection after transplantations and to follow-up HTLV-III positive patients. PMID- 3012195 TI - Cerebrospinal fluid ferritin in patients with central nervous system tumors. PMID- 3012196 TI - [Disinfection in LAV/HTLV-III infections]. PMID- 3012197 TI - [Hospital hygiene. Notice of the Expert Federal Commission for AIDS on procedures to adopt in regard to patients with AIDS and to persons carrying anti-LAV/HTLV III antibodies]. PMID- 3012198 TI - Impairment and recovery of the clipped kidney in two kidney, one clip hypertensive rats during and after antihypertensive therapy. AB - Earlier experiments have shown that in sodium depleted hypertensive rats with bilaterally constricted renal arteries the arterial pressure normalized after blockade of the renin-angiotensin system; simultaneously acute renal failure occurred. In hypertensive rats with unilateral renal artery stenosis an impaired excretory function of the clipped kidney can be expected, but may not be detectable by conventional tests of renal function. Male Wistar rats with chronic two kidney, one clip hypertension were fed a low sodium diet. After 7 days the rats were treated with vehicle, with the vasodilator dihydralazine, or with the angiotension converting enzyme inhibitor MK 421 for 2 weeks. During the 14-day treatment period a continuous blood pressure reduction was achieved in dihydralazine and MK 421 treated rats. Overall excretory kidney function (plasma creatinine concentration) was well maintained in all three groups until the end of the antihypertensive drug treatment. At the end of drug therapy mean glomerular filtration rates of the left clipped kidneys were significantly lower in both treated groups compared to hypertensive controls, and mean glomerular filtration rate of the left clipped kidneys of dihydralazine treated rats was significantly higher than in MK 421 treated rats: controls (N = 6) 1.03 +/- 0.03, dihydralazine-group (N = 10) 0.28 +/- 0.07, MK 421-group (N = 9) 0.03 +/- 0.01 ml/min. Renal blood flows were comparable in both treated groups. Only the left kidneys of rats treated with MK 421 showed a prominent tubular atrophy. Seven days after declipping of the left renal artery and right nephrectomy a considerable restitution of the tubular structure had occurred in the MK 421 group. The recovery of tubular epithelial cells was paralleled by a rise in glomerular filtration rate: MK 421 group (N = 7) 1.25 +/- 0.08 ml/min. Thus, the clipped kidney in two kidney, one clip hypertensive rats showed functional and morphological signs of impairment when systemic arterial pressure was reduced to the normal range. The alterations of the clipped kidney were most pronounced in rats with renin-angiotensin system-blockade. PMID- 3012199 TI - Humoral hypercalcemia of malignancy in nude mouse model of a canine adenocarcinoma derived from apocrine glands of the anal sac. Biochemical, histomorphometric, and ultrastructural studies. AB - A serially transplantable tumor line, designated CAC-8, has been developed in nude mice from a spontaneously occurring adenocarcinoma of the anal sac from a hypercalcemic dog. Nude mice with transplanted CAC-8 developed hypercalcemia (mean 16.3 +/- 0.6 mg/dl) and moderate hypophosphatemia without bone metastasis. Urinary excretion of calcium and hydroxyproline were increased 6- and 2.3-fold, respectively. Urinary excretion of cAMP was moderately increased but phosphorus excretion was not significantly altered. Serum 1,25-dihydroxycholecalciferol was increased significantly in tumor-bearing nude mice in proportion to the magnitude of tumor-induced hypercalcemia. Histomorphometric evaluation of lumbar vertebrae from nude mice with CAC-8 revealed decreased total and cortical bone volume, a 3.3-fold increase in bone resorption rate and a 2.5-fold increase in bone formation rate at the tissue level. The transplanted CAC-8 has maintained the histologic pattern of the original carcinoma up to the present sixth passage. Ultrastructural evaluation of transplanted tumor cells revealed 150-250-nm secretory-like granules. The granules did not stain by using an ultrastructural cytochemical (uranaffin) stain specific for neuroendocrine secretory granules. Ultrastructurally, the parathyroid glands of nude mice with CAC-8 appeared inactive with large intracytoplasmic whorl of agranular membranes. These data suggest the transplanted carcinoma secreted a humoral factor which resulted in hypercalcemia. The tumor line (CAC-8) propagated in nude mice represents an animal model of humoral hypercalcemia of malignancy that shares many features with the syndrome described in human patients. Unique features of this transplanted carcinoma associated with hypercalcemia include increased serum dihydroxycholecalciferol, increased rate of bone formation as well as bone resorption, an absence of bone metastases, and evidence of parathyroid gland suppression. PMID- 3012200 TI - Carcinoma of the lung: one hundred consecutive cases. PMID- 3012202 TI - Clinical oral pathology--oral medicine. Presentation III. PMID- 3012201 TI - Scanning spectrophotometry for the dynamic study of tissue respiration in intact organs. AB - For use with intact perfused organs a spectrophotometer system has been developed, both for dual-wavelength absorption measurements and for spectral scanning. A monochromator is used for illumination and scanning in the visible and near infrared. Optic fibres conduct light to the specimen under examination and from the specimen to a detecting photomultiplier. System control is exercised by a microcomputer, which also processes the collected data. The performance of the system on isolated perfused rat heart is demonstrated, in the spectral scanning mode and in the dual-wavelength mode, by studying simultaneously the kinetics of cytochrome aa3, and myoglobin oxidation-reduction. PMID- 3012203 TI - Hepatocellular carcinoma presenting as bone pain. AB - Hepatocellular carcinoma is an uncommon malignancy in the western world, and its presentation as symptomatic skeletal metastasis is rare. The following describes a patient whose initial complaint was arm pain that was subsequently found to be due to metastatic hepatocellular carcinoma. A review of the literature describing skeletal involvement by hepatocellular carcinoma is included. PMID- 3012204 TI - [The effects of nicergoline on the heart rate in the normotensive or spontaneously hypertensive rat. Possible participation of central alpha-1 receptors]. AB - The cardiovascular effects of nicergoline, a preferential alpha 1-adrenoceptor blocking drug, were studied in anaesthetized normotensive or spontaneously hypertensive (SH) rats. Nicergoline (300 micrograms/kg, i.v.) significantly reduced blood pressure and heart rate in control, bivagotomized or beta-blocked normotensive or SH rats. In bilaterally vagotomized and beta-blocked rats, nicergoline reduced mean blood pressure but did no longer modify heart rate. Thus, it is postulated that nicergoline could reduce the sympathetic tone and increase the vagal nerve activity, possibly by inhibiting central alpha 1 adrenoceptors. The nicergoline--induced bradycardia was greater in bivagotomized SHR than in normotensive ones. Intracerebroventricular injections of nicergoline (30 micrograms/kg) did not modify heart rate in normotensive control, bivagotomized or beta-blocked rats. On the contrary, nicergoline (30 micrograms/kg) injected into the cisterna magna induced a significant bradycardia in the three groups of normotensive rats. Blood pressure was reduced in the same way in all groups centrally treated by nicergoline. In conclusion, it seems that nicergoline reduces blood pressure by peripheral alpha-adrenoceptor blockade and modulates the autonomic nervous activity by inhibiting alpha 1-adrenoceptors mainly localized in the brainstem. PMID- 3012205 TI - Stimulation of p-nitrophenylphosphatase activity of mung bean shoot extracts. Preliminary observations as a possible screening test for anticonvulsant drugs. AB - The p-nitrophenylphosphatase (pNPPase) activity of mung bean shoot extracts has similar characteristics to that of animal tissue. Mung bean shoot pNPPase activity is maximal between pH 7.8 and 8.2 and at a substrate concentration between 10 and 20 mM and is inhibited by sodium ions. Mung bean shoot pNPPase activity is competitively inhibited by ATP with a Ki value of 2.8 mM. A wide range of anticonvulsant drugs tested all stimulated the pNPPase activity of the mung bean shoot extracts. The nonanticonvulsant drugs tested did not have this effect, with the exception of pemoline, which, although not acknowledged to be an anticonvulsant drug, did show anticonvulsant activity when tested using conventional methods involving animals. In double-blind trials, 12 out of 15 compounds were correctly classified as anticonvulsants or nonanticonvulsants. It is proposed that the pNPPase activity of mung bean shoot extracts may be useful as a preliminary screen for anticonvulsant drugs. PMID- 3012206 TI - A model for the interaction of muscle cross-bridges with ligands which compete with ATP. AB - A model is presented to describe the inhibition of muscle fiber contraction by ligands that compete with MgATP. Two ligands, adenosine 5' (beta, gamma-imido) triphosphate (AMPPNP) and pyrophosphate (PPi), decrease the force developed in isometric contractions and act as weak competitive inhibitors of the maximum velocity of contraction (Pate & Cooke, 1985). These observations provide information on the energetics of actomyosin ligand states at the end of the power stroke where MgATP dissociates the myosin cross-bridge from actin, and they are analysed in terms of a seven state model of cross-bridge kinetics. The model can reconcile the observations that these ligands bind tightly to fibers, Kd = 10(-4) M, while they are only weak inhibitors of fiber velocity, Ki = 2 X 10(-3) M. It provides a reasonable fit to the data and leads to several conclusions concerning the properties of the cross-bridge states. The states with bound ligand are shifted axially so that they occur earlier in the power-stroke than the nucleotide-free rigor state. This shift also explains the axial lengthening seen upon addition of ligands to rigor fibers. We can conclude that these ligands cause small perturbations in the cross-bridge configuration rather than large shifts. A second conclusion is that cross-bridges do not detach from actin during their power-strokes. Instead they traverse the entire length of the power stroke and are detached only at the end, leading to the suggestion that the cycling of bridges in isometric fibers is due to fluctuations in the relative positions of thick and thin filaments. With some further assumptions, the model also explains many of the rate constants and equilibrium constants of the actin-myosin-ligand interaction that have been measured in solution. PMID- 3012207 TI - Theory of the rotational contribution to facilitated diffusion. AB - Gros and others have recently shown experimentally that the facilitated diffusion of protons carried by a form of haemoglobin is enhanced by rotational diffusion of the carrier, whereas facilitated diffusion of oxygen by the same carrier is not. In this paper the theory of facilitated transport by rotating carriers is developed from first principles. The theory confirms Gros's findings that (i) the rotational contribution appears only when the angle of rotational diffusion over the average time the proton remains bound is small and (ii) under these conditions rotation enhances the normal translational contribution by a factor 1/2 at the lowest carrier concentrations. The theory also shows that there must be a rotational boundary layer. PMID- 3012208 TI - Evolution of a defective virus from a cellular defense mechanism. AB - Adeno-associated virus is a defective DNA virus, requiring the presence of a helper virus in order to replicate. In this paper we consider its origin in light of several observations, most notably the following: its own replication inhibits that of the helper virus; its DNA structure resembles that of transposable (moveable) elements; and extrachromosomal circles of DNA, about the size of adeno associated virus DNA, have been found recently in eukaryotic cells. We have arrived at a hypothesis consisting of two main ideas: (1) that cells may use transposable DNA as a mechanism of defense against viral attack, and (2) that adeno-associated virus may have evolved directly from this cellular defense mechanism. PMID- 3012209 TI - Deficiency of myeloperoxidase and abnormal chromosome 1 occurs in variant (HL60) promyelocytes. AB - Maturation of normal polymorphonuclear neutrophils is characterized by successive periods of granule synthesis, a process which frequently is abnormal in leukemia. Recently, the human leukemic cell line HL60, displaying a promyelocytic phenotype, has been used to study granulocyte maturation. We describe a variant line of HL60, called HL60-A7, resulting from growth in actinomycin D, which contains atypical large azurophilic granules deficient in myeloperoxidase. The products of in-vitro translation of A7 RNA contained less than 5% of the immunoreactive MPO found in the parent line. Electrophoresis of plasma membrane polypeptides radioiodinated by the lactoperoxidase technique revealed several differences. Karyotypic analysis identified a consistent chromosome 1q+ abnormality which was not found in any of the parental cells examined. This constellation of differences between HL60 and HL60-A7, i.e. MPO deficiency, abnormal granule morphology, cell surface changes, and further cytogenetic abnormalities, may point to a common site sensitive to altered regulation in some leukemic promyelocytes. PMID- 3012210 TI - In-vitro effects of antineoplastic prostaglandins on human leukemic cell growth and normal myelopoiesis. AB - The effects of prostaglandin (PG)E1, PGD2 and 9-deoxy-delta 9-PGD2 (PGJ2) on the clonogenic growth of six kinds of human leukemic cell lines (K562, KG1, HL60, U937, THP1 and Molt4) and normal human myeloid progenitor cells (CFU-GM) were studied using semisolid agar cultures. While the degree of suppression of leukemic growth by PGE1 varied from cell line to cell line, PGD2 and PGJ2 equally suppressed the growth of all leukemic cell lines. The potency of growth inhibition was as follows: PGJ2 greater than PGD2 greater than PGE1. The increase of cellular cAMP level induced by prostaglandin treatment did not parallel their cytotoxic potency. Normal myeloid colony formation was also suppressed by PGE1, PGD2 or PGJ2. In contrast to the preferential inhibition of macrophage colony formation by PGE1, such lineage-selective suppression was not observed for PGD2 or PGJ2. These findings suggest that PGD2 and PGJ2 potently inhibit the leukemic growth by a different mechanism from that of PGE1 and by a cAMP-independent mechanism. These prostaglandins seem to be promising chemotherapeutic agents for acute leukemia. PMID- 3012211 TI - Isolation from the spleen of myeloproliferative sarcoma virus-infected mice of a myelomonocytic tumor cell line secreting hematopoietic colony stimulating factors. AB - Clonogenic tumor cells were detected in the spleen of DBA/2 mice infected with the myeloproliferative sarcoma virus (MPSV). These cells could be isolated because of their ability to proliferate on the greater omentum of sublethally irradiated isogenic recipient mice. We have established a permanent suspension myelomonocytic cell line in vitro (TE8), from a tumor obtained after subcutaneous transplantation of the omental tumor masses. The TE8 cells are clonogenic in semi solid medium containing agarose. The colonies thus obtained gave rise to myelomonocytic cell lines growing in suspension in liquid medium. One of those cloned cell lines, C1(1011) was studied in details. It is exclusively composed of initial donor cells, it is tumorigenic in vivo, clonogenic in vitro and produces MPSV. It also secretes a colony stimulating activity (CSA), and an activity termed mixed colony promoting activity (MPA), which enables pluripotent hematopoietic stem cells to proliferate and differentiate. It differentiates through the granulomacrophage cell line independently of exogenous factors other than those which may be present in the serum. PMID- 3012212 TI - Analysis of anti-HTLV-I antibody by strip radioimmunoassay--comparison with indirect immunofluorescence assay, enzyme-linked immunosorbent assay and membrane immunofluorescence assay. AB - Antibodies against human T-cell lymphotropic virus type I (HTLV-I) in the sera from 60 patients with adult T-cell leukemia and 21 carriers who were suspected of having HTLV-I infection were investigated by indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), membrane immunofluorescence assay (MIA) and strip radio immunoassay based on the western blotting technique (SRIA). The sera of 2 of the carriers who were seropositive in IFA and ELISA were negative in MIA and did not react with virus-specific proteins by SRIA. Two sera were negative in IFA and ELISA. These sera were positive in MIA and reacted with only the envelope-related glycoprotein (gp46) and not with gag-related proteins (p28, p24, p19) by SRIA. These findings suggest that the main antigens defined by IFA and ELISA are gag-related proteins and some sera which do not contain anti HTLV-I antibodies give false-positive results because of the reaction to unknown cellular components. Also some sera may have antibodies against only envelope glycoproteins, and these sera may give false-negative results in IFA and ELISA. PMID- 3012213 TI - Study of differentiation of fresh myeloid leukemic cells by physiologic agents that induce a human promyelocytic leukemic line (HL-60) to differentiate. AB - A promyelocytic cell line known as HL-60 can be induced to mature to granulocytes or monocytes after exposure to a variety of physiologic agents including 1-alpha 25 dihydroxy Vitamin D3 (Vit D), retinoic acid, cyclic AMP cell permeant compounds, and stimulators of adenylate cyclase. These compounds were used in primary culture of blast cells from 12 patients with acute nonlymphocytic leukemia. Maturation was assessed by morphology, superoxide production, development of esterase activity and chemotactic peptide receptor expression. Morphologic maturation and superoxide production correlated with chemotactic peptide receptor expression. The majority of blast cells treated with inducer showed no significant change in morphologic or functional markers compared to the blast cells cultured in fresh media alone. Chemotactic peptide receptor expression increased 3 to 30-fold in 13 of 14 cases studied. In 4 patients, the highest receptor expression was without inducer and in 4 patients the highest increase was with dibutyryl cyclic AMP treatment. Our study suggests that physiologic inducers of HL-60 differentiation do not consistently have the same effect on primary suspension culture of freshly isolated human leukemia cells. PMID- 3012214 TI - Establishment of B-cell lines from tumor of enzootic bovine leukosis. AB - Two lymphoid cell lines were established from enzootic bovine leukosis tumor cells. Suspension cell cultures of these cell lines have been maintained in vitro for over 2 yr. The cell grew as floating cells without attaching to the glass surface. These 2 cell lines have B-cell surface marker, tumor-associated antigen on the cell surface and bovine leukemia provirus in the genomes. PMID- 3012216 TI - Abdominal fluid cytology in patients with gastrointestinal malignant lesions. AB - In a group of 76 patients with various gastrointestinal malignant lesions, we found that peritoneal washings contained tumor cells in 43% of patients with gastric cancer, 22% of those with pancreatic cancer, and 3% of those with colonic cancer. Aside from tumor site, we were unable to identify any criteria that would help to predict the presence of malignant cells in peritoneal fluid specimens. We found no malignant cells on cytology in patients with early localized cancer. The ease of obtaining such data, coupled with the fact that the test is inexpensive, makes cytologic assessment attractive. Furthermore, the results of cytology have been shown to bear a direct relationship to prognosis in some cancers and may serve as an indication for more intensive therapy. The results of sequential cytology tend to support the theory that tumor manipulation may be a source of intraperitoneal spread in certain tumors. PMID- 3012215 TI - Therapeutic embolization angiography for extra-axial lesions in the head. AB - Percutaneous transcatheter arterial embolization has played an increasingly important role in the management of vascular lesions in the head. Embolization can promote thrombosis within vascular tumors and malformations, reduce bleeding and decrease the need for transfusion intraoperatively, and facilitate surgical approaches to otherwise unresectable lesions. It is important for the clinician to be aware of this interventional technique because many of the patients who are considered for embolization are triaged through several different clinical areas, and much can be gained from the collaboration of the clinician, the surgeon, and the angiographer. We performed 31 therapeutic particulate embolization procedures for extra-axial head lesions in 23 patients by using flow-directed techniques. Of these procedures, 11 resulted in vascular occlusion and 15 resulted in 80 to 95% obstruction, as demonstrated by angiography. In 14 patients, embolization was performed preoperatively both to decrease blood loss and to occlude inaccessible or unresectable portions of a lesion. In nine patients, embolization was the sole means of treatment for occluding an abnormal vascular shunt. Two patients (9%) experienced a minor transient neurologic change after the procedure. PMID- 3012217 TI - Hepatic duct stricture after radical radiation therapy for biliary cancer: recurrence or fibrosis? AB - Two patients with biliary cancer received radical radiation therapy. After treatment, both patients experienced episodes of biliary obstruction without definite evidence of progression of the tumor. These cases emphasize the importance of including radiation-induced biliary fibrosis in the differential diagnosis of hepatic duct stricture after radical radiation therapy. PMID- 3012218 TI - Ilioinguinal neuropathy after iliac crest biopsy. PMID- 3012219 TI - Glutathione metabolism in red cell aging. AB - Normal human red cells were centrifugally separated according to age by discontinuous density gradient of Percoll. Reduced glutathione (GSH), GSH stability and glucose-6-phosphate dehydrogenase (G6PD) activity in fractionated red cells decreased with age, while oxidized glutathione (GSSG) and methemoglobin (MetHb) increased with age. PMID- 3012220 TI - Filopodia number increases with age and quiescence in populations of normal WI-38 cells, and is correlated with drug-induced changes in proliferation in both normal and transformed populations. AB - Filopodia in log and stationary phase populations of human fetal lung fibroblasts (WI-38) at low and high population doubling levels (PDLs) and of SV40 transformed WI-38 cells (VA13A), were observed and counted under different conditions of in vitro growth by scanning electron microscopy. Cells from old non-vigorously growing WI-38 populations (those at a high PDL) had more filopodia than younger populations (those at a lower PDL) at all times after seeding, and for any given population stationary phase cells (those entering, or in, quiescence), had more than log phase cells. Hydrocortisone (HC, 14 microM), which stimulates proliferation and increases life span of WI-38 cells, was associated with a marked decrease in filopodia. Conversely, retinoic acid (RA, 10 microM), which inhibits growth and decreases life span of WI-38 cells, was associated with an increase in filopodia. Since old cell populations have lower saturation densities than young, it is suggested that cell contact signaling growth cessation in these populations may be mediated by filopodia. The HC-associated decrease in filopodia may thus be possibly interpreted as a decrease in filopodia-mediated "density dependent inhibition," and the increase in filopodia with RA as a possible increase in this "inhibition." Both HC and RA inhibit growth and are associated with an increase in filopodia in VA13A cultures. PMID- 3012221 TI - An altered response in the induction of cell membrane (Na + K)ATPase by thyroid hormone is characteristic of senescence in cultured human fibroblasts. AB - There have been numerous investigations of thyroid function during senescence in humans. However, very little information is available on thyroid hormone action at the cell level during senescence. Therefore, we have investigated thyroid hormone induction of cell membrane (Na + K)ATPase during human senescence using three experimental fibroblast cell culture systems: (1) cells from premature aging syndrome, progeria; (2) aging in vitro; and (3) early passage cells from aged patients. In all cases senescence is associated with a dramatic alteration from the normal dose-dependent thyroid hormone induction of (Na + K)ATPase. Senescent cells depleted of thyroid hormones demonstrated an elevated activity of (Na + K)ATPase, while non-senescing cells exhibit the characteristic basal enzyme activities in the hypothyroid state. These results indicate that human senescence is associated with extreme alterations in thyroid hormone regulation of (Na + K)ATPase; and may suggest a more general change in thyroid hormone action at senescence. These changes may be associated with important alterations in cell metabolism and intracellular ionic environment during senescence. PMID- 3012223 TI - [Prevalence of anti-LAV/HTLV-III in prostitutes in Seville]. PMID- 3012224 TI - [Systematic detection of anti-LAV/HTLV-III antibodies in blood donors and hemophiliacs]. PMID- 3012222 TI - Receptor for epidermal growth factor retains normal structure and function in aging cells. AB - Cellular responsiveness to epidermal growth factor (EGF) and the structure of the receptor for epidermal growth factor (EGF-R) were compared in young and senescent human fibroblast (HF) cells. Biosynthetic labeling of HF cells with [35S] methionine followed by immunoprecipitation with EGF-R antibody revealed the presence of Mr 170 000 EGF-R in cells from both stages. Autophosphorylation of EGF-R in response to EGF was identical in young and senescent cells. Phosphoamino acid analysis of the autophosphorylated EGF-R indicated that tyrosine residues were phosphorylated in each preparation. Two-dimensional peptide mapping of [125I]EGF-R from young and senescent cells showed essentially the same pattern, indicating that EGF-R does not apparently undergo detectable changes in senescent human fibroblasts. The responsiveness of aging HF cells to EGF for the induction of ornithine decarboxylase activity and for the production of secretory proteins was measured. Young and senescent HF cells showed about a three-fold induction of collagenase activity upon addition of EGF. Ornithine decarboxylase activity was also stimulated by EGF to a comparable level in young and senescent cells. Our results indicate that the responsiveness of HF cells to EGF for these two biochemical parameters does not decline with the loss of proliferative activity. PMID- 3012225 TI - Cardiopulmonary resuscitation. A current perspective. AB - Cardiopulmonary resuscitation is effective if established early and coupled with specific therapeutic interventions. Most cardiopulmonary arrest is due to ventricular fibrillation and early defibrillation offers the highest probability of success. External cardiac compression alone is inadequate to provide adequate perfusion to vital organs and, therefore, cannot sustain life unless coupled with advanced therapeutic interventions. Many new techniques for increasing flow have been developed, but have not been established clinically. The American Heart Association guidelines for CPR are still valid and are the basis for our current CPR. A practical perspective is presented whereby the therapeutic interventions are pursued systematically in an expeditious and coordinated fashion so that the key interventions are made within the first 10 to 15 minutes of the arrest. PMID- 3012226 TI - Congestive heart failure. AB - A review of the epidemiology, pathophysiology, and treatment of congestive heart failure is presented, with particular attention given to newer modalities of therapy. PMID- 3012227 TI - Modulatory effect of glucans on the functional and biochemical activities of guinea-pig macrophages. AB - The particulate and soluble glucan preparations isolated from Saccharomyces cerevisiae and Candida albicans affected various activities of guinea-pig macrophages. The stimulation of INT reduction and superoxide production were observed in the presence of all insoluble and some soluble glucans in vitro, but most of the preparations under study had an inhibitory effect on phagocytic activity. On the other hand, the stimulation of both metabolic and functional activities was obtained in vivo. Macrophages from guinea-pigs treated with glucans exerted an increased ability to reduce INT and to produce superoxide. Their candidacidal capacity was rapidly elevated, and peritoneal macrophages had raised phagocytic activity as well. A more pronounced stimulatory effect was observed in the presence of insoluble rather than soluble glucans, and this was more expressive in vitro than in vivo. PMID- 3012228 TI - [Electric stimulation of a sensory nerve with 70-micrometer electrodes]. AB - Using the device of an implant (Fig. 1) for direct stimulation of the eighth cranial nerve (Zwicker et al., 1986) measurements have been performed after implantation of one electrode in the nervus suralis of the first author. The results show that threshold of sensation is reached at voltage amplitudes of about 600 mV for sinusoidal stimuli almost frequency independent in the range between 100 Hz and 3 kHz (Fig. 2). The impedance of the electrode (Fig. 3) was found to be remarkably smaller compared with values measured by Zollner (1982) in Ringer's solution. No clear relation between threshold value or impedance and active electrode area could be detected. However, there was a clear dependence of the threshold voltage on the angle between the direction of puncture of the electrode and the direction of the nerve. Parallel puncturing resulted in a 13 dB less sensitive threshold in relation to perpendicular puncturing. The practical dynamic range between the threshold of sensation and the threshold of pain was found to be 10 to 12 dB. PMID- 3012229 TI - [Preliminary studies of deaf patients assessed for cochlear implantation]. AB - Two techniques were employed to assess the suitability of deaf patients for cochlear implantation: electrical stimulation by means of ear canal electrode and promontorium electrode. The results of the ear canal tests indicate that the patients may be divided into three groups: I. non-usable dynamics, II. usable dynamics in low-frequency range, and III. usable dynamics over a wider frequency range. Comparable results were obtained via the promontory tests. Since the promontory tests showed a more accurate description of the subjective sensations, it is recommended that this test be performed with those patients who have been preselected on the basis of ear canal stimulation. PMID- 3012230 TI - [Papilloma viruses in benign and malignant tumors of the mouth and upper respiratory tract]. AB - A total of 113 benign and malignant tumours of the mouth and upper respiratory tract have been analysed for the presence of papilloma virus DNA. Thirty-four (63%) of 54 papillomatous lesions were found to contain such DNA. Seventy-two percent of the laryngeal papillomas contained HPV 6 or HPV 11 DNA, whereas the oral papillomatoses harboured HPV 6 (15.6%), HPV 11 (9.4%), HPV 7 (9.4%), HPV 13 (12.5%) and HPV 32 (9.4%) DNAs. The high number of HPV 7 DNA-positive lesions was unexpected. In most of the malignant tumours no papilloma-viral DNA was found, with the exception of 3 tongue-base carcinomas, and in a metastasis from one of the tumours, which contained HPV 16 (3 biopsies) and HPV 2 related sequences (1 biopsy). PMID- 3012231 TI - Effect of ethanol on the binding of 35S-T-butylbicyclophosphorothionate to mouse brain membranes. AB - The effect of in vitro addition of ethanol (0.02-1.0 M) on the binding of 35S TBPS was examined in brain membranes from cerebellum and cortex of naive or chronically ethanol-treated C57B1 mice. In brain membranes of untreated animals, increasing concentrations of ethanol produced a dose-related inhibition of 35S TBPS binding in the brain areas investigated. Additional studies showed that this effect of ethanol was due to a decreased affinity of 35S-TBPS for its binding sites. Chronic treatment of the animals with ethanol, which produced tolerance to and dependence on ethanol, did not alter ethanol's ability to inhibit the binding of 35S-TBPS. In naive animals, the in vitro addition of GABA or pentobarbital produced a pronounced inhibition of 35S-TBPS, both drugs being more potent in the cerebellum than in the cortex. Picrotoxin also produced a dose-dependent inhibition at 35S-TBPS, but was equally potent in the brain areas investigated. The inhibition by GABA or pentobarbital was not influenced by in vitro addition of a physiologically relevant concentration of ethanol (100 mM), whereas ethanol produced a significant increase in the IC50 values for picrotoxin both in the cortex and in the cerebellum. Furthermore, the inhibitory effects of GABA or pentobarbital on 35S-TBPS binding remained unchanged in animals chronically treated with ethanol for 7 days. Our data indicate that ethanol may affect the GABA receptor system through a rather specific interaction with the 35S-TBPS recognition site, but that this action of ethanol is not altered by the development of tolerance to and dependence on ethanol. PMID- 3012232 TI - Hyperprolactinemia in nonpregnant women due to pituitary tumors. AB - The human prolactin molecule has been isolated and its structure characterized. This anterior pituitary hormone plays an important function in the induction and maintenance of lactation in the post-partum nursing mother. Prolactin-producing tumors cause inappropriate lactation in the nonpregnant woman. Bromocriptine, an ergot derivative, mimics the action of dopamine in the anterior pituitary gland and does not cure the underlying pathology. Prior to the development of bromocriptine, there was no effective treatment for the symptoms of amenorrhea and galactorrhea. Although the methods of therapy are more sophisticated today, there remain a number of unanswered questions. The unknown long-term risks of bromocriptine therapy must be balanced against the potential risk of osteopenia. PMID- 3012233 TI - Distribution of endogenous benzodiazepine receptor ligand-monoamine oxidase inhibitory activity (tribulin) in tissues. AB - The distribution of monoamine oxidase inhibitor-benzodiazepine receptor binding inhibitor, extractable into ethyl acetate at pH 1, was examined in a range of rat tissues. Great variation in the activity of both inhibitors was found in the different tissues, the highest being present in superior cervical ganglion, and lowest in adrenal gland. There was a highly significant correlation between the distribution of the two activities in different tissues, supporting the concept that they both derive from the same molecule (tribulin). The level of inhibitory activity in some of the tissues was such that variations might conceivably play a significant role in vivo. PMID- 3012234 TI - Alpha-2 adrenergic receptor subtypes indicated by [3H]yohimbine binding in human brain. AB - Pharmacologic characterization of mammalian alpha-2 adrenergic receptors in various tissues and species has provided evidence for the existence of two alpha 2 adrenergic receptor subtypes. Prazosin and oxymetazoline have been shown to differentiate between the receptor subtypes as defined in rat tissues. In order to determine the relative proportions of these two receptor subtypes in human brain, the inhibition of the binding of the alpha-2 adrenergic antagonist [3H]yohimbine by oxymetazoline and prazosin was studied in membranes from three brain regions. Inhibition curves in membranes from the cerebral cortex and cerebellum were consistent with a single class of receptor binding sites suggesting that these two brain regions contain only one of the two subtypes. This subtype has the pharmacologic characteristics of the alpha-2A adrenergic subtype (yohimbine greater than oxymetazoline much greater than prazosin). In contrast, inhibition curves for both ligands in the human caudate nucleus were consistent with a model of two classes of binding sites in approximately equal proportions, suggesting that this tissue contains approximately equal densities of the alpha-2A and alpha-2B adrenergic receptor subtypes. PMID- 3012235 TI - The effects of glutaraldehyde cross-linking on the function of the adenylate cyclase complex of turkey erythrocytes. AB - Glutaraldehyde appears to preferentially effect the activation processes of the adenylate cyclase complex of turkey erythrocyte membranes. The primary effect of low concentration (0.01 percent, 0.05 percent, and 0.1 percent) glutaraldehyde membrane treatment is to decrease catecholamine-stimulated cAMP formation. The effect can be blocked by prior activation of the system with isoproterenol + p[NH]ppG. 0.6 percent glutaraldehyde treatment of membranes has substantial effects on both F(-)- and catecholamine-stimulated cAMP production. The effects are blocked by prior activation of the adenylate cyclase complex with NaF, but not by isoproterenol + p[NH]ppG. Glutaraldehyde at these concentrations has no effect on Mn++-stimulated cAMP formation. The data is discussed with respect to the organization of the major macromolecular components of the adenylate cyclase complex as it exists within the native membrane prior to and following activation of the system. PMID- 3012237 TI - [Gamma-topographic study of patients with pathological orbital processes]. AB - A gamma-topographic study of the orbit was performed in 64 patients with unilateral exophthalmus using 99m Tc-pertechnetate. Some peculiarities in the RP distribution in the focus of lesion in malignant and benign neoplasms and inflammatory processes in the orbit were revealed. Correlation between RP accumulation intensity in the focus and the degree of RP vascularization and volume was shown. The method does not allow assessment of the morphological nature of a process however its use extends the diagnostic potentialities in specifying the genesis of unilateral exophthalmus and facilitates the choice of appropriate therapy. PMID- 3012236 TI - Endogenous ligands for sigma opioid receptors in the brain ("sigmaphin"): evidence from binding assays. AB - Two endogenous ligands which interact preferentially with the sigma opioid receptors were identified from the guinea-pig brain extract in a Sephadex G-50 fractionation. These two ligands inhibited more potently the binding of [3H]SKF 10047 to sigma opioid receptors than [3H]naloxone to mu opioid receptors, [3H]ethylketocyclazocine to kappa opioid receptors and [3H]DADLE to delta opioid receptors. In the phencyclidine receptor assay, these two ligands were almost inactive. Incubation of these ligands with trypsin destroyed at least 50% of the activities in the sigma opioid receptor assay. Both ligands inhibited the sigma binding in a dose-dependent manner. The inhibition could be eliminated when the two ligands were removed from incubation media by extensive washings. It is therefore concluded that sigma opioid receptors are not phencyclidine receptors and that endogenous ligands for sigma opioid receptors may exist in the brain. PMID- 3012238 TI - [Radiation doses to organs and tissues outside the irradiation field during radiotherapy of nephro- and neuroblastoma in children]. PMID- 3012239 TI - [Comparative evaluation of the long-term results of treatment of cervical cancer using radiocolloids]. AB - The author provided a comparative analysis of the results of therapy of 127 cervical cancer patients. 198Au-(75 patients) and 90Y-colloids (52 patients) were administered intraparametrially for adjuvant radiotherapy. The summary absorbed dose in combined modality radiation therapy was 90-100 Gy at the point A and 40 50 Gy at the point B. The author presented 5- and 10-year survival rates which were higher with 90Y-colloid as compared to those with 198Au-colloid. Early radiation reactions and late radiation injuries developed somewhat more frequently with 90Y. However statistical differences were insignificant. Thus, combined modality radiation therapy of cervical cancer using 90Y-colloid for adjuvant radiotherapy considerably enhances therapeutic efficacy without increasing the number of radiation complications. PMID- 3012240 TI - An alternative building material. PMID- 3012241 TI - Effects of antimalarial drugs on oxygen consumption by erythrocytes infected with Plasmodium berghei: an ESR study. AB - Oxygen consumption in mouse erythrocytes infected with Plasmodium berghei has been followed by electron spin resonance (ESR) spectroscopy of nitroxide radical spin probes. The parasitized red cell suspension is mixed with the spin probe CTPO (3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy) in a closed chamber. Oxygen consumption is monitored by the increasing resolution of the superhyperfine splittings of the spin label. The antimalarial drugs quinacrine, primaquine, and quinine are shown to decrease the rate of oxygen consumption of the parasitized erythrocyte suspensions. The spin-label method offers advantages over conventional polarographic and spectrophotometric assays for highly parasitized cell populations where cells are fragile and contain oxidized hemoglobin as well as hemoglobin-derived pigments. PMID- 3012242 TI - [Biological effect of fibrous mineral dust. I. Fibrogenic properties of the products of the thermal degradation of chrysotile]. AB - Chrysotile asbestos samples, on comminution, were exposed to temperatures typical for asbestos products exploitation. Subsequently, in an experiment on white rats the effects of temperature upon asbestos fibrous effects were evaluated. The highest aggressiveness was that of the sample heated at 600 degrees C. All the animals died as soon as 50 or 25 mg of dust had been administered intratracheally. Only 3 samples could be administered intratracheally at a dose of 50 mg: non-heated. heated at 400 degrees C and heated at 800 degrees C samples. The doses heated at 400 and 800 degrees C had be reduced by half. Samples heated at 150 to 800 degrees C induced higher hydroxyproline increases than the non-heated sample. The fibrogenic activity of the dust heated at 1200 degrees C was very similar to the fibrogenic activity of the non-heated dust. PMID- 3012243 TI - [Chronic manganese poisoning]. PMID- 3012244 TI - Bone resorption and hypercalciuria in calcium stoneformers. AB - The circadian rhythm of urinary total hydroxyproline (THP) excretion was determined in matched groups of ten male idiopathic calcium stoneformers and ten normal subjects in order to determine whether enhanced resorption of bone might contribute to hypercalciuria in these patients. THP increased progressively in normal subjects in successive eight-hour urine collections from period 1 (8 AM to 4 PM) to period 3 (12 midnight to 8 AM), the nocturnal high level in period 3 being significantly greater than in period 1 (P less than 0.01) and in period 2 (P less than 0.05). By contrast, no significant circadian rhythm was observed in THP excretion in the stoneformers. Their THP excretion was similar to that of normal subjects in period 3, but was significantly higher than that of normal subjects in period 1 (THP/creatinine ratio(mg/mg): 0.026 +/- 0.003 v 0.017 +/- 0.001; P less than 0.05. Indices of parathyroid hormone activity were not significantly different between stoneformers and normal subjects; mean serum 1,25(OH)2 vitamin D levels were higher in the stoneformers than the normal subjects (44 v 37 pg/mL) but the difference was not significant (P greater than 0.05). These studies suggest that increased bone turnover may contribute to hypercalciuria in these calcium stoneformers. PMID- 3012245 TI - Enhancement of cyclic adenosine monophosphate content in bone cells by the factor extracted from a pancreatic cancer associated with hypercalcemia. AB - A woman with exocrine pancreatic cancer presented a syndrome of humoral hypercalcemia of malignancy (HHM). Either urea extract or acid/ethanol extract of the tumor showed a dose-dependent activity to elevate cyclic adenosine monophosphate (AMP) level in rat bone cells in primary culture. When each population obtained by the sequential digestion of rat fetal calvaria was cultured individually and cyclic AMP responses to parathyroid hormone (PTH), calcitonin, and tumor extract were examined, tumor extract-sensitive cells showed a similar distribution to PTH-sensitive cells. Tumor extract and PTH, but not calcitonin, increased cyclic AMP in osteogenic cell line MC 3T3-E1. PTH receptor mediated increase of cyclic AMP was indicated by an antagonistic action of PTH analogue, (3-34) hPTH, on increase of cyclic AMP in MC 3T3-E1 elicited by tumor extract. Human breast cancer derived cell line MCF-7 had calcitonin-sensitive adenylate cyclase, but neither PTH nor tumor extract increased cyclic AMP in the cells. On Bio-Gel P-60 column, the activity to stimulate bone cell cyclic AMP was eluted as a single peak at the molecular size between 6.5 K and 12.4 K. It was concluded that pancreatic cancer, although rather exceptional as a cause of HHM, produced a factor very similar to that reported in representative HHM tumors of human and animal models. PMID- 3012246 TI - Skeletal alkaline phosphatase activity as a bone formation index in vitro. AB - These studies were intended to examine the relationship between skeletal collagen formation and skeletal alkaline phosphatase (ALP) activity in vitro. Embryonic chick calvaria were exposed to skeletal effectors (including high and low pH, a range of [pi] and [Ca], PTH, NaF, etc), and collagen formation was assessed by the incorporation of 3[H]-proline as 3[H]-hydroxyproline (3[H]-hyp). ALP activity was measured in the serum-free conditioned medium and in 20% butanol extracts of the bones. We found that ALP activity and 3[H]-hyp incorporation were coordinately increased from pH 5.5 to pH 7.2 (r = .99, P less than 0.001). Calvarial ALP was not increased in response to low [Pi], but low [Ca] increased ALP and coordinately decreased collagen formation (r = -.81, P less than 0.05). Although calvarial ALP and 3[H]-hyp incorporation were coordinately increased by NaF, vanadate, PGE2, calcitonin, and insulin, the slopes of the correlations were not the same for all effectors (eg, NaF: r = .97, P less than 0.01, slope = 0.90; vanadate, r = .95, P less than 0.005, slope = 0.20), indicating differential actions on ALP and 3[H]-hyp incorporation. When a variety of effectors, including low [Ca], were used to treat different groups of calvaria, ALP activity was not correlated with 3[H]-hyp incorporation (r = .35), but when the exposure to effectors was limited to a preincubation, or when the low [Ca] data were excluded, a correlation was observed (r = .87, P less than 0.001, and r = .64, P less than 0.02, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012247 TI - Solubilization of neuropeptide receptors. PMID- 3012248 TI - Solubilization and characterization of opiate receptors. PMID- 3012249 TI - Luciferin derivatization of ligands for development of nonisotopic binding assays. PMID- 3012251 TI - Synthesis and use of colloidal gold-coupled receptor ligands. PMID- 3012250 TI - Use of vaccinia virus as a neuropeptide expression vector. PMID- 3012252 TI - Detection and measurement of secretion from individual neuroendocrine cells using a reverse hemolytic plaque assay. AB - Recent advances in technology have dramatically increased the resolution with which we may examine many features of biological systems. Intracellular recording and tracer injection techniques allow one to study the function of individual neurons and later characterize the same cells morphologically. In situ hybridization techniques can give us information about messenger RNA levels in single cells. More established techniques such as immunocytochemistry and electron microscopy also provide information at the cellular and even subcellular level. With each of these technological advances we have learned more about the mechanisms underlying cell function. We are also beginning to appreciate the role of heterogeneity among cells in relation to the function of the whole organism. Application of the reverse hemolytic plaque assay to the study of hormone or neurotransmitter secretion should help clarify this role. This technique permits accurate quantitation of hormone secreted from a large number of cells. Thus while cells can be studied individually they can also be categorized into functional subpopulations. As discussed in this chapter, many other techniques may be applied on cells which have already been functionally defined with the plaque assay. This should result in a clearer understanding of the roles of secretagogue binding and internalization, activation of second messenger systems, protein synthesis, and the cytoskeleton in hormone secretion. In the plaque assays described in this chapter individual pituitary cells are isolated in culture free from possible interactive effects coming from other cells. While these interactions are no doubt critical to the understanding of the function of the organism as a whole they can result in totally uninterpretable results. In fact, when we have gained some understanding into the functioning of individual cells it should be possible using the plaque assay to study the interactions among cells in a controlled fashion. PMID- 3012253 TI - Preparation and use of biotinylated neuroendocrine peptides. PMID- 3012254 TI - Corticotropin-releasing factor receptors in the pituitary gland and central nervous system: methods and overview. AB - Studies with the radioiodinated oCRF analog, Nle21, 125I-Tyr32-oCRF have identified, characterized, and localized high affinity binding sites for CRF in anterior and intermediate lobes of rat pituitary, in anterior lobe of human pituitary, and in rat, monkey, and human brain. The pharmacology and distribution of Nle21, 125I-Tyr32-oCRF binding in the pituitary gland correlate well with the biological potency and sites of action of CRF and suggest that these CRF binding sites represent specific receptors that mediate the well-established actions of CRF on the anterior pituitary and on the intermediate lobe of the pituitary. The studies in adrenalectomized rats demonstrating that endogenous CRF is capable of modulating its receptor density provide additional evidence that the radioligand labels the functional CRF receptor. The areas of distribution of Nle21, 125I Tyr32-oCRF binding sites in the rat CNS correlate well with the immunohistochemical distribution of CRF pathways and the pharmacological sites of action of CRF. These data confirm the established role of CRF in regulating secretion of POMC-derived peptides from the pituitary gland. In addition, the data support a physiological role for endogenous CRF in regulating CNS activity and suggest the importance of this neuropeptide in integrating endocrine and visceral functions and behavior, especially in response to stress. Studies to characterize CRF receptors and CRF-containing pathways in the brain provide a means for better understanding the various functions of this neuropeptide in different areas of the CNS. Finally, the ability to map CRF receptors in postmortem human tissue provides a basis for studying the role of CRF in a variety of endocrine, neurological, and psychiatric disorders. PMID- 3012255 TI - Autoradiographic localization of brain receptors for peptide hormones: angiotensin II, corticotropin-releasing factor, and gonadotropin-releasing hormone. PMID- 3012256 TI - Use of secretion cloning vectors for guiding the localization of proteins in Escherichia coli. PMID- 3012257 TI - Use of transposons to isolate and characterize mutants lacking membrane proteins, illustrated by the sugar transport systems of Escherichia coli. PMID- 3012258 TI - Isolation of highly coupled heart mitochondria in high yield using a bacterial collagenase. PMID- 3012259 TI - Alkyl glycoside detergents: synthesis and applications to the study of membrane proteins. PMID- 3012260 TI - Mitochondrial outer membrane channel (VDAC, porin) two-dimensional crystals from Neurospora. PMID- 3012261 TI - Reconstitution of ADP/ATP translocase in phospholipid vesicles. PMID- 3012262 TI - Fluorescent nucleotide analogs as active site probes for the ADP/ATP carrier and the uncoupling protein. PMID- 3012263 TI - Techniques for NMR and ESR studies on the ADP/ATP carrier. PMID- 3012264 TI - Fluorescent probes of the mitochondrial ADP/ATP carrier protein. PMID- 3012265 TI - ADP/ATP carrier: analysis of transmembrane folding using pyridoxal phosphate. PMID- 3012266 TI - Chemical modifications and active site labeling of the mitochondrial ADP/ATP carrier. PMID- 3012267 TI - Isolation and reconstitution of the phosphate transport protein from mitochondria. PMID- 3012268 TI - Evidence in a nematode for regulation of transposon excision by tissue-specific factors. AB - The transposable element Tc1 in Caenorhabditis elegans undergoes an excision reaction, which can be detected in a Southern hybridization as the appearance of empty chromosomal insertion sites. This excision reaction is under tissue specific regulation in that it occurs at much higher frequency in somatic cells than in the germ line. We show here that this regulation is likely to be due to the action of tissue-specific factors that either promote excision in somatic tissues or repress it in the germ line. The rate of excision of elements at five distinct chromosomal sites has been measured by a method that avoids ambiguities due to cell division. All these elements are found to undergo excision at closely similar rates during the L1 larval stage. No distinct difference exists among the elements at different sites that would suggest regulation by flanking sequences. PMID- 3012269 TI - Suppression of the Escherichia coli dnaA46 mutation by amplification of the groES and groEL genes. AB - A lambda hybrid phage (lambda Sda1), containing an 8.1 kb EcoRI DNA fragment from the Escherichia coli chromosome, was selected on the basis of its ability to suppress bacterial thermosensitivity caused by the dnaA46 mutation. We have shown that this suppression is due to a recA+-dependent amplification of the 8.1 kb fragment; consistent with this observation, cloning of the 8.1 kb fragment into a high copy number plasmid (pBR325) leads also to suppression of dnaA46. In the suppressed strains growing at high temperature, bidirectional replication starts in or near the oriC region and requires the presence of the DnaA polypeptide. These findings suggest that the overproduction of a gene product(s), encoded by the cloned 8.1 kb fragment, can restore dnaA-dependent initiation of replication at high temperature in the oriC region. Genetic mapping shows that the groES (mopB) and groEL (mopA) genes are located on the 8.1 kb suppressor fragment. Further analysis, including in vitro mutagenesis and subcloning, demonstrates that the amplification of the groES and groEL genes is both necessary and sufficient to suppress the temperature sensitive phenotype of the dnaA46 mutation. PMID- 3012270 TI - A DNA fragment containing the groE genes can suppress mutations in the Escherichia coli dnaA gene. AB - An 8.2 kb fragment of E. coli chromosomal DNA, when cloned in increased copy number, suppresses the dnaA46 mutation, and an abundant protein of about 68 kd (60 kd when measured by us), encoded by the fragment, is essential for the suppression (Takeda and Hirota 1982). Mapping experiments show that the fragment originates from the 94 min region of the chromosome. It encodes several proteins but only one abundant polypeptide of the correct size, the product of the groEL gene. Suppression by the fragment is allele specific; those mutations which map to the centre of the gene are suppressed. Other initiation mutants including dnaA203, dnaA204, dnaA508, dnaAam, dnaC, dnaP and dnaB252 are not suppressed. Most suppressed strains are cold-sensitive suggesting an interaction between the mutant proteins (or their genes) and the suppressing protein or proteins. PMID- 3012271 TI - A molecular genetic approach to the functioning of the immunity protein to colicin A. AB - A plasmid (pColAF1), derived from pColA, and lacking the region encoding Cai (colicin A immunity protein) and Cal (colicin A lysis protein) has been constructed. The strains carrying pColAF1 produce normal amounts of colicin A which remains in the cell cytoplasm and does not result in loss of viability. Similar results have also been obtained for transposon insertion mutants lacking Cai. Structure prediction analysis indicates that four peptide regions of Cai might span the cytoplasmic membrane. Since the NH2- and COOH-terminal regions are charged, this analysis suggests a topology of the 178 residues polypeptide chain in which regions 38 to 70 and 124 to 143 might be exposed at the outer side of the cytoplasmic membrane. With mutants constructed using recombinant DNA techniques, we could demonstrate that the removal of a 30 residue COOH-terminal region, and mutations altering the surface exposed loop comprised of aminoacid residues 124-143 abolish the protecting function of Cai. PMID- 3012272 TI - Physical and genetic structure of the glpD-malT interval of the Escherichia coli K-12 chromosome. Identification of two new structural genes of the glp-regulon. AB - A transducing lambda phage carrying glpD''lacZ, glpR, and malT was isolated from a strain harboring a glpD''lacZ fusion. Comparison of restriction endonuclease cleavage patterns of DNA isolated from this phage with that of the previously cloned malT region (Raibaud and Schwartz 1980) facilitated the construction of recombinant plasmids carrying different portions of the glpD-malT region. Results of minicell analysis and complementation studies showed that this region of the chromosome encodes at least five polypeptides. These included the previously identified glpD, glpR, and malT gene products. In addition, two new structural genes of the glp regulon (glpE and glpG) located between the glpD and glpR genes were identified. Hybrid plasmids carrying glpD''lacZ and glpR''lacZ fusions were constructed. Restriction endonuclease cleavage analysis of these two plasmids demonstrated that glpD and glpR are divergently transcribed. PMID- 3012273 TI - Organization of transcription units around the Drosophila melanogaster rudimentary locus and temporal pattern of expression. AB - The molecular organization of 90 kb of DNA derived from a region of the X chromosome that encompasses the rudimentary locus of D. melanogaster is presented. This segment spans the cytogenetic region 14F2-3 to 15A1-2, and there are, in addition to the rudimentary gene several transcription units present, whose functions are still unknown. We have determined the pattern of expression of all these genes at several stages of development, and found that they all show a different temporal modulation of their activity. The accumulation of the r product correlates well with the enzymatic activity determined for the protein product of the gene, being highest in very early embryos and adult females. PMID- 3012274 TI - Shadow promoters in the F plasmid transfer operon. AB - The in vivo transcription start site for PYZ, the promoter of the F plasmid transfer operon, has been located. When transcription start sites for PYZ are deleted, two additional shadow promoters, upstream of PYZ, are activated in vivo. The shadow promoters correspond to those previously identified by in vitro run off transcription experiments. The activated shadow promoters are stimulated by TraJ protein, a positive regulator of the transfer operon. PMID- 3012275 TI - Illegitimate recombination mediated by T4 DNA topoisomerase in vitro. Recombinants between phage and plasmid DNA molecules. AB - Illegitimate recombination dependent on T4 DNA topoisomerase in a cell-free system has recently been described. In that work, recombinants between two phage lambda DNA molecules were produced by the topoisomerase alone, without an Escherichia coli extract. In this paper, it is shown that recombination between phage lambda and circular plasmid DNA molecules can also be detected in the presence or absence of an E. coli extract but at frequencies two or three orders of magnitude lower than that observed in the phage-phage cross. The frequency is probably lower because multiple recombination is required in the case of the phage-plasmid cross. PMID- 3012277 TI - Cloning of the arg-12 gene of Neurospora crassa and regulation of its transcript via cross-pathway amino acid control. AB - The arg-12 locus of Neurospora crassa encodes ornithine carbamoyl transferase, which is one of many amino acid synthetic enzymes whose activity is regulated through cross-pathway (or general) amino acid control. We report here the use of probes derived from the functionally equivalent arg-B gene of Aspergillus nidulans to identify and clone a 10 kb Neurospora DNA fragment carrying the arg 12 gene. Short Neurospora DNA probes derived from this fragment were used to identify a 1.5 kb polyA+ transcript of the arg-12 region. Arg-12 transcript levels increased approximately 20 fold under conditions of arginine or histidine limitation in strains having normal cross-pathway regulation (cpc-1+) but showed no such response in a cpc-1 mutant strain impaired in this regulation. Time course studies in cpc-1+ strains revealed a rapid response (within 10 m) of arg 12 transcript levels following inhibition of histidine synthesis by 3 amino 1,2,4 triazole, but a delayed response following arginine deprivation of an arginine requiring strain. In contrast to the behaviour of arg-12 mRNA, the level of the Neurospora am gene transcript (specifying NADP dependent glutamate dehydrogenase) was unaffected either by amino acid limitation or by the cpc-1 mutation. A possible role for the cpc-1+ product as a positive regulator of transcription of genes subject to cross-pathway control is discussed. PMID- 3012276 TI - Transcription termination within the Escherichia coli origin of DNA replication, oriC. AB - Initiation of DNA replication from the Escherichia coli origin, oriC, is dependent on an RNA polymerase-mediated transcription event. The function of this RNA synthetic event in initiation, however, remains obscure. Since control of the synthesis of this RNA could serve a key role in the overall initiation process, transcription regulatory sites within and near oriC were identified using the galK fusion vector system. Our results confirm the existence of a transcription termination signal within oriC, first identified by Hansen et al. (1981), for the 16 kd transcript that is transcribed counterclockwise towards oriC. Termination is shown to be 92% efficient. A similar approach led to the detection of transcription termination within the chromosomal replication origin of Klebsiella pneumoniae. Approximately 50% of the E. coli 16 kd transcripts appear to terminate before reaching oriC between the XhoI (+416 bp) and the HindIII (+243 bp) sites. The predominant 3' ends of RNA that enter oriC, as determined by SI nuclease mapping, were located at positions +20 +/- 2, +23 +/- 2, +37, +39, +52, +66, +92, and +107. These termination sites, which map cl to RNA . DNA junctions identified by Kohara et al. (1985), appear as triplets and quadruplets. The E. coli oriC Pori-L promoter described in in vitro transcription studies by Lother and Messer (1981) was not detected in this study in either wildtype cells or isogenic dnaA mutants at the nonpermissive temperature. A new promoter activity, Pori-R1, was identified within the E. coli origin in the clockwise direction. PMID- 3012278 TI - The sequences of the traJ gene and the 5' end of the traY gene of the resistance plasmid R1. AB - The traJ gene and the 5' end of the neighbouring traY gene of the resistance plasmid R1 were sequenced. Both structural genes show relatively little homology with the corresponding sequences of the related F plasmid. At the amino acid level sufficient homology is detected to allow an assignment of the two genes in R1. In traJ two methionine codons have to be regarded as potential chain initiation signals. Because of the analogy with the F plasmid sequence the second ATG is believed to be the main translational start site. The traJ gene codes for 228 amino acids. The 5' untranslated region of the traJ gene of R1 is highly homologous to the corresponding sequence in F indicating that it fulfills an important role in regulation. The transcription of the traY-Z operon starts in the structural gene of traJ. The amino terminal part of the TraY protein shows only limited homology with the F factor counterpart. However, the few conserved amino acids are a strong indication that our sequence contains the traY gene of R1. PMID- 3012279 TI - Control of replication of the broad host range plasmid RSF1010: the incompatibility determinant consists of directly repeated DNA sequences. AB - A 500 bp DNA fragment located in the vicinity of the origin of replication of plasmid RSF1010 was cloned into the plasmid vector pBR322 and shown to exhibit incompatibility against parental RSF1010. The rightmost region of this fragment contains three perfect 20 bp direct repeats and a fourth half-repeat of 11 bp, as shown by DNA sequencing. Deletion of the four repeats from the cloned fragment resulted in complete loss of incompatibility whereas partial deletion of the repeated sequence resulted in a concurrent decrease in the expression of incompatibility. We conclude that the incompatibility determinant of RSF1010 is defined by the four repeats and also that the incompatibility expressed is not very strong, since the presence of about 1.5 times as many copies of the repeated sequence as are normally in a cell does not cause a total switch off of RSF1010 replication, but only a 40% reduction in the rate of replication. PMID- 3012280 TI - Molecular cloning and characterization of the STA2 glucoamylase gene of Saccharomyces diastaticus. AB - The Saccharomyces diastaticus structural gene STA2, encoding an exracellular glucoamylase (1,4-alpha-D-glucan glycohydrolase, EC 3.2.1.3.), has been cloned by complementation of a stao strain. A genomic library was initially constructed from a STA2 yeast strain in the yeast Escherichia coli shuttle cosmid vector pYCl. The Sta+ complementing function was further delimited to an 8.3 kb BglII fragment whose restriction map was found to be similar to related genomic regions of STA1 and STA3. Fusions of several DNA fragments derived from the 8.3 kb BglII fragment with a truncated E. coli beta-galactosidase gene resulted in two overlapping fragments that could direct the production of large fusion proteins in E. coli. These fusion proteins were immunoprecipitable by anti-glucoamylase II antibodies, confirming that the Sta+ complementing fusion was due to the expression of a gene that coded for a yeast glucoamylase. Measurements of the STA1, STA2 and STA3 RNA transcripts by RNA-DNA hybridization using an internal fragment of the cloned STA2 gene as the probe indicated that a common transcript of 2.5 kb is produced by each of the STA genes. Integrative disruption of the STA2 gene through homologous recombination was achieved by transforming a STA2 yeast strain to Sta- using an in vitro constructed donor DNA fragment that has the URA3 gene inserted within the coding region of the cloned glucoamylase gene. This was confirmed by tetrad analysis of crosses between strains carrying a disrupted STA2 and a functional STA2. Southern blot analysis using BamHI digested genomic DNA from 15 tetrads demonstrated consistent co-segregation and Mendelian inheritance of the Sta- phenotype with STA2::URA3. These data further confirm that the cloned DNA that showed Sta+ complementing activity carries a functional STA2 gene that encodes the yeast extracellular glucoamylase II. PMID- 3012281 TI - Cloning and expression in Escherichia coli of a recA-like gene from Vibrio cholerae. AB - A library containing more than 80% of the Vibrio cholerae genome was constructed by cloning BamH1 restriction fragments into pBR322. Using interspecific complementation of an Escherichia coli recA mutant with plasmids containing the gene bank of V. cholerae, a recA-like gene was identified. The recombinant plasmid, designated as pDP145, contained a 1.45 kb segment of V. cholerae DNA which codes for a protein of molecular weight 39,000. The product of this gene confers methyl methane sulphonate resistance on the E. coli recA mutant, suppresses its ultraviolet (UV) light sensitive phenotype and has proteolytic activity on the phage lambda repressor. Induction of a 39,000 dalton protein in UV-irradiated V. cholerae cells was demonstrated. PMID- 3012282 TI - Molecular cloning and characterization of sequences from the regulatory cluster of the Pseudomonas plasmid alk system. AB - Alkane oxidation functions encoded by the Pseudomonas plasmid CAM-OCT are positively regulated by one or more products of a locus designated alkR. To characterize this locus in greater detail, molecular cloning and restriction mapping of sequences covering the alkR region have been carried out in Escherichia coli, followed by mobilization to Pseudomonas recipients for analysis of genetic content. Inserts from Pseudomonas (CAM-OCT) strains were cloned into vectors pLAFR1, the pLAFR1::Tn7S derivative pXJS5403, and the transposon vector Tn3 delta 596. This has made it possible to: (1) construct a detailed restriction map of cloned fragments and the alkR region of CAM-OCT; (2) map insertion sites of the transposon Tn7S into alkR cistrons; and (3) analyze the ability of cloned sequences to complement or effect marker rescue of alkR nitrosoguanidine- and Tn7S-induced mutations. In addition, transcription of an alkB'-lacZ transcription fusion in the presence of a cloned 18.5 kb EcoRI alkR fragment and an inducer of the alk system confirmed that our cloned sequences contain functional alkR cistrons. The complementation/marker rescue results indicate that alkR is a complex locus and that the products of at least three cistrons are required for the complete AlkR+ phenotype. One of these cistrons is identified by mutations which alter a component of the inducer recognition system. PMID- 3012283 TI - A 2.6 kb DNA sequence of Streptomyces coelicolor A3(2) which functions as a transposable element. AB - Streptomyces coelicolor A3(2) contains CCC DNA molecules, 2.6 kb in size, with an average copy number of less than one per ten chromosomes. Southern hybridisation revealed, in addition, two linear, integrated copies (A and B) of this "mini circle" sequence per chromosome. The two integrated copies have similar (if not identical) ends and are present in the same locations in various S. coelicolor A3(2) derivatives. The mini-circle sequence is absent from S. lividans 66 and S. violaceolatus ISP5438 and from several Streptomyces species less closely related to S. coelicolor A3(2). None of a variety of Streptomyces plasmids tested contained homology to the mini-circle sequence. When a 1.8 kb fragment of the mini-circle lacking the ends of the integrated copies was inserted into KC515 (a derivative of the temperate phage phi C31 which is unable to lysogenise host strains by the natural route because the phage attachment site has been deleted) the resulting phage lysogenized S. coelicolor A3(2) (integrating into the genome of this host by homologous recombination with resident minicircle sequences) but not S. lividans or a variety of other phi C31 hosts. In contrast, a KC515 derivative (KC591) carrying the entire 2.6 kb mini-circle sequence linearized at its single BclI site (and therefore containing the integration site of the free mini-circle) lysogenized not only S. coelicolor A3(2) but also S. lividans 66 and most other strains normally lysogenized by phi C31. The KC591 lysogens of the eight Streptomyces species tested contained a linear, integrated prophage with termini apparently identical to those of the linear mini-circle copies of S. coelicolor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012285 TI - Immunoglobulin M to the membrane of uninfected fibroblasts in primary human cytomegalovirus infections. AB - Thirty-two patients with primary human cytomegalovirus (HCMV) infection were examined for presence of antibody to the membrane (Me) of uninfected human embryonic lung fibroblasts (HELF) using an anti-complement immunofluorescence (ACIF) test. Patients were divided into three groups according to age: less than or equal to 12 months (11 cases), 2-6 years (8 cases), greater than or equal to 13 years (13 cases). Convalescent-phase sera from 13 out of 13 of the older and 6 out of 8 of the middle-aged group gave positive fluorescence staining of the membrane of living uninfected HELF monolayers, whereas only one out of 11 younger patients showed antibody reactivity to HELF Me. ACIF performed on IgM and IgG fractions of randomly selected Me antibody-positive sera, showed a reactivity to HELF Me restricted to antibody of the IgM class (Me IgM). HELF Me IgM were first detected during the second week after the onset of symptoms of primary HCMV infection and disappeared usually within 4-6 months. Me IgM were not detected in sera from patients with HCMV infections other than primary (i.e. recurrent or congenital), or with other herpesvirus infections and were not detected in sera from patients with severe immunological diseases. HELF Me IgM were shown not to be related to rheumatoid factor and their reactivity was shown to be restricted to HELF cells, for it was not observed in any of the other cell types tested. The determination of HELF Me IgM antibody appears to be a new, practical and useful serological tool for the diagnosis of primary infections in patients over 2 years. PMID- 3012284 TI - Cloning of Candida pelliculosa beta-glucosidase gene and its expression in Saccharomyces cerevisiae. AB - Candida pelliculosa var. acetaetherius is a strain of yeast which can utilize cellobiose as the carbon source. From a gene library prepared from this yeast, the beta-glucosidase gene has been cloned in a S. cerevisiae host using a chromogenic substrate, 5-bromo-4-chloro-3-indolyl-beta-glucoside as an indicator. It was proved by Southern analysis that the DNA fragment carrying the beta glucosidase gene originated from C. pelliculosa. beta-Glucosidase produced by S. cerevisiae transformants was secreted into the periplasmic space. In Candida, beta-glucosidase was not induced by cellobiose but was derepressed by lowering the concentration of glucose. The regulation of beta-glucosidase synthesis in S. cerevisiae carrying the cloned beta-glucosidase was not clear compared with that in Candida, however, the enzyme activity in low glucose medium (0.05%) was reproducibly higher than in high glucose medium (2%). We have found the sequence that controls the expression of the beta-glucosidase gene negatively in S. cerevisiae. PMID- 3012286 TI - Does restriction enzyme analysis reveal changes in herpes simplex virus DNA after latency in mouse ganglia? AB - Possible changes in the herpes simplex virus (HSV) genome after latent infection in mice were studied by restriction enzyme analysis. Lumbosacral ganglia of C 57 Bl mice, genitally infected with a low passage strain of HSV-1 were explanted 3-8 month post infectionem and cocultivated with human embryo fibroblasts. Four virus isolates reactivated from the ganglia were compared with the original virus inoculated into the mice. The cleavage patterns yielded by 7 restriction enzymes i.e. Eco RI, Hind III, Bam HI, Kpn I, Xba I, Bg1 II, HpaI - did not indicate any differences. PMID- 3012287 TI - A study on the sensitivity of bovine rotavirus to some chemical agents. AB - The behaviour of bovine rotavirus, strain 81/36 F, to some chemical agents was studied. The chemicals tested were all more or less effective, depending on their concentration and time of exposure under room temperature. It is suggested therefore, that they could be used as disinfectants in the case of rotaviral contamination. PMID- 3012288 TI - Plaque enhancement effect of sodium thiosulfate for foot-and-mouth disease viruses. AB - A plaque enhancement effect by the addition of sodium thiosulfate for foot-and mouth disease viruses was demonstrated when this salt was incorporated in agar and in agarose overlay media. Most of the mechanism is obscure, however, as one of the effects is that sodium thiosulfate seems to interact in a reversible manner against the plaque inhibitor action of neutral red in cellular cytoplasm. A plaque inhibitor contained in agar could be removed in some degree by the addition of this salt. PMID- 3012289 TI - Transmission of bovine leukemia virus (BLV) to immunocompromised monkeys: evidence for persistent infection. AB - Splenectomized or irradiated adult and untreated baby macacques were infected with bovine leukemia virus from continuously virus producing fetal lamb kidney cell cultures. Persisting high antibody titers were followed for 4 years in the adult and 16 months in the baby monkeys, using an ELISA technique. With the exception of a persistent leukocytosis in one adult monkey, the animals remained healthy throughout the observation period. Transfer of lymphocytes from this animal to a seronegative recipient led to anti-BLV seroconversion with a lag of 5 months. All other transfer experiments showed a negative result. Virus could be recovered from the newly infected baby macacques by means of lymphocyte/fetal lamb kidney cell cocultivation for up to 4 weeks after inoculation. All other attempts to recover virus from the infected animals at a later point were unsuccessful. PMID- 3012290 TI - Genomic and antigenic comparison of an equine herpesvirus 1 (EHV 1) isolate from the 1983 Lippizan abortion storm with EHV 1 reference strains. AB - An EHV 1 isolate from the Lippizan Stud at Piber, which caused the abortion and paresis outbreak in 1983, was investigated using 3 known subtype 1 and 2 subtype 2 strains for comparison. Broad-scale restriction enzyme analysis as well as cross-neutralization with hyperimmune sera produced in rabbits were performed, and SDS-PAGE of infected cell proteins was conducted on a limited scale. The Piber isolate was clearly classified as a subtype 1 strain of EHV 1, and showed closest resemblance in its restriction patterns with a British EHV 1 strain, which originated from an outbreak with paretic symptoms. A second Piber isolate from the same outbreak examined to limited extent only was practically indistinguishable from the first one, as could have been expected. A thoroughly controlled systematic vaccination program with existing commercial vaccines against EHV 1 should protect the endangered Lippizan horses population against the abortigenic and less certainly against the paretic syndromes caused by this virus. According to data presented, a protection against respiratory disease is less probable. PMID- 3012291 TI - Sequential changes in cytomegalovirus antigenic pattern during infection of renal transplant patients. AB - Changes in the antigenic pattern of cytomegalovirus occurring during the course of infection were studied in two renal transplant patients. The virus was isolated several times from each patient. After two to five passages in vitro, the immune precipitate formed either with guinea-pig antiserum raised to Davis reference strain, or with human serum taken from the same patient during the acute phase of the infection, was analysed by SDS-polyacrylamide gel electrophoresis. Isolates from each patient showed a slightly different antigenic pattern with both human and experimental antisera. These differences possibly reflect modulation of the expression of proteins during the course of viral infection in the patients and during adaptation to cell culture in vitro. PMID- 3012292 TI - Protection of mice against herpes simplex virus infection by a Lactobacillus casei preparation (LC 9018) in combination with inactivated viral antigen. AB - Heat-killed Lactobacillus casei YIT 9018 (LC 9018) cells enhanced the resistance to herpes simplex virus type 1 (HSV-1) in adult mice, but not significantly. The protection of mice against HSV-1 infection and the production of neutralizing antibodies were significantly enhanced by the administration of LC 9018 in combination with inactivated HSV-1 antigen. The optimal enhancement of resistance was seen in mice 14 days after the simultaneous administration of these substances. The resistance to HSV-1 infection in mice could be transferred with peritoneal exudate cells from syngeneic mice previously treated with LC 9018 alone and LC 9018 in combination with inactivated HSV-1 antigen or with thioglycollate broth, whereas the transfer of peritoneal exudate cells induced by thioglycollate broth alone and of spleen cells induced by LC 9018 in combination with thioglycollate broth or by thioglycollate broth alone was not effective. These results suggest that mouse peritoneal macrophages induced by the administration of LC 9018 in combination with inactivated HSV-1 antigen may play an important role in host defense mechanisms against HSV-1 infection. PMID- 3012293 TI - Nuclear scanning in the diagnosis and localization of parathyroid adenomas. AB - Technetium-thallium nuclear scanning was performed in 17 patients whose clinical and biochemical findings were suggestive of the presence of hyperparathyroidism. An adenoma was located by scanning in 12 patients. Ten of these 12 patients underwent surgery; the scan had located the adenoma correctly in all these patients. One patient with a negative result of a scan examination subsequently had an adenoma removed at operation. Thyroid pathology interfered with the interpretation of the scan. This technique is recommended as a useful preoperative procedure for the detection of parathyroid adenomas, and its role in the rapid evaluation of hypercalcaemia seems promising. A prospective study to compare the sensitivity and specificity of this technique with computerized tomographic scanning and ultrasound is warranted. PMID- 3012294 TI - Acute HTLV-III infection. A case followed from onset to seroconversion. AB - An acute febrile illness with mouth ulcers and a skin rash was observed in a 37 year-old homosexual man. Acute human T-lymphotropic virus type III (HTLV-III) infection was suspected. The clinical features were documented prospectively, and striking changes in circulating T lymphocytes were demonstrated shortly after the onset of the illness. Seroconversion for HTLV-III antibody occurred between 46 and 78 days after the onset of the illness. PMID- 3012295 TI - Human papilloma virus and cervical cancer. PMID- 3012296 TI - The glycaemic index of foods. AB - The glycaemic index is a measure of the extent to which the carbohydrate in a food can raise the blood glucose concentration and helps to identify foods which may be beneficial to a diabetic patient. This paper reviews the results that have been obtained so far with the glycaemic index approach, the factors that affect the glycaemic response, the problems that are associated with its measurement and its value in planning diabetic diets. Individual variation and variation among individuals in glycaemic responses are also discussed. PMID- 3012297 TI - Epidemiology of AIDS retrovirus in South Australia. PMID- 3012298 TI - Dietary fibre and fluid in the control of constipation in a nursing home population. AB - A 12-month study showed that constipation in institutionalized elderly patients can be eliminated effectively and safely by the planned use of dietary fibre and a controlled fluid intake. The diet of a group of volunteer nursing home patients (average age, 83.5 years) was supplemented to provide a daily dietary fibre intake of 25 g per person. Aperient use was almost eliminated, bowel function improved, and there appeared to be no adverse effects on body weight, or on nutritional or mineral status. PMID- 3012299 TI - Bacterial fermentation in the human large bowel. Time to change from the roughage model of dietary fibre? PMID- 3012300 TI - Acute myelogenous leukemia following complete remission of small cell carcinoma of the lung. AB - The treatment of patients with small cell carcinoma of the lung (SCCL) with combination chemotherapy and radiation has dramatically improved survival in the past decade. With this increased survival, long term complications of therapy are becoming apparent. We report a patient who died of acute myelogenous leukemia (AML) while in complete remission from SCCL. Review of the literature indicates that there may be an increased incidence of AML following successful induction of complete remission in patients with SCCL. PMID- 3012302 TI - [Effectiveness of opoka dust against fleas--plague vectors in the territory of the Ural and Emba interfluvial area]. PMID- 3012301 TI - Successful chemotherapeutic decompression of epidural malignant germ cell tumor. AB - A patient with a sacrococcygeal malignant germ cell tumor developed epidural spinal cord compression syndrome 32 days after surgical resection of tumor. Rapid resolution of symptoms was observed after chemotherapy with vinblastine, bleomycin, and cisplatinum. There was no radiation therapy or laminectomy for spinal compression. The patient had an excellent neurological recovery without any sequelae from spinal compression. Chemotherapy alone is an excellent approach to the management of epidural compression by malignant germ tumor. PMID- 3012303 TI - [Adenoviral pneumonia in children]. PMID- 3012304 TI - [Acyclovir therapy of herpesvirus infections in childhood]. PMID- 3012305 TI - [Experimental in vitro research on the elimination of metal ions from non-range 2 amalgams]. PMID- 3012306 TI - [Nephroblastoma. A clinical anatomical study of a case with mandibular metastases]. PMID- 3012307 TI - Clinical genetics of dementing illness. AB - This article considers the molecular aspects, population genetics, and familial versus nonfamilial aspects of Huntington's disease, Alzheimer's disease, Down's syndrome, and Pick's disease. Single-family pedigree studies are discussed. PMID- 3012308 TI - Chronic fatigue possibly related to Epstein-Barr virus--Nevada. PMID- 3012309 TI - Recommendations for providing dialysis treatment to patients infected with human T-lymphotropic virus type III/lymphadenopathy-associated virus. PMID- 3012310 TI - Gastroenteritis outbreaks on two Caribbean cruise ships. PMID- 3012311 TI - Transfusion-associated human T-lymphotropic virus type III/lymphadenopathy associated virus infection from a seronegative donor--Colorado. PMID- 3012313 TI - [Superoxide production of polymorphonuclear leukocytes in malnourished patients with cancer of the digestive organs]. AB - In malnourished patients, little is known about production of superoxide anion which plays an important role in bactericidal activities of polymorphonuclear leukocytes (PMNs). In this study, superoxide production of RMNs was assayed in 98 malnourished patients with cancer of digestive organs, in preoperative and untreated state, in order to evaluate the bactericidal capacity, and following results were obtained. Superoxide production of PMNs in patients with cancer was significantly decreased in comparison with healthy controls, especially in advanced cancer patients. Furthermore, superoxide production of PMNs in cancer patients who were suffered from postoperative septic complications was significantly decreased in comparison with the controls and no complication group. Patients with advanced gastric cancer were evaluated as a state of malnutrition in nutritional assessment. Superoxide production of PMNs in malnourished patients with cancer of digestive organs was depressed. In gastric cancer patients, there were no differences in superoxide production of PMNs among clinical stages in well-nourished patients. On the other hand only in stage IV group of malnourished patients low values were presented. These results may suggest that the decrease in superoxide production of PMNs in patients with cancer contributes to high susceptibility to postoperative infection and is induced by malnutrition. PMID- 3012312 TI - [An analysis of prognostic examination in cirrhotic portal hypertension and hepatoma]. AB - With an increasing number of patients with advanced liver cirrhosis, the discrepancy between the preoperative examination and results of surgery for bleeding varices is widening. To correct this discrepancy, additional prognostic examinations to Child's criteria and routine hepatic laboratory tests were studied in our 246 cirrhotic patients with esophageal varices. These included wedged hepatic vein pressure, clearance and maximal removal rate of indocyanine green, and hepaplastin test. We performed the endoscope assisted terminal esophagoproximal gastrectomy with the EEA stapler gun with devascularization and splenectomy. No operative death and complications developed when the results of following 4 preoperative examinations were: wedged hepatic vein pressure below 400 mm of saline, peripheral disappearance rate (K) of indocyanine green above 0.04 min-1, maximal removal rate (Rmax) of the dye above 0.3mg/kg/min and hepaplastin test more than 40%. It is necessary for these indicators to be satisfied simultaneously prior to performing this surgery. In addition, these values should be changed a little in their critical limits when these cirrhotic patients also had hepatoma and were candidates for hepatectomy. PMID- 3012314 TI - [A resected case of collision tumor of hepatocellular carcinoma and primary liver rhabdomyosarcoma]. AB - Primary liver sarcoma is rare and especially its coexistence with hepatocellular carcinoma is extremely rare. We have collected eight cases of their coexistence in the literatures. The patient was a 62-year-old male complaining of right hypochondralgia and general malaise for about one month. CT scanning revealed the liver tumor and right hepatic lobectomy was performed. The tumor was sized in 14 by 9 by 9 cm and composed of hepatocellular carcinoma and rhabdomyosarcoma, which were were collided. Apparent striations in the rhabdomyosarcoma cells was observed under light-microscope. Alphafetoprotein, of which serum level was extraordinarily high, was detected only in hepatocellular carcinoma by special stains. He discharged one month after operation. He was readmitted due to recurrence and received trans-arterial embolisation therapy and systemic chemotherapy. Though transient effect was obtained. He died forty-five days after operation. Autopsy revealed that recurrent liver tumors consisted of only hepatocellular carcinoma and that no other tumorous lesion was found in the other parts of the body. Rhabdomyosarcoma was proved to be primary tumor the liver. PMID- 3012315 TI - [Hemodynamic change in the liver following hepatic resection in the dog. A comparative study by the 198Au colloid method, KICG and the H2 clearance method]. AB - On the basis of our hypothesis that blood flow in the residual hepatic parenchyma is a determinant factor of posthepatectomy liver function, measurements were made of blood flow in the residual liver after 40% hepatic resection in dog. Adult mongrel dogs were divided into two groups, i.e., normal control and 40% hepatectomy, and evaluated for hepatic hemodynamics by 198Au colloid method, KICG and H2 clearance method. Blood biochemistry and, histology of the liver were also examined. The results are as follows: In the control group, hepatic blood flow by 198Au colloid method, KICG and H2 clearance method was found to correlate significantly with each other. In the 40% hepatectomy group, the hepatic blood flow index by both 198Au colloid method and KICG decreased in proportion to the amount of resected hepatic tissue initially and then increased as the liver regenerated. H2 clearance method showed that hepatic blood flow per unit weight of tissue decreased in the face of reduced hepatic volume in early stage of posthepatectomy period and returned to the preoperative level as the liver regenerated. PMID- 3012316 TI - [Effects of DBcAMP on galactosamine-induced acute liver failure]. PMID- 3012317 TI - Agonist-mediated regulation of alpha 1- and beta 2-adrenergic receptor metabolism in a muscle cell line, BC3H-1. AB - We have compared the metabolism of alpha 1- and beta 2-adrenergic receptors which are both expressed in BC3H-1 muscle cells. During growth of the cells to confluence, the number of alpha 1-receptors per mg of membrane protein increases, whereas that of the beta 2-receptors remains constant. Experiments using cycloheximide and irreversible alpha 1- and beta 2-receptor antagonists, phenoxybenzamine and N-[2-hydroxy-3-(1-naphthoxy)-propyl]-N' bromoacetylethylenediamine , respectively, yield disparate turnover rates (t1/2) for the two receptors: alpha 1 congruent to 25 hr, beta 2 congruent to 200 hr. These experiments suggest that synthesis of beta 2-receptors virtually ceases in confluent cells. Maximally effective doses of agonists down-regulated both receptor types 80-90% and enhanced the rates of loss of both receptors (t1/2 = 1 5 hr). The rates of down-regulation were not affected by cycloheximide, implying that agonists enhance receptor clearance rather than decrease receptor appearance. The rank orders of potencies of agonists for promoting receptor down regulation were those characteristic of alpha 1- and beta 2-receptors. However, concentrations of agonists that resulted in down-regulation of each receptor subtype were 10- to 100-fold lower than those required for occupancy of receptors as assessed in radioligand binding studies. Receptor recovery following removal of agonists was blocked by cycloheximide and was much faster than the recovery that followed treatment of cells with irreversible antagonists. Therefore, protein synthesis (but perhaps not receptor synthesis per se) appears necessary for recovery from down-regulation. In addition, the rates of recovery of alpha 1- and beta 2-receptor-mediated functions (phosphatidylinositol turnover and cyclic AMP synthesis, respectively) following receptor down-regulation or irreversible blockade parallel the rates of receptor recovery. These data indicate that basal metabolism of alpha 1- and beta 2-receptors in BC3H-1 cells is substantially different, but that agonist-mediated changes in metabolism of the two receptor subtypes are similar. Thus, common mechanisms appear to mediate the regulation by agonists of alpha 1- and beta 2-receptors in these cells. PMID- 3012318 TI - AHN 086: an irreversible ligand of "peripheral" benzodiazepine receptors. AB - AHN 086, a derivative of 4'-chlorodiazepam (Ro 5-4864) containing an isothiocyanate moiety, binds irreversibly to "peripheral" benzodiazepine receptors in the kidney with an IC50 of approximately 1.3 nM. Using standard incubation conditions (50 mM potassium phosphate buffer, pH 7.0, 0 degrees), AHN 086 reacted rapidly with "peripheral" benzodiazepine receptors, whereas a time dependent inhibition of [3H]Ro 5-4864 binding by AHN 086 could be demonstrated by decreasing the pH of the reaction mixture, suggesting the presence of a histidine residue at or near the locus at which AHN 086 reacts. At concentrations up to 1 microM, AHN 086 did not inhibit [3H]flunitrazepam binding to "central-type" benzodiazepine receptors. Thus, AHN 086 appears to be a specific, high affinity, irreversible ligand of "peripheral" benzodiazepine receptors. PMID- 3012319 TI - Structure-activity studies of morphiceptin analogs: receptor binding and molecular determinants of mu-affinity and selectivity. AB - In this study we report the systematic investigation of conformational profiles and electronic properties of a series of analogs of the mu-selective opioid peptide, morphiceptin, together with receptor-binding studies of some of these analogs. In particular, we have investigated the effect of: substitution in the second position, substitution of D-Pro for L-Pro in the second and fourth positions, the addition of an N-methyl group at the third position, and variations in the carboxyl end group. The binding studies confirm the preference of these analogs for mu- versus delta-receptor-binding sites and also indicate differences in mu-receptor affinity among them. The theoretical analyses allow identification of a preferred conformation leading to high mu-receptor affinity and two reliable indicators of relative mu-receptor affinities. These properties are the energy required to obtain the candidate mu-binding conformer and the extent to which each compound overlaps with the highest affinity compound in this conformation. In addition, electronic interactions deleterious to high affinity mu-binding are identified. PMID- 3012320 TI - A potent and selective inhibitor of cyclic AMP phosphodiesterase with potential cardiotonic and antithrombotic properties. AB - Some biochemical and pharmacological properties of a novel, potent inhibitor of cyclic AMP phosphodiesterase, N-cyclohexyl-N-methyl-4-(7-oxy-1,2,3,5 tetrahydroimidazo[2,1-b] quinazolin-2-one) butyramide (RS-82856), were investigated. RS-82856 selectively inhibits the high affinity form of cyclic AMP phosphodiesterase (type IV) isolated from human platelets (Ki = 0.5 nM) with only weak effects on both the nonspecific and cyclic GMP-sensitive phosphodiesterases. The inhibitor reduces both maximum velocity and substrate affinity of the type IV enzyme. This mixed pattern of partial competitive and noncompetitive inhibition was also obtained with one of the two high affinity forms of phosphodiesterase found in dog heart (Ki = 0.75 nM). Of several human and dog tissues examined, RS 82856 exhibits marked selectively for the platelet high affinity enzyme. It also has significant inhibitory effects on cardiac membrane-bound phosphodiesterase. RS-82856 inhibits the aggregation of human platelets in response to adenosine 5' diphosphate (IC50 = 0.11 microM) in vitro and is active ex vivo for at least 2 hr following oral administration (10 mg/kg) to rhesus monkeys. Administration of RS 82856 to instrumented, anesthetized dogs by either intravenous or intraduodenal routes increases cardiac contractile force and reduces afterload. These data suggest that RS-82856 may be useful as an agent to increase cardiac output in the treatment of congestive heart failure. PMID- 3012321 TI - Inhibition of herpes simplex virus-induced DNA polymerases and cellular DNA polymerase alpha by triphosphates of acyclic guanosine analogs. AB - The triphosphates of the antiherpes acyclic guanosine analogs (R)- and (S) enantiomers of 9-(3,4-dihydroxybutyl)guanine [BCVTP and (S)-DHBGTP], 9-(4 hydroxybutyl)guanine (HBGTP), and 9-(2-hydroxyethoxymethyl)guanine (ACVTP) were investigated for their effects on partially purified DNA polymerases of herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) as well as cellular DNA polymerase alpha of calf thymus and Vero cells. The triphosphates of the four analogs were all competitive inhibitors when dGTP was the variable substrate with both the viral and the cellular DNA polymerases with activated calf thymus DNA or poly(dC)oligo(dG)12-18 as template. No inhibition was observed with deoxythymidine 5'-triphosphate as substrate and poly(dA)oligo(dT)12-18 as template. All analogs were preferential inhibitors of the viral DNA polymerases. Ordering the compounds according to their decreasing binding affinities, as reflected by their increasing inhibition constants for the viral DNA polymerases, gave ACVTP greater than HBGTP greater than BCVTP greater than (S)-DHBGTP. The DNA polymerase from the HSV-1 mutant, CI(101)P2C5, resistant to ACV, showed a stronger decrease in sensitivity for ACVTP and HBGTP than for BCVTP compared to the effects on DNA polymerase from the wild-type strain CI(101). The analogs were not able to support DNA synthesis in the absence of the competing substrate dGTP. A decrease in the ability of calf thymus DNA to serve as primer template for HSV 2 DNA polymerase was observed after preincubation with the triphosphates of the acyclic guanosine analogs. The analogs showed a progressive inhibition of the HSV 2 DNA polymerase activity with incubation time, and the inhibition could be reversed by high concentrations of dGTP both with and without addition of fresh enzyme or fresh template. However, no reversion was obtained when fresh enzyme or template was added if dGTP was omitted. The data indicate that these analogs inhibited the DNA polymerases by a similar mechanism and that the inhibition was reversible. PMID- 3012323 TI - Mn2+-probe ESR method for the analyses of the dissociation of charged residues on the surface of immunoglobulins. AB - Dissociation of charged residues on the surface of immunoglobulins was analysed by an Mn2+ probe ESR method that has been developed in our previous work. Several kinds of IgG proteins and their Fab and Fc fragments were used for the experiments. The pH dependence of the intensity of ESR signals was analysed. It was shown that the number of Asp, Glu and His residues on the surface of Fc is about twice as many as that of Fab. The accessible surface area of amino acid residues calculated using X-ray crystallographic data is quite consistent with the present ESR experiments. This indicates that the number of the Asp, Glu and His residues on the surface of IgG molecules in solution is similar to that in the crystal. The Mn2+-probe ESR method was also applied to other classes of immunoglobulins, i.e. IgA and IgM. It was demonstrated that the IgA protein, which is known to lack the ability to bind Clq, has on the surface of it a smaller number of Asp, Glu and His residues as compared to IgG and IgM proteins. On the basis of these results obtained by the Mn2+-probe ESR method, we suggest that the Clq molecule, which is a basic protein, interacts favorably with the Fc portion whose surface is more negatively charged with Asp and Glu residues, compared to the Fab portion. Fine adjustment of fitting of the head of the Clq molecule into the CH2 domain of the Fc portion presumably follows for optimum binding. It was also demonstrated that Ser and Thr residues are much more abundant on the surface of Fab than in the case of Fc. We suggest that the Ser and Thr residues on the surface of Fab play an important role for binding of C4b upon activation of the complement system. PMID- 3012324 TI - Fine-specificity analysis of antibodies directed to the C-terminal peptides of cytochrome c recognized by T-lymphocytes. AB - Recognition of cytochrome c by T-lymphocytes seems to involve the amino acid residues in the C-terminal region of the molecule. Lys-99 has particularly been identified as one of the critical residues in the recognition process. We have now raised antibodies against the C-terminal region of the cytochrome c molecule to map the residues that may be recognized by B-lymphocytes. These antibodies were generated in high-responder B10.A mice against either the 81-104 CNBr fragment of pigeon cytochrome c or against the synthetic spliced fragment (86-90) (94-103) of the tobacco hornworm moth cytochrome c. A good antibody response was obtained for both fragments as measured by solid-phase radioimmunoassay. A series of peptides related to these fragments were synthesized for competitive inhibition to assess the antigenic sites on these molecules. In spite of substantial homology between the moth (86-90)-(94-103) and pigeon (81-104) fragments, the antibody populations raised against each fragment differed in their recognition patterns. Residues 99 (Lys), 103 (Ala) and 104 (Lys) were found to be crucial for binding of the anti-pigeon antibody to the pigeon 81-104 fragment. The fine specificity mapping of the antigenic sites on the moth (86-90) (94-103) fragment indicated that along with some of the residues in the N terminus (86-90), residue 99 (Lys) was involved in recognition of the moth (86 90)-(94-103) fragment by its antibody. This residue (Lys-99) also acts as a T cell receptor contact site for both pigeon and moth cytochrome c. We therefore conclude that common patterns of recognition must exist between T and B-cells that recognize the C-terminal region of cytochrome c. Since Lys-99 is also present in mouse cytochrome c, the antigenic site must involve both self and non self residues. PMID- 3012325 TI - [Effect of various artificial and natural sweeteners on glucide metabolism in the gingiva of rats]. PMID- 3012326 TI - Recessive mutant genes predisposing to human cancer. PMID- 3012327 TI - CDC reports caution obstetric personnel, patients about AIDS virus. PMID- 3012328 TI - Superficial peroneal nerve conduction studies for electromyographic diagnosis. AB - A new standardized surface technique for conduction studies of the superficial peroneal nerve was developed and applied to 35 normal subjects and 63 patients with peroneal mononeuropathies, lumbar radiculopathies, sciatic neuropathies, lumbosacral plexopathies, or peripheral neuropathies. The response amplitude was greater than 5 microV and the response peak latency was less than 4.1 msec in all normal subjects; however, 8.6% of normal subjects had at least one unelicitable superficial peroneal nerve response (6% of all limbs tested). These data were compared with those of previous studies. Although this technique did not improve the diagnosis of peroneal neuropathy, it did distinguish lumbosacral plexopathy and sciatic neuropathy from L5 radiculopathy and served as a second, easily approachable, sensory conduction study of the leg. PMID- 3012322 TI - Transcription of eukaryotic ribosomal RNA gene. AB - Remarkable advances have been made in the identification of the promoter regions for the ribosomal RNA genes from lower and higher eukaryotes. There has been some progress in the elucidation of the factors that control transcription of the ribosomal RNA gene. The characterization of the transcription factors are crucial for the understanding of the molecular mechanisms of ribosomal gene expression. PMID- 3012329 TI - Muscle membrane excitation and impulse propagation velocity are reduced during muscle fatigue. AB - In order to determine whether or not impulse propagation was impaired during muscle fatigue, evoked muscle compound potentials (MCP) and twitches were recorded, both before and after fatigue, from the first dorsal interosseus (FDI), adductor pollicis (AP), and anterior tibialis (AT) muscles following supramaximal ulnar and peroneal nerve stimulation, respectively. The muscles were fatigued by maintaining maximum voluntary isometric, index finger abduction, thumb adduction, or ankle dorsiflexion for 1-5 minutes. FDI was most markedly altered, with reduced MCP amplitude (mean 32%) and increased MCP duration (mean 47%) after only 1 minute. After fatigue of longer duration (3-5 minutes), there were corresponding reductions in both the MCP amplitudes and the twitch tensions recorded from both the FDI and ankle dorsiflexors. We conclude that (1) a reduction in both the level of excitation and impulse propagation velocity of muscle membranes occurs during muscle fatigue, and (2) the magnitude of this reduced membrane function and its contribution to the mechanisms underlying fatigue depend both on the duration and degree of fatigue, as well as on the intrinsic properties of the particular muscle. PMID- 3012330 TI - Cryptococcus neoformans: rapid detection in the spleen of an AIDS patient using DAPI-fluorochrome. PMID- 3012331 TI - The activity of ketoconazole and itraconazole against Aspergillus fumigatus in mixed cultures with macrophages or leukocytes. PMID- 3012332 TI - Absence of substrate channeling in the glycosome of Trypanosoma brucei. AB - Glycolytic enzymes in the purified glycosomes of Trypanosoma brucei brucei bloodstream forms were crosslinked to form a large protein complex by the bifunctional reagent dimethyl suberimidate [Aman, R.A., Kenyon, G.L. and Wang, C.C. (1985) J. Biol. Chem. 260, 6966-6973]. The crosslinked enzyme complex was found capable of catalyzing the chain reactions leading from glucose to the formation of alpha-glycerophosphate in the presence of ATP and NADH. To determine whether the crosslinked enzyme complex exhibits multiple substrate channelings, these chain reactions were investigated in the crosslinked complex as well as in a preparation of solubilized native glycosomes. When glucose was present at a relatively high concentration (20 mM), production of alpha-glycerophosphate by the crosslinked complex had no apparent lag phase whereas the free enzyme mixture did. However, when the glucose level was lower (0.5-5.0 mM) the difference between the two enzyme preparations disappeared, a lag phase was found in both cases. Formation of sugar phosphates from radiolabeled glucose, followed by high performance liquid chromatographic analysis, showed no significant difference in the diluting powers of exogenous, unlabeled sugar phosphates added to the crosslinked complex or free enzymes. Exogenous fructose 1,6-diphosphatase demonstrated the same effectiveness in disrupting the chain reactions catalyzed by the crosslinked complex and the free enzyme mixture. In situ generation of 2 deoxyglucose-6-phosphate from 2-deoxyglucose and ATP was observed in the crosslinked complex, but the product had no amplified inhibitory effect on the phosphoglucose isomerase activity in the complex. All results suggest an absence of substrate channelings among the glycolytic enzymes in the glycosome of T. b. brucei bloodstream form. PMID- 3012333 TI - Characterization of a ribosomal DNA clone of Brugia malayi. AB - The structure of ribosomal DNA (rDNA) clone pBmr7 from microfilariae of the human parasite Brugia malayi has been examined in detail by Southern blot analysis and S-1 mapping techniques. The results demonstrate that this clone contains regions homologous to 28S, 18S and 5.8S rDNAs. A noncoding or 'spacer' region lies between the 3' end of 28S rDNA and the 5' end of 18S rDNA. An AccI-Sau3AI fragment of approximately 900 bp from this spacer region cross-hybridizes to genomic DNA fragments of different sizes from Brugia pahangi and Dirofilaria immitis. The differences observed in hybridization suggest that this rDNA fragment can be used to differentiate between various filariid species. PMID- 3012334 TI - Intrauterine infection with varicella-zoster virus after maternal varicella. AB - We investigated the consequences of maternal infection with varicella-zoster virus in a prospective study of 43 pregnancies complicated by varicella and 14 pregnancies complicated by herpes zoster. Nine of 43 pregnant women with varicella had associated morbidity--pneumonia (4 women), death (1), premature labor (4 of 42), premature delivery (2 of 42), and herpes zoster (1). Intrauterine varicella infection was identified on the basis of clinical evidence (anomalies characteristic of the congenital varicella syndrome, acute varicella at birth, or herpes zoster in infancy) or immunologic evidence (IgM antibody to varicella-zoster in the neonatal period, persistent IgG antibody to varicella zoster at one to two years of age, or in vitro lymphocyte proliferation in response to varicella-zoster virus antigen). The congenital varicella syndrome occurred in 1 of 11 infants of women with first-trimester varicella. Immunologic evidence of intrauterine varicella infection was present in 7 of 33 infants tested; 4 of these infants were asymptomatic. According to clinical or immunologic criteria, 8 of 33 infants had evidence of intrauterine varicella infection. These observations show that varicella during pregnancy was associated with maternal morbidity and evidence of fetal infection, but that herpes zoster was not. PMID- 3012335 TI - Acquisition of genital herpes from an asymptomatic sexual partner. PMID- 3012336 TI - Importance of confirmatory tests after strongly positive HTLV-III screening tests. PMID- 3012337 TI - Disseminated herpes zoster or varicella following herpes zoster: restriction endonuclease digestion analysis. PMID- 3012338 TI - Mevalonic aciduria--an inborn error of cholesterol and nonsterol isoprene biosynthesis. AB - A two-year-old boy presented with severe failure to thrive, developmental delay, anemia, hepatosplenomegaly, central cataracts, and dysmorphic features. Quantitative analyses of urinary organic acids revealed massive excretion of mevalonic acid, a metabolic precursor of cholesterol and nonsterol isoprenes: 46,000 to 56,200 mmol per mole of creatinine, as compared with 0.2 to 0.3 mmol per mole in normal children. The mevalonic acid concentration in plasma was also greatly increased at 440 mumol per liter (normal, less than 0.05). The activity of mevalonate kinase, the enzyme that catalyzes the first step in mevalonate metabolism, was severely deficient in the patient's fibroblasts, lymphocytes, and lymphoblasts. In the subsequent pregnancy of the patient's mother, gas chromatography-mass spectrometry demonstrated a marked elevation of mevalonic acid in the mother's urine and a 3000-fold elevation, as compared with control levels in the amniotic fluid, suggesting that the fetus was affected. The diagnosis was confirmed by demonstration of the deficiency of mevalonate kinase in amniocytes and ultimately in liver from the abortus. Intermediate activities of the enzyme in both parents indicated an autosomal recessive mode of inheritance. These observations identify an inherited disorder of cholesterol and nonsterol isoprene biosynthesis in humans. PMID- 3012340 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 26-1986. A 62-year-old man with progressive polyneuropathy. PMID- 3012339 TI - Biologic effects of transdermal estradiol. AB - We conducted a dose-response study in 23 postmenopausal women to compare the physiologic effects of transdermal estradiol and oral conjugated equine estrogens. The doses studied were 25, 50, 100, and 200 micrograms of transdermal estradiol per 24 hours, and 0.625 and 1.25 mg of oral conjugated estrogens. Transdermal estradiol increased circulating concentrations of estradiol and estrone. Oral conjugated estrogens also raised the levels of estrogen, particularly estrone. Both preparations lowered gonadotropin levels, decreased the percentages of vaginal parabasal cells, increased the percentage of superficial cells, and lowered urinary calcium excretion. The effects of 0.625 and 1.25 mg of oral estrogens were similar to those of 50 and 100 micrograms of transdermal estradiol per 24 hours, respectively. Oral estrogens significantly increased circulating levels of renin substrate, sex-hormone-binding globulin, thyroxine-binding globulin, and cortisol-binding globulin; transdermal estradiol had no effect. The higher dose of oral estrogens had favorable effects on concentrations of low-density and high-density lipoproteins, but transdermal estradiol did not. Neither preparation affected any of the four clotting factors studied. These data indicate that transdermal estradiol can elicit many of the desirable actions of estrogen while avoiding the pharmacologic effects of oral estrogens on hepatic proteins. PMID- 3012341 TI - HTLV-III and vacuolar myelopathy. PMID- 3012342 TI - Metabolic activity of phagocytes in experimental sporotrichosis. AB - Phagocytosis plays an important role as a protective mechanism against infections, since polymorphonuclear leukocytes (PMN) and macrophages are the first cellular lines opposed to aggressive microorganisms. In patients with sporotrichosis a diminished capability of killing engulfed yeast by their PMN has been described, but the origin of this deficiency remains unknown. In this work, partial aspects of the oxidative metabolism of PMN leukocytes and peritoneal macrophages of mongolian gerbils experimentally infected with sporotrichosis were studied. For this purpose the nitroblue tetrazolium (NBT) test as described by Baehner and Nathan (1) and myeloperoxidase activity measured according to Kaplow's method were utilized. The PMN and macrophages of mongolian gerbils infected with sporotrichosis showed increased reduction of NBT when compared with the phagocytic cells of normal ones, as is usually observed in most infections. Myeloperoxidase activity was diminished in both PMN and macrophages, but this diminution was statistically significant only in PMN leukocytes. These results show that part of the oxidative mechanisms of phagocytic cells can be impaired in experimental sporotrichosis, and could be correlated with the diminished fungicidal activity of PMN leukocytes obtained from patients infected with sporotrichosis. PMID- 3012343 TI - Characterization of phencyclidine and sigma receptor-binding sites in brain. PMID- 3012344 TI - Psychopharmacology of phencyclidine. PMID- 3012345 TI - Further evidence of phencyclidine/sigma opioid receptor commonality. PMID- 3012346 TI - Isolation and identification of an endogenous ligand for the phencyclidine receptor. PMID- 3012347 TI - Agonistic and antagonistic effects of PCP-derivatives and sigma opioids in PCP behavioral and receptor assays. PMID- 3012348 TI - Electroencephalographic (EEG), psychopharmacological, and receptor-binding profiles of 'phencyclinoids'. PMID- 3012349 TI - Vaccine engineering. Violation of rules alleged. PMID- 3012350 TI - Physiology. The expanding physiological roles of atrial natriuretic factor. PMID- 3012351 TI - Correlations between T-cell specificity and the structure of the antigen receptor. AB - The derived amino-acid sequences of the heterodimeric antigen receptors expressed by a series of murine T-cell clones are presented. A comparison of the receptor sequences indicates that several mechanisms for generating receptor diversity can influence T-cell specificity, including junctional diversity, combinatorial joining, and combinatorial chain associations. PMID- 3012352 TI - A novel type of human papillomavirus associated with genital neoplasias. AB - The role of human papillomaviruses (HPVs) in the development of genital neoplasias has been well documented. The genomes of two HPV types, HPV16 and HPV18, have been found to be associated with about 70% of invasive carcinomas of the uterine cervix. As, under non-stringent hybridization conditions, HPV DNA sequences have been detected in about 90% of cervical carcinomas, it seems likely that additional HPV types are associated with these tumours. Here we report the molecular cloning and characterization of a novel HPV type, tentatively named HPV33, whose sequences have been detected in 4-8% of biopsies of genital intra epithelial neoplasias and cervical invasive carcinomas, usually as free monomeric or oligomeric molecules. However, in one specimen of vulvar Bowen's disease, HPV33 DNA sequences were integrated in the host-cell genome. Thus, HPV33 probably represents an additional type of potentially oncogenic genital HPV. PMID- 3012353 TI - A mutated polyoma virus enhancer which is active in undifferentiated embryonal carcinoma cells is not repressed by adenovirus-2 E1A products. AB - Enhancers stimulate transcription of several eukaryotic genes (for review see ref. 1). While some enhancers, like that of simian virus 40 (SV40), are active in a wide range of cell types, others are more cell-specific. For example, the polyoma virus (Py) enhancer is not active in undifferentiated embryonal carcinoma (EC) cells, such as F9 cells, while it is active in differentiated cells. In contrast, the SV40 enhancer is active in both undifferentiated and differentiated EC cells. One possible explanation for this difference is that the two viral enhancers interact with different positively or negatively acting transcription factors, a notion supported by in vitro experiments showing that the Py enhancer interacts with proteins that do not bind to the SV40 enhancer. Some viral and cellular enhancers, including the Py and SV40 enhancers, can be negatively regulated by the products of the E1A transcription unit of adenovirus-2. As it has been postulated that undifferentiated F9 cells contain an E1A-like activity, it is possible that the latter is responsible for the lack of activity of the Py enhancer in these cells. We show here that the E1A products do not repress a point mutant of the Py enhancer (Py ECF9.1; ref. 11 and references therein) that is active in undifferentiated F9 cells. This result is consistent with the idea that undifferentiated F9 cells contain a cellular repressor that blocks the Py enhancer and that this repressor has the same target sequence as the E1A proteins. PMID- 3012354 TI - AIDS virus: more and better trans-activation. PMID- 3012355 TI - A second post-transcriptional trans-activator gene required for HTLV-III replication. AB - Evidence is provided for the existence of a seventh gene in the genome of human T lymphotropic virus type III/lymphadenopathy-associated virus. The gene is necessary for replication and acts post-transcriptionally to relieve negative regulation of the messenger RNA for the virion capsid and envelope proteins. These observations suggest mechanisms for regulating both the latent and lytic phases of the virus life cycle. PMID- 3012356 TI - Amino-acid sequence of the beta-subunit of the (Na+ + K+)ATPase deduced from a cDNA. AB - The sodium/potassium-dependent ATPase [(Na+ + K+)ATPase], which establishes and maintains the Na+ and K+ gradients across the plasma membrane of animal cells, consists of two subunits, alpha and beta. Complementary DNA clones encoding the catalytic (alpha) subunit of sheep kidney and Torpedo californica electroplax enzymes have previously been isolated and characterized. However, there is little information concerning the primary structure of the beta-subunit, a glycoprotein of unknown function and relative molecular mass (Mr) approximately 55,000 (ref. 3). Here we describe the isolation and characterization of a cDNA clone containing the entire coding region of the beta-subunit of the sheep kidney (Na+ + K+)ATPase. We also discuss structural aspects of the protein and present evidence for a possible evolutionary relationship with the KdpC subunit of the Escherichia coli K+-ATPase. PMID- 3012357 TI - Isolation of cDNA clones encoding the 20K non-glycosylated polypeptide chain of the human T-cell receptor/T3 complex. AB - The antigen receptor on human T lymphocytes consists of two variable immunoglobulin-like glycoproteins, alpha and beta, which occur in association with three invariable T3 membrane proteins. In humans two of these proteins, T3 gamma and T3-delta, are glycoproteins of relative molecular mass (Mr) 25,000 (25K) and 20,000 (20K), respectively, while the third, T3-epsilon, is a 20K non glycosylated protein. On the surface of murine T cells, a non-glycosylated protein dimer composed of 17K subunits (T3-zeta) is found associated with the T cell receptor alpha and beta chains and the three T3-like polypeptide chains. It is generally accepted that major histocompatibility complex-restricted antigen recognition is a function of the alpha-beta heterodimer. This has led to the postulation that the proteins of the T3 complex are involved in the signal transduction that immediately follows antigen recognition via the antigen receptor. Events believed to be involved in early T-cell activation, such as rapid increases in phosphatidylinositol turnover and free intracellular calcium, can be triggered by antibodies directed against either the T3 complex or the clonotypic receptor. We have previously reported our findings on the cloning of the complementary DNA and genomic structure encoding both the human and murine 20K glycoprotein, T3-delta (refs 11-13). We now present our results on the cloning of the cDNA encoding the human 20K non-glycosylated chain, T3-epsilon. PMID- 3012359 TI - Specific inhibition of herpesvirus ribonucleotide reductase by synthetic peptides. AB - Ribonucleotide reductase is an essential enzyme for DNA synthesis in all prokaryotic and eukaryotic cells; it catalyses the reductive conversion of ribonucleotides to deoxyribonucleotides. Several herpesviruses including herpes simplex virus type 1 (HSV-1), HSV-2, pseudorabies virus (PRV), equine herpesvirus type 1 (EHV-1) and Epstein-Barr virus (EBV) have been found to induce novel ribonucleotide reductase activities. There is evidence that the HSV-1 ribonucleotide reductase activity is virus-encoded and essential for virus replication. This makes herpesvirus ribonucleotide reductases potential targets for antiviral chemotherapy. The HSV-1-encoded enzyme consists of two subunits: V136, the large subunit of relative molecular mass (Mr) 136,000 (136K) (RR1), which has been shown to be essential for enzyme activity, and V38, the small subunit (RR2) which forms a complex with the large subunit and is also likely to be essential for enzyme activity. Two particular features of the enzyme make it an attractive antiviral target. First, there is evidence for a common, highly conserved herpesvirus ribonucleotide reductase and second, the interaction between the large and small subunits may itself be exploitable. Here we identify a synthetic peptide which specifically inhibits the activity of virus-induced enzyme. We deduce that the mechanism of inhibition involves interference with the normal interaction between the two types of subunit. PMID- 3012358 TI - Isolation of an HTLV-III-related retrovirus from macaques with simian AIDS and its possible origin in asymptomatic mangabeys. AB - Acquired immune deficiency syndrome (AIDS) has become a worldwide epidemic, so the development of vaccines and antiviral agents effective against the causative agent, human T-lymphotropic virus type III (HTLV-III), is vital. This work would be greatly simplified if a suitable animal model could be developed. Here we report the isolation of an HTLV-III-related retrovirus, STLV-III/Delta, from rhesus macaques (Macaca mulatta) with transmissible simian AIDS (SAIDS) and from asymptomatic sooty mangabeys (Cercocebus atys). SAIDS was initially diagnosed in several macaques previously inoculated with tissue homogenates of mangabey origin. Western blot analysis of both the mangabey and macaque sera demonstrated the presence of antibody cross-reactive primarily with the HTLV-III proteins p24 and p61. In a related experiment, analysis of these same sera revealed simian antibody to STLV-III/Delta proteins similar, but not identical, to those of HTLV III with estimated relative molecular masses (Mrs) of 16,000 (16K), 26K, 35K, 45K, 60K and 110K. Infection of the mangabey, an African primate, with an HTLV III-related virus may provide a clue to the origin of HTLV-III in humans. The apparent difference in susceptibility to SAIDS-like disease between infected macaques and mangabeys suggests that these species may respond differently to STLV-III infection. PMID- 3012360 TI - Specific inhibition of herpesvirus ribonucleotide reductase by a nonapeptide derived from the carboxy terminus of subunit 2. AB - Ribonucleotide reductase, an essential enzyme for the synthesis of deoxyribonucleotides, is formed by the association of two nonidentical subunits in almost all prokaryotic and eukaryotic cells. The same model probably holds for the herpes simplex virus (HSV)-encoded ribonucleotide reductase; two polypeptides of relative molecular mass 136,000 (136K; H1) and 40K (H2) (referred to elsewhere as RR1 and RR2; see for example, Dutia et al.) have been associated with the viral enzyme by both genetic and immunological studies. Furthermore, DNA sequence analyses have shown significant stretches of amino-acid homology between these viral polypeptides and those of, respectively, subunit 1 (ref. 12) and subunit 2 (ref. 13) of the Escherichia coli and mammalian enzymes. To assess the involvement of the 40K polypeptide in reductase activity, we synthesized a nonapeptide corresponding to the sequence of its carboxy terminus with the intention of raising neutralizing antibodies specific for the viral activity (E.A.C. et al., in preparation). We report here the unexpected finding that the nonapeptide itself specifically inhibits the HSV ribonucleotide reductase activity in a reversible, non-competitive manner, and we suggest that it does this by impairment of the correct association of the two subunits. This phenomenon emphasizes the potential usefulness of synthetic peptides in probing critical sites involved in macromolecular interactions. PMID- 3012361 TI - Re-routing of a secretory protein by fusion with human growth hormone sequences. AB - Cells with electron-dense secretory vesicles use them to store only specialized secretory products such as peptide hormones; other types of secreted proteins are externalized by an alternative, constitutive route. One possible mechanism for such segregation is that proteins destined for dense secretory vesicles contain unique 'sorting domains' that allow for selective targeting. Here, we set out to determine whether a constitutively secreted protein could be diverted to the dense secretory vesicles by attachment to a peptide hormone sequence. We made use of the ability of the mouse pituitary tumour cell, AtT-20, to correctly sort exogenous secretory proteins introduced into them by DNA transfection. We constructed a plasmid encoding a hybrid protein in which a constitutively secreted viral protein was fused to the carboxy terminus of human growth hormone (hGH). Cells expressing the hybrid protein were found to target it to dense secretory vesicles with an efficiency close to that observed for the parental hGH. These results support the hypothesis that sorting domains on peptide hormones direct their packaging into dense secretory vesicles. The results also suggest that proteins secreted by the constitutive pathway either do not contain any sorting domain, or their sorting signals can be overridden by those which direct peptide hormones. PMID- 3012362 TI - NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones. AB - Excitatory amino acids act via receptor subtypes in the mammalian central nervous system (CNS). The receptor selectively activated by N-methyl-D-aspartic acid (NMDA) has been best characterized using voltage-clamp and single-channel recording; the results suggest that NMDA receptors gate channels that are permeable to Na+, K+ and other monovalent cations. Various experiments suggest that Ca2+ flux is also associated with the activation of excitatory amino-acid receptors on vertebrate neurones. Whether Ca2+ enters through voltage-dependent Ca2+ channels or through excitatory amino-acid-activated channels of one or more subtype is unclear. Mg2+ can be used to distinguish NMDA-receptor-activated channels from voltage-dependent Ca2+ channels, because at micromolar concentrations Mg2+ has little effect on voltage-dependent Ca2+ channels while it enters and blocks NMDA receptor channels. Marked differences in the potency of other divalent cations acting as Ca2+ channel blockers compared with their action as NMDA antagonists also distinguish the NMDA channel from voltage-sensitive Ca2+ channels. However, we now directly demonstrate that excitatory amino acids acting at NMDA receptors on spinal cord neurones increase the intracellular Ca2+ activity, measured using the indicator dye arsenazo III, and that this is the result of Ca2+ influx through NMDA receptor channels. Kainic acid (KA), which acts at another subtype of excitatory amino-acid receptor, was much less effective in triggering increases in intracellular free Ca2+. PMID- 3012364 TI - AIDS virus nomenclature. PMID- 3012363 TI - Finding the defective gene. PMID- 3012366 TI - Epstein-Barr virus. Infection and tumour induction. PMID- 3012365 TI - Energy and signal transduction by transmembrane protein complexes. PMID- 3012367 TI - Do mammals need P elements? PMID- 3012368 TI - Portuguese role in spread of HTLV-I virus. PMID- 3012369 TI - Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid. AB - A variety of hypophysiotropic peptides or proteins have been reported to be present in mammalian gonads. Inhibin, a hormone that under most circumstances selectively suppresses the secretion of follicle-stimulating hormone (FSH) but not luteinizing hormone (LH), has been isolated from the gonadal fluids of several species and characterized as a heterodimeric protein consisting of alpha- and beta-polypeptides associated by disulphide bonds. The complete amino-acid sequences of the precursors of porcine and human inhibin alpha-subunits and two distinct porcine inhibin beta-subunits (beta A and beta B) have been deduced from complementary DNA sequences. Gonadotropin releasing peptides have also been found in the gonad and have generally been shown to be active in radioreceptor assays for gonadotropin releasing hormone (GnRH) but to exhibit different chromatographic and immunological characteristics from those of GnRH. During our purification of inhibin from porcine follicular fluid, we noted fractions that could stimulate the secretion of FSH by cultured anterior pituitary cells. We report here the purification of an FSH releasing protein (FRP) and its characterization by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions and by partial sequence analysis. Our results indicate that porcine gonadal FRP is a homodimer consisting of two inhibin beta A-chains linked by disulphide bonds. FRP is highly potent (50% effective concentration (EC50) approximately 25 pM) in stimulating the secretion and biosynthesis of FSH but not of LH or any other pituitary hormone. In contrast to the effects of GnRH and other reported gonadal gonadotropin releasing fractions, the action of FRP is not mediated by GnRH receptors. PMID- 3012370 TI - Sequence organization and genomic complexity of primate theta 1 globin gene, a novel alpha-globin-like gene. AB - The alpha-like and beta-like globin genes have provided a paradigm for the study of molecular evolution and regulation of multigene families in eukaryotes. The human alpha-globin gene cluster, which is on chromosome 16 (ref. 1), consists of six genes arranged in the order 5'-zeta(embryonic)-psi zeta-psi alpha 2-psi alpha 1-alpha 2(adult)-alpha 1(adult)-3'. DNA sequencing data have demonstrated that zeta (ref. 6) and alpha 2 (or alpha 1, refs 7-9) are the embryonic and adult genes, respectively, while psi zeta (ref. 6), psi alpha 2 (ref. 5) psi alpha 1 (ref. 10) are all inactive pseudogenes. Restriction mapping analysis has shown that the structure of this locus in several anthropoid primates is nearly identical to that of the human. Recently, we have isolated the adult alpha-globin gene region from orang-utan, olive baboon and rhesus macaque by molecular cloning. We report here the complete nucleotide sequence of a gene located immediately downstream from the adult alpha 1-globin gene of the orang-utan, along with its flanking DNA. We designate this gene as theta 1, and show that it contains the essential sequence elements required for an expressive gene. The putative polypeptide is 141 amino acids long, identical to that of the alpha- or zeta-globin, but its predicted amino-acid sequence is nearly as different from the orang-utan alpha-globin (55 differences) as the human zeta-globin is from the human alpha-globin (59 differences), suggesting an ancient history for the theta 1-globin gene. Results of blot hybridization experiments using the cloned orang utan theta 1 gene sequence as probe demonstrate a similar alpha 2-alpha 1-theta 1 linkage map existing in the human genome. Furthermore, multiple copies of sequences homologous to the theta 1 gene are detected in both human and orang utan. These results cast a new light on the primate alpha-globin gene family, and have intriguing implications for the existence of previously unreported, functional globin-like gene(s) in the primate genomes. PMID- 3012371 TI - Endplate channel actions of a hemicholinium-3 analog, DMAE. AB - The effect of the hemicholinium-3 analog, DMAE, on endplate currents (EPC) was investigated in the transected cutaneous pectoris muscle of the frog using a conventional two-microelectrode voltage clamp. At a low concentration (5 microM), DMAE produced a long-lasting decrease in the rate constant of decay (alpha) and an increase in the peak current amplitude (Ip). At higher concentrations (10--100 microM), DMAE produced biphasic changes characterized by a transient, marked decrease of alpha and increase of Ip followed by a long-lasting marked increase of alpha and decrease of Ip. When DMAE was removed from the bath recovery from block was asymmetrical in that alpha recovered more quickly than did Ip. Pretreatment with neostigmine or collagenase partially antagonized the initial effects without affecting the steady state effects of DMAE, indicating that the initial effects of DMAE may be, at least in part, due to inhibition of the enzyme acetylcholinesterase. The drug reverses the normal voltage dependence of alpha without altering the single exponential nature of decay of the EPC. The inward EPC was more markedly blocked than outward EPC, resulting in a highly non-linear current-voltage relation with Ip decreasing with increasing hyperpolarization. This effect may indicate that DMAE causes a voltage-dependent block of closed acetylcholine-activated ion channels. PMID- 3012372 TI - Agonist-mediated conformational changes of beta-adrenoceptors could occur independent of functional coupling to Ns. AB - Agonist and antagonist binding characteristics of beta-adrenoceptors in turkey erythrocyte ghosts were determined at different temperatures ranging between 7 degrees C and 42 degrees C. [3H]-DHA saturation binding experiments revealed that the antagonist-receptor interaction is entropy-driven with a small enthalpic contribution. Isoproterenol/[3H]-DHA competition binding followed the law of mass action at all the investigated temperatures. The agonist-receptor interaction is enthalpy driven with a small unfavorable decrease in entropy. This is consistent with the agonist's ability to favor an endoenergetic transconformation of the receptors. Only part of the agonist-bound receptors can undergo functional coupling to the stimulatory component of the adenylate cyclase system (Ns). This coupling process is associated with "locking-in" of the agonist and becomes persistent in the presence of the alkylating reagent N-ethylmaleimide. The number of agonist/N-ethylmaleimide-sensitive sites (i.e. coupling-prone receptors) increases with the temperature until it reaches a plateau value of 50% between 27 32 degrees C. Qualitatively similar data were obtained for rat lung and turkey erythrocyte membranes. These observations suggest that the whole receptor population can undergo agonist-mediated conformational changes but that only part of them can couple to Ns. PMID- 3012373 TI - Proglumide (gastrin and cholecystokinin receptor antagonist) inhibits insulin secretion in vitro. AB - CCK-8 and its desulfated analog (des-CCK-8) increase insulin secretion from isolated rat pancreatic islets in the presence of 8.3 mM glucose in a concentration-dependent manner. Proglumide (DL-4-benzamido-N,N-dipropylglutaramic acid), a gastrin and cholecystokinin (CCK) receptor antagonist, inhibits the synergistic effect of CCK on insulin release in the presence of 8.3 mM glucose; its EC50 (half-maximal effective concentration) was 1.2 +/- 0.4 mM. Its effect is specific in that it does not inhibit the glucose- or GIP (glucose dependent insulinotropic peptide) induced insulin secretion to a major degree. CCK-8, des CCK-8 and proglumide compete for binding of 125I-CCK-33 to rat pancreatic islets; the IC50 of proglumide was 0.8 mM. The affinity of proglumide is in the range of both its EC50 for inhibition of insulin secretion and its IC50 in other in vitro systems tested so far (exocrine pancreas, gall bladder, cortex). Its inhibitory effect presumably is not a gastrin antagonizing effect since gastrin does not stimulate insulin secretion. The data therefore indicate that proglumide should be monitored for diabetic effects in vivo. PMID- 3012376 TI - [Cimetidine or ranitidine?]. PMID- 3012374 TI - Vasoconstriction induced by noradrenaline and angiotensin II is antagonized by eicosapentaenoic acid independent of formation of trienoic eicosanoids. AB - The isolated perfused rabbit ear was used to investigate the effect of eicosapentaenoic acid (EPA) on the vasoconstriction induced by noradrenaline (NA) and angiotensin II (A II). EPA (0.1, 1.0, 10.0 micrograms/ml) was shown to reduce the vasoconstriction (inhibition of flow) induced by NA in a dose-dependent manner. The dose-response curves of NA were shifted to the right. Emax of NA was reduced in particular at the highest concentration of EPA. The maximum effect of EPA developed slowly and reached a steady state after 150 min (0.1 microgram/ml EPA), 120 min (1.0 microgram/ml EPA), 100 min (3.0 microgram/ml EPA) and 60 min (10 micrograms/ml), respectively. In the presence of indomethacin (3 micrograms/ml), the vasoconstrictor effect of NA was not significantly influenced. The effect of EPA (3 micrograms/ml) was not reduced by indomethacin. In the presence of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 micrograms/ml), the vasoconstrictor effect of NA was not significantly influenced. The effect of EPA (3 micrograms/ml) was not reduced by NDGA. Concomitant addition of indomethacin (3 micrograms/ml) and NDGA (10 micrograms/ml) also did not reduce the effect of EPA. EPA (3.0, 10.0 micrograms/ml) was shown to reduce also the vasoconstrictor effect of A II in the absence and the presence of the cyclooxygenase and lipoxygenase inhibitors. Determination by radioimmunoassay showed that the levels of thromboxane B2 (TXB2) like immunoreactivity were below the detection limit in the perfusate of the ear. The levels of 6-keto-PGF1 alpha- and PGE2-like immunoreactivity in the perfusate were low. EPA reduced the concentration of 6-keto-PGF 1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012375 TI - Action of AD6 (8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxycarbonylmet hox y coumarin) on human platelets in vitro. AB - The action of AD6 was tested in vitro on human platelets by measuring beta thromboglobulin (BTG), platelet factor 4 (PF4) and thromboxane B2 (TXB2) release as well as aggregation. BTG and PF4 release from blood anticoagulated with sodium citrate was inhibited by AD6 during a 3 h incubation. Platelet rich plasma (PRP) was stimulated with ADP, collagen, sodium arachidonate, PAF, A23187 and epinephrine, while resuspended washed platelets (WP) were stimulated by thrombin. AD6 (5-100 microM) inhibited dose dependently aggregation, BTG, PF4 and TXB2 release induced by threshold concentration of all the tested aggregating agents; however AD6 action could be overcome by increasing the concentration of the stimulating agents. After cyclo-oxygenase blockade by acetylsalicylic acid (ASA), PRP was stimulated by a supramaximal concentration of PAF. Under these circumstances we could observe a reversible aggregation and a partial release of BTG and PF4, AD6 was able to further reduce aggregation and release. Cyclic AMP accumulation induced in WP by prostacyclin was not modified by AD6 (100 microM), while theophylline greatly potentiated prostacyclin action. We conclude that AD6 is an inhibitor of platelet activation in vitro. Its mode of action is different from cyclo-oxygenase blockade and provides inhibition of platelet activation by a number of different stimuli. PMID- 3012378 TI - Atypical, cutaneous fibrous histiocytoma with early metastasis. PMID- 3012377 TI - [Diagnosis and clinical aspects of the TORCHES syndrome]. PMID- 3012379 TI - [Effect of kainic acid on synaptic transmission in the electroreceptors (ampullae of Lorenzini) of the skate]. AB - Superfusion of the basal membrane of the ampullae of Lorenzini of the skate Raja clavata with 10(-6)-10(-9) M kainic acid produced significant and reversible changes in background firing rate depending on its initial level. When synaptic transmission was abolished by high Mg2+ solution, succeeding superfusion with kainic acid and Mg2+ containing solution restored the background and evoked activity in the afferent fibres. It is suggested that the observed effects of kainic acid on the activity of the ampullae of Lorenzini may be explained mainly by its presynaptic action. PMID- 3012380 TI - [Effect of stimulation of the superior colliculi of the tectum mesencephali on the motor neurons of the neck muscles of the cat]. AB - Postsynaptic potentials evoked by stimulation of three points of the superior colliculus in motoneurons of neck muscles were studied in experiments on cats under chloralosenembutal anesthesia. Stimulation of ipsilateral superior colliculus evoked EPSP with latencies ranging from 1.5 to 3.5 ms in 49 motoneurons. Stimulation of contralateral superior colliculus evoked EPSP with latencies ranging from 1.5 to 3.0 ms in 63 motoneurons and IPSP with latencies ranging from 2.6 to 5.0 ms in 10 motoneurons. It is suggested that the recorded postsynaptic potentials are mono- and disynaptic. PMID- 3012382 TI - [Electrophysiologic study of descending effects of field 5b of the association cortex of the brain of the cat on the neurons of the ventroposterolateral nucleus of the thalamus]. AB - Responses of 65 thalamic ventroposterolateral (VPL) nucleus neurons to stimulation of the associative cortex (area 5b) were studied in acute experiments on cats anesthetized with ketamine (25 mg/kg) and immobilized with myorelaxine (2 mg/kg). Neurons studied were identified on the basis of peculiarities of their responses evoked by primary somatosensory cortex and medial lemniscus stimulation. Three neurons responded to stimulation of the area 5b antidromically. Primary orthodromic excitation and inhibition were observed in 17 (26.2%) VPL neurons. The role of the area 5b descending influences on the somatosensory impulses transmission through VPL relay neurons is discussed. PMID- 3012381 TI - [Synaptic interactions of individual motor neurons of the spinal cord of the carp]. AB - The interaction between motoneurons was studied in the isolated spinal cord of the fish (Cyprinus carpio) by the recording of elementary EPSPs evoked in a motoneuron by intracellular stimulation of adjacent motoneuron. These EPSPs are mediated mainly by electrical transmission as evidenced by their short or negligible latencies, stable amplitudes and bidirectional transmission between motoneurons. In some cells double-component elementary EPSPs were revealed which indicate dual (electro-chemical) mode of transmission between some motoneurons. PMID- 3012383 TI - [Gamma-aminobutyric acid receptors in the central nervous system of mammals]. AB - The review describes properties of gamma-aminobutyric acid (GABA) receptors in the central nervous system of mammalians. Two pharmacologically different receptors--GABAA and GABAB--are involved into GABA-ergic inhibition. GABA receptors (bicuculline-sensitive) regulate chloride permeability of membranes and can be functionally enhanced by benzodiazepine and barbiturate. GABA receptors (bicuculline-insensitive) are suggested to regulate calcium permeability. PMID- 3012385 TI - Tumor growth enhancement and systemic responses of Ehrlich ascites carcinoma following intermittent whole body hyperthermia. AB - Intermittent whole body hyperthermia 42 degrees C (WBH(I) administered for 30 min daily during 7 days caused significant enhancement of the growth of Ehrlich ascites carcinoma. The tumoricidal effects of hyperthermia were evident from the cytological picture as well as from the increased activity of lysosomal enzyme of the tumor cells. Elevated plasma corticosteroid levels, adrenal hypertrophy, thymus involution and lymphocytopenia were observed in both normal and tumor bearing mice exposed to hyperthermia. The alterations were more marked in the tumor bearing groups. Withdrawal of treatment caused gradual restoration of stress effects. It appears that the tumoricidal effects of WBH(I) were counteracted and surpassed by the growth stimulatory effects induced probably by hormone mediated immunosuppression. PMID- 3012384 TI - Properties of Na+/K+ ATPase and alkaline phosphatase alter during spontaneous and radiation-induced leukemogenesis in mice. AB - We characterized properties of Na+/K+ ATPase and alkaline phosphatase in thymocytes or thymoblasts from mice of two strains: AKR, in which thymoma developed spontaneously and C57Bl, in which the development was induced by X irradiation (total dose: 5.4 Gy in 3 fractions). We found that before thymoma could be discerned morphologically--properties of the two enzymes altered. There was a decrease in 86Rb uptake and in the rate of ATP hydrolysis per cell (both strains) as well as an increase in alkaline phosphatase activity per cell (C57Bl mice). In both spontaneous and radiation-induced thymomas 86Rb uptake, ATP hydrolysis and 3H-ouabain binding per cell were higher than in normal thymuses. Likewise, alkaline phosphatase activity per cell was higher in thymomas than in thymuses; this increase was accompanied by the appearance of additional isoenzyme(s) (1 in AKR, 2 in C57Bl). These changes were compared with cAMP content and 3H-thymidine incorporation, taken as indicators of the proliferative activity, and their high correlation in both AKR and C57Bl mice allowed to distinguish a pre-leukemic period. In that period thymoblasts clearly differed from the normal ones in Na+/K+ ATPase and alkaline phosphatase properties, as well as proliferation, although the morphology of the thymus was still unchanged. PMID- 3012387 TI - [Quantitative evaluation of EMG records based on 2 digital indicators]. AB - The author presents a new concept and the results of simple assessment of EMG records by means of a universal set of parameters represented by two digital indices. The first descriptor determines the mean value of a single unit potential and it was obtained as a product of the mean values of potential duration and amplitude. The second descriptor determines the mean value of the interference tracing during maximal contraction of the muscle and it is the ratio of the mean amplitude to the mean density. The use of these two diagnostic indices makes possible an immediate evaluation of EMG records during the examination. The method is very sensitive and diagnostically reliable. PMID- 3012386 TI - [cAMP and cGMP in the cerebrospinal fluid of patients with multiple sclerosis]. AB - The authors studied the changes in cAMP and cGMP concentrations in the cerebrospinal fluid in 38 patients with multiple sclerosis. A statistically significant fall of cAMP was observed with a simultaneous rise of cGMP in the initial stage of the disease as compared with the control group. In the later course of the disease and development of various clinical forms of multiple sclerosis the pattern of these changes was not characteristic. PMID- 3012388 TI - [Hereditary motor and sensory neuropathy. I. Principles of classification and clinical picture]. AB - The clinical picture was analysed in two types of hereditary motor-sensory neuropathy isolated on the ground of electrophysiological criteria. Type I comprised 34 patients with the conduction velocity in median nerve below 38 m/sec. Type II 19 patients with the conduction velocity above 38 m/sec. The age of onset was similar in both types and cases with onset below the age of 5 years prevailed. The assessment of the clinical picture using a acoring system failed to show any significant differences between type I and type II. Cases of type I shows, however, a considerable variability of the clinical picture and the course of disease process. Cases of type II were more homogeneous. PMID- 3012389 TI - [Classification of pituitary adenomas in terms of immunohistochemistry and ultrastructure]. PMID- 3012390 TI - [Clinicopathological study of supratentorial tumors with multipotential differentiation in childhood. Primitive neuroectodermal tumor]. AB - Three cases of supratentorial tumor in childhood were studied clinico pathologically in an attempt to clarify its histological character. Case 1: A 3 year-old boy. Carotid angiogram revealed avascular lesion in the left parietal lobe. Twice operations and radiotherapy were performed. Ten months after the second operation, he died. Surgical specimen at the first operation was composed mainly of round tumor cells. The tumor tissue contained many collagen fibers. At the periphery of this tissue, medulloblastomatous areas consisting of closely aggregated hyperchromatic small round cells were found. There were perivascular rosettes and Homer Wright rosettes. In part, tubular and papillary arrangement of cells was also present. Astrocytomatous and oligodendrogliomatous structures were also present. Surgical specimens at the second operation showed the predominance of sarcomatous areas consisting of spindle-shaped cells with abundant argyrophilic fibers. Case 2: A 10-year-old girl. CT scan revealed a heterogeneous enhanced mass with a cyst and calcification in the right parietal lobe. Operation and radiotherapy were performed. Twelve months after operation, she is still alive. Most of the surgical specimens showed sarcomatous structure with abundant argyrophilic fibers. In these tissues, there were medulloblastomatous and ependymomatous features. Papillary arrangement of cells was also present. In part, there was oligodendrogliomatous structure. Case 3: A 2-year-old girl. CT scan revealed a heterogeneous enhanced mass in the right frontal lobe. The tumor tissue was composed of an aggregation of undifferentiated small round cells with Homer Wright rosettes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012391 TI - [A case of growth hormone-producing adenoma presenting as a non-functioning tumor at recurrence]. AB - The authors report a case of recurrent pituitary adenoma, which changed its endocrinological function from GH producing to non-functioning. A 37-year-old woman was admitted to our hospital complaining of headaches, amenorrhea and acromegalic features. Skull X-rays showed marked ballooning of the sella turcica and mild thickening of the calvarium. X-rays of the hands and feet revealed moderate acromegalic changes. On pneumoventriculography, the tumor elevated the floor of the third ventricle. The serum GH level was 29.3 ng/ml, which did not respond to insulin induced hypoglycemia. Radical removal of the tumor was performed through a right frontal craniotomy. Histologically, it was diagnosed as a pituitary eosinophilic adenoma. Immunostains revealed the presence of many GH positive cells in the adenoma. Since the post-operative GH levels were still high (12-16 ng/ml), irradiation to the sellar region was carried out. The serum GH concentration gradually decreased to the normal level in one year after the irradiation. At that time no sellar tumor could be found on CT scans. The patient had been well for six years until she noticed hearing impairment of her right ear. She was re-admitted about seven years after the first admission because of cerebellar ataxia and hearing loss. CT scans revealed a recurrent tumor extending from the sellar region to the right cerebello-pontine angle. Serum GH levels on admission were within normal range (3-4 ng/ml). The tumor was partially removed by suboccipital craniectomy. Pathologically, the tumor was reported as a pituitary chromophobe adenoma. With immunostains, no GH positive cells could be found in the adenoma.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012393 TI - Chronic estradiol treatment decreases angiotensin II receptor density in the anterior pituitary gland and adrenal cortex but not in the mesenteric artery. AB - Chronic estrogen treatment has been shown to produce a marked reduction in anterior pituitary angiotensin II (AII) receptor density. In order to determine whether this effect is generalized, we studied the effect of chronic estradiol treatment on AII receptor density in the anterior pituitary gland, adrenal cortex and mesenteric artery of ovariectomized (OVX) rats. Treated rats were injected daily with 25 micrograms of estradiol valerate while controls received only the vehicle. Binding affinity and density of AII receptors were measured using the AII antagonist [125I]-Sar1Ile8 AII ([125I]-SARILE). Following 7-, 14- or 28-day treatments, AII receptor density decreased by approximately 80% in the anterior pituitary; 30% in the adrenal cortex and remained the same in mesenteric artery particulate fractions. In all 3 target tissues, dissociation constants (KD) for binding of [125I]-SARILE were in the nanomolar range and were the same between control and treated rats. Using conscious rats, estradiol treatment for 7 days was also shown to block the release of aldosterone by low dose infusions of AII (10 ng/min, 30 min). Plasma AII and plasma renin activity were also the same or slightly decreased following estradiol treatments. This study suggests that estrogens may be important modulators of the AII receptor and may be directly involved in modulating target cell responsiveness to AII as expressed through differential down-regulation of AII receptors. PMID- 3012392 TI - Effect of various blockers of arachidonic acid metabolism on release of beta endorphin- and adrenocorticotropin-like immunoreactivity induced by phospholipase A2 from rat adenohypophysis in vitro. AB - Anterior pituitary quarters were incubated in vitro and the release of beta endorphin-like (beta-End-IR) and adrenocorticotropin-like immunoreactivity (ACTH IR) was determined. The effect of phospholipase A2 as well as the effect of various compounds known to influence arachidonic acid metabolism under certain conditions were examined. Phospholipase A2 increased the release of beta-End-IR and ACTH-IR. This effect was reversible, concentration-dependent (1-400 ng/ml) and inhibited in calcium-free medium and in the presence of CoCl2 (5 mM) or phospholipase A2 inhibitors (p-bromophenacylbromide, 21 microM; mepacrine, 1 mM). The phospholipase A2-induced beta-End-IR release was accompanied by the release of prostaglandin E2. Inhibition of cyclooxygenase activity by indomethacin (14 or 140 microM) did not change beta-End-IR release induced by phospholipase A2 (5 ng/ml). The effects of blockers of lipoxygenase (nordihydroguaiaretic acid, NDGA; AA861) or lipoxygenase plus cyclooxygenase (BW755C; eicosatetraynoic acid, ETYA) on phospholipase A2-induced release of beta-End-IR were diverse. BW755C (up to 250 microM) and AA861 (up to 100 microM) produced no effect. However, NDGA or ETYA inhibited phospholipase A2-induced beta-End-IR release. NDGA (100 microM) produced a maximum inhibition by about 40% (p less than 0.05), whereas ETYA (100 microM) produced a maximum inhibition by about 85% (p less than 0.001). These data are consistent with the view that phospholipase A2 releases endogenous arachidonic acid which is transformed into products which stimulate ACTH and beta endorphin release from the corticotrophs; the metabolizing enzyme (possibly a lipoxygenase or epoxygenase) is sensitive to NDGA and especially to ETYA. PMID- 3012394 TI - A V1 vasopressin receptor antagonist has nonspecific neurodepressant actions in the spinal cord. AB - In the present investigation, we injected d(CH2)5Tyr(Me)AVP, a specific VI vasopressin antagonist, into the intrathecal space of the spinal cord to determine whether spinal vasopressin-containing neurons contribute to the hemodynamic effects produced by stimulation of the paraventricular nucleus (PVN). The intrathecal antagonist reduced the cardiovascular effects of PVN stimulation, but also reduced the effects produced by stimulating 2 other brain sites which do not contain vasopressin cell bodies. In addition, intrathecal administration of the vasopressin antagonist had a similar effect in Brattleboro rats which do not produce vasopressin. In conscious rats with indwelling intrathecal catheters, the vasopressin antagonist produced reversible hindlimb paralysis. These data suggest that d(CH2)5Tyr(Me)AVP has nonspecific actions within the spinal cord not related to the blockade of vasopressin receptors. PMID- 3012395 TI - Receptor-mediated actions of corticotropin-releasing factor in pituitary gland and nervous system. AB - High-affinity corticotropin-releasing factor (CRF) receptors which mediate the actions of the hypothalamic peptide on adrenocorticotropic hormone (ACTH) release have been identified in the rat anterior pituitary gland. Occupancy of the pituitary receptor by CRF agonists stimulates ACTH release via activation of adenylate cyclase and cyclic adenosine monophosphate dependent protein kinase. In the regulation of ACTH secretion, the effects of CRF on the corticotroph are integrated with the stimulatory actions of cyclic adenosine monophosphate independent stimuli such as angiotensin II, vasopressin and norepinephrine, and the inhibitory effects of glucocorticoids and somatostatin. In contrast to the major importance of the inhibitory effect of glucocorticoid feedback on ACTH secretion, somatostatin has relatively little effect on CRF-stimulated ACTH release in the normal rat corticotroph. Following adrenalectomy, the progressive elevation of plasma ACTH levels is accompanied by a concomitant decrease in pituitary CRF receptors. The postadrenalectomy loss of CRF receptors, which is prevented by dexamethasone treatment, is caused by a combination of occupancy and processing of the pituitary sites during increased secretion of the hypothalamic peptide. Recently, specific receptors for CRF have been localized in the rat and monkey brain and adrenal medulla, where they are also coupled to adenylate cyclase. Brain CRF receptors are most abundant in the cerebral and cerebellar cortices and in structures related to the limbic system and control of the autonomic nervous system. The actions of CRF on the central and peripheral nervous systems, as well as on the pituitary gland, emphasize the role of CRF as a key hormone in the integrated response to stress. PMID- 3012396 TI - Meptazinol: unusual in vivo opioid receptor activity at supraspinal and spinal sites. AB - Systemic (1-10 mg/kg, s.c.), intracerebroventricular (i.c.v. 20-80 micrograms) and spinal intrathecal (i.t., 5-20 micrograms) administration of meptazinol hydrochloride produced dose-related inhibition of reflex contractions of the urinary bladder, recorded isometrically in urethane-anesthetized rats. The effects of meptazinol were reversed by naloxone administered by the same route. Indeed, this was achieved with intracerebroventricular or intrathecal administration of naloxone (2 micrograms), which also selectively antagonized the mu-receptor ligand [D-Ala2, MePhe4, Gly(ol)5]enkephalin (DAGO). However ICI 174,864 (3 micrograms, i.c.v. or i.t.), a delta-opioid receptor antagonist, did not affect the actions of meptazinol given intracerebroventricularly or intrathecally though it consistently abolished the equieffective actions of a selective delta-receptor ligand (2-D-penicillamine, 5-L-penicillamine) enkephalin (DPLPE). Naloxonazine (5 micrograms, i.c.v. or i.t.), an irreversible mu 1-opioid receptor antagonist, produced prolonged antagonism of the effects of DPLPE and meptazinol. The effects of DPLPE partially or completely recovered by 24 hr, indicating that naloxonazine produced prolonged antagonism of delta-opioid receptors. The effects of maptazinol however only recovered after 72 hr, suggesting that antagonism by naloxonazine of this ligand was irreversible and was mediated through a unique opioid receptor interaction. Subthreshold doses of meptazinol (10 micrograms, i.c.v.; 3 micrograms, i.t.) consistently antagonized the effects of morphine given intracerebroventricularly or intrathecally but not the equieffective doses of DPLPE or DAGO. These observations suggest that meptazinol inhibited reflex contractions of the bladder by supraspinal and spinal mu-opioid receptor activation. Furthermore, its agonistic effect and its antagonistic actions were compatible with interactions at a subpopulation of opioid receptors, possibly mu 1-receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012397 TI - Interaction between [3H]ethylketocyclazocine and [3H]etorphine and opioid receptors in membranes from rat brain. A kinetic analysis. AB - Scatchard analysis of the binding to opioid receptors of [3H]ethylketocyclazocine ([3H]EKC) and [3H]etorphine at equilibrium yielded biphasic plots and computer fitting of the data resulted in a minimal model of two independent saturable binding sites. The KD values for the high- and low-affinity sites were 0.58 and 38 nM for [3H]EKC, and 0.13 and 22 nM for [3H]etorphine. The corresponding density of binding sites was 157 and 418 fmol/mg protein for [3H]EKC, and 220 and 289 fmol/mg protein for [3H]etorphine. The KD values calculated from the association and dissociation rate constants corresponded to those observed at equilibrium. In the course of equilibrium binding, various opioids competed with [3H]EKC and [3H]etorphine preferentially at the high-affinity opioid receptor sites. No difference between the competition patterns of putative mu and kappa ligands was observed. The kinetics of association and dissociation of [3H]EKC and [3H]etorphine revealed that the apparently homogeneous high-affinity binding site observed at equilibrium consisted of two components characterized by their fast and slow equilibrium times, respectively. While none of the mu and kappa opiates investigated altered the rate of dissociation of [3H]EKC or [3H]etorphine, in the presence of sodium ions the rapidly dissociating binding component of [3H]etorphine became refractory to inhibition by mu but not kappa agonists. The results underline the advantages of evaluating both equilibrium binding and the kinetics of ligand-receptor interactions. PMID- 3012398 TI - Regional cerebral blood flow during enkephalin-induced seizures in the rat. AB - Blood flow, determined by the radioactive microsphere technique during epileptiform seizures induced by [D-Ser2,Leu5]enkephalyl-Thr (DSLET), a specific delta-opioid receptor agonist, was examined in different areas of the brain of the rat at various time intervals. An increase in blood flow to the hippocampus and brain stem was observed 2.5 min after administration of DSLET into the left lateral ventricle. An additional increase in flow occurred in the striatum and cerebellum 2.5 min later (5 min after the injection), at which time both the neural and vascular effects of the drug were most marked. Ten minutes after the administration of the drug, cerebral blood flow in all regions except the hippocampus, returned to the respective baseline values. Since the time-course and the magnitude of functional activity and blood flow in the hippocampus showed a good correlation, it is suggested that this region of the brain may play an essential role in triggering and maintaining the seizure phenomena induced by enkephalin. PMID- 3012399 TI - Reversal of behavioral depression by infusion of an alpha-2 adrenergic agonist into the locus coeruleus. AB - This experiment demonstrated that behavioral depression produced by exposure of rats to strong uncontrollable shocks could be reversed by infusion of the alpha-2 adrenergic agonist clonidine into the region of the locus coeruleus (LC). A 20 min infusion, through bilateral cannulae, into the locus coeruleus of clonidine, piperoxane (alpha-2 antagonist) or inactive vehicle (0.85% saline), was given beginning 70 min after the animals were removed from the stress situation. The dose and volume of drug given in the infusion (0.16 microgram/microliter, 0.1 microliter/min) had been previously shown to produce effects specific to the locus coeruleus (Weiss, Simson, Hoffman, Ambrose, Cooper and Webster, 1986; Neuropharmacology 25: 367-384). At the conclusion of the infusion, active behavior of animals was measured in a 15-min swim test. Results showed that stressed animals infused with vehicle exhibited significantly less active behavior in the swim test than did non-stressed animals infused with vehicle, thereby showing the usual behavioral depression seen after exposure to an uncontrollable stress. Stressed animals infused with clonidine showed no difference in active behavior in comparison to non-stressed animals infused with vehicle and showed significantly more activity than did the stressed animals infused with vehicle. Stressed animals infused with piperoxane showed no significant difference in activity in comparison to the stressed animals infused with vehicle and were significantly less active than either the non-stressed animals infused with vehicle or the stressed animals infused with clonidine. Thus, infusion into the locus coeruleus of the alpha-2 agonist clonidine, but not the alpha-2 antagonist piperoxane, eliminated behavioral depression.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012400 TI - Influence of excitatory amino acid receptor antagonists and of baclofen on synaptic transmission in the optic nerve to the suprachiasmatic nucleus in slices of rat hypothalamus. AB - Electrical stimulation of the optic nerve evoked two positive waves with short latency, followed by a large negative wave in the suprachiasmatic nucleus of slices of hypothalamus of the rat. The latency to peak of the two positive waves and the large negative wave were 2.7 +/- 0.1, 6.1 +/- 0.1 and 10.3 +/- 0.5 msec, respectively. Only the large negative wave disappeared in low calcium Ca2+-high magnesium (Mg2+) Krebs solution and with the addition of tetrodotoxin (1 microM) all the waves disappeared. Baclofen inhibited the large negative wave in a dose dependent manner but not the two positive waves. Excitatory amino acid antagonists also inhibited only the large negative wave, i.e. it was reduced to about 70% by 1 mM glutamic acid diethyl ester and to about 50% by both 1 mM 2 amino-4-phosphonobutyric acid and 1 mM DL-2-amino adipic acid. All waves were unaffected by 0.1 mM atropine, hexamethonium and curare. These results indicate that two positive waves, induced by stimulation of the optic nerve are attributed to nerve conduction and the large negative wave to the neurons of the suprachiasmatic nucleus, and that the neuronal pathway from the optic nerve to the suprachiasmatic nucleus may include aspartate and/or glutamate as an excitatory neurotransmitter. PMID- 3012401 TI - L-cycloserine: behavioural and biochemical effects after single and repeated administration to mice, rats and cats. AB - L-Cycloserine dose-dependently inhibited the activity of gamma-aminobutyric acid (GABA)-transaminase (GABA-T) and elevated the level of GABA in whole mouse brain with a peak effect 3-4 hr after a single intraperitoneal injection. At a dose (30 mg/kg) which elevated the level of GABA almost 4-fold, L-cycloserine moderately increased the content of alanine and slightly reduced that of aspartate, glutamate and glycine in the brain. L-Cycloserine (10-30 mg/kg, p.o. or i.p.) prevented tonic seizures induced by 3-mercaptopropionic acid (3-MPA) and audiogenic seizures in DBA/2 mice, without affecting those evoked by pentylenetetrazol, bicuculline and electroshock. Similarly small doses of L cycloserine reduced the level of cGMP in the cerebellum of rats, prevented its elevation by 3-MPA and attenuated the hypothalamically-elicited rage reaction in cats. Larger doses of L-cycloserine (greater than 30-100 mg/kg) impaired the performance of mice in the rotarod, chimney and horizontal wire tests, and reduced spontaneous locomotor activity of rats. Upon repeated administration the inhibitory effect of L-cycloserine on the activity of GABA-T and on seizures elicited by 3-MPA in mice increased. In contrast, the depressant action of L cycloserine on motor performance and locomotion declined in subchronically treated mice and rats. The levels of amino acids in brain after repeated administration did not differ markedly from those in acutely-treated mice. It is suggested that small doses of L-cycloserine, probably by increasing GABAergic inhibition, reduce hyperexcitability in the brain in acute- and subchronically treated animals. Larger doses of L-cycloserine, possibly by inducing multiple neurochemical changes, evoke central depressant effects which diminish during subchronic treatment. PMID- 3012402 TI - Comparison of direct and indirect depressant actions of ketamine on dorsal horn cells in rabbits. AB - The neurophysiological mechanism for the depressant action of ketamine on nociceptive transmission in the spinal cord was examined in rabbits with an intact spinal cord and those with a transected or cold-blocked spinal cord. Ketamine depressed the nociceptive responses in both intact and transected spinal cord groups dose-dependently. The depressant effects of ketamine were significantly greater and longer in the intact group than in the transected group, particularly with 2 and 5 mg/kg of ketamine. The depressant effects produced by ketamine on activity induced by bradykinin were partially reversed by 1 mg/kg of naloxone. Compared to the reversible cold block of the upper part of the spinal cord, the depressant effects produced by both 2 and 5 mg/kg of ketamine on activity induced by bradykinin in the intact spinal cord were significantly greater, and 10 mg/kg of ketamine depressed the nociceptive responses to similar levels in both states. These results suggest that in small to moderate doses, the indirect depressant action of ketamine from the brain stem is more important than the direct action. On the other hand, at a large dose, the direct depressant action becomes predominant. PMID- 3012403 TI - Prolonged CNS hyperexcitability in mice after a single exposure to delta-9 tetrahydrocannabinol. AB - A single exposure to delta-9-tetrahydrocannabinol (THC) resulted in a "rebound" hyperexcitability in the CNS in mice, which was assessed in terms of the susceptibility of the CNS to electrically-induced convulsions. The magnitude of the hyperexcitability was dose-related (25-150 mg/kg, i.p.), as measured 24 hr after treatment. The time-course study of the effect indicated a peak-effect at 24 hr after administration of the drug, with a duration of the effect for as long as 196 hr. The time course of the rebound hyperexcitability to THC was compared to that for phenobarbital, which peaked at 48 hr after administration of the drug and returned to the control value by 96 hr. Tolerance developed rapidly to the motor-toxic effect of THC, but after 23 days of daily treatment there was no evidence of tolerance to the rebound hyperexcitability. The functional significance of the hyperexcitable state was assessed in two tests; electrical kindling to minimal convulsions was enhanced, even when the kindling procedure was initiated 120 hr after exposure to the drug; and the anticonvulsant activity of phenytoin was blocked when mice were treated with the anticonvulsant 96 hr after a single exposure to THC. The results suggest that the rebound response from a single exposure to THC represents a functionally significant prolonged increase in excitability of the CNS. PMID- 3012404 TI - Simultaneous determination of radio-immunoassayable methionine-enkephalin and radioreceptor-active opiate peptides in CSF of chronic pain suffering and non suffering patients. AB - Radio-immunoassayable methionine-enkephalin (ME) and radioreceptor-active opiate peptide levels (OP) were determined in CSF from patients, both with and without chronic pain, under investigation for vertebral disk disease. This study showed: that there was no direct correlation between ME and OP levels in CSF; OP levels were negatively correlated with the ME/OP ratio; migraine patients had higher levels of ME; ME concentrations were reduced in patients receiving anti inflammatory drugs (nonsteroidal): patients with chronic pain (non migraine, no anti-inflammatory drug therapy) had lower ME levels than patients without pain. The data are discussed in relation to animal models of chronic pain. PMID- 3012405 TI - Chronic iminodipropionitrile (IDPN) causes no changes in the rat brain phencyclidine (PCP) receptor. AB - Chronic IDPN treatment leads to a persistent stereotypic and dyskinetic behavioral syndrome which is reminiscent of that caused by PCP in mammals. Since the neuropharmacological profile of the two syndromes are very similar, the status of the PCP binding site was studied in rats who were suffering from the IDPN-induced syndrome. The characteristics of the receptor were not altered in either the striata or the hippocampi of the animals. These results suggest that the development of chronic stereotypies is not intimately linked to any perturbation of the PCP binding site in rat brain. PMID- 3012406 TI - Selective route of doxorubicin administration improves outcome in experimental malignant epidural cord compression. AB - Epidural spinal cord compression was produced in adult Fischer rats by injection of 10(6) viable cells of malignant fibrous histiocytoma anterior to the T-13 vertebral body. Using a tracer dye, it was demonstrated that a portion of the inoculum was always present in the anterior epidural space at the time of inoculation. Paraplegia and incontinence occurred consistently on Days 14 to 27 (median, 23 +/- 3.0). By sequential computed tomographic scans, the growth of the paravertebral tumor was documented and its volume was calculated. A single dose of cisplatin (i.p., 6 mg/kg) or doxorubicin (DXR, i.v. jugular, 6 mg/kg) was administered on Day 1. On Day 18, tumor volume was significantly reduced by DXR (P less than 0.001) and cisplatin (P less than 0.01), but paraplegia continued to occur as in the untreated rats. Comparison of treatment outcome with DXR administered via the jugular vs. the tail vein revealed that both were equally effective in retarding the growth of the paravertebral tumor, and both caused a similar transient leukopenia. However, only the tail DXR brought about a significant delay in the onset of paraplegia (P less than 0.004) and significantly increased the survival (P less than 0.001). It is suggested that the lack of efficacy of systemic chemotherapy for tumors located in the epidural space is probably related to inadequate drug exposure. The improved outcome with tail DXR infusion may be explained by the regional spinal venous perfusion, which allows an increase in the local drug concentration during its first passage through the epidural venous plexus. PMID- 3012407 TI - Non-N-methyl-D-aspartate receptors mediating synaptic transmission in the avian cochlear nucleus: effects of kynurenic acid, dipicolinic acid and streptomycin. AB - We have examined the effects of a number of excitatory amino acid antagonists on transmission at the cochlear nerve-nucleus magnocellularis synapse in the chicken. Using an in vitro preparation and bath application of drugs, we studied the effects of kynurenic acid and several related substances, streptomycin and a selective N-methyl-D-aspartate receptor antagonist, DL-alpha-aminosuberate. The last compound had no effect on evoked transmission. Of the various kynurenic acid related compounds tested, only kynurenic and dipicolinic acid selectively altered responses in nucleus magnocellularis. Quinolinic acid, a kynurenic acid analogue that is structurally akin to dipicolinic acid but which acts selectively at N methyl-D-aspartate receptors, was without effect. The effect of kynurenic acid was solely inhibitory, completely blocking postsynaptic responses with a potency dependent on the frequency of nerve stimulation. No such frequency dependence was seen with dipicolinic acid although this compound also completely suppressed evoked responses. In addition dipicolinic acid potentiated postsynaptic responses at concentrations only slightly lower than those causing inhibition. Streptomycin inhibited responses in nucleus magnocellularis but this effect seems to result partially from the ability of the drug to inhibit presynaptic calcium influx. Our finding that selective antagonists of N-methyl-D-aspartate receptors were ineffective while antagonists of both receptor types, such as kynurenic and dipicolinic acids, inhibited evoked responses reinforces the conclusion that postsynaptic receptors mediating transmission at this synapse are of the non-N methyl-D-aspartate type [Nemeth et al. (1983) Neurosci. Lett. 40, 39 44].(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012408 TI - Calcium-independent release of gamma-aminobutyrate from nerve processes in the developing rabbit retina. AB - Retinas from 3- and 10-day-old rabbits, and from young (29 days), or adult animals were used to study in parallel the development of synaptic vesicles in amacrine cells and the Ca2+ dependence of the K+-stimulated [3H]gamma aminobutyrate release from them. Few synaptic vesicles were observed in the amacrine cell processes in retinas from the 3-day-old rabbits. The number of vesicles significantly increased between 3 and 10 days and increased further between day 10 and the adult animal. The Ca2+ dependence of the K+-stimulated release decreased with increasing age. There is thus a poor correlation between the Ca2+ dependent transmitter release and the number of synaptic vesicles in the nerve terminal, favouring the existence of a Ca2+ dependent nonvesicular process for the [3H]gamma-aminobutyrate release in the rabbit retina. PMID- 3012409 TI - GABAA and GABAB receptors on porcine pars intermedia cells in primary culture: functional role in modulating peptide release. AB - A primary culture of porcine pars intermedia cells with particularly high yields has been developed. The cells, grown in monolayers, secrete the pro opiomelanocortin-derived peptide alpha-melanocyte-stimulating hormone over several weeks. The patch-clamp technique has been used to demonstrate the presence of gamma-aminobutyrateA (GABAA) receptors on the cells. GABA or the selective GABAA receptor agonist isoguvacine produced a depolarizing increase in chloride conductance that desensitized rapidly. The response was antagonized by bicuculline and by the aminopyridazine derivative of GABA (SR 95103), a novel GABAA receptor antagonist. The effects of specific agonists for each receptor were tested on peptide release from cells maintained in a perfusion system. Isoguvacine (10 microM) potentiated Ba2+-evoked release of alpha-melanocyte stimulating hormone, whereas (-)-baclofen (50 microM) decreased both basal and stimulated hormone release. This negative effect on peptide secretion was reproduced when GABA (50 microM) was perfused in the presence of bicuculline (10 microM) to block GABAA receptor activation. The possible mechanisms underlying these GABAA and GABAB effects on stimulus-secretion coupling in this neuroendocrine model are discussed. PMID- 3012410 TI - Receptors on individual neurones. AB - Membrane potential or ionic conductance of neurones of the mammalian central or peripheral nervous system maintained in vitro can be measured over periods of several hours. Drugs or transmitters which change potential or conductance can be applied repeatedly under equilibrium conditions, and pharmacological null methods used to characterize the receptors with which they interact. The method offers an advantage over ligand binding studies on nervous tissue because both agonist and antagonist affinities can be estimated on individual functioning cells. The results to date suggest the hypothesis that a given receptor subtype is always associated with the same change in ion conductance, and the corollary that distinct ion conductances affected by the same transmitter result from interactions with different receptor subtypes. PMID- 3012411 TI - Vitamin E deficiency in neuropathy of abetalipoproteinemia. PMID- 3012412 TI - Progressive polyradiculopathy in acquired immune deficiency syndrome. AB - We studied three patients with acquired immune deficiency syndrome (AIDS) and progressive polyradiculopathy. Postmortem examination of one patient disclosed extensive necrosis, inflammatory infiltrates, and focal vasculitis of spinal roots. Typical cytomegaloviral (CMV), intranuclear, and intracytoplasmic inclusions were noted within enlarged endoneurial and endothelial cells. Progressive polyradiculopathy is an unusual complication of AIDS; CMV may be the causative agent in certain cases. PMID- 3012413 TI - Case for diagnosis. AIDS. PMID- 3012414 TI - [Variations in serum kininase II activity in acute jaundice]. AB - An assay of Kininase II activity in the serum of 92 jaundice patients revealed a significant difference between the group with viral hepatitis (268.48 +/- 70.93 U/ml), that with biliary obstructions (133.05 +/- 37.64 U/ml) and with cirrhosis (173.76 +/- 79.56 U/ml). The increase encountered in patients with medical jaundice correlates well with total bilirubinaemia but not with cytolysis enzymes. This suggests failed demolition of ACE by the hepatocyte during cellular stress. PMID- 3012415 TI - Adenosine A1-receptors in human brain: characterization and autoradiographic visualization. AB - The characteristics and distribution of adenosine A1-receptors in human brain tissues were examined, using [3H]N6-cyclohexyladenosine ([3H]CHA) as a ligand. The binding of [3H]CHA had the pharmacological characteristics of an A1-receptor site and was similar to those of adenosine A1-receptors in rat brain tissue examined in parallel. Autoradiographic localization of adenosine A1-receptors in human brain tissues revealed a heterogeneous anatomical distribution with high levels, particularly in the hippocampal formation, striatum, neocortex and some thalamic nuclei. The distribution of receptors was similar to that seen in the rat brain. However, in some regions, as for example the cerebellar cortex, clear differences were seen. PMID- 3012416 TI - Gangliosides modulate glutamate receptor binding in rat brain synaptic plasma membranes. AB - The influence of gangliosides on the binding of L-[3H]glutamate (Glu) to its receptor(s) on isolated rat brain synaptic plasma membranes was investigated in two different buffer systems using an in vitro filter binding assay. It was found that the tested pure monosialogangliosides GM1 and GM2 as well as the polysialogangliosides GT1b and GD1a enhanced binding by up to about 100%, but only in the presence of 5 mM calcium. The binding site involved was sensitive to quisqualate but insensitive to N-methyl-D-aspartate and kainate. GM1 did exert its stimulating effect by increasing the number of binding sites on the membranes whereas the receptor's affinity for L-Glu was unchanged. Regional differences in rat brain were found: while the hippocampus and cortex exhibited significant Ca/GM1-induced stimulation of Glu binding, the cerebellum was unaffected. Our results suggest modulation of at least one subtype of the Glu receptor by gangliosides. PMID- 3012417 TI - Alpha 1-adrenergic receptor activation releases vasopressin and oxytocin from perfused rat hypothalamic explants. AB - Norepinephrine and the alpha-agonist phenylephrine in concentrations of 10(-5) to 10(-3) M prompted the release of radioimmunoassayable vasopressin (up to 150 pg/min) and oxytocin (up to 20 pg/min) from intraarterially perfused explants of rat basal forebrain. Drug effects were markedly reduced or abolished in the presence of the non-specific alpha-antagonists phentolamine and phenoxybenzamine, and the specific alpha 1-antagonist prazosin. In concert with recent in vivo and in vitro electrophysiological observations, these data imply that endogenous noradrenergic pathways to magnocellular neurosecretory cells are excitatory, mediated through activation of their alpha 1-receptors, thereby enhancing the release of both vasopressin and oxytocin in the neurohypophysis. PMID- 3012419 TI - High gain transmission of single impulses through dorsal column nuclei of the cat. AB - Paired recordings were made in the cat from neurones of dorsal column nuclei and from intact pacinian sensory fibres of the hindlimb interosseous nerve. Direct evidence is presented for central neurones being driven by single impulses arriving over just one sensory nerve fibre. Transmission through this sensory relay appears to be optimized for the detection of minimal sensory inputs. Two mechanisms operate for the amplification of such inputs. First, individual sensory fibres can exert divergent, suprathreshold actions on multiple target neurones, and second, a single impulse coming over one input fibre can induce pairs or bursts of output spikes from its target neurones. PMID- 3012418 TI - Some primary olfactory axons project to the contralateral olfactory bulb in Xenopus laevis. AB - Cobaltous lysine and horseradish peroxidase (HRP) were used to trace primary olfactory axons to their terminations. The tracers were taken up in the olfactory mucosa and transported to the olfactory bulb. The results suggest that either HRP or cobaltous-lysine methods are useful and simple tools for studying such connections. In addition to the ipsilateral projection, we report here for the first time a projection to the medial part of the contralateral olfactory bulb. The overlaps of ipsi- and contralateral inputs to the medial bulb provide further evidence that its functions in frogs may be different from the lateral part, because other connections differ as well. PMID- 3012420 TI - Depression of spike adaptation and afterhyperpolarization by 4-aminopyridine in hippocampal neurons. AB - In guinea pig hippocampal slices, 4-aminopyridine (4-AP) in concentrations of 100 500 microM reduced the adaptation of CA3 pyramidal neurons to depolarizing stimuli, resulting in a prolongation of repetitive firing during injection of long-lasting depolarizing currents. Concurrently, there was a decrease in the 'sag' of potential after spike bursts. Furthermore, 4-AP decreased or abolished the hyperpolarizing potential (the afterhyperpolarization) which normally followed repetitive firing of the neurons. The findings suggest that 4-AP could interfere with the Ca2+-activated K+ current in hippocampal CA3 pyramidal neurons. PMID- 3012421 TI - Opioid binding sites in the nigrostriatal system of the guinea pig are not located on nigral dopaminergic neurons. AB - Unilateral 6-hydroxydopamine injection into the compact zone of the guinea pig substantia nigra was used to destroy nigral dopamine neurons. The glyoxylic acid method for monoamine fluorescence was employed to ascertain the loss of dopaminergic fibers in the striatum and substantia nigra. It could be shown that there was no change in the number of [3H](-)-bremazocine binding sites in the substantia nigra or the striatum on the lesioned side of these guinea pigs. PMID- 3012423 TI - Cystosarcoma phyllodes with liposarcomatous stroma in the female breast. PMID- 3012422 TI - Collagenase digestion alters the organization and turnover of junctional acetylcholine receptors. AB - Acetylcholine receptors at the vertebrate neuromuscular junction are highly organized and metabolically very stable. We report here that digestion of adult rat skeletal muscle with collagenase disorganizes junctional receptors and increases their turnover rate. PMID- 3012424 TI - Biochemical and physicochemical determinations in a premyelin fraction obtained by zonal centrifugation in normal mouse and in dysmyelinating mutants (quaking, shiverer, and myelin-deficient). AB - Myelin and premyelin material denser than myelin were obtained from quaking (Qk), shiverer (Shi), and myelin-deficient (mld) mutant and control mice, using zonal centrifugation on zonal rotor. On these fractions, we performed biochemical analysis (lipids and fatty acid), and, in parallel, we determined the physical structure of membranes by the spin-label method. The hyperfine splitting constant (2 Tll) was used to determine the order of membranes and their rigidity, and frequency of rotation (Vc) was used to measure fluidity. In control mice, the premyelin material contained a lesser amount of sphingolipids than pure myelin, but the relative proportions between hydroxy- and nonhydroxy-cerebroside and sulfatides were similar in the premyelin material and in pure myelin. The premyelin material contained half the alkanes found in the pure myelin and much less very-long-chain fatty acids. The (2 Tll) was lower in the premyelin material, but the (Vc) was similar in myelin and premyelin material. In mutants, the amount of material recovered in the premyelin fraction was reduced in qk, and increased in both shi and mld. The relative amount of sphingolipids were normal in mld, but not in shi mutants, especially in cerebrosides formed with alpha hydroxylated fatty acids and sulfatides formed with unsubstituted fatty acids. The absolute amounts of sphingolipids were nearly normal in both shi and mld. In the premyelin fraction from qk mutants, both relative and absolute amounts of sphingolipids were drastically altered. In percentage, cerebrosides and sulfatides formed with nonhydroxyfatty acids were dramatically reduced, and, conversely, cerebrosides and sulfatides formed with hydroxyfatty acids were increased. In terms of absolute amount, only cerebrosides and sulfatides formed with nonhydroxyfatty acids were dramatically reduced. In the premyelin fraction, polyunsaturated fatty acids were increased in shi and mld, but decreased in qk. In this mutant, lignoceric (24:0) and nervonic (24:1) acids were drastically reduced. The amount of alkanes in the premylin material from qk and mld was reduced by 50%. The shi fraction was nearly free of alkanes. The maximal apparent coupling constant (hyperfine splitting constant, 2 Tll) was not affected in the mld and qk mutant, but was reduced in the shi mutant premyelin fraction. The Vc was dramatically increased in the qk, slightly decreased in the shi, and close to control in the mld. This work provides additional data on premyelin material prepared in various neurological mutants using continuous gradients in zonal rotor.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3012426 TI - Dietary fish oil alters leukotriene generation and neutrophil function. PMID- 3012427 TI - Sequential bilateral germ cell tumors of the testis. PMID- 3012425 TI - Antagonism of dithiobiuret toxicity in rats. AB - Daily administration of dithiobiuret (DTB, 1 mg/kg X 6 days, ip) produced delayed onset muscle weakness in rats as indicated by failure in a treadmill test. In nerve-muscle preparations from DTB-intoxicated rats neuromuscular toxicity was manifested as contractile fatigue during tetanic nerve stimulation. As muscle weakness developed, feed intake decreased and the animals lost body weight. Water intake was not altered during this time, but urine output was increased concomitant with the development of muscle weakness and resulted in a state of negative water balance. Daily administration of d-penicillamine (d-PEN) antagonized DTB-induced treadmill failure in a dose-dependent fashion. A daily dose of d-PEN (1 mMol/kg, ip) that completely antagonized treadmill failure also antagonized the contractile fatigue, reduced feed intake, weight loss and negative water balance caused by DTB administration. In rats already intoxicated with DTB, initiating daily d-PEN treatment or discontinuing further DTB administration, caused the animals to recover normal treadmill performance after a latent period of five days. A single dose of d-PEN (1 mMol/kg, iv) was not effective in reversing treadmill failure or contractile fatigue in rats already intoxicated with DTB. Thus, continuous daily administration of d-PEN was necessary for it to be effective. A single dose of d-PEN (1 m Mol/kg, ip) administered one hr after [14C]-DTB (1 mg/kg, ip) did not affect the plasma and tissue concentrations of DTB-derived radioactivity or their corresponding elimination kinetics. Cumulative urinary and fecal excretion of DTB-derived radioactivity were also unaffected by d-PEN administration as were the relative proportions of DTB and two of its metabolites, monothiobiuret and thiuret, in urine. Other agents that produced dose-dependent antagonism of DTB toxicity were diethyldithiocarbamate, disulfiram, cysteamine and 2,2'-dipyridyl. Considering the chemical and biological properties of DTB and its antagonists, a mechanism of antagonism involving an alteration of the thiol-disulfide and/or divalent metal cation status of motor axon terminals is postulated. PMID- 3012428 TI - Pleomorphic adenoma of the breast with positive estrogen receptors. PMID- 3012429 TI - Adenovirus pericarditis. PMID- 3012430 TI - A method of radionuclide angiography and comparison with contrast aortography in the assessment of aorto-iliac disease. AB - Forty-four patients with symptoms of lower limb arterial insufficiency were examined by radionuclide angiography using 99Tcm in vivo red cell labelling and by contrast aortography. The results of the study indicate that radionuclide angiography may well have a useful role to play in determining the type of contrast study most appropriate for delineating the aortic-iliac segment. PMID- 3012431 TI - The role of the radioisotope phallogram in the investigation of vasculogenic impotence. AB - Patients complaining of impotence were investigated through a multi-disciplinary approach to define the factors involved. Radioisotope phallography was performed on all the patients using 99Tcm-labelled red blood cells (RBCs) and dynamic records of the variation in activity over the penis were obtained. During the course of the study an intravenous injection of a vasodilator (isoxsuprine HCl) was administered. An analysis of the data provided quantitative parameters for measuring the changes in penile blood flow and penile blood volume in response to the vasodilator injection. The results indicate that the radioisotope phallogram is useful both in the diagnosis of vasculogenic impotence and in indicating the method of treatment. PMID- 3012432 TI - Scintigraphic phase analysis of left anterior hemiblock. AB - Left anterior hemiblock (LAHB) is a relatively common disturbance of ventricular conduction which can be an indicator of early conduction system disease. In an effort to better understand this condition, phase analysis of resting radionuclide ventriculograms (RVG) was used to evaluate five patients with LAHB and six normal patients with particular reference to the phase angle difference between the septum and the postero-lateral wall. All patients had normal ejection fractions and visually normal wall motion on RVG. Visual analysis of phase images showed significant differences between the LAHB and normal patients' LV contraction synergy (p less than 0.03) with a delay in septal contraction versus the postero-lateral wall. Four of five patients with LAHB were outside 2 standard deviations of the normal range. Regional quantitative analysis of phase angle differences between posterolateral and septal walls tended to show this difference between normals and LAHB (p = 0.08) as well. Three of five patients with LAHB were outside 2 standard deviations from the normals' mean. There were no significant differences between the standard deviations, skewness, or kurtoses of phase angle histograms of LAHB versus normal patients. Phase analysis can identify some patients with LAHB by both visual and quantitative analysis. The ability to detect and possibly quantitate subtle conduction abnormalities such as LAHB may result in a better understanding of such conduction system diseases. PMID- 3012434 TI - Computerized tomography and Trotter's syndrome in the diagnosis of maxillofacial pain. AB - Trotter's syndrome is a clinical triad of unilateral deafness, neuralgia affecting branches of the trigeminal nerve, and defective mobility of the soft palate, which is caused by malignant tumors involving the lateral pharyngeal recess (Rosenmuller's fossa). It is an ominous presentation, which can masquerade as dental or masticatory pain. Computerized tomography (CT) can be used not only to explain the anatomic basis of Trotter's syndrome but also to determine the extent and distribution of the malignant tumor involved. The advantages of CT over conventional radiography are illustrated by a case of adenoid cystic carcinoma that presented as Trotter's syndrome. Perineural invasion by tumor is shown on the gross level for the first time with CT, and important diagnostic considerations, which may aid in the early diagnosis of future cases, are discussed. PMID- 3012433 TI - Your role as AIDS educator. PMID- 3012435 TI - Familial amelodentinal dysplasia. PMID- 3012436 TI - Leukotrienes: inflammatory mediators--a review. AB - This article reviews a group of inflammatory mediators called leukotrienes. It includes their historical background, chemical pathway of formation, presence in cells and fluids, and biologic activity. These potent substances may be involved in pulpal and periradicular disease, but such involvement has not yet been reported. PMID- 3012437 TI - [A radioimmunological method in the study of the hormonal regulation of reparative osteogenesis (review of the literature)]. PMID- 3012438 TI - The effect of alpha-adrenergic stimulation and blockade on perfusion of myocutaneous flaps. AB - A porcine myocutaneous flap model was utilized to assess the development of denervation adrenergic hypersensitivity and to determine the effects of the alpha adrenergic blocking agent--phenoxybenzamine--on flap blood perfusion. During intravenous administration of norepinephrine, blood flow to the flaps and control skin was monitored simultaneously, using laser Doppler velocimetry and dermofluorometry. A relative decrease in myocutaneous flap blood flow, as compared to control skin in response to norepinephrine infusion, was observed at between 2 and 7 days following flap elevation. This is the same time period during which norepinephrine content of skin flaps is diminished, and suggests development of an increased sensitivity to adrenergic stimulation. Administration of phenoxybenzamine blunted norepinephrine-induced pressor responses and blocked development of adrenergic hypersensitivity in the porcine myocutaneous flap model. Phenoxybenzamine significantly increased flap blood perfusion (as measured by dermofluorometry). PMID- 3012439 TI - Perioperative evaluation and care of patients with lesions involving the skull base. AB - Within the past quarter of a century, contributions to the surgical literature have steadily reflected an increasing interest in the surgical extirpation of lesions involving the skull base. While optimistic reports demonstrate a variety of operative techniques and clinical experiences, there has been a paucity of discussion regarding the intraoperative and perioperative management of these complex cases. Cognizant of the fact that the care of these patients is of paramount importance, we herein review our rationale and practiced protocol for management of this unique patient population. The removal of lesions that also require the sacrifice of multiple cranial nerves results in significant postoperative morbidity. With the significant cardiopulmonary challenges attendant upon long procedures, these patients require constant multisystem Infection surveillance, wound care, cardiopulmonary support, fluid and nutritional monitoring, vocal and pharyngeal rehabilitation, along with psychological support, manifest in a complicated array of managerial decisions, require an organized approach to optimize patient care. PMID- 3012440 TI - Marijuana smoking--possible cause of head and neck carcinoma in young patients. AB - Six cases of advanced head and neck cancer in young patients, who were regular marijuana users, are presented. Numerous carcinogens, as well as respiratory irritants, are found in marijuana smoke. The active euphoria-producing agent, delta-9 tetrahydrocanabinol, has been implicated in altered DNA, RNA, and protein synthesis and consequent chromosomal aberrations. PMID- 3012441 TI - Effect of intrathecal and subepineural capsaicin on thermal sensitivity and autotomy in rats. AB - In 79 Sprague-Dawley rats, we determined the effect of either intrathecal or subepineural capsaicin injection on: latency of withdrawal of the hind foot to a nociceptive thermal stimulus (50 +/- 1 degree C hot plate) and the onset and severity of putative behavioral evidence of chronic pain in the rat (autotomy) which commonly appears following sciatic nerve section. Capsaicin (50 micrograms) was suspended in 5 microliters of vehicle (10% Tween-80 in 0.9% saline) then injected either intrathecally at the level of the L4-5 vertebral interspace or subepineurally in the sciatic nerve at the level of the midfemur. Subepineural capsaicin consistently and efficiently produced thermal analgesia in the rat, while intrathecal capsaicin had no significant analgesic effect. In chronically denervated rats, both subepineural and intrathecal capsaicin decreased the latency to onset of first autotomy, and intrathecal capsaicin increased the severity of this behavior significantly. These data are consistent with the hypothesis that autotomy is the rat's response to abnormal sensations perceived in the denervated hind limb. Deafferentation of dorsal horn neurons appears to be of paramount importance in the production of autotomy while the relevance of peripherally originating spontaneous neuroma discharges to autotomy behavior is questioned. PMID- 3012443 TI - The potential of using recombinant DNA species-specific probes for the identification of tropical Leishmania. PMID- 3012442 TI - Hypothalamic inhibition of rat dorsal horn neuronal responses to noxious skin heating. AB - The responses of single lumbar dorsal horn neurons to noxious radiant heat stimuli (42-54 degrees C, 10 sec, 1/2 min) applied to glabrous hind paw skin were recorded in rats anesthetized with sodium pentobarbital. Unit responses to 50 or 52 degrees C stimuli were constant over time and were consistently and powerfully inhibited during bipolar stimulation (three 100 msec trains/sec at 100 Hz, 200 microA) in the medial hypothalamus. Inhibition was also evoked by stimulation in medial and ventrobasal thalamic nuclei, lateral hypothalamus and adjacent cerebral peduncle, and amygdala. Inhibition increased with graded increases in intensity of hypothalamic stimulation, with a mean inhibitory threshold of 71 +/- 43 (S.D.) microA for 13 units. The responses of dorsal horn units to graded increases in the temperature of noxious heat stimuli were inhibited during hypothalamic stimulation, such that slopes of the linear temperature-response functions were reduced with no change in response threshold (mean: approximately 44 degrees C). Inhibition was blocked or reduced in 4/7 units following systemic administration of the 5-hydroxytryptamine (5-HT) antagonist methysergide. The results confirm and extend previous work in the cat and are discussed in relation to analgesic mechanisms. PMID- 3012444 TI - The application of recombinant DNA technology to problems of helminth identification. PMID- 3012445 TI - An introduction to recombinant DNA technology. PMID- 3012447 TI - A practical guide to the diagnosis of congenital infections in the newborn infant. AB - Every attempt should be made to identify an etiologic agent in infants with suspected congenital infection. Some of the infections are treatable, such as herpes simplex, syphilis, and toxoplasmosis. For other infectious agents (i.e., CMV and rubella), close follow-up will enable early detection of hearing impairment and permit early educational intervention. In addition, appropriate isolation measures will reduce the risk of nosocomial infections due to herpes simplex, rubella, hepatitis B, and CMV. Rational use of the available diagnostic tools will enable detection of most symptomatic congenitally infected neonates at a reasonable cost. PMID- 3012446 TI - [Cytological-histological correlations in stomach cancer]. PMID- 3012448 TI - Common day-care diseases: patterns and prevention. PMID- 3012449 TI - Bronchial carcinoid tumor in a 12-year-old child. AB - Symptoms (hemoptysis, recurrent pulmonary infections), diagnostic work-up (roentgenology, bronchoscopy with biopsy), and treatment (surgical resection of a bronchial carcinoid tumor in a 12 year old girl) are discussed. Special attention was paid to the tumor histochemistry, showing serotonin containing granules. Levels of circulating hormones and vasoactive agents, including serotonin, were within normal limits. PMID- 3012450 TI - Herpes simplex virus-stimulated gamma-interferon production by newborn mononuclear cells. AB - Blood mononuclear cells from newborns and from adults, immune or nonimmune to herpes simplex virus, were cultured with IL 2 and herpes simplex virus and the amount of gamma-interferon in the supernatant measured after 3 days. The newborn and nonimmune adult cells made equivalent trace amounts of gamma-interferon in cultures containing either herpes simplex virus or IL 2 alone and there was a 5- to 10-fold increase in cultures containing both. Experiments in which the Leu 11+ cells were either depleted or enriched suggest that this subset of natural killer cells is both necessary and sufficient for gamma-interferon production in the absence of immune T cells. PMID- 3012451 TI - Leukotriene B4 biosynthesis in polymorphonuclear leukocytes from blood of umbilical cord, infants, children, and adults. AB - The biosynthesis of 5(S), 12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid, leukotriene B4, by human polymorphonuclear leukocytes was examined in relation to age. The leukotriene B4 production by polymorphonuclear leukocytes from umbilical cords, infants, and adults was assayed using high pressure liquid chromatography. The specificity of the leukotriene B4 assay was examined by gas chromatography mass spectrometry. Polymorphonuclear leukocytes from 10 umbilical cords, 24 infants and children, and 10 adults were examined for their ability to synthesize leukotriene B4, in vitro after stimulation by the ionophore A23187 or platelet-activating factor. Among the infants and children, there was a slight age-dependent increase of leukotriene B4 production by polymorphonuclear leukocytes in response to ionophore A23187, but it was not statistically significant. Leukotriene B4 production by polymorphonuclear leukocytes in the umbilical cords and infants was not significantly lower than that of polymorphonuclear leukocytes in adults in response to both ionophore A23187 and platelet-activating factor under our experimental conditions. PMID- 3012452 TI - Intellectual development in school-aged children with asymptomatic congenital cytomegalovirus infection. AB - Congenital cytomegalovirus infection occurs in about 1% of live births. Although symptomatic congenital infection often results in severe developmental deficits and mental retardation, about 90% have asymptomatic infection. Previous studies of the intellectual development in children with asymptomatic congenital cytomegalovirus have resulted in mixed findings. To control for the effects of hearing impairment (which occurs in about 15% of asymptomatic children) on intelligence scores, we tested 18 prospectively followed, normally hearing, school-aged children with asymptomatic congenital cytomegalovirus (15 black, ten male) and 18 controls matched for age, sex, race, school grade, and socioeconomic status. Children were tested via the Wechsler Intelligence Scale for Children Revised, the Kaufman Assessment Battery for Children, and the Wide Range Achievement Test. Multivariate analysis revealed no differences between groups on intelligence scores or subscales, achievement scores, or incidence of learning disabilities (defined as significant discrepancy between intelligence and achievement), and mean scores for both groups were very close to national norms. It is concluded that the 25,000 children born in the United States each year with asymptomatic congenital cytomegalovirus and normal hearing are not likely to be at increased risk of mental impairment. PMID- 3012453 TI - Coxsackievirus B2 infection in a neonate with incontinentia pigmenti. AB - Because of the concern for herpes simplex virus infection in the neonate, the presence of neonatal vesiculobullous lesions is a critical finding. However, there are other etiologies for these lesions. A case of a neonate with a vesicular rash and meningoencephalitis which was initially thought due to herpes is presented. The infant was ultimately determined to have incontinentia pigmenti and a concomitant coxsackievirus B2 infection. PMID- 3012454 TI - Constipation. PMID- 3012455 TI - Day care and illness: evidence, cost, and public policy. AB - Parents, pediatricians, social scientists, and policymakers have become increasingly concerned that infants and children in day care, especially those younger than 3 years of age, are at risk for morbidity associated with several types of acute illness. We have examined the empirical evidence on the impact of day-care attendance on frequency of respiratory illnesses, diarrhea, hepatitis A, meningitis, and cytomegalovirus disease in children, day-care staff, and household contacts. The short- and long-term costs of day-care-associated illnesses were assessed, wherever possible within a benefit-cost framework. Available evidence suggests that children in day care, and sometimes their teachers and household contacts, have higher rates of diarrhea, hepatitis A, meningitis, and possible otitis media than children not in day care. There is only weak or moderate evidence that children and their families are at risk for high rates of respiratory illness (other than otitis media) or cytomegalovirus infection. Taken together, the excess of these illnesses among children in day care may impose moderate net costs on families and on society. Revisions of state regulatory policy regarding health practices in day care and policy initiatives designed to provide parents with more information and authority are recommended to protect the health and development of children in day care. PMID- 3012456 TI - [Various clinico-virological and morphological aspects of the diagnosis of herpetic encephalitis in children]. PMID- 3012457 TI - Electrical and molecular coupling between sodium and proton fluxes in basolateral membrane vesicles of rat liver. AB - Mechanisms of Na+-H+ exchange in the hepatocyte were studied utilizing isolated basolateral membrane vesicles prepared by two different methods: Evidence was obtained for the existence of molecular coupling of Na+ and H+ fluxes (Na+/H+ antiport) which exhibits saturation kinetics (Km 7 mmol/l Na+) and is inhibited by amiloride (1.0 mmol/l). Although the two membrane preparations showed differences with respect to ionic permeabilities, our data suggest that a relatively high H+ conductance exists in the basolateral plasma membrane. Hence, electrical coupling of conductive H+ and Na+ fluxes in the opposite direction could contribute to net Na+-H+ exchange across the basolateral hepatocyte plasma membrane. PMID- 3012458 TI - Junctional transmission in renin-containing and smooth muscle cells of the afferent arteriole. AB - Intracellular recordings were done in renin-containing juxtaglomerular (JG) and vascular smooth muscle (VSM) cells of the mouse kidney afferent arteriole. Both cell types exhibited a membrane potential around -75 mV and spontaneous depolarizing transients resembling spontaneous excitatory junction potentials (SEJPs) in the arterioles of other organs. The amplitude distribution of these randomly occurring transients was skewed in both cell types with a modal value of 1.2-1.9 mV. Activation of presumably postjunctional alpha 1-, P2-, ANG II- and AVP-receptors depolarized JG and VSM cells. Application of the P1-purinoceptor agonist 2-chloroadenosine strongly increased frequency and amplitude of the SEJP like events, whereas these transients were abolished by the P1-purinoceptor antagonist 8-phenyltheophylline, both substances presumably acting on prejunctional receptors. The SEJP-like events were completely depressed by reserpine treatment, but not abolished by alpha 1-, alpha 2-, and P2-antagonists. At present, it cannot be decided, whether norepinephrine is the sole transmitter in the afferent arteriole, acting on specialized junctional adrenoceptors with the P2-purinoceptors being irrelevant for junctional transmission, or whether both substances are co-transmitters. Except norepinephrine and ATP, all other transmitter candidates tested were ruled out for various reasons. PMID- 3012459 TI - Aplastic crisis and erythema infectiosum (fifth disease) revealing a hereditary spherocytosis in a familial human parvovirus infection. AB - We report two clinical observations on a father and son, who both presented an aplastic crisis at the same time during a human parvovirus (HPV) infection. This erythroblastopenia revealed hereditary spherocytosis in both patients. The son also had a clinical status of erythema infectiosum (fifth disease). This data shows that HPV may be responsible for these two different pathologies in the same person. PMID- 3012460 TI - Highly selective chemical modification of cruciform loops by diethyl pyrocarbonate. AB - Diethyl pyrocarbonate reacts with the single-stranded loops of cruciform structures with great selectivity. Adenine bases are carbethoxylated, as a result of which the backbone may be cleaved with piperidine, and the level of chemical modification at each base may be determined. We have studied the ColE1 and (A T)34 cruciforms of pColIR315 and pXG540. In each case we observe maximal modification at the most central adenosine of the loop, and an overall pattern of modification corresponding to a total loop size of about six bases. The results may be interpreted in terms of a model in which the loop has a defined tertiary structure. No modification was detected at either cruciform four-way junction, suggesting that this region is fully base-paired. PMID- 3012461 TI - Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targeting presequence. AB - A cDNA clone spanning the entire amino acid sequence of the nuclear-encoded uncoupling protein of rat brown adipose tissue mitochondria has been isolated and sequenced. With the exception of the N-terminal methionine the deduced N-terminus of the newly synthesized uncoupling protein is identical to the N-terminal 30 amino acids of the native uncoupling protein as determined by protein sequencing. This proves that the protein contains no N-terminal mitochondrial targeting prepiece and that a targeting region must reside within the amino acid sequence of the mature protein. PMID- 3012463 TI - RecBC, sbcB independent, (AT)n-mediated deletion of sequences flanking a Xenopus laevis beta globin gene on propagation in E. coli. AB - Plasmids containing sequences 3' of the adult beta 1 globin gene of Xenopus laevis are unstable on propagation in a range of E. coli host strains. Up to 300 bp of Xenopus DNA are lost by rec A independent recombination between (AT)37 and (AT)17 sequences. Additionally, smaller deletions occurring in or around the (AT)37 sequence are observed. Deletion of these potential cruciform structures occurs in the absence of exonuclease I, exonuclease V and exonuclease VIII as the same pattern of deletion events is observed in recA recBC sbcB and recBC sbcA recE strains. PMID- 3012462 TI - Structure and expression of three light-harvesting chlorophyll a/b-binding protein genes in Arabidopsis thaliana. AB - The genome of Arabidopsis thaliana is exceedingly small, in part because it lacks the large middle repetitive DNA component characteristic of other plants. In this paper we have characterized a member of the low copy DNA component: the gene family for the light-harvesting chlorophyll a/b-protein. This gene family is unusual in that it contains far fewer members than the 7-16 coding sequences for this protein found in other plants. We used cross-hybridization with a Lemna gene encoding a light-harvesting chlorophyll a/b-protein to isolate 3 genes from Arabidopsis, all of which are clustered on an 11-kb genomic clone. Southern blot analysis suggests that there is a fourth related gene in Arabidopsis. Sequence analysis of the three genes demonstrates that within the translated region the nucleic acid sequence homology is 96%, the deduced amino acid sequence of the mature proteins is identical for the three genes, and two of the genes have a high degree of sequence homology in both their 5' and 3' immediate flanking regions. The genes have regulatory sequences typical of eukaryotic genes upstream of the translation start sites. However, not all of these genes are equally expressed in plants grown under normal light-dark conditions. PMID- 3012464 TI - Identification and location of nine T5 bacteriophage tRNA genes by DNA sequence analysis. AB - Sequence analysis of two DNA fragments generated from bacteriophage T5 DNA by restriction with Hpa I and Hae III has resulted in the detection and localization of nine tRNA genes (His, two Ser genes, Leu, Val, Lys, fMet, Pro, and Ile). The genes which code for tRNAs His and Leu are partials, whereas the remaining genes are complete. A majority of the tRNA genes are located in close proximity to one another. A unique feature of the Pro and Ile genes is that their DNA sequence overlap. PMID- 3012465 TI - DNA sequence of the herpes simplex virus type 1 gene encoding glycoprotein gH, and identification of homologues in the genomes of varicella-zoster virus and Epstein-Barr virus. AB - We have determined the sequence of herpes simplex virus type 1 DNA around the previously mapped location of sequences encoding an epitope of glycoprotein gH, and have deduced the structure of the gH gene and the amino acid sequence of gH. The unprocessed polypeptide is predicted to contain 838 amino acids, and to possess an N-terminal signal sequence and a C-terminal transmembrane sequence. Temperature-sensitive mutant tsQ26 maps within the predicted gH coding sequence. Homologous genes were identified in the genomes of two other herpesviruses, namely varicella-zoster virus and Epstein-Barr virus. PMID- 3012466 TI - Acinetobacter calcoaceticus encoded mutarotase: nucleotide sequence analysis of the gene and characterization of its secretion in Escherichia coli. AB - The nucleotide sequence of the mutarotase gene from Acinetobacter calcoaceticus has been determined. It reveals an open reading frame of 381 amino acids. The codon usage of A. calcoaceticus for this gene is similar to E. coli except for the amino acids Leu, Ala, Glu, and Arg where major differences exist. This did not interfere drastically with high level expression in E. coli. The regulatory sequences for the initiation of translation are similar to the ones described for E. coli. The N-terminal 20 amino acids, which are not found in the mature enzyme, show homology to signal sequences of exported proteins. In A. calcoaceticus and E. coli mutarotase is specifically secreted into the periplasmic space. Processing of the signal sequence occurs at identical sites in both organisms. The mature mutarotase consists of 361 amino acids and has a calculated molecular weight of 38457 Da. Expression of mutarotase at a high level in a recombinant E. coli destabilizes the outer membrane. This results in coordinated leakage of mutarotase and beta-lactamase into the culture broth. PMID- 3012468 TI - Variation in number of long, open reading frames among the members of copia, a Drosophila retrotransposon, and cDNAs of copia-related RNA in virus-like particles. PMID- 3012467 TI - Nucleotide sequence and transcriptional analysis of the E. coli ushA gene, encoding periplasmic UDP-sugar hydrolase (5'-nucleotidase): regulation of the ushA gene, and the signal sequence of its encoded protein product. AB - The DNA sequence of the ushA gene, encoding UDP-sugar hydrolase (5' nucleotidase), has been determined. The amino-terminal sequence encodes a signal peptide whose predicted processing site is confirmed by N-terminal amino acid analysis of purified mature UshA protein. The signal sequence contains a concentration of rare codons in comparison with the mature sequence. The origins of transcription from the ushA promoter have been determined, using primer extension. Three transcripts, originating within a 6 bp region, were identified and might be related to three overlapping potential -10 hexamers in the ushA promoter region. There was a discernable change in the relative proportion of these transcripts during growth-phase regulation of the ushA gene. PMID- 3012469 TI - Pvu II RFLP in the 5' of the human apolipoprotein B gene. PMID- 3012470 TI - Two RFLPs generated by Taq I at the human HRAS1 locus. PMID- 3012471 TI - A frequent polymorphism for the cytosolic thymidine kinase gene, TK1, (17q21-q22) detected by the enzyme TaqI. PMID- 3012472 TI - Hydrolysis by restriction endonucleases at their DNA recognition sequences substituted with mismatched base pairs. AB - Restriction endonucleases were tested for their ability to catalyze the cleavage of mismatch-containing recognition sites in DNA. These mismatched base pairs were T.G, U.G, or A.C in covalently closed, circular heteroduplexes prepared by in vitro extension of chemically synthesized oligonucleotide primers annealed to a bacteriophage M13-derived viral DNA. None of the restriction enzymes was able to completely cleave the mismatch-containing recognition sites under standard conditions. However, three of them, SmaI, SalI, and SstI, catalyzed partial digestion leading to an accumulation of DNA singly nicked at the mismatched recognition site. The ability of SmaI and SstI to partially cleave at a mismatch was shown to depend on the nature and position of the mismatch within the corresponding recognition site. In contrast, little or no digestion was obtained with AccI, HincII, HindIII, and KpnI at mismatch-containing sites. Therefore, in some cases a transition-type substitution in only one strand of a recognition site inhibits restriction endonuclease-catalyzed digestion at that site although in others partial digestion occurs. PMID- 3012473 TI - Osteonectin mRNA: distribution in normal and transformed cells. AB - Overlapping cDNA clones encoding bovine osteonectin were isolated from a lambda gt11 expression library constructed from bovine bone cell mRNA. The longest clone, lambda On 17 (insert size 2.0 kb) was studied in detail. The clone was shown to encode osteonectin by hybrid select translation experiments and by DNA sequence analysis. Northern analysis of bone cell RNA showed the length of the osteonectin mRNA to be 2.0 kb. Osteonectin message was found in bone but not in soft tissue (liver and brain) preparations consistent with the distribution of the protein in these tissues. On the other hand, osteonectin message was observed in tendon, a tissue in which little or no osteonectin protein is found in vivo. Hybridization of osteonectin cDNA was detected in cells from a number of species including human, rat, mouse and chick. The level of osteonectin mRNA was drastically decreased in chick embryo fibroblasts transformed by Rous sarcoma virus. PMID- 3012474 TI - A new polymorphism in the factor VIII gene for prenatal diagnosis of hemophilia A. AB - A restriction fragment length polymorphism (RFLP) has been found in the gene for clotting factor VIII. Defects in this gene are the cause of hemophilia A. The DNA polymorphism affects an XbaI site in intron 22 of the gene. Two alleles occur in a frequency of 59 and 41 percent of the X chromosomes tested. Furthermore, about 25 percent of females who are homozygous for the previously reported BclI RFLP in the factor VIII gene are heterozygous for the XbaI polymorphism. This new RFLP thus represents a significant addition to available probes for the DNA-based prenatal diagnosis and carrier detection of this disease. PMID- 3012475 TI - A calculation of fragment lengths obtainable from human DNA with 78 restriction enzymes: an aid for cloning and mapping. PMID- 3012476 TI - 3'-Terminal sequence of human parainfluenza virus 3 genomic RNA. PMID- 3012477 TI - A polymorphism of the human von Willebrand factor (vWf) gene with BamHI. PMID- 3012478 TI - DNA replication is required for abundant expression of a plasmid-borne late US11 gene of herpes simplex virus type 1. AB - During herpes simplex virus type 1 (HSV-1) infection, the appearance of true-late gene products is severely reduced under conditions of DNA synthesis inhibition. This report describes the use of a plasmid-borne promoter of a true-late HSV-1 gene (US11), linked to the rabbit beta-globin gene, to study the requirement of DNA replication for late gene expression. The activity of the plasmid-borne US11 promoter in constructs containing or lacking an HSV-1 origin of replication (ORIS) was analysed by quantitative S1 mapping of correctly initiated hybrid transcripts. Following HSV-1 superinfection of transfected HeLa cells, the US11 promoter in ORI+ plasmids was expressed with similar kinetics to the viral US11 promoter. US11 promoter activity was first detected at the same time as the onset of DNA template replication. Expression of US11 RNA was detectable from non replicating ORI- plasmids, although transcript accumulation was reduced by greater than 90%. Sequences containing the IE-5 promoter (a 3' co-terminal gene whose transcription starts 5' of US11) also played a positive role in achieving normal US11 gene expression. PMID- 3012480 TI - Target sites for the transposition of rat long interspersed repeated DNA elements (LINEs) are not random. AB - The long interspersed repeated DNA family of rats (LINE or L1Rn family) contains about 40,000 6.7-kilobase (kb) long members (1). LINE members may be currently mobile since their presence or absence causes allelic variation at three single copy loci (2, 3): insulin 1, Moloney leukemia virus integration 2 (Mlvi-2) (4), and immunoglobulin heavy chain (Igh). To characterize target sites for LINE insertion, we compared the DNA sequences of the unoccupied Mlvi-2 target site, its LINE-containing allele, and several other LINE-containing sites. Although not homologous overall, the target sites share three characteristics: First, depending on the site, they are from 68% to 86% (A+T) compared to 58% (A+T) for total rat DNA (5). Depending on the site, a 7- to 15-bp target site sequence becomes duplicated and flanks the inserted LINE member. The second is a version (0 or 1 mismatch) of the hexanucleotide, TACTCA, which is also present in the LINE member, in a highly conserved region located just before the A-rich right end of the LINE member. The third is a stretch of alternating purine/pyrimidine (PQ). The A-rich right ends of different LINE members vary in length and composition, and the sequence of a particularly long one suggests that it contains the A-rich target site from a previous transposition. PMID- 3012481 TI - DNA sequence of the control region of phage D108: the N-terminal amino acid sequences of repressor and transposase are similar both in phage D108 and in its relative, phage Mu. AB - We have determined the DNA sequence of the control region of phage D108 up to position 1419 at the left end of the phage genome. Open reading frames for the repressor gene, ner gene, and the 5' part of the A gene (which codes for transposase) are found in the sequence. The genetic organization of this region of phage D108 is quite similar to that of phage Mu in spite of considerable divergence, both in the nucleotide sequence and in the amino acid sequences of the regulatory proteins of the two phages. The N-terminal amino acid sequences of the transposases of the two phages also share only limited homology. On the other hand, a significant amino acid sequence homology was found within each phage between the N-terminal parts of the repressor and transposase. We propose that the N-terminal domains of the repressor and transposase of each phage interact functionally in the process of making the decision between the lytic and the lysogenic mode of growth. PMID- 3012479 TI - Nuclease recognition of an alternating structure in a d(AT)14 plasmid insert. AB - The nuclease reactivity and specificity of a cloned tract of poly X (dA-dT) X poly(dA-dT) has been explored. Digestion with DNAse I, Mung Bean nuclease, S1 nuclease, DNAse II, and copper (1,10-phenanthroline)2 on a 256 base pair restriction fragment containing d(AT)14A revealed a dinucleotide repeat structure for the alternating sequence. Furthermore, conditions which wind or unwind the linear DNA had little effect on the reactivity of the AT insert. These preferred cleavages offer insights to structural alterations within the DNA helix which differ from A, B, or Z-DNA. Nucleation into flanking sequences by this structural alteration was not observed. PMID- 3012482 TI - Characterization of 3'----5' exonuclease associated with DNA polymerase of silkworm nuclear polyhedrosis virus. AB - 3'----5' Exonuclease specific for single-stranded DNA copurified with DNA polymerase of nuclear polyhedrosis virus of silkworm Bombyx mori (BmNPV Pol). BmNPV Pol has no detectable 5'----3' exonuclease activity on single-stranded or duplex DNA. Analysis of the products of 3'----5' exonucleolytic reaction showed that deoxynucleoside monophosphates were released during the hydrolysis of single stranded DNA. The exonuclease activity cosedimented with the polymerase activity during ultracentrifugation of BmNPV Pol in glycerol gradient. The polymerase and the exonuclease activities of BmNPV Pol were inactivated by heat with nearly identical kinetics. The mode of the hydrolysis of single-stranded DNA by BmNPV Pol-associated exonuclease was strictly distributive. The enzyme dissociated from single-stranded DNA after the release of a single dNMP and then reassociated with a next polynucleotide being degradated. PMID- 3012483 TI - Analysis of LINE-1 family sequences on a single monkey chromosome. AB - The structure of LINE-1 (L1Ca) family members present on African green monkey chromosome CAE-19 is compared with that of the entire set of L1Ca sequences present in the monkey genome. The analysis involved annealing of cloned subsegments of monkey L1 family members to DNA-blots containing restriction endonuclease digests of either total monkey liver DNA or DNA isolated from a monkey/mouse somatic cell hybrid carrying the single monkey chromosome. In addition, L1Ca segments cloned from hybrid cell DNA were characterized by restriction endonuclease mapping and hybridization. The data indicate that, taken as a whole, the set of L1Ca sequences on CAE-19 tends to differ in characteristic ways from the set present in the total monkey genome. PMID- 3012484 TI - There exists a distinct stage during mammalian DNA synthesis immediately after joining of replication intermediates. AB - We describe an approach, using alkaline cell lysis and digestion with nuclease S1, which permits to distinguish between newly ligated DNA and the DNA of mature chromatin. When cells with steady-state labelled DNA (mature DNA) are analyzed, the results show labelled "nucleosomal-sized" DNA. However, when DNA of cells pulse-labelled with thymidine for 45 seconds is examined one can detect only large DNA. The newly ligated DNA is not reduced to "nucleosomal-sized" DNA by nuclease S1. When the large DNA is denatured in formamide one can detect 10 kb DNA fragments. Furthermore in pulse-chase experiments there appear, after formamide-treatment, increasing amounts of "nucleosomal-sized" DNA with a parallel decrease in the amount of 10 kb DNA fragments. Hence the newly ligated, large, DNA differs from mature DNA and represents a distinct stage during DNA replication. PMID- 3012485 TI - Role of fat, fiber, nitrate, and food additives in carcinogenesis: a critical evaluation and recommendations. AB - This review presents a critical, select evaluation of the amount and type of fat or fiber in nutritional carcinogenesis, with the emphasis being on cancer development in the mammary gland and large bowel. The role of nitrate and nitrosation is described in relation to risk for cancers of the head and neck (especially the esophagus) and cancers of the stomach and the liver. Systematic tests of increasing complexity to delineate possible carcinogenic risk in food additives and contaminants are described. Specific recommendations stemming from these evaluations are made as to dietary recommendations designed to reduce cancer risk. PMID- 3012486 TI - Acebutolol: a review of its pharmacology, pharmacokinetics, clinical uses, and adverse effects. AB - Acebutolol is a new hydrophilic, cardioselective beta-adrenergic-blocking agent that possesses partial agonist and membrane-stabilizing activities. In the treatment of mild to moderate essential hypertension, once-daily acebutolol as monotherapy provides effective control in a large majority of patients and produces a further reduction in blood pressure when used concomitantly with diuretics. Acebutolol is as effective as other beta-blocking agents, and in a large, double-blind, parallel study against propranolol was found to cause less reduction in heart rate, and fewer neurologic side effects and patient withdrawals due to adverse effects. Oral acebutolol is also effective in suppressing premature ventricular contractions, and in small numbers of patients generally beneficial results were obtained in supraventricular and ventricular arrhythmias with intravenous administration. These salutary effects are attributable to beta blockade. Controlled clinical trials documented the antianginal actions of oral acebutolol in chronic stable angina pectoris; its efficacy in this regard is comparable to that of other beta-blocking agents. The drug produces smaller decreases in heart rate and cardiac output and alterations in peripheral vascular hemodynamics than beta-blocking drugs without partial agonist activity, and because of its cardioselectivity, it may be used cautiously in patients with bronchospastic disease. Acebutolol has minimal metabolic effects and does not elevate levels of blood lipids during long-term therapy; high density-lipoprotein cholesterol increased with acebutolol in a small number of patients. PMID- 3012487 TI - ACTH1-4 potentiates alpha-MSH-induced melanophore dispersion and excessive grooming. AB - The biological activity and a possible modulatory role of the N-terminal tetrapeptide Ser-Tyr-Ser-Met from alpha-MSH/ACTH was tested in the Anolis melanophore assay, the Xenopus melanophore assay, tyrosinase stimulation in mouse melanoma cells and in excessive grooming in the rat. ACTH1-4 did not exhibit biological activity in any of these four assays nor did it have modulatory properties in the Xenopus and the melanoma cell assay. However, in the Anolis assay ACTH1-4 potentiated pigment dispersion induced by alpha-MSH, alpha-MSH5-13 and ACTH1-24 by a factor of about 2. In the grooming assay ACTH1-4 potentiated the effects of alpha-MSH, alpha-MSH5-13, ACTH1-16 and ACTH5-16, but not those of ACTH1-24. Oxidized ACTH1-4 was without biological activity and potentiating properties in all four assays. This study shows that small fragments of the pro opiomelanocortin precursor, which are devoid of biological activity, can modulate peripheral and central actions of alpha-MSH/ACTH. PMID- 3012488 TI - Antinociceptive effects in rodents of the dipeptide Lys-Trp (Nps) and related compounds. AB - Intracerebroventricular administration of the synthetic dipeptide derivative Lys Trp (Nps) (LTN) elicits a potent and naloxone-sensitive antinociceptive effect in mice and in rats using heat and electrical current respectively as the noxious stimuli. LTN does not induce analgesia by directly acting on opioid receptors but the peptidase inhibiting activity of the new compound may account in part for the behavioral effect. LTN produces also a marked decrease in the met-enkephalin content of the periaqueductal gray suggesting a possible enkephalin releasing property. Structure-activity studies with different analogs of LTN indicate that replacement of Lys by other basic amino acids results also in compounds with a potent antinociceptive effect whereas replacement by neutral or acidic amino acids leads to a complete loss of activity. PMID- 3012489 TI - Distribution of epidermal growth factor binding sites in the adult rat anterior pituitary gland. AB - The distribution of epidermal growth (EGF) binding sites was studied in the pituitary gland using light and electron microscope autoradiography which was performed at different time intervals (2 to 60 min) after intravenous (IV) injection of [125I]EGF into adult rats. At the light microscopic level, the labeling was found over cells of the anterior pituitary gland. The time-course study performed by light microscope autoradiography showed that the maximal values were reached at the 2 min time interval. At this time interval, most silver grains were found at the periphery of the target cells. After, the number of silver grains decreased progressively and the localization of silver grains in the cytoplasm indicated the internalization of [125I]EGF. Electron microscope autoradiography showed that labeling was mostly restricted to mammotrophs and somatotrophs. Control experiments indicated that the autoradiographic labeling was due specific interaction of [125I]EGF with its binding site. These results indicate that EGF binding sites are present in at least two anterior pituitary cell types and suggest that EGF can exert a physiological role in the pituitary gland. PMID- 3012491 TI - Effects of corticotropin-releasing factor, corticotropin and cortisol on gastrointestinal motility in dogs. AB - Gastrointestinal motor activity following intracerebroventricular (ICV) and intravenous (IV) administration of corticotropin releasing factor (CRF), corticotropin (ACTH) and cortisol was investigated in fasted dogs with strain gauge transducers chronically implanted on the antrum and proximal jejunum. ICV but not IV administration of CRF (20 to 100 ng/kg) suppressed the gastric cyclic migrating motor complex (MMC) for 3 to 6 hours without affecting the jejunum. Similar disruptive effects on the gastric MMC were observed after ICV administration of ACTH (0.5 U/kg) or cortisol (0.1 micrograms/kg) but not after IV administration of 10 times higher doses. These results suggest that in dog CRF may be involved in the central control of the interdigestive gastric motility, these effects were not probably due to the release of ACTH and cortisol the other hormones of the pituitary adrenocortical system change the gastric motility when centrally administered through a possible feed-back mechanism affecting brain CRF level. PMID- 3012490 TI - Neuropeptide Y receptors in rat brain: autoradiographic localization. AB - Neuropeptide Y (NPY) receptor binding sites have been characterized in rat brain using both membrane preparations and receptor autoradiography. Radiolabelled NPY binds with high affinity and specificity to an apparent single class of sites in rat brain membrane preparations. The ligand selectivity pattern reveals strong similarities between central and peripheral NPY receptors. NPY receptors are discretely distributed in rat brain with high densities found in the olfactory bulb, superficial layers of the cortex, ventral hippocampus, lateral septum, various thalamic nuclei and area postrema. The presence of high densities of NPY and NPY receptors in such areas suggests that NPY could serve important functions as a major neurotransmitter/neuromodulator in the central nervous system. PMID- 3012492 TI - Opioid and other peptides as inhibitors of leumorphin (dynorphin B-29) converting activity. AB - A thiolprotease from rat brain membranes was shown to convert synthetic dynorphin B-29 (Dyn B-29, "leumorphin") to the tridecapeptide dynorphin B (Dyn B, "rimorphin"). This represents a "single-arginine cleavage" between threonine-13 and arginine-14 of the substrate. The dynorphin converting activity displayed typical Michaelis-Menten kinetics with an apparent Km for the substrate of 0.58 microM. Surprisingly, a synthetic peptide, Dyn B-29-(9-22), which contains the cleavage site, did not inhibit the activity. Dyn A inhibited the activity competitively with an apparent Ki of 3.7 microM. The converting activity was also inhibited by Dyn A-(6-17) but not by Dyn A-(8-17), suggesting a role of Arg6-Arg7 in the inhibition of converting activity. Bovine adrenal medulla Peptide E inhibited the converting activity substantially whereas metorphamide did not, suggesting the importance of COOH-terminal residues in recognition. Beta Endorphin was an effective inhibitor of converting activity, and [alpha-N acetyl]beta-endorphin was not, indicating a crucial role of the free NH2-terminus in recognition by the enzyme. ACTH inhibited the activity competitively with an apparent Ki of 39 nM. The converting activity was also inhibited substantially by ACTH-(1-13) but not by alpha-MSH, again indicating a requirement of the free NH2 terminus for recognition. The above results suggest that the converting enzyme recognizes peptides of the three known opioid gene families. PMID- 3012493 TI - [Computerized tomography and selected problems of visualization of the genitals]. PMID- 3012494 TI - [Secretory function of the heart atrium]. PMID- 3012495 TI - Solitary osseous myeloma with polyneuropathy. Case report and review of the literature. AB - Solitary osseous myeloma is an uncommon malignancy of bone which is distinguished from multiple myeloma by being localized and without associated monoclonal gammopathy or Bence-Jones proteinuria. This disease may present with a progressive symmetrical sensorimotor neuropathy. A recent case of localized myeloma with polyneuropathy is presented along with a review of the literature pertinent to orthopedic surgeons. PMID- 3012496 TI - Fatal adenovirus pneumonia in two newborn infants, one case caused by adenovirus type 30. AB - Adenovirus rarely causes pneumonia in the newborn infant. We added 2 cases of fatal adenovirus neonatal pneumonia to the 3 cases previously reported. One of our cases was caused by adenovirus type 30, which is not previously known to be a pathogen. While the pneumonia could have been acquired in the nursery, the presence of chorioamnionitis and mixed infection with group B beta-hemolytic streptococcus suggests that an ascending infection from the birth canal might be another mode of transmission for neonatal adenovirus pneumonia. PMID- 3012497 TI - Microbial mucocutaneous infections in acute adult leukemia. Results of an 18-year inpatient study. PMID- 3012499 TI - Lactic acidosis and small cell carcinoma of the lung. AB - Two patients with small cell carcinoma of the lung who presented with lactic acidosis are described. Hepatocellular failure due to extensive metastases may be the cause of acute lactic acidosis. PMID- 3012498 TI - Nutritional therapy in diabetes. Rationale and recommendations. AB - The cornerstone of diabetes management is an appropriate diet. Various diets, including one low in carbohydrate, have been recommended in the past. The American Diabetes Association currently recommends a diet high in complex carbohydrate and fiber. The diet plan should be individualized. Caloric content is based on the patient's body build, weight, energy needs, and activity and may be adjusted according to individual eating habits, type of insulin regimen, and metabolic derangements. The proportion of caloric sources (fat, carbohydrate, protein) may be altered depending on the presence of secondary disorders. Several small daily meals, including a bedtime snack, are advised. Diabetic diets are often cost-effective, contrary to common belief. Patients should be encouraged to exercise to achieve optimum diabetic control. Active patient participation in the management program is an essential component of therapy. PMID- 3012500 TI - [Neuron-specific enolase--a selective marker of small cell bronchial cancer]. PMID- 3012501 TI - [Lung disease in acquired immunologic deficiency syndrome (AIDS)]. PMID- 3012502 TI - [Cardiac metastases from primary bronchial carcinoma. A retrospective autopsy study]. PMID- 3012503 TI - [Immunohistochemical detection of nuclear estrogen receptors with monoclonal antibodies in different types of breast cancer]. PMID- 3012504 TI - [Intermediate filaments as a criterion in the diagnosis of skin tumors]. PMID- 3012505 TI - [Infectious endocarditis surgically treated during the acute phase. 26 cases]. AB - Twenty-six patients with infective endocarditis were operated upon during the active phase. The endocarditis was native in 24 cases and developed on cardiac valve prosthesis in 2 cases. Depending on the valve involved, the patients were divided into 3 groups: Ao (aortic valve, n = 13), M (mitral valve, n = 10) and T (tricuspid valve, n = 3). The overall mortality rate was 26% (group Ao 20%, group M 20%); death was due, in most cases, to haemodynamic failure. The duration of pre-operative antibiotic therapy, the functional stage of the disease and the cardiothoracic ratio had no influence on post-operative prognosis. In contrast, the presence of vegetations (notably on the aortic valve) at echocardiography and the pumping and aortic clamping times played a role in operative mortality. Twelve patients were followed up for a mean period of 23.9 months. They are all in stage I or II with significant decrease in cardiothoracic index. In Africa, where bacteriological facilities are often inadequate and cardiac valve diseases are diagnosed at a late stage, infective endocarditis is active in many cases. Under these conditions, early surgery is justified when heart failure is present and the infection is not clinically controlled. PMID- 3012506 TI - [Absence of anti-LAV antibodies in connective tissue diseases]. PMID- 3012507 TI - [Polychemotherapy of undifferentiated small cell bronchial cancer. Long-term results]. AB - Long-term results of 2 studies combining etoposide and adriamycin with cisplatin (CAV) or cyclophosphamide (AVE) in small cell lung cancer indicate a 2-year survival rate of 22% and 18% respectively. The complete response rate 2 years after the onset of chemotherapy was 8% (8/98) : 11% (4/36) with CAV and 5% (4/62) with AVE. Among these 8 patients, 2 had a late relapse and one died of an unrelated cause. The actual long-term survival rate was 6% with CAV and 5% with AVE. PMID- 3012508 TI - [Role of the polyol pathway in the occurrence of degenerative complications of diabetes]. AB - The activity of the polyol pathway--a non insulin-dependent metabolic pathway of glucose--is increased in diabetic patients. Polyol accumulation is involved, by a myoinositol-dependent mechanism, in the pathogenesis of some degenerative complications of diabetes. Thus, sorbitol accumulation in the eye lens and in nerves seems to be an important factor in the development of cataract and in the slowing down of nerve conduction. Recent studies suggest that the polyol pathway may play a role in early structural abnormalities of retinal and renal microangiopathy. Synthetic aldose reductase inhibitors could be used for a physiopathogenic treatment of these complications, but the first trials in diabetic neuropathy proved disappointing. Further studies, prolonged and well controlled, are necessary to pronounce on the future of this new category of drugs. PMID- 3012509 TI - [Intrathoracic chemodectoma. 2 new cases]. PMID- 3012510 TI - [Chin neuropathy in vasculitis]. PMID- 3012511 TI - [Hemolytic anemia disclosed by parvovirus infection]. PMID- 3012512 TI - [Effect of treatment by selective transsphenoidal adenomectomy in Itsenko-Cushing disease]. PMID- 3012513 TI - [Hormonally-active tumors of the pancreas (lecture)]. PMID- 3012514 TI - [Participation of catecholamines in the regulation of an induced wave of gonadotropins in ovariectomized rats]. AB - The influence of various pharmacological agents (agonists or antagonists of catecholamine action) on the LH-RH content in certain discrete areas of the hypothalamus (in the preoptic area, arcuate nuclei and median eminence) and the LH and FSH levels were studied using a model of an induced wave of gonadotropins in ovariectomized rats receiving sex steroids. The administration of alpha adrenoreceptor antagonists blocked a rise of the level of gonadotropins in the blood. The wave was restored when mesaton (alpha-adrenoreceptor stimulator) was administered in the presence of blockers. At the same time activation of the dopaminergic system with apomorphine was ineffective. Changes in LH-RH concentration at the hypothalamus level were recorded in the preoptic area and median eminence in the gonadotropin wave blockade and in wave restoration using various pharmacological drugs which may reflect changes in the rate of metabolic processes and transport of this neuropeptide. It was concluded that the role of catecholamines was not restricted only to the activation of LH-RH release from the terminals at the level of the median eminence but was also necessary at the level of LH-RH producing cells of the hypothalamus. PMID- 3012516 TI - [Phylogenesis and ontogenesis of receptors]. PMID- 3012515 TI - [Experimental and clinical basis for using the preparation nacom in the treatment of Itsenko-Cushing disease]. AB - The paper is concerned with the results of an experimental study of the nacom (NC) effect on the activity of the hypothalamohypophysioadrenal system and the results of its application to 29 patients with Icenko-Cushing's disease with curative purposes. In rats the concentration of corticotropin and aldosterone was determined with a radioimmunoassay, that of corticosterone with a competitive protein binding method. The dopamine content in the hypothalamus and cerebral cortex was studied by fluorimetry. To define the hypothalamohyphysioadrenal system in the patients a study was made of blood corticotropin, cortisol and aldosterone basal levels as well as response of the system to insulin hypoglycemia induced stress. Dopaminergic NC was shown to be dose-related with the activity of the hypothalamohypophysioadrenal system. The NC effect was associated with an increase in the dopamine level in the hypothalamus and cerebral cortex. Clinical investigations proved NC efficacy for therapy of Icenko Cushing's disease permitting one to recommend it as a pathogenetic agent for therapy of this disease. PMID- 3012517 TI - [Biological activity of Bulgarian zeolite rocks]. AB - An evaluation of the biological activity of Bulgarian zeolite rocks was made by the methods in vivo and in vitro. The results pointed out to specific biological effect--cytotoxic and fibrogenic--most strongly expressed for clinoptilolite followed by mordenite and erionite. The clinoptilolite is characterized by strong cytotoxic effect in comparison with dust of calibrated quartz DQ-12 which confirms the lack of correlation between cytotoxicity and fibrogenicity for some types of dust. On the basis of the results a temporary hygienic norm for clinoptilolite dust--2 mg/m3 is proposed. PMID- 3012518 TI - [Prostaglandins and cyclic nucleotides in patients with surgical intervention in the lungs]. PMID- 3012519 TI - [Giant-cell pneumonia in a female bronchiectasis patient]. PMID- 3012520 TI - The A+T-rich genome of Herpesvirus saimiri contains a highly conserved gene for thymidylate synthase. AB - Herpesvirus saimiri (HVS) is the prototype member of a distinctive subset of lymphotropic herpesviruses (the gamma 2 subgroup) with A+T-rich coding sequences. In this paper, we show that cells productively infected with HVS contain high concentrations of a virus-specified thymidylate synthase (5,10 methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45); we identify the active polypeptide and present the sequence of the virus gene. The predicted amino acid sequence of the 294-residue subunit of the virus enzyme is 70% homologous with the sequence of the human enzyme and about 50% homologous with prokaryotic thymidylate synthases, illustrating the remarkable structural constraints imposed by the thymidylate synthase function. However, the presence of the enzyme is not a conserved property of herpesviruses. We find no evidence for a virus-encoded thymidylate synthase activity (or a homology to a thymidylate synthase sequence) in G+C-rich representatives of alpha 1 (e.g., herpes simplex viruses, 66-68% G+C), beta (i.e., human cytomegalovirus, 58-59% G+C), and gamma 1 (i.e., Epstein-Barr virus, 60% G+C) herpesvirus subgroups. The production of excess thymidylate by a virus thymidylate synthase in cells infected with an A+T rich herpesvirus would provide one plausible source of biased mutations by the virus-encoded replicative enzymes, which we have previously suggested as the likely general cause of differences in the mean nucleotide compositions of herpesvirus genomes. PMID- 3012521 TI - Transient replication of bovine papilloma virus type 1 plasmids: cis and trans requirements. AB - A transient assay has been used to study bovine papilloma virus type 1 (BPV-1) replication. We show that BPV-1 early replication occurs faster than cellular DNA synthesis. Initial replication events are dependent on a gene product(s), encoded by the BPV-1 E1 open reading frame. Mutational analysis of the viral upstream regulatory region shows the requirement of two domains in cis for replication. Domain one, located outside of the viral 69% transforming fragment, is an enhancer-like activity and can be replaced by other known viral enhancers. Domain two lies within sequences previously defined as plasmid maintenance sequence 1. The apparent requirement for a proximal enhancer function for replication may explain why certain BPV-1 constructions, when linked to bacterial plasmid sequences, can be maintained extrachromosomally while others cannot. PMID- 3012523 TI - Regulated expression of Sindbis and vesicular stomatitis virus glycoproteins in Saccharomyces cerevisiae. AB - cDNAs encoding either the structural proteins (capsid and glycoproteins E1 and E2) of Sindbis virus or the glycoprotein of vesicular stomatitis virus (VSV) were fused to the Saccharomyces cerevisiae galactokinase gene (GAL1) promoter and inserted into a yeast shuttle vector. After addition of galactose to yeast transformed with this vector, 2.5-3% of total yeast protein synthesis was detected as virus proteins by specific anti-virus protein antibodies. In cells containing the Sindbis virus structural genes, the virus capsid protein was effectively released from the nascent polypeptide and two endoglycosidase H sensitive glycoproteins were produced. One of these was identical in its gel mobility to E1 and the other appeared to be p62, a precursor to E2. A low level of E1 protein was detected on the cell's surface membranes. A single molecular weight species of glycosylated VSV glycoprotein was produced and half of the total protein could be detected at the surface membranes of yeast. Addition of long mannose chains and acylation of the virus proteins with fatty acids were not observed. Formation of virus proteins was also examined in yeast secretory mutants; one of these (sec53) failed to glycosylate the virus proteins. PMID- 3012522 TI - Expression of the v-mos gene alters a Mr 55,000 protein during acute infection by Moloney murine sarcoma virus. AB - Infection of the rat myoblast cell line, L6E9, with Moloney murine sarcoma virus (Mo-MuSV) clone 124, altered a cellular protein of Mr 55,000 (P55) within 2 days of infection. The alteration of P55 was observed as a reduction in its steady state level in cell extracts. The reduction of P55 correlated with the appearance of p37mos in infected cells. Except for P55 and one other protein, no change was detected in the total protein pattern of infected cells compared to uninfected cells, as judged by either immunoblots of one-dimensional NaDodSO4 gels or direct two-dimensional gel analysis. P55 levels were unchanged when L6E9 cells were infected with Moloney murine leukemia virus or several different transforming retroviruses. To determine the specificity of this v-mos-induced effect on P55, L6E9 cells were acutely infected with a temperature-sensitive variant (ts110) of Mo-MuSV. When these cells were shifted from 39 degrees C to 33 degrees C, which activates the gag-mos gene product, the P55 level dropped by greater than 50% within 2-3 hr. Conversely, with a shift in temperature from 33 degrees C to 39 degrees C, the cells' P55 level returned to normal within 5 hr, starting at 30 min after shift. These results clearly show that v-mos expression in acutely infected L6E9 cells alters the cellular protein, P55. PMID- 3012525 TI - Isolation and characterization of the gene encoding Drosophila DNA topoisomerase II. AB - We have isolated the gene coding for the Drosophila type II DNA topoisomerase by immunochemically screening a Drosophila cDNA library constructed with a phage lambda expression vector, lambda gt11. The identity of the cloned gene is confirmed by the analysis of an antigenic fusion protein produced in Escherichia coli and by the in vitro translation of its RNA. The gene is 5.1 kilobases in length, the expected size for a gene encoding topoisomerase II (Mr 170,000), and it is divided into five exons. By in situ hybridization to the polytene chromosomes from salivary glands, we have mapped it to chromosome 2L at 37D. PMID- 3012524 TI - Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene. AB - A 5400-base-pair segment of the vaccinia virus genome was sequenced and an open reading frame of 938 codons was found precisely where the DNA polymerase had been mapped by transfer of a phosphonoacetate-resistance marker. A single nucleotide substitution changing glycine at position 347 to aspartic acid accounts for the drug resistance of the mutant vaccinia virus. The 5' end of the DNA polymerase mRNA was located 80 base pairs before the methionine codon initiating the open reading frame. Correspondence between the predicted Mr 108,577 polypeptide and the 110,000 purified enzyme indicates that little or no proteolytic processing occurs. Extensive homology, extending over 435 amino acids, was found upon comparing the DNA polymerase of vaccinia virus and DNA polymerase of Epstein-Barr virus. A highly conserved sequence of 14 amino acids in the carboxyl-terminal regions of the above DNA polymerases is also present at a similar location in adenovirus DNA polymerase. This structure, which is predicted to form a turn flanked by beta-pleated sheets, may form part of an essential binding or catalytic site that accounts for its presence in DNA polymerases of poxviruses, herpesviruses, and adenoviruses. PMID- 3012526 TI - Membrane tolerance to ethanol is rapidly lost after withdrawal: a model for studies of membrane adaptation. AB - The structural properties of liver microsomes and erythrocytes obtained from rats that had been chronically administered ethanol were examined by electron spin resonance (ESR) following ethanol withdrawal for 1-10 days. Membranes obtained from control animals exhibited considerable molecular disordering upon the addition of ethanol in vitro (50-100 mM). Conversely, microsomal and erythrocyte membranes from alcoholic animals were resistant to this disordering by ethanol (membrane tolerance). These membrane properties were also apparent in lipid bilayers comprised of either total lipids or phospholipids isolated from the control and alcoholic animals. While several weeks of ethanol administration were required for both erythrocytes and microsomes to develop membrane tolerance, erythrocytes from alcoholic animals were disordered by ethanol in vitro after the animals had been withdrawn from ethanol for only 1 day. The same rapid loss of tolerance was observed in microsomes after 2 days of withdrawal. The same time course for the loss of tolerance was observed in lipid bilayers prepared from the total lipid and phospholipid extracts. No significant differences in the cholesterol/phospholipid ratio were observed between the microsomal or erythrocyte membranes isolated before and after withdrawal. Thus, alterations in the microsomal and erythrocyte phospholipids, and not cholesterol content, were responsible for conveying membrane tolerance. Membrane structural properties can be rapidly adjusted in a mammalian system in response to the withdrawal of the external membrane perturbant ethanol. The withdrawal model, which begins with established membrane tolerance and leads to rapid and complete loss of tolerance, provides a model to analyze the compositional changes responsible for this tolerance to disordering by ethanol. PMID- 3012528 TI - Redox pathways in electron-transfer proteins: correlations between reactivities, solvent exposure, and unpaired-spin-density distributions. AB - The relative reactivities toward reduction by free flavin semiquinones of cytochromes (c-type cytochromes, cytochrome b5, c'-type cytochromes) iron-sulfur proteins (high-redox-potential ferredoxins, rubredoxins, low-redox-potential ferredoxins), and blue copper proteins (plastocyanin, azurins) are shown to correlate with calculations of the solvent exposure of the various prosthetic groups. In the case of the c-type cytochromes, one of the major centers of exposure is the sulfur atom of the thioether bridge that covalently links heme ring C to the protein. Charge-iterative extended Huckel calculations on a heme c model indicate that both porphyrin pi and Fe(III)d pi orbitals can delocalize onto the bridging sulfur atom. Unpaired spin densities are comparable to those obtained for individual aromatic porphyrin ring carbon atoms. Thus, the exposed sulfur of ring C may act to facilitate electron transfer. PMID- 3012527 TI - Exon-Alu recombination deletes 5 kilobases from the low density lipoprotein receptor gene, producing a null phenotype in familial hypercholesterolemia. AB - Among patients with familial hypercholesterolemia, half of the mutant alleles at the low density lipoprotein (LDL) receptor locus produce no immunologically detectable protein. To determine the molecular basis for one such null allele, we have cloned an abnormally short restriction fragment from the genomic DNA of one patient. The DNA sequence revealed a 5-kilobase deletion that joins a coding sequence in exon 13 to an Alu repetitive element in intron 15. The deletion joint is flanked by two inverted repeats that could potentially form a double stem-loop structure that might have predisposed to this deletion. A similar double stem loop structure can be drawn for a previously described deletion in the LDL receptor gene and for a deletion in the beta-globin gene cluster. We speculate that such double stem-loop structures might contribute to the formation of large deletions in the human genome. PMID- 3012529 TI - Reduced hormone-stimulated adenylate cyclase activity in NIH-3T3 cells expressing the EJ human bladder ras oncogene. AB - Recent studies have shown that the 21-kilodalton protein (p21) Ha-ras gene product shares sequence homology with and may exhibit biochemical properties similar to the mammalian guanine nucleotide-binding proteins. These data suggested that one of the biochemical functions of p21 in the vertebrate cell may be to regulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. We determined both in intact NIH-3T3 murine cells and in membranes isolated from these cells that the hormone-stimulated adenylate cyclase activity of cells expressing the EJ human bladder carcinoma oncogene (EJ-ras) is significantly reduced compared with control cells. Thus, the levels of cAMP measured in the EJ-ras-transformed cells by radioimmunoassay are reduced 78% and 93% after prostaglandin and isoproterenol stimulation, respectively, compared with the levels in control cells. Treatment of the EJ-ras-transformed cells with pertussis toxin or cholera toxin did not correct the alterations in adenylate cyclase activity. Cells expressing the normal human Ha-ras gene displayed intermediate levels of adenylate cyclase hormone sensitivity; these levels of adenylate cyclase activity were greater than those in the EJ-ras-transformed cells but lower than in control cells. Hormone-stimulated adenylate cyclase activities in cells transfected with Rous sarcoma virus DNA were similar to those in control cells. These data support the hypothesis that both the normal and mutated Ha-ras p21s are related to guanine nucleotide-binding proteins. PMID- 3012530 TI - Substrate free radicals are intermediates in ligninase catalysis. AB - The H2O2-requiring ligninase of the basidiomycete Phanerochaete chrysosporium oxidatively cleaves both lignin and lignin model compounds between C alpha and C beta (C-1 and C-2) of their aliphatic side chains. Previous work has demonstrated a reaction mechanism by which ligninase oxidizes aromatic substrates to their cation radicals, which then undergo side chain cleavage to yield carbon-centered free radicals. These carbon-centered radicals add O2 to give substrate peroxyl radicals that react further to yield the hydroxylated and carbonylated end products usually seen in experiments with ligninase. To investigate this radical mechanism, we have now designed three dimeric lignin models: 1-(3,4 dimethoxyphenyl)-2-phenylethanol (I), 1-(3,4-dimethoxyphenyl)-2-phenylpropanol (II), and 1-(3,4-dimethoxyphenyl)-2-methyl-2-phenylpropanol (III). The following results were obtained when these models were oxidized by ligninase: methyl groups at C beta of the substrate favored C alpha-C beta cleavage versus C alpha oxidation to the ketone. GC/MS and HPLC analysis showed that II gave a radical coupling dimer, 2,3-diphenylbutane, as a major (26% yield) reaction product under anaerobic conditions. The anaerobic oxidation of III yielded 2,3-dimethyl-2,3 diphenylbutane. Spin-trapping experiments with nitrosobenzene showed that model II oxidation produced alpha-methylbenzyl radicals, whereas model III oxidation gave alpha, alpha-dimethylbenzyl radicals. TLC and iodometric determinations showed that III gave cumene hydroperoxide as a major (21% yield) reaction product in air. These findings demonstrate that carbon-centered and peroxyl radicals at C beta are major products of C alpha-C beta cleavage by ligninase. PMID- 3012531 TI - Chromatin binding of epidermal growth factor, nerve growth factor, and platelet derived growth factor in cells bearing the appropriate surface receptors. AB - We analyzed the uptake and intracellular distribution of 125I-labeled epidermal growth factor, nerve growth factor, and platelet-derived growth factor in different cell lines that express or do not express the respective surface receptors for these factors. After 1 hr of incubation, all three growth factors were detected in the cytoplasmic fraction and in the nucleus, tightly bound to chromatin. The amount of chromatin-bound growth factors continued to increase during the incubation, and analysis at 48 hr revealed each chromatin-bound labeled growth factor in a nondegraded form. After limited digestion of chromatin with DNase II (10-20% digested sequences), specific release of all three growth factors was detected only after 1 hr of incubation but not after 24 and 48 hr, suggesting that the DNA regions involved in growth factor binding became nuclease resistant. Binding of labeled epidermal growth factor and nerve growth factor to isolated chromatin was inhibited by monoclonal antibodies specific for the respective growth factor receptor. The data suggest that chromatin binding may represent an important step in the pathway of growth factor action. PMID- 3012532 TI - The active immunoglobulin kappa chain gene is packaged by non-ubiquitin conjugated nucleosomes. AB - To elucidate the molecular features of active chromatin, we have mapped, by two dimensional electrophoresis, the protein composition of nucleosomes that package the immunoglobulin kappa chain gene of mouse plasmacytoma cells. Nucleoprotein particles that possess the active kappa chain gene comigrate with bulk mononucleosomes that contain high mobility group proteins HMG-14 or -17 but lack histone H1. High electrophoretic resolution of the underlying core particles, after removal of ubiquitin by isopeptidase treatment, reveals that these nucleosomes are nonubiquitinated, even though they coincidently migrate with bulk ubiquitinated particles. This distinctive electrophoretic behavior may be correlated with the presence of histone H2A.X. Nucleosomes exhibiting these unusual properties appear to span at least 10 kilobases, in both transcribed and nontranscribed regions, suggesting that mechanisms independent of transcription exist to initiate, maintain, and propagate a common chromatin phenotype over long distances along the kappa chain locus. PMID- 3012533 TI - Human fibroblast collagenase: glycosylation and tissue-specific levels of enzyme synthesis. AB - Human skin fibroblasts secrete collagenase as two proenzyme forms (57 and 52 kDa). The minor (57-kDa) proenzyme form is the result of a partial posttranslational modification of the major (52-kDa) proenzyme through the addition of N-linked complex oligosaccharides. Human endothelial cells as well as fibroblasts from human colon, cornea, gingiva, and lung also secrete collagenase in two forms indistinguishable from those of the skin fibroblast enzyme. In vitro tissue culture studies have shown that the level of constitutive synthesis of this fibroblast-type interstitial collagenase is tissue specific, varies widely, and correlates with the steady-state level of a single collagenase-specific mRNA of 2.5 kilobases. The tumor promoter, phorbol 12-myristate 13-acetate, apparently blocks the control of collagenase synthesis resulting in a similarly high level of collagenase expression (approximately equal to 3-7 micrograms of collagenase per 10(6) cells per 24 hr) in all examined cells. The constitutive level of synthesis of a 28-kDa collagenase inhibitor does not correlate with that of the enzyme. Phorbol 12-myristate 13-acetate stimulates the production of this inhibitor that in turn modulates the activity of collagenase in the conditioned media. As a result, the apparent activity of the enzyme present in the medium does not accurately reflect the rate of its synthesis and secretion. PMID- 3012534 TI - Platelet-derived growth factor modulates epidermal growth factor receptors by a mechanism distinct from that of phorbol esters. AB - Platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA) decrease high affinity binding of 125I-labeled epidermal growth factor (EGF) and potentiate mitogenesis in BALB/c 3T3 cells, and both have been shown to induce the phosphorylation of the EGF receptor at threonine residues. These similarities suggest that the actions of PDGF on EGF binding may be mediated by protein kinase C, the cellular effector of PMA. We show that in density-arrested BALB/c 3T3 cells PDGF and PMA induce a rapid, transient, cycloheximide-independent loss of EGF binding activity. As has been previously shown for PDGF, the ability of PMA to reduce EGF binding was enhanced by cholera toxin, a potent activator of adenylate cyclase. In contrast to PMA, however, PDGF induced a further reduction in EGF binding that was strictly dependent upon continued protein synthesis. Furthermore, PDGF effectively reduced EGF binding in cells refractory to PMA. Cells desensitized to PMA, presumably due to the loss of protein kinase C activity, also remained mitogenically responsive to PDGF. These data suggest that the mechanism by which PDGF modulates EGF binding differs from that of PMA and thus, at least in part, is independent of protein kinase C. PMID- 3012535 TI - Properties of the earliest clonogenic hemopoietic precursors to appear in the developing murine yolk sac. AB - We have found that a variety of clonogenic hemopoietic cells can be obtained in a viable state from mouse conceptuses as early as day 7 of gestation when tissues are disaggregated in a crude collagenase solution containing fetal bovine serum. Examination of the time course of colony formation, and the ultimate size and lineages represented in colonies produced in semisolid medium containing methylcellulose, together with analysis of individual erythroid colonies stained with rabbit antisera specific for adult (HbA) and embryonic (HbE) mouse hemoglobins, revealed the presence on days 7 and 8 of gestation (but not later) of erythropoietic progenitors that give rise to mature erythroid colonies containing up to 100 HbE-containing erythroblasts after 4-6 days of growth in culture. These progenitors are highly sensitive to the disaggregation conditions used. Clonogenic progenitors of exclusively HbA-positive erythroblasts can also be detected in the day-7 conceptus. Assays of progenitors from separately disaggregated yolk sacs and embryos from day-8 conceptuses yielded colonies only from yolk sac suspensions, and again these contained either HbE- and HbA-positive erythroblasts or only HbA-positive erythroblasts. These findings demonstrate the very early appearance in the yolk sac of a population of erythroid progenitors with a number of unique properties. Although most of these yield HbE-positive erythroblasts in vitro, some produce erythroblasts containing HbA only. Such a developmental pattern is consistent with the hypothesis that definitive erythropoiesis in the mammalian fetal liver is derived from stem cells that originate in the yolk sac blood islands. PMID- 3012536 TI - Insertion and/or deletion of many repeated DNA sequences in human and higher ape evolution. AB - The total numbers of copies of two repeat families, L1 (Kpn I) and Alu, have been measured in the DNA of four higher apes by an accurate titration method. The number of members of the Alu family repeats in the four genomes are as follows: human, 910,000; chimpanzee, 330,000; gorilla, 410,000; orangutan, 580,000. For the Kpn I family (3'-ward higher frequency region) the number of copies in these genomes are as follows: human, 107,000; chimpanzee, 51,000; gorilla, 64,000; orangutan, 84,000. Thermal stability measurements show that, although the families of repeats are moderately divergent in sequence, little net sequence change has occurred during the evolution of the higher apes. Most or all of the members of these families of repeats are interspersed throughout the genome. Therefore, a large number of events of insertion and/or deletion of these DNA sequences has occurred during higher primate evolution. PMID- 3012537 TI - Evidence for selection as a mechanism in the concerted evolution of Lycopersicon esculentum (tomato) genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. AB - The nuclear gene sequences encoding RBCS, the small subunit of ribulose-1,5 bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from several plants show extensive interspecific divergence but little intraspecific divergence, suggesting that these genes are evolving in concert within a genome. In this study, the nucleotide sequences of two tomato (Lycopersicon esculentum) RBCS genes and a cDNA clone containing the entire coding region of a third tomato RBCS gene were determined. The three genes, designated Rbcs-1, Rbcs-2A, and Rbcs-3A, each belong to a different one of the three RBCS loci in the tomato genome. The nucleotide sequence of Rbcs-1 differs from that of Rbcs-2A and Rbcs-3A by 13.9% and 13.1%, respectively. Rbcs-2A and Rbcs-3A differ from each other by 10.7%. A recently published RBCS gene sequence from tobacco (Nicotiana tabacum) [Mazur, B. J. & Chui, C.-F. (1985) Nucleic Acids Res. 13, 2373-2386] differs by 10.6% and 11.3% from Rbcs-2A and Rbcs-3A, respectively, and by 15.0% from Rbcs-1. Thus the tobacco gene seems to be phylogenetically as closely related to the tomato genes Rbcs-2A and Rbcs-3A as the latter two are to each other, and more closely related to them than Rbcs-1 is. However, the mature part of the polypeptide encoded by the tobacco RBCS gene differs by five and six amino acids from the corresponding region in the polypeptides encoded by Rbcs-2A and Rbcs-3A, respectively, while these two tomato RBCS polypeptides differ from each other in the mature part by a single amino acid. Rbcs-1, whose nucleotide sequence shows higher divergence from both the tobacco RBCS gene and Rbcs-2A and Rbcs-3A, encodes a polypeptide whose mature part differs by eight amino acids from the corresponding region in the tobacco polypeptide but only by three and four amino acids from the corresponding regions of Rbcs-2A- and Rbcs-3A-encoded polypeptides, respectively. Thus, it appears that in the tomato selection has maintained near uniformity of the coding information in the portion of the RBCS genes encoding the mature polypeptides. PMID- 3012538 TI - A phage P1 function that stimulates homologous recombination of the Escherichia coli chromosome. AB - Recombination between two different defective lacZ genes in the Escherichia coli chromosome (lac- X lac- recombination) was stimulated 2- to 8-fold by prophage P1, depending on the nature of the phage c1 repressor. The P1 BamHI restriction fragment B8 in a lambda-P1:B8 hybrid phage, stimulated lac- X lac- recombination 90-fold in the absence of P1 repressor. A gene necessary for recombination enhancement, designated ref, was localized to one end of B8. Ref expression from lambda-P1:B8 was repressed in trans by a P1 c+ prophage. Two P1 regulatory mutations, bof and lxc, derepressed prophage expression of ref and depressed a prophage function that complemented an E. coli mutant (ssb) deficient in the single-stranded DNA binding protein. Ref stimulation was dependent on preexisting E. coli recombination functions (RecA-RecBC and RecA-RecF). However, other (phage and plasmid) recombination processes involving these functions were not stimulated. ref::Tn5 phages plated and formed lysogens normally. Thus ref appears to be an integral, but not essential, phage gene that stimulates recombination of the host chromosome specifically. PMID- 3012539 TI - Construction of a map of the short arm of human chromosome 6. AB - Four DNA sequences that reveal restriction fragment length polymorphisms (RFLPs) on the short arm of human chromosome 6 have been identified. Two of these sequences were isolated from recombinant DNA libraries enriched for DNA from human chromosome 6, one was isolated as a subclone from a human DNA segment having homology to an HLA class I sequence, and one was isolated by virtue of its homology to part of the insulin gene. Genetic linkage was determined among these four polymorphic sequences and several genes known to be on chromosome 6: glyoxylase I (GLO1), HLA-DR alpha, HLA-DQ alpha, and HLA-B. Additionally, two of the four RFLPs were regionally localized by using 53 deletion cell lines that had been typed for HLA-A, -B, and -DR, and for glyoxylase I. A genetic map of the short arm of chromosome 6 has been constructed on the basis of linkage studies with the eight markers. The map spans a minimum of 60 recombinational units and will be useful in the study of HLA-associated diseases. PMID- 3012540 TI - Sequential expression of protooncogenes during lectin-stimulated mitogenesis of normal human lymphocytes. AB - The proliferation of non-neoplastic T lymphocytes is regulated, in part, by the coordinated expression of genes encoding T-cell growth factor (interleukin 2, IL2), IL2 receptors (IL2R), and transferrin receptors (TFR). In addition to growth factors and their receptors, protooncogenes may regulate lymphocyte proliferation. We used cloned cDNAs homologous to 21 different protooncogenes to screen for their expression at the mRNA level in human peripheral blood mononuclear cells (PBMC) stimulated with the mitogenic lectin phytohemagglutinin (PHA), and we compared the time course of accumulation of mRNAs for these protooncogenes to that of mRNAs for the IL2, IL2R, TFR, and histone H3 genes. mRNAs for c-abl, c-ets, c-yes, and N-ras were present in unstimulated PBMC. After stimulation of PBMC by PHA, we detected marked increases within 10 min in the levels of mRNA for c-fos and c-myc; within 6 hr for IL2 and IL2R mRNAs; within 14 hr for c-myb, p53, N-ras, and TFR mRNAs; and within 24-36 hr for H3 mRNA. Expression of c-abl, c-ets, and c-yes increased gradually following stimulation with PHA. None of the other protooncogenes tested was expressed in PBMC. Addition of the protein synthesis inhibitor cycloheximide, before the addition of PHA to cultures, abolished the PHA-induced accumulation of mRNAs for c-myb, N-ras, and TFR, but not of mRNAs for c-fos, c-myc, IL2, and IL2R. These data indicate that c fos, c-myc, IL2, and IL2R belong to a group of genes expressed early, whereas c myb, N-ras, and TFR belong to a group of genes expressed later in PHA-activated PBMC, and that the products of the c-fos and c-myc protooncogenes are not required for expression of IL2 or IL2R genes. Addition of purified IL2 augmented the expression of the later-expressed genes c-myb, p53, N-ras, and TFR in PHA stimulated cultures of PBMC, as well as of the early genes c-myc and IL2R, but not of c-fos and IL2, thus suggesting that PHA and IL2 stimulate the expression of overlapping, but nonidentical, sets of genes in PBMC. PMID- 3012541 TI - Monoclonal antibodies to a particulate superoxide-forming system stimulate a respiratory burst in intact guinea pig neutrophils. AB - Monoclonal rat antibodies were produced against a subcellular preparation of phorbol 12-myristate 13-acetate (PMA)-stimulated guinea pig neutrophils that retains NADPH-oxidase activity. Two antibodies, 1A10.4 and IG4, were isolated that bind to a surface antigen restricted to guinea pig neutrophils from bone marrow and peritoneal exudate and to macrophages and that trigger a respiratory burst in neutrophils in the presence of cytochalasin B. Intact antibody 1A10.4, subclass IgG2c, can trigger superoxide anion release directly; F(ab')2 fragments of 1A10.4 and intact IG4 require further cross-linking by F(ab')2 fragments of anti-rat immunoglobulin antibody. Both antibodies recognize the same antigen, a proteolipid of apparent molecular mass 10 kDa. Immunoprecipitation of solubilized oxidase activity with 1A10.4 brings down this activity as part of a macromolecular complex. Surface expression of the antigen is increased on treatment of cells with both PMA and cytochalasin B. 1A10.4 also triggers release of the granule enzyme beta-glucuronidase. Triggering of a respiratory burst by the antibodies appears distinct from the PMA and fMet-Leu-Phe signalling systems. These studies indicate that the antigen defined by antibodies 1A10.4 and IG4 becomes associated with the superoxide anion-generating system of neutrophils but may play a more general role in signal transduction in phagocytic cells. PMID- 3012542 TI - Herpes simplex virus immediate early infected-cell polypeptide 4 binds to DNA and promotes transcription. AB - In herpes simplex virus (HSV)-infected cells, there is a sequential expression of viral genes. In vivo experiments have implicated the Mr 175,000 immediate early protein ICP4 (infected-cell polypeptide 4) in the regulation of viral RNA synthesis, but the mechanism whereby ICP4 regulates transcription of viral genes is at present unknown. In this report we describe experiments with an in vitro transcription system and a purified preparation of ICP4 (estimated 5% of total protein). Using DNA from the HSV glycoprotein D gene (gD) as the template, we have observed that specific binding occurs between ICP4 and DNA sequences adjacent to the gD gene promoter and ICP4 stimulates initiation of transcription from the gD gene. The degree of stimulation depends on the amount of ICP4 present in the incubation. The kinetics of RNA synthesis demonstrate that the protein acts at the initiation step of transcription. These results identify ICP4 as a viral transcription factor whose presence on DNA facilitates the formation of transcription complexes. PMID- 3012544 TI - Nucleoside kinases in T and B lymphoblasts distinguished by autoradiography. AB - Nucleoside kinases catalyze the initial step leading to the accumulation of deoxypurine nucleotides that occurs in patients with inherited deficiencies of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1). This accumulation is thought to interfere with DNA synthesis in lymphocytes and, thus, to cause the immune defects associated with these enzymopathies. However, there is controversy about the identity of the nucleoside kinases that are responsible for intracellular phosphorylation of deoxyadenosine in adenosine deaminase deficiency and deoxyguanosine in purine nucleoside phosphorylase deficiency. To distinguish the nucleoside kinases present in T and B lymphoblastoid cells, we have coupled discontinuous PAGE with autoradiography. This procedure showed that deoxycytidine kinase (NTP:deoxycytidine 5' phototransferase, EC 2.7.1.74), deoxyadenosine kinase (ATP:deoxyadenosine 5' phosphotransferase, EC 2.7.1.76), and adenosine kinase (ATP:adenosine 5' phosphotransferase, EC 2.7.1.20) are all present in both T and B lymphoblasts. While adenosine kinase is expressed at nearly equal levels in B and T cells, the deoxynucleoside kinases are expressed at much lower levels in B cells than in T cells. The autoradiographic data agreed with assays of the nucleoside kinase activities. Molecular weights were determined by using 5-10% polyacrylamide gels. Mr values were 29,000 for adenosine kinase, 41,000 for deoxyadenosine kinase, and 53,000 for deoxycytidine kinase and its isozyme. The reduced expression of deoxycytidine and deoxyadenosine kinases in B lymphoblasts may account for the lower accumulation of deoxypurine nucleotides in B cells as compared with T cells. PMID- 3012543 TI - Gonadotropin-releasing hormone differentially regulates expression of the genes for luteinizing hormone alpha and beta subunits in male rats. AB - Gonadotropin-releasing hormone (GnRH) and gonadal steroids regulate synthesis and release of luteinizing hormone (LH). GnRH is secreted intermittently by the hypothalamus, producing pulsatile LH release, and a pulsatile GnRH stimulus is required to maintain LH secretion. We report the regulatory effects of GnRH pulse injections on pituitary concentrations of LH alpha and beta subunit mRNAs in a castrated/testosterone-replaced male rat model. Replacement with physiologic amounts of testosterone decreased concentrations of both LH subunit mRNAs. GnRH pulse injections (10-250 ng per pulse given every 30 min for 48 hr) increased both mRNA concentrations, but the dose response patterns were markedly different. alpha subunit mRNA was increased by all GnRH doses but not the levels seen after castration alone. In contrast, LH beta subunit mRNA concentrations showed a marked dependence on GnRH dose. Maximal responses, to values similar to those in castrates, occurred after 25-ng GnRH pulses, and larger doses produced a smaller increase in LH beta subunit mRNA. Both the acute LH secretory response to GnRH and the number of GnRH receptors followed a pattern similar to the LH beta subunit mRNA concentration and were maximal after the 25-ng GnRH dose. These results show that GnRH can differentially regulate LH subunit mRNAs and suggest that concentrations of LH beta subunit mRNA may be a limiting factor in GnRH stimulated LH release. PMID- 3012546 TI - Variable expression of the translocated c-abl oncogene in Philadelphia-chromosome positive B-lymphoid cell lines from chronic myelogenous leukemia patients. AB - The consistent cytogenetic translocation of chronic myelogenous leukemia (the Philadelphia chromosome, Ph1) has been observed in cells of multiple hematopoietic lineages. This translocation creates a chimeric gene composed of breakpoint-cluster-region (bcr) sequences from chromosome 22 fused to a portion of the abl oncogene on chromosome 9. The resulting gene product (P210c-abl) resembles the transforming protein of the Abelson murine leukemia virus in its structure and tyrosine kinase activity. P210c-abl is expressed in Ph1-positive cell lines of myeloid lineage and in clinical specimens with myeloid predominance. We show here that Epstein-Barr virus-transformed B-lymphocyte lines that retain Ph1 can express P210c-abl. The level of expression in these B-cell lines is generally lower and more variable than that observed for myeloid lines. Protein expression is not related to amplification of the abl gene but to variation in the level of bcr-abl mRNA produced from a single Ph1 template. PMID- 3012547 TI - Mapping second messenger systems in the brain: differential localizations of adenylate cyclase and protein kinase C. AB - [3H]Forskolin and [3H]phorbol 12,13-dibutyrate have been used to map the adenylate cyclase and phosphatidylinositol systems respectively in brain slices by light-microscopic autoradiography. [3H]Forskolin binding to brain sections is displaced potently by forskolin (KD approximately equal to 15 nM) and is enhanced by fluoride and GTP analogs, agents which activate the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Highest [3H]forskolin binding occurs in the corpus striatum, substantia nigra, hippocampus, and molecular layer of the cerebellum. Lesion studies demonstrate that binding sites in the substantia nigra are associated with striatal afferents, while hippocampal sites are localized to granule cell dendrites and mossy fiber terminals, and the intense binding in the cerebellar molecular layer is largely associated with granule cell axons and terminals. Protein kinase C mediates the activity of hormones and neurotransmitters, which act through the phosphatidylinositol cycle, and is labeled with high affinity by [3H]phorbol 12,13-dibutyrate. At many synapses, maps of adenylate cyclase and protein kinase C reveal reciprocal distributions, which may have implications for second messenger regulation of synaptic transmission. PMID- 3012545 TI - Increased expression of p53 protein in human leukemia cells. AB - We examined synthesis of the cellular phosphoprotein p53 in fresh bone marrow or peripheral blood cells from normal donors and from patients with leukemia, preleukemia, or other hematopoietic disorders. Lysates of cells labeled with [35S]methionine were immunoprecipitated with monoclonal antibodies to p53, and the immunoprecipitates were analyzed by NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. Bone marrow or peripheral blood cells from 8 of 33 patients with hematopoietic disorders showed increased p53, seven of the eight occurring in cells of patients with preleukemia or acute myelogenous leukemia. Increased p53 synthesis was not associated with p53 gene amplification, as shown by Southern blot analysis. Synthesis of p53 was not increased in any of nine normal human bone marrow samples or eight normal human peripheral blood granulocyte, macrophage, and lymphocyte samples. The hematopoietic cells of patients in remission or with chronic forms of leukemia did not generally synthesize elevated levels of p53. In addition, we found negligible p53 mRNA and protein expression in a variety of human myeloid leukemia lines blocked at different stages of differentiation. Southern blot analysis showed that, except for the HL-60 cells, the p53 gene of the myeloid cell lines was intact. In view of recent evidence implicating p53 in transformation of cultured cells, our results using fresh leukemia cells suggest that p53 may contribute to the phenotype of certain leukemias in vivo. PMID- 3012548 TI - Blood--brain barrier sodium/potassium pump: modulation by central noradrenergic innervation. AB - The active transport of Na+ and K+ across the blood--brain barrier by the membrane-bound enzyme Na+/K+-activated ATPase of brain microvessel endothelial cells has a major role in the maintenance of brain water and electrolyte homeostasis. To test whether the putative noradrenergic innervation of cerebral microvessels from the nucleus locus ceruleus contributes to the regulation of Na+/K+-ATPase activity of the blood--brain barrier, specific [3H]ouabain-binding studies were performed on cerebral microvessels and crude cortical membranes obtained from Wistar rats with unilateral 6-hydroxydopamine lesion of the nucleus locus ceruleus. Such lesion depleted norepinephrine by about 90% in the ipsilateral cerebral cortex without affecting the contralateral cortex. [3H]Ouabain binding to membranes of cerebral cortex and the cerebral microvessels was specific and saturable. The maximal ouabain-binding capacity in microvessels of the ipsilateral, norepinephrine-depleted, cerebral cortex was reduced by about 40%, without change in the affinity of binding. [3H]Ouabain binding to crude membrane fractions of the cerebral cortex was not significantly affected by locus ceruleus lesion. The results suggest that Na+/K+-ATPase activity of cerebral microvessels, and the consequent transport of Na+ and K+ across the blood--brain barrier, is modulated by noradrenergic innervation from the locus ceruleus. PMID- 3012549 TI - Mapping of brain areas expressing RNA homologous to two different acetylcholine receptor alpha-subunit cDNAs. AB - We have used an in situ RNA X RNA hybridization technique to determine, in the central nervous systems of the mouse and rat, the distribution of RNA homologous to cDNA clones encoding the alpha subunit of a putative neural nicotinic acetylcholine receptor and the alpha subunit of the muscle nicotinic acetylcholine receptor. Hybridization of the neural alpha-subunit probe was strongest in the medial habenula but was also detected consistently in the compact part of the substantia nigra and ventral tegmental area, in the neocortex, and in certain parts of the thalamus and hypothalamus. The in situ hybridization technique makes it possible to compile a map of brain regions containing cell bodies expressing RNA coding for a specific receptor type and subsequently to apply the techniques of molecular biology to study these brain receptors. PMID- 3012550 TI - [3H]xanthine amine congener of 1,3-dipropyl-8-phenylxanthine: an antagonist radioligand for adenosine receptors. AB - An amine-functionalized derivative of 1,3-dipropyl-8-phenylxanthine has been prepared in tritiated form as a xanthine amine congener ([3H]XAC) for use as an antagonist radioligand for adenosine receptors. [3H]XAC has higher receptor affinity, higher specific activity, lower nonspecific membrane binding, and more favorable hydrophilicity than 1,3-diethyl-8-[3H]phenylxanthine, the xanthine commonly used for adenosine receptor binding. In rat cerebral cortical membranes, [3H]XAC exhibits saturable, specific binding with a Kd of 1.23 nM and a Bmax of 580 fmol/mg of protein at 37 degrees C. N6-(R-Phenylisopropyl)adenosine is a more potent inhibitor of [3H]XAC binding than is 5'-N-ethylcarboxamidoadenosine, indicating that binding is to an A1-adenosine receptor. In the absence of GTP, the inhibition curves for adenosine agonists versus [3H]XAC binding are biphasic, indicating that [3H]XAC is binding to low- and high-affinity agonist states of the A1 receptor. In the presence of GTP, adenosine analogs exhibit monophasic, low-affinity inhibition of binding of [3H]XAC. Inhibition of [3H]XAC binding by theophylline or by various 8-phenylxanthines is monophasic, and the potencies are commensurate with the potencies of these xanthines as adenosine receptor antagonists. The receptor sites in calf brain membranes exhibit a higher affinity (Kd = 0.17 nM) for [3H]XAC, whereas sites in guinea pig exhibit a slightly lower affinity (Kd = 3.0 nM). Densities of [3H]XAC binding sites are similar in brain membranes from all species. PMID- 3012551 TI - Induction of nerve growth factor receptor in Schwann cells after axotomy. AB - We have discovered that axotomy of sciatic nerve induces Schwann cells distal to the lesion to express de novo, or at greatly increased levels, receptors for nerve growth factor (NGF). Surgical transection of sciatic nerve was performed on adult Sprague-Dawley rats, and, at various times after the operation, the following tissues were dissected for quantitation of NGF receptor: L4 and L5 dorsal root ganglia, sciatic nerve proximal to the transection, sciatic nerve distal to the transection, tibialis anterior muscle, and skin of the dorsum of the foot. The NGF receptor content of these samples was determined by labeling receptor molecules with radioiodinated NGF (125I-NGF) and then specifically immunoprecipitating the 125I-NGF-receptor complexes with 192-IgG, a monoclonal antibody directed against the rat NGF receptor. We observed a time-dependent increase in the amount of radioligand-labeled NGF receptor proteins found in the distal segment of transected sciatic nerve; by 7 days the density of receptor (crosslinked 125I-NGF molecules per mg of homogenate protein) had increased at least 50-fold. The number of receptor molecules in tibialis anterior muscle and dorsal foot skin, two structures denervated by the transection, also increased, with time courses parallel to that of distal sciatic nerve. There was little increase in the density of NGF receptors in the sciatic nerve proximal to the lesion and in the dorsal root ganglia. Immunohistochemical examination of the distal portion of transected sciatic nerve and of the muscle, with 192-IgG as the primary ligand, revealed prominent and exclusive staining of apparently all Schwann cells of the endoneurium, indicating that these peripheral neuroglial cells were expressing NGF receptors. These results show that axonal damage can induce the expression of NGF receptors in the population of sheath cells thought to promote neuronal regeneration. This dramatic increase in NGF receptors may be a mechanism to facilitate the regeneration of NGF-responsive neurons. PMID- 3012552 TI - Potentiation of muscarinic and alpha-adrenergic responses by an analogue of guanosine 5'-triphosphate. AB - Ca2+-dependent K+ and Cl- currents were recorded in isolated and dialyzed rat lacrimal gland cells by use of the tight-seal whole-cell recording technique. Under control conditions, application of acetylcholine (0.5-1.0 microM) resulted in the full activation of both types of current. When 50-200 microM guanosine 5' [gamma-thio]triphosphate (GTP[S], a nonhydrolyzable GTP analogue) was added to the intracellular solution, activation of both currents was seen with 1 nM acetylcholine, a dose 1/100th that needed under control conditions. Dialysis with solutions containing 200 microM GTP or cAMP had no, or only slight, potentiation effects. The effects of GTP[S] were obtained only when ATP was included in the intracellular solution. The potentiated responses to acetylcholine were blocked by increasing 10-fold the intracellular Ca2+-buffering capacity and were not dependent on external Ca2+. Thus, the potentiated responses appeared to result from a release of Ca2+ from internal stores. GTP[S] also greatly potentiated the Ca2+-dependent adrenergic (norepinephrine) response of this preparation. In addition, GTP[S] elicited in some cells transient responses without application of acetylcholine or norepinephrine. Finally, rapid and sustained responses were seen as soon as the cells were dialyzed with inositol trisphosphate (20 microM). These findings are discussed in terms of a possible role of a GTP-binding protein as a link between activation of muscarinic or adrenergic receptors and initiation of Ca2+ release by inositol trisphosphate. PMID- 3012553 TI - An atypical insulin receptor with high affinity for insulin-like growth factors copurified with placental insulin receptors. AB - Insulin receptors purified from human placenta by sequential affinity chromatography on wheat germ lectin-agarose and insulin coupled to 1,1' carbonyldiimidazole-activated agarose (CDI-agarose) retained full binding activity but bound a greater than predicted amount of 125I-labeled insulin-like growth factor I (IGF-I). IGF-I and multiplication-stimulating activity (MSA; the rat homologue of IGF-II) were equipotent in displacing either 125I-labeled IGF-I or 125I-labeled MSA from the purified receptors; insulin was 5-15 times more potent. Competitive binding studies indicated that this IGF binding activity could not be explained by cross-reaction with classical insulin receptors or by coelution of IGF-I or IGF-II receptors. Instead, it was due to a minor population of discrete atypical insulin receptors (6-18% total insulin receptors) with moderately high affinity (Kd = 2-4 X 10(-9) M) for IGF-I and MSA. These receptors were not an artifact of insulin-CDI-agarose chromatography, since they were present in wheat germ lectin-agarose-purified preparations and could also be purified from insulin-succinyldiaminodipropylamino-agarose. Affinity labeling with 125I-labeled MSA revealed that these atypical receptors had the same binding subunit (Mr 140,000) as classical insulin and IGF-I receptors. They displayed intermediate reactivity with polyclonal and monoclonal antibodies to the insulin and IGF-I receptors. It is therefore likely that insulin receptors purified by immunoadsorption would also contain atypical insulin receptors. The finding of more than one type of insulin receptor might relate to the slight variations in the cDNA nucleotide sequences and the multiple mRNA species reported for the insulin receptor [Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., Ou, J.-H., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A. & Rutter, W. J. (1985) Cell 40, 747-758]. PMID- 3012555 TI - Inhibition of replication and expression of human T-cell lymphotropic virus type III in cultured cells by exogenous synthetic oligonucleotides complementary to viral RNA. AB - The possibility of using oligodeoxynucleotides complementary to viral RNA or proviral DNA to inhibit the replication of human T-cell lymphotropic virus type III (HTLV-III) [the etiological agent of acquired immunodeficiency syndrome (AIDS)] in cultured human cells was addressed by studying the association of 32P labeled oligodeoxynucleotides with mammalian cellular components. The results indicated that exogenous oligodeoxynucleotides at 20 microM became associated with the membrane/cytosol fractions of the cell in amounts approximating 1.5 microM. Oligodeoxynucleotides complementary to a region close to the tRNALys primer binding site on HTLV-III RNA and others complementary to HTLV-III mRNA donor or acceptor splice sites inhibited viral replication (assayed as reverse transcriptase) and gene expression (assayed as virus-encoded proteins p15 and p24) by as much as 95%. Use of control (random) oligodeoxynucleotides suggests that the antiviral effects were specific. Although these results pertain to HTLV III-infected cells in tissue culture, rather than to AIDS patients, they nevertheless point to a therapeutic potential of the complementary oligodeoxynucleotide ("hybridization competition" or "hybridon") approach in the treatment of patients with AIDS and AIDS-related complex. PMID- 3012554 TI - Purification to apparent homogeneity of a mu-type opioid receptor from rat brain. AB - A mu-opioid-specific receptor was purified to apparent homogeneity from rat brain membranes by 6-succinylmorphine affinity chromatography, gel filtration, wheat germ agglutinin affinity chromatography, and isoelectric focusing. The purified receptor had a molecular weight of 58,000 as determined by NaDodSO4/polyacrylamide gel electrophoresis and was judged to be homogeneous by the following criteria: (i) a single band was detected by autoradiography after NaDodSO4/polyacrylamide gel electrophoresis of 125I-labeled receptor and (ii) the purified preparation had a specific opioid-binding activity of 17,720 pmol/mg of protein, close to the theoretical value. In addition, the Mr 58,000 value agrees closely with that determined by covalently labeling purified receptor with bromoacetyl-[3H]dihydromorphine or with 125I-labeled beta-endorphin and dimethyl suberimidate. PMID- 3012556 TI - Human glucose-6-phosphate dehydrogenase: primary structure and cDNA cloning. AB - The X-chromosome-linked glucose-6-phosphate dehydrogenase (D-glucose-6 phosphate:NADP+ oxidoreductase, EC 1.1.1.49) of humans and other mammals consists of a subunit with a molecular weight of about 58,000. The enzyme plays a key role in the generation of NADPH, particularly in matured erythrocytes, and the genetic deficiency of the enzyme is associated with chronic and drug- or food-induced hemolytic anemia in humans. The enzyme was purified to homogeneity from human erythrocytes. The complete amino acid sequence of the subunit, consisting of 531 amino acid residues, was determined by automated and manual Edman degradation of tryptic, chymotryptic, thermolytic, and cyanogen bromide peptides obtained from the enzyme. Based on the amino acid sequence data thus obtained, a 41-mer oligonucleotide with unique sequence was prepared. Two cDNA libraries constructed in phage lambda gt11--i.e., a human liver cDNA library and a human hepatoma Li-7 cDNA library--were screened with the synthetic nucleotide probe. Two positive clones, lambda G6PD-19 and lambda G6PD-25, were obtained from the hepatoma library. lambda G6PD-19 contained an insertion of 2.0 kilobase pairs (kbp), and encoded 204 amino acid residues that were completely compatible with the COOH terminal portion of the enzyme. The insertion of the clone had a 3' noncoding region of 1.36 kbp. The other clone, lambda G6PD-25, had an insertion of 1.8 kbp and encoded 362 amino acid residues of G6PD. Southern blot analysis of DNA samples obtained from cells with and without the human X chromosome indicated that the cDNA hybridizes with a sequence in the X chromosome. PMID- 3012557 TI - Characterization of leukotriene A4 synthase from murine mast cells: evidence for its identity to arachidonate 5-lipoxygenase. AB - Leukotriene A4 synthase was purified from the cytosolic fraction of murine mast cells. The enzyme converted 5-hydroperoxy-6-trans-8,11,14-cis-icosatetraenoic acid (5-HPETE) to leukotriene A4. This unstable product was identified by demonstration of two epimers of 6-transleukotriene B4, methanol trapping, as well as further transformation to leukotriene B4 by leukotriene A4 hydrolase. Leukotriene A4 synthase stereospecifically eliminated the D-hydrogen at C-10 (pro R) in the synthesis of leukotriene A4 when incubated with [10D-3H;3-(14)C]5 HPETE. The purified enzyme also exhibited 5-lipoxygenase activity toward arachidonic acid and 8-lipoxygenase activity towards 8,11,14-cisicosatrienoic acid. All of these activities required Ca2+ and ATP for their maximal velocities. The effects of heat treatment and of several lipoxygenase inhibitors on these enzyme activities as well as coelution in various chromatographic systems strongly suggest that lipoxygenase and leukotriene A4 synthase activities reside in the same enzyme molecule. PMID- 3012558 TI - Leader sequences of murine coronavirus mRNAs can be freely reassorted: evidence for the role of free leader RNA in transcription. AB - Mouse hepatitis virus (MHV), which replicates in cytoplasm of infected cells, contains an identical leader RNA sequence at the 5' end of each of the virus specific mRNAs. Previous studies suggested that the synthesis of these mRNAs does not involve conventional RNA splicing and may instead require priming by a free leader RNA. In this communication, we demonstrate that, during a mixed infection with two different MHVs, the leader RNA sequences from one virus could be detected on the mRNAs of the coinfecting virus at a high frequency, as if the leader sequence and mRNAs were joined together from two randomly segregating RNA segments. This finding demonstrates that MHV mRNA transcription utilizes independently transcribed leader RNA species that possess the trans-acting property. This study thus provides further evidence in support of the unique model of "leader-primed transcription" for coronavirus mRNA synthesis. PMID- 3012559 TI - Deprotonation of the Schiff base of rhodopsin is obligate in the activation of the G protein. AB - Photolysis of rhodopsin leads to the formation of an activated intermediate that activates a G protein, thus beginning the visual cascade. This activated form of rhodopsin appears coincident in time with the spectroscopically defined intermediate, metarhodopsin II. Metarhodopsin I, the precursor of metarhodopsin II, contains a protonated Schiff base, whereas metarhodopsin II does not. The question of whether the deprotonation of the protonated Schiff base is obligate in the formation of activated rhodopsin was addressed by monomethylating the active-site lysine of permethylated rhodopsin and determining whether this pigment can activate the G protein upon photolysis. The photolysis of the new pigment, which absorbs at 520 nm, led to the formation of a relatively stable metarhodopsin I-like intermediate with a lambda max of approximately equal to 485 nm, with no apparent formation of either metarhodopsin II- or metarhodopsin III like intermediates. The only probe available to detect formation of the active form of rhodopsin is G protein activation. Photolysis of the pigment in the presence of the G protein did not lead to measurable activation of the GTPase activity of the latter. These studies establish a functional link between Schiff base deprotonation and activation of the G protein. It is concluded that proton transfer from the protonated Schiff base of rhodopsin is obligate for the initiation of visual transduction. PMID- 3012560 TI - Autophosphorylation reversibly regulates the Ca2+/calmodulin-dependence of Ca2+/calmodulin-dependent protein kinase II. AB - Ca2+/calmodulin-dependent protein kinase II contains two subunits, alpha (Mr 50,000) and beta (Mr 60,000/58,000), both of which undergo Ca2+/calmodulin dependent autophosphorylation. In the present study, we have studied the mechanism of this autophosphorylation reaction and its effect on the activity of the enzyme. Both subunits are autophosphorylated through an intramolecular mechanism. Using synapsin I as substrate, Ca2+/calmodulin-dependent protein kinase II, in its unphosphorylated form, was totally dependent on Ca2+ and calmodulin for its activity. Preincubation of the enzyme with Ca2+, calmodulin, and ATP, under conditions where autophosphorylation of both subunits occurred, converted the enzyme to one that was only partially dependent on Ca2+ and calmodulin for its activity. No change in the total activity, measured in the presence of Ca2+ and calmodulin, was observed. The nonhydrolyzable ATP analog adenosine 5'-[beta, gamma-imido] triphosphate did not substitute for ATP in the preincubation. Moreover, dephosphorylation of autophosphorylated Ca2+/calmodulin dependent protein kinase II with protein phosphatase 2A resulted in an enzyme that was again totally dependent on Ca2+ and calmodulin for its activity. We propose that autophosphorylation and dephosphorylation reversibly regulate the Ca2+ and calmodulin requirement of Ca2+/calmodulin-dependent protein kinase II. PMID- 3012562 TI - Recombinant retroviruses that transduce individual polyoma tumor antigens: effects on growth and differentiation. AB - We have constructed infectious retroviral vectors, derived from Moloney murine leukemia virus, that efficiently transduce the polyoma virus tumor (T) antigens individually. The parental vector we have chosen [pZIP-NeoSV(X)1] expresses a dominant selectable marker for neomycin resistance and is a shuttle vector capable of propagation in both eukaryotic and prokaryotic cells, thus facilitating its use in structure-function studies. To address the relationship between polyoma T-antigen tumorigenesis and the effects of individual T antigens on growth control and differentiation, we used these vectors to introduce and stably express large, middle-sized, or small T antigens into mouse fibroblasts and preadipocytes. All cDNAs introduced into the vector are expressed stably even in the absence of selective pressure. The stable expression of small T antigen is noted particularly because cell lines expressing small T antigen have not been readily available prior to the use of retroviral vectors. Small T antigen-induced increase in saturation density of NIH 3T3 cells can be scored on the basis of the morphology of drug-resistant colonies. Middle-sized T antigen eliminates the growth requirement of NIH 3T3 cells for epidermal growth factor in a defined medium and permits growth in platelet-poor plasma, indicating elimination of the platelet-derived growth factor requirement as well. Large T antigen suppresses mouse preadipocyte (3T3-F442A) differentiation. These vectors and these functional assays of T-antigen activity permit genetic analysis of the relationship between tumorigenesis by T antigens and the alteration of cellular growth and differentiation. PMID- 3012563 TI - Activation of the endogenous metalloproteinase, gelatinase, by triggered human neutrophils. AB - Triggered human neutrophils degraded denatured type I collagen (gelatin) by releasing and activating the latent metalloenzyme, gelatinase. The ability of the neutrophil to activate this enzyme was significantly, but not completely, inhibited by agents known to inhibit or scavenge chlorinated oxidants generated by the H2O2/myeloperoxidase/chloride system. A direct role for chlorinated oxidants in this process was confirmed by the ability of reagent HOCl to activate the latent enzyme in either the cell-free supernatant or in a highly purified state. Gelatinase activity was also expressed by triggered neutrophils isolated from patients with chronic granulomatous disease. The amount of gelatinolytic activity expressed by the patients' cells was similar to that released by normal neutrophils that were triggered in the presence of antioxidants. Thus, human neutrophils have the ability to activate gelatinase by either an HOCl-dependent process or an uncharacterized oxygen-independent process. The ability of the neutrophil to directly regulate this enzyme suggests an important role for the metalloproteinase in physiologic and pathophysiologic connective tissue metabolism. PMID- 3012561 TI - Two related but distinct forms of the Mr 36,000 tyrosine kinase substrate (calpactin) that interact with phospholipid and actin in a Ca2+-dependent manner. AB - A method was devised that allows the identification of proteins related to the Mr 36,000 tyrosine kinase substrate calpactin based on their ability to interact with actin and phospholipid in a calcium-dependent manner. Two distinct proteins, detected in human A431 cells and fibroblasts, were resolved by two-dimensional gel electrophoresis. One of these proteins (calpactin I) appears identical to the Mr 34,000-39,000 substrate of the pp60src tyrosine kinase and the second (calpactin II) reacts with antibodies to the Mr 35,000 substrate of the epidermal growth factor receptor. Both proteins interact with phospholipid and actin, are rather basic, and share structural and antigenic determinants. A major difference between the two proteins is noted in their state of association with the Mr 10,000 light chain; i.e., calpactin I is associated with the light chain while calpactin II is not. PMID- 3012564 TI - Oocyte-specific gene expression: molecular characterization of a cDNA coding for ZP-3, the sperm receptor of the mouse zona pellucida. AB - The mouse zona pellucida genes are expressed uniquely during oogenesis and are developmentally regulated in the absence of cell division. Little is known about the mechanisms that control the expression of these germ-line-specific genes that play crucial roles in early mammalian development. We have constructed a lambda gt11 cDNA library from ovarian poly(A)+ mRNA and have isolated clones coding for ZP-3, the mouse sperm receptor. The identity of the clones was confirmed by comparing their DNA sequence with an amino acid sequence obtained from an isolated ZP-3 peptide. The ZP-3 gene is transcribed as a 1.7-kilobase poly(A) mRNA that is detected exclusively in ovarian tissue. This germ-line-specific expression is reflected in the observed hypomethylation of the ZP-3 locus in ovarian but not liver or brain DNAs. The ZP-3 gene is otherwise identically organized in somatic and germ-line DNA where it appears to be present as a low copy-number or single-copy gene. Despite the fact that the mouse sperm receptor demonstrates species specificity, the ZP-3 cDNA cross-hybridized with DNA from a variety of mammalian species, including rat, rabbit, dog, pig, cow, and human. PMID- 3012565 TI - Evidence for a common evolutionary origin of brain and pancreas cholecystokinin receptors. AB - An evolutionary basis for the distinct forms of cholecystokinin (CCK) receptors in the mammalian brain and pancreas was examined. The brains and pancreases of ratfish, frog, snake, and chicken contained saturable, high-affinity binding sites for iodinated porcine CCK-33. In the ectothermic species, the brain and pancreas CCK receptors exhibited nearly the same relative specificities for various CCKs and gastrins. Sulfated CCK-8 and sulfated gastrin-17 were the most potent while their nonsulfated analogs and gastrin-4 were less potent. By contrast, in the chicken, the specificities of brain and pancreas CCK receptors closely resembled their mammalian counterparts. We conclude that brain and pancreas CCK receptors with new specificities for binding CCKs and gastrins evolved at the level of the divergence of endotherms (birds and mammals) from reptiles. We propose that the prior evolution of gastrin provided the selection pressure for these changes. The endotherm pancreas receptor arose in evolution by narrowing its requirement for the position of a sulfated tyrosine residue in its ligands from either the sixth or seventh position from the carboxyl terminus to the seventh position only. The endotherm brain receptor arose in evolution by losing its requirement for a sulfated ligand and by transferring its high affinity binding domain from the tyrosine residue in the carboxyl termini of CCK and gastrin to the carboxyl-terminal tetrapeptide active site common to CCKs and gastrins. PMID- 3012566 TI - Locus specificity in the mutability of L5178Y mouse lymphoma cells: the role of multilocus lesions. AB - Mouse L5178Y strain LY-S and its parental strain LY-R differ in their comparative sensitivities to the cytotoxic effects of various mutagenic agents--i.e., strain LY-S has been found to be more sensitive, less sensitive, or similarly sensitive to individual agents in comparison to strain LY-R. Nevertheless, strain LY-S has been found to be uniformly less mutable than strain LY-R at the hypoxanthine (guanine) phosphoribosyltransferase (Hprt) locus following treatment with x radiation, UV radiation, or alkylating agents. In the present work we have isolated subclones of strains LY-R and LY-S that are heterozygous at the thymidine kinase (Tk) locus (chromosome 11). We have found that a heterozygous LY S Tk+/Tk- strain shows as high or higher mutability at the Tk locus than do heterozygous LY-R strains following treatment with x-radiation, UV radiation, or ethyl methanesulfonate. Mutability of all heterozygous strains at the Tk locus is much higher than at the Hprt locus following treatment with these mutagenic agents, with the exception of one heterozygous LY-R strain that possesses only one chromosome 11 and that is poorly mutable at the Tk locus by x-radiation. On the basis of these results, we have suggested that because of a repair deficiency, multilocus lesions are formed in the DNA of LY-S strains following treatment with radiation or alkylating agents; multilocus lesions lead to poor recovery of viable mutants when the target locus is closely linked to essential genes and situated on a hemizygous chromosomal region (e.g., the Hprt locus on the X chromosome or the Tk locus in strains monosomic for chromosome 11); and x radiation is a relatively poor mutagen at loci situated on hemizygous chromosomal regions, in repair-efficient and repair-deficient cells, because of its tendency to form multilocus lesions. PMID- 3012567 TI - Isolation of molecular probes associated with the chromosome 15 instability in the Prader-Willi syndrome. AB - Flow cytometry and recombinant DNA techniques have been used to obtain reagents for a molecular analysis of the Prader-Willi syndrome (PWS). HindIII total-digest libraries were prepared in lambda phage Charon 21A from flow-sorted inverted duplicated no. 15 human chromosomes and propagated on recombination-proficient (LE392) and recBC-, sbcB- (DB1257) bacteria. Twelve distinct chromosome 15 specific probes have been isolated. Eight localized to the region 15q11----13. Four of these eight sublocalized to band 15q11.2 and are shown to be deleted in DNA of one of two patients examined with the PWS. Heteroduplex analysis of two of these clones, which grew on DB1257 but not on LE392, revealed stem-loop structures in the inserts, indicative of inverted, repeated DNA elements. Such DNA repeats might account for some of the cloning instability of DNA segments from proximal 15q. Analysis of the genetic and physical instability associated with the repeated sequences we have isolated from band 15q11.2 may elucidate the molecular basis for the instability of this chromosomal region in patients with the PWS or other diseases associated with chromosomal abnormalities in the proximal long arm of human chromosome 15. PMID- 3012569 TI - High-resolution analysis of the human HLA-DR polymorphism by hybridization with sequence-specific oligonucleotide probes. AB - The human major histocompatibility complex class II antigens of the HLA-D are highly polymorphic, surface proteins essential in the cellular interactions necessary for an immune response. The analysis of this polymorphism is crucial for (i) histocompatibility matching for transplantation and (ii) understanding the association between HLA-D and certain important diseases. The polymorphism of certain HLA-D haplotypes may escape detection by current methodologies. Analysis at the genomic level of the polymorphism of one of the HLA-D subregions HLA-DR, using oligonucleotide probes specific for the polymorphic regions, is capable of distinguishing single nucleotide differences. The DRw6 haplotype was analyzed in view of the lack of DRw6 specific sera. On the basis of nucleotide sequence analysis, the DRw6 haplotype consists of at least two subtypes. When analyzed with oligonucleotide probes, this split identifies new polymorphic groups that differ from the DRw6 serological subgroups. PMID- 3012568 TI - Functional role of HLA class I cell-surface molecules in human T-lymphocyte activation and proliferation. AB - This investigation addressed the role of major histocompatibility complex-encoded class I molecules in the activation and proliferation of human lymphocytes. We studied the effect of antibodies specific for HLA-A and HLA-B locus gene products on mitogen-stimulated peripheral blood mononuclear cell (PBMC) subpopulations. Three individually derived, well-characterized anti-HLA class I monoclonal antibodies were demonstrated to inhibit the proliferation of human PBMC stimulated by either OKT3 or the calcium ionophore ionomycin. The antibody directed against HLA-A, -B, and -C locus gene products (W6/32) and the antibody directed against HLA-B locus gene products (4E) inhibited proliferation induced by either mitogen by 70-90%. The HLA-A locus-specific antibody (131), though inhibiting ionomycin-induced proliferation by 80-90%, was much less effective when OKT3 was the stimulus. The inhibition affected T4+ and T8+ cells and was not mediated by DR+ accessory cells. The inhibitory effect of these antibodies was associated with a decrease in the level of interleukin 2 activity present in culture supernatants, decreased interleukin 2 receptor expression, and decreased transferrin receptor expression and was not overcome by the addition of exogenous interleukin 2. Our results suggest that HLA class I molecules are directly involved in the early critical events of human lymphocyte activation and proliferation. PMID- 3012570 TI - Lack of class I H-2 antigens in cells transformed by radiation leukemia virus is associated with methylation and rearrangement of H-2 DNA. AB - Transformation of murine thymocytes by radiation leukemia virus is associated with reduced expression of the class I antigens encoded in the major histocompatibility complex (MHC) and increased methylation and altered restriction enzyme patterns of MHC DNA. These changes may play a role in host susceptibility to virus-induced leukemogenesis and accord with the notion that viral genomes play a regulatory function when they integrate adjacent to histocompatibility genes. PMID- 3012571 TI - Adult T-cell leukemia/lymphoma not associated with human T-cell leukemia virus type I. AB - We describe five patients with adult T-cell leukemia/lymphoma (ATL) with neither integration of human T-cell leukemia virus type I (HTLV-I) into their leukemia cells nor anti-HTLV-I antibody in their sera. These findings indicate that HTLV-I may not have been involved in leukemogenesis in these patients. The clinicohematological, cytopathological, and immunological features of HTLV-I negative ATL were exactly the same as those of HTLV-I-associated ATL. Leukemia cells with pleomorphic nuclei, generalized lymphadenopathy, hepatosplenomegaly, skin lesions, hypercalcemia, and elevated lactate dehydrogenase levels, all of which are characteristic features of typical ATL, were also seen in these patients with HTLV-I-negative ATL. Leukemia cells expressed T3, T4, and pan-T cell antigens in three cases, and T3 and pan-T-cell antigens in two. All five patients had lived in ATL-nonendemic areas. The finding of HTLV-I-negative ATL suggests that factor(s) other than HTLV-I infection may be involved in ATL leukemogenesis. PMID- 3012572 TI - Hepatocellular carcinoma in ground squirrels persistently infected with ground squirrel hepatitis virus. AB - Although persistent infection with hepatitis B virus and woodchuck hepatitis virus has been associated with development of hepatocellular carcinoma in the host, little has been known of such an association with ground squirrel hepatitis virus (GSHV), which is closely related to the woodchuck virus. Colonies of GSHV infected and -uninfected Beechey ground squirrels were observed for tumors for a period of 5 years. Tumors developed in seven squirrels after a minimum of 2.4 years of observation per animal; each of the seven animals was over 4 years old when the tumor was detected. The predominant type of tumor was hepatocellular carcinoma, which appeared in 2 of 28 GSHV-bearing animals studied and in 1 of 23 squirrels with antibody to the virus. No hepatocellular carcinoma appeared in 24 GSHV marker-free squirrels. Integrated GSHV DNA was found in the hepatocellular carcinoma tissue of the one carrier animal examined, paralleling the frequent findings of integrated hepatitis B and woodchuck hepatitis viral DNA in human and woodchuck hepatocellular carcinoma. Although the incidence of liver carcinoma reported here in carrier ground squirrels is neither as great as that in carrier woodchucks nor statistically different from the incidence in noncarrier squirrels, the data presented suggest that persistent infection with GSHV may also be associated with hepatocellular carcinoma. PMID- 3012574 TI - Inhibition of leukotriene actions by the calcium channel blocker, nisoldipine. AB - Nisoldipine, a calcium channel blocker having a highly potent effect on vascular smooth muscle relative to cardiac muscle, was tested to determine its anti leukotriene properties. Nisoldipine, at concentrations from 1 to 300 ng/ml, significantly attenuated the vasoconstrictor effects of both LTC4 and LTD4 in isolated perfused cat coronary arteries and in isolated Langendorff perfused cat hearts. In isolated perfused coronary arteries, nisoldipine exerted a greater percentage inhibition of LTC4- and LTD4-induced constriction than of the constriction induced by the thromboxane analog, carbocyclic thromboxane A2 (CTA2). In isolated cat lung fragments, higher concentrations of nisoldipine were required to inhibit leukotriene formation (i.e., 10-200 microM). These concentrations of nisoldipine markedly inhibited the formation of the chemotactic leukotriene (LTB4) as well as the peptide leukotrienes (LTC4 and LTD4) stimulated by A-23187. Both types of leukotrienes were inhibited to a comparable degree. Thus, nisoldipine has significant anti-leukotriene actions. At normally employed concentrations, nisoldipine inhibits leukotriene actions on vascular smooth muscle, and at higher concentrations, it inhibits leukotriene formation. PMID- 3012573 TI - Human serum Cohn fraction IV (alpha-globulin [correction of globin] enriched) inhibits ligand binding at neurotransmitter receptors in human brain. AB - Human serum proteins are found in significant density in the neuropil in brains of demented individuals. The functional significance of these abnormally distributed proteins has been unknown. We now report that alpha-globulin-enriched fractions of human serum decrease the specific binding of [3H]spiroperidol at its binding sites in postmortem human frontal cortex and caudate. The substances in this serum fraction apparently exert their effect by a direct action on the binding site. Since [3H]spiroperidol labels serotoninergic and dopaminergic among other neurotransmitter receptors, these results suggest that components of human serum inhibit the binding of ligands at neurotransmitter receptors. PMID- 3012575 TI - Effects of viral infection on contraction of the diaphragm in mice. AB - Isometric contractile properties of isolated phrenic nerve-diaphragm muscle preparations were used to study the effects of picornavirus infections on diaphragm muscle function. Properties of muscles from virus-inoculated and control mice were similar during brief contractions. However, when subjected to a series of fatiguing contractions by indirect or direct stimulation, muscles of mice inoculated with a paralytic variant of encephalomyocarditis (EMC) virus showed a greater rate of fatigue and a reduced capacity to recover from fatigue than did muscles from uninoculated control mice or muscles from mice inoculated with a nonparalytic coxsackievirus B3 (CVB3). Mice paralyzed by EMC virus infection had high titers of virus in the brain and similar titers of virus in diaphragm muscle as found in diaphragm muscles of CVB3-inoculated mice. The results indicate that EMC virus infection of mice leads to increased fatigability of the diaphragm muscle and that there are both neural and muscular components of this enhanced fatigue. PMID- 3012576 TI - Head and neck squamous cell carcinoma cell lines as a model system for the study of oncogene expression during tumor progression and metastasis. PMID- 3012577 TI - The biochemistry of cell activation as related to the putative actions of flavonoids. PMID- 3012578 TI - Inhibition of mitochondrial respiration and production of superoxide and hydrogen peroxide by flavonoids: a structure activity study. PMID- 3012579 TI - Effect of quercetin on ATP-driven pumps and glycolysis. PMID- 3012580 TI - The effects of flavonoids on cyclic nucleotide phosphodiesterases. PMID- 3012581 TI - The effect of quercetin on pp60v-src kinase activities. PMID- 3012582 TI - Effect of quercetin on serine/threonine and tyrosine protein kinases. PMID- 3012583 TI - Antiviral activity of flavones and flavans. PMID- 3012584 TI - Antiviral activity of 3-methoxyflavones. PMID- 3012585 TI - Formation of hepoxilin A4, B4 and the corresponding trioxilins from 12(S) hydroperoxy-5,8,10,14,17-icosapentaenoic acid. AB - 12S-Hydroperoxy-5,8,10,14,17-icosapentaenoic acid (12S-HPEPE), prepared by incubating rat platelet preparations with 14C-labelled icosapentaenoic acid and purified by HPLC, was transformed into hepoxilins A4 and B4 by treatment with bovine hemin in phosphate buffer. The identity of the products, purified by HPLC as methyl esters, was confirmed by GCMS analysis of the Me tBDMSi derivatives using both El and Cl detection and of the PFB TMSi derivatives using NlCl detection. In similarity to hepoxilin A3 and B3 derived from arachidonic acid, hepoxilin A4 and B4 also possess insulin secretagogue activity. This finding contrasts with observations in the prostaglandin series where products derived from icosapentaenoic acid have been reported to possess less potent biological actions. PMID- 3012586 TI - Prostaglandin binding to different cell types of human uterus: quantitative light microscope autoradiographic study. AB - Five human uteri of the menstrual cycle were analyzed by quantitative light microscope autoradiography to determine which uterine cell types contain prostaglandin (PG) binding sites. The results showed that stromal cells, glandular epithelium, elongated and circular smooth muscles, arterioles and erythrocytes in lumen of arterioles contained numerous PGE and very few or no detectable PGF2 alpha binding sites. The grains which were present when incubated with [3H]PGE2 alone completely disappeared following coincubation with excess unlabeled PGE2 but not with excess unlabeled PGA1. Analysis of grain count data revealed that PGE binding sites in arterioles were lower than those in other uterine cell types and PGE binding sites in endometrial cells of proliferative phase uteri were higher than those in secretory phase uteri (P less than 0.05). Thus the present results demonstrate that all the cell types, including arterioles of human uterus of the menstrual cycle, contain PGE and minimal PGF2 alpha binding sites. This suggests that PGE binding sites in different cell types of human uterus subserve diverse effects of PGEs. PMID- 3012587 TI - Radioimmunoassay of LTB4 and 6-trans LTB4: analytical and pharmacological characterisation of immunoreactive LTB4 in ionophore stimulated human blood. AB - A sensitive RIA for LTB4 (which cross-reacts 60% with 6-trans LTB4) has been developed with a minimal detectable mass of 7.4 X 10(-15) mole. The properties of a second RIA more specific for 6-trans LTB4 are also described. The latter has utility in the measurement of the enzymatic and non-enzymatic transformations of LTA4 and in the characterisation of inhibitors of LTA4 epoxide hydrolase. Conditions for the direct RIA of immunoreactive LTB4 in human plasma are described. Addition of calcium ionophore, A23187, to human blood increased basal immunoreactive LTB4 levels from less than 100pg ml-1 to 259 +/- 23ng ml-1 (mean +/- SEM, n = 16). Extraction and RPHPLC confirmed that greater than 90% of immunoreactive LTB4 co-eluted with synthetic and [3H]LTB4. Pharmacological characterisation of immunoreactive eicosanoids revealed that in vitro the NSAIDs: aspirin, indomethacin, flurbiprofen and benoxaprofen did not inhibit LTB4 but inhibited TXB2, consistent with cyclo-oxygenase inhibition whereas the prototype mixed inhibitor BW755c inhibited both LTB4 and TXB2. This experimental paradigm may be used in the clinical measurement of the bio-availability of novel agents that inhibit eicosanoid biosynthesis. PMID- 3012588 TI - Measurement of reaction times in tasks of varying complexity: an indication of the degree and evolution of mental deterioration in the elderly. AB - In a group of 40 elderly patients with intellectual deterioration, slowing of behaviour was measured with an original reaction time apparatus. Three types of measurements in ascending degree of complexity were evaluated against each other. Intercorrelations, made with indicators of physical, mental and behavioural functioning, demonstrated that ascending complexity in reaction time measurements can be used as an evaluation of the degree of mental deterioration. An example of the use of the apparatus in the evaluation of the improvement after treatment with suloctidil is given. PMID- 3012590 TI - Comparison of the effects of the benzodiazepine midazolam and three serotonin antagonists on a consummatory conflict paradigm. AB - A consummatory conflict procedure that involves an abrupt reduction in magnitude of an expected reward (negative contrast) has been shown to be particularly sensitive to the effects of anxiolytic agents. As previously reported with chlordiazepoxide, another benzodiazepine (BDZ), midazolam released suppressed consummatory performance in a dose-dependent manner. This effect was not due to a general appetitie stimulatory effect of the drug. The effects of three 5-HT antagonists on negative contrast were examined to evaluate the role serotonin may play in the anxiolytic action of BDZ. Methysergide was found to be ineffective, cinanserin tended to reduce contrast at two intermediate doses, and cyproheptadine eliminated the contrast effect in a similar fashion as midazolam. The effectiveness of cyproheptadine may not be attributed to its anticholinergic or antihistaminergic actions since scopolamine and pyrilamine did not produce similar effects. The results are discussed in terms of the role serotonin may play in the anti-conflict action of BDZ, as well as possible interactional effects of GABA. PMID- 3012589 TI - Norepinephrine infusions into the medial preoptic area inhibit lordosis behavior. AB - Neurotransmitters, including norepinephrine, have been implicated in the mediation of ovarian steroid induced lordosis behavior in ovariectomized rats. In this study we have found that norepinephrine (NE) infusions into the medial preoptic area (MPOA) reduced lordosis frequencies of estrogen-progesterone treated (0.5 micrograms estradiol benzoate for three days followed by 500 micrograms progesterone 4-5 hours before testing) receptive rats. Norepinephrine doses of 2 micrograms or more per animal infused into the MPOA significantly reduced lordosis levels within five minutes. Infusions of 10 and 20 micrograms doses of NE suppressed lordosis levels for 15 minutes after infusion. At the lowest inhibitory dose (2 micrograms/animal) simultaneous infusion of 5 micrograms/microliter of the noradrenergic antagonist yohimbine, but not of phentolamine or propranolol, blocked the reduction in lordosis resulting from NE infusion. Preoptic infusions of epinephrine and clonidine were also effective in reducing lordosis quotients, while methoxamine, phenylephrine and isoproterenol did not alter receptivity. These findings are consistent with the conclusion that the direct effect of norepinephrine infusions into the MPOA is inhibition of lordosis responding. There is some evidence that this inhibitory influence is mediated by alpha 2-noradrenergic receptors. PMID- 3012591 TI - GABAergic control of masculine sexual behavior. AB - Drugs affecting the GABAergic transmission were injected into the medial preoptic anterior hypothalamic area (MPOA) and the masculine sexual behavior analyzed. Antagonizing GABAergic neurotransmission by (+) bicuculline methiodide (30 ng/cannula), or 3-mercaptopropionic acid (10 or 20 micrograms/cannula) resulted in a drastic shortening of the postejaculatory intervals and a shortening of the ejaculation latency. Injection of compounds causing an increase in GABAergic activity, muscimol (25 ng/cannula) or ethanolamine-O-sulphate (80 micrograms/cannula) depressed masculine sexual behavior. Systemic treatment or injection into the nucleus caudatus putamen of compounds affecting the GABAergic transmission did not cause any alteration in the mating pattern. It is suggested that the GABAergic neurotransmission is involved in inhibitory processes underlying the masculine sexual behavior. PMID- 3012592 TI - Neuronal membrane enzymes in rat lines selected for differential motor impairment by ethanol. AB - Neuronal membrane enzyme activities were determined in naive and ethanol-treated (30 min after 2 g/kg) male and female rats of lines developed for more (ANT) and less (AT) ethanol-induced motor impairment. Ethanol did not affect acetylcholinesterase, (Na+K)-ATPase or 5'-nucleotidase activities, but adenylate cyclase activities were lowered in both cerebellum and cerebrum. Cerebral acetylcholinesterase activities were higher in ANT than AT rats. No consistent line difference was observed regarding (Na+K)-ATPase activities. Slightly higher cerebellar 5'-nucleotidase activities were found in the ANT line. Cerebellar adenylate cyclase levels were substantially higher in the AT line. No line differences were displayed in the activation of adenylate cyclase activity by dopamine or norepinephrine. It is concluded that ethanol in vivo may inhibit neuronal adenylate cyclase activity and that cerebellar phosphorylation may be a regulator of motor impairment. Cholinergic mechanisms may also be connected to the ethanol-induced motor impairment. PMID- 3012593 TI - The effects of REM sleep deprivation on striatal dopamine receptor sites. AB - 3H-Spiroperidol binding to dopamine receptor sites of rat striatal tissue was studied following 24, 48, 72 and 96 hr of rapid eye movement sleep deprivation (REM dep.). The density of dopamine receptor binding sites (Bmax) was decreased after 48, 72, and 96 hr of REM dep. The apparent dissociation constant (KD) decreased after 96 hr, indicating an increase in apparent affinities. The control experimental animals also presented a time-dependent decrease of Bmax and KD as compared to unhandled controls. These results suggest that dopaminergic mechanisms may indeed be involved in the effects of REM sleep deprivation and/or stress. PMID- 3012594 TI - Elevations in plasma angiotensin II with prolonged ethanol treatment in rats. AB - Chronic alcohol consumption frequently leads to hypertension in humans. While previous reports have implicated the renin-angiotensin system as a potential mediator of this effect, plasma angiotensin II (AII) levels were either not measured or yielded negative results. The present investigation noted significant elevations in circulating AII in rats intubated daily with ethanol (4 g/kg) for 50 days. Animals administered ethanol only once evidenced AII concentrations equivalent with water intubated controls. Radioligand binding assay data indicated no differences in the number or affinity of Sar1,Ile8-AII binding sites in the thalamus, septum-anterior ventral third ventrical region or adrenal gland comparing those groups chronically treated with ethanol to water intubated controls. These results may support a role for the vasoconstrictive hormone AII in the etiology of alcohol-induced hypertension. PMID- 3012595 TI - The effects of beta-carboline carboxylic acid ethyl ester and its free acid, administered ICV, on the anticonvulsant activity of diazepam and sodium valproate in the mouse. AB - The effects of intracerebroventricular (ICV) beta-carboline carboxylic acid ethyl ester (beta-CCE) and its free acid on the protective effects of diazepam against leptazol- and R05-3663-induced convulsions were investigated in mice and compared with their effects on the antileptazol effect of sodium valproate, in an attempt to demonstrate a specific central effect of beta-CCE on benzodiazepine function. The results show that a small dose (1 microgram) of beta-CCE but not its free acid (in doses of up to 100 micrograms) was able to reverse the protective effects of diazepam against leptazol- and R05-3663-induced convulsions, whereas the effects of sodium valproate, a nonbenzodiazepine anticonvulsant, could not be reversed by these beta-carboline derivatives. PMID- 3012596 TI - Methylnalorphinium fails to reverse naloxone-sensitive stress-induced analgesia in mice. AB - Exposure of mice to cold-restraint stress markedly decreased the number of abdominal constrictions induced by IP acetic acid. Naloxone pretreatment significantly attenuated the antinociceptive effect of cold-restraint stress, suggesting a partial mediation by opioid mechanisms. Pretreatment with the quaternary opioid antagonist methylnalorphinium did not reverse analgesia in stressed mice. Also, nociception in both stressed and non-stressed mice was not modified by pretreatment with the selective alpha 2-adrenoceptor blocker yohimbine. The results suggest that cold-restraint stress promotes analgesia in mice which is mediated in part by opioid but not alpha 2-adrenoceptor mechanisms. Furthermore, the results do not substantiate a peripheral analgesic role for circulating opioids in this model of stress. PMID- 3012597 TI - Opiate blockade inhibits saccharin intake and blocks normal preference acquisition. AB - Recent evidence indicates a close connection between oral sensory function and opioid effects on feeding. Not only is gustatory motivation influenced by opiate drugs but apparently gustatory stimuli can also activate central opiate receptor systems. In 3 experiments we studied the effect of opiate receptor blockade on drinking motivated by the sweet taste of saccharin. Experiment 1 established a dose-response function for inhibition of intake by naloxone (NAL) in short (60 min) 2-bottle tests. This experiment demonstrated the extreme sensitivity of nondeprived, nonstressed animals to NAL and estimated the MED50 at less than 0.1 mg/kg (SC), well below the threshold for effects due to illness or general motor disturbance. Experiment 2 further demonstrated that NAL's effectiveness depends on saccharin concentration. In particular, the lowest NAL dose studied was effective near the threshold for saccharin preference but not at higher concentrations. These data suggest that endogenous opioid systems may be activated by taste stimuli in a graded fashion. Finally, experiment 3 showed that the typical acquisition of preference for a moderate saccharin concentration can be effectively blocked by daily pre-test NAL injection. Together these experiments further demonstrate the close functional relationship between opioid systems and gustatory sensory systems. PMID- 3012598 TI - Benzodiazepine-receptor mediated convulsions in infant rats: effects of beta carbolines. AB - The effects of anticonvulsant and proconvulsant benzodiazepine-receptor ligands were studied in infant rats. The agonist flurazepam increased myoclonic twitching of the limbs as has previously been reported. In contrast, the convulsant beta carbolines DMCM and beta-CCM did not produce twitching, but did produce marked increases in locomotor activity and whole body shakes. The standard convulsants pentylenetetrazol and bicuculline similarly increased locomotor activity and shaking. These findings suggest that the effects of agonist benzodiazepines cannot be interpreted as convulsant-type behaviour. In addition, the finding that DMCM and beta-CCM have equivalent effects despite showing preferential affinities for benzodiazepine-receptor subtypes argues against one particular subtype having proconvulsant effects. PMID- 3012599 TI - Bremazocine-induced backwards walking behavior in rats is mediated via opioid kappa receptors. AB - Bremazocine dose-dependently induced backwards walking behavior in rats after its SC injection. Only the (-) but not the (+) enantiomer induced backwards walking. Pretreatment with either naloxone or MR 2266 reduced the bremazocine-induced backwards walking. MR 2266 was at least ten times more potent than naloxone. These findings suggest that bremazocine-induced backwards walking is mediated via an agonistic action of the drug with opioid kappa receptors. The data may contribute to the discussion concerning opioid kappa receptors and the psychotomimetic effects of some opioid analgesic drugs. PMID- 3012600 TI - Morphine-elicited feeding: diurnal rhythm, circulating corticosterone and macronutrient selection. AB - The present study examined the feeding response elicited by morphine, injected either intraperitoneally (IP), or into the paraventricular nucleus (PVN), as a function of diurnal cycle and also in adrenalectomized rats with or without peripheral corticosterone replacement. In animals maintained on a single diet of chow, milk and sugar, a diurnal rhythm in both the peripheral and central morphine-induced feeding responses was observed, with a stronger eating effect occurring in the early dark hours compared with the responses obtained in the early light period. Adrenalectomy significantly reduced the feeding induced by morphine injected IP or into the PVN, and acute corticosterone replacement restored the response. Rats maintained on a self-selection feeding paradigm, with carbohydrate, protein and fat simultaneously available, exhibited a significant increase in total caloric intake after morphine injected IP, along with a preferential increase in the consumption of protein and fat. Adrenalectomy nearly abolished this stimulatory effect of morphine on total intake and altered the diet preference pattern. These findings underscore the importance of corticosterone in the feeding response of morphine injected peripherally or specifically into the PVN. The present findings suggest that corticosterone plays an important role in determining the diurnal rhythm of opiate-induced feeding and the function of endogenous opioids in the regulation of energy balance. PMID- 3012601 TI - Differential antagonism of diazepam-induced loss of the righting response. AB - Ethyl-beta-carboline-3-carboxylate (beta-CCE), inosine and Ro 15-1788 are antagonists of several actions of the benzodiazepines. These compounds can be differentiated, however, according to their ability to reverse the loss of the righting response induced by diazepam. Ro 15-1788 completely reversed effects of diazepam on the righting response of pigeons and squirrel monkeys but was ineffective against comparable effects produced by pentobarbital. Pretreatment with Ro 15-1788 protected against diazepam-induced righting loss. Neither inosine nor beta-CCE reversed diazepam-induced righting loss or acted prophylactically against this effect. Since beta-CCE has been characterized as an inverse agonist at the benzodiazepine receptor, the absence of antagonism we report would suggest that beta-CCE lacks specific pharmacological activity which opposes suppression of the righting response by diazepam. Research with these preferentially-acting antagonists may lead to the development of anxiolytics devoid of the sedative hypnotic properties inherent in the drugs currently in a clinical use. PMID- 3012602 TI - Comparison of circling induced by unilateral intrastriatal microinjections of haloperidol, clozapine and CCK-8 in rats. AB - The existence of the neuropeptide cholecystokinin (CCK) within a subpopulation of central dopamine (DA) neurons has led to speculations that the peptide may serve as an endogenous modulator of DA functions. To test this possibility, the present study examined the pharmacological action of CCK-8 by comparing its effects on DA mediated circling behavior with those of a typical (haloperidol; HAL) and an atypical (clozapine; CLZ) dopamine antagonist neuroleptic drug. Rats received unilateral intrastriatal infusions of either sulfated CCK-8 (1, 2, or 8 micrograms), HAL (5 micrograms) or CLZ (5 or 20 micrograms) 15 minutes after systemic injection of d-amphetamine (1 mg/kg). Animals were then placed into rotational chambers where the number and direction of complete 360 degree turns was automatically recorded over a 1 hour session. HAL produced strong and almost exclusive ipsilateral circling while the responses after CLZ and CCK-8 were reliably more variable in rotational direction. More specifically, the results suggest that CLZ is only a weak antagonist of behaviors mediated by striatal DA activation while CCK seems to be devoid of antidopaminergic properties in the striatum. PMID- 3012603 TI - Stress induced desensitization of lymphocyte beta-adrenoceptors in young and aged rats. AB - The effects of different times of immobilization stress on intact lymphocyte beta adrenoceptors and plasma corticosterone were compared in 3-month and 24-month-old rats. In young animals after 30 min restraint 3H-dihydroalprenolol specific binding was significantly reduced (61% of control value) and plasma corticosterone significantly raised (186% of control). The effect on beta adrenoceptors was due changes in receptor number (Bmax) without any effect on affinity (KD). In aged rats both effects were only seen after 180 min restraint and were less pronounced. Isoproterenol treatment in vitro reduced beta adrenoceptors on lymphocytes. This effect was less pronounced in lymphocytes from aged rats. Corticosterone in vitro increased 3H-dihydroalprenolol specific binding. We therefore suggest that the decrease of beta-adrenoceptors reflects an adaptive response to the stress-induced catecholamine release and that corticosterone could play a role in reversing this effect. This adaptive response to stress seems to be impaired in aged animals. PMID- 3012604 TI - Differential interactions between chlordiazepoxide, pentobarbital and benzodiazepine antagonists Ro 15-1788 and CGS 8216 in a drug discrimination procedure. AB - Rats were trained to discriminate either chlordiazepoxide (CDP, 4 mg/kg, IP, N = 8) or pentobarbital (PB, 10 mg/kg, IP, N = 8) from saline in a two-lever food reinforced procedure. CDP and PB dose-dependently substituted for each other (greater than or equal to 90% drug lever responses); indicating that their discriminative stimulus properties were closely similar. However, discriminative stimulus control induced by CDP and PB differentially was affected by the proposed benzodiazepine (BDZ) antagonists Ro 15-1788 (0.08-20 mg/kg, IP) and CGS 8216 (2.5-20 mg/kg, IP) in each experimental group; suggesting that the discriminative stimulus properties of CDP and PB are mediated by different mechanisms of action. When administered alone, Ro 15-1788 (5 and 20 mg/kg), but not CGS 8216, induced CDP like discriminative effects, suggesting that Ro 15-1788 may have partial (BDZ like) agonist properties, not shown by CGS 8216. Additional evidence for a behavioral difference between Ro 15-1788 and CGS 8216 is suggested by differential effects of both compounds on response rate. The results may reflect differential interactions of the compounds with the BDZ receptor-GABA receptor-Cl- ionophore complex. PMID- 3012605 TI - Pharmacokinetics and metabolism of delta 1-tetrahydrocannabinol and other cannabinoids with emphasis on man. PMID- 3012606 TI - 3H-muscimol binding sites within guinea pig ovary: a histoautoradiographic study. AB - The distribution of 3H-muscimol within guinea pig ovary was studied using combined radioreceptor binding studies and histoautoradiographic technique performed on frozen sections of ovary mounted on microscope slides. 3H-muscimol was bound by sections of ovary in a manner consistent with the existence of specific GABA receptors. The binding was reversible, saturable, of high affinity (KD was 38 +/- 6 nmol/l and Bmax was about 378 +/- 61 fmol/mg protein, and inhibited by GABA receptor interfering drugs. 3H-muscimol binding sites were found in the blood vessel wall, in the follicle and in the oocytes. The number of oocyte and follicular receptors gradually decreases during the development of ovarian follicles. PMID- 3012608 TI - Low frequency perforant path stimulation as a conditioned stimulus demonstrates correlations between long-term synaptic potentiation and learning. AB - Stimulation of the perforant path with impulse trains of 15 cps and 670 msec duration was used as a conditioned stimulus in a two-way shuttle box avoidance on rats. Field potentials in the dentate area evoked by test stimuli were measured after the training sessions until the 7th day. Foot-shock and unconditioned escape elicited only a transient slight depression of the population spike amplitude (P) and increased also slightly the slope function (SF) of the population EPSP of the evoked test potentials. The control stimulation of the perforant path without pairing with foot-shock as in conditioning did only slightly increase SF of test potentials, but produced a strong transient inhibition followed by a long lasting moderate depression of P. After conditioning, all animals exhibited the same initial inhibition of P as shown in control stimulation of the perforant path. However during the following 4 hours, good learners with a relearning index greater than 30% developed a significant potentiation of P lasting until the second training session 24 hours later, which resulted in a further enhancement. SF of the evoked test potentials increased in good learners with a similar time course after conditioning but without initial depression. After 7 days P showed still enhanced but non-significant values. Poor learners with a relearning index less than 10% did not develop a potentiation of P after conditioning and initial inhibition, but a long-term depression. Also SF of test potentials decreased in poor learners during 4 hours after conditioning and returned almost to baseline until the following day. After 7 days, P and SF did not differ from baseline. The analysis of the observed synaptic changes by E S curves demonstrated the post-tetanic LTP seems to differ in some ways from post conditioning LTP in good learners. The latter exhibits a clear tendency of a right shift contrary to the left shift commonly occurring after tetanization. Furthermore poor learners do not only fail to produce long-term potentiation, but fail to show a change in the opposite direction with a left shift of the E-S curves. The observed correlation of LTP in the conditioning pathway with the learning ability suggests an involvement of LTP at least in the acquisition and early retention of this learned behavior. The results do however not finally clarify the role of LTP in long-term retention. PMID- 3012607 TI - Cholecystokinin, diet palatability, and feeding regulation in rats. AB - Rats ate less food than normal on cyclic-ratio schedules following cholecystokinin and lithium chloride injections. Nevertheless, they defended this lower eating rate in the same way as under control conditions. The pattern of effects produced by cholecystokinin and lithium chloride resembled those following diet adulteration with citric acid and sucrose octa acetate and differed from the effects produced by increases in body weight. Cholecystokinin and lithium chloride injections also produced similar changes in the free-feeding patterns of non-deprived rats: Both meal size and intermeal intervals decreased in manner similar to the effects of citric acid and sucrose octa acetate adulteration. Interpreted in terms of a static regulatory model, these results suggest that cholecystokinin and lithium chloride suppress feeding by degrading the palatability of food, not by promoting satiety, discomfort, or illness. PMID- 3012609 TI - Temporal characteristics of electrical self-stimulation reward: fatigue rather than adaptation. AB - Using a Y-maze preference test paradigm, we examined the temporal characteristics of the neural network subserving self-stimulation reward. Rats were given a choice between two pulse trains of stimulation, which varied in duration and pulse frequency. The results showed that increasing the pulse frequency decreases the duration at which the rewarding effectiveness of brain stimulation reaches an asymptote. The data also indicated that when prolonged stimulation is delivered at a high pulse frequency, the initial pulses contribute the most to the rewarding effect. Later pulses are affected by the reduced ability of the neurons or synapses to transmit signals along the neural network due to fatigue. The data are explained in terms of an improved model of summation involving more than one integrator and fatigue. PMID- 3012610 TI - Hyperendorphinemia in obesity and relationships to affective state. AB - Eight obese patients (exceeding ideal body weight by 50% or more) with no endocrinological or metabolic disorders and 8 healthy, age-matched, normal-weight volunteers were submitted to an overnight short dexamethasone (DXM) suppression test and to a psychological assessment through various psychometric scales. Plasma B-Endorphin (B-EP), B-Lipotropin (B-LPH), ACTH and cortisol concentrations were evaluated in basal conditions, as well as 9 and 17 hours after late night administration of 1 mg DXM in both groups. All hormones were measured by radioimmunoassay, either directly in the plasma (ACTH and cortisol) or after silicic acid extraction and Sephadex G-75 column chromatography (B-LPH and B-EP). In obese patients, plasma B-EP levels in basal conditions were three times higher than in normal weight controls and remained unaltered by DXM suppression. ACTH and B-LPH, in contrast, were within the normal range and were significantly reduced by DXM. In 3 of the 8 patients, plasma cortisol concentrations at 17 hours post-DXM were greater than 50 ng/ml indicating an early escape from the suppression. Psychometric evaluations revealed a prevalence of depressive personality in obese patients. These data indicate an hypersecretion of B-EP in obese patients, which is only partially dependent on hypothalamic control. PMID- 3012611 TI - pHG165: a pBR322 copy number derivative of pUC8 for cloning and expression. AB - During the construction of the Messing pUC plasmid series, the rop(rom) gene of pBR322 which mediates the activity of RNAI was deleted. This has resulted in an elevated copy number for the pUC plasmids which makes the expression of beta galactosidase activity constitutive in a host containing the Iqtss lac repressor. We describe the construction of a new series of vectors which retain the pUC multiple cloning site (MCS) but in which copy number control has been recovered. In addition, the lac alpha/lac promoter expression region has been inserted into a HpaI cassette. This facilitates the movement of recombinant DNA clones within the MCS. It also increases the complementation activity of the lac alpha peptide by an order of magnitude, allowing selection of recombinants by their Lac- phenotype on MacConkey agar. PMID- 3012612 TI - pHG276: a multiple cloning site pBR322 copy number vector expressing a functional lac alpha peptide from the bacteriophage lambda PR promoter. AB - The bacteriophage lambda early promoter PR has been used to direct the synthesis of lac alpha in a plasmid containing the multiple cloning site of pUC8. The copy number of the plasmid is controlled by the rop(rom) gene and the plasmid incorporates the cI857 gene for temperature regulation of lac alpha expression. Isolation of recombinant derivatives with DNA inserts in the multiple cloning region can be identified by insertional inactivation of lac alpha and consequently, a Lac- phenotype in a host carrying the M15 deletion of lac. The potential of pHG276 as a fully regulated expression vector is examined. PMID- 3012613 TI - The minimal replicon of a streptomycete plasmid produces an ultrahigh level of plasmid DNA. AB - A functional map of Streptomyces coelicolor plasmid SCP2* was deduced from derivatives constructed by in vitro deletions. Functions were analyzed on bifunctional shuttle plasmids that contained pBR322 for selection and replication in Escherichia coli and fragments of SCP2* for replication in Streptomyces griseofuscus C581 and strains of Streptomyces lividans. The aph gene for neomycin resistance from Streptomyces fradiae and the tsr gene for thiostrepton resistance from Streptomyces azureus were incorporated as selectable antibiotic resistance markers in streptomycetes. An 11.8-kb sequence bounded by EcoRI and KpnI restriction sites contains the information for self-transfer and normal replication of the plasmid. A 5.9-kb EcoRI-SalI fragment contains all of the information for normal replication. Partial digestion generated a 2.2-kb Sau3A fragment that is sufficient for replication but it produces ten times higher plasmid copy number than the basic replicon. pHJL400 and PHJL401 are useful shuttle vectors containing the moderate-copy-number streptomycete plasmid combined with the E. coli plasmid pUC19. A 1.4-kb BclI-Sau3A fragment with an additional internal BclI site contains the minimal replicon but it produces 1000 times higher plasmid copy number than the basic replicon. pHJL302 is a useful shuttle vector containing the ultrahigh-copy-number streptomycete plasmid combined with the E. coli plasmid pUC19. PMID- 3012615 TI - Identification of regions of the Streptococcus faecalis plasmid pCF-10 that encode antibiotic resistance and pheromone response functions. AB - The conjugative plasmid pCF-10 (58 kb) of Streptococcus faecalis has been mapped with restriction enzymes. By restriction mapping and Southern hybridization analysis, a 16-kb segment of the plasmid was shown to resemble closely the conjugative tetracycline resistance transposon, Tn916. Mutagenesis of the plasmid with the erythromycin resistance transposon Tn917 was used to localize a tetracycline resistance determinant and several regions involved in conjugal transfer. Fifty Tn917 insertions (outside the region of the plasmid homologous to Tn916) affecting mating behavior and the ability of donor cells to respond to the sex pheromone cCF-10 were mapped to nine distinct segments, or tra regions. Insertions into tra regions 1-3 and 7-9 led to an enhanced transfer ability of mutant plasmids relative to the transfer frequency obtained for the wild-type plasmid. Cells carrying these mutant plasmids differed in colony morphology or growth in broth culture from cells carrying pCF-10. Insertions into tra regions 4 6 resulted in reduced plasmid transfer, or completely eliminated the mating potential of donor cells. Insertions generating transfer-defective plasmids could be grouped further according to the ability of strains harboring the mutant plasmids to respond to cCF-10. HindIII fragments of pCF-10 coding for transfer functions have been cloned into Escherichia coli. PMID- 3012614 TI - Plasmids related to RSF1010 from Bordetella bronchiseptica. AB - Six out of 14 Bordetella bronchiseptica isolates from U.K. pigs each contained one plasmid, of 8.7-44 kb. All plasmid-containing isolates were sulfonamide resistant, and this property was shown to be plasmid-encoded. Five of the plasmids were related; two were indistinguishable from the broad-host-range plasmid, RSF1010. The other three, two of which appeared to be identical, were shown to have regions of homology with RSF1010. One of these regions encompassed the sulfonamide resistance determinant while the other contained oriV, which also determines plasmid incompatibility. None of the plasmids could be associated with virulence or phase variation, and it appears likely that they have been acquired in response to antibiotic pressure. PMID- 3012617 TI - Analysis of a 1.6-micron circular plasmid from the yeast Kluyveromyces drosophilarum: structure and molecular dimorphism. AB - A new plasmid has been found in the yeast Kluyveromyces drosophilarum. It is a double-stranded circular DNA, 1.6 micron in length (4.8 kilobase pairs). As in the case of Saccharomyces 2 mu circles, this plasmid occurs in two isomeric forms corresponding to the inversion of a segment between two 346-bp-long inverted repeats within the molecule. Each form has been separately cloned into bacterial plasmids. The new yeast plasmid, called pKD1, contains sequences that allow its replication in Saccharomyces cerevisiae. PMID- 3012616 TI - Multiple mutations in the transferred regions of the Agrobacterium rhizogenes root-inducing plasmids. AB - Agrobacterium rhizogenes induces root formation at the site of inoculation in plants and inserts fragments of its Ri plasmid into the plant nuclear DNA. The transferred region (T-DNA) of the Ri plasmid of the A. rhizogenes strain A4 is made of two fragments, namely TL and TR; the latter harbors a sequence homology with the tms loci (responsible for auxin synthesis) of A. tumefaciens. On Daucus carota slices, single insertion mutations on the TL region of A. rhizogenes do not confer a mutant phenotype while an insertion-deletion in the TR region do confer a Basatt phenotype. Six double mutants with a single insertion in the TL region and the same deletion-insertion of the TR region were constructed. Three of these double mutants were avirulent on D. carota which indicates that in A. rhizogenes A4 the TL and the TR regions cooperate to confer a full infectious phenotype. PMID- 3012618 TI - Restriction fragment length polymorphism of the human beta-globin gene cluster: analysis of a beta-thalassemic family in Taiwan. AB - We have analysed seven polymorphic restriction sites of the human beta-globin gene cluster of six members of a Chinese family with a beta +-thalassemic sibling. The seven polymorphic sites analysed are the HincII site at the 5'-end of the epsilon-globin gene, the HindIII sites in the two gamma-globin genes, two HincII sites within and at the 3'-end of the psi beta 1 pseudogene, the AvaII site in the beta-globin gene and the BamHI site located at the 3' side of the beta-globin gene. The beta thal chromosome has been identified to have a haplotype of +----++ with respect to these seven polymorphic sites. This is also the most predominant haplotype associated with beta +-thalassemia in Mediterranean and Chinese populations (Chen et al., 1984; Orkin et al., 1982). Of the seven sites analysed in this family, four will be useful in prenatal diagnosis of beta-thalassemia in subsequent pregnancies in the family. PMID- 3012619 TI - The search for an AIDS vaccine. PMID- 3012620 TI - Key epidemiologic questions about AIDS and infection with HTLV-III/LAV. PMID- 3012621 TI - Isolation of ducts from the pancreas of copper-deficient rats. AB - A technique is described, involving tissue dissociation and micro-dissection, for the isolation of interlobular ducts from the pancreas of copper-deficient rats. The average length and outside diameter of the isolated ducts were 589.0 +/- 18.6 and 78.1 +/- 1.6 micron (mean +/- S.E.M., n = 425) respectively. Between twenty and fifty ducts could be obtained from each pancreas. Frequently, the smaller intralobular ducts, which had outside diameters of between 15 and 25 micron, were observed as branches of the interlobular ducts. Light and electron microscopy showed that the isolated ducts were structurally intact, and that the epithelial cells possessed all the typical ultrastructural features of duct cells within the gland of copper-replete rats. The isolated ducts consumed oxygen at a rate of 2.27 +/- 0.55 ml O2/min X 100 g wet weight duct epithelium (n = 6). The concentrations of ATP, ADP and AMP in the ducts were 3.78 +/- 0.81, 0.68 +/- 0.19 and 0.41 +/- 0.13 mmol/l duct epithelium (n = 8) respectively. These data give values for ATP:ADP and ATP:AMP ratios of 5.6:1 and 9.2:1 respectively, and an energy charge of 0.85 +/- 0.01 (n = 8) suggesting that the epithelial cells are healthy and in a stable metabolic state. In the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.67 mM), the basal concentration of cyclic AMP in the isolated ducts was 17.4 +/- 0.7 mumol/l duct epithelium (n = 3). Secretin (0.1 nM-1 microM) caused a dose-related increase in cyclic AMP content up to a maximum of 376.0 +/- 85.3 mumol/l duct epithelium (n = 4). This indicates that the epithelial cells possess secretin receptors, and that these receptors can be functionally linked to adenylate cyclase. PMID- 3012622 TI - Epstein Barr virus-specific immune defects in patients with persistent symptoms following infectious mononucleosis. AB - In a number of patients recovery from infectious mononucleosis (IM) following primary Epstein Barr virus (EBV) infection, is complicated by the persistence of symptoms for months or years. Normally recovery from infectious mononucleosis is associated with the development of EBV-specific antibodies and memory cytotoxic T cells, which are present in the peripheral blood of all normal seropositive individuals. We studied four patients who had persistent symptoms for more than two years after infectious mononucleosis to determine if this abnormality was associated with a defect in EBV-specific or non-specific immune responses. All four patients had normal immunoglobulin concentrations, T- and B-cell numbers, T cell proliferative responses and natural killer cell activity. However three of the four had reduced or absent antibodies to the EBV nuclear antigen (EBNA) although other EBV-specific antibody titres were normal. All four also had reduced EBV-specific cytotoxic T-cell activity as measured by the EBV regression assay. This defect was probably EBV-specific as alloreactive cytotoxic T-cell responses were normal. In addition, three of three patients tested had reduced in vitro antibody synthesis following pokeweed mitogen stimulation. These studies indicate that the syndrome of persistent symptoms following EBV mononucleosis may be associated with a defect in EBV-specific immunity, and thus suggest a possible immunological basis for the syndrome. PMID- 3012623 TI - [Immunoenzyme reactions, immunofluorescence and complement fixation: evaluation in the study of anti-cytomegalovirus antibodies. Technical note]. AB - A comparative study of CF, IF and ELISA was carried out in order to comparatively evaluate the characteristics of sensitivity and specificity of the tests. The results seem to confirm the validity of both IF and ELISA in the laboratory diagnosis of CMV infections. PMID- 3012624 TI - Radiation sensitization by oxygen of in vitro mammalian cells: is .O-2 involved? AB - Oxygen is a potent sensitizer of cells exposed to ionizing radiation, and, although the exact chemical mechanisms are not fully understood, some evidence suggests that this sensitization may involve the formation of superoxide anion radicals (.O-2) [F. Lavelle, A. M. Michelson, and L. Dimitrijevic, Biochem. Biophys. Res. Commun. 55, 350-357 (1973); A. Petkau and W. S. Chelack, Int. J. Radiat. Biol. 26, 421-426 (1974); L. W. Oberley, A. L. Lindgren, S. A. Baker, and R. H. Stevens, Radiat. Res. 68, 320-328 (1976)] To test this hypothesis, we compared the sensitivity of Chinese hamster V79 cells irradiated in O2/N2 and O2/N2O gas mixtures with and without the addition of other radical scavenging agents. In these tests, although oxygen was present, be blocked the radiation induced reactions of O2 which produce .O-2. We found that the total amount of biological damage depends simply on the concentration of O2 that is present; the overall sensitivity is not reduced when .O-2 cannot be formed. Thus radiation sensitization by O2--at least of this cell line--does not require the formation of superoxide anion radicals. PMID- 3012626 TI - [New methods of digital evaluation of thermograms of breast cancer]. PMID- 3012625 TI - Synthesis, biodistribution, and autoradiography of radiolabeled S-2-(3 methylaminopropylamino)ethylphosphorothioic acid (WR-3689). AB - 35S- and 3H-labeled S-2-(3-methylaminopropylamino)ethylphosphorothioic acid (WR 3689) have been synthesized in our laboratory and used to study organ and cellular level distribution in C3H/Km mice bearing RIF-1 tumors. Tissue biodistributions obtained with 35S-WR-3689 showed that blood levels peak at 15 min postinjection and decline gradually over 60 min. At 30 min after drug injection the highest uptake is in kidney and submandibular salivary gland, with lowest levels in brain and moderate to low levels in the RIF-1 tumor, comparable to levels in skin and muscle. High resolution diffusible substance autoradiography with 3H-WR-3689 reveals a homogenous distribution of label over cells in liver and lung and nonuniform distribution of silver grains over the cytoplasm of cells in the kidney cortex, parotid and submandibular salivary glands, and small intestine. There are no indications of preferential nuclear location of label from protective drug in any tissue. Correlations of biodistribution and autoradiography data with measures of radioprotection in different tissues will be useful in interpreting mechanisms of radioprotection with this phosphorothioate. PMID- 3012627 TI - [Adrenal gland scintigraphy]. AB - The exact localization of adrenal lesions can be achieved by noninvasive procedures. Whereas radiological methods reflect morphological changes, scintigraphy of adrenal cortex and medulla depends on function. - Radiolabeled 6 beta-methyl-19-norcholesterol is used for adrenocortical scintigraphy in primary aldosteronism, Cushing's syndrome and hyperandrogenism. By dexamethasone suppression a correct classification of adrenocortical lesions by scintigraphy can be observed in about 89% with a specificity of 86%. 123-I- and 131-I metaiodobenzylguanidine is used for specific scintigraphy of the adrenal medulla. This method is a safe and reliable method for localization of adrenal and extraadrenal pheochromocytomas. PMID- 3012629 TI - [Reversible computed tomographic changes following brain tumor irradiation induced by the "early-delayed reaction" after radiation]. AB - We analyzed the incidence, timing and appearance of reversible CT findings in 55 patients undergoing radiation therapy (dose 60 Gy) for brain tumors. Three patients had "early delayed reactions" within three months of irradiation. CT studies showed increased size of the central zone of necrosis with contrast enhancement in the form of rings or garlands. Differentiation between these findings and recurrence of tumor was possible only through follow-up studies. PMID- 3012628 TI - Magnetic resonance imaging of the adrenal. AB - Magnetic resonance imaging (MRI) of the adrenals was performed on 50 subjects: 5 normal volunteers, 6 Cushing patients with bilateral adrenal hyperplasia, 14 patients with adrenal adenomas, 3 with adrenal carcinomas, 15 with pheochromocytomas and 7 with metastatic disease to the adrenal. The normal and hyperplastic adrenal glands were imaged in all cases. Using the signal intensity of the adrenals on a T2 weighted image, various forms of adrenal pathology could be differentiated. A ratio of signal intensity of the adrenal mass to the liver was utilized and allowed the differentiation of adrenal adenomas from adrenal carcinomas, pheochromocytomas and metastases. Using the same ratio, metastases could be distinguished from pheochromocytomas as well. MRI appears to be particularly valuable in distinguishing clinically silent adrenal metastases from nonfunctioning adrenal adenomas. PMID- 3012630 TI - Skeletal metastases from hepatoma: frequency, distribution, and radiographic features. AB - Over the past 6 years, the authors evaluated 300 patients with hepatoma as part of phase 1 and 2 treatment protocol trials. Analysis of the available clinical data and radiographic studies revealed 22 patients (7.3%) with skeletal metastases demonstrated by radiography, computed tomography (CT), and/or nuclear scintigraphy. The plain film appearance of skeletal metastases from hepatoma was osteolytic in all cases. CT scanning best demonstrated the expansile, destructive nature of these metastases, which were often associated with large, bulky soft tissue masses. Skeletal metastases from hepatomas demonstrated increased radiotracer uptake on standard bone scans and were gallium avid, similar to the hepatoma itself. In addition, they could be targeted therapeutically with I-131 antiferritin immunoglobulin. The most frequent sites of skeletal metastases were the ribs, spine, femur, pelvis, and humerus. An initial symptom in ten patients was skeletal pain corresponding to the osseous metastases. In five patients, pathologic fractures of the proximal femur or humerus developed and required total hip replacement or open-reduction internal fixation. Patients with long standing cirrhosis or known hepatocellular carcinoma who also have skeletal symptoms should be evaluated for possible osseous metastases. PMID- 3012631 TI - Biliary dilatation: defining the level and cause by real-time US. AB - In a 15-month period, 110 patients with subsequently proved biliary dilatation were evaluated with ultrasound (US). The level of dilatation was defined as pancreatic, suprapancreatic, or at the level of the porta hepatis. Causes of dilatation included pancreatitis, choledocholithiasis, neoplasm, and stricture. The distal duct was examined initially on transverse scans obtained with the patient in a semierect right posterior oblique position; the proximal duct was then examined on longitudinal scans obtained with the patient in a supine left posterior oblique position. When this scanning technique was used, US indicated the level of dilatation in 91.8% of cases and suggested the correct cause in 70.9%. Because this approach markedly improves US visualization of the intrapancreatic bile duct, distal obstructing lesions, which are the most common, can be optimally examined. PMID- 3012632 TI - Peripheral low-density area of hepatic tumors: CT-pathologic correlation. AB - To aid in the distinction between colorectal cancer metastasis to the liver and hepatocellular carcinoma, findings on computed tomographic (CT) scans taken more than 5 minutes after contrast material administration ("late-enhanced CT scans") and pathologic findings were compared. Late-enhanced CT scans of metastatic adenocarcinoma showed a peripheral low-density area (PLDA) that corresponded to viable tumor and a central high-density area that represented fibrous connective tissue. This phenomenon was recognized in 15 of 20 (75%) patients with metastatic adenocarcinoma and in one of 50 (2%) patients with hepatocellular carcinoma. Late enhanced CT scans may be useful in distinguishing between metastatic nonmucinous colorectal cancer and hepatocellular carcinoma. PMID- 3012633 TI - Posttraumatic cardiac dysfunction: assessment with radionuclide ventriculography. AB - The authors studied 54 patients with multisystem trauma, including blunt chest injury, using combined dynamic first-pass and electrocardiographically (ECG) gated radionuclide ventriculography (RNV) to evaluate for posttraumatic myocardial dysfunction. Twenty-six of 54 (48%) patients had abnormalities of ventricular wall motion. The ventricular dysfunction was confined to the right ventricle in 92% of cases. In general, abnormalities consisted of right ventricular dilatation and diffuse hypokinesia, although in seven cases there were localized wall-motion abnormalities. The right ventricular ejection fraction of those patients with wall-motion abnormalities was significantly lower than those with normal studies. Left ventricular ejection fraction did not differ significantly between these groups. ECG changes were not associated with the cardiac dysfunction demonstrated scintigraphically, nor was there a relationship between the number or type of extrathoracic or thoracic injuries demonstrated by RNV. Follow-up studies obtained in 15 cases showed a significant overall improvement in cardiac function by 3 weeks after injury. Combined first-pass and ECG-gated RNV is useful for the identification and follow-up of patients with posttraumatic cardiac dysfunction. PMID- 3012634 TI - Calmodulin and protein phosphorylation: implications in brain ischemia. PMID- 3012635 TI - Mechanisms and consequences of ion transport in the liver. PMID- 3012637 TI - Hepatitis A. PMID- 3012636 TI - Mechanisms of experimentally induced intrahepatic cholestasis. PMID- 3012638 TI - The molecular analysis of hepatitis B virus. PMID- 3012639 TI - Hepatitis B virus and polyalbumin receptors. PMID- 3012640 TI - Persistent hepatitis B virus infection and hepatocellular carcinoma. PMID- 3012641 TI - Human lipoprotein metabolism and the liver. PMID- 3012642 TI - Environmental chemicals in hepatocarcinogenesis: the mechanism of tumor promoters. PMID- 3012644 TI - Vitamin A and the liver. PMID- 3012645 TI - [Expression of pX gene of HTLV-I and its possible involvement in ATL leukemogenesis]. PMID- 3012643 TI - The role of membrane lipids in the regulation of membrane-bound enzymes. PMID- 3012646 TI - [Chemically bonded HPLC stationary phase based on silica gel]. PMID- 3012647 TI - [T4 polynucleotide kinase]. PMID- 3012648 TI - [Receptor and cell membrane damaging action of cytolytic toxin]. PMID- 3012649 TI - [Structure and function of the src gene product]. PMID- 3012650 TI - [Ribonuclease H]. PMID- 3012651 TI - NaCN inhibits the stereospecific depletion of LTB4 by guinea pig polymorphonuclear leukocytes. AB - Caseinate elicited suspension of guinea pig peritoneal PMNs synthesized LTB4, 6t LTB4, 12-epi-6t-LTB4 and 5HETE after incubations with A23187 and arachidonic acid. Concentrations of LTB4 peaked in 3 minutes and were then rapidly depleted. 6t-LTB4 and 12-epi-6t-LTB4 also peaked in concentrations in 3 min but were depleted slower than LTB4. NaCN inhibited the depletion of LTB4 in a dose dependent fashion without dramatically affecting biosynthesis. PMID- 3012652 TI - Characterization and regulation of prostaglandin and leukotriene receptors: an overview. PMID- 3012653 TI - 5-lipoxygenase from rat PMN lysate. AB - The products of arachidonic acid metabolism in the 15,000xg supernatant of sonicated rat PMN are described. Only products derived from 5-lipoxygenase are observed. These products are 5-HETE and products derived from hydrolysis of LTA4, particularly LTB4. Some minor products derived from decomposition of 5-HPETE are also observed. The dependence of the activity of 5-lipoxygenase on enzyme and on substrate concentrations is presented and discussed in terms of a kinetic model that includes enzyme inactivation during turnover and substrate inhibition. The 5 lipoxygenase activity is stimulated by Ca++ and ATP. PMID- 3012654 TI - [Familial deficiency of B lymphocytes and myeloperoxidase in neutrophils coexistent with skin changes in a female patient]. PMID- 3012655 TI - [Adrenal reserve in patients with alopecia areata]. PMID- 3012656 TI - Fractionation studies with WR-2721: normal tissues and tumour. AB - We have studied the ability of WR-2721 to protect skin, kidney and an anaplastic murine tumour against single or fractionated X-ray treatments. Skin reactions, four different kidney assays, regrowth delay and local control of tumours have been used to construct dose-response curves from which the degree of radioprotection can be quantified as a protection factor. Low doses of WR-2721 (0.2-0.3 mg X g-1) were used before each of 1, 5 or 10 fractions. The degree of protection was similar in all three systems and it did not change significantly with fractionation. PMID- 3012657 TI - Bombesin-like peptides in human small cell carcinoma of the lung. AB - The nature of bombesin-like immunoreactive peptides was studied in extracts of small cell carcinoma of the human lung. Three peaks, I, II and III, designated by their increasing retention times, were separated by reversed-phase high performance liquid chromatography (HPLC) with trifluoroacetic acid (TFA) as counter ion. None of the peaks corresponded to bombesin. Peak III was eluted at the same position as porcine gastrin releasing peptide (GRP) but was separated from it in another reversed-phase system using heptafluorobutyric acid (HFBA). Peak II material eluted in the position of bombesin in the HFBA system but not in the TFA system. The elution position of Peak I corresponded to that of the carboxyl terminal fragments of GRP, i.e. GRP18-27 and GRP19-27. This correspondence was observed in each of the reversed-phase and gel filtration systems used. The Peak III peptide was converted to peak I after incubation with trypsin. It was reasoned that this conversion could be one of the steps in the processing of bombesin-like peptides in human small cell carcinoma. PMID- 3012658 TI - Comparison of mammalian VIP bioactivities in dispersed acini from guinea pig pancreas. AB - Mammalian VIP is identical in pig, cow, human, rat, dog and goat but differs in the guinea pig (GP) in positions 5, 9, 19, and 26. We now demonstrate that GP, goat, rat and synthetic mammalian VIP are indistinguishable in their inhibition of binding of 125I-labelled synthetic VIP to dispersed acini from GP pancreas and that GP, pig, dog, goat and synthetic VIP are also similar in their efficacy and potency in stimulating amylase release from these acini. Thus in spite of the differences in amino acid sequence, GP VIP appears to have full biologic potency in its action on dispersed acini from GP pancreas. PMID- 3012661 TI - [Causes of attenuation of the sound waves in neoplasms of the breast. Histologic and echographic correlation study]. AB - The purpose of our study was to compare the ultrasound findings in malignant breast masses (which underwent biopsy) with their histological appearance. In our activity of breast sonography, we observed that the same histological type of breast cancer often shows a different ultrasonic image. The main difference concerns the sonic attenuation or increase through the neoplastic mass. The histological examination took into account the amount of stroma and cells and the architectural pattern of the lesion. Comparing these features with the ultrasonic image, it has been shown that sonic transmission is related more to the architectural pattern than to the fibrous content of the neoplastic tissue. PMID- 3012659 TI - Oxytocin inhibits ACTH and peripheral catecholamine secretion in the urethane anesthetized rat. AB - Oxytocin (OT) generally has a stimulatory effect on ACTH secretion both in vitro and in vivo. As part of a study of ACTH-releasing factors in hypophysial portal blood, the effects of i.v. OT administration on plasma ACTH levels were tested in urethane-anesthetized rats. Surprisingly, i.v. injection of 10 micrograms OT lowered plasma ACTH levels by about 35% (P less than 0.01). It was reasoned that this paradoxical inhibition of ACTH secretion by OT might be mediated by inhibition of the unusually high rate of peripheral catecholamine secretion in this model. Measurement of plasma catecholamines before and after i.v. administration of 10 micrograms OT revealed a 53% inhibition of EPI (P less than 0.01) and 43% inhibition of NE (P less than 0.05). Administration of the beta adrenergic antagonist propranolol (400 micrograms) 15 min before the beginning of the experiment completely blocked the inhibitory effects of OT on ACTH secretion and in fact unmasked the stimulatory effects of OT normally seen in conscious animals and in vitro. Superfused bisected adrenal glands exposed to 10(-6) M OT for 10 min secreted more than 30% less EPI and NE than control adrenals suggesting that the inhibition of EPI and NE secretion by OT in vivo occurs, at least in part, directly at the level of the adrenal. The data support the hypothesis that peripheral catecholamines may at times be directly involved in the control of ACTH secretion and also suggest that OT, which has recently been identified in the adrenal medulla, may have important paracrine functions in the regulation of adrenal catecholamine secretion. PMID- 3012660 TI - [Estimation of 99mTc-dimercaptosuccinic acid renal uptake using single photon emission computed tomography]. AB - Renal function was assessed by measurement of 99mTc-dimercaptosuccinic acid (DMSA) uptake by the kidney based on the transsectional tomographic image obtained by single photon emission computed tomography (SPECT). The renal uptake was expressed as percentage of the total radioactivity detected in the kidney, the volume of which was measured by convolution method, against the amount dosed. Absorption was corrected by GE-STAR method using cut off level of 42%. In order to determine normal range, measurement was made for 40 kidneys of each of 10 male and female volunteers confirmed of having normal kidneys both morphologically and functionally. The average volume of the kidney was 220.4 ml for the right and 239.3 ml for the left for males, and 205.9 ml and 236.5 ml, respectively for females. The renal uptake of radioactivity (at 2 h after injection), was 26.8% for the right and 27.6% for the left for males, with corresponding figures for females being 26.4% and 27.9%, respectively. Distribution range of renal volume and renal uptake was obtained by bivariate analysis with 90% and 95% probability. From these results, our method of renal function determination based on renal uptake of 99mTc-DMSA obtained from renal transactional tomogram by SPECT is considered to be accurate and potentially useful for clinical purpose. PMID- 3012662 TI - [Magnetic resonance in the study of cerebral gliomas]. AB - Forty surgically proved gliomas have been studied by MR: 21 were low-grade gliomas and 19 were anaplastic astrocytomas and glioblastomas. The examination involved the use of T1-weighted (IR and/or SE, TR 500 ms, TE 50 ms) and T2 weighted scans (SE, TR 1000 ms, TE 50 with multiple echos). All patients underwent CT and surgical removal or stereotactic biopsy whose findings were retrospectively compared with those of MR. MR findings were similar in low-grade and anaplastic astrocytomas, but quite different from imaging of glioblastomas. Tumoral cyst and areas of necrosis were recognized on MR studies and confirmed by surgical findings. Differentiation between tumor and oedema was difficult. The MR images did not allow a more specific diagnosis of nature or of malignancy than available with CT up to now, but offer major advantages, such as a superior depiction of tumor extent and anatomic relationships. PMID- 3012663 TI - [Measurements of skin resistance in detecting activity of the sympathetic nervous system in spinal anesthesia]. AB - The skin resistance and temperature of the surface of the skin were measured in more than 20 patients by means of mobile measuring equipment. The phasic skin resistance activities of anesthetized parts were compared with unanesthetized regions at the extremities. Considerable phasic skin-resistance activity was shown in both anesthetized and unanesthetized regions. The two measured signals were partly identical in time and amplitude behavior. Temperature signals served as additional indicators of the activity of the sympathetic nervous system. To explain the results, an interpretation is proposed: the phasic components of the sympathetic activity can still exist and be measured below a nerve block - even if the tonic component of the sympathetic activity below this block is strongly reduced or vanishes, which is indicated by an increase in the surface temperature of the skin, as observed. This theory also holds regarding measurements of a patient who underwent a sympathectomy. Total lack of tonic and phasic components of sympathetic activity can be observed only under a high spinal analgesia (T3) or intubation anesthesia. PMID- 3012664 TI - [The current status of composite resins. I. Composition and structure]. PMID- 3012666 TI - [A 39-year-old male with pain in the right leg and a picture of general malaise]. PMID- 3012665 TI - [Disseminated intravascular coagulation induced in rats: effects on plasma concentration of fibronectin]. PMID- 3012667 TI - [Persistent polyadenopathy syndrome and immunity in parenteral drug addicts infected with HTLV-III]. PMID- 3012668 TI - [Extragonadal germinal tumor syndrome]. PMID- 3012669 TI - [A glomus jugulare chemodectoma producing catecholamines. Report of a new case]. PMID- 3012670 TI - [Comparison between i.a. DSA and cineangiography for evaluating the coronary arteries and the left ventricle]. AB - In twenty-two patients with coronary heart disease, selective coronary angiography and laevo-cardiography were carried out by means of conventional cine angiography and intra-arterial DSA. For evaluating left ventricular function, intra-arterial DSA and cine angiography were of equal value (r = 0.97). A significant advantage of DSA was the fact that it is easily reproducible. On the other hand, DSA is less good for quantifying coronary stenoses and for demonstrating the peripheral vessels. PMID- 3012671 TI - [I.v. DSA in the diagnosis and follow-up of thoracic aortic dissection]. AB - Intravenous DSA was performed in 53 patients with suspected dissection of the thoracic aorta and in 13 patients following surgery for aortic dissection. In 36 patients, the suspected diagnosis could be excluded definitely and, in 14 cases out of 17, a dissection was correctly diagnosed. All 11 type B dissections were correctly diagnosed. Of six type A dissections, only three were adequately demonstrated by IV DSA. In type B dissections, IV DSA is reliable, but in type A dissection with massive aortic insufficiency or pericardial tamponade the findings are not reliable. In all 13 patients who had surgery for dissection, IV DSA proved suitable for showing the anastomosis and progress of the disease. PMID- 3012672 TI - [Digital subtraction angiography and other non-invasive procedures for evaluating renal circulation and hypertension]. AB - Nuclear medical techniques (such as isotope nephrograms, renal scintigrams) which are suitable as screening methods, have been unable to improve the diagnosis of reno-vascular hypertension. Amongst conventional procedures, the excretion urogram following a bolus injection was the most informative, particularly if performed together with nephro-tomography. Although ultrasound is of some use in evaluating the kidneys, the excretion urogram remains essential for the diagnosis of reno-vascular hypertension. Additional exposures and modifications (e.g. early phase urogram only add unnecessary radiation and cost without providing additional information. On the other hand, it would be useful to obtain digital subtraction angiograms of the renal arteries during the contrast injection. In this way, reliable information can be obtained on the cause of reno-vascular hypertension in more than 90% of patients. Similar results can be obtained by photographic subtraction (ISA). This should be used where DSA apparatus is not available. Angio-CT and sequential CT is not reliable for the diagnosis of renal artery stenosis. On the other hand, these methods provide density measurements or time-density curves of selected areas in the cortex or medulla of the kidney, which indicated abnormalities of the circulation and provide a comparison of the two sides. PMID- 3012673 TI - [DSA and conventional angiography compared in the angiographic diagnosis of impotence]. AB - Twenty-three patients complaining of impotence were examined angiographically as part of their investigation. In all patients, DSA as well as conventional angiography was carried out. The purpose of this investigation was to compare the two methods with regard to their diagnostic value. By means of sequential analysis it was shown that, with an error level of 10%, DSA was superior to conventional angiography. PMID- 3012674 TI - [Pass-through technic for translumbar catheter angiography]. AB - A new introducer has been developed for translumbar catheter angiography using a 4-F catheter. This combines the advantages of catheter angiography and translumbar aortic puncture. CT examination following the angiogram showed retroperitoneal haematomas of about the same size as result from conventional translumbar aortography. PMID- 3012675 TI - [Value of phlebo-scintigraphy for clarifying thromboembolic processes in the pelvis and legs]. AB - In 64 patients suspected of having had pulmonary emboli due to thrombo-embolic disease, radionuclide venography (RNV) and contrast phlebography were carried out (84 examinations of extremities, 64 examinations of extremities and pelvis). Phleboscintigraphy proved informative in the thigh and in the pelvis. Around the knee, RNV was sufficiently accurate as a screening method. In the calf, RNV was of diagnostic value only in the presence of positive findings on scintigraphy. PMID- 3012676 TI - [Experience with the inhalation of a 33% xenon-(stable-)oxygen mixture in relation to a new method of measuring local cerebral circulation]. AB - We describe a new non-invasive method for measuring local blood flow in the brain. Xenon (stable)-nl CBF-CT-method has been used in our clinic for the first time in Europe. It is superior to all other methods, such as 133Xenon, SPECT and PET in terms of anatomical accuracy and its resolution. We describe the method and its side effects. Inhalation of Xenon in sub-narcotic doses (33% for five minutes) produced no significant complications. Of 95 examinations, 74 were free of any incidents. The most common side effect was euphoria. The method appears to be suitable for general clinical use. PMID- 3012677 TI - [Pneumonitis with fatal pulmonary fibrosis (Hamman-Rich syndrome) due to parathion-(E-605-) poisoning]. AB - A patient with chronic Parathione (E 605) poisoning was observed over a period of 55 days. During that time he developed progressive changes, which were identical to those of progressive idiopathic pulmonary fibrosis. The rapid development of an alveolitis, followed by a lethal pulmonary fibrosis, differed in no way, macroscopically nor microscopically, from the lung changes in paraquat poisoning (paraquat lung). The radiological course has been correlated with the clinical and post mortem findings. PMID- 3012678 TI - Pulmonary sequestrations of the upper lobe in children: three presentations. AB - Pulmonary sequestrations are congenital abnormalities where nonfunctioning lung tissue receives its vascular supply from the systemic circulation (thoracic or abdominal aorta). It is necessary to establish the diagnosis in childhood when the lesions are uncomplicated. The authors present three cases of sequestration of the apex (2 extralobar and 1 atypical) with the main clinical and radiological features. Sequestrations in the upper lobe are rare, and the usual site is the left lower lobe. Plain x-rays show a dense opacity, sometimes air-filled and sometimes with an air-fluid level: angiography is currently the best mean for definitive diagnosis; however, computed tomography will probably be very useful in the future. Differential diagnosis includes tumours of the superior mediastinum (neurogenic tumours, digestive duplication, bronchogenic cysts, pheochromocytoma and hydatid cysts). PMID- 3012680 TI - [Course and differential diagnosis of McCune-Albright syndrome]. AB - The McCune-Albright syndrome consists of a combination of fibrous dysplasia of bone and endocrine lesions with abnormalities of pigmentation. The condition is rare and may be missed, as happened in our case. The case showed extreme skeletal deformities, menarche at one year and peculiar histological appearances. PMID- 3012679 TI - [Potentialities and limitations of sonographic diagnosis of the chest in childhood]. AB - The advantages and limitations of the sonographic examination of the chest in 16 children are described. Partial opacification of the chest has been found in nine, a total "white" hemithorax in 7 children. Sonographic guided taps of the chest were performed in 3 children. Necessity, extent and yield of additional radiological examinations, especially computed tomography of the thorax, are discussed in detail. PMID- 3012681 TI - [Intraoperative sonographic demonstration of intraspinal tumors]. AB - Four examples are given to demonstrate the problems, methods and advantages of intra-operative sonography for intra-spinal tumours. The value of intra-operative sonography for localising the tumours and for judging their extent is demonstrated and related to other imaging methods, such as myelography, CT and MR. PMID- 3012682 TI - [Noninvasive imaging procedures in the diagnosis and therapy control of varicoceles. I: Sonography and thermography in the diagnosis of varicoceles]. AB - 69 patients with clinically suspected varicoceles were examined thermographically and sonographically prior to testicular phlebography. The combination of sonography and thermography permitted precise differentiation into normal findings (7%), left-sided varicoceles (86%) and bilateral varicoceles (7%). Thermography was advantageous with an accuracy of 97.3%. Sonography had an accuracy of 90.5%. No clear correlation was found between varicocele size and degree of hyperthermia. The combination of sonography and thermography affords a high degree of accuracy in the diagnosis of varicoceles, including subclinical and infantile varicoceles. PMID- 3012683 TI - [Ultra-high-strip-density grids for mammography: an alternative to grid mammography?]. AB - New stationary ultra high resolution grids have been proposed as an alternative to the conventional moving grid techniques for mammography. The physical performance of the new grids resembles those of conventional grids with 1:5 grid ratio. They fail during mammography because of their inability to render fine detail and, in particular, the important shapes of micro-calcification. PMID- 3012684 TI - [Nuclear magnetic resonance tomography of the breast: diagnosis, differential diagnosis, problems and possible solutions. II: Diagnosis]. AB - Eighty-five MR examinations were carried out on both breasts of 72 patients. A special surface coil was used. The results were compared with the findings of mammography and with the post-operative findings. There were 16 fibrocystic mastopathies, seven isolated cysts, 22 fibro-adenomas and 25 carcinomas. Differentiation between fibro-adenomas and carcinomas is only possible on the basis of morphological criteria. Cysts and fibrocystic mastopathies can be identified with certainty. Typical patterns of the various pathological conditions are described, but these will have to be confirmed by the study of a larger number of patients, in order to make them statistically significant. The advantage of MR consists in the simultaneous demonstration of the thoracic wall and the axilla. PMID- 3012685 TI - [Nuclear magnetic resonance tomography of the temporomandibular joint]. AB - Because of its position, the temporomandibular joint is difficult to demonstrate by conventional radiological methods. Even the use of complex methods, such as arthro-tomography or CT, does not result in the satisfactory demonstration of the soft tissues and, in particular, of the articular disc. Magnetic resonance was carried out in 24 patients; it was possible to differentiate functional from morphological changes in the cartilage and these are discussed. Measurements were carried out during progressive opening of the mouth. This permits direct demonstration of reversible and irreversible cartilage displacement and of other changes in the joint and cartilages. PMID- 3012686 TI - [Comparative animal experiment study of aseptic abscesses using MRI: use of Gd DTPA]. AB - Sterile, chemically induced abscesses in the liver, kidney and muscles of 15 rats were investigated by MRI using Gd-DTPA as intravenous contrast media. Before and after administration of Gd-DTPA in a dose of 0.2 mmol/kg, the intensity, T1 and T2 values of both healthy and diseased tissues were recorded. The appearance of the lesions were compared visually. In all three organs the lesions were detectable by MRI without application of contrast media on T2-weighted images. The pattern of contrast enhancement was different in muscle compared to liver and kidney. In muscle typically a ring enhancement pattern was seen, whereas in liver and kidney the lesions appeared homogeneous after contrast media application. We conclude that the application of Gd-DTPA is helpful in defining abscesses in the evaluated organs. PMID- 3012687 TI - [Effect of types of field used in nuclear magnetic resonance tomography on core and surface temperature in the human body. Results of in vitro and in vivo experiments]. AB - Deep and superficial body temperature was measured by in vitro and in vivo experiments, using a fluoro-optic procedure and a variety of magnetic and electromagnetic fields in the course of magnetic resonance tomography. The in vitro experiments had shown that measured temperature changes resulting from a static magnetic field were reversible and could be reproduced readily. Temperature measurements in the human body were carried out centrally (oesophageal and rectal measurements) and at the periphery (intravascular). In vivo experiments on 30 experimental subjects showed no significant changes (p = 0.05) in central or surface temperatures as a result of static or dynamic magnetic fields or electromagnetic high frequency fields. PMID- 3012689 TI - [Simulation of a retroperitoneal mass by a rare anomaly in the path of the ureter]. PMID- 3012688 TI - [Tissue characterization with T1, T2 and proton density: dream and reality]. AB - A survey of the measurement values T1 and T2 based on radiological studies of the last two years shows a high degree of variation in these measurement values with regard to normal and pathological tissues of different organs and regions of the body. T1, which is dependent on field strength, is not suitable for interinstitutional comparison. At present the methods used for T2 measurements are so different from one another that the potential comparability of T2 cannot be realised. The combinations of T1 and T2--as presented in various radiological studies -show signs of tissue characterisation. The expected high degree of selectivity caused by using different measurement methods has not yet been confirmed. The measurement of T1 and T2 in lesions under drug and radiotherapeutic treatment seems to be informative in respect of prognosis. PMID- 3012690 TI - [Unilateral renal duplication with contralateral renal agenesis--a rare angiographic finding]. PMID- 3012691 TI - Pseudodilatation of the ascending aorta as a manifestation of absence of the right superior vena cava. PMID- 3012692 TI - Myelolipoma of the liver. PMID- 3012693 TI - [Sonographic, computed tomographic and nuclear magnetic resonance tomographic detection of thrombosis of the internal jugular vein]. PMID- 3012694 TI - [Comparative imaging of tuberous sclerosis of the brain in the computed tomogram and the nuclear magnetic resonance tomogram]. PMID- 3012695 TI - [Selective blood sampling from the inferior petrosal sinus using digital subtraction angiography]. AB - Simultaneous bilateral venous sampling of blood from the inferior petrosal sinuses helps in the differentiation between peripheral and central ACTH hypersecretion. One can also locate the site of a hormonally active hypophyseal micro-adenoma that cannot be demonstrated by other methods. The authors have experience with fifteen patients and discuss the indications, technique and problems as well as the advantages of using digital subtraction angiography. PMID- 3012697 TI - [Magnetic resonance tomography in the diagnosis of cerebral and spinal masses in children]. AB - The results of the first 71 magnetic resonance studies in 57 children are analysed. Indications, advantages and disadvantages of MR in this age group are described and compared with the results reported in recent literature. PMID- 3012696 TI - [Nuclear magnetic resonance tomography of the spine and spinal cord compared with computed tomography and myelography]. AB - In cases of syringomyelia MR is superior to CT and myelography in visualisation and delineation of the extent of the process. In diagnosing spinal tumours MR is a more sensitive method than CT and myelography. MR provides additional information on sagittal and frontal planes regarding the extent of the tumour. In diagnosis of disc prolapse MR seems to be as accurate as CT or myelography. We obtained additional information in diagnosis of degenerated disc tissue. Spinal stenosis is easily recognisable. CT was superior in differentiation of bony and disc protrusion. The results show that MR has opened up new possibilities in the diagnosis of spinal diseases and will result in a reorientation of the diagnostic approach. PMID- 3012698 TI - [Interstitial pulmonary emphysema in mechanically ventilated newborn infants. Study of the course of the disease]. AB - 106 (15.7%) of 675 artificially ventilated newborn developed interstitial pulmonary emphysema (PIE). Basic lung diseases were: IRDS, neonatal pneumonia, shock lung, meconium aspiration, hypoplasia of the lungs and other miscellaneous disorders of the chest. PIE developed in 68% of patients within 8 hours following artificial respiration. At the beginning of PIE both lungs were concerned in 41.5% of patients, one lobe of both lungs was affected in 32.1%. PIE was located in one lung in 8.5% and in only one lobe in 17.9%. Maximum of PIE was seen within 5 days after initiating respiration in 76.7% of the patients. Persistent PIE developed in 28.7% of the patients. Persistent PIE of both lungs was seen in 11 cases, PIE of one lung in 8 cases and persistent lobar emphysema in another 8. Pulmonary pseudocysts developed in 22 (20.8%) of the patients. PMID- 3012699 TI - [Puncture biopsy of the lung. A retrospective evaluation of 127 observations]. AB - On the grounds of a suspected localised tumorous lesion of the lung core biopsy (Tru-cut-needle) was performed in 127 patients to obtain a histologic diagnosis. This was successful on an average in 71.6% of the cases. The smaller lung lesions (less than 2.5 cm) showed a lower accuracy (65.2%) than the larger tumours (73.1%). Correct judgement of tumour status was effected in 88.2%. But in these cases also correct assessment of tumour status was less successful with the smaller tumours (69.6%) than with the larger ones (92.3%). In our case material core biopsy had a high complication rate: pneumothorax resulted in 54.3%, requiring special treatment in 9.4%. Haemoptisis was observed in 22.9% of which 2.4% had a severe and life-threatening clinical course. A correlation between these complications and the patient's age, the depth of the tumour in the lung and the frequency of the punctures was established. Pleural fluid was seen in 16.5% but in all cases it was negligible and no treatment was required. The pleural reaction showed no correlation to the age, to the tumorous lesion and to the frequency of puncture. The diagnostic advantage and the high accuracy of the core biopsy are evident. Nevertheless, we gave up this method in routine application in our lung tumour patients in favour of needle biopsy techniques. Core biopsy is still being used in examining special problems only. PMID- 3012700 TI - [Rounded atelectasis in the computerized tomographic image]. AB - The rounded (helical) atelectasis is a benign alteration of the lung that can be diagnosed by radiography. Besides the criteria of the chest x-ray film and of the conventional tomogram (shadow close to the pleura, located mainly in the inferior lobe, with "comet-sign" and pleural thickening) the most important finding in computed tomography is the "octopus-sign". PMID- 3012701 TI - [Non-invasive imaging procedures in the diagnosis and control of therapy of varicoceles. 2. Importance of thermography and sonography for therapy control following sclerotherapy of varicoceles]. AB - In 41 patients with varicoceles, a combined thermographic and sonographic examination of the scrotum was carried out pretherapeutically as well as 14 days and 3 months after percutaneous sclerotherapy. In the differentiation of a successful sclerosing or a varicocele persistence, thermography shows a higher sensitivity than sonography with 34 posttherapeutic normal findings and 7 persisting hyperthermias. The sonographic sign of a successful varicosclerosation is the absence of venous dilation of the pampiniform plexus in an upright position with Valsalva's manoeuvre in 36 patients. After proximal sclerosing of the testicular vein sonography reveals a reduction in vascular size; thrombosed veins of the pampiniform plexus are demonstrated only after distal sclerosing. There are no sonographically detectable disorders of the testes after varicosclerosation. Thermographic and sonographic control after percutaneous sclerotherapy or surgical ligation of varicoceles is indicated in children and in doubtful clinical or spermatological cases. PMID- 3012702 TI - [Present role of urethrocystography in the diagnosis of female stress incontinence]. AB - Urethrocystography was performed in 72 women with stress urinary incontinence (SUI). The radiological findings were compared with the clinical diagnosis. 8 Patients with a normal radiological study had SUI grade I by clinical criteria. Explanations of this discrepancy are discussed. In 4 cases the interpretation interfered with a large cystocele. In 60 patients (83.5%) the radiological study confirmed the clinical diagnosis and supported the gynaecologist in the indication for operation on SUI. However this indication cannot be based on the radiological study by itself; it must in fact take into account all the other aspects of the disease. PMID- 3012704 TI - [Correlation of scintigraphic and x-ray findings in Marie-Bamberger secondary hypertrophic osteoarthropathy]. AB - The radiological and scintigraphic findings in the rare condition of hypertrophic osteoarthropathy (Marie-Bamberger) were followed up for a period of two years. They are described and compared. Skeletal scintigraphy shows the abnormalities in the skeleton at an earlier stage than does radiology and also shows more extensive manifestations. The specific symmetrical distribution of abnormally increased uptake in the diaphyses and metaphyses of the long bones of the extremities, with sparing of the axial skeleton, produces highly specific appearances which can be considered as diagnostic. PMID- 3012703 TI - [1.5 Tesla nuclear magnetic resonance tomographic studies of bladder cancer]. AB - MR tomography was performed in 15 patients with urologically prediagnosed carcinoma of the urinary bladder. A field strength of 1.5 Tesla yields excellent morphological resolution of site and contrast. The results are compared with CT and--wherever available--with the pathological anatomic preparations. MR is often superior to x-ray computed tomography in demonstrating polypous carcinomas of the bladder and those producing thickening of the wall, since MR offers the possibility of performing coronary and sagittal cuts. In individual cases, MR can supply definite information on the depth of infiltration into the bladder wall and into perivesical structures; such findings agree with those obtained with cystectomy preparations. The contrast behaviour of the tumours and adjacent structures depends strongly on the measurement parameters employed with the high strength field technique of 1.5 Tesla used in this study. MR echo sequences using different measurement parameters are useful in delineating the tumour contours against adjacent structures such as prostate, seminal vesicles, perivesical fat, urine and to differentiate the tumour from the healthy bladder wall. PMID- 3012705 TI - [Bone marrow scintigraphy using radiocolloids in bone metastases. Sensitivity, specificity, reliability and indications]. AB - The sensitivity and specificity of bone marrow scintigraphy in demonstrating skeletal metastases was examined in 40 patients with focal metastases. Radiology and MDP scintigraphy were used as reference methods. Sensitivity depends on the region of the skeleton. False negatives are the rule in parts of the skeleton containing little bone marrow. In relation to the entire bone marrow content, sensitivity is 0.64. The high proportion of false negatives (36%) in the presence of confirmed metastases and the incomplete demonstration of the bone marrow makes marrow scintigraphy unsuitable as a screening method. Occasionally lesions confined to the marrow can be demonstrated when radiographs and bone scintigrams are still negative. In advanced cases, marrow scintigraphy can demonstrate the extent of destruction of the bone marrow. Demonstration of displacement or of an 'empty bone' is evidence of invasion of the bone marrow in patients with tumours. In patients with reduced haematopoiesis of unknown origin or unidentified diffuse skeletal uptake, bone marrow scintigraphy may provide valuable information. PMID- 3012706 TI - [Optimization of conditions for detecting density variations in focal activity in SPECT using the rotating gamma camera. Model calculations, phantom measurements and clinical applications]. AB - The signal to noise ratio (quotient of contrast and mean noise) is a quality parameter for identifying activity gaps during single photon emission computed tomography (SPECT). Contrast and mean noise level are site-dependent on the reconstruction plane which is influenced in a complex manner by absorption conditions (strong or weak absorption), modes of rotation (full angle or partial angle imaging), interdependencies of opposing partial projections (arithmetic or geometric mean), magnitude of defect (relative to the resolution of the imaging system), geometry of the object to be measured, and scatter. In the present study the authors performed analysis and graphical representation of the SPECT imaging properties on the basis of model calculations illustrated by phantom measurements. The optimal conditions of examination or evaluation and the diagnostic criteria in SPECT of the heart, liver, brain and pelvis are discussed by comparative calculation of signal to noise ratios in defect identification. PMID- 3012708 TI - [Portal circulation following Warren's operation. Long-term control using computerized tomography]. AB - Computed tomography with contrast injection was carried out in 18 patients who had undergone a Warren procedure for portal hypertension due to cirrhosis of the liver more than five years previously. The results show that it is not possible to drain only a part of the venous portal territory. The portal circulation does not consist of two portions, with different pressure relationships. Pressure difference across the splenorenal anastomosis is greater than that into the mediastinal veins. Postoperative development of a hepatofugal circulation continues for a long period and is not confined to the early phase only. This phenomenon is, however, not uniform. In particular, there are variations in the extent of the collateral circulation and in the maintenance of liver blood flow. PMID- 3012707 TI - [Functional heterogeneity of liver metastases in 3-phase computerized tomograms]. AB - Ninety patients with liver metastases (68 colorectal carcinomas, 22 breast carcinomas) were examined by triphasic angio-CT. This included demonstration of the entire liver after a bolus-like injection of contrast. Originally, the metastases were hypodense, but showed four patterns of contrast enhancement. Quantitative evaluation of the mammary carcinomas showed a marked increase in density during the bolus phase, with similar contrast values in the liver and at the centre and edge of the metastasis at ten minutes after the injection. Colorectal carcinomas showed only slight increase in density after contrast injection. The difference in density between the centre and the periphery of the metastasis was still present on later images. This finding indicates that there are differences in the vascularisation of these metastases. PMID- 3012709 TI - [Effect of signal-to-noise ratio and window width on image information in transvenous DSA of different vascular regions]. AB - The diagnostic quality of DSA images depends on numerous factors related to the apparatus and the technique of examination. An improvement in image can be brought about by correct choice of the mask and injected frames, by subsequent correct manipulation of the images and by the choice of the signal-to-noise ratio and window width. In the present study, the effect of these factors was demonstrated on image quality of venous DSA studies in various vascular regions. Practical advice is given for the examination of particular regions and for various diagnostic problems. PMID- 3012710 TI - [Percutaneous transluminal irradiation of biliary tract cancer using iridium]. AB - In ten patients with biliary carcinomas, percutaneous biliary drainage was instituted and intracavitary irradiation performed, using 192iridium with a dose of 40 to 80 Gy. Eight of these patients had been found to be inoperable at laparotomy and two had had a recurrence. In half the patients there was a reduction in the degree of stenosis due to the tumour, but in only one patient was it possible to remove the draining catheter. Survival time in eight patients was between one and eight months, average 5 months. Two patients survived for eleven and twelve months respectively. The relatively poor results are due to extensive metastases. On the other hand, there can be no doubt about the effectiveness of this treatment on locally confined tumours, in view of the reduction in the obstruction caused by the tumour. PMID- 3012711 TI - Intracystic carcinoma of the breast. PMID- 3012712 TI - [Cholecystolithiasis with a double gallbladder]. PMID- 3012713 TI - [Calculus in a bladder diverticulum]. PMID- 3012714 TI - [Portal vein aneurysm. Sonographic and computerized tomographic diagnosis]. PMID- 3012715 TI - [Digital subtraction angiography--a valuable method for detecting osteoid osteoma]. PMID- 3012716 TI - [Hemihyperplasia of thoracic vertebral segments and aggressive fibromatosis]. PMID- 3012717 TI - [A contrast medium reaction with fatal outcome--and its consequences]. PMID- 3012718 TI - Effect of neonatal testosterone administration and beta adrenergic stimulation on adult prolactin levels. PMID- 3012720 TI - [Invasive aspergillar pneumopathy during the treatment of microcellular bronchial cancer. Efficacy of early antifungal treatment]. AB - The invasive aspergillus pneumonias have been described particularly in chemotherapy for patients with haematological disorders. In respiratory disorders such cases are exceptional. The authors report a case of invasive aspergillus pneumonia, occurring during treatment of a small cell cancer; the rapid commencement of anti-fungal treatment by Amphotericin "B" and Flucytosine enabled an apparent cure of the tumour by radiotherapy and chemotherapy. The authors stress the difficulty of definitive diagnostic criteria at the beginning of the disorder and also the need to start anti-fungal treatment as soon as possible. PMID- 3012719 TI - [Na+ K+ ATPase activity in mast cells from the pleural and peritoneal cavities of the rat]. AB - ATPase activity in rat mast cells was studied, assuming that pleural and peritoneal mast cells are different populations. Mast cells were purified with Percoll. Enzymatic activity was found to be 36% higher in pleural cells that in peritoneal cells. Moreover, for both populations results show a Na+-K+ ATPase activity either low or slightly inhibited by ouabain. PMID- 3012721 TI - [Behavior of several parameters in a young population with borderline hypertension. Pathogenetic aspects and prevention]. PMID- 3012722 TI - [Crystal structure of carbonate-bearing hydroxyapatites from Durango, Mexico]. PMID- 3012724 TI - Effects of diethyldithiocarbamate and selected analogs on lead metabolism in mice. AB - Diethyldithiocarbamate (DDTC) and certain of its N,N-disubstituted analogs were evaluated for effectiveness in reducing organ concentrations and whole body burdens of lead (Pb) in mice which had received 0.06 mg of Pb(II) acetate along with 2.0 microCi of 210Pb 9 days earlier. CaNa2EDTA was used as a positive control for studies in which the compounds were administered ip, and meso-2,3 dimercaptosuccinic acid (DMSA) was used as a positive control for po studies. DDTC was more effective than EDTA in lowering the whole body Pb burdens after 5 ip injections, while N-methyl-N-dithiocarboxyglucamine (MDCG) was less active than EDTA. The other 4 analogs tested were inactive. When DDTC (1.5 mmoles/kg) was coadministered with EDTA (1.5 mmoles/kg), the reduction of total body Pb burdens was significantly greater than that attained with either compound alone at 3.0 mmoles/kg. DDTC was particularly effective in reducing hepatic and splenic Pb concentrations, but was less active than EDTA in lowering the Pb content of bone, kidneys, and brain. Excretion of Pb following ip treatment with EDTA, DDTC, or MDCG was predominantly by the fecal route. DDTC was significantly more effective than DMSA at an equimolar dose in reducing whole body Pb burdens when each was given po. However, po administration of DDTC caused a substantial increase in brain Pb levels. PMID- 3012723 TI - [Accumulation of cyclic adenosine monophosphate in the ovary of the eel (Anguilla anguilla L.) in vitro under the effect of carp gonadotropin or ovine lutropin: kinetics and thermodependence]. AB - Cyclic AMP (cAMP) in pieces of eel ovary was greatly increased in vitro by the gonadotropin (cGTH) of carp, another teleost fish; after one hour at 20 degrees C, maximal stimulation = 31.7 and E.D. 50 = 0.08 micrograms/ml. Ovine lutropin (oLH) had less effect (maximal stimulation: 2.35; E.D. 50: 1.42 micrograms/ml); its action suggested that it involved a subfraction (oLH/cGTH RAc) of the receptor-adenylate cyclase (RAc) systems which mediate the action of cGTH. Another difference was the percentage of total cAMP accumulated under hormonal stimulation and released into the incubation medium; this percentage was much higher with oLH than with cGTH (47 vs 8% after one hour at 20 degrees C). This result might be explained by a localization of oLH/cGTH RAc in cells (theca ?) situated on the outside of the follicles and/or by a relative lack of cAMP binding proteins in the case of cAMP produced by oLH/cGTH RAc. Kinetic and thermodependence studies also disclosed hormone-dependent differences; at 5 degrees C, cAMP concentration was maximal after 40 min with oLH, whereas it was still increasing after 3 h with cGTH. Differences in the properties of phosphodiesterases and/or in the clearance rate of hormone-receptor (HR) complexes could explain these results. The set of RAc systems in eel ovary recognizing fish gonadotropin would then be heterogeneous; some of them would be endowed with original properties concerning receptor specificity and cAMP diffusion as well as associated phosphodiesterase activity and/or HR metabolism. We suggest that at a stage of evolution when a single sensu stricto GTH is present (instead of two in tetrapods), "isoreceptors", differing in specificity and in their fate after hormone binding, could be an important element in the fine regulation of gonadal functions. PMID- 3012725 TI - Inhibition of carbon tetrachloride induced hepatotoxicity by dantrolene sodium. AB - A 50 mg/kg dose of dantrolene sodium decreased the hepatotoxicity of carbon tetrachloride in fed and fasted rats, as indicated by lower levels of SGPT following a toxic dose of carbon tetrachloride; however, the dantrolene sodium pretreatment did not inhibit the induction of lipid peroxidation by carbon tetrachloride. The dantrolene sodium did inhibit superoxide production by the hepatic endoplasmic reticulum in fasted rats. Also, the dantrolene sodium inhibited covalent binding of [14C] carbon tetrachloride to the hepatic endoplasmic reticulum in fasted rats, but not in fed rats. PMID- 3012728 TI - Alpha 2-adrenergic receptors on canine platelet membranes characterized by [3H] yohimbine binding. AB - Alpha 2-adrenoceptors on canine platelet membranes were characterized by [3H] yohimbine binding. Binding of [3H]-yohimbine to the membranes was rapidly saturated, stable and reversible with high affinity. Dissociation constant (KD) calculated from Scatchard analysis of the equilibrium experiments was 1.89 +/- 0.20 nM which was in good agreement with KD (1.97 +/- 0.60 nM) obtained in the kinetic experiments. Maximal binding sites of the ligand (Bmax) was 249 +/- 20 fmoles/mg protein. Adrenergic antagonists inhibited the ligand binding in following rank order of potency; yohimbine greater than phentolamine greater than phenoxybenzamine greater than corynanthine greater than prazosin. This order was in good correlation with that for these drugs in the inhibition of platelet aggregation. Hill coefficients for the displacement curves of the ligand binding by adrenergic agonists were less than 1.0 which were converted into near unity in the presence of 5'-guanylyl-imidodiphosphate (100 microM). These results demonstrate that canine platelet membranes have alpha 2-adrenoceptors and suggest that the binding of these receptors to adrenergic agonists is regulated by guanine nucleotides. PMID- 3012726 TI - Binding characteristics of the regulatory guanine nucleotide binding protein, and the activation of the enzyme adenylate cyclase, present in a bovine skeletal muscle membrane preparation. AB - The binding of [3H]GppNHp (beta, gamma-imido[8(-3)H] guanosine 5'-triphosphate) to membrane particles of a bovine skeletal muscle preparation, the m.trapezius, and the subsequent activation of adenylate cyclase in this preparation have been studied, both in the presence and absence of (-)-isoprenaline. Specific binding of [3H]GppNHp could be best explained on the assumption of a two-sites model, in which the binding sites displaying the higher affinity, appeared to be associated with the activation of adenylate cyclase, and are likely to include the regulatory guanine nucleotide binding protein (G/F or NS). The apparent excess of high affinity [3H]GppNHp binding sites over the number of beta-adrenoceptors in this preparation with respect to its possible physiological relevance is discussed. PMID- 3012727 TI - Aminoglycoside toxicity: pH dependent inhibition of ADH response. AB - The effect of aminoglycoside antibiotics on the response of the isolated toad urinary bladder to antidiuretic hormone (ADH) was investigated. Gentamicin and neomycin both acidify the serosal bathing solution and cause a dose-dependent inhibition of the hydroosmotic response to ADH, while streptomycin has minimal effect on media pH and causes no inhibition of the response to ADH. Detailed studies employing gentamicin indicate that acidification stimulates production of PGE2, a known inhibitor of the hydroosmotic response of the toad bladder to ADH. When media pH is rigidly controlled or PGE2 production is inhibited by indomethacin, the inhibitory effect of gentamicin on the response to ADH is ameliorated. These studies suggest that the defect in renal concentrating ability seen as part of aminoglycoside nephrotoxicity could be due, in part, to an acidification-induced, prostaglandin-mediated resistance to the action of ADH. PMID- 3012729 TI - Increase of serum angiotensin-converting enzyme level after exposure to cigarette smoke and nicotine infusion in dogs. AB - Smoking is known to have various effects on metabolic function as well as on the respiratory function in the lung. We report here that exposure to smoke causes a rapid elevation of the level of serum angiotensin-converting enzyme in dogs in vivo. In 7 dogs, the level of serum angiotensin-converting enzyme increased from a basal level of 11.0 +/- 5.0 to 13.9 +/- 4.0 U/ml after 60 min of exposure (mean +/- SD, p less than 0.01). A similar elevation of serum angiotensin-converting enzyme level was noted in dogs that received an intravenous administration of nicotine. The possible mechanism of the change is discussed. PMID- 3012730 TI - Computer simulation of cardiopulmonary resuscitation: computer analysis of a simple electrical model of the circulation. AB - Extensive research is being conducted to study the mechanism of blood flow during cardiopulmonary resuscitation (CPR). Recently, work has been published using a simple electrical model of the circulation to simulate the hemodynamics of CPR. This analog was a hard-wired circuit consisting of the heart and great vessels modeled as a resistive-capacitive network, pressure as voltage, blood flow as current, blood inertia as inductance and vascular valves as diodes. Such a model is useful for examining the physiology of various methods and techniques of CPR administration. In this investigation, a general purpose circuit simulation program, SPICE Version 2G.6, was used to analyze previously published CPR models. With minor modifications, the program was fully able to simulate the hard-wired circuits. The program is very flexible, allowing for easy model modification and a wide range of parameter values. In addition, the program offers the advantages of increased accuracy and low cost. Suggested future applications are for rapid evaluation of new CPR concepts. PMID- 3012731 TI - Lethal adult respiratory distress syndrome after meningococcal septicemia biochemical markers in bronchoalveolar lavage. AB - A lethal case of Adult Respiratory Distress Syndrome (ARDS) consequent to meningococcal septicemia is clinically and physiologically described. Very high levels of eosinophil cationic protein and lactoferrin in bronchoalveolar lavage were observed in spite of peripheral eosinopenia and neutropenia. These findings provide support for the hypothesis that activated granulocytes are involved in the pathogenesis of septic-induced ARDS. PMID- 3012733 TI - Fluid resuscitation of hypotensive emergency patients with and without an algorithm. AB - Seventy-seven consecutive hypotensive (mean arterial pressure (MAP) less than 80 mmHg) surgical emergency patients were resuscitated according to either physicians' individual orders (38 patients) or an algorithm (39 patients). The shock was mainly caused by accidental injuries or acute gastrointestinal bleeding. The patients of the algorithm group were given more plasma expanders than the patients of the control group, while the total amount of fluids administered was similar in both groups. The primary goal of the resuscitation (MAP greater than 80 mmHg) was reached within 30 min in three cases in the control group and in seven cases in the algorithm group. The treatment times at the emergency department and the intensive care unit were similar for the groups. The number of severe and moderate pulmonary disturbances was the same, but mild disturbances were significantly more common in the control group. Renal failure was somewhat more common in the control group and the renal function disturbances were significantly more severe among the control patients. The results suggest that the physicians in some extent altered their practices in fluid resuscitation when the algorithm was put to use, and that this change, perhaps, produced the somewhat better outcome of the patients. The authors recommend the algorithm to be used as a basis of shock treatment and particularly in those emergency departments where the resuscitation of hypotensive patients is performed by junior or inexperienced physicians. PMID- 3012732 TI - The effect of carbon dioxide, lidoflazine and deferoxamine upon long term survival following cardiorespiratory arrest in rats. AB - This study examined the effect of carbon dioxide, lidoflazine and deferoxamine therapy upon the 10-day survival incidence and subsequent neurologic function of rats subjected to 7 min of cardiorespiratory arrest with resuscitation. Cardiac arrest (asystole) was induced at time zero by injection of cold, 1% KCl into the left ventricle of ketamine-anesthetized rats pretreated with succinylcholine. Positive pressure ventilation was discontinued at time zero. Cardiopulmonary resuscitation (CPR) was begun at 7 min, and animals with return of spontaneous circulation were entered into the study. Twenty treated rats were ventilated for 1 h with 7% carbon dioxide-93% oxygen and given lidoflazine (2.0 mg/kg, i.v.) and deferoxamine (50 mg/kg, i.v.) 5 min after CPR. Twenty control rats were ventilated for 1 h with 100% oxygen and given lidoflazine vehicle and deferoxamine vehicle. Lidoflazine treatment (1.0 mg/kg) for the treated group, or lidoflazine vehicle for the control group, was repeated at 8 h postresuscitation. At 2 days postresuscitation, 75% of treated rats vs. 25% of control rats were alive (CHI2 = 10.0, d.f. = 1, P less than 0.01), and at 10 days, 60% of treated rats vs. 25% of control rats were alive (CHI2 = 5.01, d.f. = 1, P less than 0.05). There was no detectable neurologic deficit among survivors in either group at 15 days. The combination of carbon dioxide, lidoflazine and deferoxamine, administered after return of spontaneous circulation, is a simple and easily administered treatment regimen that improves the survival incidence without neurologic deficits in this animal model of cardiorespiratory arrest and CPR. PMID- 3012734 TI - Early use of naloxone in shock--a clinical trial. AB - Naloxone hydrochloride (N) 0.4-1.2 mg i.v. was administered during 10 episodes of shock (8 septic and 2 cardiogenic) in 9 adult patients. Shock was defined as systolic blood pressure (SBP) less than or equal to 90 mmHg and urine output less than 0.5 ml/h and signs and symptoms of hypoperfusion lasting for greater than or equal to 30 min, despite fluid loading to a CVP 5 cmH2O above baseline. N was given as early as 30 min after onset of shock and resulted in an increase of SBP from a mean of 75 +/- 10 to a mean of 130 +/- 25 mmHg maximum (P less than 0.01). Within 10-60 min urine output increased from 16 +/- 12 to 122 +/- 56 ml/h, heart rate, CVP and arterial blood gas tensions remained unchanged. No side effects were observed. Naloxone, even in small doses, may improve hemodynamic parameters in human shock, provided it is administered very early. PMID- 3012735 TI - Repetitive visual images in severe war head injuries. AB - Out of 20 young inpatients who suffered missile head injuries, two (10%) presented repetitive visual images (RVI). The RVI appeared during wakefulness when relaxed with closed eyes and also at sleep stage I, II and REM, at sleep onset and at sleep end. When the two patients were compared neurologically and psychologically with the other eighteen, the coexistence of combat stress syndrome, diffuse brain lesions, right non-dominant associative area damage and homonymous hemianopia characterised the two RVI patients. RVI do not appear in the absence of the combat stress syndrome even in the presence of the other 3 factors. The sleep may be secondarily contaminated by wakefulness RVI. The presence of disturbed REM temporal distribution and short REM latency indicate the depressive state of these patients. It is hypothesised that a similar brain activity in wakefulness and REM sleep explain the wake dreaming or wakefulness RVI of those patients. PMID- 3012736 TI - Death in the emergency room: Beijing, China. PMID- 3012737 TI - [Leukocyte proteases and the lung]. PMID- 3012738 TI - [Atrial natriuretic polypeptide]. PMID- 3012739 TI - [Incidence of recurrence in pleomorphic adenoma]. PMID- 3012740 TI - [Diffuse pulmonary fibrosis: behavior and responsibility of the collagen in its pathogenesis]. PMID- 3012742 TI - Use of Tc-99m DMSA and Tc-99m DTPA in reflux. PMID- 3012741 TI - [Small-cell bronchial anaplastic cancer (I)]. PMID- 3012743 TI - [Spherocytosis complicated by transient red-cell aplasia. Role of a parvoviral etiology]. PMID- 3012744 TI - [Nutrition and cancer]. PMID- 3012745 TI - [Activation of latent infections caused by the Epstein-Barr virus in systemic lupus erythematosus]. PMID- 3012746 TI - [Anaplastic small-cell bronchial carcinoma of the composite type]. AB - The authors report an anatomico-clinical case (with ultrastructural study) of bronchial anaplastic carcinoma with composite, triple differentiation oat-cell subtype. The patient underwent early surgical excision followed by radiotherapy and chemotherapy. With the help of data from the literature concerning this composite subtype which suggests new theories on the histogenesis of bronchial cancer, the authors emphasize the value of surgical treatment for this unusual subtype of anaplastic carcinoma. PMID- 3012748 TI - [Current status and outlook of the tuberculosis problem in Romania]. PMID- 3012747 TI - [Surgery of microcellular bronchial cancer: retrospective multicenter study apropos of 110 cases]. AB - The results of a multicentric retrospective study of 110 cases of small cell bronchial cancer are reported. In 57 of these patients the histological diagnosis was unknown before surgery. Among the remaining 53 patients, 22 were operated upon immediately and 31 after chemo-and/or radiotherapy (12 full responders, 10 partial responders, 5 no change and 4 in relapse). Operative data were as follows: 100 excisions and 10 exploratory thoracotomies; 19 perioperative complications, including 12 deaths; excision considered complete in 78 cases; pericardial involvement in 14 cases; invasion of the hilar lymph nodes in 57 cases, of the mediastinal lymph nodes in 39 cases; positive bronchial section in 16 cases. Overall median survival was 13.8 months for all patients and 18.3 months (perioperative deaths excluded) for patients whose tumour had been excised. At the moment, 46 patients have relapsed with recurrence at the initial site of malignancy alone in 6 cases (13%) and both at this site and at one or several metastatic sites in 10 cases (21.7%). Nineteen patients have survived for more than 2 years. An analysis of the subgroups in this population showed that the longest survivals were obtained in patients who had undergone preoperative chemotherapy. PMID- 3012749 TI - [Current bacteriological diagnosis of tuberculosis. I. The efficiency of bacteriological methods in identifying sources of infection]. PMID- 3012750 TI - [Acquired immunodeficiency syndrome (AIDS)--a new pathology involving the respiratory system]. PMID- 3012752 TI - [Tuberculosis among the teaching personnel and students at the Central University of Cluj-Napoca (1926-1983)]. PMID- 3012751 TI - [Cardiotonic and diuretic medication in the treatment of chronic cor pulmonale]. PMID- 3012753 TI - [Results of combining polychemotherapy including Riaval with surgical treatment of bronchopulmonary cancer]. PMID- 3012754 TI - [Clinical aspects of pulmonary aspergilloma (31 cases)]. PMID- 3012755 TI - [Cases of tuberculosis of the respiratory system in patients over 60]. PMID- 3012757 TI - [Post-caustic esophageal stricture and consecutive disease states: bacilliferous pulmonary tuberculosis, esophagotracheal fistula, secondary amenorrhea (a case report)]. PMID- 3012756 TI - [Tuberculous meningoencephalitis occurring in an adult with cold pulmonary miliary tuberculosis]. PMID- 3012758 TI - Experimental models for the sequential analysis of chemically-induced renal carcinogenesis. AB - In order to discriminate non-specific toxicity from the early precursor lesions of neoplasia, emphasis in these studies has been on the use of models requiring only a single administration of chemical. Our interests have focussed on three neoplastic entities in the kidney, renal mesenchymal neoplasia, renal cell carcinoma, and nephroblastoma. Dimethylnitrosamine administered as a single intraperitoneal injection to immature female Wistar rats, pre-conditioned for several days with a no-protein/sugar-only diet, has been used for investigating the complex morphological nature of renal mesenchymal tumors, their pathogenesis and the development of cell culture correlates. The near 100% tumor incidence and its facility for cell culture manipulation makes this a particularly potent model for studying chemical carcinogenesis and the evolution of cell transformation. Discovery that the rat kidney response to DMN was biphasic with respect to the time of treatment led to the subsequent development of a high incidence system for inducing renal adenocarcinoma, using older rats. Renal cell carcinomas could also be induced in mice by a single intravenous injection of streptozotocin. The tumor frequency in female CBA/H/T6J mice was almost 100%, providing a new model for the investigation of renal carcinogenesis in this species. Nephroblastoma has been a poorly comprehended neoplasm in both lower animals and man because of the lack of a high incidence model in conventional laboratory mammals. Recently, we have exploited an increased spontaneous predisposition of the Nb rat to nephroblastoma using a single intraperitoneal dose of N-ethylnitrosourea in pregnant females on day 18 of gestation, producing a frequency of 50% for this tumor type. More potent however, was a system which utilized the partially inbred IIIVO/J strain of rabbit using the same carcinogen and transplacental route of administration. The resultant incidence of nephroblastomas in the progeny was in excess of 90%, and like their counterparts in man, the neoplasms developed rapidly and had a potential for distant metastasis. Each one of these animal models is suitable for the sequential tracing of tumor pathogenesis, and in depth analysis of the biochemical and molecular mechanisms involved in the initiation and formation of different types of renal cancer. PMID- 3012759 TI - Lymphocyte sodium efflux in normotensive and borderline hypertensive subjects with and without heredity for hypertension. PMID- 3012761 TI - Vascular effects of altered sodium, potassium pump activity. PMID- 3012760 TI - The sodium, potassium-pump. PMID- 3012762 TI - Sodium transport in vascular smooth muscle cells from resistance vessels. PMID- 3012763 TI - Membrane transport of ions in hypertension: a review. PMID- 3012764 TI - Evidence for a circulating Na,K-pump inhibitor in essential hypertension. PMID- 3012765 TI - Cell membrane disturbance and the pathogenesis of essential hypertension: several hypotheses. PMID- 3012766 TI - Role of exogenous factors in alterations of red cell Na+-Li+ exchange and Na+-K+ cotransport in essential hypertension, primary hyperaldosteronism, and hypokalemia. PMID- 3012768 TI - Inhibitory action of omeprazole on acid formation in gastric glands and on H+,K+ ATPase isolated from human gastric mucosa. AB - The inhibitory effect of omeprazole on acid formation has been studied in vitro in gastric glands and partly purified H+,K+-ATPase, prepared from mucosa obtained either from healthy subjects by gastroscopic biopsy or from gastric ulcer patients during antrectomy. The effect of omeprazole was compared with the inhibitory pattern of the H2-antagonist cimetidine. Acid production in the glands was determined by measuring the accumulation of 14C-aminopyrine. In glands isolated from patients, omeprazole inhibited acid production maximally stimulated by histamine, db-cAMP, and potassium in a dose-dependent manner, with an IC50 value of about 50 nM irrespective of the agonist used. In contrast, cimetidine inhibited only histamine-induced aminopyrine accumulation, with an IC50 of about 30 micron. The inhibitory effect of omeprazole in db-cAMP-stimulated glands from healthy volunteers was of the same magnitude as seen in glands from gastric ulcer patients. Basal aminopyrine accumulation in glands from both patients and healthy volunteers was almost totally inhibited by omeprazole, whereas cimetidine was without effect. Omeprazole also concentration-dependently inhibited the H+,K+ ATPase activity in isolated gastric membrane vesicles. The estimated IC50 value was 4 micron. PMID- 3012767 TI - Epidemiology of polyps in the rectum and sigmoid colon. Evaluation of nutritional factors. AB - Epidemiological studies have suggested an association between diet and colorectal cancer. Case/control studies, however, have been scarce, and studies based on interview with cancer patients who have symptoms from their cancer are inevitably prone to bias. An endoscopic population screening study for detection of colorectal adenomas enabled a double-blind registration of diet during 5 consecutive weekdays. Neither the participant nor the dietitian was informed of the findings at endoscopy. The estimation of 23 nutritional components was based on analysis of local commercial food and on the composition of foods in Norway. Results showed increasing consumption of fat and decreasing consumption of fiber and cruciferous vegetables in the presence of increasing neoplastic changes. The present material will form the basis for dietary-related follow-up studies. PMID- 3012769 TI - Serum levels of lactoferrin and myeloperoxidase in chronic idiopathic and secondary neutropenia. A preliminary report. AB - In 20 patients with chronic neutropenia, serum lactoferrin (S-LF) and serum myeloperoxidase (S-MPO) levels were assessed. By immunofluorescence, granulocyte bound immunoglobulins were detected in 12 patients, whereas circulating immune complexes were found in the blood of 8 patients by the 125-I-C1q-binding test (C1q-BT). In both groups of patients, there was a relative increase of S-LF and a relative or sometimes absolute increase of S-MPO. In the latter group, results of the C1q-BT correlated positively with S-MPO but negatively with neutrophil counts. No correlations between S-LF or S-MPO and the results of the granulocyte immunofluorescence test were found. Our results suggest that S-LF and S-MPO levels may be helpful in the further study of patients with chronic neutropenia, to gain more insight into the pathogenetic mechanisms operative in this disease. PMID- 3012771 TI - [Clinical aspects of cytomegalovirus infection in nonimmunosuppressed adults]. AB - The histological changes in cytomegalovirus (CMV) infection were first described by RIBBERT in 1881, and for years the virus was dreaded as the agent of infection in newborns. An infectious mononucleosis-like disease with negative heterophil antibodies in otherwise healthy adults was described in 1965. We present six previously healthy adults with CMV mononucleosis observed in 1984. The diagnosis was established by CMV-IgM-ELISA. All patients were febrile for an average of 20 days. The general state of health was reduced in three patients; one patient suffered from headache and another from abdominal pain. Physical examination showed splenomegaly and mild tonsillitis in one patient each, but in no case lymphadenopathy. All patients had lymhocytosis with reactive forms (virocytes). Elevation of transaminases was seen in four cases. Compared to Epstein-Barr virus mononucleosis, fever in CMV mononucleosis lasts significantly longer and lymphadenopathy is evidently rarer. The combination of fever of unknown origin, a negative heterophil antibody titer and the presence of virocytes prompts suspicion of CMV mononucleosis. PMID- 3012770 TI - Cell-mediated immune response in the diseased joints in patients with reactive arthritis. AB - To evaluate the level of lymphocyte activation in reactive and rheumatoid arthritis, density gradient-isolated, synovial fluid mononuclear cells were stained with a panel of antisera directed at lymphocyte activation markers using an avidin-biotin-peroxidase complex (ABC) method. More specifically, we studied the expression of immune response-associated class II HLA antigen (Ia), of receptors for interleukin 2 (Tac) and transferrin (T9), as well as of gp 40/80 glycoprotein (4F2). Although Ia+ cells formed about 60% of all the synovial fluid mononuclear cells in both disease conditions, the proportion of Tac+ (33 +/- 4% vs 3 +/- 1%, P less than 0.001), T9+ (34 +/- 4% vs 5 +/- 2%, P less than 0.001), and 4F2+ (48 +/- 6% vs 3 +/- 2%, P less than 0.001) cells was high only in reactive arthritis. All the patients who had reactive arthritis followed a favourable clinical course during the 4-month-long prospective follow-up, whereas disease activity was stable in patients with rheumatoid arthritis. These findings suggest that the diseased joints in reactive arthritis are a site for an active, but normally down-regulated, cell-mediated immune response. PMID- 3012772 TI - [Viral respiratory infections in children: new diagnostic methods for early detection. Initial results of a pilot project in Switzerland]. AB - The new methods of rapid viral diagnosis make it possible to specify a number of the most prevalent agents of respiratory tract infections within 24 hours. The techniques are based on the immunological detection of antigens of respiratory syncytial (RSV), adeno, parainfluenza type 1, 2 and 3, as well as of influenza A and B viruses in nasopharyngeal secretions. During a one-year period we have used these methods to evaluate diagnostically 1541 outpatients presenting with upper and lower respiratory tract infections. The patients included babies, infants and children under 16. In about 50% of all sick babies below the age of three months a definite viral infection could be established, and in approximately 30% of infants and children aged up to 4 years. RSV was most frequently observed, accounting for 53.6% of all infections (80% of all babies below the age of 3 months, in whom specified agents could be identified, had RSV infection). The next most frequent pathogens were parainfluenza type 3 (18,8%), influenza A (11,3%) and, finally, adenoviruses (10.2%). The epidemiological and clinical characteristics of these infections are summarized. In addition, the results of these antigen detecting assays have been compared with those of concomitantly conducted virus isolation techniques in cell cultures. This comparative analysis most impressively revealed the time saved by attempting an etiological diagnosis using the antigen detecting system: in only 6% was a specific diagnosis established on the basis of virus isolation, whereas the delay was equal or more than 8 days in 36% of all patients enroled.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012773 TI - [Serologic overview study of the transmission of bovine coronavirus in Switzerland]. PMID- 3012775 TI - Identification of a missense mutation in the factor VIII gene of a mild hemophiliac. AB - DNA probes derived from the cloned factor VIII gene can be used to detect mutations in the factor VIII gene of hemophiliacs. DNA hybridization analysis led to the identification of two contrasting point mutations in the same codon. In a severe hemophiliac with no detectable factor VIII activity, the normal arginine codon (number 2307) is converted to a stop codon, while in a mild hemophiliac with 10 percent of normal activity, this same codon is converted to glutamine. PMID- 3012774 TI - Binding of the Sp1 transcription factor by the human Harvey ras1 proto-oncogene promoter. AB - Members of the ras gene family encode proteins that when overproduced or mutated can transform immortalized mammalian cells. It is therefore important to understand the mechanisms by which the ras genes are regulated. The promoter region of the human Harvey ras proto-oncogene c-Ha-ras1 initiates RNA transcription at multiple sites and contains repeated copies of the hexanucleotide GGGCGG and its inverted complement CCGCCC, referred to as GC boxes. These GC boxes consist of sequences identical to those found in the SV40 early promoter, where the human cellular transcriptional factor Sp1 binds. Footprinting analysis with deoxyribonuclease I was used to show that Sp1 binds to six GC box sequences within the c-Ha-ras1 promoter. An in vivo transfection assay showed competition between the 21-base pair repeats of the SV40 promoter and the c-Ha-ras1 promoter for common regulatory factors. In this system the presence of Sp1 is apparently required for c-Ha-ras1 transcription. Analysis of deletions of the c-Ha-ras1 promoter region by means of a transient expression assay revealed that the three Sp1 binding sites closest to the RNA start sites were sufficient for full transcriptional activity. PMID- 3012776 TI - Human immunodeficiency virus. PMID- 3012777 TI - Ground water ills: many diagnoses, few remedies. PMID- 3012778 TI - Genetic variation in HTLV-III/LAV over time in patients with AIDS or at risk for AIDS. AB - In a study of genetic variation in the AIDS virus, HTLV-III/LAV, sequential virus isolates from persistently infected individuals were examined by Southern blot genomic analysis, molecular cloning, and nucleotide sequencing. Four to six virus isolates were obtained from each of three individuals over a 1-year or 2-year period. Changes were detected throughout the viral genomes and consisted of isolated and clustered nucleotide point mutations as well as short deletions or insertions. Results from genomic restriction mapping and nucleotide sequence comparisons indicated that viruses isolated sequentially had evolved in parallel from a common progenitor virus. The rate of evolution of HTLV-III/LAV was estimated to be at least 10(-3) nucleotide substitutions per site per year for the env gene and 10(-4) for the gag gene, values a millionfold greater than for most DNA genomes. Despite this relatively rapid rate of sequence divergence, virus isolates from any one patient were all much more related to each other than to viruses from other individuals. In view of the substantial heterogeneity among most independent HTLV-III/LAV isolates, the repeated isolation from a given individual of only highly related viruses raises the possibility that some type of interference mechanism may prevent simultaneous infection by more than one major genotypic form of the virus. PMID- 3012779 TI - Calcium modulation activates Epstein-Barr virus genome in latently infected cells. AB - In many viral infections the host cell carries the viral genome without producing viral particles, a phenomenon known as viral latency. The cellular mechanisms by which viral latency is maintained or viral replication is induced are not known. The modulation of intracellular calcium concentrations by calcium ionophores induced Epstein-Barr viral antigens in lymphoblastoid cell lines that carry the virus. When calcium ionophores were used in conjunction with direct activators of protein kinase C (12-O-tetradecanoyl phorbol-13-acetate and a synthetic diacylglycerol), a greater induction of viral antigens was observed than with either agent alone. Activation of protein kinase C may be required for the expression of the viral genome. PMID- 3012780 TI - Viral shedding and transmission between hosts determined by reovirus L2 gene. AB - Two reovirus isolates (type 1 Lang and type 3 Dearing) differ in their transmissibility between littermates of newborn mice. They also differ in the amounts of virus excreted by the gastrointestinal tract. With the use of reassortant viruses, these properties were mapped to the L2 gene. Thus environmental spread of reovirus is a genetic property. PMID- 3012781 TI - The product of the human c-erbB-2 gene: a 185-kilodalton glycoprotein with tyrosine kinase activity. AB - Antibodies were raised against a synthetic peptide corresponding to 14 amino acid residues at the COOH-terminus of a protein deduced from the human c-erbB-2 nucleotide sequence. These antibodies immunoprecipitated a 185-kilodalton glycoprotein from MKN-7 adenocarcinoma cells. Incubation of the immunoprecipitates with (gamma-32P)ATP resulted in the phosphorylation of this protein on tyrosine residues. These results indicate that the human c-erbB-2 gene product is the 185-kilodalton glycoprotein that is associated with tyrosine kinase activity. Although the c-erbB-2 protein was predicted to encode a protein very similar to epidermal growth factor (EGF) receptor, EGF did not stimulate this kinase activity either in vivo or in vitro. PMID- 3012783 TI - Hepatitis A virus: clones, cultures, and vaccines. PMID- 3012782 TI - Evolving concepts of the clinical and serologic consequences of hepatitis B virus infection. AB - Measurement of conventional markers has provided a general understanding of HBV infection, and conceptual advances in knowledge of the clinical and serologic events associated with persistent HBV infection have resulted from the development and application of new methodologies. These studies suggest that persistent HBV infection is a dynamic process, with replicative and nonreplicative phases, that nonreplicative infection may be the precursor of PHC, and that the natural history of persistent infection can be modified by a variety of factors, including viral reactivation and viral superinfections. Further understanding of these phenomena may lead to the development of new therapeutic approaches. PMID- 3012784 TI - Non-A, non-B hepatitis: evolving epidemiologic and clinical perspective. AB - Evidence for the existence of human hepatitis agents besides HAV and HBV is compelling. Transmitted predominantly by transfusion and percutaneous inoculation, the type of NANB hepatitis encountered most frequently is epidemiologically similar to type B hepatitis. NANB hepatitis accounts for more than 90% of TAH, but can be transmitted by nonpercutaneous routes as well. Approximately 15 to 30% of sporadic hepatitis cases are attributable by serologic exclusion to NANB hepatitis agents, and, in addition, there is an epidemic form of NANB hepatitis that resembles hepatitis A epidemiologically in its transmission by the enteric route. Clinical features of the predominantly percutaneously transmitted forms of NANB hepatitis are similar to those of hepatitis B, but tend to be less severe during acute illness, on the one hand, but to lead more frequently to chronic hepatitis, on the other; 40 to 60% of patients with TAH have chronic elevations of aminotransferase activity, often in an episodic, fluctuating pattern. CAH can be identified histologically in a majority of patients with chronic NANB TAH. Despite a relatively quiescent course, progression of such chronic cases may be quite insidious; cirrhosis occurs in 10 to 20% of patients with chronic hepatitis after acute TAH. The frequency of chronic liver disease after nonpercutaneously acquired sporadic NANB hepatitis tends to be lower, on the order of 10% or less, and chronic hepatitis has not been recorded after the epidemic type of NANB hepatitis. Evidence supports the existence of an asymptomatic chronic NANB hepatitis carrier state that is several-fold more frequent than the chronic HBV carrier state. Neither viruses nor virus markers have been described that fulfill accepted criteria reproducibly for a specific causal association with NANB hepatitis; on the other hand, evidence suggests (but does not prove) the existence of two different blood borne NANB hepatitis agents and, in addition, an enterically transmitted NANB hepatitis agent. Effective therapy for and immunoprophylaxis against NANB hepatitis are lacking. Until specific screening tests are developed, interim screening based on indirect, nonvirus-specific tests may be the only practical approach to minimizing the frequency of NANB hepatitis after transfusion. Identification of virus-specific serologic markers remains a high priority. PMID- 3012785 TI - Cytomegalovirus infection as a complication of blood transfusion. PMID- 3012786 TI - Design and application of photolabile intracellular probes. PMID- 3012787 TI - Intrauterine fetal demise associated with enterovirus infection. AB - We have presented a case of fetal intrauterine enterovirus (coxsackievirus A) infection at 36 weeks' gestation. Viral infection should be suspected when there is a history compatible with viral illness in the mother during pregnancy. Although an increased level of IgM antibodies in cord blood suggests a previous intrauterine infection, the diagnosis of fetal or neonatal viral infection ultimately depends upon the isolation of the viral agent. PMID- 3012788 TI - Extramammary Paget's disease of the scrotum: need for early biopsy. AB - We have described a patient with Paget's disease of the scrotum, with a two-year history of erythematous and indurated skin lesion. Biopsy established the diagnosis of Paget's disease. The patient's symptoms were relieved after wide excision of the affected scrotal skin. PMID- 3012789 TI - Epidemiology of liver cancer in Indonesia. AB - Hepatocellular carcinoma (HCC) in Indonesia ranks as the 9th most common of cancers. The ratio of male and female in HCC 2.5:1. HCC were found mostly in the 4th to 7th decade of life. HCC is frequently accompanied with cirrhosis and close relationship with HBV. The positivity of HBV marker is 60% in CLD and 67% in HCC. The patients usually come late to the doctors, and early detection is still a problem. Ultrasonography every 4 months and serologic test every month in CLD is recommended. Ultrasonically guided fine needle aspiration biopsy is quite promising for diagnosis and early detection of HCC. PMID- 3012790 TI - Chemo-radiotherapy in nasopharyngeal carcinoma at Ramathibodi Hospital, Bangkok. AB - Combined chemotherapy (Cis-platinum and 5 FU) and radiation therapy were given to 11 patients with stage IV (except 1) nasopharyngeal carcinoma. None had distant metastasis. Mean duration of follow-up was 16.2 months. Objective response (CR+PR) at the primary lesion were 10 out of 11 (90.9%), while CR was 7 out of 11 (63.6%). CR+PR at the regional node were 10 out of 10 (100%), while CR was 9 out of 10 (90%). There has been no recurrence so far. One patient died of hepatocellular carcinoma. Side effects, mainly from radiation therapy, were clinically acceptable. One had transient cervical myelitis, which improved after medical treatment. There was no significant myelosuppression. PMID- 3012791 TI - Thai experience in lung cancer treatment. PMID- 3012792 TI - Chemotherapy for trophoblastic neoplasms in the Philippines. AB - This paper presents a protocol currently used in the diagnosis and management of trophoblastic disease, amongst participating hospitals within the Greater Manila Area, Philippines. Using the protocol as a general guide, the results of the study of several authors were presented. The commonly used chemotherapeutic agent are methotrexate and actinomycin D depending upon local availability and price affordability. Prophylactic chemotherapy is resorted to, in view of the inconsistent monitoring of the HCG and patients either in danger of being lost to follow-up or actually fail to report for follow up. The results of the studies generally point to the facts that: trophoblastic neoplasms is indeed a problem amongst Filipino women; the success of chemotherapy is dependent upon early diagnosis, availability of a more sensitive HCG monitoring system; a wide selection of available and affordable chemotherapeutic agents and above all faithful patient compliance. PMID- 3012794 TI - [Neurological masking of hypoglycemia]. PMID- 3012793 TI - Wilms' tumor: analysis of thirty cases. AB - Thirty cases of Wilms' tumor who had been treated at the Ramathibodi Hospital from January 1970 to December 1982 were analysed retrospectively. There were 14 boys and 16 girls, aged 6 months to 7 years (mean age was 2 years). The right kidney was involved in 12 cases, the left side involvement in 18 cases. Other than the abdominal mass, the common signs and symptoms were hematuria (30%) and hypertension (13.3%). The congenital anomalies were found in 2 cases. There was an increase in VMA in three of the six cases determined for VMA:creatine ratio and VMA in 24 hours urine. Seven cases (23.3%) had nephrectomy done in other hospitals. Ninety percent of the patients came in with the stage II-IV, only 10% had stage I. The treatment consisted of surgery, radiation therapy, actinomycin D, vincristine and adriamycin. Eight patients (26.6%) were lost to follow-up. The cure rate in stage II, III and IV were 71.4%, 50% and 29% respectively. The serious surgical complications include a case of shock due to excessive bleeding and another case of sudden death during the operation due to the tumor emboli from the inferior vena cava to the main pulmonary and both bronchial arteries. PMID- 3012795 TI - [Bactericidal components of neutrophil peroxidasosomes in abdominal typhus]. PMID- 3012796 TI - [Etiology and pathogenesis of lymphogranulomatosis]. PMID- 3012797 TI - Human apolipoprotein B: chromosomal mapping and DNA polymorphisms of hepatic and intestinal species. AB - Apolipoprotein B (apoB) is the major protein component of low-density and very low-density lipoproteins. We have recently isolated nonoverlapping cDNA clones for apoB and confirmed their identity by sequence comparisons. We now report the mapping of the human apoB gene (APOB) to the p23-p24 region of chromosome 2 by examination of human-mouse somatic cell hybrids and by in situ hybridization to human chromosomes. Thus, APOB is unlinked to members of the dispersed gene family encoding other apolipoprotein species or to the gene encoding the low-density lipoprotein receptor. Hybridization analysis with genomic DNA and liver and intestinal mRNA suggests that APOB encodes both the high-molecular-weight form of apoB (apoB100) incorporated into very-low-density lipoproteins in liver and the lower-molecular-weight form (apoB48) incorporated into chylomicrons in intestine. Restriction fragment length polymorphisms of APOB have been identified and should prove useful in examining the possibility that genetic variations of APOB are involved in dyslipoproteinemias and atherosclerosis. PMID- 3012798 TI - Somatic mutations at a heterozygous autosomal locus in human cells occur more frequently by allele loss than by intragenic structural alterations. AB - A human B-cell lymphoblastoid cell line heterozygous at the thymidine kinase (TK) locus (i.e., carrying one functional and one nonfunctional thymidine kinase allele) was used to study the molecular nature of mutations leading to loss of TK activity. A total of 113 mutant clones, both spontaneous and induced, were examined by restriction enzyme mapping and by the use of a restriction fragment length polymorphism (RFLP) at the TK locus. A majority (71%) of all mutant clones examined had lost the entire functional TK allele, becoming either homozygous or hemizygous for the nonfunctional allele. The remaining mutants had either no detectable changes (26%) or had obvious structural alterations (less than 5%) in the active TK gene. These results emphasize the importance of allele loss, presumably by mitotic chromosomal mechanisms, in mutagenesis at autosomal loci, and suggest that in vitro models for recessive somatic mutation which are based at hemizygous loci may ignore a large category of genetically significant events. PMID- 3012800 TI - Human liver fatty acid binding protein gene is located on chromosome 2. AB - The human gene for liver fatty acid binding protein was assigned to chromosome 2 using human-rodent somatic cell hybrids and Southern filter hybridization of cell hybrid DNA. The filters were hybridized to a radiolabeled cloned cDNA which covers 485 bases. Hybridization of the DNA to a human-specific 7.5-kb DNA fragment in HindIII-digested DNA was used to identify the human liver fatty acid binding protein gene. Results from two cell hybrids with chromosomal rearrangements suggest that the gene is on the short arm of chromosome 2. PMID- 3012799 TI - Immunological studies with different classes of mutants affected at the adenosine kinase locus in CHO cells. AB - Adenosine kinase (AK) from CHO cells has been purified to homogeneity and specific antibodies to it have been raised in rabbits. Using this antibody, the presence of a specific cross-reacting protein (CRP) in cell extracts of different classes of mutants resistant to purine nucleoside analogs which are affected in AK has been investigated by the immunoblotting technique. Results of our studies show that 31 of the 32 independently selected class A AK- mutants (obtained at high frequency in presence of adenosine analogs toyocamycin, tubercidin, 6 methylmercaptopurine riboside, or pyrazofurin and containing no measurable activity of AK in cell extracts) contained similar amounts of a specific CRP as seen in the parental AK+ cells. The CRP in the parental and different mutant cell lines has the same relative molecular mass as purified AK. Similar results were obtained with two mutants each of the class B and C type (selected in presence of C-nucleosides formycin A and formycin B), which are also affected in AK but show novel properties. The presence of equivalent amounts of the CRP in the vast majority of the class A mutants strongly indicates that the high frequency of those mutants in CHO cells is not a result of an epigenetic or deletion type of event, but that such mutants may contain missense types of mutations at a presumed "mutational hot spot" within the structural gene for adenosine kinase. PMID- 3012801 TI - [Selective effect of antiepileptics on the cortex and medulla of the adrenal glands]. PMID- 3012803 TI - AIDS virus pathogenesis. PMID- 3012802 TI - Analysis of the diets of 12-year-old children in Cape Town. AB - As very few data are available regarding the nutrient intake of South African children, a dietary survey was conducted among 843 12-year-old children in Cape Town. Trained interviewers used a 24-hour-recall questionnaire developed for this purpose, inserting the quantity and types of foods consumed during different periods of the day on both a weekday and Sunday. The intake of carbohydrate, protein and fat was always greatest for white males and lowest for black males. This difference was emphasized by the high weekday energy intake of white boys (10 577 kJ), in contrast with the low energy intake of the black boys (6 457 kJ). The findings on kilojoule, carbohydrate, protein, fat and fibre content of the diets of these subjects are presented. PMID- 3012804 TI - Pleomorphism in HTLV-III, the AIDS virus. PMID- 3012806 TI - Early case of acquired immunodeficiency syndrome in a child from Zaire. AB - An eight-year-old child from Zaire died in Sweden in 1982 after a clinical course compatible with the acquired immunodeficiency syndrome (AIDS). In 1975, at the age of 5 months, the infant had an acute viral infection with a rash; this illness was followed by a chronic cough. During the course of the disease he had recurrent septicemia, fever (frequently with miliary lung infiltrates), disseminated lymphadenopathy, hepatosplenomegaly, candidiasis, and diarrhea. Late in the illness the child developed lethal disseminated disturbances of the central nervous system. Immunologic investigations revealed a pronounced hypergammaglobulinemia, normal C3 but low C4 values, decreased number of T lymphocytes, and decreased lymphocyte stimulation with T-cell and B-cell mitogens. Samples of serum taken in 1981 and 1982 were analyzed and found to be positive for antibodies to HTLV-III virus. The course of the disease in this child was more prolonged than most of the pediatric cases described earlier. It is likely that this child developed AIDS early in 1975, long before the AIDS epidemic was apparent in the United States. PMID- 3012805 TI - Prevalence of genital pathogens among female prostitutes in New York City and in Rotterdam. AB - The authors studied the prevalence of genital microorganisms among 300 female prostitutes in brothels in New York City and 60 female prostitutes attending a sexually transmitted diseases clinic in Rotterdam, The Netherlands. Rates of isolation of Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum in the two cities were 9.3% and 8.3%, 25.3% and 16.6%, 57.3% and 74.9%, and 73% and 79%, respectively. Trichomonas vaginalis was detected in 3.6% of New York prostitutes and in 16.6% of those in Rotterdam. Nonspecific vaginitis was found in 33% of prostitutes examined in New York. In New York, Asian prostitutes were more likely to be infected with C. trachomatis (33 of 102; 32.3%) than were prostitutes of other ethnic backgrounds (44 of 194; 21.5%; P less than .05. PMID- 3012807 TI - Analysis of DNA from recurrent genital herpes simplex virus isolates by restriction endonuclease digestion. AB - In a blind study, DNA from 40 clinical isolates of herpes simplex viruses was analyzed by restriction endonucleases to determine whether serial isolates from an individual patient could be identified and whether exogenous reinfection occurred within this population. Five of the 40 isolates served as controls. Based on restriction patterns obtained following Bam H1 cleavage of DNA, 35 isolates were assigned to 15 patients. Isolates from two patients displayed variation in the electrophoretic mobility of certain Bam H1 fragments. However, the isolates from one patient were identical when digested with four additional enzymes. One of three sequential isolates from a patient, when cleaved with Eco R1 and Hind III, showed variable fragments in the terminal and joint regions of the S segment of the genome. The appearance of the fragments was not due to the addition of a known restriction site. We conclude that exogenous reinfection with a genetically distinct strain of herpes simplex virus type 2 (HSV-2) did not occur among these 15 patients with recurrent genital HSV infections. PMID- 3012808 TI - Comparative clinical study of Sulbactam and ampicillin and clindamycin and tobramycin in infections of soft tissues. AB - A prospective, randomized, double-blinded study was undertaken to evaluate the effectiveness of Sulbactam (a new semisynthetic, injectable penicillanic acid sulfone) to inhibit beta-lactamase activity of bacteria in infections of the soft tissue. Sixty patients with documented soft tissue infections were prospectively randomized. One-half received 1 gram of Sulbactam per 2 grams of ampicillin every six hours. The other half received 600.0 milligrams of clindamycin every six hours and 1.5 milligrams per kilogram of tobramycin every eight hours. Patient groups were similar in age, sex, associated medical problems and bacteriologic flora of wounds. Sulbactam and ampicillin showed a 93 per cent cure rate or improvement as compared with 81 per cent in the clindamycin and tobramycin group. Eradication of organisms was better in the Sulbactam and ampicillin group (67 versus 35 per cent). Antibiotic activity of ampicillin was significantly augmented by the addition of Sulbactam. Of the 223 total bacteriologic isolates, 38 per cent were sensitive to ampicillin alone. Addition of Sulbactam improved sensitivity to 70 per cent. The Sulbactam and ampicillin combination is an effective combination for the treatment of soft tissue infections. PMID- 3012809 TI - [Irradiation of grades III and IV astrocytomas with new types of radiation]. AB - Until 1983, more than 300 astrocytomas of degree III and IV have been irradiated all over the world with new types of radiation such as neutrons, protons, alpha particles, heavy ions, and pions. Up to now a therapeutic progress could not be achieved. Radiobiologic experimentations suggest that the dense ionisation is unfavorable in the irradiation of malignant cerebral tumors. The clinical radiotherapy research with protons should therefore be intensified and hyperfractionation should be critically reviewed. PMID- 3012810 TI - [Radiotherapy of carcinoma of the paranasal sinuses]. AB - Carcinomas of the paranasal sinuses are usually advanced when diagnosed and present a therapeutic challenge. During the period between February 1970 and June 1981 44 patients were treated. 22 received postoperative irradiation, seven in combination with chemotherapy. 18 patients were treated with radiation alone, eleven with concomitant chemotherapy. Four patients received preoperative irradiation, three in combination with chemotherapy. The three-year survival is 43% and the five-year survival 33%. For those 26 patients who were irradiated pre or postoperatively with or without concomitant chemotherapy the five-year survival is 45%. We believe the patient will be afforded the greatest opportunity for cure with the combined efforts of the radiotherapist and the surgeon. The combination of chemotherapy and radiotherapy did not provide better results but increased acute and chronic toxicity of the therapy. PMID- 3012811 TI - The significance of microcalcifications without palpable mass in the diagnosis of breast cancer. AB - Microcalcifications constitute an important part of nonpalpable breast lesions and may be the first sign of a breast carcinoma. Between 1975 and 1984, 150 consecutive patients with clusters of at least five microcalcifications without palpable findings as the only indication for biopsy were treated. One hundred seventy-three groups of microcalcifications were excised and 51 malignancies were detected (29.5%). Most of the malignant lesions were noninfiltrating (56%). Axillary or distant metastases occurred in 11% of the fully evaluable cases. This warrants the expectation that these patients have a very favorable prognosis. Breast biopsy for nonpalpable-clustered microcalcifications is a feasible and valuable procedure. Close cooperation is required between the surgeon, radiologist, and pathologist. PMID- 3012812 TI - A new approach to massive blood transfusion during pediatric liver resection. AB - Morbidity and death during liver resection in children are due to hemorrhage and the consequences of massive transfusion. To overcome these problems, a new rapid method of blood transfusion was used in four children (8 to 35 months, 8.6 to 13 kg) undergoing extensive hepatic resection for tumor (tumor weight, 440 to 1625 gm). The rapid infusion device consisted of a roller pump and a bubble oxygenator warmer circuit primed with washed packed red cells resuspended in fresh-frozen plasma and calcium-free balanced salt solution (Plasmalyte). The infusate was warmed, oxygenated, and buffered before it was administered. An average of 5130 ml per patient of this reconstituted blood was infused at an average rate of 122 +/- 45 ml/min, with peak infusion rates sometimes as great as 1 L/min. Cardiac output, pulmonary artery wedge pressure, body temperature, urine output, blood gases, blood chemistries, and coagulation factors remained unchanged during and after these massive transfusions. Blood transfusion at rapid rates required during pediatric liver resection can be accomplished safely if the storage lesion of the bank blood is previously corrected. PMID- 3012814 TI - [Effect of specific immunosorption on the sensitivity of beta-adrenergic reception of peripheral blood lymphocytes to specific allergens in patients with atopic bronchial asthma]. PMID- 3012813 TI - [Various metabolic systems of lymphocytes in bronchial asthma]. PMID- 3012815 TI - [Disorders of cholinergic regulation in patients with bronchial asthma]. AB - A parallel study of the activity of serum cholinesterase and excretion of cyclic guanosine monophosphate made it possible to distinguish 3 types of disorders of cholinergic regulation in exacerbation of bronchial asthma: cholinergic compensation, subcompensation and decompensation. The nature of correlations of the degree of disturbance of cholinergic regulation, intensity of allergic reaction and its type was established. The role and place of the choline blocking agents in multiple modality therapy of bronchial asthma were defined. PMID- 3012816 TI - Two stage maxillary ridge augmentation using Durapatite. PMID- 3012817 TI - Direct evidence for the inhibition of platelet aggregation and release by intracellular cyclic AMP produced with a new photoactivatable derivative. AB - An increase in platelet cyclic AMP (cAMP) via stimulation of adenylate cyclase is thought to be the underlying mechanism by which potent prostaglandins i.e. PGD2, PGI2, inhibit platelet functions. We report here new and direct evidence for the inhibitory effects of cAMP on platelet aggregation and serotonin release. Washed platelets from rat were incubated with a new photoactivatable cAMP analogue (4,5 dimethoxy-2-nitrobenzyl ester); this compound is almost physiologically inert before irradiation and liberates free cAMP ("cAMP jumps") following light flashes. A single flash, delivered after 2 min incubation in 100-200 microM of the analogue, dramatically inhibited thrombin-induced aggregation, as compared with controls. Endogenous serotonin release, measured in the same samples by means of an electrochemically treated carbon electrode was undetectable after the cAMP jump. Pre-irradiated solutions added to platelets had no effect. The kinetics of the flash-induced effects were also studied. From these results we can conclude that: i) the photoactivatable cAMP derivative has to permeate through the platelet membrane; ii) the analogue remains photolabile; and, iii) intracellular cAMP, resulting from photolysis dramatically inhibits platelet aggregation and serotonin release. It is possible that cAMP exerts its effects by regulating cytoplasmic free calcium concentration and/or other actions affecting platelet activation. PMID- 3012818 TI - Modification of platelet functions by monobromobimane, a fluorescent thiol group label. AB - Monobromobimane (mBBr, bimane), a compound that penetrates cells and forms a fluorescent adduct with thiol groups, was used to asses the significance of thiols in platelet function. Exposure of washed platelets for 1 min to 100 microM mBBr abolished ADP-induced aggregation; shape change was not inhibited by 500 microM mBBr. The nonpenetrating compound monobromotrimethylammoniobimane was ineffective. Established ADP-induced aggregation was reversed by bimane, and fibrinogen binding to ADP-stimulated platelets was inhibited, an effect mainly due to decreased number of binding sites. Aggregation stimulated by A23187 and arachidonate was less effectively inhibited whereas epinephrine- and collagen induced aggregation were abolished by 50 microM mBBr. Similar effects on aggregation and secretion were observed in platelet-rich plasma except that higher mBBr concentrations were usually necessary. Aggregation and 14C-serotonin secretion stimulated by 0.1 U/ml thrombin were partially inhibited by pretreatment with bimane. With lower thrombin concentrations, they were often enhanced, as was 3H-arachidonate release. Bimane inhibited epinephrine-induced arachidonate release in gel-filtered platelets, possibly because it abolished the primary aggregation necessary for this release. mBBr did not elevate cyclic AMP but enhanced the increase induced by PGE1 and prevented the subsequent decrease typically caused by ADP. Examination of SDS polyacrylamide gels with ultraviolet light showed that mBBr reacted with many platelet proteins but not with GP IIb or IIIa. This observation, and the fact that bimane did not inhibit the fibrinogen induced aggregation of DTT- or chymotrypsin-treated platelets suggest that it reacts with thiol group(s) that are involved in "exposing" the fibrinogen receptor. PMID- 3012819 TI - Thromboxane receptor blockade versus cyclooxigenase inhibition: antiplatelet effects in patients. AB - In a randomized pilot study we compared the antiplatelet effects of aspirin and BM 13.177 in two groups of 7 patients each undergoing PTCA. As compared with the pretreatment values template bleeding time was prolonged and collagen induced aggregation was inhibited in PRP and WB in all patients. In the course of angiography and PTCA a rise in platelet factor 4 and beta thromboglobulin was observed in both groups, followed by a decrease below the baseline levels. Thromboxane B2 in plasma and serum decreased in the aspirin group but remained unchanged during BM 13.177 treatment. In PRP and WB aggregation induced by U 46 619 was inhibited after ingestion of BM 13.177 but not following ASA. After three months a control coronary angiography was done. There was no difference in regard to the degree of restenosis between both groups. Medication was well tolerated, compliance was good and no side effects were noted. PMID- 3012820 TI - Fibrinolytic study in a homozygous protein C deficient patient. AB - The fibrinolytic system was evaluated in a patient with homozygous protein C deficiency as well as in several members of his family with a partial deficiency of this protein. Before anticoagulant therapy the patient showed skin lesions which quickly disappeared after administration of fresh plasma. After anticoagulant treatment, the propositus suffered two clinical episodes of "ecchymotic" lesions, which were controlled with fresh plasma. The patient has remained free of new lesions and other clinical episodes up to the present date. The fibrinolytic activity of both the propositus and his family was normal. The patient's father showed adequate release of tissue plasminogen activator after controlled physical exercise. According to clinical and analytical data from our patient and his family, it is suggested that, in spite of the preservation of the fibrinolytic system in this case, a localized deficiency in fibrinolysis could exist in view of the clinical behaviour of the skin lesions described. PMID- 3012821 TI - Effect of heparin on platelet aggregation inhibited by PGI2, trifluoperazine and verapamil. AB - The enhancement of platelet aggregation by heparin in the presence of certain inhibitors of aggregation was investigated in an attempt to discern the mechanism through which heparin alters platelet function in plasma. These studies were performed by adding prostaglandin I2 (PGI2), verapamil, or trifluoperazine to platelet-rich plasma (PRP) in the presence or absence of heparin. Adenosine diphosphate (ADP), collagen, or arachidonic acid were used for induction of platelet aggregation. The inhibitory agents reduced platelet aggregation to 5 to 20% of control in the absence of heparin. When present in the reaction mixture along with the inhibitor, heparin restored aggregation to approximately 57 to 92% of control depending on the inhibitor and aggregating agent. This proaggregatory action of heparin was observed when heparin and PGI2 were preincubated together or separately for 20 min prior to the addition of PRP and ADP. Results were similar regardless of the sequence in which PGI2 and heparin were added to PRP, and irrespective of the time of incubation of platelets with PGI2. No suppression of platelet cyclic AMP concentration was observed with heparin alone. Heparin also failed to reduce the magnitude of platelet cyclic AMP accumulation promoted by PGI2, forskolin, or a mixture of PGI2 and forskolin. These observations suggest that heparin promoted platelet aggregation and partially overcame the effect of certain inhibitory agents by mechanism(s) that did not involve a reduction of platelet cyclic AMP. PMID- 3012822 TI - Effects of silica on the composition of the pulmonary extracellular lining. AB - The effects of intratracheally injected silica on lung weights and on alveolar macrophages, polymorphonuclear leukocytes, phospholipid, protein, N-acetyl-beta glucosaminidase, and alkaline phosphatase of the extracellular lining of rat lungs were studied as functions of dose and time. All of these parameters increased with time up to 12 days after a single injection of silica (200 mg/kg) and showed a dose dependence in their responses. Extracellular soluble protein increased 19.8-fold from 1.9 to 37.6 mg/pair of lungs. The composition of the extracellular soluble protein was very similar to that found in normal lungs as determined with two-dimensional-polyacrylamide gel electrophoresis. Although most of the soluble proteins in lavage effluents were similar to those found in serum, several serum proteins were absent, indicating that the selectivity of the lungs for certain serum proteins was maintained after treatment with silica. Increases in extracellular soluble proteins could not be accounted for by damage to the blood/air barrier. Extracellular phospholipid increased 12.1-fold from 1.74 to 21.1 mg/pair of lungs. The phosphatidylcholine content of this phospholipid resembled that of normal pulmonary surfactant but was different from that in free cells lavaged from the lungs of control and silica-treated rats. Increases in extracellular phospholipid were probably due to silica effects on the surfactant system and not to destruction of or release by free cells in the alveoli. N Acetyl-beta-glucosaminidase and alkaline phosphatase increased approximately 33- and 6-fold, respectively, in response to silica. The number of alveolar macrophages and polymorphonuclear leukocytes increased 1.5- and 75-fold, respectively. Calculation of partial correlations revealed statistically significant relationships among extracellular phospholipids, soluble proteins, and the two hydrolytic enzymes, suggesting that these components were being released into the lung lining from a common source or by a common mechanism. PMID- 3012823 TI - Effect of amitraz on heart rate and aortic blood pressure in conscious dogs: influence of atropine, prazosin, tolazoline, and yohimbine. AB - The effect of amitraz on heart rate (HR) and mean aortic blood pressure (MAP) were studied in five conscious male dogs. An iv injection of amitraz (1 mg/kg) caused a decrease in HR, which was accompanied by sinus arrhythmia for at least 60 min. Administration of amitraz also caused an increase in MAP for 20 min. Atropine sulfate (0.045 mg/kg, iv) increased HR and prevented amitraz-induced bradycardia. In addition, atropine potentiated amitraz-induced hypertension for 45 min. Yohimbine, an alpha 2-adrenoreceptor antagonist, given iv at 0.1 mg/kg, prevented hypertension, bradycardia, and sinus arrhythmia induced by amitraz. Tolazoline, a nonselective alpha-adrenoreceptor antagonist, given iv at 5 mg/kg, reduced the bradycardia and sinus arrhythmia caused by amitraz administration but did not change amitraz-induced hypertension. Tolazoline alone also increased both HR and MAP. Prazosin, an alpha 1-adrenoreceptor antagonist, given iv at 1 mg/kg, did not affect the cardiovascular actions of amitraz. The results suggest that (1) alpha 2-adrenoreceptors mediate amitraz-induced bradycardia and hypertension, and (2) yohimbine, but not atropine, can be used to control the untoward reactions of amitraz. PMID- 3012824 TI - Aluminum impairs glucose utilization and cholinergic activity in rat brain in vitro. AB - The effects of AlCl3 on the production of 14CO2 from [U-14C]glucose and high affinity choline transport in rat brain synaptosomes, and on carbachol-stimulated hydrolysis of phosphoinositides in cortical slices were studied. In buffer containing either high K+ (50 mM) or low K+ (4.9 mM), 1 mM AlCl3 significantly depressed the synaptosomal production of 14CO2 from [U-14C]glucose to 54% and 44% of control rates, respectively. At a concentration of 0.1 mM, AlCl3 depressed the evolution of 14CO2 from [U-14C]glucose from synaptosomes incubated in the high K+ buffer, but did not significantly change 14CO2 production from synaptosomes in the low K+ buffer. Aluminum chloride also inhibited high affinity choline transport in synaptosomes prepared from rat cortex and from hippocampus with an IC50 of approximately 0.5 mM. In brain slices the carbachol-stimulated hydrolysis of phosphoinositides was inhibited by AlCl3 in a dose-dependent manner. One millimolar, 0.5 mM and 0.1 mM AlCl3 inhibited the carbachol-stimulated release of inositol phosphates by 75%, 44% and 33%, respectively. These same concentrations of AlCl3 inhibited the incorporation of [3H]inositol into phospholipids. This inhibitory effect was not dose-dependent as all 3 concentrations of AlCl3 inhibited phospholipid labelling to the same extent (27-37%). These results are discussed in relation to the in vivo neurotoxicity of aluminum. PMID- 3012825 TI - Evaluation of the cytotoxicity and genotoxicity of the spermicides nonoxynol-9 and octoxynol-9. AB - The cytotoxic and genotoxic potentials of the spermicidal agents, nonoxynol-9 (N 9) and octoxynol-9 (0-9), were evaluated in in vitro test systems using rat liver cells. N-9 was also tested for the induction of sperm abnormalities in mice. Dose related cytocidal effects were seen after the addition of N-9 and O-9 to the culture medium for 24 h. The mean concentrations of N-9 and O-9 necessary to decrease the number of viable cells by 50% (LC50) were 24 and 43 micrograms/ml of media, respectively. The spermicides neither induced DNA repair in freshly isolated hepatocytes, nor caused any mutations at HGPRT locus in the T51B rat liver cell line. There was also a lack of malignant transformation response in the low-calcium assay. Further, the germinal cells of mice remained unaffected by N-9. PMID- 3012826 TI - Polychlorinated dibenzofurans as 2,3,7,8-TCDD antagonists: in vitro inhibition of monooxygenase enzyme induction. AB - 2,4,6,8- and 1,3,6,8-tetrachlorodibenzofuran (TCDF) competitively displace [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from the rat cytosolic receptor protein and their EC50 values were 1.5 X 10(-6) and 1.25 X 10(-7) M, respectively. In contrast to their relatively high binding avidities these TCDF isomers were poor inducers of benzo[a]pyrene hydroxylase and ethoxyresorufin O deethylase in rat hepatoma H-4-II E cells in culture (EC50 greater than 10(-5) M). Coadministration of different concentrations of 2,4,6,8- and 1,3,6,8-TCDF (10(-5), 10(-6) and 10(-7) M) with 2 X 10(-10) M, 2,3,7,8-TCDD (a dose which elicits 80% of the maximal induction response) resulted in significant decreases in the expected (additive) induction of benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase by the mixture. Thus the partial agonists, 1,3,6,8- and 2,4,6,8-TCDF, antagonize the receptor-mediated enzyme induction activity of 2,3,7,8-TCDD presumably via competitive displacement of 2,3,7,8-TCDD from the receptor protein. In contrast, coadministration of 2,3,7,8-TCDF and 2,3,7,8-TCDD gave additive enzyme induction responses. The identification of the 2,3,7,8-TCDD antagonists represents a new class of halogenated aryl hydrocarbons. PMID- 3012828 TI - Cytotoxicity of phenazine methosulfate in isolated rat hepatocytes is associated with superoxide anion production, thiol oxidation and alterations in intracellular calcium ion homeostasis. AB - The metabolism of phenazine methosulfate (PMS) by isolated rat hepatocytes is associated with superoxide anion production, and with a substantial decrease in intracellular levels of reduced glutathione, most of which is oxidized to GSSG. A marked loss of protein-free sulfhydryl groups also occurs when intracellular glutathione is depleted, and cytotoxicity follows. These effects are associated with the inhibition of the plasma membrane Ca2+-ATPase and with intracellular accumulation of calcium ion which is preferentially sequestered in mitochondria. Maintenance of protein sulfhydryl groups in the reduced state by dithiothreitol (DTT) prevents the alterations in intracellular calcium homeostasis and protects against toxicity. PMID- 3012829 TI - [Tumors of the minor salivary glands]. PMID- 3012827 TI - Concentration-related changes in blood and tissue parameters of hepatotoxicity and their interdependence in rats exposed to bromobenzene and 1,2 dichlorobenzene. AB - Liver damage resulting from 4 h exposure to bromobenzene (BB) (146-957 ppm) and 1,2-dichlorobenzene (DCB) (245-739 ppm) as model toxicants was evaluated in rats. The modifications considered were the increases in serum glutamate dehydrogenase (GLDH) and sorbitol dehydrogenase (SDH) activities and the decreases in centrolobular liver-cell glucose-6-phosphatase (G6-Pase) staining intensity. A linear inverse relationship was established between the logarithmic values of blood enzyme activities and liver G6-Pase staining intensity. In addition, the levels of exposure to each test chemical were found to be linearly related to liver G6-Pase staining intensity and to the logarithmic values of blood enzyme activities. PMID- 3012830 TI - The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis. AB - Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions. PMID- 3012831 TI - Major cerebral infarction from tumor embolus. AB - A major right hemispheric infarct developed in a 31-year-old man within forty eight hours of lung resection for metastatic synovial-cell sarcoma. Post mortem exam revealed tumorous occlusion of the right internal carotid artery. Major stroke from cerebral tumor embolus should be seriously considered in patients with primary or metastatic lung cancer who have had a very recent pneumonectomy, especially when there are symptoms and signs of multi-organ or extremity ischemia. PMID- 3012832 TI - Passive transfer of cytomegalovirus by cardiac and renal organ transplants in a rat model. AB - Passive transfer of latent rat cytomegalovirus (R-CMV) infection by means of vascularized organ transplants was examined in inbred rat strains. LEWIS (LEW) rats 4-5 weeks old were infected with RCMV and used as donors at 5 months of age when the infection had become latent. Well-perfused LEW hearts and kidneys were transplanted into unmodified or 500-rad x-irradiated syngeneic or allogeneic Brown Norway (BN) recipients; recipients were sacrificed 3 weeks after transplantation, and RCMV virus from various organs was quantitated by means of a plaque assay. Passive transfer of latent infection could be accomplished with renal allografts (60%) and renal isografts (40%). When BN hosts were x-irradiated LEW renal allografts invariably transferred the latent infection (100%); cardiac allografts rarely did so (8%). X-irradiation of syngeneic hosts did not enhance the capacity of LEW kidneys to transfer the latent infection. The latent infection could not be transferred with thoracic duct lymphocytes. Results show the passive transfer of latent infection with well-perfused vascularized organ allografts to be a relative organ-specific phenomenon. PMID- 3012833 TI - Cellular immunity to vaccinations and herpesvirus infections after bone marrow transplantation. AB - The cellular immune response to herpesviruses was studied in 46 recipients of marrow grafts (23 autologous, 23 allogeneic). That study was performed in vitro by evaluating the degree of lymphocyte proliferative responses to herpes simplex virus (HSV), cytomegalovirus (CMV), and varicella zoster virus (VZV). No primary infections with any of those viruses were noted after bone marrow transplantation (BMT). The incidence of active infection in seropositive patients was significantly lower after autologous BMT than after allogeneic BMT (HSV, 2/22 vs. 11/22 patients, respectively, P = 0.007; CMV, 4/12 vs. 9/10 patients, respectively, P = 0.02; VZV, 3/23 vs. 11/23 patients, respectively, P = 0.02). After autologous BMT, the restoration of cellular immunity to the three viruses occurred at a clearly faster rate than after allogeneic BMT. That pattern may have contributed to the low incidence of active infections with those viruses after autologous BMT. Recipients of allogeneic marrow from donors with a positive lymphocyte proliferation test to HSV had a significantly increased incidence of active HSV infection post-BMT (8/9 patients) than those who received marrow from donors with a negative test (3/13 patients; P = 0.008). Acute or chronic graft versus-host disease (GVHD) decreased the cellular immune response to the three herpes viruses, but not significantly. Our program of vaccinations with diphtheria and tetanus toxoids started in the fourth month post BMT. Chronic GVHD patients who were vaccinated had a clearly impaired cellular immune response to both toxoids as compared with those without chronic GVHD. PMID- 3012834 TI - Infections in heart-lung transplant recipients. AB - Infectious episodes were analyzed in 14 heart-lung transplant recipients who survived more than one week after transplantation. These patients had higher rates of infection than heart transplant recipients at our institution (P less than 0.01) and greater than 90% of all infections were potentially life threatening. A total of 67% of all infections involved the lung or thoracic cavity as a primary site, and most of the rest were disseminated viral or fungal infections. Pneumocystis carinii infections occurred in six patients and were more common in this group than in patients who received heart transplants in the same period (P less than 0.005). Two patients followed more than one year developed a syndrome of chronic sputum production and bronchial colonization with Pseudomonas aeruginosa, which required recurrent treatment with i.v. antibiotics for symptomatic relief. The high rate of pulmonary infections in these patients presents a challenge to clinical management, and suggests that intensive and invasive monitoring for pulmonary infection is desirable. PMID- 3012835 TI - Increased oxidative burst potential exhibited by macrophages during graft-versus host reactions in mice. AB - Graft versus host reactions (GVHR) in mice are accompanied by macrophage activation. Since macrophage oxidative burst (OB) was found to increase in activated macrophages (MPs), we examined the OB of peritoneal and spleen MPs from mice during GVHR. Parental spleen lymphocytes, were injected i.p. into F1 hybrids (BALB/cXC57Bl/6) and at different intervals following injection we monitored the number of peritoneal exudate cells (PEC), spleen enlargement, and the OB of both peritoneal and spleen adherent MPs. Macrophage OB was assessed by measuring O-2 and H2O2 production, following stimulation with the phorbol ester TPA. The spleen to-body weight ratio increased by 40-70% in mice with GVHR during the period of 6 20 days post-injection and decreased to almost normal levels after 27 days. In such mice with GVHR the number of PEC returned to normal (approximately equal to 6 X 10(6) cells/animal) after 20 days compared with 5 days in control mice (injected with F1 spleen cells). Adherent peritoneal MPs from control mice and mice with GVHR exhibited increased OB activity 3 days following treatment. However, in the control mice the OB response returned to normal within 6 days, while in the experimental mice it showed a slight decrease after 6 days, increased again within 9-17 days, and finally decreased 20-27 days after treatment. Adherent spleen MPs from mice with GVHR generated a high response 6 days posttreatment, which gradually returned to the basal level after 17 days. These findings suggest that during GVHR in mice, macrophage OB was potentiated, giving rise to cytotoxic products such as O2- and H2O2, which may contribute to tissue damage during GVHR. PMID- 3012836 TI - The effects of donor pretreatment on the transmission of murine cytomegalovirus with cardiac transplants and explants. PMID- 3012837 TI - Ethical considerations in solid organ pediatric transplants. PMID- 3012838 TI - [Properties of the energy-allowance enzymes of the common frog in different physiological body states. I. Adenosine triphosphatase and succinate dehydrogenase activities]. AB - The activity of SDG, Na, K-ATPase and Mg-ATPase of the grass frog was determined in January, March and May, the number of animals examined being 30-40 in either series of experiments. In May (period of reproduction) the average activity of the above enzymes was higher than in January and March. This was observed both in males and females. A correlation between enzymatic activities of each single organism in March and May significantly increased. The role of these changes in the increase of viability of the organism is discussed. PMID- 3012839 TI - [Epidemiology of palpable abdominal masses in children in Morocco. Apropos of 1084 cases]. PMID- 3012840 TI - [Palpable abdominal masses in children 0 to 14 years old. Apropos of 97 cases]. PMID- 3012841 TI - Crystals in a jugulotympanic paraganglioma. AB - The morphologic features of a jugulotympanic paraganglioma are reported. The tumor showed the usual histology of paragangliomas. No sustentacular cells were identified. In the tumorous chief cells there were typical neurosecretory dense core granules 60-180 nm in diameter. Granules averaging 400 nm in diameter were also observed, sometimes with a regular rhomboid core or crystallized content. Rhomboid crystals were seen in the cytoplasm in membrane-bound spaces and in telolysosomes. The crystals had a substructure consisting of alternating light and dark lines with a periodocity of 5-10 nm. Such crystals had not been reported previously in paragangliomas. The possible origin of the crystals is discussed. PMID- 3012842 TI - Metastatic tumor of unknown origin. PMID- 3012843 TI - [Biological value of various bone substitutes in the treatment of bone defects. Animal experiment studies]. AB - In clinical practice, biologic and synthetic substances have been used more and more as bone substitutes during the last few years. Different properties of the implant material are required according to the various fields of application. This implies a precise investigation of the biologic value by adequate experimentations on animals. It has been demonstrated in a bioassay with several synthetic implant materials, especially ceramics made of calcium phosphate, powders and granulated materials of tricalcium phosphate, and hydroxylapatite with different pore sizes and numbers, that there is only poor or even not any biologic activity in these substances which would produce an orthotopic stimulation of osseous formation. The application of these substances as bone substitutes can only be recommended in an adequate transplantation bed. However, all implant materials tested by different experimentations on animals showed an incomplete absorption in the histomorphological investigation even after twelve months. The various biologic bone substitutes (autologous and homologous spongiosa, demineralized homologous spongiosa, different forms of bone gelatin) show different degrees of biologic activity. Hitherto an inducing heterotopic effect could be demonstrated for bone gelatin in rats, dogs, and sheep. The inducing effect of bone gelatin combined with soluble calcium phosphate ceramics in rats was increased as compared with autologous spongiosa. Regarding our own results as well as various communications in literature, we consider it absolutely necessary to give more attention to the objectifying criteria when describing the biologic activity of "biologic and synthetic bone substitutes". PMID- 3012845 TI - Selective properties of trains of real and complex RF micropulses in slice selective spin-echo MRI. AB - The spin nutation properties of frequency selective (space selective in combination with a magnetic field gradient) trains of radiofrequency micropulses were studied in a numeric model. Two cases were considered, one simulating the 90 degrees excitation pulse in spin-echo MRI, the other the 180 degrees spin inversion pulse. Image reconstruction according to the 2-D Fourier transform technique requests that the effect of the 180 degree pulse is independent of the initial phase of the spin vector relative to the radiofrequency field. It was found that with a train of phase-stable radiofrequency micropulses with an envelope of the traditional 'sinc' type the result of the spin inversion pulse was depending on the initial phase whereas this was not the case for a train of phase-shifted (complex) micropulses. The complex train of radiofrequency micropulses also had better selective properties both for excitation and spin inversion than the real one. PMID- 3012846 TI - Extragonadal retroperitoneal germ cell tumors without apparent testicular involvement. A search for the source. AB - Extratesticular germ cell neoplasms without apparent testicular primary tumors are rare. The origin of these neoplasms has been debated in the literature for decades. With the advent of effective chemotherapy for extratesticular germ cell neoplasms, the origin of these tumors becomes more than academic. We report on 3 cases of retroperitoneal germ cell neoplasms with microscopic intratesticular primary tumors. All patients with extragonadal germinal cell tumors of the testes should undergo thorough investigation for an occult testicular primary tumor despite a normal testis examination. We review previously reported cases of extratesticular germ cell neoplasms without an apparent testicular primary tumor. PMID- 3012844 TI - [Diagnosis of deep venous thrombosis of the leg using scintigraphy with 99m technetium-labeled erythrocytes]. PMID- 3012847 TI - Predictors of recurrent clinical stage I nonseminomatous testicular cancer. A prospective clinicopathologic study. AB - A prospective clinicopathologic study of 60 patients with clinical Stage I nonseminomatous testicular cancer (NSTC) has been reported. Of 60 patients with clinical Stage I NSTC who underwent retroperitoneal lymphadenectomy (RPLA), 6 proved to be Stage II, a staging error of 10 per cent. In 4 patients of the remaining 54, metastases developed in the lungs. In 1 patient metastases developed both in the lung and in retroperitoneal lymph nodes. There was no death in these groups of patients. These 10 patients with staging error and/or recurrence after RPLA have been analyzed for the causes of treatment failure utilizing a set of prognostic criteria (tumor cell type, vascular or lymphatic invasion in the primary tumor, extension to the spermatic cord, and size of the primary tumors). It has been concluded that embryonal carcinoma (P less than 0.001), vascular invasion (P less than 0.001), and extension of the tumor to the spermatic cord (P less than 0.001) are significant predictors of metastases and/or recurrence after RPLA in Stage I NSTC. A plan of management is suggested based on these criteria. PMID- 3012848 TI - Endometrial carcinoma of prostate. PMID- 3012849 TI - Transmission of feline leukaemia virus in the milk of a non-viraemic cat. AB - The possibility of the transmission of feline leukaemia virus (FeLV) from latently infected cats was studied. Five female cats with latent infections were examined for evidence of transmission of the virus to their kittens. One of the cats infected members of four consecutive litters of kittens which subsequently became persistently viraemic and transmitted the virus to other susceptible kittens by contact. Shortly after birth its kittens were apparently FeLV-free since neither viral antigen nor infectious virus was detected in their blood and no virus was found in cell cultures made from aspirates of bone marrow. The kittens became viraemic from 45 days of age onwards at a time when their passively acquired colostral FeLV neutralising antibodies were no longer detectable. Transmission of the virus occurred via the milk since both FeLV antigen and infectious virus were found in milk samples taken six weeks after kittening and the virus was transmitted to a fostered kitten. Eleven weeks after the birth of the fourth litter the cat became viraemic. The intermittent presence of FeLV antigens detected by the Leukassay F test, but not infectious virus, in the plasma of this cat over the previous months and a low level of serum neutralising antibodies distinguished it from four other latently infected queens which did not transmit infection to their kittens. These factors may indicate a risk of milk transmission and reactivation of latent virus. PMID- 3012850 TI - Observations on the use of an inactivated canine parvovirus vaccine. AB - Data are presented on studies of field and experimental use of a formalin inactivated canine parvovirus vaccine. There was an absolute correlation between a single successful vaccination and subsequent protection against clinical disease. Unsuccessful vaccinations were consistently associated with the presence of maternal antibody at the time of vaccination. The vaccine induced an antibody response within two days and anamnestic responses within 24 hours. It is suggested that a single successful vaccination probably protects against clinical parvovirus disease for life. PMID- 3012851 TI - Poxvirus infection in the domestic cat: some clinical and epidemiological observations. AB - The clinical findings from 30 cases of feline poxvirus infection in the UK are reviewed and some epidemiological observations described and discussed. In most cases the clinical signs consisted of skin lesions only, although systemic signs were also occasionally seen. Over half the cats had a history of a single recent lesion, assumed to be primary, on the head, neck or a forelimb. Twenty-nine of 30 cats developed more widespread secondary skin lesions. Cat-to-cat transmission was apparently rare. More cases were recognised in the autumn than at other times of the year. The possibility of a wild mammal reservoir of infection is discussed. PMID- 3012852 TI - Herpesvirus in red deer. PMID- 3012853 TI - Reactions to equine influenza vaccine. PMID- 3012854 TI - Feline leukaemia virus antigen in cats from Beirut, Lebanon. PMID- 3012855 TI - Viruses associated with turkey rhinotracheitis in Great Britain. PMID- 3012856 TI - Differences in the lymphoproliferative response of cattle and sheep to bovine leucosis virus infection. AB - Lymphoblastic leukaemia, preceded by a significantly increasing percentage of prolymphocytes in peripheral blood smears for from 12 to 68 weeks before death was a feature of sheep which developed lymphosarcoma following inoculation with the Australian strain of bovine leucosis virus (BLV). Lymphocytosis and/or the appearance of immature cells were a reliable predictor of tumour formation in sheep, but not in cattle. There was a terminal lymphoblastic leukaemia in only 43 of 84 cattle with lymphosarcoma. Differences in the morphological appearance and glycogen content of the leukaemic lymphoblasts of sheep and cattle were observed. In spite of these differences the high frequency of lymphocytosis and lymphosarcoma in experimentally infected sheep suggests that they could be a useful model for studying the pathological and immunological responses to BLV infection. PMID- 3012857 TI - To compare the incubation period following intratracheal and subcutaneous inoculation of bovine leukosis virus infected lymphocytes and to study their clearance from the circulation. AB - The migration of fluorescein isothiocyanate labelled lymphocytes through the tracheobronchial mucosa has been studied in cattle. Following intratracheal inoculation of labelled non-infected autologous lymphocytes and bovine leukosis virus (BLV) infected heterologous (presumed allogeneic) lymphocytes, the labelled lymphocytes appeared in the blood circulation between 4 and 7 days post inoculation. Following intravenous inoculation of labelled autologous lymphocytes, the cells could be detected in the circulation for 10 days post inoculation whereas BLV infected and non-infected heterologous lymphocytes could be detected for only 2 days. The migration of BLV-infected heterologous lymphocytes through the tracheobronchial mucosa caused a delay in the appearance of labelled lymphocytes in the circulation and a corresponding delay in the appearance of BLV antibodies. Comparison was made of the effect of two different routes of inoculation, subcutaneous and intratracheal on the incubation period as indicated by the detection of antibody. Subcutaneous inoculation of 1 X 10(4), 5 X 10(3), 1 X 10(3) of lymphocytes from a BLV infected cow caused seroconversion whereas 5 X 10(2) cells did not. Intratracheal inoculation of 5 X 10(3) cells caused sero-conversion. One animal did not develop BLV antibody until 30 weeks after inoculation although BLV could be isolated from the blood at 24 and 26 weeks post inoculation. PMID- 3012858 TI - Some antigenic and cultural properties of border disease and bovine viral diarrhoea viruses. AB - In vitro comparison of the replication cycles of three strains of border disease virus and one strain of bovine viral diarrhoea virus showed similar growth curve patterns and a tendency of cell-free virus to exceed cell-associated virus. Each virus was antigenically distinguishable from the others in cross-neutralisation tests. PMID- 3012859 TI - The effect of feeding pellets of different types of roughage on the incidence of lesions in the abomasum of veal calves. AB - Pellets of three types of roughage were fed to veal calves, to determine their effect on the frequency of lesions (erosions or ulcers) of the abomasal mucosa. The number was increased in the calves fed roughage. Pellets produced from corn silage caused a greater number of lesions than pellets produced from barley straw or lucerne hay. It is concluded that the nature of the roughage plays a role in the pathogenesis of abomasal lesions. PMID- 3012860 TI - Cellular differentiation and prognosis in embryonal rhabdomyosarcoma. A report from the Cooperative Soft Tissue Sarcoma Study 1981 (CWS 81). AB - Sixty-four cases of embryonal rhabdomyosarcoma (eRMS) were investigated for cellular differentiation by light microscopy. Of these 64 cases 20 were studied by means of immunohistochemistry. Histologically, three subgroups could be distinguished: primitive (less than 10% rhabdomyoblasts), intermediate (10-50% rhabdomyoblasts) and well differentiated (greater than 50% rhabdomyoblasts) eRMS. Vimentin-positive cells predominated in the primitive eRMS. Intermediate eRMS showed large proportions of desmin-positive cells but vimentin containing cells were also numerous. Myoglobin could only be demonstrated in well differentiated eRMS. Primitive and well differentiated eRMS mainly occurred in the head and neck area, whereas intermediate eRMS were predominantly located in the abdomen. Stage III and IV tumours predominated in cases of primitive eRMS, whereas lower stages were noted in cases of intermediate and well differentiated eRMS. Response to chemotherapy, evaluated after seven weeks of treatment, was achieved in 10/15 (66%) cases of primitive, in 16/19 (84%) cases of intermediate and 5/5 cases of well differentiated eRMS. It is concluded from the current study that the three subgroups of eRMS differ not only by cytological differentiation but also by site of predilection, stage at time of diagnosis and response to chemotherapy. PMID- 3012862 TI - Lymphoreticular infiltrates in invasive ductal breast cancer. A histological and immunohistological study. AB - Fifty-two invasive ductal breast cancers were investigated histologically and immunohistologically to assess localization and composition of the lymphoreticular infiltrates. The tumour-infiltrating cells were mainly located in the intervening stroma, whereas tumour foci often exhibited lower numbers of lymphoreticular cells. Macrophages (Mono 1+ and KiM 6+) and helper/inducer cells bearing the T4 surface antigen (Leu-3a+) regularly constituted the majority of the tumour-infiltrating lymphoreticular cells. In more than 80% of cases large numbers of macrophages were found, and many T4 cells occurred in about 60%. Next in frequency were the T lymphocytes (Leu-1+) which were mostly observed in high (46%), or in moderate (39%) numbers. In about 2/3 of the cases moderate numbers of T8 (suppressor/cytotoxic) lymphocytes (Leu-2a+) were detected. B lymphocytes (T0 15+) and natural killer cells (Leu-7+) were generally encountered in very low numbers, while eosinophilic granulocytes were virtually absent from the lymphoreticular infiltrates. Tissue mast cells and plasma cells were present in very low numbers in about one half of the tumours but cases with low, moderate or - rarely - even high numbers of infiltrating cells also occurred. It must be emphasized that an in situ histomorphological analysis of the cellular part of the stromal reaction of invasive ductal breast cancers allows only limited conclusions concerning the functional properties of the tumour-infiltrating lymphoreticular cells. From the present study, macrophages and T4 cells but also T8 lymphocytes might be of significance in immunooncological reactions "against" clinically detectable stages of invasive breast cancer. PMID- 3012861 TI - Pulmonary adenocarcinoma of fetal type: alternating differentiation argues in favour of a common endodermal stem cell. AB - Pulmonary adenocarcinoma exhibit different cell types that ultrastructurally, appear to be related to the different epithelial cell types occurring in the terminal lung lobule. The present paper describes the light-microscopical, ultrastructural and immunohistochemical features of a newly defined type of pulmonary adenocarcinoma that resembles an early stage of lung differentiation. The alternating epithelial differentiation within this tumour speaks in favour of a common endodermal stem cell for the different types of epithelial cells within the lung, including endocrine cells. The relationship of this tumour to pulmonary blastoma is discussed. PMID- 3012863 TI - Cloning and characterization of an equine cutaneous papillomavirus. AB - Equine papillomaviruses (EqPV) from naturally occurring cases of cutaneous papillomatosis in several ponies and one horse were isolated, cloned, and characterized. Group specific papillomavirus structural antigens were detected in sections of the papillomas by the peroxidase-antiperoxidase technique, and virions were observed in the in the nuclei of cells in the stratum granulosum and corneum. Negatively stained virions purified from papilloma homogenates by isopycnic CsCl centrifugation were 55 nm in diameter and had typical papillomavirus morphology. The entire viral genomes of two separate isolates were cloned at a single BamHI site into pBR322. A detailed restriction map of the viral genome is presented. Using nick-translated subgenomic fragments of BPV-1 as probes in Southern blot hybridizations, the organization of the EqPV genome was established. Southern blot analysis under various conditions of stringency revealed that EqPV shares relatively more homology with the late region of the BPV-1 genome and with the E2 region of the HPV-1 genome than with other parts of the same viral DNAs. Papillomavirus-specific sequences were found in papillomas from other anatomic sites using the EqPV DNA as a probe in Southern blot hybridizations. Genomes detected in DNA from penile papillomas had a different restriction pattern and hybridized to the EqPV probe only under nonstringent conditions. PMID- 3012864 TI - Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus. AB - The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens ("initiators") as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. As reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells. PMID- 3012865 TI - Coding content and expression of the EBV B95-8 genome in the region from base 62,248 to base 82,920. AB - The DNA sequence of a 20,672-base region of the EBV B95-8 genome from the BamHI site separating fragments BamHI-F and BamHI-Q to the EcoRI site separating fragments EcoRI-G2 and EcoR1-F has been determined. S1 mapping and northern blotting with probes from M13 recombinants have been used to search for RNAs from this region. Five mRNAs have been identified and correlated with five open reading frames in the DNA sequence. Two of these open reading frames encode ribonucleotide reductase subunits. A further two open reading frames are present including one for a predicted protein of 340 kDa for which no transcript has yet been detected. PMID- 3012866 TI - Conserved structures among the nucleocapsid proteins of the paramyxoviridae: complete nucleotide sequence of human parainfluenza virus type 3 NP mRNA. AB - The nucleotide sequence of the mRNA coding for the nucleocapsid protein (NP) of the paramyxovirus, human parainfluenza virus type 3 (PIV-3), has been determined. The NP mRNA was found to contain 1642 bases, excluding poly(A), and encode a protein of 515 amino acids, with a molecular weight of 57,823. Amino acid residues 1 through 420 of PIV-3 NP protein showed extensive sequence homology with the corresponding amino acids of Sendai virus nucleocapsid protein. There was virtually no homology between the last 95 amino acids. Comparison of the NP proteins of PIV-3, Sendai virus, measles virus, and canine distemper virus revealed, from amino acid residues 160 through 390, some conserved areas between the corresponding proteins of these paramyxoviruses. The 5' terminal sequence of PIV-3 NP mRNA (5'-AGGATTAAAG-3') was similar to the conserved sequence (formula; see text) found at the 5' termini of Sendai virus mRNAs. Both PIV-3 NP and Sendai virus mRNAs had a common 3' terminal tetranucleotide (5'-TAAG-3') preceding the poly (A) tail. PMID- 3012867 TI - Identification and structure of the gene encoding gpII, a major glycoprotein of varicella-zoster virus. AB - The genome of varicella-zoster virus (VZV) encodes three major families of glycoproteins (gpI, gpII, and gpIII). mRNA from VZV-infected cells was hybrid selected using a library of VZV recombinant plasmids and translated in vitro; polypeptide products were immunoprecipitated by polyclonal monospecific guinea pig antibodies to gpII. The mRNA encoding a 100-kD polypeptide precipitable by anti-gpII antibodies mapped to the HindIII D fragment near the center of the UL region. DNA sequence analysis of this region of the VZV genome revealed a 2.6-kbp open reading frame (ORF) potentially encoding a 98-kDa polypeptide possessing the characteristics of a glycoprotein. The 100-kDa polypeptide was specified by mRNA isolated by hybrid selection using a plasmid containing part of the 2.6-kbp ORF, and immunoprecipitation of this protein by anti-gpII antibodies and by convalescent zoster serum was blocked specifically by purified gpII. We conclude that the 2.6-kbp ORF encodes gpII. The imputed primary amino acid sequence of gpII shows a high degree of homology to that of herpes simplex virus type 1 (HSV 1) gB, a result consistent with the equivalent map locations of the respective genes in the HSV and VZV genomes and with the recently reported serological cross reactivity of HSV-1 gB and VZV gpII. Unlike the mature gene products of gB, those of gpII have been described as a pair of glycoproteins with approximate molecular weights of 60 kDa in reducing gels, products of a single glycoprotein species with approximate mol mass of 125-140 kDa in nonreducing gels. Amino-terminal sequences of purified gpII were determined and compared to the imputed amino acid sequence. This comparison implies that the primary translational product is cleaved approximately into halves in vivo and suggests that mature gpII is a disulfide-linked heterodimer. PMID- 3012868 TI - 3-Methylquercetin is a potent and selective inhibitor of poliovirus RNA synthesis. AB - 3-Methylquercetin (3MQ) is a natural compound isolated from Euphorbia grantii that selectively inhibits poliovirus replication, but has no effect on encephalomyocarditis virus. When the compound is present from the beginning of infection, the bulk of viral protein synthesis is prevented, but the shut-off of host protein synthesis still occurs. Addition of 3MQ 3 hr after infection has a slight effect on viral protein synthesis, suggesting that this compound blocks a step of viral replication different from translation. Indeed, poliovirus RNA synthesis is potently blocked by 3MQ, i.e., 50% inhibition at 2 micrograms/ml (6.3 X 10(-6) M). No effect on encephalomyocarditis, nor on cellular RNA synthesis is observed even at 20 micrograms/ml. The inhibitory effect of 3MQ is reversible, since cells treated with this compound from the beginning of infection start to synthesize viral RNA and proteins when the compound is removed. Strikingly, other natural compounds structurally related to 3 methylquercetin such as quercetin, naringenin, naringin, morin, catechin, kaempferol, myricetin, phloretin, phlorizdin, and rutin do not block poliovirus replication. PMID- 3012869 TI - Fusion glycoprotein of human parainfluenza virus type 3: nucleotide sequence of the gene, direct identification of the cleavage-activation site, and comparison with other paramyxoviruses. AB - The complete sequences of the fusion (F) mRNA and protein of human parainfluenza virus type 3 (PF3) were determined from overlapping cDNA clones. To confirm the cDNA sequence, the complete sequence of the F gene was determined independently by dideoxynucleotide sequencing of genomic RNA using synthetic oligonucleotide primers. The mRNA contains 1845 nucleotides, exclusive of poly (A), has an unusually long (193-nucleotide) 5' nontranslated region, and encodes an F0 protein of 539 amino acids. The site within F0 of the proteolytic cleavage that activates fusion activity was established by direct amino acid sequencing of the NH2 terminus of the F1 subunit. The PF3 F0 protein shares major structural features with the previously sequenced F0 proteins of Sendai virus (murine parainfluenza type 1) and simian virus 5 (SV5, canine parainfluenza type 2), including: similarity in overall length; similarity in location of the site of the activating proteolytic cleavage; the presence of an NH2-terminal signal peptide and COOH-proximal membrane anchor; strong conservation of the sequence at the NH2 terminus of the F1 subunit; and nearly exact conservation in the number and positions of cysteine residues. Alignment of the F0 protein sequences of PF3 with those of Sendai, SV5, and respiratory syncytial virus (RSV) using a matrix that scores both amino acid matches and mismatches provided highly significant statistical evidence that all four proteins are related. The order of decreasing relatedness to PF3 was found to be: Sendai virus, SV5, and RSV. PMID- 3012870 TI - Chromosomal sites for Marek's disease virus DNA in two chicken lymphoblastoid cell lines MDCC-MSB1 and MDCC-RP1. AB - In situ hybridization of viral DNA fragments to metaphase chromosomes of two chicken T-lymphoblastoid cell lines (MDCC) demonstrated that Marek's disease virus DNA was located in the short arm of chromosome 2 of MDCC-MSB1 and MDCC-RP1 cells and in the short arm of chromosome 4 of MDCC-MSB1 cells. The MDV DNA sequences in these chromosomes are not in the vicinity of oncogenes or integrated retroviruses that have been previously identified in chicken chromosomes. PMID- 3012871 TI - Susceptibility of avian B lymphocytes to retroviral infection parallels that of fibroblasts. AB - The ability of conventional virus receptors to mediate infection and transformation of immature B cells by the avian leukosis virus (ALV) was analyzed in chimeric chickens whose bursa of Fabricius contained a mixture of B cells from subgroup A virus receptor-positive and -negative chickens. The data indicate that factors that determine resistance of fibroblasts to ALV infection also apply to bursal cells and that other receptors expressed by B cells, i.e., antigen or mitogen receptors, do not initiate infection or transformation, at least not independent of conventional virus receptors. PMID- 3012872 TI - The AIDS-associated retrovirus is not sensitive to lysis or inactivation by human serum. AB - Unheated human serum does not lyse the AIDS-associated retrovirus (ARV), change the density of the virus, or suppress its ability to infect human peripheral mononuclear cells. The results indicate that ARV and the human oncovirus HTLV-I, unlike other animal retroviruses, are resistant to the effect of human serum. These two human viruses coming from different retrovirus subfamilies may be pathogenic because of this lack of sensitivity to human complement components. PMID- 3012873 TI - Induction of the autonomous stage of transformation in erythroid cells infected with SFFV: helper virus is not required. AB - The erythroleukemia induced by the Friend spleen focus-forming virus (SFFV) in mice exemplifies a multistep oncogenic process. Its sequential steps include a rapid polyclonal hyperplastic stage and a more slowly developing malignant stage characterized by autonomous erythroid cells. We report here that the helper virus normally present in mice infected by SFFV is not required for development of the second stage of transformation. In this study, mice were infected with a polycythemia-inducing variant of SFFV which was prepared as a helper-free stock (L. Wolff and S. Ruscetti, 1985, Science 228, 1549). Highly malignant cells could be detected in helper-free SFFV-infected mice by their transplantability into the omentum of sublethally irradiated mice, and erythroleukemia cell lines, typical of previously isolated Friend murine erythroleukemia cell lines, could be established from diseased spleens. Like their helper virus-containing counterparts, the lines established with helper-free SFFV are inducible for hemoglobin synthesis with a variety of chemicals, but not erythropoietin, and express p53, a marker of malignant transformation. Although the cells expressed SFFV encoded proteins, none expressed gene products of replication competent murine leukemia viruses. PMID- 3012874 TI - Location of the structural gene of pseudorabies virus glycoprotein complex gII. AB - The glycoprotein gII, one of the major glycoproteins of pseudorabies virus (PRV), is represented by a complex of three related glycopolypeptides. There is evidence that two of them, linked by disulfide bonds, arise by proteolytic cleavage of the larger precursor glycoprotein. Using specific antisera and a monoclonal antibody against the glycoprotein complex one single nonglycosylated in vitro translated precursor polypeptide with mol wt 110,000 was identified. Mapping of the gene coding for this polypeptide was achieved by hybrid selection of late viral RNA on cloned DNA fragments. The structural gene for the gII complex was found to reside in the long unique part of the PRV genome on BamHI fragment 1 and SalI subfragments 1A and G (map units 0.105 to 0.130). A 3.5-kb mRNA was identified as the probable gII-specific transcript. In addition, further polypeptides encoded in the BamHI fragment 1 were described and RNAs were characterized by Northern blot hybridizations with the cloned SalI subfragments. PMID- 3012875 TI - Activation of purified simian virus 40 virions by free amino-group containing phospholipid liposomes. AB - The infectivity of the simian virus 40 (SV40) virions purified after treatment with sodium deoxycholate is activated by mixing, prior to infection, the virions with the liposomes composed of phosphatidylserine or a mixture of phosphatidylethanolamine and phosphatidylcholine (H. Shimura, and G. Kimura (1985), Virology 144, 268-272). The sucrose-CsCl cushion sedimentation analysis of the virions mixed with the liposomes revealed that the density of the radiolabeled virions became lower and that of the radiolabeled liposomes became higher to give a similar range, suggesting the binding of virions with the liposomes. Electron microscopy revealed the side-to-side association of virions with liposomes. The efficiency of adsorption of the virions to monkey kidney BSC 1 cells varied depending on phospholipid types mixed with virions and did not always become high. In the case of phosphatidylethanolamine liposomes, the free amino group in the phospholipid molecule was essential for the activation of the virion infectivity, because mono- and di-methylated phosphatidylethanolamine failed to activate the infectivity. Fluid nature of phospholipids seemed to be necessary also for the infectivity activation, because dipalmitoyl and distearoyl phospholipids did not activate virion infectivity at 37 degrees, the temperature at which the liposomes of these phospholipids are supposed to be in a solid state. Presence of free amino groups and difference in acyl groups of the phospholipids did not influence the adsorption of the virions to cells. These results suggest that events which occur after adsorption of virions to cells are responsible for the activation of the SV40 virion infectivity by the liposomes composed of free amino-group containing phospholipids. PMID- 3012877 TI - Electrical feedback mechanism in the processing of signals in the outer plexiform layer of the retina. AB - In any chemical synapse there is an electrical feedback initiated by the electrical current generated by the postsynaptic neurone. We argue that this feedback can be rather effective in the case of invaginating synapses in the retina. A model of a dyad synapse (cone-horizontal cell-bipolar cell) is developed, in which the transfer function between the cone and bipolar cell is modulated by the synaptic current of the horizontal cell. This modulation originates by the change of the potential drop along the intercellular gap between the cone and the horizontal cell. The model takes into account the electrical coupling between the horizontal cells as well as the nonlinearity of their nonsynaptic (somatic) membrane. The model reproduces qualitatively the steady-state responses of an hyperpolarizing bipolar cell to the light spot and an annulus. It gives also the adaptational (with a light background) shift of the cone-bipolar cell transfer function. The model can be applied to depolarizing bipolar cells and to C-type horizontal cells. PMID- 3012878 TI - [Characteristics of the surgical treatment of chemodectomas]. PMID- 3012876 TI - Antibodies against small nuclear ribonucleoproteins immunoprecipitate complexes containing ts110 Moloney murine sarcoma virus genomic and messenger RNAs. AB - Small nuclear ribonucleoproteins (snRNPs) are believed to play a role in processing premessenger RNAs. In this study, snRNPs were immunoprecipitated from extracts of cells infected with ts110 Moloney murine sarcoma virus (ts110 MoMuSV). Both the unspliced 4.0 kb and the spliced 3.5-kb ts110 MoMuSV specific RNA species were found in the immunoprecipitates obtained with monoclonal antibody anti-Sm and polyclonal anti-Sm, anti-(U1) RNP and anti-La sera. Although only a portion of the total ts110 RNAs was present in these immunoprecipitates, immune recognition by the anti-snRNPs was specific and not due to contaminating anti-RNA (at least for the anti-Sm sera) or, to anti-viral protein activities. Genomic 8.3-kb RNA and subgenomic 3.0-kb spliced env mRNA from Moloney murine leukemia virus (MoMuLV) infected cells as well as the cellular actin mRNA were also detected in immunoprecipitates obtained with the same antisera. The fact that pre-mRNAs and mature mRNAs of different origin can be recovered from immunoprecipitates formed with anti-snRNP sera establishes their tight association and confirms the role of snRNPs in mRNA processing. PMID- 3012879 TI - [Malignant glomus tumor of the stomach (1 case)]. PMID- 3012880 TI - [Ultrastructure of small-cell lung cancer]. AB - The research was concerned with electron microscopic analysis of 34 lung tumors histologically identified as small cell lung cancer. It was shown that ultrastructural studies can reliably identify histologic pattern of small cell lung cancer. In most cases, this type of cancer was represented by one of the following histologic patterns: epidermoid, glandular or neuroendocrine, the latter being predominant. An ultrastructural classification of small cell lung cancer is not advisable until a clinico-morphological analysis has been carried out. PMID- 3012881 TI - Anti-HTLV-III screening in multitransfused thalassemia patients. PMID- 3012882 TI - [Differential diagnosis of insuloma]. PMID- 3012883 TI - [Secondary thrombosis of the renal veins in thrombosis of the inferior vena cava]. AB - On the occasion of the diagnosis secondary thrombosis of both renal veins made during one's life, resulting from the progressive thrombosis of v.cava inferior, the attention is drawn on that possibility in case of nephrotic syndrome with edema of the lower limbs, showing asymmetry to a certain extent. Diagnostic precision is recommended in such cases, looking for alterations in venous urography, INF or dynamic chamber scintigraphy with 131I-hippuran, perfusion angioscintigraphy with 99Tc-pertechnetate and in computer tomography. PMID- 3012884 TI - AIDS: the search for clues. PMID- 3012885 TI - Ulnar nerve abscess in Hansen's disease. PMID- 3012887 TI - [Primary bronchial cancer in patients under 40]. PMID- 3012886 TI - Near-fatal coagulopathy associated with Epstein-Barr virus hepatitis. PMID- 3012889 TI - [A 61-year-old male with a high-grade duodenal stenosis]. PMID- 3012888 TI - [Biochemical disorders in psoriasis]. PMID- 3012890 TI - Recommended health-based limits in occupational exposure to selected mineral dusts (silica, coal). Report of a WHO Study Group. PMID- 3012891 TI - Groundwater quality today and tomorrow. PMID- 3012892 TI - [Incidence of neuropathies in so-called rheumatoid vasculitis]. AB - 40 patients with so-called rheumatoid vasculitis, a special form of the course of rheumatoid arthritis with high rheumatoid titres and inclination to extraarticular organ manifestation were clinico-neurologically and electro diagnostically examined. Neurologically in 10 patients a neuropathy was found, in which case polyneuropathies numerically prevailed. By means of the additional electromyographic and electroneurographic examination in altogether 75% of the patients pathological findings and thus a participation of the peripheral nervous system could by proved. The aetiology of the individual disturbance of the nerve function remains open. Questions of the pathogenesis are discussed. PMID- 3012894 TI - [HTLV-III infection. New routes of transmission?]. PMID- 3012893 TI - [Peripartal monitoring of an HTLV III-infected, heroin-dependent pregnant patient]. AB - In the past the acquired immune deficiency syndrome (AIDS) could be mainly observed in homosexual and drug addicted persons. But at present one has to deal with this problem in obstetrics as well. In this paper a report is given on the late pregnancy and delivery in a heroin addicted gravida with positive HTLV III antibodies. We describe the useful tests to characterize the immunologic status of the patient and give recommendations for the clinical management of pregnancy and delivery. PMID- 3012895 TI - [Bowenoid papulosis and its relation to Bowen's disease and atypical condyloma]. AB - All the 5 sexual partners of male patients suffering from bowenoid papulosis and bearing the HPV 16 genome showed cervical lesions induced by HPV. 2 of them proved to be noncondylomatous infections, 3 cases were cervical condylomas, and 2 patients revealed severe dysplasia or carcinoma in situ (CIS). 3 out of 4 women suffering from multicentrical pigmented Bowen's disease of the vulva showed plain condylomas of the cervix, with severe dysplasia or CIS in 2 cases and slight dysplasia in one case. Thus bowenoid papulosis as well as multicentrical pigmented Bowen's disease of the vulva are high risk factors for cervical HPV infection and carcinoma, both for the patients themselves and their sexual partners. Atypical pigmented genital condylomas induced by HPV 6 also bear a risk for cervical HPV infection of the sexual partners. Malignant transformation, however, seems to depend on the type of the infecting virus (mainly HPV 16). PMID- 3012896 TI - [Bowenoid papulosis of the genito-anal region]. AB - Bowenoid papulosis is a new and rather frequent entity affecting young men and women. Clinically, the lesions consists of multiple red or brown papules in the genitoanal region. Histologically, the papules shows alterations of the epidermis similar to those in Bowen's disease. The course of the disease, however, is benign. Recent investigations by zur Hausen, Ikenberg et al. have demonstrated the presence of HPV type 16-DNA in bowenoid papulosis. PMID- 3012897 TI - [Kaposi sarcoma within the scope of AIDS]. AB - The disseminated form of Kaposi's sarcoma (KS) is an important cutaneous manifestation of acquired immune deficiency syndrome (AIDS). Since 1983, we observed 5 patients suffering from this rare multifocal cancer associated with AIDS. We report on clinical picture, histopathologic changes, and immunologic parameters. PMID- 3012898 TI - AIDS: clinical signposts for the Virginia physician. PMID- 3012899 TI - [Myeloperoxidase activity in neutrophils of patients with disseminated psoriasis]. PMID- 3012900 TI - [Specific and nonspecific neuronal mechanisms in learning]. PMID- 3012901 TI - [Physiology of higher nervous activity: developmental perspective]. PMID- 3012902 TI - [Reflection of the plastic properties of the simplest neuronal associations in statistical characteristics of the spike activity of their elements. Polysynaptically linked neurons]. AB - Changes of crosscorrelation histograms of trains of action potentials and mean interspike intervals of polysynaptically connected neurones were studied by means of mathematical modelling of synaptic neuronal interaction at changes of efficiency of interneuronal monosynaptic connections, at changes of neuronal excitability, and at changes of total action on them of independent disorderly afferent synaptic inflows. Increase of amplitude of the main maximum (minimum) of the normalized crosscorrelation histogram of trains of action potentials accompanied by reduction of mean interspike intervals of both neurones, was shown to be a unsignificant indication of an increase of efficiency of polysynaptic excitatory (inhibitory) connections between the neurones (due to modification of synapses or to a change of the functional state of interneurones). PMID- 3012903 TI - [Phytic acid and cereals and cereal products. I: Phytic acid and phytase in rye and rye products]. AB - Phytic acid in food is considered to be responsible for a reduced bioavailability of essential dietary minerals; its detrimental effects can be diminished by hydrolysis with phytase during processing. The average phytic acid content was 8.18 mg/g and 3.44 mg/g and average phytase activity was 3.7 U/g and 2.6 U/g in rye kernels and in flour (Type 997, 1.09 ash content), respectively. Phytate and Phytase were about equally distributed between the two kernel halves (cross sections). During the early stages of germination (3 days) phytase activity did not change, and phytic acid content was reduced to 67%. After milling most of the phytic acid and phytase activity were found in the bran fractions. It is concluded that substrate and enzyme are present in the same kernel structures but separate within the cells. Cooking of ground rye caused a phytate hydrolysis which was the more effective 1.) the smaller the particle sizes were, 2.) the more water was added, and 3.) the longer phytase worked at optimum temperature. Extrusion cooking of the rye whole flour at up to 100 degrees C did not influence the phytic acid level but caused a 23% reduction at 170 degrees C. Phytase activity was reduced by 80% by extrusion cooking at 80 degrees C. PMID- 3012905 TI - [The role of disturbed DNA synthesis in the genesis of cervical carcinomas. Results of histoautoradiographic studies]. AB - Histoautoradiographic and histopathologic studies are presented for normal and atypical cervical squamous epithelium from biopsies in 129 women. Labelling indices were obtained using in vitro labelling of biopsy specimens with H3 thymidine. In 38 cases with normal squamous epithelium, different indices were observed depending on the indication for operation. The index for normal epithelium with nearby preneoplastic changes was 5.3 +/- 1.8; for normal epithelium of patients with other diseases (uterus myomatosus, descensus uteri) it was 2.8 +/- 1.3. The index for virus-induced koilocytic dysplasias (6.3 +/- 2.9) lay between those of mild (5.3 +/- 1.4) and moderate dysplasias (7.2 +/- 1.8) without histopathological signs of virus infection. Carcinoma-in-situ (11.7 +/- 4.0) and microcarcinomas (12.6 +/- 4.2) showed higher values. There were no significant differences between the labelling indices of the microcarcinomas and preneoplastic lesions. Early stages of tumor development may histoautoradiographically offer hints of proliferative activity. In later stages the proliferative heterogeneity of the tumors limits the interpretability of the indices. The results are discussed in light of the possible role played by papilloma virus in the genesis of cervical cancer. PMID- 3012904 TI - Acute effects of carbon monoxide and cyanide on hepatic mitochondrial function. AB - The effects of carbon monoxide and cyanide on the hepatic redox state and energy charge were investigated. Rats were used for the experiment under pentobarbital anesthesia. Immediately after laparotomy, a rat was placed in an animal chamber made of a transparent plastic box and exposed to a test gas for 3 min. Every test gas was produced in a gas chamber connected to the animal chamber with a flexible tube. HCN was produced from NaCN and H2SO4. In the CO inhalation experiment, various amounts of CO were introduced into the gas chamber. Immediately after an exposure, about 2 g liver was frozen in situ with a precooled clamp. Oozed blood from the wound surface was sampled. Concentrations of ATP, ADP, AMP, acetoacetate, and beta-hydroxybutyrate in hepatic mitochondria were determined, and the redox state and the energy charge were calculated. For cyanide as well as CO, significant negative correlations were found between the concentration in the blood and the redox state. The same held true for the energy charge. The redox state showed a slight increase at low concentrations of both gases; however, thereafter it began to decrease sharply with increases in concentrations. When concentrations of the toxicant in the blood reached certain levels, a kind of turning point, beyond which the redox state does not decrease any more, was observed. It was about 40% for HbCO and about 2.0 micrograms/ml for cyanide, and the points seemed to be related to the concentrations, beyond which cells are irreversibly damaged. On the other hand, the energy charge did not change at low concentrations. With an increase in toxicant concentrations, the energy charge decreased drastically. The rate of decrease in the energy charge became higher when blood concentrations exceeded certain levels. It was about 40% for HbCO and 2.0 micrograms/ml for cyanide. The presence of low levels of blood cyanide did not affect the relationship between the energy charge and the HbCO concentration. PMID- 3012906 TI - [2D-histograms of the nucleus area and nucleus shape in the diagnosis of astrocytomas and glioblastomas]. AB - 8 astrocytomas grade 1-4 and 2 glioblastomas were investigated by a system of automated microscope picture analysis. Histograms of nuclear area and shape from more than 1,000 measured nuclei per tumor were analysed. Both one- and two dimensional histograms show findings which correlate with the malignant potential of the gliomas. The nuclear area rises with increasing tumor malignancy. On the other hand the measurements of nuclear shape factor are inversely related to the level of malignancy, indicating substantial irregularities in the nuclear structure. The 2D-histograms of both measured parameters also show changes which are correlated with the grade of tumor malignancy and which can be described as shift, loss of compactness and enlargement. The histogram method is discussed as an objectifiable process characterizing important cell and nuclear attributes connected with tumor malignancy. Histograms of both, nuclear area and shape can be helpful in the classification and grading of astrocytomas and glioblastomas. PMID- 3012907 TI - [Adenosine triphosphatase activity in the osmoregulatory organs of vertebrates]. AB - Studies have been made on the activity of cation- and anion-stimulated ATPases, as well as succinic dehydrogenase in homogenates and subcellular fractions from osmoregulatory organs of marine (elasmobranch and teleost) and freshwater (teleost) fishes, amphibians, reptiles, birds and mammals. The activity of Na+, K+-ATPase was found to be rather similar in almost all osmoregulatory organs of the species investigated. The highest level of Cl-stimulated ATPase was found in microsomal fraction of the kidneys from birds and mammals. Succinic dehydrogenase activity is significantly higher in the renal tissue of mammals, both in total homogenates and in mitochondrial fraction. PMID- 3012908 TI - [Transmission in the sensorimotor synapses of the lumbar spinal cord in Rana ridibunda tadpoles]. AB - In experiments on preparations of isolated spinal cord of the tadpoles, intracellular studies have been made on the synaptic potentials evoked in the lumbar motoneurones during total activation of the fibers within the 9th dorsal root. It was shown that primary afferents form monosynaptic contacts with motoneurones at stages XIV-XXV. During larval development, the number of motor cells in which monosynaptic EPSPs are recorded increases, whereas the number of motoneurones with only polysynaptic reactions decreases. From the moment of formation of monosynaptic contacts, transmission in direct sensory-motor synapses is realised by a dual (electrical-chemical) mode. The data obtained are discussed in relation to the problem of evolution of synaptic transmission between heterotypic neurones in vertebrates. PMID- 3012909 TI - [Action of plant extracts on the natural immunity indices of animals]. AB - The influence of extracts from oak bark, St. John's-wort leaves and pine buds on natural immunity characteristics of mice has been studied. The injection of these extracts into mice has been found to enhance their resistance to infection with Staphylococcus aureus and Bordetella pertussis virulent cultures, to decrease the enzymatic activity of 5'-nucleotidase in the peritoneal exudate macrophages of mice and to increase the level of lysozyme in their blood. The action of these extracts has proved to depend on their dosage and the time of observation. PMID- 3012910 TI - [Diagnostic significance of indices of somatosensory evoked potentials in painful radicular and reflex syndromes in patients with osteochondrosis of the lumbar portion of the spine]. AB - Forty patients with vertebrogenic painful syndromes related to radicular, muscular-tonic and neuro-dystrophic damage to the peripheral nervous system were examined for somatosensory evoked potentials of the projection zone of the cortex. The authors have shown changes in the latent periods of late components II180 and H225 depending on the nature of the syndrome and the possibility of the differential diagnosis of pain afferentation. PMID- 3012911 TI - [Effect of decimeter waves on the functional status of spinal alpha-motor neurons in patients with neurologic syndromes of lumbar osteochondrosis]. AB - Patients with neurological syndromes of lumbar osteochondrosis were subjected to clinico-electromyographic examination to study whether it is possible to regulate the function of spinal alpha-motoneurons by decimetric waves (DMW). The effect of DMW on the excitability of alpha-motoneurons was found to depend on their initial function. The neurophysiological mechanisms underlying the therapeutic action of DMW in this pathology are analyzed. PMID- 3012912 TI - [Peripheral neuropathies caused by antibiotics during their production and administration]. AB - The authors consider the clinical picture and pathogenesis of damage to the peripheral nervous system in patients suffering from occupational disease induced by exposure to antibiotics. In clinical terms it is most often manifested in sensory polyneuropathy and less frequently in isolated damage to neural stems (facial, auditory, sciatic, external femoral skin nerves). Allergic vasculitis due to exposure to antibiotics is responsible for this pathology of the peripheral nervous system. PMID- 3012914 TI - [Effect of 3',5'-cAMP on the function of Ehrlich ascites tumor cell membranes in vivo]. PMID- 3012913 TI - [Prospects for using formed elements in the blood as an extracerebral model for the study of receptors in the central nervous system (review)]. PMID- 3012915 TI - [The biological effects of sodium butyrate on cultured human stomach cancer cells and esophageal cancer cells]. PMID- 3012917 TI - [Application of electron spin labeling to investigate the effects of gamma irradiation on erythrocyte membranes]. PMID- 3012916 TI - [The status of steroid receptors and hormone response in 7,12-dimethylbenz (a) anthracene (DMBA) induced rat mammary tumor]. PMID- 3012918 TI - Current problems of the diagnostics of the retroperitoneum. AB - The authors discussed the examinations yielding optimal results in revealing the pathological retroperitoneal processes. Among those were bilateral dorsopedal lymphography, ultrasonography and computed tomography. They evaluated these examinations in detecting the retroperitoneal lymph node metastases of 15 non seminoma patients. They describe their 4 cases of extragonadal germ-cell and testicular tumours and speculate on their development. They suppose that the 'unexplainable' renal colics of the older male patients can possibly be due to primary retroperitoneal germ-cell tumours. PMID- 3012919 TI - Insulinoma of the pancreatic head: results from two surgical strategies. AB - A rare location of insulin-secreting pancreatic tumour, which presents technical difficulties, is centrally in the pancreatic head close to the duodenal wall. Local excision or enucleation gives very high postoperative morbidity, with pseudocyst and fistula, and more extensive surgery such as pancreatoduodenectomy has a high mortality rate in these patients. At Linkoping University Hospital, five patients with insulinoma at this particular site were diagnosed and operated on in 1978-84. After excision of the tumour, the enucleation cavity was treated with one of two surgical strategies--closure and drainage (3 cases) or drainage only (2 cases). The postoperative course differed greatly between the two groups. The experience from these cases suggests that endocrine tumours at this site in the pancreas can be excised locally, with strict safeguarding of the pancreatic duct, but that the cavity in the pancreas should be left open with long-term drainage. PMID- 3012920 TI - Mullerian inclusions in peritoneal washings. Potential source of error in cytologic diagnosis. AB - Mullerian inclusions in peritoneal washings from female patients may be mistaken for adenocarcinoma. Such findings were studied in the peritoneal washing cytology specimens from eight cases. The inclusions usually presented as tubular or papillary structures, often forming a single layer of epithelium surrounding psammoma bodies. The cells forming these structures often displayed some degree of atypia. Recognition of this entity in peritoneal fluids is important to avoid a misdiagnosis of disseminated cancer. A general outline is proposed for interpreting such findings in peritoneal washings, based on the cytomorphology of these structures as well as the microscopic features of the primary neoplasms. PMID- 3012921 TI - Morphologic spectrum of hepatocellular carcinoma in fine needle aspiration biopsies. AB - The light microscopic features of fine needle aspiration smears from 52 primary hepatocellular carcinomas were reviewed. Cytologic grading of these tumors, which indicated three grade I, 29 grade II and 20 grade III carcinomas, was compatible with grading done by histologic examination. In addition to the general cellular characteristics, other cytologic features observed in the smears included bile (48% of the cases), large cytoplasmic vacuoles (23%), small cytoplasmic vacuoles (46%), eosinophilic cytoplasmic inclusions (8%), basophilic cytoplasmic inclusions (48%) and intranuclear cytoplasmic inclusions (71%). The significance of these features is briefly discussed. PMID- 3012922 TI - New and prospective developments in antifungal drugs. AB - In recent years many new antifungal compounds have been developed. Some of them are currently under investigation as orally or topically active drugs in human mycotic infections. The 3 main groups in this development are imidazoles, triazoles and allylamines. The group of imidazoles consists mainly of topical drugs with an activity comparable to that of miconazole, clotrimazole e.a. Besides ketoconazole, some other orally active imidazoles are currently under development e.g. SC38390. The chemical group of antifungals receiving the most attention at present is the tiazole family. Both topical (e.g. terconazole) as oral (e.g. R 51 211, Bay 7133) drugs are currently investigated. A third group consists of allylamines in which SF86-327 is developed for both oral and topical use. PMID- 3012923 TI - Impaired insulin secretion in human diabetes mellitus. Effect of pharmacological activation of gamma-aminobutyric acid system. AB - To evaluate whether the gamma-aminobutyric acid (GABA)ergic system plays a role in the defective insulin secretion in human diabetes mellitus, 15 non-insulin dependent diabetics with fasting hyperglycemia above 140 mg/dl were submitted to two consecutive i.v. glucose tolerance tests (IVGTT) (0.33 g/kg b.w.), in basal conditions and after pharmacologic activation of the GABA system with baclofen and sodium valproate. Baclofen, a synthetic analogue, was given to 8 diabetics in two divided doses of 10 mg each 8h and 1h before the post-treatment test; sodium valproate, a drug that increases endogenous GABA activity, was given orally (800 mg) 60 min before the performance of the post-treatment IVGTT. Neither treatment brought about significant changes in insulin, C-peptide, glucagon or growth hormone responses to i.v. glucose nor did they significantly change glucose disappearance rates. These results seem to indicate that GABA does not play a major role in the pathogenesis of defective insulin secretion in non-insulin dependent diabetes mellitus. PMID- 3012924 TI - The GABAergic control of prolactin release is not affected by insulin-dependent diabetes mellitus: evidence from studies with sodium valproate. AB - In order to evaluate the influence of the GABAergic system in the regulation of PRL secretion in patients with IDDM, serum PRL levels were measured in 7 diabetics and in 7 normal men with sodium valproate (400 mg per os), a drug capable of increasing cerebral GABA concentrations. A significant decrease of serum PRL concentrations was observed between 30 and 120 min after sodium valproate administration in both control and diabetic subjects. The time course and magnitude of the sodium valproate effect were similar in all subjects. These data confirm the inhibitory control of the GABAergic system on PRL secretion in man as evidenced by the GABAergic drug sodium valproate. It is suggested that this system is not altered in diabetic patients. PMID- 3012925 TI - Ovine corticotrophin-releasing factor in dogs: dose-response relationships and effects of dexamethasone. AB - Dose-response relationships between iv bolus injections (0, 0.1, 1 or 10 micrograms/kg) of synthetic ovine corticotropin-releasing factor (oCRF) and plasma immunoreactive (i) ACTH and cortisol concentrations were examined in healthy, conscious dogs. All doses of oCRF resulted in elevated plasma iACTH and cortisol levels over those of the controls. Maximum (or Peak) plasma iACTH concentrations were generally observed 20-30 min after oCRF and the magnitude of these peaks was a linear function (P less than 0.001) of the logarithm of the oCRF dose. The time of peak cortisol concentrations was more variable but the peak cortisol level was also linearly related (P less than 0.001) to the logarithm of the oCRF dose. An estimate for the response areas for both hormones demonstrated a quadratic (P less than 0.05) relationship with the logarithm of the oCRF dose. The relationship between oCRF and the iACTH response suggested a progressively greater response at increasing oCRF doses while a maximally effective oCRF dose was predicted in the cortisol response area relationship. Graded (0, 0.01, 0.1 or 1 mg/kg) bolus doses of dexamethasone produced a dose dependent (P less than 0.03) decline in baseline plasma iACTH levels and a non dose-dependent suppression in baseline plasma cortisol. Pretreatment with 0.001 mg dexamethasone/kg 4 or 8 h before injection of 1 microgram oCRF/kg did not alter the plasma iACTH or cortisol response; however, 0.1 mg dexamethasone/kg administered at these times totally abolished the responses to oCRF.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012926 TI - Effect of insulin-like growth factors on human foetal, adult normal and tumour pituitary function in tissue culture. AB - To determine the direct effects of insulin-like growth factors (IGFs) on hormone release by the human pituitary gland, human foetal, adult normal and tumour pituitary tissues were maintained in culture for 2 to 4 weeks and tested with acute (3 h) exposures to different preparations of IGF peptides. Adult normal pituitaries and adenomas were tested with a semipurified preparation of IGFs, free of immunoreactive insulin, containing IGF-I and IGF-II in a ratio of approximately 1:4. Human foetal pituitaries were tested with the semipurified IGFs as well as more purified preparations of IGF-I and IGF-II. Culture media were assayed for hGH, hPrl, hACTH and hLH using specific radioimmunoassays. Both foetal (n = 16 (No. of pituitaries), 33 (No. of observations] and normal adult (n = 3, 16) human pituitaries cultures responded to the semipurified IGFs (2-25 ngEq/ml for foetal and 2-4 ngEq/ml for adult pituitaries) with a significant decrease in hGH release compared to basal (P less than 0.01) whereas the GH secreting pituitary tumours showed no effect when tested with from 2 to 25 ngEq/ml (n = 8, 129, NS). The effect of IGFs on human foetal somatotrope activity was dose-related for both the semipurified IGFs (2-25 ngEq/ml, n = 16, 33) and IGF-I or IGF-II (10-100 ng/ml; n = 3, 18).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012927 TI - Alpha globin gene triplication in severe heterozygous beta thalassemia. AB - In an Algerian family, three sibs with an unusually severe heterozygous beta thalassemia and two sibs with a typical heterozygous beta-thalassemia were found. Both conditions were transmitted vertically. Globin chain synthesis and DNA restriction enzyme analysis showed that the unusual severity of heterozygous beta thalassemia observed in this family is related to an overproduction of alpha globin chains originating from an alpha-globin gene triplication. PMID- 3012928 TI - Lipid content and ESR determination of plasma membrane order parameter in Candida albicans sterol mutants. AB - Sterol intermediates of ergosterol biosynthesis in seven sterol mutants of Candida albicans were determined by gas-liquid chromatography. Only one of them could synthetize ergosterol, while in the others sterol biosynthesis was blocked beyond zymosterol. Alterations in sterol composition were correlated with a slight increase in saturation and a decrease in the chain length of fatty acids, and increases in phosphatidylinositol and phosphatidic acid, and decreases in phosphatidylcholine and phosphatidylserine contents. During exponential growth, as measured on their protoplasts using the intercalated fatty acid spin probe, 5 doxylstearic acid, these single mutants exhibited higher plasma membrane order parameters than their ergosterol-producing parental strain, designated 33 erg+, as follows: erg-12 greater than erg-16 greater than erg-37 much greater than erg 2 greater than erg-20 greater than erg-40 greater than erg-41 greater than 33 erg+. The mutants displayed significantly higher phase-transition temperatures, measured in a reconstituted lipid-water dispersion, than their parental strain. PMID- 3012929 TI - Effect of amino acids on the expression of antiviral activity of different types of human interferon. I. Effect of single amino acids. AB - It has been observed that certain amino acids may influence the antiviral activity of different types of human interferon (IFN). The effect may be enhancing or inhibitory. A qualitative survey is provided on all three types of human IFN using 23 different amino acids. A more detailed study was carried out with 10 amino acids which proved to be active towards HuIFN-alpha. Their influences on the synergistic antiviral effects of HuIFN-alpha and HuIFN-gamma were also investigated. The dose-dependence curves of their effects on HuIFN alpha were established. PMID- 3012930 TI - Effect of amino acids on the expression of antiviral activity of different types of human interferon. II. Effects of various amino acid pairs. AB - Seven amino acids which influence the antiviral activity of IFN-alpha were tested in various pairs on human leukocyte and immune IFN and on their synergistic antiviral effect. Different kinds of interactions were observed between amino acids, ranging from extinction through competition to synergism. In some cases, different types of interactions could be observed in relation to HuIFN-alpha and HuIFN-gamma. It has been reported that certain amino acids influence the antiviral activity of human interferons (IFNs). Some of them are even capable of affecting synergistic cooperation between HuIFN-alpha and HuIFN-gamma. In our experiments we tested whether these effects can be modified further by applying two amino acids simultaneously. We hoped to shed light on the problem of their mode of action by analysing their interactions. PMID- 3012931 TI - Production of high titre human interferon-gamma in primed leukocyte cultures. AB - Con A-stimulated leukocytes previously treated with IFN-alpha (1500 IU/ml for 4 h) were very suitable for the production of IFN-gamma with a high titre even under large-scale conditions. The properties of the IFN produced under such conditions were identical to those of IFN-gamma. The IFN-gamma produced in primed cultures showed two peaks of antiviral activity corresponding to molecular weights of 51 000 and 23 000 daltons. PMID- 3012932 TI - A clinicopathological review of 12 autopsied cases of adult T-cell leukemia. AB - Twelve autopsied cases with adult T-cell leukemia (ATL) were reviewed clinicopathologically. The prognosis of three cases who had suffered from severe cutaneous lesions was much better than that of the other nine cases with no or negligible cutaneous lesions. The surface marker of leukemic cells from six cases was ordinary inducer/helper phenotype (OKT4+ and 8-), but in one case leukemic cells showed OKT4+ and 8+. In another case, a significant amount of leukemic cell infiltration was found in the thymic cortex. Calcium content in the bone of ATL cases was lower than that of the patients without ATL (control group), and six cases with ATL (50%) were complicated by severe hypercalcemia. Neither adenoma nor hyperplasia of the parathyroid glands was found in any case. In most severely hypercalcemic patients, bone trabeculae were actively absorbed by numerous osteoclasts and partly replaced by fibrous tissues. In two normocalcemic patients, skeletal calcium content was also markedly reduced by osteoporosis, but the activation of osteoclasts was inconspicuous. It was speculated that the manner of bone resorption in ATL cases was diverse and there were some clinicopathological subtypes in ATL from the viewpoints of cutaneous lesions, hypercalcemia, and bone lesions. PMID- 3012933 TI - Gastric leiomyosarcoma with massive myxoid degeneration. A histochemical and ultrastructural study. AB - Histochemical and ultrastructural features of a gastric myxoid leiomyosarcoma from a 55-year-old man were examined. At autopsy, the tumor was located mainly in the greater omentum and was directly connected to a coinsized gastric tumor. Multiple hepatic metastases and peritoneal disseminations were noted. Light microscopically, the tumor was composed of a prominent myxoid stroma and ovoid or rounded tumor cells. The myxoid stroma was stained weakly basophilic with hematoxylin and eosin, and was mainly composed of hyaluronic acid. Tumor cells in the stomach were spindle-shaped and apparently myogenic. Ultrastructurally, the gastric tumor cells showed a loose cohesion with junctional apparatuses, pinocytotic vesicles, basal laminae, and cytoplasmic filaments with focal densities. Tumor cells in the omentum and the liver, however, were poorly differentiated and showed an epithelioid nature in part. This unique leiomyosarcoma is reported with some differential diagnoses from other myxoid sarcomas. PMID- 3012934 TI - Breast cancer with reactive multinucleated giant cells: report of three cases. AB - We report here three cases of breast cancer with reactive multinucleated giant cells. The patients were among the 605 patients with breast cancer seen in the past 17 years at Tenri Hospital; the incidence of this variety of breast cancer was 0.5%. Enzyme histochemical and electron microscopic examination suggested that the giant cells were of histiocytic origin. However, results of immunohistochemical technique, S-100 protein, lysozyme, nonspecific cross reacting antigen with carcinoembryonic antigen, alpha-1-antitrypsin, and alpha-1 antichymotrypsin, all currently used as markers of histiocytes, were negative. Because of the rarity of this variety of breast cancer, the biological significance of these unusual findings remains unknown. PMID- 3012935 TI - Neutrophil aggregation--factors modulating stimulus-specific responses. AB - After stimulation with various soluble stimuli, e.g. fMLP and LTB4, human neutrophils aggregated in a characteristic and stimulus-specific way. They formed clumps of cells, mainly pairs and triplets. Their calculated total light obscuring cell area corresponded well with the recorded aggregation response assessed as light transmittance, when not only neutrophil clumping but also cell swelling and shape changes were taken into account. Changes of neutrophil concentrations had only marginal effects on maximal responses and their kinetics. Lowering cell temperature to 26 degrees C decreased the responses to fMLP, whereas LTB4 responses were more inhibited by increasing temperatures to 42 degrees C. At 26 degrees C, both LTB4 and fMLP induced biphasic aggregation waves with two peaks. Cytochalasin B enhanced the response both to LTB4 and fMLP, and LTB4 responses became biphasic with an early peak preceding the major response. The presence of high concentrations of platelets potentiated aggregation responses, a phenomenon that could not be ascribed to generation of cyclo oxygenase products. Thus, the formation of neutrophil aggregates and the subsequent change in light transmission can be modulated by several experimental factors which influence the stimulus-specific responses, and the effects on fMLP induced aggregation under some experimental conditions are different from those on LTB4. PMID- 3012936 TI - [Solubilization of opiate receptor and the effect of dilution on binding activity]. PMID- 3012937 TI - [Study on ternary ion-associate formation between tetraiodofluorescein and benzalkonium in the presence of quinine]. PMID- 3012938 TI - [Studies of lignans from Schisandra rubriflora Rhed et Wils]. PMID- 3012939 TI - [Isoquercitrin increases brain cGMP levels and potentiates electroacupuncture (EA) analgesia]. PMID- 3012940 TI - [Irreversible ligands of opioid receptors and their use in the study of these receptors]. PMID- 3012941 TI - The xanthine derivative 1-(5'-oxohexyl)-3-methyl-7-propyl xanthine (HWA 285) enhances the actions of adenosine. AB - The effect of two closely related xanthine derivatives, pentoxifylline and HWA 285, on cyclic AMP accumulation in rat hippocampal slices and on adenosine uptake in erythrocytes was examined. Pentoxifylline was a weak competitive antagonist of adenosine effects on cyclic AMP accumulation. HWA 285, by contrast, had a small stimulatory effect per se and also potentiated the effect of adenosine (10-30 microM). Neither pentoxifylline nor HWA 285 significantly affected the cyclic AMP accumulation induced by the stable adenosine analogue NECA or by alpha- or beta adrenoceptor activation. HWA 285 was a much more potent inhibitor of adenosine uptake into human erythrocytes than pentoxifylline and other examined xanthines including thiocaffeine, 8-p-sulphophenyltheophylline, theophylline, caffeine and enprofylline. It is suggested that HWA 285 may potentiate, rather than antagonize, the effects of endogenous as well as exogenous adenosine, partly as a consequence of adenosine uptake inhibition. PMID- 3012942 TI - Effect of serum, alpha-1 acid glycoprotein, lipoproteins and albumin on human mononuclear leucocyte beta-adrenoceptors. AB - The effects of serum, alpha-1 acid glycoprotein (AAG), serum lipoproteins (SLP) and human serum albumin (HSA) on 3H-(-)-dihydroalprenolol (3H-(-)-DHA) binding and (-)-isoproterenol ((-)-IPR) induced cyclic AMP (cAMP) elevation in human peripheral blood mononuclear leucocytes (MNL) were investigated. The saturable binding of 3H-(-)-DHA was decomposed into two classes of binding sites with maximum binding capacity of approximately 1400 and 30000 sites/cell and with dissociation constants (Kd) of approximately 0.7 and 65 nM. Stimulation of the MNL beta-adrenoceptors by (-)-IPR caused a concentration dependent cAMP accumulation (EC50 approximately 0.2 microM) with maximum level approximately 250% above basal. For all single leucocyte preparations, 30-35 min. exposure to serum, AAG and SLP increased the number of beta-adrenoceptors with 100-200% and the maximal responsiveness to (-)-IPR with 30-90%. The presence of proteins did not change the Kd or the EC50. (-)-Alprenolol inhibited concentration dependently the serum induced increment in (-)-IPR-responsiveness. Serum, AAG and SLP did also increase the number of low affinity binding sites with 25-40% without effect on the Kd. HSA had no consistent effect on beta-adrenergic binding or stimulation. The present study shows that serum, AAG and SLP influence the number and function of MNL beta-adrenoceptors in vitro. PMID- 3012944 TI - Some central effects of angiotensin II. Interactions with dopaminergic transmission. AB - The effects of angiotensin II (AT II) administered intracerebroventricularly (i.c.v.) on some behavioural reactions were studied. AT II increased the electroshock convulsive-seizure threshold in mice. Sulpiride antagonized this effect of AT II. AT II increased the exploratory behaviour of rats in open field. This effect was potentiated by nomifensine (a dopamine uptake blocker) and was blocked by haloperidol. AT II increased apomorphine stereotypy and the effect was antagonized by saralasin and haloperidol and partly by alpha-methyl-para-tyrosine (alpha-MpT) (an inhibitor of dopamine synthesis). AT II decreased the locomotor activity of mice. At doses of 0.5 and 1 microgram per mouse AT II did not change haloperidol (5 mg/kg)- or tetrabenazine (25 mg/kg)-induced catalepsy. It is assumed that the behavioural effects of AT II are realized through angiotensin receptors and through interactions between these receptors and dopaminergic transmission in the brain structures involved in the behavioural reactions studied. PMID- 3012945 TI - "Endogenous ligands" of benzodiazepine receptors, their structural analogs. Development of new psychotropic drugs. AB - This paper presents a review of literature data and results from investigations designed to determine the relation between structure, affinity to benzodiazepine receptors and pharmacological activity of possible endogenous ligands of benzodiazepine receptors and their electronic structural analogs. Structural variations of endogenous ligands of benzodiazepine receptors are substantiated to manifest themselves as active psychotropic drugs. On the basis of these considerations and experimental data, compounds with psychotropic activity and electronic structure, resembling that of "endogenous ligands" of benzodiazepine receptors, were developed. PMID- 3012943 TI - Vagally mediated stimulation of gastric acid secretion by intravenously administered adenosine derivatives in anaesthetized rats. AB - The effects of adenosine and some of its analogues on gastric acid secretion were investigated in rats. These compounds increased gastric acid secretion in anaesthetized rats after intravenous injection, the order of potency being 5'-N ethylcarboxamidoadenosine (NECA) greater than (-)N6-phenylisopropyladenosine (L PIA), N6-cyclohexyladenosine (CHA) greater than (+)N6-phenylisopropyladenosine (D PIA) greater than 2-chloroadenosine (2-CADO) greater than adenosine (ADO). The stimulation of acid secretion by L-PIA in anaesthetized rats was antagonized by theophylline and 8-phenyltheophylline and totally prevented by vagotomy and atropine. Intracerebroventricular administration of L-PIA had no effect on gastric acid secretion in anaesthetized rats, whereas in conscious rats it inhibited the output of acid, pepsin and fluid. These results indicate that adenosine derivatives stimulate gastric acid secretion in anaesthetized rats by activating the vagus nerve via adenosine receptors in afferent pathways. PMID- 3012946 TI - Water-electrolyte balance and hypothalamo-pituitary-adrenocortical function in rats with inherited diabetes insipidus (Brattleboro strain). AB - Brattleboro rats, homozygous for hypothalamic diabetes insipidus (DI), compared to their Long Evans (LE) controls, revealed typical changes in water-sodium potassium balance: hypernatremia and hyperosmolality and hypokalemia. Plasma renin activity was significantly increased (79%) and plasma concentration of aldosterone was significantly lower (--36%) in DI rats than in LE rats. Concomitantly aldosterone excretion in DI rats was increased sevenfold. Adrenal blood flow rates were not statistically different in both groups of rats but the aldosterone concentrations in the adrenal venous effluent were significantly lower (--66%) in DI rats than in LE rats, suggesting that the in vivo production rate of aldosterone was reduced in DI rats. Plasma concentration of ACTH was significantly decreased (by 25%) in DI rats. The reasons for the dissociation between the changes of aldosterone production and the variations of renin angiotensin system activity were discussed. PMID- 3012947 TI - Effects of the methylxanthine derivative pentoxifylline on benzodiazepine and muscarinic binding sites in rat cerebral cortex. AB - The subcutaneous injection of the methylxanthine derivative pentoxifylline (3,7, dimethyl-1-(5-oxo-hexyl)-xanthine) was able to induce, 3 h later, a significant reduction of benzodiazepine binding sites in rat cerebral cortex. When tested in vitro, pentoxifylline displaced 3H-flunitrazepam from specific binding sites in a competitive manner. For this effect pentoxifylline was about 10 times more potent than caffeine. When given in two daily injections for 5 days, pentoxifylline brought about a significant elevation in the number of cortical benzodiazepine binding sites. Neither acute nor chronic pentoxifylline treatment modified cortical muscarinic cholinergic binding sites. Pentoxifylline has negligible affinity for cortical muscarinic receptors in vitro. These results suggest that pentoxifylline is able to affect the cortical benzodiazepine receptors differentially, depending on time of drug administration. PMID- 3012948 TI - Effects of adenosine and two stable adenosine analogues on blood pressure, heart rate and colonic temperature in the rat. AB - Adenosine exerts effects via receptors of the AI- and A2-subtype. L phenylisopropyl adenosine (L-PIA) is more potent than N-5'-ethylcarboxamido adenosine (NECA) at the A1-subtype receptor whereas the potency order is reversed at the A2-subtype receptor. Adenosine analogues have been shown to decrease blood pressure and heart rate and to induce a marked hypothermia. In the present series of experiments adenosine, L-PIA and NECA were given i.p. or i.v. to rats, and blood pressure, ECG and colonic temperature were recorded. The NECA was the most potent of the compounds in reducing blood pressure (EC50 2 micrograms kg-1 i.v.), followed by L-PIA (EC50 approximately 30 micrograms kg-1 i.v.) and adenosine (EC50 approximately 300 micrograms kg-1 i.v.). In contrast, L-PIA and NECA were equally active in reducing heart rate (EC50 approximately 6 micrograms kg-1 i.v.). and considerably more potent than adenosine (EC50 approximately 300 micrograms kg-1 i.v.). It is suggested that simultaneous measurement of blood pressure and heart rate could be a simple in vivo model for comparison of A1- and A2-receptor subtype mediated effects. Colonic temperature was markedly reduced after i.p. administration of the adenosine analogues. Thus, 100 micrograms NECA kg-1 reduced colonic temperature from 37.8 to 26 degrees C. A 5 degrees C temperature drop was obtained by 10 micrograms kg-1 NECA, by 200 micrograms kg-1 L-PIA and by 200 mg kg-1 adenosine. The fall in colonic temperature was associated with a loss of muscular activity, as determined by needle electrodes or by palpation, indicating an inhibition of shivering.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3012950 TI - Effects of spinal cord lesions and rhizotomies on cholinergic and opiate receptor binding sites in rat spinal cord. AB - Changes in the distribution of [3H]quinuclidinylbenzilate ([3H]QNB), [3H] alpha bungaro-toxin ([3H]alpha-Btx) and [3H]etorphine binding sites were studied autoradiographically, and cholinacetyltransferase (ChAT) activity radioenzymatically, in the C6-7 segments of rats 1-20 days after combined dorsal and ventral C3-8 rhizotomies and spinal cord lesions at C3. After dorsal and ventral rhizotomies the number of [3H]QNB, [3H]alpha-Btx and [3H]etorphine binding sites were reduced ipsilaterally in the dorsal horn and those of [3H]QNB and [3H]alpha-Btx in the ventral horn. In the ventral horn ChAT activity was significantly reduced. After a unilateral spinal cord lesion at C3, ChAT activity was reduced in the ipsilateral ventral horn at C6-7 caudal to the lesions, whereas no change in receptor binding sites was observed. PMID- 3012949 TI - Altered cardiovascular responsiveness to adrenaline in endurance-trained subjects. AB - The influence of physical training on responses to i.v. adrenaline infusions and to exercise were investigated in 10 endurance-trained men (mean age: 35 y; VO2max: 61.9 ml kg-1 min-1) and 10 age-matched and sedentary controls (36 y, 37.5 ml kg-1 min-1). The untrained subjects were reinvestigated after a 4 month training period which increased their VO2max by 18%. Resting heart rate and diastolic blood pressure were significantly lower in the trained state. The venous plasma adrenaline concentrations attained during infusions (4 dose levels, 8 min each) were lower in the well-trained than in the untrained subjects (2.15 vs. 3.59 nmol l-1 at the highest dose level, P less than 0.01). The adrenaline induced increases in heart rate and in plasma cAMP and decreases in pre-ejection period (PEP) and PEP/LVET ratio were not dependent on the training state. The adrenaline-induced decrease in diastolic blood pressure was more pronounced (P less than 0.05) in the well-trained than in the untrained group and tended (0.05 less than P less than 0.1) to be enhanced by training in the latter group. The increases in systolic blood pressure were greater in the well-trained subjects (P less than 0.01) but training did not alter this response in the untrained subjects. The plasma noradrenaline response to maximal cycle ergometer exercise (VO2max test) was significantly greater in the well-trained than in the untrained subjects, while no difference was seen for adrenaline. The submaximal exercise systolic blood pressure was similar in all training conditions when related to the absolute rate of work. In summary, the present results indicate that both the vasodilator and systolic pressor responses to adrenaline are enhanced in endurance-trained subjects. The cardiac chronotropic and inotropic effects of adrenaline seem, however, to be independent of the training state. PMID- 3012951 TI - Hormone pattern and post-treatment attitudes in patients with major depressive disorder given electroconvulsive therapy. AB - As a follow-up study of a psychoendocrinological investigation of 33 patients with major depressive illness undergoing ECT, attitudes towards ECT were examined and hormones measured in remission. Two thirds of the group had a positive attitude towards ECT. Cortisol, prolactin and TSH levels differed significantly from the depressive state. In contrast, there was no difference in ACTH levels. PMID- 3012952 TI - Therapeutic methods in diagnostic radiology. PMID- 3012953 TI - Primary breast cancer. Complications of axillary management. AB - The complications following surgery and postoperative radiation therapy in the management of the axilla in 187 patients with primary breast cancer treated between 1978 and 1982 have been studied. Although no difference in complication rate could be detected between the three different postoperative radiation schedules utilised there was a strong and positive correlation between complication rate and increasing extent of surgical intervention. When the groups were sub-divided according to the extent of surgery performed, no differences in regional recurrence rates were observed but complication rates (defined as significant lymphoedema of the arm and/or restriction of shoulder movements) were significantly different (p less than 0.001) at 30 months between those who had no surgical intervention (25%), those who had had 'sampling' performed (50%) and those who had had formal dissection performed (84%). PMID- 3012954 TI - Radiographic evaluation of pulmonary fibrosis following mantle field irradiation in Hodgkin's disease. AB - Sixty-nine patients given mantle field radiation therapy for Hodgkin's disease and considered relapse-free and possibly cured were re-examined for pulmonary fibrosis as observed on antero-posterior and lateral chest radiographs. A method is described for the systematic evaluation of these late stage changes, which are observed following irradiation, in a numerical form. The method gave reproducible results in this series, the correlation coefficient being 0.72/0.81 and 0.73 respectively for the intra-observer and the inter-observer variations. The method thus may facilitate comparison between different patient materials. PMID- 3012955 TI - Computed tomography in the assessment of pancreatic tumor response after intraoperative radiation therapy. AB - Computed tomography was used in 7 patients given intraoperative electron beam therapy for advanced carcinoma of the pancreas. The local tumor response was studied quantitatively by defining the tumor contour in consecutive CT scans and then estimating the tumor volume. The maximum diameters of the tumor in 3 planes (X, Y and Z) were also estimated. There was evidence of initial tumor regression in all patients during the first few months after the treatment. No specific behaviour of the diameters in the X, Y and Z planes could be detected. Later on, regrowth of the tumor could sometimes be observed, preferably in one of the 3 planes. On the whole, CT was found to be a useful tool for assessing tumor response to this form of therapy. PMID- 3012956 TI - Early stage carcinoma of the uterine cervix. Effects of intracavitary radium treatment on lymphoid cells in blood and pelvic lymph nodes. AB - Sixteen patients with early stage carcinoma of the uterine cervix treated with primary radical hysterectomy and pelvic lymphadenectomy were compared with 17 patients who four to six weeks before the operation received intracavitary treatment with radium. The calculated radiation dose to the pelvic wall was approximately 10 Gy. The distribution of lymphoid cells in blood and pelvic lymph nodes was studied by an indirect immunofluorescence technique using monoclonal antibodies. The radium treated group showed a significant reduction of circulating OKT4+ (T helper) and OKT8+ (T suppressor/cytotoxic) lymphocytes. The number of Leu7+ (natural killer) cells and 1D5+ cells (monocytes) was not changed, but the ratio between monocytes and T cells was increased after radium therapy. In cell suspensions obtained from the pelvic lymph nodes, the radium treatment induced a significant reduction of the OKT4+ cell fraction. It is concluded that this low dose rate regimen of intracavitary treatment induces changes in the immune system which are of the same type as those seen after external field irradiation. PMID- 3012957 TI - Evaluation of the pulse wash sampling technique for screening of uterine cervical carcinoma. AB - The efficacy of a new sampling technique performed for early detection of cervical carcinoma is compared with Pap smears with the swab-and-wooden spatula technique in 312 women. In this new method, sampling of cytologic material is achieved by using a pulse wash instrument described in a previous article. Cells are rinsed and detached by liquid jets of 0.2 mm in diameter which are produced by a spray nozzle connected with a pressure hose to a high pressure pump. The liquid molecules pass through the spray nozzle at a speed of 20 m/s thus creating a successful rinsing effect on cervical epithelium due to high kinetic energy. Rinsed cells are mixed with the small amount of the flushing liquid. The suspension of cells and liquid accumulated in the speculum is then transported to a small container by a suction pump. The results of this work suggest that the pulse wash technique gives a more representative cell sample than the Pap smear sampling technique, thus offering a simple method to decrease false negative diagnoses in the detection of carcinoma of the uterine cervix. Samples by the new technique give an abundance of cells for slide preparation for cytodiagnostic techniques as well as for additional cytochemical, immunocytochemical and microbiologic diagnostic techniques. PMID- 3012959 TI - Cytogenetic damage induced in human lymphocytes by low doses of 60Co gamma rays delivered at high and low dose rates. AB - Chromosomal aberrations were investigated in human peripheral blood lymphocytes after exposure to low doses of 60Co gamma rays delivered acutely or at low dose rates (0.1 or 0.03 Gy/h). Chromosome analysis was performed in cells collected after a 44 to 46 hour culture time in order to avoid scoring of second division cells, and the dose-related induction of aberrations was analysed by the maximum likelihood method. In all cases, the induction of dicentrics was well described by a linear-quadratic dose response model (Y = aD + bD2), the data obtained at a low dose rate being equally well fitted to a linear equation. According to earlier findings on the mechanisms of aberration formation, the two lesions originating from single ionizing tracks have to be produced within a period of approximately 5 hours, in order to interact and to give rise to dicentric aberrations, which could explain the decrease in the quadratic term at the low dose rate since the highest doses were delivered over a period of more than 5 hours. PMID- 3012958 TI - Value of hydroxyproline measurements in the assessment of late radiation enteropathy. AB - In female Wistar rats roentgen irradiation of a 10 cm long temporarily exteriorized mid small intestinal segment was performed. Hydroxyproline, proline, aspartic acid and threonine were measured in intestinal samples 2 to 44 weeks after single or fractionated irradiation, and compared with a histopathologic radiation injury score in order to determine their value as assays of late radiation enteropathy. Two to 4 weeks after irradiation there was good correlation between the histopathologic injury score and the content of hydroxyproline. During the late observation period, there was no difference in hydroxyproline concentration between irradiated and sham-irradiated animals. There was no difference in hydroxyproline concentration in samples from the 1, 2 or 3 fractions groups, although the relative amount of non-collagen protein seemed to increase with fractionation. Determination of the hydroxyproline concentration may be used as an assay of early radiation injury, but it seems to be less suitable for late radiation enteropathy assessment. PMID- 3012960 TI - Influence of 90Sr, adult thymectomy and antilymphocyteglobulin on haematopoietic tissues and peripheral blood leucocytes in CBA mice. AB - The role of long-time immune suppression in carcinogenesis induced by the long lived internal emitter 90Sr, is investigated in an ongoing study. The experimental design is based on the assumption that impaired immune responsiveness, by other means than 90Sr, might increase the neoplastic response in exposed individuals, and thus reflect a protective function, if existing. Intercomparison is made of the tumour yield in mice exposed to different single doses of 90Sr and simultaneously subjected or not to long-term immune suppression by adult thymectomy (ATx) and/or antilymphocyteglobulin (ALG) treatment. Information on the general condition and responsiveness of the immune system, in the respective models, during tumour expectancy time, is essential for a conclusive evaluation of the results. To meet these demands the present paper reports on histopathologic alterations in immune organs and changes in white blood cell counts, induced by the different combinations of 90Sr, ATx and ALG treatment. The results confirm the prediction, that ATx + ALG is an efficient and, with respect to the purpose of the study, suitable treatment for additive long-term depression of the immune system in 90Sr irradiated mice, evidenced in particular by increased depletion of monomorphonuclear cells (MNC) in lymphoid organs and peripheral blood. Subsequent reports will deal with functional immune parameters. PMID- 3012961 TI - Strontium retention in mouse foetuses at different intervals after contamination of the dam. AB - Pregnant CBA mice were intravenously contaminated with radiostrontium (85Sr) on either the 14th, 16th or 18th day post coitum. The radiostrontium content in the foetuses and corresponding placentas as well as that in the femurs of the mothers was measured separately and individually on the 19th day post coitum, i.e. after intervals of 5, 3 and 1 day, respectively. The retained amount of strontium in the foetuses successively increased by the time of contamination, i.e. the later during pregnancy the contamination occurred. Expressed as per cent of the injected amount of 85Sr, the figures ranged from 4.6 per cent in foetuses contaminated on the 18th day to 0.7 per cent for those contaminated already on the 14th day after coitus, with a significantly negative correlation between time and retained amount of radiostrontium. The amount of strontium in the femurs of the mothers was reduced within the same intervals, due partly to decalcification of the skeleton of the mother during the latest part of the pregnancy. PMID- 3012962 TI - Stereotactic radiation therapy of intracranial lesions. Methodologic aspects. AB - A technique for stereotactic radiation therapy of cerebral tumours and arteriovenous malformations using a linear accelerator (6 MV photons) is proposed. Treatment relies on a fixation system that permits a precise use of the coordinates estimated at stereotactic angiography or stereotactic computed tomography. The field of treatment can be exactly outlined in the CT images during repeat examinations, thus facilitating the recognition of changes induced by radiation. The system also allows the extent of the arteriovenous malformation, as seen at angiography, to be accurately traced in the CT sections thus enabling evaluation of possible radiation damage to surrounding brain structures. The precision of the method as well as its hypothetical merits and disadvantages are discussed. The number of patients treated is still small and the follow-up time is too short in the majority of cases to allow definite conclusions. Examples of preliminary results are given. PMID- 3012963 TI - Treatment of non-Hodgkin lymphomas in the nasal cavities and paranasal sinuses. A failure analysis. AB - Twenty-five patients with sinonasal lymphoma were treated mainly with irradiation. All were non-Hodgkin lymphomas of diffuse type. Twenty patients had stage IA, 2 had stage IB, 1 stage IIA, 1 stage IIIA, and 1 stage IVA disease. Relapse developed in 16 (64%) of the 25 patients, with a failure rate of 64 per cent in the stage I patients (14/22). Most patients with failures had distant spread of the disease with or without local recurrence. Only one patient had local recurrence alone at the first relapse. Histologic classification according to the new working formulation seemed to be a reliable prognostic indicator for relapse: failure rates for low, intermediate, and high grade lymphomas were 0 per cent (0/2), 46 per cent (6/13), and 100 per cent (10/10), respectively. Computed tomography was valuable for planning of radiation therapy and for follow-up. PMID- 3012964 TI - Geographic variations of breast carcinoma incidence in Sweden. Are the differences real? AB - The validity of the reported geographic variations of breast carcinoma incidence in Sweden was assessed by examination of two possible sources of bias: non notification to the Cancer Registry of diagnosed carcinoma cases and 'biologically benign' breast carcinoma, i.e. with a low disease-specific lethality, e.g. detected accidentally at autopsy. No significant geographic differences in registration deficit were found even though non-notification tended to be slightly higher for old patients in low-incidence areas. Autopsy cases were estimated to account for less than one per cent of all cases and tended to be more frequent in high-incidence areas but the regional differences were generally small and not significant. An analysis of the relationship between 10-year relative survival and age-standardized incidence in 27 different regions revealed no significant correlation, whereas there was a significant positive correlation between age-standardized incidence and mortality. These findings indicate that non-lethal breast carcinoma cases do not explain the variations of incidence. In conclusion, no evidence was found suggesting that the geographic differences were artifactual. Registration deficit and autopsy cases, however, may have slightly increased the variations among elderly women. PMID- 3012965 TI - Establishment and characterization of an adriamycin-resistant subline of human small cell lung cancer cells. AB - An adriamycin (ADM)-resistant subline was established by continuous exposure of the SBC-3 cells, a cell line of human small cell lung cancer, to increasing concentrations of ADM, followed by the cloning procedure. The resistant sublines (SBC-3/ADM) thus established were 30-fold more resistant to ADM than the parent SBC-3 cells, in terms of the 70% lethal dose determined by soft agar clonogenic assay. The doubling times of the SBC-3 and SBC-3/ADM cells were 36 h and 22 h, respectively. When transplanted into athymic nude mice, the parent as well as resistant cells formed tumors, and serial passage was successful. Although the transplanted tumors from the two cell lines were very similar in histology, the resistance of the SBC-3/ADM cells to ADM developed in vitro was maintained in serially transplanted tumors. The uptake studies with [3H]daunomycin revealed decreased influx and enhanced active efflux of the drug in the resistant cells, whereas cytogenetic analysis showed that the cell lines had an identical karyotype. These results indicate that ADM resistance may be attributed to alternations in membrane transport, resulting in reduced intracellular accumulation of the drug. PMID- 3012966 TI - In vitro chemosensitivity and radiosensitivity of an adriamycin-resistant subline of human small cell lung cancer cells. AB - Using a cell line (SBC-3/ADM) of human small cell lung cancer, which is 30-fold more resistant to adriamycin than the parent cell line (SBC-3), the activity of a variety of anticancer agents was analyzed by soft agar clonogenic assay to search for a means of circumventing drug resistance. The SBC-3/ADM cells were markedly resistant to some anthracycline antibiotics in comparison with the SBC-3 cells: 28-fold for daunomycin, 26-fold for 4'-epiadriamycin, 18-fold for THP-adriamycin, and 8.4-fold for aclarubicin. However, the cells were as sensitive to mitoxantrone, one of the anthraquinone derivatives, as the parent cells. The cells were resistant to structurally or pharmacodynamically unrelated compounds such as vincristine, mitomycin C, and an active form of ifosfamide, whereas they were susceptible to cisplatin to some extent. The in vitro radiosensitivity of both cell lines was also evaluated, and they were found to be equally sensitive to X-ray. These results suggest that mitoxantrone and cisplatin may exert sufficient activity for small cell lung cancer which has acquired resistance to adriamycin, and that consolidative chest irradiation may be clinically useful after combination chemotherapy including adriamycin. PMID- 3012967 TI - Type IV collagen-degrading enzyme activity in hepatocellular carcinoma. AB - Type IV collagen-degrading enzyme activity was measured in liver homogenate obtained from 10 patients with hepatocellular carcinomas. Type IV collagen, the enzyme substrate, was extracted from human placenta with pepsin digestion, and labeled with [1-14C] acetic anhydride. The homogenate was preincubated with p aminophenylmercuric acetate to activate the latent form of the enzyme, and then the enzyme activity was measured at pH 7.5 by adding a substrate mixture. Referring to previous reports, the enzyme measured seemed to be a neutral metalloprotease. The enzyme activity of the homogenate was markedly reduced by omitting the p-aminophenylmercuric acetate pretreatment, indicating that the enzyme was present mainly in the latent form. The activity seemed to be higher in the peripheral portion of hepatocellular carcinoma than in the center. Further the activity was found to be the highest in a hepatocellular carcinoma patient with many metastatic nodules in the lung. The results might suggest that type IV collagen-degrading enzyme participates in tumor invasion and intrahepatic or remote metastasis. PMID- 3012968 TI - Fibronectin in differential diagnosis of primary hepatomas and carcinoma metastases in the liver. AB - Human liver specimens with primary hepatocellular and cholangiocellular carcinoma and liver metastases of cancers from different organs were studied for the distribution of fibronectin (Fn) by immunoperoxidase staining. With the exception of an undifferentiated type, all six well and moderately differentiated primary hepatocellular carcinomas contained Fn in the intercellular spaces and on the surface of certain cells of the tumour parenchyma, while in the case of three cholangiocarcinomas Fn could only be detected in the loose reactive connective tissue stroma. No Fn was observed around individual tumour cells in any of eleven carcinoma metastases in the liver. These findings indicate that hepatocellular but not cholangiocellular carcinomas synthesize Fn, while carcinoma metastases from other organs do not contain Fn in their parenchyma only in the reactive stroma. In this way the presence of Fn in the pericellular matrix of the tumour parenchyma may help to distinguish primary hepatomas from metastases. Metastases with strong fibroplastic character and pericellular fibrosis may, however, imitate Fn production. PMID- 3012969 TI - Thymidine kinase in extracts of human brain tumours. AB - Deoxythymidine kinase (TK) is an enzyme involved in DNA synthesis. It can be used as a marker of cell proliferation. TK was measured in extracts of human brain tumours and non-neoplastic brain tissue. In astrocytomas the mean TK activity increased with increasing grade of malignancy. Oligodendrogliomas showed higher TK activity than astrocytomas. The highest activities were noted in menigiomas, of which the recurrent ones exceeded the primary in TK activity. In non neoplastic brain tissue lower TK activity was found. It is concluded that TK can be measured in human brain tumour extracts, and is potentially of use for studies on tumour cell proliferation. PMID- 3012970 TI - Interaction of the antibiotic adriamycin with the plasma membrane. AB - The antitumor antibiotic adriamycin was found to be a potent modulator of the human erythrocyte discocyte echinocyte transition. Incubation of discocytes for 10 min with 10 microM adriamycin inhibited calcium-induced echinocytosis by 90 per cent. Adriamycin itself had no effect on erythrocyte morphology, a feature which distinguished it from other amphipaths which bring about the formation of a cupped cell morphology. Additionally, adriamycin differed from amphipaths such as the phenothiazines in that concentrations which prevented echinocytosis had no effects on calmodulin, as measured by effects on calmodulin-stimulated 45Ca2+ uptake into inside-out red cell vesicles. Adriamycin, paradoxically, appeared to cause a fall in the levels of erythrocyte polyphosphoinositides, but prevented further breakdown induced by calcium loading. This fall in inositides may be apparent rather than real, as the drug did not cause breakdown of the inositides to either inositol di- or triphosphates in red cell vesicles. Instead, it inhibited breakdown. It is possible that adriamycin may complex out the inositides and thus maintain levels of the inositide polyphosphates, congruent with the maintenance of the discocyte morphology. Interference with inositol lipid metabolism may be an important aspect of the pharmacology of adriamycin. PMID- 3012971 TI - Interaction of DNA polymerase and nucleotide analog triphosphates. AB - The properties of virus and host DNA polymerases are important factors in determining the selectivity of deoxynucleotide analogs used in antiviral chemotherapy. The high affinity of herpes DNA polymerase for nucleotide analogs may be particularly important in CMV and EBV-infected cells, since these viruses do not induce the synthesis of a virus-specified thymidine kinase. In general, the effect of nucleotide analog incorporation into DNA may be summarized as follows: analogs with modifications at the base moiety do not affect the rate of DNA chain elongation whereas those modified at the sugar moiety will inhibit the rate of chain elongation. ACGTP and DHPGTP competitively inhibit incorporation of dGTP into DNA; however, steric freedom of the acyclic phosphate may allow these nucleotides to bind virus enzyme in a conformation similar to that assumed by dGTP only at the transitional stage of the enzyme reaction. This may explain the high affinity of virus enzyme for these inhibitors. The interaction of aphidicolin with virus enzyme differs from that with host enzyme. These differences suggest new strategies for antiviral chemotherapy using aphidicolin derivatives. PMID- 3012972 TI - Bacterial protein toxins with latent ADP-ribosyl transferases activities. PMID- 3012973 TI - Molecular actions of steroid hormones. PMID- 3012974 TI - Steroid receptor activation: the glucocorticoid receptor as a model system. AB - The glucocorticoid receptor has been used as a model for steroid receptor activation. Because of recent evidence for the essentially nuclear location of the unoccupied receptors of 1,25-dihydroxycholecalciferol and 17 beta-estradiol, the significance of the activation mechanism converting unactivated receptor complexes to DNA-binding forms is unclear for some receptors. Up to now the weight of evidence favors a cytoplasmic location of the unactivated glucocorticoid receptor. In this article we describe studies on the nature of the activation mechanism and of regulatory factors. PMID- 3012975 TI - The new world primates as animal models of glucocorticoid resistance. AB - Many New World primate species have greatly increased plasma cortisol concentrations, decreased plasma cortisol binding globulin capacity and affinity, marked resistance of the hypothalamic-pituitary-adrenal axis to suppression by dexamethasone, and no biological evidence of glucocorticoid excess. These primates also have high levels of circulating progesterone, estrogen, mineralocorticoid, androgen and vitamin D. The glucocorticoid target tissues that have been examined (circulating mononuclear lymphocytes and cultured skin fibroblasts) have normal concentrations of glucocorticoid receptors with decreased affinity for dexamethasone. Transformation of B-lymphocytes with the Epstein-Barr virus leads to glucocorticoid receptor induction that is less than that observed with cells from Old World primates. The receptor in these cells has a low affinity for dexamethasone. The low affinity leads to an increased loss of specific bound ligand during thermal activation. Meroreceptor generation is normal. The molecular weight of the receptor, determined by SDS-PAGE, is similar to that of Old World primates (approximately 92,000) and the activation pattern per se, examined in vitro by heating cytosol and performing phosphocellulose chromatography, appears similar to that of human controls. The ratios of nuclear to cytosolic hormone-receptor-complexes and of cytosolic activated to unactivated receptor complexes in intact cells are similar to Old World primates. Results from mixing studies do not support the hypothesis that a binding inhibitor(s) or a deficient cytosolic positive modifier(s) of binding underlies the findings in these primates. The New World primates, unlike men with the syndrome of primary cortisol resistance, have compensated for their condition with intra-adrenal and mineralocorticoid receptor adaptations. Thus, unlike Old World primates, cortisol in New World primates has only weak sodium-retaining potency because the aldosterone receptor has a low affinity for cortisol. The common element that would explain the apparent resistance to six steroid hormones in New World primates remains unknown. PMID- 3012977 TI - Pseudohypoaldosteronism: a review and report of two new cases. PMID- 3012976 TI - Models of aldosterone action on sodium transport: emerging concepts. PMID- 3012978 TI - "Defective" receptors in steroid-resistant conditions may be proteolytic artifacts. AB - The specific question addressed in this report is whether the resistance to steroid treatment of certain tissues or tumors which appear to contain a normal quantity of steroid-binding sites may be due to structural defects in the receptors. This question may be seen as part of the more general question of whether there are intrinsic variations in the structures of receptors for a given class of steroids in different healthy tissues, in healthy vs. malignant tissues or in different types of tumors. Our experimental approach to these questions has involved the stabilization and precise physicochemical characterization of the receptors. To date, we have studied the estrogen and progestin receptors from human breast cancers and benign and malignant gynecologic specimens and the glucocorticoid receptors from several healthy and malignant rodent tissues and from normal human lymphocytes and various types of leukemic cells. Chromatographic and ultracentrifugal analyses in buffers of low ionic strength, containing 20 mM Na2MoO4 as the stabilizer, have revealed each of these receptors to be a large, oligomeric complex, characterized by remarkably similar values of the Stokes radius, sedimentation coefficient, molecular weight and axial ratio. In the absence of adequate stabilization, however, we found that the receptors for three classes of steroids in extracts of some healthy, steroid-responsive tissues, such as rat kidney and human uterine endometrium, are invariably degraded by endogenous proteinases. The extent of such cleavage is increased considerably by freezing the tissues prior to homogenization. Studies designed to distinguish the intact receptors from the products of proteolysis have included the characterization of receptors in cytosols prepared from mixtures of rat liver and kidney. The results strongly support the interpretation that the smaller size of the receptors detected in kidney cytosol reflects their cleavage by the more active proteinases in that tissue. The sizes and shapes of the receptors in cytosols from various tissues were found to be correlated with the activities of specific endopeptidases, assayed fluorometrically with peptidyl derivatives of 7 amino-4-methylcoumarin (AMC). These studies suggested that the receptors are vulnerable to cleavage by "lysine-specific" endopeptidases, detected with t butyloxycarbonyl-L-valyl-L-leucyl-L-lysyl-AMC. An enzyme of this specificity was partially purified from rat kidney cytosol and tested for its ability to digest the glucocorticoid receptors from rat liver cytosol.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3012979 TI - The metabolism and functions of vitamin D. AB - Vitamin D functions by stimulating intestinal calcium and phosphorus absorption, by stimulating bone calcium mobilization, and by increasing renal reabsorption of calcium in the distal tubule. These functions on bone and possibly kidney, but not intestine, require the parathyroid hormone. As a result of these functions, serum calcium and phosphorus concentrations are elevated to supersaturating levels required for the mineralization of bone to prevent rickets, osteomalacia, and hypocalcemic tetany. Recent experiments demonstrate that maintaining serum calcium and phosphorus levels in vitamin D-deficient rats in the normal range results in normal bone growth and mineralization. However, increased calcification results because bone resorption by osteoclasts is a vitamin D dependent process. Thus, bone resorption, modeling and remodeling must be considered vitamin D-dependent processes. Vitamin D must be metabolized to 25 hydroxyvitamin D3 by the liver and subsequently by the kidney to 1,25 dihydroxyvitamin D3 before function. 1,25-Dihydroxyvitamin D3 is metabolized to a C-23 carboxylic acid (calcitroic acid) but the pathway is unknown. Although 25 hydroxyvitamin D3 is metabolized to 24R,25-dihydroxyvitamin D3, 25,26 dihydroxyvitamin D3 and 25-hydroxyvitamin D3-26,23-lactone, these pathways play no role in the function of vitamin D as shown by appropriate fluoro analogs of 25 hydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 binds to a specific receptor in the intestinal nuclei to elicit a stimulation of calcium transport. 1,25 Dihydroxyvitamin D3 plus the receptor causes transcription of specific genes that code for calcium and phosphorus transport proteins. Only one protein, the calcium binding protein, has been identified as being vitamin D dependent. Two others have been described, but no clear description of the molecular mechanism of action of 1,25-dihydroxyvitamin D3 is yet available. PMID- 3012980 TI - 1,25-Dihydroxyvitamin D3 receptors: altered functional domains are associated with cellular resistance to vitamin D3. AB - 1,25-Dihydroxyvitamin D3 receptors are cytosoluble proteins detectable in a variety of tissues responsive to 1,25(OH)2D3. They are DNA binding-proteins analogous to other steroid receptors and it is this functional property which is likely involved in the activation of hormone-sensitive genes. Utilizing 1,25(OH)2D3 and DNA binding assays, as well as anti-receptor monoclonal antibodies, we have probed the relationship between the 1,25(OH)2D3 receptor binding domains after selective cleavage with trypsin. These studies reveal that the hormone and DNA binding regions are separable, and are consistent with the finding that tissue resistance to 1,25(OH)2D3 is a result of structural defects in these domains. Recently, a primate model, the LLC-MK2 monkey kidney line, has been uncovered which may exemplify a hormone-binding defect. Here, 25 hydroxyvitamin D3-24-hydroxylase induction, a 1,25(OH)2D3 bioresponse, requires 100-fold higher concentrations of the hormone for maximal response. Concomitantly, this cell contains a variant receptor form which displays a correspondingly lowered apparent affinity for the hormone despite its seemingly normal DNA binding characteristics. Taken together, these studies suggest that the 1,25(OH)2D3 receptor is a macromolecule with multiple domains each of which may produce modified cellular resistance to 1,25(OH)2D3 if structurally altered. PMID- 3012981 TI - Clinical features of hereditary resistance to 1,25-dihydroxyvitamin D (hereditary hypocalcemic vitamin D resistant rickets type II). PMID- 3012982 TI - The molecular basis for resistance to 1,25-dihydroxyvitamin D: studies in cells cultured from patients with hereditary hypocalcemic 1,25(OH)2D3-resistant rickets. PMID- 3012983 TI - The common marmoset as an animal model for vitamin D-dependent rickets, type II. PMID- 3012984 TI - Mechanisms of glucocorticoid hormone action. AB - This report summarizes our studies, in context with the results of other laboratories, of the molecular mechanisms of glucocorticoid hormone action. The receptors for these steroids are comprised of single polypeptide chains of about 90,000 molecular weight. Binding of agonist steroids to the receptor induces a conformational change to an active receptor form that is followed by a second change in the glucocorticoid-receptor complex, termed activation, that alters the charge of the complex and results in its binding to specific sites on the DNA termed glucocorticoid regulatory elements (GREs). The GRE on the human metallothionein-IIA gene is located in the 5'-flanking DNA. It can function independently of the gene's promoter, and when ligated upstream from the herpes simplex virus (HSV) thymidine kinase (TK) gene promoter, can activate it. The binding of the glucocorticoid-receptor complex to the GRE probably alters chromatin structure over a limited span to facilitate RNA polymerase action. The regulation by glucocorticoids of growth hormone gene expression is more complex. The steroid appears to elicit both transcriptional and posttranscriptional influences that are also affected by thyroid hormone. Also the glucocorticoid influences appear to be exerted in part through DNA structures located downstream from the transcriptional initiation site. A GRE has been defined in intron A of the hGH gene through gene transfer and DNA binding experiments. Finally, gene transfer experiments suggest that pituitary-specific factors influence the ability of glucocorticoids to affect GH gene expression. PMID- 3012986 TI - Cortisol resistance in man. AB - Primary cortisol resistance in man is a familial disease characterized by increased plasma cortisol concentrations, high urinary free cortisol excretion, a normal circadian pattern of cortisol secretion, resistance to adrenal suppression by dexamethasone and absence of the clinical stigmata of Cushing's syndrome or signs of adrenal insufficiency. In its severe form, hypertension and hypokalemic alkalosis are present, owing to increased secretion of the sodium-retaining corticoids, corticosterone and deoxycorticosterone. In subjects with a less severe resistance to cortisol, there are no clinical abnormalities and the disease is revealed only by detailed examination of several parameters of cortisol metabolism or by glucocorticoid receptor studies. In whole-cell glucocorticoid receptor assays (peripheral mononuclear leukocytes, fibroblasts, or B-lymphocytes transformed with the Epstein-Barr Virus) low receptor affinity for dexamethasone could be demonstrated conclusively only in the severely affected subject. When affected cells are transformed with the Epstein-Barr virus, receptor induction is less than that of normal cells. The decreased affinity of the receptor for its ligand is reflected in an increased rate of loss of specific bound ligand during thermal activation. The molecular weight of the receptor, determined by SDS-PAGE, is similar to that from normal cells (approximately 92,000). Only in the severely affected patient was the proportion of activated receptor remaining in the cytosol of thermally activated intact cells reduced. At saturating concentrations of dexamethasone, nuclear binding appears normal in cells from both the severe and the asymptomatic forms of this condition, providing an explanation for the apparently complete compensation of the target tissue resistance to glucocorticoids by the high plasma cortisol levels. The clinical manifestations of the disorder (hypertension, hypokalemia) can be corrected with high doses of dexamethasone (3mg/day). PMID- 3012985 TI - Glucocorticoid physiology, pharmacology and stress. AB - Basal levels of glucocorticoids maintained by negative feedback regulation are known to modulate a wide range of physiological processes, through a variety of effects such as those on carbohydrate metabolism and "permissive" actions on effects of other hormones. Glucocorticoid levels increase sharply in response to the stress of any kind of threat to homeostasis. The increased levels have traditionally been ascribed the function of enhancing the organism's resistance to stress. How known physiological and pharmacological effects of high levels of glucocorticoids might accomplish this function, however, has been a mystery. A generalization that is beginning to emerge is that many of these effects may be secondary to modulation by glucocorticoids of the actions of numerous intercellular mediators, including established hormones, prostanoids, neutral proteinases, and cytokines such as interferon. These mediators participate in physiological mechanisms--endocrine, renal, immune, neural, etc.--that mount a first line of defense against such challenges to homeostasis as hemorrhage, metabolic disturbances, infection, anxiety, and others. Contrary to the traditional view that the role of glucocorticoids in stress is to enhance these defense mechanisms, it has become increasingly clear that glucocorticoids at moderate to high levels generally suppress them. This paradox first emerged when glucocorticoids were discovered to be antiinflammatory agents, and had remained a major obstacle to a unified picture of glucocorticoid function. We have suggested that stress-induced increases in glucocorticoid levels protect not against the source of stress itself but rather against the body's normal reactions to stress, preventing those reactions from overshooting and themselves threatening homeostasis. This hypothesis, the seeds of which are to be found in many earlier discussions of glucocorticoid effects, immediately accounts for the paradox noted above, and provides glucocorticoid physiology with a unified conceptual framework that can accommodate such apparently unrelated physiological and pharmacological effects as those on carbohydrate metabolism, inflammatory processes, shock and water balance. It also leads us to propose that some enzymes rapidly induced by glucocorticoids detoxify mediators released during stress-induced activation of primary defense mechanisms; those mediators could themselves cause damage if left unchecked. PMID- 3012987 TI - Isolation of a bovine adenovirus from fallow deer (Dama dama). PMID- 3012988 TI - Experimental infection of laying hens with an adenovirus isolated from ducks showing EDS symptoms. PMID- 3012989 TI - Desensitization in aspirin-sensitive asthmatics. AB - Tolerance to aspirin was induced in thirteen asthmatics with aspirin-intolerance. Starting with the lowest aspirin doses eliciting bronchospasm, the dose of aspirin was progressively increased up to a 1000 mg tolerance level. Tolerance was rapidly achieved when initial threshold doses were greater than 150 mg. Daily aspirin administration led to prolonged tolerance. Desensitization to aspirin may be useful in aspirin-sensitive asthmatic patients who require antiinflammatory or antithrombotic treatment. PMID- 3012990 TI - Performance of asbestos fiber counting laboratories in the NIOSH proficiency analytical testing (PAT) program. AB - Asbestos fiber counting data reported in the NIOSH Proficiency Analytical Testing (PAT) Program are used in this study to evaluate the analytical performance of participating laboratories and to determine if overall performance has improved during a ten-year period. PAT laboratories have achieved intralaboratory precision of 0.18 to 0.28 relative standard deviation (RSD), and interlaboratory precision of 0.33 to 0.44 RSD. In addition, there was higher variability between PAT laboratories from 1974 to 1978, when the program underwent considerable change and growth than the variability found during previous or subsequent time periods. The improvements in interlaboratory precision by approximately one-third since 1974 and the tendency of laboratories with little PAT experience to have poorer interlaboratory precision than more experienced laboratories raises a concern that interlaboratory precision may deteriorate as large numbers of new laboratories start to enroll in the PAT Program with the increased emphasis on asbestos removal in public buildings. PMID- 3012991 TI - Breath hydrogen and methane: poor indicators of apparent digestion of soy fiber. AB - To examine whether end-alveolar breath hydrogen and methane could be used as indicators of fiber digestion in humans, 16 male subjects were fed four fiber free, complete liquid diets with 0, 30 g (heat processed), 30 g, and 60 g/day soy polysaccharide. Breath hydrogen was measured hourly and breath methane every 4 h on days 6 and 8 of each study period. Feces were collected, homogenized, dried, and analyzed for neutral detergent fiber (NDF). NDF in diets was determined and apparent NDF digestibility calculated. NDF from soy was extensively fermented, greater than 80%, on the fiber-containing diets. No significant relationship was found between breath-gas excretion and fiber digestion, although breath-gas values varied greatly. Breath hydrogen and methane were not significantly different when subjects consumed diets containing 0, 30, or 60 g soy polysaccharide. PMID- 3012992 TI - Prognostic significance of nuclear DNA analysis in histological sections in mammary carcinoma. AB - The nuclear DNA content in tumor cells from invasive ductal breast carcinomas was analyzed in 26 patients who survived more than 10 years and in 23 patients who died within 2 years after operation. The DNA content of individual tumor cells was measured in sections from the originally paraffin-embedded specimens. In short-term survivors, a large proportion of cells with very high DNA values was found in all tumors except one. Only two patients of the long-term survivors had tumors that exhibited such high DNA values. Prognostic information obtained by DNA analysis compared with histologic malignancy grading showed that the specificity using DNA analysis was distinctly higher. The data thus suggest that analysis of DNA content of tumor cells in breast adenocarcinomas can be a useful supplement to histologic malignancy grading to obtain prognostic guidance in individual patients. PMID- 3012993 TI - A transient hypotensive episode (Bezold-Jarisch effect) occurring in a patient treated with microwave hyperthermia. AB - A case report of a hypotensive episode occurring during a microwave hyperthermia treatment is reported. Microwave hyperthermia was delivered using 915 MHz externally applied energy. The tumor being treated was either a superficial lymph node or local recurrence in the subdigastric region overlying the carotid artery. During the patient's treatment, she reported symptomatology consistent with a neuropathy which is characterized by its association with microwave energy. As the treatment progressed the patient developed syncopy, hypotension, and bradycardia similar to a Bezold-Jarisch reflex. All of these conditions promptly self-corrected when the microwave energy was discontinued. This event can be explained by a transient microwave induced neuropathy specifically involving the carotid body or sinus. PMID- 3012994 TI - Two problems: diagnosing the EEC/EECUT syndrome and recommending dietary fiber. PMID- 3012996 TI - Monozygotic twins discordant for the acquired immunodeficiency syndrome. AB - Monozygotic twin girls discordant for acquired immunodeficiency syndrome were born to parents with antibodies to human T-cell lymphotropic virus type III. One twin had clinical evidence of the syndrome with tests positive for antibody, whereas the other at the age of 3 years was clinically, serologically, and virologically normal. PMID- 3012995 TI - Human T-cell lymphotropic virus type III (HTLV-III) embryopathy. A new dysmorphic syndrome associated with intrauterine HTLV-III infection. AB - Twenty infants and children with positive serologic tests for the human T-cell lymphotropic virus type III (HTLV-III) were noted to have similar features including growth failure (75%), microcephaly (70%), and craniofacial abnormalities consisting of ocular hypertelorism (50%); prominent box-like appearance of the forehead (75%); flat nasal bridge (70%); mild upward or downward obliquity of the eyes (65%); long palpebral fissures with blue sclerae (60%); short nose with flattened columella and well-formed, triangular philtrum (65%); and patulous lips (60%). These features constitute a new and distinct dysmorphic syndrome, the HTLV-III embryopathy. PMID- 3012997 TI - Delay in the diagnosis of pediatric brain tumors. AB - The interval from the onset of symptoms to the diagnosis in 79 children with primary brain tumors was compared with that in 45 children with Wilms' tumor and 123 children with acute leukemia. The patients with brain tumors had a significant delay from symptom onset to diagnosis. Only 38% of primary brain tumors were diagnosed within the first month after the onset of symptoms. In contrast, 84% of Wilms' tumors and 80% of cases of acute leukemia were diagnosed within one month of the onset of symptoms. Early detection of brain tumors is important as it may have a significant bearing on clinical outcome. PMID- 3012998 TI - Digestion and absorption of fiber carbohydrate in the colon. AB - Most dietary carbohydrates are digested and absorbed in the small bowel. However, fiber carbohydrate and other carbohydrates can be metabolized by the normal flora of the colon. The substrate for bacterial fermentation includes compounds for which small bowel digestive and absorptive mechanisms may, or may not, exist and soluble and some insoluble fiber. Products of fermentation include gases and volatile fatty acids which may be absorbed or nourish the colon mucosa. Total body nutrition and metabolism may also be affected by the products digested and absorbed in the colon. PMID- 3012999 TI - Cytomegalovirus primo-infection in a patient with idiopathic proctitis. AB - A young heterosexual man, suffering from a low-grade, idiopathic proctitis, who developed severe rectal ulcerations at the time of a primary cytomegalovirus (CMV) infection is described. CMV was cultured from the rectum on two occasions. Sero-conversion and the appearance of a specific IgM antibody response to CMV were documented, suggesting that this was a case of a primo-infection by CMV, and not one of reactivation of latent CMV infection. PMID- 3013000 TI - Hepatitis A in Peru. The role of children. AB - A serologic survey in 1983-1984 evaluated the presence of hepatitis A antibody (anti-HAV) and hepatitis A immunoglobulin M antibody (anti-HAV IgM) in 3,251 adults and 811 children in the jungle and coastal areas of Peru. All subjects were asymptomatic. Adults had a 98% positive anti-HAV rate except for naval cadets, who had a 76% rate. Children had an 82% positive anti-HAV rate, increasing from 30% at one year of age to 100% at eight years of age. Anti-HAV IgM was present in 27% of children one to four years of age who had antibody and was not present in those older than 12. The vast majority of Peruvian adults are immune to hepatitis A, and children with asymptomatic infection play a significant role in the transmission of this disease. PMID- 3013002 TI - Re: "Total energy intake: implications for epidemiologic analyses". PMID- 3013001 TI - A foodborne outbreak of Norwalk virus gastroenteritis. Evidence for post-recovery transmission. AB - From November 10-16, 1982, 220 (57%) of 383 attendees at eight banquets for which food had been prepared at a single hotel restaurant and the employees of the hotel had onset of Norwalk virus gastroenteritis. Epidemiologic investigation of the three largest banquets confirmed consumption of potato and fruit salads (banquet A), coleslaw (banquet B), and tossed salad (banquet C) to be significantly associated with illness. Between November 8-19, similar illness occurred in seven (54%) of 13 hotel kitchen employees. The foods implicated in banquets A and B were prepared by one salad worker during her acute illness and up to 48 hours following her recovery. A second salad worker prepared the implicated tossed salad for banquet C 24 hours following her recovery. To the authors' knowledge, this is the first foodborne outbreak investigation demonstrating Norwalk viral excretion and transmission by a food handler after recovery from illness and either person-to-person or vehicle-borne transmission between food handlers with subsequent transmission by more than one food handler to patrons. PMID- 3013003 TI - Ig gamma restriction fragment length polymorphisms indicate an ancient separation of Caucasian haplotypes. AB - This investigation was undertaken to study genetic variation in the human immunoglobulin gamma heavy-chain (IgG) genes using Southern blot hybridization techniques to identify restriction fragment length polymorphisms (RFLPs). A genomic Ig gamma-1 clone was used as a probe, and variants were identified with two restriction enzymes (R.E.), each of which defined RFLPs at two separate IgG loci. Once alleles and haplotypes were determined, molecular localization of the alleles was made through genetic analysis of recombinant haplotypes and through the use of regional specific subclones. Linkage between the newly defined RFLPs and switch region variants as well as protein allotypic markers (Gm) was complete. This analysis included markers for Ig Mu, Alpha 1, Alpha 2, Gamma 1, Gamma 2, Gamma 3, and Pseudo Gamma. The picture that emerges from the molecular study of two common haplotypes, each with many rare variants resulting from recombination or mutation, confirms and extends the earlier immunological observations. The accumulated differences between the two major Caucasian IgG haplotypes indicate that their separation may be ancient and maintained through heterozygote advantage. PMID- 3013004 TI - Chromosomal assignment of the genes for human aldehyde dehydrogenase-1 and aldehyde dehydrogenase-2. AB - Chromosomal assignment of the genes for two major human aldehyde dehydrogenase isozymes, that is, cytosolic aldehyde dehydrogenase-1 (ALDH1) and mitochondrial aldehyde dehydrogenase-2 (ALDH2) were determined. Genomic DNA, isolated from a panel of mouse-human and Chinese hamster-human hybrid cell lines, was digested by restriction endonucleases and subjected to Southern blot hybridization using cDNA probes for ALDH1 and for ALDH2. Based on the distribution pattern of ALDH1 and ALDH2 in cell hybrids, ALDH1 was assigned to the long arm of human chromosome 9 and ALDH2 to chromosome 12. PMID- 3013005 TI - The immunological detection of a 21-OH deficiency mutation HLA supratype. AB - Previous studies have shown that the late-onset and cryptic forms of 21 hydroxylase deficiency are highly associated with the HLA supratype HLA B14,C4A2,C4B1/2,DR1. Since cells from a number of unrelated normal individuals from different ethnic backgrounds expressing the DR1 associated with this supratype failed to stimulate two different DR1-restricted T-cell clones that proliferated in the presence of most other DR1 cells, we decided to test the hypothesis that cells with this supratype express "abnormal" DR1 molecules that have been affected in some way by the chromosomal mutation responsible for B14,DR1-associated 21-hydroxylase deficiency (21-OH-defL). The results showed an association between "abnormal" DR1 and 21-OH-defL (elevated rates of 17 alpha hydroxyprogesterone [17-OHP] increase and elevated peak 17-OHP values following ACTH stimulation). The presence of the B14,DR1 supratype can be used to predict the presence of "abnormal" DR1 and the clinical status of individuals not previously known to be 21-OH-defL carriers. PMID- 3013006 TI - Nonuniform recombination within the human beta-globin gene cluster. PMID- 3013007 TI - Change in attitudes toward presymptomatic testing in Huntington disease. PMID- 3013008 TI - Primary carcinoma of the fallopian tube. AB - From 1964 through 1983, 47 patients with a mean age of 63 years underwent primary treatment of fallopian tube carcinoma. Vaginal bleeding, abdominopelvic pain or pressure, and a palpable pelvic mass (or masses) were frequent retrospective clinical associations. Survival analysis demonstrated a rapid initial rate of patient attrition with stabilization after 3 years, resulting in an overall 5 year survival rate of 41%. While independent of age, histologic grade, or original tumor size, patient longevity was significantly decreased (p less than 0.01) by the presence of disease beyond the pelvis, neoplastic cells in the peritoneal washings, and size of residual disease. Therapy consisted of primary surgical resection, usually followed by adjunctive radiotherapy or chemotherapy. Initial sites of relapse were predominantly (88%) within the intraperitoneal cavity. The study supports definitive surgical staging and tumor reduction, as well as more selective adjuvant therapy based on surgical stage and residual tumor size and location. PMID- 3013009 TI - Chorionic villus sampling in continuing pregnancies. II. Cytogenetic reliability. AB - Cytogenetic analysis was performed on 103 chorionic villus samples. Analysis of the 103 samples revealed six abnormalities. In three of the six the abnormalities were confirmed in fetal or neonatal tissue (47,XY, + 13; 46,XY, t(13q13q); 45,X). In three samples the abnormalities detected were not confirmed; in two of the three the abnormalities were detected only in long-term cultures, whereas in the other samples the abnormality was restricted to direct analysis of the villi after overnight incubation. Our initial experience leads us to conclude that certain abnormalities in chorionic villus sampling may not be indicative of fetal abnormalities; 45,X/46,XX or 45,X/46,XY mosaicism is such a complement. Discrepancies between cytogenetic analysis of intact villi processed soon after sampling and of cells grown in culture can be managed by adhering to several suggested guidelines and by liberal use of confirmatory amniocentesis. PMID- 3013011 TI - Pupillary-block glaucoma associated with childhood cystinosis. AB - Cystinosis is a rare autosomal recessive metabolic disorder that results in the widespread accumulation of cystine crystals in ocular tissues as well as in bone marrow, liver, spleen, lymph nodes, and kidneys. We treated a case of pupillary block glaucoma in a 19-year-old woman caused by cystine accumulation in the iris stroma. Trabeculectomy and iridectomy relieved the pupillary block and decreased the intraocular pressure. Histologic examination disclosed the presence of crystals in the conjunctival and iris stroma and in the iris pigment epithelium. Crystals were also found within conjunctival mast cell granules, confirming the lysosomal nature of cystinosis. PMID- 3013010 TI - Use of the erythrocyte rosette test to screen for excessive fetomaternal hemorrhage in Rh-negative women. AB - The possibility of Rh immune globulin failure exists when a fetomaternal hemorrhage exceeds 25 to 30 ml of whole blood and only one 300 micrograms vial of Rh immune globulin is administered. In this prospective study of 1000 consecutive Rh-negative women who were delivered of Rh-positive newborn infants, the presence of fetal erythrocytes in maternal blood was identified with use of both the Du test read microscopically and the erythrocyte rosette test. All positive tests prompted fetomaternal hemorrhage quantification with use of a modified Kleihauer Betke acid elution test. Nineteen patients demonstrated a positive rosette test, and the only positive Du tests were in five of these 19. Six of the nineteen had levels of greater than 30 ml of whole blood for an incidence of 0.6% for fetomaternal hemorrhage exceeding the protective capabilities of the standard Rh immune globulin dosage. In experiments with simulated fetomaternal hemorrhage, all 79 samples, containing from 2.5 to 70 ml of fetal whole blood, were positive according to the erythrocyte rosette test. Applying the Du test to the same samples resulted in a 30% false negative rate at the level of a 30 ml simulated hemorrhage. Based on sufficient sensitivity, ease of interpretation, and reasonable cost, the rosette test appears to be a superior screening test for excessive fetomaternal hemorrhage in Rh immune globulin candidates. PMID- 3013013 TI - Peripheral synovial sarcoma metastatic to the temporal bone. AB - Peripheral synovial sarcoma rarely metastasizes to the head and neck. A case report of such an occurrence involving the ear is presented, along with a review of the subject. The case report is unique, as it is the only reported instance of synovial sarcoma involving the ear. PMID- 3013012 TI - Differentiation of human retinoblastoma in vitro into cell types with characteristics observed in embryonal or mature retina. AB - The capacity of a primitive human retinoblastoma cell line (Y-79) to differentiate into several cell types of normal human retina was investigated. Cells were studied in suspension and monolayer cultures, in serum-free or serum supplemented medium, and in the presence or absence of differentiating agents such as N6O12-dibutyryl adenosine 3',5'-cyclic monophosphate (dbc-AMP) and sodium butyrate (Nabut). Electron microscopy, immunohistochemistry for detection of myelin basic protein (MBP), and formaldehyde-induced fluorescence (FIF) for catecholamines were performed. Treated cells exhibited morphologic characteristics supportive of differentiation toward photoreceptors, conventional neurons and glial cells, increased FIF reactivity, and MBP expression. Growth in serum-free medium without differentiating agents led to a similar but less enhanced morphologic differentiation. These results confirm the concept that human retinoblastoma originates from a primitive neuroectodermal multipotential cell. PMID- 3013014 TI - Acquired immunodeficiency syndrome (AIDS). PMID- 3013016 TI - Receptor-independent sequestration of beta-adrenergic ligands by alveolar type II cells. AB - Numerous studies indicate that synthesis and secretion of surfactant by type II pneumocytes are modulated by the interaction of beta-adrenergic agonists with specific cell surface receptors. Two 125I-labeled beta-adrenergic ligands, l iodopindolol (IPIN) and l-iodocyanopindolol (ICYP), were thus employed to investigate the properties of type II cell beta-receptors. Saturable, high affinity, stereospecific binding to crude membrane fractions from whole rat lungs was exhibited by both ligands (IPIN, KD 283 pM, Bmax 508 fmol/mg protein; ICYP, KD 18 pM, Bmax 404). Type II cell membranes obtained by N2 cavitation also revealed stereospecific, saturable, high-affinity binding. In intact cells (37 degrees C) however, rapid, highly concentrative (cell/media greater than 1000), nonspecific ligand uptake compromised estimates of specific binding (specific/total binding less than 0.1). Total ligand uptake was inhibited at 4 degrees C, by decreasing pH within the physiological range (7-8) and by the lysosomotropic compound chloroquine (50-200 microM), without a detectable change in specific binding. Other basic drugs were also inhibitory at similar concentrations; acidic drugs had no effect. Even at 4 degrees C, specific binding remained low, as IPIN and ICYP were displaced less than 30% by l-alprenolol (1 microM). Physicochemical properties of IPIN and ICYP considered with the above studies suggest that passive ligand entry into intact pneumocytes and subsequent trapping of the protonated species in a cellular compartment of low pH may account for high nonspecific ligand uptake. PMID- 3013015 TI - Two different heavy chains are found in smooth muscle myosin. AB - Two putative myosin heavy chains designated SM1 and SM2 were detected on a 3.5% polyacrylamide-sodium dodecyl sulfate gel electrophoresis system loaded with homogenates of several mammalian smooth muscles. The two polypeptides were present in nearly equal amounts in all smooth muscle tissues tested and in myosin purified from swine carotid media and stomach. Both proteins were equally stained by smooth muscle-specific myosin antibodies. The smaller of the polypeptides had a mobility nearly identical to that of the single heavy chain observed in purified fast-twitch skeletal myosin. Electrophoresis of pyrophosphate extracts from swine carotid media, swine stomach, rabbit thoracic aorta, and guinea pig taenia coli on nondenaturing pyrophosphate gels revealed a single protein band. When subsequently electrophoresed on a sodium dodecyl sulfate gel, the native bands from swine tissue extracts revealed the two putative heavy chains in nearly equal amounts, as well as a large amount of a higher molecular weight peptide whose properties reflect those of filamen. Sodium dodecyl sulfate gel analysis of the myosin band from pyrophosphate gels of purified swine stomach myosin showed exclusively the two heavy chains in a nearly 1:1 ratio. Smooth muscle myosin migrates homogeneously on pyrophosphate gels, and the virtual equality of the two heavy chains may reflect the presence of large amounts of a myosin isoenzyme, which is a heavy-chain heterodimer. PMID- 3013017 TI - Location of major antibody binding domains on alpha-subunit of dog kidney Na+-K+ ATPase. AB - The locations of binding sites on the alpha-subunit of dog kidney Na+-K+-ATPase for both monoclonal antibodies and antibodies from polyclonal antisera have been determined. Three distinct regions of the alpha-subunit, all located within the amino terminal half of the polypeptide, were recognized by the antibodies: a region near the amino terminus of the polypeptide and two regions that are separated by a site for trypsin cleavage of the ATPase in KCl. No significant binding of antibodies to the carboxy terminal region of the alpha-subunit was detected. The binding sites for the antibodies are located within regions of the polypeptide predicted to be exposed within the cytoplasm of the cell (P. L. Jorgensen, S. J. D. Karlish, and C. Gitler, J. Biol. Chem. 257: 7435-7442, 1982). This prediction was verified by the demonstration that the antibodies did not react with Na+-K+-ATPase in tight right-side-out vesicles but would bind to the protein after the vesicles had been disrupted with detergent. A model for the folding of the alpha-subunit through the membrane, based on these data, is presented. PMID- 3013018 TI - Hypertonic cell volume regulation in mouse thick limbs. I. ADH dependency and nephron heterogeneity. AB - Differential interference contrast microscopy was used in combination with standard electrophysiological techniques in the in vitro perfused mouse medullary (mTALH) and cortical (cTALH) thick ascending limbs of Henle to evaluate the cell volume responses of these nephron segments to sudden increases in peritubular osmolality and to assess the role of antidiuretic hormone (ADH) and net NaCl absorption on hypertonic volume regulation. In the absence of CO2/HCO3- in external media, the cells of the mTALH behaved in a simple osmometric fashion, with an osmotic space equivalent to 70-80% of the total cell volume. However, in CO2/HCO3- -containing media, the cells of the mTALH, but not the cTALH, were able to increase their cell volume to the original volume after shrinkage in peritubular media made hypertonic with either NaCl or mannitol. This volume regulatory increase response (VRI) in the mTALH was mediated by an increase in intracellular osmoles, and required peritubular ADH, at concentrations that stimulate maximally the rate of net NaCl absorption. This ADH effect on VRI could be mimicked by addition of dibutyryladenosine 3',5'-cyclic monophosphate to the bath in the absence of hormone. However, 10(-4) M luminal furosemide, a concentration that abolishes ADH-dependent NaCl absorption in the mTALH, had no effect on the VRI response. These results indicate that the cells of the mTALH, but not the cTALH, are capable of hypertonic volume regulation, that ADH (via adenosine 3',5'-cyclic monophosphate) is required for expression of the VRI response in the mTALH, and that the effects of ADH on net NaCl absorption and the VRI response in the mTALH are completely dissociable. Thus these results are consistent with a role for ADH in hypertonic VRI in the mammalian mTALH, which may operate to maintain constant cell volume in this nephron segment during antidiuresis. PMID- 3013019 TI - Hypertonic cell volume regulation in mouse thick limbs. II. Na+-H+ and Cl(-)-HCO3 exchange in basolateral membranes. AB - Differential interference contrast microscopy and standard electrophysiological techniques were used to evaluate the transport processes involved in antidiuretic hormone (ADH)-dependent hypertonic cell volume regulation in the in vitro perfused mouse medullary thick ascending limb of Henle. Hypertonic cell volume regulation appeared to involve NaCl uptake into cells, since the cell volume increase after osmotic shrinkage in hypertonic media could be abolished either by symmetrical removal of Na+ from external solutions or by bath Cl- omission. The volume-regulatory process also required CO2/HCO3- in external media and could be abolished by the lipophilic carbonic anhydrase inhibitor, ethoxzolamide, in the presence of CO2/HCO3-. In addition, ADH-dependent hypertonic cell volume regulation was reduced or abolished by 10(-4) M amiloride, 10(-3) M ouabain, or 10(-4) M 4-acetamido-4'-isothiocyanostilbene-2,2-disulfonic acid in peritubular media or by cooling to 15 degrees C. In contrast, lumen Cl- omission or 10(-4) M amiloride addition to the perfusate had no effect on cell volume regulation in hypertonic peritubular media. These data suggest that ADH-dependent, hypertonic cell volume regulation in the mouse medullary thick limb depends on cell NaCl uptake via a secondary active transport process involving parallel Na+-H+ and Cl( )-HCO3- exchangers in basolateral cell membranes. Finally, luminal furosemide (10(-4) M) abolished bath ouabain-mediated, rapid cell swelling in isotonic media containing ADH. Thus these exchangers do not appear to be active in the resting, isotonic state. The specific role of ADH in this NaCl transport process remains to be defined. PMID- 3013020 TI - Proton inhibition of chloride exchange: asynchrony of band 3 proton and anion transport sites? AB - The inhibition of chloride exchange at 0 degrees C by protons at the cytoplasmic and the extracellular surface of the band 3 protein of human erythrocytes was measured between pH 4.6 and 7.6. At constant external pH and chloride concentration, internal protons were a mixed inhibitor of chloride flux, with the apparent pK2 = 6.1 for protonation of the inward-facing empty transporter conformation and the apparent pK3 = 5.7 for protonation of the chloride transporter complex. The activation of chloride exchange by external chloride was inhibited by internal protons, and internal protonation of the externally facing empty conformation had a pK1 = 6.1. External protons were also a mixed inhibitor of chloride exchange with the apparent pK1 = 5.0 for the empty outward-facing transporter conformation. Because of the pHo dependence of self-inhibition, the value of pK3 on the outside for chloride could not be accurately determined, but the apparent pK3 for protonation of the iodide-transporter complex on the extracellular surface was 4.9. The data support a mechanism with a single proton binding site that can alternatively have access to the cytoplasmic and extracellular solutions. It appears that this proton binding and transport site can be coupled to the single anion transport site for cotransport, but the two sites can be on opposite sides of the membrane at the same time and thus can be asynchronously transported by conformational changes of band 3. PMID- 3013021 TI - Stimulation of corticosterone secretion in vitro by brief ACTH exposure. AB - We examined the relationship between ACTH concentration and exposure duration on stimulation of corticosterone (B) secretion in vitro using perifused enzymatically dispersed rat adrenocortical cells. A modular perifusion apparatus was used that permitted evaluation of 20-24 cell chambers per experimental session. In expt 1, 20-1000 pg/ml concentrations of synthetic ACTH-(1-24) were presented to cells for 1 min. In expt 2, 100 pg ACTH-(1-24) was presented to adrenal cells in five dose-duration regimens ranging from 5 pg/min for 20 min to 100 pg/min for 1 min. Perifusal rate was 1 ml/min in all sessions. B was determined by radioimmunoassay. In expt 1 (constant-duration paradigm), 1-min presentation of ACTH-(1-24) produced log-linear dose-response effects across these concentrations (r = 0.926, P less than 0.01). In expt 2 (constant-mass paradigm), identical masses administered in different dose-duration regimens had different steroidogenic efficacies (P less than 0.02): low-dose long-duration regimens provoked greater total release than high-dose short-duration regimens. Overall, every dose-duration regimen was associated with stimulation of B secretion. These results indicate that very brief exposure to physiological concentrations of ACTH-(1-24) is a significant stimulus for corticosteroid secretion; variations in the dose-duration regimen over the physiological range modifies both the maximum rate of secretion and the duration of secretion, but not the response latency; and ACTH-(1-24) presentation mass is not the sole determinant of B secretion. PMID- 3013022 TI - Distribution of epidermal growth factor binding sites in the adult rat liver. AB - The localization of epidermal growth factor (EGF) receptors was studied in the liver by use of light microscope autoradiography performed at different time intervals (2-60 min) after intravenous injection of 125I-EGF into adult rat. The results revealed a substantial binding of EGF to parenchymal cells of the liver. Control experiments indicated that the autoradiographic labeling was due to specific interaction of 125I-EGF with its receptor. A very steep portal-to central lobular concentration gradient for 125I-EGF uptake was observed. The time course study performed on parenchymal cells of periportal regions of lobules showed that, at the 2-min time interval, most silver grains were found at the periphery of these cells and that the maximal values were reached 7 min after injection. After, the number of grains decreased progressively and their localization in the cytoplasm indicated the internalization of the ligand. The present results demonstrate that EGF injected into the general circulation preferentially binds to hepatocytes located in the periportal area and is rapidly internalized. PMID- 3013023 TI - Na+-H+ exchange in rat colonic brush-border membrane vesicles. AB - To demonstrate the presence of a Na+-H+ exchange process in brush-border membrane vesicles from rat colonocytes, the fluorescence response of acridine orange was used to monitor the formation and dissipation of pH gradients. An inwardly directed Na+ gradient stimulated the outward flux of H+, whereas proton influx was stimulated by an outwardly directed Na+ gradient. Since the ionophore valinomycin in the presence of a K+ gradient did not alter Na+-stimulated proton efflux, the interrelationship of Na+ and H+ movement could not be explained solely on the basis of a membrane potential. Na+-stimulated proton efflux was saturable with a Km for Na+ of 20.1 +/- 1.6 mM. Inwardly directed Li+ gradients also stimulated proton efflux, and the Km for Li+ was 30.2 +/- 1.7 mM. In contrast, impermeant cations failed to stimulate the outward flux of H+. Amiloride (1 mM) inhibited both Na+-stimulated proton efflux and influx. Therefore, Na+-H+ exchange is present in rat colonic brush-border membranes and has characteristics similar to other Na+-H+ antiporters. This exchange process may be an important mechanism for Na+ absorption in the large intestine. PMID- 3013024 TI - Inhibition of ATP levels by quinidine in a human colonic epithelial cell line. AB - The influence of quinidine, a putative K+ channel blocker, on Cl- secretion induced by vasoactive intestinal polypeptide (VIP) was investigated. Quinidine inhibited Cl- secretion induced by VIP in T84 cell monolayers. A similar inhibitory effect of quinidine on Cl- secretion was observed in an isolated human colon. However, in the isolated human colon, which absorbs Na+ avidly, inhibition of Na+ absorption predominated. In the T84 cell, the half-maximal inhibition by quinidine occurred at 60 microM, while 300 microM almost completely inhibited the VIP-induced Cl- secretion. Mucosal addition of quinidine was at least equally effective compared with serosal addition, suggesting that quinidine acts on the apical membrane or intracellularly. Quinidine had little or no effect on VIP stimulated Cl- efflux in the first 15 min after its addition, suggesting that blockage of the Cl- exit pathway on the apical membrane is an unlikely mechanism. Similarly, quinidine did not inhibit the K+-recycling mechanism on the basolateral membrane in the first 15 min after its addition. The initial inhibitory action of quinidine corresponded better with its ability to decrease cellular ATP levels. Our study suggests that the depletion of cellular ATP levels may explain the initial inhibitory action of quinidine on electrolyte transport in the intestine, while the late effect is multifactorial. PMID- 3013025 TI - Alpha 1-adrenergic constriction limits coronary flow and cardiac function in running dogs. AB - Modulation of coronary blood flow and cardiac function by alpha 1-adrenergic receptors was examined in dogs during strenuous exercise. Fifteen dogs were chronically instrumented to measure left circumflex blood flow, heart rate, regional left ventricular function (systolic shortening, and rate of shortening), and global left ventricular function (left ventricular pressure, and dP/dt). The specific postsynaptic alpha 1-receptor blocker prazosin (0.5 mg) and nonselective alpha-receptor blocker phentolamine (1.0 mg) were injected through an indwelling circumflex artery catheter to produce local adrenergic blockade of the posterior left ventricular region during exercise. Exercise significantly increased heart rate, left ventricular systolic pressure, dP/dt, segment shortening and rate of shortening, and coronary blood flow. Both prazosin and phentolamine caused similar additional increases in dP/dt by 21 +/- 4%, in rate of shortening in the posterior region by 37 +/- 6%, and in myocardial O2 consumption by 26 +/- 11%, which were associated with a 21 +/- 3% increase in coronary flow during exercise but no change in O2 extraction. Similar results were obtained when dogs were beta blocked with either atenolol (1.0 mg ic) or propranolol (1.0 mg ic) prior to exercise. These data suggest that an alpha 1-vasoconstriction modulates O2 delivery to myocardial tissue and limits both coronary vasodilation and cardiac function during exercise. PMID- 3013026 TI - Prolonged responsiveness to ouabain in hypertrophied rat heart: physiological and biochemical evidence. AB - The inotropic effect of ouabain on cardiac hypertrophy was evaluated on an isolated Langendorff rat heart preparation with performances registrated by means of an intraventricular balloon. These effects were compared with the drug action on the sarcolemma-bound Na+-K+-ATPase activity. On both normal and pressure overload induced hypertrophied rat hearts (ventricular wt-to-body wt ratios of 2.1 and 3.3, respectively) the inotropic effect of ouabain (10(-9)-10(-4) M) was evaluated at 0.25 mM external Ca2+. Compared with normal hearts, the recovery of a normal contractile function after the inotropic response was significantly slower in hypertrophied hearts. This was valid with the two protocols applied. During a 30-min washout period, the inotropic response remained nearly unchanged in hypertrophied hearts, whereas it was almost completely reversed in control groups. Sarcolemmal vesicles from both heart groups exhibited high Na+-K+-ATPase activities (sp. act.: 105 +/- 16 mumol X h-1 X mg-1). In both normal and hypertrophied cardiac sarcolemmal preparations, the Na+-K+-ATPase was heterogeneous, with high- and low-sensitivity forms. Their relative proportion was two-to-one. In both heart groups, their respective apparent affinities for ouabain were similar (inhibitory concentration of 50% = 10(-8) and 10(-6) M, respectively). The release of ouabain from these two sites was measured, in washout experiments, by the rates of enzyme relief from inhibition. High- and low sensitivity forms in hypertrophied heart preparations released ouabain at seven- and threefold lower rates, respectively, than the corresponding forms present in normal cardiac sarcolemmal vesicles.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013027 TI - Methylene blue and ETYA block flow-dependent dilation in canine femoral artery. AB - Flow-dependent dilation of the canine femoral artery is endothelial cell dependent and is not mediated by prostaglandins, adrenergic or cholinergic receptors, an ascending message from the microcirculation, or by myogenic mechanisms. We investigated the mechanism of flow dilation in 38 pentobarbital anesthetized dogs. A femoral artery-jugular vein shunt was constructed, and femoral artery diameter was continuously measured (sonomicrometer crystals) during control and maximum flow (1 l/min). Inhibition of prostaglandin formation by indomethacin did not alter the dilation response to increased flow, but the lipoxygenase-cyclooxygenase inhibitor 5, 8, 11, 14 eicosatetraynoic acid (ETYA) irreversibly inhibited the dilation response to increased flow. The guanylate cyclase inhibitor, methylene blue, caused a dose-dependent decrease in the dilation response to increased flow. Pretreatment with the H1 receptor antagonist tripelennamine sensitized the vessel to the inhibitory effects of methylene blue. Both methylene blue and ETYA shifted the ED50 for acetylcholine relaxation two orders of magnitude to the right, but did not alter the ability of the vessel to dilate or constrict to other stimuli. These data suggest that both cyclic GMP and a non-prostaglandin metabolite of arachidonic acid are involved in flow dilation. We propose that endothelial cells release a metabolite of arachidonic acid that stimulates vascular smooth muscle guanylate cyclase leading to relaxation. The role of histamine in this system is unknown. PMID- 3013028 TI - Nucleus paragigantocellularis lateralis and lumbar sympathetic discharge in the rat. AB - Two classes of medullospinal units with inhibitory baroreceptor inputs [nucleus paragigantocellularis lateralis (PGCL) units] were recorded in PGCL (conduction velocity 3.1 and 0.67 m/s, respectively). Single-pulse stimulation of PGCL produced two peaks of activation of the lumbar sympathetic chain [sympathetic nerve discharge (SND)] with latencies of 98 and 206 ms. After substracting the delay due to impulse propagation between cord and sympathetic chain (64 ms), a conduction velocity of 2.8 and 0.67 m/s could be estimated for the medullospinal portion of the two sympathoexcitatory pathways stimulated in the PGCL. R wave triggered activity histograms of PGCL unit discharge and lumbar SND exhibited a similar pattern with one major trough, occurring 108 ms later at the level of the sympathetic chain. It is concluded that PGCL contains two classes of medullospinal neurons with sympathoexcitatory function, the fast-conducting type being predominantly responsible for the cardiac-related rhythm of lumbar SND. Finally, averaging techniques revealed a large respiratory rhythm of lumbar SND in spontaneously breathing rats, but little or no such rhythm could be detected at the level of most PGCL sympathoexcitatory cells. PMID- 3013029 TI - Regulation of interacting populations during endocytosis: models of growth factor tumor promoter dynamics. AB - A model of growth factor-cell receptor interactions, including internalization, sorting, recycling, and degradation and their modulation by tumor promoters, is developed, analyzed, and tested. In keeping with data and concepts based on a large number of systems, the main assumption is that after receptor-ligand binding the complex associates with a second membrane protein, localized in coated pits, and that this event is a necessary condition for receptor-mediated endocytosis and subsequent intracellular processes. As a consequence of the model, ligands having distinct receptors interfere at the cell surface through competition between their receptor complexes for a limited pool of coated pit proteins. The utility of the model is illustrated by a detailed analysis of binding, endocytosis, and degradation of epidermal growth factor (EGF) and their modulation by phorbol esters. The analysis permits quantitative characterization of the dynamics of the endocytic processes and leads to the following conclusions. The Scatchard plot changes from linear to nonlinear as the ratio of the number of coated pit proteins to the number of receptors decreases. Competition between phorbol ester and EGF-bound receptors for coated pit proteins predicts, in agreement with observation, conversion of nonlinear EGF Scatchard plots to linear plots subsequent to reincubation with phorbol esters. The postulated competition suggests a local homology between the phorbol ester receptor and the EGF receptor. Homologous and heterologous downregulations observed in numerous systems are natural consequences of the model. Preincubation with the heterologous ligand increases the time lag between ligand binding and lysosomal degradation and alters intracellular sorting. PMID- 3013031 TI - Identifying lithium-responsive bipolar depressed patients using nuclear magnetic resonance. AB - Proton T1 relaxation times of red blood cells were significantly higher in six bipolar depressed patients than in matched normal control subjects before lithium treatment. Times decreased in five of the six patients following 1 week of lithium therapy. PMID- 3013030 TI - Renal opiate receptor mediation of renin secretion to renal nerve stimulation in the dog. AB - The present study was designed to evaluate renal opiate receptor mediation of the renin secretion response to electrical stimulation of the renal nerves in the pentobarbital sodium-anesthetized dog by use of the opiate agonist leucine enkephalin (Leu-enk) and the opiate antagonist naloxone. In all animals studied, left kidneys were pump perfused at a constant renal blood flow. Renal perfusion pressure (RPP) and glomerular filtration rate (GFR) were unaltered at a stimulation frequency of 1.0 Hz; however, renin secretion rate (RSR) increased significantly in the nontreated group. High-frequency renal nerve stimulation (10 Hz) increased RPP and decreased GFR. RSR at the high-frequency stimulation was significantly augmented in the nontreated group. Renal arterial infusion of either Leu-enk (25 micrograms X kg-1 X min-1) or naloxone (7 micrograms X kg-1 X min-1) did not alter base-line levels of renal hemodynamics and RSR and did not produce significant changes in these variables even when renal nerves were stimulated at the low frequency; however, Leu-enk inhibited RPP and RSR responses to the high-frequency stimulation, and naloxone augmented these responses. Phentolamine (13 micrograms X kg-1 X min-1) prevented renal hemodynamic responses to the renal nerve stimulation, whereas RSR responses to the stimulation were unaffected. Propranolol (8 micrograms X kg-1 X min-1) resulted in decreases in RSR at the renal nerve stimulation despite the presence of changes in renal hemodynamics similar to the other groups. The results indicate that intrarenal opiate receptors may participate in inhibiting renal secretion of renin mediated by the renal nerves when renal vasoconstriction and reduction of GFR occurred at the high-frequency stimulation. PMID- 3013032 TI - The corticotropin-releasing hormone stimulation test in patients with panic disorder. AB - Eight patients with panic disorder had significantly lower ACTH and cortisol responses to corticotropin-releasing hormone and a significantly lower ratio of ACTH to cortisol response than 30 normal control subjects. These responses resemble those previously reported for depressed patients except that they occurred in the face of significantly elevated basal cortisol and ACTH levels. These results suggest that patients with panic disorder have an element of chronic hypercortisolemia, like depressed patients, but also a more acute perturbation in ACTH secretion, not previously seen in depressed patients. PMID- 3013033 TI - Essential fatty acid supplementation in tardive dyskinesia. AB - Preclinical and clinical observations suggest that enhancement of prostaglandin activity inhibits catecholamine release and may have antidyskinetic effects. A double-blind therapeutic trial with prostaglandin precursor essential fatty acids was conducted in 16 patients with tardive dyskinesia. No beneficial effects were seen. PMID- 3013034 TI - Hepatocellular adenoma of the placenta. AB - A unique primary placental tumor, designated "hepatocellular adenoma of the placenta," is reported. The tumor was composed of cells resembling fetal hepatocytes. The hepatocellular nature of the tumor cells was supported by ultrastructural and immunohistochemical findings. Histogenetically, this tumor most likely represents a highly specialized monodermal teratoma. PMID- 3013035 TI - [Characteristics of the structure of erythrocyte membranes in pregnant women with late toxemia]. PMID- 3013036 TI - [Role of various neurohumoral factors in the pathogenesis of late pregnancy toxemia]. PMID- 3013037 TI - Chronic infectious mononucleosis syndrome, pancytopenia, and polyclonal B lymphoproliferation terminating in acute lymphoblastic leukemia. AB - A 17-year-old previously healthy girl is reported who developed acute infectious mononucleosis followed by progressive ill health over 20 months, associated with pancytopenia and a polyclonal B-lymphoproliferation, terminating in acute lymphoblastic leukemia (ALL). Epstein-Barr virus (EBV) was recovered from the patient's nasopharyngeal secretions; serologic titers of antibodies to EBV associated antigens were compatible with a chronic persistent EBV infection. Plasma interferon levels were markedly elevated. EBV-specific cell-mediated immunity, as well as Natural killer (NK) cell activity were markedly deficient. Other studies of cell-mediated immunity revealed notable abnormalities, including abnormalities in T-cell subset ratios, and a serum blocker of autologous mitogen induced lymphoproliferation. Humoral (plasma)-mediated, but not cell-mediated, suppression of hemopoiesis was demonstrated using in vitro erythroid and myeloid colony culture techniques. Immunophenotyping of the patient's bone marrow cells preterminally was consistent with ALL. Autopsy revealed pathologic changes of ALL in marrow and multiple organs. We conclude that our patient developed an EBV driven lymphoproliferative disorder, with associated defective cell-mediated immunity and hemopoiesis. Ultimately, the patient's documented polyclonal lymphoproliferative state was superimposed by acute lymphoblastic leukemia. PMID- 3013038 TI - Pseudo-Chediak-Higashi anomaly in a child with a hepatic vascular malformation. AB - A case of a congenital hepatic vascular malformation in a child complicated by disseminated intravascular coagulation and hemolytic anemia is presented. Examination of the peripheral blood disclosed the presence in the leukocytes of giant intracytoplasmic inclusions resembling those of the Chediak-Higashi anomaly. Ultrastructural analysis characterized those inclusions as phagocytosed red cell debris, a result of mechanical destruction of the red cells. PMID- 3013039 TI - Cloning of pediatric malignancies for drug sensitivity testing in the human tumor cloning assay. AB - In a 4-year period 168 tumor specimens received from 119 pediatric patients were plated in a soft agar cloning system. Overall, 47 or 28% of the tumor specimens had growth adequate enough for drug sensitivity testing. Drug sensitivity testing was performed using a variety of standards as well as investigational anticancer agents. Overall, 129 evaluable in vitro drug sensitivity tests were performed with 33 (26%) of the tests showing sensitivity to an agent. The most active drugs in vitro included the standard agents doxorubicin and cis-platinum as well as the investigational agents mitoxantrone and m-AMSA. From these data it is clear the cloning assay can be used to study pediatric malignancies. However, before the cloning assay can be applied to clinical pediatric oncology practice, the assay must be improved in terms of tumor growth and validated with prospective clinical trials. PMID- 3013040 TI - Preoperative chemotherapy in the treatment of Wilms' tumor diagnosed with the aid of fine needle aspiration biopsy. PMID- 3013041 TI - Thrombocytopenic purpura following varicella-zoster vaccination. AB - Acute thrombocytopenia developed 3 weeks after the vaccination of a child with leukemia with V-Z vaccine. This is the first case of postvaccinal thrombocytopenia to be reported following V-Z vaccine. The risk to leukemic children of postvaccinal thrombocytopenia would appear to be less than 1%. Note added in proof: The patient remained seronegative until the eighth month following vaccination. Her FAMA titer then rose to 1:32, probably as a result of exogenous reinfection with V-Z virus. PMID- 3013042 TI - Selective fetal malnutrition: effect of acute and chronic ethanol exposure upon rat placental Na,K-ATPase activity. AB - Intrauterine growth retardation (IUGR) is characteristic of the fetal alcohol syndrome (FAS). This IUGR is partly due to the toxic effect of ethanol upon placental function, including amino acid transport. Amino acid transport is dependent, in part, upon Na,K-ATPase; therefore, plasma membrane activity of Na,K ATPase was measured in rat placentas following acute or chronic ethanol exposure. Acute (A) animals were chow fed and gavaged with ethanol on day 20 of gestation and killed 2 hr later; controls (A-C) received sucrose. Binge (B) animals were gavaged on gestation days 18 and 19; controls (B-C) received sucrose. Chronic animals were fed ethanol in a liquid diet containing 2% (CHR-2%) or 6% (CHR-6%) ethanol by volume and killed on day 20. Controls (CHR-2%-C or CHR-6%-C) were isocalorically pair fed. Maternal blood ethanol levels were 197.1 +/- 29.7 mg/dl (mean +/- SE) for A dams and 128.2 +/- 15.2 for B (drawn at time of death); 12.6 +/- 2.2 for CHR-2% and 195.0 +/- 26.0 for CHR-6% (drawn weekly and at time of death). Placental weight was increased and fetal weight decreased in the CHR-6% animals. A, B, and CHR-2% placental and fetal weights were unaffected. Na,K ATPase specific activity was increased in B placentas: B = 134.7 +/- 16.5 versus B-C = 50.0 +/- 7.4 nmol of Pi/mg of protein/min (p less than 0.01). Conversely, CHR-6% treatment diminished enzyme activity: CHR-6% = 37.0 +/- 4.5 versus CHR-6% C = 58.5 +/- 4.4 (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013043 TI - The requirements for transferrin-dependent adherence of human granulocytes to pollen grains. AB - Human granulocyte/pollen binding protein (GPBP), previously identified as serum transferrin, promoted prolonged firm adherence of neutrophils to Timothy grass pollen. Some characteristics of this adherence reaction are reported. GPBP induced binding was time-, temperature- and concentration-dependent. Maximal adherence was observed by 2 h and was only slightly decreased at 18 h. The optimal temperature for adherence was 37 degrees C. Concentrations of GPBP as low as 1.25 microgram/ml gave significantly greater binding than the albumin or lactoferrin control. Eosinophils, monocytes and lymphocytes did not appear to participate in GPBP-induced pollen binding reactions at concentrations up to 300 micrograms/ml. In the presence of GPBP, neutrophils adhered to a range of grass, weed and tree pollens. These included timothy, meadow, false oat, rye, giant and short ragweed, plantain, silver birch and ash. GPBP did not facilitate the adherence of granulocytes to inert particles of similar size such as Sephadex beads and agarose. The adherence was Mg++- but not Ca++-dependent and was not inhibited by a monoclonal antibody to the transferrin receptor (OKT9). Transferrin/GPBP did not bind to either neutrophils or pollen grains. A purified commercial transferrin reacted in all respects like GPBP in these pollen binding studies. These observations indicate that GPBP/transferrin-induced adherence of granulocytes to pollen grains is a hitherto unrecognized property of transferrin which appears unrelated to iron transport or the conventional transferrin receptor. PMID- 3013044 TI - [Intra- and postoperative interactions between the 2 opioids fentanyl and buprenorphine]. AB - In order to demonstrate pharmacokinetic and pharmacodynamic interactions between fentanyl and buprenorphine, 3 groups of patients (n = 30) were compared, receiving either fentanyl (0.005 mg/kg b.w.) or buprenorphine (0.01 mg/kg b.w.) or both opioids as analgesic during surgery for disc protrusion. For a period of 4 h haemodynamic parameters were monitored and blood samples were taken for determination of the following concentrations: ADH, ACTH, cortisol, glucose, unbound glycerol, fentanyl and buprenorphine. Blood gas analyses were performed up to 2 h postoperatively. Although in all groups haemodynamic parameters were constant, there was an increase in factors related to operative stress (cortisol, glucose, unbound glycerol, postoperative acidosis) after the combination of both opioids, while postoperative ventilatory parameters in this group were not improved by the partial agonist buprenorphine. Plasma levels were not affected by combined application, except for a slight elevation of buprenorphine concentrations during additional use of fentanyl. Buprenorphine, at least in higher dosages, seems to antagonize analgesia induced by fentanyl, although respiratory depression is even more pronounced. It may be assumed, that with partial agonists the relation of agonistic and antagonistic activity may be different, depending on the dosage used and on the respective pharmacologic effect observed during investigation. PMID- 3013045 TI - High-performance gel permeation chromatography of proteins and peptides on columns of TSK-G2000-SW and TSK-G3000-SW: a volatile solvent giving separation based on charge and size of polypeptides. AB - Trifluoroacetic acid (0.1% w/v) is an excellent solvent for polypeptides, is volatile, and has a low absorbance of ultraviolet light of low wavelength. Polypeptides subjected to chromatography on columns of TSK-G2000-SW or TSK-G3000 SW in this solvent were eluted as sharp peaks. Retention volume was dependent not only on molecular weight but also on the number of formal charges per molecule. For polypeptides with a similar molecular weight, that with the highest proportion of basic amino acid residues was eluted earliest. For the TSK-G2000-SW column, the degree of deviation from a linear relationship between elution volume and logarithm of molecular weight was directly proportional to the molecular weight to the power 2/3 divided by the number of positive charges per molecule. Inclusion of 0.25 M sodium chloride in the solvent increased both the upper and lower limits of the molecular weight range over which separation occurred. The use of 0.1% trifluoroacetic acid as the mobile phase is thus particularly applicable to the separation of peptides of low molecular weight. PMID- 3013046 TI - Application of progress curve analysis to in situ enzyme kinetics using 1H NMR spectroscopy. AB - The steady-state kinetics of enzymes in tissues, cells, and concentrated lysates can be characterized using high-resolution nuclear magnetic resonance spectroscopy; this is possible because almost invariably there are differences in the spectra of substrates and products of a reaction and these spectra are obtainable even from optically opaque samples. We used 1H spin-echo NMR spectroscopy to study the hydrolysis of alpha-L-glutamyl-L-alanine by cytosolic peptidases of lysed human erythrocytes. Nonlinear regression of the integrated Michaelis-Menten expression onto the progress-curve data yielded, directly, estimates of Vmax and Km for the hydrolase; a procedure for analyzing progress curves in this manner was adapted and compared with a commonly used procedure which employs the Newton-Raphson algorithm. We also performed a sensitivity analysis of the integrated Michaelis-Menten expression; this yielded equations that indicate under what conditions estimates of Km and Vmax are most sensitive to variations in experimental observables. Specifically, we showed that the most accurate estimates of the steady-state parameters from analysis of progress curves are obtained when the initial substrate concentration is much greater than Km. Furthermore, estimates of these parameters obtained by such an analysis are most sensitive to data obtained when the reaction is 60-80% complete, having started with the highest practicable initial substrate concentration. PMID- 3013047 TI - Spectrophotometric determination of acetaminophen, oxyphenbutazone and salicylamide by nitration and subsequent complexation reactions. PMID- 3013048 TI - Adenine nucleotides and some related enzyme activities (adenylate kinase and phosphoglycerate kinase) in normal and abnormal human semen. AB - Adenine nucleotides (ATP and ADP) and enzyme activities of Adenylate Kinase (AK) and Phosphoglycerate Kinase (PGK) were assayed on semen samples from normal and infertile men. In accordance with the values obtained, samples could be classified into two groups: one group with levels of either adenine nucleotides and enzymes assumed as normal; a second group with high levels of all the parameters. The latter group comprised the samples with low sperm density (less than 40 million/ml). When the samples were subdivided according to sperm motility, the above distinction into two groups appeared less evident. Moreover, comparing biochemical parameters with morphological findings some contrasts were seen. Nevertheless, we suggest comparing both biochemical and morphological data for a more detailed classification of human semen abnormalities. PMID- 3013049 TI - Vasculitic purpura in vinyl chloride disease: a case report. AB - Vinyl chloride (VC), a volatile substance mostly used for polyvinyl chloride (PVC) synthesis, is a systemic toxicant particularly noxious to endothelium. Angiosarcoma of the liver, Raynaud's phenomenon, scleroderma-like lesions, acroosteolysis and neuritis are known to be typical vinyl chloride-associated manifestations (VC disease). A so far unknown feature of the disease is purpura. This was first observed by the authors in a worker of a PVC-producing plant. The skin eruption was characterized by small purpuric maculae with tiny, palpable spots and papulae, mostly concentrated on the lower part of the legs, changing into bullae, pustules and crusts and tending to spontaneous regression after withdrawal from VC exposure. A skin biopsy revealed marked inflammatory reaction with a mostly lymphocytic and histiocytic infiltration around and in the walls of dermal arterioles. The finding of increased circulating immune complexes and anti smooth muscle autoantibodies strengthens the hypothesis that immunologic changes play a role in the appearance of "vinylic purpura." PMID- 3013050 TI - Restriction fragment length polymorphism among Israeli Holstein-Friesian dairy bulls. AB - Israeli Holstein-Friesian dairy bulls were screened for restriction fragment length polymorphisms by hybridizing cloned DNA probes for bovine growth hormone, for chymosin, and for rat muscle beta-actin to restriction endonuclease-digested DNA immobilized on nitrocellulose filters. The population proved to be polymorphic at the growth hormone locus, with evidence consistent with the phenotypes being inherited in allelic fashion. A low level of polymorphism was also observed at one of the beta-actin gene family loci. The chymosin locus was monomorphic with the restriction enzymes utilized. The results illustrate the power of restriction fragment length polymorphism methodology in visualizing genetic variability in dairy cattle populations. PMID- 3013051 TI - Forestomach epithelial receptor activation by rumen fluids from sheep given intraruminal infusions of volatile fatty acids. AB - Forty-four reticuloruminal epithelial receptors were tested with rumen fluids obtained from 12 sheep before they were intraruminally infused with 4.0M acetic acid (8 sheep) or 4.0M butyric acid (4 sheep; preinfusion rumen fluid) and with rumen fluids obtained at the onset of ruminal stasis (abolition rumen fluid). The preinfusion rumen fluids from the 8 acetic acid-infused sheep (mean pH, 6.55) contained 1.7 mM nondissociated volatile fatty acids (VFA)/L and excited none of the 25 receptors tested. Preinfusion rumen fluids from the 4 butyric acid-infused sheep (mean pH, 6.98) contained 0.3 mM nondissociated VFA/L and also did not evoke responses in any of the 19 receptors tested. Abolition rumen fluids from sheep treated with acetic acid excited 17 of the 25 receptors tested and contained 89.4 mM nondissociated VFA/L, of which nondissociated acetic acid comprised 85.0 mM/L. Abolition rumen fluids from sheep treated with butyric acid activated 14 of the 19 receptors tested and contained 61.1 mM nondissociated VFA/L, of which 38.7 mM/L was nondissociated butyric acid. Preinfusion rumen fluids whose pH values were adjusted to that of abolition rumen fluids with HCl contained nondissociated VFA levels ranging from 16.3 mM/L (acetic acid-treated sheep) to 20.6 mM/L (butyric acid-treated sheep) and elicited responses in 4 of 30 receptors tested. Preinfusion rumen fluids whose pH values were adjusted to the pH value of abolition rumen fluid with acetic acid contained 29.5 mM nondissociated VFA/L and excited 7 of 13 tested receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013052 TI - Cloning and characterization of a canine oral papillomavirus. AB - A papillomavirus, isolated from oral papillomas in young Beagles, was used to produce a live-virus vaccine. After the IM use of this vaccine, some dogs developed squamous cell carcinomas at the inoculation site. The virus was isolated from the original vaccine and was cloned into pBR322. A detailed restriction map of the viral genome was generated. PMID- 3013053 TI - Nitrogen metabolism of calves inoculated with bovine adenovirus-3 or with infectious bovine rhinotracheitis virus. AB - Beef calves were inoculated with bovine adenovirus-3 or infectious bovine rhinotracheitis virus. After inoculation, plasma fibrinogen increased, serum phosphorus decreased, and nitrogen and phosphorus digestibility decreased compared with preinoculation values. Urinary N excretion increased when calves developed rectal temperatures greater than 39.7 C. Results indicated that clinical infection of calves with infectious bovine rhinotracheitis virus increases urinary N excretion and reduces N and phosphorus balance, and that clinical and subclinical infections with either virus reduce dietary N digestibility. PMID- 3013054 TI - A canine model of bronchial injury induced by nitric acid. Lung mechanics and morphologic features. AB - We created a model of airway obstruction that avoids long-term toxic exposure. Bronchial injury was induced in 7 dogs by exposure to nebulized 1% nitric acid (HNO3) on alternate days for 4 wk. Lung mechanics were measured prior to exposure and after 2 and 4 wk. Both TLC and VC decreased and the FRC/TLC ratio increased. Expiratory flows decreased while breathing air and an 80% helium-20% oxygen mixture; the volume of isoflow increased. Pulmonary resistance increased and dynamic compliance decreased. On single-breath nitrogen washout, there were increases in the slope of phase III and in closing capacity. Histologically, there was widespread chronic airway inflammation, slight epithelial changes, slight peribronchiolar fibrosis, and an increase in smooth muscle. Pathologic scores were significantly higher in the HNO3-exposed group than in the control group. Scores for peripheral and central airway pathology were correlated with results of tests of airway obstruction. PMID- 3013055 TI - Genetic relatedness among strains of the Mycobacterium tuberculosis complex. Analysis of restriction fragment heterogeneity using cloned DNA probes. AB - Chromosomal DNA isolated from 3 reference and 2 clinical isolates of Mycobacterium tuberculosis, 2 reference strains each of M. bovis and M. bovis BCG, and 1 reference strain of M. kansasii, was digested with 9 restriction endonucleases. The restriction fragments were analyzed by agarose gel electrophoresis. With each enzyme tested the patterns of the strains of M. tuberculosis, M. bovis, and M. bovis BCG were indistinguishable but clearly distinct from those of M. kansasii. A library of M. tuberculosis H37Rv DNA was prepared by cloning Bam HI digest fragments into lambda 1059. Eight randomly selected clones, having multiple Bam HI sites, were used to probe restriction digests of DNA from the strains of the tuberculosis complex. Two of the cloned segments hybridized with homologous fragments in 4 different enzyme digests of all strains. With 2 clones, hybridization differed among the strains; however, some homologous sequences were detected. With 4 clones, efficient hybridization occurred only with M. tuberculosis H37Rv DNA. These hybridization results indicate that some regions of the chromosome are highly conserved among members of the tuberculosis complex, whereas others are diverged. Selected DNA probes were useful in detecting differences among strains of the tuberculosis complex. PMID- 3013056 TI - Local generation of sulfidopeptide leukotrienes upon nasal provocation with cold, dry air. AB - In order to assess whether sulfidopeptide leukotrienes are generated following nonimmunologic stimulation of inflammatory cells in vivo, 11 subjects complaining of symptoms of rhinitis when exposed to cold and dry environments were challenged by nasal breathing, first with warm, moist air and then with cold, dry air. Nasal lavages with normal saline were performed before and after each exposure. Immunoreactive leukotriene in the lavage fluids was significantly increased following cold, dry air exposure (2.6 ng/ml) compared with that after warm, moist air exposure (0.7 ng/ml) or at baseline (0.4 ng/ml) (p less than 0.01 in both instances). Six more subjects, denying cold-air sensitivity, were subjected to the same protocol and had no mediator and symptom score changes after cold, dry air challenge. Leukotriene changes after cold, dry air were highly concordant with increments in histamine, prostaglandin D2,N-alpha-tosyl-L-arginine methyl ester (TAME) esterase(s) activity and symptom scores (p less than 0.001). Separation of leukotrienes by high performance liquid chromatography in the nasal washes of 3 subjects showed variable amounts of LTC4, D4, and E4, suggesting metabolism of the former to the latter 2. To our knowledge, this is the first in vivo demonstration of leukotriene production in response to a physical stimulus, and it suggests a possible role of these and other inflammatory mediators in pathologic conditions, such as exercise-induced asthma, that involve physical causative factors. PMID- 3013057 TI - Isoniazid, rifampin, and hepatotoxicity. PMID- 3013058 TI - The natural history of asbestosis in former crocidolite workers of Wittenoom Gorge. AB - The course of pulmonary asbestosis and its determinants have been examined in 280 applicants for compensation among former workers of the crocidolite mine and mill at Wittenoom Gorge, Western Australia. Serial chest radiographs accrued over more than 3 decades were graded for parenchymal disease separately by two observers according to the 1980 ILO Classification of Radiographs for Pneumoconioses and without knowledge of exposure histories or compensation details. In 136 subjects whose median duration of exposure was 37 months, radiographic asbestosis appeared between 1 and 34 yr after initial exposure and then progressed continuously. Total exposure to asbestos and time from first exposure to the appearance of definite radiographic asbestosis were significant determinants of the rate of progression of profusion of radiographic abnormality. Asbestosis should be considered to be an active disease even 3 decades after exposure has ended. PMID- 3013059 TI - Nomenclature: human immunodeficiency virus. PMID- 3013060 TI - Cimetidine, ranitidine, and Epstein-Barr virus infection. PMID- 3013061 TI - Reports of DHPG treatment of cytomegalovirus retinitis. PMID- 3013062 TI - Theophylline-induced hypercalcemia. AB - Sixty patients with theophylline toxicity were hospitalized during a 2-year period. Eleven patients had hypercalcemia; their calcium levels returned to normal as theophylline levels fell to therapeutic or subtherapeutic levels. Serum calcium levels also fell significantly in three additional patients with theophylline toxicity, although the initial serum calcium concentration was not outside normal limits. A significant increase in serum calcium levels associated with therapeutic levels of theophylline in normal volunteers was reversed by propranolol. It appears that theophylline causes elevation of serum calcium by a system subject to beta-adrenergic regulation. PMID- 3013063 TI - [Prenatal diagnosis of hemophilia A by analysis of DNA]. AB - Early ante-natal diagnosis of haemophilia A and the detection of female carriers is now possible in some cases by analysis of DNA. The diagnosis may be established with a 100 p. 100 reliability in subjects with large deletion by direct analysis, and in 40 p. 100 of haemophiliac families by linkage studies with the intra-genic polymorphism revealed by the restriction enzyme BcII. Intensive research indicates that this percentage will increase in the near future. In the meantime, indicative studies are possible in other families. They consist in studying extra-genic restriction polymorphism. Over 90 p. 100 of families with haemophilia A may benefit from these studies using the probes currently available. Recombination, although not yet described, remains possible, and therefore ante-natal diagnoses made by the extra-genic probe should be controlled by foetal blood sampling at the 20th week of pregnancy. PMID- 3013064 TI - The evolutionary origins of intercellular communication and the Maginot Lines of the mind. AB - By extending the evolutionary age of the vertebrate hormones from the vertebrates to include the metazoans, we expand their phyletic distribution about 30-fold. By tracing these molecules into the unicellular range including both eukaryotes and prokaryotes, the distribution of these molecules becomes very wide indeed. While "universal" or "ubiquitous" is probably not yet warranted, their recognition as "cosmic" molecules rather than "parochial" molecules does seem appropriate. Interestingly, the breakdown of the barriers for the hormonal molecules between the vertebrates and the rest of the metazoans, between the metazoans and the unicellular organisms, between the eukaryotes and prokaryotes, or the eubacteria and archebacteria is concordant with findings in multiple other systems. For example, hemoglobin or myoglobin is present in higher plants, Protozoa, and insects. The photosynthetic proteins of higher plants have their homologues in the photosynthetic bacteria, and the heat shock proteins of eukaryotes have their equivalents in the prokaryotes as well. PMID- 3013066 TI - Sampling of gaseous pollutants on silica gel with 1400 mg tubes. PMID- 3013065 TI - Recent studies on opioid receptors: heterogeneity and purification. PMID- 3013067 TI - Fibre release from filtering facepiece respirators. PMID- 3013068 TI - Evaluating dust exposures in foundries by a screening test. PMID- 3013071 TI - [Pharmacochemistry through the history of benzodiazepines and their binding sites (2)]. PMID- 3013070 TI - Hormonal receptor site alterations in the etiopathogenesis of otosclerosis. AB - The authors have studied calcium 45 uptake and cAMP intracellular levels in normal and otosclerotic bone cell cultures after stimulation with calcitonin in the presence or absence of propranolol. The results seem to demonstrate that poststimulatory 45Ca incorporation is slightly different in normal and otosclerotic cell cultures, being slower but longer lasting in the latter. Propranolol administration markedly inhibits 45Ca uptake in normal cells, while in otosclerotic cells a massive intracellular penetration becomes evident after an initial inhibitory phase. Also cAMP intracellular levels behave differently; a marked increase, followed by a rapid decrease, can be detected in normal cells after stimulation with calcitonin, while in otosclerotic cells, the increase is slower and followed by a long lasting reduction. Adding propranolol reduces cAMP levels in normal cells, while it increases levels in otosclerotic cells. The different behavior of calcium metabolism and cAMP levels after stimulation with calcitonin, depending upon the presence or absence of propranolol, seems to indicate an alteration of the transducing mechanism between stimulus, receptor, and cellular effector in otosclerotic cells. PMID- 3013069 TI - Variation in fibre and dust counts in an asbestos mine and mill. PMID- 3013072 TI - Selenium in food and nutrition in Finland. An overview on research and action. AB - For geochemical reasons Finland is a low-selenium area. In the 1960's several diseases associated with serious Se deficiency were observed in domestic animals. Selenium medication of animals and selenium supplementation of animal feeds from 1969 effectively eliminated these diseases. An extensive study of the trace element content of foods consumed in Finland in the 1970's demonstrated that the dietary intake of selenium was exceptionally low (25 micrograms/day/10 MJ) during the years when domestic grains were used. A study carried out in 1981 showed that supplementation of healthy middle-aged men with high selenium wheat or yeast or selenate double the glutathione peroxidase activity in platelets. Prospective epidemiological studies based on cohorts that were followed in the 1970's suggested that low selenium (less than 45 ng/ml serum) might be a risk factor for cardiovascular diseases and cancer. Technologies to increase the selenium content of foods and feeds were developed and an official decision was reached to add, starting in 1984, sodium selenate to the main fertilizers to increase the selenium content of domestic grain to about 100 micrograms/kg. This measure will increase the average selenium intake above 50 micrograms/d even in the years when grain with a high selenium content is not imported. PMID- 3013073 TI - Selenium fertilizers and foliar application, Danish experiments. AB - In 1963 Se-deficiency was observed for the first time in Denmark in a few sheep in West Jutland. The sheep were cured by injection of "Tokosel", and a survey of the Se-status of Danish fodder crops was initiated. A comprehensive set of data was produced during the early 70's, using the fluorometric method. The survey showed a general Se-deficiency in the whole country, and series of experiments were carried out to elucidate the possibility of raising the selenium level in plants from the native 0.02-0.04 ppm to more than the desired minimum of 0.05 ppm. Three different methods of application were tested: seed pretreatment, fertilizer enrichment, and foliar application. Seed pre-treatment has some disadvantages while the two other methods proved to be efficient and safe in a series of experiments and in tests on a large number of farms all over Denmark. These experiments and tests are discussed in detail. It is concluded that about 120 g Se/ha as sodium selenite, 10 g Se/ha as sodium selenate - both added through PK- or NPK-fertilizers, or foliar application of about 5 g Se/ha are sufficient yearly treatments to raise the native Se content of the Danish crops to levels of 0.05-0.1 ppm. PMID- 3013074 TI - Serum thymidine kinase in transplant patients: its relation to cytomegalovirus activity, renal transplant rejection and its use for monitoring of antiviral therapy. AB - Twenty patients, cadaveric renal transplant recipients, were retrospectively analysed for serum levels of deoxythymidine kinase. Special reference was made to the thymidine kinase level in relation to rejection, and viral infection. Seven of the patients experienced clinically suspected cytomegalovirus infection. All these patients had elevated levels of serum thymidine kinase during the period of clinical disease. Usually the thymidine kinase level parallelled the severity of the disease. All patients with irreversible rejection had increased levels of serum thymidine kinase, but normally not as high, as seen in patients with severe cytomegalovirus infection. There was also some correlation between clinically suspected rejection, that lead to rejection treatment, and moderate increase in thymidine kinase. However, not all rejection episodes were accompanied by a thymidine kinase increase. Serum thymidine kinase was analysed and compared in patients with self-healing cytomegalovirus disease, and those treated with phosphonoformate. A rapid decline in thymidine kinase level was found in connection with successful antiviral therapy, when compared to the decline in untreated patients. Some bone marrow transplanted patients were also included in this analysis. PMID- 3013075 TI - Damage in canine hearts following defibrillator shocks. AB - Experiments were performed to investigate possible differences in potential myocardial cell damage following the use of two clinically available difibrillators. One had a damped sine wave (DSW), and the other a truncated exponential waveform (TEW). The latter, therefore, had a lower peak current and voltage. After pilot studies to determine damage potential, an energy content of 10 Joules per kg was selected with three transthoracic shocks at 30 second intervals, delivered with paddles, by both defibrillators. Thirteen dogs were shocked with DSW (10 sacrificed at 24 hours and three at 72 hours). Eleven dogs were shocked with TEW (eight sacrificed at 24 hours and three at 72 hours). Three were used as untreated controls. Observations included ECG monitoring, technetium99m pyrophosphate (Tc-PyP) uptake, and gross and microscopic observations including electron microscopy. This report will concentrate on the morphologic changes. Eleven of 13 dogs shocked with DSW developed ventricular tachycardia, transient heart block, Tc-PyP uptake and myocardial cell death with acute necrosis, and aberrant contraction patterns associated with cell death. Progression of the lesions occurred from 24 to 72 hours. Only two (one at 24 hours and one at 72 hours) of the dogs shocked with TEW showed microscopic foci of necrosis. One was a chance finding (24 hours); the other was associated with an overlying "yellow streak". In neither case were arrhythmias or Tc-PyP uptake observed. The results indicate that in dogs at equivalent energy levels, TEW caused significantly less myocardial damage than DSW. PMID- 3013076 TI - Uptake of radiolabeled ions in normal and ischemia-damaged brain. AB - The regional concentrations of nine radiochemicals were measured in rat brain after induction of cerebral ischemia to identify tracers concentrated by brain undergoing selective neuronal necrosis. Transient (30 minute) forebrain ischemia was produced in the rat; 24 hours after cerebral recirculation the radiochemicals were injected intravenously and allowed to circulate for 5 hours. The brain concentrations of the radiochemicals in dissected regions were determined by scintillation counting. Forebrain ischemia of this nature will produce extensive injury to striatal neurons but will spare the great majority of neocortical neurons at 24 hours. The regional concentrations of these radiochemicals varied considerably in both control and ischemic animals. In postischemic animals, 4 radionuclides (63Ni, 99TcO4, 22Na, and [3H]tetracycline) were concentrated in the irreversibly damaged striatum in amounts ranging from 1.4 to 2.4 times greater than in normal tissue. The concentrations of 65Zn, 59Fe, 32PO4, and 147Pm in postischemic brain were similar to or less than those in normal brain. The concentration of [14C]EDTA was increased in injured and uninjured brain of postischemic rats. Autoradiographic analysis of the distribution patterns of some of these ions in normal animals showed that 99TcO4, 22Na, 65Zn, and 59Fe were distributed more uniformly throughout the brain than were 32PO4, 63Ni, and 147Pm. At 24 or 48 hours after ischemia, 63Ni, 99TcO4, and 22Na were preferentially concentrated in the damaged striatum and hippocampus, whereas 65Zn, 59Fe, 32PO4, and 147Pm did not accumulate in irreversibly injured tissue. Of the radiochemicals tested to date, Ni, TcO4, and tetracycline may be useful for diagnosing ischemic brain injury in humans, using positron emission tomography. PMID- 3013077 TI - Traumatic injury alters opiate receptor binding in rat spinal cord. AB - Recent studies with dynorphin (an endogenous ligand for the kappa-opiate receptor) and receptor-selective opiate antagonists have indicated a role for the kappa-receptor in the pathophysiology of spinal cord injury. However, no studies have specifically examined opiate receptor binding in relation to spinal injury. In the present experiments, opiate receptor binding was measured in spinal cord after traumatic injury in rats using the selective radioligands [3H] [D-Ala2, D Leu5]enkephalin (delta-receptor agonist); [3H] [D-Ala2,MePhe4,Gly (ol)5]enkephalin (mu-receptor agonist); and [3H]ethylketocyclazocine (kappa receptor agonist). The specific binding of ethylketocyclazocine, but not the other agonists, showed a significant, time-dependent, and localized increase at the injury site. Since dynorphin, which has been implicated as an injury factor after spinal trauma, shows similar localized increases after spinal injury, the present data are consistent with the hypothesis that up-regulation of the kappa receptor after injury may contribute to the subsequent secondary injury process. PMID- 3013078 TI - Pharmacological aspects of metabolic processes in the pulmonary microcirculation. PMID- 3013080 TI - Opioid peptide processing and receptor selectivity. PMID- 3013079 TI - Comparative toxicology and mechanism of action of polychlorinated dibenzo-p dioxins and dibenzofurans. PMID- 3013081 TI - [Mechanism of the interaction of the anticancer antibiotic adriamycin (doxorubicin) with the anion radical O2]. AB - Interaction of the O2. anion radical with adriamycin in water-acetonitrile mixtures was studied by UV-spectroscopy. It was shown that with changing of the water content in the reaction mixture from 0 to 90 per cent O2. practically irreversibly reacted with adriamycin to form the same product. The spectrum of the reaction product was shifted to the long-wave region as compared to that of adriamycin and depended on the solvent composition. In the presence of the proton donors the "regeneration" of the absorption spectrum of the starting antibiotic was observed. To investigate the mechanism of the reaction product formation, electrochemical reduction of adriamycin and its reaction with benzosemiquinone and alkali were studied. Formation of the same compound under the different conditions showed that the primary step of O2. interaction with adriamycin was one electron reduction of the antibiotic. The study suggested the reaction mechanism involving formation of adriamycin semiquinone as a result of the electron transfer from the O2. molecule, splitting out of the sugar moiety and reduction of deoxyaglycone semiquinone into anion of deoxyaglycone tautomer. The possible role of the oxygen anion radical in the mechanism of activation of adriamycin and other anthracycline antibiotics is discussed. PMID- 3013082 TI - Efficacy of the acyclic guanosine analog buciclovir [(R)-9-(3,4 dihydroxybutyl)guanine] in experimental genital herpes. AB - The efficacy of the anti-herpesvirus drug buciclovir [(R)-9-(3,4 dihydroxybutyl)guanine] was investigated in guinea pigs and mice infected intravaginally with herpes simplex virus type 2. Topical treatment initiated early after infection was efficacious, in contrast to topical treatment delayed 24 h or more. Systemic treatment of infected mice could not prevent the spread of virus to the brain and mortality. Systemically administered buciclovir had an effect in guinea pigs, even after delayed onset of treatment, but this effect required high doses of the drug. Our results suggest that buciclovir has only a limited effect against herpesvirus infections once the virus is present in the nervous systems of infected animals. PMID- 3013083 TI - Polymyxin B nonapeptide inhibits mating in Saccharomyces cerevisiae. AB - Polymyxin B nonapeptide enhanced susceptibility of yeast cells to various hydrophobic antibiotics and to mating pheromones. At much lower concentrations, the nonapeptide severely inhibited mating. The inhibition was caused by interference with sexual agglutination. PMID- 3013084 TI - Antirhinovirus activity of purine nucleoside analogs. AB - A wide variety of purine nucleoside (mainly tubercidin and adenosine) analogs, which had previously been shown to inhibit the replication of a broad spectrum of RNA viruses, were evaluated for their antirhinovirus activity in human diploid (WI-38) fibroblasts. Tubercidin, 5-(1-hydroxyethyl)tubercidin, 5-(2-buten-1 yl)tubercidin, toyocamycin, and sangivamycin emerged as the most potent inhibitors. These compounds inhibited the replication of rhinovirus types 1A, 1B, and 9 at an MIC well below 1 microgram/ml. However, these compounds proved cytotoxic for the uninfected host cells at concentrations which were only slightly higher (3- to 10-fold, on the average) than those required for inhibition of rhinovirus replication. The most selective inhibitor of rhinovirus replication was 3-deazaguanine, with a selectivity index of 50. None of the carbocyclic and acyclic analogs of adenosine tested exhibited a potent or selective antirhinovirus activity. PMID- 3013085 TI - Interaction of polycationic antibiotics with Pseudomonas aeruginosa lipopolysaccharide and lipid A studied by using dansyl-polymyxin. AB - A fluorescent derivative of polymyxin B (dansyl-polymyxin) was used to study the interaction of polycations with lipopolysaccharide (LPS) and lipid A from Pseudomonas aeruginosa. Dansyl-polymyxin became bound to LPS and lipid A sites, including Mg2+-binding sites, resulting in a 20-fold enhancement of fluorescence. A Hill plot of the binding data showed that the binding of dansyl-polymyxin to LPS was cooperative (n = 1.98) and of high affinity (S0.5 = 0.38 microM). The maximal binding capacity of LPS was approximately four molecules of dansyl polymyxin per mol of LPS. The dansyl-polymyxin interaction with lipid A displayed similar kinetics (n = 2.26; S0.5 = 0.38 microM), and the maximal binding capacity was approximately 2 mol of dansyl-polymyxin per mol of lipid A. A variety of polycationic compounds, including gentamicin, streptomycin, and polymyxin B, as well as Mg2+, were able to displace dansyl-polymyxin bound to LPS or to lipid A. Marked differences both in terms of the degree of displacement and in terms of the amount of competing polycation required to displace a given amount of dansyl polymyxin were observed. Addition of excess polymyxin B resulted in displacement of all of the dansyl-polymyxin, demonstrating that only polymyxin-binding sites were being probed. Our data demonstrate that polymyxin B binds to multiple sites on LPS, including sites which bind aminoglycoside antibiotics and other polycationic compounds. PMID- 3013086 TI - A novel type of resistance plasmid in Haemophilus influenzae. AB - Resistance plasmids of a novel type were found in two Haemophilus influenzae clinical isolates. pPJ301 and pPJ302 are 10.0 kilobases in size, carry a Tn2-like transposable element, and are related only by their common beta-lactamase genes to the other two types of resistance plasmids known to occur in H. influenzae. PMID- 3013087 TI - Effect of ribavirin and tributylribavirin on argentine hemorrhagic fever (Junin virus) in guinea pigs. AB - Subcutaneous injections of ribavirin into guinea pigs infected intraperitoneally or intracerebrally with Junin virus significantly increased the mean time to death but did not enhance survival of the animals. We found similar results with tributylribavirin. Virus replication was delayed, but not prevented, in ribavirin treated infected guinea pigs. The animals usually died with high virus titers in their brains and frequently were paralyzed. PMID- 3013088 TI - Comparison of susceptibilities of varicella-zoster virus and herpes simplex viruses to nucleoside analogs. AB - The susceptibilities of varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) to 17 nucleoside analogs were compared by a plaque reduction assay with human embryonic lung fibroblast cells. The susceptibility of VZV to certain nucleoside analogs was different from that of HSV-1. Against VZV the 5-halogenovinyl-arabinosyluracils were the most potent of the compounds tested. PMID- 3013089 TI - Determination of mezlocillin and its penicilloate and penilloate by high performance liquid chromatography and stability of mezlocillin at different temperatures. AB - A method is described for the determination of mezlocillin and its metabolites penicilloic acid and penilloic acid in biological fluids by high-performance liquid chromatography. The stability of mezlocillin in serum and buffer was studied at different temperatures (4, -20, -70, and -196 degrees C) over a period of 6 months. Mezlocillin remained stable at -70 and -196 degrees C, whereas degradation was observed at -20 and 4 degrees C in serum and buffer. PMID- 3013090 TI - The solution structure of mucous glycoproteins: proton NMR studies of native and modified ovine submaxillary mucin. AB - The solution structure of native and systematically modified ovine submaxillary mucin (OSM) has been probed by proton NMR spectroscopic methods. Most of the resonances in the spectra have been tentatively assigned to the peptide and O linked disaccharide, alpha-N-acetylneuraminic acid 2----6 alpha-N acetylgalactosamine protons. On the basis of the observed chemical shifts, spectral resolution, and behavior of the exchangeable protons it is concluded the mucin possesses internal segmental flexibility and exists in solution as a random coil peptide. No long-lived interresidue peptide or carbohydrate hydrogen bonds were detected. The removal of (i) the C8 and C9 carbons of the sialic acid residue, (ii) the entire sialic acid residue, and (iii) the complete disaccharide side chain resulted in no significant changes in peptide core conformation. A limited set of proton spin coupling constants and nuclear Overhauser enhancements has been obtained for the threonine glycopeptide side chains in native and modified mucin. The results are consistent with the previously reported conformations for the (1----6) linkage in oligosaccharides and the threonyl glycosidic linkage in glycopeptides. The OSM disaccharide may exist as a extended linear structure with rotational freedom about the GalNAc C5-C6 bond, while the threonine glycosidic linkage appears to be sterically constrained, although multiple conformations about the threonine C beta-O gamma bond may be allowed. The small chemical shift perturbations detected in the glycosylated threonine methyl protons and the GalNAc carbons upon removal of the terminal sialic acid residue are consistent with this model. PMID- 3013091 TI - Determination of apo and holo retinoic acid-binding protein levels in retinoid responsive transformed cells by high-performance size-exclusion chromatography. AB - A method to measure the endogenous levels of apo and holo cellular retinoic acid binding proteins was developed using calf testis cytosol as the source of retinoic acid-binding protein. [3H]Retinoic acid-retinoic acid-binding protein complexes were assayed by high-performance size-exclusion chromatography. Preincubation of cytosol with 10 mM p-hydroxymercuribenzoate at 4 degrees C resulted in complete inhibition of retinoic acid binding to apo retinoic acid binding protein. In addition, total dissociation of preformed holo retinoic acid binding protein complexes was noted within 20 min after mercurial addition. Thus, p-hydroxymercuribenzoate converted the total pool of cellular retinoic acid binding protein (apo plus holo) to mercurial-protein complexes unable to bind retinoic acid in vitro. Mercurial inhibition of retinoic acid-retinoic acid binding protein complex formation was totally reversed upon the addition of 50 mM dithiothreitol. Total cytosolic retinoic acid-binding protein was determined from specific retinoic acid binding after treatment with p-hydroxymercuribenzoate and dithiothreitol. Apo cellular retinoic acid-binding protein concentration was measured by determining specific radioligand binding prior to p hydroxymercuribenzoate treatment, and correcting for exchange of endogenously bound retinoid with exogenous tritiated retinoic acid. Holo cellular retinoic acid-binding protein concentration was derived from the difference between total and apo retinoic acid-binding protein concentrations. Using this method, we have demonstrated that retinoid-responsive EJ and T24 human bladder carcinoma cell lines and AT3A and AT3B rat pancreatic acinar carcinoma cell lines lack detectable levels of either apo or holo cellular retinoic acid-binding protein. These results established that retinoid inhibition of transformed bladder and acinar cell proliferation in culture was mediated by a cellular retinoic acid binding protein-independent mechanism. PMID- 3013092 TI - Examination of the secondary structure of the kringle 4 domain of human plasminogen. AB - The structure of a small region of human plasminogen (F4), consisting of amino acid residues Val354-Ala439 and containing its kringle 4 (K4) domain (residues Cys357-Cys434), has been predicted from Chou-Fasman calculations and hydropathy profiles, and compared to circular dichroism (CD) measurements on the isolated fragment. Calculations, by the Chou-Fasman method, of the probabilities of various types of secondary structures that exist in this region reveal that no helical structures are present. Of the total of 86 amino acid residues present in this K4-containing peptide region, 37% can adopt conformations of beta-pleated sheets, 48% of the amino acids can exist in beta-turns, and 15% of the residues can be present as coils. The structure of F4 in dilute aqueous solution has been experimentally evaluated by CD measurements. At pH = 7.4, in dilute salt solutions, a total of 64% beta-structures, 30% beta-turns, and 6% coiled structures is estimated to be present in this peptide region. Consideration of the marginal stability of many of the conformational regions of F4, as predicted by Chou-Fasman calculations, suggests that secondary structural flexibility is present in this fragment, which could result in ready adoption of new conformations. The hydropathy profile of F4 has been determined and suggests that this polypeptide is highly hydrophilic, especially in the regions of residues His387-Tyr396 and Cys406-Lys413. Thus, it appears as though a large portion of the surface of F4 can be exposed to solvent in its native conformation. PMID- 3013093 TI - Transfer of retinoic acid from its complex with cellular retinoic acid-binding protein to the nucleus. AB - Cellular retinoic acid-binding protein (CRABP), a potential mediator of retinoic acid action, enables retinoic acid to bind in a specific manner to nuclei and chromatin isolated from testes of control and vitamin A-deficient rats. The binding of retinoic acid was followed after complexing [3H]retinoic acid with CRABP purified from rat testes. The binding was specific, saturable, and temperature dependent. If CRABP charged with nonlabeled retinoic acid was included in the incubation, binding of radioactivity was diminished, whereas inclusion of free retinoic acid, or the complex of retinol with cellular retinol binding protein (CRBP) or serum retinol binding protein had no effect. Approximately 4.0 X 10(4) specific binding sites for retinoic acid were detected per nucleus from deficient animals. The number of binding sites observed was influenced by vitamin A status. Refeeding vitamin A-deficient rats (4 h) with retinoic acid lowered the amount of detectable binding sites in the nucleus. CRABP itself did not remain bound to these sites, indicating a transfer of retinoic acid from its complex with CRABP to the nuclear sites. Further, CRBP, the putative mediator of retinol action, was found to enable retinol to be bound to testicular nuclei, in an interaction similar to the binding of retinol to liver nuclei described previously. PMID- 3013094 TI - Alkylacetylglycerophosphocholine effects on the metabolism of phospholipids in rabbit platelets: effects of extracellular Ca2+ and prostacyclin. AB - Alkylacetylglycerophosphocholine (AGEPC) stimulation of 32P-labeled lysophosphatidic acid formation in washed rabbit platelets was dependent on extracellular Ca2+. Its accumulation was slower and required a higher concentration of AGEPC in comparison to the degradation of inositol phospholipids and production of phosphatidic acid induced by the same agonist. These results suggest that the formation of lysophosphatidic acid is not directly related to the primary activation of rabbit platelets by AGEPC. AGEPC elicited a preferential degradation of inositol phospholipids in the following order: phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4 phosphate greater than phosphatidylinositol. The degradation of inositol phospholipids and subsequent production of phosphatidic acid were affected by pretreatment of platelets with prostacyclin or ethylene glycol bis (beta aminoethyl ether) N,N'-tetraacetic acid (EGTA). Synergistic inhibitions of these metabolic changes were observed in the platelets pretreated with both prostacyclin and EGTA. These results were compared with effects of prostacyclin and EGTA on serotonin release induced by AGEPC, and the possible roles of metabolic changes in phospholipids induced by AGEPC are discussed with respect to the mechanism of platelet activation. PMID- 3013095 TI - [Paraneoplastic endocrine syndromes]. AB - In some instances, tumors can produce signs and symptoms at a distance from the tumor or its metastases. These are defined as paraneoplastic syndrome or humoral syndrome associated with neoplasms. Paraneoplastic syndromes can arise from circulating substances secreted by tumors. The most well-recognized and frequent concomitant of neoplasms is the production of hormones by nonendocrine tumors. These are usually called ectopic hormone-producing tumors and bring about clinically endocrinologic manifestations secondary to hormone excess in patients with nonendocrine tumors. Paraneoplastic endocrine syndromes frequently observed are Cushing's syndrome due to ectopic production of ACTH, SIADH due to ectopic production of ADH, hyper-calcemia, hypoglycemia and so on. In order to establish a paraneoplastic etiology for alteration in hormone production, evidence that the hormone is produced by the tumor must be proved. Paraneoplastic endocrine syndromes should be distinguished from hormone production by benign cells, hormone production by a malignancy of an endocrine organ or alterations in hormone production being due to infiltration into the endocrine organ by a primary tumor. The treatment of ectopic endocrine syndromes should be directed primarily at the tumor. Because the course of this type of syndrome usually runs parallel to the course of the underlying tumor, the ectopically produced hormone can be a useful monitoring marker of the disease. PMID- 3013096 TI - [Hematological disorders in malignancy]. AB - We examined hematopoietic disorders in 74 patients with various malignancies. The proportions of patients with anemia in the case of esophageal carcinoma, gastric carcinoma, colorectal carcinoma, hepatoma, and pancreatic carcinoma were 75%, 87.5%, 77%, 64% and 87.5%, respectively. Hypochromic microcytic anemia was mainly observed in showed patients with carcinoma of the gastro-intestinal tract which showed severe bleeding. The major proportion of patients with hepatoma and pancreatic carcinoma showed normochromic normocytic anemia. The WBC count was usually within the normal range except for patients with liver cirrhosis or generalized metastasis who showed decreased WBC counts. The mean lymphocyte count in the peripheral blood, which is thought to be correlated with the prognosis of cancer patients, was less than 1,500/microliter except for patients with gastric carcinoma, whose five-year survival rate was 28.6%. Monocytosis was mainly observed in patients with colorectal carcinoma and pancreatic carcinoma, accounting for 24.5% of the total cases. This finding may suggest some relationship between cancer and monocytes. Thrombocytosis was seen in patients with severe bleeding, but thrombocytopenia was seen in patients with liver cirrhosis. Most cancer patients showed normo-cellular marrow and normal M/E. We also examined the ferrokinetics and ferritin levels in cancer patients. PMID- 3013097 TI - [Internal malignancies and skin lesions]. AB - In the early stage of internal malignancies, it is important to detect the relation between the malignancy and accompanying skin lesions. Skin lesions are commonly classified according to their origin as follows; skin lesions induced by internal malignancies, skin lesions as a part of a syndrome, nonspecific lesions and complications of internal malignancies, but with an unknown relationship so called Bowen's disease, and Paget's disease. These skin lesions consist of various kinds and are multiple. Commonly, the disease which is detected seems to be an immunological reaction to internal malignancy; malignant acathosis nigricans, and dermatomyositis are examples in point. A description of internal malignancies and skin lesions is presented, including those which are most commonly encountered together with a description of such cases experienced at the National Cancer Center Hospital. PMID- 3013098 TI - [Clinical significance of the measurement of serum neuron-specific enolase levels in patients with lung cancer]. AB - Subjects were comprised of 100 healthy adults, 85 patients with primary lung cancer, 20 with benign lung disease and 4 with metastatic lung cancer. Serum neuron-specific enolase (NSE) levels were estimated by means of an NSE RIA kit produced by Eiken Radiopharmaceutical Co., Ltd. The normal range of serum NSE level was between 4.5 and 10.30 (mean: 6.81) ng/ml in the 100 healthy adults. The serum NSE level in patients with small cell carcinoma was significantly higher than the mean in patients with other histological types. Positive rates of serum NSE levels were 80% in patients with small cell carcinoma, 54% in patients with adenocarcinoma, 52% in patients with squamous cell carcinoma and 1% in healthy adults, respectively. According to the progress of staging in lung cancer patients, serum NSE levels became increased. Serum NSE level seems to be specific marker in patients with small cell lung cancer and to be useful for diagnosis and the monitoring of cancer treatment. PMID- 3013099 TI - [Combination chemotherapy with cis-platinum, adriamycin and mitomycin C (PAM) in the treatment of non-small cell carcinoma of the lung]. AB - Twenty-three patients with inoperable non-small cell lung cancer were treated with a combination chemotherapy of CDDP 100 mg/m2, ADM 30 mg/m2 and MMC 8 mg/m2 (PAM). Ten cases were adenocarcinoma, 9 cases were squamous cell carcinoma and 4 cases were large cell carcinoma. In 21 evaluable cases, partial response was obtained in 47.6%. (The response rates were 40.0% in patients with adenocarcinoma, 50.0% in those with squamous cell carcinoma and 66.7% in those with large cell carcinoma.) Leukocytopenia of less than 4,000/mm3 occurred in 100% of cases, thrombocytopenia of less than 100,000/mm3 occurred in 81.0%, and anemia(fall in hemoglobin over 2.0 g/dl) occurred in 66.7%. A transient elevation of Cr (over 1.5 mg/dl) and/or BUN (over 30 mg/dl) was observed in 23.8%. Nausea and vomiting occurred in almost all patients. No death occurred due to toxicity of PAM. These results demonstrate that PAM is an effective combination chemotherapy in patients with non-small cell carcinoma of the lung. PMID- 3013100 TI - [Cytotoxicity of lymphocytes against autologous cultured primary lung cancer cells in relation to histological type and clinical stage]. AB - The cytotoxicity of lymphocytes against autologous cultured primary lung cancer cells was examined. A total of 88 patients aged from 34 to 81 years, 71 males and 17 females, were evaluated. These consisted of adenocarcinoma (n = 42), squamous cell carcinoma (n = 33), large cell carcinoma (n = 5), small cell carcinoma (n = 5), carcinoid (n = 2) and primary lung sarcoma (n = 1) cases. There was no correlation between cytotoxicity and age, or with disease stage. The mean values of the cytotoxicities of adenocarcinoma, squamous cell carcinoma, large cell carcinoma and small cell carcinoma, were 33.9%, 32.5%, 21.6% and 3.5%, respectively. By tentatively defining cases with a cytotoxicity exceeding 15% as positive, the percentage positive rates of each of the four most common histological types were 71.4%, 75.8%, 60.0% and 20.0%, respectively. The mean value of cytotoxicity and the percentage positive rate of small cell carcinoma cases were lower than those of both adenocarcinoma and squamous cell carcinoma. When the patients were divided into 3 groups according to their N (lymph node) and T (Tumor) factor, a significant difference was recognized. That is, the mean value of cytotoxicity and percentage positive rate of the group in which the N factor exceeded the T factor minus 1, such as T1N2, T1N1 and T2N2 (N-predominant group), were significantly lower than those of the group in which the T factor minus 1 exceeded the N factor, such as T3N0, T3N1 and T2N0 (T-predominant group) and the intermittent group (T3N2, T2N1 and T1N0). PMID- 3013101 TI - Concepts of wart regression. PMID- 3013102 TI - Plane warts under spontaneous regression. Immunopathologic study on cellular constituents leading to the inflammatory reaction. AB - Immunohistologically, cellular infiltrates in regressing plane warts were mainly composed of lymphocytes and mononuclear phagocytes. There were many infiltrating T lymphocytes. Immunoelectron microscopic observation demonstrated that both helper/inducer and suppressor/cytotoxic phenotypes of T lymphocytes infiltrated in the lesions. OKT6-positive cells were observed in the dermis as well as in the epidermis. Moreover, as noted in allergic contact dermatitis, the apposition of T lymphocytes to Langerhans' cell-like cells could be seen. Lymphocytes and a small number of mononuclear phagocytes were found adjacent to damaged keratinocytes in the epidermis, the picture of which has been described as satellite cell necrosis, a hallmark of cytotoxic reaction by aggressors. These findings suggest that specific cell-mediated immunity against virus-infected keratinocytes takes place in the process of regressing plane warts. PMID- 3013103 TI - Inexplicable infantile cataracts and partial maternal galactose disorder. PMID- 3013105 TI - Retroviruses in rheumatoid arthritis. PMID- 3013104 TI - Nabilone: an alternative antiemetic for cancer chemotherapy. AB - A prospective randomised double blind crossover trial was conducted comparing the new synthetic cannabinoid nabilone with oral domperidone in a group of children receiving repeated identical courses of emetogenic chemotherapy for a variety of malignant diseases. Eighteen of 23 consecutive eligible children, aged 10 months to 17 years, completed the trial. When taking nabilone they experienced significantly fewer vomiting episodes and less nausea, and two thirds expressed a preference for the drug. The most common side effects of treatment with nabilone were somnolence and dizziness, with one patient being disturbed by hallucinations. The results indicate that nabilone is an effective antiemetic for children having chemotherapy, even for young children. It seems to be superior in this respect to domperidone, and although it has a higher incidence of side effects, these are mostly acceptable to patients. It can be recommended as an alternative to conventional antiemetic treatment throughout childhood. PMID- 3013106 TI - Primary chest wall tumors: factors affecting survival. AB - Between 1955 and 1975, chest wall resection was done in 90 patients for primary chest wall tumors. Ages ranged from 8 to 96 years (mean, 44.3 years). A painful mass was the most common sign and symptom. Eighty-two tumors (91.1%) were located in the lateral chest wall and eight, in the anterior thorax. The tumor was malignant in 71 patients (78.9%) and benign in 19. All patients with benign tumors had complete excision and are currently free from disease. Malignant fibrous histiocytoma, chondrosarcoma, and rhabdomyosarcoma constituted 62% of the malignant neoplasms. Most malignancies were treated by wide resection. There were no thirty-day operative deaths. Overall 1-, 5-, and 10-year survival was 89%, 57%, and 49%, respectively. Recurrent tumor developed in 37 patients (52%); 5 year survival, however, was only 17% after recurrence. Cell type and extent of invasion significantly influenced survival. Both chondrosarcoma and rhabdomyosarcoma had a better prognosis than malignant fibrous histiocytoma (p less than 0.05). We conclude that early resection is the treatment of choice for primary malignant chest wall tumors and that development of recurrent disease is an ominous event. PMID- 3013107 TI - Bronchoalveolar cell carcinoma of the lung. AB - A multivariable analysis was performed of all patients registered and confirmed to have bronchoalveolar cell carcinoma of the lung in the Tumor Registry of Thomas Jefferson University Hospital between 1969 and 1983. These 122 patients were reviewed for age, sex, smoking history, occupational exposure, symptoms, radiographic findings, methods of diagnosis, clinical and pathologic staging, methods of treatment, survival, and complications of treatment. No correlation could be found in this series between a patient's age, sex, smoking history, or occupational exposure and the incidence or outcome of the disease. Seventy-one of the 122 patients in this series were asymptomatic, and the carcinoma was discovered in them by routine chest roentgenogram. Of these asymptomatic patients, 50 were seen with pathologic stage I disease. Of the 51 symptomatic patients, 32 (65%) were seen with stage IIIm0 or IIIm1 disease. Despite medical evaluations, 77% of the T1 and T2 lesions required thoracotomy for diagnosis. The overall five-year survival rate was 42.3%, ranging from 75% for those with stage I disease to 8.7% for those with stage IIIm1 disease. PMID- 3013109 TI - Mechanism of cough with angiotensin-converting enzyme inhibition. PMID- 3013108 TI - Effects of clonidine and yohimbine on plasma cyclic nucleotide levels in clonidine-naive and clonidine-treated mice. AB - Effects of clonidine and yohimbine on plasma cyclic nucleotide levels were investigated in both clonidine-naive and clonidine-treated male mice. Clonidine increased plasma cyclic GMP but decreased slightly cyclic AMP levels in clonidine naive mice. Clonidine treatment for 10-14 days in the drinking water did not decrease the cyclic GMP response to clonidine indicating that no tolerance develops to the effect of clonidine on plasma cyclic GMP. alpha 2-Agonists, such as clonidine, oxymetazoline and naphazoline, were more potent than phenylephrine, an alpha 1-agonist, in increasing cyclic GMP, although azepexole, a weak alpha 2 agonist, had no effect. Inhibition of clonidine-induced increase in plasma cyclic GMP by yohimbine, hexamethonium and atropine, but not by prazosin suggests that the effect of clonidine is mediated by the central alpha 2-adrenoceptors, activating the muscarinic receptor-linked guanylate cyclase through the stimulation of vagal activity. Yohimbine increased plasma cyclic AMP levels in clonidine-naive mice. Inhibition of this effect by hexamethonium and propranolol suggests that yohimbine increases plasma cyclic AMP through increasing the sympathetic tone. The increase in plasma cyclic AMP elicited by yohimbine was potentiated by chronic clonidine treatment. Enhancement of the cyclic AMP effect of yohimbine found in clonidine-treated mice may be regarded as a precipitated withdrawal symptom and indicate development of dependence on clonidine. PMID- 3013111 TI - Environmental illness and patients with multiple unexplained symptoms. PMID- 3013110 TI - Hemorrhagic cystitis associated with adenovirus infection in bone marrow transplantation. AB - A retrospective study of bone marrow transplant recipients shedding adenovirus type 11 in the urine was carried out to determine the association between viral shedding and hemorrhagic cystitis in this population. Weekly urine virology surveillance cultures were obtained during the first 100 days following transplantation. Adenovirus type 11 was cultured from five of 502 bone marrow transplant recipients from 1977 through 1984. In four of these five patients there was associated hemorrhagic cystitis. This contrasts with an overall incidence of hemorrhagic cystitis of 20% in this bone marrow transplant population. A case of hemorrhagic cystitis occurred in a patient following bone marrow transplantation. Recognition of a viral origin of hemorrhagic cystitis may explain the occurrence of late hemorrhagic cystitis in patients despite interventions designed to prevent cyclophosphamide-induced hemorrhagic cystitis. Hemorrhagic cystitis may be the presenting sign of a lethal adenoviral infection. PMID- 3013112 TI - Histoenzymological studies on the distribution of certain phosphatases in the retinae of owlet (Athene brama) and house sparrow (Passer domesticus). AB - A comparative study on the distribution of alkaline phosphatase (AlP), acid phosphatase (AcP) and 5'-nucleotidase (5-N) amongst the different constituents of retinae of owlet and house sparrow revealed some interesting aspects of the localization of such phosphatases in both the cases. The outer segment of photoreceptors, where light strikes first, are positive for all the phosphatases. Further, areas composed of synapses, reveal activity of the three enzymes. Another interesting aspect is related to the total absence of the activity of AlP and 5-N in the ganglion cells of both the animals. Other sites of phosphatases in various layers have been also identified. The possible metabolic roles of various phosphatases at different sites have been discussed. PMID- 3013113 TI - Inhibition of wall autolysis of staphylococci by sodium polyanethole sulfonate "liquoid". AB - Liquoid (polyanethole sulfonate) was neither capable of influencing the growth nor the viability of staphylococci. But liquoid induced a suppression of the activity of different autolytic wall systems of normally growing staphylococci, i.e., autolysins which participate in cross wall separation as well as autolysins which are responsible for cell wall turnover. Additionally, the lysostaphin induced wall disintegration of staphylococci was inhibited by liquoid. However, no indication could be found for a direct inhibition of lytic wall enzymes by liquoid; rather an interaction of liquoid with the target structure for the autolytic wall enzymes, the cell wall itself, was postulated. On the basis of the experimental data with the teichoic acid- mutant S. aureus 52A5 the sites of wall teichoic acid were supposed to be an important target for the binding of liquoid to the staphylococcal cell wall. PMID- 3013114 TI - Enterotoxin A production in Staphylococcus aureus: inhibition by glucose. AB - In this study, we investigated the relationship between carbohydrate metabolism and repression of staphylococcus enterotoxin A (SEA) in Staphylococcus aureus 196E and a pleiotrophic mutant derived from strain 196E. The mutant, designated at strain 196E-MA, lacked a functional phosphoenolpyruvate phosphotransferase system (PTS). The mutant produced acid, under aerobic conditions, from only glucose and glycerol. The parent strain contained an active PTS, and aerobically produced acid from a large number of carbohydrates. Prior growth in glucose led to repression of SEA synthesis in the parent strain; addition to the casamino acids enterotoxin production medium (CAS) led to more severe repression of toxin synthesis. The repression was not related to pH decreases produced by glucose metabolism. When S. aureus 196E was grown in the absence of glucose, there was inhibition of toxin production as glucose level was increased in CAS. The inhibition was related to pH decrease and was unlike the repression observed with glucose-grown strain 196E. The inhibition of SEA synthesis in mutant strain 196E MA was approximately the same in cells grown with or without glucose and was pH related. Repression of SEA synthesis similar to that seen with glucose-grown S. aureus 196E could not be demonstrated in the mutant. In addition, glucose-grown S. aureus 196E neither synthesized beta-galactosidase nor showed respiratory activity with certain tricarboxylic acid (TCA) cycle compounds. Glucose-grown strain 196E-MA, however, did not show suppressed respiration of TCA cycle compounds; beta-galactosidase was not synthesized because the mutant lacked a functional PTS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013115 TI - Gluconate accumulation and enzyme activities with extremely nitrogen-limited surface cultures of Aspergillus niger. AB - Batch cultures of Aspergillus niger grown from conidia on a medium with high C/N ratio accumulated gluconate from glucose with a yield of 57%. During almost the whole time of accumulation there was no net synthesis of total protein in the mycelium but the activity per flask and the specific activity of glucose oxidase (EC 1.1.3.4) in mycelial extracts increased whereas both values decreased for glucose dehydrogenase (EC 1.1.99.10) 'gluconate 6-phosphatase' (cf. EC 3.1.3.1, 3.1.3.2), gluconokinase (EC 2.7.1.12), glucose 6-phosphate and phosphogluconate dehydrogenases (EC 1.1.1.49, EC 1.1.1.44), phosphoglucomutase (EC 2.7.5.1), and most enzymes of the Embden-Meyerhof pathway and the tricarboxylic acid cycle. Gluconate dehydratase (EC 4.2.1.39), gluconate dehydrogenase (EC 1.1.99.3) and enzymes of the Entner-Doudoroff pathway could not be detected. By cycloheximide the increase of glucose oxidase activity was inhibited. It is concluded that the high yield of gluconate was due mainly to the net (de novo) synthesis of glucose oxidase which occurred during protein turnover after the exhaustion of the nitrogen source, and which was not accompanied by a net synthesis of the other enzymes investigated. Some gluconate may also have been formed by hydrolytic cleavage of gluconate 6-phosphate. PMID- 3013117 TI - [Orbital tumors in children]. PMID- 3013116 TI - [Bilateral nephroblastoma with aniridia]. AB - Bilateral nephroblastoma may be associated with congenital bilateral aniridia in children. A partial deletion of the short arm of chromosome 11 has been reported in several cases of polymalformation syndrome with associated catalase deficiency. We report one case and review the recent genetic and pathogenic data. PMID- 3013118 TI - [LAV infections and the acquired immunodeficiency syndrome in infants]. AB - The clinical and immunologic abnormalities of 7 infants with Lymphadenopathy Associated Virus (LAV) infection are reported. Indicative immune changes, positive anti-LAV serologic test and/or virus isolation led to the diagnosis. In a case with a particularly severe form of the disease, whose mother died from AIDS, serologic tests and virus isolation were negative. Beside a case associated with blood transfusion, the familial and ethnic context helped the diagnosis. Clinical and biochemical features were close to those in adult AIDS, including hepatosplenomegaly, polyadenopathy, a decreased absolute number of OKT4(+) lymphocytes and hypergammaglobulinemia. The importance of cellular and sometimes also humoral immune deficiency was highly variable. For 4 patients, the severity of the immune deficiency allowed the diagnosis of AIDS. Occurrence of such an infection in the first months of life suggests a fetomaternal viral transmission; however, a postnatal contamination cannot be ruled out. Long-term prognosis is difficult to assess. It depends, among other factors, on the importance of the immune deficiency. PMID- 3013119 TI - Breast carcinoma with osteoclastlike giant cells. AB - Seven carcinomas of the breast with osteoclastlike giant cells were studied. Recurrences and lymph node metastases also contained osteoclastlike giant cells. Judging from ultrastructural and histochemical studies, the osteoclastlike giant cells are not epithelial, but are of mesenchymal origin. They may appear in a variety of different types of carcinoma. PMID- 3013120 TI - Pancreatoblastoma in an adult. AB - A 37-year-old man with a history of abdominal pain, diarrhea, and weight loss was found to have an 8-cm-diameter tumor in the head of the pancreas, a biopsy specimen of which revealed histologically and ultrastructurally typical pancreatoblastoma. The tumor was unresectable and demonstrated no response to chemotherapy; however, substantial tumor shrinkage resulted from intraoperative and external beam radiation therapy. This rare tumor, to our knowledge not previously reported in an adult, should be considered in the differential diagnosis of adults as well as children with pancreatic epithelial tumors. PMID- 3013121 TI - Immature renal tissue in colonic wall of patient with caudal regression syndrome. AB - We report an incidental microscopic finding of ectopic renal tissue in a newborn with multiple congenital anomalies. The ectopic renal tissue was located in the wall of the distal blind end of the colon. The tissue was composed of undifferentiated renal blastema with primitive and well-formed glomeruli and tubules. The potential for malignant transformation of this lesion into extrarenal Wilms' tumor is not known. PMID- 3013122 TI - Physical and hormonal evaluation of transsexual patients: a longitudinal study. AB - The physical and hormonal characteristics of 60 male-to-female transsexuals and 30 female-to-male transsexuals were measured before or during treatment with commonly used forms and dosages of hormones. Only two patients (both female-to male) had either a congenital defect in hormonal production or abnormal genital development. Patients were seen at 3- to 6-month intervals for an average of 18 months. The response to therapy was examined over time; physical parameters, hormonal concentrations, liver function tests, lipids, and glucose were measured. Three patients were changed from ethinyl estradiol to conjugated estrogen because of liver enzyme elevations. Ethinyl estradiol (0.1-0.5 mg/day) was equal to conjugated estrogen (7.5-10 mg/day) in its ability to suppress testosterone and gonadotropins and to promote breast growth. Maximum breast growth required 2 years of therapy. During treatment with testosterone, female-to-male transsexuals had a significant mild elevation of cholesterol and triglyceride. The female-to male transsexuals receiving testosterone cypionate, 200 mg every 2 weeks, ceased to have menstrual periods and became progressively masculinized. A mean maximal clitoral length of 4.6 cm which achieved by 1 year of therapy. Based on the data generated by this study, we recommend as hormonal therapy 0.1-0.5 mg/day of ethinyl estradiol or 7.5-10 mg/day of conjugated estrogen for male-to-female transsexuals, and intramuscular testosterone cypionate, 200 mg every 2 weeks, for female-to-male transsexuals. PMID- 3013123 TI - Continuous absorbable vs interrupted nonabsorbable fascial closure. A prospective, randomized comparison. AB - A prospective, randomized comparison of continuous, absorbable, No. 2, coated, polyglycolic acid suture (Dexon-Plus) vs interrupted, nonabsorbable, No. 28 monofilament stainless steel wire suture was performed in 105 patients for midline fascial closure following gastric surgery for morbid obesity. The preoperative weight, sex distribution, and type of operation were not significantly different between the two groups. No significant difference was found in the wound complication rate between the two closure methods (7/54 for wire and 8/51 for polyglycolic acid). There were one dehiscence and five incisional hernias in the wire group and five hernias in the polyglycolic acid group. Continuous closure was accomplished in significantly less time (21 +/- 8 minutes) than interrupted closure (43 +/- 19 minutes). An additional 121 patients underwent continuous No. 2 polyglycolic acid fascial closure after the end of the randomized trial, with 13 wound complications, including ten incisional hernias. In conclusion, continuous, absorbable suture closure for laparotomy wounds is recommended for its economy of time and the lack of significant difference from an interrupted, nonabsorbable wound closure. PMID- 3013124 TI - Hepatocellular carcinoma presenting with intractable diarrhea. A radiologic pathologic correlation. AB - A 44-year-old woman with hepatocellular carcinoma presented with intractable watery diarrhea and her condition was evaluated angiographically. Surgical ablation of the tumor resulted in complete resolution of the diarrhea. The tumor cells of the hepatocellular carcinoma were found to contain vasoactive intestinal polypeptide, gastrin, and prostaglandinlike immunoactivity. To our knowledge, this is the first report of such an association. PMID- 3013125 TI - Evolution of enterovirus 70 in nature: all isolates were recently derived from a common ancestor. AB - The data of large RNase T1-resistant oligonucleotide mapping of enterovirus 70 (EV 70) previously reported (Takeda et al., Virology 134, 375-388, 1984) were subjected to further genetical analysis to estimate the evolutionary rate of genome RNA of EV 70 and to clarify the phylogenetic relationship among isolates. A proportion of common spots between strains decreased as the year elapsed and eventually, only seven spots were common to all the 16 isolates tested, indicating that the substitution is scattered throughout the genome. On the other hand, some specific sets of spots were conserved among geographically or epidemiologically related strains. Base sequence variation of the isolates was deduced according to Aaronson et al. (Nucleic Acids Res. 10, 237-246, 1982) from pariwise comparison of the common spots and used as a genetic distance between them. The base substitution rate of virus genome was estimated by regression analysis of the genetic distance of the isolates against the sampling time. A fairly constant and rapid rate was obtained; it was 1.83 X 10(-3)/base/year. Based on the substitution rate, genetic distance and sampling time of the strains, the phylogenetic tree of EV 70 was constructed using Unweighted Pair Group Method Using Arithmetic Averages (UPGMA) (Nei, Molecular Population Genetics and Evolution, North Holland, Amsterdam, 1975). The tree supports the previous hypothesis that evolution of EV 70 started from a single common ancestor. The time of its emergence was estimated to be 1967 +/- 15 months. The virus branched into many strains early during the first pandemic and has evolved in a divergent fashion, yielding genetically polymorphic viruses in the world. PMID- 3013126 TI - Rabbit kidney cells abortively infected with human cytomegalovirus are arrested in mitotic phase. AB - In rabbit kidney epithelial cells (RK13) abortively infected with human cytomegalovirus (HCMV), DNA synthesis at 1 or 2 days post-infection was enhanced 4 to 5 fold, compared to mock-infected cells. DNA analysis by isopycnic centrifugation revealed that the DNA newly synthesized in the virus infected RK13 cells was of cellular origin. HCMV infection also caused a marked increase in the mitotic activity of RK13 cells. When semi-confluent RK13 cells were infected more than 20 per cent of cells demonstrated mitosis at 72 hours post-infection although the rate of cell growth was considerably reduced compared to that of uninfected cells. The most frequent chromosomal change observed was fragmentation although other aberrations, gap, break, deletion etc. occurred also. Two immediate-early viral polypeptides with apparent molecular weights 72,000 (72K) and 76,000 (76K) daltons were produced in both RK13 cells and human embryonic lung cells (HEL) by 3 hours post-infection. Synthesis of the 76K polypeptide was greater than that of the 72K polypeptide in non-permissive RK13 cells whereas the reverse occurred in permissive HEL cells. Furthermore, of three early polypeptides which were expressed in productively infected HEL cells two, 88K and 80K, were not detected in abortively infected RK13 cells. These results suggest that the arrest in mitosis of the abortively infected RK13 cells and the subsequent chromosomal changes are associated with the altered expression of immediate-early or early virus functions in these cells. PMID- 3013127 TI - Isolation of two lapine rotaviruses: characterization of their subgroup, serotype and RNA electropherotypes. AB - Rotaviruses were detected by an ELISA test in stool specimens from diarrheic rabbits in two commercial rabbitries and cultured in MA 104 cells. Their identity was confirmed by electron microscopy and indirect immunofluorescence. They were found to belong to subgroup I by testing with monoclonal antibodies and to serotype 3 by neutralization with homologous and heterologous antisera. Although both viruses were neutralized by antiserum to human serotype 3 the ALA rabbit rotavirus was minimally neutralized by antiserum to the C11 rabbit rotavirus. Electrophoresis of viral RNA revealed 11 segments characteristic of rotavirus, however both rabbit rotaviruses had unusual electropherotypes. They differed from each other with greatly reduced mobility of the tenth segment in one virus and the eleventh segment in the other virus. PMID- 3013128 TI - Changes in membrane permeability in cells infected by human cytomegalovirus. AB - To analyze whether human cytomegalovirus could induce changes in membrane permeability we studied both Hygromycin B (HyB) inhibition of protein synthesis and glycine and choline uptake during viral replication. A higher sensitivity to HyB was observed both immediately after virus adsorption and in concomitance with the release of viral progeny. HyB sensitivity immediately after virus adsorption was not associated with changes in membrane permeability to glycine and choline while 60 hours p.i. a remarkable increase in choline uptake was detected. PMID- 3013129 TI - Persistent infections with Sendai virus and Newcastle disease viruses. AB - Persistent infections (Pi) were established in two host-cell systems [Madin-Darby bovine kidney (MDBK) and Madin-Darby canine kidney (MDCK)] with Sendai virus and three strains of NDV, to test the influence of different viruses and host-cell systems. Virus was recovered from the persistently infected cells. An RNA- ts mutant was recovered from a Pi of MDBK cells, but no Pi could be established in MDCK cells with the three strains of NDV. Additionally, the Pi was established exclusively by a virulent strain, NDV-Milano. On the other hand, Sendai virus could establish Pi in MDBK and MDCK cell-systems. Several ts mutants were recovered from "late" passages of Pi, and from an accidental infection, a ts mutant with an altered P polypeptide. Ten other ts mutants were tested, however, the specific ts lesion could not be identified. From three Pi in MDCK cells, host range mutants (ts-f1, ts-f2, and ts-f3) were recovered. One of the mutants (ts f1) has an altered M (matrix) protein. The host range mutants undergo a productive infection in MDBK and MDCK cells, which are nonpermissive for wild type Sendai virus. The possible significance of the results are discussed. PMID- 3013130 TI - A new type of atypical rotavirus in pigs. AB - Two independent isolates of an unusual type of rotavirus were obtained from over 900 faecal samples obtained from piglets with diarrhoea. Genome profiles, containing eleven segments, were not characteristic of group A electropherotypes and corresponded to an atypical virus. The profiles were, however, distinct from those of other previously described atypical porcine rotaviruses. Serological comparisons showed that the two isolates were related, but unrelated to other known groups of porcine rotavirus. The new isolates were passaged in gnotobiotic piglets in which they caused mild clinical signs of enteritis. A survey of porcine sera from the United Kingdom indicated a widespread distribution of antibody to this virus in pigs aged 10 weeks and over. PMID- 3013131 TI - A comparison of the biological properties of type 2 plaque-producing agent derived from the Cal-1 strain of Marek's disease virus with other related viruses. AB - The serological and biological properties of the type 2 plaque-producing agent (PPA) derived from the Cal-1 strain of Marek's disease virus (MDV) were compared with those of reference strains of the three serotypes of MDV and herpesvirus of turkeys (HVT) groups; namely JM, HPRS-24 strains of MDV and the FC-126 strain of HVT for serotype 1, 2, and 3, respectively. By agar gel precipitation (AGP), indirect fluorescent antibody and virus neutralization tests, type 2 PPA was related but not identical to the FC-126 strain. By the AGP test, type 2 PPA showed a poor ability to synthesize B antigen and the A antigen was different from that of strain FC-126. To compare the virological characteristics of type 2 PPA with the reference strains, the release of cell-free virus into supernatants of infected cell cultures and titers of cell-free virus extracted sonically from infected cell cultures were examined. Cell-free type 2 PPA virus was easily detected in the supernatants and extracted from infected cell cultures. These properties were similar to reference strains of serotype 2 and 3. Next, the structural similarities of viral DNAs of type 2 PPA and strain FC-126 were examined by Southern blot hybridization. The restriction endonuclease-cleavage patterns of DNA of type 2 PPA were very similar but not identical to those of the FC-126 strain. In chickens inoculated with type 2 PPA, splenic lymphocytes supported a non-productive latent infection as did also those from chickens inoculated with the FC-126 or HPRS-24 strains. From these results, type 2 PPA appears to belong to serotype 3 of MDV and HVT groups. The origin of type 2 PPA is discussed. PMID- 3013132 TI - Studies on rat cytomegalovirus induced structural and non-structural proteins present at (immediate-)early and late times of infection. AB - Radioactive-labelled virions and nucleocapsids of rat cytomegalovirus (RCMV) were purified from the supernatant and subcellular fractions of infected rat embryo fibroblasts (REF) and analyzed by SDS-polyacrylamide gel electrophoresis. Nuclear nucleocapsids contain one major protein of 138 kD, which is considered to be the basic invariant structural element of RCMV. Enveloped virions consist of 28 protein species, five of which were clearly identified as glycoproteins (58, 64, 76, 112 and 118 kD). Using pulse labelling procedures on RCMV-infected REF cells, after removal of a previously established translation block by cycloheximide, two RCMV-induced immediate-early (IE) protein species (71 and 85 kD) were both detected in the nuclear and cytoplasmic cell fractions. When pulse-labelling was performed for 16 hours in presence of Actinomycin D, only the 85 kD IE protein was detected in the nucleus. The results indicate that the 85 kD IE polypeptide is of importance in early transcriptional events of viral genes. Protein metabolism studies revealed that late in the course of RCMV infection (at 3 days p.i.) protein synthesis has been dramatically changed. Some cellular proteins are greatly suppressed while other cellular proteins are clearly enhanced. Moreover, active synthesis of 8 new cytoplasmic proteins and 9 new nuclear proteins occurs. Most of these proteins were identified as structural constituents of virions. PMID- 3013133 TI - Biochemical and antigenic characterization of feline herpesvirus-1-like isolates from dogs. AB - The DNA and polypeptide patterns of feline herpesvirus-1 (FHV-1), a virus usually associated with feline respiratory infections, were compared to those of five herpesvirus isolates from dogs. These canine isolates had been shown to be antigenically similar to FHV-1 by cross neutralization tests. DNA from FHV-1 (C 27 strain) and each canine isolate was digested with either Bam HI, Eco R 1 or Sal I and analyzed on 0.8 per cent agarose gels. The restriction digest patterns of the canine isolates were nearly identical to C-27 for all three restriction enzymes. Interestingly, all of the canine isolates showed a small extension of the largest Bam HI fragment (14.5 kb) that was not present in the C-27 strain. Bam HI digested FHV-1 DNA from clinical cases in cats had digest patterns that were very similar to the canine isolates and also showed an extension of the 14.5 kb fragment. Southern blotting experiments revealed that DNA from the canine isolates has extensive homology to C-27 DNA. SDS-PAGE analysis of radiolabeled polypeptides from C-27 and the canine isolates showed identical virion-associated polypeptide profiles. In addition, a goat anti-FHV-1 antiserum precipitated three glycoprotein antigens from the canine isolates with migration patterns that were identical to the three major antigenic glycoproteins found on C-27. Hind III and Eco R1 digestion patterns of canine herpesvirus DNA showed no similarity to C-27 DNA. In addition, canine herpesvirus DNA had no homology to C-27 DNA under the stringent conditions used. PMID- 3013134 TI - Immunogenicity of purified glycoprotein gB of herpes simplex virus. AB - The efficacy of a herpes simplex virus (HSV) component vaccine consisting of viral glycoprotein gB was examined in a mouse system. Immunization of mice with HSV type 1 (HSV-1) gB emulsified in Freund's complete adjuvant or with HSV-1 gB adsorbed to aluminum gel was fully protective against subsequent challenge with HSV-1 or HSV type 2. Latent infection in the trigeminal ganglion was also prevented by immunization with gB. PMID- 3013135 TI - [Characterization and functioning of beta adrenergic receptors in the WI-38 cell line]. PMID- 3013136 TI - [Ultrastructure of a granular-cell breast tumor]. AB - Electron microscopic examination of a rare granular cell tumour of the mammary gland that provoked difficulties in the diagnosis was performed. Ultrastructural organization of the tumour allowed one to establish a proper diagnosis and to confirm the hypothesis on the origin of this tumour from Schwann's cells. PMID- 3013137 TI - Relapsing central and peripheral demyelinating diseases. Unusual pathologic features. AB - We treated a patient who had a demyelinating peripheral neuropathy and a central nervous system inflammatory demyelinating disease. The unusual pathologic feature of dense infiltrates of atypical macrophages was observed in many areas of the brain; otherwise the process had several features in common with either multiple sclerosis or chronic relapsing experimental allergic encephalomyelitis. The illness followed "swine-flu" inoculation; exacerbation followed pneumococcal vaccination. PMID- 3013138 TI - Dissolution and precipitation reactions in human tooth enamel under weak acid conditions. AB - Slices of enamel were demineralized in weak acid solutions at pH 5. The solutions were analysed for Ca, P, Na and Mg. A substantial increase of the Ca/P ratio in the solution after about 6 h of demineralization was ascribed to brushite formation. The ratios of liberated Ca/Na, P/Na, Ca/Mg and P/Mg were always lower than the correspondent ratios in sound enamel. It was concluded that precipitation of brushite, and a preferential dissolution of Na and Mg compounds from the enamel both play a role in the dissolution-precipitation reactions in dental enamel during acid attack. PMID- 3013139 TI - Cyclic-AMP-dependent protein phosphorylation in the soluble fraction of parotid glands from rats with drug-induced hypothyroidism. AB - Cyclic-AMP-dependent protein kinase from the soluble fraction of parotid glands of hypothyroid rats was partially purified. Its isozyme distribution and kinetic properties were similar to those of euthyroid rats. Electrophoresis of 100 microliters portions at 20 mA per slab revealed that an endogenous protein (mol. wt 68,000) was specifically phosphorylated in hypothyroidism; this protein was not found in euthyroid rats. In the presence of cyclic AMP, there was stimulated phosphorylation of euthyroid-soluble proteins with molecular weights of 115,000, 98,000, 57,000, 50,000, 44,000, 33,000 and 19,000, and of proteins from hypothyroid rats with weights of 115,000, 98,000, 60,000, 50,000, 33,000 and 19,000. Tolbutamide reduced incorporation of 32Pi into soluble proteins from both groups. However, cyclic AMP still induced phosphorylation in euthyroid preparations in the presence of tolbutamide, but its effect was markedly reduced in the hypothyroid state. These differences in endogenous protein phosphorylation may have different effects on amylase release induced by beta-adrenergic stimulation. PMID- 3013140 TI - Effects of elevated levels of cyclic AMP on the embryonic chick mandible in vitro. AB - Mandibles were cultured in the presence of dibutyryl cyclic AMP (1 mM) and theophylline (1 mM) and in control medium. Controls differentiated normally but the timing of events differed from that in ovo and feathers did not form. With elevated intracellular cyclic-AMP levels, membrane bone did not form in the earlier stages tested and was inhibited or reduced in older ones; chondrogenesis was inhibited only in young explants (HH stage 20) and, in certain instances, it was enhanced. There was precocious and hyperplastic differentiation of mucous cells within oral epithelium but the aboral epithelium was unchanged. PMID- 3013141 TI - Effects of dibutyryl cyclic AMP and theophylline on in-vitro development of the secondary palate in the embryonic chick. AB - The avian secondary palate is normally cleft and does not show a rise in intracellular cyclic AMP like that of the fused mammalian palate. The secondary palate of the embryonic chick (HH stages 25-34) was exposed in vitro to dibutyryl cyclic AMP and theophylline to determine whether raising intracellular cyclic-AMP levels would alter medial-epithelial development. Differentiation of the medial epithelium was not altered but there was mucous-cell hyperplasia in nasal epithelium and inhibition of membrane-bone formation in the mesenchyme. Thus the developmental factors that cause the avian palate to be cleft and the mammalian palate to be fused are more complex than differences in intracellular cyclic-AMP levels. PMID- 3013142 TI - Intracranial glioblastoma invading the orbit. PMID- 3013143 TI - Glucocorticoid incubation of human ACTH-producing pituitary tumours in vitro. A study on ACTH secretion and cell morphology. AB - ACTH-producing pituitary adenomas obtained from two patients with Cushing's disease were maintained in organ culture for 2 weeks. After 4-6 days of incubation, hydrocortisone (0.1, 1, and 5 mg/ml) was added to the culture medium. Addition of cortisol to the media in concentrations which generally inhibit the ACTH secretion in vivo in patients with Cushing's disease (1 mg/ml) failed to affect the ACTH secretion in vitro. A decrease of ACTH secretion occurred only with 5 mg/ml after 5 days of incubation. A similar effect on growth hormone (GH) release was seen when cortisol (10 mg/ml) was added to cultures from GH-producing pituitary adenoma. No ultrastructural changes were found in cultured cells that could be attributed to the addition of 0.1 mg/ml cortisol in the culture medium. With higher concentrations, (1 mg/ml and 5 mg/ml) minor ultrastructural changes were suspected. Our present findings implicate the hypothalamus as the target area for the feedback control of ACTH in Cushing's disease but could not reveal whether or not the primary lesion (i.e. the tumor) was of pituitary or hypothalamic origin. Our organ culture system appears to be a suitable model for investigating possible ACTH-regulating factors. PMID- 3013145 TI - Chemicals at work: clinical aspects. PMID- 3013144 TI - Recurrence of invasive moles and choriocarcinomas. PMID- 3013146 TI - Biochemical characterization of collagenase activity from middle ear effusions. AB - Collagenolytic enzyme activity has been demonstrated in middle ear effusions from patients suffering from otitis media with effusion. Collagenase activity was characterized using different biochemical techniques. The enzyme was found to have a higher specific activity in mucoid effusions and had characteristics similar to granulocyte derived collagenase from human leucocytes. PMID- 3013148 TI - In situ carcinoma of the pancreas. AB - Two cases of pre-invasive in situ carcinoma of the pancreas treated by surgery are reported. In each case the resection was carried out because of the probable diagnosis of invasive carcinoma of the pancreas. In situ carcinoma of the pancreas is not included in standard clinical classifications of pancreatic cancer. This is an important omission because of the prognostic implications of this diagnosis, and because of the difficulties in interpreting fine needle aspiration cytology or histology on needle biopsy in such cases. PMID- 3013147 TI - Aspiration cytology and ultrasonography of cold thyroid nodules. AB - Over an 18 month period, 50 'high risk' patients with solitary or dominant cold thyroid nodules on 99m Tc-pertechnetate scanning, have undergone fine needle aspiration biopsy cytology (ABC) under general anaesthetic prior to thyroidectomy. Histological malignancy was confirmed in only four patients (8%). Cytological malignancy was suspected or confirmed in six patients, each having a false positive rate of 4%. There were no false negative reports. Ultrasonography, performed pre-operatively in 37 of the patients, did not significantly add to the overall patient management. ABC appears to be safe, simple and sufficiently accurate to incorporate its use routinely in the pre-operative assessment of thyroid nodules. PMID- 3013149 TI - Perianal mucinous adenocarcinoma: a clue to its pathogenesis. AB - The case of a 49 year old male patient who presented with perianal mucinous adenocarcinoma is presented. This is a rare anal tumour with a low grade, well differentiated histological pattern. Its pathogenesis remains obscure, although a long antecedent history of fistula in ano and associated perianal sepsis is characteristic. The exact etiological relationship with anal fistula is not clearly established. The upper rectum is usually spared. Perianal Paget's disease is often seen in association with the tumour. Metastases occur late and spread is usually to the inguinal group of lymph nodes. Clinical diagnosis is often delayed and difficult. Treatment is abdominoperineal resection with block dissection of the inguinal lymph nodes if the glands are involved. PMID- 3013150 TI - The malignant potential of papillomavirus. PMID- 3013151 TI - The ultrasonic and angiographic appearances of Wilms tumours in adults. PMID- 3013152 TI - Alpha-receptor constriction induced by atrial fibrillation during maximal coronary dilatation. AB - The mechanism of coronary vasoconstriction induced by atrial fibrillation during maximal coronary dilatation was studied in 19 chloralose-urethane anesthetized dogs. Maximal coronary dilatation was achieved by carbochromene (5 mg/kg i.v.) or dipyridamole (0.2 mg/kg i.v.). Left circumflex coronary blood flow was measured with an electromagnetic flowmeter. Atrial fibrillation was compared with rhythmic atrial pacing at similar heart rates (207 +/- 12 vs. 204 +/- 12 beats/min). During maximal coronary dilatation, coronary resistance was 0.38 +/- 0.05 mm Hg X min X 100 g/ml (RU) at sinus rhythm, 0.41 +/- 0.06 RU at atrial pacing, and 0.52 +/- 0.07 RU at atrial fibrillation, that was significantly (p less than 0.005) higher than during sinus rhythm and atrial pacing. Accordingly, coronary oxygen extraction was 14 +/- 1% at sinus rhythm, 17 +/- 1% at atrial pacing (p less than 0.005 vs. sinus rhythm) and 27 +/- 2% at atrial fibrillation (p less than 0.001 vs sinus rhythm and atrial pacing). Beta-adrenoceptor blockade with propranolol (1 mg/kg i.v.) did not prevent this coronary vasoconstrictive effect. Following alpha-blockade with phenoxybenzamine (10 mg/kg i.v.), however, coronary resistance was 0.52 +/- 0.08 RU during sinus rhythm, 0.54 +/- 0.10 RU during atrial pacing and 0.57 +/- 0.09 RU during atrial fibrillation. The data suggest coronary vasoconstriction induced by atrial fibrillation mediated by an alpha adrenoceptor mechanism. PMID- 3013153 TI - Genetic differences in the resistance of rats to isoprenaline-induced heart lesions. AB - Two strains of rats were obtained by selective breeding: the IR strain, resistant to isoprenaline-induced myocardial lesions and the IS strain, sensitive to this damage. The IR rats grew more slowly, the weight of their adipose tissue was higher and the weight of m. soleus was less than that of the IS rats. The IR rats had a higher content of triglycerides in the serum and a lower isoprenaline stimulated lipolytic activity of adipose tissue in vitro. The basal NEFA level in the serum and its rise after the administration of isoprenaline in vivo did not differ between the strains. The IR rats had a higher content of glycogen in the heart and in the muscle. After the administration of isoprenaline the glycogen content decreased more slowly in IR rats. The findings indicate a considerable importance of the glycogen stores in the heart for the resistance of myocardium to damage. PMID- 3013154 TI - Myocardial H2O2 production in the unanaesthetized rat. Influence of fasting, myocardial load and inhibition of superoxide dismutase and monoamine oxidase. AB - Myocardial H2O2 production was studied by means of the in vivo administration of aminotriazole (AT), which inactivates the catalase-H2O2 complex compound I. Measurements of the residual catalase activity in male and female rats indicate that beta-oxidation of fatty acids in peroxisomes does not contribute in a substantial way to energy production in response to fasting or to an increased myocardial load, despite previous data on peroxisomes in myocardium. Superoxide dismutase (SOD) inhibition by diethyldithiocarbamate demonstrates that SOD participates in the production of H2O2 in physiological conditions. Such a role was not demonstrated for monoamine oxidase through inhibition by phenelzine. PMID- 3013156 TI - Identification and characterization of both the cytosolic and particulate forms of cyclic GMP-stimulated cyclic AMP phosphodiesterase from rat liver. AB - Two enzymes displaying cyclic GMP-stimulated cyclic AMP phosphodiesterase activity were purified from rat liver to apparent homogeneity: a 'particulate enzyme' found as an integral membrane protein associated with the plasma membrane, and a 'soluble' enzyme found in the cytosol. The physical properties of these enzymes were very similar, being dimers of Mr 134,000, composed in each instance of two subunits of Mr = 66,000-67,000. Both enzymes showed similar kinetics for cyclic AMP hydrolysis. They are both high-affinity enzymes, with kinetic constants for the particulate enzyme of Km = 34 microM and Vmax. = 4.0 units/mg of protein and for the cytosolic enzyme Km = 40 microM and Vmax. = 4.8 units/mg of protein. In both instances hydrolysis of cyclic AMP appeared to show apparent positive co-operativity, with Hill coefficients (happ.) of 1.5 and 1.6 for the particulate and cytosolic enzymes respectively. However, in the presence of 2 microM-cyclic GMP, the hydrolysis of cyclic AMP obeyed Michaelis kinetics (happ. = 1) for both enzymes. The addition of micromolar concentrations of cyclic GMP had little effect on the Vmax. for cyclic AMP hydrolysis, but lowered the Km for cyclic AMP hydrolysis to around 20 microM in both cases. However, at low cyclic AMP substrate concentrations, cyclic GMP was a more potent activator of the particulate enzyme than was the soluble enzyme. The activity of these enzymes could be selectively inhibited by cis-16-palmitoleic acid and by arachidonic acid. In each instance, however, the hydrolysis of cyclic AMP became markedly more sensitive to such inhibition when low concentrations of cyclic GMP were present. Tryptic peptide maps of iodinated preparations of these two purified enzyme species showed that there was considerable homology between these two enzyme forms. PMID- 3013155 TI - Regulation of the branched-chain 2-oxo acid dehydrogenase complex in hepatocytes isolated from rats fed on a low-protein diet. AB - Hepatocytes isolated from rats fed on a chow diet or a low-protein (8%) diet were used to study the effects of various factors on flux through the branched-chain 2 oxo acid dehydrogenase complex. The activity of this complex was also determined in cell-free extracts of the hepatocytes. Hepatocytes isolated from chow-fed rats had greater flux rates (decarboxylation rates of 3-methyl-2-oxobutanoate and 4 methyl-2-oxopentanoate) than did hepatocytes isolated from rats fed on the low protein diet. Oxidizable substrates tended to inhibit flux through the branched chain 2-oxo acid dehydrogenase, but inhibition was greater with hepatocytes isolated from rats fed on the low-protein diet. 2-Chloro-4-methylpentanoate (inhibitor of branched-chain 2-oxo acid dehydrogenase kinase), dichloroacetate (inhibitor of both pyruvate dehydrogenase kinase and branched-chain 2-oxo acid dehydrogenase kinase) and dibutyryl cyclic AMP (inhibitor of glycolysis) were effective stimulators of branched-chain oxo acid decarboxylation with hepatocytes from rats fed on a low-protein diet, but had little effect with hepatocytes from rats fed on chow diet. Activity measurements indicated that the branched-chain 2 oxo acid dehydrogenase complex was mainly (96%) in the active (dephosphorylated) state in hepatocytes from chow-fed rats, but only partially (50%) in the active state in hepatocytes from rats fed on a low-protein diet. Oxidizable substrates markedly decreased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had much less effect in hepatocytes from chow-fed rats. 2-Chloro-4-methylpentanoate and dichloroacetate increased the activity state of the enzyme in hepatocytes from rats fed on a low-protein diet, but had no effect on the activity state of the enzyme in hepatocytes from chow-fed rats. The results indicate that protein starvation greatly increases the sensitivity of the hepatic branched-chain 2-oxo acid dehydrogenase complex to regulation by covalent modification. PMID- 3013157 TI - Phosphorylation of fibrinogen by casein kinase 2. AB - Casein kinase 2 from rat liver cytosol phosphorylated human fibrinogen in a reaction that was not stimulated by Ca2+ or cyclic AMP, but was markedly inhibited by heparin, and proceeded at a similar rate when either ATP or GTP was used as phosphate donor. Analysis of casein kinase 2 by glycerol-density-gradient centrifugation showed that the activities towards fibrinogen, casein, phosvitin, high-mobility-group protein 14 and glycogen synthase coincided. Maximal incorporation into fibrinogen by casein kinase 2 averaged 1 mol of phosphate/mol of protein substrate, most of it in the alpha-chain, although some phosphorylation of the beta-chain was also detected. Analysis of phosphorylated alpha-chain revealed that most of the phosphate was incorporated on serine. Phosphorylation of human fibrinogen was also performed by casein kinase 2 from human polymorphonuclear leucocytes, lymphocytes and platelets. PMID- 3013158 TI - A cyclic AMP-independent protein kinase from Candida albicans. AB - A cyclic AMP-independent protein kinase which phosphorylates casein was purified to homogeneity from Candida albicans by affinity and ion-exchange chromatography. This protein kinase exhibits maximal activity with casein as substrate and is not stimulated by cyclic AMP or cyclic GMP. The Mr of the purified enzyme is 115,000, as determined by h.p.l.c. It migrates as a single band on gel electrophoresis and has three non-identical subunits, of Mr 44,000, 28,500 and 26,000, as determined by SDS/polyacrylamide-gel electrophoresis. This enzyme is insensitive to heparin, but is inhibited by polyamines. Furthermore, it is sensitive to thermal denaturation and to thiol reagents. PMID- 3013162 TI - The effects of polymyxin B, a protein kinase C inhibitor, on insulin secretion from intact and permeabilized islets of Langerhans. AB - Polymyxin B (0.01-1 mM), a polyamine antibiotic, inhibited both phorbol ester- and glucose-stimulated insulin secretion from isolated rat islets of Langerhans. This inhibition was rapidly reversible. Assay of the cytosolic protein kinase C by measurement of incorporation of labelled phosphate into a histone substrate demonstrated the presence of activity in islet extracts which could be stimulated by 12-O-tetradecanoylphorbol-13-acetate and inhibited by polymyxin B. These results suggest that protein kinase C plays a role in glucose-induced insulin secretion. PMID- 3013160 TI - Transient kinetics of subunit-III-depleted cytochrome c oxidase. AB - Cytochrome c oxidase from ox heart was depleted of subunit III and its transient kinetic properties studied by stopped-flow and flash photolysis. It was found that the overall mechanism of electron transfer is very similar for subunit-III depleted and native oxidase, although significant differences in some kinetic parameters have been detected. These include the second-order rate constant for cytochrome c oxidation and the rate-limiting step of the overall process. Moreover, at low cytochrome c/oxidase ratios (where the number of reducing equivalents is insufficient), the rate of reoxidation of cytochrome a was found to be very slow, even in air, and in fact for the subunit-III-depleted enzyme is even slower than for the native oxidase. The stability of reduced cytochrome a excludes the likelihood that removal of subunit III leads to a new O2-binding site, and the result may be relevant to the lowered vectorial H+/e- stoichiometry. The subunit-III-depleted oxidase can be pulsed under appropriate conditions and its combination with CO is unchanged, as shown by kinetic experiments and difference spectroscopy. PMID- 3013159 TI - Activation of muscarinic receptors in PC12 cells. Correlation between cytosolic Ca2+ rise and phosphoinositide hydrolysis. AB - The intracellular signals generated by carbachol activation of the muscarinic receptor [release of inositol phosphates as a consequence of phosphoinositide hydrolysis and rise of the cytosolic Ca2+ concentration ([Ca2+]i, measured by quin2)] were studied in intact PC12 pheochromocytoma cells that had been differentiated by treatment with nerve growth factor. When measured in parallel samples of the same cell preparation 30 s after receptor activation, the release of inositol trisphosphate and of its possible metabolites, inositol bis- and mono phosphate, and the [Ca2+]i rise were found to occur with almost superimposable carbachol concentration curves. At the same time carbachol caused a decrease in the radioactivity of preloaded phosphatidylinositol 4,5-bisphosphate, the precursor of inositol trisphosphate. Neither the inositol phosphate nor the [Ca2+]i signal was modified by preincubation of the cells with either purified Bordetella pertussis toxin or forskolin, the direct activator of adenylate cyclase. Both signals were partially inhibited by dibutyryl cyclic AMP, especially when the nucleotide analogue was applied in combination with the phosphodiesterase inhibitors RO 201724 and theophylline. The latter drug alone profoundly inhibited the carbachol-induced [Ca2+]i rise, with only minimal effect on phosphoinositide hydrolysis. Because of the diverging results obtained with forskolin on the one hand, dibutyryl cyclic AMP and phosphodiesterase inhibitors on the other, the effects of the latter drugs are considered to be pharmacological, independent of the intracellular cyclic AMP concentration. Two further drugs tested, mepacrine and MY5445, inhibited phosphoinositide hydrolysis at the same time as the 45Ca2+ influx stimulated by carbachol. Taken together, our results concur with previous evidence obtained with permeabilized cells and cell fractions to indicate phosphatidylinositol 4,5-bisphosphate hydrolysis and [Ca2+]i rise as two successive events in the intracellular transduction cascade initiated by receptor activation. The strict correlation between the carbachol concentration curves for inositol trisphosphate generation and [Ca2+]i rise, and the inhibition by theophylline of the Ca2$ signal without major effects on inositol phosphate generation, satisfy important requirements of the abovementioned interpretation. PMID- 3013163 TI - Expression of hepatitis B surface antigen by yeast actin gene promoter. AB - The promoter sequence (from position -500 to -21) of the yeast actin gene was subcloned into the multiple cloning site of pUC12 to generate a new recombinant plasmid pYAP12. The actin gene promoter can therefore be readily excised from pYAP12 by several restriction enzymes and subsequently placed upstream of the desired protein coding sequence. Results of expression of the hepatitis B surface antigen controlled by the actin gene promoter of pYAP12 in yeast suggest it is a strong functional promoter. To our knowledge, this is the first demonstration of the potential application of the yeast actin gene promoter for expression of foreign proteins in yeast. PMID- 3013161 TI - Platelet activating factor and U44069 stimulate a GTPase activity in human platelets which is distinct from the guanine nucleotide regulatory proteins, Ns and Ni. AB - Platelet-activating factor (PAF, 2-acetyl-1-alkyl-sn-glycero-3-phosphocholine) and the stable thromboxane-receptor agonist U44069 (9 alpha, 11 beta epoxymethanoprostaglandin H2) stimulated GTPase activity in platelet membranes in a dose-dependent fashion, yielding Ka values of 12 nM and 27 nM respectively. The degree of GTPase activation elicited by these agents was found to be additive with the GTPase activation due to either the stimulatory (Ns) or inhibitory (Ni) guanine nucleotide regulatory proteins when activated by prostaglandin E1 and adrenaline (+propranolol) respectively. Treatment of membranes with either cholera or pertussis toxins, which inhibited markedly the receptor-mediated stimulation of the GTPase activities of Ns and Ni respectively, had no or only a small effect, respectively, on the GTPase activity stimulated by PAF and U44069. It is suggested that PAF and U44069, which stimulate inositol phospholipid metabolism in platelets, exert actions through a guanine nucleotide regulatory protein which is distinct from Ns and Ni. PMID- 3013165 TI - The occurrence of cyclic AMP in archaebacteria. AB - Cyclic AMP was found in species representative of the three major groups of the archaebacteria. In Methanobacterium thermoautotrophicum starvation for H2 led to a significant increase in cellular cAMP. The findings suggest that the occurrence of cAMP antedates the divergence of the major kingdoms of biology; the observations also imply that cAMP constitutes a very early regulatory molecule. PMID- 3013164 TI - Alpha-adrenergic receptors in rat skeletal muscle. AB - Sarcolemma-enriched preparations from muscles rich in slow oxidative red fibres contained specific binding sites for the alpha 1 antagonist, prazosin (e.g. soleus Kd 0.13 nM, Bmax 29 fmol/mg protein). Binding sites for prazosin were almost absent from white muscle. Displacement of prazosin binding from sarcolemma of soleus muscle (phentolamine greater than phenylephrine greater than idazoxan greater than yohimbine) suggested that the receptors were alpha 1. Binding sites for dihydroalprenolol (beta antagonist) were also more concentrated on red than white muscle and outnumbered prazosin sites by approx. 10:1. Binding sites for idazoxan (alpha 2 antagonist) were undetectable. Contamination of sarcolemma enriched preparations by endothelial tissue indicated by the activity of angiotensin converting enzyme did not correlate with prazosin binding. It is concluded that post-synaptic alpha 1 adrenergic receptors are present on the sarcolemma of slow oxidative red fibres of rat skeletal muscle. The presence provides the mechanistic basis for apparent alpha-adrenergic effects to increase glucose and oxygen uptake in perfused rat hindquarter. PMID- 3013166 TI - Ca2+-dependent cysteine proteinase, calpains I and II are not phosphorylated in vivo. AB - To clarify phosphorylation of calpains I and II in vivo, we purified both calpains concurrently from the [32P] metabolic-labeled human chronic myelogenous leukemia cell line K-562. By Ultragel AcA34 column chromatography, enzymatic activity of calpain I was separated from [32P] radioactivity. Whereas calpain II activity was closely associated with [32P] radioactivity on Ultragel AcA34 and Blue Sepharose CL-6B column chromatographies. By the above purification procedures, calpain I was purified 1300-fold from the crude extract and calpain II was 920-fold from the original sample, respectively. Autoradiographies of purified calpains I and II from [32P] labeled K-562 cells revealed that both calpains were not specifically phosphorylated in vivo. The autophosphorylation in vitro on calpains and modulation of their proteolytic activities reported recently thus may not occur within cells. PMID- 3013167 TI - N6-functionalized congeners of adenosine with high potency at A2-adenosine receptors: potential ligands for affinity chromatography. AB - Adenosine analogues substituted at N6 with spacer arms designed for attachment to soluble macromolecules or to solid supports for affinity chromatography are agonists at the A2-adenosine receptor that mediates coronary vasodilation in the dog. The most active analogues had spacer arms terminating in -NH2, -NHCH3 or in a biotin residue. Comparisons of coronary vasoactivity with affinity for brain A1 adenosine receptors identified one biotin-containing analogue as relatively selective for coronary A2 receptors. The complex of this analogue with avidin retained coronary vasoactivity. PMID- 3013168 TI - Mycobacteria possess a surprisingly small number of ribosomal RNA genes in relation to the size of their genome. AB - DNAs from Mycobacterium tuberculosis, M. intracellulare, M. phlei and M. smegmatis were digested by restriction enzymes and hybridized with three probes consisting of the 5' (16S rRNA), the middle (16S and 23S rRNA), and the 3' (23S and 5S rRNA) portions of the Escherichia coli rrnB operon. The resulting hybridization patterns indicate that slow-growing Mycobacteria species (i.e., M. tuberculosis and M. intracellulare), with genome size 3.13 - 4.29 X 10(9) daltons, appear to possess only one rRNA operon, whereas fast-growing species (i.e., M. phlei and M. smegmatis), with genome size 4.30 - 5.20 X 10(9) daltons, appear to possess two rRNA operons. PMID- 3013169 TI - Tyr1-substituted and fluorescent Pya1-enkephalins bind strongly and selectively to mu and delta opiate receptors. AB - The fluorescent enkephalins in which an essential Tyr1 residue is replaced by L-1 pyrenylalanine (Pya) were synthesized and examined in the receptor binding assays. [Pya1, Leu5]Enkephalin and its methyl ester showed binding characteristics specific for the opiate receptors, exhibiting a potent inhibition of Tyr1-containing enkephalins. Surprisingly, the methyl ester displayed almost the same potencies to those of DAGO-enkephalin. This analog bound 24-fold more strongly to mu than to delta-receptors. C-terminal free analog Pya1-Enk-OH was delta-preferential with a fairly good affinity. These results indicate that Tyr1 in enkephalin is not necessary to recognition of the opiate receptors. PMID- 3013170 TI - Morphine-mimetic anti-paratypic antibodies: cross-reactive properties. AB - Polyclonal, anti-idiotypic, rabbit antibodies have been raised against four murine monoclonal anti-morphine Fab fragments. The antibody preparations, after affinity purification, have been shown to contain an anti-paratypic fraction which reversibly inhibits morphine binding to the anti-morphine antibodies and to cellular opiate receptors. Using some of the unique properties of this system, for the first time cross-reactivities of anti-paratypic antibodies with the monoclonal anti-morphine IgGs have been examined including competition for the same binding sites by a classical opiate agonist/antagonist pair. PMID- 3013171 TI - Evidence that phosphoinositide response is mediated by alpha 1-adrenoceptor stimulation, but not linked with excitation-contraction coupling in cardiac muscle. AB - Phosphoinositide metabolism is known to be associated with neuronal or humoral stimulation of excitable cells. The present study examined whether the phosphoinositide response is involved in such events using isolated rat papillary muscles labeled with [3H]inositol. It was found that neither increase in the stimulation frequencies (0-2 Hz) nor prolongation of the pulse duration (10-70 msec) altered the labeling of phosphoinositides and the accumulation of [3H]inositol phosphates in this preparation. However, phenylephrine, a known alpha 1-agonist, was capable of provoking the breakdown of phosphoinositides associated with a positive inotropic effect in this preparation. We report the evidence that phosphoinositide response is mediated by alpha 1-adrenoceptor stimulation, but not linked with excitation-contraction coupling in cardiac muscle. PMID- 3013172 TI - There are two gene sequences for human apolipoprotein CI (apo CI) on chromosome 19, one of which is 4 kb from the gene for apo E. AB - A cDNA probe corresponding to the mRNA sequence for apolipoprotein E (apo E) was used to screen two independently-constructed human genomic libraries. Two recombinants (lambda E-2, and lambda E2-1), isolated using the apo E cDNA probe, also contain part or all of the apo CI gene. Hybridisation studies using both apo E and apo CI cDNA probes show that these two genes are in the same orientation and separated by 4 kb. PMID- 3013173 TI - Studies on binding of toromycin, an antitumor antibiotic, to DNA. AB - Toromycin, an antitumor, bactericidal and antiviral compound, was found to bind to DNA in such a way as to interfere with the dissociation of double helix at an elevated temperature. The antibiotic did not introduce strand scission into DNA. Single-strand-specific nuclease S1-susceptibility of negatively supercoiled DNA was not influenced by its binding. The antibiotic was shown to bind to both of the alternating purine-pyrimidine copolymers, poly(dG-dC):poly(dG-dC) and poly(dA dT):poly(dA-dT). The unique C-glycoside molecule of toromycin interacted with single-stranded DNA, but was found to have no affinity for RNA. PMID- 3013174 TI - Cooperation of mitogenic growth factors with polyoma virus middle T antigen in transformation of secondary cultured rat cells. AB - Platelet derived growth factor cooperated with middle T antigen in inducing growth in agarose medium of secondary cultured rat embryo cells transfected with a polyoma virus middle T antigen cDNA clone. In contrast, epidermal growth factor and a conditioned medium containing transforming growth factor did not stimulate the colony-forming efficiency of such cells in the agarose medium. PMID- 3013175 TI - Atrial natriuretic peptide elevates cGMP contents in glomeruli and in distal tubules of rat kidney. AB - Effect of synthetic rat atrial natriuretic peptide (1-28) (ANP) on the cGMP content was studied using defined nephron segments of rat kidney. ANP elevates cGMP contents in glomeruli in a concentration and time-dependent manner. The increase of cGMP was observed in glomeruli, distal convoluted tubule (DCT) and cortical collecting tubule (CCT) (delta %; 279 +/- 35, 148 +/- 10 and 152 +/- 18, respectively), and no effect was observed in proximal convoluted (PCT) and straight tubule (PST). These results suggest that ANP may act directly on the tubular cells as well as glomeruli. In glomeruli, effects of ANP and carbamylcholine on cGMP contents were additive suggesting that these two agents may act on different receptors. Angiotensin II and norepinephrine failed to affect the ANP-induced cGMP production in the glomeruli. PMID- 3013176 TI - Protein kinase C promotes the phosphorylation of immunoprecipitated middle T antigen from polyoma-transformed cells. AB - Stimulation of protein kinase C in polyoma virus-transformed cells increased the phosphorylation of tyrosine residues of the viral middle T (mT) antigen in mT:pp60c-src complexes precipitated by anti-mT antibodies. This increase might have been due to a stimulation of the complex's pp60c-src tyrosine kinase activity or to an increased ability of the mT protein to be phosphorylated by pp60c-src. These observations suggest that cellular protein kinase C might control the ability of polyoma virus to transform its host cell. PMID- 3013177 TI - In vivo stimulation by antitumor drugs of the topoisomerase II induced cleavage sites in c-myc protooncogene. AB - Several antitumor drugs including DNA intercalative and non intercalative agents induce in vitro and in vivo double-stranded DNA breaks by stabilization of a topoisomerase II-DNA complex. In order to locate cleavage sites in an actively transcribed oncogene, N417 cells, originating from a human small cell lung carcinoma and containing 45-50 copies of c-myc oncogene, were treated with mAMSA, 9 hydroxyellipticine and VM 26. The presence of DNA lesions in c-myc was investigated by Southern blot hybridization with a human c-myc probe. In addition to normal bands, DNA patterns of drug treated-cells revealed the presence of new bands most likely corresponding to topoisomerase II-mediated cleavage as these bands were not found in untreated control DNA and in DNA treated with oAMSA, a biologically inactive stereoisomer of mAMSA. Major cleavage sites induced by drugs in the N417 cell c-myc locus were located in the 5' end of the c-myc exon 1 closely to some DNAse I hypersensitive sites which are assumed to reflect an activity of the gene. Therefore our data suggest that TopoII-mediated drug activity correlates with gene activity. PMID- 3013178 TI - Phylogenetic distribution of [3H]cyclohexyladenosine binding sites in nervous tissue. AB - The specific binding of the A1 adenosine receptor ligand, [3H]CHA, was investigated in membrane fractions prepared from brains of eleven vertebrate species and ganglia of four invertebrate species. Substantial amounts of specific [3H]CHA binding sites were demonstrated in brain membranes of all vertebrate species examined; however, [3H]CHA binding sites were not detectable in nervous tissue of the invertebrate species studied. The densities of [3H]CHA binding sites in vertebrate brains increase in higher vertebrates. Moreover, the pharmacological characteristics of the site labeled by [3H]CHA in two divergent classes of vertebrates were similar. The broad phylogenetic distribution of A1 adenosine receptors in primitive as well as advanced vertebrate species suggests a fundamental role for adenosine in neuronal modulation. PMID- 3013179 TI - Lymphocyte-Salmonella interaction: energy dependence and thiol group involvement. AB - In order to gain better insight into lymphocyte-Salmonella interaction investigation has been carried out on energy-dependence and involvement of thiol groups in this process by using a modified rosette test. Binding frequency, number of bound bacteria/number of binding lymphocytes and the number of bacteria binding sites/lymphocyte were found to be enhanced by externally added ATP and decreased by both uncouplers and electron transfer chain inhibitors. Treatment of either bacteria or lymphocytes with thiol reagents, such as mersalyl or N-ethyl maleimide, prevents lymphocyte-Salmonella adherence, thus showing the presence of thiol groups involved in the binding mechanism in both bacteria and cells. Consistently, as a result of mersalyl inhibition, a decrease in the number of bacteria-binding sites/lymphocyte was also found. PMID- 3013180 TI - Fc:Fc interactions revealed by spin-labeled IgG heterosaccharides in model immune complexes. AB - Dynamic properties of spin-labelled heterosaccharides in the Fc-region of murine monoclonal antihapten immunoglobulin G were studied in model immune complexes (IC) as a function of the IC size. Model IC dimers, trimers and oligomers were formed using bivalent photoaffinity antigens. The ESR spectrum exhibits two components. The rotational correlation time of the less-immobilized species is shorter than 10(-10) sec, and that of the more-immobilized component is in the order to 10(-9) approximately 10(-8) sec depending on the IC size. Fraction of the more-immobilized spin labels increases, and the mobility of this component decreases with increase in IC size (i.e., mobility: monomers approximately equal to dimers greater than trimers much greater than immune-complex precipitates). These data strongly suggest the existence of Fc:Fc interactions in IC, and provide the basis for a model in which such interactions underlie the initial mechanism by which the information of antigen binding to Fab region is transferred into organized Fc:Fc association structure for IgG effector activities. PMID- 3013181 TI - Decreased expression of liver epidermal growth factor receptors in rats with alloxan and streptozotocin diabetes. AB - Male rats (200 g) were rendered diabetic with one intraperitoneal injection of alloxan (150 mg/kg) or streptozotocin (60 mg/kg). In hyperglycemic animals within 3 hours after the injection, the binding of EGF to liver membranes decreased by 43-52%; the maximal drop was by 70% and persisted for the 20 days of the experiment. EGF receptors decreased in number with almost no changes in their affinity. Autophosphorylation of the receptors decreased parallel to the ligand binding. In animals that received lower doses and did not develop diabetes and in animals in whom diabetes was prevented by the injections of glucose (before alloxan) or nicotinamide (before streptozotocin) the binding of EGF to liver receptors remained normal. We conclude that the decreased expression of EGF receptors was caused by diabetes and not by the toxic effects of the diabetogenic compounds on the liver. PMID- 3013182 TI - Generation of hybridomas producing human monoclonal antibodies against human cytomegalovirus. AB - In vitro stimulation of human lymphocytes were studied in connection with cell fusion. When splenic lymphocytes were stimulated with human cytomegalovirus (CMV), they produced IgG but not IgM antibody against CMV. The stimulation with 50 ng/ml of CMV antigen induced the maximum antibody response, and higher concentrations of CMV antigen decreased antibody response and increased nonspecific IgG production. Human splenic lymphocytes were stimulated for 6 days with CMV antigen (50 ng/ml) and/or B-cell growth factor (BCGF), and then fused with mouse myeloma cells. Stimulation with a combination of antigen and BCGF were able to generate CMV-specific hybridomas synergistically. Two of these hybridomas were cloned by limiting dilution. The human monoclonal antibodies produced by them, C1 and C23, bound to CMV but not to other herpesviruses. C23 neutralized virus infectivity C1 did not at all. This method for generation of hybridomas producing human monoclonal antibodies against a predefined antigen may be applicable to a variety of viral antigens. PMID- 3013183 TI - Scavenger receptor for malondialdehyde-modified high density lipoprotein on rat sinusoidal liver cells. AB - We report here the presence of a membrane-associated receptor which mediates endocytic uptake of malondialdehyde-modified high density lipoprotein (MDA-HDL) on sinusoidal liver cells. Binding of [125I]MDA-HDL to the cells was followed by internalization and degradation in lysosomes. The binding and lysosomal degradation of [125I]MDA-HDL were effectively inhibited by unlabeled MDA-HDL and acetyl-HDL. However, formaldehyde-treated serum albumin or low density lipoprotein modified either by acetylation or malondialdehyde, ligands known to undergo receptor-mediated endocytosis by sinusoidal liver cells, did not affect the binding of [125I]MDA-HDL to the cells. These results indicate that a receptor for MDA-HDL is described as a distinct member among the scavenger receptors for chemically modified proteins. PMID- 3013184 TI - Inhibition of cytochrome c oxidase by psychosine (galactosylsphingosine). AB - Pi uptake and acetoacetate formation were suppressed by psychosine (galactosylsphingosine) in rat liver mitochondria. Besides, reduced form of cytochrome c increased in the reaction mixture which contained psychosine. Using reduced form of cytochrome c as substrate, less than 5 microM of psychosine (0.1 mumoles/mg of mitochondrial protein) inhibited cytochrome c oxidase by more than 50%. The inhibition was completely reversed by 1% human serum albumin. Thus, a lipid which is produced in the brain has a powerful and yet reversible inhibitory effect on an enzyme of cellular respiration. PMID- 3013185 TI - NADP improves the efficiency of cholera toxin catalyzed ADP-ribosylation in liver and heart membranes. AB - Guanine nucleotide binding proteins (G-proteins) can be identified by their ability to be ADP-ribosylated using [32P]NAD as the substrate and bacterial toxins as catalysts. This labelling, when performed in liver and sarcolemma membrane preparations, can be complicated by competing enzymes which degrade NAD, making it unavailable to participate in the desired reaction. The addition of NADP in reaction mixtures markedly slows the degradation of NAD, but does not compete with NAD in cholera toxin labelling of stimulatory G-protein. The efficiency of cholera toxin labelling is improved to the extent that saturation curves may be constructed, allowing the quantitation of ADP-ribosylation sites in membranes. PMID- 3013186 TI - Leukotriene B4 production by stimulated whole blood: comparative studies with isolated polymorphonuclear cells. AB - A new method was developed to study leukotriene B4 (LTB4) production by stimulated whole blood. The calcium ionophore A23187 and serum-treated zymosan induced LTB4 production, measured by radioimmunoassay, in a dose- and time dependent manner. The pattern of LTB4 production by whole blood differed markedly from that observed with isolated, purified polymorphonuclear leukocytes. Higher levels of LTB4 were reached and maintained in whole blood. The system allowed to detect drug effects on LTB4 synthesis in vitro. This new method to study the synthesis of LTB4 takes into account the complex interactions between different cell types which can modulate LTB4 metabolism. PMID- 3013187 TI - Odorant- and guanine nucleotide-stimulated phosphoinositide turnover in olfactory cilia. AB - Isolated olfactory cilia from the channel catfish (Ictalurus punctatus) exhibited phosphatidylinositol-4,5-bisphosphate phosphodiesterase (E.C.3.1.4.11) activity. The phosphodiesterase activity was stimulated in the presence of an odorant for the catfish, namely the amino acid L-alanine. The enzyme activity was also stimulated in the presence of GTP and its nonhydrolyzable analogues. The activation of the phosphodiesterase by guanine nucleotides, in combination with the identification of guanine nucleotide-binding protein(s) in the isolated cilia, indicate the probable participation of a guanine nucleotide-binding protein in stimulation of phosphoinositide turnover in the olfactory receptor neuron. PMID- 3013188 TI - Superoxide dismutase mimetic activity of cytokinin-copper(II) complexes. AB - Dissociation constants of cytokinins, derivatives of purine which form complexes with cupric ion, were determined by spectrophotometry and the stability constants of their copper complexes by pH titration. The values found for kinetin were 3.76, 9.96, 7.8, and 15.3 for pK1, pK2, logk1, and log beta 2, respectively, and those for 6-benzylaminopurine were, in the same order, 3.90, 9.84, 8.3, and 15.9. The copper(II) complexes with kinetin and 6-benzylaminopurine had superoxide dismutase mimetic activity, and the reaction rate constants with superoxide, which were determined by polarography, were 2.3 X 10(-7) M-1 s-1 for kinetin and 1.5 X 10(-7) M-1 s-1 for 6-benzylaminopurine at pH 9.8 and 25 degrees C. PMID- 3013189 TI - Transformation of Ob17 cells promotes proliferation and differentiation of Ob17 preadipocytes via distinct extracellular intermediates. AB - Conditioned serum-free medium of Ob17 cells transformed by the middle-T-only gene of polyoma virus (Ob17MT cells) is able to support growth and adipose conversion of the parental Ob17 cells. Conditioned media from 3T3-F442A cells (untransformed preadipocyte clonal line) and MTT4 cells (middle-T-transformed non-preadipocyte clonal line) are inactive. The serum-free conditioned medium of Ob17MT cells is also active on growth and adipose conversion of 3T3-F442A cells. The morphological differentiation of Ob17 cells is accompanied by the expression of early (lipoprotein lipase, LPL) and late (glycerol-3-phosphate dehydrogenase, GPDH) biochemical markers of adipose conversion. Bio-Gel P-60 chromatography and SDS-PAGE have allowed characterization of a mitogenic fraction of apparent MW approximately equal to 28 Kd distinct from an adipogenic fraction of apparent MW less than 10 Kd. This adipogenic fraction is only required for the acquisition of the GPDH activity and is therefore active on terminal differentiation. PMID- 3013190 TI - Rundown of GH3 cell K+ conductance response to TRH following patch recording can be obviated with GH3 cell extract. AB - GH3/B6 pituitary cells release prolactin (PRL) in response to thyrotropin releasing hormone (TRH). Electrophysiological assays of individual GH3 cells with sharp high-resistance microelectrodes have revealed complex effects of TRH on membrane excitability consisting of a transient hyperpolarization (1), which is thought to result from activation of Ca-dependent K+ conductance (2), followed by a prolonged phase of spontaneous, Ca-dependent action potential activity (3). Using the whole-cell patch recording (WCR) technique (4), we have found that these TRH actions on GH3 excitability rapidly rundown following patch recording. When the supernatant from osmotically lysed GH3 cells was added to the WCR patch pipette, the K+ conductance response was not only promoted but well-maintained. The results indicate that diffusible factors mediate these TRH actions and further, that the WCR technique should be useful in identifying different second messengers and elucidating their roles in membrane excitability and PRL secretion. PMID- 3013191 TI - Cholera toxin, a potent inducer of epidermal hyperplasia but with no tumor promoting activity in mouse skin carcinogenesis. AB - Intracutaneous injection of cholera toxin into mice induced epidermal hyperplasia to a greater extent than 12-O-tetradecanoylphorbol-13-acetate. It also induced adenylate cyclase and though weakly, ornithine decarboxylase of the epidermis. Cholera toxin, however, showed no tumor promoting activity in mouse skin carcinogenesis. In the single stage promotion, cholera toxin (50 ng) was injected once a week for 10 weeks into the skin of SENCAR mice initiated with 25 micrograms 7,12-dimethylbenz[a]anthracene, but no tumors developed. In the two stage promotion test, cholera toxin (10-100 ng) was injected for one or two weeks into the initiated skin and then mezerein (4 micrograms) was applied twice a week for 18 weeks, but the toxin did not increase incidence or numbers of papillomas. PMID- 3013192 TI - The inhibition of neutrophil responsiveness caused by phorbol esters is blocked by the protein kinase C inhibitor H7. AB - The relationship between the inhibition of neutrophil responsiveness to chemoattractants caused by preincubation with phorbol esters and the activation of protein kinase C was investigated using the protein kinase antagonist H7. The latter compound was found to inhibit the phosphorylation of the 50 kDa protein kinase C substrate stimulated by phorbol 12-myristate 13-acetate (PMA). On the other hand, H7 was found not to affect the quin2 and secretory responses of the neutrophils to fMet-Leu-Phe and leukotriene B4. In addition, pretreatment of the cells with H7 blocked the ability of PMA to inhibit the latter two responses to the addition of the chemoattractants. Taken together, these results provide strong evidence for the involvement of protein kinase C in the inhibition of neutrophil--and probably also other cells--responsiveness brought about by preincubation with phorbol esters. Additionally, they invite a reevaluation of the role of protein kinase C in the excitation-response coupling sequence of these cells directed more towards a negative, modulatory, role than that of a critical element in its initiation. PMID- 3013193 TI - Evidence that the subunit structure of gonadotropin receptor is preserved during regression of rat corpus luteum. AB - The level of hCG/LH receptor has been shown to undergo marked changes during the life span of rat corpus luteum. To evaluate whether these fluctuations are due to changes in the receptor subunit structure or receptor protein content, the 125I hCG binding activity and the receptor subunit structure were determined during different time periods of pseudopregnancy. The maximum 125I-hCG binding activity was observed on day 7, after which it decreased by 20 and 45% on day 11 and day 14, respectively. The Scatchard analysis of 125I-hCG binding data showed that the decrease in binding activity was caused by a change in the number of binding sites rather than a change in the binding affinity. The LH/hCG receptor in ovarian membranes obtained on days 7, 11 and 14 were then characterized by the method of affinity cross-linking. All four subunits of the LH/hCG receptor were detected in the ovarian membranes at all stages while the intensity decreased parallel to a decrease in hCG binding from day 7 to day 14. These results suggest that the decrease in 125I-hCG binding activity in rat ovarian membranes from day 7 to day 14 of pseudopregnancy is due to a decrease in receptor concentration rather than a change in the receptor subunit structure. PMID- 3013194 TI - Characterization of the insulin and insulin-like growth factor receptors and responsitivity of a fibroblast/adipocyte cell line before and after differentiation. AB - TA1 cells, like 3T3-L1 cells, undergo a differentiation process in vitro from a fibroblast to an adipocyte phenotype. The TA1 pre-adipocytes were found to have low numbers of insulin receptors but high numbers of receptors for insulin-like growth factors (IGF) I and II. Also, the pre-adipocytes were more responsive to IGF than insulin as measured by either stimulation of glucose or amino acid uptake. After differentiation, the adipocytes had higher numbers of insulin receptors and a better responsitivity to insulin than to IGF-I. These results indicate that insulin-like growth factors are the primary regulators of the pre adipocytes whereas insulin regulates the adipocytes. PMID- 3013195 TI - Phosphorylation of bovine cardiac calcium-activated neutral protease by protein kinase-C. AB - Protein kinase C prepared from rat brain was used to phosphorylate a calcium activated neutral protease, purified from bovine cardiac muscle. Attempts to phosphorylate the enzyme in the presence of calcium were unsuccessful, unless the protease inhibitor leupeptin was also present. Phosphorylation of the 74K subunit of the protease was completely inhibited in the absence of phosphatidylserine and diolein, indicating that phosphorylation of the enzyme was catalysed by the calcium and phospholipid-dependent protein kinase C. PMID- 3013196 TI - Further characterization of the low and high affinity binding components of the thyrotropin receptor. AB - Following cross-linking with disuccinimidyl suberate and analysis by SDS-PAGE and autoradiography, both the high- and low-affinity TSH binding components exhibited two similar 125I-TSH-labeled bands, with Mr values of 80,000 and 68,000. IgG fractions from patients with Graves' disease inhibited 125I-TSH binding to both components, while normal IgG had no effect. Although not entirely conclusive, these results suggest that the high- and low-affinity components share similar subunit composition and antigenic determinants. PMID- 3013197 TI - Allopurinol can act as an electron transfer agent. Is this relevant during reperfusion injury? AB - The xanthine oxidase inhibitor allopurinol markedly enhances myocardial function and decreases ventricular irritability during myocardial reperfusion. In the present report, we have evaluated the molecular mechanism of allopurinol action. First, allopurinol was shown to be a weak radical scavenger. Second, allopurinol was found to act as an electron transfer agent from ferrous iron to ferric cytochrome c. The results suggest that the beneficial effect of allopurinol might partially result from its facilitated electron transport during reperfusion when the lipid components of the chain can be expected to be disordered. PMID- 3013198 TI - Activation of (Na++K+)-ATPase by long-chain fatty acids and fatty acyl coenzymes A. AB - Long-chain unsaturated fatty acids and fatty acyl CoA derivatives activated (Na++K+)-ATPase at suboptimal, but not optimal, ATP concentrations. Activation was obtained within a narrow range of fatty acid concentrations; higher acid levels inhibited the enzyme. The various CoA esters, however, activated with K0.5 values in the range of 0.15-10 microM; and with no inhibitory effects at concentrations up to 100 microM. Palmitoyl CoA, binding reversibly to a regulatory site, reduced K0.5 of ATP from 0.37 mM to 0.17 mM; and changed the Hill coefficient of the substrate-velocity curve from 0.86 to 0.63. These compounds may be physiological regulators that desensitize the function of this enzyme to diminishing ATP levels. PMID- 3013199 TI - Effect of doxycycline on oxygen-dependent killing mechanisms of human neutrophils. AB - The effects of doxycycline on neutrophil adhesivity, ingestion rate, and oxidative burst by particle and soluble compounds have been analyzed. The rate of bacterial ingestion by neutrophils as well as its subsequently particle-induced oxidative burst comprising oxygen uptake, hydrogen peroxide and superoxide anion productions, and iodination were all inversely correlated to doxycycline concentration included in the assay medium. The neutrophil oxidative burst induced by phorbol myristate (a soluble stimulant) was also inversely correlated to doxycycline concentration. Drug effect was observed at lower concentrations when the neutrophil stimulant was a soluble compound than when it was particles. In contrast doxycycline did not affect neutrophil adhesivity to either nylon fibers or Petri dishes. Further studies are needed to assess whether the activity of the drug on the neutrophil is due only to its ability to chelate calcium and magnesium or to other properties. PMID- 3013200 TI - Effect of repeated administration of 11-hydroxy-delta 8-tetrahydrocannabinol, an active metabolite of delta 8-tetrahydrocannabinol, on the hepatic microsomal drug metabolizing enzyme system of mice. AB - The effects of delta 8-tetrahydrocannabinol (delta 8-THC) and its major and active metabolite, 11-hydroxy-delta 8-tetrahydrocannabinol (11-OH-delta 8-THC), on the hepatic microsomal drug-metabolizing enzyme system were studied in mice. The repeated administration of 11-OH-delta 8-THC (5 mg/kg/day, i.v.) for 3 or 7 days increased significantly the activities of aniline hydroxylase and p nitroanisole O-demethylase. By the same treatment, cytochrome P-450 content (3 days) or NADPH-cytochrome c reductase activity (7 days) was also increased significantly. The treatment with delta 8-THC for 7 days (5 mg/kg/day, i.v.) significantly increased aniline hydroxylase only. 11-OH-delta 8-THC increased the Vmax, but not the Km, values for both drug-metabolizing enzymes, whereas delta 8 THC decreases significantly the Km value (270 microM) for p-nitroanisole O demethylase as compared with the control (398 microM). Repeated administration of these cannabinoids for 7 days also increased the metabolism of delta 8-THC by hepatic microsomes; this was attributed to an enhanced formation of 11-OH-delta 8 THC. In contrast, microsomal formation of 7 alpha-OH-delta 8-THC was decreased significantly by treatment with delta 8-THC. 11-OH-delta 8-THC, but not delta 8 THC, treatment increased the metabolism of 11-OH-delta 8-THC by hepatic microsomes. These findings indicate that delta 8-THC and 11-OH-delta 8-THC treatment can induce hepatic microsomal drug-metabolizing enzymes and affect differently the catalytic properties of the enzymes. PMID- 3013201 TI - Induction potential of antifungals containing an imidazole or triazole moiety. Miconazole and ketoconazole, but not itraconazole are able to induce hepatic drug metabolizing enzymes of male rats at high doses. AB - Male Wistar rats were dosed with miconazole, ketoconazole and itraconazole by gastric intubation once daily for up to 7 days. A dose- and time-dependent induction of the hepatic drug metabolizing enzyme system was observed for miconazole and ketoconazole, while itraconazole proved to be devoid of inductive properties even at the highest dose studied (160 mg/kg). No effect on drug metabolizing enzymes could be demonstrated for either drug at a dose level of 10 mg/kg, which is just above the antifungally active dose. At a dose of 40 mg/kg, miconazole, but not ketoconazole, significantly increased cytochrome P-450 content. At the highest dose of 160 mg/kg, both miconazole and ketoconazole increased the relative liver weight, the cytochrome P-450- and b5-content and NADPH-cyt c-reductase. Furthermore, miconazole, but not ketoconazole, increased specific microsomal aminopyrine and N,N-dimethylaniline N-demethylase activity, p nitroanisole O-demethylase activity and UDP-glucuronyltransferase activity towards 4-nitrophenol while the specific aniline hydroxylase activity was unaffected. Ketoconazole at 160 mg/kg only induced O-demethylase activity and UDP glucuronyltransferase activity, while it lowered the specific activities towards the other substrates. Miconazole was a relatively more potent inducer when compared to ketoconazole. Both drugs displayed biphasic effects on the mixed function oxidase activities, which were lowered after acute administration (160 mg/kg, 1 hr before death) and were induced when determined after 23 hr had elapsed or after multiple dosage. Both drugs bound strongly to their respective induced cytochromes, giving rise to type II difference spectra, and inhibited the O-demethylase activity of the induced microsomes with an I50 of 5.2 microM for miconazole and 15.1 microM for ketoconazole. On the basis of a comparison of the enzymatic activities induced by both antimycotics with those induced by PB or 3 MC, it was concluded that miconazole behaved as a PB-type inducer, whereas ketoconazole did not belong to either category of inducers. A comparison of electrophoretograms of microsomes from different origins on SDS-PAGE revealed that miconazole increased the concentration of several proteins, whereas ketoconazole selectively induced a protein with Mr of 47,800. The protein pattern in the 50 kDa region of miconazole-induced microsomes resembled that of PB microsomes qualitatively. PMID- 3013202 TI - Chronic exposure of human cells in culture to the tricyclic antidepressant desipramine reduces the number of beta-adrenoceptors. AB - The effects of the antidepressant drug desipramine (DMI) on the density of beta adrenoceptor sites were studied on intact cultured human cells: skin fibroblasts, lung fibroblasts and macrophages. Direct binding studies were performed with the radioligand 3H-CGP 12177, a hydrophilic beta-adrenergic antagonist. The confluent cell cultures were exposed to DMI and all three cell types showed a dose dependent decrease in the number of beta-adrenergic binding sites. This receptor desensitisation was only seen after chronic exposure of the cells to DMI. The extent of desensitisation was comparable to that seen in brain following chronic treatment of rats with DMI. The affinity of the binding sites to the radioligand was not affected by the antidepressant drug action. From these results we suggest that the in vivo effect of antidepressant drugs on postsynaptic beta-adrenoceptor density, at least in part, reflects a primary drug action and not only an adaptive change to presynaptic events. PMID- 3013204 TI - Studies of the digestion of bradykinin, Lys-bradykinin, and des-Arg9-bradykinin by angiotensin converting enzyme. AB - We have studied the degradation of bradykinin, lysyl bradykinin and des-Arg9 bradykinin by the angiotensin converting enzyme. Bradykinin was cleaved at two sites to produce the pentapeptide Arg-Pro-Pro-Gly-Phe plus dipeptides Ser-Pro and Phe-Arg. Lysyl bradykinin was cleaved similarly to release the same dipeptides plus the hexapeptide Lys-Arg-Pro-Pro-Gly-Phe. The tripeptidase activity of ACE was observed when des-Arg9-bradykinin was digested. A single cleavage yielded the above pentapeptide plus Ser-Pro-Phe. Although des-Arg9-bradykinin was the most rapidly digested, when mixtures of des-Arg9-bradykinin and bradykinin or lysyl bradykinin were tested, virtually all of the bradykinin and most of the lysyl bradykinin was digested prior to the onset of digestion of des-Arg9-bradykinin. This was shown to be due to inhibition of des-Arg9-bradykinin cleavage by kinins and kinin-degradation products. The order in terms of potency was bradykinin greater than lysyl bradykinin greater than Ser-Pro much greater than Phe-Arg greater than Arg-Pro-Pro-Gly-Phe. The concentration of chloride ion was an important parameter which affected the rate of digestion of each substrate examined. des-Arg9-bradykinin was not digested by ACE in the absence of sodium chloride and the rate of digestion increased as the chloride concentration was increased to 100-150 mM. On the other hand, increasing NaCl concentration was inhibitory for bradykinin digestion. The rate of Lys-bradykinin digestion was increased from 0 to 1 mM NaCl and decreased thereafter up to physiologic concentration. A half-maximal rate was seen at 100-150 mM NaCl compared to no salt. Of the divalent cations examined, cupric ion inhibited further digestion of des-Arg9-bradykinin at physiologic concentrations. Our data indicate that the rate of degradation of kinins and the nature of the stable final cleavage products in plasma or serum (studied in vitro) are dependent upon the effects of chloride ion, metal ions, and the kinetic effects of multiple metabolites produced by at least two kininases. PMID- 3013203 TI - Nucleotide interactions with 5-HT1A binding sites directly labeled by [3H]-8 hydroxy-2-(DI-n-propylamino)tetralin ([3H]-8-OH-DPAT). AB - Nucleotide interactions were examined at 5-hydroxytryptamine1A (5-HT1A) binding sites labeled by [3H]-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). At a 10(-4) M concentration, GTP and GDP decreased specific binding of 0.4 nM [3H]-8 OH-DPAT to 47 +/- 4 and 61 +/- 1% of control values respectively. This nucleotide effect was significantly greater (P less than 0.005) than observed at total 5-HT1 binding sites labeled by 1.5 nM [3H]-5-HT. GMP and adenine nucleotides had a minimal effect on [3H]-8-OH-DPAT binding at concentrations less than 10(-3) M. Saturation experiments demonstrated that 10(-4) M GTP increased the KD of [3H]-8 OH-DPAT for 5-HT1A binding sites (0.79 to 2.7 nM) without changing the number of binding sites (1.98 to 1.93 pmoles/g tissue). The Ki values of classic and novel putative 5-HT agonists were increased 2- to 4-fold in the presence of 10(-4) M GTP. Affinities of 5-HT antagonists for the [3H]-8-OH-DPAT site were not affected by the addition of 10(-4) M GTP to the binding assay. PMID- 3013205 TI - Effects of short-chain alcohols and norepinephrine on brain (Na+,K+)ATPase activity. AB - (Na+,K+)ATPase activity in synaptic membranes from whole brains of mice was inhibited by a series of short-chain aliphatic alcohols (ethanol through pentanol). The relationship of inhibitory potency to alcohol chain length and to alcohol membrane:water partition coefficient suggested that the inhibitory effect of the alcohols does not depend totally on their interaction with neuronal membrane lipids. Although partitioning into the membranes is important for this inhibitory effect, a direct interaction of alcohol with the enzyme protein may also be involved in the inhibition. Norepinephrine did not significantly potentiate inhibition of (Na+,K+)ATPase activity by low concentrations of ethanol in preparations of either mouse or rat brain. Thus, under our conditions, ethanol, at levels which can be reached in vivo, only slightly inhibited enzyme activity, and the possible importance of this inhibition in mediating the in vivo acute or chronic effects of ethanol on the CNS remains open to question. PMID- 3013206 TI - Pancreatic islet arachidonic acid turnover and metabolism and insulin release in response to delta-9-tetrahydrocannabinol. AB - Isolated pancreatic islets from the rat secrete insulin in response to glucose or delta-9-tetrahydrocannabinol (THC). THC stimulated the basal release of insulin and also potentiated the secretory response to glucose. The exposure of control or glucose-stimulated islets to THC inhibited the incorporation of [14C]arachidonic acid (AA) into phospholipids. However, in islets prelabeled with [14C]AA, THC enhanced the glucose-induced loss of AA from phospholipids. The enhanced AA release from islet phospholipids in response to glucose and THC was accompanied by increased synthesis of 12-L-[5,6,8,9,11,12,14,15-3H(N)]-hydroxy 5,8,10,14-eicosatetraenoic acid (12-HETE) and prostaglandin E2. The lipoxygenase inhibitor 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline hydrochloride (BW755C) inhibited 12-HETE synthesis and insulin release in glucose and THC-challenged islets; nordihydroguaiaretic acid also inhibited insulin release in THC-treated islets. In contrast, the cyclooxygenase inhibitor, indomethacin, stimulated insulin release. In homogenized islet preparations, THC inhibited acyl-CoA acyltransferase, while it stimulated phospholipase A2 activity. The stimulatory effects of THC on islet cell AA hydrolysis from phospholipids, lipoxygenase product formation, and secretion suggests that these biochemical sequelae in cell activation are important modulators of insulin release. PMID- 3013207 TI - Contributions of superoxide, hydrogen peroxide, and transition metal ions to auto oxidation of the favism-inducing pyrimidine aglycone, divicine, and its reactions with haemoglobin. AB - The influence of O2-, H2O2 and metal ions on the auto-oxidation of divicine, a pyrimidine aglycone, was studied. In air at pH 7.4, the hydroquinonic form oxidized within a few minutes. Superoxide dismutase (SOD) markedly decreased the initial rate, giving a lag phase followed by rapid oxidation. Although catalase or diethylenetriamine-penta-acetic acid (DTPA) alone had little effect, each in the presence of SOD further slowed the initial rate and increased the lag. H2O2 decreased the lag time, as did Cu2+, Fe2+ or haemoglobin. GSH substantially increased the lag phase, but it eventually reacted with the divicine to form a 305 nm-absorbing adduct. These results indicate that an O2(-)-dependent mechanism of divicine auto-oxidation normally predominates. Auto-oxidation can also occur by a mechanism involving H2O2 and transition metal ions or haemoglobin, and if both these reactions are prevented by SOD and DTPA or catalase, a third mechanism, requiring build-up of an autocatalytic intermediate, becomes operative. Oxyhaemoglobin did not react directly with divicine, but reacted with the H2O2 produced by divicine auto-oxidation to give mainly an oxidized derivative presumed to be ferrylhaemoglobin. Divicine was shown to reduce ferylhaemoglobin to methaemoglobin, and this reaction was probably responsible for the acceleratory effect of haemoglobin on divicine oxidation. These results indicate that O2 rather than oxyhaemoglobin is likely to initiate divicine oxidation in the erythrocyte. Haemolytic crises, which are thought to result from this oxidation, occur only sporadically in glucose-6-phosphate dehydrogenase deficient individuals following ingestion of fava beans. A characteristic of the crises is acute depletion of erythrocyte GSH, and the vulnerability of these cells could relate to the ability of GSH, in combination with SOD, to protect against the autocatalytic mechanism of divicine auto-oxidation. Our demonstration of a variety of auto-oxidation pathways also suggests possible areas of individual variation. PMID- 3013208 TI - Radical activation of carbon tetrachloride in foetal and maternal rat liver microsomes. PMID- 3013209 TI - Transducing signals involved in the activation of resting tonsillar B cells. PMID- 3013210 TI - [Nucleotide sequence of the ADE 1 gene of the yeast Saccharomyces cerevisiae]. AB - The yeast ADE 1 gene has been cloned and sequenced. The primary structure deduced from the nucleotide sequence demonstrated that phosphoribosylaminoimidazole succinocarboxamide synthetase is a protein with molecular weight of 34 500 D. PMID- 3013211 TI - Biochemical studies with the new thienotriazolo-diazepine brotizolam. AB - Brotizolam (2-bromo-4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f]-1,2,4-triazolo [4,3-a]-1,4-diazepine, We 941, Lendormin) is a newly developed thienotriazolo diazepine with distinct hypnotic effects. It was biochemically characterized by performing various receptor binding studies, uptake and release studies in vitro and determining homovanillic acid brain concentrations in vivo. Using [3H] flunitrazepam as a radioligand, brotizolam showed an extremely high affinity for specific benzodiazepine binding sites in the rat brain, demonstrated by an IC50 value of 1.0 nmol/l. Using both [3H]-flunitrazepam and [3H]-brotizolam as radioligands, a strong correlation between the IC50 values of brotizolam and various benzodiazepines could be shown. [3H]-Flunitrazepam as well as [3H] brotizolam bound with similar affinities to cerebellar and hippocampal synaptosomal membranes. In contrast to [3H]-flunitrazepam binding, however, [3H] brotizolam had twice the number of specific binding sites in the hippocampus. According to the maximum binding of [3H]-flunitrazepam, the inhibition curve of brotizolam also increased in the presence of 10(-4) mol/l gamma-aminobutyric acid and decreased remarkably in the presence of 10(-4) mol/l bicuculline. Using [3H] muscimol as a ligand, a modulating effect of brotizolam could be shown by increasing the low affinity with no change in the number of binding sites. From various further receptor binding studies, as well as from in vitro uptake and release studies of [3H]-noradrenaline, [3H]-serotonin and [3H]-dopamine, no indication was found of another biochemical effect of brotizolam. There was also no change in the homovanillic acid concentration in the rat brain striata in vivo after brotizolam treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013212 TI - Pharmacological action of some known and possible metabolites of brotizolam. AB - The two major metabolites of brotizolam (2-bromo-4-(2-chlorophenyl)-9-methyl-6H thieno [3,2-f]-1,2,4-triazolo [4,3-a]-1,4-diazepine, We 941, Lendormin) in man are the hydroxylation products We 964 (hydroxylation in the methyl group at 9 position) and We 1061 (hydroxylation in 6-position). A structural isomer originating from the latter, We 1064, was found in the urine of dogs. In the same species the demethylation product We 956 was identified. In line with this demethylation the oxidation product Web 1175 might also be formed but was not yet detected. The dihydroxylated product Web 1073 which could arise either from We 964 or from We 1061 was discovered in monkeys. All compounds were investigated with respect to their pharmacological and acute toxicological properties in various experimental situations in mice and rats, and exhibited a profile of action quite similar to brotizolam. Effects of other kinds did not occur. None of the examined metabolites had a longer duration of action than the parent substance. The strength of activity in the various tests was, with very minor exceptions, less than that of brotizolam. All findings favor the conclusion that the various actions of brotizolam are mainly caused by the latter itself and not by its active metabolites. Also, the duration of action of brotizolam is not substantially determined by the pharmacokinetics of its metabolites. PMID- 3013213 TI - Pasteurization as an efficient method to inactivate blood borne viruses in factor VIII concentrates. AB - Heat treatment at 60 degrees C for 10 h in solution (pasteurization) was introduced into the manufacturing process of factor VIII concentrate (Haemate P) in order to considerably reduce the risk of transmission of human pathogenic viruses to haemophiliacs. The results of experimental and clinical studies with regard to hepatitis B, non-A, non-B hepatitis, acquired immune deficiency syndrome (AIDS) and herpes virus infections are reviewed. From this data it is concluded that pasteurization of factor VIII results in a product which is safe with regard to these viral infections. Furthermore, it was shown that pasteurization does not form new antigenic determinants on the factor VIII molecule and compared with the native product does not alter the physiological properties of this protein in patients. In comparison to these advantageous properties of the pasteurized product a slight loss of coagulant activity seems to be acceptable. This loss of yield, however, does not influence the quality or the amount of factor VIII in the final container used for the therapy of haemophilia A patients. PMID- 3013214 TI - [Antiviral agents. 28. Aliphatic substituted chlordihexylamino-1,3,5-triazine]. AB - The nucleophilic substitution of one chlorine atom in 2,4-dichloro-6 (dihexylamino)-1,3,5-triazine (1) by primary (2a-b) or secondary (2c) amines leads to the aliphatically substituted chloro-dihexylamino-1,3,5-triazines (3a-c) Structures of type 3 may be characterized by the spectroscopic data. In the mass spectra, the complete degradation of the alkyl groupings is striking and manifested in a series of consecutive peaks with differences of 14 between each other. Antiviral activity is exhibited by 3a through an inhibitory effect towards vaccinia virus and by 3b as interferon inductor towards encephalomyocarditis virus. Moreover, 3a and 3b exhibit a strong activity towards Trichomonas vaginalis. Additionally, 3a is capable of exerting temperature lowering and anthelminthic effects. PMID- 3013216 TI - Primary culture of human aortic intima cells as a model for testing antiatherosclerotic drugs. Effects of cyclic AMP, prostaglandins, calcium antagonists, antioxidants, and lipid-lowering agents. AB - Smooth muscle cells isolated from atherosclerotic lesions of human aorta retain in primary culture their intrinsic in vivo characteristics: namely, enhanced proliferative activity and high lipid levels. We have tested the effect of different compounds on [3H]thymidine uptake and on the levels of phospholipids, triglycerides, cholesterol, and cholesteryl esters in cultured aortic cells. Effects, such as the inhibition of cellular proliferation and/or lowering of the intracellular lipid levels which would be regarded as antiatherosclerotic if exerted in vivo, were observed in vitro by the following compounds: dibutyryl cyclic AMP, cholera toxin, forskolin, methylisobutylxanthine, stable prostacyclin analogues, prostaglandins E2 and D2, verapamil, reserpine, alpha-tocopherol, butylated hydroxytoluene, lipostabil, and high density lipoproteins. In this paper, we discuss the possibility of using a primary culture of smooth muscle cells from an atherosclerotic human aorta for testing drugs for possible antiatherosclerotic activity. PMID- 3013215 TI - Retention of antilipemic activity by periodate-oxidized non-anticoagulant heparins. AB - Heparin preparations with different anticoagulant and antilipemic (fat-clearing) activities were oxidized with periodate under conditions of cleavage of all the C(2)-C(3) bonds of non-sulfated uronic acid residues, while preserving the original molecular weight of the polysaccharide. Periodate-oxidised heparins (oxyheparins, O-HEP) and the corresponding borohydride-reduced products (reduced oxyheparins, RO-HEP) were compared with the original heparins for their content in trisulfated disaccharide sequences (as determined by 13C-nuclear magnetic resonance) and in active sites for antithrombin-III (as determined indirectly by affinity chromatography), and for their anticoagulant and antilipemic (lipoprotein lipase-releasing) activities. The drop of anticoagulant activity induced by periodate oxidation was paralleled by a substantial decrease of affinity for antithrombin, and is thought to arise from glycol splitting at the level of the D-glucuronic acid residue that is part of the active site for antithrombin. The trisulfated disaccharide sequences and the associated antilipemic activities were substantially unaffected by periodate oxidation. The residual anticoagulant activity of periodate-oxidized heparins obtained from preparations - such as those from beef lung - rich in trisulfated disaccharide sequences is discussed in terms of the influence of charge density on heparin protease interactions not mediated by antithrombin. PMID- 3013217 TI - Occurrence of anti-HTLV-III antibodies in Danish high-risk homosexuals in 1982-83 -seroconversion rate and risk of AIDS. PMID- 3013218 TI - Diagnosis of HTLV-III infection by ultrastructural examination of germinal centers in lymph node--a case report. AB - Serum from a patient suspected of having AIDS showed positive ELISA tests but the diagnostic p 41 band was absent and the p 24 band was barely discernible on a Western blot. Before another Western blot was performed lentivirions were detected between dendritic reticulum cells in a lymph node biopsy. It is suggested that samples of biopsies of lymph nodes from patients with or at risk for AIDS should be embedded in resin for future ultrastructural study if indicated. PMID- 3013219 TI - Decision analysis of the HTLV-III screening test for blood donors. AB - Recent research has identified the human T-lymphotropic virus type III (HTLV-III) as a probable etiologic agent of the acquired immune deficiency syndrome (AIDS). This has prompted the U.S. Public Health Service to recommend that all blood used for transfusions or in the manufacture of blood products be screened. An enzyme linked immunosorbent assay (ELISA) has been approved for use as a screening test. From the perspective of the low-risk blood donor, however, our analysis indicates that the expected utility of no-testing may exceed that of testing. This is primarily due to the risk of a false-positive test result. It follows that informed low-risk individuals may be hesitant to donate blood. We support the discarding of blood that tests positive on ELISA but, to decrease donor risk, a positive confirmatory test, such as the Western blot, should be considered as necessary before the testing outcome is treated as positive from the donor's perspective. Additionally, individuals should be given the option to donate blood without being told the testing outcome. PMID- 3013220 TI - Relationship of exposure to human T lymphotropic virus and T helper cells in homosexual men. AB - The detection of serum antibodies (Ab) against HTLV-III in individuals with AIDS and related symptoms (ARC) has unambiguously defined the association of the virus infection to AIDS. This study was done to determine the extent of exposure to HTLV-III in homosexual men by measuring (Ab) and relating it to the stage of disease and T cell subsets. We found Ab in 89.5% of the 492 men with the median titers by stage of disease being 1600 for symptom-free, 6400 for ARC or Kaposi's sarcoma, and 4800 for opportunistic infection (OI), respectively. There was no correlation between Ab titers and either absolute or relative T helper cells (T4+), even though these cells decreased with disease severity. More specifically, however, symptom free patients had a normal distribution of the helpers of suppression (T4+/Leu8+), whereas, in symptomatic men, there was a significant decrease suggesting that the target cell for the virus is a subpopulation of the T helper cell. PMID- 3013221 TI - Antibodies to AIDS-associated retrovirus (HTLV-III/LAV) in drug addicts from Vizcaya, northern Spain. AB - Serum samples from 313 asymptomatic intravenous (IV) drug users from Bilbao (Vizcaya, Vasque Country, Spain) were tested for antibodies to HTLV-III/LAV virus, the probable etiologic agent of the acquired immune deficiency syndrome (AIDS). Viral antibodies were assayed by ELISA test. 41.9% of the sera gave positive reactions. No seropositivity was detected among 22 normal blood donors, 58 chronic alcoholics, or 20 members of the Drug Control Center personnel. Virus specific reactions were confirmed by indirect immunofluorescence using an HTLV III/LAV producer cell line, and by Western blotting. 55% of the ELISA-positive sera were also positive in Western blot assay. No differences in seropositivity by age or sex were observed but it increased with the period of parenteral drug use. Presence of antibody statistically correlated with the frequency of syringe sharing, confirming the transmission of viral infection by blood products. Altered T4/T8 ratios and lower number of T4 positive lymphocytes were detected among HTLV-III/LAV positive drug addicts. PMID- 3013222 TI - Surgical considerations in the diagnosis and management of AIDS. PMID- 3013223 TI - Antibodies to acquired immune deficiency syndrome (AIDS)-associated virus (HTLV III/LAV) in Venezuelan populations. AB - Serum samples from 850 individuals from Venezuela were tested for the presence of antibodies to HTLV-III/LAV virus, the probable etiological agent of acquired immune deficiency syndrome (AIDS). At the time of the study, none of the individuals tested had symptoms indicative of AIDS or related disorders. Viral antibodies were assayed by indirect immunofluorescence (IF) assay, using a chronically infected, HTLV-III/LAV producer cell line CEM/LAV-NIT established in our laboratory. Twenty individuals (2.5%), 8 of them (40%) female, were seropositive by IF and by confirmatory Western blotting and radioimmunoprecipitation assays. The seropositivity rate ranged from 2.4% (11 of 465) in the general healthy population, 4% (2 of 50) among patients with Chagas' disease, and up to 29.2% (7 of 24) among patients with acute malaria infection. The titers of HTLV-III/LAV antibodies ranged from 1:40 to 1:640. In addition, 2 of 36 patients with hemophilia A (5.5%) also had antibodies to HTLV-III/LAV. Two of 7 patients with acute malaria had specific antibodies both to HTLV-III/LAV and HTLV-I, as determined by IF and Western blotting. None of over 169 randomly chosen, healthy blood donors from seven major Venezuelan cities, as well as none of 99 patients with leukemia/lymphoma, had antibodies to HTLV-III/LAV. The presence of specific antibodies among various Venezuelan populations indicates that HTLV-III/LAV, or a closely related cross-reactive virus, is indigenous in Latin American subjects as was previously indicated for tropical populations of central Africa. Isolation and characterization of this virus will help to understand the origin and etiology of AIDS. PMID- 3013224 TI - Determination of antibodies against LAV/HTLV III: comparative evaluation of four different commercial test kits. AB - Four different, commercially available ELISA tests for the detection of antibodies against LAV/HTLV III were evaluated for specificity and sensitivity. The relative specificity of the kits was determined by investigating a test panel of 76 sera collected from asymptomatic or symptomatic homosexual men. Completely concordant results were obtained for sera from asymptomatic male homosexuals (11% positive for Anti-LAV/HTLV III) or from patients with the AIDS-related complex (91% positive for Anti-LAV/HTLV III). Differences between the ELISA test kits, however, were observed with sera obtained from patients with AIDS. While Anti LAV/HTLV III was detected by the Abbott, Electro-Nucleonics, and Organon test in all 17 sera from AIDS patients, the Pasteur test failed to detect Anti-LAV/HTLV III in 7 consecutive sera from an individual patient with late-stage AIDS. The relative sensitivity of the ELISA tests was determined by endpoint titration of confirmed Anti-LAV/HTLV III positive sera from donors of different risk groups for AIDS. The titration experiments demonstrated that the Abbott test clearly was the most sensitive of the ELISA tests studied, followed by the Electro Nucleonics, Pasteur, and Organon test. The results further indicate that most of the differences of specificity and sensitivity observed between the Anti-LAV/HTLV III tests could be abolished by a modified definition of minimum positive absorbance values. PMID- 3013225 TI - [Value of the cytological examination of the bronchoalveolar lavage fluid in patients with acquired immunodeficiency syndrome and related syndromes]. AB - In AIDS a variety of severe pulmonary disorders may occur. The authors report 110 cases of bronchoalveolar lavage (BAL) in 43 AIDS and 41 ARC. In AIDS P. carinii pneumonia is the major cause of respiratory illness. BAL alone is a safe and valuable tool for diagnosis of P. carinii pneumonia and others opportunistic infections. Moreover, pulmonary hemorrhage diagnosed by the finding of hemosiderin laden macrophages, is very suggestive of broncho-pulmonary Kaposi' sarcoma. Finally, BAL demonstrates a severe depletion of T4 lymphocytes and an increased number of T8 lymphocytes. The T8 lymphocytosis is observed whatever the pulmonary involvement (nonspecific alveolitis, opportunistic infections, Kaposi's sarcoma), and is also found in ARC, and lymphocytosis, open lung biopsy shows a lymphoid interstitial infiltration with respect of the alveolar septa, thus differing from the classical lymphoid interstitial pneumonia described by Carrington. The prognosis of lymphocytosis in ARC remains unknown. PMID- 3013227 TI - [Disseminated histoplasmosis caused by Histoplasma capsulatum and sarcoidosis. Apropos of a case]. AB - Histoplasma capsulatum can, sometimes, simulate sarcoidosis, clinically and pathologically. A case is reported in which disseminated histoplasmosis is proved only by fungus granulomatous tissue. Another similar cases of the literature incite to propose the practice of special stains, above all Gomori-Grocott technical, before sarcoid-like lesions. Lesions pathogeny was discussed. In our case it's not possible to eliminate the role of primary sarcoidosis in disseminated histoplasmosis. PMID- 3013226 TI - [Immunohistochemical localization of angiotensin converting enzyme in epithelioid sarcoidosis granulomas]. AB - An immunohistochemical study of the localization of Angiotensin one Converting Enzyme in sarcoidosis granulomas was made in 20 cases of sarcoidosis. Immunoreactive Angiotensin one Converting Enzyme was found in 92% of these cases. At the cellular level the staining was observed on the plasma membrane. No correlation was observed between the serum Angiotensin one Converting Enzyme value and the immunostaining intensity. PMID- 3013229 TI - The AIDS epidemic. PMID- 3013228 TI - Histopathological changes resembling human immunodepressive conditions observed in sheep inoculated with leucocytes from B.L.V. infected cattle. PMID- 3013230 TI - Diet and chemoprevention in NCI's research strategy to achieve national cancer control objectives. PMID- 3013231 TI - The role of the nuclear matrix in the organization and function of DNA. PMID- 3013232 TI - Citrate synthase: structure, control, and mechanism. PMID- 3013233 TI - Neurological involvement in systemic lupus erythematosus: immunological and clinical aspects. PMID- 3013234 TI - The periaqueductal gray: a prerequisite for ACTH-induced excessive grooming. AB - The periaqueductal gray is known to be involved in the expression of a variety of behaviours such as aggression, beta-endorphin-induced immobility and peptide induced excessive grooming. In order to establish whether the periaqueductal gray (PAG) is indispensible for peptide-induced excessive grooming, lesions were placed in the dorsal part of this structure. Subsequently, the grooming-inducing abilities of adrenocorticotropin (ACTH), beta-endorphin and bombesin were tested. The lesioned animals did not display excessive grooming after intracerebroventricular injection of ACTH. beta-Endorphin administration into the lesioned animals resulted in an extreme display of immobility. Local injection of bombesin into the PAG resulted in reduced scratching behaviour followed by immobility. It was hypothesized that excessive grooming (elicited by ACTH) may be mediated through a non-opioid primary target site-situated in the lesioned region of the PAG-while excessive scratching and immobility (elicited by bombesin or beta-endorphin, respectively) may be mediated through an opioid primary target site (situated in the remaining part of the PAG). Furthermore, the analysis of social behaviour of lesioned animals revealed that these animals reacted towards an unfamiliar partner predominantly with freezing behaviour. The increase of beta endorphin-induced immobility and socially induced freezing (which is morphologically very similar to beta-endorphin-induced immobility) in lesioned animals supports the hypothesis that the release of opioid peptides such as beta endorphin in the PAG plays a role in the regulation of social behaviour. PMID- 3013235 TI - Nucleoside phosphotransferase from malt sprouts. I. Isolation, characterization and specificity of the enzyme. AB - The nucleoside phosphotransferase of barley was isolated in four steps from dried malt sprouts and was obtained homogeneous according to several criteria. The enzyme transfers the phospho group from the 3'- or 5'-position of a nucleotide to the 5'-OH group of another nucleoside. Activated phosphomonoesters (e.g. 4 nitrophenyl phosphate) can be used as donors as well. In the absence of nucleosides the enzyme shows pronounced phosphatase activity towards 3'- and 5' nucleotides as well as towards activated phosphomonoesters. 2'-Nucleotides are not hydrolysed. Studies of the phosphatase activity with a number of modified nucleotides demonstrate that the nucleotide substrates are bound by the phosphate group and by the base. The base can be extensively modified compared to naturally occurring nucleobases. Obviously, only a hydrophobic interaction with the enzyme is required; base-specific hydrogen bonds are of no importance. In the transferase reaction the acceptor nucleoside interacts only with the base which has a much more restricted specificity. Nucleotides which are donors do not need to be acceptors when they are nucleosides, while all acceptor nucleosides are efficient donors. Due to the pre-orientation of the base, the phosphate groups of the 3'- and 5'-nucleotides are in different positions relative to the groups of the enzyme that are involved in the catalysis. This is reflected in the kinetic parameters which are interpreted on the basis of a mechanism with a phospho intermediate as postulated for the enzyme of Ives et al. [(1979) J. Biol. Chem. 254, 4339; (1982) J. Biol. Chem. 257, 4931]. Evidence is obtained that Km represents the ratio of the rate constants for the decay and the formation of the intermediate and that the kcat/Km-value can be used as indicator for the formation step. PMID- 3013236 TI - Nucleoside phosphotransferase from malt sprouts. II. Studies on the active site and the phospho-intermediate. AB - The phospho-intermediate formed in the reaction of the nucleoside phosphotransferase is an acyl phosphate, the phosphorus bound to the gamma carboxylate group of a glutamic acid. Reduction of this intermediate with sodium cyanoborotritiide yields labeled 2-amino-5-hydroxyvaleric acid after hydrolysis of the protein. Nucleophilic trapping of the intermediate with hydroxylamine during the reaction with substrates leads to N-phosphohydroxylamine, which is the only reaction product at a higher concentration of hydroxylamine. Evidence is obtained from modification experiments that in addition to the carboxylate group a histidine is involved in the reaction. The pKa-value for the histidine derived from the photoinactivation of the enzyme is 7.6, indicating that this group forms a salt bridge to a carboxylate group, probably that group attacking the phosphorus. The acceptor nucleosides are bound only by hydrophobic interactions of the base, a conclusion obtained from fluorescence studies and quenching experiments. The hydrophobic interaction obviously does not involve pi interactions to tyrosine and tryptophan residues, since their fluorescence is not affected by addition of nucleotide inhibitors. Modification of these residues leads only to unspecific inactivation. From the Scatchard plot of the titration of the enzyme with 1,N6-ethenoadenosine 5'-phosphoramidate, an efficient inhibitor (Kd = 1.2 X 10(-5) M), it can be concluded that there is only one binding site on the dimeric enzyme. PMID- 3013237 TI - Nucleoside phosphotransferase from malt sprouts. III. Studies on metal ion requirement, solvent effects, kinetics and reaction mechanism. AB - The nucleoside phosphotransferase from malt sprouts contains one Mg2 per dimeric enzyme molecule. This cation can be removed by EDTA, while other chelating agents are less efficient. The metal-free apoenzyme is inactive. Activity can be restored to its initial value by Mg2 or Co2 and to a minor extent by Mn2, Zn2, Cu2, Ni2 and Fe2. Thermal stability is reduced when the metal is removed but can be restored by addition of Mg2. Adenosine 3',5'-phosphate (cAMP) and arsenate, strong competitive inhibitors, reduce the rate of inactivation by EDTA considerably but do not reduce the rate for the reconstitution with Mg2. We therefore conclude that the phosphate group interacts electrostatically with Mg2 and that the inhibitor is not bound to the apoenzyme. The Sp-isomer of adenosine 3',5'-thionophosphate is a hundred times stronger inhibitor than the Rp-isomer and ten times stronger than cAMP; this allowed us to determine the relative position of the Mg2 in the active site. The imidazolium cation, previously detected as an essential part of the active site, obviously forms a salt bridge to the carboxylate group which attacks the phosphorus opposite to the leaving alcohol group. This conclusion is derived from the fact that organic solvents increase the rate of formation of the phospho-intermediate considerably, while higher concentrations of salt decrease it strongly. In addition, the imidazolium cation seems to polarize the P = O bond and to stabilize the negative charge at the phosphoryl oxygen in the pentacoordinate transition state; this is followed from the pH-dependence of the hydrolysis reaction. The kinetic results reveal that Km does not represent a binding equilibrium, but is the ratio of the rates for decay and formation of the covalent intermediate, while kcat/Km is an indicator for the formation step of the acyl phosphate. On the basis of all these informations it should be allowed to formulate a reaction mechanism, in which all steps of the transferase reaction have to be microscopically reversible. PMID- 3013238 TI - Isoleucyl-tRNA synthetase from baker's yeast. Influence of substrate concentrations on aminoacylation pathways, discrimination between tRNAIle and tRNAVal, and between isoleucine and valine. AB - The influence of substrate concentrations on aminoacylation pathways and substrate specificities was investigated in the acylation reaction catalyzed by isoleucyl-tRNA synthetase from yeast. For the cognate substrates isoleucine and tRNAIle two Km values each differing by a factor about five were determined; the higher values were observed at concentrations higher than 1 microM, the lower values below 1 microM isoleucine or tRNAIle, respectively. At substrate concentrations below 1 microM also kcat values of the isoleucylation reaction are lowered. With the noncognate substrates valine and tRNAVal such differences could not be detected. The substrate ATP did not show any change of its Km value as far as the reaction was measurable. Under six different new assay conditions orders of substrate addition and product release followed sixtimes a sequential ordered ter-ter steady-state mechanism with ATP as the first substrate to be added, isoleucine as the second, and tRNAIle as the third one; pyrophosphate is the first product to be released, isoleucyl-tRNA the second, and AMP the third one. In one case this mechanism was modified by a rapid equilibrium segment for addition of ATP and isoleucine. From kcat and Km values and from AMP formation rates discrimination factors for discrimination between tRNAIleII and tRNAValI as well as between isoleucine and valine were determined. In the first case discrimination factors can vary up to a factor of thirty by changes of tRNA or amino-acid concentrations, in the second case discrimination factors are practically invariant. The two different Km values are hypothetically explained by assumption of anticooperativity in a flip-flop mechanism. Two hypothetical catalytic cycles are postulated. PMID- 3013239 TI - Cyanylation of 3-hydroxybutyrate dehydrogenase. The "essential" sulfhydryl group is not involved in catalysis. AB - 3-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with an absolute requirement of lecithin for function. The enzyme contains two sulfhydryl groups per monomer. Modification of the more reactive sulfhydryl group with N ethylmaleimide resulted in inactivation of the enzyme and modification of coenzyme-binding characteristics [McIntyre, J. O., Fleer, E. A. M. and Fleischer, S. (1984) Biochemistry 23, 5135-5141]. The present study further investigates the function of the sulfhydryl groups by utilizing chemical derivatization techniques. The reactive sulfhydryl was derivatized first with 3,3'-dithiobis(6 nitrobenzoic acid) (Ellman's reagent) to form the S-(carboxynitrophenylthio) derivative which could then be replaced with cyanide to form the S-cyanylated enzyme. We find that derivatizing the essential sulfhydryl group leads to some loss of activity. The effect appears to be steric since a larger derivatizing group gives greater loss of activity. The normal enzyme is inhibited approximately 50% in excess substrate. Derivatization of the reactive sulfhydryl group results in loss of this substrate inhibition, the modified enzyme being at least three-fold more active at high substrate concentrations; the activity increases from 18% to 54% and from 1% to 4% of maximal activity for the S cyanylated and S-(carboxynitrophenylthio) enzyme derivatives, respectively. Cyanylation results in complete loss of fluorescence energy transfer from tryptophan to NADH at low salt concentration but is normal in the presence of 100mM NaCl. However, the binding constant of the coenzyme is decreased only several-fold in the cyanylated enzyme as studied by fluorescence quenching. The cyanylated enzyme formed tight ternary complexes (spin-labeled NADH monomethylmalonate) (spin-labeled NAD-sulfite) similar to that formed by the normal enzyme. The spin label is highly immobilized, but the hyperfine splitting values differ somewhat from the normal enzyme. We conclude that the reactive sulfhydryl is close to the active site of 3-hydroxybutyrate dehydrogenase but is not involved in the catalytic mechanism. PMID- 3013240 TI - Salivary gland tumors. Fine-needle aspiration vs frozen-section diagnosis. AB - We examined the relative accuracy of fine-needle aspiration biopsy (FNAB) and frozen section (FS) in the diagnosis of salivary gland tumors; FNAB completely and accurately diagnosed 35 (88%) of 40 cases, including ten (100%) of ten nonneoplastic lesions, 20 (87%) of 23 benign, and five (71%) of seven malignant tumors. No complications were encountered with this procedure. These results compare favorably with previously published reports. Twenty-one of 40 tumors diagnosed by FNAB and FS at surgery. Sixteen (76%) of 21 of these were correctly diagnosed by FNAB, and 15 (71%) of 21 by FS. Cystic lesions gave the most diagnostic difficulties both on FNAB and FS. Worldwide, FNAB has been demonstrated to be a cost-effective, accurate, and safe procedure. Furthermore, the use of FNAB allows for better preoperative management and overall treatment planning. PMID- 3013241 TI - Shave excision and dermabrasion of midline angiofibroma in tuberous sclerosis. AB - We encountered a rare case of massive angiofibroma involving the midface. Treatment consisted of shave excision of long slightly vascular "shag-rug" papules, followed by repeated deep dermabrasion. The methods of treatment will vary, depending on size and vascularity of the lesions. Indications for treatment include facial hygiene, cosmetic reasons, and the cessation of chronic bleeding. Prognosis is that the angiofibroma may be expected to slowly recur and thus require further treatment. PMID- 3013242 TI - An immunochemical study of Neurospora nucleases. AB - Nucleases derived from Neurospora crassa mycelia with neutral single-strand (ss) endodeoxyribonuclease activity have been examined by immunochemical techniques and by sodium dodecyl sulfate - DNA gel electrophoresis. All of the intracellular nucleases, which have different divalent metal ion requirements, different strand specificities with single- and double-strand DNA, different modes of action on DNA and RNA, and other distinguishing characteristics, are immunochemically related to Neurospora endo-exonuclease. The evidence indicates that these enzymes are derived from one or more related large, inactive (precursor?) polypeptides that are first converted to 75- to 80-kdalton active polypeptide(s) which are very protease sensitive. Further limited proteolysis results in the production of the various active forms of nuclease studied here. Some proteolytic conversions may occur in a controlled manner in vivo in different cell compartments, but others are very likely artifacts resulting from uncontrolled proteolysis during extraction and isolation. The intracellular forms of Neurospora endo-exonuclease are immunologically cross-active with ss-DNA-binding nucleases isolated from Aspergillus nidulans and Saccharomyces cerevisiae. They are not immunochemically related to two extracellular Neurospora nucleases, the pancreatic DNase-I-like DNase A and a ss-specific exonuclease, and they are also not related to other fungal and plant nucleases with ss-specific endonuclease activity such as the S1 nuclease of Aspergillus oryzae, the P1 nuclease of Penicillium citrinum, and mung bean nuclease. PMID- 3013243 TI - Metabolism of lysopolyphosphoinositides by rat brain and liver microsomes. AB - Lysophosphatidylinositol 4,5-bisphosphate has been reported to form ion conducting channels in artificial membranes. If formed in vivo, mechanisms for its removal from cellular membranes would be required. Thus, possible pathways were explored in rat brain and liver microsomes. Since neither lysophosphatidylinositol 4-phosphate nor lysophosphatidylinositol 4,5 bisphosphate were acylated in experiments with [3H]arachidonic acid or [14C]oleoyl CoA, polyphosphoinositides do not participate directly in a deacylation-reacylation cycle as proposed for the postsynthesis enrichment of phosphatidylinositol with arachidonic acid. Similar enrichment in polyphosphoinositides can occur only via the rapid phosphorylation dephosphorylation cycle linking all three phosphoinositides. Lysophosphatidyl[2 3H]inositol 4,5-bisphosphate and lysophosphatidyl[2-3H]inositol 4-phosphate were rapidly dephosphorylated to 1-acyl-sn-glycero(3)phospho(1)-D-myo-inositol by microsomes from both tissues. Appearance of only trace quantities of radioactive lysophosphatidylinositol monophosphate during the catabolism of lysophosphatidyl[2-3H]inositol 4,5-bisphosphate indicated that the second dephosphorylation step, which was cation independent, was at least as fast as the first step which required Mg2+. In the presence of ATP, CoA, and arachidonic acid, the lysophosphatidylinositol was converted to phosphatidylinositol. This acylation reaction was rate limiting in brain microsomes. Dephosphorylation of lysophosphatidylinositol 4,5-bisphosphate was rate limiting in liver microsomes. Neither the lysopolyphosphoinositides nor the lysophosphatidylinositol produced from them in the reactions were degraded by acyl hydrolases or phosphodiesterases in microsomes from either tissue. Therefore, any lysopolyphosphoinositide formed in vivo would probably be removed by dephosphorylation and recycled to phosphatidylinositol. PMID- 3013244 TI - Effect of saturated phosphatidylcholines on the functional properties of reconstituted cytochrome oxidase. AB - Cytochrome oxidase was incorporated into liposomes, at various protein/lipid ratios, composed of either a phosphatidylcholine of varying chain length and symmetry or asolectin. Catalytic activity and respiratory control were assayed at two temperatures. All preparations showed higher activity at low protein/lipid ratios, but only asolectin showed respiratory control. A spectroscopic determination of the vectorial orientation of oxidase molecules showed that, for proteoliposomes with saturated lipids, 100% of oxidase molecules could be reduced by external substrate as compared with 75% for asolectin proteoliposomes. Freeze fracture electron microscopy confirmed that oxidase was incorporated into these proteoliposomes and differential scanning calorimetry indicated that the protein induces significant disruption in the long range packing of the saturated phospholipids. We propose that the oxidase molecules in proteoliposomes formed from saturated phosphatidylcholines do not display respiratory control because they are unable to assume the transmembrane orientation necessary for full vectorial activity. PMID- 3013245 TI - Inhibition of protein synthesis enhances transformation of primary cells by viral DNA. AB - Inhibition of protein synthesis by cycloheximide after transfection and subsequent removal of the drug increased the transformation efficiency of primary cells by plasmids containing the left 4.5, 6.7, or 16% of the adenovirus (Ad) genome. The enhancement factor ranged from 2 to as much as 70 depending on the size of the viral DNA fragments used. Addition of cycloheximide before or at the time of transfection inhibited transformation, suggesting that viral protein synthesis is important during the early phase of transformation. Transient expression assays showed that cells treated with cycloheximide post-transfection contained as much as three times the amount of viral RNA transcribed from regions E1A and E1B. Conversion of a rat cell line lacking thymidine kinase activity (TK ) to the TK+ phenotype by a plasmid containing the herpes TK gene was severely inhibited by the drug treatment, suggesting that the enhancement effects of cycloheximide on transformation may be specific for Ad DNA. Cycloheximide treatment also increased the number of transformants induced by a transformation defective E1B mutant of Ad12 (cyt mutant). Plasmid containing only the E1A region of Ad12 transformed primary rat kidney cells with very low efficiency. The inclusion of E1B in the transfecting DNA fragments increased the transformation frequency by more than 400-fold, much higher than that achieved by cycloheximide. Thus, cycloheximide cannot replace E1B functions in transformation efficiency. PMID- 3013246 TI - Enhancer-facilitated expression of prokaryotic and eukaryotic genes using human histone gene 5' regulatory sequences. AB - We examined the structural and functional properties of a human H3 histone gene promoter. The complete nucleotide sequence of an H3 structural gene and 515 nucleotides of 5' and 100 nucleotides of 3' flanking sequences were determined. The upstream region of this cell cycle dependent H3 histone gene, designated pST519, contains consensus sequences typical of genes transcribed by RNA polymerase II. To address promoter function directly, we determined the capability of the 5' flanking sequences to direct the transcription of two genes which are not functionally or structurally related. Fusion genes were constructed using the 5' flanking sequences of this human H3 histone gene and either human beta-globin or bacterial chloramphenicol acetyltransferase (CAT) coding sequences. Both of these fusion genes were expressed when transfected into HeLa cells. Under control of the pST519 histone gene promoter, a beta-globin mRNA transcript was initiated at the appropriate H3 (bp) enhancer, inserted upstream from the histone promoter in both fusion constructs, increased levels of beta globin and CAT expression. Expression of the pST519 H3 histone gene in COS cells in the absence of the SV40 72-bp enhancer confirmed that the sequences required for promoting transcription reside within the 750-bp 5' flanking sequences and that the exogenous enhancer facilitates, but is not a prerequisite for, transcription. Enhancer-facilitated expression of a cell cycle dependent human H4 histone gene was also observed following transfection into mouse L cells and indicates that the regulatory sequences of human histone genes and transcription factors of mouse cells are compatible. PMID- 3013249 TI - Electron paramagnetic resonance studies of heme c and its nitrosyl derivative in Vibrio (Achromobacter) fischeri nitrite reductase. AB - Interactions of Vibrio (formerly Achromobacter) fischeri nitrite reductase were studied by electron paramagnetic resonance spectroscopy. The spectrum of the oxidized enzyme showed a number of features which were attributed to two low-spin ferric hemes. These comprised an unusual derivative peak at g = 3.7 and a spectrum at g = 2.88, 2.26, and 1.51. Neither heme was reactive in the oxidized state with the substrate nitrite and with cyanide and azide. When frozen under turnover conditions (i.e., reduction in the presence of excess nitrite), the enzyme showed the spectrum of a nitrosyl heme derivative. The g = 2.88, 2.26, and 1.51 signals reappeared partially on reoxidation by nitrite, indicating that the nitrosyl species which remained arose from the g = 3.7 heme. The nitrosyl derivative showed a 14N nuclear hyperfine splitting, Az = 1.65 mT. The nitrosyl derivative was produced by treatment of the oxidized nitrite reductase with nitric oxide or hydroxylamine. Exchange of nitric oxide between the nitrosyl derivative and NO gas in solution was observed by using the [15N]nitrosyl compound. A possible reaction cycle for the enzyme is discussed, which involves reduction of the enzyme followed by binding of nitrite to one heme and formation of the nitrosyl intermediate. PMID- 3013248 TI - Purification and characterization of a gelatinase produced by fibroblasts from human gingiva. AB - An endopeptidase which digests denatured collagen to small, dialysable fragments was purified 2675-fold from medium that had been conditioned by the culture of fibroblasts grown from explants of human gingiva. This enzyme was inhibited by chelating agents, but not by phenylmethylsulphonyl fluoride nor by N ethylmaleimide, and is therefore probably a metalloproteinase. It showed no demonstrable activity against native collagen or ovalbumin, while alpha-casein was digested slowly, if at all. It therefore belongs to the group of enzymes which have been called tissue gelatinases. This gelatinase was secreted in a latent form or forms and could be activated by proteolysis with trypsin. The active enzyme had an apparent molecular weight of 69 000 (gel chromatography) or 72 000 (gel electrophoresis in sodium dodecyl sulphate) and an apparent isoelectric point of 4.15. PMID- 3013247 TI - Production of an antibody against rat liver nuclear T3 receptor. AB - Rabbits were immunized with rat liver nuclear L-triiodothyronine (T3) receptor purified by preparative sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis using bromoacetyl[125I]T3 as an affinity label. SDS-PAGE confirmed the presence of two receptor forms of the apparent molecular weights 57,000 and 45,000. We describe here a specific antibody, raised against the 57,000 receptor type, which reacts with both receptor forms as assessed by electroimmunoblotting and immunoprecipitation in liquid medium. PMID- 3013250 TI - Effects of fatty acids on phosphatidylcholine biosynthesis in isolated hamster heart. AB - The effects of stearic, oleic, and arachidonic acids on phosphatidylcholine biosynthesis in the hamster heart were investigated. When hamster hearts were perfused with labelled choline in the presence of fatty acids, biosynthesis of phosphatidylcholine was stimulated only by stearic acid. Stearic acid was found to accumulate in unesterified (free) form in the hamster heart after perfusion. The stimulation by stearic acid was mediated in vivo by an enhancement of CTP:phosphocholine cytidylyltransferase activity in the microsomal fraction of the hamster heart and the enzyme activity in the cytosolic fraction was not affected. In contrast with the observations in rat hepatocytes, cytidylyltransferase from the hamster heart was not stimulated directly by stearic acid. The selective activation of the microsomal enzyme when the heart was perfused with stearic acid suggests that activation of the enzyme was mediated via the modification of the membrane by stearic acid. PMID- 3013252 TI - [Expression in human cells of the gene for glycoprotein B from herpes simplex virus inserted in a eukaryotic vector]. PMID- 3013251 TI - [Plasma levels of beta-endorphin and ACTH during critical increase in arterial pressure in man]. PMID- 3013253 TI - Subcellular distribution of nucleoside phosphotransferase. Some properties of the enzyme purified from nuclear fraction of chick brain. PMID- 3013254 TI - [Purification and partial molecular characterization of peroxidases in the rat intestine (EC 1.11.1.7)]. PMID- 3013255 TI - [Hypothesis on the probable mechanism of auto-limitation of acute attacks of gout]. PMID- 3013256 TI - [Response of polymorphonuclear neutrophils to monosodium urate crystals: production of oxygen free radicals]. PMID- 3013257 TI - Effects of abnormal metabolites of maple syrup urine disease on neurotransmitter receptor binding. AB - The deficiency of keto acid decarboxylase in maple syrup urine disease results in the accumulation of branched chain amino acids and their corresponding keto acids in tissues and body fluids. The effects of abnormal metabolites were investigated on neurotransmitter receptor binding in rat brain. alpha-Keto acids caused selective in vitro decrease in alpha-adrenergic, beta-adrenergic receptor binding in synaptosomal preparations from rat brain. No significant changes were observed in binding of cholinergic, GABA, and dopamine receptors binding in appropriate rat brain preparations. These results indicate that selective inhibition of adrenergic receptor binding by branched chain keto acids may presumably account for neural abnormality in maple syrup urine disease. PMID- 3013258 TI - Aflatoxin inhibition of rat liver mitochondrial cytochrome oxidase activity. AB - Aflatoxins B1, B2, G1, G2, and M1 have been evaluated for activity toward cytochrome oxidase in isolated rat liver mitochondria employing ferrocytochrome c and p-phenylene diamine as reductants. The aflatoxins inhibited the cytochrome oxidase activity to a greater extent when monitored by O2 uptake measurements than by substrate oxidation. AFG2 and AFM1 were the most potent (50-70%). Using oligomycin and 2,4-DNP as respiratory inhibitor and uncoupler, respectively, the aflatoxins appear to inhibit e- rather than energy transfer reactions. These toxins did not uncouple cytochrome oxidase activity. PMID- 3013259 TI - Postanoxic recovery of spontaneously beating isolated atria: pH related role of adenylate kinase. AB - The functional activity, adenine nucleotides, and creatine phosphate content of spontaneously beating isolated rabbit atria were measured prior to anoxia, after 1 hr anoxia, and at the end of 1 hr reoxygenation at pH 6.7 and 7.2 During anoxia at pH 7.2 there was 13.3% loss of adenine nucleotides pool, 35.2% loss of ATP, 36.2% increase in ADP, 200% increase in AMP, and a decrease to 8.8% of CP assayed to the beating atria in oxygen. At pH 6.7 there was almost the same decrease in CP, about 10% decrease in ATP, no change in total adenine nucleotides, no change in AMP and a higher increase in ADP (88.7%). The postanoxic recovery was much more complete when the pH was 6.7 during anoxia, and the first 40 min of reoxygenation. The extent of recovery of functional activity correlated well with the level of ATP in all cases not CP. Since the adenylate kinase and ATPase activity both decrease at acidic pH, their combined diminution would tend to preserve the adenine nucleotide pool and thus the better recovery at pH 6.7, because of a decrease in energy demand and unavailability of AMP for the degradation process. This study also supports the notion of compartmented adenine nucleotides connected by the creatine phosphate-creatine energy shuttle. PMID- 3013260 TI - [A case of congenital hypomyelination polyneuropathy]. PMID- 3013261 TI - [Hypercoagulability and cerebral infarction]. PMID- 3013262 TI - [In vivo measurements of regional cerebral hemocirculation and metabolism in gliomas using 15O and 18F-fluorodeoxyglucose positron emission tomography]. AB - A high resolution PET-HEADTOME III has been employed to quantitate regional cerebral hemodynamics and metabolism in gliomas using O-15 and 18FDG tracers, and to evaluate histological malignancy preoperatively. Hemodynamic and metabolic indices of regional cerebral blood flow (rCBF), cerebral blood volume (rCBV), oxygen consumption (rCMRO2), oxygen extraction (rOEF), and glucose consumption (rCMRGl) were studied on thirteen preoperative gliomas involving one recurrent case. Regions of interest (ROIs) on PET were placed over the corresponding lesions in CT scans. In high grade gliomas, ROIs were also focused on the corresponding parts of central low density area, contrast enhanced area, and peritumoral low density area. rCBF and rCBV were variable and unrelated in both low and high grade gliomas. rCMRGl values were 5.99 +/- 3.99 mg/100 ml/min for high grade gliomas, and 3.01 +/- 0.48 mg/100 ml/min for low grade gliomas. Lesions with high uptake of radioisotope were proved to be high grade gliomas. lesions with low uptake being low grade gliomas. In comparison with the contralateral gray matter around the sylvian fissure, rCBV was significantly higher(p less than 0.02) in high grade gliomas, and rCMRGl was lower (p less than 0.05) in low grade gliomas. rCMRO2 and rOEF values were reduced markedly (p less than 0.01) in both low and high grade gliomas. These results support that anaerobic glycolysis increased with malignancy in the metabolism of gliomas. The hemodynamics and metabolism of central low density area and peritumoral low density area reflect the pathophysiological state of necrosis and edema respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013263 TI - [Flow cytometric analysis of the effect of cAMP on the cell cycle of the 9 L rat glioma in vitro]. AB - Perturbed cell kinetics in 9 L rat brain tumor treated with dibutyryl cyclic AMP (DBcAMP) and theophylline were studied. The methods include a procedure for simultaneous flow cytometric measurements of cellular DNA and amount of bromodeoxyuridine (BrdU) incorporated into cellular DNA and a procedure for flow cytometric measurements of cellular DNA and RNA. Propidium iodide and monoclonal antibody against BrdU were used as a fluorescent probe for total cellular DNA and for BrdU incorporated into cellular DNA, respectively. For analysis of cellular DNA and RNA, acridine orange staining was also applied. The results were as following; cells in S and G2 phase at the time of drug administration (1 mM, each) can undergo mitosis even though a considerable prolongation of G2 phase was apparent. However, cells in G1 at the time of drug administration were arrested in that phase, whereas those cells in S or G2 were able to complete one mitosis before becoming arrest in the G1 phase. These results have been inferred from the study by autographic method with 3H-thymidine. However, using these new methods, these were clearly demonstrated and furthermore, flow cytometric analysis with acridine orange revealed that DBcAMP induced 9 L cells into resting position (G0 cells). PMID- 3013264 TI - Reversible renal failure after combined treatment with enalapril and frusemide in a patient with congestive heart failure. AB - A patient with congestive heart failure and moderate renal insufficiency developed severe reversible non-oliguric renal failure while on frusemide and enalapril. Renal failure developed when enalapril was given in the presence of pronounced sodium depletion. When positive sodium balance was restored the plasma creatinine concentration began to fall while angiotensin converting enzyme inhibition remained effective and blood pressure was stable. These observations suggest that the degree of sodium depletion plays an important role in the tendency for angiotensin converting enzyme inhibitors to induce renal failure in patients with congestive heart failure and moderate renal insufficiency. Restoration of a positive sodium balance promotes the recovery of renal function after the combined administration of angiotensin converting enzyme inhibitors and diuretics. PMID- 3013265 TI - Enalapril in moderate to severe hypertension: a comparison with atenolol. AB - Patients with moderate to severe essential hypertension (mean untreated supine blood pressure 190/112 mm Hg) received once daily enalapril 20-40 mg or atenolol 50-100 mg, supplemented if required by hydrochlorothiazide 25-100 mg, in a randomized observer-blind trial. Both regimens produced a highly significant reduction in supine and standing blood pressure. There was no significant difference in the antihypertensive effects of enalapril and atenolol when they were used as monotherapy. After hydrochlorothiazide was added to patients not achieving 'target' blood pressure, the fall in systolic pressure was significantly greater in the enalapril group than in the atenolol group, despite similar dosage of hydrochlorothiazide in the two groups. At the end of 6 months' treatment, a supine diastolic blood pressure of 90 mm Hg or below was achieved in 74% of patients on enalapril plus hydrochlorothiazide and 56% of patients on atenolol plus hydrochlorothiazide. This difference was not statistically significant. A small rise in plasma urea and creatinine was observed in the enalapril group and a small rise of urea only in the atenolol group. These changes were statistically significant but of uncertain clinical importance. This study confirms that once daily enalapril and atenolol, both alone and in combination with hydrochlorothiazide, are effective drugs in the management of moderate to severe hypertension. PMID- 3013266 TI - Smoking and lung cancer with special regard to type of smoking and type of cancer. A case-control study in north Sweden. AB - The aetiologic role of tobacco smoking was elucidated in a case-control study comprising 579 cases of male lung cancer registered during 1972-1977 in northern Sweden. The population aetiologic fraction attributable to smoking was about 80% in this series. Pipe smoking was as common as cigarette smoking and gave similar relative risk. The pipe smoking cases, however, had significantly higher mean age and mean smoking years at the time of diagnosis than the cigarette smoking cases. An obvious dose-response relation was found for both cigarette and pipe smoking. In ex-smokers, the relative risk gradually decreased from five years after cessation of smoking. This decrease was, however, much less pronounced in ex-pipe smokers than in ex-cigarette smokers. High relative risks were obtained for small cell and squamous cell carcinomas. For adenocarcinomas the relative risk was considerably lower but still significantly increased. Two types of controls were used, i.e. decreased and living. Comparison with living controls gave generally higher risk estimates than comparison with deceased controls. PMID- 3013268 TI - The 'gastrointestinal syndrome' after chemotherapy: inferences from mouse survival time, and from histologically- and clonogenically-defined cell death in intestinal crypts. PMID- 3013267 TI - The in vivo malignant transformation of mouse fibroblasts in the presence of human tumour xenografts. AB - During the routine serial passage of over 30 human tumour xenografts in athymic (nu. nu.) mice over a period of 6 years the induction of murine fibrosarcomas at the site of transplantation has been observed on three occasions. In two cases it has been possible to follow the development of these tumours over successive transplant generations. These sarcomas had growth rates, tumour karyotypes and isoenzyme patterns which clearly distinguished them from the original human xenografts. PMID- 3013269 TI - Observations on rat spleen reticulum during the development of syngeneic hepatocellular implants. AB - August rat hepatocytes isolated by collagenase perfusion were implanted directly into the spleens of syngeneic recipients. Graft development was monitored by a combination of staining techniques and when surviving hepatocytes were difficult to recognize by routine histology, indirect immunofluorescence permitted their rapid identification. Liver cells were found up to 21 months after transplantation, thus confirming the ability of the spleen to support hepatocellular grafts. The reticular framework of the spleen appears to play an important role. The reticulum mesh of the red pulp traps injected cells and there is rapid generation of reticulum fibres around them within 4 days. The subsequent proliferation and organization of liver cells into recognizable hepatic plates, glandular rosettes and acini was accompanied by the development of a rich and distinctive reticular scaffold with a pattern resembling that of regenerating splenic tissue. PMID- 3013270 TI - Human pulmonary alveolar macrophages with smokers' inclusions: their relation to the cessation of cigarette smoking. AB - Bronchoalveolar lavage (BAL) was performed in 47 patients (24 smokers, 18 ex smokers and 5 who never smoked). Cytocentrifuged preparations were stained with May-Grunwald-Giemsa (MGG) and differential counts of pulmonary alveolar macrophages (PAM) with smokers' inclusions were performed. The data supported an exponential reduction in this percentage and indicated that about 3 years elapsed before this percentage approximated to the values in patients who had never smoked. Electron microscopy was performed on eight BALs and the results supported those obtained by light microscopy. PMID- 3013271 TI - Itraconazole, a new orally active antifungal, in the treatment of pityriasis versicolor. AB - Itraconazole, a new orally active triazole antifungal, has been tested in patients with pityriasis versicolor. A comparison of different dose schedules was carried out in 73 patients. A regime of 100 mg itraconazole daily for 15 days gave a 100% response rate; 200 mg daily for 5 days gave an 80% response rate. Two patients, who had received 50 mg itraconazole for 14 days, relapsed within 2 months of finishing treatment. Only two patients reported minor side-effects. PMID- 3013272 TI - Ultrastructure and cytogenetics in seven cases of acute promyelocytic leukaemia (APL). AB - In the seven cases of acute promyelocytic leukaemia (APL) presented here we have studied the ultrastructural and cytogenetic features which are thought to be of particular significance in this disease. On the basis of our findings from the seven cases of APL described in detail, and our unreported results obtained for a large number of myeloid leukaemias other than APL, we conclude the following. Stellate rough endoplasmic reticulum, certain inclusion bodies and Auer rods having a tubular substructure are, if present, probably diagnostic of APL. However, these structures are not always observed in APL. Inflated rough endoplasmic reticulum is highly indicative of APL while slender cytoplasmic projections, convoluted or lobed nuclei and conspicuous bundles of cytoplasmic fibrils are very common in the abnormal promyelocytes of this disease. There is a strong correlation between the presence of conspicuous bundles of cytoplasmic fibrils and convoluted or lobed nuclei. Most of the APL cases showed the characteristic translocation t(15;17) and we could find no ultrastructural difference between the cases with the translocation and the single example of a normal karyotype. PMID- 3013273 TI - The molecular basis of HbH disease in Greece. AB - Globin gene mapping in 16 Greek individuals with HbH disease and their parents has demonstrated the occurrence of several HbH genotypes brought about by the interaction of two alpha zero-thalassaemia and two alpha+-thalassaemia haplotypes. Eight of the 16 patients had the genotype - -Med/-alpha 3.7, four the genotype -(alpha)20.5/-alpha 3.7 and three the genotype - -Med/alpha alpha T. In one patient the restriction data are consistent with two possible genotypes alpha alpha T/alpha alpha T or - -/alpha alpha T. It is demonstrated that HbH disease in Greece is heterogeneous, with the deletion haplotypes - -Med and -alpha 3.7 being more prevalent than the -(alpha)20.5 and non-deletion (alpha alpha T) haplotypes. PMID- 3013274 TI - HTLV-III antibody and T-cell subset ratios in haemophiliacs and their spouses. AB - 44% of 63 British patients with either haemophilia A or B were HTLV-III antibody positive (HTLV-VIII+). HTLV-III+ was more frequent in high factor VIII concentrate users and 75% of severely affected haemophilia A patients were HTLV III+. All eight patients who were exposed to factor IX concentrate were HTLV-III . 17 haemophilia A patients who received only British made factor VIII concentrate (average 12,000 units/year) were HTLV-III-. Two of 63 patients had evidence of a pre-AIDS type symptom complex and both were HTLV-III+. Information from a cohort of 21 Liverpool haemophiliacs suggests that HTLV-III was first introduced into this country in 1981. OKT4/T8 ratios were abnormal in 52% of 21 patients studied but this finding was not confined to either HTLV-III+ or HTLV III- individuals. The spouses of 14 HTLV-III+ haemophiliacs were all HTLV-III-. PMID- 3013275 TI - Human parvovirus induced cytopenias: a report of five cases. PMID- 3013276 TI - Vibration induced injury. PMID- 3013277 TI - Erythrocyte arginase, pyrimidine 5'-nucleotidase (P5N), and deoxypyrimidine 5' nucleotidase (dP5N) as indices of lead exposure. AB - The activities of three erythrocyte (rbc) enzymes, arginase, pyrimidine 5' nucleotidase (P5N), and deoxypyrimidine 5'-nucleotidase (dP5N), were compared in 16 lead workers and 14 age matched controls as correlates of blood lead (PbB) and unextracted zinc protoporphyrin (EP) concentrations. Subjects with PbB of 0.9-2.5 microM (19-52 micrograms/dl) had 6.5 +/- 0.6 IU of P5N activity with uridine monophosphate (UMP) as substrate, significantly less (p less than 0.001) than the 12.0 +/- 0.7 IU activity of controls with PbB 0.3-0.6 microM (6-12 micrograms/dl). The mean activity of rbc dP5N with either deoxyuridine monophosphate (dUMP) or thymidine monophosphate as substrate, and of rbc arginase, did not differentiate the two groups. The correlation coefficients of ln PbB with the selected substrates for P5N and dP5N were: UMP, r = -0.75; dUMP, r = -0.61; TMP, r = -0.23. The correlation coefficient of ln PbB and arginase activity was -0.03. Rbc P5N (UMPase) is a significant correlate of PbB, equivalent to rbc protoporphyrin. HPLC assay of rbc UMPase activity is a sensitive and rapid assay that appears to meet criteria for a reliable clinical laboratory index of blood lead concentrations. PMID- 3013279 TI - Radiological survey of past and present vermiculite miners exposed to tremolite. AB - Chest radiographs taken by a standard technique were obtained from 173 current employees (164 men, 9 women) of a vermiculite mine in Montana, from 80 of 110 past employees resident within 200 miles, and from 47 men from the same area without known exposure to dust. In 43 of the 80 and 24 of the 47 an earlier chest x ray film was retrieved from the hospital archives. All 367 films were assessed blind and independently by three experienced readers using the ILO 1980 classification. Median radiographic assessment scores were analysed in relation to estimated cumulative exposure to the amphibole fibres that contaminate the vermiculite. Logistic regression analyses showed independent effects of age, smoking, and exposure on the prevalence of small opacities and of age and probably of exposure on pleural thickening. Overall, the data suggest that by retirement age the increase in prevalence of small opacities (greater than or equal to 1/0) lies between 5% and 10% per 100 f/ml years. This gradient may be somewhat steeper than for chrysotile miners and millers, but not much so. PMID- 3013278 TI - Cohort study of mortality of vermiculite miners exposed to tremolite. AB - A cohort of 406 men employed before 1963 for at least one year in a vermiculite mine in Montana was followed up until July 1983. The vermiculite ore as fed to the mill contained 4-6% of amphibole fibre in the tremolite series. Vital status was established in all but one of the 406 and death certificates were obtained and coded for 163 of the 165 men who died. Compared with white men in the United States, the cohort experienced excess mortality from all causes (SMR 1.17), respiratory cancer (SMR 2.45), non-malignant respiratory disease (SMR 2.55), and accidents (SMR 2.14). Four deaths were from malignant mesothelioma (proportional mortality 2.4%). Compared with Montana death rates, the SMR for respiratory cancer was somewhat higher (3.03). Man-year analyses of respiratory cancer and estimated cumulative exposure gave a relation that did not depart significantly from linearity. The results of this and case-referent analyses indicate an increased risk of mortality from respiratory cancer in this cohort of about 1% for each fibre year of exposure. In relation to estimated exposure the mortality experienced by the cohort from both lung cancer and mesothelial tumours was higher than in chrysotile mining. PMID- 3013281 TI - Development of an immunofluorescence test for the serodiagnosis of herpes zoster ophthalmicus. AB - An indirect immunofluorescence test has been developed and evaluated for the serodiagnosis of herpes zoster ophthalmicus (HZO) by the detection of antivaricella zoster virus (VZV) antibody. The results show that, in patients with HZO, anti-VZV IgG antibody titre usually rises rapidly after onset. One hundred and seven of the 134 sera (80%) from patients with a clinical diagnosis of HZO had an anti-VZV IgG titre of greater than or equal to 256, and IgM antibody at a level of 1 in 8 was present in six of them. In comparison only two of the 216 sera (1%) from patients with a clinical diagnosis of ocular infections other than those caused by VZV had such IgG titres. It was concluded that, on the basis of results of a single sample of serum, it is possible to make a provisional diagnosis of HZO with a high degree of confidence. PMID- 3013280 TI - Prevalence of radiographic asbestosis in crocidolite miners and millers at Wittenoom, Western Australia. AB - An estimate has been made of the prevalence of unrecognised pneumoconiosis in former crocidolite workers from Wittenoom, Western Australia. All plain chest radiographs relating to a one in six random sample (1025 men) of all former Wittenoom workers who had never entered a compensation claim to the Pneumoconiosis Medical Board of Western Australia were sought from Perth teaching hospitals and from the Perth Chest Clinic where compulsory examination of all workers in the mining industry takes place. Radiographs were recovered for 83% of the men and read independently by two observers. By means of logistic regression analysis a current prevalence of parenchymal abnormality (defined as a radiographic profusion of small opacities of category 1/0 or greater on the ILO classification) of nearly 20% was calculated after adjustment for age, time since first exposure, and cumulative exposure level. One hundred men randomly selected from those known to be alive in the sample were invited to attend for a new radiographic examination. Seventy four men attended and the predicted prevalence was confirmed. It is estimated from these data that there were between 450 and 900 former Wittenoom workers in Australia at the end of 1980 who had radiographic abnormality consistent with pneumoconiosis but had not claimed compensation or had asbestosis diagnosed. The data are consistent with there being no threshold dose of crocidolite exposure for the development of radiographic abnormality in this group. PMID- 3013283 TI - A comparative study of corneal incisions induced by diamond and steel knives and two ultraviolet radiations from an excimer laser. AB - This paper reviews the potential role of excimer lasers in corneal surgery. The morphology of incisions induced by two wavelengths of excimer laser radiation, 193 nm and 248 nm, are compared with the morphology of incisions produced by diamond and steel knives. Analysis suggests that ablation induced by excimer laser results from highly localised photochemical reactions and that 193 nm is the optimal wavelength for surgery. The only significant complication of laser surgery is loss of endothelial cells when incisions are within 40 micron of Descemet's membrane. PMID- 3013282 TI - Immunoassay of tear lysozyme in acute adenovirus conjunctivitis. AB - The tear lysozyme levels were measured by immunoassay in 92 healthy subjects and 98 patients with acute adenovirus conjunctivitis. They were found to be significantly decreased during the acute phase of the disease. The extent of this decline in the tear lysozyme level was correlated with increased severity of disease. There was no significant difference in the tear lysozyme level in viral isolation-positive and isolation-negative patients. The tear lysozyme level showed return to normal levels with clinical improvement. PMID- 3013285 TI - A review of tumours of the deep lobe of the parotid salivary gland. AB - Tumours of the deep lobe of the parotid gland may present as a swelling in the oropharynx. They are uncommon when compared with those of the superficial lobe, are frequently misdiagnosed and subjected to per-oral biopsy which is hazardous and predisposes to seeding of the tumour. Investigation and diagnosis are discussed with particular reference to the role of computed tomography. The surgical approach to these inaccessible tumours is illustrated by reference to patients treated. Modifications to the technique of Cooke and Ranger (1969) for excision of parapharyngeal tumours are suggested, which are applicable to tumours of the deep lobe extending medial to the mandible and presenting in the side wall of the pharynx. PMID- 3013284 TI - The monomorphic clear cell tumor: a report of two cases. AB - Two cases of the monomorphic clear cell tumour are described. The histology, incidence, high recurrence rate and clinical management are discussed. PMID- 3013286 TI - Anomalous driving force for renal brush border H+/OH-transport characterized by using 6-carboxyfluorescein. AB - The pH, delta pH, and membrane potential dependences of H+/OH-permeability in renal brush border membrane vesicles (BBMV) were studied by using the entrapped pH indicator 6-carboxyfluorescein (6CF). Quantitative H+/OH-fluxes (JH) were obtained from a calibration of the fluorescence response of 6CF to intravesicular pH using vesicles prepared with varying intravesicular and solution pHs. Intravesicular buffer capacity, determined by titration of lysed vesicles, increased monotonically from 140 to 260 mequiv/L in the pH range 5-8. JH was measured by subjecting voltage-clamped BBMV (K+/valinomycin) to preformed pH gradients over the pH range 5-8 and measuring the rate of change of intravesicular pH. For small preformed pH gradients (0.4 pH unit) JH [6 nequiv s 1 (mg of protein)-1] was nearly independent of pH (5-8), predicting a highly pH dependent H+ permeability coefficient. JH increased in a curvilinear manner from 6 to 104 nequiv s-1 (mg of protein)-1 as delta pH increased from 0.4 to 2.5. JH increased linearly [1.6-7.3 nequiv s-1 (mg of protein)-1] with induced K+ diffusion potentials (21-83 mV) in the absence of a pH gradient. These findings cannot be explained by simple diffusion of H+ or OH- or by mobile carrier models. Two mechanisms are proposed, including a lipid diffusion mechanism, facilitated by binding of H+/OH- to fixed sites in the membrane, and a linear H2O strand model, where dissociation of H2O in the membrane fixes H+ and OH- concentrations in strands, which can result in net H+/OH- transport. PMID- 3013287 TI - Thermoinactivation and aggregation of alpha beta units in soluble and membrane bound (Na,K)-ATPase. AB - Stability and conformational transitions of soluble and fully active alpha beta units of (Na,K)-ATPase in n-dodecyl octaethylene glycol monoether (C12E8) are examined. Sedimentation equilibrium centrifugation gave a molecular weight of 143 000 for the alpha beta unit eluting from TSK 3000 SW gel chromatography columns. Fluorescence analysis and phosphorylation experiments show that E1-E2 transitions between both dephospho and phospho forms of soluble (Na,K)-ATPase are similar to those previously observed in the membrane-bound state. The two conformations can also be identified by their different susceptibilities to irreversible temperature-dependent inactivation. E1 forms of both soluble and membrane-bound (Na,K)-ATPase are more thermolabile than E2 forms. Gel chromatography on TSK 3000 SW and 4000 SW columns shows that thermal inactivation of soluble (Na,K)-ATPase at 40 degrees C is accompanied by aggregation of alpha beta units to (alpha beta)2 units and higher oligomers. The aggregates are stable in C12E8 but dissolve in sodium dodecyl sulfate. Similar aggregation accompanies inactivation of membrane-bound (Na,K)-ATPase at 55-60 degrees C. These data suggest that inactivation both in the soluble and in the membrane-bound state involves exposure of hydrophobic residues to solvent. The instability of the soluble E1 form may be related to inadequate length of the dodecyl alkyl chain of C12E8 for stabilization of hydrophobic protein domains that normally associate with alkyl chains of phospholipids in the membrane. Interaction between alpha beta units does not seem to be required for the E1-E2 conformational change, but irreversible aggregation appears to be a consequence of denaturation of (Na,K) ATPase in both soluble and membranous states. PMID- 3013288 TI - Apocytochrome c binding to negatively charged lipid dispersions studied by spin label electron spin resonance. AB - The interaction of apocytochrome c with aqueous dispersions of phosphatidylserine from bovine spinal cord and with other negatively charged phospholipids has been studied as a function of pH and salt concentration by using spin-label electron spin resonance (ESR) spectroscopy and chemical binding assays. The ESR spectra of phospholipids spin-labeled at different positions on the sn-2 chain indicate a generalized decrease in mobility of the lipids, while the characteristic flexibility gradient toward the terminal methyl end of the chain is maintained, on binding of apocytochrome c to phosphatidylserine dispersions. This perturbation of the bulk lipid mobility or ordering is considerably greater than that observed on binding of cytochrome c. In addition, a second, more motionally restricted, lipid component is observed with lipids labeled close to the terminal methyl ends of the chains. This second component is not observed on binding of cytochrome c and can be taken as direct evidence for penetration of apocytochrome c into the lipid bilayer. It is less strongly motionally restricted than similar spectral components observed with integral membrane proteins and displays a steep flexibility gradient. The proportion of this second component increases with increasing protein-to-lipid ratio, but the stoichiometry per protein bound decreases from 4.5 lipids per 12 000-dalton protein at low protein contents to 2 lipids per protein at saturating amounts of protein. Apocytochrome c binding to phosphatidylserine dispersions decreases with increasing salt concentration from a saturation value corresponding to approximately 5 lipids per protein in the absence of salt to practically zero at 0.4 M NaCl.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013289 TI - Native or nativelike species are transient intermediates in folding of alkaline iso-2 cytochrome c. AB - Titration to high pH converts yeast iso-2 cytochrome c to an inactive but more stable alkaline form lacking a 695-nm absorbance band [Osterhout, J. J., Jr., Muthukrishnan, K., & Nall, B. T. (1985) Biochemistry 24, 6680-6684]. The kinetics of absorbance-detected refolding of the alkaline form have been measured by dilution of guanidine hydrochloride in a stopped-flow instrument. Fast-folding species (tau 2) are detected, as in refolding to the native state at neutral pH. An additional kinetic phase (tau a) is observed with an amplitude opposite in sign to the fast phase. The amplitude of this phase increases and the rate increases with increasing pH. Comparison to pH-jump measurements of the fully folded protein shows that phase tau a has the same sign, rate, and pH dependence as the alkaline isomerization reaction, suggesting that this new phase involves isomerization of native or nativelike species following fast folding. Absorbance difference spectra are taken at 5-s intervals during refolding at high pH. The spectra verify that nativelike species--with a 695-nm absorbance band--are formed transiently, before conversion of the protein to the alkaline form. Refolding in the presence of ascorbate shows that the transient, nativelike species are reducible, unlike alkaline iso-2. Thus, (1) refolding to the alkaline form of iso 2 cytochrome c proceeds through transient native or nativelike species, and (2) a folding pathway leading to native or nativelike forms is maintained at high pH, where native species are no longer the thermodynamically favored product.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013290 TI - High-resolution mapping of carcinogen binding sites on DNA. AB - We have used a photochemical method to map covalent binding sites of the carcinogen benzo[a]pyrenediol epoxide (BPDE) within DNA from the transcriptional control region of the chicken adult beta-globin gene. Our preliminary low resolution mapping has demonstrated that this region contains highly preferred BPDE binding sites [Boles, T.C., & Hogan, M.E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5623-5627]. Here, we find that BPDE binding at individual G residues in this region is influenced by nearest-neighbor interactions and also by longer range interactions that may be attributable to sequence-specific variation of DNA secondary structure. Our findings suggest that long poly(dG) sequences should be preferred sites for BPDE action in other genes. PMID- 3013292 TI - Rat growth hormone gene expression is correlated with an unmethylated CGCG sequence near the transcription initiation site. AB - The methylation status of the rat growth hormone (GH) gene was compared in DNA obtained from GH-producing and GH-nonproducing sources by digestion with three methylation-sensitive restriction enzymes. GH gene expression was correlated with an unmethylated ThaI site (CGCG) 144-bp upstream of the GH RNA transcription initiation site. This ThaI site was unmethylated in nine GH-producing subclones of the rat pituitary tissue culture cell line GH3 and in greater than 50% of the DNA isolated from rat anterior pituitary, a gland containing GH-producing somatotroph cells as well as GH-nonproducing cells. In DNA prepared from GH nonproducing tissues, e.g., rat spleen, kidney, liver, and brain, this ThaI site was entirely methylated. Furthermore, this site was entirely methylated in hybrid cells formed by the fusion of GH3 cells with mouse fibroblasts in which GH production has been extinguished. DNA methylation at 10 other restriction sites located throughout the rat GH gene region failed to correlate with GH expression in GH-producing subclones of GH3 cells as well as in GH-nonproducing GH3 X LB82 hybrid cells. We suggest that the conserved absence of methylation 144 bp 5' of the RNA transcription initiation site of transcribed GH genes identifies a potential GH gene control region. PMID- 3013291 TI - Identification of the fibroblast growth factor receptor of Swiss 3T3 cells and mouse skeletal muscle myoblasts. AB - Two distinct fibroblast growth factors (FGF) were purified to homogeneity from bovine brain on the basis of their ability to stimulate skeletal muscle myoblast proliferation. These growth factors are also mitogenic for Swiss 3T3 cells and appear to be closely related to or identical with previously isolated anionic and cationic fibroblast growth factors. The half-maximum concentrations (EC50) for stimulation of myoblast DNA synthesis by the anionic and cationic growth factors were 30pM and 1pM, respectively. In contrast, an EC50 of 45 pM was observed for stimulation of 3T3 cell DNA synthesis by both growth factors. Binding of 125I labeled anionic FGF was saturable with apparent Kd values of 45 pM and 11 pM and approximately 60 000 and 2000 receptor sites per cell for 3T3 cells and MM14 murine myoblasts, respectively. Unlabeled anionic and cationic FGF equally displaced 125I-labeled anionic FGF from 3T3 cells while cationic FGF was more potent than anionic FGF for displacement from skeletal muscle myoblasts, demonstrating that a single receptor binds the two distinct growth factors. Binding was specific for these factors since platelet-derived growth factor, insulin, insulin-like growth factor 1, epidermal growth factor, and nerve growth factor were unable to displace bound 125I-labeled anionic FGF from Swiss 3T3 cells. Chemical cross-linking of specifically bound 125I-labeled anionic FGF to 3T3 cells and MM14 myoblasts identified a single detergent-soluble FGF receptor with an apparent molecular weight of 165 000. PMID- 3013294 TI - Vesicular stomatitis virus binds and fuses with phospholipid domain in target cell membranes. AB - Fusion of vesicular stomatitis virus with some cells (HELR 66, KB, and human erythrocytes, both intact and trypsinized) and liposomes made of various natural and synthetic lipids was studied with spin-labeled phospholipid. Binding of virus was assayed separately with radiolabeled and spin-labeled virus. Binding to cells and liposomes was small at neutral pH but enhanced at acidic pHs. Fusion with cells and liposomes was negligibly small at neutral pH but greatly activated at acidic pHs lower than 6.5. Activation of fusion occurred at lower pH values than enhancement of binding. Fusion occurred rapidly and efficiently, reaching a plateau at 50-80% after 3 min at 37 degrees C. Binding and fusion with cells were enhanced by pretreatment of cells with trypsin. Binding to liposomes was dependent on the head group of the phospholipid, stronger to phosphatidylserine than to phosphatidylcholine, but not much dependent on the acyl chain composition. On the other hand, cis-unsaturated acyl chains were required for the efficient fusion, but there was only a small, if any, requirement for the head group. Cholesterol enhanced the fusion further. High fusion efficiency with cis unsaturated phospholipids cannot be ascribed to the membrane fluidity but may be related to higher tail-to-head volume ratios. Possible mode of interaction of viral G glycoprotein with phospholipid is discussed. The virus cell entry mechanism is suggested as binding to the phospholipid domain in the cell surface membranes, endocytosis, and followed by fusion with the phospholipid domain in endosomes upon acidification. PMID- 3013293 TI - 1H NMR (500 MHz) identification of aromatic residues of gene 32 protein involved in DNA binding by use of protein containing perdeuterated aromatic residues and by site-directed mutagenesis. AB - Preparation of gene 32 protein containing perdeuterated tyrosyl and phenylalanyl residues has allowed the resolution of separate 1H NMR signals for the Tyr and Phe residues of the protein by NMR difference spectra. Upfield shifts in the chemical shifts of a number of aromatic protons previously observed to accompany deoxyoligonucleotide complex formation with gene 32 protein [Prigodich, R. V., Casas-Finet, J., Williams, K. R., Konigsberg, W., & Coleman, J. E. (1984) Biochemistry 23, 522-529] can be assigned to five Tyr and two Phe residues that must form part of the DNA binding domain. Site-directed mutation of Tyr-115 to Ser-115 results in the disappearance of a set of 2,6 and 3,5 tyrosyl protons that are among those moved upfield by oligonucleotide complex formation. These findings suggest that the amino acid sequence from Tyr-73 to Tyr-115 which contains six of the eight Tyr residues of the protein forms part of the DNA binding surface. PMID- 3013295 TI - Biochemical characterization of phosphorylated beta-adrenergic receptors from catecholamine-desensitized turkey erythrocytes. AB - Isoproterenol-induced desensitization of turkey erythrocyte adenylate cyclase is accompanied (1) by a decrease in the mobility of beta-adrenergic receptor proteins, specifically photoaffinity labeled with 125I-(p-azidobenzyl)carazolol (125I-PABC), on sodium dodecyl sulfate (SDS)-polyacrylamide gels and (2) by a 2-3 fold increase in phosphate incorporation into the beta receptor [Stadel, J.M., Nambi, P., Shorr, R. G. L., Sawyer, D. F., Caron, M. G., & Lefkowitz, R. J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3173]. Analysis of 32P-labeled beta receptors partially purified by affinity chromatography and subsequently hydrolyzed in 6 N HCl revealed that the beta receptor from control erythrocytes contained only phosphoserine and that agonist-promoted phosphorylation of the receptor in desensitized cells occurred on serine residues. Comparison of limited digest peptide maps of 125I-PABC-labeled beta receptors from control and desensitized erythrocytes reveals distinctly different sensitivities of the two beta receptors to cleavage by chymotrypsin and Staphylococcus aureus protease. The altered mobility of the 125I-PABC-labeled beta receptor from desensitized erythrocytes was eliminated when 5 M urea was included in the SDS-polyacrylamide gels. Limited-digest peptide mapping of 32P-labeled beta receptors from control and desensitized cells with the protease papain identified a unique phosphorylated peptide in desensitized preparations. Our results are consistent with the hypothesis that the altered mobility of beta-receptor proteins on SDS gels following desensitization is due to changes in conformation promoted by prolonged exposure to agonists. PMID- 3013296 TI - Nicotinamide mononucleotide adenylyltransferase. Molecular and enzymatic properties of the homogeneous enzyme from baker's yeast. AB - Nicotinamide mononucleotide (NMN) adenylyltransferase has been purified to homogeneity from baker's yeast crude extract. The purification procedure is relatively simple and consists of high-salt extraction of enzyme activity and precipitation with poly(ethylenimine), followed by ion-exchange and dye ligand chromatography separations. The final enzyme preparation is homogeneous as judged by a single Coomassie blue stainable band when run on nondenaturating and denaturating polyacrylamide gels. The native enzyme shows a molecular weight of about 200 000, calculated by gel filtration and sucrose gradient centrifugation. The protein possesses quaternary structure and is composed of four apparently identical Mr 50 000 subunits. The absorption spectrum shows a maximum at 280 nm and a minimum at 253 nm. The isoelectric point is 6.2. Amino acid composition analysis shows the presence of 28 half-cystine residues. The same result has been obtained by titrating the enzyme in denaturating conditions with Ellman's reagent after incubation with sodium borohydride. NMN adenylyltransferase is a glycoprotein containing 2% sugar, 2 mol of alkali-labile phosphate per mole of enzyme, and 1 mol of adenine moiety per mole of enzyme. Therefore, the possibility that the enzyme is ADP-ribosylated exists. The Km values for ATP, NMN, and nicotinate mononucleotide are 0.11 mM, 0.19 nM, and 5 mM, respectively. Kinetic analysis reveals a behavior that is consistent with an ordered sequential Bi-Bi mechanism. The pH optimum is in the range 7.2-8.4. PMID- 3013297 TI - Activation of ribonuclease L by (2'-5')(A)4-poly(L-lysine) conjugates in intact cells. AB - Molecular hybrids were synthesized by coupling (2'-5')(A)n oligoadenylates or 2 5A, an intracellular mediator involved in antiviral activity of interferons (IFNs), with poly(L-lysine) used as a membrane carrier. (2'-5')(A)n in its free form was not taken up by cells, probably because of its ionic character. Conjugation with the polypeptide carrier overcame this problem and enabled its pharmacological properties to be developed. The alpha-glycol group of individual (2'-5')(A)n oligomers was oxidized by periodate oxidation and conjugated by an amino reductive reaction to poly(L-lysine), Mr 14 000, in a molar ratio of 5:1. These hybrid molecules left the biologically active 5' end moiety of the (2' 5')(A)n molecule unchanged, and in particular its triphosphate group, and stabilized the molecule by increasing its resistance to phosphodiesterase hydrolysis. A dose-dependent inhibition of virus growth was observed on concomitant incubation of (2'-5')(A)n-poly(L-lysine) conjugates with vesicular stomatitis virus infected L1210 cell cultures. This was a result of the activation of the (2'-5')(A)n-dependent endoribonuclease (RNase L) by intracellularly delivered (2'-5')(A)n as in some IFN-treated virus-infected cells. Indeed, (2'-5')(A)n-poly(L-lysine) conjugates bind RNase L effectively as can be seen from their ability to compete with authentic (2'-5')(A)n in a cell free radiobinding assay. Moreover, (2'-5')(A)n-poly(L-lysine) conjugates promote transient inhibition of protein synthesis and a characteristic cleavage pattern of ribosomal RNAs in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013298 TI - Specific overproduction and purification of the cytochrome b558 component of the cytochrome d complex from Escherichia coli. AB - In Escherichia coli strain GR84N[pNG10], the cloned gene for subunit I of the membrane-bound cytochrome d complex resulted in the overproduction of cytochrome b558 and facilitated purification of this cytochrome. Extracting membranes with 1% Triton X-100 followed by two chromatographic steps yielded a single band on sodium dodecyl sulfate-polyacrylamide gels corresponding to subunit I (Mr 57 000). Purified cytochrome b558 was in its native state as determined by difference absorption spectroscopy and by potentiometric analysis. Both the membranes of strain GR84N[pNG10] and the purified subunit I lacked the other two spectroscopically defined cytochromes, b595 (previously "a1") and d, of the cytochrome d complex. Reconstitution of cytochrome b558 in phospholipid vesicles demonstrated that cytochrome b558 can be reduced by ubiquinol but that it does not reduce molecular oxygen. Heme extraction of cytochrome b558 yielded an extinction coefficient of 22 000 M-1 cm-1 for the wavelength pair of 560 and 580 nm in the reduced-minus-oxidized spectrum. The mutation on pNG10 that eliminates subunit II was mapped to a 250 base pair DNA fragment. PMID- 3013299 TI - Coulometric and spectroscopic analysis of the purified cytochrome d complex of Escherichia coli: evidence for the identification of "cytochrome a1" as cytochrome b595. AB - Coulometric and spectroscopic analyses were performed on the three cytochrome components (cytochrome d, cytochrome b558, and the cytochrome previously described as cytochrome a1) of the purified cytochrome d complex, a terminal oxidase of the Escherichia coli aerobic respiratory chain. On the basis of heme extraction, spectroscopic, and coulometric data, the "cytochrome a1" component was identified as a b-type cytochrome: cytochrome b595. The pyridine hemochromogen technique revealed the presence of two molecules of protoheme IX per cytochrome d complex. This quantity of protoheme IX fully accounted for the sum of the cytochrome b558 and cytochrome b595 components as determined coulometrically. The renaming of cytochrome a1 as cytochrome b595 was further indicated by the lack of any heme a in the complex and by its resolved reduced minus-oxidized spectrum. The latter was found to be similar to that of cytochrome c peroxidase, which contains protoheme IX. Coulometric titrations and carbon monoxide binding titrations revealed that there are two molecules of cytochrome d per complex. A convenient measurement of the amount of cytochrome b558 was found to be the beta-band at 531 nm since cytochrome b558 was observed to be the only component of the cytochrome d complex with a peak at this wavelength. By use of this method and the extinction coefficient for the purified cytochrome b558, it was estimated that there is one molecule of cytochrome b595 and one of cytochrome b558 per cytochrome complex. PMID- 3013300 TI - D-lactate oxidation and generation of the proton electrochemical gradient in membrane vesicles from Escherichia coli GR19N and in proteoliposomes reconstituted with purified D-lactate dehydrogenase and cytochrome o oxidase. AB - The respiratory chain in the cytochrome d deficient mutant Escherichia coli GR19N is a relatively simple, linear system consisting of primary dehydrogenases, ubiquinone 8, cytochrome b-556, and cytochrome o oxidase. By use of right-side out and inside-out membrane vesicles from this strain, various oxidase activities and the generation of the H+ electrochemical gradient were studied. Oxidation of ubiquinol 1 or N,N,-N',N'-tetramethyl-p-phenylenediamine, which donate electrons directly to the terminal oxidase, generates a H+ electrochemical gradient comparable to that observed during D-lactate oxidation. In contrast, D lactate/ubiquinone 1 or D-lactate/ferricyanide oxidoreductase activity does not appear to generate a membrane potential, suggesting that electron flow from D lactate dehydrogenase to ubiquinone is not electrogenic. Moreover, proteoliposomes reconstituted with purified D-lactate dehydrogenase, ubiquinone 8, and purified cytochrome o catalyze D-lactate and ubiquinol 1 oxidation and generate a H+ electrochemical gradient similar to that observed in membrane vesicles. Strikingly, in inside-out vesicles, NADH oxidation generates a H+ electrochemical gradient that is very significantly greater than that produced by either D-lactate or ubiquinol 1; furthermore, NADH/ubiquinone 1 and NADH/ferricyanide oxidoreductase activities are electrogenic. It is suggested that the only component between D-lactate dehydrogenase or ubiquinol and oxygen in GR19N membranes that is directly involved in the generation of the H+ electrochemical gradient is cytochrome o, which functions as a "half-loop" (i.e., the oxidase catalyzes the scalar release of 2 H+ from ubiquinol on the outer surface of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013302 TI - Light- and nucleotide-dependent binding of phosphodiesterase to rod disk membranes: correlation with light-scattering changes and vesicle aggregation. AB - Under conditions in which large guanosine cyclic 3',5'-phosphate (cGMP)- and phosphodiesterase (PDE)-dependent changes in near-infrared transmission and vesicle aggregation and disaggregation occur, we have observed a striking change in the binding of PDE to rod disk membranes. The change in PDE binding is nucleotide and light dependent as are the light-scattering changes. The cGMP- and PDE-dependent light-scattering signal can be produced by a 500-nm light flash which bleaches 1/(1 X 10(7] rhodopsin molecules. Mg ions are an essential cofactor for the nucleotide-dependent PDE binding and light-scattering changes. 3 Isobutyl-1-methylxanthine and other competitive inhibitors of PDE hydrolytic activity support increased PDE binding to the disk membrane, vesicle aggregation, and the light-scattering signal. However, treatments which block GTP-dependent activation of PDE hydrolytic activity (colchicine, GDP, or ethylenediaminetetraacetic acid) also block these phenomena. Thus, GTP-dependent activation of PDE rather than its hydrolytic activity appears to be correlated with the light-scattering signal. PMID- 3013301 TI - Triton X-100 induced dissociation of beef heart cytochrome c oxidase into monomers. AB - Purified beef heart cytochrome c oxidase, when solubilized with at least 5 mg of Triton X-100/mg of protein, was found to be a monodisperse complex containing 180 molecules of bound Triton X-100 with a protein molecular weight of 200 000, a Stokes radius of 66-72 A, and an s(0)20,w = 8.70 S. These values were determined by measurement of the protein molecular weight by sedimentation equilibrium in the presence of D2O, evaluation of the sedimentation coefficient, S(0)20,w, by sedimentation velocity with correction for its dependence upon the concentration of protein and detergent, and measurement of the effective radius by calibrated Sephacryl S-300 gel chromatography. The monomeric complex was judged to be homogeneous and monodisperse since the effective mass of the complex was independent of the protein concentration throughout the sedimentation equilibrium cell and a single protein schlieren peak was observed during sedimentation velocity. These results are interpreted in terms of a fully active monomeric complex that exhibits typical biphasic cytochrome c kinetics and contains 2 heme a groups and stoichiometric amounts of the 12 subunits normally associated with cytochrome c oxidase. With lower concentrations of Triton X-100, cytochrome c oxidase dimers and higher aggregates can be present together with the monomeric complex. Monomers and dimers can be separated by sedimentation velocity but cannot be separated by Sephacryl S-300 gel filtration, probably because the size of the Triton X-100 solubilized dimer is not more than 20% larger than the Triton X-100 solubilized monomer.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013303 TI - Purification of labeled cyanogen bromide peptides of the alpha polypeptide from sodium ion and potassium ion activated adenosinetriphosphatase modified with N [3H]ethylmaleimide. AB - Sodium ion and potassium ion activated adenosinetriphosphatase, isolated from canine kidney, was reacted with N-[3H]ethylmaleimide while it was poised in three different conformations, ostensibly E2-P, E2, and E1, respectively. These assignments were made from a consideration of the particular concentrations of ligands in the respective alkylation mixtures. After a 30-min reaction, the remaining enzymatic activity was found to vary among these three different samples from 90 to 30% of that of unalkylated controls. In all cases, the alpha polypeptide was purified and subjected to digestion with cyanogen bromide, and in each digest the same two distinct radioactive peptides were identified and purified by gel filtration on a column of Sephadex LH-60. The incorporation of N [3H]ethylmaleimide into one of these two peptides correlated closely with enzymatic inactivation, while the incorporation into the other was most extensive when the portion of the active site to which ATP binds was unoccupied. Alkylation of the residue within the latter peptide, however, does not result in inactivation of the enzyme. Both peptides were further purified by high-pressure liquid chromatography, and their amino-terminal sequences were determined by manual dansyl Edman or solid-phase techniques. The peptide containing the sulfhydryl protected by ATP has, as its amino terminus, the lysine that reacts exclusively with fluoresceinyl 5'-isothiocyanate [Farley, R. A., Tran, C. M., Carilli, C. T., Hawke, D., & Shively, J. E. (1984) J. Biol. Chem. 259, 9532 9535]. PMID- 3013304 TI - cDNA and protein structure for the alpha subunit of human liver alcohol dehydrogenase. AB - Two cDNA clones for human liver alcohol dehydrogenase (ADH) were identified, together covering 1450 nucleotides that contain the cDNA sequence of the ADH1 locus and include a coding region of 1122 nucleotides for the alpha subunit of the enzyme. In parallel, direct peptide analyses of the carboxymethylated protein also established most of the amino acid sequence. Nucleotide and peptide data were in complete agreement and show exchanges at 24 positions in the alpha relative to the beta subunit. One of the cDNA clones had a 139-nucleotide internal deletion at a position of possible interest in relation to mRNA processing, ancestral connections, or DNA replication. The structure of the alpha subunit is homologous to that of the beta and gamma subunits but has many exchanges, also of functionally important residues, explaining the different enzymatic properties. In total, 35 of 374 amino acid residues differ between the class I isozymes, and the substitutions add an extra SH group in the alpha subunit. Only in the beta-pleated sheet region of the coenzyme-binding domain is almost complete lack of substitutions noted, illustrating the importance of this region. In contrast, the active site region is far less conserved. However, similar exchanges of functional significance have also been found in distantly related alcohol and polyol dehydrogenases. PMID- 3013305 TI - Activity of copper-substituted carboxypeptidase A toward oligopeptides and depsipeptides. AB - Cu(II)-substituted carboxypeptidase A catalyzes the hydrolysis of oligopeptides and their depsipeptide (ester) analogues. Stopped-flow fluorescence assays demonstrate that relative to the zinc enzyme the Cu enzyme can have kcat/Km values up to 24% toward esters but only up to 2.5% toward the corresponding peptides. Adding Zn(II) to the copper enzyme reveals a slow exchange process that correlates with an increase in peptidase activity and with changes in the Cu(II) electron paramagnetic resonance spectra. Low concentrations of 1,10 phenanthroline (OP) (0.1-2.5 microM) markedly increase activity toward furanacryloyl-Phe-Phe (up to 8% of the zinc enzyme), but higher concentrations inhibit, resulting in complete inhibition at 0.8 mM OP. The non-metal-binding, hydrophobic analogues m- and p-phenanthroline are only activators of peptide hydrolysis, even at 1 mM. Activation is likely due to a modifier binding to a hydrophobic locus and either displacing an inhibitory peptide binding mode or inducing a conformational change in the active site. PMID- 3013306 TI - Ligand-controlled dissociation of Chromatium vinosum cytochrome c'. AB - Carbon monoxide binding to Chromatium vinosum ferrocytochrome c' has been studied by high-precision equilibrium methods. In contrast to the CO binding properties of Rhodospirillum molischianum cytochrome c' [Doyle, M. L., Weber, P. C., & Gill, S. J. (1985) Biochemistry 24, 1987-1991], CO binding to C. vinosum cytochrome c' is found to be unusual in the following ways. The binding curve is found to be cooperative with typical Hill coefficients equal to 1.25. The shape of the binding curve is asymmetrical. The heat of CO ligation is measured by two independent methods, both of which yield large endothermic values of approximately 10 kcal [mol of CO(aq)]-1. The overall affinity for CO increases as the concentration of cytochrome c' decreases. These observations suggest the CO binding properties of C. vinosum cytochrome c' are complicated by CO-linked association-dissociation processes. Further investigation by gel filtration chromatography shows that at micromolar concentrations the dimeric state is tightly associated in both the reduced and oxidized forms of the cytochrome but addition of saturating concentrations of CO causes the reduced ligated dimer to dissociate largely into monomers. A model is presented that quantitatively fits the data, involving a ligand-linked dimer-monomer dissociation reaction. In this model, CO binds to the dimer form noncooperatively with an intrinsic affinity constant equal to 5600 +/- 1200 M-1 at 25 degrees C. The unligated dimer form is tightly associated, but addition of CO causes dissociation of the dimer into the monomer with a monomer-dimer association constant equal to 450 +/- 200 M 1.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013307 TI - Interleukin 2 increases T lymphocyte membrane mobility before the rise in cytosolic calcium concentration. AB - Using stopped-flow fluorometry with three different fluorescence probes [2-[(1 pyrenyl-butyryl)oxy]stearic acid, chlortetracycline and Quin 2], we have studied initial stage of T lymphocyte activation after interleukin 2 (IL-2) binding to a specific cell-surface receptor. After IL-2 binding to cytotoxic T lymphocyte (IL 2-dependent mouse LC7 and CTLL-2 cells), membrane mobilities of the cells increased first (4.5 +/- 0.3 s-1 for LC7 and 3.8 +/- 0.2 s-1 for CTLL-2), then calcium was released from intracellular stores into the cytoplasm (1.6 +/- 0.1 s 1 for LC7 and 2.1 +/- 0.1 s-1 for CTLL-2), and lastly, calcium was transported from the external medium into the cytoplasm (1.3 +/- 0.1 s-1 for LC7 and 1.5 +/- 0.1 s-1 for CTLL-2). The slowest process, the calcium influx from the external medium, was suppressed in the presence of a calcium channel blocking agent (verapamil). These observations give us a new information to discuss a model in T lymphocyte activation after IL-2 binding to cell-surface receptors. PMID- 3013308 TI - Outside-inside translocation of aminophospholipids in the human erythrocyte membrane is mediated by a specific enzyme. AB - When human erythrocytes are incubated with spin-labeled analogues of sphingomyelin, phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine, with a short beta chain (C5) bearing a doxyl group at the fourth carbon position, the labeled lipids incorporate readily in the outer monolayer. The incorporation is followed in fresh erythrocytes by a selective inward diffusion of the amino derivatives. This observation led us to postulate the existence of a selective ATP-dependent system that would flip aminophospholipids from the outer to the inner monolayer [Seigneuret, M., & Devaux, P. F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3751-3755]. This study further examines the nature of this selective transport and demonstrates that it is mediated by a specific membrane protein. By measurement of the initial rate of transverse diffusion of spin-labeled lipids incorporated at various concentrations in the membrane outer leaflet of packed erythrocytes, apparent Km values were determined for the phosphatidylserine and phosphatidylethanolamine analogues. A ratio of approximately equal to 1/9.4 [corrected] was obtained (KmPS/KmPE). Using spin-labels bearing either a 14N or a 15N isotope, we have carried out competition experiments allowing us to measure simultaneously the transport of two different phospholipids. By this procedure, we show that phosphatidylserine and phosphatidylethanolamine compete for the same transport site but that phosphatidylserine has a higher affinity, in agreement with a lower apparent Km. On the other hand, the slow diffusion of the phosphatidylcholine or sphingomyelin analogues has no influence on the transport of phosphatidylserine or phosphatidylethanolamine. Experiments carried out in ghosts loaded with ATP enabled us to determine the activation energies for phosphatidylserine and phosphatidylcholine transverse diffusion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013309 TI - Characterization of glucagon receptors in Golgi fractions of rat liver: evidence for receptors that are uncoupled from adenylyl cyclase. AB - Glucagon receptors have been identified and characterized in intermediate (Gi) and heavy (Gh) Golgi fractions from rat liver. At saturation, plasma membranes bound 3500 fmol of hormone/mg of membrane protein, while Gi and Gh bound 24 and 60 fmol of 125I-glucagon/mg of protein, respectively. Half-maximal saturation of binding to plasma membranes, Gi, and Gh occurred at approximately 4, 10, and 20 nM 125I-glucagon, respectively. Trichloroacetic acid precipitation of intact, but not degraded, glucagon was used to correct binding isotherms for hormone degradation. After such correction, half-maximal saturation of binding to plasma membranes, Gi, and Gh was observed in the presence of approximately 2, 7, and 14 nM hormone, respectively. After 90 min of dissociation in the absence of guanosine 5'-triphosphate (GTP), 86% of 125I-glucagon remained bound to plasma membranes, whereas only 42% remained bound to Golgi membranes. GTP significantly increased the fraction of 125I-glucagon released from plasma membranes but only slightly augmented the dissociation of hormone from Golgi fractions. 125I Glucagon/receptor complexes solubilized from plasma membranes fractionated by gel filtration as high molecular weight (Kav = 0.16), GTP-sensitive complexes and lower molecular weight (Kav = 0.46), GTP-insensitive complexes. 125I-Glucagon complexes solubilized from Golgi membranes fractionated almost exclusively as the lower molecular weight species. The lower affinity of Golgi than plasma membrane receptors for hormone, the ability of glucagon to stimulate plasma membrane, but not Golgi membrane, adenylyl cyclase, and the near absence of high molecular weight, GTP-sensitive complexes in solubilized Golgi membranes demonstrate that plasma membrane contamination of Golgi fractions cannot account for the 125I glucagon binding.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013310 TI - Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides. AB - Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of 125I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of 125I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of 125I-labeled human FSH to testis receptor. PMID- 3013311 TI - Agonist-induced isomerization of the alpha 1-adrenergic receptor: kinetic analysis using broken-cell and solubilized preparations. AB - The affinity of agonists but not antagonists at hepatic membrane alpha 1 adrenergic receptors is temperature dependent; a 100-fold higher affinity is observed at 4 degrees C than at 37 degrees C. The relationship between these two agonist affinity states was investigated by using a strategy that allows the kinetics of this transition to be examined under equilibrium conditions. When competition assays are performed at 37 degrees C for varying intervals and the reaction mixture is then rapidly cooled by freezing, allowed to thaw, and further equilibrated at 4 degrees C, a rapid and progressive decrease (t1/2 of 1-2 min) in agonist affinity occurs, the extent of which is directly related to the incubation time at 37 degrees C. This decrease in agonist affinity is sustained as long as agonist is present but can be reversed by its subsequent removal. In contrast, no change in affinity is seen in identical experiments when antagonists are employed as the competing ligand. High-affinity binding of agonists is also demonstrated in short-term nonequilibrium experiments, indicating that the low temperature incubations do not induce, but rather stabilize, a receptor conformation of high affinity for agonists. These findings suggest that the predominantly low-affinity binding of agonists to alpha 1-adrenergic receptors demonstrated in equilibrium studies at physiological temperatures may be the result of a ligand-driven decrease in affinity. Since the transition in receptor affinity for agonists occurs not only in broken-cell preparations but also after detergent solubilization of the membrane receptor, it most likely is due to an agonist-induced change in the conformation of the receptor protein per se.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013312 TI - Direct formation of complexes between cytochrome P-450 and nitrosoarenes. AB - The mechanism of the formation of the complexes between various nitrosobenzenes and cytochrome P-450 has been investigated. We have observed the formation of these complexes by a new and, as yet, undescribed route. Nitrosobenzene (NOB) itself reacts with cytochrome P-450 in the iron(III) state, in the absence of any exogenous reducing agent, to produce the iron(II)-NOB complex. Apparently, NOB is a ligand that is capable of causing the spontaneous autoreduction of the iron. The reduction of the iron may occur via ligand-induced oxidation of the axially bound thiolate of cytochrome P-450. PMID- 3013314 TI - Beta-adrenergic stimulation enhances translocation, processing and synthesis of lipoprotein lipase in rat heart cells. AB - Cells isolated from newborn rat hearts were cultured for 10-14 days, and lipoprotein lipase activity was present in an intracellular and heparin releasable pool. Treatment of the cultures with 10(-7) M isoproterenol for 3 min resulted in a 3-fold increase in heparin-releasable lipoprotein lipase and a concomitant decrease in residual cellular enzyme activity. Similar results were obtained by treatment with dibutyryl cAMP. Treatment with isoproterenol or dibutyryl cAMP for 2 h affected glycosylation of immunoadsorbable lipoprotein lipase, so that the ratio of [3H]galactose to [14C]mannose in the heparin releasable enzyme increased from 3.8 (control) to 13.0 (isoproterenol-treated). The change in the ratio of the sugars in the cellular fraction of the enzyme was from 3.1 to 9.9. 2 h treatment with isoproterenol did not enhance new enzyme synthesis, as determined by incorporation of [3H]leucine into immunoadsorbable lipoprotein lipase. 24 h after addition of either isoproterenol or dibutyryl cAMP to the culture medium, stimulation of enzyme synthesis was demonstrated. The present results permit three effects of isoproterenol on lipoprotein lipase to be distinguished: stimulation of translocation from a cellular to heparin-releasable pool; enhanced processing of mannose residues and terminal glycosylation; stimulation of synthesis of enzyme protein. PMID- 3013313 TI - Regulation of Na+-H+ exchange by transmethylation reactions in rat colonic brush border membranes. AB - Incubation of rat colonic brush-border membrane vesicles with 200 microM S adenosyl-L-[Me-3H]methionine resulted in the labeling of both membrane phospholipids and proteins. This labeling was decreased approximately 50% by the methylation inhibitor S-adenosyl-L-homocysteine (2 mM). Utilizing the pH sensitive fluorescent dye, acridine orange, as a means of determining Na+-H+ exchange, S-adenosyl-L-methionine (200 microM) significantly increased sodium stimulated proton efflux in these vesicles at all concentrations of sodium (2.5 50 mM) tested. Examination of the kinetic parameters for sodium-stimulated proton efflux in the presence and absence of 200 microM S-adenosyl-L-methionine revealed that the methyl donor increased the Vmax for this exchange mechanism (expressed in arbitrary fluorescence units) by approx. 36% but did not influence its Km for sodium. S-Adenosyl-L-homocysteine (2 mM) inhibited S-adenosyl-L-methionine mediated stimulation of this exchange process. The results demonstrate that methylation of membrane phospholipids and/or proteins can modulate Na+-H+ exchange in rat colonic brush-border membrane vesicles. PMID- 3013315 TI - Comparison of the enzyme activities of phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol synthesis in freshly isolated type II pneumocytes and whole lung from the adult rat. AB - We compared the activities of enzymes of phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol synthesis in whole lung tissue and freshly isolated type II pneumocytes from adult rats. The activities of 1 acylglycerophosphocholine acyltransferase and CDPdiacylglycerol-glycerol-3 phosphate 3-phosphatidyltransferase were 2.9- and 4.4-fold higher, respectively, in type II cell sonicates than in whole lung homogenates. There was little difference between the type II cells and whole lung in the activities of choline kinase, choline-phosphate cytidyltransferase, cholinephosphotransferase, phosphatidate phosphatase, phosphatidate cytidylytransferase or CDPdiacylglycerol inositol 3-phosphatidyltransferase. Since the type II cell is the source of pulmonary surfactant, and disaturated phosphatidylcholine and phosphatidylglycerol are major components of surfactant, it is of interest that this cell is enriched in the activities of enzymes exclusively involved in the synthesis of these lipids. In view of possible proteolytic damage during isolation we compared freshly isolated type II cells with those cultured for 1 day. The rates of incorporation of [methyl-3H]choline and [2-3H]glycerol into phospholipids, L-[U-14C]phenylalanine into protein and [methyl-3H]thymidine into DNA were the same in the freshly isolated and cultured cells. The composition of the phospholipids synthesized from [2-3H]glycerol and sodium [1-14C]acetate were also the same. The freshly isolated cells were at least 90% pure and did not release significant amounts of lactate dehydrogenase. Since use of freshly isolated cells avoids cell loss during culture they provide an attractive alternative, particularly in studies requiring large amounts of material. PMID- 3013316 TI - Polyphosphoinositides undergo charge neutralization in the physiological pH range: a 31P-NMR study. AB - The charge state of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5 bisphosphate was determined as a function of pH by way of 31P-NMR spectroscopy. The pK values for the first protonation of the phosphomonoester residues in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were found to be 6.2 and 6.6, respectively, for the 4-phosphate moiety, and 7.7 for the 5-phosphate moiety. PMID- 3013317 TI - In vitro formation of bile acids from di- and trihydroxy-5 beta-cholestanoic acid in human liver peroxisomes. AB - The conversion of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-[3H]cholestanoic acid into cholic acid and 3 alpha,7 alpha-dihydroxy-5 beta-[3H]cholestanoic acid into chenodeoxycholic acid has been studied in subcellular fractions of human liver. The products were separated from the substrates by high-pressure liquid chromatography and identified by combined gas chromatography-mass spectrometry. The highest rates of conversion were found in the light mitochondrial fraction. This fraction also contained the highest amount of the marker enzymes for peroxisomes. The maximal rates of cholic acid and chenodeoxycholic acid formation were 1.3 and 1.8 nmol/mg protein per h, respectively. The presence of KCN in the incubation medium stimulated the formation of bile acids. Peroxisomes were prepared from the light mitochondrial fraction by sucrose-gradient centrifugation. By use of different marker enzymes, it was confirmed that the major part of the activity for cholic acid formation in the light mitochondrial fraction was located in the peroxisomes. It is concluded that liver peroxisomes are important for the oxidative cleavage of the C27 steroid side chain in bile acid formation in man. PMID- 3013318 TI - The role of calcium in the secretion of surfactant by rat alveolar type II cells. AB - Beta adrenergic agonists, tetradecanoylphorbol acetate, and the ionophore A23187 all stimulate surfactant secretion in type II cells isolated from rats. We found that combinations of these agonists cause augmented secretion, suggesting that the agonists may effect different steps in the secretory process. Previous studies have shown that cAMP is likely to be an intracellular 'second messenger' in type II cells. A23187, which has been reported to increase cAMP in some cell systems, did not increase the cAMP content of type II cells. We investigated the possible role of Ca2+ as another 'second messenger' by studying cellular 45Ca fluxes and the effect of extracellular calcium depletion on secretion. Depletion of extracellular calcium for as long as 3 h did not alter stimulated secretion, although basal secretion was increased. Secretagogues did not stimulate 45Ca influx from extracellular sources. A23187 and, to a lesser extent, terbutaline caused an acceleration of 45Ca efflux from type II cells. The addition of terbutaline or tetradecanoylphorbol acetate to A23187 further accelerated 45Ca efflux, suggesting that these agonists may act on separate calcium pools or by different mechanisms on the same calcium pool. Although secretion from type II cells is not inhibited by extracellular calcium depletion, the studies on 45Ca efflux suggest that Ca2+ plays a role in the regulation of surfactant secretion from isolated type II cells. PMID- 3013319 TI - Adipose conversion of cultured rat primary preadipocytes by hypolipidemic drugs. AB - Cultured rat epididymal preadipocytes exposed for 24-72 h to either bezafibrate or clofibrate added to the culture medium were extensively converted to fat loaded adipocytes. Adipocyte conversion increased during the first 5-7 days following plating, reaching a level of 100% and 60% conversion with bezafibrate and clofibrate, respectively, as compared to 10% conversion in their absence. Adipocyte conversion in culture was a saturable function of the hypolipidemic effectors and was associated with an increase in the incorporation rate of exogenous palmitate into triacylglycerols, in glycerol-3-phosphate dehydrogenase and hormone-sensitive lipase activities but not in lipoprotein lipase activity. Adipocyte conversion by hypolipidemic drugs was much more prominent than that exerted by dibutyryl cAMP, and the relative conversion efficiency of the two fibrate drugs did not correlate with their respective cAMP content of the culture. Hence, hypolipidemic drugs and dibutyryl cAMP appear to act independently in initiating adipose conversion in primary epididymal preadipocytes. PMID- 3013321 TI - Study of the superactivity of equine follicle-stimulating hormone in in vitro stimulation of rat Sertoli cells. AB - We have previously shown that equine follicle-stimulating hormone (FSH) stimulates plasminogen activator secretion in Sertoli cells at much lower concentrations than would be expected from its relative binding activity. We have introduced the term 'superactivity' to designate this particular behavior. In the present study, we show that equine FSH triggers a long-lasting (20 h) plasminogen activator secretion, whereas rat, porcine and ovine FSH as well as equine LH and equine choriogonadotropin (CG) provoke a short-term response (2.5 h). Moreover, equine FSH was also shown to be superactive in the stimulation of estradiol secretion and cyclic AMP production. This indicates that the step responsible for the long-term stimulation by equine FSH is not located beyond cAMP accumulation. Equine and porcine FSH were found to be equally stable during incubation with the cells demonstrating that equine FSH superactivity was not due to higher stability. Besides, phosphodiesterase inhibition led to a similar increase in the responses to both hormones. This rules out the possibility that equine FSH superactivity is due to less stimulation of phosphodiesterase activity. All these data strongly suggest that equine FSH exhibits superactivity in rat Sertoli cells by stimulating adenylate cyclase activity for a much longer period of time than do all other gonadotropins. The molecular mechanism of this outstanding behavior remains to be elucidated. PMID- 3013320 TI - Protein kinase C of human erythrocytes phosphorylates bands 4.1 and 4.9. AB - Addition of 10 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) to intact human erythrocytes results in rapid phosphorylation of two cytoskeletal components, bands 4.1 and 4.9. The synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, shows a similar effect, while the biologically inactive phorbol ester, 4 alpha-phorbol didecanoate, fails to enhance phosphorylation. That TPA and 1-oleoyl-2 acetylglycerol stimulate this phosphorylation suggests that protein kinase C is being activated. In the presence of TPA, bands 4.1 and 4.9 incorporate 1.5 mol Pi/mol protein and 1.2 mol Pi/mol protein, respectively. The pattern and extent of phosphorylation shows that it is not due to cAMP-dependent protein kinases, which also phosphorylate bands 4.1 and 4.9. Ca2+-phospholipid-dependent protein kinase activity is demonstrable in the soluble fraction of erythrocytes, and has been partially purified (2200-fold) from the hemolysate by affinity chromatography (Uchida and Filburn, 1984. J. Biol. Chem. 259, 12311-12314). The affinity purified erythrocyte kinase has a 42 A Stokes' radius and phosphorylates purified bands 4.1 and 4.9 in vitro in a Ca2+- and phospholipid-dependent manner. These results show that human erythrocytes contain protein kinase C, and that band 4.1 and 4.9 are the major endogenous substrates for this kinase. PMID- 3013322 TI - Heterologous desensitization by 1,25-dihydroxyvitamin D-3 of cyclic AMP response to parathyroid hormone in osteoblast-like cells and the role of the stimulatory guanine nucleotide regulatory protein. AB - The influence of 1,25-dihydroxyvitamin D-3 on the cAMP response to parathyroid hormone was studied in the osteoblast-like rat osteosarcoma cells ROS 17/2.8. The stimulation by parathyroid hormone of cAMP production in intact cells and of adenylate cyclase activity in isolated plasma membranes was attenuated by 1,25 dihydroxyvitamin D-3 treatment. This was associated with a reduction of the stimulatory guanine nucleotide regulatory protein, as demonstrated by a lower response to NaF and guanosine 5'-[beta, gamma-imido]triphosphate, and by a lower activity of solubilized plasma membrane extracts in the reconstitution assay. 1,25-dihydroxyvitamin D-3 blunted also the cAMP response to parathyroid hormone in cells incubated with the glucocorticoid dexamethasone, where a higher activity of the adenylate cyclase catalytic unit was observed. Thus, the two steroids appear to affect distinct levels of the adenylate cyclase system. Furthermore, the two hormones also showed an antagonistic effect upon the production of osteocalcin, an osteoblast-specific extracellular matrix protein. The release of this non-collagenous matrix protein by ROS 17/2.8 cells was increased by 1,25 dihydroxyvitamin D-3 and decreased by dexamethasone. PMID- 3013323 TI - [Computer modeling of ESR spectra of the plasma in patients with Down's syndrome]. AB - ESR technique at 77 degrees K was used for studying the blood plasma ESR signals of patients with Down syndrome and of healthy people. It was observed that the first exhibited a tendency towards a decrease of ESR signal with g = 4.3 and increase of the ratio of ceruplasmin (g = 2.05) and transferrin (g = 4.3) ESR signal amplitude. A computer simulation has been carried out by means of special mathematical program of experimental ESR spectra of the blood plasma. PMID- 3013324 TI - [Formation of free radicals during interaction of adriamycin and carminomycin with xanthine oxidase]. AB - Xanthine oxidase reduces carminomycin and adriamycin to the semiquinones which have been detected by ESR technique. The steady state carminomycin semiquinone concentration is some tens times higher than the corresponding value for adriamycin. This effect appears to be a result of carminomycin semiquinone stabilization due to internal hydrogen bonding. PMID- 3013325 TI - Long term memory storage capacity of multiconnected neural networks. AB - Quantitative expressions of long-term memory storage capacities of complex neural network are derived. The networks are made of neurons connected by synapses of any order, of the axono-axonal type considered by Kandel et al. for example. The effect of link deletion possibly related to aging, is also considered. The central result of this study is that, within the framework of Hebb's laws, the number of stored bits is proportional to the number of synapses. The proportionality factor however, decreases when the order of involved synaptic contact increases. This tends to favor neural architectures with low-order synaptic connectivities. It is finally shown that the memory storage capacities can be optimized by a partition of the network into neuron clusters with size comparable with that observed for cortical microcolumns. PMID- 3013326 TI - Polymer-hydroxyapatite composites for biodegradable bone fillers. AB - A number of composites made from biodegradable polymers and hydroxyapatite were studied in vivo and in vitro in an attempt to develop biodegradable artificial bone fillers. Histological observation in rats revealed that polylactic acid, of low molecular weight (PLAoligomer), was rapidly resorbed and replaced by newly formed bone tissue when incorporated with hydroxyapatite and this suggested that the incorporated hydroxyapatite seemed to play an active role in the new bone formation. In vitro testing revealed that the solubility of hydroxyapatite was markedly enhanced when mixed with PLAoligomer. PMID- 3013327 TI - [Suitability of piezo-quartz as a sensor for a frequency-adapting pacemaker system]. PMID- 3013328 TI - Scanning fluorescence correlation spectroscopy. II. Application to virus glycoprotein aggregation. AB - Scanning fluorescence correlation spectroscopy is a new approach to measuring changes in the state of aggregation of cell membrane proteins. Measurements of the mean number of aggregates of virus glycoproteins from Sindbis virus and vesicular stomatitis virus agree with the findings of a recent fluorescence photobleaching recovery study on the same systems (Johnson, D.C., M.J. Schlesinger, and E.L. Elson, 1981, Cell, 23:423-431). Sindbis Virus glycoproteins are immobilized and cannot be induced to aggregate further by antibody cross linking. In this study, we find that Sindbis virus glycoprotein is more highly aggregated than vesicular stomatitis virus glycoprotein, which can be patched further with antibody. These measurements demonstrate the potential of scanning fluorescence correlation spectroscopy in studies of aggregation problems in membranes of cultured cells. PMID- 3013329 TI - Saturation transfer electron paramagnetic resonance study of the mobility of myosin heads in myofibrils under conditions of partial dissociation. AB - The rotational motion of rigidly spin-labeled myosin heads of glycerinated myofibrils as reflected in saturation-transfer EPR spectra behaves to a first approximation as though the heads consist of two populations with different rotational motions. An immobilized fraction has a correlation time (tau 2) of approximately 0.5 ms, comparable to that of spin-labeled subfragment-1 (S1) bound to thin filaments, while a mobile fraction has a tau 2 of 10 microseconds, comparable to that of the heads of purified myosin filaments. The effects of nonhydrolyzable ATP analogues, potassium pyrophosphate (PPi), or adenylyl imidodiphosphate, Ca2+, temperature, or ionic strength on the spectra can be analyzed in terms of the fraction of myosin heads immobilized by attachment to thin filaments, without requiring changes in the motion of either attached or detached heads. PMID- 3013332 TI - Water molecule dynamics in hydrated lysozyme. A deuteron magnetic resonance study. AB - Proton nuclear magnetic resonance relaxation investigations of water dynamics in hydrated protein powders have the serious drawback that protein-water intermolecular dipolar interactions make the unambiguous interpretation of the results difficult. To circumvent this difficulty, deuteron spin-lattice and spin spin relaxation times in lysozyme powder hydrated with deuterium oxide were measured as a function of temperature and at two frequencies. Although the deuteron relaxation results are compatible with a water molecule dynamics model based on either a bimodal distribution of correlation times or anisotropic motion, a comparison of the present results with proton data suggests than an anisotropic motion model is more likely to provide a reasonable description of the water molecule motion. An analysis based on an anisotropic motion model that uses two correlation times to characterize the motion shows that most of the water molecules rotate about their twofold axis of symmetry at a rate that is only approximately 100 times smaller than the rate of isotropic diffusion in the bulk liquid. The reorientation of the twofold axis of symmetry itself is characterized by a correlation time of approximately 10(-7) s. PMID- 3013331 TI - Applications of new saturation transfer electron paramagnetic resonance methodology to the rotational dynamics of the Ca-ATPase in sarcoplasmic reticulum membranes. AB - The presence of small amounts of weakly immobilized probes can result in large systematic errors in the measurement of correlation times (tau r) from saturation transfer EPR spectra. However, we have recently developed experimental methodology to minimize these errors (Squier and Thomas, Biophys. J., 49:921 935). In the present study we have applied this methodology to the measurement of the rotational motion of the Ca-ATPase in sarcoplasmic reticulum. This analysis involves the estimate of tau r from line-shape parameters (spectral line-height ratios) and intensity parameters (spectral integral), coupled with digital subtractions to remove spectral components corresponding to weakly immobilized probes. We have analyzed the ST-EPR spectra of the Ca-ATPase over a range of temperatures and find that, unlike line-shape parameters, intensity parameters are little affected by the subtraction of the weakly immobilized spectral component (W). Thus, tau r values from intensity parameters are a more reliable measurement of rotational motion. As reported previously, an analysis with line shape parameters yields a nonlinear Arrhenius plot of protein mobility. However, the plot is linear when intensity parameters or corrected spectra are used, consistent with the theory for the hydrodynamic properties of a membrane protein of unchanging size and shape in a fluid bilayer. An analysis with line-shape parameters yields different effective tau r values in different spectral regions, and these tau r values are temperature-dependent. However, correction of spectra for W yields temperature-independent tau r ratios, indicating that the motional anisotropy is temperature-independent. Obtaining a good match for the weakly immobilized spectral component remains a major difficulty in the quantitative analysis of ST-EPR spectra using line-shape parameters. This study shows that intensity parameters can be used to avoid this problem, making the ST-EPR technique applicable in cases that were previously resistant to analysis. PMID- 3013330 TI - Methodology for increased precision in saturation transfer electron paramagnetic resonance studies of rotational dynamics. AB - Microsecond rotational motions of nitroxide spin labels are measured primarily with saturation transfer electron paramagnetic resonance (ST-EPR). In the present study we have used model system experiments to quantitatively evaluate different ST-EPR spectral parameters, both in-phase and out-of-phase, with an emphasis on techniques for suppressing the interference from weakly immobilized probes. Analyses of both systematic and random errors show that maximum sensitivity to small changes in correlation time and minimum ambiguity of interpretation are best achieved by combining measurements of both spectral line-shape, i.e., the ratio of line-heights, and spectral intensity, i.e., the absolute amplitude of either a position within a spectrum or a spectral integral. Errors in the measurement of correlation times for the two types of parameters tend to be complementary. Integrated intensity parameters are particularly useful in measuring microsecond probe motions in the presence of weakly immobilized components. We confirm that integrated intensity parameters are sometimes effective in rejecting signals from weakly immobilized probes, but the effectiveness of this rejection is more limited than previously supposed and depends on the type of parameter being measured. We describe procedures for evaluating and minimizing errors due to weakly immobilized probes, emphasizing the advantages of a new kind of intensity parameter obtained from integrated in phase spectra. We provide detailed descriptions of experimental procedures, along with calibration plots of the most useful spectral parameters vs. rotational correlation time, which should make it possible for workers in other laboratories, using different instruments and sample geometries, to reproduce spectra quantitatively and to make accurate correlation time measurements. PMID- 3013333 TI - Spin relaxation of iron in mixed state hemoproteins. AB - In hemoproteins the relaxation mechanism of iron is Orbach for high spin (HS) and Raman for low spin (LS). We found that in met-hemoglobin and met-myoglobin, under conditions in which the two spin states coexist, both the HS and the LS states relax to the lattice through Orbach-like processes. Alos, very short (approximately 1 ns) and temperature independent transverse relaxation times T2 were estimated. This may result from the unusual electronic structure of mixed states hemoproteins that allows thermal equilibrium and interconversion of the spin states. PMID- 3013335 TI - Specific binding of the C-peptide of proinsulin to cultured B-cells from a transplantable rat islet cell tumor. AB - Specific binding of the C-peptide of proinsulin was evaluated using a transplantable NEDH rat islet cell tumour predominantly composed of insulin secreting B-cells. Cultured tumour B-cells exhibited greater than 90% viability assessed by trypan blue exclusion, and retained the ability to form tumours with accompanying hypoglycaemia and hyperinsulinaemia after reimplantation. During binding experiments with synthetic rat C-peptide I and iodinated tyrosylated rat C-peptide I, tumour B-cells exhibited 54 +/- 6% specific binding. Displacement of tracer increased with increasing concentrations of unlabelled rat C-peptide I (0.25-1,000 ng/ml), and the specificity of binding was substantiated by reduced displacement with human C-peptide. Scatchard analysis of specific C-peptide binding revealed a curvilinear plot with upward concavity. The demonstration of specific C-peptide binding to insulin-secreting B-cells provides evidence for a physiological role of proinsulin C-peptide. PMID- 3013334 TI - Protein HMG1 is different from a DNA helix unwinding protein in calf thymus. AB - A number of criteria were used--chromatography on columns with single-stranded and double-stranded DNA, electrophoresis, peptide analysis, immunological tests and thermal denaturation of DNA--to show that protein (high mobility group) HMG1 and an unwinding protein from calf thymus are two distinct, unrelated proteins. While both proteins are thought to be related to DNA replication this might involve different mechanisms of action. PMID- 3013336 TI - Representation of DNA sequences by use of helical wheels to identify pattern formation with protein binding potential. AB - Similar to the protein segments with helical potential the three-dimensional structures of DNA sequences can be represented by a two-dimensional projection along the axis of the double helix. We will call this projection according to Schiffer and Edmundson "helical wheel". In the first place the wheels are employed here as graphical restatements of known sequences which represent binding sites of restriction-modification enzymes. Specific recognition of these sites has to be based on sequence related multi contact interactions of weak bonds. Because of the spatial and directional characteristics of H-bonds we will utilize the pattern formation of H-bond donor--and acceptor groups protruding into the major groove of the helix to identify the particular sequences. The characteristics of these patterns can be readily visualized and compared by examination of these wheels. PMID- 3013337 TI - Kinetic behavior of the multienzyme system of blood prostanoid synthesis. AB - A kinetic scheme of the prostacyclin-thromboxane system has been evolved on the basis of our own experimental material and the results described elsewhere. The kinetic behavior of the model has been analysed with the aid of computer technology by varying the following parameters: phospholipase activities, free arachidonic acid exchange rates between platelets and endothelium, prostaglandin H (PGH) synthetase biosynthesis rates, velocities of arachidonic acid pathways other than cyclooxygenase ones. It has been demonstrated that the biological system is capable of sustaining prostacyclin and thromboxane concentrations at steady fixed levels within a wide range of kinetic parameters. PMID- 3013338 TI - Receptor-level interrelationships of amino acids and the adequate amino acid type hormones in Tetrahymena: a receptor evolution model. AB - Histidine stimulates the phagocytosis of Tetrahymena to the same extent as histamine, and also stimulates its division, which histamine does not. Tyrosine and diiodotyrosine equally stimulate the growth of the Tetrahymena. Both amino acids inhibit the characteristic influence of the adequate amino acid hormone when added to Tetrahymena culture 72 h in advance of it. Primary interaction with diiodotyrosine and tyrosine notably increases the cellular growth rate. Histamine has a similar, although less notable effect than histidine. In the light of these experimental observations there is reason to postulate that the receptors of the amino acid hormones have developed from amino acid receptors. PMID- 3013339 TI - Cytomegalovirus-specific lymphocyte proliferation and in vitro cytomegalovirus IgG synthesis for diagnosis of cytomegalovirus infections after bone marrow transplantation. AB - With new techniques 19 bone marrow transplant (BMT) recipients were monitored for lymphocyte proliferation and specific IgG production in vitro by cytomegalovirus (CMV) antigen in solid phase. Twelve patients got a reactivated CMV infection as defined by virus isolation or serum IgG conversion. Lymphocyte proliferation and in vitro IgG production responses were significantly stronger in these 12 patients than in seven without ongoing CMV infection (P = .02). CMV infection was indicated by the lymphocyte responses at a mean of 45 days after BMT as against a mean of 79 days that passed before CMV growth in culture was detected (P less than .05). Lymphocyte proliferation and in vitro IgG production may thus be used as tools for diagnosis and for monitoring of CMV infections in BMT recipients. PMID- 3013340 TI - 3A1 (CD7) expression precedes T beta gene rearrangements in precursor T (lymphoblastic) neoplasms. AB - The phenotypes of early stages of T cell maturation are reflected by precursor T (lymphoblastic) neoplasms. In the present study, a series of such neoplasms was analyzed to reveal the developmental association of the expression of stage related cell surface markers and T cell receptor gene rearrangement. Rearrangements of the T cell receptor beta-chain (T beta) gene were found in most, but not all, cases (88%) of T cell lymphoblastic neoplasms. T beta gene rearrangement preceded surface expression of the T cell receptor-linked molecular complex T3. Of all monoclonal anti-T cell antibodies tested, only antibody 3A1 was capable of reacting with neoplastic cells from all cases irrespective of the occurrence of T cell receptor gene rearrangements. In contrast, markers T1 and T11, normally expressed by mature T cells, were absent from the neoplastic cells in many cases (73% and 60% positive cases, respectively). Thus, antibody 3A1 is a valuable probe for the identification of T lymphoblastic neoplasms since its target antigen is consistently expressed and does not require prior T beta gene rearrangement. Furthermore, expression of 3A1 prior to T beta gene rearrangement suggests that it may be a cell surface protein that participates in the triggering of T cell receptor gene rearrangement and expression. It is concluded that precursor T cell neoplasms display an early T cell development hierarchy that, in sequence, consists of 3A1 expression, T beta gene rearrangements, and surface T3 expression. PMID- 3013342 TI - Human T lymphotropic virus type III infection of human alveolar macrophages. AB - The human T-cell lymphotropic virus type III (HTLV-III) is the etiologic agent of the acquired immunodeficiency syndrome (AIDS) and preferentially infects T4 lymphocytes. Other cell types, notably B lymphocytes and other nonlymphoid cells, also have been reported to be infected in vitro by HTLV-III. We now report on the susceptibility of human pulmonary macrophages to infection with HTLV-III in vitro. Alveolar macrophages infected with HTLV-III produced low levels of virus that could be transferred to allogeneic human peripheral blood mononuclear leukocytes as long as 2 weeks after initiation of infection. Unlike HTLV-III infection of T lymphocytes, macrophages appeared more resistant to viral-mediated cytopathic effects. Primary cultures of pulmonary macrophages from two of four patients with AIDS spontaneously produced low levels of virus detected as precipitable reverse transcriptase activity, suggesting that these cells were infected in vivo. Because tissue macrophages are long-lived cells, they may act as a reservoir of HTLV-III, capable of transmitting the virus to other susceptible cells such as T lymphocytes, causing periodic low-level viremia. Macrophage infection with HTLV-III may be one mechanism for the establishment of viral persistence in infected hosts. PMID- 3013341 TI - Synthesis of eosinophil-associated enzymes in HL-60 promyelocytic leukemia cells. AB - Eosinophils derived from HL-60 cells share many of the abnormalities of granule histochemistry and morphology frequently seen in eosinophils of patients with certain malignancies, especially those seen in acute myelomonocytic leukemia with abnormal eosinophils (FAB class M4eo). In order to understand the pathogenesis of these abnormalities, four enzymes, characteristic of the eosinophil, were studied in HL-60 promyelocytic leukemia cells at various stages of eosinophilic differentiation. Using biochemical and ultrahistochemical techniques, the following differences from normal eosinophil development were demonstrated. First, both myeloperoxidase and eosinophil peroxidase coexisted in the population of maturing HL-60 eosinophils. Second, the granules formed from the condensation of material in vacuoles which were derived from dilated segments of the endoplasmic reticulum; the role of the Golgi apparatus in processing of peroxidase appeared minimal. Third, low levels of lysophospholipase and arylsulfatase were present in the cells compared to normal eosinophils. Finally, crystallizations resembling precursor structures of Auer rods appeared in the granules of about 5% of the cells. These findings suggest that several disorders of the control of protein synthesis and processing exist in HL-60 eosinophils which may be responsible for the abnormal granule morphology and histochemistry. PMID- 3013343 TI - Cytochemical, immunologic, chromosomal, and molecular genetic analysis of a novel cell line derived from Hodgkin's disease. AB - A novel cell line, KM-H2, was established from the pleural effusion of a patient with Hodgkin's disease of mixed cellular type. Multiple phenotypic studies were carried out with this cell line. Acid phosphatase and nonspecific esterase activities were detected. Rosette formation with T lymphocytes and the receptors for C3b and Fc portion of IgG were positive. Among the antigens tested with a total of 22 monoclonal antibodies defining hematopoietic cell subsets or lineages, Ki-1, Leu-M1, MCS1, HLA-DR, and OKT9 antigens were found to be positive. The other antigens reportedly specific for T cells, B cells, natural killer (NK) cells, monocytes, interdigitating reticulum (IR) cells and dendritic reticulum cells were negative. These phenotypic features were identical to those of the Sternberg-Reed (SR) and Hodgkin (H) cells in the fresh materials reported by other researchers. Moreover, the KM-H2 cells and the parental pleural effusion cells shared several structural chromosome anomalies. These findings indicated that the KM-H2 cells are derived from the SR and H cells. Molecular genetic analysis of the KM-H2 cells disclosed that the human immunoglobulin JH gene was rearranged but not the JK gene, and that the human T cell receptor beta chain gene was of the germline type. Based on these properties of the KM-H2 cells, Hodgkin's disease may be derived from a cell lineage other than T cell or B cell. PMID- 3013344 TI - Natural history of primary infection with LAV in multitransfused patients. By the AIDS-Hemophilia French Study Group. AB - In the course of a prospective study of asymptomatic, multitransfused subjects, seroconversion to human lymphadenopathy-associated virus (LAV/HTLV-III) occurred in 34 hemophilic and in two thalassemic patients. In subjects treated with procoagulant concentrates, primary infection, as evidenced by the development of antibodies to LAV, was a clinically silent event apart from moderate lymph node enlargement in 21% of cases. Concomitant immunologic disturbances mainly affected T lymphocyte subsets. This pattern contrasted with the major lymphadenopathy syndrome observed in the thalassemic patients who received washed erythrocytes from single donors positive for LAV antibodies. Four to 10 months after seroconversion, the incidence of lymphadenopathy reached 46% and the immunologic profile associated inverted T4+/T8+ lymphocyte ratio and markedly increased serum levels of IgG. In multitransfused hemophiliac patients, primary infection with LAV appears to provoke the following simplified sequence of events: decrease of T4+ and increase of T8+ cell counts preceding or concomitant with the occurrence of IgG LAV antibodies. Polyclonal elevation of IgG and lymph node enlargement occur weeks or months later. PMID- 3013345 TI - Effects of sodium vanadate on various types of vascular smooth muscles. AB - Effects of sodium vanadate on various vascular smooth muscles of guinea pigs, rabbits, and Wistar Kyoto rats (WKY) were studied. Sodium vanadate of concentrations higher than 10(-5) M induced contractions in the aortae of all animals. The contractile effects varied among vascular smooth muscles, and mesenteric arteries showed no or only weak contractile response to the drug, while aortae showed higher contractile responses. In the portal veins, potentiation of spontaneous contractions was observed by the application of sodium vanadate. These responses were not blocked by treatments with adrenergic blocking agents or indomethacin, indicating the direct action of the drug on vascular smooth muscles. Treatment with 4,4'-diisothiocyanostilbene-2,2' disulfonic acid (DIDS) blocked completely the contractile effects of sodium vanadate, whereas it showed no effect on K-contractures. Partial depolarization of the membrane by elevations of K concentration potentiated sodium vanadate induced contractions and minimized the variations of responses among preparations. In K-depolarized preparations, sodium vanadate often induced relaxation of preparations. The contractile effects of sodium vanadate were not blocked by treatment with ouabain, though ouabain also showed contractile actions in a number of preparations. It was suggested that vanadate acts directly on vascular smooth muscles and causes contractions without relation to the inhibition of Na,K-ATPase. It may cause contractions inhibiting Ca-ATPase of sarcoplasmic reticulum and/or of cell membrane, and cause relaxation by inhibiting ATPase of contractile proteins. The variations of the responses may be explained by differences of membrane permeability to vanadate. PMID- 3013346 TI - Immune thrombocytopenic purpura associated with hepatitis A. AB - A 23-year-old man developed thrombocytopenic purpura at the end of the second week of the clinical evolution of hepatitis A confirmed by viral markers. The bone marrow of this patient showed megakaryocytic hyperplasia. Circulating in his serum immune complexes were demonstrated by solid phase conglutinin enzymo immunoassay. Platelet-reactive serum factors were also detected by an indirect immunofluorescence test using fresh donor platelets as targets. The evolution of both the hepatitis and the purpura were benign with no therapy other than bedrest. Platelet count normalized within five weeks of the onset of purpura, and IgM antibodies against hepatitis A virus as well as circulating immune complexes dropped to normal levels. It is postulated that the thrombocytopenia of this case was caused by nonspecific deposition of immune complexes at the platelet surface. PMID- 3013347 TI - Advanced breast cancer after subcutaneous mastectomy and immediate prosthetic reconstruction for benign breast disease--a rare observation and a diagnostic problem. PMID- 3013348 TI - 8th San Antonio Breast Cancer Symposium--Plenary lecture. Autocrine and paracrine growth regulation of human breast cancer. AB - We consider the hypothesis that estrogen control of hormone dependent breast cancer is mediated by autocrine and paracrine growth factors secreted by the breast cancer cells themselves. Though we show direct, unmediated effects of estrogen on specific cell functions, we also provide evidence that human breast cancer cells secrete a collection of growth factors (IGF-I, TGF alpha, TGF beta, a PDGF-like competency factor, and at least one new epithelial colony stimulating factor). Some of these are estrogen-regulated in hormone dependent cells, and are constitutively increased in cells which acquire independence either spontaneously or by ras transfection. Collectively, the secreted growth factors are capable of promoting tumor formation by MCF-7 cells in nude mice, though not to the same extent as estrogens. There would seem to be potential for clinical intervention in the autocrine and paracrine control of breast cancer cells, including some cells which are no longer dependent on estrogens. PMID- 3013349 TI - Transferrin receptor is inversely correlated with estrogen receptor in breast cancer. AB - The transferrin receptor (TfR) has been identified as a marker for proliferation in cells in culture and can be accurately quantitated by specific radioimmunoassay. This study directly quantifies levels of TfR and compares them to levels of estrogen receptor (ER) and progesterone receptor (PgR) in biopsy material obtained from patients with infiltrating ductal carcinoma of the breast. A comparison of ER and TfR levels displayed an exponential distribution which was log-normalized to yield a linear inverse relationship (r = -.44). Although ER was strongly correlated with PgR, there was no correlation pattern between TfR and PgR. Multiple regression analysis indicated that 73% of ER levels could be predicted by a combination of the other two markers, PgR (representing degree of differentiation) and TfR (representing growth rate). Transferrin receptor levels were also found to be correlated (p less than .05) with menopausal status, with tumors from premenopausal patients exhibiting higher levels, whereas the opposite pattern was shown for estrogen receptor levels (p less than .02). Neither steroid receptor nor transferrin receptor levels were correlated to stage of disease or presence of nodal involvement. Addition of TfR level as an independent marker for proliferation may facilitate the decision-making process in the treatment of individual cases of carcinoma of the breast. PMID- 3013350 TI - Class distribution of immunoglobulin-containing plasma cells in the stroma of medullary carcinoma of breast. AB - A class distribution of plasma cells associated with the stroma in twenty-eight cases of medullary carcinoma of the breast was investigated by an unlabeled immunoperoxidase method. The stroma of the medullary carcinomas tested was found to contain predominantly IgG plasma cells except in two cases. Stroma of the other types of breast carcinoma, including ten cases of papillo-tubular carcinoma, five cases of scirrhous carcinoma, and six cases of medullary tubular carcinoma, contained predominantly IgG plasma cells, although few plasma cells were associated with carcinoma tissues in the latter group. Plasma cells associated with control specimens, including normal breast, fibroadenoma, cystic disease, and intraductal papilloma, were found to be predominantly of IgA type. Few carcinomatous epithelial cells contained secretory components in the cytoplasm, while a number of cells positive for secretory components were observed in acinar and ductular epithelia of normal breast tissues and in benign proliferative lesions of the breast. It is suggested that the lymphoid cells infiltrating the stroma of medullary carcinoma represent a sign of host immune response against the carcinoma cells which is related to the well-known favorable prognosis associated with this tumor. PMID- 3013352 TI - Understanding leukotrienes. AB - The leukotrienes of white cells are powerful pharmacological mediators that are implicated in many immunological reactions. The most powerful effects (bronchoconstriction and mucus secretion) are seen in asthma. The circulatory effects are local, except perhaps in endotoxin shock. Apart from corticosteroids, there are as yet no inhibitors in use in therapeutics. PMID- 3013353 TI - Reducing the mortality from breast cancer. AB - The mortality from breast cancer could be reduced if the disease could be prevented, cured by treatment not yet developed or identified at such an early stage that it could be cured by existing treatments. Although many aspects of breast cancer remain controversial, there are grounds for optimism in improving prognosis by early identification and treatment of the disease. This paper deals with "early" breast cancer, not advanced disease. While striking, even dramatic, remissions may be achieved by treating patients with advanced breast cancer, such treatment is palliative rather than curative, and reduction in mortality is unlikely to occur. PMID- 3013354 TI - Volatile substance abuse: a review of possible long-term neurological, intellectual and psychiatric sequelae. AB - The possibility that chronic abuse of volatile substances can cause permanent neurological, psychiatric, and intellectual sequelae is critically reviewed. Toluene, present in the commonly used adhesives, is most often implicated in 'glue sniffing'; this review focuses on its potential long-term effects. Many criticisms--particularly poor matching of control samples and inability to distinguish between acute and chronic effects--can be levelled at the available studies, while no adequate follow-up studies have been performed. In the light of present knowledge, the possibility that permanent structural brain damage, with accompanying psychiatric manifestations, results from solvent abuse remains inconclusive. PMID- 3013351 TI - Cytochemical localization of acid phosphatase and trimetaphosphatase activities in Kurloff cells. AB - After stimulation of guinea pigs with estradiol to increase their Kurloff cell number, spleen imprints were prepared in order to detect non-specific acid phosphatase (AcPase) activity by light microscopic cytochemistry using naphthol AS-BI phosphate as substrate and pararosanilin or fast garnet GBC as coupler. For ultracytochemistry, Kurloff cells were prepared from spleens by filtration through a homogeneizer screen followed by repeated centrifugation. AcPase and trimetaphosphatase activities were tested using beta-glycerophosphate, cytidine 5'-monophosphate and inorganic trimetaphosphate as substrates. Significant enzymatic activities were demonstrated with all the substrates used in the cytoplasmic inclusion body of the Kurloff cells. PMID- 3013355 TI - Primary signet ring cell adenocarcinoma of the urinary bladder with calculi. PMID- 3013356 TI - Peripheral neuropathy associated with dysproteinaemia, skin changes, and endocrinopathy. PMID- 3013359 TI - Epidemic of AIDS related virus infection among intravenous drug abusers. PMID- 3013358 TI - Oral contraceptives and hepatocellular carcinoma. PMID- 3013357 TI - Contribution of Gardnerella vaginalis to vaginitis in a general practice. AB - In a study of 154 adult women who presented to their general practitioner with vaginal symptoms 30 (20%) had Gardnerella vaginalis on its own and 51 (33%) had G vaginalis in combination with anaerobes or known pathogens. Thirty one (20%) patients were culture negative. Those who were culture negative had fewer symptoms and signs of vaginitis than those with G vaginalis alone or G vaginalis plus anaerobes. Those with known pathogens had more symptoms and signs than those with G vaginalis alone or G vaginalis plus anaerobes. Those with known pathogens plus G vaginalis had the most severe signs and symptoms of vaginitis. It is concluded that G vaginalis can cause vaginitis on its own, and it makes vaginitis worse when present with other organisms. G vaginalis was also found in 30 (21%) of the 138 control patients who, although they presented "asymptomatically," had worse signs than control patients without G vaginalis. It seems that G vaginalis can occur in a spectrum ranging from the uncomplaining patient to those with severe vaginitis. PMID- 3013360 TI - A neuronal model of vowel normalization and representation. AB - A speculative neuronal model for vowel normalization and representation is offered. The neurophysiological basis for the premise is the "combination sensitive" neuron recently documented in the auditory cortex of the mustached bat (N. Suga, W. E. O'Neill, K. Kujirai, and T. Manabe, 1983, Journal of Neurophysiology, 49, 1573-1627). These neurons are specialized to respond to either precise frequency, amplitude, or time differentials between specific harmonic components of the pulse-echo pair comprising the biosonar signal of the bat. Such multiple frequency comparisons lie at the heart of human vowel perception and categorization. A representative vowel normalization algorithm is used to illustrate the operational principles of the neuronal model in accomplishing both normalization and categorization in early infancy. The neurological precursors to a phonemic vocalic system is described based on the neurobiological events characterizing regressive neurogenesis. PMID- 3013361 TI - Effects of chronic dietary administration of the cholinergic false precursor N amino-N,N-dimethylaminoethanol on behavior and cholinergic parameters in rats. AB - The choline analog, N-amino-N,N-dimethylaminoethanol (NADe), was fed ad libitum (chloride salt; 0.5%) to weanling rats in a low choline, low methionine synthetic diet. Control rats were fed choline chloride (0.5%) in place of NADe. Initial observation and behavioral screen tests of grasp strength, startle reflex, righting reflex, analgesia (hot plate test) and body temperature did not reveal any toxic effects caused by NADe, although both experimental and control groups gained weight more slowly than rats fed standard lab chow. After 25 days on the diet, the performance of rats fed NADe in a one-trial passive avoidance test was significantly impaired compared to control rats. There was no difference between experimental and control rats in sensitivity to foot shock or in activity monitored in a closed field. A subjective, 6-component behavioral rating scale indicated rats fed NADe were resistant to handling but not aggressive. These behavioral results were similar in two separate feeding experiments using deuterium-labeled and unlabeled NADe. The twitch response of isolated rat phrenic nerve-diaphragms during stimulation did not show any impairment of neuromuscular function in rats fed NADe. Receptor binding experiments indicated there were no differences between experimental and control rats in tritiated quinuclidinyl benzilate [( 3H]QNB) binding capacity in cortex, heart and ileum. Competitive [3H]QNB binding with carbachol indicated there was no difference in the IC50's measured in cortex homogenates. Acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activities in cortex were similar in experimental and control groups.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013362 TI - Induced homotypic sprouting of serotonergic fibers in hippocampus. II. An immunocytochemistry study. AB - The dorsal hippocampus of the rat normally receives its 5-HT innervation from two homologous groups of cells in the median raphe nucleus via the cingulum bundle induseum griseum (CB-IG) and the fornix-fimbria (FF) (J. Comp. Neurol., 179 (1978) 641-667 and Brain Res. Bull., 10 (1983) 445-451). 5-HT immunoreactive (IR) fibers are distributed in a laminar pattern in the hippocampus. These fibers have large varicosities and are densely distributed in the infragranular layer of dentate gyrus, in the stratum lacunosum-moleculare of the cornu Ammonis and in the area fasciola cinerea (FC). The present study provides evidence that the density of the 5-HT-IR fibers in the dorsal hippocampus is greatly decreased but maintained a similar laminar pattern 3 days after lesioning by microinjection of 4 micrograms of 5,7-dihydroxytryptamine (5,7-DHT) into the CB-IG. An apparently normal density and distribution pattern of the 5-HT-IR fiber is seen by 42 days postlesion. The FC in the hippocampus is among the first regions reinnervated by 5-HT-IR fibers with very dense and large varicosities. The restitution of 5-HT-IR fibers in the dorsal hippocampus after the 5,7-DHT lesion in the CB-IG is accompanied by a marked increase in the number and intensity of 5-HT-IR fibers in the FF. No evidence of a regeneration of 5-HT-IR fibers is seen distal to the injection site in the CB-IG. These observations provide direct evidence for homotypic collateral sprouting in the CNS induced by removal of a single fiber type. PMID- 3013363 TI - Activation of GABA receptors in the hypothalamus stimulates secretion of growth hormone and prolactin. AB - Localized intracerebral microinjections of GABA, muscimol, picrotoxin and bicuculline were made in the anterior and basal hypothalamus to determine possible sites of action of GABA in the regulation of prolactin and growth hormone (GH) secretion. Studies were carried out in unanesthetized male rats with chronic indwelling atrial cannulae and intracerebral guide cannulae which permitted stress free blood sampling and intrahypothalamic injections, respectively. Preoptic/anterior hypothalamic area. (PO/AHA) injection of muscimol (0.16 nmol) stimulated both prolactin and GH secretion. GABA (1600 nmol) stimulated prolactin. Bicuculline (0.016 and 0.16 nmol) inhibited GH secretion. Medial basal hypothalamic (MBH) injection of muscimol (0.1 and 1.0 nmol) and GABA (1000 nmol) stimulated prolactin but had no effect on GH secretion. Picrotoxin and bicuculline did not stimulate GH. These findings indicate that activation of PO/AHA GABAergic receptors facilitates secretion of GH and prolactin and activation of MBH GABAergic receptors stimulates secretion of prolactin. It is proposed that GABA inhibits somatostatin neurons in the PO/AHA to facilitate GH and inhibits tuberoinfundibular dopamine or GABA neurons in the MBH to stimulate prolactin. PMID- 3013364 TI - Autoradiographic localization of high affinity GABA, benzodiazepine, dopaminergic, adrenergic and muscarinic cholinergic receptors in the rat, monkey and human retina. AB - High affinity gamma-aminobutyric acid, benzodiazepine, strychnine (glycine), dopamine, spirodecanone, alpha 1-adrenergic, alpha 2-adrenergic, beta-adrenergic and muscarinic cholinergic binding sites were localized by semiquantitative autoradiography in rat and, in some instances, in monkey and human retinae using [3H]muscimol, [3H]flunitrazepam, [3H]strychnine, [3H]spiperone, [3H]prazosin, [3H]para-aminoclonidine, [3H]dihydroalprenolol and [3H]quinuclidinyl benzylate, respectively. In nearly every case, the inner plexiform layer (IP) contained a high receptor density. The distribution of alpha 1 sites was unusual in that binding was concentrated in the outer plexiform layer (OP). Dopaminergic and, to a lesser extent, beta-adrenergic binding was diffusely distributed in the outer nuclear layer, the OP, the inner nuclear layer and the IP. The ganglion cell layer displayed significant benzodiazepine binding. The intraretinal distribution of pre- and postsynaptic markers of these neurotransmitters is discussed. PMID- 3013365 TI - In vivo and in vitro studies on the regulation of cholinergic neurotransmission in striatum, hippocampus and cortex of aged rats. AB - Young (3 months) and senescent (23 months) rats were challenged with oxotremorine both in vivo, to determine its effects on acetylcholine content in hemispheric regions, and in vitro, to assess its action on K+-evoked release of ACh from brain synaptosomes. The drug failed to inhibit KCl-induced [3H]ACh release from the P2 fraction of striatal and hippocampal homogenates of the senescent animals, whereas it was less efficient in increasing striatal ACh content. In contrast, oxotremorine was still able to stimulate an increase in ACh in the hippocampus and cerebral cortex of the aged rats to the same extent as it did in the young ones. The [3H]ACh output from striatal synaptosomes was lower in old rats with respect to young ones at low KCl depolarizing concentrations but was equal in the two groups at a high depolarizing concentration. In the hippocampus of the senescent rats, the release was significantly lower at each concentration of KCl used, resulting in a parallel downward-shift in the concentration-release plot. We also measured cholinergic muscarinic receptor binding in rat hemispheric regions using the radioligand [3H]dexetimide, a classical non-selective muscarinic receptor antagonist. It was found, in conformity with some of the literature, that receptor binding was decreased by about 32% in striatum of aged female rats as compared to younger rats. Changes were not observed in cortex and hippocampus. Analysis of the binding data indicated that the observed decrease in specific ligand binding was due to a decrease in the number of binding sites without a change in affinity. The results favor, once again, the cholinergic hypothesis for geriatric dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013366 TI - Autoradiographic localization of calcitonin gene-related peptide binding sites in human and rat brains. AB - 125I-calcitonin gene-related peptide (CGRP) binding sites were mapped in the human brain and rat brains by in vitro macroautoradiography, and compared to each other. Binding experiments were made to characterize 125I-CGRP binding on the human and rat brains. Scatchard analysis of saturation experiments from slide mounted sections of the human and rat cerebellum displayed 125I-CGRP binding sites with a dissociation constant (Kd) of 0.17 nM and 0.11 nM, respectively, and a maximal number of binding sites (Bmax) of 96.8 fmol/mg and 23.0 fmol/mg protein. 125I-CGRP binding was time-dependent, reversible and saturable with high affinity in the brains. Autoradiograms showed a discrete distribution of 125I CGRP binding sites throughout the brains of human and rat with patterns similar to each other. In the human brain, the highest binding was seen in the cerebellum, inferior olivary nuclear complex, certain parts of the central gray matter, arcuate nuclei of the medulla oblongata and dorsal motor nucleus of the vagus, and densities of CGRP-binding sites were high in the nucleus accumbens, amygdala, tail of the nucleus caudatus, substantia nigra, ventral tegmental area, medial portion of the inferior colliculus, medial pontine nuclei, locus coeruleus, inferior vestibular nucleus, substantia gelatinosa of the spinal trigeminal nucleus, nucleus of the solitary tract and nucleus cuneatus lateralis. In the rat, high densities were found in the hippocampus pars anterior, nucleus accumbens, ventral and caudal portions of the nucleus caudatus-putamen, central and basolateral nuclei of the amygdala, caudal portion of the insular cortex, medial geniculate body, superior and inferior colliculi, certain portions of the central gray matter, locus coeruleus, inferior olivary nuclei, vagal complex, nucleus cuneatus lateralis and cerebellum. In contrast, in both species, most of the cortical areas including the hippocampus, most of the thalamus, and hypothalamus exhibited few binding sites. In addition, high quantities of the binding sites were seen on the pia mater and on walls of blood vessels in the brain and subarachnoidea. These results revealed essentially homologous locations of CGRP binding sites in the human and rat central nervous systems and well corresponding distributions of binding sites and endogenous CGRP-like immunoreactivity. PMID- 3013367 TI - Correlation between pentobarbital suppressed cerebellar cyclic GMP and performance of a swimming task. AB - Ovariectomized female Sprague-Dawley rats were trained to swim a 2.5 m course. On the day of the experiment the time required for each animal to swim the course was determined, and then the animal received either saline or one of 4 dosages of sodium pentobarbital, intravenously. Four minutes after treatment the animal's swim time was again recorded, and the animal was immediately killed by microwave irradiation. The cerebellum was collected for subsequent determination of cyclic guanosine monophosphate (cGMP). Pentobarbital caused a dosage-dependent suppression of cerebellar cGMP which was highly correlated with a drug-induced increase in the time required to swim 2.5 m. These data provide the first evidence of a correlation between a barbiturate-induced effect on a neurochemical parameter and impaired performance of a learned behavior. PMID- 3013368 TI - Effect of ACTH-like peptides on morphine-induced hypothermia in unrestrained guinea pigs. AB - Peripheral treatment with adrenocorticotropin (1-24) (ACTH1-24), at different doses and sequences, consistently antagonized the decrease in body temperature produced by morphine in the freely moving guinea pig, whereas adrenocorticotropin (4-10) (ACTH4-10), which lacks corticotrophic activity, was partially effective only when it was administered in a high dose 24 h prior to morphine. Centrally administered ACTH1-24 completely prevented the hypothermic effect of intracerebroventricularly (i.c.v.)-injected morphine. Likewise, the i.c.v. administration of ACTH4-10 was equally effective in blocking the i.c.v. morphine induced hypothermia. Neither ACTH1-24 nor ACTH4-10 did produce changes in body temperature. These results suggest that peripherally administered ACTH1-24 antagonizes indirectly the actions of morphine through the release of adrenal corticosteroids, whereas centrally injected ACTH1-24 or ACTH4-10 act as direct antagonists of morphine effects through opioid receptors. PMID- 3013369 TI - Decrease in rat striatal dopamine synthesis and metabolism in vivo by metabolically stable adenosine receptor agonists. AB - The administration of the stable adenosine analog (R)-N-(1-methyl-2 phenylethyl)adenosine (R-PIA) caused a dose-dependent decrease in the accumulation of striatal dihydroxyphenylalanine (DOPA) levels following DOPA decarboxylase inhibition, with the minimum effective dose being 0.2 mg/kg, i.p.; 5'-deoxy-5'-(ethylamino)-5'-oxoadenosine (NECA) at 0.5 mg/kg, i.p., was also active indicating that in vivo R-PIA and NECA were decreasing striatal dopamine (DA) synthesis. Both R-PIA and NECA also decreased striatal levels of the DA metabolite 3-methoxytyramine (3-MT), indicating a decreased release of DA which was consistent with their effects on DA synthesis. N-Cyclohexyladenosine (CHA) also decreased 3-MT levels. The S-isomer of PIA at an equipotent dose did not affect either DA synthesis or release. R-PIA (3 mg/kg, i.p.) antagonized the pargyline-induced increase in striatal 3-MT levels as did gamma-butyrolactone, further confirming a decreased release of striatal DA. The adenosine receptor antagonist 8-cyclopentyltheophylline antagonized the R-PIA induced-decrease in striatal DA synthesis, suggesting that the latter was mediated via adenosine receptors. It is concluded that the stable adenosine analogs R-PIA and NECA, at behaviorally active doses, are decreasing in vivo the rate of DA synthesis and release from rat striatal DA nerve terminals by an adenosine receptor-mediated effect. PMID- 3013370 TI - A comparison of 2-amino-4-phosphonobutyric acid (AP4) receptors and [3H]AP4 binding sites in the rat brain. AB - The glutamate analogue 2-amino-4-phosphonobutyric acid (AP4) is a potent antagonist at several synapses where an excitatory amino acid appears to be the neurotransmitter. Previous studies identified a Cl-/Ca2+ dependent [3H]glutamate binding site in synaptic plasma membrane (SPM) preparations that was also labeled by [3H]AP4 and exhibited a pharmacology similar to the AP4 receptor. This report examines the pharmacological specificity in both biochemical and electrophysiological preparations in greater detail. Several compounds are identified which readily interact with the apparent binding site in membranes, but neither mimic nor inhibit the action of AP4 in electrophysiological studies. The rate of dissociation of [3H]AP4 from SPMs is shown to increase in the presence of added AP4 and increasing the osmolarity in the SPM binding assay decreases the level of observed [3H]AP4 binding. These findings indicate both a heterogeneous population of binding sites and the occurrence of transport. It is concluded that much of the AP4 binding observed in SPM preparations is to a site other than the AP4 receptor. The results provide a further pharmacological description of AP4 receptors which should facilitate the identification of the receptor in biochemical preparations. PMID- 3013371 TI - Regulation of lordosis behaviour in the female rat by corticotropin-releasing factor, beta-endorphin/corticotropin and luteinizing hormone-releasing hormone neuronal systems in the medial preoptic area. AB - The role of corticotropin-releasing factor (CRF) and opiocortin neuronal systems and a possible functional relationship between the two in the control of luteinizing hormone-releasing hormone (LH-RH) activity in the medial preoptic area (MPOA) for the regulation of lordosis behaviour were assessed in ovariectomised oestrogen-progesterone-treated female rats. Lordosis behaviour (assessed as the lordosis quotient) triggered by male mounting was significantly inhibited by either CRF or beta-endorphin infused into the MPOA in animals treated with normal doses of oestradiol benzoate (OEB) (5 micrograms) and progesterone (500 micrograms). Saline-treated animals exhibited high levels of lordosis. The inhibition of lordosis produced by either CRF or beta-endorphin could be reversed by LH-RH microinfusions into the MPOA. While naloxone pretreatment of the MPOA site prevented the inhibitory effects of beta-endorphin, neither the opiate antagonist nor anti-beta-endorphin-gamma-globulin (even in high concentrations) infused into the MPOA was effective in completely preventing the inhibition of lordosis produced by CRF. These findings suggest that the inhibition of LH-RH neuronal activity and lordosis behaviour by CRF may be due to a direct action and may not be the result of activation of beta-endorphin release. The possibility that the two peptidergic systems may act in a synergistic fashion is supported by the data showing that combined CRF-beta endorphin treatment in the MPOA completely abolished lordosis. This is further supported by the finding that CRF totally abolished lordosis in animals pretreated with anti-corticotropin (ACTH-gamma-globulin although this result could suggest that CRF could preferentially stimulate the release of ACTH in the MPOA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013372 TI - Influence of deuterium oxide on circadian activity rhythms of hamsters: role of the suprachiasmatic nuclei. AB - The period of the free-running circadian activity rhythm of Syrian hamsters was measured before and during treatment with 10% deuterium oxide (D2O). Deuteration increased period length by approximately 0.5 h per cycle both pre- and postoperatively in hamsters sustaining complete, incomplete or no unilateral lesions of the suprachiasmatic nuclei (SCN). Neither coupling between the bilaterally paired SCN, nor elimination of 50% of SCN tissue affected period length during D2O treatment. However, variability of the response to D2O was much greater in lesioned than in intact hamsters. We propose that a small percentage of the normal complement of SCN neurons is sufficient to permit full responsiveness of the circadian system to D2O and that there is substantial redundancy in the neural system that responds to deuterium. Stability of the circadian system appears to be increased by the full complement of SCN neurons. PMID- 3013373 TI - Behavioral evidence for a crossed ascending pathway for pain transmission in the anterolateral quadrant of the rat spinal cord. AB - Thresholds of two behavioral responses to noxious pressure of the hindpaws (withdrawal, vocalization) were analyzed before and 3 weeks after a lesion of various quadrants of the spinal cord at the cervical level. The threshold of the spinal reflex could not be modified. Threshold of vocalization elicited by the pressure of one hindpaw was significantly increased when, and only when, the opposite ventrolateral quadrant was cut. These results emphasize the role of the lateral spinothalamic tract in the transmission of noxious messages. PMID- 3013374 TI - Percentage of relay and intrinsic neurons in two sensory thalamic nuclei projecting to the non-cortical telencephalon in reptiles Caiman crocodilus. AB - Large injections of horseradish peroxidase were placed in the non-cortical telencephalon in a reptile, Caiman crocodilus. The number of retrogradely labeled and unlabeled neurons in two sensory thalamic nuclei, nucleus rotundus (vision) and nucleus reuniens pars centralis (audition) were counted. The percentage of unlabeled cells in each nucleus was less than 1%. These data suggest that each of these thalamic nuclei contain few, if any, intrinsic or local circuit neurons. PMID- 3013375 TI - Distribution of kainic acid receptor binding sites in the goldfish CNS: evidence for the existence of intracellular sites. AB - The density of binding sites for kainic acid was measured both in fresh slices and in the particulate fraction of the homogenate from various zones of the CNS of goldfish. Binding in the homogenate fraction was always found higher than in the corresponding slices, which should be representative of receptors located on the outer surface of the cellular membrane. The hypothesis is discussed that the sites demonstrated by homogenization are located in the intracellular compartment. PMID- 3013376 TI - Benzodiazepine receptor involvement in the control of receptive field size and responsiveness in primary somatosensory cortex. AB - Microiontophoretic ejection of 3 benzodiazepines (BZDs), flurazepam, diazepam and midazolam, and of Ro 15-1788 to primary somatosensory cortical neurons altered the nature of the responses evoked by natural stimulation of the cutaneous surface. BZDs raised the thresholds to tactile stimulation and produced a decrease of the receptive field areas of neurons situated in rapidly adapting submodality regions of S1 cortex; these effects of the BZDs were exerted at different apparent potencies. Ro 15-1788 antagonized the BZD-induced receptive field size decreases and threshold changes. When administered alone, this substance sometimes lowered the threshold for tactile activation and slightly enlarged the receptive field size. PMID- 3013377 TI - The vestibulothalamic connections in the rat: a morphological analysis using wheat germ agglutinin-horseradish peroxidase. AB - The vestibulothalamic connections were studied in the rat using wheat germ agglutinin-horseradish peroxidase (WGA-HRP). The distributions of anterograde labelling of fibers and terminals in the brainstem and the thalamus were analyzed by injecting WGA-HRP into the superior (SVN) and lateral (LVN) vestibular nuclei, and the medial (MVN) and inferior (IVN) vestibular nuclei. The distributions of retrograde labelling of cells were analyzed in the vestibular nuclear complex by injecting WGA-HRP into the thalamus centered in the central lateral nucleus (CL), ventral posterolateral nucleus (VPL), and rostral part of the dorsal medial geniculate nucleus (rMGd). The vestibular projection to the CL via the medial longitudinal fasciculus (MLF) and the ascending tract of Deiters (ATD) originates mainly in the contralateral MVN and ipsilateral SVN. The vestibular projections to the VPL and the ventral lateral nucleus (VL) via MLF, ATD and superior cerebellar peduncle (SCP) originate mainly in the MVN and SVN, bilaterally. The projection to the rMGd via the lateral lemniscus-inferior collicular brachium, and MLF (and SCP) originates in the contralateral IVN. PMID- 3013378 TI - Alpha adrenergic system in medial preoptic area involved in sleep-wakefulness in rats. AB - The study is aimed at investigating the possible involvement of adrenergic mechanisms in the medial preoptic area (mPOA) for modulation of sleep-wakefulness in rats. In this study, saline, norepinephrine (NE), phenoxybenzamine (PBZ) and propranolol (PROP) were injected in the mPOA in different groups of male rats during the day and night. NE and PBZ were injected, during the day and the night respectively, in some control areas adjoining the mPOA in two other groups of animals. Arousal was produced by NE, and sleep by PBZ when they were applied in the mPOA. All other procedures, including application of NE and PBZ in the control areas and beta blocker (PROP) in the mPOA, did not produce alterations in sleep-wakefulness. These findings provide support for a physiological role played by the alpha adrenergic system in the mPOA for arousal, and area specificity of action of this system. PMID- 3013379 TI - The influence of capsaicin sensitive neurons on stress-induced release of ACTH. AB - Changes in the plasma levels of ACTH in response to cold exposure or restraint stress were measured in adult rats which had been pretreated with capsaicin or vehicle as neonates. There was no difference in basal ACTH levels between capsaicin and vehicle pretreated animals. Following restraint stress, ACTH levels rose similarly in vehicle and capsaicin pretreated rats, indicating that the pituitary-adrenal system is not impaired by capsaicin pretreatment. However, following cold exposure ACTH levels rose only in control animals whereas no change was observed in capsaicin pretreated animals. It is concluded that capsaicin-sensitive afferent neurons participate in the cold stress-induced increase of plasma ACTH levels. PMID- 3013380 TI - [Direct interaction of tricyclic antidepressants with opiate binding sites in the bovine adrenal medulla]. AB - This note reports the interaction of three currently used tricyclic antidepressant drugs (clomipramine, imipramine and amitriptyline) with delta, mu and kappa opioid binding sites in the bovine adrenal medulla. Clomipramine was the only drug interacting with delta and mu sites. On the contrary, all three drugs showed a significant interactions with subtypes of the kappa binding site. Clomipramine was the most active on the kappa 2 and kappa 3 subtypes while amitriptyline showed the highest interaction with the kappa 1 subtype. On the contrary the tricyclic cyproheptadine did not present any interaction with opioid binding sites in our system. This interaction between tricyclic antidepressants and opioid binding sites might be the origin of their analgesic action. PMID- 3013382 TI - [LAV type II: a second retrovirus associated with AIDS in West Africa]. AB - A second retrovirus, named LAV-II, has been isolated from two West-African patients with AIDS. By its genomic sequences and its proteins, this virus is different from the LAV-I/HTLV-III, isolated from U.S.A., Europe and Central Africa. It differs also from STLV-III, isolated from Rhesus Macaques with AIDS, but displays an antigenic relationship with the latter virus, at the level of its external envelope protein. The tropism of LAV-II for T4 lymphocytes and the induction of cytopathic effect in infected cells are similar to those of LAV-I. PMID- 3013381 TI - [Mammalian lignans: possible involvement in endogenous digitalis activity]. AB - Lignans are natural products, some of which were recently discovered in animal urines, semen and blood plasma. We investigated the actions of animal lignans obtained by total synthesis or extracted from urines of pregnant women on Na+, K+ ATPase in human red cells and human and guinea-pig heart cell membranes. Some of the tested lignans (enterolactone, prestegane B and 3-O-methyl enterolactone) inhibited Na+, K+-pump activity in human red cells with IC50 ranging from 5 to 9 X 10(-4) M. The IC50 for ouabain (7 X 10(-7) M) was not modified by addition of lignans. Enterolactone inhibited Na+, K+-ATPase activity in human and guinea pig heart membranes. It also displaced [3H]-ouabain binding from human heart with IC50 = 1.5 X 10(-4) M. The apparent dissociation rate constants (kd) of [3H] ouabain were not different in presence of digoxin or enterolactone. Enterolactone exhibited a poor cross reactivity against antidigoxin antibodies. The aglycones of the lignans studied here were slight inhibitors of the Na+, K+-ATPase. However, we cannot exclude that a glycosyl- (and/or butenolide-) derivative of enterolactone could be one "endogenous ouabain-like" factor. PMID- 3013383 TI - [Adrenocortical function and metastatic breast cancer]. PMID- 3013384 TI - Reorientable electric dipoles and cooperative phenomena in human tooth enamel. AB - A preliminary investigation of electric dipole reorientability in human tooth enamel (TE) in comparison to that in hydroxyapatite (OHAp) has been made with the fractional-polarization form of the thermally stimulated currents (TSC) method. The reorientable dipoles are the structural OH-ions. The OHAp exhibited compensation phenomena at 211.5 degrees C and at 356 degrees C which are associated here with the hexagonal form becoming quasi-statically stabilized and dynamically stabilized, respectively, against the monoclinic form. TE specimens were pretreated at various temperatures. All showed the onset of cooperative motions that could quasi-statically stabilize the hexagonal form at the same temperature, approximately 212 degrees C, as did OHAp, even though the TE was already statically stabilized in the hexagonal form. Parts of the TSC spectra that did not conform to the 212 degrees C compensation changed progressively with pretreatment temperature. Loss of incorporated H2O is identified as the most probable cause of most of these changes. This work shows considerable promise for TSC as a tool for further quantitative investigation of TE. PMID- 3013385 TI - Calcium homeostasis. II: The sodium-potassium pump. PMID- 3013386 TI - Effects of marijuanna on schizophrenics. PMID- 3013387 TI - Susceptibility of Haemophilus aegyptius to trooleandomycin: lack of taxonomic value. AB - Two hundred and nine strains of Haemophilus influenzae and Haemophilus aegyptius were screened for trooleandomycin susceptibility. Four strains were shown to be sensitive to the drug. Of these four, two were Haemophilus aegyptius (ATCC 11116, NCTC 8134), and the other two were Haemophilus influenzae biotype I (1-605) and IV (80-212. One strain of Haemophilus aegyptius (NCTC 8135) was resistant to trooleandomycin. Restriction enzyme assays and DNA homology were carried out to establish relationships between the strains. It is concluded that trooleandomycin susceptibility has no taxonomic value to differentiate between Haemophilus aegyptius and biotype III Haemophilus influenzae. PMID- 3013388 TI - Malignant intracranial fibrous histiocytomas. Histologic, ultrastructural and immunohistochemical studies of two cases. AB - Two cases of malignant intracranial fibrous histiocytoma are presented. In Case 1 the tumour arose from the meninges and showed a disseminated spread throughout the neuroaxis. In the second case the tumour appeared to arise from within the brain substance. In this case surgical intervention and radiotherapy appeared to have achieved a cure, since no residual tumour was found at autopsy. The tumours were examined using ultrastructural and immunohistochemical techniques, which appeared advantageous in delineating this rare tumour from other intracranial neoplasms. PMID- 3013389 TI - Cell features and patterns in fine-needle aspirates of hepatocellular carcinoma. AB - The values of the cytologic features of individual cells and cellular patterns in aspirated materials in the diagnosis of 49 hepatocellular carcinomas (HCC) were investigated. Excellent cytologic specimens were obtained by percutaneous aspiration biopsy with a heparinized fine 22-gauge needle. In the well differentiated type of HCC, a correct diagnosis of malignancy was difficult from the cytologic features of individual cells because of their resemblance to normal hepatocytes. In contrast, in moderately differentiated and poorly differentiated types of HCC, a correct diagnosis of malignancy was easily made from the features of individual cells, but there was little or no cytologic evidence of the hepatic origin of the cells. Comparison of histologic and cytologic findings in aspirated materials obtained from the same patients showed that the cellular patterns seen in cytologic specimens faithfully reflected the histologic structures of HCC. Various characteristic cellular patterns were recognized only in specimens obtained from patients with HCC, but not in those from patients with benign liver diseases. These cellular patterns were very useful not only for diagnosis of malignancy, but also for identification of the hepatic origin of cells. A combination of the features of individual cells and of characteristic cellular patterns raised the diagnostic rates for well-, moderately, and poorly differentiated types of HCC to 90.5%, 100%, and 100%, respectively. PMID- 3013390 TI - Transcatheter hepatic arterial embolization followed by hepatic resection for the spontaneous rupture of hepatocellular carcinoma. AB - In three cases of the spontaneous rupture of hepatocellular carcinoma, emergent transcatheter hepatic arterial embolization was performed for controlling the intraperitoneal hemorrhage. No more transfusions were required after embolization. Operation was carried out on the 10th, 5th, and 5th day, respectively, after liver function studies returned to the pre-embolization level. Hepatic resection of left hemi-hepatectomy was performed in Case 1, and segmentectomy in Case 2 and Case 3 each. Transcatheter hepatic arterial embolization followed by hepatic resection is a rational treatment in the management of the spontaneous rupture of hepatocellular carcinoma. PMID- 3013391 TI - Diagnostic accuracy of ultrasonography for hepatocellular carcinoma. AB - Accuracy of ultrasonography for the diagnosis of hepatocellular carcinoma (HCC) was examined. The subjects were 5339 patients who underwent ultrasonography of the upper abdomen, during the year 1980 at our hospital. Out of 5339, 113 cases were found to be hepatocellular carcinoma by a 3-year follow-up study, checking against the records of regional cancer registry. Diagnostic accuracy based on 6 months of follow-up was as follows: for identification of liver tumors, sensitivity, specificity, and overall accuracy were 94.9%, 98.7%, and 98.9% respectively; for diagnosis of HCC, sensitivity, specificity, and overall accuracy were 58.9%, 99.9%, and 99.3% respectively. In conclusion, the ability of ultrasonography to detect hepatocellular carcinoma is adequately high, and thus, ultrasonography can be employed as a diagnostic procedure for mass screening. PMID- 3013392 TI - The objectives and importance of operative staging of children with cancer. AB - Modern multidisciplinary treatment of childhood cancer has made extent of disease evaluation important for proper treatment planning. Accurate staging is essential to cooperative group studies and for comparing treatment modalities at different centers. Operative staging plays an important role where clinical or imaging methods are limited, as in abdominal Hodgkin's disease or regional nodal metastasis. Operative staging is carried out either as a special diagnostic procedure, as in lymphoma, or as part of a planned surgical resection of a solid tumor. For lymphomas: Operative staging of abdominal Hodgkin's disease is required where protocols include involved field irradiation and sparing of normal growing tissue in the child. In non-Hodgkin's lymphoma, bulky abdominal tumor may be surgically evaluated after intensive chemotherapy either in delayed primary surgery or in second look procedures. Residual tumor may be excised or tagged with clips for localized irradiation to the tumor sparing normal abdominal organs. For solid tumors: During surgical resection of neuroblastoma, Wilms' tumor and rhabdomyosarcoma, the correct procedure involves regional staging either by formal node dissections or by multiple biopsies to determine extent of spread. Regional node dissections are often part of a correct cancer operation for cure, but also give staging information unobtainable by other methods. The surgeon must plan every procedure carefully with the aim of curing the patient and also deriving maximum information from the operation to enable correct planning of further treatment. PMID- 3013393 TI - Advances in the care of the child with cancer. The importance of histologic subclassification of tumors. AB - The importance of histologic subclassification of tumors lies principally in the correlation between nosology and tumor biology, including response to therapy. Subclassifications that are practically achieved, reproducible and uniquely predictive are of great value. As new methods are engaged and new clinical data are reported, classifications may be changed. Subclassifications of solid tumors with proven therapeutic application are exemplified by renal tumors and lymphomas that are commonly referred to as "favorable" or "unfavorable histology." Subclassification of soft tissue sarcomas, neuroblastoma and tumors of the central nervous system are being investigated, but are presently of undetermined relevance. Classifications and subclassifications of solid tumors of children are presented in the context of prognostic relevance. PMID- 3013394 TI - Genetics and cytogenetics of pediatric cancers. AB - It is clear that genetic factors play an important role in the development of some human cancers. These factors may be particularly influential in the pediatric age group because environmental exposures have been minimal. Several pediatric solid tumors, including retinoblastoma and Wilms' tumor, are hereditary. Specific constitutional chromosome abnormalities have been found in these patients, thus implicating certain gene regions as being involved in tumorigenesis. Molecular genetic studies have provided insight into the events occurring at the DNA level in these gene regions. The role of genetics in the development of sporadic pediatric malignancies is also beginning to be elucidated as specific acquired chromosome abnormalities are being discovered in the malignant cells of these otherwise karyotypically normal individuals. This paper will review selected hereditary and nonhereditary pediatric cancers that demonstrate the importance of genetic considerations in the diagnosis and management of children with cancer. PMID- 3013395 TI - Advances in pediatric radiotherapy in the last ten years and future proposals. AB - Advances made can be divided into five main categories. Firstly, the problem of geographic miss which has been reduced by delivering effective radiation doses with greater precision. This has been accomplished with more sophisticated diagnostic and therapeutic equipment, immobilization techniques and computerized treatment planning. Second is the recognition of the interplay of radiation and chemotherapy on normal tissue tolerance and local tumor control. This interaction has necessitated reduction in both dose and volume of irradiation. Third is the use of wide field irradiation as a systemic treatment. Fourthly, the utilization of cooperative group trials to define the role of irradiation. Finally, with the improvement in survival has come the recognition of late effects of irradiation in the growing child and the means of reducing such effects. The current role of radiation therapy in childhood malignancies is summarized, controversies are identified, and future prospects explored. PMID- 3013396 TI - Peripheral neuropathy associated with high-dose Ara-C therapy. AB - Central nervous system toxicity associated with high-dose cytosine arabinoside (Ara-C) therapy (HD Ara-C) is well known. The authors report the case of a severe isolated peripheral polyneuropathy due to HD Ara-C. Electrophysiologic changes and histologic observations were consistent with axonal degeneration and scattered destruction of myelin sheaths. This observation emphasizes the need for careful complete neurologic evaluation for patients receiving HD Ara-C treatment. PMID- 3013397 TI - HTLV-I seroprevalence in patients with malignancy. AB - Since many malignancies often occur in patients with smoldering type adult T-cell leukemia (ATL) (5 of 18 cases in this report), the relationship between HTLV-I (human T-cell leukemia virus type I) infection, which is closely associated with ATL, with other malignancies in an HTLV-I endemic area was examined. Among the 394 patients with malignancies and who had not had blood cell transfusions, 61 (15.5%) tested positive for HTLV-I antibody. The prevalence was significantly higher in males older than age 40 years and females of all ages compared to age- and sex-matched healthy individuals. The overall seroprevalence (26.1%) in 291 patients with malignancies and who had had blood cell transfusions was higher than that of those who had not had blood transfusions. There was no significant correlation between the site of malignancy and antibody prevalence. These results suggest the possibility that development of malignancy may contribute to expression of latent HTLV-I infection and that HTLV-I infection may contribute to the risk of other malignancies. PMID- 3013398 TI - Malignant testicular germ cell tumors in a father and two sons. Case report and literature review. AB - The authors report the 17th case of primary malignant testicular tumors in father son pairs, the 61st case occurring in male first-degree relatives, and the first case identified in a father and two sons. The father had bilateral seminomas at ages 31 and 44 years. His oldest son developed left testicular teratoma with elements of seminoma and embryonal carcinoma at age 29 years. The second son developed pure seminoma of the right testicle at age 26 years. The father had mumps orchitis at age 17 years. None of the three had a history of cryptorchism, trauma, or hernia. Literature reports of familial testicular neoplasia are becoming more frequent, and evidence is presented that family history may represent a risk factor independent of cryptorchism for the development of testicular cancer. Aggressive follow-up of closely-related male relatives is advocated. PMID- 3013399 TI - Spontaneous 19-year regression of oat cell carcinoma with scalene node metastasis. AB - This is the first case report of a patient with oat cell carcinoma of the lung with scalene node metastasis who, without treatment, is alive 19 years after the original diagnosis was made by biopsy. Seven years before the publication of this report, and at the age of 66 years, he underwent coronary bypass surgery and there was no gross evidence of malignancy present. PMID- 3013400 TI - Phase II trials of alpha-difluoromethylornithine, an inhibitor of polyamine synthesis, in advanced small cell lung cancer and colon cancer. AB - alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, blocks polyamine synthesis and has demonstrated antitumor activity against small cell lung cancer and colon cancer in cell culture and animal tumor models. To evaluate clinical efficacy and further define toxic effects of this new agent, phase II trials of DFMO were performed in previously treated patients with advanced small cell lung cancer and previously untreated patients with metastatic colon cancer. Oral DFMO was administered at a dose of 2.25 g/m2/day every 6 hours continuously to patients with small cell lung cancer. The same dose was given to patients with colon cancer but on a schedule of "3 weeks on, 1 week off" to avoid hearing loss. Evaluation of toxicity indicated that thrombocytopenia was seen only in patients receiving continuous DFMO who had received prior chemotherapy, while reversible hearing loss and gastrointestinal side effects occurred on both intermittent and continuous schedules in previously treated and untreated patients. PMID- 3013401 TI - Phase I study of continuous-infusion cisplatin. AB - Cisplatin was administered to 21 patients as a continuous infusion over 5 days. Doses of 20, 25, and 30 mg/m2/day were studied. When administered in 3 L of normal saline/day, continuous-infusion cisplatin at a dose of 30 mg/m2/day was associated with neuropathy in two of nine patients and creatinine elevations (greater than 2 mg/dl) in four. No neurotoxicity or nephrotoxicity was observed at lower doses. Severe thrombocytopenia (less than 50,000/microliter) was unrelated to dose and occurred in 19% of the courses. Leukopenia was minimal. Nausea and vomiting were easily controlled. No toxicity was seen in 38% of the courses. Nineteen patients were evaluable for response. Six patients achieved partial response (32%), five had minimal response (25%), and eight had progressive disease (42%) in this group. Seven patients had received cisplatin previously as a bolus treatment (20 mg/m2/day for 5 days) and failed on chemotherapy. Among these patients there was one with partial response, three with minimal response, and three with progressive disease. It is concluded that continuous-infusion cisplatin, as given in this protocol, is well-tolerated at a dose of 25 mg/m2/day and can result in responses in patients failing bolus cisplatin at high doses. PMID- 3013402 TI - Ifosfamide plus N-acetylcysteine in the treatment of small cell and non-small cell carcinoma of the lung: a Southeastern Cancer Study Group Trial. PMID- 3013403 TI - Hepatitis B and primary liver cancer. PMID- 3013404 TI - Fluoride uptake from an anti-calculus NaF dentifrice in vitro. PMID- 3013405 TI - An opioid mechanism modulates central and not peripheral dopaminergic control of ciliary activity in the marine mussel Mytilus edulis. AB - Opioid receptors and enkephalinergic neurons in the central nervous system of Mytilus edulis have been reported. Also known is that the lateral epithelium of the gill is innervated by serotonergic, cilioexcitatory neurons and dopaminergic, cilioinhibitory neurons. The aim of the present report is to look for an effect of opioid agonists on the nervous control of the lateral cilia. Dopamine applied to the cerebral ganglion inhibited the activity of lateral cilia in the gill. This effect was blocked by the application of several opioids to the visceral ganglion. The block was reversed by the application of naloxone to the visceral ganglion. Dopamine applied to the visceral ganglion also inhibited lateral ciliary activity as shown earlier. Opioids applied to the visceral ganglion partially blocked this effect but this was overcome by higher concentrations of dopamine. Preparations with low endogenous rates of ciliary beating were stimulated by the application of opioids to the visceral ganglion. Naloxone blocked this effect. Preparations with high endogenous rates of ciliary beating were inhibited by the application of naloxone to the visceral ganglion. Electrical stimulation of the cerebrovisceral connective produced excitatory and inhibitory effects depending on the rate of stimulation. Morphine applied to the visceral ganglion diminished the cilioinhibitory effects and enhanced the cilioexcitatory effects of electrical stimulation. Morphine applied to the gill had no effect on the cilioinhibitory action of dopamine applied to the visceral ganglion. There was no observable effect of opioids applied to the gill and no alteration in the cilioinhibitory effect of dopamine or the cilioexcitatory effect of serotonin applied directly to the gill in the presence of opioids. Specific opioid binding sites were found in the visceral ganglion but were not found in gill, palp, mantle, or visceral mass tissue. A dopamine-stimulated adenylate cyclase activity was again found in the visceral ganglion and the gill. Etorphine reduced the dopamine stimulation of cyclase in the ganglion but not in the gill. It is postulated that a cilioinhibitory, dopaminergic mechanism includes nerves running from the cerebral ganglion to the gill with synaptic transmission in the visceral ganglion that can be modulated by opioids. PMID- 3013406 TI - [Interindividual changes in the specific binding of 3H-dihydroalprenolol by beta 2-adrenergic lymphocytic receptors in essential hypertension]. PMID- 3013407 TI - [Molecular biology of viruses associated with AIDS and current attempts to prevent its spread]. PMID- 3013408 TI - [Differential diagnosis of hepatitis A by immunoenzyme determination of early antiviral antibodies]. PMID- 3013410 TI - Localisation of [3H] clonidine binding in rat neurohypophysis by means of electron-microscopic autoradiography. AB - Electron-microscopic autoradiography of rat neurohypophyses treated with [3H] clonidine, an alpha 2-agonist, showed that binding apparently occurred preferentially at the neurosecretory endings and blood vessels rather than on the pituicytes. Since it is known that clonidine has a high affinity for plasma proteins, the distribution over the neurosecretory nerve endings would suggest the existence of presynaptic alpha 2-binding sites on neurosecretory neurones, which could indicate a regulatory function for catecholamines in neurohypophysial hormone release. PMID- 3013409 TI - Immunogold electron-microscopic localisation of calpain I in skeletal muscle of rats. AB - By using a double-affinity-purified first antibody and colloidal gold-conjugated second antibody, it is shown that calpain I (a cysteine proteinase activated by micromolar concentrations of Ca2+) has a predominant intracellular location in the I-band region of the extensor digitorum longus (EDL) muscle of the rat, but is not exclusively associated with the Z-line. PMID- 3013411 TI - Ultrastructural and cytochemical studies of acid phosphatase and trimetaphosphatase in rat peritoneal mast cells developing in vivo. AB - The ultrastructural and cytochemical features of peritoneal mast cells of the rat were studied. Immature mast cells show specific cytoplasmic granules of different sizes, the smaller ones localized in the Golgi region. The rough endoplasmic reticulum and Golgi apparatus are well developed, and mitochondria are numerous. Nuclei show deep indentations. Acid phosphatase is present in the Golgi saccules, in GERL (Golgi apparatus-endoplasmic reticulum-lysosome) and in some small granules. It is not present in mature granules. Trimetaphosphatase is present in the Golgi saccules, in GERL, in most immature granules and in some mature granules. These enzymes appear to be transported and packaged into granules by the Golgi apparatus, suggesting that the specific mast cell granules may be a form of lysosome. The results of this study are consistent with the hypothesis that peritoneal mast cells may be derived from macrophage-like precursors. PMID- 3013412 TI - Immunohistochemical and ultrastructural study of anterior pituitary cells in the female Afghan pika, Ochotona rufescens rufescens. AB - An immunohistochemical study of the anterior pituitary gland of the female Afghan pika was carried out to distinguish the ultrastructural features of GH, PRL, ACTH, TSH and LH cells. The histochemically identified GH cells resembled ultrastructurally oval or round GH cells of the rat laden with large, dense secretory granules. PRL cells were divided into three subtypes based on differences in the diameter of their spherical secretory granules. They lacked polymorphic or irregularly shaped secretory granules. ACTH cells resembled ultrastructurally, in some respects, Siperstein's "corticotrophs" of the rat with peripheral arrangement of secretory granules. However, they were not always stellate, but elongate or angular in shape. The dense secretory granules were concentrated in the peripheral area of cytoplasm. TSH cells were non-stellate, but usually oval in shape, containing the smallest spherical secretory granules (100-200 nm in diameter). Almost all LH cells reacted also with FSH antiserum. They were irregular in shape, sometimes in contact with or surrounded the GH cells. They contained an abundance of medium-sized secretory granules (140-260 nm in diameter) which were larger than those in the LH cells of the female rat throughout the estrous cycle. Large secretory granules in the LH cells of the female pika seemed to be related to the endocrine state of persistent estrus. PMID- 3013413 TI - Multicellular complexes of thymocytes and different types of thymic stromal cells in the mouse. AB - The isolation of multicellular complexes of thymocytes and different types of thymic stromal cells from the mouse thymus is described. Isolated complexes were examined by light microscopy. Stromal cells, binding thymocytes at their surface, were identified using electron microscopy, and three types of epithelial cells, macrophages and interdigitating-like cells (IDC-like cells) were distinguished from their morphological characteristics. The epithelial cell types correspond morphologically to epithelial cells present in situ in various thymic regions. The type of thymocyte-contact with epithelial cells, macrophages and IDC-like cells indicated that the formation of multicellular complexes is common. PMID- 3013414 TI - [Hepatitis A virus in the feces of patients with hepatitis A]. PMID- 3013415 TI - Genetic variation in AIDS viruses. PMID- 3013416 TI - An underlying repeat in some transcriptional control sequences corresponding to half a double helical turn of DNA. AB - Transcription factor IIIA, which binds to the internal control region of the Xenopus 5S RNA gene has a novel structure consisting of nine tandemly repeated structural units. It was proposed by us that each unit interacts with about 5 bp of DNA. We show here that there is a periodicity on this scale in the DNA sequence and, by fine scale probing with nucleases, a corresponding structural repeat. Similar sequence periodicities are found in the internal control regions of other genes transcribed by RNA polymerase III, and also in the SV40 promoter and a monkey gene region to which the transcription factor Sp1 binds. We propose that transcription factor IIIA is the type of a novel class of transcription factors offering combinatorial possibilities for the specific recognition of DNA. PMID- 3013417 TI - The early region of human papovavirus JC induces dysmyelination in transgenic mice. AB - Transgenic mice containing the early region of human papovavirus JC were produced. Some of these mice exhibited a shaking disorder similar to the previously described mutant mice jimpy or quaking. Neuropathological analysis indicated a dysmyelination in the central nervous system, but not the peripheral nervous system. A high level of JCV T-antigen mRNA was present in the brains of the mice exhibiting the myelin disorder. JC virus is associated in humans with a degenerative demyelinating disease: progressive multifocal leukoencephalopathy. The JCV-containing transgenic mice may therefore provide an animal model for studying this disease. PMID- 3013418 TI - Retroviruses as probes for mammalian development: allocation of cells to the somatic and germ cell lineages. AB - Preimplantation mouse embryos were infected with a recombinant retrovirus, which serves as a genetic marker for the progeny of an infected blastomere. Quantitative analysis of proviruses carried in mosaic animals indicated that the molarity of individual proviral bands was equal in almost all tissues. The cells that give rise to the embryo proper must therefore intermingle extensively before final tissue allocation to ensure equal contribution of founder cells to all somatic tissues. The distribution of molarities of individual proviruses suggested that somatic lineages are derived from at most eight founder cells. About half of the proviruses were present in the germ line and the somatic tissues of mosaic animals, while the remaining proviruses were found either in the germ line or in the somatic tissues, but not in both. Our results suggest that at least three cells form the germ line and are set aside prior to somatic tissue allocation. PMID- 3013419 TI - Control of erythroid differentiation: possible role of the transferrin cycle. AB - A monoclonal antibody to the chicken transferrin receptor (JS-8) blocked temperature-induced and spontaneous differentiation of avian erythroid cells transformed by ts- and wt-retroviral oncogenes. In cells committed to differentiate, JS-8 caused an arrest at the erythroblast or early reticulocyte stage, followed by premature cell death, whereas proliferation of noncommitted erythroid cells or other hematopoietic cells remained unaffected. JS-8 had no effect on transferrin binding or internalization, but blocked subsequent receptor recycling resulting in reduced iron uptake. Restoration of high intracellular iron levels neutralized the action of JS-8, whereas an inhibitor of porphyrine biosynthesis (4,6-dioxoheptanoic acid) closely mimicked the effect of JS-8. This suggests that erythroid differentiation might involve coordinate synthesis of erythrocyte proteins subject to regulation by hemin or hemoglobin. PMID- 3013420 TI - Development of activatable adenylate cyclase in the preimplantation mouse embryo and a role for cyclic AMP in blastocoel formation. AB - Forskolin- and cholera toxin-activated adenylate cyclase activity increases between the morula and blastocyst stages of mouse preimplantation development, as assessed by the ability of these agents to increase embryonic cAMP levels. Development of activatable adenylate cyclase requires transcription but is independent of the fifth nuclear replication, cell division, and compaction. Early cavitating embryos treated with cholera toxin and forskolin or with N6 monobutyryl-cAMP display an increase in the rate of fluid accumulation in comparison with untreated controls. The stimulatory effect is specific for cAMP, since neither the inactive cAMP analogue N6-monobutyryl-2'-deoxy-cAMP nor N2 monobutyryl-cGMP stimulates the rate of fluid accumulation. These results constitute the first report of a possible physiological function for cAMP in preimplantation development, namely, in blastocoel formation. PMID- 3013421 TI - The biology of platelet-derived growth factor. PMID- 3013422 TI - Two human 35 kd inhibitors of phospholipase A2 are related to substrates of pp60v src and of the epidermal growth factor receptor/kinase. AB - We have purified two 35 kd phospholipase A2 inhibitors from human placenta, which we refer to as lipocortin I and II. Both proteins exhibit similar biochemical properties and occur in placenta at about 0.2% of the total protein. By peptide mapping, sequence, and immunological analyses, we show that lipocortin I and the 35 kd substrate for the EGF-receptor/kinase from A431 cells are the same protein. By similar criteria, we determine that lipocortin II is the human analogue of pp36, a major substrate for pp60src, which has been characterized in chicken embryo fibroblasts and in bovine brush border preparations. The amino acid sequences of lipocortin I and II that we deduced from cDNA clones share 50% homology, indicating that they probably evolved from a common gene. PMID- 3013423 TI - The cDNA sequence for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain) reveals a multidomain protein with internal repeats. AB - We have isolated and sequenced a full-length cDNA clone for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain). This sequence predicts a 339 amino acid (Mr 38,493) protein containing an N-terminal region of 20 amino acids, known to interact with a 10 kd protein (light chain), and a C-terminal region, found to contain two Ca2+/phospholipid-binding sites, that can be aligned as four 70 amino acid repeats. A single p36 gene was detected in the mouse genome, and a major p36 mRNA of 1.6 kb was found to be expressed in different mouse tissues. Unexpectedly, p36 and the phospholipase A2 inhibitor lipocortin I were found to be 50% identical in sequence over the C-terminal 300 residues. The function of p36 and its relation to other proteins are discussed. PMID- 3013424 TI - Glucocorticoid responsiveness of the transcriptional enhancer of Moloney murine sarcoma virus. AB - The long terminal repeat of Moloney Murine Sarcoma Virus (MoMSV) contains an imperfect direct repeat that serves as a strong transcriptional enhancer. The strength of the MoMSV enhancer is strongly dependent on the presence of glucocorticoid hormone. Mapping studies in combination with DNAase I footprinting experiments define the presence of glucocorticoid regulatory elements at the promoter-proximal ends of each enhancer repeat. These elements behave like inducible enhancers: their regulatory activity is independent of position and orientation when they are linked in cis to a heterologous promoter. These data demonstrate the modular nature of the MoMSV enhancer and identify the glucocorticoid receptor as one of the trans-acting factors that interact with the MoMSV enhancer and mediate its transcriptional activity. PMID- 3013426 TI - Studies on host-virus genome relationship in Epstein-Barr virus immortalized lymphoblastoid cell lines. AB - Five human lymphoblastoid cell lines immortalized in vitro with the B95-8 EBV strain, chosen to have a low number of copies of EBV genome, were examined to detect variations in electrophoretic mobility of viral restriction fragments and in the karyotype. Patterns of mobility detected with different viral probes are always the same as those obtained with fragments from purified virus-plasmidic DNA, with one exception. This "non-plasmidic" pattern occurs with a probe containing the termini of the linear virion DNA and consists in an increase of the molecular weight and in the appearance of more than one band. Cytogenetic studies carried on the same cell populations used as source of DNA, early after immortalization, showed a diploid modal chromosome number and no G banding rearrangements. PMID- 3013428 TI - [The role of prostaglandins in the regulation of coronary circulation and the activity of the heart]. PMID- 3013425 TI - Induction by immunomodulatory agents of a macrophage antigen recognized by monoclonal antibody 158.2 and correlation with macrophage function. AB - MA158.2, a rat monoclonal antibody with binding specificity for cells of the monocyte-macrophage lineage, reacts with an antigen (158.2) whose expression is enhanced on mononuclear cells activated to the tumoricidal phenotype by treatment with lymphokine supernatant containing macrophage activating factor (MAF). The functional relevance of enhanced expression of this antigen has been examined in mouse peritoneal macrophages treated with a variety of immunomodulatory agents and assayed for augmented macrophage-mediated defense reactions, including O-2 production, microbicidal, and tumoricidal activity. An interferon-gamma (IFN gamma) preparation produced by recombinant DNA technology induced a dose dependent increase in expression of the 158.2 antigen in inflammatory macrophages which was accompanied by acquisition of microbicidal activity against Listeria monocytogenes. However, these cells did not express tumoricidal activity and induction of this property required concomitant exposure to lipopolysaccharide (LPS). Similar results were obtained using macrophages elicited with pyran copolymer. Exposure to LPS alone induced enhanced expression of antigen 158.2 but did not elicit microbicidal activity. Macrophages challenged with IFN-alpha, IFN beta, MDP, and bestatin did not exhibit increased 158.2 and also failed to acquire tumoricidal activity when treated concomitantly with LPS. Collectively, these data indicate that the MA 158.2 antibody recognizes an antigen expressed by macrophage populations displaying the so-called primed phenotype in which microbicidal activity is expressed but in which induction of tumoricidal activity requires the addition of a second signal such as LPS. PMID- 3013429 TI - [The effect of nutrition on the activity of cholesterol 7-alpha-hydroxylase]. PMID- 3013430 TI - [Tympanic chemodectoma]. PMID- 3013427 TI - [New directions in the pathogenesis and treatment of dysmenorrhea-- leukotrienes and calcium channel blockers]. PMID- 3013431 TI - [Synpolydactyly with a triphalangeal thumb--a new type of synpolydactyly?]. PMID- 3013432 TI - In vitro evaluation of myelotoxicity induced by antineoplastic drugs. AB - Myelotoxicity is one of the most important side effects of antineoplastic drugs. Even in using the same dosages, the gravity of this toxicity varies greatly among different patients. With the aim of evaluating if an in vitro test may predict such an effect we have measured in bone marrow samples taken from 15 patients undergoing chemotherapy for small-cell lung cancer the in vitro uptake of 3H thymidine in the presence or absence of the cytostatic drugs used for the clinical treatment of these patients. We did not find a clear correlation between the in vitro results and the myelotoxicity observed during the clinical course. PMID- 3013433 TI - Current status of hormone receptor measurements in cancer patients. AB - Despite increasingly sophisticated techniques, improvements in the correlation between laboratory findings and tumor response to endocrine therapy have not been obtained by hormone receptor studies. A possible explanation is that present knowledge of the mechanisms of the endocrine stimulus is incomplete. Some aspects of the present model, (elevated conjugated steroid levels, multiplicity of the plasma proteins capable of binding hormones, pulsatility of the plasma protein bond and of the receptor system for steroids), are still unclear and thus are not used in diagnosis. By evaluating these factors it will probably be possible to correlate better laboratory data with clinical findings. PMID- 3013434 TI - Chemotherapy of primary and secondary liver cancer: is it useful? AB - The therapeutic efficacy of three different antiblastic regimens was tested in patients affected by primary or secondary liver cancer. The association of cyclophosphamide and adriamycin gave the best results, despite no partial remission observed with any treatment. As regards the efficacy of antiblastic therapy in this type of tumor, we must conclude that the only detectable effect is a transient improvement of the course of the disease. PMID- 3013435 TI - Electron spin resonance spin trapping studies on the photolytic generation of halocarbon radicals. AB - The technique of free radical spin trapping has been used to study the photolytic behaviour of anaerobic solutions of aliphatic halocarbons such as CCl4, CBrCl3 and halothane in either toluene or water at room temperature. Use of the spin traps N-tert-butyl-alpha-phenylnitrone (PBN) and 2-methyl-2-nitrosopropane provides evidence for photolytic cleavage of carbon-halogen bonds yielding radicals such as .CCl3 (from CCl4 and CBrCl3) and .CHClCF3 (from halothane) which are readily trapped; this method of generating biologically important radicals may be of use in studying model reactions of these species. Under aerobic conditions evidence is obtained, by use of the spin trap PBN, for the production of the corresponding halocarbon peroxyl radicals (such as CCl3O2.) by addition of oxygen to the initially produced halocarbon radical. Though the direct detection of the peroxyl radical adducts to the spin trap proved impossible under these experimental conditions, the observed oxidation products of the spin trap (acyl nitroxides) are shown to be indicative of the presence of such species. PMID- 3013436 TI - Photosensitization by the trypanocidal agent crystal violet. Type I versus type II reactions. AB - The photoreduction of crystal violet to a carbon-centered radical was detected directly by electron spin resonance (ESR) spectroscopy under anaerobic conditions. The linewidth (0.9 G) of this radical was less broad than the linewidth (11.0 G) of the free radical obtained in Trypanosoma cruzi incubations. No crystal violet radical could be detected under aerobic conditions. However, crystal violet was found to convert oxygen to superoxide anion and hydrogen peroxide in the presence of light. This superoxide anion and hydrogen peroxide formation was greatly enhanced by reducing agents such as NAD(P)H. In addition, irradiation of crystal violet did not generate detectable amounts of singlet oxygen. PMID- 3013437 TI - Peroxidase activation of 1-naphthol to naphthoxy or naphthoxy-derived radicals and their reaction with glutathione. AB - 1-Naphthol was metabolised by horseradish peroxidase (HRP) in a H2O2-dependent reaction to methanol-soluble and covalently bound products. Spectrophotometric and electron spin resonance (ESR) studies established that HRP catalysed the one electron oxidation of 1-naphthol to naphthoxy or a naphthoxy-derived radical. Inclusion of glutathione (GSH) in the reaction caused a dose-dependent inhibition of covalent binding and an increase in the amount of unmetabolised 1-naphthol present at the end of the incubation. gamma-Radiolysis studies suggest that this is due to the reduction of naphthoxy radicals by GSH yielding 1-naphthol and GS.. In agreement with this, HRP-catalysed-oxidation of 1-naphthol in the presence of GSH, was found to stimulate oxidised glutathione (GSSG) formation. PMID- 3013438 TI - Inhibition of SV40 DNA replication by benzo[a]pyrene diol epoxide adducts: two recovery modes. AB - Anti-benzo[a]pyrene diol epoxide (BPDE) adducts produced in vitro in SV40 initially inhibit SV40 DNA replication in vivo, in cells unexposed to BPDE. A single adduct in a replicon is probably sufficient to block DNA replication. The recovery process appears to begin immediately after infection. The rate of recovery of replicative capacity is inversely related to the initial adduct number. Holding the infected cells temporarily under conditions that prevent viral DNA replication results subsequently in increased recovery, proportional to the holding time. The mechanism of recovery appears to be constitutive and prereplicative. In addition, there is a second mode of recovery which is induced by pretreatment of the host cells with BPDE before infection. The effect of pretreatment is similar to that of extending the holding time before replication: the first molecules begin to replicate earlier but the subsequent rate of recovery is unchanged. The induced mechanism may be either a limited stoichiometric repair process or a slow replicative bypass. PMID- 3013439 TI - Induction of cytochrome P-450 in congenic C57BL/6J mice by isosafrole: lack of correlation with the Ah locus. AB - Isosafrole induction of cytochrome P-450 was compared in congenic strains of C57BL/6J mice, one of which expresses normal levels of the Ah receptor [B6(Ahb)], and another that does not contain a measurable receptor concentration [B6(Ahd)]. Using sucrose gradient analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding, an Ah receptor concentration of 69.1 +/- 3.8 fmol/mg protein was measured in the hepatic cytosol from B6(Ahb) mice, while no receptor could be detected in the cytosol from B6(Ahd) mice. Isosafrole treatment (75 mg/kg X 3 days) increased the total hepatic microsomal cytochrome P-450 content to the same extent in the two congenic strains. The level of microsomal monooxygenase induction in the isosafrole-treated B6(Ahd) mice was greater than that of B6(Ahb) mice for ethylmorphine N-demethylase and isosafrole metabolite-complex formation, the latter a measure of cytochrome P2-450. In the case of 7-ethoxycoumarin O deethylase only the isosafrole-treated B6(Ahd) mice had elevated microsomal activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) also revealed a similar induction pattern for the two congenic strains, following isosafrole treatment. Thus, the isosafrole treated B6(Ahd) mice produced an equivalent or slightly larger induction of cytochrome P-450 than the B6(Ahb) mice, suggesting that there is no direct role for the Ah receptor in the regulation of these cytochrome P-450 monooxygenase activities by isosafrole. PMID- 3013440 TI - Studies on organic fluorine compounds. L. Synthesis and biological activity of 2 alpha-fluorovitamin D3. PMID- 3013442 TI - Maintenance chemotherapy for anaplastic small cell carcinoma of the bronchus: a randomised, controlled trial. AB - Since March 1980, 309 patients with anaplastic small cell carcinoma of the bronchus (ASCB) have received remission induction therapy prior to randomisation to maintenance (M) or no maintenance (NM) chemotherapy. Induction therapy consisted of six courses of vincristine, doxorubicin and cyclophosphamide (VAC) given IV every 3 weeks. Those with limited disease also received mediastinal irradiation. Consenting patients with no unequivocal residual disease were randomised to have no further treatment until relapse or a further eight courses of VAC, at a lower dosage, every 4 weeks. Patients failing to achieve randomisation status received palliative treatment only. The median survival for all patients with limited disease (LD) is 363 days and that for patients with extensive disease (ED) is 272 days (P less than 0.00001). Sixty-one patients with ED were randomised. Those having maintenance chemotherapy lived significantly longer (median 372 days) than those who did not continue therapy (median 259 days) (P = 0.006). An imbalance in the proportion of 'complete remitters' randomised to maintenance therapy does not account for this difference. There is no significant difference between the M and NM groups in the 32 randomised LD patients. Continuing treatment during remission with agents used to induce the remission can prolong survival in patients with extensive stage ASCB. PMID- 3013441 TI - Cell surface markers in leukemia: biological and clinical correlations. AB - Recent advances in analysis of leukemic cell phenotypes using cell surface markers have provided important insights into leukocyte differentiation and the cellular origin of leukemia. In addition to the traditional cell surface markers, i.e., surface membrane immunoglobulin and receptors for sheep erythrocytes that define B and T lymphocytes, highly specific monoclonal antibodies have been developed that discriminate various stages of human lymphocyte and granulocyte differentiation. Explorations of the detailed phenotypes of leukemic cells in relation to normal hemopoietic differentiation reveal that consistent, composite phenotypes of different subclasses of lymphoid malignancies closely mimic those of corresponding normal cells at equivalent levels of maturation. This is exemplified in lymphoma cells (chronic lymphocytic leukemia of B or T type, Sezary Syndrome, immunocytoma) that resemble mature and immunocompetent T and B cells, in T cell acute lymphoblastic leukemia (T-ALL) (equivalent to thymus cells) and in non-T ALL (corresponding to lymphoid progenitor cells in the bone marrow). The major phenotypes documented in different leukemias represent the level of maturation arrest imposed on the dominant subclone; this is determined by, but not necessarily synonymous with, the target cell and associated clonogenic cell population in the leukemia. The clinical significance of immunodiagnosis of leukemia cell types becomes best evidenced in acute leukemias. Besides the improvement of diagnosis by using objective criteria, clinically useful subclassifications became evident: five major subtypes of ALL are now recognized, including unclassified or null ALL, common ALL, pre-B-ALL, B-ALL and pre-T/T-ALL. In addition to disclosing that ALL is an heterogeneous disease, such classifications have proved to be prognostically significant. This is exemplified in 248 children and 145 adults with ALL which were analysed for cell type and clinical data. In addition to their utility in leukemia classification, monoclonal antibodies that identify leukemia associated antigens are becoming used therapeutically, e.g., to lyse residual leukemia cells from remission bone marrows removed from leukemia patients before reinfusion. New approaches to the treatment of leukemia in which the objective is to encourage maturation of leukemia cells rather than to achieve leukemia eradication, can be monitored by phenotyping the alterations of the cell surface, and cell markers may hopefully be useful in identifying cell types that can be induced to differentiate. PMID- 3013443 TI - Effects of tumour promoters on metabolic cooperation between human hepatoma cells. AB - Metabolic cooperation between cells from three human hepatoma cell lines was studied by the clonogenic method and by autoradiography. It was found that human HGPRT+/HGPRT- SK-HEP-1 cells only, showed a metabolic cooperation capacity that was inhibited by tumour promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phenobarbital, and was not inhibited by the non-promoter 4-O-methyl TPA, provided suitable experimental conditions (short exposure times) were used. This biological system might be the basis of a new in vitro short-term screening test for potential tumour-promoting chemicals. PMID- 3013444 TI - (Na-K)ATPase activity along the nephrons in normal and adrenalectomized rats measured by quantitative cytochemistry. AB - A cytochemical method was used to measure total, ouabain insensitive and specific (Na-K)ATPase activities along the rat nephron. Enzyme activity was expressed as per cent of mean integrated extinction with reference to a calibrated filter. The lowest mean values of total, ouabain-insensitive, and (Na-K)ATPase activities were found in the proximal convoluted tubule (PCT). In the distal convoluted tubule (DCT), total and ouabain-insensitive activities (77.8 per cent and 45.8 per cent, respectively) were significantly higher than in the medullary thick ascending limb (MAL) (66.0 per cent and 24.6 per cent, respectively). Mean values of (Na-K)ATPase activity were significantly lower in DCT than in MAL (32.0 per cent and 41.3 per cent, respectively). Using Lineweaver-Burk plots, the KM ATP value for total ATPase activity was found to be 2.33, 1.79, and 3.63 mM in DCT, MAL, and PCT respectively. Maximal velocity was lower in PCT than in MAL and DCT. For (Na-K)ATPase, the smallest KM value was found in MAL (0.95 mM) and was 2.73 and 5.71 mM in DCT and PCT respectively. Maximal velocity was the highest in MAL (49.3 per cent), lower in DCT (36.1 per cent) and least in PCT (22.5 per cent). ATPase was measured in the MAL and DCT from rats fed a normal (N-Na+) or a high (Hi-Na+) sodium diet, and from Hi-Na+ rats one week after adrenalectomy (ADX). In the MAL, (Na-K)ATPase tended to be higher in Hi-Na+ than in rats, but was significantly lower in ADX than in Hi-Na+.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013445 TI - Partial immunochemical identity between (Na+ + K+)-ATPase and a membrane component extractable with chloroform: methanol. AB - An IgG fraction prepared from an antiserum against a holoenzyme preparation of (Na+ + K+)-ATPase precipitated a single antigen when samples of holoenzyme were subjected to crossed immunoelectrophoresis but precipitated an additional, immunochemically-related antigen when a plasma membrane-enriched fraction was subjected to crossed immunoelectrophoresis under the same conditions. The immunochemically-related antigen could be extracted from the plasma membrane fraction with CHCl3:CH3OH. PMID- 3013446 TI - Stimulation by glucose and carbamylcholine of phospholipase C in pancreatic islets. AB - Phosphoinositide hydrolysis in intact pancreatic islet cells was investigated in an indirect but dynamic manner by monitoring the efflux of radioactivity from islets prelabelled with [3H]inositol. A rise in glucose concentration provoked a rapid, modest but sustained increase in effluent radioactivity, this phenomenon being abolished in the absence of extracellular Ca2+ or presence of verapamil. The release of [3H]inositol was also stimulated at high extracellular K+ concentration, but not by gliclazide. Whether in the presence or absence of glucose, carbamylcholine provoked a marked increase in effluent radioactivity. The response to the cholinergic agent was decreased in the presence of verapamil or absence of extracellular Ca2+ and abolished in the presence of atropine or LiCl. These results suggest that an increase in cytosolic Ca activity, as caused by glucose or membrane depolarization, may cause activation of phospholipase C. In response to cholinergic agents, however, the enzymic activation, although modulated by Ca2+ availability, may result directly from the occupation of muscarinic receptors. PMID- 3013447 TI - Human bone cell cultures: a new model for studying the mechanism of action of calcitonin. AB - An investigation on cell cultures obtained from temporal human bone fragments showed that they provide a suitable model for studying the mechanism involved in calcitonin action on bone cells. Furthermore they demonstrated: a transitory increase in 45Ca uptake that returned to control values ten minutes after the hormone was added; a relation between 45Ca uptake and increased cAMP concentrations when these were measured at the same time intervals; a reproduction of the salmon calcitonin (sCT) effect after incubation of the cultures with either db-cAMP or db-cGMP and inhibition of 45Ca uptake and parallel decrease in cAMP levels with propanol. These results suggest that in human bone cell cultures, sCT acts as a temporary promoter of 45Ca uptake, probably by activating an adenylate-cyclase system through a beta-receptor. PMID- 3013448 TI - [Na-K]ATPase activity in proximal and distal tubules of the rat kidney: modification and application of a quantitative cytochemical technique. AB - A modified cytochemical assay for [Na-K]ATPase in cryostat sections of kidney was further characterized and used to quantify activity in seven functionally distinct sites along the rat nephron. The activity of [Na-K]ATPase was defined as the difference in ATPase activity in specifically identified tubules contained in serial sections incubated with and without ouabain. Preincubation of sections with ouabain was required for maximal inhibition of [Na-K]ATPase activity in several distal sites. The concentration of ouabain necessary for maximal inhibition of activity was 3.0 mM and half-maximal inhibition was obtained in all regions with 30-100 microM ouabain. In distal sites, [Na-K]ATPase formed a higher proportion of total ATPase activity (60-80 per cent) than in proximal sites (20 40 per cent). Enzyme activity was quantified using two different methods. The first measured activity over the basal region of tubules and gave an index of the concentration of [Na-K]ATPase over the basal lateral infoldings of cells composing the tubule. The second read activity over the entire cross section of tubules and provided an estimate of [Na-K]ATPase per length of tubule. The highest activities over the basal basal region were obtained from tubules of the distal nephron including the inner (MALin) and outer (MALout) medullary ascending limb, distal convoluted tubule (DCT) and connecting segment (CS). Lower activities were obtained in proximal convoluted (PCT) tubules, proximal straight (PS) tubules and the papillary collecting duct (PD). Distal convoluted tubules contained the highest activity per length of tubule. Other sites contained lower levels of activity in the following order: MALin greater than MALout greater than PCT greater than PD greater than PS. The modifications introduced increase the sensitivity and precision of this assay and permit the application of this technique to studies of [Na-K]ATPase activity in the major functional regions of the rat nephron. PMID- 3013449 TI - Heterogeneity of angiotensin II receptors in membranes of developing rat metanephros. AB - Specific and high affinity binding sites for angiotensin II were demonstrated in the membranes of the developing rat metanephros during the second half of pregnancy and in the newborn by binding studies with 125I angiotensin II. Only one type of angiotensin receptor was found during intrauterine life while after birth two classes of angiotensin receptors were present in the membranes of the cortical renal tissue. PMID- 3013450 TI - Studies on rat liver mitochondria. 6. The effect of contaminating particles in mitochondria stored at 0-4 degrees C. AB - Rat liver mitochondria were stored at 0-4 degrees C for several days using an appropriate medium and energy source. The elimination of the majority of microsomes and lysosomes, that normally contaminate isolated mitochondria, had a positive effect in preservation of respiratory control, P:O ratio, and monoamine oxidase activity during long term storage. PMID- 3013451 TI - Differences among primates in defence against infection: sensitivity of polymorphonuclear leukocytes to fMet-Leu-Phe. AB - The sensitivity of polymorphonuclear leukocytes (PMN) to N-formyl-methionyl leucyl-phenylalanine (fMet-Leu-Phe) for chemotaxis and for lysosomal enzyme release was examined using the PMN of four primate species, human (H. sapiens), chimpanzee (P. troglodytes), rhesus monkey (M. mulatta), and cotton-headed tamarin (S. (O) oedipus). The 50 per cent effective concentrations (EC50) of fMet Leu-Phe for chemotaxis were 2.5 X 10(-9) M in human, 10(-9) M in chimpanzee, 8 X 10(-8) M in rhesus monkey, and 3.3 X 10(-6) M in tamarin. The EC50 values of fMet Leu-Phe for myeloperoxidase (MPO) release were 10(-8) M in human, 4 X 10(-8) M in chimpanzee, 4 X 10(-8) M in rhesus monkey, and 10(-6) M in tamarin and those for beta-glucuronidase release were 4 X 10(-9) M, 6.4 X 10(-8) M, 1.8 X 10(-7) M, and 1.6 X 10(-6) M, respectively. Thus, the sensitivity to fMet-Leu-Phe for chemotaxis was in the order: chimpanzee congruent to human greater than rhesus monkey greater than tamarin, and that for the release of lysosomal enzymes, MPO and beta-glucuronidase, was in the order: human greater than chimpanzee greater than rhesus monkey greater than tamarin. These results appear to indicate that the sensitivity to fMet-Leu-Phe increases in the order of evolution of primates toward the human, and suggest that the sensitivity of PMN in the defence function against infection also increases in the same order. PMID- 3013452 TI - The effect of in vivo endotoxin on myocardial function in vitro. AB - Isolated perfused working hearts from male adult Sprague Dawley rats were used to examine myocardial performance following acute in vivo endotoxin administration (LD50-6-hour). Three hours after endotoxin administration, cardiac output and peak systolic pressure in the isolated perfused working heart were depressed 25 50% over a range of left atrial filling pressures (preload) from 10 to 30 cm of water. Linear regression analysis of the relationship between myocardial work indices, oxygen uptake, and glucose oxidation indicated that the hearts from the endotoxin-treated animals required more oxygen and glucose to perform the same amount of work. Levels of cyclic 3'5' adenosine monophosphate were 72% higher in ventricular tissue from the endotoxin-treated group compared to the hearts of controls. Isoproterenol (10(-9) mol/min) raised levels of the nucleotide to the same final concentration in both groups of hearts, whereas myocardial pressure work in hearts from endotoxin-treated rats was only 70% that of control. Provision of isoproterenol, while increasing mechanical work by the hearts in the endotoxin-treated group, did not induce an increase to the same performance levels as that of control hearts not treated with isoproterenol. These data are consistent with the concept that endotoxemia produces an intrinsic defect in myocardial mechanical performance. PMID- 3013453 TI - Myocardial sodium pump activity in endotoxin shock. AB - The effect of endotoxin administration on the integrity of sodium pump was studied in canine heart myocardium. Sodium pump activity was determined based on the 86Rb+ uptake by ventricular muscle strips. The results show that 4 hr following endotoxin administration the ouabain-sensitive 86Rb+ uptake was inhibited by 44-70%, and the inhibition was noncompetitive with 86Rb+ ions and was independent of Na+ concentrations. The sensitivity of sodium pump toward verapamil inhibition was unaffected by endotoxin administration, suggesting that the endotoxin-induced decrease in sodium pump activity is not mediated through alteration in either calcium or slow sodium channel. The sensitivity of 86Rb+ uptake to ouabain inhibition was decreased approximately sixfold in endotoxin injected dogs. These data suggest that sodium pump is greatly impaired in dog hearts 4 hr after endotoxin administration. The impairment in sodium pump might have physiological significance in the development of myocardial dysfunction during endotoxic shock. PMID- 3013454 TI - Hepatic alpha 1-adrenergic receptor alteration in a rat model of chronic sepsis. AB - Catecholamine therapy is often ineffective in reversing the peripheral vasodilatation and hypotension of septic shock. This suggests that catecholamines might not be able to activate alpha 1-adrenergic receptors to cause vasoconstriction. Despite elevations in endogenous catecholamines, hypoglycemia is also a complication of human sepsis, suggesting that among many other causes, hepatic alpha 1-receptors might be altered. To better understand the pathophysiologic basis for this pharmacologic dilemma, we studied the effect of experimental sepsis on alpha 1-adrenergic receptors in hepatic tissue, a rich source of alpha 1-receptors, from septic and control Sprague-Dawley rats. alpha 1 adrenergic receptors were measured with [3H]-prazosin and data analyzed by a computerized nonlinear least-square regression algorithm. Twenty-four hours following cecal ligation with puncture, a decreased number of alpha 1-adrenergic receptors was noted in crude and purified plasma membrane fractions (23 and 40% reductions respectively) from septic animals. No changes in either agonist or antagonist affinity for receptors from septic animals were noted. These data indicate that the catecholamine refractoriness seen in septic shock may be a result of alterations in alpha 1-adrenergic receptor number or receptor-effector coupling. PMID- 3013455 TI - A dual function for adenosine 5'-triphosphate in the regulation of vascular tone. Excitatory cotransmitter with noradrenaline from perivascular nerves and locally released inhibitory intravascular agent. AB - A dual function for adenosine 5'-triphosphate in the regulation of vascular tone is considered. Adenosine 5'-triphosphate can cause vasodilation, acting via P2 purinoceptors located on vascular endothelial cells to release an endothelium derived relaxing factor which diffuses to the vascular smooth muscle and induces vasodilation. The main source of intraluminal adenosine 5'-triphosphate is likely to be endothelial cells, and its release can be measured during pathophysiological conditions such as ischemia and hypoxia, in amounts likely to be sufficient to activate endothelial P2-purinoceptors. Adenosine 5'-triphosphate can also be released during intravascular platelet aggregation and from intact and damaged vascular smooth muscle cells, and so may play a role in the complex physiological mechanisms controlling local vascular tone under normoxic conditions and during vessel injury. Evidence is also presented for adenosine 5' triphosphate acting as an excitatory cotransmitter with noradrenaline from sympathetic perivascular nerves, to cause vasoconstriction via excitatory P2 purinoceptors located on vascular smooth muscle. The postjunctional mechanical and electrical responses of a number of blood vessels following perivascular nerve stimulation contain a component that is resistant to blockade of the alpha adrenoceptor. This nonadrenergic response is mimicked by adenosine 5' triphosphate and can be blocked by selective desensitization of the P2 purinoceptor by alpha,beta-methylene adenosine 5'-triphosphate. Vesicular storage of adenosine 5'-triphosphate and its release from sympathetic perivascular nerves has also been demonstrated. The functional significance of adenosine 5' triphosphate acting intraluminally as a vasodilator and extraluminally as a vasoconstrictor neuronal agent in the control of vascular tone is discussed. PMID- 3013457 TI - The energetics of myocardial stretch. Creatine kinase flux and oxygen consumption in the noncontracting rat heart. AB - Previous studies have suggested that flux through the creatine kinase reaction is coupled to cardiac performance and to the rate of adenosine triphosphate synthesis in the intact, beating heart. To define the effect of passive myocardial stretch on creatine kinase kinetics, we measured the rate constants and chemical fluxes for both directions of the creatine kinase reaction with the 31P-nuclear magnetic resonance technique of magnetization transfer in isolated, arrested rat hearts at four levels of left ventricular pressure and volume. Adenosine triphosphate synthesis was estimated from oxygen consumption measurements. As left ventricular pressure rose from 0 to 24 mm Hg and oxygen consumption increased by 20%, we observed a twofold increase in the rate constants and fluxes (mean +/- SD, n = 4-7) for the creatine kinase reaction. The forward rate constant increased from 0.33 +/- 0.03 to 0.80 +/- 0.08, and the reverse rate increased from 0.34 +/- 0.11 to 0.74 +/- 0.32/sec. The forward and reverse fluxes for the creatine kinase reaction increased from 12.0 +/- 3.4 to 26.5 +/- 5.8 and from 9.1 +/- 3.4 to 19.1 +/- 3.4 mumol/g dry weight per sec, respectively. At each level of left ventricular pressure, the forward and reverse rate constants were the same. However, as left ventricular pressure increased, the ratio of the forward to the apparent reverse fluxes for the creatine kinase reaction increased. The relationships between the rate constant or flux through the creatine kinase reaction vs. left ventricular pressure were linear. In addition to providing further support for the coupling between creatine kinase and mitochondrial adenosine triphosphate synthesis, these results suggest that flux through the creatine kinase reaction and adenosine triphosphate synthesis increases with myocardial stretch in the intact, noncontracting heart. PMID- 3013456 TI - Oxygen-dependent tension in vascular smooth muscle. Does the endothelium play a role? AB - We investigated a hypothesis that an oxygen sensor involved in hypoxia-induced relaxation of vascular smooth muscle may reside in endothelial cells. We also determined the oxygen dependence of hypoxia-induced decreases in cyclic guanosine 3',5'-monophosphate concentrations in vascular smooth muscle rings. Rings of canine femoral artery, rabbit thoracic aorta, and lamb ductus arteriosus, either with an intact endothelium or with damaged or absent endothelium, were studied using organ baths that allowed changes in PO2 without a change in pH. Hypoxia induced relaxations of rabbit thoracic aorta, lamb ductus arteriosus, and canine femoral artery were not dependent on an intact endothelium. The magnitude of hypoxia-induced relaxations was unchanged in rings of canine femoral artery without intact endothelium compared to rings with endothelium. Quasi-steady state organ bath PO2-mechanical tension relationships were unchanged in rings of canine femoral artery without endothelium over an organ PO2 range of 200-20 mm Hg. With rabbit thoracic aorta, magnitudes of hypoxia-induced relaxations were significantly smaller in rings without endothelium. Quasi-steady state plots, where mechanical tension was given as percentage of maximal relaxation, were similar in rings either with or without intact endothelium. Cyclic guanosine 3',5'-monophosphate concentrations were shown to be oxygen-sensitive, decreasing during hypoxia-induced relaxations with a threshold PO2 of 80-100 mm Hg with canine femoral artery, and 60-80 mm Hg with rabbit thoracic aorta rings, but this finding seems unrelated to the mechanism of hypoxia-induced relaxation. PMID- 3013458 TI - Characterization of the human lymphocyte beta-adrenergic receptor by photoaffinity labeling. Alterations with desensitization. AB - Desensitization of the leukocyte beta-receptor system has been associated with a functional uncoupling of the components of the beta-receptor complex. In order to determine whether desensitization and uncoupling of the leukocyte beta-receptor is associated with any structural alterations in the beta-receptor, we studied labeling of lymphocytes using the photoactive beta-adrenergic antagonist p-azido m-[125I]iodobenzylcarazolol. Labeled peptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected using autoradiographic techniques. In broken cell preparations, specific labeling was demonstrated in two major peptide bands: mol wt approximately equal to 68,000 and mol wt approximately equal to 55,000. Inhibition of photolabeling was stereospecific and demonstrated an order of potency for agonists consistent with labeling of a beta 2-receptor. Preincubation of cells with the beta-agonist, isoproterenol, resulted in a reduction in beta-adrenergic-mediated adenylate cyclase activity to 60% of control, but no change in total binding sites as determined by [125I]iodocyanopindolol binding. In photolabeling studies, desensitization was associated with a reduction in proportional labeling of the 55,000 mol wt band as compared to the 68,000 mol wt band to 58 +/- 3% of control and a reduction in mobility of the upper band. These studies suggest that structural alterations in the human lymphocyte beta-receptors occur with desensitization, analogous to changes in several other beta-receptor model systems. Also, since the techniques described can identify alterations in human beta-receptor structure, these methods may be exploited to determine whether structural alterations in lymphocyte beta-receptors may occur in human disease states. PMID- 3013459 TI - Decreased number and affinity of rat atrial natriuretic peptide (6-33) binding sites in the subfornical organ of spontaneously hypertensive rats. AB - Binding sites for rat atrial natriuretic peptide (6-33) were quantified by incubation of brain sections with (3-[125I]iodotyrosyl28) rat atrial natriuretic peptide (6-33), followed by autoradiography with computerized microdensitometry. Spontaneously hypertensive rats present lower numbers and lower affinity of binding sites than normotensive controls, Wistar-Kyoto rats, in the subfornical organ (binding capacity 61.7 +/- 8.9 and 124.3 +/- 10.7 fmol/mg protein; affinity constant 4.25 +/- 0.55 and 11.10 +/- 1.67 X 10(9) M-1, respectively). In the choroid plexus, hypertensive rats have lower numbers of sites than normotensive rats (binding capacity 72.7 +/- 10.5 and 173.6 +/- 22.8 fmol/mg protein, respectively), but there was no difference in the binding affinity (affinity constant 6.28 +/- 0.82 and 7.60 +/- 2.06 X 10(9) M-1, respectively). Our results suggest that discretely localized brain binding sites for rat atrial natriuretic peptide (6-33) may have a physiological function in genetically hypertensive rats. PMID- 3013460 TI - Phorbol diester modulates alpha-adrenergic receptor-coupled calcium efflux and alpha-adrenergic receptor number in cultured vascular smooth muscle cells. AB - The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate was used to probe the role of protein kinase-C in the modulation of alpha-adrenergic receptor-coupled calcium efflux in cultured vascular smooth muscle cells derived from rabbit aorta. Exposure of cells to 12-O-tetradecanoyl phorbol-13-acetate for 6 minutes caused a concentration-related (maximum effect at greater than or equal to 10 nM) increase in calcium-45 efflux from preloaded cells. Maximum calcium-45 efflux stimulated by 12-O-tetradecanoyl phorbol-13-acetate was equivalent to maximum norepinephrine-stimulated calcium-45 efflux, and maximally effective concentrations of 12-O-tetradecanoyl phorbol-13-acetate and norepinephrine together were no more potent than either drug alone. Exposure of cells to 12-O tetradecanoyl phorbol-13-acetate for periods of 1-24 hours resulted in complete loss of norepinephrine-stimulated calcium-45 efflux and a much slower, concentration-related reduction in alpha-adrenergic receptor number, with a maximum reduction of 50-60% at 12-O-tetradecanoyl phorbol-13-acetate concentrations greater than or equal to 10 nM. Twenty-four hours of exposure to 12-O-tetradecanoyl phorbol-13-acetate (10 nM) and norepinephrine (10 microM) together caused no greater reduction in [3H]prazosin binding than did norepinephrine alone. 12-O-tetradecanoyl phorbol-13-acetate had no effect on [3H]prazosin-binding affinity. These data suggest an important role for protein kinase-C in both the acute excitation-contraction coupling of vascular smooth muscle alpha-adrenergic receptors, and in the long-term modulation of vascular alpha-adrenergic receptor responsiveness. PMID- 3013461 TI - Cyclic guanosine monophosphate inhibition of contraction may be mediated through inhibition of phosphatidylinositol hydrolysis in rat aorta. AB - The purpose of this study was to determine whether cyclic guanosine monophosphate inhibits contraction through inhibition of phosphatidylinositol hydrolysis. Sodium nitroprusside and atriopeptin II, agents which activate soluble and particulate guanylate cyclase, respectively, inhibited norepinephrine-induced contraction and accumulation of inositol monophosphate, a measure of phosphatidylinositol hydrolysis. Acetylcholine, an agent which elevates smooth muscle cyclic guanosine monophosphate levels through release of an endothelial derived relaxing factor, induced similar inhibitory effects on contraction and inositol monophosphate accumulation in the presence but not absence of the endothelium. The cyclic nucleotide analogue 8-bromo cyclic guanosine monophosphate also inhibited contraction and inositol monophosphate accumulation. These results suggest that cyclic guanosine monophosphate may inhibit contraction through inhibition of phosphatidylinositol hydrolysis. Furthermore, the inhibition of phosphatidylinositol hydrolysis was independent of the mechanism by which cyclic guanosine monophosphate elevation occurred. PMID- 3013462 TI - Interaction between angiotensin II and the baroreceptor reflex in the control of adrenocorticotropic hormone secretion and heart rate in conscious dogs. AB - The present studies were designed to examine the effect of angiotensin II on baroreflex control of adrenocorticotropic hormone secretion and heart rate in conscious dogs. Baroreflex function was assessed by examining the relationship between blood pressure and plasma corticosteroid concentration (used as an index of adrenocorticotropic hormone secretion) and between blood pressure and pulse interval (the inverse of heart rate). Blood pressure was varied by intravenous infusion of four doses (0.3, 0.6, 1.5, and 3.0 micrograms/kg per min) of the vasodilator nitroprusside. Nitroprusside infusion produced an increase in plasma corticosteroid concentration and a decrease in pulse interval, both of which were linearly related to the fall in arterial blood pressure. During infusion of angiotensin II (10 ng/kg per min), however, the lines relating blood pressure to plasma corticosteroid concentration or blood pressure to pulse interval were shifted to a higher pressure level, suggesting that angiotensin II resets the baroreceptor reflex. Angiotensin II blockade with saralasin, an angiotensin II antagonist, or with captopril, a converting enzyme inhibitor, in sodium-depleted dogs with elevated plasma angiotensin II levels produced a shift in the opposite direction in these relationships. Because baroreflex function in sodium-depleted dogs before angiotensin II blockade was similar to that in sodium-replete dogs, despite the increased plasma angiotensin II levels, and because treatment of sodium-replete dogs with saralasin did not affect baroreflex function, these results suggest that endogenous angiotensin II is necessary for the maintenance of normal baroreflex control of adrenocorticotropic hormone secretion and heart rate during sodium depletion. Previous studies designed to evaluate the physiological significance of angiotensin II in the control of adrenocorticotropic hormone secretion showed that high, perhaps supraphysiological, levels of exogenous angiotensin II are required to increase adrenocorticotropic hormone secretion. An additional finding of the present study was that exogenous angiotensin II produces a larger increase in adrenocorticotropic hormone secretion when the pressor effect of angiotensin II was eliminated with simultaneous infusion of the vasodilators nitroprusside or hydralazine. This result suggests that experiments that evaluate the physiological role of angiotensin II in the control of adrenocorticotropic hormone secretion with infusions of exogenous AII may underestimate the importance of endogenously produced angiotensin II, which is normally released without an increase in pressure. PMID- 3013463 TI - Effects of calcium channel blockade on renal vascular resistance responses to changes in perfusion pressure and angiotensin-converting enzyme inhibition in dogs. AB - We conducted these experiments to evaluate the selectivity of calcium channel blockade on the renal autoregulatory mechanism and on angiotensin II-mediated renal vasoconstriction. Experiments were performed in anesthetized dogs in which renal arterial pressure, renal blood flow, and glomerular filtration rate were measured at normal and reduced renal arterial pressure. At control arterial pressures, renal arterial infusions of verapamil increased renal blood flow and glomerular filtration rate significantly. The decreases in renal vascular resistance elicited with verapamil (n = 13) and nifedipine (n = 4) occurred only at renal arterial pressure levels within the normal autoregulatory range. Renal blood flow autoregulatory efficiency was markedly attenuated, and the pressure flow relationship obtained during calcium channel blockade approached that of a passive system. Systemic infusions of an angiotensin-converting enzyme inhibitor (captopril) during continued verapamil infusion caused further vasodilation at all renal arterial pressure values, as evidenced by an increase in slope of 27% of the pressure-blood flow relationship. This response was reversed by angiotensin II infusions. This shift indicates a reduction in minimal vascular resistance elicited by captopril, not obtainable with verapamil alone, and sensitive to angiotensin II. The effects of verapamil and nifedipine on renal blood flow autoregulation suggest a specific effect at preglomerular sites of potential operated membrane calcium channels in the autoregulatory phenomenon. The additional vasodilation elicited with captopril and reversed by angiotensin II indicates the presence of an angiotensin-sensitive postglomerular resistance component which is not influenced by calcium entry blockers. PMID- 3013464 TI - Relative contribution of various expressions of cAMP excretion to other indices of parathyroid function, as tested by discriminant multivariate linear regression analysis. AB - We evaluated the relative contribution to the diagnosis of hyperparathyroid disease from current laboratory indices of parathyroid function--plasma calcium (I), phosphate (II), carboxy-terminal (III) and predominantly amino-terminal (IV) radioimmunoassays of parathyrin, the urinary excretion ratios of cyclic adenosine monophosphate (cAMP) to creatinine (V) or to glomerular filtrate (VI), and the ratio of the nephrogenous fraction of cAMP to glomerular filtrate (VII)--in 224 subjects: 40 with surgically proven hyperparathyroid disease, the others normoparathyroid. The decreasing order of sensitivity was I greater than VI greater than VII greater than V greater than III greater than IV greater than II; all these indices differed significantly between normoparathyroid and hyperparathyroid patients. The decreasing order of specificity was VII, III greater than I greater than IV greater than V, II greater than VI. Discriminant multivariate linear regression analysis was performed in a subset of 58 subjects (17 hyper- and 41 normoparathyroid) from the population studied here, chosen because all of the laboratory indices were determined for each subject. The classification accuracy was 98.3% for combining I, VII, and III (r = 0.908), or I and V (r = 0.893), or I and VII (r = 0.889). The other variables did not add to the precision of classification. PMID- 3013465 TI - The Stratus immunofluorometric assay system evaluated for quantifying human choriogonadotropin in serum. AB - We evaluated the Stratus (American Dade, Miami, FL), an automated immunofluorometric assay system, for the quantification of human choriogonadotropin (hCG) in serum or plasma. The assay is based on the "sandwich" (two-site) immunoassay methodology: use of two monoclonal antibodies, one specific for the alpha subunit and the other for the beta subunit, results in an assay that is specific for the intact hCG molecule. Results for the first sample are obtained in 7 min; subsequent additional values are produced at 1-min intervals. Inter-run precision (CV), estimated from replicate determinations of sera, was 4.5% at an hCG concentration of 38 int. units/L, 4.9% at 114, and 6.1% at 194. Intrarun CV was less than 2% at all three concentrations. Correlations of results for 127 specimens analyzed in duplicate with the Stratus (y) and by a radioimmunoassay (x) for beta hCG (Gamma Dab M [cf931125I] beta-hCG, Travenol Genentech Diagnostics, Cambridge, MA) yielded the following regression equation: y = 0.969x - 6.0 (r = 0.995). The Stratus immunofluorometric system provides a rapid and convenient assay of hCG in serum or plasma. PMID- 3013466 TI - Preclinical determination of AIDS disease by a series of tests. PMID- 3013467 TI - Estimation of vitamin D3 and 25-hydroxyvitamin D3 in muscle and adipose tissue of rats and man. AB - An assay is described based on a high pressure liquid chromatography system for measurement of vitamin D3 and 25-hydroxyvitamin D3 concentration in adipose tissue and muscle. The sensitivity of the assay is less than 1 ng/g of tissue. Neither vitamin D3 nor 25-hydroxyvitamin D3 were found in tissues of vitamin D deficient rats using this method. 25-Hydroxyvitamin D3 could not be detected in adipose tissue and very little was found in muscle of normal rats. The mean vitamin D3 concentrations in 15 samples of human perirenal adipose tissue was 45.3 ng/g +/- 22.2 (SD) and in 6 samples of axillary tissue 115.6 ng/g +/- 52.4 (SD). Human adipose tissue contains a substantial amount of vitamin D3 and its contribution to the maintenance of vitamin D status is discussed. PMID- 3013468 TI - Adrenal and gonadal androgen secretion in normal females. AB - Both the adrenal and the ovary contain the biosynthetic pathways necessary for androgen synthesis and secretion. The fetal ovary is not very active but the fetal adrenal is an important source of DHAS. However the secretion of DHAS declines markedly after birth and until puberty there is little androgen secretion by either the adrenal or the ovary. Post-pubertally, the adrenal secretes DHAS, DHA, delta 4-A and T from the reticularis and probably the fasciculata. This secretion is under ACTH control, at least in part, but apparently also under control of another pituitary polypeptide tentatively called 'adrenal androgen secretory hormone'. THe adrenal secretion rates are in the range of 7-14 mg/day for DHAS, 3-4 mg/day for DHA, 1-1.5 mg/day for delta 4-A and 50 micrograms/day for T. Androgen secretion from the ovary arises in part from the theca cells of the follicle, the corpus luteum and the stromal cells, under LH control, and will vary somewhat during the normal menstrual cycle. The ovarian secretion rate in the follicular phase is 1-2 mg/day for DHA, 1-1.5 mg/day for delta 4-A and about 50 micrograms/day for T. In the peri-ovulatory period the secretion rate of delta 4-A can rise to 3-3.5 mg/day but there appears to be little change in the secretion of DHA and T. The normal ovary does not secrete significant amounts of DHAS. In about 50% of post-menopausal women the ovaries continue to secrete some T but little delta 4-A or DHA. PMID- 3013469 TI - Perspectives on cutaneous T cell lymphoma. PMID- 3013470 TI - Autoradiographic analysis of (-)-[125I]-CYP binding in mouse kidney. AB - The distribution and binding characteristics of the radioligand (-)-[125I] cyanopindolol (CYP) have been examined in slide mounted mouse kidney sections, using the technique of in vitro labelling and autoradiography. (-)-[125I]-CYP binding to sections was of high affinity (KD = 55.8 pmol/l, s.e.m. = 8.1, n = 4) to a single population of non-interacting sites (nH = 0.95, s.e.m. = 0.01, Bmax = 0.74 fmol/section, s.e.m. = 0.12, n = 4) and stereoselective with respect to the (-)- and (+)-isomers of both propranolol and pindolol. Autoradiographic studies showed that (-)-[125I]-CYP binding was localized to areas in the renal cortex and medulla. Both cortical and medullary binding were abolished by the inclusion of ( )-propranolol (1 mumol/l) in the incubation medium, whereas (-)-isoprenaline (200 mumol/l) selectively abolished cortical binding. Medullary binding could be prevented by the inclusion of the lipophilic compounds cinanserin (10 mumol/l), haloperidol (10 mumol/l) or phentolamine (10 mumol/l), either alone or together or by washing at 37 degrees C. These results suggest that medullary binding sites are lipid rather than receptor-related. In conclusion, in mouse kidney sections, (-)-[125I]-CYP binds to discrete areas in the cortex and medulla. Cortical binding sites have the molecular characteristics of beta-adrenoceptors while medullary binding sites are lipid-related. Caution should therefore be exercised when defining non-specific binding of lipophilic radioligands. The autoradiographic technique is useful for discriminating between receptor and non receptor binding sites. PMID- 3013471 TI - The epidemiology of pediatric acquired immunodeficiency syndrome. AB - HTLV III infection of children exhibits a relatively narrow spectrum in which the majority of patients develop clinical manifestations before the age of 2 years. In most instances the disease is transmitted in utero, whereby 35-65% of HTLV III positive women give birth to an infected child. Discordant infection in twins is described. In fewer cases the disease is acquired through blood transfusions. In exceptional cases transmission via sexual abuse or use of needles may occur in young children. We have no evidence of intrafamilial horizontal disease transmission from child to child. The number of children with the disease may increase sharply. Around 2000 pregnancies in HTLV III-infected women are projected in New York City in the next year. Should all these pregnancies be completed, several hundred additional cases of pediatric AIDS may be expected in 1986. PMID- 3013472 TI - Laboratory investigation of pediatric acquired immunodeficiency syndrome. AB - Unique laboratory abnormalities, found in pediatric patients with clinical features of immunodeficiency, led to the original observation that a syndrome of acquired immunodeficiency (AIDS) was also occurring in pediatric populations. Initial observations which demonstrated the nonspecific findings of polyclonal hypergammaglobulinemia and T-cell deficiency were followed by confirmatory findings when testing for the AIDS retrovirus became available. In the pediatric population availability of antibody testing and viral isolation became critical in differentiating primary immunodeficiency disorders which involved both the B- and T-cell systems from AIDS associated with retrovirus infection. At this time based upon clinical, epidemiologic, immunologic, and serologic studies, the syndrome of pediatric AIDS can be distinguished from other primary and secondary pediatric immunodeficiency disorders. PMID- 3013473 TI - Suppression of concanavalin A-induced blastogenesis by HTLV-III-infected H9 cells. AB - The ability of cells infected with human T lymphotropic virus type III (HTLV-III) to suppress lymphocyte responses to concanavalin A (ConA) was evaluated. Thirty homosexual men, both HTLV-III seropositive and seronegative, and 11 seronegative laboratory personnel were studied. Peripheral blood lymphocytes from all groups had lower responses to ConA in the presence of HTLV-III-infected H9 cells than in the presence of uninfected H9 cells. Among HTLV-III-seropositive males, responses to ConA in the presence of infected or uninfected H9 cells correlated with the number of T4 cells present. The studies suggest that HTLV-III-infected cells can suppress normal lymphocyte responses. Possible mechanisms of this suppression are discussed. PMID- 3013475 TI - Genetic studies on murine susceptibility to herpes simplex keratitis. AB - We studied the influence of lgh-linked genes on the development of keratopathy after corneal inoculation with herpes simplex virus (HSV). Using congenic strains of mice, we found that the lgh-1 gene locus, or genes closely linked to it, influence the clinical expression of HSV infection. Mice with the lgh-1e or lgh 1d allotype routinely developed severe keratopathy after HSV corneal inoculation, whereas congenic strains with lgh-1a or lgh-1b allotype were less susceptible. Cell-mediated immune responses to HSV also differed between susceptible and resistant murine strains. We interpret our results to imply a genetic influence on cell-mediated, acquired immune responses to HSV infection. PMID- 3013474 TI - Neutralizing antibody in Celebes black macaques recovering from infection with simian acquired immunodeficiency syndrome retrovirus type 2. AB - Neutralizing antibodies that block the ability of simian acquired immunodeficiency syndrome (SAIDS) retrovirus type 2 (SRV-2) to induce syncytium formation in cultures of Raji cells have been found in the serum of nonviremic Celebes black macaques (Macaca nigra). Serum from Celebes macaques that are viremic have little or no neutralizing activity. The neutralizing antibodies were shown to block viral infectivity. The group of monkeys with neutralizing antibodies in their serum exhibited a dramatic improvement in their health from 1982 to 1984. The correlation of neutralizing antibodies with clinical improvement suggests that neutralizing antibodies may play a critical role in limiting the pathogenic effects of SAIDS retrovirus infection and in helping eliminate the infection. PMID- 3013476 TI - Previous immunization of mice with herpes simplex virus type-1 strain MP protects against secondary corneal infection. AB - Herpes simplex virus (HSV)-induced ocular disease is occurring in epidemic proportions throughout the world, and is the number one cause of unilateral corneal blindness in all developed countries. We have found, in a mouse model of herpes simplex keratitis (HSK), that products encoded by the Igh-1 locus on chromosome 12 exert a profound influence on the immune/inflammatory response in the cornea after HSV inoculation in the cornea. Thus, mice with Igh-1c or Igh-1d phenotype routinely develop extreme keratopathy and loss of corneal clarity after HSV encounter in the eye, while congenic strains expressing other Igh-1 phenotypes develop substantially less keratopathy. We examined the effect of previous subcutaneous immunization with the mutant, less virulent, MP strain of HSV on the development of keratitis and encephalitis after secondary corneal inoculation with strains MP, mP, F, and KOS. A/J mice (Igh-1c), 5-6 weeks old, were injected sc with live HSV-1 strain MP. Controls were injected with culture media without virus. Three weeks later both immunized and control nonimmunized animals were challenged in the cornea with HSV-1, strains MP, mP, F, and KOS. The animals were clinically scored for keratitis and encephalitis at regular intervals for 21 days following corneal challenge. None of the immunized animals challenged in the cornea with strain MP, 5 X 10(4) plaque-forming units (PFU), developed clinical signs of encephalitis compared to 86% of unimmunized controls. Of the immunized animals challenged in the cornea with strain MP, 5 X 10(4) PFU, only 18% developed a mild keratitis, while 96% of unimmunized controls developed severe keratitis. Mice immunized subcutaneously with MP and subsequently challenged corneally with other HSV-1 strains (mP, F, or KOS) were also protected from development of severe keratopathy. PMID- 3013477 TI - Impairment of phagocyte oxidative metabolism in hemodialyzed patients with iron overload. AB - In order to study the influence of iron overload on the polymorphonuclear leucocyte (PMN) metabolism of patients on chronic hemodialysis, generation of superoxide anion (O2-) by PMN in whole blood was compared in two groups of hemodialyzed patients: group A consisted of twenty-one individuals with serum ferritin levels above 1000 ng/ml and group B of nineteen individuals with serum ferritin levels below 1000 ng/ml. Whereas basal production of O2- was similar in the two groups (6.3 +/- 4.6 vs 11.5 +/- 8.3 nmoles O2- 10(6) granulocytes-1 15 min-1) (mean +/- s.e.m.), PMN response to opsonized zymosan was significantly lower in group A as compared with group B (86.5 +/- 6.3 vs 120.4 +/- 8.2 nmoles O2- 10(6) granulocytes-1 15 min-1) (p less than 0.01). Superoxide anion generation induced by the dialysis procedure was reduced in eight patients from group A (89.2 +/- 32.1) as compared with eight patients from group B (374.3 +/- 100.0 nmoles O2- 10(6) granulocytes-1 15 min-1) (p less than 0.05). These data suggest that iron overload may be involved in the impairment of neutrophil phagocytosis in patients on chronic hemodialysis. PMID- 3013479 TI - Intracytoplasmic inclusion of Hirano type in Purkinje cells. AB - Intracytoplasmic, rod-shaped and eosinophilic inclusions were recognized only in Purkinje cells in a case of Crow-Fukase syndrome. The ultrastructure of the inclusions was similar to that of the Hirano body and was suspected to have some relation to neurofilaments. PMID- 3013478 TI - Host-mediated effectors of tumor invasion: role of mast cells in matrix degradation. AB - The role of collagenolytic enzymes in tumor invasion and metastasis has been emphasized, but the source of enzyme activity has remained unclear. Degradation of stromal connective tissue is a common feature of invasive neoplasia, and host tumor cell interactions are probably important for localized collagenolysis. We have examined the role of mast cells in malignant cell invasion using cells derived from the rat mammary adenocarcinoma 13762NF. Histologic studies have shown increased numbers of mast cells at the zone of tumor invasion. Mast cell products and conditioned medium from such cells stimulated the production of collagenolytic enzymes by stromal fibroblasts as well as certain subpopulations of tumor cells in vitro. The tumor cell response to mast cell-mediated stimulation of collagenolysis appears to be related to the metastatic potential of the tumor cell. A subpopulation of host fibroblasts derived from the invading tumor zone was also found to be more responsive to mast cell factors than normal fibroblasts, as judged by collagenase production. Thus the mast cell has the potential to induce collagenolytic activity from both host fibroblasts and tumor cells. PMID- 3013480 TI - Scintiscan for acute intrascrotal conditions. AB - The efficacy and merit of testicular imaging, utilizing Tc-99m pertechnetate, were studied prospectively in a group of patients who presented with acute onset of scrotal pain. Consecutive admissions were studied. All were managed according to the likelihood of the problem being testicular torsion, which was determined from the clinical history, physical examination and the routine laboratory data. The final diagnostic outcome, whether by surgical exploration or clinical progress with conservative treatment, is collated with the preoperative scintigraphic interpretations, made with respect to predefined criteria. Analysis of the pretreatment images obtained in 57 patients shows that the radionuclide study is highly reliable in cases of testicular torsion and epididymo-orchitis. It appears to be much less dependable, however, in the other acute scrotal conditions. Torsions that are intermittent in nature or corrected manually apparently can have variable presentations. Certain difficulties and potential pitfalls encountered in interpreting the scintigraphic studies are discussed. PMID- 3013481 TI - Technetium-99m DMSA imaging and the obstructed kidney. AB - Although several authors have claimed that the function of an obstructed kidney could be overestimated on Tc-99m DMSA imaging, the clinical importance of such an overestimation has not been well documented. Partial obstruction of one ureter was created in a rat, and a relative Tc-99m DMSA uptake was obtained 4 hours after intravenous injection. By puncture of the isolated obstructed kidney, it was shown that the function of that kidney was overestimated by at least 17%. PMID- 3013483 TI - Scintigraphic findings in infantile hemangioendothelioma. AB - The scintigraphic findings on sulfur colloid liver-spleen imaging, Tc-99m labeled RBC blood pool imaging, and Tc-99m MDP bone imaging in four patients with infantile hemangioendothelioma are described. Thirteen radionuclide studies were performed, with serial sulfur colloid images obtained in three patients, allowing interval assessment of liver size and tumor involvement. Findings of Tc-99m MDP uptake in the livers of two patients with hemangioendothelioma and diffuse increase in hepatic RBC labeled blood pool activity in one patient also are reported. PMID- 3013482 TI - Graves' disease. Initial presentation with exophthalmos and solitary hot nodule. AB - A 52-year-old man presented with left exophthalmos. A thyroid scan showed a right lobe hot nodule with suppression of the remainder of the gland. Thyroid function tests were normal. In less than two years, the patient had worsening of the exophthalmos. Thyroid indices then revealed hyperthyroidism and the thyroid image had markedly altered (with evidence of diffuse function). This change, initially showing a hot nodule and then diffuse thyroid overactivity, has been reported previously in three cases (all women). Characteristics of the disorder in these four individuals were reviewed. It is possible that the patients had two distinct diseases, separated temporally. PMID- 3013484 TI - A demonstration of simultaneous thyroid and parathyroid adenomata, situated in the same anatomic level. PMID- 3013485 TI - Technetium-99m DMSA abnormal uptake by metastatic lesion of a renal cell carcinoma. PMID- 3013486 TI - Nuclear medicine and esophageal surgery. AB - The principal radionuclide procedures involved in the evaluation of esophageal disorders that are amenable to surgery are illustrated and briefly described. The role of the radionuclide esophagogram (RE) in the diagnosis and management of achalasia, oculopharyngeal muscular dystrophy and its complications, tracheoesophageal fistulae, pharyngeal and esophageal diverticulae, gastric transposition, and fundoplication is discussed. Detection of columnar-lined esophagus by Tc-99m pertechnetate imaging and of esophageal carcinoma by Ga-67 citrate and Tc-99m glucoheptonate studies also is presented. PMID- 3013487 TI - The role of cardiac beta-1 receptors in the hemodynamic response to a beta-2 agonist. AB - The role of beta 1-receptors in the hemodynamic response to beta 2-stimulation was assessed in seven healthy subjects by infusion of the selective beta 2 agonist terbutaline both with and without selective beta 1-blockade by atenolol (50 mg). Infusion of terbutaline increased heart rate (+28 bpm) and indices of left ventricular (LV) performance associated with a marked decrease in LV end systolic wall stress. The LV end-diastolic dimension remained unchanged despite the tachycardia, suggesting that venous return had increased. Systolic blood pressure increased, whereas total peripheral resistance and diastolic blood pressure decreased. Atenolol pretreatment caused the hemodynamic changes expected of beta 1-blockade but did not blunt the effects of terbutaline on heart rate, peripheral resistance, or venous return. Increases after terbutaline in LV performance and systolic blood pressure were significantly blunted by atenolol. Stimulation of beta 1-receptors therefore appears to play no role in the chronotropic and only a moderate role in the inotropic response after infusion of a beta 2-agonist. Alternative mechanisms for the cardiac changes with terbutaline include (1) withdrawal of vagal tone, (2) decrease in afterload, and (3) stimulation of cardiac beta 2-receptors. PMID- 3013488 TI - The use of the signal averaging computer for evaluation of peripheral nerve problems. AB - Utilization of signal averaging computers to analyze evoked nerve potentials generated by an electrical stimulus is a useful adjunct to clinical evaluation. Currently, somatosensory evoked responses are useful for the evaluation of the peripheral nerve with suspected compression syndromes, neuropathies of unknown etiologies, post-traumatic injuries, neuromas-in-continuity, and brachial plexus injuries. Further refinements in the system will result in increased clinical application of this useful modality. Present data indicate that neuromas with a conduction velocity of less than 10 meters per second or an amplitude of less than one fourth of normal should be considered unfavorable neuromas, and if clinically correlated, resection and repair anticipated. Conduction velocity slowing of greater than 20 meters per second across a potential site of compression neuropathy at the wrist using short-segment study should also be viewed as undesirable and will usually correlate with clinical findings on an abnormal von Frey test. The somatosensory response in compression neuropathy usually correlates better with the clinical finding than with an EMG. PMID- 3013489 TI - The role of smooth muscle and its possible involvement in diseases of the lower urinary tract. PMID- 3013490 TI - Reduced number of angiotensin II receptors on platelets in insulin-dependent diabetes. AB - Angiotensin II receptors on platelets were studied in 13 patients with uncomplicated type I diabetes mellitus and in 15 age-matched normal subjects. Receptor density on cells from the diabetic patients was 15% lower than the normal subjects (5.2 +/- 0.8 SD sites/platelet in diabetic patients and 6.4 +/- 0.8 in normals, P less than 0.001), but there were no differences in receptor affinity as measured by Kd (4.9 +/- 1.5 X 10(-10) mol/l in diabetic patients and 5.4 +/- 1.4 X 10(-10) mol/l in normals). Plasma concentrations of renin and angiotensin II were similar in both groups. The reduced density of angiotensin II receptors on platelets from patients with insulin-dependent diabetes may reflect a generalized abnormality of angiotensin II receptor regulation. PMID- 3013491 TI - Adenosine monophosphate for postherpetic neuralgia. PMID- 3013492 TI - [Dietary fiber]. PMID- 3013493 TI - Tracheal smooth muscle. AB - Contraction of tracheal smooth muscle requires the binding of Ca2+ to calmodulin, which then binds to and activates MLCK. The Ca2+-calmodulin-MLCK complex catalyzes the phosphorylation of myosin, which causes contraction by stimulating actin-activated Mg2+-ATPase activity of myosin. Myosin phosphorylation appears to be a transient event that is responsible for a high velocity of shortening. The mechanism responsible for maintenance of isometric force is unknown, although a second Ca2+-dependent mechanism with a greater sensitivity to Ca2+ than the activation of MLCK has been hypothesized. Force would be maintained through the slow cycling of nonphosphorylated cross-bridges or a small population of phosphorylated cross-bridges. Tracheal smooth muscle utilizes both extracellular and intracellular pools of Ca2+ for contraction. Moreover, the membrane channels through which extracellular Ca2+ passes have been subdivided into potential dependent channels (PDCs) and receptor-operated channels (ROCs) independent of membrane potential. The relative extent to which extracellular and intracellular sources of Ca2+ as well as PDCs and ROCs are utilized depends on the agonist used for contraction, its concentration, and the type and location of the smooth muscle being investigated. Calcium antagonists such as verapamil and nifedipine, which reportedly block PDCs but not ROCs, are much better inhibitors of tracheal smooth muscle contractions induced by serotonin than those induced by acetylcholine, histamine, and leukotriene D4, indicating an effect of these latter three agents on ROCs. Relaxation of tracheal smooth muscle following stimulation of beta-adrenergic receptors most likely results from an increase in cAMP that stimulates a cAMP-dependent protein kinase to catalyze a protein phosphorylation that leads to relaxation by decreasing the intracellular concentration of Ca2+. The primary mechanisms whereby cAMP is thought to reduce intracellular Ca2+ to effect relaxation include: activation of a calmodulin sensitive Ca2+ ATPase in the plasma and sarcoplasmic reticulum membranes, and extrusion of Ca2+ by a Na+-Ca2+ exchange mechanism coupled to Na+-K+-ATPase in the cell membrane. A more controversial mechanism for relaxation that bypasses Ca2+ might involve the dephosphorylation of myosin. Leukotrienes are released by various stimuli, including immunologic challenge, and have been considered as important mediators of bronchoconstriction in allergic asthma.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3013494 TI - Crystal deposition disease in the elderly. PMID- 3013496 TI - Evaluation of five cell types for the isolation of herpes simplex virus. AB - Five cells were evaluated in a comparative analysis for sensitivity, specificity, and rapidity in detecting the presence of herpes simplex virus HSV-1 and HSV-2. Included in this study were human embryonic kidney (HEK), rabbit kidney (RK), MRC 5, mink lung (ML), and Microtus agrestis (UMMA). A total of 274 specimens from genital, throat, skin, or other sources that were submitted for HSV isolation were used in the study. The sensitivity of the different cells was assessed by the total number of positive cultures detected by all the cells under evaluation. At 48 hr, HEK and RK detected 80% of the positives, ML detected 79%, MRC-5 detected 73%, and UMMA detected 60%. All cells tested were satisfactory; however, the choice of which cell to use for isolation of HSV depends upon the needs of the specific laboratory. PMID- 3013495 TI - Varicella-zoster-specific immune responses in acute herpes zoster during a placebo-controlled trial of oral acyclovir therapy. AB - During a placebo-controlled trial of oral acyclovir therapy for acute zoster in immunocompetent patients, we examined the blastogenic response of peripheral blood mononuclear cells and antibody titers in both placebo and acyclovir recipients to determine whether the drug affected the cell-mediated or humoral immune responses. Proliferative responses to mitogens and two dilutions of varicella-zoster virus antigen were not inhibited when fresh peripheral blood mononuclear cells were simultaneously tested in autologous sera collected before and on day 7 of a 10-day course of 2 g/day of oral acyclovir (plasma drug levels averaged 4.6 microM). Using cryopreserved cells from study days 0, 3, 7, 14, and 30, thawed and tested simultaneously, there was no significant difference at the p less than or equal to 0.05 level between the net proliferative responses at each time point for the two groups. On day 14, however, the proliferative response of the acyclovir group was approximately 50% lower than that of the placebo group. Geometric mean antibody titer rises to varicella-zoster virus were also lower among drug recipients but not significantly so. Although this dose of acyclovir did not have a statistically significant effect on lymphocyte proliferative responses to varicella-zoster virus antigen or antibody titers, the lower values in drug recipients may be a reflection of the ability of acyclovir to terminate viral replication, thus reducing the patient's antigenic burden. PMID- 3013498 TI - The promotion and inhibition of collagen-breakdown in organ cultures of pig synovium: the requirement for serum components and the involvement of cyclic adenosine 3':5'-monophosphate (cAMP). AB - In many pathological situations connective tissue cells acquire the ability to degrade the macromolecular components of their extracellular matrix. To study the destruction of collagen we used organ cultures of porcine synovial tissue. In the presence of 15% rabbit serum explants shrink considerably during 10-14 days, owing to early loss of interfibrillar material followed by retraction and local destruction of collagen fibres, partly by phagocytosis. These changes, and the release of latent collagenase into the medium, are largely inhibited by cortisol and partially by indomethacin. Collagen destruction can be greatly accelerated by the addition to the culture medium of one of the following: sodium fluoride, 3 isobutyl-1-methylxanthine, dibutyryl cyclic adenosine 3':5'-monophosphate or forskolin; these agents are known to affect cyclic adenosine monophosphate metabolism and our results suggest strongly that a change in the intracellular levels of cyclic adenosine monophosphate is a key-step in the process leading to the increased catabolism of collagen. With these compounds the destruction of collagen is largely extracellular; the histological changes and the increased levels of collagenase associated with the destruction can be prevented by cortisol and, except in the case of dibutyryl cyclic adenosine monophosphate, at least partially by indomethacin. Without serum only 3-isobutyl-1-methylxanthine sometimes causes drastic breakdown of collagen. This model system should be of great benefit in exploring the mechanisms involved in collagen destruction. PMID- 3013497 TI - Relative sensitivity of cell culture systems in the detection of herpes simplex viruses. AB - Guinea pig embryonic fibroblasts were more sensitive and McCoy and Hep-2 cells were less sensitive than human foreskin fibroblasts in parallel titrations of herpes simplex virus. No difference in sensitivity was found for five lines of human fetal lung fibroblasts (including WI-38 cells), two lines of human embryo fibroblasts, one line of human foreskin fibroblasts, cells from a human fetal kidney, amnion cells from a human placenta, the Chang liver cell, the HeLa cell, and a line of mink cells. The cell doubling level of human or guinea pig fibroblast lines did not affect their sensitivity. One hundred ninety-five clinical specimens submitted for herpes simplex virus isolations were tested in parallel in primary rabbit kidney, guinea pig embryo, and human fetal lung fibroblast cultures. The percentages of positive or false-negative cultures were essentially the same for the three types of cells. PMID- 3013499 TI - Relationship between endogenous cyclic AMP production and steroid hormone secretion in chick adrenal cells. AB - Dispersed chick adrenal cells were incubated with either ACTH, cholera toxin or forskolin. All three agents stimulated cyclic AMP accumulation and secretion of corticosterone and aldosterone by the dispersed cells. The dose-response to ACTH was similar for cyclic AMP and corticosterone but aldosterone secretion appeared to be more sensitive to ACTH stimulation. Concentrations higher than 10(-8) M of ACTH caused suppression of corticosterone output but not of cyclic AMP accumulation or aldosterone secretion. A significant cyclic AMP accumulation occurred within 30 min of exposure to ACTH whereas significant increases in steroid secretion were observed only after 30 min. An early increase (within 30 min) in cyclic AMP accumulation with both cholera toxin and forskolin was not accompanied by any significant stimulation of steroid secretion, which occurred only after 120 min. The results with the avian adrenal cells are consistent with the thesis that steroid production in the adrenocortical cells is stimulated by cyclic AMP-dependent pathways, whereas steroid release may be modulated by others. PMID- 3013500 TI - Studies of sanguinarine in bone resorption models. PMID- 3013501 TI - Near-infrared spectrophotometric monitoring of oxygen distribution to intact brain and skeletal muscle tissues. AB - During continuous near-infrared optical monitoring of brain cortex and hindlimb skeletal muscles, anesthetized, ventilated cats were exposed either to progressive alveolar hypoxia, or to acute hemorrhage followed in some cases by resuscitation. Hypoxia decreased cytochrome a,a3 oxidation state in muscle more than in brain, while tissue blood volume increased in brain and decreased in muscle. At a PaO2 of 25 torr, cytochrome a,a3 oxidation level in the brain was sufficient to support EEG activity, but the cytochrome a,a,3 oxidation state in resting, innervated hindlimb muscle was near zero. Hemorrhagic hypotension invariably decreased cytochrome a,a3 oxidation state and tissue blood volume more in muscle than in brain, and muscle cytochrome a,a3 was completely reduced at about a 25-ml/kg blood loss. These observations, supported by noninvasively measured changes in near-infrared absorption in the tissues during serial intravascular injections of indocyanine green dye, indicate that different cytochrome responses to hypoxia and oligemia in muscle vs. brain tissue are attributable to different regional circulatory adjustments. PMID- 3013502 TI - Outer-membrane permeability of bacteria. AB - Gram-negative bacteria evolved to survive under the conditions in which a number of hazardous compounds are abundant. The outer membrane which protects the cell interior acts as a barrier against such hazardous agents, yet the cells must incorporate the chemicals that are essential for the cellular activity. The devices that Gram-negative bacteria developed to incorporate such essence are the transmembrane pores. These pores could be subdivided into three categories: (1) pore made of porins has a weak solute selectivity; (2) pore made of lamB protein and tsx proteins hold intermediate solute specificity. and (3) pores for the diffusion of vitamin B12 and ferric ion-chelator complexes have a tight solute specificity. Porins are identified from a number of Gram-negatives and from the outer membrane of mitochondria of various sources. Studies on the diffusion properties of these outer-membrane proteins provided essential information to understand membrane transports. PMID- 3013503 TI - Interferon effects on herpes simplex virus in rabbit and human cell cultures. AB - The effects of four subtypes of recombinant human alpha interferon (RIFN alpha), (A,B,D, and the hybrid A/D) were tested on six strains of herpes simplex virus (HSV). RIFN alpha -D was the most effective subtype in rabbit kidney cells, which is consistent with our previous in vivo results in the rabbit herpetic keratitis model. In human corneal cells, however, RIFN alpha -D was one of the least effective IFN subtypes tested. Conversely, RIFN alpha-A appeared to be relatively more effective in the human corneal cells than in the rabbit kidney cells, but RIFN alpha -B and RIFN alpha -A/D were the most effective interferon subtypes in human corneal cells. Different strains of HSV had different susceptibilities to the various IFN subtypes tested. PMID- 3013504 TI - Age alters ADPase positive dendritic (Langerhans) cell response to P. aeruginosa ocular challenge. AB - The morphology, distribution and quantitation of dendritic (Langerhans) cells (LC) was determined by analysis of ADPase stained epithelial flat mounts from 6-8 week young adult (resistant) and 24 month old (susceptible) aged mice before and after experimental infection with P. aeruginosa topically applied to the scarified cornea. The contralateral eye (controls) was also scarified and phosphate buffered saline applied similarly. This study has examined the changes in ADPase positive cell populations of the conjunctival limbal epithelium and corneal epithelium of naturally resistant mice (Swiss-Webster and CD2F1) following corneal infection with Pseudomonas aeruginosa at two different ages, young adult (8 week old) and aged (24 month old). The young adult mice recover from their infection and restore corneal clarity while the aged mice have extensive ocular destruction and corneal scarring. Conjunctival limbal dendritic cell numbers in young adult mice were found to be significantly increased at day seven post infection and then returned to baseline levels. In contrast, conjunctival limbal dendritic cell numbers in aged mice were found to increase slowly and to peak at fourteen days after infection. Other differences between the two ages (young adult and aged) included an initial increase in dendritic cells five hours post infection in the young adult groups and an initial decrease at five hours in the aged groups of mice. PMID- 3013506 TI - Major histocompatibility-restricted cytolytic T-lymphocyte precursors from the thymus of in vivo primed mice: increased frequency and resistance to anti-Lyt-2 antibody inhibition. PMID- 3013505 TI - Small cell bronchogenic carcinoma. AB - We have attempted to highlight the most important aspects of SCBC in this review. The significant strides made in a variety of areas have been associated with increased response rates and survival as well as with a prolonged disease-free interval in a fraction of patients. The consensus is that 50% or more of patients with LD can achieve a CR, with an overall objective response rate of 80% or greater and a median overall survival of 14 months or longer. Furthermore, 15% to 20% of such patients may expect a disease-free interval of at least three years that appears to be associated with cure in at least some of these patients. Patients with ED may experience a 20% or greater CR, an 80% or greater objective response, and have a median overall survival of at least seven months. Extensive research is ongoing in a variety of areas. Further refinements in developing more effective chemotherapeutic regimens are likely, as is obtaining new information concerning the intensity, duration, and selection of chemotherapeutic agents and their role in relationship to radiotherapy. Improvement in radiotherapy techniques may lead to improved therapeutic results. Only recently has a reevaluation of the role of surgery in SCBC begun to take place. Also, several new areas of investigation are on the horizon, ranging from improved staging with thoracic and abdominal computed tomography to the role of warfarin, monoclonal tumor antibodies, and several currently investigational chemotherapeutic and biologic response modifier agents. PMID- 3013508 TI - Lithium chloride as an enhancing agent for metaphase preparations from rodent lymphocytes. AB - Circulating lymphocytes of many mammals, especially rodents, have proven refractory to PHA stimulation of mitotic activity. Addition of small amounts of lithium chloride to the culture medium removes the refractoriness and increases the yield of metaphase cells 7 fold. This simple procedure makes possible the routine use of circulating lymphocytes in rodent karyology. PMID- 3013507 TI - Assignment of pancreatic ribonuclease gene to mouse chromosome 14. AB - A pancreatic ribonuclease cDNA was used as a probe for Southern blot hybridization of genomic DNA from recombinant inbred strains of mice. The results indicated that the gene coding for pancreatic ribonuclease (Rib-1) can be assigned to mouse chromosome 14. Analysis of the congenic strain B10.D2(57N)Sn confirmed this assignment and indicated that Rib-1 is closely linked to the genes encoding the T-cell receptor alpha subunit (Tcra) and nucleoside phosphorylase-2 (Np-2). PMID- 3013509 TI - Physical mapping of the factor VIII gene proximal to two polymorphic DNA probes in human chromosome band Xq28: implications for factor VIII gene segregation analysis. AB - Genomic DNA segments for the coagulation factor VIIIc gene (F8C), which exhibits only limited restriction length polymorphism, map to the proximal region of band Xq28 by somatic cell hybridization analysis and in situ hybridization. Using somatic cell hybrids, we have obtained data which place probes DX13 (used to detect locus DXS15) and St14 (used to detect DXS52) distal to F8C, within band Xq28. Previous studies have mapped the factor IX gene (F9) and probe 52A (used to detect DXS51) proximal to F8C, in Xq26----q27 and Xq27, respectively (Camerino et al., 1984; Drayna et al., 1984; Mattei et al., 1985). Thus, the relative order of genetic marker loci in the Xq27----qter region is most likely cen-F9-DXS51-F8C (DXS15, DXS52)-Xqter. The collection of these molecular probes is thus potentially useful in three-factor crosses of factor VIII gene segregation. PMID- 3013510 TI - Noninvasive evaluation of mediastinal metastases in bronchogenic carcinoma with 67Ga scanning. PMID- 3013511 TI - Bronchoalveolar lavage as the exclusive diagnostic modality for Pneumocystis carinii pneumonia. A prospective study among patients with acquired immunodeficiency syndrome. AB - Pneumocystis carinii pneumonia (PCP) is the most common life-threatening opportunistic infection among patients with the acquired immunodeficiency syndrome (AIDS). Because retrospective studies suggested that bronchoalveolar lavage (BAL) compared favorably to lung biopsy in the diagnosis of PCP, we prospectively evaluated the utility of BAL in 40 consecutive patients with AIDS or risk of AIDS who presented with respiratory complaints. The BAL revealed P carinii in 36 of 42 episodes of pneumonia (86 percent) among 40 patients. Clinical follow-up of the six patients whose BAL was negative for PCP suggested only one possible false negative BAL for PCP. Therefore, BAL detected PCP in 36 of 37 patients for a sensitivity of 97 percent. BAL detected cytomegalovirus in 15 of 38 patients, as well as Mycobacterium avium-intracellulare and Cryptococcus (each in one patient). By virtue of accuracy and lack of morbidity demonstrated in our study, BAL should supplant lung biopsy techniques in the evaluation of AIDS patients with pulmonary symptoms. PMID- 3013512 TI - [Congenital soft tissue dysplasia. A new anatomo-clinical entity]. PMID- 3013513 TI - [Burkitt's disease. Apropos of apparently surgical forms]. PMID- 3013514 TI - [Pelvic arteriography in the diagnosis of uterine malignant trophoblastic disease]. PMID- 3013515 TI - [Chromosomal studies in patients with trophoblastic diseases before and after chemotherapy]. PMID- 3013517 TI - Papillomaviruses. PMID- 3013516 TI - Heterochromatic DNA in Triturus (Amphibia, Urodela). I. A satellite DNA component of the pericentric C-bands. AB - We have studied the structure, genome organization, chromosomal location, conservation across species and transcription on lampbrush chromosomes, of an AT rich satellite DNA component of the newt, Triturus vulgaris meridionalis. The satellite (Sat G), originally isolated by gradient centrifugation, represents about 2% of the vulgaris genome and comprises a highly repetitive sequence family (HindIII family), whose monomers have been cloned. The repeat units are about 330 bp long, as measured on gels, and a cloned unit (pTvm1) is 310 bp long, as shown by sequencing. Abundant clusters of the HindIII family sequences are located within the pericentric heterochromatin (i.e. the C-bands placed at both sides of, and at a certain distance from, the centromeres) in most chromosomes. Both the sequence family and its overall pattern of chromosomal distribution are conserved within the genus Triturus, despite a few species-specific differences. The great majority of the HindIII family sequences are unexpressed on lampbrush chromosomes; they reside within pericentric, condensed segments of the chromosome axis ("loopless bars"). Only a few sequences are transcribed on some loops, suggesting that transcription promotion does not depend on the satellite sequences themselves. PMID- 3013518 TI - Criteria for establishing that a virus is oncogenic. AB - To prove that a particular infectious agent causes a disease is much more difficult in human subjects than in other animals for both ethical and practical reasons. Where the disease is a malignant tumour with a long latent period the situation is even more difficult. For these reasons, it is often necessary to concentrate in the first instance on association of the virus with the disease, and this is discussed in the context of papillomaviruses. Association of a virus with a tumour may occur for a variety of reasons other than the virus being the cause of the tumour. This is illustrated by several examples of parvoviruses and DNA tumour viruses. Conversely, the absence of any sign of virus or viral nucleic acid in a tumour does not prove that the tumour was not induced by a virus. Apart from association of a virus with a tumour it is also necessary to show that the virus in question is oncogenic. Again this cannot normally be done directly, so that indirect evidence from animal experiments or from in vitro transformation is likely to be the best available alternative. In the final analysis the best proof of oncogenicity may be the effectiveness of intervention directed at the virus. PMID- 3013519 TI - Papillomavirus infection in cattle: viral and chemical cofactors in naturally occurring and experimentally induced tumours. AB - Six different types of bovine papillomavirus (BPV-1 to BPV-6) have been identified and classified into two subgroups: subgroup A, which induce fibropapillomas, and subgroup B, which induce true epithelial papillomas. BPV-4, a member of subgroup B, is the aetiological agent of papillomas of the upper alimentary canal, which can become a focus for transformation to squamous-cell carcinomas in animals feeding on bracken fern. Strong circumstantial evidence suggests that the progression to malignancy is due to the interplay between BPV-4 and carcinogen(s) present in the fern. The carcinomas of the upper alimentary canal are often accompanied by adenomas and adenocarcinomas of the lower bowels, and by carcinomas and hemangiosarcomas of the urinary bladder. Bracken-grazing animals are also heavily immunosuppressed. Florid papillomatosis of the upper alimentary canal and cancers of the urinary bladder have been experimentally reproduced in animals either kept on a diet of bracken or immunosuppressed with azathioprine. Several bladder cancers contained multiple episomal copies of BPV-2 DNA, suggesting that this virus, or its genome, can be present in a latent form, and that it can be implicated in malignant transformation. Further indication of latent infection is provided by the onset of skin warts in papillomatosis-free animals. These warts developed at sites of damaged skin and harboured either BPV 1 or BPV-2. BPV-4 DNA has not been found in the naturally occurring cancers of the upper alimentary canal and of the lower bowels, except in one tongue carcinoma and one transforming papilloma, indicating that the viral genome is not required for the maintenance of the malignant state in the alimentary canal. PMID- 3013520 TI - Immunization against bovine papillomavirus infection. AB - The two large open reading frames denoted L1 and L2 in the non-transforming region of the bovine papillomavirus type 1 (BPV-1) genome have been molecularly cloned to expression in Escherichia coli. Antisera against the E. coli-derived L1 and L2 protein reacted with BPV-1 in both enzyme-linked immunosorbent assays and immunoprecipitation reactions. Neutralization of BPV-induced transformation of mouse C127 cells was demonstrated most consistently with antisera against the L1 protein. E. coli-derived L1 protein protected calves against BPV-1 challenge after vaccination. PMID- 3013521 TI - Epidermodysplasia verruciformis: a model for understanding the oncogenicity of human papillomaviruses. AB - The first evidence for the oncogenic potential of human papillomaviruses (HPVs) was obtained through the study of epidermodysplasia verruciformis (EV). This rare skin disease is characterized by disseminated, refractor, pityriasis versicolor like lesions as well as flat wart-like lesions, and by the development of skin carcinomas in about 30% of the patients. EV is a multifactorial disease involving genetic, immunological and extrinsic (actinic) factors, in addition to infection with specific HPV types. A number of HPVs (at least 15 types) have been characterized in benign EV lesions. HPV DNA sequences are regularly detected in EV carcinomas but, in contrast to benign lesions, the types associated with cancers are usually restricted to HPV-5 and, less frequently, HPV-8, an HPV-5 related type. HPV-5 genomes are usually found as free monomeric or oligomeric DNA molecules in EV carcinomas, and frequently contain deletions. This is in contrast with HPV DNA sequences in genital cancers, which are often integrated into the host DNA. Evidence for the transcription of HPV-5 genomes in primary and metastatic EV carcinomas has recently been obtained. The available data indicate that HPV-5 and some HPV-5-related types have an oncogenic potential and play a role in the malignant transformation of EV lesions. Infection by these HPVs must be considered a major risk factor for the development of cancers in EV patients. EV HPV DNA sequences have only rarely been detected in premalignant or malignant lesions of the skin in the general population. This further stresses the role of genetic, immunological and extrinsic factors in the abnormal susceptibility of EV patients to a set of specific HPV types. PMID- 3013522 TI - Persistence and expression of human papillomavirus DNA in genital cancer. AB - There is mounting evidence that certain types of human papillomaviruses (HPV types 16 and 18) are associated with human genital cancer. Other virus types, such as HPV-6 or HPV-11, are more regularly found in benign genital warts. Since all viruses can be present in putative precancerous lesions of the uterine cervix (dysplasia, cervical intraepithelial neoplasia) it has been postulated that individual HPV types have different 'oncogenic potential'. The molecular basis for this difference is not known. The question of the natural reservoir for the oncogenic viruses is discussed. Expression of parts of the early region of the HPV genome in cell lines established from genital cancer supports the hypothesis that papillomaviruses are involved in inducing and/or maintaining the transformed phenotype of cancer cells. PMID- 3013524 TI - Classification of the papillomaviruses--mapping the genome. AB - Papillomaviruses form one genus of the Papovaviridae family. They share common antigenic determinants and their DNAs cross-hybridize under conditions of low stringency. The classification of papillomaviruses is at present based on the host range and the relatedness of the nucleic acids. Isolates are considered independent types if there is less than 50% cross-hybridization in the liquid phase according to a standard protocol. At least 31 human and six bovine papillomavirus types can be differentiated on this basis. The host range does not reflect the natural relationship between the viruses. Subgenera, which differ in biological properties, can be distinguished in outline. Data on overall sequence homology are insufficient for a meaningful classification because two types of virus may be closely related within one genome region and rather heterogeneous in other areas. Some new isolates appear as intermediates between previously well separated types and complicate the system. A reasonable classification of such types of papillomavirus should be based on homologies between genes that are relevant for differences in the biology of the viruses. A functional mapping of the rather uniformly organized, colinear genomes of papillomaviruses has been started. Genetic studies with bovine papillomavirus type 1 have assigned functions in replication, transformation, gene expression and capsid synthesis to individual open reading frames. PMID- 3013523 TI - Organization and expression of the genome of bovine papillomavirus type 1. AB - The viral mRNAs present in C127 cells transformed by bovine papillomavirus type 1 (BPV-1) have been mapped by a variety of techniques, including S1 nuclease analysis, Northern blot analysis, primer extension and electron microscopic heteroduplex analysis. The results reveal a very complex mRNA pattern, comprising at least five types of spliced cytoplasmic mRNAs. Both unspliced and partially processed nuclear RNA species have also been identified. The transforming region of BPV-1 contains several promoter regions. A major cap site is located at coordinate 1 and another putative cap site at coordinate 31. A third candidate cap site maps around coordinate 39. PMID- 3013525 TI - Papillomavirus transforming functions. AB - The bovine papillomavirus type 1 (BPV-1) has served as a model for unravelling the molecular genetics of the papillomaviruses. BPV-1 transformation of rodent cells in tissue culture has provided a means to study the viral functions involved in latent infection of cells and in the induction of cellular proliferation functions. BPV-1 has been shown to encode two independent transforming genes, each of which can induce cellular transformation in susceptible rodent cells. These two genes apparently act synergistically in transforming mouse C127 cells. Deletion mutagenesis studies have shown that the expression of one of these genes (E6) is required for efficient tumorigenesis and anchorage independence. BPV-1 also encodes functions which may act indirectly to affect transformation. BPV-1 contains transcriptional enhancers which can act in a position-independent and orientation-independent manner to increase the transcriptional activity of a heterologous gene. One of these elements, which is located in a non-coding region of the genome, can be trans-activated by a specific viral gene product encoded by the E2 open reading frame. Mutations which eliminate this trans-activation function also have a dramatic effect on transformation and on stable plasmid maintenance. PMID- 3013526 TI - The bovine papillomavirus replicon. AB - The bovine papillomavirus genome contains two cis-acting sequences which can serve as signals for replication. At least three virally encoded genes seem to be involved in plasmid replication: E6, E6/7 and E1. Mutations in either the E6 or the E7 open reading frame create plasmids that are maintained at a low copy number per cell. Mutations in the E1 open reading frame are absolutely lethal to replication. Complementation experiments show that these mutations define separate genes. Experiments are described which show that cells harbouring plasmids with mutations in either the E6 or the E7 open reading frame acquire an immunity to high copy-number plasmids. We suggest that either the cell or the virus encodes a repressor. The positive action of E6 and E6/7 modulates the activity of this repressor to allow for the high copy-number state. Though the viral oncogenes are capable of transforming cells separately when they are expressed as part of certain recombinant DNA expression systems, it is clear that, in the context of the entire viral replicon, interactions between the transforming functions and replication functions must exist. PMID- 3013527 TI - Production of spliced DNA copies of the cottontail rabbit papillomavirus genome in a retroviral vector. AB - The early region of the cottontail rabbit papillomavirus (CRPV) genome has been introduced into a retroviral vector and recombinant retroviruses, produced upon transfection of the psi 2 packaging cell line, have been used to infect NIH 3T3 cells. Spliced derivatives of the CRPV early region can be rescued from the infected cells. Sequence analysis demonstrates that the major splicing event observed in RNA in tumours is faithfully reproduced in this system. This splice generates a polycistronic mRNA that contains in its 5' portion the E7 open reading frame, or both E6 and E7, and at its 3' end a reading frame with codons for three amino acids from the N-terminus of E1 linked to codons for 100 amino acids from the C-terminus of the E4 region. Recombinant retroviruses containing intact or spliced CRPV sequences can now be used to introduce the viral genes efficiently into a variety of cell lines. PMID- 3013528 TI - Methods for diagnosing papillomavirus infection. AB - The morphology of the lesion and the site in which the lesion is found are the initial clues in classifying papillomavirus-induced neoplasia. Human papillomavirus (HPV) types have limited site-specificity and differ in their association with benign or malignant neoplastic development. Cytopathology, electron microscopy, antigen detection and molecular hybridization all play a role in the armamentarium of diagnostic methods. Although nitrocellulose blotting procedures provide the most accurate and sensitive method for detecting and characterizing viral nucleic acid sequences, recent improvements in cytological hybridization methods allow for rapid detection of virus and analysis of HPV type directly in biopsied tissue and in cervical smears. In particular, these in situ hybridization procedures facilitate retrospective studies of stored specimens. PMID- 3013529 TI - [A study on etiology and epidemiology of 1446 patients with diarrhea]. PMID- 3013530 TI - Primary inflammatory malignant fibrous histiocytoma of the colon. AB - A unique case of inflammatory malignant fibrous histiocytoma of the large bowel is presented. This lesion occurred in the colon of an elderly man suffering from weakness, anemia, anorexia, and weight loss. A right hemicolectomy was performed, and six months later, on follow-up, he was found to be well. The literature on visceral involvement by malignant fibrohistiocytic tumors is reviewed. PMID- 3013532 TI - Appraisal and cytomorphologic analysis of common carcinomas of the breast. AB - This work is based on 15 years experience with more than 9000 needle aspiration biopsies from the breast performed by a number of clinicians without syringe guns. From 1981 through 1983, 329 carcinomas were detected with a sensitivity of 90%. A positive or suspicious report was issued in 17 of the 32 minimal carcinomas. There were no false-positive diagnoses. A retrospective comparative study was made on the aspiration biopsy cytology specimens from 65 histologically verified carcinomas: 30 infiltrating ductal, 16 infiltrating lobular, 10 medullary, and 10 colloid carcinomas. Parameters included the pattern, major malignant criteria, and cell measurements by calibrated ocular micrometry. The classic features of each carcinoma and the differential cytomorphology are described. PMID- 3013533 TI - Psammoma bodies and optically clear nuclei in bronchiolo-alveolar cell carcinoma. Diagnosis by fine needle aspiration biopsy with histologic and ultrastructural confirmation. AB - The fine needle aspiration cytology of two cases of bronchiolo-alveolar cell carcinoma of the lung having unusual features is reported. One case demonstrated numerous psammoma bodies in the cytologic smears, whereas the other case showed an abundance of cells with optically clear nuclei. Both peripherally located tumors were resected and confirmed as primary bronchiolo-alveolar cell carcinoma by histologic and ultrastructural examination. We believe this to be the first report describing these unusual features of bronchiolo-alveolar cell carcinoma diagnosed by fine needle aspiration cytology. Presented is a discussion of psammoma bodies and optically clear nuclei seen in primary and metastatic tumors of the lung. This will aid in the diagnosis of these cases. PMID- 3013531 TI - Improved antigen detection in ethanol-fixed cytologic specimens. A modified avidin-biotin-peroxidase complex (ABC) method. AB - A new amplification technique is described for the detection of small amounts of antigen in ethanol-fixed cytologic specimens and formalin-fixed tissues. By adding a third antibody layer to the already sensitive avidin-biotin immunoperoxidase system, we have demonstrated a considerable increase in both the quantitative and qualitative aspects of this immunostaining method. In evaluating this technique, we employed a previously defined small cell lung carcinoma cell surface antigen system and monoclonal antibodies. PMID- 3013534 TI - Ultrastructural studies of malignant cells in fluids. AB - An ultrastructural study was performed on 104 sequential fluids in which more than eight malignant cells per ten high-power fields were found by routine light microscopy. The study included fluids associated with mesotheliomas, melanomas, lymphomas, squamous-cell carcinomas, small-cell anaplastic (oat-cell) carcinomas, and adenocarcinomas. Electron microscopic examination reliably separated lymphoid from epithelial malignancies and benign from reactive and malignant mesothelial cell proliferations. It also suggested or identified a primary site for the adenocarcinomas. Ultrastructural examination of fluids can be a valuable adjunct to routine light microscopy of cytology specimens. No false-positive diagnoses were encountered. Sampling was the most significant limitation for this technique. PMID- 3013535 TI - Herpetic tracheobronchitis detected at bronchoscopy: cytologic diagnosis by the immunoperoxidase method. AB - Herpes simplex virus (HSV) infection of the respiratory tract requires rapid specific diagnosis to avoid late complications and maximize the efficacy of available drug therapy. We report a method of accomplishing this that was tested in 33 cytologic specimens derived from sputum, washings, or brushings from 25 debilitated elderly males suffering from a variety of underlying neoplastic and nonneoplastic chronic diseases. All specimens had shown either single cells (54%), multinucleated groups (8%), or both (38%); they displayed the ground-glass appearance (86%), discrete nuclear inclusions (4%), or both (10%), as appreciated by the Papanicolaou stain. The specimens were processed by the peroxidase antiperoxidase technique utilizing anti-HSV-1 antibody and 3,3'-diaminobenzidine (DAB) as the chromogen. In six cases, aminoethylcarbazol (AEC) was used for comparison. Twenty-nine of the 33 specimens (88%) stained positively for HSV-1 as did control sections of herpetic encephalitis and esophagitis; there were no false positives with appropriate negative controls. All 12 bronchoscopic specimens revealed virocytes with HSV immunopositivity; in contrast, 17 of 21 (80%) sputum specimens were positive for HSV. The strongest positivity was noted in bronchial brushings and washings whereas the only four negative smears were from sputum specimens. The DAB immunostain was coarser and stronger at the periphery of the cytoplasm, and the hematoxylin counterstain permitted a clear identification of nuclear viral changes. With AEC, the immunostain was more vivid and evenly distributed, but counterstaining was impaired due to the greater solubility of the chromogen. The technique is sensitive, reproducible, and less expensive and time-consuming than electron microscopy (EM) or viral cultures. PMID- 3013536 TI - Differential diagnosis of the pleomorphic aspiration biopsy sample of nonepithelial lesions. AB - The major problem confronting the cytopathologist in interpretation of the pleomorphic aspiration biopsy sample (ABS) of nonepithelial lesions is to provide a reasonable differential diagnosis, since the presence of cellular pleomorphism alone does not always denote malignancy. One must be fully cognizant of a vast number of pathologic processes to make a correct interpretation. Some indication of the extent of the problem and the potential diagnostic pitfalls are outlined in selection of nonneoplastic processes and benign and malignant tumors of mesenchymal origin presenting as pleomorphic ABSs. PMID- 3013537 TI - What does the spiral fibril of Eberth mean? PMID- 3013538 TI - Diagnosis of primary hepatic neoplasms by fine-needle aspiration cytology. AB - This article describes the cytologic features of various primary hepatic neoplasms as seen in fine-needle aspirates. Hepatocellular carcinoma can be differentiated from metastatic carcinoma by its tendency to recapitulate the characteristics of normal hepatocytes, namely, resemblance of the neoplastic cells to liver cells, growth in trabeculae, and bile production. Fibrolamellar hepatocellular carcinoma is characterized by larger, polygonal tumor cells with clearly defined cell outline, deeply eosinophilic granular cytoplasm, and extremely large solitary nucleoli. Lamellae of fibrocytes are seen dividing the tumor cells into small groups. Hepatocellular adenoma and focal nodular hyperplasia exhibit cells that are benign-appearing or minimally atypical. Cholangiocarcinoma is an adenocarcinoma and cannot be differentiated from metastatic adenocarcinoma on purely morphologic grounds. Primary hepatic sarcoma is exceptionally rare and shows malignant spindle cells. Some inflammatory conditions such as abscess, cysts, and tuberculoma often present as space occupying lesions and should be included in the differential diagnosis of hepatic neoplasm. PMID- 3013539 TI - Insulin and the regulation of isolated nuclei and nuclear subfractions: potential relationship to mRNA metabolism. PMID- 3013540 TI - Receptor defects in patients with extreme insulin resistance. PMID- 3013541 TI - Insulin receptor kinase and its mode of signaling membrane components. PMID- 3013542 TI - Insulin receptors and the molecular mechanism of insulin action. PMID- 3013543 TI - The cytoskeleton and insulin secretion. AB - One of the central, unresolved problems in our understanding of insulin secretion is the way in which stimulus recognition and its associated metabolic events are translated into the mechanical processes of insulin-storage granule movement and extrusion from the cells by exocytosis. In the present article we have examined the structural organization of the B-cell cytoskeleton in detail and have reviewed how drugs that affect the cytoskeleton alter insulin secretion. Available information about the interactions of tubulin, actin, myosin, and actomyosin with insulin-secretory granules is summarized, and a tentative model is proposed to explain how stimulus-effector system coupling might be achieved. PMID- 3013545 TI - Efficient constitutive production of human IFN-gamma in Chinese hamster ovary cells. AB - A human genomic DNA segment of 5.6 kb containing the entire gene for immune interferon-gamma was fused through its 5'-untranslated region to the corresponding region of the simian virus 40 (SV40) T-antigen gene. The SV40 early promoter used contained a modified transcriptional enhancer element with a 93-bp repeat. Supercoiled plasmid DNA was used to transfect Chinese hamster ovary (CHO) cells, the selectable marker being a SV40-dihydrofolate gene construct. Constitutive expression of the IFN-gamma gene in primary transformants was high, especially if a Harvey murine sarcoma virus long terminal repeat (LTR) was present in addition to the SV40 promoter. After gene amplification by methotrexate selection, CHO-gamma cell lines were obtained that produce 1.5-2 million units of IFN-gamma per million cells and per day (200,000 molecules per cell per minute). Metabolic labeling showed that over 90% of the protein secreted by such cells is human IFN-gamma. A one-step immuno-affinity chromatography on monoclonal antibodies yielded pure IFN-gamma with 1-2 X 10(8) units/mg protein. Like IFN-gamma from human white blood cells, the IFN-gamma from CHO-gamma cells is a mixture of two glycoproteins of 26,000 and 20,000 daltons with traces of the unglycosylated 17,000-dalton polypeptide. Large-scale cultures in 1% serum routinely yield over 600,000 units of human IFN-gamma/ml culture per day. PMID- 3013544 TI - The insulin cell: its cellular environment and how it processes (pro)insulin. PMID- 3013546 TI - The effects of transcriptional regulatory sequences introduced into a retroviral genome. AB - Chimeric plasmids in which the herpes thymidine kinase (tk) gene replaced portions of the Rous sarcoma viral genome were used to assess the relationship between viral transcription and that of an exogenous gene located within the viral genome. The entire tk gene and portions of the gene were positioned in both orientations within the viral gag and pol genomic region (which serves as intron for viral env mRNA). Microinjection assays then determined the amount of viral genomic transcription by quantitation of the amount of viral env mRNA produced. Separate injections also assayed for the presence of tk mRNA. Both mRNAs were produced unless the 3' region of the tk gene was present within the viral genome and in the same transcriptional sense. In this case viral env mRNA production was nearly abolished. PMID- 3013548 TI - cDNA clones for liver cytochrome P-450s from individual aroclor-treated rats: constitutive expression of a new P-450 gene related to phenobarbital-inducible forms. AB - Differential hybridization and screening with cloned inserts was used to identify two families of cytochrome P-450 cDNA clones in libraries prepared from total liver poly(A)+RNA of individual Aroclor-treated rats. One family has cDNA inserts for the major phenobarbital-inducible P-450s, P-450b and P-450e. Two types of P 450e inserts were identified. In addition, irregular inserts were characterized from two clones (PB23 and PB24) of this group. The other family has cDNA inserts for the major 3-methylcholanthrene-inducible species, P-450c and P-450d. No coding sequence restriction site variants were detected among 26 P-450d and P 450c inserts analyzed. The restriction map of the irregular 2.2-kb PB23 insert has a P-450b-like portion, followed by a 3' extension that hybridizes to RNAs of 2.7 and 4.8 kb, which are also detectable with a classical P-450b probe. The PB23 insert and the 2.7- and 4.8-kb RNAs presumably represent 3' extensions of P 450b/P-450e mRNAs, polyadenylated at downstream sites. The 858-bp sequence of the PB24 insert encodes the carboxy-terminal portion of a P-450b/P-450e-like protein. There is approximately 20% divergence at the polypeptide level between the PB24 and P-450b/P-450e sequences; nevertheless, they share many essential features. A PB24-specific probe hybridizes to a 1.9-kb RNA species which is present in the liver of untreated rats and which is not appreciably induced by phenobarbital or Aroclor. The PB24 cDNA most likely represents a constitutive cytochrome P-450, related to phenobarbital-inducible forms. PMID- 3013547 TI - A neuropeptide precursor expressed in Aplysia neuron L5. AB - Aplysia abdominal ganglion neuron L5 is immunoreactive with an antiserum generated against the tetrapeptide Phe-Met-Arg-Phe-amide (FMRFamide); however, the specificity of this immune reagent is limited to the sequence Arg-Phe-amide. We isolated cDNA clones homologous to mRNAs specifically expressed in L5 and demonstrated that these clones do not hybridize to a previously characterized gene encoding FMRFamide. The nucleotide sequence of one of these clones, L5-67, does not encode any FMRFamide peptides but does reveal a Gly-Lys-Arg cleavage site following the amino acids Arg-Phe. This data predicts that neuron L5 expresses a peptide ending in Arg-Phe-amide, consistent with the FMRFamide immunoreactivity. PMID- 3013549 TI - Characterization of signals promoting gene expression on the Staphylococcus aureus plasmid pUB110 and development of a gram-positive expression vector system. AB - The transcriptional and translational initiation signals of a portion of the Staphylococcus aureus plasmid pUB110 were analyzed. An Mbo I-Pvu II fragment was sequenced and the site of transcriptional initiation was determined by in vitro mapping. To convert the plasmid into a cloning vector, a multilinker was introduced in different positions relative to a detected reading frame. The Gram negative beta-galactosidase gene and the Gram-positive chloramphenicol acetyltransferase (cat) gene were fused and the level of expression was determined in Bacillus subtilis. Hybrid proteins consisting of corresponding CAT polypeptides were produced in each translational reading frame. Therefore this vector system can be used to express cloned DNA in the Gram-positive host Bacillus subtilis. Furthermore a derived lacZ fusion plasmid may be used for rapid screening of inserts. PMID- 3013550 TI - Isolation and characterization of cDNA clones from mouse skeletal muscle actin mRNA. AB - The sequence corresponding to approximately 98% of mouse skeletal muscle actin mRNA was determined from cDNA clones isolated from a library of recombinants in pBR322. One of these clones contains DNA corresponding to the complete amino acid coding region and a large part of the 5' and 3' untranslated regions of the mRNA. Comparison of the mouse coding region (conserved at the amino acid level) and noncoding regions with the corresponding regions of the rat skeletal muscle actin gene indicates that the noncoding regions have also been under selective pressure during evolution. PMID- 3013551 TI - A simple nonisotopic method for restriction mapping in single-stranded DNA cloning vectors based on taking timepoints during primed Klenow synthesis. AB - A fast, simple, and nonisotopic method for restriction mapping inserts in single stranded cloning vectors (such as M13 or single-stranded plasmids) is presented. The procedure uses a commercially available oligonucleotide sequencing primer to initiate Klenow-mediated, unidirectional DNA synthesis along the single-stranded insert DNA. Aliquots taken at very short timepoints from this reaction are quick frozen, heat-inactivated, and restriction-digested with the restriction enzyme or enzymes of interest. When the samples are run on an agarose gel and stained with ethidium bromide, the restriction bands appear in the order of their proximity to the priming site. The method's advantages are that it is fast, unidirectional and thus relatively unambiguous, requires neither isotope nor elaborate DNA handling or extraction procedures, and resolves the ambiguities due to "near doublets" that often plaque double-digest mapping and partial-digest mapping. Tetranucleotide restriction maps extending up to 5 kb can be determined from a single priming experiment; more infrequent hexanucleotide restriction sites can be mapped over longer distances. Also, a single aliquot taken at an early timepoint can be restriction-digested to establish the orientation of cloned inserts. PMID- 3013552 TI - A sensitive, nondestructive assay for transfected genes. AB - We have adapted the fibrin overlay assay for plasminogen activators (Jones et al., 1975) into a gene transfer expression assay which has the advantage of being very sensitive and nondestructive. In this assay plasminogen activators convert plasminogen to plasmin, which then degrades fibrin, resulting in clearings in a fibrin overlay. Furthermore, the assay can be used as a signal indicating the efficiency of gene transfer or the loss of introduced genetic elements in unstable cell lines. PMID- 3013553 TI - Diabetic Chinese hamsters: metabolic effects of long term guar gum consumption. AB - The effect of 13 weeks of guar gum or cellulose diet consumption upon metabolic parameters was examined in diabetic and control adult Chinese hamsters. Diabetic hamsters displayed typical diabetic metabolic profiles. Both 8% guar gum and 8% cellulose diets maintained body weights in all 4 groups during the study. Diabetic and control hamsters fed guar gum drank less water as the study progressed. At weeks 9 and 13, diabetic hamsters fed guar gum excreted less urine compared to those fed cellulose. Diabetic hamsters fed guar gum had reduced urinary glucose excretion at weeks 1, 9 and 13 compared to those fed cellulose. Control hamsters fed either diet had normal urine volumes with only traces of glucose. Similar fasting plasma glucose levels were measured initially for all diabetic hamsters; all 3 subsequent measurements revealed lower levels for the group fed guar gum. Control hamsters had normal fasting plasma glucose levels. Comparable fasting plasma insulin levels were measured for all diabetic hamsters; these levels increased during the study. Control hamster fasting plasma insulin levels were 3 times higher and did not change. Throughout the study, diabetic hamsters fed guar gum consistently had healthier metabolic profiles than those fed cellulose. PMID- 3013554 TI - Effect of phorbol esters on glucagon secretion from a glucagon-secreting clonal cell line. Synergistic effects of A23187 and theophylline. AB - The cell line In-R1-G9 is one of the clones from the hamster insulinoma cell line, In-111-R1, and it produces glucagon. Phorbol esters markedly enhanced glucagon secretion and the stimulatory effect was found to be correlated to their biological activity as tumor promoters. At a concentration of 200 nM, 12-O tetradecanoylphorbol 13-acetate (TPA) stimulated glucagon secretion 13-fold more than the control in 10 min. The effect of TPA was not influenced by actinomycin D, cycloheximide, colchicine or vincristine. Depletion of calcium from the incubation medium inhibited TPA-induced glucagon secretion by approximately 50% and dibucaine also suppressed glucagon secretion to 67.4%. An addition of A23187 to TPA induced 150% enhancement over the TPA-stimulated glucagon level, and the maximum secretory response was observed when the cells were stimulated with the simultaneous addition of TPA, A23187 and theophylline. PMID- 3013555 TI - Genetic predisposition to diabetes mellitus is associated with impaired humoral immunity to coxsackievirus B4. AB - Experiments were performed to determine whether genetic predisposition to diabetes mellitus (DM) or clinical DM or both exert an influence on the production of neutralization antibodies to coxsackievirus B4 (CB4). The homozygous diabetic mutant mouse db+/db+, on the inbred C57BL/KsJ genetic background, develops a diabetes-like disease when maintained on ad libitum diet but restriction of excess food intake prevents overt disease. The doubly heterozygote db+/+m or the homozygote +m/+m misty coat color mutant, on the C57BL/KsJ genetic background, do not develop DM and served as controls. Animals infected with one-half a previously determined LD50 of CB4 were bled prior to infection and at 3, 5, 7, 14, 21 days and at 1, 2, 3, 4 and 5 months after infection. Serum neutralization antibody (NA) levels were determined from the percent CB4 plaque reduction. Until 2 months following infection, NA levels were not significant in either of the homozygous diabetic mutant groups, db+/db+. In the diabetic mutant group db+/db+, without overt disease, neutralization of CB4 when observed, was low, short-lived, and apparently not specific. However, in the homozygous diabetic mutants with spontaneous diabetes, CB4 NA became evident at 2 months after infection. By 3 months post-infection, serum NA levels were sufficient to cause 90% virus plaque reduction. These observations demonstrate that hereditary DM as characterized by the mutation diabetes, db, in the C57BL/KsJ mouse, is associated with a marked impaired humoral immune response to a diabetogenic human CB4. Specifically, there is an inability to develop an adequate level of anti-CB4 antibodies. The type and degree of immunological impairment are apparently different prior to and after onset of diabetes mellitus. PMID- 3013556 TI - [Construction and properties of the recombinant plasmid intended for the expression of a gene fragment of HTLV-III virus surface protein in Escherichia coli cells]. PMID- 3013557 TI - [Interaction of cobra venom cytotoxin with oriented phospholipid multi-bilayers]. PMID- 3013558 TI - [Effect of rubomycin on the iron-sulfur center 2 of NAD-H-dehydrogenase in rat liver mitochondria]. PMID- 3013559 TI - Effect of amantadine on rhabdovirus infection. AB - Enveloped viruses enter host cells by fusion or viropexis. The latter mechanism is the prevalent entry pathway of rhabdoviruses into susceptible cells. Amantadine, a lysosomotropic agent, inhibits the multiplication of various groups of viruses. The effect of this drug was investigated on vesicular stomatitis virus and rabies fixed virus strain replication in fibroblasts. Amantadine was added to cells before, during and after infection to detect the phase of viral replication affected by the drug. Cells were inoculated with viruses at 4 degrees C and the incubation temperature was progressively raised to 37 degrees C in order to observe the effect of amantadine on attachment and early stages of viral replication. Experimental results indicated that the compound inhibited rhabdovirus infection in CER cells. Viral attachment and penetration did not appear to be affected by the drug, while later steps were inhibited, probably at the level of uncoating when the virus is released from the lysosomes into the intracytoplasmic compartment. PMID- 3013560 TI - Substituted benzaldehyde thiosemicarbazone with antiviral activity against poliovirus. AB - A series of eight benzaldehyde thiosemicarbazone derivatives with a variable number of -OH substituents at different positions on the aromatic ring were prepared and evaluated for in vitro antiviral activity against poliovirus types I, II and III. Some of the compounds significantly inhibited the replication of poliovirus at a mean ED50 of 2, 5.7, 10.5 and 9.7 micrograms/ml. A comparison between chemical structure and biological activity suggests that the inhibitory effect depends on the relative position of the -OH and the thiosemicarbazone group. The compounds possess antiviral action only when the two groups are distant enough to prevent the formation of intramolecular hydrogen bonds. PMID- 3013561 TI - Epirubicin pharmacokinetics after intrahepatic arterial and intraperitoneal administration. AB - The high hepatic clearance of the new doxorubicin analogue epirubicin (4' epidoxorubicin, epiDX) suggests a possible use of this drug in local and regional therapy where a first pass through the liver is required before the drug can reach systemic circulation. EpiDX pharmacokinetics was followed in advanced cancer patients with liver metastases or a primary tumour after single bolus administration in the hepatic artery, through a surgically implanted catheter and subcutaneous access port. The first-pass effect through the liver was appreciable and only a relatively low fraction of the drug reached systemic circulation. Mild leucopenia and alopecia were observed only in a patient with a hepatopulmonary shunt; this subject was actually exposed to higher epiDX plasma levels. Low intraperitoneal doses of epiDX were administered in a weekly schedule to advanced cancer patients with peritoneal metastases and ascites. Drug concentrations were monitored in the ascitic effusion and in plasma. A high concentration gradient was present between the peritoneal cavity and peripheral circulation. No relevant local or systemic toxicity was observed. PMID- 3013562 TI - In vitro activity of coumermycin alone or in combination against Staphylococcus aureus and Staphylococcus epidermidis. AB - The in vitro activity of coumermycin was tested against oxacillin-susceptible and resistant strains of Staphylococcus aureus and Staphylococcus epidermidis, and compared with that of penicillin G, oxacillin, minocyclin, erythromycin, vancomycin, teicoplanin, rifampicin and LM 427. The MICs were measured using the agar dilution method with an inoculum of 10(5) cfu/spot. Coumermycin was the most active antibiotic with MIC90 of 0.025-0.2 mg/l. The MICs of coumermycin remained unchanged by further incubation for 48 h. The combination of coumermycin with ciprofloxacin, an inhibitor of subunit A of the DNA gyrase, was studied by the time-kill curve method and resulted in a synergistic effect when subinhibitory concentrations of either antibiotic were used. The combination of coumermycin with rifampicin or LM 427 was antagonistic. PMID- 3013563 TI - A study on structure-activity relationships of nucleoside analogues. AB - A selected series of nucleoside analogues were tested for inhibitory activity vs DNA and/or RNA synthesis at L1210 cells. The structure-activity relationship was studied among derivatives of 5-fluorouracil and arabinosylcytosine (araC), compounds clinically used in cancer chemotherapy. 5-Fluorouridine was found to be a selective, most potent inhibitor of RNA synthesis. Arabinosylcytosine was the most potent selective inhibitor of DNA synthesis. Only cyclocytidine stands very close to the carcinostatic antibiotics in its strong simultaneous inhibition of both DNA and RNA synthesis. A biologically interesting new group of 5'-deoxy derivatives of arabinosylcytosine was investigated. The inhibitory activity vs DNA and/or RNA synthesis is compared with the activity against growth of E. coli and against herpes simplex virus type I (HSV). The system for inhibition of NA synthesis is proposed as a preliminary first step: an in vitro screening method promising for the discovery of new types of potential carcinostatic agents. Both transport and biotransformation processes of nucleoside analogues were studied in the isolated everted rat jejunum with a continuous perfusion technique. The 5' substituted derivatives of araC and 5'-chloro-5-fluorouridine exhibited higher transport rates and higher metabolic stability during intestinal penetration than the parent nucleosides. There was found to be no correlation between lipophilicity and transport rate, but there is a correlation between lipophilicity and metabolic alterations among the nucleoside derivatives studied. PMID- 3013564 TI - [Anti-LAV/HTLV-III antibodies in gamma globulin preparations destined for intravenous administration]. PMID- 3013565 TI - [Neurologic manifestations in acquired immunodeficiency syndrome (AIDS)]. PMID- 3013566 TI - Sulbactam/ampicillin versus metronidazole/gentamicin in the treatment of severe pelvic infections. AB - The clinical efficacy and safety of sulbactam/ampicillin versus metronidazole/gentamicin were compared in 39 patients with severe pelvic infections. 30 patients had severe acute pelvic inflammatory disease with peritonitis, 3 tubo-ovarian abscesses, 4 endomyometritis, and 2 posthysterectomy pelvic cellulitis. Aerobic and anaerobic cultures from the sites of infection yielded 259 micro-organisms from 38 patients; an average of 6.8 bacteria per infection (3.9 anaerobes and 2.9 aerobes). The most frequent isolates were Bacteroides spp. (21), B. bivius (13), B. disiens (8), Fusobacterium spp. (9), Peptostreptococcus anaerobius (15), P. asaccharolyticus (8), anaerobic Gram positive cocci (17), Gardnerella vaginalis (24), Neisseria gonorrhoeae (14), alpha-haemolytic streptococci (6) and Escherichia coli (3). Clinical cure was noted in 19 of 20 patients treated with sulbactam/ampicillin and 16 of 19 treated with metronidazole/gentamicin. The sulbactam/ampicillin failure was a patient with pelvic inflammatory disease with a positive Chlamydia trachomatis culture who required antichlamydial therapy. The metronidazole/gentamicin failures included a patient with a tubo-ovarian abscess requiring surgical drainage and 2 patients with pelvic inflammatory disease requiring antichlamydial treatment. No adverse haematological, renal, or hepatic effects were noted with either regimen. PMID- 3013567 TI - A comparison of parenteral sulbactam/ampicillin versus clindamycin/gentamicin in the treatment of pelvic inflammatory disease. AB - 60 hospitalized patients with pelvic inflammatory disease entered a randomised study to compare the therapeutic efficacy and tolerability of parenteral sulbactam/ampicillin with that of clindamycin/gentamicin. All 49 pathogens isolated at entry from 21 evaluable patients in the sulbactam/ampicillin group were eradicated. 34 out of 35 pathogens isolated from 18 evaluable patients in the clindamycin/gentamicin group were eradicated. All pathogens resistant to ampicillin in vitro were eradicated. The bacteriological, clinical and overall responses for the sulbactam/ampicillin group were 100%, 85.7% and 85.7%, respectively, compared with 97.1%, 94.4% and 94.4%, respectively, for the clindamycin/gentamicin group. The sulbactam/ampicillin combination was well tolerated. PMID- 3013569 TI - Sulbactam/ampicillin for treatment of polymicrobial pelvic infections. AB - The increasing number of beta-lactam antibiotic-resistant bacteria observed in many strains of aerobic and anaerobic Gram-positive and Gram-negative bacteria, including Bacteroides species, has been well documented. Semisynthetic synthesis of penicillins and cephalosporins with increased resistance to beta-lactamase enzyme hydrolysis has not solved the problem. An alternative to therapy with newer agents is combination of an irreversible, suicide-type, beta-lactamase enzyme inhibitor such as sulbactam with a beta-lactam antibiotic such as ampicillin. Women with a variety of acute polymicrobial pelvic infections have been treated with the above combination, metronidazole or clindamycin combined with aminoglycoside, or cefoxitin in prospective trials. The clinical efficacy of 92.4%, in vitro bacteriological efficacy of 96.6%, and safety of sulbactam/ampicillin were comparable to that observed in women given comparative therapy. Penetration of pelvic tissues by sulbactam and ampicillin was excellent. Sulbactam/ampicillin is a viable alternative for the treatment of women with acute pelvic infections. PMID- 3013568 TI - Sulbactam/ampicillin versus cefoxitin in the treatment of obstetric and gynaecological infections. AB - Preliminary results of a randomised trial comparing parenteral sulbactam 1g plus ampicillin 2g every 8 hours and cefoxitin 2g every 8 hours in 75 patients with gynaecological infection are reported. Clinical and bacteriological cure were achieved in 87% and 91% of patients treated with sulbactam/ampicillin compared with 83% and 59% treated with cefoxitin. Both treatments were well tolerated. PMID- 3013570 TI - Concentration of sulbactam and ampicillin in serum and the myometrium. AB - Sulbactam 1g and ampicillin 2g were administered intravenously to 12 patients, and concentrations of the drugs were determined in the serum and myometrium for up to 120 minutes after infusion. The ratio of sulbactam to ampicillin was about 1:2 in the serum and myometrium. Both drugs penetrated well into the myometrium, and median concentrations of sulbactam and ampicillin were 65% and 68%, respectively, of those in the serum. Sulbactam/ampicillin was effective for perioperative prophylaxis and should be effective as therapy for pelvic infections. PMID- 3013571 TI - Treatment of acute salpingitis with sulbactam/ampicillin. Comparison with cefoxitin. AB - Sulbactam/ampicillin and cefoxitin were compared in the treatment of 20 patients with acute salpingitis diagnosed during laparoscopy. Results were evaluated during laparoscopic follow-up at 2 months. Sulbactam/ampicillin appeared to be a better treatment, producing better results in tubal patency, adhesions and persistent inflammation than cefoxitin. In addition, the combination of sulbactam/ampicillin with doxycycline appears to provide better results than the combination of cefoxitin with doxycycline in chlamydial salpingitis. It is concluded that sulbactam/ampicillin is an effective treatment for nonchlamydial salpingitis. PMID- 3013572 TI - Responsiveness of astrocytes in serum-free aggregate cultures to epidermal growth factor: dependence on the cell cycle and the epidermal growth factor concentration. AB - Serum-free aggregating cell cultures of fetal rat telencephalon treated with low doses (0.5 nM) of epidermal growth factor (EGF) showed a small, transient increase in DNA synthesis but no significant changes in total DNA and protein content. By contrast, treatment with high doses (13 nM) of EGF caused a marked stimulation of DNA synthesis as well as a net increase in DNA and protein content. The expression of the astrocyte-specific enzyme, glutamine synthetase, was greatly enhanced both at low and at high EGF concentrations. These results suggest that at low concentration EGF stimulates exclusively the differentiation of astrocytes, whereas at high concentration, EGF has also a mitogenic effect. Nonproliferating astrocytes in cultures treated with 0.4 microM 1-beta-D arabinofuranosyl-cytosine were refractory to EGF treatment, indicating that their responsiveness to EGF is cell cycle-dependent. Binding studies using a crude membrane fraction of 5-day cultures showed a homogeneous population of EGF binding sites (Kd approximately equal to 2.6 nM). Specific EGF binding sites were found also in non-proliferating (and nonresponsive) cultures, although they showed slightly reduced affinity and binding capacity. This finding suggests that the cell cycle-dependent control of astroglial responsiveness to EGF does not occur at the receptor level. However, it was found that the specific EGF binding sites disappear with progressive cellular differentiation. PMID- 3013573 TI - [New infectious diseases in Finland]. PMID- 3013575 TI - AIDS pandemic. PMID- 3013574 TI - [Infections in immunosuppressed patients]. PMID- 3013576 TI - [AIDS--etiology, pathogenesis and epidemiology]. PMID- 3013577 TI - [Virological diagnosis of HTLV-III infections]. PMID- 3013578 TI - The midfacial degloving approach to the central skull-base. PMID- 3013579 TI - Triple antibiotic spray application to umbilical cords. AB - A trial in 402 newborns demonstrated that application of 'Tribiotic' spray either as a single dose at birth or repeated daily resulted in a significant reduction of umbilical cord infections compared with no antibiotic application. 'Tribiotic' spray was well tolerated but led to delayed cord separation. PMID- 3013580 TI - Inclusion bodies: formation and degeneration of the oocytes in the fish Channa punctatus (Bloch) in response to ammonium sulfate treatment. AB - In the adult Channa punctatus exposed to 500 ppm of the commonly used fertilizer ammonium sulfate for 6 months from January to June, ovarian growth was retarded significantly. The inhibition of the ovarian growth is reflected on the gonadosomatic index which was significantly reduced as compared with control. Apart from this, in treated fish, several stage-I oocytes exhibited proteinaceous extra- and intranuclear "inclusion bodies" which are apparently due to cumulative toxic effect of ammonium sulfate. These oocytes ultimately degenerate. PMID- 3013582 TI - [Virus-specific proteins in cells transformed by the chicken sarcoma virus D6]. AB - The structural proteins and the oncogene product of the avian sarcoma virus (ASV) D6 were analyzed by the radioimmunoprecipitation. ADV D6 was obtained from the chemically induced tumour. ASV D6 contains all the structural proteins found in RSV, but some of them (p19, gp 85) differ from those of RSV. The product of ASV D6 oncogene is pp65src. The protein kinase activity of this src protein is identical to that of wt pp60src. The nature of some other proteins, which can be phosphorylated in this reaction, is discussed. PMID- 3013581 TI - [Epidemiology of hormone dependent malignant tumors--ovulation inhibitors and liver tumors]. AB - Since the first descriptions of liver tumours associated with the intake of steroid contraceptives, the literature has presented an increasing number of case reports as well as epidemiological studies on the relationship between benign liver tumours and the utilization of these drugs. We report 11 benign cases and one malignant case of liver tumours in connection with the intake of these steroid preparations. PMID- 3013583 TI - Antagonism of beta-endorphin-induced prolactin release by alpha-melanocyte stimulating hormone and corticotropin-like intermediate lobe peptide. AB - The ability of two proopiomelanocortin-derived peptides, alpha MSH and corticotropin-like intermediate lobe peptide (CLIP) [ACTH (18-39)] to antagonize the stimulation of PRL secretion by beta-endorphin (beta EP) was studied in the rat. When 50 ng beta EP were injected into the lateral cerebral ventricle, plasma PRL rose from a mean baseline of 1.87 +/- 0.43 ng/ml (+/- SEM) to a peak of 23.0 +/- 3.67 ng/ml 10 min after the injection. When the same animals received 500 ng alpha MSH together with 50 ng beta EP, the peak concentration of PRL was reduced by 74% to 6.05 +/- 1.43 ng/ml (P less than 0.005). After the injection of 500 ng CLIP together with 500 ng beta EP, the peak concentration of PRL was reduced by 47% to 12.8 +/- 3.09 ng/ml (P less than 0.01). Total PRL release, determined by calculating the areas under the plasma PRL concentration curves, was also significantly reduced by the injection of alpha MSH or CLIP. A dose of 100 ng alpha-MSH or CLIP also antagonized the stimulation of PRL secretion by 50 ng beta EP. PRL release was reduced by 62% after administration of 100 ng alpha MSH (P less than 0.001) and by 43% after 100 ng CLIP (P less than 0.05). When 100 ng alpha MSH and 100 ng CLIP were injected together, there was an additive effect in blocking the stimulation of PRL release by beta EP, and the peak plasma PRL concentration was reduced by 81%. Des-acetyl alpha MSH, the predominant form of alpha MSH in the hypothalamus, was also very effective in antagonizing beta EP induced PRL release. The peak PRL concentration was reduced by 52% after administration of 100 ng des-acetyl alpha MSH plus 50 ng beta EP compared with that after beta EP alone (P less than 0.005). We conclude that relatively low doses of both alpha MSH and CLIP can effectively antagonize the actions of beta EP on pituitary PRL release. These findings suggest the possibility that differential posttranslational processing of proopiomelanocortin may serve as a regulator of anterior pituitary function. PMID- 3013584 TI - Induction of polyphosphoinositide breakdown in rat corpus luteum by prostaglandin F2 alpha. AB - The present study examines the possibility that, in the rat corpus luteum, an initial action of prostaglandin F2 alpha (PGF2 alpha) is to induce a ligand stimulated breakdown of membrane inositol phospholipids. Luteal cells in primary culture were prepared from immature rats after PMSG and human CG priming. In 32P prelabeled cells, PGF2 alpha caused a rapid decrease in the level of radiolabel found in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5 bisphosphate, as early as 20 sec after addition of the hormones. At 1 and 2.5 min, the effect of 10(-6) M PGF2 alpha on phosphatidylinositol 4,5-bisphosphate was significantly greater than that caused by 10(-6) M LHRH in identical cell cultures. By contrast, the levels of the 32P-prelabeled phosphatidylinositol and phosphatidic acid were increased at 5 min by PGF2 alpha or LHRH. Concomitant with the alterations in cellular levels of 32P-prelabeled phospholipids, PGF2 alpha markedly enhanced the accumulation of 3H-labeled inositol phosphates, i.e. inositol 1-phosphate, inositol diphosphate, and inositol triphosphate, during a 5 min incubation. A significant increase of radiolabeled inositol diphosphate was seen as early as 1 min after the addition of either PGF2 alpha or LHRH; PGF2 alpha was more effective than LHRH in this regard. The stimulatory effect of LHRH on inositol phosphate accumulation could be blocked completely by the concomitant presence of a potent LHRH antagonist, and at the concentration used (10(-6) M) the effects of PGF2 alpha and LHRH were not additive. Interestingly, the addition of an exogenous phospholipase C also caused a similar enhancement of inositol phosphate accumulation in identical cell cultures. For the first time, these data suggest that, at the level of the corpus luteum, hydrolysis of phosphoinositides may immediately follow PGF2 alpha (and to a lesser extent LHRH) receptor binding, and this in turn may lead to the generation of 1,2-diacylglycerol and inositol phosphates, resynthesis of phosphatidic acid and phosphatidylinositol, and mobilization of Ca2+. PMID- 3013585 TI - Regulation of beta-endorphin, corticotropin-like intermediate lobe peptide, and alpha-melanotropin-stimulating hormone in the hypothalamus by testosterone. AB - Ovarian steroids have previously been shown to regulate the hypothalamic content of beta-endorphin (beta EP) and its release into hypophyseal portal blood. Although the hypothalamic content of beta EP in cycling female rats was unchanged by ovariectomy, chronic treatment of ovariectomized rats with estradiol lowered hypothalamic beta EP levels. In this study, the hypothalamic content of beta EP was compared in male and cycling female rats, and the effects of orchiectomy and testosterone replacement on hypothalamic beta EP were examined. The beta EP content of the medial basal hypothalamus (MBH) was significantly higher in female rats compared to that in males of either the same weight (175-200 g) or the same age (65 days; P less than 0.025). When male rats were studied 4 weeks after castration, the beta-EP content of the MBH increased from a value of 2100 +/- 103 fmol in the controls to 2680 +/- 126 fmol (P less than 0.005). The hypothalamic beta EP content in the castrated males was similar to that in the intact females (2700 +/- 158 fmol). The increase in hypothalamic beta EP induced by castration was blocked by testosterone replacement. When orchiectomized animals were treated for 4 weeks with Silastic capsules filled with testosterone, there was a significant fall in the hypothalamic content of beta EP compared to that in the unreplaced animals. beta EP fell from 3180 +/- 115 to 2033 +/- 53 fmol in the MBH (P less than 0.001), from 1693 +/- 122 to 934 +/- 80 fmol in the anterior hypothalamus (P less than 0.001), and from 148 +/- 26 to 90.3 +/- 11 fmol in the median eminence (P less than 0.05). Testosterone replacement was also associated with a significant decline in the hypothalamic content of corticotropin-like intermediate lobe peptide and alpha MSH. Corticotropin-like intermediate lobe peptide fell from 2400 +/- 53 to 1560 +/- 84 fmol in the MBH (P less than 0.001) and from 1200 +/- 74 to 805 +/- 94 fmol in the anterior hypothalamus (P less than 0.01). alpha MSH fell from 1660 +/- 162 to 884 +/- 75 fmol in the MBH (P less than 0.001) and from 823 +/- 106 to 544 +/- 92 fmol in the anterior hypothalamus (P less than 0.05). Thus, testosterone, as well as estradiol, affects the hypothalamic content of several proopiomelanocortin-derived peptides. The effect on brain peptide content, however, depends on whether the steroids are secreted relatively constantly, as in the male, or fluctuate, as in the cycling female. PMID- 3013586 TI - Receptor binding and gonadotropin-releasing activity of a novel chicken gonadotropin-releasing hormone ([His5, Trp7, Tyr8]GnRH) and a D-Arg6 analog. AB - Receptor binding and gonadotropin-releasing activity was compared for mammalian GnRH, [Gln8]GnRH (chicken I GnRH), [His5, Trp7, Tyr8]GnRH (chicken II GnRH), [Trp7, Leu8]GnRH (salmon GnRH), and [D-Arg6] chicken II GnRH. The mean ED50 values for mammalian GnRH, chicken I GnRH, chicken II GnRH, and salmon GnRH in stimulating LH release from dispersed chicken pituitary cells were 0.27 nM, 0.28 nM, 0.055 nM, and 0.11 nM, respectively. The relative potencies of the peptides compared in the same assay were 0.93, 1.0, 5.6, and 2.5. The ED50 values for chicken I GnRH, chicken II GnRH, and salmon GnRH in stimulating FSH release were 0.37 nM, 0.034 nM, and 0.18 nM, and the relative potencies were 1.0, 13.5, and 1.8. Chicken II GnRH was, therefore, more potent than chicken I GnRH and mammalian GnRH in releasing LH and appeared to have an even greater relative FSH releasing activity than chicken I GnRH or mammalian GnRH. Introduction of D-Arg6 into chicken II GnRH enhanced the activity of this analog 4- and 2-fold relative to chicken II GnRH in LH- and FSH-releasing activity, respectively. The ED50 values of mammalian GnRH, chicken I GnRH, chicken II GnRH, and salmon GnRH in releasing LH from cultured sheep pituitary cells were 2.9 nM, 96 nM, 22 nM, and 104 nM, respectively. The relative potencies were 1.0, 0.016, 0.084, and 0.047. Introduction of D-Arg6 into chicken II GnRH enhanced activity 9-fold. In a rat pituitary receptor binding assay the ED50 values of mammalian GnRH, chicken I GnRH, chicken II GnRH, and salmon GnRH were 2.9 nM, 1480 nM, 19 nM, and 258 nM, respectively. [D-Arg6]Chicken II GnRH was 46 times more active than the natural chicken II GnRH peptide. The results show: 1) chicken II GnRH is more potent than chicken I GnRH, which is equipotent with mammalian GnRH in releasing LH from chicken pituitary cells. Chicken II GnRH is even more potent at releasing FSH. 2) Salmon GnRH is also more potent than chicken I GnRH and mammalian GnRH in stimulating gonadotropin release from chicken pituitary cells. It appears, therefore, that Trp in the 7 position contributes to the enhanced activity of salmon and chicken II GnRH. 3) The low activity of chicken I GnRH, chicken II GnRH, and salmon GnRH in the sheep pituitary cell bioassay and rat pituitary receptor binding assay confirms that Arg8 in mammalian GnRH is important for activity.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3013587 TI - Dibutyryl guanosine 3',5'-monophosphate inhibits cholecystokinin potentiation of insulin release in the isolated perfused rat pancreas. AB - (Bu)2cGMP is known to act as a specific competitive inhibitor for gastrin and cholecystokinin (CCK) peptides. We have examined the effects of (Bu)2cGMP on CCK octapeptide (CCK-8) stimulation of insulin release in the isolated perfused pancreas and compared them with those on protein output. Addition of (Bu)2cGMP after a 20-min perfusion with 100 pM CCK-8 resulted in two distinctly different phases of insulin suppression. There was a sharp initial decline in insulin release for 3 min, followed by transient recovery toward the control level for 5 min, and then a small decline until termination of (Bu)2cGMP infusion. (Bu)2cGMP produced a concentration-dependent inhibition of both phases of insulin decrement. (Bu)2cGMP also produced a concentration-dependent inhibition of protein output. Addition of 1 mM (Bu)2cGMP rapidly and completely abolished CCK-8 stimulated protein output. Since CCK is released by meal intake and exogenous CCK stimulates insulin release and augments glucose-induced insulin release, it is possible that endogenous CCK plays an important role in the enteroinsular axis. The present findings of blockade of CCK-8-induced insulin release by selective antagonist of the action of CCK provide evidence for CCK as a mediator in the enteroinsular axis. PMID- 3013588 TI - Inhibition of the preovulatory prolactin surge in the rat by catechol estrogens: functional and temporal specificity. AB - Catechol estrogens, administered iv to cycling rats on the morning of proestrus, were able to block the preovulatory PRL surge on the afternoon of the same day. Only catechol estrogens with a low affinity for the estrogen receptor, such as 2 hydroxyestrone and 2-hydroxyestradiol-17 alpha were effective in this respect, while the estrogenic catechol estrogen 2-hydroxyestradiol was unable to influence the PRL surge. The effectiveness of the PRL surge abolition was highly dependent on the state of the endogenous estradiol levels at the time of administration. Only doses given just before the peak secretion of estradiol were effective in blocking the PRL surge. Despite similarities in the inhibition of the preovulatory LH and PRL surges by catechol estrogens, these are considered to occur by different mechanisms because the LH secretion is blocked by all catechol estrogens, while the PRL surge is affected only by catechol estrogens with no estrogen agonist properties. The catechol estrogen blockade of the PRL surge may have physiological parallels and provides a useful probe of the mechanisms of the estrogen-PRL axis. PMID- 3013590 TI - Direct thyroid hormone activation of mitochondria: the role of adenine nucleotide translocase. AB - A presumptive mitochondrial T3 receptor previously reported from this and other laboratories appears capable of accounting for the activation of liver mitochondrial oxidative phosphorylation within 30 min after iv bolus injection of nanogram doses of T3 into hypothyroid rats. The inner mitochondrial membrane carrier adenine nucleotide translocase (AdNT) catalyzes the exchange between the extra- and intramitochondrial ADP and ATP, and has been shown by measurements of flux control coefficients to exert a significant measure of control over the rate of mitochondrial oxidative phosphorylation. The activity of this carrier had been reported to be depressed below normal in hypothyroid rats and restored to normal by hormone replacement. Preparations of AdNT from beef heart mitochondria were found to exhibit high affinity, low capacity binding of [125I]T3. The findings make the mitochondrial carrier AdNT a strong candidate for the initiating site for thyroid hormone stimulation in mammalian species. PMID- 3013589 TI - Modulation of the estradiol-induced luteinizing hormone surge by progesterone or antiestrogens: effects on pituitary gonadotropin-releasing hormone receptors. AB - The present study examined the question of whether modulation of estradiol induced LH surges by progesterone or antiestrogens in the immature rat might be related to changes in the concentration of pituitary GnRH receptors (GnRH-R). Rats (28 days old) that received estradiol implants at 0900 h had LH surges approximately 32 h later. Administration of progesterone or nafoxidine (U-11,100 A; 1-(2-[P-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]pyrrolidine hydrochloride) concomitantly with estradiol led to blockade of these LH surges (progesterone or nafoxidine inhibition), while progesterone treatment 24 h after estradiol brought about premature and enhanced LH release (progesterone facilitation). GnRH-R-binding capacity was determined by saturation analysis in homogenates of single pituitaries from immature rats treated with estradiol and progesterone or nafoxidine and controls treated only with estradiol using [125I]iodo-(D-Ala6,Des-Gly10)GnRh ethylamide. The affinity of GnRH-R for this analog ranged from 8.2-15.1 X 10(9) M-1 and was not affected by in vivo steroid or antiestrogen treatment. The number of GnRH-R in gonadotrophs from untreated 28 day-old rats (57.2 +/- 2.6 fmol/pituitary or 177 +/- 11 fmol/mg protein) was comparable to values previously reported for 30 day-old females. GnRH-R levels were first measured 1, 8, 24, 32, and 48 h after estradiol treatment. The pituitary content of GnRH-R paralleled changes in total pituitary protein (nadir at 24 h, rebound at 32 h, continued increase at 48 h), while their concentration (femtomoles per mg protein) was highest at 8 h. Next, GnRH-R levels were examined at 1200 h and at hourly intervals (1400-1800 h) on the afternoon of the LH surge. While GnRH-R concentrations were significantly lower at 1400 and 1700 h than at 1200 or 1800 h in animals treated with estradiol in the progesterone facilitation model, they did not change over time in the other two paradigms. There was no significant difference in pituitary content or concentration of GnRH-R at any time between immature rats treated with estradiol and progesterone or nafoxidine and their respective estradiol-treated controls. These results suggest that changes in GnRH-R levels in pituitary gonadotrophs do not play a major role in enhancement of LH surges by progesterone or in their suppression by progesterone or nafoxidine in the immature rat; therefore, these compounds may affect the pituitary at a site distal to the GnRH receptor. PMID- 3013591 TI - Pathogenesis of hypercalcemia in nude mice bearing a human renal carcinoma. AB - When grown as sc tumors in the nude (nu/nu) mouse, cells of the established human renal carcinoma cell line 786-0 produce hypercalcemia; this has an apparent humoral basis because it is reversed by resection of the primary tumor. We have investigated the pathogenesis of hypercalcemia in this model. Tumor-bearing mice were hypercalcemic (13.4 +/- 0.9 vs. 9.52 +/- 0.13 mg/dl in control mice) and hypophosphatemic (10.0 +/- 0.8 vs. 13.8 +/- 1.5 mg/dl in control mice; all values are mean +/- SEM). The serum concentration of 1,25-dihydroxyvitamin D was increased in tumor-bearing animals (70.0 +/- 9.3 vs. 43.8 +/- 4.8 pg/ml in control animals). Urinary excretion of cAMP was similar in control (33.7 +/- 1.4 nmol/mg creatinine) and tumor-bearing mice (38.2 +/- 4.7 nmol/mg creatinine). However, in the latter, the acute response of urinary cAMP to PTH was blunted. Although intestinal calcium transport in everted duodenal sacs in vitro was increased in tumor-bearing mice, hypercalcemia was unaffected by feeding the animals for 8 days a diet containing less than 0.02% calcium. Hence, absorption of dietary calcium did not play a significant role in maintenance of hypercalcemia. In hypercalcemic animals, the calcium content of the humerus was decreased (2.95 +/- 0.08 vs. 3.29 +/- 0.13 mg in controls; P less than 0.05). Quantitative histomorphometric analysis of the distal femoral metaphysis disclosed a significant reduction in trabecular bone volume in tumor-bearing mice (12.0 +/- 1.1% vs. 16.1 +/- 1.1% in controls; P less than 0.02). A strong trend for increased osteoclast surface and number was observed, suggesting that bone resorption was increased. Osteoblast surface and number were also somewhat increased, as was the rate of mineral apposition (2.55 +/- 0.14 vs. 1.91 +/- 0.04 micron/day in controls; P less than 0.01). Thus, the decrease in trabecular bone volume was associated with high turnover of bone, with an apparent net increase in bone resorption. We conclude that hypercalcemia in the nude mouse bearing human renal carcinoma cells is associated with increased bone resorption, high bone turnover, hypophosphatemia, and increased serum levels of 1,25 dihydroxyvitamin D. PMID- 3013592 TI - Binding of thyroid hormones to nuclear extracts of thyroid cells. AB - Binding of T3 and T4 to soluble nuclear extracts of FRTL-5 cells, rabbit thyroid glands, and rat liver was studied. [125I]Iodo-T3 or [125I]iodo-T4 in concentration ranges of 100-fold (10-fold on each side of measured Kd) was incubated with extract at 4 C, pH 8.2, and the quantity of bound hormone was determined by collection on nitrocellulose filters. The results were corrected for nonspecific binding. Steady state (equilibrium) binding was achieved by 36 h. Apparent dissociation constants (Kd) were determined from Scatchard analysis of data pertaining to extent of binding at 36 h as a function of hormone concentration and were also calculated from kinetics of binding as the ratio of rate constants. A single class of saturable, high affinity hormone-binding sites was found. Kd values for T3 and nuclear extracts of FRTL-5 cells, rabbit thyroid gland, and rat liver were, respectively, 3.9 X 10(-11) M, 2.8 X 10(-11) M, and 4.3 X 10(-11) M from Scatchard analysis; when calculated from kinetics of hormone association, the value was 3.6 X 10(-11) M for both FRTL-5 cell and rat hepatic nuclear extract. No analysis of the time course of binding of T3 to rabbit thyroid nuclear extract was made. Kd values for T4 and FRTL-5 cell extract were 6.2 X 10(-10) M from Scatchard analysis and 5.0 X 10(-10)M from kinetic data. Half-times (t1/2) of dissociation of T3 from FRTL-5 cell and rat liver nuclear extract, calculated from association curves, were 7 and 5 h, respectively, while corresponding values determined directly and experimentally were 10.5 and 13 h. For T4 and FRTL-5 cell extract, the t1/2 of dissociation calculated from kinetics of association was 5 h; no direct experimental determination of the value was made. Numbers of T3-binding sites of FRTL-5 cell, rabbit thyroid gland, and rat liver nuclear extracts were, respectively, 71 X 10(-15), 62 X 10(-15), and 208 X 10(-15) mol/mg protein. For T4 and FRTL-5 cell extract, the value was 70 X 10( 15) mol/mg protein. The data indicate that the reaction of T3 and T4 with the various nuclear extracts can be described as reversible and bimolecular. The presence in thyroid cells of thyroid hormone nuclear binding sites suggests that they may be receptors that mediate cellular actions of these hormones within the gland itself. PMID- 3013593 TI - Synthesis of the cholesterol side-chain cleavage enzymes in cultured rat ovarian granulosa cells: induction by follicle-stimulating hormone and dibutyryl adenosine 3',5'-monophosphate. AB - The effects of FSH and (Bu)2cAMP on synthesis of the components of the cholesterol side-chain cleavage (SCC) enzyme complex, namely SCC cytochrome P-450 (P-450scc), the iron-sulfur protein adrenodoxin (ISP), and NADPH:ISP reductase (Red), were investigated in granulosa cells obtained from ovaries of immature estrogen-primed rats cultured for up to 72 h in defined medium in the presence or absence of FSH and (Bu)2cAMP. The cells were lysed, and proteins were subjected to polyacrylamide gel electrophoresis, followed by immunoblotting using antibodies specific to bovine adrenocortical P-450scc, ISP, and Red. A time dependent increase was observed in the specific contents of these three components of SCC, but not of the reference mitochondrial protein, F1-ATPase, upon treatment with FSH or (Bu)2cAMP. The increase in the content of these three enzymes was accompanied by a rise in progesterone and 20 alpha hydroxyprogesterone production. The synthesis of P-450scc, ISP, and Red increased 3- to 4-fold with time upon FSH or (Bu)2cAMP treatment respectively, as evidenced by pulse labeling of the cell proteins with [35S]methionine, followed by immunoprecipitation. Immunoprecipitation of P-450scc and ISP from an in vitro translation system programmed by RNA isolated from cultured cells revealed that treatment with FSH or (Bu)2cAMP resulted in an increase in the levels of translatable mRNA specific for these proteins, and that the initial products of translation were precursor forms of cytochrome P-450scc and ISP, similar to those observed in bovine adrenal and granulosa cells. It is concluded that in cultured rat ovarian granulosa cells, FSH induces the synthesis of cytochrome P-450scc, ISP, and Red by increasing the content of translatable mRNA coding for the precursor forms of these enzymes and that this action is mediated by cAMP. Furthermore, the effects of FSH and (Bu)2cAMP provide an explanation for the action of these compounds to stimulate progestin synthesis in cultured ovarian cells. PMID- 3013594 TI - Specific receptor and cardiovascular effects of calcitonin gene-related peptide. AB - Specific binding sites for calcitonin gene-related peptide (CGRP) were demonstrated in the rat heart and spleen. Autoradiography revealed rat [125I]iodo CGRP binding associated with the intima and media of the aorta, the coronary arteries and the heart valves, and the red pulp of the spleen. Half-maximal inhibition of rat [125I]iodo-CGRP binding to membranes of the rat atria and the spleen was obtained with, respectively, 5 and 0.35 nM unlabeled rat CGRP; these values correspond to EC50 values of 3 and 0.14 nM for activation of adenylate cyclase by CGRP. In the isolated, spontaneously beating right atrium, the EC50 values of stimulation of the force and rate of contraction by rat CGRP were 120 and 70 nM, respectively. Rat CGRP caused relaxation of splenic strips, precontracted with noradrenaline; the EC50 was 50 nM. The beta-adrenergic blocking agent metoprolol, while obliterating the increase in the force and rate of contraction evoked by noradrenaline in the right atrium, did not significantly change the action of CGRP. Similarly, preserved action of CGRP in the presence of indomethacin as well as mepyramine and cimetidine argues against a role of prostaglandins or histamine in the functional responses of CGRP. Much like CGRP, capsaicin, which releases mediators from sensory neurons, caused stimulation of the force and rate of contraction of the isolated right rat atrium. After tachyphylaxis to CGRP, the response to noradrenaline was intact, while the positive chronotropic and inotropic effects of capsaicin were suppressed. The results indicate that the cardiac effects of capsaicin may be due to the release of endogenous CGRP through a local mode of action. PMID- 3013595 TI - 1,25-Dihydroxyvitamin D3 binding in estrogen-responsive rat breast tumor. AB - Receptors for 1,25 dihydroxyvitamin D3 [1,25-(OH)2D3] have been reported in breast tissue; however, the presence of multiple binding sites and limited availability of human tumor tissue have precluded complete biochemical characterization of the receptor in breast cancer. In the present study, binding proteins for 1,25-(OH)2D3 in breast tumor tissue were analyzed using a rat model of breast cancer. Breast tumors were induced in adult female rats with the carcinogen nitrosomethylurea. Such tumors previously have been shown to possess high levels of estrogen receptors and are estrogen dependent. Binding proteins for 1,25-(OH)2D3 in 0.3 M KCl extracts of tumor tissue were analyzed on sucrose density gradients. Two binding proteins were detected: one sedimenting at 5-6 S representing binding of 1,25-(OH)2D3 to the 25 hydroxyvitamin D3 (25OHD3) binding protein and a second moiety sedimenting, like the rat intestinal receptor, at 3.3 S. Binding of the dihydroxy metabolite to the faster sedimenting protein could be eliminated by inclusion of radioinert 25OHD3 in the incubation medium. Receptor content of rat breast tumor was investigated using an hydroxylapatite assay by incubating tumor extracts with a saturating concentration of 1,25-(OH)2-[3H]D3, plus unlabeled 25OHD3 to eliminate binding of the hormone to the 5-6 S species. Scatchard analysis of 1,25-(OH)2D3 binding to the tumor extracts yielded an apparent dissociation constant (Kd) of 0.33 nM. In summary, breast tumors induced in rats by nitrosomethylurea were shown to contain high affinity 1,25-(OH)2D3 receptors with properties very similar to those reported for the receptor in other mammalian target organs. The presence of receptors for 1,25-(OH)2D3 in these rat breast tumors implies that the tissue is potentially responsive to the hormone. PMID- 3013596 TI - Estrogens directly down-regulate receptors for angiotensin II in anterior pituitary cell culture without decreasing target cell responsiveness. AB - Chronic estradiol treatment in vivo has been shown to reduce the density of receptors for angiotensin II (ANG II) in the anterior pituitary lobe (AP). We studied whether estradiol is directly involved in the down-regulation of ANG II receptors, using AP cells in culture. Binding affinity and density of ANG II receptors were measured in disrupted AP cells with the radiolabeled antagonist [125I]Sar1, Ile8-ANG II ([125I]SARILE). Estradiol treatment (10 nM) for either 48 or 96 h caused a marked reduction (approximately 70%) in the density of receptors for ANG II in cultured AP cells, with no change in the dissociation constant of [125I]SARILE (Kd, 0.5 +/- (SE) 0.1 nM). In the AP, specific binding sites for ANG II are present in lactotrophs and ANG II has been shown to release prolactin (PRL). In AP cells treated with estradiol for 48 h, dose-response curves revealed that ANG II still increased PRL release (P less than 0.01). The average net PRL release (ANG II-stimulated minus basal) was greater in estradiol-treated cells than in controls, whereas the half-maximal stimulation dose (ED50) of ANG II was the same (0.07 +/- 0.04 nM). These results suggest that estrogens are directly involved in the modulation of ANG II receptors in the AP, causing marked receptor down-regulation without decreasing target cell responsiveness. PMID- 3013597 TI - Isoproterenol reduces insulin stimulation of hexose uptake by rat adipocytes via a postinsulin binding alteration. AB - The present studies have investigated the acute changes previously reported in insulin binding and insulin action that occur in fat cells after a 30-min treatment with isoproterenol. We find that the marked reduction in high affinity insulin binding can be explained by a drop in the pH of the incubation medium from 7.4 to 6.9, a change associated with the accelerated production of FFA. The alteration in insulin binding may explain the rightward shift in the insulin dose response for the stimulation of glucose transport. When the incubation medium is modified to prevent the pH change, isoproterenol stimulation fails to reduce insulin binding. In addition, the tyrosine kinase activity of the insulin receptor isolated from isoproterenol-treated cells is not altered, at least as measured by the phosphorylation of tyrosine residues on the artificial substrate, Glu80Tyr20. There are, however, changes produced in the insulin stimulation of 2 deoxy-D-glucose uptake. The response of the cells to a maximum effective concentration of insulin is reduced 35% by a 30-min treatment with 0.1 microM isoproterenol. This change occurs in parallel with a moderate rightward shift in the insulin dose-response curve. Thus, isoproterenol treatment rapidly alters the ability of the adipocyte hexose transport system to respond to insulin, but the responsible alteration(s) is located beyond the insulin-binding site and possibly beyond the insulin receptor itself. PMID- 3013598 TI - Isolation, characterization, and corticotropic activity of rabbit adrenocorticotropin. AB - Rabbit (r) ACTH was extracted from 600 pituitaries, and 2 forms of immunoreactive ACTH were identified with the least polar form accounting for approximately 90% of the total. Peptide mapping and sequence analysis indicated that three tryptic peptides had retention times identical to those obtained from human (h) ACTH. The least polar tryptic fragment from rACTH had a shorter retention time than the corresponding one from hACTH. Sequence analysis indicated that rACTH differed from hACTH at three different loci, namely, an Asn in place of Asp in position 29; a Val in place of Leu in position 37, and a Val in place of Phe in position 39. Biological activity of the ACTH was compared with synthetic hACTH in 2 bioassays with adrenals from 10-day-old pups, the first using dispersed rabbit adrenal cells and the second using monolayer adrenal cells in culture. The biological potencies of the two ACTH preparations were identical with respect to corticosterone (B) release in the short term bioassay, with an ED50 value of 1.67 X 10(-10) M. The ED50 value for cortisol (F) release for rACTH and hACTH were 1.1 X 10(-10) M and 1.67 X 10(-10) M, respectively, which were not statistically different. The biological potency of rACTH in the monolayer adrenal cell system for both F and B was significantly greater than the hACTH, and the ED50 values were 4.4 X 10(-10) M and 8.9 X 10(-10) M, respectively. There was a progressive decrease in the B/F ratios with increasing concentrations of ACTH in both the bioassay systems suggesting that ACTH stimulated the 17 alpha-hydroxylase activity even when the exposure of cells to ACTH was as short as 2 h. PMID- 3013599 TI - Vitamin D3 improves impaired glucose tolerance and insulin secretion in the vitamin D-deficient rat in vivo. AB - It has previously been shown in this laboratory that vitamin D3 is essential for normal insulin secretion from the perfused rat pancreas. In this present study, the influence of vitamin D status on insulin secretion in vivo was investigated. Intravenous glucose tolerance tests were performed on conscious vitamin D deficient rats (-D), vitamin D-replete rats fed ad libitum (+D AL), and vitamin D replete rats pair fed to the D-deficient animals (+D PF). Vitamin D deficiency, easily recognizable by low daily dietary intake and depressed plasma calcium levels, was found to impair plasma glucose clearance as characterized by an elevated KG value (representing a function of the area beneath the tolerance curve). KG values for the +D AL, +D PF, and -D groups were 504 +/- 15, 480 +/- 46, and 641 +/- 28, respectively. The increase in KG corresponded to a significant reduction in glucose-mediated insulin secretion as compared to the +D animals. This difference appeared not to be related to the increase caloric intake associated with vitamin D repletion, since +D rats which had been pair fed to the -D animals also exhibited restored plasma insulin levels in response to glucose. Plasma phosphorus concentrations were comparable in all three groups, and thus this parameter is also unlikely to be a contributory factor in the observed phenomenon. Additional experiments were conducted to evaluate the involvement of hypocalcemia in the observed impaired glucose tolerance. Normalization of plasma calcium levels (from 4.8 mg/100 ml to 9.6/100 ml) of the D rats, by dietary calcium and phosphorus manipulation, failed to improve glucose clearance (KG for -D normocalcemic rats = 639 +/- 61) or insulin secretion. These results support the concept that vitamin D plays a physiological role in insulin secretion, acting, at least in part, independently of nutritional factors and the prevailing plasma calcium and phosphorus concentrations. PMID- 3013601 TI - Characterization of regulation of thyrotropin (TSH) receptor function in thyroid plasma membrane: interaction between TSH receptor function and activation of adenosine 3',5'-monophosphate-dependent protein kinase. AB - The changes in the characteristics of thyrotropin (TSH) binding to thyroid plasma membranes during the activation of cyclic AMP-dependent protein kinase in the membranes were studied. Preincubation of thyroid plasma membranes with TSH or cyclic AMP reduced the maximal binding capacity but increased the association rate for TSH binding. In double reciprocal analysis, a marked reduction of the total number of binding sites and association constant was observed in the membranes treated with cyclic AMP. These reductions were also observed in the membranes preincubated with buffer alone. The degree of these reductions, however, was greater in the membranes pretreated with cyclic AMP. During incubation of the membranes with buffer alone, cyclic AMP formation (activation of adenylate cyclase) was observed though the degree of the formation was lower than that induced by TSH. The results suggested that not only TSH receptor release from thyroid plasma membrane but also the modification of TSH binding activity in the membrane is produced by cyclic AMP-dependent protein kinase. PMID- 3013600 TI - A case of partial hypopituitarism with empty sella following normal course of pregnancy and delivery. AB - A 28 year-old woman was admitted to Jichi Medical School Hospital because of amenorrhea, cold intolerance, easy fatigability and body weight loss. She was pregnant at the age of 26 years. She delivered a 3230 g healthy girl at full term without any complications. However, she did not have any lactation or recurrence of menstruation after the delivery. Serum cortisol was 0.7 micrograms/dl, and plasma adrenocorticotropic hormone (ACTH) was less than 10 pg/ml. Both hormones failed to increase in response to insulin-induced hypoglycemia or exogenous arginine vasopressin. However, serum cortisol and urinary excretion of 17 hydroxycorticosteroids (17-OHCS) were significantly increased by the repeated administration of ACTH. Serum prolactin was 2.2 ng/ml and the level did not rise after the administration of thyrotropin releasing hormone (TRH). Responses of release of adenohypophysial hormones including gonadotropins, growth hormone and thyroid stimulating hormone (TSH) were normal. Serological studies showed an antibody to the pituitary gland which was demonstrated by an indirect immunofluorescence technique. Plain skull X-ray film and brain computerized tomography revealed an empty sella of the normal size. These results indicate the presence of partial deficiency of ACTH and prolactin, and that autoimmune disorders may be involved in the pathogenesis of her hypopituitarism. PMID- 3013602 TI - Corticotropin-releasing factor (CRF) and vasopressin concentration in major brain regions after adrenalectomy and their involvement in adrenalectomy-induced ACTH elevation. AB - CRF and vasopressin concentrations in major brain regions after bilateral adrenalectomy and their involvement in adrenalectomy-induced ACTH secretion were investigated. At 5, 14 and 28 days after bilateral adrenalectomy, the plasma ACTH level was greatly elevated, whereas hypothalamic CRF content was reduced at 5 days and was not changed at 14 and 28 days after adrenalectomy. The CRF concentration in the medulla oblongata was reduced at 2-4 weeks after adrenalectomy. On the other hand, the arginine vasopressin (AVP) concentration was significantly elevated 2-4 weeks after adrenalectomy. An intrajugular administration of anti-ovine or anti-rat CRF serum significantly suppressed the elevated plasma ACTH level in adrenalectomized, freely moving rats, whereas anti AVP serum or antipressor AVP antagonist, dpTyr(Me)AVP did not suppress the ACTH level. These results indicate that CRF played an important role in the adrenalectomy-induced ACTH elevation but that vasopressin was not involved. PMID- 3013603 TI - Granular cell tumor in upper GI-tract endoscopy. Five cases of esophageal location. AB - Five cases of esophageal granular cell tumor are reported with characteristics similar to those of benign tumors described previously. In contrast to previous reports these tumors are suitable for removal by endoscopic means, provided they are small and are strictly submucosal in location. This view is further supported by the still open discussion as to histogenesis, and the contradictory opinions on its biological significance. Owing to the increasingly widespread use of fiberoptic instruments the diagnosis "granular cell tumor" will become more common. The diagnosis must always be confirmed histologically. Since the tumor may invade adjacent tissue, or even show a tendency to malignant degeneration in late stages, endoscopic removal seems necessary. PMID- 3013604 TI - Arginine vasopressin in the testis: an intragonadal peptide control system. PMID- 3013605 TI - Structural features of prolactins and growth hormones that can be related to their biological properties. PMID- 3013606 TI - Steroid hormone-producing tumors in man. PMID- 3013607 TI - Aluminum lactate treatment alters the lung biological activity of quartz. AB - Previous studies of surface modification of quartz particles have suggested that the biological activity of silica is at least in part related to its surface properties. In the present study, we exposed the tracheal lobe of 8 sheep to either 100 ml saline (saline group), 100 mg of quartz (Minusil-5) in 100 ml saline (SI group) or 100 mg of Al lactate treated quartz in 100 ml saline (SI-Al group). The 24 sheep were studied by bronchoalveolar lavage at days 0, 12, 24, 40, 60 and by autopsy at day 60. In the saline group, BAL analyses were as previously reported [1]. In the SI group, we found significant and sustained increases in total BAL cells (x 2, P less than 0.05), macrophages and lymphocytes (x 2, P less than 0.05), neutrophils (x 5-10, P less than 0.01), IgG (x 1.2-1.8, P less than 0.05), fibronectin (x 2-3, P less than 0.05), lactate dehydrogenase (x 3, P less than 0.01) and alkaline phosphatase (x 2, P less than 0.05). Histologically, a macrophagic and lymphocytic alveolitis was observed at day 60. In the SI-Al group, these changes were significantly attenuated and in the above parameters, SI-Al group did not differ from saline group after day 24. These data of BAL and histology of the sheep tracheal lobe model document clearly that aluminum lactate treatment alters the biological activity of quartz. PMID- 3013608 TI - Suppression of penicillin-induced focal epileptiform activity by locus ceruleus stimulation: mediation by an alpha 1-adrenoceptor. AB - Application of penicillin to the cerebral cortex of anesthetized rats by pressure ejection from a micropipette resulted in the appearance of focal epileptiform activity with low rates of penicillin release and focal penicillin spikes with higher rates. Electrical stimulation of the locus ceruleus (LC), a major norepinephrine-containing nucleus in the brainstem, or of its axons projecting to the forebrain, the dorsal noradrenergic bundle, suppressed penicillin-induced focal epileptiform activity but was less effective in suppressing focal penicillin spikes. Depletion of monoamines with reserpine blocked the suppressant effect of LC stimulation. Neither the selective depletion of 5-hydroxytryptamine with p-chlorophenylalanine nor administration of methysergide reduced the effectiveness of LC stimulation, suggesting that 5-hydroxytryptamine probably does not mediate the suppression. Pimozide partially antagonized the suppression of focal epileptiform activity induced by LC stimulation, which is consistent with antagonism of alpha-adrenoceptors but not dopamine receptors. beta-Receptor antagonists did not block the suppression of focal epileptiform activity by LC stimulation, suggesting that beta-receptors are not important in the observed suppression. Prazosin, a selective alpha 1-antagonist, at low doses blocked the suppression of focal epileptiform activity by LC stimulation whereas yohimbine, an alpha 2-antagonist enhanced the stimulation-induced suppression. Taken together, the data are consistent with LC and dorsal bundle stimulation releasing norepinephrine, which in turn suppresses focal epileptiform activity by an action mediated by an alpha 1-adrenoceptor. PMID- 3013609 TI - Hormonal regulation of four urea cycle enzymes in postnatal rat liver in organ culture. AB - The administration of hydrocortisone to 3- to 15-day-old rats increased the levels of hepatic argininosuccinate synthetase (ASS) and arginase. In 13-day-old rat liver explants maintained in organ culture, ornithine carbamoyltransferase (OTC), carbamoylphosphate synthetase (CPS) and arginase were stimulated by betamethasone. Actinomycin D prevented the responses of the latter two enzymes. Dibutyryl cyclic AMP raised OTC, CPS, ASS and arginase in vitro. The responses of the latter three enzymes were blocked by cycloheximide and puromycin and partially inhibited by actinomycin D. The simultaneous presence of betamethasone and dibutyryl cyclic AMP in the culture medium raised CPS and OTC in an additive manner. The sequential treatment of the cultures with betamethasone followed by dibutyryl cyclic AMP increased CPS and arginase synergistically and amplified the response of ASS to dibutyryl cyclic AMP. PMID- 3013610 TI - Yeast pre-messenger RNA splicing efficiency depends on critical spacing requirements between the branch point and 3' splice site. AB - In the yeast Saccharomyces cerevisiae the 5' and 3' splice junctions and the internal branch acceptor site (TACTAAC box) are highly conserved intron elements. Analyses of mutants have demonstrated the importance of each of these elements in the splicing process. In the present report we show by three different analytical approaches (splicing-dependent beta-galactosidase expression, in vitro splicing assays and in vivo RNA analyses) that at least two of these elements (the TACTAAC and 3' splice signals) also have to fulfill certain spacing requirements to allow efficient splicing to occur. In particular, the spacing of the 3' splice site from the 2'-5' branch site is a critical factor in determining the efficiency for completion of the final reactions of splicing, intron release and exon-exon joining. Whereas insertions within this region have little or no effect on the first reactions in splicing (the 5' cleavage and 2'-5' branch formation), they dramatically affect the efficiency of the final reactions. In contrast, a 15-base deletion between these two sites has no detectable effect on splicing efficiency. We also show that the 5' cleavage and branch formation can take place, albeit inefficiently, in transcripts in which all of the yeast sequences downstream of the branch site have been replaced by Escherichia coli sequences. We conclude from these studies that, in yeast, the 5' and 3' splice sites are recognized independently from one another, but always in conjunction with the TACTAAC signal. PMID- 3013611 TI - Conserved sequence elements upstream of the gene encoding yeast ribosomal protein L25 are involved in transcription activation. AB - Previous studies have revealed the occurrence of two closely linked conserved sequence elements, designated as HOMOL 1 and RPG box, in front of most yeast ribosomal protein genes examined. To investigate whether these conserved nucleotide elements play a role in the regulation of ribosomal protein gene expression, we performed deletion analysis of the DNA region upstream of the gene encoding ribosomal protein L25. To that end we constructed a hybrid gene consisting of the pertinent 5'-flanking sequence and the Escherichia coli galK marker gene. The effects on the transcription of this fusion gene of Bal31 generated deletions were measured by Northern analysis of RNA isolated from the respective transformed yeast cells. The results demonstrate that removal of one box has a detrimental effect on the level of transcription, whereas after the deletion of both boxes hardly any transcription can be observed. Subsequently we inserted synthetic oligonucleotides in the upstream region of an L25 gene from which the original boxes had been removed. Expression of the inactivated hybrid gene turned out to be restored even by insertion of one RPG element. Moreover, the RPG box functions in both orientations, though not with equal efficiency. PMID- 3013612 TI - Two tandemly linked identical genes code for the glycosomal glyceraldehyde phosphate dehydrogenase in Trypanosoma brucei. AB - Trypanosoma brucei contains two isoenzymes for glyceraldehyde-phosphate dehydrogenase (GAPDH); one enzyme resides in a microbody-like organelle, the glycosome, the other one is found in the cytosol. We show here that the glycosomal enzyme is encoded by two tandemly linked genes of identical sequence. These genes code for a protein of 358 amino acids, with a mol. wt of 38.9 kd. This is considerably larger than all other GAPDH proteins studied so far, including the enzyme that is located in the cytosol of the trypanosome. The glycosomal enzyme shows 52-57% homology with known sequences of GAPDH proteins from 10 other organisms, both prokaryotes and eukaryotes. The residues that are involved in NAD+ binding, catalysis and subunit contacts are well conserved between all these GAPDH molecules, including the trypanosomal one. However, the glycosomal protein of T. brucei has some distinct features. Firstly, it contains a number of insertions, 1-8 amino acids long, which are responsible for the high mol. wt of the protein. Secondly, an unusually high number of positively charged amino acids confer a high isoelectric point (pI 9.3) to the protein. Part of the additional basic residues are present in the insertions. We discuss the genomic organization of the genes for the glycosomal GAPDH and the possibility that the particular features of the protein are involved in its transfer from the cytoplasm, where it is synthesized, into the glycosome. PMID- 3013613 TI - Expression of whole and hybrid genes in Trypanosoma equiperdum antigenic variation. AB - A rapid technique involving the S1 nuclease resistance of RNA:DNA duplexes has been used to screen four Trypanosoma equiperdum variant surface glycoprotein (VSG) genes for evidence of hybrid gene structure in their transcribed regions. The results suggest that VSGs appearing early in a chronic infection each have a complete co-linear basic copy (BC) of their expressed gene while VSGs appearing later in infection are particularly associated with BC genes which are recombined before being expressed. The intensities of the S1-protected bands from hybrid VSGs indicate that the basic and expression linked copies are present in equivalent gene dosages. In addition, studies are presented on the expression of two additional VSG genes in T. equiperdum, VSG 4 and VSG 78, which (i) show that the basic copies are not located on telomeres even though one (VSG 4) is expressed early in infection and (ii) emphasize the role of a predominant expression site in T. equiperdum while nevertheless confirming the presence of multiple expression sites. PMID- 3013614 TI - The use of incomplete genes for the construction of a Trypanosoma equiperdum variant surface glycoprotein gene. AB - The expression of Trypanosoma equiperdum variant surface protein (VSG) 78 is accomplished by the duplicative transposition of silent basic copy (BC) genes into a telomer-linked expression site to form an expression-linked copy (ELC). In two independent isolates expressing VSG 78, the ELC is a composite gene. The analysis of VSG 78 cDNA clones from these two Bo Tat 78 isolates and the respective BC genes revealed that both ELCs were constructed from the same three BC genes, a 3' BC which donated the last 255 bp of each ELC and two closely related 5' BCs. Although sequences of both 5' BC genes were found in each ELC, the junction with the 3' BC was provided by the same 5' BC in both cases. This 5' BC is an incomplete gene with insufficient open reading frame to code for a complete VSG and thus can only be used when joined to a competent 3' end. Furthermore, both 5' BC genes lack a conserved 14 nucleotide sequence found on all VSG mRNAs. These results support a model in which composite gene formation plays a role in the determination of the order of VSG expression. They also illustrate similarities between immunoglobulin gene and VSG gene construction. PMID- 3013615 TI - Conserved TAAAT motif in vaccinia virus late promoters: overlapping TATA box and site of transcription initiation. AB - We have characterized the vaccinia virus 11-kd late promoter through 5' and 3' deletions and site-directed mutagenesis. The promoter function appears to be contained within an approximately 30-bp fragment, which after translocation is able to direct RNA synthesis late in infection at a reduced level. We demonstrate that a TAAAT sequence in the proximal part of the promoter is essential for its function. This cis-acting element is highly conserved within vaccinia virus late promoters and overlaps the site of transcription initiation. Deletions or mutations within this conserved element completely inactivate the promoter. The evidence indicates that the TAAAT motif functions as a TATA box. The region immediately upstream of the TAAAT motif determines the promoter strength. PMID- 3013616 TI - Regulation of the operon encoding ribonucleotide reductase in Escherichia coli: evidence for both positive and negative control. AB - The ribonucleotide reductase genes (nrd) are induced by thymine starvation. Deletion analysis of the sequences upstream of the cloned nrd genes was used to identify several regulatory regions. The start of transcription (nrdP) was mapped 110 bp upstream of nrdA, the first structural gene. A site required for positive regulation of nrd was mapped 135 bp upstream of nrdP in a region with two direct repeat sequences as well as potential secondary structure. Two other sites (one upstream of nrdP, the other downstream) were identified as sequences whose deletion markedly increase expression. These latter sites show sequence homology and probably interact since the effects of their individual deletion are not additive when combined. PMID- 3013618 TI - T7 RNA polymerase directed expression of the Escherichia coli rrnB operon. AB - Plasmids having Escherichia coli ribosomal DNA sequences under control of a promoter for T7 RNA polymerase have been constructed. Transcription of the rDNA sequences is dependent on T7 RNA polymerase because the tandem promoters for E. coli RNA polymerase, normally used to direct transcription of these sequences, have been removed. The entire 16S, 23S and 5S coding sequences from the rrnB operon can be efficiently transcribed by T7 RNA polymerase in vitro to yield full length 30S precursor RNA. When such plasmids are placed into an E. coli strain containing a chromosomal copy of the gene for T7 RNA polymerase under control of the lac UV5 promoter, high-level synthesis of rRNAs from the plasmid can be induced by adding IPTG to exponentially growing cells. Subsequent addition of rifampicin to inhibit further initiation of transcription by E. coli RNA polymerase provides a simple method to study the fate of plasmid-coded rRNAs in the complete absence of host-coded rRNA synthesis. Gel electrophoretic analysis demonstrated that the rRNAs synthesized by T7 RNA polymerase in the presence of rifampicin are processed to their mature forms and assembled into ribosomal particles for at least 35 min after rifampicin addition. T7 RNA polymerase is also capable of efficient transcription of the entire rrnB operon in the reverse direction. PMID- 3013619 TI - Heme regulates the expression in Saccharomyces cerevisiae of chimaeric genes containing 5'-flanking soybean leghemoglobin sequences. AB - The TM1 yeast mutant was transformed with a 2 micron-derived plasmid (YEp24) which carries a chimaeric gene containing the Escherichia coli chloramphenicol acetyl transferase (CAT) gene fused to the 5'- and 3'-flanking regions of the soybean leghemoglobin (Lb) c3 gene. Expression of the chimaeric CAT gene is controlled specifically by heme at a post-transcriptional level, most likely by regulating the efficiencies of translation. Expression of another chimaeric gene consisting of the neomycin phosphotransferase (NPTII) gene fused to only the 5' flanking region of the Lbc3 gene is regulated by heme in a similar way. Thus, in yeast, heme modulates the translation of the chimaeric mRNAs through interactions with the 5' Lbc3 non-coding region. PMID- 3013617 TI - Cloning of the regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. AB - The areA gene, which mediates nitrogen metabolite repression in the fungus Aspergillus nidulans, lies sufficiently close to a telomere that no indispensable gene can be distal to it. We were able therefore to exploit the existence of a near terminal pericentric inversion to devise a method for cloning areA plus the region beyond it towards the telomere. In crosses heterozygous for this inversion a class of duplication-deficient progeny lacking areA and the region centromere distal to it is obtained. We, therefore, sought clones from an A. nidulans gene library in lambda Charon 4 able to hybridize to total genomic DNA from a wild type strain but not to that from a duplication-deficiency strain. A clone, containing an 11.6-kb insert, which hybridised weakly to duplication-deficiency DNA, overlapped chromosome breakpoints of three different aberration-associated areA alleles and was able to transform an areA mutant to areA+. Southern blotting and genetic analysis established that the transforming sequence had integrated in the region centromere distal to areA. The cloning method yielded other clones from the region centromere-distal to areA which were used to show that the translocation associated with a mutant areA allele is reciprocal rather than non reciprocal, a fact which could not be established by classical genetics. Finally, analysis of the cloned portion of the dispensable region centromere-distal to areA indicates that this region contains at least 0.5% of the A. nidulans genome. PMID- 3013620 TI - Conjugation of [125I]ubiquitin to cellular proteins in permeabilized mammalian cells: comparison of mitotic and interphase cells. AB - [125I]Ubiquitin introduced into permeabilized hepatoma tissue culture (HTC) cells rapidly forms conjugates with endogenous proteins. A characteristic pattern of low mol. wt conjugates is obtained which includes the ubiquitinated histone, uH2A, and unknown molecular species with MrS of 14, 23, 26 (two bands) and 29 kd. A broad spectrum of higher mol. wt conjugates is also produced. The formation of all conjugates is absolutely dependent on ATP, and upon depletion of ATP they are rapidly broken down. The 14, 23 and 29 kd species are found in all subcellular fractions examined. uH2A is located exclusively in the nuclear fraction. The pair of 26 kd bands is specifically associated with the ribosome fraction. A considerable percentage of the higher mol. wt conjugates sediments with the small particle (100,000 g) fraction in the ultracentrifuge but is solubilized with deoxycholate, indicating that there are many membrane-associated conjugates. The pattern of ubiquitin conjugation in interphase and metaphase cells was compared. The incorporation of ubiquitin into uH2A was markedly reduced in metaphase cells whereas its incorporation into other low mol. wt conjugates and into high mol. wt conjugates was affected slightly, if at all. This shows that the known decrease of uH2A levels in metaphase is due to a specific effect on histone ubiquitination and not to a general decrease in ubiquitination activity or increase of isopeptidase activity. Changes in the levels of uH2A during mitosis measured by immunoblotting were similar to those estimated in permeabilized cells. These experiments indicate that permeabilized cells provide a useful approach to the study of rapidly turning over ubiquitin conjugates in mammalian cells. PMID- 3013621 TI - The kinase activity of SV40 large T antigen is mediated by a cellular kinase. AB - Large T antigen (large T) extracted from SV40-infected or transformed cells exhibits an in vitro protein kinase activity, whose origin and biological significance up to now had been obscure. We have addressed the questions of whether this activity is intrinsic to large T or arises by association with a cellular kinase, and, furthermore, whether this activity might play a biological role in vivo. Instead of analyzing large T from whole-cell lysates, where non specific association of a cellular kinase(s) with large T might easily occur, we analyzed individual cellular subclasses of large T, isolated from their in vivo locations. In contrast to large T isolated from whole-cell lysates which was always kinase positive, none of the cellular subclasses of large T prepared by in situ fractionation of SV40-transformed mKSA cells exhibited detectable in vitro kinase activity. We could demonstrate that our fractionation conditions neither inactivated the large T-associated kinase activity nor dissociated it from large T when they were applied to kinase-positive large T isolated from whole-cell lysates. We conclude that large T does not contain an intrinsic kinase activity. This conclusion was further supported by our finding that it was possible to remove the large T-associated kinase activity from kinase-positive large T preparations and to reconstitute it by incubating the kinase-negative large T with cell lysates from various cell lines. Therefore, the simplest way of interpreting our results is that the in vitro kinase activity measured with large T preparations from whole-cell lysates is the result of an in vitro association of a cellular kinase(s) with large T during certain conditions of cell lysis. PMID- 3013622 TI - A negative transcriptional control element located upstream of the murine c-myc gene. AB - We have investigated the nature of regulatory sequences within the vicinity of the murine c-myc locus by analyzing the expression of myc-chloramphenicol acetyl transferase (CAT) vectors transfected into a human lymphoblastoid cell line (BJAB) and a monkey fibroblast line (COS). CAT enzymatic assays and S1 nuclease protection experiments reveal that a negative element resides 428-1188 bp 5' of the first c-myc promoter, P1. This 760-bp segment of 5'-flanking c-myc DNA dramatically inhibits CAT gene expression in the pSV2CAT vector when placed in either orientation approximately 1.7 kb 3' (and approximately 3.2 kb 5' on the circular plasmid) from the SV40 promoter region. By employing this strategy, we were unable to identify an analogous DNA segment that is closer to or within the first c-myc exon. We propose that this 5' c-myc region be termed a 'dehancer' since this negative element has the opposite properties of a transcriptional enhancer. PMID- 3013623 TI - Translocation of c-myc into the immunoglobulin heavy-chain locus in human acute B cell leukemia. A molecular analysis. AB - We report the molecular analysis of primary cells from four cases of human B-cell malignancies each with an 8;14 chromosomal translocation involving the c-myc proto-oncogene and the immunoglobulin (Ig) gene cluster. In two cases of B-cell acute lymphocytic leukemia (B-ALL) the c-myc is truncated, rearranged into the Ig C alpha 1 locus and over-expressed in two abnormal mRNAs of approximately 2.0 and 2.8 kb. Conversely, in two cases of B-cell lymphoma progressed into leukemia the c-myc locus was translocated intact in its coding and 5'-flanking region into an Ig region different from C alpha 1, and over-expressed in two normal mRNA species. Cloning and sequencing of the breakpoint region on chromosome 14q+ from one of the two B-ALL cases showed that the myc gene is truncated 1077 nucleotides upstream from the translation start site, and rearranged in the opposite transcriptional orientation into an Ig class-switch segment approximately 4.8 kb upstream from the C alpha 1 gene. The c-myc anti-sense strand contains two class switch recombination consensus sequences in the immediate boundaries of the breakpoint on chromosome 8: this allows us to postulate that an erroneous, class switch-like recombination between Ig and myc sequences gave rise to the chromosomal translocation. Furthermore, we report 13 point mutations clustered in a region spanning from the first intron to the second exon of the translocated c myc gene, five of which cause amino acid changes leading to an abnormal myc protein. This is the first evidence of mutations in a translocated c-myc in primary tumor cells. PMID- 3013624 TI - Sequence, topography and protein coding potential of mouse int-2: a putative oncogene activated by mouse mammary tumour virus. AB - A major proportion of carcinomas induced by mouse mammary tumour virus (MMTV) show evidence for proviral activation of a cellular gene, int-2, on chromosome 7. The sequence of 7869 bp of DNA spanning the transcription unit of int-2 was determined and compared with that of a series of int-2-specific cDNA clones derived from mammary tumour RNA. The predicted positions of intron-exon boundaries, established by alignment of cDNA and chromosomal DNA sequences, indicate that the gene comprises at least three exons. An open reading frame capable of encoding a protein of 245 amino acids with an estimated mol. wt of 27 kd, is flanked by substantial non-coding segments at both 5' and 3' ends. Comparison of the chromosomal DNA sequence and the predicted amino acid sequence with available data-bases has revealed no homology to other known genes. These results are discussed in relation to the status of int-2 as a candidate proto oncogene. PMID- 3013625 TI - Stage-specific expression of a homeo box-containing gene in the non-segmented sea urchin embryo. AB - Hybridization of Drosophila homeo box DNA probes to Southern transfers of genomic DNA from the Hawaiian sea urchin Tripneustes gratilla has revealed that the sea urchin genome contains at least five homeo boxes. Examination of the DNA from several individuals shows that the sequences flanking these homeo boxes exhibit little restriction fragment length polymorphism, indicating they are more highly conserved than the majority of sea urchin DNA. Several clones in a T. gratilla genomic DNA library which hybridized with Drosophila homeo box probes were identified, and one found to be transcribed during embryogenesis was selected for further study. Southern transfer hybridizations showed the cloned gene to be single-copy. DNA sequencing of the sea urchin gene defined a homeo box 70-73% homologous to the Drosophila homeo box probes and an encoded homeo domain 78-88% homologous to those encoded by the probes. Hybridization of DNA probes from the sea urchin homeo box-containing gene to Northern transfers of embryonic RNA demonstrated that the gene produces two transcripts of 6.9 kb and 7.7 kb. Transcripts first accumulate at blastula stage and increase to a maximum level at gastrula stage before decreasing considerably in abundance by pluteus stage. PMID- 3013626 TI - Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface. AB - Polyclonal and monoclonal antibodies were raised against a synthetic peptide containing the 15 carboxy-terminal amino acids (497-511) of vesicular stomatitis virus glycoprotein (VSV-G). The polyclonal antibodies (alpha P4) reacted with epitopes distributed along the 15-residue peptide, whereas the monoclonal antibody (P5D4) reacted with one epitope containing the five carboxy-terminal amino acids. Both types of antibodies recognized the cytoplasmic domain of VSV-G synthesized by tissue culture cells infected with the temperature-sensitive 045 VSV mutant (ts045-VSV). They recognized immature forms of VSV-G in the rough endoplasmic reticulum (RER) and Golgi complex, as well as mature VSV-G at the cell surface and in budding virus. The effect of these antibodies on intracellular transport and maturation of VSV-G was studied by microinjection. Both divalent antibodies (alpha P4 and P5D4) blocked transport of VSV-G to the cell surface. Monovalent Fab' fragments of alpha P4 (alpha P4-Fabs) also interfered with the appearance of VSV-G at the cell surface; Fab fragments of P5D4 (P5D4-Fabs), however, had no inhibitory effect. These results suggest that accessibility of a cytoplasmic domain, located within the sequence of amino acids 497-506 of the carboxy-terminal tail, is essential for transport of VSV-G to the cell surface. PMID- 3013627 TI - A single nucleotide difference at the 3' end of an intron causes differential splicing of two histocompatibility genes. AB - The murine histocompatibility class I genes, H-2 Kb and Kk, display considerable homology at their 3' ends. In fact, from exon 5 to the termination codon, only two nucleotides differ between the two genes, one at the 5' end and the other at the 3' end of intron 7. Despite this similarity, the gene products have distinctly different mol. wts as determined by SDS-PAGE. By constructing two hybrid genes, pC2 and pC4, we demonstrated that it is the cytoplasmic parts of the antigens (encoded by exons 6-8) which are responsible for the major difference in mol. wt. We have used site-directed mutagenesis to change the two nucleotides in intron 7 of the H-2 Kk gene to those present in the H-2 Kb gene. S1 nuclease mapping has been used to identify the actual splice site of the authentic Kb and Kk genes, the hybrid genes and the mutagenized genes. We have shown that it is the 3' nucleotide difference, nine nucleotides upstream of the 3' splice site, which causes the different excision of intron 7 of the Kb gene. The 5' nucleotide difference does not alter the splicing. The choice of branch points and 3' splice signals for intron 7 of five H-2 class I genes, is discussed. PMID- 3013629 TI - Seroimmunity to poliovirus in Poitiers, France. PMID- 3013628 TI - Clinical uses of nalidixic acid analogues: the fluoroquinolones. PMID- 3013630 TI - Epstein-Barr virus-specific immunoglobulin A in patients with infectious mononucleosis, an age-dependent factor. PMID- 3013631 TI - Overview of clinical experience with ciprofloxacin. AB - Ciprofloxacin is a new 4-quinolone antibacterial agent with an extended antibacterial spectrum, enhanced potency and the ability to produce therapeutic serum, tissue and urine concentrations after oral administration. Unlike earlier 4-quinolones, it is active against gram-positive cocci and opportunistic organisms such as Pseudomonas aeruginosa. This overview demonstrates that the oral formulation has been shown to be clinically effective in a broad range of urinary and respiratory infections, gonorrhoea, gastro-intestinal infections including typhoid fever, surgical infections, skin and soft tissue sepsis and in a variety of infections caused by Pseudomonas aeruginosa, notably cystic fibrosis. Adverse reactions are infrequent and in almost every case have proved mild and transient. Ciprofloxacin has great potential for the oral therapy of infections which have traditionally required parenteral chemotherapy. PMID- 3013632 TI - A functional link between the dark Mg-ATPase activity and the light-induced enzymatic cascade in rod outer segments. AB - The characterization of a light-induced scattering change in suspensions of rod fragments, which requires previous swelling of the disks by the dark Mg-ATPase described by Uhl et al. [FEBS Lett. 107, 317-322 (1979)] is reported here. Reconstitution experiments demonstrate that this signal is dependent on the presence of G-protein, GTP and cGMP phosphodiesterase. Fast reversal associated with regenerability requires in addition the presence of some protein(s) of the cytoplasm (probably the rhodopsin kinase) and ATP. The amount of excited rhodopsin which saturates the signal is the same as that which saturates the previously described 'dissociation signal' [Kuhn et al. (1981) Proc. Natl Acad. Sci. USA 78, 6873-6877] associated with the formation of the phosphodiesterase activator G alpha GTP (alpha subunit of the G-protein with GTP bound). The kinetics of the signal is slightly slower than that of the dissociation signal and its amplitude is proportional to the extent of swelling of the disks. These results suggest that the interaction between G alpha GTP and the phosphodiesterase modifies some structural feature of the disks and provide evidence for the existence of a functional link between the dark Mg-ATPase and the light-induced enzymatic cascade. PMID- 3013633 TI - Two-dimensional gel analysis of repetitive nuclear DNA sequences in the genome of Physarum polycephalum. Developmental regulation of the ribosomal gene number. AB - The composition of repetitive sequences in restriction patterns of nuclear DNA of Physarum polycephalum was determined by high-resolution gel analysis. Three types of repeated DNA fragments in the size range of (0.2-2) X 10(3) base pairs could be identified as discrete spots on the gels and distinguished by their abundance and above-average base composition of either guanine and cytosine (G + C) or adenine and thymidine (A + T). On comparing the DNA composition from exponentially growing plasmodia with that of starved plasmodia, which have become competent to sporulate and have lost 80% of their nuclei, no change was detected among the (A + T)-rich repeat fractions, whereas several of the (G + C)-rich fractions revealed fewer copies in the DNA prepared from starved cells. As shown by hybridization under saturating conditions, the reduction of several (G + C) rich repeated sequences in the restricted nuclear DNA in sporulation-competent cells can be explained by a 64% elimination of the extrachromosomal nucleolar ribosomal DNA sequences. PMID- 3013634 TI - CT diagnosis of adrenal abnormalities in patients with primary non-adrenal malignancies. AB - Fifty-seven patients with primary non-adrenal malignancy were found to have unsuspected adrenal abnormality on CT. In 33, comparison of histopathologic findings and/or the patients' hospital course or follow-up lead to the diagnosis of adrenal metastases (23), benign non-functioning adenomas (7), metastasis with hyperplasia (1), benign hyperplasia (1), and fatty infiltration (1). The analysis of CT findings indicated that: I) A heterogeneous adrenal mass showing contrast enhancement was always metastatic, II) Nonfunctioning adenomas were always 3 cm or smaller in diameter, III) Bilateral adrenal masses and growth of adrenal mass on follow-up CT or regression on treatment indicated metastases, and IV) metastatic disease could not be excluded purely on the basis of the size of the adrenal mass. PMID- 3013635 TI - Computed tomography of the scapula. AB - Twenty eight patients with bony lesions involving the scapula were investigated by CT. Three different patient positions were tested for optimal visualisation of the scapula. An asymmetric patient positioning is recommended. CT is of value in the management of lesions involving the scapula and in the demonstration of the extent of the tumour within bone and adjacent soft tissue. PMID- 3013636 TI - Liquid crystal thermography of the breast. AB - In a retrospective study 19461 patients were examined. 2002 patients were clarified by biopsy and histological examination; 500 patients underwent aspiration cytology. The accuracy rate of thermography is 67.8% for malignant and 70.6% for benign lesions. False negative findings were given in 13.9% and false suspicious in 6.8%. False suspicious findings were mostly seen in inflammation, cysts and/or proliferation. The criteria of malignancy by "Tricoire" were examined. It can be shown, that their predictive value is very good. This especially if the "disturbance of thermoregulation" is examined carefully. PMID- 3013637 TI - Measurement of knee tendon reflex latencies in lumbar radicular syndromes. AB - The latency of the knee tendon reflex (KTR) in the vastus medialis muscle was measured by eliciting the reflex manually with a metal hammer. Normal values were obtained in a reference group of 35 subjects. The difference in right and left KTR was used as the parameter in the investigation of 20 patients with a proven L3 or L4 monoradicular syndrome; it was abnormal in 65% of the cases. In 10 patients an asymmetrical KTR was the only relevant abnormality in the combined electromyography and reflex study. Measurement of the KTR seems to be useful in the assessment of patients suspected of a lumbar radicular syndrome. PMID- 3013638 TI - Gastric stress ulcer of the rat: relative contribution of the pyloric sphincter, HCO3- bile reflux and mucosal blood flow. AB - Gastric ulceration has been induced after stress, combining 24 h of fasting and 48 h of restraint in 9 groups of 20 rats with or without a pyloroplasty or a pylorojejunostomy combined with atropine and gastric infusion of NaHCO3 or taurocholic acid. After death or sacrifice at 48 h, ulcer index and blood in the jejunum were determined. Gastric mucosal blood flow was measured semi continuously by a laser Doppler velocimeter. There were 45% deaths after 48 h of restraint alone, and 70% in the group combining pylorojejunostomy with taurocholic acid. Mortality was lower (p less than or equal to 0.01) pylorojejunostomy alone and more significantly so (p less than or equal to 0.001) when associated with NaHCO3. There was no death when NaHCO3 and atropine were combined with restraint. The mucosal blood flow increased significantly during the first 12 h of restraint in the taurocholic acid group. Both groups with NaHCO3 had mucosal blood flows similar to the controls. Gastric acid and gastric emptying, mucosal ischemia and bile reflux are joint factors inducing gastric stress ulcer. The 100% survival and the low ulcer index after a treatment by atropine and gastric infusion of NaHCO3 suggest that these well-known drugs should be used more frequently. PMID- 3013639 TI - Current status in the management of proximal bile duct carcinoma. AB - The confluence of the biliary duct constitutes the most common location of the bile duct neoplasms. Resection of the tumor is the only procedure which has met with long-term survival rates (more than 5 years). An enhanced exposure of the tumor in surgical intervention contributes to increasing the number of resectable cases. The transhepatic approach through the principal incision offers the best possibility to explore the tumors of the proximal bile duct confluence, using this approach the resection rate is higher than that of other routes. The surgical management of confluence biliary duct carcinoma can be curative if the diagnosis is made in early stages of the disease and if at that time resection is possible. PMID- 3013640 TI - Differentiation of human mature thymocytes: existence of a T3+4-8- intermediate stage. AB - A T3 complex-bearing subpopulation was characterized within an in vivo cycling T4 8- early thymocyte compartment which contains cells constitutively expressing interleukin 2 and transferrin receptors. We show differentiation in vitro of both mature subsets of thymocytes (T3+4+8- and T3+4-8+) from the above T4-8- compartment, their appearance being preceded by cells in a T3+4-8- intermediate stage. Furthermore, those mature thymocytes generated in vitro contain functionally competent cells which use T3, T4 and T8 structures for their cytolytic activity. The finding of T3+4-8- thymocytes in vivo, together with the observation that T3 antigen expression precedes that of T4 or T8 molecules in vitro, shows that T3 (and presumably Ti) is present early in ontogeny, and suggests that T3+4-8- cells constitute an "intermediate" stage relevant to the connection between early precursors and mature thymocytes during T lymphocyte ontogeny. PMID- 3013641 TI - T lymphocytes activated by interleukin 2 alone acquire permissiveness for replication of herpes simplex virus. AB - Not only mitogen stimulation or mitogen stimulation in combination with interleukin 2 (IL2) was capable of causing susceptibility of human T lymphocytes to herpes simplex virus (HSV) infection, but also selective stimulation with recombinant Il2-induced permissiveness of T lymphocytes to HSV infection. Replication of HSV in such IL2-stimulated T cell cultures was shown to be restricted to a T cell subset not exceeding 5% of the total population. Furthermore, IL2 stimulation was sufficient to obtain virus replication in T cells previously infected by HSV and cultivated for several days. This could not be achieved by stimulation with mitogens such as phytohemagglutinin. The level at which virus replication was restricted in nonpermissive T cells was determined to be before immediate early gene expression as assessed by indirect immunofluorescence with monoclonal antibodies against viral proteins expressed at different stages of the viral replicative cycle. PMID- 3013642 TI - Antigen presentation by liposomes: inhibition with antibodies. AB - The involvement of both antigen and class II major histocompatibility complex (MHC) molecules in T cell activation by liposome-bound antigens was investigated. We used a pigeon cytochrome c (PCC)-specific Ek-restricted T cell hybridoma that can be activated to produce interleukin 2 by liposomes carrying either PCC and Ek molecules, or a high concentration of PCC alone. We demonstrated that the MHC restricted response of this hybridoma to liposomes is specifically blocked by both anti-MHC and anti-PCC monoclonal antibodies, whereas unrestricted activation is only inhibited by PCC-specific antibody. PMID- 3013643 TI - Inhibition and enhancement of in vitro helper activity of apocytochrome c specific murine T cell populations and clones by anti-apocytochrome c-specific monoclonal antibodies. AB - A murine monoclonal antibody (mAb) specific for apocytochrome c was found to be able to either inhibit or enhance the helper activity of mouse apocytochrome c specific T cell clones and populations in a hapten (trinitrophenyl)-carrier (apocytochrome c) system of T-B cell cooperation. This effect of the mAb was carrier specific, could not be ascribed simply to a shift in the kinetics of the antibody response and was observed using apocytochrome c T helper cells of different mouse haplotypes. In addition, the anti-apocytochrome c mAb was able to inhibit specific T helper cell activity even when the T cells were triggered with antigen-presenting cells pulsed with antigen. Taken together, these results suggested that the mAb was inhibiting helper activity due to its ability to modify the interaction between T cells and antigen-presenting cells. PMID- 3013644 TI - Triggering of the lethal hit in human cytotoxic T lymphocytes: a functional role for a 103-kDa T cell-specific activation antigen. AB - A monoclonal antibody (CB.1) is described that defines a new triggering signal for human cytotoxic T lymphocytes (CTL). The antibody precipitates a 103-kDa surface antigen from activated normal human T cells. The antigen is undetectable or present in only low amounts on resting T lymphocytes but its expression increases strongly after activation and proliferation on T4+ and T8+ T lymphocytes. Binding of antibody CB.1 to CTL results in triggering of the lethal hit. This induction of cytotoxicity is dependent on cross-linking of CTL and an Fc receptor-bearing target cell with CB.1 and requires Ca2+ like antigen-specific triggering. CB.1-induced triggering can be specifically inhibited by binding of antibodies to the T8 or T4 molecules on T8+ or T4+ CTL. PMID- 3013645 TI - B cell growth and differentiation factors interact with receptors distinct from the interleukin 2 receptor. AB - Human B lymphocytes preactivated with Staphylococcus aureus Cowan strain I can proliferate and differentiate to Ig-secreting cells when cultured in the presence of recombinant interleukin 2 (IL2). We have compared 2 different B cell growth factors (BCGF) and a B cell differentiation factor (BCDF) to IL2 in the regulation of human B lymphocyte growth and differentiation. Utilizing a competitive binding assay, neither a high molecular weight BCGF (HMW-BCGF) nor a low molecular weight BCGF (LMW-BCGF) competitively inhibited the binding of radiolabeled IL2. Blocking studies with the anti-Tac monoclonal antibody demonstrated that B cell proliferation to IL2 was inhibited while a crude supernatant containing BCGF and IL2 was only partially inhibited. B cell Ig secretion induced by IL2 was also inhibited by anti-Tac while a crude supernatant and partially purified BCDF were not. Furthermore, IL2 plus BCGF was shown to enhance B cell proliferation better than either factor alone and IL2 plus a BCDF enhanced B cell Ig secretion better than either factor alone. By separating activated B cells into Tac-positive and Tac-negative fractions, much of the previously noted enhancement with the 2 factors was found to be secondary to the differential sensitivity of the 2 populations to BCGF and IL2 or BCDF and IL2. Thus, LMW-BCGF, HMW-BCGF and a partially purified BCDF appear to interact with receptors distinct from the IL2 receptor in mediating their effects on B cell growth and differentiation. PMID- 3013646 TI - Discrete stages of human thymocyte activation and maturation in vitro: correlation between phenotype and function. AB - Using light scatter, flow cytometric and functional analyses, three major stages in the in vitro activation of human thymocytes were defined. The earliest stage (day 0-2) was characterized by the induction of the interleukin 2 (IL2) and transferrin receptors, the T cell lineage specific Ta1 antigen, and increased reactivity with anti-T8 antibody. At this time, major changes in nuclear morphology but not cell size were observed. Early-stage thymocytes were immature with regard to T3 and cytotoxic capacities. The second stage (day 3-4) of in vitro culture was distinguished by loss of T6, maximal activation (both in cell size and nuclear morphology) and maximal expression of both transferrin and IL2 receptors. At this stage, nearly all thymocytes expressed T3, 30-70% of thymocytes were T4+T8+, and functionally, only small increases in cytotoxic capacities were observed. The third stage of maturation (day 5-7) represented thymocytes with reduced levels of activation as measured by forward and right angle light scatter analysis and declining IL2 and transferrin receptor expression. However, these thymocytes exhibited high levels of T3 antigen density, loss of T4, T8 coexpression and pronounced cytotoxic and detectable inducer function capabilities. PMID- 3013647 TI - B cell activation by the nontransforming P3HR-1 substrain of the Epstein-Barr virus (EBV). AB - The P3HR-1 substrain of Epstein-Barr virus does not transform B cells. This defect is known to be determined by the loss of the coding sequence for the nuclear antigen EBNA-2. The virus can attach to and enter resting B cells. The initial events after EBV infection are reminiscent of those induced by polyclonal B cell activators. Similar to the effect of these, P3HR-1 virus lowers membrane IgD expression on B cells and abrogates the transient elevation of activation markers BB-1 and LB-1 induced by the culture conditions. An important event of B cell activation is the acquisition of competence to respond to specific growth factors produced by T cells. This was induced by the P3HR-1 virus. The infected B cells had elevated [3H]thymidine incorporation when exposed to the supernatant of PHA-treated T cells. The EBV receptor is identical with the complement receptor CR2. Ligand binding to CR2 has been shown both with mouse and human B cells to deliver certain activation signals. Therefore, it is possible that the early step of activation by EBV is initiated through the binding to the receptor and is thus a cell surface event. PMID- 3013648 TI - myo-Inositol reverses Li+-induced inhibition of phosphoinositide turnover and ornithine decarboxylase induction during early lymphocyte activation. AB - One of the earliest detectable responses by T lymphocytes in vitro to various mitogenic ligands is the rapid induction of ornithine decarboxylase (ODC) activity (Scott et al., Eur. J. Immunol. 1985. 15: 783). This early activation is measurable within minutes after the addition of the mitogen. The T cell mitogen concanavalin A also induces rapid breakdown of phosphoinositides in T lymphocytes. The inositol phosphates formed are recycled and used in synthesis of new phosphoinositides. Li+ interrupts this cycle by inhibiting the enzyme inositol-1-phosphatase, which produces the inositol needed for phosphoinositide synthesis. Here we report that when human blood lymphocytes are kept in an inositol-deficient medium for 30 min in the presence of 1 mM Li+, the cells become unable to respond to mitogens by inositide breakdown and rapid induction of ODC activity. Addition of 1 mM myo-inositol restores the inducibility of these responses within a few minutes. These findings represent a novel aspect of the activation of resting lymphocytes and implies a new role for the inositol turnover during signal transduction. PMID- 3013649 TI - Localization of the effects of electroconvulsive shock on beta-adrenoceptors in the rat brain. AB - Chronic (10 days) application of electroconvulsive shock (ECS) to male and female rats results in a significant but highly localized decrease in beta-adrenoceptor number. Saturation studies on homogenized tissue revealed a decrease in Bmax in the frontal cortex and hippocampus but not in caudate or cerebellum of both sexes. Quantitative autoradiographic analysis showed the changes in the hippocampus to be in the CA1 and dentate gyrus but not CA3. No effect was seen in caudate, globus pallidus, substantia nigra, cerebellar molecular and granular layers and several thalamic and hypothalamic nuclei. PMID- 3013651 TI - The effect of imipramine treatment on brain serotonin receptors and beta adrenoceptors and on pineal beta-adrenergic function in adult and aged rats. AB - The effect of age and of imipramine treatment on cortical serotonin and beta adrenergic binding sites and on pineal N-acetylserotonin and melatonin were examined in Fischer-344 rats. Cortical serotonin-1 and -2 receptor binding was reduced by 15 and 22.5% respectively, in 24 vs. 6 months old animals. Ten single daily imipramine (10 mg/kg) injections in the aged animals resulted in reductions in both types of serotonin sites, while in adult animals significant binding reduction occurred only at the 5HT2 site. Cortical beta-adrenoceptor binding was also diminished in the aged rats. Imipramine treatment elicited a significantly greater decrease in these adrenergic sites in the aged (39.2%) than in the adult (27.6%) animals. In the pineal gland, N-acetylserotonin and melatonin content were reduced by age; imipramine treatment induced decreases in both indoles in the adult animals and a reduction in N-acetylserotonin in the aged animals. These age-related effects of imipramine on cortical serotonin receptor and beta adrenoceptor binding and on pineal indoles may be a consequence of the higher drug and metabolite (desmethylimipramine) blood and tissue concentrations which were found in the aged animals. PMID- 3013650 TI - Protective effect of a new prostacyclin analogue OP-2507 against cerebral anoxia and edema in experimental animals. AB - Protective effects of OP-2507 [15-cis-(4-propylcyclohexyl)-16,17,18,19,20 pentanor-9-deoxy -9 alpha, 6-nitrilo-PGF1 methyl ester] against cerebral anoxia and edema were investigated in a variety of experimental models in mice and rats. OP-2507 given s.c. or p.o. led to a consistent and dose-dependent prolongation of survival time against cerebral anoxia in hypobaric and normobaric hypoxia, KCN induced anoxia and decapitation-induced gasping. Furthermore, treatment with 0.03 0.1 mg/kg s.c. or 0.3 mg/kg p.o. of OP-2507 was found to be effective against the changes of cerebral energy metabolites and cyclic nucleotides in hypoxic brain. In brain ischemia induced by bilateral ligation of common carotid arteries of rats, a reduction in specific gravity of cortex and an increase in water content of brain were observed, in accordance with changes of cerebral energy metabolites. These edematous and biochemical changes were prevented by the treatment with 0.01-0.03 mg/kg s.c. of OP-2507. These results indicate a potential usefulness of OP-2507 in protecting the brain from oxygen insufficiency resulting from cerebral ischemia. PMID- 3013652 TI - The mu, kappa and delta properties of various opioid agonists. AB - Using the delta-enriched mouse vas deferens and the kappa-enriched guinea-pig ileal longitudinal muscle preparations, a large variety of so-called 'mu, kappa, delta and sigma' opioid agonists were tested for their mu, kappa and delta properties. Buprenorphine and U50,488H appeared to be the most highly selective agonists for mu and kappa opioid receptors, respectively. Most ligands identified as specific agonists are in fact only selective and appear to interact at more than one receptor type. PMID- 3013653 TI - Angiotensin-converting enzyme responses following enalapril in the sodium deficient rat. AB - The relationship between blood pressure lowering activity and inhibition of plasma and tissue angiotensin-converting enzyme (ACE) has been studied in the sodium deficient normotensive rat at 24, 48 and 96 h after the administration for 21 days of enalapril (MK-421, 10 mg/kg per day p.o.). Blood pressure was reduced and plasma ACE activity inhibited at 24 and 48, but not 96 h, after cessation of dosing. Tissue ACE activity (aorta, lung, mesenteric bed) was inhibited up to 96 h post dose when blood pressure had returned to control values. ACE production (activity following removal of inhibitor) was increased in plasma at 24, 48 and 96 h post dose but in tissues (adrenal glands, renal arteries and mesenteric bed) only at 48 h post dose. There was no tendency for ACE production to increase in the lung, the largest source of the enzyme in the rat. Thus it appears that inhibition of ACE activity in both plasma and tissue contributes to the blood pressure lowering activity of enalapril in the sodium deficient normotensive rat. PMID- 3013654 TI - Arylazido aminopropionyl ATP (ANAPP3): interaction with adenosine receptors in longitudinal smooth muscle of the guinea-pig ileum. AB - Arylazido aminopropionyl ATP (ANAPP3), an ATP-receptor antagonist containing a photosensitive arylazido moiety coupled to the 3' hydroxyl of ATP, inhibited the twitch response of electrically stimulated ileal longitudinal muscle strips in a dose-dependent manner. These agonist responses to ANAPP3 were attenuated by the enzyme adenosine deaminase and antagonized by the adenosine receptor antagonist 8 phenyltheophylline. Schild analysis yielded similar pA2 values for ANAPP3 and adenosine suggesting a common receptor site. Several 3'-ribose-modified adenosine analogs were tested for agonist activity and found to be inactive. Results suggest that ANAPP3 interacts at the presynaptic adenosine receptor of the ileum following its metabolism to adenosine, which explains the lack of antagonism at adenosine receptors of ileal smooth muscle following photolysis of ANAPP3. PMID- 3013655 TI - Beta-adrenoceptor density in fetal striatal transplants. PMID- 3013656 TI - Biphasic effect of GABAA receptor agonists on prolactin secretion: evidence for two types of GABAA receptor complex on lactotrophes. AB - The effects of GABA receptor agonists on prolactin secretion in vitro was examined using a rapid superfusion system. GABA and muscimol caused a biphasic effect on prolactin secretion, both components of which were antagonised by bicuculline methiodide, while baclofen had no effect on basal or stimulated secretion, demonstrating the GABAA receptor specificity of both components. Homocarnosine caused only inhibition of secretion, and a range of partly rigid GABA analogues were relatively poor at causing stimulation of secretion. Both effects of muscimol were antagonised by low-chloride medium and the anion channel blocker DIDS, but strychnine and picrotoxinin were both potent and selective antagonists of the stimulatory effect. These results demonstrate a novel biphasic effect of GABAA agonists or prolactin secretion, the two components of which appear to be independent and mediated by different types or states of GABAA receptor/chloride channel complex. PMID- 3013657 TI - Morphine tolerance increases mu-noncompetitive delta binding sites. AB - In light of more recent knowledge concerning endogenous opioid peptides and their multiple opiate receptors, we reevaluated the effects of morphine tolerance on opiate receptor binding parameters. Rats were implanted with morphine or placebo pellets, and [3H][D-Ala2,D-Leu5]enkephalin ([3H]DADL) was used to label brain membranes. Utilizing the technique of binding surface analysis, we observed a selective 47% up-regulation of lower affinity [3H]DADL binding sites (mu noncompetitive delta binding sites) in morphine pelleted rats. To corroborate these results, we treated brain membranes with the site directed alkylating agent FIT (N-phenyl-N-[1-(2-p-isothiocyanato)phenyl-ethyl)-4-piperidinyl] propanamide), which results in membranes highly enriched with the lower affinity [3H]DADL binding site. Scatchard plots of [3H]DADL binding to FIT-treated membranes also revealed that chronic morphine treatment produced a 60-65% up-regulation of the mu-noncompetitive delta binding site. These data indicate that chronic morphine alters a selective subpopulation of opiate receptors that may play a role in the mechanisms of opiate tolerance and physical dependence. PMID- 3013658 TI - Sigma receptors on NCB-20 hybrid neurotumor cells labeled with (+)[3H]SKF 10,047 and (+)[3H]3-PPP. AB - (+)[3H]SKF 10,047 and (+)[3H]3-PPP label a homogeneous population of sites in NCB 20 cell membranes that apparently represent benzomorphan specific binding sites previously reported for this cell line. Their drug specificity indicates that these sites are very similar to sigma receptor binding sites labeled in brain tissues by these ligands and do not represent PCP receptors. PMID- 3013659 TI - Effects of atriopeptins on relaxation and cyclic GMP levels in human coronary artery in vitro. AB - The effects of atriopeptins on relaxation and cyclic GMP levels were examined in human coronary artery. alpha-Atrial natriuretic polypeptide and atriopeptins I, II and III all induced relaxation. Relaxations to atriopeptin I were of a smaller magnitude. The atriopeptins elevated cyclic GMP levels from 2- to 3-fold. These studies suggest that atriopeptins released from the heart may dilate the vasculature of this organ and increase coronary blood flow through the formation of cyclic GMP. PMID- 3013660 TI - Caprolactam-barbiturate interaction at the GABAA receptor complex in the guinea pig intestine. AB - 4,6,6-Trimethylcaprolactam antagonised GABAA receptor-mediated contractile responses in guinea-pig isolated ileum, displacing the GABA dose-response curve to the right in a non-parallel manner, and causing a depression of the maximum response. Pentobarbitone not only potentiated the GABAA receptor-mediated contractions but also reversed this non-competitive antagonism by 4,6,6 trimethylcaprolactam, shifting the dose-response curve for GABA to the left and restoring the maximum response. It is conclude that this caprolactam acts at the picrotoxin-barbiturate site on the Cl(-)-ionophore complex. PMID- 3013661 TI - Labelling of human platelet alpha 2-adrenoceptors with the full agonist [3H]( )adrenaline. AB - [3H](-)Adrenaline has been used to characterize alpha 2-adrenoceptors on human platelets. Although (-)adrenaline is a good substrate for the platelet enzyme MAO B, enzymatic inhibition was not a prerequisite to quantify the specific binding of the radioligand to platelet membranes. At 25 degrees C the binding was rapid (t1/2 of association: 10.3 min), reversible (t1/2 of dissociation: 4.0 min) and linearly dependent on the amount of protein present in the assay. The binding sites for [3H](-)adrenaline showed the specificity required for an alpha 2 adrenoceptor. The rank order of potency of inhibitors of [3H](-)adrenaline binding was oxymetazoline greater than idazoxan congruent to phentolamine congruent to clonidine congruent to (-)adrenaline greater than (-)noradrenaline greater than yohimbine much much greater than phenylephrine much greater than prazosin greater than (+)propranolol. Moreover, the nucleotide guanosine triphosphate (GTP) inhibited in a concentration-dependent manner (10(-9)-10(-4) M) the specific binding of [3H](-)adrenaline, suggesting that the radioligand preferentially labelled the high affinity state of the alpha 2-adrenoceptor. Linear (Scatchard) and non-linear analyses of [3H](-)adrenaline binding indicated the existence of a single population of non-interacting sites (KD = 2.5-2.7 nM; Bmax = 49-53 fmol/mg protein). The binding characteristics for [3H](-)adrenaline were not affected by age and sex of the donors or by freezing of platelet-rich plasma. In the same subjects alpha 2-adrenoceptor density (Bmax) for the full agonist [3H](-)adrenaline was 2.9-fold lower than that quantitated by the selective antagonist [3H]yohimbine. The inhibition constants (Ki) of adrenergic drugs and of various antidepressant drugs in competing with [3H](-)adrenaline were correlated with the inhibition constants of these drugs in competing with [3H]clonidine (r = 0.96; P less than 0.001) which suggests that both radioligands labelled the same alpha 2-adrenoceptor on the human platelet. The binding of the full agonist [3H](-)adrenaline to human platelet membranes might be a useful tool for the study of dysfunctions related to the high affinity state of the alpha 2 adrenoceptor. PMID- 3013663 TI - Intercellular communication of transformed and non-transformed rat liver epithelial cells. Modulation by TPA. AB - Gap-junctional intercellular communication of transformed and non-transformed rat liver epithelial cell lines was compared using a dye transfer method in the presence and absence of 12-O-tetradecanoylphorbol 13-acetate (TPA). Whereas non transformed cells (IAR 20, non-tumorigenic in newborn rats and in nude mice) showed very high communication capacity throughout a culture period of 3 weeks, transformed cells (IAR 6-1, tumorigenic in newborn rats and in nude mice) were less able to communicate. Similar correlation between intercellular communication and expression of transformed phenotypes were also found in newly cloned epithelial cell lines, IAR 27E and IAR 27F. When TPA was added to culture medium at 100 ng/ml, intercellular communication in all lines tested was reduced within 60 min. However, communications recovered completely from the effect within 10 h after addition of TPA. Further addition of TPA to the cultures every 24 h for 3 weeks had no effect on intercellular communication (measured 30 min after each TPA addition), suggesting that a single application of TPA made these cells refractory to further doses. A known stimulator of gap-junctional communication, db-cAMP, also increased dye transfer in IAR 20 and IAR 6-1 cells. TPA added to db cAMP-treated cultures of IAR 20 and IAR 6-1 cells inhibited intercellular communication, suggesting that cAMP is not an antagonist of the effect of TPA on intercellular communication in these cell lines. These results are in sharp contrast to those obtained with the fibroblast cell line BALB/c 3T3, in which db cAMP antagonized TPA effect and inhibition by TPA of intercellular communication was transient only when administered during their growth phase, and was stable and continuous when TPA was applied at confluence, and suggest that TPA may not be an effective tumour promoter in rat liver. PMID- 3013662 TI - Liver endothelium mediates the uptake of iron-transferrin complex by hepatocytes. AB - We have previously shown that in the liver, transferrin (TF) receptors are limited to endothelial cells, and hepatocytes and Kupffer cells do not have TF receptors. To study the transport of iron into hepatocytes, we fractionated liver cell suspensions into endothelium and hepatocyte fractions. At 4 degrees C liver (but not umbilical cord) endothelium bound Fe-TF with a saturable kinetics. At 37 degrees C, the endothelial uptake was followed by its gradual release. Transendothelial transport of TF was visually demonstrated by perfusion of liver using colloidal gold-labeled TF. The released Fe-TF acquired the potential for binding to fresh target hepatocytes and binding was not inhibited by excess cold TF but was inhibitable by asialofetuin, suggesting galactosyl receptors and not TF receptors as a recognition mechanism. Isoelectrofocusing of the supernate after preincubation for 90 min at 37 degrees C with endothelial cells, demonstrated the presence of a newly generated band which co-migrated with asialotransferrin. We conclude that Fe-TF is initially removed by liver endothelium where it is modified probably by desialation to expose the galactosyl residues of the glycoproteins. The modified molecule is subsequently released and recognized by hepatocytes through a TF receptor-independent mechanism which may involve galactosyl receptors of hepatocytes. The findings indicate a key role for endothelium in the transport of Fe-TF into the liver and may suggest a physiological function for galactosyl receptors on hepatocyte surface. PMID- 3013664 TI - Endocytosis via galactose receptors in vivo. Ligand size directs uptake by hepatocytes and/or liver macrophages. AB - We followed the intrahepatic binding and uptake of variously sized ligands with terminal galactosyl residues in rat livers. The ligands were administered to prefixed livers in binding studies and in vivo and in situ (serum-free perfused livers) in uptake studies. Gold sols with different particle diameters were prepared: 5 nm (Au5), 17 nm (Au17), 50 nm (Au50) and coated with galactose exposing glycoproteins (asialofetuin (ASF) or lactosylated BSA (LacBSA)). Electron microscopy of mildly prefixed livers perfused with LacBSA-Au5 in serum free medium showed ligand binding to liver macrophages, hepatocytes and endothelial cells. Ligands bound to prefixed cell surfaces reflect the initial distribution of receptor activity: pre-aggregated clusters of ligands are found on liver macrophages, single particles statistically distributed on hepatocytes and pre-aggregated clusters of particles restricted to coated pits on endothelial cells. Ligand binding is prevented in the presence of 80 mM N-acetylgalactosamine (GalNAc), while N-acetylglucosamine (GlcNAc) is without effect. Electron microscopy of livers after ligand injection into the tail vein shows that in vivo uptake of electron-dense galactose particles by liver cells is size-dependent. Using a LacBSA-Au preparation with heterogeneous particle diameter (2.2-11.7 nm) we found that hepatocytes take up only ligands up to the size of 7.8 nm, whereas particles of all sizes available in this experiment are found in liver macrophages and endothelial cells. ASF-Au17 and LacBSA-Au17 are endocytosed by liver macrophages and endothelial cells, but not by hepatocytes. ASF-Au50 is taken up by liver macrophages only. In vivo uptake by liver macrophages is mediated by galactose-specific recognition as shown by inhibition with GalNAc. Some 52-65% inhibition was measured in in vivo experiments and 78% inhibition in in situ experiments. GlNAc showed no inhibitory effect. Furthermore, we measured uptake of [125J]ASF and of [125J]ASF adsorbed to Au17 by the different cell populations of rat livers in vivo. While the bulk of the molecular ligand is found in the hepatocyte fraction, the particulate ligand is located in the sinusoidal fraction. PMID- 3013665 TI - Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes. AB - Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, we treated oocytes with activators of this enzyme. The active phorbol esters 12-O-tetra decanoyl phorbol-13-acetate (TPA) and 4 beta-phorbol 12,13-didecanoate (4 beta PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC8). An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), did not inhibit GVBD. We then examined whether protein kinase C activators inhibit a step in the cAMP modulated pathway that regulates resumption of meiosis. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC8 partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis. Finally, we compared the effects of db-cAMP and protein kinase C activators on polar body emission following GVBD. TPA, 4 beta-PDD or diC8, but not 4 alpha-PDD or db-cAMP, inhibited polar body emission in a dose-dependent manner. The morphology and cytology of oocytes in which polar body emission was inhibited by TPA or 4 beta PDD differed from that of oocytes treated with diC8. Thirty to 60% of the former were round in shape and exhibited a clump of chromosomes but no spindle; the remainder were distended in shape and exhibited a metaphase I spindle. All oocytes treated with diC8, however, were round, had dispersed chromosomes, and no spindle. These results suggest that, in contrast to resumption of meiosis, polar body emission is inhibited by activation of protein kinase C but not cAMP dependent protein kinase. PMID- 3013666 TI - Melatonin: parallels in pineal gland and retina. AB - It is apparent that several relationships exist between the pineal gland and retina. The similarities in development and morphology have been obvious for many years. A recent resurgence of interest in this field has led to a further understanding of many functional similarities between these two organs. A notable feature of the pineal gland and retina is their common ability to synthesize the indolamine hormone, melatonin. Many investigators suspect that the cyclic rhythm of retinal melatonin synthesis may be related to other cyclic events which normally occur in the retina. PMID- 3013667 TI - ATPases of ciliary epithelium: cellular and subcellular distribution and probable role in secretion of aqueous humor. AB - The distribution of ion-stimulated ATPases of the ciliary epithelium has been examined in tissues from bovine and rabbit eyes. In homogenates of tissues from both species, both Na,K- and anion-stimulated enzyme activities were found, but no K,H-stimulated activity was detected. The anion ATPase had a broad specificity for a number of anions, and was strongly inhibited by thiocyanate. Following separation of pigmented (outer) and non-pigmented (inner) layers of the bovine ciliary epithelium and isolation of the two cell types on density gradients, higher activities of both Na,K- and anion ATPases were found in the non-pigmented cells. Subcellular fractionation of a mixed population of cells showed that the anion ATPase was almost exclusively associated with a mitochondrial fraction, rather than with the plasma-membrane fraction containing the Na,K-ATPase. These results confirm histochemical studies of the distribution of Na,K-ATPase in the ciliary epithelium and support the concept that the inner, non-pigmented cell layer is chiefly responsible for the active transport of ions into the posterior chamber. It is concluded that this transepithelial transport can be driven only by the energy derived via the Na,K-ATPase, and that any subsequent anion or proton transport in the formation of aqueous humor is driven by the sodium gradient through exchange mechanisms. PMID- 3013668 TI - Intranuclear rodlets in the rabbit retina following treatment with MPTP. AB - Retinas from pigmented rabbits treated with N-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP, a chemical inducer of Parkinsonism in man and monkeys) were studied using light- and electron microscopy. The nuclei of many cells in the inner nuclear layer and the ganglion cell layer of the treated retinas contained crystalloids (intranuclear rodlets) of varying length (0.5-8 microns) which were composed of bundles of 12 nm filaments and which were seen only rarely in untreated retinas. The induced rodlets are oval to round cylinders, 15-20 filaments across (although they are smaller in untreated retinas). Similar intranuclear inclusions have been described under varying conditions in neurons and glia in the central nervous system of several animal species. In rabbits injected acutely with MPTP, most of the affected cells are in the inner nuclear layer in the position of bipolar cells, while in the chronically injected animals, clearly identifiable amacrine cells, and the nuclei of some cells in the ganglion cell layer also contained the inclusions. Evidence is presented that the rodlet-containing cells in the ganglion cell layer include both ganglion cells and displaced amacrine cells. These anatomic findings are relevant to understanding the physiological and biochemical effects of the drug on the retina which we reported previously (Wong, Ishibashi, Tucker and Hamasaki, 1985). PMID- 3013669 TI - Plasmodium berghei: reaction of sporozoites with chemically and enzymatically modified hepatoma cells. AB - Plasmodium berghei sporozoites were observed to react with human hepatoma (HepG2) target cells which had been fixed with methanol, formaldehyde, or glutaraldehyde. The reaction consisted of attachment of sporozoites to the fixed target cells and the release of circumsporozoite protein which bound to target cell areas adjacent to the attachment sites. Treatment of fixed target cells with 0.1 N H2SO4 at 80 C, neuraminidases, neuraminidase plus galactose oxidase or inclusion of transferrin, orosomucoid, their asialo forms, or various monosaccharides in the incubation medium had no significant effect on target cell reactivity with sporozoites. Fixed cells oxidized with periodate or cells extracted with methanol or chloroform-methanol were reactive but lost activity if allowed to air dry after treatment. Treatment with papain or chymotrypsin at levels producing heavy cell structure damage caused a major loss of activity. PMID- 3013670 TI - Trypanosoma rhodesiense: mitochondrial proteins of bloodstream and procyclic trypomastigotes. AB - One- and two-dimensional gel electrophoresis of the solubilized mitochondrial proteins of bloodstream and procyclic trypomastigote Trypanosoma brucei rhodesiense and radiolabeling of proteins in the presence of cycloheximide were used to identify proteins synthesized in the trypanosome mitochondrion. The proteins which comprise the mitochondrion were found to be very similar in both bloodstream and procyclic trypomastigotes, but do differ in their level of synthesis. A protein putatively identified as subunit II of cytochrome oxidase (EC 1.9.3.1) was detected in mitochondria from both the procyclic and bloodstream organisms. The presence of this protein in bloodstream trypomastigotes and the overall similarity of protein content in the trypanosome mitochondria is noteworthy in view of the fact that bloodstream trypomastigotes have a repressed mitochondrion with no detectable tricarboxylic acid cycle or cytochrome electron transport chain. PMID- 3013671 TI - Experimental eosinophilia and inflammation--the effect of various inflammatory mediators and chemoattractants. AB - An experimental subcutaneous inflammation was produced in guinea pigs with peripheral blood eosinophilia. The eosinophilia resulted from two subsequent infections with Trichinella spiralis larvae. One group of guinea pigs served as non-infected control. Inflammation was induced by carrageenan, bradykinin, histamine, platelet activating factor and eosinophilotactic factors of lymphocytic or neutrophilic origin. Whereas in the control group no eosinophil granulocytic response was observed, this response was seen in the group with peripheral blood eosinophilia. The inflammatory substances and mediators (carrageenan, bradykinin, histamine, platelet activating factor) did not attract eosinophils alone, but also neutrophils. Under peripheral blood eosinophilia within the time course of the inflammatory reaction a second emigration with a shifted neutrophil/eosinophil ratio in favour of eosinophils was found. This could be due to a generation of chemoattractants by the injected substances themselves or more probably, by already emigrated granulocytes. Neither histamine, bradykinin, carrageenan, nor the eosinophilotactic factors (ECF's) in the concentrations used did release the cytotoxic major basic protein from eosinophils. Platelet activating factor exhibited a release of major basic protein from some eosinophils but no release of the peroxidase under our experimental conditions. The immigration of sufficient numbers of eosinophils into inflammatory areas might be one cause of the reduction of the inflammatory edema found in a previous investigation under similar conditions. PMID- 3013672 TI - Effects of mistletoe (Viscum album L.) extracts on cultured tumor cells. AB - Bacterially fermented mistletoe preparations (BFMP) were tested on rat hepatoma tissue culture (HTC) cells and human leukemia Molt 4 cells. A dose-dependent inhibition of the growth rate of the cells was observed. For both cell lines, cytostatic concentrations, expressed in weight of fresh plant, were 0.5 mg/ml culture medium for oak BFMP and 1 mg/ml for apple tree BFMP. However, the action of the two preparations was markedly different on each cell line. Non-viable HTC cells were not stained by trypan blue while non-viable Molt 4 cells were fully colored by this reagent. A lysis of cellular membranes of HTC cells was observed by electron microscopy. Furthermore, oak BFMP inhibited the growth of virus transformed 3T3-SV40 cells more than that of non-transformed 3T3 cells. In contrast to BFMP, non-fermented extracts and a purified mistletoe lectin showed a greater inhibition of the growth of Molt 4 cells than of HTC cells. Samples withdrawn at different times during fermentation gradually lost their inhibitory effect on the growth of Molt 4 cells while their action on HTC cells increased up to the 4th day of fermentation. These results are discussed in relation to the cytotoxic substances of mistletoe already characterized. PMID- 3013674 TI - Morphometric analysis of the endocrine cell composition of rat pancreas following treatment with streptozotocin and nicotinamide. AB - Using immunohistochemistry and linear scanning, a morphometric analysis was made of the composition of the rat endocrine pancreas at sequential intervals after combined injections of streptozotocin (SZ) and nicotinamide (NA). One week after treatment, the volume of islet tissue was significantly higher than that of the corresponding, saline-injected controls, probably as the result of acute hyperplasia of insulin- and somatostatin-positive cells. However, at all time periods thereafter (6, 20, and 36 weeks), the drug-treated rats showed decreased islet volumes compared to controls. Analysis of aggregate (total) volumes of hormone producing cells at various time periods after drug treatment indicated that decreases in insulin (B-cell) volumes only partially accounted for the observed changes in total islet volume. There were, in addition, early decreases in glucagon (A-cell) and increases in somatostatin (D-cell) volumes. The results suggest that SZ/NA treatment caused limited islet B-cell destruction and transient changes in the proportions of islet A and D cells. Microscopic endocrine tumors were observed at 20 weeks, and both gross and microscopic tumors were observed 36 weeks after SZ/NA treatment. When islet and tumor tissues were included in computation, aggregate volumes of insulin and somatostatin-positive cells were markedly increased, with no significant changes in glucagon-positive cell volumes compared to controls, indicating that the tumors were rich in B and D cells, but poor in A cells. These results are discussed in relation to changes in glucose tolerance and serum insulin levels, and to islet cell volumes following treatment with a diabetogenic dose of streptozotocin alone. PMID- 3013673 TI - Pituitary gonadotropin releasing hormone (GnRH) receptor levels in intact and ovariectomized-adrenalectomized female golden hamsters on a short photoperiod. AB - Both intact and ovariectomized + adrenalectomized hamsters on a short photoperiod, had a daily surge in plasma LH at approximately 16.00-18.00 h. The number of pituitary GnRH receptors was generally lower in ovariectomized + adrenalectomized hamsters than in intact animals, but both intact and ovariectomized + adrenalectomized hamsters had a decrease in the number of receptors just prior to the LH surge. These results show that gonadal steroids are not involved in regulating the pre-LH surge fall in the number of GnRH receptors. PMID- 3013675 TI - [GABA-ergic system and blood circulation regulation]. PMID- 3013676 TI - [Immunotropic activity of the cAMP phosphodiesterase inhibitors caffeine and theophylline]. AB - On mice immunized with ram erythrocytes it was shown that caffeine and theophylline (15 to 240 mg/kg thrice at different time intervals with respect to the antigenic stimulus) possess identical immunotropic activity. High doses of methylxanthines injected a few days before immunization increased and being administered during immunization decreased the indices of humoral immunity. Low doses of the drugs stimulated immunity but only when administered at the time close to the antigenic stimulus application. PMID- 3013677 TI - Cytoskeletons of ADP- and thrombin-stimulated blood platelets. Presence of a caldesmon-like protein, alpha-actinin and gelsolin at different steps of the stimulation. AB - Comparative analyses of the cytoskeletons of resting and stimulated platelets point out the involvement of a 79 kDa polypeptide in the activation step and its increased incorporation during aggregation. It appears as a doublet and cross reacts with an antibody to chicken gizzard caldesmon, whereas no 150 kDa immunoreactive form was detected. alpha-Actinin and gelsolin were detected only in the aggregation step. PMID- 3013679 TI - Identification of a 68 kDa protein which copurifies with type-1 protein phosphatase as albumin. AB - Proteins of 60-70 kDa copurify with some preparations of type-1 or type-2 phosphatases. In our system chromatography on polylysine-Affi-Gel 10 separates a 68 kDa protein from rabbit muscle glycogen particle phosphorylase phosphatase. The separation affects neither the activity nor the size of the phosphatase. The 68 kDa protein, although pure by SDS gel electrophoresis criteria, still displays phosphatase activity of approx. 6-8 U/mg. However, rechromatography either on Bio Gel A-0.5 m or on Blue Sepharose CL-6B followed by gel filtration shows that the activity is due to a contamination with phosphatases of type 1 and type 2, displaying a molecular mass of 35 kDa, which can be totally removed from the 68 kDa protein. The amino acid composition of the 68 kDa protein is identical to that of rabbit serum albumin, within the limits of variation for the method. Furthermore, the sequence of the 38 N-terminal amino acids is the same in the isolated 68 kDa protein and in rabbit serum albumin. PMID- 3013678 TI - Is thermostability of glucose-6-phosphatase indeed dependent on a stabilizing protein? AB - Partial purification of glucose-6-phosphatase from rat liver microsomes by solubilization of the membranes with the non-ionic detergent Triton X-114 at pH 6.5 and the removal of inactivating detergent by hydrophobic chromatography results in a thermostable enzyme protein which is not dependent on stabilizing phospholipids or proteins. The readdition of low amounts of detergent immediately causes a conversion into a thermo-unstable phosphohydrolase protein. Thus these findings present evidence that heat instability of partially purified glucose-6 phosphatase derives from traces of inactivating detergent changing the structural properties of the phosphohydrolase rather than from the absence of the postulated specific stabilizing protein. PMID- 3013680 TI - Inhibition of catalytic unit of adenylate cyclase and activation of GTPase of Ni protein by beta gamma-subunits of GTP-binding proteins. AB - A protein factor which inhibited adenylate cyclase was purified to apparent homogeneity from rat brain and identified as the beta gamma-subunits of the GTP binding regulatory proteins of adenylate cyclase. (i) The beta gamma-subunits (protein factor) inhibited the partially purified catalytic unit of adenylate cyclase in the presence of an activator, forskolin or the stimulative regulatory protein (Ns), to 60 and 40% of the control, respectively; inhibition of the catalytic unit in the presence of forskolin required no guanine nucleotides. (ii) The subunits enhanced the GTPase activity of the purified alpha-subunit of the inhibitory regulatory protein (Ni alpha) 3.8-fold. (iii) The subunits stimulated ADP-ribosylation of Ni alpha catalyzed by islet-activating protein (pertussis toxin). ADP-ribosylation had no effect on the GTPase activity of Ni alpha in the presence of the beta gamma-subunits. The results suggest that direct inhibition of the catalytic unit by the beta gamma-subunits liberated from Ni is essential for the receptor-mediated inhibition of adenylate cyclase. PMID- 3013681 TI - Phosphorylation of phosphatidylinositol by transverse tubule vesicles and its possible role in excitation-contraction coupling. AB - Phosphorylation of phosphatidylinositol to phosphatidylinositol 4-monophosphate and to phosphatidylinositol 4,5-bisphosphate was demonstrated in transverse tubule membranes isolated from frog skeletal muscle using [gamma-32P]ATP as substrate. At millimolar concentrations of Mg2+ both phosphorylation reactions were completed within 15 s at 25 degrees C. Isolated sarcoplasmic reticulum vesicles phosphorylated phosphatidylinositol to phosphatidylinositol 4-phosphate with a lower specific activity than the transverse tubules, and lacked the ability to produce phosphatidylinositol 4,5-bisphosphate. These findings show, for the first time, that isolated transverse-tubule membranes carry out one of the steps required to sustain a role for inositol trisphosphate as the physiological messenger in excitation-contraction coupling in skeletal muscle. The finding that 0.5 mM tetracaine apparently inhibits the phosphorylation of phosphatidylinositol 4-phosphate to phosphatidylinositol 4,5-bisphosphate also supports a role for these intermediates in excitation-contraction coupling. PMID- 3013682 TI - Effects of seminiferous tubule secreted factor(s) on Leydig cell cyclic AMP production in mature rat. AB - The effects of spent media from seminiferous tubules (STM) on Percoll-purified rat Leydig cells were investigated. Intracellular and extracellular cyclic AMP (cAMP) accumulation and testosterone production were measured. After a 5 h incubation period, STM reduces both the basal and LH-dependent cAMP levels (38 and 20%, respectively for intra- and extracellular cAMP) while, simultaneously, a stimulation of testosterone production is observed (47 to 50%, respectively in the absence or presence of LH). The reduction of cAMP levels observed after 5 h is likely to be due to the potentiating effect of the STM factor on the LH dependent initial rise of the cAMP level which, in turn, induces a desensitization of the Leydig cell adenylate cyclase. This substance is a thermolabile protein (Mr greater than 50 000) produced by the Sertoli cell, independent of FSH and testosterone controls, and different from the LHRH-like substance. PMID- 3013683 TI - Impairment of Na+/H+ exchange underlies inhibitory effects of Na+-free media on leukocyte function. AB - Inhibition of activation has been reported when neutrophils are suspended in Na+ free media. We considered the possibility that impairment of cellular pH (pHi) regulation due to elimination of Na+/H+ exchange underlies this effect. In the absence of Na+, the phorbol ester-induced respiratory burst was partially inhibited and a concomitant cytoplasmic acidification recorded. Using nigericin/K+ to clamp pHi we demonstrated that the acidification accounts for the inhibition of O2 uptake. Moreover, in Na+-free media, relieving the acidification by means of ionophores restored maximal O2 consumption. It was concluded that Na+ is not directly involved in signal transduction during stimulation. Instead, omission of Na+ affects neutrophils activation indirectly, by impairing pHi regulation. PMID- 3013684 TI - Thyrotropin and norepinephrine stimulate the metabolism of phosphoinositides in FRTL-5 thyroid cells. AB - Hormone-induced changes in phospholipid metabolism were examined in a functioning rat thyroid cell line (FRTL-5). Stimulation of FRTL-5 cells, prelabeled with 32P, with TSH or NE resulted in a rapid decrease in the radioactivity of both phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4 monophosphate (PIP). The effects of TSH on phospholipid metabolism and calcium mobilization are independent of those on adenylate cyclase. This suggests that the TSH receptor may be unique in that it activates enzyme cascades involved in cAMP production and Ca2+ mobilization. PMID- 3013685 TI - Several lines of evidence demonstrating that Plasmodium falciparum, a parasitic organism, has distinct enzymes for the phosphorylation of choline and ethanolamine. AB - In Plasmodium falciparum-infected erythrocyte homogenates, the specific activity of ethanolamine kinase (7.6 +/- 1.4 nmol phosphoethanolamine/10(7) infected cells per h) was higher than choline kinase specific activity (1.9 +/- 0.2 nmol phosphocholine/10(7) infected cells per h). The Km of choline kinase for choline was 79 +/- 20 microM, and ethanolamine was a weak competitive inhibitor of the reaction (Ki = 92 mM). Ethanolamine kinase had a Km for ethanolamine of 188 +/- 19 microM, and choline was a competitive inhibitor of ethanolamine kinase with a very high Ki of 268 mM. Hemicholinium 3 inhibited choline kinase activity, but had no effect on ethanolamine kinase activity. In contrast, D-2-amino-1-butanol selectively inhibited ethanolamine kinase activity. Furthermore, when the two enzymes were subjected to heat inactivation, 85% of the choline kinase activity was destroyed after 5 min at 50 degrees C, whereas ethanolamine kinase activity was not altered. Our results indicate that the phosphorylation of choline and ethanolamine was catalyzed by two distinct enzymes. The presence of a de novo phosphatidylethanolamine Kennedy pathway in P. falciparum contributes to the bewildering variety of phospholipid biosynthetic pathways in this parasitic organism. PMID- 3013686 TI - Electrogenic proton exchange between cytochrome a3 active center and M-aqueous phase. Evidence for cytochrome a3-associated input proton well. AB - The rate of cyanide binding with the oxidized cytochrome-c oxidase in proteoliposomes is controlled by ionization of a protein group with pK approximately 6.7, the ligand reacting with the protonated enzyme only [(1983) Bioorg. Chem. (USSR) 9, 216-227]. As shown here, the kinetics of cyanide binding depends on the pH inside the proteoliposomes. The reaction rate is affected by the electrical potential difference across the proteoliposome membranes as if the a3-linked ionizable group exchanged H+ with the proteoliposome interior electrogenically. The data corroborate a hypothesis on the existence of a proton well communicating cytochrome oxidase O2-reducing center with the M-aqueous phase. PMID- 3013687 TI - On the analogy in the structure of the spleen green heme protein and granulocyte myeloperoxidase. AB - The molecular structure of the spleen green heme protein was reinvestigated by gel-permeation, SDS-polyacrylamide gel electrophoresis, and amino acid analysis. The results showed that the enzyme is a tetramer (Mr 1.5 X 10(5)) with two heavy subunits (Mr 6 X 10(4) with a single prosthetic group per subunit) and two light subunits (Mr 1.5 X 10(4)), and that the tetramer structure is maintained by disulfide bond(s). The amino acid composition of the spleen green heme protein is similar to that of granulocyte myeloperoxidase. The present results contradict the data of Davis and Averill [(1981) J. Biol. Chem. 256, 5992-5996], who reported the enzyme as a monomeric peroxidase with an Mr of 57 000. PMID- 3013688 TI - Increased sensitivity of fat cell adenylate cyclase to stimulatory agonists during fasting is not related to impaired inhibitory coupling system. AB - In rat adipocytes, inhibition of the forskolin-stimulated cyclic AMP response by nicotinic acid and N6-phenylisopropyladenosine was unaltered by a 72 h fasting. Under assay conditions favouring inhibition, basal and forskolin-stimulated adenylate cyclase responses to inhibition by GTP and nicotinic acid were also unimpaired by fasting. Under the same conditions, however, low GTP concentrations elicited a clear activatory effect in membranes from fasted but not from fed rats. Fasting failed to alter the incorporation of [32P]ADP ribose into the alpha i-subunit of Ni and the attenuation of nicotinic acid inhibitory action that are both induced by pertussis toxin. These results, demonstrating unimpaired inhibitory control of adenylate cyclase in starved rat adipocytes, suggest that the permissive effect of fasting on the action of stimulatory receptor agonists in fat cells reflects a specific increase in the activity of the adenylate cyclase stimulatory coupling system. PMID- 3013690 TI - The complete primary structure of GTP:AMP phosphotransferase from beef heart mitochondria. AB - To complete the amino acid sequence of GTP:AMP phosphotransferase (MgGTP + AMP in equilibrium with MgGDP + ADP) from beef heart mitochondria it was necessary to sequence an intermediate region of about 33 residues after position 102 [(1984) Eur. J. Biochem. 143, 331-339] and find a suitable overlap with the rest of the protein. The required peptides were obtained by cleaving the enzyme with endoproteinase Lys-C. One peptide, covering the region from residue 79 to 144, was sequenced up to residue 124. Another peptide, extending from residue 79 to 169, was subcleaved with Staphylococcus aureus V8 protease and provided the fragment from residue 99 to 139 which was sequenced. Several other peptides from endoproteinase Lys-C cleavage were used to check large sections of the previously published sequence work. The complete sequence contains 225 amino acids and has an Mr of 25 469. PMID- 3013689 TI - Protein kinase C negatively modulated by phorbol ester. AB - Pretreatment of protein kinase C with 12-O-tetradecanoylphorbol-13-acetate (TPA) and phospholipid resulted in complete inhibition of ATP/phosphotransferase activity, irreversibly. The inactivation by TPA required the phospholipid, and TPA alone did not cause inactivation. Ca2+ and diacylglycerol mimicked TPA. This action of TPA was not general for all protein kinases as it did not accelerate the inactivation of the catalytic subunit of cAMP-dependent protein kinase by phospholipid. The addition of MgATP to the reaction mixture completely protected protein kinase C from being inactivated by TPA, in the presence of phospholipid. The nucleotide-binding site of the enzyme was probably influenced by the binding of TPA and phospholipid. PMID- 3013691 TI - Binding of ATP to nucleoside-diphosphate kinase: a kinetic study. AB - The binding of nucleotides to pig heart nucleoside-diphosphate kinase was studied using Rose Bengal as an optical probe. ATP, in the absence of Mg2+, binds slowly to the enzyme, with a second order rate constant of about 3000 M-1 . s-1, whereas in its presence the binding is much faster. This finding suggests the regulation of the nucleoside-diphosphate kinase activity by uncomplexed ATP, and that ATP binds normally to the enzyme via a metal ion bridge. PMID- 3013692 TI - Expression, secretion and processing of hirudin in E. coli using the alkaline phosphatase signal sequence. AB - A DNA fragment coding for the E. coli phoA signal peptide was synthesized and inserted into the expression vector pKK223-3. A single HindIII restriction site is located just at the end of the signal sequence. A gene coding for the proteinase inhibitor hirudin, which has previously been synthesized, was inserted into this HindIII site. The hybrid protein was expressed under control of the tac promoter and secreted into the periplasm of E. coli. From the periplasmic fraction two processed proteins were isolated. One of these was identical with desulfatohirudin and also had similar biological properties. PMID- 3013693 TI - Microvesicular secretion, a mode of cell secretion associated with the presence of an ATP-diphosphohydrolase. PMID- 3013694 TI - Antiarrhythmic drugs and the modulation of autonomic control of heart rate in rabbits. AB - A model of the components of autonomic control of heart rate was developed and used for the evaluation of quantitative contribution of sympathetic and vagal tone to cardiac function. In conscious rabbits, sequential inhibition of muscarinic and beta receptors was produced and the relative contributions of vagal and sympathetic tone were characterized. Based on the model, the magnitude of presynaptic interaction between the vagal and sympathetic nerve endings was evaluated. From data in the literature, similar analysis of the control of heart rate was performed for the rat, dog, and human subject and compared with that of the rabbit. The results show that the resting rabbit heart is under less vagal tone than sympathetic tone as compared with other species. The effects of acute administration of amiodarone on the sympathetic and parasympathetic control of heart rate as well as intrinsic heart rate were investigated. Amiodarone decreased the heart rate, which resulted from a direct effect on the sinoatrial (SA) node. In addition, it attenuated the vagal as well as the sympathetic effects on the SA node. The effect on vagal component was greater. Further, the effects of other antiarrhythmic drugs on the electrocardiographic PP and PR intervals were studied. The usefulness of this model for the analysis of the effects of antiarrhythmic drugs is presented. PMID- 3013695 TI - Lymphadenopathy associated virus/human T-cell lymphotropic virus III antibodies in homosexual men with and without sperm antibodies. AB - For the evaluation of a possible relationship between antisperm antibodies and LAV/HTLV-III antibodies, both markers were determined in the sera of 89 homosexual men. Thirty-one of 89 men (35%) had sperm antibodies in their sera, and 21 of 89 men (24%) had LAV/HTLV-III antibodies. There was no significant relationship between the occurrence of both kinds of antibodies, so that infection with the LAV/HTLV-III retrovirus in homosexual men seems not be influenced by the presence of antisperm antibodies. PMID- 3013697 TI - AIDS: a challenge for contemporary nursing, Part I. PMID- 3013696 TI - [Effect of exogenous cAMP on the development of immunologic memory to allo- and heteroantigens]. PMID- 3013698 TI - [Activity of presynaptic alpha-adrenoreceptors during prolonged perfusion with adrenaline]. AB - The release of 3H-noradrenaline and contractile response to transmural stimulation of the rat vas deferens decreased according to the concentration of the perfused noradrenaline. In the course of continuing perfusion with noradrenaline and clonidine no increase in the release of the transmitter in response to the stimulation occurred which would suggest a desensitization, i. e. a lowered sensitivity of presynaptic alpha-adrenoreceptors. Possible reasons of the absence of desensitization of presynaptic alpha-adrenoreceptors under a prolonged action of adrenomimetics, are discussed. PMID- 3013699 TI - [Mechanism of stimulation of the motor activity of the stomach by the greater splanchnic nerve]. AB - The activating effect of the splanchnic nerve on motor activity of the gastro intestinal tract could not be eliminated either with bilateral vagotomy or separate or joint administration of ornid and benzohexonium, but could be well prevented with atropin blockade of the vegetative ganglia M-serotoninergic receptors as well as diprazin blockade of the smooth muscle D-serotoninergic receptors. The data obtained reveal non-cholinergic and non-adrenergic fibers in the thoracic portion of the major splanchnic nerve which exert obvious activating effect on the stomach contractile function, serotonin being their transmitter. PMID- 3013700 TI - [Role of alpha- and beta-adrenoreceptors of the jejunum and ileum in displays of their contractile reactions]. AB - Acute experiments were performed on isolated segments of the cat small intestine vessels. The contractile activity of the jejunum and ileum was estimated by the maximal isometric tension. Blocking of the alpha-adrenoreceptors with phentolamine induced a reinforcement of the contractile reactions, whereas blocking of the beta-adrenoreceptors with propranolol led to inhibition of both jejunum and ileum contractile responses either to exogenous acetylcholine or electrical stimulation of vagal efferent fibers. The subthreshold activation of alpha-adrenoreceptors with noradrenaline induced mostly inhibition, whereas activation of beta-adrenoreceptors with isopropylnoradrenaline caused a potentiation of the contractile response to exogenous acetylcholine. The data obtained suggest existence of excitatory beta-adrenoreceptors with a presynaptic localization in the jejunum and ileum. PMID- 3013701 TI - [Changes in the activity of neurons of the visual cortex during stimulation of midbrain raphe nuclei]. PMID- 3013702 TI - Virus-specific nucleotide sequences in duck cells transformed by chicken and duck adapted Rous sarcoma virus. AB - Adaptation of PR-RSV-C on duck cells results in successful and efficient replication of the adapted virus in duck cells. The adapted variant, daPR-RSV-C, was compared with the parental chicken-cell derived PR-RSV-C. No differences in the efficiency of integration and in the number of integrated proviral copies in duck cells were found. However, the structure of proviral DNA of the adapted virus was different. Whereas EcoRI and HindIII digestion showed no differences between chicken-cell derived PR-RSV-C and the daPR-RSV-C, a new restriction site was found for BamHI endonuclease, which is probably located at the 3' end of the env gene. PMID- 3013703 TI - Effect of hyperthermia on morphology and histochemistry of spinal cord in the rat. AB - Studies were performed on mature Wistar strain rats, subjected to 43 degrees C environmental temperature for 4 h at a relative air humidity of 60-70%. Spinal cords of rats sacrificed 1, 24, 48, 72 h and one week after hyperthermia served as a material for the studies. Routinely stained preparations (H+E, Nissl) and enzymatic activity of some phosphatases and esterases was estimated as well in sections subjected to Feulgen reaction karyo- and cytophotometric measurements were performed on cell nuclei of anterior horns neurocytes, anterior columns oligodendrocytes and on anterior funicle astrocytes of the spinal cord lumbar segment. The hyperthermia resulted in rat spinal cords in several morphological and histochemical alterations. Signs of diffuse spinal cord lesion of vascular origin were present with degenerative alterations of neurocytes, oligodendroglia proliferation and astroglia hyperplasia. In histoenzymatic studies changes in enzymatic activity of NsE, AChE, ChE, AcP, ATPase and TPPase were noted. They were dependent upon the time which elapsed after hyperthermia. Karyo- and cytophotometric measurements demonstrated cell nuclei oedema in neurocytes, oligodendrocytes and astrocytes associated with a decrease in the relative DNA level and changes in density and concentration of nuclear chromatin. The observed morphological, histoenzymatic and cytophotometric changes were of a reversible type and majority of them vanished within a week after hyperthermia. PMID- 3013705 TI - Regulation of adenylate cyclase by beta-melanotropin in the M2R melanoma cell line. AB - We have examined adenylate cyclase (AC) in the M2R melanoma cell line, a novel clone of transplantable B16 melanoma cells. It has been found that activity of this enzyme is highly responsive to beta-melanotropin (beta-MSH) and other hormones possessing melanotropic activity (e.g., alpha-melanotropin (alpha-MSH) and adrenocorticotrophic hormone (ACTH1-24)). beta-MSH stimulation of adenylate cyclase, both in the intact cell and in a plasma membrane-enriched fraction derived thereof, was shown to be saturable and dose-dependent. In addition, prostaglandin E1 (PGE1) was found to be a potent stimulator of AC activity in these cells. Hormone stimulation of enzyme activity in the intact cell was strongly potentiated by forskolin which not only enhanced maximal AC activity 3 fold, but lowered by 40-fold the concentration of beta-MSH required for half maximal stimulation. Using biologically active [125I]iodo-beta-MSH prepared in our laboratory we have examined the specificity of beta-MSH binding to its receptor in both intact M2R cells and plasma membranes derived thereof. Among a series of hormones tested only alpha-MSH and ACTH1-24 competed with [125I]iodo beta-MSH for binding to the melanotropin receptor in accordance with the results obtained with AC. In contrast to the strong effect on cyclic 3',5'-adenosine monophosphate (cAMP) accumulation in M2R cells forskolin has no effect on [125I]iodo-beta-MSH binding. It appears that the kinetic properties of beta-MSH binding and beta-MSH stimulation of adenylate cyclase activity are essentially identical, the half-maximal effects of which are demonstrated at approximately 20 nM beta-MSH. PMID- 3013704 TI - Increased arachidonic acid level in diabetic platelets following improvement in diabetic control. AB - A group of non-insulin dependent diabetics taking oral hypoglycaemic therapy were studied to determine the effect of improved diabetic control on the platelet levels of the prostaglandin precursor fatty acids. The patients were randomised to receive either a low carbohydrate diet (35% of total energy) for six weeks or a high carbohydrate diet (55% of total energy), high fibre diet for six weeks and the diets were then reversed. At the end of the high carbohydrate, high fibre diet the mean fasting blood glucose (6.6 mmol section 1) and mean percentage glycosylated haemoglobin (8.2%) were significantly lower than after the low carbohydrate diet (7.8. mmol/l, p less than 0.05: 9.9%, p less than 0.01). Mean platelet phospholipid arachidonic acid percentage was significantly higher after the high carbohydrate, high fibre diet (24.2%) than after the low carbohydrate diet (19.9%, p less than 0.01). There were no significant changes in the other platelet fatty acids. PMID- 3013706 TI - Inactivation of peroxidase and glucose oxidase by H2O2 and iodide during in vitro thyroglobulin iodination. AB - Thyroglobulin iodination and thyroxine synthesis in vitro require the presence of peroxidase, H2O2 and iodide. H2O2 is usually continuously generated by glucose oxidase (GO) and glucose. The aim of this study was to investigate whether the two enzymes could possibly be inactivated by a particular concentration of H2O2 or iodide present during incubation. The results revealed that both enzymes were indeed inactivated under two distinct conditions: Lactoperoxidase and thyroid peroxidase were inactivated by modest concentrations of H2O2 accumulating during incubation. Glucose oxidase was inactivated by an oxidized species of iodine or singlet oxygen produced in the catalytic cycle. The results may explain some hitherto unsolved discrepancies between different iodination procedures. Moreover they may have an impact on the regulation of in vivo thyroglobulin iodination and hormone synthesis. PMID- 3013707 TI - Altered pathogenesis in encephalomyocarditis virus (D variant)-infected diabetes susceptible and resistant strains of mice. AB - The D variant of encephalomyocarditis virus (EMCV-D) induces a diabetes mellitus like disease in male SJL/J mice. Other inbred strains, while resistant to the diabetogenic effect, exhibit strikingly different responses to this virus. In these studies, infection of diabetes resistant C3H mice with the D variant produces massive acute pancreatitis with little apparent direct islet cell involvement. This exocrine tropism is not altered when C3H mice with an inherent macrophage defect are infected, and appears to be a gender-specific phenomenon, with female C3H mice resistant to this exocrine involvement. Long-term infection of both male and female C3H mice does not change their response to the virus. Castration of male C3H mice, using a protocol that has been reported to block the diabetogenic effect of this virus, does not alter the development of this acinar lesion. The B variant of EMCV does not induce acinar destruction, nor is it diabetogenic. However, preinfection with the B variant 3 days prior to infection with the D variant does protect against the development of the exocrine lesion. Coinfection with equal doses of the two variants also protects against this lesion, as does coinfection with a lower dose of B variant. Therefore, the host response that is generated against the B variant appears to be responsible for this protection from D variant exocrine destruction. Due to the short time frame, it is unlikely that this protection is the result of an antibody response. Rather, this data is more consistent with an interferon response generated against the B variant that would inhibit replication of the D variant. PMID- 3013709 TI - [Hormone immunocytochemical studies of 46 endocrine tumors of the pancreas in 24 patients]. AB - The aim of this retrospective study was to correlate the results of hormonal immunocytochemistry of 46 endocrine tumors to the corresponding clinical syndromes in 24 patients. They were divided as following: 14 cases of insulinoma, 3 cases of Zollinger-Ellison syndrome, 1 case of glucagonoma, 1 case of carcinoid syndrome and 5 cases without any obvious endocrine manifestations. Each tumor was tested with anti-insulin, anti-glucagon, anti-pancreatic polypeptide, anti vasoactive intestinal peptide, anti-gastrin immune sera according to the peroxidase-antiperoxidase method. The presence of insulin was proved in 13 of 14 cases of insulinomas and the presence of gastrin in 2 of 3 cases of Zollinger Ellison syndrome. Among the 5 asymptomatic cases, a somatostatinoma and a vipoma were individualized. More than 50 p. 100 of the tumors showed plurihormonal secretion with one predominantly secreted hormone responsible for the clinical syndrome. This study demonstrated the diversity of the hormonal secretion by some tumors and their metastasis in the same patient. Malignant insulinomas correspond either to poorly secreting tumors or to plurihormonal tumors secreting gastrin and glucagon as well. PMID- 3013708 TI - Effects of a transplantable insulinoma upon regulatory peptide concentrations in the gastrointestinal tract of the rat. AB - The rapid growth (0.8 +/- 0.3 g/day) of a transplantable insulinoma, which also contained substance P (2.9 +/- 2.3 pmol/g) and gastrin-releasing peptide (3.2 +/- 2.1 pmol/g), resulted in the development of hyperphagia, hyperinsulinaemia and hypoglycaemia in rats (n = 8). After a 14-day growth period, the insulinoma bearing rats showed an increase (49%; p less than 0.01) in the weight of the small intestine but no significant change in stomach weight compared with control animals. The content (pmol/organ) of somatostatin, substance P, neurokinin A and vasoactive intestinal peptide in the stomachs of the tumour rats was unchanged. A depletion in the content (53% p less than 0.01) and concentration (57%; p less than 0.01) of gastrin-releasing peptide, however, suggested either hypersecretion, possibly mediated through hypoglycaemia-induced vagal stimulation, or inhibition of synthesis. The concentration and content of glucagon-like immunoreactivity (enteroglucagon) in the small intestine of the insulinoma rats increased markedly (47%; p less than 0.01 and 120%; p less than 0.01). This increase is consistent with a proposed role of this peptide as a factor trophic to the intestinal mucosa. No significant changes in the concentrations of somatostatin, substance P, neurokinin A, vasoactive intestinal peptide and gastrin-releasing peptide in the small intestine were observed. However, the increase in gut weight resulted in a greater content of vasoactive intestinal peptide (40%; p less than 0.01) and substance P (37%; p less than 0.05) in the insulinoma rats. PMID- 3013710 TI - Effect of dietary cholesterol on biliary cholesterol content and bile flow in the hypothyroid rat. AB - The hypothyroid rat model was used to investigate the effect of dietary-induced hypercholesterolemia on biliary cholesterol content and bile flow. Rats were divided into four dietary groups--diet A: Rat Chow; diet B: Rat Chow plus 0.1% propylthiouracil; diet C: Rat Chow plus 0.1% propylthiouracil, 0.3% taurocholate, 5% lard; diet D: Rat Chow plus 0.1% propylthiouracil, 0.3% taurocholate, 5% lard, and 1% cholesterol. After 6 wk, bile was collected and livers were excised for the preparation of membranes. In cholesterol-fed animals, biliary cholesterol content was increased. However, because of a significant decrease in the rate of bile flow that occurred in these animals, biliary cholesterol output was unchanged from the cholesterol output observed in control animals. Dietary cholesterol also caused a threefold increase in liver membrane cholesterol content and a 64% decrease in the activity of sodium-potassium-stimulated adenosine triphosphatase (Na+,K+-ATPase). In a separate group of animals, microsomes prepared from livers of control rats were incubated with phosphatidylserine liposomes, liposomes containing cholesterol, or buffer. The activity of Na+,K+-ATPase was increased in microsomes incubated with phosphatidylserine liposomes. However, when the cholesterol content of the microsomes was increased twofold by incubating the membranes with liposomes containing cholesterol, the stimulation of Na+,K+-ATPase activity was significantly decreased. The data suggest that in the cholesterol-fed hypothyroid rat, biliary cholesterol content is significantly increased; however, because of a decrease in the rate of bile flow, biliary cholesterol output is not changed. The decrease in bile flow is associated with an accumulation of cholesterol and a decrease in the activity of Na+,K+-ATPase in hepatic membranes. PMID- 3013711 TI - Effect of ethanol on acid secretion by isolated gastric glands from rabbit. AB - Isolated gastric glands from rabbit, as well as basolateral and microsomal membranes derived therefrom, were used to examine the effect of ethanol on several parameters related to acid secretion. Low concentrations of ethanol, 0.2% 5% (vol/vol), had no effect on basal aminopyrine accumulation by isolated gastric glands but significantly potentiated aminopyrine accumulation stimulated by histamine. In contrast, this dose range of ethanol inhibited aminopyrine accumulation stimulated by forskolin or dibutyryl-cyclic adenosine monophosphate. This dose range of ethanol produced a similar effect on adenylate cyclase activity of basolateral membranes from isolated gastric glands, with potentiation of histamine stimulation and inhibition of forskolin stimulation. Low-dose ethanol was found to produce increased proton permeability of the apical membrane of the parietal cell but had no effect on hydrogen-potassium-stimulated adenosine triphosphatase activity. Ethanol (10%) significantly inhibited all parameters of acid secretion studied. Ethanol has a biphasic effect on acid secretion with potentiation of histamine-stimulated aminopyrine accumulation and adenylate cyclase activity at low doses and inhibition of all parameters of acid secretion at high doses. PMID- 3013713 TI - [Role of arachidonic acid and cAMP-dependent systems in thrombocyte aggregation induced by staphylococcal toxin and ADP]. PMID- 3013712 TI - [Clinical significance of human papilloma virus (HPV) infections of the lower genital tract]. AB - Using filter in situ hybridisation for HPV-DNA detection we found among 217 women with positive cervical cytology a positive result in 152 cases (70%). The distribution of the different HPV types showed an association of HPV 6/11 mainly with benign lesions and of HPV 16/18 with obligatory precancer and invasive cervical cancer. In 2652 swabs of cytologically negative patients HPV-DNA was identified in 9.5%. The infection rate for HPV 16/18 in pregnant women was 6.4% against 2.3% in nonpregnant women. In a number of the patients with positive cervical cytology we additionally examined smears from the vagina and vestibulum for HPV-DNA: in 42% of the cases a positive HPV result was obtained in these areas as well. In about 50% of 39 male partners peniscopy revealed penile lesions and HPV-DNA was found in penile smears. A prospective cytological and virological study of cytologically positive patients showed a clear association of HPV 16/18 with progression of cervical lesions. In a cytologically negative group, follow up examinations revealed HPV-DNA in 31%. PMID- 3013714 TI - Nifedipine: more than a calcium channel blocker. AB - Nifedipine exhibits a greater incidence of side effects than the other currently marketed calcium channel antagonists. In addition to those effects attributable to calcium channel blockade, nifedipine produces side effects similar to the effects of adenosine. It is probable that nifedipine exerts part of its physiological actions through potentiation of adenosine. Adenosine, an endogenous calcium channel blocker, modifies synaptic events throughout the nervous system and causes sedation, smooth and skeletal muscle relaxation, anticonvulsion, hypotension and hypothermia, all reversible by caffeine or theophylline administration. Nifedipine inhibits adenosine uptake from, and release into, the extracellular space and binds at an adenosine receptor. Both nifedipine and adenosine interact with benzodiazepine binding sites. Interaction between nifedipine and adenosine should be kept in mind when treating patients with nifedipine. PMID- 3013715 TI - Identification and characterization of human erythrocyte muscarinic receptors. AB - Cholinergic muscarinic but not nicotinic receptors could be detected on the outer surface of the human red blood cell membrane by direct labelled ligand binding. This 3H-QNB binding could be inhibited by atropine. The erythrocyte cholinergic receptor was similar to brain muscarinic receptor in regard to stimulation of cGMP production. PMID- 3013716 TI - Differential effects of antidepressant drugs on [3H]dihydroalprenolol and [3H]imipramine ligand recognition sites in olfactory bulbectomized and sham lesioned rats. AB - In the olfactory bulbectomy rat model of major depression, the binding of [3H]imipramine is increased by 60% in the midbrain, reduced by 30% in the pons and by 20% in the hippocampus, and unchanged in the hypothalamus 6 weeks after the bulbectomy. Binding of [3H]dihydroalprenolol is unchanged in the midbrain but is increased by 30% in the pons and 15% in the hippocampus. The i.p. administration of the antidepressants amitriptyline, mianserin, tranylcypromine (all at a dose of 10 mg/kg) or iprindole (25 mg/kg) for 28 days followed by a 5 day drug washout period, alters brain part [3H]imipramine and [3H]dihydroalprenolol binding in a manner that is a function of the particular drug, brain part and lesion effect. Only in the hippocampus did the lesion increase beta-binding that was reduced by all four antidepressant drugs. PMID- 3013717 TI - Cerebellar cyclic nucleotides and the development of convulsion, with reference to the anticonvulsant activity of diazepam. AB - Dibutyryl cyclic GMP (DbcGMP) or dibutyryl cyclic AMP (DbcAMP) given to rats intracerebellarly in a dose of 200 nmol/head produced electroencephalographic convulsive changes. Intracerebellar (i.c.) administration of lower doses (100 nmol/head) of DbcGMP and DbcAMP and 200 nmol/head of norepinephrine (NE) and glutamate (Glu) facilitated the pentylenetetrazol (PTZ)-induced convulsions. Diazepam (100 nmol/head, i.c.) suppressed the PTZ-induced convulsions. GABA (400 nmol/head, i.c.) did not affect the PTZ-induced convulsions. These results suggest that DbcGMP, DbcAMP and Glu inhibit seizure control mechanisms in the cerebellum, and that one of the sites of the anticonvulsant action of diazepam is located in the cerebellum. PMID- 3013718 TI - The failure of morphine to attenuate spinal cord nociceptive transmission through supraspinal actions in the cat. AB - Morphine sulphate was perfused between the third ventricle and cisterna magna in alpha-chloralose anaesthetized cats while recording from multireceptive dorsal horn neurones activated by peripheral noxious radiant heat. Morphine concentrations of 10(-5) and 10(-4) M were without effect, while 10(-3) M resulted in an increase in the spinal cord neuronal responses. The results do not support the notion that morphine activates a supraspinal mediated descending inhibition which antagonizes spinal cord nociceptive transmission. PMID- 3013719 TI - [Prevalence of antirotavirus antibodies (IgG) in 2 different Venezuelan populations]. PMID- 3013720 TI - Direction of travel of RecBC recombinase through bacteriophage lambda DNA. AB - We examined linkage relationships for RecBC-mediated recombination in lytic cycle crosses of lambda phages bearing two cohesive end sites (cos) oriented in the same direction. The relationships obtained imply that a given recombinant tends to be packaged from the cos site that is the nearer one to the right of the exchange. In view of the previously established coupling of entry of a recombinase at a cos cut and initiation of DNA packaging by that cos cut, these results imply that the recombinase (presumably the RecBC gene product) enters lambda's chromosome at the right end. PMID- 3013721 TI - Genetic and molecular analysis of the GAL3 gene in the expression of the galactose/melibiose regulon of Saccharomyces cerevisiae. AB - During the galactose adaptation period of a Saccharomyces cerevisiae strain bearing a naturally occurring gal3 allele, we found a longer induction lag and slower rate of accumulation of GAL10 and MEL1 RNAs compared to wild-type strains. A strain of genotype gal3 gal1 gal7 is noninducible for MEL1 gene expression, but this expression block is bypassed by overexpression of the GAL4 gene or by deletion of the GAL80 gene, either of which causes a constitutive phenotype. An otherwise wild-type strain that bears a chromosomal gal3 gene disruption mutation does not produce wild-type GAL3 RNA and exhibits induction comparable to a strain bearing the naturally occurring gal3. Based on this array of results, we conclude that the GAL3 gene product executes its function at a point before GAL4 mediated transcription of the GAL1-10-7 and MEL1 genes. Thus, the data are consistent with the previously advanced hypothesis that the GAL3 gene product functions to synthesize the inducer or coinducer molecule. In experiments in which the presence of either the plasmid-carried cloned GAL3 gene or the plasmid-carried cloned GAL1-10-7 genes allows MEL1 induction of a gal3 gal1 gal7 cell, we find that loss of the plasmid results in the shutoff of MEL1 expression even when galactose is continuously present. Either GAL3 function or GAL1-10-7 functions are therefore required for both the initiation and the maintenance of the induced state. Since the strains bearing either the naturally occurring gal3 allele or the gal3 disruption (null) allele do induce, the plasmid loss experiments indicate the existence of two completely independent induction initiation maintenance pathways, one requiring GAL3 function, the other requiring GAL1-10-7 function. Finally, Northern blot analysis reveals two major GAL3 transcripts that differ in size by approximately 500 nucleotides. PMID- 3013722 TI - Suppressors of the ras2 mutation of Saccharomyces cerevisiae. AB - Saccharomyces cerevisiae contains two members of the ras gene family. Strains with disruptions of the RAS2 gene fail to grow efficiently on nonfermentable carbon sources. This growth defect can be suppressed by extragenic mutations called sra. We have isolated 79 independent suppressor mutations, 68 of which have been assigned to one of five loci. Eleven additional dominant mutations have not been assigned to a specific locus. Some sra1 and SRA4 and all SRA3 mutations were RAS independent, allowing growth of yeast cells that lack a functional RAS gene. Mutations in sra1, SRA3, SRA4 and sra6 are linked to his6, ino1, met3 and ade6, respectively. Some sra mutants have pleiotropic phenotypes that affect glycogen accumulation, sporulation, viability, respiratory capacity and suppression of two cell-division-cycle mutations, cdc25 and cdc35. The proposed functions of many of the suppressor genes are consistent with the model in which RAS activates adenylate cyclase. PMID- 3013723 TI - The underlying bases of gene expression differences in stable transformants of the rosy locus in Drosophila melanogaster. AB - This report represents a continuation of our laboratory's effort to understand the major phenomena associated with P-M dysgenesis-mediated transformation in Drosophila. A group of stable transformants are characterized with respect to rosy gene expression. Stable, true-breeding, line-specific variants in gene expression are described. These are shown to be associated with single transposons present in each line, and the lines are free of functional P elements. The effects on expression are cis-acting, and there are no identifiable rosy DNA sequence lesions associated with these transposons. Evidence is presented that demonstrates that two features of the transformation experimental system are responsible for such variation. The first relates to the fact that the transposons insert at numerous genomic sites. Both heterochromatic and euchromatic position effects are characterized. The second relates to the fact that transformation involves dysgenic mobilization of a P-element transposon. This process is mutagenic, and such a mutation is characterized. PMID- 3013724 TI - Southern analysis of genomic alterations in gamma-ray-induced aprt- hamster cell mutants. AB - The role of genomic alterations in mutagenesis induced by ionizing radiation has been the subject of considerable speculation. By Southern blotting analysis we show here that 9 of 55 (approximately 1/6) gamma-ray-induced mutants at the adenine phosphoribosyl transferase (aprt) locus of Chinese hamster ovary (CHO) cells have a detectable genomic rearrangement. These fall into two classes: intragenic deletions and chromosomal rearrangements. In contrast, no major genomic alterations were detected among 67 spontaneous mutants, although two restriction site loss events were observed. Three gamma-ray-induced mutants were found to be intragenic deletions; all may have identical break-points. The remaining six gamma-ray-induced mutants demonstrating a genomic alteration appear to be the result of chromosomal rearrangements, possibly translocation or inversion events. None of the remaining gamma-ray-induced mutants showed any observable alteration in blotting pattern indicating a substantial role for point mutation in gamma-ray-induced mutagenesis at the aprt locus. PMID- 3013726 TI - Intramolecular transposition of IS102. AB - It has been postulated that deletions mediated by transposable elements are intramolecular transposition events. An implication of this hypothesis is that the deleted fragment may be recovered if it is capable of autonomous replication. We report here the characterization of the products of intramolecular transposition of the element IS102 in bireplicons. We show that when two origins (ori's) (of pSC101 and R6-5) generate the same copy numbers, two dissociated replicons are recovered as well as the inversions. On the contrary, when two ori's (of pSC101 and pBR322) have different copy numbers, intramolecular transposition results essentially in inversions. However, the very low frequency (5 X 10(-8)) at which intramolecular transpositions in the bireplicons occurs, as compared to the single replicon (10(-4)), suggests that a complete transposition reaction may not be necessary to generate deletions. PMID- 3013725 TI - Maize mitochondrial plasmid S-1 sequences share homology with chloroplast gene psbA. AB - The linear, 6397-base pair (bp), mitochondrial S-1 DNA molecule from maize contains a 420-bp segment that is homologous with the chloroplast gene (psbA) that codes for the quinone binding protein of photosystem II. This is the first report of a chloroplast sequence in a naturally occurring viral-like or plasmid DNA. The complete sequence of the S-1 chloroplast segment has been compared with homologous regions of six different chloroplast genes. The S-1 segment has diverged from the other genes both by length mutation and base substitution. Several of the length mutations are exact adjacent tandem duplications of 4 and 5 bp similar to "footprints" left after excision of transposable elements in maize nuclear DNA. PMID- 3013727 TI - An improved technique for the efficient construction of gene libraries by partial filling-in of cohesive ends. AB - For the preparation of gene libraries, DNA from lambda EMBL3 phage was digested with SalI and EcoRI, and the cohesive ends partially filled-in by addition of dTTP, dCTP and Klenow fragment of DNA polymerase I (PolIk). Genomic DNA was cleaved partially with Sau3A and subsequently incubated with dATP, dGTP and PolIk. The phage and genomic DNAs were then mixed and ligated. The recombinant DNAs were packaged in vitro. The efficiency of packaging was 10(5)-10(6) of infectious phage lambda particles per microgram of the genomic DNA (as compared to approx. 10(7) per microgram for the wild-type lambda DNA). This procedure is very rapid and requires only microgram quantities of genomic DNA for preparing an entire gene library. The other important advantage is that multiple independent insertions of genomic DNA cannot occur in a single recombinant phage and self ligation of phage DNA is blocked. It is also applicable for other SalI-containing vectors. PMID- 3013728 TI - Cloning and amplified expression in Streptomyces lividans of a gene encoding extracellular beta-lactamase from Streptomyces albus G. AB - A 4.9-kb DNA fragment containing the bla gene for the extracellular beta lactamase (BLA) of Streptomyces albus G was cloned in Streptomyces lividans using the conjugative, low-copy-number plasmid pIJ61 as vector. No expression of bla was observed when this DNA fragment was introduced into Escherichia coli HB101 on a plasmid vector. A 1.5-kb PstI-SstI fragment containing the bla gene was cloned in S. lividans on the nonconjugative, high-copy-number plasmid pIJ702. A tenfold higher yield of BLA was obtained from S. lividans carrying this plasmid than from S. albus G grown under optimal production conditions. The BLA from the clone reacts with beta-iodopenicillanate according to a branched pathway which is characteristic of the original S. albus G BLA enzyme. PMID- 3013729 TI - Cloning and expression of the lepidopteran toxin produced by Bacillus thuringiensis var. thuringiensis in Escherichia coli. AB - The Bacillus thuringiensis var. thuringiensis strain 3A produces a proteinaceous parasporal crystal toxic to larvae of a variety of lepidopteran pests including Spodoptera littoralis (Egyptian cotton leaf worm), Heliothis zeae, H. virescens and Boarmia selenaria. By cloning of individual plasmids of B. thuringiensis in Escherichia coli, we localized a gene coding for the delta-endotoxin on the B. thuringiensis plasmid of about 17 kb designated pTN4. Following partial digestion of the B. thuringiensis plasmid pTN4 and cloning into the E. coli pACYC184 plasmid three clones were isolated in which toxin production was detected. One of these hybrid plasmids pTNG43 carried a 1.7-kb insert that hybridized to the 14-kb BamHI DNA fragments of B. thuringiensis var. thuringiensis strains 3A and berliner 1715. This BamHI DNA fragment of strain berliner 1715 has been shown to contain the gene that codes for the toxic protein of the crystal (Klier et al., 1982). No homologous sequences have been found between pTNG33 and the DNA of B. thuringiensis var. entomocidus strain 24, which exhibited insecticidal activity against S. littoralis similar to that of strain 3A. PMID- 3013730 TI - [Status of laboratory animals after a long-term stay in an organic glass chamber]. PMID- 3013731 TI - [Evaluation of the carcinogenic activity of zeolite and clinoptilolite]. PMID- 3013732 TI - [Working conditions and health status of workers in the synthetic detergent industry]. PMID- 3013733 TI - Evaluation of urinary cytology of male sexual partners of women with cervical intraepithelial neoplasia and human papilloma virus infection. AB - Condylomas of the genital tract can be found in 70% of patients who have cytologic evidence of human papilloma virus (HPV) infection or mild epithelial dysplasia (CIN I). Most male sexual partners of women with overt or subclinical HPV infections have no visible condylomas. Presently, there is no therapy for subclinically infected male partners. A screening test capable of detecting such HPV infections in males would be of value should effective therapy be discovered. Fifty-four men who were the current sexual partners of women with visible condylomata acuminata or with cytologic evidence of subclinical HPV infection or cervical neoplasia were asked to give a urine specimen for cytologic examination. The cytologist had no knowledge of the cytologic or histopathologic findings in the female partners. Of the 54 women, 39 (72%) had either visible genital condylomas or cytologic evidence of mild dysplasia or of HPV infection with or without mild dysplasia (CIN I). Twenty-five (63%) had histologic evidence of HPV infection with or without mild dysplasia. With one exception, urinary cytologic preparations from the study and from the control males were negative. No correlation could be found between cervical cytology and histology in the females and urinary cytologic findings in their current male partners. At the present time there is no screening test that can be utilized to detect male carriers with subclinical disease. PMID- 3013735 TI - [Primary hypoparathyroidism and elevated muscle enzymes]. PMID- 3013734 TI - [Morphology and classification of cleft hands]. AB - It is the intention of this study to present a more profound investigation of the morphology of cleft hands and to provide a new classification based on the results of that investigation. After a short review of the literature, which shows the different opinions regarding heredity, pathogenesis and classification of cleft hand, the authors demonstrate their own patients with 35 cleft hands: The deformities were mostly bilateral and associated with cleft feet. In unilateral cases the right side was more common. Males were in the majority. This paper puts emphasis on the analysis of X-ray morphology. The authors are able to demonstrate that the cleft hand shows several peculiarities which have not been yet sufficiently respected. It was found out, that, apart from aplasia of the bones and soft tissue, synostosis is often the origin of clefting. In 40% of our cases the cleft was caused exclusively by synostosis, in a further 34% it derived partly from synostosis of the phalanges and the metacarpal bones. In the carpus we found osseous deformities surprisingly often, a feature which has hardly been mentioned in former studies. Among the numerous associated malformations emphasis must be placed on the osseous syndactylies and the central polydactylies, because they are closely related to the cleft hand as shown by Ogino. 18 of our own cases belong to this group. Our investigations lead us to the following classification: Cleft hand type 1: Cleft hands with osseous defects (aplasias) Cleft hand type 2: Cleft hands with synostosis Cleft hand type 3: Cleft hands with aplasias and synostosis Hands with central polydactyly and synostosis as preforms of the cleft hand could be classified in type 4. These phenomena form the beginning of the teratological row towards the completely developed cleft hand. In combination with Blauth's distribution of cleft hands, who distinguished the median and medio lateral form (1976, 1978) this new classification enables each cleft hand to be placed into one of the different types, which are analysed: Type 1 mostly shows a medio-lateral form, is always combined with cleft feet and shows heredity in 50% of the cases. It cannot be classified by the Ogino method. Type 2 mostly shows a median form, is not frequently combined with cleft feet, heredity occurs in one third of the cases. This type can very often be classified by the Ogino method. Type 3 varies from case to case because of the different items of defects.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3013736 TI - [Malignant fibrous histiocytoma of bone]. PMID- 3013737 TI - [Pharmacological study of nicergoline. (I): Protective effect against anoxic brain damages in animals]. AB - The protective effects of nicergoline (NCG) against anoxic brain damages in animals were compared with those of dihydroergotoxine (DHE) and phentolamine (PTA). NCG (16 mg/kg, i.p.) prolonged the survival time of mice under hypobaric hypoxia, but DHE and PTA shortened the time. NCG (1-16 mg/kg, i.p. and 16-64 mg/kg, p.o.), like DHE, dose-dependently prolonged the survival time of mice after a lethal dose of KCN (3 mg/kg, i.v.), but PTA did not. NCG (8-128 micrograms/kg, i.v.), like DHE, dose-dependently protected against disappearance of EEG of rats in histotoxic anoxia induced by a sublethal dose of KCN (1.5 mg/kg, i.v.), but PTA did not. Its protective effect was 10 times or more stronger than that of DHE. NCG (1-16 mg/kg, i.p.) dose-dependently promoted recovery from behavioral disorders and disturbance of cerebral energy metabolism of mice in histotoxic anoxia induced by a sublethal dose of KCN (1.8 mg/kg, i.v.). NCG (100 microM), like DHE, showed antagonistic action against inhibition of cerebral cytochrome oxidase activity by KCN, but PTA did not. The ED50 values of NCG, DHE and PTA for the protective effect against adrenaline-induced death in mice were 1.18, 0.27 and 0.35 mg/kg (i.p.), respectively. 7) These results suggest that NCG may show protective effects against the anoxic brain damages due to its ameliorating action on cerebral energy metabolism, mainly contributed by an activation of cerebral cytochrome oxidase, without relation to its alpha blocking action. PMID- 3013738 TI - [Neurological complications and therapy in herpesvirus diseases]. AB - An overview on the diversity of neurologic complications of infections with different human herpes virus strains is given. Encephalitis, meningoencephalitis and the Guillain-Barre-syndrome are of major importance. Affection of single cranial nerves and mononeuropathies occur in a lesser frequency, while myelitis and isolated disturbances of the autonomic nervous system are rare complications. Therapeutically the application of acyclovir in herpes simplex and varicella zoster virus infections has given encouraging outlooks, whereas no convincing results exist with respect to cytomegalovirus and Epstein-Barr virus infections. PMID- 3013740 TI - A rare source of occult gastrointestinal bleeding: jejunal filiae secondary to metastatic lung carcinoma. AB - A case of occult gastrointestinal bleeding due to jejunal metastases of a primary lung carcinoma in a 53-year-old man is reported. When after healing of a large gastric ulcer melena persisted, a subsequently performed double contrast enema of the small bowel revealed evidence of several jejunal tumors. This was confirmed by angiography of the superior mesenteric artery and computed tomography of the abdomen. After resection of the tumor-bearing jejunal loop, histological evaluation revealed metastases secondary to a large-cell bronchogenic carcinoma which had been resected 1 year previously. PMID- 3013739 TI - Diagnosis, treatment and prognosis of small hepatocellular carcinoma. AB - Diagnosis, treatment and prognosis of hepatocellular carcinoma (HCC) of small size, not larger than 5 cm in diameter, were studied in forty-three patients with underlying cirrhosis, who were detected among one-hundred-and-sixty-five HCC cases over a period of 4.5 years from 1981 to 1985. The patients included fifteen cases with tumors smaller than 3 cm in diameter which were diagnosed HCC mostly during the follow-up period of liver cirrhosis. Among various imaging procedures, real-time linear scan ultrasonography (US) had a 91% positive HCC detection rate, hepatic angiography 93% and computed tomography (CT) 88%. Surgical treatment including partial resection, subsegmentectomy and segmentectomy, was carried out in fifteen HCC cases with well-compensated cirrhosis. Transcatheter arterial embolization (TAE) was performed in nineteen cases with severe liver dysfunction and multiple location of tumors. Three-year survival was 80% in twelve patients with hepatic resection (performed since 1981) and 19% in the TAE cases; none of the other cases survived. PMID- 3013741 TI - [Hormone-producing pituitary adenoma]. PMID- 3013742 TI - Biological, functional and morphological characteristics of human bone and soft tissue tumors in nude mice. AB - Since 1975, we have performed experiments on transplantation of bone and soft tissue tumors obtained, from 115 patients into nude mouse. In the present study, we analyzed biological, functional and morphological characteristics of the tumors transplanted in nude mice. The incidence of tumor take in the initial transplantation was 55.6% (40 of 72 cases) for malignant bone tumors, 42.9% (15 of 35 cases) for malignant soft tissue tumors and 0% for benign bone and soft tissue tumors. In particular, the incidence of tumor take was high for osteosarcoma (60%, 36 of 60 cases) and for rhabdomyosarcoma (70%, 7 of 10 cases). There were 22 tumors transplanted serially for more than 5 passages. The mean survival time for mice with serially transplantable bone and soft tissue tumors was 76.0 days and mean tumor growth index was 0.25. Serum LDH levels were as high as more than 5,000 Wrob. U for 8 of 12 lines tested. Serum ALP levels were normal for all tumors other than osteosarcoma. In mice transplanted with osteosarcoma, serum ALP levels were higher than 10 KAU for 8 of 13 lines tested. Pulmonary metastasis was seen in 5 of 9 osteosarcomas examined. For many of serially transplantable tumors, tumorigenicity was observed in nude mice by various transplantation routes including the subcutaneous, intraperitoneal and intravenous routes via the tail vain. Serially transplantable osteosarcomas were morphologically classified into three groups based on osteoid formation; those in which osteoid formation disappeared immediately, those in which osteoid formation decreased gradually, and those in which osteoid formation was unchanged or even increased. Osteoid was found for 6 of 13 lines examined. For 4 of the 6 tumors zone phenomenon was maintained during passages. For 2 lines among those in which osteoid disappeared, histological type was changed into quite another type from the original type of the tumor. PMID- 3013743 TI - Discordances in the temporal pattern of the ACTH/beta-endorphin releasing effects of synthetic CRFs and partially purified bovine CRF in a superfusion system. AB - Temporal characteristics of ACTH and beta-endorphin secretion induced by bovine hypothalamic CRF-A (void volume) and CRF-B (Kav = 0.583) separated by Sephadex G 100 were compared to those of synthetic ovine or rat CRF, sauvagine and vasopressin. Dispersed cells or minced fragments of rat adenohypophyses perifused in a column were exposed to various secretagogues, and ACTH and/or beta-endorphin concentrations of the effluent were measured by radioimmunoassays. CRF-A or CRF-B induced an immediate brisk rise of ACTH and beta-endorphin within 1 min after initiation of CRF perifusion. The maximum rate of ACTH or beta-endorphin secretion was reached 1-2 min later. Hormone secretion persisted throughout a 10 min exposure to these secretagogues. More than 80% of the total ACTH or beta endorphin secretion induced by 10-min perifusion with bovine CRF occurred during exposure to CRF. With 10-min perifusion with 300 ng/ml ovine or rat CRF, the onset of the major CRF-stimulated ACTH or beta-endorphin secretion was markedly delayed compared to that following bovine CRF. During perifusion with ovine or rat CRF, a modest slow increase in ACTH or beta-endorphin secretion was observed. More than 60-70% of the total ACTH or beta-endorphin secretion induced by 10-min perifusion with rat or ovine CRF occurred after CRF withdrawal. The ACTH secretory patterns for sauvagine were very similar to those for synthetic rat or ovine CRF. These results suggest some qualitative differences between partially purified bovine CRF and synthetic ovine or rat CRF. PMID- 3013744 TI - Subcellular localization and binding of 125I-triiodothyronine in calf thyroid. AB - The subcellular distribution of 125I-T3 was studied in calf thyroid slices, under the same experimental conditions where T3 inhibits protein and RNA synthesis, labelled hormone was found mainly in the 20,000 X g supernatant. The specificity of each subcellular localization was determined by incubating the slices with 10( 5)M T3. Only in the purified nuclei a significant decrease was found, indicating a specific localization of the labelled hormone. When slices were incubated with 125I both labelled T3 and T4 were found in purified nuclei, indicating that endogenously synthesized hormones can reach thyroid nuclei. Purified thyroid nuclei were incubated with labelled T3 and increasing amounts of cold hormone. Specific binding reached a plateau after 90 min of incubation at 20 degrees C. When the displacement curves were analysed by a Scatchard plot a binding site with a Ka of 5.2 X 10(7) M-1 and a capacity of 3.0 X 10(-15) moles/microgram DNA was observed. Digestion of nuclei with trypsin and protease abolished completely the binding of 125I-T3 thus indicating the protein nature of the receptor. The hormone-receptor complex could be extracted with 0.4M KCI and eluted in the void volume after Sephadex G-25 column chromatography, similar to peripheral tissues nuclear T3 receptors. The present studies provide the first evidence for the existence of nuclear receptors for T3 in the thyroid, an event probably related to the autoregulatory mechanism. PMID- 3013745 TI - Indirect evidence of impairment of platelet desaturase enzymes in diabetes mellitus. AB - We measured the platelet total phospholipid fatty acid profiles of 20 insulin treated (Type I) diabetics, 20 non-insulin treated (Type II) diabetics and 20 matched non-diabetic controls to determine the relationship between the omega 6 and omega 3 series of fatty acids in diabetes. A significant inverse correlation between linoleic acid and arachidonic acid occurred in the normal subjects (r = 0.61; P less than 0.001) but was not seen in the Type I diabetics (r = -0.13; P = NS) or in the Type II diabetics (r = -0.27; P = NS). No significant correlation was seen between linolenic acid and eicosapentaenoic acid in the normal controls (r = -0.34; P = NS) or in the Type I diabetics (r = 0.21; P = NS) or in the Type II diabetics (r = -0.20; P = NS). The results suggest that a functional impairment of platelet delta 5 and delta 6 desaturase may occur in diabetes which disrupts the normal equilibrium between linoleic acid and arachidonic acid. However, the level of eicosapentaenoic acid appears to be less dependent on conversion from linolenic acid. Our findings are of importance to studies designed to reduce platelet aggregation in diabetics and non-diabetics by manipulation of the levels of the precursor fatty acids of thromboxane. PMID- 3013746 TI - Cellular heterogeneity in lung cancer. AB - Sixty-six lung carcinomas have been examined by light and electron microscopy, as well as by immunocytochemical techniques using a panel of monoclonal antibodies. There was considerable heterogeneity with regard to cell type and in only 18 cases was it possible to classify the tumour as a solely small cell, squamous or adenocarcinoma. In the remaining cases there was evidence of two or three cell types. These findings support the thesis that all lung cancers are derived from a pluripotential basal or reserve cell in the bronchial mucosa which may proliferate along one or more lines of differentiation. This view of the histogenesis of lung cancer would account for the heterogeneous appearance of many tumours and the difficulty experienced in placing them in one of the standard classifications. PMID- 3013747 TI - Sclerosing haemangiomas of the lung. AB - A series of 29 pulmonary sclerosing haemangiomas is analysed. Included is one case with multiple tumours and another with metastatic growth in a hilar lymph node. Histochemical and electron microscopic studies show that type 2 pneumocytes are an important constituent cell type though bronchial structures, tumorlets and angiomas also occur within these tumours. They are considered to be pulmonary hamartomas formed from distal lung structures. They are of slow growth and have been confused in the past with pulmonary histiocytomas and plasma cell granulomas. PMID- 3013748 TI - Ultrastructural and immunocytochemical definition of component neoplasms in an unusual gastro-oesophageal collision tumour. AB - An unusual collision tumour (concrescence of two neighbouring independent neoplasms) is reported. One tumour was a small cell undifferentiated (oat cell) carcinoma of the lower oesophagus and the other was a gastro-oesophageal adenocarcinoma. There was little intermingling of the two patterns. The adenocarcinoma stained strongly positive for mucin and carcinoembryonic antigen (CEA), but the small cell carcinoma was negative for both and also argyrophil negative; both were negative for neurone-specific enolase and common leukocyte antigen. Ultrastructural study showed extra-cellular glandular lumina lined by cells with apical microvilli and junctional complexes in the adenocarcinoma; primitive cells without tonofilaments or dense-core granules, and joined by rudimentary desmosomes were seen in the small cell carcinoma. Collision carcinomas may result from a carcinogenic stimulus affecting two neighbouring regions of mucosa or may simply be the chance apposition of two unrelated tumours. PMID- 3013749 TI - Benign sclerosing 'haemangioma' of the lung. PMID- 3013750 TI - Affective disorders and violence in adolescents. AB - Many adolescent affective disorders are rooted in biological vulnerability to stress and a predisposition to mood variations that are latent in childhood. As these stress-sensitive children encounter the normative helplessness and struggle for autonomy of adolescence, they may use violence, explosive rage, self starvation, grandiose self-idealization, drug abuse, and suicidal behavior to relieve psychological helplessness and tension. They may begin a lifelong cycle of failure, disruption, and rejection by family and schools. The author describes adolescent depression and its biological underpinnings, deprivation syndromes, and manic-depressive illness, as well as the concept of affective violence in organic affective syndromes and episodic dyscontrol syndromes. These disorders call for multimodal treatment in which appropriate medication facilitates psychosocial intervention. PMID- 3013751 TI - Intramitochondrial lamellar bodies in acute myeloblastic leukemia. AB - Intramitochondrial lamellar bodies were observed in three cases of acute myeloblastic leukemia. Two of the patients had M1 leukemia and the remaining patient M4 leukemia, by the FAB classification. In all three cases neoplastic cells contained dilated mitochondria that varied in size and shape and contained decreased numbers of cristae. Some mitochondria contained lamellar structures that resembled myelin figures and, occasionally, primary granules; these structures were more conspicuous in the central portion of the mitochondria. Regardless of the proliferating cell type (lymphoblasts, myeloblasts, or monoblasts), there are common ultrastructural changes that represent abnormal metabolic function, such as disorders of intramitochondrial protein synthesis. The exact meaning of these findings is not known; adequate interpretation will require further investigation of the biology of these neoplastic processes. PMID- 3013752 TI - Congenital cytomegalovirus infection presenting as massive ascites with secondary pulmonary hypoplasia. PMID- 3013753 TI - Human DNA sequences isolated with an immunoglobulin switch region probe: sequence, chromosomal localization, and restriction fragment length polymorphisms. AB - We have screened a human genomic DNA library with an immunoglobulin (Ig) derived switch (S) region specific probe for homologous sequences. Five Ig independent phage clones were isolated and characterized. The S sequence homologous DNA fragments are short compared to the S region sequences. Ig independent S sequences are flanked by highly repetitive DNA elements and perfect inverted repeats can be demonstrated in their close vicinity. Using subclones of S homologous sequences restriction fragment length polymorphisms were shown within DNA of different T cell leukemias, Burkitt lymphomas, lymphoblastoid cell lines, and DNA of healthy individuals. One of the five clones isolated with the S region probe was evidently localized to chromosome 2 and/or 10 and showed a complex hybridisation pattern with several different human DNAs. S homologous sequences of another clone are most likely localized on chromosome 1. It is possible that these Ig independent S sequences have arisen by amplification and transposition and that they are involved in genetic recombination. PMID- 3013754 TI - Restriction fragment length polymorphisms associated with immunoglobulin heavy chain gamma genes in Tunisians. AB - DNA polymorphisms in the human immunoglobulin gamma (gamma) region have been studied in random Arabo-Berber Tunisians and in a large Tunisian Berber kindred. Haplotypes have then been designated, based on variation in the BamHI restriction fragments containing the C gamma 1, C gamma 2, C gamma 4, and C psi gamma genes. Two new haplotypes, in addition to the four previously described, have been observed. These new haplotypes, designated H5 and H6, were confirmed by family studies. The H5 haplotype was associated with black African Gm haplotypes (Gm1,17;..;5,6,11 and Gm1,17;..;5,11) (Gma,z;..;blc3bo and Gma,z;..;blbo) and probably represents a common haplotype in the black population. The haplotype H6 may be derived from H5. One of 39 random Tunisians was homozygous for a multigene deletion. DNA polymorphisms of the C gamma genes, in conjunction with Gm markers, provide highly variable genetic markers important for the characterization of human populations. PMID- 3013756 TI - Use of catalase polymorphisms in the study of sporadic aniridia. AB - Catalase is known to map at chromosome 11p13. It is one of the closest known markers to the WAGR locus. Restriction fragment length polymorphisms (RFLP) of the catalase gene may be invaluable for studying rearrangements in somatic tumours, linkage in cases of familial Wilms tumour, and the relationship between sporadic and familial aniridia. We describe a catalase RFLP with two different enzymes and use these polymorphisms to exclude deletion of the catalase gene in patients with sporadic aniridia, including one who is known to have a deletion and another suspected of having a deletion. PMID- 3013755 TI - Bloom's syndrome. XIII. DNA-polymerase activity of cultured lymphoblastoid cells. AB - The biochemical defect in Bloom's syndrome (BS) remains unknown, but two characteristic features of BS cells point to a disturbance of DNA replication, namely, an excessive number of sister-chromatid exchanges (SCEs) in bromodeoxyuridine (BrdU)-substituted cells and an abnormally slow rate of replicon elongation. The hypothesis of an abnormal DNA polymerase as the explanation for these observations was tested using an in situ assay system for DNA polymerase activity and to estimate molecular weights in cellular extracts of cultured BS cells. DNA polymerase subunits in cellular extracts from the BS cells when separated electrophoretically on polyacrylamide gels showed the same mobilities (i.e., molecular weights) as the controls and were equally effective at promoting the incorporation of isotopically labeled nucleosides. It is concluded that the genetic defect in BS has no direct effect on either DNA polymerase activity or the amounts and molecular weights of the different forms of the enzyme. PMID- 3013758 TI - A rapid and sensitive culture test for the laboratory diagnosis of genital herpes in women. AB - A rapid and sensitive cell culture test has been developed to detect herpes simplex virus (HSV) in women with genital herpes. The virus is cultured by inoculation and centrifugation of cell monolayers, and the virus inclusions are detected using an indirect immunofluorescence test. The test takes only 48 hours to complete compared with the conventional cell culture test, which may take up to eight days. Of a total of 2100 cervical specimens collected from unselected women attending a sexually transmitted diseases (STD) clinic and inoculated in parallel, HSV was isolated from 55 specimens by either or both tests. Of these 55 positive specimens, 54(98%) were positive by the rapid test but only 24(44%) by the conventional test (McNemars test; p less than 0.001). PMID- 3013757 TI - DNA-diagnosis of sickle cell anemia from chorionic villi: possible influence of maternal cell contamination. AB - The admixture of maternal tissue is a possible hazard of prenatal diagnosis from chorionic villi. Therefore the tolerable degree of maternal DNA contamination in prenatal diagnosis of sickle cell anemia was investigated by mixing various amounts of DNA from HbS carriers with DNA from normal individuals and sickle cell anemia patients. The results indicate that lower rate of admixed maternal DNA does not prevent an exact direct DNA-diagnosis of sickle cell anemia. PMID- 3013759 TI - An asymmetric two electrode cuff for generation of unidirectionally propagated action potentials. PMID- 3013760 TI - Cytotoxic factor produced by human blood lymphocytes of low density upon exposure to autologous B cells. AB - The mechanism of cytolysis by natural killer cells has been shown to involve a soluble lytic factor - the natural killer cytotoxic factor (NKCF). We have investigated the generation of NKCF on exposure of subpopulations of human blood lymphocytes to autologous B lymphocytes and EBV infected B lymphocytes (BEBV), and the sensitivity of these lymphocytes to the cytotoxic factor. We present evidence that the low density lymphocytes representing majority of NK cells, can be stimulated to produce NKCF by the autologous B/BEBV cells. Allogeneic B/BEBV cells did not induce NKCF production. Recognition of the autologous B/BEBV cells did not seem to involve Dr antigens. Although the autologous B/BEBV cells could stimulate NKCF production, the NKCF was lytic only to K562 targets. The autologous B/BEBV cells could neither be lysed by the factor produced, nor could these cells absorb the factor. PMID- 3013761 TI - Epstein-Barr virus-transformed B cells can present acetylcholine receptor to autologous autoreactive T cells. AB - Epstein-Barr virus- (EBV-)transformed B cells were obtained from a patient with myasthenia gravis, who was also the donor of a T cell line specific for acetylcholine receptor (AChR). After three months of culture, the EBV-transformed B cells could effectively present native membrane-bound AChR to autologous AChR specific T cells. PMID- 3013762 TI - A region flanking H-2K is duplicated to a distant site in most mouse t haplotypes. AB - Mouse t haplotypes contain at least one inversion, which encompasses the major histocompatibility complex, relative to their wild-type counterparts. A DNA probe for a single copy sequence which flanks the H-2K region in inbred strains was found to have undergone further rearrangements in the t haplotypes. In most t haplotypes, this sequence is duplicated at a distant site, and the two regions show 1% recombination. The length of homology shared by the two sites is likely to be at least 10-15 kb. Three different alleles, as defined by restriction fragment length polymorphisms, were found for each of the two sites among different t haplotypes. These may reveal evolutionary relationships among these chromosomes. PMID- 3013763 TI - Polymorphisms within the HLA-DR3 haplotypes. I. HLA-DR polymorphisms detected at the protein and DNA levels are reflected by T-cell recognition. AB - HLA-DR molecules were isolated from eight different HLA-DR3 homozygous B-cell lines by immunoprecipitation with monoclonal antibodies, and they were subsequently analyzed by two-dimensional gel electrophoresis. We found that HLA DR3 homozygous B-cell lines of consanguineous origin express two types of HLA-DR molecules. One type of HLA-DR molecule was present in all the cell lines tested, whereas the second DR molecule appears to be polymorphic. DNA isolated from the different HLA-DR3 homozygous cell lines was studied by Southern blot analysis to determine whether any DR beta restriction fragment length polymorphism could be observed. Polymorphisms detected at both the product and genomic level have been compared to each other, and their relations to the serological (HLA-DR) and cellular (HLA-D and LB-Q1) typing data will be discussed. PMID- 3013764 TI - The murine lymphocyte differentiation antigen MALA-1 is associated with the Ly-6 complex. PMID- 3013765 TI - Role of alpha 2-adrenoceptors of nucleus ambiguus & lateral medullary pressor area in hypotensive & bradycardiac effects of clonidine. PMID- 3013766 TI - Is low-dose hydrochlorothiazide effective? AB - In a double-blind crossover study, 13 patients with pretreatment diastolic blood pressure between 95 and 109 mm Hg received nadolol, 80 mg/day, plus placebo of hydrochlorothiazide and nadolol, plus three different doses of active hydrochlorothiazide. Patients remained on each active regimen for 3 weeks, with an intervening placebo period of 2 to 4 weeks. With 12.5 mg of hydrochlorothiazide daily plus nadolol, there was no greater reduction of blood pressure than with nadolol alone. A dose of 25 mg of hydrochlorothiazide was associated with a significantly greater decrease in systolic but not diastolic pressure, as compared with nadolol alone. A significantly greater reduction in both systolic and diastolic blood pressure was obtained only with the 50 mg/day dose of hydrochlorothiazide. Extension to 6 weeks of treatment with 12.5 mg/day failed to lower the blood pressure more than the level seen at 3 weeks. These results suggest that in combination with nadolol, 12.5 mg of hydrochlorothiazide per day has no significant antihypertensive effect. There was no evidence of a flat dose-response curve in the daily dose range of 12.5 to 50 mg. For most patients, a dose of 50 mg of hydrochlorothiazide was required to lower both systolic and diastolic blood pressure significantly below the level obtained with nadolol alone. PMID- 3013768 TI - Preliminary observations on abnormalities of membrane structure and function in essential hypertension. AB - To test the hypothesis that structural abnormalities exist in the cell membrane in persons with essential hypertension and that these abnormalities affect membrane-related cellular functions, we examined several membrane-dependent phenomena and membrane lipid composition in the blood cells of subjects with essential hypertension. We analyzed platelet aggregability, membrane fluidity, membrane fatty acid composition, and erythrocyte deformability in four normolipidemic subjects with untreated essential hypertension and in five age matched normotensive controls. As compared with the controls, the subjects with essential hypertension had platelets that aggregated at lower concentrations of adenosine 5'-diphosphate, platelet membranes that were less fluid, and erythrocytes that were more deformable. Lipid analysis of the membranes of platelets from the two study groups showed that although the cholesterol content was identical, the membranes from the essential hypertension group contained significantly less linoleic acid (18:2) than did those from the normotensive controls. Given the known effects of cis-unsaturated fatty acyl composition on membrane fluidity and membrane-related cellular functions, these data suggest that one factor contributing to essential hypertension is an inherent structural membrane abnormality that alters the physical and functional properties of the cell membrane. PMID- 3013767 TI - Role of inositol lipid breakdown in the generation of intracellular signals. State of the art lecture. AB - Many hormones, neurotransmitters, and secretagogues act by increasing the intracellular free Ca2+ concentration in target cells. The initial event following binding of agonists to specific receptors in the plasma membrane involves a receptor-mediated activation of a guanosine nucleotide-binding protein (G protein), which induces a Ca2+-independent activation of phospholipase C. This novel, presently uncharacterized G protein is inactivated by pertussis toxin catalyzed adenosine 5'-diphosphate ribosylation in some but not all cell types. Phospholipase C catalyzes the breakdown of inositol lipids, notably phosphatidylinositol 4,5-bisphosphate, with the production of inositol phosphates and 1,2-diacylglycerol. Inositol 1,4,5-trisphosphate (IP3) is responsible for a rapid mobilization of intracellular Ca2+ by activating Ca2+ efflux from a subpopulation of the endoplasmic reticulum. The properties of this process are consistent with its being a ligand-activated ion channel with electrogenic Ca2+ efflux being charge-compensated by K+ influx. Sustained hormonal responses require extracellular Ca2+ and a prolonged elevation of the cytosolic free Ca2+. This is brought about by hormone-mediated changes of Ca2+ flux across the plasma membrane involving both an inhibition of Ca2+ efflux and an activation of Ca2+ influx. This review summarizes recent findings concerning the role of G proteins in receptor coupling to phospholipase C; the regulation of enzymes of phosphoinositide metabolism; the evidence for IP3 being a Ca2+-mobilizing second messenger and its mechanism of action; the formation of new inositol phosphates and their possible significance; the relation of intracellular Ca2+ mobilization and plasma membrane Ca2+ fluxes to the kinetics of the hormone-induced cytosolic free Ca2+ transient; and the possible roles of protein kinase C in influencing the hormone-mediated functional response. PMID- 3013769 TI - Cardiovascular hypertrophy in hypertension. Arthur C. Corcoran Memorial Lecture. PMID- 3013770 TI - High salt diet sensitizes cardiopulmonary baroreflexes in Dahl salt-resistant rats. AB - Compared with Dahl salt-resistant (R) rats, Dahl salt-sensitive (S) rats on a low salt diet have impaired cardiopulmonary baroreflex control of sympathetic nerve activity. The purpose of this study was to examine the sensitivity of cardiopulmonary baroreflex function in both strains of Dahl rats when they are challenged with a high salt diet. We studied Dahl R and S rats after 6 weeks of low and high salt diets. To assess cardiopulmonary baroreflex function, we measured decreases in splanchnic sympathetic nerve activity produced by increases in left ventricular end-diastolic pressure during graded volume expansion (dextran 75) after bilateral sinoaortic denervation. A given amount of infused volume produced comparable increases in left ventricular end-diastolic pressure in Dahl R rats on low and high salt diets but significantly greater decreases in sympathetic nerve activity in the high salt group than in the low salt group. Thus, the high salt diet augmented the sympathoinhibitory response to volume expansion in the Dahl R rats. In contrast, among the Dahl S rats equivalent increases in left ventricular end-diastolic pressure during volume expansion produced smaller sympathoinhibitory responses in the high salt group than in the low salt group. The gain of the cardiopulmonary baroreflex, expressed as the percentage decrease in sympathetic nerve activity per mm Hg increase in left ventricular end-diastolic pressure, was significantly increased by a high salt diet in Dahl R rats but tended to be decreased by a high salt diet in Dahl S rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013771 TI - Biological actions of leukotrienes. State of the art lecture. AB - Leukotrienes are novel mediators derived from arachidonic acid through the 5 lipoxygenase enzyme system. Leukotriene B4 has potent effects on leukocyte function and in vivo induces leukocyte accumulation and changes in vascular permeability and modulates pain responses. Peptido-lipid leukotrienes are potent smooth muscle--contracting agents. They may have important cardiovascular actions through mechanisms involving either vasoconstriction or indirect vasodilatation. Evidence for leukotriene production has been found in subjects with allergic conditions and psoriasis, indicating a putative role for these substances in human disease. PMID- 3013772 TI - Platelet binding sites for atrial natriuretic factor in humans. Characterization and effects of sodium intake. AB - Platelets bear receptors for vasoactive peptides such as angiotensin II and vasopressin. The presence of binding sites for another vasoactive peptide, atrial natriuretic factor, was therefore investigated in human platelets. 125I-labeled synthetic atrial natriuretic factor bound specifically to human platelets. Steady state and kinetic experiments demonstrated the existence of one class of high affinity low-capacity binding sites for atrial natriuretic factor in platelets with a dissociation constant of 30 pM. The order of potency of atrial natriuretic factor fragments showed that the structural requirements of the high-affinity binding site detected were similar to those of receptors for atrial natriuretic factor in rat blood vessels and adrenal zona glomerulosa. To study the regulation of these binding sites by sodium, normal young men were subjected successively in random order to a low-sodium (40 mmol per day) and high-sodium (300 mmol per day) diet for 4 days. Binding of atrial natriuretic factor to platelets was higher with the low-sodium diet (10.3 +/- 1.0 sites per cell) than with the high-sodium diet (7.1 +/- 0.7 sites per cell). In conclusion, human platelets bear binding sites for atrial natriuretic factor, the density of which may be modulated by sodium intake. The platelet is a useful model for investigating atrial natriuretic factor receptors in different physiopathological conditions in humans. PMID- 3013773 TI - Significance of the vascular renin-angiotensin pathway. PMID- 3013774 TI - Vascular renin-angiotensin system in two-kidney, one clip hypertensive rats. AB - The possible role of the renin-angiotensin system in the maintenance of hypertension in two-kidney, one clip hypertensive rats was studied. Plasma renin activity rose rapidly and markedly in association with the elevation of blood pressure and then decreased gradually, although blood pressure remained high. Renin activity in the lung, aorta, and mesenteric artery also increased with the development of hypertension and then decreased in a way similar to that of plasma renin activity at the chronic stage of hypertension. Plasma angiotensin converting enzyme activity did not change significantly until 16 weeks after unilateral renal artery clipping, whereas vascular angiotensin converting enzyme activity significantly increased at the chronic, but not the acute, stage of hypertension. In chronically renal hypertensive rats, 1-sarcosine, 8-isoleucine angiotensin II or enalapril, an angiotensin converting enzyme inhibitor, lowered the blood pressure and enalapril also lowered the angiotensin converting enzyme activity of vascular tissues. The constrictor effect of angiotensin I was greater in isolated arteries from chronically hypertensive rats than in those from age matched normotensive rats. These results suggest that the vascular renin angiotensin system plays an important role in the maintenance of two-kidney, one clip hypertension. Elevated vascular angiotensin converting enzyme activity appears to increase local production of angiotensin II, which results in vasoconstriction by acting directly and indirectly through adrenergic nerves on vascular smooth muscle. PMID- 3013775 TI - Up-regulation of renal prostaglandin receptors in genetic salt-dependent hypertension. AB - Development of hypertension in Dahl salt-sensitive rats (DS) is accompanied by reduced renomedullary prostaglandin synthesis, which may be responsible for their lower natriuretic capacity. To examine the changes in renomedullary prostaglandin E2 synthesis, the effects of high (8.0%) and normal (0.6%) NaCl diets were examined in DS and in Dahl salt-resistant rats (DR). In response to an 8.0% NaCl diet, the number of prostaglandin E2 receptors in the renal outer medulla of DR increased (2.97 +/- 0.2 vs 2.18 +/- 0.2 pmol/mg on 0.6% NaCl diet) while no change was noted in their affinities (Kd, 9.5 +/- 0.2 vs 9.4 +/- 0.3 nM). Receptor number and affinity in the renal cortex, inner medulla, and liver of DR were not affected. In contrast, renomedullary receptors of DS had a lower affinity than those of age-matched DR (Kd, 13.9 +/- 0.2 nM on 0.6% NaCl diet and 14.0 +/- 0.3 nM on 8.0% NaCl diet) and did not increase in number after a high salt diet. This apparent inability of DS to modulate prostaglandin receptors may contribute to their susceptibility to salt-induced hypertension. PMID- 3013776 TI - Monoclonal antibodies against human renin. Blood pressure effects in the marmoset. AB - The in vivo effects of two anti-human renin monoclonal antibodies with a high binding affinity for primate renin were studied in conscious, volume-depleted marmosets. These antibodies, R-3-17-7 and R-3-36-16, both have high binding activity for renin, but only R-3-36-16 inhibits the enzymatic activity of renin in vitro. In vivo, R-3-17-7 did not affect blood pressure after intravenous injection of doses up to 100 micrograms/kg, although plasma renin activity was partially reduced. In contrast, R-3-36-16 induced a reduction in blood pressure and an inhibition of plasma renin activity at a threshold dose of 3 micrograms/kg. The maximum fall in blood pressure and complete inhibition of plasma renin activity were observed after R-3-36-16, 10 micrograms/kg; these effects persisted for up to 2 hours. Pretreatment with a converting enzyme inhibitor or nephrectomy prevented the hypotensive effects of R-3-36-16. Conversely, pretreatment with R-3-36-16 prevented the hypotensive effects of a converting enzyme inhibitor. These findings indicate that the hypotensive response induced by R-3-36-16 is due entirely to blockade of the renin angiotensin system. Thus, R-3-36-16 appears to be a specific, potent, and long acting inhibitor of primate renin. Such monoclonal antibodies provide interesting tools for studying the effects of acute and chronic renin blockade. PMID- 3013777 TI - Plasma digitalislike activity in essential hypertension or end-stage renal disease. AB - Plasma extracts from 119 subjects showed a digitalislike activity, as evidenced by the ability of these extracts to inhibit ouabain binding to the Na+-K+ pump. High levels of the digitalislike compound were found in 18 of 54 untreated hypertensive subjects, 7 of 21 normotensive subjects with a family history of hypertension, and 10 of 14 patients with end-stage renal failure. Dialysis significantly reduced the activity of this compound. These results suggest 1) that endogenous digitalislike factor is not directly linked to hypertension but rather is related to sodium balance and 2) that it neither originates nor is activated by renal tissue, as it was present in four of six anephric patients. PMID- 3013778 TI - Oxidative and nonoxidative killing of Actinobacillus actinomycetemcomitans by human neutrophils. AB - Actinobacillus actinomycetemcomitans is a facultative gram-negative microorganism which has been implicated as an etiologic agent in localized juvenile periodontitis and in subacute bacterial endocarditis and abscesses. Although resistant to serum bactericidal action and to oxidant injury mediated by superoxide anion (O2-) and hydrogen peroxide (H2O2), this organism is sensitive to killing by the myeloperoxidase-hydrogen peroxide-chloride system (K.T. Miyasaki, M.E. Wilson, and R.J. Genco, Infect. Immun. 53:161-165, 1986). In this study, we examined the sensitivity of A. actinomycetemcomitans to killing by intact neutrophils under aerobic conditions, under anaerobic conditions, and under aerobic conditions in the presence of the heme-protein inhibitor sodium cyanide. Intact neutrophils killed opsonized A. actinomycetemcomitans under aerobic and anaerobic conditions, and the kinetics of these reactions indicated that both oxidative and nonoxidative mechanisms were operative. Oxidative mechanisms contributed significantly, and most of the killing attributable to oxidative mechanisms was inhibited by sodium cyanide, which suggested that the myeloperoxidase-hydrogen peroxide-chloride system participated in the oxidative process. We conclude that human neutrophils are capable of killing A. actinomycetemcomitans by both oxygen-dependent and oxygen-independent pathways, and that most oxygen-dependent killing requires myeloperoxidase activity. PMID- 3013781 TI - A rapid, flexible method for biochemical assays using a microtiter plate reader and a microcomputer. Application for assays of protein, Na,K-ATPase and K-p nitrophenylphosphatase. AB - A computerized system which greatly accelerates and eases the collection, storage, and analysis of data has been applied to several standard biochemical assays. The system uses a commercially available microtiter plate reader connected to an Apple IIe microcomputer via a standard serial port. Data are transmitted automatically from the reader to the microcomputer, where they can be viewed, printed, further analyzed immediately, or stored on a diskette for later retrieval and processing. Some or all of the data may be entered manually. The program calculates a linear least squares best fit to a standard curve after correcting all data for blanks, then determines the quantities of substrate or product contained in each well of a microtiter plate. Data from two plates may be combined, enabling calculation of enzyme specific activities. This system can be adapted to any assay whose final step can be performed by a microtiter plate reader. Its use is described for determination of protein concentration, Na,K ATPase activity, and K-stimulated p-nitrophenylphosphatase activity. PMID- 3013779 TI - Killing of Actinobacillus actinomycetemcomitans by the human neutrophil myeloperoxidase-hydrogen peroxide-chloride system. AB - Actinobacillus actinomycetemcomitans is a facultative gram-negative coccobacillus associated with periodontal disease and nonoral infections. This organism is resistant to serum bactericidal mechanisms but is nevertheless killed by human neutrophils under aerobic and anaerobic conditions. Most of the killing attributable to oxidative mechanisms is inhibited by sodium cyanide, which suggests that the myeloperoxidase-hydrogen peroxide-chloride (MPO-H2O2-Cl-) system may be a key factor in the oxidative killing process. In this report, we examine whether the isolated MPO-H2O2-Cl- system is bactericidal against A. actinomycetemcomitans. We found that three major chromatographic forms of MPO were capable of killing A. actinomycetemcomitans at sublethal concentrations of H2O2 and that both catalase-positive and catalase-negative strains of this organism were sensitive to killing by the MPO-H2O2-Cl- system. We conclude that the isolated MPO-H2O2-Cl- system is bactericidal for A. actinomycetemcomitans independent of other neutrophil granule constituents and may be an important component of the oxygen-dependent bactericidal activity of the neutrophil with respect to this periodontopathic organism. PMID- 3013782 TI - Resistance of line 6(3) chickens to reticuloendotheliosis-virus-induced bursa associated lymphomas. AB - Chickens of lines 6(3) and 151(5) X 7(1) were inoculated with the chick syncytial strain of reticuloendotheliosis virus (REV) or with the Rous-associated virus-I of avian leukosis virus (ALV) at hatching. At 4, 10, 16, and 36 weeks post inoculation (PI), chickens were tested for REV- and ALV-induced viremia and antibody. The incidence of REV- or ALV-induced bursa-associated lymphomas in line 6(3) chickens was compared with that in line 151(5) X 7(1) chickens. Inoculation of REV at hatching resulted in immunological tolerance to the virus in line 6(3) but not in line 151(5) X 7(1) chickens. Between 70% and 100% of line 6(3) chickens remained viremic and lacked REV antibody throughout the experimental period of 36 weeks. In contrast, ALV-inoculated chickens of both lines had antibody by 16 weeks PI. The frequencies of REV- and ALV-induced bursa-associated lymphomas in line 6(3) chickens were significantly lower than in line 151(5) X 7(1) chickens. Further, the incidence of bursa-associated lymphomas induced by REV in line 151(5) X 7(1) chickens was significantly lower than that induced by ALV. These results suggest that: (1) the genetic constitution of the host may influence the immunological response to REV infection; (2) chickens resistant to ALV-induced bursa-associated lymphomas are equally resistant to such lymphomas induced by another unrelated avian retrovirus, REV; and (3) ALV is a more potent inducer of bursa-associated lymphomas than REV. PMID- 3013780 TI - Purification and characterization of an enzyme produced by Treponema denticola capable of hydrolyzing synthetic trypsin substrates. AB - An enzyme from Treponema denticola that hydrolyzes a synthetic trypsin substrate, N-alpha-benzoyl-L-arginine-p-nitroanilide (BAPNA), was purified to near homogeneity, as judged by gel electrophoresis. The molecular weight of the enzyme was estimated to be ca. 69,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ca. 50,000 by gel filtration on Sephadex G-100. The pH optimum for the hydrolysis of BAPNA was around 8.5. The enzyme was heat labile and irreversibly inactivated at low pH values. Enzyme activity was enhanced by Ca2+, Mg2+, and Ba2+ but inhibited by Mn2+, Hg2+, Co2+, and Zn2+. Metal chelators and sulfhydryl reagents had no effect on this activity. The enzyme was inhibited by certain protease inhibitors such as diisopropyl fluorophosphate, N-alpha-p tosyl-L-lysine chloromethyl ketone, phenylmethylsulfonyl fluoride, L-1-tosylamide 2-phenylethylchloromethyl ketone, alpha-1-antitrypsin, and soybean trypsin inhibitor. The Km values for BAPNA and N-alpha-benzoyl-L-arginine ethyl ester were 0.05 and 0.12 mM, respectively, and the Vmax values were higher than those observed with trypsin. Although the purified enzyme hydrolyzed some low-molecular weight synthetic trypsin substrates, it did not hydrolyze casein, hemoglobin, azocasein, azocoll, bovine serum albumin, or gelatin. Thus, this enzyme is probably not a protease but is capable of hydrolyzing ester, amide, and peptide bonds involving the carboxyl group of arginine and lysine. PMID- 3013783 TI - Small-cell lung carcinoma: a systemic disease. PMID- 3013784 TI - Studies in healthy human T-cell-leukemia lymphoma virus (HTLV-I) carriers from the Caribbean. AB - Six healthy relatives of 3 adult T-cell leukemia lymphoma (ATLL) patients and 6 members of a Caribbean family immigrant to the UK have been investigated for the presence of HTLV-I and expression of interleukin 2 (IL-2) receptors. Serum antibodies to HTLV-I were detected in all but 4 samples. Four to 10% of circulating cells from 3/4 seropositive donors studied displayed IL-2 receptors (anti-Tac+) and were shown to be convoluted lymphocytes by light microscopy morphology and immunoelectronmicroscopy. After 5 to 28 days in culture, cells from 4 seropositive donors reacted with monoclonal antibodies (MAbs) against the HTLV-I core proteins, p19 and p24, and released retrovirus particles. Similar experiments with blood from 3 seronegative donors from the same families and 4 normal controls proved negative. Our findings indicate that seropositive individuals harbour the virus in a population of T-lymphocytes which may then acquire receptors for IL-2. These individuals are at risk of developing ATLL. PMID- 3013785 TI - Increased incidence of IgA antibodies to the Epstein-Barr virus-associated viral capsid antigen and early antigens in patients with chronic lymphocytic leukemia. AB - Antibody titers to Epstein-Barr virus (EBV)-associated early antigens (EA) and the viral capsid antigen (VCA) were determined by ELISA on 263 sera obtained from healthy donors, patients with Hodgkin's disease (HD), non-Hodgkin lymphomas (NHL), infectious mononucleosis (IM), Burkitt's lymphoma (BL), and nasopharyngeal carcinoma (NPC). As expected, most lymphoma patients showed markedly elevated anti-VCA IgG and anti-EA IgG antibody titers. Only one patient in the NHL group (n = 56) consisting of patients with lymphomas other than chronic lymphocytic leukemia (CLL) and hairy-cell leukemia (HCL), and 3 patients with HCL (n = 19) had high antibody titers of the IgA class to VCA and EA. Seventeen out of 48 patients (36%) with CLL had high IgA anti-VCA titers and 10 of these sera (21%) also contained IgA anti-EA. The geometric mean titer (GMT) of IgA anti-VCA was 2,510, the GMT of IgA anti-EA was 780. These antibody titers were about 10 times lower than the corresponding GMT of the NPC patients investigated in this study. The elevated IgG and IgA antibody titers to VCA and EA in CLL and HCL patients seem to reflect an immunodeficiency secondary to the malignant disease leading to reactivation of latent EBV infection. The possibility that at least some of these B-cell lymphomas are associated with EBV cannot be excluded. PMID- 3013786 TI - Post-exposure treatment with monoclonal antibodies in a retrovirus system: failure to protect cats against feline leukemia virus infection with virus neutralizing monoclonal antibodies. AB - We have attempted to protect kittens against oronasal infection with FeLV (strain A/Glasgow-1) by treatment with a mixture of two virus-neutralizing (VN) MAbs (IgGl, K) directed against the same epitope on the viral glycoprotein gp70. Ten SPF 9-week-old kittens were infected on day 0 with 10(6) ffu FeLV and subsequently inoculated i.m. with MAbs every 2 days over a 20-day period at different times after infection. The results clearly show that no protection was achieved. It is unlikely that the amount of VN antibodies, the mode and route of their application or the infectious dose of FeLV used can account for the failure to protect cats against infection. Other possibilities which may explain the lack of protective effect are that the restricted epitope specificity of the MAb preparation used may have led to selection of neutralization-resistant virus mutants, or that other mechanisms than virus neutralization (complement-mediated lysis, antibody-dependent cell cytotoxicity), that may be involved in protection, function less efficiently with MAb. However, in the light of our finding that an early anti-idiotypic response is observed in all cats following administration of the MAb preparation, the rapid clearance of anti-FeLV MAb from the circulation is a more likely explanation. The data presented support our hypothesis that by administration of MAb-as compared to polyclonal antibody-a more vigorous anti idiotypic response is elicited due to the presentation of only a limited set of idiotopes. This potential drawback of rapid clearance of MAbs as a consequence of an anti-idiotypic response might be overcome by the use of mixtures of MAbs resulting in a more heterogeneous set of idiotypic determinants. PMID- 3013787 TI - The usefulness of an endomyocardial biopsy in heart disease of unknown etiology. AB - Light-, electron microscopic and enzyme histochemical examinations (phosphorylase, LDH, NADH:TR, SDH and 3-HBDH) were performed on endomyocardial biopsies of 26 patients with heart diseases of unknown etiology. On the basis of the clinical findings the patients were grouped into hypertrophic cardiomyopathy patients), dilated-congestive cardiomyopathy (8 patients), latent cardiomyopathy and small vessel disease (11 patients) and myocarditis (4 patients). Morphologic changes which might characterize the pathogenesis, were found in 7 patients: small vessel disease in 3 patients, nonspecific myocarditis in 1, iron storage disease in 1, adriamycin cardiomyopathy in 1 and cardiomyopathy with inclusions typical of Fabry's disease in 1 patient. In the other patients the morphologic changes were not sufficiently characteristic to be indicative of an etiopathogenesis. Several pathologic alterations did, nonetheless, appear to have a certain prognostic value such as endocardial and interstitial fibrosis, myofibrillolysis, myolysis, mitochondrial degeneration and increased lipid content in the muscle fibers. The frequency of these changes was evaluated partly semiquantitatively, partly by means of the point-counting method and graded with 1-3 points. Three patients with congestive cardiomyopathy scored at least 7 points. Two of them died within 8 weeks, 1 patient with adriamycin cardiomyopathy recovered after discontinuation of the therapy but he died 4 years after the biopsy. Six to 50 months after the biopsy (mean 31.5, median 6.5) the score was less than 7 in the other patients and all these patients were still alive. The histochemical changes manifested as an increase and/or a decrease of the enzymatic activities, involving scattered muscle fibers or their segments. A decrease of the activities of all dehydrogenases examined appeared to be prognostically ominous, correlating with a score of 7 or higher. A decrease of SDH activity in 7 cases, in combination with a decrease of the HBDH activity in 4 of them, was indicative of a disturbance in the Krebs cycle and lipid metabolism in the absence of ischemic damage. The alterations in the phosphorylase activity did not, however, appear to have a prognostic significance. Normal activity of the phosphorylase seemed to be prognostically favorable. PMID- 3013788 TI - Paget's disease of the nipple. PMID- 3013789 TI - Dapsone-induced peripheral neuropathy. AB - A young man with dermatitis herpetiformis developed fatigue and neurologic complaints 4 years after he began oral dapsone therapy. Neurologic examination and nerve conduction studies confirmed the presence of a combined motor and sensory peripheral neuropathy. The symptomatic improvement reported by the patient was supported by improvement in the nerve conduction studies after cessation of dapsone therapy. Substitution of sulfapyridine did not adversely affect the resolution of his neuropathy. PMID- 3013790 TI - Ovarian fibroma with Leydig cell hyperplasia of the adjacent stroma: a light and electron microscopic study. AB - A virilizing left ovarian tumor removed from a 58-year-old woman was studied by light and electron microscopy. Histologically, the tumor was an ovarian fibroma around which Leydig cells, but no Sertoli cells, proliferated at a distance from the hilus. Although the fibroma itself did not contain Leydig cells, several Leydig cells were observed intermingled with ovarian stromal cells in the cortical tissue compressed by the fibroma. Ultrastructurally, in addition to mature Leydig cells with typical steroid-producing--cell features, immature cells with less-developed smooth endoplasmic reticulum and elementary tubular inclusions were identified. These light and electron microscopic findings suggest that the Leydig cells may have differentiated from ovarian stromal cells surrounding the fibroma. If so, this case should be distinguished from neoplastic disorders such as hilar cell tumors or stromal-Leydig cell tumors and be classified in the category of ovarian tumors with functioning stroma containing Leydig cells. PMID- 3013791 TI - Seasonal variation in food intake, pattern of physical activity and change in body weight in a group of young adult Dutch women consuming self-selected diets. AB - The effect of season on the energy balance was examined in 114 young adult Dutch women consuming self-selected diets. Energy intakes and patterns of physical activity were assessed monthly 14 times with the 24-h recall method. After this period of 14 months, in the second year the same estimates were made with intervals of 2-3 months to check if the seasonal variations observed were not accidental. The study did not demonstrate seasonal variations in the mean energy intake of the group under study. However, the intake of fat appeared to be higher in the winter and spring than in the summer and autumn, whereas for the intake of mono- and di-saccharides the reverse seemed to be true. The intake of dietary fibre was higher in the autumn than in the summer, with intermediate values for winter and spring. Small seasonal fluctuations were observed in body weight and time spent on various physical activities. On the one hand, these fluctuations are too small to indicate physiological significance, on the other hand they are wide enough to be taken into account in the design of many longitudinal studies on the relation between diet and disease. PMID- 3013792 TI - Defective post-replication recovery and u.v. sensitivity in a simian virus 40 transformed Indian muntjac cell line. AB - The responses to u.v. of two cell lines derived from the Indian muntjac are described. The u.v. sensitivity of the diploid cell falls within the range of most normal mammalian cells while the other, a heteroploid cell, transformed by SV40, is much more sensitive to killing. This hypersensitivity cannot be explained by defective excision repair: the two cell types are indistinguishable in this activity as judged by inhibitor-associated DNA break accumulation and unscheduled DNA synthesis. Rather, the SV40 transformed cells have a pronounced inability to recover normal DNA replication after u.v. These cells are, therefore, defective in a post-replication recovery mechanism and in this respect resemble the behaviour of the variant form of xeroderma pigmentosum. Their limited ability to recover normal levels of RNA synthesis after u.v. hints at the complexity of the phenotype. PMID- 3013793 TI - Chromosome aberrations induced in human lymphocytes by 8.7 MeV protons and 23.5 MeV helium-3 ions. AB - This paper describes the irradiation of thin samples of blood with 8.7 MeV protons and 23.5 MeV helium-3 ions in the track segment mode. Chromosome aberrations in human lymphocytes have been scored. The relationship between dicentric yield and dose in Gy was Y = 0.044 D + 0.058 D2 for protons and Y = 0.394 D for helium ions. These results are compared with data from other laboratories using protons and an attempt is made to reconcile differences. An unexpected observation was that the ratio of the linear coefficients for helium ions and protons was about 9 whereas the ratio of the l.e.t. values was 4.5. This disagrees with current theory which predicts that the linear coefficients should be proportional to l.e.t. Possible sources of error in our experiments are discussed but do not adequately account for the discrepancies. PMID- 3013794 TI - Aftercare residential facility for ex-psychiatric patients--the Australian and American perspectives. AB - The decline in the number of inpatients in psychiatric hospitals over the last decade has resulted in a strong demand for community based mental health services, and a search for alternative accommodation for ex-psychiatric patients. However, for any comprehensive planning for residential services for ex psychiatric patients to be effective, there must be a recognition of the variety of patient needs and resources and the range of service models available. This paper attempts to review aftercare residential facilities in America and Australia, to identify the underlying care philosophy and to propose various supervision approaches. PMID- 3013795 TI - Membrane receptors for retinol-binding protein in cultured human retinal pigment epithelium. AB - The retinal pigment epithelium (RPE) is responsible for transport of retinol from the choroidal circulation to the photoreceptors. In the intact eye, this process is mediated by membrane receptors for plasma retinol binding protein (RBP) distributed basolaterally on the RPE cells. We have shown that cultured human RPE expresses this receptor. A binding curve exhibiting saturation was generated by incubating enzymatically detached epithelial sheets with increasing concentrations of 125I-labelled RBP. 125I-RBP binding experiments also show that the receptor is expressed at a high level in first passage subcultures, suggesting de novo synthesis, and that basally oriented receptors predominate over those associated with the apical surface, reflecting the polarization characteristic of RPE in vivo. Cultured RPE can internalize 3H-retinol carried by RBP, resulting in synthesis of labelled retinyl palmitate. Production of labelled retinyl ester is competitively inhibited when incubations include an excess of holo-RBP containing non-radioactive retinol. These results indicate that RBP not only binds to the receptor specifically, but also that this interaction is functional, effecting uptake of retinol by the RPE cells. The expression of this property of differentiated RPE favors the use of cultured RPE as a model system for studying vitamin A transport and metabolism. PMID- 3013796 TI - Acceleration of scrapie disease in mice by an adenovirus. AB - Coinfected mice were examined for a possible interaction between the scrapie agent and an adenovirus. A low titer (10(2) TCD50) of mouse adenovirus (MAdV) caused a significant acceleration of clinical signs of scrapie in mice infected 128 days previously with scrapie. In this experiment, the coinfected mice died 19 days earlier than mice infected with scrapie alone. When a higher titer of MAdV (10(4)-10(5) PFU) was used, a more drastic acceleration of scrapie disease was seen in mice infected 85 and 110 days previously with scrapie. At 85 days, coinfection caused mice to die 37 days earlier than mice infected with scrapie alone, whereas at 110 days, coinfection caused mice to die 52 days earlier than mice infected with scrapie alone. MAdV alone caused no clinical disease in normal mice. The brains of coinfected mice and mice that had been infected with scrapie alone showed a histopathology consistent with scrapie. A possible explanation for these findings is that the replication of the scrapie agent is accelerated by adenovirus. Defective parvoviruses are known to be helped by adenoviruses. Spleens from coinfected mice but not from mice infected with MAdV alone yielded, in cultures of BALB 3T3 cells, infectious MAdV and one or two smaller agents with the dimension and shape of a parvovirus. PMID- 3013797 TI - VC11: an actinophage virulent to Streptomyces cattleya and Streptomyces olivaceus. AB - Five soil samples were screened for the presence of a virulent actinophage. Phage VC11 was found to be virulent on Streptomyces olivaceus, S. cattleya, S. chartreusis, S. griseus (all important beta-lactam antibiotic producers), S. ambofaciens, S. parvulus, S. alboflavus, S. aureofaciens, and S. lividans TC10. Although restriction-modification systems have been observed in S. olivaceus and S. cattleya, the phage EOP on these hosts remained relatively constant, indicating that these systems do not affect VC11. PMID- 3013798 TI - Isolation and characterization of a tupaia rhabdovirus. AB - Liver tumor biopsies of a 9-year-old moribund Tupaia (tree shrew) were explanted and cultured in vitro. The cell cultures degenerated spontaneously. A virus was isolated from the cell-free supernatant of these cultures and subsequent electron microscopy revealed rhabdovirus-like particles. Negative staining showed typical bullet-shaped particles 125-220 nm in length with a diameter of 68 nm studded with a dense layer of surface projections 9-11 nm in length. One end of the virion was flat, the other end was open; a distinct ribonucleoprotein (RNP) core was visible. The pitch of the RNP was 4.5 nm. Virus was assembled and matured by budding primarily into regions of dilated endoplasmic reticulum. The dimensions of the virion also were determined in ultrathin sections: the diameter and length of the virion were 52 and 125-255 nm, respectively, those of the RNP core were 39 and 120-240 nm. Only tupaia embryonic fibroblasts and kidney cells were susceptible to the rhabdovirus. The virus, when plaque-assayed on tupaia embryonic fibroblasts, grew to a titer of 1 X 10(6) PFU. PMID- 3013799 TI - Junin virus isolation from lympho-mononuclear cells of patients with Argentine hemorrhagic fever. AB - Detection of viremia was attempted by three different methods in 30 cases of Argentine hemorrhagic fever (AHF). Cocultivation of peripheral blood mononuclear cells (PBMC) with Vero cell monolayers was the most sensitive, detecting Junin virus (JV) in 96% of the cases. Inoculation of whole blood into suckling mice and on Vero cells rendered 53 and 46% of positive isolations, respectively. The results presented suggest that PBMC are infected with JV during the acute period of AHF. JV was isolated with decreasing frequency up to 3 days after treatment with immune plasma, but no virus was recovered from PBMC during early convalescence. PMID- 3013800 TI - Therapeutic trials of multiple sclerosis and intrathecal IgG production. AB - In our clinical trial we have compared the effect of high doses of prednisone, ACTH alone or ACTH and cyclophosphamide on plasma and CSF albumin and IgG levels. We have found that the treatment with high doses of prednisone is more effective in the suppression of CNS IgG synthesis than ACTH, or cyclophosphamide. However the significance of this immunological phenomenon in the pathogenesis of MS is a very complex problem, since we have not observed any correlation between the depression of the intrathecal IgG synthesis and clinical results of MS treatment. PMID- 3013802 TI - Malignant duodenocolic fistula. A case report. AB - Malignant duodenocolic fistula is a rare condition in which radical surgical treatment is seldom possible. A personal case treated by pancreaticoduodenectomy is presented with a review of the literature. PMID- 3013803 TI - Femoral neuropathy from iliac muscle hematoma complicating anticoagulant therapy: a surgical emergency. Report of a case. AB - A case of femoral neuropathy from iliac muscle hematoma occurring during heparin administration, successfully treated by early surgical decompression is reported. Following the evaluation of anatomy, possible pathogenesis and CT scanning findings, the role of early diagnosis is emphasized in the assessment of prognosis and for successful operative decompression. PMID- 3013804 TI - [False-positive results in the detection of anti-LAV/HTLV-III antibodies by enzyme immunoassay]. AB - Two groups of sera were investigated with two different enzyme immunoassays for the detection of anti-LAV/HTLV-III-antibodies: 50 sera collected from patients with lupus erythematosus and 5 sera containing anti-HLA-DR4 antibodies. Of the 50 sera of the lupus erythematosus patients, 2 were positive in each of the tests. Of the 5 sera that contained anti-HLA-DR4 antibodies, one was positive. In addition, 11 sera showed positive results after heat treatment (56 degrees C, 1 h) in one of the tests. However, all sera in this group gave negative results in the other test. The positive results could not be confirmed with the immunoblot. These findings show that one must be aware of false-positive results when the sera of certain types of patients are investigated in anti-LAV/HTLV-III enzyme immunoassays. The sera of patients with lupus erythematosus, as well as that of polytransfused persons or multiparous women, that have been sensitized with HLA DR4 antigens; heat treatment in one test has an effect on sera. PMID- 3013805 TI - Accuracy of frozen-section diagnosis in salivary gland neoplasms. AB - A retrospective review of 100 patients with major or minor salivary gland neoplasms was conducted to ascertain the accuracy and effect on therapy of frozen section diagnosis. Of these patients, 23% had malignant and 77% benign neoplasms. Twelve patients benefited by further surgery during the initial operation, and no treatment delay occurred as a result of frozen-section diagnosis. There were four incorrect diagnoses of clinical significance, two false positives (benign tumor called malignant on frozen section) and two false negatives (malignant tumor called benign on frozen section). The accuracy of frozen section for specific pathologic diagnosis was 92%. No unnecessary radical surgery was performed. Frozen-section diagnosis of salivary gland neoplasms in our institution was found to be accurate and useful. PMID- 3013806 TI - [Familial type of hypophyseal nanism. Apropos of 7 families in western Algeria]. AB - Eighteen pituitary dwarfs belonging to 7 different West Algerian families were studied. Eleven patients from 4 families presented isolated growth hormone deficiency, 7 patients from 3 families had multiple pituitary hormone deficiencies. Serum GH levels before and after standard pharmacological stimulations were below 2 ng/ml in all cases. Three of 10 hGH treated patients increased significantly their growth rate (8.5 +/- 0.5 cm/year) during the first year of treatment; growth was moderate (5.3 +/- 0.8 cm/year) in 3 patients and poor (4.5 cm/year) in 2 patients. In 2 cases the follow-up is insufficient. PMID- 3013807 TI - Phosphatase cytochemistry with cerium as trapping agent. Verification of acid phosphatase and glucose-6-phosphatase reactive sites. AB - Lead is prevalently replaced by cerium as trapping agent in phosphatase cytochemistry to prevent non-specific precipitation. Recently, substrate specific but artefactual lead precipitates have been described in the nuclear envelope (NE) and rough endoplasmic reticulum (RER) due to a local matrix effect. In the present study a verification was carried out of the localization of acid phosphatase and glucose-6-phosphatase in the NE and RER of rat peritoneal macrophages and hepatocytes respectively with cerium. It appeared that precipitates of cerium phosphate in NE and RER of peritoneal macrophages do not represent sites of acid phosphatase activity but are due to the matrix effect. However, in rat hepatocytes these organelles demonstrate true reactive sites for glucose-6-phosphatase. PMID- 3013801 TI - Antiepileptic drug toxicity: definition and mechanism of action. AB - A detailed review of the adverse reactions of anticonvulsants is given, focusing on the definitions of drug toxicity, sources of information, patterns of drug utilization, pharmacokinetic variables and different mechanisms of action. The information available in the literature provides a wide spectrum of drug toxicity with no attempt at a practical definition of the reported events. This favors uncertainty among practising physicians, who are led to use the individual items with different attitudes. Suggestions are given for the evaluation, prevention and treatment of anticonvulsant drug toxicity. PMID- 3013808 TI - Avidin-HRP conjugates in biotin-avidin immunoenzyme cytochemistry. AB - Avidin-HRP conjugates were prepared, analysed and tested for avidin-biotin immunocytochemistry. Suitable biotinylation of enzymes, antigens and antibody was obtained by reacting biotin at equimolar ratio to epsilon aminogroups in proteins. The avidin-biotin interaction was used for immunocytochemical detection of phenomena in the field of immunology, i.e. immune complex trapping, specific antibody forming cells and in serology for the cytochemical detection of human auto-antibodies to basement membrane components. Avidin-HRP conjugation using the two step glutaraldehyde method gave a very small amount of monomeric, low molecular weight conjugate with excellent performance. Avidin-HRP conjugation using the periodate method was modified at two points. The first modification concerns the molar ratio of avidin to HRP in the reaction mixture which was brought to about equimolarity. The second modification concerns the periodate concentration which was decreased five fold, ten fold and twenty fold. Decreasing the periodate concentration decreased the amount of polymeric conjugate. Optimal amounts of monomeric, low molecular weight conjugate were obtained with a ten fold decrease of the periodate concentration. Comparable cytochemical results were obtained with monomeric conjugates obtained using both preparation methods. PMID- 3013810 TI - Dissimilar responsiveness of cultured corticotrophs and melanotrophs to tripeptide aldehydes. AB - Cultured cells from adult rat anterior pituitaries or intermediate lobes were treated with the proteinase inhibitor tripeptide aldehydes BOC-DPhe-Pro-Arg-H (Boc-fPRH) and DPhe-Pro-Arg-H (fPRH), ovine corticotropin-releasing factor (oCRF), and bromocriptine. One millimolar fPRH stimulated basal, and slightly enhanced oCRF-induced ACTH release by melanotrophs in short-term experiments. The basal release of alpha-MSH was also stimulated by the drug. In long-term experiments, fPRH elevated markedly both the release and the intracellular level of ACTH; BOC-fPRH caused an increased alpha-MSH release. Tritiated fPRH had no preference for POMC-producing cells and BOC-fPRH or fPRH were harmless to the cell morphology. In anterior pituitary cell cultures, fPRH diminished slightly basal and oCRF-induced ACTH release. Bromocriptine was ineffective on corticotrophs, however, in melanotrophs it inhibited ACTH release markedly with or without fPRH in the medium. The dissimilar responsiveness of the corticotrophs and melanotrophs to the peptide aldehydes may be interpreted in terms of their differing membrane receptors or intracellular mechanism of stimulus-secretion coupling. PMID- 3013809 TI - Superoxide production by polymorphonuclear leukocytes. A cytochemical approach. AB - Phagocytosis by polymorphonuclear leukocytes triggers a burst of oxidative metabolism resulting in hydrogen peroxide and superoxide production, and these active oxygen species function in the killing of microorganisms. A new cytochemical technique, based on a manganese dependent diaminobenzidine oxidation, has been developed to detect superoxide in these cells. It has been shown that superoxide generation is associated with the plasma membrane in cells activated by particulate (zymosan) and non-particulate (phorbol myristate acetate) stimuli. This membrane activity is maintained during invagination such that reduced oxygen is generated within the endocytic vacuoles. Reaction product is absent from unstimulated cells; additionally, formation of precipitate is blocked by omission of Mn++, low temperature, glutaraldehyde prefixation, and the presence of superoxide dismutase in the incubation medium. PMID- 3013811 TI - Ultrastructural ultracytochemical investigation of human gastric carcinomas. AB - We describe the ultrastructure of various types of gastric carcinoma cells as well as their histochemical properties as visualized at the electron-microscope level. The histochemical properties of tumour cells were compared with those of homologous normal epithelial cells. The localization and activity of ATPase, IDPase, acidic phosphatase and alkaline phosphatase as well as of the oxidoreductases (cytochrome oxidase, succinate dehydrogenase and NADH dehydrogenase) were studied. Our findings demonstrated that, in tumour cells, a complicated process of structural-functional restructuring takes place. It seems that a number of ultracytochemical properties may be preserved or may disappear altogether; also, such properties may become enhanced or weaker. This heterogeneity of the histochemical properties of tumour cells is discussed with regard to the role of the stem (polypotent) cell in the process of the histogenesis (cytogenesis) of human gastric carcinomas. PMID- 3013812 TI - Gastric K+-stimulated p-nitrophenylphosphatase cytochemistry. AB - A cytochemical study of gastric K+-stimulated p-nitrophenylphosphatase (K-NPPase) activity, corresponding to a K+-stimulated phosphoprotein phosphatase of H-K ATPase system, has been made by a new cytochemical method. Sections of fixed guinea pig gastric mucosa in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde, were incubated with the incubation medium (1.0 M glycine-0.1 M KOH buffer, pH 9.0, 2.5 ml; 1.1 M KCl, 0.5 ml; 10 mM lead citrate dissolved in 50 mM KOH, 4 ml; levamisole, 6.0 mg; dimethyl sulfoxide, 2.0 ml; 0.1 M p nitrophenylphosphate (Mg-salt), 1.0 ml; ouabain, 73.0 mg) for 30 min at room temperature. Under a light microscope the specific gastric K-NPPase reaction was distributed only in the parietal cells of the fundic glands. The electron microscopic cytochemistry showed that the gastric K-NPPase activity was localized on the membrane lining the apical surfaces, secretory canaliculi and tubulovesicles. On the other hand, ouabain-sensitive K-NPPase activity (Na-K ATPase) was demonstrated to localize only in the basolateral membrane of parietal cells with Mayahara's method. These findings support the interrelationships between the apical surface membrane, secretory canalicular membrane and tubulovesicles, and the functional differentiation of the membrane between the secretory membrane and basolateral membrane. PMID- 3013814 TI - The proliferative immune response to autologous Epstein-Barr virus transformed lymphoblastoid cells. II. Studies with HLA class II loss variants demonstrate a role for gene products other than DR and DQ. AB - The Epstein-Barr virus (EBV) transformed lymphoblastoid cell line (LCL-721) and some of its HLA loss mutant derivatives were used to study the immune specificity of the autologous proliferative T cell response to antigens expressed as a result of EBV infection. We have measured secondary and tertiary proliferative responses to well-characterized variants that lack expression of some or all known class II gene products (DR, DQ, and DP). These experiments prove that the region mapping between DR/DQ and glyoxalase I (GLO) of one haplotype controls at least one specific restriction element which is recognized in the autologous response to LCL-721. Furthermore, specific proliferative responses to variants lacking expression of all known class II gene products indicate the recognition of determinants other than DR, DQ, and DP. PMID- 3013813 TI - Use of DNA probes from the 5' flanking region of the HLA-B gene to examine polymorphism at the HLA-B locus. AB - Two DNA probes isolated from the region 5' to the HLA-B gene are described. One of these, pCH6, detects a TaqI polymorphism which is correlated with serological alleles of HLA-B. Both probes are used to show a high level of DNA sequence homology between the 5' flanking region of HLA-B and HLA-C. PMID- 3013815 TI - Southern hybridization analysis of DNA polymorphism in the HLA-D region. AB - Restriction fragment-length polymorphisms (RFLP) were systematically analyzed by Southern hybridization with restriction endonuclease-digested genomic DNA from 28 HLA-homozygous B cell lines with Dw1-Dw19 specificity using the DR beta and DQ beta chain cDNAs as probes. These probes detected polymorphic fragments unique to each HLA-DR specificity. Furthermore, the DQ beta chain probes permitted us to distinguish between different Dw specificities with an identical DR type much more efficiently than with the DR beta chain probe. Distribution analysis of restriction fragments hybridizing to DR beta in relation to the DR and DQ specificities showed several sets of them forming ten clusters, some of which correlate with DRw53, DQw1, and DR alleles. This DNA typing technique allows the direct definition of HLA types at the gene level and provides a powerful tool for isolating genes controlling HLA-associated diseases. PMID- 3013816 TI - Clinical study of the radioprotective effects of Amifostine (YM-08310, WR-2721) on chronic radiation injury. AB - We have previously reported that Amifostine, a radioprotective agent, was effective in treating acute radiation mucositis in the head and neck region. We found that when a considerable amount of Amifostine accumulates in the salivary glands, it may be useful in preventing chronic disturbances of salivary secretion. We have observed an increase in the uptake of Ga-67-citrate to the salivary glands when they were irradiated. In this paper, the radioprotective effects of Amifostine, in treating chronic radiation injury of the salivary glands, were studied, using the cessation of an increase in uptake of Ga-67 citrate after radiotherapy as the criterion. The subjects were 105 patients, (280 salivary glands in Ga-scintigrams) with malignancy of the head and neck region treated by irradiation from 1978 to 1984. Ga-negative glands were recognized in 97%, that is, 36 out of 37 glands, before irradiation, and the figure decreased to 19%, seven out of 37, within 1 to 2 weeks (10Gy less than or equal to) after the start of radiotherapy. In patients who were irradiated with more than 30 Gy and in whom scintigraphy was performed at 6 months or more after radiotherapy, Ga negative glands were recognized in 18 out of 41 glands, 44%, with Amifostine, compared with 13%, four out of 32 glands, without Amifostine. A difference was recognized between these two groups in the negative change in Ga-67 uptake after radiotherapy (p less than 0.05). These facts suggest that Amifostine may have a radioprotective effect on chronic radiation injury. PMID- 3013819 TI - Dietary sodium bicarbonate for exertional rhabdomyolysis. PMID- 3013818 TI - Clinical and pathologic features of thyroid tumors in 26 dogs. AB - Thyroid tumors were diagnosed in 26 dogs between 1977 and 1984. A total of 23 of the 26 tumors were carcinomas, and 3, detected as incidental findings at necropsy, were adenomas. The median patient age was 9.5 years. Dogs of the Beagle breed were affected most commonly (5 dogs). The most common physical abnormalities in carcinoma patients were cervical swelling, dyspnea, and coughing. A total of 25 of 26 dogs were clinically euthyroid. Aspiration cytology provided diagnostic information in 8 of 17 cases. In dogs with thyroid carcinoma, a cervical soft tissue lesion was identified consistently by use of radiography and scintigraphy with sodium pertechnetate. Pulmonary metastases were detected radiographically in 8 of 21 dogs with thyroid carcinoma. Thoracic nuclear imaging confirmed the radiographic findings in 11 of 14 dogs. Surgical excision of the thyroid mass was the primary treatment for 17 dogs with carcinoma. Eight dogs died within 2 years (median, 7 months) of surgery because of primary tumor regrowth or metastases. Four dogs were alive at a range of 3 to 48 months after surgery, and 4 dogs died from unrelated causes. Necropsy of 7 dogs with thyroid carcinoma revealed neoplastic infiltration of the cervical blood vessels and pulmonary metastases in each dog. The most common histologic patterns of thyroid carcinoma were solid or compact cellular (11 dogs) and mixed solid-follicular tumors (8 dogs). Dogs with a solid carcinoma had a median survival time of 10.5 months (6 dogs), and dogs with a mixed solid-follicular tumor had a median survival time of 8 months (3 dogs). PMID- 3013817 TI - Detection of specific anti-antibodies in patients treated with radiolabeled antibody. AB - Over 100 patients have received cyclic treatment with polyclonal 131I labeled anti-ferritin and anti-carcinoembryonic antigen (CEA) antibodies from different animal species (rabbit, pig, cynomolgous monkey, bovine, and baboon). Because survival was prolonged from original cyclic treatment, retreatment with original antibodies (recycling) became a necessary consideration. An assay using autoradiography of Ouchterlony gels, with diffusion of patients' sera against the varied radiolabeled antibodies, was developed to detect anti-antibody precipitin bands. Anti-antibody could be detected with a sensitivity to the 60 ng level. Sera from 35 patients given from 1 to 7 separate cycles (2 injections/week, total antibody 6 mg/cycle) of radiolabeled foreign antibody were studied for the production of anti-antibodies. Anti-antibodies were detected in 11 of 22 primary hepatoma patients studied, 3 of 4 intrahepatic biliary cancer patients, and 0 of 9 Hodgkin's disease patients. In all but two of the patients, the anti-antibodies produced were specific for the species used in the treatment of the patient. Eight patients were reinjected (recycled) with previously used antibodies and the presence or absence of precipitin bands correlated with the ability of these antibodies to deposit in the tumor or to be rapidly degraded. The importance of this assay is its simplicity, sensitivity, and the rapid detection of anti antibody activity for patients requiring treatment with radiolabeled antibodies. PMID- 3013820 TI - Interepizootic survival of porcine parvovirus. AB - Porcine parvovirus (PPV) was transmitted by direct contact between experimentally infected and susceptible pigs at 1 and 2 weeks, but not at 4, 8, 16, or 25 weeks, after experimental infection. In contrast, PPV was found to remain infectious for at least 14 weeks in uncleaned rooms previously vacated by experimentally infected pigs. These findings suggest that facilities contaminated by secretions and excretions of infected pigs may provide the major means by which PPV survives between episodes of acute infection. PMID- 3013821 TI - Radioautographic localization of beta-adrenergic receptors in the rat ventral prostate. AB - Previous studies performed with crude homogenates have demonstrated the presence of beta-adrenergic receptors in the rat ventral prostate. The precise localization of these receptors in prostatic tissue, however, has not been determined. The present study describes the localization of beta-adrenergic receptors using in vitro radioautography. beta-adrenergic receptors are present exclusively in the epithelial cells, while no receptors could be detected in the stromal cells. The silver grains mostly are associated with the apical pole of the glandular cells and are much less concentrated in the basal nuclear region of the epithelium. Very low concentrations of grains are found in the lumen of the acini. Castration caused a dramatic fall in the receptor concentration, while treatment with dihydrotestosterone reversed these effects. The specific presence of beta-adrenergic receptors in prostatic epithelial cells and their control by androgens suggest that they could play a physiologic role in androgen action in prostatic tissue. Moreover, the level and activity of beta-adrenergic receptors could be used as parameters of prostatic activity. PMID- 3013822 TI - Effects of extracochlear direct current stimulation on the ensemble auditory nerve activity of cats. AB - The influence of direct current applied by round window stimulation on the whole nerve response of the auditory nerve of the cat has been studied. Effects on acoustically driven activity (CAP) and on the ensemble spontaneous activity of the nerve were observed. Stimulation with positive current suppressed driven and spontaneous activity. The strength and spread of suppressive effects was a function of the applied current level. After a period of positive electrical stimulation, driven and spontaneous activity rapidly returned to normal values. A rebound effect was sometimes observed, marked by a brief increase in spontaneous activity above the normal level. Negative current initially produced an increase in the amplitude of driven and spontaneous responses. Prolonged stimulation with negative current (greater than 30 s) resulted in a subsequent, graded reduction of neural activity, until a profound suppression of spontaneous and evoked neural activity was attained. The amplitude/latency relationship of CAPs was altered during passing of negative currents but not during passing of positive currents. Recovery from the suppression generated by negative currents was commonly prolonged for anything from a few seconds to many minutes; prolongation was dependent on stimulus amplitude, duration and duty cycles. PMID- 3013823 TI - Representation of amplitude modulation in the auditory cortex of the cat. I. The anterior auditory field (AAF). AB - The ability of cortical neurons to follow amplitude modulation (AM) of tones was examined in the anterior auditory cortical field (AAF) of anesthetized cats using multiple-unit recording techniques. Sinusoidal and rectangular modulations (100%) of a monaural carrier tone at the characteristic frequency of each location were presented to study the degree of response synchronization and changes in firing rate as a function of the modulation frequency. All investigated locations were tuned to a 'best modulation frequency' (BMF) as determined by synchronization measures. Almost all locations (94%) were tuned to a BMF as determined by spike rate. Maximal binaural-interaction strength was observed for modulation frequencies close to the BMF of neurons. For sinusoidal AM, a correlation (r = 0.63, P less than 0.01) between BMF and CF of neurons in AAF could be demonstrated for the synchronization of the response. PMID- 3013824 TI - Subsurface material in outer hair cells. AB - Tannic acid stains a homogenous material inside the outer hair cells of the organ of Corti of the guinea pig. This material is always placed between the plasma membrane and the first layer of subsurface cisterns, but only in those areas along the lateral surface of the outer hair cell lining the spaces of Nuel. The possibility that this material is related to some particular function of outer hair cell lateral face is discussed. PMID- 3013825 TI - Effect of naloxone on serum luteinizing hormone, cortisol and prolactin concentrations in anestrous beef cows. AB - Two experiments were conducted with the opioid antagonist naloxone to determine the effect of opioid receptor blockade on hormone secretion in postpartum beef cows. In Exp. 1, nine anestrous postpartum beef cows were used to measure the effect of naloxone on serum luteinizing hormone (LH), cortisol and prolactin concentrations. Cows received either saline (n = 4) or 200 mg naloxone in saline (n = 5) iv. Blood samples were collected at 15-min intervals for 2 h before and after naloxone administration. Serum LH concentrations increased (P less than .01) in naloxone-treated cows from 1.8 +/- .04 ng/ml before treatment to 3.9 +/- .7 ng/ml and 4.2 +/- .5 ng/ml at 15 and 30 min, respectively, after naloxone administration. In contrast, LH remained unchanged in saline-treated cows (1.6 +/ .3 ng/ml). Serum cortisol and prolactin concentrations were not different between groups. In Exp. 2, 12 anestrous postpartum beef cows were used to examine the influence of days postpartum on the serum LH response to naloxone. Four cows each at 14 +/- 1.2, 28 +/- .3 and 42 +/- 1.5 d postpartum received 200 mg of naloxone in saline iv. Blood samples were taken as in the previous experiment. A second dose of naloxone was administered 2 h after the first, and blood samples were collected for a further 2 h. Serum LH concentrations increased (P less than .01) only in cows at 42 d postpartum.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013827 TI - Plasmids of Staphylococcus aureus associated with live and processed poultry. AB - A series of 23 strains of Staphylococcus aureus originally isolated from processed poultry was screened for the presence of plasmids. Plasmids were more common in strains of Staph. aureus characteristically associated with live poultry than with strains endemic in poultry plants and strains of human origin. Two plasmids with sizes of 1.65 and 18.2 kilobase pairs (kBp) were present in three strains considered typical of Staph. aureus forma specialis 'altilis' and two plasmids with sizes of 1.65 and 17 kBp were present in three of four strains of Staph. aureus var. gallinae. A 1.65 kBp plasmid was present in all seven strains of these poultry biotypes and in three of 14 'endemic' strains. All the 1.65 kBp plasmids were shown by blot hybridization to share sequence homology. There was also some sequence homology between the 18.2 kBp and 17 kBp plasmids. These results were supported by restriction enzyme digest analyses. A study of cured derivatives of strain PS221 f.sp. 'altilis' suggested that the 18.2 kBp plasmid encoded the genetic determinant(s) responsible for caseolysis. Both the 1.65 and the 18.2 kBp plasmids also exerted an effect on the production of acid from lactose. In no other characteristic did cured strains resemble the plasmid free 'endemic' strains. This was therefore consistent with the notion that the genetic determinants associated with the cultural characteristics of endemic strains are chromosomally located. PMID- 3013826 TI - Effects of diet concentrate level and sodium bicarbonate on site and extent of forage fiber digestion in the gastrointestinal tract of wethers. AB - Four adult wethers (45 kg) with permanent ruminal and abomasal cannulae were used in a repeated measures Latin-square arrangement of treatments to quantitate the effects of diet concentrate level and sodium bicarbonate (NaHCO3) on site and extent of forage fiber digestion in the gastrointestinal tract. Experimental diets consisted of Kentucky-31 tall fescue hay, soybean meal and a semi-purified concentrate mixture in ratios of 95:5:0, 76:4:20, 57:3:40 and 38:2:60; NaHCO3 represented 0 or 7.5% of the concentrate mixture. Ruminal digestion (% of intake) of neutral detergent fiber (NDF) and hemicellulose decreased linearly (P less than .05), whereas acid detergent fiber (ADF) digestion responded in a cubic (P less than .05) fashion to increasing concentrate level; NaHCO3 improved ruminal digestion of NDF (P less than .10) and ADF (P less than .05), but not hemicellulose. Post-ruminal digestion (% of rumen non-degraded) of NDF and ADF tended to increase, whereas hemicellulose digestion responded in a cubic (P less than .05) fashion to increasing concentrate level; NaHCO3 decreased (P less than .05) post-ruminal digestion of all fiber fractions. Total tract digestion of NDF and ADF showed a cubic (P less than .05) response, whereas hemicellulose digestion responded in a quadratic (P less than .05) fashion to increasing concentrate level; NaHCO3 had no effect on total tract digestion of any fiber fraction. Correlations of ruminal hemicellulose digestion with mean pH (r = .33; P = .07) and minimum pH (r = .30; P = .09) were attained in a 24-h feeding cycle.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013828 TI - Transposable resistance to trimethoprim and 0/129 in Vibrio cholerae. AB - Vibrio cholerae biotype el tor strain BM2508, resistant to trimethoprim, 0/129, streptomycin and spectinomycin was isolated from the faeces of a child with severe diarrhoea. Resistance to trimethoprim and 0/129 was due to a dihydrofolate reductase type I and resistance to streptomycin-spectinomycin to a 3'',9 aminoglycoside-aminocyclitol adenylyltransferase. The resistance genes were not transferable to Escherichia coli and, as inferred from ultracentrifugation in cesium chloride-ethidium bromide and agarose gel electrophoresis of crude bacterial lysates, were located on the chromosome. The resistance genes were transposed to multiple sites of plasmids belonging to incompatibility groups 6-C and P, introduced in BM2508 and were subsequently transferred to E. coli (rec-), Salmonella typhimurium, V. cholerae and V. parahaemolyticus strains where they re transposed into the chromosome. Analysis of plasmid DNA from the transconjugants by agarose gel electrophoresis following digestion with HindIII and by Southern hybridization using a ColEl::Tn7 probe indicated the presence of a 14-kilobase transposon, Tn1527, closely related to Tn7. The emergence of Tn1527 in V. cholerae may lead to prophylactic and therapeutic failures due to trimethoprim resistance and to bacterial misidentification because of cross resistance to 0/129. PMID- 3013829 TI - Trimethoprim resistance amongst urinary pathogens in south India. AB - Two hundred and eighty four strains of Enterobacteriaceae, responsible for significant bacteriuria, were isolated, over a three month period, in Vellore, India. Sixty-four per cent of these strains were resistant to 10 mg/l of trimethoprim. Moreover, this population was dominated by high level resistance (minimum inhibitory concentration greater than 1000 mg/l) and these accounted for 57.3% of all strains studied. Over half of the resistant strains were able to transfer trimethoprim resistance to standard Escherichia coli strains. However, the high incidence of transferable resistance did not result from the spread of one plasmid type as 58 different plasmid types were identified. These results are in marked contrast to recent findings in Europe where the incidence of high level transferable trimethoprim resistance is falling. PMID- 3013830 TI - How well mixed is inert gas in tissues? AB - The washout of inert gas from tissues typically follows multiexponential curves rather than monoexponential curves as would be expected from homogeneous, well mixed compartment. This implies that the ratio for the square root of the variance of the distribution of transit times to the mean (relative dispersion) must be greater than 1. Among the possible explanations offered for multiexponential curves are heterogeneous capillary flow, uneven capillary spacing, and countercurrent exchange in small veins and arteries. By means of computer simulations of the random walk of gas molecules across capillary beds with parameters of skeletal muscle, we find that heterogeneity involving adjacent capillaries does not suffice to give a relative dispersion greater than one. Neither heterogeneous flow, nor variations in spacing, nor countercurrent exchange between capillaries can account for the multiexponential character of experimental tissue washout curves or the large relative dispersions that have been measured. Simple diffusion calculations are used to show that many gas molecules can wander up to several millimeters away from their entry point during an average transit through a tissue bed. Analytical calculations indicate that an inert gas molecule in an arterial vessel will usually make its first vascular exit from a vessel larger than 20 micron and will wander in and out of tissue and microvessels many times before finally returning to the central circulation. The final exit from tissue will nearly always be into a vessel larger than 20 micron. We propose the hypothesis that the multiexponential character of skeletal muscle tissue inert gas washout curves must be almost entirely due to heterogeneity between tissue regions separated by 3 mm or more, or to countercurrent exchanges in vessels larger than 20 micron diam. PMID- 3013831 TI - Surgery of hepatoma with intracavitary cardiac extension. AB - A case of primary liver carcinoma with intracavitary cardiac extension is presented. A 36-year-old female was admitted to our surgical clinic with dyspnea and generalized edema. Echocardiography and superior vena cavography demonstrated a large filling defect in the right atrium. After a diagnosis of acute cardiac failure due to an intracardiac tumor, the patient was operated upon immediately. A right atriotomy exposed a large yellow mass within the right atrium, which was not adherent to the atrial wall. The mass was in continuity with similar material in the inferior vena cava and right hepatic vein. With a suspicion of hepatic malignancy, the atrial tumor was removed, and debulking of the mass in the inferior vena cava and right hepatic vein was performed. A postoperative histological examination of the tumor showed hepatocellular carcinoma. Her postoperative course was uneventful, and she was discharged from the hospital. Intracardiac extension of hepatoma is rarely encountered. In this clinical setting, long-term survival cannot be anticipated from any surgery, but palliative clearing of the atrium and inferior vena cava may be of value in preventing cardiac arrest causing sudden death. PMID- 3013832 TI - Evidence of homology between the pectate lyase-encoding pelB and pelC genes in Erwinia chrysanthemi. AB - The genes for two of several pectate lyase isozymes produced by the phytopathogenic enterobacterium Erwinia chrysanthemi 1237 were subcloned and compared by DNA-DNA hybridization, and the encoded proteins were analyzed. The borders of the genes were located on a restriction map by incremental exonuclease III deletions. DNA-DNA hybridization studies revealed a low percentage of mismatch (7 to 17%) between pelB and pelC. No homology was detected between pelC and other regions of the E. chrysanthemi 1237 chromosome, in which three other isozyme genes apparently reside. The pectate lyase isozymes were readily purified by chromatofocusing or granulated-gel bed isoelectric focusing from the periplasmic shock fluids of Escherichia coli subclones. The molecular weights of PLb and PLc were 30,000 and 33,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Their isoelectric points were 7.6 and 8.1, respectively, as determined by equilibrium isoelectric focusing in ultrathin polyacrylamide gels. The Km values for PLb and PLc were 0.20 and 0.32 mg/ml, respectively, with polygalacturonate as a substrate. Thin-layer chromatography of reaction products and viscometric assays revealed little difference between the two isozymes. All our data indicate that the genes are duplicates and that the proteins are isofunctional. PMID- 3013833 TI - Predominance of gluconate formation from glucose during germination of Bacillus megaterium QM B1551 spores. AB - Metabolic pathways of glucose during germination of Bacillus megaterium QM B1551 spores were studied by using specifically labeled glucose and gluconate. The Embden-Meyerhof pathway, the pentose cycle, and the direct oxidation route of glucose to gluconate (the gluconate pathway) were all operative at this stage; among those, gluconate accumulation was most predominant, especially in the early stage. Potassium fluoride, an enolase inhibitor, abolished the catabolism by the Embden-Meyerhof pathway totally without affecting gluconate accumulation. Under these conditions glucose was exclusively oxidized to gluconate. Gluconate thus accumulated could be metabolized further via phosphorylation by gluconate kinase. Remarkable gluconate accumulation was also demonstrated in several other spores requiring alanine as an effective germinant. NADH formed by the direct glucose oxidation may serve as a initial ATP source to phosphorylate glucose in germinating spores. PMID- 3013834 TI - Cloning and expression in Escherichia coli of the gene for 10 formyltetrahydrofolate synthetase from Clostridium acidiurici ("Clostridium acidi urici"). AB - The gene for 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) from the purinolytic anaerobic bacterium Clostridium acidiurici ("Clostridium acidi urici") was cloned into Escherichia coli JM83 with plasmid pUC8. A C. acidiurici genomic library was prepared in E. coli from a partial Sau3A digest and screened with antibody against the synthetase. Of 10 antibody-positive clones, 1 expressed a high level of synthetase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis demonstrated that the protein synthesized in E. coli had the same subunit molecular weight as the C. acidiurici enzyme. The gene was located on an 8.3-kilobase genomic insert and appeared to be transcribed from its own promoter. Analysis of genomic digests with a fragment of the synthetase gene indicated that one copy of the gene was present in the C. acidiurici chromosome. PMID- 3013835 TI - Regulation of lateral flagella gene transcription in Vibrio parahaemolyticus. AB - Two distinctly different organelles of locomotion are produced by Vibrio parahaemolyticus. The polar flagellum is responsible for motility in a liquid environment (swimming), and the lateral flagella enable the bacteria to move over surfaces (swarming). Synthesis of lateral flagella occurs when V. parahaemolyticus is grown on agar media but not when it is grown in liquid media. We used lux (luminescence gene) fusions to conveniently and sensitively analyze the factors which influence transcription of lateral flagella genes (laf). Transposon mini-Mu lux was used to mutagenize V. parahaemolyticus and to generate laf::lux transcriptional fusions. Mutants with insertions of mini-Mu lux in laf genes were defective in the swarming phenotype and produced light when the bacteria were propagated on agar media, but not when cells were grown in liquid media. Thus, surface-dependent expression of lateral flagella synthesis is controlled by regulation of transcription. Such fusion strains were also used to further define the environmental conditions which induce laf gene expression. Cultivation on media solidified by gelling agents other than agar also induced light production in fusion strains, as did growth on a variety of hydrophilic membrane filters suspended over liquid media. Growth at an air-surface interface was not necessary for expression since embedding the fusion strains in agar was also effective. Furthermore, induction of laf gene transcription could also be accomplished by increasing the viscosity of the liquid medium by the addition of a high-molecular-weight polymer such as polyvinylpyrrolidone. Increase in luminescence of the fusion strains was detected within 30 min of initiation of the inducing circumstance, and reversal of induction, e.g., by dilution of the viscous medium, resulted in a rapid decline in the rate of increase in luminescence. Conditions that induced luminescence in the fusion strains also induced the synthesis of lateral flagella in wild-type V. parahaemolyticus. The growth environment of the genes, and it appears that the signal that triggers laf expression is physical rather than chemical in nature. Possibilities for a sensing mechanism are discussed. PMID- 3013836 TI - Requirement for two or more Erwinia carotovora subsp. carotovora pectolytic gene products for maceration of potato tuber tissue by Escherichia coli. AB - Several genes encoding enzymes capable of degrading plant cell wall components have been cloned from Erwinia carotovora subsp. carotovora EC14. Plasmids containing cloned EC14 DNA mediate the production of endo-pectate lyases, exo pectate lyase, endo-polygalacturonase, and cellulase(s). Escherichia coli strains containing one of these plasmids or combinations of two plasmids were tested for their ability to macerate potato tuber slices. Only one E. coli strain, containing two plasmids that encode endo-pectate lyases, exo-pectate lyase, and endo-polygalacturonase, caused limited maceration. The pectolytic proteins associated with one of these plasmids, pDR1, have been described previously (D. P. Roberts, P. M. Berman, C. Allen, V. K. Stromberg, G. H. Lacy, and M. S. Mount, Can. J. Plant Pathol. 8:17-27, 1986) and include two secreted endo-pectate lyases. The second plasmid, pDR30, contains a 2.1-kilobase EC14 DNA insert that mediates the production of an exo-pectate lyase and an endo-polygalacturonase. These enzymes are similar in physicochemical properties to those produced by EC14. Our results suggest that the concerted activities of endo-pectate lyases with endo-polygalacturonase or exo-pectate lyase or both cause maceration. PMID- 3013837 TI - Manganese reduction by a marine Bacillus species. AB - Mature dormant spores of marine Bacillus sp. strain SG1 catalyze the oxidation of Mn(II) to MnO2. We report that vegetative cells of the same strain reduced MnO2 under low-oxygen conditions. The rate of reduction was a function of cell concentration. The process had a pH optimum of 7.5 to 8.0 and was inhibited by HgCl2, by preheating of the cells at 80 degrees C for 5 min, by antimycin A, and by N-heptyl-hydroxy-quinoline-N-oxide. At a nonlimiting O2 concentration, little MnO2 reduction was observed. Under these conditions, the process could be induced by the addition of NaN3. Spectrophotometric analysis of the Bacillus cells indicated the presence of type b and c cytochromes. Both types can be oxidized in situ by addition of MnO2 to the cells. PMID- 3013838 TI - Regulation of glycerol kinase by enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system. AB - Wild-type glycerol kinase of Escherichia coli is inhibited by both nonphosphorylated enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system and fructose 1,6-diphosphate. Mutant glycerol kinase, resistant to inhibition by fructose 1,6-diphosphate, was much less sensitive to inhibition by enzyme IIIGlc. The difference between the wild-type and mutant enzymes was even greater when inhibition was measured in the presence of both enzyme IIIGlc and fructose 1,6-diphosphate. The binding of enzyme IIIGlc to glycerol kinase required the presence of the substrate glycerol. PMID- 3013839 TI - Characterization of anguibactin, a novel siderophore from Vibrio anguillarum 775(pJM1). AB - Anguibactin, a siderophore produced by cells of Vibrio anguillarum 775 harboring the pJM1 plasmid, has now been isolated from the supernatants of iron-deficient cultures. This iron-reactive material was purified by adsorption onto an XAD-7 resin and subsequent gel filtration on a Sephadex LH-20 column. The resulting neutral compound produced an ion at m/z 348 in mass spectrometry and contained one sulfur, four oxygen, and four nitrogen atoms as determined by elemental analysis. Its strong UV absorbance and blue fluorescence were suggestive of a phenolic moiety. In colorimetric reactions anguibactin behaved like a catechol. The catechol assignment was supported by the appearance of a new absorption band at 510 nm in the ferric complex and by the appearance of peaks at 1,367, 1,447, 1,469, and 1,538 cm-1 in the resonance Raman spectrum. In addition, the infrared spectrum gave evidence of a secondary amide function, but no free carboxylic acid or hydroxamic acid groups were observed. A third iron-ligating group was suggested by the liberation of three protons during iron binding; mass spectrometry of the resulting material yielded a molecular ion characteristic of a 1:1 complex of ferric anguibactin. The purified anguibactin exhibited specific growth-promoting activity under iron-limiting conditions for a siderophore deficient mutant of V. anguillarum 775(pJM1). A novel structure for anguibactin was indicated by the failure of a large number of known siderophores and synthetic chelators to yield a similar type of specific cross-feeding in the V. anguillarum bioassay. PMID- 3013841 TI - Cloning and expression of the Escherichia coli glgC gene from a mutant containing an ADPglucose pyrophosphorylase with altered allosteric properties. AB - A mutant strain of Escherichia coli K-12, designated 618, accumulates glycogen at a faster rate than wild-type strain 356. The mutation affects the ADPglucose pyrophosphorylase regulatory properties (N. Creuzat-Sigal, M. Latil-Damotte, J. Cattaneo, and J. Puig, p. 647-680, in R. Piras and H. G. Pontis, ed., Biochemistry of the Glycocide Linkage, 1972). The enzyme is less dependent on the activator, fructose 1,6 bis-phosphate for activity and is less sensitive to inhibition by the inhibitor, 5'-AMP. The structural gene, glgC, for this allosteric mutant enzyme was cloned into the bacterial plasmid pBR322 by inserting the chromosomal DNA at the PstI site. The glycogen biosynthetic genes were selected by cotransformation of the neighboring asd gene into an E. coli mutant also defective in branching enzyme (glgB) activity. Two recombinant plasmids, pEBL1 and pEBL3, that had PstI chromosomal DNA inserts containing glgC and glgB were isolated. Branching enzyme and ADPglucose pyrophosphorylase activities were increased 240- and 40-fold, respectively, in the asd glgB mutant, E. coli K-12 6281. The E. coli K-12 618 mutant glgC gene product was characterized after transformation of an E. coli B ADPglucose pyrophosphorylase mutant with the recombinant plasmid pEBL3. The kinetic properties of the cloned ADPglucose pyrophosphorylase were similar to those of the E. coli K-12 618 enzyme. The inserted DNA in pEBL1 was arranged in opposite orientation to that in pEBL3. PMID- 3013840 TI - Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. AB - Using physical and genetic data, we have demonstrated that Rhizobium meliloti SU47 has a symbiotic megaplasmid, pRmeSU47b, in addition to the previously described nod-nif megaplasmid pRmeSU47a. This plasmid includes four loci involved in exopolysaccharide (exo) synthesis as well as two loci involved in thiamine biosynthesis. Mutations at the exo loci have previously been shown to result in the formation of nodules which lack infection threads (Inf-) and fail to fix nitrogen (Fix-). Thus, both megaplasmids contain genes involved in the formation of nitrogen-fixing root nodules. Mutations at two other exo loci were not located on either megaplasmid. To mobilize the megaplasmids, the oriT of plasmid RK2 was inserted into them. On alfalfa, Agrobacterium tumefaciens strains containing pRmeSU47a induced marked root hair curling with no infection threads and Fix- nodules, as reported by others. This plant phenotype was not observed to change with A. tumefaciens strains containing both pRmeSU47a and pRmeSU47b megaplasmids, and strains containing pRmeSU47b alone failed to curl root hairs or form nodules. PMID- 3013842 TI - Chemical and spectroscopic evidence for the formation of a ferryl Fea3 intermediate during turnover of cytochrome c oxidase. AB - When partially reduced cytochrome c oxidase samples are reoxidized with dioxygen, an EPR-silent dioxygen intermediate, which is at the three-electron level of dioxygen reduction, is trapped at the dioxygen reduction site. The intermediate has novel spectral features at 580 and 537 nm. Combined optical and EPR results reveal that this intermediate reacts rapidly with CO at 277-298 K causing the abolition of the 580/537 mm features and the appearance of a rhombic CuB EPR signal. A ferryl Fea3, or an intermediate at the same formal level of oxidation, is proposed to oxidize CO to CO2 producing an EPR-detectable CuB adjacent to a low-spin ferrous Fea3-dioxygen (or carbon monoxide) adduct. PMID- 3013843 TI - Dephosphorylation of cAMP-dependent protein kinase regulatory subunit (type II) by calmodulin-dependent protein phosphatase. Determinants of substrate specificity. AB - Calmodulin-dependent protein phosphatase purified from bovine cardiac muscle catalyzed the rapid dephosphorylation of Ser-95 of bovine cardiac cAMP-dependent protein kinase regulatory subunit (RII). The kinetic constants determined for the reaction (Km = 20 microM; Vmax = 2 mumol min-1 mg-1) are comparable to those determined for other good substrates of this phosphatase. Because little is known about the determinants of substrate specificity for the calmodulin-dependent phosphatase, various phosphopeptides were used to investigate the structural features important for substrate recognition. Limited proteolysis of phospho-RII with trypsin and chymotrypsin yielded fragments (residues 93-400 and 91-400, respectively) that were poor substrates, whereas digestion with Staphylococcal aureus V8 protease produced three phosphopeptides that were all dephosphorylated as rapidly as intact RII. The sequence of the shortest phosphopeptide produced by S. aureus V8 protease was determined by sequence analysis to be Asp-Leu-Asp-Val Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser-Val-Cys-Ala-Glu, corresponding to residues 81-99 of RII. Synthetic phosphopeptides corresponding to residues 81-99, 85-99, 90-99, and 91-99 were prepared to determine the minimum sequence necessary for substrate recognition. Only the 19-residue peptide (81-99) was dephosphorylated with kinetics comparable to RII (Km = 26 microM, Vmax = 1.7 mumol min-1 mg-1). Structural analysis of this peptide indicates that an amphipathic beta-sheet structure may be an important structural determinant for some substrates of the calmodulin-dependent phosphatase. PMID- 3013844 TI - Upper and lower limits of the proton stoichiometry of cytochrome c oxidation in rat liver mitoplasts. AB - The stoichiometry of vectorial H+ translocation coupled to oxidation of added ferrocytochrome c by O2 via cytochrome-c oxidase of rat liver mitoplasts was determined employing a fast-responding O2 electrode. Electron flow was initiated by addition of either ferrocytochrome c or O2. When the rates were extrapolated to level flow, the H+/O ratios in both cases were less than but closely approached 4; the directly observed H+/O ratios significantly exceeded 3.0. The mechanistic H+/O ratio was then more closely fixed by a kinetic approach that eliminates the necessity for measuring energy leaks and is independent of any particular model of the mechanism of energy transduction. From two sets of kinetic measurements, an overestimate and an underestimate and thus the upper and lower limits of the mechanistic H+/O ratio could be obtained. In the first set, the utilization of respiratory energy was systematically varied through changes in the concentrations of valinomycin or K+. From the slope of a plot of the initial rates of H+ ejection (JH) and O2 uptake (JO) obtained in such experiments, the upper limit of the H+/O ratio was in the range 4.12-4.19. In the second set of measurements, the rate of respiratory energy production was varied by inhibiting electron transport. From the slope of a plot of JH versus JO, the lower limit of the H+/O ratio, equivalent to that at level flow, was in the range 3.83-3.96. These data fix the mechanistic H+/O ratio for the cytochrome oxidase reaction of mitoplasts at 4.0, thus confirming our earlier measurements (Reynafarje, B., Alexandre, A., Davies, P., and Lehninger, A. L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7218-7222). Possible reasons for discrepancies in published reports on the H+/O ratio of cytochrome oxidase in various mitochondrial and reconstituted systems are discussed. PMID- 3013845 TI - Suppression of protein tyrosine kinase activity of the epidermal growth factor receptor by epidermal growth factor. AB - Epidermal growth factor (EGF) receptor protein kinase activity, estimated by the use of peptide substrates, was reduced by as much as 70% after the treatment of intact A431 human carcinoma cells with EGF. The apparent decrease in protein kinase activity was observed after immunoprecipitation of the receptor or after purification of the receptor by lectin chromatography. By the use of [35S]methionine, it was determined that the total amount of receptor obtained was the same whether or not cells were treated with EGF. EGF stimulated the purified receptor protein kinase activity in vitro; however, the EGF-stimulated activity of receptor from EGF-treated cells continued to be reduced by as much at 70% compared to the EGF-stimulated activity from untreated cells. The reduction in receptor protein kinase activity induced by EGF may represent a feedback mechanism by which responsiveness to the growth factor is regulated. PMID- 3013846 TI - Amines as inhibitors of iron transport in rabbit reticulocytes. AB - The effect of the known inhibitors of iron uptake, n-butylamine and NH4Cl, was examined at the molecular level to more precisely define the mechanisms by which these lysosomotropic agents block iron uptake by rabbit reticulocytes. Utilizing a rapid pulse-chase technique to follow the handling of a cohort of 59Fe, 125I transferrin bound to rabbit reticulocytes, both amines were observed to have no effect on the cell-mediated release of 59Fe from internalized transferrin. The results indicated, however, that both agents acted to 1) retard the internalization of transferrin bound to transferrin receptors on the plasma membrane of reticulocytes, 2) retard the externalization of internalized transferrin, and 3) block the transport into the cytosol of iron released from transferrin. PMID- 3013848 TI - Rabbit red blood cell hexokinase. Decay mechanism during reticulocyte maturation. AB - In rabbit reticulocytes, the hexokinase (EC 2.7.1.1)-specific activity is 4-5 times that of corresponding mature red cells. Immunoprecipitation of hexokinase by a polyclonal antibody made in vitro shows that this maturation-dependent hexokinase decay is not due to accumulation of inactive enzyme molecules but to degradation of hexokinase. A cell-free system derived from rabbit reticulocytes, but not mature erythrocytes, was found to catalyze the decay of hexokinae activity and the degradation of 125I-labeled enzyme. This degradation is ATP dependent and requires both ubiquitin and a proteolytic fraction retained by DEAE cellulose. Maximum ATP-dependent degradation was obtained at pH 7.5 in the presence of MgATP. MgGTP could replace MgATP with a relative stimulation of 0.90. 125I-Hexokinase incubated with reticulocyte extract in the presence of ATP forms high molecular weight aggregates that reach a steady-state concentration in 1 h, whereas the degradation of the enzyme is linear up to 8 h, suggesting that the formation of protein aggregates precedes enzyme catabolism. These aggregates are stable upon boiling in 2% sodium dodecyl sulfate, 3% mercaptoethanol and probably represent an intermediate step in the enzyme degradation with hexokinase and other proteins covalently conjugate to ubiquitin. That hexokinase could be conjugated to ubiquitin was shown by the formation of 125I-ubiquitin-hexokinase complexes in the presence of ATP and the enzymes of the ubiquitin-protein ligase system. Thus, the decay of hexokinase during reticulocyte maturation is ATP- and ubiquitin-dependent and suggests a new physiological role for the energy dependent degradation system of reticulocytes. PMID- 3013847 TI - Biochemical responses in activated human neutrophils mediated by protein kinase C and a Ca2+-requiring proteinase. AB - Low concentrations of phorbol 12-myristate 13-acetate (PMA) elicit a specific response in human neutrophils, characterized by the production of oxygen radicals and the release into the medium of a membrane-bound serine proteinase (Pontremoli, S., Melloni, E., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., Damiani, G. and Horecker, B. L. (1986) Proc. Natl. Acad. Sci. U. S. A., 83, 1685-1689). The following evidence indicates that this response is mediated by membrane-bound protein kinase C: 1) it is blocked by inhibitors of protein kinase C; and 2) it is enhanced in cells preloaded with leupeptin which prevents proteolysis of protein kinase C and its subsequent dissociation from the cell membrane. This response is not accompanied by significant exocytosis of granule enzymes. With higher concentrations of PMA, and more particularly on stimulation with formylmethionyl-leucyl-phenylalanine (fMLP) plus cytochalasin B, a substantial exocytosis of constituents of both specific and azurophil granules is observed. With fMLP, exocytosis of granule enzymes is the predominant event, with little production of H2O2 and negligible release of membrane-bound serine proteinase. Exocytosis promoted either by a high concentration of PMA or by fMLP is inhibited by leupeptin, indicating that it is due to the action of an intracellular Ca2+-dependent thiol proteinase (calpain), either directly or by conversion by calpain of membrane-bound protein kinase C to the soluble Ca2+/phospholipid-independent form. Intracellular mobilization of Ca2+ is also observed following stimulation with either PMA or fMLP, but only the latter results in a net increase in the intracellular concentration of free Ca2+; under these conditions maximum exocytosis of granule contents is observed. PMID- 3013849 TI - Structure and mobility of actin filaments as measured by quasielastic light scattering, viscometry, and electron microscopy. AB - Actin filaments of different lengths were prepared by polymerizing actin in the presence of various concentrations of gelsolin, a protein which accelerates actin polymerization by stabilizing nuclei from which filaments grow and which binds to their fast growing ends. The lengths of the actin filaments following polymerization were measured by electron microscopy and showed that the number average filament length agreed with the predicted length if each gelsolin molecule acted as a seed for the growth of an actin filament. The distribution of lengths was independent of the actin:gelsolin ratio and was similar to that of actin filaments polymerized in the absence of gelsolin (Lw/Ln = 1.8). The mobility of these filaments in solution was studied by quasielastic light scattering and by viscometry. The translational diffusion constant determined by quasielastic light scattering was in agreement with the infinite dilution values calculated from the dimensions and the distribution of lengths determined by electron microscopy for relatively short filament lengths. Under conditions where overlap of the rotational domains of the filaments would be expected to occur, the measured diffusion rates deviated from their predicted dilute solution values and the solution viscosity increased abruptly. The dependence of the diffusion constant and the solution viscosity on the length of the actin filaments can be explained in terms of a theory that describes the restraints on diffusion of independent rigid rods in semi-dilute solution. The results suggest that the rheology of actin filaments can be accounted for by steric restraints. The length of cytoplasmic actin filaments in some cell types is such that these steric constraints are significant and could produce large changes in physical properties with small changes in filament length. PMID- 3013850 TI - Processing of a newly identified intermediate of human myeloperoxidase in isolated granules occurs at neutral pH. AB - Myeloperoxidase is a major component of specialized lysosomes known as azurophil granules in polymorphonuclear leukocytes or neutrophils. The processing of myeloperoxidase in human HL-60 promyelocytic leukemia cells was studied by pulse labeling cells in culture with [35S]methionine followed by immunoprecipitation and identification of myeloperoxidase polypeptides from cell fractions after various chase intervals. These studies revealed the presence of a previously unidentified intermediate with Mr 74,000 which kinetically followed the appearance of a larger Mr 81,000 intermediate. Using an in vitro lysosomal preparation the newly identified Mr 74,000 intermediate was directly converted within protected granules to mature forms of myeloperoxidase (Mr 63,000 and 60,000). This conversion occurred optimally at pH 7.5 and was not inhibited by lysosomotropic agents (chloroquine, NH4Cl) or protonophores (monensin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone). Furthermore, the uptake of radiolabeled amines indicated a neutral intragranular environment (pH 7.35-7.67) which remained unchanged in the presence and absence of 1 mM ATP or 2.5 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone. We conclude that, in contrast to other lysosomal pathways, the final proteolytic cleavage of myeloperoxidase does not require an acidic environment. PMID- 3013851 TI - Isolation of cDNA clones coding for the alpha-subunit of human beta hexosaminidase. Extensive homology between the alpha- and beta-subunits and studies on Tay-Sachs disease. AB - The lysosomal beta-hexosaminidases (N-acetyl-beta-glucosaminidase, EC 3.2.1.30) occur as two major isozymes, hexosaminidase A (alpha beta a beta b) and hexosaminidase B (2(beta a beta b)). To facilitate the investigations of the biosynthesis and structure of the enzymes and the nature of mutation in Tay-Sachs disease, we have isolated cDNA clones coding for the alpha-subunit. The polypeptide chains of hexosaminidase A (30 mg) were digested with trypsin, and peptides were isolated by reverse phase high pressure liquid chromatography and their amino acid sequences determined. One of alpha-chain peptides contained a string of seven amino acids from which two sets of oligonucleotides were specified. They were used to screen the SV40-transformed human fibroblast cDNA library of Okayama and Berg. Three cDNA clones, designated pHexA, identified from among 5 X 10(5) clones screened, contained the deduced amino-acid sequences of five alpha-chain peptides. Genomic DNA homologous to pHexA cDNA mapped to human chromosome 15 in somatic cell hybrids, as expected for the pre-alpha-polypeptide. Two of the clones contained identical polyadenylation sites, while the third was polyadenylated about 450 base pairs downstream. The two types of clones were found to correspond to a major 2.0-kilobase pair and a minor 2.3-kilobase pair mRNA species. Blot hybridizations of mRNA and DNA from Tay-Sachs variant fibroblasts revealed absence or reduction of levels of both mRNA species among infantile and juvenile variants, but no observable DNA alterations. Alignment of the pre-alpha- and pre-beta-polypeptides revealed 55% nucleotide and 57% amino acid homology. These data suggest a common origin of the HEXA and HEXB genes and account for the similar substrate specificities of the alpha-dimer subunit, hexosaminidase S, and hexosaminidase B. PMID- 3013852 TI - [3H]bumetanide binding to duck red cells. Correlation with inhibition of (Na + K + 2Cl) co-transport. AB - Bumetanide is a potent inhibitor of cation-chloride co-transport systems in many cell types, including duck red cells. We studied equilibrium binding of [3H]bumetanide to intact duck red cells under a number of conditions known to affect (Na + K + 2Cl) co-transport in these cells. Saturable [3H]bumetanide binding to duck red cells is markedly stimulated by addition of norepinephrine or cell shrinkage, conditions which similarly stimulate co-transport. In the presence of norepinephrine and saturating concentrations of extracellular sodium, potassium, and chloride for the co-transporter, we found approximately 1000 [3H]bumetanide-binding sites/red cell, and measurement of 24Na+ influx on the same cells yielded a turnover number of approximately 4000/s for the co transporter. 24Na+ influx was negatively correlated with the amount of bound [3H]bumetanide, and both saturable binding and inhibition of influx were half maximal at approximately 10(-7) M [3H]bumetanide. Binding of [3H]bumetanide to duck red cells is stimulated in a saturable manner by increasing extracellular sodium and potassium. Chloride has a biphasic effect on [3H]bumetanide binding; increasing [Cl-]o (by replacement of methylsulfate) from 0 to 32 mM markedly enhances binding, whereas further increasing [Cl-]o to 160 mM inhibits binding. This behavior is similar to that reported for bumetanide inhibition of duck red cell (Na + K + 2Cl) co-transport (Haas, M., and McManus, T. J. (1983) Am. J. Physiol. 245, C235-C240; Haas, M., and McManus, T. J. (1982) Biophys. J. 37, 214a) and [3H]bumetanide binding to membranes from dog kidney outer medulla (Forbush, B. III, and Palfrey, H. C. (1983) J. Biol. Chem. 258, 11787-11792). PMID- 3013853 TI - Structure of the murine serum amyloid A gene family. Gene conversion. AB - Serum amyloid A (SAA) is an apolipoprotein produced by the liver in response to inflammation; the levels of SAA mRNA and SAA protein increase at least 500-fold within 24 h. We have obtained clones of all three genes and pseudogene that make up the murine SAA gene family. Two of the genes have 96% sequence homology over their entire length, including introns and flanking sequences 288 base pairs (bp) 5' and 443 bp 3' to the genes: an overall length of 3215 bp. The sharp boundaries between homologous and nonhomologous sequences and the absence of interspersed repeated sequences there suggest that conversion has occurred between these two genes. The homologous regions are bounded by short inverted repeats containing alternating purine and pyrimidine residues, as described for other gene conversion units. The third SAA gene has evolved separately, although all are closely linked on chromosome 7. Comparison of the upstream regions of the SAA genes with those of the rat fibrinogen genes, whose expression is also induced by inflammation, reveals sequences common to all six genes which are very improbable on a random basis. PMID- 3013854 TI - Identification of initiation sites for transcription of Xenopus laevis mitochondrial DNA. AB - The sites of transcription initiation for Xenopus laevis mtDNA have been mapped using in vitro capping and primer extension analysis of RNA isolated from oocytes. Transcription of the heavy strand initiates predominantly at a site 33 nucleotides upstream of the tRNAPhe gene. This promoter is bidirectional, with transcription of the light strand initiating only one base pair away. A second, more predominant light strand promoter is located 70 nucleotides away from the major heavy strand promoter. The overall organization of the transcription initiation sites with respect to the tRNAPhe gene and the 5' termini of major stable D-loop DNA strands resembles that of the human mtDNA genome. Analysis of the sequences surrounding the Xenopus start sites suggests that the occurrence of a conserved sequence element, ACPuTTATA, around the start sites may be important for promoter recognition and transcription initiation. This sequence is not found in human mtDNA promoters. PMID- 3013855 TI - Characterization of two inducible periplasmic c-type cytochromes from Paracoccus denitrificans. AB - When grown on methanol or methylamine, Paracoccus denitrificans synthesized three periplasmic soluble c-type cytochromes, cytochrome c550 and two additional cytochromes which were not present during growth on succinate and have not previously been characterized. These cytochromes have been separated from each other and their physical properties have been determined. The inducible cytochromes, c551i and c553i, exhibit Mr and pI values of 22,000 and 3.5, and 30,000 and 3.8, respectively. Cytochrome c553i binds CO. None of these cytochromes accepted electrons directly from methylamine dehydrogenase, but each accepted electrons from amicyanin, the electron acceptor for methylamine dehydrogenase. Cytochrome c551i was the most efficient electron acceptor for amicyanin, exhibiting a half-time of reaction at least 6 and 15 times faster than those observed for cytochromes c553i and c550, respectively. PMID- 3013856 TI - Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells. AB - A simple enzymatic method for the quantitation of the mass of sn-1,2 diacylglycerol (DAG) present in crude lipid extracts was developed to assess the function of DAGs as intracellular "second messengers" of extracellular agents and of oncogene products. The assay employed Escherichia coli DAG kinase which constituted approximately 15% of the membrane protein of a plasmid-bearing strain and defined mixed micellar conditions to solubilize the DAG present and allow its quantitative conversion to [32P]phosphatidic acid. The assay was proportional with the amount of DAG added over the range of 25 pmol to 25 nmol. The rapid rise of DAG in platelets stimulated with thrombin (210% over basal) and in hepatocytes stimulated with vasopressin (230% over basal) was quantitated and the values agreed with previous measurements. The amounts of DAG in normal rat kidney (NRK) cells grown at 34 and 38 degrees C, respectively, were 0.47 and 0.61 nmol/100 nmol of phospholipid. In K-ras-transformed NRK cells grown at 34 or 38 degrees C, DAG levels were elevated 168 or 138%, respectively. When a temperature-sensitive K-ras NRK cell line was investigated, the amount of DAG present was elevated at the permissive but not at the restrictive temperature. These data are consistent with the K-ras protein functioning in transmembrane signalling by activating phospholipase C. Protein kinase C (Ca2+/phospholipid-dependent enzyme) activation by DAG may play an important role in cellular transformation. PMID- 3013857 TI - Effect of the bacterial DNA gyrase inhibitors, novobiocin, nalidixic acid, and oxolinic acid, on oxidative phosphorylation. AB - When incubated with isolated intact rat liver mitochondria, novobiocin and nalidixic acid act as uncouplers of oxidative phosphorylation; they stimulate oxygen uptake and inhibit ATP synthesis. Novobiocin is about as powerful an uncoupler as is 2,4-dinitrophenol, nalidixic acid is somewhat less powerful, and oxolinic acid exerts no inhibition whatsoever at the concentrations used. The three inhibitors are without effect on oxidative phosphorylation in Escherichia coli nor does novobiocin affect this process in a novobiocin-permeable mutant of yeast. While it would appear that oxolinic acid may be a relatively specific tool for the manipulation of the superhelicity of DNA in complex systems such as mammalian mitochondria and intact mammalian cells, the specificity of each of these inhibitors may depend upon the particular conditions and species used and such experiments require adequate controls on oxidative phosphorylation. PMID- 3013858 TI - Inhibitors of Na+/H+ exchange block epinephrine- and ADP-induced stimulation of human platelet phospholipase C by blockade of arachidonic acid release at a prior step. AB - The ability of epinephrine or ADP to cause an increase in the production of phospholipase C products (diacylglycerol and inositol phosphates) in human platelets is blocked by perturbants of Na+/H+ exchange, i.e. ethylisopropylamiloride, decreased extraplatelet pH, or removal of extraplatelet Na+. These perturbants do not, however, block inositol phosphate production in response to 0.2 unit/ml thrombin, indicating that inhibition of Na+/H+ exchange does not inhibit the phospholipase C enzyme directly. Since the cyclooxygenase inhibitor indomethacin and the endoperoxide/thromboxane antagonist SQ29548 block epinephrine- and ADP-induced inositol phosphate production, it can be concluded that these agonists activate phospholipase C secondary to mobilization of arachidonic acid and production of cyclooxygenase products. This conclusion is consistent with the observation that the endoperoxide analogue U46619 causes inositol phosphate production. Furthermore, the effect of U46619 is not blocked by inhibitors of Na+/H+ exchange. The initial pool of arachidonic acid mobilized by epinephrine can be measured using negative ion gas chromatography/mass spectrometry and is sensitive to inhibition of Na+/H+ exchange. The present data suggest that epinephrine and ADP cause mobilization of a small pool of arachidonic acid by a pathway involving Na+/H+ exchange. The cyclooxygenase products derived from this pool subsequently activate phospholipase C. Since the same treatments that block epinephrine- and ADP-induced diacylglycerol and inositol phosphate production also block epinephrine- and ADP-induced dense granule secretion, it appears that activation of phospholipase C, albeit indirectly via cyclooxygenase products, may be required for epinephrine and ADP to evoke platelet secretion. PMID- 3013859 TI - Evidence that Na+/H+ exchange regulates receptor-mediated phospholipase A2 activation in human platelets. AB - Data in the previous paper suggest that epinephrine can mobilize a small pool of arachidonic acid via an enzymatic pathway distinct from phospholipase C and that this pathway is blocked by perturbations that block Na+/H+ exchange. The present studies demonstrate that epinephrine and ADP stimulate a phosphatidylinositol hydrolyzing phospholipase A2 activity in human platelets. This occurs even when measurable phospholipase C activation, platelet secretion, and secondary aggregation are blocked with the thromboxane A2 receptor antagonist SQ29548. Furthermore, perturbants of Na+/H+ exchange diminish lysophosphatidylinositol production in response to epinephrine, ADP, and thrombin, but not to the Ca2+ ionophore A23187. Artificial alkalinization of the platelet interior with methylamine reverses the effect of the Na+/H+ antiporter inhibitor, ethylisopropylamiloride, on thrombin-stimulated lysolipid production, suggesting that the alkalinization of the platelet interior which would occur secondary to activation of Na+/H+ exchange might play an important role in phospholipase A2 activation. In addition, treatment of platelets with methylamine increases the sensitivity of phospholipase A2 to activation by the Ca2+ ionophore A23187, suggesting that changes in pH and Ca2+ may regulate phospholipase A2 activity synergistically. Finally, epinephrine causes a prompt decrease in platelet chlortetracyclin fluorescence even in the presence of cyclooxygenase inhibitors, suggesting that epinephrine is able to mobilize membrane-bound Ca2+ independent of phospholipase C activation. Taken together, the data suggest that epinephrine provoked stimulation of phospholipase A2 activity may occur as a result of Ca2+ mobilization and a concomitant intraplatelet alkalinization resulting from accelerated Na+/H+ exchange. PMID- 3013861 TI - Biosynthesis of bacterial glycogen. Primary structure of Escherichia coli 1,4 alpha-D-glucan:1,4-alpha-D-glucan 6-alpha-D-(1, 4-alpha-D-glucano)-transferase as deduced from the nucleotide sequence of the glg B gene. AB - The nucleotide sequence of the glg B gene, coding for branching enzyme (EC 2.4.1.18), was elucidated. It consists of 2181 base pairs specifying a protein of 727 amino acids. The deduced amino acid sequence was consistent with the amino acid analysis that was obtained with the pure protein as well as with the molecular weight determined from sodium dodecyl sulfate-gel electrophoresis. The deduced amino acid sequence was also consistent with the amino-terminal amino acid sequence and the amino acid sequence analysis of various peptides obtained from CNBr degradation of purified branching enzyme. PMID- 3013860 TI - Insulin stimulates cellular iron uptake and causes the redistribution of intracellular transferrin receptors to the plasma membrane. AB - Insulin stimulates the accumulation of iron by isolated fat cells by increasing the uptake of diferric transferrin. Analysis of the cell-surface binding of diferric 125I-transferrin indicated that insulin caused a 3-fold increase in the cell surface number of transferrin receptors. This result was confirmed by the demonstration that insulin increases the binding of an anti-rat transferrin receptor monoclonal antibody (OX-26) to the surface of fat cells. The basis of this effect of insulin was examined by investigating the number of transferrin receptors in membrane fractions isolated from disrupted fat cells. Two methods were employed. First the binding isotherm of diferric 125I-transferrin to the isolated membranes was studied. Second, the membranes were solubilized with detergent, and the number of transferrin receptors was measured by immunoblotting using the monoclonal antibody OX-26. It was observed that insulin treatment of intact fat cells resulted in an increase in the number of transferrin receptors located in the isolated plasma membrane fraction of the disrupted fat cells. Furthermore, the increase in the number of plasma membrane transferrin receptors was associated with a concomitant decrease in the transferrin receptor number in a low density microsome fraction previously shown to consist of intracellular membranes. This redistribution of transferrin receptors between cellular membrane fractions in response to insulin is remarkably similar to the regulation by insulin of glucose transporters and type II insulin-like growth factor receptors. We conclude that insulin stimulates fat cell iron uptake by a mechanism that may involve the redistribution of transferrin receptors from an internal membrane compartment (low density microsomes) to the cell surface (plasma membrane). PMID- 3013862 TI - Na+/HCO3-co-transport in basolateral membrane vesicles isolated from rabbit renal cortex. AB - Recent studies suggest that the major pathway for exit of HCO3- across the basolateral membrane of the proximal tubule cell is electrogenic Na+/HCO3- co transport. We therefore evaluated the possible presence of Na+/HCO3- co-transport in basolateral membrane vesicles isolated from the rabbit renal cortex. Imposing an inward HCO3- gradient induced the transient uphill accumulation of Na+, and imposing an outward Na+ gradient caused HCO3- -dependent generation of an inside acid pH gradient as monitored by quenching of acridine orange fluorescence, findings consistent with the presence of Na+/HCO3- co-transport. In the absence of other driving forces, generating an inside-positive membrane potential by imposing an inward K+ gradient in the presence of valinomycin caused net Na+ uptake via a HCO3- -dependent pathway, indicating that Na+/HCO3- co-transport is electrogenic and associated with a flow of negative charge. Imposing transmembrane Cl- gradients did not appreciably affect HCO3- gradient-stimulated Na+ influx, suggesting that Na+/HCO3- co-transport is not Cl- -dependent. The rate of HCO3- gradient-stimulated Na+ influx was a simple, saturable function of the Na+ concentration (Km = 9.7 mM, Vmax = 160 nmol/min/mg of protein), was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (I50 = 100 microM), but was inhibited less than 10% by up to 1 mM amiloride. We could not demonstrate a HCO3- -dependent or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive component of Na+ influx in microvillus membrane vesicles. This study thus indicates the presence of a transport system mediating electrogenic Na+/HCO3- co transport in basolateral, but not luminal, membrane vesicles isolated from the rabbit renal cortex. Analogous to the use of renal microvillus membrane vesicles to study Na+/H+ exchange, renal basolateral membrane vesicles may be a useful model system for examining the kinetics and possible regulation of Na+/HCO3- co transport. PMID- 3013864 TI - Vasopressin and/or glucagon rapidly increases mitochondrial calcium and oxidative enzyme activities in the perfused rat liver. AB - Mitochondria were prepared by a method including a Percoll purification step after the rapid homogenization of livers of fed rats which had been perfused either under unstimulated conditions or in the presence of vasopressin and/or glucagon. The two hormones separately or together increased the total calcium content of the mitochondria. This enhancement was accompanied by parallel increases in activities of the Ca2+-sensitive intramitochondrial enzymes pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. The effects of the two hormones on total mitochondrial calcium and on the activities of the oxidative enzymes were additive. The persistent enhancements of mitochondrial calcium content and enzyme activities were partially reversed by the addition of Na+ ions to the mitochondrial incubations; these effects of Na+ were blocked by diltiazem, a selective inhibitor of Na+-induced Ca2+ release. Mitochondria from control livers were incubated in vitro with CaCl2 to achieve various calcium content, and mitochondrial enzyme activities and calcium content were measured. A good correlation was obtained between the total calcium content and the activities of pyruvate dehydrogenase and oxoglutarate dehydrogenase. The results obtained are consistent with the hypothesis that vasopressin and glucagon additively cause increases in intramitochondrial [Ca2+] and so bring about the activations of these key enzymes of mitochondrial oxidative metabolism. PMID- 3013863 TI - Mechanism of activation of fructose-2,6-bisphosphatase by cAMP-dependent protein kinase. AB - The bisphosphatase reaction sequence of rat liver 6-phosphofructo-2 kinase/fructose-2,6-bisphosphatase involves a phosphoenzyme intermediate. Catalysis is activated in vitro by cAMP-dependent protein kinase-catalyzed phosphorylation. We investigated the mechanism of this activation by studying the effect of protein kinase-catalyzed phosphorylation on the formation and breakdown of the phosphoenzyme intermediate. The significant findings were as follows. 1) Phosphorylation decreased the rate of phosphoenzyme formation. 2) More importantly, phosphorylation increased the much slower rate of phosphoenzyme breakdown both in the absence and presence of the regulatory ligands, inorganic phosphate and alpha-glycerol phosphate. The increase in the rate of phosphoenzyme breakdown correlated with the degree of activation of the bisphosphatase; both were increased about 2-fold. 3) The potent inhibition of phosphoenzyme breakdown by fructose 6-phosphate indicates that, in the catalytic sequence, the release of nascent fructose 6-phosphate from the active site precedes phosphoenzyme breakdown and Pi release. 4) Phosphorylation reduced the fructose 6-phosphate inhibition of phosphoenzyme breakdown both in the absence and presence of phosphate and alpha-glycerol phosphate. 5) Phosphorylation decreased the potent substrate inhibition which occurs at physiological substrate concentrations. It appears that protein kinase-catalyzed phosphorylation activates fructose-2,6 bisphosphatase by promoting the dissociation of fructose 6-phosphate and fructose 2,6-bisphosphate from the same phosphoenzyme intermediate, hastening its exposure to water and thereby relieving both product and substrate inhibitions. PMID- 3013865 TI - Effects of beta-adrenergic stimulation on 1-O-hexadecyl-2-acetyl-sn-glycero-3 phosphocholine-mediated vasoconstriction and glycogenolysis in the perfused rat liver. AB - The beta-adrenergic agonist isoproterenol inhibited the glycogenolytic response of platelet-activating factor (AGEPC, 1-O-hexadecyl-2-acetyl-sn-glycero-3 phosphocholine) in perfused livers derived from fed rats. AGEPC-stimulated hepatic vasoconstriction, measured by increases in portal vein pressure, also was inhibited by prior isoproterenol infusion. Isoproterenol-mediated inhibition of these hepatic responses to AGEPC was not apparent when isoproterenol (10 microM) was coinfused with the beta-receptor antagonist propranolol (75 microM) or when isoproterenol was replaced with the alpha-adrenergic agonist phenylephrine (10 microM). alpha-Agonist-induced glycogenolysis and vasoconstriction in the perfused liver was unaffected by isoproterenol infusion. Glucagon (2.3 nM) had no effect on the glycogenolytic or vasoconstrictive responses of the liver to AGEPC despite the fact that glucagon increased hepatic cAMP levels to a far greater extent than isoproterenol. Additionally, inhibition of the hepatic responses to AGEPC by isoproterenol occurred in perfused livers from mature rats (i.e. greater than 300 g) in which liver parenchymal cells lack functional beta-adrenergic receptors. The data presented in this study illustrate a specific inhibition of AGEPC-induced hepatic glycogenolysis and vasoconstriction by beta-adrenergic stimulation of the perfused liver. This inhibition appears to be mediated by interaction of isoproterenol with nonparenchymal cells within the liver. These findings are consistent with the concept that AGEPC stimulates hepatic glycogenolysis by an indirect mechanism involving hepatic vasoconstriction. PMID- 3013866 TI - ATP-dependent calcium transport in rat parotid basolateral membrane vesicles. Modulation by agents which elevate cyclic AMP. AB - ATP-dependent Ca2+ transport was studied in basolateral membrane vesicles prepared from rat parotid gland slices incubated without or with agents which increase cyclic AMP. Isoproterenol (10(-5) M), forskolin (2 X 10(-6) M) and 8 bromocyclic AMP (2 X 10(-3) M) all increased ATP-dependent 45Ca2+ uptake 1.5- to 3-fold. The effect of isoproterenol was concentration-dependent and blocked by the beta-adrenergic antagonist propranolol. Enhanced uptake did not appear an artifact of vesicle preparation as apparent vesicle sidedness, 45Ca2+ efflux rates, specific activity of marker enzymes and equilibrium Ca2+ content were identical in vesicle preparations from control and 8-bromocyclic AMP-treated slices. Kinetic studies showed the ATP-dependent Ca2+ transport system in vesicles from 8-bromocyclic AMP-treated slices displayed a approximately 50% increase in Vmax and in Km Ca2+, compared to controls. The data suggest that physiological secretory stimuli to rat parotid acinar cells, which involve cyclic AMP, result in a readjustment of the basolateral membrane ATP-dependent Ca2+ pump. PMID- 3013867 TI - Role of glycosylation in transport of vesicular stomatitis virus envelope glycoprotein. A new class of mutant defective in glycosylation and transport of G protein. AB - A temperature-sensitive mutant (ts gamma 1) of the Cocal serotype of vesicular stomatitis virus synthesizes at the permissive temperature (32 degrees C) a glycoprotein G whose size is smaller (Mr 68,000) than the wild-type (Mr 71,000) and that renders the virion thermolabile. At the nonpermissive temperature (39 degrees C), reduced amounts of noninfectious virus-like particles deficient in G protein were produced. The size of the intracellular G protein was further decreased (Mr 64,000) at the nonpermissive temperature. Biochemical studies including sugar labeling, tryptic peptide analysis, and NH2-terminal sequence analysis of the various glycoproteins suggest that at 32 degrees C a G protein containing a single glycosidic moiety is synthesized. The G protein containing only 1 oligosaccharide residue is transported to the cell surface and is incorporated in infectious virus particles. In contrast, the G protein synthesized at 39 degrees C is nonglycosylated and fails to reach the cell surface. These results suggest that glycosylation of G protein is essential for its transport to the cell surface, and the presence of a single carbohydrate chain is sufficient for this purpose. PMID- 3013868 TI - Subunit structure and properties of the glycogen-bound phosphoprotein phosphatase from skeletal muscle. AB - A high molecular weight phosphoprotein phosphatase was purified approximately 11,000-fold from the glycogen-protein complex of rabbit skeletal muscle. Polyacrylamide gel electrophoresis of the preparation in the absence of sodium dodecyl sulfate showed a major protein band which contained the activity of the enzyme. Gel electrophoresis in the presence of sodium dodecyl sulfate also showed a major protein band migrating at 38,000 daltons. The sedimentation coefficient, Stokes radius, and frictional ratio of the enzyme were determined to be 4.4 S, 4.4 nm, and 1.53, respectively. Based on these values the molecular weight of the enzyme was calculated to be 83,000. The high molecular weight phosphatase was dissociated upon chromatography on a reactive red-120 agarose column. The sedimentation coefficient, Stokes radius, and frictional ratio of the dissociated enzyme (termed monomer) were determined to be 4.1 S, 2.4 nm, and 1.05, respectively. The molecular weight of the monomer enzyme was determined to be 38,000 by polyacrylamide gel electrophoresis. Incubation of the high molecular weight phosphatase with a cleavable cross-linking reagent, 3,3' dithiobis(sulfosuccinimidyl propionate), showed the formation of a cross-linked complex. The molecular weight of the cross-linked complex was determined to be 85,000 and a second dimension gel electrophoresis of the cleaved cross-linked complex showed that the latter contained only 38,000-dalton bands. Limited trypsinization of the enzyme released a approximately 4,000-dalton peptide from the monomers and dissociated the high molecular weight phosphatase into 34,000 dalton monomers. It is proposed that the catalytic activity of the native glycogen-bound phosphatase resides in a dimer of 38,000-dalton subunits. PMID- 3013869 TI - Trimeric structure and localization of the major lipoprotein in the cell surface of Escherichia coli. AB - A hybrid gene consisting of the ompF promoter, the coding regions for the signal peptide, and the Ala-Glu residue of the OmpF NH2 terminus and the coding region for the major outer membrane lipoprotein devoid of the NH2-terminal cysteine residue was constructed. Escherichia coli carrying the cloned gene produced the predicted hybrid protein that is the same as the major lipoprotein except that the diacyl glycerylcysteine residue at the NH2 terminus is replaced by the Ala Glu residue. The hybrid protein was localized in the periplasmic space as a trimer with a noncovalent interaction in addition to the previously known covalent interaction with the peptidoglycan. These results strongly indicate that the major lipoprotein exists as a trimer in the periplasmic space with covalent and noncovalent interactions with the peptidoglycan layer through the protein domain on one side and with the hydrophobic interaction with the outer membrane through the lipid domain on the other side. The trimeric structure of the lipoprotein was directly demonstrated by the chemical cross-linking of the native lipoprotein with both cleavable and uncleavable reagents. The cross-linking study also revealed interaction between the lipoprotein and the OmpA protein, a major outer membrane protein. PMID- 3013870 TI - Structure and complete nucleotide sequence of the chicken alpha-smooth muscle (aortic) actin gene. An actin gene which produces multiple messenger RNAs. AB - The alpha-smooth muscle (aortic) actin gene is a distinct member of the actin multigene family which is expressed in vascular smooth muscle cells. We have determined the complete nucleotide sequence of 11 kilobase pairs of genomic DNA encoding the chicken alpha-smooth muscle actin gene. This single copy gene specifies a protein identical in sequence to the major alpha-actin from bovine aorta. The protein-coding sequences are interrupted by seven introns which are at codons specifying amino acid residues 41/42, 84/85, 121/122, 150, 204, 267, and 327/328. An eighth intron was found in the mRNA 5' untranslated region. The 5' flanking sequences contain elements which are conserved in other chicken muscle actin genes. Additional sequences at the 5' end of the gene may be conserved in at least one human actin gene. We have identified at least four messenger RNAs ranging in size from approximately 1370 to 2700 nucleotides (excluding poly(A) tails) which are transcribed from the alpha-smooth muscle actin gene. These RNAs differ in the length of their 3' untranslated regions, probably as a result of the utilization of alternative polyadenylation signals. This is the first report of an actin gene with multiple mRNA transcripts. PMID- 3013872 TI - Eukaryotic DNA diverges at a long and complex pyrimidine.purine tract that can adopt altered conformations. AB - A domain exhibiting major sequence divergences among three cloned repeat units of a complex satellite DNA of the Bermuda land crab contains a repetitive polypyrimidine.polypurine segment consisting of a long C.G tract embedded between runs of CCT.AGG and CGCAC.GTGCG and their variations. The domain adopts at least two types of altered conformations that are markedly affected by pH and negative superhelical density; only one is sensitive to ionic strength. Supercoil dependent distortions in helical structure are most pronounced at points of interruption in compositional bias in this domain and a similar although less extensive, divergent domain nearby. Since the domain is the site of major sequence divergences among individual satellite repeat units, the altered conformations may be involved in site-specific recombination between repeat units, either those arranged in tandem or those scattered throughout the genome. PMID- 3013871 TI - In vitro and in vivo activation of the insulin receptor kinase in control and denervated skeletal muscle. AB - Skeletal muscle rapidly develops severe insulin resistance following denervation, although insulin binding is unimpaired. Insulin-stimulated receptor tyrosyl kinase activity was studied in intact and 24-h denervated rat hind limb muscles using three preparations: (a) solubilized insulin receptors incubated +/- insulin with gamma-[32P]ATP and histone H2b; (b) soleus muscles prelabeled in vitro with [32P]phosphate with subsequent insulin-stimulated phosphorylation of the receptor in situ; (c) assessment of in vivo activation of muscle receptor tyrosyl kinase by insulin. The latter was achieved by solubilizing muscle insulin receptors in the presence of phosphoprotein phosphatase and kinase inhibitors and measuring receptor-catalyzed histone H2b phosphorylation in the presence of limiting (5 microM) gamma-[32P]ATP. Receptors isolated 5 and 30 min after intravenous insulin injection catalyzed 32P incorporation into histone H2b twice as fast as those from saline-treated controls; insulin stimulated histone H2b labeling exclusively on tyrosine. In vivo activation was demonstrated using solubilized and insulin agarose-bound receptors. Autophosphorylation of the beta-subunit and receptor tyrosyl kinase activity toward histone H2b was stimulated by insulin in denervated muscles as in controls, although the biological response to insulin, in vitro and in vivo, was markedly impaired after denervation, suggesting a postreceptor defect. The method developed to assess insulin-stimulated receptor activation in vivo seems useful in characterizing mechanisms of insulin resistance. PMID- 3013874 TI - Large complex globular domains of type VII procollagen contribute to the structure of anchoring fibrils. AB - Type VII collagen, in the form of an antiparallel dimer, is a major protein component of anchoring fibrils. The ultrastructural appearance of these fibrils suggests that they may serve to anchor the basement membrane zone to the underlying connective tissue matrix. We report here the identification and initial characterization of Type VII procollagen, recovered from the media of epidermoid carcinoma cell cultures. Immunoblotting using monospecific antibodies to Type VII procollagen identifies a single, homogeneous band of at least Mr 320,000 following disulfide bond reduction. This chain contains 170 kDa of collagen triple helix and 150 kDa of non-helical domain at the carboxyl terminus. Pepsin digestion of this material yields Type VII collagen identical to that isolated from whole tissue and a series of quasi-stable peptides derived from the carboxyl-terminal region. Cell extracts contain procollagen chains identical in size to those secreted into the media. There is no evidence for processing of this material in cell culture. Partial purification by velocity sedimentation and transmission electron microscopic observation following rotary shadowing reveals both monomers (426 nm) and dimers (785 nm). Dimers are antiparallel and interact through 60-nm overlap, with amino-terminal globular domains present at the ends of the overlap. The multi-domain carboxyl-terminal region appears as three similar arms originating from a centralized globular region adjacent to the collagen helix. The carboxyl globular domain is present in whole tissue and may participate in the unique fibril form of this collagen. The amino-terminal globule may function in the antiparallel assembly of dimers. PMID- 3013875 TI - The type II insulin-like growth factor receptor is internalized and recycles in the absence of ligand. AB - Recent studies have demonstrated that ligand-bound insulin-like growth factor (IGF)-II receptors on the adipocyte cell surface are rapidly internalized into an intracellular membrane fraction prior to recycling to the plasma membrane (Oka, Y., Rozek, L. M., and Czech, M. P. (1985) J. Biol. Chem. 260, 9435-9442). In order to evaluate whether these subcellular movements of IGF-II receptors in fat cells require their binding to ligand, cell surface IGF-II receptors of insulin treated fat cells were iodinated with Na125I and lactoperoxidase at 15 degrees C. IGF-II receptors were then localized by immunoadsorption from solubilized cell surface plasma membranes and intracellular low density microsomes derived from labeled cells. When fat cells were homogenized immediately after iodination, most of the labeled IGF-II receptors were associated with the plasma membrane fraction. However, when iodinated fat cells were incubated at 37 degrees C for various times before homogenization, labeled IGF-II receptors progressively decreased in the plasma membrane fraction and concomitantly increased in the low density microsome fraction with a half-time of about 5 min. The rate of increase of radiolabeled IGF-II receptors appearing in the low density microsomes of labeled fat cells incubated with insulin was not changed by the addition of a saturating concentration of IGF-II. These results indicate that cell surface IGF II receptors are rapidly internalized and recycled even in the absence of ligand binding in insulin-treated adipocytes. PMID- 3013873 TI - Identification of serine 24 as the unique site on the transferrin receptor phosphorylated by protein kinase C. AB - Addition of tumor-promoting phorbol diesters to [32P]phosphate-labeled A431 human epidermoid carcinoma cells caused an increase in the phosphorylation state of the transferrin receptor. The A431 cell transferrin receptor was also found to be a substrate for protein kinase C in vitro. Tryptic phosphopeptide mapping of the transferrin receptor resolved the same two phosphopeptides (X and Y) after either protein kinase C phosphorylation in vitro or treatment of labeled A431 cells with phorbol diesters. [32P]Phosphoserine was the only labeled phosphoamino acid detected. Phosphopeptide X was shown to be an incomplete tryptic digestion product which could be further digested with trypsin to generate the limit tryptic phosphopeptide (Y). Radiosequence analysis of [32P]phosphopeptide Y demonstrated that the [32P]phosphoserine was the second residue from amino terminus of the peptide. This receptor phosphopeptide was found to co-migrate with the synthetic peptide Phe-Ser(P)-Leu-Ala-Arg (where Ser(P) is phosphoserine) during reverse-phase high pressure liquid chromatography and two-dimensional thin layer electrophoresis and chromatography. The peptide Phe-Ser(P)-Leu-Ala-Arg is an expected tryptic fragment of the cytoplasmic domain of the transferrin receptor corresponding to residues 23-27. We conclude that the major site of protein kinase C phosphorylation of the transferrin receptor in vivo and in vitro is serine 24. This phosphorylation site is located within the intracellular domain of the transferrin receptor, 38 residues away from the predicted transmembrane domain. PMID- 3013876 TI - Transferrin receptor number, synthesis, and endocytosis during erythropoietin induced maturation of Friend virus-infected erythroid cells. AB - Erythropoietin (EP) responsive Friend virus-infected erythroid cells had 200,000 steady-state binding sites for transferrin at 37 degrees C when isolated from the spleens of Friend virus-infected mice. Upon culture of these cells with EP, the synthesis of transferrin receptors increased 4- to 7-fold and the number of transferrin-binding sites per cell doubled after 24 h. However, the rate of uptake of 59Fe from transferrin remained constant at approximately 35,000 atoms of 59Fe per minute per cell during this period in culture. The amount of 125I transferrin internalized during the steady-state binding did not change during this culture period while the transferrin bound to the surface increased 3-fold. At all stages of erythroid maturation, the maximum rate of endocytosis was determined to be 18,000 molecules of transferrin per minute per cell, and the interval that 125I-transferrin remains in the interior of the cell was calculated to be 6.9 min. After 48 h of culture with EP, the number of steady-state transferrin-binding sites was reduced in part due to the sequestration of surface receptors within the cell. The uptake of iron from transferrin was limited by the level of endocytosis of transferrin during the initial phase of culture and the number of transferrin receptors at the cell surface during the latter stages of erythroid maturation of these cells. PMID- 3013877 TI - The nuclear-coded subunits of yeast cytochrome c oxidase. The amino acid sequences of subunits VII and VIIa, structural similarities between the three smallest polypeptides of the holoenzyme, and implications for biogenesis. AB - The complete amino acid sequences of subunits VII and VIIa from yeast cytochrome c oxidase are reported. Subunits VII and VIIa are 57 residues (Mr = 6603) and 54 residues (Mr = 6303) in length, respectively. Both polypeptides are amphiphilic, have an internal hydrophobic section and hydrophilic NH2 and COOH termini, and terminate at their COOH termini with a basic amino acid. This structural motif is similar to that possessed by subunit VIII of yeast cytochrome c oxidase. All three polypeptides have hydrophobic sections which are long enough to span the inner membrane; all three polypeptides lack methionine at their NH2 termini; and all three polypeptides have COOH termini which could result from proteolysis by a protease with trypsin or cathepsin B-like activity. These observations raise the interesting possibility that subunits VII, VIIa, and VIII are transmembranous polypeptides which are processed at both their NH2 and COOH termini during their biogenesis. PMID- 3013878 TI - Purification of N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes. AB - An N-ethylmaleimide-sensitive ATPase was purified 100-fold from chromaffin granule membranes. The purification procedure included solubilization with polyoxyethylene 9 lauryl ether, chromatography on hydroxylapatite and DEAE cellulose columns, and glycerol gradient centrifugations. Inclusion of phosphatidylserine and a mixture of protease inhibitors during the purification procedure was necessary to maintain the activity of the preparation. The purified preparation contained four major polypeptides with molecular masses of about 115, 72, 57, and 39 kDa, which were copurified with the ATPase activity. The 115-kDa subunit binds [14C]dicyclohexylcarbodiimide and the subunits of 115 and 39 kDa bind [14C]N-ethylmaleimide. The ATP-dependent proton uptake activity of chromaffin granule membranes is inhibited 50% with about 20 microM N ethylmaleimide, while over 5 mM concentrations of the inhibitor were required to block the ATPase activity of the membranes. The ATPase activity of the purified enzyme was inhibited via two different affinities: a high affinity site with a Ki in the microM range and a low affinity site in the mM range, each contributing to about 50% inhibition of the enzyme. It is concluded that the proton-ATPase of chromaffin granule membranes contains at least four subunits with the 115-kDa polypeptide being the main subunit having the active site for the ATPase activity of the enzyme. PMID- 3013879 TI - The glycosylated seed storage proteins of Glycine max and Phaseolus vulgaris. Structural homologies of genes and proteins. AB - Considerable information is now available concerning the 7 S seed storage proteins of legumes and the genes that encode them. Our study compares the gene encoding a beta-type subunit of phaseolin (Pvu beta), the 7 S protein of common bean (Phaseolus vulgaris), with the gene encoding an alpha'-subunit of beta conglycinin (Gma alpha'), the 7 S protein of soybean (Glycine max). The comparison involves 2880 base pairs of Pvu beta and 3636 base pairs of Gma alpha' and includes approximately 1 kilobase pair of 5'-flanking sequences, and 5' and 3' untranslated sequences, as well as the six exons and five introns that are found to occur in similar positions in both genes. Conserved sequences in the 5' flanking regions of these genes are discussed in light of their potential regulatory role. Published sequences for 7 S genes of pea (Pisum sativum) permit the inference of the nature and direction of evolutionary change and, in particular, show that the major size difference between the large Gma alpha' polypeptide and the smaller polypeptides of pea and common bean is due to a large insertion in the first exon of Gma alpha'. Comparisons of protein primary structure, potential glycosylation sites, and predicted protein hydropathy show that strongly conserved features of 7 S proteins cut across exon boundaries and that nonconserved regions exist that may have potential for protein modification. PMID- 3013880 TI - 24-Hydroxylation of 1,25-dihydroxyergocalciferol. An unambiguous deactivation process. AB - 1,24,25-Trihydroxyergocalciferol was isolated from bovine kidney homogenates incubated with 1,25-dihydroxyergocalciferol and from chick kidney homogenates incubated with 24,25-dihydroxyergocalciferol. The identity was established by ultraviolet absorbance, sensitivity to periodate, nuclear magnetic resonance, and mass spectrometry. The new metabolite had an affinity equal to 1,24,25 trihydroxycholecalciferol for the bovine-thymus and chick-intestinal 1,25 dihydroxyvitamin D receptor and had an affinity twice that of 1,24,25 trihydroxycholecalciferol for the rat-intestinal receptor. It was 3- and 6-fold less competitive than either 1,25-dihydroxycholecalciferol or 1,24,25 trihydroxycholecalciferol, respectively, for the rat plasma vitamin D transport protein. 1,24,25-Trihydroxyergocalciferol was at least 10-fold less active than 1,25-dihydroxycholecalciferol, 1,25-dihydroxyergocalciferol, and 1,24,25 trihydroxycholecalciferol at stimulating intestinal-calcium transport and was also relatively ineffective at stimulating bone-calcium resorption in rats. Moreover, in rats, [3H]1,24,25-trihydroxyergocalciferol was cleared from plasma approximately 40% faster than [3H]1,24,25-trihydroxycholecalciferol. These data suggest that C-24 hydroxylation of 1,25-dihydroxyergocalciferol represents a significant in vivo deactivation step, whereas equivalent deactivation of 1,25 dihydroxycholecalciferol seems to involve metabolic steps subsequent to C-24 hydroxylation (C-24 ketonization). C-24 ketonization of 1,25 trihydroxyergocalciferol would not be anticipated due to the presence of the 24(S)-methyl group. These results reveal further dissimilarities between ergocalciferol and cholecalciferol metabolism in mammals and suggest a mechanism for the lesser tendency of ergocalciferol to cause hypercalcemia relative to cholecalciferol. PMID- 3013882 TI - Enhanced catabolism of low density lipoproteins in rat by lactosaminated Fab fragment. A new carrier of macromolecules to the liver. AB - Proteins conjugated with lactose residues exhibit enhanced hepatic uptake mediated by the galactose receptor. In this study, we demonstrate that lactosaminated Fab fragments (lac-Fab) of IgG can induce hepatic catabolism of specific antigens, especially low density lipoproteins (LDL). lac-Fab and human LDL-lac-Fab complex exhibited specific uptake in isolated rat hepatocytes. In vivo in the rat, lactosamination enhanced plasma clearance of Fab fragments 2 fold and hepatic localization 20-fold. Fab fragments retained their affinity after lactosamination. Hepatic uptake of rat 125I-IgG complexed in vitro with anti-rat lac-Fab was increased almost 5-fold, compared to rat 125I-IgG alone. Injection of rats with anti-LDL lac-Fab induced plasma clearance and hepatic uptake of tracer amounts of previously injected human 125I-LDL, which decreased 50% 10 min after injection of lac-Fab, with 30% present in the liver. Asialofetuin completely inhibited these processes. After a bolus of 6 mg of human LDL, administration of anti-LDL lac-Fab reduced the serum cholesterol of rats to basal values within 2.5 h. These findings suggest that lactosaminated Fab fragments of specific IgGs are effective reagents for inducing hepatic uptake of macromolecules through the galactose receptor. lac-Fab specific for LDL may be an effective hypocholesterolemic agent in vivo. PMID- 3013881 TI - Insulin-like growth factor II binding to the type I somatomedin receptor. Evidence for two high affinity binding sites. AB - We have previously shown that the antireceptor antibody alpha IR-3 inhibits binding of 125I-somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) to the 130-kDa alpha subunit of the type I receptor in human placental membranes, but does not block 125I-insulin-like growth factor II (IGF-II) binding to a similar 130-kDa complex in these membranes. To determine whether the 130-kDa 125I-IGF-II binding complex represents a homologous receptor or whether 125I-IGF-II binds to the type I receptor at a site that is not blocked by alpha IR-3, type I receptors were purified by affinity chromatography on Sepharose linked alpha IR-3. The purified receptors bound both 125I-Sm-C/IGF-I and 125I-IGF-II avidly (KD = 2.0 X 10(-10) M and 3.0 X 10(-10) M, respectively). The maximal inhibition of 125I-Sm C/IGF-I binding by the antibody, however, was 62% while only 15% of 125I-IGF-II binding was inhibited by alpha IR-3. In the presence of 500 nM alpha IR-3, Sm C/IGF-I bound with lower affinity (KD = 6.5 X 10(-10) M) than IGF-II (KD = 4.5 X 10(-10) M) and IGF-II was the more potent inhibitor of 125I-Sm-C/IGF-I binding. These findings suggest that the type I receptor contains two different binding sites. The site designated IA has highest affinity for Sm-C/IGF-I and is blocked by alpha IR-3. Site IB has higher affinity for IGF-II than for Sm-C/IGF-I and is not blocked by alpha IR-3. PMID- 3013884 TI - Regulation of the ompC gene of Escherichia coli. Involvement of three tandem promoters. AB - ompC expression in Escherichia coli K-12 is known to be regulated by the ompB locus, comprising the ompR and envZ genes, and the OmpR protein is believed to act as a positive transcriptional factor. We examined the transcriptional capability of the ompC gene in vitro and found that RNA polymerase could transcribe ompC without a requirement for other transcriptional factors. Furthermore, transcripts from three tandem promoters in ompC were identified in vitro. We employed oligonucleotide-directed site-specific mutagenesis to dissect the promoter region of the gene and assayed the promoters separately for transcriptional ability using fusions to the lacZ gene. The levels of beta galactosidase indicate that ompC expression in vivo is dependent on the function of at least one of the upstream promoters. The function of OmpR appears to be the enhancement of a basal level of ompC expression. From the results of our experiments, the site of action of OmpR was deduced to be in the vicinity of the upstream promoters of ompC. PMID- 3013883 TI - Membrane damage by hemolytic viruses, toxins, complement, and other cytotoxic agents. A common mechanism blocked by divalent cations. AB - Hemolytic viruses, bacterial and animal toxins, the components of activated complement, cationic proteins, and detergents induce a sequence of permeability changes at the plasma membrane that are in every case sensitive to changes in ionic strength and to divalent cations. Individually, each agent exhibits positive cooperativity; when two agents are present together, they show synergy. It is concluded that such cytotoxic agents damage membranes by a common mechanism. Hence permeability changes are unlikely to depend on the formation of specific, protein-lined channels, as previously envisaged in the case of activated complement or certain bacterial toxins. PMID- 3013885 TI - Activation of 45Ca2+ influx and 22Na+/H+ exchange by epidermal growth factor and vanadate in A431 cells is independent of phosphatidylinositol turnover and is inhibited by phorbol ester and diacylglycerol. AB - Both epidermal growth factor (EGF) and vanadate can activate 45Ca2+ influx into A431 epidermal carcinoma cells, without a detectable lag period possibly via a voltage-independent calcium channel. 22Na+/H+ exchange and 45Ca2+ uptake are mutually independent. Neither EGF nor vanadate induce any significant change in the steady-state levels of [1,3-3H]glycerol-labeled diacylglycerol, myo-[2 3H]inositol-labeled inositol trisphosphate or in 32P-labeled polyphosphoinositides or phosphatidic acid over the first 10 min of treatment, suggesting that the EGF receptor is not directly coupled to phosphatidylinositol turnover and that the two ion fluxes are not induced via a kinase C-dependent pathway. An increase in turnover of polyphosphoinositides can be detected in EGF stimulated cells by nonequilibrium labeling with [32P]phosphate, but the increase shows a lag of about 1 min under the conditions used to detect 45Ca2+ influx. Chelation of free Ca2+ decreases but does not abolish the EGF-stimulated turnover. Preincubation with tetradecanoylphorbol acetate or 1-oleoyl-2 acetylglycerol inhibits the increase in 45Ca2+ uptake by both EGF and vanadate. Tetradecanoylphorbol acetate alone does not alter the basal rate of influx when added together with 45Ca2+. Surprisingly, the activation by vanadate and its inhibition by phorbol 12-myristate 13-acetate are unaffected by down-regulation of the EGF receptors through prior incubation with growth factor. Therefore, in A431 cells the activation of Na+/H+ exchange and Ca2+ influx appear to be independent of phosphatidylinositol turnover, and the EGF receptor does not itself function as a Ca2+ channel. Vanadate apparently activates influx through a mechanism distinct from or distal to the EGF receptor. PMID- 3013886 TI - Coupling of the thyrotropin-releasing hormone receptor to phospholipase C by a GTP-binding protein distinct from the inhibitory or stimulatory GTP-binding protein. AB - Thyrotropin-releasing hormone (TRH) stimulated a rapid rise in inositol trisphosphate (IP3) formation and prolactin release from 7315c tumor cells. The potencies (half-maximal) of TRH in stimulating IP3 formation and prolactin release were 100 +/- 30 and 140 +/- 30 mM, respectively. Pretreatment of the cells with pertussis toxin (for up to 24 h) had no effect on either process. Pretreatment of the cells with cholera toxin (30 nM for 24 h) also failed to affect basal or TRH-stimulated IP3 formation. TRH was also able to stimulate IP3 formation with a half-maximal potency of 118 +/- 10 nM in a lysed cell preparation of 7315c cells; the TRH-stimulated formation of IP3 was enhanced by GTP. 5'-Guanosine gamma-thiotriphosphate (GTP gamma S) and 5'-guanylyl imidodiphosphate (Gpp(NH)p), nonhydrolyzable analogs of GTP, stimulated IP3 formation in the absence of TRH with half-maximal potencies of 162 +/- 50 and 7500 +/- 4300 nM, respectively. In contrast to the lack of effect of pertussis toxin on the TRH receptor system, treatment of 7315c cells with pertussis toxin for 3 h or longer completely abolished the ability of morphine, an opiate agonist, to inhibit either adenylate cyclase activity or prolactin release. During this 3-h treatment, pertussis toxin was estimated to induce the endogenous ADP ribosylation of more than 70% of Ni, the inhibitory GTP-binding protein. GTP gamma S and Gpp(NH)p inhibited cholera toxin-stimulated adenylate cyclase activity (presumably by acting at Ni) with half-maximal potencies of 25 +/- 9 and 240 +/- 87 nM, respectively. Finally, Gpp(NH)p was also able to inhibit the [32P]ADP ribosylation of Ni with a half-maximal potency of 300 nM. These results suggest that a novel GTP-binding protein, distinct from Ni, couples the TRH receptor to the formation of IP3. PMID- 3013887 TI - Presence of endogenous calcium ion and its functional and structural regulation in horseradish peroxidase. AB - The endogenous calcium ion (Ca2+) in horseradish peroxidase (HRP) was removed to cause substantial changes in the proton NMR spectra of the enzyme in various oxidation/spin states. The spectral changes were interpreted as arising from the substantial alterations in the heme environments, most likely the heme proximal and distal sides. The comparative kinetic and redox studies revealed that these conformational changes affect the reduction process of compound II, resulting in the decrease of the enzymatic activity of HRP. It is also revealed from the ESR spectrum and the temperature dependences of the NMR and optical absorption spectra of the Ca2+-free enzyme that the heme iron atom of the Ca2+-free enzyme is in a thermal spin mixing between ferric high and low spin states, in contrast to that of the native enzyme. These results show that Ca2+ functions in maintaining the protein structure in the heme environments as well as the spin state of the heme iron, in favor of the enzymatic activity of HRP. PMID- 3013888 TI - The porcine ovarian luteinizing hormone/human chorionic gonadotropin receptor II. Is the purified receptor an oligomer of identical subunits? AB - Purified porcine luteinizing hormone/human chorionic gonadotropin receptors were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis following reduction and thermal denaturation and stained with Coomassie Brilliant Blue. A major protein of Mr = 77 +/- 4 X 10(3) and a minor protein of Mr = 66 +/- 4 X 10(3) were observed. Iodoreceptor proteins were resolved into a major component of Mr = 77 +/- 3 X 10(3) and a minor component of Mr = 62 +/- 5 X 10(3) after reduction and thermal denaturation. In the absence of reduction, the iodoreceptor had a major component of Mr 63 +/- 3 X 10(3). Purified human chorionic gonadotropin specifically transferred part of the iodoreceptor from the Mr = 63 X 10(3) species to an Mr = 110-120 X 10(3) species. Purified receptors were analyzed by nondenaturing polyacrylamide gel electrophoresis and identified by specific binding of iodo-human chorionic gonadotropin. Three binding species with approximate Mr = 60 X 10(3), 130 X 10(3), and 260 X 10(3) were identified. Iodoreceptors co-migrated with the Mr = 60 X 10(3) species under the same conditions. Similar results were obtained following renaturation of receptors separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis without reduction and thermal denaturation. These results suggest for the first time that the porcine corpus luteum luteinizing hormone/human chorionic gonadotropin receptor may be a hormone binding monomer of Mr = 60-65 X 10(3), and that the monomer may associate to form hormone binding polymeric receptor complexes. PMID- 3013889 TI - Electron spin resonance studies on a flavoprotein in neutrophil plasma membranes. Redox potentials of the flavin and its participation in NADPH oxidase. AB - Plasma membrane fractions of stimulated and resting cells were isolated from pig blood neutrophils. The midpoint redox potential (Em) of the membrane-bound flavin was determined potentiometrically by analysis of the flavin free-radical signal by electron spin resonance (ESR) spectroscopy. In both stimulated and resting cells, a peak position of the titration curve gave an Em value of -280 mV at pH 7.0 (Em7). The flavin free radical showed an ESR spectrum at g = 2.004 with a peak to peak width of 19 G, which indicates that the redox intermediate is a neutral semiquinone. Redox titrations were anaerobically examined at 25 degrees C with NADPH in place of dithionite. Addition of NADPH to plasma membranes of stimulated cells resulted in a rapid change in potential, accompanied by the formation of the ESR signal of flavin free radical. Computer simulation of the titration points gave an ambient midpoint potential of -280 mV (Em7). In contrast, those of resting cells showed a very slow change in potential and no g = 2.00 signal formation. Power saturation behavior of the ESR signal showed a marked difference between those of stimulated and resting cells. ESR characteristics of the flavin are discussed in relation to the membrane-bound NADPH oxidase. PMID- 3013890 TI - Isolation and characterization of genomic clones for two chicken phenobarbital inducible cytochrome P-450 genes. AB - A cDNA clone specific for a chicken phenobarbital-inducible cytochrome P-450 was used to screen a chicken genomic library. Twenty-nine clones were isolated, restriction mapped, and divided into two non-overlapping groups. The cDNA clone hybridized to 12 kilobases of DNA from both groups. Both groups contained restriction fragments which hybridized to both 5' and 3' fragments of the cDNA clone, and it was concluded that the two groups were derived from two separate genes. Southern transfer analysis of individual chicken DNAs and quantitative hybridization analysis indicated that these two genes are independent and are present as single copies/haploid genome. Comparison of restriction digests of the cloned DNAs and total genomic DNA discounted the possibility that other closely related P-450 genes are present in the chicken genome. PMID- 3013891 TI - The rat ovarian lutropin receptor. Purification, hormone binding properties, and subunit composition. AB - The luteinizing hormone/human choriogonadotropin (hCG) receptor from superovulated rat ovary was purified to homogeneity. A novel scheme based on reverse immunoaffinity chromatography using immobilized antibodies to membrane proteins from receptor down-regulated ovary and subsequent two-step affinity purification on hCG-Sepharose was used to isolate homogeneous receptor. The purification method was also compared to an alternate scheme involving lectin affinity chromatography followed by hCG affinity chromatography. The purified receptor obtained by the latter method was heterogeneous and highly aggregated. The hormone binding properties, molecular size, and subunit composition of the purified receptor obtained by either method were identical. The stability of the receptor during and following solubilization was markedly improved by using 20% glycerol. The pure receptor consists of four nonidentical subunits of molecular weight 79,300 (alpha), 66,400 (beta), 55,300 (gamma), and 46,700 (delta) as indicated by polyacrylamide gel electrophoresis under reducing conditions. All receptor subunits generally, but occasionally excepting the alpha-subunit, were specifically labeled with iodinated hCG in membrane and soluble receptor preparations using bifunctional cross-linking agents. Analysis of the cross linked hormone-receptor complexes under nonreducing conditions showed the molecular mass of the undissociated receptor to be 268,000 daltons. Hormone binding studies demonstrated that the isolated receptor retained all of the specific binding characteristics expected for the luteinizing hormone/hCG receptor. In combination, these results indicate that the functional and structural properties of the receptor were not altered during purification. PMID- 3013892 TI - Isolation and sequence analysis of cDNA clones for the small subunit of rabbit calcium-dependent protease. AB - We have isolated and sequenced cDNA clones for the small subunit (30-kDa subunit) of rabbit calcium-dependent protease (Ca2+-protease) using synthesized oligodeoxynucleotide probes based on the partial amino acid sequence of the protein. A nearly full-length cDNA clone containing the total amino acid coding sequence was obtained. From the deduced sequence, the following conclusions about possible functions of the protein are presented. The kDa subunit comprises 266 residues (Mr = 28,238). The N-terminal region (64 residues) is mainly composed of glycine (37 residues) and hydrophobic amino acids and may interact with the cell membrane or an organelle. The sequence of the C-terminal 168 residues is highly homologous to the corresponding C-terminal region of the large subunit (80-kDa subunit) which has been identified as the calcium-binding domain. This region of the 30-kDa subunit contains four E-F hand structures and presumably binds Ca2+, as in the case of the 80-kDa subunit. Thus, the 30-kDa subunit may play important roles in regulating enzyme activity and/or possibly in determining the location of the Ca2+-protease. The marked sequence homology of the C-terminal regions of the two subunits may indicate that the calcium-binding domains have evolved from the same ancestral gene. PMID- 3013894 TI - Replication from one of the three origins of the plasmid R6K requires coupled expression of two plasmid-encoded proteins. AB - The minimal beta-replicon of plasmid R6K contains an open reading frame for a 151 amino acid protein in addition to the seven 22-base pair direct nucleotide sequence repeats, the structural gene (pir) for the pi initiation protein, and the beta-origin sequence that have been shown to be required for replication activity. In this work, a site-specific mutation by linker insertion in this putative coding sequence, designated bis, resulted in a nonfunctional beta replicon. The nonfunctional beta-replicon was complementable in trans and the protein coded by the bis sequence was detected in an immunoblot assay as a hybrid product from a bis-lac z fused gene. The bis gene is not required for a functional alpha or gamma origin replication origin of R6K. A site-specific mutation in the upstream pir gene was shown to lead to a loss of synthesis of the bis product and inactivation of the beta-replicon. Trans-complementation of this mutation for beta-replicon activity required the wild-type sequence of the pir gene joined to the intact bis sequence. These results indicate that the bis product is required for activity specifically of the beta-origin, and its synthesis is coupled in cis to the expression of pi protein from an unaltered pir gene. PMID- 3013893 TI - Mechanism of guanine nucleotide regulatory protein-mediated inhibition of adenylate cyclase. Studies with isolated subunits of transducin in a reconstituted system. AB - The retinal nucleotide regulatory protein, transducin, can substitute for the inhibitory guanine nucleotide-binding regulatory protein (Ni) in inhibiting adenylate cyclase activity in phospholipid vesicle systems. In the present work we have assessed the roles of the alpha (alpha T) and beta gamma (beta gamma T) subunit components in mediating this inhibition. The inclusion of either a preactivated alpha T . GTP gamma S (where GTP gamma S is guanosine 5'-O (thiotriphosphate)) complex, or the beta gamma complex, in phospholipid vesicles containing the pure human erythrocyte stimulatory guanine nucleotide-binding regulatory protein (Ns) and the resolved catalytic moiety of bovine caudate adenylate cyclase (C) resulted in inhibition of the GppNHp-stimulated (where GppNHp is guanyl-5'-yl imidodiphosphate) activity (by approximately 30-60 and 90%, respectively, at 2 mM MgCl2). The inhibitions by both of these subunit species are specific for the Ns-stimulated activity with neither alpha T . GTP gamma S nor beta gamma T having any direct effect on the intrinsic activity of the catalytic moiety. Increasing the MgCl2 concentration in the assay incubations significantly decreases the inhibitions by both alpha T . GTP gamma S and beta gamma T. Similarly, when the pure hamster lung beta-adrenergic receptor is included in the lipid vesicles with Ns and C, the levels of inhibition of the GppNHp-stimulated activity by both alpha T . GTP gamma S and beta gamma T are reduced compared to those obtained in vesicles containing just Ns and C (but not stimulatory receptor). These inhibitions are reduced still further under conditions where the agonist stimulation of adenylate cyclase activity is maximal, i.e. when stimulating with isoproterenol plus GTP. In these cases the alpha T . GTP gamma S inhibitory effects are completely eliminated and the inhibitions observed with holotransducin can be fully accounted for by the beta gamma T complex. The ability of the beta-adrenergic receptor to relieve these inhibitions suggests that the receptor may remain coupled to Ns (or alpha s) during the activation of the regulatory protein and the stimulation of adenylate cyclase. These results also suggest that under physiological conditions the beta gamma subunit complex is primarily responsible for mediating the inhibition of adenylate cyclase activity. PMID- 3013895 TI - In vitro attachment of mono- and oligosaccharides to surface glycoconjugates of intact cells. AB - We have synthesized glycosylhydrazines of various mono- and oligosaccharides and coupled these to periodate- or galactose oxidase-treated human red cells and K562 erythroleukemia cells. The optimal conditions for this carbohydrate modification of cells have been established. This method makes it possible to specifically elongate oligosaccharide chains of cell surface glycoconjugates with desired carbohydrates. In this way, new antigenic and receptor properties can be conferred to cells, and the functional roles of carbohydrates in cell surface glycoconjugates can be studied. The method has been used to make red cells of blood group O reactive with anti-A and anti-B sera, and in rendering K562 cells or red cells of blood group O agglutinable with the alpha-N-acetylgalactosamine specific Helix pomatia lectin. PMID- 3013896 TI - Recurrent hepatoma with CT, MRI, and angiographic correlation. AB - Two patients who underwent successful resections for uninodular hepatomas in non cirrhotic livers were readmitted with late recurrences. Both had multiple imaging procedures prior to treatment including computed tomography, magnetic resonance imaging, and angiography. I recommend periodic ultrasonography and alphafetoprotein assay in the follow-up of patients who have had a hepatoma resected and I indicate when additional imaging methods are required. PMID- 3013897 TI - Unusual radiologic presentations of bronchioloalveolar carcinoma. AB - Bronchioloalveolar carcinoma appears with variable radiologic features. The usual findings include a single nodule, multiple nodules, or areas of consolidation in lung. An air bronchogram in an area of nodular or mass-like density is also a well-known feature. We here report six patients whose disease illustrates interesting or unusual aspects of this neoplasm. The findings include: Minimal radiological signs despite diffuse disease found on CT scan or at surgery (two patients). Thickening of the wall of a preexisting lung cavity: presumed bronchogenic cyst. Expansile lobar consolidation without an air bronchogram simulating pleural disease. An elongated lobulated area of mass-like density resembling mucoid impaction. Homogeneous lobar atelectasis without an air bronchogram. Consideration of these varied features may aid in the radiological diagnosis of bronchioloalveolar carcinoma. PMID- 3013898 TI - Molecular cloning of cDNA for rat liver gap junction protein. AB - An affinity-purified antibody directed against the 27-kD protein associated with isolated rat liver gap junctions was produced. Light and electron microscopic immunocytochemistry showed that this antigen was localized specifically to the cytoplasmic surfaces of gap junctions. The antibody was used to select cDNA from a rat liver library in the expression vector lambda gt11. The largest cDNA selected contained 1,494 bp and coded for a protein with a calculated molecular mass of 32,007 daltons. Northern blot analysis indicated that brain, kidney, and stomach express an mRNA with similar size and homology to that expressed in liver, but that heart and lens express differently sized, less homologous mRNA. PMID- 3013900 TI - Transferrin receptors recycle to cis and middle as well as trans Golgi cisternae in Ig-secreting myeloma cells. AB - The recycling itinerary of plasma membrane transferrin receptors (TFR) was charted in IgG-secreting mouse myeloma cells (RPC 5.4) by tagging surface receptors with either bound anti-transferrin receptor antibodies (anti-TFR) or Fab fragments thereof and determining the intracellular destinations of the tagged receptors by immunocytochemistry. By immunofluorescence, TFR tagged with either probe were seen to be rapidly internalized and translocated from the cell surface to the juxtanuclear (Golgi) region. When localized by immunoperoxidase procedures at the electron microscopic level, the anti-TFR-labeled receptors were detected in all cisternae (cis, middle, and trans) of the Golgi stacks as well as in endosomes and trans Golgi reticular elements. There was no difference in the routing of TFR tagged with monovalent Fab and those tagged with divalent IgG. Tagged receptors were detected in Golgi stacks of approximately 50% of the cells analyzed. The position of the labeled cisternae within a given stack was found to be quite variable with cis and middle cisternae more often labeled at 5 min and trans cisternae at 30 min of antibody uptake. The finding that recycling plasmalemmal TFR can visit all or most Golgi subcompartments raises the likely possibility that any Golgi-associated posttranslational modification can occur during recycling as well as during the initial biosynthesis of plasmalemma receptors and other membrane proteins. PMID- 3013899 TI - Membrane traffic in animal cells: cellular glycoproteins return to the site of Golgi mannosidase I. AB - The recycling of cellular glycoproteins to the site of Golgi mannosidase I, an enzyme of asparagine-linked oligosaccharide synthesis, was studied in K562 human erythroleukemia cells. Cells were metabolically labeled in the presence of deoxymannojirimycin, a reversible inhibitor of Golgi mannosidase I. This generates glycoproteins with immature oligosaccharides in their normal locations. Transport to the mannosidase I compartment was then assessed by testing for the conversion of oligosaccharides into mature forms during reculture without deoxymannojirimycin. Transferrin receptor (TfR) was acted on by mannosidase I during reculture, suggesting that it returned to the region of the Golgi complex where this enzyme resides. The slow rate of this transport (t1/2 greater than 6 h) implies that it is probably different than TfR movement during transferrin internalization (t1/2 = 10-20 min) and TfR transport to the sialyltransferase compartment in the Golgi complex (t1/2 = 2-3 h) (Snider, M. D., and O. C. Rogers, 1985, J. Cell Biol., 100:826-834). The total cell glycoprotein pool was also transported to the mannosidase I compartment with a half-time of 4 h. Because this transport is 5-10 times faster than the rate of de novo glycoprotein synthesis in these cells, it is likely that most of the glycoprotein traffic through the Golgi complex is composed of recycling molecules. PMID- 3013902 TI - Demonstration of the specific binding of bovine transferrin to the human transferrin receptor in K562 cells: evidence for interspecies transferrin internalization. AB - Specific binding of ferric bovine transferrin to the human transferrin receptor was investigated using K562 cells propagated in serum-free medium without transferrin supplemented with 10(-5) elemental iron. Affinity chromatography of solubilized extracts of K562 cells surface-labeled with 125I was performed using bovine transferrin- and human transferrin-Sepharose 4B resins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of resin eluates reveal that bovine transferrin specifically binds a Mr = 188,000 protein which dissociates into a Mr = 94,000 protein under reducing conditions, a finding identical to what is seen with human transferrin. The Mr = 94,000 reduced protein isolated by bovine transferrin resin shows an identical one-dimensional partial proteolytic digestion map with that of the human transferrin receptor. Unlabeled bovine transferrin was shown to specifically compete 125I-labeled human transferrin from the human transferrin receptor on the surface of K562 cells at 4 degrees C in a similar manner as unlabeled human transferrin; however, approximately a 2,000 fold higher concentration of bovine ligand was required to achieve comparable competition (50% inhibition of binding). Indirect immunofluorescence cytolocalization of bovine transferrin in K562 cells grown in serum-free medium supplemented with ferric bovine transferrin reveal patterns similar to those seen for human transferrin (both focal perinuclear and diffuse cytoplasmic fluorescence). Monensin treatment results in a dramatic accumulation of bovine ligand in perinuclear aggregates, suggesting that it is recycled through the Golgi, as is human transferrin. K562 cells grown in serum-free medium supplemented with either 300 micrograms/ml of ferric human or ferric bovine transferrin were found to demonstrate superimposable growth curves. PMID- 3013901 TI - Epidermal growth factor receptors associated to cytoskeletal elements of epidermoid carcinoma (A431) cells. AB - The structural interaction of the epidermal growth factor (EGF) receptor and the cytoskeleton of A431 cells has been studied using a monoclonal anti-EGF receptor antibody. This has been done with immunogold labeling using a variety of electron microscopical preparation procedures and EGF binding studies. By providing an image of the membrane-associated cytoskeleton, the dry cleavage method reveals a preferential localization of EGF receptors superimposed upon cytoskeletal filaments. The colocalization of gold particles with cytoskeletal filaments is not affected when pre-labeled cells are extracted with the non-ionic detergent Triton X-100, as visualized by dry cleavage. Using surface replication, this treatment results in visualization of the cytoskeleton. In these latter preparations, it is also observed that EGF receptor-coupled gold particles remain associated with cytoskeletal elements. Moreover, Triton extraction performed before immunogold labeling of EGF receptors demonstrates that isolated cytoskeletons contained binding sites for anti-EGF receptor antibodies. Using stereo micrographs of replica's obtained from these isolated cytoskeletons, it is shown that gold-labeled EGF receptors are exclusively present on the cortical membrane-associated region of the cytoskeleton and not on more intracellular located filaments. Scatchard analysis of EGF binding to cells fixed with glutaraldehyde and treated with Triton X-100 before and after EGF binding indicates that a high affinity EGF binding site is associated with the Triton X 100 insoluble cytoskeleton. PMID- 3013903 TI - Regulation of inositol phosphate levels by prostaglandins in cultured endometrial cells. AB - Stimulation of cultured rabbit endometrial cells by one of the rabbit endometrial cell culture proliferation factors, prostaglandin F2 alpha (PGF2 alpha), resulted in a very rapid increase in the intracellular levels of [3H]-inositol triphosphate (IP3), [3H]-inositol biphosphate (IP2), and [3H]-inositol monophosphate (IP1) in cells prelabeled with [3H]-inositol. These increases in inositol phosphate levels were detected in periods of stimulation as short as 30 seconds, reached a maximum by 1 1/2-2 min and declined to control levels by 6-10 min. The stimulation was dose-dependent with maximal increases observed near 10( 6) M PGF2 alpha. The cholinergic agent, carbachol, also led to time and dose independent increases in IP3. Lithium, cadmium, silver, copper, and zinc ions had no effect either on the breakdown of IP3 or on the accumulation of IP1. In contrast, vanadate at 10(-6) or 10(-5) M did lead to a decrease in the breakdown of IP1 and a concomitant increase in IP1, IP2, and IP3. PGF2 alpha was found previously to induce an increase in rabbit endometrial cell DNA synthesis which was inhibited by concomitant or prior addition of prostaglandin E1 (PGE1). PGE1, in a dose-dependent manner, was found to inhibit the observed IP3 increase by PGF2 alpha at 1 1/2 min of stimulation. PGF2 alpha treated and control cultures did not differ in cAMP or cGMP levels, cellular 45Ca uptake, nor cellular 22Na uptake. We propose that IP3 may be one of the intracellular messenger(s) synthesized following the treatment of rabbit endometrial cell cultures with the proliferation agent PGF2 alpha and that it may play a crucial role with cAMP in growth regulation. PMID- 3013904 TI - Electrophysiological responses of osteoclasts to hormones. AB - Electrophysiological measurements were carried out on osteoclasts in vitro. Such isolated osteoclasts are able to resorb bone in vitro and contract in response to calcitonin (CT). Our measurements show that individual osteoclasts respond to CT with a significant transient hyperpolarization of membrane potential. Application of parathyroid hormone (PTH) and dibutyryl cAMP produced a transient hyperpolarization in some osteoclasts. Measurements on an osteoblastlike line (ROS 17/2.8) showed a sustained hyperpolarizing response to CT, which is similar to but smaller than the hyperpolarizing response to PTH and dibutyryl cAMP in this and some other osteoblastlike lines. In contrast to osteoblastlike cells, the osteoclasts have no long term membrane potential response to CT, to PTH, or to dibutyryl cAMP. These results show that there are distinct differences between osteoclasts and osteoblasts in their ion transport responses to hormones. PMID- 3013905 TI - Volume changes in activated human neutrophils: the role of Na+/H+ exchange. AB - The apparent volume of neutrophils, as measured electronically with the Coulter counter, has been reported to increase upon treatment with chemotactic factors. The occurrence of a volume change was confirmed by forward angle light scattering and by isotopic measurements of intracellular water space in cells treated with 12-O-tetradecanoylphorbol 13,acetate (TPA) or formyl-methionyl-leucyl phenylalanine (FMLP). Cell swelling was associated with an increase in the osmotic content of the cells, determined from Boyle-van't Hoff plots, and with an increase in Na+ content, measured by flame photometry. The volume change was inhibited by replacement of extracellular Na+ with K+ or N-methyl-D-glucamine+, or by addition of amiloride. Swelling was also inhibited by the 5-N-substituted analogs of amiloride, which are potent specific inhibitors of the Na+/H+ antiport. This pathway is activated in neutrophils by both TPA and FMLP. Activation of Na+/H+ exchange, determined as a Na+ -dependent and amiloride sensitive cytoplasmic alkalinization, was also found when neutrophils were treated with hypertonic solutions. The hypertonic activation of the antiport was similarly followed by cell swelling, detectable by electronic sizing. The results indicate that activation of Na+/H+ exchange can lead to significant cell swelling in neutrophils. PMID- 3013906 TI - Hormonal control of the replication of human fetal fibroblasts: role of somatomedin C/insulin-like growth factor I. AB - Sparse cultures of fetal and postnatal human fibroblasts were equivalent in their responsiveness to the mitogenic action of somatomedin C/insulin-like growth factor I (SM-C/IGF-I). At both developmental stages, the addition of SM-C/IGF-I (100 ng/ml) increased cell number at day 3 1.4-fold in serum-free medium and 2 fold in the presence of 0.25% human hypopituitary serum. Furthermore, dose response curves indicated that there was no difference in the sensitivity of fetal and postnatal fibroblasts to the growth-promoting effects of SM-C/IGF-I, with a half-maximal response occurring at 6 ng/ml SM-C/IGF-I. This biological action of SM-C/IGF-I correlated with SM-C/IGF-I binding to fetal and postnatal fibroblast monolayers. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) also stimulated replication of fetal and postnatal fibroblasts. The mitogenic effects of SM-C/IGF-I, EGF, and PDGF were additive. Dexamethasone, which alone had no effect, was synergistic with SM-C/IGF-I in stimulating replication of postnatal fibroblasts. The combination of SM-C/IGF-I (100 ng/ml), dexamethasone (10(-7) M), EGF (10 ng/ml), and PDGF (5 ng/ml) had the same mitogenic effectiveness as 10% calf serum (CS) in postnatal cells. In marked contrast, there was no mitogenic interaction between SM-C/IGF-I and dexamethasone in fetal fibroblasts. In fetal cells, SM-C/IGF-I + EGF + PDGF +/- dexamethasone could only account for 50% of the activity of 10% CS. Moreover, fetal cells were 50-100% more responsive than postnatal cells to the proliferative effect of serum. PMID- 3013908 TI - A set of stage-dependent embryonic antigens expressed in cell cultures of BALB/c mouse embryos and in transformed cell lines. AB - A rabbit antiserum (A2) directed against the detergent-solubilized fraction of the simian virus 40-transformed mouse embryo fibroblast cell line VLM detects common antigens in primary cell cultures from BALB/c mouse embryos and in transformed cell lines from various species. Positively reacting cell cultures show a set of polypeptides with molecular weight species p86, p74, p68, p46, p42, p40, and p35. As tested by Western blotting procedures, all immunoprecipitated proteins carry immunologically reactive determinants. By analysis with two dimensional gel electrophoresis, all precipitated polypeptides show charge heterogeneities. Concerning the two major members of the protein set, p40 consists of at least four subspecies with isoelectric points in the range of pH 6.2-6.8, whereas p35 is composed of two subspecies focusing between pH 6.4 and pH 7.2. By comparison of the two-dimensional patterns of p35 of various transformed cell lines, a basic (pH 6.6-7.2) and an acidic (6.4-6.6) charge type of p35 could be observed. Comparative analyses of primary cell cultures from 12-16-day mouse embryos show the immunoprecipitated set of polypeptides only in the 16-day embryo cell cultures. After six further propagations, these cells express the immunoreactive proteins as strongly as the primary cell cultures. In embryonic cell cultures of day 14 of gestation the expression of this set of antigens is induced only when cells are propagated at least six times. Under identical conditions these proteins could not be induced in cell cultures of 18-day-old mouse embryos. None of the polypeptides could be immunoprecipitated from primary mouse kidney cell cultures of 12-day-old mice even when the cultures were propagated at least 15 times. This set of polypeptides is also present in simian virus 40-transformed cells of hamster, rat, monkey, and human origin. These findings suggest that in simian virus 40-transformed mouse cells, in addition to p53, the synthesis of other embryonic antigens is reactivated. The presence of the described set of polypeptides in polyoma virus-transformed cells of rat and mouse origin and in cell lines derived from malignant human tumors might indicate common functions in metabolic patterns of transformed cells. PMID- 3013907 TI - Calcitonin and vasopressin affect epithelial properties in a renal cell line. AB - We have used a stable clonal variant (D + Sc), isolated from the LLC-PK1 pig kidney-derived cell line and selected for its extensive capacity to form domes, in order to study the hormonal modulation of epithelial permeability in culture. Calcitonin, vasopressin, and other agents that raise intracellular adenosine 3',5'-cyclic monophosphate levels caused a rapid and dramatic decrease in the size and number of domes. This effect was independent of RNA and protein synthesis, and thus appeared unrelated to the production of urokinase, a proteinase synthesized by the cells in response to these agents. Calcitonin caused a decrease in transepithelial electrical resistance, suggesting that the effect of the hormone on domes was due to an increase in the permeability of a paracellular pathway. Thus, in addition to the wellknown effects of vasopressin on collecting duct permeability, part of the in vivo effect(s) of calcitonin and vasopressin on the renal tubule might also involve alterations of epithelial permeability related to those described here. PMID- 3013909 TI - Ligand-receptor interactions: detailed evaluation of occupancy-dependent affinity. AB - The exact nature of the curvilinearity of Scatchard plots derived from hormonal and nonhormonal binding systems has not been definitively resolved. Such plots are compatible with heterogeneous receptors with different but fixed affinities and with negatively interacting binding sites resulting in occupancy-dependent affinity. In the current study we examined in detail the effect of receptor occupancy by the ligand on receptor affinity under a variety of experimental conditions. We chose the human lymphocyte-leukoagglutinin (LPHA) system, which closely mimics the IM9-insulin model. Reliable estimates of total binding capacity (728 ng/10(6) cells) essential to our report were calculated from a wide database by the least-squares model. At occupancies greater than or equal to 0.085, receptors are associated with low and fixed affinity (1.5 X 10(6) M-1), whereas at occupancies less than or equal to 0.085, affinity is high and fixed (1.8 X 10(8) M-1) or high but variable (1 X 10(7) M-1 to 1.5 X 10(6) M-1) depending on whether the binding is assumed to be noncooperative or cooperative, respectively. Calculation of receptor-ligand complex dissociation velocity over a wide range of occupancies (0.01-0.40) suggested that occupancy exerts an inversely proportional effect on affinity that is rapid and sustained. Cell activation (DNA synthesis) is initiated at receptor occupancy of approximately equal to 0.004 and is magnified as ligand binding to high affinity receptors increases up to approximately equal to 0.07 occupancy (functional sites), beyond which point further binding (to low affinity sites) becomes increasingly ineffective and cytotoxic (redundant sites). These findings suggest that occupancy influences affinity as postulated by the hypothesis of negative cooperativity. Through this effect occupancy may play a significant role in regulating ligand-induced cell responses. PMID- 3013910 TI - Shape control in the human red cell. AB - When the human red cell consumes its ATP, the cell loses its discoid character in favour of a spiculated and eventually a spherical form. This discocyte-echinocyte transformation parallels both degradation of phosphatidylinositol 4,5 bisphosphate and phosphatidic acid but not dephosphorylation of cytoskeletal proteins. Dephosphorylation of both spectrin and band 3 lags behind metabolic crenation. Exogenous vanadate accelerates both shape changes and lipid dephosphorylation in a parallel manner during metabolic depletion. In contrast to its effect on lipids, vanadate reduces the rate of protein dephosphorylation. These observations strongly support a shape control mechanism in the red cell, based on phosphoinositide metabolism and compatible with a bilayer-couple model. PMID- 3013911 TI - [Free radicals]. AB - Oxygen in absolutely necessary to life but it is also a toxic gas. 1 to 2% of molecular oxygen undergoes an univalent reduction which produces very reactive and very cytotoxic species. Against them there are different protector antioxidant systems, called scavengers. Pathologically four points are fundamental: Free radicals have a main role in inflammation and fight against bacteria. In carcinogenesis, they have a key role in promotion. The cellular ageing appears to be imputable to a defect of the scavengers. Reflow following ischemia involves toxic free radicals. To prevent the tissue injury due to reperfusion pre treatment by SOD, catalase, allopurinol are at their beginning but the first results are hopeful for skin, kidney, heart and pancreas. PMID- 3013913 TI - Influence of sample ions on the isotachophoretic separation of cerebrospinal fluid. PMID- 3013912 TI - Synthesis of cation-exchange stationary phases using an adsorbed polymeric coating. AB - We have prepared several silica-based cation-exchange materials that were suitable for the high-performance liquid chromatography of basic proteins. Two synthetic routes were examined. Central to both procedures was the adsorption of a low molecular weight polyamine. One method crosslinks the adsorbed polyamine with a multifunctional oxirane, which is then extensively derivatized with a monomeric cyclic anhydride. The second involves an adsorbed uncrosslinked polyethyleneimine layer which is reacted with polyacrylic anhydride, thereby crosslinking and imparting anionic character simultaneously. The resulting media prepared by either of these methods bound more than 40 mg of hemoglobin per gram of support depending on the reaction conditions. These cation-exchange stationary phases also exhibited good chromatographic performance, successfully resolving (horse heart) cytochrome c and lysozyme. Two of the more promising support materials were effectively used to isolate cytochrome c553 from a crude extract of cyanobacteria. PMID- 3013914 TI - The synthesis and characterization of chemically bonded silica-based zwitterion exchangers for HPLC. AB - The synthesis and characterization of a zwitterionic stationary phase bonded onto microparticulate silica is described. The bonded zwitterionic phase was characterized by elemental analysis, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), and quantitative analysis of the ligands by high performance liquid chromatography (HPLC) following chemical cleavage from the silica backbone. Chromatographic evaluation of this novel bonded phase indicates that it functions as a weak cation exchanger at pH values above 4.5, an anion exchanger at pH values below 7, and as a zwitterionic phase between these two values. The simultaneous separation of a mixture of cationic, anionic and zwitterionic solutes with this novel bonded phase is shown. Using nucleotides as model compounds, a correlation was developed between maximum solute retention and the pH values corresponding to maximum solute/stationary phase zwitterion overlap. The possibility for a quadrupolar retention mechanism of the bonded zwitterionic phase for zwitterionic solutes is explored. PMID- 3013915 TI - Analysis of various classes of drugs by capillary supercritical fluid chromatography. AB - Three classes of drugs were screened for analysis feasibility by capillary column supercritical fluid chromatography. These included steroids, therapeutic antibiotic drugs, and drugs of abuse, such as cannabinoids. Supercritical fluid carbon dioxide was used as the mobile phase in conjunction with a methylpolysiloxane stationary phase capillary column and a flame ionization detector. All compounds considered were analyzed either as single component solutions, simple mixtures, or in actual complex mixtures. PMID- 3013916 TI - Rapid identification of herpesvirus simiae (B virus) DNA from clinical isolates in nonhuman primate colonies. AB - A rapid, simple technique, based on restriction endonuclease analysis of radioactively labeled infected cell DNA, is described for identification of Herpesvirus simiae (B virus) infection in clinical isolates from nonhuman primates. Isolates can be screened within 2-3 days from the time of collection of a specimen from a suspect lesion to final viral identification. Isolates were obtained from eight animals with suspected B virus infections. The results indicated the presence of B virus in each of the eight animals, each isolate unique from the others, but with the dominant prototypic pattern of the laboratory strain of B virus (E2490) and not HSV-1 (KOS), HSV-2 (186), or SA8 (3264). PMID- 3013917 TI - Responsiveness of hypophyseal-adrenocortical axis to repetitive administration of synthetic ovine corticotropin-releasing hormone in patients with isolated adrenocorticotropin deficiency. AB - The primary lesion site in isolated ACTH deficiency was studied in three patients by examining the responses of immunoreactive ACTH to insulin-induced hypoglycemia, lysine vasopressin, and synthetic ovine corticotropin-releasing hormone (CRH). In all patients, no significant changes in immunoreactive ACTH followed insulin-induced hypoglycemia or lysine vasopressin. Fifty micrograms (greater than or equal to 1 microgram/kg BW) of CRH administered as an iv bolus dose daily for 6 consecutive days elicited no significant increase in plasma immunoreactive ACTH, beta-lipotropin, or cortisol levels in all patients. Eight iv bolus injections of 0.63 microgram/kg BW CRH at 4-h intervals also failed to induce a significant response of immunoreactive ACTH to an iv bolus dose of 1 microgram/kg CRH at 36 h in one patient. In contrast, a single bolus dose of 50 micrograms CRH induced a response of plasma immunoreactive ACTH in a patient with Cushing's disease and a patient with Addison's disease. The present results suggest that the primary lesion of isolated ACTH deficiency is not the hypothalamus, but, rather, is located in pituitary ACTH-secreting cells. PMID- 3013918 TI - The antiproliferative effect of calcitriol on human peripheral blood mononuclear cells. AB - Activation of lymphocytes leads to the expression of receptors for the calcitropic hormone calcitriol [1,25(OH)2D3], and calcitriol is a potent inhibitor of interleukin-2 (IL-2) and of lymphocyte proliferation. We used peripheral blood mononuclear cells (PBM) activated in vitro with phytohemagglutinin to study 1) the relationship between 1,25(OH)2D3 receptor expression, IL-2 production, and 1,25(OH)2D3-induced inhibition of PBM proliferation in connection with the cell cycle; 2) the effect of 1,25(OH)2D3 on PBM activation and on the expression of activation-related molecules including the IL-2 receptor, and 3) the role of calcium in the antiproliferative effect of the hormone. 1,25(OH)2D3 receptor expression occurred when PBM entered the G1a phase of the cell cycle. The concentration of the receptor protein reached a peak at G1b and declined during the S phase. 1,25(OH)2D3 inhibited cell proliferation by blocking PBM at the G1a-G1b border. The antiproliferative effect of calcitriol was not caused by hormonal interference with the calcium-dependent activation process nor with the expression of activation-related molecules including the IL 2 receptor. Moreover, this effect was not influenced by extracellular calcium, suggesting that the hormonal action cannot be due to calcium translocation. These findings support the contention that 1,25(OH)2D3-induced inhibition of PBM proliferation is mediated through selective inhibition of IL-2 production. PMID- 3013919 TI - Clinical and biological phenotypes in late-onset 21-hydroxylase deficiency. AB - We analyzed data from 20 patients with late-onset 21-hydroxylase deficiency (LOHD). Three clinical phenotypes could be distinguished among the 18 women. Seven (39%) presented with clinical features suggesting polycystic ovarian disease (PCOD). However, despite androgen levels similar to those of patients with typical PCOD, high serum LH to FSH ratios were not consistently found. Seven other women (39%) presented with isolated hirsutism, suggesting idiopathic hirsutism. The remaining 4 women (22%) had no manifestations of androgen excess and were considered to have the cryptic form of LOHD. Serum 17 hydroxyprogesterone (17-OHP) and androgen levels were similar in the 3 phenotypes, suggesting that the clinical expression of LOHD in women is modulated by individual factors, such as androgen sensitivity. The 2 men were detected by family study and were clinically normal. Since clinical diagnosis of LOHD is impossible, we concentrated on hormonal data with the aim of providing guidelines for the biological diagnosis of LOHD. Assay of basal serum 17-OHD at 0800 h in both sexes and in the early follicular phase in women was sufficient to establish the diagnosis of LOHD in most patients. If doubtful results are obtained, i.e. serum 17-OHP levels between 2 and 5 ng/ml, an ACTH test must be performed. Post ACTH serum 17-OHP levels exceeding 10 ng/ml confirm the diagnosis of LOHD. Such results should avoid confusion with heterozygotes for 21-hydroxylase deficiency, whose frequency is high within the general population and may be even higher in patients with idiopathic hirsutism or PCOD. PMID- 3013920 TI - High incidence of somatostatin receptors in human meningiomas: biochemical characterization. AB - Thirteen human meningiomas were tested for their content of specific somatostatin (SRIH) receptors using an in vitro binding assay with meningioma homogenates as well as receptor autoradiography. All tumors had measurable amounts of somatostatin receptors. Receptor density, however, greatly varied among the tumors, ranging from low levels to more than 400 fmol/mg protein. Seven tumors, biochemically characterized in detail, had saturable and high affinity receptors [mean Kd, 1.10 +/- 0.42 (+/- SEM) nM], with pharmacological specificity for SRIH resembling the noncentral nervous system type of SRIH receptor. There was no correlation between the density of SRIH receptors and the density of progestin receptors measured in parallel. The presence of SRIH receptors in meningiomas was completely unexpected, and their role unknown. If the receptors can mediate antiproliferative properties in meningiomas, as has been suggested to be the case for such receptors in other endocrine tumors, the present data could be of potential therapeutic interest. PMID- 3013921 TI - Uninhibited thyroidal uptake of radioiodine despite iodine excess in amiodarone induced hypothyroidism. AB - Iodine excess is associated with a low thyroidal radioiodine uptake due to dilution of the radioisotope by the increased stable iodide pool. We studied thyroidal uptake of radioisotopes in cardiac patients with iodine excess due to amiodarone treatment. 99mTc-pertechnetate scintigraphy was performed in 13 patients receiving long term amiodarone therapy. Five patients had a clearly visible thyroid gland, and 8 patients had no or a very faint thyroid image. All patients with positive scans had an increased plasma TSH level, whereas all patients with negative scans had a normal or absent TSH response to TRH. Thyroidal uptake and discharge of 123I were studied in 30 other patients. Group I (n = 11) had normal plasma TSH responses to TRH and no iodine excess, group II (n = 7) had normal TSH responses to TRH and excess iodine from metrizoate angiography in the previous month, group III (n = 7) had normal or decreased TSH responses to TRH while receiving long term amiodarone therapy, and group IV (n = 5) had increased TSH responses to TRH while receiving long term amiodarone therapy. The mean radioiodine uptake value in group I [5.4 +/- 0.8% (+/- SE) at 60 min] was higher than those in group II (2.3 +/- 0.7%; P = 0.009) and group III (0.8 +/- 0.3%; P = 0.0005), but not different from that in group IV (5.3 +/- 1.2%; P = NS). Radioiodine discharge after perchlorate (expressed as a percentage of the 60 min uptake) in group I (10.1 +/- 2.2%) was lower than those in group II (24.9 +/- 10.6%; P = 0.05) and group III (28.8 +/- 5.3%; P less than 0.005), whereas discharge in group IV (58.0 +/- 6.1%) was greater than those in group II (P less than 0.05) and group III (P less than 0.01). In conclusion, 1) thyroid visualization by 99mTc-pertechnetate and thyroid radioiodine uptake during iodine excess are decreased in euthyroid and hyperthyroid patients, but preserved in hypothyroid patients. 2) The organification defect induced by iodine excess is greater in iodide-induced hypothyroidism than in eu- or hyperthyroidism. These findings may be explained by the increased TSH secretion in hypothyroidism and/or by decreased thyroidal concentration of an unknown specific iodinated compound (whose concentration and action vary with the total organic iodine content of the thyroid) that mediates the inhibition of iodide transport. PMID- 3013922 TI - Impaired neonatal natural killer-cell activity to herpes simplex virus: decreased inhibition of viral replication and altered response to lymphokines. AB - Human adult natural killer (NK) cells were recently demonstrated to inhibit herpes simplex virus (HSV) replication in vitro. In this study we compared the ability of newborn and adult NK cells to inhibit HSV replication. Cord blood mononuclear cells (MNCs) from healthy, term newborns and MNCs from adults were analyzed for their percentage of Leu-11+ cells and compared in vitro for their NK cell activity against HSV-infected fibroblasts and the tumor cell line K562. Cord blood MNCs, compared with adult MNCs, had significantly lower percentages of Leu 11+ cells (5 vs 11%; P less than 0.01), less anti-K562 NK activity (6 vs 54 lytic units/10(7) cells; P less than 0.001), and less anti-HSV NK activity (5 vs 52% HSV plaque inhibition; P less than 0.02). Comparing individual neonates and adults with equal percentages of Leu-11+ cells, neonatal MNCs still had less NK activity against either target. When Leu-11+ MNCs were isolated using the fluorescence-activated cell sorter, neonatal Leu-11+ MNCs still inhibited HSV replication less than adult Leu-11+ MNCs (P less than 0.01). MNCs from some neonates had significant anti-K562 NK activity but poor anti-HSV NK activity, suggesting either nonidentical NK-cell subpopulations or specific suppression. Whereas neonatal NK activity against K562 was always augmented by prior exposure to either interferon (IFN) or interleukin-2 (IL-2), the neonatal NK activity against HSV-infected cells was only augmented for half of the neonates tested. Endogenous production of alpha-IFN and gamma-IFN by MNCs exposed to HSV-infected fibroblasts was the same for cells from neonates or from HSV-seronegative adults.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013923 TI - High IgM antibody to human T-lymphotropic virus type I in systemic lupus erythematosus. AB - Twenty-six percent of 53 systemic lupus erythematosus sera had high levels of IgM antibody to human T-lymphotropic virus Type I, significantly more than the 5% of normal controls. Neither IgG antibodies to Type I virus nor IgM or IgG antibodies to Type II virus were increased in lupus. Further analysis using competition immunoassay and Western blot techniques also suggested that the IgM Type I antibodies in lupus sera were directed against viral antigens but did not completely exclude a nonviral reaction. Other studies also have not found IgG antibodies to the Type I virus but have not tested for IgM antibodies. Our study suggests that human T-lymphotropic virus Type I or a related virus may be involved in the pathogenesis of some cases of systemic lupus erythematosus. PMID- 3013924 TI - Chediak-Higashi syndrome: immunological responses to Epstein-Barr virus studies in gene heterozygotes. AB - Immunologic studies were performed in five fathers and nine mothers of patients with Chediak-Higashi Syndrome (CHS). Antibody response to Epstein-Barr virus capsid antigen was higher than in normal controls. Antibodies to diffuse component of the early antigen were not detected and serum antibodies to the restricted component of the early antigen were observed in 64% of the subjects studied. Low natural killer activity and increased proportions of OKT8 positive cells were increased. These data indicate that immunologic alterations similar to those seen in CHS patients can be observed in their asymptomatic parents. PMID- 3013926 TI - Immunoglobulin class-specific serological responses to adenovirus in respiratory infections of young adult men. AB - Complement fixation and enzyme immunoassay (EIA) were used for measuring viral antibodies in 103 military conscripts with pneumonia and 98 conscripts with other respiratory infections. Diagnostic rises in antibody to adenovirus were found in 23 (22%) patients with pneumonia and in 42 (43%) patients with other respiratory infections. EIA detected more diagnostic rises than did complement fixation (22 versus 17 for pneumonia and 42 versus 40 in other infections). Adenovirus antibodies were analyzed for different immunoglobulin classes by EIA. Diagnostic rises in immunoglobulin G (IgG) and IgA isotypes were observed in 89 and 77% of the cases, respectively. IgM antibodies were positive in 39% of the cases. In five cases (8%), the demonstration of adenovirus antibodies of the IgM class was the only serological evidence for adenovirus infection. The present study demonstrates that the immunoglobulin class-specific EIA is a sensitive method for the diagnosis of respiratory adenovirus infections. PMID- 3013925 TI - Rapid diagnosis of respiratory adenovirus infections in young adult men. AB - Rapid viral diagnosis was attempted in 106 military conscripts with pneumonia and in 101 military conscripts with other types of respiratory infections. Nasopharyngeal suction specimens (NPS) were assayed for viral antigens by immunofluorescence and enzyme immunoassay (EIA). Sputum specimens from 97 pneumonia patients were assayed for viral antigens by EIA. Also, 71 NPS and 13 sputum specimens were examined for the presence of adenovirus DNA by a sandwich hybridization (HYB) method. The reference test was adenovirus isolation in cell culture from the NPS. Adenoviruses were isolated from 6 pneumonia patients and from 20 patients with other respiratory infections. Of these 26 NPS, rapid diagnosis was successful in 13, 16, and 14 cases by EIA, immunofluorescence, and HYB, respectively. Four antigen-positive specimens were found among the 181 specimens which were negative by virus isolation. Sputum was found to contain adenovirus antigen by EIA in 5 of 97 tested specimens. Of these 97 specimens, 13 were selectively tested in HYB, and a positive signal was observed in 4 cases. Serological testing of paired sera revealed 23 adenovirus infections in the pneumonia group and 42 in the group with other respiratory infections. Other viral infections were found only sporadically. All rapid virus detection methods showed excellent specificity but had a lower sensitivity (60%) than virus isolation. Our results show that rapid methods for diagnosing respiratory adenovirus infections can be successfully used in selected groups of adults. PMID- 3013927 TI - Characterization of clinical strains of Staphylococcus aureus associated with pneumonia. AB - A total of 5 Staphylococcus aureus strains from patients with postinfluenzal staphylococcal pneumonia, 7 from burn patients with staphylococcal pneumonia, and 21 from the nasopharynx of carriers were phenotypically characterized. All or most strains produced coagulase, clumping factor, DNase, thermostable DNase, protease, gelatinase, lipase, and pigment; the strains were low to moderate producers of extracellular protein A, fibrinolysin, and alpha-hemolysin. All strains were sensitive to mercury, half were sensitive to arsenate and cadmium, and 67 to 92% were resistant to penicillin. Differences between strains were not statistically significant. Cell surface hydrophobicity was determined by measuring percent adsorption to hexadecane. Hydrophobicity of postinfluenzal staphylococcal pneumonia strains was significantly lower than that of pneumonia strains from burn patients and carriers (P less than 0.005). Immunoblot experiments with sera immune to one clinical test strain allowed the separation of all strains into three groups based on probe-positive reactions with primarily four staphylococcal polypeptides (154,200, 130,000, 77,100, and 64,400 molecular weight). The difference in distribution of clinical and carrier strains was highly significant (P = 0.007). PMID- 3013929 TI - Indirect immunofluorescence assay for the detection of hepatitis A virus-specific serum immunoglobulins. AB - Hepatitis A virus-specific BSC-1 cells were used for the detection of serum immunoglobulins to hepatitis A virus by indirect immunofluorescence. Of 150 serum samples tested, specific immunoglobulin M was detected only in patients with serologically confirmed acute hepatitis A, while specific immunoglobulin G was detected in patients with acute or past clinical hepatitis A as well as many patients with no known history of hepatitis. PMID- 3013928 TI - The vector homology problem in diagnostic nucleic acid hybridization of clinical specimens. AB - Nucleic acid hybridization techniques using cloned probes are finding application in assays of clinical specimens in research and diagnostic laboratories. The probes that we and others have used are recombinant plasmids composed of viral inserts and bacterial plasmid vectors such as pBR322. We suspected that there was material homologous to pBR322 present in many clinical samples. because hybridization occurred in samples which lacked evidence of virus by other techniques. If the presence of this vector-homologous material was unrecognized, hybridization in the test sample might erroneously be interpreted as indicating the presence of viral sequences. In this paper we demonstrate specific hybridization of labeled pBR322 DNA with DNA from various clinical samples. Evidence is presented that nonspecific probe trapping could not account for this phenomenon. In mixing experiments, it is shown that contamination of clinical samples with bacteria would explain such a result. Approaches tested to circumvent this problem included the use of isolated insert probes, alternate cloning vectors, and cold competitor pBR322 DNA in prehybridization and hybridization mixes. None proved entirely satisfactory. We therefore emphasize that it is essential that all hybridization detection systems use a control probe of the vector alone in order to demonstrate the absence of material with vector homology in the specimen tested. PMID- 3013930 TI - Detection of specific immunoglobulin M antibodies to cytomegalovirus by using monoclonal antibody to immunoglobulin M in an indirect immunofluorescence assay. AB - The detection of immunoglobulin M (IgM) antibodies to cytomegalovirus-induced late antigens by an indirect immunofluorescence assay was improved by the use of monoclonal antibodies to human IgM. Nonspecific background fluorescence was absent, facilitating the reading of the slides and the detection of a specific fluorescence reaction in sera with low levels of specific IgM. Moreover, the indirect immunofluorescence assay with monoclonal antibodies to IgM proved more sensitive than the indirect immunofluorescence assay with polyclonal antibodies to IgM. The absorption of human sera on staphylococcal protein A avoided false reactions due to the presence of rheumatoid factor and high levels of specific IgG in test sera. PMID- 3013931 TI - Genetic heterogeneity of recent isolates of adenovirus types 3, 4, and 7. AB - Restriction endonuclease analysis was carried out on adenovirus types 3, 4, and 7 (Ad3, Ad4, and Ad7, respectively) isolated during a 16-month epidemic period. Most of the isolates were associated with respiratory or ocular infection or both. Forty-four strains of the subtype Ad7b were identified with SmaI; these strains could be further subdivided into three distinct genome groups, designated Ad7b1, Ad7b2, and Ad7b3, by digestion with PvuII, BglI, and SstII. These Ad7b variants occurred at approximately similar frequencies throughout the epidemic and were associated with similar clinical illnesses. Nine distinct intratypic genome groups were identified among 29 Ad3 strains with HindIII, PstI, and SmaI; adenoviruses similar to subtype Ad3a, formerly reported to be uncommon in Europe, were isolated most frequently (41% of strains), and the previously predominant prototype (Ad3p) was not found. Considerable similarities among the genomes of some strains of Ad3 and Ad7 could be demonstrated, and clinical and epidemiological presentations were very similar. The four strains of Ad4 isolated fell into two distinct patterns, i.e., the previously recognized prototype (Ad4p) and a new subtype designated Ad4b. Both of these subtypes were associated with follicular conjunctivitis. Thus we were unable to produce evidence that particular genome types of Ad3, Ad4, and Ad7 are associated with particular clinical presentations. However, restriction endonuclease analysis should prove useful for epidemiological studies on these viruses. PMID- 3013932 TI - Two outbreaks of herpes simplex virus type 1 nosocomial infection among newborns. AB - Two outbreaks of herpes simplex virus type 1 (HSV-1) infection occurred in three newborns at each of two hospitals, and two of the infants in each case died of disseminated HSV-1 infection. Restriction endonuclease profiles of HSV-1 DNAs isolated from the three in each instance were essentially identical, indicating that they were epidemiologically related. In the first instance, each of three infants born in the same hospital at intervals of approximately 2 years or 1 year was infected with HSV-1. In this case, it was suggested that periodically reactivated HSV-1 strains from hospital personnel had been transmitted to three newborns. In the second instance (infants G-36, G-37, and G-38), the radiant warmer that had been occupied by infant G-36, who was infected with HSV-1, was used for infant G-37. Infant G-38 was in another radiant warmer 2 m from the radiant warmer occupied by infant G-37. Therefore, it was suggested that the virus had possibly been transmitted via radiant warmer and by hospital personnel in the two instances. PMID- 3013933 TI - Unusual expression of new low-level-trimethoprim-resistance plasmids. AB - In a survey, 42% of trimethoprim-resistant clinical members of the family Enterobacteriaceae were able to transfer trimethoprim resistance to standard Escherichia coli strains when selection was made on complex bacteriological media. When transfer experiments were performed with minimal medium, another 16% of the clinical strains were shown to have transferred trimethoprim resistance. Twelve transconjugants produced negligible trimethoprim resistance in complex media but were resistant in minimal medium. The methionine, glycine, and purine components of complex media appeared to be responsible for the reduced expression of trimethoprim resistance in these strains. PMID- 3013934 TI - Rapid dot enzyme immunoassay for the detection of antibodies to cytomegalovirus. AB - Cytomegalovirus (CMV) antigen was coated onto a white opaque plastic card as small dots inside circles marked in the microtiter plate well pattern. The card with antigen dots could be cut according to the number of test samples to be assayed. Small drops of undiluted serum samples, goat antibodies to human immunoglobulin G labeled with alkaline phosphatase, and finally substrate (5 bromo-4-chloro-3-indolyl phosphate) were sequentially added to the antigen spots and incubated in the open air at room temperature for 5 min each. The antigen dots showed blue color for sera with immunoglobulin G antibodies to cytomegalovirus but no color for those without. The developed antigen dots could be rinsed with water and kept as permanent records. For the assay of a large number of serum samples, a modified procedure with serum diluted 1:10 and longer first two incubations (20 min each) was found to be more comfortable to perform. The results of this assay for 123 undiluted and 256 diluted serum samples revealed very good correlations with those obtained by a commercially available test kit for immunoglobulin G antibodies to cytomegalovirus with 97 and 99% agreement, respectively. This dot test was very reproducible and required no instrumentation. The reagents, including coated antigen dots, are stable at room temperature for at least 2 months and are ready for use. PMID- 3013935 TI - Inhibition by human thrombomodulin of factor Xa-mediated cleavage of prothrombin. AB - Human thrombomodulin significantly inhibited the rate of prothrombin conversion to thrombin by Factor Xa in the presence of phospholipid or platelets, calcium, and Factor Va. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 125I prothrombin activation revealed that thrombomodulin reduced the rate of prothrombin activation but did not alter the cleavage pattern. The inhibition was reversed by the inclusion of a highly specific rabbit antithrombomodulin antibody. If thrombomodulin was replaced by hirudin, the rate of thrombin generation was not decreased excluding the possibility that the inhibition by thrombomodulin was secondary to the binding of small amounts of thrombin formed early in the reaction and the prevention of feedback breakdown of prothrombin by thrombin. The inhibitory activity of thrombomodulin was overcome by increasing the concentration of Factor Xa and specific, saturable binding of thrombomodulin to Factor Xa was demonstrated. These results indicate that thrombomodulin binds to Factor Xa and thereby inhibits the activity of the prothrombinase complex. PMID- 3013936 TI - Effects of the putative neutrophil-generated toxin, hypochlorous acid, on membrane permeability and transport systems of Escherichia coli. AB - Titrimetric addition of hypochlorous acid (HOCl) or chloramine (NH2Cl) to suspensions of Escherichia coli decreases their ability to accumulate 14C-labeled glutamine, proline, thiomethylgalactoside, and leucine in a manner that approximately coincides with loss of cell viability; quantitative differences in cellular response are observed with the two oxidants. Inhibition of beta galactosidase activity in E. coli ML-35, a strain lacking functional lactose permease, is complex and also depends upon the identity of the oxidant. Membrane proton conductivities and glycerol permeabilities are unchanged by addition of HOCl or NH2Cl in excess of that required for inactivation. The combined results are interpreted to indicate that the locus of HOCl attack is the cell envelope, that HOCl inactivation does not occur by loss of membrane structural integrity, that loss of transport function can be identified with either selective oxidative inhibition of the transport proteins or loss of cellular metabolic energy, and that different mechanisms of inactivation may exist for HOCl and NH2Cl. PMID- 3013938 TI - Electrophysiologic effects of intracellular lysophosphoglycerides and their accumulation in cardiac lymph with myocardial ischemia in dogs. AB - Lysophosphatidylcholine (LPC) accumulates in ischemic tissue, and exogenous LPC (20-100 microM) induces electrophysiologic alterations in vitro. However, to determine whether compartmentalization is critical, intracellular pressure microinjection of LPC was performed with simultaneous recording of the transmembrane action potential. Intracellular LPC in concentrations as high as 500 microM (n = 18), calculated based on calibration of injectate volume and cellular volume, did not induce electrophysiologic alterations. The concentrations and efflux of phospholipids and lysophospholipids were assessed in lymph obtained from the supracardiac lymph vessel in anesthetized dogs to assess the extent of extracellular accumulation. Prior to ischemia, phosphatidylcholine (PC) was the major phospholipid in lymph (79 +/- 2%) with substantial quantities of sphingomyelin (11 +/- 2%) and LPC (6 +/- 1%). With ischemia, the concentration of LPC increased by 18%, and net efflux of LPC increased by 24% (P less than 0.01) with no net efflux of PC or other assayed phospholipids. The calculated concentration of LPC increased from 84 to 197 microM in lymph within the ischemic region, a concentration sufficient to induce electrophysiologic derangements. PMID- 3013937 TI - Desensitization of adenosine receptor-mediated inhibition of lipolysis. The mechanism involves the development of enhanced cyclic adenosine monophosphate accumulation in tolerant adipocytes. AB - Adipocytes contain adenosine receptors, termed A1 receptors, which inhibit lipolysis by decreasing adenylate cyclase activity. The inhibition of lipolysis by adenosine agonists in vivo acutely suppresses the plasma concentrations of free fatty acids (FFA) and triglycerides. We have found that infusions of the adenosine receptor agonist phenylisopropyladenosine (PIA) initially decreases plasma FFA concentrations; however, with prolonged exposure (6 d), rats become very tolerant to the effects of the drug. Adipocytes isolated from epididymal fat pads from PIA-infused rats have altered lipolytic responses. When lipolysis is stimulated with a relatively high concentration of isoproterenol (10(-7) M), PIA does not inhibit lipolysis in adipocytes from the infused animals. However, PIA inhibits isoproterenol-stimulated cyclic AMP (cAMP) accumulation in adipocytes from the infused rats although with decreased sensitivity compared with controls. The explanation for the impaired antilipolytic effect appears to be due to the fact that isoproterenol-stimulated cAMP accumulation is markedly increased in cells from infused rats. Indeed, basal lipolysis and lipolysis stimulated with lower concentrations of isoproterenol (10(-9), 10(-8) M) are effectively inhibited by PIA. cAMP accumulation is greatly increased in adipocytes from infused rats when stimulated by isoproterenol, ACTH, and forskolin. The results have some striking analogies to changes induced in nerve cells by prolonged exposure to narcotics. These data suggest that tolerance to PIA develops in adipocytes as a consequence of enhanced cAMP accumulation. PMID- 3013939 TI - Hypophysiotropic and neuromodulatory regulation of adrenocorticotropin in the human fetal pituitary gland. AB - Synthetic human corticotropin-releasing factor (hCRF) stimulated ACTH secretion by human fetal pituitaries in superfusion and dispersed human fetal pituitary cells cultured on an extracellular matrix in static incubation from 14 to 23 wk gestational age. The action of hCRF in vitro was potentiated by arginine vasopressin (AVP) at all ages studied. 8-Br-cAMP induced a response similar to hCRF. The AVP effect on ACTH was synergistic with both CRF and 8-Br-cAMP. hCRF mediated secretion of ACTH was noncompetitively inhibited by 24-h pretreatment, or by 3-h concomitant treatment, with dexamethasone. Neither oxytocin, catecholamines, prostaglandins, nor indomethacin exerted significant effects on ACTH secretion, either alone or in combination with hCRF or AVP during the gestational ages studied. These results support a physiologic role for CRF in the regulation of secretion by corticotropic cells as early as 14 wk gestation, by which time corticotropes and ability to secrete ACTH have been demonstrated. PMID- 3013941 TI - Normalization of hypercalcemia associated with a decrease in renal calcium reabsorption in Leydig cell tumor-bearing rats treated with WR-2721. AB - The Rice-500 Leydig cell tumor (LCT) in Fischer rats is a model of humoral hypercalcemia of malignancy (HHM). In this model, the elevation of plasma calcium (Ca) does not result merely from an increased bone resorption, but also from an enhanced tubular Ca reabsorption (TRCa). We investigated the hypocalcemic response to WR-2721 [S-2,2-(3-aminopropylamino)-, ethylphosphorothioic acid] in LCT-bearing Fischer rats. WR-2721 is a potent inhibitor of normal and aberrant parathyroid hormone (PTH) secretion. Moreover, it exerts a PTH-independent inhibitory effect on TRCa. In hypercalcemic LCT-bearing rats WR-2721 induced a fall in plasma Ca from 3.24 +/- 0.12 to 2.66 +/- 0.23 mmol/liter within 2 h after one single injection of 0.7 mmol/kg body wt. The decrement in plasma Ca was associated with a marked increase in urinary Ca excretion, indicating an inhibition of TRCa. The elevated urine cyclic AMP of LCT-bearing rats, however, was not altered by WR-2721 treatment. These results suggest that in this HHM model, WR-2721 can normalize calcemia through its PTH-independent inhibitory effect on TRCa. WR-2721 could therefore be an effective drug to treat human hypercalcemia of malignancy, particularly in those tumors wherein a markedly enhanced renal Ca reabsorption contributes to the elevation of the plasma Ca level. PMID- 3013943 TI - New look at mesoblastic nephroma. AB - Thirty eight mesoblastic nephromas were studied. The age range of the patients was between the neonatal period and 18 months. The presence of cartilage is consistent with a mesoblastic origin, but squamous epithelium was a feature in three tumours. Particular attention was given to the adjacent renal tissue in which various histological features were noted: vacuolated and dysplastic tubules; cysts; and subcapsular epithelial tumourlets. The findings had aspects in common with both dysplastic kidneys and nephroblastoma. Classification of the tumours as normocellular and hypercellular was attempted, but there was considerable overlap. The behaviour of the tumour was good in all cases, although follow up was relatively short on some patients, and deaths from non-neoplastic causes occurred. PMID- 3013940 TI - Angiotensinogen gene is expressed and differentially regulated in multiple tissues of the rat. AB - To define the role of local synthesis of angiotensinogen in tissue angiotensin production, we have quantitated angiotensinogen messenger RNA (mRNA) levels in 17 different tissues of four groups of rats: control rats, nephrectomized rats, rats given dexamethasone, ethynylestradiol, and triiodothyronine, and nephrectomized rats given dexamethasone, ethynylestradiol, and triiodothyronine. Angiotensinogen mRNA was identified in 12 tissues: liver, kidney, brain, spinal cord, aorta, mesentery, atria, lung, adrenal, large intestine, stomach, and spleen. Angiotensinogen mRNA was not identified in pituitary, ventricle, testis, small intestine, or pancreas. When expressed per gram tissue wet weight, angiotensinogen mRNA levels of extrahepatic tissues were less than 4% of hepatic levels. However, when expressed per milligram total RNA, angiotensinogen mRNA levels of brain, spinal cord, aorta, and mesentery were 26-42% of hepatic levels. Regulation of angiotensinogen mRNA levels was tissue specific. This demonstration of a widespread tissue distribution of angiotensinogen mRNA may indicate a similarly widespread distribution of local angiotensin systems that are independent of the circulating renin-angiotensin system. PMID- 3013942 TI - Platelet protein phosphorylation, elevation of cytosolic calcium, and inositol phospholipid breakdown in platelet activation induced by plasmin. AB - Studies have been performed on the biochemical mechanism of platelet activation induced by the fibrinolytic protease plasmin. In washed human platelets, greater than or equal to 1.0 caseinolytic units (CU/ml plasmin induced aggregation. Platelet [14C]serotonin release was stimulated by 1.0 CU/ml plasmin to an extent comparable to that induced by 1.0 U/ml thrombin. A dose- and time-dependent phosphorylation of the platelet 47,000- and 20,000-kD proteins was noted in 32PO4 labeled platelets incubated with plasmin; phosphorylation was not affected by extracellular Ca2+, but was completely inhibited by an increase in platelet cyclic AMP. Phosphorylation of these platelet proteins suggested that plasmin may act on platelets by stimulating a rise in cytosolic calcium concentration ([Cai2+]) and activating inositol phospholipid-dependent phospholipase C and protein kinase C. Using both quin2 fluorescence and aequorin luminescence as indicators, plasmin was found to elevate platelet [Cai2+] in the presence or absence of extracellular Ca2+. Phospholipase C activation was shown by the generation of [3H]diglyceride in [3H]arachidonic acid-labeled platelets and [32P]phosphatidic acid in 32PO4 labeled platelets exposed to plasmin. Plasmin did not induce formation of thromboxane A2 (TXA2). Only small amounts of this eicosanoid were detected late in the time course after plasmin stimulation. Our results indicate that plasmin causes platelet aggregation and secretion associated with phosphorylation of the 47,000- and 20,000-kD proteins, Ca2+ mobilization, and phospholipase C and protein kinase C activation. PMID- 3013944 TI - Crystal structure of Auer rods in acute myeloblastic leukaemia (AMyL). AB - Ultrathin sections containing Auer rods from cases of acute myeloblastic leukaemia (AMyL) were tilted in the goniometer stage of the electron microscope and the resulting series of electronmicrographs analysed in an optical diffractometer illuminated by laser. The results showed that Auer rods of AMyL show a truly three dimensional crystal structure. Measurements from the optical diffraction patterns were consistent with a monoclinic unit cell, the unit cell edge lengths a, b, and c being 6.6 [SD) 0.5) nm, 8.6 (0.2) nm, and 9.6 (1.0) nm, respectively; the angle between a and c being 120 (7) degrees. This structure was quite distinct from the "tubular" substructure reported by others in the Auer rods of acute promyelocytic leukaemia (APL), although it was consistent with periodicities measured by others in Auer rods of AMyL. A complete understanding of the three dimensional structures of Auer rods in the different types of acute myeloid leukaemia (AML) could well prove to be of considerable diagnostic importance. PMID- 3013945 TI - New approach to assessing lung tumours in man. AB - One hundred and four surgically resected lung tumours were labelled in either cryostat or freeze dried sections with a monoclonal antibody (Ki67), which reacts with a nuclear antigen expressed by proliferating cells. The tumours were categorised semiquantitatively into four proliferative grades, a classification that can be performed rapidly and reproducibly by the pathologist. In keeping with previous cell kinetic studies all small cell carcinomas had high proliferation rates, whereas the carcinoid tumours were in the lowest grade. In contrast, the adenocarcinomas (27 cases) and squamous cell carcinomas (63 cases) varied widely in their proliferative state, in keeping with their heterogeneous, morphological, and clinical behaviour. Immunocytochemical labelling of lung tumour biopsy specimens with antibody Ki67 is a simple technique within the scope of routine surgical pathology laboratories, which might enable these tumours to be classified according to their proliferative status and treatment to be selected accordingly. PMID- 3013947 TI - Current status of indices of plaque. AB - Microbial plaque has been described by the Council on Dental Therapeutics of the American Dental Association in their recently drafted Guidelines for acceptance of products for the management of dental plaque and gingivitis. The choice of an index system to be used in plaque trials should be made in terms of the objective of the trial, the size of the population, the period of the study, and the type and extent of change anticipated. The indices currently in use estimate the quantity of plaque in terms of tooth area covered or the thickness of material in the area measured. The Silness and Loe and the modified Turesky methods have been suggested as acceptable indices for the estimation of cleansing ability. PMID- 3013946 TI - Abnormal steroid excretion in gestational trophoblastic disease complicated by ovarian theca-lutein cysts. AB - Serum and urine steroids were examined in two subjects with trophoblastic disease accompanied by large ovarian theca-lutein cysts and compared with those from 10 patients with trophoblastic disease but without palpable cysts. In the patients without cysts normal values were obtained for serum oestradiol, progesterone, 17 alpha-hydroxyprogesterone and androstenedione, and for urinary total oestrogens, pregnanediol, pregnanetriol, and 17-oxosteroids. Nineteen urinary steroid metabolites, quantified by capillary gas-liquid chromatography, were either within reference limits or marginally raised. In several cases relatively minor increases in serum testosterone and cortisol and urinary free cortisol were observed. In contrast, the subjects with cysts showed pronounced excesses of androgen metabolites, 17 alpha-hydroxypregnenolone, pregnanediol, and pregnanetriol, and both exhibited a similar pattern of unusual additional metabolites. The profiles superficially resembled those seen in 21-hydroxylase deficiency adrenogenital syndrome, but there were important discrepancies reflecting known differences in ovarian and adrenal steroid metabolism. Chemotherapy led to decline of human chorionic gonadotrophin concentrations, regression of the cysts, and return to normal of the steroid profile. Excess steroids in the patients with cysts may have originated in the ovary rather than in the trophoblastic tissue. PMID- 3013948 TI - Patterns of afferent synaptic contacts in the alligator lizard's cochlea. AB - Afferent synapses on both free-standing and tectorial hair cells in the alligator lizard's cochlea are described quantitatively. Semiserial sections were photographed with a transmission electron microscope. Hair cells together with their afferent nerves were reconstructed and morphometrically analyzed with the aid of a computer. Each afferent nerve forms many synapses with its hair cell. Tectorial afferents make more than twice the number of synapses with their hair cells as do free-standing afferents. This suggests a possible neuroanatomical basis for the physiological difference in synchrony reported in these two types of auditory nerve fibers; namely, the greater the number of synapses the better a fiber is able to follow faithfully the response of its hair cell. PMID- 3013949 TI - Laminar distribution of receptors in monkey (Macaca fascicularis) geniculostriate system. AB - We have examined the laminar distributions of eight types of receptor in the primary visual cortex (area 17) and the lateral geniculate nucleus (LGN) of the macaque monkey. The receptor populations and subpopulations examined included those selective for gamma-aminobutyric acid (GABA) (using [3H]-muscimol as ligand), L-glutamate-related receptors (using [3H]-L-glutamate and [3H]-AMPA), muscarinic acetylcholine (using [3H]-quinuclidinyl benzilate--QNB and [3H]-N methyl scopolamine--NMS), cholecystokinin (CCK) (using [3H] pentagastrin), benzodiazepine (using [3H]-flunitrazepam), and adenosine (using [3H] cyclohexyladenosine--CHA). Each of the receptors examined exhibited characteristic and differing laminar patterns of binding in the striate cortex. Perhaps reflecting the high density of cell bodies and synapses in layer 4C, most receptors, except those labelled by [3H]-L-glutamate or [3H]-AMPA, showed dense concentrations in this layer. Layers 4B and 5, which contain relatively few cell bodies and heavy myelin concentrations, were in general lightly labelled. Layer 6 showed relatively heavy labelling when [3H]-AMPA (quisqualate) or [3H] pentagastrin (CCK) were used as ligands. The superficial layers of the cortex were zones of relative concentration of GABA, benzodiazepine, acetylcholine, glutamate-related, and adenosine receptors. In general, the binding patterns resembled those previously described for cat visual cortex, but there were also some clear differences. The distributions of all of these receptors likely reflect the differential input substances to different laminae of the visual cortex. Of the receptors examined, only those for GABA, benzodiazepine, and acetylcholine were found in substantial concentration in the LGN. Of these, GABA and benzodiazepine receptors showed especially dense binding in the magnocellular layers of the LGN compared to the parvicellular layers. PMID- 3013950 TI - Central afferent connections and origin of efferent projections of the facial nerve in the chicken (Gallus gallus domesticus). AB - The central afferent connections and origin of efferent projections of the facial nerve in the adult domestic chicken were studied by anterograde and retrograde transport of horseradish peroxidase from the geniculate ganglion. Ipsilateral afferent projections were traced caudal to the level of entrance of the facial nerve and into tractus solitarius (TS), located dorsomedial to the spinal trigeminal nuclear complex. At several rostrocaudal levels in the medulla, fibers exited from TS and terminated in n. sensorius N. facialis (SVII), and nn. ventrolateralis anterior (Vla) and intermedius anterior (Ia) solitarii. Some axons were followed to n. presulcalis anterior (Pas) solitarii. A separate component terminated in subnucleus interpolaris (ip) of n. descendens nervi trigemini or its medially adjacent reticular formation either by exiting from TS or coursing caudally through the trigeminal complex from entering facial rootlets. Another diffuse component of facial axons ascended dorsally and rostrally from the level of entrance of the facial nerve; these projections dissipated in the pons--some on the dorsomedial border of n. principalis N. trigemini (PrV). Ipsilateral efferent projections were traced through the main genu of the facial nerve to retrogradely labelled somata of pars dorsalis (FMd), pars intermedia (FMi), and pars ventralis (FMv) of n. motorius nervi facialis. A separate group of smaller, multipolar, and spindle-shaped cells (8-25 microns) wedged between n. olivaris superioris (OS) and the caudal end of FMv, rostrally, and extending caudally in ventrolateral medulla were labelled. These small cells contrast with the larger (21-45 microns), oval, round, and multipolar somata of FMv and may correspond, in part, to a parasympathetic n. salivatorius (Sal). PMID- 3013952 TI - A technique to obviate the risk of inadvertent infection of cell cultures with bovine viral diarrhoea virus. AB - When bovine embryonic kidneys collected at the Gorgie Abattoir, Edinburgh were examined for evidence of infection with bovine viral diarrhoea virus (BVDV), 11 out of 26 kidneys were found to be positive. A technique that detected the presence of inadvertent BVDV in cell cultures consisted of processing and digesting the kidneys to produce cell suspensions, adding dimethyl sulphoxide and dispersing the suspensions in small aliquots that were stored frozen at - 114 degrees C. One aliquot was cultured and screened for BVDV by indirect immuno fluorescence and interference tests. Bovine embryonic kidney cells so processed retained their viability and virus susceptibility for 15 to 18 months. Selected stocks of "clean" cells only are then used for vaccine production or diagnostic purposes. The cytopathic NADL strain of BVDV multiplied in naturally infected cell cultures but the titres attained were significantly lower than in "clean" cell cultures. PMID- 3013951 TI - Synaptic connections of callosal projection neurons in the vibrissal region of mouse primary motor cortex: an electron microscopic/horseradish peroxidase study. AB - Reciprocal axonal projections between homotypic areas of the vibrissal region of mouse primary motor cortex (MsI) (Porter and White: Neurosci. Lett. 47:37-40, '84) suggested the existence of reciprocal synaptic connections between callosal projection neurons and callosal afferents. In the present study, the retrograde transport of horseradish peroxidase (HRP) was combined with lesion-induced degeneration to identify synapses between callosal afferents and callosal neurons in the corresponding region of the contralateral cortex. The procedure was as follows: MsI was injected with HRP and aspirated on the following day. After 4 days, the animals were perfused and motor cortex was processed for HRP according to a variation of the Adams (Brain Res. 176:33-47,'77) technique, and postfixed in OsO4. The methods used consistently filled fine dendritic branches and spines with dense reaction product, thus allowing examination of synaptic contacts with these processes. All callosal projection neurons were identified as pyramidal neurons, having somata in cortical layers II/III and V. Labeled cells from each of the two levels were prepared for electron microscopy, and that part of each cell's apical dendrite that traversed the superficial cortical layers, where most callosal axons terminate, was cut in an unbroken series of thin sections. Micrographs were taken of all labeled profiles in each thin section, and tracings of the profiles were assembled to reconstruct the apical dendrites. Data on the distribution, type, and amount of callosal and other synapses with the shaft and spines of the apical dendrites were obtained by examining the reconstructions. In addition, profiles of basal dendrites of layer II/III cells were examined in thin sections to ascertain the numbers of callosal and other synapses formed with their shafts and spines. The proportion of synapses that each dendrite formed with callosal axon terminals was compared to the concentration of callosal afferents in the neuropil. Dendrites of both layer II/III and layer V pyramidal cells synapsed with callosal axon terminals. The apical and basal dendrites of layer II/III neurons formed a similar proportion of their synapses with callosal afferents, and this was similar to the concentration of callosal synapses in the surrounding neuropil. Segments of apical dendrites belonging to layer V and layer II/III neurons course through neuropil containing nearly the same concentration of degenerating callosal terminals, but the layer V cells form fewer callosal synapses.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3013953 TI - A malignant mixed salivary tumour and a mammary carcinoma in a young wild eastern spotted native cat Dasyurus viverrinus (Marsupialia). AB - This report contains the first description of a salivary gland tumour in a dasyurid marsupial; the same animal also had a mammary carcinoma. All the previously described neoplasms have been in animals held in captivity for varying periods of time, whereas the case reported here was a young animal trapped in the wild and killed three days later. The development of tumours in the natural environment is important aetiologically. PMID- 3013954 TI - An ultrastructural study of acute and long-term lung response to commercial diatomaceous earth. AB - The acute pulmonary effects of intratracheally instilled particles of calcined diatomaceous earth were found to include a pronounced neutrophil invasion of the bronchioles by 4 h after exposure which remained well developed through 1 day post-exposure. The number of macrophages and neutrophils in the alveoli continued to increase through 1 day post-exposure and remained above control values through 7 days post-exposure. The number of macrophages, many of which contained diatomaceous earth, remained elevated for the duration of the experiment. Most phagocytosis of the particles was carried out by macrophages, with minor participation by neutrophils. Many of the reactive macrophages in the groups with post-exposure periods longer than 2 h showed various types of pathological alterations. A few particles were found in type I epithelial cells. Oedematous changes were observed in some type I epithelial cells and proliferation of type II epithelial cells was evident in some alveoli, particularly those near the respiratory bronchioles. Mild diffuse fibrosis was first observed at 6 months and was still present at 15 months but remained confined to the areas containing the diatomaceous earth. PMID- 3013955 TI - Herpes zoster: a possible early clinical sign for development of acquired immunodeficiency syndrome in high-risk individuals. AB - Zoster is uncommon before the age of 50 years in immunologically normal individuals, but it occurs with increased frequency in people who are immunosuppressed. A retrospective review of 300 patients with acquired immunodeficiency syndrome associated with Kaposi's sarcoma, revealed that 8% had prior zoster, a rate that is sevenfold greater than historic controls of the same age. We prospectively examined forty-eight patients, with no known immunodeficiency or signs of AIDS or AIDS related complex (ARC), who presented with zoster localized to the thoracic region. Forty-one patients had known risk factors for AIDS and thirty-five had antibody to the AIDS-associated virus (AAV) at the time of presentation. One seropositive subject had no known risk factors. Absolute lymphocyte counts, lymphocyte OKT4/OKT8 ratios, and lymphocyte mitogen responses were all depressed in subjects with antibody to AAV when compared with seronegative individuals. Seven of thirty-three AAV antibody-positive subjects, who could be followed longitudinally, developed AIDS from 1 to 28 months (mean = 13) after zoster. One antibody-negative subject seroconverted to become AAV seropositive 16 months after zoster and developed Kaposi's sarcoma 1 month later. These eight subjects had persistently low lymphocyte OKT4/OKT8 ratios and elevated beta-2 microglobulin. In patients at risk for AIDS, the occurrence of zoster may be one sign that heralds the marked depression of cellular immunity associated with AIDS or ARC. PMID- 3013956 TI - Giant glomangioma. PMID- 3013957 TI - Atopic eczema unresponsive to evening primrose oil (linoleic and gamma-linolenic acids) PMID- 3013959 TI - Ultrastructural study of calcinosis universalis with dermatomyositis. AB - Calcinosis universalis associated with dermatomyositis occurred in a 58-year-old woman. Tissues removed from the sublingual region in the patient were studied by ordinary microscopy, electron microscopy, and an electron-microanalytic method. The calcified materials were distributed on collagen fibers and seemed to have a relationship with foci of fibrinoid degeneration. Moreover, globular and/or membranous structures, considered to originate from the degenerate cells of the stroma, were observed in these calcified zones. Some of them contained electron dense materials. Therefore, the globular and/or membranous structures were thought to be concerned with initial calcification in this case. Furthermore, irregular bone tissue was formed adjacent to the calcified masses. In addition, the calcified materials were identified by X-ray diffraction examination and electron microscopy as hydroxyapatite. PMID- 3013960 TI - Effects of dietary protein on milk, rumen, and blood parameters in dairy cattle fed low fiber diets. AB - Eighteen multiparous and 9 primiparous Holstein cows were used to determine the effects of a 13 and 23% crude protein concentrate on milk fat depression during early lactation. Beginning on d 22 postpartum, cows were fed a high fiber diet (27% acid detergent fiber) for 3 wk and then switched to a low fiber diet (9 to 10% acid detergent fiber) for 6 wk. Crude protein percentages calculated from dry matter consumption were 13.5 and 17.9% during the high fiber period and 12.7 and 22.3% during the low fiber period. Daily milk and fat yields for both primiparous and multiparous cows were greater for the high protein treatment. The magnitude of decline in milk fat percentage (from high to low fiber) was greater for the low protein treatment, as determined by nonlinear regression. The high protein treatment was more effective in reducing the severity of milk fat depression in primiparous cows than in multiparous cows. Dietary crude protein had no effect on milk protein or solids-not-fat percentages, rumen volatile fatty acid molar proportions, or serum acetate concentration. The mechanism by which the high protein ration minimized the fat depression response to low fiber rations by primiparous cows is unknown. PMID- 3013958 TI - Converging adrenergic and cholinergic mechanisms in the inhibition of Cl secretion in fish opercular epithelium. AB - The rate of Cl secretion (Isc) by the opercular epithelium of Fundulus heteroclitus is stimulated by elevations in cyclic AMP (cAMP) levels elicited via beta 1-adrenergic receptor activation, and inhibited by both alpha 2-adrenergic and muscarinic cholinergic receptor activation via mechanisms presently unknown. A comparison of these two inhibitory responses was made using clonidine, an alpha 2-adrenergic agonist, and acetylcholine (ACh), a cholinergic agonist. The dose required for maximum inhibition was 100 times greater for ACh, but in all other respects the responses elicited by both agonists were statistically indistinguishable. Adrenergic antagonists did not diminish the ACh inhibition, and cholinergic antagonists did not diminish the clonidine inhibition, indicating that the two receptor types were distinct from each other. In control tissues and tissues pretreated with agents that increase cAMP levels (isoproterenol, IBMX, forskolin), both ACh and clonidine had no effects on cyclic AMP levels, indicating an inhibitory mechanism independent of adenylate cyclase. Neither Ca free media nor a variety of calcium antagonists diminished the ACh or clonidine inhibitions. These results suggest that the alpha 2-adrenergic and muscarinic cholinergic pathways converge into a common pathway to inhibit Cl secretion by a mechanism not involving adenylate cyclase or the mobilization of either extracellular or intracellular calcium stores. PMID- 3013961 TI - Impact of dietary fiber and physical form on performance of lactating dairy cows. AB - Two trials were conducted to study the effects of forage intake and physical form on lactating cow performance. In trial 1, four cows in a 4 X 4 Latin square were fed long alfalfa hay at 28, 36, 45, and 53% of total dry matter plus concentrate. Total dry matter intake was not affected by forage percent. Total chewing time and milk fat percentage increased linearly with increasing forage consumption. Maximum 4% fat-corrected milk production occurred when diets contained 27% neutral detergent fiber and 18% acid detergent fiber. In trial 2, four cows in a 4 X 4 Latin square were fed diets of chopped alfalfa hay and concentrate in proportions to supply 27.4% total ration neutral detergent fiber. Mean particle length measured with an oscillating screen particle separator of the chopped hay was .26, .46, .64, and .90 cm. Total dry matter and forage dry matter intakes and total chewing were not influenced by forage mean particle length. Mean particle length did not affect actual milk or 4% fat-corrected milk production. Depression of milk fat percentage was prevented when forage mean particle length was greater than or equal .64 cm. Apparent digestibility of dietary constituents and rate of passage of hay and concentrate was not influenced by forage intake or physical form. PMID- 3013962 TI - Deuterium oxide dilution kinetics to predict body composition in dairy goats. AB - Body composition and D2O dilution kinetics were studied in 15 female goats ranging from 38.0 to 70.1 kg live weight. Infrared spectrophotometric analyses of blood samples drawn during the 4 d following D2O injections were used to estimate D2O space. All does were slaughtered without shrinking and analyzed for dry matter, fat, nitrogen, and ash content. Estimates of D2O space from the late slope of the dilution curve, together with live weight, were used to predict body composition. Conclusions were 1) deuterium oxide space with live body weight accounts for about 90% of the variation in dairy goat empty body fat, empty body nitrogen, and empty body dry matter; 2) less than half the variation in empty body ash is related to live weight and D2O space; and 3) D2O space estimates would be biased by accelerations in water turnover. PMID- 3013963 TI - Influence of pecan shells and hulls as a roughage source on milk production, rumen fermentation, and digestion in ruminants. AB - In Experiment 1, 20 lambs (36 kg) were fed five diets containing 0, 5, or 10% pecan shells or hulls to evaluate digestion and nitrogen balance. Digestion was not depressed by diets containing 5% shells. Protein digestibility was not reduced and nitrogen balance was higher for lambs fed 5% hulls than for lambs in other groups. In Experiment 2, 8 Holstein cows (29.3 kg milk/d) were assigned to two diets: basal and basal with 5% shells in the grain mix. Cows fed diets containing shells produced the same amount of milk and milk fat as control cows. In Experiment 3, 12 Holstein cows (27.3 kg milk/d) were assigned to the same two diets used in Experiment 2 and a third treatment received 5% pecan hulls in the grain mix. Cows fed shells or hull diets reduced concentrate intake and milk production. In Experiment 4, 12 Hereford X Angus steers (474.5 kg) were fed diets used in Experiment 3 to examine rumen fermentation, digestion, and passage rates. Steers fed hulls had lower rumen ammonia N and higher rumen pH compared with steers fed the basal diet. Total rumen volatile fatty acid concentration was not different among treatments. Generally, rumen fluid from steers fed hulls had higher proportions of acetate and lower proportions of butyrate. Rumen fluid and particulate passage rates and digestion measurements were not affected by addition of shells or hulls. PMID- 3013964 TI - Alpha 2-adrenergic receptor function in patients with unipolar and bipolar affective disorders. AB - Alpha 2-adrenergic receptor function was measured in platelets from unipolar (UP) depressed, bipolar (BP) depressed, and bipolar euthymic patients and normal control subjects. Only the platelets from UP depressed patients were different from control in having an increased number of alpha 2-receptors, a decreased percent norepinephrine inhibition of prostaglandin E1 (PGE1)-stimulated cyclic AMP (cAMP) production, and a decrease in PGE1 stimulation of cAMP production. Platelets from BP patients, depressed or euthymic, were not significantly different from control subjects. These preliminary data suggest that alpha 2 adrenergic receptor function and PGE1 stimulation of cAMP production are diminished in UP patients. PMID- 3013965 TI - Pre- and post-dexamethasone plasma ACTH levels in depressed patients and normal controls. AB - Levels of plasma ACTH in relation to dexamethasone administration were evaluated in 16 medication-free patients with a major depressive episode and 39 normal controls. Depressed patients had significantly higher levels of plasma ACTH than controls both at 4:00 p.m. predexamethasone and 4:00 p.m. postdexamethasone. The significance of this finding is discussed. PMID- 3013966 TI - Gluten-sensitive enteropathy: a nonallergic immune hypersensitivity of the gastrointestinal tract. PMID- 3013967 TI - Single oral high-dose vitamin D3 prophylaxis in the elderly. AB - A poor vitamin D status is common in the elderly during the winter months. Because it is possible that hypovitaminosis D may be a cause of senile osteopenia, a simple method of prophylaxis would be useful. The single, oral, high-dose method was tested in two old-age homes, and the efficacy of vitamin D3 was compared with that of 25-hydroxyvitamin D3 (25-OHD3). The trials showed that 25-OHD3 caused a higher peak value in the serum 25-OHD levels in the second week than did vitamin D3. However, follow-up after four to five months showed that in those patients who received a single, oral dose of 25-OHD3, the serum 25-OHD levels had returned to the baseline low values, whereas in those patients who had had oral vitamin D3, the serum 25-OHD levels still remained significantly raised compared with the baseline values and were within normal limits. It is concluded that the single, oral, high-dose method using vitamin D3 is a safe and simple method of prophylaxis and could be used easily in large populations of elderly persons. PMID- 3013968 TI - Pre- and neonatal exposure of the Dahl rat to NaCl: development and regional distribution of myocardial alpha 1-adrenergic and cholinergic receptor sites. AB - The prenatal and/or postweaning effects of a hypertensinogenic high NaCl containing diet (8.0% NaCl, w/w) on (1) the regional distribution of alpha 1 adrenoceptors and muscarinic cholinergic receptor sites in the heart and (2) the predisposition/resistance to hypertension (HT) were assessed in the inbred Dahl HT-sensitive (S/JR) and HT-resistant (R/JR) rat. The density of alpha 1 adrenoceptors was reduced in the left ventricle but not consistently affected in the ventricular septum, right ventricle, or atria of S/JR offspring with NaCl induced HT. Both normotensive and hypertensive S/JR rats also displayed a significantly greater density of cholinergic receptor sites in the atria but few consistent alterations in other regions of the heart, compared to R/JR rats. Maternal diet had no effect on the predisposition/resistance to salt-induced HT and little effect on the regional development of alpha 1-adrenoceptors and cholinergic receptor sites. The results of this study suggest that the reduced density of ventricular alpha 1-adrenoceptors in the S/JR strain is a consequence of HT while the elevated density of cholinergic receptors in the atria may be related to the genetic predisposition/resistance to HT. PMID- 3013970 TI - Superoxide radical from xanthine oxidase acting upon lumazine. AB - The univalent and divalent reductions of dioxygen were measured using lumazine as a low turnover substrate and both xanthine and acetaldehyde as high turnover substrates. These measurements were made in solutions equilibrated with air and with 100% O2. The univalent route of dioxygen reduction predominated with the low turnover substrate and was increased by raising pO2 and by lowering substrate concentration. These results support the view that electron egress from heavily reduced xanthine oxidase occurs by divalent transfers, while that from the partially reduced enzyme is by univalent transfers. Xanthine oxidase, acting as lumazine, is a convenient source of O2-. PMID- 3013969 TI - Role of metals in oxygen radical reactions. AB - Partially-reduced forms of dioxygen or "oxy-radicals" (superoxide, O2-/HO2; hydrogen peroxide, H2O2; hydroxyl radical X OH) and oxidants of comparable reactivity are implicated in an increasing number of physiological, toxicological, and pathological states. Transition metal catalysis is recognized as being integral to the generation and the reactions of these activated oxygen species. Factors such as pH and chelation govern the reactivity of the transition metals with dioxygen and "oxy-radicals" and therefore influence the apparent mechanisms by which oxidative damage to phospholipids, DNA, and other biomolecules is initiated. In biological systems the concentrations of redox active transition metals capable of catalyzing these reactions appears to be relatively low. However, under certain conditions metal storage and transport proteins (ferritin, transferrin, ceruloplasmin, etc.) may furnish additional redox active metals. PMID- 3013972 TI - Iron-induced lipid peroxidation in spinal cord: protection with mannitol and methylprednisolone. AB - The ability of the free radical scavenger, mannitol, and the synthetic glucocorticoid, methylprednisolone sodium succinate (MPSS) to reverse the effects of iron catalyzed free radical induced lipid peroxidation was assessed in the feline spinal cord. Ferrous chloride (100 mM) was infused into the gray matter of lumbar spinal cord, the region frozen in situ, removed, and homogenates of the gray matter analyzed for activity of Na+,K+-ATPase and levels of malondialdehyde (MDA). ATPase activity had declined to approximately 30% of control by 2 h after FeCl2 infusion and remained at this level through 24 h. Malondialdehyde values were elevated almost twofold at 2 h. Mannitol essentially reversed the effects of FeCl2 infusion on Na+,K+-ATPase activity and MDA production. These results may implicate the hydroxyl radical (. OH), or an oxidizing species with . OH-like reactivity, as the initiating radical species in this model of lipid peroxidation. Similarly, MPSS prevented the decline in spinal cord Na+,K+-ATPase activity and rise in MDA levels that were induced by FeCl2 infusion. This demonstrated that at the dosage levels used in this study, MPSS was an effective antioxidant. This finding provides presumptive evidence suggesting that, at least in experimental animals, the effectiveness of MPSS in preventing the tissue necrosis and paralysis that is the sequelae of spinal cord trauma may reside, in part, in the capacity of this glucocorticord to quench peroxidative reactions in the injured tissue. PMID- 3013971 TI - Treatment of lymphocyte cultures with a hypoxanthine-xanthine oxidase system induces the formation of transferable clastogenic material. AB - Culture medium of lymphocyte cultures that have been exposed to the superoxide generating system hypoxanthine plus xanthine oxidase (X-XO) contains substances with chromosome damaging properties. This is demonstrated by the ability of ultrafiltrates of such culture media to induce chromosomal aberrations and sister chromatid exchanges in the lymphocytes of blood test cultures. Culture medium becomes active about 15 hours after the addition of X-XO and stimulation by phytohemagglutinin. Concomitant with the accumulation of clastogenic material, assays for conjugated dienes and thiobarbituric acid-reactive material which measure lipid-peroxidation become positive in the culture media. When cells are pretreated with superoxide dismutase or glutathione peroxidase before the addition of X-XO neither clastogenic substances nor lipid peroxidation products are detected. Catalase is a less efficient protector. PMID- 3013973 TI - Molecular heterogeneity in chronic granulomatous disease: a human model of defective phagocyte superoxide production. AB - Chronic granulomatous disease (CGD) is a genetically transmitted disorder thought to result from defect(s) in the activation or turnover of the NADPH dependent O2- generating oxidase enzyme system of human neutrophils and monocytes. The normal oxidase may be a flavoprotein-cytochrome b559 complex; therefore, these components of the oxidase were quantitated in the neutrophils from patients and family members of two unrelated CGD kindreds. The male propositus from an X linked recessive kindred had a neutrophil oxidase fraction with low FAD content (26 pmol/mg protein) and undetectable cytochrome b559 (less than 5 pmol/mg protein). The male propositus from an autosomal recessive kindred had a neutrophil oxidase fraction with low FAD content (34 pmol FAD/mg protein), but normal cytochrome b559 content (170 pmol cytochrome b559/mg protein). Both parents of this latter CGD patient had normal FAD and cytochrome b559 content in their neutrophil oxidase fraction. We conclude that the carrier state in certain X-linked recessive female carriers of CGD can be detected by partial deficiencies of both flavoprotein and cytochrome b559 components of the oxidase, whereas presumed heterozygous carriers of certain autosomal recessive CGD kindreds cannot be detected by this means. PMID- 3013975 TI - Ethylene production from alpha-keto-4-thiomethylbutyric acid by isolated rat liver cells, suspension medium, and perfusates in the absence and presence of iron. AB - Experiments were carried out to evaluate the production of hydroxyl radical-like species by intact rat liver cells by assaying for the production of ethylene from alpha-keto-4-thiomethylbutyric acid in the absence and presence of added iron. In the absence of iron, a low rate of ethylene production, which was not sensitive to superoxide dismutase, catalase, or competitive scavengers was observed. Ethylene was produced when KMBA was incubated with perfusates of rat liver or the suspension medium obtained after incubating liver cells for varying periods of time, followed by removal of the liver cells. Boiling the perfusate or suspension medium had no effect on ethylene production. This ethylene production does not appear to reflect an oxygen radical-mediated event. The addition of ferric-EDTA, but not ferric-desferrioxamine, increased ethylene production by the hepatocyte suspensions in a reaction sensitive to inhibition by catalase, ascorbate oxidase, and competitive scavengers, but not superoxide dismutase. The sensitivity to externally added catalase and ascorbate oxidase suggested that the ethylene production reflected an extracellular oxygen radical generating system. Ferric alone and several ferric chelates, for example, ferric-ATP, ADP, AMP, histidine, and citrate stimulated ethylene production using perfusates of liver or suspension medium after removal of the hepatocytes. The sensitivity of the added iron system to ascorbate oxidase suggested that during perfusion or incubation of liver cells, efflux of ascorbate occurs, followed by reduction of the iron and subsequently, extracellular production of oxygen radicals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3013974 TI - Failure of desferrioxamine to modify the toxicity of paraquat in rats. AB - The feasibility of using desferrioxamine (DF), an iron chelator, as a therapeutic agent against paraquat (PQ++) toxicity in male Sprague-Dawley rats was explored, based on the rationale of limiting toxic hydroxyl radical production from hydrogen peroxide by removing redox-active iron. Body weights, mortality, and lung histopathology were followed for periods up to 14 days after intraperitoneal injection of PQ++ (20 or 25 mg/kg body weight) with or without concurrent daily subcutaneous injections of DF (300 mg/day). Animals receiving PQ++ showed the expected typical patterns of mortality and of lung histopathology, namely: marked edema, subpleural hemorrhage, acute inflammation, perivascular mononuclear cell infiltrates, sloughing of alveolar and bronchiolar lining cells, and diffuse interstitial fibrosis. Desferrioxamine alone was non-toxic. Surprisingly, results when both PQ++ and DF were administered indicated a failure of DF to ameliorate toxic effects of PQ++ in the lung, and even suggested an accentuation of PQ++ induced damage by DF. Mortality data showed that PQ++/DF animals died in greater numbers (20 mg PQ++/kg) or died earlier (25 mg PQ++/kg) than animals receiving DF alone. Qualitative histopathology in PQ++/DF animals was comparable to PQ++ animals in early stages, but damage was more severe in both incidence and severity of lesions in PQ++/DF animals, particularly at the 25 mg PQ++/kg dose level. After 14 days, surviving animals receiving PQ++ alone showed almost complete resolution of previous inflammation and other acute effects, whereas in the only surviving PQ++/DF animal initial fibrosis had persisted and become more generalized. Thus, chelation therapy with DF may not be straightforward in its effects on PQ++ toxicity. PMID- 3013976 TI - Efficiency of chelated iron compounds as catalysts for the Haber-Weiss reaction. AB - The oxidation of formate to CO2 has been used to quantify . OH yields produced in oxygenated solutions by chelated iron salts reacting either directly with H2O2 in the Fenton reaction, or as catalysts in the Haber-Weiss reaction between H2O2 and radiolytically generated superoxide. This system involves a chain sequence since . OH regenerates O2- when producing CO2. Kinetic studies have been employed to show that catalysis by Fe-EDTA occurs by reduction of Fe3+-EDTA by O2- followed by its reoxidation by H2O2, and to show how O2- is ultimately consumed. At pH 7.3 more than 50 . OH radicals can be produced per molecule of Fe-EDTA and CO2 yields can exceed five per molecule of radiolytically generated O2-. Iron chelated with pyrophosphate, DTPA, citrate, ATP or ADP in phosphate or Tris buffer at pH 7.3 has less than 7% of the catalytic ability of Fe-EDTA (considerably less in most cases) even though all these ferrous chelates give appreciable yields of . OH in the Fenton reaction. Unchelated iron has no catalytic ability. Catalysis of the Haber-Weiss reaction in homogenous solution by iron salts, either free or chelated with nucleotides or citrate, is evidently a very inefficient process, and its possible role in superoxide toxicity must be viewed with these reservations. PMID- 3013977 TI - Oxygen toxicity: loss of lung macrophage function without metabolite depletion. AB - Hyperoxia inhibited concanavalin A stimulated O2- release (respiratory burst) of alveolar macrophages obtained by bronchoalveolar lavage from rats. After 36 h of normobaric 100% O2, a partial reversal (48%) of the inhibition was produced by addition of glucose. Since oxidant-induced, reversible NADPH depletion correlates with reversible inhibition of the respiratory burst, intracellular NADPH was assayed to determine whether irreversible inhibition of the respiratory burst was related to persistent changes in this metabolite. The cellular concentrations of ATP, glutathione, and ascorbate were also measured. After 36 h of hyperoxia, NADPH concentration in alveolar macrophages rose slightly while ATP and glutathione content remained at control levels. Ascorbate levels fell significantly but were not responsible for respiratory burst inhibition. Thus, irreversible loss of cellular function in hyperoxia is not due to persistent alterations in these metabolites. Significant amounts of both glutathione and ascorbate were found in extracellular fractions of lung washings, indicating high concentrations in the aqueous subphase in the lung fluid lining. There was no change in total content of these extracellular antioxidants following O2 exposure. PMID- 3013978 TI - The reaction of ferrous EDTA with hydrogen peroxide: evidence against hydroxyl radical formation. AB - Radiolytically generated hydroxyl radicals degrade cytochrome c. In contrast, ferrocytochrome c is oxidized in the Fe-EDTA-H2O2 system, without loss of any cytochrome c. It is concluded that in the latter system no hydroxyl radicals are formed and that oxidation takes place via a FeO(II)EDTA or Fe(II)-H2O2-EDTA complex. PMID- 3013979 TI - Further studies of the mechanism of the enhancement of NADH oxidation by vanadate. AB - O2-., whether generated photochemically, or introduced as a solution of KO2 in a nonprotic solvent, caused rapid oxidation of NADH in the presence, but not in the absence of vanadate. Superoxide dismutase inhibited this vanadate-stimulated oxidation of NADH, while catalase had no effect. This NADH oxidation appears to be a free-radical chain reaction whose average chain length was estimated to be 15 in the photochemical system. Vanadate stimulation of NADH oxidation by biological membranes can now be viewed as a sensitive indicator of O2-. production by those membranes. PMID- 3013980 TI - Rat liver microsomal NADPH-dependent release of iron from ferritin and lipid peroxidation. AB - Microsomes prepared by the usual method of differential centrifugation were found to contain ferritin, superoxide dismutase (SOD), and catalase which could be removed by chromatography on Sepharose CL-2B. Addition of purified rat liver ferritin to chromatographed microsomes resulted in a significant stimulation of NADPH-dependent lipid peroxidation which was inhibited by exogenously added SOD. Iron release from ferritin by these microsomes was also inhibited by SOD. Ferritin did not promote NADPH-dependent microsomal lipid peroxidation when added to microsomes isolated in the usual manner, presumably due to the endogenous SOD present in the microsomes. Accordingly, only very low rates of iron release from ferritin were observed with these microsomes. Paraquat (PQ), which generates superoxide O2-. via redox cycling, greatly stimulated iron release from ferritin and lipid peroxidation in chromatographed microsomes. Paraquat had no effect on iron release from ferritin or lipid peroxidation in microsomes. which were not chromatographed unless they were first treated with CN- to inhibit endogenous SOD. These studies indicate that the majority of microsomal iron is contained within ferritin and that following release by O2-. this iron serves to promote the peroxidation of microsomal lipids. PMID- 3013981 TI - Excited species generation in horseradish peroxidase-mediated oxidation of glutathione. AB - Photoemissive excited species are produced by the horseradish peroxidase (HRP) catalyzed oxidation of reduced glutathione (GSH), without exogenously added hydroperoxide under aerobic conditions. The emitted low-level chemiluminescence consisted of two phases. Light emission occurred at wavelengths beyond 610 nm (greater than or equal to 90% intensity), indicative of singlet oxygen 1O2. Deuterium oxide enhanced photoemission 4.4-fold. Ascorbate inhibited chemiluminescence completely. In the absence of GSH or when GSH was replaced by the disulfide, no red chemiluminescence was observed. The glutathionyl radical GS. is most likely to be involved in both phases of light emission. Further, the superoxide radical plays a role, as substantiated by the inhibitory effect of superoxide dismutase. Both phases of photoemission were abolished by glutathione peroxidase; thus hydroperoxides are regarded as essential intermediates for the formation of excited species. Catalase abolished phase I and did not affect phase II. In contrast, glutathione S-transferase 1-2 (showing peroxidase activity towards organic hydroperoxides but not towards H2O2) inhibited phase II, whereas phase I was still present. Glutathione sulfonate and the disulfide GSSG were detected as oxidation products from GSH under conditions where phase II chemiluminescence was observed. HRP Compound III accumulated during the reaction. It is concluded that phase I is dependent on exogenously added or endogenously generated H2O2, whereas phase II does not require H2O2 but an organic peroxy species. A mechanism based on chain reactions involving oxygen addition to the thiyl radical is proposed. Sulfenyl peroxy species are suggested as transient intermediates in reactions finally leading to the generation of excited states such as singlet molecular oxygen. PMID- 3013982 TI - Biosynthesis of superoxide dismutase in Saccharomyces cerevisiae: effects of paraquat and copper. AB - Growth of Saccharomyces cerevisiae in the presence of paraquat caused an increase the intracellular flux of superoxide radicals and in superoxide dismutase biosynthesis. The addition of copper to the growth medium also elicited an increase in superoxide dismutase levels. A cytochrome c deficient mutant strain was found to be more responsive than the wild type strain to paraquat and/or copper by increasing its copper-zinc superoxide dismutase. Catalase activity in both strains was not significantly affected by paraquat and/or copper. PMID- 3013983 TI - Age-related changes of beta-adrenoceptors in aging inbred mice. AB - The density (Bmax) and antagonist dissociation constant (KD) of beta adrenoceptors were determined on spleen and brain of three different inbred strains of mice--BALB/cJ, C3H/HeJ, and C57BL/6J. Receptor densities (Bmax) differed with strain and declined in both spleen and cortical receptor populations as mice became older. Age-related changes in KD were found on spleen cells of BALB/cJ and in the cortex of C3H/HeJ and C57BL/6J. Bmax and KD in different organs of the same strain changed at different rates. changed at different rates. PMID- 3013984 TI - Synovial cell sarcoma: a case report with a comment on 27 years of treatment. AB - The long-term survival of patients after surgical removal of soft tissue sarcomas is dependent on many factors. This case report shows that a postsurgical clinical "cure" may depend more on the biologic behavior of the neoplasm than on any specific operative modality. PMID- 3013985 TI - Human lymphoblastoid interferon. In vitro and in vivo studies in hepatocellular carcinoma. AB - We have examined the growth inhibitory effects of human lymphoblastoid interferon (IFN) on the human hepatocellular carcinoma (HCC) cell line PLC/PRF/5. In vitro, PLC/PRF/5 cells were sensitive to the antiproliferative effects of IFN, growth inhibition being noted at concentrations as low as 1.25 i.u. ml-1. Athymic mice with xenografted tumours derived from the PLC/PRF/5 cell line were treated daily with IFN or a saline control. An IFN dose of 2 X 10(5) i.u./day was found capable of significantly slowing tumour growth rate and prolonging mouse survival. Further studies to examine the mechanisms involved in growth inhibition in vivo demonstrated that IFN was capable of inducing the activity of the enzyme 2,5 oligoadenylic acid (2,5 A) synthetase, a potent inhibitor of protein synthesis, in tumour xenografts but not in mouse tissue, and that IFN significantly enhanced the membrane display of HLA class I glycoproteins on tumour cells, though histology did not reveal any increase in tumour infiltration by host lymphocytes. We conclude that IFN exerts potent growth inhibitory effects on the HCC cell line PLC/PRF/5 both in vitro and in vivo and its mode of action in this animal model system appears to be predominantly mediated by a direct antiproliferative effect on tumour cells. PMID- 3013986 TI - Phospholipid transmethylation and choline phosphotransferase in microsomal fraction of human diseased liver. AB - The activities of three enzymes catalyzing the production or degradation of phosphatidylcholine, a major structural phospholipid of cell membranes, were assessed in hepatocyte membrane microsomal preparation from patients with various types of liver disease. Choline phosphotransferase activity of preparation from patients with chronic aggressive, chronic active, chronic persistent, alcoholic hepatitis and cirrhosis accompanied by marked necrosis and relatively slight fibrosis was markedly decreased, compared with normal liver; the activity from patients with fatty liver and chronic inactive hepatitis was slightly decreased. Specimens from patients with acute transient hepatitis were not significantly different from normal. Methyltransferase and phospholipase A2 activities tended to parallel that of choline phosphotransferase, although the degree of changes was generally less marked. Our studies indicate that enzyme activities that are critical for hepatic cell membrane integrity and activity are attenuated in liver specimens from patients with disease in which there is marked hepatic cell necrosis. PMID- 3013987 TI - Alcohol--a major risk factor for hepatocellular carcinoma? PMID- 3013988 TI - Rapid assay for antibodies to Varicella-Zoster virus (VZV) using a VZV-HEM preparation. AB - A freeze-dried, formalized-erythrocytes-bound VZV antigen for indirect haemagglutination, VZV-HEM, was prepared. It was used to test serologically 46 children, all of them patients of Prague paediatric clinics, with a known history of chickenpox. For comparison, the same sera were tested by the indirect haemagglutination reaction with freshly prepared VZV antigen and by ELISA. All three serological tests gave congruent results in 97.8% of cases. The advantages of the VZV-HEM assay are results obtainable within 2 hours of serum dilution and simplicity. The assay is especially suitable for emergency testing the immunity of persons at risk. PMID- 3013989 TI - Structural homology between the human 4F2 antigen and a murine cell surface glycoprotein associated with lymphocyte activation. PMID- 3013991 TI - Differential expression of ecto-5' nucleotidase activity by functionally and phenotypically distinct subpopulations of human Leu-2+/T8+ lymphocytes. AB - Subsets of Leu-2+/T8+ cytotoxic/suppressor T lymphocytes were isolated by using various methods of purification and were investigated for expression of ecto-5' nucleotidase (5'NT) enzyme activity by radiochemical, cytochemical, and ultrastructural techniques. By using both the radiochemical and the cytochemical methods. T4-OKM1- cells displayed higher 5'NT activity in comparison with the entire T4- subpopulation. Analyses of the subpopulations of T4- (and predominantly Leu-2+) cells defined by the Leu-15 or Lyt-1 (9.3) monoclonal antibodies demonstrated that T4-Leu-15- and T4-Lyt-1+ cells displayed high 5'NT activity, whereas virtually no activity was present in T4-Leu-15+ and T4-Lyt-1 cells. At the ultrastructural level, the 5'NT reaction product was detected on the plasma membrane of a proportion of nongranular Leu-2+/T8+ lymphocytes, but no activity was found on cells with a granular lymphocyte (GL) morphology. 5'NT activity was also analyzed in peripheral blood mononuclear cells from one patient with expanded numbers of GL and two patients with GL leukemia. The enzymatic activity was significantly lower in these patients than in normal controls. This study provides new cytochemical evidence demonstrating the heterogeneity of Leu 2+/T8+ cells, and indicates that the population with the suppressor phenotype and function (Leu-15+/Lyt-1-, GL morphology) displays low or absent 5'NT activity, whereas the population composed of cytotoxic cell precursors (Leu-15-/Lyt-1+, nongranular morphology) has high 5'NT activity. Implications of these data for the interpretation of low 5'NT activity described in several immunodeficiency states and lymphoproliferative disorders are discussed. PMID- 3013990 TI - Antigen reactive memory T cells are defined by Ta1. AB - Ta1 is a 105,000 dalton protein that is weakly expressed on a small fraction of resting human peripheral blood T cells but strongly expressed in vitro on T cell clones and a substantial proportion of activated T cells. Unlike receptors for growth factors such as IL 2, the Ta1 antigen is present on T cell lines and clones irrespective of cell cycle. The function of Ta1 was investigated after separation of T lymphocytes into Ta1-enriched and Ta1-depleted subpopulations that were obtained from normal human subjects. Although Ta1-enriched T cells constitute only 10 to 15% of the E rosette-positive lymphocyte population, most, if not all, of the anamnestic response to the recall antigens tetanus toxoid and mumps reside in the Ta1+ population. Both Ta1-enriched and -depleted cells responded equally well to the mitogen PHA. The autologous mixed lymphocyte response was also greater in the Ta1-enriched subpopulation but not to the degree seen with soluble antigen. Increased proliferation was not due simple to increased inducer cell function within the Ta1+ subpopulations because both Ta1- and Ta1+ cells induced similar amounts of Ig synthesis in the presence of PWM. Additionally, increasing numbers of Ta1- cells did not suppress the enhanced proliferative responses of Ta1+ cells, and thus Ta1- cells do not appear to be functioning as suppressor cells. The Ta1 antigen appears to be a marker for previously activated T cells in peripheral blood, and this subpopulation appears to include T memory cells. PMID- 3013992 TI - Lack of reactivity of rheumatoid arthritis synovial membrane DNA with cloned Epstein Barr virus DNA probes. AB - Rheumatoid arthritis (RA) synovial membranes contain a 62,000 dalton (62 Kd) molecule that shares an antigenic epitope with the EBNA-1 antigen of Epstein Barr virus (EBV). This cross-reactive epitope(s) is defined by a monoclonal anti-EBNA 1 antibody (MoAb P135) and by a rabbit antibody directed against a (glycine alanine)-containing synthetic peptide from the internal repeat region-3 (IR-3) of EBNA-1. To determine whether this 62 Kd protein may result from EBV infection of RA synovial membranes, we used cloned DNA probes from the Bam M, Bam V, and Eco D regions of EBV. These probes did not show detectable reactivity with RA synovial membrane DNA in Southern blotting or slot blotting experiments. Reconstruction experiments performed with purified EBV DNA demonstrated the ability to detect 0.03 pg of viral DNA per 20 micrograms of normal genomic DNA, or approximately 1 EBV copy per 100 cells. In contrast, we found a low but significant level of reactivity of RA synovial membrane DNA with the EBV-encoded Bam K probe that encodes the EBNA-1 antigen. However, this probe also was reactive with normal genomic DNA to a similar extent. These results suggest that the 62 Kd antigen in RA synovial lining cells is probably encoded by cellular genes similar to the IR 3 region of EBV and does not result from EBV infection of the RA synovial membrane. PMID- 3013993 TI - Enhancement of glomerular mesangial cell neutral proteinase secretion by macrophages: role of interleukin 1. AB - We have examined the ability of rat mesangial cells to regulate neutral proteinase production in vitro. Mesangial cells constitutively produced gelatinase when cultured in serum-free medium, and enzyme production by these cells was inhibited by cycloheximide. Coculture with thioglycollate-elicited rat peritoneal macrophages resulted in enhanced gelatinase production. The increase in enzyme released correlated directly with the number of macrophages added. Conditioned medium from LPS-activated peritoneal macrophages also enhanced gelatinase production in a dose-dependent manner. Fractionation of these macrophage supernatants on Sephacryl S-200 revealed a predominant fraction of gelatinase-enhancing activity in a m.w. range between 10,000 and 20,000. These data suggested that the enhanced mesangial cell gelatinase production was mediated through the action of interleukin 1. This was confirmed by the finding that purified interleukin 1, prepared from LPS-stimulated rat peritoneal macrophages, stimulated mesangial cells to secrete gelatinase in a dose-dependent manner. These findings may be of significance in the understanding of the pro inflammatory role of macrophages in immune-mediated glomerulonephritis. PMID- 3013994 TI - Effect of interleukin 1 on the expression of interleukin 2 receptor (Tac antigen) on human natural killer cells and natural killer-like cell line (YT cells). AB - The effect of interleukin 1 (IL 1) on the expression of interleukin 2 receptor (IL 2R/Tac antigen) on human natural killer (NK) cells and the NK-like cell line, YT was studied with the use of a fluoresceinated anti-IL 2R monoclonal antibody and a Spectrum III flow cytofluorometer. IL 2R was expressed on approximately 10% of NK cells. The expression of IL 2R on NK cells was increased to approximately 25% by the in vitro culture with monocytes or IL 1 and to a less extent by the culture with IL 2 or interferon-gamma (IFN-gamma). IL 2R was expressed on approximately 50% of YT cells without any stimulations. The expression of IL 2R on YT cells was increased up to almost 100% by the culture with IL 1 or monocytes, but not with IL 2, IFN-alpha, IFN-beta, IFN-gamma, or lectins such as concanavalin A and phytohemagglutinin-P. IL 1 absorbed with YT cells or murine thymocytes lost both IL 1 activity detected by the stimulation of murine thymocyte proliferative response and enhancing activity of IL 2R expression on YT cells, suggesting that IL 1 has both activities. However, the assay system of the expression of IL 2R on YT cells is much more sensitive than the stimulation of murine thymocyte proliferative response. By the kinetic study, the enhancement of IL 2R expression was induced by only a 2-hr incubation of YT cells with IL 1. This enhancement did not proceed at 4 degrees C or by the treatment of YT cells with actinomycin D or cycloheximide, suggesting that this enhancement is energy dependent and requires the synthesis of RNA and protein but not DNA. Thus IL 1 plays an important role for the regulation of the expression of IL 2R on NK cells, and IL 1-dependent IL 2R expression on YT cells may give us a good model for the study of the molecular mechanism of the regulation of IL 2R expression. PMID- 3013995 TI - Interleukin 3 (IL 3) regulates the in vitro proliferation of both blood monocytes and peritoneal exudate macrophages: synergism between a macrophage lineage specific colony-stimulating factor (CSF-1) and IL 3. AB - The effect of interleukin 3 (IL 3) on regulation of macrophage proliferation was examined. Although IL 3 alone stimulates the colony formation in bone marrow cells, it fails to stimulate the colony formation by both peritoneal exudate macrophages (PEM) and blood monocytes. However, IL 3 greatly enhances the proliferative capacity of both PEM and monocytes in responding to suboptimal concentrations of CSF-1. At supraoptimal concentrations of CSF-1, IL 3 did not increase the number of colonies, but greatly increased colony size. Kinetic studies showed that IL 3 enhances CSF-1-induced macrophage proliferation by shortening the cell doubling time. Monocytes were more sensitive to the action of IL 3 and possessed higher proliferative potential than PEM. Binding studies with radioactive labeled CSF-1 (125I-CSF-1) showed that IL 3 treatment induced an increased expression of CSF-1 receptor activity by PEM which appears to be a result of increased number of available receptor sites. The effect of IL 3 on the expression of receptor activity is both dose- and time-dependent. IL 3 also augments the rate of receptor-mediated CSF-1 endocytosis by PEM which appears to be a direct result of increased expression of CSF-1 binding sites. These results demonstrate that, in addition to stimulating the growth and differentiation of several blood cell lineages by hemopoietic stem cells, IL 3 also possesses the ability to modulate CSF-1 receptors, thereby affecting proliferation of more mature blood monocytes and tissue-derived macrophages. PMID- 3013996 TI - Production of idiotypic and anti-idiotypic antibodies by BALB/c mice in response to immunizations with glucagon, vasopressin, or insulin: supporting evidence for the network concept. AB - After immunizations with glucagon or vasopressin, either conjugated to keyhole limpet hemocyanin or adsorbed to polyvinylpyrrolidone, both anti-hormone and anti receptor activities were detectable in the serum of injected mice. Anti-hormone activity was identified by ELISA techniques; anti-receptor activity, by determining the ability of serum samples to compete with labeled hormone for glucagon or vasopressin receptors on rat liver plasma membranes. Anti-receptor activity appeared only after the peak anti-hormone response to each immunogen had been established, and required intensive immunizations (six to nine monthly injections). The presence of anti-idiotypic antibodies in serum samples containing glucagon or vasopressin anti-receptor activity was confirmed by demonstrating selective binding of such samples to corresponding rabbit idiotypic antibodies. Serum from mice immunized with insulin also contained anti-hormone activity, as determined by ELISA, and anti-receptor activity, as determined by noting insulin-mimicking properties in stimulating glucose transport in rat adipocytes. The anti-insulin receptor activity developed after only one boost with the hormone. These results are consistent with Jerne's network hypothesis in that the glucagon, vasopressin, and insulin anti-receptor activity may be attributed to antibodies produced in mice as part of an idiotypic-anti-idiotypic network. PMID- 3013999 TI - Reactivity of E. coli-derived trans-activating protein of human T lymphotropic virus type III with sera from patients with acquired immune deficiency syndrome. AB - Randomly sheared DNA fragments from HTLV-III proviral DNA were cloned into an E. coli open reading frame (ORF) expression vector. The inserted ORF DNA was expressed in E. coli transformants as a polypeptide fused to the lambda CI protein at the amino terminus and to beta-galactosidase at the carboxyl terminus. The reactivity of the recombinant peptides with antibodies from sera of AIDS patients was determined by the Western blot technique. The coordinates of the DNA inserts of the immunoreactive clones were then determined by DNA sequencing. A clone, ORF 628, was found to contain a short DNA segment located between the sor and env genes (nucleotide positions 5367 to 5597), a region previously thought to be noncoding. Inspection of the DNA sequences of this clone and of other HTLV-III isolates revealed the presence of a small ORF located between nucleotide position 5411 and 5625, capable of encoding a polypeptide of 72 amino acids. The biosynthesis of the polypeptide of ORF 628 initiates from an ATG codon within the HTLV-III insert. The fusion protein of ORF 628 was partially purified by affinity chromatography on CH Sepharose 4B coupled to a beta-galactosidase ligand, and tested against a panel of sera from AIDS patients by Western blot analysis. Approximately 35% of the sera from patients with AIDS or ARC contained antibodies reactive with the peptide. The DNA region spanned by ORF 628 is now thought to be the major functional element of the trans-activator gene, tat. PMID- 3013998 TI - In vitro locomotion of allosensitized T lymphocyte clones in response to metabolites of arachidonic acid is subset specific. AB - The effects of the arachidonic acid metabolites prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) on the in vitro random migration of cloned murine T lymphocytes (derived from limiting dilution analysis of a C57BL/6 anti-DBA/2 mixed leukocyte culture) were examined. Experiments were also performed to study the effects of the cyclooxygenase inhibitor indomethacin on both random lymphocyte migration and lymphocyte migration in the presence of PGE2. The responses of cloned lymphocytes to PGE2 and LTB4 were compared with those of unsensitized lymph node lymphocytes. PGE2 at 100 ng/ml significantly inhibited (p less than 0.001) the in vitro migration of helper clones of T lymphocytes, but had no effect on random migration of cytotoxic T cells or helper independent cytotoxic (HIT) cloned cells. In contrast, LTB4 significantly (p less than 0.001) enhanced the random locomotion of helper, cytotoxic, and "HIT" cloned cells at 0.1 and 0.3 ng/ml. The effects of both PGE2 and LTB4 were found to be completely reversible by cell washing. Indomethacin (10(-7) M) did not alter random migration of any of the clones, and in particular, did not affect the inhibition of helper lymphocyte migration induced by PGE2. Unsensitized bulk lymph node lymphocyte migration was not affected by either PGE2 or LTB4. The results suggest that modulation of lymphocyte locomotor function by environmental stimuli may depend on cellular activation, and the locomotor responses of activated lymphocytes to arachidonic acid metabolites may be subset specific. PMID- 3014000 TI - Human retroviral env and gag polypeptides: serologic assays to measure infection. AB - Humoral antiviral responses to human retrovirus infections identify persistently infected individuals and can be used to characterize virus-host interactions. Antibodies to native viral polypeptides have been reliably measured, although quantitation of env antibodies is difficult due to a lack of purified antigens. To quantitate antibodies to env antigens, bacterially expressed cloned env polypeptides from the transmembrane regions of human T lymphotropic virus types I and III were applied to nitrocellulose filters in an immunodot assay. A combination of the sensitivity of the Western blot procedure and the specificity of peptides from defined viral sequences was used to detect 49/49 HTLV-III/LAV infected individuals previously defined as seropositive by radioimmunoprecipitation sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of these HTLV-III/LAV envelope seropositive people, 22% lacked antibody to p24 in a radioimmunoassay. In contrast, the sensitivity of antibody detection to HTLV-I env antigens and p24 were comparable. Antibodies to HTLV-I and HTLV-III/LAV env transmembrane peptides were not cross-reactive. Levels of antibody to env antigens of both HTLV-I and HTLV-III/LAV persisted without change for at least 26 mo, suggesting that most infections represent stable virus-host interactions. The use of bacterially expressed env peptides offers a rapid serologic approach for distinguishing human retroviral infections and can be used to define immune responses to specific regions of the viral genome. PMID- 3013997 TI - Aberrant activation and regulation of the oxidative burst in neutrophils with Mol glycoprotein deficiency. AB - Patients whose cells are deficient in the glycoproteins LFA-1, Mol, and p150,95 have recurrent infections and pronounced abnormalities in neutrophil adherence, aggregation, chemotaxis, and phagocytosis. We characterized activation and regulation of oxidative metabolism of Mol-deficient neutrophils. These cells failed to depolarize or to produce O2- or H2O2 normally when stimulated by opsonized zymosan. The chemotactic peptide formyl methionyl-leucyl-phenylalanine depolarized Mol-deficient neutrophils normally but caused supernormal production of O2- and H2O2, a result of a prolonged burst in oxidative metabolism. Phorbol myristate acetate depolarized Mol-deficient neutrophils at a nearly normal rate but evoked release of significantly less O2- and H2O2 than from normal PMN. The aberrant activation and regulation of the oxidative burst in Mol-deficient neutrophils are considered in light of recently emerging concepts in the cell biology of this process, and the possibility that these abnormalities reflect a defect in the cytoskeleton-membrane interaction is discussed. PMID- 3014001 TI - Analysis of neoplasms induced by Cas-Br-M MuLV tumor extracts. AB - Cas-Br-M is an ecotropic murine leukemia virus isolated from wild mice that induces a wide spectrum of hematopoietic neoplasms, including T and B cell lymphomas, myelogenous leukemias, and erythroleukemias. The purpose of this study was to determine if the induction of neoplasms belonging to multiple lineages was due to the ecotropic virus itself or to the generation of cell lineage-specific recombinant viruses. The results demonstrate that in some instances (two of 12 tumor extracts tested), recombinant viruses can be recovered from primary Cas-Br M-induced tumors that will induce lymphomas of single lineages in mice inoculated as newborns. One of these viruses is a recombinant mink cell focus-forming virus that induces T cell lymphomas, and the other is a replication-defective, fibroblast-transforming virus that induces early B lineage lymphomas in mice. Histologic and flow microfluorometric cell surface antigen analyses of primary and in vitro adapted tumors are presented in support of a modified scheme of hematopoietic cell development. PMID- 3014003 TI - The murine gamma-chain of the T cell receptor is closely linked to a spermatocyte specific histone gene and the beige coat color locus on chromosome 13. AB - With the use of standard genetic techniques, we have mapped the T cell gamma chain gene locus (Tcrg) to the proximal end of mouse chromosome 13. Close genetic linkage was demonstrated between a restriction fragment length polymorphism in the Tcrg locus and a locus (HlS) encoding a spermatocyte-specific histone electrophoretic variant (HlS) by using recombinant inbred strains. Because HlS is closely linked to the dominant visible marker extra toes (Xt), Tcrg must be within 5 cM of the beige (bg) locus. Mice homozygous for the recessive mutation beige (bg/bg) show diminished pigmentation, enlarged lysosomes in granulocytes, and a selective deficiency of natural killer cell function. The bg mutation in mice is an animal model for humans with Chediak-Higashi syndrome; we speculate that the human gamma-chain may be closely linked to the human mutation and may be a useful diagnostic tool. Seven restriction enzymes identify five haplotypes of the highly polymorphic gamma-chain in mice. PMID- 3014002 TI - Anti-Ig induces release of inositol 1,4,5-trisphosphate, which mediates mobilization of intracellular Ca++ stores in B lymphocytes. AB - Evidence from a variety of laboratories indicates that crosslinking of B cell mIg induces a rapid increase in intracellular free calcium (Ca++i). This mobilized Ca++ appears to act in concert with diacylglycerol (DAG; also released upon mIg cross-linking) to optimally activate Ca++/phospholipid-dependent protein kinase C, which plays a pivotal role in B cell activation. Here we report analysis of the source of this mobilized calcium and the mechanism responsible for its release into the cytosol. We observed the cross-linking of mIg induces the release of inositol 1,4,5-trisphosphate (InsP3), presumably as a result of action of phospholipase C on plasma membrane phosphatidylinositol 4,5-bisphosphate (PtdInsP2). The release of InsP3 and the elevation of Ca++i are coincidental, suggesting that they may be causally related. Finally, we demonstrate that submicromolar doses of InsP3 induce release of Ca++ from permeabilized cells that had preaccumulated 45Ca++ in the endoplasmic reticulum. On the basis of these findings we suggest that mIg cross-linking leads to mobilization of Ca++, in part by causing hydrolysis of PtdInsP2, yielding InsP3, which in turn causes release of calcium from the endoplasmic reticulum. PMID- 3014004 TI - Identification and characterization of the phosphatidylinositol kinase in membranes of murine T lymphocytes. AB - Polyphosphoinositide metabolism appears to be a widely employed mechanism by which membrane receptors transduce the signal to activate cells. This pathway has been implicated in the activation of T lymphocytes. Unlike many receptor systems in which decreased levels of polyphosphoinositides are observed, T lymphocytes demonstrate increased concentrations of polyphosphoinositides as well as phosphoinositols (particularly IP3) on activation by mitogens. To understand the early biochemical events involved in T cell activation, we sought to identify and characterize the enzyme responsible for phosphorylating phosphatidylinositol (PI) to phosphatidylinositol 4-phosphate (PIP) in murine T lymphocytes. This kinase was found to be an integral membrane protein of T lymphocytes. It was found to be Mg2+ (apparent Km = 5 mM)- or Mn2+-dependent, whereas Ca2+ was noted to be a competitive inhibitor of Mg2+. ATP is the preferred substrate over GTP (apparent Km = 0.14 mM and 0.62 mM, respectively). The kinase is inhibited by both PIP and PI 4,5-bisphosphate (PIP2), but not by other phospholipids or angiotensin II (a substrate for tyrosine kinase). The enzyme activity has identical characteristics in membranes derived from a T cell hybridoma, thymocytes, and T cell clones. The enzyme migrated predominantly as a single peak with an apparent m.w. of approximately 60,000 on gel filtration chromatography. The relationship of this enzyme to viral oncogene products such as pp60v-src and p68v-ros is discussed. PMID- 3014005 TI - The detection of endothelial cell antigens in cutaneous tissue using methacarn and periodate lysine paraformaldehyde fixation. AB - The use of monoclonal antibodies with endothelial cell specificity has prompted a search for methods of fixation which combine the morphology of paraffin-embedded tissue, with preservation of labile membrane antigens. Immunohistochemical staining using a variety of endothelial cell markers was compared in tissue fixed in formalin, methacarn, periodate lysine paraformaldehyde (PLP) and in frozen tissue. Whilst lectin-binding with Ulex europaeus agglutinin I (UEA) and localisation of Factor VIII-related antigen (FVIII RA) and laminin was well visualised in methacarn-fixed and PLP-fixed tissue, fixation in PLP was necessary for the two monoclonal antibodies, PAL-E and EN4. PLP fixation has considerable potential for investigating the histogenesis of vascular tumours, particularly in Kaposi's sarcoma where frozen tissue represents a biological hazard. The normal staining pattern of human dermal vasculature is described in relation to the above endothelial cell antigens. PMID- 3014006 TI - Virus receptors and cell tropisms. PMID- 3014007 TI - Primary infection of Japanese infants with adult T-cell leukaemia-associated retrovirus (ATLV): evidence for viral transmission from mothers to children. AB - Primary infection with adult T-cell leukemia virus (ATLV) was investigated by follow-up studies on 16 ATLV-seropositive mothers and their breastfed infants in an ATLV-endemic area of Japan. Maternal antibody to ATLV decreased in all the infants, and was detectable in only three of 12 infants tested 6 months after birth. Reappearance of the antibody 9-18 months after birth was observed in only four of the 16 infants. The ATLV-bearing cells in peripheral blood were detected in all 16 mothers after delivery. None of the 16 infants showed ATLV-bearing cells in peripheral or cord blood sampled at birth, or 1, 3 or 6 months after birth. However, virus-bearing cells in the blood became detectable 9-18 months after birth in 13 of the 16 infants. Maternal antibody and virus-bearing cells were never detected in a control group of seven infants of ATLV-seronegative mothers. These findings provide evidence for the high incidence of primary ATLV infection during early infancy among infants born to ATLV-seropositive mothers and suggest maternal viral transmission. Furthermore, samples of breast milk from all 12 seropositive mothers examined contained cell-associated ATLV capable of being transmitted to peripheral leucocytes of neonates. This finding suggests that one of the possible maternal transmission routes of ATLV is via breast milk. PMID- 3014008 TI - Identification of C5ades arg and an anionic neutrophil-activating peptide (ANAP) in psoriatic scales. AB - Scales from patients with nonpustular psoriasis were investigated for the presence of peptides capable of activating functional activities in human polymorphonuclear leukocytes (PMNL). Two compounds with similar molecular weight (12,500 and 15,000) were isolated which markedly stimulated PMNL functional activities including chemotaxis, generation of superoxide radical anion (O-2), and liberation of beta-glucuronidase as a marker enzyme. As revealed by ion exchange and subsequent radioimmunoassay followed by chromatofocusing, one peptide proved to be the desarginated form of the complement split product C5ades arg. No C5a was detectable. As a second psoriatic scale chemotaxin we isolated an anionic neutrophil-activating peptide (ANAP) which shows a single isoelectric point at pH 6.8. This peptide shares some of the characteristics of epidermal cell-derived thymocyte-activating factor and interleukin 1 and, as shown by deactivation experiments, it cross-reacts with a monocyte-derived cytokine. The 2 newly described neutrophil-activating peptides (C5ades arg and ANAP) may play an important role in the psoriatic tissue reaction. PMID- 3014009 TI - Fecal excretion of Greek strains of hepatitis A virus in patients with hepatitis A and in experimentally infected chimpanzees. AB - The presence of hepatitis A virus (HAV) in stool samples was determined in 36 children (mean age, 8.9 years) and 38 adults (mean age, 19.9 years) with acute type A hepatitis. Three stool samples, taken on admission and thereafter at three to-five-day intervals, were collected from each patient. The first day of dark urine was considered to be the onset of illness. Molecular hybridization of cloned HAV cDNA to fecal extracts was used to detect HAV RNA; radioimmunoassay was used to detect HAV antigen. In all of the samples tested, HAV RNA was detected significantly more frequently than HAV antigen (28.4% vs. 8.1%, P less than .001). HAV RNA was detected with equal frequency in both children and adults during the first week of illness. However, HAV RNA was detected more frequently in children than in adults during the second week of illness (45.7% vs. 18.9%, P less than .05). Among patients with HAV RNA, detection in multiple samples was more frequent in children than in adults (38.9% vs. 7.9%, P less than .01), especially among males. PMID- 3014010 TI - Centrifugation-augmented solid-phase immunoassay (CASPIA) for the rapid diagnosis of infectious diseases. AB - We have devised centrifugation-augmented solid-phase immunoassays (CASPIAs) for the detection of microbial pathogens in human body fluids. The CASPIA format combines several features of latex agglutination and solid-phase immunoassay systems to produce an assay system that is inexpensive and simple to perform. We found that CASPIA systems for the detection of rotaviral, group A streptococcal, and Haemophilus influenzae type b antigens were significantly more sensitive than latex agglutination assays and at least as sensitive as enzyme immunoassay systems using analogous reagents. Because CASPIA reactions could either be read macroscopically, recorded with a standard office photocopying device, or quantitated by means of a microplate colorimeter, the assays were applicable under a wide range of clinical and laboratory conditions. PMID- 3014011 TI - Detection of human cytomegalovirus in urine by DNA-DNA and RNA-DNA hybridization. AB - A diagnostic hybridization assay for detecting human cytomegalovirus (HCMV) DNA in urine specimens was developed by using cloned viral DNA and in vitro synthesized RNA probes. Both probes detected 3 pg of homologous DNA and hybridized with DNA of HCMV but not with other viral or human cellular DNA tested. In 95 urine specimens simultaneously tested by cell culture, the sensitivity of hybridization was at least 83%, and the specificity was at least 92%. This assay will be useful for rapid viral diagnosis with wide clinical applications such as screening of immunocompromised patients and quantitation of viral shedding in patients with primary or reactivated HCMV infection who may be receiving antiviral therapy. PMID- 3014012 TI - Growth of Leishmania donovani amastigotes in the continuous human macrophage cell line U937: studies of drug efficacy and metabolism. AB - We have developed a simple and reproducible system for infecting a human macrophage cell line (U937) with stationary-phase Leishmania donovani promastigotes. Four days after infection, greater than 90% of the promastigotes had transformed to amastigotes. The antileishmanial agents allopurinol riboside, formycin B, 9-deazainosine, and sodium stibogluconate effectively inhibited the growth of L. donovani amastigotes in this cell line. To study the capability of amastigotes in the U937 cell line to carry out biochemical reactions that could be monitored experimentally, we incubated the cells with radiolabeled 9 deazainosine. This purine analogue underwent metabolism in the amastigote phase similar to that occurring in the promastigote phase. This cell line should be useful for studies of parasite maturation and differentiation, parasite-human interactions, and antiparasitic drugs. PMID- 3014013 TI - The effect of asymptomatic infection with HTLV-III on the response of anogenital warts to intralesional treatment with recombinant alpha 2 interferon. PMID- 3014014 TI - A longitudinal study of varicella immunity in pediatric renal transplant recipients. PMID- 3014015 TI - Kinetics of the antibody response to BamHI-K nuclear antigen in uncomplicated infectious mononucleosis. PMID- 3014016 TI - [Radionuclide ventriculographic assessment of cardiac function based on the analysis of cardiac reserve during dynamic exercise]. AB - To evaluate the contribution of myocardial contractility and preload to increase cardiac output during supine bicycle exercise, quantitative radionuclide ventriculography was performed at rest (R) and during peak exercise (Ex) in 43 patients with coronary artery disease (CAD) and 13 normal subjects. Myocardial contractility was estimated from the ratio of peak systolic pressure to end systolic volume index (P/V index). During Ex in normal subjects, P/V index invariably increased and its percent change from R to Ex averaged 98 +/- 46 percent. Stroke index (SI) in normal subjects increased from 48 +/- 9 to 57 +/- 7 ml/m2 during Ex (p less than 0.001) without an increase in end-diastolic volume index (EDVI) (76 +/- 11 vs 78 +/- 11 ml/m2, NS). Ten of 43 patients with CAD, whose percent increase in P/V index was more than 40 percent, showed a significant increase in SI during Ex (44 +/- 5 vs 51 +/- 12 ml/m2, p +/- 0.05) without an increase in EDVI (86 +/- 14 vs 87 +/- 15 ml/m2, NS). In 16 of 43 patients with CAD whose percent increase in P/V index was less than 40 percent, SI increased from 44 +/- 10 to 51 +/- 15 ml/m2 (p less than 0.01) during Ex with an increase in EDVI (102 +/- 24 vs 117 +/- 29 ml/m2, p less than 0.001). In the remaining 17 patients with CAD whose P/V index decreased during Ex, SI did not increase during Ex (48 +/- 14 vs 44 +/- 12 ml/m2, NS) despite an increase in EDVI (80 +/- 19 vs 94 +/- 18 ml/m2, p less than 0.01). These results indicate that the Frank-Starling mechanism operates under limited augmentation in myocardial contractility, and that its compensatory function may have limitations under the severely depressed reserve of myocardial contractility. PMID- 3014017 TI - Alteration of some leukocyte functions following in vivo and in vitro exposure to recombinant bovine alpha- and gamma-interferon. AB - Bovine interferons (BoIFNs) produced by recombinant DNA technology are currently being evaluated for their prophylactic effect against virus-induced respiratory disease in cattle. In this context experiments were conducted to compare blood levels of recombinant bovine interferon-alpha 1 (rBoIFN-alpha 1) and -gamma (rBoIFN-gamma) following intravenous and intramuscular injection to healthy calves, and to assess the effect on the immune response. Maximum serum level of IFN obtained with rBoIFN-gamma was less than 20% of that seen with rBoIFN-alpha 1 regardless of whether it was administered intravenously or intramuscularly. Nevertheless, administration of rBoIFN-gamma had a greater effect on leukocyte functions than rBoIFN-alpha 1, both with respect to level and duration of changes. Migration by polymorphonuclear neutrophils (PMN) became suppressed and their generation of O2- was enhanced following rBoIFN treatment. In vitro both rBoIFNs also suppressed migration, whereas the effect on O2- generation was minimal with suppression seen only at very high doses. Lymphocyte proliferation was also suppressed 24 h after IFN injection, and this effect could be reversed by exogenous interleukin-2 (IL-2) added to the cultures. In experiments designed to elucidate the mechanism of lymphocyte-suppression, it was found that in vitro treatment with rBoIFNs can induce suppressor cells, which may act by competing for IL-2. The combination of in vivo and in vitro experimental models used here could prove useful in additional studies to further delineate the mechanisms involved in the immunomodulatory effects of IFNs. PMID- 3014018 TI - Interferon-beta ser as prophylaxis against experimental rhinovirus infection in volunteers. AB - The first test of intranasal recombinant human interferon-beta ser (IFN-beta ser) as prophylaxis against common colds is reported. IFN-beta ser was cleared from the nose like IFN-alpha. A total of 10 volunteers were each given a total of 2.6 X 10(7) units of IFN-beta ser as 13 doses administered three times daily over 4 days and there were negligible symptoms that were not significantly different from those in 10 given placebo. Twenty-seven volunteers were then given the same regime and challenged after the fourth dose with rhinovirus types 9 and 14. Compared with 27 volunteers given placebo and virus, there were significant reductions in the mean total clinical scores, the amount of nasal secretion, and the frequency of virus excretion. It is concluded that IFN-beta shows antiviral activity in the human respiratory tract and should be tested to determine whether it is tolerated on continued administration. PMID- 3014019 TI - Heterotransplantation of human pleomorphic adenomas to nude mice. AB - The purpose of the present study was to establish a model for in vivo studies of human pleomorphic adenomas by heterotransplantation of tumour tissue to nude mice. Tissue from 7 tumours was transplanted to a total of 34 mice. Take with obvious growth occurred in 12 mice, and survival of the tissue was seen in an additional 8 mice. The overall histological picture was unchanged from the donor tumours to the transplanted tissue. The possibilities of the model are discussed. PMID- 3014020 TI - [Studies on the chemiluminescence of peripheral phagocytic cells in the patients with liver cirrhosis]. PMID- 3014022 TI - Intracranial interstitial radiation. AB - Primary malignant brain tumors are fatal, with 90% of patients having these tumors dying within two years following diagnosis. Cranial interstitial radiation therapy, a technique under investigation to control these tumors, involves implantation of radioactive iodine 125 seeds into the tumor bed by stereotaxic technique. The interstitial radiation technique, monitoring of radiation, and nursing care of patients are discussed. Case histories are presented, along with discussion of results attained using this therapy, and its future. PMID- 3014023 TI - [Results of bronchoplastic surgery in patients with bronchogenic carcinomas]. PMID- 3014021 TI - Na+/K+ ATPase activity in mouse lung fibroblasts and HeLa S3 cells during and after hyperthermia. AB - The ouabain-sensitive ATP-hydrolysing activity, representing the Na+/K+ ATPase capacity, of isolated membranes and whole cells during and after hyperthermia treatments was investigated. In isolated membranes no heat damage after treatments up to 46 degrees C during 45 min or up to 6 h at 44 degrees C could be detected. The ATP hydrolysing activity of Na+/K+ ATPase seems not to be impaired by direct heat attack in the range of commonly used hyperthermic temperatures (39 46 degrees C). Heat effects on the ATP hydrolysing activity of Na+/K+ ATPase of whole mouse fibroblasts could only be detected after heat doses (greater than 40 min at 44 degrees C) necessary to yield over 99 per cent dead cells. Potassium influx, measured with 86RB+ as the K+ tracer, was initially enhanced during incubation at 44 degrees C proportionally with the enhancement of the ATP hydrolysing activity after raising the temperature. Replacement of non-lethally (10 min at 44 degrees C) and lethally (40 min at 44 degrees C) treated mouse fibroblasts to 37 degrees C showed complete reversibility of the enhanced activity at 44 degrees C to the control level at 37 degrees C. For comparison, the ATP-hydrolysing activity of Na+/K+ ATPase of HeLa S3 cells growing as monolayer was also tested. The activity after heat treatments up to 60 min at 44 degrees C was also found to be unchanged in these experiments. No indication of irreversible damage to the ATP-hydrolysing capacity of mouse fibroblasts and HeLa S3 cells, or K+ pumping activity of mouse fibroblasts by heat treatments up to 40 min at 44 degrees C was found. PMID- 3014025 TI - [A case of malignant fibrous histiocytoma of the epicardium]. PMID- 3014024 TI - [Malignant fibrous histiocytoma of the left atrium: a case report and a review of the literature]. PMID- 3014026 TI - [Clinical studies on photoradiation therapy with hematoporphyrin derivatives (HpD) and argon dye laser]. AB - Photoradiation therapy (PRT), in which hematoporphyrin derivatives (HpD) are activated by an argon-dye laser, was performed on superficial or subcutaneous tumors in 4 vulvar carcinoma, 1 vaginal carcinoma, 1 invasive cervical carcinoma and 8 carcinoma in situ (CIS) of the uterine cervix. In PRT with intravenous administration of HpD, PRT obtained complete response (CR) in one vaginal carcinoma with follow up to 6 months. Histological examination, 7 days after PRT, revealed swelling and edema of the tumor, showing degenerated cells with pyknotic nuclei or weakly stained, swollen nuclei. In PRT with local injection or local attachment of HpD, performed on 9 cervical carcinoma, PRT obtained CR in 2 CIS and PR in 7 carcinoma. A side effect with PRT was found in a patient who received intravenous administration of HpD. All patients who received PRT with local administration of HpD were free of side effects. PRT could be used as a primary treatment or upon tumor recurrence following conventional modalities such as surgery, chemotherapy and radiation therapy. In carrying out PRT, selection of patients and improvement of photosensitizing agents and laser equipment were most important. PMID- 3014028 TI - [Effect of porous hydroxylapatite granules on the healing of periodontal bone defects]. PMID- 3014027 TI - Mechanism of phagocytosis of mycobacteria by Schwann cells and their comparison with macrophages. AB - Factors influencing the phagocytosis of mycobacteria by 33B rat Schwannoma cells and rat peritoneal macrophages were studied. Uptake of 14C-acetate-labeled Mycobacterium w by these cells was used to set up a radiometric phagocytosis assay. Incubation at 4 degrees C and treatment with sodium azide (0.2% to 1%), colchicine (10(-7) to 10(-3) M), cytochalasin B (0.2 micrograms/ml to 25 micrograms/ml), and dibutyryl cyclic AMP (10(-7) to 10(-3) M) inhibited the phagocytosis by both cell types in a similar manner. These experiments demonstrate similarities in the mechanism of phagocytosis of mycobacteria by Schwann cells and macrophages. PMID- 3014029 TI - S-100 protein and myoepithelial neoplasms. AB - Besides advancing our knowledge of histogenesis, immuno-cytochemical studies of salivary gland tumors have added an important objective component to the diagnosis and classification of the tumors. Cellular localization of S-100 protein now allows identification of myoepithelial cells and tumors composed of these cells. The four tumors presented in this report illustrate the diagnostic advantages afforded by S-100 protein immunocytochemistry. PMID- 3014031 TI - Fine structure of crystalloid inclusions in the nucleus of thyroid medullary carcinoma cells. PMID- 3014030 TI - Transfacial access to the retromaxillary area. AB - A case of angiofibroma of large proportions is presented which was manifest in various regions difficult of access. Using conventional techniques marked mutilation would have been expected. Consequently the technique of temporary disarticulation of the maxilla attached to the cheek with a transfacial access to the retromaxillary area was applied and will be described. PMID- 3014032 TI - Control of rectal gland secretion in the dogfish (Squalus acanthias): steps in the sequence of activation. AB - We measured the venous and arterial pressure, as well as the rate of secretion and content of cyclic AMP and high energy phosphate compounds, of the rectal gland of the anaesthetized dogfish, Squalus acanthias (L.). Intravenous infusion of isotonic solutions produced a very large increase in the rate of secretion by the rectal gland. The increase in secretion was preceded by an increase in venous blood pressure, but arterial blood pressure was not modified. Injections of small doses of veratridine stimulated gland secretion when given in the vicinity of the heart but not when given in the dorsal aorta. During volume expansion the creatine phosphate and ATP content of the gland were markedly reduced, while ADP and AMP as well as cyclic AMP content were increased. We conclude that: volume expansion leads to the release of a message that activates adenyl cyclase in the gland; the increased venous pressure may be the initial signal in the sequence that leads to the release of the activating messenger; there is a receptor mechanism in the atrial and cardiac region that triggers the sequence that activates glandular secretion; the reduction in the content of high energy phosphate compounds during volume expansion is caused by an increase in energy expenditure, probably due to gland secretion. PMID- 3014033 TI - Requirement for HLA-DR+ accessory cells in natural killing of cytomegalovirus infected fibroblasts. AB - The role of HLA-DR+ cells in NK activity against CMV-infected FS4 foreskin fibroblasts and K562 erythroleukemia cells was examined. When nonadherent PBMC were depleted of either HLA-DR+ or Leu-11b+ cells by treatment with mAbs plus C, NK activity against CMV-FS4 target cells was markedly reduced. In contrast, depletion of HLA-DR+ cells had no effect on NK activity against K562 target cells. When HLA-DR-depleted cells were added to Leu-11b-depleted cells, NK activity against CMV-FS4 was restored. Negative selection experiments indicated that the HLA-DR+ cells contributing to NK activity against CMV-FS4 are not B or T cells, while negative and positive selection experiments excluded a role for monocytes. Experiments in which HLA-DR- and Leu-11b- cells were mixed in varying proportions indicated that NK(CMV-FS4) is mediated by Leu-11b+ cells, while HLA DR+ cells provide an accessory function. Irradiation (50 GY) abolished the NK effector function of Leu-11b+ cells, but not the accessory function of HLA-DR+ cells. The NK activity against CMV-FS4 of HLA-DR- cells was restored by the addition of rIFN-alpha or of cell-free supernatants generated by coculturing PBMC or Leu-11b- cells with CMV-FS4. The ability of these supernatants to restore NK activity of HLA-DR- cells was completely abrogated by the addition of neutralizing amounts of antibody to IFN-alpha. In related experiments, neutralization of IFN-alpha in NK assays had little or no effect on NK activity against CMV-FS4, suggesting that the accessory function of HLA-DR+ cells might be mediated by alternative mechanisms in addition to the secretion of extracellular IFN-alpha. PMID- 3014034 TI - Immunization with SV40-transformed cells yields mainly MHC-restricted monoclonal antibodies. AB - Recognition of antigens on cell surfaces only in the context of the MHC-encoded alloantigens of the presenting cell (self + X) has classically been considered the province of T cells. However, evidence from several sources has indicated that B cells and antibodies can exhibit self + X-restricted recognition as well. This report concerns the mAb response to SV40-transformed H-2b fibroblast cell lines. The specificities of the antibodies obtained have been analyzed for binding to a panel of SV40-transformed H-2-syngeneic, H-2-allogeneic, and H-2b mutant fibroblast cell lines, as well as cell lines not bearing cell surface SV40 transformation-associated antigens. A large proportion of primary C57BL/6 (71%) and BALB/c (68%) splenic B cells responding to in vitro stimulation with SV40 transformed H-2b cells recognize cell surface antigens associated with SV40 transformation only when coexpressed with MHC antigens of the immunizing cell, particularly the Kb molecule, on transformed cells. To extensively define the nature of antigen recognition by these antibodies, we have generated and characterized nine hybridoma antibodies specific for SV40-transformed H-2 syngeneic cell lines. Seven of these hybridoma antibodies recognize SV40 associated transformation antigens in the context of H-2b molecules. Six of these are restricted by the Kb molecule and discriminate among a panel of SV40 transformed Kb mutant cell lines, thus confirming the participation of class I MHC-encoded molecules in the recognition by B cells of cell surface antigens. PMID- 3014035 TI - Characterization of the cell surface receptor for human granulocyte/macrophage colony-stimulating factor. AB - 125I-labeled recombinant human GM-CSF was used to identify and characterize receptors specific for this lymphokine on both a mature primary cell, human neutrophils, and on the undifferentiated promyelomonocytic leukemia cell line, HL 60. Human GM-CSF also bound to primary human monocytes and to the myelogenous leukemia cell line, KG-1, but not to any of the murine cells known to express the murine GM-CSF receptor. In addition, although some murine T lymphomas can express the GM-CSF receptor, none of the human cell lines of T cell lineage that we examined bound iodinated human GM-CSF. Binding to all cell types was specific and saturable. Equilibrium binding studies revealed that on all cell types examined, GM-CSF bound to a single class of high affinity receptor (100-500 receptors per cell) with a Ka of 10(9)-10(10)/M. More extensive characterization with neutrophils and HL-60 cells showed that in both cases, binding of GM-CSF was rapid at 37 degrees C with a slow subsequent dissociation rate that exhibited marked biphasic kinetics. Among a panel of lymphokines and growth hormones, only human GM-CSF could compete for binding of human 125I-GM-CSF to these cells. GM CSF can not only stimulate the proliferation and differentiation of granulocyte/macrophage precursor cells, but can modulate the functional activity of mature granulocytes and macrophages as well. No significant differences in the kinetic parameters of receptor binding were seen between mature neutrophils and the undifferentiated promyelocytic leukemia cell line HL-60, indicating that maturation-specific responses to GM-CSF are not mediated by overt changes in the binding characteristics of the hormone for its receptor. PMID- 3014036 TI - Biological and biochemical characterization of a cloned Leu-3- cell surviving infection with the acquired immune deficiency syndrome retrovirus. AB - Leu-3- cells that survive infection with the acquired immune deficiency syndrome (AIDS) retrovirus can be induced with IUdR to express infectious virus. A cellular clone (8E5), isolated by limiting dilution of a mass culture of survivor cells, was found to contain a single, integrated provirus that was constitutively expressed. Although IUdR treatment of 8E5 cells failed to induce infectious virus, cocultivation with Leu-3+ cells generated the characteristic syncytia associated with acute AIDS retrovirus infection. The single integrated copy of proviral DNA directs the synthesis of all major viral structural proteins except p64, as monitored by immunoblotting. The relationship of the 8E5 clone to viral latency and persistence is discussed. PMID- 3014037 TI - Detection of a second t(14;18) breakpoint cluster region in human follicular lymphomas. AB - Our results indicate that there are two major breakpoint cluster regions in chromosome 18 DNA for t(14;18) translocations in follicular lymphomas. The absence of a pFL-1 homologous transcript in a cell line containing a pFL-2 detectable translocation suggests that there may be two different pathogenetic consequences of t(14;18) translocations. One possibility is that, despite the distances between them (greater than 20 kb), breakpoints in the two cluster regions in some way affect transcription of the same gene product, which has not yet been identified. Alternatively, two separate transcriptional units may be involved. The availability of DNA probes for each of the two t(14;18) breakpoint cluster regions will allow further studies regarding the biologic significance of these two genetically distinct classes of t(14;18) translocations. PMID- 3014038 TI - An HLA-D region restriction fragment length polymorphism associated with celiac disease. AB - This study is the first to describe a molecular marker that distinguishes the celiac disease HLA-D region haplotype from a serologically identical haplotype in unaffected controls. Using a DQ beta chain cDNA probe and the restriction endonuclease Rsa I, we have detected a polymorphic 4.0 kb fragment which, in DQw2 individuals, is associated with a 40-fold increased relative risk of developing celiac disease. This finding should permit the identification of the celiac disease susceptibility gene(s) in the HLA-D region and facilitate a more precise dissection of the molecular and immunogenetic mechanisms involved in the pathogenesis of that disease. PMID- 3014039 TI - Specific genomic markers for the HLA-DQ subregion discriminate between DR4+ insulin-dependent diabetes mellitus and DR4+ seropositive juvenile rheumatoid arthritis. AB - HLA-DR4, Dw4-associated haplotypes associated with IDDM and JRA were compared using genomic DNA restriction fragment analysis to distinguish among DQ beta and alpha alleles linked to DR4. DQ beta polymorphisms that subdivide the HLA-DQw3 specificity into DQ3.1 and 3.2 alleles were identified. More than 90% of DR4+ IDDM patients express one of these alleles, DQ3.2; restriction enzyme mapping indicates that the presence of this allele also accounts for the genomic fragment patterns previously reported in IDDM. Furthermore, haplo-identical siblings of DQ3.2 IDDM patients also carry the DQ3.2 allele, regardless of clinical presentation. In contrast, DR4+ JRA patients show no allelic preference at DQ beta, implicating different HLA genetic contributions in these two DR4-associated diseases. PMID- 3014040 TI - Identification of a restriction fragment length polymorphism by a CR1 cDNA that correlates with the number of CR1 on erythrocytes. AB - A genetic basis for the regulation of the number of CR1 on E of different normal individuals was investigated by probing Southern blots of their genomic DNA with a 0.75-kb fragment of CR1 cDNA. Using Hind III, we observed a RFLP involving fragments of 7.4 kb and 6.9 kb that correlated with the number of CR1 on E. 32 individuals having only the 7.4-kb restriction fragment had a mean of 661 +/- 33 (SEM) CR1/E, 11 donors having both restriction fragments had a mean of 455 +/- 52 CR1/E, and 7 individuals having only the 6.9-kb fragment had a mean of 156 +/- 13 CR1/E, all means being significantly different (p less than 0.005). Cosegregation in a normal family of the Hind III restriction fragments with the S, F, and F' structural allotypes of CR1 confirmed that the regulatory element identified by these fragments is linked to the CR1 gene. Moreover, an analysis of the relative expression on E of these structural allotypes in association with either the 7.4 kb Hind III fragment or the 6.9-kb fragment showed that this regulatory element is cis-acting. In contrast, quantitation of CR1 of B lymphocytes and neutrophils revealed no differences in total CR1 expression between individuals homozygous for the 7.4-kb and 6.9-kb Hind III fragments. Thus, we have identified a genomic polymorphism that is linked to the CR1 gene and is associated with a cis-acting regulatory element for the expression of CR1 on E. PMID- 3014041 TI - A single germline VH gene segment of normal A/J mice encodes autoantibodies characteristic of systemic lupus erythematosus. AB - These experiments tested the hypothesis that unmutated germline genes from normal mice can encode autoantibodies. We found that the unmutated VHIdCR gene segment, which encodes a large proportion of antiarsonate antibodies in A/J mice, also encodes antibodies with the ability to bind to DNA and cytoskeletal proteins. After Ars immunization, at a time when the VHIdCR gene segment mutates and antibody affinity for the hapten increases, reactivity with the autoantigens was lost. Six antibodies obtained after immunization with Ars bound both the Ars and DNA. Results of competitive inhibition assays suggested that the same variable region site in the antibodies bound to both Ars and DNA. The properties of the individual germline-encoded antibodies, which include reactivity to both DNA and cytoskeletal proteins, suggest that autoantibodies characteristic of SLE might be a subset of antibodies encoded by unmutated germline V genes. PMID- 3014042 TI - A potassium-proton symport in Neurospora crassa. AB - Combined ion flux and electrophysiological measurements have been used to characterized active transport of potassium by cells of Neurospora crassa that have been moderately starved of K+ and then maintained in the presence of millimolar free calcium ions. These conditions elicit a high-affinity (K1/2 = 1 10 microM) potassium uptake system that is strongly depolarizing. Current-voltage measurements have demonstrated a K+-associated inward current exceeding (at saturation) half the total current normally driven outward through the plasma membrane proton pump. Potassium activity ratios and fluxes have been compared quantitatively with electrophysiological parameters, by using small (approximately 15 micron diam) spherical cells of Neurospora grown in ethylene glycol. All data are consistent with a transport mechanism that carries K ions inward by cotransport with H ions, which move down the electrochemical gradient created by the primary proton pump. The stoichiometry of entry is 1 K ion with 1 H ion; overall charge balance is maintained by pumped extrusion of two protons, to yield a net flux stoichiometry of 1 K+ exchanging for 1 H+. The mechanism is competent to sustain the largest stable K+ gradients that have been measured in Neurospora, with no direct contribution from phosphate hydrolysis or redox processes. Such a potassium-proton symport mechanism could account for many observations reported on K+ movement in other fungi, in algae, and in higher plants. PMID- 3014043 TI - Extracellular calcium transients and action potential configuration changes related to post-stimulatory potentiation in rabbit atrium. AB - Extracellular calcium transients were monitored with 2 mM tetramethylmurexide at low calcium (250 microM total, 130 microM free), and action potentials were monitored together with developed tension at normal calcium (1.3 mM) during the production and decay of post-stimulatory potentiation in rabbit left atrial strips. At normal calcium, the contractile potentiation produced by a brief burst of 4 Hz stimulation is lost in three to five post-stimulatory excitations, which correlate with a negative staircase of the late action potential. At low calcium, stimulation at 4 Hz for 3-8 s results in a net extracellular calcium depletion of 5-15 microM. At the subsequent potentiated contraction (1-45 s rest), total extracellular calcium increases by 4-8 microM. The contractile response at a second excitation is greatly suppressed and results in little or no further calcium shift; the sequence can be repeated immediately thereafter. Reducing external sodium to 60 mM (sucrose replacement) enhances post-rest contractions, suppresses the late action potential, nearly eliminates loss of contractility and net calcium efflux at post-rest excitations, and markedly reduces extracellular calcium depletion during rapid stimulation. 4-Aminopyridine (1 mM) markedly suppresses the rapid early repolarization of this preparation at post-rest excitations and the loss of contractility at post-rest stimulation from the rested state; during a post-stimulatory potentiation sequence at low calcium, replenishment of extracellular calcium takes several post-stimulatory excitations. Ryanodine (10 nM to 5 microM) abolishes the post-stimulatory contraction at rest periods of greater than 5 s. If the initial repolarization is rapid, ryanodine suppresses the late action potential, calcium efflux during quiescence is greatly accelerated, and subsequent excitations do not result in an accumulation of extracellular calcium. A positive staircase of the early action potential correlates with the magnitude of net extracellular calcium depletion. These findings demonstrate that negative contractile staircases at post-rest stimulation correspond closely to an accumulation of extracellular calcium at activation and a negative staircase of the late action potential; the correlation of these three events suggests that electrogenic sodium-calcium exchange is the common underlying mechanism. PMID- 3014045 TI - The biology of hepadnaviruses. PMID- 3014044 TI - In situ cGMP phosphodiesterase and photoreceptor potential in gecko retina. AB - The possible involvement of phosphodiesterase (PDE) activation in phototransduction was investigated in gecko photoreceptors by comparing the in situ PDE activity with the photoreceptor potential. In the dark, intracellular injection of cGMP into a gecko photoreceptor caused a long-lasting depolarization. An intense light flash given during the depolarization phase repolarized the cell with a short latency comparable to that of the light-evoked hyperpolarizing response, which indicates that the activation of PDE in situ is rapid enough to generate the photoreceptor potential. PDE activity in situ was estimated quantitatively from the duration of the cGMP-induced depolarization, since it was expected that the higher the PDE activity, the shorter the duration. Under steady illumination, the enzyme exhibited a constant activity. On exposure to a light flash, PDE became activated, but recovered in the dark with a time course that was dependent on the intensity of the preceding stimulus. When PDE activity and photoreceptor sensitivity to light were measured in the same cell after a light flash, both recovery processes showed similar kinetics. Theoretical analysis showed that the parallelism in the recovery time courses could be explained if cGMP is the transduction messenger. These results suggest that PDE activation is involved not only in the generation but also in the adaptation mechanisms of the photoreceptor potential. PMID- 3014046 TI - A filamentous distribution for the herpes simplex virus type 2-encoded major DNA binding protein. AB - Monoclonal antibodies reacting with the herpes simplex virus (HSV)-encoded major DNA-binding protein defined an intracellular filamentous network. This network was associated predominantly with the infected cell nucleus and occurred in cells infected with HSV type 2. It did not co-distribute with microfilaments, microtubules or intermediate filaments, and DNA synthesis was required for its formation. We suggest explanations for the occurrence and function of this novel filamentous network structure. PMID- 3014047 TI - Molecular cloning and restriction endonuclease mapping of the rat cytomegalovirus genome. AB - Rat cytomegalovirus (RCMV) DNA was cleaved by restriction endonuclease EcoRI into 24 fragments ranging in mol. wt. from 34 X 10(6) to 0.20 X 10(6), of which 18 fragments could be cloned in plasmid pACYC 184. Restriction endonuclease XbaI cleaved the RCMV genome into 28 fragments, ranging in size from 44 X 10(6) to 0.81 X 10(6), of which 24 fragments were cloned in plasmid pSP62-PL. Among the restriction fragments that could not be cloned were two major terminal colinear fragments, EcoRI-A (34 X 10(6)) and XbaI-A (44 X 10(6)). Thus, the complete sets of recombinant plasmids spanned about 70% of the RCMV genome. Our mapping results including determination of the termini of the genome, characterization of double digestion products of restriction fragments and cross-hybridization of 35S labelled (cloned) EcoRI and XbaI fragments to Southern blots of EcoRI-, XbaI- or BglII-cleaved RCMV DNA, allowed us to construct the EcoRI and XbaI restriction maps of RCMV DNA. Since no cross-hybridization between internal fragments was seen, it is concluded that the RCMV genome consists of a long unique sequence of 224 kilobases without internal inverted repeat sequences, which is similar to the structures of murine and guinea-pig CMV DNA but unlike that of human CMV DNA. In a minor population (approx. 20%) of the RCMV DNA, one terminus was found to be larger by 0.35 X 10(6) mol. wt. The nature of this fragment is unclear at the moment. PMID- 3014049 TI - The myeloproliferative sarcoma virus retains transforming functions after introduction of a dominant selectable marker gene. AB - The dominant neomycin resistance gene (neoR) was introduced into the genome of the myeloproliferative sarcoma virus (MPSV), a replication-defective retrovirus carrying the mos oncogene. The resulting selectable neoR-MPSV virus did not lose its acute transforming property, unlike the results of attempts by other groups to insert marker genes into oncogenic viruses. NeoR-MPSV DNA was used to generate infectious virus by transfection followed by rescue with Friend or Moloney murine leukaemia virus. Infection of fibroblasts with this virus resulted in morphologically transformed cells which were resistant to the neomycin analogue G418. Segregation of the two functions (transformation and G418 resistance) was not observed in more than 500 independent viral transfers to fibroblasts. Furthermore, neoR-MPSV retained the leukaemogenesis-inducing properties of the wild-type virus. Myeloproliferation and G418-resistance transfer did not segregate after passage in mice. PMID- 3014048 TI - Conservation of potential phosphorylation sites in the NS proteins of the New Jersey and Indiana serotypes of vesicular stomatitis virus. AB - A full length cDNA copy of the NS mRNA of the Missouri strain (Hazelhurst subtype, New Jersey serotype) of vesicular stomatitis virus (VSV) has been cloned and sequenced. The mRNA is 856 nucleotides long (excluding polyadenylic acid) and encodes a protein of 274 amino acids (mol. wt. 31 000). Comparison with the NS gene of the Ogden strain (Concan subtype, New Jersey serotype) showed 15% difference at the nucleotide level and 10% difference at the amino acid level; the majority of the changes were located in the 3' half of the mRNA. Comparison with the NS genes of two strains representing the Indiana serotype showed about 50% nucleotide and 33% amino acid sequence homology between the serotypes. In a four-way comparison of the proteins, two regions of higher homology were noted which may be of functional importance. Eighteen potential phosphorylation sites (Ser or Thr) were conserved between the four proteins; five of these sites correspond to the residues which have been suggested to be constitutively phosphorylated and may be essential for NS activity. PMID- 3014050 TI - Structural analysis of p28 adult T-cell leukaemia-associated antigen. AB - The 28,000 mol. wt. polypeptide (p28) of adult T-cell leukaemia-associated antigen encoded by the 24S defective human T-cell leukaemia virus (HTLV-I) is associated with protein kinase activity. We have determined the nucleotide sequence of this defective HTLV-I provirus and found that it contains a portion of the gag gene (p19 and part of p24), the pX region, and two long terminal repeats, one at each end. The predicted p28 gag-pX fused protein consists of 190 amino acids and its mol. wt. was calculated as 21,055. The results of peptide mapping analysis showing that p28 contains p19 supported the nucleotide sequence data. That p28 was encoded by this defective provirus was also demonstrated by transient expression of p28 polypeptide in COS 7 cells transfected with a recombinant plasmid containing a simian virus 40 early promoter and the p28 coding region of the 24S HTLV-I. PMID- 3014051 TI - Identification of a 17000 molecular weight antigenic polypeptide in transmissible gastroenteritis virus-infected cells. AB - Pulse labelling of cells with [35S]methionine at different times after infection followed by SDS-PAGE was used to resolve and to identify polypeptides designated as specific to transmissible gastroenteritis virus (TGEV)-infected swine testicular (ST) cell cultures. The major TGEV structural proteins, with apparent molecular weights of 200,000 (200K), 47K and 30K were detected in radiolabelled cell extracts by 6 h postinfection. Additionally, a 17K major polypeptide was present in infected cells but not in mock-infected control cultures. Labelling with [3H]glucosamine revealed only the 200K and 30K proteins to be glycosylated. TGEV-primed porcine lymphocytes, secondarily stimulated in vitro with sucrose gradient-purified virus, produced antibody only to the two glycoproteins (gp) indicating that the 17K polypeptide is not a surface feature of the virion. Two pigs were infected oronasally with the virulent Miller strain of TGEV and their sera were analysed by immunoprecipitation. At 25 days postinfection convalescent sera responded strongly to gp30 and gp200 and there was a weak initial response to the 17K polypeptide. Serum immunoglobulins at 60 days postinfection reacted strongly to the 17K protein while the antibody response to gp30 was significantly reduced and that to gp200 was slightly reduced. PMID- 3014052 TI - Infectious bronchitis immunity: its study in chickens experimentally infected with mixtures of infectious bronchitis virus and Escherichia coli. AB - The live infectious bronchitis (IB) vaccine, H120, protected chickens against intranasal challenge with a mixture of Escherichia coli strains (E. coli Pool) and IB virus (IBV) strains of the same (Massachusetts) serotype as H120; it usually also protected against challenge with the E. coli Pool and IBV strains of other serological types. When these challenge strains were themselves used as vaccines they usually protected against challenge with a mixture of the E. coli Pool and an IBV strain of the Massachusetts serotype (VF69-149) or an IBV strain not of the Massachusetts serotype (HVI-116). Poor protection, when observed, was most common in those experiments involving a minority of the IBV strains that had been incriminated in recent outbreaks of disease in vaccinated flocks of chickens. Much lower concentrations of IBV strain VF69-149 and E. coli O18 were found in the nose, trachea and spleen of H120-vaccinated chickens killed at different times after they were given a mixture of these organisms than were found in these sites in similarly challenged unvaccinated chickens. Some protection against challenge with IBV and the E. coli Pool was also observed in chickens vaccinated with an inactivated IBV strain; it was much less effective than that obtained following vaccination with the corresponding live IBV strain. PMID- 3014053 TI - Coronavirus IBV: virus retaining spike glycopolypeptide S2 but not S1 is unable to induce virus-neutralizing or haemagglutination-inhibiting antibody, or induce chicken tracheal protection. AB - Avian infectious bronchitis coronavirus (IBV) inactivated by beta-propiolactone induced partial protection of the trachea in up to 40% of chickens following one intramuscular inoculation 4 to 6 weeks prior to challenge. Retention of an intact tracheal ciliated epithelium 4 days after challenge was the criterion of protection. There was no correlation between protection and serum titres of virus neutralizing (VN) and haemagglutination-inhibiting (HI) antibody, which were maximal at about 4 weeks after inoculation. Virus from which the S1 but not the S2 (spike-anchoring) spike glycopolypeptide had been removed by urea did not induce protection or VN or HI antibody. Four intramuscular inoculations of monomeric S1 induced VN and HI antibody in two and four chickens respectively. These results indicate that VN and HI antibodies are induced primarily by S1, that intact spikes are a major requirement for the induction of protective immunity and that this property is probably associated with S1. PMID- 3014054 TI - Coronavirus IBV: removal of spike glycopolypeptide S1 by urea abolishes infectivity and haemagglutination but not attachment to cells. AB - Urea has been used to remove the S1 spike glycopolypeptide from avian infectious bronchitis virus (IBV) strains M41 and Beaudette, without removing the S2 spike anchoring glycopolypeptide. Reduction of the pH to 2.9 did not cause release of S1 although some S1 was released spontaneously from IBV Beaudette at pH 7.4. Virus that lacked S1 was no longer infectious or able to cause haemagglutination (HA). However, radiolabelled IBV that lacked S1 attached to erythrocytes and chick kidney cells to the same or similar extent as did intact virus. Treatment of IBV with a phospholipase C preparation, required to make IBV cause HA, did not increase binding of IBV to erythrocytes. The results indicate that while the attachment to cells of virus that lacks S1 is qualitatively different from that of intact virus, the decline in infectivity is the consequence of the loss of some other spike function. PMID- 3014055 TI - Gibbon ape leukaemia virus RNA in leukaemic T-lymphoid cell lines: expression of a novel RNA transcript. AB - Fibroblast cell lines infected in vitro with different strains of gibbon ape leukaemia virus or the related woolly monkey virus (SSAV) synthesized two RNA species of approximately 8.4 kb and 2.9 kb. The former, a complete RNA, represents the gag-pol mRNA, while the latter is a spliced transcript lacking gag and pol, and represents the env mRNA. In contrast, RNA from one T-lymphoid cell line derived from a gibbon ape T-lymphocytic leukaemia (UCD-144) expressed a viral mRNA in addition to gag-pol and env mRNA. This RNA is 6.4 kb and lacks at least 3.0 kb of sequences derived from the internal region of the viral genome, including most or all of the pol gene. These data, as well as data from Southern blots of UCD-144 DNA, suggest that the 6.4 kb mRNA could represent a transcript from a defective recombinant provirus and may contain cell-derived sequences. PMID- 3014056 TI - Characterization of glycoprotein complexes present in human cytomegalovirus envelopes. AB - Three disulphide cross-bridged glycoprotein complexes were immunoprecipitated from purified human cytomegalovirus envelopes using a monoclonal antibody with a specificity for a glycoprotein of mol. wt. 52 X 10(3). These complexes were isolated by electroelution after polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions. Compositional analysis of each complex by PAGE under reducing conditions showed that at least two distinct complexes, one containing glycoproteins with mol. wt. of 52 X 10(3) and 95 X 10(3) and the other with glycoproteins of 52 X 10(3) and 130 X 10(3), were present. The results obtained indicated that one of these complexes could also exist as a dimer. PMID- 3014057 TI - Fc receptor(s) induced by human cytomegalovirus bind differentially with human immunoglobulin G subclasses. AB - The IgG subclass specificity of Fc receptor(s) induced on cells by infection with human cytomegalovirus (HCMV) was studied in a binding assay by using infected cells and purified iodinated IgG of various subclasses from HCMV seronegative healthy adult donors. All four human IgG subclasses bound to HCMV-infected cells, with the following relative magnitudes: IgG1 greater than or equal to IgG4 greater than IgG2 greater than IgG3. The IgG subclass specificity of the Fc receptor was further analysed in an inhibition assay by using fragments prepared from purified human IgG by papain digestion, and using unlabelled subclass proteins. Fc but not Fab fragments inhibited the binding of 125I-labelled human IgG to HCMV-infected cells. The biological role of the Fc receptor in HCMV infection is discussed. PMID- 3014058 TI - Persistence of BK virus in human foetal pancreas cells. AB - High multiplicity BK virus (BKV) infection of primary cells derived from human foetal pancreas resulted in massive cytopathology and subsequent outgrowth of cells. Intranuclear BKV T-antigen was present in all cells and viral antigen was detected in 10 to 30% of these cells. The subcultured cells yielded BKV in the supernatant (approx. 10(5) TCID50/ml) and in the cells free viral DNA was present (approx. 10% of total cellular DNA content). Analysis of the viral DNA indicated the presence of deleted and rearranged BKV DNA molecules. Although all cells continuously expressed BKV T-antigen they did not exhibit the transformed phenotype. This persistent infection of human foetal pancreas cells represents a novel type of in vitro interaction between BKV and human cells which may correspond to the in vivo findings on BKV tropism for pancreatic cells. PMID- 3014060 TI - Hepatitis A virus among natives and expatriates in Saudia Arabia. PMID- 3014061 TI - An outbreak of epidemic diarrhoea in adults caused by a new rotavirus in Anhui Province of China in the summer of 1983. AB - Shexian County of Anhui Province in East China experienced a severe outbreak of nonbacterial diarrhoea in May and June of 1983. Over 20,0000 cases were reported. Most were adults of 20 to 50 years old. The isolated virus was morphologically indistinguishable from ordinary infantile rotavirus, but CF and ELISA tests showed that the new virus lacked the common group-A antigen shared by ordinary rotavirus. Nine of 10 convalescent-phase and three paired sera of patients showed antibody positive, with a greater than four-fold antibody rise against new rotavirus in the CF test. Seventeen faecal samples from patients showed identical, and special, electropherotype. They all had a double-stranded RNA with 11 discrete segments. The RNA profiles were quite different from those of reference rotavirus Wa strain and ordinary infantile rotaviruses. Third and 4th segments were near to each other, 5th and 6th segments were very close, but 7th, 8th, and 9th segments were widely separated. This study indicates the new virus can be identified as part of a new group of rotaviruses. This new rotavirus has been incriminated as the causative agent of the epidemic. PMID- 3014059 TI - Prevalence of HTLV-III/LAV antibodies in Italian asymptomatic hemophiliacs given commercial concentrates of factors VIII and IX. AB - The prevalence of HTLV-III/LAV antibodies was evaluated in 222 Italian asymptomatic hemophiliacs treated exclusively with commercial factor concentrates made with American plasma. An increase of HTLV-III/LAV seropositivity from 1983 to 1985 was evident. This was independent of the type of hemophilia (A or B) but directly correlated to the amount of concentrate administered in the previous year. Immunological data (T4 and T8 lymphocyte subsets) were evaluated in relation to seropositivity to HTLV-III/LAV. The presence of antibodies was found to be significantly associated with a low T4/T8 ratio and to a reduced T4 subpopulation, whereas increased levels of T8 lymphocytes correlated weakly with seropositivity. Data from 47 hemophiliacs tested both in 1984 and 1985 showed that 100% of those negative in 1984 and highly transfused in the previous year seroconverted up to 1985, whereas 33% of those not highly transfused did so. PMID- 3014062 TI - Indirect immunofluorescence, serum neutralization, and viremia responses of rhesus monkeys (Macaca mulatta) to Machupo virus. AB - Although indirect immunofluorescent antibody tests (IFAT) have been developed for several arenaviruses, none has been applied to the rhesus monkey model for Bolivian hemorrhagic fever (caused by the arenavirus Machupo). We infected eight rhesus monkeys with Machupo virus and bled them weekly for determination of viremia and for serum antibody detection by IFAT and serum neutralization (SN) testing. Viremia peaked day 14 post-inoculation (PI), when two of eight animals had low IFAT titers. At day 21 PI, the six surviving monkeys had elevated IFAT titers and diminished viremias. SN titers were not observed until 28 days PI, when three of four survivors had low titers. Results of the IFAT were available more rapidly than the SN, and detected increased serum antibody titers earlier than the SN. Acetone fixation did not completely inactivate BHF antigen spot slides. PMID- 3014063 TI - A comparative study of soluble calcium-dependent proteolytic activity in brain. AB - Recent studies have shown that soluble calcium activated proteases (calpains) in brain degrade proteins associated with the cytoskeleton and vary markedly in activity across regions and as a function of development. It was suggested that the observed differences in calpain activity reflect differences in the turnover rate of structural elements. The present study extends this analysis by measuring the properties and activity of calpain in representatives of the five classes of vertebrates with particular emphasis on the mammals. No evidence for proteolysis was found in soluble fractions of fish brains at neutral pH in the presence or absence of added calcium. A substantial calcium-independent proteolytic activity was found in amphibian brains--the effects of a variety of protease inhibitors indicated that it is also a neutral thiol (cysteine) protease. Reptilian brains exhibited both calcium-independent and calcium-dependent proteolytic activity. Virtually all proteolytic activity in birds (5 species) and mammals (9 species) measured at neutral pH was calcium-dependent. The endogenous substrates for the calcium activated proteases were very similar in several species of birds and mammals as were the effects of a variety of protease inhibitors. However, the activity of the enzyme, expressed per mg of soluble protein, was highly and negatively correlated with brain size in the mammals. The allometric expression for this relationship was similar to that found for the density of neurons in cerebral cortex as a function of absolute brain size. These results indicate that soluble proteolytic enzymes in brain are differentially expressed among classes of vertebrates and suggest that the turnover of cytoskeletal elements in birds and mammals differs in important ways from that found in fish and amphibians. The results obtained for mammals raise the possibility of a relationship between brain size and the rate at which structural elements are broken down and replaced in this vertebrate class. PMID- 3014064 TI - 5'-nucleotidase localization in the brains and spinal cords of adult normal and dysmyelinating mutant (shiverer) mice. AB - Immunocytochemical staining with the antibody against mouse liver 5'-nucleotidase revealed 5'-nucleotidase antigenicity in myelinated fibers in the brains and in myelinated fibers and some interfascicular oligodendroglia in the spinal cords of normal adult mice. Although the 5'-nucleotidase specific activity in adult shiverer mouse CNS tissue homogenates had been shown to be normal, immunocytochemical staining with anti-mouse-5'-nucleotidase could be demonstrated in CNS tissue sections from only 2 out of 10 of the mutant animals. In tissue from these animals the staining, which was relatively faint, was localized specifically to cell-bodies, usually arranged in rows, and to material oriented parallel to nerve fibers. This pattern of immunostaining with anti-5' nucleotidase resembled the immunostaining with anti-carbonic anhydrase but not with anti-glial-fibrillary-acidic-protein. This suggested that the rows of cells were oligodendrocytes, not astrocytes, and that the material parallel to nerve fibers might consist of oligodendrocyte processes wrapped loosely around axons. The antibody against rat 5'-nucleotidase, as distinguished from mouse, immunostained only the blood vessels in the shiverer mouse CNS, a finding similar to a previous observation in the normal mouse CNS. From these findings it was inferred that the primary loci of 5'-nucleotidase in the shiverer mouse CNS were interfascicular oligodendrocytes, their processes, and blood vessels, and in the normal mouse CNS, the myelin in some tracts, the blood vessels, and some interfascicular oligodendrocytes. PMID- 3014065 TI - Blood-cerebrospinal fluid barrier permeability to serum IgG subfractions and measurement of intrathecal IgG synthesis. AB - CSF/serum gradients of IgG subfractions separated by isoelectric focusing (IF) have been measured by high resolving laser densitometry. In patients with normal blood-CSF barrier permeability (N.25) and with barrier damage due to acute idiopathic polyneuropathy (N.15) and to medullary compression (N.17), the CSF/serum gradients of IgG subfractions were negatively correlated with their pI. This electrostatic selectivity appeared to be reverted in barrier damage due to acute meningoencephalitis (N.15). In a series of multiple sclerosis patients (N.31), the CSF/serum gradients of IgG subfractions lacking CSF oligoclonal bands have been used to assess the overall barrier permeability to serum IgG. All intra BCB synthesized IgG subfractions could be measured by densitometry, whereas with other quantitative formulae, 23-26% of the results were false negatives; the total intrathecal IgG amount ranged from 0.01 to 11 mg/dl. The most frequent and prominent fractions appeared to be cathodic. Electrostatic and steric barrier selectivity must be taken into account when the amount of intrathecal IgG synthesis has to be measured. PMID- 3014066 TI - Edema and increased endoneurial sodium in galactose neuropathy. Reversal with an aldose reductase inhibitor. AB - Galactose neuropathy was produced in rats by feeding a diet containing 30% D galactose. After 12 weeks of galactose ingestion, all rats developed bilateral cataracts, polydypsia and polyuria. These galactose-intoxicated animals were divided into two groups that both continued with the galactose diet: animals that were treated with the aldose reductase inhibitor, ICI 128,436, for 4-6 weeks, and a control group of animals that received just excipient. At the end of the study, endoneurial fluid pressures, nerve water contents and endoneurial fluid electrolyte concentrations were determined from sciatic nerves of treated and untreated animals. The extent of neuropathy in each animal was evaluated by light microscopy. Treatment of galactose-intoxicated rats with ICI 128,436 restored to normal levels the elevated endoneurial sodium concentration, increased water content and interstitial fluid pressure characteristic of galactose neuropathy. These results, obtained with an agent that blocks the sorbitol pathway, associate elevated sodium with an osmotic force contributing to edema and increased endoneurial fluid pressure in galactose neuropathy and suggest that endoneurial sodium levels are linked to blood-sugar concentration. PMID- 3014067 TI - Anterior horn changes of motor neuron disease associated with demyelinating radiculopathy. AB - Morphologic study of the spinal cord of a patient with generalized motor deficits revealed changes in the anterior horns characterized by the selective loss of large motor neurons, gliosis and the abnormal accumulation of 10 nm filaments which appeared as argyrophilic spheroids in the perikarya and axons of motor neurons. The ventral roots were predominantly affected and showed a variable loss of axons. The remaining axons displayed prominent onion-bulb formations, frequent axonal sprouting and occasionally evidence of active demyelination. The coexistence of a demyelinating motor radiculopathy and anterior horn changes simulating those of amyotrophic lateral sclerosis (ALS) may contribute to our understanding of the unresolved question of whether the neuronal perikaryon or its axon is the primary target in the pathogenesis of ALS. These observations also indicate that a rigid separation of pathogenetic mechanisms into neuronopathy, axonopathy and myelinopathy may not be always possible. PMID- 3014068 TI - Effect of cyclic AMP on ammonia-induced alterations in primary astrocyte cultures. AB - Current evidence suggests that astrocytes may be the target of ammonia toxicity. Consistent with this view are recent investigations which have shown morphologic alterations in primary astrocyte cultures following exposure to ammonia. In the present study, these alterations became severely aggravated when the cultures were not grown or maintained in dibutyryl cyclic adenosine monophosphate (AMP). Cyclic AMP analogues and agents that increase intracellular cyclic AMP levels significantly inhibited the toxic effects of ammonia. The exact mechanism responsible for this apparent protective effect of cyclic AMP on ammonia-treated astrocytes is not known. The possible means by which cyclic AMP may serve to ameliorate ammonia-induced toxicity are discussed. PMID- 3014069 TI - Peripheral nervous system demyelination with herpes simplex virus. AB - Inoculation of the cornea or footpad with herpes simplex virus Type I (HSV) has been shown to produce subsequent encephalitis or myelitis respectively. Although Schwann cells become infected, there is no destruction or demyelination in the peripheral nervous system (PNS). Demyelination only occurs in the central nervous system. Previous studies have shown that the Schwann cells infected with HSV do not produce enveloped viral particles. The studies presented here demonstrate that microinjection of HSV into the sciatic nerve of mice causes focal mononuclear cell infiltration and demyelination seven days after injection. The Schwann cells in this model produced enveloped virus. These studies demonstrate that when HSV is introduced into the extracellular space of the PNS, demyelination occurs. PMID- 3014071 TI - Locally synthesized antibodies in cerebrospinal fluid of patients with AIDS. AB - Twelve patients with AIDS were examined by enzyme-linked immunosorbent assay for antibody activity of IgG in serum and CSF. Two patients were only anti-HTLV III antibody carriers (stage I), two had lymphadenopathy syndrome (stage II) and eight had manifest AIDS (stage III). Eight of the 12 patients had 2- to 8-fold higher antibody titres in CSF than in serum, indicating that anti-HTLV III antibodies were produced in the nervous system. One of these patients with obviously locally synthesized anti-HTLV III antibodies in CSF belonged to stage I, two to stage II and five to stage III. Only four of these eight patients also showed locally synthesized IgG in CSF as measured by laser-nephelometry. In contrast, 61 controls with normal CSF (12), impaired blood-CSF barrier (12) multiple sclerosis (12) and various infections of the CNS other than HTLV III (25), the last two groups with locally synthesized IgG in CSF, all revealed negative results. It appears possible that locally synthesized anti-HTLV III antibodies in CSF are a sensitive and early indicator of an HTLV III infection of the nervous system. PMID- 3014070 TI - Diagnosis of brachial root and plexus lesions. AB - The diagnosis and management of lesions of the brachial roots and of the brachial plexus is improved by appropriate investigation, both in acute and chronic disorders. The choice of investigation should be determined by the clinical problem. Since they are relatively non-invasive, electrophysiological investigations are particularly useful. In this review the role of these investigations is considered in relation to diagnosis and management. PMID- 3014072 TI - Glioblastoma multiforme in three family members, including a case of true multicentricity. AB - Three cases of glioblastoma multiforme are described, occurring in two brothers and the sister of their father. One of the cases was found at autopsy to represent true multicentric glioblastoma, with five widely separated lesions affecting both hemispheres. It is postulated that there may be an association between factors responsible for multiple foci of transformation and a familial tendency to develop glioma. PMID- 3014074 TI - The effect of an intravenous calcium load on serum total and ionized calcium in normotensive and hypertensive subjects. AB - Altered regulation of serum calcium level was proposed to be associated with arterial hypertension and to be dependent on a renal calcium leak or an altered calcium binding to plasma proteins and cell membrane described in human and experimental hypertension. The aim of this study was to analyze the regulation of serum total and ionized calcium levels during an intravenous calcium infusion (0.25 mmol calcium/kg body weight/hr for 2 hours) in a group of untreated essential hypertensives and a comparable normotensive group. Basal serum calcium concentrations did not differ between the two groups, whereas parathyroid activity and urinary calcium were significantly increased in hypertensive subjects. During the calcium load, serum calcium rose almost linearly in all subjects but with a reduced slope in the hypertensive group, which showed serum total and ionized calcium levels significantly lower than those of the controls at the end of the infusion. Our data indicate that hypertensive patients have an altered regulation of serum calcium concentrations, probably due to a different body distribution of calcium, rather than to an altered calcium binding to plasma proteins. PMID- 3014073 TI - Urinary excretions of kininase I and kininase II activities in essential hypertension. A sensitive and simple method for its kinin-destroying capacity. AB - To further clarify the role of the renal kallikrein-kinin system in essential hypertension, a sensitive and simple method for the determination of both human urinary kininase I and kininase II was established, and the system components were determined in patients. In the measurement of kininase activity, desalted urine samples were incubated with synthetic bradykinin, and the reaction was terminated with kininase inhibitors, ethylene diamine tetraacetic acid and phenanthroline. Thus, kininase activity was determined as the kinin-destroying capacity. Moreover, the specific inhibitor for kininase II, SQ14225, was applied for the separation of kininase I and kininase II activities. Daily urinary excretions of total kininase and kininase I activities were significantly higher in essential hypertensive patients than those in normotensive subjects, whereas no difference was observed in kininase II activity. As reported previously, daily excretions of urinary kallikrein and kinin simultaneously determined in these patients were significantly lower than excretions in normotensive subjects. From these results, it was suggested that not only decreased renal kallikrein, but also increased kininase activity, may play an important role in the suppression of the renal kallikrein-kinin system through the reduction of active kinin level in essential hypertension. PMID- 3014075 TI - Rest and exercise hemodynamic and adrenergic responses to enalapril, hydrochlorothiazide, and combination treatment in patients with systemic hypertension. AB - The effects of enalapril (10-20 mg twice daily), hydrochlorothiazide (25-50 mg twice daily), and combination enalapril-hydrochlorothiazide therapy (10-20 mg enalapril/25-50 mg hydrochlorothiazide in combination tablet twice daily) were evaluated and compared to no therapy (control) in eight patients with mild to moderate hypertension at rest and during treadmill exercise. All active treatments reduced standing blood pressure in patients at rest compared to the control group (p less than 0.05); however, none produced significant reductions of standing blood pressure in patients at peak exercise. Standing heart rates of patients at rest and at peak exercise were not changed with active therapy. However, standing heart rate in patients at rest was lower with enalapril than with hydrochlorothiazide and combination therapy (p less than 0.05). Heart rate of patients on hydrochlorothiazide was higher than with control and other therapies at Stage I of exercise (p less than 0.01). Supine norepinephrine levels in patients at rest were elevated with both hydrochlorothiazide and combination therapy when compared to that in patients with enalapril and control (p less than 0.05). Treatment with enalapril alone produced no changes in plasma catecholamine levels compared to control. There were no differences between control and all treatment regimens in peak exercise levels of catecholamines. Thus, enalapril, hydrochlorothiazide, and combination therapy, although effective in lowering resting blood pressure, may not be effective in blunting the blood-pressure response to exercise. The drugs do not appear to have any significant effects on catecholamine levels in patients at peak exercise. PMID- 3014076 TI - An overview of labetalol and adrenergic effects. PMID- 3014078 TI - Potassium metabolism and hypertension. PMID- 3014077 TI - Beta-adrenergic receptors on polymorphonuclear cell membranes in essential hypertension. AB - Beta adrenergic receptor binding sites were determined and characterized by specific binding of (+/-)[125I] iodocyanopindolol to membranes obtained from circulating polymorphonuclear leukocytes. No difference was found in the number of receptor sites and in their dissociation constants (Kd) between patients with untreated essential hypertension (EH), EH treated with drugs other than beta blockers, and in normotensive controls. The group with EH receiving treatment with beta blockers had a significantly higher receptor density and Kd as compared with all the other groups (p less than 0.05). It is concluded that the beta adrenergic system of patients with essential hypertension at the receptor level is not different from normotensive subjects and responds to beta blockers by up regulation. PMID- 3014079 TI - Renal insufficiency during angiotensin-converting enzyme inhibitor therapy in hypertensive patients with no renal artery stenosis. AB - Worldwide experience with captopril and enalapril showed that angiotensin converting enzyme (ACE) inhibitor monotherapy in hypertensive patients rarely caused renal dysfunction. The ACE inhibitors in combination with potent vasodilating drugs and diuretics may produce sudden systemic normotension or hypotension that may impair glomerular filtration at reduced renal perfusion pressure. Reversible renal insufficiency developed during the 13th week of hydrochlorothiazide-enalapril-alpha methyldopa therapy in patient 1 and during the 6th week of hydrochlorothiazide-enalapril treatment in patient 2. Systemic hypotension in patient 1 and routine biochemical monitoring in patient 2 was the first clue of renal insufficiency. Renal angiography was normal in both patients. Renal insufficiency resolved after stopping all drugs temporarily and did not recur on other antihypertensive drug regimens. These data suggested the importance of systemic arterial blood pressure as the best clinical determinant of renal function in hypertensive patients receiving an ACE inhibitor in combination with other antihypertensive agents. PMID- 3014080 TI - Combination therapy with enalapril and hydrochlorothiazide: optimal dose, renin response, and prostaglandin excretion. PMID- 3014082 TI - Mortality and morbidity in long-term surviving patients treated with chemotherapy with or without irradiation for small-cell lung cancer. AB - Mortality and morbidity was investigated in a consecutive series of 72 patients with small-cell lung cancer (SCLC) who were found to be disease-free at restaging after 18 months of treatment. These patients were all the long-term survivors among 874 patients included in one of six trials between 1973 and 1981. All studies used combination chemotherapy with or without irradiation. Follow-up of the patients varied between 4 and 11 years. The estimated 5-year survival rate subsequent to discontinuation of therapy was 0.24, corresponding to a death rate of 0.25 per year or ten times greater than the expected mortality for persons of the same age group. This high mortality was primarily related to recurrent SCLC, the estimated cumulative risk of relapse reaching 46% at the time of the latest recurrence 5 years from diagnosis. The risk of relapse was generally independent of the pretreatment disease stage although it was reduced in patients with resectable disease and was greater in those with pretreatment liver or bone marrow metastases. Equal risks of relapse were related to the use of regimens with and without radiotherapy. The cumulative risk of relapse in patients surviving 3 years from initiation of the treatment was less than 15% and accordingly, 3 years of follow-up seems sufficient for comparison of long-term results obtained in different trials. The second factor resulting in death or disease was second cancer, for which the cumulated risk increased to 32%, the latest occurring 5.4 years from the diagnosis of SCLC. Five of these cases were non-small-cell lung cancers and three were secondary leukemias. The estimated mortality related to non-neoplastic conditions was just significantly greater than expected. In spite of the increased mortality in this series, 38 of 54 2 year disease-free survivors and 20 of 22 5-year survivors resumed a lifestyle similar to that before diagnosis of SCLC. PMID- 3014081 TI - Long-term effects of enalapril monotherapy and enalapril/hydrochlorothiazide combination therapy on blood pressure, renal function, and body fluid composition. AB - Enalapril, a potent long-acting angiotensin-converting enzyme inhibitor, was prescribed either alone (n = 9) or in combination (n = 20) with hydrochlorothiazide for 96 weeks in essential hypertensive subjects. Blood pressure was well controlled following both monotherapy or combination therapy. Plasma renin activity was stimulated in all subjects; plasma aldosterone concentration was stimulated only in subjects receiving combination therapy. Glomerular filtration rate (assessed by inulin clearance) was unchanged in subjects with initial clearances greater than 80 ml/min/1.73 m2 but was significantly improved (55%) following either form of therapy in subjects with initial clearances less than or equal to 80 ml/min/1.73 m2. Neither monotherapy nor combination therapy adversely affected 24-hour urinary protein excretion, sodium excretion, or body fluid composition. These results suggest that enalapril, either alone or in combination with hydrochlorothiazide, is effective therapy for mild to moderate hypertension. There are no adverse effect on renal function; indeed, enalapril has the capability of improving renal function in those subjects whose renal function was initially impaired from long-standing hypertension. PMID- 3014083 TI - A phase II study of combined 5-fluorouracil, doxorubicin, and cisplatin in the treatment of advanced upper gastrointestinal adenocarcinomas. AB - In a phase II study of 67 patients with upper gastrointestinal carcinomas and measurable disease without previous chemotherapy, we have evaluated the combination of intensive course 5-fluorouracil (5-FU) (300 mg/m2/d for five days) doxorubicin (40 mg/m2 on day 1), and cisplatin (60 mg/m2 on day 1). Courses were repeated every 5 weeks. Among 26 patients with gastric carcinoma, a 50% regression rate was obtained with a median survival for all patients of 9 months. Among 29 patients with pancreatic carcinoma, the regression rate was 21% and the median survival was 4 months. Regressions were also observed in smaller numbers of patients with carcinomas of the gallbladder and ampulla of Vater, as well as in cholangiocellular carcinoma of the liver. Toxic reactions were usually clinically tolerable and consisted primarily of nausea, vomiting, stomatitis, diarrhea, leukopenia, and alopecia. Phase III studies are in progress to place the value of this experimental regimen into clinical perspective. PMID- 3014084 TI - Central nervous system sarcoidosis. PMID- 3014085 TI - Advantage of indium-111 leukocytes over ultrasound in imaging an infected renal cyst. AB - Indium-111-labeled leukocyte scanning is a highly sensitive and specific method of detecting abscesses. This report describes a patient with polycystic kidneys and a single infected cyst. Ultrasound could not determine which cyst was infected, but the infected cyst could be localized by [111In]leukocyte imaging in conjunction with a [99mTc]DMSA renal scan. The two radionuclide studies were used to identify an infected renal cyst and direct ultrasound guided aspiration. PMID- 3014087 TI - Potential pitfall of DMSA scintigraphy in patients with ureteral duplication. AB - A 5-wk-old male presented with radiographic findings of a duplicated collecting system. A [99mTc]DMSA scan was requested to evaluate cortical function. Images obtained immediately. postinjection showed activity restricted to the upper poles; in contrast, delayed images at 4 hr showed activity in the bladder and throughout both kidneys. Catheterizing the patient drained the activity from the bladder but had little effect on the refluxed renal activity. The early [99mTc]DMSA images were critical in making the proper interpretation. Technetium 99m DMSA is excreted into the urine and this fact needs to be considered when interpreting scans of patients with possible reflux or obstruction. When DMSA scans are obtained in pediatric patients with possible reflux, catheterization prior to the study and early images prior to the appearance of DMSA in the collecting system are recommended. PMID- 3014086 TI - Poor results with technetium-99m (V) DMS and iodine-131 MIBG in the imaging of medullary thyroid carcinoma. AB - The value of [99mTc(V)]DMS and [131I]MIBG in imaging medullary carcinoma of the thyroid was investigated in five patients. Results with [99mTc(V)]DMS were negative in all five patients as well as with [131I]MIBG in four patients; however, there was significant tumor uptake in one patient with [131I]MIBG. PMID- 3014088 TI - Effect of Sn(II) ion concentration and heparin on technetium-99m red blood cell labeling. AB - While convenience and economy favor the use of in vivo methods for labeling red blood cells (RBCs) with [99mTc]pertechnetate, previous reports suggested that patient medication such as heparin might interfere and thus result in inferior quality images. In this study, using a canine model, the role of stannous Sn(II) ion in in vivo and in vitro labeling of RBCs both in the presence and absence of a therapeutic dose of heparin was investigated. Our results showed that Sn(II) ion concentration of 20 micrograms/kg body weight levels provided better than 80% in vivo labeling efficiency enabling high quality blood-pool images even in the presence of therapeutic doses of heparin. PMID- 3014090 TI - Retention of iron by rat intestine in vivo as affected by dietary fiber, ascorbate and citrate. AB - The effects of pH, ascorbate, citrate and dietary fiber on retention of ferrous and ferric iron by jejuno-ileal segments of rat intestine were examined in vivo. Iron was introduced in an isosmotic solution of sodium chloride and dextrose buffered by 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid (BES) and acetate. Stabilization of the iron solutions was aided by use of iron concentrations less than or equal to 1 microgram/ml injected into the intestine for 10-min periods. Iron retention was optimal over a broad pH range from 5 to 7.8. Inclusion of ascorbic acid in the solution injected (5, 25 or 75 micrograms/ml) did not increase retention of iron in either valence state. A low concentration of sodium citrate (2 mM) had no effect on iron retention, but increasing the concentration to 5 mM released iron from the mucosa. Maize and wheat fibers decreased the retention of ferrous iron by binding and by promoting autoxidation and formation of poorly soluble iron polymers. Bound ferrous iron was released completely at pH below 5. Retention of ferric iron was also lowered in the presence of fiber, presumably as a result of polymerization. Retention of iron by the rat in the absence of ligands was independent of valence state. PMID- 3014089 TI - Technetium-99m DMSA uptake by metastatic carcinoma of the prostate. PMID- 3014091 TI - Effects of NaCl on calcium balance, parathyroid function and hydroxyproline excretion in prednisolone-treated rats consuming low calcium diet. AB - The short-term effects of dietary sodium chloride supplementation on calcium balance were examined in an animal model of corticosteroid-mediated osteoporosis. Changes in calcium and phosphate balance, parathyroid function and bone resorption elicited by salt supplements alone (8 g/100 g diet), prednisolone alone (2.2 mg/kg body wt per day) and both salt and prednisolone in combination were measured in rats consuming a low calcium diet (0.1% calcium) for 10 d. Parathyroid function was monitored by measuring urinary cyclic AMP excretion. Bone resorption was monitored by measuring urinary hydroxyproline excretion. Salt alone raised urinary calcium, cyclic AMP and hydroxyproline; prednisolone alone depressed net calcium absorption and urinary hydroxyproline but had no effect on urinary calcium. Salt and prednisolone each depressed calcium retention independently and together produced an additive adverse effect on calcium balance. Thus high dietary salt intakes augment urinary calcium loss, raise parathyroid activity, increase bone resorption and adversely affect calcium balance in prednisolone-treated growing rats with a restricted dietary calcium intake. These findings support the view that high salt intakes may exaggerate bone loss during chronic glucocorticoid therapy. Because people also develop osteoporosis during glucocorticoid therapy and respond to dietary salt supplements by increasing urinary calcium excretion and parathyroid hormone, high salt intakes may accelerate bone loss in patients receiving chronic glucocorticoid therapy. The beneficial effects of dietary salt restriction on the conservation of bone mass warrant investigation in these patients. PMID- 3014092 TI - Effect of dietary zinc on endogenous free radical production in rat lung microsomes. AB - The objective of this study was to investigate the effect of dietary zinc on endogenous production of free radicals in lung and liver microsomes. Male weanling rats were fed a zinc-deficient basal diet containing less than 1.1 ppm zinc, or were pair-fed or fed ad libitum a zinc-adequate diet supplemented with 100 ppm zinc. The isolated microsomes (100,000 X g precipitate) of lung and liver were incubated with 0.1 M PBN (spin trap) and 0.3 mM NADPH (cofactor) at 37 degrees C for 1.0 h. A carbon-centered free radical (aN = 16.0 G, aH beta = 3.4 G) was trapped in both lung and liver microsomes. There was a significant increase in the concentration of carbon-centered free radicals generated in lung microsomes in animals fed a zinc-deficient diet. Dietary zinc status did not significantly affect the concentration of free radicals in liver microsomes. The amount of free radicals generated is proportional to microsomal protein concentration and is linear with protein concentration between 5 and 20 mg per milliliter of incubate. The free radicals formed in the microsomal system were dependent on the presence of NADPH. Carbon monoxide inhibited 40-50% of the free radical production in both lung and liver microsomes. The results suggest that dietary zinc deficiency stimulates the production of endogenous free radicals in rat lung microsomes by an NADPH- and cytochrome P-450-dependent system. PMID- 3014094 TI - Dietary fat and cancer: specific action or caloric effect. PMID- 3014093 TI - Effect of dietary saturated fatty acids on hormone-sensitive lipolysis in rat adipocytes. AB - The objective of this work was to examine the mechanism by which dietary saturated fatty acids, as compared with polyunsaturated fatty acids, lower hormone-sensitive lipolysis in rat adipocytes. Rats were fed a purified diet containing 14% of a fat with a high concentration of either saturated fatty acids (coconut oil or beef fat) or polyunsaturated fatty acids (safflower oil) as a control. In addition, each diet contained 2% corn oil. The animals were fed these diets for 4 wk. Norepinephrine-stimulated lipolysis was 50% lower when diets rich in saturated fatty acids, regardless of their chain length, were fed than when a diet containing a high concentration of polyunsaturated fatty acids was fed. The specific activities of adenylate cyclase, 3',5'-cyclic nucleotide (cAMP) phosphodiesterase and hormone-sensitive lipase were lower when saturated fatty acids were fed than when polyunsaturated fatty acids were fed. Accumulation of cAMP upon stimulation with 10(-5) M norepinephrine was lower when saturated fatty acids were fed than when polyunsaturated fatty acids were fed. Moreover, adipocytes were larger when saturated fatty acids were fed than when polyunsaturated fatty acids were fed. The data obtained suggest that dietary saturated fats exert their inhibitory effect on hormone-stimulated lipolysis by influencing several points in the lipolytic cascade. PMID- 3014096 TI - Effect of synthetic atrial natriuretic peptide on rat renal juxtaglomerular cells. AB - We examined the effect of synthetic atrial natriuretic peptide (ANP) on unstimulated renin release from rat renal juxtaglomerular cells. Using renal cortical cell cultures containing 80-90% juxtaglomerular cells, we found that ANP strongly inhibited renin release from the cells in a dose-dependent fashion. Half maximal inhibition was observed at 10(-11) mol/l ANP. Furthermore, ANP produced a rise in the intracellular concentration of cyclic guanosine monophosphate (cGMP) and a fall in the concentration of cyclic adenosine monophosphate (cAMP). Data indicated that the inhibition of renin release by ANP was related to the rise in cGMP and not to the fall of cAMP. Atrial natriuretic peptide (10(-10) mol/l) had no influence on the transmembrane calcium influx nor did it alter the intracellular calcium concentration of the juxtaglomerular cell. We found, however, that the inhibitory effect of ANP on renin release could be attenuated by the calcium channel blocker verapamil. Our results suggest that ANP inhibits renin release from juxtaglomerular cells by a mechanism that is mediated by cGMP; the mechanism is not linked to any alteration in intracellular calcium concentration but requires a normal level of calcium. PMID- 3014095 TI - Effects of dietary cellulose and xylan on absorption and tissue contents of zinc and copper in rats. AB - This investigation was undertaken to determine the effects of purified cellulose and xylan on the apparent absorption and tissue concentration of zinc and copper in male weanling Sprague-Dawley rats. The control group was fed a fiber-free diet and six other groups were fed a diet containing 3, 6 and 12% cellulose or xylan. All diets contained 10 ppm Zn and 4 ppm Cu. Four 14-d collections of feces were made during wk 3 and 4, 10 and 11, 17 and 18 and 24 and 25. The balance technique was used to measure the apparent absorption of zinc and copper. After 26 wk of consuming the test diets, rats were killed and plasma liver, kidney and mucosa analyzed. There were no significant differences between groups in regard to weight gain, feed intake and feed efficiency. The average amounts of ingested cellulose and xylan that survived the passage of the intestinal tract were 86 and 18%, respectively. Inclusion of 12% cellulose in the diet resulted in a significantly lower apparent absorption of both zinc and copper, whereas at 6% cellulose only the absorption of zinc was lower. The addition of cellulose also resulted in lower plasma zinc and liver and kidney copper concentrations. In contrast to cellulose, xylan did not exert a significant influence on apparent absorption and tissue concentrations of the minerals. Regardless of the type and level of fiber, the apparent absorption of zinc and copper declined as rats grew older. PMID- 3014097 TI - Carcinoma in a pleomorphic adenoma. Review of the literature and report of a case. PMID- 3014098 TI - Alveolar ridge maintenance with hydroxylapatite ceramic cones in humans. AB - The efficacy of solid cones of hydroxylapatite ceramic implanted in fresh extraction sockets for the preservation of alveolar bone was evaluated. Ten experimental subjects (70 implants) and eight control subjects (63 extractions) were treated. After extraction, hydroxylapatite ceramic cones were inserted into the sockets at least 1 mm below the alveolar crest in the experimental group. Alveolar ridge resorption was measured on lateral cephalometric radiographs, and statistical analysis was performed. The follow-up periods ranged from 12 to 24 months (mean, 20.6 months). Thirty-seven of the 70 hydroxylapatite ceramic cone implants (53%) became exposed, and 19 cones (27%) had to be removed. It was concluded that hydroxylapatite ceramic cone implants placed in fresh extraction sockets do not significantly preserve alveolar bone. PMID- 3014099 TI - Identification of human papillomavirus types in oral verruca vulgaris. AB - Eleven oral verruca vulgaris specimens were examined for the presence of papillomavirus structural antigens by reaction with antibody to type-common antigens and detection by the avidin-biotin-peroxidase complex method. The specimens were also examined by in situ hybridization with biotin-labelled human papillomavirus (HPV) DNA to determine the specific HPV types present in the lesions. Six of the 11 specimens were positive for papillomavirus structural antigens. Of these 6, 5 hybridized to the HPV Type 2 (HPV2) probe and one to the HPV4 probe. PMID- 3014100 TI - Prevalence of neuroendocrine granules in small cell lung carcinoma. Usefulness of electron microscopy in lung cancer classification. AB - Fifty-four lung carcinomas submitted for routine electron microscopy under the light microscopical diagnosis of small cell carcinoma were investigated. In 42 or of 45 evaluable cases, neuroendocrine granules were considered definitely or probably present, the modification 'probably' being necessary in suboptimal material. In three cases, squamous cell differentiation was seen, but no neuroendocrine granules were found. On revision of these three cases, the light microscopical diagnosis was changed to squamous cell carcinoma in one instance, and neither of the other two cases was considered classical for small cell carcinoma. Patient follow-up of these three cases showed tumour behaviour indicative of non-small cell carcinoma in two evaluable cases, the third case yielding no significant data. These results indicate that neuroendocrine granules can generally be found in small cell lung carcinomas, provided the material is sufficient for evaluation. When these granules are absent, and other differentiation is found on electron microscopy, the final classification of the tumour should incorporate this finding, to warn the clinician that the tumour will not necessarily behave as a small cell carcinoma. PMID- 3014101 TI - The 'grey area' between small cell and non-small cell lung carcinomas. Light and electron microscopy versus clinical data in 14 cases. AB - We studied 14 lung tumours which on light microscopy had posed difficulties on classification as either small cell or non-small cell carcinomas. The light and electron microscopical features were compared with patient follow-up data. Electron microscopy showed neuroendocrine granules in 12 cases, and adeno- and squamous cell differentiation but no neuroendocrine granules in the remaining two cases. The latter two cases showed prolonged patient survival (both patients alive after 2 1/2 and 2 years, respectively). Ten of the cases with neuroendocrine granules showed a rapid course of disease (death between 2 1/2 weeks and 15 months after diagnosis) and marked initial response to multiagent chemotherapy. Thus, the clinical impression of these cases was that of small cell carcinoma. The remaining two cases with neuroendocrine granules showed a more protracted course, with death after 1 1/2 and 2 1/2 years. These two tumours did not show the light microscopical features of atypical carcinoid. The results illustrate the value of electron microscopy in predicting clinical behaviour of carcinomas difficult to place into small cell or non-small cell carcinoma groups. They also point to the existence of neuroendocrine carcinomas other than carcinoids with a more protracted course than small cell carcinomas. PMID- 3014102 TI - Thrombocytopenia as the presenting manifestation of human T-lymphotropic virus type III infection in infants. AB - Three infants between 8 and 9 months of age developed thrombocytopenia resulting from immune-mediated platelet destruction, as evidenced by the presence of serum antibody to platelets and elevated platelet-associated immunoglobulin G in two patients, and abundant bone marrow megakaryocytes in all patients. The patients had a satisfactory response to corticosteroid therapy, and platelet counts have remained normal during observation after therapy. All patients had serum antibody to human T-lymphotropic virus type III, and HTLV-III was isolated from the peripheral blood lymphocytes in two patients. The HTLV-III infections were presumably acquired via blood transfusions in the neonatal period; none of the patients' mothers belonged to a risk group for HTLV-III infection, and all were HTLV-III seronegative. Although thrombocytopenia was the major clinical manifestation, the patients had a number of immunologic abnormalities characteristic of HTLV-III infection; these included hyperimmunoglobulinemia, a decreased proportion of peripheral blood T cells, and a marked reduction in the proportion of peripheral blood T helper-inducer lymphocytes. We conclude that the patients had immune-mediated thrombocytopenia caused by HTLV-III infection. PMID- 3014103 TI - Cytomegalovirus transmission in a Midwest day care center: possible relationship to child care practices. AB - The epidemiology of cytomegalovirus (CMV) infection and transmission in a large Iowa day care center was studied. Over the 9 months of the study the overall CMV prevalence rates were 21% to 22%, with rates as high as 71% in toddlers. Titers of CMV in the urine or saliva of infected children were as high as 3 X 10(4) plaque-forming units of CMV per milliliter, similar to titers of CMV observed in some congenitally infected infants. Restriction enzyme analysis of CMV isolates from children in the center demonstrated two major clusters with similar patterns, one among 2- and 3-year-old children and another among infants. The clustering of similar CMV isolates among nonambulatory infants suggests that child care or hygienic practices may contribute to the spread of CMV infection in day care centers. Furthermore, the relatively high prevalence of CMV excretion in this center and the low seropositivity rates to CMV among adults in Iowa suggest that adults in the Midwest who have contact with children in day care centers may be at risk for primary CMV infection. PMID- 3014104 TI - Diffuse hepatic fibrosis in congenital cytomegalovirus infection. AB - Liver dysfunction in congenital cytomegalovirus (CMV) infection is common but usually self-limited. We describe a patient with congenital CMV infection that resulted in diffuse (noncirrhotic) hepatic fibrosis and liver failure. Morphologic patterns of liver disease in congenital CMV are briefly discussed. PMID- 3014105 TI - Treatment of murine coccidioidal meningitis with fluconazole (UK 49,858). AB - Male ICR mice were challenged intracerebrally with endospores of Coccidioides immitis and then treated with water (control), fluconazole, amphotericin B (Fungizone), or ketoconazole (Nizoral). All three drugs markedly prolonged survival, and all three drugs lowered brain colony counts of C. immitis. Survival of mice treated orally with fluconazole at the high dose was longer than in the ketoconazole treated group. Amphotericin B was more efficacious than fluconazole. Further investigations are needed to determine the efficacy of fluconazole in treatment of coccidioidal meningitis. PMID- 3014106 TI - Azole resistance in Candida albicans. AB - An isolate of Candida albicans from a patient with chronic mucocutaneous candidosis who relapsed during ketoconazole treatment was compared with a number of other azole-sensitive and azole-resistant isolates by tests in vitro and in three animal models of vaginal or disseminated infection. In-vitro tests indicated that the isolate was cross-resistant to all imidazole and triazole antifungals tested. In the animal models, treatment with miconazole, ketoconazole, itraconazole or fluconazole failed to influence the infection. PMID- 3014107 TI - Attenuation of the development of hypertension in spontaneously hypertensive rats by chronic oral administration of eicosapentaenoic acid. AB - Chronic effects of highly purified eicosapentaenoic acid (EPA) on systolic blood pressure in spontaneously hypertensive rats (SHR) and normotensive rats were studied. Daily oral administration of 30 to 300 mg/kg EPA for eight weeks significantly decreased the development of hypertension in SHR dose-dependently. Eight weeks treatment of 30, 100, and 300 mg/kg EPA reduced mean systolic blood pressure by 23, 29, and 32 mmHg, respectively, compared to untreated rats. Hypotensive effect of EPA progressed slowly and was reversible after the termination of the treatment. However, daily administration of EPA to normotensive rats did not affect the systolic blood pressure. EPA may be useful as a hypotensive agent for treatment of hypertension. PMID- 3014108 TI - Effects of dehydroepiandrosterone sulphate on the production of collagenase and gelatinolytic metalloproteinase by rabbit uterine cervical cells in primary cultures. AB - Cells from pregnant rabbit uterine cervix in primary cultures produced specific collagenase and gelatinolytic metalloproteinase, both in latent forms. The two enzymes were separated by CM-52 cellulose chromatography. Dehydroepiandrosterone 3-sulphate (DHAS) stimulated the production of the latent collagenase by the cells in a dose-dependent manner and the maximal effect, which was about 2-fold of control cultures, was observed when 1 X 10(-6) M DHAS was added to the medium containing 10% (v/v) foetal calf serum (FCS). DHAS also stimulated the production of the gelatinolytic metalloproteinase. The significant stimulative effects were also confirmed when the cells were incubated with 1 X 10(-7) M DHAS in the medium containing 9% (v/v) dextran-coated charcoal treated FCS and 1% (v/v) FCS. The uptake of [3H]DHAS by the cells from pregnant rabbits was 5-10-fold greater than that from nonpregnant rabbits. When confluent cultures were incubated with 6.5 X 10(-8) M [3H]DHAS for 2 d, 3.4% and 0.03% of total radioactivity added were accumulated as [3H]oestradiol-17 beta ([3H]E2) in the medium and cell layer, respectively. Nevertheless, the addition of 1 X 10(-9) M and 1 X 10(-11) M E2 or dehydroepiandrosterone (DHA) to the confluent cultures caused no significant effect on the collagenase production. These results strongly suggest that the stimulative effect of DHAS on the production of collagenase and gelatinolytic metalloproteinase by the rabbit uterine cervical cells was due to unchanged DHAS and not due to E2 or DHA converted from DHAS. PMID- 3014109 TI - Diurnal variations of benzodiazepine binding in rat cerebral cortex: disruption by pinealectomy. AB - In a previous work, pinealectomy was found to depress benzodiazepine (BZP) receptor binding in cerebral cortex membranes of rats killed at noon. In order to assess the effect of pineal removal on diurnal variations of BZP binding site concentration and affinity, groups of intact, pinealectomized, or sham pinealectomized rats (subjected to surgery 2 wk earlier) were killed at six different time intervals during the 24-h cycle. BZP binding was assessed by Scatchard analysis of 3H-flunitrazepam high-affinity binding to cerebral cortex membranes. In intact and sham-pinealectomized rats, a maximum in BZP receptor concentration was found at midnight. Pinealectomy blunted the nocturnal peak of receptor concentration and caused a significant depression of binding site number at noon. No changes in the affinity of the binding sites for the radioligand were detected as a function of time of day or following surgery. In a dose-response experiment for melatonin ability to restore the depressed BZP receptor concentration of cerebral cortex membranes of pinealectomized rats killed at noon, a minimal effective dose of 25 micrograms/kg body weight was obtained. These results further support a link between pineal activity and brain BZP receptors in rats. PMID- 3014110 TI - Effect of food on the bioavailability of lisinopril, a nonsulfhydryl angiotensin converting enzyme inhibitor. AB - A randomized, two-way, crossover study was performed on 18 normal volunteers to assess the influence of food on the bioavailability of lisinopril, (1-[N2-[(S)-1 carboxy-3-phenylpropyl]-L-lysyl]-L-proline), a long-acting nonsulfhydryl angiotensin converting enzyme inhibitor. A single, 20-mg oral dose of lisinopril was administered to volunteers in the fasting state or following a standardized breakfast. Treatment periods were separated by 2-week intervals. No significant differences existed between fasting and fed regimens in the mean +/- SD area under the serum concentration-time curve (AUC0-120h; 1231 +/- 620 versus 1029 +/- 254 ng X h X ml-1), peak lisinopril serum concentration (86 +/- 48 versus 69 +/- 19 ng/mL), or time to peak lisinopril serum concentration (6.2 +/- 1.1 versus 6.8 +/- 1.0 h). Five-day urinary excretion of lisinopril was not altered by food (5.3 +/- 3.0 versus 5.1 +/- 2.0 mg). Based on the urinary data, the mean +/- SD bioavailability of lisinopril was not different following fasting or fed regimens (27 +/- 15 versus 26 +/- 10%). Unlike with captopril, food did not affect the bioavailability of lisinopril. PMID- 3014111 TI - Alpha-1 and alpha-2 adrenoceptor: response coupling in canine saphenous and femoral veins. AB - The aim of the present study was to analyze alpha-1 and alpha-2 adrenoceptor response coupling in isolated canine blood vessels. Rings of saphenous and femoral veins and of femoral arteries were suspended for isometric tension recording in modified Krebs-Ringer bicarbonate solution, gassed with 95% O2-5% CO2 and maintained at 37 degrees C. Dissociation constants for the alpha-1 adrenergic agonists, phenylephrine and cirazoline, and the alpha-2 adrenergic agonist, UK 14,304, were determined by analysis of concentration-effect curves to the agonists under control conditions and after partial inactivation of alpha adrenoceptors by phenoxybenzamine. The dissociation constant of phenylephrine for alpha-1 adrenoceptors in saphenous veins was approximately 10-fold higher than that obtained for the agonist in femoral arteries or femoral veins. Similarly the dissociation constant for cirazoline in the saphenous vein was higher than that obtained in other alpha-1 adrenergic systems. Dissociation constants were used to determine alpha adrenoceptor occupancy-response relationships. The alpha-1 adrenergic responses evoked by high intrinsic-efficacy agonists (cirazoline and phenylephrine) were associated with a very large receptor-reserve in the saphenous vein, but no, or only a limited receptor-reserve in the femoral vein. The dissociation constant for UK 14,304 in saphenous veins was significantly lower than that obtained for alpha-2 adrenergic stimulation by norepinephrine. There was no alpha-2 adrenoceptor reserve in the saphenous vein for these putative high intrinsic-efficacy agonists. The differences in receptor-reserve between alpha-1 adrenoceptors in canine saphenous and femoral veins and between alpha-1 and alpha-2 adrenoceptors in saphenous veins may help to explain the differential modulation of adrenergic responses in these blood vessels. PMID- 3014112 TI - Effect of cooling on alpha-1 and alpha-2 adrenergic responses in canine saphenous and femoral veins. AB - Experiments were designed to determine the effects of cooling on alpha-1 and alpha-2 adrenergic responses in isolated canine veins. Rings of saphenous and femoral veins were suspended for isometric tension recording in modified Krebs Ringer bicarbonate solution, gassed with 95% O2 and 5% CO2. Cooling (from 37-24 degrees C) augmented contractions to norepinephrine in saphenous but caused depression in femoral veins. Cooling (to 24 degrees C) had no effect on alpha-1 adrenergic responses evoked by phenylephrine in saphenous veins but caused depression in femoral veins. Alpha-2 adrenergic responses produced by UK 14,304 were augmented by cooling in the saphenous but were virtually abolished by cooling in femoral veins. Cooling decreased the dissociation constant (i.e., increased affinity) of corynanthine for alpha-1 adrenoceptors in saphenous and femoral veins (approximately 3-fold), and the dissociation constant of rauwolscine for alpha-2 adrenoceptors in saphenous veins (approximately 7.5 fold). The influence of cooling on alpha adrenoceptor responsiveness was analyzed using computer-generated receptor-models. The results suggest that the differential sensitivity of cutaneous and deep blood vessels to cooling results from differences in efficiency of alpha-1 and alpha-2 adrenoceptor response coupling. In the saphenous vein, there is a large alpha-1 adrenoceptor reserve which buffers the alpha-1 adrenergic response from the inhibitory influence of cooling. This coupled with a cooling-induced increase in alpha-2 adrenoceptor affinity ensures that cooling augments the response to norepinephrine. In the femoral vein, there is no alpha-1 adrenoceptor reserve and cooling therefore depresses alpha-1 adrenergic responses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014113 TI - Uptake of N-[1 (S)-carboxy-(4-OH-3-125I-phenyl)ethyl]-L-Ala-L-Pro, an inhibitor of angiotensin-converting enzyme by rabbit lungs in situ. AB - Single pass extraction of a new iodinated inhibitor of angiotensin-converting enzyme (ACE) was measured by means of indicator-dilution techniques applied to rabbit lungs, perfused in situ at 20 ml/min with Krebs bicarbonate solution containing 3% bovine serum albumin. A bolus containing the inhibitor, N-[1(S) carboxy-(4-OH-3-125I-phenyl)ethyl]-L-Ala-L-Pro (CPAP), and an intravascular marker, [14C]dextran, was injected and extraction calculated at the peak of the resulting venous outflow-time curve. In 13 of 21 lungs used, a synthetic substrate for ACE, [3H]benzoyl-phenylalanyl-alanyl-proline (BPAP), was added to the bolus and appearance of its hydrolysis product, [3H]benzoyl-Phe, measured in effluent samples. When low amounts (0.15 nmol) of [125I]CPAP were injected, pulmonary extraction (E) of CPAP was 67 +/- 14% (X +/- S.D; n = 21) and metabolism (M) of BPAP was 56 +/- 9% (n = 13). Addition of unlabeled CPAP (3, 34 or 340 nmol) to the Addition of unlabeled CPAP (3, 34 or 340 nmol) to the injected bolus caused dose-dependent reduction of E(CPAP) and M(BPAP) that was no longer evident 10 min after the largest dose of CPAP. Coadministration of the ACE inhibitor, captopril (3, 6, 8 and 28 nmol), also caused dose-dependent, reversible depression of both E(CPAP) and M(BPAP). Accordingly, extraction of CPAP by perfused rabbit lung is saturable. Inasmuch as CPAP inhibits ACE activity (as reflected by BPAP metabolism) and CPAP uptake is inhibited by captopril (which also inhibits BPAP hydrolysis), it appears that a large portion of this saturable process probably reflects binding to vascular ACE.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014115 TI - Elevated cytosolic calcium in rat hepatocytes exposed to carbon tetrachloride. AB - CCl4 rapidly and severely inhibits hepatic endoplasmic reticulum calcium (Ca++) sequestration in rats exposed to this hepatotoxin in vivo. As a consequence, it is possible that cytosolic Ca++ concentrations become elevated in liver cells. In this study, the authors examined intracellular Ca++ concentrations in cultured rat hepatocytes exposed to CCl4 by monitoring the activity of phosphorylase a. Glycogen phosphorylase is converted to its a form in response to increases in cytoplasmic Ca++. Elevated phosphorylase a activity was observed within 2.5 min and was maintained for at least 30 min after exposure of hepatocytes to CCl4. Endoplasmic reticulum Ca++ pump activity decreased in a parallel manner. Phosphorylase activation was cyclic AMP independent and did not require extracellular Ca++. Cytoplasmic enzyme was released from hepatocytes within 30 min after CCl4 addition. Thus it was confirmed that exposure of hepatocytes to CCl4 causes release of Ca++ from an intracellular store (likely endoplasmic reticulum) and resultant activation of a Ca++-responsive cytosolic enzyme. From a calibration curve, it was estimated that cytosolic Ca++ is elevated up to 100 fold in rat hepatocytes exposed to the model hepatotoxin CCl4. It is postulated that prolonged elevation of intracellular Ca++ concentrations may trigger excessive stimulation of Ca++-sensitive enzymes capable of initiating irreversible liver cell injury. PMID- 3014114 TI - Benzodiazepine interactions with central thyroid-releasing hormone binding sites: characterization and physiological significance. AB - Thyrotropin-releasing hormone (TRH) and several TRH analogs were examined in the [3H]-3-Me-His2-TRH ([3H]MeTRH) receptor-binding assay in rat amygdala, striatal and cortical membranes. The benzodiazepine, chlordiazepoxide, as reported in the literature was found to displace [3H]MeTRH with an IC50 value of 3.6 X 10(-7) M in amygdala membranes. Midazolam was, however, identified as being 6-fold more active than chlordiazepoxide with an IC50 value of 6.3 X 10(-8) M. The effect of these benzodiazepines on [3H]MeTRH binding did not appear to be related to their anxiolytic activity because the novel pyrazoloquinoline nonsedating anxiolytic, CGS 9896 was without effect on [3H]MeTRH binding at concentrations up to 1 X 10( 5) M. Chlordiazepoxide had similar activity in cortical membranes whereas midazolam was some 5 times less active in this preparation than in amygdala. Both compounds were weak displacers of [3H]MeTRH binding in striatal membranes, being at least two orders of magnitude less potent than in amygdala. In contrast TRH and its analogs, RX 77368 and DN-1417, were approximately 2 to 8 times more active in striatum than amygdala membranes. TRH and DN-1417 were less active in cortical membranes whereas RX 77368 was some three times more active than in striatum and amygdala. In three test procedures indicative of TRH agonist activity; thyroid-stimulating hormone release, reversal of pentobarbital sleeping time in mice and elevation of cerebellar cyclic GMP levels, the benzodiazepines were found to be devoid of activity, whereas TRH and related compounds produced their expected responses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014117 TI - Limitations of Schild plots in a two-receptor system: alpha adrenoceptors of vascular smooth muscle. AB - The contractile response of vascular smooth muscle to l-norepinephrine is mediated almost exclusively by alpha-1 adrenoceptors in some blood vessels, and by a mixture of alpha-1 and postsynaptic alpha-2 receptors in others. To learn the sensitivity of the Schild plot as a test for the presence of more than one receptor type in such tissues, we constructed the plots to be expected from the dissociation constants of l-norepinephrine and prazosin for alpha-1 and alpha-2 adrenoceptors, using values determined by radioligand methods, and examining the effects of varying the proportions of the two types. The principles involved can be applied to other drugs and other two-receptor systems. Two occupation-response models were tested, one perfectly linear and the other convex toward the response axis. No receptor reserve was assumed, in accordance with experimental observations on vascular adrenoceptors. Schild plots over the range of antagonist concentrations customarily used did not deviate significantly from a straight line with a slope between 0.95 and 1.05 unless the second type amounted to more than 35% of the total receptors in the linear model, or more than 15% in the nonlinear model. Inasmuch as 95% CL of +/- 20% are often reported for experimental estimates of the slope, it may be difficult in practice to recognize from Schild plots the presence of a second receptor type unless it amounts to at least one-third of the total receptor pool. PMID- 3014116 TI - Evaluation of alpha-1 and alpha-2 adrenoceptor-mediated vasoconstriction in the in situ, autoperfused, pulmonary circulation of the anesthetized dog. AB - Alpha-1 and alpha-2 adrenoceptor-mediated vasoconstriction was studied in the in situ, autoperfused pulmonary circulation of the open-chest anesthetized dog utilizing selective alpha adrenoceptor agonists and antagonists. Animals were pretreated with propranolol (1 mg/kg i.v.) to eliminate beta adrenoceptor mediated effects in the pulmonary circulation. Blood was withdrawn from the right femoral artery and transferred, via a peristaltic pump, to the pulmonary arterial branch supplying the left diaphragmatic lobe of the lung. The flow rate of the pump was set so that the perfusion pressure in the lobe was equal to resting diastolic pulmonary artery pressure (10 +/- 1 mm Hg). Under conditions of constant left atrial pressure and pulmonary blood flow, intralobar administration of alpha adrenoceptor agonists elicited increases in perfusion pressure of the lobe, reflecting changes in pulmonary vascular resistance. Intralobar administration of the selective alpha-1 adrenoceptor agonist methoxamine and the selective alpha-2 adrenoceptor agonist B-HT 933 elicited dose-dependent increases in lobar perfusion pressure, as did the nonselective alpha adrenoceptor agonist norepinephrine. Prazosin (100 micrograms/kg i.v.), a selective alpha-1 adrenoceptor antagonist, inhibited pulmonary vasopressor responses to methoxamine and norepinephrine without altering significantly the response to B-HT 933. Rauwolscine (100 micrograms/kg i.v.), a selective alpha-2 adrenoceptor antagonist, inhibited the response to B-HT 933 and norepinephrine with little effect on methoxamine. Intralobar administration of tyramine to evoke the release of endogenous norepinephrine resulted in dose-dependent increases in lobar perfusion pressure. The response to tyramine was inhibited selectively by prazosin with little effect of rauwolscine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014118 TI - Studies that question the existence of alpha-2 adrenoceptors in tail arteries of normotensive Sprague-Dawley rats. AB - Many pharmacological studies have demonstrated two distinct types of alpha adrenoceptor in the vasculature; these receptors have been named alpha-1 and alpha-2. In the present study, using isolated perfused tail arteries from normotensive Sprague-Dawley rats, we have demonstrated two types of alpha adrenoceptor but neither of these could be classified as an alpha-2 adrenoceptor. Dose-dependent contraction of rat tail arteries was produced by the following alpha adrenoceptor agonists or agonist-antagonist combination: phenylephrine (PE alpha-1), clonidine (alpha-1 and alpha-2), clonidine in the presence of 10(-7) M prazosin (alpha-2) and BHT-920 (alpha-2). The ED50 values for PE and clonidine were four orders of magnitude lower than those for clonidine plus prazosin and BHT-920. In addition, the action of PE was faster in onset than that of BHT-920, reached a higher maximum (5-fold) and attenuated more rapidly than that of BHT 920. The specific alpha-2 adrenoceptor antagonist yohimbine, in concentrations as high as 10(-6) M, did not antagonize arterial responses to BHT-920. However, responses to BHT-920 were antagonized by the alpha-1 adrenoceptor antagonist prazosin, in concentrations as low as 10(-10) M, and by the serotonin/alpha-1 adrenoceptor antagonist ketanserin (10(-7) M). These results suggest that the two alpha-adrenoceptor types in isolated rat tail arteries are both of the alpha-1 type. We also found that whereas responses to PE were stable and reproducible between 2 and 5 hr of arterial perfusion, responses to BHT-920 increased progressively over 5 hr. The latter effect probably resulted from a gradual disappearance of the arterial endothelium. PMID- 3014119 TI - Phosphatidylinositol response to cholinergic agonists in airway smooth muscle: relationship to contraction and muscarinic receptor occupancy. AB - Hydrolysis of membrane phosphatidylinositol (PI) and polyphosphonoinositides (PPI) may be the coupling mechanism between receptor stimulation and the rise in intracellular calcium concentration that leads to smooth muscle contraction. In bovine tracheal smooth muscle, we correlated PI/PPI turnover, contraction and muscarinic receptor occupancy by carbamoylcholine (10(-9) to 10(-2) M). Inositol monophosphate formation after agonist stimulation, in the presence of lithium, provided a direct measurement of PI/PPI breakdown, and receptor occupancy was determined by [3H]quinuclidinyl benzylate binding. Carbamoylcholine caused a concentration-dependent contraction (EC50 = 7.4 X 10(-8) M), PI/PPI response (EC50 = 3.8 X 10(-5) M) and [3H]quinuclidinyl benzylate displacement (with high and low affinity binding sites have dissociation constants (Kd) of 3 X 10(-7) and 6 X 10(-4) M, respectively). This indicates the presence of spare receptors as maximal contraction is obtained when less than 20% of receptors are occupied. The concentration of carbamoylcholine inhibiting 50% of the PI/PPI response and 50% of maximal receptor occupancy (IC50) were similar for atropine (IC50 = 1 X 10(-9) and 5.3 X 10(-9) M, respectively), and for pirenzepine (IC50 = 3 X 10(-6) and 2.3 X 10(-6) M, respectively); the pA2 of the contraction was 8.3 +/- 0.12 for atropine and 7.2 +/- 0.08 for pirenzepine, indicating that M2 receptors may be largely predominant among bovine tracheal smooth muscle muscarinic receptors. Bovine tracheal smooth muscle may be a useful model to study the effects of other spasmogens, as it allows comparison of functional effects, PI breakdown and receptor occupancy in the same preparation. PMID- 3014120 TI - Effects of the cyclic GMP lowering agent LY83583 on the interaction of carbachol with forskolin in rabbit isolated cardiac preparations. AB - Cyclic GMP (cGMP) has been proposed to be involved in mediating negative inotropic responses to muscarinic agonists in the presence of cyclic AMP (cAMP) generating agents in the heart. In order to investigate this hypothesis, the effects of the novel cGMP lowering agent, LY83583, on carbachol-induced increases in cGMP levels and decreases in tension were measured in rabbit isolated left atria and right ventricular papillary muscles, in the presence and absence of the adenylate cyclase activator, forskolin. In vehicle-treated preparations, negative inotropic responses to 3 microM carbachol in the presence of 3 microM forskolin were accompanied by significant increases in cGMP levels. Carbachol had no significant effect on forskolin-induced increases in cAMP levels. LY83583 (10 microM) reduced basal tension and basal cGMP levels, and completely abolished carbachol-induced increases in cGMP both in left atria and in papillary muscles. The LY83583 significantly reduced the magnitude of the negative inotropic responses of papillary muscles to carbachol in the presence of forskolin, but had no effect on these responses in left atria. Although a causal relationship has not been established, these data suggest that cGMP may be involved in negative inotropic responses to muscarinic stimulation in the presence of cAMP-generating agonists in ventricular muscle, but not in atria. PMID- 3014121 TI - Renal Na,K-adenosine triphosphatase transport rate limits transcellular NaCl reabsorption in distal nephrons of volume-expanded dogs. AB - To examine whether the adenosine triphosphatase (Na,K-ATPase) transport rate regulates transcellular NaCl reabsorption, experiments were performed on anesthetized volume-expanded dogs. Ouabain was injected into the renal artery in doses inhibiting 10 to 80% of the renal Na,K-ATPase activity. Acetazolamide was administered before ouabain to render the NaHCO3 reabsorption and associated NaCl reabsorption constant during variations in the glomerular filtration rate. Ouabain reduced sodium reabsorption significantly after inhibiting 20% of the Na,K-ATPase. By inhibiting 80% of the Na,K-ATPase, NaCl reabsorption was reduced by 40 to 50% without affecting NaHCO3 reabsorption. During mechanical constriction of the suprarenal aorta, the remaining NaCl reabsorption was constant until the glomerular filtration rate was lowered by about 50%. Bound ouabain and the remaining Na,K-ATPase activity were distributed between the cortex and medulla in proportion to the Na,K-ATPase activity before ouabain injection. The reduction in NaCl reabsorption and ouabain binding were correlated (r = 0.90), the slope suggesting a turnover for ATP similar to the in vitro turnover of 5700 ATP min-1 estimated from the relationship between the remaining Na,K-ATPase activity and bound ouabain (r = 0.95). We conclude that transcellular reabsorption of NaCl in the distal nephron reaches a maximum in volume-expanded dogs by saturating the sodium sites of Na,K-ATPase because even a small dose of ouabain inhibits NaCl reabsorption and because the calculated turnover for Na,K ATPase activity is similar to in vitro maximum estimates. The Na,K-ATPase transport rate, therefore, limits transcellular NaCl reabsorption in volume expanded dogs. PMID- 3014122 TI - Effects of epinephrine, isoproterenol and IPS-339 on sympathetic transmission to the dog heart: evidence against the facilitatory role of presynaptic beta adrenoceptors. AB - The autoregulation of norepinephrine (NE) release mediated by presynaptic alpha and beta adrenoceptors on sympathetic nerve terminals in the heart of pentobarbital-anesthetized dog was studied. NE overflow elicited by left cardiac sympathetic nerve stimulation was determined from the coronary sinus blood, by using high-performance liquid chromatography with electrochemical detection. Intracoronary infusion of epinephrine (1,3 and 10 micrograms/min) into the left circumflex artery increased basal left ventricular dp/dt maximum (LV dp/dt max) and coronary sinus blood flow. The epinephrine infusion decreased coronary sinus output of NE (NE output) during left cardiac sympathetic nerve stimulation. Intracoronary infusion of isoproterenol (0.03, 0.1 and 0.3 microgram/min) increased the basal LV dp/dt max and coronary sinus blood flow, whereas the stimulation-induced increases in NE output, LV dp/dt max and coronary sinus blood flow were not altered by its infusions. Intravenous injection of IPS-339 (0.03, 0.1 and 0.3 mg/kg), a selective beta-2 adrenoceptor antagonist, diminished the stimulation-induced increases in LV dp/dt max and coronary sinus blood flow in a dose-dependent manner, whereas it did not decrease the stimulation-induced increase in NE output. Intracoronary infusion of yohimbine (10, 30 and 100 micrograms/min), a preferentially selective alpha-2 adrenoceptor antagonist, facilitated the stimulation-induced increases in NE output, LV dp/dt max and coronary sinus blood flow. There was no significant difference in the facilitation of the stimulation-induced increases in NE output, LV dp/dt max and coronary sinus blood flow between intracoronary infusion of both isoproterenol (0.1 microgram/min) and yohimbine (100 micrograms/min) and the infusion of yohimbine alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014123 TI - Quantitative analysis of the selectivity of radioligands for subtypes of beta adrenergic receptors. AB - In tissues with two classes of binding sites for a drug, it is common to estimate the proportion of each class of binding site by inhibiting the binding of a radioligand with a selective unlabeled ligand. Accurate estimates of the density of each class of binding site, however, will be obtained only if the radioligand is nonselective or used at a concentration that saturates both classes of binding sites. A method of simultaneous regression analysis of multiple inhibition curves, using the program MLAB on the PROPHET system, was used to quantify the selectivity of radioligands for beta-1 or beta-2 adrenergic receptors. The selectivity of [125I]iodopindolol, [125I]iodocyanopindolol, [125I]iodohydroxybenzylpindolol and [3H]dihydroalprenolol for beta-1 and beta-2 adrenergic receptors was assessed by inhibiting the binding of each radioligand with the beta-1-selective unlabeled ligand ICI 89,406 at increasing concentrations of the radioligand, using membranes prepared from C6 glioma cells, which have both beta-1 and beta-2 adrenergic receptors. Scatchard plots for all four radioligands were linear, with correlation coefficients greater than 0.95. [125I]Iodopindolol and [125I]iodocyanopindolol were 3.2- and 2-fold selective, respectively, and [125I]iodohydroxybenzylpindolol and [3H]dihydroalprenolol were 5.8- and 2.3-fold selective, respectively, for beta-2 adrenergic receptors. Values obtained for the densities of beta-1 and beta-2 adrenergic receptors and the affinities of the receptors for ICI 89,406 were independent of the radioligand used.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014124 TI - Role of receptor reserve in the inhibition of alpha-1 adrenoceptor-mediated pressor responses by calcium antagonists in the pithed rat. AB - The effect of the calcium channel antagonists nifedipine and FR 34235 on the vasopressor response to alpha-1 adrenoceptor stimulation in the pithed normotensive rat was investigated. The maximal pressor response elicited by the full alpha-1 adrenoceptor agonist SK&F l-89748 was slightly but significantly reduced by 1-mg/kg doses of nifedipine (21 +/- 2%) and FR 34235 (34 +/- 4%). In comparison, the maximal pressor response to alpha-1 adrenoceptor stimulation by the partial alpha-1 agonist SK&F 88444 was markedly inhibited by nifedipine (51 +/- 1%) and FR 34235 (65 +/- 3%). Partial inactivation of the postsynaptic alpha 1 adrenoceptors with phenoxybenzamine (0.1 mg/kg) resulted in a maximal increase in diastolic pressure to alpha-1 adrenoceptor activation by SK&F l-89748 less than that induced by SK&F 88444. After phenoxybenzamine treatment, nifedipine and FR 34235 produced even greater reductions in the maximal vasopressor response to alpha-1 adrenoceptor stimulation by SK&F l-89748 (77 +/- 8 and 85 +/- 1%, respectively). Moreover, an inverse linear correlation (r = 1.00) was observed between the sensitivity of the maximal vasopressor response to nifedipine and FR 34235 and the magnitude of the maximal pressor response. The data suggest that the sensitivity of the alpha-1 adrenoceptor-mediated pressor response to inhibition by calcium antagonists in the pithed rat is inversely related to the magnitude of the pressor response, and they are consistent with the notion that the presence of "spare" alpha-1 adrenoceptors may determine the sensitivity of the pressor response to calcium antagonists. PMID- 3014125 TI - Presence of a noradrenaline uptake system on a ligated cat sympathetic nerve. AB - [3H]noradrenaline [( 3H]NA) uptake studies were carried out in cat hypogastric nerves ligated in vivo, 2 cm distal to the inferior mesenteric ganglion, for different time periods. Atria from the same animals served as controls to determine the uptake of the amine by the U1 uptake system present in noradrenergic nerve terminals. The net uptake of [3H]NA by hypogastric nerves increased with time of ligation, reaching a maximum 24 h after ligation in the segment of nerve immediately proximal to the ligature (P1 segment, neurosome). No further increase in uptake was observed at 48 or 72 h. Segments distal (D1) to the ligature also retained significant amounts of [3H]NA. In both cases the uptake was blocked by cocaine (3 microM). Reserpine pre-treatment (2 mg/kg I.M.) markedly decreased the endogenous NA content to 1-2% of untreated cats and the net uptake of [3H]NA was lowered to 25% both in cat hypogastric nerve and atria. The uptake was further decreased in the presence of cocaine (3 microM). 6 Hydroxydopamine (100 microM) did not modify the [3H]NA uptake by ligated cat hypogastric nerves but almost abolished the [3H]NA net uptake by right atria from the same animals. After collagenase pre-treatment (0.05% for 15 min) the net uptake of [3H]NA was not altered in the atrium. However, collagenase-pre-treated ligated nerves, took up almost twice as much [3H]NA; under these conditions, 6 hydroxydopamine produced a marked decrease in [3H]NA net uptake. These data suggest the presence in the perineurium of a diffusion barrier for very polar substances, including 6-hydroxydopamine. In conclusion, our data demonstrate that the cat hypogastric nerve ligated in vivo has a cocaine-sensitive system for NA uptake which resembles the NA uptake mechanism (U1) present in noradrenergic nerve terminals. Our data further support the view that the ligated cat hypogastric nerve (neurosome) could be considered as a model of noradrenergic nerve terminal free of effector cell. PMID- 3014126 TI - Botulinum toxin prevents stimulus-induced backfiring produced by neostigmine in the mouse phrenic nerve-diaphragm. AB - The origin of motor nerve antidromic activity (backfiring) induced by anticholinesterase treatment was examined in the mouse phrenic nerve hemidiaphragm preparation. Botulinum toxin was used to determine whether backfiring is due to (a) a direct effect of the cholinesterase inhibitor on the nerve terminal, or (b) an indirect effect via the prolongation of the action of acetylcholine. In previously untreated control preparations, neostigmine produced spontaneous and stimulus-induced antidromic activity in the phrenic nerve when rapidly introduced into the diaphragm via its vasculature. This activity could be reversibly blocked by d-tubocurarine and decamethonium, but not by atropine. Neostigmine-induced backfiring did not occur in preparations in which transmitter release was blocked with botulinum toxin. Infusion of a small bolus of a high concentration of acetylcholine following neostigmine treatment resulted in a short-term increase in the incidence of antidromic activity, followed by block, in both controls and botulinum toxin-treated preparations. It is concluded that transmitter release is necessary for the production of backfiring following cholinesterase inhibition since neostigmine alone does not elicit antidromic activity in botulinum toxin-treated preparations at concentrations which are effective in controls. Our results support the hypothesis that the effects of neostigmine on the motoneurone terminal are mediated by the prolonged action of acetylcholine that occurs with inhibition of acetylcholinesterase. PMID- 3014127 TI - Effect of trifluoperazine on rabbit cortical collecting tubular response to vasopressin. AB - Anuran membrane studies suggest that the calcium-binding protein calmodulin is necessary for arginine vasopressin (AVP) to exert a hydro-osmotic effect. We therefore examined the effect of trifluoperazine and N-(6-aminohexyl)-5-chloro-1 naphtholene sulphonamide (W-7), chemically dissimilar calmodulin inhibitors, on hydraulic conductivity (Lp) response to AVP in rabbit cortical collecting tubules perfused in vitro. Trifluoperazine but not W-7 increased basal Lp in rabbit cortical collecting tubules. When cortical collecting tubules were pre-treated with either trifluoperazine or W-7, the effect of AVP to increase Lp was significantly inhibited. To determine the site of this inhibition, Lp responses to exogenous cyclic adenosine 3',5'-phosphate (AMP) were studied. Both trifluoperazine and W-7 pretreatment significantly inhibited the effect of a cyclic AMP analogue to increase rabbit cortical collecting tubule Lp. These results suggest that calmodulin may be an important mediator of the hydro-osmotic response to AVP in the mammalian cortical collecting tube by acting at a site or sites distal to cyclic AMP formation. PMID- 3014128 TI - Collagen tube containers: an effective means of controlling particulate hydroxyapatite implants. AB - HA has been used successfully as a hard tissue graft in alveolar ridge augmentation; however, its particulate nature limits its application to select cases. A study in rats was done to examine the feasibility of a combined system of HA encased in collagen film as a hard tissue graft. Gross, radiographic, and histologic examination showed that collagen film helped to shape and contain the HA particulate during healing for as long as 4 weeks. The collagen did not interfere with the normal tissue response around the HA particulate. PMID- 3014129 TI - Gain control in the electrosensory system: a role for the descending projections to the electrosensory lateral line lobe. AB - The responses of E-cells, basilar pyramidal cells, of the electrosensory lateral line lobe (ELLL) were studied in normal animals (Apteronotus leptorhynchus) and in fish in which a component of the descending input from the midbrain n. praeeminentialis to the ELLL was interrupted by lesions or by application of local anesthetics. This treatment increased the responsiveness of these neurons by 100 to 300%. A method is described by which the animal's electric organ discharge (EOD) can be increased or decreased in amplitude. Responses of E-cells to a brief stationary electrosensory stimulus and to moving electrolocation targets were studied in normal and in lesioned animals with normal and altered EOD amplitudes. Large reductions in EOD amplitude, approximately 50%, result in no significant changes in the average size of E-cells' responses to either type of electrosensory stimulus in normal animals. Interruption of the descending input, however, results in a loss of the E-cells' ability to maintain constant response size when the EOD amplitude is reduced. Increases in EOD amplitude cause reductions in the size of E-cell responses to the moving electrolocation targets and to the stationary stimulus. The effects of increased EOD amplitude are present in normal animals and in animals in which the descending input is interrupted. The descending input to the ELLL seems to function as a gain control mechanism that is capable of compensating for losses in stimulus strength resulting from reduced EOD amplitude. The component of the descending input studied here does not seem to play a role in the response of the system to increases in EOD amplitude. These results are discussed in conjunction with the known details of the ELLL circuitry and its connections with other brain areas. PMID- 3014130 TI - Inhibitory effect of irradiation of DNA with near ultraviolet light in the presence of 8-methoxypsoralen on cleavage by restriction endonucleases. PMID- 3014131 TI - Inactivation of rabbit mammary prolactin receptor by N-acetylimidazole. AB - Treatment of rabbit mammary prolactin receptor with N-acetylimidazole resulted in loss of prolactin binding activity. Loss of activity of the particulate receptor was time and concentration dependent with 100 mM reagent causing total inactivation in 10 min. Similar results were obtained with solubilized receptor preparations, but at lower reagent concentrations. The loss of binding activity was due to loss in the number of binding sites. Incubation of the reagent inactivated membranes or soluble receptor with hydroxylamine for 3 hr resulted in 80-90% reactivation of the prolactin binding activity. These results indicate the possible involvement of tyrosyl residue(s) on the receptor in the prolactin receptor interaction. PMID- 3014132 TI - Bacterial toxins and the role of ADP-ribosylation. AB - Studies on the pathogenesis of "Whooping Cough" and cholera have resulted in the discovery of important pathways in the regulation of cellular metabolism leading to the realization of a complex family of proteins that appear to play central roles in the regulation of hormonal responses and which utilize guanine nucleotides in their mechanism of action. The fact that these bacterial toxins interfere so precisely with the complex regulation of eukaryotic cellular metabolism and the discovery of analogous enzymes within the cytosol of eukaryotic cells suggests that ADP-ribosylation may be an important pathway through which the cell can establish its responsiveness to its environment. Clearly, future work directed towards the role of ADP-ribosylation and towards the mechanisms of the regulation of these endogenous ADP-ribosyltransferases and lyases may provide great insights into the mechanisms of hormone action. PMID- 3014134 TI - Current understanding of the natural history of genital herpes simplex infections. AB - Considerable variations in the disease make an accurate portrayal of genital herpes simplex virus (HSV) infections quite difficult. Primary genital HSV infections classically produce ulcerated lesions lasting a mean of 16-21 days in men and 10-16 days in women. HSV shedding has been documented for a mean of 9.1 11.6 days in men and 8.0-14.7 days in women. Probably many adults have a less severe initial genital infection, however, and it may be so mild as to be misdiagnosed or undetected. Studies of recurrent genital HSV infections are often misleading because they overestimate the frequency and severity of recurrences. By necessity, these studies have recruited and evaluated those adults with the worst and most frequent recurrences. Data from these studies show even greater variations than do data on initial episodes of genital HSV infection. The frequency and clinical importance of asymptomatic genital HSV shedding are being recognized and documented only now. Further studies of this phenomenon and of the natural history of all genital HSV infections are needed urgently. PMID- 3014133 TI - Metabolism of radiolabelled energy-yielding substrates by rat Sertoli cells. AB - The rates of metabolism in vitro of 3H- or 14C-labelled glucose, pyruvate, glutamine and leucine by Sertoli cells from immature rats were estimated. The overall rate of glucose utilization exceeded by far the rates of oxidation of pyruvate (derived from glucose) via the citric acid cycle and glucose metabolism via the oxidative branch of the pentose phosphate pathway. This pattern of glucose metabolism was not markedly altered after stimulation of glucose metabolism by FSH. The rate of oxidation of exogenous pyruvate indicated that the energy yield from glucose metabolism by Sertoli cells could be dependent on the extracellular concentrations of pyruvate and lactate. There is no evidence that a high rate of aerobic glycolysis is of vital importance for Sertoli cells. In medium containing glucose and all amino acids, 14C-labelled glutamine and leucine were converted to 14CO2 at considerable rates. It was calculated that the oxidation of glutamine and leucine in addition to glucose and fatty acids can yield much of the required energy of Sertoli cells. PMID- 3014135 TI - Clinical features of herpes genitalis. AB - In a recent survey reported on by the Centers for Disease Control, herpes genitalis was the presenting complaint behind more than 35,000 visits to two clinics in major cities. Herpes genitalis is probably more prevalent today than any other sexually transmitted disease. Type 2 virus accounts for most infections seen; however, type 1 infections are reported more and more often as time passes. Also, there is now evidence suggesting that subclinical infection occurs in women with no antibodies to herpes simplex virus type 1. Meningitis and encephalitis are infrequent sequelae to primary infection, and when encephalitis occurs, the mortality has been reported to be as high as 70% of cases. Mortality in disseminated herpes simplex infection during pregnancy can reach 50% for mother and fetus. In general, signs and symptoms of recurrent infection are milder than those of primary infection. PMID- 3014136 TI - Diagnostic techniques for evaluating herpes simplex virus infections. Laboratory considerations. AB - Recent advances in the management and treatment of genital herpes simplex virus (HSV) infections underscore the importance of timely and accurate diagnosis of the disease. Moreover, increased physician awareness of the importance of accurately diagnosing herpes has led to a better understanding of its natural history. Like all diagnostic medical devices, microbiologic tests used to diagnose HSV infections are subject to governmental regulations. Based on the current status of available testing and our knowledge of the nature of HSV, clearly there is still much to be learned about HSV infections. The answers to some key questions will provide advances in diagnostic expertise and, ultimately, in the effective treatment and prevention of HSV-associated illness. PMID- 3014137 TI - Proper methods of culturing herpes simplex virus. AB - Viral isolation is the most sensitive and specific technique for establishing the diagnosis of herpes simplex virus (HSV) infection. The most important factor influencing the ability to isolate HSV in the laboratory is the method of specimen collection. Selection of appropriate specimens, culturing of vesicle fluid or ulcers early in the course of the infection, avoidance of calcium alginate swabs, use of appropriate transport media and storage at 4 degrees C (not freezing) until inoculation are techniques that enhance HSV recovery. Over 50% of HSV isolates are recovered within one day, 80% by two days and approximately 90% by three days. Laboratory confirmation of HSV infection is critical in many clinical settings, and isolation of the virus in tissue culture remains the gold standard for laboratory diagnosis. PMID- 3014138 TI - Characterization of the tumor-associated 38-kd protein of herpes simplex virus type 2. AB - The BglII N DNA fragment of herpes simplex virus type 2 (HSV 2), which is capable of oncogenically transforming cells in vitro, encodes a 37,800-dalton (38-kd) protein that has been seroepidemiologically associated with uterine cervical carcinoma. Polyclonal monospecific antiserum was produced against electrophoretically purified 38 kd from HSV 2-infected cells and used to identify antigenic and biochemical characteristics of the protein as well as to probe transformed cells for the expression of viral 38 kd. The HSV 2 type specificity of the 38-kd protein, previously shown using anti-HSV 2 serum and monoclonal antibodies, was confirmed using anti-38-kd serum. The 38-kd protein of HSV 2 produced in vivo and in vitro displayed type specificity and showed no evidence of posttranslational processing. The 38-kd protein has a relative isoelectric point of 9.1, is synthesized at a maximum level four hours after infection and appears to be a component of the virion. When 35S-methionine radiolabeled 38 kd was immunoprecipitated by anti-38-kd serum, high-molecular-weight proteins (118 140 kd) were also present. However, if prior to reacting with the anti-38-kd serum the high-molecular-weight proteins were separated from 38 kd with sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the only reaction observed with immunoblot was with 38 kd. Therefore, the observed coprecipitation appears to result from the formation of a complex between the proteins and is not the result of shared antigenic determinants. Cells transformed by inactivated HSV 2 were examined for the expression of the 38-kd protein using immunoenzymatic staining. The viral 38-kd protein was not consistently found, but since the protein is reported to be a component of the viral enzyme complex ribonucleotide reductase, it cannot be excluded from possible HSV 2 transformation. PMID- 3014139 TI - Genital herpes simplex virus infections complicating pregnancy. Natural history and peripartum management. AB - The current practice of obtaining weekly herpes simplex virus cultures from 34 weeks of gestation until the onset of labor is costly, traumatic, anxiety producing to patient and physician and directed at the wrong population; also, it increases the rate of cesarean section but has not arrested the rising rate of neonatal herpes. Instead, the entire obstetric population should receive a careful history, speculum examination of the cervix, tactile examination of the labia and herpes simplex virus cultures of the cervix and labia at the onset of labor. PMID- 3014140 TI - Antibodies to type IV collagen in rheumatic diseases. AB - Rheumatic disease sera were examined by a sensitive and specific enzyme linked immunosorbent assay (ELISA) for antibodies to native and to denatured type IV collagen from basement membranes of bovine anterior lens capsules or human placenta. The frequency of antitype IV collagen antibodies depended on the antigen and the disease studied. No controls had human type IV collagen antibodies and only 5.6% had antibody to bovine type IV collagen. Antibody to one or more of our 4 antigens was seen in 20% of children with juvenile rheumatoid arthritis (JRA), 35% of patients with mixed connective tissue disease (MCTD), 40% of those with juvenile dermatomyositis (DM), 52% of adults with rheumatoid arthritis (RA), 56% of patients with scleroderma and 60% of patients with systemic lupus erythematosus (SLE). Antibodies to native human type IV collagen were rare (0-10%) except in SLE (45%). Antibodies to denatured human type IV collagen were commoner in RA, scleroderma and SLE. Antibodies to native bovine type IV collagen occurred in 8-20% of patient sera, and to denatured antigen in 25-26% of scleroderma, MCTD and DM patients. Antibovine type IV collagen activity measured by ELISA could be absorbed from positive sera by preincubation of the sera with bovine type IV collagen but not bovine type I collagen or native human placental type IV collagen, indicating that the antibodies were specific for bovine type IV collagen. PMID- 3014141 TI - Intraarticular T lymphocytes in monoarticular and oligoarticular inflammatory joint diseases. Normal subset distribution and less numbers of activated T cells indicate major differences as compared to rheumatoid arthritis. AB - T lymphocyte subpopulations and the expression of T cell activation antigens were determined in peripheral blood, synovial fluid and/or synovial tissues of patients with recurring monoarticular arthritis and patients with HLA-B27 associated oligoarthritis in comparison to patients with rheumatoid arthritis (RA). In individuals with monoarthritis or oligoarthritis, there was a normal T cell subset distribution, both in peripheral blood and in intraarticular sites, with only a small number of T cells bearing Ia antigens. This was in marked contrast to the patient group with RA that demonstrated a significantly decreased ratio of T helper/inducer to T suppressor/cytotoxic cells in addition to large numbers of Ia+ T cells in intraarticular sites. The expression of the Tac antigen was similar in all disease groups. PMID- 3014142 TI - Effect of auranofin (SK&F 39162) on water and electrolyte flux in canine small bowel: a possible diarrheogenic mechanism. AB - Auranofin (AF) a new antiarthritic gold compound effective when administered orally, frequently causes diarrhea with abnormal stool electrolyte content. Studies were designed to determine the mechanism of the diarrhea caused by AF. In perfused canine Thiry-Vella loops, AF caused significant elevations in effluent volume, osmolarity, and sodium concentration and a significant decrease in potassium concentration. In mucosal homogenates of rat small bowel, AF inhibited sodium, potassium ATPase in a concentration dependent manner. AF did not alter canine colonic smooth muscle activity in vitro. We suggest that AF induced diarrhea results from interruption of normal water and electrolyte absorption by inhibition of enterocyte sodium, potassium ATPase activity. PMID- 3014143 TI - Calcium pyrophosphate dihydrate (CPPD) crystal deposition in the trochanteric bursa of a patient with hip osteoarthritis. PMID- 3014144 TI - Bee venom and arthritis. PMID- 3014145 TI - Transsphenoidal surgery for Cushing's disease. AB - A series of 101 patients with Cushing's disease underwent transsphenoidal surgery. Diagnosis was fundamentally based on dynamic testing, mainly on the dosage-dependent suppression of cortisol after dexamethasone. The effect of surgery was monitored by intraoperative ACTH measurements. In 96 out of 101 patients a microadenoma of the pituitary was identified and removed selectively. In 74% of patients there was a clinical and endocrinological remission of Cushing's disease. Four 'operative failures' after selective adenomectomy underwent hypophysectomy in a second operation and each remitted. Thus the overall remission rate was 77%. In general, bilateral adrenalectomy was performed in patients who had failed to remit after selective adenomectomy. Although there is a considerable mortality and morbidity in patients with Cushing's syndrome, complications attributed to surgery were low. Two patients died postoperatively. In general, an improvement of disturbed pituitary function was noted after selective adenomectomy. PMID- 3014146 TI - Plasma constituents in Nguni cows over forty-eight hours. AB - Plasma constituents were investigated at ninety minute intervals over a forty eight hour period on four unrestrained Nguni cows (in small cubicles) in order to study the presence or absence of rhythms. No circadian, ultradian or diurnal rhythm was found for any of the parameters investigated. Basal values over a forty-eight hour period were obtained for certain plasma constituents. These values are of use for studies relating to the effects of stress in cattle. PMID- 3014147 TI - DNA polymorphic haplotypes on the short arm of chromosome 11 and the inheritance of type I diabetes mellitus. AB - The linked polymorphic loci 5' to the insulin gene and 3' to the c-Harvey-ras-1 (c-Ha-ras) gene, both localised to the short arm of chromosome 11, have been studied in 14 type I diabetic pedigrees. The use of a cloned gene probe corresponding to the polymorphic locus adjacent to the insulin gene, in combination with the restriction endonuclease PvuII, has permitted an improvement in the resolution of sizes of insert at this locus. An MspI restriction fragment length polymorphism at the c-Ha-ras proto-oncogene locus (4 cM upstream from the insulin gene) was used to identify parental insulin gene related alleles unambiguously, and subsequently a pedigree analysis was performed to determine whether subclasses of inserts at this locus track with insulin dependent diabetes. Segregation analysis demonstrated no linkage between the polymorphic loci 5' to the insulin gene, nor 3' to the c-Ha-ras, and type I diabetes. However, a similar analysis confirmed an association between the HLA locus chromosome 6 and insulin dependent diabetes. PMID- 3014149 TI - Type II syndactyly or synpolydactyly. AB - A new family with syndactyly type II or synpolydactyly is described with 16 affected members in six generations. No other major skeletal or extraskeletal malformations were present, but the association with minor local anomalies may be a common feature. Various metacarpal or metatarsal abnormalities may be part of this type of syndactyly. The family pedigree confirms the autosomal dominant mode of inheritance with incomplete penetrance and the frequent occurrence of non manifesting heterozygotes resulting in 'skipped generations'. PMID- 3014148 TI - Exclusion of close linkage between the parathyroid hormone gene and a mutant gene locus causing idiopathic hypoparathyroidism. AB - A family is presented in which the mother has transmitted primary hypoparathyroidism with early onset and serum PTH (44-68) and C terminal deficiency to her two sons. Restriction enzyme analysis of allelic variation at the PTH gene locus revealed that the disease and the PTH alleles segregate independently. It is therefore concluded that the primary molecular defect leading to this form of hypoparathyroidism is not located within the PTH gene itself. PMID- 3014150 TI - Coxsackie B virus-specific IgM antibody and myocardial infarction. AB - The ELISA technique was shown to be group-specific for the detection of IgM antibodies against coxsackie B viruses, and probably against a wider range of enteroviruses. No evidence was obtained that recent coxsackie B-virus infection predisposes to myocardial infarction. PMID- 3014151 TI - Quantitative estimation of islet cell surface antibodies in sera of patients with diabetes mellitus, using BK virus-induced insulinoma, rat islet or fish islet cells and 125I-antihuman IgG antibody. AB - Using a radioligand assay with 125I-antihuman IgG antibody as well as an 125I protein A binding assay, we quantitatively evaluated islet cell surface antibodies (ICSA) in the sera of diabetic patients already proven to contain antibodies by the immunofluorescence method (IF). It was possible to use not only rat islet but also BK virus-induced hamster insulinoma cells and yellow tail fish (Serolia quinqueradiata) islet cells as target cells in these radioassays. 7 out of 8 diabetic subjects, positive for ICSA by IF, showed levels of binding of 125I antihuman IgG antibody greater than 2SD above the mean for normal controls. Additionally 3 diabetic subjects negative for ICSA by IF showed levels of binding of 125I-antihuman IgG antibody less than 2SD above the mean for normal controls, and 2 subjects negative for ICSA by IF had levels slightly higher than 2SD above the mean for normal controls. The quantitative ICSA radioligand assay reported here is both sensitive and specific in the detection of the IgG of ICSA. It is also more convenient to use than other methods, and may use any of several different kinds of target cells, each of which may be easily and reproducibly obtained. These radioimmunological methods for the detection of ICSA appear to be ideal for screening large numbers of patients for ICSA. PMID- 3014152 TI - Raised serum levels of IgM-rheumatoid factor and anti-F(ab')2 autoantibodies in patients with active inflammatory bowel disease. AB - The serum levels of IgM-Rheumatoid Factor and of anti-F(ab')2 autoantibodies were investigated in patients with inflammatory Bowel Disease (IBD) by sensitive radioimmunoassays. Serum levels of the 2 autoantibodies were significantly increased in active IBD. In patients with Crohn's Disease raised titers of the 2 antibodies appeared to be also related to colonic involvement. There was, in Crohn's Disease, a significant association between concordantly positive results with the 2 assays and the occurrence of systemic complications. Immunocomplexes detected by the C1q-SP method were higher in sera of Crohn's Disease patients with raised IgM-RF than in the others. Data from the present investigation indicate that in active IBD and particularly in Crohn's Disease autoantibodies directed against different parts of the immunoglobulin molecule may be produced. These findings add support to the concept that an in vivo polyclonal B-cell activation may occur in these patients. PMID- 3014154 TI - Conserved sequences and cell-specific DNase I hypersensitive sites upstream from the co-ordinately expressed alpha I- and alpha II-globin genes of Xenopus laevis. AB - The globin gene family of Xenopus laevis comprises pairs of closely related genes that are arranged in two clusters, each pair of genes being co-ordinately and stage-specifically expressed. To get information on putative regulatory elements, we compared the DNA sequences and the chromatin conformation 5' to the co ordinately expressed adult alpha-globin genes. Sequence analysis revealed a relatively conserved region from the cap site up to position -289, and further upstream seven distinct boxes of homology, separated by more diverged sequences or deletions/insertions. The homology boxes comprise 22 to 194 base-pairs showing 78 to 95% homology. Analysis of chromatin conformation showed that DNase I preferentially cuts the upstream region of both genes at similar positions, 5' to the T-A-T-A and the C-C-A-A-T boxes, only in chromatin of adult erythroblasts and erythrocytes, where adult globin genes are expressed, but not in chromatin of adult liver cells or larval erythrocytes, where these genes are silent. This suggests that cell- and stage-specific activation of these genes coincides with specific changes in chromatin conformation within the proximal upstream region. No difference was found in the nucleotide sequence within the DNase I hypersensitive region proximal to the adult alpha 1-globin gene in DNA from embryonic cells, in which this gene is inactive, and adult erythrocytes, expressing this gene. PMID- 3014155 TI - Characterization of a defective phage system for the analysis of bacteriophage T4 DNA replication origins. AB - We have developed a defective phage system for the isolation and analysis of phage T4 replication origins based on the T4-mediated transduction of plasmid pBR322. During the initial infection of a plasmid-containing cell, recombinant plasmids with T4 DNA inserts are converted into fully modified linear DNA concatamers that are packaged into T4 phage particles, to create defective phage (transducing particles). In order to select T4 replication origins from genomic libraries of T4 sequences cloned into the plasmid pBR322, we searched for recombinant plasmids that transduce with an unusually high efficiency, reasoning that this should select for T4 sequences that function as origins on plasmid DNA after phage infection. We also selected for defective phage that can propagate efficiently with the aid of a coinfecting helper phage during subsequent rounds of phage infection, which should select for T4 sequences that can function as origins on the linear DNA present in the defective phage. Several T4 inserts were isolated repeatedly in one or both of these selective procedures, and these were mapped to particular locations on the T4 genome. When plasmids were selected in this way from genomic libraries constructed using different restriction nucleases, they contained overlapping segments of the T4 genome, indicating that the same T4 sequences were selected. The inserts in two of the selected plasmids permit a very high frequency of transduction from circular plasmids; these have been shown to contain a special type of T4 replication origin. PMID- 3014153 TI - Biosynthesis and metabolic degradation of receptors for epidermal growth factor. PMID- 3014156 TI - Functional and binding evidence of taurine inhibition of alpha-adrenoceptor effects on guinea-pig ventricle. AB - The effect of taurine on alpha- and beta-mediated positive inotropic effects was investigated in guinea-pig ventricular strips. Loading with taurine dose dependently antagonized the phenylephrine-induced positive inotropic effect while it was ineffective on beta-mediated positive inotropic effect. The superfusion with a taurine-free medium determined a loss of intracellular taurine, while the loading with 20 mM taurine prevented this depletion. Preincubation of cardiac membranes with different concentrations of taurine (1 to 20 mM) decreased 3H prazosin binding, and the saturation curve obtained after preincubation of cardiac membranes with 20 mM taurine showed that taurine had a more marked effect on the number of binding sites. In both experimental models, beta-alanine did not mimic any taurine effects, suggesting that taurine actions might be specific. PMID- 3014157 TI - Regulation of beta-adrenergic receptor density in the non-innervated and denervated embryonic chick heart. AB - Factors which regulate beta-adrenergic receptor density in non-innervated and denervated embryonic tissues have not been fully elucidated. In the present study, the effects of exposure to isoproterenol and propranolol on beta adrenergic receptor density in the non-innervated (preneural) and partially innervated (neural) embryonic chick heart are examined. In addition, the effects of chemical and surgical sympathectomy on cardiac beta-adrenergic receptor density in the chick embryo are investigated. (125I)-pindolol was used as a receptor probe. The density of beta-adrenergic receptors in the embryonic chick heart peaks on incubation day 9 (664 fmols/mg protein) and then decreases by more than 80% by incubation day 19. Administration of propranolol or isoproterenol on incubation days 4 to 6 results in no change or a decrease (to 62% of control), respectively, in receptor density on incubation day 7. Administration of propranolol on incubation days 10 to 19 causes an increase (to 230% of control) in beta-adrenergic receptor density on day 20. Administration of isoproterenol on incubation days 10 to 16 results in a decrease (to 26% of control) in receptor density on day 17. Neither chemical sympathectomy, produced by administration of 6-hydroxydopamine, nor surgical sympathectomy, produced by removal of premigratory neural crest cells, significantly alters cardiac beta-adrenergic receptor density from control values. Administration of adrenergic drugs produces a greater change in beta-adrenergic receptor density after sympathetic innervation (day 10) than in preneural hearts. This indicates that sympathetic nerves influence the properties of beta-adrenergic receptors during embryonic development. PMID- 3014158 TI - Binding sites for glycosaminoglycans on developing sympathetic neurones. AB - Recent studies have suggested that cell to cell communication in the immune system is mediated by cell surface receptors for glycosaminoglycans (GAGs) [Parish et al, 1984]. The intention of this study was to see whether similar recognition molecules for GAGs are present on sympathetic neurones. A mechanical dissociation technique was used to isolate neurones from superior cervical ganglia (SCG) of rats aged between gestational day 19 and postnatal day 21. Receptors for GAGs on sympathetic neurones were detected by the ability of neurones to form rosettes with sheep red blood cells coupled with one of 12 different GAGs. It was found that SCG cells bind to all the GAGs tested. In addition, a range of developmental binding patterns for the various GAGs was found. PMID- 3014159 TI - Chick embryo myotubes contain transferrin receptors and internalize and recycle transferrin. AB - Embryonic chick skeletal myotubes grown in cell culture require transferrin to provide iron for proliferation and differentiation. We demonstrate here that cultured myotubes contain transferrin receptors as demonstrated by the finding of specific, saturable, and reversible high-affinity binding sites. Scatchard analysis of equilibrium binding data indicates an apparent Kd of 37 nM and one muscle cell equivalent contains 7,500 transferrin receptors. Myotubes exhibit a Kd 100 times higher for apotransferrin than for iron-saturated transferrin. Internalization of specifically bound transferrin is temperature dependent and occurs rapidly at 37 degrees C with a steady state reached after 10 min. Internalization studies using either 125I-ovotransferrin or 55Fe-ovotransferrin suggest that transferrin is internalized, depleted of iron, and recycled intact to the extracellular medium as shown in other cell systems. Autoradiography of muscle cell cultures incubated with 125I-ovotransferrin at 4 degrees C reveals clusters of receptors along the myotubes. The possible mechanisms by which transferrin is supplied to muscle in vivo are discussed in light of the evidence that motor neurons contain transferrin. PMID- 3014160 TI - Purification and characterization of plasma membranes obtained from rat neurons prepared by bulk-isolation. AB - The properties of neuronal plasma membranes are highly specialized, since they include the receptors by which many hormones, neurotransmitters, drugs, and toxins initiate their actions, as well as the components necessary for receiving synaptic input from a variety of sources and for generating the appropriate pattern of action potentials. To be able to study plasma membranes of neurons, it is necessary to separate sufficient quantities of neurons from the other cell types, processes, and myelin found in brain. The methodology for obtaining neurons by bulk-isolation from 10-day-old rat brain has been established, and techniques for purifying the plasma membranes are presented here. The procedure involves swelling the cells in a buffered solution, homogenizing the cells vigorously, and separating the membrane fragments on discontinuous sucrose gradients. The plasma membrane fraction is enriched in plasma membrane marker enzymes and has low activity for contaminating enzymes for subcellular organelles. Electron micrographs show that the fraction consists of membrane vesicles and profiles of various sizes. The protein composition reveals over 60 proteins with molecular weights as high as 100,000 to a low of 14,000. This plasma membrane fraction now can be the focus for studying receptors, glycoproteins, and cell-specific markers of neurons in health and disease. PMID- 3014161 TI - Clinico-pathological changes induced in rats treated with amine-curing agent for epoxy resin, bis(4-amino-3-methylcyclohexyl)methane. AB - Amine-curing agent for epoxy resin, bis(4-amino-3-methyl-cyclohexyl)methane (commercial name; Laromin C) has been suspected to have induced in the workers some toxic signs such as collagen disease like scleroderma or polymyositis. Subacute toxicity of this agent was studied in rats following repeated oral administration. The agent was given orally at 5 dose levels (25 mg/kg to 100 mg/kg per one dose) for periods ranging from 10 days to 4 weeks. After the completion of administration, clinico-biochemical tests and histopathological examinations were carried out. In a few cases, skeletal muscles and choroid plexus of the brain were examined by electronmicroscopy. Clinico-biochemical tests revealed some elevation of muscle-derived components such as GOT and CPK as seen in the myopathic diseases. Histologically, various degrees of atrophy, degeneration and regeneration of muscle fibers and a numerical increase of interstitial cells were observed in the skeletal muscles. Electronmicroscopical examination of the gastrocnemius muscle revealed intrasarcoplasmic osmiophilic round-shaped inclusion bodies, sometimes with lamellar structure, which were suggestive of some lipidosis. The epithelial cells of choroid plexus in the brain ventricles represented various degrees of vacuolar changes lightmicroscopically, which were suggested to be dilated smooth endoplasmic reticulum electronmicroscopically. Although scleroderma-like changes were not observed in our experiments, the results suggest that this amine-curing agent for epoxy resin could be one of the causative agents which induced toxic lesions like some collagen diseases including muscle lesions in the workers. In addition, it is considered that the agent may have systemic toxic effects. PMID- 3014162 TI - Renal disease associated with toluene inhalation. AB - A 32-year old woman developed severe quadriparesis, hypokalemia, and distal renal tubular acidosis following paint sniffing for one week. A review of literature indicated that a spectrum of renal diseases may develop in association with inhalation of toluene-containing substances. Toluene inhalation should be considered in the differential diagnosis in any young patient who presents with an unexplained renal disorder. PMID- 3014163 TI - Clinicopathological study on submucosal injection of collagenase in the treatment of submucous fibrosis in the oral cavity. PMID- 3014164 TI - Treatment of paracoccidioidomycosis with itraconazole in a murine model. AB - A new triazole, itraconazole, was studied as oral therapy of paracoccidioidomycosis in a murine model. The Paracoccidioides brasiliensis isolate, susceptible to itraconazole in vitro, was given by intranasal challenge, producing acute pulmonary and disseminated disease. Therapy was given twice daily over 4 weeks, and animals observed over 2 months. The infection was lethal for 70 80% of controls (untreated or polyethylene glycol diluent), whereas all treated animals, given 10-200 mg kg-1 day-1, survived. Itraconazole was ineffective in eradicating lung disease in survivors, though effective in treatment of disseminated sites. Since the highest doses did not give a better response than the lower doses, pharmacokinetic studies were performed. These showed irregular curves and small increases in peak serum concentrations and total area under the serum concentration-time curves, which were not proportional to the dose. This non-linearity appears to be best explained by poor absorption. Itraconazole, from these studies, appears to have promise for the therapy of human paracoccidioidomycosis but possibly with a different formulation. PMID- 3014165 TI - A prospective evaluation of patients with nasopharyngeal carcinoma: an overview. AB - One hundred and eighty-two patients in North America with nasopharyngeal carcinoma underwent initial and interval estimations of serum viral capsid antigen (VCA) and early antigen (EA), and the sera were also titrated for antibody to the Epstein-Barr virus induced membrane antigen complex by using the antibody-dependent cellular cytotoxicity (ADCC) assay. The serologic findings differentiated the World Health Organization (WHO) type 1 tumors (keratinizing squamous cell carcinomas) from the WHO types 2 and 3 tumors (nonkeratinizing and undifferentiated carcinomas) which were often small, submucosal and hard to detect. Prospective evaluation of the predictive value of staging variables has shown that the extent of tumor within the nasopharynx, involvement of lower neck nodes, WHO tumor type, ADCC titer, the number of symptoms and the patient's age are the most important. PMID- 3014166 TI - Influence of induced disease states on the disposition kinetics of imidocarb in goats. AB - The influence of fever, induced by different agents, on the disposition kinetics of imidocarb was determined in goats. Escherichia coli endotoxin (0.2 microgram/kg), Trypanosoma evansi (10(7) in 1 ml sterile glucose citrate), and Infectious Bovine Rhinotracheitis virus (10(6.5)TCID50) were the agents administered to induce the febrile state. In control and febrile animals the two compartment model was used to describe the disposition kinetics of the drug. Fever caused significant changes to occur in the apparent volume of distribution and the body (systemic) clearance of imidocarb, but the half-life remained unchanged. The statistical significance of the changes in these pharmacokinetic parameters varied with the etiology of the febrile state. E. coli endotoxin and IBR virus caused corresponding decreases in apparent volume of distribution and clearance of imidocarb, while fever induced with T. evansi caused highly significant increases in both pharmacokinetic parameters. It was concluded that the alterations in the disposition kinetics of imidocarb that occurred in the febrile goats were related not only to the febrile reaction per se but also to the pathophysiology of the disease condition. PMID- 3014168 TI - Congenital obstructive uropathy and nodular renal blastema. AB - The occurrence of nodular renal blastema and renal dysplasia was determined in a retrospective study of 75 cases of congenital obstructive uropathy. Nodular renal blastema was present in 3 upper pole nephrectomy specimens removed as a consequence of nonfunction owing to ectopic ureterocele; none was dysplastic. A more differentiated type of nodular renal blastema was present in 3 other total nephrectomy specimens, bilateral involvement in a case of posterior urethral valves and unilateral nodular renal blastema associated with ureteral atresia. This subset of differentiated nodular renal blastema was associated with renal dysplasia. PMID- 3014167 TI - Sertoli cell production of mullerian inhibiting substance in vitro. AB - Sertoli cell cultures were monitored for mullerian inhibiting substance with a solid phase radioimmunoassay. Sertoli cells from newborn calf testes were placed in defined media free of serum in monolayer culture after treatment with trypsin collagenase followed by gravity separation. Immunoreactive mullerian inhibiting substance was detected in the culture media of Sertoli cells but not National Institutes of Health 3T3 cells. To verify that the Sertoli cells were intact, cyclic adenosine monophosphate levels were determined after follicle-stimulating hormone stimulation. Cyclic adenosine monophosphate levels were elevated in Sertoli cells but not National Institutes of Health 3T3 cells. The newborn calf Sertoli cell culture provides a useful system in which to study factors affecting mullerian inhibiting substance production and release, and documents this substance as another reliable marker for the Sertoli cell. Mullerian inhibiting substance levels also could be measured in media beneath testicular fragments in organ culture, and were increased and prolonged in comparison to mullerian inhibiting substance release from Sertoli cell monolayer cultures. Modulation of mullerian inhibiting substance release from monolayer and organ cultures then was attempted. Neither gonadotropin nor steroid additions affected the release of mullerian inhibiting substance during 24 to 72 hours in organ or tissue culture. We are using the mullerian inhibiting substance radioimmunoassay to monitor attempts to immortalize a Sertoli cell line capable of continuous mullerian inhibiting substance production using viral deoxyribonucleic acid transfection techniques. PMID- 3014170 TI - The John Lattimer lecture. Wilms tumor and other renal tumors of childhood: an update. PMID- 3014169 TI - Bilateral nephrectomy for Wilms tumor. AB - Bilateral nephrectomy was performed in 4 patients with bilateral Wilms tumor. The current philosophy regarding the need to preserve maximum renal parenchyma is discussed. New guidelines suggested by the National Wilms Tumor Study group discourage unilateral nephrectomy and partial nephrectomy at initial exploration for bilateral Wilms tumor. Alternatively, it is recommended that continued treatment with chemotherapy and/or radiation therapy followed by second and third look operations to maximize preservation of renal parenchyma be done with bilateral nephrectomy as a last resort option. PMID- 3014171 TI - Elusive vesicoureteral reflux in children with normal contrast cystograms. AB - Recurrent pyelonephritic episodes in children with normal contrast cystograms pose a difficult management problem, since the lack of demonstrable reflux adversely affects the treatment. A total of 10 children with clinical symptoms of pyelonephritis in whom normal contrast cystograms had been performed properly subsequently had significant degrees of reflux detected by isotope cystography. These findings indicate that isotope cystography should be included in the evaluation of patients with pyelonephritis in whom reflux is not confirmed by conventional contrast medium techniques. PMID- 3014172 TI - Leads from the MMWR. Prevention and control of influenza. PMID- 3014173 TI - Laboratory and epidemiologic evaluation of an enzyme immunoassay for antibodies to HTLV-III. AB - The enzyme immunoassays (EIAs) for antibody to human T-cell lymphotropic virus type III (HTLV-III) were rapidly adopted for screening donated blood and plasma. To evaluate the significance of a positive EIA reaction, test performance was examined in a blood bank screening program. Specimens were tested by EIA, Western blot assay, and HTLV-III/lymphadenopathy-associated virus (LAV) culture. The EIA was positive in 0.25% of 67 190 blood donations. Specimens were categorized and 57.3% had low (weak) reactivity, 12.7% had moderate reactivity, and 30.0% had high reactivity. Highly reactive specimens were strongly associated with a positive Western blot or culture (86.7%) in contrast to moderately and weakly reactive specimens (1.9%). Twenty-five of 29 donors interviewed with a highly reactive EIA had risk factors for HTLV-III/LAV infection. Risk factors were not identified for 74 of 75 interviewed donors with specimens of lower reactivity. The minimum calculated specificity was 99.82%. The use of the HTLV-III EIA has virtually eliminated the use of blood and plasma from HTLV-III/LAV infected donors. PMID- 3014174 TI - Leads from the MMWR. Gastroenteritis on two Caribbean cruise ships. PMID- 3014175 TI - Leads from the MMWR. Transfusion-associated human T-lymphotropic virus type III/lymphadenopathy-associated virus infection from a seronegative donor- Colorado. PMID- 3014176 TI - Leads from the MMWR. Recommendations for providing dialysis treatment to patients infected with human T-lymphotropic virus. PMID- 3014177 TI - Serological markers as indicators of sexual orientation in AIDS virus-infected men. PMID- 3014178 TI - Prevalence of HTLV-III/LAV in household contacts of patients with confirmed AIDS and controls in Kinshasa, Zaire. AB - Household members of 46 patients with confirmed acquired immunodeficiency syndrome (AIDS) and 43 human T-cell lymphotropic virus type III/lymphadenopathy associated virus (HTLV-III/LAV)-seronegative controls from Kinshasa, Zaire, were identified and sought for serologic testing for evidence of HTLV-III/LAV infection. Twenty (9.8%) of 204 case-household members and three (1.9%) of 155 control-household members were HTLV-III/LAV seropositive (relative risk = 5.1; 95% confidence interval, 1.7 to 15.2). Eleven (61.1%) of 18 spouses of patients with AIDS were HTLV-III/LAV seropositive, compared with one (3.7%) of 27 control spouses (relative risk = 16.5; 95% confidence interval, 3.7 to 75.0). Except for spouses, the rate of HTLV-III/LAV seropositivity did not differ significantly between case and control households. Furthermore, for adults in case households who were not spouses, the number seropositive for HTLV-III/LAV was identical to that predicted from sex- and age-specific HTLV-III/LAV seroprevalence rates. These data from Zaire confirm the results of US and European studies of household contacts of infected hemophiliacs and pediatric patients with AIDS. PMID- 3014180 TI - [Primary adenoid cystic carcinoma of the esophagus]. AB - A case of primary adenoid cystic carcinoma of the esophagus is reported, and 45 cases are reviewed. The light and electron microscopic appearances are described in a 77-year-old woman. This patient manifested the intraepithelial spread of cancer cells, which suggested that the tumor arose from the mucosal epithelium of the esophagus. The patient died of mediastinal metastasis 11 months after resection. Adenoid cystic carcinoma of the esophagus is a relatively rare lesion, which exhibits a clinically aggressive behavior. Earlier resection and more intensive chemotherapy will be required. PMID- 3014179 TI - [Diagnostic variations among eight pathologists in the histologic classification of lung cancer]. AB - In order to estimate the magnitude of variations among pathologists in histological diagnoses of lung cancer, eight doctors were asked to independently diagnose 73 preparations of the lung (16 biopsied cancers, 27 resected specimens, 21 autopsied specimens and nine benign autopsied cases). The rates of correct diagnoses by the pathologists ranged from 81.7% to 100%, averaging 91.8%. There was no significant difference between the lowest rate and mean or median of the rates. The variation of the diagnosis tended to be more prominent in large cell carcinoma than in adenocarcinoma or small cell and squamous cell carcinoma. It was suggested that the diagnostic criteria for large cell carcinoma varied to some extent among pathologists. PMID- 3014181 TI - [HTLV-I associated T-cell lymphoma terminating in B-cell lymphoma]. PMID- 3014182 TI - [A case of intrahepatic bile duct carcinoma misdiagnosed as choledocholithiasis]. PMID- 3014183 TI - [Diagnostic imaging of glucagonoma and transcatheter arterial embolization in hepatic metastases--report of a case]. PMID- 3014185 TI - [Immunohistochemical localization of Cu, Zn-SOD in the liver with primary hepatocellular carcinoma]. PMID- 3014184 TI - Comparison of the antitumor effects of human natural and recombinant interferons (alpha and beta) on human cancer cell lines. AB - The antitumor effects of 5 different subtypes of human type I interferons (IFN alpha(Le), rIFN-alpha A, rIFN-alpha D, IFN-beta, and rIFN-beta) were studied on 3 human cancer cell lines (Daudi lymphoma, PLC/PRF/' hepatoma, and G361 melanoma). IFN-alpha(Le) was the most potent against Daudi cells. The effects of IFNs were cytostatic. On the other hand, 500 IU/ml of IFN-alpha(Le), IFN-beta and 5000 IU/ml of rIFN-beta showed cytocidal effect against PLC/PRF/5 cells. The G361 cells were the least sensitive to the IFNs tested. This is the first report on the antitumor effect of rIFN-beta isolated from insect cells. The effect of this rIFN-beta was similar to that of IFN-beta. PMID- 3014186 TI - The probability of parentage exclusion based on restriction fragment length polymorphisms. PMID- 3014187 TI - [Clinical diagnosis in obstetrics. 13. Diagnosis of trophoblastic tumor]. PMID- 3014188 TI - [Progress in cancer therapy. 4. Liver cancer]. PMID- 3014189 TI - Intraocular penetration of new antiviral agent, hydroxyacyclovir (BW-B759U). AB - 9-[(2-hydroxyl-1-(hydroxymethyl)ethoxy]methyl)guanine (hydroxyacyclovir) is a nucleoside analogue with a high degree of efficacy against herpes simplex virus types 1 and 2 and cytomegalovirus (CMV). This antiviral agent demonstrates a low toxicity towards uninfected cells. Intraocular levels in rabbits were evaluated following subconjunctival, intravenous, intravitreal and topical administration. Retinal toxicity studies were performed. Hydroxyacyclovir may have applications in the treatment of deep herpetic infections, CMV retinitis, and acute retinal necrosis. PMID- 3014191 TI - Gustatory signal processing in the glossopharyngeo-hypoglossal reflex arc of the frog. AB - The relationship between the gustatory input and motor output in the glossopharyngeo-hypoglossal reflex was analyzed on the basis of neuronal activities in the solitary tract and hypoglossal motor nuclei of bullfrogs. Concentration-response relations for NaCl, quinine and acetic acid, obtained from the glossopharyngeal (IXth) nerve and simultaneously recorded from the hypoglossal (XIIth) nerve, were expressed relative to the response of each nerve to 1 M NaCl. Compared with a relatively small amount of the afferent input for acid, the reflex motor output was much larger in the relative value. A similarly high output relation was obtained for warmed saline but not for quinine and cooled saline. Although the responsiveness of the nucleus tractus solitarius neurons to 1 M NaCl and 1 mM quinine was not significantly different from that of the hypoglossal motoneurons, responses to 10 mM acetic acid were greater in the latter neurons than in the former by a factor of about 5.2. These phenomena were consistent with those in the peripheral nerves. The solitary tract neurons responsive to NaCl, quinine and acid showed both the phasic and tonic components of discharges. According to classification by a transiency index, the discharge mode became more phasic for the hypoglossal motoneurons responsive to NaCl and quinine, but more tonic for those responsive to acid. The above-mentioned chemoreflex is thus regulated by the intrinsic neural network which sends signals to the XIIth nerve after modifying not only the amount but also the temporal pattern of gustatory nerve signals for a particular taste. PMID- 3014190 TI - 2',3'-cyclic nucleotide 3'-phosphohydrolase activity in rat visual pathways with experimental allergic encephalomyelitis. AB - The enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) is one of the important markers for myelin synthesis or demyelination. We measured the CNPase activities in the visual pathway tissues of Lewis strain rats with experimental allergic encephalomyelitis (EAE) induced by inoculation of myelin basic protein from the brain. The enzyme activities in the retina, optic nerve, optic chiasma and lateral geniculate body were reduced to about 50-80% of that of controls at 15 days after myelin basic protein inoculation when symptoms of EAE were most severe. However, the activities recovered at 19 days except in the retina when the symptoms of EAE disappeared. Furthermore, the activities of the optic nerve and chiasma increased to a level higher than that of the controls at this time. By histopathological study, infiltration of inflammatory cells and focal demyelination were found in the optic nerve and lumbar spinal cord at 15 days after myelin basic protein inoculation. It was considered that the reduction of CNPase activity and its recovery and increase reflect demyelination and activation of oligodendroglia for remyelination, respectively. PMID- 3014192 TI - Luteinizing hormone releasing hormone modulates the cholinergic transmission in frog neuromuscular junction. AB - The effects of luteinizing hormone releasing hormone (LHRH) on cholinergic transmission were studied at the neuromuscular junction of the frog. Brief application of LHRH produced a prolonged increase in the amplitude of end-plate potentials (e.p.p.s), which lasted 20 to 30 min after removal of LHRH. LHRH (0.4 1 microM) increased in the quantal content of the e.p.p. dose-dependently, while having no effect on the quantal size. LHRH (0.4-1 microM) did not affect the frequency and the amplitude of miniature end-plate potential (m.e.p.p.). At a high concentration (8 microM), however, LHRH consistently produced an increase in the frequency and a decrease in the amplitude of m.e.p.p. The acetylcholine induced end-plate current (ACh current) produced by iontophoretic application of ACh was reversibly and dose-dependently reduced by LHRH (4.6-46 microM). An analysis with a dose-response curve of the ACh current revealed that LHRH decreased the sensitivity of the nicotinic receptor in a noncompetitive manner. These results suggest that LHRH at low concentrations facilitates neuromuscular transmission by increasing ACh-release from the presynaptic nerve terminals, while at higher concentrations it depresses transmission post-synaptically. Possible mechanisms of these LHRH actions are discussed. PMID- 3014193 TI - Omental transposition and skin graft in patients for advanced or recurrent breast cancer. AB - Nine patients, including 4 with primary advanced breast cancer (stage IV) and 5 with local recurrent cancer, underwent chest wall reconstruction using an omental flap and mesh skin grafting. In 2 of these patients the defect of bony chest wall was reconstructed with an acryl-resin plate and omental flap. The postoperative course in all patients was uneventful, except for a slight necrosis on the transposed mesh skin. Flail chest or dyspnea did not occur in those with a bony chest wall reconstruction. The immediate postoperative performance status in 6 of 9 patients and also quality of life improved. PMID- 3014194 TI - [Heterogeneity of human renal cell carcinoma. III. 1 alpha,25-dihydroxyvitamin D3 receptor and inhibition on the growth of renal carcinoma cell lines]. PMID- 3014195 TI - Transmissible lymphoma and simian acquired immunodeficiency syndrome in rhesus monkeys. AB - Four rhesus monkeys (Macaca mulatta) were inoculated with a homogenate of a cutaneous lepromatous leprosy lesion from a mangabey monkey (Cercocebus atys). One died of B-cell lymphoma, and another died of an immunodeficiency syndrome. Cell suspensions prepared from the tumor and spleen of the monkey with lymphoma induced lymphoma or an immunodeficiency syndrome when inoculated into additional young rhesus monkeys. The immunodeficiency syndrome was similar to simian acquired immunodeficiency syndrome and consisted of opportunistic infections, lymphoid hyperplasia or atrophy, wasting, and syncytial cell formation. Mitogen responses and percentages of T4- and T8-positive lymphocytes were normal until the animals were moribund. Lymphoblastoid cell lines became established in vitro from tumor cell suspensions. These cells were infected with a herpesvirus related to Epstein-Barr virus. In addition, a retrovirus morphologically similar to human T-cell lymphotrophic virus type III (HTLV-III) and simian T-lymphotrophic virus type III (STLV-III) was isolated from one of the lymphoblastoid cell lines (LCL). Type D retroviruses could not be demonstrated in the monkeys in the transmission study; however, a retrovirus similar to that in the LCL was isolated from 4 animals by coculture of peripheral blood lymphocytes with the human cell line H9. These results suggest that this retrovirus, STLV-III/Delta, may be associated with the immunodeficiency syndrome in these macaques and may be of mangabey origin. PMID- 3014196 TI - Induction of gastric carcinomas in nonhuman primates by N-ethyl-N'-nitro-N nitrosoguanidine. AB - N-Ethyl-N'-nitro-N-nitrosoguanidine [(ENNG) CAS: 63885-23-4] was administered to 5 Macaca monkeys (Macaca mulatta and M. irus) at a concentration of 200 or 300 micrograms/ml for 11-26 months in their drinking water. Gastric carcinomas in the pyloric region were observed in all 5 monkeys between experimental months 11 and 38. Histologically, these carcinomas were mainly poorly differentiated adenocarcinomas and signet-ring cell carcinomas, and a few moderately and well differentiated adenocarcinomas were also found. The macroscopic and histologic appearances of these carcinomas were similar to those in humans. PMID- 3014197 TI - In vitro binding of potent mutagenic pyrolysates to intestinal bacteria. AB - The ability of 22 strains of intestinal bacteria to bind the mutagenic pyrolyzates--3-amino-1,4-dimethyl-5H-pyrido-[4,3-b]indole [(Trp-P-1) CAS: 62450 06-0], 3-amino-1-methyl-5H-pyrido [4,3-b]indole [(Trp-P-2) CAS: 62450-07-1], 2 amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole [(Glu-P-1) CAS: 67730-11-4], 2 aminodipyrido[1,2-a:3',2'-d]imidazole [(Glu-P-2) CAS: 67730-10-3], 2-amino-3 methylimidazo[4,5-f]quinoline [(IQ) CAS: 76180-96-6], 2-amino-3,4 dimethylimidazo[4,5-f]quinoline [(MeIQ) CAS: 77094-11-2], and 2-amino-3,8 dimethylimidazo[4,5-f]quinoxaline [(MeIQx) CAS: 77500-04-0]--was investigated and compared to their ability to bind to some dietary fibers (corn bran, apple pulp, soy bean fiber, cellulose, chitin, and chitosan). The pyrolyzates are potent mutagenic and carcinogenic heterocyclic amines formed during cooking. Solution of these amines was mixed with aqueous suspension of bacterial cells or dietary fibers, and removal of these amines from the reaction mixture by centrifugation was defined as the binding. Trp-P-1 and Trp-P-2 were effectively bound to all gram-positive and some gram-negative bacterial cells, corn bran, apple pulp, and soy bean fiber. Binding of Trp-P-1 and Trp-P-2 to Escherichia coli, Klebsiella pneumoniae, and cellulose was moderate, and to chitin and chitosan it was little. None but corn bran bound Glu-P-1 and Glu-P-2 effectively. Corn bran effectively bound all mutagens tested. The quantity of the binding of IQ, MeIQ, and MeIQx was dependent on the strain of bacteria and the kind of fiber. The mechanism of binding of Trp-P-2 to freeze-dried feces, Lactobacillus casei YIT 9018 (LC9018), and corn bran was investigated. The binding was pH dependent, occurred instantaneously, and was inhibited by the addition of metal salts. These results indicate that the binding was mostly due to a cation-exchange mechanism, but some irreversible binding of Trp-P-2 was observed, most notably to freeze-dried feces. The mutagenicity of Trp-P-2 for Salmonella typhimurium TA98 in the presence of S9 mix was inhibited by the addition of LC9018 or corn bran to the reaction mixture. The results indicate that bound Trp-P-2 did not cause mutation under the assay conditions. PMID- 3014199 TI - Secular trends in histologic types of lung cancer. AB - The histology pattern of lung cancer in Los Angeles County was reviewed for a 10 year period, 1972-81. In men, the total lung cancer incidence has been fairly constant, but there has been a shift in the histology pattern with an increase in adenocarcinoma and a decrease in "other" cell type (i.e., carcinoma not otherwise specified, large-cell and undifferentiated tumors). This changing histology pattern may be partly due to changes in diagnostic standards and practices. With the assumption that these changes are comparable in men and women, the "true" annual rate of change was estimated for each lung cancer cell type in women. All lung cancer types have increased in women; of the cell types squamous cell carcinoma, small-cell carcinoma, and adenocarcinoma, small-cell carcinoma showed the largest rate of annual increase and adenocarcinoma, the smallest. PMID- 3014198 TI - Effects of diethylnitrosamine on hepatic receptor binding and autophosphorylation of epidermal growth factor and insulin in rats. AB - Carcinogen-mediated alterations in hepatic membrane growth factor receptor activity were investigated with the use of the hepatocarcinogen diethylnitrosamine [(DENA) CAS: 55-18-5]. For the separate assessment of carcinogen-induced acute membrane receptor changes from changes associated with carcinogen-mediated alterations in growth, the receptor activity was measured both acutely after, and several months after, a single ip dose of DENA in male F344 rats. An acute dose-dependent decrease was observed in ligand binding and autophosphorylation of the epidermal growth factor (EGF) and insulin receptor. Binding decreased sharply by 36 hours and recovered by 30 days in Golgi fractions and more slowly in plasma membranes. Scatchard analysis revealed a decrease in receptor number but not affinity. Chronic DENA administration also decreased insulin and EGF binding. Hepatocellular carcinomas, induced by single DENA injections 1 year previously, had decreased EGF receptor binding and autophosphorylation compared to those seen in age-matched controls and also less extensive insulin receptor changes. Single DENA doses thus have two effects: an acute reversible receptor change and a delayed, apparently permanent change associated with altered growth. PMID- 3014200 TI - Enhancement of pulmonary metastases induced by decreased lung natural killer cell activity. AB - Administration of repeated high doses of 7.5 mg chlorozotocin [(CZT) CAS: 54749 90-5]/kg to syngeneic WAG rats bearing the rhabdomyosarcoma 9-4/0 enhanced the incidence of spontaneous metastasis compared to its incidence in untreated rats. This enhancement was observed concomitantly with an increase in the survival of 9 4/0 rhabdomyosarcoma and P 77 fibrohistiocytoma tumor cells, labeled with [125I]5 iodo-2'-deoxyuridine or 51Cr and injected iv. Within the first 24 hours after P 77 cell injection, the lungs retained 10% of the cells while the lungs of controls or of rats given one CZT injection only retained 0.06%. The natural killer (NK) cell cytotoxicity of cells flushed out from lung capillaries [lung intracapillary cells (LIC)] was studied concomitantly in a 4-hour 51Cr release assa against YAC-1 and P 77 target cells. A large reduction was again observed in NK cytotoxicity but only after repeated injections of 7.5 mg CZT/kg. Lung defenses were gradually restored after treatment stopped. Administration of polyinosinic-polycytidylic acid with CZT restored the NK cell cytotoxicity of LIC and inhibited lung metastases amplification. The close relationship between metastasis and NK activity indicates the need for caution as regards the effects of chemotherapy on NK activity. PMID- 3014201 TI - Effects of antihypertensive treatment in one-clip, two kidney hypertension in rats. AB - In order to investigate the consequences of antihypertensive therapy on hormonal and renal parameters in one-clip, two kidney renovascular hypertension, we compared the effects of converting enzyme inhibition (CEI) with those of tripletherapy (clonidine, dihydralazine and furosemide) in this experimental model in rats. The treatment period was initiated four weeks after application of the clip and was continued for five weeks. In plasma, renin was increased and renin substrate was negatively correlated to plasma renin. Hypertension was associated with activation of the renin angiotensin system in both plasma and kidney. The degree of activation of the renin-angiotensin system in the clipped kidney and its suppression in the unclipped kidney was evaluated by two methods, renal renin content and semi-quantification of juxtaglomerular hyperplasia by immunofluorescent renin. These two methods were correlated. During the treatment period, average systolic blood pressure was 144 +/- 13 mmHg in the CEI treated group (HT1) which was not significantly different from the value found in the sham-operated group (139 +/- 4 mmHg; C2). Blood pressure, however, was lowered only to 173 +/- 18 mmHg in the group treated with tripletherapy (HT2). In control hypertensive animals, the wt of the clipped kidney did not decrease whereas significant hypertrophy was present in the unclipped kidney. Tripletherapy did not alter this relationship, whereas converting enzyme inhibition decreased kidney wt in the clipped kidney and increased further the hypertrophy of the contralateral unclipped kidney. A histological examination revealed that hypertensive microangiopathy was a predominant feature in the unclipped kidney of the untreated hypertensive group and of the group treated with tripletherapy, these lesions were completely absent in the CEI treated group. In the CEI treated group, however, ischemic lesions during this treatment were found to be decreased in the contralateral unclipped kidney and increased in the clipped kidney by comparison with untreated hypertensive rats. These renal lesions observed in the clipped kidney were most likely related to the normalization of blood pressure or to a disturbance of intrarenal mechanisms normally mediated by the renin angiotensin system during stenosis of a renal artery. PMID- 3014202 TI - [Significance of immunohistologic methods in the differential diagnosis of solid tumors in childhood]. AB - We have investigated 56 histologic and 13 cytologic specimens of malignant round cell tumors of childhood. The immunohistological detection of intermediate filament polypeptides, neuron specific enolase (NSE) as well as leukocyte common antigen (LCA) allows the histological classification of such tumors. Neuroblastomas demonstrate a positive reaction with antibodies to neurofilaments and NSE independent of differentiation. Rhabdomyosarcomas could be labeled by the desmin antibody, while Ewing sarcomas, malignant lymphomas as well as nonmuscular sarcomas only express vimentin. In nephroblastomas the intermediate filament specific antibodies reveal expression of keratin and vimentin in blastema cells, while tubules are only labeled by the keratin antibody. In undifferentiated nephroblastomas, which lack formation of tubules blastema cells are keratin negative and vimentin positive. Thus antibodies to intermediate filaments, LCA and NSE seem to be useful tools to distinguish the so called "round cell tumors" of childhood. PMID- 3014203 TI - [Bone marrow transplantation in acute leukemia, chronic myeloid leukemia, severe aplastic anemia and stage IV neuroblastoma. Effect of antiviral prevention with anti-CMV-hyperimmunoglobulin and acyclovir]. AB - Bone marrow transplantation was performed between IV/82 and X/85 in 64 patients with acute leukemia (n = 36), chronic myelogenous leukemia (CML; n = 13), severe aplastic anemia (n = 12), and neuroblastoma stage IV (n = 3). Of these patients 57 received allogeneic marrow from HLA-ABCDR identical, MLC-negative sibling donors. Six transplants were performed with syngenic marrow and one with autologous marrow. Of the 64 patients 48 survived 40-1,250 days after transplantation, resulting in a survival rate (SR) of 75% and a survival probability (SP) of 71%. Of the 36 patients suffering from acute leukemia (SR = 64%, SP = 51%), patients with acute myelogenous leukemia (AML) in first complete remission (n = 11; SR = 81%, SP = 76%), as well as patients with acute lymphatic leukemia (ALL) in 1st to 4th complete remission at the time of transplantation (n = 14; SR = 81%, SP = 76%) show a favorable prognosis. A poor survival rate was seen for patients with AML when transplanted in second or partial remission (1/5; SR = 20%), as well as for patients suffering from ALL and transplanted during relapse or partial remission (1/6; SR = 16%). Of 13 patients suffering from CML 12 survived the transplantation free of relapse (SR = 93%, SP = 92%), and one patient died from varicella zoster pneumonia. Of the transplanted patients with severe aplastic anemia, 12 of 13 are surviving with complete hematologic reconstitution; one patient, however, died on day 10 from a sepsis. In our patient group, the SR as well as the SP has been improved through changes in the irradiation protocol concomitant with prophylactic application of anti-CMV hypergammaglobulin, as well as through additional oral medication of Azyklovir. The 41 patients (BMT No. 7-47) with total body irradiation at one time show an SR of 44% and an SP of 41%. The following 46 patients (BMT No. 48-93) have reached an SR of 83% and an SP of 74% under the regimen of fractionated total body irradiation, plus prophylaxis with anti-CMV hypergammaglobulin and Azyklovir. Within this group, no fatal CMV pneumonia was encountered as opposed to six patients lost from CMV pneumonia in the first group. PMID- 3014205 TI - [The theory of adequate nutrition]. PMID- 3014207 TI - [Irritable colon syndrome]. PMID- 3014206 TI - [Clinical significance of the bactericidal systems in endogenous infections]. PMID- 3014204 TI - [Regulation of acinar cell receptors of the pancreas by peptides]. AB - Peptides may act on the same receptor they regulate or on another receptor by causing regulations via receptor interactions. These receptor regulations include changes of receptor affinity and capacity. Receptor capacity is regulated by internalization, recycling, degradation, synthesis, and modification of bioavailability without migration of the receptor. Examples for those regulations, mostly based on experiments with isolated pancreatic acini from the rat, mouse, or guinea pig, are given. For the CCK receptor these examples include complex regulations of this receptor by CCK itself, bringing into discussion the hypothesis of negative cooperativity and the two-site receptor model, desensitization of the receptor by CCK, in vivo CCK influences on its receptor, and insulin receptor/CCK receptor interactions. For the insulin receptor the physiological significance of "up and down regulation" of this receptor by insulin itself is discussed. For the IGF receptors and the EGF receptor CCK induced, Ca2+-mediated regulation of receptor internalization are another type of regulation with unknown physiological and pathophysiological significance. Finally CCK-induced, Ca2+-mediated regulation of somatostatin receptor capacity and affinity are mentioned. It is postulated that those regulations play an important role in influencing the biological effect of hormones and that knowledge about them may improve our understanding of pathophysiology. PMID- 3014208 TI - [Status of the cardiovascular system in para-influenza patients]. PMID- 3014209 TI - Relationship of mitogen reactivity to type D retrovirus infection in Celebes black macaques (Macaca nigra). AB - The Celebes black macaque (Macaca nigra) colony at the Oregon Regional Primate Research Center has a high incidence of an immunodeficiency syndrome characterized by recurrent diarrhea and the development of retroperitoneal fibromatosis (RF). We have examined the relationship of type D viral infection to the immunodeficiency syndrome by surveying the colony for viral infection and for mitogen reactivity. Type D virus-positive monkeys (28% of the colony) have a higher prevalence of diarrhea, splenomegaly, lymphadenopathy and weight loss than do virus-negative monkeys, and RF has been found to occur only in virus-positive animals. Comparison of the concanavalin A (con-A) and phytohemagglutinin reactivities of the virus-positive and -negative populations has revealed no significant difference. However, within the virus-positive population, those with RF have reduced con-A reactivity and there are both high and low mitogen responders in the groups lacking RF. Thirty-two percent of the virus-positive monkeys are free of clinical symptoms, 40% have clinical symptoms but no RF, and 27% have clinical symptoms and RF. Five of the six monkeys with RF are older than the RF-free monkeys but monkeys are susceptible to type D retrovirus infection regardless of age or sex. The progressive nature of this immunodeficiency syndrome, its broad age range, and the probability that the etiological agent is also a type D retrovirus and the similarity of RF to Kaposi's sarcoma make this a potentially useful model for human AIDS. PMID- 3014210 TI - Diagnosis of murine infections in relation to test methods employed. AB - Comparative investigations of Sendai virus, pneumonia virus of mice (PVM), mouse encephalomyelitis virus (mouse polio), minute virus of mice (MVM), and reovirus type 3 (Reo 3) infected murine colonies revealed a 30% higher incidence of positive sera when enzyme-linked immunosorbent assay (ELISA) was employed instead of hemagglutination inhibition (HI) tests. Equivalent sensitivity as in the ELISA was obtained when the same sera were investigated by indirect immunofluorescence (IF) tests. The virus purification techniques described resulted in highly suitable antigens for all indirect ELISA established. Since IIF requires no purified antigens, this test is recommended as an alternative to ELISA as well as to HI and complement fixation (CF) tests for laboratories lacking the necessary equipment for high speed centrifugation. A high incidence of false positive HI reactions was found particularly in Reo 3 routine serology. An updated survey of seromonitoring showed that European murine colonies appeared to be infected far less with Reo 3 if ELISA or IIF tests were employed. During 1982-1984, only 13% of the mouse colonies screened possessed Reo 3 positive sera whereas no natural Reo 3 infection was found in rat colonies. Mouse hepatitis virus (MHV) and the coronaviruses of rats exhibited the highest incidence in murine colonies. A total of 60% of mouse and 41% of rat colonies were found to be infected by these viruses. In comparison with earlier serological surveys, the relative incidence of other murine infections was similar. Antibodies against Bacillus piliformis (Tyzzer's disease) were detected by the IIF test in 41% of the rat colonies screened. PMID- 3014211 TI - Family studies of the Lesch-Nyhan syndrome: the use of a restriction fragment length polymorphism (RFLP) closely linked to the disease gene for carrier state and prenatal diagnosis. PMID- 3014213 TI - Development of a protocol for newborn screening for disorders of the galactose metabolic pathway. AB - The protocol evaluated in this paper employs an enzymatic assay of galactose metabolites, thin layer chromatography, and an assay of galactose-1-phosphate uridyl transferase on a single sample of blood collected routinely for newborn screening. Its effectiveness was tested by a retrospective study of known galactosemic blood samples, and also by a prospective study of 207,000 newborn samples from which 6 infants with severe transferase deficient galactosaemia and 2 infants with red cell epimerase deficiency were identified. The detection rate for severe transferase deficiency in the newborn population was 1:35,000. Advantages include low false positive rate, definitive diagnosis within 6 hours of sample receipt, and the use of technically simple and robust procedures. This protocol overcomes the difficulties encountered with previously described procedures. PMID- 3014212 TI - Enzyme activities and phospholipid storage patterns in brain and spleen samples from Niemann-Pick disease variants: a comparison of neuropathic and non neuropathic forms. AB - Phospholipid levels and enzyme activities were measured in brain and spleen samples from patients with the three major variants of Niemann-Pick disease. Accumulations of sphingomyelin and bis(monoacylglycero)phosphate were demonstrated in spleen from types A and B and group C Niemann-Pick disease, whereas only in type A Niemann-Pick brain was the sphingomyelin concentration increased. Sphingomyelinase activity was markedly deficient in type A Niemann Pick brain and spleen but residual activity of approximately 12% of control was measured in type B Niemann-Pick brain. Normal or raised sphingomyelinase and beta glucosidase activities were measured in group C Niemann-Pick brain and spleen. Significant (17% of control) residual beta-glucosidase activity was also measured in non-neuropathic Gaucher brain. Normal levels of neutral sphingomyelinase activity were measured in brain samples from the three variants of Niemann-Pick disease. Acid sphingomyelinase activity in group C Niemann-Pick brain appeared normal with respect to enzyme extraction, pH optimum (pH 5.0) and apparent Km (approximately 0.4 mmol/L). Isoelectric focusing of brain sphingomyelinase revealed a degree of heterogeneity with activity peaks between pI 4.5 and 6.5. No defect was observed in group C Niemann-Pick brain and, although attenuated, all peaks were present in type B Niemann-Pick brain. PMID- 3014215 TI - Vascular cell interactions with special reference to the pathogenesis of atherosclerosis. PMID- 3014214 TI - Lymphoproliferative syndrome in an immunodeficient rhesus monkey naturally infected with an HTLV-III-like virus (STLV-III). AB - A rhesus monkey with a naturally acquired STLV-III infection developed immunosuppression and a lymphoproliferative syndrome characterized by progressive lymphadenopathy and widespread visceral mononuclear cell infiltration. On microscopic examination, diffuse sheets of plasmacytoid lymphoblasts obliterated the sinuses and follicles of the nodes, replaced normal cellular elements of the spleen, bone marrow, and thymus, and infiltrated the lung, liver, kidney, salivary gland, pancreas, thyroid, stomach, and tongue. Immunohistologic studies indicated that the predominant cell in these infiltrates was a B lymphocyte, of oligo- or polyclonal origin. Similar but less extreme lymphoproliferative abnormalities were seen at necropsy in a substantial number of other animals with naturally occurring macaque immunodeficiency syndrome. The present case represents the first prospectively studied monkey with a naturally acquired simian T lymphotropic virus type III infection and illustrates an important manifestation of disease associated with such an infection. PMID- 3014217 TI - Myasthenia gravis associated with small cell carcinoma of the lung. PMID- 3014216 TI - Clinical evaluation of occupational toxicity of methylmethacrylate monomer to dental technicians. PMID- 3014218 TI - Effects of chronic administration of LH releasing hormone on age-dependent activities of testicular 4-ene-5 alpha-reductase and 17 beta-ol-dehydrogenase in mice. AB - Male (WB X C57BL/6)F1 hybrid mice of 16, 26 and 66 days of age, 4 in each group, were injected daily with 0.2 micrograms/10 g body weight of LH releasing hormone (LHRH) or saline for 14 days. Testicular homogenates were incubated with [14C]4 androstene-3,17-dione and enzyme activities were examined. In mice treated with saline, testicular 17 beta-ol-dehydrogenase activity increased with age but 4-ene 5 alpha-reductase (5 alpha-reductase) activity decreased with age. LHRH treatment for 14 days starting from day 26 resulted in a delay in sexual maturation, as evidence by significant decreases (P less than 0.05) in seminal vesicle weight and testicular 17 beta-ol-dehydrogenase activity and by a significant increase (P less than 0.05) in 5 alpha-reductase activity. However, LHRH treatment starting from day 66 had no significant effect on these testicular enzyme activities. PMID- 3014219 TI - Prophylactic oophorectomy in premenopausal women with stage II breast carcinoma. AB - Prophylactic oophorectomy, as an additional treatment for stage II breast cancer, is controversial. In a retrospective study, a group of 37 premenopausal women with stage II infiltrating duct carcinoma and one to three positive axillary lymph node involvement after modified radical mastectomy and bilateral oophorectomy were compared to a matched group of 34 women treated by modified radical mastectomy only. Prophylactic oophorectomy prolonged the disease free interval significantly as compared to the control group. However, it did not prolong survival. This raises the question whether the prolongation of survival achieved by late oophorectomy in women with advanced breast cancer is preferable to an improvement in quality of life resulting from longer disease free intervals. PMID- 3014220 TI - Neoplasms in skin and subcutis over the breast, simulating breast neoplasms: case reports and literature review. AB - Twenty cases of neoplasms in skin and subcutaneous tissue over the breast were reviewed. There were 17 women, from 15 to 70 years of age, and three men, from 25 to 66 years of age. Among the benign skin neoplasms, superficial leiomyoma, granular cell tumor, and eccrine acrospiroma were misdiagnosed clinically as primary breast carcinoma. Among the malignant neoplasms in subcutaneous tissue, there were three metastatic malignant melanomas, one metastatic epidermoid bronchogenic carcinoma, and two malignant lymphomas. It is interesting that four of these six patients had no prior history of malignant lesion, the subcutaneous nodule presenting as the first manifestation of an occult primary. It is concluded that histological diagnosis of such tumors may lead to avoidance of unnecessary radical surgery. PMID- 3014221 TI - Efficacy of polyglycolic acid mesh sling in keeping the small bowel in the upper abdomen after abdominal surgery: a 12-month study in baboons. AB - The purpose of this study was to determine if a "sling" made of polyglycolic acid (PGA) would be a reliable method of preventing small bowel descent into the pelvis following abdominal surgery. Baboons were used, as they respond to infection and ambulate similarly to humans. Animals had the small bowel mobilized to the upper abdomen and had the PGA "sling" sewn into place. Documentation of small bowel position was evaluated by upper gastrointestinal series over the 12 month study. Small bowel descent into the pelvis was prevented by utilization of the PGA "sling." Animals were sacrificed and autopsied, and sections of small bowel were taken. There was no evidence of mesh, obstruction, sepsis, fistulae, or herniation in animals at autopsy. Small bowel sections were considered normal histologically. Utilization of PGA sling appears to be a safe and reliable method of preventing small bowel descent into the pelvis after abdominal surgery. PMID- 3014222 TI - Liposarcoma arising within a cystosarcoma phyllodes. AB - A liposarcoma arising within a cystosarcoma phyllodes in a 17-year-old black female was treated by total mastectomy without the need of radiation and/or chemotherapy. The patient is alive and well with no evidence of recurrence or metastasis at 20 months recent follow-up. The clinicopathological features of malignant cystosarcoma phyllodes-liposarcoma are discussed. PMID- 3014224 TI - Melanotic primitive neuroectodermal (neuroepithelial) tumor of medulla. AB - A 69-year-old man had a melanotic primitive neuroectodermal tumor of the medulla displaying various neuroepithelial elements including undifferentiated neuroepithelial cells forming Homer Wright's rosettes as well as neoplastic neuroglia resembling those seen in medulloblastoma. The neuroglial tumor cells were verified by demonstrating glial fibrillary acidic protein (GFAP) in the cells. These findings support the concept that the primitive neuroectodermal tumor and medulloblastoma are similar neoplasms. They have been described by such diverse names as melanotic medulloblastomas and progonomas. Review of 18 reported cases of intracranial melanotic primitive neuroectodermal tumors, including the present one, reveals that they have common pathologic features, are most frequent in the cerebellum and fourth ventricle, often metastasize widely within the neuraxis or even systemically, occur more frequently in children than adults, and strike males more often than females. PMID- 3014223 TI - The demonstration of papilloma virus in cervical dysplasia and/or neoplasia. AB - Thirty-three random biopsy specimens of uterine cervix were studied by immunoperoxidase technique to evaluate the presence of HPV antigen in dysplastic and neoplastic cells. Four cases of mild dysplasia, nine cases of moderate to severe dysplasia, two cases of carcinoma in situ and one case of invasive carcinoma were associated with condylomatous lesions. Intranuclear dark-brown precipitates were observed in superficial and intermediate zones of the dysplastic epithelium. Positive reaction was seen in condylomatous epithelium associated with neoplasia. No nuclear precipitates were observed in carcinoma cells, but one noncondylomatous lesion with moderate dysplasia showed positive intranuclear staining. These preliminary findings suggest that papilloma viruses may play a role in the development of human cervical neoplasias. PMID- 3014225 TI - Cystic islet cell tumor of the pancreas. AB - Presented is the fifth case of a cystic islet cell tumor of the pancreas. The diagnosis is usually made postoperatively but should be considered in a patient with "recurrent pseudocyst" or a history incompatible with pseudocyst in combination with a cystic area in the pancreas. Computed tomography is preferred for diagnosis, and resection is the preferred therapy. PMID- 3014226 TI - Myoepithelioma of the breast: histologic, immunologic, and electromicroscopic appearance. AB - This report describes the histologic, immunologic, and ultrastructural features of a distinctive spindle-cell tumor of the female breast interpreted as pure myoepithelioma. By light microscopy, the tumor showed the mammary parenchyma replaced by bundles of fusiform cells, which cytoplasms contained myosin and actin, demonstrated immunologically. Ultrastructurally, the spindle cells were joined by mature desmosomes and presented parallel bundles of microfilaments and remnants of basal lamina. PMID- 3014227 TI - Dysplastic nevus syndrome: association with multiple primary neoplasms. AB - A 65-year-old white man with dysplastic nevus syndrome is presented. The patient also developed an extramammary Paget's disease of the scrotum, two malignant melanomas of the skin of the arm and abdomen, two squamous cell carcinomas in the mouth, and several benign tumors such as lentigo maligna, dermatofibroma, and a cavernous hemangioma. Besides the well-established tendency of patients with the dysplastic nevus syndrome to develop malignant melanomas of the skin, their possible propensity to develop other primary malignant neoplasms is discussed. PMID- 3014228 TI - Adenocarcinoma of the breast associated with silicone injections. AB - A 42-year-old woman developed inflammatory breast cancer in a breast with "silicone mastitis" 12 years after bilateral breast augmentation with liquid silicone injections. Despite aggressive local and systemic therapy, the patient died of her disease. Breast cancer arising in silicone-injected breasts is reported infrequently, and physicians caring for patients with silicone breast injection augmentation should be aware of this potentially fatal association with breast cancer. PMID- 3014229 TI - A pleomorphic hepatocellular carcinoma with biochemical features of fibrolamellar hepatocellular carcinoma. AB - A case of pleomorphic hepatocellular carcinoma with biochemical characteristics similar to those of fibrolamellar carcinoma is described. During chemotherapy there was a marked disorder of vitamin B12 metabolism. PMID- 3014230 TI - Prevention of ischemia-induced myocardial platelet deposition by exogenous prostacyclin. AB - The antithrombotic effects of prostacyclin infusion on myocardial platelet deposition were studied in a canine model during and after global ischemia. Eleven isolated heart preparations were subjected to 1 hour of cardioplegic arrest under moderate hypothermia (27 degrees to 28 degrees C), including a control group (n = 7) and a prostacyclin-treated group (n = 4). The hearts of four other dogs were continuously perfused for 180 minutes. Platelet deposition was measured at 15 minute intervals throughout the 3 hour study. Serial full thickness myocardial biopsy specimens were analyzed for activity of 111In-labeled platelets with 99mTc-labeled erythrocyte correction for tissue blood content. The pattern of platelet distribution was determined by scintiscans of each heart, taken with a gamma camera at the end of the 60 minute reperfusion period. Substantial myocardial platelet deposition was found in the control hearts after ischemia but not in the prostacyclin-treated group (p less than 0.05). Furthermore, prostacyclin infusion had a significant disaggregatory effect on intracoronary platelet deposits when the precardioplegic and postcardioplegic biopsy specimens were analyzed (p less than 0.05). Three hours of continuous perfusion did not increase tissue 111In-labeled platelet activity. Ex vivo images showed platelet deposition to be a diffuse patchy process with significantly more 111In activity in the endocardium than in the epicardium after global ischemia (p less than 0.05). These data show the potent antithrombotic properties of prostacyclin in preventing and disaggregating ischemia-induced intracoronary platelet deposition during and after cardioplegic arrest. PMID- 3014232 TI - Altered lymphatic circulation at the site of melanoma excision. AB - Local excision of malignant melanoma promotes both disruption and regeneration of regional lymphatics. These disturbances in local lymphatic drainage favor escape of residual melanoma cells either locally or in transit from more distal sites. Accordingly, a wide tridimensional resection to eradicate all local tumor and circumvent interstitial entrapment and migration of melanoma cells is still advocated. Changes in lymph vessels after excision also demand caution when distal endolymphatic isotopes are administered. Lymph leakage and trapping with overconcentration of the isotope may result in excess local irradiation and skin breakdown. PMID- 3014231 TI - Serum angiotensin-converting enzyme and lysosomal enzymes in asbestosis. PMID- 3014233 TI - Effects of magnesium deficiency on the pathogenesis of myocardial infarction. AB - The death rate due to myocardial infarction appears to vary with dietary consumption of Mg. This could be due to effects on atherosclerosis, coronary artery spasm, altered pathogenesis of myocardial infarction, increased vulnerability to arrhythmia, or some combination of these. Mg deficiency (MD) has been found to increase the severity of a coronary occlusive event in terms of the amount of necrosis produced by a given occlusion. MD is also associated with increased likelihood of arrhythmia development. In addition, reduced extracellular magnesium concentration (Mgo) is associated with contraction of vascular smooth muscle that may be the equivalent of arterial spasm. In hamsters, MD leads to fibrinoid necrosis thought to be secondary to Ca overload. These 3 effects: coronary artery spasm, cardiac arrhythmia, and increased vulnerability to myocardial necrosis following coronary occlusion, may all be dependent on changes in myocardial and vascular smooth muscle electrolyte metabolism that follow from the reduced Mgo that is associated with MD. PMID- 3014234 TI - Magnesium deficiency in the pathogenesis of mitral valve prolapse. AB - Idiopathic mitral valve prolapse (MVP) is the commonest valvular disorder in industrialized nations. It is predominantly a familial condition, showing Mendelian dominance with delayed and variable penetrance. Although hyperkinesis and hypertrophy of the left ventricle have been described in MVP, its histopathology, somatic morphology and genetics support the leading theory that MVP results from a hereditary disorder of connective tissue. Latent tetany (LT) due to chronic Mg deficit (Mg-D) occurs in over 85% of MVP cases; MVP complicates 26% of LT. Mg-D can explain many clinical features of the MVP syndrome which are not easily explained by its genetics. Mg-D hinders the mechanism by which fibroblasts degrade defective collagen, increases circulating catecholamines, predisposes to cardiac arrhythmias, thromboembolic phenomena and dysregulation of the immune and autonomic nervous systems. Mg therapy provides relief of MVP symptoms. PMID- 3014235 TI - Autonomic interactions in the aging heart: age-associated decrease in muscarinic cholinergic receptor mediated inhibition of beta-adrenergic activation of adenylate cyclase. AB - The ability of the muscarinic receptor agonist, carbachol, to inhibit beta adrenergic activation of adenylate cyclase was examined in cardiac membranes from 6-month (young adult) and 24-month (aged) old rats in an effort to assess the effect of aging on adrenergic-cholinergic interactions in the heart. At varying concentrations (0.1-100 microM) of carbachol, GTP plus isoproterenol-stimulated adenylate cyclase activity was inhibited 5-39% in cardiac membranes from 6-month old rats; this inhibition was statistically significant at all but the lowest concentration (0.1 microM) of carbachol used. In contrast, in cardiac membranes from 24-month-old rats, the inhibition of GTP plus isoproterenol-stimulated adenylate cyclase activity by carbachol was very weak (3-20% with 0.1-100 microM carbachol), and statistically insignificant. The muscarinic receptor antagonist, atropine, blocked the inhibition of GTP plus isoproterenol-stimulated enzyme activity by carbachol showing that the observed inhibitory effect of carbachol was muscarinic receptor dependent. The basal adenylate cyclase activity (which showed no significant age-related difference) was unaffected by carbachol. No significant age-related differences were evident in: (a) the concentration of carbachol required for half-maximal inhibition of GTP plus isoproterenol stimulated adenylate cyclase activity; (b) the density of muscarinic receptor sites; and (c) their agonist and antagonist binding affinities. The GTP plus isoproterenol-stimulated cyclase activity measured in the absence of carbachol was approximately 70% lower in cardiac membranes from 24-month-old, compared to 6 month-old rats, confirming an age-associated decline in beta-adrenergic activation of the cyclase observed in our previous study [Mech. Ageing Dev., 19: (1982) 127-139]. The above findings suggest an apparent age-related decline in the postsynaptic antiadrenergic action of cholinergic stimulus in the heart; thus, exaggerated cholinergic antagonism of beta-adrenergic stimulus does not seem to contribute to the impaired adrenergic control of the heart in aging. On the other hand, autonomic imbalance, due to excessive weakening of the antiadrenergic influence of cholinergic stimulus, may compromise the ability of the cholinergic system to counteract the tendency of unrestrained adrenergic drive to increase ventricular vulnerability to fibrillation; this, in turn, may favor the high incidence of cardiac arrhythmias in aging. PMID- 3014236 TI - Free radicals and anti-inflammatory drugs. AB - It is widely accepted that oxygen radicals and other activated oxygen species are potent mediators or modulators of acute and chronic inflammation. They are common products of cellular metabolism, where their concentrations are controlled by different protective mechanisms such as superoxide dismutase, catalase etc. In addition to their destructive effects on various macromolecules, oxygen radicals or their products are beneficial e.g., in killing bacteria. Oxygen radicals are also closely related to arachidonic acid metabolism, prostanoids (cyclo-oxygenase pathway) and leukotrienes (lipoxygenase pathway) as well as to lipid peroxidation in general. Also, the classical mediators of inflammation, histamine and bradykinin, may be connected with the release of oxygen radicals. In addition to the earlier described inhibition of formation of prostanoids, non-steroidal anti inflammatory drugs can inhibit production of free radicals or scavenge those already formed. Antirheumatic penicillamine and allopurinol used in the treatment of gout also act on oxygen radicals. New anti-inflammatory compounds with antioxidant properties will be developed in the near future. PMID- 3014237 TI - Diethyldithiocarbamate, but not disulfiram, inhibits alloxan-induced dye accumulation of isolated mouse islet beta-cells. AB - The diabetogenic action of alloxan on pancreatic beta-cells is thought to be mediated by hydroxyl radicals. The initial attack of the radicals is probably at the plasma membrane level. Diethyldithiocarbamate (DDTC) and its dimer disulfiram (Antabuse) have recently been shown to protect against damage by free radical generating agents. The ability of DDTC and disulfiram to inhibit alloxan-induced dye accumulation of isolated ob/ob mice islet beta-cells was therefore studied. Evans blue was used as an indicator of plasma membrane permeability. DDTC (100 microM 1 mM) but not disulfiram (100 microM 1 mM) inhibited alloxan-induced dye uptake of beta-cells. The effect of DDTC on oxygen consumption in a mixture of reduced glutathione (GSH), alloxan and FeSO4 was studied with a Clark-type oxygen electrode. DDTC (20, 100 microM) had no effect on the oxygen consumption of this mixture. It is suggested that the DDTC inhibition of alloxan-induced dye uptake of isolated beta-cells takes place at a step beyond the generation of free radicals. PMID- 3014238 TI - [Usefulness of the determination of anti-HTLV-III antibodies in parenteral drug addicts]. PMID- 3014239 TI - [HTLV-III in patients in a hemodialysis program]. PMID- 3014240 TI - [High-dosage melphalan followed by bone marrow autotransplant in solid tumors of childhood: our experience]. PMID- 3014241 TI - [Hypoglycemia--a report on 5 patients with insulinoma and 1 with pleural mesothelioma]. PMID- 3014242 TI - [Molecular mechanisms in the development of cancer]. PMID- 3014243 TI - Inositol phosphates turnover, cytosolic Ca++ and pH: putative signals for the control of cell growth. AB - The signals that induce a cell to divide are usually external and in the form of a binding of growth factors. We focussed our attention in defining the sequence of events which occurs after the binding of the mitogens to their surface receptors. One of the early membrane events stimulated by growth factors is a Na+ flux coupled to a H+ efflux that is typically inhibited by amiloride. The importance of this event and of the consequent cytoplasmic alkalinization for the cell proliferation is discussed. Recent data indicate that mitogens increase intracellular Ca++ levels and activate protein kinase C by inducing the hydrolysis of membrane phosphoinositides. A role for Ca++ and protein kinase C in activating Na+/H+ A role for Ca++ and protein kinase C in activating Na+/H+ exchange system is discussed. Finally a model is presented that illustrates the first membrane events stimulated by the growth factors. The model reveals an intimate interconnections between phosphoinositide metabolism, cytosolic Ca++ rise, protein kinase C and cytoplasmic alkalinization. PMID- 3014244 TI - Demonstration and characterization of angiotensin-converting enzyme in human pituitary tissue. AB - High activity of angiotensin-converting enzyme was demonstrated in human pituitary tissue. This activity required the presence of chloride ion and was almost completely inhibited by a specific converting enzyme inhibitor captopril (10 nM), indicating that the activity measured is indeed angiotensin-converting enzyme. The specific activity of the enzyme was 1.68 +/- 1.20 nmol hippuric acid generated mg of protein-1 min-1 (mean +/- SD, for 11 specimens). The biochemical features of the enzyme were closely related to the well-characterized human lung converting enzyme, such as molecular weight (290,000), optimum pH (8.0-8.5), the presence of glycoprotein residues, and dependence on chloride ion concentration. These results provide definitive evidence for the presence of angiotensin converting enzyme in human pituitary tissue. PMID- 3014245 TI - Inhibition of the glycogenolytic effects of alpha-adrenergic stimulation and glucagon by cobalt ions in perfused rat liver. AB - Cobalt ions (2 mM) inhibited the glycogenolysis induced by phenylephrine and glucagon in perfused rat liver. Cobalt ions also inhibited 45Ca++ efflux from prelabelled livers induced by phenylephrine and glucagon. In addition, they inhibited the rise in tissue levels of cyclic AMP caused by glucagon, but did not inhibit the stimulation of 45Ca++ efflux or glycogenolysis by cyclic AMP or dibutyryl cyclic AMP. The specific binding of glucagon and alpha-agonist to hepatocytes was not inhibited by cobalt ions. These data suggest that cobalt ions, presumably through their high affinity for calcium binding sites on membranes inhibit the stimulation of glycogenolysis by phenylephrine and glucagon in distinct ways; one by inhibiting calcium mobilization and the other by inhibiting cyclic AMP production. Therefore, it is conceivable that membrane bound calcium plays an important role in stimulating Ca++ mobilization by phenylephrine, and cyclic AMP production by glucagon. PMID- 3014246 TI - Long-term amitriptyline treatment alters the affinity state of alpha 2 adrenoceptors in rabbit hindbrain. AB - The effects of 7 and 21 days amitriptyline treatment on brain stem alpha 2 adrenoceptor number and affinity state and on the in vivo depressor response to intracisternal clonidine were examined in male New Zealand white rabbits. PMID- 3014247 TI - Role of ADH in ethylketocyclazocine-induced diuresis: studies in the Brattleboro rat. AB - Kappa opioids produce diuresis presumably through ADH. We investigated further the role of ADH in kappa-induced diuresis by utilizing the Brattleboro rat, a strain lacking endogenous ADH. Ethylketocyclazocine (EKC), a kappa opioid prototype, increased urine formation in Sprague-Dawley, but not in Brattleboro rats. Furthermore, EKC pretreatment abolished the antidiuretic response to ADH administered exogenously to Brattleboro rats. Our study suggests that, in addition to a fall in plasma ADH reported previously, kappa opioids have direct effects on the renal response to ADH. PMID- 3014248 TI - Is Metaphit a phencyclidine antagonist? Studies with ketamine, phencyclidine and N-methylaspartate. AB - The dissociative anaesthetics, phencyclidine and ketamine, block excitation of central neurones by N-methylaspartate. Using the technique of microelectrophoresis on rat spinal neurones in vivo Metaphit, a phencyclidine receptor acylating agent, was tested to see whether it would antagonise this effect of dissociative anaesthetics. The predominant effect of Metaphit was, however, to reduce N-methylaspartate induced excitation. It is concluded that Metaphit has mixed agonist/antagonist effects at the phencyclidine receptor. PMID- 3014250 TI - Stereospecific effects of d- and l-pentazocine on contractions of the mouse vas deferens. AB - The effects of the d- and l-isomers of pentazocine were compared to that of racemic pentazocine on contractions of the mouse isolated vas deferens. L pentazocine inhibited electrically evoked contractions of the mouse vas deferens (MVD) in a dose-dependent manner (ID50 0.37 +/- 0.04 microM). In contrast, d pentazocine augmented field stimulated contractions dose-dependently; per cent increases in contractions at 10 and 30 microM were 57.8 +/- 18.0 and 98.0 +/- 15.1%, respectively. Racemic pentazocine produced an intermediate effect between the two isomers. The effect of 1-pentazocine was antagonized by naloxone, whereas that of d-pentazocine was not. L-pentazocine did not effect the response of the MVD to exogenous norepinephrine at any concentration tested, while d-pentazocine depressed the response of the MVD to exogenous norepinephrine at one dose (0.3 microM). These findings demonstrate that d- and l-pentazocine produce opposite effects on the MVD. The effects of l-pentazocine are opioid mediated, while those of d-pentazocine are not. In the racemic mixture the opposing effects of the two isomers modulate each other, resulting in a diminished effect. PMID- 3014249 TI - Vascular vasopressin receptors mediate phosphatidylinositol turnover and calcium efflux in an established smooth muscle cell line. AB - Vasopressin-induced phosphatidylinositol turnover and mobilization of intracellular Ca2+ was studied using an established smooth muscle cell line (A 10). The cells were subcloned to ensure a monoclonal cell population. The accumulation of inositol mono-, di-, and tris-phosphates (IP1, IP2, and IP3, respectively), and the mobilization of intracellular Ca2+ were dependent on the time of incubation and the concentration of arginine vasopressin (AVP). IP1, IP2, and IP3 were significantly elevated after 15 sec and remained elevated for up to 2 hr. The concentrations of AVP required for half-maximal stimulation of IP1, IP2, and IP3 formation were 2, 12, and 4 nM, respectively. LiCl was required to observe the accumulation of inositol phosphates in response to AVP. Significant 45Ca2+ efflux was observed within 15 sec after exposure to AVP. By employing the vasopressin receptor subtype selective antagonists [d(CH2)5Tyr(Me)AVP, V1; d(CH2)5D-Tyr(Et)VAVP,V1/V2; d(CH2) 5D-IleVAVP,V2] and agonists [AVP, V1/V2; dDAVP, V2; dVDAVP, V2], we found that the vasopressin-induced stimulation of phosphatidylinositol turnover and 45Ca2+ efflux were mediated by receptors of the vascular V1 subtype. Pertussis toxin pretreatment partially inhibited vasopressin induced phosphatidylinositol turnover. These data demonstrate that activation of V1 receptors of vascular smooth muscle cells resulted in enhanced phosphatidylinositol turnover and mobilization of intracellular Ca2+. PMID- 3014251 TI - A tetrapeptide isolated from hamster embryo with central opiate properties on gastrointestinal motility but not on pain perception. AB - The effects of centrally administered kentsin (H-Thr-Pro-Arg-Lys-OH) on intestinal motility and on pain perception were investigated in rats chronically equipped with lateral ventricle catheters. Intestinal motility was recorded electromyographically from electrodes placed on the duodeno-jejunum; analgesia was evaluated by the hot-plate and tail-flick tests. Kentsin (4.0 ug/kg), injected intracerebroventricularly (ICV) 2 hours after the beginning of a meal, restores the "fasted" i.e. the migrating myoelectric complex of intestinal motility, while a 5 times higher dose administered subcutaneously was inactive. The ICV effect of kentsin was blocked by previous ICV administration of naloxone (400 ug/kg). In contrast, kentsin administered ICV (40 ug/kg) or SC (200 ug/kg) did not affect significantly (P greater than 0.05) the time latency in the two analgesic tests during 90 minutes after its administration and did not significantly modify the analgesic effects of (D5-Ala2, Met5) enkephalinamide. We conclude that kentsin when centrally administered acts on opiate receptors to alter gastrointestinal motility but without effects on pain perception. PMID- 3014252 TI - Effect of chronic uremia on the cardiovascular alpha 1 receptor. AB - Adrenergic dysfunction in uremia has been well described. Several lines of evidence suggest disorders of blood pressure regulation and myocardial response may occur secondary to adrenergic dysfunction; attenuated pressor response to norepinephrine (NE) in uremia; attenuated chronotropic responses during dialysis induced hypotension. Since the adrenergic receptors are the effector component of the adrenergic nervous system, we have employed the partially nephrectomized uremic rat, to examine the effect of chronic uremia (4-6 weeks) on the binding properties of alpha 1 receptors in rat mesenteric artery and myocardial tissue. The results indicate that moderate levels of uremia alter the binding properties of both the alpha 1 vascular and myocardial receptor. PMID- 3014253 TI - Neuro-endocrine aspects of the dexamethasone suppression test in psychiatry. PMID- 3014254 TI - High concentrations of catecholamines selectively diminish the sensitivity of CRF stimulated ACTH release by cultured rat pituitary cells to the suppressive effects of dexamethasone. AB - ACTH-release by primary cultures of rat anterior pituitary cells in response to CRF, vasopressin, epinephrine, norepinephrine and VIP is readily suppressible by dexamethasone. Rat hypothalamic extract-induced ACTH release is less sensitive to the inhibitory effect of dexamethasone than that elicited by CRF and the other secretagogues mentioned above. In studying the additive and potentiating effect on ACTH release of CRF in combination with vasopressin, VIP and the catecholamines it became evident that only the combination of micromolar concentrations of epinephrine or norepinephrine together with nanomolar concentrations of CRF will make ACTH release significantly less sensitive to the suppressive effect of dexamethasone. Other combinations of CRF and vasopressin or CRF and VIP will render ACTH release as suppressible to dexamethasone, as that elicited by each of these compounds by itself. This observation in the rat might explain at least in part the observation that a diminished suppressibility of the pituitary-adrenal axis to dexamethasone can be found in patients with psychiatric disease, especially depression. PMID- 3014256 TI - [Does diabetes affect the probability of cure and prolongation of life of oncological patients?]. AB - The authors present an analysis of the results of therapy of 236 breast cancer patients, 212 patients with cancer of the uterine body, 269 patients with bladder cancer and 386 lung cancer patients. The frequency of diabetes mellitus is compared among cancer patients of different age groups. Cancer patients with diabetes mellitus were, on an average, more advanced in age than patients without it. A possible metabolic effect of glucose and acidification of tumors on the patients' cure is discussed. PMID- 3014255 TI - Influence of dietary fiber on lipids and aortic composition of vervet monkeys. AB - A semipurified, cholesterol-free diet containing 40% carbohydrate can produce aortic sudanophilia or aortic atherosclerosis in vervet monkeys (Cercopithecus aethiops pygerethrus) depending on the particular carbohydrate fed. Four groups of vervet monkeys (three males and three females per group) were fed semipurified diets containing lactose. Two of the groups were also fed 15% cellulose (C) or 15% cellulose plus 0.1% cholesterol (CC); the two other groups were fed 15% pectin (P) or 15% pectin plus 0.1% cholesterol (PC). The average serum total cholesterol and low density lipoprotein cholesterol levels over the entire feeding period (mg/dl +/- SEM) were, for C, 156 +/- 14 and 95 +/- 5; for P, 173 +/- 15 and 112 +/- 8; for CC, 187 +/- 27 and 122 +/- 21; and for PC, 155 +/- 11 and 108 +/- 7. Cholesterol levels at autopsy (mg/dl +/- SEM) were, for C, 103 +/- 6; for P, 108 +/- 16; for CC, 92 +/- 9; and for PC, 106 +/- 7. Aortic sudanophilia (percentage of area) was, for C, 5.9 +/- 2.7; for P, 13.5 +/- 9.4; for CC, 5.3 +/- 2.1; and for PC, 21.6 +/- 10.3. Dietary pectin led to more severe sudanophilia (increased by 129% in the absence of cholesterol and by 308% in its presence) than did cellulose. Analysis of aortic glycosaminoglycans (GAG) revealed that dermatan sulfate levels fell in both cholesterol-fed groups, and chondroitin sulfate fell in aortas of group CC. Heparan sulfate levels were unaffected by cholesterol feeding. Hexuronic acid, galactosamine and hexosamine levels were elevated in the pectin-fed monkeys, but levels were unaffected by dietary cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014257 TI - [Radionuclide lympho- and venography in the diagnosis of radiation edema of the lower extremities]. AB - Lymphography and venography were used for diagnosis of radiation edemas of the lower limbs in patients receiving radiation or combined therapy for malignant tumors of the female genital organs. It was established that 99mTc-lymphocys could be used to detect lymphostasis, to determine the level and nature of the lymphatic trochlea. An additional study of the venous blood flow made it possible to determine the type of disorder of the lymph flow. Radionuclide methods of the investigation of the lymph flow and venous blood flow are easy to use, atraumatic and quite informative, therefore they should be recommended for the investigation of patients with radiation edemas of the limbs to determine indications for surgery. PMID- 3014258 TI - [Radionuclide diagnosis of retroperitoneal tumors in children]. AB - In static scintigraphy with 99m Tc-pertechnetate of primary lesions of the retroperitoneal space in children 4 types of scintigraphic pictures were singled out for differential diagnosis of Wilms' tumors, avascular benign lesions of the retroperitoneal space and neurogenic retroperitoneal tumors. The sensitivity of diagnosis of Wilms' tumor was 92.6%, specificity 90.9%. The most objective and complete information on the kidney involvement in tumor process could be obtained in static scintigraphy with 99m Tc-pertechnetate combined with scintigraphy with 99m Tc-gluconate or 99m Tc-pyrophosphate. The sensitivity of diagnosis of secondary lesions of the lymph nodes of the retroperitoneal space was 76.8%. PMID- 3014259 TI - [Effectiveness of super-fractionated irradiation in the radiotherapy of inoperable lung cancer]. AB - The paper is concerned with the results of gamma-beam therapy of 120 patients with inoperable lung cancer using the fractionation of a common daily dose of 2 2.2 Gy into 2 equal fractions as a variant of super-fractionated irradiation used in clinical practice. The direct and short-term therapeutic results were improved: a complete tumor regression rate determined by x-ray was 26.7%, a 2 year survival rate 19.2%. In the group of patients who were irradiated by the commonly used methods, the same values were lower. Improved tolerance of therapy with fractionation of a common daily dose was noted. PMID- 3014260 TI - [Kaposi's sarcoma in Burundi and the Central African Republic in the framework of acquired immunodeficiency syndrome (AIDS)]. AB - The authors carried out in 1985 a survey in two French speaking States in Central Africa, namely Burundi and Central African Republic (C.A.R.), in order to study the links between Kaposi sarcoma (K.S.) and A.I.D.S. In Burundi the prospective study conducted in Bujumbura, lead to collect in one year 25 cases of K.S. out of them 24 linked to A.I.D.S. No group at risk has been identified. The 24 K.S. linked to A.I.D.S. present a stage IV (cutaneous and visceral form) in 21 cases. 20 of them got an associated affection, 5 being tuberculosis bacteriologically confirmed. All of them present a cellular immunity deficiency. Evolution was fatal in 22 cases out of 24, average presumption of survival was 10 months. In C.A.R., retrospective survey conducted in Bangui made possible to find out 24 cases in 4 years, of which 20 having had a L.A.V. antibodies research, were considered. 9 of them were linked to A.I.D.S. No group at risk. 7 patients presented a sporadic form, 6 an African endemic form, 7 an epidemic form with associated infection. Out of 9 LAV positive patients, 5 deceased. Out of 11 LAV negative patients, 3 deceased with a A.I.D.S. clinical aspect. This survey carried out in Burundi and in C.A.R. demonstrates that K.S. is significantly in increase in these two countries. In Burundi it is significantly linked to A.I.D.S. In C.A.R., classical African K.S. do exist (sporadic, endemic), as well as K.S. linked to A.I.D.S., as underlined recently in Bayley's publications in Zambia. Since A.I.D.S. has been detected, it does exist an outbreak and a new clinical form of K.S. in Central Africa. PMID- 3014261 TI - [Waterworks and public health in Ivory Coast]. AB - In Ivory Coast, the provision for good drinking water has received priority attention since 1973. The National Water-works Programme which started in 1975, is progressing satisfactorily. All over the country, villages and towns are being equipped with hydraulic machines and other structures for the production and distribution of good drinking water. This has contributed in no small way to the control of certain diseases related to water pollution. The authors think however that a lot still needs to be done especially to inform and to educate the village communities and to control the quality of the drinking water. These actions should check water pollution and encourage people in the rural areas to drink portable pipe-borne water. PMID- 3014262 TI - Specific high affinity binding and degradation of high density lipoproteins by isolated epithelial cells of human small intestine. AB - The interaction of high density lipoproteins (HDL) with isolated epithelial cells of human small intestine (enterocytes) was studied. 125I-HDL3 (density = 1,125 to 1,126 g/cm3) exhibit a high-affinity (Kd = 8.3 X 10(-8). Bmax = 886 ng/mg cell protein), saturable and reversible binding to isolated enterocytes. In the presence of excess unlabeled HDL3, the cell surface-bound 125I-HDL3 are released into the medium nondegraded. Treatment of cells with pronase does not affect 125I HDL3 binding. The binding is accompanied with internalization and degradation of 125I-HDL3. Chloroquine inhibits the degradation and increases 125I-HDL3 uptake. A threefold excess of HDL3 and HDL2 inhibits the binding and degradation of 125I HDL3 by 60%, whereas a 20-fold excess of low density lipoproteins (LDL), only by 20%. HDL3 (20 to 1,000 micrograms/mL) stimulates the synthesis of sterols and inhibits sterol ester synthesis in enterocytes. The obtained results make it possible to assume that epithelial cells of the small intestine may participate in the catabolism of HDL in human organism. PMID- 3014263 TI - Optimal strategies for developing human-human monoclonal antibodies. AB - Human monoclonal antibodies are desirable, especially as therapeutic agents, but the best means of producing them is still a matter of investigation. It is clear that human antibodies of predicted specificity from patients with autoimmune disease can be derived, and this may help unlock some of the mysteries of these illnesses. Human monoclonal antibodies against tumor-specific antigens for use in in vivo diagnosis and therapy remain desirable goals. Problems involved in their routine development include the lack of available, adequately immunized, and differentiated lymphocytes and the nature and paucity of the available human "myeloma" cell lines. These lines have been compared now by a number of authors who have reached similar conclusions. Our study directly compared the greatest number of cell lines and found UC729-6 and HF2 to be the best; on the other hand, our success in developing IgG-secreting hybridomas from U-266, using hyperimmunized lymphocytes, suggests that this line may only be capable of secretion with the more differentiated cell, the human equivalent of those hyperimmunized murine spleens. Hence both sides of the fusion equation must be made optimal. Two new approaches to circumvent this problem involve the use of either a human-murine myeloma chimera as the parental myeloma line or, more recently, genetic engineering techniques to substitute human constant regions for the murine while retaining the murine hypervariable region, preserving the binding specificity of the murine antibody. PMID- 3014265 TI - The EBV-hybridoma technique. PMID- 3014264 TI - Comparative phenotypic analysis of available human hybridoma fusion partners. PMID- 3014266 TI - Generation of human monoclonal antibodies by fusion of EBV-activated B cells to a human-mouse hybridoma. PMID- 3014267 TI - Improved hybridoma technology: spleen cell separation and soluble growth factors. PMID- 3014268 TI - In vitro immunization for the production of antigen-specific lymphocyte hybridomas. PMID- 3014269 TI - Separation of IgG idiotypes by high-performance hydroxylapatite chromatography. PMID- 3014270 TI - Intra- and extracellular modifications of apolipoproteins. AB - This chapter outlined the methods used to study intra- and extracellular modifications of apolipoproteins. These and other related studies have shown that several of the apolipoproteins undergo a series of intra- and extracellular modifications as follows: All apolipoproteins studied contain an 18-26 long signal peptide which is cleaved cotranslationally by the signal peptidase of the rough endoplasmic reticulum. ApoE is further modified intracellularly with carbohydrate chains containing sialic acid and is secreted in the modified form designated apoEs. The modified apoE is subsequently desialated in plasma. ApoA-I is secreted in a proapoA-I form, which consists of 249 amino acids. The N terminal hexapeptide of proapoA-I is cleaved extracellularly by a proapoA-I to plasma apoA-I converting protease. This cleavage generates the plasma apoA-I form which consists of 243 amino acids. Other known apolipoprotein modifications include the modification of apoB, apoC-III, and apoD with carbohydrate chains that contain sialic acid and the proteolytic cleavage of the proapoA-II segment. At the present time we are able to distinguish several isoprotein forms for a particular apolipoprotein. In addition, we began to understand the biochemical changes which lead to a few of these isoproteins. Future research should be directed toward a better understanding not only of the structure but most importantly of the physiological significance of the different apolipoprotein forms. PMID- 3014272 TI - Isolation of cDNA and genomic clones for apolipoprotein C-II. PMID- 3014271 TI - Characterization of the apolipoprotein A-I-C-III gene complex. PMID- 3014273 TI - Cloning and expression of the human apolipoprotein E gene. PMID- 3014274 TI - Receptor-mediated transport of cholesterol between cultured cells and high density lipoproteins. PMID- 3014275 TI - Lipoproteins and steroid hormone-producing tissues. PMID- 3014276 TI - Isolation, characterization, and assay of lecithin-cholesterol acyltransferase. PMID- 3014277 TI - Protective effects of nasal immunization in mice with various kinds of inactivated Sendai virus vaccines. AB - The immunoprophylactic effects of nasal vaccination with 13 different kinds of inactivated Sendai virus vaccines were compared by contact exposure to infector mice. Efficacies of the vaccines were evaluated on the basis of the presence of virus-infected cells by immunofluorescent examination of the entire respiratory tract, including the nasal mucosa. A single or double inoculations of B propiolactone (0.5%)-vaccine promoted the infection in the respiratory tract, particularly in the nasal mucosa, whereas three inoculations of B-propiolactone (0.2%)-vaccine provided considerable protection throughout the respiratory tract with only slight development of serum HI titer. Formalin (0.1%)-vaccine and UV irradiated-vaccine strongly protected the nasal mucosa from infection, but did not sufficiently safeguard the lower respiratory tract even with three vaccinations despite adequate development of serum antibody. Nearly complete protection of the entire respiratory tract was induced with six to eight inoculations of a vaccine treated excessively with both UV rays and 1% formalin, without significant development of serum antibody. Out of eight thermal vaccines, five (inactivated at 23 C, 30 C, 37 C and 7 C, and 30 C and 7 C) provided strong protection against infection when inoculated three times. The others inactivated at higher temperatures (37 C, 50 C, or 60 C) were not so protective. High serum HI titers developed, on the whole, with the drop in the temperature required for inactivating the virus. In eight immune mouse groups in which infection was strongly suppressed in the entire respiratory tract, most of the mice harbored less than 50 viral antigen-positive cells in their nasal mucosa in the postexposure period. The number of the cells was assumed to be a useful criterion for evaluation of vaccine efficacy. PMID- 3014278 TI - Massive induction of Staphylococcus aureus unstable L-forms in a liquid medium. PMID- 3014279 TI - Tubular structures detected in the nuclei of human embryonal lung fibroblasts infected with human cytomegalovirus. PMID- 3014280 TI - [Detection of antibodies against TORCH agents during pregnancy]. AB - The antibody levels against TORCH agents in sera samples from pregnant women who admitted to Hacettepe Medical Faculty Hospital Obstetrics and Gynecology Department were tested. The measurements of IgG antibodies by ELISA against Toxoplasma gondii in 301 sera was given 47.8% sero-positivity. Rubella ELISA-IgG test performed for 226 sera given 89.8% seropositive value. 128 sera were also tested for the presence of Cytomegalovirus (CMV) IgG antibodies and 87.5% seropositivity is found. Complement fixation test is employed for the detection of Herpes simplex virus type 1 (HSV) antibodies in 65 sera samples and 87.5% of sera tested were given positive result in various titers. These results are discussed in terms of seronegativity of this population, sensitivity of ELISA test and the distribution of antibody levels. PMID- 3014281 TI - [Molecular biology technics in the clinical microbiology laboratory]. PMID- 3014282 TI - A deficiency in dietary gamma-linolenic and/or eicosapentaenoic acids may determine individual susceptibility to AIDS. AB - We hypothesize that a relative deficiency in gamma-linolenic and eicosapentaenoic acids and in their derivatives may contribute to the development of AIDS. These polyunsaturated fatty acids (PUFAs) may be the source of natural endogenous agents against AIDS by preventing the spread of viral infection due to their ability to destroy enveloped viruses, by controlling cancer development either directly due to their cytostatic and cytotoxic effects on cancer cells or indirectly by modulating the immune response and by protecting from genetic damage. Supplementation of these dietary PUFAs in the prevention, and possibly in the treatment of AIDS, is considered. PMID- 3014283 TI - Absence of antibodies to HTLV-III/LAV virus in family members who administer coagulation products in home treatment programmes. PMID- 3014285 TI - [Warts in humans. Pathogens--clinical aspects--therapy]. PMID- 3014284 TI - Absence of antibodies to HTLV-III on Bougainville (Papua New Guinea) PMID- 3014286 TI - Studies on the mechanism of lymphocytic choriomeningitis virus homologous interference. PMID- 3014288 TI - Progressive extension of the endemic area and changing incidence of Argentine Hemorrhagic Fever. PMID- 3014289 TI - Heterogeneity of Junin virus strains. PMID- 3014287 TI - Relationship between Junin virus infection of thymus and the establishment of persistence in rodents. PMID- 3014290 TI - Current status of the treatment of Argentine Hemorrhagic Fever. PMID- 3014291 TI - Damage of human polymorphonuclear leukocytes by Junin virus. PMID- 3014293 TI - Arenaviruses. Fourth International Symposium of the Heinrich-Pette-Institut fur Experimentelle Virologie und Immunologie an der Universitat Hamburg. Hamburg, 23 26 September 1985. PMID- 3014292 TI - Effect of ribavirin and immune serum on Junin virus-infected primates. PMID- 3014294 TI - Photodynamic inactivation of Junin virus. PMID- 3014295 TI - Cross reactivity between Junin and Tacaribe viruses as determined by neutralization test and immunoprecipitation. PMID- 3014296 TI - Toward an automated analysis system for nuclear magnetic resonance imaging. II. Initial segmentation algorithm. AB - Image segmentation algorithms based on hierarchical clustering have been developed for analysis of T1 and T2 nuclear magnetic resonance images. Application of these algorithms to simultaneous T1-T2 images of healthy volunteers extracted fundamental tissue types in the brain. These algorithms also were used both to identify the extent of the region of involvement of a subject with a history of a grade 3 astrocytoma of the right frontal lobe of the brain, and to characterize the tissue within the region of involvement. These results suggest that a simple segmentation algorithm can produce reasonable clustering of tissue types within the brain. PMID- 3014297 TI - Comments on "Dental enamel as an in vivo radiation dosimeter". PMID- 3014298 TI - Differential endocytosis of lipoproteins by capillary endothelial vesicles. AB - Plasma levels of lipoproteins are believed to be controlled largely by lipoprotein receptors on the surfaces of tissue cells which facilitate their internalization and degradation. This presupposes transit of lipoproteins across the walls of blood vessels in order to gain access to the receptor sites. The endothelium of tissue capillaries may therefore constitute an additional regulatory barrier for lipoproteins. In order to test this hypothesis, we have measured the uptake of fluorescent-labeled lipoproteins into endothelial vesicles of capillaries isolated from fat tissue. HDLs are ingested at more than twice the efficiency of LDLs and VLDLs are excluded from vesicular ingestion. This represents a decreased efficiency of ingestion with an increase in molecular diameter of lipoproteins. This phenomenon correlates well with the dimensions of endothelial vesicles and caveolae which may restrict entry of very large serum lipoproteins. Selective transport of lipoproteins by capillary endothelial vesicles on the basis of molecular size may therefore serve to regulate blood tissue interchanges of lipid. PMID- 3014299 TI - Response of the forelimb vasculature to vasoactive agents: effects of ouabain. AB - The effect of the local intra-arterial infusion of ouabain (11.8 micrograms/min.) on the response of the forelimb to vasoactive agents was examined. In seven dogs, bolus injections of CaCl2, MgSO4, KCl, norepinephrine, adenosine, acetylcholine, PGE1 and saline were made into the forelimb perfused at constant flow before and three times during ouabain infusion. Ouabain blocked potassium vasodilation and changed the response to CaCl2 from vasoconstriction to vasodilation. The response of the forelimb to the other vasoactive agents was initially unaffected by ouabain but with time the forelimb vasculature became less sensitive to all agents studied. These changes were not seen in a series of 5 saline infused control animals. In a third series of animals steady-state dose responses to CaCl2, Ca-gluconate and KCl were explored by infusing solutions intrabrachially at three dosages. Before ouabain, forelimb resistance increased as a function of Ca++ and decreased as a function of K+. Ouabain completely blocked potassium vasodilation and on the average blocked Ca++ vasoconstriction although a number of animals evidenced vasodilation to Ca++ during ouabain infusion. These data indicate that K+ vasodilation is Na+, K+-ATPase dependent and that Na+, K+-ATPase inhibition unmasks a vasodilatory action of locally applied Ca++. PMID- 3014300 TI - Reduced parathyroid hormone response to peroral phosphate in osteoporotic patients. AB - To determine whether PTH secretion can be stimulated in osteoporosis by peroral phosphate (1.5 g phosphorus), we have compared serum PTH and urinary cyclic AMP responses in 8 osteoporotic patients with vertebral compression fractures to those of 7 age-matched control subjects. While there was no statistically significant difference in the rise of serum phosphate and urinary phosphate excretion and in the fall of serumionized calcium concentrations, serum PTH and urinary cyclic AMP were increased in control subjects, but not in osteoporotic patients (osteoporotics vs. control group: PTH, p less than 0.01; cyclic AMP/glomerular filtrate, p less than 0.05). Moreover, plasma levels of 25 hydroxyvitamin D and 1,25-dihydroxyvitamin D were the same in the 2 groups of subjects. Serum levels of the carboxyl-terminal flanking peptide (PDN-21) encoded by the calcitonin gene and cosecreted with calcitonin were similar in normal and osteoporotic subjects before and 3 min after 1-min intravenous calcium infusions (2 mg/kg body weight), and of calcitonin after the calcium infusions. We conclude that calcitonin and PDN-21 responses to intravenous calcium are the same in normal and osteoporotic subjects, whereas stimulation of PTH secretion by peroral phosphate is impaired in some osteoporotic patients. PMID- 3014301 TI - [Renal tubular acidosis and deafness. Description of a case]. PMID- 3014302 TI - Acquired immunodeficiency syndrome. Recommendations to prevent transmission for hospitals and health care workers. PMID- 3014303 TI - The decision to confront a chemically dependent colleague. PMID- 3014304 TI - Computers and education. PMID- 3014305 TI - Human T-lymphotropic virus type III/lymphadenopathy-associated virus antibody prevalence in U.S. military recruit applicants. PMID- 3014306 TI - Spontaneous coupling of the beta-adrenergic receptor to Ns in mammalian cardiac membranes. AB - The beta-adrenergic receptor in membranes from cat, rat, and guinea pig hearts appears to undergo spontaneous coupling to the stimulatory nucleotide-regulatory protein, Ns. This was demonstrated by incubating cardiac membranes from acutely reserpinized animals with the alkylating reagent N-ethylmaleimide (NEM), which is known to "freeze" the beta-receptor Ns-complex. The concentration of norepinephrine in these membrane preparations was less than 0.1 nM. Cat heart membranes were preincubated for 10 min at 30 degrees with NEM (10(-7)-10(-3) M) and subsequently incubated with (-)-[125I]iodopindolol (IPIN) (18 min, 30 degrees). NEM caused a concentration-dependent decrease in specific IPIN binding with a maximal reduction of about 20% at 0.1 mM. This decrease occurred even in the absence of MgCl2 (0.5 mM EDTA added as well). A comparable reduction was also observed in myocardial membranes from rat and guinea pig. This fall reflects a decrease in the number of beta-adrenergic receptor sites, as demonstrated by saturation binding experiments with IPIN. The equilibrium dissociation constant of the radioligand for the remaining receptors was not affected. When increasing concentrations of GTP were included in the preincubation mixture, it resulted in a dose-dependent protection of NEM-induced decrease in IPIN binding. The protection was complete at 0.1 mM GTP. In addition, GTP reversed the NEM effect when added to the incubation mixture 10 min after NEM. The apparent reduction in cardiac beta-adrenergic receptor number by NEM (in the absence of beta-receptor agonist) is compatible with a model in which part of the receptor population is able to undergo spontaneous coupling to Ns. PMID- 3014307 TI - Acute and chronic effects of ethanol on receptor-mediated phosphatidylinositol 4,5-bisphosphate breakdown in mouse brain. AB - Phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown was stimulated by agonists acting at muscarinic cholinergic and alpha 1-adrenergic receptors in mouse brain. Ethanol, in vitro, inhibited basal cerebral cortical PIP2 breakdown with a threshold concentration of 75-100 mM. Basal PIP2 breakdown in hippocampus and striatum was less sensitive to ethanol. A high concentration of ethanol (500 mM) increased the EC50 for carbachol stimulation of PIP2 breakdown in all three brain areas, but had no effect on the EC50 for norepinephrine. Following chronic ingestion of ethanol by mice, the EC50 for carbachol stimulation of PIP2 breakdown in cortex was decreased, and there was no change in striatum. These effects were consistent with previously observed increases in quinuclidinylbenzilate (QNB) binding in cortex, but not striatum, of mice fed ethanol chronically. However, in hippocampus, where chronic ethanol ingestion had also induced an increase in QNB binding, the EC50 for carbachol stimulation of PIP2 breakdown was increased. Binding studies using the specific M1 muscarinic cholinergic receptor antagonist, pirenzepine, revealed that the number of pirenzepine-binding sites was increased in cortex, but not hippocampus (or striatum) of ethanol-fed mice. These results support the hypothesis that high affinity pirenzepine-binding sites are coupled to PIP2 breakdown in mouse cortex. The changes in cerebral cortex represent one of the first demonstrations of a functional correlate of a change in receptor density in ethanol-treated animals. Increased sensitivity to cholinergic agonists in cortex may contribute to particular signs of ethanol withdrawal. PMID- 3014308 TI - Role of sodium-calcium exchange and effects of calcium entry blockers on endothelial-mediated responses in rat isolated aorta. AB - Acetylcholine relaxes rat aorta and increases aortic cyclic GMP levels by a mechanism (or mechanisms) dependent on the endothelium and on extracellular calcium. Therefore, the effects of representatives of different subclasses of calcium entry blockers, verapamil, nifedipine, diltiazem, and bepridil, on maximal acetylcholine (1 microM)-induced increases in cyclic GMP levels were investigated in rat isolated aorta. None of these compounds, at a concentration (3 microM) sufficient to maximally inhibit agonist-stimulated Ca2+ influx into vascular smooth muscle cells, significantly affected either the basal or the acetylcholine-stimulated tissue cyclic GMP levels. On replacing all but 20 mM Na+ by choline, a condition that might be expected to limit or even abolish Na+-Ca2+ exchange, or in the presence of amiloride (1 mM), an inhibitor of Na+-Ca2+ exchange, acetylcholine-stimulated increases in tissue cyclic GMP levels were abolished or inhibited by about 80%, respectively. In choline containing solution acetylcholine relaxant responses were abolished. The presence of amiloride, or the replacement of Na+ by choline, had no effect on increases in cyclic GMP levels evoked by sodium nitroprusside (0.3 microM), an agent that stimulates cyclic GMP formation in smooth muscle without intervention of the endothelium. Replacement of Na+ by Li+ but not the other treatments depressed basal tissue cyclic GMP levels by about 45% but did not abolish either acetylcholine- or sodium nitroprusside-induced relaxant responses. However, the time course of relaxant responses elicited by both these relaxant agonists in precontracted rat aortic rings with endothelium was altered by Li+ replacement; the half-time to relaxation to acetylcholine was increased by about 70-fold. It is concluded that calcium channels, as characterized in smooth muscle and cardiac tissue, are not involved in the stimulated liberation of an endothelial-derived relaxant factor by acetylcholine, but that an Na+-Ca2+ exchange process may be of importance. PMID- 3014310 TI - Regulatory effects of S-100 protein and parvalbumin on protein kinases and phosphoprotein phosphatases from brain and skeletal muscle. AB - In the eluted fractions of histone-treated crude extracts separated by Sephadex G 200 filtration, multiple protein kinase (PK) activities, including three from brain and two from skeletal muscle, were augmented by both S-100 protein and parvalbumin on the phosphorylation of endogenous substrates. One additional PK activity suppressed by both S-100 and parvalbumin was also found in muscle. In comparison, phosphoprotein phosphatases (PPase), which were also prepared by the same procedure of initial step of histone-treatment followed by the steps of Bio Gel P-6DG for brain and DNA-cellulose for muscle, were all activated by S-100 while inhibited by parvalbumin and phosphatidylserine. PMID- 3014312 TI - [Structural analysis of alphoid DNA of primates. I. Heterogeneity of nucleotide sequence of alphoid repeats in human DNA]. AB - The nucleotide sequence of two cloned fragments of human alphoid DNA was established. These fragments were earlier characterized in our laboratory as molecular markers of the 3rd (pHS05) and 11th (pHS53) chromosomes. Fragment pHS53 (2546 bp) contains alphoid repeats tandemly arranged and organized into three highly homologous pentamers. The heterogeneity of monomeric sequences within individual pentamers reaches 24-33%. Structural analysis of EcoRI subfragment pHS05 showed that this alphoid tetramer consists of two dimers 340 bp long. These dimers differ up to 16% from each other and from the so-called consensus sequence of the EcoRI-340 bp-restriction fragments family reported earlier by Wu and Manuelidis. The primary structure of four cloned fragments of EcoRI-340 bp-family was established. The data show that human alphoid DNA is highly heterogeneous. This conclusion is opposite to the view suggesting that alphoid DNA is a highly homogeneous class of reiterated sequences of the human genome. PMID- 3014311 TI - AMP deaminase in Dictyostelium discoideum: increase in activity following nutrient deprivation induced by starvation or hadacidin. AB - AMP deaminase, the activity that catalyzes the deamination of AMP to form IMP and NH3 has been measured in Dictyostelium discoideum. A new procedure to assay the activity of this enzyme was developed using formycin 5'-monophosphate, a fluorescent analog of AMP as the substrate, and ion-paired reverse phase HPLC to separate the reactants and products. Quantitation of the formycin containing compounds was accomplished at 290 nm. At this wavelength adenosine containing compounds were not detected and activity could be monitored in the presence of its activator ATP. The AMP deaminase activity in vegetative cells was 7.4 nmols/min/mg proteins while the activity in cells measured at 2 and 6 hrs after starvation-induced growth-arrest was 376 nmols/min/mg protein...a 51-fold increase. When vegetative cells were treated with hadacidin, a drug that restricts de novo AMP synthesis and pinocytosis, the activity of the AMP deaminase was 511 nmols/min/mg protein...a 70-fold increase compared to that in untreated vegetative cells. Smaller increases were noted following the inhibition of growth with the drugs cerulenin and vinblastine, as well as after the inhibition of de novo GMP synthesis with the drug mycophenolic acid or the partial inhibition of de novo AMP synthesis with analogs of hadacidin, N hydroxyglycine and N-formylglycine. In addition, when the activity of two other enzymes involved in purine metabolism, namely adenosine kinase and hypoxanthine guanine phosphoribosyl transferase, was measured in vegetative cells, and the activity of both compared to that measured in starvation and hadacidin induced growth-arrested cells, showed no significant changes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014309 TI - The association of distinct acid phosphatases with the flagella pocket and surface membrane fractions obtained from bloodstream forms of Trypanosoma rhodesiense. AB - Cell fractionation of bloodstream Trypanosoma rhodesiense, using isopycnic sucrose gradient centrifugation, reveals acid phosphatase activities against a range of substrates to be associated, to varying degrees, with subcellular particle populations identified as derived from flagella pocket membrane and surface membrane. Using these same substrates (alpha and beta glycerophosphate, p nitrophenyl phosphate and glucose-6-phosphate) at least two distinct acid phosphatase activities can be distinguished. One is thermolabile (approximately 80% inactivated after 30 min. at 60 degrees C), sensitive to tartrate (50% inhibited at 1.8 mM Na tartrate) with a pH optimum approximately 4.5 and appears to exhibit little substrate preference. The other acid phosphatase is relatively heat stable (approximately 30% inactivated), insensitive to tartrate (greater than 5.0% inhibited using 1.8 mM Na tartrate) exhibits a somewhat higher pH optimum (approximately 6.0) and is more substrate specific (6X more active toward glucose-6-PO4 than beta-glycerophosphate). Further cell fractionation experiments reveal 85% of the tartrate sensitive acid phosphatase to be associated with flagella pocket membrane and to account for 80% of the organisms hydrolytic activity toward beta-glycerophosphate. The tartrate resistant acid phosphatase however, has a much less exclusive localization being almost equally distributed between surface membrane (40%) and flagella pocket membrane (60%). PMID- 3014314 TI - [Structural instability of co-integrates formed during interaction of plasmid R57 with pB322 and RP1. Possible role of IS1 element in the degradation of co integrates]. AB - The conjugative plasmid R57 determines resistance to ampicillin and chloramphenicol. Earlier it was shown that R57 encodes site-specific recA independent recombinase, which acts in cis and resolves IS1-mediated cointegrates arising in the Escherichia coli recA cells between R57 and pBR322. In the present work the properties of the cointegrates between R57 and pBR322 or RP1 arising in the E. coli rec+ strains were studied. It was found that the cointegrates between R57 and pBR322, obtained by mating of the respective biplasmid donors of E. coli rec+ and the rec+ recipients, lost as a result of deletion a large DNA segment of R57 containing determinant Cmr. The resulting hybrid replicons preserved determinants Apr and Tcr of pBR322 and the R57 conjugative properties and were structurally identical. By using plasmid RP1ts12, which is temperature-sensitive in replication, it was demonstrated that in cells rec+ the cointegrates between R57 and RP1 are extremely unstable. On storage they undergo structural degradation mainly affecting the RP1 replicon. The degradation products of the hydrid complex had lost their RP1 genes but preserved the R57 functional determinants. For elucidation of the observed phenomena the properties of the IS1 mediated cointegrates between pBR322:Tn9 and plasmid pBR3.1--deletion derivative of RP1 were studied. It was found that insertion of IS1 sometimes resulted in formation of unstable cointegrates capable of resolving and loosing determinant Cmr with a high frequency. It was suggested that IS1 encodes the site-specific recombinase responsible for resolution of the IS1-mediated cointegrates and deletion generation. Expression of this recombinase appears to be dependent on structure of the insertion sites. The possible role of IS1 and recombinase encoded by it in resolution and structural instability of the cointegrates between R57 and pBR322 or RP1 is discussed. PMID- 3014313 TI - [Structural analysis of alphoid DNA of primates. II. Evolution and possible origin of alphoid DNA of primates]. AB - A computer analysis of human and primate alphoid DNA was performed. The number and localization of short inverted complete repeats within alphoid DNA dimers (but not monomers) remain conserved. Thus, in spite of high heterogeneity of the primary structure the conserved secondary structure of alphoid DNA might be functionally important. The analysis of internal periodicity of the monomeric sequences of human and primate alphoid DNA revealed its potential ancient sequence, that is a simple satellite DNA with a reiterated heptanucleotide TGAAAAA, which is suggested to be the ancestor of satellite DNase of rodents. The facts reported propose the ancient origin and possible functional role of alphoid like DNA as a universal pericentromeric superfamily of DNA. PMID- 3014315 TI - [Effect of superhelical DNA on the transcription of cloned genes of the T4 phage]. AB - The effect of DNA superhelicity on the transcription of T4 DNA fragments containing early genes uvs W, Y and late genes 25-29 was studied. RNA polymerase transcribes both early and late phage genes within the supercoiled recombinant plasmid. Late genes relative transcription increases essentially when T4-modified RNA polymerase is used. DNA relaxation causes a sharp decrease of modified RNA polymerase activity, especially on the late genes. The same effect is obtained in intact cells with recombinant plasmid, which superhelicity is lowered by temperature-sensitive mutation in DNA gyrase. The data obtained prove that DNA superhelicity is required for the T4 late transcription by phage-modified RNA polymerase. It is suggested that the dependence of late transcription on the phage DNA replication in infected cells is connected with phage DNA superspiralization throughout its replication. PMID- 3014316 TI - [Comparative analysis of B1- and B2-like repetitive sequences in DNA from different organisms]. AB - Sequences homologous to short repeated elements of the mouse genome, B1 and B2, were detected in DNA of different organisms by dot-hybridization. The sequences B1 and B2 hybridized most efficiently with DNA of Myomorpha rodents and primates. The hybridization was also observed with DNA of all other eukaryotes studied, however, it is probably caused by an existence of short homologies with the B1 and B2 sequences only. The effective hybridization of the B1 sequence with DNA of primates is apparently explained by a presence of numerous copies of Alu sequence in their genomes. A repeated sequence of the human DNA that is able to hybridize with B2 sequence was cloned. It was designated as HsB2 sequence. There are about 5000 copies of this sequence in the human genome. To estimate the degree of homology, the melting temperature of hybrids of sequences B1 and B2 with DNA of rodents and some other mammals was measured. It was found that the degree of homology of B2 sequence (but not B1) correlated well with the phylogenetic relationship of the organisms. Perhaps, the difference of evolution of these sequences results from their structural and functional peculiarities. PMID- 3014317 TI - [Carnitine deficiency]. AB - Carnitine facilitates the transport of activated fatty acids across the mitochondrial membrane and regulates energy metabolism through regeneration of intramitochondrial coenzyme A. In carnitine deficiency it may be a limiting factor for fatty acid oxidation and ketogenesis. Primary myopathic carnitine deficiency is characterized by low carnitine concentrations usually restricted to muscle; whereas systemic carnitine deficiency shows decreased concentrations in other organs and plasma as well. The latter condition features recurrent metabolic crises similar to those seen in Reye's syndrome and nonketotic hypoglycemia. A therapy with L-carnitine should be undertaken, but does not always prove effective. Similar symptoms may be caused by defects in beta oxidation, Krebs cycle or respiratory chain enzymes. The conditions may be associated with secondary carnitine deficiency. Patients with organic acidurias exhibit an increased excretion of carnitine esters and an insufficiency of free carnitine. Carnitine supplementation may ameliorate the metabolic disturbance. Secondary carnitine deficiency has also been described in patients receiving chronic valproic acid therapy. Hemodialysed chronic renal patients may benefit from L-carnitine therapy and show improvement of their hyperlipidemia. Nutritional carnitine deficiency can be primarily expected in premature infants receiving a carnitine free diet, since these infants have an impaired capacity for carnitine biosynthesis. PMID- 3014318 TI - Effect of diet on dissolution of gallstones by ursodeoxycholic acid, including a comparison between ultrasonography and cholecystography. PMID- 3014319 TI - The histiocyte and the lung: a radiologic approach to classification of histiocytic pneumonopathies. PMID- 3014320 TI - Molecular basis of X-ray-induced mutation at the HPRT locus in human lymphocytes. AB - Human lymphocytes lacking functional HPRT enzyme after a dose of 300 rad X radiation were cloned and the monoclonal populations expanded so that sufficient genomic DNA was obtained for Southern analysis. A total of 33 mutant clones were analysed. Wild-type clones showed no evidence of changes to the HPRT gene resolvable by Southern banding patterns whereas 17 of 33 mutant clones showed changes. The alterations observed included total gene deletions (3 clones) and partial gene deletions with or without the appearance of novel bands (12 clones). Two clones showed the appearance of novel bands only. There were no changes observed in 16 of the 33 mutant clones. Three clones showed changes inconsistent with deletion of portions of the gene. In these clones inversion seems to have been the most likely cause of the mutation. The spectrum of gene alterations following ionizing radiation appears different to that previously observed for spontaneous mutations. Consequently, ionizing radiation or radiomimetic agents would appear to be aetiologic, at the most, for only a minor proportion of in vivo somatic mutations. PMID- 3014321 TI - Molecular analysis of chemically-induced mutations at the RpII215 locus of Drosophila melanogaster. AB - A substantial fraction, perhaps 50% or more, of spontaneous mutations in Drosophila melanogaster have been shown by molecular analyses to be associated with the presence of a transposable element (TE) inserted into the affected gene. We are interested in the molecular structure of induced mutations in Drosophila, in particular whether TEs are also responsible for a significant proportion of chemically-induced mutations. We report here the molecular analysis of 58 mutations at the RpII215 locus induced with EMS or ENU. While we find evidence for moderately sized deletions at this locus (in 3/58, or 5% of the examined mutants), we failed to detect any mutations which were associated with an insertion event. It may be the case that induced mutations are qualitatively different from spontaneous mutations. PMID- 3014322 TI - The relationship between radiation-induced and transposon-induced genetic damage during Drosophila oogenesis. AB - The combined effect of X-irradiation and transposon mobility on the frequencies of X-linked recessive lethals and dominant lethals was investigated in female hybrids in the P-M system of hybrid dysgenesis. X-linked lethals were measured in G2 hybrid dysgenic females whose X chromosome was derived from the M X P cross. To test for additivity or synergism, the mutation rate in irradiated dysgenic females was compared to that of unirradiated females as well as to irradiated nondysgenic hybrid females derived from M X M crosses. Eggs collected for 2 days after irradiation, were represented by the more radiation-sensitive A and B oocytes (about 75%) and the least sensitive C oocytes (about 25%). The production of X-linked lethal events in X-irradiated dysgenic females was 8.1%, as compared to 4.5% in dysgenic controls and 3.4% in irradiated, nondysgenic controls, demonstrating an additive effect of radiation and dysgenesis-induced genetic damage. The effect of irradiation on sterility of dysgenic hybrid females was a negative one, resulting in 20% less sterility than expected from an additive effect. The combined effect of radiation and dysgenesis on dominant lethality tested in A, B and C oocytes of the same hybrid females was synergistic. Egg broods collected for 3.5 days after irradiation showed that significantly more damage was induced in the presence of ionizing radiation in dysgenic females than in their nondysgenic counterparts. This effect was most obvious in B and C oocytes. The synergism observed may be related to the inability of cells to repair the increased number of chromosome breaks induced both by radiation and transposon mobility. PMID- 3014323 TI - The observation of some biological characteristics of transformed cells induced by MNNG in vitro. AB - The early passage diploid Syrian hamster embryo (SHE) cells were treated with N methyl-N'-nitro-N-nitrosoguanidine (MNNG). The treated cells proliferated rapidly; the doubling times shortened; colonies appeared in solid agar medium and transformed foci formed in tissue culture. All of these phenomena suggest that malignant transformation of SHE cell has occurred. Faster cell division rate and multipolar mitosis were demonstrated by time-lapse cinemicrography and scanning electron microscopy. Multipolar mitosis can occur in two forms: direct division and indirect division. The transformed cells were more abundant in microvilli, the number of which increased in accordance with the degree of malignancy. In comparison with the controls, the transformed cells expressed a greater tritiated thymidine incorporation, greater DNA contents and more chromosomes, but no difference in nuclear area. The determination of amino acid changes in media due to the growth of transformed cells showed that the decrease in arginine and increase in ornithine are significant. The results of allogenic animal inoculation suggest that the transformed cells can be characterized into several different stages in the process of transformation. PMID- 3014324 TI - Role of the radB gene in postreplication repair in UV-irradiated Escherichia coli uvrB. AB - In UV-irradiated Escherichia coli, the radB101 mutation sensitized uvrB recF cells 4-fold and uvrB recB cells 1.2-fold, but did not sensitize uvrB recB recF cells. The radB mutation had very little effect (1.2-fold or less) on the repair of UV radiation-induced DNA daughter-strand gaps in uvrB cells, but it did cause about a 3-fold deficiency in the repair of the DNA double-strand breaks that arise in association with nonrepaired daughter-strand gaps in UV-irradiated uvrB recF cells. Thus, the radB gene does not appear to be involved in the recF dependent or recF recB-independent processes for the repair of DNA daughter strand gaps, but is involved in the recB-dependent postreplication repair of DNA double-strand breaks. PMID- 3014325 TI - Enhancement of UV survival, UV- and MMS-mutability, precise excision of Tn10 and complementation of umuC by plasmids of different incompatibility groups. AB - 29 conjugative resistance and colicin plasmids from 19 different incompatibility (Inc) groups were examined for their ability to enhance post-ultraviolet (UV) survival and UV- and methyl methanesulfonate(MMS)-induced mutability in Salmonella typhimurium LT2 strains. 14 Muc+ plasmids enhanced each of the survival and mutation-related properties tested, while 14 Muc- plasmids showed no enhancing effects in any tests. One Muc+ plasmid, pRG1251 (IncH1), enhanced post UV survival and each of the mutation-related properties tested, except MMS induced mutagenesis. Two further noteworthy plasmids, R391 (IncJ) and R394 (IncT), produced apparent strain-dependent effects in S. typhimurium which differed from those reported to have been found in Escherichia coli. Plasmid R391 enhanced post-UV survival in S. typhimurium, in contrast to its UV-sensitizing effects in E. coli. In both hosts plasmid R391 enhanced UV- and MMS-induced mutagenesis. Plasmid R394 had no enhancing effects on UV survival or UV- and MMS induced mutagenesis in S. typhimurium, in contrast to its reported enhancement of MMS-induced mutagenesis in E. coli. Conjugal transfer of R394 to E. coli strain AB1157 and assays of mutagenesis-related traits supported results observed in S. typhimurium. Muc+ plasmids were found to enhance the frequency of precise excision of the transposon Tn10 when inserted within hisG or trpA in S. typhimurium strains. Precise excision could be further enhanced in S. typhimurium by UV-irradiation. Analysis of Tn10 mutants with altered IS10 ends indicated that intact inverted repeats at the ends of Tn10 were required for efficient enhancement of precise excision. PMID- 3014326 TI - Microinjection of Escherichia coli UvrA, B, C and D proteins into fibroblasts of xeroderma pigmentosum complementation groups A and C does not result in restoration of UV-induced unscheduled DNA synthesis. AB - The UV-induced unscheduled DNA synthesis (UDS) in cultured human fibroblasts of repair-deficient xeroderma pigmentosum complementation groups A and C was assayed after injection of identical activities of either Uvr excinuclease (UvrA, B, C and D) from Escherichia coli or endonuclease V from phage T4. Under conditions where the T4 enzyme was able to induce repair synthesis in both XP complementation groups in agreement with earlier observations (de Jonge et al., 1985), no effect of the UvrABCD excinuclease could be observed either when the enzymatic complex was injected into the cytoplasm, or when it was delivered directly into the nucleus. In addition, no effect of the E. coli excinuclease was found on the repair ability of normal repair-proficient human fibroblasts. We conclude that the UvrABCD excinuclease may not work on DNA lesions in human chromatin. PMID- 3014327 TI - Comparative studies of host-cell reactivation, cellular capacity and enhanced reactivation of herpes simplex virus in normal, xeroderma pigmentosum and Cockayne syndrome fibroblasts. AB - Host-cell reactivation (HCR) of UV-irradiated herpes simplex virus type 2 (HSV 2), capacity of UV-irradiated cells to support HSV-2 plaque formation and UV enhanced reactivation (UVER) of UV-irradiated HSV-2 were examined in fibroblasts from 4 patients with Cockayne syndrome (CS), 5 with xeroderma pigmentosum and 5 normals. All UV-survival curves for HSV-2 plaque formation showed 2 components. HCR was similar to normal for the XP variant strain and the 2 CS strains tested, but substantially reduced in the 4 excision-deficient XP strains. The capacity of UV-irradiated fibroblasts to support HSV-2 plaque formation was determined by UV irradiating fibroblast monolayers with various doses of UV and 48 h later, infecting the monolayers with unirradiated HSV-2. The D37 values for the delayed capacity curves so obtained were in the range 8.6-12.4 J/m2 for the normal strains, 2.8-3.2 J/m2 for the CS strains, 6.7 J/m2 for an XP variant strain and between 0.3 and 1.5 for the XP excision-deficient strains tested. These results indicate that delayed capacity for HSV-2 plaque formation is a more sensitive assay than HCR in the detection of cellular DNA-repair deficiency for XP and CS. For the examination of UVER, fibroblasts were irradiated with various UV doses and subsequently infected with either unirradiated or UV-irradiated HSV and scored for plaque formation 2 days later. UVER expression was maximum when the delay between UV-irradiation of the cells and HSV infection was 48 h. The magnitude of UVER expression was also found to be dependent on the UV dose to the cells and increased with increasing UV dose to the virus. Using a UV dose to the virus resulting in a plaque survival of about 10(-2) on unirradiated cells, the the maximum UVER factor had a mean value of 1.3 for the normal strains following a dose of 15 J/m2 to the cells. Somewhat higher UVER values were found for all the patient strains tested and resulted from lower UV doses to the cells than for normal strains. Maximum UVER factors for the CS strains ranged from 2.2 to 3.3 at a dose of 5 J/m2 to the cells, for the XP excision-deficient strains; 2.1 to 2.6 at doses of 0.5 to 2.5 J/m2 to the cells and for the XP variant strain tested; 2.5 at UV dose of 10 J/m2 to the cells. PMID- 3014328 TI - The muscle slice--a new preparation for the characterization of beta-adrenergic binding in fast- and slow-twitch skeletal muscle. AB - A new procedure is presented that characterizes the specific binding of the beta adrenergic antagonist, [3H]CGP-12177, to thick (1 mm) slices from fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) mouse skeletal muscle. Binding is reversible, saturable, stereospecific, of high affinity, and subject to agonist-induced desensitization, indicating that it is to beta-adrenoreceptors and not to other sites. In both muscles, the majority of specific binding is to the beta 2-receptor subtype. Bmax is approximately twice as high in the soleus (5.64 +/- 0.52 fmol/mg wet weight) as in the EDL (2.66 +/- 0.29 fmol/mg wet weight) (P less than 0.05), whereas affinity is higher in the fast-twitch (Kd = 0.30 +/- 0.08 nM) than the slow-twitch muscle (Kd = 0.45 +/- 0.08 nM). The minimal tissue disruption associated with this procedure, as well as its speed, simplicity and relatively low cost, suggest that the slice preparation may prove to be invaluable for the future study of beta-adrenergic receptor binding and associated responses in skeletal muscle. PMID- 3014329 TI - Induction of spheroid cytoplasmic bodies in a rat muscle by local tetanus. AB - The term "cytoplasmic body" or "spheroid body" myopathy refers to a heterogeneous group of familial or sporadic diseases characterized primarily by the presence of abundant spheroid or cytoplasmic bodies in the muscles. The morphogenesis of these inclusions remains unclear. This article describes the induction and evolution of spheroid cytoplasmic bodies (SCBs) in the rat plantaris muscle (PL) with local tetanus, which was induced in rats by the injection of a minute amount of tetanus toxin. In contrast to the tetanized soleus muscle (SOL), which developed core fibers (central cores, minicore, target fiber, targetoid fiber, and rods), the tetanized PL produced numerous SCBs with a predictable time course. They were induced in both type 1 and 2 fibers of PL, which is composed predominantly (95%) of type 2 fibers, in contrast to SOL (85% type 1 fibers). Factors inducing SCBs may include immobilization, shortening, intact innervation, and disuse atrophy. PMID- 3014330 TI - Chronic peripheral neuropathy: unusual nerve and muscle biopsy findings. PMID- 3014331 TI - Terminal galactose residues and the antigenicity of Plasmodium falciparum glycoproteins. AB - The presence of terminal alpha-D-galactosyl residues in the carbohydrate chains of glycoprotein antigens from the asexual blood stages of Plasmodium falciparum is demonstrated by the alpha-D-galactosidase sensitivity of particular parasite antigens, the use of specific glycosidases to cleave sugars from parasite glycoproteins radiolabeled with [3H]glucosamine, and the ability of Bandeirea simplicifolia lectin, which has a specificity for terminal alpha-galactosyl residues, to bind to the parasite. The carbohydrate side chains, and in particular the terminal alpha-galactosyl residues, are shown to have an important role in determining the binding of antibodies to parasite glycoproteins. PMID- 3014332 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 27-1986. A 57-year-old woman with treated ovarian carcinoma, chest pain, and dyspnea. PMID- 3014333 TI - Attempted suicide with enalapril. PMID- 3014334 TI - Association of BK viruria with hemorrhagic cystitis in recipients of bone marrow transplants. AB - Fifty-three recipients of bone marrow transplants were monitored prospectively for urinary excretion of human polyomaviruses by enzyme-linked immunosorbent assays of urinary supernatants and DNA hybridization assays of urinary cells. Excretion of BK virus was demonstrated in 47 percent of the transplant recipients and was the result of the reactivation of latent virus. Hemorrhagic cystitis of long duration (greater than or equal to 7 days) was associated with BK viruria. The disease occurred four times more frequently in patients who excreted BK virus than in those who did not, and the virus was identified in 55 percent of the urine specimens during episodes of cystitis as compared with 8 to 11 percent of the specimens during cystitis-free periods. BK viruria often preceded or coincided with the onset of the disease. Among 19 patients with BK viruria lasting seven days or longer, hemorrhagic cystitis occurred in 15. Occurrence of the disease was related to the source of marrow. The disease occurred in 50 percent of 38 recipients of allogeneic marrow and in 7 percent of 15 recipients of syngeneic or autologous marrow. Among recipients of allogeneic marrow, the disease was observed in 71 percent of the 21 patients excreting BK virus and in 24 percent of the 17 not excreting the virus. An association of BK virus with hemorrhagic cystitis was demonstrated in 16 of the 18 cases of the disease that were adequately characterized. We conclude that reactivation of BK virus may account for a substantial proportion of late-onset, long-lasting hemorrhagic cystitis in recipients of bone marrow transplants. PMID- 3014335 TI - HTLV-III antibody in East Africa. PMID- 3014336 TI - Tumor lysis syndrome after tamoxifen flare. PMID- 3014337 TI - Expression of a novel T-cell activation antigen on bile canaliculi. PMID- 3014339 TI - Cryptococcus neoformans: comparisons of in vitro antifungal susceptibilities of serotypes AD and BC. AB - Thirty-nine isolates of Cryptococcus neoformans, nineteen serotype AD and twenty serotype BC, were assayed for susceptibility to eight antifungal agents using an in vitro agar dilution assay. Media employed were Kimmig agar and yeast nitrogen base supplemented with 10% glucose. The antifungal agents used were ketoconazole, amphotericin B, 5-fluorocytosine, nystatin, miconazole, BAY N 7133, ICI 153,066, and itraconazole. No clinically significant differences in vitro minimum inhibitory concentrations were detected between serotypes AD and BC against any of the compounds tested. An adverse medium effect was observed in two of the assays, but the outcome of the AD/BC comparison was not affected. This is the first report in which the in vitro antifungal susceptibilities of Cryptococcus neoformans serotypes are analyzed. PMID- 3014338 TI - Secretion of pancreatic polypeptide in patients with pancreatic endocrine tumors. AB - Pancreatic polypeptide is often secreted by pancreatic endocrine tumors and is considered a marker for such tumors. To investigate the diagnostic value of this marker, we studied 323 patients with proved pancreatic endocrine tumors. We found plasma concentrations of pancreatic polypeptide to be elevated (more than 300 pmol per liter) in 144 patients (diagnostic sensitivity, 45 percent). However, plasma levels of pancreatic polypeptide can also be elevated in the absence of a pancreatic tumor. To ascertain whether the administration of atropine could distinguish between normal and tumor-associated polypeptide secretion, we studied 30 patients with pancreatic tumors and high plasma levels of pancreatic polypeptide, 18 patients without tumors who had elevated levels of pancreatic polypeptide, and eight normal controls. Polypeptide levels in the 18 patients without tumors were substantially lower than in the 30 patients with tumors. Atropine (1 mg intramuscularly) did not suppress polypeptide levels in patients with tumors, but did suppress plasma levels by more than 50 percent in all subjects without tumors. Thus, although its diagnostic sensitivity is low, pancreatic polypeptide appears to be a useful adjunctive marker of many pancreatic endocrine tumors, and the atropine suppression test can be used to distinguish normal from tumor-related secretion of the polypeptide. Identification of the type of pancreatic endocrine tumor still requires measurement of the hormone that is specific for the tumor. PMID- 3014340 TI - Light-dependent phosphorylation of rhodopsin by beta-adrenergic receptor kinase. AB - The structural components involved in transduction of extracellular signals as diverse as a photon of light impinging on the retina or a hormone molecule impinging on a cell have been highly conserved. These components include a recognition unit or receptor (for example, the beta-adrenergic receptor (beta AR) for catecholamines or the 'light receptor' rhodopsin), a guanine nucleotide regulatory or transducing protein, and an effector enzyme (for example, adenylate cyclase or cyclic GMP phosphodiesterase). Molecular cloning has revealed that the beta AR shares significant sequence and three-dimensional homology with rhodopsin. The function of the beta AR is diminished by exposure to stimulatory agonists, leading to desensitization. Similarly, 'light adaptation' involves decreased coupling of photoactivated rhodopsin to cGMP phosphodiesterase activation. Both forms of desensitization involve receptor phosphorylation. The latter is mediated by a unique protein kinase, rhodopsin kinase, which phosphorylates only the light-bleached form of rhodopsin. An analogous enzyme (termed beta AR kinase or beta ARK) phosphorylates only the agonist-occupied beta AR. We report here that beta ARK is also capable of phosphorylating rhodopsin in a totally light-dependent fashion. Moreover, rhodopsin kinase can phosphorylate the agonist-occupied beta AR. Thus the mechanisms which regulate the function of these disparate signalling systems also appear to be similar. PMID- 3014341 TI - No functional gamma-chain transcripts detected in an alloreactive cytotoxic T cell clone. AB - Three groups of genes that undergo rearrangements during T-cell maturation have been isolated from T cells. Two of them encode the alpha- and beta-subunits of the T-cell antigen receptor and are shared between antigen-specific, major histocompatibility (MHC) class I-restricted cytotoxic T cells and antigen specific, MHC class II-restricted helper T cells. The third group of genes, called gamma, is preferentially transcribed in cytotoxic T cells. This led to the hypothesis that the unidentified gamma-gene products could be part of a putative T-cell receptor responsible for MHC class I recognition. We report here on the isolation of three different types of gamma-gene transcripts of an alloreactive cytotoxic T-cell clone (3F9). Two are derived from two rearrangements that have occurred at the same locus (V gamma 10.8A to J gamma 10.5 and transcribed with C gamma 10.5), while the third involves a new V gamma-gene segment that is joined to J gamma 13.4 and transcribed with C gamma 13.4. All these rearrangements are abortive and lead to the formation of non-functional gamma-chain genes because the proper translational reading frame is not maintained. Because the second copy of the C gamma 13.4 gene segment is deleted and as C gamma 7.5 is considered to be a pseudogene and has not undergone any rearrangements in 3F9, we conclude that the alloreactive cytotoxic T-cell clone 3F9 does not contain a functional transcript of a known gamma-chain gene. PMID- 3014342 TI - Corticotropin releasing factor induction of leukocyte-derived immunoreactive ACTH and endorphins. AB - Human peripheral leukocytes infected by virus or treated with endotoxin will, like unstimulated mouse spleen macrophages, synthesize immunoreactive corticotrophin (ir-ACTH) and endorphins. The ir-ACTH produced appears to be identical with authentic ACTH, while enough of the material has been produced in hypophysectomized mice infected with virus to demonstrate a steroidogenic response. Because the production of ACTH by in vivo pituitary cells and by leukocytes is suppressed by dexamethasone both in vitro and in vitro, suggesting that the production of ACTH and endorphins by leukocytes is indeed controlled, we have investigated the effects of corticotropin releasing-factor (CRF), which is known to regulate the pituitary production of both ACTH and beta-endorphin. We now report that the production of ACTH and endorphins by leukocytes is indeed induced by synthetic CRF and, in turn, suppressed by dexamethasone, suggesting that, as in pituitary cells, the proopiomelanocortin (POMC) gene may be expressed and similarly controlled in leukocytes. PMID- 3014344 TI - Cancer. Cell growth control mechanisms. PMID- 3014343 TI - The beta-subunit of follicle-stimulating hormone is deleted in patients with aniridia and Wilms' tumour, allowing a further definition of the WAGR locus. AB - One in 10,000 children develops Wilms' tumour, an embryonal malignancy of the kidney. Although most Wilms' tumours are sporadic, a genetic predisposition is associated with aniridia, genito-urinary malformations and mental retardation (the WAGR syndrome). Patients with this syndrome typically exhibit constitutional deletions involving band p13 of one chromosome 11 homologue. It is likely that these deletions overlap a cluster of separate but closely linked genes that control the development of the kidney, iris and urogenital tract (the WAGR complex). A discrete aniridia locus, in particular, has been defined within this chromosomal segment by a reciprocal translocation, transmitted through three generations, which interrupts 11p13. In addition, the specific loss of chromosome 11p alleles in sporadic Wilms' tumours has been demonstrated, suggesting that the WAGR complex includes a recessive oncogene, analogous to the retinoblastoma locus on chromosome 13. In WAGR patients, the inherited 11p deletion is thought to represent the first of two events required for the initiation of a Wilms' tumour, as suggested by Knudson from epidemiological data. We have now isolated the deleted chromosomes 11 from four WAGR patients in hamster-human somatic cell hybrids, and have tested genomic DNA from the hybrids with chromosome 11-specific probes. We show that 4 of 31 markers are deleted in at least one patient, but that of these markers, only the gene encoding the beta-subunit of follicle stimulating hormone (FSHB) is deleted in all four patients. Our results demonstrate close physical linkage between FSHB and the WAGR locus, suggest a gene order for the four deleted markers and exclude other markers tested from this region. In hybrids prepared from a balanced translocation carrier with familial aniridia, the four markers segregate into proximal and distal groups. The translocation breakpoint, which identifies the position of the aniridia gene on 11p, is immediately proximal to FSHB, in the interval between FSHB and the catalase gene. PMID- 3014345 TI - AIDS. Depressing news from Paris. PMID- 3014346 TI - New HLA DNA polymorphisms associated with autoimmune diseases. AB - Certain class II determinants of the human histocompatibility locus antigens (HLA) have been implicated in the aetiology of several autoimmune diseases, including rheumatoid arthritis (RA) and insulin-dependent diabetes mellitus (IDDM). HLA-Dw4 was the first HLA determinant found to be significantly increased in RA patients compared with controls, while Dw4 and Dw3 were found to be significantly increased in IDDM patients. When the HLA-DR system was defined, RA patients were found to have an increased frequency of DR4 and IDDM patients an increased incidence of both DR4 and DR3 compared with controls. As the HLA-Dw specificities are narrower than the serologically defined DR specificities, it was of specific interest to the present study that Dw4, Dw10, Dw13, Dw14, Dw15 and DKT2 are included in DR4. We describe here new restriction fragment length polymorphisms (RFLPs) and, together with the newly described serologically defined DQ specificity TA10, test their prevalence and associations in controls and diseased patients. We find that the newly characterized DNA bands are present at a much higher frequency in RA and IDDM patients than in controls. These findings may lead to a greater understanding of the pathogenesis of such diseases. PMID- 3014347 TI - Hepatitis B virus DNA integration in a sequence homologous to v-erb-A and steroid receptor genes in a hepatocellular carcinoma. AB - Hepatitis B virus (HBV) is clearly involved in the aetiology of human hepatocellular carcinoma (HCC) and the finding of HBV DNA integration into human liver DNA in almost all HCCs studied suggested that these integrated viral sequences may be involved in liver oncogenesis. Several HBV integrations in different HCCs and HCC-derived cell lines have been analysed after molecular cloning without revealing any obvious role for HBV. From a comparison of a HBV integration site present in a particular HCC with the corresponding unoccupied site in the non-tumorous tissue of the same liver, we now report that HBV integration places the viral sequence next to a liver cell sequence which bears a striking resemblance to both an oncogene (v-erb-A) and the supposed DNA-binding domain of the human glucocorticoid receptor and human oestrogen receptor genes. We suggest that this gene, usually silent or transcribed at a very low level in normal hepatocytes, becomes inappropriately expressed as a consequence of HBV integration, thus contributing to the cell transformation. PMID- 3014349 TI - Carcinogen-specific mutation and amplification of Ha-ras during mouse skin carcinogenesis. AB - Cellular proto-oncogenes can be activated by both point mutations and chromosomal translocations, suggesting that there may be a direct link between exposure to agents which damage DNA and genetic change leading to malignancy. Several groups have therefore analysed mutations found in cellular oncogenes of tumours induced by particular physical or chemical carcinogens. Here, we have analysed the molecular changes at different stages of carcinogenesis in mouse skin tumours induced by initiating and promoting agents. Over 90% of tumours, including premalignant papillomas, initiated with dimethylbenzanthracene (DMBA) have a specific A----T transversion at the second nucleotide of codon 61 of the Harvey ras (Ha-ras) gene. The frequency of this mutation was dependent on the initiating agent used, but not on the promoter, suggesting that the mutation occurs at the time of initiation. The mutation was heterozygous in most papillomas tested, but was homozygous or amplified in some carcinomas. The development of further chromosomal changes at the c-Ha-ras gene locus is therefore a common feature of tumour progression. PMID- 3014350 TI - Enzymatic activity of the conserved core of a group I self-splicing intron. AB - Splicing of the Tetrahymena ribosomal intron was first studied by Cech et al., who subsequently demonstrated that the intron RNA catalyses its own excision from a primary transcript to yield mature ribosomal RNA. This intron shares several short conserved sequences and a common secondary structure with several other introns, some of which have also been shown to self-splice. Here I show that the conserved core of the Tetrahymena intron can act in trans to catalyse the sequence-specific cleavage and addition of guanosine to a separate RNA substrate. PMID- 3014351 TI - Analysis of human T-lymphotrophic virus sequences in multiple sclerosis tissue. AB - Several observations suggest that retroviral infection is involved in the pathogenesis of the human demyelinating disease multiple sclerosis (MS). First, lymphadenopathy-associated virus/human T-lymphotropic virus type III (LAV/HTLV III), the agent of acquired immune deficiency syndrome (AIDS), has been shown to be neurotropic in man. Second, the genetic organization of the lentivirus visna, which causes a chronic demyelinating disease of sheep, closely resembles that of LAV/HTLV-III. Recently, Koprowski and colleagues reported that MS is associated both with raised levels of circulating antibodies to HTLV-I and with the presence of HTLV-I-specific RNA within cell lines derived from the cerebrospinal fluid (CSF). Here we report that no HTLV-I-like or LAV/HTLV-III-like sequences can be detected, by in situ hybridization, in central nervous system (CNS) tissues from MS patients, and that nonspecific HTLV-I-like signal in peripheral blood mononuclear cells or in CSF cell lines is characteristic of MS. Furthermore, enzyme-linked immunosorbent assay (ELISA) analysis of circulating and CSF antibodies for HTLV-I reactivity fails to distinguish between MS and control groups. PMID- 3014348 TI - Analysis of deletions in DNA from patients with Becker and Duchenne muscular dystrophy. AB - Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder for which the biochemical defect is as yet unknown. Recently, two cloned segments of human X-chromosome DNA have been described which detect structural alterations within or near the genetic locus responsible for the disorder. Both of these cloned segments were described as tightly linked to the locus and were capable of detecting deletions in the DNA of boys affected with DMD. In an attempt to determine more precisely the occurrence of these deletions within a large population of DMD patients and the accuracy of one of the segments, DXS164 (pERT87), in determining the inheritance of the DMD X chromosome, the subclones 1, 8 and 15 were made available to many investigators throughout the world. Here we describe the combined results of more than 20 research laboratories with respect to the occurrence of deletions at the DXS164 locus in DNA samples isolated from patients with DMD and Becker muscular dystrophy (BMD). The results indicate that the DXS164 locus apparently recombines with DMD 5% of the time, but is probably located between independent sites of mutation which yield DMD. The breakpoints of some deletions are delineated within the DXS164 locus, and it is evident that the deletions at the DMD locus are frequent and extremely large. PMID- 3014352 TI - Lack of evidence for involvement of known human retroviruses in multiple sclerosis. AB - The recent report by Koprowski et al. that human T-cell lymphotropic retroviruses (HTLVs) may be involved in the development of multiple sclerosis (MS) has aroused much interest. The report was based largely on immunological evidence, using enzyme-linked immunosorbent assays (ELISAs) with viral antigens or disrupted virions. We have accordingly sought confirmation by screening sera and cerebrospinal fluid (CSF) samples from MS patients against cell lines infected respectively with adult T-cell leukaemia (ATL) virus (ATLV/HTLV-I) of Japanese cells (MT-1 and MT-2 lines), our own isolate from British black patients with ATL, the MoT cell line which produce HTLV-II, and our own T-cell line containing a local isolate of acquired immune deficiency syndrome (AIDS) virus (C-LAV/HTLV III). We have failed to find antibodies against these retroviruses in the sera or CSF. Furthermore, neither virus could be isolated from the peripheral white blood cells of two MS patients. PMID- 3014353 TI - Newly replicated DNA is associated with DNA topoisomerase II in cultured rat prostatic adenocarcinoma cells. AB - DNA topoisomerases have been proposed to function in a variety of genetic processes in both prokaryotes and eukaryotes. Here, we have assessed the role of DNA topoisomerase II in mammalian DNA replication by determining the proximity of newly synthesized DNA to covalent enzyme-DNA complexes generated by treating cultured rat prostatic adenocarcinoma cells with teniposide. Teniposide (VM-26), an epipodophyllotoxin, is known to interact with mammalian DNA topoisomerase II so as to trap the enzyme in a covalent complex with DNA. We have found that the teniposide-induced trapping of such complexes requires MgCl2, is stimulated by ATP and is inhibited by novobiocin. The formation of covalent complexes seems to be reversible on removal of teniposide. Furthermore, analysis of the covalent complexes formed between 3H-thymidine pulse-labelled DNA and topoisomerase II following teniposide treatment reveals a direct association of the enzyme with nascent DNA fragments. Our results suggest that DNA topoisomerase II may interact with newly replicated daughter DNA molecules near DNA replication forks in mammalian cells. PMID- 3014355 TI - [Encephalopathy in patients with LAV-HTLV-III infections]. PMID- 3014354 TI - [Diagnosis and treatment of sarcoidosis: a plea for responsible reticence]. PMID- 3014356 TI - [Initial Dutch experiences with heart transplantation]. PMID- 3014357 TI - [Change in subjective well being and physical complaints in multiple sclerosis patients within the scope of inpatient therapy lasting several weeks]. AB - At the beginning and the end of a 4 to 6 weeks hospital treatment 141 patients with multiple sclerosis were investigated in order to measure changes in subjective well-being and reported symptoms. For this purpose we used self-rating scales (v. Zerssen's "Befindlichkeits-Skala" (state of well-being scale) and "Beschwerden-Liste" (complaints list). Data from treatment onset, as well as changes during treatment, were correlated with age, age at illness onset, duration of illness, disability score and neurological deficit, but also with differences in treatments and with social characteristics. At the time of discharge from hospital, two-thirds of the MS patients reported relief of symptoms, and three-quarters showed a much better general sense of well-being. In general a subdepressive state was found initially, and a normal relaxed mood at time of discharge. ACTH improved the patients well-being to a much greater extent when compared with matched MS patients without ACTH therapy. Reading aids for those with impaired vision, and especially relief of bladder dysfunction (which was found in 80% of the patients) led to an enhanced general sense of well-being, while ACTH did not lead to any greater relief of illness symptoms. PMID- 3014358 TI - Pituitary apoplexy. AB - Three patients with symptomatic haemorrhagic necrosis and infarction of the pituitary gland are described. They showed a range of clinical presentation, diagnostic pitfalls and diversity of treatment. PMID- 3014360 TI - Kinetics of the 5'-nucleotidase and the adenosine deaminase in subcellular fractions of rat brain. AB - Suspensions of rat brain microsomes, synaptosomes, and synaptic vesicles were able to convert adenosine to inosine by means of adenosine deaminase. Isosbestic points of this transformation, at 222, 250 and 281 nm, remained unchanged with time-course. This fact suggests that adenosine deaminase (ADA, E.C. 3.5.4.4) is located on the surface of the vesicles whereas purine nucleoside phosphorylase (PNP, E.C. 2.1.2.4) is located inside the vesicles. Kinetic parameters of the particulate 5'-nucleotidase (5'N, E.C. 3.1.3.5) and adenosine deaminase were analogous to those of the cytosolic enzymes. These results suggest that soluble and particulate enzymes represent different pools of the same molecular species. PMID- 3014359 TI - Aging and day-night rhythms in feeding in mice: effects of the putative sigma opiate agonist, N-allylnormetazocine (SKF-10,047). AB - Day-night rhythms in feeding behavior and response to the putative sigma opiate agonist, N-allylnormetazocine (+/- SKF-10,047, 0.10-10 mg/kg), were measured in young (1-2 months), mature (8-12 months) and old (24-30 months) male CF-1 mice. The mice consumed more food at night than in the day-time, though this nocturnal peak was markedly reduced in the mature and old animals. The young mice also displayed a significant nocturnal enhancement in SKF-10,047 (0.10-1.0 mg/kg) stimulated feeding, that could, in part, be suppressed by the opiate antagonist naloxone (1.0 mg/kg). The day-night rhythm in ingestive responses to SKF-10,047 (0.10-1.0 mg/kg) was reduced in the mature animals and absent in the old animals. The old mice failed to show any significant increase in ingestive response following opiate administration. A higher dose of SKF-10,047 (10 mg/kg) had no significant ingestive effects in any of the age groups of mice; the excitatory, psychotomimetic-related effects, being also reduced in the old animals. PMID- 3014361 TI - Pipecolic acid receptors in rat cerebral cortex. AB - Identification of [14C]pipecolic acid (PA) receptors was attempted in the solubilized membrane fraction from rat cerebral cortex. Specific binding proteins for both PA and muscimol, a potent gamma-aminobutyric acid (GABA) agonist, were detected in the same preparation. Separation of labeled PA and GABA binding proteins by glycerol gradient centrifugation has shown labeled protein bands of similar sedimentation rates, suggesting that PA and GABA may be binding to identical proteins. It seems likely that the PA binding receptor either may possess the same sedimentation characteristics as that of the GABA receptor, or both GABA and PA which is an endogenous and weak GABA agonist may bind to the same receptor complex, if not to the same binding site. PMID- 3014362 TI - GABA-agonists induce the formation of low-affinity GABA-receptors on cultured cerebellar granule cells via preexisting high affinity GABA receptors. AB - The kinetics of specific GABA-binding to membranes isolated from cerebellar granule cells, cultured for 12 days from dissociated cerebella of 7-day-old rats was studied using [3H]GABA as the ligand. The granule cells were cultured in the presence of the specific GABA receptor agonist 4, 5, 6, 7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP, 150 microM) or THIP plus the antagonist bicuculline methobromide (150 microM of each) or in the absence of the agonist or antagonist. Membranes isolated from granule cells cultured in a medium without the GABA agonist revealed a single binding site for GABA with a binding constant (KD) of 7.9 +/- 0.4 nM and a Bmax of 3.42 +/- 0.08 pmol X mg-1 protein. Membranes from cells cultured in the presence of THIP had two binding sites for GABA with KD values of 6.8 +/- 0.9 nM and 476 +/- 311 nM, respectively. The corresponding Bmax values were 4.41 +/- 0.42 pmol X mg-1 and 5.81 +/- 1.20 pmol X mg-1. The effect of culturing the cells in THIP was antagonized by the simultaneous presence of bicuculline in the culture media, i.e. no significant low-affinity binding for GABA was found on the membranes from granule cells cultured in both THIP and bicuculline. The KD value (14.3 +/- 1.4 nM) for the high affinity binding site was, however, slightly increased compared to the non-treated cells. These findings suggest that the ability of THIP to induce formation of low-affinity GABA receptors is mediated by preexisting high-affinity GABA-receptors on the granule cells. PMID- 3014364 TI - Dopamine-beta-hydroxylase inhibitors modulate the concentration of functional estrogen receptors in female rat hypothalamus and pituitary gland. AB - In order to study further the regulation of steroid hormone receptors in the brain and anterior pituitary gland of ovariectomized rats by catecholamines, the influence of two dopamine-beta-hydroxylase inhibitors on the estrogen receptor system was studied. The two inhibitors, FLA 63 and diethyldithiocarbamate (DDC) both caused a biphasic effect on the concentration of cytosol estrogen receptors in the mediobasal hypothalamus without influencing the concentration of cell nuclear estrogen receptors in the absence of estradiol; the concentration of cytosol estrogen receptors first increased and then decreased after treatment. In a study of the neuroanatomical areas in which inhibition of dopamine-beta hydroxylase decreases the concentration of cytosol estrogen receptors, it was found that the concentration of cytosol estrogen receptors decreased after drug treatment in the mediobasal hypothalamus and preoptic area-septum, but not in the amygdala. This suggests that the decrease in concentration of hypothalamic cytosol estrogen receptors caused by dopamine-beta-hydroxylase inhibitors is not a nonspecific effect on all cytosol estrogen receptors. Finally, an injection of estradiol-17 beta in animals that had been pretreated with either DDC or FLA 63 resulted in less accumulation of cell nuclear estrogen receptors in those tissues in which the drug first caused a decrease in the concentration of cytosol estrogen receptors than in vehicle-injected controls. Therefore, under some conditions, pretreatment with a dopamine-beta-hydroxylase inhibitor decreased the concentration of functional cytosol estrogen receptors resulting in decreased nuclear estrogen receptor accumulation in response to an estradiol injection.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014365 TI - Role of intracerebral angiotensin receptors in the regulation of vasopressin release and the cardiovascular system. AB - In order to investigate the physiological role of the brain renin-angiotensin system in the regulation of vasopressin (ADH) release, angiotensin II (Ang II, 10 ng/kg/min) or 1-Sar-8-Ile-Ang II (50 ng/kg/min), an Ang II antagonist, was administered intracerebroventricularly to dogs (n = 42) anesthetized with urethane and chloralose after morphine sedation. The effects of the intravenous infusion of either 0.15 M or 2.5 M NaCl (0.1 ml/kg/min, 75 min) were also studied. In control dogs, artificial cerebrospinal fluid (ACSF) was administered at a rate of 10 microliter/min for 105 min. ACSF given intracerebroventricularly plus 0.15 M NaCl given intravenously did not affect ADH release, but 2.5 M NaCl given intravenously raised the plasma ADH level in parallel with the rise in plasma osmolality. Heart rate and blood pressure did not change significantly in ACSF along with 0.15 M NaCl, but heart rate increased significantly in ACSF along with 2.5 M NaCl. Ang II along with 0.15 M NaCl significantly raised plasma ADH and decreased heart rate without any changes in blood pressure. Ang II along with 2.5 M NaCl brought about a significant rise in plasma ADH level, arterial blood pressure, heart rate, and plasma osmolality. But simultaneous application of Ang II and 2.5 M NaCl did not result in a larger rise in plasma ADH than that expected from the effects of the two stimulations given separately. Namely, Ang II did not potentiate ADH release elicited by osmotic stimulation. Ang II antagonist given intracerebroventricularly neither affected ADH release and the cardiovascular system in 0.15 M NaCl nor inhibited ADH release in response to osmotic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014363 TI - Effects of pentylenetetrazol on GABA-A/benzodiazepine/picrotoxinin receptor complexes in rat brain regions. AB - The effects of acute convulsive doses of pentylenetetrazol (PTZ) on [35S]t-butyl bicyclophosphorothionate (TBPS), [3H]flunitrazepam (FNP), [3H]muscimol, and [3H]gamma-aminobutyric acid (GABA) binding sites were examined in well-washed homogenates of various brain regions of rat. Except for a significant increase in the number of striatal [35S]TBPS binding sites, no significant change in [35S]TBPS, [3H]FNP, [3H]muscimol, and [3H]GABA binding was found in various brain regions 30 min after subcutaneous injection of PTZ at 90 or 100 mg/kg. Similarly, there were no significant changes in [35S]TBPS and [3H]FNP binding to unwashed P2 membranes of cerebral cortices 30 min following administration of convulsive doses of PTZ. These experiments failed to demonstrate acute modulation of GABA A/benzodiazepine/picrotoxinin receptor complex by PTZ in the various brain regions examined except striatum. The significance of the increased [35S]TBPS binding in striatum caused by PTZ remains unclear. PMID- 3014367 TI - Niemann-Pick disease type C. Skin biopsies in parents. PMID- 3014366 TI - Down-regulation of growth hormone releasing factor receptors following continuous infusion of growth hormone releasing factor in vivo. AB - The in vivo chronic infusion of growth hormone releasing factor (GRF) results in a loss of the pituitary growth hormone (GH) response to GRF as well as in a substantial depletion of pituitary GH content. To evaluate if the loss in response is due to the down-regulation of GRF receptors the specific GRF binding capacity of pituitary homogenates prepared from rats infused with saline or GRF (1 or 15 micrograms/h for 24 h) was determined. The pituitary binding capacity of animals infused with GRF was significantly reduced as compared to animals infused with saline. PMID- 3014368 TI - The effect of intravenous L-5-HTP in the myoclonic encephalopathy of infants. AB - A 7 year old girl suffering from myoclonic encephalopathy of infants (MEI) underwent acute treatment by slow intravenous L-5-hydroxytrypthophan (L-5-HTP). The authors found a clear clinical physiological improvement of myoclonic symptoms at rest and during action, but the opsoclonus remained unchanged. The authors suggest that, at least in this case of MEI, the myoclonus was serotonin responsive, as already reported in action or intention and stimulus sensitive myoclonus. PMID- 3014369 TI - Maturation of central somatosensory conduction time in infancy and childhood. AB - To investigate the age-dependency of central conduction time somatosensible evoked potentials to median nerve stimulation were recorded in 80 infants and children from the age of one week to 20 years. It is shown that the central conduction time starts at about 14 ms in the neonatal period and then gradually declines until the 7th or 8th year of life to the normal adult value with an upper limit of 7 ms. By dividing the data into several groups with age delimiters at 0.5, 1, 3, 5 and 7 years it shows significant differences of the mean values for the central conduction times according to the Student's t-test. The graph itself can well be approximated by the exponential regression y = 6.099 + 7.55 X e-0.686 X x where y represents the CCT and x the age in years. This slow maturation kinetic is presumably due to the ongoing central myelinisation. PMID- 3014370 TI - Sydenham's chorea and psychosis. AB - The medical histories of 600 psychotic and 369 nonpsychotic subjects were examined for the occurrence of rheumatic chorea. There was significantly more rheumatic chorea in the histories of psychotic patients than in nonpsychotics (p less than 0.01, chi-square). Neuropathological associations are discussed. PMID- 3014371 TI - Sensation seeking, paired associate learning and brain catecholamines. AB - Paired associate learning performance and strategies were analyzed in terms of learning situations (training vs. 'contextual' and 'noncontextual' transfer), personality factors [Thrill and adventure seeking (TAS) and disinhibition (Dis)] and catecholamine enzyme activity [dopamine-beta-hydroxylase (DBH) and monoamine oxydase (MAO)]. Performance was better in transfer situations, in high MAO-high DBH subjects (high catecholamine turnover rate?), and as a negative function of DBH (preponderance of dopaminergic activity?). Error patterns of response selection (high omission and low intrusion error rate) were found to be a positive function of contextual transfer, TAS and DBH (high response-contingent noradrenergic activity?). PMID- 3014372 TI - Trazodone and the mental pain hypothesis of depression. AB - The mental pain hypothesis used to develop trazodone is illustrated, together with its medical and methodological implications. Trazodone possesses a well documented antidepressant activity, which indirectly confirms the above hypothesis. It lacks the aminergic effects of other antidepressants and has a marked adrenolytic activity. Therefore it is potentially indicated in depressive states with adrenergic hyperactivation, such as during alcohol withdrawal. The methodological aspects of the approach taken for trazodone are also discussed, in the context of the strategies of psychopharmacological research. PMID- 3014373 TI - Computed tomography in cervical spondylotic myelopathy and radiculopathy: visualisation of structures, myelographic comparison, cord measurements and clinical utility. AB - Sixty-nine patients with cervical spondylotic myelopathy (CSM), radiculopathy (CSR), or both (CSMR) were studied with computed tomography (CT). Computer assisted myelography (CAM) accurately determines the site and nature of spondylotic protrusions and provides good visualisation of the subarachnoid space and cord deformities even in areas with dilute metrizamide. However, excessive vertebral movement and bulging ligamenta flava with their effects on cord deformity, so easily visualised in myelograms, are completely or partially missed. In the assessment of CSM, metrizamide myelography (MM) followed by CAM should be performed, particularly when the myelographic images are unsatisfactory due to contrast dilution or blockage, when cord compression cannot be ascertained with MM and when cord atrophy is suspected. In CSR, the diagnostic information from MM and CAM is comparable. The diagnostic criteria in CAM are, however, less direct and since MM is adequate in uncomplicated cases, CAM is generally not necessary. The APD, APD/TD ratio, area and circularity are sensitive indices of cord deformity and the first two should be used more often to assist visual assessment of cord deformity. The relation between cord parameters and treatment response is better reflected in CSM cases managed conservatively and the results suggest that the degree of cord deformity is helpful in determining the outcome and hence the choice between surgical and conservative treatment. In plain CT, the osteophytes and calcified discs are adequately visualised and canal dimensions measured with accuracy, but the cervical cord and roots cannot be properly assessed and the diagnosis of CSM or CSR cannot be ascertained. At present, its role in cervical spondylosis is therefore limited. PMID- 3014376 TI - Pseudohypertrophy of the calf following S1 radiculopathy. AB - Development of unilateral calf enlargement following chronic S1 radiculopathy occurred in a 39-year-old woman. Computed tomographic examination of the leg musculature showed that the calf enlargement was due to pseudohypertrophy, as the increased area in the posterior leg muscles showed a decrease in density. PMID- 3014375 TI - Effects of intracarotid ionic and non-ionic contrast material on the blood-brain barrier in a rabbit model. AB - A rabbit model was used to assess the effects of intracarotid injections of ionic monomer (meglumine iothalamate), non-ionic monomer (iohexol, iopromide), and non ionic dimer (iotrol) contrast materials on the blood-brain barrier. The degree of blood-brain barrier damage was assessed qualitatively using Evans' blue dye, and quantitatively by calculating the difference in pertechnetate uptake between injected and non-injected hemispheres. The results showed that the non-ionic dimer, iotrol, had the least effect on the blood-brain barrier, and that although iopromide and iohexol produced greater damage than iotrol, the ionic compound, meglumine iothalamate, caused the greatest disruption to the blood-brain barrier. The implications of these findings are discussed. PMID- 3014374 TI - The blood tumour barrier in intracranial tumours studied with X-ray computed tomography and positron emission tomography using 68-Ga-EDTA. AB - Disruption of the blood brain barrier or rather blood tumour barrier in cerebral tumours was studied with CT after intravenous injection of contrast medium and with PET after intravenous administration of 68-Ga-EDTA. Histology from stereotactic biopsies or open surgery is compared with the radiologic findings and advantages of the respective methods are discussed. The material consisted of 47 patients mainly with supratentorial gliomas and a few miscellaneous tumours. Astrocytomas (Kernohan grade II) were found to have no disruption of blood tumour barrier while anaplastic astrocytomas and glioblastomas (Kernohan grade III and IV) had. PET is somewhat superior to CT in detection of disruption of the blood tumour barrier. It is concluded that the combination of CT and PET is of value in the assessment of intracranial tumours. PMID- 3014377 TI - [Arterial hypertension in alcohol withdrawal syndrome]. PMID- 3014378 TI - [Prevention of laparocele after portasystemic shunt. Randomized comparison of sutures with absorbable vs non-absorbable thread]. PMID- 3014379 TI - [Case reports of 77 HTLV III/LAV-positive patients observed during one-year period. Preliminary note]. AB - Three hundred and forty-four patients at risk for HTLV/LAV infection were examined and seventy-seven were positive at the HTLV III/LAV Ab test. The clinical and laboratory study permitted to diagnose one case of AIDS, 26 cases of ARC and 50 cases of simple positivity at the HTLV III/LAV Ab test. PMID- 3014380 TI - [Neuron-specific enolase in bronchial carcinoma. Immunohistochemical and immunoenzyme study]. AB - Serum level of neuro-specific enolase (NSE) was determined in 20 lung cancer patients. NSE concentration was detected also on neoplastic tissue and NSE positive neoplastic cells on histological sections were observed immunohistochemically. The presence of high level of NSE was showed in small cell lung cancer. PMID- 3014382 TI - [Bilateral carcinoma of the male breast. Analysis of a clinical case]. AB - The rare clinical case of bilateral carcinoma of the male mammary gland is analysed. Current knowledge of the aetiopathogenesis of the pathology is also rapidly analysed. PMID- 3014381 TI - [Bone metastasis as a sign of the existence of latent neoplasia]. AB - A series of 65 patients in whom bone metastasis was the earliest sign of tumour is examined. The diagnostic aspects of the bone metastasis from laboratory examination to radiology, from scintigraphy to bone marrow biopsy are analysed as is the problem of locating the primary tumours that remain of unknown site. The prognosis is poor for all such patients. The possibility of altering the course of the disease and the quality of the patient's life depends on the site and type of the primary neoplasm concerned. Effective systemic palliative treatment is currently only available for plasmacytomas and lymphomas in general, for cancers of the prostate, breast, thyroid and gonads and for a certain number of small cell lung cancers. In the light of the present study and data from the literature it is concluded that the approach to early bone metastasis patients should concentrate on the search for tumours responsive to treatment using simple clinical and instrumental examination techniques. PMID- 3014383 TI - [Lactic acidosis]. AB - Lactic acidosis is a metabolic disturbance characterized by an increase of the production/clearance ratio of lactate. Lactate is a catabolite of glycolysis when this takes place under anaerobic conditions. Clinically LA is characterized by: signs of acidosis, venous blood lactate greater than 5 mMol/l, arterial pH less than 7.25. LA is classified in type A, due to shock, and type B which, in turn, can be divided according to its pathogenesis in B1 correlated to particular pathologies, B2 due to exogenous substances and B3 caused by congenital metabolic diseases. LA is of particular interest in type II diabetes mellitus treated by phenformin. Current therapeutic directions, although suboptimal, are: to eliminate the causes of lactate hyperproduction by maintaining a sufficient efficiency of the cardio-vascular apparatus, to correct acidosis by using alkalinizing solutions, to remove pharmacologically or by dialysis the excess of lactate. PMID- 3014384 TI - [Cystosarcoma phyllodes of the breast]. PMID- 3014385 TI - Evidence for species differences in 'peripheral' benzodiazepine receptors: an autoradiographic study. AB - The presence, density and distribution of 'peripheral' benzodiazepine (BZ) binding sites was investigated by autoradiography in the brains of rats, mice, guinea pigs and cats and in some areas of the dog and monkey brains, using [3H]Ro 5-4864 as a ligand. Marked interspecies differences were found in the distribution and densities of these sites. Rats and mice presented a low density of binding uniformly distributed throughout the brain with high densities concentrated in the ependyma, choroid plexus and olfactory nerve layer of the olfactory bulb. In contrast, guinea pig and cat brains presented relatively high concentrations of peripheral BZ binding sites throughout the grey matter ependyma and choroid plexus, but low in the olfactory bulb. Monkey and dog brains presented low densities of peripheral BZ binding sites, including the choroid plexus. PMID- 3014386 TI - Neurotoxicity of excitatory amino acid receptor agonists in rat cerebellar slices: dependence on calcium concentration. AB - In slices of developing rat cerebellum, a 30-min application of the excitatory amino acid receptor agonist, N-methyl-D-aspartate (NMDA), led to the necrosis of differentiating granule cells and deep nuclear neurones. The corresponding effect of another agonist, kainate, was the death of Golgi cells. The toxic effects of both agonists were prevented if the concentration of calcium in the exposing solution was reduced to 0.3 mM from the control level of 2.5 mM. A lesser reduction (to 1 mM) was enough to prevent 90% of the NMDA-induced necrosis of granule cells. The results indicate that an important component of the acute neurotoxic effects of excitatory amino acids is calcium-dependent and suggest reasons why this may not have been revealed in some previous studies. PMID- 3014387 TI - Excitatory amino acid-releasing and cholinergic neurones in Alzheimer's disease. AB - Brains of normal controls and patients with primary degenerative dementia were investigated for choline acetyltransferase (ChAT) activity in the frontal, temporal and parietal cortex, hippocampus, amygdala and thalamus. A few patients with Alzheimer's disease were unusual as the cholinergic marker was unaffected, except in the amygdala. Other patients with dementia and undiagnosed neurodegenerative disorder had elevated cortical ChAT activity. The interpretations offered are: (a) the syndrome of dementia and Alzheimer pathologic change precedes significant loss of cortical cholinergic innervation; (b) denervation in dementia can occur early in olfactory areas, exemplified here by the amygdala; (c) dementia is associated with the loss of non-cholinergic structure. An indication of structures involved was given by loss of a marker of excitatory amino acid-releasing neurones. PMID- 3014388 TI - Convulsant effect of Ro 5-4864, a peripheral type benzodiazepine, on the baboon (Papio papio). AB - The effects of Ro 5-4864, a 1,4-benzodiazepine with a high affinity for the peripheral-type benzodiazepine (Bz) binding site, were investigated in the baboon (Papio papio), which is genetically predisposed to epilepsy. A proconvulsant effect of low doses (1-3 mg/kg, i.v.) of Ro 5-4864 was observed by studying its effect on the photic responses induced by intermittent light stimulation in non photosensitive baboons. Higher doses of Ro 5-4864 (10 mg/kg, i.v.) were overtly convulsant. The Bzs clonazepam and diazepam blocked these convulsant actions of Ro 5-4864 whereas neither Ro 15-1788, an antagonist of central Bz binding sites, nor PK 11 195, an antagonist of peripheral Bz binding sites, had any effect. It thus appeared that the convulsant effect of Ro 5-4864 was not mediated by Bz binding sites of either the central or the peripheral type. It is possible that Ro 5-4864 exerts its convulsant action at the picrotoxin site of the central Bz receptor - gamma-aminobutyric acid receptor-chloride ionophore complex. PMID- 3014389 TI - GABA receptor binding in rat cerebral cortex and superior cervical ganglion in the absence of GABAergic synapses. AB - High affinity binding of [3H]gamma-aminobutyric acid (GABA) to membrane containing homogenates prepared from rat cerebral cortex (neonatal or adult) or from adult rat superior cervical ganglion (SCG) was tested for sensitivity to baclofen, bicuculline and several other analogues of GABA. Since no major differences were found between the tissues free of GABAergic synapses (SCG and neonatal cortex) and GABAergic synapse-rich adult rat cortex, it is suggested that the presence of GABAergic synapses does not play a decisive role in the determination of pharmacological sensitivities of high-affinity GABA receptors. PMID- 3014390 TI - Autoradiographic localization of adenosine uptake sites in guinea pig brain using [3H]dipyridamole. AB - Tritiated dipyridamole, a specific adenosine uptake inhibitor binds in a saturable and reversible fashion to high-affinity receptor sites in guinea pig brain sections (Kd = 10 +/- 1.5 nM; Bmax = 650 +/- 100 fmol/mg prot.). The anatomical distribution of [3H]dipyridamole binding sites obtained with autoradiographic techniques shows a widespread but heterogeneous distribution of the binding sites throughout the whole guinea pig brain. Very high densities of binding sites are observed in the cerebellar cortex (molecular layer), the pyriform cortex, the superior colliculus (superficial layer), the supraoptic nucleus and the nucleus of the tractus solitarius. The anatomical characterization of the adenosine uptake site, using [3H]dipyridamole as a probe, may be useful to determine the functional role of adenosine in the brain. PMID- 3014391 TI - Changes in substantia nigra pars reticulata activity following lesions of the substantia nigra pars compacta. AB - Single unit activity of substantia nigra reticulata (SNr) neurons was recorded in normal rats and bilaterally in rats subjected to a unilateral 6-hydroxydopamine lesion of the substantia nigra compacta 2-4 weeks previously. Lesions were assessed by rotational behaviour to apomorphine. In normal rats the majority of neurons (63%) showed a regular firing pattern. Following lesion the percentage of these cells was similar in the SNr contralateral to the lesion but decreased on the lesioned side to 26%, whereas bursting activity developed (37% vs 5% in normal rats). The mean firing rate of reticulata neurons was slightly increased after lesion, but not statistically significant. PMID- 3014392 TI - Angiotensin II binding sites in the anteroventral-third ventricle (AV3V) area and related structures of the rat brain. AB - Angiotensin II binding sites were localized in the rat brain by incubation of sagittal sections with 125I-[Sar1]-angiotensin II, followed by [3H]Ultrofilm autoradiography. Binding sites were localized in the subfornical organ and the entire anteroventral-third ventricle (AV3V) area, including the subependymal area of the anterior third ventricle, the median preoptic nucleus and the organon vasculosum laminae terminalis. Binding sites were also present in the paraventricular and periventricular nuclei and in the median eminence. These findings support the hypothesis of an extensive involvement of angiotensin in the neuronal circuits connecting the subfornical organ, the AV3V area, the paraventricular nucleus and the median eminence, and in the regulation of fluid balance, blood pressure and pituitary secretion. PMID- 3014393 TI - Selective neuronal vulnerability to hypoxia in vitro. AB - Hippocampal slices were subjected to a sequence of hypoxic periods of progressively increasing duration. In all slices, synaptic transmission returned during reoxygenation in the fascia dentata, but not in CA1. Thus, the relative vulnerability of CA1 and the fascia dentata to ischemia in situ can be replicated by a purely hypoxic insult in vitro. PMID- 3014394 TI - Autoradiographic distribution of phencyclidine receptors in the rat brain using [3H]1-(1-(2-thienyl)cyclohexyl)piperidine ([3H]TCP). AB - The distribution of phencyclidine (PCP) receptors in the rat brain was determined by autoradiography using 1-(1-(2-thienyl)cyclohexyl)piperidine ([3H]TCP). [3H]TCP appeared to bind to PCP receptors as only PCP-like drugs and sigma-opioids inhibited the binding of [3H]TCP. The areas of the rat brain with the highest density of radiolabeled binding sites were the superficial layers of cerebral cortex, hippocampus and dentate gyrus. Moderate densities of binding sites were found in the medial geniculate nuclei, caudate nucleus, nucleus accumbens, interpeduncular nucleus, superior colliculus, periaqueductal gray and cerebellum. Low densities of binding sites were observed in spinal cord, most of the brainstem, the substantia nigra and most of the hypothalamus. PMID- 3014395 TI - Slow cholinergic excitation of guinea pig hippocampal neurons is mediated by two muscarinic receptor subtypes. AB - Stimulation of cholinergic fibers or bath application of carbachol (0.1-10 microM) induced a slow excitability increase in CA3 neurons and dentate granule cells of hippocampal slices. This effect which was antagonized by atropine (1 microM) was mediated by two receptor subtypes: a pirenzepine (10 microM) insensitive receptor, 'M2', and a pirenzepine (1 microM)-sensitive receptor, 'M1'. The M2-receptor activation led to a blockade of slow afterhyperpolarizations following trains of action potentials and to the occurrence of threshold-activated plateau-depolarizations associated with a conductance increase. The M1-receptor mediated a membrane depolarization sometimes associated with a conductance decrease which reversed its polarity at membrane potentials negative to -80 mV. The 'slow excitatory postsynaptic potential' which results from activation of cholinergic fibers is thus caused by the activation of two receptor subtypes. PMID- 3014397 TI - Somatostatin receptors in rat hippocampus: localization to intrinsic neurons. AB - The effect of neurotoxic chemical and electrolytical lesions on somatostatin (SS) receptor binding in the septo-hippocampal afferents, pyramidal and granule cells of the rat hippocampus was examined by autoradiography using the stable SS analogue 125I-204-090 as radioligand. Electrolytical lesions of the septum did not result in modification of SS binding in the hippocampus. In contrast, both granule cell lesion with colchicine and pyramidal or pyramidal and granule cell lesions with increasing kainic acid doses did result in a specific decrease of binding in the dentate gyrus and hippocampus (CA1 and CA3). These results suggest that SS receptors in the hippocampus are probably associated with elements from intrinsic neurons. PMID- 3014396 TI - Distinct functions for dopamine and serotonin in locomotor behaviour: evidence using the 5-HT1 agonist RU 24969 in globus pallidus-lesioned rats. AB - The locomotor effect of RU 24969, a potent 5-HT1 agonist, was tested in two experimental conditions. Firstly, 5-HT neurons were degenerated by i.c.v. infusion of 5,7-dihydroxytryptamine (5,7-DHT). Secondly, outputs of the nigrostriatal and mesolimbic dopaminergic (DAergic) systems were bilaterally disrupted by electrolytic lesion of the globus pallidus (GP). After both types of lesion, RU 24969 (1.25-5 mg/kg, i.p.) induced an intense and long-lasting hyperlocomotion which was more pronounced than in intact rats. The hyperlocomotion induced in 5,7-DHT lesioned rats as well as that in intact rats was abolished by the DA blocker haloperidol (0.5 mg/kg, i.p.). On the contrary the hyperlocomotion induced in GP-lesioned rats was either unmodified or increased both by haloperidol even at a very high dose (2 mg/kg, i.p.) and by methysergide (5 mg/kg, i.p.). It is concluded that (i) there is no DAergic link in the locomotor response to RU 24969, (ii) 5-HT1 receptors are involved in the motor execution of locomotion, (iii) DA initiates and controls the 5-HT-mediated locomotion in normal rats. PMID- 3014398 TI - Neonatal monosodium glutamate dosing alters the sleep-wake cycle of the mature rat. AB - Alterations of the sleep-wake cycle have been studied in male adult rats after neonatal administration of monosodium glutamate (MSG; 4 X 4 mg/g body wt.). Results indicated that MSG treatment caused: an almost complete disappearance of ACTH and alpha-MSH immunoreactive (IR) perikarya in the rostral part of the arcuate nucleus; an increase in total sleep duration with a more pronounced effect on paradoxical sleep. Regarding circadian rhythmicity there was a trend to a decomposition of the 24 h period into ultradian components (12 h, 8 h, 6 h harmonics). The participation of pro-opiomelanocortin peptides in sleep regulation is discussed. PMID- 3014399 TI - The cerebellorubral projection in the rat: retrograde anatomical study. AB - The cerebellorubral projections have been studied in the rat using the retrograde transport of horseradish peroxidase-wheat germ agglutinin conjugate. The lateral cerebellar nucleus projects to the parvocellular red nucleus (RN), the anterior (NIA) and posterior (NIP) interposed nuclei project to the magnocellular RN. Whereas the projections from the NIP are limited to the medial aspect of the RN, those from the NIA extend throughout the magnocellular RN. NIA-RN projections are topographically arranged: the medial NIA projects ventrally, the lateral NIA projects dorsally. Functionally, this differential distribution seems to fit the hindlimb-forelimb areas of origin of the rubrospinal tract. PMID- 3014400 TI - Two thalamo-telencephalic pathways in a urodele, Triturus alpestris. AB - Ascending thalamo-telencephalic projection systems have been investigated in an urodele, Triturus alpestris, using the horseradish peroxidase technique. Two separate dorsal thalamic projections onto the telencephalon have been identified; one arises from the posterior dorsal thalamus and terminates in the ipsilateral striatum, the other originates from anterior dorsal thalamic cells and reaches the medial pallium and a part of the dorsal pallium bilaterally. Both systems, which are spatially well segregated, might carry visual information to the telencephalon, as the posterior dorsal thalamus receives tectal, and the anterior dorsal thalamus direct retinal input. The urodele projection scheme as described here shows great similarities to the one described in anurans, although there are remarkable cytoarchitecture differences between the anuran and the urodele thalamus. PMID- 3014401 TI - Neuron-specific gamma-enolase derived from human glioma. AB - Neuron-specific gamma-enolase in human neurogenic tumors, including gliomas, transplanted gliomas, and permanent human glioma cell lines, was studied quantitatively, using newly established enzyme immunoassay methods, together with immunostaining of the tissue and cell preparations. A significantly high level of gamma-enolase was found in some glioblastomas, astrocytomas and oligodendrogliomas as well as medulloblastomas. Glioblastomas transplanted into mice and cultured cell lines derived from the same origins, as well as the permanent human glioma cell lines, also contained gamma-enolase, although the contents were low compared with findings in the original tumor tissues. Immunohistochemically, gamma-enolase stained intensely in the glioblastomatous cells. Serum gamma-enolase concentrations in some patients with gliomas and those of all the transplanted mice were enhanced. The serum gamma-enolase levels in the mice correlated well with size of the transplanted tumor tissues. These results indicate that neuron-specific gamma-enolase is produced in some neurogenic tumors of nonneuronal origin, therefore, serum gamma-enolase may be a useful biomarker for monitoring the extent of disease in patients with gliomas. PMID- 3014402 TI - Maternal riboflavin carrier protein is essential for fad synthesis in the rat fetus. PMID- 3014403 TI - Copper bioavailability: assessment and extrinsic factors. PMID- 3014404 TI - Hormonal regulation of phosphoenolpyruvate carboxykinase. PMID- 3014405 TI - Uptake, distribution, and esterification of retinol in liver. PMID- 3014406 TI - Post-discharge care--how to follow-up. PMID- 3014407 TI - AIDS and HTLV-III infection. PMID- 3014408 TI - Pregnancy rates after hysterosalpingography with oil- and water-soluble contrast media. AB - Hysterosalpingography can be accomplished with either oil or water-soluble contrast medium. This randomized prospective study compared pregnancy rates in women who had hysterosalpingography with either water- or oil-soluble contrast material and were followed for six months. Fifteen of 60 (25%) patients who received water-soluble dye conceived compared with 14 of 46 (30%) patients in the oil-soluble group, a statistically insignificant difference. Furthermore, no difference in pregnancy rates within each subgroup of fertility diagnosis was detected. Intravasation was more common in patients administered oil-based contrast materials (six of 46 versus one of 60 patients, P = .02), although no serious consequences occurred. No difference in the amount of pain as assessed by pain scoring was experienced by patients in each group. The authors conclude that pregnancy rates are similar after hysterosalpingography with oil- and water soluble contrast material, during at least the first six months after the procedure. PMID- 3014409 TI - [Pathophysiology and clinical aspects of acquired immunologic deficiency syndrome (AIDS)]. PMID- 3014410 TI - The controversial Keyes' technique. PMID- 3014411 TI - [Our experience in using collalysine]. PMID- 3014412 TI - Intraocular 9-([2-hydroxy-1-(hydroxymethyl) ethoxy] methyl) guanine levels after intravitreal and subconjunctival administration. AB - Clearance studies in rabbits following intravitreal injection of 400 micrograms of 9-([2-Hydroxy-1-(hydroxymethyl)ethoxy]methyl) guanine demonstrated levels of the antiviral at 60 hours above the in vitro ID50 for several strains of human cytomegalovirus. Intravitreal administration of this new antiviral may have therapeutic application in the treatment of acute retinal necrosis and cytomegalovirus retinitis complicating acquired immune deficiency syndrome (AIDS). PMID- 3014413 TI - Aging changes in Bruch's membrane of monkeys: an electron microscopic study. AB - Bruch's membrane from monkeys of various ages was studied by electron microscopy. In monkeys under 15 years of age, Bruch's membrane rarely contained a small amount of polymorphous material that did not appear to increase with advancing age up to 15 years. However, the polymorphous material did increase over 20 years of age. The accumulation of vesicular, granular, tubular and linear polymorphous material in Bruch's membrane was though to be a result of evagination of a minimal portion of a retinal pigment epithelial (RPE) cell between adjacent basal infoldings, and its subsequent degeneration. The plasma membrane of the evagination seemed to be the primary source of the tubular or linear material, vesicles the main source of vesicular material, and cytosol and basement membranes to be the source of the granular material. The fibrous long-spacing collagen was associated with the basement membrane of the choriocapillaris and RPE cells. The granular deposits between the plasma infoldings and the basement membrane of RPE cells may originate from the basement membrane of the RPE, and could be the initial lesion of basal linear deposits. PMID- 3014414 TI - Varicella zoster virus is a cause of the acute retinal necrosis syndrome. AB - We studied two blind eyes enucleated during the active phase of the acute retinal necrosis syndrome. Both eyes showed similar histopathologic findings of necrotizing retinitis, retinal arteritis, and optic neuropathy. A virus morphologically consistent with a herpes group virus was found on electron microscopy and immunocytopathologic stains showed this virus to be varicella zoster in both cases. Varicella zoster virus was cultured from the vitreous of one of the eyes. We conclude that varicella zoster virus retinal infection is a cause of the acute retinal necrosis syndrome. PMID- 3014415 TI - Visual outcome after proton beam irradiation of uveal melanoma. AB - Prognostic factors for visual loss following proton irradiation of uveal melanoma were evaluated for 440 eyes treated from 1975 to 1984, with visual acuity 20/200 or better before treatment. Analysis involved Kaplan-Meier survival curves and Cox proportional hazards analysis with visual outcome defined as worse than 20/200. Prognostic factors were tumor height: rate ratio (ratio of rate of visual loss for one category of the variable relative to the rate of visual loss for a reference category of that variable) of 5.26 (95% confidence interval, 2.66 10.39) for tumors greater than 5 mm compared to tumors 3.0 mm or less in height; distance of tumor from the optic disc and fovea: rate ratio 2.59 (1.63-4.11) for tumors 2DD or less from both the optic disc and fovea compared to those greater than 2 DD from these structures. Also predictive of visual loss were tumor location close to disc only, or close to fovea only, macular detachment, worse pretreatment vision, and higher radiation doses delivered to both the disc and fovea, and lens. Regression analysis using a visual acuity scale gave similar results. PMID- 3014416 TI - Prognostic factors for metastasis following proton beam irradiation of uveal melanomas. AB - Prognostic indicators for the development of metastasis following proton beam irradiation of uveal melanomas were evaluated for 510 patients treated from 1975 to 1984. Thirty-three patients developed metastasis (6.5%) from 3 to 51 months following treatment. The primary site of metastasis was the liver in 28 cases (85%). Both demographic and clinical factors were considered. The three leading predictors of survival without metastasis after proton beam irradiation in order of importance were: (1) largest diameter of the tumor; (2) location of the anterior margin of the tumor; and (3) age at treatment. Worse prognosis was associated with largest tumor diameter greater than 15.0 mm, tumor involvement of the ciliary body and age at treatment older than 59 years. PMID- 3014418 TI - Foot-and-mouth disease and the African buffalo (Syncerus caffer). 1. Carriers as a source of infection for cattle. AB - Ten pregnant buffalo cows, six of which were subsequently shown to be carriers of SAT 1, 2 and 3 viruses, were captured in the Kruger National Park (KNP) and allowed to calve in captivity. The buffalo cows and calves were separated by a fence from 6 FMD susceptible cattle but the buffalo and cattle were obliged to use common drinking troughs and hay racks. Over a period of 15 months, during which the buffalo calves lost their maternally-derived immunity, neither the buffalo calves nor the susceptible cattle became infected with FMD virus. By the end of the observation period, however, only 1 buffalo cow still had detectable virus in its oesophageal/pharyngeal specimens. PMID- 3014417 TI - Isolation of Cowdria ruminantium by means of Percoll density gradient centrifugation and detection by an enzyme-linked immunosorbent assay. AB - The isolation of Cowdria ruminantium by means of Percoll density gradient centrifugation permits the recovery of partially purified viable populations of the organism possessing distinctly different densities. These conclusions are based upon results of analyses of density fractions by intravenous inoculation into sheep, protein determination, electron microscopy and enzyme-linked immunosorbent assay. Morphological differences were observed in the density fractions obtained from infected brain tissue and Amblyomma hebraeum nymphae. PMID- 3014419 TI - Foot-and-mouth disease and the African buffalo (Syncerus caffer). II. Virus excretion and transmission during acute infection. AB - Three groups of young buffalo in captivity were infected by exposing them to similar buffalo in the acute stages of infection induced by needle inoculation with SAT 1 or 2 viruses. Clear foot lesions developed in most of the buffalo from which the relevant virus types were re-isolated. During the first week following infection virus was found in blood, nasal secretions, saliva, preputial secretions and faeces. Air samples collected in the immediate vicinity of acutely infected buffalo were also found to contain virus. However, the regularity of virus detection as well as the quantity of virus in buffalo specimens was generally lower than for cattle infected with viruses of the same type. Conversely, virus was detected in the nasal secretions or saliva of 3 buffalo up to 4 weeks after infection, a situation which has not been encountered in cattle. Susceptible cattle and impala (Aepyceros melampus) were penned together with or in the immediate vicinity of infected buffalo and shared feeding and watering facilities with the buffalo. The pattern of transmission which emerged indicated that transfer of these viruses from buffalo to other species probably occurs only in the acute stages of infection and where there is direct physical contact between the species. PMID- 3014420 TI - True malignant mixed tumors (carcinosarcoma) of salivary glands. AB - True malignant mixed tumors (carcinosarcomas) of salivary glands are of a high grade of malignancy and are distinguishable from the more frequently occurring carcinomas ex pleomorphic adenoma. Having a putative origin from a benign pleomorphic adenoma, the true malignant mixed tumor is an aggressive, often rapidly lethal neoplasm in which the sarcomatous element is most often a chondrosarcoma and the epithelial element is most often a ductal carcinoma. The twelve cases in this report represent the largest recorded series to date. PMID- 3014421 TI - Peroxidase-antiperoxidase evaluation of human oral squamous cell papillomas. AB - In human beings, the viral origin of condylomatous lesions of the mouth and cervix, multiple papillomas, and verruca vulgaris of the larynx has been established. The viral origin of papillomatosis in rabbits and dogs has also been documented. The literature, however, shows no conclusive evidence of a viral origin of human oral squamous cell papillomas (SCP). The purpose of this study was to use the peroxidase-antiperoxidase (PAP) staining technique to determine whether human papillomavirus (HPV) antigens exist in serial step sections of human oral SCP. Fifty human oral SCPs were randomly selected from the department files, and tissue sections were subjected to an indirect immunoperoxidase-PAP technique. Evidence of HPV-Type I virus antigens was found in 2 of 50 papillomas studied (2 of 194 tissue sections). This suggests that all oral SCPs are not of viral antigen. PMID- 3014423 TI - [Cytomegalovirus hepatitis]. PMID- 3014422 TI - [Importance of the determination of free thyroxine and triiodothyronine serum levels in preclinical hyperthyroidism and subclinical hypothyroidism]. PMID- 3014424 TI - [Complicated respiratory diseases with detection of adeno-, influenza and RS viruses]. PMID- 3014425 TI - Characterizing "difficult" acute leukemias. A combined electron microscopic and immunological marker study. AB - The techniques of transmission electron microscopy (TEM), including ultrastructural myeloperoxidase cytochemistry (MPO), and immunological marker analysis, have been used to classify 58 "difficult" cases of acute leukemia where a precise diagnosis could not be made on the basis of conventional light microscopy and cytochemistry. TEM with MPO proved most valuable in characterizing 15 cases of acute myeloid leukemia and its variants, as well as defining complex cellular subpopulations in 11 cases of chronic myeloid leukemia in blast crisis. Immunological marker studies provided conclusive evidence of lymphoid differentiation in 18 cases of acute lymphoblastic leukemia and related disorders. In addition, the combined techniques were used to document 14 cases of terminal transferase-positive acute myeloid leukemia. This study demonstrates that these 2 techniques provide overlapping and complementary information for accurate diagnosis and classification of morphologically difficult hematological malignancies. PMID- 3014426 TI - The syndrome of achalasia of the esophagus, ACTH insensitivity and alacrima. AB - A 7-year-old male presented with a triple A syndrome, a tirad of ACTH insensitivity, achilasia and alacrima. His clinical course is followed and the literature reviewed. PMID- 3014427 TI - Henoch-Schonlein purpura and human parvovirus infection. PMID- 3014428 TI - [Hypoglycemia and hyperlactacidemia in relation with a new case of fructose-1,6 diphosphatase deficiency. Determination of enzymatic activity in leukocytes]. AB - A 11.5 month-old girl with recurrent episodes of hypoglycemia and lactic acidosis was identified as having fructose-1,6-diphosphatase deficiency. The diagnosis may be realised with a simple way by an oral fructose tolerance test and the activity dosage of this enzyme in white blood cells. The fructose and sucrose-free diet and avoidance of prolonged fasting resulted in a decrease of hepatomegaly and normal values of lactate between the episodes. PMID- 3014429 TI - The search for the mechanism of hormone action. PMID- 3014430 TI - [Basic and clinical studies of dynamic sequential computed tomography during arterial portography in the diagnosis of hepatic cancers]. PMID- 3014431 TI - [Radiosensitivity of carcinoma of the esophagus]. PMID- 3014432 TI - [Leukotrienes as mediators in allergy]. PMID- 3014433 TI - [Icelandic research. Slow virus infections and AIDS]. PMID- 3014434 TI - [The physiological function of the Na+,K+ pump]. PMID- 3014435 TI - Cloning of the STA2 and SGA genes encoding glucoamylases in yeasts and regulation of their expression by the STA10 gene of Saccharomyces cerevisiae. AB - The Saccharomyces STA2 and SGA genes, encoding the extracellular and intracellular sporulation-specific glucoamylase respectively, have been cloned and their transcription and regulation studied. The STA2 gene differs from the SGA gene in that it contains an extra piece of DNA, which encodes the domain for exportation of the extracellular glucoamylase. The STA2 gene produces a single 2.85 kb transcript. Transcription of the SGA gene is initiated from two different sites, yielding two transcripts of 1.95 and 2.40 kb. Transcription of both STA2 and SGA genes is repressed by the STA10 gene of Saccharomyces cerevisiae. PMID- 3014436 TI - Structural variations in the Drosophila retrotransposon, 17.6. AB - More than 21 members of 17.6, a Drosophila retrotransposon, were isolated and their possible structural changes were examined by restriction mapping, blot hybridization, heteroduplex analysis and nucleotide sequence determination of long terminal repeats (LTRs). At least 7 members were found to suffer with terminal or internal long deletions. No pair of LTRs having an identical nucleotide sequence was found either within an element or between elements. Although an initiation site for the presumable genome-sized transcript of 17.6, a potential substrate for reverse transcription on translocation, was identified within the left-hand LTR, our results as a whole support the notion that the majority of 17.6s have continued to reside for a long period of time at their present chromosomal loci and hence the rate of translocation of 17.6 is very low. PMID- 3014437 TI - Preferential transcription of HTLV-I LTR in cell-free extracts of human T cells producing HTLV-I viral proteins. AB - The promoters of the adenovirus 2 major late gene, the mouse beta-globin gene, the mouse immunoglobulin VH gene and the LTR of the human T-lymphotropic retrovirus type I were tested for their transcription activities in cell-free extracts of four cell lines; HeLa, CESS (Epstein-Barr virus-transformed human B cell line), MT-1 (HTLV-I-infected human T cell line without viral protein synthesis), and MT-2 (HTLV-I-infected human T cell line producing viral proteins). LTR was preferentially transcribed in the extracts of MT-2 although the other three genes were transcribed with relatively constant efficiencies in different extracts. The results agree well with the previous in vivo studies on the promoter activity of HTLV-I LTR. Mixing of HeLa and MT-2 extracts revealed the presence of a LTR-specific stimulating activity in MT-2 extracts. PMID- 3014438 TI - Localization and DNA sequence analysis of the C gene of bacteriophage Mu, the positive regulator of Mu late transcription. AB - The C gene of bacteriophage Mu, required for transcription of the phage late genes, was localized by construction and analysis of a series of deleted derivatives of pKN50, a plasmid containing a 9.4 kb Mu DNA fragment which complements Mu C amber mutant phages for growth. One such deleted derivative, pWM10, containing only 0.5 kb of Mu DNA, complements C amber phages and transactivates the mom gene, one of the Mu late genes dependent on C for activation. The DNA sequence of the 0.5 kb fragment predicts a single long open reading frame coding for a 140 amino acid protein. Sequence analysis of DNA containing a C amber mutation located the base change to the second codon of this reading frame. Generation of a frameshift mutation by filling in a BglII site spanning codon 114 of this reading frame resulted in the loss of C complementation and transactivation activity. These results indicate that this open reading frame encodes the Mu C gene product. Comparison of the predicted amino acid sequence of the C protein with those of other transcriptional regulatory proteins revealed some similarity to a region highly conserved among bacterial sigma factors. PMID- 3014439 TI - Requirement of A-A-U-A-A-A and adjacent downstream sequences for SV40 early polyadenylation. AB - RNA mapping experiments and chloramphenicol acetyltransferase assays were used to analyze polyadenylation in COS cells of transcripts from derivatives of pSV2-neo and pSV2-cat, in which the SV40 early poly(A) signal has been modified. Neither the sequence A-A-U-A-A-A nor the sequences located immediately downstream from it in the SV40 early gene appear to function by themselves as a poly(A) signal. When combined, however, these two elements form a poly(A) signal whose efficiency and specificity closely resemble those of the wild type signal. The addition of six nucleotides between the A-A-U-A-A-A sequence and the poly(A) site has no detectable effect on the efficiency or site of polyadenylation. Deletion of the 60 nucleotides immediately upstream from the hexanucleotide also has no detectable effect on polyadenylation. Therefore, A-A-U-A-A-A and sequences downstream from it appear to be sufficient for SV40 early polyadenylation. PMID- 3014440 TI - Occurrence of 9 homologous repeat units in the external spacer region of a nuclear maize rRNA gene unit. AB - A 14 kb maize DNA fragment carrying nuclear rRNA genes and spacer regions was isolated and characterised by restriction enzyme mapping. A complete 3020 bp long external spacer region was sequenced and revealed 9 tandemly arranged 200 bp long repeat units with high homology. The repeat units lie upstream from two prominent S1 mapping signals. The sequence of a typical repeat unit is compared to a corresponding 130 bp long wheat repeat unit. The possible functional relevance of the repeat units is discussed. PMID- 3014441 TI - Genomic structure of the large RNA segment of infectious bursal disease virus. AB - The larger RNA segment of infectious bursal disease virus (IBDV: Australian strain 002-73) has been characterized by cDNA cloning and nucleotide sequence analysis. We believe IBDV is the first birnavirus to be sequenced and so have confirmed the coding region by N-terminal amino acid sequence analysis of intact viral proteins and several tryptic peptide fragments. The large RNA segment encodes in order the 37-kDa, 28-kDa and 32-kDa proteins within a continuous open reading frame and the primary translation product appears to be subsequently processed into the mature viral proteins. The large protein precursor is still processed into the 32-kDa host protective immunogen when expressed as a fusion protein in E. coli. These results are in marked contrast to the predictions from in vitro translation data that birnavirus genomes are expressed as polycistronic templates. We can now propose that birnaviruses, in particular IBDV, possess monocistronic segments and that the precursor is proteolytically processed in vivo. The sequence data presented for the 32-kDa host protective immunogen may provide the basic information needed for the production of an effective subunit vaccine against this commercially important virus. PMID- 3014442 TI - RFLPs for epidermal growth factor (EGF), a single copy sequence at 4q25-4q27. PMID- 3014443 TI - RFLPs for PstI and EcoRI in the human blood clotting factor X gene. PMID- 3014444 TI - TaqI polymorphism at the human coagulation factor XII locus (F12). PMID- 3014445 TI - NcoI RFLP at the human apolipoprotein CII gene locus. PMID- 3014446 TI - BglII dimorphism at the human atrial natriuretic peptide (ANP) gene locus. PMID- 3014447 TI - [Incidence of anti-Herpesvirus antibodies in a group of pregnant women]. PMID- 3014448 TI - [Poland-Moebius syndrome. Description of a new case]. AB - A new case of Poland-Moebius syndrome in a male is report and developmental field and developmental field defect is debate. PMID- 3014449 TI - [Herpesvirus simplex disease in the newborn infant. Clinical aspects, diagnosis, prophylaxis, therapy]. AB - Herpes Simplex virus infection (Particularly by HVS2) is one of the most important disease transmitted by mother to newborn because of the seriousness of symptoms and the high incidence of genital infection of pregnants. Transmission of congenital HVS infection occurs usually by contact with infected genital secretions during the birth process, but there is evidence that transplacenter transmission also may occur. Emphasis must be placed then on prophylaxis of genital infection of pregnants and of their partners. Specific Immunoglobulins with high antibody titre are recommended for prophylaxis. An anti HVS2 vaccine without oncogen property os still at experimental stage. This prophylaxis is inadequate for the newborn of infected mother: this one must be treated with newer antiviral agents (Acyclovir), even is absence of herpetic lesion and other symptoms. PMID- 3014450 TI - [Dynamics of changes in the titres of Herpes simplex virus type 1 antibodies in patients treated with long-term dialyses]. PMID- 3014451 TI - [Microangiopathic hemolytic anemia in stomach cancer--description of 2 cases]. PMID- 3014452 TI - Presentation of neonatal herpes simplex virus infections: implications for a change in therapeutic strategy. AB - To identify clinical signs of disease that might lead to more rapid recognition in treatment, we reviewed the time from onset of illness to diagnosis of 42 consecutive cases of neonatal herpes simplex virus (HSV) infection seen between 1965 and 1984. The first signs of illness included mucocutaneous lesions in 14, central nervous system signs in 20, fever in 6 and respiratory insufficiency in 2 infants. The median time from onset of illness to presentation to medical personnel was 1 day. The median time from presentation to medical personnel to obtaining viral cultures was 3 days (range, 1 to 11) and was similar in infants who did and did not have mucocutaneous lesions. Viral cultures were performed within 24 hours of admission on 8 of 13 noncongenitally infected infants born between 1982 and 1984 compared to 5 of 24 seen between 1965 and 1981 (P less than 0.03). However, a greater than 72-hour delay between presentation to medical personnel and obtaining viral diagnostic studies occurred in 33, 40 and 14% of infants born in the years 1965 to 1977, 1978 to 1981 and 1982 to 1984. Involvement of additional organ systems by HSV was noted in 57% of infants between the time from presentation to medical personnel and diagnosis. Neonatal HSV infection was often severe by the time patients presented to medical personnel, and the disease usually progressed rapidly. To achieve a better therapeutic outcome for infants with neonatal herpes, consideration should be given to the initiation of antiviral therapy on presumptive clinical and epidemiologic grounds. Future strategies for therapy of neonatal herpes should be directed at preventing the acquisition of disease. PMID- 3014453 TI - Molecular epidemiology of cytomegalovirus: evidence for viral transmission to parents from children infected at a day care center. AB - Based on an analysis of the DNA of 16 cytomegalovirus (CMV) isolates obtained from children attending a day care center, we had previously determined that one group of 7 and another group of 4 children were shedding common viral strains. Because the DNA of each epidemiologically unrelated strain of CMV is unique, at least 9 of these 11 children acquired CMV from other children attending the day care center. In order to assess CMV transmission between these 11 viruric children and their parents, the parents were surveyed for CMV infections. Nine of the 10 mothers of the viruric children were seropositive but only 12 of 33 mothers of nonviruric children at the day center were seropositive (P less than 0.007), Fisher's exact test). Urine and saliva specimens from each parent of the viruric children were cultured for CMV. Three parents (a father and two pregnant mothers) were excreting virus. When analyzed with restriction endonucleases, the viral DNA of the isolate from each parent was identical to that from the virus excreted by the child and from the virus excreted by other children at the day care center. The results of this survey indicate that CMV was frequently transmitted to parents from children who had acquired the virus at the day care center. PMID- 3014454 TI - Infectious complications in pediatric patients undergoing transplantation with T lymphocyte-depleted bone marrow. AB - We performed a retrospective study of the infectious complications in 14 pediatric patients undergoing transplantation with T lymphocyte-depleted bone marrow. The purpose of this study was to document the infectious complications encountered in these patients and to ascertain whether or not they appeared to be more susceptible to any particular infections. It was found that all 14 patients developed infectious complications following transplantation: there were a total of 24 bacterial infections among 11 patients, 21 fungal infections among 11 patients, 2 parasitic infections and 4 viral infections. The viral infections included two cases of infection with cytomegalovirus, one of which manifested as encephalitis and pneumonia, and one case of possible infection with Epstein-Barr virus, which included clinical findings compatible with a lymphoproliferative disorder. These patients were at an increased risk to certain viral infections and particular attention must be paid to the prevention, diagnosis and treatment of these infections. PMID- 3014455 TI - Two pseudo-outbreaks of infectious mononucleosis. AB - Two outbreaks of suspected infectious mononucleosis (IM) were investigated. In the first outbreak IM was diagnosed in nine children attending a day care center. They had been tested in physicians' offices for heterophile antibody using rapid differential slide tests; all tests had been reported positive. On retesting, none of the suspected cases had detectable serum heterophile antibody. The initial test results had been falsely positive as a result of poor laboratory technique. In the second outbreak IM had been diagnosed in 285 college students. Suspected cases had been found to have serum IgG antibody to the viral capsid antigen of Epstein-Barr virus, but most had not been tested for the presence of heterophile antibody. Retesting of 64 students within 1 month of initial testing yielded only one with heterophile antibody. With the exception of young children (less than 4 years of age), differential slide tests for heterophile antibody are sensitive and specific for recent Epstein-Barr virus infection if properly performed. Viral capsid antigen to Epstein-Barr virus (IgG) titers are of limited usefulness in diagnosing acute IM. The misdiagnosis of IM can be prevented by the appropriate selection, performance and interpretation of diagnostic laboratory tests. PMID- 3014456 TI - Epstein-Barr virus serologic testing: diagnostic indications and interpretations. PMID- 3014457 TI - Virus-associated hemophagocytic syndrome: relationship to herpes group viruses. PMID- 3014459 TI - Acute infectious diarrhea. II. Diagnosis, treatment and prevention. PMID- 3014461 TI - Radiologic case study. Malignant fibrous histiocytoma of bone. PMID- 3014458 TI - Life table analysis of children with acquired immunodeficiency syndrome. PMID- 3014460 TI - A new latex agglutination test kit for detecting rotavirus in stool from children with gastroenteritis. PMID- 3014462 TI - Neurofibromatosis and associated neuroectodermal tumors: a congenital neurocristopathy. AB - The synchronous occurrence of neurofibromatosis and neuroblastoma has been labeled in the recent literature as a chance event. We report 2 cases of newborn infants with congenital neurofibromatosis and a similar midline pattern of multiple Schwann cell and neuroblastic tumors; other types of ectomesenchymal tumor differentiation are documented, along with supportive ultrastructural and immunohistochemical studies. The tumors may take an aggressive, fatal course despite maximal multimodality antitumor therapy. These 2 cases are reported, with additional literature review, to document a clinically recognizable neurocristopathy that links neuroblastic tumors and neurofibromatosis. PMID- 3014464 TI - Lisinopril cough. PMID- 3014465 TI - Active immunization against varicella. Proceedings of a symposium. Munich, November 29 and 30, 1984. PMID- 3014463 TI - [Role of adrenergic receptors in the regulation of glycogen metabolism in the liver]. PMID- 3014466 TI - Varicella vaccine in children with chronic renal insufficiency. AB - This study reports the antibody response and clinical follow-up of uraemic children awaiting kidney transplantation after administration of the Oka-strain varicella vaccine (Varilirix). Seroconversion was observed in 20 out of 23 patients found to be seronegative when tested by the fluorescent antibody to membrane antigen technique, and an antibody booster response was observed in 41 out of 47 seropositive patients. Mild clinical varicella occurred in 5 vaccinated patients and herpes zoster in 3 initially seropositive ones. Nevertheless, a dramatic decrease in the incidence of both varicella and herpes zoster was observed in a series of 330 consecutive transplantations after the introduction of the varicella vaccine. PMID- 3014467 TI - Experience with the live Oka-strain varicella vaccine in children with solid tumours. AB - A comparative evaluation of radioimmunoassay (RIA), indirect immunofluorescence, fluorescent antibody to membrane antigen, competitive inhibition RIA, and complement fixation methods for determining the immune status to varicella-zoster virus (VZV) showed that RIA was the most sensitive test. However, this test is still not regarded as being entirely satisfactory and possible reasons are discussed. To date, 28 children, mainly with solid tumours, have been inoculated with the Oka-strain live varicella vaccine. Two of these vaccinees developed minor reactions, but only 62% developed a RIA-detectable humoral response. Approximately 46% of the responders had lost induced antibody within 12 months of vaccination. None of the 6 vaccinees that have subsequently been in contact with varicella developed any illness suggesting that they had been protected. PMID- 3014468 TI - Worldwide experience with the Oka-strain live varicella vaccine. AB - Sufficient safety and efficacy data have been obtained on a live varicella vaccine (Varilrix, Smith Kline-RIT) derived from the Oka-strain attenuated varicella-zoster virus to justify its recent licensure in several European countries for use in special indication groups. This review article summarizes the data obtained in Japan, Europe, and the United States. In addition, a survey of the published literature on clinical studies performed on as yet unlicensed Oka-strain varicella vaccines produced by the Biken Institute and Merck Sharp & Dohme is presented. PMID- 3014469 TI - Live varicella vaccine in healthy individuals. AB - Eight hundred healthy seronegative children and 32 adults were vaccinated with the Oka-strain live attenuated varicella vaccine (Varilrix), manufactured by Smith Kline-RIT, Belgium. The vaccine was safe (no side effects) in healthy individuals, highly immunogenic (dose-dependent induction of humoral antibodies and specific cell-mediated immune response), and highly protective against clinical varicella. The simultaneous administration of a measles-mumps-rubella vaccine together with the varicella vaccine is possible, although combination in the same syringe was less effective than simultaneous administration in different arms. Various observations suggest that the risk of contracting zoster in adulthood may well be reduced for varicella vaccinees. Based on our results, it is recommended that seronegative hospital staff be vaccinated. Furthermore, if the varicella vaccine should prove to be cost effective, large-scale immunization of healthy children may be indicated. PMID- 3014470 TI - Live varicella immunization in healthy non-immune nurses. AB - Thirty-four varicella-zoster virus (VZV) seronegative nurses were vaccinated with the live varicella vaccine (Varilrix) and followed for periods of up to 36 months. No major vaccine reactions were observed. At 5 and 12 months, 94% of the nurses had seroconverted but at 3 years, only 64% retained antibody activity. However, lymphocyte transformation to VZV antigen was positive in 7 seronegative nurses, all of whom had previously seroconverted. The one nurse who developed chickenpox had not seroconverted after vaccination. Two out of 11 seroconverted nurses had a subclinical reinfection, as shown by a rise in antibody, upon exposure to varicella. In contrast, 4 out of 5 seronegative nurses who had refused vaccination developed chickenpox. Varilrix is therefore safe, immunogenic, and protective in adults and can be considered for routine use in susceptible health workers. However, it is still uncertain whether lifelong immunity is obtained with this vaccine. Both cell-mediated and antibody tests are needed for long-term assessment of immunity to chickenpox. PMID- 3014471 TI - Delayed hypersensitivity skin test to detect susceptibility to varicella and zoster. AB - A crude varicella skin-test antigen corresponding to an inactivated partly purified high-titre varicella Oka-strain vaccine was prepared at Smith Kline-RIT. It was used for a delayed hypersensitivity skin test in 60 healthy adults for determination of their immune status. The findings were analysed according to the presence or absence of humoral antibodies to varicella-zoster virus (VZV), and of a specific cell-mediated immune (CMI) response as measured by the VZV-specific lymphocyte transformation test. The immune status to varicella could be determined in all subjects provided that the amount of antigenic material in the skin-test antigen was sufficiently high. The 2 tests for the CMI response, the delayed hypersensitivity skin test and the lymphocyte transformation test, gave concordant results in both seropositive and seronegative individuals. The inactivated skin-test antigen cannot replace vaccination with the live varicella vaccine since it is unable to induce seroconversion in seronegatives or an antibody booster response in seropositives. Furthermore, seropositive and seronegative subjects showing a negative response to the varicella-zoster specific lymphocyte transformation test remained negative 2 weeks after the injection of the skin-test antigen. PMID- 3014472 TI - Restoration of varicella-zoster virus cell-mediated immune response after varicella booster vaccination. AB - In older age groups, an increasing proportion of healthy adults with a positive varicella history have lost their capacity for a cell-mediated immune (CMI) response to varicella-zoster virus (VZV) antigen despite the presence of virus specific humoral antibodies. While it remains a matter of speculation whether this decline is connected with a predisposition to herpes zoster, it is known that a second zoster attack is rare in immunocompetent individuals. In order to determine whether the VZV-specific CMI response can be restored through varicella vaccination, the immune status of a group with naturally occurring herpes zoster was compared with that of a group of vaccinated subjects. Twenty zoster patients were tested for VZV antibody and VZV-specific CMI within the first 4 days of the disease. Antibodies were present in normal or moderately high titres (mean: 2,900). Lymphocyte reactivity to VZV antigen was negative or weakly positive in 17 of the 20 patients. After 6 weeks, antibody titres rose significantly (mean: 36,000) and the lymphocyte response was highly positive in 11, weakly positive in 6, and remained negative in 3 patients. Thirty-three healthy adults ranging in age from 55-65 years with a negative VZV-specific CMI, but positive VZV antibodies (mean: 190), were immunized with the Oka-strain varicella vaccine (Varilrix). After 6 weeks, the mean antibody titre had only increased slightly (mean: 400). However, a positive VZV-specific CMI response occurred in 28 out of 33 subjects, while 5 remained negative. Therefore, conversion from a negative to a positive CMI response can be achieved by varicella vaccination of seropositive subjects. This raises the question whether booster vaccination of CMI-negative subjects could prevent zoster. PMID- 3014473 TI - Stimulation of specific immune response to varicella antigens in the elderly with varicella vaccine. AB - Ageing is associated with several immune defects that probably lead to the increased incidence of zoster observed after the 5th decade. In a double-blind study to assess an eventual stimulation of the specific immune response against the varicella-zoster virus by vaccination, either the Oka-strain live varicella vaccine or a placebo was administered to 30 healthy volunteers over 70 years of age, after a 4-week pretreatment period with either zinc acetate as an immunostimulant or a placebo. Specific skin delayed hypersensitivity and antibody levels measured by the fluorescent antibody to membrane antigen method were recorded at the start of the pretreatment period, just before vaccination, and two weeks or one month after vaccination. Initially decreased delayed-type responses to varicella antigens were observed when compared with reported values for younger subjects despite normal levels of skin reactivity to common bacterial antigens (Multitest). The varicella-specific responses were markedly boosted in the 3 groups by repeated skin testing. As a result, the effects of zinc pretreatment and vaccination were masked. On the other hand, treatment with zinc acetate was shown to increase tuberculin delayed reactions. Preexisting specific antibody titres were elevated and no significant changes occurred in the 3 study groups. The data suggest that it is not the potential for an immune response that is decreased in the elderly but rather the speed of the response. This might be the defect responsible for the increased incidence of zoster in this age group. PMID- 3014474 TI - The future of varicella vaccine. AB - Based on conservative figures, an estimated 60 million varicella-zoster cases occur annually worldwide, highlighting the global significance of this disease. The development of a viable varicella vaccine, therefore, raises important questions as to the indications for its use in normal children, normal seropositive and seronegative adults, and immunocompromised patients. A review of the available data addresses the potential future role of the varicella vaccine in these groups. PMID- 3014475 TI - Management of varicella-zoster contact and infection in pregnancy using a standardized varicella-zoster ELISA test. AB - An ongoing 5-year prospective study of the outcome of contact with varicella zoster virus (VZV) and infection in pregnant women has made use of a standardized VZV enzyme-linked immunosorbent assay for the determination of immune status and as a guide for therapeutic management. Out of of a total of 778 such cases investigated to date in the Federal Republic of Germany, 93.1% have been shown to be VZV immune, whereas 6.9% were seronegative and, therefore, susceptible to infection. Those of the latter group who received zoster hyperimmunoglobulin (ZIG) between 24-96 hours after varicella contact remained symptom-free, while women receiving ZIG from the 3rd-10th day after contact developed a modified varicella. For the prevention of varicella, ZIG with known titres should be administered at doses of 0.2-0.4 ml/kg. It is also recommended for the newborn when maternal infection occurs within 4 days before until 2-4 days after delivery. The prevention and attenuation of varicella in pregnancy is justified since the outcomes may include spontaneous abortion in early pregnancy, the congenital varicella syndrome, and severe neonatal disease with a rather high mortality around term. PMID- 3014476 TI - Attenuation and laboratory markers of the Oka-strain varicella-zoster virus. AB - A live varicella vaccine was developed by serial passages of the Oka-strain varicella-zoster virus (VZV), isolated in our laboratory, in human embryonic lung cells and guinea-pig embryonic fibroblasts (GPEF). It is slightly temperature sensitive at 39 degrees C and shows a higher ratio of infectivity in GPEF to that in human embryonic fibroblasts (HuEF) than wild-type strains, indicating that it is a variant in thermosensitivity and host dependency. A DNA digest with the HpaI enzyme of the Oka strain contained a unique fragment, denoted K. Clinical studies with VZV isolated from vaccinees indicated that the GPEF/HuEF ratio of infectivity and the profiles of HpaI DNA digests are useful tests to differentiate the vaccine from wild-type strains. Prompt vaccination of household contacts proved effective in preventing spread of clinical varicella and seemed to be related to the early appearance (4-7 days after vaccination) of cell mediated immunity in vaccinees, assessed by the VZV skin test. VZV could be isolated from mononuclear blood cells of varicella patients shortly before or after appearance of rashes, whereas no virus could be detected in 27 vaccinees 4 14 days after vaccination, suggesting that replication of the vaccine virus in susceptible organs and blood cells in humans is far less than that of wild-type viruses, but sufficient to induce VZV immunity. This difference seems to be related to the attenuated nature of the vaccine strain. PMID- 3014477 TI - Laboratory markers of immunity to varicella-zoster virus. AB - Clinical and subclinical reinfection with varicella-zoster virus (VZV) in healthy persons may occur despite detectable neutralizing antibodies in serum. Subclinical reinfection is not uncommon, but clinical reinfection is rare. Therefore, detectable anti-VZV antibody correlates with a subclinical course after reinfection rather than with immunity to reinfection. Cell-mediated immunity (CMI) to VZV appears to be a better indicator of immunity. Skin testing may be a simple and reliable test for CMI, but this must be further evaluated. PMID- 3014478 TI - Production and quality control of the Oka-strain live varicella vaccine. AB - The Smith Kline-RIT live attenuated Oka-strain varicella vaccine (Varilrix) is manufactured following the seed lot principle, with each vaccine lot derived from the same working seed. Production of the vaccine basically consists in the multiplication of the working seed under standardized, well-defined conditions guaranteeing consistency of the vaccine lots. Quality control tests, determining the vaccine's purity, safety, potency, and efficacy, are carried out at each defined stage of the production process. PMID- 3014480 TI - Active immunization against varicella of children with acute leukaemia or other malignancies on maintenance chemotherapy. AB - Twenty-six patients with acute leukaemia and other malignancies susceptible to varicella were vaccinated with the Oka-strain live attenuated varicella vaccine during maintenance chemotherapy. All recipients showed no adverse clinical reactions. There was no spread of vaccine virus. Seroconversion was 94% in seronegative patients. Among those having low antibody titres before vaccination, a booster effect was demonstrable in 56%. None of the seroconverted recipients contracted varicella despite documented contact exposure. No case of herpes zoster occurred. The results suggest that, in immunocompromised children, live varicella vaccination has a protective effect against varicella infection which may result in a mortality rate of up to 7% in these patients. PMID- 3014481 TI - Clinical and pathogenetic aspects of varicella-zoster. AB - In this review article the pathogenetic mechanisms of infection with the varicella-zoster virus (VZV) and its short- and long-term clinical consequences are discussed. It is concluded that the impact of VZV is severe enough to seriously consider widespread immunization against it. PMID- 3014479 TI - Clinical experience with Oka live varicella vaccine in Japan. AB - A live varicella vaccine (Oka strain) has been developed and used since 1974 in normal and diseased children, particularly those at high risk. Children with acute leukaemia were usually vaccinated while in remission when showing a normal cell-mediated immunity as assessed with phytohaemagglutinin (PHA) or other reagents, and during suspension of all anticancer therapy, except 6 mercaptopurine from 1 week before to 1 week after vaccination. While clinical reactions were observed in only 40 out of 263 (15.2%) of these patients, they were noted in 30 out of 72 (41.7%) children immunized without suspension of chemotherapy. Symptoms were mostly mild; only a few cases of the latter group with T-cell leukaemia or malignant lymphoma developed severe symptoms. An immune response was observed in most vaccinees, but some (11%) developed clinical symptoms after exposure to natural varicella due to immunodepression caused by continuing anticancer chemotherapy. In these cases, revaccination seems advisable. The incidence and severity of zoster in vaccinated acute leukaemic children were less than in those with natural infection. Satisfactory immune responses with few concomitant clinical reactions were observed in approximately 1,500 vaccinees having nonmalignant diseases and in about 4,000 normal children. A 7-10-year follow-up study revealed that the vaccine had conferred solid immunity on the children. These results indicate that live varicella vaccine of the Oka strain is useful in preventing varicella in high-risk as well as in normal children. PMID- 3014483 TI - Early results of a trial of the Oka-strain varicella vaccine in children with leukaemia or other malignancies in Sweden. AB - Nineteen children with acute lymphoblastic leukaemia or other malignancies, aged 3-13 years, with negative histories of varicella-zoster virus (VZV) and of whom 5 were still on regular maintenance chemotherapy, were vaccinated with the Oka strain varicella vaccine. Mild systemic reactions, involving low-grade fever of a few days duration, were recorded in 4 of the vaccinees. Two of them also developed an extremely sparse rash. A mild local reaction at the vaccination site was observed in 1 child. Conversion rates for circulating VZV antibody, measured at 4-6 weeks and 4-6 months after vaccination using an indirect immunofluorescence test, were 8/12 (67%) and 5/9 (56%), respectively. Markedly lower antibody conversion rates of 5/18 (28%) and 5/14 (36%) were found by means of the enzyme-linked immunosorbent assay method. Corresponding conversion rates for cell-mediated immunity against VZV, determined using a specific lymphocyte stimulation technique, were 9/17 (52%) and 12/14 (86%), respectively. PMID- 3014482 TI - A multicentre trial of live attenuated varicella vaccine in children with leukaemia in remission. AB - Two hundred forty children with acute leukaemia in remission for at least 1 year were immunized with live attenuated varicella vaccine. All were susceptible to varicella before immunization. There was a seroconversion to varicella-zoster virus in approximately 85% after 1 dose, and in 97% after 2 doses. The major side effect was mild to moderate rash, seen mainly in children with maintenance chemotherapy suspended for 1 week before and 1 week after vaccination. Vaccinees with rash were at some risk (10%) to transmit vaccine virus to varicella susceptibles with whom they had close contact. Twenty-nine vaccinees were subsequently exposed to varicella in their households. The attack rate of clinical varicella in these vaccinees was 21%, which is significantly lower than the 80%-90% attack rate occurring in varicella susceptibles after household exposure. All these breakthrough cases of varicella were mild, even in leukaemics receiving chemotherapy. Varicella vaccine was approximately 80% effective in preventing clinical varicella in children with leukaemia and completely effective in preventing severe varicella in this high-risk group. PMID- 3014484 TI - Vaccination of children with malignant disease against varicella. AB - Nineteen seronegative children and one young adult with malignant disease in remission and on maintenance chemotherapy were immunized with the Oka-strain live attenuated varicella vaccine (Varilrix). Side effects were moderate and a rash was seen in 50% of the patients after vaccination. Humoral immune response to the vaccine was tested by the fluorescent antibody to membrane antigen (FAMA) test, a simpler indirect immunofluorescence test (IFT), and an enzyme-linked immunosorbent assay (ELISA). The seroconversion rate after immunization varied according to the method used. Seven out of 8 responded by FAMA, 15 out of 20 by IFT, and 12 out of 20 by ELISA. A decline in post-vaccination varicella-zoster virus (VZV) antibodies was seen in some responders. In 2 children, detectable levels of passively transferred VZV antibodies at the time of vaccination, due to varicella-zoster immune globulin, may have interfered with or modified the response to the vaccine. Cross-reacting antibodies to herpes simplex virus may have possibly interfered with vaccine response in a third child. Six out of 8 children receiving a second vaccine dose showed a good serological response. Specific cell-mediated immune response to the vaccine, measured by lymphocyte proliferation tests, corresponded well with the humoral response in the initial study of 8 patients. Two children who had responded to the vaccine were exposed to varicella in their families without contracting the clinical disease. PMID- 3014485 TI - Immunization of children with malignant diseases with the Oka-strain varicella vaccine. AB - Twenty children with different malignancies were vaccinated using an Oka-strain varicella vaccine (Varilrix) obtained from Smith Kline-RIT. Until now, no serious side effects to the vaccine have been observed and none of the vaccinees have had a varicella infection during this period. Nine children have been tested for cell mediated immune responses against varicella antigen. Antibodies against varicella have been measured using an enzyme-linked immunosorbent assay technique. Tests were performed 4 weeks, 6 and 12 months after vaccination; at 4 weeks, 85% of the children had seroconverted, but by 12 months, those patients who were negative before vaccination had no detectable antibodies. While the blastogenic lymphocyte response to varicella antigen gradually increased during the first year after vaccination, it was much lower than after natural infection. The results indicate the need for revaccination, probably within 6 months after the first vaccination. PMID- 3014486 TI - Live varicella vaccine in severely immunodepressed children. AB - One dose containing 3,100 PFU of a live attenuated Oka-strain varicella vaccine (Varilrix, Smith Kline-RIT, Belgium) was administered subcutaneously to 45 children, 26 of whom were suffering from acute leukaemia and 19 from solid malignant tumours. Their immunological status had been severely compromised by chemotherapy as evidenced by markedly low values for all immunological parameters. Of the 31 children seronegative for varicella at the time of vaccination, 70%, 85%, 75%, and 40% had varicella serum antibodies at 1, 2, 3, and 12 months after vaccination, respectively. Evaluation of the immunological parameters indicated that they were of no predictive value regarding the antibody response of the patients to the vaccine. Eight children (18%) developed varicella after vaccination. In one case, the disease was caused by the vaccine virus while in another, the vaccine virus was probably also responsible. The remaining six cases were caused by wild virus. The antibody response and accompanying protection against disease induced by the vaccine in severely immunodepressed patients is acceptable, but clearly lower and of shorter duration than in normal subjects. Thus, in immunocompromised patients, booster doses of the vaccine may be required at relatively short intervals. PMID- 3014488 TI - Angiotensin I-converting enzyme in human placenta. AB - Angiotensin I-converting enzyme (ACE, peptidyldipeptide hydrolase, kininase II, EC 3.4.15.I) from human placenta was purified 6297-fold and characterized. ACE could be extensively purified by affinity chromatography with Captopril (D-3 mercapto-2-methylpropanoyl-L-proline), an orally active antihypertensive agent and a potent inhibitor of this enzyme. Its molecular weight and subunit size were estimated to be 300 000 by high-performance gel permeation chromatography and 85 000 by sodium dodecyl sulphate gel electrophoresis, respectively, indicating its polymeric structure. PMID- 3014487 TI - Epidermal growth factor binding and receptor distribution in term human placenta. AB - Human placenta has a large number of epidermal growth factor (EGF) receptors when measured either by [125I]iodoEGF binding or by protein yield after purification. To localize EGF receptors in situ in normal human term placenta, two different light microscopic methods were used. To detect unoccupied, accessible EGF binding sites on the extracellular surface of placental cells in intact blocks of tissue, samples were incubated with [125I]iodoEGF, sectioned and autoradiography performed. To detect the total pool of intracellular and extracellular EGF receptors, placental tissue was sectioned, treated with detergent, and then anti EGF receptor antibody was localized by immunohistoperoxidase techniques. Both [125I]iodoEGF and anti-EGF receptor antibody methods showed that EGF receptors were primarily present on syncytiotrophoblast cells of placental villi. Smooth muscle cells of placental blood vessels also contained EGF receptors. Neither connective tissue cells within the core of terminal chorionic villi nor endothelium of fetal blood vessels had detectable [125I]iodoEGF binding or immunoreactive EGF receptors. Since the quantity of placental smooth muscle cells is only a small fraction compared to trophoblast cells, we conclude that syncytiotrophoblast cells are primarily responsible for the high levels of EGF receptors found in extracts prepared from human term placenta. PMID- 3014489 TI - In vitro secretion of peptide hormones by the human placenta: I. ACTH. AB - In order to address the question of autonomy of placental hormone secretion, fresh human term placentae were utilized for the preparation of small tissue fragments. The fragment pool was divided over four parallel chambers in a superfusion apparatus and could thus serve as both control and experiment under identical in vitro conditions. Oxygen consumption was substantial and could be maintained for at least 5 h. Adrenocorticotrophic hormone (ACTH) concentrations in the effluent buffer were estimated by radioimmunoassay and bioassay. Both non specific (membrane depolarization with 45 mM KCl) and specific (isoproterenol at 10(-7) M) stimulation increased the ACTH secretion from 10 to 20 pg/min/g to 60 to 80 pg/min/g. Propranolol blocked the adrenergic stimulation almost completely, indicating the specificity of the effect. Thus, in terms of in vitro ACTH secretion, the human placenta can be stimulated and therefore does not seem to behave in an autonomous manner. PMID- 3014490 TI - Immunohistochemical localization of follicle-stimulating hormone, luteinizing hormone, growth hormone, adrenocorticotrophic hormone and prolactin in the human placenta. AB - The sites of localization of luteinizing hormone (LH), follicle-stimulating hormone (FSH), growth hormone (GH), adrenocorticotrophic hormone (ACTH) and prolactin (PRL) within placental tissues have been studied by an immunoperoxidase technique. The syncytiotrophoblast is the sole significant site of localization of LH, FSH, GH and ACTH; PRL is found both in syncytiotrophoblast and in decidual cells. It is highly probable that the sites of localization of these peptide hormones represents their sites of synthesis in the placenta and thus that the syncytiotrophoblast is the sole site of synthesis of LH, FSH, LH and ACTH. PRL appears to be synthesized both in syncytiotrophoblast and decidua, but the latter is probably not the major site of synthesis of this hormone. Whether these placental peptide hormones have any physiological role to play during pregnancy or whether the placental capacity to synthesize such hormones is an atavistic phenomenon of no functional importance is currently a moot point. PMID- 3014491 TI - [Parvovirus B 19]. AB - Since 1981, many reports have contributed to establish that a human parvovirus (parvovirus B 19), which had been isolated only in asymptomatic blood donors, is the causative agent of transient but intense erythroblastopenia in patients with different types of chronic haemolytic anaemia. In vitro cultures of erythroid inhibitors revealed an inhibition by parvovirus B 19, abolished by convalescent serum. In healthy subjects without chronic haemolysis, first contact with parvovirus B 19 results in an inconstant influenza-like syndrome with transient erythroblastopenia which does not produce symptoms since the normal erythrocyte life span covers the effect of parvovirus B 19 on bone marrow. Parvovirus B 19 is also suspected to be the causative agent of erythema infectiosum (fifth disease) which occurs in children. PMID- 3014492 TI - [Simultaneous or successive lung cancer?]. PMID- 3014493 TI - [Serum angiotensin converting enzyme in the acquired immunodeficiency syndrome or related syndromes]. PMID- 3014494 TI - [The fasting test during sleep. A simple and inexpensive diagnostic element for insulinoma]. PMID- 3014495 TI - [Hepatocellular carcinoma and primary biliary cirrhosis. A new case]. PMID- 3014496 TI - The use of nuclear magnetic resonance in medicine. PMID- 3014497 TI - AIDS: recent events. PMID- 3014498 TI - GTPase center of elongation factor Tu is activated by occupation of the second tRNA binding site. AB - Interaction of the elongation factor EF-Tu with the antibiotic kirromycin results in activation of the GTPase center of the factor and in induction of an additional tRNA binding site (tRNA binding site II to distinguish it from the classical tRNA binding site I). Activation of the GTPase center under these conditions is stimulated by addition of tRNA. Two-fold evidence is presented that this stimulation is due to tRNA binding to site II rather than to site I. First, a strong correlation is observed between stimulation of the GTPase activity and enhancement of the reactivity of Cys-81 of EF-Tu toward N-ethylmaleimide at various concentrations of aminoacyl-tRNA, deacylated tRNA, and N-acetylaminoacyl tRNA. The latter effects signal tRNA binding to site II. Stimulation of the kirromycin-induced GTPase activity by tRNA binding to the factor also occurs when binding to site I is completely abolished. Such an abolishment was achieved by treating EF-Tu extensively with the thiol reagent L-1-tosylamido-2-phenylethyl chloromethyl ketone. EF-Tu X GTP thus treated has lost its ability to protect the ester bond of aminoacyl-tRNA. The relevance of these data for the sequence of events during protein synthesis and for control of translational fidelity is discussed. PMID- 3014500 TI - Genetic evidence that Acanthamoeba myosin I is a true myosin. AB - Acanthamoeba castellanii contains two enzymes, myosins IA and IB, that exhibit the catalytic properties of a myosin but possess very unusual physical properties, the most striking of which are their single, low molecular weight heavy chain, their globular shape, and their inability to form bipolar filaments. We have now isolated a putative myosin IB heavy chain gene from Acanthamoeba, using as a heterologous probe a portion of a sarcomeric myosin heavy chain gene from Caenorhabditis elegans. The amoeba genomic clone hybridizes to a 4250 nucleotide RNA species and hybrid-selects an mRNA encoding a 125-kDa polypeptide. This polypeptide comigrates exactly with the heavy chain of purified amoeba myosin IB and is specifically immunoprecipitated with antiserum to myosin IB. We sequenced two restriction enzyme fragments of this gene, and the deduced amino acid sequences show strong homology with the regions of muscle myosins that contain the reactive thiols and the ATP binding site. Our identification of a myosin IB heavy chain gene demonstrates that myosin IB, despite the unusually low molecular weight of its heavy chain, is a true gene product. The sequence results show that, despite its atypical physical properties, myosin IB is clearly related to conventional myosins. PMID- 3014499 TI - Preparation of [125I-Tyr27,Leu5]beta h-endorphin and its use for crosslinking of opioid binding sites in human striatum and NG108-15 neuroblastoma-glioma cells. AB - A radioligand suitable for crosslinking studies to opioid receptors has been obtained by radioiodination and purification of the monoiodotyrosine-27 derivative of the synthetic human beta-endorphin (beta h-endorphin) analogue [5 leucine]beta h-endorphin. The derivative, [27-[125I]monoiodotyrosine,5 leucine]beta h-endorphin, was crosslinked to human striatal (caudate and putamen) and NG108-15 neuroblastoma-glioma cell membranes by using disuccinimidyl suberate. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing conditions revealed four specifically labeled bands at 68, 40, 30, and 25 kDa for both human caudate and putamen, whereas NG108-15 cell membranes gave specifically labeled bands at 92, 56, 38, and 23 kDa. PMID- 3014501 TI - Replication of a plasmid bearing a human Alu-family repeat in monkey COS-7 cells. AB - Monkey COS-7 cells were transformed with BLUR8 DNA, a pBR322 plasmid containing a human Alu-family sequence at the BamHI site. Within 24 hr of transformation 2-5% of the BLUR8 molecules recovered resisted cleavage by Dpn I, indicating they had replicated. Electron microscopy revealed appropriately sized circular molecules with replication bubbles whose centers were mapped to the Alu insert. A 16-base pair deletion within the Alu sequence prevented replication. The results indicate that certain Alu sequences can serve as origins of replication in COS-7 cells. PMID- 3014502 TI - A repetitive DNA family (Sau3A family) in human chromosomes: extrachromosomal DNA and DNA polymorphism. AB - In this paper, we report a tandemly repeated DNA sequence found in human chromosomes. The DNA sequence, which is present at approximately 1000 copies per haploid genome, consists of a basic unit 849 base pairs (bp) long with a single specific restriction enzyme (Sau3AI) cutting site. The unit is further composed of five subunits, each approximately 170 bp long. When DNAs from various sources were examined by Southern hybridization using the repetitive DNA as a probe, a considerable degree of restriction fragment length polymorphism was observed. Furthermore, a substantial percentage (approximately 1.0%) of the same DNA sequence was also found extrachromosomally in the cultured human (HeLa) cells as monomers and oligomers of the basic unit in the form of covalently closed circular DNA. These results suggest that the repetitive DNA is unstable and prone to be excised from the chromosomes through homologous recombination. PMID- 3014503 TI - Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein. AB - Human papillomavirus type 16 DNA and RNA were characterized in the cervical cancer-derived CaSki cell line, which contains only integrated DNA, and in a cervical cancer, which contains predominantly plasmid DNA. In both, a major RNA can code for the early open reading frame E7 and a minor one can code for E6. The cervical cancer, but not the CaSki cell line, contains a minor RNA that can code for an intact E2 protein, and this may relate to the continued presence of plasmid DNA. The RNA mapping data suggest that the poly(A)+ RNA is transcribed from a minor fraction of the several hundred gene copies present, and in the cervical cancer these genomes appear to be integrated. The E7 protein has been identified in CaSki cells and the prevalence of its mRNA suggests a possible function in progression to, or long-term maintenance of, the malignant state. PMID- 3014504 TI - Inquiries into the structure-function relationship of ribonuclease T1 using chemically synthesized coding sequences. AB - The genes for ribonuclease T1 and its site-specific mutants were chemically synthesized and introduced to Escherichia coli. All enzymes were fusion products produced by joining the synthetic gene at specific restriction sites to the synthetic gene for human growth hormone in a plasmid containing the E. coli trp promoter. The fusion protein from this plasmid contained 66% of the amino terminal sequences of the human growth hormone, which were recognizable immunologically. RNase T1 or its mutants were cleaved from the fusion protein with cyanogen bromide. The synthetic RNase T1 endowed with the revised wild-type triad Gly-Ser-Pro, residues 71-73, was fully functional, readily hydrolyzing pGpC bonds, whereas a mutant enzyme having the originally reported, erroneous triad Pro-Gly-Ser was totally inactive. Various amino acid substitutions were also introduced to the guanosine recognition region comprised of residues 42-45, Tyr Asn-Asn-Tyr. Substitution of either of the tyrosine residues noted above with phenylalanine had no dramatic effect on the enzyme's function. Replacement of asparagine-43 with arginine or alanine also caused only a small change in the hydrolyzing activity--a mutant enzyme maintained greater than 50% of the wild type activity. In sharp contrast, when aspartic acid or alanine was substituted for asparagine-44, the activity was dramatically reduced to a few percent of the wild-type activity. PMID- 3014505 TI - DNA-binding site of major regulatory protein alpha 4 specifically associated with promoter-regulatory domains of alpha genes of herpes simplex virus type 1. AB - Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. We have previously reported that labeled DNA fragments containing promoter-regulatory domains of three alpha (alpha 0, alpha 4, and alpha 27) and a gamma 2 gene form stable complexes with proteins from lysates of infected cells as detected by a gel electrophoresis binding assay and that monoclonal antibody to alpha 4 protein reduced the electrophoretic mobility of the complex of labeled DNA and protein from infected cells. In this study we identified one monoclonal antibody, H950, from a panel of monoclonal antibodies to the alpha 4 protein that blocks the formation of specific infected cell complexes with labeled DNA fragments containing promoter and regulatory domains of alpha genes. We also report the nucleotide sequence of the alpha 0 promoter domain protected from exonuclease III digestion by alpha 4 protein in the absence of H950 monoclonal antibody but not in its presence. In addition, we identified a 59-base-pair sequence from the regulatory domain of the alpha 4 gene that binds alpha 4 protein. Deletion clones of this fragment localize sequence elements required for formation of the alpha 4 protein-DNA complex. Furthermore, deletion of the in vitro binding site of the SP1 transcription factor from the 59-base-pair fragment did not affect the formation of the alpha 4 protein-DNA complex. PMID- 3014507 TI - Transcriptional regulation of steroid hydroxylase genes by corticotropin. AB - Maintenance of optimal steroidogenic capacity in the adrenal cortex is the result of a cAMP-dependent response to the peptide hormone corticotropin (ACTH). The molecular mechanism of this action of ACTH has been examined by using five recombinant DNA clones specific for enzymes of the steroidogenic pathway (P 450scc, P-45011 beta, P-450C21, P-45017 alpha, and adrenodoxin). The presence of nuclear precursors in steady-state RNA samples derived from cultured bovine adrenocortical cells and moderate increases in the number of RNA chain initiations, as determined by in vitro nuclear run-off assays, indicate that ACTH controls the expression of the gene(s) for each of these proteins at the transcriptional level. The ACTH-mediated increase in accumulation of transcripts specific for steroid hydroxylases in nuclear RNA can be specifically blocked by inhibiting protein synthesis in bovine adrenocortical cell cultures. The steady state concentrations of nuclear RNA for control genes show no decrease upon cycloheximide treatment. These studies suggest that a primary action of ACTH in the adrenal cortex is to activate (via cAMP) the synthesis of rapidly turning over protein factors that in turn mediate increased initiation of transcription of steroid hydroxylase genes. We propose that these protein factors impart specificity of induction to genes encoding components of this pathway in steroidogenic tissues. PMID- 3014506 TI - Isolation of a Drosophila genomic sequence homologous to the kinase domain of the human insulin receptor and detection of the phosphorylated Drosophila receptor with an anti-peptide antibody. AB - A Drosophila genomic fragment has been isolated with a deduced amino acid sequence that is strikingly homologous to that of the kinase domain of the human insulin receptor. The Drosophila DNA hybridizes with an 11-kilobase mRNA that is most prominent in 8- to 12-hr embryos. An anti-peptide antibody prepared to a sequence in the human insulin receptor kinase domain that is conserved in the Drosophila sequence immunoprecipitates a single 95-kDa Drosophila protein whose phosphorylation on tyrosine residues is dependent on insulin. We conclude that the DNA sequence is that of the kinase domain of the Drosophila insulin receptor and that the 95-kDa phosphoprotein is the autophosphorylated beta subunit of that receptor. The results are compatible with our previous reports demonstrating a specific insulin-binding Drosophila glycoprotein and an insulin-dependent tyrosine protein kinase whose activity is greatest during embryogenesis. The observations suggest a role for insulin-dependent protein tyrosine phosphorylation during embryogenesis. PMID- 3014508 TI - Cloning and sequencing of cDNA of bovine N-acetylglucosamine (beta 1 4)galactosyltransferase. AB - Galactosyltransferases constitute a family of enzymes, each member of which transfers galactose from UDPgalactose to a specific acceptor molecule, generating a specific galactose-acceptor linkage. Two synthetic oligonucleotides, 27mer and 21mer, were synthesized, based on the amino acid sequences of two peptides derived from bovine milk N-acetylglucosaminide (beta 1-4)galactosyltransferase (EC 2.4.1.90), and used as hybridization probes to isolate cDNA clones for galactosyltransferase from a bovine mammary gland cDNA library. One of the plasmids, designated pLbGT-1, contains an insert of about 3.7 kilobases that hybridizes to both of the probes and encodes the amino acid sequences of five peptides obtained from bovine milk (beta 1-4)galactosyltransferase. A second plasmid, designated pLbGT-2, contains an insert of about 4.1 kilobases that hybridizes to only the 27mer and that encodes a polypeptide containing the sequence of the carboxyl-terminal 120 residues identical to the peptide encoded by pLbGT-1; the rest of the protein sequence, however, does not contain known sequences from bovine galactosyltransferase. The two cDNAs contain a 3' untranslated region of about 2.7 kilobases that includes two copies of the Alu equivalent sequences. pLbGT-1 and pLbGT-2 hybridize to mRNAs of various sizes obtained from the bovine and rat mammary gland and the human mammary tumor cell line MCF-7, with the longest mRNA from each species being around 4.5 kilobases. The results show that pLbGT-1 is a cDNA clone for bovine (beta 1 4)galactosyltransferase, and pLbGT-2 encodes a protein that is structurally and may be functionally related to transferases. PMID- 3014509 TI - Atrial natriuretic peptide inhibits renin release from juxtaglomerular cells by a cGMP-mediated process. AB - We have examined the effect of a synthetic analogue of human alpha-atrial natriuretic peptide (ANP), APII, on renin release in cultured renal juxtaglomerular cells (JGA cells). Using cell cultures containing 80-90% renal juxtaglomerular cells, we found that ANP (10(-13)-10(-9) M) strongly inhibited renin release from the cells in a dose-dependent fashion (ki, 10 pM) to about 10% of control. Inhibition of renin release by ANP was paralleled by an increase in cellular cGMP levels; while in the presence of the cGMP-phosphodiesterase inhibitor M&B 22948 (1 mM), concentrations of ANP lower by a factor of 100 were required to obtain the same effects on renin release and cGMP levels. The guanylate cyclase inhibitor methylene blue (10 microM), on the other hand, shifted the dose-response curves for renin release and cGMP levels to 100-fold higher concentrations of ANP. Neither the influx of 45Ca into the cells nor the intracellular quin-2 signal, which is a measure for changes of intracellular Ca concentration, was in any way altered by ANP. Our results suggest that ANP inhibits renin release from juxtaglomerular cells by a cGMP-dependent process that does not involve changes in intracellular calcium. PMID- 3014511 TI - Homeo box gene expression in anterior and posterior regions of the Drosophila embryo. AB - The homeo box is a 180-base-pair coding sequence that has been implicated in the control of Drosophila development. A common feature of the nine previously reported homeo box genes is their involvement in the establishment of the segmentation pattern of the embryo. In this report we describe the isolation and properties of two additional homeo box genes, F90-2 and S67. Transcripts encoded by the two genes are detected in embryonic tissues that derive from regions near the anterior and posterior poles of the embryo, which are outside the limits of expression of known homeotic genes. These results suggest that at least some homeo box genes specify positional identity along the anterior-posterior body axis that is independent of the process of segmentation. PMID- 3014510 TI - Nerve growth factor rapidly induces c-fos mRNA in PC12 rat pheochromocytoma cells. AB - The nerve growth factor (NGF)-mediated increase in c-fos gene expression in the rat pheochromocytoma PC12 cell line has been investigated. NGF treatment of PC12 cells results in an increased level of c-fos mRNA within 15 min. An approximately 100-fold increase in the level of c-fos mRNA occurs 30-45 min after exposure to NGF and the c-fos mRNA concentration returns to its basal level 2 hr after NGF treatment. Thus, the half-life of this RNA transcript is extremely short. In the presence of cycloheximide, the c-fos gene is superinduced and the increased level of c-fos mRNA persists for at least 24 hr. The induction of c-fos gene expression was further studied by utilizing a monoclonal antibody (mAb-192) that is directed against the NGF receptor but does not compete with NGF for binding to the receptor. Treatment of the cells with mAb-192 inhibits the NGF-stimulated elevation of c-fos mRNA, suggesting that the antibody may interfere with the receptor's ability to generate the signal required to stimulate the transcription of this gene. NGF is not the only agent capable of inducing c-fos gene expression in these cells; epidermal growth factor, the tumor promoter phorbol 12-myristate 13-acetate, and the calcium ionophore A23187, agents that induce the c-fos gene in other cell lines, are also effective in PC12 cells. The mRNA for the nuclear protein fos is rapidly induced by NGF and other agents to which PC12 cells respond. This supports the hypothesis that the fos gene product may play a role in signal transduction. PMID- 3014512 TI - Interaction of cAMP with the cell-surface receptor induces cell-type-specific mRNA accumulation in Dictyostelium discoideum. AB - The accumulation of many postaggregative mRNA species in Dictyostelium discoideum is dependent upon the continuous presence of elevated levels of cAMP. We have analyzed the cyclic nucleotide specificity of this requirement and show that it is similar to that of the cell-surface receptor and distinct from the specificity displayed by the cAMP-dependent protein kinase. The same specificity is displayed for the accumulation of two classes of prespore mRNAs (class I, early; class II, late) and a prestalk mRNA and for the shutoff of a growth-phase mRNA. Under conditions in which cAMP phosphodiesterase activity is competitively inhibited, half-maximal accumulation of prestalk mRNA can be obtained at cAMP concentrations of 320-520 nM, whereas a higher concentration, 1-2 microM, is required for half maximal accumulation of the prespore mRNAs and shutoff of the growth-phase mRNA. These effects of cAMP and its analogues on gene expression have been obtained under conditions in which cAMP-mediated activation of adenylate cyclase is completely inhibited. We conclude that cAMP acts to stimulate postaggregative gene expression by interacting at the cell-surface receptor. PMID- 3014513 TI - Genetic alteration of the c-myc protooncogene (MYC) in human primary breast carcinomas. AB - We have studied the genomic organization of the c-myc locus (MYC) from 121 human primary breast carcinomas. Two types of alterations were observed: (i) the c-myc protooncogene appeared to be amplified 2- to 15-fold in 38 (32%) of the carcinoma DNAs and (ii) a non-germ-line c-myc-related fragment of variable size was detected in 5 primary breast carcinoma DNAs. With three exceptions, all the tumors containing a genetic alteration of the c-myc locus were invasive ductal carcinomas. A significant correlation (P less than 0.02) was observed between patients more than 50 years old and the presence of a genetically altered c-myc. Enhanced levels of c-myc RNA were observed in 10 of 14 breast carcinomas examined. The c-myc gene was genetically altered in 6 of these 10 tumors. The frequency with which the c-myc gene is altered and its correlation with age suggest that it may play a role in the development of breast carcinomas. PMID- 3014515 TI - Human alpha-galactosidase A: nucleotide sequence of a cDNA clone encoding the mature enzyme. AB - The complete nucleotide sequence has been determined for a lambda gt11 cDNA clone (lambda AG18) containing the full-length coding region for the mature lysosomal form of human alpha-galactosidase A (alpha-Gal A; EC 3.2.1.22). The lambda AG18 insert contained a 1226-base-pair sequence with an open reading frame encoding 398 amino acids of the mature polypeptide (predicted Mr = 45,356) and the last 5 amino acids of the propeptide sequence. The poly(A) signals AATACA and ATTAAA occurred 28 and 11 nucleotides prior to the TAA stop codon, respectively. There was no 3' untranslated region as the poly(A) sequence immediately followed the TAA termination codon; a second independently cloned cDNA confirmed this finding. The predicted amino acid sequence was colinear with 86 nonoverlapping residues (22% of the mature subunit) determined by microsequencing amino-terminal, tryptic, and cyanogen bromide peptides of the purified mature enzyme. Four potential N-glycosylation sites were identified, all of which occurred at predicted beta turns in hydrophilic regions of secondary structure. RNA transfer hybridization analysis of HeLa poly(A)+ RNA demonstrated a single 1.45-kilobase band whose signal was decreased by prior immunoabsorption of polysomes with monospecific alpha-Gal A antibodies. Searches of nucleic acid and protein data bases did not reveal significant homology even with the limited sequences available for mammalian lysosomal enzymes. PMID- 3014514 TI - A long interspersed (LINE) DNA exhibiting polymorphic patterns in human genomes. AB - Several human DNAs digested with Kpn I restriction endonuclease released a 0.6 kilobase (kb) segment that varied in its intensity among human samples. A recombinant DNA clone (N6.4) of these 0.6-kb Kpn I segments was isolated and used to probe the genomic content and restriction cleavage pattern of homologous sequences. The hybridization patterns revealed a previously undescribed, moderately repetitive long interspersed (LINE) sequence family, which we have termed L2Hs (second LINE family in Homo sapiens). This LINE family exhibits both quantitative and qualitative polymorphisms in the human population. The content of L2Hs sequences in human genomes varies over a 5-fold range. Relative to the value for a human placental DNA, sequences homologous to the L2Hs family occur in lower amounts in gorilla DNA (approximately 20%) and even less in DNA from chimpanzees and other primates (less than 1%). Thus, the L2Hs sequences appear to have emerged only recently as a moderately repetitive sequence family in primate evolution. The observed restriction fragment length polymorphism of the L2Hs family members may reflect patterns of sequence rearrangements, amplifications, and/or deletions in human genomes. PMID- 3014516 TI - DNA differences found between Africanized and European honeybees. AB - The harmful en masse introduction of Africanized honeybees into the United States will occur within 5 years. Possible means of control are dependent on a reliable way to distinguish the Africanized bees from the extant European bees. Current means of identification are inadequate. Reported here are the encouraging initial results to distinguish the bees by their nuclear DNA. With 9 restriction enzymes and 16 probes, six genetic differences have been found among three samples of European bees from California. Twelve additional differences were detected between the European samples and a sample of Africanized bees from Costa Rica. PMID- 3014517 TI - Mutation affecting the expression of immunoglobulin variable regions in the rabbit. AB - We have found a variant of the allotype allele a2 in the rabbit, which presumably arose by mutation, that segregates as expected for an allele at the a locus. This allele is called "ali" and the corresponding rabbit strain is called "Alicia." In heterozygous animals (ali/a1 and ali/a3) the concentration of a2 molecules is lower by a factor of 1000 than in standard a2/a2 homozygotes. In homozygous ali/ali individuals the a2 concentration varies with age--i.e., very low in young rabbits and higher in older ones--but it never reaches normal levels. The low level of a2 is compensated by increased amounts of a-negative molecules. Southern blot analysis did not reveal any gross changes in the intron between JH and C mu (joining region of immunoglobulin heavy chain and constant region of immunoglobulin mu chain) or in the number of VH gene segments encoding a locus specificities. We suggest that the ali phenotype is due to a mutation in a control element. PMID- 3014518 TI - Modes of transmission and evidence for viral latency from studies of human T-cell lymphotrophic virus type I in Japanese migrant populations in Hawaii. AB - Human T-cell lymphotrophic virus type I (HTLV-I) seroprevalence was 20% among Hawaiian Japanese migrants (issei) and their offspring (nisei) from Okinawa compared to 35% in similarly aged men who were lifetime residents of Okinawa. A control group of migrants from a nonendemic area of Japan, Niigata, had low rates of HTLV-I antibodies, suggesting that Hawaii per se is not an endemic area for HTLV-I. Factors that were significantly associated with seropositivity in the Okinawa migrant groups were years of residence in Japan before migration (issei) and age for offspring of Okinawa migrants (nisei). Antibody titer was highest in Okinawa lifetime residents, intermediate in migrants (issei), and significantly lower in offspring of Okinawa migrants (nisei), with increasing titer observed with advancing age in the offspring of the migrant group. Based on these data, infection within the household occurring early in life appears to be a major route of HTLV-I transmission and may help to explain the curious geographic clustering of this virus in certain locales. As yet to be defined cofactors, including sexual transmission and/or environmental exposures (e.g., particularly before age 20), also may contribute to HTLV-I seropositivity. The pattern of rising seroprevalence and titer with age in the offspring of migrants who resided all of their lives in Hawaii raises the possibility that HTLV-I infection acquired early in life may become dormant and reexpressed with reactivation of latently infected T cells. The importance of this model in the process of viral leukemogenesis is supported by recent reports of adult T-cell leukemia in offspring (nisei) of Okinawa migrants. PMID- 3014519 TI - Design of potent, orally effective, nonpeptidal antagonists of the peptide hormone cholecystokinin. AB - We describe the design and synthesis of nonpeptidal antagonists of the peptide hormone cholecystokinin. Several of these compounds have high specificity and nanomolar binding affinity and are active after oral administration. To our knowledge, the design of such agents has not previously been accomplished for any peptide hormone. The structural similarities between these synthetic compounds and the anxiolytic 1,4-benzodiazepines are noted, and the potential of this structural feature for future design of ligands for other peptide hormone receptors is discussed. PMID- 3014520 TI - Biochemical and pharmacological characterization of an extremely potent and selective nonpeptide cholecystokinin antagonist. AB - 3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4- benzodiazepine-3-yl)-1H indole-2-carboxamide (L-364,718) interacted in a competitive manner with rat pancreatic cholecystokinin (CCK) receptors as determined by Scatchard analysis of the specific binding of 125I-labeled CCK. The affinity of L-364,718 for both pancreatic (IC50, 81 pM) and gallbladder (IC50, 45 pM) CCK receptors in radioligand binding assays greatly exceeded that of other reported nonpeptide CCK antagonists and was similar to that of CCK itself. In vitro functional studies utilizing CCK-induced contractions of the isolated guinea pig ileum and colon further demonstrated that L-364,718 acts as a competitive CCK antagonist, which lacks agonist activity and has a similar high affinity in these tissues (pA2, 9.9). L-364,718 exhibited a very high selectivity for peripheral CCK receptors relative to brain CCK, gastrin, and various other peptide and nonpeptide receptors in both in vitro radioligand and isolated tissue assays. In vivo, low intravenous doses of L-364,718 (0.1 mg/kg) markedly antagonized the contractions of the guinea pig gallbladder produced by intravenous administration of CCK for at least 2 hr. Administered orally, L-364,718 (ED50, 0.04 mg/kg) was highly effective as an antagonist of CCK-induced inhibition of gastric emptying in mice. The biochemical and pharmacological properties of L-364,718--namely, very high affinity and selectivity for peripheral CCK receptors, long-lasting in vivo efficacy, and oral bioavailability--makes this compound a powerful tool for investigating the physiological and pharmacological actions of CCK, and possibly its role in gastrointestinal disorders. PMID- 3014522 TI - Isolation and identification in bovine cerebral cortex of n-butyl beta-carboline 3-carboxylate, a potent benzodiazepine binding inhibitor. AB - A substance having benzodiazepine-binding inhibitory activity has been extracted from 18 kg of gray matter of bovine cerebral cortex and purified to homogeneity. This substance inhibits competitively [3H]flunitrazepam and ethyl beta [3H]carboline-3-carboxylate binding with high affinity (Ki, 3 nM), but it is inactive upon 3H-labeled Ro 5-4864, [3H]quinuclidinyl benzylate, [3H]prazosin, [3H]clonidine, [3H]dihydroalprenolol, and upon high-affinity [3H]muscimol binding. This inhibitor has been identified as n-butyl beta-carboline-3 carboxylate (beta-CCB) by fast atom bombardment mass spectroscopy (Mr, 268) and electron bombardment fragmentography, ultraviolet and fluorescence spectra, coelution in HPLC with standard beta-CCB, and by the exact correspondence in Ki with beta-CCB on the displacement of [3H]flunitrazepam binding. The possible artificial formation of beta-CCB has been discarded by a series of control experiments including addition of tryptophan to the starting homogenate, extraction from liver, isolation and purification by an alternative procedure avoiding organic solvents, and by the impossibility of making beta-CCB from beta carboline-3-carboxylic acid or its methyl ester in the conditions of our extraction and purification procedures. PMID- 3014521 TI - Activation of two different but complementary biochemical pathways stimulates release of hypothalamic luteinizing hormone-releasing hormone. AB - Evidence exists that a norepinephrine/prostaglandin E2 (PGE2)/cAMP pathway is involved in the regulation of luteinizing hormone-releasing hormone (LHRH) secretion. The aim of the present experiments was to determine if release of LHRH from the immature rat hypothalamus could also be stimulated by activation of protein kinase C. Median eminences from 28-day-old female rats were incubated in vitro with either dioctanoylglycerol (a synthetic diacylglycerol that selectively activates protein kinase C in intact cells) or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (another protein kinase C activator). Both agents increased LHRH release, the response to dioctanoylglycerol being more pronounced than that to the phorbol ester. This direct activation of protein kinase C was not accompanied by changes in PGE2 formation. Activation of the PGE2/cAMP pathway by either norepinephrine, PGE2, or forskolin (a stimulator of adenylate cyclase) increased LHRH release. Dioctanoylglycerol or phorbol ester in conjunction with either norepinephrine, PGE2 or forskolin resulted in an additive effect on LHRH release suggesting coexistence of both pathways. Phospholipase C, which activates protein kinase C via formation of diacylglycerol, increased the release of both LHRH and PGE2. This suggests that an increase in endogenous phospholipase C activity caused by neurotransmitter inputs may lead to both activation of protein kinase C and PGE2 formation. Blockade of cyclooxygenase activity by indomethacin obliterated phospholipase C-induced PGE2 release. The same treatment reduced the LHRH response by only 50% indicating that protein kinase C activation can cause LHRH release in the absence of PGE2 synthesis. It is suggested that the median eminence of the rat possesses a protein kinase C-dependent pathway that is coupled positively to LHRH release and complements PGE2/cAMP-dependent mechanisms. Norepinephrine, however, does not appear to be the neurotransmitter responsible for activating the protein kinase C pathway. Simultaneous activation of both pathways may provide a mechanism by which a large increase in LHRH secretion occurs, such as in the afternoon of first proestrus. PMID- 3014523 TI - Amino acid sequence of mammalian elongation factor 2 deduced from the cDNA sequence: homology with GTP-binding proteins. AB - Complementary DNA clones, pHEW1 and pRE2, coding for hamster and rat polypeptide chain elongation factor 2 (EF-2), respectively, were isolated and sequenced. It was shown that the cDNA insert in pHEW1 contains a 2574-base-pair open reading frame coding for an 857-amino acid polypeptide with Mr 95,192, excluding the initiation methionine. Comparative studies of sequence homology among EF-2 and several GTP-binding proteins show that five regions in the amino-terminal position of EF-2, corresponding to about 160 amino acids, show homology with GTP binding proteins, including protein synthesis elongation and initiation factors, mammalian ras proteins, and transducin. The carboxyl-terminal half of EF-2 contains several regions that have 34-75% homology with bacterial elongation factor G. These results suggest that the amino-terminal region of EF-2 participates in the GTP-binding and GTPase activity whereas the carboxyl-terminal region interacts with ribosomes. Finally, the sequence provides direct evidence that diphthamide (2-[3-carboxy-amido-3-(trimethylammonio)propyl]histidine), the site of ADP-ribosylation by diphtheria toxin, is produced by post-translational modification of a histidine residue in the primary translational product. PMID- 3014525 TI - Molecular cloning of an oncogene from a human hepatocellular carcinoma. AB - A transforming DNA, named lca (for liver cancer), was obtained from a primary human hepatocellular carcinoma (HCC) in transformation assays using NIH 3T3 cells and a calcium phosphate coprecipitation method. High molecular weight DNA obtained from the HCC tissue was employed for this purpose. This transforming DNA had a linkage to the Alu sequence and was cloned in lambda phage for further studies. Restriction enzyme analyses showed that the minimal size of the lca transforming DNA is about 10 kilobase pairs and that its cleavage profiles are different from those of any one of the previously reported human transforming genes or retroviral oncogenes. No cross-hybridization was observed between these genes and the lca DNA. Southern blot analyses of DNAs from flow-sorted human chromosomes and human-mouse somatic cell hybrids indicated that the lca DNA is located on human chromosome 2. An independently obtained transforming DNA from another HCC exhibited identical restriction enzyme cleavage profiles. Thus, lca DNA is likely to represent a commonly encountered transforming DNA in HCC. PMID- 3014524 TI - Enhanced expression of the bacterial chloramphenicol acetyltransferase gene in mouse cells cotransfected with synthetic polynucleotides able to form Z-DNA. AB - Recent studies have demonstrated that the left-handed, Z-DNA conformation is favored in polymers containing alternating purine/pyrimidine sequences that can exist in vivo and may play a role in gene expression. On the basis of this assumption, we have studied the effect of various cotransfected polynucleotides on the transient expression of the chloramphenicol acetyltransferase (CAT) gene in thymidine kinase-deficient murine L cells. Cotransfections were performed by calcium phosphate coprecipitation of CAT gene plasmids with various polymers, and the CAT enzymatic activity was measured in cell lysates after 48 hr. About 2- to 10-fold stimulation of CAT gene expression was observed when the cells were cotransfected with 10 micrograms (per 10-cm culture dish) of plasmid pSV2cat, which contains simian virus 40 (SV40) promoter and enhancer sequences, and 2-10 micrograms of polymers that can form Z-DNA, such as poly(dG-m5dC) X poly(dG-m5dC) or poly(dG-dC) X poly(dG-dC), as compared to transfection with pSV2cat alone. Further, enhanced CAT gene expression was also observed when cotransfections were performed with these polymers and two other plasmid vectors, one containing the SV40 promoter but no enhancer and the other lacking any SV40 regulatory sequences. However, poly(dA-dC) X poly(dG-dT), which can form Z-DNA, did not induce any stimulation. Similarly, no or very little stimulation was observed after cotransfection of pSV2cat with either poly(dG) X poly(dC) or poly(dA-dT) X poly(dA-dT), which do not adopt the Z conformation. These results suggest that certain polynucleotides may enhance transcription of the CAT gene. PMID- 3014526 TI - Tissue distribution, immunoreactivity, and physical properties of 6-phosphofructo 2-kinase/fructose-2,6-bisphosphatase. AB - 6-Phosphofructo-2-kinase (EC 2.7.1.105) and fructose-2,6-bisphosphatase (EC 3.1.3.46) activities were determined in various rat tissues, the latter by using a method based on the formation of a phosphorylated enzyme intermediate during the course of catalysis. Both activities from liver, skeletal muscle, lung, kidney, and testis copurified during polyethylene glycol fractionation, anion exchange and blue Sepharose chromatography, and gel filtration. The Stokes radius of these enzymes and of the liver bifunctional enzyme was 45 A. Extrahepatic tissues had only 10% or less of the kinase activity found in liver. The results indicate that a liver-type bifunctional enzyme is present in most extrahepatic tissues but that it is minimally expressed. However, the ratio of kinase to bisphosphatase activity in most extrahepatic tissues was 4- to 6-fold higher than in liver, whereas heart 6-phosphofructo-2-kinase had no associated bisphosphatase activity, although its Stokes radius was also 45 A. The heart enzyme was not precipitated by an antiserum to the liver enzyme, whereas only a fraction of the kidney and testis activities was precipitated by this antiserum. The data support the existence of a distinct form of extrahepatic 6-phosphofructo-2-kinase, most readily demonstrated in heart, which may not be bifunctional. PMID- 3014527 TI - Truncation of the c-myb gene by a retroviral integration in an interleukin 3 dependent myeloid leukemia cell line. AB - Among a series of myeloid leukemia cell lines, one (NFS-60) was found to have a rearrangement of the c-myb locus. The rearrangement involved the integration of a retrovirus into the region of the gene corresponding to the sixth exon of the avian c-myb locus. The insertion is associated with the production of a truncated RNA and the introduction of a terminator codon at the juncture of the long terminal repeat and the c-myb locus. The properties of the NSF-60 cells were compared with those of other myeloid cell lines, and the known sequence of differentiation induced by interleukin 3. Similar to other myeloid cell lines, the NFS-60 cells do not terminally differentiate in response to interleukin 3, granulocyte/macrophage, or granulocyte colony-stimulating factor suggesting that the cells are transformed with regard to their ability to differentiate. The NFS 60 cells are totally dependent on interleukin 3 for growth and maintenance of viability in vitro but also proliferate in response to granulocyte colony stimulating factor. The properties of the cells support the concept that the c myb protooncogene is involved in the control of normal differentiation of hematopoietic cells. PMID- 3014528 TI - Evidence and characterization of the receptor to eye-derived growth factor I, the retinal form of basic fibroblast growth factor, on bovine epithelial lens cells. AB - Eye-derived growth factor I (EDGF-I), the retinal form of the basic fibroblast growth factor, has been purified to homogeneity from bovine retina by heparin Sepharose chromatography. The radioiodinated EDGF-I retained full mitogenic activity and was used to study the interaction of the growth factor with bovine epithelial lens cells. We showed that 125I-labeled EDGF-I bound in a saturable and reversible manner to a specific cellular receptor. Scatchard analysis of the equilibrium binding gave a Kd of 53 X 10(-12) M with approximately equal to 20,000 binding sites per cell. Crosslinking experiments using two homobifunctional reagents induced the formation of a specific major complex with a Mr of approximately equal to 145,000, as determined by NaDodSO4/PAGE, and independent of reducing conditions. These data establish the existence of a receptor for the basic growth factor derived from neural tissues and give an estimation of the size of this receptor at Mr 130,000. PMID- 3014529 TI - Identification of conserved and divergent domains within the envelope gene of the acquired immunodeficiency syndrome retrovirus. AB - The nucleotide sequences of the envelope genes of an African and a North American acquired immunodeficiency syndrome (AIDS) viral isolate have been determined. When their deduced amino acid sequences were aligned with the envelopes of the lymphoadenopathy and AIDS-associated retrovirus isolates, conserved and divergent regions were readily identified. Hypervariable stretches of 28-74 amino acids, exhibiting 20-30% amino acid identity at each position and characterized by reciprocal insertions and deletions, were confined to the gp120 external envelope protein. The origin and clinical implications of the AIDS viral env gene diversity are discussed. PMID- 3014530 TI - Escherichia coli mutS-encoded protein binds to mismatched DNA base pairs. AB - The Escherichia coli mutS gene product is involved in mismatch correction in this organism. We have purified a biologically active form of the 97,000 Mr protein to near homogeneity from an overproducing strain. Enzymatic and chemical protection ("footprinting") experiments have demonstrated that mutS-encoded protein specifically binds to DNA regions containing a single base-pair mismatch. The protein displayed variable affinity for the limited set of mismatches tested (G-T greater than G-A approximately equal to A-C greater than T-C). PMID- 3014531 TI - Mutations of the ras gene product p21 that abolish guanine nucleotide binding. AB - We have constructed several point mutations affecting the GTP-binding site of p21, the ras-encoded protein. Both lysine (116K) and tyrosine (116Y) mutations of asparagine-116, which, by analogy with the crystal structure of elongation factor Tu (EF-Tu), has critical interactions with the guanine base, abolish GTP binding and transforming activities of p21. These activities are retained by proteins with a mutation at position 117 or 118. Both 116K and 116Y mutant p21s, when overproduced in Escherichia coli, are apparently devoid of GTP-binding and autokinase activities. Similarly, the mutant DNAs do not transform NIH 3T3 cells in a focus-forming assay. By cotransfection with pSV-neo, cell clones resistant to the neomycin analog G418 have been isolated. Cells transfected with 116K or 116Y mutant DNA are contact inhibited. In contrast to competent clones, the defective mutants have no detectable phosphorylated p21. The present results suggest that the basic structure of the GTP-binding site is conserved between p21 and EF-Tu and that this binding site is crucial for ras gene function. PMID- 3014532 TI - The fusion-related hydrophobic domain of Sendai F protein can be moved through the cytoplasmic membrane of Escherichia coli. AB - Recent work on a prokaryotic membrane protein, gene III protein (pIII) of coliphage f1, showed that polypeptide segments of sufficient hydrophobicity functioned to stop transfer of the polypeptide across the cell membrane: strings of 16 or more hydrophobic amino acids sufficed. A fusion-related hydrophobic domain (FRHD) of Sendai F protein, a sequence of 26 consecutive uncharged residues, has been implicated in the fusion of the viral membrane envelope and the target-cell membrane through a hydrophobic interaction. As it is located on the exterior of the viral membrane, this sequence must be transferred across the host-cell membrane during synthesis. We have inserted either the FRHD or the F protein membrane anchor (the COOH-terminal region of the F protein) into an internal site of a secreted pIII, which lacks its natural membrane anchor. These two hydrophobic sequences behave in the bacteria just as they do in their natural eukaryotic cell host. The F protein membrane anchor functions to stop transfer, conferring a membrane-spanning topology to the F-pIII hybrid protein; however, the FRHD is moved through the cytoplasmic membrane and derivatives carrying this sequence are secreted to the periplasm. We discuss how the FRHD is compatible with passage through the membrane and yet is still able to mediate membrane fusion through a presumed hydrophobic interaction. PMID- 3014534 TI - Enhanced expression of epidermal growth factor receptor correlates with alterations of chromosome 7 in human pancreatic cancer. AB - Recently, the gene for the epidermal growth factor (EGF) receptor has been mapped to chromosome 7p, the short arm of chromosome 7 [Shimizu, N., Kondo, I., Gamou, M. A., Behzadian, A. & Shimizu, Y. (1984) Somatic Cell Mol. Genet. 10, 45-53]. Utilizing EGF binding in saturation studies, karyology, and cDNA hybridization experiments, we have sought to determine whether there is a correlation between dosage or alteration of chromosome 7 and enhanced expression of EGF receptor in cultured human pancreatic carcinoma cells. Saturation binding studies with 125I labeled EGF were performed at 4 degrees C with four established human pancreatic cancer cell lines: T3M4, PANC-1, COLO 357, and UACC-462. Analysis of binding data revealed enhanced numbers of EGF receptors in all four cell lines. Chromosome banding analysis revealed clonal structural alterations of chromosome 7p in the cell lines T3M4, PANC-1, and COLO 357, whereas UACC-462 displayed multiple copies of chromosome 7. Hybridization studies using a radiolabeled EGF receptor cDNA probe failed to demonstrate DNA sequence amplification in any cell line but confirmed the presence of EGF receptor mRNA in these cells in approximate proportion to EGF receptor number. Our results suggest that enhanced expression of EGF receptor in human pancreatic cancer can be associated with either structural or numerical alterations of chromosome 7. PMID- 3014533 TI - In vitro regulation of cartilage matrix assembly by a Mr 54,000 collagen-binding protein. AB - In cartilage, type II collagen is present as thin, short, randomly oriented fibrils. In vitro, however, type II collagen forms fibrils of large diameter, indicating that additional factors may be involved in the regulation of collagen fibril formation. We have examined extracts of a cartilage-producing tumor for the presence of collagen-binding proteins. In addition to fibronectin and link protein, a Mr 54,000 protein was found to bind to collagen fibrils as well as to native and denatured type II collagen. Immunological studies using antibody against the protein indicate that it is a cartilage matrix protein, not present in bone or in several other tissues. In vitro studies show that the Mr 54,000 protein in combination with cartilage proteoglycan decreases the rate of type II fibril formation and causes the fibrils to be of small diameter (24 +/- 8 nm). These studies indicate that complexes between collagen and proteoglycans mediated by this protein may regulate the assembly of cartilage matrix. PMID- 3014535 TI - Topoisomerase I identified by scleroderma 70 antisera: enrichment of topoisomerase I at the centromere in mouse mitotic cells before anaphase. AB - Antibodies derived from scleroderma patients positive for the extractable 70-kDa antigen were shown to react with topoisomerase I. Topoisomerase I was identified by molecular size and by antibody inhibition of the topoisomerase I-specific relaxation of supercoiled plasmid DNA. By using in situ localization by indirect fluorescence, we found topoisomerase I preferentially located in the centromeric regions of the mouse G2-phase cells and chromosomes, while the distribution in human cells is much more dispersed. Moreover, comparison of a published consensus sequence for topoisomerase I binding with mouse satellite DNA revealed a high degree of homology. The localization of topoisomerase I in the centromeres of mouse cells in the later part of the cell cycle and prior to anaphase suggests functional involvement in mitosis. PMID- 3014536 TI - Analysis of the human alpha-globin gene cluster reveals a highly informative genetic locus. AB - Extensive molecular studies have characterized 15 dimorphic and 2 multiallelic genetic markers within the human alpha-globin gene cluster. Analysis of these markers in 9 populations has shown that the alpha-globin locus is remarkably polymorphic and is therefore an ideal marker on chromosome 16 for the construction of a human genetic linkage map. The combined analysis of 9 polymorphic markers has established alpha-globin haplotypes that provide the means to study the molecular genetics and common mutants of this cluster. The novel association of a conventional restriction fragment length polymorphism haplotype and linked, hypervariable regions of DNA should allow a comparison of the rate of change of such markers. PMID- 3014537 TI - Human chromosome 7 carries the beta 2 interferon gene. AB - A cDNA clone (pAE20-4) corresponding to the 1.3-kilobase human beta 2 interferon mRNA was used as a probe in blot-hybridization experiments of DNA from a panel of human-rodent somatic cell hybrids containing overlapping subsets of human chromosomes. The DNA hybridization experiments showed that the human beta 2 interferon gene is located on human chromosome 7. This assignment is consistent with previous experimental data in which the expression of the translationally active 1.3-kilobase beta 2 interferon mRNA was assayed in various somatic cell hybrids. Blot-hybridization experiments using DNA from different human cell strains and cell lines reveal distinct EcoRI restriction fragment length polymorphisms of the human beta 2 interferon gene. PMID- 3014538 TI - Multiple restriction fragment length polymorphisms at the insulin receptor locus: a highly informative marker for linkage analysis. AB - Although resistance to insulin action is a well-studied phenomenon in non-insulin dependent diabetes and certain genetic syndromes, the role of inherited defects of the insulin receptor in these disorders is unknown. To facilitate the evaluation of that role, restriction fragment length polymorphisms (RFLPs) were identified using various portions of the insulin receptor cDNA to examine digested DNA from American Blacks, Pima Indians, and Caucasians. Five RFLPs were identified in Caucasians. Two of these were detected with a single 1.3-kilobase probe in Rsa I digests with minor allele frequencies of 0.48 and 0.23. An additional RFLP was noted with Bgl II and two more RFLPs with Sac I using a different 1.6-kilobase probe, with minor allele frequencies of 0.17 for Bgl II and 0.12 for both Sac I RFLPs. All RFLPs except for the second Sac I RFLP were present in American Blacks, while only the Rsa I RFLPs were present in Pima Indians. Pairwise analysis showed random association between all sites except for the Bgl II and second Rsa I sites, where the disequilibrium statistic, delta, was -0.70 (different from 0 at P less than 0.001). No association of any RFLP was noted with non-insulin-dependent diabetes in a small population. These studies show that this is a highly informative locus that should be important for mapping of chromosome 19p and for linkage studies. PMID- 3014539 TI - Adenovirus type 12 early region 1A proteins repress class I HLA expression in transformed human cells. AB - The adenovirus type 12 (Ad12) early region 1A (E1A) gene is thought to play a major role in repressing class I major histocompatibility complex expression in transformed rodent cells. However, since transformation by adenovirus requires both E1A and E1B genes, it has not been demonstrated whether the Ad12 E1A gene acts alone or synergistically with the E1B gene to accomplish this effect. Moreover, it is not known whether the repression of class I antigen synthesis by Ad12-transforming gene products occurs only in rodent cells. We show that the Ad12 E1A gene, in the absence of the E1B gene, is capable of greatly reducing the levels of class I HLA antigens and mRNAs in primary human cells transformed by the E1A gene of Ad12 and the large tumor antigen (T-antigen) gene of BK virus; control cells transformed by BK virus T-antigen gene alone or the highly related simian virus 40 T-antigen gene showed no apparent alteration in class I HLA expression. Human recombinant interferon gamma was able to restore synthesis of class I HLA antigens in transformed cells that produced Ad12 E1A proteins, indicating that these cells were not deficient for class I genes. These results strongly indicate that the Ad12 E1A proteins modulate class I gene expression by similar mechanisms in both transformed rodent and human cells. PMID- 3014540 TI - Deficiency of cyclic AMP-dependent protein kinases in human psoriasis. AB - To determine possible differences in the cyclic AMP-dependent protein kinases of normal and psoriatic human fibroblasts, the levels of the regulatory subunits (RI and RII, respectively) of protein kinase I and protein kinase II were quantitated by photoaffinity labeling with 8-azido[32P]cAMP. The level of RII was significantly decreased, or was undetectable, in cytosol prepared from fibroblasts from five psoriatic subjects when compared to RII levels found with normal human fibroblasts. The level of cytosolic RI was decreased in fibroblasts from four psoriatic patients and was within the normal range for one diseased patient when compared to RI levels in normal human fibroblasts. The elution profile from a DEAE-cellulose column of protein kinase activity in the soluble fraction from two psoriatic patients also showed a decrease in type I kinase activity and the complete absence of type II kinase activity. Other results indicate that the level of RI in erythrocyte membranes from psoriatic subjects is significantly decreased when compared to that of erythrocyte membranes from eight normal subjects. A significant correlation (P less than 0.001) was observed between the severity of the cutaneous manifestation of the disease and the level of RI in psoriatic erythrocyte membranes. The changes noted in the levels of RI and RII in cell types other than those thought to be specifically involved in the proliferative epidermis disorder of the disease suggest a general protein kinase deficiency. PMID- 3014541 TI - Expression of the protein encoded by the 3' open reading frame of human T-cell lymphotropic virus type III in bacteria: demonstration of its immunoreactivity with human sera. AB - The genome of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV), the infectious agent etiologically associated with the acquired immunodeficiency syndrome, contains, in addition to the genes for the polymerase, core, and envelope proteins, several open reading frames. To investigate whether the 3' open reading frame (3' orf) located between the envelope gene and the 3' long terminal repeat is a gene expressed in vivo in infected individuals, we inserted a fragment of 3' orf in a prokaryotic expression vector. The protein product synthesized in bacteria was purified and allowed to react with sera from individuals infected with human T-cell lymphotropic virus type III as indicated by seropositivity for other viral proteins. Two-thirds of the sera, regardless of the clinical status of the individuals, reacted with the purified protein indicating that 3' orf is a viral gene the product of which is immunogenic in vivo. A polyclonal rabbit antiserum reacting against the 3' orf gene product was obtained by serial injection of rabbits with the purified bacterial protein. The antiserum recognized a 27-kDa protein in the human T-cell lymphotropic virus type III-infected lymphocytes. PMID- 3014542 TI - Isolation of a T-lymphotropic retrovirus from naturally infected sooty mangabey monkeys (Cercocebus atys). AB - Healthy mangabey monkeys in a colony at the Yerkes Regional Primate Research Center were found to be infected with a retrovirus related to human immunodeficiency virus (HIV). Virus was isolated from peripheral blood cells of 14 of 15 randomly selected mangabeys. All virus-positive animals had antibodies to the mangabey virus at the time of virus isolation and, in a retrospective study, 82% of mangabey serum samples obtained in 1981 had antibodies to the virus. The newly isolated retrovirus is (i) morphologically identical to HIV by electron microscopy; (ii) serologically related to the human virus by enzyme immunoassay, immunoblotting experiments, radioimmunoprecipitation, and neutralization; and (iii) cytopathic for human OKT4+ cells. The mangabey virus also shares these properties with the simian T-lymphotropic virus type III (STLV III) recently isolated from diseased macaques and from healthy African green monkeys (STLV-IIIAGM). However, the mangabey virus, like STLV-IIIAGM, differs from both HIV and STLV-III in that it apparently does not cause clinical immunodeficiency or disease following natural infection of the host from which it was isolated. Comparison of the virus-host interactions of these isolates may be valuable in defining determinants of pathogenicity for cytopathic retroviruses. PMID- 3014545 TI - Cyclosporin. Mechanism of action: antagonism of the prolactin receptor. PMID- 3014543 TI - Hyperalgesic properties of 15-lipoxygenase products of arachidonic acid. AB - Induction of hyperalgesia by leukotriene B4 (LTB4), a potent chemotactic factor for polymorphonuclear leukocytes (PMNLs), depends on the generation by cutaneous PMNLs of mediators that are probably derived from the 15-lipoxygenation of arachidonic acid. The capacity of dihydroxyeicosatetraenoic acid (diHETE) products of the 15-lipoxygenation of arachidonic acid in PMNL to elicit hyperalgesia was evaluated by assessing the effects of intradermal injection of synthetic diHETEs on the pressure nociceptive threshold in rats. (8R,15S) Dihydroxyeicosa-(5E-9,11,13Z)-tetraenoic acid [(8R,15S)-diHETE] produced a dose dependent hyperalgesia, as measured by decrease in threshold for paw withdrawal. The isomer (8S,15S)-diHETE antagonized in a dose-dependent manner this hyperalgesia due to (8R,15S)-diHETE but did not suppress prostaglandin E2-induced hyperalgesia. (8S,15S)-DiHETE produced a dose-dependent hypoalgesia, as reflected by an increase in nociceptive threshold, suggesting a contribution of endogenous (8R,15S)-diHETE to normal nociceptive threshold. The hypoalgesic effect of (8S,15S)-diHETE was blocked by corticosteroids but not by the cyclooxygenase inhibitor indomethacin. Neither (8R,15S)-dihydroxyeicosa-(5,15E-9,11Z)-tetraenoic acid nor (8R,15S)-dihydroxyeicosa-(5,11E-9,13Z)-tetraenoic acid exhibited any hyperalgesic or hypoalgesic activity. The stereospecificity of the effect of (8R,15S)-diHETE suggests that the induction of hyperalgesia is a receptor dependent phenomenon and that (8S,15S)-diHETE may be an effective receptor directed antagonist. The (8R,15S)-diHETE and (8S,15S)-diHETE from PMNL, keratinocytes, and other epithelial cells may modulate normal primary afferent function and contribute to inflammatory hyperalgesia. PMID- 3014544 TI - Biochemical and electrophysiological evidence of functional vasopressin receptors in the rat superior cervical ganglion. AB - Binding of radioactive vasopressin--but not of oxytocin--was detected by autoradiography and by labeling of membranes obtained from the rat superior cervical ganglion. In both instances binding could be displaced by V1 (smooth muscle-type) but not by V2 (kidney-type) agonists, indicating that the ganglionic vasopressin receptors are similar to those present on hepatocytes and vascular smooth muscle. In accordance with the V1 character of the receptors, vasopressin activated the turnover of membrane inositol lipids, and this effect was abolished by a structural analogue known to act as a vasopressor antagonist. A possible physiological role of vasopressin was suggested by intracellular recordings obtained from ganglion cells in vitro. Vasopressin induced a reduction in the amplitude of the fast excitatory postsynaptic potential evoked by electrical stimulation of the preganglionic nerve. This reduction in ganglionic transmission was antagonized by the same synthetic structural analogue that blocked the effect of vasopressin on inositol lipids. This study provides evidence for the presence of functional vasopressin receptors in a rat sympathetic ganglion and thus suggests that vasopressin may play a role in peripheral autonomic function. PMID- 3014546 TI - Mechanism of action: ciclosporin- and prolactin-mediated control of immunity. PMID- 3014547 TI - Effect of the type of dietary carbohydrate on small intestinal functions. PMID- 3014549 TI - Effects of dietary carbohydrates on blood pressure. PMID- 3014548 TI - Effects of dietary carbohydrates in metabolic disturbances in man. PMID- 3014550 TI - Analysis of the serum paraoxonase/arylesterase polymorphism in some Sudanese families. AB - The combination of several qualitative tests of paraoxonase and arylesterase activity of human serum permits a clear, unequivical designation of phenotype for nearly every individual. The qualitative tests appear to be applicable for further studies of sample populations from Africa, the near East, and the Orient. These tests should facilitate the estimation of the respective gene frequencies in various ethnic groups. It is probable that the same two major alleles determining the isozymes of paraoxonase/arylesterase of human serum are present in the European, North American and African populations. However, there appears to be a higher frequency of B alleles among African and Oriental people. Further surveys should provide more details of the ethnic differences and the extent of variation in gene frequencies in selected populations for this interesting polymorphic protein. PMID- 3014551 TI - A radioimmunoassay useful for quantitating megakaryocyte growth in vitro. PMID- 3014552 TI - Megakaryocyte biochemistry. PMID- 3014553 TI - Malignant disorders of megakaryocytes associated with primary mediastinal germ cell tumors. PMID- 3014554 TI - Changes in the patterns of patient work-up in community hospital oncology programs and in comparison hospitals. PMID- 3014555 TI - Utilization of local patient management guidelines and protocols in CHOP hospitals. PMID- 3014556 TI - The impact of the Community Hospital Oncology Program (CHOP) on the quality of data reporting. PMID- 3014557 TI - Protocol cost analysis. PMID- 3014558 TI - Changes in the patterns of patient therapy and multidisciplinary consultation in CHOP and in comparison hospitals. PMID- 3014559 TI - Fatty acid composition and arachidonic acid metabolites in ascitic fluid of patients with ovarian cancer. AB - Fatty acid composition and arachidonic acid metabolites in ascitic fluids of patients with ovarian cancer were compared to those in the peritoneal fluids of patients with benign gynecologic conditions. Substantial amounts of PGE2, PGF2 alpha, TXB2, and leukotriene B4 were detected in the fluids of the both patient groups. In the group of the cancer patients the concentrations of TXB2 were slightly smaller than those in the control group. In the percentage amounts of the eicosanoid precursor fatty acids there could not be detected differences between these two groups. However, in the peritoneal fluids of the cancer patients the percentage amount of palmitoleic acid (16:1) was significantly higher than that in the control group. PMID- 3014560 TI - Mathematical description of the interaction between prostaglandins and calcium. AB - The interpretation of the biological action of prostaglandins is very difficult because the shape of the dose-response curves in different assays is often unusual. Horrobin has attempted to explain these unusual curves by supposing special interactions between Ca and the prostaglandin receptors. This paper gives a mathematical description of his theoretical models. The results of computer simulation based on these models were in good agreement with the reported experimental observations. PMID- 3014561 TI - Protective effect of polyunsaturated fatty acid supplementation in apoferritin induced murine glomerulonephritis. AB - The effects of increasing two dietary polyunsaturated fatty acids, eicosapentaenoic and linoleic, on the glomerulonephritis induced by repeated injections of apoferritin in the mouse were studied. Urinary protein excretion was measured serially; serum creatinine, aortic and renal production of eicosanoids and kidney histology were measured at sacrifice at 8 weeks. Both high EPA and LA feedings were associated with lesser proteinuria, normalization of renal function and profound changes in the tissue production of prostaglandin and thromboxane, which may explain their protective effect in this model of renal disease. PMID- 3014563 TI - The inhibitory effect of endogenous convulsants quinolinic acid and kynurenine on the pentobarbital stimulation of [3H]flunitrazepam binding. AB - The metabolites of tryptophan-kynurenines with convulsant action quinolinic acid (QA) and l-kynurenine (l-KYN) antagonized the enhancing effect of pentobarbital (1 mM) on [3H]Flunitrazepam binding. IC50 for l-KYN were 35.9 +/- 14.8 microM and for QA 31.2 +/- 7.2 microM respectively. The inhibitory effect of KYN was stereoselective: IC50 of l-isomer was about two fold lower than IC50 of racemic form, d,l-KYN. Scatchard analysis revealed that inhibitory effect of QA and l-KYN on [3H]Flunitrazepam binding enhanced by pentobarbital is due to the decrease in affinity of benzodiazepine receptors. On the basis of these data it is proposed that QA and l-KYN possess their convulsant action interacting with barbiturate/picrotoxin sensitive sites of GABA-benzodiazepine-barbiturate complex. PMID- 3014562 TI - Dopamine neurotransmission in the nucleus accumbens may be involved in oxytocin enhanced grooming behavior of the rat. AB - Intracerebroventricular (ICV) infusion of a low dose of oxytocin enhanced novelty induced grooming in male rats. The present experiments were undertaken to investigate whether dopamine neurotransmission in the nucleus accumbens is involved in this effect. Bilateral lesions of the nucleus accumbens by microinjections of 6-hydroxydopamine (6-OHDA) totally prevented the enhancement of grooming behavior after subsequent ICV infusion of oxytocin. Furthermore, bilateral injections of the dopamine receptor antagonist, haloperidol, into the nucleus accumbens completely suppressed grooming behavior of rats infused ICV with oxytocin. These results suggest that dopamine neurotransmission in the nucleus accumbens is involved in the behavioral response enhanced by the peptide. PMID- 3014564 TI - Behavioral specificity of beta-endorphin suppression of sexual behavior: differential receptor antagonism. AB - Open field behavior was observed in conjunction with mating behavior to discern whether the effect of intraventricular (ICV) beta-endorphin (beta-END) on sexual behavior may be secondary to akinesia. Three groups of ovariectomized, estrogen progesterone-primed rats each received counterbalanced treatments of saline ICV, 2 micrograms beta-END ICV, or 2 micrograms beta-END ICV in combination with a selective opioid receptor antagonist. Receptive behavior (lordosis) and proceptive behaviors (presentation and ear wiggling) were consistently suppressed by beta-END, while ambulation was unaffected. Rearing and grooming were generally decreased, although this effect was statistically significant in only one experiment. Pretreatment with the mu-1 antagonist naloxazone (50 mg/kg intravenously) reversed the effects of beta-END on all behaviors tested. The delta receptor antagonist ICI-154,129 (12.5 and 50 micrograms ICV) only partially reversed the sexual effects of beta-END but completely reversed the open field effects. It is concluded that the suppressive effect of beta-END on sexual behavior, while not behaviorally specific, is not secondary to opioid-induced akinesia. PMID- 3014565 TI - Inhibition of penicillin-induced EEG discharges by low doses of morphine or naloxone in the rabbit. Evidence for a possible non-opioid receptor-mediated mechanism at the sensorimotor cortex. AB - In rabbits, pretreatment by intravenous (IV) and intracortical (IC) routes with low doses of morphine (250 micrograms/kg IV or 60 pmoles/rabbit IC) and naloxone (1-50 micrograms/kg IV or 0.3 pmoles/rabbit IC) antagonizes the EEG and behavioural seizures due to the IC injection of penicillin (150 Units) at the level of the sensorimotor cortex. Pretreatment with naloxone (20 micrograms/kg IV) did not alter the anticonvulsant effect of morphine (250 micrograms/kg IV). The similar anticonvulsant effect of the two drugs together with the absence of any antagonism by naloxone on the effect of morphine seem to suggest that both drugs act through a non-opioid receptor-mediated mechanism. Further, in light of the low effective doses of the drugs and of the absence of any additive effect after their combined administration, one might speculate that morphine and naloxone do not act through different pharmacological receptors. However, the presence of distinct EEG patterns with either morphine or naloxone, injected IC and IV, in animals fully protected against penicillin-induced seizures, does not seem to be in favour of the latter possibility. PMID- 3014566 TI - Effects of ACTH and related peptides on medial septal self-stimulation. AB - The effects of ACTH1-24, ACTH4-10, the ACTH4-9 analog Org 2766 and [D-Phe7] ACTH4 10 on medial septal self-stimulation were determined in the rat following intracerebroventricular (ICV) injections. Self-stimulation rates were increased by 0.01-10 micrograms ACTH1-24 or 0.1-10 micrograms ACTH4-10, but not by Org 2766 or [D-Phe7] ACTH4-10. A dose of 1 microgram ACTH1-24 ICV did not affect open field behaviour. Subcutaneous administration of 1 microgram ACTH1-24 did not influence self-stimulation in the septum. Thus, the ACTH1-24 effect appears to be a central effect and provides evidence for an influence of ACTH containing pathways on structures involved in maintaining self-stimulation behavior. A possible role of opiate receptors and dopaminergic neurons in this effect of ACTH1-24 is also discussed. PMID- 3014567 TI - DSP4-induced noradrenergic lesions increase beta-adrenergic receptors and hippocampal electrophysiological responsiveness. AB - Following profound (greater than 90%) depletions of norepinephrine (NE) by the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4), the numbers of beta-adrenergic receptors were significantly increased (20-25%) in rat hippocampal and somatosensory cortical membranes; however, the numbers of alpha 1 adrenergic receptors and the affinities of both types of receptors were unaffected. This selective up-regulation of beta-adrenergic receptors was evident 1 week after DSP4 administration and was maintained for at least 2 more weeks. In electrophysiological experiments in the hippocampal slice preparation, responses to threshold as well as maximal concentrations of isoproterenol were enhanced 150% and 33%, respectively, in the DSP4-lesioned animals. The results demonstrate that nearly complete depletion of brain NE produced by administration of DSP4, like that produced by 6-hydroxydopamine, results in increased numbers of beta- but not alpha-adrenergic receptors, and suggest that the density of the former are regulated by afferent noradrenergic fibers. Furthermore, the functional significance of the increased number of hippocampal beta-adrenergic receptors is directly manifested in a greater electrophysiological responsiveness to an exogenously administered beta-adrenergic receptor agonist. PMID- 3014568 TI - Dual effect of morphiceptin on lordosis behavior: possible mediation by different opioid receptor subtypes. AB - Intracerebroventricular (ICV) infusions of the selective mu receptor agonist morphiceptin produce a dual effect on lordosis behavior in ovariectomized, steroid-primed rats. Low doses of morphiceptin (20 ng) inhibit lordosis whereas higher doses (2000 ng) facilitate this behavior. The present experiment tested whether naloxone, an antagonist of both high- and low-affinity mu receptors, or the long-acting high-affinity mu receptor antagonist naloxazone could block the dual effect of morphiceptin on lordosis. Ovariectomized rats primed with estrogen and progesterone received naloxone, naloxazone, or a control solution prior to ICV infusions of either 0, 20, or 2000 ng of morphiceptin. Naloxone reversed both the inhibition and facilitation of lordosis produced by morphiceptin, but had no effect on lordosis when administered before control infusions. In contrast, naloxazone reversed the inhibition but not the facilitation of lordosis. These results indicate that the inhibitory effect of morphiceptin on lordosis reflects the activation of high-affinity mu receptors whereas the facilitatory effect reflects either the activation of low-affinity mu receptors or other opioid receptor subtypes. The failure of naloxone or naloxazone to affect lordosis in rats receiving control infusions of saline further suggests that endogenous opioid systems do not exert a tonic inhibitory or facilitatory action on lordosis behavior. PMID- 3014569 TI - Mechanism of the plasma cholesterol lowering effect of tazasubrate in rats: accelerated plasma cholesterol transport with increased liver lipoprotein receptor activity. AB - Tazasubrate (30 mg/kg/day) a new lipid lowering agent, chemically unrelated to fibrates, reduces plasma cholesterol in rats within 2-3 days to 50% of controls. During the early rapid decrease of plasma cholesterol, increased liver cholesterol concentrations are observed. These return to normal, once the new steady state of plasma cholesterol (at 50% of controls) has been reached. Radiotracer studies with 3H-cholesterol labelled plasma very low density lipoproteins are consistent with an accelerated plasma cholesterol turnover. Analysis of the binding to liver membranes of 125I labelled cholesterol rich very low density lipoproteins (beta-VLDL) shows a marked increase in the total binding (Bmax rising from 106 ng/mg of protein 378 ng/mg), without significant alterations in the receptor affinity. PMID- 3014570 TI - The in vitro and in vivo effects of enkephalins and beta-endorphin on ACE activity in mice. AB - The in vitro and in vivo effects of naloxone (NAL) and endogenous opioids namely methionine and leucine enkephalins (MET-ENK, LEU-ENK) and beta-endorphin (BETA END) on the brain and lung angiotensin converting enzyme (ACE) activities were investigated. All three peptides dose -dependently inhibited ACE activity in vitro except 10(-5) M concentration of BETA-END which increased the lung ACE activity. NAL which intensified the in vitro inhibitory effect of the used opioids showed an antagonistic effect on the in vivo suppressive effect of BETA END on both brain and lung ACE activities whereas it had neither antagonistic nor synergistic effect on the in vivo inhibiting effect of MET-ENK and LEU-ENK on the lung ACE activity. The results were consistent with those obtained by using morphine and NAL. As a result the possible contributory action of the excessively released endogenous opioids to overcome shock via their inhibiting effect on ACE was discussed. PMID- 3014571 TI - Inhibition of bacterial DNA-dependent RNA polymerases and restriction endonuclease by UV-absorbing components from propolis. AB - Several UV-absorbing substances inhibiting the DNA-dependent RNA polymerases of Escherichia coli and Streptomyces aureofaciens, as well as the restriction endonuclease Eco RI have been isolated from the water-soluble extract of Propolis by two-dimensional paper chromatography. The inhibition of bacterial RNA polymerases by the components of Propolis was probably due to the loss of their ability to bind to DNA. The general characteristic of the UV-absorbing component of Propolis with the most pronounced inhibitory effect upon transcription in vitro is described. PMID- 3014572 TI - [Synthesis of n-substituted amino acid-p-nitrobenzyl esters as potential ACE inhibitors]. PMID- 3014573 TI - [Inositophospholipids and hormone action]. PMID- 3014574 TI - [Antiviral activity of new pyrophosphate analog compounds]. PMID- 3014575 TI - Inhibitors of picornavirus uncoating as antiviral agents. PMID- 3014576 TI - Changes in contents of calpain and calpastatin in rat liver during growth. AB - Variation of calpain I, calpain II, and calpastatin in rat liver during growth from 0 to 14 weeks was studied by chromatographic fractionation of the liver cytosol and enzyme assays on the eluted fractions. When compared in terms of units per g wet liver, high-Ca2+-requiring calpain II always exceeded low-Ca2+ requiring calpain I in male and female rats. The level of calpain II in neonatal (0 week) rat liver was 1.9-2.9 times higher than that for the adults (7 to 14 weeks). The contents of calpastatin, calpain-specific inhibitor protein, were were always higher than those of calpain II in adult rat liver, but the difference was much less, or sometimes even reversed, in neonatal and young (1 and 2 weeks) animals. In general, the variation was more pronounced in female than in male rats. PMID- 3014577 TI - Effect of cupric ions on the permeability of erythrocyte membrane to non electrolyte spin labels. AB - The addition of cupric ions caused decreased permeability to hydrophilic molecules and increased permeability to hydrophobic molecules. These results suggest that TEMPOL penetrates the erythrocyte in a different way than TEMPO. Penetration of TEMPOL is controlled by-SH groups, while TEMPO probably diffuses through the lipid bilayer. Cupric ions increase the permeability of erythrocyte membranes to both non-electrolytes in vivo. PMID- 3014578 TI - Entrainment of rat circadian rhythms by daily injection of melatonin depends upon the hypothalamic suprachiasmatic nuclei. AB - Although pinealectomy has little effect on the generation of circadian rhythmicity by mammals, daily injections of the pineal hormone melatonin entrain free-running rats [30]. The present study was designed to determine if known components of mammalian circadian organization were necessary for melatonin entrainment. Rats received either lesions to the suprachiasmatic nuclei (SCN), sham-lesions or neurotoxic lesions to brain catecholamines or serotonin. They were then allowed to free-run in constant dim red light (DD) before each received daily injections of either 1 mg/kg melatonin or ethanol:saline vehicle for 90 days. They were allowed to free-run for 30 days afterwards. Rats which received sham-lesions or neurotoxic lesions entrained to melatonin injections but not to vehicle. Rats which received complete SCN lesions were unaffected by melatonin or vehicle. These data suggest that the behavioral effects of melatonin, like those on reproduction in seasonally breeding mammals, depend upon an intact circadian system and the SCN. PMID- 3014579 TI - Disruption of conditioned taste aversion by ECS: the role of lithium chloride. AB - Rats were taught a conditioned taste aversion (CTA) by pairing a 10% sucrose solution (CS) with lithium chloride (LiCl)-induced poisoning (UCS). The CS-UCS interval was 30 min. The LiCl dose (20 ml/kg) was either strong (0.15 M) or weaker (0.075 M). Electroconvulsive shock (ECS) (80 mA for 600 msec) was interpolated within the CS-UCS interval at either 15 or 30 min. ECS caused a significant disruption of CTA only when the aversion was established with the weaker dose of LiCl. There was also no indication that interference with CTA was dependent upon close temporal contiguity between the ECS and LiCl. In a second experiment a CTA was established with LiCl (0.15 M) which was heated to 45 degrees C. Under these conditions ECS produced a similar disruption of learning to that when the UCS was the weaker dose of LiCl (0.075 M). The results suggest that an apparent differential loss of learning within the CS-UCS interval described in a previous report was accidentally created when some groups of animals were poisoned with warm and others with cold LiCl. PMID- 3014580 TI - Substitution of natural conditioned and unconditioned stimuli by artificial stimuli does not prevent acquisition of conditioned taste aversion in rats. AB - Attempts to replace a natural conditioned stimulus (taste) by electrical stimulation and a natural unconditioned stimulus (gastrointestinal disorder) by intracranial application of harmaline in the conditioned taste aversion (CTA) paradigm are described. The taste is replaced either by electrical stimulation of taste receptors of the tongue or by intracranial self-stimulation of the lateral hypothalamus, both triggered by licking. Both stimuli lose their rewarding properties when paired with gastrointestinal distress whereas self-stimulation triggered by nose poking is not affected by the same procedure. The unconditioned stimulus was replaced successfully by intracerebral injection of harmaline hydrochloride. The effect of the injection of 3-6 micrograms harmaline into the region of the inferior olive is comparable to that of systemic injection of 10 mg/kg harmaline. Electrophysiological analysis of the effect of locally and systemically applied harmaline indicates that the drug probably elicits CTA by activation of bulbar structures including the lateral reticular nucleus and lateral vestibular nucleus. PMID- 3014581 TI - Dopaminergic influence on the effects of angiotensin II in behavioural reactions. AB - Using a neuropharmacological approach we studied the dopaminergic influence on the effects of angiotensin II (ATII) in some behavioural reactions. Haloperidol reduced the exploratory behaviour-increasing effect of ATII in the open field. Haloperidol also reduced the apomorphine stereotypy-increasing effect of ATII. Sulpiride tended to overcome the locomotor activity-decreasing effect of ATII. Seven resp. 21 days after withdrawal of pimozide (applied for 14 days) the convulsive-seizure thresholdincreasing effect of ATII was enhanced. Haloperidol impaired the retention-facilitating effect of ATII in rats performing a shuttle box active avoidance task. The results suggest close interactions between central DA-ergic transmission and ATII in the realization of the ATII effects on behaviour. PMID- 3014582 TI - Phenylpropanes and lignans of Viscum album cardioactive drugs V. PMID- 3014583 TI - [Angiotensin-converting enzyme in sarcoidosis]. PMID- 3014584 TI - [Treatment of microcellular carcinoma of the lung]. PMID- 3014585 TI - [Mechanisms of alcohol dependence in the light of biochemical and pharmacological studies]. PMID- 3014586 TI - Age of onset of schizophrenia in siblings: a test of the contagion hypothesis. AB - The possibility that schizophrenia is horizontally transmitted has been assessed in an analysis of age of onset in 264 recorded pairs of siblings with the disease. Age of onset was found to be correlated between siblings, and there was a tendency for the disease to occur at an earlier age in the younger sibling. Three explanations for this finding are considered: horizontal transmission, early detection, and ascertainment bias. An analysis by date of birth differences between siblings gives results consistent with horizontal transmission, but analysis by order of onset of illness (which shows that the age shift is not seen in elder-sibling-ill-first pairs) indicates that ascertainment bias (which arises from a tendency to include an excess of early onsets in younger siblings) is a more cogent explanation of the age shift. Although horizontal transmission is not altogether eliminated, the data suggest that age of onset is determined by genetic or prenatal factors rather than environmental precipitants in postnatal life. The retrovirus/transposon hypothesis (Crow, 1984) can accommodate the findings more readily than the gene-virus interaction hypothesis (Crow, 1983). PMID- 3014587 TI - Viruses, brain and immunosuppression. PMID- 3014588 TI - Viruses in human brains: a search for cytomegalovirus and herpes virus 1 DNA in necropsy tissue from normal and neuropsychiatric cases. AB - DNA was extracted from the brains of patients with Alzheimer-type dementia, schizophrenia, Huntington's chorea and from patients without neurological disease, and examined for the presence of herpes simplex virus type 1 and human cytomegalovirus sequences. By selecting cloned virus DNA fragments which do not hybridize to normal human DNA we were able to achieve a detection level assessed on reconstruction experiments of 1 virus genome per 50 cells. Screening at such sensitivity did not detect virus sequences in the higher CNS, except in cases of encephalitis or immunosuppression. We conclude that, at this level of sensitivity, these viruses cannot be regarded as normal residents of the higher CNS, and at the time of death they do not appear to be associated with these neuropsychiatric conditions. PMID- 3014589 TI - Premenstrual depression, cortisol and oestradiol treatment. AB - A woman with a 5-year history of frequently recurrent depressions responded poorly to the usual antidepressants. She had a raised plasma cortisol and was made worse by progesterone or by ACTH. An oestradiol/testosterone implant every 4 months abolished all symptoms for at least 8 years, and plasma cortisol returned to normal. This case is relevant to an understanding of premenstrual syndromes and the genesis of depressive illness. PMID- 3014590 TI - Midazolam cue in rats: effects of Ro 15-1788 and picrotoxin. AB - The discriminative stimulus effect of midazolam, a short-acting benzodiazepine, was used for testing the effects of drugs thought to act as antagonists at different sites in the proposed benzodiazepine receptor complex. Rats were trained in a standard two-bar operant conditioning procedure with food reinforcers delivered on a tandem schedule. The 0.4 mg/kg dose of midazolam used for training was well discriminated, typically yielding at least 95% correct responding. The benzodiazepine receptor antagonist Ro 15-1788 blocked the discriminative effect of midazolam but did not influence generalization to pentobarbitone (7.5 mg/kg). The indirect GABA antagonist picrotoxin attenuated both generalization to pentobarbitone and its response rate-reducing effect. Picrotoxin had no effect on the discriminative effect of midazolam at 0.4 mg/kg but it blocked the effect of 0.1 mg/kg. Even in doses which reduced overall response rates, nicotine did not block discrimination of midazolam (0.4 mg/kg). The results are consistent with models which postulate a GABA-linked ion channel which is a site of action for barbiturates and which is "downstream" of the benzodiazepine receptor itself. PMID- 3014591 TI - Pharmacological investigations of the mechanisms underlying the effects of peripheral 5-HT on flavour consumption and preference. AB - Systemic administration of serotonin (5-hydroxytryptamine, 5-HT) to non-deprived rats increased saline (0.9%) consumption (5-HT hyperdipsia), without altering saline preference in two-bottle test. When sodium saccharin (0.1%) was the test solution 5-HT suppressed both consumption and preference. 5-HT saline hyperdipsia was blocked by pretreatment with an angiotensin I converting enzyme inhibitor (MK421) and mimicked by isoprenaline-induced stimulation of renin production; saccharin consumption and preference were unaffected by either drug. However, methysergide (a 5-HT antagonist) attenuated the effects of 5-HT on saccharin consumption and preference, thus confirming that these effects are mediated via peripheral 5-HT receptors. It is suggested that the effects of 5-HT on saline consumption are mediated via stimulation of the renin-angiotensin system, but its effects on saccharin consumption and preference are mediated by a separate mechanism at some point subsequent to peripheral 5-HT receptors. PMID- 3014592 TI - Rolipram forms a potent discriminative stimulus in drug discrimination experiments in rats. AB - Long Evans rats were trained to discriminate 0.2 mg/kg IP (+/-)-rolipram from vehicle injection in a food-motivated two-lever operant task. Eight out of nine rats acquired the discrimination after an average of 91 sessions (min 65, max 137). The ED50 of (+/-)-rolipram was 0.06 mg/kg IP. Generalization tests with (-) and (+)-rolipram showed that the (-)-isomer was 8 times more active than (+) rolipram with an ED50 of 0.06 and 0.4 mg/kg IP respectively. The phosphodiesterase inhibitor RO 20-1724 partially (83%) generalized to (+/-) rolipram in doses of 0.6 and 1.0 mg/kg IP. IBMX 5 mg/kg IP showed 63% generalization. Tests with imipramine and the (+)- and (-)-isomer of the noradrenaline uptake inhibitor oxaprotiline suggest that NA-uptake inhibiting drugs do not form an interoceptive cue which is (+/-)-rolipram-like. dbcAMP 12.5 mg/kg SC and 100 mg/kg SC dbcGMP did not generalize to the training drug. The nature of the discriminative stimulus produced by this dose of (+/-)-rolipram in rats remains to be elucidated. PMID- 3014593 TI - Discrimination of Ro 11-6896, chlordiazepoxide and ethanol in gerbils: generalization and antagonism tests. AB - Separate groups of gerbils were trained in a T-maze to discriminate between either: (1) the 1,4-benzodiazepine Ro 11-6896 (1 mg/kg), (2) chlordiazepoxide (25 mg/kg) or (3) ethanol (2,000 mg/kg) and vehicle. Thus, the respective training condition served as a discriminative stimulus guiding choice behavior. A degree of generalization which was enhanced by increasing injection-test intervals occurred between ethanol and the benzodiazepines. Ro 15-1788 markedly attenuated the cue properties of the benzodiazepines, but not that of ethanol. Additional tests in the Ro 11-6896 group showed that this discrimination was stereoselective but not stereospecific in that the isomer Ro 11-6893 generalized to Ro 11-6896 at a dose of 10 mg/kg but not at 1 mg/kg; Ro 15-1788 attenuated this generalization. Diazepam (3 mg/kg) generalized to Ro 11-6896 whereas the structurally related Ro 5-4864 (3 mg/kg and 30 mg/kg) did not. In separate tests of open-field activity and temperature recording Ro 15-1788 significantly attenuated the effects of Ro 11-6896 (1 mg/kg) and Ro 11-6893 (10 mg/kg). PMID- 3014594 TI - 3H-clonidine and 3H-yohimbine binding to glass fiber filters: implications for studies with platelet membranes. AB - 3H-Clonidine and 3H-yohimbine were observed to bind to glass fiber filters. The binding was displaced by co-filtration with the corresponding non-radioactive ligand. Phentolamine and (--)-norepinephrine were ineffective in displacing either 3H-clonidine or 3H-yohimbine bound to filters. Failure to correct for filter binding resulted in an over-estimation of specific binding to platelet membranes. Certain published methodologies may have consequently misidentified up to 20% of the specific binding to platelets, that was actually due to displaced filter binding. Experimental conditions are described which eliminate filter binding. These results are significant for the interpretation of data from studies of platelet binding in depressed patients. PMID- 3014595 TI - Further studies of the putative serotonin agonist, m-chlorophenylpiperazine: evidence for a serotonin receptor mediated mechanism of action in humans. AB - To further evaluate the effects and mechanism of action of the putative serotonin agonist m-chlorophenylpiperazine (m-CPP) in humans, changes in plasma prolactin, cortisol, growth hormone, ACTH and body temperature were studied in a group of 10 healthy volunteers following oral administration of m-CPP (0.75 mg/kg), before and after pretreatment with the serotonin receptor antagonist metergoline (MTG). M-CPP produced transient significant increases in plasma prolactin, cortisol, ACTH and in body temperature, but did not significantly alter plasma growth hormone concentration. Moreover, pretreatment with the 5HT antagonist metergoline blocked the m-CPP-induced hormonal and temperature changes. These findings provide strong support for m-CPP's effects in humans being mediated through an interaction with 5HT receptors, and thus support the usefulness of m-CPP as a pharmacologic tool for studying disease and drug-induced alterations in serotonin function in man. PMID- 3014596 TI - Hydrochloric acid-pumice enamel surface abrasion for color modification: results after six months. PMID- 3014597 TI - [Joint disorders. Synovial sarcoma]. PMID- 3014598 TI - Hepatic lobar atrophy following obstruction of the ipsilateral portal vein from hilar cholangiocarcinoma. AB - Gross deformity of the liver associated with hilar carcinoma is rare. In 17 patients with hilar cholangiocarcinoma and intrahepatic bile duct dilatation, the relationships between lobar or segmental atrophy, compensatory hypertrophy, and patency of portal vein branches were evaluated with computed tomography (CT) and angiography. All six patients with obstructed or narrowed portal veins (group A) had lobar or segmental atrophy on CT scans and angiograms. Compensatory hypertrophy was observed in the unaffected lobe with a patent portal vein in five. In contrast, neither hepatic atrophy nor hypertrophy was demonstrated in the other 11 patients with patent portal veins. All group A patients had differences in hepatic attenuation on CT scans or dense opacification during the hepatogram phase of angiography. Biliary decompression was optimized when the bile duct selected for percutaneous drainage paralleled a patent portal vein. Knowledge of radiologic findings will assist in determining the primary site along the bile duct from which carcinoma has arisen. PMID- 3014600 TI - Boundary effects from opposed magnetization artifact in IR images. AB - A cancellation of signal intensity at the interface separating selected tissue equivalent materials is observed in inversion recovery proton MR images. The absence of signal intensity at the interface is always one pixel wide and appears only when the tissue-equivalent materials forming the interface differ substantially in their longitudinal relaxation times (T1). Images were obtained of various two-layer combinations of tissue-equivalent materials consisting of vegetable oil, animal fat, saline, aqueous Mn+2, or 2% agar doped with Mn+2. This type of boundary is compared with chemical shift artifacts, which at 0.15 T and 0.35 T produce a similar effect. A clinical example of the opposed magnetization artifact is also shown. Since tissues with substantially different T1s are found in vivo, it is expected that this effect could lead to an instrument-dependent artifact that could easily be misinterpreted. PMID- 3014599 TI - Wilms tumor in children: abdominal CT and US evaluation. AB - Computed tomographic (CT) scans and sonograms of 13 children with Wilms tumor were reviewed to determine the ability of each imaging test to characterize the tumor and determine its extent. The findings of this review were correlated with diagnoses based on surgical and pathologic evidence. Tumor necrosis and a pseudocapsule were detected more often using CT scans than sonograms. CT scanning also was more sensitive in assessing perinephric extension, lymph node involvement, and bilateral tumors. Overall, CT scans allowed better determination of the extent of a suspected tumor, enabling correct diagnosis in 77% of patients, while US study was correct in only 23%. PMID- 3014602 TI - [Kuwata technic. 1]. PMID- 3014601 TI - [Clinical testing program and 3-year follow-up results in a multi-center study of daylight hardened composite resins used for restorations in the posterior region]. PMID- 3014603 TI - [Electronic pantography]. PMID- 3014604 TI - [Posterior teeth--why no composite (yet)?]. PMID- 3014605 TI - New approaches to bronchodilator and antiallergic drug therapy. PMID- 3014606 TI - Heterocyclic analogues of GABA: chemistry, molecular pharmacology and therapeutic aspects. PMID- 3014607 TI - Genetically controlled resistance to virus infections of the central nervous system. PMID- 3014608 TI - [D-2 dopamine receptor and hormone release in the intermediate lobe of the pituitary gland]. PMID- 3014609 TI - [Physiological functions of RNaseH in Escherichia coli]. PMID- 3014610 TI - [Molecular biological approach to oncogenicity by hepatitis B virus]. PMID- 3014611 TI - Leukotrienes cause eosinophil emigration into conjunctival tissue. AB - The ability of LTB4, LTC4, the 5S,6R and 5R,6S LTD4 stereoisomers, and LTE4 to evoke leukocyte infiltration into the conjunctiva was demonstrated in the guinea pig by histological and light microscopy techniques. LTD4 and LTE4 demonstrated a dose-dependent and predominantly eosinophilic infiltrate over the selected dose range (10 ng to 1000 ng), while there was only a minimal response to LTC4. LTB4 produced marked eosinophil infiltrates only at the highest dose; scattered neutrophil infiltrates were also noted at the high dose of LTB4. The 5R,6S LTD4 stereoisomer did not evoke any leukocyte infiltration. The SRS-A antagonist, FPL 55712, abolished peptidoleukotriene-induced eosinophil emigration, and indomethacin pre-treatment had no inhibitory effect, indicating direct mediation of this response by LTs. Histamine caused a comparable eosinophilia over a dose range of 10 micrograms to 1000 micrograms. LT-induced eosinophil emigration was directed to the conjunctival epithelium; the cells appeared intact and no tissue damage was observed. These results may have relevance in the areas of allergic conjunctivitis and asthma research. PMID- 3014612 TI - Chemotaxis of human neutrophils and eosinophils towards leukotriene B4 and its 20 w-oxidation products in vitro. AB - Peripheral blood neutrophils and eosinophils from 70 patients and controls were studied for their in vitro chemotactic and chemokinetic responses towards synthetic leukotriene B4 (LTB4), 20-OH-LTB4 and 20-COOH-LTB4. All three factors induced chemotaxis and chemokinesis of cells. 20-OH-LTB4 was always less and 20 COOH-LTB4 even less active than the parent compound. Cells from patients with atopic eczema and T cell lymphoma moved less than cells from normal controls or from patients with psoriasis. In the presence of LTB4, 20-OH-LTB4 and buffer alone, more eosinophils than neutrophils moved to the lower side of the filter, while this did not occur with platelet activating factor as chemoattractant. Studies of neutrophil and eosinophil chemotaxis in the presence of LTB4 should therefore always take into account a high variability of the quantitative response which is donor and disease dependent. PMID- 3014613 TI - Leukotriene B4 potentiates airway muscle responsiveness in vivo and in vitro. AB - We studied the effects of leukotriene B4 (LTB4) on guinea pig airway muscle responsiveness in vivo and in vitro. Responsiveness in vivo was assessed by measuring specific airway resistance (SRaw) upon intravenous acetylcholine infusion in 5 unanesthetized, spontaneously breathing guinea pigs. We found that aerosolized LTB4, in a concentration that itself had no effect on baseline SRaw, caused a substantial increase in bronchial reactivity to i.v. ACh within 8 min of its administration. Responsiveness in vitro was assessed by measuring isometric contraction of the guinea pig trachealis upon stimulation by either chemical or electrical field stimuli. These studies in vitro showed that a concentration of LTB4 that itself did not cause contraction, potentiated airway muscle contraction to ACh and KCl, but not to norepinephrine. This effect of LTB4 was substantially reduced by nifedipine. Our data suggests that amounts of LTB4 that are themselves non-contractile in vivo or in vitro, may directly potentiate the responsiveness of airway smooth muscle to other bronchoconstrictors. PMID- 3014616 TI - The use of epidemiology, scientific data, and regulatory authority to determine risk factors in cancers of some organs of the digestive system. 5. Stomach cancer. AB - In 1930, stomach cancer was the leading cause of death due to cancer among men in the United States. Among women it was the third leading cause of cancer deaths. Although it is well known that death rates for stomach cancer have diminished dramatically over the past 50 years, stomach cancer still has the third poorest 5 year relative survival rate of the different cancers, after pancreatic and lung cancer. Most evidence indicates that environmental factors play an important role in the development of stomach cancer. The remarkable decrease in stomach cancer mortality over the past 50 years and the results of a variety of migrant studies support this review. Several published case-control studies have shown positive associations with preserved meat and salted and pickled food in general. However, there are negative associations with vegetables, fruits and milk; vitamin C is implicated. Milk has both positive and negative associations. This points to the possibility of a carcinogen produced by traditional preservation methods such as salting and suggests that fresh vegetables and fruits and high intakes of vitamin C may reduce the risk. What specific role/s the diet plays in the incidence of stomach cancer remains to be seen. There is a need to elaborate the relationship of such factors as age, sex, migration, geography, environmental factors, and diet to the carcinogenic process that produces cancer of the stomach. PMID- 3014617 TI - [Prevention of risks due to the use of crystallized silica in dental prosthesis laboratories]. PMID- 3014614 TI - Soluble pyrophosphate as a calculus inhibitor. PMID- 3014615 TI - A comparison of grooming behavior potencies of neurohypophyseal nonapeptides. AB - We have previously demonstrated that intracerebroventricular (ICV) administration of oxytocin (OXY) enhanced grooming behaviors in male and female rats at a 1 microgram dose. In the present study female rats were injected ICV with 1 microgram OXY or equimolar doses of other peptides. At this dose arginine vasopressin (AVP), arginine-vasotocin (AVT) and lysine-vasopressin (LVP), as well as alpha-MSH, were as effective as OXY in increasing grooming behavior. At equimolar doses, ACTH1-10, tocinoic acid (the ring structure of OXY) and Pro-Leu Gly-NH2 (the tail structure of OXY) had no significant effect on grooming behavior. The potency of AVP and AVT was determined across a 0.05-5 microgram dose range. Grooming scores increased in an apparent linear manner across a similar OXY dose range. Both AVP and AVT, however, manifested an inverted U grooming response curve. Maximum grooming scores resulted from a 0.1 microgram dose of AVT or a 0.5 microgram AVP dose. Analyses of the aspects of grooming separately found that nonapeptides OXY, AVP and AVT all elevated body grooming, washing, and scratching. Because AVT and AVP administration resulted in grooming scores significantly higher than OXY at lower doses, we concluded that the CNS is more sensitive to the effects of AVT and AVP on grooming behavior than OXY. PMID- 3014619 TI - [Relation between the presence of polymerized human serum albumin receptors and the level of virus replication in chronic hepatitis B virus infection]. PMID- 3014618 TI - [Comparative study of 2 methods in the diagnosis of rotaviruses in infants with acute and asymptomatic diarrhea]. PMID- 3014620 TI - [Identification of a strain of eastern equine encephalitis virus isolated from the pigeon Colomba livia domestica]. PMID- 3014621 TI - [Acute rotavirus enteritis]. PMID- 3014623 TI - Relationships between structure and function of lactogenic hormones. AB - Lactogenic activity of several hormone derivatives obtained by chemical modifications of lysine residues was studied by radioreceptor assay. The relationships between structure and binding to lactogenic receptors are discussed taking into account lysine residue positions liable to be involved in the location of lactogenic function. PMID- 3014622 TI - [Absence of an increase in prolactin receptors on the day of estrus in the mammary gland of the nu IPL rat]. AB - Ovine prolactin (o-PRL) binding to rat mammary gland membranes has been shown to vary during the estrous cycle in the normal rat. In this study we report the characteristics of o-PRL binding to mammary gland membranes throughout the estrous cycle in a new rat strain, IPL nude, which presents a total absence of lactation. Prolactin receptors were quantified in the 100 000 g pellet. The mean value of the affinity constant in IPL nude rat was 16.7 X 10(9) M-1 and no variation was observed throughout the estrous cycle. The binding capacity also remained unchanged and the values were situated between 11.6 +/- 1.1. fmoles/mg protein and 38.6 +/- 12.3 fmoles/mg protein, corresponding to the lowest values obtained in normal rat. On the day of estrus, the prolactin binding capacity in the mammary gland of the IPL nude rat was significantly different from that of normal rat. These findings confirm that prolactin certainly plays an important role in the induction or stimulation of the rat mammary gland function. PMID- 3014624 TI - [Effect of adrenalectomy on the capacity of brown adipose tissue for thermogenesis and the development of obesity in the fa/fa Zucker rat]. AB - This study was undertaken to examine whether adrenalectomy performed during the weaning period could correct some of the first metabolic abnormalities to develop in obese fa/fa rats: impaired thermogenesis in brown adipose tissue and hyperlipogenesis in interscapular brown and white (inguinal) adipose tissues. Pups were adrenalectomized or sham-operated at 23 days of age and studied at 30 days of age. Body weight, interscapular brown adipose tissue and inguinal white adipose tissue weight were decreased after adrenalectomy in Fa/fa and fa/fa pups. Adrenalectomy had no effect on the thermogenic capacity of brown adipose tissue (as assessed by GDP binding to mitochondria) which remained significantly lower in fa/fa than in Fa/fa rats. In both Fa/fa and fa/fa rats the lipogenic capacity of brown and white adipose tissues (as assessed by fatty acid synthetase activity) was dramatically reduced by adrenalectomy. However, in adrenalectomized rats, the fatty acid synthetase activity of brown and white adipose tissue remained 2 and 5-fold higher, respectively, in fa/fa than in Fa/fa rats. These results show that adrenalectomy at postweaning, did not affect specifically the rats bearing the fatty genotype but induced large alterations in both groups of rats. Adrenalectomized fa/fa animals remained obese as compared to the appropriate controls. PMID- 3014625 TI - Corticotropin-releasing factor receptors: autoradiographic identification. AB - Our studies on CRF receptors confirm the established role of CRF in regulating pituitary hormone secretion and support a physiological role for endogenous CRF in regulating CNS activity. Studies to characterize CRF receptors and CRF containing pathways in the brain provide a means for understanding the various functions of this neuropeptide in different areas of the CNS. Finally, the ability to identify and localize CRF receptors in postmorten human tissue provides a basis for studying the role of CRF in the etiology and pathophysiology of a variety of endocrine, neurological, and psychiatric disorders. PMID- 3014626 TI - Use of gene transfer approaches to study regulation of expression of opioid peptide genes. PMID- 3014627 TI - Protein kinases and phosphoproteins in the nervous system. PMID- 3014628 TI - [Isolation and purification of an endogenous digitalis-like compound. Physiological and pathological role]. PMID- 3014629 TI - [Inhibition of angiotensin converting enzyme as a therapeutic approach: the pros and cons]. PMID- 3014630 TI - Chemical induction of tumor cell differentiation. PMID- 3014631 TI - Spontaneous regression and cytodifferentiation of cancer in early life: the oncogenic grace period. PMID- 3014632 TI - [Conference at La Salpetriere. January 1985. Encephalopathy of rapid development with supranuclear ophthalmoplegia and peripheral neuropathy]. PMID- 3014633 TI - Pathogens that cause travelers' diarrhea in Latin America and Africa. AB - With the advent of rapid and convenient means of transportation, millions of persons travel each year from industrialized to developing countries in the tropics and subtropics. These travelers are at risk for a variety of infectious diseases that are endemic in these areas; the most frequently occurring of these is diarrhea. Studies of groups of travelers to Latin America and Africa have found that approximately one-half develop diarrhea during their stay abroad. Etiologic investigations of these illnesses have demonstrated that the important agents that cause travelers' diarrhea are similar to those that cause diarrhea in children in the developing countries. One-third of the cases are associated with enterotoxin-producing strains of Escherichia coli. Smaller proportions appear to be due to rotavirus, Norwalk virus, Shigella, Salmonella, Giardia lamblia, and Entamoeba histolytica. Although they have not been fully evaluated in travelers' illnesses in Latin America or Africa, Campylobacter jejuni, Aeromonas hydrophila, other viruses, and Cryptosporidium probably cause some of the currently unexplained cases of diarrhea. PMID- 3014635 TI - [The dietary fiber content in Polish food products. II. Dietary fiber in groats, rice, cereal flakes, spaghetti, bread and rolls and in various infant formulas]. PMID- 3014636 TI - [Differential diagnosis of local muscular hypertrophy as demonstrated by extensive multiple-site muscle involvement in malignant histiocytoma]. PMID- 3014634 TI - [Renal function in patients with mucocutaneous leishmaniasis treated with pentavalent antimony compounds]. PMID- 3014637 TI - Methods of active immunotherapy and viral oncolysis in some forms of cancer. AB - In some cancers with a high antigenicity (gastric, intestinal, mammary, laryngeal, etc.) the authors associated to the classical treatment an autovaccine prepared by combining the tumoral extract (obtained by trituration, sonication and centrifugation, and stored at -80 degrees C) with some mitogens (phytohemagglutinin) and adjuvants (ovalbumin, tuberculin, lymphocyte promotion factor, etc.) obtaining a greater survival (4-6 years). The association of viruses with an oncolytic action (mumps virus) to the active unspecific immunotherapy in some cancerous structures (peritoneal, pleuropulmonary and synovial malignant mesothelioma, malignant cholangioma of the liver, hepatic metastases of some adenocarcinomas of the colon, gastric cirrhous, clearly differentiated macrocellular carcinomas of the lung, pulmonary mucoid adenocarcinomas, traditional recurrent carcinomas of the urinary bladder), in which all other oncotherapeutic resources have been exhausted, induced slow involution up to disappearance of the tumor and a favourable evolution of the patients without signs-of recurrence after 5-7 years in 73% of cases. To obtain the expected results it is mandatory to comply strictly with certain principles, established by the authors for each of the two original methods, expounded in detail in the text. PMID- 3014638 TI - [Etiopathogenesis of malignant tumors of the otorhinolaryngologic area]. PMID- 3014639 TI - Hepatitis B antigens and antibodies in serum from 41 cases of primary carcinoma of the liver. A study from Thailand. AB - Thirty-seven patients with hepatocellular carcinoma (HCC), one with cholangiocellular carcinoma (CCC), two with mixed HCC and CCC, and one with an anaplastic primary carcinoma of the liver, all from Bangkok, Thailand, were examined for the presence of hepatitis B virus infection markers in their blood. Of the patients with HCC, 70.6% had macronodular cirrhosis. Their serum was positive for hepatitis B surface antigen (HBsAg) in 64.9%, for hepatitis B core antibody (anti-HBc) in 97.3%, and for hepatitis B e antibody (anti-HBe) in 56.8% of the cases. The serum was positive for hepatitis B surface antibody (anti-HBs) in 53.9% of the HBsAg-negative and positive for hepatitis B e antigen (HBeAg) in 16.7% of the HBsAg-positive patients. The results of the study support the hypothesis of an etiological association between hepatitis B virus infection and HCC in Thailand. PMID- 3014640 TI - Mechanism of action of omeprazole. AB - The inhibitory effect of omeprazole on gastric acid secretion in vivo and in vitro is presented. In the gastric fistula dog omeprazole was found to be about 10 times more potent than cimetidine. When omeprazole was administered in vivo, the inhibition of acid secretory rates was found to correlate with the degree of inhibition of the gastric H+K+ATPase purified from the omeprazole treated animals. The inhibitory action of omeprazole was found to depend on acid induced transformation of omeprazole into an active inhibitor of the gastric H+K+ATPase, as no inhibition was obtained when omeprazole was incubated under neutral conditions with either the isolated gastric mucosal or the H+K+ATPase preparations. A model is proposed in which the inhibition of acid formation is mediated by an inhibitory compound generated form omeprazole within the acid compartment of the parietal cell. PMID- 3014641 TI - Correlation between the phenotype and the functional capacity of activated T cells in patients with active systemic lupus erythematosus. AB - Activated T cells in the peripheral blood of patients with systemic lupus erythematosus (SLE) were determined using monoclonal antibodies against activation antigens. Elevated percentages of HLA-DR+ T cells were found in association with active disease. In contrast, we observed an increase in IL-2 receptor-bearing T cells in only six out of 16 patients with active disease. In vitro assays, like spontaneous proliferation, response to IL-2, production of IL 2, and immunoglobulin synthesis have shown that the different patterns of activation antigens are related to different functional stages of T-cell activation. The possible therapeutic consequences are discussed. PMID- 3014642 TI - Murine thymocytes mediate a natural killer-like activity against herpes virus infected target cells but not YAC-1 target cells. AB - In this report, we demonstrated a natural killer (NK)-like activity against HSV-1 infected cells mediated by CD-1 mouse thymocytes. This cytolytic activity is specific for HSV-1-infected MCN cells, since both uninfected MCN and YAC-1 target cells are not susceptible to thymocyte lysis. Antibody plus complement depletion experiments indicate that a portion of the activity is associated with the Lyt 2 /L3T4- thymocyte subpopulation. This NK-like activity cannot be enhanced by addition of interleukin 2 in vitro. PMID- 3014643 TI - [AIDS: an imported disease?]. AB - AIDS (acquired immune deficiency syndrome) has been known for 5 years. The first few cases in Switzerland were observed in persons infected outside the country, but this has changed in the last 2 years. The infectious agent is transmitted by sexual intercourse and by sharing of blood contaminated needles and syringes of drug addicts, two factors which are also responsible for most of the virus transmissions in Switzerland. From some African countries there are reports of a high transmission rate probably caused by prostitution. PMID- 3014644 TI - [The feline leukemia virus vaccine Leukocell: reference for use]. PMID- 3014645 TI - AIDS virus entry pinpointed in brain. PMID- 3014646 TI - Chimeric receptors give clues to oncogene action. PMID- 3014647 TI - Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein. AB - Mammalian cell lines have been engineered to produce a secreted form of the AIDS retrovirus envelope glycoprotein. The recombinant protein has been isolated from growth-conditioned culture media and used to immunize animals. Antibodies directed against the recombinant molecule were found to react with the envelope glycoprotein produced in virus-infected cells. Furthermore, these antibodies were able to directly inactivate the AIDS retrovirus in a neutralization assay in vitro. The expression system reported here should provide sufficient quantities of the AIDS retrovirus envelope protein for biological and vaccination studies. PMID- 3014648 TI - The role of mononuclear phagocytes in HTLV-III/LAV infection. AB - Cells with properties characteristic of mononuclear phagocytes were evaluated for infectivity with five different isolates of the AIDS virus, HTLV-III/LAV. Mononuclear phagocytes cultured from brain and lung tissues of AIDS patients harbored the virus. In vitro-infected macrophages from the peripheral blood, bone marrow, or cord blood of healthy donors produced large quantities of virus. Virus production persisted for at least 40 days and was not dependent on host cell proliferation. Giant multinucleated cells were frequently observed in the macrophage cultures and numerous virus particles, often located within vacuole like structures, were present in infected cells. The different virus isolates were compared for their ability to infect macrophages and T cells. Isolates from lung- and brain-derived macrophages had a significantly higher ability to infect macrophages than T cells. In contrast, the prototype HTLV-III beta showed a 10,000-fold lower ability to infect macrophages than T cells and virus production was one-tenth that in macrophage cultures infected with other isolates, indicating that a particular variant of HTLV-III/LAV may have a preferential tropism for macrophages or T cells. These results suggest that mononuclear phagocytes may serve as primary targets for infection and agents for virus dissemination and that these virus-infected cells may play a role in the pathogenesis of the disease. PMID- 3014649 TI - GABA receptor-mediated chloride transport in a "cell-free" membrane preparation from brain. PMID- 3014650 TI - AIDS research in new phase. PMID- 3014651 TI - Studies and perspectives of protein kinase C. AB - Protein kinase C, an enzyme that is activated by the receptor-mediated hydrolysis of inositol phospholipids, relays information in the form of a variety of extracellular signals across the membrane to regulate many Ca2+-dependent processes. At an early phase of cellular responses, the enzyme appears to have a dual effect, providing positive forward as well as negative feedback controls over various steps of its own and other signaling pathways, such as the receptors that are coupled to inositol phospholipid hydrolysis and those of some growth factors. In biological systems, a positive signal is frequently followed by immediate negative feedback regulation. Such a novel role of this protein kinase system seems to give a logical basis for clarifying the biochemical mechanism of signal transduction, and to add a new dimension essential to our understanding of cell-to-cell communication. PMID- 3014652 TI - Studies of the human c-myb gene and its product in human acute leukemias. AB - The myb gene is the transforming oncogene of the avian myeloblastosis virus (AMV); its normal cellular homolog, c-myb, is conserved across a broad span of evolution. In humans, c-myb is expressed in malignant hematopoietic cell lines and in primary hematopoietic tumors. Partial complementary DNA clones were generated from blast cells of patients with acute myelogenous leukemia. The sequences of the clones were compared to the c-myb of other species, as well as the v-myb of AMV. In addition, the carboxyl terminal region of human c-myb was placed in an expression vector to obtain protein for the generation of antiserum, which was used to identify the human c-myb gene product. Like v-myb, this protein was found within the nucleus of leukemic cells where it was associated with the nuclear matrix. These studies provide further evidence that c-myb might be involved in human leukemia. PMID- 3014654 TI - NIMH celebrates 40th birthday. PMID- 3014653 TI - Human prion protein cDNA: molecular cloning, chromosomal mapping, and biological implications. AB - A human complementary DNA whose protein product is considered to be the major component of scrapie-associated fibrils in Creutzfeldt-Jakob disease, kuru, and Gerstmann-Straussler syndrome has been identified and characterized. The extensive homology of this gene sequence to the hamster PrP 27- to 30-kilodalton prion protein complementary DNA clone, and its existence as a single copy in the human genome, leads to the conclusion that this is the human prion gene. This human prion gene has been mapped to human chromosome 20, negating a direct link between the prion protein and Down's syndrome or the amyloid of Alzheimer's disease. PMID- 3014655 TI - AIDS case dismissed on legal technicality. PMID- 3014656 TI - Brain endothelial cells infected by AIDS virus. PMID- 3014657 TI - In search of the best drugs against AIDS. PMID- 3014658 TI - Measuring antibodies may predict disease. PMID- 3014659 TI - Gene amplification of c-myc and N-myc in small cell carcinoma of the lung. AB - The relationship of the copy numbers of the c-myc and N-myc oncogenes to tumor formation and progression was studied in small cell carcinoma of the lung. When 96 neoplastic lesions from 45 patients were examined, these lesions could be grouped into three categories: high copy (tumors with greater than 3 copies of the N-myc or c-myc gene per haploid genome), middle copy (1.5 to 3 copies per genome), and normal copy. Fourteen of the patients had middle copy tumors, but this was almost always a result of chromosome duplication rather than the amplification of a small genetic locus. In contrast, five patients had high copy tumors, with the increased copy number in each case due to gene amplification. The amplification did not occur in a heterogeneous fashion within individual patients, since all metastatic lesions from patients with high copy lung tumors were also high copy, while none of 41 metastatic lesions from the other patients were high copy. These data suggest that gene amplification is an important step in neoplastic growth in a subset of patients with small cell carcinoma of the lung and that this genetic event occurs relatively early (before metastasis) in this subset. PMID- 3014660 TI - The E5 transforming gene of bovine papillomavirus encodes a small, hydrophobic polypeptide. AB - Bovine papillomavirus (BPV-1) contains two independent transforming genes that have been mapped to the E5 and E6 open reading frames (ORF's). The E5 transforming protein was identified by means of an antiserum against a synthetic peptide corresponding to the 20 COOH-terminal amino acids of the E5 ORF. The E5 polypeptide is the smallest viral transforming protein yet characterized; it had an apparent size of 7 kilodaltons. The transforming polypeptide is encoded entirely within the second half of the E5 ORF and its predicted amino acid composition is very unusual; 68% of the amino acids are strongly hydrophobic and 34% are leucine. Cell fractionation studies localized this polypeptide predominantly to cellular membranes. PMID- 3014661 TI - Tandem regions of yeast DNA topoisomerase II share homology with different subunits of bacterial gyrase. AB - The nucleotide sequence for the Saccharomyces cerevisiae gene TOP2, which encodes DNA topoisomerase II, was compared with the sequence for bacterial DNA gyrase. The amino and carboxyl terminal halves of the single-subunit yeast enzyme showed homologies with the B and A subunits of bacterial gyrase, respectively, at corresponding positions along the polypeptide chains. Although the two enzymes differ in both quaternary structure and activity, the homology between the two proteins indicates mechanistic as well as structural similarities, and a probable evolutionary relationship. PMID- 3014662 TI - Immunoregulatory feedback between interleukin-1 and glucocorticoid hormones. AB - The production and action of immunoregulatory cytokines, including interleukin-1 (IL-1), are inhibited by glucocorticoid hormones in vivo and in vitro. Conversely, glucocorticoid blood levels were increased by factors released by human leukocytes exposed to Newcastle disease virus preparations. This activity was neutralized by an antibody to IL-1. Therefore the capacity of IL-1 to stimulate the pituitary-adrenal axis was tested. Administration of subpyrogenic doses of homogeneous human monocyte-derived IL-1 or the pI 7 form of human recombinant IL-1 to mice and rats increased blood levels of adrenocorticotropic hormone (ACTH) and glucocorticoids. Another monokine, tumor necrosis factor, and the lymphokines IL-2 and gamma-interferon had no such effects when administered in doses equivalent to or higher than those of IL-1. The stimulatory effect of IL 1 on the pituitary-adrenal axis seemed not to be mediated by the secondary release of products from mature T lymphocytes since IL-1 was endocrinologically active when injected into athymic nude mice. These results strongly support the existence of an immunoregulatory feedback circuit in which IL-1 acts as an afferent and glucocorticoid as an efferent hormonal signal. PMID- 3014663 TI - Infectious mutants of HTLV-III with changes in the 3' region and markedly reduced cytopathic effects. AB - A variant of human T-lymphotropic virus type III (HTLV-III) is described that replicates but does not kill normal human T cells in vitro. This variant, designated X10-1, was derived from the genome of a cytopathic HTLV-III clone (pHXB2D) by excision of a 200-base pair segment in the 3' region of the virus, spanning the env and 3'-orf genes. Comparable variants with 55 to 109 base pairs deleted exclusively in 3'-orf produced, in contrast, virus that was extremely cytopathic. On the basis of these findings it is concluded that the 3'-orf gene is not required for cytopathogenicity or replication of HTLV-III. In addition, the results suggest that virus replication and cytotoxicity are not intrinsically coupled. Furthermore, since clone X10-1 retains the ability to trans-activate genes linked to the viral long terminal repeats, trans-activation per se is not responsible for T-cell killing by HTLV-III. These results also raise the possibility that the carboxyl terminus of the envelope gene of HTLV-III has a direct role in T-cell killing by this virus. PMID- 3014664 TI - Clinical aspects of Sjogren's syndrome. PMID- 3014665 TI - [Disorders of cell membrane function in essential hypertension]. PMID- 3014666 TI - [Prevalence of antibodies against the lymphadenopathy-associated virus/human T cell lymphotropic virus type III (LAV/HTLV) in a population of homosexuals in Mexico]. PMID- 3014667 TI - [Chronic generalized lymphadenopathy with splenomegaly associated with HTLV-III virus antibodies]. PMID- 3014668 TI - [Study of the Intragal and Biocuprum intrauterine devices for the presence of additives]. PMID- 3014669 TI - [Nuclear inclusions in plasma cells. Ultrastructural study]. PMID- 3014670 TI - [Anti-HTLV-III/LAV antibodies in hematology reactives]. PMID- 3014671 TI - [Pyrimidine 5'nucleotidase deficiency (P5N). Description of a new case]. PMID- 3014673 TI - Calcification in glioblastoma multiforme of the cervical spinal cord. AB - A patient having glioblastoma accompanied with calcification in the cervical spinal cord is presented. A calcifying lesion, detected on preoperative x-ray computed tomograms, was histologically confirmed as calcified areas in the tumor tissue. We discuss the difficulty in differentiating glioblastomas containing calcified masses from benign tumors with areas of calcification and present our hypothesis regarding the cause of calcification in the tumor tissue. PMID- 3014674 TI - Major liver resection: perioperative course and management. AB - This report investigates the perioperative course in 81 consecutive major liver resections, performed mainly because of primary liver cancer or colorectal liver secondaries. The liver was resected transabdominally with or without prior ligation of hilar structures. Intravenous nutrition consisted of 10% dextrose alone and was started preoperatively. Albumin or plasma was used rarely and only in conjunction with massive intraoperative transfusion of blood. Major complications, including four operative deaths (4.9%), consisted of bleeding and/or infection in eight (10%) patients and overt liver failure in two patients (2%) and occurred only after right and extended right lobectomies. Intraoperative blood loss was significantly larger in patients with postoperative complications than in patients with an uneventful postoperative course. The direct parenchymal approach was associated with a shorter operative time and an unchanged intraoperative bleeding. Coagulopathy and hypoalbuminemia did not cause any problems. Blood glucose levels were stable, and no patient suffered from hypoglycemia. It is concluded that major liver resection should be based on prevention of intraoperative bleeding and that preresection ligation of hilar structures offers no advantage in this respect. Infusion of hypocaloric glucose solutions should be started the day before operation, and routine administration of other nutrients does not seem necessary. PMID- 3014675 TI - The asymptomatic pancreatic islet cell tumor: a novel presentation. AB - Pancreatic islet cell tumors that secrete one or several polypeptide hormones have been suspected and diagnosed secondary to their systemic manifestations. This case report details the diagnosis and treatment of an 62-year-old man with a large pancreatic islet cell tumor without symptoms in whom the mass was found as a direct result of blunt trauma to the abdomen. The tumor contained high concentrations of both vasoactive intestinal polypeptide (VIP) and somatostatin. A discussion of VIP-containing tumors is included. PMID- 3014676 TI - [Vaginal contraception using the Today and Benzaltex tampons]. PMID- 3014672 TI - Thymic hormones--a clinical update. PMID- 3014677 TI - Synergistic actions of PAF-acether and sodium arachidonate in human platelet aggregation. 1. Studies in normal human platelet rich plasma. AB - The effect of sodium arachidonate and paf-acether in the activation of human platelet was studied. Concentrations of paf-acether which induced a reversible aggregation in normal human platelet rich plasma (0.029-0.0029 microM) and subthreshold concentrations of sodium arachidonate (0.25-0.35 mM), produced full aggregation when added together. Pre-exposition of platelets to paf-acether that renders them insensitive to paf-acether supresed the synergism. With full aggregation a markedly increase of thromboxane synthesis was detected by RIA. In vitro addition of aspirin (200 micrograms/ml) or indomethacin (12 microM) prevented aggregation and thromboxane formation by the joint action of sodium arachidonate plus paf-acether. Specific inhibition of 12-lipoxygenase by esculetin (10 microM) did not affect the synergistic action of paf-ace-ther and sodium arachidonate. These findings suggest that synergism between both agonists is mediated by active derivatives of arachidonic acid via cyclooxygenase. PMID- 3014678 TI - Cleavage site of calcium-dependent protease in human platelet membrane glycoprotein Ib. AB - Chicken muscle-derived m-type calcium-dependent protease cleaved purified glycoprotein Ib alpha-chain (GPIb alpha, Mr 130,000) from human platelets into two fragments (Mr 100,000 and Mr 38,000) in the presence of 5 mM calcium. With partially purified glycoprotein Ib (alpha beta-dimer), an appearance of a fragment of Mr 100,000 was also demonstrated after treatment with both the m-type and human platelet-derived mu-type protease. These processes in glycoprotein Ib were inhibited by inhibitors of calcium-dependent proteases, 50 muM E-64-C or 0.2 mM leupeptin and by the chelation of calcium. Using two-dimensional gel electrophoresis system, release of glycocalicin in addition to 100 kDa fragment was demonstrated by calcium-dependent proteases. Then surface-labeled platelets were stimulated with A23187 in the presence of 5mM calcium. Under this condition, endogenous calcium-dependent protease is activated. Of the labeled glycoproteins, glycocalicin and glycoprotein V but not 100 kDa fragment were released from the platelet membrane. The released glycocalicin was further digested into a fragment of Mr 100,000 by the addition of m-type calcium-dependent protease. These results showed (i) that GPIb alpha was hydrolyzed by exogenous calcium-dependent proteases in two points and glycocalicin and 100 kDa fragment were produced and (ii) that endogenous protease cleaved GPIb alpha at one point and released glycocalicin. PMID- 3014679 TI - Thrombin enhanced adhesion of platelets to von Willebrand factor substrates. AB - When exposed to thrombin, the adhesion of platelets to a von Willebrand factor (vWf) substrate, relative to a control substrate, is selectively increased. Adhesion to a vWf substrate is dependent upon the concentration of vWf, the duration of the adhesion assay, the concentration of thrombin, and the presence of divalent cations. The enhanced adhesion results from an action of thrombin on the platelets; no effect on the vWf substrate is involved. Once adherent to the substrate, the platelets undergo a profound change in morphology from the spiny sphere phenotype characteristic of activated platelets to a flattened and highly spread state. The adhesion of activated platelets to solid phase vWf is not inhibited by physiological concentrations of fibrinogen. PMID- 3014680 TI - Gamma ray-induced mutants as a tool for the production and characterisation of monoclonal antibodies against HLA-alloantigens. AB - To simplify the screening procedure for murine monoclonal antibodies specific for polymorphic HLA determinants, spleen cells from a mouse immunized with the human cell line BJAB-B95.8.6 were fused with NS1 mouse myeloma cells, and hybridoma supernatants were screened for their reactivity on BJAB-B95.8.6 and two gamma ray induced HLA-loss mutants of this line. The use of these HLA-loss mutants allowed the rapid identification of two new allospecific MOABs designated TU160 and TU161. Serological as well as biochemical studies revealed TU160 to be specific for HLA-A2, and TU161 for HLA-B13 molecules, respectively. Both MOABs were determined to be antibodies of the IgG class and were able to precipitate their antigens from lysates of radioactively labeled cells. PMID- 3014681 TI - Free lung cell response of mice and rats to mainstream cigarette smoke exposure. AB - Male C57BL mice and F-344 rats were exposed through nose only to fresh mainstream smoke from one University of Kentucky Reference cigarette (2R1) daily under standardized conditions. At different exposure points, the lungs of room control (RM), sham control (SH), and smoke-exposed (SM) animals were lavaged and the number, composition, and properties of bronchoalveolar lavage (BAL) cells were studied. Significantly elevated levels of blood COHb and pulmonary aryl hydrocarbon hydroxylase activity indicated effective inhalation of smoke by animals. The BAL cell analysis showed that cigarette smoke induced a five- to sevenfold increase in the number of BAL cells in mice following 10- to 12-week exposure. The proportion of neutrophils (PMN) increased to about 18 +/- 3% in SM mice as compared to less than 1% in controls. Cessation of smoke treatments returned the PMN levels to those of controls within 5 weeks. Unlike mice, smoke exposure for up to 32 weeks failed to induce appreciable changes in the number and proportion of macrophages and neutrophils in rats. Large brown macrophages were observed in SM groups of both species. Functional analysis demonstrated that the BAL cells from SM mice but not rats released greater amounts of superoxides than controls under resting and phagocytically stimulated conditions. Enzymatic analysis of macrophages showed that the activity of N-acetylglucosaminidase was increased in SM groups of both species. The activity of 5'nucleotidase was significantly reduced in macrophages from SM mice but not rats. Activity of leucine aminopeptidase remained unaltered in both species. These results demonstrate distinct differences in the response of mice and rats to identically generated cigarette smoke. PMID- 3014682 TI - Embryolethality of bromofenofos in rats. AB - Bromofenofos, an organophosphorus anthelmintic, was suspended in deionized water and administered once daily to pregnant rats by gastric intubation on days 8 through 15 of pregnancy in doses of 0, 2.5, 5, 10 and 20 mg/kg. The dams were killed on day 21 of pregnancy, and the number of implants, resorptions and live fetuses was counted. All fetuses were weighed and examined by routine teratological methods. As the results showed, this compound was highly embryolethal in the 20 mg/kg group; approx. 91% of the implants were resorbed at this dose level. Although none of the fetuses were externally malformed in any group, the incidence of skeletal and internal malformations was significantly increased in fetuses in the 20 mg/kg group. Skeletal malformations observed at this dose level were bipartite vertebral centra and wavy ribs. Internal malformations involved anophthalmia, hydronephrosis and hypoplasia of the uterus. PMID- 3014683 TI - Mobilization of nickel by potassium ethylxanthate in mice: comparison with sodium diethyldithiocarbamate and effect of intravenous versus oral administration. AB - Intravenous administration of 63Ni2+ (as 63NiCl2) together with potassium ethylxanthate resulted in highly increased levels of 63Ni2+ in several tissues of mice in comparison with animals given 63Ni2+ alone. However, this effect was not observed when 63Ni2+ and potassium ethylxanthate were given orally. Sodium diethyldithiocarbamate was active in increasing 63Ni2+ concentrations after both intravenous and oral administration. Both ethylxanthate and diethyldithiocarbamate can form highly lipophilic complexes with nickel and a facilitated penetration of these complexes through the cellular membranes of the tissues probably explains the increased uptake of the metal. Xanthates are unstable at acid pH and degradation in the acid milieu of the stomach probably underlies the lack of effect at oral administration. PMID- 3014684 TI - Exposure to smoke constituents by passive smoking. PMID- 3014685 TI - [Causes of sudden death in children with acute respiratory viral infections]. PMID- 3014686 TI - Assays of sulbactam in the presence of ampicillin. AB - Three assays for sulbactam, a beta-lactamase inhibitor, in serum in the presence of ampicillin were compared. A synergistic bioassay, a gas chromatographic assay with detection by mass spectrometry, and a high performance liquid chromatography assay were similar with respect to both accuracy and precision. PMID- 3014687 TI - In vitro and in vivo induction of terminal deoxynucleotidyl transferase activity in bone marrow cells by thymic humoral factors derived from a tumor cell of thymic epithelial origin. AB - Thymic epithelial tumor cell supernatant fraction 5 isolated from the cell culture medium of a tumor cell of thymic epithelial origin was tested for its ability to induce TdT expression. TdT activity could be induced in fractionated and unfractionated bone marrow cells from athymic nude rnu/rnu rats by supernatant fraction 5. Incubation with supernatant fraction of 5 rnu/rnu bone marrow cells isolated from the interface between 23% and 26% BSA restored the level of TdT activity to levels normally found in the thymus-bearing rnu/+ littermates. A does-related response was observed in vitro and in vivo in the expression of TdT. TdT induction in vitro was maximal with 5 micrograms/ml supernatant fraction 5 and in vivo with 200 micrograms supernatant fraction 5/rat/day. The effect was specific in that neither saline nor culture medium fraction 5 treated cells were induced to express TdT. The data presented in this study demonstrate that thymic epithelial tumor cell supernatant fraction 5 exerts an influence on the early maturation and differentiation of bone marrow stem cells. PMID- 3014688 TI - Isoenzyme variation in schizonts of Plasmodium vivax from Burma. AB - Fifteen isolates of blood containing Plasmodium vivax were collected from hospital in-patients in Rangoon and the schizonts were harvested for starch gel electrophoresis of the following parasite isoenzymes--glucose phosphate isomerase (GPI) (EC.5.3.1.9), NADP-dependent glutamate dehydrogenase (GDH) (EC.1.4.1.4), lactate dehydrogenase (LDH) (EC.1.1.1.27) and 6-phosphogluconate dehydrogenase (6PGD) (EC.1.1.1.43). Variation was found only in GPI. The other three isoenzymes appeared to be invariant in all the isolates. Forms of GPI were recorded in two isolates of P. vivax which differed from those reported for P. falciparum; these new forms were designated GPI-4 and GPI-5. PMID- 3014689 TI - Detection of an atypical rotavirus associated with diarrhoea in Chaco, Argentina. AB - Recently some unusual human and animal rotaviruses have been described which, although indistinguishable from standard rotaviruses by electron microscopy (EM), fail to react with antibody directed against the rotavirus group antigen. The genome of these viruses is composed of 11 double-stranded (ds) RNA segments; these RNA segments display different patterns when analysed by means of polyacrylamide gel electrophoresis (PAGE). The electrophoretic profile of the ds RNA segments of those human, antigenically different, viruses reported up to now appear to fit into two different patterns. Faecal samples from children with diarrhoea from different geographical areas of Argentina were evaluated for the presence of rotaviruses, using EM, ELISA and PAGE. During this survey a rotavirus like agent was detected in the faeces of a child with diarrhoea from Chaco province, North-East Argentine. The pattern of the ds RNA segment of this virus on PAGE analysis appeared to be related to one of the atypical rotaviruses. This is the first description of such a virus strain in Argentina. PMID- 3014690 TI - Rotavirus infection in wild marsupials (Didelphis marsupialis) of the Amazon region. AB - Rotavirus was detected by enzyme-linked immunosorbent assay in faecal specimens collected from two (1.35%) of 148 marsupials trapped in the Amazon jungle environment. The positive samples were both from the "common opossum", Didelphis marsupialis. No infections were found in the stools of 198 animals belonging to other mammalian species: the latter included small rodents, chiropterans and primates. Electron microscopic examination of one (MA 5928) rotavirus-positive specimen showed a large number of empty particles. However, both rotavirus strains grew when inoculated in MA 104 cells (foetal Rhesus monkey kidney cells) producing clear cytopathogenic effect; indirect immunofluorescence technique of these cells showed a typical granular cytoplasmic fluorescence. The electrophoretic profile of strain MA 5928 showed a high grade of homology with that of SA 11, but also showed minor differences. PMID- 3014691 TI - Antimalarial drugs and human neutrophil oxidative metabolism. AB - The effect of several commonly used antimalarial drugs on human peripheral blood neutrophil oxidative metabolism was studied. The following drugs were tested: chloroquine diphosphate, quinine HCl, mefloquine, proguanil HCl, cycloguanil, pyrimethamine, sulphadoxine, and tetracycline HCl. It was found that none of the antimalarial drugs examined, at clinically obtainable concentrations, had any inhibitory effect on neutrophil oxygen consumption, glucose oxidation, superoxide production, NBT reduction, and chemiluminescence. However, at higher concentrations chloroquine, quinine, mefloquine, and proguanil inhibited neutrophil oxidative burst. There was a slight enhancing effect on neutrophil oxidative metabolism by pyrimethamine, combination of pyrimethamine-sulphadoxine, cycloguanil and tetracycline at concentrations lower than the clinical levels. PMID- 3014692 TI - Elimination of infectious retroviruses during preparation of immunoglobulins. AB - Safety concerns for immunoglobulin preparations have led us to study partition/inactivation of two prototype retroviruses, mouse xenotropic type C and lymphadenopathy-associated virus (LAV) of the acquired immunodeficiency syndrome (AIDS), during manufacture and storage of immunoglobulins. Reduction of infectious retrovirus titers were 10(5) to 10(8)-fold through Cohn-Oncley cold ethanol fractionation from plasma to fraction II, 10(3) to 10(5)-fold through incubation at pH 4.0 and another 10(4)-fold through incubation of the purified liquid immunoglobulin preparations at 27 degrees C or 45 degrees C. The results support the clinical and epidemiological evidence that therapeutic immunoglobulin preparations do not transmit AIDS virus. PMID- 3014693 TI - Lack of seroconversion to human T-cell lymphotropic virus, type III, after intravenous immune globulin. PMID- 3014694 TI - Epstein-Barr virus infection and immunity in bone marrow transplant recipients. AB - Studies on patients for up to one year following allogeneic, HLA-matched bone marrow transplants have shown no increased incidence of salivary Epstein-Barr (EB) virus secretion and no significant rise in EB-virus-specific antibody titers. EB-virus-specific cytotoxic T cells could be detected in the peripheral blood of all patients by six months posttransplant. For up to one year posttransplantation in vitro EB virus infection of peripheral blood B lymphocytes from the majority of patients leads to an abortive infection followed by cell death, and without the establishment of continuously growing cell lines. This abnormality appeared to be due to patients' monocytes, which formed a defective feeder cell layer in culture, and it could be circumvented by culturing the EB virus-infected B cells from patients on a feeder layer of x-irradiated adherent cells from normal peripheral blood. These findings may explain the relative lack of EB-virus-associated lymphoma seen in bone marrow transplant recipients when compared with other groups of transplant patients. PMID- 3014695 TI - Marrow function reconstitution by fraction 3 of Percoll-density-gradient separated cells. AB - Eight children with advanced tumors or acute myeloid leukemia were treated either with very-high-dose multi-agent chemotherapy (4) or marrow ablative high-dose melphalan (4) followed by autologous marrow rescue, using only fraction 3 from a Percoll-discontinuous density gradient separation of bone marrow buffy coats. Each patient received less than 20% of the number of cells usually reinfused from the buffy coat. The yield of CFU-c in Percoll gradient 3 was similar to the yield obtained from whole buffy coats of bone marrow. Reconstitution of marrow function with a neutrophil count greater than 0.5 X 10(9)/L and platelet count greater than 50 X 10(9)/L occurred in 7 patients in a medium time of 15 and 16 days, respectively--a time comparable to that following infusion of whole buffy coat in 20 other patients. In one patient, hemopoietic recovery was considerably delayed, suggesting that fraction 3 from the Percoll gradient had been relatively ineffective in marrow reconstitution. We conclude that fraction 3 from marrow separated on Percoll gradients has the advantages of small volume and good recovery of marrow stem cells and can promptly reconstitute marrow function in the majority of children treated with very-high-dose chemotherapy or marrow ablative doses of melphalan. PMID- 3014696 TI - Effect of Isabgol and cellulose on the digestion and absorption of sucrose by everted sacs of adult hamster intestine. PMID- 3014697 TI - [Inorganic pyrophosphatase activity of the mouse spleen in the immune response and after treatment with bis-phosphonates]. AB - The inorganic pyrophosphatase activity was determined in different tissues of mice. The immunization of mice by sheep erythrocytes increased the inorganic pyrophosphatase activity of the spleen. The in vivo administration of bisphosphonates (40 mg per 1 g of mass), which are structural analogs of inorganic pyrophosphate (methylene bisphosphonic acid--MBPA, hydroxyethylidene bisphosphonic acid--HEBPA and aminomethylene bisphosphonic acid--AMBPA), inhibited the inorganic pyrophosphatase activity only by MBPA in the thymus and spleen but not in liver. The addition of MBPA, HEBPA as well as of phosphonoacetic acid, imidobisphosphate, bis(phosphonomethyl)-phosphonic acid, MBPA and phosphoric acid monoanhydride to cytosol from the mouse spleen led to the competitive (relative to the [Mg (PPi)2-] complex) inhibition of the inorganic pyrophosphatase activity. AMBPA didn't possess the analogous effect. PMID- 3014698 TI - [Superoxide dismutase activity of human plasma; the effect of Cu2+-complex compounds]. AB - The superoxide-dismutase (EC 1.15.1.1) activity was revealed in blood plasma after its successive treatment with alcohol, chloroform and one- or two substituted phosphate. Its value is the highest when treating plasma with KH2PO4. The boiling of the supernatant liquid obtained after addition of K2HPO4 leads to a complete loss of the activity. The treatment of plasma with KH2PO4 provides higher stability of the enzyme to thermal actions. The analysis of EPR-spectra has shown the presence of different complex compounds of copper ions in the supernatant liquid. Certain experiments with the use of aqueous solutions of CuCl2 and histidine have shown that Cu2+ inhibits the superoxide-dismutase activity in samples treated with KH2PO4 and increases it in samples treated with K2HPO4. PMID- 3014699 TI - [Isolation of liposomes containing serotonin and their effect on cAMP accumulation in the rat liver]. AB - To prolong the biological effect of serotonin the latter was enclosed into liposomes. The use of liposomes of phosphatidyl choline, cholesterol and dicetylphosphate in the 2:1:1 ratio permits increasing the level of serotonin incorporation inside the vesicles and reaching the slow release of serotonin out of them. It is shown by electron microscopy that sizes of liposomes enlarge due to the serotonin incorporation. When the liposomal form of serotonin is introduced to animals the amine effect on the cAMP accumulation in the liver is prolonged and intensified; an increase of the cAMP accumulation is observed in the prereplicative and replicative phases of hepatocytes synchronized by cycloheximide. The above mentioned permits a conclusion to be made that liposomes modify considerably the effect of serotonin on the cAMP system of the rat liver cells. PMID- 3014700 TI - Reversibility of oxygen-induced injury to the alveolar epithelium: an ultrastructural study. AB - Continuous exposure of rabbits to 100% O2 at one atmosphere (1 ATA) for 48 h damages the alveolar epithelium increasing its permeability to lipid insoluble molecules. The purpose of this study was to quantify the reversibility of this injury upon resumption of air breathing, using a cytochemical technique. Rabbits were exposed to 100% O2 at 1 ATA for either 48 or 63 h and then returned to air. Cytochrome C (Cyt) was instilled into the alveolar space and detected ultrastructurally in the different components of the blood gas barrier by its peroxidase activity. After 63 h in 100% O2 and 24 h in air, Cyt was present in the basal lamina of all instilled alveoli. After 33 or 48 h in air, there was focal replacement of type I by type II pneumocytes. Cytochrome C was identified in the basal lamina of 30% of the alveoli lined with type I but not type II cells. In contrast, after 48 h in O2 followed by 24 h of breathing air, cytochrome C was totally restricted in the alveolar space. Our studies indicate that the increased alveolar permeability of the lung to Cyt abates completely 24 h after return to air. At this time, though, the PaO2 was significantly lower than control, indicating the existence of residual pulmonary damage. In contrast, the damage to the alveolar epithelium after 63 h of continuous O2 breathing is only partially restored, even after 48 h of return to room air. PMID- 3014701 TI - Effect of dibutyryl cyclic adenosine monophosphate on canine pelviureteral discharge potentials. AB - An electromyographic study of the responses of the pelviureter to dibutyryl cyclic AMP was performed using isolated canine pelviureteral preparations. Dibutyryl cyclic AMP caused a marked increase in the amplitude of discharge potential in the pelvicalyceal pacemaker region and this was associated with a decrease in renal pelvic pressure. The frequency of the pacemaker discharge did not increase. On the other hand, in the lower pelvis and ureter, dibutyryl cyclic AMP caused a small but significant decrease in the amplitude of discharge potentials and this was associated with a marked decrease in discharge frequencies in the lower pelvis and ureter. PMID- 3014703 TI - [Treatment of ciliochoroidal melanomas with a medical narrow proton beam]. PMID- 3014702 TI - Acute hemorrhagic cystitis caused by adenovirus type 11 after renal transplantation. AB - A case of acute hemorrhagic cystitis caused by adenovirus type 11, which occurred in an allograft recipient 6 months after a living-related renal transplantation, is described. The patient lacked a neutralizing antibody to adenovirus type 11 before transplantation. Adenovirus type 11 was isolated from his urine and he developed a neutralizing and complement-fixing antibody to this virus. Although adenovirus type 11 isolates had been obtained from 2 of 18 renal allograft recipients, only 1 patient suffered acute hemorrhagic cystitis. Adenovirus type 11 may play a role in acute hemorrhagic cystitis in renal allograft recipients during immunosuppressive therapy. PMID- 3014704 TI - [Dynamics of passive humoral immunity transfer against Marek's disease in the progeny of the grandparent breeding stock of broilers]. AB - In the progeny of the grandparents of broiler type HYBRO, imported by the Dutch firm EURIBRID, neonatal humoral immunity was studied during the whole laying period, i.e. in laying hens at the age of 26-52 weeks. Precipitation test was used to examine 292 samples of sera from the parent chickens of sire stock AB and 299 samples of sera from the chickens of dam stock CD, for the presence of maternal antibodies to Marek's disease virus. The number of positive samples decreased with the growing age of the grandparent generation. In the sire stock it ranged from 41.7 to 82.6% (average number 57.5%); in the dam stock, it was from 50 to 90.9% (average number 71.6%). The average incidence of precipitation antibodies in the dam stock was significantly higher than in the sire stock (p less than 0.05). The relationship between laying hen age and percent incidence of passive antibodies in parent-generation chickens was characterized by a regression line and correlation coefficient. We discuss the problem that the incidence of heterologous passive antibodies in the produced parent-generation chickens can only minimally influence the onset of post-vaccination immunity after application of the Czechoslovak vaccine MARVAK, i.e. passive immunity in these chickens cannot be caused by insufficient prophylaxis in some of Czechoslovak multiplier flocks of broiler type. PMID- 3014705 TI - Evaluating toxin-induced hepatic injury in rats by laboratory results and discriminant analysis. AB - The ability of 14 serum biochemical assays to predict the presence of hepatic necrosis induced by carbon tetrachloride (CCl4) (centrilobular necrosis), allyl alcohol (periportal necrosis), and 1-napththylisothiocyanate (ANIT) (biliary duct necrosis) was evaluated in rats. Results of these assays were analyzed using multivariate discriminant analysis to determine: which assays have the highest predictive value for discriminating between control and treated rats, and which assays would discriminate between rats in the three treatment groups. Individual assays with the highest predictive value for CCl4-induced lesions versus controls were glutamate dehydrogenase (GDH), sorbitol dehydrogenase (SDH), and alanine aminotransferase (ALT). Assays with the highest predictive value for ANIT-induced lesions were GDH, 5'-nucleotidase (5'NT), and ALT. Assays the highest predictive value for ANIT-induced lesions were GDH, 5'-nucleotidase (5'NT), and ALT. Assays with the highest predictive value for allyl alcohol-induced lesions were an ALT/isocitrate dehydrogenase (ICD) ratio, GDH, and ALT. Canonical correlation coefficients for each assay ranged from 0.98 to 0.91 with 95-100% correct group membership predictions (treated versus control) provided by each assay. Individual assays were not highly predictive for determining group membership among all three treatment groups. A two assay combination of 5'NT and an ALT/ICD ratio provided 100% correct group membership predictions and had high canonical correlations (f1 = 0.95, f2 = 0.83). PMID- 3014707 TI - Pathology of Aujeszky's disease in mink. AB - Lesions in 21 mink which died of Aujeszky's disease included hemorrhages in lungs, heart, mediastinum, thymus, diaphragm, gastric wall, pancreas, and enteric wall. Microscopically, hyalin and fibrinoid degeneration and necrosis of vessel walls were present in cardiac muscle, brain, gastrointestinal wall and occasionally elsewhere in the body. Hemorrhages, exudation of plasma proteins and necrosis were associated with the angiopathy. Inflammation was minimal or absent. Other findings were congestion and extravasation of blood (lungs, liver), necrosis of lymphoid cells, and hemoglobinuric nephrosis. Aujeszky's disease virus was isolated from all but three animals. After experimental infection of three mink, similar though less pronounced lesions were found to those observed in the field cases. PMID- 3014706 TI - Sialodacryoadenitis virus-associated lesions in the lower respiratory tract of rats. AB - Eight- to 10-week-old outbred Wistar rats were inoculated intranasally with 10(2.9) medium mouse lethal infective doses of sialodacryoadenitis (SDA) virus. Sham inoculated control rats and challenged rats were killed at 1 day intervals for the first 8 days, then on days 10, 12, 14, and 20. Typical lesions associated with SDA were seen microscopically in the salivary and lacrimal glands of inoculated rats. In addition, laryngitis, tracheitis, bronchitis, bronchiolitis, and multifocal alveolitis were present during the acute stages of the disease. Viral antigen was demonstrated in epithelial cells lining airways by immunofluorescence microscopy. SDA virus was recovered from the lower respiratory tract from days 2 to 6 post-inoculation (PI). Serum antibodies to SDA virus, but not to Sendai virus or Mycoplasma pulmonis were present in rats tested at day 20 PI. These findings demonstrate that during the acute stages of the disease, significant lesions do occur in the lower respiratory tract of SDA virus-infected rats. PMID- 3014708 TI - Studies with an atypical avian rotavirus from pheasants. PMID- 3014709 TI - Involvement of spleen components of mature fowl in the primary and secondary humoral antibody response following experimental EDS'76 virus infection. AB - Cellular changes in spleens of mature fowl in relation to both the primary and secondary humoral antibody response following experimental EDS'76 virus infection were studied. The influence of splenectomy on humoral antibody response was also examined. Experimental fowl had been naturally infected with fowl adenovirus (FAV) but did not possess precipitins to these viruses at the time of EDS'76 virus infection. Since EDS'76 infection provokes a recall of the group antibody to FAV, this infection simultaneously induces a primary response against EDS'76 virus and a secondary response due to the recall of the group antibody to FAV. HI and precipitating antibody to EDS'76 virus (primary response) were first detected at 6 and 8 days p.i. respectively. Curves of HI, precipitating and neutralising antibody titres were biphasic; the first peak (IgM peak) occurred at 10-11 days p.i., the second (IgG peak) at 16-28 days p.i. Precipitating antibodies to FAV (secondary response) were demonstrated from 4 days p.i. The curve of these antibody titres was also biphasic, with peaks at the same times as in the primary response. Based on HI and AGP testing of primary and secondary immune response in both splenectomised and non-splenectomised fowl it is concluded that in the primary response the spleen of the adult fowl is involved significantly in only IgM secretion, while in the secondary response it is likely that both IgM and IgG are secreted in considerable amounts. Clusters of lymphoblasts and plasmablasts were observed at 3 days p.i. in the red pulp. It is very likely that antigen antibody complexes are formed from that time and circulate bound to the surface of lymphocytes. These antigen-loaded lymphocytes are 'picked up' from the blood stream by -red pulp macrophages, leading to enhanced formation of lymphoblasts in the red pulp. Great numbers of these cells (which are very probably IgM secreting cells) were present on days 6 and 7 p.i., but were no longer detectable after day 10 p.i. -macrophages of the macrophagal ellipsoidal corona (MEC), leading to significant enlargement of the periellipsoidal lymphoid tissue (PELT) by an increase of the number of lymphocytes observed from days 4-12 p.i. The MEC was significantly enlarged from 7-12 days p.i., very likely due to an increased number of macrophages. Following deposition of antigen in the white pulp, formation of follicles begins. The number of small, intact follicles including follicle precursors increased from 6 days p.i.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3014710 TI - The effect of replacement of 0.30% sodium chloride by 0.43% sodium bicarbonate in rations of fattening pigs on leg weakness, osteochondrosis and growth. AB - The effect of replacing dietary sodium chloride by sodium bicarbonate on leg weakness, osteochondrosis and growth in young fattening pigs was studied in two experiments using 104 and 126 animals. The experimental pigs were fed 0.43% dietary sodium bicarbonate, which replaced the sodium chloride (0.30%), was present in the diets of the control groups. It was found that the clinical symptoms of leg weakness could be improved significantly in the experimental group which received bicarbonate. No positive effects on osteochondrosis, however, could be shown. The treated animals even tended to have more severe osteochondral lesions. Reasons for the negative tendency are discussed. Daily weight gain and food conversion were not influenced by the experimental bicarbonate diet excluding a chloride deficiency. Differences in carcass grading were not significant, although barrows fed the NaHCO3-containing diet tended to score better, while the carcass quality of the experimental gilts was slightly less in comparison to the control animals. PMID- 3014711 TI - Enzyme linked immunosorbent assay used to monitor serum antibodies to bovine respiratory disease viruses. AB - An enzyme linked immunosorbent assay (ELISA) was applied to the detection of serum antibodies against infectious bovine rhinotracheitis (IBR), parainfluenza-3 (PI3), adenovirus type 3 (adeno 3) and bovine respiratory syncytial (BRS) viruses. Paired serum samples from calves vaccinated with live attenuated virus vaccines were tested. The ELISA compared favorably with the virus neutralization test for detecting serologic responses to IBR, BRS, and adeno 3 viruses or with the hemagglutination inhibition test for PI3 virus. The simplicity, sensitivity and rapidity of the ELISA test makes it a useful tool for immunological studies with respiratory viruses. PMID- 3014712 TI - Differentiation between virulent and avirulent strains of infectious laryngotracheitis virus by DNA:DNA hybridization using a cloned DNA marker. AB - The DNAs of virulent and avirulent strains of infectious laryngotracheitis virus (ILTV) showed greater than 96% homology by reciprocal DNA:DNA hybridization. Nevertheless, by use of selected restricted DNA fragments, it was possible to differentiate strains according to pathotype more readily than by the more laborious restriction enzyme analysis. Restricted DNA fragments were successfully cloned into Escherichia coli HB101 cells and could be used not only for pathotyping ILTV strains but also for their differentiation from other avian viruses. PMID- 3014713 TI - Foot-and-mouth disease virus (FMDV) experimental infection: susceptibility and immune response of adult mice. AB - Adult mice are susceptible to foot-and-mouth disease virus (FMDV) infection only under some experimental conditions. This paper report the results of pathogenesis studies on 4 different strains of mice (CF1, C3H, NIH-nude, BALB-c/J) infected with the cloned and uncloned 0(1)C strain of FMDV. High virus titers were detected in blood and pancreas 12-24 h after infection (p.i.); these persisted for up to 48 h p.i. in CF1 and BALB-c/J mice and 72 h p.i. in the two other mouse strains. Virus titers observed in other organs were lower than those found in blood. In pancreas, and occasionally in salivary glands, oropharynx, heart and testicles, viral antigen was detected by direct immunofluorescent assay. Circulating neutralizing antibodies appeared in CF1 and C3H mice at 72 and 96 h p.i. respectively, and their titers remained unchanged during the 30-day experimental period. Antibodies against viral infection-associated antigen (VIA) were detected for a shorter period. In animals irradiated with 1 LD 50 (total body irradiation), viremia persisted up to 14 days p.i. and a low antibody response was observed which began at the end of viremia. No differences in the response of mice to cloned or uncloned FMDV were observed. PMID- 3014714 TI - Prolonged excretion and failure of cross-protection between distinct serotypes of bovine rotavirus. AB - Four newborn calves were experimentally infected with two distinct serotypes of bovine rotavirus (BRV-1 and BRV-2). Initially, three colostrum-deprived calves were inoculated orally with either BRV-1 or BRV-2; all developed severe diarrhea and produced serotype-specific neutralizing antibodies. Fecal virus was first demonstrated by immunofluorescence the day after inoculation. The virus titers reached a maximum of 10(5.2)-10(6.6) fluorescent focus forming units g-1 of feces 2-5 days after inoculation and then decreased. Fecal virus was detected in low titers beyond 28 days after inoculation despite the development of serum neutralizing antibodies. One calf, which had acquired specific active immunity against BRV-1 following oral infection, was further infected orally with BRV-2 4 weeks later. The calf again manifested diarrhea, excreted BRV-2 and showed an increase in serum neutralizing antibody against BRV-2. These results indicated that calves infected with either BRV-1 or BRV-2 do not have cross-protection to infection with heterologous BRV, and that recurrence of the disease can occur. The possible mechanisms of the persistence of BRV in calves and its role in the epidemiology of this infection are discussed. PMID- 3014715 TI - In utero adenoviral infection of sheep. AB - The occurrence of natural in utero adenovirus infection in sheep was examined. Isolation of three adenovirus strains from the kidneys of 174 sheep foetuses is reported; all three isolates belonged to Type 2 bovine adenovirus (BAV 2) Subtype B. Five of 25 blood samples from sheep foetuses contained virus-neutralizing antibodies (1:4-1:24) against BAV 2. These data prove that transplacental transfer of adenovirus infection can occur naturally in sheep. PMID- 3014716 TI - [Rapid peroxidase test for demonstrating the bovine pestivirus in cell cultures]. AB - Different variants were tested of the immunoperoxidase method for the demonstration of the mucous disease-virus diarrhoea agent in infected cells with the use of a specific conjugated antiserum. It was found that the virus could be specifically demonstrated in infected cell cultures. The test proved rapid and readily applicable. PMID- 3014717 TI - [Experiments to culture the bovine pestivirus in heterologous cell cultures]. AB - Attempts were made to adapt a strain of the bovine pestivirus to cell cultures of swine kidney in a series of 15 passages. The adaptation of the virus was followed up through the production of a cytopathic effect, through comparative investigations, and by means of cytologic preparations. It was found that the cytopathic effect induced and the titer of the virus grew with increasing the number of the serial passages. Immunofluorescence and cytologic data showed the progressive development of the virus in cell cultures of swine kidney. It was also established that the time of incubation of the infected cultures to produce an overall cytopathic effect was shorter. PMID- 3014718 TI - Human chorionic gonadotropin in primary liver carcinoma in adults. An immunohistochemical study. AB - Production of human chorionic gonadotropin (hCG) by extragonadal tumours is not a rare phenomenon. In the liver, similar results have been reported in hepatoblastomas. The present study was attempted to survey hCG level in serum and hCG-immunoreactivity in primary liver carcinoma in adults. Although hCG was elevated in serum in 2 (22.2%) of 9 autopsied cases with hepatocellular carcinoma (HCC), the hCG-reactivity of carcinoma cells was found in 2 (2.1%) of 95 HCC cases. Carcinoma cells positive for immunoreactive hCG was found in 2 (15.4%) of 13 cases with cholangiocarcinoma (CC). The patients with hCG-immunoreactivity in carcinoma and/or elevated serum level of hCG failed to reveal distinct clinical and endocrinological disturbance due to excess hCG. The hCG-positive cells were focal within the carcinoma and showed poor histological differentiation in both HCC and CC, and there were no trophoblastic cells. It is suggested that hCG is one of the hormones produced by primary liver carcinoma in adults and can be localised immunohistochemically in a small number of poorly differentiated carcinoma cells. PMID- 3014719 TI - The effect of some asymmetric triazine derivatives on the in vitro formation of free superoxide radicals. AB - According to their chemical structure, asymmetric triazine derivatives have an activator or scavenger action in a superoxide radical generating system. Complexation with Cu2+ of one of the derivatives enhanced its scavenger activity. PMID- 3014720 TI - Insertion and deletion mutants of vaccinia virus. AB - Thirteen viable insertion mutants of vaccinia virus have been constructed. These mutants, containing coding sequences of the herpes simplex virus thymidine kinase (HSV-TK) gene, were generated by marker transfer via in vivo recombination. The mutants were identified using a replica filter plating technique by in situ hybridization using 32P-nick translated HSV-TK sequences and obtained as pure cultures by repeated plaque purification. Some of these insertion mutants were in turn used as substrates to generate viable deletion mutants of vaccinia virus in the presence of 5'-bromodeoxyuridine. An example of this approach resulting in a vaccinia virus deleted of approximately 1.5 kb of nonessential DNA is presented. Furthermore, the analysis of spontaneously occurring viable deletion mutants of vaccinia lacking approximately 21.4 kb of nonessential DNA is described. PMID- 3014721 TI - Bovine rotavirus maturation is a calcium-dependent process. AB - Bovine rotavirus-infected MA-104 cells maintained in the presence and absence of CaCl2 displayed cytopathic effects (cpe) distinct from each other. Lysates of calcium-free cultures were unable to induce cpe in subsequent passages in MA-104 cells, an observation reflected by the demonstration that virus titers of such lysates were drastically reduced. The minimum concentration of CaCl2 in the growth medium required to maintain maximum virus yield was determined to be approximately 0.17 mM. The period of calcium-dependency for infectious virus formation was between 6 and 12 hr postinfection at 37 degrees, a time corresponding to the entire log phase of virus growth. Viruses produced in the absence of calcium were found to be exclusively incomplete single-shelled particles (D particles), as determined by cesium chloride density gradient analysis, electron microscopy, and SDS-polyacrylamide gel electrophoresis. Subsequent examination of virus-specified proteins in infected cells revealed that there was a reduction in the level of the major outer capsid protein (42K) in the absence of calcium. Thus, total inhibition of mature virus production under this condition could be due to the combined effect of reduced production of the 42K protein and incomplete assembly of the virus. PMID- 3014722 TI - Tumorigenic poxviruses: analysis of viral DNA sequences implicated in the tumorigenicity of Shope fibroma virus and malignant rabbit virus. AB - The DNA sequence has been determined for a 7-kb region within the terminal inverted repeats (TIR) of Shope fibroma virus (SFV), a poxvirus which induces benign fibromas in rabbits. This region of the SFV TIR, which flanks the junction of the TIR with the unique internal sequences of the viral genome, had previously been shown to be also present in the genome of malignant rabbit virus (MRV), a hybrid poxvirus derived from a recombination event between SFV and a related leporipoxvirus, myxoma. Unlike SFV, the recombinant MRV induces an invasive profile of tumors in infected rabbits, but the capacity to induce proliferant fibromas appears to have been derived from SFV. These SFV DNA sequences have been analyzed and their genetic organization shows a unique tandem arrangement of three large open reading frames (ORFs) which share considerable homology with each other. Very short spacer sequences are present between the majority of ORFs, all of which are transcribed toward the terminal hairpins of SFV. Unusual dyad symmetries flank two of the most closely related ORFs and evidence is presented that one SFV ORF (T9-L) which maps precisely at the TIR/unique sequence boundary was truncated during transposition to the left terminus from a progenitor copy (T9-R) at the right terminus. The origin of these putative viral genes is considered in light of the recent observation (C. Upton and G. McFadden, 1986, Mol. Cell. Biol. 6, 265-276) that a subset of this region of the SFV genome is closely related to, and may have been originally derived from, an endogenous covalently closed circular plasmid species detected in uninfected rabbit cells. PMID- 3014723 TI - env genes of avian retroviruses: nucleotide sequence and molecular recombinants define host range determinants. AB - The env gene of avian sarcoma and leukosis retroviruses is allelic in the virus population permitting the virus to use different host cell receptors. This polymorphism has allowed the classification of these viruses into different subgroups. In order to understand further the role of viral sequences involved in determining this host range phenomenon, we constructed molecular recombinants between subgroup A, B, and E viruses and showed that the host range determinant defining subgroup specificity was located within a 1.1-kb region of the genome that included most of the coding region for the env gene product gp85. We also determined the nucleotide sequence of the region of the env gene encoding gp85 for virus isolates representing subgroup A and B viruses. We compared the predicted amino acid sequences of gp85 to themselves and to the previously published sequences of subgroup B, C, and E env genes. Based on these comparisons, we draw the following conclusions: Within the gp85 coding domain, there are four variable regions (VR-1 to VR-4) ranging in size from 9 to 52 amino acids. The variable regions are located in the same relative positions for each of the env gene alleles compared. The variable regions range in homology from 42% (A compared to B) to 57% (C compared to E) in pairwise comparisons; the flanking conserved domains are on average 95% homologous. The sequences of three different subgroup B virus isolates are highly homologous in both the conserved and variable regions. Secondary structure predictions suggest that gp85 is composed mostly of beta sheet topology. Hydrophilic loops within the variable regions may define sites of receptor interaction and binding sites for subgroup specific neutralizing antibodies. PMID- 3014724 TI - Transforming properties of a 15-kDa truncated Ad12 E1A gene product. AB - A mutant Ad12 E1A region (Ad12 R11E1A) was constructed, which directs the synthesis of only a 15-kDa N-terminal E1A product. When controlled by the SV40 early promoter plus enhancer region (SVR11E1A) this mutant E1A region is capable of immortalizing primary baby rat kidney (BRK) cells, showing that the information essential for immortalization is located in the N-terminal part of region E1A and is shared by the 13 S and 12 S mRNA gene products. This immortalization is thought to be an essential step in the process of oncogenic transformation. Primary BRK cells transformed by SVR11E1A in the presence of Ad12 E1B are nononcogenic. This implies that the E1A region also codes for activities required for oncogenicity. However, in the presence of an activated c-Ha-ras oncogene the SVR11E1A region can oncogenically transform primary BRK cells, showing that the c-Ha-ras oncogene not only can complement for the Ad12 E1B region, but also for the E1A function lost by the R11 deletion. PMID- 3014725 TI - Characterization of site-specific antibodies to the erbB gene product and EGF receptor: inhibition of tyrosine kinase activity. AB - Site-specific antibodies were generated against the erbB protein and epidermal growth factor (EGF) receptor by immunizing rabbits with a synthetic peptide corresponding to amino acid residues 285-296 of the predicted AEV-H erbB protein sequence. This peptide region lies within the tyrosine kinase domain of erbB and EGF receptor. Antibodies directed against this region readily identified native and denatured forms of the erbB gene product and EGF receptor as demonstrated by immuneprecipitation and immunoblot analysis. The anti-peptide antibody immuneprecipitated a functional EGF binding receptor molecule. Scatchard analysis demonstrated a KD for 125I-labeled EGF binding of 40 nM, a value consistent with that of detergent solubilized EGF receptor. Immuneprecipitates, though able to bind EGF, were unable to transfer phosphate from gamma-labeled ATP in a standard phosphorylation reaction. In detergent solubilized extracts of crude A431 microsomes, the anti-peptide antibody inhibited in a dose dependent manner the autophosphorylation of EGF receptor as well as receptor mediated phosphorylation of exogenously added substrates. In addition, this anti-peptide antibody reduced the overall level of tyrosine kinase activity present in microsomes prepared from AEV-transformed erythroblasts. This site-specific antisera should be useful for understanding the role of EGF receptor and erbB tyrosine kinase activity and their link with cell proliferation. PMID- 3014726 TI - Transactivation of BKV and SV40 early promoters by BKV and SV40 T-antigens. AB - The early promoters of BKV and SV40 plasmids were transactivated in both BKV and SV40-transformed cells which failed to support replication of these plasmids. This suggests that the T-antigen of either virus can transactivate BKV and SV40 early promoters by either increasing the availability of cellular transcription factors or by directly interacting with specific sequences which comprise the transcriptional control region of the early promoters. We also observed that removal of 8-bp on the early side of T-antigen binding site I of BKV does not alter viral-plasmid replication. PMID- 3014728 TI - Two-dimensional gel analyses of the 24-kDa cap binding protein from poliovirus infected and uninfected HeLa cells. AB - The 24-kDa cap binding protein (CBP) from uninfected, mock-infected and poliovirus-infected HeLa cell extracts was isolated by m7GTP affinity chromatography and examined by isoelectric focusing followed by SDS-PAGE. Two major species (pI 6.7 and 7.1) and two minor species (pI 6.5 and 6.8) were found in all cases. Preparations from postribosomal supernate (S200) and the supernate from 0.5 M KCl washed ribosomes (RSW) also demonstrated these same four species. We conclude that there are no detectable differences between ribosome-associated and soluble 24-kDa CBP and that the 24-kDa CBP is not detectably altered by poliovirus infection. We also report the presence of a previously undescribed 16 kDa polypeptide(s) doublet that copurifies with the 24-kDa CBP from uninfected, mock-infected and poliovirus-infected HeLa cells. PMID- 3014727 TI - Intracellular equine arteritis virus (EAV)-specific RNAs contain common sequences. AB - Equine arteritis virus (EAV) is a nonarthropod-borne togavirus. Six virus specific RNA species have been found in EAV-infected cells having the following molecular weights: 4.3 X 10(6) (RNA1), 1.3 X 10(6) (RNA2), 0.9 X 10(6) (RNA3), 0.7 X 10(6) (RNA4), 0.3 X 10(6) (RNA5), and 0.2 X 10(6) (RNA6). RNA1 comigrates with the viral genome (M. F. Van Berlo, M. C. Horzinek, and B. A. M. Van der Zeijst, 1982, Virology 118, 345-352). All RNAs hybridized with a radio-labeled cDNA probe representing RNA6, indicating that they contain common sequences. To study this homology in more detail, RNase T1 oligonucleotide fingerprinting of the RNAs was undertaken. This confirmed the presence of common sequences and showed more specifically that the intracellular viral RNAs form a nested set. The number of oligonucleotides in RNA1, however, is only one-third of the expected value. In all aspects studied the replication mechanism of EAV differs from that of other known positive-stranded RNA viruses. PMID- 3014729 TI - Moloney murine sarcoma virus encoded p37mos expressed in yeast has protein kinase activity. AB - We describe the expression of the Moloney murine sarcoma virus env-mos protein (p37mos) in yeast under the control of the yeast GAL1 promoter. Consistent with our previous results concerning the p37mos protein kinase made in virus infected mouse cells, p37mos produced in yeast possesses autophosphorylation activity in an immune complex kinase assay using anti-mos (37-35) serum. PMID- 3014730 TI - The effect of overproduction of nonstructural proteins on alphavirus plus-strand and minus-strand RNA synthesis. AB - We determined the effect of the overproduction of viral nonstructural proteins on alphavirus plus-strand and minus-strand RNA synthesis. Because alphavirus minus strand synthesis ceases normally at 3 to 4 hr postinfection and requires continuous protein synthesis [D. L. Sawicki and S. G. Sawicki, J. Virol. 34, 108 118 (1980); D. L. Sawicki, S. G. Sawicki, S. Keranen, and L. Kaariainen, J. Virol. 39, 348-358 (1981a)], we determined if the cessation of minus-strand synthesis was the result of the failure to continue synthesis of viral nonstructural proteins after 3-4 hr postinfection and if the overproduction of viral nonstructural proteins would increase the rate of plus-strand synthesis. Cells infected with ts1, an RNA-positive mutant of Semliki Forest virus (SFV) which overproduced the viral nonstructural proteins and underproduced the viral structural proteins at the nonpermissive temperature, did not cause the synthesis of increased amounts of viral minus strands relative to parental SFV and did not affect the time at which minus-strand synthesis ceased. All four viral nonstructural proteins were synthesized at early and late times after infection in the same relative proportions. The overproduction and the continued synthesis of nonstructural proteins late in infection did not increase the maximal rate of plus-strand synthesis above that in wild-type SFV-infected cells. PMID- 3014731 TI - Effect of fluoride on the phosphodiesterase of bovine photoreceptors. AB - In the absence of the specific hormone, fluoride is able to activate the adenylate cyclase because it interacts with the GTP-binding protein. It has been reported that fluoride activates also the phosphodiesterase of the light sensitive enzymatic cascade in dark-adapted retinal rod outer segments, but there is no indication that the GTP-binding protein is involved in this process or not. We show here that also in the photoreceptor system fluoride does interact with the GTP-binding protein in order to activate the phosphodiesterase in the dark. Further, we show evidences that fluoride solubilizes the GTP-binding protein in the dark and that the resulting complex activates the phosphodiesterase in dark adapted rod outer segment membranes. PMID- 3014732 TI - Immunochemical study of the cyclic nucleotide system of retinal photoreceptor membranes: antibodies raised to phosphodiesterase, its protein inhibitor and GTP binding proteins. AB - Monospecific precipitating antibodies raised to phosphodiesterase, its protein inhibitor and GTP-binding proteins of bovine retina photoreceptor membranes were obtained. The characterization of the antibodies was carried out by immunochemical methods and according to their functional properties as determined by their effect on enzyme activities. The antibodies were used to study the distribution of immunolike proteins in different animal retinas and to purify the inhibitor protein by the method of immunoaffinity chromatography. PMID- 3014733 TI - [Transport of cholecalciferol (vitamin D3) and its metabolites in the blood circulation. Plasma binding protein of vitamin D3 and its metabolites]. PMID- 3014734 TI - [Late neurologic consequence of triorthocresyl phosphate (TOCP)]. PMID- 3014735 TI - [Mechanism of Na,K-ATPase inhibition with gossypol and megasin]. AB - Kinetic patterns of megosin and gossypol effects on properties of highly purified Na+, K+-ATPase were studied. Inhibition of the enzymatic activity appears to occur due to interaction of these compounds with the enzyme probound to the intracellular side of the transport enzyme. PMID- 3014736 TI - [Heparin-induced impairment of thrombin interaction with fibrinogen and receptors of the anti-coagulation system]. AB - Administration of heparin (2 un) into rats with depression of the anticoagulation system before treatment of the animals with alpha-thrombin (8 NIH un) inhibited the enzyme interaction with blood fibrinogen, which was manifested as a distinct decrease in content of soluble fibrin in blood as compared with its concentration evaluated after the treatment with thrombin. Heparin inhibited the reaction of thrombin with specific receptors in vascular walls. The effector response of the anticoagulation system, which is specific for interaction of free alpha-thrombin with the cell wall receptors, was not observed if thrombin was administered intravenously together with heparin. The patterns of the anticoagulation system were not altered after administration of the equimolar complex of DIP (diisopropyl phosphoryl)-alpha-thrombin and heparin, although free DIP-alpha thrombin activated distinctly the anticoagulation system. The data obtained suggest that heparin, which inhibits partially the recognition site in thrombin molecule, impaired also the enzyme ability to bind to the specific receptors of vascular walls and therefore it impaired the distinct response of the anticoagulation system. PMID- 3014737 TI - [Various regulatory properties of fructose-1,6-diphosphatase of the rat liver under normal conditions and in experimental autoimmune cardiomyopathy]. AB - An increase in fructose-1,6-biphosphatase activity in liver tissue of rats with experimental autoimmune cardiomyopathy was observed. At certain concentrations of EDTA and Mg2+ the AMP-inhibition curves exhibited an intermediate plateau, which appear under different experimental, normal and pathological conditions. The occurrence of complex kinetic curves could be attributed to simultaneous existence of several enzymatic forms differing in the "structural" sites with tightly bound metals, which defines the differences in the kinetic cooperativity and sensitivity to AMP inhibition. PMID- 3014738 TI - [Comparative study of cyclic nucleotide phosphodiesterase activity in the rabbit and bovine heart]. AB - Activities of cyclic nucleotide phosphodiesterases (PDE) were studied in partially purified preparations from bovine and rabbit heart tissues. The PDE activity from rabbit heart was not increased in the course of purification and remained distinctly lower as compared with the enzymatic activity from bovine heart. Specific chelating agent Ca2+-EGTA (5 X 10(-4)M) inhibited effectively the PDE activity from bovine heart both in the ammonium sulfate fraction and in the fractions obtained by means of gel filtration on Sepharose 6B, but affected only slightly the enzyme activity from rabbit heart. The enzyme from bovine heart appears to involve mainly the Ca2+-calmodulin -dependent form, while the enzyme from rabbit heart, isolated under similar conditions, was Ca2+-calmodulin independent. Phenothiazine derivatives inhibited distinctly the PDE activity from bovine heart at 5 X 10(-5)M concentration, whereas they did not affect or affected only slightly the enzyme preparations from rabbit heart even at 1 X 10( 4)M concentration; the inhibition was of the calmodulin -unspecific type. Phenothiazine derivatives (inhibitors of the calmodulin activity) exhibited only slight effect on the Ca2+-calmodulin -independent PDE from rabbit heart. This enzyme could not be used in studies of drugs, the effect of which is realized via regulation of the enzymatic activity by means of Ca2+ and calmodulin. PMID- 3014740 TI - [Effect of enterosorption on EPR signals in the liver of rats of different ages]. AB - Liver EPR signals were studied in adult (8 months) and old (24 months) Wistar male rats after the 30 days course of enterosorption carried out daily. The signals were analyzed at the values of g = 1.94, 2.00, 2.25, 2.035, 2.16, 2.10 and 1.97. Age-dependent differences were found in liver EPR signals of intact animals, i. e. the intensity of signals of g = 1.97, 2.00, 1.97 and 2.25 was decreased in old animals. In adult animals enterosorption caused a decrease of signals of g = 2.25, 1.94, 2.00, 2.16 and 2.10. In old animals the treatment led to a dissimilar effect: the intensity of signals was decreased at the ranges of g = 2.25, distinctly increased at g = 1.97 and unaltered at the other EPR signal values studied. Geroprotective action of enterosorption is discussed considering the decrease in enzymatic activity of electron transport systems due to the treatment studied. PMID- 3014739 TI - [Removal of lipoprotein complexes of cholesterol from plasma using silochromes]. AB - Industrial silicates, silochromes, proved to be effective sorbents of cholesterol lipoprotein complexes in blood in vitro. Silochrome SH-1.5 with a diameter of pores of 180-200 nm and a capacity of 12-14 mg of cholesterol per 1 g of the sorbent exhibited the best efficiency in isolation of the complexes. At the same time, maximal unspecific binding of blood plasma proteins was observed in the systems with silochrome SH-3 with a diameter of pores of 60 nm. By use of silochrome SH-1.5 for plasmosorption the content of cholesterol was decreased from 3,600 mg/l down to 2,150 mg/l, i.e. by 40%, in blood plasma of a rabbit with experimental hypercholesterolemia. PMID- 3014741 TI - [Beta-endorphin and corticotropin in the blood of patients during the acute period of severe craniocerebral injury]. AB - An increase in content of beta-endorphin simultaneously with either low or high concentrations of corticotropin were observed in blood of patients with critical craniocerebral injury at the first day of the trauma. The content of beta endorphin was increased and the content of corticotropin was decreased in blood of the patients within one day after the surgical treatment of sub-, epi- and intracerebral hematomas. No correlation between contents of beta-endorphin and corticotropin was noted. The data obtained suggest existence of various pathways for regulation of beta-endorphin synthesis and secretion, which are independent from the corticotropin regulation processes. PMID- 3014742 TI - The use of DNA polymorphisms demonstrated by means of the HLA system. AB - The advances reached by using DNA polymorphisms (restriction fragment length polymorphisms) in blood group serology are demonstrated by means of the HLA system. The potentialities for various applications (e.g. better definition of alloantigens, association with diseases, paternity testing, forensic medicine or genetic counselling) are mentioned. PMID- 3014743 TI - Inactivation of the AIDS-causing retrovirus and other human viruses in antihemophilic plasma protein preparations by pasteurization. AB - Heat treatment at 60 degrees C for 10 h in solution (pasteurization) was introduced into the manufacturing process of antihemophilic cryoprecipitate (AHC) and factor VIII concentrates (F VIII) to reduce the risk of transmission of hepatitis to hemophiliacs. Since the acquired immunodeficiency syndrome (AIDS) may also be transmitted to hemophiliacs by antihemophilic plasma protein preparations, we have investigated inactivation of the AIDS virus HTLV III by pasteurization in AHC or F VIII and included in this study cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), poliovirus and vaccinia virus. Each of these viruses was efficiently inactivated by pasteurization although considerable differences were observed between the different viruses HTLV III was rapidly inactivated, becoming nondetectable within 30-60 min. Our findings indicate that pasteurized AHC or F VIII should have a high margin of safety regarding the transmission of AIDS or any other infectious disease caused by viruses such as those tested. PMID- 3014744 TI - Detection of adult T-cell leukemia virus (ATLV) bearing lymphocytes in concentrated red blood cells derived from ATL associated antibody (ATLA-Ab) positive donors. AB - Adult T cell leukemia associated antibody (ATLA-Ab) positive persons were screened by indirect immunofluorescence (IF) testing. Their lymphocytes were collected from concentrated red blood cells (CRC), and cultured in vitro with and without phytohemagglutinin (PHA) for 10 days. The expression of ATL virus (ATLV) positive lymphocytes during the in vitro culture was then analyzed by IF assay using mouse monoclonal antibody ATL-19 reactive to p19 core protein of ATLV. 97% of ATLA-Ab positive CRC (36 cases) demonstrated ATLV positive lymphocytes after being cultured for more than 10 days with PHA, whereas, none of ATLA-Ab negative CRC (22 cases) demonstrated ATLV positive lymphocytes. All of the 10 ATLA-Ab positive CRC that were stored for 2, 4, and 7 days contained lymphocytes which expressed ATLV after in vitro culture, while 7 of 10 CRC stored for 14 days and only 1 of 10 CRCs stored for 20 days, expressed ATLV positive lymphocytes. This data indicates that almost all of the ATLA-Ab positive blood contained ATLV positive lymphocytes, and that the in vitro appearance of these ATLV positive lymphocytes was reduced by storing the CRC for more than 14 days. PMID- 3014745 TI - Acquired loss of red-cell Wj antigen in a patient with Hodgkin's disease. AB - A patient with Hodgkin's disease became temporarily Wj-negative with alloanti-Wj in his serum. Four human autoantibodies, and 1 of 2 murine monoclonal antibodies, with serological characteristics of anti-Wj were nonreactive with his red cells, confirming that they have anti-Wj specificity. Six siblings of the patient are all Wj-positive. The patient was also temporarily Anton-negative, and cross testing between Wj and Anton red cells and antisera showed mutual compatibility, indicating that the antigens are the same. The patient and 3 of his 6 siblings are also of the rare Lu: - 13 phenotype, providing the first evidence that this is an inherited characteristic. PMID- 3014747 TI - [Epidemiological characteristics of non-A, non-B viral hepatitis with a fecal oral transmission mechanism]. AB - The analysis of verified cases of non-A-non-B hepatitis with the fecal-oral mode of transmission which had occurred in one of the districts of the Turkmen SSR, 1984-1985, revealed a this infection: an explosive nature of the incidence, number of epidemiological characteristics of this infection: an explosive nature of the incidence, even distribution in the territory of the district and within one residential area, predominant affection of 15-29-year-old subjects, high mortality among hepatitis-affected pregnant women, insignificant number of secondary cases in the families of index cases. The occurrence of these non-A-non B hepatitis cases was associated with water. The results of virological and serological studies ruled out the role of hepatitis A and B viruses in the etiology of the acute hepatitis cases occurring in the area. Anti-hepatitis A IgM was detected in the blood in only 3% of the patients in 1984 and in 2% in 1985, exclusively in young patients, and HBsAg in 11% and 9%, respectively. Immune electron microscopy revealed virus-like particles 27-30 nm in diameter in fecal extracts from the patients. The antigen of non-A-non-B hepatitis virus was detected in the first days of the jaundice period in feces of 14% of the patients in 1984 and in 11% in 1985 by an enzyme-immunoassay using a test developed at the Institute of Virology of the USSR Academy of Medical Sciences. PMID- 3014746 TI - [Electron microscopic research in non-A, non-B viral hepatitis with a fecal-oral mechanism of infection transmission]. AB - Fecal specimens collected in the early stage of the disease from hepatitis patients classified by serological, clinical, and epidemiological data as non-A non-B hepatitis were examined by means of cross immune electron microscopy. Extracts of feces from such patients were found to contain full, semi-empty, and empty virus particles without envelopes, 27-30 nm in size, forming immune complexes only in the presence of acute and convalescent sera from patients with this type of hepatitis. The lack of serological crosses with hepatitis A and non A-non-B viruses isolated in other regions suggests that this virus is antigenically distinct. The final solution requires further studies in which methods of molecular biology of viruses should be used. PMID- 3014748 TI - [Morphological characteristics of the rotaviruses and the structure of their genome]. AB - The data on human and animal rotavirus reproduction in monkey kidney cell culture, their morphology, and genome structure are presented. Both viruses, simian rotavirus SA 11 and human rotavirus K1, did not differ morphologically, in both cases two-capsid and one-capsid particles were found. The genome structure was determined by electrophoresis in 6% PAG. The electrophoretic mobility of genome fragments of both viruses was similar. PMID- 3014749 TI - [Rapid diagnosis of rotavirus infections by RNA electrophoresis on polyacrylamide gel]. AB - A method of rapid diagnosis of rotavirus infections is described. It is based on the isolation of viral RNA from the feces of patients with viral gastroenteritis and determination of RNA mobility by 6% polyacrylamide gel electrophoresis. It was demonstrated that rotavirus infection could be diagnosed after long-term storage of fecal specimens as well. The method is of interest for epidemiological and genetic studies of human rotavirus diseases. PMID- 3014751 TI - [Comparative study of the effectiveness of acycloguanosine and phosphonoacetic acid in herpetic infection in cell cultures]. AB - A comparative study of cytotoxicity and antiherpes activity of acycloguanosine (Acg) of the Soviet and American manufacture and a Soviet preparation of phosphonoacetic acid (PAA) for herpes simplex virus types 1 and 2 (HSV-1 and HSV 2) was carried out in primary and continuous cell cultures. The Acg preparation was shown not to be inferior in maximal tolerated concentration, minimal inhibiting concentration, and chemotherapeutic index (CTI) to American drug. Zovirax (Zvr), in relation to both herpes simplex virus types in primary and continuous cell lines. The CTI of Acg and Zvr was 25-30-fold higher than that of PAA for HSV-1 (Vero cells) and 80-fold higher in chick embryo fibroblast cultures (CEF), and 25-fold higher for HSV-2 in CEF. PMID- 3014750 TI - [Indices of lymphocyte functional activity in patients with recurrent cutaneous genital herpes during treatment with a herpes vaccine]. AB - The functional activity of lymphocytes of patients with severe forms of recurrent cutaneous-genital herpes was studied in the course of treatment with inactivated polyvalent herpes vaccine on the basis of lymphocyte blast-transformation response to polyclonal mitogens and herpes simplex virus antigen. In the group of patients who showed a marked therapeutic effect as a result of the treatment the initially high values of the antigen-dependent lymphocyte blast-transformation test declined and became normal. Thus, in some cases the herpes vaccine exerts an immunomodulating effect normalizing the functions of different lymphocyte subpopulations. PMID- 3014752 TI - [Characteristics of new loci of endogenous proviruses in chickens]. AB - The composition and structure of endogenous proviruses in the genome of Italian partridge chicken was studied by the blot-hybridization method using 32P-DNA of RAV-2 virus as a probe. Among 34 fowls examined, 5-contained no endogenous proviruses related to fowl leukemia viruses. DNA of the other fowls contained various combinations of 4 previously undescribed endogenous proviruses varying in structure and localization. None of them was identical in its structure with DNA of endogenous fowl virus RAV-0, all the four discovered loci have deletions. The origin, modes of genetic variability, and function of endogenous proviruses are discussed. PMID- 3014754 TI - [Isolation of the monkey pox virus from a wild African squirrel]. PMID- 3014753 TI - [Effect of antioxidants on the HeLa cell infection process in culture]. AB - A certain role in the outcome of virus infection of cells was assumed to belong to cell membrane lipids peroxidation. The influence of known inhibitors of lipid peroxidation, beta-naphthol and ionol, on HeLa cells infection with human adenovirus type 2 was studied. These antioxidants in certain concentrations were found to be capable of effectively inhibiting virus infection of HeLa cell cultures. At the same time, beyond these concentration levels these antioxidants did not inhibit but stimulated virus infection of HeLa cell cultures. It is concluded that inhibition of virus infection of cells by antioxidants in moderate concentrations is due to their blocking of membrane lipids peroxidation. The opposite effect of antioxidants in much higher or lower concentrations may be due to their membranotropic effect which enhances the development of virus infection. The results of the study attest to the possible use of antioxidants in appropriate concentrations for control of virus infection. PMID- 3014755 TI - [Fluorescent immunoglobulins to the herpes simplex virus from rat immune ascitic fluids]. PMID- 3014756 TI - Glucagonoma--an underdiagnosed syndrome? PMID- 3014757 TI - [Etiopathogenesis and treatment of Alzheimer's disease]. PMID- 3014758 TI - [Clinical analysis of serous and suppurative cerebrospinal meningitis]. PMID- 3014759 TI - [Ophthalmologic after care of premature infants]. AB - Preterm infants, owing to their relative immaturity of organs and tissues show an increased vulnerability to exogenous factors such as oxygen, which may be required as a life-saving measure. In certain cases this may lead to retinopathy of prematurity and even blindness in the most severe cases. Regular ophthalmological follow up of at-risk infants is, therefore, essential to avoid irreparable retinal damage. From 1.8. 1983 till 31.7. 1985 159 at-risk infants were investigated ophthalmologically by direct and indirect ophthalmoscopy. 14 cases (8.8%) of stage I/II and one case (0.6%) of terminal stage retinopathy of prematurity were observed. In 34 children (21.4%) other changes of lesser (dacryostenosis) or greater pathological severity (hydrophthalmus, disc abnormalities, manifest squint) were seen. Initial results in preterm infants with an improved ophthalmoscopic device fur examining the temporal periphery of the fundus, the site of predilection for early retinopathic changes, are reported. PMID- 3014760 TI - Parathyroid hormone, prostaglandins and bone resorption. PMID- 3014761 TI - [Nutritional behavior of insulin-dependent diabetes patients studied with the KALI 2.1.2 computer program]. AB - We investigated the levels of intake of essential nutrients in 51 patients with insulin dependent diabetes mellitus. The mean daily intake in males was 223 +/- 55 g carbohydrates, 111 +/- 22 g fat and 112 +/- 27 g protein. In females the mean daily intake was 166 +/- 36 g carbohydrates, 78 +/- 23 g fat and 97 +/- 25 g protein. the percentage of calories from carbohydrates, fat and protein was for males 36:40:18 and for females 37:39:21 respectively. The daily intake of dietary fiber was 32.5 +/- 8.0 g in men and 28.3 +/- 6.4 g in women. The mean linoleic acid intake was 12 +/- 5 g/day in men and 9 +/- 4 g in women. A marginal deficiency of linoleic acid was found in 15% of men and in 25% of women. The P/S ratio of the diet was 0.35. The consumption of vitamins, minerals and trace elements differed considerably from the recommended dietary allowances. PMID- 3014762 TI - [The effect of cellulose on endogenous nitrogen excretion in rats, evaluated using N-15 technics]. AB - Metabolic faecal nitrogen excretion was assessed in 8 young rats (90-100 g body weight) following an oral application of 75 mg 15N-glycine (95 atom-% 15N). Four rats received an experimental diet containing 12% cellulose, while a control diet containing 4% cellulose was fed to the remaining 4 animals. The high cellulose content induced a highly significant reduction of the N balance due to a greatly increased urinary N excretion. The metabolic faecal nitrogen was determined by measuring 15N excretion in faeces and urine from day 5 (3) to day 8 following 15N glycine administration. During this time interval 15N elimination follows an exponential curve. Increasing the dietary cellulose content from 4 to 12% produced a rise in metabolic faecal nitrogen from 13.9 to 15.7 mg/day and in total faecal nitrogen from 21.3 to 24.4 mg/day. From these values a mean true protein digestibility of 98% was calculated for both groups of rats, regardless of the difference in dietary cellulose content. The fraction of endogenous faecal nitrogen which is of bacterial origin was determined through the analysis of 2, 6 diamino-pimelic acid (DAP). The added cellulose in the experimental diet caused a rise in faecal DAP from 0.302 to 0.402 mg/day corresponding to an increase of bacterial nitrogen from 5.2 to 6.4 mg/day. Accordingly the observed rise in endogenous faecal nitrogen is largely due to increased bacterial nitrogen. PMID- 3014763 TI - [Use of roentgen diffractometry and infrared spectroscopy in the analysis of suspended dust samples for fractions of crystalline silicon dioxide]. PMID- 3014764 TI - [Propagation and detection of human rotavirus (Wa strain) in cell cultures of the line MA 104]. PMID- 3014765 TI - [Comparative studies of the detection of rotavirus antigen with electron microscopy, countercurrent electrophoresis and enzyme immunoassay]. PMID- 3014767 TI - On the problems of clear vesicles in sensory corpuscles. PMID- 3014766 TI - [Gastrointestinal involvement in acquired immunologic deficiency syndrome (AIDS)]. AB - The gastrointestinal tract is a major target organ of the acquired immunodeficiency syndrome. Opportunistic infections or Kaposi's sarcoma within the gastrointestinal tract are the two most frequent lesions. Diarrhoea, weight loss or odynophagy may be the presenting symptoms or signs for which a gastroenterological consultation is sought. In this report we present our own observations of patients with AIDS. Diagnostic and therapeutic aspects of this new syndrome are discussed. PMID- 3014768 TI - Glycogen bodies in ciliated cells of the rabbit endometrium. PMID- 3014769 TI - RNA fingerprinting of South American prototype aphthovirus strains. AB - Aphthovirus strains used in South America for vaccine production or as reference for diagnostic purposes were analysed by RNA fingerprinting (RNase T1 maps, one- and two-dimensional gels). The results obtained constitute the basis for a data bank containing available information about the genome structure of strains of aphthovirus prevalent in this continent and can be used as an adjunct to serological and immunological information. These data are currently being used in South American countries to assess the genetic stability of strains during vaccine production; to establish possible vaccine origin of field outbreaks and to monitor the origin, behaviour and fate of new strains in the field. PMID- 3014770 TI - Serological evaluation of poliomyelitis oral and inactivated vaccines in an urban low-income population at Rio de Janeiro, Brazil. AB - Oral and inactivated poliomyelitis vaccines (OPV and IPV), were given to 160 children two months old, in a low income population at Rio de Janeiro. The vaccination was repeated 2 and 4 months later, always in association with diphtheria, tetanus and pertussis (DPT) vaccine. Blood specimens were collected before vaccination at the time of the third dose of vaccine and later at the time of measles vaccination, when the children were nine months old. The serological response to two doses of IPV showed high titres of antibody in all but one child and 100% conversion after three doses. Although poliomyelitis has been controlled in Brazil by the use of OPV in large mass campaigns, the results obtained with IPV support the possibility of its use in the basic immunization schedule, providing lower costs could be achieved for the inactivated vaccine. PMID- 3014771 TI - Live varicella vaccine: the third act? PMID- 3014772 TI - Vaccines made from recombinant yeast cells. AB - Production of polypeptide and protein antigens through recombinant DNA technology in prokaryotic and certain eukaryotic cells in culture is facilitating the development of new vaccines that are safe, efficacious, and economically feasible to manufacture. A current example is that of human hepatitis B vaccine that, to the present, has been produced commercially using hepatitis B viral surface antigen (HBsAg) purified from the plasma of human carriers chronically infected with the virus. Production of plasma-derived vaccine is limited by the available supply of suitable carrier plasma and by the need to apply highly technical procedures to purify the antigen as well as to ensure inactivation of all infectious agents that might be present in human plasma. PMID- 3014773 TI - Early interactions between animal viruses and the host cell: relevance to viral vaccines. AB - Viral recognition of specific receptors in the host cell plasma membrane is the first step in virus infection. Attachment is followed by a redistribution or capping of virus particles on the cell surface which may play a role in the uptake process. Certain viruses penetrate the plasma membrane directly but many, both enveloped and non-enveloped viruses, are endocytosed at coated pits and subsequently pass into endosomes. The low pH environment of the endosome facilitates passage of the viral genome into the cytoplasm. For some viruses the mechanism of membrane penetration is now known to be linked to a pH-mediated conformational change in external virion proteins. As a consequence of infection there are alterations in the permeability of the plasma membrane which may contribute to cellular damage. Recent advances in the understanding of these processes are reviewed and their relevance to the development of new strategies for vaccines emphasised. PMID- 3014774 TI - Adjuvanticity of stearyl tyrosine on inactivated poliovirus vaccine. AB - Preliminary studies of some of the properties of stearyl tyrosine have shown that it is non-toxic, free of adverse reactions at the sites of injection, non pyrogenic, stable upon storage and easy to sterilize. Formalin inactivated poliovirus vaccine (IPV) adjuvanted with stearyl tyrosine hydrochloride induced significantly higher titres of antibodies in non-human primates, after two injections, than the non-adjuvanted vaccine. Furthermore, the adjuvanted vaccine, even when diluted 1:4, showed consistently higher antibody titres as well as a longer persistence of antibodies than the non-adjuvanted undiluted vaccine. These studies suggest that stearyl tyrosine is an excellent and cost effective adjuvant for IPV. Hence further investigation with this novel synthetic compound would be worthwhile to ascertain its adjuvanticity for IPV in human subjects. PMID- 3014775 TI - [Comparative evaluation of the combined chemotherapy and chemoradiation therapy of patients with a localized form of small-cell lung cancer]. PMID- 3014777 TI - [Detection of herpes virus in gonococci (electron microscopic study)]. PMID- 3014776 TI - [Carcinoembryonic antigen as an indicator of the effectiveness of the chemotherapy of malignant tumors]. PMID- 3014778 TI - [Klippel-Trenaunay syndrome]. PMID- 3014779 TI - Isolation and characterization of temperature sensitive mutants of Broadhaven virus, a Kemerovo group orbivirus (family, Reoviridae). AB - Eighteen stable temperature sensitive (ts) mutants of Broadhaven virus were isolated without the aid of mutagens. Spontaneous mutants were detected using 41 degrees C as the nonpermissive temperature and 36 degrees C as the permissive temperature. High frequency pairwise recombination defined five recombination groups. Four mutants belonged to group I, three to group II, six to group III, two to group IV, and two to group V. ts 7 was a possible double mutant representing lesions corresponding to those of groups III and V mutants. This is the first reported isolation of temperature sensitive mutants of a tick-borne orbivirus. PMID- 3014780 TI - Replication of varicella zoster virus in Raji cells. AB - This report provides evidence for the replication of varicella zoster virus (VZV) in Raji cells. Infection was achieved by co-cultivation of Raji cells with VZV infected human fibroblasts. Replication of VZV, as assessed by immunofluorescence using monoclonal antibodies against VZV-glycoproteins, ranged from 18 to 24% of the cells. Electron microscopy detected complete virions within the membrane bound cytoplasmic vesicles and free viral particles in the nuclear matrix as late as 12 days post-infection. Western blot analysis of infected Raji cells demonstrated VZV-specific glycoproteins. The availability of a VZV-susceptible cell line growing in suspension culture provides a useful model for future studies. PMID- 3014783 TI - Sequences in the proximal IRL of herpes simplex virus DNA hybridize to human DNA. AB - A short segment of the herpes simplex virus (HSV) DNA proximal IRL region hybridized to two doublet bands of EcoRI-digested human cellular DNA. The hybridization was abolished neither by increasing stringency conditions nor by inclusion of guanine-rich DNA in the hybridization mixture. Thus, the hybridization appeared to represent authentic base sequence homology between HSV DNA and middle repetitive human DNA. PMID- 3014782 TI - Genome analysis of human rotaviruses by oligonucleotide mapping of isolated RNA segments. AB - The genomes of adult diarrhoea rotaviruses isolated in different parts of China during winter outbreaks in 1983 and 1984 were compared by segmental oligonucleotide (ON) mapping. The RNA profiles of most of the isolates were indistinguishable but it was found that some corresponding RNA segments had identical or very closely related ON maps whereas others differed considerably. This finding can be taken to suggest that the strains compared may be genetically related by a natural reassortment event. The genomes of cocirculating group A rotaviruses isolated in Scotland during winter outbreaks in 1981/82 were also compared. The ON maps of corresponding RNA segment differed extensively irrespective of whether or not the segments comigrated on gels. PMID- 3014781 TI - Structural components of bovine leukemia virus: further biochemical and immunological characterization of major structural proteins and glycoproteins. AB - Proteins from bovine leukemia virus (BLV) were investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the presence and absence of 14C amino acids. Virus grown either in fetal lamb kidney (FLK) or bat lung (BL) cells revealed seven major proteins designated p10, p12, p15(1), p15(2), p24, gp30, and gp64. By tryptic peptide analysis, N-terminal amino acid analysis and radioimmunoassay it could be shown that p10, a basic protein with hydrophobic properties, is a cleavage product of p15(2), the acidic and slightly hydrophobic phosphoprotein of BLV. The structural protein p12 was shown to be a phosphorylated protein identifying it as a second major viral protein to contain phosphorous. Investigation of [3H]glucosamine incorporation, N-terminal amino acid analysis and hydrophobic properties by chloroform-methanol extraction confirmed properties of gp30 and gp64 predicted by nucleotide sequence data. The two p15 proteins have been characterized in more detail. PMID- 3014784 TI - The endogenous mouse mammary tumour virus locus Mtv-8 contains a defective envelope gene. AB - The Mtv-8 associated provirus, GR40, is not expressed in vivo. However, upon transfection into rat XC cells, the two MMTV specific mRNAs of 35S and 24S are transcribed. Further, the level of these transcripts is augmented when the transfected cells are grown in the presence of dexamethasone (Ponta et al., 1983). No virus can be detected in the medium of the transfected cells. Intracellular protein analysis of these transfected cells shows that although apparently authentic gag proteins are synthesized, a novel protein of 68 kDa is the only env related protein detectable. The sequence of the env gene of GR40 was determined and the predicted amino acid sequence of the env protein obtained. A premature termination codon is present 68 amino acids before the COOH-terminus of the known MMTV env precursor Pr73env. This would result in an env protein of about 68 kDa, in agreement with the size of the env protein found in GR40 transfected cells. The lack of the carboxy terminus may be responsible for the non-processing of the aberrant env precursor protein, p68. PMID- 3014785 TI - Bronchiolo-alveolar carcinoma with nuclear tubular inclusions. Case report and electron microscopy findings. AB - A case of bronchiolo-alveolar carcinoma with the ultrastructural characteristics of type II pneumocytes is presented. Tubular type nuclear inclusions were frequent. Two types of inclusions were seen: a common uncircumscribed type and a chromatin or cisterna demarcated type which resembled cytoplasmic pseudoinclusions. Both types, however, appear to be variants of the same lesion since intermediate or transitional forms can be observed. Inclusions of the same type were seen in cells from extrapulmonary metastases from this carcinoma. This excludes the possibility that cells with inclusions represented trapped non neoplastic type II pneumocytes. PMID- 3014786 TI - [Diagnostic problems and incidence distribution in primary lung tumors]. AB - Reasons for discrepancies in histopathologic findings in an international study are explored in relation to the author's own analysis of 4,485 operative or post mortem specimens. A frequency distribution of these tumours is presented according to the WHO-classification system. PMID- 3014788 TI - Occurrence and isolation in tissue culture of equine rotaviruses. PMID- 3014787 TI - [Modification of cAMP concentration of the human sperm by low temperature preservation and sperm preparation for in vitro fertilization of oocytes]. AB - The cryopreservation was accompanied by a significant decrease of the concentration of cAMP of human spermatozoa. The process of isolation of spermatozoa with the aim of the fertilization of oocytes hardly influenced the cAMP-concentration. Immediately following incubation of the spermatozoa in a basic capacitation medium led to an increase of the level of cAMP in spermatozoa. Significant differences were detectable comparing the concentration of cAMP after incubation in presence and absence of albumin. PMID- 3014790 TI - Stomach rupture following ingestion of sodium bicarbonate. AB - Spontaneous rupture of the stomach is an uncommon condition with a usually poor prognosis. The rupture occurs as a result of a closed loop obstruction with increased pressure against the stomach wall. A case of stomach rupture occurring after hyperdistention of the stomach following ingestion of sodium bicarbonate is described and the pathophysiological mechanism is discussed. PMID- 3014789 TI - [Enhanced pathenogicity of chicken anemia agent (CAA) in dual infections with Marek's disease virus (MDV), infectious bursal disease virus (IBDV) or reticuloendotheliosis virus (REV)]. PMID- 3014791 TI - Effect of an alpha-blocking agent (phenoxybenzamine) in the management of premature ejaculation. AB - Phenoxybenzamine has been extremely effective in treating patients with vesical dysfunctions, its minimal side effects include anejaculation and delay and difficulty in ejaculation. Fifteen men were treated with phenoxybenzamine (PBZ) for premature ejaculation. Eight patients reported a subjective improvement of the time from penetration to ejaculation. PBZ is a well tolerated drug. The only side effect of the treatment was dry ejaculation. The Authors suggest its use in selected patients. PMID- 3014792 TI - New vitamin K-dependent plasma proteins, protein C and protein S. PMID- 3014793 TI - Structure and activation of protein C. PMID- 3014794 TI - First case in Italy of fatal AIDS in a hemophiliac. AB - The first fatal case of AIDS in an hemophiliac observed in Italy is reported. The propositus is a 53-year-old hemophilia A patient who died on the 8th December, 1984. AIDS was documented clinically and in the laboratory by serum antibodies to HTLV-III detected by ELISA and Western blot assays. A progressive intellectual worsening of the patient due to diffuse cerebral atrophy was followed by CT scan, EEG and by evaluation of proper neurological signs and symptoms. PMID- 3014795 TI - Variant translocation t(7;922) during lymphoid blastic crisis in a case of Ph1 positive chronic myelogenous leukemia with previous EBV infection. AB - We report the case of a 19-year-old girl in whom a Ph1-positive chronic myeloid leukemia was diagnosed 10 months after an episode of infectious mononucleosis. Clinical and cytogenetical follow-up demonstrated the appearance of a variant Philadelphia chromosome translocation t(7;9;22) during a blastic crisis characterized by early B-lymphoid elements. The association between the t(7;9;22) variant Philadelphia and the lymphoid blast cells is emphasized, and the importance of Epstein-Barr virus (EBV) infection in the pathogenesis of the disease in this case is discussed. PMID- 3014796 TI - [Immunological study of adult T-cell leukemia-lymphoma cells--special reference to the effect on the proliferative response of normal human T-lymphocytes]. PMID- 3014797 TI - Health consequences of anger and implications for treatment. PMID- 3014798 TI - Sensory and motor peripheral neuropathy in olivopontocerebellar atrophy. AB - We report the findings of an electrophysiological study in 9 patients affected by olivopontocerebellar atrophy, 4 with a dominant form and 5 with a sporadic form. Superficial peroneal nerve biopsy was obtained from 2 patients. The electrophysiological alterations were signs of collateral reinnervation and loss of motor units, decrease in sensory potential amplitude and increase in distal motor latency. Only a slight reduction in motor and sensory conduction velocity was observed in some cases. Nerve biopsy showed slight reduction of the number of myelinated fibres. In the first case, fibre diameter distribution was unimodal, due to reduction of myelinated fibres of large diameter, in the second case there was no significant alteration of the fibre distribution. In both cases short internodes were present with no signs of segmental demyelination, remyelination or axonal degeneration. The alterations observed in the peripheral nervous system are probably secondary to a lesion of the posterior root ganglion and the anterior horn cell in the spinal cord. PMID- 3014799 TI - Binding of serum IgA of multiple myeloma to normal peripheral nerve. AB - Serum from a 60-year-old man with multiple myeloma (monoclonal IgA-lambda) and peripheral sensorimotor neuropathy showed immunohistochemical binding to normal human endoneurium in dilutions up to 1:6000 (IgA = 1 mg/dl). The binding was shown to be specific for IgA-lambda and it was negative for IgA-kappa, IgG and IgM. Absorption of the serum with peripheral myelin eliminated the myelin immunostaining. Peptic digestion of the serum failed to eliminate immunostaining of the endoneurium. Immunoblot analysis revealed reactivity of the monoclonal IgA with 58,000, 43,000 and 18,500 dalton components of human nerve endoneurium. Sera from two patients with monoclonal IgA without peripheral neuropathy, from one patient with peripheral neuropathy and lung cancer, from one patient with stroke and from six normal subjects failed to stain myelin in sections of peripheral nerves or to react in immunoblots at comparable serum concentrations. Axonal staining was seen with some of the control sera. The relationship of the findings to the pathogenesis of peripheral neuropathy in multiple myeloma is discussed. PMID- 3014800 TI - Dopamine-GABA interactions: evidence that GABA transmits, modulates and mediates dopaminergic functions in the basal ganglia and the limbic system. PMID- 3014801 TI - Immunohistochemical demonstration of viral antigens in Japanese encephalitis. AB - Japanese encephalitis virus antigens were immunohistochemically demonstrated in formalin-fixed paraffin sections from an autopsied brain. Glial nodules were always associated with antigen-positive cell debris. Glia shrubs in the cerebellar cortex appeared to be formed along the apical dendrite of Purkinje cells. Most, but not all, of the neurons involved in neuronophagia were viral antigen positive. Antigenic masses were occasionally encountered in the center of so-called acellular plaques. Neurons with strong viral antigens were sporadically found in normal-appearing regions in the thalamus and cerebral cortex. Viral antigens were demonstrable only in neurons and not in glial or vascular endothelial cells. PMID- 3014802 TI - Influence of clomiphene citrate in vivo on subsequent steroid formation and gonadotropic responsiveness in vitro of isolated human follicular cells. AB - Granulosa and thecal cells from human preovulatory follicles, extirpated from the ovaries of women undergoing sterilization by minilaparotomy, were incubated for 2 hours in the presence or in the absence of human gonadotropin (hCG). The follicles were obtained from 16 women, 8 of whom had previously been treated with clomiphene citrate (CC) to induce follicular maturation. The tissue contents of cyclic AMP (cAMP) and the content of progesterone (P), testosterone (T) and estradiol-17 beta (E2) in the incubation medium were measured. Granulosa and thecal cells from control and CC-induced follicles all responded to hCG in vitro with increased formation of cAMP. Both granulosa and thecal cells from CC-induced follicles produced larger amounts of P in vitro than cells from spontaneously matured follicles. There were no other differences in the patterns of steroid formation or reactivity to hCG between the cells from the two types of follicle. It is concluded that CC treatment in vivo induces the growth and maturation of preovulatory follicles with apparently normal biochemical characteristics, as far as the content of and capacity to form cyclic AMP, progesterone, testosterone and estradiol-17 beta are concerned. PMID- 3014803 TI - Natural killer (NK) cells with HNK-1 phenotype in the cervical biopsies of women followed-up for human papillomavirus (HPV) lesions. AB - Local immunocompetent cell infiltrates in 263 cervical punch biopsies of women followed-up since 1981 (16 +/- 14 months, M +/- SD) for an established Human papillomavirus (HPV) lesion with or without concomitant cervical intra-epithelial neoplasia (CIN) were analysed for presence of human natural killer (NK) cells, defined by the monoclonal antibody HNK-1 (Leu-7) using the avidin-biotin peroxidase complex (ABC) technique. HNK-1+ cells remained at a constant low level (1.8 to 3.0% of the cells) in the different types of HPV lesions (flat, inverted, or papillomatous condylomas), their percentages (range 1.3 to 2.5% of cells) remaining similarly unaffected by the severity of associated CIN. As shown to be the case with human NK cell function in the peripheral blood, the percentages of local HNK-1+ cells did not evidence any age-dependence. Furthermore, the intensity of the infiltrate did not correlate with the relative levels of HNK-1+ cells in the biopsies. Slightly (but not significantly) higher levels (2.5%) of HNK-1+ cells were found in the first biopsies of the HPV lesions found to regress during the follow-up period (28.8% of cases), when compared with those (1.7% and 1.8%, respectively) in persisting lesions (52.1% of cases) or progressing lesions (19.1% of lesions). The results are discussed in terms of the proposed immune surveillance function of human NK cells in malignant growths, and the conclusion is drawn that the present study does not provide any firm evidence to suggest that local NK cell function would contribute significantly to the clinical behavior of the cervical HPV lesions. PMID- 3014805 TI - Effect of amino acid input rates on lens metabolism in a perfusion culture system. AB - A perfusion system has been established for the organ culture of the lens. The system is designed so that a constant flow of fluid occurs past the lens and a separate channel of fluid simultaneously bypasses the lens. Lens metabolic activity can be determined by analyzing differences between these two samples of fluid. The system also maintains a level of pressure comparable to intraocular pressure. Apparatus for observation and photography of the lens is built into the system. The purpose of these studies was to determine if this perfusion system could maintain the rabbit lens close to the physiological state, to study the metabolic behavior of the lens under steady state conditions, and to determine the effect of increasing amino acid concentrations on the metabolic activity of the lens under steady state conditions. By increasing the amino acid concentrations in the medium we hoped to compensate for the lack of protein in the medium and to provide metabolic substrate to maintain the lens under perfusion conditions. The rabbit lens was cultured at a temperature of 33 degrees C for periods up to 100 h. A totally synthetic perfusion medium without proteins or antibiotics was devised. This medium imitates posterior chamber aqueous humour, except that it does not contain ascorbic acid because it was found that ascorbic acid readily auto-oxidizes and depletes the oxygen content of the medium. The amino acid concentration of the perfusion medium was adjusted so that its total nitrogen content was equivalent to the total nitrogen content of posterior chamber aqueous humour. To determine the metabolic behavior of the lens, 10 different metabolic substances were analyzed in the medium and 15 were analyzed in the lens. These substances are mainly metabolic substrates and end products. As a reference for the evaluation of the behavior of the lens in the perfusion system, the perfused lens was compared to the lens in the other eye of the rabbit. Standards for differences between the right and left lens and the first or second lens removed from the rabbit were developed in control studies. A series of experiments was devised to determine the effect of increased amino acid concentration in the medium on the metabolic behavior of the lens. Concentrations of amino acids from 0 to 7.26 times the posterior concentration of amino acids in chamber aqueous humour were studied. Seven studies were done with a flow rate of medium of about 2 g per h, with each experiment lasting from 80 to 100 h.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3014804 TI - Choroideremia. A clinical and genetic study of 84 Finnish patients and 126 female carriers. AB - The aim of this work was to identify the choroideremia families in northern Finland, form an impression of the incidence of the disease in Finland, construct a picture of its clinical progression and gather new information on relevant genetic questions. A total of 111 choroideremia patients and 188 carriers were traced, members of four families from northern Finland and one from the Savo district. Ophthalmological confirmation was obtained for 84 choroideremia cases and 126 carriers. The largest of the families from northern Finland contained 80 cases of the disease and 146 carriers in eight generations among a total of more than 3000 descendants from one ancestral mother. The clinical picture for choroideremia proved to be more variable than could have been supposed from the literature, including cases of patients under 30 years of age who were already virtually blind and of patients of over 50 who were subjectively symptom-free. Only 7 out of 105 carriers could be shown anamnestically to have had subjective symptoms, but surprisingly, as many as 21 out of 52 carriers examined had changes in the visual field and 13 out of 40 examined showed deterioration in dark adaptation. One carrier was seen to undergo an obvious decline in dark adaptation during a three-year observation period. One indirect indication of the progression of fundus changes in choroideremia carriers was obtained from the fact that these changes, and also alterations in visual field and dark adaptation, were greater in the older carriers. A progression could also be detected by fundus photography in six instances, although the changes involved were fairly mild ones. Considerable variety was noted in the fundus findings for the choroideremia carriers, there being some 80-year-old subjects with quite minor changes and some 20-year-olds with obvious, extensive changes. Practical visual acuity remained normal throughout life in the majority of the carriers, however. Diagnosis within the known choroideremia families was fairly difficult, especially at the early stages in the survey, and even later on a few cases aged up to ten years produced diagnostic problems. Quite often diagnosis was easy, however, and choroideremic fundus changes were even identified in two boys aged 3 and 8 months. No other diseases could be shown to be associated with choroideremia, and the occurrence of dominantly inherited olivopontocerebellar atrophy alongside choroideremia in one branch of a family may be regarded as a coincidence.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3014806 TI - Histological, anatomo-pathological features of the cranial nerves. PMID- 3014807 TI - Treatment of Cushing's disease in childhood and adolescence by stereotactic pituitary irradiation. AB - Eight children with Cushing's disease aged 6-18 years were treated with external radiation to the pituitary gland using 60Co gamma radiation given with stereotactic technique. The dose given varied between 50 and 70 Gy. The observation time was 2.6 to 6.75 years. Seven children had a clinical remission with normal urinary cortisol excretion. One child had insufficient effect of two irradiations and underwent bilateral adrenalectomy. In the patients in remission the growth velocity increased during the first year after treatment but growth retardation occurred again during the second year. Insufficient growth hormone secretion was demonstrated in all subjects. Two patients were given thyroxine substitution and three showed evidence for secondary hypogonadism. In conclusion, stereotactic pituitary irradiation was effective in normalizing the excessive glucocorticoid production in children with Cushing's disease. However, with the doses used, it was not possible to maintain a normal anterior pituitary function. PMID- 3014808 TI - A comparative study of size and function of the remnant kidney in patients nephrectomized in childhood for Wilms' tumor and hydronephrosis. AB - To obtain more information about the natural history of compensatory renal hypertrophy beginning in childhood we traced those who were nephrectomized in childhood for Wilm's tumor (W) and hydronephrosis (Hn) between 1950 and 1978 at one department of surgery in Stockholm. All W patients had received treatment that suppresses cellular division. None of the patients were in renal failure or treated with antihypertensive drugs. All the patients in the follow-up study (22 W, 15 Hn) had a normal contralateral kidney at nephrectomy. Five healthy adults served as controls. The kidney was enlarged in both Hn (142%) and W (125%), but significantly larger in Hn than in W. Renal compensatory growth in W was retarded during the first two years after nephrectomy. The glomerular filtration rate (GFR) was 92% of control in Hn and 82% of control in W. The GFR did not seem to decline with a longer follow-up time in any of the groups. PAH clearance was the same in Hn and W. Albumin excretion was significantly higher in Hn than in W, but not significantly higher in W than in controls. The highest albumin excretion rates were found among the Hn patients with long follow-up time. The results suggest that the large increases in size and function that follow childhood nephrectomy can be blunted by antimitotic agents. PMID- 3014809 TI - Phagocytosis-associated oxidative metabolism in human milk macrophages. AB - Evidence exists that human milk macrophages, which are the most abundant cells in milk, play an important role in the protection of the newborn infant against infection. We investigated the oxidative metabolism of milk macrophages by measuring luminol-dependent chemiluminescence, generation of superoxide anion (O2 ) and production of hydrogen peroxide (H2O2) in the resting state and after stimulation with opsonized zymosan or phorbol myristate acetate (PMA). Generally, macrophages generated luminol-dependent luminescence, O2- and H2O2 with either stimulus to a similar extent as did blood monocytes. In addition, macrophages killed Escherichia coli and Staphylococcus aureus as effectively as did blood monocytes (approximately 75%, 120 min). Acidification of macrophages in milk (pH 1, 30 min) only slightly reduced their PMA-stimulated production of oxygen radicals. Bacterial killing by macrophages preincubated at pH 1 was about 70% that from controls maintained at pH 7. When macrophages were cultured for several days in endotoxin-free medium, their ability to produce oxygen metabolites declined. By continuous treatment with bacterial LPS (10 ng/ml), milk-macrophages could be "primed" to release large amounts of oxygen intermediates. In addition, the O2- response of macrophages cultured without bacterial products could be partially restored by the addition of LPS to the culture. We conclude that milk macrophages are capable of releasing large amounts of oxygen metabolites, and could contribute to the protection of neonates against bacterial and fungal infections. PMID- 3014810 TI - Viral findings in children under the age of two years with expiratory difficulties. AB - Viral findings were prospectively studied in lower respiratory tract infections in small children with and without expiratory difficulties. On first admission, a viral aetiology was found in 71 of 127 children (56%). On re-admission, a viral etiology was found in only two of 31 cases (6%). Respiratory syncytial viruses (RSV) were responsible for 71% of the cases with viral diagnoses. A recently developed method for the direct detection of viral antigens in nasopharyngeal specimens by radio-immunoassay was more sensitive than complement fixation serology, especially in patients aged less than six months. Viral diagnosis was reached using this new method alone in 43% of infections caused by RSV and in 27% of infections caused by other viruses. In children under six months, RSV were found in 89% by direct antigen detection and in 22% by serology. We suggest that direct antigen detection should be used as the primary virological method in small children with lower respiratory tract infections. The aetiological agents were the same in cases with and without expiratory difficulties, RSV being found in about 40% of children in both instances. It is concluded that host factors are critical to the development of expiratory difficulties. PMID- 3014811 TI - Hepatocellular carcinoma and schistosomiasis japonica. A clinicopathologic study of 59 autopsy cases of hepatocellular carcinoma associated with chronic schistosomiasis japonica. AB - The association between hepatocellular carcinoma (HCC) and chronic hepatic schistosomiasis (CS) was studied by reviewing, 4,886 autopsies in adults during the past 20 years. In 229 cases of CS, 59 (25.7%) also had HCC. Among cases without CS, 399 (8.5%) had HCC. The incidence of HCC in patients with CS was significantly higher than that of other autopsy cases (p less than 0.01). Serum HBsAg was positive in 25.7% of 35 HCC cases with CS examined for hepatitis B virus (HBV) markers and anti HBs was positive in 10 of the 12 HBsAg-negative cases associated with CS, and in 62.1% of the other HBsAg-negative cases examined. Thus, most of HCC cases, including those associated with CS, probably had HBV infection at one time. Morphological examination revealed varying degrees of non-schistosomal hepatic changes, including macronodular or mixed macro-and micronodular cirrhosis, superimposed on schistosomal fibrosis in about two-thirds of the cases of HCC associated with CS. Although conclusive evidence whether or not schistosomal infection had a direct role in hepatocarcinogenesis could not be obtained, it was predicted that the additional non-schistosomal factors, particularly HBV infection, might play a synergistic role. PMID- 3014812 TI - Early cancer and related lesions in the bronchial epithelium in former workers of mustard gas factory. AB - The bronchial epithelium in stepwise transverse sections was examined histologically in 66 male autopsy cases, composed of the groups of 19 mustard gas (MG) ex-workers with lung cancer, 17 MG ex-workers with non-lung cancer, 10 non MG lung cancer cases, and 20 non-MG non-lung cancer cases. Foci of moderate or severe atypical cellular lesion or dysplasia, or of carcinoma in situ (CIS) in total slides of each group, were counted as 146 in 3,485, 72 in 2,226, 70 in 3,797, and 18 in 4,611, respectively. The relative frequency of moderate or severe dysplasia and CIS in MG exposed non-lung cancer cases resembled that found in lung cancer cases of both MG and non-MG exposed. Seven CIS lesions were detected from among all MG-exposed cases and one CIS was found in a non-MG lung cancer case. Six out of eight CIS examples were adjoined by dysplasia. A multi variate analysis revealed a significant correlation between the incidence of atypical lesions and MG exposure, though the incidence of atypical lesions was also influenced significantly by age, smoking, and chronic bronchitis. The incidence of atypical lesions was significantly higher in cases of squamous cell lung cancer than those of other histological types, particularly small cell cancer. PMID- 3014813 TI - Hepatocellular carcinoma with metastatic gastric cancer simulating Borrmann type 2 and hyperlipidemia. AB - A case of hepatocellular carcinoma with metastasis to the stomach and hyperlipidemia as a paraneoplastic syndrome was presented. The patient, a 69-year old man, was admitted to Kurobe City Hospital with a complaint of epigastralgia. He was diagnosed as having hepatocellular carcinoma by an increased plasma AFP and the abnormalities of hepatic scintigram and abdominal angiography. Endoscopic examination of the stomach revealed an ulcerative lesion suggesting Borrmann type 2 gastric cancer and the gastric mucosal biopsy was interpreted as tubular adenocarcinoma. At autopsy, the liver was enlarged and weighed 4,170 g without liver cirrhosis. Histologic finding of the liver tumor was hepatocellular carcinoma of Edmondson's grade 2 and the gastric tumor with bile production was identical to that of liver tumor. The tumor architecture of the stomach, however, was mixed with trabecular pattern and tubular pattern near the site of gastric mucosa, and was concordant with the findings of gastric mucosal biopsy. Multiple tumor thrombi in the portal system suggested that hepatocellular carcinoma retrogradely metastasized to the stomach through the portal system. PMID- 3014814 TI - Cystic brain lesion in utero. AB - Two autopsy cases of cystic brain lesion in utero are reported. One of them was a donor infant of twin transfusion syndrome. The baby died immediately after birth and showed multicystic encephalomalacia in the distribution of the anterior cerebral artery. The second baby was a stillborn infant with thanatophoric dwarfism with associated chronic periventricular leukomalacia (PVL). It was suggested that the multicystic encephalomalacia and chronic PVL found in the first and second cases were caused by persistent circulatory disturbances in utero. PMID- 3014815 TI - Influence of a carcinogenic agent, 7.12 dimethylbenz (alpha) anthracene (DMBA), on production of interferon alpha/beta in murine and human cells. AB - Interferon production in mouse and human cells induced by paramyxovirus was inhibited by pretreatment of cells with 7.12 dimethylbenz (alpha) anthracene. The inhibition was moderate but reproducible. It was most pronounced in mouse fibroblast cells, somewhat less in the mouse L-929 cell line and in human embryo fibroblast cells. Addition of the microsomal activator systems was necessary in the human system. A possible employment of this phenomenon in testing carcinogenic potential is discussed. PMID- 3014816 TI - [Comparison of the effects of spegatrine and dispegatrine on alpha adrenoceptors]. PMID- 3014817 TI - [The investigation of lignans from Sargentodoxa cuneata (Oliv) Rehd et Wils]. PMID- 3014818 TI - Trifluoperazine stimulated sodium transport by increased prostaglandin E2 synthesis in isolated frog skin (Rana esculenta). AB - Addition of trifluoperazine (TFP) to the inside bathing solution of the isolated frog skin resulted in a biphasic increase in the short-circuit current (SCC). The Na+-flux measurements showed that the TFP-induced increase of SCC was accounted for by active sodium transport. The intracellular voltage in short circuited skins changed after addition of TFP (100 microM) from a control value of -80.9 +/ 0.8 to -66.2 +/- 1.0 mV (n = 8). This depolarization indicates that TFP acts by increasing the Na+-permeability of the apical membrane. The biphasic increase in SCC is due to different mechanisms. The primary activation could be abolished by the calcium ionophore A23187, whereas the secondary activation could be abolished by the prostaglandin synthesis inhibitor indomethacin, PGE2 or A23187. Stimulation of the SCC by TFP and theophylline or antidiuretic hormone (AVT) was additive. Furthermore, TFP did not increase the cAMP level of isolated epithelia or theophylline-stimulated epithelia. These results indicate that the TFP-induced change in the Na+-permeability was not due to an enhanced cAMP level. The TFP simulated SCC requires Ca2+ in the inside bathing solution. Addition of TFP resulted in an increase in prostaglandin E2 release to the inside bathing solution from a control value of 0.31 +/- 0.04 to 5.4 +/- 1.4 pmol PGE2 h-1 cm-2 (n = 8). It is suggested that TFP induces a Ca2+-dependent PGE2 synthesis, leading to an increase in the intracellular PGE2 concentration which might increase the Na+-permeability of the apical membrane. PMID- 3014819 TI - Renal handling and effects of [3H]digoxin and interactions with quinidine in the avian kidney. AB - Renal handling and effects of tritium digoxin and interactions with quinidine in the avian kidney were studied using a modified Sperber technique. Results showed that tritium digoxin was extracted at the peritubular side of the nephron in a process competitively inhibited by increasing amounts of unlabelled digoxin. Light microscope autoradiography showed distinct concentrations of silver grains only over distal tubules in the injected kidney. Inhibition of the proximal tubular transport systems for organic anions and cations, respectively, did not change extraction. Addition of quinidine to the injection solution up to an estimated concentration of 1.4 X 10(-5) M in systemic blood significantly lowered 1 min peritubular extraction of tritium digoxin. However, when the amount of quinidine was further increased, extraction of tritium digoxin augmented significantly. Tritium recovery in urine after renal portal bolus injection of tritiated and unlabelled digoxin already showed a distinct ipsilateral peak 2 min after injection with an equally distinct peak of ipsilateral sodium excretion appearing 1 min later. When quinidine was added to the bolus ipsilateral tritium recovery in urine (0-7 min) was halved, with the true tubular excretion fraction (TTEF) lowered by two-thirds, but without changes in the magnitude of ipsilateral natriuresis. Contralateral natriuresis increased more than four-fold with quinidine in the bolus in spite of unchanged tritium recovery in the urine. Thus, our results show tritium digoxin to be extracted from peritubular blood through a specific process probably localized to the distal nephron of the avian kidney.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014821 TI - Naloxone and haemorrhagic hypotension in rats. Evidence against sympathetic nervous system as the primary mediator of improved cardiovascular haemodynamics. AB - Release of endogenous opiate-like substances seem to occur in different forms of stress. Earlier studies have shown that the opiate antagonist, naloxone, has a positive effect on cardiac performance and blood pressure in animals with haemorrhagic shock. In the present study, we have examined the involvement of the sympathetic nervous system in this response. Two groups of anaesthetized normotensive Wistar-Kyoto rats were studied. Both groups were bled rapidly (about 5 min) down to an arterial pressure of 50 mmHg and were kept at that level for 30 min. At the end of the 30-min bleeding period, naloxone 1, 2, or 5 mg kg-1 was injected i.v. in a small volume of saline. In the first group of rats (n = 6), the aortic pressure was kept constant at 50 mmHg by further bleedings after naloxone. In the other group (n = 7), the arterial pressure was allowed to rise after naloxone. As reported earlier, haemorrhagic hypotension caused a pronounced inhibition of renal sympathetic nerve activity. Naloxone injected after 30 min of hypotension caused an immediate rise in blood pressure, followed 1-2 min later by a rise in sympathetic nerve activity (SNA). In animals in which pressure was held constant by further bleeding after naloxone, only small and insignificant changes in SNA were observed. The conclusions are the following: injection of naloxone increases blood pressure in rats exposed to severe haemorrhage (Faden & Holiday 1979). The rise in aortic pressure is followed 1-2 min later by a rise in SNA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014820 TI - Inhibitory effects of chemically-different 'loop' diuretics on chloride transport across the bullfrog cornea. AB - Frog (Rana catesbeiana) corneas were mounted in an Ussing chamber, modified to facilitate dissection and to avoid edge damage to the epithelial tissue during mounting and measurements. When bathed in Conway solution of pH 7.4 the corneas displayed highly stable electrical properties, with a transepithelial potential difference (PD) of 16.6 +/- 1.O mV and a short-circuit current (SCC) of 10.3 +/- 0.8 microA cm-2. The DC resistance was 2.0 +/- 0.15 k omega X cm2 (mean +/- SE, n = 45). An increase in the intracellular cyclic AMP level induced by prostaglandin E2 and by 3-isobutyl-1-methylxanthine (IBMX) resulted in an increase in SCC. Ortho-vanadate, an inhibitor of Na+-K+-ATPase, reduced SCC. The acidic loop diuretics furosemide, bumetanide and the levorotatory form of indacrinone (MK 196) reduced SCC by about 50% at concentrations of 500, 100 and 46 microM, respectively. The (-)form of MK-196 was four times more active than the (+)form. Dimethylation of the SO2NH2 group reduced the activity of bumetanide. The basic diuretics muzolimine (Bay G 2821) and MK-447, were found, similarly, to reduce SCC by about 50% at concentrations of 500 microM. In view of their 'high ceiling' type of saluretic effects in whole animals, these basic agents should therefore be classified as 'loop' diuretics. The effects of these structurally highly different 'loop' diuretics are similar in epithelia which secrete (cornea) and in those which absorb (the renal thick ascending limb of Henle's loop; TALH) chloride ions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014822 TI - Binding of catecholamines to phospholipids isolated from adrenal medullary granules. AB - The large granules of the adrenal medullary fraction were lysed and subjected to Sepharose 2B gel filtration. Proteophospholipids (PPL) were isolated from the membrane fraction and used for binding of catecholamines (CA), such as noradrenaline (NA), adrenaline (A) and dopamine (DA). The affinity order was DA greater than NA = A. The binding capacity was dependent on amine concentration, pH and ionic strength, and amounted to about 2 and 3.5 mumol per mumol lipid phosphorus in ether and ether-ethanol, respectively. A Scatchard plot revealed only one type of binding site; Kd = 8.3 mM. The anionic detergent sodium dodesylsulphonate (SDS) moderately increased the uptake of NA and A while cationic detergents like cetyltrimethylammonium bromide (CTAB) and Hyamine 2389 strongly diminished the binding to PPL. The ionic detergent sodium deoxycholate (DOC) and the non-ionic detergent Triton X-100 were without significant effect. It is suggested that the dipolar head group of the granule lipids, particularly the lipid phosphate groups, are involved in electrostatic or complex interactions with the CA, either directly, or through water molecules within the lipid polar head group. PMID- 3014824 TI - [Changes in the renal parenchyma in children with cytomegaly]. PMID- 3014823 TI - Twenty-four-hour serum levels of T4 and T3 in relation to decreased TSH serum levels and decreased TSH response to TRH in affective disorders. AB - The serum levels of thyroxine and triiodothyronine (T4 and T3) were investigated at 10 different time points during a 24 h period in 31 inpatients meeting the RDC criteria for acute major depressive disorder. Twenty-three of these patients were also reinvestigated in a state of partial or complete remission. The results show that there was no significant difference in T4 or T3 levels during the 24 h period between depressed patients and 32 healthy controls despite significantly decreased TSH levels and TSH response to TRH administration (delta TSH) in the patient group. No indications were obtained that the patients' clinical presentation or depressive symptomatology as revealed by their CPRS scores, psychotropic medication, melatonin levels, or the outcome of the dexamethasone test, significantly influenced the T4 or T3 levels. The depressed patients who were studied longitudinally showed increased T4 levels in the acute phase compared to remission, whereas the T3 levels did not change. However, the levels of thyroid hormones were within the normal range in the acute phase as well as in remission. Furthermore, the changes in thyroid hormones between the state of relapse and remission were not significantly correlated to the corresponding increase in TSH levels and delta TSH between the two assessments. The present results are consistent with the hypothesis that the mechanism behind the impaired TSH response to TRH in acute major depressive disorder is a downregulation of the pituitary TRH receptors. PMID- 3014825 TI - [AIDS and artificial insemination by donor]. PMID- 3014826 TI - Absence of HTLV-III/LAV antibodies in ex-residents from Angola and Mozambique returning to Portugal before 1979. PMID- 3014827 TI - [Prolactin in the feto-placental unit. Immunohistochemical data]. PMID- 3014828 TI - Source and fate of circulating pyrimidines. PMID- 3014829 TI - Uridine monophosphate kinase and susceptibility to invasive Haemophilus influenzae type B disease. AB - A polymorphic genetic variant of the pyrimidine pathway enzyme, uridine monophosphate kinase-3 (UMPK-3), was positively associated with invasive Hib disease. All UMPK 3-3 homozygotes in this study were Hib cases, and we found that in cases and controls, there was no difference between UMPK phenotype and serum levels of total Hib antibody as measured by radioimmunoassay. This suggests that UMPK-3 may play a role in mediating the non-humoral immunity to Hib. However, unlike other enzyme variants in the nucleoside synthesis pathways which result in syndromes of severe immunodeficiency, this gene appears to confer a more subtle disease susceptibility. Thus, the UMPK-3 allele, although rare in Caucasians, is associated with an increased risk of invasive Hib infection in Alaskan Eskimos. PMID- 3014830 TI - Ecto-nucleoside triphosphate pyrophosphohydrolase activity and calcium pyrophosphate dihydrate crystal deposition disease. AB - Articular cartilage contains any ectoenzyme activity, NTP-PPH, which is capable of generating PPi from NTP substrates. The PPi generated is from the cleavage of the alpha-beta pyrophosphate bond of NTP and does not result from the effects of NTP catabolites. NTP-PPH activity is expressed on human skin fibroblasts in culture and is significantly increased in subjects with CPPD deposition. In addition, cultured fibroblasts from subjects with CPPD disease have higher intracellular PPi concentrations compared to cells from normals and patients with OA. These results support the hypothesis that alterations in PPi metabolism provide the metabolic basis for CPPD deposition. PMID- 3014831 TI - Insertion of hypoxanthine phosphoribosyltransferase cDNA into human bone marrow cells by a retrovirus. PMID- 3014832 TI - The human gene for adenosine deaminase. PMID- 3014834 TI - Adenylosuccinase deficiency. PMID- 3014833 TI - Analysis of the transcriptional regulatory sequences in the CHO APRT gene. PMID- 3014835 TI - The prevalence of purine metabolic disorders in Japan. PMID- 3014837 TI - A comparative study of allopurinol and pentostam in the treatment of visceral leishmaniasis. PMID- 3014836 TI - The "switch-off" mechanism of spontaneous resolution of acute gout attack. PMID- 3014838 TI - Modulation of polymorphonuclear leukocyte function by adenosine analogues. PMID- 3014840 TI - Phosphorylation of purine deoxynucleosides in human T- and B-lymphoblasts. PMID- 3014839 TI - PRPP synthetase superactivity in lymphoblast lines. PMID- 3014842 TI - Histamine H2 receptor activity and histamine metabolism in human U-937 monocyte like cells and human peripheral monocytes. AB - Functional and specific histamine H2 receptors were characterized in human peripheral monocytes and in U-937 cells, before and after retinoic acid--induced differentiation into monocyte/macrophage-like cells. The relative potencies of histamine and the H1, H2 receptor agonists and antagonists studied are remarkably similar in U-937 cells and U-937 monocytes. There is no change in histamine concentration and activity of the enzymes forming and degrading histamine during monocytic-like differentiation. The results raise the possibility that histamine H2 receptors might be involved in pathophysiological regulations (proliferation/differentiation and biological function) of normal and leukemic monocytes. PMID- 3014841 TI - Inherited phosphoribosylpyrophosphate synthetase superactivity due to aberrant inhibitor and activator responsiveness. PMID- 3014843 TI - 3H-mepyramine binding and histamine-stimulated cAMP accumulation in mammalian retina. AB - The specific binding of different amounts of 3H-mepyramine to the bovine retina revealed a quasi-hyperbolic curve which approached saturation at 3H-ligand concentration over 9-12 nM. Scatchard analysis of the binding data showed two binding sites with KD values of 0.76 nM and 7.3 nM and Bmax of 49.3 and 194.6 fmole/mg protein, respectively. In the guinea-pig brain 3H-mepyramine bound to a single population of binding sites with KD value of 1.6 nM and Bmax of 291 fmole/mg protein. Various H1-antihistamines were potent competitors of the 3H mepyramine binding: there was a big difference in potency of d- and 1 chlorpheniramine in both membrane preparations. In the rabbit retina slices histamine, in contrast to dopamine, weakly stimulated cAMP accumulation. The data suggest that the mammalian retina may possess histamine receptors. PMID- 3014844 TI - Effects of GABA agonists on Herxheimer microshock in guinea pigs. AB - In this paper we describe the first observation of GABA inhibition in an experimental model of asthma in vivo. Guinea-pigs were actively sensitized with ovalbumin i.p. and 20 days later the Herxheimer microshock was performed. GABA and (-)-baclofen injected 20 min previously significantly prevented the development of microshock. Therefore GABAergic drugs appear to modulate in vivo anaphylactic reaction. The value of this observation with regard to the physiopathology and therapy of asthma remains to be elucidated. PMID- 3014846 TI - The possible role of synovial factors in cartilage destruction. PMID- 3014845 TI - Separate purinoceptors mediate enhancement by adenosine of concanavalin A-induced mediator release and the cyclic AMP response in rat mast cells. AB - Adenosine caused a concentration-related enhancement of concanavalin A (Con A) induced 5-hydroxytryptamine (5-HT) release from rat purified peritoneal mast cells. This was accompanied by an enhancement and prolongation of the cyclic AMP response to Con A. The cyclic AMP response but not enhancement of 5-HT release was blocked by 8-phenyltheophylline suggesting the two events to be unrelated. The effects of AMP and ADP, adenosine analogues and adenosine uptake inhibitors suggest the enhancement of 5-HT release to be mediated by a P1-cell surface purinoceptor which does not show the characteristics of either A1 or A2 subtypes. PMID- 3014847 TI - A clinical study of the prognostic value of technetium dimercaptosuccinic Acid uptake in renal obstruction. PMID- 3014848 TI - [The influence of carrageenan, colchicine, 2,4-dinitrophenol, ethanol, galactose, indomethacin and liquoid on hemolytic complement activity (CH50E)]. AB - The influence of Liquoid (0.00005, 0.00001%), Carrageenan (0.001% up to 0.000001%), Colchicine (0.01% up to 0.0025%), Indomethacin (1 X 10(-4) up to 2.5 X 10(-5) mol/1), Ethanol (10% up to 1.25%), Galactose (1 X 10(-1) up to 1 X 10( 2) mol/l) and 2.4-Dinitrophenol (5 X 10(-4) up to 1 X 10(-5) mol/l) on the total hemolytic activity of complement was tested. By Liquoid, Carrageenan, Colchicine and Ethanol in all studied concentrations an remarkable inhibition of the CH50E could be estimated. Indomethacin up to an concentration of 3.75 X 10(-5) mol/l reduced the activity partially. In contrast, Galactose and 2.4-Dinitrophenol have not influenced the complement titers. PMID- 3014849 TI - Pediatric viral gastroenteritis. AB - Rotavirus and Norwalk agent cause most viral gastroenteritis in the pediatric population. Infection results in osmotic diarrhea, which can cause dehydration and acidosis. Oral rehydration therapy has been shown worldwide to be a safe and effective means of treating this disease. Formula feeding should be resumed as soon as possible; breast feeding should be maintained throughout the illness. Admission criteria are based on weight loss, serum sodium level and ability to rehydrate orally on an outpatient basis. PMID- 3014850 TI - Acute hemodynamic and hormonal effects of lisinopril (MK-521) in congestive heart failure. AB - The acute hemodynamic and hormonal effects of the oral angiotensin-converting enzyme (ACE) inhibitor lisinopril (MK-521) were assessed over a period of 96 hours in 12 patients with heart failure. This compound is the lysine analogue of enalaprilat (MK-422), is biologically active following absorption, and is cleared via the urine without any known metabolic transformation. Single doses of lisinopril, ranging from 1.25 mg to 10 mg, were administered on days 1 and 3, each followed by 48 hours of intensive hemodynamic observation. Across all doses, maximal reductions in mean arterial pressure (17.2%), mean pulmonary capillary wedge pressure (28%), and systemic vascular resistance (25.6%) were observed compared to baseline values. No significant changes in heart rate were recorded. Arterial blood was sampled at frequent intervals for angiotensin II, ACE activity, plasma renin activity, renin substrate, plasma aldosterone, and serum drug levels. Right atrial blood was sampled simultaneously for angiotensin I, thus permitting assessment of the degree of pulmonary conversion to angiotensin II. The results indicate potent inhibition of the renin-angiotensin-aldosterone system along with hemodynamic efficacy over a period exceeding 24 hours. Frequent clinical follow-up on long-term chronic therapy has revealed no adverse experience. PMID- 3014851 TI - Effect of suloctidil on tomographically quantitated platelet accumulation in Dacron aortic grafts. AB - Platelet deposition contributes to the thrombotic and embolic complications of prosthetic materials in man. To determine if the investigational platelet inhibitory drug suloctidil (200 mg 3 times daily) reduces platelet deposition on Dacron aortic grafts, a randomized, double-blind, crossover trial was conducted in 12 men with grafts that had been in place more than 9 months. Platelet deposition in the graft was assessed by quantitative analysis of planar images obtained at 24, 48 and 72 hours after injection of indium-111-labeled platelets. Also, a tomographic method of imaging and quantitating labeled platelet deposition in the graft was developed. Tomographic imaging was performed at 24 and 72 hours after platelet injection and was quantitated by a graft/blood ratio that compared indium-111 platelet activity in summed 1.8-cm-thick transaxial tomographic slices of the aortic graft to indium-111 platelet activity in well counted whole blood. Compared with placebo, suloctidil failed to decrease the tomographic graft/blood ratio at 24 hours (6.2 +/- 1.3 vs 5.7 +/- 0.8) and 72 hours (11.4 +/- 2.9 vs 10.7 +/- 2.2). Similarly, the graft/blood ratio determined by planar imaging was not different between placebo and suloctidil therapy at 24 hours (1.7 +/- 0.3 vs 1.6 +/- 0.2), 48 hours (2.2 +/- 0.4 vs 2.4 +/- 0.4) or 72 hours (2.6 +/- 0.5 vs 2.8 +/- 0.5) after labeled platelet injection. Thus, suloctidil does not significantly reduce platelet deposition on chronically implanted Dacron grafts in humans. PMID- 3014852 TI - A simple method to estimate neutral detergent fiber content of typical daily menus. AB - The purpose of this research was to develop a simple method to estimate daily intakes of the insoluble fraction of dietary fiber, that portion most effective in increasing stool weight and defecation frequency and decreasing gastrointestinal transit time. Neutral detergent fiber (NDF) contents of 53 foods were determined by analysis and used to calculate the NDF content of typical institutional menus; mean NDF content (n = 14 days) was 10.8 +/- 1.4 g/day. Average NDF content of one serving of fruits and vegetables was approximately 1 g, of the refined grain products, approximately 0.5 g. Estimating daily NDF content of the menus was simplified without a loss of accuracy by substituting the mean NDF content of one serving of refined grains, vegetables, and fruits for the actual values, and by eliminating foods present at less than 1/4 of a serving. Crude fiber was a poor predictor of the NDF in a menu. PMID- 3014853 TI - Digestion of the carbohydrates of banana (Musa paradisiaca sapientum) in the human small intestine. AB - The digestion and absorption from the small bowel of the carbohydrate of banana has been studied by feeding ileostomy subjects banana from six batches of different ripeness and measuring the amounts excreted in the effluent. Starch content of bananas depended on the ripeness being 37% of dry weight in the least ripe and 3% in the most ripe. Excretion of carbohydrate from banana in ileostomy effluent ranged from 4-19 g/day and was directly related to the starch content (r = 0.99). Up to 90% of the starch could be accounted for in the effluent. Complete recovery of nonstarch polysaccharides [NSP (dietary fiber)] was obtained. The amount of banana starch not hydrolyzed and absorbed from the human small intestine and therefore passing into the colon may be up to 8 times more than the NSP present in this food and depends on the state of ripeness when the fruit is eaten. PMID- 3014854 TI - The effect of the polysaccharide composition and structure of dietary fibers on cecal fermentation and fecal excretion. AB - The fermentation of dietary fibers gives a clue to their mode of action. Wheat bran and gum tragacanth increase stool weight but have no effect on serum cholesterol or on hydrogen excretion. Gum arabic and pectin in the form of raw carrot have no effect on stool weight but decrease serum cholesterol and are associated with an increase in breath-hydrogen excretion. Other fiber sources like gum karaya have no effect on stool weight, serum cholesterol, or breath hydrogen. There are no correlations between the chemical composition and structure of the fibers studied and their physiological effects. PMID- 3014855 TI - Effects of calcium carbonate and hydroxyapatite on zinc and iron retention in postmenopausal women. AB - We measured the effect of calcium carbonate and hydroxyapatite on whole-body retention of zinc-65 in 11 and iron-59 in 13 healthy, postmenopausal women. In a single-blind, controlled, crossover study, each subject, on three occasions, ingested a standard test meal supplemented with iron-59 or zinc-65 and capsules containing placebo or 500 mg elemental calcium as calcium carbonate or hydroxyapatite. Whole-body countings were performed prior to, 30 min after, and 2 wk after each meal. Mean (SEM) zinc retention was 18.1 +/- 1.0% with placebo (control) and did not vary significantly with calcium carbonate (110.0 +/- 8.6% of control) or hydroxyapatite (106.0 +/- 7.9% of control). Iron retention, 6.3 +/ 2.0% with placebo, was significantly reduced with both calcium carbonate (43.3 +/- 8.8% of control, p = 0.002) and hydroxyapatite (45.9 +/- 10.0% of control, p = 0.003). Iron absorption may be significantly reduced when calcium supplements are taken with meals. PMID- 3014856 TI - Validation of doubly labeled water for measuring energy expenditure during parenteral nutrition. AB - The doubly labeled water method was compared with intake-balance for measuring energy expenditure in five patients receiving total parenteral nutrition (TPN). Because parenteral solutions were isotopically different from local water, patients had to be placed on TPN at least 10 days before the metabolic period. Approximately 0.1 g 2H2O and 0.25 g H2(18)O per kg total body water were given orally. We collected saliva before, 3 h, and 4 h after the dose for measurement of total body water and urine before, 1 day, and 14 days after the dose for measurement of isotope eliminations. On day 14, total body weight was remeasured and change in body energy stores was calculated, assuming constant hydration. Intake was assessed from weights of TPN fluids plus dietary record for any oral intake. Energy expenditure from doubly labeled water (+/- SD) averaged 3 +/- 6% greater than intake-balance. Doubly labeled water method is a noninvasive, nonrestrictive method for measuring energy expenditure in patients receiving TPN. PMID- 3014857 TI - Increasing starch intake in the human diet. PMID- 3014858 TI - Intraoperative remote afterloading endocurietherapy with high-activity 60cobalt for treatment of glioblastoma multiforme. AB - An intraoperative remote afterloading endocurietherapy technique with high activity 60Co for the treatment of glioblastoma multiforme is described. The technique can be used for initial management of the unresectable tumor or for retreatment of patients with recurrent tumor who have been treated previously with surgery and postoperative radiotherapy. Neither intraoperative nor postoperative complications were encountered in our treatment of 11 patients in this Phase I toxicity study. PMID- 3014859 TI - A modified immunoperoxidase method for rapid diagnosis of herpes simplex I keratitis. AB - To study the possibility of establishing a rapid diagnosis of herpes simplex keratitis, corneas of 10 rabbits, a total of 20 eyes, were inoculated with herpes simplex virus (HSV), type I, strain PH. Epithelial keratitis developed within three days of inoculation in all the animals used. Scraping of infected corneas were smeared and examined, using a modified indirect immunoperoxidase technic. One hundred percent of the smears prepared from these corneas demonstrated positive cells. Negative findings in corneas inoculated with adenovirus 19 suggest the specificity of the reaction. To test the possibility of blockage of staining by the presumed development of circulating endogenous anti-HSV I antibodies, the corneas of eight consecutive patients who presented to the Albany Medical Center Hospital with known recurrent dendritic keratitis also were scraped and stained, using a similar procedure. Positive cells present in each of these scrapings suggest against the blocking of this immunoperoxidase method by the development of circulating anti-HSV antibodies. PMID- 3014860 TI - Proliferative sialometaplasia arising in an intraparotid lymph node. AB - A case of florid proliferative sialometaplasia arising in salivary ductal inclusions in an intraparotid lymph node is described. This lesion probably represents an exuberant response to sialadenitis. Other entities that enter into its differential diagnosis in this particular location are discussed. Except for Warthin tumors, lesions of clinical import originating from such inclusions are rare. PMID- 3014861 TI - Chronic idiopathic vitritis. Ultrastructural properties of bacteria-like bodies within vitreous leukocyte phagolysosomes. AB - In chronic idiopathic vitritis (CIV) corticosteroid treatment failures, vitrectomy is beneficial. Searching for vitreous microbial agents, 14 vitrectomy specimens from 11 corticosteroid-failing CIV patients were inoculated into numerous in vitro cultural systems; Gram's-, Giemsa-, periodic acid-Schiff- (PAS), and Dieterle-stained centrifuged sediment smears were studied with the light microscope; and the sediment was examined electron microscopically. None of the specimens demonstrated in vitro growth. However, by light microscopy the smears of ten specimens from 8 of the 11 patients demonstrated, in a background of predominantly mononuclear leukocytes, a few polymorphonuclear leukocytes with minute cytoplasmic Gram's variable coccal bodies. By electron microscopy those ten specimens showed morphologically similar 0.5-0.7-micron, thick-walled, coccal shaped, bacteria-like bodies and 0.03-micron electron-dense spheric particles within polymorphonuclear leukocyte phagolysosomes. The results suggest that CIV vitreous, sterile by contemporary laboratory technics, commonly demonstrates these phagolysosomal bacteria-like bodies. Innovative attempts should be made to cultivate these bacteria-like bodies. Animal pathogenicity studies, using these vitreous specimens as inocula, have been conducted. The results of that investigation will be the subject of another report. PMID- 3014862 TI - Recurrent abdominal pain and dietary fiber. PMID- 3014863 TI - Periorbital cellulitis complicating adenovirus infection. PMID- 3014864 TI - Sudden death due to a paraganglioma of the organs of Zuckerkandl. AB - A 20-year-old woman died suddenly in a hospital emergency room after presenting with nausea, vomiting, back pain, and hypertension. At autopsy, an extra-adrenal pheochromocytoma (paraganglioma) of the organs of Zuckerkandl was found, with microscopic focal myocardial necrosis similar to that described in death from adrenal pheochromocytomas. Tumors of the organs of Zuckerkandl are extremely rare; less than 100 such cases have been reported in the world's literature, and only six, including the present case, have presented as a sudden, unexpected death. The symptoms of catecholamine storm may mimic those of acute drug intoxications, leading to misdiagnosis by both clinical physicians and pathologists. PMID- 3014865 TI - Regional localization of DNA sequences on chromosome 21 using somatic cell hybrids. AB - We have used a panel of Chinese hamster X human somatic cell hybrids, each containing various portions of chromosome 21 as the only detectable human chromosome component, for regional mapping of cloned, chromosome 21-derived DNA sequences. Thirty unique and very low-repeat sequences were mapped to the short arm and three sections of the long arm. Three unique sequences map to the proximal part of the terminal band 21q22.3, and five to the distal part of this band. Some of these may represent parts of gene sequences that may be relevant to the pathogenesis of Down syndrome, as 21q22 is the area required to be present in triplicate for the full clinical picture. PMID- 3014867 TI - Identification and application of additional restriction fragment length polymorphisms at the human ornithine transcarbamylase locus. AB - Two additional restriction fragment length polymorphisms (RFLPs) have been identified at the human ornithine transcarbamylase (OTC) locus. Approximately 11% of women are heterozygous for an RFLP characterized by polymorphic bands at 3.7 and 3.6 kilobasepairs (kbp) observed after DNA digestion with TaqI. Twenty-nine percent of women are heterozygous for an RFLP characterized by polymorphic bands at 18.0 and 5.2 kbp observed after digestion with BamHI. Thus, in combination with the previously reported RFLPs identified using MspI, the X chromosomes in approximately 80% of women at risk for having a son with OTC deficiency are distinguishable by RFLPs at the OTC locus. Furthermore, we show that these RFLPs will be useful in families for prenatal diagnosis of OTC deficiency, carrier detection, and carrier exclusion. PMID- 3014866 TI - On the power to detect differences between male and female mutation rates for Duchenne muscular dystrophy, using classical segregation analysis and restriction fragment length polymorphisms. AB - The power to detect departures from the theoretical proportion of new mutants in X-linked lethal disorders has been analyzed for several types of segregation analysis, including methods based on completely linked restriction fragment length polymorphisms. It is shown that all methods require large sample sizes in order to detect even large differences between male and female mutation rates. Ascertainment bias is shown to have a great effect on the outcome of the segregation analysis. All reviewed studies concerning the proportion of new mutants in Duchenne muscular dystrophy, whether they claimed equality or inequality between the male and female mutation rates, give insufficient evidence because of ascertainment bias and a too low power. An ascertainment bias-free method is given, with the advantage that information from many studies can be combined. By doing so, in the long run, even moderate departures from equality in mutation rates (if present) can be detected. PMID- 3014868 TI - Relationships between the human pepsinogen DNA and protein polymorphisms. AB - Pepsinogens (PGA) are the inactive precursors of pepsin, the major acid protease found in the stomach. The PGA gene family exhibits polymorphic variation in human populations that can either be demonstrated by electrophoretic analysis of the proteins or by analysis of the respective genes with cDNA probes. Here, we describe the interrelationships between the most common pepsinogen protein phenotypes and the corresponding pepsinogen haplotypes (A, B, and C) containing different combinations of the PGA3, PGA4, and PGA5 genes. We propose that this unusual genetic variation involving haplotypes that contain three, two, and one genes, respectively, is the result of molecular evolution by gene duplication. PMID- 3014869 TI - Identification of a beta-thalassemia mutation associated with a novel haplotype of RFLPs. AB - The study of the molecular defects that result in beta-thalassemia in Mediterraneans has uncovered a large number of unique mutations. This information is already being utilized for prenatal diagnosis of pregnancies at risk. Here, we report the definitive identification, by molecular cloning, of the beta thalassemia mutation associated with a Mediterranean chromosome bearing a novel haplotype of restriction fragment length polymorphisms (RFLPs) in the beta gene cluster that has been previously designated as haplotype X. The thalassemia mutation was identified as a T----C base substitution at IVS-1 position 6, a mutation previously described in association with haplotype VI. We describe the use of the restriction enzyme SfaNI for the detection of this mutation and point out a possible pitfall that should be avoided if such an approach is used for the detection of this mutation, which appears to be a common cause of mild beta+ thalassemia in some populations. PMID- 3014870 TI - Characterization of a spontaneous mutation to a beta-thalassemia allele. AB - We have studied a nuclear family containing a single child with severe beta thalassemia intermedia, a Greek-Cypriot mother with hematological findings of beta-thalassemia trait, and a Polish father who is hematologically normal. Since both the child and her father were heterozygous for a DNA polymorphism within the beta-globin gene, it was possible to clone and sequence the beta-globin gene identical by descent from both the child and her father. A nonsense mutation in codon 121 (GAA----TAA) was found in the beta-globin gene of the child, while the same gene from her father lacked this mutation and was normal. This mutation has not been previously observed among over 200 beta-thalassemia genes characterized in Caucasians. Since the mutation eliminates an EcoRI site in the beta-globin gene, we could show that the mutation is not present in genomic DNA of the father. To rule out germinal mosaicism, sperm DNA of the father was also digested with EcoRI, and the mutant EcoRI fragment was not observed under conditions that would detect the mutation if it were present in at least 2% of sperm cells. Routine HLA and blood group testing supported stated paternity. In addition, studies with 17 DNA probes that detect multiple allele polymorphisms increased the probability of stated paternity to at least 10(8):1. These data provide evidence that the G----T change in codon 121 of the beta-globin gene in the child is the result of a spontaneous mutation that occurred during spermatogenesis in a paternal germ cell. PMID- 3014871 TI - Complete absence of serum alpha-1-antitrypsin in conjunction with an apparently normal gene structure. AB - A family in which a Pinull allele for alpha-1-antitrypsin (A-1-AT) segregates has been studied in detail. Two homozygous sisters have no detectable A-1-AT in their serum as measured with the most sensitive methods currently available. Both have airways obstruction, and one has bullous emphysema. Heterozygotes for Pinull and the common normal allele PiM1 have half-normal serum concentrations of A-1-AT. A restriction enzyme analysis of chromosomal DNA of the two homozygotes and one heterozygote demonstrated the presence of an apparently complete structural gene for A-1-AT. Thus, the genetic defect in Pinull is not a complete or partial deletion of the structural gene. A base pair change that cannot be detected by the restriction enzymes used here, of course, cannot be excluded. Another possibility is a mutation outside the structural gene that affects the synthesis of the protein. PMID- 3014873 TI - Use of sodium bicarbonate vials or syringes to buffer cardioplegic solutions. PMID- 3014874 TI - Divalent ion handling in human kidney donors with increased blood pressure after uninephrectomy. AB - In a prospective study of 21 normal human kidney donors, increases in blood pressure were found in seven of 12 males and in three of nine females within the first year after uninephrectomy. Donors with blood pressure increases were characterized by greater weight and body mass indexes. In addition, urinary phosphate excretion was positively correlated (r = 0.73, p less than 0.001) and tubular reabsorption of phosphate negatively correlated (r = -0.61, p less than 0.01) with blood pressure at the follow-up periods in which increases were observed. All donors experienced an increase in parathyroid hormone levels and urinary cyclic AMP excretion. These changes were accompanied by decreases in urinary calcium and serum phosphate values. Thus, the increase in blood pressure took place in a milieu similar to that of "normocalcemic" hyperparathyroidism. The correlation of phosphate excretion and blood pressure in normal donors suggests an important role for phosphate metabolism in the genesis of hypertension associated with loss of renal mass. PMID- 3014872 TI - The use of restriction fragment length polymorphisms in paternity analysis. AB - This paper examines the utility of restriction fragment length polymorphisms (RFLPs) for paternity analysis. While, on the average, 99% of falsely accused males can be excluded with the standard battery of blood group antigens, red cell enzymes, serum proteins, and HLA antigens, there are still mother-child pairs for whom the exclusion probability is not high. It has been suggested that additional resolution would be available with RFLPs. We have examined the strategic aspects of using RFLPs for paternity analysis, comparing the efficacy and cost of a multimarker haplotypic set with those of a comparable set of unlinked RFLPs, using published frequencies for the beta-globin complex, the serum albumin region, and the growth hormone region. There are four major findings. (1) Greater resolution is obtained with a carefully chosen set of tightly linked RFLPs producing chromosomal haplotypes than with a comparable set (same allele frequencies) of unlinked markers, but only if it is possible to establish linkage phase unambiguously. (2) Assay of linked sets is cheaper than is the assay of unlinked markers, but the cost advantage is optimized with sets of no more than two or three linked markers. (3) Also, with more than two or three tightly linked markers, the haplotypic frequencies are too poorly estimated to provide a reliable measure of the probability of paternity for unexcluded males, given the sample sizes likely to be available in the near future. (4) Optimal resolution, minimal cost, and acceptable accuracy are obtained with several independent sets of no more than two or three tightly linked RFLP markers each. With current technology, RFLP analysis is more expensive for the same level of genetic resolution than is the standard battery, but gradual replacement of the latter can be anticipated as economies of scale reduce the cost of the DNA technology. PMID- 3014876 TI - Secondary pulmonary alveolar proteinosis occurring in two patients with acquired immune deficiency syndrome. AB - This report describes two patients with acquired immune deficiency syndrome (AIDS) in whom respiratory failure and opportunistic infection associated with secondary alveolar proteinosis developed. In one patient, the alveolar proteinosis was apparently secondary to Mycobacterium tuberculosis and in the other to Pneumocystis carinii and cytomegalovirus infection. Both patients died of respiratory failure, and it was suspected that secondary alveolar proteinosis could have been a contributing cause of death. PMID- 3014875 TI - Non-small-cell lung cancer. Major cause of late mortality in patients with small cell lung cancer. AB - Among 360 patients with small cell lung cancer treated in National Cancer Institute therapeutic trials from 1973 to 1982, 40 were two-year cancer-free survivors. Of these 40 patients, six had later development of non-small-cell lung cancer at 3.5 to 8.0 years (median 5.1) after the diagnosis of small cell lung cancer. Three had the second malignant tumor in the contralateral lung, one in a different lobe, and two in the same lobe as the initial small cell lung cancer. Ten patients had relapses of small cell lung cancer at 2.1 to 6.2 years (median 3.2) from diagnosis. Three recurrences were in the same site or lobe as the initial lesion, four in the same lobe and in sites outside the thorax, and three solely in sites outside the thorax. It is concluded that these non-small-cell lung cancers usually represent second primary lung tumors and that most late small cell lung cancers represent relapses occurring up to 6.2 years from diagnosis. In this study, the risk of development of non-small-cell lung cancer after two years of disease-free survival following small cell lung cancer is 4.4 percent per person-year, approximately 10 times higher than the rate of 0.5 percent previously determined in screening studies of men at high risk for lung cancer. Non-small-cell lung cancer represents more than a third of lung cancer deaths in patients with small cell lung cancer surviving beyond two years from diagnosis and more than half of lung cancer deaths beyond three years. It is recommended that all patients treated for small cell lung cancer discontinue smoking. PMID- 3014877 TI - Susceptibility of aerobic gram-negative bacilli to aminoglycosides. Effects of 45 months of amikacin as first-line aminoglycoside therapy. AB - Amikacin was instituted as the primary empiric aminoglycoside at the San Juan Veterans Administration Medical Center in January 1982; at that time, 16 percent of the strains at the hospital were gentamicin-resistant. A prospective surveillance study was designed to correlate detection of bacterial resistance with aminoglycoside use. In the current report, the baseline period, during which gentamicin was the first-line aminoglycoside, accounting for 61 percent of overall aminoglycoside use, is compared with the period from January 1982 to September 1985, during which the first-line aminoglycoside was amikacin, accounting for 85 percent of overall use. This study is ongoing. During the two periods, the patient population did not differ with regard to aminoglycoside therapy, indications, or overall aminoglycoside use (541 versus 680 patient days per month). Among the gram-negative bacilli isolated, the percent of strains resistant to amikacin was as follows: pre-baseline period/baseline period, 0.8/0.2 percent; amikacin-usage period, 3.6 percent. Resistance to gentamicin and tobramycin during the period of amikacin use decreased from 16 to 11 percent for gentamicin and from 17 to 11 percent for tobramycin. The decrease in resistance of the gram-negative bacilli to gentamicin varied among strains: the resistance of Escherichia coli decreased from 8 to 4 percent; that of Proteus mirabilis, from 12 to 5 percent; that of indole-positive Proteus, from 19 to 12 percent; that of Acinetobacter, from 57 to 23 percent; that of Citrobacter, from 15 to 7 percent; and that of Pseudomonas aeruginosa, from 24 to 16 percent. During the amikacin-usage period, amikacin resistance was unchanged for most strains, with the exception of P. aeruginosa, the resistance of which increased from 4.5 to 7.8 percent. Of the 4,795 strains isolated, 174 were resistant to amikacin; of these, 29 Pseudomonas strains were studied for all mechanisms of resistance. Changes in permeability were exhibited by 11 of the 29 strains; 14 strains exhibited the AAC(6')-I enzyme, 10 strains exhibited the APH(3')-II enzyme, and two strains exhibited ANT(2") in addition to some other unidentified mechanism. Multiple enzyme production was found in 15 of the strains. The use of amikacin as a first line aminoglycoside is associated with a decrease in resistance to other aminoglycosides and a slight increase in overall resistance to amikacin among aerobic gram-negative bacilli. The usefulness of amikacin has not been affected at our institution. PMID- 3014878 TI - Transmission of lymphadenopathy-associated virus/human T lymphotropic virus type III in sexual partners. Seropositivity does not predict infectivity in all cases. AB - To investigate transmission of lymphadenopathy-associated virus (LAV)/human T lymphotropic virus type III (HTLV-III) in long-term sexual partners, and the relationship between lymphadenopathy-associated virus seropositivity and transmission, nine couples (five heterosexual and four homosexual) at increased risk for acquired immune deficiency syndrome (AIDS) were studied. In two heterosexual couples, transmission of lymphadenopathy-associated virus from a seropositive man at increased risk to his monogamous wife occurred. In one couple, the wife of a man with hemophilia had lymphadenopathy-associated virus antibody and decreased T helper cells; in the other couple, the wife of a bisexual intravenous drug-user had AIDS. Neither woman had a recognized AIDS risk except marriage to a seropositive man at increased risk. However, study of the other couples revealed that regular sexual contact with seropositive persons over long periods did not always lead to evidence of lymphadenopathy-associated virus infection. This study suggests that presence of lymphadenopathy-associated virus antibody does not always indicate a high degree of infectivity. PMID- 3014881 TI - Attack rate for neonatal herpesvirus. PMID- 3014879 TI - Nitrous oxide, an opioid addictive agent. Review of the evidence. AB - Pharmacologic evidence that nitrous oxide is addictive through direct interaction with the endogenous opioid system includes the possibility that it is a partial agonist and acts at the mu, kappa, and sigma opioid receptors. The medical and psychiatric complications of its abuse are discussed with special reference to the 32 cases of myeloneuropathy so far reported; other dangers are also mentioned. Notwithstanding the extremely low incidence of reported cases of nitrous oxide addiction compared with all other addictive substances, greater controls should be placed on its commercial availability to at least maintain its low level of abuse. PMID- 3014880 TI - Coxsackievirus-positive cervices in women with febrile illnesses during the third trimester in pregnancy. AB - Coxsackievirus B5 infection was demonstrated in five of seven third-trimester pregnant women with undifferentiated febrile illnesses or aseptic meningitis. Coxsackievirus B5 was recovered from the cervix and throat in four women and from the rectum in three. No obvious illnesses were evident in the babies. These findings suggest that previously unrecognized cervical enterovirus carriage or infection is common in infected pregnant women in the last trimester and that subsequent neonatal infection at delivery may result. PMID- 3014882 TI - Insulinoma complicating pregnancy: case report and review of the literature. AB - A 24-year-old woman, gravida 1, para 0, experienced recurrent attacks of headaches and bizarre behavior from the sixth week of gestation onward. Three days before confinement, she lapsed into coma and was delivered of a normal child. Plasma glucose, insulin, and C-peptide levels were diagnostic of insulinoma. Subsequently, after she spontaneously regained consciousness, a pancreatic tumor was removed at laparotomy with complete resolution of symptoms. The problems of diagnosing insulinoma during pregnancy are discussed. PMID- 3014883 TI - Prognostic value of peritoneal washings in patients with malignant mixed mullerian tumors of the uterus. AB - Peritoneal washings from 36 women with all pathologic stages of mixed mullerian tumors of the uterus (27 homologous stroma, nine heterologous stroma) were reviewed in a blinded retrospective fashion for the presence and type of malignant cells collected. Malignant tumor cells were demonstrated in the washings of 16 patients; 13 contained adenocarcinoma only, two contained adenocarcinoma and sarcoma, and one contained sarcoma cells only. Cox's logistic regression analysis showed that cytologic examination of peritoneal washings when combined with pathologic staging provides a statistically significant discriminant of disease-free survival. Patients with a favorable prognosis (pathologic Stage I and peritoneal washings free of malignancy) had sevenfold increased disease-free survival compared with survival of the patients with an unfavorable prognosis (pathologic Stages II, III, or IV or malignant cells present in peritoneal washings). Three of the 18 patients with pathologic Stage I disease manifested malignant tumor cells in the peritoneal washings and all three patients died of disease in less than 1 year. The presence of malignant cells in peritoneal washings in patients with mixed mullerian tumor apparently limited to the uterus suggests a clinical course that is similar to that of those with more advanced disease. PMID- 3014884 TI - Influence of extracellular [Ca2+] on secretory and redox responses to CCK-8 in perfused rat pancreas. AB - There is a distinct discrepancy between the dose-dependent secretory responses (pancreatic protein output and juice flow) and the dose-dependent redox responses of cytochromes aa3, b, and c+c1 to various concentrations of CCK-8: a bell-shaped relation for the secretory responses contrasts with a sigmoidal relation for the redox responses. Continuous stimulation with CCK-8 at physiological low concentrations (5-10 pM) induced an increase in pancreatic protein output with little if any reduction of cytochromes. Continuous stimulation with CCK-8 at a pharmacological intermediate concentration (50 pM), however, induced an increase in pancreatic protein output with delayed reduction of cytochromes. The secretory and redox responses were completely abolished when CaCl2 was removed from the perfusing and bathing solution. An almost linear relation was found between the magnitude of the protein output in response to 50 pM CCK-8 and extracellular [Ca2+] [( Ca2+]o) in the range of 0.5-2.5 mM. Similar relations were also found between the levels of reduction of cytochromes and [Ca2+]o. These results are compatible with the view that continuous stimulation with CCK-8 at a pharmacological or a pathological concentration may cause excess increase in intracellular [Ca2+] and intramitochondrial Ca2+ and in this way may accelerate the formation of reducing power for oxidative phosphorylation. PMID- 3014886 TI - Effects of protein reagents on electrotonic coupling in crayfish septate axon. AB - The lateral giant axons of the crayfish nerve cord are composed of segments contributed by each ganglion, which are electrotonically coupled by way of gap junctions. We have investigated the involvement of protein residues in regulating the resistance of crayfish junctional channels by determining effects of group specific protein reagents. When applied to well-coupled axons, the sulfhydryl group reagents N-ethylmaleimide (NEM) and diamide uncoupled the segments; junctional resistance (Rj) was increased without changing membrane resistance or axoplasmic pH (pHi). The uncoupling produced by NEM could be reversed by alkalinization of the cytoplasm (addition of ammonium chloride to the external medium). Another sulfhydryl reagent (p-chloromercuribenzoic acid) increased Rj to a lesser extent. A disulfide reagent and three amino and three carboxyl group reagents had no effect on the Rj of these axons. The effect of group-specific reagents on partially uncoupled axons was tested by applying the drugs to axons previously exposed to weak acids. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline recoupled partially uncoupled axons by decreasing Rj and prevented subsequent uncoupling of the junction by low pHi. Another carboxyl group reagent, as well as sulfhydryl and amino group reagents, either had no effect or uncoupled the axons further by increasing Rj. These experimental results suggest that amino acid residues, possibly containing carboxyl and sulfhydryl groups, control the opening and closing of junctional channels and may thus be associated with the channels' active sites. PMID- 3014885 TI - Parathyroid hormone inhibits phosphate transport in OK cells but not in LLC-PK1 and JTC-12.P3 cells. AB - Na+-dependent phosphate transport and its response to parathyroid hormone (PTH) has been investigated in three continuous cell lines of renal epithelial origin (LLC-PK1, JTC-12.P3, and OK). The apparent Km for phosphate was similar, but the maximal transport rate (Vmax) was markedly different in the three cell lines. PTH and forskolin produced an increase of cellular adenosine 3',5'-cyclic monophosphate (cAMP) in all cell lines, but Na+-dependent phosphate transport was inhibited exclusively in the OK cells (a threefold reduction of influx after 4 h of exposure to 10(-10) M PTH). The change in phosphate transport is accounted for by a lowered Vmax (30.8 +/- 5.3 vs. 10.2 +/- 1.1 pmol X mg-1 X 3 min-1). The reduction in phosphate transport was reversible, such that 5 h after removal of PTH the Vmax had increased threefold over the inhibited state. Addition of PTH did not alter Na+-dependent L-alanine influx in the OK cells. Experiments with apical membrane vesicles showed that the change in Vmax occurred at the membrane level. It is concluded that the regulatory event responsible for PTH-reduced phosphate transport is beyond cAMP. Of the cell lines studied, only OK cells have a complete regulatory cascade. PMID- 3014887 TI - Halothane is less suppressive than pentobarbital on reflex and neural activation of pancreatic F-cell. AB - To determine the suitability of halothane anesthesia for studies of parasympathetic control of the endocrine pancreas in dogs, we assessed the effect of halothane on reflex, direct neural, and direct chemical activation of the parasympathetic input to the islet. Levels or output of pancreatic polypeptide (PP), an islet hormone under predominant cholinergic influence, were used as an indicator of the degree of parasympathetic activation and its potential suppression by anesthesia. Reflex stimulation of the parasympathetic nervous system by 2-deoxy-D-glucose (2-DG) in dogs anesthetized with halothane (0.8%) caused a fourfold increase in plasma PP levels, equivalent to the response in conscious dogs. In contrast, 2-DG at this dose and at a threefold higher dose did not alter PP levels in dogs anesthetized with pentobarbital (30 mg/kg iv), suggesting that halothane at this dose is not suppressive and that pentobarbital is very suppressive on reflex activation of the parasympathetic nerves to the pancreas. Bilateral electrical stimulation of the cervical vagi in halothane anesthetized dogs elicited a sixfold increase in the pancreatic output of PP. The same stimulation caused only a twofold increase of PP output in pentobarbital anesthetized dogs. These data suggests that halothane is also less inhibitory than pentobarbital on either peripheral neurotransmission or pancreatic F-cell responsiveness. The effect of direct activation of the F-cell by bethanechol did not differ between the two anesthesias. Therefore, the attenuated PP response to vagal stimulation in pentobarbital-anesthetized dogs is probably due to an action of pentobarbital on peripheral neurotransmission, perhaps at the intrapancreatic parasympathetic ganglia.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014888 TI - Central vasopressin system mediation of acute pressor effect of gamma-MSH. AB - Intravenous (iv) administration of gamma-melanocyte-stimulating hormone (gamma MSH) produces central sympathetically mediated pressor and cardioaccelerator effects and increases the activity of hypothalamic vasopressinergic neurons. The autonomic actions are similar to infusion of vasopressin (Vp) into the hindbrain of 4th ventricle (Ven). To ascertain whether activation of the central Vp system is the proximate cause of the pressor effects of gamma-MSH, we investigated the effects of gamma-MSH in rats pre- and postblockade of central nervous system Vp receptors and in rats with a hereditary lack of vasopressin (Brattleboro strain). Central Vp receptor blockade significantly reduced (80%) the pressor effects of iv gamma-MSH. As a control, iv administration of the antagonist, while effective in blocking the pressor effect of iv Vp, had no effect on the gamma-MSH pressor response. When compared with their genetic controls (Long-Evans strain), Brattleboro rats also had greater than 80% reduction in their pressor response to iv gamma-MSH. The results indicate that circulating gamma-MSH activates the central Vp system to produce its sympathoexcitatory pressor effects. PMID- 3014889 TI - Rat visceral yolk sac: a target for parathyroid hormone action. AB - The stimulation of cAMP production by parathyroid hormone [human PTH-(1-34)] in fetal membranes was studied over the last 9 days of pregnancy in the rat. PTH significantly stimulated cAMP production in the placental and capsular parts of the visceral yolk sac at all days studied. In contrast PTH stimulated cAMP production in the amnion only at days 14 and 17 and had no effect in the maternal and fetal sides of the placenta nor in the parietal yolk sac before it degenerated (day 14). The biochemical characteristics of PTH-stimulated cAMP production in visceral yolk sac are similar to those reported in other systems: a) maximal stimulation was obtained with 1.2 X 10(-7) M hPTH-(1-34) and 50% stimulation achieved by 2-3 X 10(-8) M; and b) the antagonist [Nle8,Nle18,Tyr34] bovine PTH-(3-34) amide competitively inhibited the stimulation of cAMP production by hPTH-(1-34). These results strongly suggest that the rat visceral yolk sac is a target organ for PTH during the last 9 days of pregnancy. PMID- 3014890 TI - Binding of IGF I and IGF I-stimulated phosphorylation in canine renal basolateral membranes. AB - To characterize the interaction of the renal proximal tubular cell with insulin like growth factor I (IGF I), we measured binding of 125I-IGF I to proximal tubular basolateral membranes from dog kidney and induced IGF I-stimulated phosphorylation of basolateral membranes. Specific binding of 125I-IGF I to basolateral membranes was observed that was half-maximal at between 10(-9) and 10(-8) M IGF I. 125I-IGF I was affinity cross-linked to a 135,000 Mr protein in basolateral membranes that was distinct from the alpha-subunit of the insulin receptor and from the IGF II receptor. IGF I-stimulated phosphorylation of a 92,000 Mr protein was effected in detergent-solubilized membranes incubated with 100 microM [gamma-32P]ATP. The 32P-labeled protein was distinct from the beta subunit of the insulin receptor, the 32P phosphorylation of which was stimulated by insulin. We conclude that specific receptors for IGF I are present in the basolateral membrane of the renal proximal tubular cell. Physiological actions of IGF I at this nephron site may occur through the binding of this peptide circulating in plasma, to specific basolateral membrane receptors, followed by IGF I stimulated phosphorylation. PMID- 3014891 TI - Role of vasopressin in stimulation of ACTH secretion by angiotensin II in conscious dogs. AB - Three series of experiments were performed in conscious dogs to test the possibility that the stimulation of adrenocorticotropin (ACTH) release by angiotensin II (ANG II) is mediated by arginine vasopressin (AVP). In the first protocol, the effect of ANG II on ACTH release was studied in dogs in which endogenous AVP levels had been increased by water deprivation. Water deprivation for 24 h increased plasma AVP concentration from 3.0 +/- 0.5 to 7.7 +/- 0.5 pg/ml (P less than 0.01) and increased the AVP response to the highest dose of ANG II (20 ng X kg-1 X min-1). Despite these changes, water deprivation failed to increase the ACTH response to ANG II. Next, the contribution of endogenous AVP to the stimulation of ACTH release by ANG II was examined using the V1-receptor antagonist, d(CH2)5Tyr[Met]-AVP (10 micrograms/kg iv). The ACTH response to ANG II in the presence of the AVP antagonist (66.4 +/- 3.1 to 100.1 +/- 15.9 pg/ml) was not significantly less than that in its absence (53.0 +/- 4.8 to 72.2 +/- 11.1 pg/ml). Finally, ANG II and AVP were infused in combination to determine whether there is a synergism between these two peptides in the release of ACTH. In one protocol, AVP and ANG II were infused separately and in combination. The ACTH response to ANG II and AVP in combination (48.7 +/- 6.5 to 61.5 +/- 8.5 pg/ml) was not enhanced compared with the responses to ANG II (59.8 +/- 7.3 to 71.0 +/- 10.1 pg/ml) or AVP (48.8 +/- 5.7 to 55.6 +/- 6.5 pg/ml) alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014892 TI - Angiotensin II receptor alterations during pregnancy in rabbits. AB - Despite activation of the renin-angiotensin system during pregnancy, renal and peripheral vascular blood flows increase, and the systemic blood pressure and the pressor response to exogenous angiotensin II (Ang II) fall. Gestational alterations in Ang II receptors could contribute to these changes. Ang II binding parameters were determined utilizing 125I-Ang II in vascular (glomeruli and mesenteric arteries) and nonvascular (adrenal glomerulosa) tissues from 24- to 28 day pregnant rabbits. Comparisons were made utilizing tissues from nonpregnant rabbits. Binding site concentrations (N) and dissociation constants (Kd) were obtained by Scatchard analyses of binding inhibition data. In glomeruli from nonpregnant and pregnant rabbits, N was 515 +/- 84 and 300 +/- 54 fmol X mg-1 protein (P less than 0.005; n = 8), respectively. Kd did not differ (P greater than 0.05). In mesenteric artery membranes from nonpregnant (n = 3) and pregnant (n = 4) rabbits, N was 304 +/- 21 and 112 +/- 23 fmol X mg-1 (P less than 0.005), respectively. Kd did not differ. Neither N nor Kd differed in adrenal glomerulosa tissues (n = 6). Meclofenamate (M) inhibits prostaglandin synthesis, reduces plasma renin activity, and enhances the pressor response to infused Ang II in pregnant rabbits. Administration of M to pregnant rabbits increased N in glomerular and in mesenteric artery membranes from 298 +/- 16 to 381 +/- 8 fmol X mg-1 (n = 3) and from 144 +/- 13 to 218 +/- 13 fmol X mg-1 (n = 4), respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014893 TI - Relationship between nerve blood flow and intercapillary distance in peripheral nerve edema. AB - Some human and experimental neuropathies are characterized by endoneurial edema and increased intercapillary distance (ICD). This may potentially produce chronic endoneurial ischemia. To examine the relationship between nerve blood flow (NBF) and ICD we measured NBF in rats with experimental galactose neuropathy (EGN), a model where ICD is known to be increased. Simultaneous measurements of NBF in the center and subperineurial region were made in normal and edematous tibial nerves using hydrogen-sensitive microelectrodes and hydrogen polarography. NBF was significantly reduced in rats with EGN when compared with controls. A second finding was that in half the rats with EGN there was a greater reduction in NBF in the subperineurial region, a site of maximal ICD increase. In contrast, NBF was similar in central and peripheral regions in control rats. These findings support the hypothesis that an increase in ICD produces a reduction in NBF. Further support for the hypothesis is derived from a computer model of the effect of changes in ICD on endoneurial oxygen tension. We conclude that a chronic reduction in NBF may participate in the pathogenesis of edematous neuropathies. PMID- 3014894 TI - Importance of calcium in renal renin release. AB - The sequence of intracellular events that lead to renin release is incompletely defined. Accordingly, we examined the interrelationship of two important factors in the process: renin release coupled to cAMP and renin release related to a decrease in intracellular calcium activity (Cai). Rat renal cortical slices were used to study these relationships in vitro. In the initial studies, cAMP-coupled renin release was established for isoproterenol (10(-5) M), prostacyclin (PGI2; 10(-6) M), and forskolin (10(-5) M). Each agent caused an increase in renin release and tissue cAMP levels, which were inhibited by the addition of the adenyl cyclase inhibitor 2',5'-dideoxyadenosine (DDA, 10(-5) M) to the media. Diltiazem (10(-4) M) and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB 8; 0.6 X 10(-4) M) are believed to decrease Cai by different mechanisms; each of these agents caused a significant increase in renin release. Renin release stimulated by diltiazem, and TMB-8 was not inhibited by either DDA or indomethacin. The calcium ionophore A23187 (17 X 10(-6) M) and vanadate (10(-3) M) were next added to produce an increase in Cai. Both of these agents blunted renin release produced by isoproterenol, PGI2, and forskolin. These results provide strong indirect support for an inverse relationship between Cai and renin release in the juxtaglomerular cell. The results also imply that changes in Cai occupy a step that is distal to cAMP-coupled events in the sequence of intracellular events which culminate in renin release. PMID- 3014895 TI - Colonic peristaltic reflex: identification of vasoactive intestinal peptide as mediator of descending relaxation. AB - Isolated segments of rat and guinea pig midcolon were used to examine the neurotransmitters responsible for ascending contraction and descending relaxation components of the peristaltic reflex. Graded radial stretch of the extreme orad end caused only descending relaxation accompanied by significant release of vasoactive intestinal peptide (VIP) in rat (82%, P less than 0.005) and guinea pig (47%, P less than 0.05). Radial stretch of the caudad end caused only ascending contraction without VIP release. VIP antiserum (1:480 to 1:60) inhibited descending relaxation in a concentration-dependent manner at all grades of stretch (40 +/- 12% to 74 +/- 15%) but augmented ascending contraction (25 +/- 7% to 108 +/- 21%). Axonal blockade with tetrodotoxin and ganglionic blockade with hexamethonium abolished both components, indicating the participation of cholinergic neurons. Atropine and the tachykinin antagonist [D-Pro2,D Trp7,9]substance P inhibited ascending contraction but not descending relaxation; their combination abolished ascending contraction at all grades of stretch. We conclude that cholinergic neurons coupled to VIP motor neurons regulate descending relaxation and that cholinergic neurons coupled to tachykinin and cholinergic motor neurons regulate ascending contraction. PMID- 3014897 TI - Enhanced renal tubular ouabain-sensitive ATPase in streptozotocin diabetes mellitus. AB - The effect of streptozotocin-induced diabetes mellitus on rat renal ouabain sensitive ATPase in six distinct nephron segments was studied. Twenty-four hours after administration of streptozotocin, blood glucose increased threefold (P less than 0.001), and glucosuria was evident. Aldosterone levels increased almost twofold (P less than 0.001). Ouabain-sensitive ATPase increased in the proximal segments PC (proximal convoluted tubule) and PS (proximal straight tubule) by 43 and 62%, respectively, (P less than 0.001) and CD (cortical collecting duct) ouabain-sensitive ATPase increased 77% (P less than 0.001). Ouabain-sensitive ATPase in the cortical (CTAL) and medullary (MTAL) thick ascending limbs of Henle's loop and in the DC (distal convoluted tubule) remained unchanged after 24 h of streptozotocin administration. Eight days after streptozotocin administration, when glomerular filtration rate (GFR) was already markedly elevated, ouabain-sensitive ATPase remained increased in the PC, PS, and CD but was significantly less compared with the activity after 24 h (P less than 0.05), whereas in the CTAL and MTAL a marked increase in ouabain-sensitive ATPase occurred by 54% in the CTAL and 65% in the MTAL (P less than 0.001). Aldosterone levels remained elevated compared with control but less than after 24 h. Pretreatment with deoxycorticosterone acetate abolished the increase in ouabain sensitive ATPase in the CD. These findings show that streptozotocin-induced diabetes mellitus in the rat is associated with a substantial increase in ouabain sensitive ATPase activity along most of the nephron. This increase in enzyme activity may represent a mechanism of physiological adaptation of the nephron to maintain electrolyte homeostasis in diabetes in face of the increased GFR and osmotic diuresis. PMID- 3014896 TI - Actions of vasoactive intestinal peptide and secretin on chief cells prepared from guinea pig stomach. AB - Vasoactive intestinal peptide and secretin increased cellular cAMP and pepsinogen secretion in dispersed chief cells from guinea pig gastric mucosa. With each peptide there was a close correlation between the dose-response curve for changes in cellular cAMP and that for changes in pepsinogen secretion. Vasoactive intestinal peptide-(10-28) and secretin-(5-27) had no agonist activity and antagonized the actions of vasoactive intestinal peptide and secretin on cellular cAMP and pepsinogen secretion. Studies of binding of 125I-vasoactive intestinal peptide and of 125I-secretin indicated that gastric chief cells possess four classes of binding sites for vasoactive intestinal peptide and secretin and that occupation of two of these classes of binding sites correlates with the abilities of vasoactive intestinal peptide and secretin to increase cellular cAMP and pepsinogen secretion. What function, if any, is mediated by occupation by the other two classes of binding sites remains to be determined. PMID- 3014898 TI - Superficial and juxtamedullary nephron function during converting enzyme inhibition. AB - The influence of the renin-angiotensin system on whole-kidney and regional single nephron function was studied in anesthetized Munich-Wistar rats by use of a converting enzyme inhibitor (CEI), captopril (3 mg . h-1 . kg-1 body wt-1). Single-nephron glomerular filtration rate (SNGFR) was measured in superficial (S) and juxtamedullary (JM) nephrons by a micropuncture technique that blocked tubuloglomerular feedback (TGF) activity. Hydrostatic free-flow pressure (FFP) measurements were conducted in S proximal tubules, in JM loops of Henle, and in papillary vasa recta. Glomerular filtration rate (GFR) and urinary electrolyte excretion were measured in the contralateral kidney. In the control situation SNGFR of S nephrons was 35.4 +/- 2.24 nl . min-1 . g kidney wt-1 and of JM nephrons was 75.0 +/- 9.41 nl . min-1 . g-1 kidney wt-1. Thus it was more than twice as high in JM as in S nephrons when the TGF activity was blocked. In this situation administration of CEI had no additional effect on either S or JM nephrons. However, administration of CEI resulted in a significant increase in whole-kidney GFR by 25% (P less than 0.05) and in urine flow rate by 60% (P less than 0.001) under free-flow conditions. Further, intratubular FFP increased significantly in JM nephrons by an average of 2.9 +/- 0.52 mmHg (P less than 0.001), indicating an increase in tubular urine flow in JM nephrons, whereas S nephrons were unaffected. These results suggest an active preglomerular vasoconstriction in JM nephrons under normal free-flow conditions. This seems to be mediated by TGF and modulated by angiotensin II. PMID- 3014900 TI - Amiloride reduces potassium conductance in frog kidney via inhibition of Na+-H+ exchange. AB - The existence of a carrier-mediated Na+-H+ exchange has been described recently in many epithelial and nonepithelial tissues including the diluting segment of the amphibian kidney. In this preparation the Na+-H+ exchanger is dramatically stimulated by so-called K+ adaptation (chronic exposure of animals to high potassium) and completely inhibited by the diuretic drug amiloride. We performed electrophysiological experiments in diluting segments of the isolated perfused frog kidney to investigate whether amiloride affects the conductance properties of this epithelium. Amiloride dramatically increased the transepithelial resistance and the ratio of lumen over peritubular cell membrane resistance. Cell membrane potential changes, induced by luminal K+ concentration steps, were blunted by luminal application of amiloride, by luminal Na+-free perfusates, or by acidification of the kidney perfusion solution. K+ secretory net flux, measured by K+-sensitive microelectrodes, decreased by half in presence of the diuretic. The experiments reveal that amiloride reduces the K+ conductance of the luminal cell membrane of frog diluting segment via inhibition of the luminal Na+ H+ exchanger. This decreases transepithelial K+ net secretion in this nephron segment. PMID- 3014899 TI - Regulation of chloride self exchange by cAMP in cortical collecting tubule. AB - The hormonal control of Cl transport was examined in rabbit cortical collecting tubules using the lumen-to-bath 36Cl tracer rate coefficient (KCl, nm/s). Tracer movement via Cl-HCO3 exchange was minimized by using HCO3-CO2-free solutions. The electrical driving force was minimized by treating with amiloride. Under these conditions, net Cl transport was zero, yet there was a large KCl that fell 88% on removing bath (trans) Cl. These results are consistent with the mechanism of tracer flux being predominantly Cl self exchange. KCl fell spontaneously with time in vitro; after this decline KCl could be stimulated with 8-bromo-cAMP. cAMP present from the onset of perfusion prevented the time-dependent fall in KCl. When tracer movement was restricted to diffusion by eliminating Cl self exchange (0 Cl bath), cAMP had no effect on KCl. Although both isoproterenol and vasopressin are known to stimulate adenylate cyclase in this epithelium, only isoproterenol mimicked the cAMP effect on KCl. The isoproterenol effect was blocked by either propranolol or prostaglandin E2. Lumen addition of the disulfonic stilbene DIDS had no effect on KCl. Lumen addition of furosemide or trichloromethiazide had minimal or no effect. Taken together, these results indicate that Cl self exchange is regulated by beta-adrenergic agents acting via cAMP. The lack of an effect of vasopressin suggests cellular heterogeneity in this response to cAMP. PMID- 3014901 TI - Vasopressin increases cytosolic free calcium concentration in glomerular mesangial cells. AB - Cytosolic free calcium concentration ([Ca2+]f) was determined in cultured rat glomerular mesangial cells under basal conditions and after exposure to arginine vasopressin (AVP) or angiotensin II (ANG II). [Ca2+]f was determined using quin 2 or fura-2, two intracellular fluorescent probes. [Ca2+]f was measured to be 102 +/- 3 nM (n = 154) using quin 2 and 82 +/- 4 (n = 34) using fura-2. AVP and ANG II increased [Ca2+]f. Maximal levels of [Ca2+]f were achieved in less than 10 s after addition of the hormone. This peak value was followed by a rapid fall toward the base line. With fura-2 as the intracellular Ca2+ indicator, [Ca2+]f increased from 74 +/- 7 to a peak of 578 +/- 39 nM (n = 17) with 100 nM AVP. At 115 s after addition of AVP, [Ca2+]f was 125 +/- 9 nM. Similar peak levels of [Ca2+]f were observed using quin 2. The increase in [Ca2+]f was due in large part to release of Ca2+ from intracellular stores, since reduction in extracellular free [Ca2+] with EGTA did not prevent the hormone-induced increase in [Ca2+]f, although it did result in a decreased peak level and a more rapid return to base line. The AVP-induced increase in [Ca2+]f was blocked by the V1 receptor antagonist (CH2)5Tyr(Me)VDAVP. Neither isoproterenol, which increased adenylate cyclase activity, nor dibutyryl cAMP had any affect on [Ca2+]f directly or on the AVP-induced increase in [Ca2+]f. In this report we present the first direct measurements of [Ca2+]f and hormone-induced changes in [Ca2+]f in glomerular mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014902 TI - Ambulatory pulmonary arterial pressures in humans: relationship to arterial pressure and hormones. AB - Six healthy volunteers were studied by use of a continuous ambulatory recording technique to document the normal range and variability of pulmonary artery pressure (PAP) and to examine its relationship to systemic arterial pressure (SAP) both at rest and during standardized interventions. Vasoactive hormone levels were measured at frequent intervals. Over 8-10 h of study the mean PAP was 15.7/6.3 mmHg. Parallel changes in PAP and SAP were observed at rest and during exercise and eating. On the contrary, PAP rose and SAP fell with hypoxia, whereas smoking was associated with a rise in SAP but no change in PAP. Sympathetic nervous system activity, as gauged by plasma norepinephrine levels, may have contributed to pressure and heart rate changes during exercise and smoking, but activity of the renin-angiotensin system was not altered by any of the maneuvers. These results provide base-line information on the level of PAP and its variability in healthy volunteers under standardized conditions. Pressures within the systemic and pulmonary circuits change in parallel under some circumstances but move in opposite directions under other conditions. PMID- 3014903 TI - Characterization of vasopressin receptors of rat urinary bladder and spleen. AB - By use of tritiated arginine-8-vasopressin (AVP), vasopressin specific binding sites were detected on Sprague-Dawley rat urinary bladder and spleen. In both tissues, one class of high-affinity binding sites was characterized with an equilibrium dissociation constant of 1.61 +/- 0.22 and 1.91 +/- 0.16 nM and a maximal binding capacity of 155 +/- 5 and 110 +/- 11 fmol/mg of protein, for bladder and spleen, respectively. In both tissues, several experimental arguments suggest that these receptors belong to the V1-vascular type: Highly significant correlations were found between the relative agonistic vasopressor activities of eight AVP agonists and their relative abilities to inhibit [3H]AVP binding to the receptors, whereas no such relationship existed when antidiuretic activities were considered. The same profile was also observed with the antagonistic activities of five AVP antagonists. Moreover, AVP (10(-12)-10(-5) M) did not modify the basal cyclic AMP production in either tissue. As cyclic AMP is known to respond to V2 stimulation, the data suggest that the receptors measured are the V1 type. In Dahl rats the receptor characteristics were modulated by salt diet. More interestingly, the number of spleen vasopressin binding sites was always lower in Dahl salt-resistant animals than in the Dahl salt-sensitive animals receiving either a sodium deficient or a 1% NaCl or an 8% NaCl-containing diet. The exploration of vasopressin receptors regulation should facilitate the comprehension of the role played by AVP in different models of experimental hypertension. PMID- 3014904 TI - Angiotensin receptors and pressor hyperresponsiveness in renal prehypertensive rabbits. AB - This study consisted of five different experiments with conscious rabbits. In experiment 1, the angiotensin II (ANG II) antagonist [Sar1-Ala8]ANG II infused iv into one-kidney rabbits with renal artery stenosis (RAS) of 3 days' duration, at a dose that blocked pressor responses to ANG II, did not decrease the exaggerated pressor responses to norepinephrine (NE). In experiment 2, captopril infused iv into one-kidney, 3-day, RAS rabbits blocked pressor hyperresponsiveness to NE, and the concurrent infusion of [Sar1-Ala8]ANG II did not reestablish pressor hyperresponsiveness, indicating that this ANG II analogue had no agonistic action to promote hyperresponsiveness to NE. In experiment 3, infusion of ANG II at a subpressor dose (6.7 pmol . min-1 . kg body wt-1) into normal rabbits resulted in pressor hyperresponsiveness to NE, which was blocked by [Sar1-Ala8]ANG II. Experiment 4 involved infusing [Sar1-Ala8]ANG II or [Sar1-Ile8]ANG II at various doses into 3-day RAS rabbits, to determine their abilities to attenuate the pressor responses to ANG II (100 ng/kg) and the pressor hyper-responses to NE (800 ng . min-1 . kg-1). [Sar1-Ile8]ANG II decreased the ANG II pressor responses at an ID50 dose of 64 +/- 5 (SEM) pmol . min-1 . kg-1 and attenuated the NE pressor hyper-response at an ID50 dose of 65 +/- 5 pmol . min-1 . kg-1; [Sar1 Ala8]ANG II diminished the ANG II pressor response at an ID50 dose of 757 +/- 247 and the NE pressor hyper-response at an ID50 dose of 10,061 +/- 944 pmol . min-1 . kg-1.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014905 TI - Excretion of artifactual endogenous digitalis-like factors. AB - Radioimmunoassays have been used to detect digoxin-like immunoreactive factors (DLF) in the plasma and urine of hypertensive patients and rats with deoxycorticosterone acetate (DOCA)-salt hypertension. Uninephrectomized rats (n = 9), given 15 mg DOCA . kg-1 . wk-1, were fed a standard rat chow supplemented with 2% NaCl (DOCA-HS); control animals (n = 15) were given vehicle injection and a specially formulated low-salt diet (0.05% NaCl). At 4 wk, DOCA-HS rats were hypertensive (121.4 +/- 10.1 vs. 88 +/- 4.4 Torr, mean +/- SEM, P less than 0.05) and excreted more DLF (2.7 +/- 1.1 vs. 0.2 +/- 0.1 ng digoxin equivalents . day 1, P less than 0.001) compared with control rats. DLF, partially purified from DOCA-HS urine by antidigoxin antibody immunoaffinity chromatography, was found to have a molecular weight less than 2,000, was resistant to acid hydrolysis or proteases, and had many properties of the cardiac glycosides, including inhibition of Na+-K+-ATPase activity, displacement of ouabain from human erythrocyte membranes, and inhibition of 86Rb influx into red blood cells. When DOCA-HS rats were switched to the low-sodium chow, DLF excretion dropped precipitously. No measurable DLF (less than 10 pg/ml) was detected in the plasma of rats eating either chow. However, greater than 95% of the urinary DLF could be attributed to a contaminant in the standard laboratory chow; rats fed the low salt chow supplemented with 2% NaCl excreted much less DLF, and DLF was isolated from the standard chow.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014906 TI - Isoproterenol directly stimulates the Na+-K+ pump in isolated cardiac myocytes. AB - It has been suggested that catecholamines directly stimulate Na+-K+ pump activity in heart; however, these studies on multicellular preparations are confounded by possible alterations of extracellular K+ concentrations ([K+]o). We reinvestigated this problem by studying the effect of the beta-agonist isoproterenol (Iso) on intracellular Na+ activity (aiNa) in ventricular myocytes enzymatically isolated from rabbit heart. In 5 mM [K+]o, 0.1 microM Iso caused a 24.6 +/- 2.0% decrease of aiNa. Exposure to 1 microM Iso only caused a small additional decrease (27.8 +/- 2.4%), while a diminution of aiNa could already be noticed with only 10 nM Iso (12.8 +/- 1.9% diminution). Myocytes superfused with 15 mM [K+]o also exhibited a significant decrease of aiNa (22.9 +/- 3.6%) when exposed to 0.1 microM Iso. These data argue that accumulation of external K+ does not account for the effect of Iso on steady-state aiNa as postulated by Gadsby (Nature Lond. 306: 691-693, 1983). Furthermore, aiNa in myocytes superfused with 1.5 mM [K+]o decreased by only 8.7% on addition of 0.1 microM Iso. The latter observation suggests that the beta-agonist effect on aiNa regulation is directly or indirectly dependent on membrane potential and/or aiNa. Finally, kinetic analysis of the effect of 1 microM Iso on the decrease in aiNa on changing [K+]o from 1.5 to 5 mM demonstrated that the time course of aiNa recovery was accelerated by a factor of 1.9. This readily suggests that active Na+ transport is directly stimulated by Iso. The much greater relative effect of Iso on the time constant than on steady-state aiNa further indicates that Iso may also increase passive Na+ influx. PMID- 3014907 TI - Significance of production of peptide leukotrienes in murine traumatic shock. AB - We studied the formation of a leukotriene metabolite in plasma and bile during traumatic shock. Anesthetized rats subjected to Noble-Collip drum trauma developed a lethal shock state characterized by a survival time of 1.9 +/- 0.3 h, a 4.5-fold increase in plasma cathepsin D activity, and a reduction in mean arterial blood pressure to 45 +/- 2 mmHg compared with 108 +/- 5 mmHg in sham shock controls. Plasma and bile samples were analyzed by reverse-phase high pressure liquid chromatography (HPLC) for peptide leukotrienes (e.g., LTC4, LTD4, and LTE4), and their retention times were confirmed by co-elution with radioactive standards, radioimmunoassay (RIA), and UV spectrophotometry. No leukotrienes or metabolites were found in plasma. The major peptide leukotriene from bile was eluted between LTC4 and LTD4 and corresponds to a metabolite of LTE4, N-acetyl-LTE4, which is also produced during endotoxin shock. The metabolite increased nearly sevenfold in traumatic shock compared with sham trauma. The identity of the metabolite was confirmed by UV scan, which revealed a spectrum consistent with a peptide leukotriene and similar to that of previously reported spectra for N-acetyl-LTE4. In conclusion, peptide leukotrienes are rapidly cleared from the blood and appear in the bile as N-acetyl-LTE4, a metabolite of the peptide leukotrienes. These findings support a role of the peptide leukotrienes in the pathogenesis of traumatic shock. PMID- 3014908 TI - Medullary lesions eliminate ACTH responses to hypotensive hemorrhage. AB - The adrenocorticotropin (ACTH) response to hemorrhage (15 ml . kg-1 . 3 min-1) before and 30 min or 4 days after placement of bilateral electrolytic lesions of the nucleus tractus solitarius (NTS) were examined in anesthetized and in conscious rats. Two groups of rats were anesthetized with pentobarbital sodium (45 mg/kg). Femoral arterial and venous cannulas were placed acutely in the anesthetized group and chronically in the conscious group. Each rat received a hemorrhage 30 min before and 30 min after NTS lesions (in the anesthetized group) and 1 day before and 4 days after NTS lesions (in the conscious group). Plasma ACTH was determined before and 20 min after hemorrhage, and mean arterial blood pressure and heart rate were measured throughout. The baroreceptor reflex (bradycardia caused by a phenylephrine-induced rise in MABP) was determined 5 min before hemorrhage (in the anesthetized group) and 1 day before hemorrhage (in the conscious group) to assess the effectiveness of lesion. Hexamethonium was given to rats that developed hypertension postlesion and to sham-lesioned controls. Plasma ACTH did not increase after hemorrhage 30 min or 4 days after NTS lesions when compared with the other groups (sham, sham with hexamethonium, and missed lesion) and to prelesion controls. Also, lesions of the NTS had no effect on resting ACTH levels 4 days later. Mean arterial pressure and heart rate decreased during hemorrhage to similar extents before and after lesions in all groups. This study demonstrates that lesions of the NTS eliminate the ACTH response to hemorrhage immediately and 4 days after the lesions but have no effect on resting ACTH levels. The result suggests that the NTS is an essential part of the neural pathway for ACTH release after hemorrhage. PMID- 3014909 TI - ACTH, cortisol, and renin responses to arterial hypotension in sheep. AB - This study was designed to investigate adrenocorticotropin (ACTH), cortisol, and renin responses to nitroprusside-induced hypotension in adult sheep. Five sheep were surgically prepared with carotid arterial skin loops at least 1 yr before these experiments. After catheterization of the carotid arteries and external jugular veins the sheep were infused with nitroprusside intravenously at rates of 0, 10, 15, or 20 micrograms . kg-1 . min-1 for 10 min. Nitroprusside produced significantly dose-related decreases in mean arterial pressure and increases in heart rate, plasma ACTH and cortisol concentrations, and plasma renin activity. Hematocrit was significantly increased in the 10- and 20-micrograms . kg-1 . min 1 groups during nitroprusside, probably reflecting contraction of the spleen. After the end of the period of hypotension, hematocrit was significantly decreased in all nitroprusside infusion groups, probably reflecting transcapillary movement of fluid into the vascular space. A posteriori analysis of the data suggests that the ACTH response to nitroprusside infusion was better predicted by the nadir in mean arterial pressure and that the renin activity response was better predicted by the initial rate of decrease of mean arterial pressure during nitroprusside infusion. PMID- 3014911 TI - Harmful psychotherapy experience. AB - This paper reports on an interview study of 47 patients, all mental health professionals, who believed they had been harmed by psychotherapy or analysis. The harmful therapies were therapies characterized as distant, cold, unengaged, and lacking in "human quality," or therapies characterized by intense emotional and/or sexual involvement. PMID- 3014910 TI - Neurohypophysial peptide potencies in cultured anuran epithelia (A6). AB - To characterize the V2 receptor (for antidiuretic hormone), we have studied the effect of a number of neurohypophysial hormone analogues on cyclic AMP (cAMP) accumulation and short-circuit current in cultured epithelia formed by A6 cells. A6 is the designation of a continuous cell line derived from the kidney of Xenopus laevis. The order of potency for stimulating cAMP accumulation and short circuit current in A6 epithelia is like that for stimulating water permeability in toad urinary bladder. As anticipated, arginine vasotocin (AVT), the antidiuretic hormone of Amphibia, is more potent than arginine vasopressin (AVP), the antidiuretic hormone of most mammals. The two hormones differ only in the third amino acid (Phe-3 in AVP is a substitution for Ile-3 in AVT). However, there are a number of striking differences in the responsiveness of these amphibian V2 receptors and mammalian V2 receptors to changes in the 7th, 8th, and 9th amino acids where AVT and AVP are identical. 1) Substitution of Lys-8 for Arg 8 in AVP results in marked loss of potency in Amphibia, whereas there is only modest loss of potency in mammals. 2) Desglycinamide AVP is nearly as potent as AVP in Amphibia, whereas it is inactive in mammals. 2) Tocinoic acid, lacking amino acids 7, 8, and 9, has activity in Amphibia, but pressinoic acid, lacking the same three amino acids, is inactive. PMID- 3014913 TI - Mucocele-like tumors of the breast. AB - Ruptured cysts of the breast containing mucinous material may discharge secretions and epithelium into the surrounding tissues. This is a benign, little known condition analogous to mucocele of the minor salivary glands. The age at diagnosis of six women with mucocele-like lesions (MLL) of the breast averaged 40 years (range, 25-61) and all but one were premenopausal. The lesion caused a mass in five cases. In one it was an incidental finding, and this patient had a separate unrelated nonmucinous intraductal carcinoma treated by mastectomy. One woman was treated by simple mastectomy. Four patients were treated by excision. All have remained well with follow-up of 6-88 months. The histological appearance of MLL of the breast simulates colloid carcinoma and should be considered in the differential diagnosis of such lesions. This is particularly important in young, premenopausal women, among whom colloid carcinoma is very uncommon. The contents of mammary MLL may be difficult to distinguish from colloid carcinoma in an aspiration biopsy. PMID- 3014912 TI - Histologic variation in the skin lesions of the glucagonoma syndrome. AB - Three cases of glucagonoma syndrome were seen in 1 year. Study of the skin biopsies from the first two cases led to a correct diagnosis from skin biopsy of the third case, although it was not suggested clinically. In each case serum glucagon levels were high and a pancreatic tumor was found, with complete remission of symptoms in cases 1 and 3 after resection; case 2 refused surgery and has died. A total of nine skin biopsies from the three patients showed a variety of findings: epidermal necrosis; subcorneal pustules, either isolated or associated with necrosis of the epidermis; confluent parakeratosis, epidermal hyperplasia, and marked papillary dermal angioplasia; and suppurative folliculitis. The clinical lesions in this syndrome vary from bright red macules to annular superficial erosions and flaccid pustules. Similarly, several histopathologic features of the disease can occur, which may represent the progression of the disease. No single histologic feature was specific for the disease, but a combination of the features is probably diagnostic. Therefore, multiple skin biopsies are recommended when this diagnosis is suspected. PMID- 3014914 TI - Locally aggressive granular cell tumor causing priapism of the crus of the clitoris. A light and ultrastructural study, with observations concerning the pathogenesis of fibrosis of the corpus cavernosum in priapism. AB - A case of focal priapism of the clitoris caused by a microscopic granular cell tumor (GCT) is described. This neoplasm is considered locally aggressive because it invades the lumens of peripheral cavernous sinuses of the crus of the clitoris. Caverns adjacent to those invaded by tumor exhibit stasis, telangiectasia, and necrosis of the smooth muscle of the trabecular wall. These alterations lead to telescoping collapse and compression of the cavernous spaces and culminate in fibrosis. Ultrastructurally, replicated basal lamina is found surrounding clusters of granular cells. We suspect that the multilayered lamina, in addition to being produced by tumor cells, is derived from the trabecular endothelium surrounding the caverns invaded by the GCT. The replication of the basal lamina may be provoked by cycles of injury and repair to these vessels caused by repeated episodes of prolonged vascular stasis. A peculiar large vein with perforating branches was observed in the center of the cavernous spaces of the crus. This vein is not found in normal crura and, therefore, represents a morphologic adaptation created to drain the cavernous spaces. PMID- 3014915 TI - Molecular techniques in the study of Salmonella typhi in epidemiologic studies in endemic areas: comparison with Vi phage typing. AB - We examined 141 Salmonella typhi strains of known phage type isolated during ongoing epidemiologic studies in Santiago, Chile, and Lima, Peru. Plasmids were present in 12 (17%) of 70 S. typhi isolates from Santiago and 5 (7%) of 71 isolates from Lima; these plasmids were not associated with antimicrobial resistance. Identical 21 kilobase (kb) plasmids (as defined by restriction endonuclease digest pattern) were present in 13 of the 17 plasmid-containing isolates. Virtually identical digest patterns were identified when chromosomal DNA of selected strains from Santiago, Lima, and the United States was extracted and then digested with restriction endonucleases. The similarities among plasmids and chromosomal digest patterns emphasize the homogeneity and possible clonal origin of S. typhi isolates; these data also suggest that there is only a limited role for plasmid and chromosomal analysis as a substitute for phage typing in epidemiologic studies. PMID- 3014916 TI - Intermediate filaments and salivary gland tumors. PMID- 3014917 TI - Mixed-mode hydrophobic ion exchange for the separation of oligonucleotides and DNA fragments using HPLC. AB - Hydrophobic anion exchangers were formed by cobonding both ionic and hydrophobic ligands to silica gel. These phases were used to separate single-stranded oligonucleotides and double-stranded DNA restriction fragments. By varying the ratio of n-octyldimethylsilane and either 3-chloropropyldimethylsilane or 4 chlorobutyldimethylsilane added during silanization a series of mixed-ligand or mixed-mode stationary phases was created. Concentration and ratio of bonded ligands were determined using a new gas chromatography fluorination method. Total ligand coverage was found to approach 2.1 ligands nm-2 for n-octyldimethylsilane. Bonding reproducibility for mixed-mode phases was good. Nucleic acid separations were achieved under gentle mobile phase conditions by using the stationary phase as an easily modifiable variable. PMID- 3014918 TI - The adenosine triphosphate-pyrophosphate isotopic exchange reaction: a tool for determination of tryptophan. AB - Quantitative determination of tryptophan at the picomole level is described, using the ATP-[32P]PPi isotopic exchange reaction catalyzed by tryptophanyl-tRNA synthetase. Sensitivity limits of 500 fmol were obtained. The presence of other amino acids at a 1000-fold excess over tryptophan did not interfere significantly with the quantitative determination of tryptophan. The specificity of the reaction was checked using five tryptophan analogs. These analogs did not prevent the determination of tryptophan when present in the same concentration range as tryptophan. When sensitive determination of a single amino acid is needed, the ATP-[32P]PPi exchange reaction catalyzed by aminoacyl-tRNA synthetases is suggested as a general method and as an alternative to HPLC procedures. PMID- 3014919 TI - Preparation of high-specific-activity D-[3-3H]pantothenic acid. AB - High-specific-activity D-[3-3H]pantothenic acid (5 Ci/mmol) was prepared from commercially available beta-[3-3H]alanine employing Escherichia coli strain DV1 (panD2 pan F1). This strain is defective in beta-alanine synthesis and pantothenate uptake, and under appropriate growth conditions converted 85 to 90% of the input beta-[3-3H]alanine to extracellular D-[3-3H]pantothenate. The radiolabeled vitamin was purified from the medium by thin-layer chromatography followed by reverse-phase high-performance liquid chromatography. The overall yield of D-[3-3H]pantothenic acid was 30% and radiochemical purity was greater than 99%. PMID- 3014920 TI - A convenient fluorescent assay for vertebrate collagenases. AB - A versatile, convenient assay for vertebrate collagenases has been developed using the fluorescent peptide substrate dansyl-Pro-Gln-Gly-Ile-Ala-Gly-D-Arg. This sequence resembles that of collagen at the site of cleavage but includes modifications designed to eliminate nonspecific hydrolysis by contaminating peptidases. Both human skin fibroblast and bovine corneal cell collagenases cleave the substrate specifically at the Gly-Ile bond. Plasmin, thrombin, trypsin, alpha-chymotrypsin, carboxypeptidase B, and bacterial collagenase do not cleave the substrate. Elastase and angiotensin converting enzyme display 20- and 400-fold less activity than the vertebrate collagenases, respectively, and cleave the peptide at different positions. The assay is performed by incubating a 5- to 25-microliters aliquot of trypsin-activated sample with an equal volume of 2 mM substrate overnight at 33 degrees C and pH 7.5. Thin-layer chromatography then separates the fluorescent product from the substrate in less than 20 min and allows the detection of subnanogram levels of collagenase. The assay is applicable to the screening of large numbers of samples under different conditions of pH and ionic strength and is readily adaptable for use in a variety of collagenase-dependent systems, such as assays for collagenase activating and/or inducing factors. PMID- 3014921 TI - Different behavior of type I and type I-trimer collagen in neutral sodium chloride solutions. AB - Type I and type I-trimer collagen, isolated from ductal infiltrating carcinoma of the human breast, have been tested for their behavior in neutral NaCl solutions. Evident diversities in their rate of precipitation at different saline concentrations have been found, since type I-trimer collagen precipitates at low NaCl molarity while type I collagen is mostly recovered in 2.6-3.6 M NaCl solutions. The native conformation of homotrimer collagen is proved by its ability to produce segment long-spacing crystallites and native-type fibrils. PMID- 3014922 TI - Effects of halothane, enflurane, isoflurane, and nitrous oxide on somatosensory evoked potentials in humans. AB - Median nerve somatosensory evoked potentials (SSEPs) were recorded in 21 healthy subjects anesthetized with halothane, isoflurane, or enflurane (with and without nitrous oxide) for abdominal or pelvic surgery. Recordings were made prior to induction, then at 0.5 MAC increments of each volatile agent with 60% N2O up to 1.5 MAC, and, finally, at 1.5 MAC without N2O. All three volatile anesthetics produced dose-related reductions in the amplitude and increases in the latency of the cortical component of the SSEP. These changes were most pronounced with enflurane and least with halothane. At 1.5 MAC of each volatile agent, cortical latency decreased and amplitude increased when nitrous oxide was discontinued. The results suggest that in neurologically intact patients, end-tidal concentrations of 1.0 MAC halothane and 0.5 MAC enflurane or isoflurane (each in 60% N2O) can be compatible with effective SSEP monitoring. Volatile anesthetic concentrations consistent with satisfactory somatosensory-evoked potential recording may be greater if N2O is not employed. PMID- 3014923 TI - Epidural and intravenous sufentanil in the rat: analgesia, opiate receptor binding, and drug concentrations in plasma and brain. AB - Doses of sufentanil (i.e., 0.01, 0.04, 0.16, 0.63, 2.5, 10, and 40 micrograms/rat) were injected either into the lumbar epidural space or intravenously in rats weighing +/- 250 g, and in vivo pharmacologic activities (i.e., prolongation of latency to tail withdrawal in response to noxious heat, blockade of cornea and pinna reflexes, increase of skeletal muscle tone), ex vivo mu-opiate receptor binding (i.e., displacement of specific 3H-sufentanil binding in thalamus, striatum, hippocampus, cortex, mamillary body-medulla oblongata segment, medulla oblongata, and in cervical, thoracic, and lumbar spinal cord), and drug concentrations in plasma, brain, cortex, and cerebellum, were determined. An ED50 dose of intravenous sufentanil of 0.075 micrograms/rat produced analgesia. CNS-mediated in vivo side effects (i.e., blockade of pinna and cornea reflexes, muscle rigidity) were apparent at 6-28 times higher doses. Epidural sufentanil also produced analgesia at an ED50 dose of 0.08 micrograms/rat, but CNS-mediated side effects occurred only at 35 to 76 times higher doses. This greater in vivo selectivity of epidural sufentanil in producing analgesia was consistent with ex vivo binding data that showed that in most areas of brain, but not in spinal cord, more mu-opiate binding occurs with intravenous than with epidural sufentanil. The two routes nonetheless differed by no more than a factor of approximately two in producing detectable levels of sufentanil both in plasma and in brain tissue.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014924 TI - CDC guidelines for the prevention and control of nosocomial infections. Guideline for handwashing and hospital environmental control, 1985. Supersedes guideline for hospital environmental control published in 1981. PMID- 3014925 TI - [Neuroendocrine response to anesthesia with isoflurane]. AB - The effects during surgery of a new halogenated volatile anaesthetic, isoflurane, on the hypothalamo-hypophyseal-thyroid-suprarenal axis were studied. In fact, it was important to prove whether this new halogenated anaesthetic would provide better protection, for the patient, from surgery and anaesthetic stress compared with other anaesthetic agents in use. The study was carried out in 16 young class ASA I patients who were to undergo appendicectomy. Before and during operation, blood was taken to measure ACTH, cortisol, TSH, T3, T4 and PRL plasma levels. A remarkable increase of PRL, cortisol and T4 plasmatic rate was found, especially at the end of the operation. It was concluded that isoflurane, just like enflurane, did not prevent the increase of PRL, cortisol and T4 that usually takes place during surgery. PMID- 3014926 TI - Experimental bluetongue virus infection of sheep; effect of previous vaccination: clinical and immunologic studies. AB - Clinical and immunologic responses of sheep to vaccination and subsequent bluetongue virus (BTV) challenge exposure were studied and compared with those of non-vaccinated sheep. Sheep were vaccinated with inactivated BTV administered with aluminum hydroxide and cimetidine or levamisole. After sheep were vaccinated, precipitating group-specific antibodies to BTV were detected, but serotype-specific neutralizing antibodies were not detected. Cellular immune responses (lymphocyte blastogenesis) to BTV were not detected. After virulent BTV challenge exposure, vaccinated and nonvaccinated sheep developed acute clinical disease of similar severity. Clinical signs included hyperemia and petechiae of oral mucosa and coronary bands of the feet, excess salivation, nasal discharge with crusting, ulceration of the muzzle, and edema of lips and intermandibular space. Marked increases in serum creatine kinase activity were associated with stiff gait, reluctance to move, and vomiting. Fever and leukopenia were detected in most of the challenge-exposed sheep. Viremia and neutralizing antibodies were detected in vaccinated and nonvaccinated sheep after challenge exposure. Bluetongue virus-specific reaginic antibodies were not detected in sera from any of the sheep when the passive cutaneous anaphylaxis test was used. PMID- 3014927 TI - Experimental bluetongue virus infection of sheep; effect of vaccination: pathologic, immunofluorescent, and ultrastructural studies. AB - Ten sheep were inoculated with bluetongue virus (BTV) serotype 17. Six of the sheep had been vaccinated before challenge exposure, 4 sheep served as nonvaccinated challenge-exposed controls, and 2 additional sheep served as nonvaccinated, nonchallenge-exposed, contact controls. Biopsy specimens (oral labial mucosa and skin) were obtained periodically after challenge exposure. Sheep were killed 8 to 13 days after challenge exposure, and necropsy was done. Vaccination did not seem to affect the nature or severity of the lesions observed. The changes in the mucosa of the cranial portion of the digestive tract included hyperemia, edema, inflammation, petechiae, erosions, ulcers, and surface encrustations. Lesions of skeletal, cardiac, and smooth muscles included hemorrhage, edema, myofiber degeneration, and necrosis. Lesions in cardiac muscles were sometimes widespread, indicating that cardiac failure may have been the major contributor to pulmonary congestion, edema, and eventual death during acute BTV infection. Damage to esophageal musculature resulted in vomiting. Hemorrhage was observed within the base of the pulmonary artery of all challenge exposed sheep. Using immunofluorescence, bluetongue viral antigens were detected in small blood vessels of the skin, oral labial mucosa, tongue, esophagus, rumen, reticulum, urinary bladder, and pulmonary artery and in skeletal and cardiac muscles. Viral antigens were present in tissues obtained 3 to 11 days after inoculation. Ultrastructurally, changes in small-caliber blood vessels included congestion, hemorrhage, swollen degenerated endothelial cells, and occasional fibrin-platelet thrombi. Tubular structures and virus-like particles were observed within some of these endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3014928 TI - Immune response in sows given transmissible gastroenteritis virus or canine coronavirus. AB - Twelve pregnant sows were inoculated oral-nasally 8 weeks before farrowing with attenuated transmissible gastroenteritis virus (TGEV), tissue culture-adapted canine coronavirus (CCV), or fluids from mock-infected culture (controls). A 2nd dose of the same inoculum, one-half oral-nasally and one-half intramammarily, was given 6 weeks later. Neutralizing antibodies for TGEV and CCV were demonstrated in sera, colostrum, and milk whey from the virus-vaccinated sows. Homologous geometric mean neutralizing titers were generally 4-fold higher than were heterologous titers. After challenge exposure of the nursing pigs with virulent TGEV, average morbidity and mortality for the pigs were 81% and 34% (mortality range = 11% to 63%), respectively, in the TGEV-vaccinated group; 83% and 39% (mortality range = 15% to 83%), respectively, in the CCV-vaccinated group; and 97% and 84% (mortality range = 78% to 100%), respectively, in the controls. Seemingly, sera from swine exposed to CCV could test serologically positive for TGEV-neutralizing antibody, and TGEV and CCV share at least 1 common neutralizing determinant that may be involved in protection. PMID- 3014929 TI - Cellular inflammatory response in the lungs of calves exposed to bovine viral diarrhea virus, Mycoplasma bovis, and Pasteurella haemolytica. AB - Three, 5, or 7 days after inoculation with bovine viral diarrhea (BVD) virus (n = 12) or Mycoplasma bovis (n = 12), groups of calves were exposed to aerosols of Pasteurella haemolytica and were euthanatized 4 hours later. Histologic lesions in the lungs and the ratios of neutrophils to alveolar macrophages, collected by bronchoalveolar lavage, were compared with those of clinically healthy calves (n = 8) and calves inoculated with BVD virus only (n = 4), M bovis only (n = 4), or P haemolytica only (n = 2). Inoculation with BVD virus or M bovis did not have a significant (P greater than 0.05) effect on the neutrophil/macrophage ratio in the bronchoalveolar lavage. Aerosol exposure to P haemolytica induced a marked and significant (P less than 0.01) change in the neutrophil/macrophage ratio (from less than 1:9 to greater than 9:1). The reversed neutrophil/macrophage ratio in calves exposed to P haemolytica correlated well with the histologic changes in which small bronchi and bronchioles were plugged with purulent exudate. Inoculation with BVD virus did not induce gross or microscopic lesions in the lungs. Inoculation with M bovis resulted in a severe peribronchial lymphoid hyperplasia with mild exudation of neutrophils and macrophages into the cranioventral parts of the lungs. PMID- 3014930 TI - Electrophysiologic characteristics of tibial and sciatic nerves in the hen. AB - The peripheral nerve of the hen has become an increasingly important animal model in studies of peripheral neuropathy, especially that induced by organophosphorus agent exposure. However, few electrophysiologic studies have been performed, and few data on normal peripheral nerve function exist. The purpose in the present study was to measure the characteristics of the compound action potential of peripheral nerves of the healthy hen. The results showed that conduction velocities of the tibial and the sciatic nerves were 41 m/s and 60 m/s, respectively. The relative refractory period was slightly shorter in the sciatic nerve. The tibial nerve exhibited a lower threshold for stimulation and a significantly shorter chronaxie (19 microseconds) than did the sciatic nerve (25 microseconds). Variability between animals was relatively small. These data should provide useful reference points on studies in which peripheral neuropathy is induced or suspected. PMID- 3014931 TI - Cyclophosphamide modulation of bronchoalveolar cellular populations and macrophage oxidative metabolism. Possible mechanisms of pulmonary pharmacotoxicity. AB - Cyclophosphamide, an immunosuppressive alkylating agent, has been reported to cause acute and chronic pulmonary injury in both humans and animals. Cyclophosphamide is also a common component of multi-drug regimens that show high pulmonary toxic potential. Although mechanisms of pulmonary damage caused by cyclophosphamide or other cytotoxic agents are unknown, possibilities include direct toxicity to pulmonary tissue or indirect toxicity through activation of pulmonary inflammatory cells. We report here a model system for the study of acute effects of cyclophosphamide on pulmonary immune cells in rats. Our findings show that 16 h after 1 intraperitoneal dose of cyclophosphamide there is: a dose dependent release of locally produced low molecular weight chemotactic factors for blood monocytes into bronchoalveolar lavage (BAL) fluid, a pulmonary influx of immature myeloperoxidase positive macrophages in low dose cyclophosphamide treated animals, an enhancement of oxidant generation by pulmonary macrophages from low dose treated animals that correlates with the presence of myeloperoxidase positive macrophages, the presence of factors in BAL fluid of treated rats that modulate oxidant release by normal rat pulmonary macrophages, a dose dependent reduction in the percentage of BAL lymphocytes, and evidence for pulmonary injury as manifested by elevated BAL fluid albumin concentrations in low dose cyclophosphamide treated animals. These findings suggest that cyclophosphamide may induce pulmonary injury through activation of pulmonary immunocompetent cells and subsequent attraction of systemic inflammatory cells. PMID- 3014932 TI - Lung asbestos content in long-term residents of a chrysotile mining town. AB - The effects of long-term exposure to very small amounts of chrysotile asbestos are controversial. To examine this problem, the lung asbestos content from 7 long term (25 yr and greater) residents of Thetford Mines, Quebec, who were never employed in the chrysotile mining and milling industry, was analyzed. Thetford Mines is a chrysotile mining town with a demonstrated ambient atmospheric concentration of chrysotile asbestos approximately 200 to 500 times that in urban areas of North America. Data on the residents' lungs were compared with those obtained from 20 long-term (25 yr and greater) chrysotile industry workers from Thetford Mines and 20 members of the general population of Vancouver. The median concentrations of chrysotile and tremolite in the Thetford residents were only about one fiftieth of those of the chrysotile workers, but about 10 times that of the population of Vancouver. Because long fibers of asbestos are generally thought to be more dangerous than short ones are, the sizes of fibers from these 3 groups were also examined. The fiber size distribution of the asbestos from the Thetford residents was significantly longer than that of the Vancouver population, and resembled that of the chrysotile workers. Because epidemiologic studies have consistently failed to find an increased respiratory disease incidence in lifelong residents of Quebec chrysotile mining towns who were never employed in the chrysotile industry, these findings imply that even asbestos burdens much higher, and fiber size distributions much longer, than those of the general population of most North American cities, are not associated with demonstrable pathologic effects. PMID- 3014933 TI - An in vitro model for polymorphonuclear-leukocyte-induced injury to an extracellular matrix. Relative contribution of oxidants and elastase to fibronectin release from amnionic membranes. AB - Alteration of the extracellular matrix by inflammatory cells is believed to be important in both lung injury and the subsequent restoration of lung architecture. Here we describe the results of the interaction between an acellular human amnionic membrane model and stimulated human polymorphonuclear neutrophils (PMN) in vitro. Polymorphonuclear neutrophil suspensions were placed on one surface of the amnion, and either the chemotactic peptide FMLP or the cell membrane activator phorbol myristate acetate (PMA) was placed on the opposite side of the amnion. Stroma and basement membrane sides of the amnion were separately exposed to the PMN. The PMN suspension was removed and centrifuged, and the supernatant was assayed for superoxide anion (O2-.) and for elastase activity. Injury to the acellular amnion was evaluated by transmission electron microscopy and by measurement of fibronectin (FN) released from the membrane matrix. Although both stimulants cause a concentration-dependent release of O2-., only PMA stimulated elastase release. These effects were similar when either the stroma or the basement membrane side was exposed to PMN. PMA-stimulated cells and supernatants from PMA-stimulated cells caused solubilization of membrane at different incubation times. Electron microscopy confirmed the disruption of the basement membrane of the amnion by PMA-stimulated PMN. Oxidant scavengers (SOD and catalase) did not prevent matrix degradation, and elastase inhibition by a specific chloromethylketone inhibitor diminished FN release on both sides of the amnion by activated PMN supernatants, but only on the basement membrane side by intact PMN. We conclude that in this model, elastase rather than oxygen radicals solubilizes FN from the matrix. PMID- 3014934 TI - Transbronchial needle aspiration staging of bronchogenic carcinoma. AB - Transbronchial needle aspiration (TBNA) has been advocated as a reliable technique in the nonsurgical staging of patients with bronchogenic carcinoma. Some have questioned the reliability of TBNA, however. We used TBNA directed by computed tomography (CT) in 88 consecutive patients with bronchogenic carcinoma who had undergone chest CT. Chest CT was 94% sensitive, 79% specific, and 85% accurate in evaluating the mediastinum for malignant lymphadenopathy. There were 19 malignant aspirates in 44 patients with malignancy and apparent adenopathy evaluated by chest CT. No malignant carinal aspirates were obtained in any patient with a normal mediastinum evaluated by chest CT. There were 2 false positive needle aspirates. One patient with apparent right paratracheal adenopathy and malignant needle aspirate had no mediastinal neoplasm detected at surgery. The other false positive aspirate had been contaminated by tracheal debris. The overall sensitivity, specificity, and accuracy of TBNA mediastinal staging were 50, 96, and 78%, respectively. We conclude that CT scanning is a useful adjunct in the staging of patients with bronchogenic carcinoma, and that TBNA is a sensitive and highly specific staging technique that may negate the need for surgical staging in a large number of patients with bronchogenic carcinoma. PMID- 3014935 TI - Persistent airways disease caused by toluene diisocyanate. AB - The natural history of isocyanate-induced asthma is not well documented. We evaluated a patient who developed persistent shortness of breath, wheezing, and cough after a massive exposure to toluene diisocyanate (TDI). Despite no further occupational exposures to isocyanates, he continued to have symptoms of asthma and variable airway obstruction 12 yr later. A methacholine inhalation challenge test was markedly positive, and a bronchial challenge test to TDI produced a dual asthmatic response. This report demonstrates that sensitivity to TDI can persist for many years in the absence of further occupational exposure and suggests that some patients with TDI-induced asthma do not recover from their disease after being removed from isocyanate exposure. PMID- 3014936 TI - Natural mycobacteriostatic activity in human monocyte-derived adherent cells. AB - The effects of human monocyte maturity on the replication of virulent Mycobacterium tuberculosis were examined. Mycobacteria grew readily in freshly isolated, adherent peripheral blood monocytes and in monocyte-derived macrophages obtained after 7 days in culture, as measured by counts of acid-fast bacilli and colony-forming units. Monocytes cultured for only 3 days before infection, however, were less permissive for the mycobacteria than either uncultured or 7 day cells. The association between the low permissiveness of 3-day cells and superoxide production was examined. Mycobacteria induced only a slight increase in superoxide production during the first 60 min of infection in uncultured and in 3-day cells, and no increase in cells cultured for 7 days before infection. Freshly isolated adherent cells produced small amounts of superoxide in response to phorbol myristate acetate (PMA) stimulation, but PMA-induced superoxide production increased steadily for 7 days. Mycobacteria had no effect on superoxide production by PMA-stimulated adherent cells. These results suggest that the suppressive activity of the 3-day cells is not associated with the production of increased amounts of reactive oxygen species. PMID- 3014937 TI - Changes in serum angiotensin-converting enzyme during cardiopulmonary bypass in humans. AB - In 15 male patients 45 to 74 yr of age, we measured angiotensin-converting enzyme (ACE) activity during and after cardiopulmonary bypass (all values were corrected for hemodilution occurring during surgery). Baseline ACE measurements (prior to onset of bypass) were 17.6 +/- 6.7 nmol of hippuric acid formed/min/ml (mean +/- SD). After the lung was isolated, serum ACE activity fell rapidly (half-clearance time, 39.5 min) and after 20 min during bypass reached a new steady state 33% below baseline values. Within 10 min of lung reperfusion, serum ACE rose to 90% of baseline values. ACE activity fell (half-clearance time, 198.3 min) and was again significantly below baseline for the period from 60 to 300 min after the end of cardiopulmonary bypass. Serum ACE activity had again returned to baseline when assessed 24 h after surgery; ACE activity in serum fluctuates rapidly, and it is rapidly cleared from serum at a site distant from the lung, probably the liver. Secretion of ACE into the vascular compartment by the lung is impaired in the period immediately after cardiopulmonary bypass. PMID- 3014938 TI - [Type C Niemann-Pick disease in 2 siblings. Biochemical bases of its diagnosis]. AB - Two brothers, a seven-year-old male and a nine-year-old female are reported. Clinical features include scholar troubles and clumsiness, hepatosplenomegaly, vertical supranuclear ophthalmoplegia and ataxic gait. Moreover, the girl showed intention tremor. Foamy histiocytes were seen in bone marrow and some Niemann Pick type Kupffer cells were present in liver. Girl's conjunctival biopsy showed lamellar inclusions. Biochemical studies were performed in girl's skin and liver biopsies. Sphingomyelinase activity assayed with 14C sphingomieline in cultured skin fibroblasts was 26% at the mean control value. Liver lipid composition did not show an appreciable increase of sphingomyelin or cholesterol, but bis (monoacylglyceryl) phosphate was clearly elevated. These data are compatible with Niemann-Pick disease type C. PMID- 3014940 TI - Biliary tract obstruction in the acquired immunodeficiency syndrome. AB - Three patients with the acquired immunodeficiency syndrome had biliary obstruction resulting from benign strictures of the biliary tract. Stenosis of the distal common bile duct with differing degrees of irregularity of the smaller intrahepatic and extrahepatic ducts was seen in association with either cryptosporidial or cytomegaloviral infection of the biliary tree. We review cytomegaloviral and cryptosporidial infections of the biliary system, as well as possible relationships with idiopathic primary sclerosing cholangitis. Stenotic biliary tract disease appears to be yet another complication of the acquired immunodeficiency syndrome. PMID- 3014941 TI - Treatment of cytomegalovirus pneumonia with 9-[2-hydroxy-1 (hydroxymethyl)ethoxymethyl]guanine and high-dose corticosteroids. PMID- 3014939 TI - Lymphocytic hypophysitis with isolated corticotropin deficiency. AB - In a 32-year-old woman, acquired, isolated corticotropin deficiency resulted from postpartum lymphocytic hypophysitis. The literature suggests that lymphocytic hypophysitis may cause acquired deficiencies of anterior, and possibly posterior, pituitary hormones. Immunologic evaluation of our patient failed to uncover anticorticotroph antibodies. Prompt recognition of this potentially fatal condition is important because of the availability of effective treatment. PMID- 3014942 TI - Classification system for human T-lymphotropic virus type III/lymphadenopathy associated virus infections. Centers for Disease Control, U.S. Department of Health and Human Services. AB - Infection with the human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) [now human immunodeficiency virus] can manifest as a spectrum of conditions ranging from severe immunodeficiency to asymptomatic infection. Because of the rapid growth of knowledge about this virus, there is a need for a system to classify patients with the various manifestations of infection. The presented system comprises four mutually exclusive groups: I, acute infection; II, asymptomatic infection, III, persistent generalized lymphadenopathy; and IV, other HTLV-III/LAV disease (with five subgroups, A to E, and two subcategories, C-1 and C-2). The classification should be useful in disease reporting and surveillance, epidemiologic studies, prevention and control activities, and public health policy and planning. PMID- 3014943 TI - Lactic acidosis: the case against bicarbonate therapy. PMID- 3014944 TI - Cytomegalovirus infection, 9-(1,3-Dihydroxy-2-propoxymethyl)guanine, and Crohn's disease. PMID- 3014945 TI - Estrogens regulate production of specific growth factors in hormone-dependent human breast cancer. PMID- 3014946 TI - Androgen and estrogen metabolism in breast cancer patients. PMID- 3014948 TI - Early pathologic changes in experimental and human breast cancer: facts and comments. PMID- 3014947 TI - 16 alpha-hydroxylation of estradiol: a possible risk marker for breast cancer. AB - From these results we may conclude that estradiol 16 alpha-hydroxylation is highly correlated with tumor incidence, and that the reaction is partly regulated by MMTV and the rest by genetic influences. Elevated hydroxylation appears to be an autosomal dominant trait that is highly specific for estradiol. It is also pertinent that the product of the 16 alpha-hydroxyestrone reaction is a potent estrogen that is capable of binding covalently to amino acids and nucleotides, including the estrogen receptor molecule. The results obtained in these studies establish the usefulness of the mouse model for studying the interrelationship between enhanced 16-hydroxylation of estradiol and the incidence of mammary tumors. PMID- 3014949 TI - Steroid profiles and optimization of high-performance liquid chromatographic analytic procedure. AB - This paper presents a short review of the results obtained to date in our laboratory, with respect to the studies of steroid excretion profiles in both breast and endometrial cancer patients, by using gas-liquid chromatographic analysis. These data demonstrate the importance of minor estrogens, including catechol estrogens, and of their ratios with the classical ones, in studies of steroid metabolism in both breast and endometrial cancer. New data concerning postmenopausal endometrial cancer are consistent with our previous observations and demonstrate the necessity of measuring these steroids directly in tumors and examining patterns of metabolism in vitro. In order to analyze steroid metabolic patterns in vitro, however, high-performance liquid chromatography rather than gas-liquid chromatography methods are preferable on account of their selectivity, specificity, sensitivity and capacity to handle labile materials. With the aim of providing methods suitable for the complete resolution and analysis of these complex natural mixtures a method of computer-aided optimization of HPLC has been developed and its practical utility has been tested. PMID- 3014950 TI - Identification and characterization of monoclonal antibodies to prolactin receptors from the mammary gland. PMID- 3014951 TI - Regulatory sequences involved in the hormonal control of casein gene expression. PMID- 3014952 TI - Release of superoxide anion by alveolar macrophages in pulmonary sarcoidosis. AB - Biological mechanisms involving nonprotease factors mediate the alterations of the alveolar structures which lead to the interstitial fibrosis of pulmonary sarcoidosis. Thus, we have investigated the production of oxidant species by BAL cells from 50 sarcoidosis patients and 18 healthy controls using a lucigenin dependent CL method. Spontaneous and PMA-induced CL's were significantly higher in untreated patients and treated patients than in spontaneously cured patients or healthy controls (p less than .05). SOD inhibits 60 to 75% of spontaneous CL and 91 to 93% of PMA-induced CL. There was no apparent correlation between the CL of AM's and the radiological types, SACE levels, and gallium scans. In marked contrast, CL was significantly higher in patients with increased alveolar lymphocytosis (greater than or equal to 18%) than in patients with normal BAL. Since there were neither neutrophils nor eosinophils in BAL and since lymphocytes do not produce lucigenin-dependent CL, we believe that CL is produced by AM's. CL inhibition by SOD suggests that superoxide anion is involved in the production of CL. The release of both superoxide anion and related radicals may be of importance in the pathogenesis of pulmonary sarcoidosis. PMID- 3014953 TI - Activated alveolar macrophage and lymphocyte alveolitis in extrathoracic sarcoidosis without radiological mediastinopulmonary involvement. AB - Cellular characteristics of BAL were investigated in 18 patients with proved extrathoracic sarcoidosis (that is, sarcoidosis that affected the skin, eyes, parotid glands, stomach, nose, kidneys, or meninges) without clinical or radiological mediastinopulmonary involvement. Computed tomography of the thorax was performed on five patients: four patients were normal, and one had enlarged lymph nodes (these enlargements were not detectable on the patient's chest roentgenogram). The results of pulmonary function tests were normal in all patients. The total BAL cell count did not differ significantly between controls and patients. Abnormal percentages of alveolar lymphocytes (from 18 to 87%) were noted in 15 out of 18 patients. SACE levels were normal in 15 patients. No pulmonary gallium uptake was detected. The chemiluminescence of AM's, whether spontaneous or PMA induced, was increased in five out of seven patients. The percentages of T3+ lymphocytes in sarcoidosis patients did not significantly differ from those in controls. The T4+:T8+ ratio was normal in four patients and slightly increased in one. Follow-up of patients showed that alveolar lymphocytosis is as lasting as extrathoracic involvement. Our data demonstrate increased percentages of lymphocytes and activated AM's in the BAL of patients with extrathoracic sarcoidosis. This may be due to the initial involvement of the respiratory tract in extrathoracic sarcoidosis or to the diffusion of activated macrophages and lymphocytes from an extrathoracic site into the lung. PMID- 3014954 TI - Angiotensin I-converting enzyme. A marker of highly differentiated monocytic cells. PMID- 3014955 TI - Sven Lofgren Memorial Lecture. Do measurements of bronchoalveolar lymphocytes and neutrophils, serum angiotensin-converting enzyme, and gallium uptake help the clinician to treat patients with sarcoidosis? PMID- 3014956 TI - Systemic and lung protein changes in sarcoidosis. Lymphocyte counts, gallium uptake values, and serum angiotensin-converting enzyme levels may reflect different aspects of disease activity. AB - BAL lymphocyte percentages, quantitated gallium-67 lung uptake, and SACE levels have all been proposed as measures of disease activity in sarcoidosis. We analyzed 32 paired sera and BAL fluids from sarcoidosis patients by high resolution agarose electrophoresis to look for protein changes characteristic of systemic or local inflammation and compared the results with those from the above tests. Nine patients (group 1) had serum inflammatory protein changes and increased total protein, albumin, beta 1-globulin (transferrin), and gamma globulin levels in fluid recovered by BAL. Thirteen patients (group 2) had normal protein levels in sera but abnormal protein levels in BAL specimens. Ten patients (group 3) had normal protein levels in sera and in BAL specimens. Patients in groups 1 and 2 had a disproportionate increase in beta 1-globulin (transferrin) and gamma-globulin levels in their BAL specimens. The BAL lymphocyte percentage changes paralleled the BAL protein level changes, suggesting relationships among the immunoregulatory role of these cells, increased local immunoglobulin synthesis, and the pathogenesis of altered alveolar permeability. Gallium-67 uptake was highest in patients with serum inflammatory protein changes. Thus, systemic inflammation may facilitate pulmonary gallium-67 uptake, possibly by changes in BAL fluid or serum transferrin saturation and/or kinetics. SACE levels showed no relationship to changes in the levels of serum or BAL proteins. These data suggest that the various proposed measures of disease activity reflect different aspects of inflammation in sarcoidosis. PMID- 3014957 TI - Gallium-67 activity in bronchoalveolar lavage fluid in sarcoidosis. AB - Roentgenograms and gallium-67 scans and gallium-67 counts of BAL fluid samples, together with differential cell counts, have proved to be useful in assessing activity and lung involvement in sarcoidosis. In active pulmonary sarcoidosis gallium-67 scans are usually positive. Quantitation of gallium-67 uptake in lung scans, however, may be difficult. Because gallium-67 uptake and cell counts in BAL fluid may be correlated, we set out to investigate gallium-67 activity in BAL fluid recovered from patient of different groups. Sixteen patients with recently diagnosed and untreated sarcoidosis, nine patients with healthy lungs, and five patients with CFA were studied. Gallium-67 uptake of the lung, gallium-67 activity in the lavage fluid, SACE and LACE levels, and alpha 1-AT activity were measured. Significantly more gallium-67 activity was found in BAL fluid from sarcoidosis patients than in that from CFA patients (alpha = .001) or patients with healthy lungs (alpha = .001). Gallium-67 activity in BAL fluid could be well correlated with the number of lymphocytes in BAL fluid, but poorly with the number of macrophages. Subjects with increased levels of SACE or serum alpha 1-AT showed higher lavage gallium-67 activity than did normals, but no correlation could be established. High gallium-67 activity in lavage fluid may be correlated with acute sarcoidosis or physiological deterioration; low activity denotes change for the better. The results show that gallium-67 counts in BAL fluid reflects the intensity of gallium-67 uptake and thus of activity of pulmonary sarcoidosis. PMID- 3014958 TI - Assessment of gallium-67 scanning in pulmonary and extrapulmonary sarcoidosis. AB - Gallium-67 scans have been widely employed in patients with sarcoidosis as a means of indicating alveolitis and the need for corticosteroid therapy. Observation of 32 patients followed 3 or more years after gallium scans showed no correlation between findings and later course: of 10 patients with pulmonary uptake, 7 recovered with minor residuals; of 18 patients with mediastinal of extrathoracic uptake, 10 had persistent or progressive disease; of 4 patients with negative initial scans, 2 had later progression. The value of gallium-67 scans as an aid to diagnosis was studied in 40 patients with extrapulmonary sarcoidosis. In 12 patients, abnormal lacrimal, nodal, or pulmonary uptake aided in selection of biopsy sites. Gallium-67 scans and serum ACE levels were compared in 97 patients as indices of clinical activity. Abnormal gallium-67 uptake was observed in 96.3% of the tests in active disease, and ACE level elevation occurred in 56.3%. In 24 patients with inactive or recovered disease, abnormal gallium-67 uptake occurred in 62.5% and ACE level elevation in 37.5%. Gallium-67 scans have a limited but valuable role in the diagnosis and management of sarcoidosis. PMID- 3014959 TI - The stimulation of angiotensin-converting enzyme induction in cultured monocytes by T4+ and T8+ lymphocytes. PMID- 3014961 TI - Clinical study on cardiac sarcoidosis. AB - Forty-one patients with sarcoidosis were investigated for cardiac involvement. The results are summarized as follows: The focal left ventricular perfusion defect was found on the TMPS's of 5 out of 41 patients with sarcoidosis. The defect disappeared in four of these five patients over a 3-year period, but remained in the patient with fatal myocardial sarcoidosis. A decreased left ventricular ejection fraction was found in 14 out of 41 patients with sarcoidosis, and in 3 out of 5 patients with the left ventricular perfusion defect. Echocardiographic examination did not reveal left ventricular wall dysfunction at the site of the left ventricular perfusion defect, except in one patient with fatal myocardial sarcoidosis. Holter ECG's showed such abnormalities as CRBBB, heart blocks, PVC, and PSC in 13 out of 32 patients with sarcoidosis. SACE activity was elevated in all three patients with the left ventricular perfusion defect that were examined, and it could be correlated with changes in the left ventricular perfusion defect in one of them. PMID- 3014960 TI - A European survey on the usefulness of 67Ga lung scans in assessing sarcoidosis. Experience in 14 research centers in seven different countries. AB - Fifty-eight contributors from 12 European and 2 American sarcoidosis centers have collaborated in a survey to define many questions concerning the use of 67Ga lung scan in sarcoidosis. The new quantitative scoring methods based on digital evaluation seem better in detecting lung activity. In 20.1% of untreated patients, the 67Ga lung scan appeared to be the only noninvasive method with which clinical activity could be detected. 67Ga scans may be useful in guiding lung biopsy and in choosing pulmonary segments for BAL. Of 382 patients studied during follow-up (154 patients with three to nine scans at intervals of 2 to 12 months), the 67Ga scan was far more sensitive than chest radiography, both in detecting improvement and in foreseeing relapses. Steroid therapy appears to suppress ACE levels more than 67Ga uptake, and 67Ga uptake more than the alveolitis detectable by BAL. Gallium-67 uptake rebounds to positivity occur in about 40% of patients after steroid discontinuation and in about 20% of patients after steroid reduction to daily doses of 10 mg or less of prednisone. The 67Ga dose of 1.5 mCi seems appropriate for clinical purposes and is recommended for the subjective scoring method in order to reduce the cost and the radiation burden. PMID- 3014962 TI - Different mechanisms of hypercalciuria in sarcoidosis. Correlations with disease extension and activity. PMID- 3014963 TI - Pulmonary function tests, serum angiotensin-converting enzyme levels, and clinical findings as prognostic indicators in sarcoidosis. PMID- 3014964 TI - Use of budesonide in the treatment of pulmonary sarcoidosis. AB - Twenty patients with active sarcoidosis (increased serum ACE activity) and progressive pulmonary disease (stages 2-3) were treated with inhaled budesonide instead of with oral glucocorticosteroids in an open clinical study. Results after an 18-month treatment are reported. A general improvement in chest roentgenograms and FVC was noted. Serum ACE activity was normalized. The results indicate that pulmonary manifestations of sarcoidosis can be treated with the inhaled steroid, budesonide. In this way the risk of systemic side effects is considerably reduced. The final place of inhaled budesonide in the treatment of sarcoidosis must be determined via placebo-controlled and comparative, double blinded clinical studies in larger series of patients. PMID- 3014966 TI - Modulation of the kinetic parameters of microtubule assembly by MAP-2 phosphorylation, the GTP/GDP occupancy of oligomers, and the tubulin tyrosylation status. PMID- 3014967 TI - Separation of microtubule-associated cAMP and calmodulin-dependent kinases that phosphorylate MAP-2. PMID- 3014965 TI - Thermolysin-like serum metalloendopeptidase. A new marker for active sarcoidosis that complements serum angiotensin-converting enzyme. PMID- 3014968 TI - Phosphorylation of MAP-2 at distinct sites by calmodulin- and cyclic AMP dependent kinases. PMID- 3014969 TI - Location of the guanosine triphosphate (GTP) hydrolysis site in microtubules. AB - The rate for GTP hydrolysis remains approximately constant during microtubule assembly from microtubular protein. This indicates that GTP hydrolysis does not accompany tubulin-GTP subunit addition to microtubule ends. We suggest that GTP, within tubulin-GTP subunits that are incorporated into microtubules, is hydrolyzed predominantly at one or both microtubule ends at an interface of a cap of tubulin-GTP subunits and a core of tubulin-GDP subunits. PMID- 3014970 TI - Multiple morphine and enkephalin receptors: biochemical and pharmacological aspects. PMID- 3014971 TI - Hyperalgesic functions of peripheral opiate receptors. PMID- 3014972 TI - Stress-induced analgesia: its effects on performance in learning paradigms. PMID- 3014973 TI - Multiple endogenous opiate and non-opiate analgesia systems: evidence of their existence and clinical implications. PMID- 3014974 TI - American adult T cell leukemia/lymphoma: a flow cytometric and morphological study. PMID- 3014975 TI - Determination of cell cycle DNA and 5'-nucleotide phosphodiesterase in endometrial cancer. PMID- 3014977 TI - Application of EPR methods in studies of immobilized enzyme systems. PMID- 3014976 TI - Active inducer transport and regulation of microbial enzyme biosynthesis in chemostat cultures. PMID- 3014978 TI - Chemical synthesis, cloning, and expression of genes for human somatomedin C (insulin-like growth factor I) and 59Val-somatomedin C. PMID- 3014979 TI - Interaction of DNA containing phosphorothioate groups with restriction enzymes. PMID- 3014980 TI - The mechanism of action of phenylalanine hydroxylase. PMID- 3014981 TI - [Vascular purpura associated with an infection caused by human parvovirus]. PMID- 3014982 TI - [Rheumatoid purpura and infection caused by human parvovirus]. PMID- 3014983 TI - [Trophic ulcer caused by trigeminal neuropathy in children]. PMID- 3014984 TI - Primary liver cancer in Sweden. PMID- 3014985 TI - Primary liver cancer in Denmark. PMID- 3014986 TI - Natural history of liver cancer. PMID- 3014987 TI - Pathology of liver cancer. PMID- 3014988 TI - Liver transplantation in liver cancer. PMID- 3014989 TI - Systemic cytostatic drug therapy in liver cancer. PMID- 3014991 TI - Radiation therapy in liver cancer. PMID- 3014990 TI - Primary liver cancer in Norway. PMID- 3014992 TI - Unusually located granular cell myoblastoma. PMID- 3014993 TI - [Insulinoma: critical study of methods of localization and strategy]. AB - In patients with proven hyperinsulinism, localization of the underlying insulinoma may be difficult. The localization diagnosis may be performed preoperatively using different procedures, such as ultrasonography, computed tomography, selective arteriography of the pancreatic vessels and percutaneous transhepatic blood sampling in the portal venous system. At operation, insulinomas may be detected by inspection and bidigital palpation, pancreatico sonography and rapid determination of insulin concentration after sampling of blood in pancreatic veins. By discussing the advantages and disadvantages of each localization procedure, the authors propose a strategy fort the detection of pancreatic insulinomas. PMID- 3014994 TI - The AIDS dementia complex: II. Neuropathology. AB - In order to define the histopathological substrate of the dementia that frequently complicates the acquired immune deficiency syndrome (AIDS), we analyzed the neuropathological findings in 70 autopsied adult AIDS patients, 46 of whom had suffered clinically overt dementia. Less than 10% of the brains were histologically normal. Abnormalities were found predominantly in the white matter and in subcortical structures, with relative sparing of the cortex. Their frequency and severity generally correlated well with the degree and duration of clinical dementia. Most commonly noted was diffuse pallor in the white matter, which in the pathologically milder cases was accompanied by scanty perivascular infiltrates of lymphocytes and brown-pigmented macrophages, and in the most advanced cases by clusters of foamy macrophages and multinucleated cells associated with multifocal rarefaction of the white matter. However, in nearly one third of the demented cases the histopathological findings were remarkably bland in relation to the severity of clinical dysfunction. In addition, similar mild changes were noted in over one half of the nondemented patients, consistent with subclinical involvement. Vacuolar myelopathy was found in 23 patients and was generally more common and severe in patients with advanced brain pathology. Evidence of cytomegalovirus (CMV) infection was noted in nearly one quarter of the brains and was associated with a relative abundance of microglial nodules, but correlated neither with the major subcortical neuropathology nor with the clinical dementia, indicating that CMV infection likely represented a second, superimposed process. This study establishes the AIDS dementia complex as a distinct clinical and pathological entity and, together with accumulating virological evidence, suggests that it is caused by direct LAV/HTLV-III brain infection. PMID- 3014995 TI - Chronic idiopathic ataxic neuropathy. AB - Fifteen patients with chronic sensory ataxia caused by a large-fiber sensory neuropathy were studied and followed up for a period of 17.4 years (range, 4 to 41). When first seen, they had distal paresthesias and sensory ataxia of slow onset and progression, areflexia, normal strength, and a profound loss of proprioceptive and kinesthetic sensation extending up to the most proximal joints. Needle electromyogram and motor-nerve conduction velocity findings were normal in most of the patients and sensory potentials were absent in all. Nerve biopsy showed severe loss of the large myelinated fibers. Nine patients had a serum monoclonal or polyclonal gammopathy (3 with IgM kappa, 1 with IgA kappa, and 5 with a polyclonal increase of IgG, IgA, or IgM), and 8 had elevated cerebrospinal fluid gamma globulin levels in spite of low normal total cerebrospinal fluid protein levels. No circulating antibodies to ganglionic neurons were found. Therapy with immunosuppressants or plasmapheresis was unsuccessful. All patients are disabled and their conditions have continued to worsen without signs of malignancy or systemic illness during a mean follow-up period of 17.4 years. Chronic idiopathic ataxic neuropathy is a proprioceptive neuropathy, clinically indistinguishable from the one associated with carcinoma or pyridoxine abuse due to involvement of the dorsal root ganglia, and could represent a distinct form of an indolent, slowly progressive sensory neuronopathy (ganglionopathy). Although immunopathological mechanisms may play a role, especially in patients with an associated paraproteinemia, the resistance of such patients to therapy, the progressive course, and the resemblance of this disorder to other toxic neuronopathies associated with pyridoxine abuse or doxorubicin administration suggest a possible toxic etiopathogenesis. PMID- 3014996 TI - Antiidiotypic antibody to reovirus binds to neurons and protects from viral infection. AB - A syngeneic monoclonal antiidiotype directed against the idiotype of an antireovirus type 3 hemagglutinin demonstrates several of the biological actions of the original viral hemagglutinin and binds to rat and murine cortical neurons grown in dissociated cell culture. Receptor-bearing neurons appear within 24 hours of plating in cultures from mouse or rat cortex taken on embryonic day 15; these neurons are demonstrable for the duration of the culture life span (4 to 8 weeks). When cortical cultures are incubated with antiidiotype before or during exposure to reovirus, the antiidiotype protects neurons from type 3 infection without inhibiting infection of nonneuronal cells with either type 3 or type 1. Thus an antibody directed against a viral receptor can prevent infection of receptor-bearing cells without directly neutralizing the virus. PMID- 3014997 TI - N-acetyl-beta-hexosaminidase beta locus defect and juvenile motor neuron disease: a case study. AB - A patient with partial deficiency of N-acetyl-beta-hexosaminidase (Hex) developed a progressive motor neuron syndrome beginning at age 7, characterized by dysarthria, muscle wasting, fasciculations, and pyramidal tract dysfunction. Minor clinical features have included tremor and late distal sensory abnormalities. Rectal biopsy at age 24 demonstrated membranous cytoplasmic bodies in submucosal ganglion cells. Biochemical evaluation revealed nearly absent Hex B activity in serum, leukocytes, and fibroblasts, with partial Hex A activity in serum and leukocytes, and low normal Hex A activity in fibroblasts. Motor neuron disease can be a presentation of a Hex beta locus defect, in addition to the previously recognized Hex alpha locus defects. PMID- 3014998 TI - Treatment of mitochondrial myopathy due to complex III deficiency with vitamins K3 and C: A 31P-NMR follow-up study. AB - A patient with mitochondrial myopathy due to complex III deficiency who was treated with vitamin K3 (menadiol sodium diphosphate, 40 mg daily) and vitamin C showed clinical improvement. A 1-year study with phosphorus 31 nuclear magnetic resonance (31P-NMR) monitoring has shown that clinical and metabolic improvement was maintained by this therapy; increasing the dose of vitamin K3 to 80 mg daily improved the bioenergetic state of the patient's muscles at rest; postexercise recovery was less responsive to the increased dose; and a higher dose of vitamin K3 (80 mg/day) did not produce side effects. The differential therapeutic effects of vitamin K3 at rest and during exercise recovery are probably due to the differential kinetics of each metabolic state. Monitoring muscle bioenergetics with 31P-NMR is valuable in documenting therapeutic improvements in mitochondrial myopathies. PMID- 3014999 TI - [Formation of a streptothricin-group antibiotic by a Streptomyces glaucus 1136 culture]. AB - In screening of new antibiotics a streptomycete (strain 1136) was isolated from a soil sample of Armenia. It showed no antagonistic properties in streek cultures on agarized media. When grown under submerged conditions strain 1136 produced an antibiotic active against grampositive and gramnegative bacteria. By its cultural and morphological properties strain 1136 was classified as Streptomyces glaucus Agre et Preobrazhenskaya, 1983. Microbiological and chemical investigation of the antibiotic produced by strain provided its identification at the early stages of the investigation as an antibiotic of the streptothricin group. Up to now no organisms producing streptothricin antibiotics were detected among streptomycetes of the Azureus section including strain 1136. PMID- 3015000 TI - Effects of norfloxacin on DNA metabolism in Pseudomonas aeruginosa. AB - Norfloxacin is a quinolone (pyridonecarboxylic acid derivative) effective against Pseudomonas aeruginosa infections. We studied the effects of this drug on DNA metabolism in P. aeruginosa. Norfloxacin inhibits DNA replication immediately on its addition to a logarithmically growing culture of P. aeruginosa. It inhibits the ability of P. aeruginosa DNA gyrase to supercoil relaxed, closed circular DNA in vitro. At intermediate concentrations of the drug, inhibition of DNA replication in vivo is followed by secondary (recovery) synthesis. Both recovery synthesis and the bactericidal effects of norfloxacin are dependent on continued protein synthesis, suggesting that these are inducible functions. Neither norfloxacin nor nalidixic acid induces Weigle-reactivation (inducible DNA repair) or mutagenesis in P. aeruginosa. PMID- 3015001 TI - Comparative activities of UK-49,858 and amphotericin B against Blastomyces dermatitidis infections in mice. AB - UK-49,858, a new antifungal triazole derivative, was compared with amphotericin B in the treatment of pulmonary infections by Blastomyces dermatitidis in male BALB/cByJ mice. Administration of UK-49,858 in daily doses of 25 or 50 mg/kg for 21 days gave 30 and 100% survival rates, respectively. These results compared with 100% mortality in infected controls and 100% survival among mice treated with amphotericin B. UK-49,858 did not eradicate the fungus from the lungs of surviving animals, while amphotericin B effected sterilization of the lungs in 66% of the survivors. PMID- 3015002 TI - Acyclic guanosine analogs as substrates for varicella-zoster virus thymidine kinase. AB - This study was performed to obtain information on the enzymatic background to the antiviral activity of acyclic guanosine analogs. Five acyclic guanosine analogs, the (R)- and (S)-enantiomers of 9-(3,4-dihydroxybutyl)guanine, 9-(4 hydroxybutyl)guanine, 9-[(2-hydroxyethoxy)methyl]guanine, and 9-[[2-hydroxy-1 (hydroxymethyl)ethoxy]methyl]guanine, were compared in enzyme kinetic experiments using purified varicella-zoster virus and human placenta mitochondrial thymidine kinase (TK). All analogs showed competitive patterns of inhibition in the phosphorylation of thymidine by varicella-zoster virus TK, but only low affinities and phosphorylation rates were observed. No affinity for the mitochondrial TK was observed for any of the analogs. PMID- 3015003 TI - Activities of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine and its metabolites against herpes simplex virus types 1 and 2 in cell culture and in mice infected intracerebrally with herpes simplex virus type 2. AB - As measured by plaque and yield reduction assays, several metabolites of 1-(2 deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) were highly active against herpes simplex virus types 1 and 2. These metabolites included the 2' deoxy-2'-fluoroarabinosyl derivatives of 5-iodouracil (FIAU), cytosine (FAC), uracil (FAU), and thymine (FMAU). In mice inoculated intracerebrally with herpes simplex virus type 2, the relative order of potency of these compounds and licensed antiviral drugs was as follows: FMAU much greater than FIAC approximately equal to FIAU greater than acyclovir approximately equal to vidarabine much greater than FAC approximately equal to FAU. One of the main metabolites of FMAU, 2'-fluoro-5-hydroxymethyl-arabinosyluracil, was essentially inactive in vivo. FIAC-, FIAU-, FMAU-, FAC-, and FAU-resistant herpes simplex virus variants prepared in cell culture were found to be (i) devoid of viral thymidine kinase, (ii) cross-resistant to one another and resistant to drugs requiring viral thymidine kinase for activation, and (iii) sensitive to vidarabine or phosphonoformate. These results indicate that FIAC, FIAU, and FMAU require the virally encoded thymidine kinase for activation and suggest that the antiviral activity of FAU and FAC in cell cultures is also mediated by this enzyme. The interaction of the fluoroarabinosyl pyrimidine nucleosides with herpes simplex virus thymidine kinase in a cell-free system is also described. PMID- 3015004 TI - Leakage of periplasmic proteins from Escherichia coli mediated by polymyxin B nonapeptide. AB - The effects of polymyxin B and polymyxin B nonapeptide (PMBN) on cell envelope integrity in Escherichia coli were compared. Both compounds caused loss of proteins from E. coli K-12 3300(pBR322), although PMBN released less protein than did polymyxin B. The origin of the released protein was determined both by polyacrylamide gel electrophoresis and by using specific enzyme markers (beta lactamase in periplasm, beta-galactosidase in cytoplasm). The proteins released by both compounds were derived principally from the periplasm, accompanied in the case of polymyxin B by a low level of cytoplasmic proteins. Although polymyxin B and PMBN both caused release of periplasmic proteins, the individual proteins released by the compounds differed. The periplasmic fraction contained six principal polypeptides with molecular weights between 62,000 (polypeptide 1) and 29,000 (polypeptide 6). Polypeptide 6 was identified as the pBR322-encoded beta lactamase, but the other proteins were not specifically identified. Polymyxin B caused considerable release of polypeptides 1, 2, and 5 with some release of polypeptides 4 and 6. PMBN released polypeptide 1 (trace), 3, 4, and 6 (trace). Scanning electron microscopy showed that polymyxin B and PMBN both caused surface damage in E. coli. However, polymyxin B produced greater morphological changes than PMBN. PMID- 3015005 TI - Evolution and spread of IncFIV plasmids conferring resistance to trimethoprim. AB - Twenty-one IncFIV-group plasmids conferring trimethoprim resistance in Escherichia coli isolates from humans and pigs were examined. Three evolutionary lines of plasmids were identified on the basis of restriction enzyme analysis. One was found exclusively in human isolates and another was found in pig isolates, while the third line consisted of plasmids from both sources. All R plasmids readily transferred to laboratory strains, and evidence was found for transfer to other biotypes of E. coli in the environment. The Tpr genes from representatives of the plasmid lines were cloned and compared by restriction analysis and by hybridization with two characterized Tpr dihydrofolate reductase genes. The sequences flanking the Tpr genes were different for each line, but all showed homology with the type 2 dihydrofolate reductase gene, irrespective of whether they were of human or animal origin. There was no hybridization to the type 1 gene. The remarkable degree of similarity among plasmids of the third line provided clear evidence of the exchange of plasmid-bearing E. coli between humans and pigs. PMID- 3015007 TI - Transfer of amikacin resistance by closely related plasmids in members of the family Enterobacteriaceae isolated in Chile. AB - During a 9-month period when amikacin was the sole aminoglycoside used clinically in a hospital in Santiago, Chile, resistance to amikacin and other antibiotics was encountered in 42 strains of the family Enterobacteriaceae, including Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae, Serratia marcescens, and Serratia liquefaciens. Amikacin resistance was transferable by conjugation and carried by IncM plasmids ranging in size from ca. 48.4 to 58.1 kilobase pairs. The plasmids had ca. 70 to 80% of their structure in common, as judged after digestion with restriction endonucleases. The resistance was mediated by a 6' aminoglycoside acetyltransferase. We conclude that selective pressure has favored the dissemination of a wide-host-range amikacin resistance plasmid and its derivatives. PMID- 3015006 TI - Influence of acyclovir and bucyclovir on nucleotide pools in cells infected with herpes simplex virus type 1. AB - The effects of the acyclic guanosine analogs acyclovir (ACV) and (R)-9-(3,4 dihydroxybutyl)guanine (bucyclovir, BCV) on the deoxyribonucleoside triphosphate (dNTP) pools of herpes simplex type 1 (HSV-1)-infected African green monkey kidney (GMK) and human embryonic lung fibroblast (HL) cells were investigated. HSV-1 infection increased the dNTP pools in both cell types compared with those in uninfected cells. Mock-infected GMK cells showed a 10-fold-higher dTTP concentration than comparable HL cells. ACV or BCV treatment of HSV-1-infected cells yielded further increases of the dNTP pools. ACV- or BCV-treated, HSV-1 infected HL cells showed 20- to 50-fold-higher concentrations of ACV triphosphate and BCV triphosphate, respectively, than similarly treated GMK cells. This is in accord with previous results, which showed that ACV and BCV are less active in GMK cells than in HL cells. This difference in activity is attributed to the substantial deoxythymidine pools previously found in GMK cells. The results are discussed in relation to known metabolic and kinetic parameters. PMID- 3015008 TI - Enzymology and pathogenicity in mice of a herpes simplex virus type 1 mutant resistant to 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodocytosine. AB - The deoxypyrimidine nucleoside analog 2'-fluoro-2'-deoxy-1-beta-D arabinofuranosyl-5-iodocytosine (FIAC) is a potent and selective inhibitor of herpes simplex virus type 1 in vitro (C. Lopez, K. A. Watanabe, and J. J. Fox, Antimicrob. Agents Chemother. 17:803-806, 1980). Isopycnographic analysis demonstrated that 1 microM FIAC inhibited herpes simplex virus DNA replication by more than 95% but inhibited cellular DNA replication by only 32%. Mutant herpes simplex virus type 1 selected resistant to FIAC displayed linked resistance to other nucleoside analogs, including arabinosylthymine and acyclovir. Lysates derived from Vero cells infected with FIAC-resistant virus showed markedly lower levels of thymidine kinase activity and were unable to phosphorylate selectively arabinosylthymine or FIAC, in contrast to lysates from cells infected with wild type herpes simplex virus type 1. Finally, drug-resistant virus displayed a 6,000 fold decrease in pathogenicity when inoculated intraperitoneally into genetically susceptible A/J mice. These results indicate that resistance to deoxypyrimidine nucleoside analogs is due, at least in part, to alterations in viral thymidine kinase and is accompanied by decreased pathogenicity in vivo. PMID- 3015009 TI - Susceptibility to other antiherpes drugs of pathogenic variants of herpes simplex virus selected for resistance to acyclovir. AB - Cross-resistance data for a group of nine acyclovir-resistant variants of herpes simplex virus type 1 are reported. These mutants, which express either altered thymidine kinase (TK) or DNA polymerase, were all derived from the same wild-type (wt) strain after exposure to acyclovir in tissue culture. Furthermore, all variants have pathogenic properties similar to the wt parental strain as assessed using mouse model systems (G. Darby, H.J. Field, and S.A. Salisbury, Nature (London) 289:81-83, 1981; B.A. Larder and G. Darby, Virology 146:262-271, 1985). Two groups of antiherpes compounds were used: those requiring activation by TK and those whose action is independent of that enzyme. The TK substrate specificity mutants were generally resistant to the TK-activated drugs but showed wt susceptibility to phosphonoacetic acid, 9-beta-D-arabinofuranosyladenine, and aphidicolin. The DNA polymerase mutants were relatively susceptible to most TK activated drugs, although two were resistant to 5-(trifluoromethyl)-2' deoxyuridine. The polymerase mutants showed a more complex pattern of susceptibility, however, to those compounds whose mode of action is independent of TK. In general, these variants showed similar responses to phosphonoacetic acid, phosphonoformate, and 9-beta-D-arabinofuranosyladenine, a particular variant being either resistant, susceptible, or hypertensive to all three. The response of each variant to aphidicolin, however, appeared to be the inverse of its response to the other three drugs. The cross-resistance patterns are discussed, and their implications for combined or successive therapies are considered. PMID- 3015010 TI - Inhibition of herpes simplex virus type 1 replication by halothane. AB - Halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) inhibited herpes simplex virus type 1 (HSV-1) replication, although HSV-1 DNA was synthesized at normal levels in Vero cells. Viral capsids and extracellular virions were inhibited, and HSV-1 protein synthesis decreased by 50%, although no specific HSV-1 protein failed to be synthesized. Hyperbaric pressure failed to reverse the halothane-induced inhibition of HSV-1 replication. PMID- 3015011 TI - Comparative evaluation of a new beta-lactamase inhibitor, YTR 830, combined with different beta-lactam antibiotics against bacteria harboring known beta lactamases. AB - YTR 830, a new beta-lactamase inhibitor, combined with amoxicillin or carbenicillin, showed a synergistic effect similar to that observed with clavulanic acid, and generally better than that with sulbactam, against strains harboring chromosome-encoded penicillinases and broad-spectrum beta-lactamases or plasmid-determined beta-lactamases. With ampicillin, YTR 830 showed the best synergistic activity of the inhibitors against Proteus morganii, Citrobacter freundii, and Enterobacter cloacae and their mutants with a derepressed chromosome-encoded cephalosporinase. PMID- 3015012 TI - Effect of ribavirin on macromolecular synthesis in vesicular stomatitis virus infected cells. AB - Ribavirin at 200 micrograms/ml inhibited vesicular stomatitis virus (VSV) growth in Chinese hamster ovary (CHO) cells by 2.5 logs. To determine the mechanism of this inhibition, viral macromolecular synthesis was examined. VSV primary transcription remained unaffected, but overall VSV RNA synthesis decreased by 40 to 60%. When ribavirin was added 1.5 h after infection, inhibition of progeny production was partially lost, indicating that the antiviral effect was on an early stage after primary transcription. Inhibition of RNA polymerization by premature chain termination was not evident. Viral translation, on the other hand, was reduced by 95% with an inhibition of every protein species. Furthermore, viral RNA synthesized in the presence of ribavirin did not translate well in an in vitro translation system. In contrast, uninfected CHO cells treated with ribavirin showed a greater sensitivity in RNA synthesis than in protein synthesis. This suggests that the cellular translational machinery was not directly affected. Short-term treatment of cells resulted in negligible toxicity, but after 24 h there was marked alteration of cellular integrity. These results, taken together with data on other viruses, suggest that in the presence of ribavirin, dysfunctional VSV mRNA was synthesized, resulting in its failure to be translated. The selective antiviral effects of ribavirin and its relative lack of toxicity for host cells may be predicted on the basis of mRNA turnover and the requirements for de novo functional mRNA. PMID- 3015013 TI - Comparison of the modes of antiviral action of 2'-nor-deoxyguanosine and its cyclic phosphate, 2'-nor-cyclic GMP. AB - The metabolisms of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (2'NDG) and its cyclic phosphate, 9-[(2-hydroxy-1,3,2-dioxophosphorinan-5-yl) oxymethyl]guanine P oxide (2'-nor-cGMP), were compared in cultures of primary rabbit kidney cells infected with herpes simplex virus type 1 (HSV-1). 2'-Nor-cGMP was taken up by the cells essentially intact, after which it was opened to the acyclic monophosphate and phosphorylated further, ultimately to the triphosphate. Formation of the triphosphate was independent of HSV thymidine kinase expression, unlike what is observed with 2'NDG. In addition, there was a direct correlation between the antiviral activity of 2'NDG and the level of triphosphate formed in HSV-1-infected cells, whereas such a correlation was absent with 2'-nor-cGMP. In vivo experiments indicated that only a small percentage of free 2'NDG was formed in the bloodstream of mice after oral administration of 2'-nor-cGMP. Incubation of 2'-nor-cGMP with crude extracts of HSV-1-infected or uninfected HeLa cells resulted in the direct production of 2'NDG triphosphate. The possibility that the triphosphate of 2'NDG produced from 2'-nor-cGMP was the enantiomer of the triphosphate made from 2'NDG by viral and cellular kinases was investigated and disproved. Taken together, these data indicate that (i) 2'-nor-cGMP does not act simply as a prodrug of 2'NDG, (ii) 2'-nor-cGMP does not require viral thymidine kinase for its activity, and (iii) 2'-nor-cGMP may have an additional, triphosphate-independent mode of action. PMID- 3015014 TI - Characterization of a varicella-zoster virus variant with altered thymidine kinase activity. AB - A varicella-zoster virus (VZV) strain resistant to 5-iodo-2'-deoxyuridine (IdUrd) and 5-bromo-2'-deoxyuridine (BrdUrd) but sensitive to (E)-5-(2-bromovinyl)-2' deoxyuridine (BVdUrd) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVdUrd) was isolated. The 2'-deoxythymidine (dThd) kinase of this mutant (Ito) strain was characterized; it was much less efficient in phosphorylating dThd, 2' deoxycytidine, and BrdUrd than were the dThd kinases from wild-type (CaQu, Kobayashi) VZV strains. The Ito dThd kinase had a markedly decreased affinity for dThd, 2'-deoxycytidine, and BrdUrd but only a slightly decreased affinity for IVdUrd than had the wild-type VZV dThd kinase. BrdUrd was incorporated to a much lesser extent in VZV (Ito strain)-infected cells than wild-type VZV-infected cells, but IVdUrd was incorporated in Ito VZV-infected cells as efficiently as in wild-type VZV-infected cells. While resistant to IdUrd and BrdUrd, the Ito strain was susceptible to inhibitors of de novo thymidylate biosynthesis such as aminopterin. PMID- 3015015 TI - Effect of 4-quinolones and novobiocin on calf thymus DNA polymerase alpha primase complex, topoisomerases I and II, and growth of mammalian lymphoblasts. AB - The influence of ciprofloxacin, nalidixic acid, norfloxacin, novobiocin, and ofloxacin on elements of eucaryotic DNA replication was investigated in vitro. Each of the 4-quinolones, when present in amounts of more than 100 micrograms/ml, reversibly inhibited the DNA synthesis performed by the 95 DNA polymerase alpha primase complex from calf thymus. Novobiocin at 500 micrograms/ml or at higher concentrations irreversibly inactivated DNA polymerase alpha primase complex. The accuracy of in vitro DNA synthesis in the absence of repair mechanisms was determined from amber-revertant assays with phi X174am16(+) DNA as template. The antimicrobial agents did not significantly increase the frequencies of base pairing mismatches during the course of replication, indicating that the basal mutation rate is not affected by novobiocin and the 4-quinolones. The Ki values of 50% inhibition of DNA topoisomerases from calf thymus by ciprofloxacin, norfloxacin, novobiocin, nalidixic acid, and ofloxacin were 300, 400, 1,000 or more, 1,000 or more, and 1,500 or more micrograms/ml, respectively, in the case of topoisomerase I, and the Ki values were 150, 300, 500, 1,000, and 1,300 micrograms/ml, respectively, in the case of topoisomerase II. The procaryotic topoisomerase II is approximately 100-fold more sensitive to inhibition by ciprofloxacin, norfloxacin, and ofloxacin than is its eucaryotic counterpart. Growth curves of lymphoblasts were recorded in the presence of ofloxacin and ciprofloxacin. Neither 1 nor 10 micrograms of ciprofloxacin or of ofloxacin per ml affected cell proliferation. Ofloxacin and ciprofloxacin at 100 micrograms/ml inhibited cell growth; 1,000 micrograms/ml led to cell death. No correlation exists between the antimicrobial and cytotoxic activities of the 4-quinolones. PMID- 3015016 TI - Susceptibility of Haemophilus ducreyi to ampicillin and sulbactam in vitro. AB - The MICs of ampicillin, ampicillin plus sulbactam in equal proportions, and a range of ampicillin concentrations with a constant 0.5 micrograms/ml concentration of sulbactam were determined for 66 strains of Haemophilus ducreyi by an agar dilution technique with standardized inocula prepared by ultrasonication. Fifty-five strains were susceptible to ampicillin alone (MIC range, 0.06 to 1 microgram/ml). The MICs for the 11 resistant strains were (micrograms per milliliter): range, 8 to greater than 16; MIC for 50% of the strains tested (MIC50), greater than 16; MIC90, greater than 16; 8 of them produced beta-lactamase. In the presence of an equal concentration of sulbactam, the MICs for ampicillin-resistant strains were lowered to (micrograms per milliliter): range, 0.125 to 2; MIC50, 0.25; MIC90, 1. In the presence of a fixed 0.5-micrograms/ml concentration of sulbactam, the MICs for the resistant strains were (micrograms per milliliter): range, 0.125 to 2; MIC50, 0.125; MIC90, 0.25. Sulbactam-ampicillin appears to be suitable for the treatment of H. ducreyi infections, especially those caused by ampicillin-resistant strains. PMID- 3015018 TI - 2-deoxy-D-glucose inhibition of herpes simplex virus type-1 receptor expression. AB - Growth of HEp-2 cells in 2-deoxy-D-glucose (2-DOG) supplemented media decreased the cells' binding capacity for herpes simplex virus type-1 KOS(HSV-1) but not vesicular stomatitis virus. HEp-2 cells tolerated up to 30 mM 2-DOG, but 2-DOG was toxic for Vero cells over 2 mM. The reduction in binding was maintained for at least 24 h even after careful removal of the inhibitor and growth in normal media. Complete regeneration of the receptor sites on HEp-2 cells was observed 8 h after mild trypsinization of cells grown in either normal or the 2-DOG supplemented media. Specific glycoprotein characteristics of the HSV-1 binding site were indicated by its inactivation upon trypsinization (0.1 mg per 5 X 10(5) cells for 30 s) and blocking by wheat germ agglutinin but not limulin. These results suggest that 2-DOG inhibits the proper expression of cell surface glycoprotein HSV-1 receptor sites on HEp-2 cells. PMID- 3015017 TI - Comparative activities of the beta-lactamase inhibitors YTR 830, clavulanate, and sulbactam combined with ampicillin and broad-spectrum penicillins against defined beta-lactamase-producing aerobic gram-negative bacilli. AB - The in vitro synergistic activities of the beta-lactamase inhibitors YTR 830, clavulanate, and sulbactam, combined with ampicillin, ticarcillin, mezlocillin, azlocillin, piperacillin, and apalcillin, were determined against 34 strains of members of the Enterobacteriaceae family, Pseudomonas aeruginosa, Aeromonas hydrophila, and Haemophilus influenzae with characterized plasmid or chromosomal beta-lactamases or both. Strains were tested against fixed concentrations of beta lactamase inhibitors (8 micrograms/ml) combined with doubling dilutions of beta lactams. Synergy was defined as a fourfold or greater decrease in the MIC of the beta-lactam. Against Enterobacteriaceae producing Richmond and Sykes class III and V plasmid-mediated beta-lactamases, synergy was obtained against most strains with YTR 830- and clavulanate-beta-lactam combinations, with sulbactam being less effective. Against Enterobacteriaceae producing class I chromosomal beta lactamases, combinations containing YTR 830 or sulbactam were more synergistic than combinations containing clavulanate. Against strains producing class V PSE enzymes, all three inhibitors were synergistic with piperacillin and apalcillin against strains producing PSE-1, -3, and -4 enzymes, while the PSE-2-producing strain was resistant to all inhibitors. YTR 830-beta-lactam combinations were also synergistic against strains producing the novel beta-lactamases OHIO-1, TLE 1, AER-1, and ROB-1. Overall, YTR 830 with piperacillin or apalcillin was the most effective combination. PMID- 3015019 TI - Double-blind comparison of weekend and daily regimens of oral acyclovir for suppression of recurrent genital herpes. AB - The potential utility of intermittent regimens of oral acyclovir for suppression of recurrent genital herpes depends on how long the suppressive effect of the drug persists during pauses in treatment. To study this question, we admitted 38 patients in a double-blind controlled trial comparing the results of daily acyclovir treatment (200 mg t.i.d.) with treatment on weekend days only (400 mg t.i.d. on Saturday and Sunday) for suppression of recurrent genital herpes. Of the 35 patients completing the study, significantly more failures occurred in the weekend group (13/17) than in the daily group (3/18, P less than 0.001). Failures on the weekend regimen were more frequent as the week progressed (P = 0.005). The findings suggest a short-term persistence of suppression by acyclovir and hence that intermittent regimens with more closely spaced periods of treatment may be more effective than the regimen we studied. Most virus isolates studied, including all of those isolated from the patients during treatment, were sensitive to acyclovir. PMID- 3015020 TI - Effect of interferon on herpes simplex virus replication in murine macrophage like cell lines. AB - The effect of interferon on the replication of herpes simplex virus types 1 and 2 was studied in two murine macrophage-like cell lines which differ in their ability to synthesize interferon. Higher titers of herpes simplex virus type 1 occurred in PU5-1.8 cell cultures where interferon was not produced than in J774A.1 cell cultures where low amounts of interferon were produced. Herpes simplex virus type 2 replicated poorly in both types of cell cultures. Interferon synthesis was induced in J774A.1 cell cultures but not in PU5-1.8 cell cultures. Exogenously added interferon was shown to inhibit virus replication, however the restrictiveness of these cells to HSV replication was not relieved by treating cell cultures with anti-interferon serum. These results show that factors other than induced interferon regulate the replication of herpes simplex viruses in these cells and suggest that induced interferon synthesis does not affect herpes simplex virus replication in macrophages. PMID- 3015021 TI - Evaluation of mixed cell types and 5-iodo-2'-deoxyuridine treatment upon plaque assay titers of human enteric viruses. AB - Four continuous cell lines, BGM, L-132, HEL-299, and RD, were compared both when cultured separately and as mixtures for use in plaque assay titrations of human adenovirus 1 and six human enterovirus serotypes. The effect of incubating these cell cultures in media containing 5-iodo-2'deoxyuridine (IDU) prior to inoculation with virus was also studied. The use of mixed-cell cultures revealed cell line-dependent synergistic effects as well as inhibitory effects. These effects were strongly virus dependent. In particular, enterovirus 69 did not form plaques on any of the four cell lines when cultured independently. However, it did form plaques on nearly all of the cell lines when cultured as mixtures. Contrary to this effect, when BGM cells were used in combination with the other cell lines, plaque counts for adenovirus 1 were greatly reduced. The effect of IDU pretreatment was also virus and cell line specific and enabled some viruses to form plaques on cell lines when they otherwise would not. Overall, IDU pretreatment resulted in an approximate twofold increase in plaque titers over those obtained without treatment. PMID- 3015022 TI - Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta. AB - Parathion hydrolases have been previously described for an American isolate of Pseudomonas diminuta and a Philippine isolate of Flavobacterium sp. (ATCC 27551). The gene which encodes the broad-spectrum organophosphate phosphotriesterase in P. diminuta has been shown by other investigators to be located on a 66-kilobase (kb) plasmid. The intact gene (opd, organophosphate-degrading gene) from this degradative plasmid was cloned into M13mp10 and found to express parathion hydrolase under control of the lac promoter in Escherichia coli. In Flavobacterium sp. strain ATCC 27551, a 43-kb plasmid was associated with the production of parathion hydrolase by curing experiments. The M13mp10-cloned fragment of the opd gene from P. diminuta was used to identify a homologous genetic region from Flavobacterium sp. strain ATCC 27551. Southern hybridization experiments demonstrated that a genetic region from the 43-kb Flavobacterium sp. plasmid possessed significant homology to the opd sequence. Similar hybridization did not occur with three other native Flavobacterium sp. plasmids (approximately 23, 27, and 51 kb) present within this strain or with genomic DNA from cured strains. Restriction mapping of various recombinant DNA molecules containing subcloned fragments of both opd plasmids revealed that the restriction maps of the two opd regions were similar, if not identical, for all restriction endonucleases tested thus far. In contrast, the restriction maps of the cloned plasmid sequences outside the opd regions were not similar. Thus, it appears that the two discrete bacterial plasmids from parathion-hydrolyzing soil bacteria possess a common but limited region of sequence homology within potentially nonhomologous plasmid structures. PMID- 3015023 TI - Growth of Pseudomonas aeruginosa on nitrous oxide. AB - Three strains of Pseudomonas aeruginosa were grown anaerobically on exogenous N2O in a defined medium under conditions that assured the maintenance of highly anaerobic conditions for periods of 1 week or more. The bacteria were observed reproducibly to increase their cell density by factors of 3 to 9, but not more, depending on the initial amount of N2O. Growth on N2O was cleanly blocked by acetylene. Cell yields, CO2 production, and N2O uptake all increased with initial PN2O at PN2O less than or equal to 0.1 atm. Growth curves were atypical in the sense that growth rates decreased with time. This is the first observation of growth of P. aeruginosa on N2O as the sole oxidant. N2O was shown to be an obligatory, freely diffusible intermediate during growth of strains PAO1 and P1 on nitrate. All three strains used this endogenous N2O efficiently for growth. For strains PAO1 and P1, it was confirmed that exogenous N2O had little effect on the cell yields of cultures growing with nitrate; thus, for these strains exogenous N2O neither directly inhibited growth nor was used significantly for growth. On the other hand, strain P2 grew abundantly on exogenous N2O when small and growth-limiting concentrations of nitrate or nitrate (2 to 10 mM) were included in the medium. The dramatic effect of these N-anions was realized in large part even when the exogenous N2O was introduced immediately after the quantitative conversion of anion-nitrogen to N2. No evidence was found for a factor in filter-sterilized spent medium that stimulated fresh inocula to grow abundantly on N2O.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3015025 TI - Mobilization of Tn5 insertions from Rhizobium fredii by pJB3JI. AB - Techniques for in vivo cloning were used with the fast-growing nitrogen-fixing soybean microsymbiont R. fredii USDA 191. Selection for transfer of Tn5 insertions from R. fredii USDA 191 containing the gene-mobilizing plasmid pJB3JI provided recombinants at up to 400 times the background mutation level. These techniques may be useful for future genetic analysis of R. fredii. PMID- 3015024 TI - Recovery of viruses from water by a modified flocculation procedure for second step concentration. AB - A reduction in virus recovery efficiencies stemming from a change in the commercial processing of powdered beef extract was reversed by the addition of Celite analytical filter aid. Supplementing beef extract with this silicate is recommended as a modification to the organic flocculation procedure for second step concentration in monitoring for waterborne viruses. Considerable differences in virus recovery were found among lots of beef extract and Celite preparations; this indicates that the performance of each lot of these substances should be checked before use. PMID- 3015026 TI - Cloning and expression of the beta-D-galactosidase gene from Streptococcus thermophilus in Escherichia coli. AB - The beta-D-galactosidase (beta-gal) gene from Streptococcus thermophilus was cloned to isolate and characterize it for potential use as a selection marker in a food-grade cloning vector. Chromosomal DNA from S. thermophilus 19258 was cleaved with the restriction enzyme PstI and ligated to pBR322 for transformation into Escherichia coli JM108. A beta-galactosidase-positive clone was detected by its blue color on a medium supplemented with 5-bromo-4-chloro-3-indolyl-beta-D galactoside. This transformant possessed a single plasmid, designated pRH116, which contained, in addition to the vector DNA, a 7.0-kilobase (kb) PstI insertion fragment coding for beta-gal activity. An extract from JM108(pRH116) contained a beta-gal protein with the same electrophoretic mobility as the beta gal from S. thermophilus 19258. Compared with the beta-gal from E. coli HB101, the S. thermophilus beta-gal was of lower molecular weight. A restriction map of pRH116 was constructed from cleavage of both the plasmid and the purified insert. The construction of deletion derivatives of pRH116 with BglII, BstEII, and HindIII revealed the approximate location of the gene on the 7.0-kb fragment. The beta-gal gene was further localized to a 3.85-kb region. PMID- 3015027 TI - Chromatographic resolution of soluble and particulate protein phosphatases from Dictyostelium discoideum. AB - We have examined protein phosphatase activities that are present during the cellular differentiation of Dictyostelium. Utilizing differential centrifugation, ion exchange, gel filtration, and concanavalin A affinity chromatography we found a number of distinct protein phosphatase activities. Three peaks of soluble Kemptide phosphatase activity and a very broad and heterogeneous soluble histone phosphatase activity were resolved by anion exchange chromatography. Histone phosphatase was associated with the particulate fraction, while Kemptide phosphatase was not. The protein phosphatase activities were able to dephosphorylate sites that had been phosphorylated by the cyclic AMP-dependent protein kinase. Therefore it is possible that their function in vivo may be to oppose the action of the cAMP-dependent protein kinase. In addition several paranitrophenyl phosphate phosphatase activities are shown to be largely separable from the protein phosphatases. An apparent heat-stable inhibitor of histone phosphatase is shown to be artifactual in that instead of interacting with the enzyme it acts by complexing with histone. PMID- 3015028 TI - Reactions of 3-ethylcatechol and 3-(methylthio)catechol with catechol dioxygenases. AB - The reactions of 3-ethylcatechol and 3-(methylthio)catechol with catechol 1,2 dioxygenase and catechol 2,3-dioxygenase from Pseudomonas putida were examined. Both 3-substituted catechols are oxidized by catechol 2,3-dioxygenase at approximately 30% of the rate observed for catechol oxidation by this enzyme. Analysis of the products of the reactions showed that ring cleavage occurs in a normal fashion between carbons 2 and 3 of the alternate substrates. 3 Ethylcatechol is oxidized by catechol 1,2-dioxygenase at about 6% of the rate of catechol oxidation; ring cleavage occurs between carbons 1 and 2 to give 2-ethyl cis,cis-muconic acid. However, 3-(methylthio)catechol is a very poor substrate for catechol 1,2-dioxygenase (0.8% of the rate of catechol), but it is a potent competitive inhibitor (Ki = 0.6 microM). The effects of 3-(methylthio)catechol and 3-ethylcatechol on the visible and EPR spectra of catechol 1,2-dioxygenase are also reported. PMID- 3015029 TI - The efficiencies of the component steps of oxidative phosphorylation. II. Experimental determination of the efficiencies in mitochondria and examination of the equivalence of membrane potential and pH gradient in phosphorylation. AB - In the accompanying article (T.E. Gunter and B.D. Jensen, 1986 Arch. Biochem. Biophys. 248, 289-304), a method is described for measuring the efficiencies of individual steps of the process of oxidative phosphorylation. The results of applying this method to the case of state 3 phosphorylation in rat liver mitochondria are reported here. The rate of energy use (or power use) at the gradient generation, leakage, and phosphorylation steps are reported as efficiencies and energy use factors in tabular form. The limits of the degrees of coupling of the gradient generation and phosphorylation steps are also determined and under the current conditions of measurement these degrees of coupling are found to be quite close to unity. The data can be used to show that the only sets of the stoichiometric parameters noH (the charge/2e- ratio in this case from succinate to oxygen), nPH (the H+/ATP ratio), and nTH (number of protons translocated during substrate-product transport) which are simultaneously consistent with both the laws of thermodynamics and with the current data are 8, 3, 1, and 6, 3, 0. The The efficiency of the phosphorylation step which is independent of noH and nTH averages 80% for the control data analyzed. If noH is 8 (succinate to oxygen), the average value of the efficiency of generation of the electrochemical proton gradient is approximately 91 percent. Since very little power (energy) would then be left over to be coupled in parallel to phosphorylation through some other means of coupling, this would place the electrochemical proton gradient in the direct path of power flow and identify it as "an" intermediate in the process. This would suggest that any other intermediate should be considered as being "in series" with the electrochemical proton gradient. The agents butyrate and propionate have been employed to permit investigation over a range of pH gradient and membrane potential. Both butyrate and propionate decrease the efficiency of generation of the electrochemical proton gradient and increase proton leakage. In addition, butyrate activates electron transport whereas propionate inhibits it. By using butyrate to modify the values of pH gradient and membrane potential, it can be shown that the ratio of the efficiency with which the pH gradient is used in phosphorylation to that with which the membrane potential is used is 1.08 +/- 0.38. PMID- 3015030 TI - Interaction between pyridine adenine dinucleotides and bovine liver catalase: a chromatographic and spectral study. AB - Two different fractions were present in crystalline bovine liver catalase, and could be resolved using dye-ligand affinity chromatography with Red-A Matrex gel containing Procion HE 3B. The major part (alpha) was not adsorbed on this gel. The second fraction (beta) was firmly adsorbed to the gel, and could be eluted either by high salt or by NADPH in the micromolar range. Elution of catalase beta was also obtained with NADH, NADP+, and ADP at higher concentration. Fractions alpha and beta displayed no detectable difference in specific activity, stability to heat, and light absorption data. It is suggested that the difference in behavior between alpha and beta is related to the binding of NADPH to the mammalian catalase [H. N. Kirkman and G. F. Gaetani (1984) Proc. Natl. Acad. Sci. USA 81, 4343-4347], and that the beta fraction corresponds to the enzyme molecules that have at least one free site for NADPH binding. Modifications of catalase molecules in the presence of dithioerythritol (DTE) were examined using light absorption and EPR data. Thiol induced changes that corresponded to the formation of catalase complex II. They were partially reversed by NADPH at very low level, and the dinucleotide appeared to be oxidized in this process. DTE treated bovine catalase was totally adsorbed on the Red-A Matrex columns, and could be eluted as fraction beta. Similar spectral changes in the presence of DTE and NADPH were displayed by a bacterial catalase from Proteus mirabilis. This enzyme was also able to oxidize NADPH, but was not adsorbed by Red-A Matrex. This work suggests that dye-affinity chromatography provides a very convenient tool for isolating dinucleotide-depleted catalase from bovine liver, facilitating further study of the physiological function of this cofactor within the enzyme. PMID- 3015032 TI - [Intraarterial DDP and angiotensin II infusion chemotherapy]. AB - Twenty-four patients, each with advanced bladder cancer (BC) or brain tumor (BT), were treated by intraarterial infusion of angiotensin II in combination with DDP as the main drug together with other anticancer drugs. As for the frequency of treatment by the intraarterial therapy, only one course was given in 15 cases, two courses in 9 (BC), one course in 21 cases, and two courses in 3 (BT). CR was obtained in ten of 24 evaluable patients with BC and PR in eleven. Of 20 evaluable patients with BT, 3CRs and 7PRs were attained. The selective enhancement of tumoral blood flow induced by regionally infused angiotensin II was observed using continuous intraarterial infusion of krypton-81 m. PMID- 3015031 TI - [Anti-tumor effect and the mechanism of action of human recombinant TNF]. AB - TNF is cytokine derived from macrophages and holds strong promise for application to cancer therapy because of its marked antitumor effects and its high specificity to tumors. The clinical application (Phase I-II) of TNF has been started because human recombinant TNF (rH-TNF) can been produced on a large scale. In spite of notable antitumor effects, little is known concerning the mechanism of action of its cytotoxic activity. In this article, the antitumor effects of rH-TNF against human and murine tumors, the mechanism of its action and the synergistic effects of rH-TNF in combination with IFN-gamma or with cyclophosphamide are reviewed. PMID- 3015033 TI - [Systemic chemotherapy of hepatocellular carcinoma]. AB - One hundred and twenty-three patients with hepatocellular carcinoma and 4 patients with hepatoblastoma were treated with systemic chemotherapy, and then one patient with hepatocellular carcinoma and 4 patients with hepatoblastoma underwent hepatectomy. Preoperative chemotherapy was evaluated to have been successful in the patient with hepatocellular carcinoma and 3 of the 4 patients with hepatoblastoma. In the former, marked tumor necrosis was observed in the resected specimen. In the latter, a rapid decrease of serum AFP level (T1/2 was less than 13 days) was observed in the effective cases. Preoperative chemotherapy is very useful if effective drugs are available. However, the most serious problem in this field is the lack of drugs active against hepatocellular carcinoma. Our recent results of phase II studies on hepatocellular carcinoma using various drugs were as follows adriamycin 6% (1/17), tegafur 7% (1/15), UFT 4% (1/26) and VP-16 5% (1/21). Development of a new active anticancer agent is thought to be essential if treatment of hepatocellular carcinoma is to advance. PMID- 3015034 TI - [Maintenance chemotherapy by UFT after trans-arterial embolization for hepatocellular carcinoma]. AB - Of 160 cases examined from 1980 until October 1985, 60 cases were given 400 mg of UFT twice daily. Of the stage I and II cases, 28 were treated with UFT and 20 without UFT. In the cases given UFT, the one-year survival rate was 60.3% and the two-year survival rate was 51.6%. In the cases without UFT, however, the one-year survival rate was 44.4% and the two-year survival rate was 19.1%. After 22 months, the survival rate was significantly higher in cases treated with UFT than in those without UFT. Among the stage III cases, survival rate was higher, but there was no statistically significant difference between cases with and without UFT treatment. In the stage I and II cases there were only micrometastases and thrombosis of the tumor. It was therefore considered that maintenance chemotherapy by UFT, was useful for preventing micrometastasis and prolonging survival time. PMID- 3015035 TI - [Multidisciplinary therapy of hepatocellular carcinoma--TAI. TAE treatment by intra-arterial catheterization]. AB - Two hundred sixty-six cases of hepatocellular carcinoma (HCC) were treated between June 1980 and October 1985 (4 years and 4 months) at our hospital. Hepatectomy was performed in 118 patients, 82 of which had received transcatheter arterial embolization with iodized oil (Lipiodol) 58 of then with an intraarterial catheter. HCC tumors were often multiple when they were combined with liver cirrhosis and smaller than 3 cm in diameter. For this reason treatment of HCC by surgery alone has limitations for prolongation of life. A multidisciplinary treatment is therefore necessary. We have found hepatectomy and transarterial embolization to be the most effective treatment for HCC. In order to perform repeated embolizations after hepatectomy, we developed a heparinized catheter with notches to permit safe fixation. This is suitable for long-term intraarterial use. While previous arterial catheters only permitted infusion of drugs due to their small diameters, our new catheter can be used for embolizations with Lipiodol and Gelfoam and for angiography. It is inserted through the right gastroepiploic artery into the gastroduodenal artery so that its tip lies at the level of the hepatic artery. It is brought out through the abdominal skin and flushed at two-week intervals with heparin-urokinase. The indications for the use of the catheter have been repeated embolizations 1) for prevention of tumor recurrence (surgical adjuvant therapy), and 2) after absolutely non-curative operations. For the first indication, we have found that multiple tumors and tumors larger than 5 cm frequently recur within 1 year after surgery. We have, since July 1983, used the catheter treatment to prevent recurrence in 30 such cases. Embolization with Lipiodol + Adriamycin followed by Gelfoam cubes is performed at three-month intervals for one year after surgery, starting one month after surgery, as a rule. The preliminary results indicate an improved survival rate after the treatment. PMID- 3015036 TI - [CT image of the liver cancer after administration of SMANCS/lipiodol]. AB - A new type of anticancer agent with an amphiphilic nature, poly (styrene-co maleic acid)-conjugated neocarzinostatin [SMANCS], was dissolved in the lipid contrast medium Lipiodol [SMANCS/LPD]. This medium was injected intra-arterially and was found to be an invaluable tool for highly sensitive CT image analysis of tumors. Following administration, CT images revealed high-density areas which corresponded to the location and size of liver cancer, the smallest being 4 mm in diameter. The deposition pattern of SMANCS/LPD in liver cancers by CT was classified into 3 types. In type A, Lipiodol was distributed relatively evenly in the tumor lesion, while in type B it was accumulated predominantly around the tumor periphery, the central portion remaining low in density. A for cases exhibited a type C pattern which was a mixture of types A and B. Type A was found essentially in primary liver cancer, and types B and C in secondary liver cancer. Thus, the CT pattern was found to be useful for differential diagnosis. For a sufficient therapeutic effect, 0.08 ml of SMANCS/LPD per cm2 of the maximal CT cross-section of the tumor area was found to be necessary. As a routine protocol after SMANCS/LPD administration, CT scanning was recommended for primary liver cancer initially at one week and then once every month. Secondary liver cancer required more frequent CT follow-ups after administration, on the 3rd day, after one and two weeks, and every month, due to the relatively rapid disappearance of the stain than in primary liver cancer. PMID- 3015037 TI - [Treatment of hepatocellular carcinoma by alcohol injection into the tumor and irradiation of the tumor]. AB - Small hepatocellular carcinoma has come to be diagnosed by imaging modalities recently progressed. However, in patients with the carcinoma surgery is often contraindicated due to liver dysfunction. From such reason intratumor alcohol injection therapy has been developed and the efficacy has been clearly demonstrated in hepato cellular carcinoma with tumor under 3 cm is diameter. While in radiation therapy has been effective in that with tumor up to 10 cm in diameter. In treatment of hepatocellular carcinoma, it is important to choose a therapeutic method by considering the degrees of cancer progress and liver dysfunction. PMID- 3015038 TI - [Intra-tumoral injection therapy in patients with hepatocellular carcinoma]. AB - Ten patients with hepatocellular carcinoma (HCC) received intra-tumoral injection of OK-432 (6 patients), 99.5% ethanol (2 patients) or both (2 patients). Under ultrasonographic control, a PTC needle (22 G) was inserted percutaneously into the tumor and OK-432, which was prepared with a solution of Su-strain Streptococcus pyogenes A3, or 99.5% ethanol was injected. Patients were injected with OK-432 repeatedly at one-to two-week intervals (up to 5 times) for a total duration of 5 to 15 weeks. The degree of skin test reaction for Streptococcus pyogenes was increased in all patients after the treatment. Over 40% tumor regression was noted in 6 out of 9 patients who received intra-tumoral injection of OK-432. Complete regression was noted in one patient. Before treatment, Interleukin-2 (IL-2) and lymphokine-activated killer (LAK) cell activity in peripheral blood lymphocytes decreased in HCC patients. Two of 6 patients showed markedly increased activity of LAK-cells one week after treatment with OK-432. One other patient had moderately increased LAK-cell activity after treatment with OK-432. No increase in LAK-cell activity was seen in 3 patients who received intra-tumoral injection of ethanol. An especially increased response of LAK-cell activity was seen in patients with small-sized HCC (diameter below 5 cm). PMID- 3015039 TI - [Role of hepatic resection in multiple combination therapy of hepatocellular carcinoma]. AB - Recently, resectable hepatocellular carcinoma (HC-C) has been increasing. However, it has not been established whether or not hepatectomy is beneficial in achieving long survival in HCC patients. The present study was aimed at evaluating whether hepatic resection could lead to long survival by retrospectively analyzing cumulative survival rate. One hundred twenty-three cases underwent hepatic resection for HCC between 1977 and 1985. Their post operative cumulative survival rates including operative death were 65, 34 and 27% for 1, 3 and 5 years, respectively. The 3-year survival rates for stages 1, 2, 3 and 4 were 55, 48, 27 and 0%, respectively. When the survival rate was analyzed by curability, the 3-year survival rates were 68 and 28% for curative and non curative operations. In our department, 18 cases have survived over 3 years after hepatectomy. The macroscopical appearance of their tumors was characterized by encapsulation, with no tumor thrombosis in the portal vein. Tumor size or satellite lesions did not necessarily affect long survival. CONCLUSIONS: Liver resection for hepatoma patients at stage I and II can be expected to bring about long-term survival. PMID- 3015040 TI - [Epidemiological aspects of cancer and dietary habits]. AB - Causal association between dietary habits and cancer is supported by both epidemiological and laboratory studies and dietary factor is at present one of the most important risks in carcinogenesis. However, the mechanisms of long low dose exposure to dietary metabolites in carcinogenesis are little known, and there could be many other factors intervening over a long latent period, even if initiation and promotion of cancer occurred as a result of such dietary factors. The purpose of this paper was to review very preliminary reports on epidemiological studies carried out by Ogata-Segi among Japanese in Japan and by Haenszel among Japanese-Americans in Hawaii, together with those of others in the recent years and to evaluate the role of dietary factors in carcinogenesis. Recent epidemiological studies have repeatedly confirmed such dietary effects on cancers, but short-term intervention trials based on certain nutrients such as vitamins and minerals failed to demonstrate any suppressive effects among human populations. A number of other prospective studies on dietary intervention are in progress, but it appears to be difficult to obtain direct evidence from such studies on the population level, due to a lack of knowledge of the complicated metabolic processes of nutrients and their carcinogenic influence on cells and tissues. PMID- 3015042 TI - [Clinical or experimental use of an anticancer drug-oil suspension and its characteristics]. AB - The anticancer agent, Nimustine, which is a derivative of Nimustine hydrochloride (Sankyo CC, Ltd), was suspended in an oil, Lipiodol, using an ultrasonic suspender and used in experimental animals and human subjects with malignant tumor. The use of Lipiodol facilitates the fluoroscopic demonstration of the site into which the suspension has been injected. The Nimustine-Lipiodol suspension was almost stable in room air over 7 days and diffusion of suspended Nimustine into saline in vitro was still noted 4 weeks later. Remarkable regression of tumor size was observed when the Nimustine-Lipiodol suspension was locally injected into the lesion of Lewis lung cancer subcutaneously inoculated into mice. Moreover, a marked regression of tumor size and improvement of CEA level in serum were also obtained when arterial injection of the Nimustine-Lipiodol suspension was carried out in patients with metastatic liver cancer. Therefore, local or arterial injection of Nimustine-Lipiodol suspension is considered to be effective as a method of cancer targeting therapy. PMID- 3015041 TI - [Combination chemotherapy and radiotherapy in small cell carcinoma of the lung]. AB - Thirty-nine patients with small cell carcinoma of the lung were treated sequentially with induction chemotherapy, radiotherapy and then maintenance chemotherapy. Induction chemotherapy consisted of two regimens, cyclophosphamide, vincristine, methotrexate (COM) and adriamycin, ACNU, vindecine (ANV) given by randomization. Radiotherapy was given for patients with limited disease (LD) as a rule. After radiotherapy the drugs used for maintenance chemotherapy were alternated and reduced in dose. Eighteen patients were treated with a COM-ANV sequential combination and sixteen patients were treated with an ANV-COM combination. Thirteen patients had limited disease (LD) and eleven patients had extensive disease (ED). Of 12 patients with LD treated with COM-ANV therapy, 9 patients (75%) responded with 3 (25%) complete responses. Of 11 patients with LD treated with ANV-COM therapy, 9 patients (81.8%) responded to the therapy. According to disease extent, response rate was 82.6% for LD and 54.5% for ED. The median survival times were 9 months for patients with COM-ANV therapy and 12 months for those with ANV-COM therapy. Also, the median survival time was 15 months for LD patients and 5 months for ED patients. Major toxicities in ANV therapy were anorexia, nausea, and myelosuppression, and were more frequent than with COM therapy. These results showed no clear evidence of superiority in either the COM-AMV or ANV-COM regimen. PMID- 3015043 TI - [Brain metastasis from breast cancer]. AB - Thirty-seven patients with breast cancer who developed brain metastasis were analyzed. At the diagnosis of brain metastasis, all patients had widespread metastasis, and 36 patients were receiving chemotherapy. Thirty patients were treated by radiotherapy to the brain at doses of 4,000 rads. There were 6 CRs (20%) and 5 PRs (17%). The median survival time for all patients was 53 months (8 177+) from diagnosis of the primary tumor, 24 months (7-126+) from the first recurrence, and 6 months (1-47+) from diagnosis of brain metastasis. Patients who achieved CR or PR survived longer than non-responders (11+ months vs. 6 months: p less than 0.01). Several backgrounds factors were analyzed, and the results indicated that patients with better performance status survived significantly longer than those with poorer performance status (11 months vs. 4 months: p less than 0.001). PMID- 3015044 TI - [Clinical investigation of neuron-specific enolase, squamous cell carcinoma related antigen and carcinoembryonic antigen in carcinoma of the lung]. AB - Serum levels of neuron-specific enolase (NSE), squamous cell carcinoma-related antigen (SCC antigen) and carcinoembryonic antigen (CEA) were measured in 53 untreated patients with carcinoma of the lung. The positive rates of serum NSE were 17.0% in all patients with lung cancer, 53.8% in small cell carcinoma, 6.7% in adenocarcinoma, 5.0% in squamous cell carcinoma and 0% in large cell carcinoma; 0% in stage I, 14.3% in stage III and 26.7% in stage IV. The positive rates of serum SCC antigen were 45.3% overall, 70.0% in squamous cell carcinoma, 40.0% in adenocarcinoma, 23.1% in small cell carcinoma and 0% in large cell carcinoma; 42.9% in stage I, 57.1% in stage III and 46.7% in stage IV. In comparison with serum CEA, serum NSE and SCC antigen were much more specific in small cell carcinoma and squamous cell carcinoma, respectively. Moreover, serum levels of NSE and SCC antigen changed in parallel with the clinical course during the treatments. In conclusion, serum NSE and SCC antigen were considered to be very useful markers of lung cancer, especially of small cell carcinoma and squamous cell carcinoma, respectively. PMID- 3015045 TI - [Combination chemotherapy with etoposide, mitomycin C, and cyclophosphamide in advanced non-small cell lung cancer]. AB - Forty-three patients with advanced non-small cell lung cancer were treated with a combination chemotherapy regimen comprising etoposide 100 mg/m2 p.o. days 1-5, mitomycin C 10 mg/m2 i.v. day 1 and cyclophosphamide 500 mg/m2 i.v. day 1, every 4 weeks. The median age was 61, and the median initial PS-2. Fourteen patients had received prior therapy. The response rates in previously untreated patients were 25% (5/20) for adenocarcinoma, 0% (0/4) for squamous cell carcinoma, 0% (0/3) for large cell carcinoma, and 18.5% (5/27) for all patients. There were no responders among the pretreated patients. The median survival time was 7 months for previously untreated patients, 4 months for pretreated patients and 6 months for all patients. Patients with adenocarcinoma survived significantly longer (8 months) than those with squamous cell carcinoma (4 months) and large cell carcinoma (3 months). Toxicity consisted of leukopenia (74%), anemia (74%), nausea or vomiting (55%) and alopecia (94%). PMID- 3015046 TI - Pityriasis rotunda. A cutaneous marker of hepatocellular carcinoma in South African blacks. AB - Although paraneoplastic phenomena occur frequently in patients with hepatocellular carcinoma, cutaneous changes have rarely been reported. During the past two years, ten South African blacks with hepatocellular carcinoma and pityriasis rotunda have been seen in a single hospital. The rash affected the trunk, especially the lower back and buttocks. The lesions ranged in size from 1.5 to 25 cm and were always multiple. They had a characteristic circular or arcuate configuration with scaling and varying degrees of hyperpigmentation. Pityriasis rotunda may prove to be a useful cutaneous marker of hepatocellular carcinoma in South African blacks. PMID- 3015047 TI - Chemotactic lipoxygenase products in sera from patients with psoriasis. AB - Sera obtained from 12 patients with moderate to severe psoriasis were investigated for the presence of arachidonate lipoxygenase metabolites using reverse-phase high-performance liquid chromatography after extraction on silicic acid columns. Peaks which co-chromatographed with standards of synthetic leukotriene B4 (LTB4) were collected, and the material was tested for chemotactic activity. In the sera of 5 of the patients, chemotactic activity was demonstrable in these 'LTB4' peaks. Although minor peaks co-chromatographing with LTB4 were found in control sera, none of them contained chemotactic material. Isolated monocytes from the psoriasis patients showed enhanced chemotactic activity as compared to monocytes obtained from healthy controls. The results of our study support the view that abnormal 5-lipoxygenase activity is present in psoriasis. Further investigation is required to determine whether LTB4 is released from circulatory leukocytes or the skin. PMID- 3015048 TI - Anti-oxidant effects of retinoids on inflammatory skin diseases. AB - It is well known that retinoids are effective in the treatment of various dermatological disorders. It has been reported that retinoids have inhibitory effects on the generation of superoxide (O2-) by stimulated polymorphonuclear leukocytes (PMNs). In the present study, we investigated the effects of retinoids on the generation of O2- and other reactive oxygen species (ROS), including OH., H2O2 and chemiluminescence, by zymosan-stimulated PMNs and in the xanthine xanthine oxidase system, because these potent ROS may play an important role in PMN-mediated skin inflammation. It was found that some retinoids had antioxidant effects in the PMN system; however, apart from the effect of tretinoin and Ro 10 1670 on OH. generation, none of the retinoids studied inhibited ROS generation in the xanthine-xanthine oxidase system. On the basis of these results, we discuss a possible mechanism connected with the role of ROS by which retinoids have favourable effects on several inflammatory skin disorders. PMID- 3015049 TI - Generation and characterization of a neutrophil-derived inhibitor of fibroblast chemotaxis. AB - During in vitro chemotaxis, human embryonic fibroblasts migrate toward the leukotriene B4 (LTB4) contained in ionophore-induced human mononuclear-cell supernatants, but they do not migrate toward the LTB4 contained in ionophore induced neutrophil supernatants. We further analyzed and characterized this inhibitory effect. The inhibitor was found to be present in stimulated, but not in unstimulated, neutrophil supernatants. The inhibitor was also shown to be heat labile, to interfere with the chemotaxis of the tumor cell lines HT 1080 and L 929, and to be effective during chemotaxis stimulated by LTB4, conditioned medium, or fibronectin. At Sephadex-G-200 chromatography, the inhibitor eluted in a region corresponding to a molecular mass of 16,000 daltons. Preincubation experiments showed that its mechanism of action is not cell directed, and it had no effect on random migration, cell spreading, and cell attachment. Furthermore, the inhibitor does not interact with the binding of fibronectin to its specific antibody; thus, an interaction of the inhibitor with nonspecific sites which are common to several chemotactic factors must be postulated instead. The biological role of the inhibitor may be related to the regulation of cell migration during wound repair. PMID- 3015051 TI - Demonstration of human papillomavirus DNA in two keratoacanthomas. PMID- 3015050 TI - Papillomavirus DNA in warts of immunosuppressed renal allograft recipients. AB - Renal allograft recipients were shown to have an increased incidence of warts and skin cancers. We examined 148 patients for evidence of wart virus infections and tested for papillomavirus types, which are known to be associated with human malignancies. Of the 148, 36 (24.3%) patients were afflicted with warts at the end of our study period, in contrast to 5 of 148 (3.3%) before transplantation. DNA from 16 different biopsies was extracted by phenol treatment for further virological studies. DNA of human papillomavirus (HPV) 2 was detected three times, DNA of HPV 4 and 10 twice, and DNA of HPV 3 and 16 once each by blot hybridization. One probe led to strong signals with HPV types previously found only in epidermodysplasia verruciformis patients. A correlation between histology and virus type existed in cases of HPV 2, 3, 4, and 10 infections. PMID- 3015052 TI - Epidermodysplasia verruciformis (L-L, 1922) in a patient with systemic lupus erythematosus. PMID- 3015053 TI - [Pulmonary involvement in AIDS. Postmortem study of 50 cases]. PMID- 3015054 TI - [Carcinoembryonic antigen, a marker of cellular retrodifferentiation in breast carcinoma. Its role among the prognostic factors. Its integration into a coefficient of cell aggressiveness]. PMID- 3015055 TI - [5 cases of breast metastases in children. Cytologic aspects]. PMID- 3015056 TI - Serum osteocalcin in rheumatoid arthritis and other inflammatory arthritides: relation between inflammatory activity and the effect of glucocorticoids and remission inducing drugs. AB - Osteocalcin, a vitamin K dependent protein synthesised by osteoblasts, was measured in serum by radioimmunoassay in patients with rheumatoid arthritis (n = 36) and seronegative spondyloarthropathies (n = 23). The serum osteocalcin levels were decreased in both patient groups compared with the levels measured in age and sex matched healthy controls. We found no relation between serum osteocalcin and the disease duration or inflammatory activity of the patients who were without drug treatment at the first examination. After administration of glucocorticoids (20 mg prednisolone a day) circulating osteocalcin decreased significantly after one week of treatment. During gradual reduction of the steroid dosage osteocalcin returned to pretreatment values. Treatment with non steroidal anti-inflammatory drugs (NSAIDs) did not influence circulating osteocalcin. During treatment with chloroquine or penicillamine serum osteocalcin increased significantly, concomitant with a reduction of the acute phase reactants. Controversy persists about the abnormality of bone turnover in rheumatic diseases, but our data suggest that the overall bone turnover is decreased in patients with rheumatoid arthritis and other inflammatory arthritides. PMID- 3015057 TI - Idiopathic haemorrhagic rupture of the shoulder in destructive disease of the elderly. AB - The cases of two patients with hydroxyapatite crystal associated destructive disease of the shoulder are described who have developed gross haemorrhagic effusions with spontaneous joint rupture and extensive soft tissue damage. No collagenase activity was found in the synovial fluid. Other possible mechanisms of the destructive process are discussed. PMID- 3015060 TI - Assay of antimonial compounds by activation analysis: its use in monitoring the clearance of free and liposomally-entrapped sodium stibogluconate by isolated perfused rat liver. PMID- 3015059 TI - Lymphatic and local spread of T1 and T2 pancreatic cancer. A study of autopsy material. AB - Eight autopsy cases of pancreatic cancer (duct cell adenocarcinoma) with T1 and T2 primary tumors were studied histologically to examine the exact extent of lymphatic and local spread. Six of them had microscopic metastasis in grossly negative lymph nodes near the primary tumor. In addition, four of them had a few metastatic nodes in the para-aortic region. In cases with lymphatic metastases, the extent of cancer infiltration within lymphatic vessels, nerves, and/or connective tissues was almost the same as that of lymph node metastasis. Major vascular involvement was found in four cases. There was no case in which multicentricity or marked intraductal spread of cancer cells was observed in the pancreas. It has been suggested that most of T1 and T2 pancreatic cancers have a fairly widespread microscopic extension, although extremely small T1 cancers have a very limited extension. PMID- 3015061 TI - Cytoplasmic inclusions in Giardia: an electron microscopy study. AB - Ultrastructural observations of Giardia trophozoites showed two different types of cytoplasmic inclusions. In Giardia muris from hamsters, the inclusions scattered in the cytoplasm were round, about 0.2 micron in diameter, limited by a double membrane, and with some electron-dense contents. In G. duodenalis from domestic rats, the inclusions were polyhedral, about 0.2 micron in diameter, limited by a double membrane, and with less electron dense contents. These inclusions have an unknown nature, and evidences show interaction between the inclusions and the Giardia trophozoites. PMID- 3015058 TI - The importance of lipid type in the diet after burn injury. AB - The effects of different types of dietary lipids were tested in burned guinea pigs. All diets were identical except for the type of lipid, with total energy intake from lipids equaling 10%. All animals received a 30% total body surface area (TBSA) flame burn and were fed identically by pump-controlled gastrostomy feedings for 14 days. When compared to safflower oil (74% linoleic acid) as well as linoleic acid alone, fish oil (18% eicosapentaenoic acid or EPA) administration resulted in less weight loss, better skeletal muscle mass, lower resting metabolic expenditure, better cell mediated immune responses, better opsonic indices, higher splenic weight, lower adrenal weight, higher serum transferrin, and lower serum C3 levels. With the exception of better cell mediated immune responses in the animals fed linoleic acid, the administration of indomethacin made little difference. These findings can be explained by a reduction in the synthesis of the dienoic prostaglandins that are derived from the omega 6 series of fatty acids, some of which are significantly immunosuppressive. Regulation of dietary lipids may be an important therapeutic advance in nutritional support after burn injury, and controlled trials should be considered. PMID- 3015062 TI - The non-adrenergic non-cholinergic nervous system. PMID- 3015063 TI - The bronchial effects of adenosine in the rat. AB - The in vivo intravenous administration of adenosine caused bronchoconstriction in the rat. There were significant differences between inbred rat strains with regard to bronchial reactivity to adenosine. Inosine caused bronchoconstriction in the rat but was a less potent bronchoconstrictor than adenosine and there was no correlation between the bronchial reactivity to inosine and the reactivity to adenosine. Theophylline, DSCG and nedocromil were potent inhibitors of the adenosine-induced bronchoconstriction. Bilateral vagotomy and the ganglion blocker hexamethonium had no effect on the adenosine-induced bronchoconstriction. High doses of atropine had a small inhibitory effect. The bronchial adenosine receptor has the characteristics of the A2 type. PMID- 3015065 TI - Angiotensin I-converting enzyme: a marker? PMID- 3015064 TI - Human T-cell lymphotropic virus type III associated disorders. The spectrum in the heterosexual population. AB - Eleven heterosexual patients (nine women, two men) without classic risk factors for development of acquired immunodeficiency syndrome (AIDS) were seen between March 1983, and April 1985, and diagnosed as having AIDS (four), persistent, generalized lymphadenopathy (PGL) (four), or asymptomatic human T-cell lymphotropic virus type III (HTLV-III) carrier state (three). The clinical presentations and course of those with AIDS or PGL were similar to those reported in homosexual men with AIDS or PGL, with reversed T4/T8 ratio, and the presence of antibody to HTLV-III. Asymptomatic carriers had normal T4/T8 ratios, had an absence of HTLV-III antibodies, but had HTLV-III virus cultured from blood. We conclude that the heterosexual population, with or without history of sexual exposure to individuals at risk for AIDS, may develop a wide range of clinical manifestations secondary to HTLV-III, varying from AIDS to the asymptomatic carrier state. PMID- 3015067 TI - [Morphology and histochemistry of the liver of swine fetuses (from the 80th day of development) and newborn piglets]. PMID- 3015068 TI - Epstein-Barr virus and depression. PMID- 3015066 TI - Isolation and characterization of TMPD-oxidase mutants of Pseudomonas aeruginosa. AB - Mutants showing negative oxidase-reaction have been isolated from Pseudomonas aeruginosa following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. These mutants were compared to the wild type cells with respect to their respiratory activities and cytochrome contents. They exhibit lower respiration rates and contain much less cytochrome c's which are responsible for their weak or negative oxidase-reaction in these mutants. This is supported in part from an initial linear relationship observed between the measured oxidase activities and the lower cytochrome c contents in these mutants. Further evidence comes from analyzing oxidase-negative cells of P. syringae, in which low cytochrome c contents similar to these oxidase mutants account for negative oxidase activities. Cytochrome o was the sole oxidase found among these mutants as well as in the wild type cell, suggesting that cytochrome c + o complex is responsible for the tetramethyl-p-phenylenediamine-oxidase activity in these mutants as the case in the wild-type cells. From the spectral characteristics it seems that all mutants contain about the same amount and type of terminal oxidase as that of the wild-type cells. The mutation occurred which altered the oxidase activities in these mutants appears to affect cytochrome c gene(s), but not the terminal oxidase gene(s). PMID- 3015069 TI - Lectin histochemistry applied to human nerves. AB - Histologic sections of normal and pathologically altered human peripheral nerves were stained with a battery of 20 fluorescein isothiocyanate-labeled lectins to determine whether these histochemical reagents could be used to identify peripheral nerve injury. Eight plant lectins--from Canavalia ensiformis, Lens culinaris, Triticum vulgare (wheat germ), types E4 and L4 from Phaseolus vulgaris, types I and II from Ricinus communis, and Wistaria floribunda--were found to bind to normal and pathologically altered nerves. Only two lectins, from Helix pomatia and Maclura pomifera, were found to bind selectively to pathologically altered nerves. The changes recognized by lectin histochemistry were not pathognomonic of any specific type of nerve injury. This study provides baseline data on the reaction pattern of human peripheral nerve with a series of lectins and shows that lectin histochemistry could provide means for the study of peripheral nerve pathology. PMID- 3015070 TI - Multiple granular cell tumors. A familial occurrence in children. AB - A 23-year-old black woman and her 6-year-old son, both with multiple granular cell tumors, are described herein. The mother and son both presented as children with multiple granular cell tumors. This is the first reported case of multiple lesions arising in childhood in successive generations. Only two other case studies of familial granular cell tumors have been reported, but in neither of these cases did multiple tumors present initially in both family members during childhood. A preponderance of multicentric lesions is reported in blacks. The tumors recurred locally in some of the sites where there were inadequate surgical margins, emphasizing the need for complete excision. PMID- 3015071 TI - Use of an absorbable mesh to repair contaminated abdominal-wall defects. AB - When polypropylene mesh (Marlex) is used to repair contaminated abdominal-wall hernias, a high incidence of mesh-related chronic infection, drainage, erosion, and bleeding is noted. As an alternative to placing polypropylene mesh in a contaminated field, in the past 18 months we have used an absorbable polyglycolic acid mesh (Dexon) to repair contaminated abdominal-wall defects in eight patients -three with necrotizing abdominal-wall infections, one with an extensive electrical burn of the abdominal wall, three with infected polypropylene mesh from a previous repair, and one whose hernia was covered by a chronically infected scar. In seven of the eight cases, a single sheet of polyglycolic acid mesh was sewn to the fascial margins. In four cases, skin was closed over the mesh; wound packing and subsequent skin grafting were required in the other four. In follow-up studies that ranged from three to 18 months, six of the eight patients developed abdominal-wall hernias at the site of absorbable mesh placement. None of the patients required an abdominal binder. Postoperative hernia development is probable in patients whose defects are repaired with absorbable mesh. However, this complication is balanced against the more serious complications of fistula, bleeding, skin erosion, drainage, and chronic infection, which require removal of the more rigid nonabsorbable meshes in 50% to 90% of cases when the latter are placed under contaminated conditions. Placement of absorbable mesh for temporary abdominal-wall support until wound contamination resolves enhances the likelihood of subsequent successful placement of a permanent mesh. PMID- 3015072 TI - Metastasis to the choroid of the eye from carcinoma of the breast. PMID- 3015073 TI - Hepatitis B virus components produced by the human hepatoma cell line PLC/PRF/5: do they indicate virus propagation? Brief review. PMID- 3015075 TI - Hemagglutination with ovine rotavirus. Brief report. AB - The virus was grown in MA 104 cells, a stable cell line derived from embryonic rhesus monkey kidney, and tested for hemagglutination (HA) with erythrocytes of a variety of species at 4 degrees C, room temperature and 37 degrees C. HA was observed at all temperatures with chicken, sheep, rabbit, guinea pig and human erythrocytes but not with horse, cattle, goat, swine and goose erythrocytes. HA reaction was inhibited by specific antiserum. Some factors involved in the HA and HA-inhibition (HI) were investigated and standard HA and HI tests were worked out. The HI test as well as neutralization test clearly distinguished ovine rotavirus from bovine, human, simian, equine, porcine and lapine rotaviruses. PMID- 3015074 TI - Endothelial cell infection and thrombosis in paralysis caused by equid herpesvirus-1: equine stroke. AB - Eight mares were infected with equid herpesvirus-1 subtype 1 isolated from a case of equine paresis. In two mares killed at 4 d.p.i. immunofluorescence showed endothelial cell infection together with thrombosis in the rete arteriosus of the nasal mucosa and also in the spinal cord of one of these mares. Circulating platelet counts in the other six mares fell as early as 2 d.p.i. and remained depressed for seven days. Circulating immune complexes started to appear at 2 d.p.i., reached maximum levels at 10 d.p.i., but were undetectable at 28 d.p.i. Three of the six remaining mares developed varying degrees of inco-ordination at 8 and 9 d.p.i. In the two inco-ordinate mares that were killed at 9 and 10 d.p.i. the haemorrhages in the spinal cord and brain were associated with extensive endothelial cell fluorescence and thrombus formation. Clinical paresis coincided with an increase in circulating complement fixing and neutralising antibodies which in all six mares were higher against the subtype 2 isolate than subtype 1. In five yearlings infected with a subtype 2 isolate of EHV-1 platelet counts remained normal and neither immune complexes nor viraemia, nor inco-ordination were detected. PMID- 3015076 TI - Comparison of 17 isolates of the human parvovirus B 19 by restriction enzyme analysis. Brief report. AB - The genomes of 17 isolates of the human parvovirus B 19 were compared by restriction with eight endonucleases. All but four isolates proved indistinguishable. A 3.2 kb B 19 DNA fragment was cloned and used as a molecular probe. PMID- 3015077 TI - Patterns of polypeptide synthesis in human rotavirus infected cells. AB - Polypeptide analysis of three strains of human rotavirus (KUN, Wa and MO) were conducted using a hypertonic culture which suppressed host protein synthesis and unmasked rotavirus specific protein synthesis. As a result, eleven human rotavirus specific polypeptides (Vp 1--Vp 11) were detected by pulselabeling infected cells with [14C]-leucine. Among the 11 polypeptides, three polypeptides (Vp 7, Vp 10 and Vp 11) underwent post-translational processing, and two (Vp 7 and Vp 10) were glycosylated. Six polypeptides (Vp 1, 2, 3, 4, 6 and 7) were identified as viral structural proteins. Comparisons of three strains of different serotypes revealed that their polypeptide profiles differed from each other in electrophoretic mobility; in particular, profiles of the glycosylated polypeptide, Vp 7, were distinct among the three strains. PMID- 3015078 TI - Differentiation between primary and recurrent cytomegalovirus infections. AB - Two blocking tests were applied simultaneously to detect antibodies to cytomegalovirus (CMV) in human serum specimens. Two different peroxidase-labelled tracers were prepared from human sera taken either early during a primary CMV infection or late after recurrent infection. Sera of acute primary CMV cases effectively competed with the labelled antibodies of the "early" tracer but a rather weak inhibition was observed with the "late" tracer. Sera of cases with recurrent infection strongly inhibited both tracers. Immunoblot experiments were carried out to explain the mechanism of this differential inhibition. PMID- 3015079 TI - Proteins expressed by Mill Door/79 virus, a Kemerovo serogroup orbivirus transmitted by the ticks Ixodes uriae. AB - Mill Door/79 virus, an orbivirus of the Kemerovo serogroup, Great Island Complex, was shown to induce in infected cells 10 segments of double-stranded RNA with a total molecular weight of 11.46 X 10(6) daltons. Using methyl mercuric hydroxide as a denaturing agent the double-stranded RNA was translated in a cell-free system producing 11 polypeptides, 10 of which co-migrated with those produced in Mill Door/79 virus infected Vero cells. The segments were separated and individually translated in a cell-free system allowing their coding assignments to be made. These assignments were confirmed by partial proteolysis. PMID- 3015080 TI - In vitro cytopathology and pathogenicity to inbred mice shown by five variants of a laboratory strain of type 1 herpes simplex virus. AB - The in vitro cytopathology and the neurovirulence to inbred mice demonstrated by five variants originally derived from one laboratory strain (Miyama) of type 1 herpes simplex virus (HSV-1) were studied comparatively. Three of the variants are syncytial [+GC (LPV), +GC (SPV), +GC (81)] and two are non-syncytial [-GCr and -GCf]. The size of plaques produced by the five variants was found to be in the order of +GC (LPV) greater than +GC (81) greater than +GC (SPV) greater than GCf greater than -GCr. The pathogenicity of these variants was compared in three kinds of inbred mice (AKR, C 3 H/He and C 57 BL) after intraperitoneal (IP) or intracerebral (IC) inoculation. The +GC (LPV) variant was the most virulent as shown by the highest mortality of mice by either route of inoculation. The other four variants caused death of mice only after IC inoculation, and among these variants, +GC (81) was shown to be the most virulent. These data indicate that so far as these five variants of the Miyama strain of HSV-1 are concerned, neurovirulence is positively correlated with their cell fusion activity or the size of plaques which they produce. Pre-IP-inoculation with any of the less virulent variants [-GCr, +GC (SPV) and +GC (81)] protected mice from subsequent lethal infection with +GC (LPV) by the same route of inoculation. PMID- 3015081 TI - Herpes simplex virus type 1 and 2 in the adrenal glands: replication and histopathology. AB - The adrenal glands were shown to be the most severely infected organs in the early phase of HSV-1 infections (up to 10 days p.i.) after i.p. infections in mice. Virus could be isolated from the adrenal glands as early as one hour after infection with pathogenic and apathogenic strains. Infection of the adrenal glands is a result of viremia. The content of HSV-1 (5 strains) was much higher in the adrenals than in spleen and liver. It peaked at 3-4 days p.i. compared to 1-2 days in spleen and liver. Only strain 17 syn+ produced low tissue titres in the adrenal glands. Morphologic alterations by HSV-1 infections commenced with distinct foci 2 days after infection in the zona fasciculata, detected immunohistochemically by HSV-specific peroxidase-staining. Necrotic cells could be observed. The foci became confluent until day 4 and remained in this status up to day 7 p.i. During infection immunocompetent cells (macrophages, granulocytes, many T-helper--but only few T-cytotoxic/suppressor lymphocytes) could be observed. On day 10 p.i. the viral antigen had been completely eliminated. In contrast, intraperitoneal infections with 5 strains of HSV-2 resulted in infection of the adrenal glands only to a low degree. The titer of virus was low (exception: strain HG 52). This correlates well with the type of disease produced by either HSV-1 or 2. By comparing the replication of different strains of HSV-1 and 2, three types of "tropism" after i.p. infection of mice can be distinguished. PMID- 3015082 TI - A latent infection of herpes simplex virus type 2 in a human neuroblastoma cell line IMR-32. AB - Human neuroblastoma (IMR-32) cells were infected with herpes simplex virus type 2 (HSV-2) at a multiplicity of infection (MOI) of 2 plaque-forming units (PFU)/cell and were cultured at 40 degrees C for 14 days. Then neither infectious virus particles nor virus capsids were detected in these cells whereas the presence of virus-specific antigens was observed by immunofluorescent antibody staining technique in 16.9 +/- 3.2 per cent of the infected cell population. When the cultivation temperature was shifted down from 40 degrees C to 35 degrees C, reactivation of virus growth occurred after lag periods of 2-9 days. These findings indicate that the IMR-32 cells can be latently infected with HSV-2 at 40 degrees C and that virus growth may be inhibited at the level of synthesis of virus-specific macromolecules or at some step preceding nucleocapsid formation. PMID- 3015083 TI - Role of epidermal Langerhans cells in resistance to herpes simplex virus infection. AB - To investigate the role of epidermal Langerhans cells (LC) in resistance to herpes simplex virus (HSV) infection, nonadherent spleen cells taken from BALB/c mice immunized with HSV were cultured with syngeneic epidermal cells (EC) and ultraviolet light-inactivated HSV antigen. After five days of culture, T cell dependent proliferative response was determined by 3H-thymidine incorporation. Treatment of EC with anti-Iad monoclonal antibody plus complement before cultivation prevented this proliferation, which suggested that LC induced stimulation of immune T cells. When these stimulated spleen cells were transferred to intracutaneously infected nude mice, the virus titer in the skin was reduced markedly and the formation of zosteriform skin lesions was completely inhibited. On the other hand, transfer of unstimulated cells, which were cultured with HSV antigen only or with HSV antigen and EC treated with anti-Iad monoclonal antibody plus complement, resulted in delayed viral clearance and development of the lesions. These results indicate that LC are of importance as accessory cells in the control of cutaneous HSV infection. Furthermore, Lyt 1+ T cells in the stimulated population were shown to have greater protective activity than Lyt 2+ T cells. PMID- 3015085 TI - Mouse cytomegalovirus is infectious for rats and alters lymphocyte subsets and spleen cell proliferation. AB - The Smith strain of mouse cytomegalovirus (MCMV) was infectious for infant and mature DA strain laboratory rats as judged by development of neutralizing antibodies and specific spleen cell proliferation on stimulation with MCMV antigen. An i.p. inoculum of 10(6) PFU of MCMV was fatal for more than two-thirds of infant mice (1-7 days of age), and disseminated viral infection was documented by isolation of virus from body organs. In contrast, weanling and adult rats did not become ill as a result of infection with a larger inoculum of 10(7) PFU. However, these older MCMV infected rats did show transient reversals of T helper/suppressor cell ratios and alterations of immune cell function as detected by in vitro spleen cell proliferation assays. Seven days after MCMV infection, there was a generalized increase in 3H-thymidine incorporation by spleen cells in both resting (unstimulated) cultures and cultures exposed to mitogens (Con A, PHA, LPS) and to MCMV antigen. At 14 days, the spleen cell proliferation in the unstimulated cultures returned to normal but was depressed compared to controls in response to Con A. These observations show that laboratory rats are susceptible to MCMV infection and that asymptomatic infection may occur and cause transient alterations in lymphocyte subsets and in their reactivity to mitogens. PMID- 3015084 TI - Equine herpesvirus type 1 (EHV-1) induced abortions and paralysis in a Lipizzaner stud: a contribution to the classification of equine herpesviruses. AB - Out of 30 cases of abortion and perinatal deaths in a Lipizzaner stud in Austria 10 mares died after having shown central nervous system disturbances, ataxias and paralysis. The etiological agent of this "abortion storm" was equine herpesvirus type 1 (EHV-1). The restriction enzyme pattern of the DNA from 5 isolates recovered from fetuses has been analyzed and compared with the known reference strains of EHV-1, -2, -4 and an Austrian vaccine strain. The DNA restriction profiles of the Lipizzaner isolates as well as of the vaccine strain could be identified as being typical of abortigenic strains with minor variations. Such variations on the molecular biological level of the DNA do not justify characterization of the strains as neuro-variants. The vaccine strain differed from other isolates investigated with 4 restriction endonucleases (Bam HI, Bgl II, EcoRI, Kpn I) which was due to a deletion in the unique short segment of the genome. The lack of similar DNA bands in two EHV-1 viruses, causing mild respiratory disease, as well as in the vaccine strain Prevaccinol is suggestive of lowered virulence. In contrast to one Lipizzaner isolate tested (strain Austria IV) the Austrian vaccine strain proved to be of strong neurovirulence for suckling mice. PMID- 3015086 TI - Human rhinovirus protein synthesis and polyprotein cleavage in infected HeLa-RH cells. Brief report. AB - Precursor and mature polypeptides of four human rhinovirus types (HRV 30, 63, 81 and 88) were compared. The SDS-PAGE profiles of the HRV-specific polypeptides differed significantly from each other, as well as from those of the HRV types studied previously (HRV 1A, 2 and 14). Our results provide further evidence for considerable heterogeneity within the rhinovirus genus. PMID- 3015088 TI - [Progress in the study of allergic lung diseases]. PMID- 3015087 TI - Antigenic variants of Junin virus isolated from infected Calomys musculinus. Brief report. AB - Junin virus establishes a long-term persistent infection in its natural host, Calomys musculinus. Virus recovered from blood of infected animals from 14 to 61 days p.i. behaved antigenically distinct to parental virus, as shown by cross neutralization assays. The emergence of antigenic viral variants occurs during both the acute and persistent state of infection. PMID- 3015089 TI - [Beta and alpha adrenergic receptor density on cellular components of peripheral blood in patients with bronchial asthma. 1. Relationship between receptor density and airway hypersensitivity]. PMID- 3015090 TI - Neurologic complications of gastric partitioning. PMID- 3015092 TI - Peripheral dystonia. AB - We studied four patients with distal, action-induced involuntary postures of the hand that could be considered focal dystonia. All four patients had electrophysiologic findings consistent with peripheral nervous system lesions (pronator teres syndrome, radial nerve palsy, lower brachial plexus lesion, or median nerve lesion). With varying success, patients were treated with carbamazepine, trihexyphenidyl, methocarbamol, and wrist splinting. We wish to emphasize that peripheral entrapment and brachial plexopathy should be added to the causes of secondary dystonias. PMID- 3015093 TI - AIDS as a sexually transmissible disease. PMID- 3015091 TI - Analysis of JHM central nervous system infections in rats. AB - Intracerebral inoculation of murine coronavirus JHM into 2- to 3-day-old Wistar Furth rats causes an acute encephalomyelitis, while inoculations at 10 days of age usually result in hind leg paralysis. To examine the distribution of viral antigens within this infected central nervous system (CNS) tissue, we used the avidin-biotin-peroxidase method to detect monoclonal and polyclonal antibodies bound to JHM structural proteins; in addition we used the Western blot technique to detect viral proteins. Our study demonstrated the following characteristics: Infected neuronal and glial cells produced viral nucleocapsid and E2 glycoprotein. The synthesis of these viral structural proteins was not restricted to cells in any particular part of the central nervous system. While JHM E2 proteins could be detected in individual cells of JHM-infected CNS tissue, the relative level of detectable E2 protein in the total CNS tissue of infected rats was reduced by more than 13-fold compared with JHM-infected tissue culture cells. Hippocampus neuronal cells provided a sensitive indication of JHM infection. These cells invariably contained antigens in both acutely and chronically infected animals. The distribution of cells containing viral antigens differed markedly for JHM-induced acute encephalitis and chronic demyelinating disease. Acutely infected brains had large lesions containing low levels of viral antigen scattered throughout the brain. One percent to ten percent of histologically normal cells in many parts of the brain contained viral antigens; in addition, more neuronal cells than glial cells were observed to be antigen-positive. The hippocampus appeared normal with hematoxylin-eosin staining; however, a scattered infection of neuronal cells was apparent.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3015094 TI - Scintiscanning of mucoepidermoid tumor of the parotid gland. AB - 99mTc-O4 and/or 67Ga-citrate scintigraphies were preoperatively applied to 13 cases of parotid mucoepidermoid tumor. The resected specimens were histopathologically subdivided into three types: well (6 cases), moderately (one case), and poorly differentiated (6 cases). As the other parotid neoplasms except adenolymphoma and oxyphilic adenoma, all poorly differentiated tumors showed focal defect image in 99mTc-O4 scintiscanning (4/4). They indicated a focal hot image in 67Ga-citrate scanning at a high rate (4/6). Otherwise, well differentiated tumors were scarcely pointed out in cold image and were usually indicated as a symmetrical image (5/6) in 99mTc-O4 scanning. None of them showed a focal hot image in 67Ga-citrate scanning, but two cases indicated diffuse increased uptake and another case showed a focal defect image. The clinical prognosis of mucoepidermoid tumors is extremely correlated to the degree of cell differentiation in our prospective study. For that reason, the therapeutic method should be carefully selected in the tumors suspected of low grade malignancy. Our study suggests that RI (99mTc-O4 and 67Ga-citrate) scintigraphy is helpful in evaluating the malignancy grade of mucoepidermoid tumors. PMID- 3015095 TI - Vulvovaginitis of goats due to a herpesvirus. AB - Two concurrent outbreaks of genital disease in goats were associated with infection by a herpesvirus that was isolated from vulval and vaginal lesions of affected does. Serum neutralising antibody to the virus was present both in goats with the clinical disease and some unaffected goats. Of 19 goat herds examined only 4 had serum neutralising antibody positive goats with low (5%) to high (60%) incidence of infection. The virus isolate was characterised as a herpesvirus on its physico-chemical and morphological features. It contained DNA and was inactivated at low pH and by treatment with lipid solvents and trypsin. The virus particles were icosahedral, consisting of a nucleocapsid surrounded by an envelope membrane and measured approximately 150 nm in diameter. The virus was serologically related to a New Zealand isolate of caprine herpesvirus (NZ-CpHV), associated with similar genital disease, and was distinct from bovine herpes virus-1 (BHV-1) showing a one way neutralisation pattern. PMID- 3015097 TI - Fowl pox in the trachea of point-of-lay hens. PMID- 3015098 TI - Comparison of a kinetic-based enzyme-linked immunosorbent assay (KELISA) and virus-neutralization test for infectious bursal disease virus. II. Decay of maternal antibody in progeny from white Leghorns receiving various vaccination regimens. AB - A computer-assisted single-serum-dilution indirect kinetic-based enzyme-linked immunosorbent assay (KELISA) was used for quantitating the natural decay rate of infectious bursal disease virus (IBDV) maternal antibody in progeny obtained from white leghorn breeders. The KELISA results were compared with those of a standard virus-neutralization (VN) test. Pullets were subjected to two IBDV immunization regimens. Group 1 was vaccinated at weeks 0, 2, and 10 with two live vaccines in drinking water and at week 20 with an oil-emulsified (SC) IBDV vaccine, group 2 received only the first and last immunizations, and group 3 served as the unvaccinated control. Pullets were artificially inseminated at 28 weeks of age. Progeny chicks from each group were bled every other day for 47 days. Both KELISA and VN test detected linear relationship in the decay of maternal antibodies. The VN test detected no significant differences (P greater than 0.05) in the IBDV maternal antibody titers at day 1 or in the rate of decay between the progeny from groups 1 and 2. The VN maternal antibody titers decreased at a rate of 0.16 log2 titer per day. In contrast, KELISA revealed higher IBDV maternal antibody titers in day-old progeny from pullets vaccinated 4 times (log2 = 14.3). However, KELISA titers of progeny from this group decreased at a faster rate than titers of progeny from pullets vaccinated twice (0.20 vs. 0.13 log2 titer per day). PMID- 3015096 TI - Two distinct gel diffusion precipitin tests for the diagnosis of retrovirus infection in goats. PMID- 3015099 TI - Characterization of monoclonal antibodies to avian leukosis viruses. AB - Hybridoma cell lines secreting monoclonal antibody (MCA) to avian leukosis virus (ALV) structural proteins p27 and p19 have been established. In an indirect enzyme-linked immunosorbent assay (ELISA), MCA 6AL20 (IgG1 isotype) reacted with RPL-40 (ALV subgroup A), avian myeloblastosis virus (AMV) (a mixture of subgroups A and B), Rous-associated virus (RAV)-2 (subgroup B), and Carr-Zilber strain of Rous sarcoma virus (CZ-RSV) (subgroup D) but not with Prague strain of RSV (PrC RSV) (subgroup C) or the endogenous virus RAV-0 (subgroup E). MCA 6AL22 reacted as above and also reacted marginally with PrC-RSV. Both MCAs immunoprecipitated p19 from 35S-methionine-labeled chicken embryo fibroblasts (CEFs) infected with RPL-40 or RAV-1, but not from CEFs infected with RAV-0, thus identifying the viral structural protein p19 as a polypeptide with subgroup-specific epitopes. Both MCAs can be used to differentiate RPL-40 from RAV-0 infection either in an indirect antibody ELISA or by immunoprecipitation. A third MCA, 6AL42 (IgG2a isotype), reacted with the above viruses of subgroups A, B, C, and D at an antibody titer up to 1000-fold higher than with subgroup E RAV-0 virus in indirect ELISAs. MCA 6AL42 immunoprecipitated p27 from cells infected with RPL 40, RAV-1, or RAV-0. These MCAs are potentially useful in developing immunological tests for differentiation of ALV strains. PMID- 3015101 TI - Nonspecific reactions in an enzyme-linked immunosorbent assay caused by binding of immunoglobulins in situ to egg-propagated infectious bronchitis virus. AB - High levels of nonspecific background absorbance and increased variability were found in a previously optimized enzyme-linked immunosorbent assay (ELISA) for infectious bronchitis virus (IBV) antibody after changing to commercially available non-pathogen-free eggs for viral antigen production. An increase in bound viral antigen in the assay caused a proportionate increase in the nonspecific binding of the conjugate, independent of other variables, in the absence of serum. Virus was propagated in non-pathogen-free eggs, and individual viral proteins were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose. Localization of chicken IgG-virus complexes were identified by immunoprecipitation with peroxidase-conjugated anti-chicken IgG. Specific staining at molecular weights corresponding to major proteins of IBV was demonstrated in these viral preparations. Virus grown in specific-pathogen-free eggs and treated in the same manner showed only slight amounts of staining. This evidence suggests that viral antigens grown in eggs from a non-pathogen-free flock bind with maternal chicken immunoglobulins present in the allantoic cavity of eggs. This IgG caused nonspecific reactions in our chicken ELISA system and gives cause for concern in any diagnostic system requiring the propagation of agents in fertile eggs. PMID- 3015100 TI - Rapid serological profiling by enzyme-linked immunosorbent assay. IV. Association of infectious bursal disease serology with broiler flock performance. AB - Breeder and broiler flocks were serologically evaluated using a multiple enzyme linked immunosorbent assay (M-ELISA). The serologic status of two commercial broiler-breeder flocks and their progeny was monitored, and 840 sera were promptly assessed for antibodies against six infectious agents using the M-ELISA. Breeder flocks were sampled at lay, and broiler chicks were hatched from fertile eggs collected on the scheduled lay date of the breeders. The broiler chicks were placed for growout as eight separate flocks (four from each breeder), and the serologic survey of broilers included sequentially sampling each flock five times between 1 day of age and market. Association of broiler vaccination schedules, mortality, and condemnation data with the temporal serologic data obtained indicated that the earlier appearance of active antibody against infectious bursal disease (IBD) in some unvaccinated flocks was associated with subsequent higher growout mortality and with the poorer overall performance that these flocks experienced. The results of this serologic survey also demonstrated that if a constant, well-timed monitoring program had not been used, major serologic differences between flocks would not have been detected. Serologic profiles of selected broiler flocks by virus-neutralization (VN) tests for infectious bronchitis virus (IBV) and reovirus or by hemagglutination-inhibition (HI) tests for Newcastle disease virus (NDV) compared favorably with the serologic profiles obtained by M-ELISA. Comparison of vaccination histories with serologic results derived from M-ELISA, VN or HI tests indicated that response to vaccination for IBV and NDV at 1 day was either blocked or significantly delayed by moderate levels of maternal antibody and/or were suppressed by an apparent field outbreak of IBD that occurred in all eight broiler flocks. PMID- 3015102 TI - Gallid-1 herpesvirus infection in the chicken. 3. Reinvestigation of the pathogenesis of infectious laryngotracheitis in acute and early post-acute respiratory disease. AB - Specific-pathogen-free chickens were infected via the trachea when 4 weeks old with 2000 plaque-forming units (PFU) of the virulent Australian infectious laryngotracheitis (ILT) virus strain CSW-1. Titers of ILT virus in the trachea were greatest (10(7.0) PFU/ml in washings, 10(6.0) PFU/g of tissue) 2-4 days postinfection (PI). Infectivity then declined rapidly, to become undetectable by 7 days PI, although highly localized areas of ILT antigen in the tracheal epithelium were occasionally observed by fluorescent antibody staining at 7 and 8 days PI. Tracheal organ cultures established 7 and 8 days PI provided no evidence of latent ILT virus infection at this immediate post-acute stage of pathogenesis. ILT virus was not isolated from peripheral blood leukocytes or lymphoid organs (spleen, bursa, thymus). ILT virus was found in the trigeminal ganglia and/or brain in 14 of 36 chickens (40%) examined between 4 and 7 days after intratracheal inoculation, but it was not in these tissues in five chickens examined at 8 days PI. Virus was also detected at 6 days PI in the trigeminal ganglia in one of five chickens infected by the conjunctival route. These data indicate that the early pathogenesis of ILT (CSW-1) infection frequently involves the tissues of the nervous system. In acute ILT in 4-week-old chickens, interferon-alpha/beta activity was not detectable in serum or tracheal exudates within 14 days PI, but tracheal washings contained significant virus-neutralizing activity by 7 and 8 days PI. In 3-day-old chickens infected via the trachea with 200 PFU of ILT CSW-1, the clearance of ILT virus from the trachea was similar to that observed in 4-week-old chickens, but ILT virus spread systemically to the livers of 20% by 5-7 days PI. PMID- 3015103 TI - Effect of dietary histamine on broiler chickens infected with avian reovirus S1133. AB - One-day-old broiler chickens were infected with either of two vaccine strains of avian reovirus S1133 and fed diets containing 0.2% histamine dihydrochloride for 21 or 35 days. Control groups received either or neither of these treatments. The most notable virus-histamine interaction observed was increased (P less than 0.01) early mortality of chickens infected with the more virulent (pullet) vaccine virus. Histamine in the diet did not affect seroconversion rates or the incidence of stunting in virus-infected chickens. Other evidence of virus histamine interaction was proventricular enlargement and decreased (P less than 0.05) weight gains in chickens infected with the less virulent (chick) vaccine virus, but these signs were observed inconsistently. The possible clinical significance of these observations is discussed. PMID- 3015105 TI - Application of the positive/negative ratio method of analysis to quantitate antibody responses to infectious bursal disease virus using a commercially available ELISA. AB - An enzyme-linked immunosorbent assay (ELISA) test kit has been developed for infectious bursal disease virus (IBDV), and it is widely used for the detection and quantification of IBDV antibody. With results obtained using the commercial kit, a method of analysis was performed utilizing a positive/negative (P/N) ratio standard curve. A prediction equation was derived from this analysis by which the ELISA titer of a serum sample could be determined using one serum dilution. Values obtained using this method were compared with values obtained by the virus neutralization equivalent method recommended by the kit manufacturer. The results were comparable in that the trends in titer were similar. The P/N ratio standard curve method is another way of determining titer using the IBDV ELISA test kit. PMID- 3015104 TI - Influenza virus surveillance in waterfowl in Pennsylvania after the H5N2 avian outbreak. AB - During the latter stages of the lethal H5N2 influenza eradication program in domestic poultry in Pennsylvania in 1983-84, surveillance of waterfowl was done to determine if these birds harbored influenza viruses that might subsequently appear in poultry. From late June to November 1984, 182 hemagglutinating viruses were isolated from 2043 wild birds, primarily ducks, in the same geographical area as the earlier lethal H5N2 avian influenza outbreak. The virus isolates from waterfowl included paramyxoviruses (PMV-1, -4, and -6) and influenza viruses of 13 antigenic combinations. There was only one H5N2 isolate from a duck. Although this virus was antigenically related to the lethal H5N2 virus, genetic and antigenic analysis indicated that it could be discriminated from the virulent family of H5N2 viruses, and it did not originate from chickens. Many of the influenza viruses obtained from wild ducks were capable of replicating in chickens after experimental inoculation but did not cause disease. These studies show that many influenza A virus strains circulating in waterfowl in the vicinity of domestic poultry in Pennsylvania did not originate from domestic poultry. These influenza viruses from wild ducks were capable of infecting poultry; however, transmission of these viruses to poultry apparently was avoided by good husbandry and control measures. PMID- 3015106 TI - Reversion to virulence of chicken-passaged infectious bronchitis vaccine virus. AB - Serial passage of two infectious bronchitis virus (IBV) vaccine strains in chickens enhanced their capacity to increase the incidence and severity of Mycoplasma synoviae (MS) airsacculitis. Included in this report were the mild Massachusetts-type Connaught strain and the Arkansas 99 vaccine strain of IBV. The Connaught strain and one of two Ark 99 vaccine strains passaged in chickens increased the incidence of airsacculitis markedly compared with nonpassaged virus. The other Ark 99 vaccine virus already exacerbated MS airsacculitis, before passage in chickens, and its influence did not increase on passage. All IBV strains studied to date have either possessed this trait or reacquired it on passage in the natural host. PMID- 3015107 TI - An unusual case of inclusion body hepatitis in a cockerel. AB - This report describes inclusion body hepatitis in a 12-week-old leghorn-type cockerel. The cockerel had come from a specific-pathogen-free flock and was reared in a positive-pressure isolator until 6 weeks old. Grossly, the cockerel was jaundiced, and the liver was swollen and had small white foci throughout. Histologically, there were foci of hepatic necrosis and basophilic inclusion bodies in the nuclei of numerous hepatocytes. A fowl adenovirus, isolated from the liver, was found to belong to serotype 2/12. The bursa of Fabricius had some degenerative changes, but these were not consistent with infectious bursal disease, nor was there evidence of seroconversion to infectious bursal disease virus in the remaining chickens. PMID- 3015109 TI - Pathology of avian adenovirus serotypes in the presence of Escherichia coli in infectious-bursal-disease-virus-infected specific-pathogen-free chickens. AB - Ten strains of adenovirus representing 10 serotypes were administered intratracheally to 3-week-old specific-pathogen-free chickens, which also received 2.5 X 10(5) colony-forming units of a pathogenic Escherichia coli intranasally. Those birds had been inoculated by eye drop at 1 day of age with a virulent infectious bursal disease virus (IBDV). Controls consisted of groups of chickens inoculated with: (a) IBDV and E. coli, (b) IBDV only, (c) E. coli only, and (d) no virus or E. coli. Gross pulmonary alterations at 5 days postinoculation (PI) included congestion and consolidation of one or both lungs of a chick inoculated with adenovirus serotype Ind-C and another inoculated with A-2. Histopathologic alterations in the lungs were those of multifocal interstitial and occasionally diffuse pneumonia. All 10 adenovirus serotypes elicited multifocal or diffuse pneumonia and bronchiolitis in one or more of the five birds per group necropsied at 5 days PI. T-8 and A-2 serotypes induced marked to serve diffuse pneumonia within 5 days; Ind-C, Stein, Tipton, 75-1A, B 3, and X-11 incited a mild diffuse pneumonia. In all groups, the pneumonic lesions were more severe 5 days PI than 12 days PI. Tracheitis was incited by Ind C, Stein, T-8, and A-2 at 5 days PI; the lesions were minimal to marked in severity. None of the four control groups exhibited gross or histopathologic alterations except the two IBDV-infected groups, which exhibited bursal change. PMID- 3015108 TI - Future applications of biotechnology in poultry. AB - The major biotechnological advances that can be applied in the poultry industry will include molecular genetics, molecular immunology, and solid-state reactions. The elucidation of the genetic code and the development of techniques to manipulate genes offer new opportunities for changing pathogenic agents and changing chickens to reduce the effect of disease and improve productivity. The monoclonal antibody technique and the discovery that cells of the immune response communicate with one another through peptide factors will permit improved diagnostic techniques and enhanced immune responses to vaccines. Immunologic and biochemical reactions that occur on a solid substrate can be used to simplify and accelerate diagnostic tests and to purify antigens and antibodies. These advances will lead to improvements in diagnosis, disease resistance, and productivity of poultry. PMID- 3015110 TI - Isolation and serial propagation of turkey rotaviruses in a fetal rhesus monkey kidney (MA104) cell line. AB - Turkey rotaviruses from the intestinal contents of poults were isolated and serially propagated in MA104 cell monolayers by a simple procedure. The initial virus isolation was done by low-speed centrifugation of the inoculum onto the monolayers, and subsequent passages were accomplished in roller-tube monolayers using trypsin-treated virus suspensions. Each of the turkey rotavirus isolates possessed the morphologic, antigenic, and genomic attributes characteristic of turkey group A rotaviruses. Attempts to isolate and serially propagate turkey rotavirus-like viruses in MA104 cell monolayers by this procedure were unsuccessful. Turkey reoviruses also did not serially propagate in MA104 cell monolayers by this procedure. PMID- 3015111 TI - Replication-competent endogenous avian leukosis virus in commercial lines of meat chickens. AB - Group-specific (gs)-antigen-positive egg albumen in seven commercial lines of meat chickens was found to result from the presence of endogenous avian leukosis virus (ALV); these lines had resisted selection attempts to reduce the shedding rate. In two meat lines, exogenous as well as endogenous ALV contributed to the gs-antigen shedding. All hens that produced gs-antigen-positive albumen transmitted endogenous ALV to a high proportion of their embryos (20 to 100%). Hens shedding gs-antigen to albumen were negative for endogenous ALV in vaginal swabs and had no detectable antibody to subgroup E virus. Chickens hatched from these dams were negative for endogenous ALV in meconia but were viremic at 2 weeks of age. Replication-competent endogenous ALV was almost uniformly expressed in embryos of hens from nine meat lines that were negative for gs-antigen in albumen. Shedding of gs-antigen to albumen was not related to the level of endogenous ALV expression. Embryos from five meat lines tested were resistant to infection with ALV of subgroup E. The level of endogenous gs-antigen in albumen was consistently lower than the level of exogenous gs-antigen. PMID- 3015112 TI - Pathogenicity studies with plaque-purified preparations of Marek's disease virus strain CVI-988. AB - The pathogenicity of Marek's disease (MD) strain CVI-988 vaccine, eight plaque purified preparations originating from this strain, and the vaccine HVT FC126 (based on herpesvirus of turkeys) was determined by intramuscular administration of high virus doses to day-old specific-pathogen-free Rhode Island Red (RIR) chickens, which are extremely MD-susceptible. Paralysis and neuritis were observed in 88% of RIR chickens inoculated with MDV CVI-988 at the cell-passage level of the commercial vaccine. HVT FC126 caused paralysis in two of 39 RIR chickens tested, of which one had an endoneural lymphoma, and another three had endoneural inflammation. Five plaque-purified MDV CVI-988 virus preparations at various cell-culture-passage levels caused no lesions. Of another three clones, two caused inflammatory B-type lesions in the nerves of 1/10 chickens, and the third clone caused inflammatory nonneoplastic MD lesions in the liver of 1/11 chickens. PMID- 3015113 TI - Protective efficacy of Marek's disease virus (MDV) CVI-988 CEF65 clone C against challenge infection with three very virulent MDV strains. AB - Comparative 50% protective dose (PD50) assays were performed using a plaque purified preparation of Marek's disease virus (MDV) strain CVI-988 at the 65th chicken embryo fibroblast (CEF) passage level (MDV CVI-988 CEF65 clone C) and three commercial MD vaccines: herpesvirus of turkeys (HVT) FC126, MDV CVI-988 CEF35, and a bivalent vaccine composed of HVT FC126 and MDV SB-1. In addition, comparative PD50 assays were performed in groups of chickens with maternal antibody to each of the three vaccines. Three representatives of the newly emerged biovariant very virulent (vv) MDV strains-RB/1B, Tun, and Md5-were employed as challenge virus. The experiments made feasible the differentiation between virulent MDV and vvMDV strains, within serotype 1. Vaccination with CVI 988 clone C vaccine resulted in PD50 estimates of about 5 plaque-forming units (PFUs) against challenge infection with each of the three vvMDV strains. The PD50 estimate of CVI-988 clone C vaccine was 12-fold below the PD50 of HVT FC126. The protective synergism of bivalent vaccine, composed of HVT and SB-1, was confirmed by groups given the lowest vaccine doses. The bivalent vaccine, however, resulted in incomplete protection in groups given the highest vaccine doses. Homologous maternal antibodies to serotype 1 caused a fivefold increase in the PD50 estimate of CVI-988 clone C. Heterologous maternal antibodies against HVT did not interfere with efficacy of CVI-988 clone C vaccination. However, the combination of maternal antibodies against both HVT and SB-1 (serotypes 2 and 3) showed a strong adverse effect on CVI-988 clone C vaccine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3015114 TI - Chronological observations of Marek's disease-associated feather-pulp lesions in field chickens. AB - Fourteen test groups consisting of a total of 541 field chickens in six rearing farms, all of which were vaccinated with turkey herpesvirus at one day old, were investigated for chronological changes in Marek's disease (MD)-associated feather pulp lesions (FPL) and their association with nuclear-inclusion (NI) formation in the feather-follicle epithelium (FFE) and with incidence of MD. The FPL observed were categorized into R1-type (non-tumorous lymphoid lesion), R2-type (inflammatory lesion consisting of plasma cellular infiltration, edema or collagenosis of the pulp, and germinal-center formation) and T-type (tumorous lymphoproliferative lesion). In every test group, the incidence of R1-type lesions was highest at 2-4 weeks of age, in spite of the low incidence of NI formation in the FFE. The initial R1-type lesions were generally transitory and were followed occasionally by R2-type lesions. In each test group, incidence of NI formation in the FFE peaked between 9 and 16 weeks of age. The second peak of incidence of R1-type lesions coincided with the period of peak NI formation. Subsequent skin samples revealed that the incidences of R1-, R2-, and T-type lesions in individual chickens or test groups were closely related to the incidence of NI in the FFE and to the incidence of MD. PMID- 3015115 TI - A comparison between the effect of an avian reovirus and infectious bursal disease virus on selected aspects of the immune system of the chicken. AB - Reovirus 81-176 was inoculated subcutaneously into day-old specific-pathogen-free leghorns and evaluated for its effects on the immune system over a 3-week period. Structural criteria included organ weights of the bursa of Fabricius (BF) and spleen (SP), scoring of histological lesions in the BF, SP, and thymus, and hematological analyses of the circulating leukocytes. Alterations in the functional capacity of the immune system were measured using the graft-versus host reaction, the response of peripheral blood lymphocytes (PBLs) to mitogens, the ability of circulating monocytes to phagocytize latex beads, and the serological responses to Newcastle disease virus, sheep red blood cells, and Brucella abortus antigens. For comparison, infectious bursal disease virus (IBDV) was similarly evaluated by most of the same tests. Structurally, reovirus 81-176 altered BF and SP organ weights, the total numbers of white blood cells in circulation, and the degree of follicular atrophy in the BF. Functionally, reovirus inoculation reduced both the response of PBLs to the phytohemagglutinin P stimulation and monocyte uptake of latex beads. According to the protocols used here, no significant alteration in B-cell function could be detected in reovirus infected chicks. With the exception of leukocyte hematology, IBDV-infected chicks had significantly altered responses in all tests used. By way of comparison, the effects of IBDV were more persistent and pronounced than were those seen with reovirus. The graft-versus-host reaction indicated an elevated and/or uninhibited response of T-cells in the blood of IBDV-infected chicks. PMID- 3015117 TI - In vitro infection studies with infectious laryngotracheitis virus. AB - Infectious laryngotracheitis (ILT) virus strains were studied for their ability to infect chicken macrophages, lymphocytes, and kidney cells in vitro. Although macrophages were as susceptible as chicken kidney cells to infection, replication of most virus strains in macrophages was markedly restricted. Only a few isolates induced progressive infections in macrophages, and even with these the donor of the macrophages influenced replication. Thus, it appears that both cell genotype and virus genotype may help determine the extent of restriction of virus replication. Macrophages were more susceptible to an attenuated vaccine strain of ILT virus than to virulent virus strains. Spleen lymphocytes, peripheral blood lymphocytes, thymocytes, bursal lymphocytes, buffy coat leukocytes, and activated T-cells were nearly or totally refractory to infection by ILT virus. PMID- 3015116 TI - Coronaviruses associated with outbreaks of transmissible enteritis of turkeys in Quebec: hemagglutination properties and cell cultivation. AB - Coronaviruses were observed by electron microscopy in the intestinal contents of turkeys in Quebec flocks where repeated outbreaks of enteritis occurred. Three isolates could be serially propagated in turkey embryos inoculated by the amniotic route with clarified intestinal contents. Purification and concentration of viral particles contained in intestinal contents of infected embryos were achieved by precipitation with polyethylene glycol and ultracentrifugation on sucrose density gradients. Three particle types were demonstrated: intact virions with a density of 1.18 to 1.20 g/ml and incomplete particles with densities of 1.14 and 1.24 g/ml. Hemagglutination of rabbit and guinea pig erythrocytes was demonstrated with the intact viral particles; the hemagglutinin was not dependent on incubation temperature. All the isolates were antigenically related, as shown by hemagglutination-inhibition. The turkey coronaviruses did not cross-react with antisera against coronaviruses of avian infectious bronchitis, porcine transmissible enteritis, bovine neonatal calf diarrhea, or mouse hepatitis. One of the Quebec isolates was shown to induce syncytia formation on its third passage in primary chicken-embryo kidney cell cultures. Electron-microscopic examination of infected cell-culture fluids revealed characteristics coronavirus particles identical to those found in intestinal contents of infected turkeys. PMID- 3015118 TI - Pigeon herpesvirus: inclusion body hepatitis in a free-ranging pigeon. AB - An adult male pigeon (Columba livia) was presented to the Wildlife Service at the University of Pennsylvania School of Veterinary Medicine for depression, cachexia, and diarrhea. Five days after the initial presentation, the bird died and was necropsied. Gross lesions included opaque air sacs and multiple 1-mm yellow-white foci on the epicardial surface of the heart. Histopathologic lesions included a pericarditis, epicarditis, and multifocal hepatic necrosis accompanied by eosinophilic inclusion bodies. Ultrastructural examination of the hepatic inclusions revealed viral particles consistent with a herpesvirus. The gross, light microscopic, and electron microscopic findings are consistent with pigeon herpesvirus infection. PMID- 3015119 TI - Differential effects of dorsal raphe lesions and intraraphe GABA and benzodiazepines on conflict behavior in rats. AB - Both reductions in brain serotonin activity and injections of benzodiazepine drugs increase punished responding in rats, but the evidence is conflicting on the role of serotonin pathways in the benzodiazepine effect. Therefore a series of studies were carried out using a Geller-Seifter procedure with three components, to examine drug effects on rewarded, nonrewarded, and punished responding. Using male hooded Lister rats and chronic indwelling cannulae, it was found that neither chlordiazepoxide (1.5 and 5.0 micrograms in 0.5 microliter), midazolam (1.0 and 10.0 micrograms in 0.5 microliter), nor GABA (100, 500, 1000, and 5000 ng in 0.5 microliter), exerted significant anticonflict activity when injected into the dorsal raphe. Lesions of the dorsal raphe produced by injections of 5,6-dihydroxytryptamine significantly increased punished responding, and there were significant correlations between lesion size, extent of forebrain serotonin depletion, and increases in punished responding. Peripheral injections of chlordiazepoxide (5.0 and 10.0 mg/kg) and midazolam (1.25, 2.5, and 5.0 mg/kg) significantly increased punished responding both before and after raphe lesions. The increase in lesioned animals was significantly greater than after drug or lesion alone and represented a powerful additive effect which was specific to punished responding. As intraraphe benzodiazepines did not exert significant anticonflict activity, and raphe lesions did not attenuate the anticonflict activity of peripheral benzodiazepines, it is concluded that increases in punished responding seen after serotonin depletion and after benzodiazepine drugs may be dissociable. PMID- 3015120 TI - Spinal and locus coeruleus noradrenergic lesions abolish the analgesic effects of 5-methoxy-N,N-dimethyltryptamine. AB - Two experiments were performed on Sprague-Dawley rats to study the effects of noradrenaline and 5-hydroxytryptamine depletion upon the antinociceptive effects of acute 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) administration. 6 Hydroxydopamine-induced lesions following microinjections to either the locus coeruleus or the spinal cord (lumbar) abolished completely 5-MeODMT-induced analgesia in the tail-flick, hot-plate, and shock titration tests whereas 5,7 dihydroxytryptamine-induced lesions of the nucleus raphe magnus and the lumbar spinal cord attenuated 5-MeODMT analgesia in the tail-flick and shock titration tests. Thus, the experiments serve to demonstrate an important interaction between descending noradrenergic and serotonergic pathways, possibly at a spinal locus. PMID- 3015121 TI - The use of avoidance training in studies of modulation of memory storage. AB - Studies of the modulatory effects on memory of many treatments have relied in large part on the use of inhibitory (passive) avoidance training procedures. Recent critiques have questioned the validity of data obtained with the inhibitory avoidance task. This paper addresses these comments and describes many of the advantages of using the procedure in studies of processes which regulate the neurobiological mechanisms which store new information. PMID- 3015122 TI - Intracerebroventricular injection of anti-prolactin serum suppresses excessive grooming of pituitary homografted rats. AB - Rats with endogenous hyperprolactinaemia, as induced by pituitary homografts under the kidney capsule, displayed increased grooming behavior as compared to that of sham-operated animals. Twelve days after surgery, intracerebroventricular injection of anti-prolactin serum (dilution 1:100, 1 microliter) suppressed the excessive grooming of homografted rats. These observations suggest that prolactin from a peripheral source may reach the central nervous system to affect brain mechanisms involved in grooming behavior. PMID- 3015123 TI - Congener characteristics influence aversion conditioning to alcoholic beverages in rats. PMID- 3015124 TI - Photoaffinity labeling of human platelet and rabbit kidney alpha 2-adrenoceptors with [3H]SKF 102229. AB - A newly developed alpha 2-adrenergic photoaffinity ligand, 3-methyl-6-chloro-9 azido-1H-2,3,4,5-tetrahydro-3-benzazepine (SKF 102229), has been radiolabeled with tritium to a specific activity of approximately 80 Ci/mmol. Using membranes prepared from human platelets and from rabbit kidney, alpha 2-adrenoceptors have been covalently labeled following photolysis in the presence of [3H]SKF 102229. As determined by SDS-PAGE, the apparent molecular weight of alpha 2-adrenoceptors from both of these tissues was 64,000. The yield of covalent insertion of [3H]SKF 102229 into the alpha 2-adrenoceptor was very good. Thus, following photolysis up to 90% of the alpha 2-adrenoceptors could be irreversibly labeled with [3H]SKF 102229. PMID- 3015125 TI - Alpha-human natriuretic polypeptide (alpha-hANP) specific binding sites in bovine adrenal gland. AB - The effects of synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) on steroidogenesis in bovine adrenocortical cells in primary monolayer culture were investigated. alpha-hANP did not inhibit basal aldosterone secretion. alpha hANP induced a significant dose-dependent inhibition of basal levels of cortisol and dehydroepiandrosterone (DHEA) secretion and also of ACTH (10(-8) M) stimulated increases in aldosterone, cortisol and DHEA secretion. Visualization of [125I]alpha-hANP binding sites in bovine adrenal gland by an in vitro autoradiographic technique demonstrated that these sites were highly localized in the adrenal cortex, especially the zona glomerulosa. These results suggest that the adrenal cortex may be a target organ for direct receptor-mediated actions of alpha-hANP. PMID- 3015126 TI - Purification of the type I insulin-like growth factor receptor from human placenta. AB - The type I IGF receptor from human placental membranes was purified to near homogeneity by affinity chromatography on IGF I-Sepharose. SDS-polyacrylamide gel electrophoresis of the affinity purified type I IGF receptor demonstrated a high molecular weight protein with Mr greater than or equal to 300,000 under non reducing conditions. After reduction with 2-mercaptoethanol two protein bands were found of Mr = 125,000 and 95,000, representing the alpha- and beta-subunits of the receptor molecule, respectively. A co-purification of the insulin receptor through the IGF I-affinity column could be avoided by a preincubation step with insulin. PMID- 3015127 TI - A similar ribosomal protein S6 kinase activity is found in insulin-treated 3T3-L1 cells and chick embryo fibroblasts transformed by Rous sarcoma virus. AB - Insulin and transformation by Rous sarcoma virus stimulate the phosphorylation of ribosomal protein S6. Soluble fractions containing activated S6 protein kinase from insulin-treated cells and from transformed chick embryo fibroblasts were compared. Based upon several characteristics notably elution from DEAE-cellulose and sedimentation in glycerol gradients, these two S6 protein kinase activities appear to be similar enzymes. Thus insulin and retroviral transformation may activate the same enzyme to regulate the phosphorylation state of S6. PMID- 3015128 TI - Homologous up-regulation of the 1,25 (OH)2 vitamin D3 receptor in rats. AB - This study investigates the ability of vitamin D-metabolites to regulate 1,25(OH)2D3 receptors in vivo. Rats made vitamin D-deficient were treated with 1,25(OH)2D3 or vehicle for 1-5 days. In treated animals, receptors for 1,25(OH)2D3 in kidney increased dramatically compared with control levels. An increase in specific binding to 220% of control was seen after 2 doses of hormone, which reached to 336% after 5 days of treatment. Intestinal receptors increased to only 130% of control levels after 5 days of treatment. In vitamin D replete animals, the difference between control and treated groups was slightly greater when endogenously occupied sites were measured by exchange (TPCK). However, significant changes were observed only after 4 days of hormone treatment. The data indicate that homologous up-regulation of the 1,25(OH)2D3 receptor occurs in vivo. The difference in response in kidney and in intestine suggests differential importance of up-regulation in various organs. PMID- 3015129 TI - Modulation of the relaxing activity of Escherichia coli topoisomerase I by single stranded DNA binding proteins. AB - Removal of negative superhelical turns in ColE1 plasmid DNA by Escherichia coli topoisomerase I was markedly enhanced by the presence of single-stranded DNA binding protein from E. coli. A lack of species specificity makes unlikely the possibility of physical association between topoisomerase I and single-stranded DNA binding proteins. Stabilization of single-stranded regions in supercoiled DNA by single-stranded DNA binding protein would appear to be the basis of the enhancement of topoisomerase activity. PMID- 3015130 TI - GTP and cytosol stimulate phosphoinositide hydrolysis in isolated platelet membranes. AB - Hydrolysis of polyphosphoinositides by phospholipase C was examined in isolated membranes prepared from [32P]labelled platelets. In the presence of GTP gamma S, thrombin increased the release of inositol triphosphate and inositol biphosphate approximately 500%. GTP gamma S alone stimulated release 2 fold. Maximal activation of thrombin-induced phosphoinositide hydrolysis was observed at 10 uM GTP. Although addition of calcium had no effect, 2 mM EGTA completely inhibited inositolphosphate release. Addition of high speed supernatant to [32P]labelled membranes stimulated the release of inositolphosphates. This hydrolysis was further enhanced by the addition of GTP. These data demonstrate that the breakdown of polyphosphoinositides in isolated platelet membranes is dependent on GTP and stimulated by platelet cytosol. PMID- 3015131 TI - Opioid receptors in human neuroblastoma SH-SY5Y cells: evidence for distinct morphine (mu) and enkephalin (delta) binding sites. AB - Human neuroblastoma SH-SY5Y cells exhibited a heterogeneous population of mu and delta types of opioid binding sites. These specific binding sites displayed the characteristic saturability, stereospecificity and reversibility, expected of a receptor. Scatchard analysis of [3H]-D-Ala2-D-Leu5-enkephalin (DADLE) in the presence of 10(-5) M D-Pro4-morphiceptin (to block the mu receptors) and the competitive displacement by various highly selective ligands yielded the binding parameters of delta sites which closely resemble those of the delta receptors in brain and mouse neuroblastoma clones. Similarly, the high affinity binding of [3H]-dihydromorphine, together with the higher potency of morphine analogues to displace [3H]-naloxone binding established the presence of mu sites. Guanine nucleotides and NaCl significantly inhibited the association and increased the dissociation of [3H]-DADLE binding. The observed heterogeneity of opioid receptors in cultured SH-SY5Y cells would serve as an excellent model for the biochemical and pharmacological characterization of brain opiate receptors. PMID- 3015132 TI - Multiple cytochrome deficiency and deteriorated mitochondrial polypeptide composition in fatal infantile mitochondrial myopathy and renal dysfunction. AB - Mitochondria isolated from the skeletal muscle of an infant with mitochondrial myopathy and renal dysfunction were analyzed. Activities of NADH dehydrogenase, succinate dehydrogenase, ubiquinol-cytochrome c oxidoreductase, and cytochrome c oxidase were severely decreased. Cytochromes aa3 and b were not detected in patient mitochondria, and the cytochrome c+c1 content was 14% of control. Immunoblotting demonstrated that the amount of cytochrome c oxidase subunits were markedly decreased in patient mitochondria. The polypeptide profile of patient mitochondria was quite different from that of control mitochondria. These results suggest that deterioration of mitochondria in a severe case of mitochondrial myopathy involves not only cytochrome c oxidase but also other mitochondrial proteins. PMID- 3015133 TI - K dependence of the Na-K pump is abnormal in erythrocytes from patients with cystic fibrosis and obligate heterozygotes. AB - The affinity of the Na-K pump for K was significantly (P less than .001) lower in erythrocytes from patients with cystic fibrosis (Km 4.6 +/- 0.35 mM; n = 26) or from heterozygotes (Km 3.9 +/- 0.57 mM; n = 12) than in controls (Km 2.2 +/- 0.10 mM; n = 20). The affinity of the Na-K pump for K was lower in normal erythrocytes than in normal fibroblasts which may explain the variability in the severity of involvement of different organs in cystic fibrosis. We have now shown in human skin fibroblasts and erythrocytes, that the K affinity of the Na-K pump is lower in patients with cystic fibrosis than in controls. Since the abnormality is also present in erythrocytes from heterozygotes who are clinically normal, it is likely that this abnormality is closely related to the genetic defect in cystic fibrosis. PMID- 3015134 TI - Destabilization of cytoplasmic mouse mammary tumor RNA by heat shock: prevention by cycloheximide pretreatment. AB - Thermal stress of cultured mouse mammary carcinoma cells results in a rapid loss of basal and glucocorticoid-stimulated mouse mammary tumor virus (MMTV) RNA. MMTV RNA is almost nondetectable within 1-2 hr after a shift in the culture temperature to 42 degrees. Actin mRNA is stable over this time period. Pretreatment of the cells with cycloheximide prevents the decay of MMTV RNA. Nuclear associated glucocorticoid receptor is not decreased but rather increased about 95% by the heat shock. The results show that heat shock has pronounced effects on expression of a glucocorticoid-regulated gene, and suggest that a nuclease, which is subject to rapid turnover, is stimulated by thermal stress. PMID- 3015135 TI - Specific binding of atrial natriuretic polypeptide to renal basolateral membranes in spontaneously hypertensive rats (SHR) and stroke-prone SHR. AB - The binding of alpha-human atrial natriuretic polypeptide (alpha-hANP) to basolateral membranes isolated from renal cortex of spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHR-SP) was measured at 0 degree C and compared with that of Wistar-Kyoto rats (WKY). The binding of 125I-alpha-hANP in SHR and SHR-SP at 14-15 weeks old demonstrated different binding profiles compared with that in WKY, though there were no apparent differences of the profiles among WKY, SHR and SHR-SP at 5 weeks old. For the high affinity binding sites, the apparent dissociation constant and the maximal binding capacity in SHR and SHR-SP were significantly decreased in comparison with those in WKY. The present data suggest that the altered characteristics of specific receptors for atrial natriuretic polypeptide in SHR and SHR-SP may be involved in the development or maintenance of genetic hypertension. PMID- 3015136 TI - A reversible effect of low carbon monoxide concentrations on the EPR spectra of the periplasmic hydrogenase from Desulfovibrio vulgaris. AB - The effect of low concentrations of CO (0.93 - 5.58 microM) on the EPR spectrum of the periplasmic non-heme iron hydrogenase from D. vulgaris has been investigated. The "g = 2.06" EPR signal is maximally induced (0.94 spin/molecule) at 46.5 microM CO and partial induction of the EPR signal could be observed at 0.93 microM CO. This effect is reversed by removal of the CO or irradiation of the hydrogenase with white light. PMID- 3015138 TI - Occurrence of two cholecystokinin binding sites in guinea-pig brain cortex. AB - Saturation experiments of the highly potent cholecystokinin analogue [3H]Boc(diNle28,31)CCK27-33 ([3H]BNDL-CCK7, 100 Ci/mmol) with guinea pig brain cortex in a large concentration range (0.05 nM to 30 nM) show the presence of two different binding sites (A site: KD = 0.13 nM, Bmax = 35 fmol/mg; B site: KD = 6.4 nM, Bmax = 92 fmol/mg). Both sites exhibit different sensitivity to sodium ions and therefore can be selectively investigated at [3H]BDNL-CCK7 concentration lower than 1 nM for the A site in Tris buffer and in Krebs buffer for the B site. The selectivity factors KIB/KIA of various CCK related peptides vary from 58 for CCK4 to 26 for CCK8 and 4 for the antagonist (Nle28,31) CCK27-32-NH2. The occurrence of two different CCK binding sites in the brain could explain biphasic pharmacological effects of CCK8. PMID- 3015137 TI - Tumor necrosis factor provokes superoxide anion generation from neutrophils. AB - We report that tumor necrosis factor (TNF) provokes superoxide anion generation from human neutrophils. Superoxide anion generation was provoked at TNF concentration of 1 X 10(-11) M and maximal generation was attained at TNF concentration of 1 X 10(-9) M. We also show that movements of intracellular calcium may mediate the TNF-stimulated superoxide anion generation because 8 (diethylamino) octyl 3,4,5-trimethoxybenzoate hydrochloride--but not extracellular EGTA--inhibited the generation of superoxide anion. These results suggest that TNF may mediate some mechanisms of host defense by provoking superoxide anion generation from neutrophils. PMID- 3015139 TI - Localization of human SAA gene(s) to chromosome 11 and detection of DNA polymorphisms. AB - A human serum amyloid A (SAA) cDNA was used as a probe in chromosome mapping studies to detect human SAA gene sequences in DNA isolated from human/mouse somatic cell hybrids. Southern analysis of DNA from 20 hybrid cell lines, including some with translocations of human chromosomes, placed the SAA gene(s) in the p11----pter region of chromosome 11. Screening of human DNA from unrelated individuals by Southern analysis using the SAA cDNA probe revealed restriction fragment polymorphisms for HindIII and PstI. An analysis of the segregation of these polymorphisms with other markers on the short arm of chromosome 11 should more precisely map the SAA gene(s). PMID- 3015140 TI - Active fragments and analogs of the insect neuropeptide leucopyrokinin: structure function studies. AB - Evaluation of analogs of the blocked insect myotropic neuropeptide leucopyrokinin (LPK) has demonstrated its relative insensitivity to amino acid substitution in the N-terminal in contrast to the C-terminal region. Truncated analogs of LPK without the first, second, and third N-terminal amino acids retain a significant 144%, 59% and 30% of the activity of the parent octapeptide, respectively. The [2 8]LPK analog is the first fragment of an insect neuropeptide to exhibit greater activity than the parent hormone. In contrast, truncated analogs of the insect myotropic, proctolin, exhibit little or no activity. The pentapeptide fragment Phe-Thr-Pro-Arg-Leu-NH2 has been identified as the active core of LPK. PMID- 3015141 TI - Hormonal regulation of amino acid uptake by cultured 3T3-L1 adipocytes. AB - Incubation of the adipocytes for 20 hours with insulin or with Bt2cAMP plus the theophylline stimulated adipocyte uptake of AIB and MeAIB but did not stimulate the uptake of glutamine or cycloleucine. MeAIB uptake by both 3T3-L1 preadipocytes and 3T3-C2 cells was relatively unresponsive to insulin. However, MeAIB uptake by 3T3-C2 cells was stimulated by treatment with Bt2cAMP plus theophylline. Incubation of 3T3 adipocytes for 60 min with insulin yielded maximal stimulation of 2-deoxyglucose uptake but no stimulation of the uptake of AIB, MeAIB or glutamine. Responsiveness of transport to Bt2cAMP does not appear to require adipocyte differentiation. By contrast, adipocyte differentiation may be required for the development of the insulin-responsive transport systems. PMID- 3015143 TI - Initial studies on the cellular pharmacology of 2',3-dideoxycytidine, an inhibitor of HTLV-III infectivity. PMID- 3015142 TI - Renal and cardiovascular effects of atrial natriuretic factor. PMID- 3015144 TI - Aryl hydrocarbon hydroxylase activity and cytochrome P1-450 gene expression in newly established human lymphoblastoid lines. PMID- 3015145 TI - Epoxide hydrolysis in the cytosol of rat liver, kidney, and testis. Measurement in the presence of glutathione and the effect of dietary clofibrate. AB - The hydrolysis of trans- and cis-stilbene oxide and benzo[a]pyrene-4,5-oxide was measured in cytosol and microsomes of liver, kidney, and testis of control and clofibrate-fed rats. Significant levels of nonprotein sulfhydryls were detected in cytosol from liver (4.6 mM) and testis (1.5 mM). Glutathione was moderately stable in these fractions and interfered with the partition assays as conjugates were retained in the aqueous phase along with diols. When the products were separated by thin-layer chromatography, significant amounts of glutathione conjugates were found to have been formed in the cytosol of liver and testis. Overnight dialysis or preincubation of cytosol with 0.5 mM diethylmaleate eliminated conjugate formation without affecting diol production. In dialyzed cytosol from clofibrate-fed rats (0.5%, 14 days), the rates of hydrolysis of trans-stilbene oxide were 506, 171, and 96% of controls for liver, kidney, and testis, respectively, and 126% of controls in liver microsomes. Rates of hydrolysis of cis-stilbene oxide were 149, 172, and 96% of controls in microsomes and 154, 124, and 91% of controls in cytosols from livers, kidneys, and testis of clofibrate-fed rats respectively. Hydrolysis of benzo[a]pyrene-4,5-oxide was similar to that of cis-stilbene oxide. Conjugation of the cis-stilbene oxide with glutathione was detected in cytosols from all three tissues with lesser amounts in the microsomes from liver and kidneys. After clofibrate treatment, the rates of this activity were 200, 173, and 95% of controls in cytosol from liver, kidneys and testis, and 203 and 202% of controls in microsomes from liver and kidneys respectively. These results indicate that epoxide hydrolysis and conjugation in rat liver and kidney are responsive to clofibrate treatment and support other evidence which suggests that hydrolysis of cis- and trans-stilbene oxides in cytosol is catalyzed, in part, by distinct enzymes. PMID- 3015146 TI - The in vitro involvement of topoisomerase II in the activity of aza-ellipticine analogues is not correlated with drug activity on isolated nuclei. AB - Aza-ellipticines are DNA intercalative ellipticine analogues with antitumor activity that induce protein-linked DNA breaks in NIH 3T3 cells in culture. The effects of two aza-ellipticine congeners (BD-40 and BR-76) on the activity of purified Calf Thymus type II topoisomerase were studied using pUC13 DNA as substrate. DNA cleavage was stimulated by both molecules at those doses required for inducing lethal effects in cells (DE5O). This effect was reversed by high salt treatment, indicating that it was actually mediated by Topo II. Mapping of cleavage sites on linearized and 3' end-labelled pUC13 DNA showed that ellipticine and aza-ellipticines stimulated the same sites, which differed from those stimulated by m-AMSA. Decatenating activity of Topo II on Trypanosoma cruzi kDNA was both inhibited by ellipticine and BD-40 at concentrations much higher than DE50 concentrations. Activity of aza-ellipticines was also investigated on isolated nuclei. Unlike ellipticine which promoted DNA-breaking activity, BD-40 and BR-76 were repeatedly inactive. Prior treatment of DNA by Proteinase K did not reveal hidden breaks which are formed in intact cells treated with BD-40 (Vilarem et al., 1984, Nucleic Ac. Res. 12, 8653). Concordant with these data, BD 40 did not impair DNA-synthetic activity in isolated nuclei, while Ellipticine largely decreased it. These results indicate that lesions induced in DNA by Aza ellipticines are mediated by Topo II. The absence of effect of these drugs on isolated nuclei compared to that of Ellipticine may be due to some specific features of the association between Topo II and Aza-ellipticines or reflect a bioactivation step as a prerequisite for in vivo activity. PMID- 3015147 TI - Subcellular fractionation of liver organelles from phenobarbital-treated rats by counter-current partition and sucrose gradient centrifugation. AB - Counter-current partition, sucrose density gradient centrifugation and enzymic analysis were used to explore the changes in rat liver organelles induced by phenobarbital. There was a small increase in partition coefficient and marker enzyme activity of the endoplasmic reticulum. The modal density of the marker enzyme showed a significant decrease reflecting the proliferation of the smooth endoplasmic reticulum. The mitochondria showed a bimodal distribution with a small reduction of marker enzyme activity. In contrast, lysosomes and peroxisomes were relatively unaffected by phenobarbital treatment. Phenobarbital caused a small but statistically insignificant increase in gamma-glutamyl transferase activity: density gradient centrifugation studies indicated that the increased activity was predominantly in the biliary canalicular elements. In contrast, cytosolic gamma-glutamyl hydrolase activity was strikingly reduced by phenobarbital treatment. PMID- 3015148 TI - Blockade of N1E-115 murine neuroblastoma muscarinic receptor function by agents that affect the metabolism of arachidonic acid. AB - Inhibitors of arachidonate metabolism and perturbants of the oxidation-reduction state of the cell were employed to develop a pharmacologic profile for muscarinic receptor-mediated cyclic GMP formation in murine neuroblastoma cells (clone N1E 115). Several lipoxygenase inhibitors [eicosatetraynoic acid (ETYA), nordihydroguaiaretic acid (NDGA), FPL 57231, FPL 55712, BW755c, propylgallate, and AA861] blocked the elevation of [3H]cyclic GMP induced by muscarinic receptor activation. The cyclooxygenase inhibitors indomethacin and ibuprofen were two orders of magnitude less potent in blocking the muscarinic receptor-mediated [3H]cyclic GMP response than in blocking cyclooxygenase in other systems. ETYA and NDGA did not affect the muscarinic inhibition of the prostaglandin E1 mediated increases in [3H]cyclic AMP levels in N1E-115 cells. ETYA did not have a reproducible effect on the muscarinic receptor-induced release of inositol phosphates. Thus, these lipoxygenase inhibitors appeared to be selective for the effector system coupled to the low-affinity muscarinic agonist-receptor conformation, i.e. that which induces cyclic GMP formation. Other effective inhibitors of the cyclic GMP response were methylene blue, catalase, bromphenacyl bromide, retinal, dithiothreitol, quinacrine, and oxidized glutathione. The antioxidant alpha-tocopherol in the concentration range of 100 microM to 1 mM potentiated the receptor response. Arachidonic acid itself was an inhibitor of the muscarinic receptor-mediated cyclic GMP response (IC50 = 45 microM). Linoleic acid and oleic acid were less potent (IC50 = 130 and 190 microM, respectively), and stearic acid was ineffective. When arachidonic acid was air-oxidized, its inhibitory potency was increased 10-fold. Most but not all of the spontaneously produced oxidative metabolites, separable by reverse-phase high pressure liquid chromatography, were inhibitory to the receptor response. Enzymatically synthesized 12-hydroxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid inhibited the muscarinic receptor [3H]cyclic GMP response, with IC50 values of 17 and 8 microM respectively. Catalase was effective in blocking the muscarinic cyclic GMP response (IC50 = 5 microM) while having no effect on either the muscarinic receptor-induced inositol phosphate release or the reduction of cyclic AMP levels. Thus, the effector system for increasing cyclic GMP in these cells displays may of the expected characteristics for the involvement of a lipoxygenase or a related enzyme that oxidatively metabolizes arachidonate in order to activate the guanylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3015149 TI - Reversal of glucose-induced inhibition of newborn rat liver mitochondrial maturation by administration of alkylxanthines at birth. AB - A glucose injection given immediately after birth delays the maturation which normally occurs in rat liver mitochondria and which increases the rate of ATP synthesis coupled to succinate oxidation from a low value at birth to the adult value a few hours after birth [R. Meister, J. Comte, L. Baggetto, C. Godinot and D. C. Gautheron, Biochim. biophys. Acta 722, 36 (1983)]. Alkylxanthine (pentoxifylline, HWA 285) administration at birth has no effect on the maturation of mitochondria prepared from 2-hr-old rat livers while DBcAMP administration increases their RCR and their rate of ATP synthesis. On the contrary, both alkylxanthines and DBcAMP reverse the glucose-induced inhibition of mitochondrial maturation. This DBcAMP effect cannot be mimicked by butyrate and is therefore related to cAMP. The cAMP content of rat liver increases during this postnatal period in both control and glucose-treated rats, although glucose administration tends to decrease the level of cAMP. Alkylxanthine administration restores after 2 hr the cAMP level in glucose-treated animals. The variations of RCR could not be completely correlated with the level of cAMP. The possible involvement of other factors in the mitochondrial maturation and the glucose effect is discussed. PMID- 3015150 TI - Cigarette smoke-induced alterations in the release of arachidonate metabolites by pulmonary alveolar macrophage from selenium-fed and selenium-deficient rats. AB - Male weanling F-344 rats were maintained on selenium-supplemented or -deficient diets and were exposed to fresh cigarette smoke daily for 28 weeks. The deficient status of animals was demonstrated by a significant reduction in the pulmonary and hepatic glutathione peroxidase (GSH-Px) activity of rats on selenium deficient diet. Sham and smoke treatment did not influence the GSH-Px activity in either diet group. Elevated levels of blood carboxyhemoglobin and pulmonary aryl hydrocarbon hydroxylase activity in the smoke-exposed rats of both diet groups indicated effective inhalation of cigarette smoke by animals. Studies of the extracellular release of arachidonate metabolites by pulmonary alveolar macrophages (PAMs) indicated that resting cells released small amounts of prostaglandin E2 (PGE2), thromboxane B2 (TXB2) and leukotriene B4 (LTB4). Upon phagocytic challenge by opsonized zymosan particles, the release of the three metabolites was substantially increased in all diet and treatment groups. While the release of cyclooxygenase products, PGE2 and TXB2, remained unaffected by cigarette smoke, an inhibition of approximately 50% in the release of lipoxygenase product, LTB4, was observed in cells from selenium-fed animals. In selenium-deficient animals, cigarette smoke almost completely inhibited (greater than 80%) the zymosan-stimulated release of LTB4 by PAMs and additionally caused about 50% reduction in TXB2 release. These results suggest a specific inhibition of lipoxygenase pathway by cigarette smoke in PAMs of selenium-fed rats and suggest that cigarette smoke may additionally impair enzymes of the cyclooxygenase pathway in PAMs of selenium-deficient animals. PMID- 3015152 TI - [Synthesis and analysis of des-Met5-[D-Ala2]enkephalinamide analogs]. AB - The effects of N- and C-terminal oligoalanine insertions into des-Met5-[D Ala2]enkephalin amide (I) on the biological activity and spatial structure were examined. The corresponding analogues were obtained by solid-phase synthesis using Sephadex LH-20 ac a polymeric support. Biological activity was assayed via changes in the pain threshold in the rat, body temperature, and also as affinity for opiate receptors. Active analogues were obtained upon modifying the carboxylic group in the tetrapeptide I with di- and tri-D-alanyls. The CD spectra of the C-derivatized analogyes were similar to those of the starting tetrapeptide I and [Met5]enkephalin, whereas the N-derivatized analogues showed essentially different CD spectra. PMID- 3015151 TI - Gentisic acid: an aspirin metabolite with multiple effects on human blood polymorphonuclear leukocytes. PMID- 3015153 TI - [Characteristics of the interaction of restriction endonuclease BamHI with oligodeoxynucleotides]. AB - Interaction of the restriction endonuclease BamHI with a series of synthetic oligodeoxynucleotides containing the restriction site has been studied. The enzyme is shown to specifically cut the BamHI site in hexanucleotide (I) and in non-selfcomplementary deca- and octanucleotides (II)-(IV). The data obtained led to the conclusion that BamHI reacts with duplex structures, while playing an important role in their stabilization. In 14-mer (V) BamHI cuts a non-standard half-site GAA to yield the 5'-terminal tri- (rather than hepta-) nucleotide. Hypothetical mechanisms of the process are discussed basing on conception of the role of higher DNA structures in the interaction with restriction endonucleases. PMID- 3015154 TI - [Synthesis and cloning of a DNA fragment containing a probable site for eukaryotic mRNA binding to ribosome]. AB - A series of oligonucleotides, including two polynucleotides of 33 bases long, were synthesized by a solid-phase phosphotriester method. Potassium salt of 3 nitro-1,2,4-triazole in the presence of 18-crown-6 ether was used as nucleophilic catalyst. The partly complementary polynucleotides were elongated by DNA polymerase I (Klenow fragment) to the full duplex, which was digested with SalGI and was inserted into a plasmid pUR222. Phe synthesized DNA fragment precedes the gene of human gamma-interferon in the chromosome and contains the site for mRNA binding to ribosome. PMID- 3015155 TI - [Complete primary structure of DNA from the transducing bacteriophage lambda plac5]. AB - In studying molecular mechanisms of specialized transduction, primary structure of the junction between the E. coli gene lacI and the lambda phage locus Ea47 in transducing bacteriophage lambda plac5 has been established. Along with the lambda DNA and E. coli lac operon structures as well as with our earlier data on another phage-bacterial junction in lambda plac5, it lead to the complete sequence of lambda plac5 DNA, including the lac5 substitution, a wellknown segment of lambdoid cloning vehicles. The lambda plac5 DNA is shown to consist of 48645 b.p. distributed as follows: 19368 (lambda left arm) + 3924 (lac5 substitution) + 25353 (lambda right arm). The presence of the phage pbL promoter near to the right end of the lac5 insert is shown. The lacI gene distal end in lambda plac5 proved to be considerably more long-stretched than it used to be believed, coding for 224 C-terminal amino-acid residues of lac repressor. The recombination studied in this paper, similarly to the abnormal prophage excision, occurred near to a Chi-like structure, which is partly homologous to the chi+lacZ site present in lambda plac5. On the basis of the data obtained, a key role of the E. coli RecBC system and Chi sites in the formation of long-stretched deletions in the bacterial cell has been suggested. PMID- 3015156 TI - [Genes encoding the beta-subunit of bacterial RNA-polymerases. I. Primary structure of the EcoRI-C fragment of the Salmonella typhimurium gene rpoB]. AB - BglII fragment of S. typhimurium DNA, containing rpoB gene coding for the RNA polymerase beta-subunit, was cloned. The nucleotide sequence of the rpoB gene EcoRI-C fragment (2873 b.p.) was determined. PMID- 3015157 TI - Evidence for the existence of non-esterified cholesterol carried by albumin in rat serum. AB - Isolation of non-esterified [14C]cholesterol bound to albumin from rat serum, 8 days after i.p. injection of [14C]cholesterol, was achieved by affinity chromatography, using Cibacron blue F3GA bound to Sepharose 4B and by Sephadex G 150 column chromatography. Both methods permit isolation of large quantities of cholesterol-loaded albumin, free of globulins and lipoproteins. The isolated albumin-cholesterol fraction was estimated to be 4.6 mg/100 ml serum, which represents approx. the 24% of the non-esterified cholesterol present in the rat serum. Albumin-cholesterol, cholesterol glucoside, cholesterol hemisuccinate and hydroxylated derivatives of cholesterol produced a biphasic curve of changes in synaptosomal plasma membranes (SPM)-bound (Na+ + K+)-stimulated ATPase activity. Low concentrations of the ligand progressively increased the enzyme activity, while increasing the ligand concentration above that which maximally stimulated the enzyme activity, produced a progressive inhibition. Lipoproteins did not have any effect on the enzyme activity. The fluorescence anisotropy of 1,6-diphenyl 1,3,5-hexatriene-labeled SPM, increased in albumin-cholesterol derivatives treated SPM, which is consistent with a general decrease in membrane bilayer fluidity. The results provide evidence that the 'albumin-cholesterol' fraction of the serum may directly affect the cell membrane-bound enzyme activity. PMID- 3015158 TI - Effect of chronic ethanol consumption on human and animal receptor plasticity during aging. AB - Ethanol alters equilibrium between neurotransmitter availability, receptor systems and biological responsiveness. This action may contribute to the accelerating effects of ethanol on the aging process. On this line, the interaction between age and ethanol consumption was studied at laboratory and clinical levels. PMID- 3015159 TI - Evidence for the importance of the "genotype" theory in alcohol seeking behavior: a commentary. AB - Consensus of the literature points towards a neuropsychogenetic model of alcoholism. Evidence in both animals and humans tends to support the proposed "genotype" theory of alcohol-seeking behavior, whereby a predisposition to alcohol preference may be mediated in part by either innate (genetic) or environmentally (stress and/or alcohol) induced brain opioid peptide dysfunction. Potential therapeutic rationale involving the utilization of novel inhibitors of carboxypeptidase A (enkephalinase) which raise endogenous enkephalin levels and possess anti-alcohol seeking effects is emphasized. PMID- 3015160 TI - [Immunoblastic lymphoma after bone marrow graft. Apropos of a case treated by OKT3 monoclonal antibodies for an acute graft versus host reaction]. AB - The incidence of malignant lymphoma after bone marrow transplant seems very low. A 31 years old patient was treated with allogeneic bone marrow transplantation for chronic myeloid leukemia; she develops 12 days after grafting a severe graft versus-host disease (GVH) refractory to treatment with steroids and Cyclosporin A. The GVH was then treated with an anti-T lymphocyte monoclonal antibody (OKT3). The disease responded dramatically to this treatment but the GVH reappeared immediately at the end of OKT3 therapy and the patient died the 103th day after grafting. The autopsy revealed extensive lymphoid infiltrate by a monoclonal IgM K immunoblastic proliferation. After organ or bone marrow transplant, a wide spectrum of lymphoid lesions can be observed, ranging from follicular or diffuse polyclonal hyperplasia to monoclonal immunoblastic lymphoma. The criteria for monoclonality are discussed. Epstein-Barr Virus infection associated with immunosuppressive treatments play probably a major role in the occurrence of a malignant lymphoma. PMID- 3015161 TI - [Cockayne syndrome. Study of a peripheral nerve biopsy]. AB - A sural nerve biopsy was performed in a 10 year-old girl with a Cockayne syndrome. Morphometric and electron microscope studies revealed signs of a chronic demyelinating disease and some dense inclusions in Schwann cells. The significance of these abnormalities is discussed from a review of literature. PMID- 3015163 TI - Dietary fiber and colon cancer. PMID- 3015162 TI - "Giant fibroadenoma in an adolescent Puerto Rican female". PMID- 3015164 TI - [Comparison of the efficacy of 2 oral rehydration solutions: the conventional solution recommended by WHO containing sodium bicarbonate and another containing sodium citrate]. PMID- 3015167 TI - [Brain shrinkage induced by ACTH therapy in epileptic children]. PMID- 3015166 TI - [A case of neonatal herpes simplex encephalitis]. PMID- 3015165 TI - Biocompatibility of a polycarbonate membrane. A preliminary report. AB - In this preliminary study, the biocompatibility of a new dialysis membrane, polycarbonate, was studied. The bioincompatible effect of this membrane is less pronounced compared to cuprophane. PMID- 3015168 TI - [Plasma pipecolic acid concentration in epileptic children]. PMID- 3015169 TI - [Infantile case of herpes simplex encephalitis with spontaneous recovery]. PMID- 3015170 TI - The biochemical functions of ascorbic acid. PMID- 3015171 TI - Alcohol and nutrition: caloric value, bioenergetics, and relationship to liver damage. AB - Alcoholic beverages contribute an appreciable percentage (4-6%) to the total caloric intake in Western societies. The caloric value of ethanol as fuel may be dose-related. Most evidence suggests that at moderate intake levels of less than 45 g/day (3 drinks) ethanol is efficiently utilized as a fuel by the liver. At high intakes, ethanol calories may not be utilized for cellular synthesis of ATP and maintenance of weight. The exact mechanism for this inefficient utilization remains unknown but may be related, in part, to metabolism of ethanol by the microsomal ethanol-oxidizing system, a reaction that does not contribute to generation of reducing equivalents for ATP synthesis. Although ethanol is utilized for ATP synthesis after single-dose administration, chronic consumption leads to morphological changes in hepatic mitochondria and to decreased ATP synthesis. Reductions in the activities of the enzymes of the mitochondrial electron transport chain have been reported after alcohol feeding and may help to explain decreases in hepatic ATP synthesis. There is some evidence that ATP degradation by "Na-K ATPase" is increased after ethanol feeding and that hepatic O2 consumption is likewise enhanced. However, other studies have failed to demonstrate enhanced O2 consumption. Current evidence suggests that malnutrition alone is not sufficient to explain the pathogenesis of chronic liver disease in alcoholics. Although the daily amount of alcohol consumed and the duration of excessive consumption are clearly important factors in the development of alcoholic hepatitis and cirrhosis, other factors, particularly nutritional deficiencies, may modulate the risk of developing alcohol-related liver damage. The prevalence of malnutrition is exceedingly high in alcoholics with clinically severe liver disease. Nutritional deficiencies are better correlated with a clinical index of severity than with histologic severity of alcoholic hepatitis. Prognosis and outcome of patients with alcoholic liver disease may be affected by nutritional deficiencies, which thus provides a rationale for aggressive nutritional management of these patients. PMID- 3015173 TI - [A mild form of brain stem encephalitis due to herpes simplex virus]. AB - A 10-year-old boy had gait and speech disturbances 17 days after the initial symptoms of a fever, headache and cough. Four days later he was admitted to a hospital with mild disturbances of gait, speech, writing, visual acuity, left facial nerve, nystagmus and consciousness. Impairments of cranial nerves (II, III and VII), pyramidal sign and cerebellar sign were noticed. EEG showed generalized slow waves. Auditory brain stem response showed prolongation of the interval between I and V waves and poor differentiation between them. Brain CT could not find any abnormalities. Brain stem encephalitis was diagnosed. Clinical signs and symptoms continued for two weeks when steroid therapy was started and it was effective to improve the disease. He was discharged from the hospital without sequelae. Herpes simplex virus (HSV) type 1 was detected from cells in CSF on admission by fluorescence antibody method. HSV antibody titers in sera changed from 1/8 to 1/64 during three months by complement fixation test. Specific IgG and IgA by enzyme linked immunosorbent assay (ELISA) was high in CSF. Specific antibody in CSF/total antibody in CSF: specific antibody in serum/total antibody in serum for IgG and IgA classes were more than 1. Reports of mild type of HSV brain stem encephalitis seemed to be rare. Our case which was followed for several months carefully would be important to discuss. PMID- 3015174 TI - Correction of local alveolar defects by implantation of hydroxyapatite: a preliminary study. PMID- 3015172 TI - Vitamin D receptors: nature and function. PMID- 3015176 TI - Alpha receptors and ventricular tachycardia after clonidine withdrawal. PMID- 3015175 TI - Diagnosis and prognosis of right ventricular infarction. AB - The values of several non-invasive methods for the diagnosis of right ventricular necrosis in inferior myocardial infarction were compared in 51 consecutive patients who underwent serial radionuclide ventriculography, pyrophosphate scintigraphy, and cross sectional echocardiography. In addition a unipolar electrocardiographic lead V4R was recorded on admission, daily, and during episodes of further pain. Profound right ventricular dysfunction was evident in 50% of patients studied by radionuclide methods after inferior myocardial infarction but recognition on clinical groups alone was poor. Functionally important right ventricular infarction was best detected and followed serially by radionuclide ventriculography. Echocardiographic methods for evaluating right ventricular ejection fraction correlated poorly with radionuclide methods. Increased uptake of radioactivity by the right ventricle on pyrophosphate scintigraphy usually indicated poor right ventricular function, but a scan that was negative in the right ventricular territory did not exclude dysfunction. ST segment elevation in V4R was not specific for right ventricular infarction and its routine use may lead to overdiagnosis of this condition. Serial measurements suggest that profound right ventricular dysfunction persists after acute inferior infarction and is associated with considerable morbidity and mortality. Of 25 patients with severe right ventricular dysfunction, six died in the late hospital period. In the remaining 19 patients mean right ventricular ejection fraction over a two month period did not improve; six patients had persistent right ventricular dyskinesia and features of chronic right ventricular failure developed in three survivors. PMID- 3015177 TI - Prolonged infusion of suxamethonium in infants and children. AB - The neuromuscular blockade produced by a prolonged (greater than 90 min) continuous infusion of suxamethonium and measured with train-of-four stimulation was studied in 20 infants and 20 children during nitrous oxide and halothane in oxygen anaesthesia. The results were compared with a previous study in adults. Suxamethonium requirement was increased in infants and children. Mean peak infusion rates were 297 and 284 micrograms kg-1 min-1 in infants and children, compared with 134 micrograms kg-1 min-1 in adults. An initial tachyphylaxis was followed by bradyphylaxis, and the peak requirement occurred earlier in infants than in children and adults (40 v. 80-100 min). Phase II block developed during the tachyphylaxis. Recovery of neuromuscular activity commenced after stopping the infusion and was accelerated with neostigmine. PMID- 3015178 TI - Comparison of the effects of pancuronium and tubocurarine on different muscles of young and old mice. AB - In order to evaluate the sensitivity of different muscle types to neuromuscular blocking drugs, a system using mouse muscles in vitro was developed and applied to detect changes in drug sensitivity in relation to age. The effect of pancuronium and tubocurarine on initial twitch and on the ratio of fourth twitch to first twitch (T4/T1) of a train-of-four at 2 Hz were compared in fast-twitch, slow-twitch and respiratory muscles in the mouse. The muscles used were: extensor digitorum longus (EDL), soleus (SOL) and diaphragm (DIA). For both drugs the order of decreasing sensitivity was EDL greater than SOL greater than DIA. This result was the same whether first twitch or T4/T1 was used, although the latter was a more sensitive indicator. The sensitivity of neuromuscular block was less in muscles from old (30-33 month) animals than in the equivalent muscles from young (8-12 month) animals. PMID- 3015181 TI - Porphyrias, porphyrins and hepatocellular cancer. PMID- 3015179 TI - The 1986 Walter Hubert lecture. Recent studies on a vaccine to prevent EB virus associated cancers. AB - Epstein-Barr (EB) virus was discovered in 1964 (Epstein et al., 1964). In the decades since then an immense body of information has been accumulated on the virus and a great deal is now known about its general biological behaviour, its epidemiology, its molecular biology, the humoral and cellular immunological responses which it evokes, and about its relationship to human cancers. The fact that EB virus was thought from the outset to be a human tumour virus was no doubt responsible for the large number of laboratories in which it has been studied. Viruses causing tumours in animals have been known since early in the present century and affect frogs, fowl, rodents, rabbits, cats, cattle, monkeys and even fish (Klein, 1980). It was obvious that man could not be different in this respect and the finding of EB virus therefore promised to bring human tumours into line with those of other species. PMID- 3015180 TI - The in vitro genetic effects of fibrous erionite and crocidolite asbestos. AB - Epidemiologic evidence has recently identified an association between an endemic outbreak of pleural and peritoneal mesothelioma in the Urgup region of Turkey and exposure to zeolite fibres. This malignancy is usually associated with exposure to asbestos dusts whose mineralogical characteristics differ from those of zeolites. The present study further defines the in vitro biologic activity of erionite, a common zeolite fibre found in the Urgup region of Turkey. Both erionite and crocidolite asbestos fibres were clastogenic in synchronous Chinese hamster ovary (CHO) fibroblasts. Both fibres also altered CHO ploidy. Erionite, unlike crocidolite or Min-U-Sil quartz, caused a slight increase in sister chromatid exchanges in synchronous CHO cells. Neither erionite nor crocidolite was mutagenic in a human lymphoblastoid cell line. Erionite fibres thus produced in vitro cytogenic changes similar to those caused by asbestiform mineral dusts and, like asbestos fibres, did not induce mutations in human lymphoblastoid cells. PMID- 3015182 TI - Variations in steroid receptors and cyclic AMP binding proteins across human breast cancers: evidence for heterogeneity. PMID- 3015183 TI - Serum hepatitis B viral DNA in HBsAg-positive hepatocellular carcinoma treated with interferon or adriamycin. AB - Sera from 31 HBsAg-positive Chinese patients with inoperable hepatocellular carcinoma (HCC) were tested for hepatitis B virus DNA (HBV DNA) by means of dot hybridisation and Southern blot technique. HBV DNA probes were prepared from human plasma. Eighteen of the patients were HBeAb-positive, 12 were HBeAg positive and one case had neither marker. Serial specimens were obtained from 16 cases over 5-42 weeks, while the patients were treated with recombinant leukocyte A interferon (rIFN-A) or adriamycin. Seven patients (2 HBeAg-positive, 5 HBeAb positive) were positive for HBV DNA. In two patients HBV DNA and HBV DNA polymerase (DNAp) appeared in serum weeks after rIFN-A or adriamycin treatment was started. In two other cases, HBV DNA that was initially present disappeared during rIFN-A treatment. In a fifth patient HBV DNA persisting after adriamycin treatment diminished after change of treatment to rIFN-A. With one possible exception the HBV DNA detectable by Southern blot technique was composed chiefly of sequences 2.2-3.2 kb size indicating the presence of unintegrated DNA forms. DNAp activities were raised in the presence of HBV DNA in 4 patients. These findings show that HBV replication can be activated or suppressed in advanced HCC. Treatment with rIFN-A may have been effective in suppressing HBV DNA synthesis, but the number of cases studied was too small to arrive at a definite conclusion on this point. PMID- 3015185 TI - Rheumatoid pneumoconiosis in a dolomite worker. PMID- 3015186 TI - Three cases of eccrine porocarcinoma. AB - We report three cases of eccrine poroma, present for many years, in which features were seen suggesting transformation from a benign to a malignant tumour. These changes ranged from in situ Bowenoid dysplasia to frankly invasive squamous carcinoma. The most helpful diagnostic feature in distinguishing the origin of the tumours was the presence of strong cytoplasmic staining for carcinoembryonic antigen (CEA) in cells surrounding, and giving rise to, neoplastic ducts and clefts. Dermatopathologists examining eccrine poromata should examine the lesions carefully for any evidence of malignant change. PMID- 3015184 TI - Chemotherapy versus chemotherapy plus irradiation in limited small cell lung cancer. Results of a controlled trial with 5 years follow-up. AB - One hundred and forty-five patients with limited stage small cell lung cancer were included in a randomized trial to evaluate the effect of chemotherapy with or without chest irradiation. Seventy-six patients were allotted chemotherapy alone while 69 patients received the same chemotherapy plus radiotherapy, 40 Gy in split-course, administered in weeks 6 and 10 after the initiation of chemotherapy. The chemotherapy consisted of lomustine, cyclophosphamide, vincristine and methotrexate. Patients treated with chemotherapy alone survived for a median of 52 weeks compared to 44 weeks in patients receiving the combined regimen (P = 0.055). After exclusion of five early deaths and one patient refusing the irradiation plus 14 completely resected patients, the remaining 65 patients receiving chemotherapy alone and the 60 patients treated with chemotherapy plus radiotherapy were included in a new analysis. The difference in survival duration which could be ascribed to treatment with or without chest irradiation thereby diminished (P = 0.24). Eighteen months' disease-free survival was obtained in 9.2% of the 65 patients and in 9.8% of the 60 patients. The complete remission rates were 37% and 46%, respectively, (P = 0.33) and the median durations of complete remission were 40 weeks and 52 weeks (P = 0.67). Treatment failure of the primary tumour occurred in 85% of patients treated with chemotherapy alone in contrast to 61% of patients receiving the combined regimen (P = 0.005). Seventy-nine of these patients underwent autopsy at which no residual chest disease was observed in 17% and 37%, respectively (P = 0.045). The combined regimen was more toxic than chemotherapy alone resulting in significantly greater dose reductions and more pronounced thrombocytopenia. Lung and pericardial fibrosis was responsible for four deaths among the complete responders in the radiotherapy group. The combined regimen thus tended to be more efficacious with respect to tumour control at the expense, however, of increased toxicity which per se, eliminated a potential improvement of the overall therapeutical results. PMID- 3015187 TI - Gianotti-Crosti syndrome: a study of 26 cases. AB - We have studied 26 patients presenting with a symmetrical papular or papulovesicular acrolocated eruption of more than 10 days duration. Mean age at onset was 2 years (range 10 months to 5.75 years). Lymphadenopathy was noted in eight cases, and hepatomegaly in one case. In 12 cases, histopathology and direct immunofluorescence were non-contributory. Cytolytic hepatitis occurred in one case and was associated with HBs antigenemia. A history of recent immunization was given in two cases. There was serological evidence of recent Epstein-Barr virus infection in seven out of 13 cases tested. Coxsackie B viruses were isolated from three patients, and cytomegalovirus was probably associated with the syndrome in one case. We conclude that the Gianotti-Crosti syndrome is not rare in France, and that non-hepatitis B virus (HBV)-associated cases are more frequent than the classical HBV-associated papular acrodermatitis of childhood. PMID- 3015188 TI - Incontinentia pigmenti: eosinophil chemotactic activity of the crusted scales in the vesiculobullous stage. AB - To investigate the mechanisms underlying eosinophil infiltration into the epidermis in incontinentia pigmenti (IP), we studied the eosinophil chemotactic activity in extracts of the crusted scales from three patients with IP in the vesiculobullous stage. Eosinophil chemotactic activity was detected in the eluates from a Sephadex G-75 chromatography column between the vitamin B12 and phenol red markers. The chemotactic activity was heat-stable and resistant to enzyme digestion, and recovered after ether extraction at low pH. Leukotriene B4 (LTB4) was demonstrated in the fractions with high eosinophil chemotactic activity. These findings suggest that LTB4 plays an important role in the accumulation of eosinophils within the epidermis in IP, in the vesiculobullous stage. Blood eosinophilia, however, may not be induced by the eosinophil chemotactic factors in the scales. PMID- 3015189 TI - Normal levels of ATP, total nucleotides and activities of three enzymes related to nucleotide metabolism in fetal erythrocytes. AB - Pure fetal blood was obtained by direct-vision fetoscopy from 30 fetuses at 17-23 weeks gestation. The erythrocyte concentrations of ATP and total nucleotides and the activities of the enzymes pyrimidine-5'-nucleotide nucleosidase (Pyr5N), phosphoribosylpyrophosphate (PRPP) synthetase and adenylate kinase (AK) were analysed by established techniques to find the normal ranges for this gestational age. The ranges were relatively narrow and could serve as reference values for the prenatal diagnosis of defects in nucleotide metabolism. The results from the fetal erythrocytes were compared with the corresponding values from the maternal blood collected and analysed concurrently. The ATP and total nucleotide concentrations and the activity of Pyr5N in the fetal cells were substantially higher than those of the maternal blood. The activities of PRPP synthetase and AK were much lower. The significance of these findings is discussed. PMID- 3015190 TI - Molecular pathology of haemoglobin H disease in Sardinians. AB - We investigated the molecular basis for haemoglobin H disease in 50 Sardinian patients by restriction endonuclease analysis. We found that the majority (78% of the cases) are due to gene deletion (- -/- alpha). Among those with a combination of deletion and nondeletion defects (- -/alpha alpha th), the most prevalent nondeletion lesion (70% of the nondeletion defects) was the initiation codon mutation of the alpha 2 gene (alpha Nco alpha), previously discovered in this population. Of the remaining patients with the (- -/alpha alpha th) genotype, two showed the IVS-1 splice junction lesion and one a mutation in the alpha 1 gene, removing the Nco I site within the 5' part of the alpha 1 gene, which may arise from a process of gene conversion from the initiation codon mutant of the alpha 2 gene. A single patient had the homozygous state for the initiation codon mutant of the alpha 2 gene. Study of genotype-phenotype correlations indicates that the (alpha Nco alpha) haplotype is associated with a more severe defect in the alpha globin chain output than that resulting from the (-alpha) haplotype. We may conclude that restriction endonuclease analysis is a powerful method for the definition of the molecular heterogeneity of haemoglobin H disease. PMID- 3015191 TI - Red cell metabolism in hereditary pyrimidine 5'-nucleotidase deficiency: effect of magnesium. AB - Since pyrimidine nucleotides avidly bind magnesium, we tested the hypothesis that the haemolytic anaemia in hereditary pyrimidine 5'-nucleotidase (P5N) deficiency is due to a state of functional magnesium depletion in the red cell (RBC). In haemolysates from normal subjects, cytidine triphosphate (CTP) inhibited the activity of pyruvate kinase in a competitive manner for magnesium. The CTP Ki was 0.4 mmol/l. CTP inhibited the activity of hexokinase in a competitive manner for ATP (Mg-ATP2-) with a Ki of 4 mmol/l. The inhibitory effect of CTP on both enzymes was overcome by increasing the magnesium content of the test system. Since CTP appeared to inhibit enzymes which required magnesium as a cofactor or Mg-ATP2- as a substrate, we tested the effect of exogenous magnesium on the metabolism of P5N deficient RBC. The autohaemolysis test, the incubated Heinz body assay and the rate of glucose oxidation by the pentose phosphate shunt were abnormal in the intact RBC from a patient with hereditary P5N deficiency. The addition of MgCl2 (6-10 mmol/l) did not improve these abnormal in vitro measures of metabolism in the P5N deficient RBC. This lack of effect of exogenous magnesium may be due to the slow uptake of magnesium by the human RBC. PMID- 3015192 TI - Production of anti-cytomegalovirus antibody following T-cell depleted bone marrow transplant. AB - The serological status to cytomegalovirus (CMV) was examined in 24 patients with no detectable CMV excretion, following T-lymphocyte depleted bone marrow transplantation. The results show that seropositive recipients continue to produce CMV antibody regardless of the serological status of the donor but that seronegative recipients fail to produce CMV antibody even when the donor is seropositive. These findings suggest that when an effective CMV vaccine becomes available vaccination of the donor would be unlikely to confer protection on the recipient but that vaccination of the recipient may achieve this. PMID- 3015193 TI - Reduced risk of recurrent leukaemia in bone marrow transplant recipients after cytomegalovirus infection. AB - The first 72 consecutive bone-marrow transplant recipients with haematological malignancies (29 with acute nonlymphoblastic leukaemia, 31 with acute lymphoblastic leukaemia, nine with CML and three with myelofibrosis, IgA myeloma and T-cell lymphoma, respectively) were investigated for the frequency of relapses 1 year or later after bone-marrow transplantation. Seven relapses occurred from 30 to 850 d after transplantation (median 180 d). All relapses occurred in patients with acute leukaemia less than or equal to 18 years of age with a high risk for relapse, i.e. transplanted in second or later remission or with more than 10% blasts in the marrow before transplantation. Among all patients the probability of relapse was increased in patients without cytomegalovirus (CMV) infection (P = 0.001) and in patients without chronic GVHD (P = 0.049). Among leukaemic patients less than or equal to 18 years of age with a high risk of relapse all relapses occurred in patients (n = 11) without CMV infection, whereas no relapses were seen in patients (n = 13) with CMV infection (P = 0.006). Known risk factors for leukaemic relapse were comparable in both groups. PMID- 3015194 TI - Mortality of nitrate fertiliser workers. AB - An epidemiological cohort study was conducted to investigate the mortality patterns among a group of workers engaged in the production of nitrate based fertilisers. This study was designed to test the hypothesis that individuals exposed to high concentrations of nitrates might be at increased risk of developing cancers, particularly gastric cancer. A total of 1327 male workers who had been employed in the production of fertilisers between 1946 and 1981 and who had been occupationally exposed to nitrates for at least one year were followed up until 1 March 1981. In total, 304 deaths were observed in this group and these were compared with expected numbers calculated from mortality rates in the northern region of England, where the factory was located. Analysis was also carried out separately for a subgroup of the cohort who had been heavily exposed to nitrates--that is, working in an environment likely to contain more than 10 mg nitrate/m3 for a year or longer. In neither the entire cohort nor the subgroup was any significant excess observed for all causes of mortality or for mortality from any of five broad categories of cause or from four specific types of cancer. A small excess of lung cancer was noted more than 20 years after first exposure in men heavily exposed for more than 10 years. That men were exposed to high concentrations of nitrate was confirmed by comparing concentrations of nitrates in the saliva of a sample of currently employed men with control men, employed at the same factory but not in fertiliser production. The men exposed to nitrate had substantially raised concentrations of nitrate in their saliva compared with both controls within the industry and with men in the general population and resident nearby. The results of this study therefore weight against the idea that exposure to nitrates in the environment leads to the formation in vivo of material amounts of carcinogens. PMID- 3015195 TI - Vulvar fibrous histiocytoma of low grade malignancy. Case report. PMID- 3015196 TI - Cortisol concentrations in the umbilical artery and vein of breech-presenting infants at term in relation to the method of delivery. PMID- 3015197 TI - Poly(dA-dT).poly(dA-dT) two-pathway proton exchange mechanism. Effect of general and specific base catalysis on deuteration rates. AB - The deuteration rates of the poly(dA-dT).poly(dA-dT) amino and imino protons have been measured with stopped-flow spectrophotometry as a function of general and specific base catalyst concentration. Two proton exchange classes are found with time constants differing by a factor of 10 (4 and 0.4 s-1). The slower class represents the exchange of the adenine amino protons whereas the proton of the faster class has been assigned to the thymine imino proton. The exchange rates of these two classes of protons are independent of general and specific base catalyst concentration. This very characteristic behavior demonstrates that in our experimental conditions the exchange rates of the imino and amino protons in poly(dA-dT).poly(dA-dT) are limited by two different conformational fluctuations. We present a three-state exchange mechanism accounting for our experimental results. PMID- 3015198 TI - Multiple forms of ubiquitin-protein ligase. Binding of activated ubiquitin to protein substrates. AB - Enzyme activities that catalyzed the covalent attachment of ubiquitin to protein substrates (ubiquitin-protein ligase, UbL) were purified from the extracts of human red blood cells. These activities required the presence of ubiquitin activating enzyme and ATP for activity. Four fractions (UbL A, B1, B2, and C) were resolved and showed different specificities toward added substrates [carboxymethylated bovine serum albumin (CM-BSA), G-actin, lysozyme, and alpha lactalbumin]. The enzyme fractions gave different products with a given substrate. UbL A and UbL B1 were exclusively active with CM-BSA and alpha lactalbumin, respectively. UbL B2 was most active toward CM-BSA with substantial activities to G-actin and alpha-lactalbumin and with no activity to lysozyme. UbL C showed significant activities with all four substrates, having a highest activity toward CM-BSA. There were many endogenous proteins present in the erythrocyte extract which were efficient substrates for ubiquitin conjugation. In particular, a pair of substrates were identified from erythrocyte extracts which were far more efficient substrates than the denatured proteins exogenously added. PMID- 3015200 TI - Limited proteolysis of human von Willebrand factor by Staphylococcus aureus V-8 protease: isolation and partial characterization of a platelet-binding domain. AB - Purified human von Willebrand factor (vWF) was digested with Staphylococcus aureus V-8 protease, and specific domains interacting with platelets were isolated and characterized. Amino acid sequence analysis and sodium dodecyl sulfate gel electrophoresis demonstrated that the digestion proceeded primarily by a single cleavage of the native 270K subunit between an internal Glu-Glu peptide bond. This produced an integral stepwise degradation of the multimers of vWF with a concomitant accumulation of bands with mobility similar to that of the smaller molecular weight vWF multimers. The immediate precursor of the final products contained equimolar amounts of 270K subunit and of two polypeptides (170K and 110K). The cleavage of the remaining 270K subunit converted vWF into two main fragments (fragments II and III). These fragments were isolated by ion exchange chromatography, characterized, and assayed for platelet binding in the presence of ristocetin. Fragment III is a dimer of 315K composed primarily of two chains of 170K. Amino acid sequence analysis indicated that it originated from the amino-terminal portion of the 270K subunit and contained 11% of the original ristocetin cofactor activity. Also, it binds to platelets at the same specific sites as native vWF and shows a platelet binding pattern similar to that of partially reduced vWF (500K). Fragment II is a dimer of 235K composed of two identical chains of 110K. Amino acid sequence analysis indicated that it originated from the carboxyl-terminal portion of the 270K subunit and lacked ristocetin cofactor activity. Also, it does not bind to platelets or inhibit the binding of 125I-vWF in the presence of ristocetin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3015199 TI - Human von Willebrand factor: a multivalent protein composed of identical subunits. AB - A large-scale method for the isolation of von Willebrand factor (vWF) from human factor VIII concentrates was developed in order to study the structure of this protein and its platelet binding activity. vWF is composed of a number of glycoprotein subunits that are linked together by disulfide bonds to form a series of multimers. These multimers appear to contain an even number of subunits of 270K. Two minor components of Mr 140K and 120K were also identified, but these chains appear to result from minor proteolysis. The smallest multimer of vWF contained nearly equimolar amounts of the 270K, 140K, and 120K subunits, while the largest multimers contained less than 20% of the two minor components. Amino acid sequence analysis, amino acid composition, and cleavage by cyanogen bromide indicate that the 270K subunits are identical and each is a single polypeptide chain with an amino-terminal sequence of Ser-Leu-Ser-Cys-Arg-Pro-Pro-Met-Val-Lys and a carboxyl-terminal sequence of Glu-Cys-Lys-Cys-Ser-Pro-Arg-Lys-Cys-Ser-Lys. Platelet binding in the presence of ristocetin was 8-fold greater with multimers larger than five (i.e., containing more than 10 subunits of 270K) as compared to multimers less than three (containing less than six subunits of 270K). However, partially reduced vWF (Mr 500K), regardless of whether it was prepared from large or small molecular weight multimers, gave platelet binding similar to that of the smallest multimers. Likewise, partial proteolysis by elastase, thermolysin, trypsin, or chymotrypsin produced small "multimer-like" proteins with platelet binding properties similar to either partially reduced vWF or to the smallest multimers. We conclude that human vWF contains identical 270K subunits assembled into a multivalent structure. Disassembly by either partial reduction or partial proteolysis produces essentially monovalent protein with platelet binding properties similar to that of the smallest multimers. Multivalency is likely the primary factor responsible for the increase in biological activity with multimer size. PMID- 3015201 TI - Purification and characterization of a deoxyribonucleic acid dependent adenosinetriphosphatase from mouse FM3A cells: effects of ribonucleoside triphosphates on the interaction of the enzyme with single-stranded DNA. AB - There are at least four forms of DNA-dependent ATPase in mouse FM3A cells [Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., & Yamada, M. (1984) Biochemistry 23, 529-533]. One of these, ATPase B, has been purified and characterized in detail. During the purification of the enzyme, we encountered the difficulties that the enzyme could not be recovered well from the single stranded DNA-cellulose column and that the enzyme activity was distributed very broadly. The problems were resolved by the addition of ATP in the elution buffer. The ATPase has a sedimentation coefficient of 5.5 S in both high salt and low salt. The enzyme hydrolyzes rNTPs and dATP, but ATP and dATP are preferred substrates. Adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), 5'-adenylyl methylenediphosphate (AMP-PCP), and 5'-adenylyl imidodiphosphate (AMP-PNP) inhibit the enzyme activity. The enzyme is insensitive to ouabain, oligomycin, novobiocin, and ethidium bromide. A divalent cation (Mg2+ congruent to Mn2+ greater than Ca2+) as well as a nucleic acid cofactor is required for activity. Poly(dT), single-stranded circular DNA, and heat-denatured DNA were very effective. Native DNA was little effective with an efficiency of 29% of that obtained with heat-denatured DNA. In addition, the enzyme showed almost no activity with poly(dA).poly(dT) although it showed very high activity with the noncomplementary combination of poly(dT) and poly(dC), suggesting that ATPase B requires single-stranded DNA for activity. ATP altered the affinity of ATPase B for single-stranded DNA. The interaction of the enzyme with DNA was studied by Sephadex G-200 gel filtration assay.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3015202 TI - Thermostable alanine racemase from Bacillus stearothermophilus: molecular cloning of the gene, enzyme purification, and characterization. AB - The alanine racemase (EC 5.1.1.1) gene of a thermophilic bacterium, Bacillus stearothermophilus, was cloned and expressed in Escherichia coli C600 with vector plasmid pICR301, which was constructed from pBR322 and the L-alanine dehydrogenase gene derived from B. stearothermophilus. A coupled assay method with L-alanine dehydrogenase and tetrazolium salts was used to detect visually the alanine racemase activity in the clones. Alanine racemase overproduced in a clone carrying the plasmid pICR4, 12 kilobases of DNA, was purified from cell extracts about 340-fold to homogeneity by five steps including heat treatment. The overproduced enzyme was confirmed to originate from B. stearothermophilus by an immunochemical cross-reaction with the enzyme of B. stearothermophilus. The purified enzyme has a molecular weight of about 78 000 and consists of two identical subunits of Mr of 39 000. At the optimum temperature (50 degrees C), the enzyme has a specific activity of 1800 units/mg (Vmax, D- to L-alanine). Resolution and reconstitution experiments together with the absorption spectrum of the enzyme clearly indicate that alanine racemase of B. stearothermophilus is a pyridoxal 5'-phosphate enzyme. PMID- 3015204 TI - Differential effects of resact analogues on sperm respiration rates and cyclic nucleotide concentrations. AB - Analogues of resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-LeuNH2) were synthesized to determine whether or not a stimulation of sperm respiration could be obtained independent of elevations of cyclic nucleotide concentrations. Modification of the CO2-terminal leucine NH2 did not alter biological activity; however, substitution of the two cysteinyl residues by Ser or Tyr or methylation of the cysteinyl residues resulted in divergent relative potencies dependent on whether respiration rates or cyclic nucleotide concentrations were measured. [Ser1,Tyr8]resact, as an example, was approximately 40% as potent as resact at stimulating respiration rates but was 1% as effective as resact at causing cyclic GMP elevations. An NH2-terminal fragment (Cys-Val-Thr-Gly-Ala-Pro-Gly) neither stimulated respiration nor elevated cyclic nucleotide levels at concentrations up to 10 microM whereas a CO2-terminal fragment (Cys-Val-Gly-Gly-Gly-Arg-LeuNH2) had approximately 20% of the respiration activity and 0.1% of the cyclic GMP elevating activity of resact. When the CO2- and NH2-terminal fragments were added simultaneously, however, cyclic nucleotide concentrations were elevated at the same relative concentrations as observed with resact. An analogue (125I [Tyr1,Ser8]resact) was subsequently synthesized and used for receptor binding studies. Both the NH2-terminal and CO2-terminal fragments competed for binding, although they were 0.0004 and 0.025 times as effective as resact, respectively. However, in the presence of 1 microM resact-(1-7), resact-(8-14) was almost as potent as resact in the competitive binding assay.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3015203 TI - Kinetic studies of reduction of a 1:1 cytochrome c-flavodoxin complex by free flavin semiquinones and rubredoxin. AB - The kinetics of reduction by free flavin semiquinones and reduced rubredoxin of the individual components of the 1:1 complex formed between horse heart cytochrome c and Clostridium pasteurianum flavodoxin have been studied. Complex formation did not affect the rate constant for reduction of flavodoxin by 5 deazariboflavin semiquinone, indicating that the accessibility of the flavin mononucleotide (FMN) of complexed flavodoxin is the same as in the free protein. Reduction of the complexed cytochrome c by the neutral flavin semiquinones of lumiflavin and riboflavin was significantly affected by complex formation (2-3 fold rate constant decrease), indicating that there are steric constraints on the accessibility of the cytochrome heme to small exogenous reductants. Reduction of complexed cytochrome c by the negatively charged semiquinones of FMN and Cl2FMN was also characterized. A repulsive electrostatic interaction between the reductants and complexed cytochrome was observed, whereas with free cytochrome an attractive interaction had previously been found. This is consistent with the presence of negative electrostatic potential at the protein interface due to uncompensated flavodoxin carboxylates, as predicted by Matthew et al. [Matthew, J. B., Weber, P. C., Salemme, F. R., & Richards, F. M. (1983) Nature (London) 301, 169-171]. Further, pseudo-first-order rate constants for the reduction of complexed cytochrome by these flavins had a nonlinear concentration dependence, rather than obeying simple second-order kinetics. This is interpreted by using a mechanism involving a rate-determining structural isomerization of the protein complex prior to the second-order electron-transfer step. The magnitude of the decrease in the rate constant for reduction of complexed cytochrome c by the negatively charged reduced rubredoxin was approximately the same as observed for free flavins. Furthermore, simple second-order kinetics were obtained, and the apparent electrostatic interaction between rubredoxin and the complex was attractive. These results suggest that flavodoxin was partially displaced from its complex with cytochrome c by a collisional interaction with rubredoxin. The effects of complexation on the kinetics have been correlated with a solvent accessible surface representation of the computer-generated model of the flavodoxin-cytochrome c complex [Simondsen, R. P., Weber, P. C., Salemme, F. R., & Tollin, G. (1982) Biochemistry 21, 6366-6375]. The experimental observations are generally consistent with the structural model but clearly require the invocation of dynamic motions at the protein-protein interface. PMID- 3015205 TI - Structural homologies in the lutropin/human choriogonadotropin receptor and the follitropin receptor on porcine granulosa cells. AB - In order to examine the structure of the human choriogonadotropin (hCG) receptor and the follitropin (FSH) receptor on porcine granulosa cells, the hormone receptors were photoaffinity-labeled or affinity-cross-linked. The resulting hormone-receptor complexes were analyzed by alkaline cleavage of cross-links, reduction of disulfides, and peptide maps. The results revealed striking similarities in the structure of the hormone receptors. Both appear to be oligomeric; the hCG receptor has at least four components of 18, 24, 28, and 34 kDa, whereas the FSH receptor shows three distinct components of 18, 22, and 34 kDa. The 24- and the 22-kDa components are the sites for the primary photoaffinity labeling or affinity cross-linking by hCG and FSH, respectively. These components were linked by intercomponent disulfides. Reduction of cross linked complexes revealed that in the hCG receptor the 24-, the 28-, and the 34 kDa components were disulfide-linked sequentially in a linear form as were the 22 , the 18-, and the 34-kDa components in the FSH receptor. The peptide maps of cross-linked hCG-receptor and FSH-receptor complexes, however, were distinct, indicating that the hCG receptor and the FSH receptor were not identical. PMID- 3015206 TI - Evidence for two independent pathways of electron transfer in mitochondrial NADH:Q oxidoreductase. I. Pre-steady-state kinetics with NADPH. AB - The reduction of NADH:Q oxidoreductase by NADPH occurring in submitochondrial particles has been studied with the freeze-quench technique. It was found that 50% of the Fe-S clusters 2, 3 and 4 could be reduced by NADPH within 30 ms at pH 6.5. The remainder of the clusters, including cluster 1, were reduced slowly and incompletely; it was concluded that these clusters play no role in the NADPH oxidase activity. Nearly the same results were obtained at pH 8 under anaerobic conditions, demonstrating that the rate of reaction of NADPH with the enzyme was essentially the same at both pH values. The rate and extent of reduction of half of the clusters 2 by NADPH at pH 8 were not affected by the presence of O2 of rotenone. This implies a pH-dependent oxidation of the enzyme as the cause for the absence of the NADPH oxidase activity at this pH. A dimeric model of the enzyme is proposed in which one protomer, containing FMN and the Fe-S clusters 1 4 in stoichiometric amounts, is responsible for NADH oxidation at pH 8. This protomer cannot react with NADPH. The other protomer, containing only FMN and the clusters 2, 3 and 4, is supposed to catalyse the oxidation of NADPH. The oxidation of this protomer by ubiquinone is expected to be strongly dependent on pH. This protomer might also catalyse NADH oxidation at pH 6-6.5. PMID- 3015207 TI - Evidence for two independent pathways of electron transfer in mitochondrial NADH:Q oxidoreductase. II. Kinetics of reoxidation of the reduced enzyme. AB - The pre-steady-state kinetics of reoxidation of NADH:Q oxidoreductase present in submitochondrial particles has been studied by the freeze-quench method. It was found that at pH 8 only 50% of the Fe-S clusters 2 and 4 and 75% of the clusters 3 were rapidly reoxidised after transient and complete reduction by a pulse of NADH in the presence of excess NADPH. Thus, NADPH keeps 50% of the clusters 2 and 4 and 25% of the clusters 3 permanently reduced at this pH. Since NADH oxidation is nearly optimal at this pH, whereas NADPH oxidation is virtually absent, it was concluded that these permanently reduced clusters were not involved in the NADH oxidation activity. Incomplete reoxidation of the clusters 2, 3 and 4 after a pulse of NADH was also found in the absence of NADPH, both at pH 6.5 and at pH 8. A pulse of NADPH given at pH 6.5, where NADPH oxidation by oxygen is nearly optimal, caused a slow reduction of 50% of clusters 2 and 4 and 30% of the clusters 3, which persisted for a period of at least 15 s. It was concluded that these clusters were not involved in the oxidation of NADPH by oxygen, as catalysed by the particles. As a working hypothesis a dimeric model for NAD(P)H:Q oxidoreductase is proposed, consisting of two different protomers. One of the protomers, containing FMN and the Fe-S clusters 1-4 in stoichiometric amounts, only reacts with NADH, and its oxidation by ubiquinone is rapid at pH but slow at pH 6.5. The other protomer, containing FMN and the clusters 2, 3 and 4, reacts with both NADH and NADPH and has a pH optimum at 6-6.5 for the reaction with ubiquinone. PMID- 3015208 TI - A theoretical study of double-inhibitor-titration curves in free-energy transducing networks. AB - The general relationship between the double-inhibitor-titration curves and the kinetic properties of pumps in a delocalized chemiosmotic free-energy-transducing network is studied. The kinetic conditions for a delocalized system to generate the observed double-inhibitor-titration results are derived and the effectiveness of double-inhibitor experiments in discriminating between the localized and the delocalized proton-coupling mechanisms is assessed. It is found that, using simple enzymatic cycles for the kinetics of the pumps in a delocalized network, one can reproduce the experimentally measured double-inhibitor-titration curves that were widely used to argue against the delocalized mechanism. This implies that double-inhibitor-titration curves alone are not sufficient to discriminate between localized and delocalized coupling systems. Additional information concerning the kinetic responses of isolated pumps on the proton gradient across the membrane and inhibitor concentrations are required. PMID- 3015209 TI - Quantitative structure-activity relationship of carbonylcyanide phenylhydrazones as uncouplers of mitochondrial oxidative phosphorylation. AB - The dependence of the uncoupling activity in the series of 16 carbonylcyanide phenylhydrazones on their physico-chemical properties (partition coefficient, dissociation constant and rate constant for reaction with thiols) is investigated using two physiologically based models, one for protonophoric mechanism of uncoupling and the other assuming the covalent modification of a membrane constituent to be the key step in this process. As indicated by uptake experiments, at the given conditions a lipophilic-hydrophilic equilibrium is attained without any loss of the compounds via chemical reactions. Using this fact to reduce the number of adjustable parameters, a better fit to the data on stimulation of respiration is obtained with the former (protonophoric) model. PMID- 3015210 TI - Studies on the role of the oligomeric state and subunit III of cytochrome oxidase in proton translocation. AB - Anion-exchange fast protein liquid chromatography in the presence of lauryldimethylamine N-oxide (LDAO) was introduced to separate cytochrome oxidase into different complexes that either did or did not contain subunit III. Both kinds of enzyme complex exhibited H+ translocation after reconstitution into phospholipid vesicles, but with a significantly (approx. 50-60%) reduced H+/e- ratio as compared with unchromatographed enzyme. The anion-exchange FPLC fractions of the enzyme (with or without subunit III) sedimented more slowly than the control enzyme upon sucrose gradient centrifugation in the presence of cholate and a high potassium phosphate concentration. When the control enzyme was subjected to the sucrose gradient centrifugation in the presence of LDAO or Triton X-100, instead of cholate, one band containing all subunits was observed, which sedimented slowly like the FPLC fractions. Transfer of this band to cholate medium, and reapplication on the sucrose gradient (with cholate), yielded both a slow- and a fast-migrating band after centrifugation. Enzyme complexes that sedimented slowly or rapidly in the sucrose gradients revealed longer and shorter elution times, respectively, in gel filtration FPLC. This suggests that these complexes corresponds to monomers and dimers of cytochrome oxidase. Solubilization of proteoliposomes and subsequent sucrose gradient centrifugation in cholate yielded one fast-migrating band for the untreated enzyme, but both a fast- and a slow-migrating band for the anion-exchange FPLC-treated enzyme, which was exclusively slow-migrating before reconstitution into liposomes. It is suggested that dimerisation of monomeric cytochrome oxidase may be favoured when the enzyme encounters a membranous milieu, and that the dimeric structure might be necessary for proton translocation. PMID- 3015211 TI - Depolymerization of solubilized gastric (H+ + K+)-ATPase by n-octylglucoside or cholate. AB - We have previously shown that an active (H+ + K+)-ATPase can be extracted from gastric apical membranes using n-octylglucoside (Soumarmon, A., Grelac, F. and Lewin, M.J.M. (1983) Biochim. Biophys. Acta 732, 579-585). This extract contained an holomeric enzyme of 390-420 kDa and contained 68% of the K+-stimulated ATPase specific activity originally present. We demonstrate here that inactivation, induced during a more classically designed protocol, is associated with the appearance of smaller, polymorphic structures with molecular mass of 330-360 and 240-250 kDa estimated using molecular sieve chromatography and glycerol gradients. This suggests that (H+ + K+)-ATPase solubilization by n-octylglucoside is a complex process involving first extraction of the enzyme as an active polymer, with subsequent depolymerication and inactivation of this polymer. Depolymerization was specifically studied by treating the large holomeric n octylglucoside-extracted (H+ + K+)-ATPase with increasing concentrations of either n-octylglucoside or cholate. Detergent-induced changes were characterized by centrifugation on glycerol gradients. Progressive displacement of ATPase activity into three different peaks at 32%, 26% and 20% glycerol was found with increasing detergent concentrations. n-Octylglucoside inhibited enzyme activities and was more deleterious for phosphatase than for ATPase activity. Moreover, it induced the dissociation of phosphatase and ATPase distribution profiles. At concentrations of 0.2 to 1.15%, cholate induced the displacement of the glycerol gradient profiles but no loss of activities and no dissociation of phosphatase and ATPase profiles. Higher concentrations of this detergent (2.5%) also inactivated the ATPase concomitantly with the appearance of a protein peak with no related activity at 16-18% glycerol. From this study we suggest that solubilization of gastric (H+ + K+)-ATPase can be achieved through the extraction of a polymer by n-octylglucoside and through subsequent depolymerization using cholate. We suggest that the different sizes correspond to monomers, dimers, trimers and perhaps tetramers. The monomers were apparently inactive under present test conditions. PMID- 3015212 TI - H+ transport by reconstituted gastric (H+ + K+)-ATPase. AB - Gastric (H+ + K+)-ATPase was reconstituted into artificial phosphatidylcholine/cholesterol liposomes by means of a freeze-thaw-sonication technique. Upon addition of MgATP, active H+ transport was observed, with a maximal rate of 2.1 mumol X mg-1 X min-1, requiring the presence of 100 mM K+ at the intravesicular site. However, in the absence of ATP an H+-K+ exchange with a maximal rate of 0.12 mumol X mg-1 X min-1 was measured, which could be inhibited by the well-known ATPase inhibitors vanadate and omeprazole, giving the first evidence of a passive K+-H+ exchange function of gastric (H+ + K+)-ATPase. An Na+ H+ exchange activity was also measured, which was fully inhibited by 1 mM amiloride. Simultaneous reconstitution of Na+/H+ antiport and (H+ + K+)-ATPase could explain why reconstituted ATPase appeared less cation-specific than the native enzyme (Rabon, E.C., Gunther, R.B., Soumarmon, A., Bassilian, B., Lewin, M.J.M. and Sachs, G. (1985) J. Biol. Chem. 260, 10200-10212). PMID- 3015213 TI - Inhibition of ion pump ATPase activity by 3'-O-(4-benzoyl)benzoyl-ATP (BzATP): assessment of BzATP as an active site-directed probe. AB - The interaction of 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) with the renal (Na+ + K+) ATPase, the sarcoplasmic reticulum Ca-transport ATPase, and the gastric (H+ + K+) ATPase has been investigated in order to determine whether BzATP is a suitable probe for the labeling and identification of a peptide from the ATP binding sites of these ion pumps. After ultraviolet irradiation BzATP inhibited the enzymatic hydrolysis of ATP by each of the ion pumps, and also was covalently incorporated into the 100 000 dalton polypeptides of each protein. The presence of excess ATP in the reaction solution did not prevent either the inactivation of ATPase activity or the labeling of the catalytic polypeptides by BzATP. Prior modification of the ATPases with fluorescein-5'-isothiocyanate (FITC), however, prevented much of the labeling of the 100 000 dalton polypeptides by BzATP. BzATP competitively inhibited the high-affinity binding of ATP to the ion pumps, but ATP did not block the high-affinity binding of BzATP by the enzymes. BzATP binds to the membrane-bound ATPases at a high-affinity site with a Kd of 0.8-1.2 microM and a Bmax of 2-3 nmol/mg, and also binds to at least one low-affinity, high capacity site on the membranes. HPLC separation of the soluble peptides from a tryptic digest of BzATP-labeled (Na+ + K+)-ATPase revealed the presence of several labeled peptides, none of which was protected by either ATP or FITC. Although BzATP can displace ATP from a high-affinity binding site on the ion pumps, it appears, therefore, that inactivation of enzymatic activity is the result of reactions between BzATP and the proteins at locations outside this site. Thus, it is concluded from these experiments that BzATP is not likely to be a useful probe for the ATP binding sites on the ion transport ATPases. PMID- 3015214 TI - Purification and characterization of the polycation-stimulated protein phosphatase catalytic subunit from porcine renal cortex. AB - The predominant form of phosphorylase phosphatase activity in porcine renal cortical extracts was a polycation-stimulated protein phosphatase. This activity was present in extracts in a high-molecular-weight form which could be converted to a free catalytic subunit by treatment with ethanol, urea, or freezing and thawing in the presence of beta-mercaptoethanol. The catalytic subunit of the polycation-stimulated phosphatase was purified by chromatography on DEAE Sephacel, heparin-Sepharose, and Sephadex G-75. The phosphatase appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The enzyme had an apparent Mr of 35 000 on gel filtration and SDS-polyacrylamide gel electrophoresis. The purified phosphatase could be stimulated by histone H1, protamine, poly(D lysine), poly(L-lysine) or polybrene utilizing phosphorylase a as the substrate. It preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. The phosphatase was highly sensitive to inhibition by ATP. These results suggest that the renal polycation-stimulated phosphatase catalytic subunit is very similar to or identical with the skeletal muscle phosphatase form which has been previously designated phosphatase-2Ac. PMID- 3015215 TI - Cytochrome c peroxidase compound I: formation of covalent protein crosslinks during the endogenous reduction of the active site. AB - Cytochrome c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) was oxidized by hydrogen peroxide in the absence of exogenous electron donor. Higher molecular weight species were observed in the decay products at pH 4.5. Monomer and dimer were separated by gel filtration and purified by anion exchange chromatography. Peptide mapping of tryptic digests of the dimer indicated a tyrosine crosslink localized between residues 32 and 48 of the native enzyme. PMID- 3015216 TI - Kinetic properties and essential amino acids of the 2,3-bisphosphoglycerate synthase-phosphatase from pig skeletal muscle. AB - Histidine, arginine and lysine residues are essential for the multifunctional 2,3 bisphosphoglycerate synthase-phosphatase purified from pig skeletal muscle. The synthase, phosphatase and phosphoglycerate mutase activities of the enzyme are concurrently lost upon treatment with diethylpyrocarbonate, phenylglyoxal and trinitrobenzenesulfonate. The phosphatase activity shows hyperbolic kinetics. In contrast, the synthase activity shows a nonhyperbolic pattern which fits to a second-degree polynomial. The Km values for glycerate 1,3-P2, glycerate 3-P and glycerate 2,3-P2 are similar to those of the enzyme from mammalian erythrocytes. PMID- 3015217 TI - Reaction sequences for (Na+ + K+)-dependent ATPase hydrolytic activities: new quantitative kinetic models. AB - To delineate better the reaction sequence of the (Na+ + K+)-ATPase and illuminate properties of the active site, kinetic data were fitted to specific quantitative models. For the (Na+ + K+)-ATPase reaction, double-reciprocal plots of velocity against ATP (in the millimolar range), with a series of fixed KCl concentrations, are nearly parallel, in accord with the ping pong kinetics of ATP binding at the low-affinity sites only after Pi release. However, contrary to requirements of usual formulations, Pi is not a competitor toward ATP. A new steady-state kinetic model accommodates these data quantitatively, requiring that under usual assay conditions most of the enzyme activity follows a sequence in which ATP adds after Pi release, but also requiring a minor alternative pathway with ATP adding after K+ binds but before Pi release. The fit to the data also reveals that Pi binds nearly as rapidly to E2 X K X ATP as to E2 X K, whereas ATP binds quite slowly to E2 X P X K: the site resembles a cul-de-sac with distal ATP and proximal Pi sites. For the K+-nitrophenyl phosphatase reaction also catalyzed by this enzyme, the apparent affinities for both substrate and Pi (as inhibitor) decrease with higher KCl concentrations, and both Pi and TNP-ATP appear to be competitive inhibitors toward substrate with 10 mM KCl but noncompetitive inhibitors with 1 mM KCl. These data are accommodated quantitatively by a steady-state model allowing cyclic hydrolytic activity without obligatory release of K+, and with exclusive binding of substrate vs. either Pi or TNP-ATP. The greater sensitivity of the phosphatase reaction to both Pi and arsenate is attributable to the weaker binding by the occluded-K+ enzyme form occurring in the (Na+ + K+)-ATPase reaction sequence. The steady-state models are consistent with cyclical interconversion of high- and low-affinity substrate sites accompanying E1/E2 transitions, with distortion to low-affinity sites altering not only affinity and route of access but also separating the adenine- and phosphate-binding regions, the latter serving in the E2 conformation as the active site for the phosphatase reaction. PMID- 3015218 TI - Impaired hepatocyte binding, uptake and degradation of glucosylated low-density lipoproteins. AB - The catabolism of low-density lipoproteins (LDL), the major cholesterol-carrying lipoproteins in plasma, is mediated in part via a high-affinity uptake pathway in the liver. Non-enzymatic glucosylation of lysine residues of apolipoprotein B, the major protein of LDL, blocks receptor-mediated uptake of LDL by fibroblasts and endothelial cells. We investigated the effect of the degree of glucosylation on the binding, uptake and degradation of radioiodinated LDL by the human hepatoma cell line Hep G2. Human LDL was glucosylated with 250 mM glucose and 30 mM cyanoborohydride at 37 degrees C. Incubations ranging from 3 to 48 h in duration resulted in the formation of 6-27% of glucitol-lysine adducts as demonstrated by coincubation with [14C]glucose. The degree of glucose incorporation corresponded to the extent of inhibition of binding, uptake and degradation of LDL (10-90%). The data are consistent with the view that glucosylation of LDL markedly impairs their catabolism. This phenomenon may be related to the pathophysiology of the premature atherosclerosis observed in diabetes mellitus. PMID- 3015219 TI - Effects of adrenaline on the turnover of lipoprotein lipase in rat adipose tissue. AB - The mechanisms by which adrenaline brings about a reduction in the lipoprotein lipase activity of adipose tissue in vitro were investigated. The incorporation of [3H]leucine into lipoprotein lipase was measured during 1-h pulse incubations of rat epididymal fat bodies that had been preincubated for 4 h in the presence of glucose, insulin and dexamethasone. When adrenaline was added to the incubation medium at the start of the pulse, the incorporation of [3H]leucine was markedly reduced, suggesting that the rate of the enzyme's synthesis had decreased. On the other hand, the degradation of lipoprotein lipase, as measured by the loss of 3H-labelled enzyme protein during pulse-chase incubations of the epididymal fat bodies, was found to be significantly increased by the addition of adrenaline to the incubation medium at the start of the chase period. It is concluded that adrenaline is able both to inhibit the synthesis of lipoprotein lipase and to stimulate its degradation. PMID- 3015220 TI - Biochemical and ultrastructural studies on an Epstein-Barr virus-transformed lymphoid cell line from a Niemann-Pick disease type C patient. AB - Human lymphoid cell lines established from normal subjects and from a Niemann Pick disease type C patient were investigated from a triple point of view of enzymology, metabolism and ultrastructure: Sphingomyelinase activities, isoenzyme electrofocusing profiles and properties of the major enzyme were quite similar in type C and normal lymphoid cell lines. Similarly, no significant difference was observed in non-specific phosphodiesterases hydrolysing bis(methylumbelliferyl)phosphate and bis(methylumbelliferyl)pyrophosphate. The study of the lipid composition of type C cells showed no obvious accumulation of sphingomyelin or other phospholipid, but only a higher amount of glycolipids (mainly GlcCer and GbOse3Cer), as visualized by bidimensional thin-layer chromatography. Ultrastructural studies demonstrated, in type C cells, the presence of an obvious lysosomal storage of amphiphilic lipids quite similar to that observed in tissues of type C patients. These studies, which demonstrate the validity of lymphoid cell lines as an experimental model system for type C disease, agree with the current opinion that an impairment of sphingomyelin catabolism is not the primary defect in type C disease. PMID- 3015221 TI - Synthesis and secretion of lecithin-cholesterol acyltransferase by the human hepatoma cell line HepG2. AB - The human liver cell line HepG2 was investigated for its synthesis and secretion of lecithin-cholesterol acyltransferase. The cells were grown to confluency in Eagle's minimal essential medium plus 10% fetal bovine serum. At the onset of the study, fetal bovine serum was removed and cells were grown in minimal essential medium only. At 6, 12, 24, and 48 h the cells were harvested, and the culture medium collected at each time point was assayed for lecithin-cholesterol acyltransferase mass and activity, cholesterol esterification rate, and apolipoprotein A-I mass. The rate of the enzyme secretion measured by both mass and activity was linear over 24 h of culture. The enzyme mass by radioimmunoassay was 1.7, 4.1, 7.9 and 13.7 ng/ml culture medium (or 8.3, 19.9, 38.5 and 66.7 ng/mg cell protein), respectively, and enzyme activity using an exogenous source of phosphatidylcholine/cholesterol liposomes containing apolipoprotein A-I as substrate was 85, 170, 315, and 402 pmol cholesterol esterified/h per ml culture medium (or 414, 828, 1534 and 1957 pmol cholesterol esterified/h per mg cell protein) for 6, 12, 24, and 48 h of culture, respectively. The endogenous cholesterol esterification rate of the culture medium was 47, 104, 224 and 330 pmol/h per ml and apolipoprotein A-I mass was 305, 720, 2400 and 3940 ng/ml culture medium over the same time frame. In contrast to culture medium, low levels of enzyme activity (approximately 10% of that in culture medium at 24 and 48 h) were observed in the extracts of HepG2 cells. The enzyme secreted by HepG2 was found to be similarly activated by apolipoprotein A-I, apolipoprotein E, or apolipoprotein A-IV, and was similarly inhibited by phenylmethylsulfonyl fluoride, dithiobisnitrobenzoate, p-hydroxymercuribenzoate, or iodoacetate as compared to human plasma enzyme. High-performance gel filtration of the culture medium revealed that the HepG2-secreted enzyme was associated with a fraction having a mean apparent molecular weight of approximately 200,000. We concluded that human hepatoma HepG2 cells synthesize and secrete lecithin-cholesterol acyltransferase, which is functionally homologous to the human plasma enzyme. PMID- 3015222 TI - Synthesis of polyphosphoinositides in vertebrate photoreceptor membranes. AB - Rod outer segments isolated from bovine retinas incorporated 32P into phospholipids after incubation with [gamma-32P]ATP in a Mg2+-containing medium. Only phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate, and phosphatidate were labelled. The incorporation of label into lipids was detected as early as 20 s after the start of incubation and the products were stable for at least 10 min. The reactions were time, protein and ATP-concentration dependent. Entire rod outer segments showed higher diacylglycerol kinase and lower phosphatidylinositol and phosphatidylinositol 4-phosphate kinase activities than the disc membranes obtained from them. Exogenously added phosphatidylinositol (up to 1 mM) in the presence of Triton X-100 increased phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate labelling in rod outer segments (8- and 6-fold, respectively). Triton X-100 at a concentration of 0.4% stimulated phosphorylation of endogenous phosphoinositides. Diacylglycerol kinase activity was largely suppressed by the detergent, but this effect was partially reversed by addition of phosphatidylinositol. It is suggested that the rod outer segments contain phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase bound to disc membranes, as well as an active diacylglycerol kinase occurring either as a soluble or a peripherally bound protein in disc membranes. PMID- 3015223 TI - Fatty acid synthesis in the fetal lung: relationship to surfactant lipids. AB - The aims of this study were to investigate the control of fatty acid synthesis and its relationship to surfactant production in the fetal lung during alteration of hormonal and substrate conditions. Lung explants from 18 day fetuses (term = 22 days) which were cultured 2 days in the presence of 10 mM lactate showed parallel acceleration of de novo fatty acid synthesis (3H2O incorporation) and [14C]choline incorporation into disaturated phosphatidylcholine (DSPC) compared to culture of explants in glucose. Both the cultured and fresh explants were resistant to the classical short term (4 h) cAMP inhibition of fatty acid synthesis with 3 mM dibutyryl cAMP or 0.5 mM aminophylline. In the cultured explants short term cAMP elevation increased DSPC production, and long term (2 day) cAMP elevation caused a further increase in DSPC synthesis and also stimulated fatty acid synthesis. In cultured explants from 17 day fetuses, dexamethasone (0.1 microM) caused a synergistic increase with aminophylline in both fatty acid synthesis and DSPC production whereas, in explants from 18 day fetuses, dexamethasone inhibited both processes and reduced the level of stimulation of DSPC and fatty acid synthesis seen with aminophylline alone. Dexamethasone also reduced the stimulation of both DSPC and fatty acid synthesis produced in the culture of 18 day explants with bacitracin (0.5 mg/ml), whereas the combination of bacitracin and aminophylline resulted in a synergistic increase in DSPC production. Culture with glucagon (0.1 microM) also stimulated DSPC synthesis but at physiological levels insulin had no effect on either DSPC or fatty acid synthesis. These data show that lung fatty acid synthesis exhibits unique features of fatty acid synthesis regulation compared to other lipogenic tissues and also suggest a link between de novo fatty acid synthesis and surfactant production during the critical period of accelerated lung maturation. PMID- 3015225 TI - Limited proteolysis of the erythrocyte membrane skeleton by calcium-dependent proteinases. AB - The action of purified calcium-dependent proteinases on human erythrocyte membrane skeleton proteins has been examined. Preferential cleavage of proteins 4.1 a and b and band 3 and limited cleavage of alpha- and beta-spectrin occur when either calcium-dependent proteinase I or calcium-dependent proteinase II has access to the cytoplasmic side of the ghost membrane skeleton in the presence of calcium. Thus, when these proteinases are incubated with sealed ghosts they do not cleave these proteins. Leupeptin, mersalyl, the specific cellular protein inhibitor of these enzymes, and calcium chelators can inhibit proteolysis of the red cell ghost proteins by Ca2+-dependent proteinases. Each proteinase has also been loaded into erythrocyte ghosts in the absence of calcium at low ionic strength and subsequently trapped inside by resealing the ghosts. The proteinases were activated by incubating these ghosts in the presence of the calcium ionophore A23187 and calcium. Examination of the ghost proteins by electrophoresis demonstrated calcium-dependent proteolysis of Bands 4.1 and 3 and limited cleavage of alpha- and beta-spectrin similar to that observed on proteolysis of the open, leaky ghosts. In the presence of calcium each calcium dependent proteinase appears to associate with the erythrocyte ghost membrane. PMID- 3015224 TI - Preparation and properties of an improved photoaffinity ligand for the N-formyl peptide receptor. AB - A new superior photoaffinity ligand for the N-formyl peptide receptor was prepared by derivatization of N-formyl-Met-Leu-Phe-Lys with a commercially available heterobifunctional crosslinking agent. The product, N-formyl-Met-Leu Phe-N epsilon-(2-(p-azidosalicylamido)ethyl-1,3'- dithiopropionyl)-Lys was readily synthesized and radiolabelled, and had increased specificity and stability as compared to previously used photoaffinity ligands. The ligand rapidly associated with the receptor with high affinity (Kd = 0.28 nM). Once bound, it was virtually non-dissociable (in the absence of photolysis) in an experimental time-frame (t1/2 (off) = 154 min). The covalent incorporation of the photoaffinity ligand into the receptor upon irradiation was complete within 5 min, minimizing cell damage, and the efficiency of covalent incorporation was approx. 40%. The derivative had enhanced biological activity, having an ED50 for superoxide anion production of 0.23 nM, 27-fold lower than its parent peptide. This derivative of the N-formyl peptide was useful for up to 3 months after radiolabelling, showing a progressive decline in specific activity during storage in the dark at 4 degrees C. The enhanced stability, reproducibility and solubility of the photoaffinity ligand is expected to aid in the purification of the N-formyl peptide receptor and will prove a useful tool for analysing receptor mediated processes. PMID- 3015226 TI - Purification of the two forms of the high-molecular-weight neutral proteinase ingensin from rat liver. AB - Two forms of a high-molecular-weight proteinase were isolated from rat liver. The purification procedure involved homogenization of the tissue, chromatography on DEAE-cellulose, high-performance liquid chromatography (HPLC: TSK 3000 SWG) and hydroxyapatite chromatography. The breakthrough fraction from the hydroxyapatite column contained the sodium dodecyl sulphate (SDS)- and linoleic acid-activated proteinase, ingensin A, but the other form, ingensin B, which was also activated by SDS and linoleic acid, was bound to the hydroxyapatite and eluted at 200 mM phosphate. A distinct feature of ingensin A was its activation by a brief sonication procedure. The optimum pH of the two forms was 7.5-9.5, and both of them were activated by monovalent cations. Although both enzymes show similar molecular weights of 700,000 on gel filtration, ingensins A and B were separated into a major subunit of 120,000 and subunits of 25,000-35,000, respectively, under the denaturing conditions. PMID- 3015227 TI - Characterization of nucleoside-diphosphate kinase-associated guanine nucleotide binding proteins from HeLa S3 cells. AB - Nucleoside-diphosphate (NDP) kinase-associated [alpha-32P]GTP-incorporating proteins from HeLa S3 cells have been biochemically characterized. Two distinct NDP-kinases (F-I and F-II) had been partially purified from HeLa S3 cells by Sephacryl S-300 gel filtration and DEAE-cellulose column chromatography. The [alpha-32P]GTP-incorporating proteins (approx. Mr 20,000) could be separated from NDP-kinases (approx. Mr 80,000) by 5-25% glycerol density-gradient centrifugation analysis after treatment with 7 M urea in the presence of 1 mM EDTA. [alpha 32P]GTP incorporation into these two proteins (G1 and G2) from NDP-kinases required 5 mM Mg2+ and was highly inhibited by either GDP or GTP analogues, such as guanylyl imidodiphosphate and guanylyl methylenediphosphate. [3H]GDP, but no other nucleoside 5'-diphosphates, was also bound to these two proteins in the presence of Mg2+ (5 mM). Moreover, incubation of [alpha-32P]GTP with either G1 or G2 in the presence of Mg2+ (5 mM) resulted in the formation of [32P]GDP and Pi. The data presented here indicated that the guanine nucleotide-binding activity, the GTPase activity, and the molecular weight (approx. Mr 20,000) of NDP-kinase associated proteins from HeLa S3 cells are similar to those reported for ras oncogene products (p21 proteins). PMID- 3015228 TI - Isolation and characterization of thrombomodulin from bovine lung. AB - Bovine thrombomodulin was isolated from the lung by Triton X extraction, affinity chromatography on diisopropyl phosphate-thrombin-agarose, and gel filtration on Ultrogel AcA-44. The final preparation was purified 6000-fold from the membrane extract with a yield of 21%. It showed apparent Mr of 78,000 and 105,000, before and after reduction, respectively, on polyacrylamide gel electrophoresis in SDS. The activity of the thrombomodulin was stable under the conditions of 1% SDS, 8 M urea, pH 2 and 10, and heat treatment at 60 degrees C for 30 min, but was unstable against treatment with 2-mercaptoethanol. Activation of protein C by thrombin in the presence of the thrombomodulin depended on Ca2+, and an equimolar complex formation between thrombin and thrombomodulin was required for the maximum rate activation. The rate of protein C activation by thrombin was increased 900-fold by thrombomodulin. Thrombomodulin inhibited the thrombin induced fibrinogen clotting and platelet activation. However, it did not affect the inhibition of thrombin by antithrombin III with or without heparin, a protein C inhibitor or several synthetic inhibitors. These properties of bovine thrombomodulin were similar to those of rabbit thrombomodulin reported earlier. PMID- 3015229 TI - Characterization of a novel gelatin-binding 21 kDa protein secreted by cultured adherent cells. AB - A novel gelatin-binding 21 kDa protein was identified in the culture medium of fibroblastic and sarcoma cells by affinity chromatography on gelatin-Sepharose. Its affinity for gelatin was lower than that of the other gelatin-binding proteins, fibronectin and the 70 kDa protein, as judged by stepwise elution by urea and arginine. The protein bound also to spermine and to some extent to heparin but not to staphylococcal protein A, bovine serum albumin, concanavalin A or plain Sepharose 4B. In gel filtration chromatography the protein eluted in fractions differing from those of fibronectin and the Mr 70,000 protein and retained its ability to bind to gelatin-Sepharose, indicating that the binding was not mediated by the two other gelatin-binding proteins. It contains intrachain disulfide bridges, as judged by analysis under nonreducing and reducing conditions. The protein is composed of two major subtypes with pI values of 5.85-6.10 and 6.55-6.75. It was sensitive to trypsin but not to collagenase or thrombin. Antiserum was raised in rabbits against the gelatin-binding proteins isolated from serum-free conditioned fibroblast culture medium. The antiserum reacted with fibronectin, the Mr 70,000 protein and the Mr 21,000 protein in immunoprecipitation experiments. Absorption of the antiserum with human plasma fibronectin did not decrease its reactivity with the Mr 70,000 and 21,000 proteins. However, absorption with the Mr 70,000 protein abolished also the reactivity against the Mr 21,000 protein, suggesting immunological cross reactivity. The protein was synthesized independently from the Mr 70,000 protein, as shown by pulse-chase labeling experiments of cells. The production of the Mr 21,000 protein in cultured cells was enhanced by transforming growth factor-beta. PMID- 3015230 TI - Effects of ACTH on the ligand-binding properties of the glucocorticoid receptor exerted not only via elevated levels of corticosteroids. AB - Treatment of mice with ACTH not only increased plasma corticosterone levels, but also reduced the ligand-binding capacity of the glucocorticoid receptor in skeletal muscle cytosol. Non-linear Scatchard plots were obtained for the glucocorticoid receptor following ACTH treatment. This upward concave curvilinearity could not be reproduced by simply adding to the cytosol an amount of corticosterone corresponding to the increase, even though this treatment resulted in reduced binding capacity. The addition of corticosterone resulted in a slower formation of ligand-receptor complexes (lower association rate constant), which offered a partial explanation for the low binding. In addition, ACTH treatment was also found to reduce the binding capacity in adrenalectomized mice that did not respond with corticosterone elevation. However, in this case Scatchard plots were linear. PMID- 3015231 TI - Interaction of radicals from water radiolysis with melanin. AB - Melanins are considered to be natural photoprotectors in the melanocytes and keratinocytes of the skin. These pigments have also been suggested to play an important role in protection of melanin-containing cells against ionising radiation. Various mechanisms have been proposed to explain the protective role of melanin which invoke the radical scavenging properties of the polymer. In the present work the reactions of melanins with radicals generated in aqueous media by pulse radiolysis have been studied. Time-resolved changes in absorbance of the melanin or the radical species were recorded at selected wavelengths. Experiments were carried out on synthetic dopa- and 5-S-cysteinyldopa-melanins and a natural melanin in phosphate buffer (pH 7.4). Under the conditions employed, melanin reacted predominantly with either oxidising (OH., N3.) or reducing (eaq-, CO2-) species. We were also able to monitor the interaction of melanin with superoxide radical, which was reducing in this case. Detailed analysis of transient changes in melanin absorbance, detected at different wavelengths, was demonstrated to be a convenient method for studying redox processes of this substance, as shown by model experiments using ferricyanide and dithionite as oxidising and reducing agents, respectively. Among the radicals studied, OH. exhibited the strongest reactivity with melanins. Apparent rate constants for the reactions of radicals with autoxidative dopa-melanin (1.5 X 10(9) M-1 X s-1, 2.6 X 10(8) M-1 X s-1, 1.8 X 10(8) M-1 X s-1, 5 X 10(5) M-1 X s-1, 10(6)-10(7) M-1 X s-1 for OH., eaq-, N.3. O2- and CO2-, respectively) are reported. The reactivity of melanins with radicals from water radiolysis and their effect on pigment properties are discussed in terms of the structure and possible biological role of the pigments. PMID- 3015232 TI - The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat liver cells. AB - The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat hepatocytes was studied. In the presence of low levels (10-100 microM) of the cyclic nucleotide the initial rate of uptake was increased significantly, with a peak occurring at about 20 microM. In contrast, concentrations of dibutyryl cyclic AMP between 200 microM and 1 mM caused a significant decrease in the initial rate of uptake of the bile acid by the cells. Sodium-dependent transport of taurocholic acid was found to be enhanced by 20 microM dibutyryl cyclic AMP, but sodium-independent uptake appeared to be unaffected. Inhibition by 1 mM dibutyryl cyclic AMP, however, was found to occur in both the sodium-dependent and -independent components of the transport system. The initial rate of taurocholic acid uptake in hepatocytes incubated with 1.2 mM extracellular calcium was increased compared to that in calcium-depleted cells, and this increase was entirely due to enhanced sodium-dependent transport. 1.2 mM calcium and 20 microM dibutyryl cyclic AMP together did not stimulate the uptake rate to a greater extent than either treatment alone. It is concluded that calcium and low levels of dibutyryl cyclic AMP alter the rate of taurocholic acid uptake by changing the flux of sodium in the hepatocytes. The inhibitory effect of 1 mM dibutyryl cyclic AMP was not relieved by the presence of 1.2 mM calcium in the cell incubation medium. The results show that dibutyryl cyclic AMP can affect the rate of transport of bile acid into liver cells, and suggest a possible regulatory role for cyclic AMP in this process. PMID- 3015233 TI - Interrelation between gluconeogenesis and hepatic protein synthesis. AB - Acute administration of glucagon to the rat in vivo inhibits hepatic polypeptide chain elongation by about 30%. This effect was not observed in adrenalectomized rats, despite the significant increases in the hepatic content of cyclic AMP. Fatty acid administration mimics the glucagon action on protein synthesis; however, in adrenalectomized animals they were ineffective. Whether glucagon or fatty acids were administered, there was a significant increase in the state of reduction of the NAD system in normal as well as in adrenalectomized rats. This observation rules out the change in the cellular state of reduction as the mediator of their action on protein synthesis. A correlation was observed between the ability of glucagon or fatty acids to inhibit protein synthesis and to stimulate gluconeogenesis. An increased biosynthetic activity as reflected by an increased gluconeogenic flux is accompanied by a decreased phosphorylation state of adenine nucleotides that might be responsible for the inhibitory effect on protein synthesis. In adrenalectomized animals in which neither glucagon nor fatty acids stimulate gluconeogenesis, no effects on phosphorylation state or on the rate of protein synthesis were detected. PMID- 3015234 TI - Metal ion-induced activation of molecular oxygen in pigmented polymers. AB - Diamagnetic and paramagnetic metal ions enhanced the rate of production of hydrogen peroxide during autoxidation of melanin pigments, as measured using an oxidase electrode. However, redox-active metal ions, such as Fe3+ and Cu2+, caused a marked decrease in H2O2 production. Evidence for redox-active metal ion dependent formation of hydroxyl radicals during autoxidation of melanin pigments has been obtained using the electron spin resonance-spin trapping method. Evidence for direct reduction of Fe3+ by melanin polymers also has been obtained using optical spectroscopy. Mechanisms of molecular activation of oxygen induced by metal ions on melanin polymers are discussed. PMID- 3015235 TI - Interplay between calcium and activated cGMP phosphodiesterase from retinal rod outer segments. AB - Hypotonic extraction of bovine retinal rod outer segments after bleaching in isotonic buffer yielded an extract exhibiting activated cGMP phosphodiesterase properties. Since this extract was virtually devoid of other proteins involved in the rod outer segment cGMP enzymatic cascade, it was used to study phosphodiesterase catalytic activity. The hypotonic extract required Mg2+ in the range 0.1-1.0 mM for optimal cGMP hydrolysis. At these Mg2+ concentrations hydrolysis could be effectively inhibited by Ca2+ at concentrations which might be attainable in rod outer segments. Since higher Ca2+ concentrations were required to give a chosen degree of inhibition at higher Mg2+ concentrations, this inhibition was probably due to competition by Ca2+ for Mg2+ binding site(s) on the phosphodiesterase catalytic unit. Other divalent cations were also able to inhibit cGMP hydrolysis, many of them (especially those with ionic radii close to that of magnesium) more effectively than calcium. It is suggested that Ca2+ may play a role in phototransduction by participating in the control of photoreceptor sensitivity, and that this is achieved by modulating rod outer segment cGMP hydrolysis. PMID- 3015236 TI - Apparent activation of the MgATP-dependent protein phosphatase by pp60v-src. Identification of an activity like that of glycogen synthase kinase 3 in immunoaffinity purified pp60v-src preparations. AB - Immunoaffinity purified pp60v-src was found to activate the MgATP-dependent protein phosphatase in the presence of MgATP. Although preliminary evidence suggested that phosphorylation of the inhibitor-2 subunit on tyrosine residues was responsible for the activation, preincubation of the pp60v-src preparation at 41 degrees C resulted in a rapid loss of its protein kinase activities towards both casein and inhibitor-2 while its ability to activate the protein phosphatase complex was relatively insensitive to this treatment. This result demonstrated that pp60v-src was not responsible for activation of the MgATP-dependent protein phosphatase. A protein kinase activity which phosphorylated glycogen synthase on serine residues was detected in the pp60v-src preparation. The protein kinase was active in the presence of inhibitors of phosphorylase kinase, glycogen synthase kinase 5/casein kinase II, and cAMP-dependent protein kinase. It is, therefore, likely that activation of the MgATP-dependent protein phosphatase resulted from the presence of a glycogen synthase kinase 3 like activity in the pp60v-src preparation. Our results illustrate the importance of applying multiple criteria to link the phosphorylation of a protein with an observed change in its activity. PMID- 3015237 TI - Phosphoinositides provide a regulatory mechanism of surface charge and active transport. AB - Yeast cells, when grown in the presence of arsenate, are capable of accumulating phosphoinositides (PI) at the expense of inhibiting their degradation more than their synthesis. PI levels return to normal when the cells are cultured or exposed to media without arsenate. These reversible changes are employed as a tool to test the effect of inositide accumulation and dynamics on several membrane properties. In the PI-rich cells, phosphate and arsenate transport from low external concentrations (high affinity systems), as well as the transport of glycine, which enter the cells accompanied by protons, were increased. The proton ejection energized by glucose is also enhanced in the PI-rich cells that show a more efficient potassium inflow at pH 4.0-4.5. The membrane surface potential of the PI-rich cells was found to be 2-times higher than that of the normal cells, in agreement with the 2-fold increment in the PI. All the above mentioned alterations in membrane properties are reverted when the PI content of the PI rich cells is reduced to the level of normal cells. The results show the participation of the phosphoinositides in the formation, maintenance and regulation of the membrane surface potential and their possible influence upon transport mechanisms. PMID- 3015238 TI - The response of an established line of rat liver cells to thyroid hormone. AB - The response of an established line of non-transformed adult rat liver epithelial cells (ARL 15) to thyroid hormone (T3) (3,5,3'-triiodothyronine) was characterized. Exposure of confluent monolayers to 1.10(-8) M T3 for 3 days increased O2 consumption (QO2) between 14-58%, ouabain-sensitive Rb+ uptake 26%, (Na+ + K+)-ATPase activity 32%, alpha-glycerophosphate dehydrogenase activity 103% and cytochrome oxidase activity 208%. The ARL 15 cells, maintained in continuous culture, therefore, exhibit the hallmarks of an authentic physiological response to thyroid hormone. PMID- 3015239 TI - Exposure of latent prostaglandin binding sites in the rat epididymal adipocyte membrane and the effects of albumin, heating and alkylating agents. AB - Brief incubation (1 min) of the adipocyte isolated membranes at 60 degrees C caused an increase in prostaglandin E2 binding, similar to that obtained with albumin. The increase in the membranal binding capacity after a short heating of the membranes was concomitant with a substantial decline in the ability of albumin to induce a further increase in the binding capacity of the treated membranes. Pretreatment of the isolated adipocyte membranes with 10 mM N ethylmaleimide inhibited the enhancement of prostaglandin E2 binding in the presence of albumin, but did not affect the prostaglandin E2 binding in the absence of albumin. Identical treatment of the isolated membranes with glutathione-maleimide, an impermeable SH reagent with comparable alkylation reactivity, enhanced the binding of prostaglandin E2 in the absence of albumin and failed to inhibit the enhancement of prostaglandin E2 binding in its presence. In contrast to the effect of albumin on prostaglandin E2 binding to the isolated membranes, albumin failed to alter prostaglandin E2 specific binding to intact adipocytes. PMID- 3015240 TI - Effects of triiodothyronine on the synthesis of sulfolipids by oligodendrocyte enriched glial cultures. AB - Glial cultures were obtained from the brains of 1-week-old rats and were grown in a chemically defined, serum-free medium. We investigated the development of oligodendrocytes in these cultures and the synthesis of sulfolipids in the presence and absence of triiodothyronine (T3) in the medium: (1) In the presence of T3, the incorporation of [35S]sulfate into sulfolipids exhibited a developmental profile which is comparable to that found in the developing brain in vivo. A sharp peak of sulfolipid synthesis was observed at day 5 in vitro, which is equivalent to day 12 after birth. As observed in vivo, the percentage of label incorporated into sulfogalactosyldiradylglycerols decreased with time in culture. (2) Addition of T3 to the medium stimulated sulfolipid synthesis by oligodendrocytes in a dose-related manner (optimal T3 concentration, 30 nM). The hormone also enhanced the rates of cholesterogenesis and lipogenesis but to a lesser extent than sulfolipid synthesis. (3) The temporary omission of T3 from the medium resulted in lower rates of sulfolipid synthesis that could not be restored by readdition of T3. This inhibitory effect was most pronounced if the hormone was omitted from the medium on days 2 and 3 in culture. (4) Omission of T3 also resulted in the development of fewer oligodendrocytes in the cultures. Our results show that T3 is essential for the development of oligodendrocytes in our neurone-free culture system. They also indicate that the stimulation of myelination by thyroid hormones can, at least partially, be explained as a direct effect of T3 on oligodendrocytes, independent of an effect of T3 on neuronal growth. PMID- 3015241 TI - Inositol 1,4,5-triphosphate induces calcium release from a non- mitochondrial pool in amoebae of Dictyostelium. AB - Evidence is presented for two distinct CA2+ pools in amoebae of Dictyostelium discoideum. One pool, presumably mitochondrial, was sensitive to the mitochondrial inhibitors oligomycin and dinitrophenol and showed an affinity for Ca2+ in the micro M concentration range. The other Ca2+ pool, which was insensitive to these inhibitors, was of lower capacity but had higher affinity (in the nM range). Inositol 1,4,5-trisphosphate (5 micro M) added to saponin permeabilized amoebae induced a rapid release of Ca2+ from the latter pool but had no effect on the presumed mitochondrial pool. Controls using addition of inositol 1,4-bisphosphate (the hydrolytic product of IP3) induced no such CA2+ release. The results provide strong support for the involvement of IP3 in signal transmission during chemotaxis of D. discoideum. PMID- 3015243 TI - Pleiotropic hydrogenase mutants of Escherichia coli K12: growth in the presence of nickel can restore hydrogenase activity. AB - Anaerobic growth in the presence of 0.6 mM NiCl2 was able to restore hydrogenase and benzyl-viologen-linked formate dehydrogenase activities to a mutant (FD12), which is normally defective in these activities. This mutant carries a mutation located near minute 58 in the genome. Hydrogenase isoenzyme I and II activities were restored along with the hydrogenase activity that forms part of the formate hydrogen lyase system. A plasmid (pRW1) was constructed, containing a 4.8 kb chromosomal DNA insert, which was able to complement the lesion in mutant FD12. Further mutants with mutations near 58 minutes on the chromosome, and which lacked hydrogenase and formate dehydrogenase activities were isolated. These mutants were divided into three groups. Class I mutants were restored to the wild type phenotype either by growth with 0.6 mM NiCl2 or following transformation with pRW1. Class II mutants were also complemented by pRW1 but were unaffected by growth with NiCl2. Class III mutants were unaffected by both pRW1 and growth with NiCl2. The cloned 4.8 kb fragment of chromosomal DNA therefore encodes two genes essential for hydrogenase activity. Restriction analysis indicates that the cloned DNA is the same as a fragment that has previously been cloned and which complements the hydB locus (Sankar et al. (1985) J. Bacteriol., 162, 353-360). None of the three classes of mutants possess mutations in hydrogenase structural genes. PMID- 3015242 TI - Cloning of DNA fragments carrying hydrogenase genes of Rhodopseudomonas capsulata. AB - A cosmid library of Rhodopseudomonas capsulata DNA was constructed in Escherichia coli HB101 using the broad-host-range cosmid vector pLAFR1. More than ninety per cent of the clones in the bank contained cosmids with DNA inserts averaging 20 kilobase pairs in length. Mutants deficient in uptake hydrogenase (Hup-) were obtained from R. capsulata strain B10 by ethylmethylsulfonate (EMS) mutagenesis. The content of hydrogenase protein in Hup- mutant cells was tested by rocket immunoelectrophoresis. Hup- mutants (Rifr) were complemented with the clone bank by conjugation and, from the transconjugants selected by rifampicin and tetracycline resistance, Hup+ transconjugants were screened for the ability to grow photoautotrophically and to reduce methylene blue in a colony assay. The recombinant plasmid pAC57 restored hydrogenase activity in the Hup- mutants RCC8, RCC10, RCC12 and ST410 whereas pAG202 restored that of IR4. The cloned R. capsulata DNA insert of pAC57 gave 5 restriction fragments by cleavage with EcoRI endonuclease. Fragment 1 (7 kb) restored hydrogenase activity in Hup- mutant strains RCC12 and ST410 and fragment 5 (1.3 kb) in strains RCC8 and RCC10. Since the 2 cosmids pAC57 and pAG202 are different cosmids, as indicated by restriction analyses and absence of cross hybridization, it is concluded that at least two hup genes are required for the expression of hydrogenase activity in R. capsulata. PMID- 3015244 TI - Hydrogenases of phototrophic microorganisms. AB - This review surveys recent work done in the laboratory of the author and related laboratories on the properties and possible practical applications of hydrogenases of phototrophic microorganisms. Homogeneous hydrogenase preparations were obtained from purple non-sulfur (Rhodospirillum rubrum S1, Rhodobacter capsulatus B10) and purple sulfur (Chromatium vinosum D, Thiocapsa roseopersicina BBS) bacteria, and from the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum L; highly purified hydrogenase samples were prepared from the cyanobacterium Anabaena cylindrica and from the green alga Chlamydomonas reinhardii. It was shown that hydrogenases of R. capsulatus and T. roseopersicina contain Ni and Fe-S cluster. The cytochromes of the c or b type serve as native electron acceptors for the hydrogenases of the purple bacteria and cyanobacteria; rubredoxin or cytochrome c for the hydrogenase of the green sulfur bacterium; and ferredoxin for Ch. reinhardii hydrogenase. The hydrogenase of T. roseopersicina BBS reversibly activates H2 at Eh less than -290 mV (pH 7), whereas those from R. capsulatus and from C. limicola f. thiosulfatophilum exhibit their maximum activity at Eh greater than -300 mV and are thus favourable only for the H2 uptake. Hydrogenase synthesis in different phototrophs depends on pO2, H2 concentrations and organic substrates. Organic compounds, which serve as electron donors and carbon sources, repress hydrogenase synthesis in R. rubrum, R. capsulatus and in Ectothiorhodospira shaposhnikovii when present at high concentrations. The synthesis of T. roseopersicina hydrogenase is constitutive. H2 notably stimulates hydrogenase activity in R. capsulatus. The synthesis of hydrogenase in R. sphaeroides 2R occurs only in the presence of H2 and does not depend on the presence of organic compounds in the medium. PMID- 3015245 TI - Application of hydrogenase for photoinduced hydrogen evolution. AB - Various attempts have been made to develop suitable redox systems for the photochemical utilization of solar energy. Recent work has shown that the three component systems containing a photosensitizer, an electron donor, and an electron acceptor can be used to evolve hydrogen when a suitable catalyst is present. The reactions are quite general and have been demonstrated for a wide range of photosensitizers and electron carriers. Hydrogenase or colloidal platinum are widely used as catalysts, for they can catalyze the reduction of protons in the presence of a suitable electron donating agent such as reduced methylviologen. Some properties of these components and the efficiency of the photoinduced hydrogen evolution are discussed. PMID- 3015246 TI - Application of hydrogenase in biotechnological conversions. AB - Evidence will be presented in this review article that the application of hydrogenase has large biotechnological possibilities. Our investigations show: Fast reaction of hydrogenase at an electrode surface to reduce H+; Photochemical production of H2 by hydrogenase by photosensitized Ru-complexes dissolved in reversed micellar membranes and vectorial H+ transport through the membrane to the water phase; The production of fine chemicals in reversed micelles by a system containing specific enzymes, hydrogenase and H2. The rules to obtain maximal conversion rates with this system will be presented. PMID- 3015247 TI - On the novel H2-activating iron-sulfur center of the "Fe-only" hydrogenases. AB - The two hydrogenases (I and II) of the anaerobic N2-fixing bacterium Clostridium pasteurianum (Cp) and the hydrogenases of the anaerobes Megasphaera elsdenii (Me) and Desulfovibrio vulgaris (strain Hildenborough, Dv), contain iron-sulfur clusters but not nickel. They are the most active hydrogenases known. All four enzymes in their reduced states give rise to EPR signals typical of [4Fe-4S]1+ clusters but exhibit novel EPR signals in their oxidized states. For example, Cp hydrogenase I exhibits a sharp rhombic EPR signal when oxidized under mild conditions but the enzyme is inactivated by over-oxidation and then exhibits an axial EPR signal. A similar axial signal is observed from mildly oxidized hydrogenase I after treatment with CO. EPR, Mossbauer and ENDOR spectroscopy indicate that the EPR signals from the oxidized enzyme and its CO derivative arise from a novel spin-coupled Fe center. Low temperature magnetic circular dichroism (MCD) studies reveal that an EPR-silent Fe-S cluster with S greater than 1/2 is also present in oxidized hydrogenase I. From a study of all spectroscopic properties of Cp, Dv, and Me hydrogenases, it is concluded that the H2-activating site of all four is a novel Fe-S cluster with S greater than 0 and integer, which in the oxidized state is exchange-coupled to a S = 1/2 species. The data are most consistent with the S = 1/2 species being a low spin Fe(III) center. The H2-activating site is susceptible to oxidative rearrangements to yield both active and inactive states of the enzyme. We discuss the possible implications of these finding to methods of enzyme oxidation and purification procedures currently used for hydrogenases. PMID- 3015248 TI - Activation and deactivation of the membrane-bound hydrogenase from Desulfovibrio desulfuricans, Norway strain. AB - The hydrogenase from D. desulfuricans, when isolated in air, had a low activity in the hydrogen-methyl viologen reductase assay, and no activity in the hydrogen methylene blue reductase assay. The activity increased markedly during incubation under hydrogen. This process is interpreted in terms of conversion of the enzyme from a relatively inactive Unready state to the Active state. Oxidation by dichloro-indophenol caused conversion to a state in which the hydrogen-uptake activity to methyl viologen was preserved, but hydrogen-methylene blue activity was not. This form is termed the Ready state. This behaviour resembles that of the hydrogenase of Desulfovibrio gigas and thus may be a widespread property of this class of hydrogenases. The electron-spin-resonance spectra of the D. desulfuricans enzyme showed the presence of [3Fe-xS] and [4Fe-4S] clusters. Spectra were also observed in the various states of activation of the enzyme. In these respects, the hydrogenase of D. desulfuricans resembles that from D. gigas, although the latter may have an additional iron-sulphur cluster. PMID- 3015249 TI - The pH dependence of proton-deuterium exchange, hydrogen production and uptake catalyzed by hydrogenases from sulfate-reducing bacteria. AB - Different patterns have been found in the pH dependence of hydrogenase activity with enzymes purified from different species of Desulfovibrio. With the cytoplasmic hydrogenase from Desulfovibrio baculatus strain 9974, the pH optima in H2 production and uptake were respectively 4.0 and 7.5 with a higher activity in production than in uptake. The highest D2-H+ exchange activity was found also at pH 4.0 but the optima differed for the HD and the H2 components. Both similarly rose when the pH decreased from 9.0 to 4.5, but the rate of H2 evolution slowed whereas the HD evolution continued rising till pH values around 3.0 were reached. The H2 to HD ratio at pH above 4.5 was higher than one. With the periplasmic hydrogenase from Desulfovibrio vulgaris Hildenborough, the highest exchange activity was near pH 5.5, the same value as in hydrogen production. The periplasmic hydrogenase from Desulfovibrio gigas had in contrast the same pH optimum in the exchange (7.5-8.0) as in the H2 uptake. The ratio of H2 to HD was below one for both enzymes. These different patterns may be related to functional and structural differences in the three hydrogenases so far studied, particularly in the composition of their catalytic centers. PMID- 3015250 TI - Redox properties and activity studies on a nickel-containing hydrogenase isolated from a halophilic sulfate reducer Desulfovibrio salexigens. AB - A soluble hydrogenase from the halophilic sulfate reducing bacterium Desulfovibrio salexigens, strain British Guiana (NCIB 8403) has been purified to apparent homogeneity with a final specific activity of 760 mumoles H2 evolved/min/mg (an overall 180-fold purification with 20% recovery yield). The enzyme is composed of two non-identical subunits of molecular masses 62 and 36 kDa, respectively, and contains approximately 1 Ni, 12-15 Fe and 1 Se atoms/mole. The hydrogenase shows a visible absorption spectrum typical of an iron-sulfur containing protein (A400/A280 = 0.275) and a molar absorbance of 54 mM-1cm-1 at 400 nm. In the native state (as isolated, under aerobic conditions), the enzyme is almost EPR silent at 100 K and below. However, upon reduction under H2 atmosphere a rhombic EPR signal develops at g-values 2.22, 2.16 and around 2.0, which is optimally detected at 40 K. This EPR signal is reminiscent of the nickel signal C (g-values 2.19, 2.16 and 2.02) observed in intermediate redox states of the well characterized D. gigas nickel containing hydrogenase and assigned to nickel by 61 Ni isotopic substitution (J.J.G. Moura, M. Teixeira, I. Moura, A.V. Xavier and J. Le Gall (1984), J. Mol. Cat., 23, 305-314). Upon longer incubation with H2 the "2.22" EPR signal decreases. During the course of a redox titration under H2, this EPR signal attains a maximal intensity around--380 mV. At redox states where this "2.22" signal develops (or at lower redox potentials), low temperature studies (below 10 K) reveals the presence of other EPR species with g values at 2.23, 2.21, 2.14 with broad components at higher fields. This new signal (fast relaxing) exhibits a different microwave power dependence from that of the "2.22" signal, which readily saturates with microwave power (slow relaxing). Also at low temperature (8 K) typical reduced iron-sulfur EPR signals are concomitantly observed with gmed approximately 1.94. The catalytic properties of the enzyme were also followed by substrate isotopic exchange D2/H+ and H2 production measurements. PMID- 3015251 TI - Activation and active sites of nickel-containing hydrogenases. AB - Hydrogenases that contain nickel and iron-sulphur clusters also have a regulatory mechanism, by which exposure to oxidants such as oxygen prevents their reaction with hydrogen. Treatment with reducing agents then causes reactivation. In some hydrogenases from Desulfovibrio species, there is evidence that there are at least two different deactivated states, which differ in their rates of reductive reactivation. The membrane-bound hydrogenase of D. desulfuricans, Norway strain, the periplasmic hydrogenase of D. gigas and the membrane-bound hydrogenase of Alcaligenes eutrophus can be isolated in a state (termed "Unready") which requires up to several hours for full activation by hydrogen. By contrast the soluble hydrogenases of D. desulfuricans and A. eutrophus can be reactivated relatively rapidly. In all of these enzymes, with the exception of the latter one, the existence of the activated and deactivated states can be correlated with different ESR-detectable forms of nickel. The possible functions of nickel and [Fe-4S] clusters in catalysis are discussed. PMID- 3015252 TI - The redox properties of the iron-sulphur cluster in hydrogenase from Chromatium vinosum, strain D. AB - The midpoint potentials of the changes in the electron spin resonance (ESR) spectra in the region of g = 2 in hydrogenase II from Chromatium vinosum were estimated by redox titrations. As the enzyme was progressively reduced, the g = 2.02 signal increased, while the satellite lines at g = 1.98 etc. decreased. At still lower potentials the signal at g = 2.02 decreased. The midpoint potentials of the two processes were estimated to be + 100 mV and - 20 mV, respectively, at pH 8.5. The first potential showed significant pH-dependence. The titration data fitted to n = 1 curves with reasonable reversibility. The enzyme activity showed no significant changes in this potential range. The results are discussed in relation to the interaction of the iron-sulphur cluster with nickel. PMID- 3015253 TI - Redox properties and active center of phototrophic bacteria hydrogenases. AB - It is shown that the activity of phototrophic bacteria hydrogenases depends on the redox potential (Eh) of the medium. Hydrogenase from the purple sulfur bacterium Thiocapsa roseopersicina strain BBS reversibly activates H2 at Eh less than -290 mV (pH 7.0). When Eh is increased from -290 to -170 mV, the enzyme is converted into an inactive form which is accompanied by one-electron oxidation of its Fe-S cluster. In contrast, the hydrogenases of the purple nonsulfur bacterium Rhodobacter capsulatus B10 and the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum exhibit maximum activity at Eh greater than -300 mV, favourable only for H2 uptake. When Eh decreases the activities of these enzymes drop dramatically; this accounts for their unidirectional effect directed mainly towards H2 uptake. Such dependence on Eh of activity of hydrogenases from these bacteria correlates with their physiological function in the metabolism of phototrophic bacteria, i.e. with the catalysis of the H2 uptake reaction. Hydrogenases from purple bacteria contain nickel and a single Fe-S cluster. Metal chelators do not affect the activity of these enzymes, which indicates that iron and nickel are tightly bound to the apoprotein. Sulfhydryl compounds irreversibly inactivate T. roseopersicina hydrogenase by 30-40% in the presence of sulfide. Acetylene and carbon monoxide are reversible inhibitors of the enzyme. EPR and inhibitory analysis indicate a direct interaction of H2 with the nickel ion in the active center of the T. roseopersicina hydrogenase. PMID- 3015254 TI - Properties and kinetics of a neutral beta-galactosidase from rabbit kidney. AB - A neutral beta-galactosidase has been purified by concanavalin A-Sepharose affinity chromatography, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and hydroxylapatite chromatography. The enzyme was purified 126-fold with a yield of about 21%. This form has a neutral optimal pH (7.5) and it is located in the cytosolic fraction. It shows a wide pH stability from pH 4.5 to 8.0, but it is very unstable at low pH values. Its isoelectric point is 4.9 and this value does not change on neuraminidase treatment. The estimated molecular weight was 47 000. The neutral form shows beta-D-galactosidase, beta-D-fucosidase and beta-D-glucosidase activities, all of them associated in a single peak in all the purification steps. p-Nitrophenyl beta-D-galactosides, p-nitrophenyl beta-D fucosides and p-nitrophenyl beta-D-glucosides competed fully for a common active site in mixed-substrate experiments. Using gamma-D-galactonolactone as competitive inhibitor the Ki values were always coincident for the three activities. The effect of NaCl, methyl mannoside and some sugars (fucose, galactose and glucose) was studied. PMID- 3015255 TI - 2-Ketoglutarate as a possible regulatory metabolite involved in cyclic AMP dependent catabolite repression in Escherichia coli K12. AB - 2-Ketoglutarate--unlike any other derivative of the citric acid cycle--was found to strongly repress catabolite-sensitive genes, such as the lactose operon (lac) or the tryptophanase gene (tna), when added to cells grown in glycerol. 2 ketoglutarate affects the expression of these genes by decreasing cyclic AMP synthesis. Such inhibition of cyclic AMP synthesis requires the presence of enzyme III, a component of the phosphoenol pyruvate:sugar phosphotransferase transport system (PTS). Thus, it is proposed that 2-ketoglutarate is one of the catabolite repressors postulated by Magasanik in 1961. In addition, by studying the effect of 2-ketoglutarate in various mutants, we show the existence of a cyclic AMP-independent catabolite repression mechanism whose mediator is synthesized from 2-ketoglutarate. PMID- 3015257 TI - Purification and properties of the endoglucanase C of Clostridium thermocellum produced in Escherichia coli. AB - The celC gene, which codes for a new endoglucanase of Clostridium thermocellum, termed endoglucanase C, was found to be expressed when cloned in Escherichia coli. The enzyme was purified to electrophoretic homogeneneity from E. coli and its biochemical properties were studied. It differs from the previously studied endoglucanases A and B. In particular, endoglucanase C displays features common to endo- and exoglucanases, since it had a high activity on carboxymethylcellulose and on p-nitrophenyl-beta-D-cellobioside where only the agluconic bond was split. In addition, the enzyme was able to release cellobiose units from G3, G4 and G5 cellodextrins. Endoglucanase C was characterized by Western blot in a culture supernatant from C. thermocellum grown on cellulose, using an antiserum raised against the enzyme produced by E. coli. PMID- 3015256 TI - [Human apolipoprotein E: polymorphism and binding domain of the receptors]. AB - This paper summarizes present knowledge of the LDL receptor-binding domain of apolipoprotein E, with special emphasis on the influence of apolipoprotein polymorphism on the interaction with apo B/E receptors. PMID- 3015258 TI - The physiology of stringent factor (ATP:GTP 3'-diphosphotransferase) in Escherichia coli. AB - The enzyme ATP:GTP 3'-diphosphotransferase catalyzes the transfer of the beta, gamma-pyrophosphate of ATP to the 3' position of GTP or GDP. The amounts of enzyme were measured in cell extracts of a relA+ strain of E. coli grown at different growth rates between 0.4 and 1.9 generations per hour, using precipitation with specific antibodies to purify the enzyme. The amount of enzyme was found to be a constant fraction of total protein at all growth rates corresponding to about 45 molecules of enzyme per genome equivalent of DNA. The purified enzyme has little catalytic activity by itself but has to be activated either by a complex of 70S ribosomes, mRNA and uncharged tRNA or by a solvent like ethanol at a concentration of about 20%. The kinetic constants of the enzyme for the transfer pyrophosphate from ATP to GTP in the ribosome-activated state were determined. The Vmax was estimated to be 140 mumol/min X mg at 37 degrees C and the S0.5 values for GTP and ATP were 0.35 and 0.53 mM, respectively. The reaction was estimated to have an equilibrium constant of about 300. In the pyrophosphate transfer from ATP to GDP the Vmax was estimated to be 90 mumol/min X mg at 37 degrees C and the S0.5 for GDP as 0.3 mM. During amino acid starvation of a relA+ strain of E. coli the amounts of enzyme and the catalytic capacity of the enzyme are sufficient to maintain the observed ppGpp levels in the cells at all growth rates. PMID- 3015259 TI - Pulse radiolysis study of a yeast cytochrome c from Hansenula anomala. AB - The reduction of Hansenula anomala yeast cytochrome c by e-aq and CO-.2 was investigated by pulse radiolysis, at a high reductant to protein concentration ratio. The reactivity of the radicals was studied by observing absorbance changes in the cytochrome c spectrum over the wavelength range 280-600 nm. At pH 7, over the time scale of the radical decays (i.e. 0-4 microseconds for e-aq; 0-40 microseconds for CO-.2s) and beyond, the hemoprotein was reduced without any spectrally detected intermediate between ferri-and ferro-forms. This conclusion was reached by simulation studies based on the direct reduction of the yeast cytochrome c from the ferri- to the ferro-form, yielding a correct fit between experimental and calculated absorbance curves. The reduction rate constants were determined to be 1.0 +/- 01 X 10(10) M-1 S-1 for e-aq and 0.7 +/- 0.05 X 10(9) M 1 S-1 for CO-.2 at 0.16 M ionic strength, pH 7.0 and 20 degrees C, thus not significantly different from other values reported for horse heart cytochrome c. However, in the 360-390 nm region the generation of an additional radical species was noticed. The present experimental data were compared with previously published reports. PMID- 3015261 TI - [Effect of GDP on transducin interaction with cyclic nucleotide phosphodiesterase and rhodopsin from bovine retinal rods]. AB - In the presence of guanyl nucleotides and rhodopsin-containing retinal rod outer segment membranes, transducin stimulates the light-sensitive cyclic nucleotide phosphodiesterase 5.5-7 times. The activation constant (Ka) for GTP and Gpp(NH)p is 0.25 microM, that for GDP and GDP beta S is 14 and 110 microM, respectively. GDP purified from other nucleotide contaminations at concentrations up to 1 mM does not stimulate phosphodiesterase but binds to transducin and inhibits the Gpp(NH)p-dependent activation of phosphodiesterase. The mode of transducin interaction with bleached rhodopsin also depends on the nature of the bound guanyl nucleotide: in the presence of GDP rhodopsin-containing membranes bind 70 100% of transducin, whereas in the presence of Gpp(NH)p the membranes bind only 13% of the protein. The experimental results suggest that GDP and GTP convert transducin into two different functional states, i.e., the transducin X GTP complex binds to phosphodiesterase causing its stimulation, while the transducin X GDP complex is predominantly bound to rhodopsin. PMID- 3015260 TI - Chromate, molybdate, tungstate and vanadate behave as substrates of yeast diadenosine 5',5'''-p1,p4-tetraphosphate alpha, beta-phosphorylase. AB - Diadenosine 5',5'''-p1,p4-tetraphosphate (Ap4A) alpha, beta-phosphorylase from yeast Saccharomyces cerevisiae catalyzes two reactions: Ap4A cleavage and nucleoside diphosphate--phosphate (NDP-Pi) exchange. In both reactions phosphate can be substituted by arsenate, chromate, molybdate, tungstate or vanadate. In the presence of each anion, nucleoside 5'-monophosphate (NMP) always accumulates as a product of the reaction. This indicates that an unstable NMP anion is formed as an intermediate. PMID- 3015262 TI - [Structure and function of kinetoplast DNA of Trypanosomatidae]. AB - Kinetoplast DNA of trypanosomatids represents a complex associate composed of mini- and maxicircular molecules, the latter being analogous to mitochondrial DNA of other eukaryotic species. Some new data on the structural organization and transcription of trypanosomatid mitochondrial genome are given. Detailed genetic and transcriptional maps of maxicircular kinetoplast DNAs of trypanosome and leishmania are presented. The replication mechanisms of mini- and maxicircular molecules of kinetoplast DNA are analyzed. Some characteristic features of trypanosomatid mitochondrial genome organization similar and different with respect to mitochondrial genomes of other eukaryotic species are discussed. PMID- 3015263 TI - [Ability of virus SV40 T-antigen to simulate the effect of specific T lymphocyte growth factor--interleukin-2]. AB - The entering of T-lymphocytes into the DNA-synthesizing phase was marked by three consecutive signals, i.e., antigenic influence, interleukin-2, a specific T lymphocyte cell growth factor, and non-specific serum growth-promoting factors, in the first place, transferrin. This system was used for the study of effects of virus SV40 T-antigen on cell mitotic cycle. Purified T-antigen was injected consecutively into T-lymphocytes, using erythrocyte ghost vesicles instead of one of control signals. It was shown that T-antigen cannot simulate the antigenic response but simulates the effect of interleukin-2, a specific growth-promoting factor. However, both normally proliferating T-lymphocytes and T-antigen-induced lymphocytes showed an absolute requirement for transferrin and, apparently, for other nonspecific growth-promoting factors. It was assumed that the polymorphism of tumours induced by papovaviruses is determined by the ability of their "early" proteins to imitate the effects of their specific growth-promoting factors on the cells. PMID- 3015265 TI - [The role of adenylate kinase in the regulation of the rate and effectiveness of energy transfer from mitochondria to hexokinase in vitro]. AB - The effect of adenylate kinase activity on the rate and efficiency of energy transport from mitochondria to hexokinase was studied in a system containing isolated rabbit heart mitochondria, hexokinase and adenylate kinase at low concentrations of adenine nucleotides. Oxygen consumption by mitochondria and glucose-6-phosphate synthesis by hexokinase were recorded. It was found that with adenylate kinase being active both in mitochondria and in the washing solution, the rate and efficiency of glucose-6-phosphate synthesis considerably increases. The effects of adenylate kinase activity are fully abolished by diadenosine pentaphosphate, an inhibitor of adenylate kinase. The experimental results based on the use of adenylate kinase demonstrate the possibility of increasing the rate and efficiency of energy transfer between two spatially uncoupled biochemical processes in vitro with the aid of an enzymatic system. PMID- 3015264 TI - [Isolation and properties of the angiotensin-converting enzyme from human lungs]. AB - Using chromatofocusing, an angiotensin-converting enzyme (EC 3.4.15.1) has been isolated from human lung. The procedure allows for 24 300-fold purification of the enzyme. The enzyme specific activity is 36.3 u. per mg protein; Mr as determined by polyacrylamide gel electrophoresis is 150 000. The lung enzyme after solubilization by trypsin treatment was found to be heterogeneous. Four isoforms of the enzyme with pI 5.3, 4.9, 4.8 and 4.6 were identified. The pH optimum for the enzyme with respect to hippuryl-L-histidyl-L-leucine hydrolysis lies at 8.3; Km = 2.8 mM. The effect of Cl- on the enzyme activity was studied. It was found that the bradykinin-potentiating factor (SQ 20 881) inhibits the human lung angiotensin-converting enzyme (I50 = 1.6 X 10(-8) M). PMID- 3015266 TI - [NAD+-kinase from Neurospora crassa: isolation and properties]. AB - The NAD+ kinase (EC 2.7.1.23) of the filamentous fungus N. crassa is localized in cytosol. The activity in the dialyzed cell free extract has a pH optimum 8.3; it utilizes only ATP but not inorganic polyphosphates as a phosphoryl donor. A method for 200-fold purification of NAD+ kinase with a 20% yield has been developed. The procedure includes 105000 g centrifugation, fractionation with (NH4)2SO4, isoelectrofocusing in a Ultrodex layer and preparative electrophoresis in polyacrylamide gel. The molecular heterogeneity of NAD+ kinase was demonstrated by polyacrylamide gradient electrophoresis and by gel filtration through Sephadex G-200. The molecular weights of four individual forms of the enzyme are: 330000-338000, 305000-306000, 215000-229000 and 203000 Da. The Km values for the reaction catalyzed by purified NAD+ kinase for NAD+ and ATP are 3.0 X 10(-4) M and 0.9 X 10(-3) M, respectively. PMID- 3015268 TI - [Effect of thyroid hormone receptors in normal and cancerous cells on the ionic conductivity of bilayer phospholipid membranes]. AB - The effect of thyroid hormones receptors isolated from normal and cancer cells on bilayer phospholipid membranes (BPhLM) conductivity, has been studied. The receptor isolated from normal cells in complex X with the hormone selectively induces H+-conductivity of BPhLM generating transmembrane potential equal to 42 mV on the membrane at pH gradient equal to 1. In the presence of K+, Na+, Ca+, Mn2+, Sr2+, Mg2+ the changes of BPhLM are not observed. Neither hormones (T3, T4) nor receptor in free position affect the BPhLM conductivity. Thyroid hormone receptor isolated from mamalignantly transformed cells in a complex with T3 or T4 increases the BPhLM permeability for Ca2+. The transmembrane potential measured at 10fold Ca2+ ion concentration is equal to 16 mV. In the presence of H+, K+, Na+, Mn2+, Sr2+, Mg2+, Ba2+, the resistance of BPhLM doesn't change. PMID- 3015269 TI - Supersensitive endocrine response to physostigmine in dopamine-depleted rats: a model of depression? AB - Depressed patients exhibit an abnormal "supersensitive" increase in the plasma concentration of several pituitary hormones following intravenous injection of the acetyl cholinesterase inhibitor physostigmine (PHY). In the present study, we examined the effects of PHY treatments on the plasma concentrations of prolactin (PRL) and adrenocorticotrophic hormone (ACTH) in the rat. Physostigmine (0-0.6 mg/kg, s.c.) produced a dose-dependent increase in PRL and ACTH immunoreactivity in unoperated animals. Neurotoxin-induced depletion of brain dopamine (DA) or norepinephrine (NE) did not significantly alter baseline plasma PRL or ACTH values. Following depletion of brain DA, but not NE, animals exhibited a "supersensitive" increase in plasma ACTH values, which was evidenced by a sixfold left shift in the dose-response properties of PHY. These results suggest that there are intriguing parallels between the abnormal endocrine response to PHY demonstrated by depressed patients and that demonstrated by rats following depletion of central nervous system (CNS) DA levels. PMID- 3015267 TI - [Mn2+-dependent endonuclease activity of chromatin]. AB - The endogenous endonuclease activity of chromatin in isolated rat liver nuclei in the presence of Mn2+, Mg2+ and Ca2+ + Mg2+ was studied. The existence of a Mn2+ dependent endonuclease activity not coupled with the Ca2+, Mg2+-dependent endonuclease was demonstrated, which was weaker than the former one in isolated cell nuclei but higher than in the preparation of Ca2+, Mg2+-dependent nuclease obtained by gel filtration through Toyopearl HW 60F. The Mn2+-dependent splitting of chromatin predominantly occurs at linker DNA of distal parts of chromatin loops. A split-off of purified DNA was more universal than in the presence of Ca2+, Mg2+-dependent endonuclease; the hydrolysis rate of native and denaturated DNA appeared to be the same. PMID- 3015271 TI - Pathophysiology of "cholinoceptor supersensitivity" in affective disorders. AB - Phenomenological and physiological variables demonstrate supersensitive changes to cholinergic challenge in affective disorder subjects. Theorists generally assume the primary defect is the postsynaptic muscarinic receptor. However, in addition to defectiveness or up-regulation of this receptor, the appearance of postsynaptic "cholinoceptor supersensitivity" can result from abnormal presynaptic mechanisms, membrane "pathology," derangement of intrasystolic mechanisms that amplify effects of receptor-agonist coupling, or aberrant cholinergic-monoaminergic interaction. This article discusses abnormalities of the postsynaptic receptor, regulation of postsynaptic receptor density, the presynaptic muscarinic receptor, and other mechanisms regulating the release of acetylcholine, membrane dynamics, and "cascade" mechanisms-specifically the phosphatidylinositol (PI) cycle, Ca2+ mobilization, and cyclic guanosine monophosphate (GMP) generation-as causes of cholinergic system "supersensitivity." It is suggested that an approach to the topic emphasizing site of abnormality will encourage greater clarity of thought in the study of the cholinergic component of the pathophysiology of affective illness. PMID- 3015272 TI - Distribution of a seminal plasma-associated protein kinase inhibitor in normal, oligozoospermic, and vasectomized men. AB - Human sperm-free seminal plasma contains an inhibitor, which is protein in nature, of the histone kinase present in seminal plasma. Since protein kinase inhibitors have been observed to be present in spermatozoa, the objective of the present study was to determine whether this seminal plasma-associated enzyme inhibitor originates from the sperm, or whether it is a component of accessory secretion(s) comprising the seminal plasma. Sperm-free seminal plasma from normospermic (greater than 20 X 10(6) sperm/ml), oligozoospermic (less than or equal to 20 X 10(6) sperm/ml), and vasectomized donors was obtained, and inhibitor-enriched fractions were prepared by (NH4)2SO4 fractionation and gel filtration. Contamination of the sperm-free seminal plasma by spermatozoa or spermatozoan components was negligible as assessed by light microscopy, polyacrylamide gel electrophoresis, and measurement of the activity of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase. Specific (inhibitory units/mg protein) and total inhibitory activities were determined in each of the donors by constructing linear inhibition curves using various concentrations of inhibitor. The results were correlated with the initial sperm concentration. There was no apparent relationship between the amount of inhibitory activity present and the initial sperm concentration. The histone kinase inhibitor also did not appear to be associated with testicular or epididymal secretions since it was observed in the seminal plasma of vasectomized donors. It is concluded that this inhibitor of histone kinase originates from the accessory secretions comprising the human ejaculate. PMID- 3015270 TI - Noradrenergic and neuroradiological abnormalities in tardive dyskinesia. AB - Previous studies suggest that catecholaminergic overactivity and structural brain damage may contribute to the pathogenesis of tardive dyskinesia (TD). Although dopaminergic (DA) mechanisms, specifically postsynaptic receptor supersensitivity, have been extensively studied, equally plausible noradrenergic (NE) changes have been all but ignored. Likewise, the interaction of neurochemical and neuroradiological abnormalities has received little attention. Over the past 6 years, 111 inpatients were studied with a battery of neurological, behavioral, biochemical, and neuroradiological measures. Forty-one patients met specific diagnostic criteria for TD, based in part on global ratings on the Abnormal Involuntary Movement Scale. Subgroups of patients were also evaluated with the Brief Psychiatric Rating Scale and were assayed for plasma dopamine-beta-hydroxylase (DBH) activity, platelet 3H-dihydroergocryptine (3H DHE)-alpha 2 adrenergic receptor binding, lumbar cerebrospinal fluid (CSF) monoamines and metabolites [NE, 3-methoxy-4-hydroxyphenylglycol (MHPG), DA sulfate, homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC)], and CT scan indices of brain atrophy, including ventricle/brain ratio (VBR), bifrontal/bicaudate ratio, and cortical atrophy. All patients were studied in the steady state, primarily when free of neuroleptics. Patients with TD had significantly greater DBH activity than those without TD. In addition, 3H-DHE binding and CSF NE were significantly correlated with the severity of TD when present. Finally, TD patients with low DBH activities (below the mean) had significantly larger ventricles than non-TD patients with low DBH activities. Other data suggested that subcortical, rather than cortical, atrophy was more likely to be responsible for the larger VBR in the low DBH TD group. These results suggest an association of NE overactivity and TD in a portion of patients. Moreover, the presence of neuroradiological abnormalities in TD patients with low DBH activity underscores the contribution of heterogeneous factors to the pathogenesis of this disorder and may provide one possible explanation for the discrepant biochemical findings in TD reported by earlier investigators. PMID- 3015273 TI - Reproductive physiology of the clouded leopard: II. A circannual analysis of adrenal-pituitary-testicular relationships during electroejaculation or after an adrenocorticotropin hormone challenge. AB - A circannual analysis was made of serum cortisol, luteinizing hormone (LH), and testosterone concentrations in the male clouded leopard (Neofelis nebulosa). Group I males (n = 4), maintained in a standardized environment, were bled serially during a regimented anesthesia/electroejaculation episode occurring monthly (beginning in January, ending in December). Additional sampling intervals were conducted under anesthesia only (control, n = 8), anesthesia plus a single adrenocorticotropin hormone challenge (ACTH, Cortrosyn, n = 4), or anesthesia plus a single 25 micrograms injection of gonadotropin-releasing hormone (GnRH, Gonadorelin, n = 4). Group II males (n = 6) from various zoological collections were sampled serially under the same semen collection conditions on one random occasion within the year. Serum cortisol levels were 2 times greater than values measured in comparable studies involving other felid species. Cortisol concentrations were similar during electroejaculation and control (anesthesia only) episodes, and mean levels did not rise as a result of semen collection. Adrenocorticotropin caused an immediate rise in cortisol to levels at least 1.5 times greater than electroejaculated or control counterparts. Mean concentrations of basal cortisol in individual males gradually increased as the year progressed, possibly as a consequence of repeated psychogenic stress. Between seasons, there were no differences in mean LH; however, testosterone levels were greater (p less than 0.05) in the winter compared to all other seasons. There were no differences (p greater than 0.05) between individual males in secretory patterns or mean concentrations of cortisol, LH, or testosterone. Within males, distinct temporal fluctuations were observed in both LH and testosterone during the approximately 80-min sampling interval. Neither LH nor testosterone profiles appeared affected by cortisol patterns during electroejaculation or after an ACTH challenge. A bolus of GnRH induced a marked rise in serum LH and testosterone within 15 and 30 min respectively, indicating that these two hormones were coupled. Both LH and testosterone profiles in Group II males mimicked those in Group I; concentrations of cortisol in Group II males immobilized on one occasion were similar to those of Group I animals sampled from January-May but appeared to be less than values measured from June-December. These data demonstrate that the clouded leopard, compared to other felids, produces markedly elevated concentrations of cortisol, which are likely related to an aggressive behavioral temperament.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3015274 TI - Formation of myeloperoxidase compound II during aerobic stimulation of rat neutrophils. AB - We have made simultaneous spectrophotometric and O2 measurements on suspensions of rat neutrophils during activation of the respiratory burst. Under aerobic conditions an absorption increase attributable to myeloperoxidase compound II was observed in parallel with the rapid phase of O2 uptake. Identification of this compound was confirmed by analysis of a spectrum obtained with purified myeloperoxidase and H2O2. Whereas a second addition of stimulus did not increase O2 uptake any further, a second phase of myeloperoxidase release and compound II formation was observed. These results suggest that in vivo myeloperoxidase reacts with H2O2 generated via the respiratory burst to form compound II under conditions in which the chlorination reaction would be the expected major pathway. PMID- 3015275 TI - A comparison of topoisomerase I activity in normal and transformed cells. AB - Many viral oncogenes encode protein-tyrosine kinase activities. However, important in vivo substrates of these enzymes have yet to be identified. Recently, type I topoisomerases were shown to be in vitro substrates for two tyrosine kinases. Following tyrosine phosphorylation, topoisomerase I activity was reduced 10-fold (Tse-Dinh et al. Nature 312: 785-786, 1984). To determine whether topoisomerase I activity was modulated by tyrosine phosphorylation in vivo, we have measured topoisomerase I activity in nuclear lysates prepared from both normal fibroblasts and cells transformed by two different viral oncogenes (v abl, v-src). Under a variety of experimental conditions, we have found no evidence to support the notion that type I topoisomerase activity is modulated by tyrosine phosphorylation in vivo. PMID- 3015276 TI - Immunochemical characterization of a new platelet specific monoclonal antibody and its use to demonstrate the cytoskeletal association of the platelet glycoprotein IIb-IIIa complex. AB - We describe the production and characterization of a monoclonal antibody specific for platelets. This antibody reacts strongly with human and primate platelets, but does not recognise human monocytes, polymorphonuclear leucocytes, lymphocytes, erythrocytes, leukaemic nor fibroblast cell lines, nor rodent platelets. Immunoprecipitation studies using radiolabelled platelet membrane proteins showed that the monoclonal antibody binds to the platelet membrane glycoprotein IIb-IIIa complex. Affinity chromatography using immobilized monoclonal antibody allows purification of the antigen, but also co-purifies the cytoskeletal proteins actin and myosin. Our results demonstrate immunochemically that although the GP IIb-IIIa complex is an external structure, it is connected through the cell membrane to the microfilament system. PMID- 3015278 TI - [Changes in the surface potential of plasma lipoproteins in ischemic heart disease. Studied with spin probes]. AB - Changes in lipoprotein surface potentials were studied by a positively charged analog as a spin probe. Low density lipoproteins (LDL) and high density lipoproteins (subfractions HDL2 and HDL3) of patients with coronary heart disease (CHD) were studied. CHD patients have revealed a significant decrease (by 14.4 +/ 0.3 mV) in LDL and an increase (by 6.3 +/- 2.0 mV) in HDL3 negative surface potential, as compared to the control. The increase in HDL2 surface potential in CHD patients was insignificant (1.9 +/- +/- 2.5 mV). The possible role of LDL and HDL3 surface potential changes in the mechanism of interaction of these types of lipoproteins with vascular wall and blood cellular membranes and in pathogenesis of CHD and atherosclerosis is discussed. PMID- 3015277 TI - [Rapid pH changes associated with synaptic transmission in isolated slices of the mammalian hippocampus]. AB - To perform rapid optical detection of possible pH changes accompanying electrical activity hippocampal slices were stained with pH indicator--phenol red (0.2 mM). Electrical response of granular and pyramidal cells was evoked by stimulation of perforant path, Schaffer collateral and commissural afferents in the stratum radiatum. Biphasic pH changes occurred both in pyramidal and granular cells: rapid acid changes, with the maximum reached in several msec, were followed by alkaline changes lasting up to one sec. pH changes disappeared with the blocking of synaptic transmission by Mg2+ (10 mM) and were absent in antidromic stimulation of granular cells. pH changes are believed to be related to the processes accompanying synaptic transmission. PMID- 3015279 TI - [Mechanisms of the effects of Ca2+ ions on lipid peroxidation]. AB - Mechanisms underlying Ca2+ effects on lipid peroxidation (LPO) induced in liposomes (from egg yolk lecithin) and UFsomes (from linolenic acid, methyl linolenate) with the aid of O2- -system (Fe2+ + ascorbate) were studied. It was shown that stimulation of lipid peroxidation by low Ca2+ concentrations (10(-6) 10(-5) M) was due to its ability to release Fe2+-ions bound to negatively charged (phosphate, carboxylic) lipid groups (of licethin, linolenic acid), thus increasing the concentration of catalytically active Fe2+. The inhibitory effect of high Ca2+ concentrations was caused by its interaction with superoxide anion radicals and was not observed in LPO-systems, independent of O2- generation (e. g. Fe2+ + cumol hydroperoxide). PMID- 3015280 TI - [Interrelation of diurnal rhythms of recirculation of lymphocytes and their synthesis of cAMP]. AB - The correlative analysis of diurnal rhythms of cell number and adenylate cyclase cellular activity was performed in the thymus, spleen and peripheral blood of Swiss mice, intact or thymectomized at the age of 3 months. The existence of somehow synchronized diurnal rhythms of cAMP exchange and cell recirculation was found in the peripheral lymphocyte pool and in the thymus. Diurnal rhythm of cAMP synthesis in peripheral lymphocytes was regulated by the thymus. Thymectomy led to desynchronization of recirculating processes and cyclic nucleotide metabolism in immunocompetent cells. PMID- 3015281 TI - The relation of platelet density to platelet age: survival of low- and high density 111indium-labeled platelets in baboons. AB - The relationship between platelet density and platelet age has been studied using continuous linear Percoll density gradients and 111In-labeling of autologous platelets in baboons. To investigate changes in platelet density during senescence in the circulation, baboons were infused with 111In-labeled autologous platelets, and blood was collected at one hour postinfusion and twice daily thereafter for six days. Platelets were isolated from these samples in high yield (greater than 95%) and separated in continuous linear Percoll density gradients following density equilibrium centrifugation. Although at one hour postinfusion the density distribution of radiolabeled platelets coincided closely with the distribution of the total platelet population, a detectable symmetrical shift toward higher densities was observed after five days. The relative specific radioactivity (RSR) of high-density platelets (1.064 to 1.067 g/mL) decreased at a slower rate than that of the total platelet population (platelets of all densities), whereas the RSR of low-density platelets (1.053 to 1.056 g/mL) showed a more immediate and rapid decrease. These results give rise to one of two interpretations: (1) low-density platelets have a shorter survival time than more dense platelets and are therefore cleared from the circulation at a faster rate, or (2) platelets of all densities increase in density upon aging in the circulation. To determine the explanation for changing RSR of different density fractions we studied the in vivo disappearance characteristics of low- and high density 111In-labeled platelets. There were no significant differences between the mean survival times of low-density platelets (5.0 +/- 0.49 days, +/- 1 SD, n = 6), high-density platelets (4.9 +/- 0.56 days, n = 6), or control platelets representing platelets of all densities (4.9 +/- 0.38 days, n = 6). Although a slight increase in the density of all platelets during platelet senescence is indicated by these studies, we conclude that platelet density heterogeneity is not primarily a consequence of age-related changes in platelet density. PMID- 3015282 TI - Dibutyryl cyclic adenosine monophosphate reduces expression of c-myc during HL-60 differentiation. AB - The human promyelocytic leukemia cell line HL-60 is induced to differentiate along a myelocytic pathway by dibutyryl cyclic adenosine monophosphate (dbcAMP). Other cAMP analogs are ineffective as inducing agents. The effect of these compounds on expression of c-myc was investigated using a DNA probe for c-myc to detect RNA transcripts. The dose response and time to commitment for reduction in c-myc expression with dbcAMP was similar to the findings for phenotypic changes. Bromo-cyclic AMP and butyrate alone caused no changes in c-myc expression in 24 hours, but demonstrated dramatic synergism together, suggesting that butyrate contributes in part to the effects of dbcAMP. Evidence for mechanisms of action of cAMP other than activation of the cAMP-dependent protein kinase is reviewed. PMID- 3015284 TI - Platelet membrane alterations induced by the local anesthetic dibucaine. AB - Tertiary amine local anesthetics modify a variety of platelet membrane-related functions. The present study explored dibucaine (DB)-induced inhibition of platelet cohesion by examining structural and functional alterations of the human platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) and platelet Ca2+ homeostasis. Complete inhibition of ADP-induced aggregation was achieved five minutes after platelet exposure to 0.10 to 0.25 mmol/L of DB when fibrinogen binding was reduced by 50%. At higher concentrations of DB (approximately 1 mmol/L), ADP-induced fibrinogen binding was completely blocked. Scatchard analysis revealed loss of high-affinity binding sites in addition to reduction in Bmax. In contrast, chymotrypsin-treated platelets sustained 50% inhibition of fibrinogen binding when incubated with 0.4 to 0.5 mmol/L DB, and kinetic analysis showed that the high-affinity platelet-fibrinogen interactions were reduced but not absent. Fibrinogen binding to chymotrypsin-treated platelets could not be completely inhibited even at high DB concentrations (1 mmol/L). The inhibition of fibrinogen binding to chymotrypsin-treated platelets correlated with changes in binding of a monoclonal antibody (10E5) specific for an epitope on the GPIIb-IIIa complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioelectroimmunoassay of DB-treated platelets, however, showed no evidence of a reduction or degradation of GP IIb or IIIa. Platelet incubation with DB (five minutes, 0.1 to 1.0 mmol/L) was also accompanied by: increased platelet membrane associated Ca2+ involving low-affinity binding sites [Kd = 5 X 10(-5) mol/L-]; increased 45Ca2+ uptake which correlated with degradation of actin-binding protein (ABP) and digestion of GPIb as visualized on periodic-acid Schiff (PAS) stained SDS gels and as inferred from decreased binding of a monoclonal antibody (6D1) directed against this glycoprotein; and enhanced Ca2+ exchange. Thus, exposure of platelets to DB results in membrane-related alterations that may contribute to inhibition of platelet cohesion: Decreased fibrinogen receptor exposure by traditional agonists and diminished accessibility of the GPIIb-IIIa complex to extracellular ligands correlate with DB-induced inhibition of platelet aggregation; and increased calcium uptake and exchange across the platelet membrane likely leads to activation of the calcium-dependent protease(s) which was previously shown to correlate with DB-induced inhibition of ristocetin induced platelet agglutination. PMID- 3015283 TI - Separation and analysis of subcellular organelles in a human promyelocytic leukemia cell line, HL-60: application to the study of myeloid lysosomal enzyme synthesis and processing. AB - We describe a system for analysis of the intracellular pathways in the biosynthesis and packaging of functionally important proteins in human myeloid cells. The human promyelocytic cell line HL-60 was used since peripheral blood neutrophils are terminally differentiated and do not actively synthesize protein. Cells were disrupted by nitrogen cavitation and subcellular organelles in postnuclear supernatant separated on a discontinuous gradient of Percoll modified to resolve organelles important in protein synthesis. This Percoll gradient separated azurophilic granules from less dense organelles and partially separated the less dense organelles from one another. Approximate densities of organelles identified by electron microscopy and by biochemical markers are azurophilic granules, 1.102 g/mL; endoplasmic reticulum, 1.039 g/mL; Golgi apparatus, 1.032 g/mL; and plasma membrane, 1.027 g/mL. We validated the utility of this method of subcellular fractionation by examining intracellular transport of myeloperoxidase, a myeloid lysosomal enzyme present in azurophilic granules. The subunits of mature myeloperoxidase (molecular weight [mol wt] = 59,000 and 13,500) cosediment with biochemical markers for lysosomes, whereas the large-mol wt (89,000) precursor forms cosediments with biochemical markers of less dense organelles. Within the limits of assay sensitivity, the 89,000-mol wt precursor is enzymatically inactive and has no spectral evidence for a heme group, suggesting that precursors of myeloperoxidase may undergo proteolytic maturation in a prelysosomal compartment with concomitant incorporation of a heme group and acquisition of enzymatic activity. This system of analysis should be suitable for the identification, subcellular localization, and maturational analysis of other myeloid lysosomal enzymes as well as functionally important membrane proteins. PMID- 3015285 TI - Monocyte nonspecific esterase: purification and subunit structure. AB - Monocyte nonspecific esterase has been purified from cultured cells of the acute myeloid leukemia cell line, ML-1. The purified enzyme shows the characteristic properties of the monocyte neutral serine carboxyl esterase, with high sensitivity to organophosphorus inhibitors and sodium fluoride inhibitor. The enzyme is a membrane protein which in the native state exists as a monomer of a mol wt of approximately 68,000 and a trimer of mol wt 205,000. These forms exhibit a complex pattern of dissociation and reassociation based on apparent noncovalent binding of subunits. The delipidated dissociated enzyme runs as a single protein chain of a mol wt of approximately 62,000 on sodium dodecyl sulfate (SDS) gel electrophoresis. The relation of the subunits to monocyte isoenzymes seen on isoelectric focusing (IEF) and polyacrylamide gel electrophoresis at pH 9.5 (pH 9.5 PAGE) of cell extracts is demonstrated. Availability of purified enzyme allows development of monoclonal antibodies and analysis of myeloid differentiation. In addition, the substrate specificity and function of the purified monocyte ectoenzyme are being examined. PMID- 3015286 TI - High resolution of heterogeneity among human neutrophil granules: physical, biochemical, and ultrastructural properties of isolated fractions. AB - Previous studies on the fractionation of human neutrophil granules have identified two major populations: myeloperoxidase (MPO)-containing azurophil, or primary, granules and MPO-deficient specific, or secondary, granules. Peripheral blood neutrophils from individual donors were lysed in sucrose-free media by either hypotonic shock or nitrogen cavitation. Using a novel two-gradient Percoll density centrifugation system, the granule-rich postnuclear supernatant was rapidly (ten minutes) and reproducibly resolved into 13 granule fractions (L1 through L8 and H1 through H5). Granule flotation and recentrifugation experiments on both continuous, self-generated and multiple-step gradients using individual and mixed isolated fractions demonstrated that the banding patterns were isopycnic and nonartifactual. Isolated granules were intact based on the findings that biochemical latency of several granule enzymes was greater than 95%, and thin-sectioned electron micrographs demonstrated intact granule profiles. Biochemical analyses of the granule marker proteins MPO, beta-glucuronidase, lysozyme, and lactoferrin indicated that a number of the fractions were related to the major azurophil and specific granule populations. Lactoferrin was found in ten of 13 fractions (L1 through L8, H1 to H2), whereas MPO was found in every fraction. Consistent with these biochemical data, all fractions exhibited varying degrees of heterogeneity based on ultrastructural morphology and cytochemistry, including diaminobenzidine (DAB) reactivity for peroxidase and periodate thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining for complex glycoconjugates. A variable but significant percentage (23% to 70%) of the granules in fractions L1 through L8 and H1 and H2 showed DAB reactivity, while about 90% of the granules in fractions H3 through H5 were peroxidase positive. These results demonstrated that DAB-reactive granules spanned the entire range of granule size and density. Ultrastructural PA-TCH-SP staining of isolated granule fractions revealed patterns similar to those of granules in intact neutrophils at different stages of development. Granules from human acute promyelocytic leukemia cells (HL-60) exhibited a surprisingly low density compared with typical azurophil granules from normal, mature neutrophils. The data suggest that both functional and maturational differences contribute to granule heterogeneity, and provide a new practical and conceptual framework for further defining the phenomenon of neutrophil granule heterogeneity. PMID- 3015287 TI - Acquired thrombasthenia due to GPIIb/IIIa-specific platelet autoantibodies. AB - An otherwise healthy woman developed a hemorrhagic diathesis with fluctuating clinical symptoms and laboratory findings, but without thrombocytopenia, over 8 years. In periods of bad clinical condition, a platelet defect, characteristic of thrombasthenia, was found. In contrast to classic thrombasthenia, electrophoresis of the patient's platelet membranes revealed normal amounts of glycoproteins IIb alpha, IIb beta, and IIIa in the normal positions. Monoclonal antibodies, specific for GPIIIa and GPIIb/IIIa, respectively, bound normally to the P1A1 positive platelets from the patient. Although no antibody and no platelet function inhibitor were evident in the autologous plasma, an IgG1 antibody that was bound to the patient's platelets and was directed against GPIIb/IIIa could be demonstrated. After elution from the patient's platelets, this antibody immunoprecipitated GPIIb (both subunits), IIIa, and a 200-kilodalton (kd) band (probably undissociated GPIIb/IIIa complex) from solubilized normal platelets, but did not react with thrombasthenic platelets. Adding the eluate from the patient's platelets to normal platelet-rich plasma immediately caused concentration-dependent inhibition of adenosine diphosphate (ADP)-induced and collagen-induced aggregation and also strong inhibition of ADP-stimulated fibrinogen binding. Because it was very unlikely from the patient's medical history that the antibody was caused by alloimmunization, the hemorrhagic diathesis must be interpreted as acquired thrombasthenia due to an anti GPIIb/IIIa autoantibody. PMID- 3015289 TI - [Metabolism of vitamin A and retinoids]. AB - Dietary vitamin A is stored in the liver as ester derivatives; after hydrolysis it is transported through the organism bound to a Retinol Binding Protein Prealbumin complex. Intracellular metabolism is complex and involves the binding to specific receptors for retinol (CRBP) and retinoic acid (CRABP) followed by a nuclear translocation. In addition, retinol can be phosphorylated and implicated in glycosylation processes. Retinoids are characterized by their action on cellular growth and differentiation. Such properties result in anticancer activities which have been clearly put in evidence in vitro and begin to be applied in human oncology. Nevertheless there is always a need for a better comprehension on fundamental mechanisms of natural or synthetic retinoids. PMID- 3015288 TI - No evidence of LAV infection in the Republic of Liberia, West Africa, in the year 1973. AB - Sera collected 13 years ago from 592 residents of the Republic of Liberia have been tested for antibodies to LAV polypeptides. 7 sera were positive by ELISA using two commercially available test kits whereas immunoblotting did not confirm antibodies specific for LAV. PMID- 3015291 TI - Difficult acetabular revision. A preliminary report. AB - Thirteen patients (14 hips) underwent revision from conventional cemented total hip arthroplasty to uncemented acetabular threaded screw-in components and cementless femoral press-fit stems by means of the Autophor, Biofit, and Ti Thread designs. The patients suffered from all forms of primary hip disease except rheumatoid arthritis; their average age was 34 years. The average preoperative Harris hip score was 39.4; the average postoperative score was 71.2 at six months, 90.6 at one year, and 91.6 at two years. Complications included two femoral shaft fractures, one femoral nerve palsy, and one dislocation. No signs have been observed of loosening or migration as of an early (6-24 months) follow-up. PMID- 3015290 TI - [Presence of free retinol receptor (cRBP) and retinoic acid receptor (cRABP) in human skin tumors]. AB - The concentrations of cellular retinoic acid binding protein (cRABP) and free cellular retinol binding protein (cRBP) were determined by ultracentrifugation in sucrose gradient in the cytosol of 41 human skin tumours (14 melanomas, 19 basal cell carcinomas, 8 squamous cell carcinomas). cRBP was found respectively in 36%, 42% and 37% of the studied samples. On the contrary, cRABP was more frequently found in carcinomas (89% in basal cell carcinomas and 100% in squamous cell carcinomas) than in melanomas (21%) (p less than 0.001). These results are discussed according to the different embryologic origin of carcinomas and melanomas. Furthermore, the better efficiency of synthetic retinoids in carcinomas than in melanomas should be explained by a different way of action in these 2 kinds of tumours. PMID- 3015292 TI - Incidence of physical and psychosocial disabilities in chronic pain patients: initial report. AB - Chronic pain is a leading health care problem with a wide range of physical and psychosocial outcomes. This report reviews the key intake findings of the first 227 patients admitted to a comprehensive inpatient/outpatient program for the treatment of chronic pain. Key trends in the data which suggest the different presentations of the chronic pain syndrome are highlighted and indications for future research are given. PMID- 3015294 TI - Combined ankle and subtalar instability. AB - Ipsilateral ankle and subtalar instability has been alluded to in the orthopaedic literature. A case demonstrating this combined instability pattern is presented and a technique for documenting this disorder is described. PMID- 3015293 TI - Spinal configuration during lifting. AB - The change in spinal configuration of the cervical, thoracic, and lumbar regions in relation to an amount of weight lifted was determined using videophotogrammetry. Fifteen healthy male subjects, 20-38 years of age, with no previous history of back pain participated in the study. The subjects lifted a crate containing 0, 10, and 20 kg weights using the straight-legs, bent-over-back method of lifting. The results showed that cervical and thoracic spinal segment configurations were not significantly influenced by the amount of weight lifted and that the mobility of the lumbar spinal segment was significantly decreased with increasing load (p = .03). PMID- 3015295 TI - Recurrent articular spondylolisthesis: common cause of vertebral instabilities, root pain, sciatica, and ultimately spinal stenosis. Early detection and blocking of specific dislocations. PMID- 3015296 TI - Elbow injury: a new imaging approach. AB - The authors describe a new technique for evaluating traumatic conditions to the elbow: the radial head-capitellum view. This projection has proved useful in demonstrating minimally displaced or nondisplaced fractures of the radial head, capitellum, and coronoid process. The radial head-capitellum view not only delineates the fracture, it also defines the extent of displacement. PMID- 3015297 TI - A pitfall in the insertion of a sliding screw. AB - This article describes the problem that can arise when an effort is made to insert a malaligned side plate during a sliding screw fixation. PMID- 3015298 TI - Accurate determination of limb length during total hip arthroplasty. AB - The authors present a method they have devised to measure limb length intraoperatively in total hip replacement using readily available operating room materials. PMID- 3015299 TI - Median nerve compression caused by a synovial cyst. AB - A case of median nerve compression at the wrist by a synovial cyst is described. Precise localization of the source of the synovial cyst was determined by surgical exploration and visualization of the carpal tunnel space. PMID- 3015300 TI - Iliopsoas recession through a medial approach. AB - A new method of performing iliopsoas recession in patients with cerebral palsy is described. The approach to the tendon is made by means of a medial incision. The method has been used on seven hips, and the results are comparable to those obtained by a formal anterior approach. PMID- 3015301 TI - The role of Na-hylan in reducing postsurgical tendon adhesions. AB - Na-hylan, a chemically modified sodium hyaluronate jelly, was studied mechanically and histologically as a surgical device to diminish tendon adhesion in the rabbit long toe extensor three weeks after surgical abrasion. This device was found to be highly effective (55% of treated tendons formed no adhesions compared to 5% of controls, and only 18% formed severe adhesions compared to 62% of controls). No gross or histologic evidence of significant acute or chronic inflammatory reaction to the Na-hylan was found. PMID- 3015302 TI - [Secretion of proteinase F from mouse salivary glands]. PMID- 3015303 TI - Rearrangement of mammalian chromatin structure following excision repair: absence of an effect of inhibitors of poly (ADP-ribose) polymerase and topoisomerase. AB - Newly-repaired DNA in chromatin is more sensitive to micrococcal nuclease than bulk DNA, but tends to become equally sensitive with time. This rearrangement of chromatin, which had previously been observed following repair of lesions produced by UV-light and of some bulky adducts, has now been shown to occur after repair of lesions induced by hydrogen peroxide and dimethylsulfate. In both cases there was an enhanced sensitivity to nuclease digestion of newly repaired DNA followed by a rearrangement whose kinetics was very similar to that observed in UV irradiated cells. Benzamide and 3-aminobenzamide, inhibitors of the synthesis of poly (ADP-ribose) and novobiocin, an inhibitor of topoisomerase, had no effect on the initially enhanced digestibility of repaired regions or on the chromatin rearrangement that followed. Poly (ADP-ribose) polymerase and topoisomerase are known to play some role in excision repair and have also been shown to cause alterations in the chromatin structure. However, the present results show that these alterations are not involved in this kind of chromatin rearrangement. PMID- 3015305 TI - Pentavalent antimonial inhibition of the osmotic effect of oxytocin on the isolated toad bladder. AB - The inhibition of osmotic stimulated water flow in the isolated toad bladder by 0.1 mM sodium stibogluconate (pentavalent antimony) is described. Pentavalent antimony on the serosal surface significantly inhibited oxytocin-induced water flow but when the drug was added to the mucosal surface only the effect of low oxytocin concentrations was reduced. The phosphodiesterase inhibitor pentoxifylline, when present on the serosal side, blocks the effect of pentavalent antimony on stimulated water flow. No effect was detected when indomethacin was present on the serosal side. It is suggested that phosphodiesterase activation might play a role in the mechanism of pentavalent antimonial inhibition of oxytocin-induced water flow in the isolated toad bladder. The effect obtained when sodium stibogluconate on the serosal side was replaced with antimony pentachloride (SbCl5) supports the view that the metal in the molecule is responsible for the inhibition of the effect of oxytocin. PMID- 3015306 TI - [Physiology of the growth hormone in pregnancy and discovery of a human placental growth hormone]. PMID- 3015307 TI - Diabetic ketoacidosis. AB - Diabetic ketoacidosis remains a threat to the survival of an insulin-dependent diabetic. Despite improvements in the management of the condition, mortality is still substantial. In this review, we will discuss approaches to the management of the condition and ways in which incidence, as well as case fatality, might be reduced. PMID- 3015304 TI - Effect of endogenous pyrogen, corticosteroids and inhibitors of prostaglandins and leukotrienes on the plasma concentrations of haptoglobin and fibrinogen in rats. AB - Injections of endogenous pyrogen (EP) in normal rats significantly increased plasma concentration of haptoglobin and fibrinogen while in adrenalectomized animals the same treatment was ineffective. Nevertheless when cortisone was injected simultaneously with EP into adrenalectomized rats the responses of fibrinogen and haptoglobin were restored. These results suggest that biosynthesis of fibrinogen and haptoglobin is corticosteroid-dependent. Inhibition of the cyclo-oxygenase pathway with ibuprofen or the lipoxygenase pathway with BW755C had no effect on the increase of plasma fibrinogen or haptoglobin levels stimulated by either EP or endotoxin. These data indicate that neither prostaglandins nor leukotrienes are involved in the production of these acute phase reactants induced by either stimulus. PMID- 3015308 TI - Metabolic acidosis. AB - Metabolic acidosis is the most frequent acid-base abnormality observed in the critically ill. Although there are many different causes, in the absence of ketosis and renal failure lactic acidosis is the most likely underlying disturbance. Controversy continues to surround the relative roles of the liver and the kidneys in the control of acid-base balance. There is no consensus concerning the use of bicarbonate in the treatment of a life-threatening metabolic acidosis. PMID- 3015309 TI - Observations on dermal blood flow as reflected by technetium-99m pertechnetate clearance. AB - Skin blood flow as reflected by the clearance rate of intradermal injections of Technetium-99m (99mTc) was studied in the skin of human volunteers and in a variety of clinical situations. In volunteers comparisons between the effect on clearance following intradermal injections of either lignocaine or lignocaine/adrenaline showed that the latter resulted in a dramatic reduction in the clearance rate of the technetium isotope and that such clearance curves exhibited no fast component. Clinical studies in grossly ischaemic skin revealed a similar absence of the fast component of isotope clearance and this was also the case for healed split thickness skin grafts. The dermal isotope clearance was also used in pedicle skin flaps both before and after clamping the axial pedicle in order to derive clearance ratios which would reflect the degree of neovascularisation to the flap. These preliminary observations suggest that the technique is safe, and easy to use in many clinical circumstances. We consider that it offers a valuable method by which changes in dermal blood flow may be quantified, when simultaneous control areas of isotope clearance are also studied. Suggestions are made for further avenues of study. PMID- 3015310 TI - Isothiouronium compounds as gamma-aminobutyric acid agonists. AB - Analogues of gamma-aminobutyric acid (GABA) incorporating an isothiouronium salt as a replacement for a protonated amino functional group have been investigated for activity on: GABA receptors in the guinea-pig ileum; [3H]-GABA and [3H] diazepam binding to rat brain membranes; and GABA uptake and transamination. For the homologous series of omega-isothiouronium alkanoic acids, maximum GABA mimetic activity was found at 3-[(aminoiminomethyl)thio]propanoic acid. Introduction of unsaturation into this compound gave two isomeric conformationally restricted analogues. The trans isomer was inactive at GABA receptors while the cis compound ((Z)-3-[(aminoiminomethyl)thio]prop-2-enoic acid (ZAPA)) was more potent than muscimol and GABA as a GABA agonist with respect to low affinity GABA receptor sites. Both isomers were moderately potent at inhibiting the uptake of [3H]-GABA into rat brain slices. Comparison of possible conformations of the two unsaturated isomers by interactive computer graphics modelling and comparison with muscimol has led to a plausible active conformation of ZAPA, which may be a selective and potent agonist for low affinity GABA binding sites. PMID- 3015311 TI - The sulphoxide moiety of substituted benzimidazoles is essential for inhibition of parietal cell K+/H+-ATPase. AB - The antisecretory action of the benzimidazole sulphoxide derivative B 823-10, 2[(4-methoxy-3-methyl-2-pyridylmethyl)-sulphinyl]- 5-trifluoromethyl(1H) benzimidazole, was compared with the effect of the corresponding sulphide B 823 08 in several in vivo and in vitro and in vitro test systems. The sulphide B 823 08 and the sulphoxide B 823-10 were found to be equipotent in the Shay rat. The sulphide was found to inhibit H+ secretion in intact rabbit gastric glands and enriched guinea-pig parietal cells with lower potency than the corresponding sulphoxide. The relative potency in antisecretory activity (sulphide/sulphoxide) decreased in the following rank order: Shay rat: gastric glands: parietal cells. Purified K+/H+-ATPase was not blocked by the sulphide, whereas the sulphoxide inhibited the overall as well as the partial reactions of this enzyme. In all in vitro systems tested, inhibition of H+ secretion and enzyme activity by the sulphoxide, but not by the sulphide, was antagonized by SH-compounds such as dithiothreitol. It is concluded that in vivo sulphoxidation of the sulphide plays an important role in acid inhibition. In vitro an additional inhibitory mechanism of the sulphide has to be considered. PMID- 3015313 TI - The demonstration of tumours of the parapharyngeal space by magnetic resonance imaging. AB - Seven patients with parapharyngeal neck masses have been investigated by magnetic resonance imaging (MRI). These presented a wide spectrum of the tumours commonly found in this location. Magnetic resonance imaging has been shown to be superior to computed tomography in the investigation of these patients. The advantages include: better delineation of the tumour in three planes and the extent of its involvement in the head and neck, the demonstration of neck vessels without intravenous contrast, the demonstration of the vascular nature of the mass, and better soft-tissue demonstration of skull base involvement. It is concluded that CT is no longer necessary as a routine procedure for the investigation of parapharyngeal tumours when MRI is available. PMID- 3015314 TI - ABC of Resuscitation: use of sodium bicarbonate. PMID- 3015312 TI - Inhibition of neutrophil activation by p-bromophenacyl bromide and its effects on phospholipase A2. AB - In an effort to elucidate the nature of the inhibitory effects of p-bromophenacyl bromide (pBPB) on neutrophil stimulation, we have examined its effects on several stages of stimulus-response coupling. Pretreatment of rat neutrophils with pBPB resulted in a dose- and time-dependent irreversible inhibition of both N formylmethionyl-leucylphenylalanine (fMet-Leu-Phe)-induced lysosomal enzyme release and change in transmembrane potential. Inhibition of the biological responses to the chemotactic peptide fMet-Leu-Phe was not due to receptor inactivation since fMet-Leu-[3H]-Phe binding to the formyl peptide receptor was not significantly altered by pBPB pretreatment. Inhibition by pBPB of phorbol myristate acetate (PMA)-induced changes in transmembrane potential and the generation of superoxide (0-2) was also observed. pBPB treatment appeared to inhibit activation of the NADPH oxidase without a direct effect on the oxidase itself. This inhibitory effect was not accompanied by cell death or decrease in cellular titratable sulphydryl groups (at least at doses less than 20 microM). There was, however, significant inhibition of a membranous fraction of fMet-Leu Phe-induced phospholipase A2 activity by pretreatment with 10 microM pBPB, although total cellular phospholipase A2 was only minimally (less than 20% inhibition) affected. These data would indicate that pBPB inhibits an early event associated with stimulus-response coupling in rat polymorphonuclear leukocytes (i.e. change in transmembrane potential). The inhibitory effects of pBPB may be secondary to the inhibition of a critical membranous fraction of cell bound phospholipase A2 activity or its activation, necessary for the initiation of cell activation. PMID- 3015315 TI - Left brain, retrotransposons, and schizophrenia. PMID- 3015316 TI - DNA sequences of human papillomavirus types 11, 16, and 18 in lesions of the uterine cervix in the west of Scotland. AB - Punch biopsy specimens of the cervix were examined both histologically and for the presence of human papillomavirus (HPV) DNA sequences. The presence of HPV DNA sequences was sought with the Southern blot technique using radioactively labelled HPV-6, 11, 16, and 18 DNA probes, both together and separately. Twenty six biopsy specimens were examined. Histological examination showed cervical intraepithelial neoplasia grade 2 or 3 in 16 specimens, viral changes (koilocytosis) in four, and inflammation or a normal appearance in three. Eleven specimens were negative for HPV DNA sequences, 10 contained HPV-16 DNA, four contained HPV-18 DNA, and one contained both HPV-18 and HPV-11 DNA. Episomal HPV 16 DNA was detected in one case of cervical intraepithelial neoplasia grade 3 and in five cases of cervical intraepithelial neoplasia grade 2/3 with koilocytosis; and episomal HPV-18 DNA was found in two specimens classed as cervical intraepithelial neoplasia grade 2/3, one of which also contained HPV-11 DNA, and in one specimen that showed viral changes alone. Integrated HPV DNA was found in six specimens (four with HPV-16 DNA and two with HPV-18 DNA), including two cases of chronically inflamed cervix with no histological evidence of viral infection or cervical intraepithelial neoplasia. Detection of viral DNA in early lesions may identify patients at risk of malignant progression. This is the first report of HPV-18 DNA in cervical intraepithelial neoplasia in Scotland. PMID- 3015317 TI - Cytomegalovirus but not human T lymphotropic virus type III/lymphadenopathy associated virus detected by in situ hybridisation in retinal lesions in patients with the acquired immune deficiency syndrome. AB - Paraffin sections of retinal tissue from five patients who died from the acquired immune deficiency syndrome (AIDS) and retinopathy were examined by in situ hybridisation experiments with deoxyribonucleic acid (DNA) labelled with sulphur 35 of lentivirus, human T lymphotropic virus type III/lymphadenopathy associated virus (HTLV-III/LAV), and cytomegalovirus. HTLV-III/LAV ribonucleic acid (RNA) was not detected in any of the tissue sections. Cytomegalovirus RNA was identified, however, in three of the five patients. Retinopathy induced by cytomegalovirus may thus be one of the many syndromes potentiated by the immunosuppression caused by HTLV-III/LAV. PMID- 3015318 TI - Neurological complications of coronary artery bypass graft surgery: six month follow-up study. AB - As part of a major prospective study of the neurological complications of coronary artery bypass graft surgery patients were reviewed over six months to determine the clinical course and functional impact of early postoperative complications. One hundred and ninety one out of 312 (61%) patients had developed early postoperative disorders. At six months 165 of the 191 patients with early neurological complications were reviewed. Of the 165, 85 still had detectable neurological signs, but these were often minor and of little functional importance. Only 10 patients had neurological disability at six months, and this was major in only four patients, all of whom had suffered major perioperative stroke. No patient with non-disabling neurological complications in hospital became functionally impaired on returning home. Neurological disorders are not a major cause of failure to return to work by six months after coronary artery bypass surgery. Of 139 patients who were of working age and had not returned to work by six months, only four were prevented by neurological injury related to surgery. The long term prognosis for early neurological disorders after coronary artery bypass surgery is usually favourable, except in those patients who have sustained major perioperative stroke. PMID- 3015319 TI - Retrovirus infections among patients treated in Britain with various clotting factors. AB - At the end of 1984 a collaborative survey was carried out to determine the prevalence of infection with human T cell lymphotropic virus type III/lymphadenopathy associated virus (HTLV-III/LAV) and HTLV-I among 584 recipients of various blood products in Britain at that time. In 204 cases yearly point prevalence figures for infection were also obtained for 1978 to 1983. In 1984, 215 of 315 patients (68%) who had received commercial concentrate for haemophilia A were identified as positive for anti-HTLV-III/LAV as compared with only 18 of 166 patients (11%) given British concentrate alone for this disease. This difference was further emphasised by the yearly point prevalence rates: seroconversion began in 1980 among recipients of commercial concentrate, but not until 1983 did such an instance occur among recipients of British concentrate. Any conclusions must remain speculative, but possibly seropositivity among haemophiliacs may not carry so grave a prognosis as previously thought. PMID- 3015320 TI - Vitamin B6 concentrations in patients with chronic liver disease and hepatocellular carcinoma. PMID- 3015321 TI - Paralytic poliomyelitis: a forgotten diagnosis? PMID- 3015322 TI - Oral contraceptives and hepatocellular carcinoma. PMID- 3015323 TI - AIDS and swimming pools. PMID- 3015324 TI - Insulinoma producing progressive neurological deterioration over 30 years. PMID- 3015325 TI - Pharmacology: analysis and exploration. PMID- 3015326 TI - Dilated cardiomyopathy, myocarditis, and the bioptome. PMID- 3015327 TI - Remodelling of nerve structure in experimental isoniazid neuropathy in the rat. AB - The neuropathy caused by a single dose of isoniazid in rats was studied with a computer-assisted morphometric method. Scatter diagrams of the g ratio (quotient fibre diameter/axon diameter) define regenerating fibres as a distinct population, distinguishable from the surviving fibres by reduced sheath thickness and reduced axon calibre. There was also evidence of a subtle direct toxic effect on the entire fibre population, causing axon shrinkage masked by readjustment of the myelin sheath. PMID- 3015328 TI - Catecholamine metabolism in the rat locus coeruleus as studied by in vivo differential pulse voltammetry. III. Evidence for the existence of an alpha 2 adrenergic tonic inhibition in behaving rats. AB - One of the various regulations controlling the noradrenergic (NA) locus coeruleus (LC) activity has been proved to be alpha 2 adrenergic specific, on the basis of electrophysiological data obtained in anesthetized preparations. To assess, under rigorously chronic conditions, the existence of such an inhibition, recordings of LC catechol metabolic activity were performed with in vivo differential pulse voltammetry. A guiding cannula and appropriate wires were implanted under anesthesia. After 48 h of recovery a carbon fiber electrode was threaded to the LC through this cannula to monitor the LC catechol oxidation current. Piperoxane 60 mg/kg i.p. and yohimbine 10 mg/kg i.p. induced an increase in catechol oxidation current to approximately 300% of baseline (100%) values. Graded doses of piperoxane (1-100 mg/kg i.p.) induced a dose dependent increase in LC catechol metabolic activity (ED50 = 29.7 mg/kg). These changes in catechol oxidation current were confirmed either by combined electrophysiological and electrochemical recordings in the LC of an anesthetized preparation, or by postmortem HPLC catechol determinations on LC microdissections. By contrast, guanfacine 1 mg/kg and clonidine (10-200 micrograms/kg i.p.) induced a dose dependent decrease in catechol peak height. Clonidine 50 micrograms/kg reversed the effect of piperoxane 30 mg/kg i.p. On the other hand, a highly selective alpha 1 antagonist, such as prazosin (1 mg/kg i.p.), evoked only a small increase in catechol peak (11% above saline effect). This data is consistent with previously reported electrophysiological, biochemical and autoradiographic data. They confirm the presence of a tonic alpha 2 adrenergic inhibition on NA-LC cell activity, in behaving rats. PMID- 3015330 TI - Quantitative distribution of angiotensin-converting enzyme (kininase II) in discrete areas of the rat brain by autoradiography with computerized microdensitometry. AB - We report the localization of angiotensin-converting enzyme (kininase II, EC 3.4.15.1) in discrete nuclei and areas of the rat brain by a quantitative autoradiographic technique using image processing coupled to computerized microdensitometry, after incubation of brain sections with the specific converting enzyme inhibitor [125I]351A. High angiotensin-converting enzyme levels are present in circumventricular organs (organon subfornicalis and area postrema), the choroid plexus, and extrapyramidal areas (nucleus caudatus, globus pallidus and substantia nigra) with intermediate levels in selected hypothalamic, septal, habenular and brainstem nuclei. Our results support the idea that angiotensin II could be formed in specific brain areas, both outside and inside the blood-brain barrier. In other brain structures, such as the extrapyramidal areas, kininase II could be involved in the processing or metabolism of other brain peptides. PMID- 3015329 TI - Long-term potentiation in the goldfish optic tectum. AB - Field potential recordings were made in the primary retinal synaptic area, the stratum fibrosum et griseum superficiale (SFGS), of an in vitro goldfish optic tectum preparation. Stimulation of the optic nerve at frequencies of 1 and 5 Hz produced a long-term potentiation (LTP) of the synaptic response which developed gradually. No potentiation was seen with lower or higher frequencies. These results demonstrate a significant LTP with a slow time course and restricted low frequency dependence. PMID- 3015331 TI - Functional organization of the spinal reflex pathways from forelimb afferents to hindlimb motoneurones in the cat. II. Conditions of the interneuronal connections. AB - The interneuronal conditions of the descending pathways from forelimb afferents to hindlimb motoneurones were investigated by testing spatial interactions in these pathways and between these pathways and segmental lumbar reflex pathways. In high spinal unanaesthetized cats hindlimb motoneurones were intracellularly recorded and spatial interactions were tested between effects evoked by stimulation of pairs of ipsi- and contralateral forelimb nerves or pairs of a forelimb and an ipsilateral hindlimb nerve. The excitatory and late inhibitory pathways from forelimb afferents projecting to most of the hindlimb motoneurone pools, showed an interactive pattern which was distinctly different to the fast inhibitory pathway projecting specifically from ipsilateral forelimb afferents to flexor digitorum and hallucis longus (FDHL) motoneurones. Stimulation of homonymous or heteronymous pairs of two forelimb nerves of both sides evoked generally a distinct spatial facilitation of the excitatory and late inhibitory effects, while the specific early IPSPs to FDHL motoneurones were not facilitated. Paired stimulation of two forelimb nerves of one side only produced spatial facilitation of EPSPs or late IPSPs if low strength stimuli were used, using higher strength which induced larger effects, generally caused occlusion instead. In case of large IPSPs this may be due to the vicinity to the equilibrium potential. Except for an inhibition of cutaneous reflex pathways, the spatial interaction of the excitatory and late inhibitory pathways onto segmental lumbar reflex pathways was weak and variable. The fast inhibitory pathway to FDHL motoneurones showed a partial spatial facilitatory interaction with lumbar reflex pathways from cutaneous and group II muscle afferents. The second IPSP wave evoked by this pathway was inhibited by antidromic stimulation of the ventral root L7S1 and of the alpha-efferents of the antagonistic peroneal nerve. From the results conclusions are drawn on the interneuronal organization of the descending pathways from forelimb afferents to hindlimb motoneurones. PMID- 3015332 TI - Perfusion with lithium modifies neurophysiological responses in the CA1 region of the hippocampal slice preparation. AB - The effects of acute lithium exposure on extracellular electrophysiological responses in the CA1 region of the in vitro hippocampus were investigated. Field potentials were assessed while perfusing slices with normal media or media in which LiCl was substituted for NaCl in 30, 20, 10 and 2 mM amounts. Lithium concentration in the slice following 20 min perfusion with 20 mM lithium was determined to be about 14 mM. At the higher concentrations, lithium exposure depressed the presynaptic fiber volley and antidromic population spike. On the other hand, the population EPSP and orthodromic population spike were enhanced. No significant changes were found at 2 mM. The findings are compatible with one action of lithium being on the excitability of axons and synaptic terminals. Comparisons were drawn between previous studies involving chronic lithium exposure and the present results. In this acute preparation lithium effects, as reflected in the population EPSP, were in opposition to those found with chronic lithium exposure. Changes demonstrated in this preparation in fiber volley and antidromic population spike paralleled those found with chronic lithium exposure. PMID- 3015334 TI - Autoradiographic localization of angiotensin receptors in the sheep brain. AB - Binding of [125I]-(Sar1,Ile8)angiotensin II (AII) to frozen sections of sheep brain was determined by in vitro autoradiography. Greatest AII-binding occurred in the organum vasculosum of the lamina terminalis, subfornical organ, median preoptic and periventricular nuclei situated in the anterior third ventricle wall. Other binding sites included the hypothalamic supraoptic and paraventricular nuclei and the medullary nucleus tractus solitarius. These regions may be central receptor sites for AII involvement in fluid and electrolyte balance and blood pressure regulation. PMID- 3015333 TI - Effects of pimozide and naloxone on latency for hypothalamically induced eating. AB - Latency to feed in response to lateral hypothalamic electrical stimulation was assessed at a variety of stimulation frequencies using moderate stimulation intensity; shorter latencies accompanied higher stimulation frequencies. Pimozide (0.0625, 0.125, 0.25 and 0.5 mg/kg, i.p.) and naloxone (0.5, 1.0, 2.0 and 4.0 mg/kg, i.p.) increased latencies in a dose-dependent manner. At 0.5 mg/kg (4 X the lowest effective dose), pimozide blocked eating completely for most trials and in all animals. Naloxone attenuated eating at 1.0 mg/kg, but 4.0 mg/kg was not more effective than 2.0 mg/kg and did not block the behavior completely in any animal. With each drug, latencies were normal or near-normal at the beginning of each session and anorexic effects developed with repeated testing. Thus neither drug caused a simple overall impairment in performance capability. In both cases it is inferred that the proximal food cues lost the ability to maintain eating well before the distal food cues lost the ability to elicit eating. The similarity between the effects of pimozide and naloxone raises the possibility that opioid-containing and dopamine-containing neurons interact to influence feeding through common brain circuitry. PMID- 3015336 TI - Rapid, stress-induced modification of the benzodiazepine receptor-coupled chloride ionophore. AB - Rapid changes in the chloride ionophore component of the benzodiazepine-GABA receptor complex were observed in cerebral cortical membranes from rats exposed to a brief, ambient temperature swim stress. These changes were manifest as: an increase in both the efficacy and potency of chloride ions to enhance [3H]flunitrazepam binding, and an increase in both the number of [35S]t butylbicyclophosphorothionate (a ligand that binds at or near the GABAA receptor gated chloride ionophore) binding sites and the apparent affinity of this radioligand. These studies demonstrate that the GABA-gated, benzodiazepine coupled chloride ionophore, which can be considered the effector component of this 'supramolecular complex', is rapidly modulated by acute stress. Such changes could represent the compensatory response of an organism to stressful or anxiety provoking changes in the environment. PMID- 3015335 TI - Norepinephrine-stimulated cyclic AMP accumulation in rat hypothalamic slices: effects of estrous cycle and ovarian steroids. AB - Hypothalamic slices prepared from female rats in various hormonal conditions were incubated in vitro in the presence or absence of 10 microM norepinephrine (NE), and cyclic AMP content was measured. Slices from animals in late diestrus or with exogenous estrogen treatment responded to NE with marked elevations of cyclic AMP in all but the posterior hypothalamus. Slices from rats sacrificed in late proestrus or following estrogen plus progestin administration showed little or no NE-stimulated cyclic AMP accumulation. PMID- 3015337 TI - Thermal and acute-phase protein responses of guinea pigs to intrapreoptic injections of leukotrienes. AB - Although it seems probable that intrahypothalamic prostaglandin (PG) E2, a cyclooxygenase metabolite of arachidonic acid, modulates interleukin-1 (IL1) induced fever, the evidence that it plays such a role is still only circumstantial; PGE2 does not, however, centrally mediate the fever-associated, acute-phase glycoprotein response. In this study, we investigated whether lipoxygenase products of arachidonic acid, viz. leukotriene (LT) B4, C4, D4 OR E4, injected intrapreoptically (2 ng/microliter, 1 microliter bilaterally) induces, like IL1, febrile and acute-phase glycoprotein responses in guinea pigs; controls received pyrogen-free saline, IL1 or PGE2. Measurements were: core temperature (Tco) and, as indices of acute-phase glycoproteins, plasma levels of copper (Cu) and protein-bound N-acetylneuraminic acid (NANA). Unlike IL1 or PGE2, no LT caused a febrile rise in Tco. Similar to PGE2 but unlike IL1, no LT produced increases in the plasma levels of Cu and NANA. These results indicate that intrapreoptic LTs probably are not involved in initiating the febrile or acute-phase glycoprotein responses characteristics of IL1. PMID- 3015338 TI - Electrophysiological properties of neurons in the caudal ventrolateral medulla projecting to the paraventricular nucleus of the hypothalamus in rats. AB - Extracellular recordings were made from neurons in the caudal ventrolateral medulla in urethane-chloralose-anesthetized rats. Stimulation of the paraventricular nucleus (PVN) in the hypothalamus evoked antidromic action potentials in 71 neurons. On the basis of antidromic spike latencies, these neurons could be divided into fast- (24 neurons) and slow-conducting cell groups (47 neurons). Slow-conducting cells showed irregular and slow spontaneous discharges, while a majority of the fast-conducting cells did not show spontaneous discharges. The spontaneous activity of slow-conducting cells was suppressed by i.v. clonidine administration. The effects of clonidine could be consistently reversed by administration of the alpha 2-adrenergic antagonist, yohimbine. The responses by clonidine and yohimbine remained unimpaired in baroreceptor-denervated rats. Vagus nerve stimulation produced an excitation in 80% of slow-conducting cells tested. Baroreceptor activation induced by i.v. administration of phenylephrine inhibited about half of slow-conducting cells tested. Similar elevation of blood pressure in baroreceptor-denervated rats did not show any effect. These physiological and pharmacological properties of slow conducting cells were similar to those previously reported for catecholaminergic cells in other parts of the brain. The results show the existence of two different populations among neurons in the caudal ventrolateral medulla which project directly to the PVN, and suggest that the presumed A1 catecholaminergic cells are involved in the afferent pathway from cardiovascular baroreceptors and the vagus nerve to the PVN. PMID- 3015339 TI - Calcium distribution in dendritic spines of the dentate fascia varies with age. AB - Calcium distribution in dendritic spines of the dentate fascia was studied as a function of age with the oxalate-pyroantimonate precipitation technique. In postnatal ages P3, P9, P24 and P30 spines were analyzed as to the presence of the spine apparatus (SA) and as to the presence of Ca2+ deposits within the SA and within the spine cytoplasm. The percentage of spines with SA-containing precipitates declined significantly between P3 and P24. Conversely, the percentage of spines with precipitates in the spine cytoplasm was significantly increased by P24. In the absence of an SA loss, this result suggests an age related decrease in the Ca2+-sequestering capacity by the SA. These parameters were improved by P30 so that they approximated the values of P3. Such a seeming amelioration could be attributed to the fact that the mortality rate in rats sharply increases by P24, so that animals surviving this age represent a selected population in which a compensatory growth of spines has occurred and has secured functionally valid connections. PMID- 3015340 TI - Vasoactive intestinal polypeptide increases inositol phospholipid breakdown in the rat superior cervical ganglion. AB - The effects of VIP and of peptides of the VIP family: secretin, glucagon, the porcine histidine isoleucine containing peptide (PHI) and the rat hypothalamic growth hormone-releasing hormone (rhGRF) on the cyclic AMP and inositol phosphate contents of isolated rat superior cervical ganglia were investigated. We demonstrate that VIP is able to provoke a large inositol lipid breakdown by acting directly on ganglionic cells. This observation suggests the presence in rat superior cervical ganglia of a new type of receptors for VIP or for an unidentified peptide structurally related to VIP. PMID- 3015341 TI - Catecholamines, insulin and ACTH increase quantal size at the frog neuromuscular junction. AB - The size of miniature end-plate potentials (m.e.p.p.s) and miniature end-plate currents (m.e.p.c.s) at frog neuromuscular junctions was increased by a factor of two or more following treatment with norepinephrine, epinephrine, a cAMP derivative, insulin or ACTH. The increase in size was reversible. These agents do not appear to alter the ACh noise spectrum or the reversal potential of the end plate. They do not alter the binding of alpha-bungarotoxin. Therefore they may increase the quantity of acetylcholine (ACh) in a quantum. PMID- 3015342 TI - Differential effect of adenosine on pre- and postsynaptic calcium fluxes. AB - In rat hippocampal slices, stimulus-evoked field potentials and the concomitant decrease of the extracellular concentration of free Ca ions [Ca2+]o were measured with combined reference/ion-sensitive microelectrodes. By reducing [Ca2+]o from 2.0 mM to 0.2 mM, evoked synaptic transmission was blocked, but orthodromic repetitive stimulation of CA1 afferents still elicited a marked decrease of [Ca2+]o. This Ca2+ signal is attributed predominantly to Ca2+ entry into the activated axon terminals. It was significantly depressed by adenosine. The adenosine agonist, L-phenylisopropyl adenosine (L-PIA) was more effective than D PIA, indicating that the adenosine depression of presynaptic Ca2+ entry is mediated via the A1 receptor. 4-Aminopyridine (4-AP) enhanced decreases in [Ca2+]o without restoring synaptic transmission. Adenosine depressed also these Ca2+ signals. Adenosine deaminase was even more effective in the presence of 4-AP and enhanced the orthodromic Ca2+-signal by a factor of two. Antidromic stimulation of hippocampal pyramidal cells also evoked reductions in [Ca2+]o. These were less affected by adenosine and the other treatments under the conditions tested. PMID- 3015343 TI - The effects of acute intraseptal injection of haloperidol in vivo on hippocampal cholinergic function in the mouse. AB - Acute injection of haloperidol into the lateral septum in mice produced an immediate and long-lasting increase in hippocampal sodium-dependent high-affinity choline uptake. Parallel electrophysiological investigations revealed that the increased septo-hippocampal cholinergic activity augmented CA1 pyramidal cell excitability and also accelerated the extinction of a conditioned reinforcement. These results constitute further evidence that septal dopaminergic terminals, via their control of septo-hippocampal cholinergic activity play a significant role in the modulation of hippocampal function. PMID- 3015345 TI - Intrastriatal N-methyl-D-aspartate prevents amygdala kindled seizures in rats. AB - Microinfusion of an excitatory amino acid, N-methyl-D-aspartate, into the ventral mid-striatum in the rat protects from amygdala kindled seizures. This result demonstrates that excitatory activity in the striatum modulates the threshold for seizures in the limbic forebrain. PMID- 3015344 TI - Noradrenergic modulation of hypothalamic progestin receptors in female guinea pigs is specific to the ventromedial nucleus. AB - The concentration of estrogen-induced cytosolic progestin receptors (CPRs) in the hypothalamus of alpha 1-noradrenergic antagonist-treated female guinea pigs is reduced relative to non-drug-treated controls. The present study determined where within the hypothalamus this reduction occurs. Ovariectomized, estradiol-treated guinea pigs were given either the alpha 1-noradrenergic antagonist prazosin or vehicle. Microdissected brain regions were assayed for CPR levels. Prazosin caused a selective relative decrease in CPR levels of the ventromedial nucleus of the hypothalamus. No significant effects of prazosin were seen in other hypothalamic or preoptic area nuclei. PMID- 3015346 TI - Curare can reverse the failure in muscle contraction caused by an AChE inhibitor. AB - Diisopropyl fluorophosphate, which is an organophosphate inhibitor of acetylcholinesterase, caused an irreversible block of neuromuscular transmission in the rat diaphragm preparation. This block could be reversed by the addition of a competitive antagonist of acetylcholine such as D-tubocurarine. PMID- 3015347 TI - Wheat germ agglutinin-ricin A-chain conjugate is neuronotoxic after vagal injection. AB - 'Suicide transport' is a term coined to describe the use of retrogradely axonally transported toxin to produce anatomically selective neural lesions. As a first step in developing neuron type-selective, systemically non-toxic suicide transport agents, a prototype hybrid toxin consisting of ricin A-chain (RTA) disulfide coupled to wheat germ agglutinin (WGA) was synthesized by first derivatizing WGA by reaction with N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) in the presence of N-acetylglucosamine and then formation of WGA-SS-RTA by mixing the derivatized WGA with reduced RTA. The ability of this conjugate to inhibit protein synthesis was tested on two cell lines in vitro; the ID50 was 0.2 nM using the K562 hematopoietic stem cell line and 0.02 nM for the 2a neuroblastoma cell line. Suicide transport activity was assessed by microinjection of hybrid into the cervical vagus nerve of rats. Intact WGA-SS RTA, but not hybrid that was pretreated with dithiothreitol to uncouple RTA from the WGA carrier, reliably killed vagal motor neurons. Both intact and reduced hybrid killed vagal sensory neurons. Indirect peroxidase immunohistochemistry demonstrated transport of RTA to vagal sensory neurons and WGA to both vagal sensory and motor neurons. These results are the first evidence that a hybrid toxin can be active as a suicide transport agent. PMID- 3015348 TI - The effects of moderate changes of extracellular K+ and Ca2+ on synaptic and neural function in the CA1 region of the hippocampal slice. AB - The effects of moderate changes of the concentration of ions on the function of mammalian central nervous tissue have not exactly been determined. We placed tissue slices from rat hippocampal formation in an interface chamber for study in vitro. Extracellular potentials were recorded in stratum radiatum and stratum pyramidale in response to stimuli of varying intensity applied to the Schaffer collateral bundle. The overall input-output relationship of excitatory synaptic transmission was gauged by expressing postsynaptic population spike amplitude as a function of presynaptic volley amplitude. The components of the transmission process were also examined by plotting the maximal rate of rise (slope) of the focally recorded synaptic potential (fEPSP) as a function of presynaptic volley amplitude, and the population spike amplitude as a function of the fEPSP slope. Raising the concentration of K+ from the normal level of 3.5 mM to 5 mM caused an average increase of 48% in the population spike evoked by a given presynaptic volley. This was due to an increased electrical excitability of pyramidal cells, as indicated by an increase of the population spike evoked by a given magnitude of fEPSP. Conversely, lowering [K+]o from 3.5 to 2 mM caused a decrease of the population spike relative to a given magnitude of either the presynaptic volley or the fEPSP. Changing [K+]o within these limits caused no significant change of the fEPSP evoked by a given presynaptic volley. Raising [Ca2+]o from 1.2 to 1.8 mM caused a 35% increase in both the fEPSP and the population spike evoked by a given presynaptic volley, and lowering [Ca2+]o to 0.8 mM caused a decrease of both these functions. The amplitude of the population spikes evoked by given fEPSPs changed surprisingly little (but consistently) when [Ca2+]o was varied within these limits. We conclude that moderate changes of [K+]o influence mainly the electric excitability of hippocampal pyramidal cells, with little effect on transmitter release or on the response of the postsynaptic membrane to transmitter, while moderate changes of [Ca2+]o affect the release of excitatory synaptic transmitter more than they affect postsynaptic membrane function. PMID- 3015350 TI - Acute effects of GM1 ganglioside: reduction in both behavioral asymmetry and loss of Na+, K+-ATPase after nigrostriatal transection. AB - GM1 ganglioside injections (i.p.) reduce amphetamine-induced asymmetric rotation in rats 48 h after a partial unilateral transection of the nigrostriatal pathway. We found that this reduction was maximal when rats received their first GM1 injection within 2 h after surgery. Rats injected 4-12 h after surgery, or rats only pretreated with GM1, showed no significant effect on rotation. Striatal membrane Na+,K+-ATPase in rats injected with GM1 0-2 h after hemitransection showed only a 10% loss in activity (versus the untransected hemisphere) as compared to control losses of 38%. The maintenance of membrane Na+,K+-ATPase activity in GM1-treated rats may be one mechanism by which a balance between hemispheres in striatal dopaminergic transmission is preserved, resulting in reduced asymmetric rotation. The observation that there is a critical postsurgical period when GM1 administration results in optimal functional recovery supports our hypothesis that gangliosides are exerting an acute effect on damaged CNS tissue. This acute effect is further evidenced by the reduced loss of membrane Na+,K+-ATPase following injury. PMID- 3015349 TI - Characterization of the forms of beta-endorphin and alpha-MSH in the caudal medulla of the rat and guinea pig. AB - The post-translational processing of pro-opiomelanocortin (POMC) in brain remains controversial. Classically, there was thought to be a single cell group in the arcuate nucleus with long projections through limbic structures. More recently, a second cell group was discovered in the caudal medulla. This study addresses the question of POMC processing in this region. Steady-state analysis of acid extracts of dorsal caudal medulla from rat and guinea pig CNS by gel filtration chromatography and radioimmunoassay indicated that in both species the major POMC related end products are alpha-MSH-sized material and beta-endorphin-sized. In this tissue beta-LPH and ACTH represent minor end products. Analysis of the alpha MSH-sized material from both species by reverse-phase HPLC indicated that in the rat caudal medulla approximately 79% of the alpha-MSH-related material is acetylated, and in the guinea pig caudal medulla approximately 85% of the alpha MSH-related material is acetylated. Analysis of the forms of beta-endorphin isolated from the rat caudal medulla by cation exchange chromatography revealed that acetylated and non-acetylated forms of beta-endorphin are present in this region of the rat CNS. Approximately 65% of the beta-endorphin in the rat caudal medulla is N-acetylated. Analysis of the forms of beta-endorphin isolated from the guinea pig caudal medulla indicated that approximately 63% of the beta endorphin is N-acetylated in this region of the guinea pig CNS. These data indicate that the post-translational processing of POMC in the dorsal caudal medulla, the site of the nucleus tractus solitarius POMC cell group, is distinct from the processing patterns that have been reported for POMC systems in the mammalian anterior pituitary, intermediate pituitary and arcuate nucleus. PMID- 3015351 TI - Stress-induced changes in the analgesic and thermic effects of opioid peptides in the rat. AB - Stress (e.g. restraint) potentiates analgesia and alters changes in body temperature induced by morphine administered either systemically or intracerebroventricularly (i.c.v.) in rats. In order to extend the generality of this phenomenon to opioid peptides, we determined whether the analgesic and thermic effects of i.c.v. D-Ala2-D-Leu5-enkephalin (DADLE) or D-Ala2-N-MePhe4 Gly5(ol)-enkephalin (DAGO), agonists selective for delta- and mu-opioid receptors, respectively, were affected by restraint stress. Analgesia was measured in the tail-flick test and core body temperature by rectal probe. The unstressed rats exhibited a dose-dependent increase in tail-flick latencies after administration of either DAGO or DADLE. Restrained rats treated with DAGO or DADLE had a greater analgesic response to each dose of peptide than did unstressed rats; both the magnitude and duration of the drug effect were increased. The unstressed group of rats responded to all doses of DAGO and DADLE with an increase of core temperature. In contrast, restrained rats showed a decrease of core temperature following injection with either DAGO or DADLE. Thus, restraint stress can significantly modify the effects of DAGO and DADLE on analgesia and body temperature in a manner that is qualitatively and quantitatively similar to that observed previously for morphine administered by the i.c.v. route. PMID- 3015352 TI - Beta-receptor involvement in locus coeruleus-induced inhibition of spinal trigeminal nucleus neurons: microiontophoretic and HRP studies. AB - Microiontophoretic and HRP studies were performed on cats anesthetized with alpha chloralose to determine whether or not the locus coeruleus (LC)- and noradrenaline (NA)-induced inhibition of relay neurons in the subnucleus oralis of the spinal trigeminal nucleus (STN) is mediated by beta-adrenergic receptors. The inhibition of orthodromic spike generation upon intracranial trigeminal nerve stimulation by LC conditioning stimulation and microiontophoretically applied NA (100-200 nA) was antagonized during microiontophoretic application of sotalol, a beta-adrenergic antagonist, but not affected by phentolamine, an alpha-adrenergic antagonist. When HRP at doses of 300-500 nA was applied for 5-15 min to the immediate vicinity of the STN relay or interneuron, which was electrophysiologically identified by stimulating the ipsilateral trigeminal nerve and contralateral medial lemniscus, the injection site was localized to an area 0.3 mm in diameter and HRP-reactive cells were found in the ipsilateral LC, dorsal raphe nucleus and periaqueductal gray ventral to the aqueduct. These results strongly suggest that NA released from the nerve terminals of LC cells inhibits transmission in the STN relay neuron via beta-adrenergic receptors. PMID- 3015353 TI - Potent depressant action of baclofen on hippocampal epileptiform activity in vitro: possible use in the treatment of epilepsy. AB - Effects of baclofen on the cortical electrical paroxysm were examined using slices of the guinea pig hippocampus. A very low concentration of baclofen (10( 8) M) readily suppressed spontaneous epileptiform discharges of CA3 pyramidal cells, whereas synaptic transmission between mossy fibers and CA3 pyramidal cells were not affected even by 10(-4) M of this drug. Such a strikingly selective depressant action of baclofen on the epileptiform activity raises a possibility that this drug may be effective in the treatment of epilepsy. PMID- 3015354 TI - Influence of neurons of the parafascicular region on neuronal transmission from perforant pathway through dentate gyrus. AB - We have previously reported that activation of an ascending brainstem pathway by stimulation of the median raphe nucleus (MR) influences neuronal transmission from the perforant pathway through the dentate gyrus in a behaviorally dependent manner. In particular, stimulation of the MR markedly facilitated such transmission when applied during slow-wave sleep (SWS), but was ineffective when applied during the still-alert state (SAL). We present here evidence for a relay in this circuit located rostral to the MR in cells proximal to the fasciculus retroflexus (PF, parafascicular region). In contrast to stimulation of the MR, stimulation of the PF facilitates neuronal transmission from the perforant pathway through the dentate gyrus during both SWS and SAL indicating the presence of a gate at or proximal to the PF that is preferentially closed during SAL. PMID- 3015356 TI - Phencyclidine-induced stereotyped behaviors after injection of ethylketocyclazocine, Mr 2266 and naltrexone in rats. AB - The effects of ethylketocyclazocine (EKC), Mr 2266 and naltrexone on the stereotyped behaviors induced by an intraperitoneal injection of phencyclidine (PCP) were examined. PCP-induced turning, backpedalling, head weaving and sniffing were antagonized by pretreatment with EKC (0.25-4.0 mg/kg). While pretreatment with Mr 2266 (2.5 mg/kg), a kappa selective antagonist, and naltrexone (10 mg/kg), a mu selective antagonist, failed to affect the PCP induced stereotypy, Mr 2266 antagonized the suppressing effect of EKC on PCP induced stereotypy. Taken into consideration, this suggests that kappa opioid agonists such as EKC antagonize PCP-induced stereotyped behaviors through a kappa opioid mechanism, and that the mu opioid receptor may not play an important role in the PCP-induced stereotypy in rats. PMID- 3015355 TI - Lesions of dorsolateral funiculi (DLF) do not affect the depressive effects of systemic morphine upon dorsal horn convergent neuronal activities related to pain in the rat. AB - It has been shown that morphine could interact with supraspinal inhibitory controls which modulate the transmission of nociceptive messages at the spinal level. However, the way in which such interactions occur is still a subject of controversy. Based on behavioural experiments, it has been proposed that systemic morphine increases descending inhibitory controls travelling via the dorsolateral funiculus (DLF). To directly test this hypothesis from an electrophysiological standpoint, we have investigated the effects of morphine upon C-fibre responses of dorsal horn convergent neurones in rats with bilateral lesions of the DLF. Previous studies by our group have shown that 6 mg/kg morphine chlorhydrate was the intravenous mean effective dose for depressing by 50% (ED50) the C-fibre evoked responses of convergent neurones recorded at the lumbar level. In the present work, the effects of 6 mg/kg morphine were investigated under identical experimental conditions, except that a bilateral destruction of the dorsolateral funiculus (DLF) was performed previously. These lesions did not change the mean C fibre evoked responses. Following morphine administration, the C-fibre evoked responses were depressed by 47.1 +/- 10.1% (n = 13) and reversal of these effects by naloxone was always observed. The A-fibre evoked responses, concurrently recorded, were not modified by the drug. As the depressive effects observed with this dose of morphine appear to be essentially of the same magnitude as those previously found in the intact or spinal preparations, we conclude that the DLF is not involved in the depressive effects of systemic morphine on dorsal horn convergent neurones. PMID- 3015357 TI - Phorbol ester promotes growth of synaptic plasticity. AB - Iontophoretic application of the phorbol ester 12-O-tetradecanoylphorbol-13 acetate (TPA) to the intact rat hippocampus enhances potentiation produced by subsequent high frequency stimulation of the perforant path. The decay of the enhanced population spike amplitude recorded in the hilar dentate gyrus was prevented in animals receiving ejections of TPA, as compared to controls which decayed to baseline values within 2 h following high frequency stimulation. In fact, growth of the potentiated response was observed beginning at 45 min. Similar results were observed with the population excitatory postsynaptic potential slope, a measure of the synaptic response. Since tumour-promoting phorbol esters are known to translocate and activate protein kinase C (PKC) and PKC is translocated to the membrane following hippocampal potentiation, a role for membrane-associated PKC in the regulation of synaptic plasticity is suggested. PMID- 3015358 TI - Selective acrylamide-induced degeneration of color opponent ganglion cells in macaques. AB - P beta (color opponent) retinal ganglion cells in macaques were found to degenerate as a result of oral administration of acrylamide. Histological examination, wheat germ agglutinin-horseradish peroxidase transport and cytochrome oxidase histochemistry indicate that other retinal ganglion cells and other neurons in the visual pathways were spared. PMID- 3015359 TI - Ultrastructural evidence of enkephalinergic input to glucoreceptor neurons in ventromedial hypothalamic nucleus. AB - A combination of intracellular injection of horseradish peroxidase into the ventromedial hypothalamic nucleus glucoreceptor neurons and immunohistochemical staining for enkephalin allowed ultrastructural visualization of interrelations between these electrophysiologically identified neurons and enkephalin-like immunoreactive (ENK-IR) terminals. The results show that glucoreceptor neurons receive direct inputs from ENK-IR neurons. This is the first evidence that glucoreceptor neurons, identified electrophysiologically, can be affected monosynaptically by endogenous enkephalin, probably released from ENK-IR terminals. PMID- 3015360 TI - Effect of low calcium and protease inhibitors on synapse elimination during postnatal development in the rat soleus muscle. AB - The mechanisms controlling the reorganisation of synaptic inputs to developing skeletal muscle fibres was studied using electrophysiological and histological methods. In the developing rat soleus muscle there is a rapid reduction of polyneuronal innervation between 9 and 12 days. Reducing the local concentration of calcium by applying chelating agents such as EGTA or BAPTA in vivo to 9-day old rat soleus muscles over a period of 3 days slowed the rate of elimination of polyneuronal innervation. It was established that the reduction of calcium induced by EGTA or BAPTA was not sufficient to produce a detectable reduction in neuromuscular activity. The possibility that a calcium-dependent enzyme such as CANP may play a role in synapse reorganisation was therefore tested. Local application of inhibitors of calcium-activated neutral protease (CANP), leupeptin or E-64, to 9-day-old rat soleus muscles over 3 days had similar effects to those of EGTA or BAPTA, i.e. the elimination of polyneuronal innervation that usually takes place was much slower. Since the inhibition of thiol proteases had similar effects on synapse elimination as a reduction of calcium concentration, it is concluded that CANP is important in the reorganisation of the developing neuromuscular junction. PMID- 3015361 TI - [Secretion of growth hormone, prolactin, ACTH and testosterone after blockade of opiate receptors during stress in humans and rats]. PMID- 3015362 TI - [Increased excretion of total cyclic adenosine 3',5'-monophosphate in patients with insulin-dependent diabetes mellitus]. PMID- 3015363 TI - Burns after care: a booklet for parents. Your child at home after injury. PMID- 3015365 TI - Calcitonin gene-related peptide (CGRP) acts independently of calcitonin on cyclic AMP formation in clonal osteogenic sarcoma cells (UMR 106-01). AB - In several human cancer cell lines and in a subclone of rat osteogenic sarcoma cells (UMR 106-06) possessing calcitonin receptors and a calcitonin-responsive adenylate cyclase, calcitonin gene-related peptide (CGRP) behaved as a weak calcitonin agonist. In another subclone of the same osteogenic sarcoma (UMR 106 01) with no measurable calcitonin receptors or response, both rat and human CGRP were found to increase cyclic AMP formation in a dose-dependent manner. The data indicate that CGRP is capable of a weak calcitonin-like action in cells with calcitonin receptors, but also that in some cells CGRP activates adenylate cyclase itself, independently of calcitonin receptors. PMID- 3015364 TI - Avian medullary bone in organ culture: effects of vitamin D metabolites on collagen synthesis. AB - A new organ culture system for the study of bone metabolism has been developed using chicken medullary bone. The presence of viable bone cells in culture was demonstrated by histological and histochemical techniques. Incorporation of 3H proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP) was determined using purified bacterial collagenase. Collagen accounted for approximately 10-15% of the total protein labeled. The addition of 1,25 dihydroxycholecalciferol (1,25(OH)2D3) resulted in a dose-dependent inhibition of 3H-proline incorporation into CDP at doses from 10(-10)M to 10(-7)M, with maximal suppression reaching 30% of control. The effect was specific for collagen, since 3H-proline incorporation into NCP was unaffected. Hydroxyproline analysis of bone explants and culture medium revealed a 1,25(OH)2D3-induced decrease in the 3H hydroxyproline content of the system (bone + medium), suggesting that the effect of 1,25(OH)2D3 is due to inhibition of collagen synthesis rather than enhanced collagen degradation, impaired incorporation of collagen into bone matrix, or bone resorption. Medullary bone collagen synthesis was not affected by 24,25(OH)2D3, either alone or in combination with 1,25(OH)2D3. Structure-activity studies of vitamin D metabolites showed that 1,25(OH)2D3 and 1,24,25(OH)3D3 were the most potent metabolites tested, followed by 1-alpha(OH)D3. 25(OH)D3 and 24,25(OH)2D3 had no effect at concentrations as high as 10(-7)M. These results indicate a possible role for vitamin D in the regulation of medullary bone formation during the reproductive cycle of the egg-laying hen, and suggest the potential utility of medullary bone as an in vitro model for the study of bone formation. PMID- 3015366 TI - Effects of nifedipine on hepatic blood volume in cats: indirect venoconstriction and absence of inhibition of postsynaptic alpha 2-adrenoceptor responses. AB - Intravenous administration of hypotensive doses (30-200 micrograms/kg) of nifedipine to cats anesthetized with pentobarbital caused an increase in cardiac output accompanied by hepatic venoconstriction. The hepatic venoconstriction and the increase in cardiac output were abolished in animals in which the hepatic sympathetic nerves were cut, the adrenal glands were excluded, and the kidneys were removed. This contrasts with the indirect hepatic venoconstrictor action of isoproterenol which was shown previously not to be abolished by these procedures. Further experiments showed that the hepatic venoconstrictor effect of nifedipine was blocked by removal of the kidneys, but not by removal of the hepatic sympathetic nerves and adrenals. These results support the hypothesis that venoconstriction plays an important role when drugs produce increased cardiac output. In nephrectomized animals, nifedipine had no direct effects on hepatic blood volume and it did not alter the effects of infusions of norepinephrine on hepatic blood volume, which have previously been shown to be mediated through alpha 2-adrenoceptors. However, it did reduce the hepatic venous responses to hepatic sympathetic nerve stimulation by 30%. PMID- 3015367 TI - Severe coughing during captopril and enalapril therapy. PMID- 3015368 TI - Prophylactic chemotherapy for hydatidiform mole. Five to 15 years follow-up. AB - The effectiveness of the prophylactic chemotherapy was evaluated in 420 patients with molar pregnancy. All patients were followed for 5 to 15 years after the evacuation. Twenty-two (7.5%) of 293 patients with prophylactic chemotherapy and 23 (18.1%) of 127 patients without prophylactic chemotherapy (control) developed secondary trophoblastic disease. The prophylactic chemotherapy could reduce the occurrence of secondary trophoblastic disease. In these secondary trophoblastic diseases, 5 (22.7%) of 22 patients in the prophylactic chemotherapy group and 5 (21.7%) of 23 in the control had metastatic trophoblastic disease. Choriocarcinoma after the molar pregnancy developed in two patients (0.7%) of the prophylactic chemotherapy group and two (1.6%) of the control. Prophylactic chemotherapy did not eliminate the occurrence of choriocarcinoma. The complication of the prophylactic chemotherapy was seen in 27.3% of the patients. Neither severe complication nor death were related to the toxicity. PMID- 3015369 TI - Adult T-cell leukemia-lymphoma. Unusual features of two patients from a low incidence area. AB - The occurrence of adult T-cell leukemia-lymphoma in two New Orleans patients, one native-born, the other originally from Honduras, is reported. Both exhibited an unusual feature. One patient was diagnosed following an atypical episode of sinusitis and demonstrated lymphomatous infiltration of the nasopharynx. A second patient had a fulminant course complicated by severe diarrhea and was found to have both tumor involvement and cytomegalovirus inclusions in the colon. Both had high titers of antibodies to HTLV-I. A cell line with T-lymphocyte characteristics was established from the peripheral blood of the first patient. Such studies may help better establish the clinical course, detection, and epidemiologic features of this difficult disease. PMID- 3015370 TI - Small cell carcinoma of the major salivary glands. AB - Small cell carcinoma is primarily a pulmonary neoplasm that rarely arises in extrapulmonic sites including salivary glands of the head and neck. Twelve cases of small cell carcinoma of salivary gland origin were retrieved from the Armed Forces Institute of Pathology files. Six tumors occurred in the parotid gland and six in the submandibular gland. Tumors were classified into two categories: those with areas of histologically typical small cell carcinoma (7 cases) and those with areas of typical small cell carcinoma with foci of ductal differentiation (5 cases). Follow-up information was available in all 12 cases. Electron microscopy was done on eight tumors; only one demonstrated round electron dense intracytoplasmic neurosecretory granules. These observations further support evidence in the literature suggesting most of the small cell carcinomas of salivary gland origin are not true neuroendocrine ("oat cell") carcinomas, but actually are small cell ductal carcinomas. These tumors appear to have a better prognosis than small cell carcinoma of the lung or nonsalivary gland sites in the head and neck region, with an estimated 2- and 5-year survival of 70 and 46%, respectively. PMID- 3015371 TI - Colorectal adenocarcinoma in young Lebanese adults. The American University of Beirut-Medical Center experience with 32 patients. AB - Colorectal adenocarcinoma is uncommon in Lebanon. The low frequency and the low average age at the time of diagnosis, 53.7 years, is similar to that observed in other developing countries. Over a period of 40 years (1945-1985), 32 patients (5.8%) developed colorectal adenocarcinoma before age 30 years. Seventeen and 15 patients were males and females, respectively (age range, 14-29 years). The most common presenting symptoms were blood per rectum (27 patients) and abdominal pain (23 patients). The average interval from the first symptom to histologic diagnosis was 5.7 months. The only significant predisposing factors were the presence of a positive family history for colorectal carcinoma in one patient and bladder exstrophy with ureteral diversion in another. Twenty-four patients had surgery with curative intent. Colloid and signet ring adenocarcinoma were present in 22 patients (68.7%). Classification by Duke's staging system demonstrated Stage C in 15 and Stage D in 5 patients. These findings show a definite increase in carcinoma with high histologic grade and advanced stage at presentation in young Lebanese patients. PMID- 3015372 TI - Cisplatin, etoposide, and mitomycin in the treatment of non-small cell carcinoma of the lung. A pilot study. AB - A total of 39 patients with non-small cell carcinoma of the lung (NSCL) were treated with cisplatin, etoposide, and mitomycin. A major response rate (complete response + partial response) was seen in 15 patients (39%). Median survival for all patients was 340 days; median survival of the responding group was 514 days. Toxic effects included moderate hematologic toxicity, nausea, and vomiting. There were no treatment-related deaths. This regimen clearly is effective in treating NSCL. PMID- 3015373 TI - Integration of hepatitis B virus DNA in hepatocellular carcinoma. AB - Integration of hepatitis B virus (HBV) DNA into genomic DNA was investigated in 34 livers bearing hepatocellular carcinoma (HCC) by Southern blot hybridization using 32P-labeled, cloned and purified HBV DNA as a probe. Rehybridization of nitrocellulose paper with a probe containing only the cloning vector was performed after dehybridization to avoid possible false-positive results. Integrated HBV DNA was detected in all 9 hepatitis B surface antigen (HBsAg) seropositive cases and 3 out of 25 (12%) HBsAg seronegative cases. The hybridization patterns of viral DNA were the same among several cancer nodules in two HCC cases with multiple liver tumors, indicating unicentric hepatocarcinogenesis in these two cases. These results, obtained with avoidance of false-positive results, showed that only a minority of HBsAg-seronegative HCC cases in Japan had demonstrable HBV DNA in the tumors studied by the Southern blot hybridization technique. PMID- 3015374 TI - Estrogen and progesterone receptors in human breast cancer. Correlation with histologic subtype and degree of differentiation. AB - Microscopic review of 490 consecutive human breast biopsy and mastectomy specimens were correlated with estrogen and progesterone receptor content of the tissue, by subtype and degree of differentiation. Of the 4 grades of differentiation, the less differentiated Grade III and IV tumors showed significantly lower levels of estrogen and progesterone receptors in infiltrating ductal and lobular carcinoma (P less than 0.001). In contrast, patients with medullary carcinoma had the lowest tissue levels of estrogen and progesterone receptors with approximately 80% of the cases with less than 10 fmol/mg protein. Patients with mucinous carcinoma had the highest percentages of positive estrogen and progesterone receptor levels (75% and 87%, respectively). Sixty-three percent of the patients with Grade IV infiltrating ductal carcinoma were younger than 53 years of age (P less than 0.001). Patients younger than 53 years of age with Grade II and III infiltrating ductal carcinoma also had significantly lower levels of estrogen receptors, but not of progesterone receptors, than those patients older than 53 years of age (P less than 0.001). Nineteen of 20 "normal" breast tissue specimens were negative (less than 3 fmol/mg protein) for estrogen and progesterone receptors. About 50% of 17 tissue specimens from benign breast lesions (fibroadenoma, fibrocystic disease, sclerosing adenosis) showed positive estrogen (greater than 10 fmol/mg protein) or progesterone receptor values. In two patients with gynecomastia, no estrogen or progesterone receptors were detectable. PMID- 3015375 TI - The search for the primary tumor in patients with skeletal metastases of unknown origin. AB - Forty-six patients who had been evaluated because of skeletal metastases of unknown origin, were reviewed. Twenty-six of the patients were referred to an orthopedic surgeon before confirmation of the metastases by biopsy; 20 others were referred to an oncology clinic after a diagnosis of bone metastases had been established. A simple diagnostic sequence consisting of a medical history, physical examination, routine laboratory studies, chest roentgenogram, technetium 99m phosphonate bone scintigram, and intravenous pyelogram identified the site of the primary tumor in 14 patients; 7 of the primaries were lung carcinomas, 4 were hypernephromas, 2 were breast carcinomas, and 1 was a prostate carcinoma. In two other patients, the histologic findings from the biopsy study were diagnostic; one had a thyroid carcinoma and one, a prostate carcinoma. Further extensive diagnostic workups revealed the site of origin in only four additional patients; two had hypernephromas which were discovered by computed axial tomography of the abdomen; one had an ovarian carcinoma and one had a hepatoma, both of which were found at laparotomy. On the basis of this study, a simple diagnostic strategy is recommended for patients with histopathologically confirmed skeletal metastases of unknown origin: medical history, physical examination, routine laboratory studies, chest radiograph, and technetium 99m phosphonate bone scintigram, followed by computed axial tomographic examination of the abdomen and pelvis. In female patients, it may be judicious to use mammography. If this regimen fails to reveal the primary site, it is unlikely that it will be identified with further extensive diagnostic procedures. PMID- 3015376 TI - 6q- and loss of the Y chromosome--two common deviations in malignant human salivary gland tumors. AB - Nine cases of malignant human salivary gland tumors cultured in vitro were subjected to detailed cytogenetic analysis with G-banding. Together with observations from three earlier published cases, the results of 12 cases were surveyed: five adenoid cystic carcinomas, three acinic cell tumors, three adenocarcinomas, and one mucoepidermoid carcinoma. All tumors had stemlines in the diploid-near-diploid mode. The most consistent changes among the adenoid cystic carcinomas were stem lines and/or variant cells with anomalies affecting the terminal part of 6q (i.e., 6q 16-25). Deviations affecting the Y chromosome (losses) and, to a lesser extent, #6 (structural changes) and #8 (gains) characterized the early karyotypic evolution in acinic cell tumors. Two of the three analyzed adenocarcinomas showed stemlines or variant cells with loss of gonosomes. The karyotypic features of the different tumor types, including primary changes, evolutionary characteristics, and progressional pathways, are discussed. The cytogenetic relationships between benign and malignant salivary gland tumors also will be considered. PMID- 3015377 TI - DNA breakage in human lung carcinoma cells and nuclei that are naturally sensitive or resistant to etoposide and teniposide. AB - Evidence suggests that the anticancer agents etoposide (VP16-213) and teniposide (VM26) produce DNA breaks and cytotoxicity by interaction with type II topoisomerase. Therefore, levels of type II topoisomerase may influence sensitivity to VP16-213 and VM26. We have characterized four lung carcinoma derived cell lines for natural sensitivity or resistance to VP16-213 and VM26. Included in this study were two small cell lung carcinoma lines (SW900 and SW1271), an adenocarcinoma line (A549), and a large cell carcinoma (H157). SW1271 was the most sensitive line with a median inhibitory concentration for cell proliferation of 0.5 microM for VM26 and 2.7 microM for VP16-213, and SW900 was the most resistant with median inhibitory concentration values of 2.0 and 16 microM, respectively. A549 and H157 cells were intermediate in sensitivity to these drugs. Alkaline elution techniques were used to study in vivo formation and repair of single and double strand DNA breaks. Single strand DNA breaks were observed in SW1271 cells exposed to as little as 10 nM VM26 or 100 nM VP16-213 for 1 h, whereas SW900 cells required exposure to 10-fold higher concentrations of VM26 or VP16-213 to produce similar results. Single strand DNA breaks predominated only in SW1271 and A549 cells and then, only at low drug concentrations, whereas the ratios between single and double strand DNA breaks decreased at higher drug concentrations. Plots of cytotoxicity versus single and double strand DNA breakage revealed that cytotoxicity produced by both drugs was more closely related to double strand DNA break formation in all four cell lines. DNA breaks appeared rapidly upon addition of drug, reaching plateaus in DNA breaks within 30 min, and repair of both single and double strand DNA breaks occurred rapidly with time to repair one-half of the DNA breaks of 20 to 60 min in all four cell lines upon removal of drug, arguing against repair as a mechanism for drug resistance. DNA breakage was also observed in nuclei isolated from SW900 and SW1271 cells in similar magnitude to that observed in the respective cells. Results indicate that DNA breakage plateaus may reflect a steady-state equilibrium established between the drug and its nuclear target, possibly type II topoisomerase, and suggest that natural resistance to VP16-213 and VM26 may be due to different enzyme levels in sensitive and naturally resistant cells. PMID- 3015378 TI - DNA damage and cytotoxicity of 2-chloroethyl (methylsulfonyl)methanesulfonate (NSC 338947) produced in human colon carcinoma cells with or without methylating agent pretreatment. AB - 2-Chloroethyl (methylsulfonyl)methanesulfonate (ClEtSoSo) was more toxic to the BE (Mer-) cell line than to the HT-29 (Mer+) colon carcinoma. The sensitivity of the BE cells closely paralleled the induction of DNA interstrand cross-links by ClEtSoSo. No DNA interstrand crosslink formation was detected in the HT-29 cells after exposure to ClEtSoSo. Pretreatment of the HT-29 cells with methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine or streptozotocin increases their sensitivity to ClEtSoSo. Little or no increase in the toxicity of ClEtSoSo was found in BE cells after methylating agent pretreatment. Despite the increase in cell killing, no DNA interstrand cross-links were induced by ClEtSoSo after N methyl-N'-nitro-N-nitrosoguanidine pretreatment. In contrast, streptozotocin pretreatment allowed ClEtSoSo to form DNA interstrand cross-links in HT-29 cells. The production of DNA strand breaks by ClEtSoSo was observed in HT-29 cells both with and without methylating agent pretreatment. These results suggest that the mechanism of ClEtSoSo may differ from other chloroethylating agents such as the chloroethylnitrosoureas. In addition, there may be a difference in the mechanism by which streptozotocin or N-methyl-N'-nitro-N-nitrosoguanidine pretreatment causes an increased cell killing in a previously resistant human colon carcinoma cell line. PMID- 3015379 TI - Modulation of etoposide cytotoxicity and DNA strand scission in L1210 and 8226 cells by polyamines. AB - The anticancer agent etoposide (VP-16) produces DNA strand scission in intact tumor cells or isolated nuclei. This activity may be mediated by topoisomerase II, an enzyme capable of producing double strand breaks in mammalian cells. Two established tumor cell lines were examined to see whether polyamines, which alter DNA conformation and topoisomerase II activities, affected the cytotoxicity, strand scission, and antitumor efficacy of VP-16. L1210 murine leukemia and 8226 human myeloma cells were treated with alpha-difluoromethylornithine (DFMO) to reduce intracellular polyamine levels via inhibition of ornithine decarboxylase. The polyamines putrescine and spermidine were markedly reduced by a 48-h incubation with 50 microM DFMO. This DFMO concentration did not inhibit colony formation in either cell line, but did reduce the growth rate of both cultures. In contrast, VP-16 produced a dose-dependent inhibition of colony formation. This was especially marked in the 8226 cell line. This correlated with DNA single strand breaks (SSBs) detected by the alkaline elution technique. When cells previously treated with DFMO were exposed to VP-16, a synergistic inhibition of colony formation (determined by isobologram analysis) was observed. However, VP 16-induced SSBs were only marginally increased by the DFMO pretreatment. When putrescine was combined concurrently with VP-16, both the in vitro cytotoxic effects and the number of DNA SSBs in L1210 cells were significantly reduced. These results demonstrate that putrescine inhibits VP-16-induced SSBs and commensurate cytotoxic effects, while DFMO, which depletes intracellular putrescine and partially reduces intracellular spermidine, acts to produce synergistic cytotoxic effects when combined with VP-16. PMID- 3015380 TI - Effect of triamcinolone acetonide on the growth of NEL-M1 human melanoma cells cultured in the presence and absence of growth stimulatory agents. AB - The human melanoma cell line NEL-M1 proliferates in Ham's F-12 medium in the absence of serum, hormones, and growth factors. Culturing NEL-M1 cells in defined medium supplemented with insulin (5 micrograms/ml) and transferrin (5 micrograms/ml) results in a 94% increase in [3H]thymidine incorporation after 24 h and a 6- to 8-fold increase in cell number after 5 days. The addition of 17 beta-estradiol, testosterone, and progesterone to defined medium, either as single agents or in combination with insulin and transferrin, had no effect on cell growth. No specific estrogen, androgen, or progesterone receptor proteins were detected in NEL-M1 cells. In contrast, the synthetic glucocorticoid triamcinolone acetonide (10 nM) inhibited growth of NEL-M1 cells in serum-free Ham's F-12 medium by 50%. The 6- to 8-fold stimulation of cell growth by insulin and transferrin was reduced to 1.75-fold when triamcinolone acetonide (10 nM) was added to the medium. Additional studies show that medium conditioned by NEL-M1 cells stimulated [3H]thymidine incorporation into cellular DNA of NEL-M1 cells and increased the growth of NEL-M1 cells 3-fold over cells maintained in defined medium. The addition of triamcinolone acetonide (10 nM) to defined medium supplemented with conditioned medium resulted in only a 1.48-fold increase in cell number. These results show that triamcinolone acetonide inhibits growth of NEL-M1 human melanoma cells in serum-free defined medium, medium supplemented with insulin and transferrin, and medium supplemented with an endogenous growth stimulatory factor(s). Thus, NEL-M1 cells are an excellent model system to study the mechanism of action of glucocorticoids and also the interplay between exogenous hormones and growth factors and endogenous growth factors. PMID- 3015381 TI - Transformation of rat hepatocytes by transfection with simian virus 40 DNA to yield proliferating differentiated cells. AB - Cultured hepatocytes from adult Fischer 344 rats were transformed by virion or cloned simian virus 40 (SV40) DNA using the calcium phosphate method. Transformation by SV40 occurred in either serum-supplemented medium or chemically defined medium (CDM). The frequency was greatest in serum-supplemented medium but transformants did not remain differentiated. In contrast, SV40 transformants developed less frequently in CDM, but retained differentiated functions. The frequency of transformation was enhanced by treatments that stimulated cell proliferation, in particular supplementing CDM with epidermal growth factor. Hepatocytes transformed in CDM were epithelial in morphology, secreted albumin, transferrin, hemopexin, and expressed the enzyme glucose-6-phosphatase, all characteristics of normal liver. Transformants did not produce detectable levels of alpha-fetoprotein, a marker of fetal or abnormal liver. We conclude that (a) hepatocytes can be transformed by transfection with SV40 DNA; (b) the frequency of transformation is enhanced by stimulating DNA synthesis; and (c) the transformed cells retain specific functions of normal hepatocytes in situ. Using this system it will be possible to study transformation of hepatocytes by viral and cellular oncogenes and to determine their effects on hepatocellular differentiation. PMID- 3015382 TI - Mouse 3T3-L1 cell variants unable to respond to mitogenic stimulation of dihydroteleocidin B: genetic evidence for the synergism of tumor promoters with growth factors. AB - Dihydroteleocidin B, an indole alkaloid tumor promoter, stimulates confluent, quiescent mouse 3T3-L1 fibroblasts to initiate DNA synthesis and undergo cell division. Using a mitotic shakeoff technique, we have isolated 12 clones of genetic variants which are unable to respond to the mitogenic stimulation of dihydroteleocidin B from a total of 12 million cells. Biochemical characterization of these nonresponsive variants to dihydroteleocidin B revealed that there is no change in the ability to bind [3H]phorbol dibutyrate, the activity of protein kinase C, and the turnover of phosphatidylinositol. The evidence indicates that nonresponsiveness to dihydroteleocidin B is caused by several different lesions, including defects in receptors for insulin or epidermal growth factor and in the postreceptor mechanisms. The evidence also suggests that mitogenic signal transfer via the epidermal growth factor receptor system appears to share a common step with dihydroteleocidin B whereas the signal transfer for insulin seems separate from these. These results suggest that phosphatidylinositol turnover followed by protein kinase C activation alone is not sufficient for mitogenic stimulation and that the coordination of the protein kinase C system with the receptor systems for growth factors may be necessary for "full" mitogenic response. PMID- 3015384 TI - Prognostic factors in small cell lung cancer: multivariate model based on 778 patients treated with chemotherapy with or without irradiation. AB - The relationships between prognostic factors and duration of survival in small cell lung cancer were investigated in a consecutive series of 874 patients treated with combination chemotherapy with or without irradiation. The series included 443 patients with limited and 431 patients with extensive stage disease based on staging including bone marrow examination and peritoneoscopy with liver biopsy but no routine scans. The median durations of survival for the two disease categories were 48 and 30 weeks, respectively. The influence on survival of various pretreatment factors was investigated by use of univariate methods and Cox's multivariate regression model. Patients in each stage were treated according to one of three controlled trials. Variations among the applied treatment regimens did not result in significant differences in duration of survival among patients with limited disease. An alternating regimen was superior to continuous therapy in patients with extensive disease and raised serum lactate dehydrogenase. Prognosis was correlated with disease extent. Surgical resection as well as limited stage disease thus both contributed to survival. Poor performance status, reduced hemoglobin concentration, and raised values for serum lactate dehydrogenase were significantly associated with a reduced duration of survival in both stages. Females with limited disease lived significantly longer than males while advanced age was a negative prognostic factor in extensive disease. Plasma sodium and serum urate were both predictive of survival in limited disease. Proved metastatic disease affecting specific sites or total number of metastatic sites did not carry significant prognostic information in a model including a general variable characterizing stage of disease. Fifty of the 778 patients, on whom the multiple regression model was based, were alive and disease free 2 years after the start of the treatment. Two-year survival rates were strongly correlated to groupings based on prognostic factors, and information about disease extent was not mandatory for predicting the probability of long term disease-free survival. PMID- 3015383 TI - Effect of epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate on the phosphorylation of soluble acidic proteins in A431 epidermoid carcinoma cells. AB - Treatment of [32P]phosphate prelabeled intact human A431 epidermoid carcinoma cells with epidermal growth factor (EGF, 100 ng/ml) or 12-O-tetradecanoylphorbol 13-acetate (TPA, 10(-7)M) resulted in a selective enhancement in the phosphorylation of the following soluble acidic proteins: a phosphoprotein with a molecular weight of 17,000 (pp17; similar notation used throughout) pI approximately 5.5); pp27 (pI approximately 5.5); pp34 (pI approximately 6.2); and pp80 (pI approximately 4.5) as detected by two-dimensional gel electrophoresis. EGF or TPA induced a 4- to 6-fold increase in the phosphorylation of pp17 and a 2 to 4-fold increase in the phosphorylation of pp27, pp34, and pp80 within 15 min after treatment of subconfluent A431 cells. Alkali treatment of the gels removed most of the incorporated [32P] phosphate from the phosphoproteins, including pp27, pp34, and pp80; however, the phosphoester bond in pp17 was stable to alkaline hydrolysis since there was no removal of [32P]phosphate from this protein. Treatment of A431 cells with dibutyryl cyclic adenosine 3':5' monophosphate (1 mM) also increased the phosphorylation of pp17, pp27, and pp34 but not of pp80. Activation of endogenous calcium- and phospholipid-dependent protein kinase C in the cytosol of A431 cells in a cell-free system resulted in the enhanced phosphorylation of pp27, pp34, and pp80 but not of pp17 while exogenous addition of the catalytic subunit of cyclic adenosine 3':5' monophosphate-dependent protein kinase to cytosol preparations resulted in the phosphorylation of pp17, pp27, and pp34, but not of pp80. These results demonstrate that at least four soluble acidic proteins are phosphorylated in A431 cells in response to either EGF or TPA in vivo suggesting that these two agents may exert part of their biological effects on A431 cells through a biochemical pathway involving the phosphorylation of several common proteins; moreover, the studies suggest that these four acidic proteins may be substrates in vitro for protein kinase C and/or a cyclic adenosine 3':5'-monophosphate-dependent protein kinase. PMID- 3015385 TI - Immunoregulatory T-lymphocyte functions in patients with small cell lung cancer. AB - The present study was performed to elucidate the differences in immune status between patients with small cell lung cancer (SCLC) and those with non-small cell lung cancer. The study group consisted of 18 patients with SCLC and 15 with non SCLC. Two healthy volunteers and 13 patients with benign disease were also included in the present study as the non-cancer control. In the non-SCLC group, although not statistically significant, the percentages of both OKT3+ and OKT4+ T lymphocytes in the peripheral blood lymphocytes (PBL) were slightly decreased, associated with a slight increase in the percentage of OKT8+ T-cells, and a slight decrease in the OKT4+ to OKT8+ T-cell ratio. In contrast, the PBL of the SCLC patients showed significantly lower proliferative responses to phytohemagglutinin and human recombinant interleukin 2 than did the PBL of both the SCLC patients and the noncancer control group. The ability of PBL to produce lymphokines (interleukin 2 and macrophage activating factor) was significantly impaired in the SCLC group but not in the non-SCLC group. These results suggest that suppression of helper T-cell functions and/or potentiation of suppressor T cell functions should occur in patients with SCLC. PMID- 3015386 TI - Quantitative immunological detection of estrogen receptors in nuclear pellets from human breast cancer biopsies. AB - A rapid, convenient method for the quantitative determination of estrogen receptors (ER) under high salt (0.6 M KCl) conditions (such as in extracts of nuclear pellets from human breast cancer biopsies) using a commercially available kit [estrogen receptor enzyme immunoassay (ER-EIA) Monoclonal; Abbott] is described. This assay has been validated using breast tumor cytosol ER preparations. It determines total (both occupied and unoccupied) ER, it is insensitive to KCl at concentrations up to 0.8 M, and it can be used with ER preparations having very low protein concentrations. Results obtained using the ER-EIA method for breast tumor nuclear extracts have been compared to those obtained using the hydroxylapatite method, and higher values have been found using the ER-EIA method. The reasons for this discrepancy may be due to: the sensitivity of ER binding to hydroxylapatite in high concentrations of KCl; the temperature dependent degradation of the receptor complex at 30 degrees C, the temperature commonly used to achieve exchange between radioactive estradiol and the endogenously bound estradiol; and possible detection of immunoreactive, but non-ligand-binding forms of ER. The possibility that occurrence of "free" receptors in high salt extracts from nuclear pellets may be an artifact is discussed. The availability of this ER-EIA suitable for nuclear ER determinations opens the possibility of extending correlations between the clinical course of breast cancer and the levels of the ER form (nuclear) that are thought to be of greatest physiological significance. PMID- 3015387 TI - Vasoactive intestinal peptide receptor regulation and reversible desensitization in human colonic carcinoma cells in culture. AB - Vasoactive intestinal peptide (VIP) receptors are widely distributed in different tissues or carcinoma cells originating from entoderm and have been shown to regulate the growth of colonic adenocarcinoma cells through the action of cyclic AMP (cAMP). After exposure of cultured HT-29 human colonic carcinoma cells to 10( 8) M VIP, the cAMP-mediated signals in response to a new challenge with this neuropeptide were strongly attenuated as a function of time (half-life, less than 3 min) and VIP concentrations (half-maximal desensitization, 4 X 10(-9) M VIP). Desensitization is receptor mediated as indicated by: (a) the pharmacological specificity of the desensitization (VIP greater than secretin); (b) the considerable decrease of the potentiative action of VIP on forskolin-induced cAMP generation; and (c) the close temporal relationship between VIP receptor desensitization and the disappearance of the VIP binding sites from the cell surface. Desensitization is reversible upon the removal of VIP. Recovery of functional VIP receptors is insensitive to cycloheximide treatment, is critically dependent upon temperature, and in optimal conditions (37 degrees C) does not exceed 75 and 55% of the binding of 125I-VIP monoiodinated on tyrosine residue and VIP-induced cAMP production, respectively. The characteristics of the desensitization and internalization/recycling of the VIP receptors in carcinoma cells in culture are consistent with the transient action of this neurotransmitter and underline the biological significance of these processes. The study of drugs and natural agents interfering with membrane regulation of VIP receptor density and activity may be of considerable importance in intestinal cell tumor biology. PMID- 3015389 TI - Effect of O6-alkylguanine pretreatment on the sensitivity of human colon tumor cells to the cytotoxic effects of chloroethylating agents. AB - Two human colon carcinoma cell lines which differ greatly in their content of O6 alkylguanine-DNA alkyltransferase were analyzed for their response to 1-(2 chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and 2 chloroethyl(methylsulfonyl)methanesulfonate (ClEtSoSo) before and after treatment with O6-alkylguanines. HT29 cells contained about 17 times more alkyltransferase activity than BE cells. The alkyltransferase activity of HT29 cells was reduced by 60-80% by treatment for 24 h with 0.05-0.4 mM O6-methylguanine or O6-n butylguanine. Such pretreatment prior to exposure to CCNU or ClEtSoSo increased the sensitivity of HT29 cells to these drugs. The exposure to O6-alkylguanines gave a greater enhancement of the toxic effects of ClEtSoSo than of CCNU. There was no significant increase in the toxicity of these agents towards the BE cells which contained much lower levels of the alkyltransferase. When added alone neither O6-methylguanine nor O6-n-butylguanine showed any toxicity towards HT29 or BE cells at the doses used. These results provide strong evidence that the formation of adducts at the O6-position of guanine by these agents contributes significantly to their lethality and that this reaction is more critical for ClEtSoSo than CCNU. The enhancement of the activity of chloroethylating agents by pretreatment with nontoxic doses of O6-alkylguanines may be clinically useful in terms of increasing their therapeutic efficacy towards cells containing high levels of alkyltransferase. PMID- 3015388 TI - Effects of 12-O-tetradecanoylphorbol-13-acetate on epidermal growth factor receptors in BALB/c-3T3 cells: relationship between receptor modulation and mitogenesis. AB - The addition of medium containing 0.1% serum and 12-O-tetradecanoylphorbol-13 acetate (TPA) to quiescent cultures of BALB/c-3T3 cells caused a rapid decrease in the subsequent binding and accumulation of epidermal growth factor (EGF) during a 30-min exposure at 37 degrees C. With further incubation in medium containing TPA, the rate of EGF accumulation steadily increased over time, until by 24 h it had recovered to control values. The addition of TPA with low serum did not stimulate cell cycle traverse. In contrast, when TPA was added directly to spent medium or with fresh medium containing insulin, there was a marked increase in DNA synthesis. Under these conditions the initial TPA-induced decrease in EGF accumulation persisted over a 24-h period. A Scatchard analysis indicated that TPA caused an initial decrease in receptor affinity that persisted if the cells were also stimulated to enter the cell cycle. Thus TPA appeared to have multiple effects on the modulation of the EGF receptor. The pattern of binding following addition of TPA depended on both direct actions of the promoter and an indirect action which depended on whether the promoter was added under conditions in which cell cycle traverse was stimulated. PMID- 3015390 TI - Differentiation and the multiple drug resistance phenotype in human leukemic cells. AB - A common feature of mammalian cell lines selected for multiple drug resistance is the overexpression of a high-molecular-weight surface membrane glycoprotein(s). While its amount has been shown to be related to the degree of resistance of such cells, its function in this phenomenon remains obscure. Because there are some biochemical and functional similarities between drug-resistant cells and differentiated cells, we asked if resistance-associated glycoproteins were also associated with cellular differentiation. Using three monoclonal antibodies against antigens known to be associated with differentiation and three monoclonal antibodies that distinguish our multiple drug-resistant human leukemic CEM/VLB100 cells from their drug-sensitive counterparts, we found that the resistant cells were neither altered in their apparent state of differentiation nor were they altered in their ability to respond to a differentiation stimulus with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. We did find, however, that one antibody that recognizes the resistance-associated glycoprotein, Mr 180,000 glycoprotein (gp180), was only minimally altered in amount bound after treatment with the phorbol ester, but two other resistance-associated glycoproteins, Mr 155,000 glycoprotein (gp155) and, to a lesser extent, Mr 130,000 glycoprotein (gp130), were reduced in expression after this treatment. We suggest that the function of the previously described "marker" glycoprotein associated with multiple drug resistance remains unknown, but that the expression of two other resistance-associated glycoproteins also appears to be related to cellular differentiation or maturation in these cells. PMID- 3015391 TI - Binding of epidermal growth factor and insulin and the autophosphorylation of their receptors in experimental primary hepatocellular carcinomas. AB - Experimental chemical hepatocellular carcinomas that were induced in male F344 rats using three different regimens of limited exposure to the carcinogens 2 acetylaminofluorene or diethylnitrosamine were characterized by very low (as compared to peritumorous or normal tissues) binding of epidermal growth factor and decreased autophosphorylation of the epidermal growth factor receptors. Similar changes were also found in the insulin receptors. We suggest that the carcinogens 2-acetylaminofluorene and diethylnitrosamine cause an initial chemical effect on the great majority of cells. Most of them with time recover their receptor function, and only a small minority become truly initiated and retain these changed characteristics up to the tumor stage. The observed changes appear to be associated with the altered growth state induced by chemical carcinogens. Simultaneous changes observed in the two receptors raise the possibility of a common underlying mechanism. PMID- 3015392 TI - Differences in the nuclear matrix phosphoproteins of a wild-type and nitrogen mustard-resistant rat breast carcinoma cell line. AB - Using polyclonal antibodies raised against a rat liver nuclear envelope protein, lamin protein A, the nuclear matrix proteins of a Walker 256 rat mammary carcinoma wild-type (WS) and a selected cell line with acquired resistance to nitrogen mustards (WR) were found to possess antigenic determinants which were recognized by the antibodies. In one-dimensional immunoblotting analysis, the nuclear matrix protein fractions of both cell lines revealed a common band at Mr 75,000; however only the WS nuclear matrix protein fraction contained a broad band at approximately Mr 70,000. Two-dimensional gel blotting studies of these proteins showed that this Mr 70,000 WS protein had a pI of approximately 7.5. Immunoprecipitation analysis revealed that the altered mobility of this protein could be a function of phosphorylation. The nuclear matrix proteins from both WS and WR cells were shown to bind 3':5'-cyclic adenylic acid (cAMP), as judged by photoaffinity labeling and gel electrophoresis studies. The WS nuclear matrix proteins showed a quantitatively greater level of cAMP binding compared to WR, with predominant binding to proteins with molecular weights of 45,000, 55,000, and 70,000. In WR cells, there was no cAMP binding in the Mr 70,000 region. These data indicate that the Mr 70,000 nuclear matrix lamin proteins are antigenically similar in WS and WR but differ in that the WR protein is hypophosphorylated and does not bind cAMP. PMID- 3015393 TI - Effects of epidermal growth factor receptor concentration on tumorigenicity of A431 cells in nude mice. AB - To test the relationship between the concentration of epidermal growth factor (EGF) receptors and tumor growth in vivo, we measured the rate of growth of several independently isolated A431 cell lines in athymic mice. This series of A431 clonal variants with differing extents of EGF receptor gene amplification and protein expression were implanted into athymic mice and the time to solid tumor formation and rate of growth were measured. Results of these experiments indicate that the degree of gene amplification and concentration of EGF receptors are directly correlated with the growth of these cells as solid tumors in host animals. Complementary DNA hybridization analysis revealed no change in the extent of gene amplification and expression in implanted cells versus excised tumors nor any evidence of further gene rearrangement in vivo. A high concentration of EGF receptors appears to facilitate the growth of tumor cells in vivo and in vitro. PMID- 3015394 TI - Expression of epidermal growth factor receptor in human cultured cells and tissues: relationship to cell lineage and stage of differentiation. AB - Mouse monoclonal antibodies have been used to study the distribution of the epidermal growth factor receptor in human cultured cells and tissues. As expected, most epithelial cells expressed epidermal growth factor receptor (EGFr), whereas cells of hematopoietic origin were EGFr-. However, EGFr was found to be differentially expressed on cultured cells of neuroectodermal origin. Normal melanocytes and a proportion of melanomas are EGFr-, whereas a distinct subset of other melanomas, astrocytomas, and neuroblastomas is EGFr+. Expression of the EGFr in melanomas was closely related to the expression of phenotypic traits of differentiation. Less differentiated melanomas have an epithelioid morphology and are nonpigmented, Ia+, and EGFr+; in contrast, more differentiated melanomas have a dendritic morphology and are pigmented, Ia-, and EGFr-. In the melanoma cell panel used, expression of EGFr did not correlate with rate of proliferation. Expression of EGFr in tissues also showed a lack of correlation with the proliferative state of cells. Our findings indicate that expression of EGFr is related to cell lineage and specific stages of cellular differentiation, rather than only to cell proliferation. The data suggest that receptor content may be elevated in a large number of tumor cell lines. PMID- 3015396 TI - Human T-cell leukemia virus I provirus and antibodies in a captive gorilla with non-Hodgkin's lymphoma. AB - Antibodies reactive against human T-cell leukemia virus I (HTLV-I) were detected by indirect immunofluorescence assay using MT-2 as target cells, enzyme linked immunosorbent assay screen and competition assay, and Western blot analysis in three sera (one collected in 1979) from a captive gorilla which developed diffuse histiocytic lymphoma in 1983. The sera from four other healthy gorillas housed separately were HTLV-I antibody negative. All sera were negative for HTLV-III antibodies by enzyme linked immunosorbent assay. Southern blot analysis of DNA from lymphoma tissue after digestion with BamHI and using complete HTLV-I genome probe gave one 10-kilobase fragment and a characteristic 1.05-kilobase internal fragment detected in all known HTLV-I isolates. These results indicate that the gorilla was infected with HTLV-I or a closely related simian virus several years before the development of lymphoma. PMID- 3015395 TI - A novel monoclonal antibody-defined antigen which distinguishes human non-small cell from small cell lung carcinomas. AB - Spleen cells from BALB/c mice hyperimmunized with the human epidermoid lung carcinoma cell line T222 were fused with NS-1 mouse myeloma cells to produce monoclonal antibodies to human lung cancer antigens. Hybridoma culture supernatants were tested by an enzyme-linked immunosorbent assay for reactivity against a panel of human lung tumor cell lines. Supernatant from hybridoma EA1 (immunoglobulin G1) displayed strong reactivity with four of four non-small cell lung carcinomas but did not react with three of three small cell lung carcinoma (SCLC) cell lines. This hybridoma was cloned by limiting dilution and utilized to generate ascites antibody for subsequent immunohistochemical and antigen characterization studies. Evaluation of fresh frozen tumor tissue sections by immunoperoxidase staining methods revealed EA1 reactivity with the vast majority of non-SCLCs tested (21 of 21 epidermoid, 17 of 18 adenocarcinomas, four of four large cell, two of two bronchioloalveolar) and no reactivity with nine of nine small cell lung carcinomas. EA1 also stained bronchial epithelium and other benign and malignant epithelial tissues. The EA1 antigen was determined to have a molecular weight of 75,000 by immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis of human non-SCLC tumor extracts. These data imply that EA1 recognizes a novel antigen expressed by non-SCLCs and other epithelial tissues. The absence of EA1 reactivity with SCLCs suggests that this monoclonal antibody may find future application in distinguishing non-SCLC from SCLC and prove useful in furthering our understanding of the histogenesis of lung carcinomas. PMID- 3015398 TI - Invasiveness and metastatic capability of rat fibroblast-like cells before and after transfection with immortalizing and transforming genes. AB - Invasion in vitro and in vivo and spontaneous metastasis was investigated in cell lines before and after introduction of immortalizing (polyoma large-T and activated myc) genes and of transforming (polyoma middle-T and activated ras) genes in Fischer rat cells. Invasion in vitro was tested by confrontation of rat cells with embryonic chick heart fragments in organ culture. Invasion in vivo and metastasis was evaluated in nude mice and in syngeneic rats after injection of cells i.p. or s.c. in the flank and after implantation of cell aggregates s.c. in the tail. Rat cells were also analyzed for the presence of myc oncogenes, and for the expression of ras oncogenes. Cells from primary or low passage rat embryo (REF) cells were not invasive in vitro and did not produce tumors in vivo. Cell lines (LTRAT1, LTaRAT1) derived from REF cultures after transfection with plasmids encoding polyoma large-T antigens, behaved like REF cells. Cell lines (REFpEJgpt4, REFpEJmycN7) established from REF cultures after transfection with either a plasmid encoding an activated human ras protein or with the latter plasmid plus one containing an activated myc gene, were invasive in vitro and in vivo and produced invasive and metastatic tumors in syngeneic rats. Cell lines (FR3T3) established in an apparently spontaneous way were invasive in vitro and produced invasive tumors in vivo without metastasis. Derivatives of FR3T3 (FRLT1, MTT4, MMC1, and PyT21) transfected with plasmids encoding one or more of the polyoma antigens, differed from FR3T3 cells by a shorter latency period of tumor formation (less than 1 versus 1 to 3 weeks). Like FR3T3 tumors, FRLT1, MTT4, MMC1, and PyT21 tumors were invasive but not metastatic. Other spontaneously established lines (Rat1) were invasive and metastatic. Cells (Rat1pEJ6.6) derived from Rat1 cultures after transfection with a plasmid encoding an activated ras protein, showed shorter tumor latency periods (less than 1 versus 7 weeks). A thymidine kinase deficient Rat1 derivative (Rat2) was not invasive in vitro but produced invasive and metastatic tumors in vivo with long (9 to 21 weeks) latency periods. Rat2pT24B4 cells derived by us from Rat2 cells after transfection with a plasmid containing a mutated human ras gene (pT24), were invasive in vitro and in vivo as were cells derived from Rat2 tumors. We conclude from our experiments that invasiveness and metastatic capability are often acquired by established REF derived cell lines in an apparently spontaneous way.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3015399 TI - The relation of passive smoking to lung cancer. AB - To evaluate the role of passive smoking in the development of lung cancer among nonsmokers, data were pooled from three large incident case-control interview studies. Ninety-nine lung cancer cases and 736 controls never used any form of tobacco. Overall the adjusted odds ratio for lung cancer among nonsmokers ever living with a smoker was 0.8 (95% confidence interval, 0.5-1.3) rising to 1.2 among those exposed for 40 or more years. Persons living with a spouse who smoked cigarettes were at increased risk (adjusted odds ratio, 1.5; 95% confidence interval, 0.8-2.8). When adjusted for age and gender, there was a significant trend in risk with increasing amounts smoked per week by the spouse (P = 0.05) and with cumulative pack-years of exposure (P = 0.03). This effect was limited to females, especially older women whose husbands were heavy smokers. The elevated risk associated with spouse smoking was restricted to squamous and small cell carcinomas (odds ratio, 2.9; 95% confidence interval, 0.9-9.3), which provides additional evidence linking passive smoking to lung cancer. PMID- 3015397 TI - Two novel cell surface antigens on small cell lung carcinoma defined by mouse monoclonal antibodies NE-25 and PE-35. AB - Two mouse monoclonal antibodies, NE-25 and PE-35, defining novel cell surface antigens of small cell lung carcinoma (SCLC) were produced. The molecular weight of NE-25 and PE-35 antigens estimated by radioimmunoprecipitation was 25,000 and 35,000, respectively. NE-25 antigen was expressed on the majority of cell lines and tumor specimens of SCLC among lung carcinoma. These NE-25-positive cell lines showed typical growth morphology as SCLC classic lines and expressed high levels of neuroendocrine biomarkers, such as aromatic L-amino acid decarboxylase, while NE-25 antigen-negative lines lacked apparent neuroendocrine properties. This antigen was expressed also on a subset of neoplastic cells with (neuro)endocrine properties, including pulmonary carcinoid, and on various tumors of nervous tissues, such as neuroblastoma. Among the normal cells, Kulchitski cells of lung, thyroid gland, adrenal gland, Langerhans islet, and nervous tissues were positive. Thus, the expression of NE-25 antigen is closely associated with the neural and/or (neuro)endocrine differentiation state. On the contrary, PE-35 antigen was present on four major types of lung carcinomas as well as on squamous cell carcinoma and adenocarcinomas of various tissues, but it was absent from nervous tissue tumors. Thus, PE-35 antibody showed a "pan-epithelial" reactivity. Analysis by NE-25 and PE-35 antibodies provided evidence for the heterogeneities of SCLC by demonstrating four surface phenotypes, with the NE-25+/PE-35+ phenotype being most common. In addition, the results supported the current understanding that various histological types of lung carcinoma, including SCLC, are derived from a stem cell of the bronchial epithelium. PMID- 3015400 TI - Gastric and intestinal phenotypic expressions of human signet ring cell carcinomas revealed by their biochemistry, mucin histochemistry, and ultrastructure. AB - Gastric and intestinal phenotypic expression in 37 surgically obtained primary signet ring cell carcinomas, five of their metastases to lymph nodes, and three signet ring cell carcinomas transplanted into nude mice were determined by biochemical, mucin, histochemical, and ultrastructural studies. Crude extracts of cancer tissues were used for measurements of pepsinogen isozymes, sucrase, aminopeptidase (microsomal), and alkaline phosphatase. Histochemical staining of mucin by paradoxical concanavalin A, the galactose oxidase-Schiff sequence and sialidase-galactose oxidase-Schiff, and the periodate-borohydride technique/potassium hydroxide/periodic acid-Schiff procedure was performed. The procedures allowed clear definition of pyloric gland, surface mucous, small and large intestinal goblet, and intestinal absorptive cell types. Of 40 specimens examined, 19 consisted entirely of gastric-type cells, and three entirely of intestinal-type cells. The others consisted of mixtures of gastric and intestinal type cells. The observed high incidence of intestinal-type cells in signet ring cell carcinomas suggested that intestinal-type cells develop independently from intestinal metaplasia within signet ring cell carcinomas (diffuse-type gastric cancers), which probably originate from nonmetaplastic gastric mucosa. PMID- 3015401 TI - Comparison of alternating to nonalternating chemotherapy regimens for non-small cell lung cancer. AB - Forty-seven patients with stage III lung cancer from four institutions in the Denver area were entered in a study comparing two regimens of chemotherapy. The patients were randomized into two groups: Group A received lomustine, cyclophosphamide, vincristine, cisplatin, and doxorubicin monthly; Group B received the five-drug regimen on Months 1, 3, and 5 and received 5-FU by constant infusion, methotrexate, and mitomycin on Months 2, 4, and 6. The age, extent of disease, Karnofsky score, prior therapy, and average number of chemotherapy courses received in each group were comparable. The median survival in Group A was 265 days and in Group B was 163 days (P greater than 0.25). There does not seem to be an advantage in survival in patients who are treated with the eight-drug regimen over the five-drug regimen. PMID- 3015402 TI - Northern California Oncology Group protocol 6G91: response to treatment with radiation therapy and seven-drug chemotherapy in patients with glioblastoma multiforme. AB - The Northern California Oncology Group (NCOG) conducted a nonrandomized phase II study to evaluate the benefit of a seven-drug chemotherapy protocol (NCOG 6G91) in patients with glioblastoma multiforme (GM). Time to tumor progression was the primary end point of the study. The treatment consisted of 5-FU and lomustine administered after surgery and before radiation therapy, hydroxyurea and misonidazole during radiation therapy, and procarbazine and vincristine alternated with carmustine and 5-FU after radiation therapy. Ninety patients entered the study; data from the 64 patients with GM who completed radiation therapy and at least started the postradiation chemotherapy regimen and returned for follow-up examination are analyzed in this report. The median time to tumor progression in the 64 adequately treated patients was 42 weeks; the 25th percentile value was 60 weeks. A Cox multivariate analysis showed that age and extent of surgical resection were important prognostic variables in patients with GM. The results of this treatment regimen were similar to those of a previous NCOG protocol (6G61), which consisted of hydroxyurea during radiation therapy followed by chemotherapy with carmustine or a combination of lomustine, procarbazine, and vincristine. PMID- 3015403 TI - Combination chemotherapy with vindesine and cisplatin for refractory small cell bronchogenic carcinoma. PMID- 3015404 TI - Phase II evaluation of aclarubicin in lung cancer: a Southeastern Cancer Study Group Trial. PMID- 3015405 TI - Phase II trial of epirubicin in hepatoma. PMID- 3015407 TI - D-galactosyltransferase and its endogenous substrates in chick embryo fibroblasts transformed by Rous sarcoma virus. AB - UDP-D-galactose: 2-acetamido-2-deoxy-beta-D-glucopyranosyl 4-beta-D galactosyltransferase (GalTase) activity was purified, from primary chick embryo fibroblast (CEF) transformed by a temperature-sensitive, Rous sarcoma virus mutant (CEF-RSV), by chromatography on an affinity resin prepared with monoclonal antibodies to GalTase. Cellular glycopeptides from CEF, as well as CEF-RSV, maintained at permissive (37 degrees) [CEF-RSF (37 degrees)] and nonpermissive temperatures (41 degrees) [CEF-RSV (41 degrees)], were solubilized and galactosylated in vitro by incubation with purified GalTase substrates, composed of at least six discrete complex glycopeptides having bi- to tetra-antennary structures. The glycopeptides isolated from transformed cells, CEF-RSV (37 degrees), included the six types observed in nontransformed cells, but demonstrated alterations in their relative amounts, including an increase in the content of a glycopeptide containing 3 mannose and 4 glucosamine residues. Furthermore, two additional complex-type glycopeptides were isolated from CEF- but demonstrated alterations in their relative amounts, including an increase in the content of a glycopeptide containing 3 mannose and 4 glucosamine residues. Furthermore, two additional complex type glycopeptides were isolated from CEF-RSV (37 degrees). These malignant transformation-related glycopeptides were partially characterized and found to represent tri- and tetra-antennary complex glycopeptides. Endogenous galactosylation appeared to have occurred in a branched, nonspecific manner in these transformed cell-derived glycopeptides. These findings indicate that transformed cells may contain a greater preponderance of more highly branched, complex oligosaccharides which are randomly galactosylated at nonreducing termini by cellular GalTase. PMID- 3015406 TI - Effects of Mega-COMLA (cyclophosphamide, cytarabine, vincristine, and methotrexate followed by leucovorin and prednisone) plus CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) in the treatment of lymphoid neoplasms with very poor prognosis. AB - Treatment results remain very poor for some clinical and histopathologic subsets of patients with aggressive non-Hodgkin's lymphoma. We treated 21 such patients with a high-dose combination chemotherapy regimen [Mega-COMLA (cyclophosphamide, cytarabine, vincristine, and methotrexate followed by leucovorin and prednisone) + CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone)] in an attempt to improve disease-free survival. Neoplasms were classified using the Lukes-Collins system. Eight patients had T-cell lymphomas (convoluted lymphocytic lymphoma, four patients; T-cell lymphoma/leukemia, one; and peripheral T-cell lymphoma, three), eight had B-cell lymphomas (immunoblastic sarcoma, five patients; small noncleaved follicular center cell, one; and large noncleaved follicular center cell, two), and five had nontypable large noncleaved cell lymphomas. All patients were previously untreated; 18 of 21 patients had clinical stage III or IV disease. Following induction therapy (4-8 weeks' duration), 16 patients (76%) achieved complete remission, while three had partial remission. Two patients died of sepsis during induction therapy. Eleven of 16 complete responders (69%) remain in complete remission after a median follow-up of 35 months. The actuarial 3-year survival rate is 51% for the entire group. Myelosuppression with this regimen was severe and prolonged, with a median duration of neutropenia (less than 500 cells/microliter) of 14 days. Seven patients (33%) developed severe neuropathy following induction treatment. High dose induction therapy with this regimen resulted in a high complete remission rate with manageable toxicity. Survival results are encouraging when compared retrospectively to our patients with similar poor-prognosis histologies treated with standard combination chemotherapy. However, the value of this intensive therapy, relative to newer ("third-generation") regimens, can only be established by prospective randomized studies. PMID- 3015408 TI - Lysis of small cell carcinoma of the lung tumor cell lines by gamma interferon activated allogeneic peripheral blood mononuclear cells: abrogation of killing by pretreatment of tumor cells with gamma interferon. AB - Interferon has been shown to enhance the ability of nonspecific cytotoxic mononuclear cells to lyse some, but not all, tumor cells. We have examined the effect of recombinant human gamma interferon (rIFN gamma) on the cell-mediated cytolysis of tumor target cells derived from continuously cultured lines of small cell carcinoma of the lung (SCCL). Cells from the SCCL lines DMS 44, 53, 79, 92, and 406 were labeled with 51Cr and incubated with normal and rIFN gamma stimulated peripheral blood mononuclear cells for 18 h at 37 degrees C and tumor cell lysis estimated by measuring 51Cr release. Although cells from certain SCCL lines were good targets for cell mediated cytotoxicity, susceptibility to lysis was heterogeneous among the different SCCL lines. DMS 406 and 79 were, on average, maximally lysed, while DMS 44, 53, and 92 showed less susceptibility to lysis by either control or rIFN gamma-stimulated effector cells. In addition, although pretreatment with rIFN gamma increased the cytolytic capacity of normal peripheral blood mononuclear cells from several different donors, preincubation of the tumor cell lines with rIFN gamma resulted in inhibition of cytolysis mediated by both control and IFN-activated effector cells. These findings suggest that although rIFN gamma may enhance cell-mediated lysis of SCCL tumor cells, it may also decrease susceptibility to lysis. PMID- 3015410 TI - [The gamma-aminobutyric acid system (GABA)]. PMID- 3015409 TI - Emphysematous cholecystitis: complication of hepatic artery embolization. AB - We report a case of acute emphysematous cholecystitis that occurred following hepatic artery embolization for hepatocellular carcinoma but was cured by conservative therapy. In view of its pathogenesis, emphysematous cholecystitis seems likely to be a complication of hepatic artery embolization. PMID- 3015411 TI - Primary amyloid heart disease presenting as hypertrophic obstructive cardiomyopathy. AB - This report describes the unusual presentation of a patient with primary cardiac amyloidosis. Initial clinical symptoms and hemodynamic studies, including Technetium-99m-pyrophosphate scintigraphy, suggested hypertrophic obstructive cardiomyopathy, but endomyocardial biopsy revealed diffuse amyloid infiltration. Only two other cases of left ventricular outflow tract obstruction due to cardiac amyloidosis have been reported. The false-negative technetium-99m-pyrophosphate scintigram in this patient argues for the use of endomyocardial biopsy to aid in the diagnosis of left ventricular hypertrophy. PMID- 3015412 TI - Down regulation of the alpha-factor pheromone receptor in S. cerevisiae. AB - The peptide pheromone, alpha-factor, was found to elicit down regulation of receptor sites on yeast a cell targets. Cellular uptake of alpha-factor accompanied the loss of receptor sites. Receptor-deficient a cells bearing a deletion of the STE2 gene were unable to internalize alpha-factor. Cultures were found to reaccumulate receptor sites following the initial period of down regulation; reaccumulation was dependent upon protein synthesis. Pheromone resistant mutants, ste4-3 and ste5-3, retained the ability to down regulate receptors but failed to show reaccumulation. Our results suggest that alpha factor-receptor complexes enter the cell by receptor-mediated endocytosis and that receptors are continuously lost and resynthesized in the presence of alpha factor. We found no reduction of alpha-factor binding capacity in a cell cultures that had adapted to alpha-factor. PMID- 3015413 TI - Regulation of human interleukin-2 gene: functional DNA sequences in the 5' flanking region for the gene expression in activated T lymphocytes. AB - Interleukin-2 (IL-2) is a lymphokine that plays a crucial role in the immune system, especially in the growth control of T lymphocytes. Expression of this lymphokine is restricted to activated T lymphocytes. Here we demonstrate the presence of unique DNA sequences in the 5' flanking region of the human IL-2 gene that control induced T-cell-specific gene expression. We also show that the DNA sequences function in an orientation-independent manner and activate a heterologous promoter which is otherwise inert in induced T cells. The DNA, which spans about 200 bp, contains regions with sequence homology to LTR sequences of HTLV-III (or LAV) and the 5' upstream region of the IL-2 receptor and interferon gamma genes. PMID- 3015414 TI - Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus. AB - Hepatitis B virus (HBV) has not yet been propagated in vitro, and knowledge concerning its reaction with receptors on target cells remained scant. We have located within the HBV envelope proteins a sequence mediating the attachment of HBV to human hepatoma HepG2 cells. A synthetic peptide analog (PLGFFPDHQLDPAFGANSNNPDWDFNP) is recognized by both cell receptors and anti-HBV antibodies and elicits antibodies reacting with native HBV. The synthetic peptide is a promising immunogen expected to elicit protective antibodies based on the concept of the attachment blockade pathway of virus neutralization. The approach described here, based on anti-peptide antisera and the binding of peptide analogs to cell receptors is generally applicable for the delineation of cell receptor binding sites on viruses with known gene sequences. PMID- 3015415 TI - v-ras genes from Harvey and BALB murine sarcoma viruses can act as initiators of two-stage mouse skin carcinogenesis. AB - Activated Harvey murine sarcoma virus ras genes were introduced into epidermal cells in vivo by direct application of retroviruses to mouse skin. Subsequent treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced benign papillomas, some of which progressed to invasive carcinomas. Initiation with virus was irreversible for at least 4 months, since TPA treatment after this latency period produced papillomas within 4 weeks. Analysis of viral integration sites showed that carcinomas are clonal in origin. Both papillomas and carcinomas express virus-specific ras mRNA and the viral form of ras P21 protein. The results show that activated ras genes can replace chemical carcinogens in initiation of mouse skin carcinogenesis. This system presents a novel approach to in vivo analysis of the biological role of oncogenes in epithelial tumorigenesis. PMID- 3015416 TI - Linkage of prion protein and scrapie incubation time genes. AB - A single gene (Prn-i) that affects scrapie incubation period in mice has been identified. I/LnJ mice have a very long incubation period after inoculation of scrapie prions (200-385 days) and NZW/LacJ mice have a short one (113 +/- 2.8 days). (NZW X I/Ln)F1 hybrid mice had incubation times of 223 +/- 2.8 days indicating longer incubation times were dominant. Incubation periods in the backcross progeny of (NZW/LacJ X I/LnJ)F1 X NZW/LacJ segregated into two groups (64 mice, 130 +/- 1.1 d; 66 mice, 195 +/- 1.9 d) indicating single gene control. NZW/LacJ and 20 other inbred strains have the Prn-pa allele which is identified as a 3.8 kb Xbal fragment using a hamster PrP (prion protein) cDNA probe. I/LnJ and three other Prn-pb mouse strains have a 5.5 kb Xbal restriction fragment. Analysis of DNA from 66 backcross mice indicated Prn-i is tightly linked to Prn p, the structural gene for PrP. PMID- 3015417 TI - Germ line transmission of autonomous genetic elements in transgenic mouse strains. AB - Upon microinjection into fertilized mouse eggs of circular molecules of plasmid pPyLT1 carrying the gene encoding the large T protein of polyoma virus within bacterial vector sequences, autonomous circular plasmids were stably maintained in low copy numbers in transgenic strains. These plasmids could be rescued in E. coli by transfection. Integrated forms could be detected neither in somatic tissues, nor in spermatozoa. Efficiency of paternal or maternal transmission was close to 100%. The plasmids had lost or had extensively rearranged the polyoma sequences. In addition, they had acquired defined segments of genomic mouse DNA, which might be responsible for correct segregation of daughter copies at both mitosis and meiosis (centromeric function). PMID- 3015418 TI - Cohabitation of scaffold binding regions with upstream/enhancer elements of three developmentally regulated genes of D. melanogaster. AB - We find DNA fragments attached to the nuclear scaffold (SARs) both 5' and 3' of three Drosophila genes, defining looped domains ranging from 4.5 to 13 kb. For the two-promoter-containing gene Adh (alcohol dehydrogenase), we find two upstream and two downstream SARs. For Sgs-4, the 5' SAR covers 866 bp immediately upstream of the transcript, and in the case of fushi tarazu, the 5' SAR is found on a small fragment 4.8 kb upstream of the start of transcription. These four upstream scaffold-attached fragments comap with enhancer-like regulatory sequences. Sequence analysis of five upstream SARs reveals clusters of sequences closely related to the cleavage consensus of topoisomerase II, several copies of a specific 10 bp A-rich sequence (AATAAATCAAA), and another 10 bp T-rich stretch. PMID- 3015419 TI - Induction of nuclear transport with a synthetic peptide homologous to the SV40 T antigen transport signal. AB - A system was developed for the analysis of protein transport to the nucleus. Carrier proteins cross-linked to synthetic peptides were microinjected into the cytoplasm of mammalian cells, and protein transport was evaluated by immunofluorescence staining of fixed cells. A 13-mer synthetic peptide containing seven amino acids homologous to SV40 T antigen was capable of inducing nuclear transport, but no transport was observed when proteins were coupled with a synthetic peptide homologous to a nuclear-transport-defective T antigen. The largest protein-peptide conjugate efficiently transported was ferritin (Mr 465,000). The rate of transport was influenced by the number of peptides per molecule of carrier protein and, to some degree, by the size of the carrier protein. Transport of some conjugates was almost complete in 15 min at room temperature. PMID- 3015420 TI - The primary structure of the putative oncogene pim-1 shows extensive homology with protein kinases. AB - We have shown previously that the putative oncogene pim-1 is frequently activated by provirus insertion in murine leukemia virus-induced T cell lymphomas. Here we describe the structure of the pim-1 gene as determined by sequencing genomic and cDNA clones. The gene has an open reading frame, encoding a protein of 313 amino acids, extending over six exons and preceded and followed by stop codons in all reading frames. Proviruses always integrate outside the protein-encoding domain, showing a high preference for a small region in the 3'-terminal exon; integration in the 3' exon results in relatively high levels of pim-1 mRNA. Computer search reveals homology between pim-1 and protein kinases: all the domains characteristic of protein kinases are conserved in the pim-1 amino acid sequence. The highest homologies were observed with the protein-serine kinases. PMID- 3015421 TI - Intracellular sorting and polarized cell surface delivery of (Na+,K+)ATPase, an endogenous component of MDCK cell basolateral plasma membranes. AB - Madin Darby Canine Kidney (MDCK) cells grown on polycarbonate filters in a two chamber culture system were used to study the postsynthetic sorting of the alpha subunit of the (Na+,K+)ATPase, an important native protein of the MDCK cell basolateral plasmalemmal domains. The N-azidobenzoyl derivative of ouabain (NAB ouabain) and anti-ouabain antibodies were used in pulse labeling experiments to monitor the arrival of newly synthesized molecules of (Na+,K+)ATPase at the apical and basolateral cell surfaces. The results show that newly synthesized alpha-subunits bind NAB-ouabain and become substrates for immunoprecipitation only when this compound is present in the basolateral chamber. No more than 10% of the (Na+,K+)ATPase synthesized during the pulse period could appear at the apical surface without being detected by our assay. Thus, sorting of this native protein is effected intracellularly prior to its direct insertion into the basolateral plasmalemmal domain. Passage through an acidic compartment is not required for proper sorting. PMID- 3015422 TI - Transformation of dog embryo kidney cells by human herpesviruses. AB - The infection of dog embryo kidney (DEK) cells with herpes simplex virus type 2 (HSV-2) or human cytomegalovirus (HCMV) led to the development of transformed cell lines. Rapidly dividing DEK cells with unlimited division potential exhibited growth in 2% serum, contained nuclear virus antigens, and formed small (+/- 0.2 mm) colonies in 0.3% agarose. Immortal cell lines showing the same transformation properties were also obtained after transfection with purified HSV 2 or HCMV DNA. These results confirm the transforming capacity of both herpesviruses as well as the usefulness of this different type of mammalian cells in transformation studies. PMID- 3015423 TI - Sperm binding activity of the zona pellucida of immature mouse oocytes. AB - Immature oocytes taken from ovarian follicles are sometimes used in studies of sperm-zona interaction in species for which it is difficult to obtain ovulated eggs. As yet, however, there has been no quantitative comparison of the sperm binding capacities of immature and ovulated oocytes. We report here that in mice there is no significant difference in the numbers of sperm which bind to the zonae pellucidae of immature and ovulated oocytes in vitro. These results support the use of immature oocytes in studies of sperm-zona interaction. We have also analyzed the sources of variability in sperm binding assays, and we make suggestions for the most efficient design of experiments. PMID- 3015424 TI - An alteration in tubulin expression detected in SV40 transformed 3T3 cells. AB - Tubulin expression was analysed in normal and simian virus-40 (SV40) transformed 3T3 cells by two-dimensional polyacrylamide gel electrophoresis and immunoblotting studies using monoclonal antibodies raised to alpha- and beta tubulin subunits. The ratio of alpha- to beta-tubulin recognised was calculated for both cell lines and found to shift from 2.50 in normal cells to 0.52 in virally transformed cells. beta-Tubulin was thereby shown to be the predominant subunit in SV40-transformed 3T3 cells in contrast to normal 3T3 cells. PMID- 3015425 TI - Selective inhibition of stimulation responses of neutrophils by membrane modulators. AB - Polymorphonuclear leukocytes undergo a series of morphological and biochemical changes in response to various chemical stimuli. Transmembrane potential change is an early event that follows stimulation and membrane depolarization may act as a trigger for superoxide generation. To determine if there is a correlation between membrane depolarization and superoxide generation, we investigated the effects of different membrane modulators on stimulus-dependent depolarization. The membrane modulators mepacrine, chlorpromazine and cepharanthine inhibited the superoxide generation produced by chemotactic peptide, FMLP, and/or digitonin in neutrophils. Inhibitory profiles of the activation parameters, however, demonstrate that membrane depolarization is not associated with superoxide generation: FMLP-induced depolarization was inhibited by the modulators tested and was accompanied by the suppression of superoxide generation, but the depolarization produced by digitonin was stimulated somewhat by these drugs. Our results indicate that receptor-mediated membrane depolarization is not a necessary event for the activation of superoxide generation by digitonin. PMID- 3015426 TI - Fusion of SV40-induced endocytotic vacuoles with the nuclear membrane. AB - The interaction between simian virus 40(SV40)-induced endocytotic vacuoles and the nuclear membrane was investigated using cationized ferritin (CF) and concanavalin A (Con A) as cell membrane markers. These markers bound to the cell surfaces of CV-1 cells together with SV40 at 4 degrees C. Following incubation of these modified cells at 37 degrees C in serum-free medium, the cell membranes showed many invaginations. After incubation for 60 min at 37 degrees C in the same medium, many various-sized vacuoles were present that contained membrane bound CF, Con A and SV40. After 2 h of incubation at 37 degrees C, Con A was present in some areas of the perinuclear cisterna along the nuclear membrane. The control experiment, however, showed no localization of Con A-binding on the nuclear membrane. These results provide evidence that SV40-induced endocytotic vacuoles migrate toward the nucleus and fuse with its membrane. PMID- 3015427 TI - Effect of calcium ion on fatty acid-induced generation of superoxide in guinea pig neutrophils. AB - When phospholipases of plasma membranes are activated by certain stimuli, unsaturated fatty acids are liberated. Because unsaturated fatty acids enhance the transmembrane movement of calcium ions, the fatty acids released may modulate intracellular calcium homeostasis in various cells, including neutrophils. To determine the physiological function of these unsaturated fatty acids, we studied the effects of various fatty acids on superoxide generation and on changes in intracellular calcium contents of guinea pig neutrophils. Some unsaturated fatty acids, arachidonate and linoleate, stimulated the rate of superoxide generation concomitant with the increase in the amount of intracellular calcium. In contrast, the saturated fatty acid, myristate, stimulated the generation of superoxide without affecting the content of intracellular calcium. The stimulating actions of arachidonate and myristate were increased dramatically by the presence of a low concentration (1 microM) of extracellular calcium ion. The rate of superoxide generation in fatty acid-treated neutrophils was inhibited by chlorpromazine, an inhibitor of such calcium-binding proteins as C-kinase. These and other observations suggest that liberated unsaturated fatty acids increase the amount of intracellular calcium and enhance C-kinase activity also that the increased activity of the enzyme is involved in the chain of events leading to the stimulation of superoxide generation in fatty acid-treated neutrophils. PMID- 3015429 TI - Phorbol ester-induced regulation of transferrin receptors in human leukemia K562 cells. AB - The treatment of human leukemia K562 cells with 12-0-tetradecanoyl phorbol-13 acetate (TPA) caused a decrease of transferrin receptors. The mechanism of the decrease of the receptors with TPA has been investigated. In cells incubated with TPA, the rate of biosynthesis of transferrin receptors was reduced to 10-20% of that in untreated cells. Pulse-chase experiments showed that turnover of the receptors in TPA-treated cells was accelerated over that in untreated cells. These results indicated that the decrease of transferrin receptors in TPA-treated cells was caused by reduced biosynthesis and accelerated degradation of the receptors. PMID- 3015428 TI - EL-4 tumor cell-induced human and rabbit platelet aggregations. AB - EL-4 tumor cells were assayed in vitro for their ability to aggregate two kinds of platelets. An inhibition study showed that the EL-4 tumor cell can induce platelet aggregation by at least two different mechanisms. One, mediated by thrombin, was dominant with rabbit platelets because hirudin, which specifically inhibits thrombin, considerably suppressed the rabbit platelet aggregation induced by EL-4 tumor cells. In contrast, EL-4 cells induced the aggregation of human platelets even in citrated PRP. It is the apyrase-sensitive pathway that is believed to work in human platelets. The human platelet responses to EL-4 tumor cells clearly differed from those of rabbit platelets in terms of inhibition by hirudin and apyrase and of reactivity in citrated PRP. Both phospholipase A2 and dibutyryl cAMP strongly inhibited EL-4 tumor cell-induced platelet aggregation in both rabbit and human platelets. These two compounds may block a vital step in platelet aggregation that is elicited by the EL-4 tumor cells. Our results show that human platelet response to tumor cells is not necessarily deducible from experimental data obtained with animal platelets. PMID- 3015430 TI - Role of the valence of concanavalin A in the activation of guinea pig polymorphonuclear leukocytes. AB - Stimulation of polymorphonuclear leukocytes (PMN) by tetravalent concanavalin A (alpha-ConA) induces membrane depolarization preceding the onset of superoxide anion (O2-) production. Both divalent and monovalent ConA analogues were studied to evaluate the role of valence. Monovalent ConA (m-ConA) was inactive in stimulating O2- production and divalent derivatives were less active than native alpha-ConA. Similarly, membrane depolarization was dependent on the valency of ConA. m-ConA did not induce a marked change in membrane potential, whereas sustained depolarization occurred with multivalent ConA. The formation of multiple linked interactions between surface receptors may be an important early event in the activation of PMN by ConA. PMID- 3015432 TI - [Possibilities of detecting rotavirus diseases in clinical practice. Comparison of the ELISA, CIEP and latex agglutination methods for the detection of viruses by the determination of an increase in antibodies]. PMID- 3015431 TI - [Possibilities of enzymo-immunologic determination of hepatitis A virus antigens]. PMID- 3015434 TI - [Experimental studies on the mechanism of anti-platelet aggregation action of motherwort]. PMID- 3015433 TI - Synthesis and angiotensin converting enzyme inhibitory activity of 1,5 benzothiazepine and 1,5-benzoxazepine derivatives. I. PMID- 3015435 TI - [Clinical analysis of the tongue picture in 130 patients with primary liver carcinoma]. PMID- 3015436 TI - [Preliminary observation on cyclic nucleotide and other immunological parameter changes in treating chronic glomerulonephritis with replenishing qi and nourishing yin principle--a study of 41 cases]. PMID- 3015437 TI - [Chronomorphological effects of needling "yong quan point" on some rats' organs]. PMID- 3015438 TI - Micellar formation of spin-labeled fatty acyl derivatives of lipophilic muramyl dipeptides and their incorporation into liposomal membranes. AB - A lipophilic muramyl dipeptide (MDP) with a nitroxide moiety in its acyl chain (SL-MDP) and its N-methyl derivative (SL-methyl MDP) were synthesized. The SL MDPs formed micelles (cmc, 0.1-0.3 mM). The ESR spectra of the SL-MDPs in phosphatidylcholine (PC) liposomes at 25 degrees C consisted of an anisotropic signal and three sharp lines, indicating that both SL-MDPs partitioned between membranes and aqueous phase. The amounts of the SL-MDPs in membranes depended on the phospholipid species and the cholesterol (Chol) content, but no appreciable difference was observed between SL-MDPs. The SL-MDPs partitioned well at 25 degrees C into egg yolk PC liposomes but not into pure dipalmitoylphosphatidylcholine (DPPC), suggesting that the incorporation may be related to the membrane fluidity. Chol enhanced the incorporation into both phospholipids. The mobilities of the SL-MDPs in the membranes were less than that of the corresponding spin-labeled fatty acid. Comparison of the mobilities among SL-MDPs, spin-labeled ganglioside and spin-labeled galactosylceramide showed that the hydrophilicity of the polar group may influence the immobilization of their acyl chains. PMID- 3015440 TI - [Chronic hepatitis, liver cirrhosis, primary liver cancer and virus HB infection. Epidemiologic study in a sahelian hospital milieu. Apropos of 185 cases]. AB - From December 1982 to June 1985, 185 patients with signs of chronic liver disease are investigated in National Hospital (Niamey, Republic of Niger). A first group ("Hepatopathy") of 131 patients (75 males, 56 females) is made of 3 chronic liver diseases: 35 chronic hepatitis (CH), 58 hepatic cirrhosis (HC), 38 hepatocellular carcinoma (HCC). A second group ("Controls") of 54 patients (29 males, 25 females) is made of miscellaneous diseases other than CH, HC, HCC. In the first group ("Hepatopathy") serum hepatitis B virus (HBV) surface antigen (HBsAg radioimmunoassay) is present in 64.9% (85/131). In the second group ("Controls") serum HBsAg is present in 3.7% (2/54) (Chi-2 test P less than 10(-9)). Serum HBsAg prevalence is more than 50% in each group of CH, HC and HCC. A maximum value is observed in males with HCC (75.7%). These results demonstrate the importance of evolutive HBV infection in the course of CH, HC and HCC in the sahelian region. PMID- 3015439 TI - A new chemical procedure for the preparation of gangliosides carrying fluorescent or paramagnetic probes on the lipid moiety. AB - A new chemical procedure is described for preparing labelled GM1 molecular species, carrying as acyl moiety pyrene-decanoic acid, 5-doxyl-stearic acid and 16-doxyl-stearic acid. It makes use of a mixed anhydride formed by ethylchloroformate and the labelled acyl chain, as the reagent for N-acylation of a deacetylated, deacylated GM1 ganglioside, which is prepared by alkaline hydrolysis of natural GM1. The reaction performed with a unitary GM1 derivative/mixed anhydride molar ratio, occurs with a yield of above 40%. The labelled deacetylated GM1 molecular species are then N-acetylated by means of acetic anhydride with quantitative yield. The chemical process of insertion of labelled fatty acid and reconstitution of GM1 ganglioside has been confirmed by GLC-MS and NMR analyses. Fluorescence and electron spin resonance experiments indicate that the labelled gangliosides behave similarly to natural GM1, in both the aggregation properties and the capability to be transferred from micelles to vesicular dispersions of phospholipids. PMID- 3015441 TI - [Serological study of the occurrence of Herpesviridae in French Guyana]. AB - 300 samples of serum (in seven age-groups) from the "creole" population of french Guiana were tested for antibodies to the four human herpesviruses (HSV, VZV, CMV and EBV). Results show the higher prevalence of CMV and EBV in early childhood, HSV primary infection appears to take place earlier in life than in temperate and developed countries, but later than in other tropical countries. The pattern of VZV epidemiology in french Guiana is consistent with what is known in tropical countries: varicella is also a disease of adolescents and young adults. CMV appears to be more prevalent in women than in men between 21 and 40. PMID- 3015442 TI - Human viruses in sediments, sludges, and soils. AB - Recent studies have provided a greater understanding of the movement of viruses in the environment by their attachment to solids. These studies have focused on solids-associated viruses present in wastewater discharged into the ocean and on viruses in sludge and wastewater that may be retained in soil following their land disposal. Such ocean or land disposal of wastewater and sludge may result in a discharge of one or more of 120 human enteric virus pathogens including those causing poliomyelitis, viral hepatitis A and acute gastroenteritis.Solids associated viruses in effluents discharged into coastal waters accumulate in bottom sediments, which may contain 10 to 10 000 more virus per unit volume than the overlying seawater. Solids-associated viruses resuspended by water turbulence may be transported from polluted to distant non-polluted recreational or shellfish-growing water. Transmission of viruses causing hepatitis or gastroenteritis may result from contact by bathers or swimmers with these viruses in recreational waters, or from ingestion of raw or improperly cooked shellfish in which the solids-associated virus had been bioaccumulated.The land disposal of sludge and wastewater has a potential of causing infections in farm workers, contamination of crops, pollution of raw potable water sources or infiltration of ground water. Viruses retained on soils can be released by rain water and may contaminate ground water through lateral and vertical movements. PMID- 3015443 TI - Citrate can effectively replace bicarbonate in oral rehydration salts for cholera and infantile diarrhoea. PMID- 3015444 TI - Second meeting of the WHO Collaborating Centres on AIDS: Memorandum from a WHO meeting. PMID- 3015445 TI - Dissociation between induction of ornithine decarboxylase and oxidative burst by phorbol esters in a macrophage cell line. AB - In order to investigate the correlation between stimulation of superoxide generation and induction of ornithine decarboxylase (ODC) by 12-O tetradecanoylphorbol-13-acetate (TPA) we have used the macrophage cell line J774.16 and a clone derived from this line that, by contrast with the parental line, is unable to generate superoxides in response to TPA. No difference was observed between the normal and the defective cells, with respect to ODC induction by TPA over a wide range of TPA concentrations (0.2-5.0 micrograms/ml). Similar results were obtained comparing resident and caseinate-elicited mouse peritoneal macrophages. Although resident macrophages did not generate superoxides in response to TPA, they did not differ from superoxide-generating, caseinate-elicited macrophages with respect to ODC induction. These data suggest a dissociation between the stimulation of the oxidative burst by TPA and a growth factor-like effect such as ODC induction. PMID- 3015446 TI - Enhanced protein phosphorylation of carcinogen-initiated 10T1/2 cells accompanies their neoplastic transformation. AB - Retinyl acetate (RAC) prevents neoplastic transformation of carcinogen-exposed C3H/10T1/2 C18 cells, and has allowed the isolation of cells having the properties expected of initiated cells. After removal of RAC from logarithmically growing initiated cells, cultures first become confluent and growth arrested, as occurs with their normal counterparts. This is followed by an increase in thymidine labelling index and finally by morphological transformation on days 15 and 23, respectively, after seeding and drug removal. The increase in labelling index is the earliest indication yet seen of transformation taking place in these cultures. Phosphorylation of three nonionic detergent-soluble proteins was observed to be correlated with the increase in labelling index. Using two dimensional gel electrophoresis, a protein of 35 kd (pp35(1)) and one of 38 kd (pp38) were seen to become more heavily phosphorylated in association with the increase in labelling index. A second protein of 35 kd (pp35(2)) was only detected in isolated transformed cells and is phosphorylated in an alkali resistant manner consistent with phosphorylation of tyrosine residues. Alkali resistant phosphorylation of pp35(2)) could be eliminated in transformed cells by treatment with RAC. Phosphorylation of pp35(1)) appears to be a permanently acquired characteristic of these cells, and cannot be made to revert. Phosphorylation of pp38, as indicated by isoelectric point, is reduced by RAC treatment. These studies implicate changes in protein phosphorylation in loss of growth control, and suggest that the cancer chemopreventive action of retinoids may also be mediated at this level. PMID- 3015447 TI - Possible role of oxygen radicals in cell transformation by diethylstilbestrol and related compounds. AB - Diethylstilbestrol (DES) and four derivatives, viz. tetrafluoro-DES, 3'-hydroxy DES, Z,Z-dienestrol and hexestrol, were examined for their abilities to form superoxide radicals and to induce DNA strand breaks in the presence of horseradish peroxidase/hydrogen peroxide metabolism in a cell-free system. Furthermore, the induction of strand breaks by these compounds was tested in Syrian hamster embryo (SHE) cells in vitro. Formation of superoxide radicals could be demonstrated by reduction of nitro blue tetrazolium for DES but not for its derivatives. With isolated superhelical DNA, induction of strand breaks in the presence of Fe3+ was observed for DES, tetrafluoro-DES and 3'-hydroxy-DES, while hexestrol and Z,Z-dienestrol were ineffective. In SHE cells, alkaline elution technique showed that DNA strand breaks were induced by DES and all derivatives tested, although only at cytotoxic concentrations. It is concluded that DES, under conditions of peroxidative metabolism, can give rise to superoxide generation and DNA strand breaks, and that these events may play a role in the process of DES-induced cell transformation. PMID- 3015448 TI - Isolated adipocytes: an assessment of cell surface changes during their preparation. AB - A method is described, based on the detection of adipocyte-specific cell surface antigens, which allows assessment of the relative surface damage incurred by the cells when they are prepared under a variety of conditions. Using the method it is possible to develop, for any set of reagents, a set of cell isolation conditions (collagenase concentration, time of incubation) which will produce minimally damaged cells which exhibit high levels of specific cell surface immunoreactivity. Under certain conditions a recovery from limited surface damage can be achieved, although, when cells are prepared under more extreme conditions irreversible surface damage occurs. The surface morphology of the cells as revealed by scanning electron microscopy, is also clearly affected by the conditions of cell isolation. The method has been used to define the conditions necessary for the isolation of cells to be used in the study of subtle biochemical responses. PMID- 3015449 TI - Coxsackie B4 virus induces short-term changes in the metabolic functions of mouse pancreatic islets in vitro. AB - Mouse pancreatic islets cultured in vitro were infected with a tissue culture adapted or a mouse pancreas-adapted strain of Coxsackie B4 (CB4) virus. The effects of the viruses on the islets were assessed by examination of their biochemical functions. It was found that the mouse pancreas-adapted strain of CB4 induced a 'leakage' of insulin from islets incubated at a basal (2 mmol l-1) glucose concentration, both at two and four days following infection. However, at a stimulatory concentration of glucose (20 mmol l-1) the rate of insulin secretion appeared to be normal in these islets. At two days the rate of total protein synthesis in islets infected with mouse pancreas-adapted CB4, incubated at high glucose concentration, was reduced; at four days the degree of inhibition was more severe, the rate at basal glucose concentration falling to half that of the control islets and at the stimulatory glucose concentration to a quarter of the control islets. (Pro)insulin biosynthesis was also inhibited, the rate being reduced to less than half the mean control value in islets infected with mouse pancreas-adapted CB4 virus at 20 mmol l-1 glucose at two days; at four days the rate was greatly reduced at both 2 and 20 mmol l-1 glucose. It is concluded from this study that only certain strains of CB4 virus can infect mouse pancreatic islets in vitro and that infection with strains of virus tropic for the islets leads to an impairment of metabolic functions of the B-cells, and is not necessarily lytic. PMID- 3015451 TI - Synergism between cytosolic and mitochondrial oxidation: its possible relevance to the metabolism of vitamin D3 in the kidney. AB - Using frozen liver sections and quantitative cytochemistry it has been established that when cells are allowed to oxidize a cytoplasmic and a mitochondrial substrate simultaneously the resulting oxidative activity is markedly higher than the sum of the oxidation of each substrate measured separately. In the present study this type of synergistic interaction has been confirmed in the kidney, particularly in cells of the pars recta. Our results support the evidence of the influence of cytoplasmic NADPH on the intramitochondrial oxidative process and it is suggested that, in cells of the pars recta, cytosolic NADPH may be involved in intramitochondrial mixed function oxidases such as 1 alpha-hydroxylase: these results could further elucidate the mechanism responsible for the production of the hormonal form of vitamin D3. PMID- 3015450 TI - Aggregation states of cyclic nucleotide phosphodiesterase of murine thymocytes. AB - In murine thymocytes cyclic nucleotide phosphodiesterase is represented by cAMP- and cGMP-specific forms. cAMP and cGMP phosphodiesterase activities showed anomalous kinetic behaviour indicative of 'low' and 'high' affinity enzyme forms. Sucrose density gradient centrifugation resolved only 'low' affinity forms of cAMP and cGMP phosphodiesterases. Gel filtration on Ultragel Aca 34 column showed that cAMP and cGMP phosphodiesterases are probably oligomeric enzymes. Storage of enzyme preparation at 4 degrees C for 24-48 h led to a decrease of higher molecular weight form and enhancement of cAMP and cGMP phosphodiesterase activities. PMID- 3015452 TI - The role of high-energy phosphate in norepinephrine-induced acute renal failure in the dog. AB - Previous studies have demonstrated that pretreatment with mannitol, furosemide, or bradykinin can attenuate the severity of norepinephrine-induced renal functional impairment. The present studies were designed to evaluate the possibility that these agents are protective, in part, by preserving cellular metabolic integrity. The renal cortex was repetitively biopsied during the course of this study, and high-pressure liquid chromatography was used to analyze the tissue content of adenine nucleotides (expressed in nanomoles per gram of wet tissue). The adenine nucleotide charge ratio (CR) and total adenine nucleotide (TAN) content were calculated as indices of cellular metabolic integrity. In addition to the above-established protective agents, phenoxybenzamine was used to evaluate a direct toxic effect of norepinephrine on renal tissue. Inulin clearance at 3 hours post infusion (expressed as a percent of control) was 7% with norepinephrine alone and, in the protected groups, 36% with bradykinin, 61% with furosemide, 51% with mannitol, and 100% with phenoxybenzamine. There was no change in CR or TAN with phenoxybenzamine. In contrast, during norepinephrine administration CR fell significantly in all other groups. Three hours after stopping norepinephrine, CR had returned toward control values and the level of CR was significantly better in all protected groups when compared with norepinephrine alone. Similarly, the levels of TAN were significantly diminished in the norepinephrine-alone group when compared to all protected groups, and there was significantly more tubular necrosis as well. The maintenance of higher levels of TAN and the preserved ability to regenerate adenosine triphosphate in the protected groups, when compared to the norepinephrine-alone group, support the contention that these agents offer protection, at least in part, by preserving cellular metabolic integrity. PMID- 3015454 TI - Relationship between serum angiotensin-I-converting enzyme activity and free thyroid hormones: a marker of macrophage activity? PMID- 3015453 TI - Adenosine-sensitive ventricular tachycardia: evidence suggesting cyclic AMP mediated triggered activity. AB - Catecholamine-induced triggered activity is thought to be caused by intracellular calcium overload mediated by elevation of intracellular cyclic AMP (cAMP). Although shown to occur in isolated preparations, evidence supporting its clinical existence has been lacking. Electrophysiologic studies were performed in four patients with structurally normal hearts who had exertionally related sustained ventricular tachycardia (VT). Programmed stimulation reproducibly initiated and terminated VT in all patients. Induction of tachycardia was also facilitated by infusion of isoproterenol. Adenosine, an endogenous nucleoside, whose only known electrophysiologic effect on ventricular myocardium and Purkinje fibers is antagonism of catecholamine-induced stimulation of intracellular cAMP production, reproducibly terminated all episodes of VT. The tachycardia was also terminated by intravenous verapamil and by the Valsalva maneuver and/or carotid sinus massage. Beta-Adrenergic receptor blockade with propranolol either terminated or prevented induction of VT during programmed stimulation or catecholamine challenge. Adenosine was also administered during VT to 14 patients whose arrhythmias fulfilled standard criteria for reentry, two of whom also had exercise-induced VT. Adenosine, at a dose (112.5 to 225 micrograms/kg iv) sufficient to cause either sinus slowing/arrest or ventriculoatrial block during ventricular pacing, failed to slow or terminate any episode of VT in these patients. Verapamil and autonomic modulation were also ineffective in this group of patients. Adenosine, verapamil, vagal maneuvers (acetylcholine), and beta adrenergic receptor blockade are all known to decrease the slow-inward calcium current either directly by modulating calcium channels or indirectly by inhibiting production of cellular cAMP. Therefore the observation in this study that interventions that lower intracellular cAMP either terminate or prevent induction of VT in patients with structurally normal hearts and exercise-induced VT suggests that the mechanism of tachycardia may be cAMP-mediated triggered activity. PMID- 3015455 TI - Na-K ATPase activity of erythrocyte membrane correlated with urinary Na/K ratio in normotensive adolescents with a family history of hypertension. AB - In female adolescents with a normal blood pressure range, the correlation between urinary sodium excretion and Na-K ATPase activity of erythrocyte membrane was investigated. 34 subjects with a family history of hypertension showed a significantly positive correlation between body mass index and mean blood pressure (r = 0.64, p less than 0.01), and a significantly lower urinary Na/Cr and Na/K ratio compared with 37 subjects without the same family history (p less than 0.05 and p less than 0.01, respectively). The positive correlation between urinary Na/K ratio and Na-K ATPase activity of erythrocyte membrane was demonstrated in only subjects with a family history of hypertension (r = 0.41, p less than 0.05). The authors discuss these significant correlations in terms of the abnormal membrane cation fluxes associated with essential hypertension. PMID- 3015456 TI - Mitogenic response of neoplastic B cells: comparison of reactivity to Staphylococcus aureus Cowan I and anti-immunoglobulin antibodies. AB - Neoplastic B cells from two patients with hyperleucocytic hairy cell leukaemia (HCL) and 19 patients with chronic lymphocytic leukaemia of B cell Type (B-CLL) were investigated to examine the mitogenic responses to the F(ab')2 fraction of anti-human immunoglobulins (anti-Igs) and Staphylococcus aureus Cowan I (STA). Neoplastic cells from both HCL patients lacked surface Tac antigen. Mononuclear cells from the two HCL patients strongly responded to both anti-Igs and STA as measured by 3H-thymidine incorporation in vitro. Although the mononuclear cells from two patients with B-CLL showed high response to STA, cells from none of the patients with B-CLL responded to anti-Igs. Mononuclear cells as well as T-cell depleted fractions from the two HCL patients showed a strong proliferative response by anti-gamma chain antibody (anti-gamma) and the mononuclear cells from one of the patients were also induced to proliferate by anti-delta, whereas those from normal subjects responded only to a high concentration of anti-mu. Based on the difference in reactivity to anti-Ig, it is suggested that the HCL cells in this study originate from a subset equivalent to 'memory' B cells, whereas the B CLL cells originate from a subset equivalent to 'virgin' B cells. PMID- 3015457 TI - Increased frequency of antibodies to ubiquitous viruses in essential mixed cryoglobulinaemia. AB - Antibodies to ubiquitous Herpes viruses have been studied in 13 patients with type II essential mixed cryoglobulinemia (EMC), and in two different control groups. All the EMC patients had monoclonal IgM kappa in their cryoprecipitates. IgM antibodies to the viral capsid antigen (VCA) of Epstein-Barr virus (EBV) were found in the sera of 11 EMC patients but all the cryoprecipitates were negative. IgG antibodies were also present in the sera of all and in the cryoprecipitates of some patients. In contrast, the number of subjects with antibodies to Cytomegalovirus (CMV) and Hepatitis B Virus (HBV) was not higher than in controls. Possible correlations between EMC and EBV infection are discussed. PMID- 3015459 TI - Modulation of proximal tubular reabsorption by angiotensin II. AB - In shrinking-drop micropuncture studies in anaesthetized rats proximal tubular fluid reabsorption (JVa) decreased by 36% following intravenous infusion of enalapril. In a separate group of rats enalapril reduced fractional lithium clearance indicating decreased proximal fluid reabsorption. Following enalapril, GFR rose by 46% but absolute proximal reabsorption rose by 22% indicating 48% effectiveness of proximal glomerulo-tubular balance (GTB). Since renal blood flow and glomerular filtration rate (GFR) increased in parallel and arterial pressure fell, fluid uptake and proximal GTB were unlikely to have been decreased by peritubular physical forces. In anaesthetized rats proximal fluid reabsorption and proximal GTB are modulated by endogenous AII through direct stimulation of proximal tubular transport. PMID- 3015458 TI - Effects of tetradecapeptide renin substrate on isolated preparations of rat caudal artery, guinea-pig atria and on pithed rat blood pressure. AB - In rat isolated caudal artery preparations, tetradecapeptide renin substrate enhanced the responses to sympathetic nerve stimulation (0.5 Hz, 10 s). In guinea pig isolated atria, previously incubated in 3H-noradrenaline, tetradecapeptide renin substrate enhanced the stimulation-induced efflux of radioactivity. The facilitation of noradrenergic transmission in rat caudal arteries and guinea-pig atria was blocked by the angiotensin II receptor antagonist saralasin, but was not altered by converting enzyme inhibitors. In the pithed rat tetradecapeptide renin substrate increased blood pressure and this effect was reduced by the converting enzyme inhibitor, captopril. PMID- 3015460 TI - Pressor responsiveness in steroid-induced hypertension in man. AB - Pressor responsiveness to angiotensin II (AII) and phenylephrine (PE) was examined before and after 5 days of ACTH (1 mg, i.m., daily) or hydrocortisone (200 mg, orally, daily) in six normotensive men. Pulse pressure was higher prior to PE than AII infusion, presumably due to feeding. Systolic blood pressure (SBP) was increased by both ACTH and hydrocortisone treatment, but more by ACTH. There were no significant changes in AII pressor responsiveness with either ACTH or hydrocortisone. ACTH increased pressor responsiveness to PE at 1.35 and 2 micrograms/kg per min, and hydrocortisone at 0.6-2 micrograms/kg per min, with falls in pulse rate at 0.3-0.9 micrograms/kg per min. Changes in pressor responsiveness do not explain ACTH hypertension. PMID- 3015461 TI - Transient immune deficiency in patients with acute Epstein-Barr virus infection. AB - To study the effect of primary Epstein-Barr virus (EBV) infection on antigen specific antibody production, we immunized 17 college students who had developed acute infectious mononucleosis with the T-cell dependent neoantigen bacteriophage phi X174. During the early phase of infectious mononucleosis, the proportion of peripheral blood lymphocytes displaying Ia and T8 (CD8) phenotypes was increased and the T helper/suppressor (T4/T8) ratio was decreased (less than 1). These abnormalities disappeared during the convalescent phase. Correlating with EBV induced changes in T lymphocytes, we demonstrated depressed humoral immune responses to bacteriophage phi X174 both in vivo and in vitro. In vitro coculture experiments indicated that the Ia+ suppressor T cells could inhibit antibody production and isotype switch. Removal of T8+ lymphocytes from patient T cells normalized in vitro antibody synthesis. In addition, impaired B-cell function was shown to be in part responsible for deficient antibody production. These studies demonstrate that infection with EBV affects both B and T lymphocytes and causes a broad-based transient immune deficiency in patients with uncomplicated infectious mononucleosis. PMID- 3015462 TI - T-cytotoxic/suppressor cell phenotypes in a group of asymptomatic homosexual men with and without exposure to HTLV-III/LAV. AB - The number of lymphocytes bearing the Leu 2+ or T8+ (suppressor/cytotoxic) phenotype is elevated in asymptomatic homosexual men. By two-color immunofluorescence using paired monoclonal antibodies (alpha-Leu 2 and alpha-Leu 15, alpha-Leu 2 and alpha-Leu 7, alpha-Leu 7 and alpha-Leu 11), we enumerated phenotypic subpopulations that are associated with cytotoxic, suppressor, or natural killer function. Both cytotoxic (Leu 2+15-) and suppressor (bright Leu 2+15+) cell populations are elevated in homosexual men. Homosexual men who have been exposed to human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV) have higher numbers of Leu 2+15- and Leu 2+7- cells than homosexual men who have not been exposed. Phenotypic subpopulations (dim Leu 2+ Leu 15+ and Leu 7-11+) that are associated with the most potent natural killer activity (against K562 target cells) were not found to be elevated in homosexual men. PMID- 3015463 TI - Different complement and granulocyte activation in patients dialyzed with PMMA dialyzers. AB - Plasma C3a and C5a levels as well as plasma levels of granulocyte lactoferrin, granulocyte myeloperoxidase and granulocyte elastase in complex with alpha 1 proteinase inhibitor (E-alpha 1PI) were investigated in 10 patients (52.7 +/- 5.9 years) undergoing maintenance hemodialysis (39.4 +/- 12.4 months) with hollow fiber dialyzers made from polymethylmethacrylate. Plasma levels of lactoferrin increased from 166.5 +/- 28.5 to 712.5 +/- 165.9 ng/ml, myeloperoxidase from 59.0 +/- 15.3 to 210.5 +/- 33.9 ng/ml and E-alpha 1PI from 114.2 +/- 18.1 to 681.8 +/- 102.6 ng/ml during dialysis. In contrast, plasma C3a levels rose from 179.8 +/- 33.6 to maximal 276.2 +/- 45.4 ng/ml and C5a from 55.7 +/- 8.1 to maximal 101.1 +/- 14.8 ng/ml. Our data indicate that degranulation of granulocytes occurs during dialysis despite only little complement activation and mild initial granulocytopenia. PMID- 3015464 TI - Colposcopic assessment of the lower genital tract in female renal transplant recipients. AB - The incidence of epithelial or cutaneous abnormality of the lower genital tract was studied colposcopically in 31 women aged 35 (SD9) years on renal replacement therapy for one to 184 months (mean 73 months). Twenty-four women had a renal transplant and had received immunosuppressive therapy for two to 173 months (mean 61 months). Seven women (two with failed transplants) were on home hemodialysis (one to 48 months, mean 19 months) awaiting transplantation. In 22 women (17 transplant and 5 dialysis patients) no abnormality was found. One hemodialysis patient had an extensive area of vulval intra-epithelial neoplasia. Five transplant recipients had evidence of genital papilloma virus (HPV) infection. No patient had cervical intra-epithelial neoplasia or invasive genital tract malignancy. The high incidence of HPV-associated lesions in this group, and the increasing evidence that HPV changes may predispose to cervical neoplasia suggest that this high risk group require regular gynecological assessment. PMID- 3015465 TI - Arachidonate metabolism in blood cells and the vessel wall. AB - Arachidonic acid (AA) is metabolized by the cyclo-oxygenase and the lipoxygenase pathways to give a number of products, some of which have potent and sometimes opposing biological activities. Different cell types produce different metabolites, so that the chief AA metabolite produced by the platelet is the pro aggregatory thromboxane A2 (TXA2), whereas that produced by the vascular endothelium is the anti-aggregatory prostacyclin. White blood cells, on the other hand, are the chief source of the leukotrienes, which are implicated in the inflammatory process. Generation of these products may be modified in certain pathological conditions, such as atherosclerosis and diabetes, where prostacyclin synthesis is reduced and TXA2 synthesis increased, resulting in a pro-thrombotic state. Synthesis of AA metabolites may be inhibited, either totally or selectively, using drugs which inhibit different enzymes in the metabolic pathway. These drugs may be beneficial in the treatment of thrombotic disorders and inflammation. AA metabolism may also be modified by dietary substitution with eicosapentaenoic acid, a fatty acid present in fish oils. PMID- 3015466 TI - The natural anticoagulants. PMID- 3015468 TI - Pulmonary endothelium: a dynamic interface. AB - Appreciation of the cell biology of endothelial cells has contributed greatly to understanding of endothelium as a tissue. Endothelium can no longer be considered simply as an inert barrier with fixed permeabilities, nor as an unreactive expanse of non-thrombogenic surface. Pulmonary endothelium is now recognized to be a tissue composed of metabolically active, functionally responsive cells, that interact with circulating substrates and formed elements in ways that regulate the composition of systemic arterial blood, affect target organ functions, and contribute to thrombosis, hemostasis and immune reactions. Much impetus for the new appreciation of endothelial cell structures and functions has been derived from the ability to isolate and culture pulmonary endothelial cells. Improvements in culture conditions and the growing awareness that biochemical, ultrastructural, as well as physical (fluid mechanical), considerations must be taken into account should pave the way for greater understanding of the role of the endothelium in health and disease. PMID- 3015467 TI - The generation of superoxide anions by polymorphonuclear leucocytes from patients with ankylosing spondylitis in response to the stimulant f-met-leu-phe. AB - Superoxide anion (O2-) generation in response to the chemoattractant f-met-leu phe was measured in PMNL from 30 patients with ankylosing spondylitis and in PMNL from 27 controls. At a concentration of stimulant above that necessary to achieve maximal response, no differences in O2- production between the groups were noted. With a lower concentration of stimulant, cells from patients with AS or RA taking non-steroidal anti-inflammatory drugs had lower levels of O2- generation, compared both to controls and to AS patients, off medication. Results suggest any decrease in O2- generation seen in patients with AS is likely to be related to therapy and not to the disease or to possession of HLA B27. PMID- 3015469 TI - The relationship between 2-5A synthetase levels and persistent lymphadenopathy in homosexual men with antibodies to HTLV-III. AB - This study compared clinical and laboratory parameters in 37 individuals with serologic evidence of exposure to human T-cell lymphotropic virus type 3 (HTLV III) and generalized lymphadenopathy. Basal levels of the interferon-induced enzyme, 2-5A synthetase were also measured and compared with the clinical parameters. A significant linear relationship (p less than .05) was demonstrated between the logarithm of the basal synthetase level and both the number and size of palpable nodes in the posterior cervical triangles of the men studied. No other lymph node chains demonstrated such a relationship. In addition, men with more than 3 palpable nodes (the median number) in the posterior triangles had lower mean levels of lymphocytes, reduced mean mitogen responses to PWM, PHA, and ConA, elevated mean levels of IgG, IgA, and IgM, and were more frequently anergic on skin testing. These findings suggest the need for further longitudinal study of the possibility that extent of lymphadenopathy and level of 2-5A synthetase might serve as useful parameters to monitor disease activity. PMID- 3015471 TI - [A case of consciousness disturbance and polyneuropathy after exposure to paraquat and its nerve biopsy findings]. PMID- 3015470 TI - Nuclear cardiology techniques in the assessment of ischemic heart disease. AB - Invasive techniques of cardiac catheterization and angiography have become the gold standard for the diagnosis and management of patients with ischemic heart disease. More recently there has been a remarkable development of noninvasive imaging techniques which has resulted in improved ability to select patients in need of invasive investigations and in a more complete understanding of the physiological and clinical significance of information obtained from such invasive investigations. The value and limitations of the 3 most common techniques, radionuclide ventriculography, myocardial perfusion scintigraphy and acute myocardial infarction scintigraphy, are discussed in this review in relation to the assessment of patients with proven or suspected ischemic heart disease. These nuclear cardiology techniques are now available in most hospitals with nuclear medicine equipment; a good understanding of the strengths and weaknesses of each technique is essential for optimal clinical use. PMID- 3015472 TI - Evaluation of intrahepatic I-131 ethiodol on a patient with hepatocellular carcinoma. Therapeutic feasibility study. AB - This study assesses the therapeutic efficacy of radiolabeled iodized oil on a patient with hepatocellular carcinoma (HCC). An iodized oil, such as Lipiodol or Ethiodol (Savage Laboratories, Melville, NY), was retained selectively in the tumor vessels of large tumors as well as in the daughter tumors of HCC for long periods of time following intra-arterial injection into the hepatic artery proper. A small fraction of the stable iodine (1 pg of I-127) of the 37% iodine by weight in Ethiodol was replaced by the I-131 with 100% efficiency. A patient with HCC was injected with I-131 Ethiodol into the hepatic artery. Sequential imaging of organs such as the liver, lung, stomach, and thyroid over an eight-day period demonstrated a high tumor-to-normal-liver ratio and a negligible amount of radioactivity in these organs. These findings indicate that I-131 Ethiodol, or Ethiodol labeled with other pure beta emitters, such as Y-90 or P-32, will be effective delivering a high internal radiation dose to HCC with a small radiation effect to normal tissues. To evaluate its potential as a radiotherapeutic agent for HCC, the kinetics, biodistribution, determination of absolute activity in the tumor following intra-arterial injection of I-131 Ethiodol will be studied in the future. At the same time, an effort will be made to label Ethiodol with Y-90 and P-32. PMID- 3015473 TI - Beta-adrenergic receptors in the elderly are not less sensitive to timolol. AB - The elderly have been reported to be less sensitive to the beta-adrenergic blocking effect of propranolol. However, propranolol is a racemate, and age related changes in stereoselective metabolism or protein binding could confound interpretation of the data. To avoid these problems, we studied timolol in 12 young and 12 elderly healthy subjects. The dose of isoproterenol required for a heart rate increase of 25 bpm (I25) was determined before and 2 hours after an oral 10 mg dose of timolol. A dose ratio (DR) was calculated for each subject as the I25 after timolol/I25 before timolol. The binding constant for timolol binding to the receptor was calculated as the plasma timolol concentration divided by (DR-1). The I25 for the elderly group was significantly greater than the I25 for the young group, but the timolol binding constant was the same for both groups. We conclude that, although the elderly are less sensitive to isoproterenol, they are not less sensitive to timolol, and thus our data do not implicate a change in the interaction of beta-adrenoceptors with antagonists. PMID- 3015474 TI - Reflex vagal withdrawal and the hemodynamic response to intravenous isoproterenol in the presence of beta-antagonists. AB - To investigate the contribution of reflex vagal tone to the hemodynamic response after intravenous isoproterenol, 12 healthy subjects received isoproterenol by both bolus injection and continuous infusion before and after atropine, and during intravenous infusion of the beta 1-selective antagonist atenolol and the nonselective beta-antagonist, propranolol. With bolus injections, atropine displaced the heart rate dose-response curve for atenolol to the right, implying reflex withdrawal of cardiac vagal tone, but did not alter the heart rate dose response curve for propranolol. With continuous infusions of isoproterenol, atropine displaced the heart rate dose-response curves for both atenolol and propranolol to the left, implying the presence of a reflex increase rather than withdrawal in cardiac vagal tone. These reflex changes in cardiac vagal tone can be partly understood by changes in mean arterial pressure and pulse pressure. As the two methods of isoproterenol administration are associated with contrasting contributions from reflex vagal tone, dose ratios obtained for the displacement of the heart rate dose-response curve by beta-antagonists may differ. PMID- 3015475 TI - A clinical study of the anticalculus effect of a dentifrice containing soluble pyrophosphate and sodium fluoride. PMID- 3015476 TI - The effect on calculus deposits of a dentifrice containing soluble pyrophosphate and sodium fluoride. A 3-month clinical study. PMID- 3015477 TI - Computed tomography and ultrasonography of hepatoma. AB - Computed tomography (CT) scans and sonograms of 37 patients with hepatocellular carcinoma (hepatoma) were reviewed to determine the characteristics of the tumour and to compare the modalities in terms of accuracy in defining tumour morphology and ability to predict vascular invasion and extrahepatic spread. By CT, slightly over 50% of the tumours were multicentric, about 40% were solitary, and the rest were diffuse. About half of the hepatomas were heterogeneous in density before injection of contrast agent and most became enhanced in a non-uniform manner. In addition, about a quarter of the tumours either became visible or were better seen after injection of contrast agent. At sonography, approximately two-thirds of the neoplasms were thought to be solitary and one-third multicentric. The majority also had a mixed echo texture. Although the lesion was identified in all 13 patients who had both studies, sonography underestimated the extent of hepatic involvement in 38% of the cases. Sonography also failed to demonstrate lymphadenopathy that was detected by CT in two patients. In general, both techniques were effective in identifying vascular invasion. CT was very accurate in showing the extent of hepatic involvement but was unable to identify direct invasion of neighbouring structures. Because each technique has limitations in the evaluation of hepatoma, we believe that both should be performed if curative resection is being considered. PMID- 3015479 TI - Vascular masses in the middle ear. AB - High resolution computed tomography (CT) is of great value in demonstrating soft tissue masses in the middle ear cavity. However, tissue characterisation even for vascular masses after contrast enhancement has proved disappointing. Differentiation therefore depends upon the site and anatomical configuration of the mass, and in many cases angiography is mandatory for diagnosis. Examples of high ectopic jugular bulb, glomus jugulare and glomus tympanicum tumours and aberrant internal carotid artery are presented and their differential diagnosis considered. The value of CT and more traditional techniques, particularly lateral tomograms to show the spur of bone between the jugular bulb and internal carotid artery, are discussed. PMID- 3015478 TI - The radiology of fibrolamellar hepatoma. AB - Seven patients with fibrolamellar hepatoma were examined with computed tomography (CT), ultrasonography and angiography. On CT the tumours were large, of low attenuation, had a well-defined edge and some contained areas of calcification or necrosis. Ultrasonography revealed well-defined masses of mixed echogenicity, occasionally involving the portal vein. In one patient there was dilatation of the intrahepatic biliary tree. Arteriography showed vascular tumours with involvement of the portal vein in five cases and compression of the inferior vena cava in five cases. CT and ultrasonography are the most useful radiological investigations for suggesting the diagnosis of fibrolamellar hepatoma which should be considered in the case of any large solitary well-defined hepatic tumour in the noncirrhotic liver of a young person. PMID- 3015480 TI - A rapid procedure, by collagenase treatment and cytocentrifugation, for the cytological evaluation of bone marrow biopsy specimens. PMID- 3015481 TI - Heat-treated NHS factor VIII concentrate in the United Kingdom--a preliminary study. AB - Three patients who have been given intermediate purity NHS heat-treated factor VIII concentrate have been followed prospectively for 7-10 months. None had previously received more than six donor units of blood products containing factor VIII. There were no clinical side effects from concentrate administration, haemostasis was satisfactory and no patient developed clinical or laboratory evidence of hepatitis or HTLV III/LAV infection. Heat treatment resulted in the loss of slightly more than 20% of factor VIII activity but in vivo recovery of factor VIII and half disappearance times were within the expected range. PMID- 3015482 TI - Sarcoidosis. PMID- 3015483 TI - Human fibroblast tissue inhibitor of metalloproteinases: glycosylation and function. AB - The glycosylation of human fibroblast tissue inhibitor of metalloproteinases and procollagenase was examined in vivo using tunicamycin B2 and in vitro using Peptide: N-glycosidase F. In the presence of tunicamycin B2, unglycosylated inhibitor continues to be synthesized and secreted at normal or increased rates. The protein core of this collagenase inhibitor has an apparent Mr of 21,000 and possesses at least two oligosaccharide linkage sites as evidenced by the accumulation of a single 25,000 dalton intermediate. Collagenase inhibitor deglycosylated by Peptide: N-glycosidase also has an apparent molecular weight of 21,000 daltons; furthermore, deglycosylated inhibitor continues to block the activity of collagenase at a 1:1 molar stoichiometry and does not differ from the native glycoprotein in its resistance to tryptic degradation. Using these same two reagents, the characteristic doublet of human fibroblast procollagenase was found to result from glycosylation in the upper (60,000 dalton) form. Secretion of procollagenase was significantly inhibited in the presence of tunicamycin B2. PMID- 3015485 TI - A comparative study of the purine- and pyrimidine-metabolising enzymes of a range of trypanosomatids. AB - A range of trypanosomatids (amastigotes and cultured promastigotes of Leishmania mexicana mexicana, cultured promastigotes of L. m. amazonensis, L. donovani and L. tarentolae, culture forms of Crithidia fasciculata, Herpetomonas muscarum muscarum and H. m. ingenoplastis and procyclic trypomastigotes of Trypanosoma brucei brucei) have been surveyed for the presence of purine- and pyrimidine metabolising enzymes. Several common features were observed, including the presence of nucleosidases, catabolic phosphorylases, phosphoribosyltransferases, kinases and cytidine deaminase and the apparent absence of AMP deaminase, anabolic purine phosphorylase and cytosine deaminase. Significant differences between species were discovered, notably in adenine and adenosine metabolism. Nucleoside phosphotransferase active on inosine was detected in insect trypanosomatids but not in L. m. mexicana. PMID- 3015484 TI - Response of liver and kidney adenylate kinase to fasting and refeeding in three strains of mice. AB - The effects of fasting and refeeding on the AK isozymes in liver and kidney were studied in three strains of mice. Our studies showed that changes in total AK activity and AK isozyme patterns were associated with fasting and refeeding. The AK isozyme changes were strain-dependent, differing in kind and degree among the three strains. It was concluded that species, strain and individual isozyme identities should be included in studies defining changes of enzyme activity owing to changes in physiological conditions. PMID- 3015486 TI - A computer model of neuronal pathways in the basal ganglia. AB - Disorders of the basal ganglia and the extrapyramidal motor system exhibit an imbalance of neurotransmitter concentrations in affected neurons. For three synapses with dopamine, acetylcholine, and gamma-amino butyric acid (GABA), mathematical models of synaptic transmission are developed. To describe the kinetics of transmitter substances, compartment analysis is used. Membrane potential behaviour is described by the Hodgkin-Huxley equations with an additional equation accounting for a presynaptic calcium current mediating transmitter release. At the postsynaptic site, activated receptor molecules control the activity of ion channels, eliciting either inhibitory or excitatory postsynaptic potentials. A simple model of the feedback loop connecting the caudate nucleus and the substantia nigra is simulated on a digital computer using the simulation language ACSL. A comparison of the control case with a model of Parkinson's disease shows a shift of eigenvalues towards zero in the diseased state. PMID- 3015487 TI - Effect of vaginal contraceptive sponges on growth of toxic shock syndrome associated Staphylococcus aureus in vitro. AB - Toxic Shock Syndrome (TSS) is associated with certain toxin-producing strains of Staphylococcus aureus (TSS-S aureus), and with the use of some vaginal devices such as tampons or contraceptive diaphragms. The present study was designed to examine the effect of Nonoxynol-9 (N-9), and of a newly-approved vaginal contraceptive sponge (VCS) containing N-9 on the growth of TSS-S aureus in vitro. Flasks containing culture media inoculated with TSS-S aureus were incubated at 37 degrees C for 30 hours, in the presence or absence of either a VCS, or N-9 alone. At 0.5, 1, 2, 6, and 12 hours, there was suppression of TSS-S aureus colony counts in media containing VCS, compared to control. Colony counts from media containing N-9 demonstrated suppression at 0.5, 1, 2, and 6 hours. After incubation as long as 30 hours, colony counts from VCS-containing media approached, but did not exceed, counts from control media. From these data, it is concluded that this VCS containing N-9 does not enhance the growth of TSS-S aureus in vitro. Instead, an inhibition of bacterial growth for at least 12 hours is observed in media containing VCS, consistent with a bacteriostatic effect. If such an effect is also present in vivo, it would suggest that this type of VCS is unlikely to increase the risk of TSS. PMID- 3015488 TI - Coronavirus antibody detection in cats by computer-assisted kinetics-based enzyme linked immunosorbent assay (KELA): field studies. AB - A total of 2238 feline serum samples submitted to the New York State Diagnostic Laboratory over a 1-year period were tested for the presence of coronavirus antibodies, using the computer-assisted, kinetics-based enzyme-linked immunosorbent assay (KELA). Cats from which sera were obtained were categorized by sex, age, breed, and disease status, and variations in mean antibody titers for different sub-classifications within each category were analyzed by computerized statistical analysis. As expected, higher mean antibody titers were recorded for cats with feline infectious peritonitis, and for cats with a recent history of possible coronavirus exposure. However, an unexpected inverse relationship between coronavirus antibody titer and age was also found. Certain cattery-oriented pure breeds appeared to have higher mean antibody titers, because their sample populations contained a higher percentage of younger cats and cats of unknown age-groups which, over-all, had higher mean titers. Taken together, the data substantiated the efficacy of the computer-assisted KELA for routine detection of serum coronavirus antibodies in cats. PMID- 3015489 TI - Adrenergic regulation of the acute ischaemic myocardium: facts, interpretations and consequences. AB - Anoxic stress is accompanied by activation of the central and peripheral sympathetic nervous system resulting in a high local catecholamine concentration. The authors studied how myocardial cells cope with the high level of catecholamines under ischaemic conditions. The beta-adrenoceptor-adenylate cyclase system (AC) was investigated in different models of ischaemia and anoxia (global ischaemia, low-perfused hearts, coronary artery ligation) in rat hearts. It was shown that beta-receptor function is not changed up to 40 min of ischaemia. Myocardial AC function was depressed in the total ischaemic myocardium but not in the low-perfused hearts indicating a non-uniform alteration of AC function. Reduced AC activity was completely reversible by aerobic perfusion as long as the ischaemic period did not exceed 20 min. Depression of AC function during severe ischaemia was avoided by reducing Ca2+ in the extracellular fluid and by pretreatment with Ca2+ channel blockers (verapamil). Depression of AC function during severe ischaemia is caused mainly by increased intracellular Ca2+ which inhibits AC at its catalytic site. Myocardial ischaemia alters the response of myocardial cells to catecholamines and other activators of the AC system. This alteration is time-limited and turns damage to AC function from reversible to irreversible after prolongation of ischaemia to more than 30 min. PMID- 3015490 TI - Structure of transcriptionally active chromatin. AB - Transcriptionally active or potentially active genes can be distinguished by several criteria from inactive sequences. Active genes show both an increased general sensitivity to endonucleases like DNase I or micrococcal nuclease and the presence of nuclease hypersensitive sites. Frequently, the nuclease hypersensitive sites are present just upstream of the transcription initiation site covering sequences that are crucial for the promoter function. Viral or cellular transcription enhancer elements are also associated with DNase I hypersensitive sites. At least for the SV40 enhancer, it was shown by electronmicroscopic studies that the DNase I hypersensitive DNA segment is excluded from nucleosomes. It is highly plausible that the binding of regulatory proteins to enhancer or promoter sequences is responsible for the exclusion of these DNA segments from nucleosomes and for the formation of nuclease hypersensitive sites. We speculate that the binding of such proteins may switch on a change in the conformation and/or the protein composition of a chromatin segment or domain containing one to several genes. Biochemical analysis of fractionated nucleosome particles or of active and inactive chromatin fractions have revealed differences in the composition as well as in the degree of modification of histones in these two subfractions of the chromosome. However, until present it is impossible to define unambiguously what are the crucial structural elements that distinguish between particles present on active and inactive chromatin. PMID- 3015491 TI - Endogenous digitalis-like factors. AB - Recent research has demonstrated the presence of endogenous compounds in blood and urine that crossreact with antibodies raised against digoxin. Given the widespread therapeutic use of digoxin and its being monitored clinically by immunoassay, such digoxin-like immunoreactive compounds pose significant diagnostic and interpretive problems. Serum levels of this factor(s) approaching therapeutic digoxin levels have been found in digoxin-free patients in renal failure, pregnant women, and newborns. The compound is incompletely characterized; however, existing data suggest that it is a small, neutral, nonpeptidic compound. In serum it is highly protein bound, and alterations in this binding appear to give rise to the false-positive assay results. The urinary form is probably conjugated. Digoxin-like immunoreactive substances may play a role in volume homeostasis and appear associated with essential and pregnancy induced hypertension. If such roles are primary, measurement of digoxin-like immunoreactive substances may prove to be of value in and of itself. PMID- 3015492 TI - Resistance of alpha-crystallin-glutathione mixed-disulfide to tryptic digestion. AB - Protein-mixed disulfides (PSSG) were formed by interaction of glutathione disulfide (GSSG) with lens crystallins. Total water-soluble crystallins and alpha crystallin purified on a Sephacryl S-200 column were separately incubated with 0, 2, 4, and 8 mM (final concentrations) GSSG overnight and then dialyzed to remove unbound GSSG and GSH. Either TPCK-treated trypsin or TLCK-treated alpha chymotrypsin were added to about 200 micrograms crystallin samples and incubated for 20 min at room temperature. Reactions were terminated by boiling in SDS mercaptoethanol-Tris (pH 6.8) solution and subjected to electrophoresis on 10% polyacrylamide slab gels. Comparison of SDS-PAGE patterns of proteolysis with or without GSSG treatment showed that GSSG at a concentration of 2 mM or higher reduced or abolished proteolysis of alpha-crystallin by trypsin but not by alpha chymotrypsin. The protective effect of GSSG was greater with alpha-crystallin than with beta-crystallins. Addition of alpha-crystallin-mixed-disulfide to an assay system in which trypsin was hydrolyzing N-alpha-benzoyl-DL-arginine-P anilide (BAPNA) inhibited the tryptic activity. Direct addition of GSSG or native alpha-crystallin had no significant inhibitory effect on trypsin. Based on these results, it is speculated that alpha-crystallin glutathione mixed-disulfide appears to become resistant to trypsin probably by non-competetive inhibition of the enzyme. PMID- 3015493 TI - Superficial membrane -SH groups inaccessible by intracellular GSH. AB - The importance of membrane -SH groups in the epithelium and posterior fiber cells of rabbit lens was demonstrated by employing a non-penetrating sulfhydryl reagent parachloromercuribenzoate sulfonic acid (PCMBS). Both fiber cell and epithelial membrane preparations contain substantial amounts of -SH, 31 nmoles/mg membrane protein. PCMBS-treatment of anterior and posterior surfaces of the lens leads to dramatic increases in the calcium influx across both anterior and posterior surfaces, indicating that the importance of membrane -SH groups is not limited to the epithelium. When the entire lens is bathed in PCMBS (0.1 mM) for short duration and transferred to normal medium, calcium continues to increase from 0.4 mM to nearly 1 mM over a 20 hr period. At this point in time, GSH levels are normal, indicating that intracellular GSH does not gain access to PCMBS-binding sites. In contrast, external GSH or cysteine, at lower levels (5 mM) quickly reverses PCMBS binding with membrane -SH groups and leads to near normal levels of lens calcium during subsequent culture. This in addition to the fact that PCMBS is not found in the cell interior where GSH levels are undiminished, suggests that the critical -SH groups involved in control of barrier properties are externally located where little protection from intracellular GSH is afforded. These data indicate that aqueous humor GSH may play a critical role in maintaining reduced -SH groups controlling membrane permeability located on the surface of membranes. PMID- 3015495 TI - Restriction enzyme polymorphisms in V kappa and J kappa genes of inbred and wild mice. PMID- 3015494 TI - The protective effect of glucose on soluble rat lens hexokinase in the presence of oxidative stress. AB - An in vitro animal model was used to characterize the protective effect of glucose on lenses subjected to oxidative stress. Paired rat lenses were incubated in TC-199 medium for six hours in the presence of an oxidant (0.06 mM H2O2, superoxide produced from 5 mM purine, or hydroxyl radical) and 2 mM glucose (control) or no glucose (experimental). Soluble hexokinase (HK) specific activity and lactate production were measured. 0.06 mM H2O2 inactivates 48% of the hexokinase in the absence of glucose; with glucose present hexokinase activity is reduced only 26%. Control experiments without oxidants show a statistically insignificant difference between hexokinase activities in the 0 and 2 mM groups, suggesting that the changes observed are not simply due to the presence or absence of glucose. Hexosemonophosphate shunt activity increases nearly 2.5-fold in the presence of 0.06 mM H2O2 and 2.0, 4.0 or 5.5 mM glucose. This suggests that the loss of hexokinase (a -SH enzyme) in the presence of H2O2 and 0 mM glucose is due to NADPH production inadequate to offset the oxidative stress on enzyme -SH groups. FPLC analysis suggests that type II HK is more susceptible to oxidative inactivation than type I, and further studies have shown that this inactivation is localized to the capsule/epithelium. Lactate levels were measured and controls (without oxidants) were run, to obtain a baseline value for fresh lenses and assess the contribution of endogenous glucose to lactate production. H2O2 levels in superoxide and hydroxyl radical media were measured, and the protective effects of mannitol and catalase were also determined. PMID- 3015497 TI - Variable resistance to ectromelia (mousepox) virus among genera of Mus. PMID- 3015496 TI - Genetically controlled resistance to flaviviruses within the house mouse complex of species. PMID- 3015499 TI - Xenotropic and MCF related retroviral genes in wild mice. PMID- 3015500 TI - Mammary tumorigenesis in feral species of the genus Mus. PMID- 3015501 TI - Endogenous MMTV proviral genomes in feral Mus musculus domesticus. PMID- 3015498 TI - Molecular mechanism of an ecotropic MuLV restriction gene Akvr-1/FV-4 in California wild mice. PMID- 3015502 TI - Patterns of mediastinal metastases in bronchogenic carcinoma. AB - The location and frequency of metastases to the lymph nodes were documented in a review of 200 patients with bronchogenic carcinoma who underwent pulmonary resection and total lymph node resection. No nodal metastases were found in 120 patients (60 percent). Metastases were present in only lobar or hilar nodes (or both) in 32 patients (16 percent), and 34 (17 percent) had metastases in mediastinal nodes as well as in lobar or hilar nodes. Only mediastinal nodal metastases were found in 14 patients (7 percent). Previously described lymphatic pathways can explain the presence of metastases in mediastinal nodes alone. Unexplained findings were the higher prevalence of mediastinal nodal metastases in adenocarcinoma vs squamous cell carcinoma and a much higher frequency of mediastinal metastases without lobar or hilar involvement (or both) in patients with adenocarcinoma compared to those with squamous cell carcinoma. PMID- 3015503 TI - Absence of effects on platelet membrane fluidity by antibiotics in ESR spectra. AB - The effect of several antibiotics on rat platelet membrane fluidity was examined by ESR spectrometry using 5-doxyl stearate as the spin probe. Latamoxef, cefamandole, cefotaxime and carbenicillin had negligible effects on the order parameters, the indices of membrane fluidity, of platelet-rich plasma or washed platelets at high concentrations that would inhibit ADP-induced rat platelet aggregation in vitro. A further in vivo study with one of these antibiotics, latamoxef, showed no significant effect on the fluidity based on ESR data. PMID- 3015504 TI - Studies on muntiacus muntjak cells infected with poliovirus. PMID- 3015507 TI - [Radiologic-pathologic correlative study on X-ray findings on bronchotomograms in lung cancer]. PMID- 3015505 TI - Localization and characterization of recombinant DNA clones derived from the highly repetitive DNA sequences in the Indian muntjac cells: their presence in the Chinese muntjac. AB - A total of seven, highly repeated, DNA recombinant M13 mp8 clones derived from a Hpa II digest of cultured cells of the Indian muntjac (Muntiacus muntjac vaginalis) were analyzed by restriction enzymes, in situ hybridization, and DNA sequencing. Two of the clones, B1 and B8, contain satellite DNA inserts which are 80% homologous in their DNA sequences. B1 contains 781 nucleotides and consist of tandem repetition of a 31 bp consensus sequence. This consensus sequence, TCCCTGACGCAACTCGAGAGGAATCCTGAGT, has only 3 bp changes, at positions 7, 24, and 27, from the consensus sequence of the 31 bp subrepeats of the bovine 1.715 satellite DNA. The satellite DNA inserts in B1 and B8 hybridize primarily but not specifically to chromosome X, and secondarily to other sites such as the centromeric regions of chromosomes 1 and 2. Under less stringent hybridization conditions, both of them hybridize to the interior of the neck region and all other chromosomes (including chromosomes 3 and Y). The other five DNA clones contain highly repetitive, interdispersed DNA inserts and are distributed throughout the genome except for the neck region of the compound chromosome X + 3. Blot hybridization results demonstrate that the satellite DNA component is also present in Chinese muntjac DNA (Muntiacus reevesi) in spite of the very different karyotypes of the Chinese and Indian muntjacs. PMID- 3015506 TI - Human cellular sequences detectable with adenovirus probes. I. Evidence for novel repeat sequences and a possible E1a-like cellular "gene". AB - Previous studies suggesting homology between human cellular DNA and the DNAs from adenovirus types 2 and 5 are extended in the present paper. A clone (ChAdh), isolated from a human genomic DNA library using an adenovirus probe, hybridized to discrete regions of adenovirus 2 DNA, including part of the transforming genes E1a and E1b, as well as to repeated sequences within human DNA. The E1a and E1b genes both hybridize to the same 300 base pair Sau3AI fragment within ChAdh although there is no obvious homology between E1a and E1b. The Ad 2 E1a gene was also used as a probe to screen other cellular DNAs to determine whether repeated sequences detectable with Ad2 DNA probes were conserved over long evolutionary periods. Hybridization was detected to the genomes of man, rat, mouse and fruit fly, but not to those of yeast and bacteria. In addition to a "smear" hybridization, discrete fragments were detected in both rodent and fruit fly DNAs. The experiments reported suggest the existence of two different types of cellular sequences detected by Ad 2 DNA: (1) repeated sequences conserved in a variety of eukaryote genomes and (2) a possible unique sequence detected with an E1a probe different from that responsible for hybridization to repeated sequences. This unique sequence was detected as an EcoRI fragment in mouse DNA and had a molecular size of about 8.8 kb. PMID- 3015508 TI - Cephalo-adrenal interactions in the broader context of pragmatic and theoretical rhythm models. AB - Some of the literature on modeling of biological rhythms with mathematical, physical, chemical, biochemical, in vivo and in vitro oscillations is succintly annotated. The need for biologic models that account for the interaction of 3 or more periodic entities is indicated, documented and illustrated, with emphasis on the cephalo-adrenal network of rodents. Patterns of interaction, in this context, involve attenuation, no-effect and/or amplification by a third entity, the modulator, of the effect of an actor upon the reactor. Such feedsidewards lead to chronomodulation, a phenomenon accounting for qualitatively as well as quantitatively different modulatory effects of the same drug or other stimulus. Controversies of long standing can thus be resolved and novel effects uncovered. PMID- 3015510 TI - [Secondary hematologic changes in cases of small cell lung cancer and malignant lymphomas]. PMID- 3015509 TI - [Mechanism of anisodamine in the treatment of aplastic anemia]. PMID- 3015511 TI - The pineal and pubertal development. AB - The pineal gland, through its major secretory product melatonin, influences seasonal breeding in species such as the hamster and the sheep. Recent studies from our laboratory have shown that melatonin also affects sexual development in the rat. A role for melatonin in humans has not yet been found. The laboratory rat is sensitive to daily administration of melatonin at the beginning of sexual maturation. The male rat is most sensitive between day 20 and day 30 of life. Melatonin does not permanently inhibit sexual maturation, since normal but delayed sexual development occurs after 45 days of life whether melatonin administration is discontinued or maintained indefinitely. In female rats, daily injection of melatonin during the prepubertal period delays the vaginal opening and disrupts the normal cyclicity of the first oestrous cycles. In both male and female rats, the inhibitory action of melatonin is highly dependent upon the time of injection, with maximal effects when melatonin is given in the late photoperiod. The inhibitory action of melatonin is most likely exerted at the hypothalamic level, possibly through interference with the control of pulsatile secretion of gonadotropin-releasing hormone. In contrast to some published work, our experiments provide no evidence for modifications of diurnal or nocturnal melatonin secretion during puberty in humans. Our results with the rat indicate that melatonin may be an important factor for the timing of sexual maturation. PMID- 3015512 TI - Photoneural regulation of the mammalian pineal gland. AB - Mammalian pineal function appears to be controlled primarily through the release of noradrenaline from the terminals of nerves whose cell bodies lie in the superior cervical ganglia. This is the final segment of the following neural pathway: retina----retinohypothalamic projection----suprachiasmatic nuclei--- paraventricular nuclei----intermediolateral cell column----superior cervical ganglia----nervi conarii----pineal gland. Noradrenaline acts on pinealocytes through alpha- and beta-adrenoceptors in an atypical manner. Beta-Adrenergic activation is an absolute requirement for the stimulation of both cyclic AMP and cyclic GMP production, and by itself produces a sixfold increase in the former and a twofold increase in the latter. Alpha-Adrenergic activation potentiates the beta-adrenergic stimulation of cyclic AMP production 10-fold, and that of cyclic GMP production about 200-fold. The mechanism of alpha- and beta-adrenergic interaction is being examined, and progress is being made in understanding the adrenergic control of cyclic AMP. It appears that alpha-adrenergic agonists act through the alpha 1-subclass of adrenoceptors to stimulate phospholipid turnover and the production of a breakdown product of phosphatidylinositol, diacylglycerol. This compound promotes the association of protein kinase C with membranes, which leads to the marked phosphorylation of one protein. The precise identity of this protein remains a mystery. This interaction leads to a larger cyclic AMP response but does not appear to be involved in the mechanism of potentiation of the cyclic GMP response. Changes in chronic neural stimulation produce reciprocal changes in the magnitudes of cyclic AMP and cyclic GMP responses. Chronic denervation results in a supersensitive cyclic AMP response and nearly complete disappearance of the cyclic GMP response. This is termed 'see saw' signal processing. All the available evidence indicates that melatonin production is regulated by cyclic AMP. This nucleotide not only increases the activity of serotonin N-acetyltransferase (more correctly called arylalkylamine N acetyltransferase) but also stabilizes the enzyme and prevents its inactivation. PMID- 3015513 TI - Respiratory response of phagocytes: terminal NADPH oxidase and the mechanisms of its activation. AB - The chemical composition, properties and activation mechanism of the O2(-) forming NADPH oxidase of phagocytes were investigated, using partially purified enzyme preparations. Highly active NADPH oxidase was extracted as an aggregate of high Mr from the membranes of neutrophils and macrophages. The enzyme complex contained phospholipids and cytochrome b-245, very little FAD and almost no quinones or NAD(P)H-dye reductase activity. The purification of a polypeptide with a relative molecular mass of 31 500 strictly paralleled the purification of NADPH oxidase, suggesting that this polypeptide is a component of the enzyme. This protein was identified as cytochrome b -245 after dissociation of the proteolipid complex and purification of the cytochrome moiety. The 31 500 Mr protein was phosphorylated in enzyme preparations from activated but not from resting cells. The results indicate that: cytochrome b-245 is a major component of NADPH oxidase; the involvement of NAD(P)H dye reductases in the O2(-)-forming activity is questionable; the cytochrome b-245: FAD ratio in the enzyme complex is much higher than that indicated in crude preparations; the Mr of pig neutrophil cytochrome b-245 is 31 500; the activation of the O-2-forming system involves a process of phosphorylation of cytochrome b-245. PMID- 3015514 TI - Action of the colony-stimulating factor, CSF-1. AB - Colony-stimulating factor 1 (CSF-1) is a glycoprotein growth factor that specifically regulates the survival, proliferation and differentiation of mononuclear phagocytes and their precursors via a cell surface receptor selectively expressed on these cell types. The purified receptor is a single glycosylated polypeptide, Mr 165 000, which exhibits CSF-1-dependent autophosphorylation in tyrosine. CSF-1 alone regulates cells of the mononuclear phagocytic series (CSF-1-dependent colony-forming unit [CFU-C]----monoblast--- promonocyte----monocyte----macrophage). However, the presence of a multipotent haemopoietic cell growth factor, haemopoietin-1, permits CSF-1 to stimulate precursors of CFU-C to proliferate and differentiate to macrophages. Precursors of CFU-C possess low levels of the CSF-1 receptor but there is an increase in receptor levels on CFU-C just before their differentiation to adherent, proliferating mononuclear phagocytes. As the timing of this developmentally associated increase in receptor expression coincides with the acquisition of responsiveness to CSF-1 alone, it is an early indicator of determination to the mononuclear phagocytic lineage. PMID- 3015515 TI - Specificity of action of colony-stimulating factors in the differentiation of granulocytes and macrophages. AB - Four colony-stimulating factors (CSFs) (M-CSF, GM-CSF, Multi-CSF and G-CSF) can each stimulate the production of macrophages from progenitor cells in murine bone marrow or fetal liver. However, they differ in their relative selectivity for macrophage progenitor cells and in their dose-response characteristics for stimulating macrophage progenitors relative to other progenitors. It is unresolved whether distinct subsets of progenitor cells exist with a unique responsiveness to one or other CSF or whether the macrophages produced by different CSFs are all functionally equivalent. However, it is shown here that various CSFs can generate from blast progenitor cells an intermediate macrophage progenitor cell whose growth is specifically inhibited by a substance in lectin stimulated spleen cell-conditioned media. It is also shown that, for at least one myelomonocytic leukaemic cell line, differentiation to macrophages and granulocytes can be induced most effectively by G-CSF but not by M-CSF or Multi CSF. Finally, the involvement of macrophages and macrophage cell lines in the induced production of these CSFs as well as their display of specific receptors for the different CSFs is examined. PMID- 3015516 TI - Raising antibodies by coupling peptides to PPD and immunizing BCG-sensitized animals. AB - The use of PPD (purified protein derivative of tuberculin) as a carrier has several significant advantages. It provides very powerful T cell help and it gives rise to virtually no antibody response against itself. This is particularly useful if it is intended to go on to make monoclonal antibodies, where the presence of a large amount of anti-carrier antibody is a nuisance! Furthermore, unlike most comparably powerful adjuvant systems, it can be used in man. PPD coupling has been used to raise antibodies to haptens and to raise T cell responses to tumour cells. It is here reported that small peptides coupled to PPD will give rise to good titres of anti-peptide antibody. For peptides that contain no cysteine, coupling has been achieved by attaching succinimidyl 4-(N maleimidomethyl) cyclohexane-1-carboxylate (SMCC) to the alpha-amino group of the peptide and N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to the PPD and allowing an uncleavable bond to form between them. Data on immunization with the leucotactic nonapeptide of the alpha chain of the complement component C3 and with some oncogene-related peptides have been obtained. PMID- 3015518 TI - [Herpetic viral antibody in the serum and cerebrospinal fluid of patients with sporadic encephalitis]. PMID- 3015517 TI - The importance of conformation and of equilibria in the interaction of globular proteins and their fragments with antibodies. AB - Oligopeptide fragments of globular proteins often inhibit the interaction of antibodies with native protein. Although such fragments appear to possess in aqueous solution an unfolded conformation it is apparent, in those cases where the protein's three-dimensional structure is known, that the antibody-bound fragment possesses a folded conformation mimicking that of the corresponding portion of the whole protein. The probable explanation of this dichotomy is that the fragment has various conformations in equilibrium including a small proportion of molecules whose shape is recognized and stabilized by the antibody. A related situation can exist in the interaction of antibody(ies) with the whole protein. Thus, antibodies against an altered form of the protein can induce the native antigen to adopt the conformation of the altered form. In this case, it appears that localized regions of the protein's surface are flexible, adopting various conformations in equilibrium, one of which is stabilized (selected) by interaction with the appropriate antibody. In both instances interaction with antibody perturbs an equilibrium, leading to the selection of a particular conformation. Such dynamic effects have profound implications on the choice of peptides as synthetic vaccines. PMID- 3015519 TI - [An analysis of the therapeutic effects of 120 cases of lung cancer in the elderly]. PMID- 3015520 TI - Mucinous adenocarcinoma associated with long-standing fistula in the cecal region. AB - A patient with mucinous adenocarcinoma associated with long-standing chronic fistula in the cecal region is presented. Association of mucinous carcinoma with chronic fistula seems to occur not infrequently in the anal region, but is extremely rare elsewhere in the large intestine. This is the first report of an occurrence in the cecal region. PMID- 3015521 TI - Sexual diseases important to dentistry. Clinical diagnoses and treatment. PMID- 3015522 TI - Primary liver cancer. Quadrennial review lecture. AB - Primary liver cancer, particularly HCC, is increasing in certain countries, notably Japan. Although hepatitis B virus has been etiologically linked to hepatocarcinogenesis and integration of its DNA into hepatocyte chromosomal DNA has been emphasized, other etiologic factors seem to have an interplay with virus infection. Histopathology of HCC has geographic variations. An expanding encapsulated HCC is most common in Japan, whereas it is nearly nonexistent in the West; such regional differences can only be explained by differences in the major etiologic factors. Early detection of HCC is now possible with ultrasound examination combined with AFP measurement, and this strategy has been executed with success in the Far East where HCC is endemic among cirrhotics. The speed of tumor growth can be measured with accuracy by ultrasound examination. Preneoplastic or early lesions of HCC in a cirrhotic liver seem to be adenomatous hyperplastic nodules or foci, and the conventional histological criteria for malignant liver cells do not seem applicable to such lesions. Although advanced cirrhosis is a real deterrent for hepatic surgery, hepatic resection affords a better survival compared with any nonsurgical therapeutic modality. Transcatheter arterial embolization is one of the current preferences of the hepatologist for inoperable patients. Lastly, a new staging scheme has been proposed for the assessment of prognosis and for comparison of efficacy of various therapeutic modalities. PMID- 3015523 TI - Decreased numbers of platelet alpha-adrenergic binding sites in diabetes mellitus. AB - Eleven men with diabetes mellitus were compared with 45 male controls for platelet alpha-adrenergic binding sites by using [3H]dihydroergocryptine (DHE) as the radioligand antagonist. There was no difference between the two for binding affinity, but the number of sites was 430 +/- 30 (means +/- SEM) for diabetic subjects and 574 +/- 29 for controls (P = .005). Decreased sites were related to increased glycosylated hemoglobin levels (P = .002). There was no relationship between the decreased sites and catecholamine levels, duration of disease, body weight, or fasting blood sugar. Hence, binding sites were inversely related to control, but further studies are needed to define the pathophysiologic significance of this. PMID- 3015525 TI - [Transplantation model of human hepatocellular carcinoma in nude mice. II. Establishment of the LTNM2 human liver cancer model in nude mice and observation on the growth of the transplanted tumor]. AB - LTNM2 model was established by the surgical specimen of a patient with recurrent hepatocellular carcinoma (HCC) with positive AFP. Human HCC was successfully transplanted subcutaneously into nude mice (Swiss nu/nu) following a latent period of 93 days with serial passages for 7 generations. Transplantability in nude mice was 72% (42/58). Based on the observation of 42 nude mice models, it was proved that the original properties of the human HCC, such as the morphological features, cell type and degree of differentiation and the functions, synthesis of AFP, were preserved. The tumor growth was progressive. The mean value of increased geometrical mean diameter tumor growth was progressive. The mean value of increased geometrical mean diameter (GMD) was 2.5 +/- 1.1 mm/week. Necrosis, ulceration and spontaneous regression was rarely observed. These results show that the transplanted tumor in nude mice bears a strong resemblance to the parent human HCC. LTNM2 model can be used not only for basic research of liver cancer but also for experimental therapeutic study. PMID- 3015524 TI - Two cases of retinal degeneration with an unusual form of electroretinogram. AB - An unusual form of retinal degeneration is reported in 15-year-old girl and 11 year-old girl with different pedigrees, which resembles the cases reported by Gouras and associates (1983). The subjective symptoms in these patients included decreased visual acuity, photophobia, anomalous color vision and night blindness. Electroretinograms (ERGs) in these two patients were identical in substance and revealed drastic alterations in both photopic and scotopic functions. The stimulus versus intensity response curve in a single-flash ERG showed an unusual form. This peculiar supernormal response was elicited by bright stimuli although the stimulus threshold was extremely elevated. PMID- 3015526 TI - [Family aggregation of hepatocarcinoma--preliminary analysis]. AB - A pedigree study was carried out in families with hepatocarcinoma (HCC) in Qidong county. The results showed that the incidence of families with familial history of HCC was 41.59% (443/1065). By means of test for Fitness of Binomial Distribution, it was found that HCC had an evident tendency of familial aggregation (P less than 0.01). In the high risk families, the average death age of patients with HCC in filial generation was younger (38.92 years old) than that of people who die of HCC in Qidong county (49.26 years old). The age at which siblings had HCC was often similar: 31.63%, with age-difference less than or equal to 2 years and 61.22%, less than or equal to 5 years. Whether the members of high risk families lived together or not, the incidence of HCC was alike (P greater than 0.05). These results suggest that the role of genetic factors in the etiology of HCC should not be neglected. PMID- 3015527 TI - [Analysis of trace elements by proton induced X-ray emission (PIXE) in serum of patients with liver cancer and normal subjects]. AB - The levels of trace elements in serum of the patients with liver cancer and the normal subjects were determined by the PIXE technique. The significant increase of serum copper level (SCL) and the decrease of serum zinc level (SZL) in the patients with liver cancer, as compared to those of normal, were observed. Cu/Zn ratio in the patients with liver cancer was significantly higher than that of the normal (P less than 0.01). In the Cartesian coordinate graph of SCL + SZL, the liver cancer patients were separated from the normal by a line joining the intersection of abscissa and ordinate and the point of the sum of the mean value and the standard deviation of the Cu/Zn ratio of the normal subjects. The authors believe that the serum Cu/Zn ratio is likely a supplementary target in the diagnosis and prognosis of liver cancer. PMID- 3015529 TI - [Discovery of herpes simplex virus (HSV)-like particles in biopsy samples of cervical carcinoma]. AB - The tissue of cervical cancer and HSV-2 infected baby rabbit kidney (BRK) cells were studied comparatively by electron microscope. In 2 out of 12 samples from patients with cervical carcinoma, some virus-like particles were observed in the cytoplasm or intercellular space. Some particles seemed to be enveloped by endoplasmic reticulum. According to their size, structure and morphology, these particles resembled the herpes simplex virus. The relationship between the discovery of herpes simplex virus-like particles in tissue of cervical cancer and the development of this cancer is discussed. PMID- 3015528 TI - [Enhanced transformation of human lymphocytes by Chinese herbs]. AB - The enhancing effect of extracts from some Chinese herbs on transformation of lymphocytes by Epstein-Barr (EB) virus was tested in soft agar system. Daphne genkwa, Wikstroemia chamaedaphne, Wikstroemia indica, Stellera chamaejasme and Sparganium stoloniferum were found to activate early antigen (EA) of EB virus in Raji cells and also enhance transformation of lymphocytes by EB virus. But some other herbs, such as Abrus cantoniensis and Bulbophyllum inconspicuum were not able to activate EA, hence, no enhancing effect on transformation. The significance of these herbs in the enhancement of lymphocytes by EB virus and their relation to the development of nasopharyngeal carcinoma and other malignant diseases are discussed. PMID- 3015530 TI - [Ovarian tumor in the juvenile--an analysis of 77 cases]. AB - This paper analyzes 77 cases of ovarian tumor in children and adolescents treated in our hospital from Jul. 1952 to Aug. 1982. All were under 20 years and the youngest was 3 years old. The incidence was 6.0% of the total patients with ovarian tumor during the same period. Of 77 cases, 54 (70.1%) were benign (10 were lost to follow-up, 43 survived and 1 died of unrelated cause), and 23 (29.9%) were malignant (4 lost and 8 died, the mortality was 34.8%). 37 (48.0%) were considered to be of germ cell origin and the rest (52.0%), non-germ cell origin. In 18 patients before menarche, 14 had germ cell tumor, but in only 3, the tumor arose from the coelomic epithelia. It was of interest to note that 1 patient, only 3 years old, had granulosa cell tumor associated precocious puberty. The initial presenting symptoms were usually an abdominal mass or lower abdominal pain. Some of the germ cell tumors and mucinous cystadenomas could reach a considerable size. The prognosis of ovarian tumor is related to the clinical stage, pathological type as well as whether the treatment is thorough or not. The diagnosis, differential diagnosis and treatment are discussed. It is emphasized that the conservation of ovarian function and the fertility should be considered under the treatment. PMID- 3015531 TI - [Discovery and electron microscopic observation on unusual intranuclear inclusion bodies of breast carcinoma]. AB - Unusual intranuclear inclusion bodies in one case of breast carcinoma discovered and studied by electron microscope is reported. The ultrastructural feature of these inclusion bodies is very characteristic and has not been reported previously. They presented with unequal sizes, location at the margin of nuclei, and frequent separation from the nuclear chromatin by a single thin membrane. These inclusion bodies can be divided into two kinds: one with granular central portion surrounded by numerous radiating flagella-like filaments, the other assuming concentric laminated arrangements. There are transitional forms between them. The nature of these intranuclear inclusion bodies are discussed. PMID- 3015532 TI - [Study on ATPase of tumor cells--I. A comparison of several ATPase activities]. AB - The activities of several ATPases of the ascitic hepatoma cells in mice were compared. The activities of the total ATPase and mitochondrial Mg++-ATPase in normal liver cells were 50% and 140% higher than those in the hepatoma cells (P less than 0.001). However, the Na+, K+-ATPase activity in hepatoma cells was 170% higher than that in normal liver cells (P less than 0.01). The proportion of the activities of mitochondrial Mg++-ATPase, as well as Na+, K+-ATPase to their respective total ATPase in hepatoma cells was in disorder. The relation between the changes of the activity of different ATPases, their proportion and the abnormal metabolism as well as some characteristics of tumor cells are discussed. PMID- 3015533 TI - [Electrophoretic mobility of T and B lymphocytes in bone tumor patients]. AB - Electrophoretic mobility of peripheral blood T and B lymphocytes in bone tumor patients (malignant 24 and benign 16) was studied. Its variations associated with cellular immunity were evaluated in this paper. The electrophoretic mobility of cells was measured at 25 +/- 0.5 degrees C with a cytopherometer. The results showed that electrophoretic mobility of T lymphocytes and E-rosettes in malignant tumor patients were significantly low (P less than 0.05) as compared with that in the normal subjects, while those in the benign tumor patients were not significantly different (P greater than 0.05). PMID- 3015534 TI - [Primary bone and joint tumor--statistical analysis of 571 cases]. AB - This paper analyses 571 cases of primary bone and joint tumors diagnosed by histopathology, excluding the tumor-like lesions. They were divided into two groups: the benign (412 cases, 72.15%) and the malignant (159 cases, 27.85%). The susceptible ages were between 15-29 years and the susceptible location of these tumors were femur and tibia often on the right side. There were more males than the females. The frequencies in the two groups are as follows: In the benign group, osteoma had the highest incidence and then, with decreasing frequencies: osteochondroma, chondroma, synovioma, giant cell tumor, ossifying fibroma, osteoid osteoma, chondromyxoid fibroma. In the malignant group, the highest incidence was in the osteosarcoma, and then synoviosarcoma, chondrosarcoma, malignant giant cell tumor, Ewing's tumor, fibrosarcoma, osteomyeloma. The sequence of the incidence was basically similar to that reported abroad and at home. The pathogenesis of several kinds of bone tumor are discussed. PMID- 3015535 TI - [Lung cancer in the young adult and results of surgical treatment]. AB - Sixty patients with primary lung cancer and under 40 years of age were operated from Jan. 1960 to June 1983. It comprised 3.7% of 1,635 lung cancers in all during the same period. The average age was 34.4 (17-39) years old. 35 were male and 25 female with a sex ratio of 1.4:1 which was lower than that reported for all lung cancers. Of the 60 patients, 31 (51.7%) presented with cough, 27 (45%) with bloody sputum, 23 (43.3%) chest pain and 13 (21.7%) feverishness. The average delay before the first medical examination was 6.4 months. It was over 1 year in 8 patients. The misdiagnosis rate was 76.7%. According to the TNM classification, the lesions were: stage I in 16.7%, stage II in 23.3% and stage III in 60%. By pathology, 45% were adenocarcinoma, 25% squamous cell carcinoma, 23.3% undifferentiated carcinoma and 6.7% squamous-adenocarcinoma. The resection rate was 83.3% (50 patients). The 1, 3, 5, 7 and 10 year survival rates were 83.3%, 42.5%, 32.3%, 18.5% and 21.1% which show that the survival rate of lung cancer in the young adults was similar to that of all ages. Most of the patients treated only by exploration died within 1.5 years. The authors believe that early diagnosis, early resection supplemented by radiotherapy, chemotherapy and immunotherapy might improve the survival rate of lung cancer in the young adults. PMID- 3015536 TI - [Glomus tumor of the stomach--a case report on a light and electron microscopic study]. AB - This paper reports a case of glomus tumor of stomach from a male patient, 58 years old, with the chief complaint of nausea and vomiting after meal. This tumor was at the anterior wall of the antrum near the lesser curvature. By light microscopy, it was composed of glomus tumor cells and abundant blood vessels. Ultrastructurally, there were numerous filaments, dense bodies, adhesive bodies and pinocytotic vesicles in the cytoplasm. This kind of tumor should be differentiated from the leiomyoblastoma and hemangiopericytoma. PMID- 3015537 TI - [Preparation and identification of monoclonal antibody against the gastric cancer cell line MGC 803]. AB - Spleen cells of Balb/c mice, immunized with gastric cancer cell MGC 803, were fused with murine myeloma cell NS-1. After selective culture, screening and subcloning, a hybridoma PC1 which produced monoclonal antibody (McAb) against MGC 803 cells was obtained. McAb PC1 bound strongly with 3/4 gastric cancer and 1/2 hepatoma cell lines, weakly with another gastric cancer and 2/2 lung cancer cell lines, but did not bind with the autologous and allogenic lymphocytes, ABO red blood cells, human fetal lung fibroblasts and normal bone marrow cells. The binding capacity of McAb PC1 to MGC 803 decreased significantly due to the absorption by MGC 803 cells, but was not affected by lymphocytes and CEA. The corresponding antigen of McAb PC1 was expressed on the surface of MGC 803 cells. It may be a gastric cancer-associated antigen. PMID- 3015538 TI - [Cyclic-GMP-phosphodiesterase from the cattle retina. The nucleotide sequence of gamma-subunit cDNA]. PMID- 3015539 TI - [Common endocytosis pathway of the epidermal growth factor and transferrin associated with the Golgi apparatus acidic (ph 6.1) compartment of A431 cells]. PMID- 3015540 TI - In vitro effect of different ubiquinones on the scavenging of biologically generated O2-. AB - The ability of different homologues of Coenzyme Q to quench O2- was tested in vitro with three experimental systems known to generate O2-. Two of them were biological generators, namely the xanthine-xanthine oxidase system and the cyanide-insensitive NADPH oxidase of polymorphonuclear leucocytes. The third was a chemical generator of O2-, the NADH-phenazine methosulphate-nitroblue tetrazolium mixture. Short-side-chain ubiquinones were found to be the most potent scavengers of O2-, being effective at concentrations as low as 10(-7) M. This finding might be ascribed to the relatively greater water-solubility of the lower homologues of CoQ. We postulate that CoQ10 may well exert such an O2- scavenging mechanisms in vivo where it is inserted in its natural phospholipid environment. PMID- 3015541 TI - The essentiality of coenzyme Q for bioenergetics and clinical medicine. AB - Coenzyme Q is an essential component of the respiratory chain, where it represents a mobile pool between dehydrogenases and cytochromes. The fact that Q is a free component, and its concentration is not in great excess over the Km of the respiratory complexes, renders this compound potentially rate-limiting in the respiratory chain. On the other hand, the rate of lateral diffusion of Q in the mitochondrial membrane is not a limiting step under physiological conditions. Quinoid compounds, which act as inhibitors of the respiratory chain at the level of Q, besides being useful tools for investigations of electron transfer, could be important in pathology as inhibitors of respiration. PMID- 3015542 TI - Modern trends in the management of hepatocellular carcinoma. AB - No treatment of proven validity in hepatocellular carcinoma (HCC) has yet been found, with the exception of surgery in a small subset of patients. Furthermore, there is a paucity of phase I and II trials and of phase III prospective randomized trials. Radiation therapy is not considered very effective, even as a palliative procedure, but there has been renewed interest in this modality, with several ongoing trials attempting to establish optimal doses, fractionation and effectiveness. Chemotherapy, single or combination, has not increased the survival of patients with HCC, although there have been unquestionable, even spectacular, responses to chemotherapy. Adriamycin seems the most effective agent with approximately 25% objective responses. The regional administration of drugs and/or interruption of arterial supply, in this malignancy with a pattern of local confinement, yields quite high response rates but no increase in survival. Several trials with combined therapeutic modalities are now in progress. Furthermore, there have been important advances in nuclear medicine, in the totally implantable pump, in the understanding of pharmacokinetics and in microspheres, monoclonal antibodies, etc. It is remarkable that there have been so few randomized trials in such a common malignancy. To obtain valuable results any future studies should randomize patients into "treatment" and "no treatment" groups and stratify patients according to prognostic factors. It seems, however, that the combination of local and systemic treatment is the most promising answer to this virulent and fatal disease. PMID- 3015544 TI - [The hemodynamic effects of captopril]. PMID- 3015543 TI - Combination of surgery and chemotherapy for small-cell bronchial carcinoma. AB - In several test model systems using spontaneous metastasizing experimental tumours, convincing data indicate the importance of the tumour burden left after surgery for the efficacy of the combination of surgery and chemotherapy. Early removal of the primary tumour by radical surgery for cure seems to improve the conditions for chemotherapy. Since 1979, in nine different departments of thoracic surgery, patients with small-cell carcinoma of the lung (SCCL) have been randomized after surgery for cure to receive a new sequential intermittent polychemotherapy (sq.CT) of 3 different alternating drug combinations given intermittently over 1 year, or one 4-drug combination chemotherapy (CT) given intermittently over 3 years. The calculation of their life table curves at 1 August 1984 indicated an improvement in the 4-year survival rate of 23 patients receiving sq.CT to about 50%, compared with a survival rate of about 30% for 29 patients receiving CT. The number of patients is still too small for firm conclusions to be drawn, but it is concluded that surgery for SCCL seems to be an advisable measure for the efficacy of aggressive intermittent long-term polychemotherapy. However, this can only be proved in large cooperative studies. PMID- 3015545 TI - [Prevalence of antibodies against LAV/HTLV-III in patients with terminal renal insufficiency treated with hemodialysis and following renal transplantation]. AB - Sera of 1046 patients undergoing haemodialysis for terminal renal failure or after renal transplantation were tested with the ELISA screening test for anti LAV/HTLV-III and, if positive, the Western blot and ELAVIA tests. Four patients (0,38%) had "true" antibodies against LAV/HTLV-III, confirmed by the Western blot and ELAVIA tests. These patients had definite signs of cellular immune defects. They had received transplants from drug addict donors. Such kidneys should therefore no longer be used for transplantation. In addition, 29 patients (2.8%) had "false-positive" antibodies against LAV/HTLV-III in the ELISA test, unconfirmed in the Western blot and ELAVIA tests. The "false-positive" result was presumably due to cross-reaction with HLA antibodies. Sera of dialysis and transplantation patients who had received many blood transfusions should therefore be especially carefully tested before a diagnosis of infection with LAV/HTLV-III is made. PMID- 3015546 TI - [Spontaneous course of LAV/HTLV-III infection. Follow-up of persons from AIDS risk groups]. AB - 543 persons in AIDS risk groups were examined for LAV/HTLV-III infection. Antibodies against LAV/HTLV-III were demonstrated in 377, during an observation period of 3 months to 3 years. Patients were divided into 5 groups, according to clinical, serological and immunological criteria. Severity of symptoms and incidence of recurrences of the lymphadenopathy syndrome correlated positively with an increase in cellular immune deficiency. At the end of the observation period only 30 of 307 antibody-positive patients were well. The AIDS incidence of all antibody-positive patients increased with time from 3% (6-11 months) to 19.4% (24-36 months). If one takes into account only those patients who had marked immune deficiency (stage 2b) at the initial examination, the AIDS incidence during the same periods was 10 and 57%, respectively. At the same time, the condition worsened by at least one stage in half of all patients in stages 1b-2a observed for 1-2 years. A change of stage was observed in up to 80% of patients followed for longer periods. Long-time prognosis of LAV/HTLV-III infection is remarkably bad. PMID- 3015548 TI - Loss of polarization of plasma membrane domains in transformed pancreatic endocrine cell lines. AB - Using enveloped RNA viruses that bud selectively from either the apical or basolateral surface in polarized epithelial cells, we have recently provided evidence for polarization of plasma membrane domains in cultured pancreatic islet cells. In this study, we have followed the same experimental strategy to establish whether these polarized properties are maintained in transformed pancreatic endocrine cells. We find that influenza virus and vesicular stomatitis virus emerge from both the attached and free surfaces of cultured insulinoma cells (RIN cells) and SV40-transformed beta-cells (HIT cells). This demonstrates loss of polarization in transformed pancreatic endocrine cells. PMID- 3015549 TI - Effects of an acute bacterial infection on serum thyroid hormones and nuclear triiodothyronine receptors in mice. AB - Serum T3, T4, and rT3 levels as well as liver nuclear T3 receptors (NT3R) were measured in mice with a bacterial infection. Pseudomonas aeruginosa were injected into one thigh of ICR mice, resulting in a severe infection at sacrifice 15 h later. Since food intake, which influences serum thyroid hormone levels and NT3R, was 75% lower in infected than in control mice, infected mice were either fed and compared with pair-fed controls or fasted and compared with fasted and fed controls. Fasting induced a fall in serum T3 and T4 levels, which was even more pronounced in infected fasted animals. However, while fasting caused an approximately 80% increase in serum rT3 concentrations, serum rT3 levels in infected fasted animals were not different from those in fed controls. The combination of infection and fasting thus prevented the rise in serum rT3 otherwise invariably associated with fasting. NT3R measurements on isolated nuclei revealed the presence of NT3R in mouse liver similar to those reported in rat liver. The NT3R Kd (approximately 2 X 10(-10) M) was not affected by decreased food intake, infection, or a combination thereof. The NT3R maximum binding capacity (MBC) was decreased in fasted animals (460 vs. 306 pg/mg DNA). However, the MBC of infected fasted mice was not different from that of fasted mice. Similarly, no difference in MBC was found between infected fed and pair-fed control mice. In mice injected with heat-killed P. aeruginosa to evaluate potential effects of endotoxins, neither serum thyroid hormone levels nor hepatic NT3R were different from those of controls. These data show that in mice, a severe bacterial infection with P. aeruginosa has effects on serum hormone levels not explained by the disease-associated diminished food intake, whereas it has no effects on liver NT3R beyond those due to the disease-related decreased food intake. PMID- 3015547 TI - Detection, distribution and inhibition of Branhamella catarrhalis beta lactamases. AB - Beta-lactamase-producing isolates of Branhamella catarrhalis were first detected in France in 1977. The frequency of beta-lactamase producers has increased, especially since 1980. An agar iodometric test, a fast chromogenic test and an acidimetric test were used to assess the beta-lactamase-producing capabilities of 188 isolates of B. catarrhalis obtained mainly from sputum and the pharynx. Data from the first 2 procedures indicated positive beta-lactamase activity for all 49 strains of B. catarrhalis identified, but there were some discrepancies in the acidimetric test results. Evidence from a diffusion technique showed significant increases in the inhibition diameters surrounding filter discs impregnated with amoxycillin in the presence of clavulanic acid, or with ampicillin in the presence of sulbactam, compared with discs of the penicillins used alone. Two types of enzyme activity emerged from examination of isoelectric focusing patterns. Type I, having pI values of 5.35, 5.55 and 5.85, accounted for 87.2% of the enzyme-producing isolates. Type II, with pIs of 5.5, 5.9 and 6.25, occurred in 12.8% of isolates and appeared to be less widely distributed. The beta lactamase inhibitors clavulanic acid and sulbactam in combination with benzylpenicillin produced potentiated effects, as demonstrated by significant reductions in MIC (33- and 44-fold decreases, respectively). Higher concentrations of each inhibitor similarly affected the MICs of amoxycillin. A weak synergy occurred with cefoxitin, a beta-lactamase-resistant beta-lactam antibiotic, and the 2 beta-lactamase inhibitors. Because B. catarrhalis has been shown to be a beta-lactamase-producing pathogenic organism, the addition of enzyme inhibitors, such as clavulanic acid and sulbactam, to standard therapy may be beneficial. PMID- 3015550 TI - A reduced pancreatic protein secretion in response to cholecystokinin (CCK) in the obese Zucker rat correlates with a reduced receptor capacity for CCK. AB - Pancreatic membrane receptors for cholecystokinin (CCK) in obese and nonobese Zucker rats were compared with the use of a biologically active [125I]iodo-CCK-8 radioprobe. Membrane homogenates from obese rats bound half the amount of radioligand in 2 h as did membranes from lean rats (specifically bound, 7.0% vs. 14.0%; P less than 0.001). The reduced binding in membranes from obese rats did not result from kinetic effects or radioligand degradation; similar rates of association and dissociation of [125I]iodo-CCK-8 were obtained in membrane preparations from both, and no differences were found in the extent of radioligand degradation in the two membrane preparations. These differences also did not reflect an effect of cell size, as pancreatic acinar cells from obese and nonobese rats had about the same perimeters (24.6 and 26.3 micron, respectively) and areas (30.1 and 34.2 micron 2, respectively). Scatchard-type plots of competitive displacement data for CCK-binding sites on pancreatic membranes from both genotypes were curvilinear and were analyzed by a two-site binding model. The Kd values for both the high (0.56 vs. 0.45 nM) and low (9.0 vs. 14 nM) affinity sites on membranes from nonobese and obese rats, respectively, were the same (P greater than 0.1), whereas the capacities for CCK in the high (365 vs. 165 fmol/mg protein) and low (1020 vs. 360 fmol/mg protein) affinity regions were significantly different (P less than 0.025). This difference in CCK receptor capacity was reflected by a reduced pancreatic protein secretory response in the obese rat. After injections of 40, 80, 160, and 320 ng CCK/kg BW, total pancreatic protein secretion in nonobese rats increased 5, 12, 19, and 21 times above basal levels, whereas the same doses caused 2-, 6-, 12-, and 13-fold increases in obese rats. Whereas the reduced secretion in the obese rat may reflect a difference in the intracellular machinery leading to protein secretion between the two genotypes, these data are more consistent with a direct mechanism, whereby reduced numbers of pancreatic receptors for CCK are responsible for reduced protein secretion. PMID- 3015551 TI - Thyroid hormone regulation of epidermal growth factor receptor levels in mouse mammary glands. AB - The specific binding of iodinated epidermal growth factor ([125I]iodo-EGF) to membranes prepared from the mammary glands and spontaneous breast tumors of euthyroid and hypothyroid mice was measured in order to determine whether thyroid hormones regulate the EGF receptor levels in vivo. Membranes from hypothyroid mammary glands of mice at various developmental ages bound 50-65% less EGF than those of age-matched euthyroid controls. Treatment of hypothyroid mice with L-T4 before killing restored binding to the euthyroid control level. Spontaneous breast tumors arising in hypothyroid mice also bound 30-40% less EGF than tumors from euthyroid animals even after in vitro desaturation of the membranes of endogenous growth factors with 3 M MgCl2 treatment. The decrease in binding in hypothyroid membranes was due to a decrease in the number of binding sites, not to a change in affinity of the growth factor for its receptor, as determined by Scatchard analysis of the binding data. Both euthyroid and hypothyroid membranes bound EGF primarily to a single class of high affinity sites [dissociation constant (Kd) = 0.7-1.8 nM]. Euthyroid membranes bound 28.4 +/- (SE) 0.6 fmol/mg protein, whereas hypothyroid membranes bound 15.5 +/- 1.0 fmol/mg protein. These data indicate that EGF receptor levels in normal mammary glands and spontaneous breast tumors in mice are subject to regulation by thyroid status. PMID- 3015552 TI - Dephosphorylation of 19K and 21K polypeptides in response to thyroid-stimulating hormone in cultured thyroid cells. AB - Cultured dog thyroid cells incubated with [32P] phosphate contain at least two phosphoproteins of 19 and 21 kDalton (K), as determined by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Myosin light chain appears to be a component of the 19K and 21K phosphoproteins by the following criteria: 1) coextraction with myosin heavy chain from Triton-insoluble cytoskeletons with KCl-ATP, 2) coisolation with myosin heavy chain by immunoprecipitation, and 3) purification of undenatured myosin with pyrophosphate agarose gel electrophoresis. The phosphorylation state of these proteins is decreased by incubation of cells with TSH. In the basal state, the 19K and 21K proteins from Triton-insoluble cytoskeleton fractions contain 0.86 +/- 0.07 (+/- SE) mol phosphate/mol protein, which is reduced to 0.34 +/- 0.03 in TSH-treated cells. TSH-induced dephosphorylation occurs in 1 min with 2.5 mU/ml TSH and reaches a maximum at 15 min. This TSH effect appears to be mediated by cAMP, since it is mimicked by (Bu)2cAMP, forskolin, cholera toxin, and prostaglandin E1 and is potentiated by isobutylmethylxanthine. Carbamylcholine, ionophore A23187, and norepinephrine, which inhibit TSH stimulation of cAMP, have no effect on basal phosphorylation of the 19K and 21K proteins, but do inhibit the effect of TSH. PMID- 3015553 TI - Development of renal hypertrophy and increased renal Na,K-ATPase in streptozotocin-diabetic rats. AB - The effects of experimental diabetes on renal tubular Na,K-ATPase activity were measured 4, 7, 14, 21, 28, and 56 days after ip injection of streptozotocin (STZ; 60 mg/kg) in rats. Significant increases in serum and urinary glucose levels as well as urinary volume and electrolyte output were observed 24 h after STZ injection. The elevated serum and urinary glucose levels were maintained during the entire 8-week experimental period, while urinary volume and electrolyte output showed an initial rising phase, reaching a peak at approximately 2-3 weeks, followed by a stabilization phase at a level lower than the peak effect, but still significantly higher than that in the saline-citrate-treated controls. A significant increase (+25.3%) in renal outer medullary Na,K-ATPase activity was observed 4 days after the induction of STZ-diabetes, while similar increases were not observed in the cortical regions until after 7 days of experimental diabetes. These elevated renal cortical and outer medullary enzyme activities, however, were subsequently maintained during the entire 8-week experimental period. In addition, a similar time course of development of renal hypertrophy, as indicated by increases in kidney weights and kidney protein to DNA ratios, was observed after the induction of STZ-diabetes in rats. Therefore, the present data indicate that renal hypertrophy and increased renal Na,K-ATPase develop early and with a similar time course after induction of STZ diabetes in rats and may mediate the gradual amelioration of excessive renal electrolyte loss seen in this experimental condition. Since Na,K-ATPase-mediated ion transport is the major consumer of metabolic energy in the kidney and is centrally important to renal function, it is suggested that the early and pronounced increase in renal Na,K ATPase seen in diabetes is an essential component of the renal hypertrophy and hyperfunction seen in this disease and may represent an important adaptive change in the kidneys in response to glucose osmotic diuresis in the experimental diabetic animals. PMID- 3015554 TI - Rabbit uterine oxytocin receptors and in vitro contractile response: abrupt changes at term and the role of eicosanoids. AB - We examined the relation between increased uterine oxytocin receptor concentration and increased in vivo sensitivity of the rabbit uterus to oxytocin at the end of gestation. We determined oxytocin receptor concentrations in myometrium and decidua on different days near term of gestation and postpartum. We also examined the in vitro contractile response to oxytocin on days 30 and 5 days postpartum, when the uterus is unresponsive in vivo, and on day 31 (term), when the uterus is exquisitely sensitive to this hormone in vivo. In addition, we tested the role of endogenous eicosanoids and decidual oxytocin receptors in the myometrial contractile response to oxytocin by examining the contractile response in the presence of the cyclooxygenase/lipoxygenase inhibitor sodium meclofenamate or in muscle strips from which the decidua had been removed by scraping. The concentration of specific binding sites for [3H]oxytocin in myometrial and also decidual membrane preparations was determined. We demonstrate that contractile sensitivity to oxytocin increases at least 4-fold between days 30 and 31 (term) of gestation, and this is accompanied by a nearly 10-fold increase in the concentration of oxytocin-binding sites in both decidua and myometrium. The lesser sensitivity to oxytocin on day 30 was, however, only apparent in the presence of meclofenamate, which suggests that endogenous eicosanoids contribute to the preterm response to oxytocin measured in vitro. The maximal response to oxytocin (integrated area) increased 2-fold between day 30 and term. Thus, an increase in both sensitivity and maximal response to oxytocin could be demonstrated at term in vitro. Five days after parturition, maximal response and uterine sensitivity measured in the presence of meclofenamate had returned to those of the preterm uterus, and the concentration of oxytocin-binding sites had declined. In contrast, sensitivity and maximal response to the cholinergic agonist carbamylcholine declined between day 30 and term. These results support a highly regulated physiological role for oxytocin in parturition which depends primarily on changes in receptor concentration. PMID- 3015555 TI - Active and inactive forms of 3,5,3'-triiodo-L-thyronine (T3)-binding protein in rat kidney cytosol: possible role of nicotinamide adenine dinucleotide phosphate in activation of T3 binding. AB - Extraction of rat kidney cytosol with 10% charcoal at 4 C inactivated specific T3 binding. The decreased T3 binding in extracted cytosol could be restored by addition of boiled kidney cytosol. Three different factors (a, b, and c) which could increase T3 binding were identified by Sephadex G-50 column chromatography of boiled cytosol. Two factors (b and c) were eluted as relatively small molecules. Factor a was present in small amounts. Factor c was neutralized by incubation with EDTA, but factor b was not. Factor b was not destroyed by trypsin, protease, DNase, or RNase, but was destroyed by alkaline phosphatase. Factor b was destroyed by incubation with nicotinamide adenine dinucleotide phosphate (NADPH)-dependent glutathione reductase in the presence of oxidized glutathione. Although T3 binding to charcoal-extracted cytosol protein was not influenced by reduced glutathione or dithiothreitol, it was markedly increased by NADPH. Maximal activation induced by 50 microM NADPH was not further increased by further addition of endogenous factor b. The elution position of NADPH in gel chromatography corresponded to the elution position of factor b. Factor b or NADPH increased maximal binding capacity without changes in affinity constant. These observations suggest that T3-binding protein in cytosol is present in inactive and active forms and that the active form is generated by NADPH, which is present as one of the activators in cytosol. The effect of these cytosolic T3 binding proteins on nuclear T3 binding in vitro was also studied. In the absence of cytosolic T3-binding protein, [125I]T3 binding to nuclear receptor was decreased by unlabeled T3 in a concentration-dependent manner. In the presence of inactive form of cytosolic T3-binding protein, nuclear [125I]T3 binding was slightly diminished. In the presence of NADPH and cytosolic T3-binding protein, however, the amount of [125I]T3 bound to nuclei markedly decreased, which was associated with an increase of cytosolic [125I]T3 binding. NADPH alone did not influence nuclear T3 binding. These results suggest that T3 binding to nuclear receptor is regulated by an active form of cytosolic T3-binding protein in vitro. PMID- 3015557 TI - Characterization of porcine growth hormone (pGH) binding to porcine liver microsomes: chronic administration of pGH induces pGH binding. AB - The effect that GH has on regulating GH binding to its receptors has not been resolved. This report describes the characterization of porcine (p) GH binding to pig liver membranes and clarifies the issue of regulation of GH binding by measuring pGH binding to liver membranes prepared from pigs treated daily for 35 days with different doses of pGH (0, 10, 30, or 70 micrograms/kg BW). Specific binding of [125I]pGH was dependent on time, pH, and membrane protein concentration. At 23 C, pGH binding reached a steady state after 24 h. Maximal pGH binding was observed at pH 7. Binding increased linearly as membrane protein concentration was increased from 150 to 450 micrograms/tube. Specificity studies indicated that the hepatic GH receptor was somatogenic, since porcine PRL poorly inhibited [125I] pGH binding (cross-reactivity, 0.1%). Treatment of microsomes from control pigs with 4 m MgCl2 to remove endogenously bound pGH did not affect pGH binding, whereas binding was significantly increased to microsomes from pGH treated pigs. Binding of pGH increased in a linear manner with the dose of pGH given for 35 days (r = 0.79), thus establishing the inductive effect of chronic pGH administration on pGH binding in pig liver. GH binding was highly correlated with weight gain in pigs treated with pGH (r = 0.76). In addition, the serum insulin-like growth factor I (IGF-I) concentration was increased linearly (r = 0.87) by pGH. This increase in serum IGF-I was also highly correlated with the increase in pGH binding (r = 0.71). These results suggest that hepatic GH binding plays an important role in regulating pig growth, which may be mediated, in part, by an increase in hepatic IGF-I synthesis and secretion. The present report is also the first to establish that exogenous pGH induces pGH binding to pig hepatic GH receptors and to relate this increase in binding to an enhancement in pig growth. PMID- 3015556 TI - Plasma immunoreactive proopiomelanocortin peptides and cortisol in normal dogs and dogs with Addison's disease and Cushing's syndrome: basal concentrations. AB - We measured basal plasma concentrations of the immunoreactive (IR) proopiomelanocortin (POMC)-derived peptides ACTH, beta-lipotropin (beta LPH), beta-endorphin (beta END), and alpha MSH in 160 normal dogs, 32 dogs with Addison's disease, 42 dogs with adrenocortical tumors causing Cushing's syndrome, and 169 dogs with pituitary-dependent Cushing's disease. In normal dogs, plasma IR-POMC peptide levels were similar to those in man, except that IR-alpha MSH, a pars intermedia POMC product, was readily detected. In Addisonian dogs, plasma cortisol was decreased, and the IR-POMC peptides were increased, except for IR alpha MSH, which was normal. In 7 Addisonian dogs given dexamethasone, elevated plasma IR-ACTH, beta LPH, and beta END levels fell dramatically. In dogs with Cushing's syndrome due to adrenal tumors, plasma IR-ACTH, beta LPH, and beta END were decreased, and cortisol was increased, but IR-alpha MSH was normal. Dogs with Cushing's disease due to pars distalis tumors had elevated plasma IR-ACTH, beta LPH, beta END, and cortisol, but normal IR-alpha MSH; their plasma cortisol was suppressed by dexamethasone. There appeared to be 2 types of pars intermedia tumors causing Cushing's disease: 1 dexamethasone nonsuppressible and with disproportionately high plasma IR-alpha MSH levels, the other relatively dexamethasone suppressible and with normal to slightly elevated IR-alpha MSH levels. These 2 pars intermedia tumor types may arise from 2 distinct normal canine pars intermedia cell types. Canine Cushing's disease may provide a useful model for variants of the disorder in man. PMID- 3015558 TI - Evidence for tight coupling of receptor occupancy by thyrotropin-releasing hormone to phospholipase C-mediated phosphoinositide hydrolysis in rat pituitary cells: use of chlordiazepoxide as a competitive antagonist. AB - Chlordiazepoxide (CDE) has been shown to antagonize the effects of TRH to stimulate the hydrolysis of phosphoinositides and elevate cytoplasmic free calcium in rat pituitary tumor (GH3) cells. Herein, we show that CDE inhibits TRH stimulation of PRL secretion and that the effect of CDE to antagonize TRH action is caused by its ability to compete with TRH for binding to receptors on GH3 cells. We also use CDE to explore whether continued receptor occupancy is required for prolonged stimulation of cellular responses. CDE had no effect on basal PRL secretion, but caused a dose-dependent inhibition of TRH-induced PRL secretion. CDE decreased the affinity of TRH binding to intact GH3 cells without affecting the maximum binding capacity. As shown previously, CDE had no effect on phosphoinositide metabolism, which was monitored because it appears to be a mechanism for signal transduction by TRH, and when added simultaneously with TRH, caused a dose-dependent inhibition of TRH-induced phosphoinositide metabolism. When CDE was added to cells 2.5 or 5 min after TRH, CDE rapidly terminated the stimulation by TRH of phosphoinositide hydrolysis, shown as inhibition of the continued formation of inositol phosphates and inositol, and of the decrease in phosphoinositides. Lastly, when cells were stimulated with 50 nM TRH, then exposed to 100 microM CDE, and finally to 1000 nM TRH, inositol phosphate formation was stimulated, then inhibited, and then restimulated. These data demonstrate that CDE acts as a competitive antagonist of TRH action on GH3 cells by competing with TRH for binding to its receptor and that continued stimulation by TRH of phospholipase C-mediated hydrolysis of phosphoinositides is tightly coupled to receptor occupancy. PMID- 3015560 TI - Modulation by retinoic acid of 1,25-dihydroxyvitamin D3 effects on alkaline phosphatase activity and parathyroid hormone responsiveness in an osteoblast-like osteosarcoma cell line. AB - Based on the finding that retinoic acid (RA) increases 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor number in ROS 17/2 cells, we investigated the effects of RA on the ability of 1,25-(OH)2D3 to regulate alkaline phosphatase activity and PTH-responsive adenylate cyclase in these cells. A maximally effective dose of 1,25-(OH)2D3 (10(-8) M) caused a 75-80% increase in alkaline phosphatase activity and an approximately 70-75% attenuation of the cAMP response to PTH, while RA (10(-6) M) decreased alkaline phosphatase activity by 30-45% and decreased PTH stimulated cAMP levels by approximately 20%. Preincubation with RA did not enhance the 1,25-(OH)2D3-induced increases in alkaline phosphatase activity. The ED50 values for control and RA-treated cultures were approximately 8 X 10(-10) M and 6 X 10(-10) M, respectively. With regard to PTH responsiveness, the effects of RA preincubation on the 1,25-(OH)2D3 attenuation of cAMP response varied with the concentration of 1,25-(OH)2D3. At low doses (less than 10(-9) M), the effects of 1,25-(OH)2D3 and RA were additive. At higher doses of 1,25-(OH)2D3, the effects of RA and 1,25-(OH)2D3 were not additive, and there were no differences between control- and RA-treated cultures. The ED50 values for control- and RA treated cultures were 10(-10) M and 3 X 10(-11) M, respectively. None of the above effects were observed using equimolar doses of the vitamin D3 metabolites 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3. The data show that pretreating ROS 17/2A cells with RA to increase 1,25-(OH)2D3 receptors does not correspond with a concomitant increase in the cellular responsiveness to 1,25 (OH)2D3, as measured by increases in alkaline phosphatase activity and decreases in PTH-responsive adenylate cyclase. PMID- 3015559 TI - Growth hormone maintains its own receptors in rat adipocytes. AB - Hypophysectomy decreased the capacity of adipocytes isolated from epididymal fat to bind [125I]human GH [( 125I]hGH) specifically without changing the apparent affinity for hGH. Specific binding of hGH by adipocytes of both normal and hypophysectomized rats appeared saturated when incubated with 75-80 ng/ml or higher concentrations of GH regardless of whether binding was studied for 2 h at 37 C or for 16 h at 0 C. Maximum binding of hGH by normal adipocytes was approximately 0.45 ng/10(6) cells, and that by adipocytes of hypophysectomized rats ranged from 0.15-0.25 ng/10(6) cells. In cells of both normal and hypophysectomized rats, only 25-30% of the hormone specifically bound at 37 was removed by digestion with trypsin, and about 75% was displaced by incubation with 5 M magnesium chloride, suggesting that these adipocytes internalized a significant fraction of bound hormone and that hypophysectomy did not alter the extent of internalization. Previously bound hormone was lost from normal adipocytes with a half-time of about 32 min and from adipocytes of hypophysectomized rats with a half-time of about 45 min, suggesting that hypophysectomy slowed the rate of processing bound hormone. To determine which pituitary hormone(s) might be required to maintain GH binding, we measured the binding of [125I]hGH at 3 or 30 ng/ml by fat cells prepared from hypophysectomized rats after various treatment regimens. Administration of bovine GH ip at a dose of 10 micrograms/rat every 4 h for 24 h doubled the binding of [125I]hGH by adipocytes prepared 4 h after the last injection. Similar results were obtained in fat cells examined 4 h after only one injection of 60 micrograms bovine GH to rats hypophysectomized 2-4 weeks previously. When binding was measured 16-24 h after GH administration, there was no apparent effect on restoration of binding even after treatment with 100 micrograms GH/day for up to 6 days, suggesting that the effects of GH in maintaining receptor number are transient. In accord with the apparently short-lived ability of GH to maintain its receptors on fat cells, GH binding was significantly reduced in adipocytes obtained form both hypophysectomized and sham-operated rats as early as 4 h after surgery, and by 8 h after surgery, declined to a level as low as that in adipocytes of chronically hypophysectomized rats. Twenty-four hours after surgery, GH binding by cells of sham-operated animals returned to normal. Fasting for 24 h also reduced GH binding by adipocytes of normal rats to a level comparable to that in adipocytes of fed hypophysectomized animals.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3015561 TI - Identification of binding sites for an insulin-like growth factor (IGF-I) in the median eminence of the rat brain by quantitative autoradiography. AB - The microanatomical location of IGF-I binding in the rat brain was determined by in vitro autoradiography with slide-mounted sections of frozen brain. Sections incubated in 0.1 nM [125I]-iodo-IGF-I produced a dense grain concentration in regions of the autoradiographic image corresponding to the external palisade zone of the median eminence; other hypothalamic regions were not so heavily labeled. This reaction was significantly reduced in the presence of 100 nM IGF-I. Measurement of binding by computer digital image analysis of autoradiographic images showed that specific binding for IGF-I in the median eminence was 41.3 +/- 8 X 10(-3) fmol/mm2 (mean +/- SEM); nonspecific binding was 11.9 +/- 1.8 X 10(-3) fmol/mm2. In contrast, specific binding to other hypothalamic regions was uniformly lower. In a separate experiment, 1000 nM unlabeled insulin was added. Without insulin, specific binding was 23 +/- 0.9 X 10(-3) fmol/mm2; nonspecific binding was 8 +/- 0.5 X 10(-3) fmol/mm2. In the presence of 1000 nM unlabeled insulin, specific binding for [125I]-iodo-IGF-I was 23 +/- 1 X 10(-3) fmol/mm2. The results suggest that a high concentration of receptors for an IGF-I-like molecule is present in the median eminence. PMID- 3015562 TI - Comparative effect of angiotensin II, potassium, adrenocorticotropin, and cyclic adenosine 3',5'-monophosphate on cytosolic calcium in rat adrenal cells. AB - The present study compares changes in cytosolic calcium and steroidogenesis when rat adrenal cells are stimulated with potassium (K+), angiotensin II (AII), ACTH, and (Bu)2cAMP (cAMP). The calcium-sensitive fluorescent dye, quin 2, was used to determine cytosolic calcium concentrations. K+ and AII both induced parallel increases in cytosolic calcium and aldosterone output. Removal of external calcium from the incubation media or addition of nifedipine inhibited the rise in cytosolic calcium in response to these two secretagogues. Inhibition of release of intracellularly-bound calcium by incubating the cells with 8-(N-N diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride or dantrolene sodium reduced the rise in cytosolic calcium in response to these two secretagogues by 40-50%. In contrast, neither ACTH nor cAMP altered cytosolic calcium levels in the glomerulosa cells, even though quin 2-loaded cells showed a normal steroidogenic response to these agents. Thus, there was a dissociation between the cytosolic calcium response and steroidogenesis during cAMP stimulation of glomerulosa cells. Fasciculata cells incubated in the presence of increasing concentrations of cAMP, ACTH, K+, or AII failed to demonstrate an increase in cytosolic calcium, although the cells had a normal steroidogenic response to ACTH and cAMP. These results suggest that the responses of fasciculata and glomerulosa cells to secretagogues have different dependencies on calcium. The fasciculata cell has little calcium dependency while the glomerulosa cell has a variable dependency. In the glomerulosa cell, both AII and K+ induced similar responses in steroid output and cytosolic calcium, suggesting an important role for cytosolic calcium as a mediator of the steroidogenic effect of these secretagogues. Furthermore, part of the increase in cytosolic calcium induced by these agents is due to release of intracellularly bound calcium and part from increased calcium flux across the cell membrane. The absence of such dependency with cAMP suggests that an increase in intracellular calcium levels is not required for increased steroidogenesis in glomerulosa cells. PMID- 3015564 TI - Effects of norepinephrine on basal and thyrotropin-stimulated thyroid hormone secretion in the mouse. AB - The possibility that norepinephrine stimulates basal but inhibits TSH-induced thyroid hormone secretion was explored by the use of in vivo and in vitro techniques. Basal thyroid hormone secretion was defined as TSH-independent secretion, experimentally obtained in vivo by pretreatment with T4 or T3. In mice pretreated with 125I and T4, norepinephrine enhanced baseline blood radioiodine levels, but in control mice, norepinephrine failed to alter basal plasma T4 levels. To study whether the stimulated release of radioiodine truly reflects stimulated thyroid hormone secretion, the nature of the radioiodine that was elevated by norepinephrine after 125I pretreatment was investigated. In controls, the fraction of radioiodine that was bound to thyroid hormones was 24.6 +/- 0.8% (+/- SE) of the total radioiodine. This figure increased to 31.9 +/- 1.1% (P less than 0.001) in response to norepinephrine. Further, by a technique of binding the released radioiodine to specific anti-T4 antiserum, norepinephrine was found to increase secretion of radiolabeled T4 by 22 +/- 2% (P less than 0.001). Thus, it is concluded that norepinephrine stimulates basal thyroid hormone secretion in vivo, and that its failure to alter plasma T4 levels is due to the low sensitivity of the technique. In contrast, it was found that norepinephrine inhibits the thyroid stimulatory effect of TSH in vivo. Under in vitro conditions, norepinephrine was found to increase the release of radioiodine that was bound to thyroid hormones from thyroid glands of mice that had been pretreated with 125I. However, norepinephrine failed to alter the baseline thyroid content of cAMP, yet it inhibited the cAMP-accumulating effect of TSH. In conclusion, the present study demonstrates that norepinephrine exerts a dual effect on thyroid hormone secretion in the mouse; it stimulates basal but inhibits TSH-induced thyroid hormone secretion. PMID- 3015565 TI - Dependence of the mitochondrial uptake of triiodothyronine (T3) in rat kidney on cytosolic T3-binding protein. AB - The effect of cytosolic T3-binding protein (CTBP) on mitochondrial T3 uptake was investigated by using rat kidney tissue in vitro. [125I]T3 uptake into mitochondria was time dependent in the absence of CTBP. Addition of CTBP to the incubation medium significantly increased mitochondrial [125I]T3 uptake. When mitochondria were incubated with a [125I]T3-CTBP complex, [125I]T3 uptake into mitochondria continued to be time dependent, but the amount of [125I]T3 incorporated was greater than in mitochondria incubated with free [125I]T3. Although [125I]T3 uptake was not significantly inhibited by an excess of unlabeled T3, it was markedly inhibited by an excess of unlabeled T3-CTBP complex. The degree of inhibition was related to the concentration of T3-CTBP complex. However, [125I]T3 uptake produced by incubation of mitochondria with [125I]T3-CTBP complex was not inhibited by unsaturated CTBP. [125I]T3 binding to outer mitochondrial membranes was observed when the membranes were incubated with free [125I]T3 or [125I]T3-CTBP complex; the amount of [125I]T3 bound was greater in the membranes incubated with [125I]T3-CTBP complex. [125I]T3 binding to the membranes incubated with [125I]T3-CTBP complex was displaced by the addition of unlabeled T3-CTBP complex. These results suggest that mitochondrial T3 uptake is mediated by previous binding of T3 to CTBP, and that the uptake is possibly regulated by T3-CTBP complex binding to its receptor in the outer mitochondrial membrane. PMID- 3015563 TI - Vasopressin induces breakdown of membrane phosphoinositides in adrenal glomerulosa and fasciculata cells. AB - We have previously shown that vasopressin exerts a marked mitogenic effect on adrenal glomerulosa cells. In the present study, we demonstrate that vasopressin (VP) stimulates the formation of inositol monophosphate (IP), inositol diphosphate (IP2) and inositol triphosphate (IP3) in primary cultures of glomerulosa as well as fasciculata cells 5- to 8-fold over the corresponding basal values. In both cell types, the relative stimulations of IP, IP2, and IP3 formation were similar. Angiotensin II (ATII) also induced glomerulosa cells to produce a dose-dependent (up to 10-fold) increase in IP, IP2, and IP3, but had only a small effect on fasciculata cells. The dose dependencies for ATII-induced IP, IP2, and IP3 formation and aldosterone production were nearly the same. We conclude that VP- and ATII-induced formation of inositol phosphates may represent an early step in the action of these peptides on adrenal cells. However, additional elements must be involved to account for the cell specificity of VP and ATII. In glomerulosa cells, VP stimulates mitotic activity and aldosterone secretion, while ATII is only steroidogenic. On fasciculata cells, VP induces a significant increase in the formation of inositol phosphates in spite of the absence of a known biological function in these cells. PMID- 3015566 TI - Purification and characterization of estrogen-2/4-hydroxylase activity from rabbit hypothalami: peroxidase-mediated catechol estrogen formation. AB - Estrogen-2/4-hydroxylase (E-2/4-H) activity of rabbit hypothalamic tissue was previously found to be localized in the soluble subcellular fraction. In the present study, the enzymatic activity responsible for catechol estrogen formation in this subcellular fraction of the rabbit hypothalamus was purified by ammonium sulfate fractionation, ion exchange chromatography, and chromatofocusing. E-2/4-H activity was found to be associated with a group of hemoproteins with peroxidase activity. The characteristics of this hypothalamic E-2/4-H activity were reestablished in light of a peroxidatic mechanism for catechol estrogen formation. Organic hydroperoxides stimulated E-2/4-H activity, presumably by serving as oxidizing cosubstrate required for peroxidase-mediated reactions. E 2/4-H activity in a 17,500 X g supernatant of hypothalamic tissue was linear with time for at least 10 min and with protein concentration to at least 100 micrograms/150 microliter. It displayed a pH optimum of 6 and an apparent Michaelis-Menten constant (Km) of 32 microM with respect to estradiol. The amounts of 4-hydroxyestradiol formed were comparable to those of 2 hydroxyestradiol. The characteristics established in this study for the peroxidase-type E-2/4-H were distinct from those of the particulate, NADPH dependent 2-hydroxylases found in rat liver and in porcine blastocyst and ovary. These differences provide a basis for differentiating between the two types of enzymatic activity that can lead to catechol estrogen formation in vitro. PMID- 3015567 TI - Inhibition of immunoreactive corticotropin-releasing factor secretion into the hypophysial-portal circulation by delayed glucocorticoid feedback. AB - Nitroprusside-induced hypotension evokes ACTH secretion which is primarily mediated by enhanced secretion of immunoreactive corticotropin-releasing factor (irCRF) into the hypophysial-portal circulation. Portal plasma concentrations of neither arginine vasopressin nor oxytocin are significantly altered in this paradigm. Application of a delayed feedback signal, in the form of a 2-h systemic corticosterone infusion in urethane-anesthetized rats with pharmacological blockade of glucocorticoid synthesis, is without effect on the resting secretion of arginine vasopressin and oxytocin at any corticosterone feedback dose tested. Resting irCRF levels are suppressed only at the highest corticosterone infusion rate, which resulted in systemic corticosterone levels of 40 micrograms/dl. Suppression of irCRF secretion in response to nitroprusside-induced hypotension is observed and occurs at a plasma corticosterone level between 8-12 micrograms/dl. These studies provide further evidence for a strong central component of the delayed feedback process which is mediated by modulation of irCRF release. PMID- 3015568 TI - Evidence for the presence of luteinizing hormone/human chorionic gonadotropin binding sites in the porcine uterus. AB - LH/hCG-binding sites were measured in crude membrane fractions of porcine uteri. Specific high affinity and low capacity receptors for LH/hCG were found in all (n = 17) membrane preparations of myometrium but in only 5 of 17 crude membrane fractions of endometrium of porcine uteri. There was very little competition between hCG and porcine GH (pGH), bovine TSH, pFSH, and pPRL (0.5%, 0.3%, 0.2%, and less than 0.005%, respectively). Specificity of [125I]hCG binding to other tissues was determined by incubating crude membrane preparations of heart, skeletal muscle, liver, and kidney. Numbers and affinities of available LH/hCG binding sites were characterized in all samples of myometrium and 5 endometrium membrane preparations that were positive for LH/hCG receptors. The results indicate that the number of uterine LH-binding sites in myometrium (0.66 +/- 0.17 fmol/mg) is 10 times less than the receptor capacity in porcine corpora lutea (7.46 +/- 0.54 fmol/mg) when expressed per mg protein of crude membrane preparation. However, it is approximately 60 times less when expressed per mg DNA equivalent of initial homogenate (1.31 +/- 0.28 vs. 81.18 +/- 3.64 fmol/mg, respectively). Receptor affinities of uterine LH/hCG-binding sites remained comparable to those of corpora lutea receptors (Ka = 7.8 X 10(10) M-1). Concentrations of LH/hCG-binding sites in myometrium taken from gilts in the late follicular phase of the estrous cycle (0.13 +/- 0.06 fmol/mg protein; n = 5) were significantly less (P less than 0.05 and P less than 0.01) compared to those in myometrium from luteal phase (0.85 +/- 0.22 fmol/mg protein; n = 6) or early pregnancy (1.03 +/- 0.15 fmol/mg protein; n = 6), respectively. This is probably the first evidence demonstrating specific binding of [125I]hCG by LH receptors in female uterine tissue. PMID- 3015570 TI - Effect of the antileukemic agent L-asparaginase on thyroxine-binding globulin and albumin synthesis in cultured human hepatoma (HEP G2) cells. AB - L-Asparaginase (ASNase), a drug widely used in the treatment of acute lymphoblastic leukemia, has been reported to decrease serum T4-binding globulin (TBG) levels, while results of serum albumin determinations were conflicting. This effect in vivo has been attributed to depressed liver protein synthesis, but this hypothesis has not been proved. To investigate this problem, human hepatoma (Hep G2) cells were continuously labeled for 4 h with 100 microCi/ml [35S]methionine in the absence or presence of graded amounts of ASNase (from 0.1 nM to 0.1 mM). Media and cell lysates were collected, immunoprecipitated with antialbumin or anti-TBG serum and protein A, and submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were sliced, and the radioactivity was counted in a beta-counter. A dose-dependent inhibition of TBG and albumin biosynthesis (as well as of total protein synthesis) was demonstrable, but TBG appeared to be more sensitive to the action of the drug. In fact, TBG biosynthesis was reduced by 8% with 0.1 nM ASNase, while an effect on albumin was observed only at 1 nM ASNase; 50% inhibition was obtained with 30 nM ASNase in the case of TBG and with 800 nM in the case of albumin. At the highest concentration (0.1 mM), TBG biosynthesis was reduced by 94%, and albumin biosynthesis by 75%. ASNase also proved to have a time-dependent effect, as assessed by the measurement of radioimmunoassayable TBG in the media from Hep G2 cells grown in the presence of 10 nM ASNase for 1-4 days. The TBG concentration was progressively reduced, by 40% after 1 day to 85% after 4 days. In pulse-chase experiments, a reduction of total (intracellular plus secreted) immunoprecipitable TBG and, to a lesser extent, albumin was observed, suggesting that the drug also affected the catabolism of newly synthesized proteins. These results provide the first in vitro evidence that ASNase actually inhibits TBG biosynthesis. This effect is not specific for TBG, but this protein appears to be more susceptible than albumin to ASNase action. This can explain why in patients treated with ASNase for leukemia, a decrease in serum TBG concentrations has not always been associated with a reduction in serum albumin levels. PMID- 3015571 TI - Increase in the number of type II insulin-like growth factor receptors during propylthiouracil-induced hyperplasia in the rat thyroid. AB - We have observed that membranes isolated from rat thyroids contain receptors for the insulin-like growth factors (IGF). As IGFs are known to be important mediators of tissue growth, we conducted this study to determine whether modulation of thyroid IGF receptors might be involved in TSH-stimulated hyperplasia. A substantial increase in both the weight of the thyroid and its DNA content was observed within 2 days of exposing adult male rats to 0.1% propylthiouracil (PTU) in their drinking water. Serum T4 reached unmeasurable levels and serum TSH rose 3-fold over control by the tenth day of treatment. [125I]Iodo-human(h)IGF-II binding to membranes isolated from hyperplastic glands was significantly higher than control beginning at 2 days. A maximum was reached after 5 days (13.3 +/- 0.8%/25 micrograms protein vs. a control level of 6.7 +/- 0.7%, mean +/- SEM). The increase had disappeared by 15 days of PTU exposure, paralleling the drastic fall in the growth rate of the glands. This increase in binding was specific for the thyroid, as it was not seen in other organs. In both treated and control animals, the receptor involved was shown to be type II by preferential binding to IGF-II, lack of interaction with insulin, and molecular sizing. The observed increase in binding could be accounted for by an increase in receptor site number, the affinity remaining essentially the same. We conclude that the TSH-stimulated hyperplasia of the rat thyroid, induced by PTU, is associated with an increase in the binding sites of the type II IGF receptor. This observation raises the possibility that modulation of this receptor may play a role in the mediation of the mitogenic effect of TSH on the thyroid gland. PMID- 3015569 TI - Effects of pancreatic hormones and glucocorticosteroids on argininosuccinate synthetase and argininosuccinase activities of rat liver during the perinatal period: in vivo and in vitro studies. AB - The activity changes of two urea cycle enzymes, argininosuccinate synthetase (ASS) and argininosuccinase (ASL), were followed after corticosteroid and pancreatic hormone treatments in utero and in primary cultured fetal hepatocytes. The ASL activity which was induced by glucagon or by (Bu)2cAMP administration was enhanced by a treatment with streptozotocin for 2 days, although ASS was not changed under these conditions. The activity of both enzymes was enhanced by cortisol administration in utero only in streptozotocin-treated fetuses, suggesting an inhibitory effect of insulin. In cultured fetal hepatocytes, dexamethasone produced a marked increase of the two enzyme activities, which was abolished by the simultaneous addition of insulin. The parallel results obtained with these two experimental models allow one to conclude that the high plasma insulin level in late gestation might repress the development of ASS and ASL activities in utero and antagonize the effect of corticosteroids on these enzyme activities. PMID- 3015572 TI - Effect of growth hormone-releasing factor-44 upon release of concurrently synthesized hormone by perifused rat pituitary tissue. AB - We previously reported the differential stimulation of stored and newly synthesized rat (r)GH release by human GH-releasing factor-44 (hGRF-44). Those studies were performed over a 3-h period in a static in vitro incubation system. The present experiments focus on hGRF-44 effects upon release of new hormone (synthesized during tissue stimulation by the secretagogue) and were performed in in vitro perifusion to study the time course of the response. A double label ([14C], [3H]), immunoprecipitation protocol defined hormone release in relation to time of synthesis: intracellular stores of hormone were prelabeled with [14C]; subsequent exposure of prelabeled tissue to continuous concurrent [3H]leucine and 3 nM hGRF-44 defined newly synthesized hormone and associated it with the secretagogue. In a parallel set of experiments, 1 mM (Bu)2cAMP was substituted for hGRF-44. Prolonged exposure to hGRF-44 in perifusion stimulated an initial surge of stored [14C]rGH release which was followed by a rate of [14C]rGH release which declined rapidly but remained suprabasal. [14C]rGH release in response to (Bu)2cAMP was also biphasic, but the initial surge was delayed and the later stimulatory period was better sustained in comparison to responses to hGRF-44. Stimulation of stored [14C] rPRL release by hGRF-44 was observed in perifusion, confirming our observation in the static system. Release of newly synthesized, 3H labeled rGH was immediately stimulated by either hGRF-44 or (Bu)2cAMP, and that stimulation was maintained throughout exposure to either secretagogue. In contrast, whereas newly synthesized [3H]rPRL release was stimulated by (Bu)2cAMP, its release in response to hGRF-44 resembled that in control experiments. No effect upon hormone synthesis was observed during the 3h of exposure to hGRF-44. In conclusion, these experiments confirm that hGRF-44 differentially stimulates release of both newly synthesized and stored rGH, and demonstrate differential dynamics in the response as well. Specifically, hGRF-44 stimulation of new rGH release is sustained while its effect on stored hormone simultaneously wanes. Further, when expressed as a percent of intracellular hormone available for release, new hormone is released more than 10 times faster than stored hormone. These observations argue for the existence of separate intracellular paths along which newly synthesized and stored hormone are released by the somatotroph. Finally, these data confirm that hGRF-44 stimulates release of stored rPRL without altering release of newly synthesized rPRL. PMID- 3015573 TI - Extracellular calcium is required for copper-amplified prostaglandin E2 stimulation of the release of gonadotropin-releasing hormone from median eminence explants. AB - Prostaglandin E2 (PGE2) is known to stimulate the release of LHRH from hypothalamic tissue incubated under in vitro conditions. We have previously shown that a short preincubation of explants of the median eminence area with copper(II) [Cu(II)] complexed to histidine (CuHis) markedly amplifies PGE2 stimulation of LHRH release. In this study, we wished to ascertain if Cu amplified PGE2 stimulation of LHRH release is dependent on influx of extracellular calcium (Ca) and, if so, whether it is the copper and/or PGE2 action that is Ca dependent. Median eminence area explants were incubated for 5 min with 200 microM CuHis, then for 15 min with 10 microM PGE2, and finally for 45 min in buffer (Cu/PGE2). Controls were incubated with CuHis or PGE2. In the presence of Ca (1.8 mM), Cu/PGE2 stimulation of LHRH release reached a peak value within 15 min of exposure to PGE2 (accelerated phase), after which the rate of release declined, with a half-life of about 20 min (decelerated phase). In the absence of Ca, Cu/PGE2 stimulation of LHRH release was inhibited by 70% during the accelerated phase, and it was completely inhibited during the decelerated phase. When incubation was carried out in the presence of Ca and 100 microM verapamil, Cu/PGE2 stimulation of LHRH release was similar to that seen in the absence of extracellular Ca. When Ca was omitted from the incubation medium during the exposure to CuHis but was included during and after the exposure to PGE2, the response to PGE2 was fully restored. In addition, we evaluated the extracellular Ca requirement for stimulation of LHRH release by PGE2 alone (without pretreatment with CuHis) and found that release was inhibited by 65% in the absence of Ca. These results indicate that the process of PGE2 stimulation of LHRH release is partially dependent on extracellular Ca, that the process of Cu amplified PGE2 stimulation of release is highly dependent on influx of extracellular Ca, and that PGE2 action, but not Cu action, is dependent on Ca influx. Since cAMP has been implicated in Ca2+-dependent secretion in other cells, we propose that Cu amplifies the functional state of a postreceptor component involved in the Ca-cAMP secretory pathway stimulated by PGE2. PMID- 3015574 TI - Tissue distribution and subcellular localization of an endogenous substrate (pp 120) for the insulin receptor-associated tyrosine kinase. AB - The beta-subunit of the insulin receptor possesses a tyrosine-specific protein kinase activity which may play a role in coupling insulin binding to insulin action. Previously, we have identified a substrate for the receptor-associated protein kinase in a cell-free system. This endogenous substrate (pp120), which appeared to be a glycoprotein with an apparent mol wt of 120,000, was detected in rat liver microsomes. In the present work, we have demonstrated that pp120 is localized to a highly purified preparation of rat liver plasma membranes (Neville preparation). Moreover, pp120 appears to be specific to liver, having been detected in liver from rat, monkey, and rabbit, but not in rat brain, skeletal muscle, heart, kidney, or adipocytes. As a preliminary to addressing the question of whether insulin stimulates phosphorylation of pp120 in intact cells, we have sought to identify tissue culture cell lines that contain both insulin receptors and pp120. We have succeeded in identifying pp120 in two cell lines derived from rat liver: 1) H35 hepatoma cells (Reuber hepatoma) and 2) rat hepatocytes transformed with a temperature-sensitive mutant form of SV-40 (cultivated at both permissive and nonpermissive temperatures). In conclusion, pp120 appears to be a liver-specific plasma membrane glycoprotein which serves as a substrate for phosphorylation by the insulin receptor-associated protein kinase in a soluble cell-free system. The presence of pp120 in cultured cell lines will facilitate investigation of whether the phosphorylation of pp120 in intact cells is physiologically regulated in response to insulin. PMID- 3015575 TI - Angiotensin II and adrenocorticotropin release: mediation by endogenous corticotropin-releasing factor. AB - Recent reports indicate that the main effect of systemically administered angiotensin II (AII) on ACTH release is probably due to some central nervous system mechanism. The present studies were designed to investigate whether the central action of AII on ACTH release is directly mediated through CRF. In order to test the participation of endogenous CRF in the AII-induced ACTH release in vivo, intact and pharmacologically blocked (pretreated with chlorpromazine morphine-nembutal) female rats were injected iv with AII (8 nmol/100 g BW). Plasma levels of ACTH as well as CRF content in the median eminence (ME) and medial basal hypothalamus (MBH) were evaluated before and 1, 2.5, and 5 min after treatment. These responses were compared with the effect of 1 min ether exposure on hypothalamic CRF content and plasma ACTH levels in unanesthetized animals. AII injection and ether exposure increased plasma ACTH levels several-fold at both 2.5 and 5 min post treatment in intact rats. Conversely, AII failed to induce any significant increase in plasma ACTH levels in centrally blocked rats at any interval studied. On the other hand, AII injection and ether inhalation acted in similar fashion on CRF content in ME, inducing fast depletion at 1 min post treatment, recovering to control values at 2.5 min after injection, and finally, accumulating peptide at 5 min post treatment. In addition, CRF content in the MBH decreased significantly at 5 min, under both experimental conditions; AII had no effect on hypothalamic CRF content in centrally blocked rats. In vitro experiments using whole MBH (containing ME) fragments incubated with either neural peptides or high K+ solutions indicate that AII possesses a CRF releasing effect at concentrations of 10(-6) M or more. Conversely, other hypothalamic peptides, such as LHRH, TRH, and somatostatin did not induce significant release of CRF at any of the concentrations assayed (10(-7) to 10(-5) M). On the other hand, high K+ solutions released CRF in a concentration-related manner (15-60 mM). These studies suggest that the central effect of AII stimulation on ACTH release in vivo could be, at least in part, through the release of hypothalamic CRF into the portal circulation. PMID- 3015576 TI - Seasonal changes in serum thyroid hormone binding proteins in the woodchuck (Marmota monax). AB - To evaluate the role of serum T4- and T3-binding proteins in the elevation of serum T4 and T3 concentrations in the woodchuck in the fall and winter, blood was collected from woodchucks during the four seasons of the year (seasonal study) and from 2-week fasted woodchucks in the summer (fasting study) and the serum concentrations of total T4 and T3 were measured. The distribution of [125I]4 and [125I]T3 tracers among the serum binding proteins and the serum T4-binding globulin (TBG) binding capacity for T4 was determined by polyacrylamide gel electrophoresis. Plasma concentrations of both T4 and T3 were highest in the winter. The major T4-binding protein in the woodchuck is TBG. There was an increase in both [125I]T4 and [125I]T3 tracer binding to serum TBG in fall and winter, and TBG binding capacity for T4 was 2-fold higher in winter than in summer. There were increases in TBG binding and in the TBG T4 binding capacity in the 2-week starved animals. The increased binding of T4 and T3 by TBG in the fall and winter may be partially responsible for the increased serum concentrations of T4 and T3 in the fall and winter in the woodchuck, a time when secretion of T4 and T3 by the thyroid gland is very low. This may be facilitated by the low or absent food consumption at these times of the year. PMID- 3015577 TI - Biphasic inhibition of adrenocorticotropin release by corticosterone in cultured anterior pituitary cells. AB - The inhibition of ACTH secretion by glucocorticoids in vivo is biphasic, with rapid early suppression followed by transient recovery and a late inhibitory phase. To evaluate whether this biphasic effect of glucocorticoids occurs at the pituitary level, the effects of corticosterone (B) on stimulated ACTH release were analyzed in rat anterior pituitary cell cultures. Preincubation with 1 microM B inhibited the ACTH response to 10 nM CRF in a biphasic manner, with rapid inhibition after 10-40 min of preincubation, followed by partial recovery between 40-80 min, and a later phase of inhibition after 80-140 min. Preincubation with B for 40 or 120 min caused a dose-dependent suppression of CRF stimulated ACTH release, with ED50 values of 416 +/- 21 and 45 +/- 12 nM B, respectively. Pretreatment with B also caused a biphasic inhibitory effect on the stimulatory action of vasopressin, angiotensin II, and norepinephrine on ACTH release. However, addition of these stimuli in combination with CRF surmounted B inhibition of CRF-stimulated ACTH release. B also inhibited the ACTH-releasing effects of postreceptor stimuli, including 8-bromo-cAMP, phorbol 12-myristate 13 acetate, and 1,2-dioctanoylglycerol. In the presence of cycloheximide (10 microM), the early inhibitory effect of B was unchanged, but the delayed effect was decreased. Whereas preincubation with B for 40 min inhibited ACTH release, but not total intracellular plus released ACTH, preincubation for 120 min decreased both released and total ACTH. These findings demonstrate that the two inhibitory effects of B on ACTH release differ in their kinetics, steroid sensitivity, and dependence on protein synthesis. The inhibitory effect of B on ACTH responses to stimuli with different mechanisms of action suggests that the suppressive effects of B are mainly exerted at a site distal to the formation of the second messengers involved in hormonal activation of ACTH release. PMID- 3015578 TI - Luteinizing hormone triggers two opposite regulatory pathways through an initial common event, receptor aggregation. AB - LH receptor internalization was studied with an antireceptor monoclonal antibody (aLHR) which induces Leydig cells to produce testosterone. To follow receptor mediated aLHR internalization, cells were incubated with aLHR at 10 C for 3 h to generate an aLHR complex; this was followed by a second incubation with fluorescent labeled antimouse immunoglobulin at 34 C, a temperature which allows internalization. Within 15 min at 34 C, cytoplasmic fluorescent staining was detectable; this staining was strongly visible after 60 min. At no time was nuclear staining observable. Employing such an approach, it has also been possible to follow the fate of unoccupied receptors when cells are stimulated with a submaximal dose of LH. The results show that LH interactions with 20% of its receptors produces microaggregation, patching, capping, and internalization of free receptor sites. The results further demonstrate that cells with receptors in the state of capping are less sensitive to a second LH stimulation, suggesting that in this state receptors are no longer coupled to the adenylate cyclase system. PMID- 3015579 TI - Adrenal and pituitary effects of mitotane in Cushing's syndrome. PMID- 3015580 TI - Cholinergic but not serotonergic mediation of exercise-induced growth hormone secretion. AB - In order to clarify the roles of cholinergic and serotonergic neurotransmission in the mediation of exercise-induced growth hormone (GH) release, normal young volunteers of both sexes were studied. Exercise was for 20 minutes at 800 kpm for the men and 500 kpm for the women. Pretreatment with 0.4 mg atropine 1 hour prior to exercise, or with methysergide 2 mg po q 6 h for 48 hours prior to exercise, were used to evaluate the influence of cholinergic and serotonergic blockade, respectively. Five of the ten men studied failed to raise GH values with exercise, perhaps because the exercise was not vigorous enough for their high degree of fitness. Of three non-responders restudied, at the same workload, one responded on the second occasion. The mean peak GH with exercise 13.4 +/- 3.27 ng/ml, was reduced to 2.4 +/- 1.28 ng/ml (p less than 0.01) after atropine, but was unaffected by methysergide (15.2 +/- 6.58 ng/ml, p greater than 0.5). Prolactin did not rise with exercise, and was not affected by atropine, but lowered by methysergide as expected. Cholinergic neurotransmission therefore represents a key link in exercise-induced GH secretion, but serotonergic influences are probably not involved. PMID- 3015581 TI - Release of stored, pre-labeled growth hormone and prolactin from perifused rat pituitary: effect of human pancreatic growth hormone-releasing factor-44. AB - Human pancreatic growth hormone-releasing factor-44 (hpGRF-44) differentially stimulates release of stored and newly synthesized rGH without altering rGH synthesis over 3 h in static in vitro incubation; hpGRF-44 also stimulates release of stored, but not newly synthesized, rPRL. To study the time course of pre-labeled, stored hormone release without pharmacologically interrupting synthesis, the current experiments were performed in perifusion. Fifteen minute pulses of 0.1 to 10 nM hpGRF-44 stimulated stored [3H]rGH release (to 890% of base); 1.0 to 10 nM hpGRF-44 stimulated stored [3H]rPRL release (to 440% of base). Pulses of 0.1 to 1.0 mM (Bu) 2cAMP also stimulated release of [3H]rGH (to 570% of base) and [3H]rPRL (to 410% of base). However, peak [3H]rGH and [3H]rPRL responses to hpGRF-44 required 10 min, while peak responses to (Bu) 2cAMP required 25 min. Continuous hpGRF-44 stimulated an initial surge of stored [3H]rGH release which was not sustained; the diminishing release was not explained by hpGRF-44 degradation. Total radioimmunoassayable (RIA) hormone release roughly paralleled release of stored immunoprecipitable (IPn) hormone. CONCLUSIONS: in pituitary perifusion, hpGRF-44 stimulates release of both stored rGH and rPRL as shown in static incubation, but the response is biphasic: initial rapid release is followed by a progressively lesser response; and the response is both more acute and less well sustained than that resulting from exposure to (Bu) 2cAMP. PMID- 3015582 TI - Potentiation of sister chromatid exchange by 3-aminobenzamide is not modulated by topoisomerases or proteases. AB - Poly(ADP-ribose) is synthesized in response to DNA strand breaks and covalently modifies numerous intracellular proteins. We have proposed that this modification regulates, i.e., inhibits, the activity of these enzymes, e.g., topoisomerases and proteases, which could otherwise cause additional DNA damage or alterations in chromatin structure. Inhibition of poly(ADP-ribose) polymerase by 3-amino benzamide (3AB) in cells exposed to DNA-damaging agents would, according to this proposal, eliminate the regulatory role of ADP-ribosylation. When Chinese hamster ovary cells are cultured with methyl methanesulfonate (MMS) and 3AB, a synergistic increase in sister chromatid exchange frequency is observed. We investigated the regulatory role of poly(ADP-ribose) polymerase to see if topoisomerases or proteases are involved in this synergistic increase. Cells were exposed to MMS or the intercalating agent 4'-(9-acridinylamino)methanesulfon-m anisidide (m-AMSA), 3AB, and either the topoisomerase inhibitor novobiocin or the protease inhibitor antipain. Neither novobiocin nor antipain affected the synergistic response of MMS and 3AB or the additive response of m-AMSA and 3AB. These results suggest that topoisomerases or proteases do not account for the effect of 3AB on sister chromatid exchange frequency after DNA damage. PMID- 3015583 TI - The role of complement in experimental silicosis. AB - The role of the complement system in the pathogenesis of crystal-induced pulmonary inflammation and fibrosis was evaluated using a mouse model of silicosis and congenitally complement-deficient mice. Mice lacking the fifth component of complement (B10.D2/o) were compared to C5-sufficient animals (B10.D2/n) for pulmonary changes following intratracheal instillation of silica crystals. Complement-deficient mice demonstrated a significant reduction compared to complement-sufficient mice in both cell number and protein content of lung lavage fluid throughout the 12 weeks following silica exposure. Lung hydroxyproline content (indicative of collagen deposition) was equivalent for both strains and significantly higher than controls at all time points following silica instillation. Moreover, studies in vitro have shown that silica crystals are capable of activating complement via the alternative pathway. These studies indicate that the complement system may be responsible for some of the pulmonary inflammation, but not fibrosis elicited by silica exposure. PMID- 3015584 TI - Comparison of hemolytic activities of coal fly ash and its soluble and insoluble fractions. AB - Coal fly ash of a particle diameter smaller than 10 micron was collected from the precipitator of a power plant in Hong Kong. Comparison of hemolytic activities between fly ash and free silica showed that fly ash had a lower biological effect than free silica. The hemolytic activities of the soluble and insoluble fractions of fly ash were further compared by two methods: total hemoglobin method and cyanmethemoglobin method. An analysis of results showed significant differences for fly ash and its soluble fraction between methods (P less than 0.05 and P less than 0.01, respectively). Fly ash, which contained a silicate level similar to its insoluble fraction, had a hemolytic activity higher than the summation of both its soluble and insoluble fractions. This indicates that the hemolytic activity was independent of the silicate content in the fly ash samples. PMID- 3015585 TI - Equine viral arteritis: a disease of emerging significance? PMID- 3015586 TI - Association of turkey erythrocyte beta-adrenoceptors with a specific lipid component. AB - We have recently reported that the highly potent beta-adrenergic affinity label [125I]bromoacetylamino cyanopindolol ([125I]BAM-CYP) irreversibly blocks the turkey erythrocyte beta-adrenoceptor binding site by combining with a receptor associated non-protein component. In this communication, we report: lipid labelling is inhibited by beta 1-adrenergic ligands with the potency ratio and stereospecificity characteristic for the turkey erythrocyte beta 1-adrenoceptor; the tagged component is a glycolipid, probably a ganglioside; [125I]BAM-CYP blocked receptor, after solubilization in deoxycholate, can be separated from the [125I]BAM-CYP-glycolipid with restoration of the binding capacity of the beta 1 adrenoceptor protein; the tightly associated [125I]BAM-CYP-labelled glycolipid can be displaced by a glycolipid mixture extracted from turkey erythrocyte membranes but not by bovine brain gangliosides, when the blocked receptor is solubilized in digitonin. This is the first direct demonstration that a receptor protein is associated with a specific membrane lipid. The possibility that glycolipids play a role in receptor-mediated signal transduction is discussed in view of these findings and in view of data from the literature. PMID- 3015588 TI - Dissociation of cellular responses to epidermal growth factor using anti-receptor monoclonal antibodies. AB - Three biologically active monoclonal antibodies against the human epidermal growth factor (EGF) receptor (2E9, 2D11 and 2G5) have been used to analyse the interrelationship between various cellular responses to EGF. Antibody 2E9 (IgG1) is directed against the protein core of the receptor, close to or at the EGF binding site, while 2D11 (IgG3) and 2G5 (IgG2a) recognize blood-group A-related carbohydrate determinants of the receptor. These antibodies have EGF-like effects in that they can activate the receptor tyrosine kinase both in vitro and in vivo. Cross-linking of the receptor-bound antibodies by a second antibody mimics EGF in inducing a rapid aggregation of receptors on the cell surface. However, all three antibodies fail to mimic EGF in raising cytoplasmic pH and free Ca2+ and do not stimulate DNA synthesis in quiescent fibroblasts, even after external cross linking of the occupied receptors. It is concluded that EGF-R tyrosine kinase activity as well as substrate specificity can be modulated by ligands other than EGF, even if they bind to sites distinct from the EGF binding domain; activation of the receptor tyrosine kinase, receptor clustering and induction of the ionic signals are causally unrelated events; and tyrosine kinase activation and receptor cross-linking are not sufficient for stimulation of DNA synthesis. PMID- 3015587 TI - Binding of fluoresceinated epidermal growth factor to A431 cell sub-populations studied using a model-independent analysis of flow cytometric fluorescence data. AB - A method is developed for determining ligand-cell association parameters from a model-free analysis of data obtained with a flow cytometer. The method requires measurement of the average fluorescence per cell as a function of ligand and cell concentration. The analysis is applied to data obtained for the binding of fluoresceinated epidermal growth factor to a human epidermoid carcinoma cell line, A431. The results indicate that the growth factor binds to two classes of sites on A431 cells: 4 X 10(4) sites with a dissociation constant (KD) of less than or equal to 20 pM, and 1.5 X 10(6) sites with a KD of 3.7 nM. A derived plot of the average fluorescence per cell versus the average number of bound ligands per cell is used to construct binding isotherms for four sub-populations of A431 cells fractionated on the basis of low-angle light scatter. The four sub populations bind the ligand with equal affinity but differ substantially in terms of the number of binding sites per cell. We also use this new analysis to critically evaluate the use of 'Fluorotrol' as a calibration standard in flow cytometry. PMID- 3015589 TI - Spontaneous deletion formation at the aprt locus of hamster cells: the presence of short sequence homologies and dyad symmetries at deletion termini. AB - To examine the factors governing the generation of DNA sequence rearrangements in mammalian somatic cells, we have cloned and sequenced novel junctions produced by six spontaneous deletion mutations at the aprt locus of Chinese hamster ovary cells. Our analyses indicate that these rearrangements were produced by non homologous recombinational events occurring between short (2-7 bp) sequence repeats at the two termini of the deletion which leave one copy of the repeat in the mutant gene. Certain tri- and tetranucleotides recur at the deletion termini, suggesting that these may possibly be a recognition sequence for an enzyme involved in the event. No other gene structural alterations were found at the novel junctions or in neighbouring sequences. The deletions are not randomly distributed over the aprt gene; four termini clustered in a 40-bp sequence. This region of aprt is unusual as it contains both significant stretches of dyad symmetry which could potentially form stable DNA secondary structures and short direct repeats. Regions of dyad symmetry were also found at at least one terminus of all the deletions. In view of the similar properties of this set of deletions, possible mechanisms for the formation of this type of gene rearrangement are considered. PMID- 3015591 TI - Nucleotide sequence of the coding region of the mouse N-myc gene. AB - A genomic clone for the mouse N-myc gene was isolated and the total nucleotide sequence (4807 bp) of the two coding exons and an intron located between them was determined. The amino acid sequence of the N-myc protein was deduced from the DNA sequence. This protein is composed of 462 amino acids, slightly larger than human and mouse c-myc proteins, and is rich in proline like the c-myc protein. Comparison of the amino acid sequences of the mouse N-myc and c-myc proteins showed that conserved sequences are located in eight regions: four regions are in the N-terminal half of the N-myc protein and are separated from each other by regions poorly homologous to those of the c-myc protein, and the four others are located in the C-terminal half, throughout which certain homology exists. A remarkable sequence containing 13 successive acidic amino acids is present in one of the conserved regions located in the middle of the N-myc protein. PMID- 3015590 TI - Cloning and structural characterization of a human non-erythroid band 3-like protein. AB - Polypeptides which are immunologically related to the erythrocyte anion transport protein have been identified in a variety of non-erythroid cells. We describe two cDNA clones encoding a human non-erythroid band 3 protein (HKB3) and the mouse erythrocyte band 3 (MEB3) and show that these proteins are structurally similar. Comparison of the predicted amino acid sequences from HKB3 and MEB3 reveals a high degree of sequence homology (71%) and conservation of the overall topography of the transmembrane domain. Similar levels of homology are also observed in comparisons with published amino acid sequence from the human erythrocyte band 3. In addition, specific residues which have been demonstrated to be involved in erythroid anion transport are conserved in HKB3, suggesting that this non erythroid band 3 protein functions in this respect. Although protein sequence homology within the cytoplasmic domain is considerably lower (35%), three specific regions in HKB3 are conserved, one of which may represent an ankyrin binding site. Northern blot analysis reveals transcripts that cross-hybridize with the HKB3 cDNA in a variety of non-erythroid cell lines but not in cells of erythroid lineage. PMID- 3015592 TI - The structure of the mouse glutathione peroxidase gene: the selenocysteine in the active site is encoded by the 'termination' codon, TGA. AB - Glutathione peroxidase (GSHPx) is an important selenium-containing enzyme which protects cells from peroxide damage and also has a role in leukotriene formation. We report the identification of a genomic recombinant as encoding the entire mouse GSHPx gene. Surprisingly, the selenocysteine in the active site of the enzyme is encoded by TGA: this has been confirmed by primer extension/dideoxy sequencing experiments using reticulocyte mRNA. The same site of transcription initiation is used in three tissues in which the GSHPx mRNA is expressed at high levels (erythroblast, liver and kidney). Like some other regulated 'house keeping' genes, the GSHPx gene has Sp1 binding site consensus sequences but no 'ATA' and 'CAAT' consensus sequences upstream of the transcription initiation site. Moreover, there is a cluster of two Sp1 binding site consensus sequences and two SV40 core enhancer sequences in the 3' region of the gene, close to the previously mapped position of a DNase I-hypersensitive site found only in tissues expressing the GSHPx mRNA at high levels. PMID- 3015593 TI - Embryonic expression and nuclear localization of Xenopus homeobox (Xhox) gene products. AB - We have isolated a Xenopus genomic clone (Xhox-1) that contains two homeoboxes, at least one of which is highly conserved in vertebrates. Two transcripts are derived from Xhox-1 and these are present from early in development through swimming tadpole stages. A detailed analysis of one of the genes shows that it is expressed maternally and that transcription from the zygotic genome begins at mid gastrulation. Examination of exogastrula shows that the expression of these genes does not depend on neural induction. We have determined the DNA sequence of a Xhox-1 cDNA and this allows us to predict the complete sequence of a vertebrate homeobox protein. Synthetic mRNAs made from this cDNA are translated efficiently in Xenopus oocytes where the homeobox protein localizes to the oocyte nucleus. PMID- 3015594 TI - Increased levels of mitochondrial gene expression in rat fibroblast cells immortalized or transformed by viral and cellular oncogenes. AB - Steady-state levels of the mitochondrial (mt) mRNA encoding subunit II of cytochrome oxidase (COII) were increased 5-10 fold in fully transformed cell lines derived from rodent embryonic fibroblasts after transfer of polyoma virus DNA, and in immortalized cell lines established by transfer of plt (polyoma large T protein), E1A (adenovirus) and myc oncogenes. Increased mitochondrial gene expression was not related with active growth per se: it was low in fast-growing rat embryo cells, and it did not change upon serum starvation and subsequent stimulation of FR3T3 cells. The number of copies of mtDNA did not vary, and different mitochondrial mRNAs and rRNAs were increased in the same proportions, suggesting a change in the rate of accumulation of their common precursor. PMID- 3015595 TI - Characterization of the gene for the microbody (glycosomal) triosephosphate isomerase of Trypanosoma brucei. AB - To determine how microbody enzymes enter microbodies, we are studying the genes for glycosomal (microbody) enzymes in Trypanosoma brucei. Here we present our results for triosephosphate isomerase (TIM), which is found exclusively in the glycosome. We found a single TIM gene without introns, having one major polyadenylated transcript of 1500 nucleotides with a long untranslated tail of approximately 600 nucleotides. By a novel method, suitable for low abundance transcripts, we demonstrate that TIM mRNA contains the 35-nucleotide leader sequence (mini-exon) also found on several other trypanosome mRNAs. The TIM gene and a DNA segment of at least 6 kbp upstream of the gene are transcribed at an equal rate in isolated nuclei, suggesting that the gene is part of a much larger transcription unit. The predicted protein is of the same size as TIMs from other organisms and shares approximately 50% amino acid homology with other eukaryote TIMs, somewhat less with prokaryote TIMs. Trypanosome TIM is the most basic of all TIMs sequenced thus far. This is, in part, due to the presence of two clusters of positively charged residues in the molecule which may act as a signal for entry into glycosomes. PMID- 3015596 TI - One nuclear gene controls the removal of transient pre-sequences from two yeast proteins: one encoded by the nuclear the other by the mitochondrial genome. AB - The proteolytic processing of the mitochondrially encoded subunit II of cytochrome oxidase is prevented by the yeast mutation ts2858. We report that the mutant is, in addition, temperature sensitive for the processing of cytochrome b2, a protein encoded by nuclear DNA. Thus the same mutation affects the removal of pre-sequences from a mitochondrially encoded inner membrane protein and from an imported soluble protein located in the intermembrane space. The mutation blocks the second processing step of cytochrome b2. The cytochrome b2 intermediate accumulates in the mutant at 36 degrees C and assumes its enzyme activity. At 23 degrees C the conversion to the mature protein is considerably slower than in wild-type cells. The similarity of the cleavage sites Asn-Asp and Asn-Glu of the precursors for cytochrome oxidase subunit II and cytochrome b2, respectively, suggests a sequence-specific recognition by one protease or a factor activating a protease. On the other hand maturation of cytochrome c peroxidase, another enzyme of the intermembrane space, is not affected by the pet ts2858 mutation. PMID- 3015597 TI - Generation of histone mRNA 3' ends by endonucleolytic cleavage of the pre-mRNA in a snRNP-dependent in vitro reaction. AB - Incubation of SP6 generated mouse histone H4 mRNA precursors in nuclear extracts of HeLa cells yields processed mRNA species which end on the 3' adenosine of the conserved terminal ACCA sequence not unlike ten different histone mRNAs isolated from sea urchin embryos which end either on the 3' C or A. In the presence of 20 mM EDTA, the cleaved off 3' spacer portions of the RNA transcripts are readily detected, hence the 3' ends of histone mRNAs arise as a consequence of endonucleolytic cleavage(s) of the precursor RNA. The in vitro cleavage reaction is specifically inhibited by human antisera of the Sm-serotype, but not by control sera and can be rescued by the addition of a preparation of partially purified small nuclear RNPs to the antibody-depleted extract. Interestingly, the snRNP preparation is sufficient to elicit 3' processing of pre-mRNA in the absence of added nuclear extract. PMID- 3015598 TI - A chemically synthesized pre-sequence of an imported mitochondrial protein can form an amphiphilic helix and perturb natural and artificial phospholipid bilayers. AB - Subunit IV of yeast cytochrome oxidase is made in the cytoplasm with a transient pre-sequence of 25 amino acids which is removed upon import of the protein into mitochondria. To study the function of this cleavable pre-sequence in mitochondrial protein import, three peptides representing 15, 25 or 33 amino terminal residues of the subunit IV precursor were chemically synthesized. All three peptides were freely soluble in aqueous buffers, yet inserted spontaneously from an aqueous subphase into phospholipid monolayers up to an extrapolated limiting monolayer pressure of 40-50 mN/m. The two longer peptides also caused disruption of unilamellar liposomes. This effect was increased by a diffusion potential, negative inside the liposomes, and decreased by a diffusion potential of opposite polarity. The peptides, particularly the two longer ones, also uncoupled respiratory control of isolated yeast mitochondria. The 25-residue peptide had little secondary structure in aqueous buffer but became partly alpha helical in the presence of detergent micelles. Based on the amino acid sequence of the peptides, a helical structure would have a highly asymmetric distribution of charged and apolar residues and would be surface active. Amphiphilic helicity appears to be a general feature of mitochondrial pre-sequences. We suggest that this feature plays a crucial role in transporting proteins into mitochondria. PMID- 3015599 TI - Mitochondrial targeting sequences may form amphiphilic helices. AB - Twenty three mitochondrial targeting sequences have been analysed with regard to their potential for forming amphiphilic helices. It is shown that most if not all of these sequences can be expected to form helices with high hydrophobic moments in a suitable environment. In the few cases studied so far, the segments of maximal hydrophobic moment coincide closely with 'critical' regions defined by deletions and point mutations. PMID- 3015600 TI - Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide-binding site. AB - v-Ha-ras encoded p21 protein (p21V), the cellular c-Ha-ras encoded protein (p21C) and its T24 mutant form p21T were produced in Escherichia coli under the control of the tac promoter. Large amounts of the authentic proteins in a soluble form can be extracted and purified without the use of denaturants or detergents. All three proteins are highly active in GDP binding, GTPase and, for p21V, autokinase activity. Inhibition of [3H]GDP binding to p21C by regio- and stereospecific phosphorothioate analogs of GDP and GTP was investigated to obtain a measure of the relative affinities of the three diphosphate and five triphosphate analogs of guanosine. p21 has a preference for the Sp isomers of GDP alpha S and GTP alpha S. It has low specificity for the Sp isomer of GTP beta S. Together with the data for GDP beta S and GTP gamma S these results are compared with those obtained for elongation factor (EF)Tu and transducin. This has enabled us to probe the structural relatedness of these proteins. We conclude that p21 seems to be more closely related to EF-Tu than to transducin. PMID- 3015601 TI - Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector. AB - The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has played a major role in studies on the molecular biology of insect DNA viruses. Recently, this system has been effectively adapted as a highly efficient vector in insect cells for the expression of several mammalian genes. A cDNA sequence of the influenza (fowl plague) virus haemagglutinin gene has been inserted into the BamHI site of the pAc373 polyhedrin vector. Spodoptera frugiperda cells were co-transfected with this construct, pAc-HA651, and authentic AcNPV DNA. Recombinant virus was selected by adsorption of transfected cells to erythrocytes followed by serial plaque passages on S. frugiperda cells. We have determined the site of insertion of the haemagglutinin gene into the AcNPV genome by restriction enzyme cleavage and Southern blot hybridization analyses using haemagglutinin cDNA as a probe. The influenza haemagglutinin gene is located in the polyhedrin gene of AcNPV DNA. Immunofluorescent labelling, immunoprecipitation and immunoblot analyses with specific antisera revealed that S. frugiperda cells produce immune reactive haemagglutinin after infection with the recombinant virus. The haemagglutinin is expressed at the cell surface and has haemolytic capacity that has been activated by post-translational proteolytic cleavage. When chickens were immunized with S. frugiperda cells expressing haemagglutinin, they developed haemagglutinin-inhibiting and neutralizing antibodies and were protected from infection with fowl plague virus. These observations demonstrate that the haemagglutinin is processed in insect cells in a similar fashion as in fowl plaque virus-infected vertebrate cells and that it has full biological activity. PMID- 3015602 TI - Nuclear factor 1 interacts with five DNA elements in the promoter region of the human cytomegalovirus major immediate early gene. AB - The human cytomegalovirus (HCMV), a ubiquitous pathogen of the herpesvirus group, has a linear double-stranded DNA genome of 235 kb. The expression of its major immediate early gene (IE1) is entirely dependent on host factors, presumably proteins binding to DNA elements in the regulatory regions of the gene. We have identified four high-affinity binding sites for nuclear factor 1 (NF1) in the promoter upstream region of IE1 gene between nucleotides -780 and -610, and an additional, even stronger, binding site in the first intron near nucleotide +350. NF1 activity is found in a wide range of species and binds to the sequence 5' TGGC/ANNNNNGCCAA3' on double-stranded DNA, protecting approximately 25 bp from DNase I digestion; its functional importance has been found first in the necessity for adenovirus DNA replication, where it may be important in mediating the binding of other proteins. The regulatory significance of NF1 recognition elements within other genes is unknown. The NF1 binding sites in the HCMV IE1 gene coincide with regions that had been shown to be sensitive to DNase I in the active gene but not sensitive in the silent gene; there was, however, no NF1 binding in the strong and constitutively DNase I-hypersensitive transcription enhancer of the IE1 gene. This suggests that the specific protein--DNA interaction described is important in the regulated control of the IE1 gene. PMID- 3015605 TI - Demonstration of membrane receptors for human natural and recombinant 125I labeled tumor necrosis factor on HeLa cell clones and their role in tumor cell sensitivity. AB - Samples of natural and recombinant tumor necrosis factor (TNF) were labelled with 125I by use of Iodobeads and were used to study TNF receptor expression on different HeLa clones. Both labeled preparations retained cytotoxic activity after 125I-labelling and were indistinguishable from their unlabeled counterparts on sodium dodecylsulfate/polyacrylamide gel electrophoresis. They bound specifically to TNF-sensitive cells but not to HeLa clones that were resistant to TNF. The binding of 125I-TNF was competed up to 90% by the addition of non radiolabeled TNF. The binding reached a half-maximal level at 10 pM and saturation at 100 pM. These concentrations approximated those required for cell destruction. Scatchard analysis of the binding data yielded linear plots suggesting that TNF binds to homogeneous TNF receptor sites. The number of receptor sites ranged between 770 and 2200 sites on TNF-sensitive cells. The surface expression of these TNF receptors appeared to be necessary but not sufficient for the cells to become sensitive against the cytotoxic action of TNF. One of the HeLa clones was found to express the same number of TNF receptors as the most sensitive clone, but was nevertheless resistant to the toxic effect of TNF. PMID- 3015604 TI - DNA gyrase complex with DNA: determinants for site-specific DNA breakage. AB - DNA gyrase catalyses DNA supercoiling by making a transient double-stranded DNA break within its 120-150 bp binding site on DNA. Addition of the inhibitor oxolinic acid to the reaction followed by detergent traps a covalent enzyme-DNA intermediate inducing sequence-specific DNA cleavage and revealing potential sites of gyrase action on DNA. We have used site-directed mutagenesis to examine the interaction of Escherichia coli gyrase with its major cleavage site in plasmid pBR322. Point mutations have been identified within a short region encompassing the site of DNA scission that reduce or abolish gyrase cleavage in vitro. Mapping of gyrase cleavage sites in vivo reveals that the pBR322 site has the same structure as seen in vitro and is similarly sensitive to specific point changes. The mutagenesis results demonstrate conclusively that a major determinant for gyrase cleavage resides at the break site itself and agree broadly with consensus sequence studies. The gyrase cleavage sequence alone is not a good substrate, however, and requires one or other arm of flanking DNA for efficient DNA breakage. These results are discussed in relation to the mechanism and structure of the gyrase complex. PMID- 3015606 TI - Orthophosphate-pyrophosphate exchange catalyzed by soluble and membrane-bound inorganic pyrophosphatases. Role of H+ gradient. AB - A comparative study of the orthophosphate-pyrophosphate exchange reaction catalyzed by the soluble pyrophosphatase from baker's yeast and by the membrane bound pyrophosphatase of Rhodospirillum rubrum chromatophores was performed. In both systems the rate of exchange increased when the pH of the medium was raised from 6.0 to 7.8 and when the MgCl2 concentration was raised from 0.1 mM to 20 mM. For the yeast pyrophosphatase the exchange rates measured at different pH values and in the presence of 6.7 to 8.8 mM free Mg2+ superimposed as a single curve when plotted as a function of the concentrations of either HPO4(2-) or MgHPO4. This was not observed with the use of R. rubrum chromatophores. With yeast pyrophosphatase, the Km for Pi was higher than 10 mM and could not be measured when the free Mg2+ concentration in the medium was lower than 0.5 mM. There was a decrease in the Km for Pi when the free Mg2+ concentration was raised to 6.7-8.8 mM or when, in the presence of low free Mg2+, the organic solvents dimethylsulfoxide (20% v/v) or ethyleneglycol (40% v/v) were included in the assay medium. In the presence of 6.7-8.8 mM free Mg2+ the Km for total Pi was 7 mM at pH 7.0 and 12 mM at pH 7.8. For the ionic species HPO4(2-) and MgHPO4, the Km values were 5.8 mM and 4.2 mM respectively. In the presence of 0.24-0.42 mM free Mg2+ and either 20% (v/v) dimethylsulfoxide or 40% (v/v) ethyleneglycol the Km values for total Pi, HPO4(2-) and MgHPO4 were 7.6, 3.5 and 0.5 mM respectively. With R. rubrum chromatophores, the Km for Pi in the presence of 5.5 7.5 mM free Mg2+ was very high and could not be measured. In the presence of 0.24 0.45 mM free Mg2+ the ratio between the velocities of hydrolysis and synthesis of pyrophosphate measured at pH 7.8 with yeast pyrophosphatase and chromatophores of R. rubrum were practically the same. When the free Mg2+ concentration was raised to 5.5-8.8 mM this ratio decreased from 1028 to 540 when the yeast pyrophosphatase was used and from 754 to 46 when chromatophores were used. PMID- 3015603 TI - Synthetic lac operator mediates repression through lac repressor when introduced upstream and downstream from lac promoter. AB - Plasmids were constructed which carry a synthetic lac operator at various distances from the lac promoter. They were tested in vivo for function in the presence and absence of lac repressor. We found significant repression when the lac operator is situated at the 3' end of the lac I gene or at the 5' end of the lac Z gene. When lac operators are inserted at both sites, we found a greater than 150-fold repression. The complex between lac repressor and DNA carrying these two lac operators is exceedingly stable in vitro suggesting that one tetrameric lac repressor may bind to both lac operators. PMID- 3015608 TI - Demonstration of cGMP-dependent protein kinase and cGMP-dependent phosphorylation in cell-free extracts of platelets. AB - Homogenates, membranes and cytosol of rat and human platelets were found to contain cGMP-dependent protein kinase immunoreactivity. Specific cGMP-dependent protein kinase immunoreactivity was about 1.7 pmol protein kinase/mg protein for homogenates of human platelets and 0.7 pmol/mg for homogenates of rat platelets; the majority appeared to be associated with the membrane fraction. In membranes of platelets low concentrations of cAMP (0.5-2 microM) stimulated the phosphorylation of five major proteins with apparent relative molecular masses, Mr, of 240 000, 130 000, 50 000, 42 000 and 22 000 while low concentrations of cGMP (0.5-2 microM) stimulated the phosphorylation of three major proteins with apparent Mr of 130 000, 50 000 and 46 000. An affinity-purified antibody against the cGMP-dependent protein kinase was prepared which specifically inhibited the activity of cGMP-dependent protein kinase. In membranes of human platelets this affinity-purified antibody inhibited the cGMP-stimulated phosphorylation of the three proteins with Mr of 130 000, 50 000 and 46 000 while it had no effect on the cAMP-dependent and cyclic-nucleotide-independent protein phosphorylation. The results demonstrate that platelets contain a cGMP-dependent protein kinase and at least three specific substrates for this enzyme. Two of these substrates, the proteins with apparent molecular Mr of 130 000 and 50 000, are substrates for both cAMP- and cGMP-dependent protein kinase. The protein with apparent Mr of 130 000 appears to be closely related to an intrinsic plasma membrane protein of vascular smooth muscle cells which is a substrate for a membrane-associated cGMP dependent protein kinase. Therefore, cGMP-dependent protein kinase and cGMP regulated phosphoproteins may mediate in platelets the intracellular effects of those hormones, vasodilators and drugs which elevate the level of cGMP and inhibit platelet aggregation. PMID- 3015607 TI - Mechanisms of lipid peroxidation dependent upon cytochrome P-450 LM2. AB - A mechanism of lipid peroxidation dependent on the oxidase activity of cytochrome P-450 LM2 in reconstituted membrane vesicles has been investigated. The rate of lipid peroxidation, determined as the formation of thiobarbituric-acid-reactive substances, was inhibited by CO. It increased concomitantly to the production of O-2 and H2O2, when cytochrome P-450 LM2 was incorporated into vesicles containing NADPH-cytochrome-P-450 reductase, until a 1:1 molar ratio between the enzymes was reached. Also the formation of lipid hydroperoxides was dependent on the presence of cytochrome P-450 LM2 in the membranes. This lipid peroxidation was not inhibited by hydroxyl radical scavengers and not specifically inhibited by scavengers of singlet oxygen. By contrast, superoxide dismutase was a very potent scavenger of the lipid peroxidation. A half-maximal effect at 3 ng/ml enzyme was registered, whereas a 100-fold higher concentration was necessary in order to inhibit O-2 formation as detected by succinylated cytochrome c or pyrogallol. The reason for this difference might be inherent in different types of kinetics in the interaction of O-2 with different scavengers or might possibly indicate that SOD scavenges another type of reactive oxygen, different from O-2, generated by cytochrome P-450 LM2. Iron chelators inhibited the P-450-dependent lipid peroxidation, whereas iron chelate interacted with NADPH-cytochrome-P-450 reductase in the membranes giving rise to reductase-dependent lipid peroxidation. Neither superoxide dismutase nor EDTA at high concentrations, inhibited CCl4 initiated lipid peroxidation, indicating the point of action of these compounds at the initiation step in the cytochrome-P-450-LM2-dependent lipid peroxidation. Superoxide generated by pyrogallol, in three times the amount produced by P-450 LM2, could not bring about lipid peroxidation. It is suggested that the cytochrome-P-450-dependent lipid peroxidation mechanism might be of importance for intracellular oxidative damage under certain conditions. PMID- 3015610 TI - Binding of nucleotides to nucleoside diphosphate kinase monitored by rose Bengal. AB - The binding of nucleotides to nucleoside-diphosphate kinase from pig heart was studied using the dye rose Bengal as an optical probe. By difference absorption spectroscopy and by equilibrium dialysis it was shown that one dye molecule strongly bound per enzyme subunit. By competition with nucleotides it was shown that two nucleotide-binding sites exist on each subunit of either unphosphorylated or phosphorylated enzyme: one of them binds ATP or ADP tightly, the other one binds rose Bengal tightly and ADP loosely. As detected by different affinities for rose Bengal the enzyme exists in two conformations: a 'relaxed' conformation induced by the binding of ADP, and a 'tense' conformation induced by the binding of ATP or by phosphorylation. PMID- 3015609 TI - Processing of follitropin and its receptor by cultured pig Sertoli cells. Effect of monensin. AB - Immature pig Sertoli cells, cultured in a chemically defined medium, are able to maintain many of their functional characteristics for at least two weeks. This model was used to investigate the binding, internalization and degradation of 125I-labelled human follitropin (hFSH) and the effects of pig FSH (pFSH) on its own receptors. The binding of 125I-labelled hFSH was dependent on time, temperature and concentration. At 4 degrees C, the apparent steady state was reached in 8-12 h and remained constant for at least 24 h, whereas at 33 degrees C the apparent equilibrium was reached in 4-6 h. Thereafter the total binding declined and by 24 h it was less than 50% of the maximum binding. At 33 degrees C the binding for the hormone to its surface receptor was followed by internalization of the hormone (half-life approximately equal to 1 h) and its degradation (half-life approximately equal to 3 h). The receptor-mediated internalization of hFSH was blocked by phenylarsine oxide. In the presence of the ionophore monensin (20 microM) the rates of binding and internalization were not modified but the degradation rate was much lower (half-life approximately equal to 18 h). Thus, in the presence of monensin, maximum binding increased twofold to threefold, and remained constant for 24 h. This increase was mainly due to an increase of the internalized hormone. When Sertoli cells were exposed to pFSH there was a loss of its own receptor, which was both dose-dependent (ED50 = 250 ng/ml) and time-dependent (t 1/2 = 14 h). Cycloheximide did not modify the FSH induced down-regulation, whereas monensin enhanced the down-regulation process. These results show that FSH, like other ligands, is internalized and degraded by its target cells and indicate that the hormone-mediated down-regulation is related to the internalization process. However, the discrepancy between the rate of internalization and of hormone-induced down-regulation, suggests that some of the internalized receptors are recycled. PMID- 3015612 TI - Determination of the upper and lower limits of the mechanistic stoichiometry of incompletely coupled fluxes. Stoichiometry of incompletely coupled reactions. AB - A rationale is formulated for the design of experiments to determine the upper and lower limits of the mechanistic stoichiometry of any two incompletely coupled fluxes J1 and J2. Incomplete coupling results when there is a branch at some point in the sequence of reactions or processes coupling the two fluxes. The upper limit of the mechanistic stoichiometry is given by the minimum value of dJ2/dJ1 obtained when the fluxes are systematically varied by changes in steps after the branch point. The lower limit is given by the maximum value of dJ2/dJ1 obtained when the fluxes are varied by changes in steps prior to the branch point. The rationale for determining these limits is developed from both a simple kinetic model and from a linear nonequilibrium thermodynamic treatment of coupled fluxes, using the mechanistic approach [Westerhoff, H. V. & van Dam, K. (1979) Curr. Top. Bioenerg. 9, 1-62]. The phenomenological stoichiometry, the flux ratio at level flow and the affinity ratio at static head of incompletely coupled fluxes are defined in terms of mechanistic conductances and their relationship to the mechanistic stoichiometry is discussed. From the rationale developed, experimental approaches to determine the mechanistic stoichiometry of mitochondrial oxidative phosphorylation are outlined. The principles employed do not require knowledge of the pathway or the rate of transmembrane leaks or slippage and may also be applied to analysis of the stoichiometry of other incompletely coupled systems, including vectorial H+/O and K+/O translocation coupled to mitochondrial electron transport. PMID- 3015611 TI - Antibodies to the ATP-binding site of the human epidermal growth factor (EGF) receptor as specific inhibitors of EGF-stimulated protein-tyrosine kinase activity. AB - A region of the primary amino acid sequence of the epidermal growth factor receptor (EGF) protein-tyrosine kinase, which is involved in ATP binding, was identified using chemical modification and immunological techniques. EGF receptor was 14C-labelled with the ATP analogue 5'-p-fluorosulphonylbenzoyladenosine and from a tryptic digest a single radiolabelled peptide was isolated. The amino acid sequence was determined to be residues 716-724 and hence lysine residue 721 is located within the ATP-binding site. Antisera were elicited in rabbits to a synthetic peptide identical to residues 716-727 of the EGF receptor and the homologous sequence in v-erb B transforming protein from avian erythroblastosis virus. The affinity-purified antibodies precipitated human ECF receptor from A431 cells and placenta, and the v-erb B protein from erythroblasts. The antibodies inhibited EGF-stimulated receptor protein-tyrosine kinase autophosphorylation and phosphorylation of an exogenous peptide substrate containing tyrosine. The antibodies did not immunoprecipitate the transforming proteins pp60v-src or P120gag-abl or cAMP-dependent protein kinase, proteins which have homologous but not identical sequences surrounding the lysine residue within the ATP-binding site, nor did they react with the platelet-derived growth factor receptor. The antibodies had no effect on the kinase activity of purified v-abl protein in solution. The antibodies may therefore be a specific inhibitor of the tyrosine kinase of the EGF receptor. PMID- 3015614 TI - Molecular conformation of gonadoliberin using two-dimensional NMR spectroscopy. AB - Complete resonance assignments of the proton NMR spectrum of gonadoliberin (in its native amide and free acid forms) have been obtained using two-dimensional nuclear magnetic resonance spectroscopy under three different environmental conditions, namely, dimethyl sulphoxide solution, aqueous solution and lipid bound form in model membranes. The proton chemical shifts in the three cases have been compared to derive information about inherent conformational characteristics of the molecule. It has been inferred that the molecule possesses no short-range or long-range order under any of the three solvent conditions. However, there is a nonspecific increase in the linewidths when gonadoliberin is bound to model membranes, indicating a reduced internal motion in the molecule due to lipid peptide interactions. PMID- 3015613 TI - The upper and lower limits of the mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Stoichiometry of oxidative phosphorylation. AB - Determination of the intrinsic or mechanistic P/O ratio of oxidative phosphorylation is difficult because of the unknown magnitude of leak fluxes. Applying a new approach developed to overcome this problem (see our preceding paper in this journal), the relationships between the rate of O2 uptake [( Jo)3], the net rate of phosphorylation (Jp), the P/O ratio, and the respiratory control ratio (RCR) have been determined in rat liver mitochondria when the rate of phosphorylation was systematically varied by three specific means. (a) When phosphorylation is titrated with carboxyatractyloside, linear relationships are observed between Jp and (Jo)3. These data indicate that the upper limit of the mechanistic P/O ratio is 1.80 for succinate and 2.90 for 3-hydroxybutyrate oxidation. (b) Titration with malonate or antimycin yields linear relationships between Jp and (Jo)3. These data give the lower limit of the mechanistic P/O ratio of 1.63 for succinate and 2.66 for 3-hydroxybutyrate oxidation. (c) Titration with a protonophore yields linear relationships between Jp, (Jo)3, and (Jo)4 and between P/O and 1/RCR. Extrapolation of the P/O ratio to 1/RCR = 0 yields P/O ratios of 1.75 for succinate and 2.73 for 3-hydroxybutyrate oxidation which must be equal to or greater than the mechanistic stoichiometry. When published values for the H+/O and H+/ATP ejection ratios are taken into consideration, these measurements suggest that the mechanistic P/O ratio is 1.75 for succinate oxidation and 2.75 for NADH oxidation. PMID- 3015616 TI - Brain pyridoxal kinase. Purification and characterization. AB - Pyridoxal kinase has been purified 9000-fold from sheep brain. The purification procedure involves ammonium sulphate fractionation, DEAE-cellulose chromatography, affinity chromatography and Sephadex G-100 gel filtration. The final chromatography step yields a homogeneous preparation of high specific activity with a pI of 5. The molecular mass of the native enzyme was estimated to be approximately 80 kDa by 10-25% gradient polyacrylamide gel electrophoresis and Sephadex G-200 gel filtration. The subunit molecular mass was determined by sodium dodecyl sulphate (SDS)/polyacrylamide gel electrophoresis to be 40 kDa compared with a series of molecular mass standards. This indicates that pyridoxal kinase is a dimeric enzyme. Further results obtained from electron microscopy, using a negative staining technique, provide evidence that pyridoxal kinase exists as a dispherical subunit structure. PMID- 3015615 TI - Tissue-specific and light-dependent changes of chromatin organization in barley (Hordeum vulgare). AB - The DNase I sensitivity of the nuclear genes encoding the NADPH protochlorophyllide oxidoreductase, the light-harvesting chlorophyll a/b protein (LHCP), the hordeins and a 15-kDa protein of unknown function was assayed in chromatin of etiolated and green leaves and endosperm tissue of barley (Hordeum vulgare L.). A tissue-specific differentiation of chromatin structure was found for the LHCP, hordein and 15-kDa protein genes. The genes for the LHCP and the 15 kDa protein, which are expressed in leaf tissue, display DNase I sensitivity in leaves but not in endosperm. Hordein genes which are expressed solely in endosperm, were insensitive to low levels of digestion with DNase I in leaves but sensitive in endosperm. The effect of light on chromatin structure was determined by comparing leaves of etiolated plants and plants which had been grown under a day/night cycle. Only in the case of the 15-kDa protein is there a remarkable change from a DNAse-I-sensitive configuration in etiolated leaves to a more resistant one in leaves from illuminated plants. The gene for the NADPH protochlorophyllide oxidoreductase was found to be equally sensitive to DNase I in leaves and endosperm. PMID- 3015617 TI - The role of compound III in reversible and irreversible inactivation of lactoperoxidase. AB - In the presence of iodide (I-, 10 mM) and hydrogen peroxide in a large excess (H2O2, 0.1-10 mM) catalytic amounts of lactoperoxidase (2 nM) are very rapidly irreversibly inactivated without forming compound III (cpd III). In contrast, in the absence of I- cpd III is formed and inactivation proceeds very slowly. Increasing the enzyme concentration up to the micromolar range significantly accelerates the rate of inactivation. The present data reveal that irreversible inactivation of the enzyme involves cleavage of the prosthetic group and liberation of heme iron. The rate of enzyme destruction is well correlated with the production of molecular oxygen (O2), which originates from the oxidation of excess H2O2. Since H2O2 and O2 per se do not affect the heme moiety of the peroxidase, we suggest that the damaging species may be a primary intermediate of the H2O2 oxidation, such as oxygen in its excited singlet state (1 delta gO2), superoxide radicals (O-.2), or consequently formed hydroxyl radicals (OH.). PMID- 3015618 TI - Dimeric ubiquinol:cytochrome c reductase of Neurospora mitochondria contains one cooperative ubiquinone-reduction centre. AB - Dimeric ubiquinol:cytochrome c reductase of Neurospora mitochondria was isolated as a protein-Triton complex and free of ubiquinol (Q). The enzyme was incorporated into phosphatidylcholine membranes together with Q. The effects of varying the molar ratio of Q to enzyme on the electron transfer from duroquinol (DHQ2) to the cytochromes c, c1 and b were studied. The rate of electron flow from DQH2 to cytochrome c was 15 times increased by Q and was maximal when one molecule of Q was bound to one enzyme dimer. The apparent Km value for DQH2 of the Q-free enzyme was 5 microM and of the Q-supplemented enzyme 25 microM. The pre-steady-state rate of electron transfer from DQH2 to cytochrome c1 was also 15 times increased by Q and was maximal with one Q molecule bound to one enzyme dimer. This effect of Q was inhibited by antimycin. The pre-steady-state rate of electron transfer from DQH2 to cytochrome b was 5 times decreased when Q was bound to the enzyme and this effect of Q was insensitive to myxothiazol. The H+/2e- stoichiometry with DQH2 as substrate of the Q-supplemented enzyme was 3.6. These results are interpreted in accordance with a Q-cycle mechanism operating in a dimeric cytochrome reductase. Each enzyme monomer catalyses a single electron transfer from the QH2-oxidation centre to the Q-reduction centre and the two monomers cooperate in the reduction of Q to QH2 at one Q-reduction centre. This centre contains two different binding sites for Q. DQH2 does not properly react at the QH2-oxidation centre. DQH2, however, binds to the loose Q-binding site of the Q-reduction centre and reduces the Q bound to the tight Q-binding site of the centre. The QH2 thus formed at the Q-reduction centre serves as electron donor for the QH2-oxidation centre. PMID- 3015619 TI - Purification, subunit composition and regulatory properties of the ATP X Mg2+ dependent form of type I phosphoprotein phosphatase from bovine heart. AB - The ATP X Mg2+-dependent phosphoprotein phosphatase has been purified from bovine heart to near-homogeneity. It is a heterodimer (75 kDa) consisting of a catalytic (C) subunit (40 kDa) and a regulatory (R) subunit (35 kDa). The R subunit, which is identical to inhibitor-2, is transiently phosphorylated during activation of the enzyme catalyzed by phosphatase-1 kinase (FA). Maximal activation requires preincubation of the phosphatase with FA and ATP X Mg2+. However, relatively low yet definitively demonstrable basal activity can be expressed by Mg2+ alone (ranging from 3% to 10% of the FA X ATP X Mg activity, depending on the degree of endogenous proteolytic damage of the phosphatase during purification), but not by either FA or ATP alone. Limited trypsinization results in a rapid and total degradation of the R subunit and partial degradation of the 40-kDa C subunit to active proteins of 35-38 kDa. The resulting 'nicked' C subunit of 35-38 kDa is no longer dependent on FA for activation and can be fully activated by Mg2+ (or Mn2+) alone. Endogenous proteolytic damage of the R subunit also results in an increase of activity that can be expressed by M2+ alone with a concomitant decrease of the FA-dependent activation. Although Mn2+ is slightly more effective than Mg2+ in expressing the holoenzyme basal activity, the activation by Mn2+ is only about 60% of that of Mg2+ when FA and ATP are also present. In the activation by adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]), Co2+ is the most effective cofactor. The activation by ATP[gamma S] X Co2+ is more than 50% of that by ATP X Mg2+. The present studies indicate that Mg2+ is the natural divalent cation for the FA-catalyzed activation in which Mg2+ plays two distinctly different roles: it forms Mg2+ X ATP which serves as a substrate for the kinase; it acts as an essential cofactor for the catalytic function of the phosphatase. The discrepancies between the results obtained by this and other laboratories with respect to the effectiveness of Mg2+ and ATP[gamma S] in the activation of the phosphatase are discussed. PMID- 3015621 TI - Thallium-pertechnetate subtraction scintigraphy: a quantitative comparison between adenomatous and hyperplastic parathyroid glands. AB - In a prospective study of 201Tl-99mTc subtraction scintigraphy, 61 hyperparathyroid patients were investigated prior to neck exploration. At surgery, 46 adenomatous and 28 hyperplastic parathyroid glands were excised. We examined the relationship between the pathological category of these glands, their mass, uptake of 201Tl thallous chloride, and the frequency of true-positive and false-negative scintigraphic findings. The variation of sensitivity with parathyroid mass was found to be similar for both adenomatous and hyperplastic glands, with a detection threshold that lay in the range 0.3-0.8 g. The higher overall sensitivity for the detection of adenomas (85%) compared with hyperplasias (44%) was due to the smaller mean weight of the latter. When the parathyroid uptake of thallium was quantified scintigraphically, the practical detection limit of subtraction scanning was found to be an uptake of 0.015%. For glands greater than 1.5 g in weight, uptake increased linearly with mass, and specific uptakes were within the range 0.01-0.04%/g. Below lg, certain small glands had much higher specific uptakes, up to 0.2%/g. The range of specific uptakes found was similar for both adenomatous and hyperplastic categories. Multinodular or diffuse goitre was a cause of failure in 10% of investigations. In a further 5%, a solitary thyroid nodule gave rise to a false-positive result. PMID- 3015620 TI - A comparison of two radionuclide ejection-fraction techniques with contrast angiography in ischemic heart disease and valvular heart disease. AB - First-pass radionuclide angiography (FPRA) in the 30 degree right anterior oblique and equilibrium gated radionuclide angiography (EGNA) in the 45 degree left anterior oblique were used for quantitative measurements of left ventricular ejection fraction (LVEF). Equipment used was a 400T gamma-camera interfaced with a Simis III Informatek computer. The results were compared with contrast angiography (CA). The aim of this study was to determine the sensitivity of both radionuclide techniques. The present data are based on 65 patients in whom CA and EGNA were performed. In 47 patients both FPRA and EGNA were performed. Results suggested that in ischemic heart disease (IHD) and valvular heart disease (VHD) the EGNA technique is well correlated with CA (r = 0.9 and 0.73, respectively). FPRA correlated well only with CA in IHD (r = 0.86), but not in VHD (r = 0.18). This study indicates that both FPRA and EGNA are sensitive, noninvasive techniques for measuring ejection fraction in IHD, while in VHD, EGNA is more sensitive technique than FPRA. PMID- 3015622 TI - Chemical, radiochemical, and radionuclide purity of eluates from different commercial fission 99Mo/99mTc generators. AB - Seven 99Mo/99mTc generators (using fission 99Mo) obtained from seven different manufacturers were studied in 1984 and 1985 to test the quality of the eluates. We present the findings concerning the elution efficiency, radionuclide purity, 99Mo breakthrough, radiochemical purity, pH, and aluminium content of the eluates. One generator was overloaded with 99Mo by about 40%, while one generator had 99mTc yields of only about 80%. The eluates generally (although with some exceptions) exhibited a high and satisfactory radionuclidic purity and good radiochemical purity. The low-level determination of 99Mo breakthrough using a commercially available dose calibrator with a 99Mo assay shield indicated a misleadingly high 99Mo content. All of the eluates had pH values of between 5.0 and 5.5, and the aluminium content was always below the detection limit of 1 microgram per milliliter of eluate. The generators performed well and proved their capability of functioning as reliable sources of sodium pertechnetate Tc99m. In all cases, the pertechnetate produced met the requirements of the European Pharmacopeia. PMID- 3015623 TI - 99mTc-DTPA and 99mTc-DMSA renal gamma imaging in the surveillance of patients with conduit urinary diversion. AB - We studied renal anatomy and function using 99mTc-2-3 dimercaptosuccinic acid (DMSA) and 99mTc-diethylenetriaminepentaacetic acid (DTPA) in 27 patients with conduit urinary diversion. In this condition, free ureteral reflux is often associated with bacteriuria, and these factors are thought to precipitate progressive renal deterioration. Gamma-camera images provided valuable information concerning the structure of the renal parenchyma, the function of individual kidneys and possible ureteral obstruction, thus helping us to decide whether or not to instigate further treatment. The information gained using renal gamma imaging with 99mTc-DTPA and 99mTc-DMSA was complementary and partly overlapping. We preferred the use of 99mTc-DTPA because of its ability to visualise the ureters and the region of ureteroconduit anastomosis. Using diuretic medication, we were able to differentiate true ureteral obstruction from atony in 9 patients using 99mTc-DTPA. PMID- 3015624 TI - Possible polyclonal B cell activation in mucocutaneous lymph node syndrome. AB - Immunoserological studies on polyclonal B cell activation were carried out on 39 patients with mucocutaneous lymph node syndrome (MCLS) and in age-matched healthy individuals. The incidence of anti-mite, P. acnes (Kato) and EB virus antibodies, recently proposed as aetiological agents by some investigators, was increased in the patient group. Serum immunoglobulin (Ig) M level and IgM-anti-dinitrophenyl (DNP) antibodies, which are considered to be parameters of polyclonal B cell activation, were determined in MCLS cases. The level of serum IgM in MCLS was significantly elevated (0.02 less than P less than 0.05). Levels of anti-DNP antibodies in seven cases of MCLS (18%) were significantly higher than those of the controls (P less than 0.01). Nine of the ten pair sera in MCLS showed a stage dependent decrease in anti-DNP antibodies. These results suggest that polyclonal B cell activation occurs in MCLS. PMID- 3015626 TI - Further study of the epidemiology of rotavirus infection in Tokyo. PMID- 3015625 TI - Parathyroid hormone, calcitonin and vitamin D metabolites in beta-thalassaemia major. AB - Serum calcium (Ca), phosphorus (P), alkaline phosphatase (Al-P), parathyroid hormone (PTH), calcitonin (CT), 25-hydroxyvitamin D3 (25OHD3), 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) levels and urinary excretion of Ca, P, hydroxyproline (OH-P) and cyclic AMP (cAMP) were determined in summer and in winter in 13 thalassaemic children (7 aged 3-5 years-group 1-; and 6 aged 10-13 years-group 2-), who had never taken vitamin D supplements or therapy, and in two groups of 14 controls of the same age. In thalassaemics of group 1 only serum Al P levels and OH-P urinary excretion were higher than in controls (P less than 0.01). In thalassaemics of group 2 Ca (P less than 0.05), P (P less than 0.05), PTH (P less than 0.001), CT (P less than 0.001), 25OHD3 (P less than 0.05), 1,25(OH)2D3 (P less than 0.001) levels and cAMP urinary excretion (P less than 0.001) were lower, whereas Al-P (P less than 0.001) and CT (P less than 0.001) levels and urinary excretion of P (P less than 0.05) and of OH-P (P less than 0.001) were higher than in controls, both in summer and in winter. Advancing age induces in thalassaemic patients a decrease in PTH secretion and a consequent deficit in synthesis of 1,25(OH)2D3 that may explain some aspects of bone changes, which CT hypersecretion may tend to counteract. PMID- 3015627 TI - Protein phosphorylation of neutrophils from normal children and patients with chronic granulomatous disease. AB - The phosphorylation and dephosphorylation of proteins in neutrophils from normal children and patients with chronic granulomatous disease (CGD) were studied with two-dimensional gel electrophoresis and autoradiography, followed by densitometric scanning. In normal neutrophils the radioactivities of 11 spots among approximately 50 radioactive spots were changed by stimulation with phorbol 12-myristate 13-acetate (PMA) and 6 of the 11 spots were also changed by stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP) and NaF. The phosphorylation of only two spots (Mr = 48 000 and 62 000) was inhibited by 2 deoxyglucose and N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7), which inhibits superoxide production, while it was not affected by dibutyryl cAMP, KCN and ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), which do not affect superoxide production. The observation indicates that the Mr = 48 000 and 62 000 proteins may be involved in the activation process of superoxide production. When the neutrophils of four male and two female CGD patients were examined, the changes in 11 spots on stimulation were similar to those of normal children, indicating that the (de)phosphorylation of the proteins which seems to be involved in the activation process is not affected in CGD neutrophils. PMID- 3015629 TI - Demonstration of Epstein-Barr virus DNA in a previously healthy boy with fulminant hepatic failure. AB - A previously healthy 9-year-old boy died from acute liver failure during an acute Epstein-Barr virus infection. Epstein-Barr virus DNA could be demonstrated in the liver by Southern blot--and by in situ hybridization techniques. The identification of the virus in the liver suggests a causal relation between the Epstein-Barr virus and the acute massive liver cell necrosis. PMID- 3015628 TI - Evaluation of serum osteocalcin as an index of altered bone metabolism. AB - Recent evidence suggests that the protein osteocalcin is like the bone alkaline phosphatase produced by osteoblasts and circulates in human blood. With the introduction of a radioimmunoassay for serum osteocalcin it was hoped that this test would provide a useful index of altered bone metabolism. Therefore serum osteocalcin was measured in 88 controls and 112 patients with disorders of calcium and phosphate metabolism, isolated elevation of alkaline serum phosphatase in the absence of disease (isolated hyperphosphatasaemia) and children prone to osteopenia. In the controls serum osteocalcin was higher in children less than 15 years (median and range: 11.9, 7.7-15.3 ng/ml) than in adults (3.7, 2.6-5.2 ng/ml) and was highly correlated to alkaline serum phosphatase activity (r = 0.87, n = 88, P less than 0.01). Osteocalcin was elevated in primary hypoparathyroidism, low in untreated hypoparathyroidism but normal in hypoparathyroidism (including pseudohypoparathyroidism) during vitamin D treatment. The bone protein was low-normal and increased to high-normal levels during vitamin D therapy in vitamin D deficiency rickets and familial hypophosphataemic rickets, but remained low in patients with end organ resistance to 1,25-dihydroxyvitamin D. Osteocalcin (and urinary hydroxyproline) were not elevated in isolated hyperphosphatasaemia, indicating that mechanisms other than increased bone turnover may account for the markedly elevated serum alkaline phosphatase activity in these subjects. Osteocalcin was decreased in children with diabetes mellitus type I and in patients on glucocorticoid treatment, indicating decreased bone formation. It is concluded that the measurement of serum osteocalcin seems to be a reliable index of bone formation provided that the vitamin D status and renal function are normal.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3015630 TI - HTLV-III and HTLV-I infection in populations at risk in the Veneto region of Italy. AB - A seroepidemiological survey has been carried out in the Veneto region to determine the prevalence of HTLV-III and HTLV-I antibodies in subjects at risk for development of AIDS and related conditions. Serum samples were tested by ELISA and, for confirmation, by radioimmunoassay (Western blot), using disrupted virus as antigen. The results show that 22 out of 112 hemophiliacs had antibodies against HTLV-III; however disaggregation of data resulted in 22.6 and 77.8% positivity for patients with severe forms of hemophilia A and B, respectively. Two patients with hemophilia A and two with hemophilia B were positive for antibodies to HTLV-I. The prevalence of HTLV-III antibodies in the homosexual and intravenous drug abuser groups was 52 and 33% respectively. No positive cases for antibodies to HTLV-I were found in homosexuals, while 4.3% seropositivity to HTLV I was observed in drug abusers. Among patients suffering from various pathologic conditions not strictly AIDS related, only 1 with generalized non-Hodgkin lymphoma was positive for HTLV-I antibodies. In a further group of patients with clinical diagnosis of LAS and AIDS, antibodies to HTLV-III were found in 90 and 100% respectively, while seropositivity for HTLV-I was observed only in 6.4% of LAS patients. The implications of these findings are discussed, particularly in view of the potential oncogenic effect possessed by HTLV-I. PMID- 3015631 TI - Aromatase, 17 beta-hydroxysteroid dehydrogenase and intratissular sex hormone concentrations in cancerous and normal glandular breast tissue in postmenopausal women. AB - In a study of the origin of estrogens in patients with breast cancer, the concentrations of estrogens and their androgen precursors, and aromatase and 17 beta-hydroxysteroid dehydrogenase (E2DH) activities were determined in normal glandular and cancerous breast tissue. The correlation between tissue estrogens, precursor concentrations, enzyme activities and plasma levels and/or receptor status were calculated. In both normal glandular and carcinomatous breast tissue, the concentrations of androstenedione (A), dehydroepiandrosterone (DHEA), 5 androstene-3 beta, 17 beta-diol (5-Adiol), estrone (E1), estradiol (E2) and progesterone (P) were significantly higher than plasma concentrations. While testosterone (T) concentrations were similar, dehydroepiandrosterone (DHCA) and estrone sulphate (E1S) concentrations were lower in tissue than in plasma. In carcinomatous tissue androgen concentrations were lower, but estrogen concentrations were higher than in glandular breast tissue. Estradiol (E2) concentration was positively correlated with the receptor concentration with the mean E2 concentration corresponding to an estimated receptor occupancy of about 25%, probably sufficient for a submaximal biological response. Aromatase and E2DH (E2----E1) activities were observed in all breast cancer and glandular breast tissues, activities being higher in carcinoma than in glandular breast tissues; nevertheless, aromatase activity accounts probably only for a small fraction of tissue estrogen concentration. E2DH, but not aromatase activity, was significantly higher in estrogen receptor positive than in estrogen receptor negative tissues and was negatively correlated with tissue dehydroepiandrosterone (DHEA) and its sulphate (DHEAS) concentration; the latter two steroids are non competitive inhibitors of E2DH which inactivates E2 to E1. This effect of DHEA(S) may constitute a mechanism by which these androgens stimulate cancer growth and a rationale (besides suppression of estrogen precursors) for medical or surgical adrenalectomy in hormone sensitive metastatic mammary cancer. E2DH activity might constitute an additional marker of hormone dependency of mammary cancer. PMID- 3015632 TI - The physiology of static exercise. PMID- 3015634 TI - Generation of superoxide anion and hydrogen peroxide by erythrocytes from individuals with sickle trait or normal haemoglobin. AB - To test the hypothesis that the resistance of sickle trait (AS Hgb) erythrocytes (rbcs) to malaria may be mediated by increased production of activated oxygen species, the production of superoxide anion (O2-) and hydrogen peroxide (H2O2) by AS rbcs and normal (AA Hgb) rbcs was measured under defined conditions. Formation of O2- and H2O2 was time, temperature and oxygen saturation dependent. Reproducible measurement of O2- formation required the presence of 0.2 mmol l-1 KCN to inhibit a cytochrome oxidase activity found in the cytochrome C preparation used. There was an inverse relationship between cell concentration and O2- and H2O2 formation. Use of the inhibitor of superoxide dismutase (SOD), diethyldithiocarbamic acid, increased the amount of O2- measured. When rbcs from blacks with AS Hgb and with AA Hgb were incubated under standardized conditions, significantly (P less than 0.05) more O2- was formed by AS than AA cells (24.3 v. 14.5 mmol per mol Hgb). These findings show that AS rbcs can generate more O2- than AA rbcs. The increased formation of O2- by rbcs containing AS Hgb may contribute to the resistance of AS rbcs to malarial parasitism. PMID- 3015633 TI - Alterations in norepinephrine content and beta adrenoceptor regulation in myocardium bordering aneurysm in human heart: their possible role in the genesis of ventricular tachycardia. AB - On the assumption that alterations in the adrenergic system may play a role in generating ventricular tachycardia in patients with myocardial post-infarction apical aneurysm, we evaluated norepinephrine concentration, number and affinity of both beta 1 and beta 2 adrenoceptors in perianeurysmatic tissue in twelve patients operated upon for congestive heart failure and recurrent sustained ventricular tachycardia. Concentration of norepinephrine in perianeurysmatic tissue was 0.1 +/- 0.05 micrograms g-1 tissue (n = 8), this value being much lower than that found in papillary muscle (n = 10) from patients with mitral valve stenosis (0.8 +/- 0.02 micrograms g-1 tissue) (P less than 0.01). The total number of beta adrenoceptors (71.4 +/- 7.8 v. 48.0 +/- 5.1 fmol mg-1 protein; P less than 0.01) and the percentage of beta 1 subtype were found to be higher in perianeurysmatic tissue (approximately 90%) than in papillary muscle (approximately 68%). Out of twelve patients with aneurysm, beta 2 adrenoceptors had considerably decreased in three patients and were absent in the remaining nine. Decrease in the neuronally released norepinephrine associated with contrasting behaviours of beta 1 and beta 2 adrenoceptors suggests the presence of a profound alteration in the sympathetic innervation of the perianeurysmatic myocardial tissue that may contribute to the genesis of sustained ventricular tachycardia in patients with postinfarction apical aneurysm. PMID- 3015636 TI - Altered functions of peripheral blood monocytes in homosexual males and intravenous drug users with persistent generalized lymphadenopathy. AB - Persistent generalized lymphadenopathy (PGL) is observed predominantly in subjects at risk of developing AIDS. Twenty-seven individuals belonging to such groups: twelve homosexual males and fifteen intravenous drug users, were investigated for immunological abnormalities with particular attention to monocyte functions. They were compared with five AIDS patients. Twenty out of twenty-two individuals had anti-LAV/HTLV-III antibodies and most had abnormalities characteristic of AIDS: polyclonal hypergammaglobulinemia, decreased cell-mediated immunity, inverted T-cell helper/suppressor ratio and histological alterations of lymph nodes. As for peripheral blood monocyte functions, phagocytic capacity and production of O2- were normal and bactericidal capacity was decreased. Monocytes cultured in the presence of concanavalin A produced less PGE2 and more IL-1/MCF than normal monocytes. Similar abnormalities were found using monocytes from AIDS patients. These data suggest that monocytes from patients with PGL have functional alterations that may be either intrinsic or secondary to lymphocyte dysfunction(s); these alterations do not account for the decreased capacity of lymphocytes to respond to mitogens but may explain the uncontrolled activation of B cells. PMID- 3015635 TI - Protoporphyrinogen oxidase and porphobilinogen deaminase in variegate porphyria. AB - Two enzymes of the haem biosynthetic pathway were investigated in patients with variegate porphyria. Protoporphyrinogen oxidase in cultures of Epstein-Barr virus transformed lymphoblasts from twenty-seven patients showed a mean maximal velocity (Vmax) of 0.39 +/- 0.08+ nmol of protoporphyrin mg protein-1 h-1, a 52% reduction (P less than 0.001) from a non-porphyric control group (0.82 +/- 0.10). Km values (1.00 +/- 0.27 microM) did not differ significantly (P greater than 0.05) from control values in any of the patients. The mean Vmax of porphobilinogen deaminase in the cultures was 1.50 +/- 0.18 nmol of uroporphyrin mg protein-1 min-1, a 24% reduction (P less than 0.001) from controls (1.94 +/- 0.14). Mean porphobilinogen deaminase activity in the erythrocytes of twenty-one patients with variegate porphyria was 8.37 +/- 1.99 nmol of uroporphyrin 1 erythrocytes-1 s-1, a 28% reduction (P less than 0.001) from normal (11.98 +/- 2.11). The reduced activities of these two enzymes comply with the expression of variegate porphyria during its quiescent and acute phases. PMID- 3015637 TI - Intra-individual comparison of captopril and enalapril in patients undergoing regular haemodialysis. AB - The acute and long-term efficacy, tolerance and safety of two orally active angiotensin converting enzyme (ACE) inhibitors, captopril (C) and enalapril (E) were compared in patients on regular haemodialysis (RHD). C and E were successively administered for 6 months to 8 RHD patients with hypertension unresponsive to fluid withdrawal and conventional antihypertensive therapy. The fall in blood pressure after a starting dose of 25 mg C or 5 mg E was of the same magnitude. It was not correlated with the initial PRA levels, which were normal in all patients. The mean daily dose of ACE inhibitor was 45 +/- 28 mg during the C period and 19.4 +/- 17.6 mg at the end of the E period. Three patients required additional treatment, comprising beta-blockers and/or calcium antagonists. The individual daily dose of ACE inhibitor, the need for additional treatment and the antihypertensive response achieved were highly correlated during both study periods. During C administration 4 out of 8 patients presented a taste disturbance, which disappeared 2 weeks after substituting E for C. Serum electrolytes, liver enzymes, haemoglobin concentration and white cell and platelet counts remained unchanged throughout both study periods. It is concluded that RHD patients with hypertension are responsive to ACE inhibitors, C and E being equally effective. PMID- 3015638 TI - Simultaneous measurement of epinephrine-induced platelet aggregation in 14 plasma samples. AB - A method is described for the simultaneous measurement of epinephrine-induced platelet aggregation in 14 different plasma samples in 15 min. It is based upon discontinuous registration of platelet aggregation and computer evaluation of the data. The samples are placed in holders mounted over magnetic stirring bars in a 37 degrees C water bath and the extinction is measured by removing the samples one after the other, placing them in a Braun Universal Aggregometer and returning them to their holders in the water bath. The time required to reach 37% of maximal aggregation was chosen as the evaluation criterion. It sufficed for the determination of aggregation sensitivity. This method for the first time permits measurement of a complete titration curve rapidly and under identical conditions and can be used to show the influence of a wide range of aggregation inducers and the concurrent effects of inducers plus various blocking agents. A correlation between aggregation sensitivity and alpha 2-receptor binding capacity in platelets, measured by competitive radioactive binding, was established in samples from 10 healthy volunteers. One group exhibited high aggregation sensitivity coupled with high alpha 2-binding capacity and the other showed low sensitivity with low binding capacity. PMID- 3015640 TI - In vivo 'enkephalinase' inhibition by acetorphan in human plasma and CSF. AB - Thiorphan, the potent inhibitor of 'enkephalinase', has shown some analgesic properties in experimental animals and in man. The possibility that the intravenous infusion of acetorphan, a prodrug of thiorphan (26 micrograms/kg per min for 60 min), can inhibit plasma and cerebrospinal fluid (CSF) enkephalinase in man in vivo was investigated. A decrease of approximately 65% in enzyme activity was observed in both plasma and CSF. Acetorphan did not induce any significant variation of plasma angiotensin-converting enzyme activity. PMID- 3015639 TI - Morphine-induced opioid receptor down-regulation detected in intact adult rat brain cells. AB - Intact brain cells dissociated from the brains of adult rats were used to study the regulation of opioid receptors by in vivo chronic morphine treatment. When the specific binding of [3H]naloxone in a physiological iso-osmotic buffer was compared in cells obtained from sham-operated rats and in those treated for 6 days with four (2 X 2) 75 mg morphine pellets, it was found that morphine treatment resulted in a significant reduction in the density of [3H]naloxone binding sites. This receptor down-regulation was not accompanied by a change in the receptor affinity for the ligand. This effect of morphine was reversed upon the removal of morphine pellets for 18 h after tolerance induction. When similar experiments were performed using brain homogenates prepared and assayed in the same physiological buffer, there was an increase in the number of [3H]naloxone binding sites in morphine-treated animals compared to controls. On the other hand, when the binding experiments were conducted in 50 mM Tris-HCl buffer, no difference in ligand binding was apparent between control and morphine-treated groups. The present results demonstrate opioid receptor down-regulation by chronic morphine treatment measured in intact brain cells, and suggest that different conclusions may be reached when other tissue preparations are used to assess the receptor density. PMID- 3015641 TI - Rat atrial natriuretic factor stimulates renin release from renal cortical slices. AB - We investigated the effect of synthetic rat atrial natriuretic factor (ANF) on renin release from rat renal cortical slices. Rat ANF (10(-6) M) increased renin release from the slices with a concomitant increase in the levels of cGMP contents. The increase in cGMP was also prominent in the case of incubation with 10(-4) M sodium nitroprusside but was not accompanied by an enhanced release renin. 8-Bromo-cGMP did not stimulate renin release. We propose that the stimulation of renin release from rat renal cortical slices is not related to an increase in endogenous cGMP. PMID- 3015642 TI - The protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibits muscarinic (M1) receptor-mediated inositol phosphate release and cyclic GMP formation in murine neuroblastoma cells (clone N1E-115). PMID- 3015643 TI - Interactions between opiate- and dopamine-induced effects on adenylate cyclase in rat striatum. AB - In rat striatal P2 membrane preparations examined under constant experimental conditions, activation of either D2-dopamine or opiate receptors inhibited basal adenylate cyclase with a similar maximal inhibition of approximately 30%. These inhibitions were not additive. D1 dopamine receptor-mediated activation of adenylate cyclase by SKF38393 was inhibited by morphine in an additive fashion, but the opiate had minimal effects on the net activation of adenylate cyclase by dopamine which activates both D1 and D2 receptors. From these results it would appear that opiate and D1 dopamine receptor-mediated activation of adenylate cyclase summate, but that opiate and D2 dopamine receptors regulate a common pool of adenylate cyclase in striatal membranes. PMID- 3015644 TI - Thermogenic responses to adrenoceptor agonists and brown fat adrenoceptors in overfed rats. AB - Rats fed a cafeteria diet to produce hyperphagia showed increases in the maximal thermogenic responses (rise in oxygen consumption) to isoprenaline (mixed beta agonist), prenalterol (beta 1-selective agonist) and clenbuterol (beta 2 agonist), and left-shifts in the dose-response curves to the latter two. The maximal response to phenylephrine (alpha-agonist) was similar for control and cafeteria rats. Ligand binding studies revealed increases in beta-adrenoceptor density of 33-38% in brown fat cells and isolated membranes from cafeteria-fed rats, but a 30% reduction in beta-receptors in heart membranes. Cold-adaptation caused a 22% reduction in beta-receptor density in brown fat membranes, but no change in heart. The ratio of beta 1/beta 2-receptors in brown fat was reduced from 59/45 in control to 47/54 in cafeteria-fed rats, but was not significantly altered in heart (58/44) or in brown fat from cold-adapted animals (64/30). alpha Adrenoceptor density was increased above control values by 69 and 25% in brown adipose tissue from cafeteria and cold-adapted rats, respectively. PMID- 3015645 TI - Responses of isolated dog blood vessels to glucagon. AB - The responses to glucagon were compared in helical strips of different arteries or veins isolated from dogs, and the relationship between the glucagon-induced relaxation and the intracellular cyclic AMP content in the arteries was studied. Relaxations induced by glucagon followed the order renal greater than mesenteric greater than femoral greater than cerebral = coronary arteries precontracted with PGF2 alpha. Glucagon-induced relaxations were greater in renal and mesenteric arteries than in the respective veins. Removal of the endothelium from the renal artery did not alter the magnitude of relaxations induced by glucagon nor the apparent ED50 value of glucagon. Glucagon increased the intracellular cyclic AMP content in both renal and coronary arteries, but the nucleotide increments were significantly greater in renal arteries than in coronary arteries. Isoproterenol produced significant increases in the intracellular cyclic AMP content and relaxations in renal and coronary arteries. Relaxations induced by dibutyryl cyclic AMP were greater in coronary arteries than in renal arteries. It is concluded that glucagon relaxed differently a variety of dog blood vessels, possibly by different extents of production of cellular cyclic AMP. The quantity and sensitivity of receptors for glucagon in smooth muscle cells may be heterogeneous in the vessels. PMID- 3015646 TI - Evidence for an interaction between GABAB and glutamate receptors in astrocytes as revealed by changes in Ca2+ flux. PMID- 3015647 TI - Effect of adenine nucleotides on granulopoiesis and lithium-induced granulocytosis in long-term bone marrow cultures. AB - Studies were undertaken to evaluate the role of adenine nucleotides in regulating hematopoiesis using a long-term liquid culture system. In contrast to early investigations using clonogenic stem cell assays, where inhibitory effects were observed, adenosine and adenosine-5'-monophosphate (AMP) were found to stimulate myelopoiesis whereas the dibutyryl derivative of cyclic adenosine-3',5' monophosphate (dcAMP) had either a modest inhibitory effect or no effect on long term hematopoiesis. Dose effects for AMP enhancement of hematopoiesis were relatively narrow. When cultures were exposed to a broad range of concentrations (10 mM-10 nM), stimulation was only seen at a molar concentration of 1 X 10(-4) M. Stem cell assays revealed stimulation of multipotent stem cells (CFU-S), as well as committed progenitor cells (CFU-C). Lithium chloride has been shown to cause granulocytosis both in vivo and in vitro. Reductions in intracellular cAMP levels resulting from adenylate cyclase inhibition is a proposed mechanism for this stimulatory effect. However, lithium-induced granulocytosis in long-term cultures could not be blocked by the addition of dcAMP. Measurement of nucleotide levels on spent medium revealed rapid utilization and/or degradation of these reagents. This suggests that failure to abrogate the lithium effect with dcAMP may have been related to the inability to maintain constant intracellular concentrations. The varied observations regarding adenine nucleotide effects on hematopoiesis, as well as the reproducible stimulation by lithium, may be explained by our current appreciation of the complex adenylate cyclase system, which contains both inhibitory and stimulatory subunits for nucleotides and monovalent cations. PMID- 3015648 TI - No response improvement after sequential chemotherapy for small cell lung cancer. AB - In a phase II study in patients with small cell lung cancer (SCLC) the combination of cyclophosphamide, cisplatinum and etoposide was found to be active, the response rate was 91% (30% CR, 61% PR) in the whole group. In 40 limited disease patients 19 CR (48%) and 20 PR (50%) were seen, whereas in 30 extensive disease patients only 2 CR (7%) and 23 PR (77%) were reached. Adding a second combination of doxorubicin, vincristine and procarbazine resulted in response improvement in only two patients. Median response duration was 41 weeks in CR patients and 30 in PR patients (p less than 0.01). Median survival was 66 in CR and 45 weeks in PR patients (p less than 0.002). Performance score and disease stage were found to be good prognostic factors. Four patients (6%) are disease-free at 2 1/2 years. The value of sequential chemotherapy for SCLC is probably minimal in view of the lack of response improvement. PMID- 3015649 TI - Gallium-67 lung scintigraphy in patients with acquired immune deficiency syndrome (AIDS). AB - Fifteen 67Ga lung scans were obtained from 11 men with AIDS to detect opportunistic lung infection. Results were compared with clinical findings, chest radiographs, CO-transfer and transbronchial biopsies or BAL. Clinical symptoms were least helpful in diagnosing pneumonia. Chest radiographs were normal in six of eight normal 67Ga studies and abnormal in four of seven abnormal scans. In four cases the X-ray showed equivocal abnormalities, twice corresponding to a normal and twice to an abnormal scan. Once the X-ray was normal, but the scan showed diffuse abnormalities. CO-transfer in patients with a normal scan was higher (55-68%) than in patients with an abnormal scan (22-44%). Biopsy or BAL revealed P. carinii in five of six patients. Cytomegalo-virus was isolated once. All six patients had abnormal 67Ga scans. 67Ga lung scintigraphy is a sensitive indicator of active lung disease in AIDS, especially when chest X-rays are normal or equivocal. PMID- 3015650 TI - High-dose etoposide with autologous bone marrow transplantation as initial treatment of small cell lung cancer--a negative report. AB - Seven patients with small cell lung cancer were treated with high-dose etoposide (1400-2400 mg/m2), given as a single course over 3 days, in conjunction with autologous bone marrow transplantation. Five patients achieved a partial response and two had no response. All patients required further treatment. The lack of any complete responder or good partial responder dissuaded us from entering further patients into the study, and high-dose, single-agent etoposide cannot be recommended in the initial treatment of small cell lung cancer. PMID- 3015652 TI - Synaptic organization of dorsal root projections to lumbar motoneurons in the clawed toad (Xenopus laevis). AB - Synaptic connexions between dorsal root primary afferents and lumbar motoneurons have been investigated in the isolated spinal cord of the clawed toad. The study of monosynaptic actions evoked in motoneurons by 9th or 10th dorsal root stimulation or by impulses in single primary afferents provided evidence for both electrical and chemical junctional transmission at the sensory-motor synapses. The anterograde filling of the 9th and 10th dorsal roots with horseradish peroxidase (HRP) shows that afferents do project to the motoneuron field of the segments IX and X. Some of the fibres not only reach the dorsally located motoneurons, but also cross the lateral motor column (LMC) and terminate in the marginal zone of ventral horn gray matter. The projections of the 9th and 10th dorsal root fibres are most numerous in the caudal part of segment X. Simultaneous HRP labeling of single motoneurons and the whole 10th dorsal root has revealed that afferent fibres make contacts not only on the distal dendrites of the motor cells, but also on the proximal ones. This latter finding is in a good agreement with the electrophysiological data. PMID- 3015653 TI - Long-term potentiation in the interpositus and vestibular nuclei in the rat. AB - Previous unpublished experiments from this laboratory had revealed only post activation depression effects in the cerebellar cortex when its inputs were activated by high frequency trains. In the experiments reported in this paper, we found reliable long-term potentiation (LTP) effects in the deep nuclei (interpositus and vestibular) when stimulation trains were applied to the white matter at the point where inferior peduncle fibers enter the cerebellum. LTP effects were found in both acute and chronic preparations. In the chronic preparations, LTP lasted for at least 8 days in all but one animal. PMID- 3015654 TI - Position dependence of stretch reflex dynamics at the human ankle. AB - The purpose of this study was to examine the effect of ankle position on the human ankle stretch reflexes during tonically-maintained contractions over most of the range of motion. The ankle was placed at randomly selected mean positions. Target levels of triceps surae (TS) or tibialis anterior (TA) tonic contractions were generated while the ankle was displaced by small amplitude, stochastic perturbations. System identification techniques were used to identify the stretch reflex dynamics at each combination of tonic level and ankle angle. As shown previously, the TS stretch reflex was characterized by an unidirectional, velocity-sensitive impulse response function whereas the TA stretch reflex was characterized by a linear impulse response function between ankle velocity and TA EMG. TS stretch reflexes showed a strong dependence on ankle position while TA stretch reflexes did not. Thus the TS stretch reflex magnitude increased greatly as the ankle was progressively dorsiflexed. In contrast, ankle mean position had only a minor effect on the TA stretch reflex magnitude. Our results indicate that the position-dependent facilitation of the TS stretch reflex is not due to changes in the level of skeletal motoneuron excitability. Rather, this effect may be accounted for by mechanisms that modulate the efficacy of the stochastic ankle perturbation. Such mechanisms could include position-induced: modulation of monosynaptic and polysynaptic afferent inputs to skeletal motoneurons, alterations in the extent of fusimotor drive and changes in the transmission of the joint perturbation to spindle receptors. Such mechanisms are discussed in terms of the differences between TS and TA stretch reflexes. Finally, the functional significance of position-dependent reflex responses are considered. PMID- 3015656 TI - Skeletal muscle phosphoproteins: muscle-specific basal and cAMP-dependent phosphoprotein profiles. AB - To determine whether or not phosphorylation of skeletal muscle proteins is related to functional specialization of individual muscles, we examined the distribution of phosphoproteins in skeletal muscles with different functional properties. Protein phosphorylation was carried out using [gamma-32P]ATP and employing endogenous protein kinases present in skeletal muscle homogenates. Phosphoprotein bands were separated by polyacrylamide gel electrophoresis. We found distinct cAMP-stimulated and basal phosphoprotein patterns in each contraction type; indicating that the phosphoprotein profile is related to functional characteristics. Muscle-specific, cAMP-dependent phosphoproteins may permit coordinate short-term alterations in twitch characteristics of skeletal muscle fibers in response to circulating hormones or other mediators. PMID- 3015655 TI - Potassium ion channel blockade restores conduction in heat-injured nerve and spinal nerve roots. AB - Compound action potentials were recorded in vitro from rat peroneal and sural nerves and from dorsal and ventral roots of the cauda equina before and after radiofrequency heating of 5-mm-length segments of these nerves to 41 to 45 degrees C. The heating was continued for intervals sufficient to reduce response amplitude by 50%. Inflection velocity, potential duration at 1/2 peak height, and the proportion of conducting A alpha fibers were also measured. The topical application of 4-aminopyridine (4-AP) and tetraethylammonium chloride (TEA) to the previously heated segments immediately following the radiofrequency injury completely or near-completely restored amplitude height to the preheat value in all experiments. A alpha sensory fibers were the most susceptible to the conduction block. Conduction in these fibers was also the most readily restored by the application of 4-AP or TEA. The effects of TEA, but not of 4-AP, could be reversed by saline or buffer washing. Topical application of verapamil and of magnesium or calcium ions had no discernible effect on heated nerves. We suggest that the mechanism of heat-induced conduction block may be similar to that from early demyelination or stretch injury. Further, motor and sensory A alpha fibers differ both in their vulnerability to heat and in their subsequent response to the application of potassium channel blockers. PMID- 3015657 TI - Ultrastructural morphology of thalamic cytoplasmic inclusion bodies in El mouse. AB - The established hereditary El mouse, clinically characterized by intermittent seizures of the grand mal type, has been investigated mainly from biochemical or electrophysiologic points of view. The present paper provides morphologic details on neuronal inclusion bodies occurring in the thalamus of the El mouse. Numerous intracytoplasmic inclusion bodies, 1 to 10 microns in diameter, were observed in the thalamic neuronal cells even in the young or seizure-free El mouse. Although such inclusion bodies were also studied in aging normal mice, their number in the El mouse was significantly higher than the number seen in aged mice. In addition to the thalamic inclusions, there were other changes observed, such as hippocampal neuronal loss and ischemic or chromatolytic neurons in the temporal cortex. We suggest that the neuronal changes in hippocampus and cortex were not primary to the El mouse epileptogenesis. We discuss the correlation between thalamic neuronal inclusions and convulsive seizures in the El mouse. PMID- 3015651 TI - The control of retinogeniculate transmission in the mammalian lateral geniculate nucleus. AB - In the mammalian visual system, the lateral geniculate nucleus is commonly thought to act merely as a relay for the transmission of visual information from the retina to the visual cortex, a relay without significant elaboration in receptive field properties or signal strength. However, many morphological and electrophysiological observations are at odds with this view. Only 10-20% of the synapses found on geniculate relay neurons are retinal in origin. Roughly half of all synapses derive from cells in layer VI of visual cortex; roughly one third are inhibitory and GABAergic, derived either from interneurons or from cells of the nearby perigeniculate nucleus. Most of the remaining synapses probably derive from cholinergic, noradrenergic, and serotonergic sites within the brainstem reticular formation. Moreover, recent biophysical studies have revealed several ionic currents present in virtually all thalamic neurons. One is a Ca2+-dependent K+ current underlying the afterhyperpolarization (or the IAHP), which may last up to 100-200 ms following an action potential. Activation of the IAHP leads to spike frequency adaptation in response to a sustained, suprathreshold input. Intracellular recordings from other neuronal preparations have shown that the IAHP can be blocked by noradrenaline or acetylcholine, leading to an increased cellular excitability. Another ionic current results from a voltage- and time dependent Ca2+ conductance that produces a low threshold spike. Activation of this conductance transforms a geniculate neuron from a state of faithful relay of information to one of bursting behavior that bears little relationship to the activity of its retinal afferents. We propose that state-dependent gating of geniculate relay cells, which may represent part of the neuronal substrate involved in certain forms of selective visual attention, can be effected through at least three different mechanisms: conventional GABAergic inhibition, which is largely controlled via brainstem and cortical afferents through interneurons and perigeniculate cells; the IAHP, which is controlled via noradrenergic and cholinergic afferents from the brainstem reticular formation; and the low threshold spike, which may be controlled by GABAergic inputs, cholinergic inputs, and/or the corticogeniculate input, although other possibilities also exist. Furthermore, it seems likely that gating functions involving the corticogeniculate pathway are suited to attentional processes within the visual domain (e.g., saccadic suppression), whereas brainstem inputs seem more likely to have more global effects that switch attention between sensory systems.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3015658 TI - What do GABA neurons really do? They make possible variability generation in relation to demand. AB - It is proposed that GABA neurons play key roles in maintaining meaningful communications within and among neural units by making possible variability generation in relation to demand. Activities of GABAergic inhibitory projection neurons from command centers and local circuit GABAergic inhibitory interneurons allow adaptive nervous system function to take place in a manner characterized by freedom without license. Through their multiple activities and connections these neurons make possible smooth transitions between modes of nervous system function over a range of increasing demands (neural pressures), enabling organisms to explore full ranges of their options. PMID- 3015659 TI - Opiate-induced seizures: a study of mu and delta specific mechanisms. AB - Two groups of experiments were conducted to determine if morphine- and enkephalin induced seizures are specifically mediated by the mu and delta receptor, respectively. In the first experiments, designed to assess the ontogeny of mu- or delta-seizures, rats from 6 h to 85 days of age received implanted cortical and depth electrodes as well as an indwelling cannula in the lateral ventricle. Various amounts of the mu-receptor agonists, morphine and morphiceptin, and the delta agonists, D-Ala2-D-Leu5-enkephalin (DADL) and Tyr-D-Ser-Gly-Phe-Leu-Thr (DSLET), were then administered intracerebroventricularly (icv) with continuous EEG monitoring. The second experiments entailed use of the nonspecific opiate antagonist, naloxone, as well as the specific delta antagonist, ICI 154,129, against seizures induced by icv-administered morphine, morphiceptin, DADL, or DSLET. Both morphine and morphiceptin produced electrical seizure activity in rats as young as 5 days after birth. The drugs produced similar seizure activity in terms of electrical morphology, observed behavior, ontogeny, threshold dose, and reversibility with small doses of naloxone. In the pharmacologic experiments, icv naloxone blocked all opiate-induced seizures. ICI 154,129 blocked DSLET seizure, had little effect on enkephalin or DADL seizures, and no effect on morphine or morphiceptin seizures. These data indicate that DSLET seizures are delta-specific but that all other opiate-induced seizures studied may involve multiple opiate receptor-mediated mechanisms. PMID- 3015660 TI - Neuronal plasticity in the nigrostriatal system of the rat after unilateral removal of vibrissae. AB - The vibrissae of rats were shaved from one side of the face daily for 10 days. To see whether or not this treatment had an effect on crossed and uncrossed striatal afferent connections from the midbrain, the tract tracer horseradish peroxidase was applied to the caudate-putamen on day 11. When the tracer was deposited on the side opposite the vibrissae removal, more labeled cells were found in the contralateral substantia nigra than when it was applied on the same side as the vibrissae removal, or placed in animals with intact vibrissae. Unilateral removal of vibrissae did not affect uptake of the tracer by the cells which give rise to the homolateral nigrostriatal projections. These changes in HRP labeling in the crossed projection from the substantia nigra were seen after 10 days of unilateral removal of the vibrissae; i.e., at a time when the animals have had opportunity to learn to compensate for vibrissotomy-induced behavioral asymmetries. PMID- 3015661 TI - Neurochemical consequences of status epilepticus induced in rats by coadministration of lithium and pilocarpine. AB - Status epilepticus was produced in rats by administering pilocarpine (30 mg/kg, s.c.) 16 h after treatment with LiCl (3 meq/kg, i.p.). After 35 min of status epilepticus, several parameters of cholinergic activity were measured. Seizures had no effect on the in vivo concentration of acetylcholine or choline in cerebellum, cortex, hippocampus, or striatum. Synaptosomal high-affinity choline transport was also not changed by seizures in hippocampus, cortex, or striatum. Cortical slices from seizing rats had elevated concentrations of acetylcholine and released acetylcholine at a greater rate than did controls, but these effects seemed to be due to a reduction in the postmortem hydrolysis of acetylcholine. Synaptosomal 45calcium uptake during 2 to 60 s of incubation was no different from control rates in tissue prepared from seizing rats. These results indicate that presynaptic cholinergic activity is not markedly altered by 35 min of continuous seizure activity induced by lithium and pilocarpine. In contrast, the in vivo concentration of cyclic guanosine 5'-monophosphate was elevated above control values in seizing rats by 57 to 170% in cerebellum, cortex, hippocampus, and striatum. PMID- 3015662 TI - Why do hormone receptors arise? PMID- 3015664 TI - Receptor ontogeny and hormonal imprinting. PMID- 3015663 TI - Internalization of polypeptide hormones and receptor recycling. PMID- 3015665 TI - Receptors for intercellular messenger molecules in microbes: similarities to vertebrate receptors and possible implications for diseases in man. PMID- 3015667 TI - Cooperative inhibitory effect of follicular fluid and cAMP on hamster oocyte maturation. AB - Porcine or human follicular fluid inhibited the spontaneous maturation of isolated hamster oocytes in vitro during the first 1.5 h of culture. Moreover, the presence of 50% follicular fluid combined with 100 microM dbcAMP cooperatively reduced the incidence of germinal vesicle breakdown. The addition of FSH also inhibited the resumption of meiosis, and the presence of LH did not overcome the inhibitory effects of follicular fluid and tended to impede isolated hamster oocyte maturation in vitro. PMID- 3015668 TI - Hormone and forskolin-stimulated cyclic AMP accumulation in human lymphocytes: reliability of longitudinal time measurements. AB - Reliability of measurement of lymphocyte cyclic AMP synthesis in intact cells was estimated by taking 3 successive blood samples during a one-month period from 11 healthy volunteers. Isoproterenol and prostaglandin E1-stimulated cyclic AMP accumulation were used to evaluate the activity of these two receptor activities in human lymphocytes. Forskolin-stimulated cyclic AMP accumulation was used to evaluate the activity of the Ns/catalytic subunit. Only for forskolin was significant reliability observed. For isoproterenol and prostaglandin E1 significant reliability was observed only for male subjects. PMID- 3015666 TI - Development of hormone receptors: conclusion. PMID- 3015671 TI - A copper-containing protein that inhibits nitrite reductase from Pseudomonas aeruginosa. AB - A non-blue copper-containing glycoprotein was isolated from Pseudomonas aeruginosa. The protein has a molecular mass of 10 kDa and contains 1 atom of EPR detectable type II copper. The protein inhibits oxidation of both azurin and cytochrome c-551 catalyzed by nitrite reductase from Ps. aeruginosa. Thus, it may be considered as an endogenous inhibitor of nitrite reductase. PMID- 3015670 TI - Cadmium in sludges used as fertilizer. AB - In intensively populated countries efficient sewage treatment is essential to protect river quality. An inevitable by-product is sewage sludge which has to be disposed of safely and economically. Utilisation of sludge as a fertilizer of agricultural land is the most economic disposal route for inland sewage-treatment works and also benefits farmers by providing a cheap manure. Much of the cadmium in wastewater is concentrated into sludge which consequently contains higher concentration of cadmium than soil does. It is impracticable to reduce cadmium concentrations in sludge below certain levels. When sludge is used on farmland rates of application must be controlled so that cadmium concentrations in soil never reach levels that could significantly contaminate food crops. Cadmium is a principal factor limiting the use of sludge on land. Nevertheless, it is a local problem since agricultural land in general receives more cadmium from aerial deposition and phosphatic fertilizers. The significance of accumulations of cadmium in soil depends mainly on its availability for crop uptake. Investigations are described which have attempted to identify and to determine the availability of forms of cadmium in soil. There is considerable research interest in cadmium in soil solution which is likely to be directly available for crop uptake. Another area of interest is the apparent disappearance of cadmium from sludge-treated soil. Soil analysis often cannot fully account for the cadmium added in sludge. Apart from the effect of soil conditions, especially pH value, crop uptake varies according to the particular crop examined. Highest concentrations of cadmium occur in tobacco, lettuce, spinach and other leafy vegetables. Using crop uptake data from field trials it is possible to relate potential human dietary intake of cadmium, on which hazard depends, to soil concentrations of cadmium, which can be controlled by regulating applications of sludge. This provides an objective basis for limits for cadmium concentrations in soils receiving sludge. Transfer of cadmium via farm animals to meat and dairy products for human consumption is thought to be minimal, even allowing for some direct ingestion of sludge-treated soil by the animals. Evidence from these and other investigations suggests that a loading rate limit of 5 kg Cd/ha (equivalent to a soil concentration of about 3.5 mg Cd/kg) affords adequate protection to the foodchain where sludge is used on agricultural land. More research work is needed to provide a basis for predicting the long-term availability of cadmium introduced to the soil in sludge. PMID- 3015669 TI - An antiviral factor from Melia azedarach L. prevents Tacaribe virus encephalitis in mice. AB - Treatment of neonatal mice with an antiviral factor, (AVF), obtained from the leaves of Melia azedarach L. protected them against lethal encephalitis caused by Tacaribe virus inoculation. The degree of protection obtained varied from 66% to 100% depending on the virus dose. Similarly, administration of AVF to nursing mothers protected their offspring from developing virus encephalitis. AVF does not directly inactivate Tacaribe virus; it inhibits an early step (s) in the replication process in cell cultures. PMID- 3015672 TI - Interaction of GTP-binding proteins with calmodulin. AB - Two GTP-binding proteins (Gi and Go), which were the substrates for islet activating protein, pertussis toxin, were purified from bovine cerebral cortical membranes. Both Gi and Go completely inhibited calmodulin-stimulated cyclic nucleotide phosphodiesterase activity. The same concentrations of these proteins, however, had no appreciable effect on the basal phosphodiesterase activity. The isolated Gi alpha and beta gamma subunits of GTP-binding proteins were potent inhibitors of the calmodulin-stimulated phosphodiesterase activity, but Go alpha was very weak. Therefore, the beta gamma subunits were likely to be the major active molecules in the brain membranes. GTP-binding proteins were shown to bind directly to calmodulin in a Ca2+-dependent manner by a gel permeation binding experiment. PMID- 3015673 TI - Localization of dolichols in phospholipid membranes. An ESR spin label study. AB - We have used ESR methods employing spin-labeled stearates to investigate the effects of dolichol on the motion of lipid molecules in phospholipid membranes of phosphatidylethanolamine and phosphatidylcholine. The ESR spectra show that the presence of dolichol affects the motion of the spin probes at carbon-16, but not at carbon-5. Similar results are obtained with phospholipid membranes comprising only phosphatidylcholine. It is suggested that dolichol molecules are present mainly in the lipid core region of phospholipid membranes. PMID- 3015674 TI - Identification of high levels of protein phosphatase-1 in rat liver nuclei. AB - Rat liver nuclei contain a protein phosphatase that is indistinguishable from the catalytic subunit of protein phosphatase-1 in its molecular mass, sensitivity to inhibitor-1 and inhibitor-2 and specificity for the beta-subunit of phosphorylase kinase. This activity is not bound to the outer nuclear membrane, but located within the nucleus. The average level of protein phosphatase-1 activity in nuclei is at least 5-fold higher than its average extranuclear concentration. PMID- 3015675 TI - Hormonal regulation of phosphorylase phosphatase activity in rat liver. AB - The effect of glucagon and insulin on rat liver phosphorylase phosphatase activity in vivo was investigated. The activity of phosphatase was found to decrease following the administration of glucagon and increase with insulin in a reversible manner. No change was detected in the activity of heat-stable phosphatase inhibitors in the hormone-treated samples. Liver protein kinases (regulatory subunit of cAMP-dependent protein kinase and/or Ca2+-dependent phosphorylase kinase) are suggested to regulate the activity of hepatic phosphorylase phosphatase (type 1 and 2A). PMID- 3015677 TI - Stimulation of cyclic GMP synthesis in human cultured glomerular cells by atrial natriuretic peptide. AB - Recently a stimulatory effect of atrial natriuretic peptide (ANP) on the particulate guanylate cyclase system has been reported in the glomeruli from different species. Using cultures of homogeneous human glomerular cell lines, we found that rat and human ANP stimulated markedly cGMP formation in epithelial cells with a threshold dose of 1 nM. A 20-fold increase was obtained at 5 microM. Stimulation was also present but less substantial (2-fold at 5 microM) in mesangial cells. cGMP was formed rapidly and released in the medium. ANP and sodium nitroprusside, an activator of soluble guanylate cyclase, had additive effects on cGMP formation. ANP did not inhibit cAMP formation in both cell lines. These results demonstrate that, at least in the human species, epithelial cells represent the main target of ANP in the glomerulus. Synthesis of cGMP in the glomerular epithelial cells in response to ANP also suggests that the excess of urinary cGMP produced by the kidney which is observed after ANP administration is of glomerular rather than of tubular origin. PMID- 3015676 TI - Cell alkalinization is not necessary and increased sodium influx is not sufficient for stimulated superoxide production. AB - Preincubation of rabbit neutrophils for 5 min with the protein kinase C inhibitor H7 causes inhibition of the rise in intracellular pH but not the increase in Na+ influx or stimulated oxidative burst produced by the chemotactic factor formyl methionyl-leucyl-phenylalanine. On the other hand, the stimulated superoxide production, but not the increase in Na+ influx produced by phorbol 12-myristate 13-acetate, is inhibited by H7. The effect is more pronounced on the rate than the extent of the stimulated superoxide release. Furthermore, cell acidification produced by the phorbol ester but not by the chemotactic factor is decreased in the presence of H7. These results suggest that most of the stimulated Na+ influx is not coupled to H+ efflux, in the case of the chemoattractant, the rise in intracellular pH is not necessary for stimulated superoxide production, the increase in Na+ influx, in the case of the phorbol ester, is not sufficient for the stimulation of the oxidative burst, and the sources of the H+ responsible for the stimulated pH drop are the various metabolic activities of the cell, including NADPH oxidation and activation of the hexose monophosphate shunt. PMID- 3015678 TI - Activation of polyphosphoinositide phospholipase C by fluoride in WRK1 cell membranes. AB - Partially purified plasma membranes prepared from myo-[3H]inositol-prelabeled WRK1 cells exhibit a phosphatidylinositol 4,5-biphosphate (PIP2) phospholipase C activity sensitive to NaF. NaF increased the production of IP2 and IP3 in a time- and concentration-dependent manner. The maximal increase in IP2 and IP3 production rates represented 400 +/- 18 and 360 +/- 40% of the basal production rate, respectively. Half-maximum stimulation was reached with 2-4 mM NaF. The observed effect was specific for F-. Aluminium potentiated fluoride-induced IP3 and IP2 accumulation in a concentration-dependent manner. The effect of fluoride on the PIP2 phospholipase C from WRK1 cell membranes appears to be similar to the well-documented effect of F- on the well-characterized Ns. Ni and transducin GTP binding proteins. This observation constitutes an additional argument to suggest that a GTP-binding protein is involved in the process of receptor-mediated activation of PIP2 phospholipase C. PMID- 3015679 TI - ATP-induced calcium mobilization and inositol 1,4,5-triphosphate formation in H 35 hepatoma cells. AB - Addition of ATP (but not epinephrine, angiotensin II, vasopressin, or platelet activating factor) to H-35 hepatoma cells whose cellular lipids have been pre labelled with [3H]inositol, causes a rapid increase in [3H]inositol triphosphate. In H-35 cells pre-incubated in the presence of 45Ca2+, ATP causes a similarly rapid release of 45Ca2+. The concentration-effect relationships for inositol triphosphate formation and Ca2+ efflux are similar to those reported previously for differentiated hepatocytes. These results demonstrate that at least one of the Ca2+-mobilizing receptors normally found on hepatocytes is functionally retained in the H-35 hepatoma cell line and thus could provide a useful model for the study of these receptor mechanisms in liver. PMID- 3015680 TI - Complete cDNA sequence for rabbit muscle glycogen phosphorylase. AB - The cDNA for the nearly full-length rabbit muscle glycogen phosphorylase mRNA has been isolated and sequenced. The cDNA is rich in G and C nucleotides. This feature is especially striking at the 3rd position of codons, where 86% of the 843 amino acid codons terminate with G or C. Methionine, presumably the initiation residue, is found at position-1, suggesting that the removal of only a single methionine residue precedes the amino-terminal acetylation at serine. Eight differences between the deduced amino acid sequence and the previously determined protein sequence are discussed. PMID- 3015681 TI - Cyclic GMP phosphodiesterase from cattle retina. Amino acid sequence of the gamma subunit and nucleotide sequence of the corresponding cDNA. AB - The primary structure of the gamma-subunit of cyclic GMP phosphodiesterase was determined by parallel analysis of the amino acid sequence of the protein and nucleotide sequence of the corresponding cDNA. The enzyme gamma-subunit contains 87 amino acid residues, its N-terminal amino group being acetylated. PMID- 3015682 TI - Structure of the extra-membranous domain of the beta-subunit of (Na,K)-ATPase revealed by the sequences of its tryptic peptides. AB - Membrane bound dog kidney (Na,K)-ATPase was digested with trypsin. The peptides that were recovered in the supernatant were purified and sequenced. By comparing these results with the sequence of alpha- and human beta-subunits, the location of each of the peptides could be allotted. Both accessibility to trypsin and the facility of release into the water phase indicated that these peptides were derived from the exposed surface of the intact enzyme. The sequence, GXGXXG, reported in the Torpedo californica beta-subunit [(1986) FEBS Lett. 196, 315-319] was likely a mere coincidence with the sequence of the dinucleotide-binding site, since the last glycine was replaced by proline in the sequence of the dog beta subunit. A disulfide bridge was found within a peptide derived from the beta subunit. A possible model for the beta-subunit structure is proposed. PMID- 3015683 TI - A U3 RNA pseudogene in mouse: sequence and organization in genomic DNA. AB - A mouse U3 RNA pseudogene has been identified; it corresponds to a U3B full length coding sequence with a 3'-oligo(A) tail, precisely flanked at both ends by a pair of 15 bp direct repeats. These structural features suggest that it has arisen through an RNA-mediated mechanism involving an insertion at staggered nicks in the genome. Sequence data indicate that this mouse specimen has been generated by a different event as compared to the recently described rat pseudogenes. It represents the first reported case, for a pseudogene of this class, to be present at more than one copy per genome. PMID- 3015684 TI - Effects of thrombin, phorbol myristate acetate and prostaglandin D2 on 40-41 kDa protein that is ADP ribosylated by pertussis toxin in platelets. AB - Intact platelets were stimulated with thrombin and the amount of GTP-binding protein (G-protein) oligomers was assessed by measuring ADP ribosylation of 40-41 kDa protein by pertussis toxin in isolated membranes. The toxin substrate fell by 57-62% in 10-60 s, but then returned towards normal over 5 min. Recovery was greatly enhanced by removal of thrombin from receptors with hirudin. Phorbol myristate acetate increased ADP-ribosylatable protein, but only back to initial levels prior to PMA. In contrast prostaglandin D2 plus theophylline (which increase cyclic AMP) did not increase ADP ribosylation, but could completely block the fall of the toxin substrate caused by thrombin. These results indicate that activation of thrombin receptors promotes the dissociation of G-protein oligomers to release free alpha-subunits, and this effect can be modulated by protein kinase C and cyclic AMP-dependent protein kinase. The possible relationships of these findings to the regulation of stimulus-response coupling in platelets is discussed. PMID- 3015685 TI - Neuropharmacology of supraoptic nucleus neurons: norepinephrine and gamma aminobutyric acid receptors. AB - The neurosecretory neurons in the mammalian hypothalamic supraoptic nucleus receive prominent GABAergic and noradrenergic projections arising from local interneurons and the A1 cells in the ventrolateral medulla, respectively. Intracellular recordings in in vitro perfused hypothalamic explants reveal an abundance of spontaneous inhibitory postsynaptic potentials (IPSPs) and a compound IPSP after electrical stimulation in the diagonal band of Broca area. The sensitivity of both spontaneous and evoked IPSPs to intracellular chloride injection, bicuculline, and pentobarbital is consistent with a GABA-activated, chloride-mediated inhibitory synaptic input. Parallel changes in membrane voltage and conductance are present during applications of GABA and muscimol, with similar sensitivity to ionic manipulation, bicuculline, and pentobarbital. These observations contrast with the consistently excitatory effects that follow either the stimulation of A1 cells or the application of norepinephrine and alpha 1 adrenergic agonists. Norepinephrine induces membrane depolarizations and bursting activity patterns that are blocked by the selective alpha 1 antagonist prazosin. Membrane response to norepinephrine is voltage dependent and is associated with little change in conductance. GABA and norepinephrine are proposed as transmitters in the final central pathways that mediate information to supraoptic vasopressinergic neurons from peripheral baroreceptors and chemoreceptors, respectively. PMID- 3015687 TI - Spare alpha adrenoceptors in the peripheral circulation: excitation-contraction coupling. AB - Postsynaptic alpha adrenoceptors in arteries and veins represent a mixed population of alpha 1 and alpha 2 adrenoceptors, with both subtypes mediating vasoconstriction. In the peripheral arterial circulation, postsynaptic vascular alpha 1 adrenoceptors are found in the adrenergic neuroeffector junction, whereas postsynaptic vascular alpha 2 adrenoceptors are located extrajunctionally. In the venous circulation, it appears that alpha 2 adrenoceptors may be predominantly junctional, whereas alpha 1 adrenoceptors may be predominantly extrajunctional. In general, alpha 1 adrenoceptors play a more important functional role in arteries than in veins, with the converse being true for postsynaptic vascular alpha 2 adrenoceptors. The relationship between alpha-adrenoceptor occupancy and vasoconstrictor response is more favorable for postsynaptic vascular alpha 1 adrenoceptors than for alpha 2 adrenoceptors in both arteries and veins, and there is evidence for a receptor reserve in alpha 1 adrenoceptors in both the arterial and venous circulation. No reserve in postsynaptic vascular alpha 2 adrenoceptors is seen in the arterial circulation, but in isolated venous preparations, a reserve in alpha 2 adrenoceptors has been observed. It has been suggested that spare alpha 2 adrenoceptors found in veins, but not arteries, may be responsible, at least in part, for the exaggerated alpha 2-adrenoceptor mediated response of veins relative to arteries. PMID- 3015686 TI - Nature of alpha 1 and postjunctional alpha 2 adrenoceptors in the pulmonary vascular bed. AB - The subtypes of postjunctional alpha adrenoceptors in the feline pulmonary vascular bed were studied by using selective alpha-adrenoceptor agonists and antagonists. Under conditions of controlled pulmonary blood flow and constant left atrial pressure, intralobar injections of the alpha 1 agonists phenylephrine and methoxamine, and the alpha 2 agonists UK 14,304 and B-HT 933, increased lobar arterial pressure in a dose-related manner. Prazosin, an alpha 1-adrenoceptor antagonist, reduced responses to phenylephrine and methoxamine to a greater extent than responses to UK 14,304 and B-HT 933. Yohimbine, an alpha 2 blocker, decreased responses to UK 14,304 and B-HT 933 without altering responses to phenylephrine or methoxamine. The same pattern of blockade was observed in animals pretreated with 6-hydroxydopamine, an adrenergic neuronal blocking agent. However, in propranolol-treated animals, prazosin antagonized responses to phenylephrine and methoxamine without altering responses to UK 14,304 or B-HT 933, and the selectivity of the blocking effects of yohimbine were preserved. Responses to intralobar injections of norepinephrine (NE) were markedly decreased by prazosin, whereas yohimbine had only a small effect. These data suggest the presence of both postjunctional alpha 1 and alpha 2 adrenoceptors mediating vasoconstriction in the pulmonary vascular bed. These results also indicate that the vasoconstrictor responses to injected NE in the cat pulmonary vascular bed result mainly from activation of alpha 1 adrenoceptors. PMID- 3015689 TI - The alpha adrenoceptors on endothelial cells. AB - Endothelial cells release a powerful factor (endothelium-derived relaxing factor [EDRF]) that relaxes smooth muscle cells in response to some vasodilating agents such as acetylcholine. Contraction curves to norepinephrine (NE) in greyhound, mongrel dog, and pig coronary artery rings were studied in vitro in the presence of propranolol. Removal of endothelium increased the sensitivity and maximum contraction in response to NE. In other experiments pig coronary rings were precontracted with a thromboxane mimetic U 46619 in the presence of propranolol. NE relaxed these arteries only if endothelium was present. Methoxamine was without effect but the relaxation response to NE was antagonized by phentolamine, idazoxan, and yohimbine, which suggests that there are alpha 2 adrenoceptors on endothelial cells that mediate the release of EDRF. Greyhound and mongrel dog large coronary arteries relaxed to NE only if prazosin was present, which suggests that alpha 1-adrenoceptor stimulation on the vascular smooth muscle can override the relaxation response to EDRF. Comparison of NE responses in carotid, mesenteric, renal, and femoral large arteries of the pig, greyhound, and mongrel dog indicate the nonuniformity of distribution of alpha 2 adrenoceptors on endothelium and alpha 1 and alpha 2 adrenoceptors on vascular smooth muscle. The integrity of the endothelium must now be considered in interpreting the vascular responses to alpha-adrenoceptor agonists. PMID- 3015688 TI - Effects of temperature on alpha adrenoceptors in limb veins: role of receptor reserve. AB - In cutaneous veins of the dog, cooling augments the response to sympathetic nerve stimulation and exogenous norepinephrine (NE). The postjunctional alpha adrenoceptors in this blood vessel belong to both the alpha 1 and alpha 2 subtypes. Cooling augments alpha 2-adrenergic responses (presumably because of an increased receptor affinity), but depresses alpha 1-adrenergic responses (presumably because of a direct inhibitory effect on the contractile process). When agonists of high efficacy such as NE or phenylephrine are used, an alpha 1 adrenoceptor reserve is present that buffers the response from the inhibitory effect of cooling. This allows the potentiating effect of cold on the alpha 2 adrenergic component of the response to catecholamines to predominate, and the contractile response to exogenous NE and sympathetic nerve stimulation is augmented. By contrast, in deep veins of the limb, cold reduces the contractions evoked by alpha 1- and alpha 2-adrenergic activation. This can be explained best by the absence of a receptor reserve for alpha 1-adrenergic agonists of high efficacy, combined with a reduced density of postjunctional alpha 2 adrenoceptors. PMID- 3015690 TI - Molecular characteristics of receptors for atrial natriuretic factor. AB - Specific, high-affinity receptors for atrial natriuretic factor (ANF) have been identified on membranes from a variety of tissues and cultured cells. By affinity labeling procedures, radioactivity from 125I-labeled ANF was specifically incorporated into three different polypeptides of ca. 120,000, 70,000, and 60,000 daltons, which may represent the binding subunits of ANF receptors. These polypeptides were present in varying amounts in different target tissues. In rat adrenal membranes, the 120,000- and 70,000-dalton peptides were specifically labeled whereas in A10 rat smooth muscle cells, only the 60,000-dalton peptide was labeled. Membranes from rat kidney and rabbit aorta contain all three peptides. Gel filtration chromatography of solubilized receptors suggested that intact ANF receptors are large molecular complexes with apparent molecular masses in the range of 250,000-350,000 daltons. The differential labeling pattern observed with the various tissues suggested that there might be at least two different receptors composed of unique ANF-binding polypeptides. PMID- 3015691 TI - Possible mechanisms underlying the vasorelaxant response to atrial natriuretic factor. AB - Synthetic analogs of atrial natriuretic factor (ANF) have been utilized to assess possible mechanisms underlying the vasorelaxation response to this peptide. ANF is a potent relaxant of aortic smooth muscle contracted by a variety of agonists and low (e.g., 20 mM) but not high (e.g., greater than or equal to 80 mM) levels of extracellular K+. The relaxation does not require the presence of a functional endothelium and is temporally associated with the elevation of tissue levels of cyclic GMP resulting from a direct activation of particulate guanylate cyclase. The ANF-induced relaxation is not associated with membrane hyperpolarization but may be related to an alteration of Ca2+ handling by the vascular smooth muscle cell via inhibition of agonist-induced Ca2+ translocation, stimulation of Ca2+ extrusion, or interference with Ca2+ release from intracellular storage sites. ANF displays regional vasorelaxant selectivity in vitro (e.g., arteries vs. veins, central vs. peripheral arteries), which may be, in part, a function of an altered distribution of high-affinity receptors and/or particulate guanylate cyclase. These latter developments may explain the discrepancy between the potent vasorelaxant response in vitro and the modest or limited vasodilator response in whole-animal experiments. PMID- 3015692 TI - Inhibition of aldosterone synthesis by atrial natriuretic factor. AB - Atrial natriuretic factor (ANF) inhibits basal and stimulated aldosterone synthesis in adrenal glomerulosa cells. ANF probably acts through specific membrane receptors. Alterations in cyclic GMP and cyclic AMP levels do not account for ANF's inhibitory effect. ANF does not block angiotensin II (AngII) receptors nor does it interfere with phosphoinositide metabolism or calcium movements stimulated by adrenal agonists. ANF does not inhibit protein synthesis nor does it work by inhibiting NA+,K+-ATPase or depleting cell potassium. ANF decreases conversion of endogenous cholesterol to pregnenolone, the step stimulated by adrenocorticotropin and AngII. ANF does not affect the conversion of 20-alpha-hydroxycholesterol, which easily penetrates mitochondrial membranes to the site of the cholesterol side-chain cleavage enzyme. These results suggest that ANF inhibits the ability of endogenous cholesterol to reach or interact with the side-chain cleavage enzyme. ANF does not act like a calcium channel-blocking agent. However, ANF is less effective at high-calcium concentrations, which suggests that it may inhibit a step that calcium stimulates. Understanding ANF action will probably require identification of the specific biochemical changes (mediators) that it induces. Parallel efforts to understand how other agents stimulate steroidogenesis (particularly in the areas of protein synthesis, protein phosphorylation, and cholesterol movements) will further this understanding. PMID- 3015694 TI - Effect of short- and long-term dexamethasone on 3 alpha-androstanediol glucuronide in hirsute women. AB - The role of glucocorticoid therapy in regulating plasma 3 alpha-androstanediol glucuronide (3 alpha-diol G) content, a marker of androgen action, in hirsute women was unclear. A pulse injection of adrenocorticotropic hormone (0.5 U) following 1 mg of dexamethasone (DEX) at midnight significantly increased the plasma level of cortisol (P less than 0.01), 17-hydroxyprogesterone (17-OHP, P less than 0.01) in 17 hirsute women, but it had an insignificant effect on delta 4-androstenedione (delta 4A), testosterone, dehydroepiandrosterone sulfate (DHEA S), and 3 alpha-diol G. Human chorionic gonadotropin administered for 3 days produced a significant (P less than 0.01) increase only in 17-OHP. DEX administered as a single dose in the late evening failed to affect the plasma levels of these four androgens when measured at 8:00 A.M. In contrast, when the glucocorticoid was given each evening for over 2 months, plasma delta 4A, DHEA-S, and 3 alpha-diol G were suppressed (P less than 0.001) substantially, as compared with baseline values in 12 hirsute women, 7 with polycystic ovary disease and 5 with idiopathic hirsutism. These observations indicate that chronic glucocorticoid therapy suppresses androgen action in hirsute women. PMID- 3015693 TI - Prevalence of and markers for the attenuated form of congenital adrenal hyperplasia and hyperprolactinemia masquerading as polycystic ovarian disease. AB - To determine the prevalence of the attenuated form of congenital adrenal hyperplasia (CAH) and hyperprolactinemia (HPPN) relative to polycystic ovarian disease (PCOD), 100 consecutive women presenting with the classic clinical features of PCOD were evaluated by basal hormonal profiles and subsequent adrenocorticotropic hormone (ACTH) stimulation tests. The study also sought biochemical markers for CAH other than ACTH stimulation. The prevalences were found to be as follows: PCOD, 65%; PCOD with HPPN, 9%; HPPN, 3%, end-organ hypersensitivity (EOH), 4%; homozygotic CAH, 4%; and heterozygotic CAH, 15%. Other than the differential response to ACTH, the only other biochemical markers observed for homozygotic CAH were significantly higher basal levels of testosterone (T) and 17 alpha-hydroxyprogesterone (17-OHP). Luteinizing hormone/follicle-stimulating hormone ratio, androstenedione, and dehydroepiandrosterone sulfate all showed no significant differences between homozygotic CAH, heterozygotic CAH, HPPN, PCOD, and EOH. This study establishes the relative prevalences of the syndromes commonly mimicking PCOD. We also conclude that the observed low incidence of CAH does not justify routine ACTH testing on all patients presenting with features of PCOD--however, our data suggest that patients with basal serum levels of T and 17-OHP greater than 50% above the upper limit of normal should undergo this dynamic test, especially if there are also certain clinical features suggestive of CAH. PMID- 3015695 TI - [Effect of guanosine-5'-monophosphate and inosine on the isolated heart of the frog]. AB - The chronotropic and inotropic effects of guanosine-5'-monophosphate (5'-GMP) and inosine were investigated in isolated frog hearts. 5'-GMP induced biphasic chronotropic and inotropic responses: positive those were inhibited with propranolol, the negative inotropic response could not be inhibited with atropine. Inosine was able to enhance the positive inotropic response to 5'-GMP. PMID- 3015696 TI - [Age-related changes in secretion of adrenocortical steroid hormones in normal healthy men]. AB - The influence of aging on the steroid secretory capacity of the adrenal gland was evaluated by comparing data on young (age 20 to 21 years) with elderly (age 77 to 86 years) healthy male subjects. After the administration of ACTH-Z (1 mg, im) during the treatment of dexamethasone (1 mg/day, for 2 days), blood samples were taken at time 0, 1, 2, 3, 6, 12 and 24 h. The mean basal levels of pregnenolone (P5), 17-hydroxypregnenolone (170HP5), dehydroepiandrosterone (DHEA), progesterone (P4), 17-hydroxyprogesterone (170HP4), androstenedione (A-dione) and aldosterone (Ald) gradually decreased with advance in age. Dexamethasone administration to the elderly men produced no significant fall in plasma P4 and Ald. Plasma ACTH levels after ACTH-Z administration were significantly higher in the elderly men than the comparable levels in the young men. The apparent half life of ACTH-Z in plasma was prolonged in the elderly men. For 3 hours after ACTH Z injection, the responses of all plasma steroids, such as P5, 170HP5, DHEA, P4, 170HP4, A-dione, Ald and cortisol (F), were significantly lower in the elderly men. When the 24-hour secretion rates of steroid hormones were compared by delta area, which indicated the increased area for 24 hours after ACTH-Z administration, the secretion rate of F showed no significant difference between the two groups, but that of DHEA was significantly low in the elderly men. The 24 hour secretion rates of P5 and P4 were not impaired and that of 170HP4 was significantly high in the elderly men. These results indicate that the steroidogenic response to ACTH decreases with aging, and that, in the elderly men, an apparent decrease in C17,20 lyase efficiency may be related in part to the decreased secretion of adrenal androgens. PMID- 3015698 TI - The adhesion of dental resins to metal surfaces: the Kulzer technique. PMID- 3015697 TI - [The use of hydroxyapatite (HA) in periodontology]. PMID- 3015699 TI - Risk factors in gestational trophoblastic disease, and consequences for primary treatment. AB - Treatment with methotrexate (MTX) for two patients with gestational choriocarcinoma proved to be inadequate; subsequently both patients received a combination of cis-platinum, cyclophosphamide, actinomycin D and etoposide. These histories demonstrate the need for better prediction of the efficacy of MTX treatment. Baghshawe and Goldstein developed scoring systems to recognize patients requiring primary combination chemotherapy. The Dutch Working Group for Trophoblastic Tumors recently introduced a simplified scoring system to classify these patients. In order to compare these three scoring systems to predict the effect of primary treatment with MTX a retrospective study was made of 37 patients. MTX treatment failures were predictable in 8 out of 13 patients using Bagshawe's system and 6 out of 13 by the Dutch scoring system. The specificity was 88 and 92%, respectively. Goldstein's scoring system proved to be the least sensitive, but very specific. PMID- 3015701 TI - Extended X-ray absorption fine structure studies of calcification. PMID- 3015700 TI - Non-resolution of pelvic sonographic abnormality after chemotherapy for persistent trophoblastic disease--a word of caution. AB - A case of persistent trophoblastic disease (PTD) is presented in whom pelvic sonography demonstrated persistent uterine abnormality and dilated adnexal vessels after cessation of chemotherapy. Hysterectomy was performed on account of subsequent uterine bleeding. A viable tumour was not demonstrated in the hysterectomy specimen. In the absence of haemorrhagic complications persistent sonographic abnormality should not necessarily indicate hysterectomy, especially when hCG levels are normal. PMID- 3015702 TI - The independence of myogenesis and chondrogenesis in micromass cultures of chick wing buds. AB - Micromass cultures prepared from stage 23, 24, or 25 chick wing buds and cultured under identical conditions produce similar numbers of myoblasts. After treatment with the DNA synthesis inhibitor cytosine-1-beta-D-arabinofuranoside, [3H]thymidine labeling and autoradiography of the cultures show that the increase in myoblast number during the first 48 hr of culture is due primarily to cell division. Micromass cultures prepared from proximal and distal portions of stage 23 or 24 wing buds have very different chondrogenic potentials in vitro (B.J. Swalla, E.M. Owens, T.F. Linsenmayer, and M. Solursh (1983). Dev. Biol. 97, 59 69) but a similar myogenic potential under these culture conditions. Medium supplements that significantly enhance chondrogenesis by proximal cell cultures, such as low serum or 1 mM db cyclic AMP, do not affect the number of myoblasts per unit area of culture during the first 3 days. Muscle cells are eventually reduced in number in whole limb micromass cultures, yet persist as long as 6 days in proximal and distal cultures. These results suggest that myogenic cells are already committed in the early limbs but are inhibited from differentiation in situ until a later time. Myogenesis and chondrogenesis occur independently in culture, consistent with the idea that these two differentiated cells are derived from two separate cell populations. Furthermore, treatments which enhance chondrogenesis do not act indirectly by killing the myoblast population in these cultures. PMID- 3015704 TI - Safety of immune globulins in relation to HTLV-III. PMID- 3015703 TI - Major transitions in histone gene expression do not occur during development in Xenopus laevis. AB - In light of the parallels that exist in the structure of histone genes in sea urchins and in the frog, Xenopus laevis, and in the early development of these animals, it has been thought that Xenopus histone gene expression might be subject to the type of developmental regulation observed in sea urchins. We have examined the patterns of histone mRNA accumulation in Xenopus oocytes and embryos by primer extension and S1 nuclease protection techniques. The data demonstrate that histone genes which are active in Xenopus oocytes, and which contribute to large pools of histone mRNA in the absence of DNA replication, are also transcriptionally active in late embryos and in cultured cells. These results suggest that, rather than activating distinct sets of histone genes at different developmental stages, the developing frog embryo reprograms the expression of histone genes active in nondividing oocytes so that their expression becomes coupled to DNA replication subsequently during embryogenesis. PMID- 3015705 TI - [Surgical treatment of hepatocellular carcinoma in cirrhosis. Value of peroperative ultrasonography]. AB - During the past three and half years, 19 patients with hepatocellular carcinoma associated with cirrhosis were operated on. Pain was present in seven patients while 12 were asymptomatic. Alpha foeto-protein was negative in 7 patients. Intraoperative ultrasonography was performed in the last 15 patients. Three right hepatic resections, 5 left hepatic lobectomies and 11 segmentectomies or subsegmentectomies were performed. The operative mortality was 5 p. 100 (one patient). The long term survival in the 3 patients who underwent palliative resection was 6 months. Among the 15 other patients, four died from causes unrelated to their tumor; three patients with tumors larger than 8 cm died from recurrence 12 to 26 months after surgery; and the remaining 7 are still alive without evidence of recurrence 3 to 18 months after surgery. We concluded that in patients with cirrhosis, resection of limited hepatocellular carcinoma is possible, using intraoperative sonography, with low operative mortality. Early detection by repeated ultrasonic examination of the liver in patients with cirrhosis could be the best way to improve the surgical treatment of hepatocellular carcinoma. PMID- 3015706 TI - [Linitis plastica of the colon and stomach following breast cancer]. PMID- 3015707 TI - [Anti-Epstein-Barr virus IgM antibodies in viral hepatitis A and B: significance and prevention]. PMID- 3015708 TI - The role of carbonic anhydrase in gastric mucosal protection with special reference to H+ back diffusion and concomitant metabolic acidosis induced by acetazolamide. AB - It is suggested that carbonic anhydrase is implicated not only in gastric acid secretion, but in mucosal protection. It was reported that acetazolamide induced gastric mucosal lesions. But acetazolamide also caused concomitant metabolic acidosis by inhibiting H+ secretion from renal tubules. We investigated whether concomitant metabolic acidosis is implicated in gastric mucosal lesions induced by acetazolamide in vivo. We also evaluated the effect of acetazolamide on gastric H+ back diffusion in vivo. Correction of metabolic acidosis with sodium bicarbonate had no effect on the degree of the gastric mucosal lesions. Acetazolamide caused no change in gastric H+ and Na+ flux. These results suggest that metabolic acidosis induced by acetazolamide is not implicated in gastric mucosal lesions. Carbonic anhydrase has no effect on H+ back diffusion but may be implicated in mucosal protection by the disposition of back diffused H+ into the gastric mucosa. PMID- 3015709 TI - Immunohistochemical study of neuron-specific enolase and CA 19-9 in pancreatic disorders. The value of neuron-specific enolase as a marker for islet cell and nerve tissue. AB - Immunohistochemical studies of neuron-specific enolase were performed on pancreatic tissues from patients with insulinoma, nonfunctioning islet cell tumor, chronic pancreatitis, and pancreatic adenocarcinoma, and from 5 normal patients. The concentration of neuron-specific enolase was also measured in the sera of patients and in the pancreatic tissue, and the tissues were stained for carbohydrate antigen 19-9 by immunohistochemical techniques. Neuron-specific enolase was localized in nerve fibers, normal islet cells, and islet cell tumors; its concentration was elevated only in the tissue of islet cell tumors and in serum from patients with insulinoma. In the pancreatic tissue of pancreatitis or pancreatic adenocarcinoma, various changes in acini and islets were present. The altered islets stained clearly for neuron-specific enolase and could easily be distinguished from altered, unstained acini in cases of pancreatitis or pancreatic adenocarcinoma. Islets in the pancreatic tissue remained intact with various morphologic changes, although acini had degenerated severely. Carbohydrate antigen 19-9 was localized in all the carcinoma cells in the pancreatic tissue and in some of the normal pancreatic ducts. No cells were simultaneously immunostained by anti-neuron-specific enolase and anti carbohydrate antigen 19-9 antibodies. Thus, neuron-specific enolase is a good marker for islet cell tumor, and is valuable for examining islets in pancreas with various disorders both alone and in combination with other tumor markers. PMID- 3015711 TI - Intracolonic WR 2721 protection of the rat colon from acute radiation injury. AB - The radioprotective thiophosphate compound WR 2721, when given intraperitoneally, has been shown to effectively protect normal murine tissues, but not tumors, from radiation injury. Intravenous administration in humans has produced limiting nausea and vomiting at protective doses. The accessibility of the colon, coupled with the frequency of acute radiation injury to the rectum during pelvic irradiation, stimulated us to determine if WR 2721 was radioprotective when administered intracolonically. Double-blind histologic evaluation of colons from irradiated rats treated with intracolonic WR 2721 demonstrated a radioprotective effect with a dose modifying factor of 1.8 when compared with controls. A contact time of 30-60 min was optimal as was a WR 2721 dose of at least 15 mg. No systemic absorption was found. These data demonstrate that WR 2721 exerts its radioprotective effect without the coincident development of secondary tissue hypoxia and provide rationale for a clinical trial in humans. PMID- 3015710 TI - Release and characterization of cholecystokinin from isolated canine jejunal cells. AB - We have developed an isolated intestinal cell system to study the regulation of cholecystokinin release. Enzymatically dispersed canine jejunal mucosal cells were separated by counterflow elutriation to enrich cholecystokinin content 20 fold. Release of cholecystokinin from freshly isolated cells was determined by radioimmunoassay. Elevated extracellular potassium, dibutyryl cyclic adenosine monophosphate, and the diterpene derivative forskolin each stimulated an increase in cholecystokinin release over a 60-min period compared to basal secretion. L Tryptophan stimulated a dose-dependent and stereospecific increase in cholecystokinin. D-Tryptophan did not significantly alter basal cholecystokinin secretion. Carbachol inhibited L-tryptophan-stimulated cholecystokinin release in a dose-dependent manner. Analysis of extracts from intact jejunal mucosa by high pressure liquid chromatography revealed three components with cholecystokinin immunoreactivity eluting in positions with cholecystokinin 8, with cholecystokinin 33/39, and after cholecystokinin 33/39. Only the two molecular forms coeluting, respectively, with cholecystokinin 8 and cholecystokinin 33/39 were present in the elutriator-enriched jejunal cells, and these two forms of cholecystokinin immunoreactivity were released from cells upon stimulation. These data suggest that L-tryptophan directly regulates the release of cholecystokinin and that membrane depolarization and intracellular generation of cyclic adenosine monophosphate may play a role in activating cholecystokinin cells. Stimulated cholecystokinin release is inhibited by the muscarinic agonist carbachol. The molecular profile of released cholecystokinin corresponded to the two molecular components, cholecystokinin 8 and cholecystokinin 33/39, contained in dispersed cells. PMID- 3015712 TI - Noncirrhotic portal fibrosis. PMID- 3015713 TI - [Effect of beta-adrenoreceptors on thrombocytopoietin formation in rats]. PMID- 3015714 TI - [Protease-binding protein and its role in regulating the action of thrombin on cells]. PMID- 3015715 TI - Transitory derepression and the maintenance of conjugative plasmids. AB - It has been proposed that bacterial plasmids cannot be maintained by infectious transfer alone and that their persistence requires positive selection for plasmid borne genes. To test this hypothesis, the population dynamics of two laboratory and five naturally occurring conjugative plasmids were examined in chemostat cultures of E. coli K-12. Both laboratory plasmids and three of the five wild plasmids failed to increase in frequency when introduced at low frequencies. However, two of the naturally occurring plasmids rapidly increased in frequency, and bacteria carrying them achieved dominance in the absence of selection for known plasmid-borne genes. Three hypotheses for the invasion and persistence of these two plasmids were examined. It is concluded that although these two extrachromosomal genetic elements are repressed for conjugative pili synthesis, as a consequence of high rates of transfer during periods of transitory derepression in newly formed transconjugants, they become established and are maintained by infectious transfer alone. The implications of these observations to the theory of plasmid maintenance and the evolution of repressible conjugative pili synthesis are discussed. PMID- 3015716 TI - Interaction of DNA polymerase III gamma and beta subunits in vivo in Salmonella typhimurium. AB - We show that temperature-sensitive mutations in dnaZ, the gene for the gamma subunit of DNA polymerase III holoenzyme, can be suppressed by mutations in the dnaN gene, which encodes the beta subunit. These results support a direct physical interaction of these two subunits during polymerase assembly or function. The suppressor phenotype is also sensitive to modulation by the dnaA genotype. Since dnaA is organized in an operon with dnaN, and dnaA is a regulatory gene of this operon, we propose that the dnaA effect on suppression can best be explained by modulation of suppressor dnaN levels. PMID- 3015717 TI - Meiosis can induce recombination in rad52 mutants of Saccharomyces cerevisiae. AB - The RAD52 and RAD50 genes have previously been shown to be required for normal meiotic recombination and for various types of recombination occurring in mitotic cells. Recent evidence suggests that rad52 mutants might be defective in an intermediate recombination step; we therefore examined recombination during meiosis in several rad52 mutants at several different loci and in genetic backgrounds that yield efficient sporulation and synchronous meiosis. Similar to previous reports, spores from rad52 diploids are inviable and meiotic recombination is greatly reduced by rad52 mutations. However, intragenic recombinants were detected when cells were plated on selective media during meiosis; rad52 mutants experience induction of recombination between homologues under these special conditions. The frequencies of recombination at four loci were considerably greater than the mitotic controls; however, they were still at least 20 times lower than corresponding Rad+ strains. The prototrophs induced by meiosis in rad52 mutants were not typical meiotic recombinants because incubation in nutrient-rich medium before plating to selective medium resulted in the complete loss of recombinants. We propose that previously observed single-strand breaks that accumulate in rad52 mutants may be associated with recombinational intermediates that are resolved when cells are returned to selective mitotic media and that the meiosis-induced recombination in rad52 cells does not involve double-strand breaks. PMID- 3015718 TI - Analysis of meiosis-defective mutations in yeast by physical monitoring of recombination. AB - We have developed a method by which the extent of physical exchange of DNA molecules can be determined throughout meiosis in the yeast Saccharomyces cerevisiae. We have used this technique to analyze the effect of five meiosis defective mutations (rad6, rad50, rad52, rad57 and spo11) on the physical exchange of DNA molecules. In the same experiments, we have also measured other meiotic parameters, such as premeiotic DNA synthesis, commitment to intragenic recombination, haploidization, ascus formation, and viability. rad50 and spo11 diploids make an undetectable amount of physically recombined DNA and less than 1% of wild-type levels of viable intragenic recombinants. In contrast, diploids homozygous for rad52, rad6 or rad57 all yield significant amounts of novel restriction fragments which arise by recombination. rad57 diploids make nearly wild-type levels of the recombined restriction fragments, although they produce less than 10% of the wild-type levels of viable intragenic recombinants. rad52 strains are also capable of a significant (33%) amount of exchange of DNA molecules, but make less than 1% of wild-type levels of viable intragenic recombinants. rad6 diploids are also capable of undergoing a high level of exchange, as measured by the appearance of the recombined restriction fragment. In addition, rad6 diploids show an unusual allele- or locus-specific variability in the level of viable intragenic recombinants produced. Although rad6 diploids produce no viable spores, they are able to complete a significant amount of haploidization upon return to vegetative growth conditions. PMID- 3015719 TI - Genetical and molecular analyses of qa-2 transformants in Neurospora crassa. AB - Neurospora crassa qa-2+ transformants from five different donor DNA clones were analyzed by genetical and molecular techniques. None of the 32 transformants have the qa-2+ DNA replacing the qa-2- gene in linkage group VII. In one transformant, the qa-2+ DNA was inserted adjacent to the qa-2- gene. Thirty-one transformants have the qa-2+ inserts at sites not linked, or not closely linked, to the qa-2 locus in LG VII. Plasmid sequences were integrated along with the qa-2+ gene in 28 transformants. In the unlinked duplication-type transformants, catabolic dehydroquinase (the qa-2+ gene product) was induced at 5-100% of the wild-type induced enzyme activity, with 24 transformants in the 5-80% range. The reduced levels of enzyme activity may be due to "position effects" of sequences adjacent to the integration site either in the N. crassa genomic DNA or in the flanking plasmid (pBR322 or pBR325) sequences. Unexpected gene conversion-like events, in which a qa-2+ gene was changed to qa-2-, were observed in tetrads from intercrosses between unlinked duplication-type transformants and in selfings of such transformants. PMID- 3015720 TI - [Cloning and physical mapping of the ADE1 gene of the yeast Saccharomyces cerevisiae]. AB - ADE1 gene of Saccharomyces cerevisiae codes for the primary structure of SAICAR synthetase. Mutational changes of ADE1 gene result in the accumulation of red pigment in cells. Colour differences, thus, serve as a basis for the selection of mutants or transformants. ADE1 gene was cloned as a 4.0 kb HindIII fragment of yeast DNA in a shuttle vector by complementing the ade1 mutation in yeast. The study of ADE1 gene expression in Escherichia coli showed that the 4.0 kb fragment containing the ADE1 gene does not complement purC mutations in E. coli. However, prototrophic colonies appeared at a frequency of 10(-7)-10(-8) after incubating clones bearing the recombinant plasmid with ADE1 gene on selective media. The plasmid DNA isolated from such clones complements the purC mutation in E. coli and the ade1 mutation in S. cerevisiae. Structural analysis of the plasmid demonstrated that the cloned DNA fragment contained an additional insertion of the bacterial origin. Further restriction enzyme analysis proved the insertion to be the bacterial element IS1. Expression of the cloned ADE1 gene in S. cerevisiae is controlled by its own promoter, whereas in E. coli it is controlled by the IS1 bacterial element. PMID- 3015721 TI - [Two loci in the genome of the Pseudomonas aeruginosa transposable phage B39 which affect the integration process. II. Mapping of the pdeX and pdeY loci by restriction and heteroduplex analysis]. AB - The coordinate function of two loci - pdeX and pdeY - in the genome of a transposable phage (TP) provides the phage function pde+ (good growth on bacteria with Rms163 plasmid). When these two loci in hybrid phages originate from different TP, some of the hybrid phages have Pde- phenotype. To localize pdeX and pdeY, the structure of hybrid TP genomes with Pde+ and Pde- phenotype obtained in crosses between B39ts+ and PH132 were studied using restriction and heteroduplex analysis. On the basis of data obtained, pdeX and pdeY were mapped in 2.85-6.4 and 6.4-16 kbp regions, respectively. PMID- 3015722 TI - [Characteristics of the het mutations in the Escherichia coli chromosome causing an increase in Tn1 transposition frequency]. AB - Four mutations were studied which lead to increasing the frequency of transposon Tn1 translocation into different replicons. These mutations (het1, het2, het3 and het4) increase the frequency of Tn1 translocation 10-20-fold. The het1 mutation is recessive and has been localized in the 90-94.5 min region of the bacterial chromosome. The mutation effects Tn1 transposition in the presence of F plasmid only. As we have demonstrated recently, F-plasmid inhibits Tn1 transposition in Escherichia coli cells. The het1 mutation eliminates this inhibition. Unlike het2, het3 and het4 mutations, het1 is responsible for resistance to male phages f1, f2, MS2 and inhibition of conjugative transfer in F+ bacteria. PMID- 3015723 TI - [Inhibition of the reproduction of a herpes simplex I virus carrying mutations in the thymidine kinase and DNA polymerase genes]. AB - Sensitivity of Herpes Simplex Virus type I (HSV-I) mutants carrying genetic defect in the DNA polymerase and thymidine kinase genes to the action of some drugs was studied. TK- mutant of HSV-I was resistant to Ara-T and ACG and sensitive to PAA, Ara-A as well as to ribavirin and ADEA. PAAr mutant of HSV-I was resistant to PAA, Ara-A, ACG and sensitive to Ara-T, ribavirin and ADEA. A double mutant of HSV-I-TK-, PAAr was resistant to all drugs, except for ribavirin and ADEA. To inhibit reproduction of HSV with genetic defect, it is important using drugs of independent mode of action on the function of defective viral gene. PMID- 3015724 TI - [Mutants of ColE1 plasmid with altered expression of the colicin E1 immunity gene]. AB - Mutagenesis with Tn1 transposon was used to isolate mutants of ColE1 plasmid with inactivated gene responsible for immunity to colicin E1. Cells containing such mutants synthesized active colicin E1 and were sensitive to its action. Spontaneous and UV-induced colicin synthesis was strongly changed in the mutants, as compared to the control. Mutations occurring outside the immunity gene, including those within the structural gene for colicin E1, could also affect the immunity gene expression. PMID- 3015725 TI - [Coupling and rec-independence of the processes of replication and transposition of Pseudomonas aeruginosa phage D3112. The effect of the phage genes controlling the replication of DNA of D3112]. AB - D3112 phage was shown to replicate via the process of coupled replication- transposition: the phage DNA is not excised from the chromosome after prophage induction and new phage copies insert into many different sites. The transposition is controlled by two D3112 early genes--A (mapped in the 1.5-3 kbp region) and B (3-4.5 kbp), and requires intact attL site (involvement of the phage right end attR not studied). D3112 is capable to transpose RP4 plasmid into the chromosome; both the D3112 and RP4 transpositions are rec-independent. The product of the early C gene which is not required for D3112 transposition has pleiotropic effect on the development of D3112 and is necessary for the process of D3112 DNA excision from the chromosome, for cell lysis as well as for mature phage production. We suggest that this gene is responsible for positive regulation of D3112 late genes expression, similar to the C gene of Mu phage or Q gene of lambda. Mutations in four D3112 late genes ts25, ts35, ts73 and ts110 do not affect transposition or excision processes. No detectable (less than 0.02 copies per cell) amount of linear or circular D3112 DNA is formed during the replication--transposition. Hence, in the course of replication and transposition processes D3112 genome has its ends permanently bound covalently to the chromosome. The excision of the D3112 DNA takes place at late stages. PMID- 3015726 TI - [Mutagenic effect of the SV40 oncogene: induction of resistance to 6 mercaptopurine and serum independence]. AB - The mutagenic and transforming activity of SV40 DNA fragment, corresponding to its oncogene (the gene for large T antigen) was studied in Chinese hamster cells. After expression time of 3 to 4 days, the oncogene induced mutations of resistance to 6-mercaptopurine (6MP), while the DNA encoding the SV40 late genes, as well as DNA of Chinese hamster cells, were devoid of mutagenic activity. The value of induction ranged from 10(-4) to 10(-5). After the same expression time, the oncogene induced a typical character of oncogenic transformation - independence of serum growth factors (ser+). The value of induction of ser+ variants was somewhat higher than for resistance mutations. The study of 12 clones induced by the oncogene has shown the ser+ character to be hereditary, the expression of viral oncogene being not necessary for its maintenance. The data obtained support the hypothesis in favour of the participation of mutations of cellular genes in viral carcinogenesis. PMID- 3015727 TI - Oligodeoxynucleotide-directed mutagenesis using the yeast transformation system. AB - In this report we describe a highly efficient method for site-specific mutagenesis using the yeast transformation system. The method is based on the observation that Saccharomyces cerevisiae can be transformed at high frequency with single-stranded circular DNA vectors [Singh et al., Gene 20 (1982) 441-449]. The model system studied was the TRP1 gene of S. cerevisiae cloned into a derivative of the phage M13mp9 vector containing the yeast URA3 gene. ARS1, located adjacent to the TRP1 gene, allows the plasmid to replicate autonomously in yeast. Synthetic 5'P-oligodeoxynucleotides, 19 and 35 nucleotides (nt) in length, designed to produce an A----T transversion mutation within the TRP1 gene, were annealed to ss DNA of the M13 vector at a molar ratio of 30:1 and directly transformed into yeast. The intended single nt mutation was obtained at frequencies of 24 and 43%, respectively. The latter approaches the theoretical limit of 50%. In the absence of the 5'-terminal phosphate, both the transformation frequency and the efficiency of mutagenesis by the synthetic oligodeoxynucleotide (oligo) were decreased by 2-4 fold. This procedure completely obviates the need for any enzymatic manipulations in vitro after forming the heteroduplex with the oligo primer containing the desired mutation. For yeast genes, direct phenotypic selection is possible in the recipient strain. PMID- 3015728 TI - Structural and functional analysis of the goat epsilon-globin genes. AB - Since none of the vertebrate beta-globin loci studied to date has more than two functional embryonic beta-like globin genes, it would be unique if all six goat embryonic beta-globin genes were required for its survival. In this study we have asked whether all six embryonic genes in the goat are functional. This question has been addressed by examining the transient expression of these genes in HeLa cells and correlating these results with the sequence information obtained to date. Our studies show that only epsilon I and epsilon II are functional while the remaining four epsilon-globin genes are nonfunctional, i.e., pseudogenes. Interestingly, the two active epsilon-globin genes are located at the 5' end of the locus. While this unusual inactivation pattern may be the result of chance, it could also have resulted because the two duplication events, of the ancestral gene set epsilon-epsilon-psi beta-beta, did not include distally located regulatory region(s) essential for epsilon-globin gene expression. Once separated from the 5'-regulatory sequences the remaining four embryonic genes (epsilon III, epsilon IV, epsilon V and epsilon VI) accumulated mutations and became pseudogenes. PMID- 3015729 TI - Cloning and sequencing of mRNAs coding for two adult alpha globin chains of the salamander Pleurodeles waltlii. AB - A cDNA library of erythrocyte mRNAs was established from immature red blood cells of the adult amphibian, Pleurodeles waltlii (urodele; salamander). The cDNA clones corresponding to the four adult globin chains were first identified and characterized by positive selection and the cDNAs derived from the two (major and minor) alpha-globin chains sequenced. The sequences presented contain both the complete 3'-noncoding region and the coding region of both chains, with the exception of the first nine codons of the minor alpha-chain, and a portion of the 5'-noncoding region of the major chain. The amino acid sequences of the encoded alpha-globin polypeptides have been deduced and compared with those of Xenopus laevis and of man. These comparative studies suggest that the alpha-globins of Pleurodeles waltlii and Xenopus laevis may have diverged from a common ancestral gene at the time when mammalian and amphibian lines diverged, and that they then evolved separately. Duplication of the alpha-gene, which is responsible for the polypeptide heterogeneity, appears to have occurred earlier in Pleurodeles waltlii than in Xenopus laevis. PMID- 3015730 TI - A rapid, efficient method for isolating DNA from yeast. AB - A method is described for the purification of chromosomal and plasmid DNA from the yeast Saccharomyces cerevisiae. This method is rapid, gives 75% of theoretical yield, and produces DNA that can be cut with restriction endonucleases. Yeast cells are treated with zymolyase, and the resulting spheroplasts are lysed in the presence of the chaotropic agent guanidine hydrochloride. After a brief ethanol precipitation, protein is removed by treatment with proteinase K followed by phenol-chloroform extraction. After ethanol precipitation, the DNA is sufficiently pure for restriction analysis or for the transformation of Escherichia coli. PMID- 3015732 TI - Molecular cloning and genetic mapping of the DNA topoisomerase II gene of Saccharomyces cerevisiae. AB - The structural gene for DNA topoisomerase II from the yeast Saccharomyces cerevisiae has been cloned. The clones were selected from a YEp13 plasmid bank of yeast DNA by complementing a temperature-sensitive mutation (top2-1) in the topoisomerase II gene, TOP2. Chromosomal integrants of the clone were derived by homologous recombination in strains lacking the 2 mu circle plasmid. Genetic analysis of these integrants indicates that we have cloned the TOP2 gene and not an extragenic suppressor. A YEp13-TOP2 hybrid plasmid integrant was used to localize the TOP2 gene to the left arm of chromosome XIV by the 2 mu circle directed marker loss method. Results from standard meiotic mapping experiments indicate that TOP2 is about 16 centi-Morgans to the centromere proximal side of MET4. Northern blot analysis of TOP2 RNA isolated from a wild-type strain and from an rna2 mutant shows the RNA to be 4.5 kb long in both cases, thus indicating that the TOP2 gene has no large introns. PMID- 3015731 TI - Construction and uses of a new transposable element whose insertion is able to produce gene fusions with the neomycin-phosphotransferase-coding region of Tn903. AB - We describe the construction of a transposable element derived from the Mu phage that upon insertion is able to create a gene fusion between the region of Tn903 coding for neomycin phosphotransferase (NPT I), which confers resistance to aminoglycosides including kanamycin (KmR), neomycin and G418, and the control elements of the gene where the insertion occurs. A chloramphenicol (Cm) transacetylase gene (cat) that confers resistance to Cm is present in the transposon so that transposition events can be monitored even when no active fusions with the nptI coding region occur. The transposase gene is deleted and, therefore, this transposon is perfectly stable upon insertion. The properties of this new transposable element were studied by obtaining gene fusions between the Escherichia coli L-arabinose operon and 'nptI gene. In some of them the KmR phenotype is induced by arabinose. Insertions of this element in cloned fragments of the T-DNA region of Agrobacterium rhizogenes were also isolated. Some of them confer a KmR phenotype upon its E. coli carriers, which indicates that portions of the T-DNA are expressed in these cells. PMID- 3015734 TI - Spontaneous insertions into cosmid vector: a warning. AB - During the course of characterization of a human genomic library we found that some of the selected pNNL cosmid clones carried only very short inserts. In addition, several clones that did contain full-length inserts were found to be unstable and generated repeated deletions of various portions of their inserts. Moreover, all the clones examined displayed rearrangements in the vector portions. The rearranged clones that were characterized by restriction mapping were found to have deletions starting between ClaI and HindIII in the region of the cos segment of the pNNL vector used in the construction of the library. We determined the nucleotide sequence of the 1300-bp EcoRI-PvuII segment located in the cos region, and by the computer search we found that it contains Escherichia coli insertion elements IS1. PMID- 3015733 TI - The nucleotide sequence of the malT gene encoding the positive regulator of the Escherichia coli maltose regulon. AB - The molecular organization of the malT region of the Escherichia coli K-12 chromosome has been elucidated by nucleotide sequence studies. A single open reading frame of 901 codons comprises the malT gene which is separated by a repetitive extragenic palindromic unit from an unidentified gene, orfX, divergently oriented with respect to malT. The predicted Mr of the MalT protein is 102988, making it the largest transcriptional regulatory protein yet described in E. coli. By deleting in vitro the 3'-end of the gene or constructing malT-lacZ gene fusions, it was found that the integrity of the C-terminus of MalT is indispensable for the activity of the protein. Furthermore, it was found that truncated MalT proteins lacking up to 300 amino acids at the C-terminus blocked the activity of the wild type protein. No sequence homology can be found either with the other activators known in E. coli or with the other proteins of the maltose regulon. PMID- 3015735 TI - Cloning of a streptomycin-production gene directing synthesis of N-methyl-L glucosamine. AB - A Streptomyces bikiniensis DNA fragment complementing a deficiency in streptomycin (Sm) production was cloned on the plasmid vector pIJ385. Host strain S. bikiniensis SD 1 used as the recipient in cloning displayed deficiency in biosynthesis of N-methyl-L-glucosamine, one of the moieties of Sm. The cloned fragment on the multicopy plasmid pIJ385 conferred sevenfold increase in Sm production in comparison with the wild-type parental strain. By subcloning, the region complementing the Sm deficiency of SD 1 was narrowed to a 3.0-kb fragment. PMID- 3015736 TI - Genomic organization and nucleotide sequences of two corn histone H4 genes. AB - The sea urchin histone H4 gene has been used as a probe to clone two corn histone H4 genes from a lambda gtWES X lambda B corn genomic library. The nucleotide (nt) sequences of both genes showed that the encoded amino acid sequences were identical to that of the H4 of pea and one variant of wheat. The nt sequences of the coding regions showed 92% homology. 5'- and 3'-flanking regions do not show extensive nt sequence analogies. Southern blotting of the EcoRI digested genomic DNA suggests the existence of multiple H4 genes dispersed throughout the genome. PMID- 3015737 TI - Nucleotide sequence of gene F of Bacillus phage Nf. AB - The nucleotide sequence of Bacillus phage Nf gene F has been determined. The deduced amino acid sequence of gpF is very similar to that of gp4, the transcriptional activator of phage phi 29. Both proteins contain the consensus structure that is conserved for the DNA-protein interacting domain of DNA-binding proteins such as the repressor, Cro and CII proteins of phage lambda. PMID- 3015738 TI - Expression of the E2 open reading frame of papilloma viruses BPV1 and HPV6b in Escherichia coli. AB - A new expression vector, pRA10, has been constructed for the expression of the open reading frames (ORF) of bovine (B) and human (H) papilloma viruses (PV). This vector is a derivative of pAJ pi and contains 15 restriction sites proximal to the lambda PL promoter, offering considerable versatility for insertion of different ORFs. This vector was used specifically to express the E2 ORF gene products from BPV1 and HPV6b at high level in Escherichia coli. The genuine nature of these proteins was demonstrated by restriction map analysis of expression vector plasmids to insure proper orientation, nucleotide sequence analysis to demonstrate in-frame insertion, E2 ORF protein production by expression-vector plasmids, and not by appropriate controls and, immunoprecipitation of E2 proteins by antibody specific for a common N-terminal sequence derived from the expression vector. PMID- 3015739 TI - Positive-selection vectors utilizing lethality of the EcoRI endonuclease. AB - The construction and use of a series of positive-selection vectors are described. These plasmids encode EcoRI endonuclease, the synthesis of which is under the control of the lacUV5 promoter. The pKG2 plasmid encodes a wild-type EcoRI endonuclease. In the absence of EcoRI methylase, the endonuclease is lethal. Cloning into any of the unique restriction sites within the endonuclease-coding gene allows survival of the transformed EcoRI-methylase-less host. The pKGW and pKGS plasmids encode an altered EcoRI endonuclease which, when repressed in a lacIQ host, allows survival in the absence of the methylase. Induction with IPTG, however, results in cell death as a result of high-level EcoRI synthesis. Cloning into any of the unique restriction sites within the EcoRI gene of pKGW or pKGS allows survival of derepressed transformed cells. These vectors strongly select for cloning events which inactivate the endonuclease gene. PMID- 3015740 TI - Heterogeneity in the ribosomal family of the yeast Yarrowia lipolytica: genomic organization and segregation studies. AB - The cloned r-DNA units of Yarrowia lipolytica [Van Heerikhuizen et al., 39 (1985) 213-222] and their restriction fragments have been used to probe blots of genomic DNA of this yeast. Wild-type and laboratory strains were shown to contain two-to five types of repeated units, each strain displaying a specific pattern. By comparing their restriction patterns, we could localize the differences between units within their spacer region. Tetrad analysis strongly suggested a clustered organization of each type of repeat as well as the occurrence of meiotic exchanges within the r-DNA family. Chromosome loss was induced by benomyl and allowed to map several r-DNA clusters on the same chromosome. All those results indicate that the Y. lipolytica r-DNA gene family is quite different from other yeasts. PMID- 3015741 TI - Pulsed field gradient electrophoresis of DNA digested in agarose allows the sizing of the large duplication unit of a surface antigen gene in trypanosomes. AB - Intact chromosome-sized DNA molecules from eukaryotes may be prepared by performing lysis and enzymic deproteinization on cells embedded in agarose [Schwartz and Cantor, Cell 37 (1984), 67-75]. Here we show that DNA prepared by this method may be cut with restriction enzymes, or modified with site-specific methylases and cut by DpnI. As the DNA remains incorporated in the gel matrix, shear degradation of large fragments is avoided. The fragments can then be sized by conventional or pulsed field gradient gel electrophoresis. Phage lambda genomic oligomers are used as size markers, allowing the estimation of fragment sizes up to about 1200 kb. We apply these techniques to show that activation of the telomeric gene encoding variant surface antigen 1.3 in Trypanosoma brucei strain 427, involves the duplication of a DNA segment that starts between 29 and 42 kb upstream of the gene and to assign a chromosomal fragment into which the duplicated 1.3 gene may have transposed. PMID- 3015742 TI - Polypeptides specified by the mercuric resistance (mer) operon of plasmid R100. AB - Overlapping deletion mutations were constructed in chimaeric plasmids carrying the mer operon of plasmid R100. Polypeptides specified by the mutant plasmids in Escherichia coli minicells correlated with the mer genes as follows: merT, 17- and 16-kDa polypeptides; merP, 9.8- and 9.5-kDa polypeptides; merC, a 14-kDa polypeptide; merA, 65- and 62-kDa polypeptides. The products of the merR and merD genes were not identified. The revised nomenclature of the mer genes is explained. PMID- 3015743 TI - Site-specific DNA splicing: a general procedure for the creation of a restriction site at a predetermined position in a DNA sequence. AB - A method is described for creating any of a wide array of restriction sites at a predetermined position in a known DNA sequence. The method utilizes the exonuclease activity of BAL 31 and a specially designed bifunctional oligodeoxynucleotide linker. The desired restriction site is generated when the linker is ligated to those BAL 31-digested DNA fragments which end with the target sequence. The proper ligation product is then identified by a highly specific hybridization procedure. The method is versatile and specific and is especially useful in the isolation of functional elements of a gene. PMID- 3015744 TI - An amplified genome that may have resulted from recombination within bidirectionally replicating DNA. AB - Temperature shift-down of permissive mouse cells transformed by a temperature sensitive (ts) polyomavirus (Py) genome has been shown to induce the accumulation of free copies of the viral DNA. We report here on an unusual product from such induction. The structure of this product is that expected from the occurrence of recombination between growing points in a bidirectionally replicating Py-mouse DNA molecule. This observation may be relevant to the mechanism of gene amplification in mammalian cells. PMID- 3015745 TI - Type II diabetes: current nutrition management concepts. AB - Benefits of improved glycemic control and lowered blood lipids continue as patients follow high-fiber, high-carbohydrate diets over time. In our experience, body weight, insulin requirements, glycemic control, and serum lipids are well managed by such diets for up to 10 years of follow-up. Besides minimizing the need for drugs, we found high-fiber, high-carbohydrate diets were well tolerated by diabetic patients. Other family members can consume the diet as well, since it closely approximates the dietary recommendations of the American the dietary recommendations of the American Heart Association and the National Cancer Institute. PMID- 3015746 TI - Brain water and aging. AB - Brain water content and Na/K-ATPase activity have been compared in two groups of rats aged 60 +/- 5 days (group I) and 630 +/- 10 days (group II). In group II control animals, brain water content (p less than 0.001) and Na/K-ATPase activity (p less than 0.02) were found to be significantly reduced. Following a cryogenic lesion of the brain, the increase in water content was larger in group I (not significant), whereas impairment of Na/K-ATPase activity was much more pronounced in group II (p less than 0.001). PMID- 3015747 TI - [Pregnancy and labor in a woman with Klippel-Trenaunay syndrome]. PMID- 3015748 TI - Effects of a new, concentrated wheat fibre preparation on intestinal transit, deoxycholic acid metabolism and the composition of bile. AB - When the cholesterol saturation index of bile is reduced by wheat bran there is generally a fall in the deoxycholic acid content of bile. As the same effects occur with senna, bran might act on bile simply via its accelerating effect on colonic transit. We have studied the effects of a new, concentrated, wheat fibre preparation (Testa Triticum Tricum, Trifyba, which is 80% dietary fibre) upon bile composition, deoxycholic acid metabolism and intestinal transit time, and have assessed whether these effects are related. Twenty constipated volunteers were prescribed Testa Triticum Tricum in doses (10-32 g/day) sufficient to relieve their symptoms for at least six weeks. Before and at the end of this period, duodenal bile was sampled to enable measurement of deoxycholic acid pool (by isotope dilution), total bile acid pool, bile acid composition and cholesterol saturation index. Whole gut transit time fell from 120 +/- SD35 to 68 +/- 35 hours. At the same time, biliary % deoxycholic acid fell from 26.6 +/- 12.0 to 23.0 +/- 11.8 (p = 0.002), the total bile acid pool expanded from 2.36 +/ 0.88 to 2.75 +/- 0.90 g (p = 0.008) and cholesterol saturation index fell from 1.13 +/- 0.32 to 1.07 +/- 0.29 (p = 0.04). In subjects with initial cholesterol saturation index over 1.0 (n = 12), it fell from 1.33 +/- 0.25 to 1.22 +/- 0.21 (p = 0.008). There was no significant correlation between change in saturation index and change in % deoxycholic acid or deoxycholic acid pool, nor between any of these parameters and change in transit time. Testa Triticum Tricum reduces the cholesterol saturation index of supersaturated bile but this action appears to be independent of its effect on colonic transit and of changes in deoxycholic acid metabolism. PMID- 3015749 TI - Latent pulmonary involvement in Crohn's disease: biological, functional, bronchoalveolar lavage and scintigraphic studies. AB - We have investigated the following pulmonary related parameters in 22 patients with Crohn's disease who were free of clinical pulmonary symptoms and had normal chest roentgenograms and in 25 controls: serum angiotensin converting enzyme, pulmonary function tests, bronchoalveolar lavage (lymphocyte count and subpopulations, macrophage viability and superoxide anion release by macrophages) and pulmonary scannings. Serum angiotensin converting enzyme was lower in Crohn's disease (14.1 +/- 5.1) than in controls (25.2 +/- 4.7) (p less than 0.001). Twelve of 22 Crohn's disease (54%) had a bronchoalveolar lavage lymphocytosis (greater than 18% alveolar lymphocytes). Bronchoalveolar lavage lymphocytes subpopulations were quite variable. Twelve of 17 Crohn's disease (71%) had an increase spontaneous and/or stimulated superoxide anion production by alveolar macrophages. Six of 12 Crohn's disease (50%) had an increase physiologic dead space in the upper part of their lung against one of 11 controls (9%). These data suggest that most patients with Crohn's disease have a latent pulmonary involvement. PMID- 3015751 TI - [Infantile myoclonic encephalopathy]. PMID- 3015750 TI - [Effect of enalapril on blood pressure and renal function in mild hypertension]. PMID- 3015752 TI - Histochemical reactions of arylosulphatases and beta-glucuronidase in the intestinal epithelium in rats under conditions of changed ACTH and steroid hormones level. PMID- 3015753 TI - [Inhibitory effect of nafamostat mesilate (FUT-175) on O2- production in rat polymorphonuclear leucocytes]. AB - Effect of nafamostat mesilate (FUT-175), a serine protease inhibitor, having anti inflammatory effects was studied on superoxide (O2-) production in rat polymorphonuclear leucocytes (PMN) and compared with those of other serine protease inhibitors and typical anti-inflammatory agents. 1) O2- productions in rat PMN stimulated with concanavalin A (Con A) and cytochalasin B (Cyt B) were too weak to observe. With NADH, however, strong O2- production was induced by Con A and Cyt B. 2) FUT-175 at 10(-6) and 10(-5) M inhibited O2- production in rat PMN induced by Con A and Cyt B with NADH in a concentration-dependent manner. 3) The serine protease inhibitor L-tosylamido-2-phenylethyl-chloromethyl ketone (TPCK) and soybean trypsin inhibitor (SBTI) inhibited O2- production at 10(-5) M and 10(-4) M, respectively, while aprotinin, chymostatin and leupeptin did not. 4) Neither indomethacin nor dexamethasone, typical anti-inflammatory agents, inhibited O2- production. Mepacrine, a phospholipase A2 inhibitor, strongly inhibited it. 5) O2- production in PMN prepared from the rat administered FUT 175, 200 mg/kg, p.o., was significantly decreased in comparison with that of the control rat. 6) FUT-175 had no effect on O2- production by hypoxanthine-xanthine oxidase. These results showed FUT-175 had a strong inhibitory effect on O2- production in rat PMN which other typical anti-inflammatory agents did not have. PMID- 3015754 TI - [Primary malignant fibrous histiocytoma of the maxilla and mandible. Clinical aspects and pathology--2 case reports]. PMID- 3015755 TI - [Importance of congenital melanotic neuroectodermal tumors in the area of the jaw face based on their clinical behavior and structural and histochemical organization]. PMID- 3015756 TI - Enzymes of adenosine metabolism of sheep adipose tissue: changes in activity with season, pregnancy and lactation. AB - Activities of enzymes involved in the synthesis and degradation of adenosine have been measured in samples of adipose tissue from unmated sheep at various times of the year between January and October and from pregnant and lactating sheep. 5' Nucleotidase activity increased during the spring in unmated sheep but this increase was suppressed in lactating sheep. Neither adenosine kinase nor adenosine deaminase activities varied significantly between January and October in unmated sheep. Lactation resulted in a rise in adenosine deaminase activity and a small decrease in adenosine kinase activity. Pregnancy had no obvious effect on the activities of any of the three enzymes noted above. Changes in adipocyte mean cell volume and number per g tissue and the concentrations of DNA and protein of the tissue are also described. Results are discussed in relation to changes in the capacity for lipid synthesis and mobilization which occur in response to season, pregnancy and lactation in sheep. PMID- 3015757 TI - A possible competition between 5'-monodeiodination of thyroxine and the respiratory burst in human granulocytes. AB - Thyroxine (T4) pretreatment of A 23187-stimulated human granulocytes in 10(-5) 10(-6) M concentration range inhibited the superoxide anion production of these cells. T4 increased the level of oxidized form of glutathione, whereas the intracellular level of the reduced form decreased. A similar alteration in the ratio of the oxidized to reduced forms of glutathione was detected in granulocytes during yeast cell phagocytosis. In addition, conversion of T4 to triiodothyronine (T3) was also inhibited during phagocytosis. A possible competition between 5'-monodeiodination of T4 and the oxidative burst of human granulocytes is discussed. PMID- 3015758 TI - Serum calcitonin levels in hepatocellular carcinoma and other liver diseases. PMID- 3015759 TI - Comparison between polyclonal and first and second generation monoclonal radioimmunoassays in the detection of hepatitis B surface antigen in patients with hepatocellular carcinoma. AB - Serum from 221 black patients with hepatocellular carcinoma was tested for HBsAg using a polyclonal radioimmunoassay, and first and second generation monoclonal radioimmunoassays designated M1-RIA and M2-RIA. These monoclonal radioimmunoassays have a lower limit of detection of about 55 and 15 pg of HBsAg associated epitopes per ml of serum. Correlations were made with other hepatitis B virus serologic markers such as anti-HBc and anti-HBs, using polyclonal radioimmunoassays. We found 47.5% (105/221) of the patients were HBsAg positive by conventional polyclonal radioimmunoassay; all of these patients were also reactive by monoclonal radioimmunoassay. Of the 116 polyclonal radioimmunoassay negative patients, 4 (3.4%) were reactive by M1-RIA. These four patients were all positive for anti-HBc and/or anti-HBs antibodies. There were 106 of 112 patients negative for HBsAg by polyclonal radioimmunoassay and M1-RIA available for testing by the M2-RIA; 11 (10.4%) were found to be positive only by this test. Thus, with the use of M1- and M2-RIAs, the number of hepatocellular carcinoma patients negative for HBsAg by polyclonal radioimmunoassay was reduced by 14% in this population. More importantly, of the 11 M2-RIA-positive patients, six demonstrated anti-HBc and/or anti-HBs antibodies whereas the remaining five had no serologic evidence of recent or past hepatitis B virus exposure. Finally, of the 21 patients in this series with no markers of hepatitis B virus infection using polyclonal radioimmunoassay and M1-RIA, five (24%) were reactive for HBsAg associated epitopes by M2-RIA.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3015760 TI - The fluidity of liver plasma membranes from patients with different types of liver injury. AB - The physical properties of cell membranes play an important role in cellular homeostasis. Previous studies with animal models have shown that the hepatocyte membrane fluidity has important effects in cellular function. In order to further investigate the relationship between physical properties of membranes and liver disease, the present study was performed. Twenty-nine patients with different degrees of liver injury and 10 control individuals were studied. A lower membrane fluidity--as assessed by diphenylhexatriene fluorescence polarization--was observed in membrane preparations from patients, as compared to controls. These alterations are positively correlated with the severity of liver damage and could be accounted for by a higher cholesterol content of patients' membrane preparations. PMID- 3015761 TI - Primitive neuroectodermal tumour of the central nervous system associated with malignant rhabdoid tumour of the kidney: report of a case. AB - Malignant rhabdoid tumour of the kidney is a recently reported tumour presenting in young children. Irrespective of stage and despite intensive chemotherapy these tumours have a poor prognosis, with death usually occurring within a matter of months. A recent report has shown the association of second embryonal tumours of the central nervous system occurring in patients with the renal tumour; most of these second tumours have occurred in the posterior fossa. We report here an infant who presented with a mass in the right groin, showing features of a poorly differentiated sarcoma, possibly rhabdomyosarcoma. Further investigations revealed a tumour in the lower pole of the right kidney which was subsequently shown to be a malignant rhabdoid tumour. The child was given chemotherapy but re presented at 10 months of age with hydrocephalus, irritability and spasms leading to death. At autopsy a large tumour was found filling the right lateral and third ventricles; histology showed a primitive neuroectodermal tumour with focal astrocytic differentiation. Residual rhabdoid tumour was restricted to a few para aortic lymph nodes and focal lymphatic micrometastases in lungs. The association of two embryonal neoplasms of possible similar histogenesis is discussed. PMID- 3015762 TI - NIMH to launch major campaign on recognition and treatment of depression. PMID- 3015763 TI - Using electroconvulsive therapy for patients with neurological disease. AB - In the United States electroconvulsive therapy (ECT) is not commonly used with patients who have conditions affecting the structure or function of the brain. Many clinicians may be unaware, therefore, that ECT has been used safely to treat patients with combined major depression and central nervous system disorders; patients with organic mental syndromes, particularly delirium; and patients who have psychiatric disorders that mimic or are distorted by brain disease. The author discusses the successful use of ECT with such patients as well as potential dangers of the treatment through a review of worldwide experience with ECT and presentation of case examples. He concludes by suggesting possible mechanisms through which ECT may benefit both depression and organic mental syndromes. PMID- 3015765 TI - Sclerosing adenosis in the breast of a man with pulmonary oat cell carcinoma: report of a case. AB - A case of sclerosing adenosis of the male breast is reported. This very unusual lesion was found at autopsy in a man with pulmonary oat cell carcinoma. In females, sclerosing adenosis is a well-characterized entity. In males, however, it does not normally occur because of the physiologic lack of lobular development. The possible pathomechanism of this lesion is briefly discussed, with an emphasis on presumed lobular stimulation by tumor-elaborated ectopic hormone. PMID- 3015764 TI - Inclusion body myositis: a chronic persistent mumps myositis? AB - Among the generalized chronic idiopathic inflammatory myopathies, inclusion body myositis (IBM) has emerged as a clinico-pathologic variant during the past two decades. It occurs primarily in elderly persons (in approximately the sixth decade of life), but young adults (in approximately the second decade of life) may also be affected. Slowly progressive weakness of distal as well as proximal muscle groups in IBM is usually not associated with skin rash, malignancy or collagen-vascular disease, and is refractory to treatment with steroids or other immunosuppressants. Exceptions to each of these general rules have been found. Muscle biopsy and electromyography may suggest a neurogenic process mixed with myopathic features. Rimmed vacuoles with basophilic granules in cryostat sections stained with hematoxylin-eosin are strongly suggestive of IBM if accompanied by the histopathologic triad of polymyositis. The presence of eosinophilic intranuclear or cytoplasmic inclusions in affected myofibers is further suggestive of IBM. The ultimate diagnosis, however, depends on ultrastructural demonstration of characteristic microtubular filaments resembling the nucleocapsids of the paramyxovirus group. Recent reports of immunostaining of the inclusions for mumps virus antigen strongly suggest a chronic persistent mumps virus infection as the cause of IBM. IBM is considered to be pathologically related to both distal myopathy (DM) and oculopharyngeal muscular dystrophy (OPMD). PMID- 3015766 TI - The human ribosomal RNA genes: structure and organization of the complete repeating unit. AB - The complete repeating unit of the human ribosomal RNA gene has been reconstructed by the cloning of approximately 27 kilobases (kb) of non transcribed spacer. The structure of this tandemly repeated gene can now be studied in its entirety. We report the analysis of spacer DNA by molecular cloning and its organization in the genome by Southern transfer analysis. These studies reveal both length and sequence variation of the spacer. Sequence variations are distributed throughout the spacer while the length variations exist near the 5' end of the transcript and just beyond the 3' end. The human spacer shares extensive homology with primates but little with other mammals. Within the primates the degree of homology reflects the rapid evolutionary changes characteristic of the primate group. PMID- 3015767 TI - Different zeta globin gene deletions among black Americans. AB - Four types of chromosomes with a deletion between the human embryonic zeta and psi zeta globin genes were identified among 2.8% of 321 Black Americans from Georgia. Two deletions of approximately 11 kb which differed by about 300 bp occurred on chromosomes with or without a polymorphic Xba I site 5' to the zeta globin gene [(X+) or (X-)]. The deletions are identifiable in Xba I digests of genomic DNA using an alpha or a zeta globin gene probe which yield fragments of 23 kb from (X+)-zeta alpha alpha chromosomes or 27 kb from (X-)-zeta alpha alpha chromosomes. Digestion with other enzymes and probing with both alpha and zeta probes gave fragments typical of the two zeta globin gene deletions previously identified in Polynesians. Among Black Americans, these zeta globin gene deletions have been found in combination with alpha globin gene deletions in trans but not in cis. Homozygotes have not been found. Hematologic data on carriers of the zeta globin gene deletions in association with Hb AS, SS, and SC suggest that these deletions have no effect on the function of the adult alpha globin genes. PMID- 3015768 TI - Direct regional assignment of the gene for vitamin D binding protein (Gc globulin) to human chromosome 4q11-q13 and identification of an associated DNA polymorphism. AB - Using a characterized human vitamin D binding protein (DBP) cDNA probe and a panel of rodent X human somatic cell hybrids, we established the chromosomal location of the structural gene for DBP on human chromosome 4. In situ hybridization of 3H-labeled DBP cDNA to human metaphase chromosomes confirmed this assignment and allowed regional localization to bands 4q11-4q13. A restriction fragment length polymorphism associated with the DBP gene that should prove useful in future linkage studies was identified with the enzyme BamHI. PMID- 3015769 TI - Haplotypes of the human apoprotein AI-CIII-AIV gene cluster in coronary atherosclerosis. AB - Restriction fragment length polymorphisms of the apoprotein AI-CIII-AIV gene cluster (on the long arm of chromosome 11) were investigated in a group of Caucasian survivors of myocardial infarction, using genomic hybridisation analysis. Four common haplotypes were identified at this locus, M1P1S1, M2P1S1, M1P2S1, and M2P1S2; where M1 and M2 are the common and uncommon alleles defined using the restriction enzyme MspI, P1 and P2 are the common and uncommon alleles defined by the enzyme PstI, and S1 and S2 are the common and uncommon alleles defined by the enzyme SstI. Seven genotype combinations were observed of approximate frequencies; a/a 0.70 (33/47), a/d 0.15 (7/47), a/b 0.04 (2/47), d/d 0.04 (2/47), a/c 0.02 (1/47), b/c 0.02 (1/47), and c/d 0.02 (1/47). In contrast the corresponding values for normotriglyceridaemic Caucasian controls, without a personal or family history of atherosclerotic heart disease were; 0.83 (40/48), 0.02 (1/48), 0.06 (3/48), 0, 0.04 (2/48), 0.04 (2/48), and 0. The relative incidence of the d haplotype, characterised by the presence of a cleavage site for the enzyme SstI in the fourth exon of the ApoCIII gene, was significantly higher in the patient group (P less than 0.01). However, because of the tight linkage between the polymorphic loci studied, it was not possible to identify haplotypes associated with any greater risk of premature atherosclerosis than when the SstI polymorphism was considered in isolation. PMID- 3015770 TI - Mapping of human X-linked hypophosphataemic rickets by multilocus linkage analysis. AB - Eleven families with X-linked dominant hypophosphataemic rickets (HPDR) have been typed for a series of X chromosome markers. Linkage with probe 99.6 (DXS41) was demonstrated with a peak lod score of 4.82 at 10% recombination. Multilocus linkage analysis showed that HPDR maps distal to 99.6; this probe has previously been located at Xp22.31-p21.3 by in situ hybridisation. In the mouse hypophosphataemia (Hyp) maps to the distal part of the X chromosome; our location in man is consistent with a scheme which relates the mouse and human X chromosomes by two rearrangements. No marker has yet been found which shows no recombination with HPDR. PMID- 3015771 TI - X-linked dominant hypophosphatemia is closely linked to DNA markers DXS41 and DXS43 at Xp22. AB - Two families with X-linked dominant hypophosphatemia (McKusick No. *30780) were investigated for linkage of the disease locus with several marker genes defined by cloned, single-copy DNA sequences derived from defined regions of the X chromosome. Close linkage was found with DNA markers DXS41 (p99-6) and DXS43 (pD2) at Xp22, suggesting a location of the HPDR gene on the distal short arm of the X chromosome. PMID- 3015772 TI - Factors affecting seropositivity to human T cell lymphotropic virus type III (HTLV-III) or lymphadenopathy associated virus (LAV) and progression of disease in sexual partners of patients with AIDS. AB - Fifty four sexual partners of homosexual men with the acquired immune deficiency syndrome (AIDS) were studied, of whom 32 were seropositive and 22 seronegative for human T cell lymphotropic virus type III or lymphadenopathy virus (HTLV III/LAV) antibody, which showed that repeated exposure by anal intercourse does not necessarily lead to seroconversion. Seropositivity to HTLV-III/LAV was not associated with the absolute number of sexual partners, receptive anal intercourse, or the use of recreational drugs, but was associated with a history of other sexually transmitted diseases (STDs), particularly in the year preceding the patient's initial examination. Acquisition of an STD after the date of last sexual contact with a person with AIDS was strongly associated (p less than 0.001) with the development of persistent generalised lymphadenopathy (PGL). Concurrent or recent STDs would seem to be an important cofactor in developing antibody to HTLV-III/LAV and in the progression of infection from a person being asymptomatic to having PGL. PMID- 3015773 TI - Screening to detect asymptomatic shedding of herpes simplex virus (HSV) in women with recurrent genital HSV infection. AB - To investigate the asymptomatic shedding of herpes simplex virus (HSV) from women with recurrent genital herpes infection, and to assess whether inapparent shedding could occur, eight such women were examined thrice weekly for one month. At each visit colposcopy was performed and multiple sites sampled for HSV. During the study four women had no recurrence of HSV infection, but four had at least one positive viral culture. One of these patients was asymptomatically shedding HSV on nine of her 11 clinic visits. Two episodes of urethral shedding were detected. In this group of patients the presence of inguinal lymphadenopathy was appreciably associated with the isolation of HSV from the urogenital tract. PMID- 3015774 TI - Antibodies to cytomegalovirus in heterosexual and homosexual men in Cardiff. PMID- 3015775 TI - Purulent penile ulcers of patients in Singapore. AB - In 80 patients with painful purulent penile ulcers who attended the outpatient service of Middle Road Hospital in Singapore, Haemophilus ducreyi was isolated from 18 (22%), herpes simplex virus type 2 from nine (11%), and Neisseria gonorrhoeae from eight (10%). Primary pathogens were not isolated from 45 (57%) men. Painful purulent penile ulcers were more common in uncircumcised men, and patients had often acquired the disease after sexual intercourse with prostitutes in Singapore. PMID- 3015776 TI - Enzyme and chemical composition of plasma membranes of virulent and avirulent strains of Acanthamoeba culbertsoni. PMID- 3015777 TI - Nonfunctioning islet cell tumour of the pancreas--a case report. PMID- 3015778 TI - Primary malignant fibrous histiocytoma of ethmoid sinus. PMID- 3015780 TI - Immune protection against foot-and-mouth disease virus studied using virus neutralizing and non-neutralizing concentrations of monoclonal antibodies. AB - Monoclonal antibodies (MAb) against sequential or conformational epitopes on foot and-mouth disease virus (FMDV) passively protected neonatal syngeneic (BALB/c) mice at dilutions at which they could not neutralize virus infectivity in vitro. The B2, D9, 1C6 and 4C9 MAb, against the Group 1 (sequential) and Group 2 (conformational) epitopes, protected the mice at an antibody:virion molar ratio of between 38:1 and 84:1 (12-18 times lower than that required for neutralization of virus infectivity in vitro). The 3C8 (Group 3) and 6C3 (Group 4) MAb were, respectively, between 5 and 12 times, and between 18 and 40 times, less efficient at protection. There was no consistent correlation between the efficiency of neutralization of virus infectivity in vitro and the protection of neonatal mice against the virus pathogen. Thus, immune protection against FMDV must use mechanisms other than the direct neutralization of virus infectivity by antibody. Complement did not increase the virus neutralization titre of the MAb, but pepsin digestion of the MAb abrogated the enhanced in vivo protection over in vitro neutralization, with little effect on their capacity to neutralize virus infectivity. It is therefore likely that opsonization to a minimum affinity, and subsequent rapid phagocytosis, play a major role in the immune defence against FMDV. This is discussed in terms of the natural host for FMDV and the induction of immunological protection. PMID- 3015779 TI - Depression of delayed-type hypersensitivity in mice with hypothalamic lesion induced by monosodium glutamate: involvement of neuroendocrine system in immunomodulation. AB - Delayed-type hypersensitivity (DTH) was depressed in mice that had been treated with monosodium glutamate (MSG) in their suckling period. Analysis of the DTH depression by use of the macrophage migration inhibition assay showed dysfunction of DTH effector T cells. The neuronal loss of nuclei in the hypothalamus, which elaborates the corticotropin-releasing factor and the hypersecretion of adrenocorticotrophic hormone, was observed in the MSG-treated mice. Therefore, DTH response may be modulated by the neuroendocrine system. PMID- 3015782 TI - Regulation of antibody production by an antigen-specific EBV-transformed B lymphoblastoid cell line: effect of high-dose antigen and antigen-pulsed T cells. AB - A B-cell line (C1B2) secreting monoclonal IgG antibody to influenza virus haemagglutinin (HA3) was obtained by Epstein-Barr virus (EBV) transformation of human tonsillar B cells activated in vitro to influenza A/X31. Antibody secretion by C1B2 was completely inhibited by purified HA3 at concentrations above 100 ng/ml. By contrast, high doses of HA3 had no effect on EBV-transformed B-cell lines making antibody of unrelated specificity. Inhibition of specific antibody secretion by HA3 continued for at least 3 days after the removal of soluble antigen, but this could be partially reversed by treatment with pronase, suggesting that inhibition was due to 'effector cell' blockade by binding of antigen to surface Ig receptors. T cells pulsed with high doses of antigen also suppressed antibody secretion by C1B2, but this effect was probably due to a tolerogenic signal delivered to the B cell by HA3 complexed to the T-cell membrane rather than suppression by antigen-induced Ts, or carryover of free antigen. These experiments demonstrate two independent mechanisms of high-dose tolerance in vitro, and show that monoclonal B-lymphoblastoid lines of known specificity can be used to study regulation of specific antibody production at the level of the B cell. PMID- 3015781 TI - The role of complement in monoclonal antibody-mediated protection against virulent Semliki Forest virus. AB - Monoclonal antibodies (MAs), specific for either the E1 or E2 glycoproteins of Semliki Forest virus (SFV), and belonging to various immunoglobulin subclasses (IgM, IgG2a, IgG2b and IgG3), effected lysis of SFV-infected L cells in co operation with guinea-pig complement. In this antibody-dependent complement mediated cytolysis (ADCMC) test, IgG1 MAs were not effective although these antibodies recognize the viral antigens on the surface of SFV-infected L cells. The latter was shown with horseradish peroxidase (HRPO)-labelled MAs in a direct enzyme immunoassay. The binding reactivities of HRPO-labelled MAs to infected L cells at selected time-intervals after infection correlated well with the amount of cytolysis in a parallel ADCMC test. Cytolysis was dependent on the duration of incubation with antibodies: more cytolysis was measured after a 4-hr incubation period with MA, starting at 4 hr after infection, compared to a 1-hr incubation period starting after 7 hr of infection. However, in the latter case (1-hr period) the amount of cytolysis measured correlated better to neutralization and/or protection by MAs than after the extended period (4 hr) of incubation. Complement (C3) depletion by cobra venom factor treatment led to a higher mortality and viraemia of mice prophylactically injected with critically protective doses of either the neutralizing MA UM 8.4 (IgM) or the non neutralizing MA UM 4.2 (IgG2a). The results suggest a co-operative role of MA with complement in mediating protection against SFV. Passive immunization by administration of low amounts (0.1 micrograms/mouse) of neutralizing MA UM 5.1 resulted in protection of normal mice against a lethal infection with SFV. Mice immunosuppressed by cyclophosphamide were not protected by these doses. If the doses were increased however, these mice were protected both prophylactically and therapeutically. These results indicate that, using critical doses of MAs, an intact immune system ensures survival in normal mice after infection with virulent SFV. PMID- 3015784 TI - Effect of human recombinant interleukin 2 on natural killing of low density Percoll fraction cells. AB - Human recombinant IL-2 (R IL-2) was used to validate the ability of this lymphokine to increase NK cell activity. It was found that R IL-2 was able to augment the K562 lytic activity of phagocyte-depleted mononuclear cells of low density in a dose dependent manner. This phenomenon was detectable after 24 h of incubation. Low concentrations of R IL-2 were sufficient for the activation of natural killing when the incubation time was longer than 24 h. Furthermore, IL-1 exerted an additive effect on R IL-2 induced augmentation of natural killing after 24 h, but not after longer incubation periods. Lymphocyte culture derived (L) and R IL-2 had similar effects on the K562 cytolytic potential of cells contained in low density fractions. The highest cytolytic activity was observed when Leu11a+ cells were incubated with IL-2. IL-2 induced K562 cytolytic activity was also seen after incubation of low density Leu11- cells for 48 h, 72 h, or 7 days. In cultures of low density Leu11- cells incubated with IL-2 for 7 days, a proportion of cells became Leu11+. From these findings we conclude that low density Leu11- cells are pre-NK cells which acquire natural killing potential under the influence of IL-2. PMID- 3015783 TI - Two phenotypically distinct T cells (Ly1+2- and Ly1-2+) are involved in ultraviolet-B light-induced suppression of the efferent DTH response to HSV-1 in vivo. AB - We have previously demonstrated that the delayed-type hypersensitivity (DTH) response to herpes simplex virus type 1 induced by subcutaneous injection with live virus was suppressed by irradiating mice with a low dose (96 mJ/cm2) of ultraviolet-B (UV-B) light 3 days before sensitization. In order to determine the nature of the suppression, cells from mice irradiated with UV-B before immunization with live virus were transferred to syngeneic animals that had been sensitized with live virus without prior UV-B exposure. Efferent suppression of DTH to HSV-1 was shown to be due to T lymphocytes, and two phenotypically distinct T cells were involved: an Ly1+2- subset and an Ly1-2+ subset. PMID- 3015785 TI - Administration of silica or monoclonal antibody to Thy-1 prevents low-dose streptozotocin-induced diabetes in mice. AB - Multiple injections of low doses of streptozotocin induce an experimental diabetes in mice. We have analyzed in two inbred strains whether the development of hyperglycaemia can be influenced by administration of macrophage-toxic silica particles or by a monoclonal antibody to Thy-1.2. Mice received streptozotocin (30 or 40 mg/kg) on five consecutive days (day 0-day 4) and in addition either silica particles (starting at day 0) or anti-Thy-1.2 (starting at day -2 or -3). In both strains mice receiving streptozotocin alone became hyperglycaemic within two weeks. Additional treatment with silica almost fully prevented diabetes development. Anti-Thy-1.2 administration was similarly effective in C57B1/Ks and partially protective in C57BL/6 mice. Histological analysis of pancreatic islets showed that a large fraction of beta cells had been spared from destruction by this treatment. The data indicate a role for both macrophages and Thy-1 positive cells in the pathogenesis of low-dose streptozotocin-induced diabetes. PMID- 3015786 TI - Proliferative responses of T-cell lines grown from joint fluids of patients with rheumatoid arthritis and other arthritides. AB - T-cell lines were established by culturing, in the presence of IL-2, mononuclear cells obtained from synovial fluids (SF) of most (77%) patients with rheumatoid arthritis (RA). By contrast, SF from patients with osteoarthritis and crystal synovitis rarely yielded lines. The cell lines were identified as T-cells because they express the CD3 surface antigen and either CD8 or CD4. The ability of the T cell lines to react with putative antigens was tested using autologous Epstein Barr virus transformed B-cells (EBV-B) as antigen presenting cells. IgG failed to stimulate proliferation of any RA patients' T-cell lines while Type II collagen stimulated T-cell lines from one RA patient and from one with osteoarthritis. Some T-cell lines (17/26) took up more tritiated thymidine after three days culture with a mixture of autologous SF and irradiated autologous EBV-B than with either alone. The effect could not be ascribed to a mitogen in the RA SF as the fluids failed to stimulate blood mononuclear cells. We consider that RA SF contain a T-cell growth factor which can only exert its effect through an antigen presenting cell (EBV-B). PMID- 3015787 TI - Structure and evolution of the HLA class II SX beta gene. AB - The class II major histocompatibility complex antigens are cell-surface heterodimers consisting of an alpha and a beta chain. Cosmid cloning has shown that the three families of class II antigens, DR, DQ, and DP, are encoded within the HLA-D region of chromosome 6 as a series of discrete gene clusters. The DP cluster contains two pairs of alpha and beta genes, one of which encodes the biochemically-defined DP antigen. In order to assess whether the other two genes, SX alpha and SX beta, are also expressed, potential coding regions have been subcloned and sequenced. The SX3 beta gene is shown to contain regions closely homologous to all six exons of DP beta. A 1 bp deletion in the beta 2 exon, also observed for the SX4 beta allele, causes a translational frameshift, suggesting that SX beta is a pseudogene. However, all the other exons, as well as their splice sites and the putative promoter region, appear to be intact. PMID- 3015788 TI - Characterization of three HLA-DR beta genes isolated from an HLA-DR 3/4 insulin dependent diabetic patient. AB - Three HLA-DR beta genes were isolated from a Swedish HLA-DR3/4 insulin-dependent diabetes mellitus (IDDM) patient and characterized by restriction endonuclease mapping and nucleotide sequence analysis. Two out of the three DNA sequences differed from those of published DR beta-chain sequences. A DR beta-gene probe prepared from exon 4 and flanking sequences was used in a Southern blot analysis of blood donors' DNA and DNA from HLA-DR3/4 IDDM patients and HLA-DR-matched healthy control subjects. This probe differentiated HLA-DR3/4 IDDM patients and HLA-DR-matched controls in the Scandinavian population but not in the North American Caucasoid population. PMID- 3015789 TI - Southern blot and microfingerprinting analysis of two DR7 haplotypes. PMID- 3015790 TI - DNA restriction fragment analysis of E alpha genes in E-negative H-2 recombinant mouse strains. PMID- 3015791 TI - Comparative in vitro inhibitory activities of novel beta-lactam compounds against multiresistant beta-lactamase producing Staphylococcus aureus. PMID- 3015792 TI - Role of opioid receptors in stress induced gastric ulceration in rats. PMID- 3015794 TI - Differential renal function during angiotensin converting enzyme inhibition in renovascular hypertension. AB - Renal function was measured sequentially in 32 patients with proven renovascular hypertension who were treated with the oral angiotensin converting enzyme inhibitor captopril. Renal function was assessed by serial measurement of serum creatinine. Six patients showed acute rises in serum creatinine concentration compatible with acute renal failure. Acute renal failure was confined to those patients with stenosis to a solitary kidney (transplant or native, occurring in 3 of 8 patients) or bilateral renal artery stenosis (occurring in 3 of 13 patients). No rise in serum creatinine concentration was observed in 11 patients with unilateral renal artery stenosis during long-term angiotensin converting enzyme inhibitor therapy. Acute renal failure during angiotensin converting enzyme inhibitor therapy was not related to the degree of blood pressure fall or the plasma angiotensin II level. Eleven patients with renovascular hypertension were followed prospectively with estimation of renal function by 99mTc diethylenetriaminepentaacetic acid (DTPA) clearance (determined by computer analysis of scintillation camera renography). In six patients with unilateral renal artery stenosis, total 99mTc-DTPA clearance and serum creatinine level remained constant following angiotensin converting enzyme inhibitor therapy, while in five patients with bilateral renal artery stenosis 99mTc-DTPA clearance fell from 40 +/- 9 to 27 +/- 5 ml/min (p less than 0.05). Split renal function studies revealed that 99mTc-DTPA clearance fell in most kidneys with stenosed arteries during angiotensin converting enzyme inhibition, including the stenosed kidney from patients with unilateral renal artery stenosis (16 stenosed kidneys studied; change in Tc-DTPA clearance, -7.5 +/- 2.7 ml/min).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3015795 TI - Enhanced platelet cyclic AMP response to prostaglandin E1 in essential hypertension. AB - Platelets provide an accessible and homogeneous cellular system for investigative studies on hypertension. Hypertension-associated abnormalities of cyclic adenosine 3',5'-monophosphate (AMP) metabolism were studied in human platelets. Platelets from hypertensive subjects had an enhanced cyclic AMP accumulation response to prostaglandin E1 (twofold increase in prostaglandin E1 sensitivity). The degree of adenylate cyclase activation in response to both prostaglandin E1 (receptor-mediated) and forskolin (non-receptor-mediated) was greater in hypertensive than normotensive subjects, and prostaglandin E1-stimulated and forskolin-stimulated adenylate cyclase activity correlated directly (r = 0.71, p less than 0.001, n = 26). This finding suggests that the catalytic subunit of the enzyme is the rate-limiting step of this hormonal information transduction. Platelets from hypertensive subjects were more sensitive to epinephrine-induced inhibition of the stimulatory effects of prostaglandin E1 on both cyclic AMP accumulation (fourfold) and activation of cyclic AMP-dependent protein kinase. These findings suggest that the enhanced cyclic AMP metabolic response to prostaglandin E1 in platelets from subjects with established essential hypertension may function as a negative feedback mechanism to protect the cells against calcium overload and to reduce their stimulated participation in hemostatic and thrombotic processes. PMID- 3015793 TI - Laboratory decontamination and destruction of carcinogens in laboratory wastes: some antineoplastic agents. International Agency for Research on Cancer. PMID- 3015796 TI - Dexamethasone-suppressible hyperaldosteronism. Adrenal transition cell hyperplasia? AB - Dexamethasone-suppressible hyperaldosteronism is a rare familial syndrome in which hypokalemia, suppression of plasma renin concentration, and elevated aldosterone secretion are corrected by treatment with glucocorticoids. Regulation of adrenocortical function and body electrolytes was studied in two affected brothers. Both were hypertensive (210/128 and 160/106 mm Hg) with hypokalemia (3.3 and 3.5 mM) and low plasma renin concentrations. Aldosterone was elevated intermittently with levels as high as 45 ng/dl (normal range, 4-16 ng/dl). Cortisol concentrations were normal but were correlated with aldosterone levels (r = 0.9 and 0.7). Concentrations of 11-deoxycorticosterone (19 and 21 ng/dl; normal range, 4-16 ng/dl) and 18-hydroxycortisol (1000 and 950 ng/dl; normal range, 34-150 ng/dl) were elevated, and diurnal changes in both were the same as those seen with aldosterone. Infusion of adrenocorticotropic hormone (ACTH) caused exaggerated increases of aldosterone, 11-deoxycorticosterone, and 18 hydroxycortisol; cortisol response was normal. A 4-week trial of dexamethasone normalized blood pressure and caused a natriuresis, a fall in aldosterone, and a rise in plasma renin. Administration of ACTH after dexamethasone treatment again caused exaggerated increases of aldosterone. Aldosterone did not respond to angiotensin II before dexamethasone therapy (r = 0.01), but it showed a normal response after therapy (r = 0.8, p less than 0.01). Neither administration of dopamine (1 microgram/kg/min) nor long-term therapy with bromocriptine (2.5 mg t.i.d. for 4 weeks) affected aldosterone biosynthesis. Thus, loss of dopaminergic inhibition of mineralocorticoid biosynthesis does not account for hyperaldosteronism in this condition.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3015797 TI - Malignant hepatic tumors in childhood. PMID- 3015798 TI - Oxidative inactivation of Actinobacillus actinomycetemcomitans leukotoxin by the neutrophil myeloperoxidase system. AB - The leukotoxin of Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of inflammatory periodontal disease. We examined a potential mechanism for detoxification of this microbial product by the neutrophil myeloperoxidase system. Exposure to myeloperoxidase, H2O2, and a halide resulted in marked inactivation of leukotoxin, an effect which required each component of the myeloperoxidase system. Toxin inactivation was blocked by agents which inhibit heme enzymes (azide, cyanide) or degrade H2O2 (catalase). Reagent H2O2 could be replaced by the peroxide-generating enzyme system glucose oxidase plus glucose. The latter system, in fact, was more potent than reagent H2O2 in terms of the capacity to inactivate high concentrations of toxin. Toxin inactivation was complete within 1 to 2 min at 37 degrees C. These observations suggest a possible role for oxidative inactivation of leukotoxin by secretory products of neutrophils. PMID- 3015799 TI - Augmentation of host defense by a unicellular green alga, Chlorella vulgaris, to Escherichia coli infection. AB - Protection against Escherichia coli inoculated intraperitoneally into mice was enhanced by intraperitoneal, intravenous, or subcutaneous administration of a water-soluble, high-molecular-weight fraction extracted from a dialyzed hot-water extract from a strain of Chlorella vulgaris (CVE-A). The enhancing effect was detected with doses over 2.0 mg/kg and when doses were administered 1, 4, or 7 days before the infection. The elimination of bacteria from the spleen of CVE-A treated mice was increased, and this enhanced elimination may have been related to the acceleration of superoxide generation and chemokinesis in polymorphonuclear leucocytes by CVE-A treatment. A cyclophosphamide-induced decrease in protection against E. coli could be prevented by subcutaneous administration of CVE-A. PMID- 3015800 TI - Acid tolerance, proton permeabilities, and membrane ATPases of oral streptococci. AB - Differences in acid tolerance among representative oral streptococci were found to be related more closely to the dynamic permeabilities of the bacteria to protons than to differences in the sensitivities of cell membranes to gross damage caused by environmental acidification. For Streptococcus mutans GS-5, Streptococcus sanguis NCTC 10904, and Streptococcus salivarius ATCC 13419, gross membrane damage, indicated by the release of magnesium from whole cells, occurred at pH values below about 4 and was rapid and extensive at pH values of about 3 or less. A more aciduric, lactic acid bacterium, Lactobacillus casei ATCC 4646, was more resistant to environmental acidification, and gross membrane damage was evident only at pH values below 3. Assessments of the movements of protons into S. mutans cells after an acid pulse at various pH values indicated that permeability to protons was minimal at a pH value of about 5, at which the average half time for pH equilibration across the cell membrane was about 12 min. The corresponding values for the less aciduric organism S. sanguis were pH 7 and 8.2 min, and the values for the intermediate organism S. salivarius were pH 6 and 6.6 min. The ATPase inhibitor dicyclohexylcarbodiimide acted to increase markedly the permeability of each organism to protons, and this action indicated that permeability involved not only the passive inflow of protons but also active outflow through the proton-translocating membrane ATPase. Membranes were isolated from each of the bacteria, and pH profiles for ATPase activities indicated pH optima of about 7.5, 7.0, 6.0, and 5.0 for S. sanguis, S. salivarius, S. mutans, and L. casei, respectively. Thus, the pH profiles for the enzymes reflected the acid tolerances of the bacteria and the permeabilities of whole cells to protons. PMID- 3015801 TI - Identification of Shigella sonnei form I plasmid genes necessary for cell invasion and their conservation among Shigella species and enteroinvasive Escherichia coli. AB - A series of Tn1 insertions in pSS120, the 120-megadalton form I plasmid of Shigella sonnei, were constructed by a Tn1-mediated conduction system previously described (H. Watanabe and A. Nakamura, Infect. Immun. 48:260-262, 1985, and screened for cell invasion in a tissue culture assay. The analysis of Tn1 insertion sites of seven noninvasive mutants suggested that four separate HindIII fragments were necessary for cell invasion. HindIII fragments including Tn1 of mutant plasmids were cloned into a vector plasmid, pACYC184. The DNA was used as a DNA probe to identify the corresponding, parental HindIII fragments. We identified one contiguous molecule of 2.6- and 4.1-kilobase pair (kb) HindIII fragments as being responsible for restoring cell invasiveness to the three mutant plasmids, pHW505, pHW510, and pHW511. Polypeptide analysis in minicells demonstrated that the contiguous HindIII fragments of 2.6 and 4.1 kb coded for at least four polypeptides, of 38, 41, 47, and 80 kilodaltons (kDa). A comparison of polypeptides synthesized by parental and mutant plasmids strongly suggested that the 38-kDa protein was essential for cell invasion. The 4.1-kb DNA which encoded the 38-kDa protein was conserved among plasmids of Shigella species and enteroinvasive Escherichia coli. PMID- 3015802 TI - Degradation of Chlamydia trachomatis in human polymorphonuclear leukocytes: an ultrastructural study of peroxidase-positive phagolysosomes. AB - We have previously shown that human polymorphonuclear leukocytes (PMNs) killed organisms belonging to both human biovars of Chlamydia trachomatis. However, the mechanism of destruction was still unclear. We therefore conducted an ultrastructural and cytochemical study to investigate the mechanism of chlamydial degradation. PMNs were inoculated with the trachoma serovar B (B/TW-5/OT) or with the lymphogranuloma venereum serovar L2 (L2/434/Bu) for 15, 30, 60, or 120 min and then fixed and processed for transmission electron microscopy. Diaminobenzidine, a cytochemical marker, was used to demonstrate the localization of intracellular peroxidase. Ultrastructural evidence is presented showing the progressive degradation of chlamydiae over a 2-h period within peroxidase positive phagolysosomes. Pretreatment of organisms with normal or immune serum was not required for the process of degradation. PMID- 3015803 TI - Epidemiological correlations between African AIDS and AIDS in Europe. PMID- 3015805 TI - Prospective study on the relationship between cervical neoplasia and herpes simplex type 2 virus. III. Presence of herpes simplex type-2 antibody in sera of subjects who developed cervical neoplasia later in the study. AB - Paired sera taken from 63 subjects who developed cervical neoplasia in the course of a prospective study on this disease were examined for the presence of herpes simplex virus type 2 (HSV-2) antibody. The first serum was taken at enrollment into the study, while the second was obtained after development of the disease, i.e. 2-4 years later. Simultaneously, paired sera from a group of control subjects, who remained free of any cytological and colposcopical abnormalities throughout the study, were also investigated. Controls were matched with patients by age, age at first intercourse, number of sexual partners, smoking habits and history of diathermoelectrocoagulation of ectopic epithelium and transformation zone of the cervix. The first sera from these subjects were obtained at enrollment while the second ones were taken at the end of the study, i.e. 5 to 7 years later. Antibody titres were remarkably stable in both patients and control subjects. Seroconversions from HSV-2 negativity to HSV-2 positivity as measured by the increase in the II/I ratio or development of antibody reactive with HSV-2 specific glycoprotein G were rare and no significant differences between the patients and control subjects were detected. This indicates that the development of the disease was apparently not followed by immediate or early activation of latent HSV-2 infection. PMID- 3015804 TI - Inhibition of the platelet activating factor mediated component of guinea pig anaphylaxis by receptor antagonists. AB - The role of platelet activating factor (PAF) and its inhibition by specific receptor antagonists was studied in hypersensitivity reactions in guinea pig lung parenchymal strips and in guinea pigs in vivo. Immunological challenge of isolated lung parenchymal strips with ovalbumin resulted in a contractile response of 184 +/- 16 mg (n = 38). Pretreatment of the strips with a combination of the antihistamine diphenhydramine, the dual lipoxygenase and cyclooxygenase product synthesis inhibitor BW-755C, and the PAF receptor antagonist kadsurenone totally inhibited the increase in tension. The bronchoconstrictor effects induced by immunological challenge were also totally inhibited when the leukotriene receptor antagonist FPL-55,712 was used to replace BW-755C or another PAF receptor antagonist, alprazolam was used to replace kadsurenone. Ovalbumin challenge in sensitized guinea pigs in vivo resulted in airway constriction, circulatory failure and an abrupt fall in continuously measured platelet count by 75 +/- 12%, followed by death of all animals within 5-8 min. Pretreatment with diphenhydramine, BW-755C and kadsurenone (or alprazolam) improved survival to 64% (i.e., 7 of 11 animals) compared to a survival rate of 0% (i.e., 0 of 8 vehicle treated controls). Despite the improved survival, the severe thrombocytopenia was unaltered with the combined therapy. PAF, histamine and peptide leukotrienes appear to act in concert in mediating the anaphylaxis-induced airway constriction and circulatory failure in guinea pigs, but are not apparently responsible for the accompanying thrombocytopenia. PMID- 3015806 TI - Case-control study on colorectal cancer and diet in Marseilles. AB - A case-control study of cancer of the colon and rectum was conducted in the Marseilles region of southern France. Cases (399) and a corresponding number of controls, matched for age and sex, were included, the controls being selected from patients undergoing functional re-education for injuries or trauma which reduced their mobility. A dietary history questionnaire was used to determine the usual eating habits during the year preceding diagnosis for cases, or preceding interview for controls. The cases reported lower consumption of vegetables and oil than controls, but no differences were seen in the consumption of meat, bread, eggs or butter. The intake of several nutrients, particularly vitamins B2, B6, C, potassium, iron, magnesium and vegetable fibre, was lower among cases. However, when all these nutrients were analysed jointly and adjusted one for the other, only potassium retained a significant effect. This may be due to the high degree of colinearity between the estimated intake of many nutrients. No association was seen with fat or fibres from cereals. PMID- 3015807 TI - A virion-like particle (VLP) released from an Epstein-Barr virus-producing cell sub-line of P3HR-1: identification of a specific cellular DNA contained within VLPs. AB - A virion-like particle (VLP) was isolated from the culture fluid of P3HR-I cells. VLPs were separable from the enveloped Epstein-Barr virus (EBV) through rate velocity sedimentation by virtue of their greater density. VLPs comprised electron-dense nucleoid material and the surrounding round or oval envelope which ranged from 100 to 150 nm in diameter. The polypeptide constitution of VLPs was very similar to that of enveloped EBV by SDS-polyacrylamide gel electrophoresis. VLPs contained reasonably specific cellular DNA (VLP DNA) with a sedimentation coefficient of 48S, which was cleaved into 5, 8, and at least 30 specific DNA fragments by digestion with BamHI, SalI, and EcoRI, respectively. Blot hybridization data suggested a limited heterogeneous genomic organization of VLP DNA and the presence of a VLP DNA-related molecule(s) in human cord lymphocytes. PMID- 3015808 TI - Calcium and ATPase activity during hepatic intoxication with thioacetamide. AB - The effects of acute thioacetamide (TAA) intoxication on the calcium, magnesium, sodium and potassium content of liver and on its ATPase activities were studied in rats. Samples of liver were analyzed by atomic absorption spectrometry for their cationic content 6, 24 and 48 h after administration of TAA through a stomach tube. After sonification other samples were assayed for (Na+ + K+) dependent, (Ca2+ + Mg2+)- and Ca2+-dependent ATPases. No apparent correlation has been found between the increased hepatic calcium after TAA treatment and the variations of ATPase activity. Both calcium movement and ATPase activity appear sex-determined. These results bring support to the hypothesis that an interaction between TAA or its metabolites with the cell membrane provokes an increased influx of extracellular calcium. The relationship between this calcium increase and the changes in ATPase activity with the events triggering the neoplastic transformation is discussed. PMID- 3015809 TI - Pharmacokinetics of amitriptyline influenced by oral charcoal and urine pH. AB - The effects of orally given activated charcoal, sodium bicarbonate and ammonium chloride on the pharmacokinetics of amitriptyline were studied in 6 volunteers in a randomized, cross-over study. The serum and urine concentrations of amitriptyline and nortriptyline were determined by HPLC for up to 72 h. Activated charcoal (50 g), given within 5 min of the amitriptyline hydrochloride dose (75 mg), reduced its absorption by 99%. When given in repeated doses from 6 h on, 50 g followed by 12.5 g at 6-h intervals, charcoal shortened the serum half-life of amitriptyline by 20% and that of nortriptyline by 35% (p less than 0.05). The renal excretions of amitriptyline and nortriptyline increased 1000-fold by the acidification of urine pH to 4. However, the cumulative excretion of amitriptyline and nortriptyline even into acidic urine only accounted for up to 5% of the dose during 72 h. Since urinary pH has a great influence on the ratio of urinary versus serum amitriptyline and nortriptyline concentrations, pH should be taken into consideration, when the clinical significance of their concentrations in urine is evaluated. Activated charcoal in adequate doses very effectively prevents the absorption of that fraction of amitriptyline which is in the stomach at the time of charcoal administration. Furthermore, given in repeated oral doses, charcoal increases, to some extent, the rate of elimination of amitriptyline and nortriptyline, probably by interrupting their enterohepatic or enteroenteric circulation. PMID- 3015810 TI - Influences of sodium diethyldithiocarbamate, DTC (imuthiolR) on T cell defective responses of aged BALB/c mice. AB - The effect of chronic imuthiol treatment, 25 mg/kg weekly for 4 months initiated at the age of 12 months, on T-cell functions of aged female BALB/c mice was investigated. Imuthiol restored to normal value the impaired response to Concanavalin A (Con A) and enhanced the proliferative response to phytohemagglutinin (PHA). Impaired cytotoxic T-cell activity (CTL), was restored near to the value of young controls by imuthiol. Serum thymic factor (FTS) levels in serum of treated aged animals outpassed those of untreated young mice. Delayed type hypersensitivity (DTH) reaction to oxazolone was increased. In contrast, the graft-vs-host (GVH) mortality induced by injecting H-2 histoincompatible cells to irradiated recipients, which GVH was impaired by aging, was not significantly modified by imuthiol. The excessive cytotoxicity for chicken red cells of macrophages (ADCC) from aged mice was reduced, as well as macrophage cytotoxicity for tumor cells. Natural killer cell activity remained unchanged. This finding confirms that imuthiol enhanced effectively T cell-dependent responses but the data on GVH reaction suggest that its effects are under a complex mode of action. Restoration of a normal production of FTS may be one mechanism by which imuthiol acts on the reinduction of the T-cell differentiating pathway in aged mice. PMID- 3015811 TI - Alpha-2-adrenoreceptor density on intact platelets and adrenaline-induced platelet aggregation in endurance- and nonendurance-trained subjects. AB - Alpha-2-adrenoreceptor density on intact platelets was evaluated in 17 sportsmen, 8 of whom were endurance-trained athletes. Receptor density was determined as being equivalent to 3H-Yohimbine, specifically bound on intact platelets. Adrenaline-induced aggregation of platelets in vitro was additionally ascertained. Bmax (maximal binding) and KD (dissociation constant) were significantly lower in endurance-trained athletes (148 fmol per 10(9) platelets, KD = 0.92 nmol) than in the nine nonendurance-trained individuals (284 fmol, KD = 1.79 nmol, P less than 0.01), amounting to 89 receptors per cell (endurance trained subjects) and 171 (nonendurance-trained subjects). No significant change of receptor density or affinity was observed subsequent to exhaustive exercise in the group of eight endurance-trained individuals. Bmax values and oxygen uptake capacity correlated negatively (r = -0.78, P less than 0.001) and Bmax values and induced platelet aggregation in vitro positively (r = 0.79, P less than 0.001). Data may indicate a reduced sensitivity of platelets in endurance-trained subjects. PMID- 3015812 TI - Synthesis and receptor binding affinity of both E- and Z-dehydrophenylalanine4 enkephalins. AB - The dehydrophenylalanine4-enkephalin having the E-configuration (delta EPhe; phenyl and C = 0, cis) was prepared by photoisomerization of the Z-isomer with 3100 A light, followed by reversed-phase HPLC separation of the resulting mixture of the Z- and E-isomers. In the radioligand receptor binding assays, the E-isomer of [D-Ala2, delta Phe4, Leu5]enkephalin exhibited an extremely diminished affinity as compared with the Z-isomer, namely 150-260-fold loss of affinity for the delta and mu opiate receptors. The results indicate that the interrelationship of the Tyr1 and Phe4 residues in the enkephalin molecule seems to be of great importance in receptor recognition. PMID- 3015813 TI - Histological assessment of periodontal osseous defects following implantation of hydroxyapatite and biphasic calcium phosphate ceramics: a case report. PMID- 3015814 TI - Six month evaluation of Calcitite (hydroxyapatite ceramic) in periodontal osseous defects. PMID- 3015815 TI - Human clinical and histological responses to a Calcitite implant in intraosseous lesions. PMID- 3015816 TI - Influence of metal ions on the transport of ascorbate across membranes. AB - The effect of Fe2+, Cu2+, Ca2+, and Mg2+ on the permeation of ascorbate across membranes of dipalmitoyllecithin (DPPC) vesicles was investigated. As in previous experiments, the spin label I (1,14) was used for monitoring this transport. While Mg2+ and Ca2+ exerted a small effect only, Fe2+ and Cu2+ increased considerably the reduction of the spin label. In the case of Fe2+, this is not only due to a higher permeation rate of ascorbate but is also caused by a permeation of Fe2+. As a matter of fact, the largest reduction of the spin label was obtained if Fe2+ alone was added to the spin labelled DPPC solution. The reduction of the spin label seems to depend on the redox potentials of the constituents. PMID- 3015817 TI - Post prandial thermogenesis in different pathophysiological conditions. AB - Post Prandial Thermogenesis (PPT) is defined as the net increase in energy expenditure following the ingestion of a meal whereas diet-induced thermogenesis (DIT) indicates the overall influence of nutrients on energy expenditure. This paper reviews available data on PPT under specific clinical and dietetic conditions, e.g. the findings on PPT from pregnant and ovariectomized women, for elderly people, or the effects after MCT or LCT supplemented meals. PPT can be considered a suitable method to evaluate possible abnormalities of regulatory thermogenesis under certain clinical conditions and for the study of the acute effects of dietary components on energy expenditure. PMID- 3015818 TI - Infection control considerations for in vitro fertilization and embryo transfer programs. PMID- 3015819 TI - Benign fibrous histiocytoma: a rare endobronchial neoplasm. AB - Fibrous histiocytomas are rarely found within the lung and have presented as a mass within a major airway only twice previously. This is the third reported case of such a tumor visualized roentgenographically in an airway on the frontal chest film of a patient presenting with recurrent pneumonia. PMID- 3015820 TI - Operated bronchial carcinoma: a review of 230 cases. AB - Two hundred and thirty patients, treated by resection for bronchial carcinoma, were analysed. The histological examination showed in 80% a squamous cell carcinoma, in 11.3% an adenocarcinoma, in 5.3% a large cell and in 3.4% a small cell carcinoma. There was a great difference between preoperative and postsurgical TNM-classification: 90% stage I preoperatively and only 68.3% after resection with mediastinal lymph node dissection. Twenty-four patients (10.4%) died during the first 30 days after operation. The main cause of death was cardiac failure or respiratory insufficiency. Forty-four patients (19.1%) had non fatal complications. Atelectasis and pneumonia predominated. Survival without regard to stage and cell type was 27.6% at 5 years. As expected survival rate in T1N0M0 was best (40%). Therefore early detection of bronchial carcinoma is essential. PMID- 3015821 TI - [Therapy of chronic heart failure with positive inotropic drugs]. PMID- 3015822 TI - [Therapy of chronic heart failure with vasodilator agents]. PMID- 3015823 TI - The development of an improved murine iontophoresis reactivation model for the study of HSV-1 latency. AB - The present study reviews the development of an effective murine iontophoresis reactivation model for the study of HSV-1 latency. In a series of experiments, Balb C mice latently infected with HSV-1 McKrae strain were iontophoresed with epinephrine X 3 days (EPI X 3/ION) or 6-hydroxydopamine X 1 day followed by topical epinephrine (6-HD ION/EPI). Reactivation and recovery of latent HSV-1 was determined by daily ocular swabs, titration, and neutralization. Additional studies determined the effect of topical ocular steroids on viral recovery rate. The results demonstrated no recovery of McKrae strain in Balb C (0%) with EPI X 3/ION, and no enhancement with topical steroids. 6-HD ION/EPI demonstrated a low recovery rate in mice (8%). However, the recovery rate was significantly increased to 50% by the addition of topical steroids to form the 6-HD ION/EPI/STEROID model, a useful experimental tool. The substitution of a clinical isolate, W strain, for McKrae strain further improved the model. The results demonstrated that, following the acute infection in mice, W strain was associated with a significantly higher (P = .001) survival rate than McKrae strain (81% vs. 52%). There was no statistically significant difference between the two strains, W vs McKrae, in Balb C mice comparing keratitis, establishment of latency (by co cultivation), spontaneous shedding rate, or induced ocular shedding following iontophoresis. The development of an effective murine iontophoresis model offers an economical method which is uniquely suited for immunological and genetic studies of HSV-1 latency. PMID- 3015824 TI - The degradation of alpha-crystallin at its carboxyl-terminal portion by calpain in bovine lens. AB - Bovine lens calpain (Ca2+-dependent cysteine proteinase; EC 3.4.22.17) was shown to catalyze limited proteolysis of A and B chains of alpha-crystallin in vitro. The sites of cleavages were determined by isolating and analyzing the peptide fragments formed using several different methods, including high-performance liquid chromatography, cyanogen bromide cleavage, and carboxypeptidase digestion. The results indicated that calpain cleaved both A and B chains at their respective carboxyl-terminal regions. A chain was cleaved at A(Arg163-Glu164) bond and A(Ser162-Arg163) bond, the former being split with 2.4 times preference over the latter. B chain was cleaved at B(Thr170-Ala171) bond and B (Arg163 Glu164) bond, the former being preferred 6.5 times. Peptide cleavage at any other sites were not detected by the present method of analysis. PMID- 3015825 TI - Apical and basal-lateral Na/K-ATPase in rat lacrimal gland acinar cells. AB - The distribution of Na/K-ATPase in rat exorbital lacrimal gland was studied using immunofluorescent localization of an antibody raised against rat kidney Na/K ATPase. In cryostat sections, intralobular ducts were strongly immunoreactive and acinar cells showed localization on both apical and basal-lateral surfaces. Acinar cells also had strong intracellular reactivity in apical regions. These results are discussed in light of current models of exocrine gland electrolyte secretion. PMID- 3015826 TI - The effects of bisantrene on human platelets. AB - Bisantrene, (9,10-anthracenedicarboxaldehyde bis [(4,5-dihydro-1H-imidazol 2yl)hydrazone] dihydrochloride) is one of a series of anthracene dicarboxaldehyde compounds in Phase II trials. Preliminary studies suggested that bisantrene has anti-platelet activity. Therefore, in vitro studies of its effects on platelets were undertaken. Bisantrene in clinically attainable concentrations of 0.625 - 10 microM, caused a 95 +/- 1% decrease in maximal platelet aggregation to collagen (1 microgram/ml), and epinephrine (5-40 microM) and 90 +/- 10% inhibition of arachidonic acid (50 micrograms/ml) induced aggregation. Collagen induced platelet shape change was not affected. Aggregation to calcium ionophore A23187 was inhibited by 10-30%. No effect on ADP induced aggregation was seen at clinically relevant bisantrene concentrations. This inhibition was time dependent, reaching a maximum when platelets were preincubated with bisantrene for 10 minutes before exposure to agonist. Inhibition persisted after bisantrene was removed by washing. To determine the mechanism of platelet inhibition, platelet prostaglandin metabolism, oxygen consumption and release reaction were measured. In the presence of drug: normal cyclooxygenase activity was demonstrated by O2 consumption studies and normal secondary wave ADP (3.2 microM) aggregation; lipoxygenase activity, by O2 consumption was also normal. There was 30-50% inhibition of thromboxane A2 synthesis induced by arachidonic acid or collagen; ADP, collagen and AA induced release of serotonin was decreased by 30 60% but was never abolished. No effect on basal platelet cAMP levels nor additive effect on PGE1 induced elevation of platelet cAMP was detectable. These data demonstrate that bisantrene has potent antiplatelet activity probably mediated by several different mechanisms. The inhibition may have important clinical and theoretical consequences. PMID- 3015827 TI - Phase II trial of nimustine (ACNU; 3-[4-amino-2-methyl-5-pyrimidinyl) methyl]-1 (2-chloroethyl)-1-nitrosourea hydrochloride) in patients with small cell carcinoma of the lung after failure on combination chemotherapy. AB - Thirty-nine previously treated patients received Nimustine in a phase II trial to test the therapeutic activity in refractory small cell lung cancer. Nimustine was given as a direct i.v. injection of 100 mg/m2 with treatments repeated every six weeks. Three partial remissions of 56, 123 and 355 days duration were noted among 34 evaluable patients. Thrombocytopenia was prominent with a median platelet nadir of 47,000/microliter. We conclude that Nimustine has minor antitumor activity in heavily pretreated patients with small cell lung cancer. The definitive value of Nimustine in the treatment of small cell lung cancer, as well as its superiority over its parent compounds remains to be established. PMID- 3015828 TI - The detection of vesicoureteral reflux in the child. AB - Reflux is often an important phenomenon in the urinary tract of a child, and many methods have been advocated to detect it. The single most sensitive, accurate, and efficient way currently available to detect and characterize reflux is direct VCUG under fluoroscopic control with the contrast material instilled through a urethral catheter. Nuclear cystography is the most efficient method for screening for reflux in appropriate situations, for making sure the reflux has disappeared after corrective surgery, and for following reflux that is likely to subside spontaneously. PMID- 3015829 TI - Transcatheter renal arterial embolization with the mixture of ethanol and iodized oil (Lipiodol). AB - We induced experimental renal arterial embolization with the radiopaque solution of ethanol and iodized oil (Lipiodol) in 35 rabbits to evaluate the capability of the mixture to induce renal ablation. Serial renal angiography and plain abdominal radiography were performed immediately, one week and two weeks after embolization. Neither pure Lipiodol nor a 50% ethanol solution caused renal embolization. However, infusion of 50% and 75% ethanol-Lipiodol solutions resulted in embolization equal to that caused by absolute ethanol. The 50% ethanol-Lipiodol solution was so radiopaque that we could easily observe the embolization process during fluoroscopy. Renal arterial embolization with the 50% ethanol-Lipiodol solution was successful in three patients with hypernephroma. Our results suggest that a 50% ethanol-Lipiodol solution is radiopaque and an effective agent for renal arterial embolization. PMID- 3015830 TI - Cellular DNA synthesis associated with activation of latent herpes simplex virus in cell culture. AB - Herpes simplex virus type 1 (HSV-1) latency was established in human embryo lung (HEL) cells by treatment with (E)-5-(2'-bromovinyl)-2'-deoxyuridine and human leukocyte interferon and subsequent temperature increase (from 37 degrees to 40.5 degrees). This in vitro system was used to study reactivation after temperature shift-down to 37 degrees. Four methods known to induce cell DNA synthesis, superinfection with human cytomegalovirus, increased serum concentration in the medium, wound production, and superinfection with simian virus 40 (SV40), accelerated activation and replication of HSV-1. In contrast, reduced serum concentration resulted in postponement of virus reactivation and lower yield. In control experiments with mock-infected HEL cells treated with inhibitors and maintained for prolonged periods of time at 40.5 degrees, [3H]-thymidine incorporation was stimulated by increased serum concentration in the medium, wounding and SV40 superinfection, and hampered by reduced serum concentration in the medium. The data seem to indicate that cell DNA synthesis, the processes associated with it, or both, play a key role in the activation of latent HSV-1 and subsequent virus replication in this system. PMID- 3015831 TI - Detection of human papillomavirus DNA in genital warts, cervical dysplasias and neoplasias. AB - Biopsies from human genital lesions in Japan (108 samples), including condyloma acuminata, squamous metaplasia, dysplasia, and cervical cancer, were screened for the presence of human papillomavirus (HPV) 6-, 11-, 16-, and 18-related DNAs by spot hybridization and Southern blot hybridization under stringent conditions. By spot hybridization, HPV 6/11-related DNA was found in 92.9% (13/14) of condyloma acuminata and in 6.7% (1/15) of cervical cancer biopsies. HPV 16/18-related DNA was found in 37.7% (5/14) of cervical cancer biopsies and was exclusively associated with invasive squamous cell cancer, 66.7% (8/12), and with cervical carcinoma in situ, 50% (9/18). Some sample DNAs were further characterized by restriction enzyme digestion followed by Southern blot analysis, and we confirmed the presence of HPV-specific DNA fragments and mixed infection with both HPV 6/11 and HPV 16/18-related HPVs in one lesion. PMID- 3015832 TI - Stable stem-loop and cruciform DNA structures: isolation of mutants with rearrangements of the palindromic sequence at the simian virus 40 replication origin. AB - With the objective of generating DNA molecules that form stable stem-loop structures or cruciform structures in solution, we have altered the palindromic sequence at the Simian Virus 40 (SV40) replication origin. These alterations include: deletion of 18 of the 27 base pairs (bp) in the 13-bp inverted repeat; deletion of 26 of the 27 bp; substitution of the entire 27 bp with a totally different 26-bp sequence containing a 13-bp inverted repeat; and substitution of the 27 bp with an 8-bp sequence containing a 4-bp inverted repeat. The DNA from these mutants was purified. Mutant DNAs were hybridized to wild-type SV40 DNA or to each other, and the heteroduplexes were purified. The predicted structures were verified by S1 and restriction endonuclease digestion. The mutants - the heteroduplexes have not been tested - are capable of complementing tsA mutants and/or transforming mouse cells. PMID- 3015833 TI - Restriction endonuclease cleavage patterns of commercial and serially passaged isolates of Heliothis baculovirus. AB - The restriction endonuclease patterns of three Baculovirus isolates (Br, Vh, El) from Heliothis zea having different passages and production histories were compared. Digestion of the ds DNA genomes of the three isolates with EcoRI, HindIII, and XhoI showed no major difference in the cleavage patterns, although submolar fragments were detected. One of the commercial isolates (Vh) was serially passaged 20 times through larvae of H. zea and a substitute host, H. virescens. EcoRI cleavage profiles of the ds DNA showed the absence of a band in the 3-megadalton region that was present in the original isolates. In addition, the cleavage pattern of the DNA from Vh passed in H. zea showed additional submolar fragments in the 15- and 6-megadalton regions. Differences also were observed in the HindIII and XhoI restriction patterns. No significant differences in virulence between the original isolate and passage isolates were detected after 20 serial passages in larvae of either H. zea or H. virescens. PMID- 3015834 TI - An unusual case of lead neuropathy. PMID- 3015835 TI - Inversion of T/TMP ratio in ALS: a specific finding? AB - Thiamine (T) and thiamine monophosphate (TMP) levels were determined by an electrophoretic fluorometric method in the CSF of patients with typical sporadic ALS (50 cases), in other motor neuron diseases (MND) (14 cases) and in patients with upper and/or lower motor neuron lesions of varying origin (disseminated sclerosis, polyneuropathy, spondylotic myelopathy). T/TMP ratio was greater than or equal to 1 in a high percentage of patients with typical sporadic ALS (94%), in 35.7% of cases with other MND, while it was below 1 in the all other patients. The decrease of TMP with the inversion of the T/TMP ratio is a finding highly specific to typical sporadic ALS. PMID- 3015836 TI - The identification of neuroapraxia, axonostenosis and trigger zone in facial nerve pathology. AB - The classical electromyographic investigations and simultaneous recordings of voluntary activity and M responses from muscles of both the upper and lower branch of the facial nerve were carried out in 5 patients with Bell's palsy. R1 responses were also recorded. All investigations were extended to the healthy side. With the aim of localizing the point of axonostenosis with axonal atrophy we investigated the following parameters: conduction velocity (c.v.) in the fallopian canal, c.v. in the external facial nerve, side difference of R1-M. In one case of amyotrophic lateral sclerosis with previous Bell's palsy signs of collateral sprouting and of ephaptic transmission were detected. PMID- 3015837 TI - Segmental amputation with re-implantation in the treatment of malignant bone tumours of the limbs. AB - Stage 2 B malignant skeletal tumours are currently treated by amputation or disarticulation of the limb. The present paper describes a surgical technique for complete removal of the neoplasm and surrounding soft tissues by resection of a cylinder of the limb complete with skin. The resection of bone and adjacent soft tissues is extended sufficiently proximal and distal to the neoplasm to ensure complete removal of neoplastic tissue. The authors describe two cases; an osteosarcoma of the distal third of the femur and a malignant fibrous histiocytoma of the lower radius. After removing the affected cylinder of the limb, osteosynthesis is performed by one of a variety of methods. The main vessels and nerves are dealt with according to the findings revealed by pre operative investigations or per-operative findings. If they have to be sacrificed, end to end suture is performed, but if main nerves can safely be spared (as in Case 1) it greatly enhances the functional prognosis. The value of this operation is that it is as radical as amputation while the aesthetic and functional results are equivalent to those of a resection-arthrodesis. The operation has therefore been designated segmental amputation. PMID- 3015838 TI - [Retroviruses and the immune system]. AB - Three examples are presented to illustrate the importance of retroviruses and associated oncogenes in neoplasms of the immune system. In adult T-cell leukemia/lymphoma the HTLV I-provirus is often integrated in the human genome on chromosome 8 adjacent to the c-myc protooncogene. Cytogenetic studies of Burkitt's lymphoma (translocation of a chromosome 8 fragment) and a case of mycosis fungoides (trisomy of chromosome 8) indicate that the same oncogene may possibly be activated not only by retroviruses, but also by chromosomal aberrations. PMID- 3015840 TI - [Dermatologic diseases in patients with AIDS]. AB - This short review deals with the cutaneous manifestations of the acquired immune deficiency syndrome (AIDS), which comprise Kaposi's sarcoma, as an important marker disease for AIDS, as well as various skin infections caused by bacteria, fungi and viruses. In patients at risk of acquiring AIDS, an extensive immunological investigation should be performed if skin infections are established. In addition, a large number of cutaneous complications encountered in AIDS patients have an immunological background, e.g. an extreme hypersensitivity to trimethoprim-sulfamethoxazole. Therefore, the dermatological investigation of patients at risk of acquiring AIDS is urgently recommended as a regular part of the general check-up. PMID- 3015841 TI - [Herpesvirus infections in animals]. AB - Eighty different types of animal herpesviruses are known that affect mammals, birds, fishes, reptiles and molluscs. During evolution the viruses adapted to each animal species, and thus interspecies transmission of an infection does not occur. An exception is the Aujeszky virus, which has a wide host spectrum. In addition to morphology and a typical virus-host relationship herpesviruses of man and animals possess such common characteristics that their overall aspects can be viewed. On this basis, epidemiology, pathogenesis, clinical manifestations, immunology, therapy and prophylaxis are discussed. PMID- 3015839 TI - [Properties and transmission of the AIDS virus]. AB - The acquired immunodeficiency syndrome (AIDS) is caused by a retrovirus. LAV/HTLV III is mainly transmitted by blood and sperm. More than 90% of the newly infected persons belong to four risk groups: male homosexuals, drug addicts, persons with multiple changing sex partners, and bisexuals. Infected individuals carry the virus and antiviral antibodies simultaneously in their blood. The antibodies can be demonstrated by the ELISA and immunofluorescence tests, but positive and questionable test results must be confirmed by the immunoblot. Not all of the persons carrying the virus will develop clinical symptoms, but the percentage of those suffering from severe illnesses rises with time and reaches 40% after 8 years. At present, there is no effective therapy and no vaccine will be ready in the near future. Therefore, it is necessary to take all precautions possible to prevent new infections. PMID- 3015842 TI - [Human T-cell leukemia (lymphoma) virus (HTLV-I) antibodies in Danish patients with cutaneous T-cell lymphoma]. AB - HTLV-I antibodies were found in 10 out of 68 patients with cutaneous T-cell lymphoma (14%). HTLV antibodies were found in the earliest pre-diagnostic stage [Mycosis fungoides I (MF I), histologically non-diagnostic] as well as in the later stages (MF-III), where tumour cells are histologically apparent and the skin lesions have progressed from the plaque stage to the tumour stage. Of 4 patients with lymphomatoid papulosis, only 1 had positive HTLV antibody sera. Sequential studies of 6 patients demonstrated that the presence of HTLV-I antibodies was independent of the clinical remission - relapse stages in these patients. The present results show that HTLV-I or a related virus is found in Denmark and probably more widely distributed than was previously thought. PMID- 3015843 TI - Assessing the risks of Rn exposure: the influence of cigarette smoking. AB - The principal hazard associated with exposure to Rn progeny is lung cancer. However, most lung cancer is caused by smoking, which raises a dual problem of deriving Rn-progeny cancer risk estimates from miner populations who, in large part, are smokers and applying these estimates to the general population whose lung cancer risk, in large part, is determined by smoking habits. We examine current risk estimates for Rn-progeny-induced lung cancer using a cohort life table methodology. Estimates of lifetime probability of dying of lung cancer, average loss in life expectancy due to premature lung cancer death, and loss in life expectancy per premature lung cancer death are calculated for the general population for 1969 and 1978, nonsmokers, and smokers. These calculations demonstrate that such risk estimates are affected by smoking, and by trends in smoking habits, in several ways. Major smoking-related factors in this interaction are the proportion of smokers in the mining population used to derive lung cancer risk estimates, the proportion of smokers in the "general" population, and the assumed interaction (additive or multiplicative) between lung cancer risk, Rn-progeny exposure, and smoking history. At this time the data are not sufficient to recommend one particular modeling approach. However, our evaluation demonstrates that broad statements about Rn-progeny lung cancer risk such as "x cancers/10(6) person working level month," while informative, are incomplete without further specification. Any risk assessment must clearly state the population assumed to be at risk and the risk model assumed to be operating. Finally, the caveats appropriate to these assumptions should also be enunciated. PMID- 3015844 TI - Pre-operational survey of a U mine near Val Vedello, Italy. AB - Results obtained from radiometric research carried out from 1980-81 in the region of U mines in the Val Vedello are provided. Data are presented on 222Rn, 226Ra and natural U concentrations in spring and surface waters. Concentrations were found to be relatively low. There is some increase in the vicinity of the exploratory U mine but this is, at least up to the present, only of a local character. Data are presented on external gamma exposure, 210Pb and 210Po in foodstuffs and in the urine of normal and exposed population as well as 226Ra in milk and vegetable samples. The aim of this study is to outline a preliminary map of natural radioactivity on the site before starting the mining activity. This will enable us later to detect changes caused by the U mining, milling and mill tailing disposal operations. PMID- 3015845 TI - Computer simulations of nuclear reactions by protons with incident energies of 250, 300 and 500 MeV in a human body. AB - Computer simulations of nuclear reactions by protons in a human body were carried out at the incident energies of 250, 300 and 500 MeV. About 20% of the incident protons are absorbed by the body with nuclear interactions and the rest of the protons pass through the body at these energies. Radiation of gamma rays from the body and radioactivity of the body were estimated as a function of the time after irradiation with the proton beam. PMID- 3015846 TI - Angiotensin I converting enzyme in human intestine and kidney. Ultrastructural immunohistochemical localization. AB - The localization of immunoreactive angiotensin I-converting enzyme (ACE) has been investigated at the optical and ultrastructural level with anti-human ACE antibodies in the human kidney and small intestine. In both tissues ACE was found in blood vessels and in extravascular situation in the absorptive epithelial cells of intestinal mucosa and renal proximal tubules. Ultrastructural immunohistochemistry showed that in intestinal and renal proximal tubular cells ACE was prominent in microvilli and brush borders. In the kidney ACE was also present on the basolateral part of the plasmalemmal membrane, where it may contribute to the regulation of angiotensin II-dependent absorption processes. Intracellular positivities were also observed inside the renal vascular endothelial and proximal tubular cell in endoplasmic reticulum and nuclear envelope reflecting the synthesis and the cellular processing of ACE. The intestinal microvascular endothelium was strongly labeled suggesting that the mesenteric circulation is an important site for the production of angiotensin II. Vascular endothelial ACE was also detected in the peritubular but not glomerular capillaries of the kidney. PMID- 3015847 TI - Interhemispheric transmission delays in human strabismics. AB - Experimentally induced strabismus in cats and monkeys leads to a variety of anatomical, physiological, and perceptual deficits. Given the similarities between the perceptual deficits of human strabismics and those of animal models, it is reasonable to suggest similarities in the underlying anatomical and physiological deficits. In cat, one anatomic anomaly associated with strabismus is a disruption of the normal development of the posterior corpus callosum. The present study provides evidence that interhemispheric transmission in human strabismics may be disrupted also. Simple unimanual reaction times were used to examine the transmission of information through callosal fibres. Estimates of cross-callosal transmission time for strabismics were consistently longer than for individuals with no history of strabismus. This difference was found only when targets were presented within 5 degrees of the fixation point. The results suggest that human strabismics may have abnormalities in those callosal fibres which are involved in the interhemispheric transmission of visual information. PMID- 3015848 TI - Comments on evaluation of diagnostic test results. PMID- 3015849 TI - Molecular mechanisms involved in increasing epidermal growth factor receptor levels on the cell surface. AB - Various tissues and cell lines were analyzed to determine factors which regulate the epidermal growth factor (EGF) receptor level on the cell surface. At least 3 mechanisms are postulated to potentially control the EGF receptor level: gene amplification, receptor mRNA levels, and receptor half-life. The abnormal activation of any or all of these mechanisms may contribute to the high level of EGF receptors observed in squamous cell carcinomas. PMID- 3015850 TI - Narrow host range of AIDS-related retroviruses (YU-1, 2, 3, 4) isolated from Japanese hemophiliacs: inability to infect H9, Molt-4, and MT-4 cells. AB - Infectivity was assayed by infecting human T-lymphocytes, H9, Molt-4, and MT-4 cells with different strains of AIDS viruses (HTLV-III, LAV, ARV, and YU viruses). Human T-lymphotropic virus type-III (HTLV-III), lymphadenopathy associated virus (LAV), and AIDS-associated retrovirus (ARV) were able to infect all kinds of target cells tested and to induce virus-specific antigens, whereas Japanese isolates, YU viruses, infected only interleukin-2-dependent human T lymphocytes. Long-term propagation of the YU viruses and pretreatment of the cells with Polybrene did not allow the YU virus to produce viral antigens by infecting H9, Molt-4, and MT-4 cells. Thus, YU viruses have a rather narrow host range as compared with HTLV-III, LAV, and ARV. These different infectivities may be reflected in the progress and symptoms of AIDS in vivo. PMID- 3015851 TI - Liposoluble platinum(II) complexes with antitumor activity. AB - Seventeen liposoluble bis(carboxylato)-cyclohexane-1,2-diammineplatinum(II) and bis(carboxylato)-cis-diammineplatinum(II) complexes were synthesized and tested for antitumor activity against leukemia L1210 cells in mice. The former complexes had excellent antitumor activity without any toxicity to the host at the therapeutic dose when used with lipiodol as a carrier solvent. The latter complexes had neither antitumor activity nor toxicity in vivo. The former complexes were gradually released from lipiodol to saline in vitro; the latter were not. The activity depended on the chain length of the carboxylato residue and also on the molecular shape of the ligand part of the complexes. PMID- 3015852 TI - Cloning and characterization of the streptothricin-resistance gene which encodes streptothricin acetyltransferase from Streptomyces lavendulae. AB - The streptothricin-resistance gene of Streptomyces lavendulae No. 1080 was cloned in S. lividans using pIJ41 as a vector. From subcloning experiments, the 1.6 kb BamH I fragment was determined to encode the structural gene. The cell extracts of S. lividans carrying the gene on the plasmid pKS7 had activity to inactivate streptothricin in the presence of S-acetyl coenzyme A, indicating that the gene product was streptothricin acetyltransferase. PMID- 3015853 TI - Potential prodrugs of 6-acetylmethylenepenicillanic acid (Ro 15-1903). AB - The synthesis and biological activities of a series of non-classical penicillins are described. These compounds were synthesized by treating the pivaloyloxymethyl ester of 6-acetylmethylenepenicillanic acid (Ro 15-1903) with various nucleophiles. They were found to be less active against the beta-lactamases from Proteus vulgaris 1028, Escherichia coli 1024, Klebsiella pneumoniae NCTC 418 and E. coli RTEM than the parent compound. Nevertheless, synergy with ampicillin against whole bacterial cells producing beta-lactamases was evident, although the single compounds did not exhibit antibacterial properties. With the compounds 2a and 2b, synergistic interaction with ampicillin could also be demonstrated in mice. PMID- 3015854 TI - Albothricin, a new streptothricin antibiotic. PMID- 3015855 TI - Neurotransmission in the inner ear. AB - The present view on cochlear neurotransmission can be summarized as follows: There are two main types of synapses on cochlear hair cells, afferent and efferent ones. Afferent synaptic structures are abundant on inner hair cells whereas similar structures on the outer hair cells are less frequent and appear to be rudimentary. Presynaptic vesicles seem to be rare in outer hair cells. For the inner hair cell--afferent terminal--the presence of a chemical transmission mechanism is generally accepted. The transmitter substance has not yet been unequivocally demonstrated. Glycine, catecholamines, GABA and 5-HT can be eliminated as candidates as these compounds do not activate afferent fibres. There are good reasons, however, to consider amino acids. Most of the experimental results support glutamate as the transmitter (e.g. effectiveness of glutamate, kainic acid, glutamate diethylester). Aspartate is less likely. It is not yet well understood, however, why glutamate has to be applied in concentrations of up to 10(-3) M intracochlearly in order to activate afferent fibres and why elevated glutamate levels could not be demonstrated in perilymph collected during acoustical stimulation, whereas this same perilymph was able to activate afferent nerve terminals when applied intracochlearly. Efferent endings use acetylcholine as a transmitter. Enzymes for synthesis and breakdown of acetylcholine are present; acetylcholine is effective at the synaptic junction, as are cholinergic compounds and specific blockers. However, there may be different types of efferent endings in both the cochlear and vestibular organs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3015856 TI - Spike activity recorded from the organ of Corti. AB - During microelectrode passes through the inner hair cell region of the organ of Corti, spike activity is frequently encountered. We have obtained both extracellular and intracellular records from afferent terminals or dendrites. The response characteristics of all-or-none spike trains mimic those recorded from primary auditory axons. EPSPs may be identified, either associated with the spikes or in the absence of a spike. Inferences may be made about the nature of neurotransmitter quantum release from inner hair cells. PMID- 3015857 TI - Molecular mechanisms of drug-induced hearing loss. AB - Although the ototoxic actions of a variety of drugs have long been documented, the biochemical mechanisms underlying such toxicity largely remain to be established. For example, recent advances have provided us with information about the actions of salicylates (aspirin) and diuretics (furosemide) but we are not yet able to specify the mechanisms by which these drugs damage the cochlea. On the other hand, the considerable amount of biochemical and pharmacological data on the effects of aminoglycosides (streptomycin, neomycin, gentamicin and related compounds) has enabled us to formulate a rational hypothesis of their mechanism of action. We have previously presented evidence for an involvement of polyphosphoinositides in the ototoxic actions of aminoglycosides. Recent electrophysiological and pharmacokinetic studies have shown in addition that aminoglycosides occupy at least two distinct compartments in the course of their actions. Further studies of drug uptake in vitro and of drug toxicity in cochlear perfusions suggested the involvement of an active (energy-requiring) aminoglycoside transport system. These and other data are compatible with the following multi-step model of aminoglycoside toxicity: The initial step in the reaction sequence is an electrostatic interaction of aminoglycosides with the plasma membrane. The resulting displacement of calcium accounts for acute effects but the action is reversible and antagonized by divalent cations. An energy dependent uptake process is required for the expression of toxicity. It can be prevented by select metabolic blockers. A crucial step in subsequent intracellular drug actions is the binding of aminoglycosides to phosphatidylinositol bisphosphate inhibiting its hydrolysis and preventing its physiological function.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3015858 TI - Care of the HBV positive mother and her infant. PMID- 3015859 TI - Effects of human chorionic gonadotropin and cyclic adenosine monophosphate on progesterone secretion by the ovine placenta. AB - Progesterone production by the ovine placenta was investigated between d 80 and 115 of gestation. Serum progesterone concentrations in ewes ovariectomized (ovx) on d 75 of gestation were measured throughout the remainder of gestation, and after the ewes were injected with human chorionic gonadotropin (hCG) or saline on d 80 or 115. In addition, cotyledonary tissue was collected from intact ewes sacrificed on d 80 or 115 and progesterone accumulation was determined during 2 h incubation with or without pregnenolone supplementation and in the presence or absence of hCG or dibutyryl cyclic adenosine monophosphate (dbcAMP). Serum concentrations of progesterone in ovx ewes increased from 3.5 +/- .4 ng/ml on d 80 to 16.4 +/- 2.1 ng/ml on d 115 (P less than .05). That increase was coincident with a 1.5-to 4.5-fold increase in progesterone output by placental tissue in vitro. Addition of pregnenolone enhanced progesterone accumulation in all tissue incubations. Addition of dbcAMP increased progesterone accumulation in the incubation medium only when supplemented with pregnenolone. Human chorionic gonadotropin did not increase placental progesterone secretion in vivo or in vitro. The results confirm the enhanced secretion of progesterone by the ovine placenta between d 80 and 115 of gestation and indicate that the increase results primarily from increased secretory capability per unit of placenta. The tropic mechanism controlling the placental secretion of progesterone remains unclear, but the mechanism may involve elevation of intracellular cAMP. PMID- 3015860 TI - Ventilatory effect of an adenosine analogue in unanesthetized rabbits during development. AB - The effect of an adenosine analogue N6-L-(R-phenylisopropyl)adenosine (R-PIA) on respiration was studied in rabbit pups (1-8 days old). Respiration was monitored by a noninvasive barometric method during natural sleep. The adenosine analogue was given by an indwelling intraperitoneal catheter. R-PIA given in a dose of 0.1 mumol/kg (380 micrograms/kg) body wt caused a decrease of the ventilation. The respiratory decrease could be reversed or prevented by pretreatment with theophylline (10 mg/kg). R-PIA caused a considerably more pronounced effect in 1- to 3-day-old animals than in 8-day-old animals. This effect was seen both when the ambient temperature was held at 28 (P less than 0.01) and 32 degrees C (P less than 0.05). Determination of R-PIA receptors in whole brains of rabbit pups of various ages showed that R-PIA bound with higher affinity to membranes from newborn animals (Kd 0.53 nM) than older animals (Kd 0.7-1.26). Since adenosine is released during hypoxia, it may be involved in "hypoxic depression" of respiration in neonates and apnea of prematurity. This might also explain the potent therapeutic effect of the adenosine antagonist theophylline on recurrent apnea in preterm infants. PMID- 3015861 TI - Inherent resistance of HeLa cell derivatives to paromomycin. AB - The human tumor-derived cell line HeLa S3 and nuclear and mitochondrial gene mutants derived from it are resistant to the aminoglycoside antibiotic, paromomycin (PAR). Other carcinoma-derived cells, SV40-transformed cells, and four human diploid fibroblast cell lines are all sensitive to PAR. Sensitivity is dependent on cell density, and at cell numbers greater than 400/cm2 sensitive cells will proliferate in PAR. The resistance to PAR is inherited in a dominant manner in cell-to-cell fusion hybrids, but is not transferred in cytoplast-to cell fusions. PAR resistance is therefore encoded by a nuclear gene(s). Resistance to PAR is not caused by changes in the response of mitochondrial or cytoplasmic protein synthesis to PAR in vitro. The uptake of PAR is similar in resistant and sensitive cells, and dimethyl sulfoxide does not render resistant cells more sensitive. Thus, HeLa cell PAR resistance is unlike previously reported ribosomal mutations and may derive from differences in the intracellular metabolism of PAR. PMID- 3015862 TI - Susceptibility of endothelial cells derived from different blood vessels to common viruses. AB - We examined whether endothelial cells derived from different blood vessels vary in their susceptibility to viral infection. Five common viral pathogens of humans (herpes simplex 1, measles, mumps, echo 9, and coxsackie B4 viruses) were evaluated for growth in endothelial cells derived from bovine fetal pulmonary artery, thoracic aorta, and vena cava. All five viruses replicated in each type of endothelial cell. There were apparent differences in the quantities of measles and mumps viruses produced in pulmonary artery endothelium compared with thoracic aorta and vena cava when endothelial cells were obtained from different animals. However, when pulmonary artery endothelial cells were compared with vena cava cells from the same animal, growth of each virus was similar in the two cell types. Four of the viruses replicated in the various endothelial cells without producing appreciable changes in cell morphology. These results indicate that endothelial cells from different blood vessels are equally susceptible to the human viruses evaluated, and that viral replication can occur without major alterations in cell morphology. Endothelial cells could serve as permissive cells permitting viruses to leave the circulation and initiate infection in adjacent tissues, including subendothelial smooth muscle cells. PMID- 3015863 TI - Laminin: an initiating role in retinoblastoma cell attachment and differentiation. AB - The effect of laminin (LN) on the attachment and differentiation of a human retinoblastoma cell line (Y-79) was investigated. We found that 10 micrograms/ml LN in the serum-free culture medium for 3 to 4 d induces 20 to 30% of the cells to firmly attach to a plastic substratum. This effect seems to be complex because the short-term effect of LN is inhibition of cell attachment. The specificity of LN may be indicated because antilaminin antibody counteracted this effect. The attached cells form small processes immediately after attachment and continue to proliferate, forming small colonies. Treatment of these cells with 4 mM dibutyryl cyclic AMP (db-cAMP) or 2 mM sodium butyrate starting on the 3rd or 4th d of culture results in extensive differentiation of all the attached cells. Db-cAMP provokes the formation of long ramifying neuritelike processes whereas butyrate induces the appearance of epithelial-like cells with a flat morphology. Thus, laminin seems to act in concert with other agents promoting attachment and potentiating differentiation. PMID- 3015865 TI - Clinical profile of the Japanese encephalitis epidemics in Goa in 1982-83. PMID- 3015866 TI - An unusual case of phosphate dependent myopathy. PMID- 3015864 TI - Effect of insulin on cyclic nucleotide levels and promotion of mitosis by insulin and ionophore A23187 in cultured newt blastemata. AB - The levels of cyclic GMP (cGMP) and cyclic AMP (cAMP) were assayed, using radioimmunoassay, in newt blastemata cultured with and without insulin. Our observations show that insulin significantly increases the levels of cGMP over the control values, whereas the levels of cAMP remain unaltered. Our in vitro studies also show that Ca2+-carrying ionophore A23187, albeit capable of promoting blastema cell proliferation, is unable to replace the insulin effect. The possible role of cGMP and Ca2+ as mediators of insulin action in regeneration is discussed. PMID- 3015867 TI - Growth hormone response to oral glucose before and after transfrontal pituitary surgery and radiotherapy in acromegaly. PMID- 3015868 TI - cis-acting proteins. PMID- 3015869 TI - Regulation of envelope protein composition during adaptation to osmotic stress in Escherichia coli. AB - Adaptation to osmotic stress alters the amounts of several specific proteins in the Escherichia coli K-12 envelope. The most striking feature of the response to elevated osmolarity was the strong induction of a periplasmic protein with an Mr of 31,000. This protein was absent in mutants with lambda plac Mu insertions in an osmotically inducible locus mapping near 58 min. The insertions are likely to be in proU, a locus encoding a transport activity for the osmoprotectants glycine betaine and proline. Factors affecting the extent of proU induction were identified by direct examination of periplasmic proteins on sodium dodecyl sulfate gels and by measuring beta-galactosidase activity from proU-lac fusions. Expression was stimulated by increasing additions of salt or sucrose to minimal medium, up to a maximum at 0.5 M NaCl. Exogenous glycine betaine acted as an osmoregulatory signal; its addition to the high-osmolarity medium substantially repressed the expression of the 31,000-dalton periplasmic protein and the proU lac+ fusions. Elevated osmolarity also caused the appearance of a second periplasmic protein (Mr = 16,000), and severe reduction in the amounts of two others. In the outer membrane, the well-characterized repression of OmpF by high osmolarity was observed and was reversed by glycine betaine. Additional changes in membrane composition were also responsive to glycine betaine regulation. PMID- 3015870 TI - Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product. AB - TOL plasmid pWW0 specifies enzymes for the oxidative catabolism of toluene and xylenes. The upper pathway converts the aromatic hydrocarbons to aromatic carboxylic acids via corresponding alcohols and aldehydes and involves three enzymes: xylene oxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase. The synthesis of these enzymes is positively regulated by the product of xylR. Determination of upper pathway enzyme levels in bacteria carrying Tn5 insertion mutant derivatives of plasmid pWW0-161 has shown that the genes for upper pathway enzymes are organized in an operon with the following order: promoter-xylC (benzaldehyde dehydrogenase gene[s])-xylA (xylene oxygenase gene[s])-xylB (benzyl alcohol dehydrogenase gene). Subcloning of the upper pathway genes in a lambda pL promoter-containing vector and analysis of their expression in Escherichia coli K-12 confirmed this order. Two distinct enzymes were found to attack benzyl alcohol, namely, xylene oxygenase and benzyl alcohol dehydrogenase; and their catalytic activities were additive in the conversion of benzyl alcohol to benzaldehyde. The fact that benzyl alcohol is both a product and a substrate of xylene oxygenase indicates that this enzyme has a relaxed substrate specificity. PMID- 3015872 TI - RNA-DNA hybridization analysis of transcription of the plasmid ColV-K30 aerobactin gene cluster. AB - Plasmid pABN1 contains the genetic determinants for the aerobactin iron uptake system of plasmid ColV-K30. Transposon Tn1000 mutants of pABN1 defective in synthesis of a 50,000-dalton polypeptide were found neither to secrete nor to accumulate aerobactin, but were not impaired in iron transport functions, clearly indicating a role for this polypeptide in aerobactin biosynthesis. RNA-DNA hybridization studies with probes spanning the entire aerobactin gene cluster showed that the system is regulated at the transcriptional level by the availability of iron in the external medium. When induced by low-iron stress, all five genes of the cluster were transcribed at a uniformly high level. When repressed by excess iron, transcripts of the four biosynthesis genes were some 30 fold less abundant in the case of the parental ColV-K30 plasmid and 10-fold less for the recombinant plasmid pABN1, whereas the receptor gene in either plasmid was transcribed at only about a third of the induced level. PMID- 3015871 TI - Translational control of exported proteins in Escherichia coli. AB - We recently described the suppression of export of a class of periplasmic proteins of Escherichia coli caused by overproduction of a C-terminal truncated periplasmic enzyme (GlpQ'). This truncated protein was not released into the periplasm but remained attached to the inner membrane and was accessible from the periplasm. The presence of GlpQ' in the membrane strongly reduced the appearance in the periplasm of some periplasmic proteins, including the maltose-binding protein (MBP), but did not affect outer membrane proteins, including the lambda receptor (LamB) (R. Hengge and W. Boos, J. Bacteriol., 162:972-978, 1985). To investigate this phenomenon further we examined the fate of MBP in comparison with the outer membrane protein LamB. We found that not only localization but also synthesis of MBP was impaired, indicating a coupling of translation and export. Synthesis and secretion of LamB were not affected. The possibility that this influence was exerted via the level of cyclic AMP could be excluded. Synthesis of MBP with altered signal sequences was also reduced, demonstrating that export-defective MBP which ultimately remains in the cytoplasm abortively enters the export pathway. When GlpQ' was expressed in a secA51(Ts) strain, the inhibition of MBP synthesis caused by GlpQ' was dominant over the precursor accumulation usually caused by secA51(Ts) at 41 degrees C. Therefore, GlpQ' acts before or at the level of recognition by SecA. For LamB the usual secA51(Ts) phenotype was observed. We propose a mechanism by which GlpQ' blocks an yet unknown membrane protein, the function of which is to couple translation and export of a subclass of periplasmic proteins. PMID- 3015875 TI - Genetic rearrangements of a Rhizobium phaseoli symbiotic plasmid. AB - Different structural changes of the Sym plasmid were found in a Rhizobium phaseoli strain that loses its symbiotic phenotype at a high frequency. These rearrangements affected both nif genes and Tn5 mob insertions in the plasmid, and in some cases they modified the expression of the bacterium's nodulation ability. One of the rearrangements was more frequent in heat-treated cells, but was also found under standard culture conditions; other structural changes appeared to be related to the conjugal transfer of the plasmid. PMID- 3015873 TI - Expression in Escherichia coli and function of Pseudomonas aeruginosa outer membrane porin protein F. AB - The gene encoding porin protein F of Pseudomonas aeruginosa was cloned onto a cosmid vector into Escherichia coli. Protein F was expressed as the predominant outer membrane protein in a porin-deficient E. coli background and was clearly visible on one-dimensional sodium dodecyl sulfate-polyacrylamide gels in a porin sufficient background. The identity of the protein F from the E. coli clone and native P. aeruginosa protein F was demonstrated by their identical mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoretograms, 2-mercaptoethanol modifiabilities, and reactivities with monoclonal antibodies specific of two separate epitopes of protein F. In the course of gene subcloning, a 2-kilobase DNA fragment was isolated, with an apparent truncation of the part of the gene encoding the carboxy terminus of protein F. This subclone produced a 24,000 molecular-weight, outer membrane-associated, truncated protein F derivative which was not 2-mercaptoethanol modifiable and which reacted with only one of the two classes of protein F-specific monoclonal antibodies. The 2-kilobase fragment was used in Southern blot hybridizations to construct a restriction map of the cloned and subcloned fragments and to demonstrate with restriction digests of whole P. aeruginosa DNA that only one copy of the protein F gene was present in the P. aeruginosa chromosome. The protein F produced by the original cosmid clone in a porin-deficient E. coli background was purified. To demonstrate retention of porin function after cloning, the protein F from the E. coli clone was incorporated into black lipid bilayer membranes. Two major classes of channels were revealed. The predominant class of channels had an average conductance of 0.36 nS in 1 M KCl, whereas larger channels (4 to 7 nS) were seen at a lower frequency. Similar channel sizes were observed for porin protein F purified by the same method from P. aeruginosa outer membranes. PMID- 3015874 TI - Transcriptional regulation of nitrogen fixation by molybdenum in Azotobacter vinelandii. AB - Multiple genomic regions homologous to nifH were found in the diazotroph Azotobacter vinelandii. The nifHDK gene cluster, located on a 12.8-kilobase (kb) XhoI fragment and two additional XhoI fragments (7.4 and 8.4 kb) hybridized to a nifH-specific DNA template but the 7.4- and 8.4-kb fragments did not hybridize to nifD- or nifK-specific DNA probes. In vivo transcription of the nifHDK gene cluster was ammonia-repressible and required the presence of at least 50 nM molybdenum in the derepression medium. Three mRNA species were transcribed from the nifHDK gene cluster, a 4.2-kb transcript homologous to nifH-, nifD-, and nifK specific DNA templates, a 2.6-kb transcript homologous to nifH- and nifD-specific DNA templates, and a 1.2-kb transcript homologous only to the nifH-specific DNA template. In strain CA11, a nifHDK deletion mutant, the nifHDK-specific transcripts were not produced and the strain was unable to grow in N-free medium in the presence of Na2MoO4 at concentrations of 50 nM or higher. However, at concentrations of 25 nM Mo or less, growth occurred in N-free medium. Under these conditions two nifH-homologous (but not nifD- or nifK-homologous) transcripts were observed (1.2 and 1.8 kb). Presumably these were transcribed from the additional nifH-homologous sequences present in the genome. These results are consistent with the existence of two N2 fixation systems in A. vinelandii which are regulated by molybdenum at the level of transcription. PMID- 3015876 TI - Role of ner protein in bacteriophage Mu transposition. AB - The Ner protein of bacteriophage Mu negatively regulates transcription initiated at the early promoter and at the major repressor promoter. The construction and isolation of a Ner- mutant of Mu is described. Ner is an essential function for Mu, because the mutant phage only forms plaques when complemented for Ner. Mutations in the repressor protein did not abolish the need for Ner. However, when transcription of the repressor gene c was blocked by deleting the major repressor promoter, Ner was no longer essential for normal Mu development. PMID- 3015878 TI - Chromosomal insertions of Tn917 in Bacillus subtilis. AB - We describe 46 insertions of the Streptococcus faecalis transposon Tn917 into the chromosome of Bacillus subtilis. These insertion mutations were mapped genetically. Some caused auxotrophic requirements, and others were cryptic. These insertions were scattered around the B. subtilis chromosome. The mutant strains were useful in several ways for mapping and cloning B. subtilis genes and were added to the Bacillus Genetic Stock Center collection. Among the auxotrophic markers were a new serine auxotrophy and deletion-insertions that caused auxotrophy in one case for homoserine and threonine, in another case for uracil and either cysteine or methionine, and in a third case for leucine, isoleucine, and valine. PMID- 3015877 TI - Characterization and mapping of regions encoding clindamycin resistance, tetracycline resistance, and a replication function on the Bacteroides R plasmid pCP1. AB - The Bacteroides drug resistance plasmid pCP1 encodes clindamycin resistance (Clr) and a cryptic tetracycline resistance (Tcr) determinant that is expressed in Escherichia coli cells grown aerobically, but not anaerobically, and is not expressed phenotypically in Bacteroides spp. Localization of genetic functions on pCP1 was facilitated by the construction of hybrid shuttle plasmids containing portions of pCP1 ligated to pDG5, a pBR322 derivative carrying the RK2 transfer origin. pDP1 delta 4 is a BglII deletion derivative of pCP1 linked to pDG5 and can be maintained in both E. coli and Bacteroides fragilis. By using Tn5 mutagenesis and subcloning, we localized the Clr and Tcr regions on the EcoRI B fragment between the 1.2-kilobase direct repeats of pCP1. The Clr and Tcr determinants are distinct and appear to be transcribed separately. Control of the Tcr phenotype is unusual in that expression is constitutive and is enhanced by a region encompassing the adjacent direct repeat. In addition, a region of pCP1 required for replication in Bacteroides spp. has been identified in the neighboring EcoRI A fragment. PMID- 3015879 TI - Construction of a host-vector system in Candida maltosa by using an ARS site isolated from its genome. AB - To construct a host-vector system in an n-alkane-assimilating yeast, Candida maltosa, the isolation of an ARS site from its genome which replicates autonomously in C. maltosa was attempted. Leu- mutants of C. maltosa were transformed with a gene library prepared by using YEp13 (LEU2+) as a vector, and Leu+ transformants were obtained at a high frequency. A plasmid named pCS1 was isolated from the recipient cells. pCS1 contained a 6.3-kilobase (kb) fragment of the C. maltosa genome, and a 3.8-kb fragment with ARS activity was subcloned and designated the TRA (transformation ability) region. Vectors (pTRA1 and pTRA11) for C. maltosa J288 were constructed that contained this 3.8-kb fragment, pBR322, and the LEU2 gene of Saccharomyces cerevisiae. Transformation of C. maltosa J288 with these plasmids was successful by both spheroplast and lithium acetate methods. Southern blot analysis suggested that the copy number of pTRA1 in C. maltosa was between 10 and 20, and it was stably maintained during growth without selective pressure in the medium. It was also found that these vectors could transform S. cerevisiae leu2- to LEU2+, suggesting that the TRA region contained an ARS site(s) that was specific not only for C. maltosa but also for S. cerevisiae. PMID- 3015880 TI - Linear plasmidlike DNA in the plant pathogenic fungus Fusarium oxysporum f. sp. conglutinans. AB - Double-stranded, 1.9-kilobase-pair (kbp) DNA molecules were found in 18 strains representing three pathogenic races of Fusarium oxysporum f. sp. conglutinans. The DNA element (pFOXC1) from a race 1 strain and the DNA element (pFOXC2) from a race 2 strain were shown by restriction endonuclease mapping to be linear. pFOXC2 was found in mitochondrial preparations and appears to have blocked 5' termini, as it was sensitive to 3'----5' exonuclease III but insensitive to 5'----3' lambda exonuclease. The major 1.8-kbp BglII restriction endonuclease fragment of pFOXC2 was cloned in plasmid pUC12. The recombinant plasmid (pCK1) was not homologous to the mitochondrial or nuclear genomes from F. oxysporum f. sp. conglutinans. This suggests that pFOXC2 is self-replicating. pCK1 was homologous to all 1.9-kbp DNA elements of race 2 but was not homologous to those of race 1 or race 5. All race 1 and 5 elements were also shown to share common DNA sequences. PMID- 3015881 TI - Identification and characterization of recD, a gene affecting plasmid maintenance and recombination in Escherichia coli. AB - We isolated mutations that reduce plasmid stability in dividing cell populations and mapped these mutations to a previously undescribed gene, recD, that affects recombination frequency and consequently the formation of plasmid concatemers. Insertions of the transposable element Tn10 into recD resulted in increased concatemerization and loss of pSC101 and ColE1-like replicons during nonselective growth. Both concatemer formation and plasmid instability in recD mutants require a functional recA gene. Mutations in recD are recessive to recD+ and map to a small region of the Escherichia coli chromosome located between recB and argA. Although the recD locus is distinct from loci encoding the two previously identified subunits of the RecBC enzyme, mutations in recD appear to affect the exonuclease activity of this enzyme. PMID- 3015883 TI - Cloning of multiple genes involved with cobalamin (Vitamin B12) biosynthesis in Bacillus megaterium. AB - An effective shotgun cloning procedure was developed for Bacillus megaterium by amplifying gene libraries in Bacillus subtilis. This technique was useful in isolating at least 11 genes from B. megaterium which are involved with cobalamin (vitamin B12) biosynthesis. Amplified plasmid banks were transformed into protoplasts of both a series of Cob mutants blocked before the biosynthesis of cobinamide and Cbl mutants blocked in the conversion of cobinamide into cobalamin. Amplification of gene libraries overcame the cloning barriers inherent in the relatively low protoplast transformation frequency of B. megaterium. A family of plasmids was isolated by complementation of seven different Cob and Cbl mutants. Each plasmid capable of complementing a Cob or Cbl mutant was transformed into each one of the series of Cob and Cbl mutants; many of the plasmids isolated by complementation of one mutation carried genetic activity for complementation of other mutations. By these criteria, four different complementation groups were resolved. At least six genes involved in the biosynthesis of cobinamide are carried on a fragment of DNA approximately 2.7 kilobase pairs in length; other genes involved in the biosynthesis of cobinamide were located in two other complementation groups. The physical and genetic data permitted an ordering of genes within several of the complementation groups. The presence of complementing plasmids in mutants blocked in cobalamin synthesis resulted in restoration of cobalamin biosynthesis. PMID- 3015882 TI - Cloning and DNA sequence analysis of the wild-type and mutant cyclic AMP receptor protein genes from Salmonella typhimurium. AB - The crp gene from Salmonella typhimurium, as well as two mutant adenylate cyclase regulation genes designated crpacr-3 and crpacr-4, were cloned into the EcoRI site of plasmid pUC8. Initially cloned on 5.6-kilobase fragments isolated from EcoRI digests of chromosomal DNA, these genes were further subcloned into the BamHI-EcoRI site of plasmid pBR322. When tested, Escherichia coli crp deletion strains harboring the clones regained their ability to pleiotropically ferment catabolite-repressible sugars. Also, the crpacr-containing strains displayed sensitivity to exogenous cyclic AMP (cAMP) when grown on eosin-methylene blue medium with xylose as the carbon source. The proteins encoded by the S. typhimurium wild-type and mutant crp genes were found to have similar molecular weights when compared with the wild-type cAMP receptor protein (CRP) from E. coli. DNA sequence analysis of the wild-type crp gene showed only a three nucleotide difference from the E. coli sequence, suggesting little divergence of the crp gene between these organisms. The crpacr sequences, however, each contained single nucleotide changes resulting in amino acid substitutions at position 130 of the CRP. Based on the site at which these substitutions occur, the crpacr mutations are believed to affect CRP-cAMP interactions. PMID- 3015885 TI - DNA environment of the aerobactin iron uptake system genes in prototypic ColV plasmids. AB - The aerobactin iron uptake system genes in the prototypic plasmid pColV-K30 are flanked by inverted copies of insertion sequence IS1 and by two distinct replication regions. To address the question of how these flanking regions may facilitate the maintenance and spread of the aerobactin system among the plasmids and chromosomes of enteric species, we investigated the DNA environment of 12 ColV plasmids. We found that the aerobactin system-specific genes are conserved in every plasmid phenotypically positive for the aerobactin system. The upstream IS1 and its overlapping replication region (REPI) are also conserved. This replication region was cloned from several ColV plasmids and found to be functional by transforming these cloned derivatives into a polA bacterial host. In contrast, the downstream flanking region is variable. This includes the downstream copy of IS1 and the downstream replication region (REPII). We infer from these results that sequences in addition to the two flanking copies of IS1, in particular the upstream region including REPI, have been instrumental in the preservation and possible spread of aerobactin genes among ColV plasmids and other members of the FI incompatibility group. PMID- 3015884 TI - Nucleotide sequence analysis of the gene specifying the bifunctional 6' aminoglycoside acetyltransferase 2"-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities. AB - The gene specifying the bifunctional 6'-aminoglycoside acetyltransferase [AAC(6')] 2"-aminoglycoside phosphotransferase [APH(2")] enzyme from the Streptococcus faecalis plasmid pIP800 was cloned in Escherichia coli. A single protein with an apparent molecular weight of 56,000 was specified by this cloned determinant as detected in minicell experiments. Nucleotide sequence analysis revealed the presence of an open reading frame capable of specifying a protein of 479 amino acids and with a molecular weight of 56,850. The deduced amino acid sequence of the bifunctional AAC(6')-APH(2") gene product possessed two regions of homology with other sequenced resistance proteins. The N-terminal region contained a sequence that was homologous to the chloramphenicol acetyltransferase of Bacillus pumilus, and the C-terminal region contained a sequence homologous to the aminoglycoside phosphotransferase of Streptomyces fradiae. Subcloning experiments were performed with the AAC(6')-APH(2") resistance determinant, and it was possible to obtain gene segments independently specifying the acetyltransferase and phosphotransferase activities. These data suggest that the gene specifying the AAC(6')-APH(2") resistance enzyme arose as a result of a gene fusion. PMID- 3015887 TI - In vivo phosphorylation of Saccharomyces cerevisiae ribosomal protein S10 by cyclic-AMP-dependent protein kinase. AB - Using wild-type Saccharomyces cerevisiae strains and mutants which are defective in the regulatory subunit of cyclic-AMP-dependent protein kinase (bcy1) and phosphoprotein phosphatase activity (ppd1), we demonstrated that a cyclic-AMP dependent protein kinase phosphorylated the S. cerevisiae ribosomal protein S10 in vivo. S10 was not dephosphorylated in bcy1 or ppd1 mutants after heat shock. The phosphorylated forms of S10 were diminished during the stationary phase in bcy1 and ppd1 mutants as well as in wild-type cells. PMID- 3015886 TI - Transposition of the gram-positive transposon Tn917 in Escherichia coli. AB - The streptococcal transposon Tn917 was demonstrated to transpose in Escherichia coli from the Bacillus subtilis-E. coli shuttle plasmid pHK1207 into an F' plasmid derivative. Subsequently, a second round of transposition from the F' plasmid into pACYC184 could be readily demonstrated. These results represent the initial demonstration of the transposition of a gram-positive transposon in a gram-negative bacterium at a relatively high frequency. PMID- 3015888 TI - Transposition of Tn917 in Bacillus megaterium. AB - Transposon Tn917, carried on plasmid pTV1, was introduced into Bacillus megaterium and transposed efficiently and apparently randomly. Insertional mutations included at least eight different auxotrophic loci, two carbon source loci, and sporulation loci. One trp::Tn917 mutation was further verified as an insertion by both reversion and transduction. PMID- 3015889 TI - Temperate Bacillus bacteriophage SP16 genome is circularly permuted and terminally redundant. AB - The physical nature of temperate Bacillus bacteriophage SP16 DNA was analyzed by electron microscopy, exonuclease digestion, denaturation-renaturation experiments, and restriction enzyme analysis. The SP16 genome is a linear molecule 60.0 +/- 2.0 kilobases in length without cohesive ends. Electron micrographs of denatured and renatured SP16 DNA showed that the DNA is circularly permuted. The genome possesses terminal redundancy, as demonstrated by electron microscopy of exonuclease III-digested DNA. PMID- 3015890 TI - Iron respiration-driven proton translocation in aerobic bacteria. AB - Washed cell suspensions of Aquaspirillum magnetotacticum MS-1, A. itersonii E12639, Bacillus subtilis 6633, and Escherichia coli CSH27 translocated protons in response to the added oxidant O2 or NO3-, with triphenylmethylphosphonium bromide as the permeant ion. Iron respiration-driven proton translocation was observed in A. magnetotacticum MS-1, B. subtilis, and E. coli but not in a nonmagnetic strain of A. magnetotacticum (strain NM-1A) or with A. itersonii. Proton translocation to Fe3+ was totally inhibited by 500 microM NaN3 or 0.5 microM carbonyl cyanide m-chlorophenylhydrazone. PMID- 3015891 TI - Electron-paramagnetic-resonance spectroscopy of Bacillus subtilis cytochrome b558 in Escherichia coli membranes and in succinate dehydrogenase complex from Bacillus subtilis membranes. AB - Cytochrome b558 of the Bacillus subtilis succinate dehydrogenase complex was studied by electron-paramagnetic-resonance (EPR) spectroscopy. The cytochrome amplified in Escherichia coli membranes by expression of the cloned cytochrome gene and in the succinate dehydrogenase complex immunoprecipitated from solubilized B. subtilis membranes, respectively, is shown to be low spin with a highly anisotropic (gmax approximately equal to 3.5) EPR signal. The amino acid residues most likely forming fifth and sixth axial ligands to heme in cytochrome b558 are discussed on the basis of the EPR signal and the recently determined gene sequence (K. Magnusson, M. Philips, J.R. Guest, and L. Rutberg, J. Bacteriol. 166:1067-1071, 1986) and in comparison with other b-type cytochromes. PMID- 3015893 TI - Plasma levels of cortisol, corticotropin, and beta-endorphin in patients with major depression. AB - Efforts to elucidate the abnormal mechanism of corticotropin and beta-endorphin in major depression have yielded conflicting findings. The relationship of plasma levels of cortisol, corticotropin, and beta-endorphin in 42 patients with a Research Diagnostic Criteria diagnosis of major depression, endogenous subtype was examined. Following the DST, 32 patients were nonsuppressors and 10 were suppressors. The differences between the median values for plasma corticotropin and beta-endorphin immunoreactivity were not significant at any time of measurement after the DST. PMID- 3015894 TI - A perspective on Q-cycles. AB - An examination is made of both the Q-cycle and b-cycle formulations of electron transfer and energy conservation in the cytochrome bc1 complex. A working hypothesis for the complex is presented, based upon the Q-cycle notion of vectorial reaction sites, but incorporating the b-cycle feature of semiquinone movement between these sites. PMID- 3015892 TI - Cloning of the aroP gene and identification of its product in Escherichia coli K 12. AB - The aroP gene of Escherichia coli K-12 was located in a ca. 1.2-kilobase region of DNA. The aroP gene product was identified as a membrane-bound protein with an apparent molecular weight of approximately 37,000. PMID- 3015895 TI - The semiquinone cycle. A hypothesis of electron transfer and proton translocation in cytochrome bc-type complexes. AB - The Q cycle and the b cycle are the main current models of action of the cytochrome bc-type complexes of mitochondria, bacteria, and chloroplasts. Both are based on the concept, proposed in 1972, of two sequential one-electron oxidations of (ubi)quinol along two discrete pathways which operate at different redox potentials, and with bound semiubiquinone as an intermediate. The models differ in two respects, viz. in the pathway of electron transfer and the principle of linkage of electron transfer to proton translocation. In this article we outline a new model, called the semiquinone or, simply, SQ cycle, which is based on the electron transfer principles of the b cycle but which incorporates the Q cycle concept of direct coupling between electron transfer and proton translocation through action of ubiquinone. PMID- 3015896 TI - The pathway of electron transfer in the dimeric QH2: cytochrome c oxidoreductase. AB - The experimental data currently available suggest that QH2:cytochrome c oxidoreductase functions according to a Q-cycle type of mechanism. The molecular weight of the enzyme in a natural or artificial phospholipid bilayer or in solution corresponds to that of a dimer. The pre-steady state kinetics of reduction of the prosthetic groups indicate that the enzyme is functionally dimeric. A double Q cycle is proposed, describing the pathway of electron transfer in the dimeric QH2:cytochrome c oxidoreductase. According to this scheme, the two monomeric halves of the enzyme act in a cooperative fashion to complete the catalytic cycle. It is proposed that high-potential cytochrome b-562 and low-potential cytochrome b-562 act cooperatively, viz. as a functional pair, in the antimycin-sensitive reduction of ubiquinone to ubiquinol. PMID- 3015897 TI - Phorphorylative electron transport chains lacking a cytochrome bc1 complex. AB - Electron transport-coupled phosphorylation with fumarate as terminal acceptor in Wolinella succinogenes yields less than 1 ATP/2 electrons. The delta mu H generated by the electron transport is 0.18 V and the H+/electron ratio is 1. The electron transport chain is made up of two dehydrogenases (hydrogenase and formate dehydrogenase) that catalyze the reduction of menaquinone, and fumarate reductase which catalyzes the oxidation of menaquinol. C-type cytochromes are not involved. The phosphorylative electron transport with sulfur as terminal acceptor in W. succinogenes or Desulfuromonas acetoxidans does not involve known quinones. The ATP yields should be even smaller than those with fumarate. Succinate oxidation by sulfur, which is a catabolic reaction in D. acetoxidans, is accomplished by reversed electron transport. PMID- 3015899 TI - Rapid synergistic interaction between thyroid hormone and carbohydrate on mRNAS14 induction. AB - Recent studies have shown that hepatic mRNAS14 responds rapidly to thyroid hormone administration. Moreover, this mRNA is known to increase in mass with the administration of a high carbohydrate fat-free diet. Therefore, it appears to share many of the same properties of the known hepatic lipogenic enzymes. Because the lipogenic enzymes display a synergistic interaction between thyroid hormones and carbohydrates, we investigated the kinetics of response of mRNAS14 to carbohydrate feeding, as well as its interaction with triiodothyronine (T3). We found that mRNAS14 responds rapidly to the dietary administration of sucrose in euthyroid rats, with a 2-fold increase within 30 min, and a 25-fold increase by 4 h. On the other hand, when given to hypothyroid rats, sucrose ultimately lead to only a 2-3-fold increase in the level of mRNAS14, attaining a level less than that found in starved euthyroid rats. The diminished response of mRNAS14 to sucrose in hypothyroidism could not be enhanced by insulin administration. However, administration of replacement doses of T3 (400 ng/100 g of body weight) immediately restored the rapid response to sucrose feeding. The response of sucrose and T3 was synergistic. Dose-response studies with T3 indicated that the rapid interaction between T3 and sucrose was limited by the occupancy of the T3 nuclear receptor. A similar synergistic response to T3 and glucose was noted in primary hepatocyte cultures, thus indicating that the synergism between these two stimuli is not due to changes in extrahepatic hormones or metabolites. Our data are most consistent with the hypothesis that the T3-nuclear receptor complex multiplies a signal generated by carbohydrate metabolism to induce hepatic mRNAS14. The interaction does not appear to require the preliminary induction of carbohydrate-metabolizing enzymes and their mRNAs. PMID- 3015900 TI - Molecular basis for two forms of the G protein that stimulates adenylate cyclase. AB - Most cells contain two forms of the alpha subunit of the G protein (Gs) that stimulates adenylate cyclase; their apparent molecular weights are 45,000 and 52,000. Two cDNAs that correspond to distinct mRNAs for the alpha subunit of Gs have been cloned from a bovine adrenal library and sequenced. The sequences of the two cDNAs, designated pGs-l and pGs-S, are identical except for a single stretch of 46 nucleotides in the coding region, where four are altered and 42 are deleted in pGs-S. Expression of pGs-S and pGs-l in COS-m6 cells yields protein products with apparent molecular weights of 45,000 and 52,000, respectively, based on their mobility in sodium dodecyl sulfate-polyacrylamide gels. We conclude that pGs-S and pGs-l encode the 45- and 52-kDa forms of Gs alpha, respectively, and propose that the mRNAs encoding these proteins arise from a single gene by internal alternative RNA splicing. PMID- 3015898 TI - Experimental observations on the structure and function of mitochondrial complex III that are unresolved by the protonmotive ubiquinone-cycle hypothesis. AB - The current model of the protonmotive ubiquinone cycle as applied to mitochondrial ubiquinol-cytochrome c reductase complex (Complex III) is able to explain a number of previously puzzling observations concerning electron-transfer and proton translocating functions of the complex. However, a number of pertinent experimental observations concerning the structure and function of this complex cannot as yet be incorporated into the present version of the ubiquinone cycle. The yet unresolved problems of electron transfer uncovered by these observations include some kinetic and thermodynamic problems, uncertainties in the binding site(s) and mode of binding of ubiquinol and inhibitors, the observed multiple spectroscopic, electrochemical, and kinetic forms of cytochromes b, iron-sulfur protein, and cytochrome c1, the multiple and overlapping effects of inhibitors, and the functional role of conformational changes in the complex. It is concluded that although the Q cycle is a valuable base for the design of future experiments, its mechanism must be reconciled with the above uncertainties as well as with the accumulated evidence that Complex III can exist in two or more interchangeable forms, exhibiting different properties with respect to electron transfer pathways, inhibitor binding, and spectral and electrochemical properties of the electron-carrier subunits. PMID- 3015901 TI - Oxidation of chloride and thiocyanate by isolated leukocytes. AB - Peroxidase-catalyzed oxidation of chloride (Cl-) and thiocyanate (SCN-) was studied using neutrophils from human blood and eosinophils and macrophages from rat peritoneal exudates. The aims were to determine whether Cl- or SCN- is preferentially oxidized and whether leukocytes oxidize SCN- to the antimicrobial oxidizing agent hypothiocyanite (OSCN-). Stimulated neutrophils produced H2O2 and secreted myeloperoxidase. Under conditions similar to those in plasma (0.14 M Cl , 0.02-0.12 mM SCN-), myeloperoxidase catalyzed the oxidation of Cl- to hypochlorous acid (HOCl), which reacted with ammonia and amines to yield chloramines. HOCl and chloramines reacted with SCN- to yield products without oxidizing activity, so that high SCN- blocked accumulation of chloramines in the extracellular medium. Under conditions similar to those in saliva and the surface of the oral mucosa (20 mM Cl-, 0.1-3 mM SCN-), myeloperoxidase catalyzed the oxidation of SCN- to OSCN-, which accumulated in the medium to concentrations of up to 40-70 microM. Sulfonamide compounds increased the yield of stable oxidants to 0.2-0.3 mM by reacting with OSCN- to yield derivatives analogous to chloramines. Stimulated eosinophils produced H2O2 and secreted eosinophil peroxidase, which catalyzed the oxidation of SCN- to OSCN- regardless of Cl- concentration. Stimulated macrophages produced H2O2 but had low peroxidase activity. OSCN- was produced when SCN- was 0.1 mM or higher and myeloperoxidase, eosinophil peroxidase, or lactoperoxidase was added. The results indicate that SCN- rather than Cl- may be the physiologic substrate (electron donor) for eosinophil peroxidase and that OSCN- may contribute to leukocyte antimicrobial activity under conditions that favor oxidation of SCN- rather than Cl-. PMID- 3015902 TI - Characterization of the phosphoenolpyruvate carboxykinase (GTP) promoter regulatory region. I. Multiple hormone regulatory elements and the effects of enhancers. AB - Transcription of the gene for cytosolic Phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) from rat liver is increased by cAMP and glucocorticoids and decreased by insulin. A PEPCK-thymidine kinase (TK) chimeric gene was transfected into FTO-2B rat hepatoma cells, which were TK-deficient. Previous studies showed that a cAMP regulatory element is located at the 5' end of the PEPCK gene. In this report, we demonstrate that the 5' end of the gene also contains a glucocorticoid regulatory element, but not one for insulin. Regions of the PEPCK gene that contain these regulatory elements were attached to the Herpes simplex virus TK structural gene containing its own promoter. The hormone regulatory elements within the 5' flanking region of the PEPCK gene conferred cAMP and glucocorticoid responsiveness on the TK gene after transfection into FTO-2B cells. Like viral enhancer elements, these regulatory elements functioned properly when placed in either orientation at various positions 5' or 3' to TK. The presence of the SV40 enhancer element upstream from the PEPCK-TK gene had little effect on the basal level of expression or hormonal regulation of the chimeric gene. PMID- 3015903 TI - Characterization of the phosphoenolpyruvate carboxykinase (GTP) promoter regulatory region. II. Identification of cAMP and glucocorticoid regulatory domains. AB - Hormonal regulatory elements within the phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) promoter region were mapped using a series of 5' deletions linked to the amino-3'-glycosyl phosphotransferase structural gene. These deletion mutants were stably transfected into the genome of FTO-2B hepatoma cells. A 47-base pair region of the PEPCK promoter was identified which was essential for stimulation by dibutyryl cAMP. A 12-base pair core sequence (CTTACGTCAGAG) within this region shows significant homology with sequences in four other cAMP-regulated genes. There are two glucocorticoid regulatory elements within the promoter, as well as an inhibitory element which depresses the level of basal gene transcription. The deletion of this inhibitory sequence prevents the induction of the chimeric gene by dexamethasone. The existence of the hormone regulatory domains within the PEPCK promoter was confirmed by attaching these elements upstream of the heterologous Herpes simplex virus thymidine kinase structural gene, containing its own promoter. PMID- 3015904 TI - Functional coupling between enzymes of the chromaffin granule membrane. AB - The reactions of cytochrome b561 with other redox-active components of the adrenal chromaffin granule were examined using optical difference spectroscopy. It was shown that there is no direct electron transfer between the cytochrome and dopamine beta-hydroxylase, but that in the presence of ascorbate, turnover of dopamine beta-hydroxylase causes an oxidation of the cytochrome, which is partially reversed by the action of the mitochondrial NADH:A-. oxidoreductase. Thus, these three proteins may be functionally coupled via ascorbate. A quantitative study of the relationship between the redox state of the cytochrome and the ascorbate radical concentration measured by EPR showed that ascorbate reduces the cytochrome in a one-electron transfer reaction. Generation of a proton electrochemical gradient across the granule membrane causes only a small (20 mV) increase in the cytochrome midpoint potential suggesting the cytochrome is not a proton pump. The data are consistent with a model in which cytochrome b561, by reacting with ascorbate or ascorbate free radical on either side of the granule membrane, could couple the ascorbate-consuming reaction of the dopamine beta-hydroxylase inside the chromaffin granule to the ascorbate-regenerating reaction of the NADH:A-. oxidoreductase on the outer mitochondrial membrane. The H+-ATPase of the granule membrane could both drive the flow of electrons in the direction from cytosol to granule and replenish protons consumed by the turnover of dopamine beta-hydroxylase inside the granule. PMID- 3015905 TI - Electron transfer across the chromaffin granule membrane. Use of EPR to demonstrate reduction of intravesicular ascorbate radical by the extravesicular mitochondrial NADH:ascorbate radical oxidoreductase. AB - A two-compartment electron paramagnetic resonance system has been developed in which the membrane-impermeable spin probe Ni(en)2+3 is used to selectively eliminate the EPR signal from extravesicular ascorbate radical, such that radicals in intra- and extravesicular compartments can be distinguished. Using this system, we have shown that an increase in ascorbate radical in the extravesicular medium is reflected by an increase in ascorbate radical within resealed chromaffin granule ghosts containing trapped ascorbate but has no effect on radical concentrations inside liposomes containing ascorbate. This indicates that the chromaffin granule membrane contains a component, not present in liposomes, that allows equilibration between the intra- and extravesicular ascorbate/ascorbate radical couples. This component is probably cytochrome b561. We further show that activation of the mitochondrial NADH:ascorbate radical oxidoreductase in the extravesicular medium causes a decrease in intravesicular ascorbate radical in chromaffin granule ghosts but not in liposomes. These data provide direct experimental evidence for the hypothesis that the adrenal medullary mitochondrial NADH:ascorbate radical oxidoreductase could drive the re reduction of ascorbate free radical generated inside the chromaffin granule by the turnover of dopamine beta-hydroxylase, without the ascorbate radical ever having to leave the granule. PMID- 3015907 TI - Binding of ADP and ATP analogs to cross-linked and non-cross-linked acto X S-1. AB - We previously determined the binding constants of ADP, adenylyl imidodiphosphate (AMP-PNP), and inorganic pyrophosphate (PPi) to acto . myosin subfragment 1 (acto X S-1) by measuring the dissociation of acto X S-1 as a function of ATP analog concentration (Greene, L.E., and Eisenberg, E. (1980) J. Biol. Chem. 255, 543 548). In the present study, we reinvestigated this question by measuring the extent to which these ATP analogs inhibit the acto X S-1 ATPase activity using both cross-linked actin X S-1 and non-cross-linked proteins. No significant difference was found between the cross-linked and non-cross-linked acto X S-1 complexes in their affinity for either ADP or AMP-PNP. The binding constant of ADP to acto X S-1 determined by the inhibition method was in excellent agreement with that obtained previously by the dissociation method, both techniques giving values of about 7 X 10(3) M-1. However, this was not the case for AMP-PNP and PPi, with the inhibition method giving about 10-fold weaker binding constants than those determined previously by the dissociation method. Upon redoing our dissociation experiments over a wider range of actin concentrations than we used previously, we now find that the dissociation method gives much weaker values for the binding constants of PPi and AMP-PNP to acto X S-1, i.e. values on the order of 4 X 10(2) M-1. The very weak binding of these ATP analogs to acto X S-1 makes it difficult to obtain these values with great accuracy. Nevertheless, they seem to be in good agreement with the binding constants determined by the inhibition method. The weak binding of AMP-PNP and PPi to acto X S-1 is consistent with the recent fiber studies of Pate and Cooke (Pate, E., and Cooke, R. (1985) Biophys. J. 47, 773-780) and Schoenberg and Eisenberg (Schoenberg, M., and Eisenberg, E. (1986) Biophys. J. 48, 863-872). PMID- 3015906 TI - Reduced ouabain inhibition of Na,K-activated adenosine triphosphatase in cultured cell recipients of the ouabain-resistance gene. AB - Using sequential DNA-mediated gene transfer, a mouse DNA sequence (ouaR) that confers resistance to the cytotoxic effects of ouabain in cultured cells was recently isolated. To determine the basis of this resistance, we examined the ouabain-resistant phenotype of cells from each stage of the transfers that led to isolation of the ouaR gene, as well as the recipient of the isolated gene. Membranes prepared from the original DNA donor, the two intermediate stages of the sequential gene transfer, and the final isolated gene recipient contained at least two functionally distinct forms of the Na,K-ATPase. In addition to a form indistinguishable in ouabain affinity from that in the parental lines, these ouabain-resistant cells contained a form characterized by a low affinity for the glycoside. That the low-affinity form contributed to ouabain resistance was suggested by the correlation between its relative abundance and the ability to limit the increase in intracellular Na+ content when exposed to ouabain. Maintenance of the final recipient of the isolated ouaR gene for 24 h in 10 microM ouabain had no effect on the specific activity of the resistant form of the Na,K-ATPase, implying that the low-affinity form was not uniquely inducible by ouabain. These results suggest that the ouabain-resistant phenotype conferred by ouaR is attributable to expression of a Na,K-ATPase with a low affinity for the glycoside. PMID- 3015908 TI - Preparation of 32P-labeled murine immune interferon and its binding to the mouse immune interferon receptor. AB - Murine immune interferon (Mu-IFN-gamma) can be radiolabeled with [gamma-32P]ATP by the catalytic subunit of cAMP-dependent protein kinase. The resulting 32P labeled Mu-IFN-gamma (32P-Mu-IFN-gamma) with high radiological specific activity (60-260 muCi/micrograms) retains biological activity. Acid hydrolysis of 32P-Mu IFN-gamma or 32P-labeled human IFN-gamma leads to the release of [32P]phosphoserine but not phosphothreonine or phosphotyrosine. With 32P-Mu-IFN gamma, we have demonstrated that there are 5 X 10(3) to 1.5 X 10(4) receptors per cell on several murine cell lines of diverse origin and that the Kd at 24 degrees C for these cells is in the range of 1 X 10(-10) to 1 X 10(-9) M. Covalent binding of 32P-Mu-IFN-gamma to its receptor results in the formation of several specific high-molecular weight products, the major one of which has an apparent molecular weight of 90,000-100,000. If this represents a 1:1 complex of Mu-IFN gamma and its receptor (or its binding subunit), the murine interferon gamma receptor has a molecular weight of 75,000-85,000. PMID- 3015909 TI - Molecular cloning and nucleotide sequence of a complete human uroporphyrinogen decarboxylase cDNA. AB - We have cloned and sequenced a full-length cDNA coding for human uroporphyrinogen decarboxylase. The deduced 367-amino acid sequence is consistent with the molecular weight, the partial amino acid sequence of cyanogen bromide peptides, and the total amino acid composition of the purified enzyme. Southern analysis of human genomic DNA shows that its gene is present as a single copy in the human genome, and Northern analysis demonstrates the presence of a single size species of mRNA in erythroid and non-erythroid tissues and in several cultured cell lines. We have also demonstrated that the level of uroporphyrinogen decarboxylase mRNA is markedly increased in tissues or cell lines of erythroid origin and that this is due to a tissue-specific transcriptional activation of the uroporphyrinogen decarboxylase gene. PMID- 3015910 TI - Direct demonstration of adrenocorticotropin-induced changes in cytoplasmic free calcium with aequorin in adrenal glomerulosa cell. AB - Effects of adrenocorticotropin (ACTH) on cytoplasmic free calcium concentration, [Ca2+]c, have been measured in adrenal glomerulosa cells using a calcium sensitive photoprotein, aequorin. ACTH causes a rapid and transient increase in [Ca2+]c. Dose response study demonstrates that 1 pM ACTH induces an elevation of [Ca2+]c and that effect of ACTH appears to be saturated at 100 pM. ACTH action is greatly inhibited but not abolished by removal of extracellular calcium and is completely blocked in medium containing no added calcium and 1 mM EGTA. Under similar conditions, angiotensin II induces a remarkable rise in [Ca2+]c. ACTH action is not affected by pretreatment with dantrolene, which considerably decreases angiotensin II action on [Ca2+]c. One micromolar forskolin, which mimics 1 nM ACTH-mediated elevation of intracellular cAMP, does not increase [Ca2+]c nor modulates changes in [Ca2+] induced by a low dose of ACTH. One hundred micromolar forskolin or 1 mM 8-bromo-cAMP, however, increases [Ca2+]c even in calcium-free medium containing 1 mM EGTA. When glomerulosa cells are co loaded with aequorin and quin2, angiotensin II-induced change in aequorin signal is greatly reduced, and ACTH-induced change is abolished. Quin2 loading results in accumulation of calcium in the cell under both unstimulated and stimulated conditions. These results indicate that ACTH increases [Ca2+]c by cAMP independent mechanism, that ACTH action on [Ca2+]c is exclusively dependent on extracellular calcium, and that quin2 is unable to detect the rapid change in [Ca2+]c because of its calcium chelating activity. PMID- 3015911 TI - Quaternary structure of the calf testis follitropin receptor. AB - The quaternary structural relationships between subunits of the follitropin (FSH) receptor were determined through the use of the reversible, homobifunctional, chemical cross-linking reagent bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES) after formation of covalent 125I-azidobenzoyl-FSH-receptor subunit complexes by photoaffinity labeling. This experimental approach resulted in the formation of high molecular mass complexes (116, 172, and 320 kDa) as detected by autoradiography. After reversal of the BSOCOES cross-links, a second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of these complexes demonstrated that two lower molecular mass complexes, 64 and 84 kDa, identified previously as specific components of the FSH receptor by photoaffinity labeling alone (Smith, R. A., Branca, A. A., and Reichert, L. E., Jr. (1985) J. Biol. Chem. 260, 14297-14303) were contained within the 116- and 172-kDa complexes. In addition, the 116-kDa complex was not found to be a component of the 172-kDa complex. Since the high molecular weight complexes have molecular weights large enough to contain additional unlabeled proteins, these data indicate the possibility that there are several distinct FSH receptor subunits. Furthermore, the observation of the 320-kDa band, taken together with the observed structural relationships, suggests that the FSH receptor may contain two each of three distinct subunits with approximate molecular weights of 32,000, 48,000, and 86,000, respectively. PMID- 3015912 TI - Four Ca2+-dependent proteinase activities isolated from crustacean muscle differ in size, net charge, and sensitivity to Ca2+ and inhibitors. AB - Four Ca2+-dependent proteinase activities in lobster claw and abdominal muscle have been resolved by high-performance liquid chromatography on gel filtration and ion-exchange columns. These activities, which do not appear to be generated by autolytic or other degradative processes, differed from each other in molecular weight (peak I, Mr = 310,000; peak IIa, Mr = 125,000; peak IIb, Mr = 195,000; peak III, Mr = 59,000) and net charge, as indicated by elution from an ion-exchange column with a NaCl gradient. Although optimum activity occurred at 5 10 mM Ca2+ at pH 6.8, the enzymes differed in activation at lower Ca2+ concentrations. The concentrations required for half-maximal activation were 0.6 mM for peak III, 1 mM for peak I, 1.5 mM for peak IIa, and 2 mM for peak IIb. Only the peak III proteinase was active at 100 microM Ca2+; none were active at 10 microM and below. Although the lobster Ca2+-dependent proteinases were all inhibited, from 75 to 98%, by the cysteine proteinase inhibitors leupeptin, N-[N (L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine, and iodoacetamide, they showed differential responses to the aspartic proteinase inhibitor pepstatin and the serine proteinase inhibitor phenylmethanesulfonyl fluoride. Peak I was moderately (26%) inhibited by phenylmethanesulfonyl fluoride, whereas peaks IIb and III were inhibited 26 and 90%, respectively, by pepstatin. This is the first description of multiple forms of Ca2+-dependent proteinase that require Ca2+ at millimolar levels in any tissue, either vertebrate or invertebrate. PMID- 3015914 TI - Biosynthesis, processing, and secretion of M and Z variant human alpha 1 antitrypsin. AB - The Z genetic variant of human alpha 1-antitrypsin (alpha 1AT) is associated with decreased serum alpha 1AT levels, hepatic inclusion bodies, and an increased risk of lung and liver disease. We studied the biosynthesis, processing, and secretion of normal and Z variant alpha 1AT in cell-free translation systems, reconstituted in vitro processing systems, and in the Xenopus oocyte secretory system. Human liver mRNA was prepared from normal subjects (PiMM) and from individuals homozygous for alpha 1AT deficiency (PiZZ). Cell-free translation resulted in the synthesis of 49,000-Da preproteins with a 23-amino acid signal sequence. The genetic variants were synthesized at comparable levels and could be distinguished on the basis of charge. The majority of the amino acids in the ZZ signal peptide were identified and found to be the same as those comprising the MM signal sequence. These proteins were co-translationally processed with similar efficiency by dog pancreas microsomes, producing 52,000-Da glycoproteins which were completely translocated across the endoplasmic reticulum membrane. When the human liver RNA preparations were injected into Xenopus oocytes, both of the alpha 1AT variants were synthesized intracellularly and alpha 1AT was detected in the medium of all oocytes injected with MM RNA. However, the Z variant accumulated within the microsomal vesicles of the cell and was undetectable or present at decreased levels in the medium. We conclude that the single amino acid substitution in the Z variant of alpha 1AT does not affect its synthesis or co translational processing but that it strongly affects its transport from the rough endoplasmic reticulum through the secretory pathway. PMID- 3015913 TI - Eukaryotic topoisomerase II. Characterization of enzyme turnover. AB - While the binding of adenyl-5'-yl imidodiphosphate (App(NH)p) to Drosophila melanogaster topoisomerase II induces a double-stranded DNA passage reaction, its nonhydrolyzable beta,gamma-imidodiphosphate bond prevents enzyme turnover (Osheroff, N., Shelton, E. R., and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9536 9543). Therefore, this ATP analog was used to characterize the interactions between Drosophila topoisomerase II and DNA which occur after DNA strand passage but before enzyme turnover. In the presence of App(NH)p, a stable post-strand passage topoisomerase II-nucleic acid complex is formed when circular DNA substrates are employed. Although noncovalent in nature, these complexes are resistant to increases in ionic strength and show less than 5% dissociation under salt concentrations (greater than 500 mM) that disrupt 95% of the enzyme-DNA interactions formed in the absence of App(NH)p or under a variety of other conditions that do not support DNA strand passage. These results strongly suggest that the process of enzyme turnover not only regenerates the active conformation of topoisomerase II but also confers upon the enzyme the ability to disengage from its nucleic acid product. Experiments with linear DNA molecules indicate that after strand passage has taken place, topoisomerase II may be able to travel along its DNA substrate by a linear diffusion process that is independent of enzyme turnover. Further studies demonstrate that the regeneration of the enzyme's catalytic center does not require enzyme turnover, since topoisomerase II can cleave double-stranded DNA substrates after strand passage has taken place. Finally, while the 2'-OH and 3'-OH of ATP are important for its interaction with Drosophila topoisomerase II, neither are required for turnover. PMID- 3015915 TI - Regulation of the (Na+ + K+)-ATPase in cultured chick skeletal muscle. Modulation of expression by the demand for ion transport. AB - The levels of (Na+ + K+)-ATPase expression during muscle development and in response to modulation of demand for ion transport were studied in chick skeletal muscle cells in culture. The number of (Na+ + K+)-ATPase molecules on the myogenic cell surface, quantified with 125I-labeled monoclonal antibodies, increased 20-fold during muscle differentiation, with a substantial increase in (Na+ + K+)-ATPase molecules/unit area of membrane. The demand for sodium ion transport by the (Na+ + K+)-ATPase was modulated by activating voltage-sensitive sodium channels with veratridine or exposing cultures to low [K+]o (0.5 mM). Exposure to veratridine (10 microM) resulted in a 60-100% increase in cell surface and a smaller increase in intracellular (Na+ + K+)-ATPase over a 24-36-h period. Neither high [K+]o (50 mM) nor Ca2+ ionophore A23187 (1 microM) produced any such change, suggesting that neither membrane depolarization nor elevated cytosolic calcium was mediating the effect of veratridine. Veratridine stimulated up-regulation was specific for the (Na+ + K+)-ATPase, blocked by tetrodotoxin, and completely reversible. The kinetics of the reversal (down-regulation) process were much faster (t1/2 = 3 h) than those of up-regulation (t1/2 = 18 h). Up regulation of the (Na+ + K+)-ATPase by veratridine occurred by a combination of two mechanisms: the first an early phase involving a stimulated biosynthesis of the (Na+ + K+)-ATPase and a later phase in which the biosynthetic rate returned to approximately control levels while the degradation rate slowed (t1/2 control = 31 h, t1/2 veratridine = 64 h). PMID- 3015916 TI - A phospholamban protein phosphatase activity associated with cardiac sarcoplasmic reticulum. AB - Canine cardiac sarcoplasmic reticulum vesicles contain intrinsic phospholamban protein phosphatase activity, which is also effective in dephosphorylating phosphorylase a. The phosphatase associated with sarcoplasmic reticulum membranes was solubilized with Triton X-100 and subjected to chromatography on Mono Q HR 5/5 and polylysine-agarose. A single peak of phosphatase activity was eluted from each column and it was coincident for both phospholamban and phosphorylase a, used as substrates. Thermal denaturation of the enzyme resulted in progressive and coincident loss of both phospholamban and phosphorylase a phosphatase activities. Enzymic activity was partially inhibited by protein phosphatase inhibitor 1. Migration of the enzyme during sucrose density gradient ultracentrifugation corresponded to a globular protein with an apparent Mr of 46,000. This enzyme preparation could dephosphorylate both the calcium-calmodulin dependent as well as the cAMP-dependent sites on phospholamban. Thus, dephosphorylation of phospholamban by this sarcoplasmic reticulum-associated phosphatase may participate in modulating sarcoplasmic reticulum function in cardiac muscle. PMID- 3015917 TI - Action of insulin on the subcellular metabolism of polyphosphoinositides in isolated rat hepatocytes. AB - The effect of insulin on 32Pi incorporation into phospholipids in various subcellular sites of isolated rat hepatocytes was investigated. After labeling the phospholipids of hepatocytes from rats previously starved for 24 h with 32Pi (10 mu Ci/10(6) cells) for 90 min, either saline or insulin (32 nM) was added. Following incubations of 1, 5, and 30 min, chilled cells were rapidly washed, homogenized in the presence of inhibitors of phospholipid degradation, and fractionated into the major subcellular organelles. Phospholipids were extracted from plasma membranes, microsomes, lysosomes, mitochondria, and nuclei with acidic chloroform:methanol. The aqueous deacylation products were separated by anion exchange high performance liquid chromatography, and the 32Pi incorporated into all the major diacylglycerophospholipids was determined. In parallel experiments, the specific radioactivity of 32Pi and [gamma-32P]ATP was determined. The results revealed that insulin had no effect on the turnover of the major phospholipids, including the polyphosphoinositides, of all subcellular compartments analyzed relative to the control. In addition, there were no significant differences in the amount and 32P labeling of cellular orthophosphate between saline- and insulin-treated cells. The specific radioactivity of [gamma 32P]ATP was increased by 20% after 30-min treatment with insulin, requiring appropriate correction of 32P-labeled phosphatidic acid, phosphatidylinositol 4 phosphate, and phosphatidylinositol 4,5-bisphosphate for estimation of mass changes at near steady-state labeling of cellular ATP. PMID- 3015918 TI - Effects of protein kinase C activation after epidermal growth factor binding on epidermal growth factor receptor phosphorylation. AB - The possible role of epidermal growth factor (EGF) receptor phosphorylation at threonine 654 in modulating the protein-tyrosine kinase activity of EGF-treated A431 cells has been studied. It has been suggested that EGF could indirectly activate a protein-serine/threonine kinase, protein kinase C, that can phosphorylate the EGF receptor at threonine 654. Protein kinase C is known to be activated, and threonine 654 is phosphorylated, when A431 cells are exposed to 12 O-tetradecanoylphorbol-13-acetate (TPA). The protein-tyrosine kinase activity of EGF receptors is normally evidenced in EGF-treated cells by phosphorylation of the receptor at tyrosine. This is inhibited when TPA-treated cells are exposed to EGF. We now show that receptor phosphorylation at threonine 654 can also be detected in EGF-treated A431 cells, presumably due to indirect stimulation of protein kinase C or a similar kinase. Some receptor molecules are phosphorylated both at threonine 654 and at tyrosine. Since prior phosphorylation at threonine 654 inhibits autophosphorylation, we propose that protein kinase C can phosphorylate the threonine 654 of autophosphorylated receptors. This provides evidence for models in which protein kinase C activation, consequent upon EGF binding, could reduce the protein-tyrosine kinase activity of the EGF receptor. Indeed, we find that 12-O-tetradecanoylphorbol-13-acetate, added 10 min after EGF, further increases threonine 654 phosphorylation and induces the loss of tyrosine phosphate from A431 cell EGF receptors. PMID- 3015919 TI - Reconstitution of the muscarinic acetylcholine receptor. Guanine nucleotide sensitive high affinity binding of agonists to purified muscarinic receptors reconstituted with GTP-binding proteins (Gi and Go). AB - Muscarinic acetylcholine receptors purified from porcine brain were reconstituted with two kinds of GTP-binding proteins (Gi and Go). The binding of agonists was affected by guanine nucleotides when the receptor was reconstituted with either Gi or Go, but not in the absence of one of the GTP-binding proteins. The displacement curves with agonists for the [3H]quinuclidinyl benzylate [( 3H]QNB) binding were explained by assuming there are two sites with different affinities for a given agonist. The proportion of the high affinity site increased with increasing concentrations of the GTP-binding proteins, and the maximum value represented 50-70% of the total [3H]QNB-binding sites. Reconstitution of the receptor with both Gi and Go did not increase the proportion any further. These results indicate that Gi and Go interact with the same site, which rules out the possibility that there are two kinds of muscarinic receptors, one interacting with Gi and the other with Go. GDP as well as GTP decreased the affinity for the agonists of the muscarinic receptors reconstituted with Gi or Go. The conversion of GDP to GTP during the incubation was less than 1%, indicating that the effect of GDP is not due to its conversion to GTP, and that the binding of either GTP or GDP with the GTP-binding proteins suppresses their interaction with the receptor. PMID- 3015920 TI - Thyrotropin-releasing hormone stimulation of polyphosphoinositide hydrolysis in GH3 cell membranes is GTP dependent but insensitive to cholera or pertussis toxin. AB - Thyrotropin-releasing hormone (TRH), like numerous other Ca2+-mobilizing agonists, has been found to stimulate polyphosphoinositide hydrolysis in responsive cells. The present studies further clarify the mechanism of action of this peptide hormone by demonstrating direct in vitro effects of TRH on polyphosphoinositide hydrolysis in GH3 pituitary cell membranes. Membranes from [3H]myoinositol-labeled cells were found to generate inositol bis- and tris- but not monophosphate upon incubation. Inositol polyphosphate generation was stimulated 2-3-fold by nanomolar concentrations of TRH in a reaction which was potentiated by micromolar concentrations of GTP; hormone-stimulated hydrolysis observed in the absence of GTP was fully antagonized by guanosine 5'-O-(2 thiodiphosphate). Guanosine 5'-O-(3-thiotriphosphate), Ca2+, and sodium fluoride also activated phosphoinositide hydrolysis in vitro. Stimulated inositol polyphosphate generation was accompanied by stimulated 1,2-diacylglycerol formation. Evidence that both phosphatidylinositol 4,5-bisphosphate as well as phosphatidylinositol 4-phosphate served as substrates for the activated phosphoinositide phosphodiesterase is presented. Pretreatment of GH3 cells with cholera or pertussis toxin did not influence stimulated hydrolysis in membranes. It is concluded that the TRH receptor directly regulates polyphosphoinositide hydrolysis in GH3 cell plasma membranes by a GTP-dependent process. The GTP dependence does not appear to be mediated through a cholera or pertussis toxin substrate and may involve a novel GTP-binding protein (NP). PMID- 3015921 TI - Guanine nucleotide effects on catecholamine secretion from digitonin permeabilized adrenal chromaffin cells. AB - The nonhydrolyzable GTP analogue guanosine 5'-(beta, gamma-imido)triphosphate (GMP-PNP) produced an ATP-dependent but Ca2+-independent stimulation of [3H]norepinephrine release from permeabilized chromaffin cells. This stimulation of secretion was 25-35% of the secretion induced by 10 microM Ca2+. A similar Ca2+-independent stimulation was produced by other non-hydrolyzable GTP analogues. No effect was seen with a variety of other nucleotides, including GTP. The GMP-PNP effect was specifically inhibited by low concentrations of guanine nucleotides. Addition of cAMP did not mimic the Ca2+-independent GMP-PNP effect, but did slightly enhance Ca2+-dependent secretion. Pretreatment with pertussis toxin had no effect on Ca2+-dependent secretion or on the GMP-PNP effect. There was no detectable diglyceride or inositol phosphate produced during GMP-PNP treatment, and addition of diglyceride and inositol trisphosphate did not induce secretion. Guanosine 5'-(beta-thio)diphosphate (GDP-beta-S), in addition to its ability to inhibit the GMP-PNP effect, partially inhibited Ca2+-dependent secretion. At 10 microM free Ca2+, the effects of GMP-PNP and Ca2+ were nonadditive. In fact, secretion in the presence of both GMP-PNP and 10 microM Ca2+ was slightly less than secretion due to Ca2+ alone. These data suggest that a guanine nucleotide-dependent process interacts in some way with one or more components of the normal Ca2+-dependent secretory pathway. However, it may not be an intrinsic part of the mechanism underlying Ca2+-dependent secretion. PMID- 3015922 TI - Hepatic Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Partial stabilization by molybdate. AB - In structure and general mode of action, the Ah receptor is very similar to the receptors for steroid hormones. Molybdate previously has been shown to be highly effective at preserving ligand-binding function in steroid receptors during their exposure to elevated temperature or high ionic strength and at stabilizing steroid receptors as high molecular weight oligomeric complexes. Since such stabilization by molybdate can be very useful during characterization and purification of receptors, we tested the effects of molybdate on the Ah receptor to determine if the Ah receptor, like the receptors for steroid hormones, might be stabilized. In hepatic cytosols from C57BL/6N mice and Sprague-Dawley rats, molybdate concentrations up to 30 mM in homogenizing and analysis buffers did not alter the concentration of specific Ah receptor sites detected by binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin. However, inclusion of 20 mM molybdate in the homogenizing buffer did significantly protect unliganded Ah receptor from thermal inactivation at 20 degrees C and from KCl-induced loss of ligand-binding ability. In accord with previous reports, 20 mM molybdate in homogenizing and analysis buffers greatly increased the concentration of detectable glucocorticoid receptor in rat hepatic cytosol and estrogen receptor in rat uterine cytosol. Exposure to 0.4 M KC1 caused the glucocorticoid receptor from rat liver to shift sedimentation from approximately equal to 8 S to approximately equal to 4 S and caused a severe loss of specific glucocorticoid binding. Presence of 20 mM molybdate stabilized the glucocorticoid receptor as a single discrete peak sedimenting at approximately equal to 8 S. In contrast, the Ah receptor from rat liver exposed to 0.4 M KC1 in the presence of molybdate sedimented as biphasic peaks; one peak (approximately equal to 9.5 S) corresponded to the form of Ah receptor observed at low ionic strength, while the other peak (approximately equal to 5.5 S) corresponded to the form of Ah receptor seen in cytosol treated with 0.4 M KC1 in the absence of molybdate. Addition of heparin to hepatic cytosols from mice or rats shifted sedimentation of Ah receptor from approximately equal to 9.5 S to approximately equal to 5.5 S. Molybdate, again, provided stabilization in the approximately equal to 9.5 S form, but only for about one-half the total Ah receptor content in both rat and mouse hepatic cytosols. In sum, molybdate is far less effective at stabilizing rodent Ah receptors than it is at stabilizing steroid receptors in the same species. PMID- 3015923 TI - Mechanism of ubiquitin carboxyl-terminal hydrolase. Borohydride and hydroxylamine inactivate in the presence of ubiquitin. AB - Ubiquitin (Ub) carboxyl-terminal hydrolase (E) catalyzes the hydrolysis, at the Ub-carboxyl terminus, of a wide variety of C-terminal Ub derivatives. We show that the enzyme is inactivated by millimolar concentrations of either sodium borohydride or hydroxylamine, but only if Ub is present. We have interpreted these results on the assumption that the hydrolase mechanism is one of nucleophilic catalysis with an acyl-Ub-E intermediate. The borohydride inactivated enzyme has the following properties. It is a stoichiometric complex of E and Ub containing tritium from sodium boro[3H]hydride. This complex is stable at neutral pH in 5 M urea and can be isolated on the basis of size on a sieving column, but a labeled product the size of Ub is released under more strongly denaturing conditions. The "Ub" released in acid is Ub-carboxyl-terminal aldehyde, based on the observations that: it contains the tritium present in the reduced complex and it is able to form the inactive enzyme from a stoichiometric amount of fresh enzyme, and inactivation is accompanied by E-Ub adduct formation; it has chemical properties expected of an aldehyde: after a second reduction of the Ub released with boro[3H]hydride and complete acid hydrolysis, tritium counts are found in ethanolamine (the carboxyl-terminal residue of Ub is glycine). These results suggest that enzyme and Ub combine in an equilibrium reaction to form an ester or thiol ester adduct (at the Ub-carboxyl terminus), and that this adduct is trapped by borohydride to give a very stable inactive E-Ub (thio) hemiacetal which is unable to undergo a second reduction step and which can release Ub aldehyde in mild acid. Inactivation in the presence of hydroxylamine of hydrolase occurs once during hydrolysis of 1200 molecules of Ub-hydroxamate by the enzyme. The hydrolysis/inactivation ratio is constant over the range of 10-50 mM hydroxylamine showing that forms of E-Ub with which hydroxylamine and water react are different and not in rapid equilibrium. The inactive enzyme may be an acylhydroxamate formed from an E-Ub mixed anhydride generated from the E-Ub (thiol) ester inferred from the borohydride study. A direct radioactive assay for the hydrolase has been developed using the Ub-C-terminal amide of [3H]butanol-4 amine as substrate. PMID- 3015924 TI - The involvement of iron in lipid peroxidation. Importance of ferric to ferrous ratios in initiation. AB - Intense lipid peroxidation of brain synaptosomes initiated with Fenton's reagent (H2O2 + Fe2+) began instantly upon addition of Fe2+ and preceded detectable OH. formation. Although mannitol or Tris partially blocked peroxidation, concentrations required were 10(3)-fold in excess of OH. actually formed, and inhibition by Tris was pH dependent. Lipid peroxidation also was initiated by either Fe2+ or Fe3+ alone, although significant lag phases (minutes) and slowed reaction rates were observed. Lag phases were dramatically reduced or nearly eliminated, and reaction rates were increased by a combination of Fe3+ and Fe2+. In this instance, lipid peroxidation initiated by optimal concentrations of H2O2 and Fe2+ could be mimicked or even surpassed by providing optimal ratios of Fe3+ to Fe2+. Peroxidation observed with Fe3+ alone was dependent upon trace amounts of contaminating Fe2+ in Fe3+ preparations. Optimal ratios of Fe3+:Fe2+ for the rapid initiation of lipid peroxidation were on order of 1:1 to 7:1. No OH. formation could be detected with this system. Although low concentrations of H2O2 or ascorbate increased lipid peroxidation by Fe2+ or Fe3+, respectively, high concentrations of H2O2 or ascorbate (in excess of iron) inhibited lipid peroxidation due to oxidative or reductive maintenance of iron exclusively in Fe2+ or Fe3+ form. Stimulation of lipid peroxidation by low concentrations of H2O2 or ascorbate was due to the oxidative or reductive creation of Fe3+:Fe2+ ratios. The data suggest that the absolute ratio of Fe3+ to Fe2+ was the primary determining factor for the initiation of lipid peroxidation reactions. PMID- 3015925 TI - Demonstration of two distinct transferrin receptor recycling pathways and transferrin-independent receptor internalization in K562 cells. AB - The endocytosis and recycling of the human transferrin receptor were evaluated by several experimental modalities in K562 cells perturbed with 10(-5) M monensin. The work presented is an extension of a previous study demonstrating both complete inhibition of release of internalized human transferrin and a 50% reduction in the number of cell surface transferrin binding sites in K562 cells treated with monensin (Stein, B. S., Bensch, K. G., and Sussman, H. H. (1984) J. Biol. Chem. 259, 14762-14772). The data directly reveal the existence of two distinct transferrin receptor recycling pathways. One pathway is monensin sensitive and is felt to represent recycling of transferrin receptors through the Golgi apparatus, and the other pathway is monensin-resistant and most likely represents non-Golgi-mediated transferrin receptor recycling. A transferrin-free K562 cell culture system was developed and used to demonstrate that cell surface transferrin receptors can be endocytosed without antecedent ligand binding, indicating that there are factors other than transferrin binding which regulate receptor internalization. Evidence is presented suggesting that two transferrin receptor recycling pathways are also operant in K562 cells under ligand-free conditions, signifying that trafficking of receptor into either recycling pathway is not highly ligand-dependent. PMID- 3015926 TI - Mechanism of interaction between Ku protein and DNA. AB - The mechanism of interaction between the Ku autoantigenic protein, a heterodimer of noncovalently linked 70,000- and 80,000-dalton subunits, and DNA was studied using immunoaffinity-purified Ku protein and a 300-base pair EcoRI fragment from HeLa cell DNA. In the nitrocellulose filter-binding assay, the Ku protein bound 32P-labeled double-stranded DNA, and much less efficiently single-stranded DNA. The binding of Ku to DNA was dependent on ionic strength and prevented by IgG from patient sera containing anti-Ku antibodies. In competitive assays, using unlabeled nucleic acid competitors, the DNA binding of Ku was not inhibited in the presence of yeast tRNA, synthetic copolymer of poly(A)-poly(dT), or circular plasmid pBR322 DNA, but was inhibited when the plasmid DNA was cleaved with appropriate restriction endonucleases. The inhibitory activities of cleaved plasmid DNA were independent of the configuration or nucleotide sequences at ends but proportional to the number of recognition sites of restriction enzymes used. Footprint analysis demonstrated that Ku protein protected both 3'- and 5' terminal regions of double-stranded DNA from DNase I digestion. When Ku protein was fractionated electrophoretically, transferred to nitrocellulose filter, and probed with 32P-labeled DNA, only the 70,000-dalton subunit exhibited DNA binding. Thus, the Ku protein appears to recognize selectively ends of double stranded DNA molecules. Possible functions of the Ku autoantigen in eukaryotic cells are discussed. PMID- 3015927 TI - Oligovanadate binding to sarcoplasmic reticulum ATPase. Evidence for substrate analogue behavior. AB - Solutions of vanadate were controlled through concentration and pH adjustment to give specific compositions of mono- and oligovanadates. By monitoring the EPR spectrum of iodoacetamide spin-labeled ATPase, it is shown that decavanadate and the oligovanadate species present at neutral pH exhibit behavior typical of a substrate analogue. This is seen in terms of Ca2+ binding site affinity (microM), outward Ca2+ site orientation, and conformational effects on the enzyme normally associated with enzyme activation. In contrast, monovanadates exhibit behavior identical to that observed with Pi, with one exception: the vanadoenzyme is stable to Ca2+ in the concentration range of high affinity binding at the vanadate concentrations used here (200 microM). It is further demonstrated that Ca2+ binding in the 100 microM range directly induces enzyme devanadation of the monovanadate enzyme complex through Ca2+ binding to internal sites. Extensive array formation of dimeric ATPase units is found only with decavanadate in the absence of Ca2+, and then stoichiometric amounts are sufficient. Electron micrographs of dimeric arrays show evidence of increased penetration into the lipid bilayer, including freeze-fracture replicas which show evidence of corresponding "pits" in the inner leaflet of the bilayer. In turn, EPR spectra provide a means of following vanadate binding to the ATPase per se, as well as monitoring Ca2+-induced changes in the vanadoenzyme conformation, as only binding to specific sites on the enzyme affect the EPR spectrum. PMID- 3015928 TI - The nonhistone chromosomal protein, H2A-specific protease, is selectively associated with nucleosomes containing histone H1. AB - We have determined the distribution of the nucleosomal bound nonhistone chromosomal protein, H2A-specific protease, in calf thymus and liver chromatin. The protease was unevenly distributed in chromatin with domains containing histone H1 being selectively complexed with the enzyme. Moreover, the protease had a preference for the less compact chromatin domains enriched in the H1 subtypes H1a and -c. We have demonstrated that ubiquitinated H2A is a substrate of the H2A-specific protease and that the enzyme is a serine protease which can be inactivated with protease inhibitors only after it is released from the nucleosome. Possible functions of the protease in modulating chromatin structure are discussed. PMID- 3015929 TI - Proofreading function associated with the RNA-dependent RNA polymerase from influenza virus. AB - The influenza virus-associated RNA polymerase cleaves capped RNA in an endonucleolytic manner and the transcription is initiated by the addition of GMP, the first substrate to be polymerized under the direction of viral RNA template, onto 3'-termini of resulting capped RNA fragments. In the presence of high concentrations of GTP as a sole substrate, multiple GMP residues were polymerized onto the primers. By the addition of the second substrate CTP, excess GMP residues, other than the 1st residue, were removed prior to elongation. The result may suggest that the RNA-dependent RNA polymerase carries a proofreading function. PMID- 3015930 TI - Turnover of the catalytic subunit of Na,K-ATPase in HTC cells. AB - A polyclonal antibody to the catalytic subunit of rat kidney Na,K-ATPase has been raised in rabbits and used to analyze the turnover of the subunit in the rat hepatoma cell line HTC. It had been shown previously (Baumann, H., and Doyle, D. (1978) J. Biol. Chem. 253, 4408-4418) that the membrane proteins of these cells displayed multicomponent turnover kinetics, the minority of the surface proteins turning over with a half-time of about 20 h and the remainder with a half-time of about 100 h. That the antibody precipitated both the alpha (catalytic) and beta (glycosylated) subunits of the Na,K-ATPase from Triton extracts of HTC cells could be demonstrated following metabolic labeling of the cells with either [3H]leucine or a mixture of [3H] mannose and [3H]fucose, but following labeling with [35S]methionine radioactivity was found only in the alpha subunit of the precipitates. Incorporation of [35S]methionine into the alpha subunit could be detected 2 min after addition of the isotope to the cell suspension. Then and at all times thereafter the label was recoverable only from the particulate fraction of a 150,000 X g 60-min centrifugation; no labeled alpha subunit was ever detected in the supernatant fraction. By quantitative densitometry of radioautographs of sodium dodecyl sulfate-polyacrylamide gels of labeled antibody precipitates, it could be shown in pulse-chase experiments that the specific activity of the alpha subunit remained unchanged for 3-4 h (transit time) after the pulse was initiated and that the activity subsequently decayed exponentially with a half-time of 18 h. In a population growing with a generation time (tG) of 33 h, this decay corresponds to a turnover rate constant of 0.49/tG. The catalytic subunit is among those membrane proteins with a rapid turnover rate. PMID- 3015931 TI - Developmental and thyroid hormone regulation of two molecular forms of Na+-K+ ATPase in brain. AB - In the brain there are two isozymes of Na+-K+-ATPase differing in their catalytic subunits: alpha, indistinguishable from the kidney form of alpha, and alpha +, found in axolemma. The time course of the increase in each alpha during development was described by quantitating the abundance of each form, studied in unpurified membranes resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with specific antibodies and with fluorescein 5'-isothiocyanate. Both the alpha and alpha + subunits, quantitated with antibodies, increased 10 fold in abundance from 18 days gestation to 20 days of age, with alpha + increasing more rapidly than alpha early in development. A 10-fold increase in enzyme activity was also observed during this period. Using fluorescein 5' isothiocyanate to quantitate the two alpha subunits, a similar increase in alpha + was observed with less of an increase in alpha. The ratio of alpha + to alpha increased from 0.75 at 18 days gestation to 3 at 3 days of age remaining at this ratio to 20 days of age. The possibility that thyroid hormone, a known regulator of brain Na+-K+-ATPase during development, differentially regulated the two forms was tested using 15-day-old hypothyroid rats. The abundance of both forms of alpha was similarly decreased: alpha + to 69% and alpha to 48% of control values. Na+-K+-ATPase activity was 70% of control. We conclude that both alpha and alpha + abundance increase in the brain during pre-and neonatal development and that the increase in both alpha subunits is regulated, directly or indirectly, by thyroid hormones. PMID- 3015932 TI - Isolation and characterization of Limulus C-reactive protein genes. AB - Three homologous genes coding for Limulus C-reactive protein (CRP) have been isolated and characterized from a lambda phage EMBL-3 library containing genomic DNA sequences from Limulus amebocytes. The genes have a typical promoter region with a CAAT (nucleotides 50-53) and a TATAA (nucleotides 77-81) box located, respectively, 178 and 149 base pairs 5' upstream from the initiation codon ATG. The polyadenylation site AATAAA is situated within 300 base pairs downstream from the stop codon TAG. Nucleotide sequence analysis reveals a 24-residue signal peptide preceding a coding region of 218 amino acids. Significant differences were found between the genes coding for human and Limulus CRPs. In the human CRP gene there is an intron separating the signal peptide and the coding region. In Limulus this intervening sequence is missing. The Drosophila heat shock consensus sequence CTnGAAnnTTnAG (Simon, J. A., Sutton, C. A., Lobell, R. B., Glaser, R. L., and Lis, J. T. (1985) Cell 40, 805-817), found in the genes of human (Woo, P., Korenberg, J. R., and Whitehead, A. S. (1985) J. Biol. Chem. 260, 13384 13388) and rabbit (Syin, C., Gotschlich, E. C., and Liu, T.-Y. (1986) J. Biol. Chem. 261, 5473-5479) CRP at the 5' end, is not found in the Limulus CRP genes. Whereas a single CRP gene was found in the human, multiple genes were found for the Limulus CRPs. All CRPs exhibit calcium-dependent phosphorylcholine ligand binding properties. The coding regions of the Limulus and human CRP genes share approximately 25% identity and two stretches of highly conserved regions, one of which falls in the region proposed as the phosphorylcholine binding site, while the other site is very similar to the consensus sequence required for calcium binding in calmodulin and related proteins. The nucleotide sequence analysis provides convincing evidence to support the evolutionary relatedness of the human and Limulus CRPs. PMID- 3015933 TI - Glutamyl-tRNA synthetase of Escherichia coli. Isolation and primary structure of the gltX gene and homology with other aminoacyl-tRNA synthetases. AB - The gltX gene encoding the glutamyl-tRNA synthetase of Escherichia coli and adjacent regulatory regions was isolated and sequenced. The structural gene encodes a protein of 471 amino acids whose molecular weight is 53,810. The codon usage is that of genes highly expressed in E. coli. The amino acid sequence deduced from the nucleotide sequence of the gltX gene was confirmed by mass spectrometry of large peptides derived from the glutamyl-tRNA synthetase. The observed peptides confirm 73% of the predicted sequence, including the NH2 terminal and the COOH-terminal segments. Sequence homology between the glutamyl tRNA synthetase and other aminoacyl-tRNA synthetases of E. coli was found in four segments. Three of them are aligned in the same order in all the synthetases where they are present, but the intersegment spacings are not constant; these ordered segments may come from a progenitor to which other domains were added. Starting from the NH2-end, the first two segments are part of a longer region of homology with the glutaminyl-tRNA synthetase, without need for gaps; its size, about 100 amino acids, is typical of a single folding domain. In the first segment, containing sequences homologous to the HIGH consensus, the homology is consistent with the following evolutionary linkage: gltX----glnS----metS----ileS and tyrS. PMID- 3015934 TI - Some gene variants for 5 S RNA are dispersed in the rat genome. AB - In the course of studies on genes for small nuclear RNAs, seven lambda phage clones containing sequences homologous to 5 S RNA were plaque purified from a rat genomic library. The seven clones were found to be from six different genomic loci. When the 5 S RNA hybridized to these clones was digested by T1 RNase, only clone 5S-2 protected the RNA completely. Moreover, clone 5S-2 which has five nucleotide substitutions in the internal control region was transcribed 10 times more efficiently than a bonafide Chinese hamster 5S gene. The other clones were less efficiently transcribed than a bonafide 5S gene or not transcribed at all. The number of gene variants for 5 S RNA in the rat genome was approximately 3000. In contrast to the clustering of 5S genes and gene variants found in Xenopus, Drosophila, hamster, mouse, and human cells, the 5S gene variants in the rat genome are dispersed and most contained conserved 3'-flanking sequences. These naturally occurring 5S gene variants may be useful in binding transcription factors that affect 5S genes. PMID- 3015935 TI - Nucleotide sequence of the purM gene encoding 5'-phosphoribosyl-5-aminoimidazole synthetase of Escherichia coli K12. AB - 5'-Phosphoribosyl-5-aminoimidazole synthetase (EC 6.3.3.1), encoded by the purM gene of Escherichia coli, catalyzes the synthesis of 5'-phosphoribosyl-5 aminoimidazole from 5'-phosphoribosylformylglycinamidine. The purM gene was subcloned from the Clarke and Carbon (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99) plasmid pLC1-41 and the nucleotide sequence determined. The mature protein, as deduced from the purM structural gene sequence, contains 344 amino acid residues and has a calculated Mr of 36,726. The 5' end of the purM mRNA was determined by mung bean nuclease mapping to be 44-45 nucleotides up-stream of the proposed GTG translation initiation codon. A C-G-rich region characteristic of stringently controlled promoters is located immediately in front of the proposed purM promoter region. Comparison of the upstream sequences of the purM and the coregulated purF loci revealed a highly conserved (33 of 39 base pairs are identical) sequence. The presumptive purM promoter is located in this region, thus suggesting that both purine loci share a common mechanism of regulation mediated through this sequence. PMID- 3015936 TI - Sequence of two related P-450 mRNAs transcriptionally increased during rat development. An R.dre.1 sequence occupies the complete 3' untranslated region of a liver mRNA. AB - Polyclonal antibody against a cytochrome P-450 expressed in adult male rats was utilized to screen a lambda gt-11 library. Two cDNAs were isolated and sequenced. The cDNA and deduced protein sequence revealed that these P-450s correspond to previously isolated P-450 PB1 (Waxman D. T., Ko, A., and Walsh, C., (1983) J. Biol. Chem. 258, 11937-11947) and P-450f (Ryan, D. E., Iida, S., Wood, A. W., Thomas, P. E., Lieber, C. S., and Levin, W., (1984) J. Biol. Chem. 259, 1239 1250). Both P-450s contain 490 amino acids and share 75% similarity with each other and 50% similarity with P-450b and P-450e. Southern blot analysis indicates that these enzymes are part of a small P-450 gene subfamily composed of two or three members. An unusual feature of P-450 PB1 is the presence of a rat brain identifier-type repetitive sequence (R.dre.1) that encompasses the complete 3' untranslated region of the mRNA. This sequence is absent from P-450f mRNA. A cDNA from an apparent allelic form of P-450 PB1 mRNA was isolated and sequenced. This cDNA was identical to the other P-450 PB1 cDNA except for a single base substitution in the R.dre.1 repeat and the presence of a repeat (AAATAAA)13 at the 3' end of the mRNA. By use of a 3' specific probe for P-450f mRNA and a probe common to P-450 PB1 and P-450f mRNAs, the mechanism by which these P-450s are elevated during rat development was studied. Both RNAs are virtually absent in newborn rats and become elevated between 1 and 4 weeks after birth. Maximal levels of P-450f mRNA levels continue to increase up to 12 weeks after birth, and 2-fold higher levels of P-450f mRNA are reached in 12-week-old female rats compared with male rats. P-450 PB1 mRNA reached maximal levels at 4 weeks and no sex difference in mRNA level was detected for P-450 PB1. Nuclear run-on transcription assays confirmed that the increase in these mRNAs is due to transcriptional activation. PMID- 3015937 TI - Kinetic properties of the sodium bicarbonate (carbonate) symport in monkey kidney epithelial cells (BSC-1). Interactions between Na+, HCO-3, and pH. AB - BSC-1 kidney epithelial cells derived from the African green monkey are known to express a Na+HCO3- symport (Jentsch, T. J., Schill, B. S., Schwartz, P., Matthes, H., Keller, S. K., and Wiederholt, M. (1985) J. Biol. Chem. 260, 15554-15560). In the present work, 4,4-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) sensitive 22Na+ uptake into confluent monolayers of BSC-1 is measured in the presence of ouabain (10(-4) M) and amiloride (10(-3) M) to define the interactions between Na+ and HCO3- binding and pH. Dependence of DIDS-sensitive 22Na+ fluxes on either Na+ or HCO3- can be described by Michaelis-Menten kinetics. External apparent Km for HCO3- decreases with increasing Na+ concentration (Km app (HCO3-) = 36 +/- 10, 18 +/- 5, and 9 +/- 3 mM at 20, 45, and 151 mM Na+o, respectively (pHo = 7.4)). Similarly, external apparent Km for Na+ decreases with increasing HCO3- concentration (Km app (Na+) = 73 +/- 22, 28 +/- 8, and 14 +/- 4 mM at 6, 17, and 56 mM HCO3o-, respectively (pHo = 7.4)). Vmax app remains constant within the experimental error. When data are replotted as a function of calculated NaCO3- concentration, they can be approximated by a single Michaelis-Menten equation. DIDS-sensitive uptake at constant Na+ and HCO3- displays a broad pH optimum in the range between 7.2 and 7.6. The data are compatible with the ion pair model in which the transported species, NaCO3-, binds to the transport site with Km = 15.3 +/- 4 microM. However, the data may also be fitted by either a random or ordered bireactant system. Sets of parameters necessary for these fits are given. PMID- 3015938 TI - Identification of multiple cyclic AMP-binding proteins in developing Dictyostelium discoideum cells. AB - We have purified two cAMP-binding proteins from developing Dictyostelium discoideum cells, which we designate as CABP-1 and CABP-2. Purified CABP-1 consists of two polypeptides of Mr 41,000 and 36,000, which we refer to as CABP 1A and CABP-1B, respectively. Although CABP-1 exhibited specificity for cAMP, it was not labeled at a detectable level when mixed with 8-azidoadenosine 3':5' monophosphate (8-N3[3H]cAMP). Unlike CABP-1, CABP-2 was labeled efficiently with 8-N3[3H]cAMP. Purified CABP-2 has a molecular weight of 41,000 and an isoelectric point of 5.8-6.0. The physical and biochemical properties of CABP-2 suggest that it is the regulatory subunit of cAMP-dependent protein kinase described by others (de Gunzburg, J., Part, D., Guiso, N., and Veron, M. (1984) Biochemistry 23, 3805 3812; Majerfeld, J. H., Leichtling, B. H., Maligeni, J. A., Spitz, E., and Rickenberg, H. V. (1984) J. Biol. Chem. 259, 654-661). Although CABP-1A and CABP 2 have the same molecular weight, they appear to be encoded by different genes. Two-dimensional gel electrophoresis revealed that the two polypeptides had different isoelectric points. Moreover, monoclonal antibodies raised against CABP 1 did not cross-react with CABP-2. Also, in vitro translation followed by immunoprecipitation showed that these two polypeptides were derived from primary translation products. Our finding of a novel cAMP-binding protein, CABP-1, suggests that cAMP-dependent protein kinase may not be the only intracellular regulator mediating the effects of cAMP in developing D. discoideum cells. PMID- 3015939 TI - Differential regulation of glucokinase activity in pancreatic islets and liver of the rat. AB - The differential tissue-specific regulation of glucokinase activity in liver and pancreatic islet cells was investigated in the insulinoma-bearing rat. A transplantable insulinoma caused hyperinsulinemia and hypoglycemia in the host by 2-3 months after implantation. Suppression of the pancreatic B-cells by the high insulin and/or low glucose manifested itself by a decrease of insulin in islet tissue. Removal of the tumor initiated transient insulin deficiency and hyperglycemia with extremes of these changes at 24 h after tumor resection. These conditions markedly affected glucose phosphorylation in the islet cells: glucokinase activity was reduced 71% in islet samples from insulinoma-bearing rats, and the enzyme fully recovered within 24 h after tumor resection. Hexokinase activity, by contrast, was not affected by these manipulations. To evaluate the relative contributions of hypoglycemia and hyperinsulinemia in islet glucokinase adaptation, glucose was intravenously infused to insulinoma-bearing rats; glycemia in excess of 150 mg/100 ml combined with excessive hyperinsulinemia resulted in a partial recovery of islet glucokinase activity, first apparent after 9 h of glucose infusion and with doubling of the activity after 24 h after glucose loading. In contrast, liver glucokinase was increased nearly 4-fold at the time of extreme hypoglycemia and hyperinsulinemia and rapidly fell to control rates following tumor removal. Intravenous infusion of glucose for 24 h into the tumor-bearing rat (i.e. hyperglycemia combined with excessive plasma insulin) had no influence on liver glucokinase activity. Liver hexokinase was not influenced by any of these experimental manipulations. The data indicate that the activities of pancreatic islet and liver glucokinase are regulated in a differential manner. Insulin is apparently the primary determinant of liver glucokinase and glucose seems to control islet glucokinase. Biochemical mechanisms for differential organ-specific regulation of glucokinase activity seem to have evolved such that this enzyme may play a dual role in glucose homeostasis, namely to serve as insulin-dependent glucose sensor in the B-cells and as insulin-sensitive determinant of hepatic glucose use. PMID- 3015940 TI - Rous sarcoma virus-transformed baby hamster kidney cells express higher levels of asparagine-linked tri- and tetraantennary glycopeptides containing [GlcNAc-beta (1,6)Man-alpha (1,6)Man] and poly-N-acetyllactosamine sequences than baby hamster kidney cells. AB - The alterations in complex-type N-linked oligosaccharides that can occur when an animal cell line is transformed by two dissimilar viruses were examined by comparing the N-linked oligosaccharides of baby hamster kidney (BHK) cells, metabolically radiolabeled with [2-3H]mannose, to the same class of oligosaccharides from BHK cells separately transformed by Rous sarcoma virus (RS BHK), an RNA retrovirus, and polyoma virus (PY-BHK), a DNA papovavirus. Based on experiments that utilized serial lectin affinity chromatography, glycosidase digestions, and methylation analyses, both RS-BHK and PY-BHK cells demonstrated a significant increase in the relative amounts of tri- and tetraantennary complex type N-linked oligosaccharides containing the branching sequence, [GlcNAc beta(1,6)Man-alpha(1,6)Man], compared to the nontransformed BHK cells. In addition, almost all of the poly-N-acetyllactosamine sequence, [GlcNAc-beta(1,3) Gal-beta(1,4)], was expressed on the tri- and tetraantennary N-linked oligosaccharides from BHK and RS-BHK cells that contain the sequence, [GlcNAc beta(1,6)Man-beta(1,6)Man]. The increase in the relative amounts of this latter sequence in the transformed cells, therefore, most likely results in an increase in the amount of poly-N-acetyllactosamine sequence on the N-linked glycopeptides of these cells. The analysis of the degree of sialylation of the complex-type N linked oligosaccharides from BHK and RS-BHK cells by ion exchange chromatography revealed no apparent differences, and in both of these cell types approximately 3% of the glycopeptide fraction radiolabeled with mannose was recovered in a highly negatively charged fraction that was identified by keratanase digestion to be keratan sulfate. PMID- 3015941 TI - Nucleotide sequence of the gene for the ferrienterochelin receptor FepA in Escherichia coli. Homology among outer membrane receptors that interact with TonB. AB - We have determined the nucleotide sequence of the Escherichia coli fepA gene, which codes for the outer membrane receptor for ferrienterochelin and colicins B and D. The predicted FepA polypeptide has a molecular weight of 79,908 and consists of 723 amino acids. A 22-amino acid leader or signal peptide preceded the mature protein. With respect to overall composition, hydropathy, net charge and distribution of nonpolar segments, the FepA polypeptide was typical of other E. coli outer membrane proteins, except that FepA contained 2 cysteine residues. Comparison of the deduced amino acid sequence of FepA with that of three other TonB-dependent receptors (BtuB, FhuA, and IutA) revealed only a few regions of sequence homology; one of these included the amino-termini. An amino acid substitution within the conserved amino-terminal region of BtuB resulted in production of a receptor that had normal binding functions but was incapable of energy-dependent transport of vitamin B12. This result suggests that the amino terminal end of these four polypeptides is involved in interaction with the TonB protein or another step of energy transduction. Three other regions of homology were shared among the four proteins: one near residues 50 to 70, another at about residue 100 to 140, and the last between 20 and 40 amino acid residues from the carboxyl terminus. The function of these three regions remains speculative. PMID- 3015942 TI - Kinetics of receptor modification. The multiply methylated aspartate receptors involved in bacterial chemotaxis. AB - A method for determining the extent of methyl esterification of each of the four potential sites on the aspartate receptors involved in chemotaxis in Escherichia coli and Salmonella typhimurium is presented. In this procedure, radioactive methyl esters are incorporated into the receptors, the receptors are cleaved by trypsin and the V8 protease from Staphylococcus aureus, and the four fragments containing sites of methylation are separated by high performance liquid chromatography. Using this technique, we find that the rate of methyl esterification increases at all four sites after stimulation with the "attractant" aspartate, suggesting that all four sites of modification are involved in adaptation to aspartate. We also find that the rate of methyl esterification at each site is correlated with the homology between the protein sequence at that site and the "consensus" sequence, Glu-Glu-X-X-Ala-Thr/Ser. PMID- 3015943 TI - The nucleotide sequence of three genes participating in the adipose differentiation of 3T3 cells. AB - When 3T3 cells undergo adipose differentiation, they are reprogrammed by changes in gene expression of sufficient magnitude to greatly alter the protein composition of the cells. Three participating genes encode glycerophosphate dehydrogenase, a lipid-binding protein, and a serine protease. These three genes have now been cloned and sequenced. Their exon/intron structures are described, together with some interesting peculiarities and some regions of common sequence. It remains to be demonstrated whether the information for common participation of the genes in the program of differentiation resides in controlling elements within the regions sequenced. PMID- 3015944 TI - Photoaffinity cross-linked A1 adenosine receptor-binding subunits. Homologous glycoprotein expression by different tissues. AB - Mammalian A1 adenosine receptor-binding peptides can be visualized by covalently labeling them with the photoaffinity cross-linking ligand N6-2-(4-amino-3-[125I] iodophenyl)ethyladenosine followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/autoradiography. The proteins comprising the A1 adenosine receptor-binding subunit of rat brain and fat migrate with Mr 38,000. In this study, the glycoproteins representing the radiolabeled A1 adenosine receptor binding subunit expressed in each of these tissues (brain and fat) were compared through the use of peptide mapping and exo- and endoglycosidase treatments. Peptide mapping studies with several enzymes demonstrate that the protein component of the radiolabeled A1 adenosine receptor-binding subunit is conserved between different tissues. Both labeled receptor peptides demonstrate a sensitivity to neuraminidase as evidenced by increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the receptors contain complex-type carbohydrate chains. Insensitivity to alpha-mannosidase suggests a lack of high mannose-type carbohydrate chains. Deglycosylation of the labeled receptor-binding subunits with endoglycosidase F results in a single labeled polypeptide of Mr 32,000 for both systems. These data suggest that the A1 adenosine receptor-binding subunits expressed in the rat brain and fat are similar glycoproteins as evidenced by similar overall molecular weights, identical peptide maps, and equivalent responses to endo- and exoglycosidase treatment. PMID- 3015945 TI - Selective cleavage of human ACTH, beta-lipotropin, and the N-terminal glycopeptide at pairs of basic residues by IRCM-serine protease 1. Subcellular localization in small and large vesicles. AB - We present a study of the cleavage specificity of IRCM-serine protease 1 from frozen porcine pituitary neurointermediate lobes using polypeptide substrates representing different segments of human pro-opiomelanocortin. Using 125I-labeled ACTH(11-24) and a 125I-labeled model beta-lipotropin (beta-LPH) peptide, the preference of this protease for cleavage C-terminal to the pairs of basic residues Lys-Arg and Lys-Lys was clearly seen. This study was extended to larger unlabeled natural human polypeptides including ACTH(1-39), beta-LPH(1-89), and the N-terminal glycopeptide (1-76), which are known to serve as substrates for further cleavage in vivo. In these substrates IRCM-serine protease 1 cleaved C terminal to all pairs of basic residues known to be cleaved in vivo. In addition, the enzyme cleaved between two pairs of basic amino acids found in NT(1-76) which are also known to be cleaved in vivo. Many potential "tryptic-like" cleavage sites were not cleaved by the enzyme. However, IRCM-serine protease 1 cleaved C terminal to Phe-Arg in the three melanocyte-stimulating hormone sequences of pro opiomelanocortin. In order to better understand the physiological role of IRCM serine protease 1, differential centrifugation was used to study the subcellular distribution of the enzyme from porcine pituitary anterior lobe homogenates. We present evidence that the active enzyme form, isolated from the subcellular fractions, possesses a similar molecular architecture as the enzyme isolated from frozen tissue (Mr 38,000 catalytic domain linked via disulfide bridge(s) to another polypeptide chain(s) to form an Mr 88,000 monomeric structure). The majority of IRCM-serine protease activity is found to be associated with small vesicles (150,000 X g for 5 h) of as yet undetermined nature. In addition, a latent activity was found to be associated with a 27,000 X g (15 min) pellet containing the majority of mature secretory granules. If IRCM-serine protease 1 participates in prohormone maturation in vivo, we propose a model in which this protease is present in an enzymatically active form in small vesicles, possibly within clathrin-coated structures (prosecretory granules) which are then transformed to mature secretory granules by a process which would also inactivate most of the enzyme. PMID- 3015946 TI - The binding of catabolite activator protein and RNA polymerase to the Escherichia coli galactose and lactose promoters probed by alkylation interference studies. AB - The Escherichia coli galactose and lactose promoter regions have been studied by alkylation interference experiments. The data reveal those bases or phosphate groups which, when modified, prevent the binding of the catabolite activator protein (CAP) or RNA polymerase and hence are presumably in contact with the proteins. Interference contacts made by CAP at its primary binding sites at gal and lac are quite similar, indicating that CAP-cAMP uses the same mode of binding at these two operons. RNA polymerase, when bound in the presence of CAP-cAMP, exhibits contacts at the gal and lac P1-10 regions very much like those of the lac UV5 and T7 A3 promoters (Siebenlist, U., Simpson, R. B., and Gilbert, W. (1980) Cell 20, 269-281). CAP, therefore, does not detectably alter the structure of the open complex. The binding sites for CAP and RNA polymerase at lac, as deduced from interference experiments, do not overlap. However, at gal a CAP molecule is found much closer to the enzyme, and there is competition for a set of mutual contacts. These experiments thus reveal both similarities and differences in the mechanisms whereby CAP activates transcription at catabolite sensitive operons. PMID- 3015947 TI - Uncoupling of the DNA breaking and rejoining steps of Escherichia coli type I DNA topoisomerase. Demonstration of an active covalent protein-DNA complex. AB - DNA topoisomerases have been shown to cleave DNA phosphodiester bond and simultaneously become linked to the DNA at the cleavage site via a phosphotyrosine linkage (Tse, Y.-C., Kirkegaard, K., and Wang, J. C. (1980) J. Biol. Chem. 255, 5560-5565). For prokaryotic DNA topoisomerases, this is observed only when denaturant or protease is added to the topoisomerase-DNA incubation mixture. Previous attempts to reform DNA phosphodiester bonds from the covalent protein-DNA complex have been unsuccessful. Using oligonucleotides as substrates, the cleavage reaction of Escherichia coli DNA topoisomerase I occurs spontaneously (Tse-Dinh, Y.-C., McCarron, B. G. H., Arentzen, R., and Chowdhry, V. (1983) Nucleic Acids Res. 11, 8691-8701). Upon reaction with oligo(dA) labeled with 32P using terminal transferase and [alpha-32P]dATP, the enzyme becomes covalently linked to the 32P-labeled oligonucleotide. This 32P label can then be transferred to the 3'-OH end of a linear or nicked duplex DNA molecule subsequently added to the reaction mixture. This phosphodiester bond rejoining reaction can occur at a recessed, blunt, or protruding 3'-end of double-stranded DNA. It requires magnesium ions. These observations suggest that the covalent protein-DNA complex is a true intermediate during topoisomerization. Implications on the structure of prokaryotic type I DNA topoisomerases as compared to their eukaryotic counterparts are discussed. PMID- 3015948 TI - A study of roles of evolutionarily invariant proline 30 and glycine 34 of cytochrome c. AB - The previous studies (Juillerat, M. A., and Taniuchi, H. (1986) J. Biol. Chem. 261, 2697-2711), using a three-fragment complex (1-25)H X (28-38) X (39-104) of horse cytochrome c, have shown that invariant leucine 32 and partially invariant leucine 35, both buried in the interior, exhibit a striking difference in perturbation of binding fragment (28-38) by substitution with isoleucine. Then the idea has been proposed that the energy states of leucine 32, the Met-80-S heme-Fe bond and other distant residues such as tryptophan 59 would be coupled to generate extra force while leucine 35 would be less important for such coupling if it were involved. In the present studies we synthesized three (28-38) analogs substituting invariant proline 30 with glycine or invariant glycine 34 with alanine or serine. Thermodynamic and kinetic studies and UV CD and biological activity measurements were carried out on binding of the analogs to complex (1 25)H X (39-104). The results with the ferric form show that perturbations of delta G, delta H, and delta S associated with formation of the intermediate complex and with the ensuing process by the Gly34----Ala or Ser substitution result in weakening the Met-80-S-heme-Fe bond formed in the latter process; in contrast, perturbation by the Pro30----Gly substitution is small. However, the biological activity is more perturbed by the Pro30----Gly substitution than by the Gly34----Ala or Ser; and in the Gly34----Ala or Ser substitution the complex appears to be more readily activated for both formation and disruption of the Met 80-S-heme-Fe bond at 20 degrees C and below than without substitution. In all cases reduction of the heme strengthens the binding of fragment (28-38). However, striking are the increases in perturbation (less negative) of both delta H and delta S for binding of fragment (28-38) to form the ground state on reduction of the heme in the Pro30----Gly, Gly34----Ala or Ser (the present studies), and Leu32----norvaline (the previous studies) substitutions. It is known that fluctuation of the atomic positions of most residues of tuna ferrocytochrome c including Pro30, Leu32, and Gly34 increases on oxidation of the heme and that these three residues are among those showing the least fluctuating atomic positions (Takano, T., and Dickerson, R.E. (1982) in Electron Transport and Oxygen Utilization (Ho, C., ed) pp. 17-26, Elsevier/North-Holland Biomedical Press, New York).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3015950 TI - Pyridoxal 5'-phosphate-dependent histidine decarboxylase. Nucleotide sequence of the hdc gene and the corresponding amino acid sequence. AB - The nucleotide sequence of a 1.3-kilobase NaeI fragment from Morganella morganii AM-15 that contains the gene for histidine decarboxylase has been determined. The gene was initially identified among total chromosomal digests using a mixed sequence oligonucleotide probe corresponding to amino acids 11-16 of histidine decarboxylase and then cloned on a 5.5-kilobase PstI fragment. The structural gene contains 1131 nucleotides and encodes 377 amino acids with the sequence: (sequence: in text). The independently determined NH2-terminal sequence of this enzyme (Tanase, S., Guirard, B. M., and Snell, E. E. (1985) J. Biol. Chem. 260, 6738-6746) and the amino acid sequences of two tryptic peptides reported in the accompanying paper (Hayashi, H., Tanase, S., and Snell, E. E. (1986) J. Biol. Chem. 261, 11003-11009) are localized in the sequence presented here; the lysine that binds pyridoxal phosphate is situated at residue 232, whereas the serine that binds the adduct formed between pyridoxal phosphate and the inhibitor alpha fluoromethylhistidine is positioned at residue 322. PMID- 3015949 TI - Mechanism of activation by anions of phosphoglycolate phosphatases from spinach and human red blood cells. AB - Phosphoglycolate phosphatases from spinach and human red blood cells show a number of common features not often found in enzymes. Both enzymes are activated more than 50-fold by millimolar concentrations of Cl-. Other inorganic anions and a number of carboxylic acids also activate. Each enzyme has limited substrate specificity yet each hydrolyzes P-glycolate and ethyl-P with the same maximal velocity. L-P-lactate is only a good substrate for the red cell enzyme. With both enzymes initial rate data obtained by varying both the P-glycolate and Cl- give parallel line double reciprocal plots. Similar experiments with ethyl-P as substrate give intersecting lines with both enzymes. The likelihood that both classes of substrates are acting at the same site is strengthened by the results of inhibition studies with alternative substrates and the constancy of inhibition constants for glycolate with all substrates for a given enzyme. For each substrate the experimentally observed variation in V/Km with different activators is small, suggesting that the enzyme has an ordered mechanism with the phosphorylated substrate reacting first. A mechanism that is consistent with all of the data is presented. PMID- 3015952 TI - Regulation of diacylglycerol kinase biosynthesis in Escherichia coli. A trans acting dgkR mutation increases transcription of the structural gene. AB - The mechanism of a trans-acting mutation, dgkR1, which causes a 7-fold elevation of diacylglycerol kinase activity in membranes (Raetz, C. R. H., Kantor, G. D., Nishijima, M., and Jones, M. L. (1981) J. Biol. Chem. 256, 2109-2112) was investigated by direct measurement of diacylglycerol kinase polypeptide by high performance liquid chromatography and by construction of fusions of the dgkA promoter to beta-galactosidase and galactokinase. The dgkR1 mutation was demonstrated to act by increasing the transcription of the structural gene for diacylglycerol kinase, dgkA. Additionally, sn-glycerol-3-phosphate acyltransferase activities were shown to be decreased 30-50% in membranes from dgkR1 mutant strains. Increased diacylglycerol levels occurred when cells were grown on low osmolarity media. This did not affect dgkA expression. In a dgkR+ background, enhanced expression of sn-1,2-diacylglycerol kinase activity in cells containing a high copy number plasmid bearing dgkA decreased sn-1,2 diacylglycerol levels. However, overproduction of diacylglycerol kinase in a dgkR1 genetic background did not affect diacylglycerol levels, suggesting that the dgkR1 mutation affects diacylglycerol metabolism by mechanisms additional to enhancement of dgkA transcription. PMID- 3015951 TI - The interaction of phosphate with uteroferrin. Characterization of a reduced uteroferrin-phosphate complex. AB - The interaction of phosphate with reduced uteroferrin has been re-examined in light of disagreements on the oxidation state of the binuclear iron cluster (Keough, D. T., Beck, J. L., de Jersey, J., and Zerner, B. (1982) Biochem. Biophys. Res. Commun. 108, 1643-1648; Antanaitis, B. C., and Aisen, P. (1985) J. Biol. Chem. 260, 751-756). Our results based on Mossbauer observations and the kinetics of spectral change and activity loss show clearly that phosphate binds to reduced uteroferrin to form a reduced uteroferrin-phosphate complex. This complex exhibits a pair of quadrupole doublets at 119 K with parameters typical of a high spin ferric and a high spin ferrous center, respectively, but distinct from those of the native reduced enzyme. The reduced phosphate complex exhibits a pH-dependent visible absorption maximum ranging from 530 to 561 nm. In air, the reduced phosphate complex converts to the oxidized phosphate complex with a first order rate constant of 4 X 10(-3) min-1, as monitored by spectral changes and loss of enzyme activity. PMID- 3015953 TI - Analysis of glucocorticoid unresponsive cell variants using a mouse glucocorticoid receptor complementary DNA clone. AB - We have used a glucocorticoid receptor cDNA isolated from a mouse lymphoma cell line to characterize receptor mRNA and genomic sequences present in wild type and mutant rat hepatoma (HTC) and mouse thymoma (S49 and WEHI7) cells. Wild type rat and mouse cell lines contain two receptor mRNAs, 5 and 7 kilobase pairs (kb) in length, which differ in the length of their 3'-untranslated regions. Levels of receptor mRNA present in mutant HTC, WEHI7, and S49 cells of the r- (receptorless) phenotype are decreased compared to wild type cells. This decreased level of receptor mRNA parallels the decreased level of total immunoreactive receptor protein found in these cells. S49 nt- (nuclear transfer minus) cells contain receptor mRNA levels which parallel their hormone binding and immunoreactive receptor levels. Cells of the r- and nt- phenotype contain no detectable deletions or rearrangements of the receptor gene. We conclude that r- cells have lesions which affect the expression of receptor mRNA. Surprisingly, HTC cells of the r- phenotype differ from WEHI7 and S49 r- cells in that HTC r- cells contain a lower level of receptor DNA than does their parental wild type cell line. Although these cells may contain multiple lesions, it appears that loss of receptor genomic sequences is responsible, in part, for the phenotype of the HTC r- cells. The S49 nti (nuclear transfer increase) cells produce glucocorticoid receptors of molecular weights 40,000 and 94,000. These cells produce, in addition to the wild type 5- and 7-kb receptor mRNAs, two other receptor messages 5.5 and 3.5 kb in length. RNA blot analysis using various portions of our receptor cDNA indicates that these are 5' truncated messages and suggests that the 40-kDa nti receptor is truncated at its NH2-terminal end. These data also indicate that the hormone and DNA-binding regions of the receptor are located in the COOH-terminal half of the protein. PMID- 3015954 TI - Kinetics of platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3 phosphocholine-induced fibrinogen binding to human platelets. AB - Platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF acether) triggers exposure of fibrinogen binding sites on platelets via binding to specific receptors. Comparison of [3H]PAF-acether binding with 125I-fibrinogen binding shows that the rate with which PAF-acether binds to a number of receptors and not the degree of receptor occupancy determines how much fibrinogen binds. At low concentrations of PAF-acether (0.1-1.0 nM) binding site exposure is incomplete and parallels the rate of formation of the PAF-acether-receptor complex. Fibrinogen binding then primarily depends on the concentration of PAF acether. At a high concentration of PAF-acether (500 nM) binding site exposure is complete within 2-5 min. Fibrinogen binding then depends on the concentration of fibrinogen. Exposure of binding sites in the absence of fibrinogen leads to disappearance of accessible binding sites. At 500 nM PAF-acether, this disappearance is exponential in nature and shows the same characteristics after 5 15 min incubation with fibrinogen as after 60 min. Exposure of binding sites is then complete within 5 min and their disappearance is not disturbed by other processes. At 0.5 nM PAF-acether, the same characteristics are found after 60 min incubation with fibrinogen, but shorter incubation times reveal an ongoing binding site exposure that interferes with the disappearance process. These results demonstrate close coupling between the PAF-acether receptors and fibrinogen binding sites and indicate that the rate of formation of the PAF acether-receptor complex is a major factor in the regulation of binding site exposure. PMID- 3015956 TI - NMR studies of pig gastric microsomal H+,K+-ATPase and phospholipid dynamics. Effects of ethanol perturbation. AB - The effects of ethanol on the gastric H+,K+-ATPase activity and the degree of mobility of various microsomal phospholipids were assessed using 31P and 1H NMR. This illuminated the role of lipid-protein association in the function of pig gastric microsomes. Treatment of gastric microsomes with 15% ethanol for 1 min at 37 degrees C inactivated the H+,K+-ATPase activity, which could largely be reconstituted by supplementation with phosphatidylcholine isolated from the gastric microsomes. Under similar conditions, the 1H NMR profile of the microsomal +N(CH3)3 choline moiety showed dramatic enhancement of peak intensity as well as a break point at 25 degrees C which was restored to the untreated control value after reconstitution. This break, together with the dramatic enhancement in the overall lipid profile, compared to the control and reconstituted microsomes, suggested a greater degree of freedom of movement of the microsomal lipids following ethanol perturbation. The data demonstrate the unique ability that a combined approach using 31P and 1H NMR holds as a noninvasive probe to study the structure-function relationship of biomembranes. PMID- 3015955 TI - Photoaffinity labeling of the human erythrocyte glucose transporter with 8 azidoadenosine. AB - 8-Azidoadenosine was employed as a possible covalent probe of the erythrocyte nucleoside transporter. 8-Azidoadenosine was shown to enter human erythrocytes by a saturable mechanism (apparent Km for influx 80 microM) that was inhibited by nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport, and competitively inhibit uridine influx and NBMPR binding. Irradiation with UV light of human erythrocyte membranes or a partially purified preparation of the nucleoside transporter in the presence of [3H]8-azidoadenosine and dithiothreitol (as a free radical scavenger) resulted in selective covalent incorporation into the band 4.5 region of sodium dodecyl sulfate-polyacrylamide gels (Mr 66,000 45,000). Covalent labeling of band 4.5 was inhibited by adenosine, uridine, and inosine, but NBMPR had no effect. Surprisingly, D-glucose and cytochalasin B, but not L-glucose and cytochalasin E, blocked covalent attachment of the ligand. No incorporation of radioactivity into membranes from rabbit and pig erythrocytes was observed, cells which transport nucleosides rapidly, but have little or no functional glucose carrier. Limited treatment with trypsin of unsealed human erythrocyte membranes photolabeled with [3H]8-azidoadenosine yielded a single radioactive fragment of Mr 19,000, a pattern identical to that obtained with [3H]cytochalasin B-labeled membranes. These results suggest that, despite 8 azidoadenosine being a permeant for the nucleoside transporter, under photoactivation 8-azidoadenosine preferentially labeled the glucose carrier. PMID- 3015957 TI - Resonance Raman study on cytochrome c peroxidase and its intermediate. Presence of the Fe(IV) = O bond in compound ES and heme-linked ionization. AB - Resonance Raman spectra of ferrous and ferric cytochrome c peroxidase and Compound ES and their pH dependences were investigated in resonance with Soret band. The Fe(IV) = O stretching Raman line of Compound ES was assigned to a broad band around 767 cm-1, which was shifted to 727 cm-1 upon 18O substitution. The 18O-isotopic frequency shift was recognized for Compound ES derived in H218O, but not in H216O. This clearly indicated occurrence of an oxygen exchange between the Fe(IV) = O heme and bulk water. The Fe(IV) = O stretching Raman band was definitely more intense and of higher frequency in D2O than in H2O as in Compound II of horseradish peroxidase, but in contrast with this its frequency was unaltered between pH 4 and 11. The Fe(II)-histidine stretching Raman line was assigned on the basis of the frequency shift observed for 54Fe isotopic substitution. From the intensity analysis of this band, the pKa of the heme linked ionization of ferrocytochrome c peroxidase was determined to be 7.3. The Raman spectrum of ferricytochrome c peroxidase strongly suggested that the heme is placed under an equilibrium between the 5- and 6-coordinate high-spin structures. At neutral pH it is biased to the 5-coordinate structure, but at the acidic side of the transition of pKa = 5.5 the 6-coordinate heme becomes dominant. F- was bound to the heme iron at pH 6, but Cl- was bound only at acidic pH. Acidification by HNO3, H2SO4, CH3COOH, HBr, or HI resulted in somewhat different populations of the 5- and 6-coordinate forms when they were compared at pH 4.3. Accordingly, it is inferred that a water molecule which is suggested to occupy the sixth coordination position of the heme iron is not coordinated to the heme iron at pH 6 but that protonation of the pKa = 5.5 residue induces an appreciable structural change, allowing the coordination of the water molecule to the heme iron. PMID- 3015958 TI - Isolation of D-myo-inositol 1:2-cyclic phosphate 2-inositolphosphohydrolase from human placenta. AB - We have isolated D-myo-inositol 1:2-cyclic phosphate 2-inositolphosphohydrolase (EC 3.1.4.36) from human placenta. This enzyme catalyzes the conversion of inositol 1:2-cyclic phosphate to inositol 1-phosphate. The enzyme was purified 1300-fold to apparent homogeneity from the soluble fraction of human placenta. The enzyme requires Mn2+ or Mg2+ ions for activity, has an apparent Km for inositol 1:2-cyclic phosphate of 0.15 mM and forms 2.2 mumol of inositol 1 phosphate/min/mg protein. The enzyme does not utilize the cyclic esters of inositol polyphosphates as substrates. The molecular weight determined by gel filtration chromatography is approximately 55,000. Upon electrophoresis in polyacrylamide gels in sodium dodecyl sulfate, the molecular weight was found to be 29,000 both in the presence and absence of beta-mercaptoethanol. The enzyme was inhibited by inositol 2-phosphate (IC50 = 4 microM) and to a lesser degree by inositol 1-phosphate (IC50 = 2 mM) and inositol (IC50 = 4 mM). Zn2+ is a potent inhibitor of enzyme activity (IC50 = 10 microM). Neither Li+ nor Ca2+ had any effect on enzyme activity. This enzyme may serve to generate inositol from inositol cyclic phosphate metabolites produced by the phosphoinositide signaling pathway in cells. PMID- 3015959 TI - Cloning and structure of the Bacillus subtilis aspartate transcarbamylase gene (pyrB). AB - The Bacillus subtilis gene (pyrB), which encodes aspartate transcarbamylase (ATCase), was cloned on a HindIII restriction endonuclease fragment inserted into the pUC13 plasmid vector. B. subtilis pyrB was expressed in Escherichia coli, as judged by complementation of E. coli pyrB mutants and production of enzyme that was specifically inhibited by antibody directed against B. subtilis ATCase. The extent of expression was strongly dependent on the orientation of the inserted DNA in the vector, which suggested that transcription was initiated from vector borne (rather than B. subtilis) promoters. The entire 1098-base pair HindIII fragment of B. subtilis DNA was sequenced by the Maxam-Gilbert method. The amino acid sequence of B. subtilis ATCase was deduced from a 305-codon open reading frame and agreed very well with analyses of the purified enzyme. Comparison of the sequence of B. subtilis ATCase with that of E. coli ATCase catalytic subunit, for which the three-dimensional structure is known, revealed many homologous residues of probable importance in catalysis and structural folding of ATCases. The significance of homology to E. coli ornithine transcarbamylases was also analyzed. The sequences of the 5' and 3' flanking regions to pyrB encode open reading frames in both cases which overlap with pyrB by eight and six codons, respectively. It is probable that these open reading frames encode other enzymes of a coordinately regulated unit. The sequence 5' to pyrB also encodes an mRNA bearing a pyrimidine-rich sequence followed by a typical sequence for a rho independent transcription terminator. The presence of these elements and the 5' open reading frame suggest that B. subtilis pyrB, like E. coli pyrBI, is regulated by an attenuation mechanism. PMID- 3015961 TI - Effect of estradiol on the amino acid-accepting activity of uterine tRNAs and their participation in protein synthesis. AB - Estradiol (E2) induces a complementary increase in both the amount of mRNA and the rate of translation of the mRNA in the uterus of ovariectomized mature rats. The mechanism of the translational effect was evaluated by measuring the functional capacity of uterine tRNA isolated from control, E2 (1 h)- and E2 (14 h)-treated ovariectomized rats to support amino acid acceptor activity and uterine protein synthesis. The specific amino acid acceptor activity (SAA) of deacylated tRNA for 18 individual amino acids was determined using a tRNA dependent rat liver tRNA synthetase preparation. The SAA was the same for all amino acids for uterine tRNA from control and E2 (1 h)-treated rats but was increased for uterine tRNA from E2 (14 h)-treated rats to levels that were 1.4 4.3 times the SAA of uterine tRNA from control rats. When uterine tRNA from control and E2 (14 h)-treated rats was incubated with purified tRNA nucleotidyltransferase, the SAA for all amino acids was increased an average of 1.6-fold for control tRNA and 0.3-fold for tRNA from E2 (14 h)-treated rats. The ability of uterine tRNA to support maximal rates of protein synthesis in tRNA dependent uterine ribosome protein synthesis assay was increased by either in vivo treatment of the rats with estradiol or by in vitro repair of the 3'-CCA terminus of this tRNA by nucleotidyltransferase. These observations suggest that E2 may increase the rate of mRNA translation in the uterus, in part, by increasing the proportion of certain tRNAs with intact and functional 3'-CCA acceptor termini. PMID- 3015960 TI - Purification and characterization of a novel 4-methyleneglutamine synthetase from germinated peanut cotyledons (Arachis hypogaea). AB - A newly detected amide synthetase, designated 4-methyleneglutamine synthetase, has been partially purified from extracts of 5- to 7-day germinated peanut cotyledons (Arachis hypogaea). Purification steps include fractionation with protamine sulfate and ammonium sulfate followed by column chromatography on Bio Gel and DEAE-cellulose; synthetase purified over 300-fold is obtained. The enzyme has a molecular weight estimated to be approximately 250,000 and a broad pH optimum with maximal activity at approximately pH 7.5. Maximal rates of activity are obtained with NH+4 (Km = 3.7 mM) as the amide donor and the enzyme is highly specific for 4-methylene-L-glutamic acid (Km = 2.7 mM) as the amide acceptor. Product identification and stoichiometric studies establish the reaction catalyzed to be: 4-methyleneglutamic acid + NH4+ + ATP Mg2+----4 methyleneglutamine + AMP + PPi. PPi accumulates only when F- is added to inhibit pyrophosphatase activity present in synthetase preparations. This enzymatic activity is completely insensitive to the glutamine synthetase inhibitors, tabtoxinine-beta-lactam and F-, and is only partially inhibited by methionine sulfoximine. It is, however, inhibited by added pyrophosphate in the presence of F- as well as by certain divalent metal ions (other than Mg2+) including Hg2+, Ni2+, Mn2+, and Ca2+. All data obtained indicate that this newly detected synthetase is distinct from the well-known glutamine and asparagine synthetases. PMID- 3015962 TI - Interaction of milk xanthine oxidase with folic acid. Inhibition of milk xanthine oxidase by folic acid and separation of the enzyme into two fractions on Sepharose 4B/folate gel. AB - Inhibition of xanthine oxidase by folic acid was reexamined after complete removal of the contaminant which was responsible for time-dependent inactivation (Lewis, A. S., Murphy, L., Mcalla, C., Fleary, M., and Purcell, S. (1984) J. Biol. Chem. 259, 12-15; Spector, T., and Ferone, R. (1984) J. Biol. Chem. 259, 10784-10786). From turnover experiments using stopped flow equipment with a limited amount of xanthine and excess oxygen, and from kinetic analyses with an oxygen electrode, folic acid was found to be an inhibitor of xanthine oxidase. The inhibition was competitive with xanthine with a Ki value of 4.2 X 10(-5) M. From the behavior of the enzyme in affinity chromatography using a Sepharose 4B/folate column, folic acid was also confirmed to be a competitive inhibitor of xanthine oxidase. When enzyme which had been pretreated with oxipurinol was applied to the affinity column, two fractions of xanthine oxidase were separated. The first fraction was found to contain the fully active form (double-active dimers) from the analyses of spectral changes on addition of xanthine, oxipurinol titration, and ESR slow signal, whereas the second fraction was assumed to contain mixed dimers and double-inactive dimers. The ratio of the content of the first fraction to that of the second fraction supports the hypothesis that there are three enzyme species and that there is no interaction either in catalytic activity or in sulfuration or desulfuration reactions between the two subunits. PMID- 3015963 TI - Functional domains of assimilatory NADH:nitrate reductase from Chlorella. AB - Assimilatory nitrate reductase from Chlorella is a homotetramer which contains one of each of the prosthetic groups FAD, heme, and molybdenum per subunit. Besides the reduction of nitrate by NADH, nitrate reductase also catalyzes the partial activities NADH:cytochrome c reductase, NADH:ferricyanide reductase, and reduced methyl viologen:nitrate reductase. Incubation of native nitrate reductase with either trypsin, Staphylococcus aureus V8 protease, or a natural inactivator protease from corn results in a loss of NADH:nitrate reductase and NADH:cytochrome c reductase activities but no loss of reduced methyl viologen:nitrate reductase activity. Incubation of nitrate reductase with V8 protease or corn inactivator protease resulted in two different products, each of which retained a different partial activity. Reduced methyl viologen:nitrate reductase activity was associated with a homotetrameric fragment of about 260 kDa which contained heme and molybdenum but no FAD. The molecular mass of native nitrate reductase determined under the same conditions was 375 kDa. NADH:ferricyanide reductase activity was associated with a monomeric species of approximately 30 kDa which contained FAD and the NADH-binding site. These results are consistent with a structure-function model of nitrate reductase which has the following features: FAD/NADH-binding domains exposed on the surface of the molecule, a protease-sensitive hinge region which connects the nitrate-reducing and NADH dehydrogenase moieties, and the quaternary structure maintained via association sites on the heme/molybdenum domain. PMID- 3015964 TI - A highly bent fragment of Crithidia fasciculata kinetoplast DNA. AB - Kinetoplast DNA minicircles from Crithidia fasciculata contain a single major region of bent helix. Restriction fragments containing this bent helix have electrophoretic behavior on polyacrylamide gels which is much more anomalous than that of previously studied bent fragments. Therefore, the C. fasciculata fragments probably have a more extreme curvature. Sequencing part of a cloned minicircle revealed an unusual structure for the bent region. In a sequence of 200 bases, the bent region contains 18 runs of 4-6 As with 16 of these runs in the same strand. In some parts of this sequence the A runs are regularly spaced with a periodicity of about 10 base pairs. This spacing is nearly in phase with the twist of the DNA helix. This same sequence arrangement has been observed in other bent fragments, but the number of A runs is much greater in this C. fasciculata sequence. It is likely that there are small bends associated with each A run which, because of their periodic spacing, add up to produce substantial curvature in this molecule. In addition to having highly anomalous electrophoretic behavior, the fragment has unusual circular dichroism spectra. Its spectrum in the absence of ethanol is that of B DNA, but ethanol in the concentration range of 51-71% (w/w) induces changes to forms which are different from those of any well characterized DNA structure. The C. fasciculata bent helix is neither cleaved by S1 nuclease nor modified by bromoacetaldehyde under conditions in which other unusual DNA structures (such as cruciforms or B-Z junctions) are susceptible to attack by these reagents. Finally, a two dimensional agarose gel analysis of a family of topoisomers of a plasmid containing the bent helix revealed no supercoil-induced relaxation. PMID- 3015965 TI - Sensitization of the Escherichia coli cyclic AMP receptor protein to trypsin cleavage by polydeoxyribonucleotides and polyribonucleotides. AB - In the absence of cAMP the cyclic AMP receptor protein (CRP) is relatively resistant to trypsin whereas the cAMP X CRP complex is attacked yielding N terminal core fragments of 14,300 and 18,500 Da which still bind cAMP. The DNA X CRP complex formed at low ionic strength in the absence of cAMP is cleaved by trypsin with the formation of 9,700- and 6,000-Da fragments and the concomitant loss of cAMP binding activity. DNA X CRP remains as resistant to attack by subtilisin, clostripain, and the Staphylococcus aureus V8 protease as unliganded CRP but is slowly digested by chymotrypsin. All of the double-stranded polydeoxyribonucleotides and several of the single-stranded polydeoxyribonucleotides and polyribonucleotides tested render CRP sensitive to cleavage by trypsin. CRP is less rapidly cleaved by trypsin in the presence of d(A)n, d(I)n, and r(C)n indicative of a weaker affinity of CRP for these polynucleotides. The 9,700-Da fragment is N-terminal in CRP and probably terminates at Lys-89. The loss of cAMP binding activity following trypsin cleavage of DNA X CRP indicates that regions beyond this residue are important in the function of the cAMP-binding domain of CRP. The 6,000-Da fragment extends from Val-131 to Arg-185 or Lys-188 and contains part of the F helix involved in DNA binding by CRP. PMID- 3015966 TI - Similar control mechanisms regulate the insulin and type I insulin-like growth factor receptor kinases. Affinity-purified insulin-like growth factor I receptor kinase is activated by tyrosine phosphorylation of its beta subunit. AB - Insulin-like growth factor I (IGF-I) receptors are partially purified from human placenta by sequential affinity chromatography with wheat germ agglutinin-agarose and agarose derivatized with an IGF-I analog. Adsorption specificity to this affinity matrix demonstrates that low coupling ratios of IGF-I analog to agarose yield preparations that are highly selective in purifying IGF-I receptor with minimal cross-contamination by the insulin receptor present in the same placental extracts. Incubation of the immobilized IGF-I receptor preparation with [gamma 32P]ATP results in a marked phosphorylation of the receptor beta subunits, which appear as a doublet of Mr = 93,000 and 95,000 upon electrophoresis on dodecyl sulfate-polyacrylamide gels. The 32P-labeled receptor beta subunit doublet contains predominantly phosphotyrosine and to a much lesser extent phosphoserine and phosphothreonine residues. The immobilized IGF-I receptor preparation exhibits tyrosine kinase activity toward exogenous histone. The characteristics of the IGF-I receptor-associated tyrosine kinase are remarkably similar to those of the insulin receptor kinase. Thus, prior phosphorylation of the immobilized IGF-I receptor preparation with increasing concentrations of unlabeled ATP followed by washing to remove the unreacted ATP results in a progressive activation of the receptor-associated histone kinase activity. A maximal (10 fold) activation is achieved between 0.25 and 1 mM ATP. The concentration of ATP required for half-maximal (30 microM) activation of the IGF-I receptor kinase is similar to that of the insulin receptor kinase. Like the insulin receptor kinase, the elevated kinase activity of the phosphorylated IGF-I receptor is reversed following dephosphorylation of the receptor beta subunit with alkaline phosphatase. Furthermore, the phosphorylation of the IGF-I receptor beta subunit doublet is enhanced by 7-8-fold when reductant is included in the reaction medium, as is observed for the insulin receptor kinase. Significantly, the dose responses of both receptor types to reductant are identical. Both of the 32P labeled IGF-I receptor beta subunit bands are resolved into six matching phosphopeptide fractions when the corresponding tryptic hydrolysates are resolved by reverse phase high pressure liquid chromatography. Significantly, four out of the six phosphopeptide fractions derived from the trypsinized IGF-I receptor beta subunits are chromatographically identical to those from the tryptic hydrolysates of 32P-labeled insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3015967 TI - Unusual DNA structures in the adenovirus genome. AB - More than 80% (approximately 29 kilobase pairs) of the adenovirus serotype 2 genome was surveyed for the presence of unusual DNA conformations. Seven recombinant DNAs containing the largest HindIII fragments of AD2 DNA were analyzed for the presence of negative supercoil-dependent S1 nuclease-sensitive sites. Four plasmids each contained a specific site of S1 nuclease sensitivity whereas the other three showed no reaction. Further investigation was focused on a plasmid containing one of the positively reacting fragments (fragment C) which contained the major late promoter at coordinate 16.4 on the genome; three serotypes (Ad2, Ad7, Ad12) were studied. Fine mapping studies revealed the S1 sensitive sites to be a small region (approximately 6 base pairs) located at the TATA box of the major late promoter in all three cases. Other determinations (supercoil relaxation, T7 gene 3 product sensitivity, bromoacetaldehyde reactivity, anomalous gel mobility, the influence of negative superhelical density on nuclease sensitivity) led to the conclusion that the B-helix deformation was not due to a previously recognized DNA conformation (left-handed Z-DNA, cruciform, bent DNA), but may be accounted for by the homopurine X homopyrimidine nature of this region. PMID- 3015968 TI - Restriction enzyme digestions identify discrete domains in the chromatin around the promoter of the mouse alpha 2(I) collagen gene. AB - We have examined the chromatin structure around the +1 transcriptional start site of the mouse alpha 2(I) collagen gene by studying the accessibility of DNA to several restriction enzymes as well as to DNase I. In NIH 3T3 cells, which express high levels of alpha 2(I) collagen mRNA, we detect a DNase I hypersensitive site from -240 to +110 relative to the start site of transcription at +1. By digesting chromatin with restriction enzymes, which cleave naked DNA at multiple sites within the -2000 to +1000 region, a considerably more complex picture was revealed. DNA sequences upstream of around -550 and downstream of +150 are much less accessible to restriction enzymes than the region between these sites and are, therefore, probably packaged in a more compact conformation. The region from around -550 to -240 although not within the DNase I hypersensitive domain is nevertheless accessible to restriction enzymes and, therefore, presumably in a relatively "open" conformation. In addition, beginning 5' to -100 there is a gradual decrease in restriction enzyme accessibility as one approaches +150. Of particular interest is the finding that although sites at +65 and +126 are relatively accessible, a HinfI site at +113 is resistant in chromatin. In v-mos transformed NIH 3T3 cells which express alpha 2(I) collagen at much lower levels than untransformed NIH 3T3 cells, the DNase I-hypersensitive site as well as the majority of the chromatin restriction enzyme accessibility patterns are similar to those found in untransformed NIH 3T3 cells. However, a SphI site at +58 appears less accessible in the transformed cells. We also examined the chromatin of a myeloma cell line which does not synthesize alpha 2(I) collagen at detectable levels. In the nuclei of these cells the DNA of the alpha 2(I) collagen promoter is inaccessible to DNase I and to all restriction enzymes. PMID- 3015969 TI - 3'-O-(4-benzoyl)benzoylcytidine 5'-triphosphate. A substrate and photoaffinity label for CMP-N-acetylneuraminic acid synthetase. AB - A photoreactive, radiolabeled pyrimidine nucleotide, 3'-O-(4 benzoyl)benzoylcytidine 5'-triphosphate was synthesized from benzoylbenzoic acid and radiolabeled CTP. Benzoylbenzoyl-[5-3H]CTP could substitute for CTP, in an enzymatic reaction with N-acetylneuraminic acid catalyzed by Escherichia coli or rat liver CMP-NeuAc synthetase, to yield radiolabeled benzoyl-benzoyl-CMP-NeuAc. E. coli CMP-NeuAc synthetase could be specifically radiolabeled using benzoylbenzoyl-[alpha-32P]CTP as a photoaffinity label. This specific covalent binding occurred using enzyme preparations of different degrees of purity. These results suggest that benzoylbenzoic acid derivatives of pyrimidines should be of general use in the identification and active site mapping of pyrimidine-requiring proteins and enzymes. These include glycosyltransferases, sugar nucleotide synthetases, and transporters, and enzymes participating in the conjugation of bile acids and biosynthesis of nucleic acids and choline nucleotides. PMID- 3015970 TI - Study of barley endonucleases and alpha-amylase genes. AB - We have identified an endonuclease(s) that preferentially cleaves the internucleosomal linker regions in the aleurone chromatin producing mono- and oligonucleosomes. This enzyme(s) has been designated as a "linker"-specific nuclease(s). This nuclease does not require divalent cations for activity, and therefore it is not the "Ca2+-Mg2+-DNase" found in mammalian cells. The linker specific nuclease activity is not detectable in the dry aleurone tissue and in the tissue treated with 0.5 mM cordycepin. The endonuclease activity of the aleurone tissue incubated with gibberellic acid is higher than the level of this endonuclease in tissue treated with abscisic acid or water alone. Nuclei isolated from embryos have lower levels of endonuclease activities compared to those from aleurone tissue. Digestion of the nuclei from embryos with micrococcal nuclease revealed the subunit structure of chromatin. In Southern blots of the HindIII digests of DNA from embryos, five DNA bands hybridized to a nick-translated alpha amylase cDNA clone. In similar autoradiograms with aleurone DNA, particular bands are less visible, notably in the DNA isolated from the tissue treated with gibberellic acid. This is the first report of the presence of a linker-specific nuclease activity in plant cells. PMID- 3015971 TI - The major antigen of elastin-associated microfibrils is a 31-kDa glycoprotein. AB - The major antigen derived from elastic fiber microfibrils was identified as a Mr = 31,000 glycoprotein, using immunoblotting and immunohistochemical techniques with antisera raised to "reductive guanidine extracts" of fetal bovine nuchal ligament, and to subfractions of these. A second, elastic fiber-derived, but unidentified, antigen of large molecular size (Mr greater than 200,000) was present in these extracts. Antisera raised to the purified 31-kDa glycoprotein were shown, by immunoelectron microscopy, to localize specifically to the elastin associated microfibrils. Thus, the macro-molecule was called "microfibril associated glycoprotein" or MAGP. MAGP is an acidic glycoprotein with a distinctive amino acid composition, being exceptionally rich in glutamic acid, rich in cystine, and low in glycine. MAGP was extractable from tissue homogenates using NaCl, urea, or guanidine hydrochloride solutions, only if a strong reducing agent was present. Thus, disulfide bonding is important for the strong association of MAGP with elastic fibers. Immunoblotting with anti-MAGP antiserum identified two additional reactive species, of Mr = 60,000 and Mr approximately 300,000, in tissue extracts. As only the 31-kDa species was detected in fibroblast culture medium, these additional species were probably aggregates, rather than precursors. MAGP did not react with antilysyl oxidase antiserum on immunoblots or by enzyme-linked immunosorbent assay. MAGP is the first macromolecule to have been established to be a constituent of elastin-associated microfibrils in both developing and mature elastic tissues. PMID- 3015972 TI - Autoradiographic evidence for a heterogeneous distribution of alpha 1 adrenoreceptors labelled by [3H] prazosin in rat, dog and human kidney. AB - The selective alpha1-adrenoreceptor antagonist radioligand [3H] prazosin has been used to localize alpha1-adrenoreceptors in slide mounted sections of rat, dog and human kidney. The biochemical characteristics of [3H] prazosin binding to rat kidney sections were examined and found to be saturable, reversible, stereoselective, with a KD of 0.12 nM and Bmax of 6.72 +/- 0.2 fmoles/section. 3H Ultrofilm images of [3H] prazosin binding to rat, dog, and human kidney revealed binding to the vasculature but in the rat additional receptors were confined to the renal cortex. In the rat kidney autoradiography using emulsion coated coverslips showed that binding in the renal cortex was largely to proximal tubules. In all three species the autoradiographic studies support a role for alpha1-adrenoreceptors in control of renal blood flow. In the rat the location of alpha1-adrenoreceptors suggests that they can also have an important influence on fluid and electrolyte balance, gluconeogenesis and production of prostanoids. PMID- 3015973 TI - A study on the postjunctional excitatory alpha-adrenoreceptor subtypes in the mesenteric arterial bed of the rat. AB - The nature of the postsynaptic adrenoreceptor subtypes which mediate vasoconstriction in the mesenteric arterial bed of the rat was investigated using mixed and selective alpha 1, alpha 2 and beta-agonists and antagonists. Phenylephrine (PE) an alpha 1-selective agonist and noradrenaline (NA) a mixed alpha1 and alpha 2-agonist, produced a rise in perfusion pressure (vasoconstriction). The responses to NA remained stable with time whereas responses to PE considerably increased. UK14304 an alpha 2-selective agonist at low doses (10(-8)-10(-7) moles), caused small, slow contractions in most preparations. Repeated administration of these doses or slightly higher ones, densensitized the tissue to this compound but not to NA or PE. Finally, UK14304 given simultaneously with NA or PE, at doses higher than 5 X 10(-7) moles, reduced contractions to the latter compounds and this effect was not altered by 10(-7) M rauwolscine, an alpha 2-selective antagonist. Prazosin, an alpha1 selective antagonist, as expected, reduced contractions to NA considerably at 10( 10)-10(-8) M and abolished contractions to UK14304 at 2 X 10(-9) M. Rauwolscine, at 10(-8) M, potentiated contractions to NA and at 10(-6) M reduced contractions to both NA and PE (when compared to time controls). When propranolol (10(-6) M), a beta-antagonist was included in the perfusion fluid, rauwolscine no longer potentiated responses to NA but reduced them at all concentrations. Under the same conditions rauwolscine affected the responses to PE in a similar direction to that observed in the absence of propranolol. These results suggest that in the rat mesenteric arterial bed: rauwolscine exerts an effect additional to alpha2 adrenoreceptor antagonism.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3015974 TI - Localization of somatomedin-C binding to bovine growth-plate chondrocytes in situ. AB - The somatomedins are a family of low-molecular-weight peptides that are thought to mediate the stimulatory effect of growth hormone on skeletal growth. The cells that are directly responsible for skeletal growth are the chondrocytes of the epiphyseal growth plate, and these are the presumed skeletal target cells for somatomedin. As with other peptide growth factors, the cellular effects of the somatomedins are initiated by the interaction of the growth factor with specific receptors on target-cell surface membranes. Chondrocytes that have been isolated from bovine growth plates were previously found to possess specific surface receptors for the principal growth-hormone-dependent somatomedin, somatomedin-C. These studies indicated that the interaction of growth-plate chondrocytes with somatomedin-C involves specific receptor-binding followed by somatomedin-C internalization by the cell, a process identical to that identified in the mechanism of action of other peptide growth factors in other cells. These studies, however, left unanswered the questions of whether there are differences in binding of somatomedin-C by the different cell populations within the physis and whether somatomedin-C has access to cells in intact tissues. The current studies address these issues and indicate that bovine physeal chondrocytes in situ are accessible to exogenous somatomedin-C, that they specifically bind somatomedin-C in situ, and that cells of different physeal zones bind somatomedin C differently. Labeled somatomedin-C is specifically bound by cells of all physeal zones. However, the binding is greatest for those cells undergoing active synthesis of DNA in the proliferative zone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3015976 TI - Neural fibrosis and the effect of neurolysis. AB - Thickening of the fibrous element of a peripheral nerve may be caused by repeated friction, traction, constriction, ischaemia or partial rupture. The sequel may be a conduction disorder and a clinical condition such as an entrapment neuropathy or a tardy nerve palsy. Neural fibrosis is typically associated with a pseudoneuroma in continuity which has resulted from scarring and adhesions around the nerve as well as proliferation of the fibrous element within the nerve; the fibrosis may be classified as extraneural, intraneural or dispersive. We report 17 cases treated by external neurolysis, with 14 satisfactory results, and 42 patients treated by internal neurolysis with success in 37. Seven of the eight failures were in cases of dispersive fibrosis. A technique of internal neurolysis is described. PMID- 3015975 TI - Porous hydroxyapatite as a bone-graft substitute in metaphyseal defects. A histometric study. AB - Porous hydroxyapatite (Interpore 500) formed by conversion of the Porites goniopora coral exoskeleton has pores averaging 600 micrometers and pore interconnections averaging 260 micrometers in diameter. In the proximal tibial metaphysis of eight dogs, a defect one cubic centimeter in size was created unilaterally and was fitted with a block of Interpore 500. Both proximal tibial metaphyses were retrieved at two, four, six, and twelve months. Stained undecalcified sections were examined by light microscopy and quantitated by histometric methods. The implant-side specimens contained compact bone along the external surface and trabecular bone interiorly. The interior of these specimens was composed of 51.9 +/- 1.3 per cent soft tissue, 13.0 +/- 1.2 per cent bone, and 35.1 +/- 1.2 per cent Interpore 500 (mean and standard error). The interior of the normal specimens was composed of 79.7 +/- 1.4 per cent soft tissue and 20.2 +/- 1.4 per cent bone. The allocation of implant pore space between bone and soft tissue was proportional to that of bone and soft tissue in the normal tibiae. The stereological distribution of regenerated bone in the porous hydroxyapatite was also the same as in the normal tibiae. The appositional process of incorporation of the implant was confirmed by the finding that 66.5 per cent of the surface of the Interpore 500 was covered with bone ingrowth at twelve months. PMID- 3015978 TI - Pattern of fibronectin distribution in human lung cancer. AB - The pattern of fibronectin (FN) distribution in human lung cancer was studied by indirect immunofluorescent staining, and by the peroxidase-antiperoxidase method in a total of 60 surgical specimens. They comprised 8 small cell carcinomas, 4 large cell carcinomas, 19 squamous cell carcinomas, 28 adenocarcinomas, and 1 adenoid cystic carcinoma. Of the 60 specimens 13 were FN-positive. They included 4 large cell carcinomas, 4 small cell carcinomas, 3 poorly differentiated squamous cell carcinomas, and 2 poorly differentiated and 1 moderately differentiated adenocarcinomas. On the other hand, none of the well differentiated carcinomas was FN-positive around tumor cells. Our data suggest that undifferentiated, or poorly differentiated carcinomas of the lung tend to be FN-positive. PMID- 3015979 TI - Calcitonin in human gastric mucosa and carcinoma. AB - The localization of immunoreactive calcitonin (IR-CT) in the human gastric mucosa and tumor tissues was studied using an immunohistochemical peroxidase antiperoxidase method. A small number of IR-CT-containing cells were observed in both infant and adult gastric antral mucosa and the ratio of IR-CT-containing cells to G cells was about 1:50-100. Moreover, tissue content of IR-CT in normal antral mucosa was 2.37 +/- 0.35 ng/g wet weight. IR-CT-containing cells and G cells decreased with the progress of chronic atrophic gastritis and were totally absent in intestinal metaplastic glands. IR-CT was detected in G cells, suggesting a paracrine relation between gastrin and CT. IR-CT was not found in tumor cells of 35 gastric adenomas and 40 well differentiated adenocarcinomas. On the other hand, it was demonstrated in a very small number of tumor cells in 4 of 46 poorly differentiated adenocarcinomas, and in a good number in 3 of 7 scirrhous argyrophil cell carcinomas. IR-CT in plasma could serve, therefore, as a tumor marker of scirrhous endocrine cell carcinoma, and its production in cancer cells was considered to be eutopic rather than ectopic. PMID- 3015977 TI - The epidemiology of primary liver cancer in a West German population: the Saarland. AB - The present study is based on the data of 321 cases of primary liver cancer (PLC); 225 hepatocellular carcinomas (HCC), 54 cholangiocellular carcinomas (CCC) and 35 unclassified carcinomas autopsied between 1963 and 1982 or reported to the Saarland Cancer Registry between 1967 and 1981. The age standardized incidence rate for the Saarland was determined as 1.1. We noticed an increase in incidence for both HCC and CCC. The HCC rise was based on a significant (p less than 0.05) increase for women. The incidence maximum in the successive birth cohorts is shifting into younger age groups. The highest rates were observed in men and women born between 1900 and 1909. The regional distribution of PLC in the Saarland shows an accumulation in the urban areas. The mean survival time from diagnosis to death was 3.1 months. Prognosis was only influenced by the grade of differentiation. 88.3% of the HCC, but only 28.6% of CCC occurred in cirrhotic livers. Orcein staining of 55 liver specimens showed evidence of previous HBV infection in 12 out of 38 cases of HCC (31.6%) and no evidence of HBVB in the 17 cases of CCC studied. PMID- 3015980 TI - Reexamination of histological findings of ileal sarcomas induced in rats given diet containing bracken fern. AB - The ileal sarcomas induced in rats given diet containing bracken fern were examined microscopically and electron microscopically. These ileal sarcomas, which were induced at high incidence (80%-44%), have previously been identified as spindle cell fibrosarcomas. However, studies showed that they should be diagnosed as malignant fibrous histiocytomas (MFH) being classified into the following four histological types; fibrous, pleomorphic, giant cell and myxoid type. No types of sarcomas other than MFH were found in the ileum. PMID- 3015981 TI - Large deletions in the cytoplasmic kinase domain of the epidermal growth factor receptor do not affect its laternal mobility. AB - The lateral diffusion coefficients of various epidermal growth factor (EGF) receptor mutants with increasing deletions in their carboxy-terminal cytoplasmic domain were compared. A full size cDNA construct of human EGF receptor and different deletion constructs were expressed in monkey COS cells. The EGF receptor mutants expressed on the cell surface of the COS cells were labeled with rhodamine-EGF, and the lateral diffusion coefficients of the labeled receptors were determined by the fluorescence photo-bleaching recovery method. The lateral mobilities of three deletion mutants, including a mutant that has only nine amino acids in the cytoplasmic domain, are all similar (D approximately equal to 1.5 X 10(-10) cm2/s) to the lateral mobility of the "wild-type" receptor, which possess 542 cytoplasmic domain of EGF receptor, including its intrinsic protein kinase activity and phosphorylation state, are not required for the restriction of its lateral mobility. PMID- 3015983 TI - Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. II. Autoradiographic evidence for its uptake into motor nerves by acceptor-mediated endocytosis. AB - Using pharmacological (Simpson, L.L., 1980, J. Pharmacol. Exp. Ther. 212:16-21) and autoradiographic techniques (Black, J.D., and J.O. Dolly, 1986, J. Cell Biol., 103:521-534), it has been shown that botulinum neurotoxin (BoNT) is translocated across the motor nerve terminal membrane to reach a postulated intraterminal target. In the present study, the nature of this uptake process was investigated using electron microscopic autoradiography. It was found that internalization is acceptor-mediated and that binding to specific cell surface acceptors involves the heavier chain of the toxin. In addition, uptake was shown to be energy and temperature-dependent and to be accelerated by nerve stimulation, a treatment which also shortens the time course of the toxin-induced neuroparalysis. These results, together with the observation that silver grains were often associated with endocytic structures within the nerve terminal, suggested that acceptor-mediated endocytosis is responsible for toxin uptake. This proposal is supported further by the fact that lysosomotropic agents, which are known to interfere with the endocytic pathway, retard the onset of BoNT induced neuroparalysis and also affect the distribution of silver grains at nerve terminals treated with 125I-BoNT. Possible recycling of BoNT acceptors (an important aspect of acceptor-mediated endocytosis of toxins) at motor nerve terminals was indicated by comparing the extent of labeling in the presence and absence of metabolic inhibitors. On the basis of these collective results, it is concluded that BoNT is internalized by acceptor-mediated endocytosis and, hence, the data support the proposal that this toxin inhibits release of acetylcholine by interaction with an intracellular target. PMID- 3015985 TI - Increased level of cAMP-dependent protein kinase in aging human lung fibroblasts. AB - Regulation of the expression of cAMP-dependent protein kinase in cellular aging was studied using the IMR-90 diploid human lung fibroblasts. The level of cAMP dependent protein kinase present in cell extracts was monitored by 1) photoactivated incorporation of 8-N3-[32P]cAMP into the 47,000- and 54,000-dalton regulatory subunits of the type I and type II cAMP-dependent protein kinases, respectively; 2) cAMP-dependent phosphorylation of histone II AS catalyzed by the catalytic subunit of the kinase; and 3) fractionation and analysis of the type I and type II cAMP-dependent protein kinase by DEAE-Sephacel column chromatography. Our results showed an approximately two- to threefold increase in the level of the type I cAMP-dependent protein kinase and a somewhat smaller increase in the type II kinase in extracts of the "old" IMR-90 cells (population doubling greater than 48) as compared to that of the "young" cells (PDL 22-27). The timing of the increase in cAMP-dependent protein kinase coincided with a significant decrease in the proliferative potential of the cells. This result together with previously demonstrated effects of cAMP in the control of cell growth and differentiation and the increased expression of cAMP-dependent protein kinase during terminal differentiation of the murine preadipocytes (3T3-L1) and myoblast (L-5, L-6, and C2C13) suggests that regulation of the levels of cAMP and cAMP-dependent protein kinase plays a significant role in the control of cell growth and differentiation. PMID- 3015982 TI - Cyclosporine A inhibits Ca2+-dependent stimulation of the Na+/H+ antiport in human T cells. AB - The cyclic undecapeptide cyclosporine A (CsA) is a potent immunosuppressive agent that inhibits the initial activation of T lymphocytes. This agent appears to be most effective in blocking the action of mitogens such as concanavalin A and the calcium ionophore A23187, which cause an influx of Ca2+, but not those that may act by alternate mechanisms. These observations suggest that CsA may block a Ca2+ dependent step in T cell activation. We have shown that stimulation of the T3-T cell receptor complex-associated Ca2+ transporter activates the Na+/H+ antiport (Rosoff, P. M., and L. C. Cantley, 1985, J. Biol. Chem., 260: 14053-14059). The tumor-promoting phorbol esters, which are co-mitogenic for T cells, activate the exchanger by a separate pathway which is mediated by protein kinase C. Both the rise in intracellular Ca2+ and intracellular pH may be necessary for the successful triggering of cellular activation. In this report we show that CsA blocks the T3-T cell receptor-stimulated, Ca2+ influx-dependent activation of Na+/H+ exchange, but not the phorbol ester-mediated pathway in a transformed human T cell line. CsA inhibited mitogen-stimulation of interleukin-2 production in a separate cell line. CsA also inhibited vasopressin stimulation of the antiporter in normal rat kidney fibroblasts, but had no effect on serum or 12-O tetradecanoyl phorbol 13-acetate stimulation. CsA did not affect serum or vasopressin or serum stimulation of normal rat kidney cell proliferation. CsA also had no effect on lipopolysaccharide or phorbol ester stimulation of Na+/H+ exchange activity or induction of differentiation in 70Z/3 pre-B lymphocytes in which these events are initiated by the protein kinase C pathway. These data suggest that mechanisms of activation of Na+/H+ exchange that involve an elevation in cytosolic Ca2+ are blocked by CsA but that C kinase-mediated regulation is unaffected. The importance of the Na+/H+ antiport in the regulation of growth and differentiation of T cells is discussed. PMID- 3015984 TI - Mitogenic activity of hydroxyapatite: requirement for somatomedin C. AB - Synovial hyperplasia is a feature of the chronic synovitis associated with basic calcium phosphate crystals [hydroxyapatite (HA), octacalcium phosphate, tricalcium phosphate] and calcium pyrophosphate. Each of these crystals stimulated mitosis of cultured human skin fibroblasts or canine synovial fibroblasts in a concentration-dependent fashion. We examined the effect of pure somatomedin C (Sm-C) on HA crystal induced mitogenesis. Confluent cultures of human fibroblasts were rendered quiescent by incubation in the presence of 1% platelet-poor-Sm-C free plasma (PPSCFP) for 24 hours. HA crystals stimulated thymidine incorporation 2.3-fold over control value. Addition of Sm-C significantly augmented the effect of HA crystals (P less than 0.01). Nearly identical effects were observed in the presence of 100 micrograms/ml HA crystals or 15 ng/ml PDGF. Monoclonal antibodies against Sm-C had little effect on the basal 3H thymidine uptake by control cells incubated in 1% PPSCFP but blocked over 50% of the HA crystal or PDGF-induced 3H thymidine incorporation both in the presence or absence of Sm-C. The incomplete blocking suggested either the presence of other "progression" factors, such as insulin-like growth factor II in the conditioned media or the possibility that HA or PDGF in high enough dosage enabled cells to escape their dependence on Sm-C for DNA synthesis. PMID- 3015986 TI - Energy-dependent volume regulation in primary cultured cerebral astrocytes. AB - Cell volume regulation and energy metabolism were studied in primary cultured cerebral astrocytes during exposure to media of altered osmolarity. Cells suspended in medium containing 1/2 the normal concentration of NaCl (hypoosmotic) swell immediately to a volume 40-50% larger than cells suspended in isoosmotic medium. The cell volume in hypoosmotic medium then decreases over 30 min to a volume approximately 25% larger than cells in isoosmotic medium. In hyperosmotic medium (containing twice the normal concentration of NaCl), astrocytes shrink by 29%. Little volume change occurs following this initial shrinkage. Cells resuspended in isoosmotic medium after a 30 min incubation in hypoosmotic medium shrink immediately to a volume 10% less than the volume of cells incubated continuously in isoosmotic medium. Thus, the regulatory volume decrease (RVD) in hypoosmotic medium involves a net reduction of intracellular osmoles. The RVD is partially blocked by inhibitors of mitochondrial electron transport but is unaffected by an inhibitor of glycolysis or by an uncoupler of oxidative phosphorylation. Inhibition of RVD by these metabolic agents is correlated with decreased cellular ATP levels. Ouabain, added immediately after hypoosmotic induced swelling, completely inhibits RVD, but does not alter cell volume if added after RVD has taken place. Ouabain also inhibits cell respiration 27% more in hypoosmotic medium than in isoosmotic medium indicating that the (Na,K)-ATPase coupled ion pump is more active in the hypoosmotic medium. These data suggest that the cell volume response of astrocytes in hypoosmotic medium involves the net movement of osmoles by a mechanism dependent on cellular energy and tightly coupled to the (Na,K)-ATPase ion pump. This process may be important in the energy-dependent osmoregulation in the brain, a critical role attributed to the astrocyte in vivo. PMID- 3015987 TI - ATP-induced cell contraction in dermal fibroblasts: effects of cAMP and myosin light-chain kinase. AB - When 1 mM ATP is added to human dermal fibroblasts (DF) in monolayer culture permeabilized by glycerol, they undergo a rapid reduction in length and their intracellular actin filaments aggregate. This process is referred to as cell contraction. Treating glycerol-permeabilized DF with alkaline phosphatase before adding 1 mM ATP should cause dephosphorylation. Dephosphorylated preparations do not undergo cell contraction initiated by ATP. When myosin light-chain kinase (MLCK) isolated from turkey gizzard is added with cofactors to cells dephosphorylated by alkaline phosphatase treatment, contraction is restored. DF incubated for 24 h with db cAMP or cholera toxin show elevated intracellular concentrations of cAMP and little cell contraction. Contraction is reestablished when MLCK with cofactors is incubated with these preparations before ATP is added. Fibroblasts from Epidermolysis Bullosa dystrophica recessive patients produce excess cAMP. Those cells show minimal contraction, however; treating them with MLCK and cofactors renews contraction brought about by ATP. When DF are incubated with trifluoperazine to block calmodulin-dependent enzyme reactions, cell contraction is inhibited. Adding cytochalasin B disrupts microfilaments and also inhibits contraction. This work supports the idea that myosin ATPase is critical to cell contraction. Myosin ATPase is dependent on the phosphorylation of the regulatory peptide, myosin light chain. Elevating intracellular concentrations of cAMP or treatment of permeabilized cell preparations with alkaline phosphatase may inhibit myosin ATPase activity. The restoration of phosphorylation by adding MLCK with cofactors served to reestablish cell contraction. PMID- 3015988 TI - Comparative studies of the binding and growth-supportive ability of mammalian transferrins in human cells. AB - The ability of human-derived cells in culture to bind, remove iron from, and grow in the presence of transferrins (Tf) isolated from the sera of species commonly included in tissue culture medium was investigated. Kinetic studies on HeLa cells reveal apparent first-order association rate constants of 0.43 min-1 for human Tf and 0.15 min-1 for equine Tf. Labeled chicken ovo-Tf and fetal bovine Tf were not recognized by the HeLa cells. Competition experiments with HeLa cells that use either isolated Tf or parent serum confirm these findings. Equilibrium binding experiments performed on HeLa cells at 37 degrees C in the presence of 2,4 dinitrophenol to prevent iron removal indicate 1 X 10(6) Tf bound/cell with a dissociation constant (K'D) of 28 nM for human Tf and 182 nM for equine Tf. Equilibrium binding performed at 0 degrees C to prevent endocytosis reveals 4.1 6.7 X 10(5) Tf binding sites/cell with a K'D of 8.3 nM for human Tf and 41.5 nM for equine Tf. Parallel experiments in normal human diploid fibroblast-like MRC-5 cells indicate expression of 0.82-2.78 X 10(5) Tf binding sites/cell with a K'D of 8.2 nM for human and 39.1 nM for equine Tf. Thus, the results of equilibrium binding studies of a more differentiated cell type are consistent with those found for HeLa cells. Fetal bovine Tf was found to compete weakly with labeled human Tf for human receptor on HeLa cells in a soluble receptor assay, with an approximately 500-fold excess needed to reduce binding to half maximal. Iron uptake experiments show an iron donating hierarchy where human greater than horse greater than calf, suggesting that the rate of iron uptake depends on the affinity of receptor for transferrin. Growth experiments involving HeLa cells in chemically defined serum-free medium demonstrate that bovine Tf will support growth as well as human Tf, but at concentrations much higher than are required of human Tf. PMID- 3015989 TI - Transformation of diploid human fibroblasts by DNA transfection with the v-sis oncogene. AB - The simian sarcoma virus (SSV) oncogene (v-sis) has a high degree of homology to the cellular gene coding for the B peptide of human platelet-derived growth factor (PDGF), a potent fibroblast mitogen. The cellular homolog of v-sis is activated in some mesenchymal human tumors and cell lines derived from them. To determine the phenotype produced by v-sis in diploid human fibroblasts, we constructed plasmids containing the SSV provirus and drug-resistance markers and transfected them into early-passage human cells. Fibroblasts that had integrated the plasmid were selected for drug resistance and shown to contain and express the v-sis oncogene by DNA and RNA hybridization. The v-sis-expressing cells grew to higher saturation densities than control cells transfected with the vector plasmid alone and formed large, well defined foci. This allowed selection of transfectants directly for focus formation. The v-sis transformed cells continued to grow well in the absence of serum, whereas age-matched, vector-transfected control cells ceased replicating under these conditions so that the final difference in density between the two populations was tenfold. Incorporation of thymidine in serum-free medium by the v-sis-transformed cells was independent of exogenous PDGF. In contrast, PDGF increased thymidine incorporation in such medium by the control cells to the level found in the v-sis-transformed cells with or without added PDGF. These results suggest that expression of the v-sis oncogene in diploid human fibroblasts causes sufficient endogenous synthesis of the B chain of PDGF to allow transformants to grow to abnormally high cell densities. When individual v-sis-transformed cells were grown on a background of normal cells, this higher cell density at confluence could be visualized as a focus. PMID- 3015990 TI - Localization of human histone gene transcripts predominantly in the nonmatrix nuclear fraction. AB - Using S1 nuclease protection assays, we have examined the representation of cell cycle-dependent H4 histone RNAs in the nuclear matrix and nonmatrix nuclear fractions of human cells. Cytoplasmic and nuclear fractions were prepared from exponentially growing HeLa S3 cells by double detergent (sodium deoxycholate and NP40) lysis. The nuclear matrix and nonmatrix nuclear fractions were then prepared by digestion of nuclei with RNase-free DNase I and subsequent high-salt [0.4 M (NH4)2SO4] extraction. Subcellular fractions were characterized by 1) DNA, RNA, and protein composition; 2) electrophoretic analysis of the proteins in each fraction; 3) the representation of 45S ribosomal RNA precursors and processed 18S and 28S ribosomal RNAs; and 4) the presence of mitochondrial RNAs. In contrast to ribosomal and messenger RNA precursors, which are largely associated with the nuclear matrix, the human H4 histone RNAs in the nucleus were found predominantly in the nonmatrix nuclear fraction. The presence of H4 histone RNA in the nonmatrix nuclear fraction appeared to be coupled to DNA replication, since inhibition of DNA synthesis by hydroxyurea resulted in a loss of histone RNA from the nucleus. Our results suggest either that the association of histone RNAs with the nuclear matrix is very transient or that posttranscriptional modifications of the rapidly processed histone gene transcripts do not involve the nuclear matrix. PMID- 3015991 TI - Functional and structural characteristics of EGF and its receptor and their relationship to transforming proteins. AB - Epidermal growth factor (EGF) is a peptide which effects the growth and/or differentiated functions of many cell types. Several pieces of evidence indicate that EGF and its receptor may play a role in carcinogenesis. Functional and structural characteristics of EGF and its receptor and their relationship to transforming proteins are discussed. EGF has extensive homology with alpha transforming growth factor (alpha-TGF), which may actually be an embryonic form of EGF. Nevertheless, both EGF and alpha-TGF elicit transformation-associated phenotypes in target cells under certain conditions. EGF effects are mediated by a receptor present on the plasma membrane. The EGF receptor is a highly complex protein having several functions in addition to binding EGF in a highly specific manner. One of these functions is to phosphorylate tyrosyl residues on certain proteins. This activity is similar to that expressed by the src family of oncogene-encoded proteins. Besides sharing functional homology the EGF receptor also exhibits structural homology to several oncogene-encoded proteins. The v-erb B-transforming protein has a striking extent of homology (95%) to the cytoplasmic portion of the EGF receptor. These data support the concept that some aspect of EGF-stimulated metabolism is involved in cellular transformation. PMID- 3015992 TI - The bacterial phosphotransferase system: kinetic characterization of the glucose, mannitol, glucitol, and N-acetylglucosamine systems. AB - The kinetic mechanisms by which the glucose, glucitol, N-acetylglucosamine, and mannitol enzymes II catalyze sugar phosphorylation have been investigated in vitro. Lineweaver-Burk analyses indicate that the glucose and glucitol enzymes II catalyze sugar phosphorylation by a sequential mechanism when the two substrates are phospho-enzyme III and sugar. The N-acetylglucosamine and mannitol enzymes II, which do not function with an enzyme III, catalyze sugar phosphorylation by a ping-pong mechanism when the two substrates are phospho-HPr and sugar. These results, as well as previously published kinetic characterizations, suggest a common kinetic mechanism for all enzymes II of the system. It is suggested that all enzymes II and enzyme II-III pairs arose from a single (fused) gene product containing two sites of phosphorylation and that phosphoryl transfer from the second phosphorylation site to sugar can only occur when the enzyme II-III pair is present in the associated state. PMID- 3015993 TI - Characterization of a specific antiserum for mammalian collagenase from several species: immunolocalization of collagenase in rabbit chondrocytes and uterus. AB - A specific antiserum to rabbit bone collagenase was raised in a sheep and shown to react with collagenase from several mammalian species. This antiserum was used to demonstrate that only active collagenase binds to and can be immunolocalized on collagen fibrils and, by indirect immunofluorescence, the secretion of latent collagenase by stimulated rabbit chondrocytes and cells of the post-partum involuting rabbit uterus. The ionophore monensin was used to demonstrate intracellular accumulation of collagenase in the Golgi apparatus of both stimulated chondrocytes and involuting uterine cells. Collagenase was not detectable in either normal cartilage or non-gravid uterus. These results are discussed in relation to other studies of collagenase immunolocalization reported in the literature. PMID- 3015994 TI - Adenosine receptors of cerebral microvessels and choroid plexus. AB - We studied, by ligand binding methods, the two adenosine receptors, A1 and A2, in rat and pig cerebral microvessels and pig choroid plexus. Ligand binding to cerebral microvessels was compared with that to membranes of the cerebral cortex. [3H]Cyclohexyladenosine and [3H]L-phenylisopropyladenosine were the ligands used for A1-receptors, and [3H]5'-N-ethylcarboxamide adenosine ([3H]NECA) was used to assess A2-receptors. We report that cerebral microvessels and choroid plexus exhibit specific [3H]NECA binding, but have no appreciable A1-receptor ligand binding sites. Specific binding of [3H]NECA to cerebral microvessels, choroid plexus, and cerebral cortex was saturable and suggested the existence of two classes of A2-receptor sites: high-affinity (Kd approximately 250 nM) and low affinity (Kd approximately 1-2 microM) sites. The Kd and Bmax of NECA binding to cerebral microvessels and cerebral cortex were similar within each species. Our results, indicating the existence of A2-receptors in cerebral microvessels, are consistent with results of increased adenylate cyclase activity by adenosine and some of its analogues in these micro-vessels. PMID- 3015995 TI - Preparation and evaluation of inorganic anion-exchange sorbents not based on silica. AB - The use of alternate macroporous (greater than 200 A) inorganic support materials in the preparation of pellicular anion-exchange packings was explored. Alumina, magnesia, titania, and a zirconia-coated silica were chosen for comparison with silica. The stationary phase attached to the support surfaces was an adsorbed polyethyleneimine, crosslinked with 1,4-butanediol diglycidyl ether. Packing materials were characterized by static elemental analyses, chromatographic retention, static loading capacity and pH stability. Titania, alumina, and zirconyl-clad silica packings were found to be substantially more stable under alkaline conditions than silica-based materials. The data show that the stationary phase was successfully bonded in all cases and functioned in anion exchange chromatography. When the surface area and pore diameter of these alternate materials is equivalent to silica, there is little difference in chromatographic properties. PMID- 3015996 TI - Metal chelate-interaction chromatography of proteins with iminodiacetic acid bonded stationary phases on silica support. AB - An iminodiacetic acid (IDA)-bonded stationary phase on a wide-pore microparticulate silica support was used for the chromatography of amino acids and proteins at pH 5.0 and 6.0. Without chelated metal the retention behavior of the stationary phase parallelled that of other silica-bound cation exchangers used in high-performance liquid chromatography of proteins. In metal chelate interaction chromatography (MCIC) with IDA, chelated by Cu(II), Zn(II), Ni(II), Fe(II), or Fe(III), amino acids were most strongly retained on Cu(II)-IDA, whereas all metal chelates separated the proteins under investigation but with different selectivity. The effect of salt concentration in the eluent on protein retention was investigated and the pertinent electrostatic and hydrophobic interaction parameters were evaluated. The proteins were separated by MCIC with increasing salt gradient and, using the same column, by hydrophobic-interaction chromatography with decreasing salt gradient. In MCIC the addition of methanol to the mobile phase had disparate effect on protein retention, whereas addition of histidine or glycine, which acted as competing ligands, reduced the retention. PMID- 3015997 TI - Separation of DNA restriction fragments by ion-pair chromatography. AB - The separation of restriction endonuclease fragments of DNA on columns of Pharmacia PepRPC (C2/C18) has been studied. The effect of different concentrations of triethylammonium or tetrabutylammonium salts as ion-pairing reagents, as well as of physical parameters, such as flow-rate and sample load, has been investigated. With the use of triethylammonium buffers, removed by evaporation under vacuum, separated fragments were recovered in yields of 68%. Isolated fragments were accessible to further cleavage with restriction enzymes. Resolution of fragments ranging from 10 to 3000 base pairs depended primarily upon molecular size. PMID- 3015998 TI - Thermal behavior of proteins in high-performance hydrophobic-interaction chromatography. On-line spectroscopic and chromatographic characterization. AB - The thermal behavior of a series of standard proteins in hydrophobic interaction chromatography on a previously developed weakly hydrophobic ether-bonded phase column has been studied. Depending on the temperature and protein, conformational changes can occur in the chromatographic system. Methods for recognizing these conformational effects are presented, including retention and peak width variations with temperature, and Z values (the slope of the plot of log k' vs. log %B solvent. The Z value is shown to be a general index characterizing protein retention as a function of salt concentration. In addition, on-line UV spectroscopic analysis, (absorbance ratios and second derivative spectroscopy) with a photodiode array detector, is shown to corroborate chromatographic trends. Lysozyme maintains a stable conformation over the temperature range 10-40 degrees C, whereas beta-lactoglobulin A has a conformational transition between 25 degrees C and 40 degrees C. Calcium-depleted alpha-lactalbumin, a rather labile species, maintains a stable conformation up to ca. 20 degrees C, and then undergoes structural changes. Finally, cytochrome c appears to be relatively stable up to ca. 65 degrees C. Since conformational changes for this protein occur at ca. 35 degrees C on more hydrophobic phases, the extent of hydrophobicity of the stationary phase is important for maintenance of the native state. Based on this work, hydrophobic-interaction chromatography at sub-ambient temperatures appears promising. PMID- 3015999 TI - Size-exclusion high-performance liquid chromatography of Sendai virus membrane proteins in different detergents. A comparison of different columns. AB - Four column packings for size-exclusion high-performance liquid chromatography (HPLC) of proteins with particle sizes from 3 to 13 micron were compared, using 0.1% sodium dodecyl sulphate in the solvent. Their suitability for the purification of hydrophobic membrane proteins was studied with Sendai virus proteins as a model. The calibration curves of the two 13-micron column packings were linear up to high molecular weights. In contrast to this, large proteins (greater than 100-150 kD) were eluted later than expected from the 3- and 6 micron packings. Peak capacities for proteins larger than 20 kD ranged from 4.7 to 5.5. Therefore, purification of complex mixtures of membrane proteins will often require rechromatography by a different mode of HPLC. Non-ionic detergents are suitable for further ion-exchange chromatography. The effect of addition of 0.1% of five non-ionic detergents (three gluco-methylalkanamide detergents, octylglucoside, and decyl-polyethyleneglycol-300) to the solvent was investigated and decyl-polyethyleneglycol-300 was found to be most suitable. Size-exclusion HPLC with this detergent resulted in the separation of micelles of three different sizes, of which the larger two contained exclusively the Sendai virus F protein. PMID- 3016000 TI - Comparison of porous silica packing materials for preparative ion-exchange chromatography. AB - Although analytical high-performance liquid chromatographic columns have been successfully used for purification of milligram amounts of proteins, they do not appear to be ideal for preparing gram or kilogram quantities because of cost and load capacities. In this paper the development of preparative weak anion-exchange materials is described. These materials possess similar chromatographic characteristics to analytical 5-10 micron materials, yet also have high load capacities. A number of inorganic packings were examined to determine which had the best combination of high load capacity, good resolution, stability, and low cost. When appropriate flow-rates and gradient shapes were used, 30-50 micron materials produced resolution of components of a commercial ovalbumin sample that was comparable to that achieved on a 6-micron material. An amount of 3 g of a protein could be loaded onto a 250 X 21 mm-I.D. column with adequate resolution to separate it from some of its impurities. PMID- 3016001 TI - Isolation of high-specific-activity subunits of cholera toxin by reversed-phase high-performance liquid chromatography. AB - Facile, rapid procedures for the separation of active cholera toxin subunits were developed, based on high-performance liquid chromatography (HPLC) with a Nucleosil C8 reversed-phase column. These procedures were capable of completely resolving subunits A and B as well as S-carboxymethylated or reduced alpha-, gamma-, and beta-chains. The binding of HPLC-purified B subunit to GM1 ganglioside was essentially identical to that of cholera toxin when compared on a molar basis. The adenosine 5'-diphosphate-ribosyltransferase activity of HPLC purified A subunit, reduced alpha-chain, or carboxymethyl alpha-chain was also determined to be reasonably high compared to that of cholera toxin or commercially prepared A subunit. PMID- 3016002 TI - Rapid purification of topoisomerase I from human breast cancer cells by high performance liquid chromatography. AB - The DNA regulatory enzyme topoisomerase I (TpI) from human breast cancer cells has been analyzed by high-performance liquid chromatography (HPLC) for the first time. Cells were homogenized in Tris buffer and TpI activity was extracted with 0.5 M sodium chloride. Negatively supercoiled plasmid pBR322 was used as the substrate to monitor TpI activity, as judged by relaxed products, analyzed on 1% agarose gels. HPLC in the anion-exchange mode (HPIEC) provided an approximately 6 fold purification of the enzyme. Enhanced purification was subsequently obtained by chromatography of a HPIEC eluate on size-exclusion columns (30- to 60-fold). Recovery of TpI from size-exclusion columns, whether used in multistep analysis or as the first step, was dependent on inclusion of organic solvent, 1-propanol (0.5%, v/v), in the mobile phase. Marked resolution of TpI activity was observed with HPIEC on a SynChrom CM-300 column. Enzyme activity was noted in the void volume, at 150-200 mM phosphate and at 250-350 mM phosphate. TpI purification was 10- and 120-fold in the latter two peaks, respectively. Silver-stained polyacrylamide gels of TpI-containing activity, eluted from a CM-300 column, showed considerable purification of all but the void volume fraction. A distinct protein band at approximately 88-90 kD was seen in the peak eluted from the CM 300 column with 250-350 mM phosphate. These results indicate that HPLC is useful for rapid purification of the labile enzyme, TpI, in the analysis of its structure-function relationship. PMID- 3016003 TI - Multimodal liquid chromatography columns for the separation of proteins in either the anion-exchange or hydrophobic-interaction mode. AB - Several high-performance stationary phases suitable for protein chromatography were synthesized. Columns packed with these materials could be operated independently in either the anion-exchange or hydrophobic-interaction mode. Two approaches were used to prepare these materials. In the first method, a polyamine was adsorbed on the surface of macroporous silica and then crosslinked with a multifunctional oxirane. The hydrophobicity of the crosslinking agent and the extent of interconnection were used to modulate the electrostatic and solvophobic interactions. The second approach also utilized a crosslinked polyamine stationary phase; however, the forces of interaction were attenuated through controlled acylation of surface amines with a small anhydride molecule. The resolving ability of these columns, functioning in either mode, was comparable to commercial high-performance liquid chromatographic columns, designed to operate by a single retention mechanism. Column selectivity for proteins was completely different in each mode. Protein fractions collected from a multimodal column, operated in the anion-exchange mode, could be further purified by rechromatographing them on the same column in the hydrophobic-interaction mode. Utility of the multimodal column was demonstrated with the fractionation of several cytochromes and ferredoxins from the cyanobacterium Microcystis aeruginosa. PMID- 3016004 TI - Purification of the M-type phosphoglyceromutase from rabbit muscle. AB - A new method for the purification of M-type phosphoglyceromutase from rabbit muscle involving affinity chromatography on dye ligand media in the presence of 2,3-diphosphoglycerate is described. The method is rapid and technically simple. The purity of the enzyme was verified by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate and by Cellogel electrophoresis. Immunological techniques showed that the anti-M antibodies are specific and do not cross-react with the B isozyme. PMID- 3016005 TI - High-performance ion-exchange chromatographic separation of cytidine monophosphate-N-acetylneuraminic acid and cytidine nucleotides. PMID- 3016006 TI - Identification of anabolic stilbene derivatives in bovine urine by high performance liquid chromatography and off-line chemiluminescent immunochemical detection. PMID- 3016007 TI - Seasonal variations in the stability of monoamines and their metabolites in perchloric acid as measured by high-performance liquid chromatography. AB - The stability in acid medium of dopamine, dihydroxyphenylacetic acid (DO-PAC), homovanillic acid (HVA), serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) was investigated. The stability of 5-HT and 5-HIAA was poor, but could greatly be improved by the addition of sodium bisulphite and disodium edetate. Under these conditions, dopamine, DOPAC, HVA, 5-HT and 5-HIAA showed good stability over 24 h at room temperature throughout the year when stored in capped vials. In uncapped vials, the stability of 5-HT and 5-HIAA was reasonable during the winter months, but was poor during the summer months. PMID- 3016008 TI - Studies on steroids. CCXIX. Separation and determination of 4-hydroxyoestriol monoglucuronides and monosulphates in biological fluids by high-performance liquid chromatography with electrochemical detection. AB - The separation and determination of 4-hydroxyoestriol monoglucuronides and monosulphates by high-performance liquid chromatography with electrochemical detection on a reversed-phase column has been carried out. The effects of the salt, composition and pH of the mobile phase on the resolution were investigated with a Develosil ODS-5 column. Each group of isomeric monoglucuronides and monosulphates of 4-hydroxyoestriol was efficiently resolved on this column when 0.5% sodium acetate-acetonitrile and 0.5% sodium acetate-tetrahydrofuran acetonitrile were used as mobile phases, respectively. The use of the present method revealed that 4-hydroxyoestriol orally administered to the rat was excreted as 4-, 3-, 16-glucuronides and 4-sulphate in bile. PMID- 3016009 TI - Analysis of dihydroxyeicosatetraneoic acids by gas chromatography-mass spectrometry. PMID- 3016010 TI - Comparative study of solid phase extraction techniques for isolation of leukotrienes from plasma. AB - We have compared five commercially available absorbent materials (i.e. C18 Sep Pak, C18 J.T. Baker, Amberlites XAD-7, XAD-2 and XAD-4) for their applicability as effective tools for extraction of leukotrienes from plasma samples. Leukotriene C4 (LTC4) and B4 (LTB4) were selected as representatives of peptidic and non-peptidic leukotrienes, respectively. These leukotrienes were added to 1 ml of plasma and passed through columns containing the above described adsorbent materials. The recovery was determined for each material using different combinations of solvents. XAD-4 gave the highest recovery for LTB4 (90%), whereas XAD-4 and XAD-2 gave identical recoveries for LTC4 (90%) when an eluting solvent mixture of pyridine-water--dimethylformamide (50:45:5) was used. The efficiency of the other three solid adsorbent materials for leukotriene extraction were in order of decreasing magnitude, C18 J.T. Baker greater than XAD-7 greater than C18 Sep-Pak. XAD-7 was shown to be more efficient for LTB4 than for LTC4, whereas the octadecylsilane C18 materials gave approximately similar recoveries for both of the leukotrienes. In addition to very good extraction properties of XAD-4 and XAD 2 as compared to octadecylsilane silica, these solid adsorbent materials retained less plasma impurities than the C18 materials, giving cleaner chromatograms for leukotrienes extracted from plasma. Therefore, XAD-4 or XAD-2 are the best overall choice for extraction of leukotrienes from plasma for reversed-phase high performance liquid chromatographic analysis. PMID- 3016011 TI - Rapid and sensitive on-line precolumn purification and high-performance liquid chromatographic assay for disulfiram and its metabolites. AB - The disulfiram (Antabus) metabolites diethyldithiocarbamate, diethyldithiocarbamate methyl ester, carbon disulphide and bis(diethyldithiocarbamato) copper complex were quantitatively analysed from directly injected heparin plasma by reversed-phase high-performance liquid chromatography. The highly volatile metabolite, carbon disulphide, was converted to the methyl ester of dimethyldithiocarbamate before chromatography. The analytical procedure is simple and does not require sample preparation or addition of an internal standard, and the compounds are eluted from the columns in 15 min. After automated on-line precolumn enrichment, the parent compound and biotransformation products could be back-flushed and chromatographed on an ordinary reversed-phase column. The influence of plasma protein binding on the constituents in the precolumn enrichment step was also investigated. Dissociation and partition of constituents from plasma proteins gave a complete retardation on the precolumn. The time courses of diethyldithiocarbamate and its methyl ester were followed in patients receiving therapeutic doses of disulfiram. PMID- 3016013 TI - Use of lectins to detect and differentiate subtypes of Marek's disease virus and turkey herpesvirus glycoproteins in tissue culture. AB - Biotinylated lectins in conjunction with an avidin-biotin-peroxidase complex were used for the first time to reveal glycoproteins in chicken and duck embryo fibroblasts infected with three prototype members of the avian herpesvirus group, Marek's disease virus serotypes 1 and 2 and turkey herpesvirus. By using a panel of 10 lectins, a pattern of reactivity emerged which was both group- and type specific. Morphological details of the lectin-stained cells include cytoplasmic granulation, capping and bleb-like protrusions of the cell membrane. Although no antibody is necessary for the reaction, this novel approach allows specific detection of the glycan moieties of viral glycoproteins as they are synthesized during infection. PMID- 3016012 TI - Determination of 5-lipoxygenase activity in human polymorphonuclear leukocytes using high-performance liquid chromatography. PMID- 3016014 TI - Antigenic analysis of yellow fever virus glycoproteins: use of monoclonal antibodies in enzyme-linked immunosorbent assays. AB - A sensitive enzyme-linked immunosorbent assay with biotin and streptavidin (B/SA ELISA) was developed for the specific detection of yellow fever (YF) viruses. Monoclonal antibodies (MCA) specific for the envelope (E) glycoprotein or the non structural glycoprotein (NV3) of YF virus-infected cell lysates were tested by antigen and antibody capture B/SA ELISA against YF viruses and a large number of other flaviviruses. In an antigen capture assay, with suckling mouse brain antigens, MCA directed against the envelope glycoprotein clearly differentiated YF viruses from any other flaviviruses, wild-type YF viruses from YF vaccine viruses and YF17D-204 substrains from other YF vaccine viruses. Specific MCA could identify YF viruses in an antibody capture assay with concentrated YF infected tissue culture supernatant medium as the solid phase. On the basis of the principles established above, MCA binding profiles of purified YF virus strains were compared. No qualitative differences in immunoreactivity could be detected between closely related strains of YF virus. An antibody capture assay on infected tissue culture monolayers was developed to enable in situ detection of YF viruses using MCA specific for both the envelope glycoprotein and the non structural NV3 glycoprotein. PMID- 3016015 TI - Passive hemagglutination for cytomegalovirus antibody: specificity and stability of reagents. AB - Glutaraldehyde fixed red cells coated with CMV antigen were lyophilized in the presence of stabilizers such as glycerol, lactose, sorbitol and sucrose. These cells maintained their sensitivity for anti-CMV detection for at least 6 mth when held at 4 degrees C. Some rheumatoid factors containing sera react non specifically with crude CMV antigen coated cells. The agglutinins in these sera could be effectively removed by adsorption with red cells coated with polymerized IgG. PMID- 3016016 TI - Aujeszky's disease: blocking ELISA for the detection of serum antibodies. AB - A blocking ELISA for the detection of serum antibodies to Aujeszky's disease virus has been developed. The test was designed in particular with a view to examining large numbers of blood samples. It has been found to be sensitive, specific and precise. In comparison with the serum neutralization test it was superior in detecting low levels of antibodies in pig sera. The blocking ELISA gave higher titre values in serum samples taken early after experimental infection of pigs with Aujeszky's disease virus, than did the serum neutralization test. PMID- 3016017 TI - Determination of vaccine-induced and naturally acquired class-specific mumps antibodies by two indirect enzyme-linked immunosorbent assays. AB - Paired sera from 46 vaccinees and 22 patients with clinically typical or atypical parotitis were tested for class-specific mumps antibodies by two different indirect enzyme-linked immunosorbent assay (ELISA) procedures. Both ELISAs appeared suitable, specific and more sensitive than neutralization (NT) and complement-fixation (CF). However, the macro-ELISA (M-ELISA) method, using beads as antigen-coated solid phase, showed a higher sensitivity than micro-ELISA (m ELISA), performed on microplates. Diagnostic rises in mumps IgG antibodies and mumps IgA antibodies were detected more frequently by M-ELISA, mostly in post vaccination sera. In addition, higher mean OD values of mumps IgG, IgA and IgM antibodies were generally found by M-ELISA. Nevertheless, m-ELISA appeared more convenient for evaluating class-specific mumps antibodies in large-scale studies, since the procedure is simpler, more rapid and less expensive than that of M ELISA. Conversely, M-ELISA may be considered the test of choice for detecting low class-specific antibody levels. However, the determination of class-specific mumps antibodies appeared as an essential tool for evaluating vaccine-induced or naturally acquired mumps immunity. PMID- 3016018 TI - Electrophoretic separation of the plus and minus strands of rotavirus SA11 double stranded RNAs. AB - The genome of the rotaviruses consists of eleven segments of completely double stranded RNA (dsRNA). To provide a method for separating and identifying the complementary plus and minus strand RNAs within these segments, we have characterized their migration patterns under denaturing conditions on agarose urea gels. Virion-derived 3H-labelled dsRNAs were resolved by electrophoresis on a polyacrylamide gel and the individual genome segments recovered by electroelution. Upon electrophoresis in a low pH 1.75% agarose gel containing 6 M urea, the dsRNAs were denatured with complementary plus and minus strands migrating at different rates. Our results showed that, like cytoplasmic polyhedrosis virus but unlike human reovirus (Smith et al., 1981), rotavirus plus strand RNA migrates faster than its complementary minus strand on agarose-urea gels. PMID- 3016020 TI - Characterization of human thyroid peroxidase purified by monoclonal antibody assisted chromatography. AB - A murine monoclonal antibody (mAb) to human thyroid peroxidase (TPO) was produced and used for purification of the enzyme. This report describes studies done to characterize the mAb and the purified human TPO. The mAb bound to human TPO at a molar ratio of 1:2 or 1:1 and to human thyroid microsomes with a dissociation constant of 1.2 nM. The mAb had no effect on TPO activity to catalyze guaiacol oxidation. As immunoglobulin G, the mAb consisted of a light chain (kappa) of 26 kilodaltons (kDa) and a heavy chain (gamma 1) of 53 kDa; its pI value was 5.9. Using an mAb immunoaffinity column, 85% of TPO activity was recovered from an ammonium sulfate precipitate of a human thyroid microsomal preparation solubilized with deoxycholate. The purified TPO had a specific activity of 164 guaiacol U/mg protein and an A413 nm/A280 nm ratio of 0.26. The enzyme showed bands only at 107 and 100 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified TPO was very stable at alkaline pH, and its ability to catalyze iodide oxidation and tyrosine iodination was increased to the same extent as its ability to catalyze guaiacol oxidation. For comparison with a conventional method, the immunoaffinity-purified TPO was trypsinized and rechromatographed on the immunoaffinity column. The trypsinized enzyme had a specific activity of 336 U/mg and an A413 nm/A280nm ratio of 0.65. The absorption spectrum of the enzyme suggested that the prosthetic group of human TPO is protoheme IX. These results indicate the value of the immunoaffinity procedure for human TPO purification. The major advantages of this purification procedure are not only high yield and speed, but also recovery of intact enzyme. PMID- 3016019 TI - Detection of Marek's disease virus antigens and DNA in feathers from infected chickens. AB - Two novel tests, enzyme-linked immunosorbent assay (ELISA) and dot-blot hybridization, were developed to detect and quantify the antigens and DNA of Marek's disease virus (MDV) in feather tips from infected chickens. In both methods, buffered extracts of the feathers served as the same test material. The ELISA technique was compared to the conventional agar-gel precipitation (AGP) test, using the same convalescent serum from a MDV-infected bird. Of 86 feather samples tested, 34 were negative by both methods, while 6 out of 52 were ELISA positive but AGP negative. Viral antigen detection by the AGP and ELISA methods was compared with the detection of MDV DNA by the dot-blot DNA hybridization technique. At an ELISA reading (OD 405) of 0.3 and above, only 5 out of 48 DNA extracts failed to hybridize with the MDV-DNA probe. The use of the radioactively labelled MDV-DNA probe for hybridization with DNA extracts from feather tips of MDV-infected chickens was both sensitive and specific, and there was good correlation among the different tests. PMID- 3016021 TI - The neuroendocrine response of luteinizing hormone to estrogen administration in heterosexual, homosexual, and transsexual subjects. AB - The neuroendocrine response of LH to estrogen administration may be related to sexual dimorphism of the brain, and therefore, homosexual and especially transsexual individuals may differ from heterosexual individuals in their responses. This study failed to find such differences among groups of female heterosexuals, homosexuals, and transsexuals. Specifically, after single dose estrogen administration, all subjects had an initial decline in serum LH levels, followed by a brisk rise of equal magnitude. Among males, the type of response was less uniform. After an initial fall in serum LH levels, the individual responses varied. In 12 of 23 male homosexuals, 10 of 15 male heterosexuals, and all 6 genetic male transsexuals studied, serum LH levels remained below pretreatment levels. In the remaining 11 male homosexuals and 5 of the heterosexuals, serum LH levels increased to values exceeding those before treatment, resembling the response found in the 3 groups of women. Those homosexual and heterosexual men with a rise in serum LH levels to above pretreatment values also had the greatest fall in testosterone levels after estrogen administration, while these same men had the lowest testosterone response to hCG stimulation. I conclude from these results that 1) the similarity of LH responses to estrogen administration in all groups of women studied does not support a theory of brain androgenization as a factor in the establishment of gender identity of sexual orientation; and 2) individual differences in men in the type of LH response to estrogen administration can be satisfactorily explained by endocrine factors, such as Leydig cell function, and need not be related to gender identity, sexual orientation, or other possible causes. PMID- 3016022 TI - Insulin-like growth factor I regulates growth hormone secretion and messenger ribonucleic acid levels in human pituitary tumor cells. AB - GH secretion and mRNA levels were measured in cultured human GH adenoma cells incubated in serum-free medium for up to 48 h. A human recombinant insulin-like growth factor I (IGF-I) analog, Thr-59-IGF-I (6.5 nM), inhibited basal GH secretion by up to 60% in tumor cell cultures. The 30-50% stimulation of GH secretion by GH-releasing hormone (GHRH) was prevented by simultaneous exposure of the cells to IGF-I (6.5 nM). Gel electrophoresis of total RNA derived from GH cell adenoma tissue, followed by transfer and hybridization with 32P-labeled human GH cDNA, revealed a distinct mRNA species of about 1.0 kilobases. Using cytoplasmic dot blot hybridization, IGF-I inhibited the levels of human GH mRNA sequences in these cells and also prevented the GHRH-induced stimulation of GH mRNA. A monoclonal antibody to the type I IGF-I receptor (alpha IR3) prevented the inhibitory effects of IGF-I on basal and GHRH-stimulated GH secretion. This antibody also prevented the IGF-I-induced suppression of GH mRNA sequences. PRL secretion in these cells was not altered by IGF-I. Furthermore, relative levels of beta-actin mRNA were unaltered by IGF-I. Thus, IGF-I suppresses basal and GHRH stimulated GH secretion and GH mRNA levels in pituitary adenoma cells, indicating that IGF-I acts selectively on the somatotroph to directly regulate GH gene expression. PMID- 3016023 TI - Myosin light chain phosphorylation in intact rat uterine smooth muscle. Role of calcium and cyclic AMP. AB - Myometrial strips from oestrogen-primed rat uterus were exposed to various treatments, isometric contraction was measured, and the extent of myosin light chain phosphorylation determined after rapid freezing in liquid nitrogen. Two dimensional electrophoresis revealed five spots having the same molecular weight as the light chain, with isoelectric points comprised between 5.15 and 4.95. Two of these spots (pI 5.09 and 5.00) were not present in pure uterine myosin, whether prepared from incubated or nonincubated tissue; they do not represent light chain isoforms or electrophoresis artefacts but rather degradation products appearing during the treatment. Two spots (pI 5.15 and 5.06) were identified as the nonphosphorylated and the phosphorylated forms of the light chain. The fifth minor spot (pI 4.95) may represent a diphosphorylated myosin species. Strips incubated in a normal Ca2+-medium 0.8 mM) exhibited basal contractions and an incorporation of 0.2 mol phosphate per mol light chain. Removal of Ca2+ resulted in almost complete dephosphorylation, coincident with a total relaxation of the muscle. Exposure of the myometrium to carbachol caused tetanic contractions with an increase to 0.5 mol phosphate per mol light chain. Isoproterenol, a beta adrenergic agonist elevated intracellular cyclic AMP and induced uterine relaxation. Addition of isoproternol to a resting myometrium caused a slight but significant decrease in phosphorylation; its addition prior to carbachol markedly prevented the increase in myosin phosphorylation normally induced by the cholinergic effector. Forskolin (1 microM) increased intracellular cyclic AMP, caused relaxation and a concomitant decrease in basal myosin phosphorylation. Prostaglandin E2-induced elevation in intracellular cyclic AMP was however accompanied by an increase in contraction together with an increase in light chain phosphorylation. The data imply that light chain phosphorylation dephosphorylation, regulated by Ca2+-dependent mechanisms, is essential for both uterine contraction and relaxation but question the role of cyclic AMP in exclusively mediating relaxation and myosin dephosphorylation in intact myometrium. PMID- 3016024 TI - Murine coronavirus-induced encephalomyelitis in rats: analysis of immunoglobulins and virus-specific antibodies in serum and cerebrospinal fluid. AB - The humoral intrathecal immune response in coronavirus-induced demyelinating encephalomyelitis in rats associated with an autoimmune reaction to brain antigen, was analysed. The CSF of these animals revealed immune reactions which were directed against coronavirus and other, unknown, antigens. In general, no direct correlation between the disease, the state of the blood-brain barrier (BBB), intrathecal synthesis of Ig and the presence of virus-specific antibodies was detectable, suggesting that the humoral, in contrast to the cellular, immune response does not play a significant pathogenetic role in this CNS disease. PMID- 3016025 TI - DNA restriction fragment length polymorphism of HLA-DR2: correlation with HLA-DR2 associated functions. AB - HLA-DR2 can be divided into at least 3 distinct HLA-D clusters which correlate with structural differences within the HLA-D region. Further, a functional counterpart of this subdivision has been previously identified. The presence of a particular DR beta 2 polypeptide chain correlated precisely with the susceptibility of measles virus-infected HLA-DR2 homozygous typing cell lines to lysis by measles virus-specific, HLA class II-restricted CTL clones. To determine if a genetic basis for these functional differences could be detected, the degree of polymorphism at the DNA level within the serologically defined HLA-DR2 haplotype has been examined. By using DNA probes for DR beta and DQ beta 4 of the 5 HLA-D clusters of HLA-DR2 could be distinguished and a RFLP pattern was identified which correlates with known immunological functions associated with these various D types. In addition, this technique of 'molecular genotyping' was used to investigate a limited panel of patients with multiple sclerosis (MS) who were HLA typed as either HLA-DR2,2 or HLA-DR2,blank. The RFLP profile of the HLA DR2 Dw2 D type was found in all of these MS patients. PMID- 3016026 TI - Abundant calcitonin receptors in isolated rat osteoclasts. Biochemical and autoradiographic characterization. AB - Calcitonin receptors have been characterized for the first time in isolated osteoclasts. These receptors have been demonstrated by autoradiographic and biochemical methods, and the cells have also been shown to respond to calcitonin with a dose-dependent increase in cyclic AMP. The receptors in rat osteoclasts are specific and of high affinity (dissociation constant, Kd, 1 to 6 X 10(-10) M), and are present in greater numbers than in any cell previously studied (greater than 10(6) per cell). Chemical cross-linking of 125I-labeled salmon calcitonin to osteoclasts using disuccinimidyl suberate resulted in identification of a receptor component with a relative molecular weight of 80,000 90,000. By counting grains in autoradiographic experiments, we found that greater than 80% of specifically bound radioactivity was associated with multinucleate osteoclasts and the remainder was associated with mononuclear cells that are not osteoblasts, but that may be osteoclast precursors. PMID- 3016027 TI - Binding and metabolism of platelet-activating factor by human neutrophils. AB - Human polymorphonuclear neutrophils rapidly incorporated radiolabeled platelet activating factor, 1-O-[hexadecyl-9, 10-3H2]-2-acetyl-sn-glycero-3-phosphocholine ([3H]PAF), and then metabolized it into its sn-2-fatty acyl derivative. Fractionation of radiolabel-pretreated cells over Percoll gradients revealed that virtually all of the intact [3H]PAF was located in nongranule membranes that were enriched with alkaline phosphatase and cell surface glycoproteins. While still membrane associated, the ligand was rapidly converted to its acyl derivative and then more slowly transferred to specific granules and, to a lesser extent, azurophilic granules. In contrast, neutrophils did not metabolize [3H]PAF at 4 degrees C but rather gradually accumulated it in their alkaline phosphatase enriched membrane subfractions. These same subfractions contained receptors for the ligand, as determined by their capacity to bind [3H]PAF specifically. Binding was readily saturated, partially reversible, and fit a two receptor model; dissociation constant (Kd) values for high and low affinity sites were 0.2 and 500 nM, respectively. Receptors with similar affinities were detected in whole cells. Furthermore, the potencies of several structural analogues in inhibiting binding of [3H]PAF to membranes correlated closely with their respective potencies in stimulating degranulation responses. Finally, quantitative studies suggested all or most of the cell's receptors were membrane associated. We conclude that PAF rapidly enters cellular membranes to bind with specific receptors that trigger function. The intramembranous ligand is also deacetylated, acylated, and then transferred to granules. This metabolism may be sufficiently rapid to limit ligand-receptor binding and distort quantitative analyses of receptors. PMID- 3016028 TI - Mechanisms of B cell activation in patients with acquired immunodeficiency syndrome and related disorders. Contribution of antibody-producing B cells, of Epstein-Barr virus-infected B cells, and of immunoglobulin production induced by human T cell lymphotropic virus, type III/lymphadenopathy-associated virus. AB - Patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) have hyperimmunoglobulinemia and increased numbers of circulating immunoglobulin-secreting cells. In this paper, we studied the basis for this B cell hyperactivity. Limiting dilution studies of B cells from seven patients with ARC and four with AIDS revealed that some B cells spontaneously produced antibodies to human T cell lymphotropic virus, type III/lymphadenopathy associated virus (HTLV-III/LAV) (39:10(6) and 7:10(6) B cells, respectively), suggesting that chronic antigenic stimulation by HTLV-III/LAV was one contributing factor. The patients also had an increased number of spontaneously outgrowing B cells than did normals (6:10(6) vs. less than 2:10(6) B cells), suggesting that they had an increased number of Epstein-Barr virus (EBV)-infected B cells. However, fewer B cells from patients were immortalized by exogenously added EBV than were B cells from normals. In additional studies, HTLV-III/LAV induced immunoglobulin secretion (mean 2,860 ng/ml) by peripheral blood mononuclear cells from normals; this HTLV-III/LAV-induced immunoglobulin secretion required the presence of both B and T cells. Thus, antigenic stimulation by HTLV-III/LAV, increased numbers of EBV-infected B cells, and HTLV III/LAV-induced T cell-dependent B cell activation all contribute to the B cell hyperactivity in patients with HTLV-III/LAV disease. PMID- 3016029 TI - Cell pH in the rat proximal convoluted tubule. Regulation by luminal and peritubular pH and sodium concentration. AB - To examine the relative roles of apical and basolateral membrane transport mechanisms in the regulation of cell pH in the proximal convoluted tubule, cell pH was measured in the in vivo microperfused rat tubule using fluorescence. Decreasing luminal pH by 0.7 pH units caused cell pH to decrease by 0.08 pH units, whereas a similar decrease in peritubular pH caused cell pH to decrease by 0.32 pH units. Inhibition of basolateral membrane bicarbonate transport with peritubular 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS) enhanced the response to luminal fluid acidification. Removal of luminal sodium caused a small transient acidification which was followed by a late alkalinization. Peritubular SITS increased the magnitude of the transient acidification, and eliminated the late alkalinization. The acidification was partially inhibited by luminal amiloride. The results demonstrate sodium-coupled processes on both the apical (Na/H antiport) and basolateral (Na/HCO3 symport) membranes. Basolateral membrane transporters are more important determinants of cell pH. PMID- 3016031 TI - Myeloperoxidase as an effective inhibitor of hydroxyl radical production. Implications for the oxidative reactions of neutrophils. AB - Hydroxyl radicals have been generated from hydrogen peroxide and superoxide (produced with xanthine oxidase), and an iron (EDTA) catalyst, and detected with deoxyribose, or in some cases with benzoate or alpha-keto-gamma-methiolbutyric acid. Purified myeloperoxidase, and neutrophils stimulated with fMet-Leu-Phe and cytochalasin B, strongly inhibited this hydroxyl radical production in a concentration-dependent manner. Supernatants from stimulated cells also inhibited, and inhibition by cells or supernatant was prevented by azide. There was much less inhibition by myeloperoxidase-deficient neutrophils. Inhibition thus was due to myeloperoxidase released by the cells. With neutrophils stimulated with phorbol myristate acetate, which release very little myeloperoxidase, hydroxyl radical production was enhanced due to the additional superoxide produced by the cells. It is concluded that under conditions where neutrophils release myeloperoxidase as well as superoxide and hydrogen peroxide, breakdown of hydrogen peroxide by myeloperoxidase would make conditions unfavorable for hydroxyl radical production. PMID- 3016030 TI - Changes in the phenotype of human small cell lung cancer cell lines after transfection and expression of the c-myc proto-oncogene. AB - Small cell lung cancer growing in cell culture possesses biologic properties that allow classification into two categories: classic and variant. Compared with classic small cell lung cancer cell lines, variant lines have altered large cell morphology, shorter doubling times, higher cloning efficiencies in soft agarose, and very low levels of L dopa decarboxylase production and bombesin-like immunoreactivity. C-myc is amplified and expressed in some small cell lung cancer cell lines and all c-myc amplified lines studied to date display the variant phenotype. To investigate if c-myc amplification and expression is responsible for the variant phenotype, a normal human c-myc gene was transfected into a cloned classic small cell lung cancer cell line not amplified for or expressing detectable c-myc messenger RNA (mRNA). Clones were isolated with one to six copies of c-myc stably integrated into DNA that expressed c-myc mRNA. In addition, one clone with an integrated neo gene but a deleted c-myc gene was isolated and in this case c-myc was not expressed. C-myc expression in transfected clones was associated with altered large cell morphology, a shorter doubling time, and increased cloning efficiency, but no difference in L dopa decarboxylase levels and bombesin-like immunoreactivity. We conclude increased c myc expression observed here in transfected clones correlates with some of the phenotypic properties distinguishing c-myc amplified variants from unamplified classic small cell lung cancer lines. PMID- 3016032 TI - Production of 1,25-dihydroxyvitamin D3 by human T cell lymphotrophic virus-I transformed lymphocytes. AB - The human T cell lymphotrophic virus type I (HTLV-I) has recently been identified in a T cell lymphoma associated with hypercalcemia and increased bone turnover. Since increased serum concentrations of 1,25-dihydroxyvitamin D have been reported in this disease, we have examined the capacity of HTLV-I-infected cord blood lymphocytes to metabolize 25-hydroxyvitamin D3. Our results demonstrate that HTLV-I-infected cells have the capacity to metabolize 25-hydroxyvitamin D3 to a substance that co-migrates with 1,25-dihydroxyvitamin D3 by high performance liquid chromatography over a silica column using either 12% isopropanol in hexane or 5% isopropanol in dichloromethane. The metabolite binds to the 1,25 dihydroxyvitamin D3 receptor in rat osteosarcoma cells and stimulates bone resorption in cultures of fetal rat long bones. Mass spectrometric analysis of the metabolite confirmed the presence of 1,25-dihydroxyvitamin D3. Production of 1,25-dihydroxyvitamin D by lymphoma cells may contribute to the pathogenesis of the hypercalcemia seen in patients with HTLV-I-associated T cell lymphomas. PMID- 3016034 TI - Study of childhood renal tumours using peroxidase conjugated lectins. AB - Six peroxidase conjugated lectins were used to compare their ability to bind to formalin fixed paraffin embedded tissue sections of childhood renal tumours (Wilms' tumour, mesoblastic nephroma, renal carcinoma, rhabdoid renal tumour, and bone metastasising renal tumour of childhood (BMRTC) with fetal and normal children's kidney. Lectins were found to be helpful in the differential diagnosis of renal tumours. Another important finding was that the mesenchyme of renal tumours showed differences in its reactivity among various types of kidney tumours. The results of lectin binding were not helpful in establishing the origin of kidney tumours. PMID- 3016033 TI - Polyacrolein microspheres as a new solid phase for radioimmunoassay. AB - Polyacrolein (PA) microspheres contain reactive aldehyde groups through which ligands containing primary amino groups such as proteins and drugs can be covalently bound in a single step at physiological pH. Antibodies against cyclic AMP, digoxin and rabbit serum were thus coupled to PA microspheres. The immuno microspheres were kept in suspension or freeze-dried, with insignificant decrease in their binding capacity. The conjugates were used in the respective radioimmunoassay (RIA) systems to facilitate the separation of the free and the antibody-bound 125I ligands, in comparison with precipitation of Protein A of Staphylococcus aureus. Cyclic-AMP was assayed using PA microspheres coupled either with the primary antibody or with anti-rabbit serum as a secondary antibody, in a buffer system, in chick plasma, in urine and in media in which avian dispersed kidney cells had been stimulated by various agents. The results obtained using the immuno-microspheres and the bacterial separation methods were indistinguishable. Other 125I-ligands, such as digoxin in buffer system or thyroxine and triiodothyronine in chick plasma, were assayed in the picogram range. Owing to the solubility of non crosslinked microspheres conjugates in toluene-based scintillation fluids, both the free and the bound fractions could be counted when using 3H-ligands. Corticosterone was assayed using this technique. PMID- 3016035 TI - Functional heterogeneity in the myenteric plexus: demonstration using cytochrome oxidase as a verified cytochemical probe of the activity of individual enteric neurons. AB - The cytochemical technique for the demonstration of cytochrome oxidase has been used to locate the sites of chronically active neurons in the CNS. The current experiments were undertaken to validate the use of this technique for the identification of active neurons and ganglia in the myenteric plexus of the gut. The excitotoxin veratridine was used to cause repetitive firing of action potentials and to depolarize myenteric neurons over an 8-hour test period in vitro. The effects of veratridine were monitored electrophysiologically and correlated with the ability of the drug to affect the density of the cytochrome oxidase reaction product, as well as the proportion of reactive myenteric neurons. The action of veratridine on cytochrome oxidase activity was evaluated by means of video microdensitometry and computer-assisted morphometry. Effects of veratridine were considered to be specific if they were blocked by the inclusion of tetrodotoxin (TTX), which antagonizes the action of veratridine on voltage dependent Na+ channels. Veratridine (1.0 microM) depolarized the membranes of myenteric type II neurons and greatly increased both the average density of the cytochrome oxidase reaction product in individual neuronal cell bodies and the proportion that were reactive. The concentration-effect relationship for veratridine was biphasic. As the concentration of veratridine was raised, neurons became sufficiently depolarized such that they ceased to fire action potentials. This absence of spiking was associated with a decline in the stimulation of cytochrome oxidase activity by veratridine. At still higher concentrations of veratridine the degree of neuronal depolarization continued to increase. This further depolarization was associated with a secondary increase in cytochrome oxidase activity. All effects of veratridine were blocked by TTX. It is concluded that the cytochemical demonstration of cytochrome oxidase reflects the recent metabolic history and thus the physiological activity of myenteric neurons. In control preparations, fixed without exposure to veratridine, considerable variation in cytochrome oxidase was observed between ganglia and between individual neurons within ganglia. This suggests that there are significant differences in the activity of individual ganglia and neurons of the myenteric plexus. These observations support the hypothesis that there is a functional heterogeneity that corresponds to an anatomical heterogeneity (previously demonstrated) of the myenteric plexus. PMID- 3016036 TI - Anatomical organization of the visual system of the mink, Mustela vison. AB - The organization of the retinogeniculocortical visual system of the mink was studied by anterograde and retrograde tracer techniques, by physiological mapping, and by direct recordings from axonal terminals after injection of kainic acid. In the lateral geniculate nucleus, retinogeniculate afferents are segregated according to eye of origin between the two principal layers, A and A1. Within each of these layers there is a further parcellation according to functional type: on-center afferents terminate in the anterior leaflets of A and A1, and off-center afferents in the posterior leaflets. This separation is preserved in area 17: geniculocortical afferents terminate in ocular dominance patches in layer IV, and these patches coexist with an alternating, partially overlapping set of patches for on-center and off-center inputs that we have demonstrated previously (McConnell and LeVay: Proc. Natl. Acad. Sci. USA 81:1590 1593, '84). In both the lateral geniculate nucleus and in area 17, the contralateral eye predominates to a much greater extent than in the cat. Visual cortical areas corresponding to the cat's areas 17, 18, and 19 can be identified in the mink, but they are shifted posterolaterally in the hemisphere, and they show less emphasis on the representation of central retina. Mapping studies also revealed the existence of a fourth visual area in the splenial sulcus (area SV) adjacent to the representation of the far periphery in area 17. This area differs from the corresponding region in the cat in that it receives direct projections from the lateral geniculate nucleus and from areas 17 and 18. The lateral geniculate nucleus projects to each of the four cortical areas that were mapped. The bulk of the projection to area 17 is derived from the principal layers, A and A1, while most cells projecting to areas 18 and SV are found in the C-layer complex. The recurrent projection from area 17 to the lateral geniculate nucleus arises from pyramidal neurons in layer VI, and terminates through all layers of the lateral geniculate nucleus, but most densely in the interlaminar zones. Areas 18 and SV project predominantly to the C layers. Areas 17, 18, and SV are reciprocally connected with the claustrum and the LP-pulvinar complex, and project to the superior colliculus. All four visual cortical areas are mutually interconnected; these associational projections arise from both the supragranular and infragranular layers. PMID- 3016037 TI - The topography of expanded uncrossed retinal projections following neonatal enucleation of one eye: differing effects in dorsal lateral geniculate nucleus and superior colliculus. AB - The topographic organization of the uncrossed retinal projections to the dorsal lateral geniculate nucleus (dLGN) and superior colliculus (SC) was studied in normal adult hooded rats and in rats subjected to unilateral ocular enucleation on the day of birth. Sections were stained for anterograde degeneration products following discrete retinal lesions at various locations. The projection from the temporal crescent to the dLGN in neonatally enucleated rats had an expanded but topographically normal organization, with the nasotemporal and dorsoventral retinal axes displaying polarities identical to those in normal adults. Neonatal enucleation permits the remaining uncrossed retinogeniculate projection to extend primarily along the "lines of projection" into neuropil normally recipient of binocularly conjugate crossed projections. In the SC, the dorsoventral axis of the temporal crescent showed a normal polarity, but the nasotemporal axis failed to display any topographic organization. Retinal loci in the temporal crescent projected throughout the rostrocaudal extent of the ipsilateral SC. Retinal lesions placed outside the temporal crescent failed to produce any substantial degeneration in ipsilateral dLGN or SC. These topographically distinct effects in dLGN and SC following unilateral eye removal on the day of birth are discussed in the context of differing constraints upon axonal ingrowth and connectivity during early development, which may normally bring about the characteristically distinct features of retinogeniculate and retinocollicular organization. PMID- 3016039 TI - Ultrastructure and origin of adenocarcinomas detected in the lungs of three cows. AB - Three types of bovine adenocarcinoma were found in the lungs of 3 cows. Case 1 was a well differentiated adenocarcinoma with mucin granules and microvilli with core filaments seen ultrastructurally. In Case 2, the neoplastic tissue showed a sarcoma-like growth with scattered tubular structures. Many cilia were observed in the tubular structures and a few cilia were found in the anaplastic tissues. Case 3 had epithelial and undifferentiated tissues. A few mucin granules and cilia were observed in the tumours in both lung and kidney. The relationships between these neoplastic cells and bronchial epithelial cells are discussed. PMID- 3016040 TI - Seasonal adaptation of brown adipose tissue in the Djungarian Hamster. AB - The composition and oxidative capacity of brown adipose tissue (BAT) were investigated in Djungarian hamsters kept under natural photoperiod, either indoors at neutral Ta (23 degrees C) or under outdoor conditions. BAT comprises up to 5% of the body weight in summer/indoor hamsters, with lipid representing 86% of the total tissue mass. Tissue mass and thermogenic capacity are inversely related during seasonal adaptation: 30% decrease of total DNA, accompanied by extensive lipid depletion, reduces the amount of BAT by almost 60% during acclimatization from summer/indoor to winter/outdoor conditions. Mitochondrial protein in BAT is increased by a factor of 2.6 concomitantly, and by a factor of 4 when related to body weight (body weight reduction 36%). Cytochrome oxidase activity in different brown fat deposits varies by up to 150% in summer/indoor hamsters; depending on the fat pad, the enzyme activity is increased 200%-700% during adaptation to winter/outdoor conditions. Natural photoperiod is decisive in determining the seasonal adaptation of DNA content in BAT and of body weight. Short photoperiod alone may lead to depletion of lipid content of BAT and thus decrease the tissue mass practically to the lowest seasonal level, even though both parameters may be also influenced by Ta. One third of the maximum adaptive increase of tissue mitochondria may be attributed to seasonal changes in photoperiod and up to two thirds to Ta. Photoperiod establishes a fixed fundament of slow-reacting functional adaptation of BAT, whereas the effect of decreased Ta depends on the rate and duration of cold influence. PMID- 3016038 TI - Mapping of an olfactory receptor population that projects to a specific region in the rat olfactory bulb. AB - An anatomically distinct group of glomeruli, termed the modified glomerular complex (MGC), is present in the posterior dorsomedial portion of the main olfactory bulb. This region has been strongly implicated as part of the pathway that processes odor cues for suckling in neonatal rat pups. We studied the distribution pattern of olfactory receptor neurons that project to the MGC region after ionophoretic injections of WGA-HRP into the olfactory bulbs of 12-day-old rat pups. HRP label was confined to an identifiable localized region in the MGC of the main olfactory bulb. Label extended over 2-7% of the glomerular sheet of the main olfactory bulb, including the MGC. Olfactory receptor neurons within the olfactory epithelium of the nasal cavity were labeled with HRP ipsilateral to the injected side. Maps constructed of the olfactory epithelium revealed that the labeled neurons occurred within topographically defined regions. Anteriorly, labeled olfactory neurons were confined to a narrow strip medial to the dorsal recess, and, more posteriorly, this strip widened medially along the septal wall and laterally onto a limited area on the nasal turbinates. Only a portion of the receptor population within a region was labeled. The boundaries between labeled and unlabeled regions were sharp. These findings support the concept that the olfactory epithelium is an anatomical mosaic in which receptors with different glomerular projections sites are intermingled. In conjunction with previous evidence on the functional specificity of the MGC, and staining of receptor neuron subgroups with monoclonal antibodies, these findings further suggest that olfactory receptor neurons form a functional mosaic within the olfactory epithelium. PMID- 3016041 TI - Prolactin binding sites in the brain and kidneys of the toad, Bufo arenarum Hensel. AB - (125I)-ovine prolactin (oPRL) binding was found in several brain areas of the toad, Bufo arenarum Hensel. The olfactory bulb, cerebral hemispheres, and both dorsal and ventral mesencephalic regions showed saturable, high affinity, (125I) oPRL binding, ranging between 5.6 to 29.9 fmol/mg protein, while the association constant (Ka) by Scatchard analysis was between 4.0 to 8.7 x 10(9) M-1. This binding was compared with the Scatchard plot of the kidney, which has been already described by other groups, and gave 41.7 fmol/mg protein and Ka 2.5 x 10(9) M-1. Liver showed no binding and in the cerebral hemispheres (125I)-oPRL was not displaced by non-lactogenic hormones, indicating that binding was hormone and tissue specific. PMID- 3016042 TI - Arrhenius parameters of mitochondrial membrane respiratory enzymes in relation to thermoregulation in endotherms. AB - The relationship between the body temperature (Tb), the Arrhenius critical temperature (T*), and the apparent activation energy above T* (Ea1), of liver and heart mitochondrial respiratory enzymes from eleven homeothermic and eight heterothermic species was determined using a linear regression analysis. An inverse relation was observed between T* and Ea1 during torpor and hibernation. In all thermoregulatory states, T* decreased with Tb and T* was equal to or below Tb. During torpor Ea1 increased in a linear manner as Tb was lowered. It appears that the above Arrhenius parameters are closely linked to the thermoregulatory state of endotherms and thus may represent an adaptation for function at low Tb's. PMID- 3016043 TI - CT demonstration of pulmonary effects of tangential beam radiation. AB - Breast cancer is sometimes treated with an excisional biopsy and a radiation portal limited to the breast and the adjacent chest wall, especially in patients with negative lymph node dissections. The beam passes through this portion of the chest wall tangentially. Such radiation can result in changes in the included lung parenchyma that are pleural-based and sharply demarcated from the normal lung on CT lung windows. In our experience CT lung windows were more sensitive than chest radiography in showing these changes. Such changes must be recognized and differentiated from pleural metastases. Computed tomography also resulted in a more specific diagnosis than chest radiography because it better localized the abnormality to the radiation portal. PMID- 3016044 TI - MR imaging of the breast: comparison with mammography and ultrasound. AB - Fifty-seven biopsy proven breast masses have been examined at our institution by magnetic resonance (MR), mammography, and ultrasound. The three techniques have been evaluated concerning their capability to visualize the lesion and the diagnostic information obtained. Magnetic resonance proved comparable to mammography and superior to sonography in the fatty to medium dysplastic breasts but inferior to the combined examination by mammography and sonography in the dense breasts. In some selected cases, however, special advantages of MR have been found. PMID- 3016045 TI - CT volumetrics of primary liver cancers. AB - A new computer algorithm is described for liver and tumor volume determinations for patients with hepatoma and primary hepatic cholangiocarcinoma. The algorithm is based on global histograms of CT numbers of the liver and primary liver cancers. The algorithm includes computer-assisted definition of the liver boundary in each CT slice. Liver and tumor volumes of 10 patients calculated by the histogram method were compared with volumes obtained from CT slices that were manually contoured by experienced observers. A correlation coefficient of 0.995 was determined for these two methods of volume computations. Mean values of the differences in volumes obtained by the two methods were 6.7 and 8.0% for the liver and tumor, respectively. The computer algorithm was tested on CT scans for an additional 46 patients by highlighting regions corresponding to normal liver and tumor tissues in each CT slice and determined to be accurate by experienced observers. The computer software is being used clinically to assess tumor response in a new treatment program for primary liver cancers that includes radiolabeled antibodies. PMID- 3016046 TI - Inflammatory lymphadenoid reactions with dermatofibroma/histiocytoma. AB - Lymphoid nodules were found associated with 44 of 1,506 dermatofibroma/histiocytoma tumors of the skin. The lymphoid nodules were usually in the adjacent fat. Germinal center formation occurred, and perinodular and perivascular plasmacytosis was frequently associated. PMID- 3016047 TI - Phosphorylation of rabbit muscle glycogen synthase by casein/glycogen synthase kinase-1 (CK-1). Stoichiometry and distribution of the phosphorylation sites on the glycogen synthase subunit. AB - The stoichiometry of the phosphorylation of rabbit muscle glycogen synthase by casein/glycogen synthase kinase-1 (CK-1) depended on the concentration of protein kinase in the assay and reached values of 7-8 mol/mol subunit at high concentrations. Phosphorylation by CK-1 above 4 mol/mol subunit promoted a further decrease of glycogen synthase activity when determined by the low glucose 6-phosphate/high glucose-6-phosphate activity ratio assay. Analysis by limited proteolysis with trypsin and chymotrypsin showed that all of the regions in glycogen synthase phosphorylated by casein/glycogen synthase kinase-2 (CK-2), the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase), FA/glycogen synthase kinase-3 (FA/GSK-3) and phosphorylase b kinase were also phosphorylated by CK-1. Digestion with CNBr of glycogen synthase phosphorylated by CK-1 revealed the presence of the two phosphopeptides also labeled by the other protein kinases, the largest phosphopeptide (CB2) containing more phosphorylation sites for CK-1 than the smallest one (CB1). Three phosphopeptides (CB2-c, CB2-d and CB2 e) were obtained by trypsinization of CB2 phosphorylated by CK-1. None of them coincided with those labeled by A-kinase, a fact that was confirmed by the additivity of the effect of both protein kinases. In contrast, CB2-d comigrated with the peptide phosphorylated by FA/GSK-3, and CB2-e with that labeled by CK-2, whereas CB2-c would correspond to a new phosphopeptide. PMID- 3016048 TI - Inhibition of thyrotropin-stimulated adenosine 3',5'-monophosphate formation in rat thyroid cells by an adenosine analog. Evidence that the inhibition is mediated by the putative inhibitory guanine nucleotide regulatory protein. AB - Addition of N6-(L-2-phenylisopropyl)-adenosine (PIA) to cultured FRTL-5 rat thyroid cells led to a concentration-dependent inhibition of TSH-stimulated cAMP formation. Half-maximal inhibition was attained with approximately 0.5 nM PIA. Forskolin and cholera toxin-stimulated cAMP production were also inhibited by PIA. 3-Isobutyl-methylxanthine inhibited the effect of PIA. These results are consistent with the presence of inhibitory adenosine receptors (Ri). Ri-sites were further demonstrated by the binding of 3H-cyclohexyl-adenosine to FRTL-5 plasma membranes. High (Kd = 0.50 +/- 0.07 nM) and low affinity (Kd = 5.95 +/- 2.33 nM) binding sites were observed. Pretreatment of FRTL-5 cells with pertussis, but not cholera, toxin effectively antagonized the inhibitory effects of PIA on cAMP production. ADP-ribosylation of FRTL-5 membranes with [32P]-NAD in the presence of cholera or pertussis toxin specifically labeled a 45,000 and 41,000 Mr species, respectively, which correspond to the alpha subunit of the stimulatory and inhibitory guanine nucleotide regulatory proteins. These results demonstrate that PIA inhibits TSH-stimulated cAMP production via Ri-sites on FRTL 5 thyroid cells. PIA appears to exert its inhibitory effects through the inhibitory guanine nucleotide regulatory protein. PMID- 3016049 TI - Interaction of salivary fibronectin with oral streptococci. AB - Immunoreactive Fibronectin (Fn) has been demonstrated in stimulated human parotid saliva by western blot analysis and also found to be a component of the artificial tooth pellicles derived from hydroxyapatite (HA) beads coated with parotid saliva. Saliva depleted of gelatin-binding components showed a significantly lower degree of reactivity with anti-Fn antibodies than did the control saliva when tested by and enzyme-linked immunosorbent assay (ELISA). Depletion of gelatin-binding components from saliva was also found to affect the degree of saliva-mediated aggregation of four of the seven oral streptococci tested [Streptococcus mutans strains GS-5 and OMZ 176, S. sobrinus, and S. rattus]. Similarly, the adherence of the same four micro-organisms to the artificial tooth pellicles (derived form saliva which had previously been depleted of gelatin-binding component) was significantly inhibited (37-53%) when compared with the control saliva-coated HA beads. Pre-treatment of streptococci with 100 micrograms of soluble Fn also caused a 34-57% inhibition of adherence of the same oral streptococci to saliva-treated HA beads. Quantitation of Fn in human parotid saliva showed that the amounts of immunoreactive Fn varied form 2 to 6 micrograms/mL of parotid saliva. Furthermore, the Fn from parotid saliva was found to be adsorbed onto the bacterial surfaces, as demonstrated by immunofluorescence and ELISA. The presence of Fn in parotid saliva and its ability to bind to HA beads (artificial pellicles), in conjunction with the ability of soluble Fn to inhibit the adherence of streptococcal strains to the artificial tooth pellicles, suggest that the microbial ecology of the oral cavity may, in part, be influenced by the interactions mediated by salivary fibronectin. PMID- 3016051 TI - Antimicrobial properties of hydrogen peroxide and sodium bicarbonate individually and in combination against selected oral, gram-negative, facultative bacteria. AB - The topical application of hydrogen peroxide (H2O2) and sodium bicarbonate (NaHCO3), individually and in combination, has been used empirically in the treatment of periodontal diseases. In this study, we examined both minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of these disinfectants individually and in combination against selected facultative, Gram-negative oral bacteria in a microtiter dilution assay. The bacteria studied included Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Eikenella corrodens, and Capnocytophaga gingivalis. These bacteria exhibited MBC (one hr) values ranging from 75 mumol/L to greater than 10 mmol/L and MIC from less than 5 to 500 mumol/L for H2O2. The tested bacteria exhibited MIC values for NaHCO3 of from 23 to 182 mmol/L, and the MBC (one hr) exceeded 728 mmol/L for most of the strains examined. At sublethal (sub-MIC) concentrations, sodium bicarbonate antagonized the ability of H2O2 to inhibit bacterial growth in MIC assays, but sublethal concentrations of H2O2 had no effect on the MIC values of NaHCO3. Lethal concentrations of H2O2 and NaHCO3 exhibited synergistic antimicrobial activity in combination in one-hour bactericidal assays. Since the bactericidal properties of these antimicrobial agents are synergistic, we conclude that it may be rational to use them in combination to treat certain forms of periodontal disease. Also, lower and perhaps safer concentrations of H2O2 can be used in combination with NaHCO3 when oxidative antimicrobial chemotherapy is indicated. PMID- 3016050 TI - Effects of neutral salts in a bench-scale caries model. AB - In an earlier paper on bench-scale simulation of the caries process, it was shown that the passage of ions through ion-permselective barriers could have profound effects on the composition of the solution within the "lesion" at steady state. As indicated in earlier papers, these changes are produced by unequal rates of diffusion of Ca and PO4 ions prior to reaching steady state. Comparable effects are attributable to F ions when present. Here, we used the same two-compartment diffusion apparatus and membranes, as described in the earlier paper, to show that a neutral salt, such as NaCl, disproportionates under the influence of membrane potential. Thus, although the Na and Cl concentrations are nearly equal in the "plaque-saliva" compartment, they become very different in the "lesion" solution. An excess of Na over Cl is equivalent to the presence of the component NaOH, and an excess of Cl over Na is equivalent to the presence of HCl. With the highly permselective commercial membranes used in these experiments, the Ca/P ratio in the "lesion" solution changed from an initial value of 1.6 to a value as high as 53 or as low as 0.1 at steady state. These phenomena are relevant to the events taking place in a caries lesion and must be taken into account in devising physicochemical models of the caries process. A valid caries model, in turn, offers the possibility of identifying steps in the caries mechanism which might be blocked to prevent tooth decay. PMID- 3016053 TI - A theoretical study of in vivo lesion repair using a controlled-release device. AB - A theoretical model of lesion remineralization predicts that in vivo caries repair can be effected in a five-day period by means of a controlled- and sustained-release device containing calcium, phosphate, and fluoride. The calculations detail an 80% repair before the remineralization process stops. However, the fluoridated hydroxyapatite formed is calculated to be about 20% more resistant to future attack than is hydroxyapatite, making effective repair complete. Optimal flux (W) conditions for repair of 100 microns lesions are WCa = 1.0 X 10(-6) kg/m2s, stoichiometric phosphate, and WF = 2.0 X 10(-9) kg/m2s. PMID- 3016052 TI - Modulation of the kinetics of induced neutrophil superoxide generation by fluoride. AB - Fluoride (F-) is unique in that, depending upon concentration, it can either stimulate or inhibit the synthesis of the neutrophil's major intracellular microbicidal product, superoxide anion (O2-). While a number of studies have delved into F- induction of O2- generation, little has been reported on the effect of F- on the synthesis of O2- induced by other agents. In this report, we show that F- inhibits the activation and activity of neutrophils induced either by formyl-methionyl-leucylphenylalanine (FMLP) or by phorbol myristate acetate (PMA). The extent of increase in lag period and decrease in velocity of O2- synthesis was found to be directly related to the concentrations of F- and of hydrogen ion in the medium and to the length of contact time between the neutrophil and F-. Decreasing the pH of the medium, while maintaining a constant concentration of F-, disproportionately increased the inhibitory effect of the anion over the effect of pH alone. Fluoride inhibited the responsiveness of neutrophils to FMLP more than it did their responsiveness to PMA. A gradual but partial reversal of F-'s inhibition of O2- synthesis could be achieved by transferring inhibited cells to F(-)-free medium. Our study shows that the suppression of both FMLP- and PMA-induced synthesis of O2- by neutrophils is most dramatic at a reduced pH and is probably related to the intracellular accumulation of F-.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016055 TI - [Punctualism, nonadapationism, neutralism and evolution]. PMID- 3016054 TI - Basal cell carcinoma and collagenase. AB - The interaction of connective tissue stroma and epithelial cutaneous cancer is an active area of investigation in dermatology. Studies summarized here explore the role of collagenase in basal cell carcinoma (BCC) invasiveness. Evidence is presented to support the role of a cytokine or cytokines secreted by BCCs that stimulate collagenase production by surrounding stromal fibroblasts. Prospects for further research in this area are proposed. PMID- 3016056 TI - [Characteristics of the action of heavy metals on the membrane of Escherichia coli]. PMID- 3016057 TI - [EPR study of the denitrosation of alkylnitrosoureas in vivo]. PMID- 3016058 TI - [Pathways of 2,4,6-trinitrotoluene biotransformation]. PMID- 3016059 TI - [Effect of lipid peroxidation on the membrane structure of the sarcoplasmic reticulum]. PMID- 3016061 TI - Platelet prostacyclin binding in coronary artery disease. AB - Reduced responsiveness of platelets to prostacyclin, reported in vitro in patients with coronary artery disease, has been thought to be a factor predisposing toward coronary thrombosis and vasospasm as a result of enhanced in vivo release of cyclic endoperoxides and thromboxane A2 by the platelets. In this study, specific binding of prostacyclin to intact platelets was determined in patients with coronary artery disease by direct binding studies using 9-3H prostacyclin sodium salt. In addition, the inhibitory effect of prostacyclin on primary aggregation induced by adenosine diphosphate and cyclic adenosine monophosphate (cyclic AMP) accumulation stimulated by prostacyclin was examined. Twenty patients with angiographically documented coronary artery disease and stable angina, 8 patients with acute myocardial infarction, 14 healthy volunteers and 10 patients with normal angiograms were studied. In patients with stable angina, binding capacity and affinity of platelet prostacyclin binding sites and prostacyclin-induced cyclic AMP accumulation were not different from those of control subjects. In patients with acute myocardial infarction, however, binding capacity of platelet prostacyclin receptors was significantly reduced (0.69 +/- 0.45 versus 1.35 +/- 0.37 pmol/10(9) platelets, p = 0.001) and the postreceptor response, represented by platelet responsiveness to prostacyclin and prostacyclin induced cyclic AMP synthesis, was impaired. Because all patients with myocardial infarction were receiving intravenous heparin and nitroglycerin, which might interfere with platelet prostacyclin binding, competition experiments were performed in vitro. Neither heparin (3 to 250 IU/ml) nor nitroglycerin (0.8 to 22 microM) displaced specifically bound 9-3H-prostacyclin. L-Epinephrine in concentrations up to 10 microM also exhibited no competition with specific platelet prostacyclin binding.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016062 TI - Effect of 8-bromo-cyclic guanosine monophosphate (cGMP) on coronary artery constriction in isolated rabbit hearts. AB - The vasodilator 8-bromo-guanosine 3':5'-monophosphate (8-bromo-cGMP) effectively counteracts vasopressin-induced coronary artery constriction in a supported perfused working rabbit heart. In this preparation, the coronary arteries remain in contact with the beating heart. The obtuse marginal artery and portions of the left anterior descending coronary artery were deprived of endothelium. Perfusion was carried out with Krebs-Henseleit solution, oxygenated with a disposable infant oxygenator. The internal diameter of large coronary arteries was determined by color arteriography (injection of patent blue dye and gated photography). The effect of vasopressin with and without the addition of 8-bromo cGMP on cardiac performance (cardiac output, left ventricular systolic pressure, left ventricular end-diastolic pressure, maximal rate of rise in left ventricular pressure [dP/dtmax], mean aortic pressure) and large coronary vessel and total coronary vascular resistance was determined in nine experiments. In addition, changes in coronary sinus partial pressure of carbon dioxide (PCO2) and pH were observed. Vasopressin alone caused a significant decline in coronary flow, myocardial oxygen consumption and coronary sinus pH. Cardiac performance declined, probably because of myocardial ischemia. Large coronary vessel and total coronary vascular resistance rose. The vasodilator 8-bromo-cGMP strongly inhibited the vasoconstrictor action of vasopressin, counteracted the increase in large and total coronary vascular resistance, prevented the fall in myocardial oxygen consumption and eliminated changes in pH or PCO2 of coronary sinus effluent. Because of the elimination of myocardial ischemia by 8-bromo-cGMP, cardiac performance was normalized.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016064 TI - Is there any physiological significance to the enterohepatic circulation of vitamin D sterols? PMID- 3016063 TI - Beta-adrenergic receptor properties of canine myocardium: effects of chronic myocardial infarction. AB - To determine the effects of chronic myocardial infarction on beta-adrenergic properties of canine myocardium, the hearts of nine mongrel dogs were studied 3 weeks after acute myocardial infarction. Infarction was produced by ligating the left anterior descending coronary artery in five dogs and the circumflex artery in four dogs. The heart was divided into normal and infarct zones (either anterior or posterior, depending on the vessel ligated) and marginal zones (septal and lateral), each zone being subdivided into epicardial and endocardial portions. Myocardial blood flow (microsphere technique) was markedly reduced in the infarct zone. In eight endocardial infarct samples after left anterior descending ligation, the maximal number (+/- SD) of binding sites assessed by 125I-iodocyanopindolol was 3.9 +/- 1.9 pmol/mg deoxyribonucleic acid (DNA) and was reduced from normal endocardial values (9.7 +/- 9.4 pmol/mg DNA, p less than 0.05). The dissociation constant (Kd), which is a measure of the affinity of the iodinated antagonist for the receptor, did not differ (304 +/- 222 versus 338 +/- 219 pM, p = NS). In the epicardium, the maximal number of beta-adrenergic receptors was also reduced (p less than 0.05), without a change in Kd. In the lateral and septal zones neither the maximal number of binding sites nor Kd values differed from those of normal endocardium. In nine endocardial infarct zones, (-)-isoproterenol-stimulated adenylate cyclase activity was reduced compared with control (34,870 +/- 29,430 versus 88,660 +/- 63,640 pmol/mg DNA/30 minutes, p less than 0.01), but the ratio of (-)-isoproterenol-stimulated to maximal (sodium fluoride-stimulated) adenylate cyclase activity was unchanged between normal and infarct zones.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016060 TI - Role of thromboxane, prostaglandins and leukotrienes in endotoxic and septic shock. AB - Intravenous bolus endotoxin elicits a marked but transient increase in plasma TxB2 and 6-keto-PGF1 alpha in a large number of species. A smaller, delayed and more prolonged increase in TxB2 and 6-keto-PGF1 alpha are reported in animals with septic shock, i.e., those with fecal peritonitis or cecal ligation. Thromboxane synthetase inhibitors or antagonists attenuate endotoxin-induced acute cardiopulmonary changes, the delayed increase in serum lysosomal enzymes, fibrin/fibrinogen degradation products and the thrombocytopenia in a number of species. While these drugs increase survival of rats or mice following endotoxin they do not alter survival of rats in septic shock. These results support the hypothesis that TxA2 exerts a pathophysiologic effect in shock following bolus endotoxin. In contrast, nonsteroidal antiinflammatory drugs (NSAID) and dietary essential fatty acid deficiency increase survival of rats subjected to endotoxin shock, and survival time in models of septic shock. These results also suggest that some other cyclooxygenase product(s) is involved in septic shock due to fecal peritonitis or cecal ligation. Preliminary experimental studies indicate salutary effects of leukotriene inhibitors and antagonists in endotoxin shock and in models of acute pulmonary injury. Clinical studies have demonstrated elevated plasma TxB2 and 6-keo-PGF1 alpha concentrations in patients with septic shock, and elevated LTD4 in pulmonary edema fluid of patients with the adult respiratory distress syndrome. In view of these clinical and experimental results, clinical trials of NSAID and/or leukotriene inhibitors/antagonists should be considered. PMID- 3016065 TI - Allergy and the syndrome of chronic Epstein-Barr virus infection. PMID- 3016066 TI - Correlation between allergy and persistent Epstein-Barr virus infections in chronic-active Epstein-Barr virus-infected patients. AB - Forty-six anti-Epstein Barr nuclear antigen-positive allergic patients, 11 of whom having clinical and laboratory evidence of chronic-active Epstein-Barr virus (CA-EBV) infections, were characterized by EBV serology, percentages of T cells, B cells, and IgE+ cells, serum levels of IgE, and allergen-induced responsiveness of lymphocytes. Results demonstrated patients with CA-EBV have significantly increased responsiveness toward specific allergens, responses toward greater numbers of allergens, numbers of IgE+ T and B cells, and levels of background DNA activity in nonstimulated lymphocytes than do subjects who suffer from allergies in the absence of the CA-EBV syndrome. Further comparison between subjects with laboratory-determined mild and moderate allergy and those with CA-EBV demonstrated a progressive increase in the serum levels of IgE as the degree of allergy increased, no difference in concentrations of T and B cells, and titers of anti-viral capsid antigen and anti-early antigen to be significantly greater in patients with CA-EBV. Statistical analysis demonstrated that patients with CA EBV could be separated from subjects with allergies by metabolic and immunologic variables. The data suggested that allergen-induced responses may contribute to the CA-EBV syndrome. PMID- 3016067 TI - Specific allergen-induced Epstein-Barr nuclear antigen-positive B cells from patients with chronic-active Epstein-Barr virus infections. AB - Enriched B cells and peripheral blood lymphocytes from patients with chronic active Epstein-Barr virus (CA-EBV) infections and subjects with mild and moderate allergies were cultured in vitro with specific allergens known to cause allergic reactions. A significant increase in Epstein-Barr nuclear antigen+ cells occurred only in the B cells obtained from patients with CA-EBV when cells were stimulated with the specific antigen. Results indicate an association between EBV transformed cells and B cells with idiotypic expressions and may help to explain the association between CA-EBV and allergy in these patients. PMID- 3016068 TI - Methods of determining fiber. PMID- 3016069 TI - Stool cyclic AMP in diarrhoea due to different causative organisms. PMID- 3016070 TI - Electropherotypes of ds-RNA of rotavirus in infants and young children with gastroenteritis in Bangladesh. PMID- 3016071 TI - Changes in the blood gas concentration caused by secretin and cholecystokinin. Indication of a vasomotor effect? AB - The assumption that secretin has a general vascular effect led to an investigation of the blood gas level in the peripheral veins and the acid/base balance under the influence of secretin (1 CU/kg/h, 0-120 min) and cholecystokinin (CCK) (1 IU/kg/h, 60-120 min) in a group of 10 volunteers. Six of the volunteers were subjected to a randomized, cross-over NaCl infusion study. With a secretin infusion alone (0-60 min) there was a transient, significant rise in PO2 (p less than 0.01) and oxygen saturation (p less than 0.05), which was no longer detectable after 60 min (p greater than 0.05). With an additional administration of CCK (60-120 min) and in the follow-up phase of observation (120 180 min) there was a significant fall in both parameters (120 min: p less than 0.05, 180 min: p less than 0.01). PCO2, HCO-3, and pH remained unaffected. The isolated increase in PO2 and O2 saturation may be attributed to vasodilation induced by secretin. The drop in both parameters under the influence of an additional infusion of CCK and in the follow-up phase of observation is linked to a possible vascular effect of CCK. PMID- 3016072 TI - Phosphatase localization in the endomembrane system of the dinoflagellate Crypthecodinium cohnii. AB - The distribution of four enzymes within the endomembrane system of the protist Crypthecodinium cohnii has been determined using cytochemical localizations with lead as a capture agent. Nucleoside diphosphatase (NDPase) activity, using inosine diphosphate (IDP) and thiamine pyrophosphate (TPP) as substrates, was observed in the Golgi apparatus, with a gradient of increasing reaction product noted in some cells from the cis to trans cisternae. Tubules and vesicles associated with the trans cisternae also contained reaction product. The endoplasmic reticulum exhibited a high activity of glucose-6-phosphatase [with glucose-6-phosphate (G-6-P) as substrate]. Traces of reaction product were also observed in the cis-most and trans-most cisternae of the dictyosomes. Activity of acid phosphatase (AcPase) was observed in Golgi cisternae as well as in associated cytoplasmic vesicles. Heaviest deposition was localized in medial and trans dictyosome cisternae. The cytoplasmic system of flattened vesicles subtending the surface membranes in these cells did not exhibit reactivity with any of the substrates used. The distribution of these enzymes in this algal cell appears similar to that observed in animal cells and suggests that these enzymes may represent markers for algal cell endomembrane compartments. PMID- 3016074 TI - Histochemical localization of cathepsin B, dipeptidyl peptidase I, and dipeptidyl peptidase II in rat bone. AB - The histochemical distribution of the thiol proteases cathepsin B and dipeptidyl peptidase I and the serine protease dipeptidyl peptidase II was examined in rat bone and joint using amino acid derivatives of 4-methoxy-2-naphthylamine (MNA). The liberated MNA was then visualized by simultaneous coupling with fast blue B. Cathepsin B was examined with CBZ-Arg-Arg-MNA, dipeptidyl peptidase I (DPP I) with Gly-Arg- or Pro-Arg-MNA, and dipeptidyl peptidase II (DPP II) with Lys-ALA- or Lys-Pro-MNA. Bright red reaction product indicative of proteolytic activity was observed in most cell types associated with bone and its surrounding connective tissues, including osteocytes, osteoblasts, chondrocytes, chondroblasts, fibroblasts, and macrophages. Surprisingly, protease activity in osteoclasts could not be established with certainty, and it was concluded that these enzymes are either absent, present in very low amounts, or secreted as soon as they are synthesized rather than stored within the cell. The cells of the resting zone of the growth plate were intensely reactive for DPP II but were only moderately reactive for cathepsin B and DPP I. The reverse was true of the proliferating and hypertrophic layers. The protease activity observed in bone, cartilage, tendon, ligament, and synovium would be expected to contribute significantly to normal protein metabolism as well as to pathological destruction in these tissues. PMID- 3016073 TI - A triple ultrastructural immunogold staining method. Application to the simultaneous demonstration of three hypophyseal hormones. AB - The triple ultrastructural immunocytochemical labeling technique described here is based on the use of three different antisera raised in two different animal species: rabbit anti-corticotropin (R-ACTH), guinea pig anti-prolactin (GP-LTH) and rabbit anti-gonadotropin (R-LH beta). Staining is carried out on both sides (A and B) of the same tissue section. First, side A is incubated with a mixture of R-ACTH and GP-LTH and then with a mixture of the two corresponding species specific immunoglobulins (IgG) adsorbed respectively to 5 and 20 nm gold particles: goat (G) anti-R IgG 5 + G anti-GP IgG 20; second, side B is incubated with R-LH beta, followed by species-specific secondary antibodies adsorbed in 10 nm gold particles; G anti-R IgG 10. With this technique, we demonstrated, on the same thin section, ACTH, LTH, and LH cells. The immunocytochemical procedure used has proved useful for simultaneous ultrastructural localization, on the same thin section, of three different antigenic sites. This technique, applied to other materials, could provide interesting information in several biological fields. PMID- 3016075 TI - Geographic distribution of restriction types of Mycobacterium bovis isolates from brush-tailed possums (Trichosurus vulpecula) in New Zealand. AB - DNA restriction endonuclease analysis was used for intra-specific typing of Mycobacterium bovis isolates from 83 brush-tailed possums (Trichosurus vulpecula) obtained between 1982 and 1984 from the three major regions in New Zealand with endemic bovine tuberculosis. All the isolates were found to be genetically very similar. Differentiation of the isolates into 33 restriction types was achieved by using high-resolution electrophoresis and the combined results from separate digestions with the restriction enzymes Bst EII, Pvu II and Bcl I. The typing system was entirely reproducible. Isolates of the same type were usually found in adjacent localities and were always limited to one of the three major regions. In some cases, isolates of the same type were found in both 1982 and 1984. The phenotypic significance of the small genetic differences identified between different isolates is unknown. The typing system will be useful for monitoring the transmission of M. bovis to other species and the future spread of different M. bovis types through possum populations. PMID- 3016076 TI - Poliovirus antibody in Northern Greece. AB - In order to study the serological status of the Northern Greek population to poliovirus, 881 sera from healthy people were examined for neutralizing antibody by the micrometabolic inhibition test. The people under examination were aged from 1 day to 70 years old. Overall, of the 881 sera examined, 704 (80%) had antibodies (titre greater than or equal to 4) to poliovirus 1, 742 (84%) had antibodies to poliovirus 2 and 715 (81%) had antibodies to poliovirus 3. Fifty five per cent of the sera had antibodies to all three polioviruses while 3.3% had no poliovirus antibody at all. There was no statistically significant difference in the rates of seropositivity to the various poliovirus types or between males and females. However the rates of seropositivity did vary with age. PMID- 3016077 TI - Humoral response of pregnant sows to foot and mouth disease vaccination. AB - Four groups of sows were inoculated, either once or twice, with O1BFS 1860 foot and mouth disease oil-emulsion vaccine during pregnancy and samples of serum, for analysis, were collected at intervals for greater than 300 days. The pregnant sows responded well to vaccination regardless of their state of gestation. Single vaccination produced protective levels of antibody (greater than 1.53 log10SN50) in 3 out of 4 sows while double vaccination produced protective levels in all 6 sows tested. Anti-FMD IgM antibodies could be detected for 40-60 days after vaccination or revaccination. Anti-FMD IgG antibodies appeared within 10 days of vaccination and persisted, in each sow, for the duration of the study. The anti FMD IgA response observed was less easy to characterize due to significant animal to animal variation. Although there was no evidence of a fall in the neutralizing antibody titres over one year post vaccination the anti-FMD IgG antibody population did show signs of a change in its heterogeneity and avidity. PMID- 3016078 TI - Age-dependent prevalence of BK virus IgG and IgM antibodies measured by enzyme linked immunosorbent assays (ELISA). AB - Enzyme immunoassays (ELISA) have been developed for the detection of BK virus IgG and IgM-antibodies. Specific IgG is detected by an antigen-coated solid phase test; IgM by an antibody capture method. These methods have been used to study the age-distribution of BK virus antibodies in Tromso county in Northern Norway. The serum panels tested were: 60 sera from paediatric patients aged 0-1 year; 220 sera from healthy persons aged 1-82 years; 74 sera from healthy blood donors; 107 sera from healthy pregnant women. The age-distribution of BKV-IgG antibodies showed that primary infections took place predominantly between the ages of 1 and 6 years, and that there were no sex differences, either in the age-specific prevalence or in the level of BKV-IgG. We found no significant differences in the prevalence of BKV-IgM antibodies in healthy children and adults and pregnant women. BKV-IgM was detected in 26 of the 461 sera tested (5.6%). PMID- 3016079 TI - Recovery of bluetongue virus serogroup from sera collected for a serological survey from apparently healthy cattle, from the Sudan. AB - Virus of the bluetongue (BT) serogroup was recovered from 11% of cattle sera collected from apparently healthy animals in Khartoum Province for the sole purpose of screening for BT antibodies. Since these sera did not contain BT antibodies, the donor cattle could have been scored as BT free in the serological survey. Virus was initially isolated in chicken embryos inoculated intravascularly, and was further adapted to Vero cell cultures. Isolates were identified as belonging to the BT serogroup using the agar gel immunodiffusion (AGID) and complement fixation (CF) tests. The results indicated that cattle in the Sudan could harbour BT virus without showing symptoms of the disease. Such an observation necessitates further work to clarify the role of cattle in the epidemiology of BT in the Sudan. PMID- 3016080 TI - Adenovirus type 8 keratoconjunctivitis--an outbreak and its treatment with topical human fibroblast interferon. AB - An outbreak of keratoconjunctivitis is described which involved at least 186 people; adenovirus type 8 was identified in 50 of the cases. Topical human fibroblast interferon was assessed in a double-blind, placebo-controlled study in which 34 patients participated. Seventeen of the 34 trial patients yielded adenovirus type 8; three were infected with adenovirus type 7. The outbreak was curtailed by control of infection measures: principally careful hand-washing by medical personnel between cases and by discouraging attendance of new cases at the Eye Infirmary. Consequently the trial numbers are small. In addition there was a wide interpatient variation in the severity of infection. Therefore it was not possible to make any statistically valid conclusions concerning the recovery rate of patients receiving interferon or placebo. PMID- 3016082 TI - Chemical disinfection of human rotavirus-contaminated inanimate surfaces. AB - Fomites may play a role in the transmission of rotavirus infections, and in view of this, 27 disinfectants were evaluated for their ability to inactivate human rotavirus (HRV) on contaminated non-porous inanimate surfaces. Disks of stainless steel, glass and two types of plastics were contaminated with about 10(7) plaque forming units of HRV suspended in faecal matter. The inoculum was allowed to dry and an equal volume of the product under test was applied to the contaminated surface. After contact for 1 min, the action of the disinfectant was stopped by dilution. Surviving infectious virus on the disks was determined by plaque assay in MA-104 cells. A product was considered to be effective if it could reduce the virus titre by at least 3 log10. Only 33.3% (9/27) of the formulations tested proved to be effective. Further testing of the effective products, which included antiseptics, instrument soaks and hard-surface disinfectants, showed that all of them could, in fact, reduce the virus titre on contaminated surfaces by at least 6 log10. These findings show the relative resistance of HRV to a wide range of chemical disinfectants in common use, and also emphasize the need for a more thorough evaluation of the virucidal potential of formulations regularly employed in attempts to prevent and control outbreaks of rotaviral diarrhoea. PMID- 3016083 TI - The inactivation of a bovine enterovirus and a bovine parvovirus in cattle manure by anaerobic digestion, heat treatment, gamma irradiation, ensilage and composting. AB - A bovine enterovirus and a bovine parvovirus seeded into liquid cattle manure were rapidly inactivated by anaerobic digestion under thermophilic conditions (55 degrees C), but the same viruses survived for up to 13 and 8 days respectively under mesophilic conditions (35 degrees C). The enterovirus was inactivated in digested liquid manure heated to 70 degrees C for 30 min, but the parvovirus was not inactivated by this treatment. The enterovirus, seeded into single cell protein (the solids recovered by centrifugation of digested liquid manure), was inactivated by a gamma irradiation dose of 1.0 Mrad, but the parvovirus survived this dose. When single cell protein seeded with bovine enterovirus or bovine parvovirus was ensiled with cracked corn, the enterovirus was inactivated after a period of 30 days, while the parvovirus survived for 30 days in one of two experiments. Neither the enterovirus nor the parvovirus survived composting for 28 days in a thermophilic aerobic environment when seeded into the solid fraction of cattle manure. It was concluded that, of the procedures tested, only anaerobic digestion under thermophilic conditions appeared to be reliable method of viral inactivation to ensure the safety of single cell protein for refeeding to livestock. Composting appeared to be a suitable method for the disinfection of manure for use as a soil conditioner. PMID- 3016081 TI - Chemical disinfection of human rotaviruses: efficacy of commercially-available products in suspension tests. AB - Suspension tests were conducted on 69 commercial and 7 non-commercial disinfectant formulations to determine which classes of chemicals were most active against human rotavirus (HRV). Virus samples, in the presence of varying levels of organic matter, were exposed to the disinfectants for 1 min. The levels of remaining infectious virus were determined by plaque assay. Products were rated by their ability to reduce the levels of infectious virus by more than 3 log10 in the presence or absence of tryptose phosphate broth (peptides and inorganic salts) or fecal matter. Of the commercially-available products tested, only 25% were rated as highly and 7% as moderately effective. The remaining 68% were either effective only in the absence of any additional organic matter (48%) or were completely ineffective (20%). The majority (64%) of the moderately and highly effective products were further examined for their ability to inactivate greater than 6 log10 of infectious HRV in the presence of fecal matter or tryptose phosphate broth. With one exception, all these products were still effective. Products potentially suitable as topical antiseptics, hard surface disinfectants and instrument soaks were identified. The results emphasize the care that should be exercised in the selection of disinfectants for the control and prevention of rotaviral infections. PMID- 3016085 TI - Blood pressure, erythrocyte sodium and potassium concentrations and Na+K+ATPase activity in children with hypertensive mothers. AB - Fifty-four children aged 10-15 years were studied. Twenty-two children had mothers who had sustained hypertension after a hypertensive pregnancy (HT) and 17 had normotensive mothers who had previously had a hypertensive pregnancy (NT). A control group consisted of 15 children with normotensive mothers (C). Blood pressure was significantly higher in the HT group in comparison with the C group (P less than 0.01). Erythrocyte sodium (Na+) and potassium (K+) concentrations were similar in all groups and not related to blood pressure. The Na+K+ATPase activity in erythrocyte membranes was lower in the HT group than in the NT group (P less than 0.02), but not significantly different from the C group. There was no statistically significant correlation between Na+K+ATPase activity and blood pressure. PMID- 3016086 TI - Investigation of membrane proteins in rat erythrocytes in spontaneous hypertension by means of spin-label technique. AB - It has been suggested that alterations in cell membrane proteins may play a role in changes of erythrocyte membrane structure and function in hypertension. In order to characterize the structure of membrane proteins of erythrocytes from spontaneously hypertensive rats (SHR) the spin-label technique with a maleimide spin-label was used. A significant difference was observed in the characteristic electron-spin resonance (e.s.r.) spectrum of the label between samples from normotensive rats and SHR. The difference was eliminated and the spectrum significantly changed after treatment of the labelled membrane with EDTA followed by washing out the EDTA extracts, whereas the same treatment with EDTA without the following washing had no effect on the e.s.r. spectrum. It is concluded that the EDTA extracts different substances in the different rat groups. The spin label technique is a useful method for distinguishing cell membrane properties in SHR and normotensive rats. PMID- 3016084 TI - The effect of oxygen-dependent antimicrobial systems on strains of Legionella pneumophila of different virulence. AB - Four strains of Legionella pneumophila of different virulence as identified by ability to produce pneumonia and death in guinea-pigs infected by a fine-particle aerosol were examined for factors which may intracellularly influence virulence. Possible bactericidal mechanisms possessed by alveolar phagocytes were examined. A relationship could be established between resistance to H2O2, catalase activity and virulence amongst the strains. Virulent strains resisted the bactericidal activity generated by the xanthine oxidase system; avirulent strains did not. Incorporation of various specific inhibitors of the xanthine oxidase system indicated that the main bactericidal activities were associated with the production of H2O2 and hydroxyl radicals (.OH). All strains of L. pneumophila were susceptible to the bactericidal activity generated by the myeloperoxidase H2O2-halide system, confirming earlier observations that polymorphonuclear neutrophil leucocytes (PMNLS) are able to kill both virulent and avirulent strains of L. pneumophila. PMID- 3016087 TI - Functional modifications of alloreactive T cell clones infected with HTLV-I. AB - Two alloreactive T cell clones with anti-HLA-DR1 specificity showed significant alterations of cognitive and functional characteristics after infection with human T cell leukemia virus (HTLV-I). Similar to HTLV-I-infected lines derived from immunologically uncommitted lymphocytes, the transformed clones displayed blastogenic mixed lymphocyte culture (MLC) responses to cells carrying any of the allelic variants of human HLA-D/DR antigens, including self. Although these two clones were originally able to provide allospecific help only to B cells expressing the DR1 antigen, after infection with HTLV-I they stimulated B cells of any HLA-D/DR phenotype to produce immunoglobulin in cultures. The helper inducer activity of the transformed clones remained susceptible to the effect of monoclonal antibody anti-LDA1 that inhibits the helper function of normal human T cells. One of these clones (207TK), which before infection specifically killed DR1-positive target cells, lost its killing ability. The other clone (19TK) although originally noncytotoxic, acquired natural killer-like function after transformation. Study of the rearrangement of the genes coding for the beta-chain of the T cell antigen receptor revealed no differences between the wild (noninfected) and mutant (infected) clones. There was, however, an increased level of this message, as well as of the message encoded by the beta-chain gene of HLA-DR in the mutant clones. Such changes may be related to transacting transcriptional effects induced by the human T cell lymphotropic virus HTLV-I. PMID- 3016088 TI - Vaccines consisting of periodate-cleaved oligosaccharides from the capsule of Haemophilus influenzae type b coupled to a protein carrier: structural and temporal requirements for priming in the human infant. AB - Reducing oligosaccharides from the Haemophilus influenzae type b capsular polymer (PRP) coupled by reductive amination to diphtheria toxoids (DTd) had been shown to elicit potentially protective serum anti-PRP antibodies (Ab) in infants too young for an adequate response to PRP vaccine. Here we report that cleavage of PRP with periodate gives antigenic oligosaccharides that couple with high efficiency. DTd-coupled saccharides of mean length eight or 20 repeat units (Dpo8 and Dpo20, respectively) were tested for immunogenicity in young adults (single injection) and in infants 9 to 15 mo old who received a sequence of primary (1 degree) and secondary (2 degrees) injections. Both vaccines consistently induced high anti-PRP Ab responses in adults. In infants, Dpo8 elicited only modest anti PRP responses, whereas Dpo20 gave consistently high titers; post-2 degrees responses were higher when the interval between 1 degree and 2 degrees injections was 6 to 14 wk than with an interval of 2 to 4 wk. Thus with this type of immunogen, priming responses in infancy has more stringent structural requirements than does triggering responses in adults, and the priming appears to maximize more slowly than the Ab level. PMID- 3016089 TI - Effect of human syncytiotrophoblast plasma membrane-soluble extracts on in vitro mitogen-induced lymphocyte proliferation. A possible inhibition mechanism involving the transferrin receptor. AB - Transferrin (Tf) binding to lymphocytes and to syncytiotrophoblast plasma membranes (STPM) and their soluble extracts (STPM-SE) was examined, as well as the effect of the latter on mitogen-induced lymphoproliferation. Lymphocytes only express Tf receptors (Rtf) after mitogen (phytohemagglutinin or concanavalin A) stimulation, and the percentage of Tf bound by stimulated lymphocytes increased as a function of contact time with the mitogen, reaching a maximum at 72 hr. Studies of Tf binding to STPM-SE showed that the percentage of bound Tf increased proportionally to the protein concentration, but was additionally enhanced by a factor of three when STPM were pretreated with 3 M KCl. Scatchard analysis of Tf binding to lymphocytes cultured for 72 hr in the presence of mitogen, as well as to STPM and STPM-SE, revealed that this binding was specific and occurs via a single category of identical and independent receptors the numbers and affinity constants (Ka) of which have been determined. The results obtained by using STPM indicate that the Ka does not vary significantly from one preparation to another, but that the number of sites per milligram of protein increases by a factor of 10 when the STPM are pretreated with 3 M KCl (KCl-STPM). Finally, STPM-SE inhibited the mitogen-induced lymphoproliferative response whether or not they were treated with 3 M KCl. This inhibition was not due to lymphocytotoxicity, was dose dependent regardless of the preparation used, but was maximized with the KCl-STPM SE fraction. The correlation between the inhibitory capacities of the soluble STPM extracts and the numbers of RTf sites present on their membranes leads to the hypothesis that the observed inhibition could involve the RTf. This effect may help in protecting the fetus from the maternal immune system at the time of the semi-allogenic fetal graft. PMID- 3016090 TI - Concanavalin A induces Ca2+ mobilization, but only minimal inositol phospholipid breakdown in mouse B cells. AB - Concanavalin A (Con A) induces DNA synthesis in bulk cultures of murine T cells. In contrast, the lectin has been shown to stimulate purified B cells to leave G0, but not to proliferate. We demonstrate here that Con A induces comparable increases in intracellular Ca2+ levels in the two cell types. In B cells this appears to be due to Ca2+ influx. As expected, the lectin also provokes significant degradation of inositol phospholipids in T cells. However, it causes only minimal release of inositol phosphates in B cells. We therefore postulate that Con A may stimulate B cells by causing influx of Ca2+. In line with this, activation of B cells by the lectin is inhibited by cyclosporine, which preferentially affects stimulation of lymphocytes by Ca2+ mobilizing agents. PMID- 3016091 TI - Amiloride inhibition of DNA synthesis and immunoglobulin production by activated human peripheral blood mononuclear cells is independent of sodium/hydrogen antiport. AB - Activation of sodium/proton (Na+/H+) antiport activity has been shown to occur as an early event in mitogenesis. Because amiloride inhibits Na+/H+ antiport activity, it is hypothesized that mitogenesis may be inhibited by amiloride. In this work, we examined the effect of amiloride on DNA synthesis as measured by [3H]thymidine uptake and immunoglobulin (Ig) production as measured by an ELISA system in human peripheral blood mononuclear cells (PBM). Amiloride at 100 microM concentration inhibited irradiated Raji cell (*R)-activated and phytohemagglutinin-P (PHA-P)-stimulated DNA synthesis by 50 +/- 11% and 72 +/- 12%, respectively. IgG production was inhibited by 71% at 100 microM amiloride concentration in *R-activated PBM. This concentration of amiloride inhibited Na+/H+ antiport activity by 92%. Because amiloride is known to inhibit other pre replicative cellular functions such as protein synthesis, we used an amiloride analogue, dimethylamiloride, which inhibited Na+/H+ antiport activity by 90% at a concentration of 1 microM without inhibition of PBM Ig or DNA synthesis. Furthermore, neither PHA-P nor *R-stimulated PBM demonstrated an intracellular alkalinization even after 6 hr of stimulation. Similarly, T cell-enriched or B cell-enriched populations did not show intracellular alkalinization after PHA-P or *R activation. Thus, it appears that Na+/H+ antiport activation is not an early event in PBM mitogenesis. The inhibition of mitogenesis by amiloride may be due to abrogation of premitotic events such as protein synthesis. PMID- 3016093 TI - The mechanism of action of lymphokines. IX. The enzymatic basis of hydrogen peroxide production by lymphokine-activated macrophages. AB - The purpose of this study was to elucidate the biochemical basis of the enhanced hydrogen peroxide (H2O2) production by guinea pig peritoneal macrophages (MP) cultured in lymphokine (LK)-containing medium. The markedly augmented H2O2 generation by these cells, demonstrable by the horseradish peroxidase (HRP) catalyzed oxidation of phenol red, is distinguished by its lack of dependence on a second stimulus. We demonstrate that H2O2 production is truly spontaneous and is not caused by a stimulant present among the H2O2 assay reagents. The principal candidate for such a role was HRP type II (a mixture of five isoenzymes) that was reported to be capable of eliciting an oxidative burst in MP. Four distinct HRP isoenzymes that were found incapable of provoking an oxidative response were nevertheless adequate for demonstrating H2O2 production by LK-activated MP. Blocking the MP receptor for mannose by the addition of mannan to the assay system resulted in enhanced detection of H2O2 by low concentrations of HRP type II and by three out of four HRP isoenzymes. Treatment of MP with LK-containing medium for 72 hr did not result in a significant change in the activity of cellular superoxide dismutase (SOD) compared with MP cultured for the same length of time in control medium. By using the specific inhibitor of copper, zinc containing SOD, sodium diethyldithiocarbamate (DDC), and the universal SOD inhibitor, sodium nitroprusside, we found that the predominant enzyme in guinea pig peritoneal MP is probably manganese-containing SOD. Incubation of LK activated MP with nitroprusside resulted in almost total inhibition of H2O2 production and a simultaneous switch to superoxide (O2-) liberation. Similar exposure to DDC had no effect. These data indicate that H2O2 produced by LK activated MP is derived exclusively by enzymatic dismutation of O2- mediated by a manganese-containing SOD. The increase in spontaneous H2O2 production induced by LK is therefore secondary to augmented O2- production that occurs at a cellular location where O2- is accessible to SOD. The enzymatic basis of the enhanced oxygen radical production was investigated by determining the kinetic parameters of the O2- -forming NADPH oxidase of resting LK-treated MP in a cellfree system in which O-2 production was induced by sodium dodecyl sulfate. The Km for NADPH and the Vmax of the enzyme of LK-treated MP were not different from those of the enzyme of MP incubated in control medium. We conclude that LK treatment of MP does not modulate the NADPH oxidase itself but, most likely, a process related to activation of the enzyme. PMID- 3016092 TI - Selective inhibition of human neutrophil chemotaxis to N-formyl-methionyl-leucyl phenylalanine by sulfones. AB - The therapeutic efficacy of the sulfones, dapsone, and sulfoxone in neutrophilic dermatoses may be related to the effects of these drugs on neutrophil function. Therefore we determined whether neutrophil chemotactic migration to various chemoattractants could be inhibited by sulfones in vitro. The chemotactic responses of human neutrophils from healthy donors were tested by using N-formyl methionyl-leucyl-phenylalanine (F-met-leu-phe), purified human C5a, and leukocyte derived chemotactic factor (LDCF). Therapeutic concentrations of sulfones selectively inhibited neutrophil chemotaxis to F-met-leu-phe, but did not affect neutrophil chemotaxis to LDCF or C5a. Inhibition of neutrophil chemotaxis to F met-leu-phe was induced by both dapsone and sulfoxone at a concentration of 10 micrograms/ml without affecting random migration, and the inhibition was reversed by washing the neutrophils. When dapsone- and sulfoxone-treated neutrophils (100 micrograms/ml) were stimulated with F-met-leu-phe, neutrophil superoxide generation was not inhibited. Sulfapyridine (10 micrograms/ml) also selectively inhibited neutrophil chemotaxis to F-met-leu-phe; however, sulfamethoxazole and sulfisoxazole did not affect chemotaxis. The inhibitory effects of dapsone, sulfoxone, and sulfapyridine could not be demonstrated with granulocytes from rabbits or guinea pigs nor with human monocytes. Experiments with radiolabeled dapsone showed rapid, nonspecific, and reversible binding of dapsone to human neutrophils. These data suggest that a mechanism of action of sulfones in neutrophilic dermatoses may be a selective inhibition of neutrophil migration to as yet undefined chemoattractants in the skin. PMID- 3016094 TI - Immunity to varicella-zoster viral glycoproteins, gp I (gp 90/58) and gp III (gp 118), and to a nonglycosylated protein, p 170. AB - Humoral and cellular immunity against two major glycoproteins (gp) of varicella zoster virus (VZV), gp I (gp 90/58) and gp III (gp 118), and against a nonglycosylated phosphoprotein (p 170) was demonstrated in human subjects. Primary VZV infection was accompanied by the development of IgG to gp I (mean titer 1:200), gp III (mean titer 1:132), and p 170 (mean titer 1:331). Increased IgG antibody production to each of the VZV proteins occurred during recurrent VZV infection with mean titers to gp I of 1:29512, to gp III of 1:15848, and to p 170 of 1:15848. Persistent high titers to gp III (mean titer 1:891) and to p 170 (mean titer 1:2238) were observed in 75% and 88% of VZV-immune subjects, respectively. T lymphocytes which proliferated on stimulation with gp I, gp III, and p 170 developed with primary VZV infection. VZV-immune subjects had mean transformation indices of 4.2 +/- 0.70 SE to gp I, 4.7 +/- 1 SE to gp III, and 3 +/- 0.39 SE to p 170. Among individual subjects, humoral and cellular immunity was not always detected to all three of the VZV proteins. Resolution of primary VZV infection and maintenance of VZV latency did not require a host response to each of these major viral proteins. PMID- 3016095 TI - Epstein Barr virus binding induces internalization of the C3d receptor: a novel immunotoxin delivery system. AB - Epstein Barr virus (EBV) infection of human B lymphocytes is initiated by selective binding of the virus to the C3d receptor (EBV/C3d receptor) on the cell surface and results in polyclonal proliferation of infected cells. In these studies we examined the fate of the EBV/C3d receptor during viral infection by using an immunotoxin made from a monoclonal antibody (HB5) reactive with the receptor and the potent toxin, gelonin. Binding of the HB5-gelonin conjugate to the EBV/C3d receptor before EBV infection (at concentrations as low as 10(-11) M) significantly inhibited the subsequent polyclonal proliferation of virus-infected B lymphocytes. HB5 antibody and gelonin alone did not inhibit proliferation. Because internalization of gelonin-antibody conjugates is required to cause cytotoxicity, these results indicate that infection of B lymphocytes with EBV selectively induced endocytosis of the EBV/C3d receptor with concomitant internalization of the immunotoxin. Proliferation of B lymphocytes that were activated by prior infection with EBV, or activated by cross-linking of their surface immunoglobulin molecules, was not inhibited by the antibody-toxin conjugate even at concentrations as high as 10(-7) M. Also, the growth of B lymphoblastoid cell lines cultured in the presence or absence of infectious EBV was not inhibited by HB5-gelonin. Thus, our results suggest that the EBV/C3d receptor is internalized only during the infection of normal B lymphocytes by EBV, with co-internalization of immunotoxin, and indicate that internalization of the EBV/C3d receptor-immunotoxin complex does not occur simply as a consequence of activation and proliferation of B lymphocytes. The use of a ligand to induce endocytosis of its receptor offers a new strategy for the selective delivery of immunotoxins to cells and may be more generally applicable. PMID- 3016097 TI - Counterimmunoelectrophoresis for detection of human serum antibody to HTLV-III. AB - We examined the usefulness of a counterimmunoelectrophoresis (CIE) technique for detecting antibodies to HTLV-III using sera that previously had been assessed for antibodies to HTLV-III by the standard enzyme-linked immunosorbent assay (ELISA). We selected a subset of 53 sera from patients with the acquired immune deficiency syndrome (AIDS) or the generalized lymphadenopathy syndrome (GLS) in which 81.1% were initially ELISA-positive, and 96.2% were positive by Western blot technique. In our standard HTLV-III CIE technique, 58.5% were positive and repeat testing increased the yield to 67.9%. Varying several parameters of the standard CIE assay did not improve sensitivity. We also studied 20 ELISA-negative and 10 ELISA borderline sera from normal controls; all were negative by CIE. These results indicate that CIE may be used for detection of human serum antibodies to HTLV III, but that the present assay was less sensitive than the HTLV-III ELISA. PMID- 3016096 TI - Preparation of Fab fragments from a mouse monoclonal IgM. AB - A procedure is described for preparing and purifying Fab fragments from a mouse monoclonal IgM. The IgM was digested with trypsin in the presence of the reducing agent, cysteine. The resulting Fab fragments were alkylated with iodoacetamide and purified using a Sephacryl S-200 column. The Fab fragments had a molecular weight of 48 000 as determined by SDS polyacrylamide gel electrophoresis and molecular sieve chromatography. This procedure has been successfully used with five different mouse monoclonal IgM. The Fab fragments bound antigen with the same specificity as the whole IgM. PMID- 3016098 TI - A DNA restriction fragment length polymorphism in the complement region of the human MHC shows an absolute correlation with polymorphism of complement factor B(Bf) defined by isoelectric focusing. AB - The gene coding for properdin factor Bf is located in the human major histocompatibility complex and is closely linked to the genes coding for the complement components C2 and C4. Recently, by Southern blotting techniques, a restriction fragment length polymorphism was identified using the endonuclease Taq I, which subdivides haplotypes carrying the F allele of factor Bf. The F allotype has also been subdivided at the protein level by isoelectric focusing into two subtypes Fa and Fb. We have investigated the DNA of 41 healthy unrelated individuals with known BfF subtypes using the 2.3 kb factor Bf cDNA probe to determine if there is any correlation between the Taq I polymorphism and F subtype. We have found that in 23 individuals who carried the Fb subtype a 6.6 kb Taq I fragment was present. The remaining 18 individuals carried the Fa subtype and showed only the 4.5 kb Taq I fragment on Southern blotting (P = 10(-12). This striking correlation (r = 1) between the Fb protein and DNA polymorphism is surprising especially as the 4.5 kb and 6.6 kb Taq I fragments overlap the Bf and C2 genes and the polymorphic Taq I site is located within the C2 gene. PMID- 3016099 TI - Human parvovirus and thalassaemia. AB - The human parvovirus (HPV) is responsible for aplastic crises in patients with chronic haemolytic anaemia. We describe the cases of four children with aplastic crises in various types of thalassaemia (alpha and beta thalassaemias, major and intermediate forms). In all four patients, specific anti-human parvovirus IgM was detected in their serum, thereby indicating recent infection. PMID- 3016100 TI - Concurrent cytomegalovirus and Coxsackie B virus infections in a heart-lung transplant recipient. AB - A 35-year-old woman with fibrosing alveolitis was given a combined heart and lung transplant at Papworth Hospital, Cambridge. Although her immediate post-operative course was satisfactory, she subsequently experienced concurrent primary cytomegalovirus and Coxsackie B virus infections associated with significant morbidity including pancreatitis. She made a full recovery and has remained well for more than 2 years after transplantation. PMID- 3016101 TI - Progressive nodular fibrosis of the skin: altered procollagen and collagenase expression by cultured fibroblasts. AB - A 33-year-old man presented with a spontaneous progressive cutaneous tumor-like fibrosis involving the right leg and buttock. Histologically the deep dermis was composed of numerous fibroblasts and dense bands of collagen, suggesting that the lesion might be related to an abnormality in collagen metabolism. Fibroblast cultures were established from the affected and normal-appearing skin. The growth rate of the lesional cells was essentially equal to that of control cells. The synthesis of procollagen was approximately 3.5-fold increased in the cells derived from the nodules when compared with control fibroblasts (p less than 0.001). The increase in procollagen synthesis was reflected by an approximate 6 fold increase in both type I and type III procollagen mRNA abundance in the lesional fibroblasts (p less than 0.001), thus suggesting an aberration in the pretranslational level of procollagen gene expression. In contrast, the synthesis of collagenase, the enzyme required for the initiation of collagen degradation, was decreased to approximately 25% of control values (p less than 0.0025), although the enzyme was catalytically normal. The data indicate that these cells are characterized by an increased synthesis of procollagen and decreased synthesis of collagenase, 2 phenotypic characteristics that could account pathophysiologically for the lesions. The unusual reciprocal nature of these biochemical parameters in 2 proteins important in connective tissue homeostasis suggests that this progressive tumor-like condition may have resulted from the expansion of a clonal population of cells. PMID- 3016103 TI - Butcher's wart virus (HPV 7) infections in non-butchers. AB - The analysis of a total of 654 benign and malignant lesions of the skin, genitalia, lungs and bronchi, intestine, kidneys, bladder, mammae, and of the head and neck region, resulted in the identification of human papilloma virus 7 (HPV 7) infections in 3 individuals. One of these was an ordinary "butcher's wart," whereas the other 2 patients have never been involved in meat-handling or farming. One of the latter revealed extensive verrucosis of hands, feet, axilla, neck, and face, persisting for about 27 years, with new lesions arising in the neck region. Particularly the new lesions showed filiform morphology. The second patient has, for a period of more than 2 years, been showing filiform papillomas in the face which recurred after surgical removal. These 2 patients appear to represent the first cases of HPV 7 infections in non-butchers or non meathandlers. PMID- 3016102 TI - An investigation of the ability of antipsoriatic drugs to inhibit calmodulin activity: a possible mode of action of dithranol (anthralin). AB - Epidermal calmodulin (CaM) has been reported to be elevated in psoriasis and to decrease following clearance of psoriasis with treatment. We set out to investigate whether any of the principle drugs used in the treatment of psoriasis had inherent CaM antagonist activity. Utilizing a CaM-activated phosphodiesterase we have demonstrated that even at very high concentrations, the systemic drugs etretinate, methotrexate, and 8-methoxypsoralen, and the topical agents hydrocortisone and crude coal tar showed minimal CaM inhibitory activity. Dithranol (anthralin), however, whether freshly prepared or oxidized, produced substantial inhibition of CaM activity and was demonstrated to be a potent competitive antagonist of CaM, suggesting another possible therapeutic mode of action of dithranol in psoriasis. PMID- 3016104 TI - Quantitative evaluations of the effect of UV irradiation on the infectivity of HTLV-III (AIDS virus) with HTLV-I-carrying cell line, MT-4. AB - The effect of UV irradiation on HTLV-III was quantitatively studied to evaluate the dosage of UV irradiation which inactivates the virus for sterilization of blood products and for laboratory decontamination. In order to estimate the biologic activity and quantitation of the virus, induction of HTLV-III-specific antigens and inhibition of DNA synthesis in MT-4 cells infected by UV-irradiated HTLV-III were detected by indirect immunofluorescence technique and proliferation assay using [3H]thymidine uptake, respectively. Furthermore, plaque-forming assay was performed to count the infectious viral particles. Results showed that HTLV III was completely inactivated by 5000 J/m2 UV irradiation. Cloned UV-irradiated HTLV-III (UV-1) was obtained from a plaque that was formed by 2000 J/m2 UV irradiated virus. When MT-4 cells were infected by the clone UV-1, ballooning degeneration of cells was predominantly induced. These ballooning cells were not usually observed in MT-4 cells infected by unirradiated HTLV-III. The resistance to UV was not different between clone UV-1 and unirradiated HTLV-III. PMID- 3016105 TI - Characterization of insulin-like growth factor-I/somatomedin-C receptors on human keratinocyte monolayers. AB - A membrane receptor for insulin on cultured human keratinocyte monolayers has recently been reported. It has also been established by previous investigators that the receptors for insulin and the somatomedin peptide, insulin-like growth factor-I/somatomedin-C (IGF-I), have similar oligomeric composition. However, these 2 receptors can be distinguished by their high affinity for their respective peptides. The present study was undertaken to identify and characterize IGF-I receptors on human keratinocytes, using time courses at different temperatures, competitive displacement of [125I]IGF-I by unlabeled IGF I and insulin, and comparative binding of IGF-I, IGF-II, and insulin. The binding of [125I]IGF-I was time and temperature dependent, achieving steady state after 6 h of incubation at 15 degrees C. Specific binding at 15 degrees C averaged 4.33% for 0.8-1.0 million cells. Competition for binding was observed at IGF-I concentrations as low as 1 ng/ml, with half maximal displacement of [125I]IGF-I at IGF-I concentration of 17 ng/ml. Insulin, on the other hand, was 100-fold less potent than IGF-I in displacing [125I]IGF-I. Scatchard analysis of the competitive binding data revealed a curvilinear plot, with a calculated Kd = 1.4 X 10(-9) mol/liter. When the binding of [125I]IGF-I, -IGF-II, and -insulin was compared in the same experiment, the specific binding of [125I]IGF-I was 3.43% as compared with 5.60% for [125I]insulin and 0.90% for [125I]IGF-II. The finding of specific IGF-I receptors on cultured human keratinocytes suggests a possible role for this mitogenic peptide in epidermal cell proliferation. PMID- 3016106 TI - Gelatinase expression in generalized epidermolysis bullosa simplex fibroblasts. AB - The use of gelatinase expression in dermal fibroblast cultures as a marker for generalized epidermolysis bullosa simplex (D-EBS-Kobner) has been tested. None of the 6 Kobner patients tested (from 3 families) produced reduced amounts of gelatinase compared with their healthy relatives and other control groups. This shows that a reduced production of gelatinase from dermal fibroblasts is not uniformly a marker for D-EBS-K. PMID- 3016107 TI - Monocyte localization of elevated cAMP phosphodiesterase activity in atopic dermatitis. AB - Patients with atopic dermatitis (AD) manifest a number of immune abnormalities which correlate with in vitro defects including lymphocyte transformation, chemotaxis, and cytotoxicity. Past studies have shown reduced leukocyte cyclic 3',5'-adenosine monophosphate (cAMP) levels after exposure to adenylate cyclase active agonists, and we have demonstrated that this results from increased catabolism due to elevated cAMP-phosphodiesterase activity. These results were obtained in preparations containing mixtures of lymphocytes and monocytes. In order to determine more precisely the cellular site of the defect we have separated the leukocytes into lymphocyte- and monocyte-enriched preparations using either Percoll-gradient centrifugation or adherence isolation. Both techniques yielded over 93% pure lymphocytes, whereas the former yielded 64% monocytes compared with the latter method which generated 94% pure monocytes. Atopic monocytes, obtained by either technique, consistently showed elevated phosphodiesterase activity compared with those of the nonatopic monocytes. Such differences were not evident in lymphocyte preparations from normal and atopic subjects. In spite of the increased rate of cAMP degradation in atopic leukocytes, the resting cAMP levels do not differ from those of normal subjects. We questioned whether this is caused by increased cAMP synthesis and evaluated cellular adenylate cyclase activity. We found no evidence in AD cells for an increased rate of adenylate cyclase catalysis, either basal activity or after stimulation by forskolin. Therefore, the resting cAMP levels must have been compensated by other mechanisms. Impaired cyclic nucleotide metabolism in atopic monocytes may affect a number of immunologic and inflammatory reactions and could account for many of the clinical abnormalities in atopic diseases. PMID- 3016108 TI - Cytokine from basal cell carcinomas stimulates collagenase production. PMID- 3016110 TI - Early immune response in healthy and immunocompromised subjects with primary varicella-zoster virus infection. AB - Events in pathogenesis and immunity during primary varicella-zoster virus (VZV) infection were examined in 64 healthy subjects and 21 immunocompromised patients. Activation of the interferon system and activation of circulating T lymphocytes were early immune responses that occurred during the incubation period in some healthy subjects. Elevated levels of 2-5A synthetase in peripheral blood mononuclear cells and detection of serum alpha interferon (IFN-alpha) and gamma interferon (IFN-gamma) were present in the majority of healthy subjects who had acute primary VZV infection. Expression of HLA-DR antigen occurred on circulating T lymphocytes from subjects with acute VZV infection. The early production of VZV specific IgG or IgM antibodies did not correlate with the severity of the clinical infection, but the detection of T lymphocyte proliferation to VZV antigen within three days after the appearance of the varicella exanthem was associated with milder illness. The mean VZV-specific lymphocyte transformation for subjects with less than 100 lesions/m2 was 7.5 +/- 10.43 SD compared with 1.4 +/- 1.85 SD for those with greater than 400 lesions/m2 (P less than .05). Only one (7.7%) of 13 immunocompromised patients had early VZV-specific lymphocyte transformation compared with 19 (42%) of 45 healthy subjects (P less than .05). The rapid host response to primary VZV infection was associated with rapid termination of viremia in healthy subjects; VZV was isolated from only 11% of peripheral blood mononuclear cell samples cultured within 48 hr after the appearance of the exanthem. PMID- 3016109 TI - The role of parvovirus B19 in aplastic crisis and erythema infectiosum (fifth disease). AB - In 1984, simultaneous outbreaks of aplastic crisis and erythema infectiosum occurred in northeastern Ohio. Sera were analyzed from 26 patients with aplastic crisis: 24 had IgM specific for parvovirus B19, five had B19-like particles by electron microscopy, and 13 had DNA from B19; no sera from 33 controls had evidence of recent infection with B19 (P less than .0001). DNA from B19 was also detected in specimens of throat gargle and urine from two patients with aplastic crisis. Sera from 36 of 51 children with erythema infectiosum had B19-specific IgM, compared with serum from one of 42 susceptible controls (P less than .0001). DNA from B19 was detected in sera from only two of 51 patients who had erythema infectiosum. The secondary attack rates among susceptible contacts decreased with age (overall total, 49.6%). Differential rates of asymptomatic infection were observed among black (68.8%) and white (20.0%) household members (P = .003). These were the first identified simultaneous outbreaks of aplastic crisis and erythema infectiosum. Their occurrence provided an opportunity to study the epidemiology and spectrum of B19 infection with geographically and temporally matched comparison groups; our results support the hypothesis that infection with parvovirus causes these two clinical entities. PMID- 3016111 TI - Prevention of experimental coronavirus colds with intranasal alpha-2b interferon. AB - Fifty-five volunteers treated with either intranasal recombinant interferon (rIFN; 2 X 10(6) IU/day) or placebo for 15 days were exposed to coronavirus by direct intranasal inoculation on the eighth day of treatment. Symptom scores were recorded, and cultures of virus were taken daily for all volunteers for seven days after inoculation. Nineteen (73%) of the 26 placebo recipients met symptom score criteria for a cold, compared with 12 (41%) of the IFN recipients (P = .02). The mean nasal symptom scores in the placebo and IFN groups were 9.2 and 5.4, respectively (P = .03), and the mean total symptom scores in the two groups were 23.2 and 9.4, respectively (P = .003). The mean number of days with a total symptom score greater than 4 was 1.6 in the placebo recipients and 0.5 in the rIFN recipients (P = .02). Prophylactic intranasal rIFN effectively shortened the duration and reduced the severity of coronavirus cold symptoms. PMID- 3016112 TI - Antigenic characterization and ELISA detection of adult diarrhea rotaviruses. AB - Recently, severe epidemics of diarrhea among both adults and children in China have been associated with an agent called adult diarrhea rotavirus (ADRV). We have studied ADRV from two areas of China (Jinzhou and Guangxi) and compared them with prototype group A, B, and C rotaviruses. The viral genomes were compared by electrophoresis of the RNA genome segments in polyacrylamide gels, and the antigenic relatedness of these viruses was examined by immune electron microscopic studies using virus preparations of either double- or single-capsid particles. Hyperimmune antisera (guinea pig and rabbit) to one strain of ADRV reacted with high titers with other strains of ADRV and with single-shelled capsids of a porcine group B rotavirus. The antisera to ADRV did not react with either group A or group C rotaviruses. Antisera to either bovine or porcine group B rotaviruses reacted with ADRV but not with the group A or group C viruses. Antisera to group A or group C virus reacted only with their respective homologous virus. These results and those of immunofluorescence studies place the human ADRV rotaviruses from China among the group B rotaviruses. We also report the development of an ELISA to detect ADRV; this ELISA should be useful for defining the epidemiology of these recently described rotaviruses. PMID- 3016113 TI - Release of 5'-guanosine monophosphate and adenine by Brucella abortus and their role in the intracellular survival of the bacteria. AB - Brucella abortus releases two components (fractions 3b and 10) that inhibit the myeloperoxidase-H2O2-halide antibacterial system of bovine neutrophils by inhibiting degranulation. Fractions 3b and 10 were analyzed by high-performance liquid chromatography and thin-layer chromotography and compared with nucleotide and base standards. These investigations indicated that fraction 3b coeluted and comigrated with 5'-guanosine monophosphate (GMP), whereas fraction 10 coeluted and comigrated with adenine. We determined the biologic effects of GMP, adenine, and B. abortus fractions 3b and 10 on neutrophil functions. Iodination activity of neutrophils was inhibited to approximately 65% of control by GMP (0.04 mg/ml) and by fraction 3b (0.04 mg/ml). Iodination was also suppressed to approximately 80% of control by adenine (0.04 mg/ml) and by fraction 10 (0.04 mg/ml). These results suggest that B. abortus produces GMP and adenine and that these substances contribute to the intracellular survival of the bacteria. PMID- 3016114 TI - Macrophage maturity and modulation of response to Junin virus in infected rats. AB - Replication of Junin virus in peritoneal macrophages from newborn rats was greater for prototype strain XJ than for strain XJC13, whereas in cells from adult animals viral multiplication proved minimal. Transfer of peritoneal adherent cells from normal adult to strain XJ-infected newborn rats lowered mortality significantly. Silica blockade of macrophages protected two-day-old strain XJ-infected animals and depressed brain titers of virus significantly, whereas treatment had no effect on strain XJC13-infected rats. Macrophages from infected two-day-old rats caused illness and death in recipient animals. Silica blockade rendered macrophage-mature 11-day-old rats partially susceptible to infection with Junin virus. Thus pathogenicity of strain XJ in the two-day-old rat by the intraperitoneal route depends on the ability to replicate in peritoneal macrophages, whereas strain XJC13 is nonlethal because it fails to multiply efficiently in these cells. Macrophage maturity seems essential to inhibit viral replication and thus confer protection on the adult host. PMID- 3016115 TI - Isolation of cytomegalovirus from toys and hands in a day care center. PMID- 3016116 TI - Transmission of cytomegalovirus from husband to wife. PMID- 3016117 TI - Antigen detection in the diagnosis of Norwalk virus gastroenteritis. PMID- 3016118 TI - [Theoretical analysis of spread of the infection with Japanese encephalitis virus. III. Forecasting of Japanese encephalitis epidemic]. PMID- 3016119 TI - Immediate palatal reconstruction following surgery for tumours of the intra-oral accessory salivary glands. PMID- 3016120 TI - [Two cases of primary HCG-secreting lung cancer]. PMID- 3016122 TI - [A comparative study of the peri-focal stromal reaction in endometrial carcinoma and internal endometriosis]. PMID- 3016121 TI - [Pregnancy after chemotherapy of invasive moles]. PMID- 3016123 TI - [Anti-tumor effect of transferrin-neocarzinostatin conjugate which is taken up by cells with receptor medicated endocytosis]. PMID- 3016124 TI - [New approach in chemoembolization of hepatocellular carcinoma--lipiodol, cisplatin sandwich therapy]. PMID- 3016126 TI - [A clinical study on alveolar ridge augmentation using calcium phosphate hydroxylapatite]. PMID- 3016125 TI - [Studies on collagenase activity in patients with rheumatoid arthritis- relationship of immune complex, IgG-rheumatoid factor and prostaglandin E2 level]. PMID- 3016127 TI - Successful treatment of murine cytomegalovirus disease does not prevent latent virus infection. AB - 9-(1,3-Dihydroxy-2-propoxymethyl)guanine (DHPG), a nucleoside analogue, inhibits the replication of human and murine cytomegalovirus (MCMV) in cell culture. We studied the effects of treatment with DHPG on acute MCMV infection in mice and assessed the impact of drug therapy on the eventual development of latent viral infection. In virus-susceptible Balb/c mice, DHPG treatment limited dissemination of virus infection and prevented death. In sublethal infection of both Balb/c and virus-resistant C3H/St mice, DHPG prevented recovery of infectious virus from visceral organs, including the spleen. Despite these effects of drug treatment on virus replication during acute infection, latent MCMV could be reactivated in vivo by immunosuppression and in vitro by spleen explantation in virtually all mice. These results indicate that successful treatment of MCMV infection and marked suppression of viral replication do not prevent establishment of viral latency. PMID- 3016128 TI - Platelet prostacyclin binding in smokers. AB - Binding capacity (Bmax) and dissociation constant (KD) of platelet prostacyclin (PGI2) receptors in 23 male smokers and 14 nonsmokers have been determined by direct binding studies to investigate whether platelet prostacyclin binding is altered in smokers. In addition, the inhibitory effect of PGI2 (IC50) on adenosine diphosphate (ADP)-induced platelet aggregation was measured. As confirmed by discriminant analysis, 69% of the smokers had significantly different Bmax and KD. Binding capacity was increased in 12 smokers (Bmax + 74%), with a concomitant decrease in affinity (KD + 94%). In these volunteers, the postreceptor responses (PGI2-induced cyclic adenosine monophosphate [AMP] accumulation in platelet-rich plasma as well as IC50) did not differ from those of controls. In contrast, four smokers with considerably reduced PGI2 binding capacity (Bmax - 65%) exhibited a lower antiaggregatory effect (IC50 + 74%), although the affinity was slightly increased (KD - 61%). Nicotine, L-epinephrine, and prostaglandin E2 did not significantly compete with the binding of 9-3H-PGI2 sodium salt. The antiaggregatory effect of PGI2 on ADP-induced platelet aggregation, however, was inhibited by L-epinephrine (Ki 26 nmol/L) and prostaglandin E2 (Ki 230 nmol/L). Our data suggest that with respect to platelet PGI2 binding and in vitro responsiveness to PGI2, smokers are a heterogeneous population. Although increased binding capacity was observed in 50% of the smokers investigated, our data provide no evidence that a biologically relevant upregulation, for example with concomitant enhanced postreceptor reaction, occurs in smokers. PMID- 3016129 TI - Characterization of lipoproteins produced by the human liver cell line, Hep G2, under defined conditions. AB - Confluent monolayers of the human hepatoblastoma-derived cell line, Hep G2, were incubated in serum-free medium. Conditioned medium was ultracentrifugally separated into d less than 1.063 g/ml and d 1.063-1.20 g/ml fractions since very little VLDL was observed. The d less than 1.063 g/ml fraction was examined by electron microscopy; it contained particles of 24.5 +/- 2.3 nm diameter, similar in size to plasma LDL; a similar size was demonstrated by nondenaturing gradient gel electrophoresis. These particles possessed apoB-100 only. The d less than 1.063 g/ml fraction had a lipid composition unlike that of plasma LDL; unesterified cholesterol was elevated, there was relatively little cholesteryl ester, and triglyceride was the major core lipid. The d 1.063-1.20 g/ml fraction was heterogeneous in size and morphology. Electron microscopy revealed discoidal particles (14.9 +/- 3.2 nm long axis and 4.5 +/- 0.2 nm short axis) as well as small spherical ones (7.6 +/- 1.4 nm diameter). Nondenaturing gradient gel electrophoresis consistently showed the presence of peaks at 13.4 11.9, 9.7, and 7.4 nm. The latter peak was conspicuous and probably corresponded to the small spherical structures seen by electron microscopy. Unlike plasma HDL, Hep G2 d 1.063-1.20 g/ml lipoproteins contained little or no stainable material in the (HDL3a)gge region by gradient gel electrophoresis. Hep G2 d 1.063-1.20 g/ml lipoproteins differed significantly in composition from their plasma counterparts; unesterified cholesterol and phospholipid were elevated and the mole ratio of unesterified cholesterol to phospholipid was 0.8. Cholesteryl ester content was extremely low. ApoA-I was the major apolipoprotein, while apoE was the next most abundant protein; small quantities of apoA-II and apoCs were also present. Immunoblot analysis of the d 1.063-1.20 g/ml fraction after gradient gel electrophoresis showed that apoE was localized in the larger pore region of the gel (apparent diameter greater than 12.2 nm); the apoA-I distribution in this fraction was very broad (7.1-12.2 nm), and included a distinct band at 7.4 nm. Immunoblotting after gradient gel electrophoresis of concentrated medium revealed that a significant fraction of apoA-I in the uncentrifuged medium was in a lipid poor or lipid-free form. This cell line may be a useful model for investigating the metabolism of newly formed HDL. PMID- 3016130 TI - Inhibition of lysosomal protein degradation inhibits the basal degradation of 3 hydroxy-3-methylglutaryl coenzyme A reductase. AB - The effect of inhibiting lysosomal protein degradation on the activity of 3 hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was determined using a mouse mammary cell line (TS-85) which expresses a temperature-sensitive mutation in the ubiquitin degradative pathway. Incubating cells for 18 hr in medium containing 20 mM NH4Cl did not alter total protein synthesis or cell growth, but it did inhibit the rate of total protein degradation by 19%, which is consistent with the known inhibitory effect of NH4Cl on lysosomal protein degradation. NH4Cl treatment also resulted in an increase (81% +/- 20) in HMG-CoA reductase activity. The increase in reductase activity was not correlated with changes in the phosphorylation state of the enzyme or with alteration in the relative rate of reductase synthesis. However, the basal degradation rate of the reductase was significantly inhibited, and after NH4Cl treatment, the half-life of the enzyme increased from 4.0 +/- 0.4 hr to 8.3 +/- 0.8 hr. The change in the rate of reductase degradation can account completely for the increase in reductase activity observed in NH4Cl-treated cells. The accelerated degradation of HMG-CoA reductase induced by 25-hydroxycholesterol treatment was not affected by either NH4Cl or by inactivation of the ubiquitin degradative pathway. Therefore, two different mechanisms may be responsible for the accelerated degradation and basal degradation of HMG-CoA reductase. The latter can be inhibited by NH4Cl and may imply that under basal conditions the enzyme may be degraded in lysosomes. PMID- 3016131 TI - Effect of epinephrine and other lipolytic agents on intracellular lipolysis and lipoprotein lipase activity in 3T3-L1 adipocytes. AB - 3T3-L1 adipocytes were used to test the hypothesis that hormone-sensitive lipolysis and lipoprotein lipase activity might be regulated in a reciprocal manner. Intracellular lipolysis was stimulated by catecholamine, dibutyryl cAMP, and ACTH, but not by glucagon. The effects of epinephrine on lipolysis were blocked by the beta-antagonist propanolol but not by the alpha-antagonist phentolamine. Hormone-stimulated lipolysis was not changed by acute (45 min) or chronic (2 days) treatment of the cells with insulin whereas the latter treatment augmented lipoprotein lipase activity about fivefold. Epinephrine did not affect the lipoprotein lipase activity of insulin-stimulated cells. Withdrawal of glucose from the medium decreased lipoprotein lipase activity and the effect of epinephrine on lipolysis. Effects of lipolytic agents on activity of lipoprotein lipase were variable and concentration-dependent. Lipoprotein lipase activity was decreased only by concentrations of epinephrine greater than those inducing maximal intracellular lipolysis, and the decrease in activity occurred about 30 min after the increase in glycerol release. There seems to be no relationship between the level of activity of lipoprotein lipase and the maximal rate of hormone-stimulated lipolysis in 3T3-L1 cells. Unlike in adipose tissue and adipocytes of rats, hormone-stimulated lipolysis and lipoprotein lipase activity in murine 3T3-L1 adipocytes appear to be regulated independently. PMID- 3016132 TI - Cholesterol metabolism is altered by hydrolytic metabolites of prostacyclin in arterial smooth muscle cells. AB - Cholesteryl esters are the major lipids that accumulate in arteries during atherogenesis. The mechanisms responsible for this lipid accretion have not been completely defined. Our previous experiments have shown that prostacyclin (PGI2) enhances cholesteryl ester catabolism by increasing cyclic AMP in cultured arterial smooth muscle cells. However, PGI2 is rapidly degraded under physiologic conditions and endogenous levels of PGI2 in the human circulation are extremely low. These findings suggest that it is not a circulating hormone. We tested the hypothesis that stable PGI2 metabolites alter cholesteryl ester metabolism and cellular lipid accumulation. Ten to 100 nM dinor-6-keto PGF1 alpha, 13,14-dihydro 6,15-diketo PGF1 alpha, and 6,15-diketo PGF1 alpha increased cyclic AMP levels significantly two- to threefold with a concomitant enhancement of both lysosomal and cytoplasmic cholesteryl ester hydrolytic activities. Cholesteryl ester synthesis was unchanged by the PGI2 metabolites. When cyclic AMP concentrations were maintained at basal levels by an adenylate cyclase inhibitor, no effect on cholesteryl ester hydrolysis was observed following addition of PGI2 metabolites to the cells. Furthermore, addition of PGI2 metabolites during a 1-week culture period reduced free and esterified cholesterol by 50%. These data suggest that PGI2 metabolites: 1) decrease intracellular cholesterol accumulation by increasing cholesteryl ester catabolism; 2) act via enhancement of cyclic AMP; and, 3) may represent circulating regulators of arterial cholesteryl ester metabolism. PMID- 3016133 TI - Effect of epidermal growth factor on the membrane potential of cultured porcine thyroid cells. AB - Cultured porcine thyroid cells maintained in media containing TSH exhibited a membrane potential of -50 mV, and hyperpolarized by about 10 mV within 1 h of the addition of epidermal growth factor (EGF; 10 ng/ml). Follicle cells had depolarized to -45 mV after 4 h of exposure to EGF. Cells maintained in dibutyryl cyclic AMP (dbcAMP) did not alter their membrane potential when exposed to EGF for up to 4 h. Cultures washed to remove the TSH or dbcAMP hyperpolarized to -75 mV within 30 min, and a reversible depolarization to -60 mV was observed on addition of EGF. It was concluded that EGF acts as a physiological antagonist of TSH and also exerts a separate depolarizing influence on cultured thyroid cells. PMID- 3016134 TI - Dopamine effects on adrenocorticotrophin-stimulated aldosterone, cortisol, corticosterone and 11-deoxycorticosteroid concentrations in sodium-replete and sodium-deplete man. AB - The effect of dopamine (1 microgram/kg per min) on corticosteroid response to ACTH (0.1, 1 and 10 ng/kg per min) was compared with that of a placebo in sodium replete (150 mmol/day) and -deplete (10 mmol/day) normal man. Dopamine had no effect on aldosterone, cortisol or corticosterone responses in either dietary phase, but increased deoxycorticosterone (897.0 +/- 126.4 (S.E.M.) vs 590.0 +/- 84.3 pmol/l, normal Na+; 1264.2 +/- 84.3 vs 764.5 +/- 84.3 pmol/l, low Na+) and deoxycortisol (6.033 +/- 0.583 vs 5.048 +/- 0.680 nmol/l, normal Na+; 5.112 +/- 0.600 vs 4.130 +/- 0.367 nmol/l, low Na+) levels during ACTH administration (all P less than 0.01). Deoxycorticosterone and corticosterone responses to ACTH were greater during sodium depletion than repletion (both P less than 0.01). Dopamine therefore increased 11-deoxycorticosteroid concentrations during ACTH-stimulated steroidogenesis. This may reflect action of dopamine to increase extra-adrenal formation of 11-deoxycorticosteroids. PMID- 3016135 TI - Purification of dispersed rat adrenal zona glomerulosa cells by Percoll density gradient centrifugation and the isolation of a population of cells highly responsive to adrenocorticotrophin. AB - Percoll density gradient centrifugation is a simple, inexpensive and convenient method to eliminate contaminating zona fasciculata (ZF) cells from unpurified rat adrenal capsular glomerulosa (ZG) cell preparations (with less than 0.1% ZF cells in the final cell preparation). Basal steroid (aldosterone and corticosterone) output by the purified (PG) cells was unchanged. These purified cells, although free from ZF contamination, were more highly responsive than expected to ACTH (3 nmol/l). When PG cells were further separated by Sephadex column filtration, the filtered PG cells exhibited the steroidogenic response of ZG cells purified by unit gravity sedimentation and Sephadex column filtration, i.e. reduced basal steroid output and an ACTH response reduced to that stimulated by K+ (8.4 mmol/l). Although the cells retained in the column resembled the filtered PG cells ultrastructurally, they showed unchanged basal steroid output and a high ACTH response with increased late-pathway activity (the conversion of corticosterone to aldosterone). By combining Percoll density gradient centrifugation and Sephadex column filtration we have a method for the isolation and study of both the high- and low-response rat ZG cells which are free from ZF contamination. PMID- 3016136 TI - A modulatory role for cyclic AMP in the control of thyrotrophin release: studies with forskolin and dibutyryl cyclic AMP. AB - We have studied the effects of cyclic AMP (cAMP) on TSH secretion by cultured rat pituitary cells, using forskolin and dibutyryl cAMP (dbcAMP) to raise the cellular cAMP content by different mechanisms. Forskolin (10 mumol/l), a stimulator of adenylate cyclase, raised the cAMP content within 10 min, but had a more delayed effect on TSH release, with no significant stimulation for at least 6 h, but a clear dose-dependent effect at 24 h. Incubation with dbcAMP likewise increased TSH release after 6-24 h. By contrast, high cellular cAMP levels induced by either forskolin or dbcAMP augmented the TSH response to TRH at an early stage, before any detectable change in unstimulated TSH release. Pretreatment of cells with forskolin led to a parallel upward shift in the subsequent TRH dose-response curve, without a significant change in median effective dose or any change in cellular TSH content. These findings suggest that cAMP acts to increase the availability of TSH for acute release by TRH by modulation of an intracellular releasable hormone pool, and indicate synergistic interactions between the adenylate cyclase system and the phospholipid-calcium stimulus-release coupling mechanism of TRH. PMID- 3016137 TI - Effect of adrenocorticotrophin on cortisol and androstenedione secretion from dispersed cells of guinea-pig adrenal zonae fasciculata and reticularis. AB - We have studied cortisol and androstenedione secretion by dispersed cells of the outer zona fasciculata (ZF) plus zona glomerulosa, and the inner zona reticularis (ZR) plus medulla of the guinea-pig adrenal. The ZF and ZR were microdissected apart, the cells dispersed and incubated (200 000 cells/ml) for 90 min in the presence of adrenocorticotrophin (ACTH; 500 ng/l), dibutyryl cyclic AMP (dbcAMP; 1 mmol/l), pregnenolone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11 deoxycortisol and 21-deoxycortisol. The steroid concentrations were 5-25 mumol/l. Cortisol secretion was assayed by radioimmunoassay. There was no detectable cortisol secretion (less than 50 nmol/l) from the ZR in the controls (no additive) or after dbcAMP stimulation. Adrenocorticotrophin-stimulated cortisol secretion was also low (range less than 50-340 nmol/l). In contrast the ZF secreted 177-379 (control), 828-2052 (dbcAMP) and 2863-9735 (ACTH) nmol cortisol/l. There was no detectable (i.e. less than 2 nmol/l) cAMP production by ZR or ZF either basally (no ACTH) or after ACTH stimulation (500 ng/l). Challenge of the ZR cells with each cortisol precursor steroid (5 mumol/l) increased (P less than 0.05) cortisol secretion over that seen with the corresponding basal and ACTH-stimulated controls. Thus pregnenolone, 17-hydroxypregnenolone, 17 hydroxyprogesterone, 11-deoxycortisol and 21-deoxycortisol (converted directly to cortisol by 21-hydroxylase) gave rise to (mean +/- S.D., n = 4) 406 +/- 86, 680 +/- 180, 1307 +/- 111, 1141 +/- 234 and 3160 +/- 419 nmol cortisol/l respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016139 TI - Field evaluation of a pregnancy immunoassay for the detection of oestrone sulphate in goats. AB - The concentration of oestrone sulphate in whey obtained from 66 pregnant dairy goats was measured by direct radioimmunoassay. The mean time at which pregnancy was first detected was day 41 of gestation. Levels remained low (37 pmol/l-0.96 nmol/l, mean = 167 pmol/l) until week 5 of gestation when they rose rapidly. The test had an accuracy of 95.6%, was able to distinguish true from pseudopregnancy, and suggested that pregnancy in females carrying multiple fetuses could be detected earlier, possibly as a result of the fetal-placental origin of oestrone sulphate. PMID- 3016138 TI - Ontogeny of prolactin receptors in rat decidual tissue: binding by a locally produced prolactin-like hormone. AB - The objectives of this investigation were to determine whether decidual tissue possesses specific binding sites for prolactin, and to examine whether the locally produced prolactin-like hormone binds to these receptors. Characterization of the binding of prolactin to decidual tissue from rats at day 9 of pseudopregnancy revealed specific, high-affinity sites. Binding approached saturation with increasing concentrations of either the ligand or the protein. Cytosolic extracts of day-9 decidual tissue, containing various amounts of decidual luteotrophin which possesses several of the physiological and biochemical characteristics of prolactin, displaced the binding of 125I-labelled prolactin to decidual membranes in a linear fashion. Prolactin-binding sites were detectable 72 h after induction of decidualization and 48 h after the appearance of decidual luteotrophin in the decidua. Prolactin receptor concentrations increased significantly between days 8 and 9, reached a plateau between days 9 and 12 and declined abruptly on days 14 and 15, accompanied by a similar decline in decidual luteotrophin concentration in the tissue. Thus rat decidual tissue possesses specific receptors for prolactin to which decidual luteotrophin locally produced can bind, thereby suggesting an auto/paracrine role for this substance. PMID- 3016140 TI - Steroid regulation of Na+/K+-ATPase activity in the rat kidney: effect of a new 19-nor-progestagen. AB - The effects of Nomegestrol acetate (17 alpha-acetoxy-6-methyl-19-nor-4,6 pregnadiene-3,20-dione), a new 19-nor-progesterone derivative, on renal Na+/K+ ATPase activity were assessed in normal and adrenalectomized rats, and compared with the stimulatory or inhibitory actions produced by other steroids. This compound displayed an inhibitory effect which was similar to, but smaller than, that induced by progesterone and quite distinct from the stimulation produced by 19-nor-progesterone and corticosteroids. In addition, unlike progesterone, it did not antagonize the effect of aldosterone in adrenalectomized rats. This result, together with previous in-vivo and in-vitro observations on this compound indicates that additional modifications introduced in the molecular structure of 19-nor-progesterone produces a potent progestagenic substance virtually devoid of effects on renal Na+/K+-ATPase activity and sodium loss in urine. PMID- 3016141 TI - Cyclic adenosine nucleotides and growth hormone-releasing factor increase cytosolic growth hormone messenger RNA levels in cultured rat pituitary cells. AB - The cellular mechanisms involved in GH biosynthesis have been investigated by the measurement of steady-state levels of cytosolic GH messenger RNA (mRNA) in primary cultures of rat pituitary cells using an RNA-complementary DNA (cDNA) hybridization assay. Growth hormone mRNA-cDNA hybridization increased in a linear manner with increasing cytosol concentration. Cellular GH mRNA levels rose by an average of 2.4-fold (range, 1.6-3.3; n = five experiments) after exposure to GH releasing factor (GRF(1-40); 10 nmol/l) for 3 days. Treatment with GRF increased the release of GH into the culture medium, and depleted the cellular GH content by 40%. Total GH (in the medium plus cells) after GRF treatment increased by between 1.5- and 3.8-fold, a magnitude similar to the increase in GH mRNA levels. Treatment of cells with dibutyryl adenosine 3':5'-cyclic monophosphate (1 mmol/l) or forskolin (5 mumol/l) increased the levels of cytosolic GH mRNA by between 1.6 and 4.7-fold. These agents increased GH release into the medium, depleted cellular GH content and increased total GH in the system to the same extent as GRF (10 nmol/l). These data demonstrate that cyclic adenosine nucleotides may mediate the GRF induction of GH gene transcription. In addition, we have shown that increases in the levels of cellular GH mRNA are reflected by increased GH biosynthesis, suggesting that the regulation of hormone gene transcription is one cellular site for the control of hormone biosynthesis and, ultimately, hormone available for release. PMID- 3016142 TI - Reduced adrenal cortical sensitivity to ACTH in lambs with cut splanchnic nerves. AB - The possibility that the sensitivity of the adrenal cortex to endogenous ACTH may be affected by splanchnic nerve activity has been investigated in conscious, weaned, 5- to 8-month-old lambs. The animals were atropinized (0.5 mg/kg) and tested with an i.v. infusion of noradrenaline (333 ng/kg per min for 10 min), which produced a significant rise in the mean concentration of both ACTH and cortisol in the arterial plasma. In lambs tested at least 7 days after section of both splanchnic nerves, just below the diaphragm, the rise in plasma ACTH concentration was significantly greater, and that in plasma cortisol significantly less, than in control lambs. The mean plasma ACTH and cortisol concentrations were linearly related to one another in both groups (r = 0.93 and 0.92) but the sensitivity of the adrenal cortex to the steroidogenic action of ACTH appeared to have been roughly halved 1 week after bilateral splanchnic nerve section. PMID- 3016143 TI - Serum enzymes of collagen synthesis and type III procollagen amino-propeptide in Nigerian patients with sickle cell disease. AB - Serum immunoreactive prolyl hydroxylase protein, galactosylhydroxylysyl glucosyltransferase activity and the aminoterminal propeptide of type III procollagen were measured in 20 patients with sickle cell disease and the values were compared with those in 20 apparently healthy Nigerians. The means for the two enzymes and serum aminoterminal propeptide of type III procollagen were significantly higher in the sickle cell disease patients. Significant correlations were found between the values for the two enzymes and the protein serum aminoterminal propeptide of type III procollagen within the sickle cell disease patients. The data confirm that collagen formation is found in the liver, bone and other organs of patients with this disease. The measurement of serum immunoreactive prolyl hydroxylase protein, serum galactosylhydroxylysyl glucosyltransferase activity and serum aminoterminal propeptide of type III procollagen in prospective studies might be helpful in predicting hepatic, bone or diffuse fibrogenesis in sickle cell disease. PMID- 3016144 TI - Glandular kallikrein, renin and tonin in tissues of diabetic and hypertensive rats. AB - The submaxillary gland and kidney of diabetic and hypertensive rats were compared for their content of glandular kallikrein and the activities of tonin and renin. The submaxillary glands and the kidneys of both diabetic Wistar strain and hypertensive rats contained significantly less glandular kallikrein than non diabetic Wistar strain and hypertensive rats (reduction fron 40 to 76%). The renin activity of the kidney showed only a slight change in spite of diabetes, whereas the activity of the submaxillary gland decreased in parallel with the reduction of the kallikrein content when diabetes was induced. On the other hand, the tonin of the submaxillary gland, which has a potent hypertensive activity like renin, was not affected by induction of diabetes. However, the tonin activity in hypertensive rats was significantly higher (p less than 0.001) than that in the normotensive rats (His-Leu, 168.7 +/- 10.1 vs. 131.5 +/- 17.3 nmol/min X mg protein). PMID- 3016145 TI - [Effects of cyclic nucleotides on the polyamines in glioma cells]. PMID- 3016146 TI - Multilocus genotypes determined by enzyme electrophoresis of Neisseria meningitidis isolated from patients with systemic disease and from healthy carriers. AB - Variation in nine enzymes in 152 isolates of Neisseria meningitidis from Norway (118 from blood or cerebrospinal fluid of patients with systemic disease and 34 from the pharynx of healthy carriers) was analysed by starch-gel electrophoresis. All nine enzymes were polymorphic and the number of allozymes (electromorphs) identified per locus ranged from 3 to 12, with a mean of 6.1. Among the 152 isolates, 55 unique combinations of electromorphs (electrophoretic types, ETs) were distinguished. Twenty ETs were represented among the carrier isolates and 37 among the systemic isolates; hence, only two ETs were found in both groups of isolates. ET-5 was identified 67 times among the 118 systemic isolates (58%), indicating an association of this ET with invasiveness; ET-5 was also the most common type among the carrier isolates (18%). Genetic similarity between ETs was analysed by pairwise comparison of all 55 ETs with respect to the number of electromorphs by which they differed. No evidence of a general genetic difference between carrier and case isolates was found. Two well-defined clusters of ETs were observed, each including one of the two most common ETs identified among the systemic isolates (ET-5 and ET-37), together with isolates differing from them only at one or two loci. All isolates of ET-5 and ET-37, as well as their closely related variants defined by the similarity matrix, were resistant to sulphonamide, independent of their antigenic characteristics and isolation site. The extensive allozyme variation among isolates of the same serogroup demonstrated the limited value of serogrouping as an epidemiological tool. All but one isolate of serotype 15:P1.16 were electrophoretically similar, as were all the 2a:P1.2 isolates. The 15:P1.15 isolates, however, were genetically heterogeneous. The distribution of alleles in genotypes identified among the systemic isolates indicated that genetic recombination may occur in natural populations of N. meningitidis. PMID- 3016147 TI - Three new temperate phages of Bacillus subtilis. AB - Three temperate bacteriophages of Bacillus subtilis were isolated from soil samples and analysed, together with all the other known temperate phages of this organism, with respect to their host range, immunity, serology and DNA restriction pattern, and by other tests. The results show that the newly isolated phages are new members of the immunity sub-group I of the group III of B. subtilis temperate bacteriophages. We named these new phages IG1, IG3 and IG4. PMID- 3016148 TI - Synthesis of OmpA protein of Escherichia coli K12 in Bacillus subtilis. AB - We have inserted a C-terminally truncated gene of the major outer membrane protein OmpA of Escherichia coli downstream from the promoter and signal sequence of the secretory alpha-amylase of Bacillus amyloliquefaciens in a secretion vector of Bacillus subtilis. B. subtilis transformed with the hybrid plasmid synthesized a protein that was immunologically identified as OmpA. All the protein was present in the particulate fraction. The size of the protein compared to the peptide synthesized in vitro from the same template indicated that the alpha-amylase derived signal peptide was not removed; this was verified by N terminal amino acid sequence determination. The lack of cleavage suggests that there was little or no translocation of OmpA protein across the cytoplasmic membrane. This is an unexpected difference compared with periplasmic proteins, which were both secreted and processed when fused to the same signal peptide. A requirement of a specific component for the export of outer membrane proteins is suggested. PMID- 3016149 TI - Molecular cloning into Tn5 and integration in the Pseudomonas aeruginosa chromosome: a tool for heterologous gene expression. AB - The DNA primase gene of the promiscuous IncP-1 conjugative plasmid RP1, encoding two polypeptides of 118 and 80 kDa, was inserted into the transposon Tn5 in Escherichia coli. The derivative transposon, Tn2523, was then transposed to a temperature-sensitive replication mutant of the promiscuous IncP-1 conjugative plasmid R68 at permissive temperature and the plasmid transferred to Pseudomonas aeruginosa strain PAO. The latter strain was then grown at non-permissive temperature to identify transposition of Tn2523 into the P. aeruginosa chromosome. Immunological and enzymic analysis showed the expression of functional primase polypeptides in the constructed P. aeruginosa strain. This strain also restored wild-type conjugational transfer proficiency, by complementation, to mutants of the IncP-1 plasmid R18 affected in transfer from P. aeruginosa to P. stutzeri or to Acinetobacter calcoaceticus due to transposon Tn7 insertion mutations in the primase gene. This strategy of cloning into a transposon and integration into the bacterial chromosome should facilitate genetic manipulation and studies of gene expression in a range of Gram-negative bacteria. PMID- 3016151 TI - Rescue of presumptive viral information from human cells by a helper oncovirus. AB - We have attempted to rescue presumptive human endogenous retrovirus(es) by using a competent animal oncovirus as a helper. Human melanoma cells (line HMB2) were fused, using polyethylene glycol, with mouse NIH-3T3 cells which had been infected and transformed by the Harvey murine leukaemia and sarcoma virus complex (MLV and MSV). The heteropolykaryons obtained were co-cultivated with fresh NIH 3T3 cells; filtered (Millipore 0.22 micron) medium from these was used to infect further NIH-3T3 cells. In these cells after several passages, vesicular stomatitis virus (VSV) pseudotypes could be produced. These were infectious not only for mouse cells (manifesting the helper MLV), but also for human cells (HeLa, HEC human embryo fibroblasts, HMB2); they were not infectious for CCL64 (mink) or for Vero (African green monkey) cells. The presence of such VSV pseudotypes infectious for human cells indicated that a human ecotropic virus [provisionally named rescued human virus (RHV)] had been rescued by the fusion of human melanoma cells with MLV-infected mouse cells. This was supported by the following evidence. The human-specific pseudotype was neutralized by sheep antisera raised to antigens selected by VSV from human tumour cell lines HMB2, T47D and HeLa. These antisera also aggregated NIH cells infected with MLV and RHV. Mouse antisera raised to antigens present in HIH cells infected with MLV and RHV, in contrast to sera raised to NIH cells infected with MLV only, immunoprecipitated an 85,000 mol. wt. protein band from human cells (HEC, HMB2 and HeLa) surface-labelled with 125I. PMID- 3016150 TI - Effects of divalent cations and of phospholipase A activity on excretion of cloacin DF13 and lysis of host cells. AB - Induction of cloacin DF13 synthesis in Escherichia coli harbouring plasmid CloDF13 results in the release of cloacin DF13, inhibition of growth and ultimately in lysis of the host cells. Expression of the pCloDF13-encoded protein H is essential for both the release of cloacin DF13 and the lysis of the cells. The divalent cations Mg2+ and Ca2+ interfered with the mitomycin C-induced protein H-dependent lysis, but hardly affected the release of cloacin DF13. Essentially all of the bacteriocin was released from the cells before a detectable degradation of the peptidoglycan occurred, independent of the presence of mitomycin C. Experiments with phospholipase A mutants revealed that activation of detergent-resistant phospholipase A was essential for the export of cloacin DF13 across the outer membrane and the lysis of induced cells. Transport of cloacin DF13 across the cytoplasmic membrane was mainly dependent on protein H. A revised model for the excretion of cloacin DF13 is presented. PMID- 3016152 TI - Cloning and expression of a viral phosphoprotein: structure suggests vesicular stomatitis virus NS may function by mimicking an RNA template. AB - The phosphoprotein (NS) gene from the Indiana serotype of vesicular stomatitis virus (VSV; Mudd-Summers strain) was cloned and sequenced. The NS gene encodes a protein of 265 amino acids which was expressed from a simian virus 40 vector in COS cells. The post-translational modification characteristic of viral NS, the extensive phosphorylation of a cluster of serine and threonine residues, was also evident in recombinant NS protein. The NS gene displays a property common to the phosphoprotein genes of negative-strand RNA viruses: the phosphoprotein mRNA has a second open reading frame (ORF) which could encode a small (7500 mol. wt.) protein. Both measles virus and Sendai virus employ the second ORF of their phosphoprotein gene, and the resultant proteins have an amino acid composition similar to that predicted for the VSV ORF. Comparison of phosphoproteins from different VSV strains revealed two conserved domains that we propose are critical for the function of NS in transcription and replication. PMID- 3016153 TI - Human papillomavirus DNA replication mediated by simian virus 40 T antigen in trans. AB - The putative E1 gene product of papillomaviruses is thought to be involved in the initiation of viral replication, as large-T antigen (T antigen) is in the case of polyomaviruses. Mouse cell lines cloned after transformation by a plasmid consisting of the simian virus 40 (SV40) early region and the complete genome of human papillomavirus type 16 (HPV16) maintained episomal plasmid DNA. In contrast, the DNAs of either SV40 or HPV16, when employed separately in transfection experiments, were consistently integrated into the host DNA. To test the hypothesis that SV40 T antigen might be involved in the replication of the hybrid plasmids, HPV16 DNA was used in a transient replication assay for transfection of either CV-1 or COS-7 cells. The HPV16 DNA replicated to a high copy number in the T antigen-producing COS-7 cells, but failed to replicate in the CV-1 cells. To define the HPV16 sequences that were essential for the plasmid maintenance in SV40 T antigen-producing cells, restriction fragments of HPV16 were analysed for their replication capacity in COS-7 cells. Here it is reported that the presence of the coding region of the putative E1 gene product of HPV16 together with the 5' transcriptional control elements is essential and is sufficient to support plasmid replication mediated by SV40 T antigen in trans. PMID- 3016154 TI - Identification and cloning of the fowlpox virus thymidine kinase gene using vaccinia virus. AB - Using vaccinia virus as a selection and cloning vehicle, a thymidine kinase (TK) gene of fowlpox virus (FPV) has been identified. A plasmid, pF130, containing part of the HindIII-F region of vaccinia virus was used to shotgun clone EcoRI fragments of FPV DNA into TK- vaccinia virus and select for TK+ recombinants. The TK+ recombinant vaccinia virus contained a 5.5 kb EcoRI fragment of FPV. This FPV fragment was cloned into pUC9 and the presence of the TK gene in this fragment was confirmed by its ability to rescue TK+ vaccinia virus from TK- virus, when inserted into pF130. A recombinant vaccinia virus containing this FPV fragment induced TK enzyme activity in the cytoplasm of infected cells. The vaccinia virus RNA polymerase appeared able to recognize the FPV promoter sequences of the FPV TK gene since the fragment operated in the marker rescue, irrespective of its orientation to the vaccinia virus promoter in pF130. Using restriction enzyme analysis, insertion of subfragments of the 5.5 kb FPV fragment into pF130 and marker rescue, we were able to map the position of the TK gene in the 5.5 kb EcoRI fragment. This approach may facilitate identification and cloning of TK genes from other poxviruses. PMID- 3016155 TI - Antibody response, recurrence patterns and subsequent herpes simplex virus type 2 (HSV-2) re-infection following initial HSV-2 infection of guinea-pigs: effects of acyclovir. AB - The production of antibody to specific herpes simplex virus type 2 (HSV-2) polypeptides, the recurrence patterns and the susceptibility to re-infection were studied in the guinea-pig model of genital HSV-2 infection. Further, we defined the effects of acyclovir (ACV) therapy on these parameters of infection. Treatment with ACV reduced the clinical severity of the initial disease but did not affect vaginal viral shedding. Production of neutralizing antibody as well as antibody to the nucleocapsid protein, and glycoproteins B, D and G were all delayed in ACV recipients. ACV treatment of initial infection did not significantly alter the pattern of subsequent recurrence although by several criteria treated animals tended towards decreased recurrences. Re-inoculation with a second strain of HSV-2 resulted in a local cervicovaginal infection but, except for one ACV-treated animal, neural tissue was protected from re-infection. PMID- 3016156 TI - Identification of a varicella-zoster virus origin of DNA replication and its activation by herpes simplex virus type 1 gene products. AB - We have identified and characterized an origin of DNA replication in the genome of the human herpesvirus, varicella-zoster virus (VZV). This origin of replication (VZV ORIS) is located within the major inverted repeats in a position equivalent to that occupied by one of the herpes simplex virus type 1 (HSV-1) replication origins. Products encoded by both VZV and HSV-1 activate cloned copies of VZV ORIs, generating high molecular weight molecules consisting of tandem duplications of the input plasmid. The VZV ORIS region contains a tract of alternating A and T residues located at the centre of symmetry of an almost perfect palindrome of 45 bp, and the use of plasmid deletion mutants has demonstrated that this tract is an important functional element of the origin. Two sequences common to the VZV ORIS region and the regions specifying the two HSV-1 origins (ORIS, located within the TRS/IRS regions, and ORIL, located within the UL region) were identified and these may represent important recognition sites. One is an 11 bp sequence (CGTTCGCACTT), and the other is represented by the tract of alternating A and T residues. VZV does not appear to contain an origin of replication in a position equivalent to that of HSV-1 ORIL. PMID- 3016157 TI - Structure-activity relationships among alpha-(N)-heterocyclic acyl thiosemicarbazones and related compounds as inhibitors of herpes simplex virus type 1-specified ribonucleoside diphosphate reductase. AB - 2-Acetylpyridine thiosemicarbazone, a potent antiviral drug, and 13 analogues were examined as inhibitors of partially purified herpes simplex virus type 1 specified ribonucleoside diphosphate reductase. N4,N4-Azacycloheptane derivatives were more active than their N4-unsubstituted analogues. Selenosemicarbazones were similar in potency to their thiosemicarbazone congeners, whereas a related semicarbazone was much less active. Maximum inhibition was observed when an ethylidene side-chain was present in the compounds. No discernible trend in potency was observed when the pyridine moiety was replaced by quinoline or isoquinoline. Thiosemicarbazide derivatives were less potent than their unsaturated thiosemicarbazone analogues. Inhibitory potencies increased at longer incubation times consistent with the hypothesis that thiosemicarbazones inactivate the enzyme in a time-dependent manner. PMID- 3016158 TI - Biological activities and receptor binding of two human recombinant interferons and their hybrids. AB - Two human recombinant lymphoblastoid interferon-alpha subtypes, LyIFN-B (alpha 8) and LyIFN-D (alpha 1), and 10 hybrids generated therefrom were produced in Escherichia coli and purified. The antiviral and antiproliferative activities and the induction of (2'-5')oligoadenylate synthetase were compared to their receptor binding affinities. The IFN subtypes and their hybrids had similar specific antiviral activities on bovine cells. On human cells both the specific antiviral and antiproliferative activities of LyIFN-B were about 30-fold higher than those of LyIFN-D. This difference in activity could be attributed partly to the N terminal amino acids 1 to 60 and partly to amino acids 61 to 92. A third domain affecting the biological activities was found within the carboxy-proximal segment from amino acids 93 to 150. The differences in these activities were found to correlate with their ability to bind the receptor, suggesting that the differences in activity might be due to altered binding of the IFNs to the cellular receptors. In contrast, the induction of (2'-5')oligoadenylate synthetase did not follow the same activity profile. On mouse cells, the efficiency of the hybrids was affected by at least four sites on the IFN protein. A hybrid with the N-terminal segment 1 to 60 from IFN-B and amino acids 61 to 166 from IFN-D had a specific antiviral activity on mouse cells as high as on human cells corresponding to a 500- and 5000-fold increase in specific activity compared to IFN-D and IFN-B, respectively. We suggest that on mouse cells the IFN activity may be more dependent on conformational differences than on human cells, which in turn might reflect a less precise fit to the mouse receptor than to the human receptor. PMID- 3016159 TI - Regulation of interferon-induced antiviral states: contrasting profiles of decay in response to the removal of MuIFN-alpha, MuIFN-beta and MuIFN-gamma. AB - The stabilities of the antiviral states induced by different types of murine interferons (IFNs) were compared to gain insight into possible differences in the modes of regulation of their respective antiviral states. Both type I (MuIFN alpha and MuIFN-beta) and type II (MuIFN-gamma) IFNs were employed to establish antiviral states against mengovirus in mouse L-929 cells. At various times after IFN treatment, the IFNs were removed and the stability of the antiviral states was determined by single cycle mengovirus yield reduction experiments. The antiviral states induced by the two type I IFNs decayed significantly by 12 h following IFN removal. The rate of this decay was an exponential function of the level of the antiviral state induced. A transcriptional block effectively delayed the decay of the antiviral state, suggesting the involvement of a positive feedback mechanism of regulation. In contrast, the profile of the antiviral state induced by type II IFN showed a significant enhancement upon MuIFN-gamma removal. This enhancement was not dependent upon de novo transcriptional and translational activity of the cells. These data suggest that the modes of regulation of the antiviral states against mengovirus induced by type I and type II IFNs are distinctly different. PMID- 3016160 TI - The structure of the rotavirus inner capsid studied by electron microscopy of chemically disrupted particles. AB - The inner capsid structure of the OSU strain of porcine rotavirus was studied by electron microscopy of freeze-dried preparations and of negatively stained chemically disrupted virus particles. The analysis of the particles by the freeze drying technique revealed a T:13 l (laevo) symmetry for the organization of the inner capsid. Treatment of single-capsid rotavirus particles with 30% formamide or 5 M-urea resulted in their degradation, giving rise to very similar products, corresponding to isolated vertices, edges and faces of the virus icosahedron. An analysis of such structures confirmed the triangulation number and handedness of the rotavirus inner capsid, and provided evidence for the open-mesh model, in which the five- and six-coordinated axes are represented by 'holes' formed by smaller trimeric morphological subunits. PMID- 3016161 TI - Characterization of cells transformed by the human polyomavirus JC virus. AB - One unique feature of the prototype JC virus (JCV) (Mad 1) genome is the occurrence of a second TATA sequence within the early promoter region. A naturally occurring oncogenic variant of JCV (Mad 4) lacks this second TATA box. Several cell lines transformed by Mad 1, Mad 4 and simian virus 40 were characterized, in part to investigate whether the second TATA sequence is functional. S1 nuclease mapping of early JCV gene transcription products revealed a major set of start sites common to both Mad 1 and Mad 4 mRNAs. In addition, a second set of early transcripts was found exclusively in Mad 1 transformants, presumably positioned by the second TATA box. The presence of these unique mRNAs in the Mad 1-transformed cells did not appear to have any bearing on the other parameters investigated, including size and quantity of early viral proteins, integration patterns of viral DNA and growth properties of the cells. PMID- 3016162 TI - Molecular cloning of cDNA from hepatitis A virus strain HM-175 after multiple passages in vivo and in vitro. AB - Hepatitis A virus (HAV) strain HM-175 was passaged six times in marmosets, 59 times in cell culture and purified from infected cell culture supernatant fluid. The viral RNA was extracted, copied into cDNA and the cDNA:RNA hybrids were cloned into the PstI site of plasmid pBR322. The cDNA clones were authenticated by hybridization to RNA extracted from HAV-infected cells and clones representing the 3' end of the genome were identified using a previously authenticated cDNA clone. The clones represented all but 29 bases of the HAV genome. They were compared to HAV strain HM-175 cDNA cloned from viral RNA after three passages in marmosets on the basis of restriction endonuclease mapping and DNA sequencing. No differences were found in either the presence or absence of restriction endonuclease sites using 33 different restriction enzymes. Sequencing of cDNA representing bases 29 to 1002 of the HAV genome revealed eight base changes all of which were within the 5' noncoding region. PMID- 3016163 TI - Routine use of mu-antibody-capture ELISA for the serological diagnosis of Coxsackie B virus infections. AB - The role of coxsackie B viruses (CBV) in myo/pericarditis has been well documented; however, interpretation of static high neutralising antibody titres in individual patients has always been difficult. In introducing the mu-antibody capture ELISA test for the detection of CBV-specific IgM, we hoped to overcome this problem. A regimen for the routine serological diagnosis of CBV infections was introduced, using the CBV IgM ELISA as a screening test, followed by neutralisation tests (NT) to confirm the positive results. Seven hundred and sixty patients and 304 healthy adult controls were tested. The percentage CBV IgM positive in each of the clinical categories myo/pericarditis (33%) chest pain (22%), myalgic encephalomyelitis (31%), myalgia/Bornholm (19%) and controls (9%) was similar to those found in previous studies using NT alone. Cross-reactions with other enteroviruses, including hepatitis A (Enterovirus 72), were observed but did not prove to be a problem in the illness studied, since most involved adults. Both homotypic and heterotypic CBV IgM responses were found. Matching IgM and NT indicated a recent CBV infection. Positive IgM with negative NT titres suggested a recent infection with an enterovirus other than a CBV. PMID- 3016164 TI - Mu-antibody capture ELISA for the rapid diagnosis of enterovirus infections in patients with aseptic meningitis. AB - Between August and November 1985, 45 patients with suspected aseptic meningitis were investigated using conventional virus isolation procedures and the mu antibody capture Coxsackie B IgM enzyme-linked immunoabsorbent assay (ELISA) test, which is well known to cross-react with other members of the enterovirus group. An enterovirus was isolated from 22% of patients compared with 67% who were positive in the ELISA test. Not only was the rate of enterovirus detection increased by using this ELISA method, the clinician received a result within 2 days of submission of serum to the laboratory. A positive result was reassuring to the patient and helpful in clinical management. The main disadvantage of this test was its cost since Coxsackie B1-5 virus antigens were essential. Development of a single inexpensive enterovirus-specific antigen is thus desirable. PMID- 3016165 TI - A combination of four cell types for rapid detection of enteroviruses in clinical specimens. AB - Isolation in cell culture is currently the most sensitive and reliable way to demonstrate enterovirus (EV) in clinical specimens. During July-October 1982 and 1983, we studied the impact of adding two new cell lines, Buffalo green monkey kidney (BGM) and human rhabdomyosarcoma (RD), to the more traditional cell combination used for EV isolation, human embryonic lung (HEL) and primary cynomolgus monkey kidney (CMK) cells; 2,558 specimens were studied: 632 fecal, 677 respiratory, 537 CSF, and 712 blood. An EV was isolated from 417 (16%); of these, 77 (18%) were positive only in BGM or RD; 35% (146/417) of the specimens were positive in BGM, RD, or both, at least one day earlier than in the traditional cells. BGM cells were helpful in isolation of group B coxsackieviruses (CB): 99% of 121 positive specimens were detected in BGM vs 73% in CMK and 23% in HEL; 72% of the CB isolates were detected by day 2 in BGM vs 48% in CMK and 0% in HEL. RD cells were helpful in the isolation of echoviruses: 59% of the 189 positive specimens were detected in RD vs 67% in HEL and 58% in CMK. RD was the only positive cell type in 28/189 (15%) positive specimens; 31% of the echovirus isolates were detected by day 2 in RD, vs 20% in HEL and 19% in CMK. Using the cell types described, we provided the clinician with results in 42% of the EV-positive specimens by day 2 after inoculation and in 61% by day 4. PMID- 3016166 TI - Lymphocyte-associated viremia in varicella. AB - Varicella-zoster virus was isolated from mononuclear cells from seven immunocompetent children with varicella during the late incubation period and during the acute phase of the disease. The cells from which the virus was isolated were nonphagocytic and did not adhere to the surface of plates. The results indicate lymphocyte-associated viremia at the time of onset of varicella. PMID- 3016167 TI - Humoral immune response in hereditary and overt diabetes mellitus. AB - The inbred diabetic mutant mouse, C57BL/KsJ db +/db + (db +/db +), spontaneously develops diabetes mellitus when allowed food ad libitum. However, restriction of food intake prevents the expression of this genetic predisposition for diabetes. This experimental design has been used previously to demonstrate a deficient neutralizing antibody response to coxsackievirus B4 (CB4) in mutants with the genetic predisposition only. These observations demonstrate that in the genetically predisposed diabetic mutant, deficient humoral immunity extends further to a general impairment in both total IgM and IgG production after CB4 infection. Furthermore, these mice are unable to produce a virus-specific IgG response but do show a high level of nonspecific antibody suggesting a polyclonal activation following CB4 challenge. In addition, we observed an increase in the number of spleen IgM antibody-forming cells to sheep erythrocytes (SRBC) in the overtly diabetic animal following CB4 infection with little change apparent in the genetically predisposed animal after infection. These results were identical to the changes seen in total spleen cell numbers. Our animal model provides an opportunity to distinguish between the genetic predisposition to diabetes and the overt disease and suggests that some of the immune impairment found prior to diabetes onset may be partially diminished afterwards. PMID- 3016168 TI - Genital herpes and hepatitis in healthy young adults. AB - Although case reports of herpes simplex virus (HSV) causing acute hepatitis in otherwise healthy adults have appeared recently in the literature, a prospective study of the incidence of HSV-hepatitis in the general population hitherto has not been reported. In the present study, serum samples from 124 young adults attending a sexually transmitted disease clinic with either genital herpes infections (n = 86) or non-herpes sexually transmitted diseases (n = 38) (controls) were analyzed for liver enzyme abnormalities (including aspartate aminotransferase [AST] and alanine aminotransferase [ALT]). Twelve of eighty-six (14%) herpes-infected patients had mildly abnormal liver enzyme tests (less than or equal to twice the upper limit of normal) as opposed to only 1 of 38 controls (2.6%), (P less than .05). All individuals in the herpes-hepatitis group were anicteric, and only two complained of constitutional symptoms (malaise and fatigue). Liver enzyme tests were repeated in nine herpes-hepatitis patients 1 week after their genital lesions had resolved, and in six of nine patients the results had returned to within normal limits. Four patients subsequently returned at the onset of a recurrence of their genital herpes. In all four, serum ALT levels were elevated from the previous occasion, and in three of the four levels just exceeded the upper limit of normal. One patient was followed through three recurrences of his genital herpes. In that individual, the extent of liver enzyme abnormalities appeared to correlate with the presence or absence of his genital lesions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016170 TI - Neutralization test for BK virus: plaque reduction detected by immunoperoxidase staining. AB - We developed an immunoperoxidase staining test to detect structural antigens of BK virus (BKV) in Vero cell cultures. This test was used to examine the neutralizing activity of human and immunized animal sera. It was shown that sera positive for BKV antibodies measured by hemagglutination inhibition test and enzyme-linked immunosorbent assay (ELISA) were able to prevent expression of BKV structural antigens in cell cultures. The correlation between titers in the hemagglutination inhibition test, levels of BKV IgG measured by ELISA, and the titers assayed by the immunoperoxidase neutralization test was high. We suggest that this type of test may be used instead of conventional neutralization tests for other viruses with slowly developing cytopathogenic effects. PMID- 3016169 TI - Nucleic acid hybridization for detection of herpes viruses in clinical specimens. AB - A diagnostic hybridization assay for detecting varicella zoster virus (VZV), herpes simplex virus (HSV), and Epstein-Barr virus (EBV) in different clinical specimens was developed using cloned viral DNAs as probes. All probes detected at least 5 pg of homologous DNA and did not cross-react with other viral or cellular DNA. Results of cell culture, serology, and DNA assay were highly concordant. Using a simple standardized protocol for preparation of specimens, hybridization, and washing procedures, this sensitive and specific assay appears to be useful for screening clinical specimens and may be helpful in confirming the serological diagnosis of HSV encephalitis and persistent EBV infections or EBV-associated diseases. PMID- 3016171 TI - The effect of some anions on the spectral properties of bovine ceruloplasmin. AB - The effect of binding of N3-, SCN-, OCN-, and F- to bovine ceruloplasmin (Cp) has been studied in detail using absorption, circular dichroic (CD), and electron paramagnetic resonance (EPR) spectroscopies. With the addition of increasing amounts of N3-, SCN-, and OCN- to a Cp solution, the intensity of the band at 614 nm at first increased several percent and then decreased gradually as at least one type I copper was reduced and/or as the type I copper was changed to type II copper. Concomitantly, new bands appeared at 430 and 365 nm for N3-, 435 and 380 nm for SCN-, and about 390 nm for OCN-. A conformational change in the protein induced by the binding of N3-, SCN-, and OCN- to the type II and type III coppers led to the change in the CD spectra. The observed increase of the band at about 430 nm was attributed to the change occurring at the type I copper site. On the other hand, the band at about 370 nm may come from a charge transfer of coordinated anions to the Cu(II) ion. Fluoride ion did not induce the appearance of the band at around 430 and 370 nm, but the parallel component of the type II copper EPR signal was split upon the binding of two fluoride ions to the copper ion. PMID- 3016172 TI - Enhancement of gamma-aminobutyric acid binding by quazepam, a benzodiazepine derivative with preferential affinity for type I benzodiazepine receptors. AB - We evaluated the effect of the two N-trifluoroethyl benzodiazepines, quazepam and its 2-oxo metabolite SCH 15725, which possess preferential affinity for type I benzodiazepine recognition sites, on the binding of [3H] gamma-aminobutyric acid ([3H]GABA) to rat brain membrane preparations. The study also included compounds such as diazepam and N-desalkyl-2-oxoquazepam (SCH 17514), which have equal affinity for the type I and type II receptor subtypes. Binding of [3H]GABA was studied in frozen-thawed and repeatedly washed cortical membranes incubated in 20 mM KH2PO4 plus 50 mM KCl, pH 7.4, at 4 degrees C in the absence and presence of quazepam or its metabolites. Addition of 10(-6) M quazepam increased by 30% specific [3H]GABA binding; as revealed by Scatchard plot analysis, the effect was due to an increase in the total number of GABA receptors. The effect of quazepam was concentration dependent, and it was shared by its active metabolite SCH 15725. The potency of quazepam and SCH 15725 in enhancing [3H]GABA binding was similar to that of diazepam, whereas CL 218872 and SCH 17514 were less active. Moreover, the [3H]GABA binding-enhancing effect of quazepam was mediated by an occupancy of benzodiazepine receptors, because it was specifically antagonized by 5 X 10(-6) M Ro15-1788. PMID- 3016174 TI - Desensitization of beta-adrenergic receptor-coupled adenylate cyclase in cerebral cortex after in vivo treatment of rats with desipramine. AB - Continuous treatment (1-10 days) of rats with desipramine (10 mg/kg, twice per day) caused desensitization of the beta-adrenergic receptor-coupled adenylate cyclase system of cerebral cortical membranes. The decrease in the isoproterenol stimulated adenylate cyclase activity was more rapid and greater than the decrease in the number of beta-adrenergic receptors in membranes during treatment of the membrane donor rats with desipramine, indicating that the desensitization occurring at an early stage of the treatment was not accounted for solely by the decrease in the receptor number. Neither the guanine nucleotide regulatory protein (N) nor the adenylate cyclase catalyst was impaired by the drug treatment, since there was no decrease in the cyclase activity measured in the presence or absence of GTP, guanyl-5'-yl-beta-gamma-imidodiphosphate [Gpp(NH)p], NaF, or forskolin. Gpp(NH)p-induced activation of membrane adenylate cyclase developed with a lag time of a few minutes in membranes from control or drug treated rats. The lag was shortened by the addition of isoproterenol, indicating that beta-receptors were coupled to N in such a manner as to facilitate the exchange of added Gpp(NH)p with endogenous GDP on N. This effect of isoproterenol rapidly decreased during the drug treatment of rats. Thus, functional uncoupling of the N protein from receptors was responsible for early development of desensitization of beta-adrenergic receptor-mediated adenylate cyclase in the cerebral cortex during desipramine therapy. PMID- 3016173 TI - Effect of nerve growth factor on glucose utilization and nucleotide content of pheochromocytoma cells (clone PC12). AB - The effect of nerve growth factor (NGF) on the utilization and fate of uniformly labeled 14C glucose and on the content of several pyridine and purine nucleotides has been tested in the clonal cell line PC12. After incubation for 72 h with NGF, PC12 cells exhibit a 2.7-fold increase in glucose utilization and a 4.7-fold increase in CO2 release. During the same incubation period, all the nucleotides tested (NAD+, AMP, GMP, UDP-glucose, UDP-galactose, UDP, ADP, GDP, UTP, CTP, ATP, and GTP) underwent significant increments, varying from a minimum of 27% for ADP to a maximum of 90-120% for AMP, GMP, UDP-glucose, and UDP-galactose. These findings are discussed in connection with the trophic and differentiative effects of NGF in PC12 cells, which, in the presence of this factor, shifted from a neoplastic to a neuronal-like cell population. PMID- 3016175 TI - An investigation of the low intrinsic activity of adenosine and its analogs at low affinity (A2) adenosine receptors in rat cerebral cortex. AB - The potencies and intrinsic activities of adenosine analogs for stimulating cyclic AMP accumulation in slices of rat cerebral cortex were examined. 5'-N Ethylcarboxamidoadenosine (NECA) caused the greatest increase in cyclic AMP accumulation (19.2-fold). 2-Chloroadenosine (2-CAD) induced a similar increase, but adenosine and six other analogs caused much smaller increases. All agonists tested had similar potencies in activating this response. Inhibition of adenosine uptake with 10 microM dipyridamole did not affect the maximal response to any agonist, although the potency of adenosine was increased approximately threefold. Each analog was also able to block partially the stimulation of cyclic AMP accumulation caused by NECA. Levels of cyclic AMP accumulation in the presence of NECA plus another analog were similar to those observed when the analog alone was present, as expected for partial agonists. Furthermore, the EC50 value for R-(-) N6(2-phenylisopropyl)adenosine in increasing cyclic AMP accumulation was similar to the KI value for inhibiting the response to NECA. The EC50 value for adenosine was substantially higher than the KI value for inhibiting the response to NECA; however, in the presence of dipyridamole, the two values were more closely correlated. The response to NECA was blocked by 8-phenyltheophylline, 1,3-diethyl 8-phenylxanthine, and 8-p-sulfophenyltheophylline, with KI values from 1 to 10 microM. The results suggest that adenosine analogs stimulate cyclic AMP accumulation in cerebral cortex through low-affinity receptors, but that some analogs only partially activate these receptors. Adenosine itself may also be a partial agonist, or its actions may be obscured by simultaneous activation of another receptor. PMID- 3016176 TI - Disulfide bond formation between nerve growth factor and the nerve growth factor receptor from embryonic sensory neurons. AB - Recent studies with sympathetic neurons using radiolabeled nerve growth factor have indicated that a high-molecular-weight covalent complex is formed. This complex is between the nerve growth factor and the high-affinity (type I) receptor and occurs through the formation of a disulfide bond. Studies presented in the present article demonstrate a similar complex is formed on chicken embryonic sensory neurons. The formation of this complex is inhibited by the addition of unlabeled nerve growth factor, metabolic energy inhibitors (dinitrophenol and NaF), and of sulfhydryl reagents. On the other hand, formation of this complex is not inhibited by temperature, or by the addition of insulin or epidermal growth factor. The receptor involved in the covalent complex formation is the high-affinity (type I) receptor. The molecular weight of this complex is approximately 232,000 daltons. Evidence indicates that this covalent complex may be required for the biological activity of the nerve growth factor. PMID- 3016177 TI - Do secretin and vasoactive intestinal peptide have independent receptors on striatal neurons and glial cells in primary cultures? AB - Vasoactive intestinal peptide (VIP) and secretin are two related peptides that activate adenylate cyclase on membranes of striatal neurons and glial cells from embryonic mouse brain grown in primary culture. On the two cell types, the maximal activation that could be induced by secretin was only 40% above basal activity, which represented less than 15% of the maximal effect obtainable with VIP. From competition experiments performed on glial cells and the neuroblastoma X glioma hybrid, NG 108-15, a cell line known to possess both VIP and secretin sensitive-adenylate cyclase, we demonstrate that secretin does not activate VIP receptors. Furthermore, secretin has an apparent high affinity (EC50 10(-8) M) for its receptors on striatal neurons and NG 108-15 whereas an apparent low affinity (EC50 7 X 10(-6) M) was found on striatal glial cells. This suggests the existence of either two distinct secretin receptors or a desensitized form. PMID- 3016179 TI - See-saw signal processing in pinealocytes involves reciprocal changes in the alpha 1-adrenergic component of the cyclic GMP response and the beta-adrenergic component of the cyclic AMP response. AB - Pineal cyclic AMP and cyclic GMP are regulated by norepinephrine (NE) acting through alpha 1- and beta-adrenoceptors. beta-Adrenergic stimulation appears to be an absolute requirement and alpha 1-adrenergic activation amplifies beta adrenergic stimulation of the cyclic AMP response 10-fold and the cyclic GMP response 100-fold, respectively. Chronic deprivation of adrenergic stimulation, due to exposure to constant light (LL) or by surgical denervation, enhances the cyclic AMP response and diminishes the cyclic GMP response as compared to control animals in a 10:14 light/dark (LD) cycle. This phenomenon is termed see-saw signal processing. In the current study we find these changes do not reflect shifts in the time course or Ka of these responses. Dose-response studies indicate the beta-adrenergic component of cyclic AMP stimulation is enhanced and the alpha 1-adrenergic component of cyclic GMP stimulation is diminished in LL pinealocytes. Several observations indicate these changes may reflect alterations in Ca2+-sensitive postreceptor mechanisms. PMID- 3016178 TI - Enhanced noradrenergic neuronal activity increases homovanillic acid levels in cerebrospinal fluid. AB - Idazoxan, a highly specific and selective alpha 2-adrenoceptor antagonist, caused a dose-dependent increase in the concentration of homovanillic acid (HVA) a metabolite of 3,4-dihydroxyphenylethylamine, in cisternal CSF of freely moving rats. This increase in HVA level could be antagonized by the alpha 2-adrenoceptor agonist medetomidine. The increase was directly proportional to the concurrent elevation in level of 3-methoxy-4-hydroxyphenylglycol, a metabolite of noradrenaline, in the CSF of individual rats and followed a similar time course. It is suggested that the HVA level in CSF may be increased under conditions of enhanced noradrenergic activity and that, in such situations, it reflects noradrenergic rather than dopaminergic neuronal activity. Care should be taken, therefore, when changes in central dopaminergic activity are assessed by measurements of HVA level in CSF. PMID- 3016180 TI - Glycine antagonists structurally related to 4,5,6,7-tetrahydroisoxazolo [5,4 c]pyridin-3-ol inhibit binding of [3H]strychnine to rat brain membranes. AB - [3H]Strychnine binding to rat pons + medulla membranes was used as a measure of glycine receptors or glycine receptor-coupled chloride channels in vitro. A series of compounds structurally related to 4,5,6,7-tetrahydroisoxazolo[5,4 c]pyridin-3-ol (THIP), which previously were shown to antagonize glycine responses in cat spinal cord, inhibited [3H]strychnine binding in micromolar concentrations. The most potent of these glycine antagonists, 5,6,7,8-tetrahydro 4H-isoxazolo[3,4-d]azepin-3-ol (iso-THAZ), was also the most potent inhibitor of [3H]strychnine binding, with a Ki of 1,400 nM. The Ki value for strychnine was 7.0 nM, whereas the Ki value for the mixed gamma-aminobutyric acid (GABA)/glycine antagonist 3 alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (RU 5135) was only 4.6 nM. Sodium chloride (1,000 mM) enhanced the affinity of strychnine, brucine, isostrychnine, and the nonselective GABA antagonist pitrazepin for [3H]strychnine binding sites, whereas the affinities of glycine, beta-alanine, and taurine were reduced. These sodium chloride shifts, however, were not predictive of antagonist or agonist properties, since the sodium chloride shift for the glycine antagonist iso-THAZ and of the other THIP-related antagonists were similar to those of the glycine-like agonists. The various sodium chloride shifts show that different groups of ligands bind to glycine receptor sites in different ways. PMID- 3016181 TI - Expression of a plasma membrane proteolipid during differentiation of neuronal and glial cells in primary culture. AB - Plasma membrane proteolipid protein (PM-PLP) synthesis was examined in embryonic rat neurons and neonatal rat glial cells during differentiation in culture. Glial cultures were treated with 1 mM N6, O2, dibutyryl cyclic adenosine monophosphate (dbcAMP) following confluency to induce differentiation, which resulted in the elaboration of long cellular processes. However, no changes in the biosynthetic level of PM-PLP was observed during the differentiation of these cells. Neurons differentiated spontaneously in culture, forming cellular aggregates immediately following plating and elaborating a network of neurites over 7 days. The differentiation of neurons was accompanied by a seven-fold increase in PM-PLP synthesis with increases in biosynthetic increase in PM-PLP synthesis with increases in biosynthetic rate observed between days 1 and 3 and between days 3 and 7 in culture. Ultrastructural examination of neurons indicated that the Golgi apparatus was also developing during this period of time, with an increase in both the number of lamellae and generation of vesicles. The transport of PM-PLP to the plasma membrane was therefore examined in neurons at day 7 in culture by pulse labeling experiments with monensin and colchicine. Monensin (1 microM) was found to inhibit the appearance of radiolabeled PM-PLP in the plasma membrane by 63%, indicating that a functional Golgi apparatus is required for transport of PM PLP to its target membrane. Colchicine (125 microM) also inhibited the appearance of newly synthesized PM-PLP in the plasma membrane by greater than 40%, suggesting that microtubules may also be required for PM-PLP transport to the plasma membrane. PMID- 3016183 TI - D-1 dopaminergic and beta-adrenergic stimulation of adenylate cyclase in a clone derived from the human astrocytoma cell line G-CCM. AB - Clones have been isolated from the human astrocytoma cell line G-CCM. Homogenates of clone D384 contain an adenylate cyclase that is stimulated by 3,4 dihydroxyphenylethylamine (dopamine), noradrenaline, and isoprenaline with Ka apparent values of 4, 56, and 2.7 microM, respectively. The Ka apparent value for dopamine was increased by the D-1 antagonist cis-flupenthixol, 25 and 100 nM, to 23 and 190 microM, respectively, but was unaffected by propranolol (1 microM). Noradrenaline stimulation of adenylate cyclase was only partially inhibited by either propranolol (10 microM) or cis-flupenthixol (1 microM). Propranolol (10 microM), but not cis-flupenthixol (1 microM), prevented stimulation by isoprenaline. The stimulation of adenylate cyclase by dopamine and noradrenaline remained unchanged in the presence of phentolamine (1 microM) and sulpiride (1 microM). These results suggest that clone D384 contains both D-1 dopaminergic and beta-adrenergic receptors coupled to adenylate cyclase. Dopamine stimulates D384 adenylate cyclase through D-1 receptors, isoprenaline via beta-receptors, and noradrenaline through both receptors. PMID- 3016184 TI - Effect of seizures induced by intra-amygdaloid kainic acid on kainic acid binding sites in rat hippocampus and amygdala. AB - [3H]Kainic acid binding sites with a slow dissociation rate in the rat limbic system were investigated in detail. Extensively washed membranes prepared from the hippocampal formation and from the region comprising the amygdala and the piriform cortex yielded non-linear Scatchard plots. Microdissection showed that the high-affinity component (affinity constant around 1 nM) was present in the hippocampal CA3 region (4.2 fmol/mg wet tissue) and the amygdaloid complex (4.6 fmol/mg wet tissue), whereas the remaining part of the hippocampal formation and the piriform lobe contained the low-affinity component (affinity constant 5-20 nM; 11.6 and 11.3 fmol/mg wet tissue, respectively). In the lateral + medial septum we detected only the low-affinity component. Severe limbic seizures, induced by unilateral injection of 0.7 or 0.8 microgram kainic acid in 0.3 microliter of phosphate-buffered saline into the amygdala, reduced kainic acid binding sites in the ipsilateral amygdala and CA3 region. The decline of kainic acid binding sites in the injected amygdala was followed by a similar effect in the contralateral amygdala ("mirror focus") and later by a moderate loss also in the contralateral CA3 region. Kainic acid receptor autoradiography demonstrated that binding sites were lost from the stratum lucidum in hippocampus. Septal lesion had no effect on kainic acid binding sites in the hippocampus. Comparison with previous results on the histopathological changes after this lesion shows that high-affinity kainic acid binding sites are preferentially located on neurons that undergo selective degenerations after severe kainic acid-induced seizures. PMID- 3016185 TI - Cell-free desensitization of opioid inhibition of adenylate cyclase in neuroblastoma X glioma NG108-15 hybrid cell membranes. AB - When membranes from neuroblastoma X glioma NG108-15 hybrid cells were incubated in a cell-free system with opioid agonists, a time-, temperature-, and dose dependent desensitization to opioid inhibition of adenylate cyclase activity was observed. The composition of the system during the incubation was manipulated to elucidate the biochemical mechanisms of desensitization. Receptor coupling appeared to be a prerequisite for desensitization, because both magnesium and sodium, which are necessary for coupling, were required for desensitization. Removal of ATP and addition of cyclic AMP or cyclic GMP had no effect on desensitization. PMID- 3016182 TI - Brain Na+,K+-ATPase: alteration of ligand affinities and conformation by chronic ethanol and noradrenergic stimulation in vivo. AB - These experiments examined effects of chronic ethanol, repeated noradrenergic stimulation or inhibition, and ethanol combined with the noradrenergic treatments on regulation of Na+,K+-ATPase. Chronic treatment with ethanol reduced the sensitivity of K+-p-nitrophenyl-phosphatase to ethanol, increased affinity for K+, reduced the sensitivity of K+ affinity to ATP or ethanol, and reduced delta H and delta S for K+ activation and for the E1-E2 transition. These effects were all opposite to those of ethanol added in vitro. Treatment with yohimbine had the opposite effects on ethanol sensitivity, K+ affinity, K+ interactions with ethanol and ATP, and thermodynamic parameters for cation activation or conformational change. These effects were similar to those of norepinephrine in vitro. The effects of yohimbine treatment were eliminated or reduced in rats also treated with ethanol. Depletion of norepinephrine had effects opposite to those of yohimbine. These data are consistent with a reduction in membrane fluidity, at least in the vicinity of Na+,K+-ATPase, during ethanol tolerance. Exposure to norepinephrine, in vitro or in vivo, had effects on Na+,K+-ATPase that were similar to those of increased membrane fluidity. PMID- 3016186 TI - Cerebral phosphoinositide, triacylglycerol, and energy metabolism in reversible ischemia: origin and fate of free fatty acids. AB - Levels of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4 phosphate (PIP), phosphatidylinositol (PI), phosphatidic acid, diacylglycerol (DAG), triacylglycerol (TAG), and free fatty acids (FFAs), as well as their fatty acid composition, were determined in rat forebrain during ischemia and postischemic recirculation. Cerebral energy state and electroencephalograms (EEGs) were also studied. Fifteen minutes of ischemia resulted in a decrease in PIP2 and PIP contents but not in PI content, concurrent with an enlargement of the FFA and DAG pools. The latter were enriched in stearate and arachidonate. Prolongation of ischemia did not produce further changes in content of any of the inositol phospholipids, but the increase in levels of FFAs and DAG continued. At the end of 45 min of ischemia, levels of both PIP2 and PIP decreased by 45-50%, and the total phosphoinositide content (PIP2 + PIP + PI) decreased by 21%, whereas levels of FFAs and DAG increased to 14- and 3.6-fold of control levels, respectively. During ischemia, the TAG-palmitate level decreased, but the TAG arachidonate level increased; the tissue energy state deteriorated severely; and the EEG was suppressed. A 30-min recirculation period after 15 or 45 min of ischemia led to increases in PIP2, PIP, and total phosphoinositide contents, whereas levels of FFAs and DAG promptly decreased toward control values. The TAG arachidonate level peaked and the TAG-palmitate level returned to a low control value during early recirculation. The ischemic changes in tissue lipids were completely reversed within 3 h of recirculation after both periods of ischemia. Adenylates were fully phosphorylated with as little as 30 min of reflow. The EEG activity partially recovered during reflow after 15 min of ischemia, whereas it remained depressed after prolonged ischemia. Thus, phosphodiesteric cleavage of PIP2 and PIP followed by deacylation of DAG is likely to contribute to the production of FFAs in early ischemia. Deacylation of undetermined lipids plays a role for the increment in levels of FFAs in the later period of ischemia. The rapid postischemic increase in levels of PIP2 and PIP indicates active synthesis not only from existing PI, but probably also by means of accumulated FFAs and DAG. These results indicate that the impaired resynthesis of inositol phospholipids cannot be a cause of the poor EEG activity after prolonged ischemia. Degradation and resynthesis of polyphosphoinositides and formation of TAG-arachidonate may be important for modulation of free arachidonic acid levels in the brain during temporary ischemia. PMID- 3016187 TI - Benzodiazepine receptor subunits in avian brain. AB - Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of the photoaffinity-labeled benzodiazepine receptor in duck brain, two equally intensive bands of relative molecular masses of 53,000 and 54,000 were observed, thus being identical to the subunit pattern previously described for chicken brain. An attempt was made to characterize further the avian subunits. Comparison of the fluorographic subunit pattern in cerebellum and telencephalon revealed a pronounced quantitative heterogeneity. Addition of ethyl beta-carboline-3 carboxylate resulted in the selective inhibition of the 53,000 subunit. These results are compared with the mammalian subunit pattern in the corresponding brain regions. The findings are discussed in view of the apparent contradiction between the existence of regional heterogeneity when photoaffinity labeling is applied and its lack when monoclonal antibodies are used. PMID- 3016188 TI - Ectonucleotidase activities associated with cholinergic synaptosomes isolated from Torpedo electric organ. AB - Intact synaptosomes isolated from the electric organ of the electric ray Torpedo marmorata contain, at their surface, enzyme activities for the hydrolysis of externally applied nucleoside phosphates. The diazonium salt of sulfanilic acid, as a low-molecular-weight, slowly permeating, covalent inhibitory agent, selectively blocks these enzyme activities and leaves intracellular lactate dehydrogenase intact. The ectoenzymes comprise both a nucleoside triphosphate and diphosphate phosphohydrolase, as well as a 5'-nucleotidase. Activity of nonspecific ectophosphatases is absent. The nucleoside triphosphatase hydrolyzes almost equally well ATP, GTP, CTP, UTP, and ITP and is activated to a similar degree by Mg2+ or Ca2+. It has a high affinity for ATP (Km for ATP in the presence of Mg2+, 75 microM; in the presence of Ca2+, 66 microM). Maximal rates in the presence of Mg2+ and Ca2+ were very similar (34.8 and 32.5 nmol of Pi/min/mg of synaptosomal protein, respectively). Either Mg-ATP or Ca-ATP can act as a true substrate. ADP inhibits hydrolysis of ATP, but AMP is without effect. The nucleoside triphosphatase is not inhibited significantly by a number of inhibitors of mitochondrial Mg2+-ATPase or of Ca2+ + Mg2+-ATPases. It is, however, considerably inhibited by filipin and quercitin. The capacity of intact synaptosomes to hydrolyze also extracellular ADP, GDP, AMP, GMP, and IMP suggests that the nucleoside triphosphatase is part of an enzyme chain that causes complete hydrolysis of the respective nucleoside triphosphate to the nucleoside. We conclude that the cholinergic nerve terminals of the Torpedo electric organ can hydrolyze ATP released on coexocytosis with acetylcholine via an ectonucleoside triphosphatase activity that is different from known endogenous nerve terminal ATPases. The final product of the hydrolysis, adenosine, can then be salvaged by the nerve terminal for resynthesis of ATP. Other possible physiological functions of the ectonucleotidases are discussed. PMID- 3016189 TI - Comparative stereostructure-activity studies on GABAA and GABAB receptor sites and GABA uptake using rat brain membrane preparations. AB - The affinities of a number of analogues of gamma-aminobutyric acid (GABA) for GABAA and GABAB receptor sites and GABA uptake were studied using rat brain membrane preparations. Studies on the (S)-(+)- and (R)-(-)-isomers of baclofen, 3 hydroxy-4-aminobutyric acid (3-OH-GABA), and 4,5-dihydromuscimol (DHM) revealed different stereoselectivities of these synaptic mechanisms in vitro. Although (S) 3-OH-GABA and, in particular, (S)-DHM were more potent than the corresponding (R) isomers as inhibitors of GABAA binding, the opposite stereoselectivity was demonstrated for the GABAB binding sites. Thus, (R)-3-OH-GABA and (R)-baclofen were more potent than the (S)-isomers as inhibitors of GABAB binding, (R) baclofen being some five times more potent than (R)-3-OH-GABA. These two (R) isomers actually have opposite orientation of the substituents on the GABA backbones, suggesting that the lipophilic substituent of (R)-baclofen interacts with a structural element of the GABAB receptor site different from that that binds the very polar hydroxy group of (R)-3-OH-GABA. The O-methylated analogue of 3-OH-GABA, 3-methoxy-4-aminobutyric acid (3-OCH3-GABA), did not interact significantly with GABAB sites. The homologues of GABA, trans-4-aminocrotonic acid (trans-ACA), muscimol, and 3-OH-GABA, that is, 5-aminovaleric acid (DAVA), trans-5-aminopent-2-enoic acid, homomuscimol, and 3-hydroxy-5-aminovaleric acid (3-OH-DAVA), respectively, were generally much weaker than the parent compounds, whereas 2-hydroxy-5-aminovaleric acid (2-OH-DAVA) showed a significantly higher affinity for GABAB sites than the corresponding GABA analogue.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016190 TI - Phenytoin dephosphorylates the catalytic subunit of the (Na+,K+)-ATPase in C57/BL mice. AB - The effects of phenytoin, a potent antiepileptic drug, on the active transport of cations within membranes remain controversial. To assess the direct effects of phenytoin on the Na+,K+ pump, we studied the drug's influence on the phosphorylation of partially purified (Na+,K+)-ATPase from mouse brain. (Na+,K+) ATPase subunits were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phenytoin, in vitro, decreased net phosphorylation of the (Na+,K+)-ATPase catalytic subunit in a dose-dependent manner (approximately 50% at 10(-4) M). When the conversion of E1-P to E2-P, e.g., the two major phosphorylated conformational states of (Na+,K+)-ATPase, was blocked by oligomycin or N-ethylmaleimide, phenytoin had no effect. The results suggest that phenytoin acts on the phosphatasic component of the reaction cycle, decreasing the phosphorylation level of the enzyme. PMID- 3016191 TI - Fate of cyclic nucleotides in PC12 cell cultures: uptake, metabolism, and effects of metabolites on nerve growth factor-induced neurite outgrowth. AB - The fate of cyclic AMP (cAMP), dibutyryl-cAMP (Bt2-cAMP), and the (Sp)-isomer of adenosine 3',5'-monophosphorothioate [(Sp)-cAMPS] was studied in the PC12 culture medium by means of HPLC. In the absence of PC12 cells, cAMP and Bt2-cAMP were rapidly degraded by nonspecific esterases and cyclic nucleotide phosphodiesterase both originating from the serum commonly used as a culture medium ingredient, whereas (Sp)-cAMPS was completely stable. Since 5'-AMP, adenosine, inosine, and hypoxanthine appeared in the culture medium after incubation with cAMP or Bt2 cAMP, we have determined their effect on nerve growth factor (NGF)-induced neurite outgrowth. 5'-AMP, adenosine, and inosine were indeed potent agents in producing a potentiating effect on NGF-induced early neurite outgrowth at a concentration of 1 mM. Thus, cAMP metabolites had the capacity to induce an effect that has been described as cAMP-specific. In serum-free culture medium and in the presence of cells, all cyclic nucleotides were taken up by PC12 cells. Uptake was highly correlated with the hydrophobic nature of the compounds, and was accompanied by a simultaneous excretion of metabolites. On incubation with cAMP, NGF had a pronounced effect on the metabolic pattern found in the culture medium. In particular, dephosphorylation of 5'-AMP was specifically enhanced. This effect of NGF on the degradation of cAMP was also apparent when cAMP metabolites were incubated with PC12 cells. Whereas 5'-AMP degradation was greatly increased, NGF had no effect on the metabolism of the other purine compounds. PMID- 3016192 TI - Comparison of benzodiazepine receptors in cerebellum and inferior colliculus. AB - Reversible and irreversible binding of [3H]flunitrazepam was investigated in membranes from cerebellum and inferior colliculus of young and adult rats. Results indicate that in adult animals predominantly BZ1 receptors are enriched in both brain regions. In the brains of newborn animals, however, additional benzodiazepine receptor subtypes seem to exist in cerebellum as well as in inferior colliculus. PMID- 3016194 TI - NAD+ glycohydrolase of the plasma membrane prepared from glial and neuronal cells. AB - NAD+ glycohydrolase (EC 3.2.2.5) activity was detected in the plasma membrane prepared from the primary culture of rat astrocytes. The enzyme has a broad optimum pH range. From the kinetic analysis, a Michaelis constant of 91.2 microM and a maximum velocity of 0.785 mumol/min/mg protein were obtained. ADPribose exhibited a competitive inhibition with respect to NAD. The inhibition by nicotinamide was shown to be of a non-competitive type. ATP and GTP were found to be competitive inhibitors. NAD+ glycohydrolase activity was not detected in the plasma membrane prepared from the primary culture of neuronal cells of chick embryos. PMID- 3016193 TI - Neuropeptide-metabolizing peptidases in neuro-2a neuroblastoma and C6 glioma cells. AB - Mouse Neuro-2a neuroblastoma and rat C6 glioma cloned cells were screened for neuropeptide-metabolizing peptidases using a kininase bioassay combined with a time-course bradykinin-product analysis, and a fluorimetric assay for prolyl endopeptidase. The complementary peptide products Arg1----Phe5/Ser6----Arg9 and Arg1----Pro7/Phe8-Arg9 were released during bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5 Ser6-Pro7-Phe8-Arg9) inactivation by homogenates of Neuro-2a and C6 cells. The 1:1 stoichiometry of the complementary fragments and their high yields, at 10% bradykinin inactivation, demonstrated the sites of hydrolysis. The initial rate of Phe5-Ser6 bond cleavage was six-fold higher than that of the Pro7-Phe8 bond. These sites of cleavage can be attributed to enzymes similar to endopeptidase A (Phe5-Ser6) and prolyl endopeptidase (Pro7-Phe8) on the basis of the specificity and sensitivity to inhibitors of the kininase activity in Neuro-2a and C6 cell homogenates. Kininase and prolyl endopeptidase specific activities (fmol/min/cell) were 10.5 and 12.4 for Neuro-2a, and 1.5 and 2 for C6 homogenate, respectively. The recovery of kininase activity was 2.2-fold higher in the particulate than in the soluble (105,000 g for 1 h) neuronal fraction, whereas the amount of prolyl endopeptidase activity was about the same in both fractions. Kininase and prolyl endopeptidase activities in C6 cells were recovered mostly in the soluble fraction. Prolyl endopeptidase specific activity decreased 10-fold in serum-starved Neuro-2a cultured cells, with no change in activity in similarly treated C6 cells. In contrast, kininase specific activity in both cell types was essentially unaffected on serum-deprivation-induced differentiation. PMID- 3016195 TI - Detection of HSV1 DNA by in situ hybridisation in human brain after immunosuppression. AB - Human brain cells were examined for the presence of herpes simplex virus type 1 (HSV1) DNA sequences by in situ hybridisation. Viral genome was detected in immunosuppressed patients with virological evidence of past HSV infection but not in immunosuppressed patients with no such evidence. In patients who had not been immunosuppressed, no HSV DNA sequences were detectable. PMID- 3016197 TI - Axonal stimulation for end-plate jitter studies. AB - This single fibre EMG study compares the standard method of neuromuscular jitter measurement in voluntarily activated muscle to that by intramuscular electrical stimulation of motor axons in a group of normal subjects. The latter method avoids the interdischarge interval-dependent jitter, as well as a possible failure to recognise split muscle fibres. The mean MCD on axonal stimulation was only 5.2 microseconds less than in the voluntary activation study and was thus 8% more than theoretically expected for single motor end plates. The difference could be due to an axonal jitter and some other factors. Axonal stimulation has proved to be a relatively easy and reliable method for routine estimation of neuromuscular jitter, provided that the resolution of time measurement is better than 2 microseconds, so that low jitter due to occasional direct muscle fibre stimulation is not mistaken for a normal reading. The upper normal limits for the extensor digitorum communis muscle suggested by the present study are 40 microseconds (individual muscle fibres) and 25 microseconds (mean of 30 muscle fibres). PMID- 3016196 TI - Myopathy with abnormal mitochondria, transient low electron transport capacity in the respiratory chain, and absence of energy transduction at sites 1 and 2 in vitro. AB - A male adult with exercise-related myalgia and weakness from the age of 17 years, developed contractions after moderate exertion which were electrically silent. Triglyceride loading or prolonged fasting provoked excessive ketosis. His isolated muscle mitochondria had severe blockade of the respiratory chain, particularly of NADH-CoQ reductase. After 1.5 years a second biopsy was performed. The electron transport capacity of the respiratory chain was much improved, but now a lesion was observed in energy transduction of sites 1 and 2 of the respiratory chain. The unexpected abolishment of respiratory chain blockade was paralleled by only mild clinical improvement. PMID- 3016199 TI - [Postmortem study of case I of the original family of Roussy and Melle Levy]. AB - Post-mortem examination of case I of Roussy and Levy's original family shows an extensive Schwann cell proliferation with some onion bulb formations. This confirms that this family is suffering from a form of hypertrophic neuropathy. The author reaffirms his concept of Roussy-Levy syndrome as an autonomous hypertrophic neuropathy. The following criteria seem to justify the autonomy of this syndrome: dominant transmission, very precocious onset, extreme slowness of the evolution, and remarkable benignity of the general prognosis. PMID- 3016198 TI - Ethylene oxide neuropathy in rats. Exposure to 250 ppm. AB - In Wistar rats subjected to a 6-h exposure to ethylene oxide at the concentration of 250 parts per million once, 5 times a week for 9 months, histopathologic studies of myelinated fibers of the proximal sural, distal sural and peroneal nerves and of the fasciculus gracilis at the 5th thoracic and 3rd cervical segments of the spinal cord were performed to observe whether ethylene oxide of such a concentration would lead to degeneration of primary sensory neurons. Throughout the study, no definite abnormality of the gait or posture was observed in both control and test rats. Qualitative histologic studies disclosed preferential distal axonal degeneration of myelinated fibers in both sural nerves and gracile fascicles in the test rats, although the extent of the distribution and the severity of the degenerative findings were variable. Such findings are consistent with mild axonal degeneration found among patients suffering from ethylene oxide toxicity. Therefore, in rats, exposure to 250 ppm ethylene oxide produces central-peripheral distal axonal degeneration of primary sensory neurons. PMID- 3016200 TI - Methods for producing a reproducible crush in the sciatic and tibial nerve of the rat and rapid and precise testing of return of sensory function. Beneficial effects of melanocortins. AB - A procedure for placing a crush lesion in the sciatic and tibial nerve of the rat based on anatomical landmarks is described. These crush lesions are used to study the process of regeneration of peripheral nervous tissue and the beneficial effects of melanocortins on speed and quality of nerve regeneration. A new precise and rapid method for testing the return of sensory function by a locally applied electric stimulus is discussed. PMID- 3016201 TI - Somatosensory evoked potentials after experimental head injury in the awake rat. AB - Experimental acceleration concussion was induced in 30 male rats who were immobilized with curare, artificially ventilated but not anaesthetised. Serial recordings of cortical somatosensory evoked potentials (SEPs) were made from the onset of concussion and for the following 30 min. Immediately after the head blow the initial three components of the SEP (P1, N1 and P2) were all either absent or markedly increased in latency. The late negative component (N2) was always abolished. All components reappeared and returned to approximately pre-concussion latencies within 5-6 min. The most persisting abnormality was in N2 whose post concussion amplitude stabilized and remained depressed at only 50% of baseline value. The results suggest that experimental head injury produces a significant abnormality in every component of the evoked potential. The findings are therefore inconsistent with the theory that concussion differentially affects the diffuse nonspecific pathways whose activity is mediated by the reticular formation while leaving the lemniscal system comparatively intact. It is also shown that the morphology of the post-concussion SEP waveform can be quite accurately simulated by recording SEPs from normal animals at high rates of stimulation (50/s). This implies that the pathophysiology of concussion involves a temporary dysfunction in synaptic transmission although the level at which this is occurring has yet to be determined. PMID- 3016202 TI - Immunoelectron-microscopical labelling of glycolipids in the envelope of a demyelinating brain-derived RNA virus (Semliki Forest) by anti-glycolipid sera. AB - Immunoelectron-microscopical techniques using gold-labelled antibodies were used to localize the glycolipids ganglioside, glucocerebroside and galactocerebroside, and spike glycoprotein antigens, in the envelope of the RNA virus Semliki Forest which had replicated in mouse brain cell cultures. The demonstration of host cell membrane glycolipid antigens in viruses is discussed in relation to the possibility of an autoimmune reaction to central nervous system cells. PMID- 3016203 TI - Cytotoxicity of autologous Epstein-Barr virus-transformed cells mediated by interleukin-2 dependent, long-term T cell cultures is augmented by beta, but not alpha, recombinant interferon. AB - The immunomodulatory effect of two highly purified recombinant human interferons (IFN) on T cell-mediated cytotoxicity of autologous Epstein-Barr virus transformed B cells (EBV-LCL) was assessed. Interferon beta ser, but not alpha 76, potentiated the destruction mediated by interleukin-2 (IL-2) dependent, long term T cell cultures. The degree of potentiation of specific cytotoxicity was dependent on the concentration of the exogenously added IFN beta ser. In contrast to the potentiation of T cell-mediated cytotoxicity, IFN beta ser and alpha 76 did not modulate the cytotoxic activity of cytotoxic T cell clones that are specifically reactive to autologous EBV-LCL. These results indicate that the selective potentiation of EBV-LCL-reactive T cell-mediated cytotoxicity by IFN beta ser may not be due to a direct IFN effect on the cytotoxic T cells (CTLs) themselves. Rather, our results are consistent with IFN beta ser augmenting cytotoxic T cell reactivity indirectly, possibly via its influence on other immunoregulatory cells. Although the precise mechanism whereby IFN beta ser may potentiate cytotoxicity is not yet defined, the synergistic action of IFN beta ser and IL-2 on long-term CTL cultures offers an alternate means to selectively enhance a desirable cytotoxic response. PMID- 3016204 TI - New inhibitors of osteolysis: implications for hypercalcemia and bone metastases. PMID- 3016205 TI - Treatment of malignancy-associated hypercalcemia with intravenous aminohydroxypropylidene diphosphonate. AB - Treatment of malignancy-associated hypercalcemia remains unsatisfactory. We have prospectively treated 26 consecutive hypercalcemic cancer patients with intravenous (IV) aminohydroxypropylidene diphosphonate (APD). The drug was administered daily as a 15-mg two-hour IV infusion until both serum and urinary calcium had been normalized for 48 hours. Twenty-four patients were fully evaluable (eight head and neck tumors, seven breast cancers, three epidermoid tumors of the lung, and six miscellaneous neoplasms). Whereas rehydration had only inconsistent effects, APD normalized serum calcium in all patients after a mean of three daily doses: serum calcium decreased from 13.3 +/- 0.4 mg/dL (mean +/- SEM) before APD to 8.0 +/- 0.1 mg/dL at the end of treatment. Ionized calcium declined in parallel to total calcium. APD was as effective in hypercalcemia due to bone metastases as in paraneoplastic hypercalcemia. The drug was tolerated without toxicity and had a prolonged effect: serum calcium remained normal during 3+ weeks (1 + to 8 +) in 17 patients who did not receive or did not respond to antitumoral treatment. APD normalized serum calcium by inhibiting bone resorption, as evidenced by the dramatic decrease in urinary excretion of calcium and hydroxyproline. Inhibition of bone resorption was probably also responsible for the decrease in serum phosphorus from 2.9 +/- 0.2 to 2.0 +/- 0.1 mg/dL. In summary, IV APD constitutes a major advance in the treatment of malignancy associated hypercalcemia: it is very effective, well tolerated, and has a prolonged efficacy. PMID- 3016206 TI - The syndrome of inappropriate secretion of antidiuretic hormone (SIADH) in small cell lung cancer. AB - Review of clinical data from 350 patients with small-cell lung cancer (SCLC) revealed hyponatremia (sodium less than 130 mEq/L) attributable to the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) in 40 patients (11%). Although hyponatremia was severe in most instances (median, sodium 117 mEq/L), symptoms attributable to water intoxication were identified in only 27% of hyponatremic episodes. Development of SIADH showed no correlation with clinical stage, distribution of metastatic sites, sex, or histologic subtype of small-cell carcinoma. SIADH occurred most often with initial presentation (33 of 40), and resolved promptly (less than 3 weeks) with initiation of combination chemotherapy in 80% of evaluable patients. The presence of SIADH did not influence response to chemotherapy or overall survival as an independent variable. However, in five patients profound hyponatremia developed immediately following primary cytotoxic therapy (range, one to five days). Despite initial control of SIADH, dilutional hyponatremia recurred in 70% of patients with tumor progression. Our findings suggest that development of clinically demonstrable SIADH in patients with SCLC is dependent on functional properties of the neoplastic cells, rather than tumor burden or metastatic site. The potential for development of clinically significant hyponatremia early in the course of cytotoxic therapy emphasizes the need to closely monitor patients, particularly those receiving chemotherapy regimens requiring substantial intravenous hydration. PMID- 3016207 TI - Prognostic factors in disseminated germ cell tumors. PMID- 3016208 TI - Afterhyperpolarization mechanisms in cat sympathetic preganglionic neuron in vitro. AB - A long-lasting afterhyperpolarization (AHP) follows the antidromic or current induced action potential of sympathetic preganglionic neurons (SPNs) studied in slices of cat spinal cord maintained in vitro. Duration and amplitude of the AHP that follows a single spike were 2.8 +/- 0.3 s and 16.0 +/- 0.7 mV (mean +/- SE), respectively. In most cases two components could be distinguished, an initial faster and usually larger component [fast (F) AHP] followed by a slowly decaying component [slow (S) AHP]. An increase in membrane conductance was associated with the AHP. The amplitude of both components increased with membrane depolarization and decreased with hyperpolarization. Both fast and slow component were nullified at a voltage of -90 mV in 3.6 mM K+. Peak AHP amplitude decreased as K+ was increased from 1.5 to 7.0 mM. The null point for both fast (F) AHP and slow (S) AHP shifted in the depolarizing or hyperpolarizing direction when K+ was increased or decreased, respectively. These data suggest that an increase in K+ conductance is the mechanism underlying the AHP. The two components of the AHP could be separated by their differential sensitivity to superfusion with the Ca2+ channel blocker cobalt (2 mM) or with low Ca2+ (0.25 mM). These procedures resulted in an AHP of much shorter duration (330 ms, range 150-600), presumably the FAHP. These observations indicate that a Ca2+-activated K+-conductance is likely to be involved in the generation of the SAHP. The FAHP was depressed during superfusion with tetraethylammonium (TEA) (20 mM) and intracellular cesium injection. The SAHP was enhanced by TEA and enhanced or depressed by cesium. In 3.6 mM K+ the FAHP reversed in polarity at membrane voltages more negative than 90 mV. This component had an approximately linear relation of amplitude to membrane potential. The SAHP did not reverse in most cells. In the few cases in which it reversed, the change in amplitude for a given change in membrane voltage was much smaller on the negative than on the positive side of the null potential. Thus the SAHP shows voltage-dependent, nonlinear characteristics. This difference in behavior of the two components was also observed when the null point was displaced in high or low K+. In the presence of tetrodotoxin (TTX) the AHP persisted in temporal association with a high-threshold, TTX-resistant, cobalt sensitive spike. During the time course of the AHP the efficacy of synaptic input decreased, suggesting that the AHP has an important role in regulating the firing rate of the SPN. PMID- 3016210 TI - Motion selectivity in macaque visual cortex. I. Mechanisms of direction and speed selectivity in extrastriate area MT. AB - Mechanisms of direction selectivity and speed selectivity were studied in single neurons of the middle temporal visual area (MT) of behaving macaque monkeys. Visual stimuli were presented in both smooth and stroboscopic motion within a neuron's receptive field as the monkey fixated a stationary point of light. Direction selectivity, speed selectivity, and the spontaneous discharge characteristics of MT neurons in behaving monkeys were similar to those reported in previous studies in anesthetized monkeys. Stroboscopic motion stimuli were sequences of flashes characterized by the spatial and temporal intervals between each flash. The spatial and temporal intervals were systematically varied so that suppressive and facilitatory interactions could be studied in both the preferred and null directions. Suppression and facilitation were measured by subtracting the peak discharge rate elicited by a single flash from the peak discharge rate elicited by a stroboscopic train of flashes. The dominant mechanism of direction selectivity in MT was a pronounced suppression of discharge for motion in the null direction which we interpreted as inhibition. The inhibition was sufficiently potent to abolish the responses to single flashed stimuli when they were embedded in a series of flashes in the null direction, and it frequently reduced the neuronal discharge to a level below the spontaneous firing rate. Facilitation in the preferred direction was a prominent feature of the responses of some, but not all, MT neurons. The peak discharge rate for stroboscopic motion in the preferred direction was more than twice the peak rate to a single flash for approximately 50% of the neurons in our sample. The direction selectivity of most MT neurons showed the effects of both inhibitory and facilitatory mechanisms, and it was not possible to segregate MT neurons into distinct groups on the basis of these measures. Suppressive mechanisms contributed to speed tuning as well as direction tuning. The low-speed cutoff for motion in the preferred direction resulted from suppression in 82% of the neurons tested. The high-speed cutoff resulted from suppression in 32% of the neurons tested. The latter mechanism appeared to be distinct from the inhibitory mechanism which acted in the null direction in that large spatial intervals were required for its activation. PMID- 3016209 TI - Cellular and synaptic properties of amygdala-kindled pyriform cortex in vitro. AB - The evoked and spontaneous activity of neurons in the pyriform cortex of control and kindled rats was examined using a coronal slice preparation containing the amygdala-pyriform region. Electrical stimulation of the amygdala nuclei elicited synchronized burst responses in pyriform cells of slices from both control and kindled animals. The mean duration of the burst was greatly prolonged in cells from kindled preparations. The depolarizing synaptic events underlying the burst response in the kindled and control animals could be examined when Mg2+ was increased to suppress but not block synaptic transmission. Electrical stimulation evoked a short-latency graded synaptic depolarization, followed by a long-latency all-or-none depolarizing event, which appeared to be involved in generating the burst response. Norepinephrine (NE), in a 4-microM concentration, reversibly blocked the burst responses in the control preparation. Burst responses elicited from kindled preparations were also suppressed by NE. For the latter cases, higher concentrations of NE were required to produce this effect. The alpha-2 agonist clonidine mimicked the suppressive action of NE on the evoked events. In contrast the beta-agonist isoproterenol facilitated the occurrence of spontaneous synchronous bursts and prolonged evoked burst discharges in both the control and kindled preparations. NE and clonidine block the burst response by suppressing the underlying synaptic events. The facilitatory action of isoproterenol on spontaneous and evoked responses suggests that NE may also exert an excitatory effect. PMID- 3016211 TI - An intracellular study of myenteric neurons in the mouse colon. AB - Intracellular recordings have been made in vitro from the myenteric neurons of the distal colon of normal littermates of the piebald-lethal mouse. Out of a total of 90 neurons, 82 were classified as S/type 1 cells and 8 as AH/type 2 cells. Seventy-eight out of 82 S cells showed spontaneous fast excitatory postsynaptic potentials (EPSPs) sensitive to d-tubocurarine (dTC, 280 microM), and 22 S cells showed spontaneous action potentials (APs). Six S cells and 1 AH cell showed spontaneous nonnicotinic slow depolarizations associated with an increase in the input resistance of the cells; during the spontaneous slow depolarization in the S cells there was an increase in the frequency of nicotinic fast EPSPs and APs. Three S cells showed spontaneously occurring regular oscillations of the membrane potential (approximately mV in amplitude and approximately 4/min). Transmural nerve stimulation produced fast EPSPs with a wide range of latencies (3 ms to 20 s) in S cells; the fast EPSPs were blocked by dTC (280 microM) or solutions containing low Ca2+ (0.25 mM) and high Mg2+ (12 mM) but not by atropine (ATR, 14 microM). Single or repetitive transmural stimulation produced slow EPSPs in 24 S cells and 3 AH cells; these were not blocked by dTC (280 microM) nor ATR (14 microM). During the slow EPSPs there was an increase in the input resistance of the cells. In those S cells that showed slow EPSPs there were many long-latency fast EPSPs; long-latency fast EPSPs were also observed in 11 other S cells that did not show a slow EPSP following repetitive transmural nerve stimulation. Long-latency fast EPSPs may be related to the firing of other neurons during their slow EPSPs. The myenteric neurons in the mouse colon have similar properties to the myenteric neurons in the guinea pig small intestine. However, the colonic myenteric neurons show more ongoing synaptic activity and more prolonged activity after nerve stimulation than myenteric neurons in the guinea pig small intestine. This activity may be due to regional differences, species differences, or preparation differences (in this study the myenteric plexus was adherent to the underlying circular muscle layer). PMID- 3016212 TI - The activation of inositol phospholipid metabolism as a signal-transducing system for excitatory amino acids in primary cultures of cerebellar granule cells. AB - L-Glutamic, L-aspartic acids and a number of their structural analogs, including quisqualic, kainic, ibotenic, quinolinic, and N-methyl-D-aspartic (NMDA) acids, increase inositol phospholipid hydrolysis when added to primary cultures of cerebellar granule cells, as is reflected by an enhanced formation of 3H inositolmonophosphate (3H-IP1) in the presence of Li+. L-Glutamic acid also enhances the formation of the initial products of inositol phospholipid hydrolysis, 3H-inositol di-(3H-IP2) and triphosphate (3H-IP3). In the absence of extracellular Ca2+, L-glutamic acid fails to enhance 3H-IP1 formation, but still increases 3H-IP2 and 3H-IP3 formation. The stimulation of 3H-IP1 formation elicited by L-glutamic acid is reduced by DL-2-amino-5-phosphonovaleric acid (APV) and gamma-glutamylglycine and, to a lesser extent, by 2,3-cis piperidindicarboxylic acid (PDA). The stimulation of 3H-IP1 formation by kainic acid is antagonized by PDA and gamma-glutamylglycine, but it is almost unaffected by APV. The increase in 3H-IP1 formation elicited by quisqualic acid is not reduced by any of the dicarboxylic amino acid receptor antagonists that we have tested. We conclude that different subtypes of excitatory amino acid recognition sites are associated with inositol phospholipid metabolism in primary cultures of cerebellar granule cells. PMID- 3016213 TI - Concanavalin A prevents acetylcholine receptor redistribution in Xenopus nerve muscle cultures. AB - During neuromuscular junction formation ACh receptors accumulate at the nerve contact region. It has been shown that this is at least partly due to lateral migration of existing receptors in the membrane (Anderson et al., 1977). Randomly diffusing ACh receptor molecules in the membrane may be trapped at the nerve contact region to form a high receptor density area. If this were the major mechanism, cross-linking ACh receptors by tetravalent concanavalin A (Con A) should immobilize receptors and prevent nerve-induced receptor accumulation. We examined the effect of Con A on nerve-induced receptor accumulation and on the mobility of ACh receptors in cultured Xenopus muscle cells. ACh receptors were stained with tetramethyl rhodamine conjugated alpha-bungarotoxin. The cells were then treated briefly with Con A, and neural tube cells were added to these cultures. The mobility of ACh receptors was measured by the fluorescence photobleaching recovery method. The Con A treatment prevented rapid diffusion of ACh receptors as well as nerve-induced receptor accumulation. Functional synapse formation was not inhibited by this treatment. In contrast, divalent succinyl Con A did not affect the mobility of ACh receptors nor prevent nerve-induced ACh receptor accumulation. When the Con A concentration was varied, the blocking effect on the nerve-induced receptor accumulation changed in parallel with the mobile fraction of receptors. Newly inserted ACh receptors after the Con A treatment were found to be mobile and to accumulate at the nerve-contact region. In these cultures, new receptors accumulated around old, immobilized receptors in some areas along the nerve contact. This observation suggests that new receptors were inserted elsewhere and migrated to the nerve-contact region surrounding immobilized old ones. In addition to the accumulation of receptors, the nerve disperses preexisting receptor clusters prior to induction of high-density regions along the contact area, and, at this early stage, denervation disperses nerve-induced receptor clusters in Xenopus cultures (Kuromi and Kidokoro, 1984a, b). When cultures were treated with Con A, neither of these events occurred, suggesting that these are also diffusion-mediated. PMID- 3016214 TI - Electrophysiological and anatomical identification of the peripheral axons and target tissues of Aplysia neurons R3-14 and their status as multifunctional, multimessenger neurons. AB - The giant neurons R3-14 in the parietovisceral ganglion of Aplysia, originally proposed to be a homogeneous group of neuroendocrine cells, are likely candidates for a multifunctional and multiple messenger status. The studies reported here suggest that individual R3-14 giant neurons not only innervate specific target tissues but appear to operate more autonomously than previously thought. Identified members of the group were traced into peripheral tissues by electrophysiological, autoradiographic, and intracellular cobalt staining techniques. Five neurons (numbered R6, R7, R8, R11, and R14) were identified on the basis of their unique patterns of axonal projections. R6 innervates the ganglionic artery and pericardial area; R7 and R8, the heart; R11, the kidney; and R14, a large number of vascular tissues. The wide distribution of R3-14 terminals innervating a variety of vascular tissues indicates that several general and local aspects of circulatory physiology are likely to be regulated by these neurons. R3-14 contain the free amino acid glycine, a putative neuromodulator that potentiates cardiac and vascular smooth muscle contraction and several small peptides of unknown, but probably neurohormonal, function. A model is proposed in which R3-14 release glycine to modulate local (e.g., hemolymph pressure and distribution) cardiovascular performance and, indirectly, metabolic homeostasis as well. PMID- 3016215 TI - A V1-like receptor mediates vasopressin-induced flank marking behavior in hamster hypothalamus. AB - A vasopressin-sensitive mechanism within the medial preoptic area-anterior hypothalamus (MPOA-AH) appears to be essential for expression of a complex behavior involved in olfactory communication in Golden hamsters called flank marking. The present study investigated whether the induction of flank marking by arginine-vasopressin (AVP) within the MPOA-AH is mediated by a receptor that is more similar to the vasopressor (V1) or the antidiurectic (V2) AVP receptor. Adult male hamsters were anesthetized and implanted with a 26 gauge guide cannula stereotaxically aimed at the MPOA-AH and then microinjected with analogs of vasopressin, oxytocin, and selective V1 and V2 antagonists. Hamsters were tested for flank-marking behavior during a 5 or 10 min observation period following the injection of peptide in a vehicle of 100 nl of saline. None of the 15 analogs of AVP and oxytocin produced more flank marking than the 50.8 +/- 16.2 and 76.8 +/- 4.4 (mean +/- SEM; n = 4) flank marks observed following injection of AVP at the 1 or 10 ng dose, respectively. The number of flank marks produced by each analog was found to be highly related to the pressor activity of that analog at both the 1 ng (rho = +0.74, p less than 0.01) and 10 ng (rho = +0.82, p less than 0.01) doses. In contrast, no statistically reliable relationship between flank marking and the antidiuretic activity of these analogs was found at either dose (1 ng: rho = +0.07, p greater than 0.05; 10 ng: rho = +0.10, p greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016216 TI - Automated comparison of scintigraphic images. AB - New algorithms for automated comparison of scintigraphic images have been developed and are described here. The first step presented here is the registration of the images, performed by optimizing, with respect to the registration parameters (two translational shifts, one angle of rotation, the two parameters of a linear transformation of the gray levels), the stochastic sign change (SSC) criterion. The optimization of this criterion is demonstrated to be efficiently performed using the adaptative random search strategy; a more limited but less time-consuming method is also presented. The second step described is the point-by-point comparison of the registered images. The pixel-by-pixel application of Poisson variable statistical tests permits the generation of the significant image differences. From such images it is possible to detect modifications which escape visual inspection. Examples of applications are given in controlled and routine conditions. These algorithms are useful for the processing of many investigations and are proposed for implementation on all nuclear medicine data processing systems. PMID- 3016218 TI - Autogenous rib graft-hydroxylapatite augmentation of the severely atrophic mandible: preliminary report. AB - Traditional transoral rib grafts were combined with particulate hydroxylapatite (HA) for augmentation in four patients who had advanced alveolar atrophy or cortical resection of the mandible. Follow-up evaluation for up to 18 months revealed restoration of mandibular morphology, with good prosthetic function and insignificant resorption. No infection or graft loss was observed, in spite of wound dehiscence in two of the four patients, possibly due to the placement of the HA lateral and superior to the rib grafts. PMID- 3016217 TI - Hydroxylapatite blocks and particles as bone graft substitutes in orthognathic and reconstructive surgery. AB - A three-year clinical evaluation of 98 patients in whom dense hydroxylapatite in particle and block form had been placed in facial contour defects and osteotomy sites, and in cystic and reconstructive defects, alone or with autogenous bone, was conducted. The results indicate that the implants were effective in reducing operating time and potential for infection and relapse, as well as in reducing or eliminating the necessity of a donor site. The clinical response was excellent, and complications with both forms were minor, generally related to lack of initial fixation or failure to use autogenous bone in specific situations. PMID- 3016219 TI - Tissue response in dogs to dense hydroxylapatite implantation in the femur. AB - Six and eight years after the implantation of both granular and solid ceramic hydroxylapatite forms in the femurs of beagle dogs, histologic examination demonstrated that the implants were totally encased in dense mature bone. The endosteal and periosteal surfaces appeared normal, and no resorption of the solid implants was observed. However, at six years, a few granules located at the periosteal surface showed interdigitation of connective tissue stalks, with large multinucleated cells at the interface with the implant. This phenomenon may represent some limited resorptive activity on the surfaces of these few isolated granules. Initially, radiographs showed exaggerated degrees of bone deposition on the endosteal surface under the solid implants (discs), as opposed to a less pronounced endosteal response to the implants of particulate material. In some cases, particularly with the disc implants, cracks were found in the ceramic material six years after implantation. These cracks, on staining, were found to be filled with amorphous material, suggesting an osseous matrix. The results of these long-term studies indicate that such hydroxylapatite implants in bone are highly biocompatible. Bone deposition and maturation on the implant surface resulted in a homogeneous bone/implant interface in which the host tissues appeared to respond to the implant as if it were normal bone. PMID- 3016221 TI - A commentary on the pathogenesis and biochemical profile of alcohol-induced depression. PMID- 3016220 TI - Linkage analysis in a family with dominantly inherited torsion dystonia: exclusion of the pro-opiomelanocortin and glutamic acid decarboxylase genes and other chromosomal regions using DNA polymorphisms. AB - A search for the defective gene causing torsion dystonia has been carried out in a family manifesting an autosomal dominant mode of inheritance of this movement disorder. Complete neurologic examination and establishment of lymphoblast lines have been carried out for over 50 members. Linkage analysis, using cloned DNA sequences and restriction fragment length polymorphisms, was evaluated by the LOD score method with requisite assumptions for mode of inheritance, age-of-onset and incomplete gene penetrance. Genes for pro-opiomelanocortin and glutamic acid decarboxylase, which have been implicated in the etiology of the disease in rat models, were excluded as being responsible for the disease state in this family. Other regions of the genome were also excluded using DNA probes for other genes and random "unique" sequences. PMID- 3016222 TI - Contiguous gene syndromes: a component of recognizable syndromes. PMID- 3016223 TI - Ketoconazole treatment of type 1 autoimmune polyglandular syndrome: effects on pituitary-adrenal axis. PMID- 3016224 TI - Familial occurrence of malformations possibly attributable to vascular abnormalities. PMID- 3016225 TI - Structural analysis of the calcium-binding protein calmodulin from Ascaris suum obliquely striated muscle. AB - Calmodulin was purified from the obliquely striated skeletal muscle of Ascaris suum. The calmodulin had a molecular weight of 16,400 and the amino acid composition indicated it is highly similar to other purified calmodulins, showing insignificant variation in 12 of 17 residues. In the residues that showed variation, a trend towards conservative substitution was observed. Spectrophotometric absorption maxima of 276 nm and 283 nm were observed. A molar absorption coefficient of 7,800 was calculated. Calcium-dependent binding to phenothiazine Affi-Gel confirmed that calcium binding induces conformation changes characteristic of calmodulin. Double reciprocal analysis of phosphodiesterase activation by A. suum calmodulin gave a Kapp of 40 nM. PMID- 3016227 TI - Clinical associations of Norwalk-like virus in the stools of children. AB - From among 1,360 stool specimens examined during the 19-month period from March 1982 to September 1983, small round-structured viruses, morphologically indistinguishable from the Norwalk agent, were detected by electron microscopy in 42 specimens from 28 children. Diarrhoea was mild, and vomiting, although frequently observed, was not an invariable feature. Mild pyrexia occurred in three patients. Many cases occurred some time after admission to the ward; this and other features suggested a hospital-acquired infection. These cases in particular tended to have other viral infectious agents detected at the same time. Persistent diarrhoea occurred in only three cases; one with preexisting gastrointestinal pathology, and two with a combined Norwalk-like agent, astrovirus, and rotavirus infection. PMID- 3016226 TI - The effects of gaviscon and metoclopramide in gastroesophageal reflux in children. AB - Oral metoclopramide (0.5 mg/kg/24 h) and a liquid alginic acid-antacid compound were administered to infants and children with gastroesophageal reflux (GER) in a double-blind randomized controlled trial. The effect of medication was measured with 24-h intraesophageal pH monitoring. Neither metoclopramide nor the alginic acid-antacid compound decreased the frequency or duration of gastroesophageal reflux. PMID- 3016228 TI - Regulation of mannitol catabolism in Candida albicans: evidence for cyclic AMP independent glucose effect. AB - Candida albicans was examined for a glucose effect and showed typical diauxic growth on a mixture of glucose and mannitol, in which mannitol utilization occurred only after exhaustion of glucose. The activity of NAD-linked mannitol dehydrogenase was very low while glucose was present in the medium, but started to increase after consumption of glucose. This increase in activity was fully prevented by trichodermin, an inhibitor of protein synthesis. The uptake of mannitol was detected in the cells grown on mannitol, but not in those grown on glucose with or without mannitol. Mannitol uptake by mannitol-grown cells was not affected by the presence of glucose (0.2 g l-1). These findings indicate that in C. albicans glucose represses the inducible syntheses of mannitol dehydrogenase and a mannitol transport system, and that the involvement of inducer exclusion in this effect is unlikely. Fructose, and to lesser extents galactose, mannose and sucrose, also exhibited similar effects on mannitol metabolism. No correlation was found between the intracellular cyclic AMP levels and the glucose effect. PMID- 3016229 TI - Effect of nucleotides on glucan synthesis in Paracoccidioides brasiliensis. AB - The activity of Paracoccidioides brasiliensis glucan synthetase was partially inhibited by guanosine 5'-triphosphate, adenosine 3'5'-phosphate and adenosine 5' triphosphate. This inhibition was more pronounced in the mycelial system than in the yeast one, being higher at 23 degrees C than at 37 degrees C. Addition of ethene diamine tetracetic acid to the incubation mixture inhibited partially the enzymatic activity in mycelial preparations but stimulated it in the yeast system. PMID- 3016230 TI - Inhibition of superoxide generation by human polymorphonuclear leukocytes with chlorhexidine. Its possible relation to periodontal disease. AB - During the acute phase of periodontal disease, as many as 60% of the cells of the junctional epithelium may be accounted for by polymorphonuclear cells (PMNs). It is generally accepted that these cells play a dominant role in the destruction of connective tissue by virtue of the proteinases they release and the free oxygen radicals they generate. Modulation of the proteolytic activity and free radical production seems to be essential for the inhibition of tissue destruction. We have therefore studied the effect of chlorhexidine on the generation of free oxygen radicals and luminol-dependent chemiluminescence (LDCL) by stimulated human PMNs. Nontoxic chlorhexidine concentrations (0.1-1 microgram/ml) were found to inhibit superoxide production but did not affect LDCL. We therefore suggest that in addition to its antiseptic effect, chlorhexidine may also modulate the generation of free radicals by activated PMN cells in the inflamed gingiva. PMID- 3016231 TI - The effect of root detoxification on human gingival fibroblasts. AB - Comparative growth or inhibition of growth of human gingival fibroblasts on experimental root surfaces was used to assess the efficacy in vitro of the Cavitron and Prophy-Jet. Six teeth with a hopeless periodontal prognosis, slated for prosthetic replacement, were extracted and sectioned into 12 specimens. Experimental specimens were instrumented with the Cavitron or Cavitron and Prophy Jet. Four calculus-covered control specimens were not treated. Eight root specimens in fibroblast tissue culture were stained with 1.0% toluidine blue for determination of fibroblast viability after 48 hours. No fibroblast growth took place on calculus control specimens. Cavitron specimens showed light fibroblast growth and viability. Cavitron/Prophy-Jet specimens showed superior growth and vitality of fibroblasts. PMID- 3016232 TI - Pharmacological studies of imidazoline derivatives. I. A comparison of the effect on pre- and postsynaptic alpha-adrenoceptors in the rat vas deferens. AB - The effects of 8 imidazoline derivatives on pre- and postsynaptic alpha adrenoceptors and [3H]norepinephrine release were investigated in the rat vas deferens and were compared with those of reference drugs, yohimbine, phentolamine, tolazoline, and prazosin. K-6341 and K-6343 activated postsynaptic alpha 1-adrenoceptor and were antagonized by prazosin. The order of potency series as alpha 1-antagonist, prazosin greater than phentolamine greater than yohimbine greater than K-4011 greater than K-3827 greater than K-4300, tolazoline was found in antagonism against phenylephrine. Many derivatives exhibited an antagonistic action against clonidine, and the potency series as alpha 2 antagonist was revealed as follows: phentolamine greater than yohimbine greater than K-3827 greater than K-4011 greater than K-6341 greater than K-6343 greater than K-4300, tolazoline greater than prazosin greater than K-4299. The antagonistic effect was more selective on presynaptic (alpha 2) than postsynaptic (alpha 1) adrenoceptor and the rank order of selectivity (KB-post/KB-pre) was K 3827 greater than yohimbine greater than K-4011 greater than tolazoline, K-4300 greater than phentolamine greater than prazosin. Imidazoline derivative increased [3H]norepinephrine release, the actions of K-3827, K-6341 and K-4300 being particularly potent. From these results, the structure-activity relationship was summarized. First, when 2',3'-methylenedioxyphenyl group is connected with the imidazoline ring via an amino or methylene residue, these compounds become alpha 1-agonist. Secondly, when the methylenedioxyphenyl group and the imidazoline ring are connected via an amino residue, these compounds show a weak agonistic and potent antagonistic characteristics at the presynaptic alpha 2-adrenoceptor. Thirdly, an addition of a methylenedioxy radical to the phenyl ring increased the affinity of the compounds to presynaptic alpha 2-receptor. PMID- 3016233 TI - Radioimmunoassay for the quantitation of lisinopril and enalaprilat. AB - A sensitive radioimmunoassay (RIA) capable of measuring either lisinopril (1-[N2 [(S)-1-carboxy-3-phenylpropyl]-L-lysyl] -L-proline) or enalaprilat (1-[N-[(S)-1 carboxy-3-phenylpropyl]-L-alanyl] -L-proline), the active metabolite of enalapril has been developed. A suitable antiserum was raised against an immunogen prepared from conjugation of lisinopril, the lysyl analogue of enalapril, with succinoylated keyhole limpet hemocyanin. A novel radiotracer was also prepared for use in the assay by acylation of the epsilon amine group on the lysyl side chain of lisinopril with N-succinimidyl [2,3-3H]propionate. The antiserum was used at a final dilution of 1:44,500 and the sensitivity of the assay for enalaprilat was estimated at 2 pmol/mL plasma sample and 0.4 pmol/mL for lisinopril. Enalapril, the ethyl ester of enalaprilat, exhibited little cross reactivity (0.005%), and several other compounds (captopril, proline, lysine, tyrosine, hippuric acid, and tryptophan) were found not to crossreact. In rabbits given a 2.03 mumol/kg iv dose of enalapril, plasma concentrations of enalaprilat were determined by the RIA technique and compared with an estimation of the enalaprilat concentrations derived from the extent of inhibition of plasma angiotensin converting enzyme (ACE). The plasma levels estimated by ACE inhibition were less than those obtained by the RIA in the first 45 min but were always greater in the samples taken after this time. Both assay methods showed that the conversion of enalapril to enalaprilat was rapid, and also indicated that there was initial rapid clearance of enalaprilat from the plasma. PMID- 3016234 TI - Interactions in the solid state. II: Interaction of sodium bicarbonate with substituted benzoic acids in the presence of moisture. AB - The interaction of an organic acid with sodium bicarbonate in water produces an effervescent reaction. The reaction products are carbon dioxide, water, and the sodium salt of the acid. The kinetic rate-determining step for this reaction is the dehydration of carbonic acid. The solid-solid interaction with known amounts of moisture was followed by quantitatively determining carbon dioxide evolution as a function of time. The aqueous solubilities, diffusion coefficients, dissociation constants, and solid-solid interaction rates of six different substituted benzoic acids were determined. Using a model based on diffusion of the organic acid through the aqueous layer coupled with chemical reaction, predicted rates and levels of carbon dioxide production were compared with experimental results. Included in the model were the effects of the reaction products on the solution properties of the reactants. It was found that high concentrations of substituted sodium benzoate were generated very quickly and affected the solubility of the reactants, diffusion coefficient of the acid, and the carbonic acid dehydration rate constant. Moisture content was found to have a profound influence on the interaction rate. Water provides a medium for diffusion of the reacting species as well as the reaction solvent. PMID- 3016235 TI - Conformation of glyburide in the solid state and in solution. AB - Nuclear magnetic resonance spectroscopy was combined with X-ray crystallography to determine the solution and solid-state conformations of glyburide (C23H28CIN3O5S). In solution, there is apparently free rotation about several of the single bonds. Crystals of glyburide belong to space group P2(1)/n with a = 9.414(3)A, b = 17.591(5)A, c = 14.410(4)A, beta = 93.42(3) degrees, V = 2382(1)A3, R = 0.0694, Z = 4. In the solid, the conformation about all of the bonds is frozen. The torsion angles about some of the important bonds are: C(7) N(8)-C(9)-C(10), -86 degrees; N(8)-C(9)-C(10)-C(11), -179 degrees (extended); S(17)-N(18)-C(19)-N(20), 178 degrees (extended); and N(18)-C(19)-N(20)-C(21), 179 degrees (extended). Thus, except for the N(8)-C(9) bond, the conformation in the solid state is extended. The solution and solid-state NMR spectra are different as might be expected based on the differences in conformation discussed above. PMID- 3016236 TI - Desensitization of aortic smooth muscle contraction in rats harboring pheochromocytoma. AB - Desensitization of smooth muscle contraction was studied in aortic ring segments obtained from New England Deaconness Hospital rats harboring pheochromocytomas, a norepinephrine-secreting tumor. Rats were studied 5 to 6 weeks after implantation of the pheochromocytoma, by which time severe hypertension had developed. Aortic ring segments from the pheochromocytoma rats were significantly less sensitive to the alpha-1 adrenergic agonist phenylephrine [log10 (ED50) = -7.21 +/- 0.09 vs. 7.63 +/- 0.11 in controls). In addition, the maximal force of contraction induced by phenylephrine was decreased in the aortas from pheochromocytoma rats (1.31 +/- 0.15 g) compared to controls (2.17 +/- 0.23 g). The potency of the thromboxane A2 receptor agonist U-46,619 was decreased in the aortic ring segments from pheochromocytoma rats compared to controls, although it elicited a similar maximal force of contraction. Desensitization of alpha receptor-mediated contraction was prevented by treating the pheochromocytoma-bearing rats with the reversible alpha adrenergic antagonist phentolamine (200 micrograms/kg/hr) via osmotic minipumps for 2 weeks. However, phentolamine did not decrease the hypertension in these rats. Also, large doses of the irreversible alpha adrenergic antagonist phenoxybenzamine (4 mg/kg/day i.p.) did not decrease blood pressure in rats harboring pheochromocytoma or did the drug completely block aortic alpha receptors in these rats as it did in controls. The results indicate that pheochromocytoma induces heterologous desensitization of smooth muscle contraction in rat aorta. The desensitization is due to direct effects of catecholamines unrelated to hypertension. The New England Deaconess Hospital rat harboring pheochromocytoma is an interesting model to study the effects of high concentrations of plasma catecholamines on smooth muscle contraction. PMID- 3016237 TI - Kappa opiate agonists modulate the hypothalamic-pituitary-adrenocortical axis in the rat. AB - Systemic injections of opiate agonists were made in male rats to elucidate the involvement of multiple opioid receptors in the stress response. As an index of activity in the hypothalamic-pituitary-adrenocortical axis, plasma corticosterone was measured by radioimmunoassay. Rats were injected with ethylketocyclazocine (EKC), U50488H, MR2034, bremazocine or tifluadom and sacrificed 1 hr later. These kappa agonists produced potent, dose-dependent, stereospecific increases in plasma corticosterone levels at doses far below those needed to elicit analgesia. These effects were reversed by opiate antagonists, naloxone or Win 44441-3, which by themselves caused dose-dependent decreases in plasma corticosterone. Animals made tolerant to the prototype kappa agonist, U50488H, showed an attenuated response to an acute injection of the drug. However, when animals made tolerant to morphine were injected acutely with U50488H, the drug caused a dramatic increase in corticosterone levels. In hypophysectomized rats, U50488H and L-EKC did not increase plasma corticosterone. The agonist/antagonists, butorphanol and cyclazocine, when injected, behaved like kappa agonists and increased plasma corticosterone levels potently. The mu opiates, morphine and etorphine, also had similar effects but were less potent and efficacious than the kappa agonists. The delta agonist D-Ala-D-Leu enkephalin showed similar results, confirming a mu and delta opioid input into the hypothalamic-pituitary-adrenocortical axis. There were concomitant increases in plasma adrenocorticotropin in morphine-, D-Ala-D Leu enkephalin-, L-EKC- and U50488H-treated rats which were also seen in adrenalectomized rats. D-EKC and D-cyclazocine, which bind to sigma sites, had no effect on corticosterone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016238 TI - 5-Hydroxytryptamine2 receptors coupled to phospholipase C in rat aorta: modulation of phosphoinositide turnover by phorbol ester. AB - In rat aorta, 5-hydroxytryptamine (5-HT) stimulated phosphoinositide (PI) turnover and contraction (EC50 = 10 +/- 3 microM); these two responses were highly correlated (r = 0.95; P less than .01). We have characterized the inhibitory potency of a variety of 5-HT-antagonists against the stimulation of PI turnover elicited by 5-HT. Classic 5-HT2 antagonists mianserin, ketanserin, metergoline and pizotifen were found to inhibit this response in the low nanomolar range; amitryptiline and haloperidol were 10- to 20-fold less potent. The alpha-1 receptor antagonist, prazosin, was inactive in micromolar concentrations. The potency of the 5-HT2 antagonists was correlated with their ability to displace [3H] ketanserin binding from rat frontal cortex membranes (r = 0.90; P less than .05). The tumor promoter phorbol dibutyrate was found to inhibit 5-HT-stimulated PI turnover at low nanomolar concentrations whereas the biologically inactive substance 4-alpha-phorbol was ineffective. Pretreatment of rat aorta with phorbol dibutyrate at concentrations that inhibited 5-HT-induced PI turnover also attenuated the aortic contraction induced by 5-HT in the presence of a calcium channel blocker nitrendipine. Our results suggest that phorbol esters may desensitize 5-HT2-receptor-mediated PI turnover and contraction of rat aorta, possibly via an activation of protein kinase C. PMID- 3016239 TI - Rapid desensitization of the acute stimulatory effects of nicotine on rat plasma adrenocorticotropin and prolactin. AB - The dose of nicotine and the frequency of its administration appear to be essential determinants of its action on multiple systems including the neuroendocrine regulation of the adrenocorticotropin (ACTH)-corticosterone and prolactin (PRL) axes in the rat. Because desensitization to the acute depressive effects of nicotine has been observed after both acute and chronic administration, these investigations assessed whether desensitization to the stimulative effects of nicotine on ACTH and PRL secretion occurs with repetitive dosing. Extensive dose and time course experiments showed that nicotine rapidly elevates rat plasma ACTH and PRL levels with a threshold dose between 0.1 to 0.25 mg/kg b.wt. i.p. After the stimulation of PRL, levels became significantly depressed. Desensitization to the acute stimulatory effects of nicotine on both hormones was induced by a single dose of nicotine (0.5 mg/kg). One hour later nicotine (1.0 mg/kg) failed to significantly stimulate PRL levels and resulted in a modest increase of ACTH. Desensitization was maximal by 1 hr after the first dose and persisted for at least 6 hr. Adrenalectomy, performed to eliminate corticosterone-induced negative feedback, did not enhance PRL responsiveness to a second dose of nicotine but it partially restored the ACTH response. Pretreatment with corticosterone also failed to modify the PRL response to a single dose of nicotine whereas it partially suppressed the ACTH response. Rapid desensitization to the acute stimulatory effects of nicotine on plasma PRL is independent of glucocorticoid negative-feedback whereas desensitization of the ACTH response is modestly dependent. PMID- 3016240 TI - Differential inhibition of alpha-1 vs. alpha-2 adrenoceptor-mediated responses in canine saphenous vein by nitroglycerin. AB - In the present investigation, the subtype of alpha adrenoceptor that is preferentially antagonized by the noncalcium entry blocker, nitroglycerin (GTN), was studied in vitro using rings prepared from isolated canine saphenous vein (CSV). The calcium entry blocker, diltiazem (DZ), was used for purposes of comparison. Contractions were produced by the following alpha adrenergic agonists: norepinephrine (NE), a nonselective alpha adrenoceptor agonist, phenylephrine (PE), a selective alpha-1 adrenoceptor agonist and B-HT 920, a selective alpha-2 adrenoceptor agonist. The inhibitory effects of GTN on contractions produced by submaximal concentrations (EC65 to EC75) of NE, PE and B HT 920 were determined. GTN was found to inhibit preferentially the postsynaptic alpha-2 adrenoceptor-mediated responses to B-HT 920 while producing minimal effects on those produced by NE or PE. Similar results were found with DZ. However, when a portion of the alpha-1 adrenoceptor pool was inactivated by phenoxybenzamine (5 X 10(-8) to 1 X 10(-7) M), GTN and DZ both produced a significant depression of responses to PE, a full alpha-1 adrenoceptor agonist. In addition, contractions produced by l-dobutamine, a selective partial alpha-1 adrenoceptor agonist (no alpha receptor reserve), were highly sensitive to inhibition by GTN. These results suggest that the selective inhibition of contractions mediated by postjunctional alpha-2 vs. alpha-1 adrenoceptors in CSV by GTN and DZ may be due partially to the presence of a large alpha-1 adrenoceptor reserve that conceals an underlying functional antagonism to alpha-1 adrenoceptor-mediated responses.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016241 TI - Evidence for alpha adrenoceptor modulation of the nociceptive jaw-opening reflex in rats and rabbits. AB - The jaw-opening reflex (JOR) in anesthetized rats and rabbits was the pain paradigm studied. The JOR was elicited by the electrical intrapulpal (left maxillary) stimulation and quantified by the measurement of threshold values for eliciting electromyograms (dEMGs) from the ipsilateral digastric muscle which served as the experimental nociceptive index. In both species, the threshold for the JOR was significantly elevated by the systemic administration of clonidine (12.5-50 micrograms/kg i.v.) and these JOR thresholds were inversely correlated with the frequency of stimulation. The analgesia elicited by clonidine was antagonized by pre- and postdrug treatment with the alpha-2 adrenoceptor antagonist yohimbine (1 mg/kg i.v.) but not the alpha-1 adrenoceptor antagonist prazosin (1 mg/kg i.v.) or the opiate receptor antagonist naloxone (1 mg/kg i.v.). The lipophilic alpha-1 adrenoceptor agonist St587 (100-400 micrograms/kg i.v.) had no significant effect on dEMG. Yohimbine did not antagonize the increase in dEMG elicited by morphine or pentobarbital. There was no direct correlation between the antinociceptive and cardiovascular effects of clonidine. Our results suggest that in the JOR nociceptive paradigm, clonidine elicits potent analgesia by activation of alpha-2 and not alpha-1 adrenoceptors. PMID- 3016242 TI - Analysis of cardiac muscarinic receptors recognized selectively by nonquaternary but not by quaternary ligands. AB - Three muscarinic receptor antagonists, [3H]quinuclidinyl benzilate ([3H]QNB), N [3H]methylscopolamine ([3H]NMS and N-[methyl-3H]QNB ([3H]NMeQNB), each bind to an apparently homogeneous population of receptors on intact chick heart cells. [3H]QNB binds to approximately 9500 sites/cells, whereas [3H]NMS and [3H]NMeQNB bind to approximately 5000 sites/cell. Atropine and scopolamine compete with all three radioligands with a single, high affinity. Their quaternary analogs N methylatropine and NMS and the quaternary agonist carbachol also show a single affinity for [3H]NMS and [3H]NMeQNB binding sites, but have biphasic competition curves for [3H]QNB sites with low "apparent" affinity for a subpopulation of sites. When 10 nM or greater propylbenzilylcholine mustard is used to alkylate receptors virtually all [3H]NMS binding is abolished, whereas [3H]QNB still labels a significant fraction of the binding sites seen in control cells. The sites with low apparent affinity for quaternary ligands are shown to have characteristics of muscarinic receptors, but do not appear necessary for muscarinic receptor-mediated phosphoinositide hydrolysis. We suggest that a subpopulation of nonfunctional muscarinic receptors are sequestered within the membrane or otherwise inaccessible to hydrophilic or charged ligands. PMID- 3016243 TI - Evaluation of the role of alpha-1 and alpha-2 adrenoceptors in the maintenance of neurogenic tone to the hindlimb vasculature. AB - The location of two distinct subtypes of alpha adrenoceptors on blood vessels is now well established. The present study was designed to assess the relative contribution of alpha-1 and alpha-2 adrenoceptors to the maintenance of sympathetic vasoconstrictor tone to the canine hindlimb. Bilateral hindlimb perfusion was carried out at controlled flow rates where one of the hindlimbs was neurally intact and the other limb was denervated surgically. Vascular resistances in the hindlimbs were evaluated by pressure-flow curves which were constructed by recording perfusion pressures at various flow levels. Administration of alpha-1 adrenoceptor antagonist, prazosin (50 micrograms/kg), which selectively inhibited the hindlimb vasoconstrictor effect of phenylephrine but not of B-HT 933, produced a significant decrease in the hindlimb vascular resistance in the innervated limb. This was reflected in a decrease in perfusion pressure and a downward shift of the pressure-flow curve of 30% from control. No changes in resistance occurred in the denervated limb. The alpha-2 adrenoceptor antagonist, yohimbine (100 micrograms/kg), which selectively antagonized vasoconstriction to B-HT 933 but not to phenylephrine, further decreased by 27% hindlimb vascular resistance in the innervated limb when given to the same group of prazosin-treated animals. The magnitude of the decrease in vascular resistance produced by yohimbine (27%) was similar to that caused by prazosin (30%). The vascular resistance in the innervated limb was now similar to that in the denervated limb after prazosin and yohimbine. Phentolamine, when administered after prazosin and yohimbine, did not produce any changes in the hindlimb vascular resistance in these dogs.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016244 TI - Activity of mu- and delta-selective opioid agonists in the guinea pig ileum preparation: differentiation into peptide and nonpeptide classes with beta funaltrexamine. AB - Previous studies have demonstrated that pretreatment of guinea pig longitudinal muscle-myenteric plexus ileal preparations with the highly selective noncompetitive mu antagonist beta-funaltrexamine (beta-FNA) causes an increase in the Ke value for the interaction of morphine with naloxone, suggesting that beta FNA inactivates those receptors at which morphine interacts in the guinea pig ileum. The effect is selective for mu receptors since beta-FNA has no effect upon the interaction of naloxone with the kappa agonist nalorphine. In the present study, it was found that although beta-FNA attenuated the effects of morphine and other morphine-like agonists at mu receptors in the guinea pig longitudinal muscle-myenteric plexus ileal preparation, the mu-mediated actions of delta selective peptide agonists and mu-selective peptide agonists were not completely attenuated by beta-FNA pretreatment. These data suggest that morphine-like mu agonists and other mu-selective and delta-selective peptide agonists in the guinea-pig ileum preparation either interact with similar opioid receptors but in a distinguishable manner or interact with different populations of opioid receptors or the peptides studied had greater intrinsic activity than the nonpeptides. PMID- 3016246 TI - Relationships between cyclic AMP levels and lipolysis in fat cells after isoproterenol and forskolin stimulation. AB - Using the flask-incubated fat cell system, alterations in glycerol release (lipolysis) and cAMP accumulation were determined after incubation with isoproterenol or forskolin. These agents caused concentration-dependent increases in both cAMP accumulation and lipolysis. The maximum responses to forskolin for each variable were greater than the corresponding responses to isoproterenol. The maximum responses to isoproterenol for both cAMP accumulation and glycerol release were increased by the presence of either adenosine deaminase or theophylline. Under these conditions, high concentrations of isoproterenol continued to increase cAMP accumulation while having no further effect on lipolysis. These results support the concept that the maximum response to isoproterenol alone was limited by the accumulation of cAMP within the cells. The maximum response to isoproterenol in the presence of either theophylline or adenosine deaminase (and to forskolin) was limited by some step in the lipolytic process distal to cAMP accumulation. The relationships between cAMP levels and lipolysis for isoproterenol and forskolin were found to be different. A 6-fold increase in cAMP levels was sufficient to maximally increase lipolysis with isoproterenol, whereas the maximum lipolytic response to forskolin was associated with a 20-fold increase in cAMP levels. A plot of log cAMP vs. glycerol release resulted in linear relationships for both drugs. The slope of the line for isoproterenol was significantly greater than that for forskolin. At any given concentration of cAMP the corresponding lipolytic response was greater for isoproterenol than for forskolin. PMID- 3016245 TI - Lack of pharmacodynamic interactions between quinidine and digoxin in isolated atrial muscle of guinea pig heart. AB - Quinidine has been reported to have no effect on the positive inotropic action of digoxin observed in isolated cardiac muscle preparations. This is surprising because quinidine has been shown to reduce Na+ influx in cardiac muscle. The conditions which increase Na+ influx stimulate the glycoside binding to Na+- and K+-activated Mg++-dependent ATP phosphohydrolase (Na+,K+-ATPase), and therefore quinidine may be expected to have an opposite effect. Thus, the effects of quinidine on cardiac muscle and its possible interactions with digoxin were re evaluated using electrically paced left atrial muscle preparations of guinea pig heart. Quinidine caused a frequency- and concentration-dependent decrease in maximal upstroke velocity and amplitude of the action potential without altering resting membrane potential. In addition, quinidine prolonged action potential duration markedly in a frequency-dependent manner. Despite action potential prolongation, the alkaloid reduced net Na+ influx as determined by a decrease in steady-state ouabain-sensitive 86Rb+ uptake. Under these conditions, however, quinidine failed to reduce the rate of onset or the maximal positive inotropic effect of digoxin; or did it reduce digoxin binding to Na+,K+- ATPase in beating atrial muscle preparations. Benzocaine, which reduced net Na+ influx without increasing the action potential duration, also failed to affect the peak inotropic effect of digoxin or the glycoside binding. Quinidine had no direct effects on glycoside binding to isolated cardiac Na+,K+-ATPase. Moreover, [3H]ouabain binding to isolated enzyme was relatively insensitive to changes in Na+ concentrations between 1 and 8 mM although binding was stimulated clearly by Na+ above 8 mM. These results indicate that quinidine, at therapeutic concentrations, does not interact pharmacodynamically with digoxin in isolated cardiac muscle. PMID- 3016247 TI - Light microscopic autoradiographic localization of delta opioid receptors in the rat brain using a highly selective bis-penicillamine cyclic enkephalin analog. AB - Light microscopic autoradiography was used to visualize the neuroanatomical distribution of rat brain delta opioid receptors. Slide-mounted sections of rat brain were labeled with [3H]-[2-D-penicillamine, 5-D penicillamine]enkephalin([3H]DPDPE), a highly selective delta opioid agonist. Saturation isotherms of [3H]DPDPE binding to thaw-mounted brain slices gave a maximal number of binding sites of 79.9 fmol/mg of protein and an apparent dissociation constant (Kd) of 6.3 nM. DPDPE and met-enkephalin inhibited [3H]DPDPE binding with high affinity (lC50 values of 6.3 and 13.8 nM, respectively). Putative mu opioid receptor selective ligands such as morphine sulfate, Tyr-D-Ala-Gly-NMePhe-Gyl-ol and [N-MePhe3, D-Pro4]morphiceptin (PL017) were less potent inhibitors of [3H]DPDPE binding. The rat brain areas containing the highest densities of receptors were the claustrum, basolateral amygdaloid nucleus, the caudate-putamen and nucleus accumbens, the external plexiform layer of the olfactory bulb and the olfactory tubercle. Moderate receptor density was characteristic of the hippocampal formation in which grains were seen over the molecular layer of the dentate gyrus and stratum oriens (CA1), and of the different layers of cerebral cortex. Generally, low density of binding was found over the thalamus and the septal nuclei. Low specific binding was also seen in the cerebellum, medulla oblongata and in the dorsal horn of the spinal cord. There was little specific [3H]DPDPE binding over the white matter areas. PMID- 3016248 TI - Pharmacological and autoradiographic discrimination of sigma and phencyclidine receptor binding sites in brain with (+)-[3H]SKF 10,047, (+)-[3H]-3-[3 hydroxyphenyl]-N-(1-propyl)piperidine and [3H]-1-[1-(2 thienyl)cyclohexyl]piperidine. AB - The benzomorphan opioid, SKF 10,047, is the prototypical agonist for the sigma receptor. In this study, pharmacological and autoradiographic analyses reveal that (+)-[3H]SKF 10,047 labels two sites in brain: a high affinity site resembling the sigma receptor and a second site, labeled with lower affinity by (+)-[3H] SKF 10,047, similar to the phencyclidine (PCP) receptor. The drug specificity of the high affinity site for (+)-[3H]SKF 10,047 resembles that of the putative sigma receptor labeled with (+)-[3H]-3-[3-hydroxyphenyl]-N-(1 propyl)piperidine [(+)-[3H]-3-PPP], being potently inhibited by (+)-3-PPP, haloperidol and (+/-)-pentazocine, and demonstrating stereoselectivity for the (+)-isomer of SKF 10,047. In contrast, these drugs are weak in inhibiting binding of (+)-[3H]SKF 10,047 to the low affinity site, whereas PCP analogs, such as 1-[1 (2-thienyl)cyclohexyl]piperidine (TCP) and 1-[1-(m aminophenyl)cyclohexyl]piperidine (m-NH2-PCP), are potent inhibitors. No stereoselectivity for the isomers of SKF 10,047 is noted at the low affinity binding site. Autoradiographic localizations of high affinity (+)-[3H]SKF 10,047 binding sites closely resemble those of (+)-[3H]-3-PPP labeled sites with high levels of binding in the hippocampal pyramidal cell layer, hypothalamus, pontine and cranial nerve nuclei and cerebellum. By contrast, low affinity (+)-[3H]SKF 10,047 sites are most abundant in nonpyramidal layers of the hippocampus, the cerebral cortex and thalamic nuclei, similar to the distribution of [3H]TCP labeled PCP receptors. PMID- 3016249 TI - In vivo [3H]flunitrazepam binding: imaging of receptor regulation. AB - The use of [3H]flunitrazepam as a ligand to measure alterations in benzodiazepine receptors in vivo in rats was investigated. Animals were injected with [3H]flunitrazepam i.v., arterial samples of [3H]flunitrazepam were obtained and, later, the animals were sacrificed to assay brain binding. [3H]flunitrazepam enters the brain rapidly and binds to benzodiazepine receptors. About two-thirds of this binding is blocked by predosing the animals with 5 mg/kg of clonazepam. The amount of remaining (nonspecific) binding correlates very well (r = 0.88) with the amount of radioactivity found in plasma at the time of death. A series of rats were lesioned unilaterally with kainic acid in the caudate-putamen several months before the infusion of [3H]flunitrazepam. In vivo autoradiography in lesioned rats showed that benzodiazepine binding in globus pallidus and substantia nigra on the side of the lesion was increased significantly as compared to the intact side. The observed changes in benzodiazepine binding were similar to those observed previously in lesioned rats using in vitro techniques. Thus, benzodiazepine receptor regulation can be imaged quantitatively using in vivo binding techniques. PMID- 3016250 TI - Effects of antidepressant drugs injected into the amygdala on behavioral responses of rats in the forced swim test. AB - Experiments were conducted to determine if the actions of antidepressant drugs in a pharmacological screen would be localized to specific brain regions. Rats were infused in discrete brain regions with drugs of different pharmacological properties and processed in the forced swim test. Infusion of imipramine and pargyline into the amygdala produced behavioral responses similar to i.p. injections of the drugs. The regions of the amygdala from which positive responses could be elicited were highly selective. From cannula placements and diffusion studies with autoradiography it appears that a locus of action of imipramine and pargyline is confined to the central, basolateral and/or lateral nuclear regions of the amygdala. Behavioral responses of rats in which imipramine was infused into the anterior amygdala or caudate-putamen did not differ from saline controls. When infused into regions of the amygdala in which imipramine was active, iprindole, an "atypical" antidepressant, did not produce a behavioral response in the forced swim test. Two "false positives" in the forced swim test, atropine and amphetamine, were also not active when infused into the amygdala. These results indicate that the amygdala is a critical site of action for certain antidepressant drugs in the forced swim test, and that behavioral activation in the test induced by iprindole, amphetamine and atropine involve brain regions other than the amygdala. PMID- 3016251 TI - [Regulatory mechanism of contraction-relaxation cycle in smooth muscle cells]. PMID- 3016252 TI - Calcium-dependent phosphatidylinositol phosphorylation in lamellibranch gill lateral cilia. AB - Pure lateral (L) cilia may be separated from the remaining (R) cilia types of Mytilus edulis gill by serotonin activation after hypertonic shock. The two classes of cilia were permeabilized with 0.012% Triton X-100 and incubated with 32P-labeled ATP at low Ca++ (10(-7) M), where L cilia beat, or in high Ca++ (2-20 microM), where L cilia arrest but R cilia are active. The labeled cilia were separated into axoneme and membrane-matrix fractions by detergent extraction, subjected to SDS-PAGE on 5-15% gels, and autoradiographed. Neither cilia type undergoes Ca++-dependent phosphorylation of specific proteins, suggesting that neither Ca++-induced arrest in L cilia nor the Ca++ activation of other cilia is phosphorylation-dependent. However, lipid phosphorylation in L cilia is highly Ca++-dependent. Identified by thin-layer chromatography, the phospholipid that is phosphorylated in a Ca++-dependent manner is phosphatidylinositol 4-phosphate (PIP), yielding the 4,5-bisphosphate (PIP2). PIP2 increases at least 3-fold under Ca++-arrest conditions. Aequipecten gill lateral cilia, which require higher Ca++ levels for arrest, show even more striking changes. In both cases, the effect is maximal at micromolar Ca++ levels. Phosphorylation of other lipids is Ca++ independent. In the Ca++-insensitive or activated R cilia, PIP2 levels are intermediate, increasing only marginally with increased [Ca++]. The formation of PIP2 in response to Ca++, as opposed to its breakdown to form inositol 1,4,5 trisphosphate and diacylglycerol, may be characteristic of a Ca++ transport system. Mechanically sensitive, the L cilia arrest as a consequence of an inward flux of Ca++ ions, acting directly on the axoneme.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016253 TI - Cyclic AMP and calcium in the differential control of Mytilus gill cilia. AB - Lateral (L) cilia of Mytilus gill are activated by serotonin which, in molluscan systems, is known to activate adenylate cyclase. Triton-extracted models of L cells, arrested at greater than 10(-6) M Ca++, are stimulated to beat by the addition of 10(-5) M cAMP while still under Ca++ arrest conditions, suggesting that cAMP-activation is not mediated by alterations of Ca++ levels. Using isolated, permeabilized cilia, we find, independent of [Ca++], that cAMP dependent protein phosphorylation in L-cilia occurs uniquely and reversibly on three low molecular weight polypeptides of 23,000, 18,000, and 14,000 daltons. Phosphorylation is maximal at cAMP concentrations above 0.5 microM. The phosphorylated chains partially co-extract at high salt with a 14S dynein fraction and have approximately the same molecular weights as reported for dynein light chains. Such conditions mainly extract the outer dynein arm, about 40% of the Mg++-ATPase activity, and a corresponding amount of the cAMP phosphorylated chains. However, the three polypeptides sediment together at 10-11S, clearly separable from the 14S dynein ATPase. Using a gel-overlay technique, we find that calmodulin binds to axonemal polypeptides of L-cilia with molecular weights of 18,000 and 13,000, independent of Ca++, while in mixed-population cilia, only a 12,000 dalton chain binds calmodulin, in a Ca++ dependent manner. In neither case are calmodulin binding proteins found in the high salt fraction containing the cAMP-dependent phosphorylated chains, indicating that, in spite of some similarity in molecular weight, the cAMP-phosphorylated and calmodulin binding polypeptides are different. Also, double-labelling indicates that only the 18,000 dalton chains co-migrate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016254 TI - S-neurons and not L-neurons are the source of GABAergic action in the ocellar retina. AB - Electrophysiological evidence obtained with current- and voltage clamp experiments from single L-neurons of the ocellar nerve of locust (Locusta migratoria) questions a direct synaptic feedback from these neurons onto the photoreceptors. The synaptic currents recorded under voltage clamp reflected the photoresponse of the L-neuron, despite the fact it developed no synaptic activity under these conditions. This result is contrary to GABAergic feedback models proposed in the literature. Electrophysiological recordings, as well as immunocytochemistry revealing GABA and glutamate decarboxylase, indicated a possible contribution of S-neurons in such a feedback system. A population of probable S-neurons whose somas were in the pars intercerebralis adjacent to the ocellar nerve tracts was heavely labelled. About 10 fibres entered each tract and formed a dense network of fine arborizations within the ocellar plexiform layer. L-neurons showed no GABA-immunoreactivity. Based on these data a new model for GABAergic feedback is proposed and discussed. PMID- 3016255 TI - Electrical coupling of neuro-ommatidial photoreceptor cells in the blowfly. AB - A new method of microstimulation of the blowfly eye using corneal neutralization was applied to the 6 peripheral photoreceptor cells (R1-R6) connected to one neuro-ommatidium (and thus looking into the same direction), whilst the receptor potential of a dark-adapted photoreceptor cell was recorded by means of an intracellular microelectrode. Stimulation of the photoreceptor cells not impaled elicited responses in the recorded cell of about 20% of the response elicited when stimulating the recorded cell. This is probably caused by gap junctions recently found between the axon terminals of these cells. Stimulation of all 6 cells together yielded responses that were larger and longer than those obtained with stimulation of just the recorded cell, and intensity-response curves that deviated more strongly from linearity. Evidence is presented that the resistance of the axon terminal of the photoreceptor cells quickly drops in response to a light flash, depending on the light intensity. Incorporating the cable properties of the cell body and the axon, the resistance of the gap junctions, and the (adapting) terminal resistance, a theoretical model is presented that explains the measurements well. Finally, it is argued that the gap junctions between the photoreceptor cells may effectively uncouple the synaptic responses of the cells by counteracting the influence of field potentials. PMID- 3016256 TI - Significance of intracellular calcium and cyclic adenosine 3',5'-monophosphate in the mechanisms by which prostaglandins influence human sperm function. AB - The selective ability of PGE-1 and PGE-2 to enhance the capacity of human spermatozoa for hamster oocyte penetration was dependent upon high concentrations (1.7 mM) of extracellular Ca2+ and a prolonged (4 h) duration of exposure, and insensitive to the Ca2+ channel antagonist, verapamil. Studies with the intracellular calcium indicator Quin-2 indicated that exposure to PGE-1 and PGE 2, but not PGF-2 alpha, induced a significant rise in the levels of cytoplasmic Ca2+, suggesting that an ionophore-like action might be responsible for the ability of the E-series prostaglandins to influence sperm function. Stimulation of oocyte penetration with PGE-1 and PGE-2 was significantly enhanced when these compounds were presented to the spermatozoa in medium of high osmolarity (410 mosmol). A combination of PGE-1 in hyperosmotic medium did not significantly influence sperm function in cases of oligozoospermia, although it was effective with patients exhibiting idiopathic infertility. Exposure to high doses of PGE-1 and PGE-2, but not 19-hydroxy PGE or PGF-2 alpha, also induced a significant rise in the cyclic AMP content of human spermatozoa. This effect did not appear to be involved in the enhancement of fertilization rates because it did not exhibit the same absolute dependence on high levels of extracellular Ca2+ as did the fertilization responses and the enhancement of oocyte penetration and the elevation of cAMP were independent of each other within the patient population. PMID- 3016257 TI - Transient uterine refractoriness after oxytocin administration in ewes. AB - Steroid-primed, ovariectomized ewes were treated intravenously with 2 doses of 1 microgram oxytocin at intervals of 1, 2, 4 or 6 h. The initial dose resulted in increases in 13,14-dihydro-15-keto-PGF-2 alpha in the peripheral circulation from 173 to 667 pg/ml within 5 min; subsequent doses caused responses of 23 +/- 1, 23 +/- 6, 54 +/- 12 and 62 +/- 10% respectively of the initial dose. Concentrations of oxytocin receptor in myometrium, caruncular endometrium and intercaruncular endometrium were, respectively, 185 +/- 33, 128 +/- 7 and 105 +/- 14 fmol/mg protein at 2 h after saline injection and 147 +/- 27, 195 +/- 52 and 170 +/- 50 fmol/mg protein at 2 h after administration of 1 microgram oxytocin. The dose of oxytocin administered was shown to raise circulating concentrations to levels characteristic of those observed during spontaneous episodes of release of oxytocin at luteolysis. Oxytocin administration therefore results in transitory uterine refractoriness which may be due to failure of a post-receptor response and this may contribute to the episodic nature of uterine prostaglandin secretion. PMID- 3016258 TI - Interactions of antirheumatic drugs with the superoxide generation system of activated human polymorphonuclear leukocytes. AB - The effects of antirheumatic drugs on superoxide anion (O2-.) production by human polymorphonuclear leukocytes (PMNL) activated in vitro with phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) were investigated. Chloroquine (CLQ), auranofin (AF) and chlorotriethylphosphine gold (CTEP-G) at 10(-6) to 10(-4) M inhibited PMA and fMLP induced O2-. production in a dose dependent fashion. These drugs had no effect on O2-. production by the hypoxanthine/xanthine oxidase system, indicating that their inhibitory effects were directed at the O2-. generating mechanism and that they were not scavengers of the O2-. formed. AF and CTEP-G inhibited the specific binding of 3H fMPL to PMNL membrane receptors, whereas CLQ had no effect. Colchicine (COL) and gold sodium thiomalate (GSTM) on the other hand did not significantly affect PMA and fMLP induced O2-. production or the specific binding of the ligands to PMNL. The antirheumatic drugs had no effect on isolated neutrophil membrane protease, a chymotrypsin-like enzyme that has been implicated in the activation of NAD(P)H oxidase. However, several of the drugs inhibited the enzymatic activity of a subcellular preparation of PMNL NAD(P)H oxidase, the order of potency being GSTM greater than CLQ greater than penicillamine greater than COL greater than AF approximately equal to CTEP-G. The isolated NAD(P)H oxidase was also inhibited by the thiol compounds--thiomalic acid and dithiothreitol, suggesting that the mechanism of inhibition may involve sulfhydryl-disulfide interchange reactions. PMID- 3016259 TI - A spin label study of the membranolytic effects of crystalline monosodium urate monohydrate. AB - The nature of the membranolytic interaction between monosodium urate monohydrate (MSUM) crystals and phospholipid membranes was studied using electron spin resonance. Two spin probe molecules were incorporated into intact human erythrocytes and incubated with MSUM crystals. The apparent increased fluidity of 5-doxyl stearic acid incorporated erythrocytes after a 2 h incubation with MSUM was probably due to an electrostatically induced redistribution of probe from the outer more rigid layer to the fluid inner leaflet via a flip-flop mechanism. It was suggested that the MSUM induced redistribution of cationic amphiphilic probe population in the whole erythrocyte was also due to an electrostatic interaction between negatively charged MSUM crystals and positively charged probe. Possible mechanisms of MSUM induced membranolysis are discussed. PMID- 3016260 TI - The mechanism of action of the gastric acid secretion inhibitor omeprazole. PMID- 3016261 TI - Sequentially labile water-soluble prodrugs of alprazolam. AB - A 1,4-benzodiazepine analogue alprazolam (1) undergoes ring-opening hydrolysis under acidic conditions to form a triazolobenzophenone (2). At a neutral pH, 2 rapidly and quantitatively cyclizes back to 1. This facile reversible reaction was utilized in developing water-soluble prodrugs of 1 in which the "solubility anchoring" acyl moieties are formyl, acetyl, succinyl, glycyl, leucyl, and gamma aminobutyryl groups. These compounds were prepared directly from 2 and corresponding acids through DCC coupling in a 1:1 mixture of H2O and THF. They should be stable in aqueous media at room temperature. Promoieties used should be nontoxic at an intended dose level. In vitro human serum hydrolysis study showed that only glycyl and leucyl amides were able to regenerate 1 within a reasonable period of time. Ex vivo competitive receptor binding assay in mice with [3H]flunitrazepam indicated that leucyl amide prodrug should be able to produce a rapid onset of the intrinsic central nervous system activity of 1 when parenterally administered. For all compounds studied, the final outcome of the biological response appears to be kinetically controlled, rather than by an unfavorable equilibrium, particularly by the first step in which the amide bond is hydrolyzed in vivo. PMID- 3016263 TI - Synthesis and anti-herpes-virus activity of acyclic 2'-deoxyguanosine analogues related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine. AB - Several "sugar" modified acyclic nucleoside analogues related to 9-[(1,3 dihydroxy-2-propoxy)methyl]guanine (DHPG, 2) were synthesized and evaluated for antiviral activity. The preparation generally involved the condensation of the acetoxymethyl ether of alcohols 6c-g and 10-12a with diacetylguanine to give adducts 7c-g and 14-16, which were then deprotected to afford analogues 9c-g and 17-19. Alternatively, alcohols 12a and 13a were converted to iodides via their tosylates 12b and 13b and then reacted with the sodium salt of guanine to afford, after deprotection, analogues 22 and 23. A crossed aldol-Cannizzaro reaction on aldehyde 27 readily afforded 28, which was deprotected to give analogue 29. An in vitro assay against HSV-1 showed that all compounds tested were less active than DHPG, though several were good substrates for the viral thymidine kinase. The more promising acyclic nucleosides 9c, 19, and 29 were evaluated in a mouse encephalitis model and proved ineffective at preventing death at a dose of 20 mg/kg. PMID- 3016262 TI - Catechol estrogens of the 1,1,2-triphenylbut-1-ene type: relationship between structure, estradiol receptor affinity, estrogenic and antiestrogenic properties, and mammary tumor inhibiting activities. AB - 1,1,2-Triphenylbut-1-enes (26-35), which are substituted with one or two 3,4 diacetoxy groups or with one 3,4-diacetoxy and one 3- or 4-acetoxy group in two aromatic rings, were synthesized. The occurring E and Z isomers were isolated, and their identity was established by 1H NMR spectroscopy. A study on structure activity relationship was carried out with regard to estradiol receptor affinity in vitro, estrogenic and antiestrogenic properties in the immature mouse, and inhibition of the hormone-dependent MXT mammary tumor of the mouse in vivo. Among the tested compounds, most of the 1,1-disubstituted 1,1,2-triphenylbut-1-enes (29, Z-30, Z,E-31) and (E)-1-(3-acetoxyphenyl)-1-phenyl-2-(3,4 diacetoxyphenyl)but- 1-ene (E-35) as well as its respective Z isomer (Z-35) exerted antiestrogenic properties. Compounds Z-30, Z,E-31, Z-35, and E-35 inhibited the growth of the hormone-dependent MXT tumor. The best antitumor effect without estrogenic side effects during therapy was shown by E-35. PMID- 3016264 TI - Synthesis and antiviral activity of certain nucleoside 5'-phosphonoformate derivatives. AB - (Ethoxycarbonyl)phosphonic dichloride (3) was synthesized by chlorination of bis(trimethylsilyl) (ethoxycarbonyl)phosphonate with thionyl chloride. Adenosine 5'-(ethoxycarbonyl)phosphonate (4), guanosine 5'-(ethoxycarbonyl)phosphonate (5), 2'-deoxyadenosine 5'-(ethoxycarbonyl)phosphonate (18) and 2'-deoxyguanosine 5' (ethoxycarbonyl)phosphonate (19) were synthesized by coupling of compound 3 with adenosine, guanosine, 2'-deoxyadenosine, and 2'-deoxyguanosine, respectively. Alkaline treatment of 4, 5, 18, and 19 gave the corresponding adenosine 5' (hydroxycarbonyl)phosphonate (14), guanosine 5'-(hydroxycarbonyl) phosphonate (15), 2'-deoxyadenosine 5'-(hydroxycarbonyl)phosphonate (20), and 2' deoxyguanosine 5'-(hydroxycarbonyl) phosphonate (21). Treatment of 4 and 5 with methanolic ammonia resulted in the production of adenosine 5' (aminocarbonyl)phosphonate (12) and guanosine 5'-(aminocarbonyl)phosphonate (13), respectively. The nucleotide analogue 20 exhibited the most potent antiviral activity of this group of nucleotide tested in vitro and was most active against herpes viruses especially HSV-2. The nucleotide analogue 21 had lower, but significant, activity against HSV-2. All of the compounds tested were nontoxic to confluent Vero cells at concentrations as high as 5000 microM. PMID- 3016265 TI - 4-Amino-6-chloro-2-piperazinopyrimidines with selective affinity for alpha 2 adrenoceptors. AB - A series of 4-amino-6-chloro-2-piperazinopyrimidines were synthesized and evaluated for their ability to interact with alpha 1- and alpha 2-adrenoceptors in vitro in binding assays using [3H]WB-4101, [3H]clonidine, and [3H]idazoxan as radioligands. Some compounds were also tested as inhibitors of [3H]spiroperidol binding. Several members of this series showed high and selective affinity for alpha 2-adrenoceptors. The nature of the 4-amino substituent seems to be the most critical factor in determining the potency at these receptors. PMID- 3016266 TI - Conformationally defined adrenergic agents. 3. Modifications to the carbocyclic ring of 5,6-dihydroxy-1-(2-imidazolinyl)tetralin: improved separation of alpha 1 and alpha 2 adrenergic activities. AB - A series of modifications to positions 1, 2, and 4 of the tetralin ring of 5,6 dihydroxy-1-(2-imidazolinyl)tetralin (1, A-54741) succeeded in improving the separation of the potent alpha 1 and alpha 2 adrenergic agonism observed for the parent compound 1. In particular 5,6-dihydroxy-4,4-dimethyl-1-(2 imidazolinyl)tetralin (7) was found to be a specific alpha 1 adrenergic agonist, and 7,8-dihydroxy-4-(2-imidazolinyl)chroman (2) was found to have improved alpha 2 adrenergic agonistic selectivity relative to the parent compound 1. PMID- 3016267 TI - Synthesis and structure-activity relationship studies of a series of 5-aryl-4,6 dithianonanedioic acids and related compounds: a novel class of leukotriene antagonists. AB - A series of 5-alkynyl- and 5-aryl-4,6-dithianonanedioic acids and related compounds has been prepared for evaluation of leukotriene antagonist activity. The alkynyl compounds were prepared by thioacetal exchange from the corresponding acetylenic acetals. The aryl derivatives were synthesized from the appropriate benzaldehydes, most of which were prepared by one of three general routes: Meyers' oxazolin method, a palladium coupling procedure, and a hydroxybenzaldehyde alkylation. The analogues were examined in vitro for their ability to antagonize an LTD4-induced contraction of isolated guinea pig tracheal smooth muscle and to compete with [3H]LTD4 for receptor sites on guinea pig lung membrane. A number of structure-activity relationships have emerged from this study. There is an optimal chain length of 10-12 atoms (or its equivalent) in the lipid tail and two methylenes in the polar region. In the aromatic series, the ortho- and meta-substituted compounds have comparable activity, whereas the para derivatives are inactive. Substitution in the aromatic ring and lipid tail is generally well tolerated, with the terminal phenyl (6) and acetylene (33) analogues having especially good activity. Conformational restriction of either the polar region or lipid tail produced compounds devoid of activity. A number of selected analogues were also evaluated in vivo as antagonists of LTD4-induced bronchoconstriction in the guinea pig. The data established these compounds as a novel class of leukotriene antagonists with potential utility for the treatment of asthma and other immediate hypersensitivity diseases. PMID- 3016269 TI - Anticonvulsant activity of piperidinol and (dialkylamino)alkanol esters. AB - Aromatic and heterocyclic esters of 1-methyl-4-piperidinol and 1,4-dimethyl-4 piperidinol and aromatic esters of (dialkylamino)alkanols were prepared and evaluated for antiepileptic activity by the maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole seizure threshold (scMet) assays and for minimal central neurotoxicity by the rotorod ataxia test. The most potent compound, namely the 2-phenylbenzoate (57) of 3-(diethylamino)propanol, was slightly more potent than diphenylhydantoin in the MES assay, while the 2 phenylbenzoate (24) of 1-methyl-4-piperidinol and the 2-phenylbenzoate (56) of (diethylamino)ethanol displayed activity comparable to that of diphenylhydantoin. The 2-phenethylbenzoate ester (6) of 1-methyl-4-piperidinol exhibited one-third the activity of diphenylhydantoin. The 2,4,5-trimethylbenzoate 40 and 2,4,6 trimethylbenzoate 41 of 1-methyl-4-pieridinol were even less potent, but did display activity in the phenobarbital-methsuximide range. Certain compounds interact with sites associated with the GABA receptor-chloride channel complex, but their potencies as anticonvulsant agents do not correlate with interaction at sites on the channel complex. Certain analogues antagonize binding of a batrachotoxin analogue to sodium channel sites, a property indicative of local anesthetic activity. There are structural similarities between 2-phenylbenzoates 57, 56, and 24 and diphenylhydantoin, and the latter anticonvulsant also antagonizes binding of the batrachotoxin analogue. PMID- 3016270 TI - Analogues of 1,3-dipropyl-8-phenylxanthine: enhancement of selectivity at A1 adenosine receptors by aryl substituents. AB - The effect of a variety of aryl substituents on the potency and selectivity of 19 analogues of 1,3-dipropyl-8-phenylxanthine as antagonists at A1- and A2-adenosine receptors in brain tissue was determined. The 4-sulfamoylphenyl and 4 carbamoylphenyl analogues are potent and somewhat selective for the A1 receptor. None of the dihydroxyphenyl analogues are remarkably potent, but all are selective for the A1 receptor. 1,3-Dipropyl-8-(2-hydroxy-4-methoxyphenyl)xanthine is the most selective A1 antagonist of the analogues with a A1/A2 potency ratio of about 90. PMID- 3016268 TI - Topical nonsteroidal antipsoriatic agents. 1. 1,2,3,4-Tetraoxygenated naphthalene derivatives. AB - On the basis of previous observations that both 2,3-dihydro-2,2,3,3-tetrahydroxy 1,4-naphthoquinone (oxoline, 1) and 6-chloroisonaphthazarin (2) had demonstrated antipsoriatic activity in vivo, a series of structural derivatives of 2 were prepared and examined in the Scholtz-Dumas topical psoriasis bioassay. Of these six (5, 6, 9a, 10, 11a, 11b), the most effective compound was found to be 6 chloro-1,4-diacetoxy-2,3-dimethoxynaphthalene (RS-43179, lonapalene, 11a). An extensive series of 1,2,3,4-tetraoxygenated naphthalenes (16-74) incorporating variations of the ester, ether, and aryl substituents were prepared as analogues of 11a to examine the structural requirements for activity and were screened in vivo as inhibitors of arachidonic acid induced mouse ear edema, a topical bioassay capable of detecting 5-lipoxygenase inhibitors. Net lipophilicity, hydrolytic stability, and ring substitution play significant roles in determining the observed in vivo activity. Lonapalene (11a) is currently in clinical development as a topically applied nonsteroidal antipsoriatic agent. PMID- 3016271 TI - Antitumor agents. 78. Inhibition of human DNA topoisomerase II by podophyllotoxin and alpha-peltatin analogues. AB - It has been reported that the action of etoposide (VP-16) (14) as an antitumor agent is mediated through its interaction with DNA topoisomerase II which results in DNA breakage inside the cell. In order to understand the mechanism of action as well as structure-activity relationships of 14, several novel, synthetic and some naturally occurring analogues related to podophyllotoxin were examined for inhibition of the DNA topoisomerase II activity. Compound 2 exhibited enhanced activity and compound 5 slightly diminished activity relative to 14. A 4 beta substituted ether at the C ring and O-demethylation at the E ring appear to enhance activity. PMID- 3016272 TI - Activity of N-methyl-alpha- and -beta-funaltrexamine at opioid receptors. AB - The N-methyl analogues (2a, 2b) of the nonequilibrium mu opioid receptor antagonist beta-funaltrexamine (1b) were synthesized and evaluated in the guinea pig ileum preparation (GPI). These analogues are highly potent, reversible opioid agonists and possess no nonequilibrium antagonist activity. The ineffectiveness of 2b in protecting against irreversible blockage of mu opioid receptors by 1b and the fivefold lower reactivity of 2b with cysteine suggest that N-methyl substitution adversely affects both the first and second recognition steps that are essential for effective covalent blockage of opioid receptors. PMID- 3016273 TI - Characterisation of chloramphenicol resistance plasmids of Staphylococcus aureus and S. epidermidis by restriction enzyme mapping techniques. AB - Chloramphenicol resistance (Cmr) plasmids pSK2 and pSK5 from Staphylococcus aureus and pSK102 and pSK103 from S. epidermidis have been characterised and detailed restriction endonuclease cleavage maps constructed. TaqI digestion profiles illustrated the identity of pSK5 and pSK102 and also revealed a high degree of similarity between these four Cmr plasmids from Australian staphylococci and three Cmr plasmids from S. aureus strains of geographically unrelated origin. DNA-DNA hybridisation indicated that the chloramphenicol acetyltransferase determinant carried by pSK5/pSK102 could be found on other structurally-distinct Cmr plasmids. The role of S. epidermidis as a reservoir for Cmr plasmids found in S. aureus is discussed. PMID- 3016275 TI - Role of hypochlorous acid and chloramines in the extracellular cytolysis by neutrophil polymorphonuclear leukocytes. AB - The lysis of human red blood cells (HRBC) by neutrophil polymorphonuclear leukocytes (PMN), triggered with opsonized zymosan (OPZ) particles, was inhibited by azide, catalase, Cl- -free medium and amino acids indicating the involvement of myeloperoxidase (MPO), hydrogen peroxide (H2O2), Cl- ions and hypochlorous acid (HOCl) respectively. Thus, the cytolytic process depends on the following reaction: (Formula: see text). Because the oxidizing agent HOCl is also the precursor of the chloramines, a group of oxidants formed by the reaction between HOCl and PMN-derived ammonia (NH4+) or amines (R-NH2), the observed HRBC lysis can be theoretically due to HOCl and/or chloramines. Nevertheless, we found that PMN-mediated cytotoxicity occurs as an unidirectional process, being HRBC targets lysed and PMN unaffected. This finding indicates that the cytotoxin must be relatively more efficient against HRBC as compared with PMN. In fact, reagent HOCl (used at concentrations comparable to those generated by PMN) but not chloramines displayed such a type of property. Taken together, the data suggest that HRBC are killed by PMN-derived HOCl without the requirement for chloramines: this implies that NH4+ and R-NH2, released by PMN, act as down-modulators of the cytotoxic process, serving as HOCl trapping agents. PMID- 3016274 TI - Radioreceptor assay of anti-TSH receptor antibody activity: comparison of assays using unextracted serum and immunoglobulin fractions, and standardization of expression of activities. AB - A radioreceptor assay for TSH receptor antibodies developed by Shewring and Smith is described in which unextracted serum is used. The assay is simple and reproducible. TSH-binding inhibitor immunoglobulin (TBII) activity determined using unextracted serum correlated well with that determined using the immunoglobulin fraction purified with polyethylene glycol. The assay detected TSH receptor antibodies in 81 of 85 patients (95%) with untreated Graves' disease, no patients with Graves' disease in remission, and 7 of 57 patients (12%) with Hashimoto's disease. TSH-binding inhibitor activity of a human TSH preparation (Kabi Diagnostica) was not detectable at 100 mU/L, but detectable at 1,000 mU/L. Consistent with this, TBII activity was not detectable in hypothyroid patients with goitrous Hashimoto's disease, whose serum TSH concentrations were 11-520 mU/L. Of 57 patients with Hashimoto's disease positive TBII activity was detected in only 7 of 15 patients (46%) with primary atrophic hypothyroidism. Five of these had very high TBII activities (greater than 90% inhibition of labelled TSH binding), and no linear dose-response relationship was observed in dilution experiments. For exact determination of TBII activity in specimens with high activities, serum samples were diluted with normal pooled serum and activities were expressed in U/ml by comparison with values for standard serum, prepared from bovine TSH diluted with normal pooled serum. In this way, samples with TBII activities of 1-50 U/ml (20-90% inhibition of binding of labelled TSH) showed linear dose-response relations in dilution experiments, and the dilution curves of all samples examined were parallel to the standard curve. This standardization procedure was used in determining the half life of TBII. PMID- 3016276 TI - Dissociation of T8+ cell-mediated cytolytic and suppressor functions in young adults. AB - In the normal young adult population, in vitro levels of polyclonally-induced IgG secretion by mononuclear cells (MNCs) vary widely amongst individuals. Levels of IgG secretion correlate with functional suppressor activity of T8+ cells but not with their proportion within the MNCs either prior to culture, or as found in this study, at the end of the culture period. The current study also demonstrates a lack of correlation between T8+ cell-mediated suppressor (Ts) function and a second predominantly T8+ cell-mediated function, alloantigen-directed cytolytic (Tc) activity. Whether this dissociation between Ts and Tc functions reflects quantitative differences in subsets contained within the T8+ population or qualitative differences in the mechanisms required for induction or generation of Ts and Tc remains to be established. PMID- 3016277 TI - Formation of extracellular cGMP in blood mononuclear and platelet cell preparations. AB - The addition of inorganic acids to cell suspensions of mononuclear cells and platelets significantly increased concentrations of cGMP in supernatant fractions (3-6 X basal level). Increases in supernatant cGMP with the addition of 10(-4) M adrenaline (19 X basal level) 10(-5) M 5-HT (33 X) and 5 mM ascorbic acid (38 X) were detectable after acidification of the cell suspensions. Agonist and acid concentrations at termination of the incubations were important in determining cGMP concentrations in supernatants. The results suggest that cGMP is formed on the exterior surface of the cell membrane and therefore has an extracellular, rather than intracellular, role in transmission of the membrane stimulus for cell activation. PMID- 3016279 TI - Isolation of brush-border membranes from rat and rabbit colonocytes: is alkaline phosphatase a marker enzyme? AB - A method for the isolation of brush-border membranes of large intestinal epithelial cells was developed, which is based on the purification of intact brush-border caps by Percoll density-gradient centrifugation followed by separation of the vesiculated brush-border membranes on sucrose gradients. The procedure has two major advantages in comparison to known methods: its first step does not depend on the determination of marker enzymes and the method is applicable to rats as well as rabbits without major modifications. Due to the lack of an accepted marker for the colonic brush-border membrane the validity of the isolation procedure was tested by its application to the small intestine. Rat small intestinal brush-border membranes were enriched 21-fold when compared to the homogenate. The method was used to evaluate alkaline phosphatase as a marker enzyme for the colonic brush-border membrane. The results suggest that alkaline phosphatase is not exclusively localized in the brush-border membrane since this enzyme was also associated with membranes having different physical properties. PMID- 3016278 TI - Mechanisms of aldosterone action in tight epithelia. PMID- 3016280 TI - Voltage dependence of the rheogenic Na+/K+ ATPase in the membrane of oocytes of Xenopus laevis. AB - Electrophysiological experiments were performed to analyze the Na+/K+-ATPase in full-grown prophase-arrested oocytes of Xenopus laevis. If the Na+/K+-ATPase is inhibited by dihydroouabain (DHO), the resting potential of the membrane of Na+ loaded oocytes may depolarize by nearly 50 mV. This hyperpolarizing contribution to the resting potential depends on the degree of activation of the Na+/K+-ATPase and varies with intracellular Na+ activity (aiNa) and extracellular K+ (K+o). It is concluded that variations of aiNa among different oocytes are primarily responsible for the variations of resting potentials measured in oocytes of X. laevis. Under voltage-clamp conditions, the DHO-sensitive current also exhibits dependence on aiNa that may be described by a Hill equation with a coefficient of 2. This current will be shown to be identical with the electrogenic current generated by the 3Na+/2K+ pump. The voltage dependence of the pump current was investigated at saturating values of aiNa (33 mmol/liter) and of K+o (3 mmol/liter) in the range from -200 to +100 mV. The current was found to exhibit a characteristic maximum at about +20 mV. This is taken as evidence that in the physiological range at least two steps within the cycle of the pump are voltage dependent and are oppositely affected by the membrane potential. PMID- 3016282 TI - Particles and pits matched in native membranes. AB - We have obtained high resolution electron microscopic images of complementary membrane surface replicas of purified microsomal vesicles from pig kidney outer medulla containing Na+,K+-ATPase. Ultra-rapid freezing of a membrane suspension was followed by fracturing and replicating of the liquid helium cooled specimen under ultra-high vacuum conditions free of hydrocarbon contaminants. The protoplasmic fracture faces are populated with intramembrane particles while the external fracture faces reveal complementary pits. This is the first demonstration of extended precise matching of individual intramembrane particles and their corresponding pits in biological membranes containing transmembrane proteins. The data are also consistent with the theory that the majority of Na+,K+-ATPase mass is located at the protoplasmic half of the membrane. PMID- 3016281 TI - Modification of gap junctions in cells transformed by a temperature-sensitive mutant of Rous sarcoma virus. AB - Prompted by our observation that a reduction in junctional permeance is one of the earlier events in the process of neoplastic transformation of a cell line by Rous sarcoma virus, we analyzed the gap junctions from these cells to determine if the basis of the reduction is a loss of junctional channels. The cells (normal rat kidney, or NRK) are infected with a temperature-sensitive mutant of Rous sarcoma virus, allowing one easily to manipulate the cells into and out of the transformed state, and hence also to manipulate the junctional permeance. Using freeze-fracture electron microscopy, we found that the number and size of the junctions did not change in parallel with the permeance changes we had previously characterized. There is, however, a significant rearrangement of the junctional particles to a more random configuration when the cells are transformed and a reversal to the more ordered pattern when the cells are shifted back to the normal phenotype. These changes do parallel the changes in junctional permeance. We conclude that the permeance of existing junctional channels is modified and that the change in permeance may involve a change in the interaction of the junctional channels with each other and/or the surrounding lipid domain. PMID- 3016284 TI - Transcription of Escherichia coli ara in vitro. The cyclic AMP receptor protein requirement for PBAD induction that depends on the presence and orientation of the araO2 site. AB - The mechanism by which the cyclic AMP receptor protein, CRP, stimulates transcription of the Escherichia coli araBAD promoter was studied in vitro. Under one set of conditions, CRP stimulated by eightfold the rate of RNA polymerase open complex formation on supercoiled DNA template containing the normal wild type araBAD regulatory region. Since previous studies in vivo had identified an upstream site termed araO2 that is involved in both repression and in the CRP requirement for PBAD induction, we performed similar experiments in vitro. Deletion of araO2 or alterations of its orientation with respect to the araI site by half integral numbers of turns greatly reduced the CRP requirement for induction of PBAD. Linearizing the DNA has the same effect as deleting araO2 from the supercoiled DNA template. The similarity of conditions that relieve the classical repression of PBAD in vivo and the conditions that eliminate the requirement for CRP for maximal activity in vitro suggest a close relationship between repression in the ara system and the role of CRP. At lower concentrations of AraC protein and slightly different conditions than those used in the above mentioned experiments, CRP does stimulate transcription from linear or supercoiled templates lacking araO2. On linear DNA under these conditions, one dimer of AraC protein binds to linear araPBAD DNA, but is incapable of stimulating transcription without the additional binding of CRP. The responses of the ara system under the second set of conditions are unlike its behavior in vivo. PMID- 3016283 TI - Mapping of endogenous nuclease-sensitive regions and of putative topoisomerase sites of action along the chromatin of Dictyostelium ribosomal RNA genes. AB - Indirect end-labelling and the digestion patterns of endogenous and exogenous nucleases were used to analyse chromatin organization along the ribosomal RNA genes of Dictyostelium discoideum cells. A zone just upstream from the 5' end of the coding region was particularly sensitive to endogenous nucleases. In exponentially growing cells, this hypersensitive zone extended from -350 to -1600 bp relative to the transcription start. In sharp contrast, the DNA between 0 and 350 bp was strongly protected. In differentiating cells, in which the ribosomal RNA transcription rate is low, the 5' hypersensitive zone was more diffuse than in exponentially growing cells, and the protected region at the 5' end of the transcribed region was less pronounced. It is known that where DNA topoisomerase is acting on DNA, the addition of sodium dodecyl sulphate will result in cleavage of the DNA and covalent attachment of the enzyme to the cut DNA end. Treatment of nuclei from both exponentially growing cells and differentiating cells with SDS caused double-stranded cleavages at -200 (i.e. within the protected region), at 2200, and at two sites at about -17 kb. A fraction of the cleavage products appeared to be strongly associated with protein. Novobiocin, a DNA topoisomerase II inhibitor, did not inhibit the SDS-induced cleavages in vegetative cells. However, it significantly reduced the extent of nuclease cleavage within the -350 to -1600 bp hypersensitive zone. The possibility is discussed that there are two DNA topoisomerase-like activities on the ribosomal genes. One is site-specific and novobiocin-insensitive. We speculate that the other is responsible for maintaining DNA at the 5' end of the gene in a torsionally strained, nuclease hypersensitive state. PMID- 3016285 TI - Structure refinement of oligonucleotides by molecular dynamics with nuclear Overhauser effect interproton distance restraints: application to 5' d(C-G-T-A-C G)2. AB - The solution structure of the self-complementary DNA hexamer 5' d(C-G-T-A-C-G)2 is refined by restrained molecular dynamics in which 192 interproton distances, determined from pre-steady-state nuclear Overhauser enhancement measurements, are incorporated into the total energy of the system in the form of effective potentials. First the method is tested by applying an idealized set of distance restraints taken from classical B-DNA to a simulation starting off from A-DNA and vice versa. It is shown that in both cases the expected transition between A- and B-DNA occurs. Second, a set of restrained molecular dynamics calculations is carried out starting from both A- and B-DNA with the experimental interproton distances for 5' d(C-G-T-A-C-G)2 as restraints. Convergence to the same B-type structure is achieved with the interproton distances equal to the measured values within experimental error. The root-mean-square atomic difference between the two average restrained dynamics structures (less than 1 A) is approximately the same as the root-mean-square fluctuations of the atoms. PMID- 3016286 TI - FLP site-specific recombinase of yeast 2-micron plasmid. Topological features of the reaction. AB - The 2-micron plasmid of the yeast Saccharomyces cerevisiae encodes a site specific recombinase (FLP) that promotes inversion across a unique site contained in each of the 599-base-pair inverted repeats of the plasmid. We have studied the topological changes generated in supercoiled substrates after exposure to the purified FLP protein in vitro. When a supercoiled substrate bearing two FLP target sequences in inverse orientation is treated with FLP, the products are multiply knotted structures that arise as a result of random entrapment of interdomainal supercoils. Likewise, a supercoiled substrate bearing two target sequences in direct orientation yields multiply interlocked catenanes as the product. Both types of substrate seem to be able to undergo repeated rounds of recombination that result in products of further complexity. The FLP protein also acts as a site-specific topoisomerase during the recombination reaction. PMID- 3016287 TI - Metaphase chromosome structure. Involvement of topoisomerase II. AB - SCI is a prominent, 170,000 Mr, non-histone protein of HeLa metaphase chromosomes. This protein binds DNA and was previously identified as one of the major structural components of the residual scaffold structure obtained by differential protein extraction from isolated chromosomes. The metaphase scaffold maintains chromosomal DNA in an organized, looped conformation. We have prepared a polyclonal antibody against the SC1 protein. Immunolocalization studies by both fluorescence and electron microscopy allowed identification of the scaffold structure in gently expanded chromosomes. The micrographs show an immunopositive reaction going through the kinetochore along a central, axial region that extends the length of each chromatid. Some micrographs of histone-depleted chromosomes provide evidence of the substructural organization of the scaffold; the scaffold appears to consist of an assembly of foci, which in places form a zig-zag or coiled arrangement. We present several lines of evidence that establish the identity of SC1 as topoisomerase II. Considering the enzymic nature of this protein, it is remarkable that it represents 1% to 2% of the total mitotic chromosomal protein. About 60% to 80% of topoisomerase II partitions into the scaffold structure as prepared from isolated chromosomes, and we find approximately three copies per average 70,000-base loop. This supports the proposed structural role of the scaffold in the organization of the mitotic chromosome. The dual enzymic and apparent structural function of topoisomerase II (SC1) and its location at or near the base of chromatin loops allows speculation as to its involvement in the long-range control of chromatin structure. PMID- 3016288 TI - Covalent carcinogenic O6-methylguanosine lesions in DNA. Structural studies of the O6 meG X A and O6meG X G interactions in dodecanucleotide duplexes. AB - High-resolution proton and phosphorus nuclear magnetic resonance studies are reported on the self-complementary d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6meG X A 12-mer when N3 = A3 and O6meG X G 12-mer when N3 = G3), which contain symmetry-related A3 X O6meG10 and G3 X O6meG10 interactions in the interior of the helices. We observe inter-base-pair nuclear Overhauser effects (NOE) between the base protons at the N3 X O6meG10 modification site and protons of flanking G2 X C11 and G4 X C9 base-pairs, indicative of the stacking of N3 and O6meG10 bases in both O6meG X A 12-mer and O6meG X G 12-mer duplexes. We have assigned all the base and a majority of the sugar protons from two-dimensional proton-correlated and nuclear Overhauser effect experiments on the O6meG X A 12-mer duplex and O6meG X G 12-mer duplex in solution. The observed NOEs establish that the A3 and O6meG10 at the modification site and all other residues adopt the anti configuration about the glycosidic bond, and that the O6meG X A 12-mer forms a right-handed duplex. The interaction between the bulky purine A3 and O6meG10 residues in the anti orientation results in large proton chemical shift perturbations at the (G2-A3-G4) X (C9-O6meG10-C11) segments of the helix. By contrast, we demonstrate that the O6meG10 residue adopts a syn configuration, while all other bases adopt an anti configuration about the glycosidic bond in the right-handed O6meG X G 12-mer duplex. This results in altered NOE patterns between the base protons of O6meG10 and the base and sugar protons of flanking C9 and C11 residues in the O6meG X G 12-mer duplex. The phosphorus backbone is perturbed at the modification site in both duplexes, since the phosphorus resonances are dispersed over 2 parts per million in the O6meG X A 12-mer and over 1 part per million in the O6meG X G 12-mer compared to a 0.5 part per million dispersion for an unperturbed DNA helix. We propose tentative pairing schemes for the A3 X O6meG10 and G3 X O6meG10 interactions in the above dodecanucleotide duplexes. PMID- 3016290 TI - Recovery training and self help: a relapse-prevention program for treated opiate addicts. AB - Recovery Training and Self Help (RTSH) is a new form of psychosocial treatment for drug addiction. Developed as an outpatient group aftercare modality for opiate addicts in New England and Hong Kong, it significantly reduced the probability of relapse to illicit opiates and helped unemployed subjects find work. Based on a social theory of addiction and health promotion principles, the four-part program features a weekly recovery training session, a weekly self-help style session, weekend recreational and social activities, and a support network of long-term ex-addicts. Recovery training follows a 26 week sequence of didactic presentations and exercises that systematically address predictable causes of relapse, while the other clinical components provide motivation and support for continued abstinence and social reintegration. The authors believe that RTSH should have a wide range of applicability. PMID- 3016289 TI - Adenosine production and release by adult rat cardiocytes. AB - We have examined adenosine (ADO) production and transport in a preparation of isolated adult cardiocytes which attach to and form a monolayer on culture dishes. This preparation contains 85% viable cells which are greater than 50% rod shaped and maintain an ATP/ADP ratio of nine. Incubation under control conditions for 15 mins results in a net release of 240 +/- 47 pmol ADO/mg protein (final adenosine concentration in the medium = 47 +/- 9 nM). Both 0.1 mM dinitrophenol (DNP) and 10 mM iodoacetate (IAA) cause a significant increase in ADO (DNP = 1763 +/- 147 and IAA = 612 +/- 90 pmol/mg). Both 20 microM nitrobenzylthioinosine (NBMPR), an inhibitor of the purine nucleoside carrier, and 0.1 mM alpha,beta methylene adenosine diphosphate (AOPCP), an inhibitor of 5'-nucleotidase activity, attenuate DNP-stimulated ADO release (NBMPR by 62% and ADOCP by 76%). The results are consistent with the hypothesis that under the conditions of our experiments, adenosine is formed by a 5'-nucleotidase in association with transport across the cell membrane, perhaps by an enzyme-carrier complex. In addition, we have examined the effect of 0.1 mM dipyridamole on the extracellular appearance of adenosine in this preparation and found that it causes a significant increase in the amount of adenosine released. These results are consistent with the hypothesis that dipyridamole inhibits adenosine's uptake more than its release in cardiac myocytes. PMID- 3016291 TI - Interruption-deficient mutants of bacteriophage T5: analysis of single-site mutants. AB - The properties of viable mutants of bacteriophage T5 that lack, singly, each of the four major sites at which single-chain interruptions normally occur in T5 DNA are described. The mutations responsible for loss of each interruption were mapped by analysis with HhaI, a restriction endonuclease with a cleavage site (pGCGC) that occurs at the 5' termini of the major interruptions (B. P. Nichols and J. E. Donelson, J. Virol. 22:520-526, 1977). For each mutant tested, loss of a specific interruption resulted in loss of a specific HhaI cleavage site. Multiple single-site mutants were constructed to determine the effect of loss of more than one interruption on phage viability. These recombinants, including a phage that lacks the four major interruptible sites, were fully viable and did not exhibit a compensating increase in the frequency of minor interruptions. The effect of loss of a specific interruption on genetic recombination was tested in two-factor crosses with markers that occur close to, but on opposite sites of, the interruption. Loss of the interruptible site did not affect recombination frequency. PMID- 3016292 TI - Further characterization of the vesicular stomatitis virus temperature-sensitive O45 mutant: intracellular conversion of the glycoprotein to a soluble form. AB - Reexamination of the viral products of tsO45, a glycoprotein mutant of vesicular stomatitis virus, showed that at 39 degrees C there was a conversion of the glycoprotein (G) to a truncated, soluble form, Gs, which subsequently appeared in the extracellular medium. The half-life for this intracellular conversion and extracellular appearance was about 2 h at 39 degrees C. Gs was precipitated by a monoclonal antibody to the ektodomain but not by an antipeptide serum made against the first 15 amino acids at the carboxy terminus of G. Gs was also resistant to endoglycosidase H digestion. On the basis of pulse-chase experiments, the generation of Gs most probably occurred in the rough endoplasmic reticulum. This additional phenotype of the tsO45 mutant provides another approach for studying the generation and subsequent transport of a secreted protein in fibroblast cells. PMID- 3016293 TI - DNA sequence of the gene for pseudorabies virus gp50, a glycoprotein without N linked glycosylation. AB - The DNA sequence was determined for a region of the pseudorabies virus (PRV) genome to which a mutation defining resistance to a monoclonal antibody has been mapped (M. W. Wathen and L. M. K. Wathen, J. Virol., 51:57-62, 1984). This sequence was found to contain an open reading frame that did not include an amino acid sequence directing N-linked glycosylation. This open reading frame was expressed in uninfected Chinese hamster ovary cells to produce the PRV glycoprotein gp50. When PRV-infected Vero cells were incubated in the presence of tunicamycin, the gp50 that was produced had an identical molecular weight to that produced in the absence of drug. When infected cells were incubated in the presence of monensin, the molecular weight of gp50 was reduced from 60,000 to 45,000, but was not sensitive to endo-beta-N-acetylglucosaminidase H. These observations led to the conclusion that gp50 does not contain N-linked carbohydrate, as predicted from the DNA sequence. A region of the amino acid sequence and the positions of the cysteine residues of PRV gp50 are homologous to glycoprotein D of herpes simplex virus. PMID- 3016294 TI - Replication and resolution of cloned poxvirus telomeres in vivo generates linear minichromosomes with intact viral hairpin termini. AB - The covalently closed terminal hairpins of the linear duplex-DNA genomes of the orthopoxvirus vaccinia and the leporipoxvirus Shope fibroma virus (SFV) have been cloned as imperfect palindromes within circular plasmids in yeast cells and recombination-deficient Escherichia coli. The viral telomeres inserted within these recombinant plasmids are equivalent to the inverted-repeat structures detected as telomeric replicative intermediates during poxvirus replication in vivo. Although the telomeres of vaccinia and SFV show little sequence homology, the termini from both viral genomes exist as AT-rich terminal hairpins with extrahelical bases and alternate "flip-flop" configurations. Using an in vivo replication assay in which circular plasmid DNA was transfected into poxvirus infected cells, we demonstrated the efficient replication and resolution of the cloned imperfect palindromes to bona fide hairpin termini. The resulting linear minichromosomes, which were readily purified from transfected cells, were shown by restriction enzyme mapping and by electron microscopy to have intact covalently closed hairpin termini at both ends. In addition, staggered unidirectional deletion derivatives of both the cloned vaccinia and SFV telomeric palindromes localized an approximately 200-base-pair DNA region in which the sequence organization was highly conserved and which was necessary for the resolution event. These data suggest a conserved mechanism of the resolution of poxvirus telomeres. PMID- 3016295 TI - Evolutionary changes of transcriptional control region in a minute-plaque viable deletion mutant of BK virus. AB - Two plaque morphology BK virus (BKV) mutants (pm526 and pm527) rescued from a hamster pineocytoma cell line Pc13 were characterized and compared with a similarly rescued and previously characterized mutant (pm522), its derivatives (tr530, tr531, and tr532), and the wild type (501) for their biological activities and for the structures of their transcriptional control regions. The two mutants grew somewhat more slowly in human embryonic kidney cells but transformed rat 3Y1 cells more efficiently than did the wild-type BKV. BKV pm526 formed minute plaques and had the shortest transcriptional control region, having only one 68-base-pair element, which is triplicated in the wild-type BKV. BKV pm526 was unstable during repeated replication in human embryonic kidney cells and yielded large-plaque viruses with longer HindIII C segments encompassing the BKV DNA replication origin. Comparison of nucleotide sequences of the transcriptional control regions among the mutants and the wild type showed that pm526 is a parent virus, from which all the other mutants had evolved, and that the evolutionary changes of the plaque size, from minute to small to large, were due to duplications of a certain segment containing the adenovirus E1A enhancer core or the simian virus 40 enhancer core found in the BKV 68-base-pair element. The activities to enhance early transcription, as measured by the ability to direct the synthesis of chloramphenicol acetyltransferase, approximately paralleled the plaque size. The duplication containing the adenovirus E1A enhancer core did not affect the transforming capacity of the parent virus, but the duplication including the simian virus 40 enhancer core significantly lowered the transforming capacity for rat cells. PMID- 3016296 TI - Expression of molecular clones of v-myb in avian and mammalian cells independently of transformation. AB - We demonstrated that molecular clones of the v-myb oncogene of avian myeloblastosis virus (AMV) can direct the synthesis of p48v-myb both in avian and mammalian cells which are not targets for transformation by AMV. To accomplish this, we constructed dominantly selectable avian leukosis virus derivatives which efficiently coexpress the protein products of the Tn5 neo gene and the v-myb oncogene. The use of chemically transformed QT6 quail cells for proviral DNA transfection or retroviral infection, followed by G418 selection, allowed the generation of cell lines which continuously produce both undeleted infectious neo myb viral stocks and p48v-myb. The presence of a simian virus 40 origin of replication in the proviral plasmids also permitted high-level transient expression of p48v-myb in simian COS cells without intervening cycles of potentially mutagenic retroviral replication. These experiments establish that the previously reported DNA sequence of v-myb does in fact encode p48v-myb, the transforming protein of AMV. PMID- 3016297 TI - A mouse c-myc retrovirus transforms established fibroblast lines in vitro and induces monocyte-macrophage tumors in vivo. AB - Activation of the c-myc proto-oncogene, in the form of DNA rearrangements that lead to constitutive expression, has been implicated in the genesis of a wide range of tumors. Therefore, it is of great interest to determine the influence of c-myc oncogene activation on cellular growth control, especially in primary cells. To facilitate the efficient transfer of an activated c-myc oncogene, we developed a mouse retrovirus that contains the c-myc protein-coding sequences and which can be transmitted in the presence of a Moloney murine leukemia virus helper or established as a helper-free stock with a retrovirus-packaging cell line. The virus can transform established lines of mouse fibroblasts to anchorage independent growth; the transformed cells are tumorigenic in nude mice. However, the virus was not capable of inducing foci of transformed cells on confluent monolayers. In addition to studies on established cell lines, the effect of the c myc retrovirus on primary cells was examined. Infection of bone marrow cells gave rise to partially transformed mononuclear phagocytes which were entirely dependent upon an exogenous supply of the monocyte-specific colony-stimulating factor CSF-1 for proliferation. Infection in vivo induced monocyte-macrophage tumors with a latency period of 8 to 10 weeks. PMID- 3016298 TI - Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. AB - We constructed an infectious molecular clone of acquired immunodeficiency syndrome-associated retrovirus. Upon transfection, this clone directed the production of infectious virus particles in a wide variety of cells in addition to human T4 cells. The progeny, infectious virions, were synthesized in mouse, mink, monkey, and several human non-T cell lines, indicating the absence of any intracellular obstacle to viral RNA or protein production or assembly. During the course of these studies, a human colon carcinoma cell line, exquisitely sensitive to DNA transfection, was identified. PMID- 3016299 TI - Molecular basis for interference of defective interfering particles of pseudorabies virus with replication of standard virus. AB - Serial passage of pseudorabies virus (PrV) at high multiplicity yields defective interfering particles (DIPs), but the sharp cyclical increases and decreases in titer of infectious virus that are observed upon continued passage at high multiplicity of most DIPs of other viruses are not observed with DIPs of PrV (T. Ben-Porat and A. S. Kaplan, Virology 72:471-479). We have studied the dynamics of the interactions of the virions present in a population of DIPs to assess the cis functions for which the genomes of the DIPs are enriched. The defective genomes present in one population of DIPs, [PrV(1)42], replicate preferentially over the nondefective genomes present in that virion population at early stages of infection, indicating that the DIP DNA is enriched for sequences that can serve as origins of replication at early stages of infection. This replicative advantage of the DIP DNA is transient and disappears at later stages of infection. The defective DNA does not appear to be encapsidated preferentially over the nondefective DNA present in this virion population, which might indicate that it is not enriched for cleavage-encapsidation sites. However, the nondefective DNA in the DIP virion population has become modified and has acquired reiterations of sequences originating from the end of the unique long (UL) region of the genome. Furthermore, both the infectious and defective genomes present in the DIP population compete for encapsidation more effectively than do the genomes of standard PrV. These results indicate that the defective genomes in the population of virions studied are enriched not only for an origin of replication but probably also for sequences necessary for efficient cleavage encapsidation. Furthermore, the nondefective genomes present in this population of DIPs have also been modified and have acquired the ability to compete with the defective genomes for cleavage-encapsidation. PMID- 3016300 TI - cis Functions involved in replication and cleavage-encapsidation of pseudorabies virus. AB - Serial passage at high multiplicity of pseudorabies virus generates defective interfering particles (DIPs) whose genomes consist at least in part of reiterations of segments of DNA in which sequences originating from different regions of the genome have become covalently linked (F. J. Rixon and T. Ben Porat, Virology 97:151-163). To determine whether some cis functions present in these reiterated DNA sequences may be responsible for the amplification of DIP DNA, BamHI restriction fragments of this DNA were cloned. These fragments were analyzed and tested for their ability to promote the amplification of covalently linked pBR325 DNA when cotransfected into cells with helper pseudorabies virus DNA. The cloned DIP BamHI DNA fragments consisted of various combinations of sequences originating from either one or both ends as well as sequences from the middle of the unique long (UL) segment of the genome. Only plasmids with inserts consisting of segments of defective DNA originating from the middle of the UL, as well as from both ends of the genome, were able to replicate and be encapsidated autonomously. This finding indicated that signals present at both ends of the genome may be necessary for efficient cleavage-encapsidation. To confirm this observation, we constructed plasmids in which DNA segments containing an origin of replication and sequences from either one or both ends of the virus genome were linked. These experiments showed that efficient cleavage-encapsidation requires the presence of sequences derived from both ends of the genome. Two origins of replication, one at the end of the UL segment and one in the middle of the UL segment, were also identified. PMID- 3016301 TI - Structure and transforming function of transduced mutant alleles of the chicken c myc gene. AB - A small retroviral vector carrying an oncogenic myc allele was isolated as a spontaneous variant (MH2E21) of avian oncovirus MH2. The MH2E21 genome, measuring only 2.3 kilobases, can be replicated like larger retroviral genomes and hence contains all cis-acting sequence elements essential for encapsidation and reverse transcription of retroviral RNA or for integration and transcription of proviral DNA. The MH2E21 genome contains 5' and 3' noncoding retroviral vector elements and a coding region comprising the first six codons of the viral gag gene and 417 v-myc codons. The gag-myc junction corresponds precisely to the presumed splice junction on subgenomic MH2 v-myc mRNA, the possible origin of MH2E21. Among the v myc codons, the first 5 are derived from the noncoding 5' terminus of the second c-myc exon, and 412 codons correspond to the c-myc coding region. The predicted sequence of the MH2E21 protein product differs from that of the chicken c-myc protein by 11 additional amino-terminal residues and by 25 amino acid substitutions and a deletion of 4 residues within the shared domains. To investigate the functional significance of these structural changes, the MH2E21 genome was modified in vitro. The gag translational initiation codon was inactivated by oligonucleotide-directed mutagenesis. Furthermore, all but two of the missense mutations were reverted, and the deleted sequences were restored by replacing most of the MH2E21 v-myc allele by the corresponding segment of the CMII v-myc allele which is isogenic to c-myc in that region. The remaining two mutations have not been found in the v-myc alleles of avian oncoviruses MC29, CMII, and OK10. Like MH2 and MH2E21, modified MH2E21 (MH2E21m1c1) transforms avian embryo cells. Like c-myc, it encodes a 416-amino-acid protein initiated at the myc translational initiation codon. We conclude that neither major structural changes, such as in-frame fusion with virion genes or internal deletions, nor specific, if any, missense mutations of the c-myc coding region are necessary for activation of the basic oncogenic function of transduced myc alleles. PMID- 3016303 TI - In vivo transformation of human skin with human papillomavirus type 11 from condylomata acuminata. AB - Human papillomaviruses (HPVs) have been implicated in the development of a number of human malignancies, but direct tests of their involvement have not been possible. We describe a system in which human skin from various sites was infected with HPV type 11 (HPV-11) extracted from vulvar condylomata and was grafted beneath the renal capsule of athymic mice. Most of the skin grafts so treated underwent morphological transformation, resulting in the development of condylomata identical to those which occur spontaneously in patients. Foreskins responded with the most vigorous proliferative response to HPV-11. The lesions produced the characteristic intranuclear group-specific antigen of papillomaviruses. Both dot blot and Southern blot analysis of DNA from the lesions revealed the presence of HPV-11 DNA in the transformed grafts. These results demonstrate the first laboratory system for the study of the interaction of human skin with an HPV. The method may be useful in understanding the mechanisms of HPV transformation and replication and is free of the ethical restraints which have impeded study. This system will allow the direct study of factors which permit neoplastic progression of HPV-induced cutaneous lesions in human tissues. PMID- 3016302 TI - Characterization of Rous sarcoma virus-related sequences in the Japanese quail. AB - We detected sequences related to the avian retrovirus Rous sarcoma virus within the genome of the Japanese quail, a species previously considered to be free of endogenous avian leukosis virus elements. Using low-stringency conditions of hybridization, we screened a quail genomic library for clones containing retrovirus-related information. Of five clones so selected, one, lambda Q48, contained sequence information related to the gag, pol, and env genes of Rous sarcoma virus arranged in a contiguous fashion and spanning a distance of approximately 5.8 kilobases. This organization is consistent with the presence of an endogenous retroviral element within the Japanese quail genome. Use of this element as a high-stringency probe on Southern blots of genomic digests of several quail DNA demonstrated hybridization to a series of high-molecular-weight bands. By slot hybridization to quail DNA with a cloned probe, it was deduced that there were approximately 300 copies per diploid cell. In addition, the quail element also hybridized at low stringency to the DNA of the White Leghorn chicken and at high stringency to the DNAs of several species of jungle fowl and both true and ruffed pheasants. Limited nucleotide sequencing analysis of lambda Q48 revealed homologies of 65, 52, and 46% compared with the sequence of Rous sarcoma virus strain Prague C for the endonuclease domain of pol, the pol-env junction, and the 3'-terminal region of env, respectively. Comparisons at the amino acid level were also significant, thus confirming the retrovirus relatedness of the cloned quail element. PMID- 3016304 TI - Measurement of the mutation rates of animal viruses: influenza A virus and poliovirus type 1. AB - Epidemiologic and genetic evidence suggests that influenza A viruses evolve more rapidly than other viruses in humans. Although the high mutation rate of the virus is often cited as the cause of the extensive variation, direct measurement of this parameter has not been obtained in vivo. In this study, the rate of mutation in tissue culture for the nonstructural (NS) gene of influenza A virus and for the VP1 gene in poliovirus type 1 was assayed by direct sequence analysis. Each gene was repeatedly sequenced in over 100 viral clones which were descended from a single virion in one plaque generation. A total of 108 NS genes of influenza virus were sequenced, and in the 91,708 nucleotides analyzed, seven point changes were observed. A total of 105 VP1 genes of poliovirus were sequenced, and in the 95,688 nucleotides analyzed, no mutations were observed. We then calculated mutation rates of 1.5 X 10(-5) and less than 2.1 X 10(-6) mutations per nucleotide per infectious cycle for influenza virus and poliovirus, respectively. We suggest that the higher mutation rate of influenza A virus may promote the rapid evolution of this virus in nature. PMID- 3016305 TI - Molecular characterization of Marek's disease herpesvirus B antigen. AB - The Marek's disease herpesvirus (MDHV) B antigen (MDHV-B) was identified and molecularly characterized as a set of three glycoproteins of 100,000, 60,000, and 49,000 apparent molecular weight (gp100, gp60, and gp49, respectively) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after immunoprecipitation from [35S]methionine-labeled infected cells by specific rabbit antiserum directed against MDHV-B (R alpha B), as previously determined by immunodiffusion. Further identification was accomplished by blocking this immunoprecipitation with highly purified MDHV-B. The same set of three polypeptides was also immunoprecipitated from [35S]methionine- and 14C-labeled infected cells with two other sera shown to have anti-B activity, i.e., rabbit anti-MDHV-infected-cell plasma membrane (R alpha PM) and immune chicken serum from birds naturally infected with MDHV. The three herpesvirus of turkeys (HVT) B antigen (HVT-B) glycoproteins immunoprecipitated with all three sera containing anti-B activity were also shown to be identical in size to those of MDHV-B by immunoprecipitation and SDS-PAGE. These data serve to clarify the molecular identification of the polypeptides found in common between MDHV and HVT by linking them to MDHV-B, previously identified only by immunodiffusion, and to a similarly sized set of immunologically related common glycoproteins called gp100, gp60, and gp49, detected with monoclonal antibody by other workers. Tunicamycin inhibition of N-linked glycosylation resulted in either nonglycosylated or O linked glycosylated putative precursors of MDHV-B and HVT-B with apparent molecular weights of 88,000, called pr88, and 44,000, tentatively called pr44, both immunoprecipitable with all three sera. However, the relationships of these two polypeptides to each other and to the overall precursor-processing relationship of the MDHV-B complex remains to be elucidated. The three fully glycosylated B-antigen polypeptides were not connected by disulfide linkage. Collectively, the data presented here and by others support the conclusion that all three glycoproteins now identified as gp100, gp60, and gp49 have MDHV-B determinants. Finally, detection of the same three polypeptides with well absorbed R alpha PM, which was directed against purified infected-cell plasma membranes, suggests that at least one component of the B-antigen complex has a plasma membrane location in the infected cell.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3016306 TI - Site-specific alteration of murine hepatitis virus type 4 peplomer glycoprotein E2 results in reduced neurovirulence. AB - Strains of the murine coronavirus mouse hepatitis virus type 4 (MHV-4) which contained a mutation in the E2 peplomer glycoprotein were obtained by selection for resistance to neutralization by monoclonal antibodies. Characterization of six variants representing two independent epitopes on E2, E2B and E2C, by in vitro neutralization and antibody-binding assays demonstrated that selection for an alteration in epitope E2B also resulted in changes in epitope E2C and vice versa. We observed a mutation frequency of approximately 10(-4.3) to 10(-4.6), which is consistent with the expected occurrence of single point mutations. The variant virus strains were attenuated with respect to neurovirulence when compared with wild-type MHV-4. Mice normally develop encephalomyelitis and die after wild-type MHV-4 infection. Mice receiving 2- to 3-log-higher doses of the variant strains survived and developed demyelinating disease. As the disease progressed, evidence of remyelination and ongoing demyelination was observed up to 65 days after infection. Virus reisolated 15 days after infection retained the variant phenotype. The data indicate that the E2 glycoprotein plays a central role in determining the cellular tropism and virulence of MHV-4 in the mouse. PMID- 3016308 TI - Amino-terminal mutation of the vesicular stomatitis virus glycoprotein does not affect its fusion activity. AB - Earlier studies demonstrated that synthetic peptides corresponding to the amino terminus of the vesicular stomatitis virus glycoprotein (G protein) have a pH dependent hemolytic activity that is thought to be related to the fusion activity of G protein (R. Schlegel and M. Wade, J. Biol. Chem. 259: 4691-4694, 1984; R. Schlegel and M. Wade, J. Virol. 53: 319-323, 1985). A single amino acid change (lysine to glutamic acid at the amino terminus) abolishes the hemolytic activity of the peptide. Here we used oligonucleotide-directed mutagenesis to create a DNA encoding G protein with this same amino acid change at its amino terminus. The mutant protein encoded by this gene was expressed transiently in a monkey fibroblast cell line (COS) and was found to have a pH-dependent fusion activity indistinguishable from wild-type G protein. This result indicates that the hemolytic activity of the synthetic peptides was not related to the fusion activity of the G protein. PMID- 3016307 TI - Relationship between poliovirus neutralization and aggregation. AB - The interaction of mono- and polyclonal neutralizing antibodies with poliovirus was studied. In all cases, neutralization was due to antibody-mediated virus aggregation, and the unpolymerized virions accounted for the residual infectivity. The effect of papain on previously neutralized virus was to deaggregate the virus to fully infective single virions. With some antibodies, the amount of aggregated virus regressed in the region of greatest antibody excess, even though the virus remained fully neutralized. Under these conditions, noninfective, unaggregated immune complexes were formed. A mutant resistant to one of the monoclonal antibodies was selected. The mutant virions were still bound but no longer aggregated or neutralized by the selecting antibodies. PMID- 3016310 TI - Herpes simplex virus 1 recombinants with noninverting genomes frozen in different isomeric arrangements are capable of independent replication. AB - Herpes simplex virus 1 genome consists of two covalently linked components, L and S, that invert relative to each other to yield four equimolar isomeric populations designated P (prototype), Is (inversion of S component), Il (inversion of L component), and Ils (inversion of L and S components) differing in the orientation of the two components. Previous studies have yielded recombinant genomes frozen in the P isomeric arrangement, reinforcing suggestions that the four isomers may not be functionally equivalent. We report the isolation of recombinants produced by insertional mutagenesis with alpha TK mini-Mu that are frozen in Is and Ils arrangements. Thus, all isomeric forms of herpes simplex virus DNA appear to be capable of independent replication and must be considered as functionally equivalent. PMID- 3016309 TI - Highly infectious plasmids carrying poliovirus cDNA are capable of replication in transfected simian cells. AB - We examined events leading to production of infectious poliovirus upon transfection of simian cells with plasmids carrying poliovirus cDNA and simian virus 40 transcription and replication signals. The nature of the simian virus 40 promoter upstream from the poliovirus cDNA had no influence on its infectivity. A high specific infectivity was correlated with plasmid replication, dependent on expression of T antigen either encoded by the plasmid or present in host cells (COS-1). PMID- 3016311 TI - Molecular and biological properties of BK virus-IR, a BK virus variant isolated from a human tumor. AB - We describe the molecular and biological properties of BK virus (BKV)-IR, a new BKV variant isolated from a human tumor of pancreatic islets. BKV-IR bears a 253 base-pair (bp) deletion and an 80-bp insertion in the early region of the genome. The deletion abolishes the expression of small-t antigen. The inserted sequences, grouped in four clusters, produce rearrangements in the first and second enhancer elements. They are bound by 12-bp direct repeats and could form a 217-base stem loop structure suggestive of an insertion sequence. As compared with wild-type BKV, BKV-IR transformed hamster cells with a reduced efficiency and induced ependymomas in hamsters at a lower frequency and with a longer latency period. Tumors induced by BKV-IR, however, showed features of higher malignancy. The possible role of the insertion sequence-like element in transformation by BKV-IR is discussed. PMID- 3016313 TI - Serological characterization of human reassortant rotaviruses. AB - We analyzed the serological properties of two human wild-type cell culture adapted rotaviruses (strains 308 and 46) and of 308 X 46 reassortants which were previously obtained and genetically characterized. Strain 308, exhibiting a so called long RNA pattern, was found to belong to human rotavirus subgroup II, serotype 1, whereas strain 46, exhibiting a so-called short RNA pattern, represented subgroup I, serotype 2. Among the 308 X 46 reassortants we analyzed, two belonged to subgroup II, serotype 1, and exhibited short RNA patterns. This showed that the correlation observed between human subgroups I and II rotaviruses and the short and long electrophoretic patterns is not supported by any molecular basis (i.e., gene segment 10 or 11 was not involved in the subgroup specificity). PMID- 3016314 TI - Differential expression of endogenous mouse mammary tumor virus genes during development of the BALB/c mammary gland. AB - Expression of endogenous mouse mammary tumor virus sequences varied over the course of development of the mammary gland during primary pregnancy and lactation in virus-free BALB/c mice. Although RNA from all regions of the genome was detected, both the level and temporal regulation of expression were different for long terminal repeat-, env-, and gag-pol-specific RNAs. Analysis of the methylation status of proviral DNA indicated differential accessibility of the three endogenous units during development. The results demonstrated noncoordinate regulation of mouse mammary tumor virus expression with respect to provirus template utilized and specific transcripts accumulated. PMID- 3016315 TI - Sequential detection of different antigens induced by Epstein-Barr virus and herpes simplex virus in the same Western blot by using dual antibody probes. AB - A dual antibody probing technique that permitted a color-coded identification of polypeptides representing different classes of Epstein-Barr virus (EBV) antigens as well as differentiation of the polypeptides induced by different herpesviruses in the same Western blot was developed. When the nitrocellulose sheet was probed first with monoclonal antibody against EBV early antigen diffuse component (EA-D) and then stained with 4-chloro-1-naphthol, four polypeptides specific for EA-D were identified by purple bands. Subsequently, the same nitrocellulose sheet was reprobed with human serum containing antibodies against EBV early antigen, viral capsid antigen, and nuclear antigen and stained with 3,3'-diaminobenzidine. Several brown bands corresponding to early, viral capsid, and nuclear antigen polypeptides were detected. The dual antibody probing technique was used in an analysis to differentiate polypeptides resulting from either EBV or herpes simplex virus infection, either in cells infected by individual virus or in a cell line dually infected by both viruses. On the basis of different colored bands in different lanes of the same gel, 20 polypeptides with molecular weights ranging from 31,000 to 165,000 were identified as herpes simplex virus-specific proteins. These results suggested that the dual antibody probing technique may be applicable in clinical diagnosis for detecting antigens and antibodies derived from different pathogens. PMID- 3016312 TI - Herpes simplex virus (HSV)-specific human T-cell clones recognize HSV glycoprotein D expressed by a recombinant vaccinia virus. AB - Human cytotoxic T-cell (CTL) clones that lyse autologous cells infected with herpes simplex virus (HSV) type 1 or 2 were generated by stimulating lymphocytes with a recombinant vaccinia virus (recombinant vaccinia-gD-1 virus) that expresses HSV type 1 glycoprotein D (gD-1). Furthermore, CTL clones generated with HSV type 1 or with cloned gD-1 lysed autologous cells infected with the recombinant vaccinia-gD-1 virus. Our findings thus showed that gD serves as a target antigen for human CTLs and that a recombinant vaccinia-gD virus activates HSV-specific human CTL. PMID- 3016316 TI - Mutagenesis of the avian erythroblastosis virus erbB coding region: an intact extracellular domain is not required for oncogenic transformation. AB - Avian erythroblastosis virus (AEV) is an oncogenic retrovirus of birds. The AEV encoded erbB polypeptide, a transmembrane glycoprotein bearing an N-terminal domain exposed on the surface of virally transformed cells, plays a crucial role in AEV-mediated oncogenesis. We report here a characterization of a mutated form of the AEV erbB protein which lacks over two-thirds of the extracellular region of this oncogenic protein. This mutant v-erbB protein, although lacking the three possible extracellular sites of N-linked protein glycosylation, appears unimpaired in the ability to transform cells to an oncogenic phenotype. PMID- 3016317 TI - Characterization, chromosome assignment, and segregation analysis of endogenous proviral units of mouse mammary tumor virus. AB - In the course of analyzing sites of proviral integration in tumors induced by mouse mammary tumor virus (MMTV), we have isolated recombinant DNA clones corresponding to the 5' and 3' ends of four endogenous MMTV proviruses present in BALB/c and BR6 mice. This has permitted the structural characterization of each locus by detailed restriction mapping and the preparation of DNA probes specific for the cellular sequences flanking each provirus. These probes have been used to trace the segregation patterns of the proviruses, designated Mtv-8, Mtv-9, Mtv 17, and Mtv-21, in a panel of inbred strains of laboratory mice and to map Mtv-17 and Mtv-21 to mouse chromosomes 4 and 8, respectively. The unambiguous resolution of these four proviruses on Southern blots has greatly facilitated the analysis of other endogenous MMTV proviruses in these inbred mice. PMID- 3016318 TI - Amplification and chromosomal dispersion of human endogenous retroviral sequences. AB - Endogenous retroviral sequences in humans have undergone amplification events involving both viral and flanking cellular sequences. We cloned members of an amplified family of full-length endogenous retroviral sequences. Genomic blotting, employing a flanking cellular DNA probe derived from a member of this family, revealed a similar array of reactive bands in both humans and chimpanzees, indicating that an amplification event involving retroviral and associated cellular DNA sequences occurred before the evolutionary separation of these two primates. Southern analyses of restricted somatic cell hybrid DNA preparations suggested that endogenous retroviral segments are widely dispersed in the human genome and that amplification and dispersion events may be linked. PMID- 3016319 TI - Physical and genetic characterization of the genome of Lactobacillus lactis bacteriophage LL-H. AB - Bacteriophage LL-H is a virulent phage of Lactobacillus lactis LL23. A restriction map of the phage genome was constructed with various restriction endonucleases. This chromosome has a 34-kilobase size and seems to be circularly permuted. We used a bank of LL-H restriction fragments to study the expression of five of the seven main phage particle proteins. Immunoblotting experiments permitted the mapping on the chromosome of several genes coding for phage particle proteins. We also show that the gene of the main capsid protein is expressed from its own promoter in an Escherichia coli strain. PMID- 3016320 TI - Transduction of c-src coding and intron sequences by a transformation-defective deletion mutant of Rous sarcoma virus. AB - The mechanism of cellular src (c-src) transduction by a transformation-defective deletion mutant, td109, of Rous sarcoma virus was studied by sequence analysis of the recombinational junctions in three td109-derived recovered sarcoma viruses (rASVs). Our results show that two rASVs have been generated by recombination between td109 and c-src at the region between exons 1 and 2 defined previously. Significant homology between td109 and c-src sequences was present at the sites of recombination. The viral and c-src sequence junction of the third rASV was formed by splicing a cryptic donor site at the 5' region of env of td109 to exon 1 of c-src. Various lengths of c-src internal intron 1 sequences were incorporated into all three rASV genomes, which resulted from activation of potential splice donor and acceptor sites. The incorporated intron 1 sequences were absent in the c-src mRNA, excluding its being the precursor for recombination with td109 and implying that initial recombinations most likely took place at the DNA level. A potential splice acceptor site within the incorporated intron 1 sequences in two rASVs was activated and was used for the src mRNA synthesis in infected cells. The normal env mRNA splice acceptor site was used for src mRNA synthesis for the third rASV. PMID- 3016321 TI - Identification of the p53 protein domain involved in formation of the simian virus 40 large T-antigen-p53 protein complex. AB - An expression vector utilizing the enhancer and promoter region of the simian virus 40 (SV40) DNA regulating a murine p53 cDNA clone was constructed. The vector produced murine p53 protein in monkey cells identified by five different monoclonal antibodies, three of which were specific for the murine form of p53. The murine p53 produced in monkey cells formed an oligomeric protein complex with the SV40 large tumor antigen. A large number of deletion mutations, in-frame linker insertion mutations, and linker insertion mutations resulting in a frameshift mutation were constructed in the cDNA coding portion of the p53 protein expression vector. The wild-type and mutant p53 cDNA vectors were expressed in monkey cells producing the SV40 large T antigen. The conformation and levels of p53 protein and its ability to form protein complexes with the SV40 T antigen were determined by using five different monoclonal antibodies with quite distinct epitope recognition sites. Insertion mutations between amino acid residues 123 and 215 (of a total of 390 amino acids) eliminated the ability of murine p53 to bind to the SV40 large T antigen. Deletion (at amino acids 11 through 33) and insertion mutations (amino acids 222 through 344) located on either side of this T-antigen-binding protein domain produced a murine p53 protein that bound to the SV40 large T antigen. The same five insertion mutations that failed to bind with the SV40 large T antigen also failed to react with a specific monoclonal antibody, PAb246. In contrast, six additional deletion and insertion mutations that produced p53 protein that did bind with T antigen were each recognized by PAb246. The proposed epitope for PAb246 has been mapped adjacent (amino acids 88 through 109) to the T-antigen-binding domain (amino acids 123 through 215) localized by the mutations mapped in this study. Finally, some insertion mutations that produced a protein that failed to bind to the SV40 T antigen appeared to have an enhanced ability to complex with a 68-kilodalton cellular protein in monkey cells. PMID- 3016322 TI - Terminal structure and heterogeneity in human cytomegalovirus strain AD169. AB - We have characterized the heterogeneity occurring at the junction of the long (L) and short (S) segments and at the termini of the strain AD169 human cytomegalovirus (HCMV) genome by restriction endonuclease mapping and nucleotide sequence analyses. The HCMV a sequence was identified by its position at both termini and inverted orientation at the L-S junction. Heterogeneity at both termini and the L-S junction was generated by the presence of fused and tandem a sequences. Some S termini lacked an a sequence. In addition, near the L terminus and at the L-S junction there were a variable number of 217-base-pair (bp) XhoI fragments arranged in tandem. The 217-bp fragments consisted of a portion of the a and adjacent b sequences (in the L-segment repeat) bounded by the same direct repeats (DR1) found at the boundaries of the a sequence. A model for the generation of these heterogeneous fragments is presented. We also determined the sequence of seven cloned terminal fragments, five from the L terminus and two from the S terminus. All L termini contained identical terminal sequences ending with base 32 of a 33-bp DR1. The S termini differed from each other and from the L-segment termini. One S terminus lacked an a sequence and terminated within S segment repeat (c) sequences. The second S terminus contained an a sequence and terminated with bases 20 to 33 of a 33-bp DR1. A comparison of the cloned L and S terminal sequences with cloned L-S junction sequences suggested that the termini contained 3' single base extensions which were removed during the cloning. We also show that the herpesvirus conserved sequence is in a similar position relative to the termini of HCMV and several other herpesviruses, thus adding further support for the role of the sequence in the maturation of viral DNA. PMID- 3016325 TI - Immunologic selection of simian virus 40 (SV40) T-antigen-negative tumor cells which arise by excision of early SV40 DNA. AB - A clonal line of highly oncogenic spontaneously transformed mouse cells (104C) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned (106CSC). This 106CSC cell line expressed T antigen and transplantation antigen but was about 100 times less tumorigenic than the 104C parent. When 10(5) 106CSC cells were injected into immunocompetent syngeneic mice, tumors were produced. From such tumors, cell lines were established in culture, all of which were consistently negative for T antigen. We found previously by solution DNA hybridization methods that the tumor cells were depleted in the early region of SV40 DNA which codes for the T antigen. We postulated that this loss occurs through a DNA rearrangement of unknown mechanism in one or a few 106CSC cells and that the tumors are then produced from such a cell or cells, whereas all the T antigen-positive 106CSC cells are rejected by immunologic means. In this investigation we showed by the DNA transfer method using appropriately selected SV40 DNA probes that indeed the tumor cell clone (130CSCT) we selected to investigate came from one 106CSC cell in which the T-antigen-coding SV40 DNA sequences (but not all the early SV40 DNA sequences) were lost by an excision and recombination mechanism. We also showed that the 130CSCT cells, which are highly tumorigenic, could again be transformed by SV40 and that the resulting T-antigen positive cloned derivative cells became much less tumorigenic (approximately 10(5)-fold), apparently again because of immunologic recognition and rejection. Indeed, when 10(7) T-antigen-positive cloned cells were injected, all the T antigen-positive cells were rejected and the tumor was produced again from one or more T-antigen-negative cells. Thus, a one-step in vivo transplantation experiment allowed a selection (for tumorigenicity and against the SV40 T antigen) of a mutant mammalian cell with a DNA deletion at a definable site. PMID- 3016323 TI - Functional domains within the a sequence involved in the cleavage-packaging of herpes simplex virus DNA. AB - Newly replicated herpes simplex virus (HSV) DNA consists of head-to-tail concatemers which are cleaved to generate unit-length genomes bounded by the terminally reiterated a sequence. Constructed defective HSV vectors (amplicons) containing a viral DNA replication origin and the a sequence are similarly replicated into large concatemers which are cleaved at a sequences punctuating the junctions between adjacent repeat units, concurrent with the packaging of viral DNA into nucleocapsids. In the present study we tested the ability of seed amplicons containing specific deletions in the a sequence to become cleaved and packaged and hence be propagated in virus stocks. These studies revealed that two separate signals, located within the Ub and Uc elements of the a sequence, were essential for amplicon propagation. No derivative defective genomes were recovered from seed constructs which lacked the Uc signal. In contrast, propagation of seed constructs lacking the Ub signal resulted in the selection of defective genomes with novel junctions, containing specific insertions of a sequences derived from the helper virus DNA. Comparison of published sequences of concatemeric junctions of several herpesviruses supported a uniform mechanism for the cleavage-packaging process, involving the measurement from two highly conserved blocks of sequences (pac-1 and pac-2) which were homologous to the required Uc and Ub sequences. These results form the basis for general models for the mechanism of cleavage-packaging of herpesvirus DNA. PMID- 3016324 TI - Expression of simian virus 40 early and late genes in mouse oocytes and embryos. AB - Simian virus 40 (SV40) large- and small-tumor antigens (T-Ag, t-Ag) are normally synthesized early after infection of either permissive (monkey) or nonpermissive (mouse) fibroblasts, whereas an equivalent amount of viral coat protein (V-Ag) is observed late after infection of permissive cells and only after viral DNA replication has occurred. To determine whether or not expression of these genes is regulated in the same manner during early mammalian development, SV40 DNA was injected into the nuclei of mouse oocytes and one- and two-cell embryos. In oocytes, about three times more V-Ag was produced than T-Ag, and both were synthesized concomitantly in the same cells. Viral mRNA and proteins synthesized in oocytes comigrated during gel electrophoresis with the same products synthesized in SV40-infected monkey cells. Viral gene expression required circular DNA molecules injected into the nuclei of transcriptionally and translationally active cells. Injected DNA was stable and underwent conformational changes consistent with chromatin assembly. Oocytes did not replicate either polyomavirus or SV40 DNA. Thus, the temporal order of viral gene expression is circumvented in mouse germ cells, allowing these proteins to be expressed concurrently and in equivalent amounts with no requirement for DNA replication. However, in preimplantation embryos, neither T-Ag nor V-Ag was detected by immunoprecipitation although T-Ag synthesis was demonstrated as a specific requirement for SV40 DNA replication. Thus, viral gene expression in mouse embryos as early as the one-cell stage was reduced at least 500-fold relative to that in oocytes. Similarities between SV40 gene expression in mouse oocytes and that in Xenopus oocytes suggest that germ cells in higher animals share common regulatory mechanisms. PMID- 3016326 TI - Pseudorabies virus gene encoding glycoprotein gIII is not essential for growth in tissue culture. AB - We have established that in the Becker strain of pseudorabies virus (PRV), the glycoprotein gIII gene is not essential for growth in cell culture. This was accomplished by construction and analysis of viral mutants containing two defined deletion mutations affecting the gIII gene. These mutations were first constructed in vitro and introduced into Escherichia coli expression plasmids to verify structure and protein production. Each mutation was then crossed onto PRV by cotransfection of plasmid DNA and parental viral DNA by using gIII-specific monoclonal antibodies as selective and screening reagents. One resultant virus strain, PRV-2, contained an in-frame deletion of a 402-base-pair (bp) SacI fragment contained within the gIII gene. Another virus strain, PRV-10, contained a deletion of a 1,480-bp XhoI fragment removing 230 bp of the upstream, putative transcriptional control sequences and 87% of the gIII coding sequence. The deletion mutants were compared with parental virus by analysis of virion DNA, gIII specific RNA, and proteins reacting with gIII specific antibodies. Upon infection of PK15 cells, the deletion mutants did not produce any proteins that reacted with two gIII specific monoclonal antibodies. However, two species of truncated glycosylated proteins were observed in PRV-2 infected cells that reacted with antiserum raised against bacterially produced gIII protein. PRV-10 produced no detectable gIII-specific RNA or protein. PRV-10 could be propagated without difficulty in tissue culture. Virus particles lacking gIII were indistinguishable from parental PRV virus particles by analysis of infected-cell thin sections in the electron microscope. We therefore conclude that expression of the gIII gene was not absolutely essential for PRV growth in tissue culture. PMID- 3016327 TI - Human parainfluenza virus type 3: messenger RNAs, polypeptide coding assignments, intergenic sequences, and genetic map. AB - cDNA clones of mRNAs for the major nucleocapsid protein (NP), the nucleocapsid P protein plus the nonstructural C protein (P+C), and the matrix protein (M) of human parainfluenza virus type 3 (PF3) were identified by hybrid arrest and hybrid selection of in vitro translation. Previously, cDNA clones were identified and sequenced for the hemagglutinin-neuraminidase glycoprotein (HN) and the fusion glycoprotein (F) mRNAs (N. Elango, J. E. Coligan, R. C. Jambou, and S. Venkatesan, J. Virol. 57:481-489, 1986; M. K. Spriggs, R. A. Olmsted, S. Venkatesan, J. E. Coligan, and P. L. Collins, Virology 152:241-251, 1986). Synthetic oligonucleotides, designed from nucleotide sequences of the cDNAs, were used to direct dideoxynucleotide sequencing of gene junctions in PF3 genomic RNA (vRNA). From sequencing of vRNA, a sixth viral gene was detected and identified as the large nucleocapsid protein (L) gene by hybridization of a synthetic oligonucleotide to intracellular PF3 mRNAs separated by gel electrophoresis. The order of the six PF3 genes on vRNA was 3'-NP-P+C-M-F-HN-L-5'. The five intergenic regions consisted of the trinucleotide 3'-GAA. The PF3 genes initiated with semiconserved 10-nucleotide gene-start sequences and terminated with semiconserved 12-nucleotide gene-end sequences. The M gene terminated with an aberrant gene-end sequence; analysis of intracellular mRNA showed that this aberrant sequence correlated with a disproportionately high accumulation of readthrough mRNA. These studies showed that PF3 encodes six unique mRNAs (NP, P+C, M, F, HN, and L) that encode seven proteins (NP, P, C, M, F, HN, and L) and provided evidence of a close relationship between PF3 and Sendai (murine parainfluenza type 1) viruses. PMID- 3016328 TI - Ability of a T-antigen transport-defective mutant of simian virus 40 to immortalize primary cells and to complement polyomavirus middle T in tumorigenesis. AB - The oncogenic potential of polyomavirus in newborn rats could not be expressed by a genome encoding only the middle T antigen but required the presence of one of the other two viral early genes, small T or large T. The tumorigenicity defect could also be complemented by other viral or cellular genes that are known to be implicated in immortalization and establishment functions. The simian virus 40(cT)-3 mutant (R. E. Lanford and J. S. Butel, Cell 37:801-813, 1984), which fails to localize to the nucleus, has the capacity to complement polyomavirus middle T in tumorigenesis and to immortalize primary rat embryo fibroblasts when it was cotransfected in the presence of pSV2-neo. Our data suggested that under the conditions of DNA-mediated tumor induction and cotransfection with a dominant selection marker, the cellular alterations achieved by nonnuclear oncogenes such as polyomavirus small T and simian virus 40(cT)-3 were sufficient to complement polyomavirus middle T in transformation and tumorigenesis. PMID- 3016329 TI - Putative glycoprotein gene of varicella-zoster virus with variable copy numbers of a 42-base-pair repeat sequence has homology to herpes simplex virus glycoprotein C. AB - A strain variation of varicella-zoster virus that maps to the UL region of the genome was found to be due to different copy numbers of a high GC 42-base-pair repeat. DNA sequence analysis of this variable region showed the sequence to be 5 GCGGGATCGGGCTTTCGGG(A/T)AGCGGCCGAGGTGGGCGCGACG-3. Strains Scott and Webster both contain 7 and 32/42 copies of the repeat, whereas strain Oka has exactly 4 copies less. Microheterogeneity exists within the repeated sequences, depending on the strain and the repeat number. Sequencing of the entire EcoRI P fragment (which contains the repeated sequences) and part of the adjacent EcoRI M and EcoRI Q fragments from strain Scott showed that the repeats are part of a large open reading frame that could code for a polypeptide core with a molecular weight of 66,000. Several potential TATA boxes exist upstream and two polyadenylation signals are found downstream of the open reading frame. The predicted protein bears several characteristics of a glycoprotein. The region is transcriptionally active in varicella-zoster virus-infected cells, specifying at least three RNA species of 1.7, 1.95, and 2.5 kilobases, which are transcribed from the same DNA strand. Part of the predicted protein has a high degree of homology to the herpes simplex virus type 1 glycoprotein gC. PMID- 3016330 TI - Molecular cloning and characterization of gag-, pol-, and env-related gene sequences in the ev- chicken. AB - Using less stringent hybridization conditions and cloned viral DNA probes representing the avian sarcoma virus gag, pol, env, and long terminal repeat (LTR) gene sequences, we detected related sequences in two avian species purportedly lacking all endogenous avian leukosis viruses, the ev- chicken and the Japanese quail. The blot hybridization patterns obtained with the various probes suggest the presence of between 40 and 100 copies of retrovirus-related sequences in the genomes of these two species. An ev- chicken genomic DNA library was prepared and screened with gag-specific and pol-specific DNA probes. Several different clones were obtained from this library and characterized. Analysis of these clones revealed that the retrovirus-related gene sequences are linked in the order LTR-gag-pol-env-LTR, a structure indicative of a complete provirus. These data indicate the presence of previously unidentified endogenous retrovirus species in avian cells, suggesting that under the appropriate conditions of hybridization additional, more distantly evolved families of endogenous retrovirus genes may be identified in vertebrate species. PMID- 3016332 TI - Primate cytomegalovirus glycoproteins: lectin-binding properties and sensitivities to glycosidases. AB - The lectin-binding properties and glycosidase sensitivities of the virion glycoproteins of primate cytomegaloviruses (CMVs) were examined. Three simian CMV (SCMV) strains, including Colburn, and four human CMV (HCMV) strains were compared. Their proteins were separated in denaturing polyacrylamide gels and electrotransferred onto nitrocellulose, and the glycosylated species were visualized with iodinated concanavalin A or wheat germ agglutinin (WGA). Virions of both HCMV and SCMV strains contained six principal and several minor lectin reactive bands. Neuraminidase treatment abolished WGA binding and reduced the charge and charge heterogeneity of the SCMV (i.e., Colburn) virion glycoproteins and had a similar, although less dramatic, effect on those of HCMV. The specificities of concanavalin A and WGA in these assays were evaluated with endo beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F, and a combination of lectins and glycosidases was used to demonstrate that many of the primate CMV glycoproteins contain both high-mannose and complex, N-linked oligosaccharides. Results suggest that the HCMV virion glycoproteins are more extensively glycosylated or have more completely processed carbohydrate side chains, or both, than their SCMV counterparts. PMID- 3016331 TI - Cellular proteins which can specifically associate with simian virus 40 small t antigen. AB - When crude, radiolabeled extracts of various cells were applied to homogeneous simian virus 40 small t antigen-Sepharose adsorbents, three cell proteins (57, 32, and 20 kilodaltons [kDa]) bound specifically. Each also bound to an insoluble, truncated t derivative composed of the COOH-terminal 123 residues of the protein. The binding of these proteins was greatly inhibited after reduction and alkylation of the t ligand. Therefore, some element of native conformation, but not all of the primary structure of t, is necessary for this binding property, which may constitute a discrete, in vitro biochemical function of this protein. Results of cell fractionation experiments suggested that the 57- and 32 kDa proteins are nonnuclear cell constituents, whereas the 20-kDa protein was closely associated with a detergent-washed nuclear fraction. Specific immunoblotting and comparative partial proteolytic digestion analyses indicated that the 57-kDa protein is tubulin, a major component of the cytoskeleton. In this regard, t and tubulin were observed to coimmunoprecipitate from crude cell extracts after incubation with monospecific anti-t antibody. Therefore, it is possible that t and tubulin interact in vivo. PMID- 3016334 TI - Antibody to mouse alpha/beta interferon abrogates Pichinde virus-induced liver lesions in suckling mice. AB - Infection of newborn C3HeB/FeJ mice with the arenavirus Pichinde resulted in stunted growth, severe liver cell degeneration, and death. Administration of sheep anti-mouse alpha/beta interferon globulin completely abrogated liver lesions in virus-infected mice, although it did not decrease the incidence of mortality. These results indicate that endogenous interferon may be responsible for some manifestations of viral disease. PMID- 3016336 TI - DNA-binding properties of the major structural protein of simian virus 40. AB - We investigated whether the VP1 protein of simian virus 40 binds to DNA. In vitro DNA-binding experiments clearly indicate that VP1 bound strongly to double stranded and single-stranded DNA, with a higher affinity for the latter; additional experiments show that VP1 did not bind to a specific sequence of simian virus 40 DNA. PMID- 3016333 TI - Characterization of phosphoproteins and protein kinase activity of virions, noninfectious enveloped particles, and dense bodies of human cytomegalovirus. AB - Phosphorylation of the proteins of human cytomegalovirus (CMV) virions, noninfectious enveloped particles (NIEPs), and dense bodies was investigated. Analyses of particles phosphorylated in vivo showed the following. Virions contain three predominant phosphoproteins (i.e., basic phosphoprotein and upper and lower matrix proteins) and at least nine minor phosphorylated species. NIEPs contain all of these and one additional major species, the assembly protein. Dense bodies contain only one (i.e., lower matrix) of the predominant and four of the minor virion phosphoproteins. Two-dimensional (charge-size) separations in denaturing polyacrylamide gels showed that the relative net charges of the predominant phosphorylated species ranged from the basic phosphoprotein to the more neutral upper matrix protein. In vitro assays showed that purified virions of human CMV have an associated protein kinase activity. The activity was detected only after disrupting the envelope; it had a pH optimum of approximately 9 to 9.5 and required a divalent cation, preferring magnesium to manganese. In vitro, this activity catalyzed phosphorylation of the virion proteins observed to be phosphorylated in vivo. Peptide comparisons indicated that the sites phosphorylated in vitro are a subset of those phosphorylated in vivo, underscoring the probable biological relevance of the kinase activity. Casein, phosvitin, and to a minor extent lysine-rich histones served as exogenous phosphate acceptors. Arginine-rich and lysine-rich histones and protamine sulfate, as well as the polyamines spermine and spermidine, stimulated incorporation of phosphate into the endogenous viral proteins. Virions of all human and simian CMV strains tested showed this activity. Analyses of other virus particles, including three intracellular capsid forms (i.e., A, B, and C capsids), NIEPs, and dense bodies, indicated that the active enzyme was not present in the capsid. Rate-velocity sedimentation of disrupted virions separated the protein kinase activity into two fractions: one that phosphorylated exogenous casein and another that phosphorylated primarily the endogenous virion proteins. PMID- 3016335 TI - Integration of hepatitis B virus DNA in chromosome-specific satellite sequences. AB - We previously reported the cloning and detailed analysis of the integrated hepatitis B virus sequences in a human hepatoma cell line. We report here the integration of at least one of hepatitis B virus at human satellite DNA sequences. The majority of the cellular sequences identified by this satellite DNA were organized as a multimeric composition of a 0.6-kilobase EcoRI fragment. This clone hybridized in situ almost exclusively to the centromeric heterochromatin of chromosomes 1 and 16 and to a lower extent to chromosome 2 and to the heterochromatic region of the Y chromosome. The immediate flanking host sequence appeared as a hierarchy of repeating units which were almost identical to a previously reported human satellite III DNA sequence. PMID- 3016337 TI - Large T antigens of simian virus 40 and polyomavirus efficiently establish primary fibroblasts. AB - Recombinant retroviruses that transduce the simian virus 40 (SV40) large T antigen or the polyomavirus large T antigen as well as encoding resistance to antibiotic G418 were used to investigate whether these genes alone were sufficient for immortalization of primary cells. The results provided definitive evidence that either viral gene can efficiently establish primary fibroblasts. The capability of the SV40 large T antigen to establish primary fibroblasts was undiminished by a mutation that alters its binding to sequences within the origin of replication. Surprisingly, most of the primary cells established by the expression of the SV40 large T antigen did not have a transformed phenotype. This suggests that transformation by SV40 is not simply due to a high level of expression of the SV40 large T antigen and stabilization of cellular p53. PMID- 3016338 TI - Vesicular stomatitis virus N and NS proteins form multiple complexes. AB - The vesicular stomatitis virus nucleocapsid protein, N, associated specifically with the viral phosphoprotein, NS, in an in vitro system which supported vesicular stomatitis virus RNA replication. Essentially all the N protein was found complexed with NS. In addition, multiple forms of the N-NS complex were detected which differed in their sedimentation properties and ratios of N to NS. PMID- 3016339 TI - Negative regulation of early polyomavirus expression in mouse embryonal carcinoma cells. AB - Embryonal carcinoma cells are resistant to infection by polyomavirus (Py). We showed that this block was partially removed by inhibiting protein synthesis temporarily. The block was also partially removed when Py was coinfected with simian virus 40. Cycloheximide treatment of cells infected with Py mutants able to grow on PCC4 embryonal carcinoma cells led to 3- to 10-fold increases in the production of T-antigen-positive cells. At 31 degrees C, Py T-antigen expression was enhanced when the cells were treated with cycloheximide. We suggest that a negative labile regulatory protein(s) is synthesized in PCC4 cells, preventing the initiation of early Py transcription by binding to the noncoding sequence, especially the enhancer element B and perhaps also element A, and that the Py mutants retained a binding site(s). PMID- 3016341 TI - Site of attachment of encephalomyocarditis virus on human erythrocytes. AB - Previous studies of the attachment of encephalomyocarditis (EMC) virus to human erythrocytes concluded that the glycophorins, a family of human erythrocyte sialoglycoproteins, act as EMC virus receptors. Evidence is presented that the major glycophorin species, glycophorin A, is the receptor for EMC virus attachment to human erythrocytes. Comparison of the structures of glycophorins A and B and sialoglycopeptides released by chymotrypsin and trypsin treatment of erythrocytes confirmed our previous suggestion (A. T. H. Burness and I. U. Pardoe, J. Gen. Virol. 64:1137-1148, 1983) that attachment of EMC virus to glycophorin A involves the region containing amino acids 35 to approximately 70 (numbered from the NH2 terminus), four of which (amino acids 37, 44, 47, and 50) are glycosylated. In addition, we provide evidence that the segment containing amino acids 35 to 39 with an oligosaccharide side chain on threonine-37 is particularly important for EMC virus attachment. PMID- 3016340 TI - Chromosomal organization of the herpes simplex virus genome during acute infection of the mouse central nervous system. AB - After corneal inoculation, herpes simplex virus type 1 replicates in the mouse eye, trigeminal ganglia, and brainstem, producing first an acute and then a latent infection. Previous work from this laboratory focused on the structure of the viral DNA in this system. We have now examined the structure of the viral genome at the chromosome level by using micrococcal nuclease digestion. Studies with disaggregated cell preparations made from the brainstems of acutely infected mice show that the majority of the viral DNA is in a nonnucleosomal form; however, a nucleosomelike fraction was also consistently detected. A similar result was obtained for viral DNA in herpes simplex virus type 1-infected C1300 (clone NA) neuroblastoma cells (a neuronal cell line). PMID- 3016342 TI - Klippel-Trenaunay and Sturge-Weber syndromes with renal hemangioma and double inferior vena cava. AB - We describe a 3 1/2-year-old boy with the Klippel-Trenaunay and Sturge-Weber syndromes. The child had congenital superficial capillary hemangiomas, congenital glaucoma and mild hydrocephalus. During the first year of life he experienced intermittent hematuria. When he was 3 years old he presented with seizures and left hemihypertrophy first was noted. Several months later radiological examination of a large abdominal mass demonstrated its origin to be in the right kidney. Radical nephrectomy documented the presence of renal hemangioma with complicating perirenal hematoma. A double inferior vena cava was another unexpected surgical finding that complicated the course of this patient. All of these unusual features in these rare syndromes with their clinical, pathogenetic and therapeutic implications are discussed. The differential diagnosis of renal masses in these syndromes also is presented. PMID- 3016343 TI - An unusual complication of silver nitrate treatment of hemorrhagic cystitis: case report. AB - A patient with severe cyclophosphamide cystitis was treated with intravesical silver nitrate instillation to control bleeding. This resulted in apparent reflux and extravasation of the silver nitrate solution with secondary retroperitoneal inflammation. Subsequently, the patient required treatment for a small, fibrotic bladder with persistent reflux. The precautions and recommendations for treatment of such difficult patients are discussed. PMID- 3016344 TI - Recurrent penile condylomata acuminata in a 17-month-old boy. AB - Penile condyloma acuminatum is a rare pediatric entity. We report on a 17-month old boy with penile and urethral condyloma acuminatum. The presence of cervical dysplasia in a histological setting suggestive of condyloma in his mother supports congenital acquisition of the disease. Histological and histochemical evidence for a viral etiology of the disease in the mother and patient is presented. PMID- 3016345 TI - Contribution of endogenous vasoactive compounds to renal vascular resistance in neonatal chronic partial ureteral obstruction. AB - To evaluate the relative contribution of endogenous vasoactive compounds to maintenance of increased renal vascular resistance in neonatal obstructive nephropathy, cardiac output and renal blood flow were measured using radioactive microspheres in 25 +/- 3 day-old guinea pigs subjected to unilateral partial ureteral constriction within the first two days of life. Mass and renal blood flow of the obstructed kidney were significantly lower than those of the contralateral kidney. Following a control period, thromboxane synthesis was blocked by infusion of OKY-046, after which prostaglandin synthesis was inhibited by indomethacin. In a separate group of animals, angiotensin converting enzyme inhibitor, MK-422, was infused before or after administration of OKY-046. While neither OKY-046 nor indomethacin had a consistent effect on vascular resistance, infusion of MK-422 resulted in selective reduction of renal vascular resistance of the obstructed kidney compared to resistance in the intact kidney and other vascular beds. Removal of the contralateral kidney at the time of ureteral constriction in an additional group of animals resulted in hypertrophy and vasodilation of the obstructed kidney which was not altered by thromboxane or cyclooxygenase inhibition. We conclude that in the neonatal kidney subjected to ipsilateral chronic partial ureteral obstruction, vasoconstriction is mediated at least in part by angiotensin II, but not by thromboxane. Furthermore, vasodilation of the obstructed kidney resulting from contralateral nephrectomy is not dependent on prostaglandin synthesis. Renal vascular resistance of the kidney with prolonged partial ureteral constriction in early development thus appears to be inversely related to renal growth and is not significantly mediated by endogenous prostanoids. PMID- 3016346 TI - Nonseminomatous germ cell tumor of the testicle: does extensive staging of the primary tumor predict the likelihood of metastatic disease? AB - To assess the hypothesis that local extent of the primary lesion in patients with nonseminomatous germ cell tumors of the testicle can predict disseminated disease, clinicopathological correlations of the primary tumor and metastases were determined in a retrospective review of 120 patients treated at our institution from 1970 to 1982. Pathological staging was available for all 93 patients with subdiaphragmatic disease and the primary tumor was examined by routine histological techniques in all cases. Increased primary tumor stage, as evidenced by invasion into the tunica vasculosa or extension into the rete testis, epididymis and/or lower or upper spermatic cord, was associated with metastatic disease in 91, 96, 97 and 97 per cent of the patients, respectively. Only 9 per cent of the patients with pathological stage A disease had vascular invasion compared to 45 and 67 per cent of those with stages B and C disease, respectively (p less than 0.01). Furthermore, metastases and/or eventual dissemination occurred in 84 per cent of the patients with clinical stage A disease and vascular invasion, and in only 23 per cent of those without vascular invasion (p less than 0.01). Size of the primary tumor was not of predictive value. Local extension of the primary lesion was common with embryonal carcinoma but it was not demonstrated when the predominant histological type was teratoma or teratocarcinoma, although metastases were present in 37 and 46 per cent of the latter cases, respectively. The implications of these findings, especially with regard to expectant management for clinical stage A nonseminomatous germ cell testis tumors, are discussed. PMID- 3016347 TI - Polyglycolic acid mesh in experimental renal trauma. AB - We investigated the efficacy of kidney wrapping with polyglycolic acid (PGA) mesh for control of hemorrhage and preservation of renal function following extensive potentially lethal kidney lacerations in the dog. Wrapping of lacerated kidneys resulted in reapposition of the renal parenchyma and prompt, sustained hemostasis. At 21 days following injury the renal lacerations were well healed. Among five dogs with lacerations involving the entire surface of one kidney, the mean of the ratios of the creatinine clearance of the affected kidney divided by the creatinine clearance of the uninjured contralateral kidney was 0.83 +/- 0.14. Among ten dogs with lacerations confined to the lower pole of one kidney, five were treated by mesh wrapping and five by partial nephrectomy. The mean of the ratios of the creatinine clearance of the affected kidney divided by the creatinine clearance of the uninjured contralateral kidney was 0.93 +/- 0.17 for the former group and 0.58 +/- 0.06 for the latter group. Perirenal infection following kidney wrapping developed in only one dog who had an E. coli bacteriuria at the time of injury. Blood pressure was monitored in eight dogs treated with mesh wrapping. None became hypertensive. These data suggest that PGA mesh may have clinical utility in the management of selected renal injuries in humans. PMID- 3016348 TI - Antibodies to vesicular stomatitis virus in populations of feral swine in the United States. AB - From 1979 to 1985, 941 feral swine (Sus scrofa) from 53 locations in 15 states were serologically tested for antibodies to vesicular stomatitis virus (VSV). Antibodies to New Jersey serotype VSV were present in 75 swine from five locations in Arkansas, Florida, Georgia, and Louisiana. Within these populations, antibody prevalences ranged from 10 to 100%. No antibodies to Indiana serotype were detected. PMID- 3016349 TI - Nitrite poisoning in herring gulls (Larus argentatus) and ring-billed gulls (Larus delawarensis). AB - Landfill disposal of a fertilizer manufacturing waste product was associated with a die-off of gulls in New Hanover County, North Carolina. An estimated 250 herring and ring-billed gulls were found dead at the site following the initial disposal of this material. Chemical analyses revealed that the fertilizer waste contained predominately calcium (12.0 to 22.2%) and nitrite (3.0 to 15.2%). Contents of the proventriculi and gizzards of dead gulls also contained calcium (3.0 to 10.9%) and nitrite (1,730 ppm). Fertilizer waste administered orally to 16-day-old domestic turkeys resulted in acute, progressive signs of depression, respiratory distress, pallor, convulsions and death. The mean percentage methemoglobin in blood from convulsing turkeys (90.6) was significantly increased from that of normal control turkeys (3.6). The ante-mortem signs and increased blood methemoglobin concentrations in the experimental turkeys support the conclusion that the toxic principle in the fertilizer waste was nitrite, and that nitrite poisoning was the cause of the die-off of gulls. PMID- 3016350 TI - Experimental infection of vampire bats with foot and mouth disease virus. PMID- 3016351 TI - Isolation of a poxvirus from a house finch, Carpodacus mexicanus (Muller). PMID- 3016353 TI - Atypical lesions of the anal mucosa in homosexual men. AB - Recent studies suggest that there is an increased incidence of squamous cell carcinoma of the anus in male homosexuals, but a precursor lesion has not been identified. We retrospectively analyzed in a blind fashion all anal tissue removed surgically during 1984. Twelve (6.7%) of the 180 specimens from men contained lesions with foci of epithelial atypia. Only one (0.85%) of 118 specimens from women harbored similar atypia. Of seven additional file cases exhibiting atypical anal mucosa, six were from men. Of 14 men with atypical anal lesions whose sexual orientation was known, 11 (79%) were homosexuals. In the 20 cases found to have atypical mucosal lesions, three patterns of atypia were identified, with more than one often occurring in the same specimen. Anal intraepithelial neoplasia (dysplasia) was identified in seven cases (35%) and occurred primarily at the anorectal junction and in anal ducts. Atypical condyloma was found in three cases (15%). A third lesion histologically indistinguishable from Bowen's disease or bowenoid papulosis was found in 12 cases (60%). In ten of these the lesion was adjacent to a condyloma. Although the natural history of these lesions of the anal mucosa is presently unknown, it may resemble that of similar lesions in other anatomic locations. PMID- 3016352 TI - The safety of the hepatitis B vaccine. Inactivation of the AIDS virus during routine vaccine manufacture. AB - In the United States, one hepatitis B vaccine (Heptavax-B) has been licensed for the prevention of hepatitis B virus infections. Even though this vaccine has been shown to be highly effective and well tolerated in controlled trials and has been recommended for use in those at risk for acquiring infection by hepatitis B virus, many individuals have been reluctant to be immunized for fear of contracting acquired immunodeficiency syndrome (AIDS). In this study, we demonstrate that each of the three inactivation steps used in the manufacture of Heptavax-B independently will inactivate the infectivity of high-titered preparations of the AIDS virus; recipients of the hepatitis B vaccine do not develop antibodies to the AIDS virus; the hepatitis B vaccine does not contain detectable levels of nucleic acids related to the AIDS virus. These observations clearly demonstrate that vaccination with the currently available hepatitis B vaccine poses no demonstrable risk for acquiring AIDS. PMID- 3016354 TI - Possible defense against common cold: 'block that cellular receptor site'. PMID- 3016356 TI - Measures to control Chlamydia trachomatis infections: an assessment of new national policy guidelines. PMID- 3016355 TI - Leads from the MMWR. Human T-lymphotropic virus type III/lymphadenopathy associated virus antibody prevalence in US military recruit applicants. PMID- 3016357 TI - Prostacyclin generation by cultured human vascular endothelial cells with reference to angiotensin I-converting enzyme. AB - Prostacyclin (PGI2) generation has been known to be regulated by several endogenous vasoactive substances, and in this study the relationship between angiotensin I-converting enzyme (ACE) related substances and PGI2 generation was investigated using cultured human vascular endothelial cells. Addition of angiotensin I (AI) or bradykinin (BK) enhanced PGI2 generation and increased the level of ACE activity in the culture medium, while the addition of ACE inhibitor (captopril) caused a dose dependent suppression of PGI2 generation and ACE activity. The enhancement of PGI2 generation induced by AI or BK was not affected by pretreatment with captopril, and angiotensin II (AII) did not show any effect on either PGI2 generation or ACE activity. Through these experimental results, the conversion of AI to AII by ACE was considered not to cause the enhancement of PGI2 generation. Captopril solely inhibited PGI2 generation and the reported hypothesis that captopril enhances PGI2 generation by the accumulation of AI or BK via inhibition of ACE was not confirmed in this experimental system. Rather, it is proposed that AI or BK induced PGI2 generation may be regulated by the increased breakdown of AI or BK, as an autoregulation mechanism, that is derived from increased ACE activity by AI or BK. PMID- 3016358 TI - Susceptibility to ischemic insult in hypertensive rats: correlation between degree of ischemia and hypertension. AB - The Wistar rat, with a blood pressure range of 120-160 mmHg, and two strains of spontaneously hypertensive rats (SHR), stroke-prone (SHRSP, range 210-270 mmHg) and stroke-resistant (SHRSR, range 160-240 mmHg), were used to determine the degree of damage after ischemic insult induced by bilateral carotid artery ligation (BLCL). The survival rate and McGraw Stroke Index correlated well with the degree of hypertension. After BLCL, impairment of cerebral blood flow is abrupt and residual flow is near zero in rats with initial blood pressures greater than 200 mmHg. A markedly deteriorated aerobic metabolism, as measured by the concentrations of ATP, c-AMP and lactate, is seen to precipitate in rats with initial blood pressures greater than 180 mmHg and severe edema occurs if the pressure is more than 160 mmHg. The degree of hypertension that produces high vulnerability to stroke and severe damage to the brain after ischemic insult is indicated as beginning at about 180 mmHg. PMID- 3016359 TI - [Stability of high molecular weight anticancer agent SMANCS and its transfer from oil-phase to water-phase. Comparative study with neocarzinostatin]. AB - SMANCS is a conjugate protein of copolymer of styrene-maleic acid [SMA] (molecular weight: 1,500) and an antitumor protein neocarzinostatin [NCS] (molecular weight: 11,700). It has an approximate molecular weight of 15,000. We report here stability of SMANCS in oil and in water, and NCS in water, under various physical conditions such as exposure to heat, UV, pH, and ultrasonic treatment. Then, we carried out an experiment of transfer of SMANCS in lipid contrast medium [lipiodol] (oil phase) to water phase (blood and physiological saline) in vitro. Results are summarized as follows: In aqueous condition, SMANCS is far more stable than NCS against the exposure to heat and UV, though it is inactivated by excessive exposures. SMANCS in an oily medium was found much more stable even at higher temperatures than in the aqueous phase. Both SMANCS and NCS are the most stable at pH 4.9-6.0. SMANCS dissolved in oil transferred to water phase slowly, having T1/10 of 24 hours (in case of lipiodol). This helps maintaining the anticancer effect of the drug in vivo for a long period of time. SMANCS in lipiodol was found to exert its action against cultured tumor cells as in an aqueous solution. PMID- 3016360 TI - [Clinical investigation of indications in proton therapy]. AB - The indications for curative treatment with proton therapy were investigated by the clinical results of 31 cancer patients treated with proton beams between 1983 and 1985 at the Particle Radiation Medical Science Center of Tsukuba University. Many locally extended, radioresistant lesions, lesions with large volume and small radiocurable lesions in this series revealed sufficient improvement without definite late damage in the surrounding normal tissues. This clinical result suggests that proton beam treatment could be applied to lesions in a much wider range for curative purposes as compared with conventional radiotherapy. This study is to be continued further. PMID- 3016361 TI - [Massive osteoplastic bone metastasis of hepatocellular carcinoma--a case report]. AB - Massive osteoplastic bone tumor in hepatocellular carcinoma is very rare. A 48 year-old man was misdiagnosed as osteosarcoma of the right proximal tibia with dense sclerosis and marked periosteal spiculation. Histologically, there were many osteoids and immature trabeculi. Tumor cells with spindle nuclei were not atypical and had few mitoses. Three years later, he suddenly died of rupture of cerebral aneurysm. Autopsy revealed small hepatocellular carcinoma with distant metastases of the tibia, lumbar spine and lung. In this case, it was extremely difficult to decide whether or not we were dealing with primary malignant tumor. PMID- 3016362 TI - [A case of CSF (colony-stimulating factor)-producing lung cancer and a review of the literature]. AB - A 36-year-old man with large-cell carcinoma of the lung showed marked leukocytosis of up to 109,300/mm3. As high CSF activity was detected in the pleural effusion and the supernatant of cell cultures originating from the effusion, a diagnosis of CSF-producing lung cancer was made. Twenty-one CSF producing lung cancers have been reported in Japan. They comprised 13 large-cell carcinomas, six squamous cell carcinomas, one adenocarcinoma and one small cell carcinoma. PMID- 3016364 TI - [Striated strip structures in the cells of adult T-cell leukemia: ultrastructural study]. PMID- 3016363 TI - [A case of congenital stomatocytosis with normal sodium fluxes, normal enzyme activities of red cells, but with increased osmotic fragility of red cells]. PMID- 3016366 TI - [Corticotropin releasing factor-producing tumor]. PMID- 3016367 TI - [Biosynthesis of pituitary hormones. Gene organization and processing- proopiomelanocortin peptides]. PMID- 3016365 TI - [Corticotropin releasing factor tolerance test]. PMID- 3016368 TI - [Secretory control of pituitary hormones--ACTH]. PMID- 3016369 TI - [Secretory regulation of gonadotropin]. PMID- 3016370 TI - [New pituitary hormones-special reference to opioid peptides]. PMID- 3016371 TI - [Immunohistochemistry of pituitary adenoma]. PMID- 3016372 TI - [Enzyme-immuno-histo in situ hybridization]. PMID- 3016374 TI - [Transsphenoidal microadenomectomy of pituitary tumors]. PMID- 3016375 TI - [Etiology of Cushing's syndrome]. PMID- 3016376 TI - Risk of lung cancer by histologic type among smokers in Miyagi Prefecture. AB - Smoking habits of the 827 lung cancer patients aged 40 years or over who were treated in the Sendai Kosei Hospital from 1977-82 were compared with those in the general population of a non-metropolitan district in northern Japan, which we recently surveyed by mail questionnaire. The relative risks of lung cancer by histologic type adjusted by age and area of residence were as follows: 1.9 for adenocarcinoma, 4.3 for squamous cell carcinoma, 3.9 for small cell carcinoma and 3.4 for large cell carcinoma in men, and 2.9, 6.4, 4.5 and 4.0 for these histologic types, respectively, in women. These elevated risks were all statistically significant (p less than 0.05). These findings suggest that smoking habits should be carefully considered when studying the etiology of any cell type of lung cancer, even adenocarcinoma, which often has been thought to be unrelated to smoking habits. PMID- 3016377 TI - A case of small cell lung cancer successfully treated with VM26. AB - A 79-year-old man was admitted to our hospital for a close examination of an abnormal shadow on a routine chest roentgenogram. A large tumorous shadow in the right S1 segment, and hilar and mediastinal node swelling were observed. Transbronchial biopsy revealed that the histologic type of the tumor was oat cell carcinoma. The clinical stage was considered to be limited disease. Because of his age, 79 years, he was treated with VM26 as a single agent. VM26 was administered by intravenous drip infusion of 60 mg/m2 daily for five consecutive days every three to four weeks. After the third course of treatment with VM26, he achieved partial response, and then radiation therapy to the primary site and the hilar and mediastinal nodes was given at a total dose of 6,000 rad. There have been few phase II studies of VM26 in patients with small cell lung cancer (SCLC). Further phase II studies should be conducted for confirmation of the reproducibility of the phase II study of VM26 in SCLC, and the efficacy of combination chemotherapy including VM26 against SCLC should also be tested. PMID- 3016378 TI - [Role of the scintigram in the diagnosis of liver and biliary tract diseases]. PMID- 3016373 TI - [Drug and radiotherapy of pituitary tumors]. PMID- 3016379 TI - [MRI of islet cell tumors of the pancreas: preliminary results with a superconductive magnet unit]. PMID- 3016380 TI - [Fibroblast collagenase activity in some types of inherited epidermolysis bullosa]. PMID- 3016381 TI - [Clinical studies on hypoglycemia in hepatocellular carcinoma]. PMID- 3016383 TI - Familial neurogenic acro-osteolysis, type Giaccai--report of two families. PMID- 3016382 TI - [Lymphokine activated-killer cell (LAK) activity in patients with hepatocellular carcinoma and its modification by a biological response modifier]. PMID- 3016384 TI - [A gas chromatographic measurement of total body water in patients on maintenance hemodialysis using deuterium oxide]. PMID- 3016385 TI - [Determination of the right ventricular ejection fraction using the first transit clearance technic]. PMID- 3016386 TI - [Fundamental and clinical evaluations of the neuron-specific enolase radioimmunoassay kit "SD-8570"]. PMID- 3016387 TI - [An investigation of factors that induce low efficiency of 99mTc-RBC in vivo labeling--with special reference to the effect of the reaction between 99mTcO4- and SnCl2 remaining in the three-way cock]. PMID- 3016388 TI - Maternal phase control of fetal circadian oscillation during pregnancy. AB - Mother-pups interaction is essential for the normal development of individuals during ontogeny. In this review, the emphasis was on a special function in a special stage of development, i.e., fetal entrainment to pregnant mother of the circadian rhythm which is one of the fundamental autonomic functions in the organism. To this end, the basic assumptions and problems were introduced and the validity of the assumption was defended. Based on this, the various factors of postnatal life which might affect the phase angle of overt hormone rhythm were experimentally evaluated. Finally, by introducing experimental designs which were to exclude disturbing effects of the nursing mother, we found that the fetal oscillation was possibly entrainable by pregnant mother as early as in the middle of gestation. Possible mechanism of the entrainment and adaptive implications of the findings were discussed. PMID- 3016389 TI - The effects of diethyldithiocarbamate on the hepatotoxic action and antitumor activity of N-methylformamide in mice. AB - The oral administration of diethyldithiocarbamate (DTC) prevented hepatic necrosis induced by N-methylformamide (NMF) in ddY-strain mice, in more susceptible BALB/c mice and in diethylmaleate-treated mice in which NMP hepatotoxicity was potentiated, as evidenced by suppression of increases of plasma glutamic pyruvic transaminase activity and liver calcium content or by histological observations. Early depletion of liver glutathione following NMF administration was also prevented by DTC. DTC markedly delayed the in vivo metabolism of NMF as indicated by a prolonged retention of plasma and liver NMF levels and an enhancement of urinary excretion of NMF. These observations support a bioactivation mechanism for NMF hepatotoxicity, and the hepatoprotective action of DTC may be due to an inhibition of the metabolic activation of NMF. Hepatotoxic manifestations after repeated administration of NMF also tended to be ameliorated by simultaneous treatment with DTC. Cotreatment with DTC, however, decreased the antitumor activity of NMF against Ehrlich ascites tumors, and Sarcoma 180. This also implies the involvement of a bioactivation mechanism in the antitumor action of NMF, but further studies are necessary to confirm this point. The possible therapeutic value of DTC as a hepatoprotector may be diminished by the suppression of the antitumor activity of NMF. PMID- 3016390 TI - Effects of 8-(2-dimethylaminoethyl)-3-oxo-4-phenyl-1-thia-4,8-diazaspiro [4,5] decane dihydrochloride monohydrate (Y-8845) on carbon tetrachloride-induced liver injury. AB - The effects of 8-(2-dimethylaminoethyl)-3-oxo-4-phenyl-1-thia-4,8-diazaspiro [4, 5] decane dihydrochloride monohydrate (Y-8845) on carbon tetrachloride (CCl4) induced liver injury were investigated in rats. CCl4-induced attenuation of the plasma cyclic AMP (cAMP) response to glucagon stimulation was significantly prevented by pretreatment with Y-8845. Y-8845 also effectively suppressed the increases in the activities of serum transaminases as well as the decreases in microsomal glucose-6-phosphatase activity and microsomal cytochrome P-450 concentrations induced by CCl4. In rats at 72 hr after CCl4 administration, the plasma cAMP response to glucagon, microsomal glucose-6-phosphatase activity and P 450 concentration were all below the control level. Y-8845 treatment after CCl4 administration rectified these reductions to nearly normal levels. Furthermore, Y 8845 stimulated DNA synthesis during liver regeneration after CCl4 intoxication. These results demonstrate that Y-8845 has a protective effect against CCl4 induced injury in the liver and a stimulating effect on the recovery of the damaged liver. PMID- 3016391 TI - Effects of deoxyribonuclease I and neuraminidase treatments on the specific binding of 3H-prazosin and 3H-quinuclidinyl benzilate (3H-QNB) to alpha adrenergic and muscarinic receptors in rat myocardial membranes. AB - The effects of neuraminidase and deoxyribonuclease I (DNase) treatments on the specific binding of 3H-prazosin and 3H-quinuclidinyl benzilate (3H-QNB) to alpha adrenoceptors and muscarinic cholinergic receptors in the membrane of the rat myocardium was examined, and the dissociation constant (Kd) and the maximum number of binding sites (Bmax) was analyzed using the method of Scatchard analysis. Although no changes in Kd values were observed when DNA or sialic acid was removed from cardiac muscles by treatments with DNase or neuraminidase, the Bmax values of alpha-adrenergic and muscarinic receptors were markedly decreased after treatment with DNase, while neuraminidase treatment induced an increase in the Bmax values of the alpha-adrenoceptors. The possibility that these results provide the basis for elucidation of the characteristics of these receptors in the rat myocardium was discussed. PMID- 3016392 TI - Surgical clinicopathologic study of malignant fibrous histiocytoma. AB - Fifteen men and six women, ranging in age from 20 to 79 years, with malignant fibrous histiocytoma comprised this series. Amputation of the right lower extremity was performed in one patient, wide resection in 7 and marginal excision in 9, respectively. In the other 4 patients, a non-curative resection was carried out. Adjuvant chemotherapy was prescribed post-operatively for 12 patients. Histologic grade of the surgical specimens was Stage I in 3, II in 6, III in 8 and IV in 4. Among fourteen patients living from 10 months to 9 years and 9 months after the operation, the 7 subjected to a wide resection are all disease free. Seven patients died of a local recurrence or a distant metastases. The survival rate of the patients with Stages I, II is significantly higher than those with Stages III, IV. These results show that a wide resection is to be favored and that staging is useful to estimate the prognosis. PMID- 3016393 TI - [The effect of ketotifen on human lymphocyte beta-adrenergic receptors]. PMID- 3016394 TI - [2 cases of bilateral chylothorax associated with malignant tumor]. PMID- 3016396 TI - Interleukin 1-like thymocyte and fibroblast activating factors from rat alveolar macrophages exposed to silica and asbestos particles. PMID- 3016395 TI - [A case of pulmonary asbestosis induced by actinolite with bilateral pleural effusion]. PMID- 3016397 TI - Transplantable primate tumors induced by Rous sarcoma virus. I. Induction of tumors transplantable into young marmosets. AB - The tumors induced in white-lipped marmosets (Saguinus fuscicollis, S. nigricollis) by Rous sarcoma virus (RSV) of chicken origin (RSV-SR) were not transplantable to allogeneic hosts. In contrast, RSV rescued from these tumors (RSV-M) induced sarcomas that were transplantable to young but not to adult marmosets. The tumors induced by RSV-M and the transplants rapidly enlarged, metastasized to various organs, and killed the recipients 29-59 days post inoculation. Cell lines were readily established from all transplantable sarcomas. No virus expression was detected in transplantable tumor cell lines by electron microscopy or by biochemical and biological assays. However, RSV of the same subgroup as RSV-SR was rescued from both short-term and long-term tumor cell cultures by cocultivation with chicken embryo fibroblasts (CEF). The rescued viruses transformed marmoset cells 100-fold more efficiently than CEF cells, although CEF cells remained permissive for virus replication. Cytogenetic studies revealed extensive chromosome abnormalities in tumor transplants but not in RSV-M induced sarcomas. All cell lines were hyperploid and contained structurally abnormal, large metacentric and telocentric chromosomes. Immunologic studies failed to detect group-specific (gs) antigen of the avian sarcoma-leukemia complex in either RSV-M-induced, transformed cells or tumor transplants. By complement-dependent cytotoxicity assays, with the use of marmoset anti-gs serum, RSV-associated antigen could be detected on the surfaces of tumor cells. No differences in the expression of this antigen existed between transplantable and nontransplantable marmoset sarcomas. All transplantable cell lines contained abnormal amounts of lipids and glycogen in comparison to RSV-SR-induced tumors and normal marmoset cell lines. The glycogen was associated with unique cytoplasmic membrane complexes and was surrounded by either single- or double membraned vesicles. PMID- 3016398 TI - [Comparative study of glycosphingolipid levels, activity of enzymes participating in their degradation and various indicators of aggregative properties of the blood in myocardial infarction]. PMID- 3016399 TI - [Effect of complamin on the levels of ACTH, cortisol and lipids in the blood of elderly patients with ischemic heart disease]. AB - Blood ACTH, cortisol and lipid levels, as well as complamin effects thereon, were studied in 116 elderly and old coronary patients. Changes of lipid metabolism were not identical in coronary patients of different age. They were particularly unfavorable in old age. ACTH and cortisol levels and the ACTH/cortisol ratio change with age, and the highest hypercortisolemia is found in old age. Complamin regulates lipid metabolism and blood ACTH and cortisol in coronary patients of advanced age. The ACTH/cortisol ratio is however unaffected by treatment and remains stable in all age groups. PMID- 3016403 TI - [Effectiveness of chemotherapy of lung cancer after exploratory thoracotomy]. PMID- 3016401 TI - [Herpes simplex virus encephalitis in childhood]. PMID- 3016400 TI - [Participation of the endogenous opioid system in the formation of the body's response to stress]. AB - Autonomic responses to acute stressors in unanesthetized, chair-restrained baboons and macacas include elevations in heart rate, blood pressure and respiratory rate. Naloxone, in a dose of 1.0 mg/kg intravenously, as well as morphine (1.0 mg/kg) suppresses autonomic alterations produced by the introduction of conditioned and unconditioned stimuli. When given in a dose of 0.1 mg/kg, naloxone, on the contrary, facilitates autonomic responses. The autonomic changes induced by electrical stimulation of the medial hypothalamus and nucleus tractus solitarius are influenced by naloxone--in both doses--and by morphine in the opposite manner. These data, as well as those derived from experiments on the effects of micro-injections of naloxone and morphine into the medial hypothalamus and nucleus tractus solitarius suggest that the endogenous opioid system is necessary to display complete autonomic response pattern in monkeys. A hypothesis is proposed to the effect that endogenous opioid system is important for establishing a precise correspondence between the body's potentialities and behavior, and environmental demands. PMID- 3016402 TI - Studies on coordination stereo-selectivity in nucleotide base-metal complexes with biochemical activity. PMID- 3016404 TI - [Use of the myorelaxant Arduan during general anesthesia in children with congenital hip dislocation]. PMID- 3016406 TI - [Protective role of endogenous morphine-like substances in mice with acute hypoxia]. PMID- 3016405 TI - Effects of the new angiotensin-converting enzyme inhibitor, ramipril, in patients with essential hypertension. AB - The antihypertensive and hormonal effects of the new angiotensin-converting enzyme (ACE) inhibitor, ramipril, were assessed by means of a single-blind trial in ten unselected patients with mild-to-moderate essential hypertension. After a 2-week period on placebo, 5 mg ramipril was administered once daily for 2 weeks. Blood pressure returned to normal in five patients and decreased in the remaining patients, without significant changes in heart rate or orthostatic hypotension. A fall in blood pressure was apparent within 1-2 h of the first dose; the maximum decrease was reached at 4-6 h and a fall in pressure was still detectable after 24 h. At 24 h post dose angiotensin-converting enzyme activity was suppressed to 40% of the baseline. Blood pressures for the 10 h interval post dosing showed smooth through-the-day control with minimal peak/trough difference in lowering effect. The magnitude of the blood pressure decrement achieved with the inhibitor did not correlate with baseline renin levels or the rise in renin following treatment. No side-effects were noted during the 2-week observation period. The study demonstrates that ramipril, given in a once-daily regimen over a period of 2 weeks, is well tolerated and provides smooth and effective blood pressure control throughout the 24-h interval between doses. PMID- 3016407 TI - Silica-induced hypertrophy of type II cells in the lungs of rats. AB - Several investigators have reported the appearance of hypertrophic type II cells in the lungs of silica-treated rats. The purpose of this study was to isolate and characterize these hypertrophic type II cells. Lungs were digested with trypsin and the released cells were separated by using a flow gradient during centrifugal elutriation. Type II cells from control lungs were distributed in the flow gradient as a single population, whereas type II cells from the lungs of silica treated rats had a bimodal distribution suggesting the presence of two distinct populations of type II cells; one of these populations appeared hypertrophic (type IIB) and the other appeared normal (type II cells; one of these populations appeared hypertrophic (type IIB) and the other appeared normal (type IIA). These two populations of type II cells from silica-treated rats differed significantly in cell size and their lamellar body content. The mean volume of type IIA and type IIB cells was 350 +/- 38 micron 3 and 523 +/- 29 micron 3, respectively. The mean number of lamellar bodies in type IIA and type IIB cells was 77 +/- 53 and 131 +/- 84 per cell, respectively. The mean volume of lamellar bodies was 0.39 +/ 0.09 micron 3 in type IIA cells and 0.66 +/- 0.10 micron 3 in type IIB cells. Type IIA cells were not significantly different from type II cells from the lungs of untreated rats. The distribution of type II cells from silica-treated lungs was such that 2 weeks after a single intratracheal injection of silica (10 mg/rat) type IIB cells accounted for 39.2 +/- 6.4% of the total type II cells recovered after centrifugal elutriation. The general morphological appearance of the isolated type IIA and type IIB cells was similar to that observed in type II cells isolated from untreated rats. These data indicate that hypertrophic type II cells may be isolated from the lungs of silica-treated rats and separated from normal type II cells thus allowing the role of these unusual type II cells in lung injury and repair to be investigated. PMID- 3016408 TI - Conservation of the DNA binding domain and other properties between porcine and rat glucocorticoid receptors. AB - Activated glucocorticoid receptor protein (GCR) was partially purified from porcine liver cytosol by sequential chromatography on phosphocellulose and DNA cellulose using a modification of a protocol developed for purification of rat GCR. This partially purified preparation, when separated by SDS-polyacrylamide gel electrophoresis and immunoblotted, indicated that a Mr = 94,000 protein band cross-reacts with a monoclonal antibody against rat GCR. A nitrocellulose filter binding assay showed that both the partially purified porcine and rat GCRs interact specifically with a cloned synthetic 24 base pair deoxyoligonucleotide containing the GCR binding sequence in the first intron of the human growth hormone (hGH) gene. This specific protein-DNA interaction is blocked by a single base pair change in the binding site. All three putative domains of the GCR molecule: the steroid binding, immunoreactive, and DNA binding have been conserved between two divergent species. PMID- 3016409 TI - Hyperprolactinemia enhances LH-stimulated 4-ene-5 alpha-reductase activity but inhibits LH-induced 17-hydroxylase activity in testes of hypophysectomized immature rats. AB - Two pituitaries from 7-week old female rats (Sprague-Dawley strain) were grafted under the capsule of the left kidney of 21-day old male rat. The pituitary grafted and sham-operated rats were hypophysectomized at 27 days of age. The hypophysectomized rats, in groups of 4, were given daily injections of 9 micrograms NIAMDD-oLH-23 (minimum effective dose) or saline for 3 days starting from day 29. Testicular homogenates were incubated with [3H]progesterone or [14C]4-androstene-3,17-dione, and enzyme activities per testes were estimated. Testicular HCG-binding sites were also measured. Hypophysectomy caused significant decreases in activities of testicular 5 alpha-reductase, 17 hydroxylase, and 17 beta-hydroxysteroid oxidoreductase. These decreased enzyme activities were significantly stimulated by LH treatment. Although pituitary grafts alone showed no effects on these enzyme activities in the testes of the hypophysectomized rats, the grafts significantly enhanced LH-stimulated 5 alpha reductase activities but inhibited LH-stimulated 17-hydroxylase activity. Testicular LH/HCG receptors were significantly increased by the grafts, especially in the presence of LH, without affecting affinity for HCG. The present results demonstrate for the first time that hyperprolactinemia directly stimulates LH-stimulated 5 alpha-reductase activity in rat testes. The results also show that the same grafts directly inhibit LH-stimulated 17-hydroxylase activity, probably via postreceptor mechanisms. PMID- 3016410 TI - The dual effect of calcium on aromatization by cultured human trophoblast. AB - To study the effect of calcium ion on aromatization of an androgenic precursor to estradiol by the placenta, cultured term trophoblasts were used as a model system. Secretion of estradiol into the culture medium was regarded as indicating aromatization, since cells cultured with no androgenic precursors produced only insignificant amounts of estradiol. EGTA, verapamil and ionophore A23187 inhibited aromatization, while trifluoperazine, an inhibitor of the calcium calmodulin complex, interfered with the stimulatory effect of cyclic AMP on aromatization. We conclude that calcium ion has an essential role in the aromatization of 4-androstene-3,17-dione to estradiol. The calcium-calmodulin complex is required for activation of aromatase by cyclic AMP. However, when flooded with calcium by ionophore A23187, the trophoblast is unable to effectively buffer calcium, and aromatization is inhibited. PMID- 3016411 TI - Retroperitoneal malignant fibrous histiocytoma presenting with inferior vena caval obstruction. AB - A 54-year-old-man presented with inferior vena caval obstruction. At autopsy, a retroperitoneal malignant fibrous histiocytoma was noted to have invaded the inferior vena cava. Such a clinical presentation is quite unusual for a retroperitoneal sarcoma. PMID- 3016413 TI - Phylogenetic trees constructed from hydrophobicity values of protein sequences. AB - Information about conformational properties of a protein is contained in the hydrophobicity values of the amino acids in its primary sequence. We have investigated the possibility of extracting meaningful evolutionary information from the comparison of the hydrophobicity values of the corresponding amino acids in the sequences of homologous proteins. Distance matrices for six families of homologous proteins were made on the basis of the differences in hydrophobicity values of the amino acids. The phylogenetic trees constructed from such matrices were at least as good (as judged from their faithful reflection of evolutionary relationships), as trees constructed from the usual minimum mutation distance matrix. PMID- 3016412 TI - Hepatic artery ligation combined with radiotherapy for treatment of nonresectable primary liver carcinoma. AB - Clinical and experimental evidence shows that hepatic artery ligation (HAL) is an effective method for treatment of nonresectable primary liver carcinoma (PLC). Improvement of subjective symptoms in 50-70% patients has reported, although effective duration is short and 30-50% of the enlarged liver could be decreased. Usually, arterial collateral of the tumors after HAL may be evident again in 4-6 weeks. There is arteriographic evidence of the appearance of collateral flow as early as the fourth days after HAL. HAL combined with irradiation was carried out in 17 cases between October 1978 to October 1982 in order to improve the prognosis of nonresectable PLC. PMID- 3016414 TI - A simple way to look at DNA. AB - A method is presented for embedding nucleotide sequence data in a simple metric space. Computer graphical examination of spatially-represented sequences permits rapid searches for canonical patterns or interesting structures. Sequence comparisons are facilitated by plots of distance measures for homologous sequences, and the large-scale structure of the genetic code can be studied by measures such as fractal dimensionality. PMID- 3016415 TI - A comparative study of free radical scavengers in cardioplegic solutions. Improved protection with peroxidase. AB - Oxygen-derived free radicals play an important role in the myocardial injury associated with ischemia and reperfusion. This study was designed to assess whether the protection afforded by a K+ rich, Mg2+ rich cardioplegic solution could be enhanced by the addition of free radical scavengers acting at different levels of the radical generating pathway. Forty isolated isovolumic rat hearts were divided into five groups (n = 8). Four groups of hearts were subjected to 90 minutes of normothermic cardioplegic arrest followed by 45 minutes of reperfusion. Hearts were given an initial bolus of either unmodified cardioplegic solution or cardioplegic solution enriched with superoxide dismutase (200,000 U/L) reduced glutathione (0.1 mmol/L), or peroxidase (6,000 U/L). One group of hearts was aerobically perfused throughout the experimental protocol and served as nonischemic controls. Based on comparisons of postreperfusion ventricular pressure development, maximal ventricular dP/dt, left ventricular compliance and coronary flow, peroxidase-containing cardioplegic solution afforded the best myocardial protection, with values of these indicators not significantly different from those of nonischemic perfused control heart. Glutathione afforded protection slightly inferior to that of peroxidase but still markedly better than in groups receiving superoxide dismutase or unmodified cardioplegic solution. This study confirms that cardioplegic protection can be enhanced by the addition of free radical scavengers, in particular peroxidase. PMID- 3016416 TI - An approach to the effect on tumors of acupuncture in combination with radiotherapy or chemotherapy. PMID- 3016417 TI - A molecular and genetic approach to understanding the mechanisms by which fractionated X-irradiation induces leukemia in mice. AB - Our laboratory's approach to try to shed light on the question of a viral etiology for radiation-induced leukemia has focused on defining, localizing and understanding the mode of action of genes involved in susceptibility to FXI induced disease. These studies have indicated that multiple genes control the process of leukemogenesis. In addition not every mouse strain which shows some susceptibility to FXI-induced leukemia carries the susceptible gene at each of the multiple loci involved in the disease process. Thus, it is plausible to conclude that more than one mechanism of leukemogenesis can be triggered by FXI. Our studies have focused on the mode of action of one such locus Ril-1. Several reagents have been developed to help us clone and characterize this locus. Currently chromosomal "walking" and "hopping" techniques are being used in conjunction with an RFLP molecular probe which is adjacent to Ril-1. In addition a cDNA library has been prepared from a radiation-induced thymoma and subtraction hybridization analysis is being used in the search for Ril-1. PMID- 3016418 TI - Oncogene involvement in radiation- and virus-induced mouse osteosarcomas. AB - Internal irradiation of mice using bone seeking radionuclides results in the activation of endogenous retroviruses and in the subsequent development of bone tumors. Genomic DNA from an osteosarcoma cell line, derived from an 90Sr-induced bone tumor, was cotransfected with the plasmid pSV2-neo into NIH/3T3 cells and G418-resistant transfectants gave rise to colonies in soft agar. Southern blot analysis of these first cycle transformants revealed the presence of extra copies of c-ras. We have analysed the arrangement of ecotropic murine leukemia proviral sequences in seven 90Sr-induced bone tumors and one osteosarcoma cell line of CF1 mice. Integration of ecotropic and/or ecotropic recombinant proviruses seems to be involved in rearrangements of 3' provirus cellular junction fragments occurring in all tumor DNAs analysed, but no indication for site-specific integration was found. We also determined the primary structure of FBR-MuSV, a transforming retrovirus able to induce bone tumors in newborn mice. FBR-MuSV contains sequences from all four exons of the murine c-fos gene, but lacks sequences encoding the first 24 and the last 98 amino acids of the c-fos gene product. The coding region of FBR-MuSV has also undergone two small in frame deletions. Thus, the v-fosFBR-MuSV retains 236 amino acids of the 380 amino acids of the murine c-fos product. In FBR-MuSV-transformed cells two fos-containing mRNAs have been detected: a 3.3-kb full-size genomic RNA and a 2.2-kb subgenomic mRNA as revealed by both fos- and MuLV-hybridization probes. PMID- 3016419 TI - A perfused rabbit retina preparation suitable for pharmacological studies. AB - We present a method for maintaining the isolated retina-eyecup of the rabbit in a manner which permits the introduction of pharmacological agents at a controlled concentration. As judged by physiological criteria, the retina is well maintained under these conditions. The stability of the preparation is excellent; intracellular or extracellular recordings from single cells can be maintained through multiple solution changes. By using Co2+ to block synaptic transmission, we can distinguish direct from transsynaptic effects. Use of this preparation should facilitate the investigation of neurotransmission in the mammalian retina. PMID- 3016420 TI - [Prevalence of the use of cannabis in a young population]. PMID- 3016421 TI - [Acute adrenal insufficiency in a case of isolated deficit of the adeno corticotropic hormone (ACTH)]. PMID- 3016422 TI - [Acquired immune deficiency syndrome and related conditions]. PMID- 3016423 TI - [Pancoast syndrome as the initial manifestation of a hepatocarcinoma]. PMID- 3016424 TI - [Inflammatory fibrous histiocytoma: presentation of a case with onset as a fever of unknown origin and review of the literature]. PMID- 3016425 TI - [Acute hydrocyanic acid poisoning: an infrequent occupational accident]. PMID- 3016426 TI - An overview of Wellferon (interferon alfa-n1): the product. AB - Since 1959, The Wellcome Foundation Ltd. has been involved in research to develop interferon for practical use. A brief historical perspective is presented on the development and production of interferon alfa-n1. PMID- 3016427 TI - Neurologic emergencies. AB - This article considers the rapid assessment and initial management of several neurologic emergencies--altered consciousness, increased intracranial pressure, stroke, status epilepticus, acute neurogenic respiratory failure, acute autonomic instability, the neuroleptic malignant syndrome, and spinal cord compression. PMID- 3016429 TI - [Etiopathogenesis and sequelae of viral neuroinfections in children. V. Diagnosis of neuroinfection caused by herpes simplex virus]. PMID- 3016428 TI - [Etiopathogenesis and sequelae of viral neuroinfections in children. IV. Etiology and clinical aspects of neuroinfections with a severe course]. PMID- 3016431 TI - Effects of lisinopril, a new angiotensin converting enzyme inhibitor in a cryo injury model of chronic left ventricular failure. AB - The angiotensin converting enzyme inhibitor, lisinopril, was studied in a cryo injury model of chronic heart failure. This model is characterized by a progressive decrease in cardiac output starting six weeks after cryo-injury to a 30% decrease in cardiac output 10 weeks after injury. Although histological damage to the myocardial tissue, particularly in the epicardial area, was observed, no significant changes occurred in body weight, mean arterial blood pressure, heart rate or central venous pressure. Daily intraperitoneal injection of 3 mg/kg lisinopril was instituted starting four weeks after injury for a duration of six weeks. Lisinopril completely restored the cardiac output to normal values at the end of this six week period. Thus, converting enzyme inhibition appears to be an effective therapeutic agent in this model of chronic cardiac failure. PMID- 3016430 TI - The possible role of brain histamine in neuroendocrine and cardiovascular regulation. PMID- 3016432 TI - Elemental and trace element distribution in medical samples: analysis by proton induced X-ray emission. AB - The analysis of trace elements is performed by proton-induced X-ray emission. The process is most effective if the velocity of the exciting particles--protons--is similar to the velocity of the electron on its orbit in the simple atomic model of Bohr. For K-shell electrons of the elements with 15 less than or equal to z less than or equal to 40 this requires proton energies of a few MeV, available from electrostatic van de Graaf accelerator machines. After knocking out the K shell electron, the empty place is filled up by electrons jumping from higher orbits with simultaneous emission of characteristic X-rays, which are registered with a cooled Si (Li) detector. By a set of electrodes the beam can be swept across the specimen surface. Therefore this method yields an excellent correlation of trace element distribution within the morphological structure of organic tissue. In the present study the sweep went along a line perpendicular to the arterial wall layers (aortic, renal artery and heart muscle) of normotensive and spontaneously hypertensive rats. Along this line all elements and trace elements are recorded simultaneously. These are P, S, Cl, K, Ca, Fe, Cu, Zn, Br and Sr. The trace element content of the aortic wall and the renal artery, of 22 spontaneously hypertensive and 11 normotensive rats and of human heart muscle was investigated. The results demonstrate that Zn was only detected in the muscle containing layers of the arteries. There was no different distribution between hypertensive and normotensive rats. However, Ca2+ was mainly detected in the smooth muscle-containing tunica media of hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016433 TI - [Monoclonal antibody UW 21/123: clinical use and diagnostic specificity in head and neck cancers]. AB - In addition to the originally described monoclonal antibody (MAB) against laryngeal carcinoma cells (Arch. Otorhinolaryngol. 233 (1981) 161) a new MAB was induced by means of somatic cell hybridisation techniques. The MAB UW 21/123 enables recognition of a group of squamous cell carcinomas of the head and neck. An in-vitro assay was designed allowing clinical diagnosis of malignancy in 18% out of 211 tumour patients. PMID- 3016434 TI - The relationship of the herpesvirus family to sudden hearing loss: a prospective clinical study and literature review. AB - The herpesvirus family is associated with sudden hearing loss syndrome and the evidence includes clinical findings (HV-Z), temporal bone studies (CMV and HV-Z), and serologic studies. The data presented demonstrate that herpes infections, in association with sudden viral hearing loss, occur as part of a multiple viral infection in 70% of instances. This feature is unique to the herpesvirus infections when compared to other neurotropic viral agents. The study also demonstrates that the variables of viral hearing loss, such as degree of hearing loss, percentage of recovery, or the incidence of vertigo are unaffected by the presence of herpesvirus infection. Mechanisms for inner ear injury may be influenced by temporary alterations in cellular immunity secondary to simultaneous viral infections as well as the native virulence of the infecting herpesvirus. PMID- 3016436 TI - Regulation of phosphoinositide breakdown by guanine nucleotides. AB - Phosphoinositide hydrolysis is coupled to receptor systems involved in the elevation of cytosolic Ca2+ and activation of protein kinase C. In cell-free systems, guanine nucleotides are required to transduce the effects of receptor activation to phosphoinositide breakdown. Non-hydrolyzable guanine nucleotides stimulate phosphoinositide breakdown in permeabilized cells as well as membranes prepared from salivary glands, GH3 cells, neutrophils, hepatocytes and cerebral cortical tissue. In blowfly salivary gland membranes, 5-hydroxytryptamine stimulates a guanine-nucleotide dependent breakdown of both endogenous and exogenous phosphoinositide substrate through activation of phospholipase C. These data suggest that a GTP-binding protein modulates phospholipase C activity. The identity of this GTP-binding protein has not been established but may resemble other regulatory GTP-binding proteins which have been identified as transducing proteins in a variety of receptor systems. PMID- 3016435 TI - [An epidemic of hepatitis A in Pula in 1984 caused by the consumption of mussels]. PMID- 3016437 TI - Rapid down-regulation of S2 serotonin receptors by antidepressants: noradrenergic serotonergic interactions. AB - Mianserin stereoselectively decreases rat cortical S2 binding, the (+)enantiomer having higher potency. This and other data suggest that an alpha-2 receptor is unlikely to contribute to the mechanism of rapid S2 down-regulation by mianserin. Yohimbine, an alpha-2 antagonist which enhances desipramine-induced S2 decreases, was not dependent on NE or 5-HT release for its effect. Depletion of NE by 75% or 5-HT by 94% did not alter the ability of yohimbine and desipramine to decrease binding. These results raise previously unsuspected mechanisms involved in acute down-regulation of S2 binding by mianserin and yohimbine. PMID- 3016438 TI - Effects of prostaglandin E1, isoproterenol and forskolin on cyclic AMP levels and tension in rabbit aortic rings. AB - The role of cyclic AMP in the control of vascular smooth muscle tone was studied by monitoring the effects of prostaglandin E1 (PGE1), isoproterenol and forskolin on cyclic AMP levels and tension in rabbit aortic rings. PGE1, isoproterenol and forskolin all increased cyclic AMP levels in rabbit aortic rings. Isoproterenol and forskolin relaxed phenylephrine-contracted aortic rings, but PGE1 contracted the rings in the presence or absence of phenylephrine. Isoproterenol relaxed these PGE1-contracted aortic rings without further change in total cyclic AMP levels, which were already elevated by the PGE1 alone. Pretreatment with forskolin potentiated the effects of PGE1 on cyclic AMP levels. PGE1 caused contractions in muscles partially relaxed by forskolin, even though very large increases in cyclic AMP levels (30 fold) were produced by PGE1 in the presence of forskolin. Isoproterenol was able to relax these forskolin-treated, PGE1 contracted muscles with no further increase in cyclic AMP levels. Thus, there does not appear to be a good correlation between total tissue levels of cyclic AMP and tension in these experiments. Our results suggest that, if cyclic AMP is responsible for relaxation of smooth muscle, some form of functional compartmentalization of cyclic AMP must exist in this tissue. PMID- 3016439 TI - Influence of yohimbine on release of anterior pituitary hormones. AB - We used a double-blind crossover design to study the effects of alpha 2 adrenoreceptor blockade with yohimbine on levels of anterior pituitary hormones. A dose of yohimbine was used which raised plasma norepinephrine from 379 +/- 74 (S.E.) to 730 +/- 143 pg/ml and mean arterial pressure from 83 +/- 4 to 92 +/- 5 torr (p less than 0.025). This dose (125 micrograms/kg, then 1 microgram/kg/min) also altered mood when compared to saline infusion. In spite of these changes, when prolactin, cortisol, ACTH, beta-endorphin, TSH and growth hormone were measured after 45 minutes of yohimbine infusion, no changes from baseline were noted. These data suggest that in normal man, at rest, alpha 2 adrenoreceptors in the hypothalamus, adenohypophysis or other brain areas do not tonically modulate release of these hormones into the blood. PMID- 3016440 TI - The prolonged action of the LHRH agonist buserelin (HOE 766) may be due to prolonged binding to the LHRH receptor. AB - Hemi-pituitary glands of ovariectomized rats were superfused for 4 h with either LHRH or the analog buserelin (HOE 766) at several concentrations, and thereafter with medium only for another 1.5 h. In a further experiment glands were exposed for 2.5 h to LHRH or buserelin at a single concentration (5 ng/ml) and subsequently for another 2.5 h to either the same agonist (LHRH or buserelin) alone (5 ng/ml), the agonist plus an LHRH-antagonist (ORG 30093, 1000 ng/ml), the LHRH- antagonist alone, or medium alone. LHRH and buserelin stimulated gonadotropin release equally well. After cessation of this stimulation, the gonadotropin release by the buserelin-treated pituitary glands and the glands, treated with the highest dose of LHRH (1000 ng/ml), continued, while the release by the glands, treated with the lower doses of LHRH, declined. The LHRH antagonist completely blocked the release of LH, stimulated by buserelin or LHRH, as well as the prolonged activation of the release, caused by buserelin pre treatment. In a superfusion experiment with pituitary cell aggregates of 14-day old intact female rats, buserelin stimulated the release of LH much more effectively than LHRH itself. Moreover, the release caused by buserelin declined more slowly after cessation of the stimulation. Finally, in a pituitary cell monolayer culture the Kd's of LHRH, buserelin and the antagonist were determined as 4.7 X 10(-9) M, 2.4 X 10(-10) M and 4.6 X 10(-9) respectively. It was concluded that the estimates of the potencies of LHRH and buserelin depend on the choice of the test-system. It is suggested that the long duration of action of buserelin is at least partly due to prolonged binding to the LHRH-receptor. PMID- 3016441 TI - The colliculus superior modulates ACTH-induced excessive grooming. AB - In previous studies the involvement of nigrostriatal dopaminergic activity in ACTH(1-24)-induced grooming has been established. It was suggested that the dopaminergic modulation of ACTH(1-24)-induced excessive grooming is exerted through the striato-nigro-collicular pathway. To obtain further evidence it was investigated, whether local application of GABAergic agents into the colliculus superior modulates excessive grooming occurring after an intraventricular injection with ACTH(1-24). It appeared that intra-collicular picrotoxin (a GABAergic antagonist) suppressed ACTH-induced grooming, whereas muscimol (a GABAergic agonist) enhanced the grooming response. The picrotoxin-induced R(unning) F(it) B(ehavior), elicited from the colliculus superior was also seen after intraventricular administration of picrotoxin. A detailed comparison of this behavioral response seen after both routes of administration of picrotoxin suggests that intraventricularly injected picrotoxin may well induce the RFB via a direct effect on the colliculus superior. Lesions placed in the colliculus superior completely abolished picrotoxin-induced RFB, exploration and orientation behavior. Yet, these lesions did not reduce excessive grooming suggesting that although this region may be involved in the modulation of ACTH-induced grooming it is not the primary site of peptide action. PMID- 3016443 TI - Opioid receptors of human placental villi modulate acetylcholine release. AB - The human placental villous tissue contains components of the cholinergic system and opioid receptors of the kappa type. In vitro stimulation of the villous tissue releases acetylcholine in organ baths. A selective kappa agonist, ethylketocyclazocine, inhibits the release of acetylcholine. This inhibition is reversed by the antagonist Mr 2266. The antagonist alone stimulates the release of acetylcholine 18-fold over control. These results demonstrate an interaction between the placental opioid receptors and the cholinergic system in a non-neural tissue. The modulation of acetylcholine release by endogenous opioid peptides could be one of the in vivo functions of placental opioid receptors. PMID- 3016442 TI - The effect of methadone addiction on cyclic nucleotide levels in regions of rat brain. AB - Studies of tissue culture cells and tissue slices have implicated the nucleotides, cyclic AMP and cyclic GMP, in the mechanism of action of opiates. However, there are little in vivo data to corroborate this hypothesis. We addicted rats to the synthetic opiate, methadone, by providing the drug in their drinking water (dosage 2.1 mg./kg./day). The two cyclic nucleotides were measured in four brain areas which contain a high concentration of opiate receptors: amygdala, neostriatum, periventricular grey, and thalamus. Data were obtained after acute exposure of the drug (1 day), tolerance (35 days), withdrawal (35 days on drug then 1 day off drug), and readjustment (35 days on drug then 21 days off drug). Cyclic GMP levels were low (0.03 pmol./mg. tissue) in the four regions and did not differ significantly during the experiment. Cyclic AMP levels were higher (1-3 pmol./mg.) and fluctuated consistently in the four regions. After acute methadone treatment, there was a reduction in cyclic AMP, which continued at lower levels after tolerance. One day of withdrawal led to increased cAMP, which rose to near control levels. After readjustment, the levels were reduced. These data indicate an involvement of cyclic AMP in the addiction and withdrawal processes in the intact animal. PMID- 3016444 TI - Peripheral-type benzodiazepine-binding sites in platelets of schizophrenics with and without tardive dyskinesia. AB - The density of peripheral-type benzodiazepine (BZ)-binding sites was studied in platelets of 10 medicated chronic schizophrenics with tardive dyskinesia (TD), 10 medicated chronic schizophrenics without TD, 7 drug-free schizophrenics, and 10 normal controls. The age range of the study population was 36-60 years. Age and sex distribution were similar in all 4 groups. The unmedicated schizophrenics did not differ in their maximal binding capacity from the healthy controls. A significant decrease in the density of peripheral-type BZ-binding sites in platelets was observed in treated schizophrenics both with and without TD in comparison to controls and untreated schizophrenics. The reduction in [3H]PK 11195 binding was more pronounced in TD patients (31.3% of controls) than in patients without TD (21.1% of controls). However, this parameter failed to discriminate statistically between TD and non-TD medicated schizophrenics. PMID- 3016445 TI - Regional increase of cyclic GMP by atrial natriuretic factor in rat brain: markedly elevated response in spontaneously hypertensive rats. AB - Atrial natriuretic factor (ANF)-responsive areas in rat brain were examined by measuring ANF-stimulated cyclic GMP production in rat brain slice preparations. The medulla oblongata, thalamus, and pituitary gland responded most sensitively, the septum, hypothalamus, pons, midbrain and olfactory bulb responded moderately, but neocortex, cerebellum, striatum and hippocampus were unresponsive to ANF. The most responsive regions in spontaneously hypertensive rats brains showed 2 to 5 times higher cyclic GMP production than those from the control Wistar-Kyoto rats. These findings provide evidence for biological action of ANF on brain tissues, and indicate the action of ANF produced in the brain. PMID- 3016446 TI - Effects of kappa opiate agonists on neurochemical and neuroendocrine indices: evidence for kappa receptor subtypes. AB - Four kappa opiate agonists, U-50488H, MR-2034, EKC and tifluadom, elevated plasma corticosterone and decreased plasma TSH in a dose-dependent manner. These effects were naloxone-reversible. However, WIN 44441-3, a long acting narcotic antagonist, was unable to reverse the effects of U-50488H and MR-2034 upto doses of 5 mg/kg. U-50488H and MR-2034 but not tifluadom or EKC, also increased levels of DOPAC and HVA in the olfactory tubercle. This effect was also naloxone reversible but not WIN 44441-3 reversible. Tifluadom and EKC did not increase DOPAC and HVA. The differential responses of the tested kappa agonists to WIN 44441-3 antagonism and dopamine metabolism in A10 neurons suggest that the kappa agonists can be separated into two groups. This is the first physiological evidence suggestive of kappa opioid receptor subtypes. PMID- 3016447 TI - Hormonal interactions with benzodiazepine binding sites in vitro. AB - Prostaglandin A1 and hormones like corticosteroids and DL-Thyroxin (T4) inhibit binding of [3H]RO 5-4864 and [3H] Clonazepam to their respective binding sites with inhibition constants in the low micromolar range. The corticosteroid Cortisone inhibits [3H] RO 5-4864, but not [3H] Clonazepam binding in a competitive manner with an inhibition constant of 4.3 +/- 0.7 microM, Prostaglandin A1 inhibits [3H] Clonazepam, but not [3H] RO 5-4864 binding in a competitive manner with an inhibition constant of 6 +/- 1.2 microM and DL Thyroxin (T4) inhibits both [3H] RO 5-4864 and [3H] Clonazepam binding with inhibition constants of 12.1 +/- 2.2 and 1.6 +/- 0.4 microM respectively. While the inhibition of [3H] RO 5-4864 binding by DL-Thyroxin (T4) is competitive, the inhibition of [3H] Clonazepam binding is of the mixed type as indicated by Scatchard Plot. PMID- 3016448 TI - Effects of chronic administration of doxorubicin on myocardial beta-adrenergic receptors. AB - The effects of multiple doses of doxorubicin (DXR) on myocardial beta-adrenergic receptor density and dissociation constant were investigated in male Sprague Dawley rats. The rats received DXR (2 mg/kg) or vehicle weekly by the SC route for 13 weeks. One group of DXR-treated rats plus corresponding controls were sacrificed at 14 weeks, one week after the last dose. Another group of DXR treated rats plus corresponding controls were sacrificed at 19 weeks, six weeks after the last dose. The myocardial beta-adrenergic receptor was characterized by radio-ligand binding studies using [125I]iodocyanopindolol. Beta--receptor densities in DXR-treated rats of 7.0 and 7.4 fm/mg protein were unchanged from control levels of 7.2 fm/mg protein at both 14 and 19 weeks, respectively. Receptor dissociation constants in DXR-treated rats of 36.7 and 36.9 pM were increased over control levels of 24.6 and 30.0 pM at 14 and 19 weeks, respectively. However, the change in dissociation constant is only significant at 14 weeks. The increased dissociation constants suggest diminished agonist binding affinity of the myocardial beta-receptor. This impaired response of the receptor to catecholamines would tend to diminish the ability of myocardium to adequately respond to adrenergic stimuli. PMID- 3016450 TI - Ionophore A23187 stimulates phosphorylation of the 40,000 dalton protein in human platelets without phospholipase C activation. AB - The Ca2+ ionophore A23187 (0.2-5 microM) stimulates the phosphorylation of the substrates of protein kinase C (40,000 dalton protein) and myosin light chain kinase (20,000 dalton protein) in the presence or absence of cyclooxygenase inhibitors. In the presence of cyclooxygenase inhibitors or millimolar Ca2+ there is no stimulation of phospholipase C by A23187. Fingerprints of the 32P-labeled 40,000 dalton protein isolated from platelets that have been stimulated with A23187, thrombin, phorbol 12,13-dibutyrate and 1,2-didecanoylglycerol were identical. Higher concentrations of A23187 (1-5 microM) induced the loss of polyphosphoinositides through phosphomonoesterase activity. PMID- 3016449 TI - Analogs of caffeine: antagonists with selectivity for A2 adenosine receptors. AB - Several analogs of caffeine have been investigated as antagonists at A2 adenosine receptors stimulatory to adenylate cyclase in membranes from rat pheochromocytoma PC12 cells and human platelets and at A1 adenosine receptors inhibitory to adenylate cyclase from rat fat cells. Among these analogs, 1-propargyl-3,7 dimethylxanthine was about 4- to 7-fold and 7-propyl-1,3-dimethylxanthine about 3 to 4-fold more potent than caffeine at A2 receptors of PC12 cells and platelets. At A1 receptors of fat cells, both compounds were about 2-fold less potent than caffeine. These caffeine analogs have an A1/A2 selectivity ratio of about 10-20 and are the first selective A2 receptor antagonists yet reported. The results may provide the basis for the further development of highly potent and highly selective A2 adenosine receptor antagonists. PMID- 3016451 TI - Eaton-Lambert syndrome and small cell lung cancer: side effects and certainty. PMID- 3016452 TI - [Computerized emission tomography in the diagnosis of brain tumors]. AB - Computerized emission tomography of the brain is an atraumatic and highly informative technique for diagnosis of metastatic brain tumours. The above method is of major practical significance as a primary test in diagnosing metastases in the brain in cases of clinical neurological symptom complex. PMID- 3016454 TI - Polymorphisms in the apolipoprotein AI-CIII gene complex. AB - We report the presence of five restriction fragment length polymorphisms in the apolipoprotein AI-CIII (apo AI-CIII) gene complex as well as their respective allelic frequencies in a German population (pi) that we studied: an SstI (BanII) polymorphism in the 3' non-coding region of apo CIII q1(pi) = 0.13); an MspI polymorphism in the third intron of apo AI (q2(pi) = 0.12); a PvuII polymorphism in the first intron of apo CIII (q3(pi) = 0.30); and two XmnI polymorphisms in the 5' side of apo AI (q4(pi) = 0.20 and q5(pi) = 0.05). PMID- 3016453 TI - [Radiation doses to patients after the use of 99mTc-pyrophosphate and 113mIn indifor]. AB - The doses of irradiation of patients during the use of 99mTc-pyrphotech and 113mIn-indifor have been defined on the basis of their pharmacokinetics. The data has been obtained with the use of radiometer of human radiation, the method of organs screening during radiometry, and of the gamma-camera and blood samples radiometry. The calculations have been performed in accordance with the known method based on the heterogeneous model of the "standard" human. In the dose calculations for bone tissue and for the red marrow the dosimetry model is used and the values of absorbed fractions recommended by the ICRP. The absorbed and equivalent doses of irradiation of different organs and systems in human organism have been defined. The required values of quality index have been calculated using the spectrum of absorbed dose on the scale of linear transfer of energy for all radiations of the analysed nuclides. The obtained values of efficient equivalent dose are 8.6 and 16 MeV/MBq for 113mIn-indifor and 99mTc-pyrphotech correspondingly. PMID- 3016455 TI - Ataxia-telangiectasia: a human mutation giving high-frequency misrepair of DNA double-stranded scissions. AB - The ability of three normal and one radiosensitive Ataxia-telangiectasia (A-T) human cell lines to rejoin restriction endonuclease-induced double-stranded (ds) DNA scissions was investigated using gene-transfer techniques with recombinant plasmid as target DNA. The results of cellular experiments using gene transfer frequencies as a measure of DNA rejoining strongly suggested that the A-T cell line had a greatly elevated frequency of misrepair of double-stranded DNA scissions. Southern blot analysis of DNA from plasmid-transformed cells confirmed this and further suggested that the misrepair in the A-T cell line took the form of large deletions and/or rearrangements at or around the scission. We postulate a disequilibrium in A-T between rejoining and exonuclease digestion of DNA termini as a possible basis for the misrepair and discuss this mechanism in relation to the major clinical features of the disease. PMID- 3016456 TI - Characterization of the human aldolase B gene. AB - The structure of the human gene encoding the aldolase B isozyme has been determined, including the sequence of 14,887 base-pairs. The 5'- and 3'-ends have been determined by S1 mapping. There is a single gene for this enzyme in humans that was determined from the sequence and restriction enzyme digestions of genomic DNA. The gene is 14,500 base-pairs long containing nine exons. In addition, 924 and 208 base-pairs of the 5'- and 3'-flanking region, respectively, have been determined. There is a high degree of conservation of nucleic acid sequence between aldolase B genes of human, rat and chicken. The conservation extends to untranslated and flanking regions, and includes the derived protein structures. In the 5'-flanking region there are several sequence elements that are conserved in vertebrate aldolase B genes in addition to the T-A-T-A and C-C-A A-T boxes. These sequences may be involved in the co-ordinate and tissue-specific control of expression of this gene. Several possible polymorphic sites were detected in the sequence of the human gene which may be useful for linkage mapping of the human genome and diagnostic analysis of alleles in families with hereditary fructose intolerance. PMID- 3016457 TI - Organization of the T-cell receptor alpha-chain gene and rearrangement in human T cell leukaemias. AB - The structure and rearrangement of the human T-cell receptor alpha-chain gene have been analysed. The constant region segment is comprised of four exons, with the 3' non-coding region present as a separate exon. Two J alpha segments have been identified by sequence analysis about 4 X 10(3) and 15 X 10(3) base-pairs upstream from the constant region segment. Analysis of alpha-chain rearrangements in a panel of T-cells reveals the presence of at least four more J alpha segments within 19 X 10(3) base-pairs of C alpha and suggests the existence of additional J alpha segments further upstream. This dispersed organization of the J alpha region contrasts with the clustered organization observed in other lymphoid rearranging genes. The use of J alpha probes in diagnostic purposes for T-cell leukaemia is not, therefore, convenient and will require a number of different probes to ensure complete analysis of the locus. PMID- 3016458 TI - Endocrine and neurophysiologic responses of the pituitary to insulin-induced hypoglycemia: a review. PMID- 3016460 TI - Protein dynamics and hydration. PMID- 3016459 TI - Biomembranes. Part O. Protons and water: structure and translocation. PMID- 3016461 TI - Determining intracellular viscosity from the rotational motion of spin labels. PMID- 3016462 TI - A method of solvent structure analysis for proteins using D2O-H2O neutron difference maps. PMID- 3016464 TI - Proton conduction through proteins: an overview of theoretical principles and applications. PMID- 3016463 TI - Cell water viscosity. PMID- 3016465 TI - Proton polarizability of hydrogen bonds: infrared methods, relevance to electrochemical and biological systems. PMID- 3016467 TI - Proton permeation through model membranes. PMID- 3016468 TI - Lipid phase transitions: water and proton permeability. PMID- 3016469 TI - Electron paramagnetic resonance methods for measuring H+/OH- fluxes across phospholipid vesicles. PMID- 3016466 TI - Theoretical calculation of energetics of proton translocation through membranes. PMID- 3016471 TI - Localized protonic coupling: overview and critical evaluation of techniques. PMID- 3016472 TI - Measurement of density and location of solvent associated with biomolecules by small-angle neutron scattering. PMID- 3016470 TI - Application of the laser-induced proton pulse for measuring the protonation rate constants of specific sites on proteins and membranes. PMID- 3016474 TI - Bacteriorhodopsin: fourier transform infrared methods for studies of protonation of carboxyl groups. PMID- 3016473 TI - Use of hydrogen exchange kinetics in the study of the dynamic properties of biological membranes. PMID- 3016475 TI - Deuteration as a tool in investigating the role of water in the structure and function of excitable membranes. PMID- 3016476 TI - Spectroscopic methods for the determination of membrane surface charge density. PMID- 3016477 TI - Determination of the net proton-hydroxide ion permeability across vesicular lipid bilayers and membrane proteins by optical probes. PMID- 3016478 TI - Conservation and variation in the large scale organisation of the globin gene domains of duck and chicken. AB - The genomic DNA of cloned recombinants containing the duck globin genes was compared to that of the analogous domains of the chicken. A 36 kb insert including the three alpha-type globin genes was isolated from a newly prepared duck genomic library in the cosmid PJB8; another recombinant contained a 45 kb insert with the four beta globin genes. In the alpha globin gene domain, the relative positions of genes, of repetitive sequences, and of the A + T-rich segments (AT-rich linkers, ATRLs) which frame the gene cluster (Moreau et al. 1982), were found to be closely maintained between duck and chicken. Although ATRLs and repetitive sequences also frame the gene cluster in the beta globin domains of duck and chicken, there is more genetic drift in their relative positions than in the alpha domain. It is of interest that several repetitive DNA segments were detected in the chicken beta globin domain which do not exist in corresponding positions in the duck. In view of the strict conservation in both species of genes and their relative positions in the cluster, this observation seems to exclude a simple function of repetitive sequences in the control of individual genes. The data are discussed with regard to the possible significance of repetitive and AT-rich DNA segments in genome organisation and function. PMID- 3016479 TI - Characterization of a collection of deletion mutants at the 3'-end of 16S ribosomal RNA of Escherichia coli. AB - Deletions were constructed in plasmid pKK3535 in the coding region for the 3'-end of E. coli 16S rRNA. The plasmid was cleaved with restriction endonuclease Hae2 under conditions favoring the production of single cut linear plasmid DNA and deletions were produced by digestion with exonuclease Bal31. Seven different deletions were isolated ranging in size from 90 to about 200 base pairs. Transcription of ribosomal DNA, processing of ribosomal RNA and incorporation of mutant rRNA into mutant particles was studied in UV-sensitive cells using a modified maxicell labeling procedure. The different mutants were missing defined features in the secondary structure of 16S rRNA and were characterized according to their stability, ability to be processed, sensitivity to colicin E3, and ability to bind ribosomal protein S1 and to interact with 50S subunits. These analyses show that the small stem and loop structure at positions 1350 to 1372 is necessary for the stability of rRNA. The deletion of the long terminal stem structure (1409-1491) in all mutant rRNAs does not block processing of the mutant rRNAs or S1 binding, although processing of the mutant rRNAs or S1 binding, although it does prevent the association of particles containing the mutant rRNA with 50S subunits. PMID- 3016480 TI - Genetic studies on the beta subunit of Escherichia coli RNA polymerase. VII. RNA polymerase is a target for ppGpp. AB - RNA polymerase was isolated from two suppressed rpoB amber strains exhibiting relaxed control over RNA synthesis in vivo. In vitro transcription analysis of these mutant holoenzymes carrying defined substitutions at known sites in the beta subunit clearly shows that single amino acid changes in beta render RNA polymerase resistant to ppGpp. This unambiguously demonstrates that RNA polymerase is indeed the target for ppGpp. PMID- 3016481 TI - Homology-facilitated plasmid transfer in Haemophilus influenzae. AB - The 8 kbp plasmid pAT4 transformed Haemophilus influenzae Rd cells at low frequencies. Transformation was increased up to 100 times, however, when the recipient cells carried a DNA segment in either their chromosome or in a resident plasmid that was homologous to at least part of plasmid pAT4. Linearized plasmid DNA molecules did not transform cells without DNA homology; they efficiently transformed homology recipients, but only when the cuts had been made in the region of shared homology. In most cases examined the circular donor plasmid had been reconstituted from the transforming DNA; in some cases the reconstituted plasmid carried a mutation initially present in the recipient chromosome, provided the transforming plasmid had been linearized in the region of shared homology. Plasmid reconstitution was not observed in recA1 cells. We conclude that homology-facilitated plasmid transformation (transfer) is similar to that reported for Bacillus subtilis and Streptococcus pneumoniae. PMID- 3016482 TI - Intron mutations that affect the splicing efficiency of the CYH2 gene of Saccharomyces cerevisiae. AB - To define the extent of intervening sequences required for efficient splicing of the CYH2 gene in Saccharomyces cerevisiae, we have constructed a series of intron mutations. Artificial intron extensions of more than 300 bp of the natural intron lead to an inhibition of splicing whereas intron deletions lead to a drastic improvement of the splicing efficiency. It is shown that deletion of a 32 bp sequence element within the intron is responsible for this drastic improvement. PMID- 3016484 TI - Genetic selection of lambda phage clones from a gene clonotheque. AB - A genetic procedure for selection of specific lambda clones, by homologous recombination between lambda clones from a gene clonotheque and sequences cloned into a plasmid, was developed. Resulting clones are isolated in transduction experiments by plating infected Escherichia coli cells under conditions selecting for the antibiotic resistance marker carried by the plasmid. The feasibility of the method was demonstrated in a model test system as well as by isolation of alpha-interferon-specific sequences from the human gene clonotheque. PMID- 3016483 TI - Rearrangements of microinjected recombinant DNA in the genome of transgenic mice. AB - Previously, mouse zygotes were microinjected with the recombinant plasmid pMA3, which contains the Herpes simplex virus thymidine kinase gene attached to the promoter region of the Rous sarcoma virus (Gazaryan et al. 1984a). In the present work the pMA3 fragment with the flanking genomic sequences was isolated from the DNA of one transgenic Fo mouse by the "plasmid rescue" technique. The rescued plasmid (pMAR1) lacked all virus-specific sequences and retained only some pBR322 sequences. The flanking region at one end of the integrated pBR322-specific fragment contained a highly conserved mouse repetitive sequence. The possible mechanisms of rearrangement of foreign DNA in germ line cells are discussed. PMID- 3016486 TI - The acquired immune deficiency syndrome. PMID- 3016487 TI - AIDS--aspects of infection control. PMID- 3016488 TI - Cannabis: toxicological properties and epidemiological aspects. PMID- 3016485 TI - Regulation of gene expression by pH of the growth medium in Aspergillus nidulans. AB - In the fungus Aspergillus nidulans the levels of a number of enzymes whose location is at least in part extracellular (e.g. acid phosphatase, alkaline phosphatase, phosphodiesterase) and of certain permeases (e.g. that for gamma amino-n-butyrate) are controlled by the pH of the growth medium. For example, at acidic pH, levels of acid phosphatase are high and those of alkaline phosphatase are low whereas at alkaline pH the reverse is true. Mutations in five genes, palA, B, C, E and F, mimic the effects of growth at acid pH whereas mutations in pacC mimic the effects of growth at alkaline pH. palA, B, C, E and F mutations result in an intracellular pH (pHin) which is more alkaline than that of the wild type whereas pacC mutations result in a pHin more acidic than that of the wild type. This indicates that these mutations exert their primary effects on the regulation of gene expression by pH rather than on the pH homeostatic mechanism but that the expression of at least some component(s) of the pH homeostatic mechanism is subject to the pH regulatory system. It is suggested that pacC might be a wide domain regulatory gene whose product acts positively in some cases (e.g. acid phosphatase) and negatively in others (e.g. alkaline phosphatase). The products of palA, B, C, E and F are proposed to be involved in a metabolic pathway leading to synthesis of an effector molecule able to prevent the (positive and negative) action of the pacC product.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016489 TI - Illness caused by a Kokobera-like virus in south-eastern Australia. AB - Three patients who lived in south-eastern Australia and who suffered acute polyarticular illnesses in the summer months of 1983-1984 and 1984-1985 are described. Two patients lived in the southwestern plains of New South Wales and one in Bairnsdale in eastern Victoria. Serological studies implicated Kokobera virus, a flavivirus, as the likely causative agent. This would appear to be the first report to indicate the pathogenicity of Kokobera virus. PMID- 3016490 TI - Musculocutaneous neuropathy after strenuous physical activity. AB - Three cases of isolated musculocutaneous neuropathy that were related to heavy physical activity are reported. The condition was predominantly motor, and each patient presented with weakness and loss of the normal contour of the biceps muscle. Recovery was complete in two cases, while the third case was left with residual weakness of the biceps muscle. It is postulated that the neuropathy in such cases is due to entrapment or stretching of the nerve where it passes through the coracobrachialis muscle in the upper arm. The condition should be distinguished from rupture of the biceps muscle belly or tendon, from brachial plexopathy and from cervical radiculopathies. PMID- 3016491 TI - Heterogeneous distribution of adrenergic receptors in coronary arteries and their influence on coronary arterial tone. AB - In-vitro experiments, using canine large (o.d. 1-2 mm) and small (o.d. 0.4-0.8mm) coronary arteries, were undertaken to determine the functional state of alpha- and beta-adrenergic receptors and their role in regulation of coronary arterial tone. Both large and small coronary arteries of the dog possess alpha and beta adrenergic receptors. Alpha-adrenergic receptors are more numerous on the circumflex vessel than elsewhere. Functionally, beta-adrenergic receptors appear to be dominant under physiological conditions in most types of coronary arteries. In canine coronary arteries, beta-adrenergic receptors have a greater affinity for noradrenaline than adrenaline. Alpha-adrenergic receptors have a greater affinity for adrenaline than noradrenaline. It is concluded that after beta adrenergic blockade, in both large and small coronary arteries, alpha-adrenergic receptors play an important role in regulation of coronary arterial tone. PMID- 3016493 TI - Enterovirus surveillance--United States, 1986. PMID- 3016492 TI - [Adhesions of the labia minora in children. Response to local treatment with estrogens]. PMID- 3016494 TI - [Radionuclide angiography (scintiphotosplenovenography) in the evaluation of splenorenal shunt patency]. AB - Radionuclide angiography of the splenic vein (Scintiphotosplenovenography: SSV) was done in 7 patients following a distal splenorenal shunt (Warren procedure). Spleen was punctured with a 22 gauge needle under ultrasonographic guidance. One half ml of 99m TcO4- solution, which contained 10 mci of nuclide energy, was injected into the splenic parenchyma through the needle. Injected material was followed by scintillation camera. In five cases, whose esophageal varices had been endoscopically atrophic, the splenic vein, the left renal vein, the inferior vena cava and the heart were clearly viewed in a continuity within 8 seconds from the start of injection. In one case, whose varices had recurred soon after the operation, only the collaterals were delineated. Most of them seemed running upwards alongside the esophagus. In the remaining patient, whose varices had been atrophic, the splenic vein was faintly delineated within 8 seconds. However, most of the 99m TcO4- stayed at first in the spleen. Thereafter it went out into the collaterals, the lower intercostal vein and the hemiazygos vein. This method is less invasive than the conventional angiography. It can reveal not only the entire imaging of shunt flow but also the collaterals, if they exist. It seemed useful for the evaluation of shunt patency following distal splenorenal shunt. PMID- 3016496 TI - Turnover of beta 1- and beta 2-adrenergic receptors after down-regulation or irreversible blockade. AB - The turnover of beta 1- and beta 2-adrenergic receptors was measured after both isoproterenol-induced down-regulation and irreversible blockade of receptors. Changes in the density of receptors were quantified using the radioligands 125I iodopindolol and 125I-iodocyanopindolol. Treatment of intact L6 myoblasts or C6 glioma cells with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) inactivated beta-adrenergic receptors on membranes prepared from these cells. At a concentration of 100 microM EEDQ, more than 90% of beta 1- and beta 2 adrenergic receptors were inactivated within 2 hr of treatment. Recovery of beta adrenergic receptors on intact cells after inactivation by EEDQ required more than 24 hr and was prevented by cycloheximide, an inhibitor of protein synthesis. The kinetics of recovery of the density of receptors were analyzed in terms of a model that allows estimation of the rate constants for receptor appearance in and disappearance from the membrane, assuming that the rate of appearance of receptors is constant and the rate of disappearance of receptors is proportional to the number of receptors. Beta 2-Adrenergic receptors on L6 myoblasts were incorporated into the membrane at a rate of 28 fmol/mg of protein/hr and had a half-life of 12.6 hr. On C6 glioma cells, Beta 1- and beta 2-adrenergic receptors appeared at rates of 13.3 and 6.6 fmol/mg of protein/hr, respectively, with half lives of 9.4 and 6.4 hr. Recovery of receptors on C6 cells after isoproterenol induced down-regulation was inhibited by cycloheximide. The rate of recovery of beta 1- and beta 2-adrenergic receptors was reduced after treatment with isoproterenol for 8 hr when compared to recovery after treatment with EEDQ. The major effect of treatment with isoproterenol was a persistent decrease in the rate of appearance of beta 1- and beta 2-adrenergic receptors (rate of synthesis and insertion into the membrane after treatment with isoproterenol = 4.0 fmol/mg of protein/hr). Since treatment with isoproterenol did not alter the rate of cell division or total protein synthesis, the isoproterenol-induced alteration was probably a specific effect on the rate of synthesis of beta-adrenergic receptors. PMID- 3016495 TI - [An in vitro sensitivity assay for anti-cancer agents by measuring the inhibition rate of DNA synthesis (3H-thymidine uptake of cancer cells). II. Clinical study of 110 cases of breast cancer]. AB - The sensitivity of cancer cells to anti-cancer agents (ACA) was assessed in 110 cases of breast cancer (87 primary cases and 23 recurrent cases). The cancer cells were cultured with ACAs: Mitomycin C (MMC), Adriamycin (ADR), 5 Fluorouracil (5-FU), Cytosine Arabinoside (Ara-C), Carboquone (CQ), Nimustine Hydrochloride (ACNU), Cis-platinum Diammine Dichloride (CPDD) or Vincristine (VCR) for 3 days and their sensitivity was estimated by the inhibition rate (I.R.) of DNA synthesis (3H-thymidine uptake) of cancer cells. The DNA synthesis was higher in recurrent cases than in primary cases. The primary cases showed high sensitivity to ADR or CQ, and the recurrent cases showed high sensitivity to ADR. Histologically, papillotubular or medullary tubular carcinoma showed high sensitivity to CQ, and scirrhous carcinoma showed high sensitivity to ADR, CQ or 5-FU. The sensitivities of medullary tubular or scirrhous carcinoma to ADR, 5-FU and CQ in patients with stage III and IV were lower than those in patients with stages I and II. No difference of ACA sensitivity was observed between estrogen receptor (+) and (-) cases. All recurrent cases were treated with 5-FU or its derivatives. The 50% survival period in the 5-FU high sensitivity (I.R. greater than 80%) group was 7.0 months and that of the low sensitivity (I.R. less than 80%) group 3.0 months, respectively. PMID- 3016497 TI - Inhibition of chemotactic factor-activated Na+/H+ exchange in human neutrophils by analogues of amiloride: structure-activity relationships in the amiloride series. AB - The ability of a number of analogues of the diuretic, amiloride, to inhibit chemotactic factor-stimulated Na+/H+ exchange in human neutrophils was investigated. Intracellular pH (pHi) changes were measured from the equilibrium distribution of 14C-labeled 5,5-dimethyloxazolidine-2,4-dione (DMO). Exposure of cells to 10 nm N-formyl-methionyl-leucyl-phenylalanine (FMLP) caused activation of Na+/H+ exchange: in 140 mM Na+ medium (extracellular pH 7.40), the pHi rose from a resting value of approximately 7.25 to reach a new steady state of approximately 7.75 by 10-15 min. This intracellular alkalinization was sensitive to amiloride (apparent Ki approximately 75 microM), a known inhibitor of Na+/H+ countertransport. The structure-activity relationships in the amiloride series were characterized by testing the effect of these compounds on the DMO-derived pHi changes and on the FMLP-stimulated rate of 22Na+ efflux from the cells. Substitutions of the guanidino group of amiloride resulted in relatively inactive products (Ki greater than or equal to 1 mM). Replacement of the 6-Cl group of amiloride by other halogen atoms had only modest effects on drug efficacy. However, replacement of one or both H atoms of the 5-amino group by short alkyl groups led to a 10-500-fold increase in potency for inhibition of Na+/H+ exchange. Amiloride and three of its more potent derivatives (compounds I, O, and MM, the 5-N,N-dimethyl, 5-N,N-diethyl, and 5-N,N-hexamethylene analogues, respectively) caused parallel inhibition of FMLP-activated 22Na+ efflux and the rate of intracellular alkalinization, with apparent Ki values of approximately 75, 8, 1, and 0.2 microM, respectively. In each instance, the inhibitory effects of the drugs were readily reversible on washing the cells. None of the compounds altered the binding of 3H-labeled FMLP to its cell surface receptors. The development of potent derivatives of amiloride should provide powerful tools for assessing the role of FMLP-activated Na+/H+ exchange and the resultant pHi transients on stimulated neutrophil functions. PMID- 3016498 TI - Single-site model of the neuronal 5-hydroxytryptamine uptake and imipramine binding site. AB - Recently, a high affinity [3H]imipramine-binding site of protein nature that appeared to be related to the 5-hydroxytryptamine (5-HT, serotonin) uptake mechanism was demonstrated. This binding site was only part of desipramine displaceable [3H]imipramine binding, which contained a significant amount of additional binding not related to 5-HT uptake. The present study further investigates the [3H]imipramine-binding site of protein nature in the rat brain. Displacement by 5-HT and 6-methoxytetrahydro-beta-carboline (6-MeO-TH beta C) revealed monophasic displacement patterns with 60% displaceable binding. This binding fraction was abolished by protease treatment of the brain tissue prior to binding assay. Saturation studies of [3H]imipramine binding (1-30 nM) in rat cortex showed that the binding displaced by 30 microM 5-HT [Bmax 322 +/- 16 fmol/mg of protein, Kd 4.17 +/- 1.07 nM (means +/- SE)] was not different from the binding displaced by 1.0 microM norzimeldine (Bmax 349 +/- 15 fmol/mg of protein, Kd 4.47 +/- 1.07 nM) or 30 microM 6-MeO-TH beta C (Bmax 439 +/- 28 fmol/mg of protein, Kd 5.49 +/- 1.09 nM). When 100 microM desipramine was used in saturation studies, the binding was different from that displaced by 5-HT with Bmax 608 +/- 42 fmol/mg of protein and Kd 6.68 +/- 1.09 nM. Both displacement and saturation studies in which two displacing agents were combined indicated that most of the binding competed by 5-HT (30 microM) and norzimeldine (1.0 microM) is identical. Similarly, the binding displaced by 5-HT or norzimeldine is subsumed within 6-MeO-TH beta C (30 microM)-displaceable binding. Lesion studies with parachloroamphetamine, a selective toxin for 5-HT terminals, which resulted in a 83% reduction of [3H] 5-HT uptake ( [3H]noradrenaline uptake unaffected), abolished cortical [3H]imipramine binding displaced by 30 microM 5-HT or 1.0 microM norzimeldine. (greater than 80% reduction). However, with 100 microM desipramine as displacer, 40% of the binding remained in lesioned animals. The [3H]imipramine binding displaced by 30 microM 5-HT or 1.0 microM norzimeldine was sodium dependent, and an increase in NaCl concentration from 0 to 120 mM resulted in a 10-fold increase in affinity without effect on Bmax, whereas no change in binding was observed with increasing concentrations of LiCl.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3016499 TI - Homogeneous uridine kinase from Ehrlich ascites tumor: substrate specificity and inhibition by bisubstrate analogs. AB - Uridine kinase has been purified to homogeneity from Ehrlich ascites tumor cells. For the phosphate acceptor site, the enzyme shows substrate specificity only for ribopyrimidine nucleosides and is active with various analogs that have limited structural alterations; both endocyclic and exocyclic substituents can be acceptable. Of nucleosides that have been used in the chemotherapy of cancer, 5 fluorouridine, 6-azauridine, and 3-deazauridine are good substrates, whereas arabinosylcytosine is a poor substrate. No analogs are better substrates than the physiological substrates uridine and cytidine. 5', 5''' -P1, P4-Bisnucleoside oligophosphate bisubstrate analogs (e.g., Ap4U, Ap5U) were synthesized and tested as inhibitors. The most effective compound was Ap4U; with a Ki of 197 microM, it bound more tightly than ATP but no better than uridine. Ap3A, Ap4A, and Ap5A were also tested, with the result that both Ap4A and Ap4U were most effective, suggesting that this size of bisubstrate analog most closely approaches the spacing of the catalytic site. PMID- 3016500 TI - Vasopressin antagonists allow demonstration of a novel type of vasopressin receptor in the rat adenohypophysis. AB - The ligand specificity of rat adenohypophyseal vasopressin receptors was directly compared to that of peripheral receptors of the V1 and V2 types. For this purpose a series of 15 recently designed vasopressin antagonists was used. The affinities of these antagonists for rat adenohypophyseal membranes were deduced from the determination of the concentration-dependent inhibition of [3H]vasopressin binding. In parallel experiments the corticotropin (or anti-corticotropin) releasing activities of the tested peptides were determined on freshly dispersed rat adenohypophyseal cells. All peptides tested which were found to be antagonists of the vasopressor and antidiuretic responses to vasopressin in vivo behaved as antagonists of vasopressin-induced corticotropin release. There was a close correlation between the relative affinities of the analogues tested for binding to adenohypophyseal membranes and their relative potencies in inhibiting vasopressin-induced corticotropin release, indicating that the detected vasopressin-binding sites are the receptors involved in the vasopressin effect on corticotropin secretion. No correlation could be demonstrated between anti corticotropin-releasing activities and either anti-antidiuretic or antivasopressor potencies of the antagonists tested. A direct comparison of the ligand specificities of adenohypophyseal receptors on the one hand, and V1 (hepatic) and V2 (renal) receptors on the other hand, showed that most of the antagonists discriminated very efficiently between adenohypophyseal and either hepatic or renal receptors. The selectivity index reaches values as high as 260,000 for desGly(NH2)9 [1-(beta-mercapto-beta, beta cyclopentamethylenepropionic acid), 2-D-O-ethyl-tyrosine, 4-valine] arginine vasopressin. It is concluded that adenohypophyseal receptors represent a novel type of vasopressin receptors. Based on the observation that adenohypophyseal receptors, like hepatic or vascular V1 receptors, do not appear to be coupled to adenylate cyclase, we propose that adenohypophyseal receptors could be designated as V1b receptors as opposed to the V1a receptors previously characterized on liver and blood vessels. PMID- 3016501 TI - Differences in the mechanism of functional interaction between NADPH-cytochrome P 450 reductase and its redox partners. AB - Selective methylamidation of NADPH-cytochrome P-450 reductase (EC 1.6.2.4) carboxyl groups was used to assess the relative importance of these groups in the enzyme-catalyzed reduction of cytochromes c, b5, and P-450. Methylamidation of as few as 7 mol of carboxyl groups per mol of reductase caused 80% inhibition of cytochrome c reduction, 50% inhibition of rat liver microsomal RLM3 reduction, and up to 90% inhibition in the capacity of the reductase to support reconstituted monooxygenase activities of RLM3, RLM5, and LM2. In marked contrast, cytochrome b5 reduction measured under comparable conditions was stimulated by 50%. The impaired interactions between the reductase and cytochromes P-450 LM2 and RLM5 were shown not to arise from an impaired capacity for the proteins to bind each other but more likely to be due to an inhibition of a step(s) subsequent to complex formation between the oxidized proteins. These results show that the reductase interacts functionally with cytochrome c and cytochromes P-450 on the one hand and cytochrome b5 on the other through different mechanisms. PMID- 3016502 TI - Proenkephalin-A gene regulation in the rat striatum: influence of lithium and haloperidol. AB - This report explores the influence of lithium and haloperidol on (Met5) enkephalin (ME) biosynthesis in the rat striatum. Male Fischer 344 rats were treated intraperitoneally with lithium chloride (4 mEq/kg/day) for 2, 4, or 6 days and sacrificed 24 hr after the last dose; in addition, the effect of lithium at 2 and 24 hr after a single dose was studied. Serum levels increased in a time related manner on repeated administration of lithium. Lithium increased the striatal ME content (native ME) in a time-dependent fashion, reaching 160% of control following six doses; no changes in ME were observed in hypothalamus and hippocampus. ME levels recovered to control values 8 days after cessation of a 4 day course of repeated administration (4 mEq/kg/day) of lithium. In an attempt to characterize the nature of this selective increase of ME content in the striatum, the precursor content (cryptic ME) as well as the preproenkephalin mRNA abundance was determined. Lithium increased the precursor content in a time-dependent fashion and this pattern closely paralleled the increase in native ME content. The preproenkephalin mRNA abundance with respect to control was quantitated by blot-hybridization of total RNA with a 32P-labeled cDNA probe derived from rat brain. Lithium increased the mRNA abundance following repeated doses for 4 or 6 days. Concurrent administration of an opiate antagonist, naltrexone (5 mg/kg/day), for 4 days did not influence the changes induced by lithium. On repeated administration (1 mg/kg/day) for 4 days, the neuroleptic, haloperidol, increased the biosynthesis of ME which was more marked than that of lithium administered for the same period; the combination of a haloperidol and lithium regimen did not lead to an additive of synergistic effect. The results indicate that, like haloperidol, repeated injections of lithium increase the biosynthesis of ME in the basal ganglia by increasing the preproenkephalin mRNA abundance and translation process. PMID- 3016503 TI - Sodium regulation of agonist binding at opioid receptors. I. Effects of sodium replacement on binding at mu- and delta-type receptors in 7315c and NG108-15 cells and cell membranes. AB - The effects of varying the sodium concentration (at constant ionic strength) on opioid binding at mu- and delta-opioid receptors in 7315c and NG108-15 cells has been examined. The binding of [3H]etorphine to mu-receptors on 7315c cells was increased by replacing the sodium in the incubation medium with potassium or N methyl-D-glucamine. This effect was shown to be attributable to an increase in affinity, with no change in the maximum number of binding sites, both in cell membrane suspensions and in intact 7315c cells. Replacement of sodium with potassium or N-methyl-D-glucamine in NG108-15 membrane or intact cell suspensions also resulted in an increase in [3H]etorphine binding, but in these cells the effect was associated with an increase in the number of binding sites measurable under these experimental conditions. The effects of sodium on opioid inhibition of adenylate cyclase in membrane preparations from 7315c and NG108-15 cells also differed. Sodium reduced apparent agonist affinity in 7315c membranes. In NG108 15 cell membranes, sodium was essential for the demonstration of opioid inhibition of cyclase activity. Increasing the sodium concentration above 0.5 mM resulted in an increase in the fraction of total enzyme activity inhibited by opioid, but the opioid IC50 did not change. In the companion paper, it is shown that the effects of sodium removal on mu- and delta-receptor binding in guinea pig brain neural membranes were similar to those observed in the cell preparations. An increase in intracellular sodium concentration without change in extracellular concentration was effected by incubation of 7315c and NG108-15 cells with the sodium-selective ionophore, monensin. When sodium was present in the extracellular medium, monensin reduced [3H]etorphine binding by 50% or more, both at mu-receptors in 7315c cells and at delta-receptors in NG108-15 cells. In the absence of sodium, however, monensin treatment produced only a small inhibition of binding. These results suggest that sodium acts at an intracellular site to regulate opioid agonist binding at both mu- and delta-receptors, but that the mode of regulation is not identical at each site. Since a reduction in intracellular sodium concentration by removal of extracellular sodium increases agonist binding, and an increase in intracellular sodium following monensin treatment reduces agonist binding, it is probable that the intracellular sodium concentration is a critical regulator of opioid agonist binding in intact cells. PMID- 3016504 TI - Sodium regulation of agonist binding at opioid receptors. II. Effects of sodium replacement on opioid binding in guinea pig cortical membranes. AB - We have examined the effects of sodium on the binding of opioid agonists to mu-, delta-, and kappa-receptors in guinea pig cortical membranes. Concentration curves for sodium indicated that maximal inhibition of mu binding by this cation was about 60% and maximal inhibition for delta binding was about 70%, whereas that for kappa binding was only about 20%. The concentration of sodium required for half-maximal inhibition of binding to all three sites was about 10-30 mM, corresponding to the intracellular sodium concentration. The nature of the sodium effect was further characterized by saturation analysis of binding to each of the three receptor types by comparing results obtained in the presence of 120 mM sodium with those obtained with equimolar replacement of sodium by another cation. Two radiolabeled agonists with different structural characteristics were tested for each binding site. In the presence of sodium, the affinity of the labeled agonists for mu sites was approximately 2-3-fold less than in its absence, but the density of binding sites was not changed. At kappa sites, sodium reduced agonist affinity slightly but, again, did not alter the number of binding sites. In contrast, sodium reduced the apparent density of delta-binding sites while leaving the agonist affinity unchanged. Competition against antagonist binding to delta sites indicated that, in the presence of sodium, a higher proportion of sites was in a lower affinity state, as reflected by the biphasic nature of the agonist displacement curve. In contrast, the effect of sodium on displacement of antagonist from mu sites was to of sodium on displacement of antagonist from mu sites was to lower the affinity of the agonist. Competition against antagonist binding to kappa sites also showed a reduction in agonist affinity by sodium, but no change in number of receptors. The results indicate that sodium may differentially regulate agonist binding to opioid receptor types and that this regulation may occur at an intracellular site. The kappa site appears to be less sensitive to sodium than the mu and delta sites. PMID- 3016505 TI - Inhibition of DNA-dependent transcription by the leader RNA of vesicular stomatitis virus: role of specific nucleotide sequences and cell protein binding. AB - The leader RNA transcript of vesicular stomatitis virus inhibits transcription of the adenovirus major late promoter and virus-associated genes in a soluble HeLa cell transcription system. We examined the specific nucleotide sequence involved and the potential role of leader-protein interactions in this inhibition of RNA polymerase II- and III-directed transcription. Using synthetic oligodeoxynucleotides homologous to regions of the leader RNA molecule, we extend our previous results (B.W. Grinnell and R.R. Wagner, Cell 36:533-543, 1984) that suggest a role for the AU-rich region of the leader RNA or the homologous AT region of a cloned cDNA leader in the inhibition of DNA-dependent transcription. Our results indicate that a short nucleotide sequence (AUUAUUA) or its deoxynucleotide homolog (ATTATTA) appears to be the minimal requirement for the leader RNA to inhibit transcription by both RNA polymerases, but sequences flanking both sides of this region increase the inhibitory activity. Nucleotide changes in the homologous AT-rich region drastically decrease the transcriptional inhibitory activity. Leader RNAs from wild-type virus, but not from a 5' defective interfering particle, form a ribonuclease-resistant, protease-sensitive ribonucleoprotein complex in the soluble HeLa cell extract. Several lines of evidence suggest that the leader RNA specifically interacts with a 65,000-dalton (65K) cellular protein. In a fractionated cell extract, only those fractions containing this 65K protein could reverse the inhibition of DNA-dependent RNA synthesis by the plus-strand vesicular stomatitis virus leader RNA or by homologous DNA. In studies with synthetic oligodeoxynucleotides homologous to leader RNA sequences, only those oligonucleotides containing the inhibitory sequence were able to bind to a gradient fraction containing the 65K protein. PMID- 3016507 TI - P-element-induced control mutations at the r gene of Drosophila melanogaster. AB - The P-M hybrid dysgenesis system was used to produce five putative regulatory mutations at the rudimentary locus, r. All five mutations were the result of insertions at the 5' end of the gene, upstream of the proposed start of transcription. All of the mutants displayed a leaky wing phenotype, and four of the mutants showed an uncoupling of the wing and female-sterility phenotypes, suggesting that they altered the normal spatial and temporal expression of the r gene. Four of the insertions were P elements. The fifth insertion, which was larger than an intact P element, consisted of a small P element connected to non P-element DNA. Two of the mutants produced very little r transcript in adult females and were clustered 80 to 150 base pairs upstream of the start of transcription. The other three mutants had higher levels of r transcript in adult females and were clustered 440 to 500 base pairs upstream of the start of transcription. All of the data suggest that the insertions are in a 5' noncoding region of the r gene involved in the control of its spatial and temporal expression. PMID- 3016506 TI - Mapping genetic elements of Epstein-Barr virus that facilitate extrachromosomal persistence of Epstein-Barr virus-derived plasmids in human cells. AB - The Epstein-Barr virus (EBV) genome becomes established as a multicopy plasmid in the nucleus of infected B lymphocytes. A cis-acting DNA sequence previously described within the BamHI-C fragment of the EBV genome (J. Yates, N. Warren, D. Reisman, and B. Sugden, Proc. Natl. Acad. Sci. USA 81:3806-3810, 1984) allows stable extrachromosomal plasmid maintenance in latently infected cells, but not in EBV-negative cells. In agreement with the findings of Yates et al., deletion analysis permitted the assignment of this function to a 2,208-base-pair region (nucleotides 7315 to 9517 of the B95-8 strain of EBV) of the BamHI-C fragment that contained a striking repetitive sequence and an extended region of dyad symmetry. A recombinant vector, p410+, was constructed which carried the BamHI-K fragment (nucleotides 107565 to 112625 of the B95-8 strain, encoding the EBV associated nuclear antigen EBNA-1), the cis-acting sequence from the BamHI-C fragment, and a dominant selectable marker gene encoding G-418 resistance in animal cells. After being transfected into HeLa cells, this plasmid persisted extrachromosomally at a low copy number, with no detectable rearrangements or deletions. Two mutations in the BamHI-K-derived portion of p410+, a large in frame deletion and a linker insertion frameshift mutation, both of which alter the carboxy-terminal portion of EBNA-1, destroyed the ability of the plasmid to persist extrachromosomally in HeLa cells. A small in-frame deletion and linker insertion mutation in the region encoding the carboxy-terminal portion of EBNA-1, which replaced 19 amino acid codons with 2, had no effect on the maintenance of p410+ in HeLa cells. These observations indicate that EBNA-1, in combination with a cis-acting sequence in the BamHI-C fragment, is in part responsible for extrachromosomal EBV-derived plasmid maintenance in HeLa cells. Two additional activities have been localized to the BamHI-C DNA fragment: (i) a DNA sequence that could functionally substitute for the simian virus 40 enhancer and promoter elements controlling the expression of G-418 resistance and (ii) a DNA sequence which, although not sufficient to allow extrachromosomal plasmid maintenance, enhanced the frequency of transformation to G-418 resistance in EBV-positive (but not EBV-negative) cells. These findings suggest that the BamHI-C fragment contains a lymphoid-specific or EBV-inducible promoter or enhancer element or both. PMID- 3016508 TI - Isolation of cellular genes differentially expressed in mouse NIH 3T3 cells and a simian virus 40-transformed derivative: growth-specific expression of VL30 genes. AB - We constructed and screened a cDNA library made from simian virus 40 (SV40) transformed NIH 3T3 cells, and we isolated cDNAs representing genes that are differentially expressed between the parental cell and its SV40-transformed derivative. We found only a small number of cDNAs representing such genes. Two isolated cDNA clones represented RNAs expressed at elevated levels in the transformed cell line in a manner relatively independent of growth conditions. The expression of two other cDNAs was growth specific because transformed cells and nonconfluent parental cells contained higher levels of the homologous RNAs than did confluent, contact-inhibited parental cells. Another cDNA was well expressed in confluent parental and confluent transformed cells, but not in nonconfluent cells. The expression of some of these cDNAs varied strikingly in different mouse cell lines. Thus the genotype or histories of different cell lines can also affect the expression of certain genes. Interestingly, the only cDNA isolated that was expressed exclusively in the transformed cell was from an SV40 message. We focused on a growth-specific cDNA which we show is derived from a mouse endogenous retrovirus-like family called VL30. We sequenced the 3' long terminal repeat (LTR) of this transcriptionally active VL30 gene. This LTR has good homology with other VL30 LTR sequences, but differences occur, particularly upstream of the VL30 promoter. We found that VL30 gene expression varied in different mouse cell lines such that C3H cell lines had very low levels of VL30 transcripts relative to NIH 3T3 cell lines. However, Southern analysis showed that both cell lines had about the same number of VL30 genes homologous to our probe and that the position of the majority of these genes was conserved. We discuss possible explanations for this difference in VL30 expression. PMID- 3016509 TI - Mechanisms of nonhomologous recombination in mammalian cells. AB - The primary mechanism of nonhomologous recombination in transfected DNA involves breakage followed by end joining. To probe the joining step in more detail, linear simian virus 40 genomes with mismatched ends were transfected into cultured monkey cells, and individual viable recombinants were analyzed. The transfected genomes carried mismatched ends as a result of cleavage with two restriction enzymes, the recognition sites of which are located in the intron of the gene encoding the T antigen. Because the T antigen gene was split by this cleavage, the transfected genomes were inert until activated by cell-mediated end joining. Clonal descendants of the original recombinants were isolated from 122 plaques and were grouped into four classes based on the electrophoretic mobility of the junction fragment. The structures of representative junctions were determined by nucleotide sequencing. The spectrum of nonhomologous junctions analyzed here along with a large number of previously reported junctions suggest that there are two mechanisms for the linkage of DNA molecules: (i) direct ligation of ends and (ii) repair synthesis primed by terminal homologies of a few nucleotides. A paired-priming model of nonhomologous recombination is discussed. PMID- 3016510 TI - Alternative excision products originating from a single integration of polyomavirus DNA. AB - The Cyp cell line consists of mouse cells transformed by a thermosensitive polyomavirus (Py) genome and routinely propagated at 39 degrees C. Cyp cells are readily induced to synthesize free Py DNA by being transferred to 33 degrees C. In one subclone (C12/a1/S48, or S48) of this line, such induction resulted in the intracellular accumulation of three discrete species of cyclic DNA, i.e., genomic Py DNA, RmI, and RmII. RmI and RmII are Py-mouse chimeras, each of which contains a distinct set of sequences originating from the site of integration. Conceivably, genomic Py DNA, RmI, and RmII could persist at 39 degrees C as free replicating plasmids or originate from distinct populations of cells in S48 cultures. The data indicated that all three species arise at 33 degrees C from a genetically homogeneous cell population in which neither RmI nor RmII replicates at 39 degrees C. Examination of the sequence at the viral-cellular junction unique to RmII indicated that this chimera is excised from the host chromosome through a recombination event involving a complex viral sequence and a simple cellular sequence. Therefore, RmII provides another example of precise recombination occurring between nonhomologous sequences in a mammalian cell, as already observed for RmI (B. S. Sylla, D. Huberdeau, D. Bourgaux-Ramoisy, and P. Bourgaux, Cell 37:661-667, 1984). PMID- 3016511 TI - Unequal homologous recombination between tandemly arranged sequences stably incorporated into cultured rat cells. AB - Cultured rat cells deficient in endogenous thymidine kinase activity (tk) were stably transformed with a recombination-indicator DNA substrate constructed in vitro by rearrangement of the herpes simplex virus tk gene sequences into a partially redundant permutation of the functional gene. The recombination indicator DNA did not express tk, but was designed to allow formation of a functional tk gene via homologous recombination. A clonal cell line (519) was isolated that harbored several permuted herpes simplex virus tk genes. 519 cells spontaneously produced progeny that survived in medium containing hypoxanthine, aminopterin, and thymidine. Acquisition of resistance to hypoxanthine, aminopterin, and thymidine was accompanied by the rearrangement of the defective tk gene to functional configuration. The rearrangement apparently occurred by unequal exchange between one permuted tk gene and a replicated copy of itself. Recombination was between 500-base-pair tracts of DNA sequence homology that were separated by 3.4 kilobases. Exchanges occurred spontaneously at a frequency of approximately 5 X 10(-6) events per cell per generation. Recombination also mediated reversion to the tk- phenotype; however, the predominant mechanism by which cells escaped death in the presence of drugs rendered toxic by thymidine kinase was not recombination, but rather inactivation of the intact tk gene. PMID- 3016512 TI - Identification of a sequence element on the 3' side of AAUAAA which is necessary for simian virus 40 late mRNA 3'-end processing. AB - Our previous studies of the 3'-end processing of simian virus 40 late mRNAs indicated the existence of an essential element (or elements) downstream of the AAUAAA signal. We report here the use of transient expression analysis to study a functional element which we located within the sequence AGGUUUUUU, beginning 59 nucleotides downstream of the recognized signal AAUAAA. Deletion of this element resulted in (i) at least a 75% drop in 3'-end processing at the normal site and (ii) appearance of readthrough transcripts with alternate 3' ends. Some flexibility in the downstream position of this element relative to the AAUAAA was noted by deletion analysis. Using computer sequence comparison, we located homologous regions within downstream sequences of other genes, suggesting a generalized sequence element. In addition, specific complementarity is noted between the downstream element and U4 RNA. The possibility that this complementarity could participate in 3'-end site selection is discussed. PMID- 3016513 TI - Processing of p60v-src to its myristylated membrane-bound form. AB - p60src of wild-type Rous sarcoma virus is myristylated at its N-terminal glycine residue. We have shown previously that this myristylation is necessary for p60src membrane association and for cell transformation by using src mutants with alterations within the N-terminal 30 kilodaltons of p60src. In this study we analyzed the process of p60src myristylation in wild type- and mutant-infected cells. All myristylated src proteins examined lack the initiator methionine, but two mutant src proteins lacking the initiator methionine are not myristylated, indicating that removal of the initiator methionine and myristylation are not obligatorily coupled. Analysis of the kinetics of myristylation and the association of p60src with cellular proteins p50 and p90 indicated that myristylation occurs before p60src becomes membrane associated and that transient association with p50 and p90 occurs regardless of myristylation. Myristylation is required for stable association of p60src with the plasma membrane but is not sufficient for membrane association. A mutant with an src deletion of amino acids 169 through 264 has an src protein that is myristylated but not membrane bound, remaining stably associated with p50 and p90. This mutant is transformation defective. Several N-terminal deletion mutants possessing tyrosine kinase activity have myristylated and membrane-bound src proteins but are not fully active in cell transformation, suggesting that additional N-terminal functional domains exist. PMID- 3016515 TI - Structure and unusual characteristics of a new family of transposable elements in the sea urchin Strongylocentrotus purpuratus. AB - The transposable element family TU of the sea urchin Strongylocentrotus purpuratus, a higher eucaryote, has recently been described (D. Liebermann, B. Hoffman-Liebermann, J. Weinthal, G. Childs, R. Maxson, A. Mauron, S.N. Cohen, and L. Kedes, Nature [London] 306:342-347, 1983). A member of this family, TU4, has an insertion, called ISTU4, of non-TU DNA. ISTU4 is a member of a family of repetitive sequences, which are present in some 1,000 copies per haploid S. purpuratus genome (B. Hoffman-Liebermann, D. Liebermann, L.H. Kedes, and S.N. Cohen, Mol. Cell. Biol. 5:991-1001, 1985). We analyzed this insertion to determine whether it is itself a transposable element. The nucleotide sequence of ISTU4 was determined and showed an unusual structure. There are four, approximately 150 nucleotides long, imperfect direct repeats followed by a single truncated version of these repeats. This region is bounded at either side by approximately 100-nucleotide-long sequences that are not related to each other or to the repeats. Nucleotide sequences at the boundaries of ISTU4-homologous and flanking regions in five genomic clones show that ISTU4 represents a family of sequences with discrete ends, which we call Tsp elements. We showed that the genomic locus that carries a Tsp element in one individual was empty in other individuals and conclude that Tsp elements are a new and different type of transposable element. Tsp elements lack two features common to most other transposable elements: Tsp integration does not result in the duplication of host DNA, and there are no inverted repeats at their termini, although short inverted repeats are present at a distance from the termini. PMID- 3016514 TI - Synthesis of bovine growth hormone in primates by using a herpesvirus vector. AB - A strain of herpesvirus saimiri containing a bovine growth hormone (bGH) gene under the control of the simian virus 40 (SV40) late-region promoter was constructed. This strain, bGH-Z20, was replication competent and stably harbored the bGH gene upon serial passage. Nonpermissive marmoset T cells persistently infected with bGH-Z20 produced a 0.9-kilobase RNA which contained all of the bGH exon sequences and appeared to initiate within the SV40 promoter region. However, in permissively infected owl monkey kidney cells, RNAs containing growth hormone sequences appeared to initiate from herpesvirus saimiri promoters positioned upstream from the SV40-growth hormone gene. Persistently infected T cells in culture secreted 500 ng of bGH protein per 10(6) cells per 24 h during the several months of testing. The secreted protein was 21 kilodaltons, the size of authentic bGH. New World primates experimentally infected with bGH-Z20 produced circulating bGH and developed immunoglobulin G antibodies directed against bGH. Because herpesviruses characteristically remain latent in the infected host, these observations suggest a means for replacing gene products missing or defective in hereditary genetic disorders. PMID- 3016516 TI - Tsp transposons: a heterogeneous family of mobile sequences in the genome of the sea urchin Strongylocentrotus purpuratus. AB - In the preceding paper (J.B. Cohen, B. Hoffman-Liebermann, and L. Kedes, Mol. Cell. Biol., 5:2804-2813, 1985), we described the nucleotide sequence of ISTU4, which is a member of a new family of repetitive sequences, the Tsp family, present in a higher eucaryote, the sea urchin Strongylocentrotus purpuratus. We provided evidence that individual members of this family can act as transposable elements. Here we describe our structural analysis of the Tsp element family, which numbers about 1,000 members per haploid genome. Hybridization and nucleotide sequence analysis of several genomic Tsp clones demonstrate that structurally most Tsp elements resemble ISTU4. Tsp elements range in size up to about 1.3 kilobase pairs, have terminal domains that are conserved between the various examples studied, and contain a central portion of varying size, which may be extensively diverged. Structurally, however, the central portions are very similar and consist of several approximately 150-base-pairs-long, tandemly arranged, imperfect repeats, which are followed by a truncated repeat. The structural analysis is consistent with the possibility that the individual Tsp elements differ by multiples of these 150-base-pair repeats. One variant genomic clone has a solitary repeat and lacks the truncated repeat. The nucleotide sequences of different repeats of a single Tsp element can diverge extensively. The truncated repeat is divergent from most of the repeats, but in one case it is almost identical to a repeat of the same element. Comparison of the sequences from different elements enabled us to determine the boundaries of each structural domain and allows us to propose that each of these domains may be independent units of genetic information. Analysis of the population of Tsp-related sequences in the S. purpuratus genome by genomic blot hybridization suggests that most Tsp family members share the same overall structure. In addition, there is a structural element, about 70 base pairs long, that appears to interrupt the tandem arrangement of the 150-base-pair repeats at regular intervals. PMID- 3016517 TI - Genetic characterization of human c-rel sequences. AB - We isolated and sequenced a human genomic-DNA segment that is homologous to a portion of v-rel, the transforming gene of reticuloendotheliosis virus (strain T). We also localized the human rel sequences to human chromosome 2 by screening a panel of rodent X human somatic-cell hybrids with the newly described human rel segment. PMID- 3016518 TI - Murine leukemia virus long terminal repeat sequences can enhance gene activity in a cell-type-specific manner. AB - We tested the ability of sequences in the long terminal repeat (LTR) of a mink cell focus-forming (MCF) murine leukemia virus to function as an enhancer in a cell-type-specific manner. In a stable transformation assay, the MCF or Akv LTR and the simian virus 40 enhancer had similar activities in murine fibroblasts. In contrast, the MCF LTR had a significantly greater activity in murine T lymphoid cells than did either the simian virus 40 enhancer or the Akv LTR. PMID- 3016519 TI - Surprising S1-resistant trimolecular hybrids: potential complication in interpretation of S1 mapping analyses. AB - Although the technique of S1 mapping is a powerful analytical tool for the analysis of RNA, we now report a surprising complication involving a trimolecular hybrid between two RNA species and a single DNA probe molecule which, if unrecognized, can lead to misleading interpretations. We document that such trimolecular hybrids can be efficiently formed under some hybridization conditions and that the probe DNA sequence at the junction of the two RNA molecules can be remarkably stable to digestion with S1. Trimolecular hybrids can arise in any instance whenever a distal region of an end-labeled DNA probe is homologous to a moderately abundant RNA in the sample to be analyzed. This situation presents a serious, potential complication for a variety of S1 analyses, particularly those in which DNA transfection has been utilized to reintroduce in vitro-engineered genes into cultured animal cells. PMID- 3016520 TI - Surface expression of influenza virus neuraminidase, an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells. AB - We have investigated the site of surface expression of the neuraminidase (NA) glycoprotein of influenza A virus, which, in contrast to the hemagglutinin, is bound to membranes by hydrophobic residues near the NH2-terminus. Madin-Darby canine kidney or primary African green monkey kidney cells infected with influenza A/WSN/33 virus and subsequently labeled with monoclonal antibody to the NA and then with a colloidal gold- or ferritin-conjugated second antibody exhibited specific labeling of apical surfaces. Using simian virus 40 late expression vectors, we also studied the surface expression of the complete NA gene (SNC) and a truncated NA gene (SN10) in either primary or a polarized continuous line (MA104) of African green monkey kidney cells. The polypeptides encoded by the cloned NA cDNAs were expressed on the surface of both cell types. Analysis of [3H]mannose-labeled polypeptides from recombinant virus-infected MA104 cells showed that the products of cloned NA cDNA comigrated with glycosylated NA from influenza virus-infected cells. Both the complete and the truncated glycoproteins were found to be preferentially expressed on apical plasma membranes, as detected by immunogold labeling. These results indicate that the NA polypeptide contains structural features capable of directing the transport of the protein to apical cell surfaces and the first 10 amino-terminal residues of the NA polypeptide are not involved in this process. PMID- 3016521 TI - Long interspersed repeated DNA (LINE) causes polymorphism at the rat insulin 1 locus. AB - The insulin 1, but not the insulin 2, locus is polymorphic (i.e., exhibits allelic variation) in rats. Restriction enzyme analysis and hybridization studies showed that the polymorphic region is 2.2 kilobases upstream of the insulin 1 coding region and is due to the presence or absence of an approximately 2.7 kilobase repeated DNA element. DNA sequence determination showed that this DNA element is a member of a long interspersed repeated DNA family (LINE) that is highly repeated (greater than 50,000 copies) and highly transcribed in the rat. Although the presence or absence of LINE sequences at the insulin 1 locus occurs in both the homozygous and heterozygous states, LINE-containing insulin 1 alleles are more prevalent in the rat population than are alleles without LINEs. Restriction enzyme analysis of the LINE-containing alleles indicated that at least two versions of the LINE sequence may be present at the insulin 1 locus in different rats. Either repeated transposition of LINE sequences or gene conversion between the resident insulin 1 LINE and other sequences in the genome are possible explanations for this. PMID- 3016522 TI - Differential transformation of C3H10T1/2 cells by v-mos: sequential expression of transformation parameters. AB - Extremely small quantities of the product of the transforming gene v-mos of Moloney murine sarcoma virus are able to efficiently transform cells. Recent data indicate the existence of a threshold level for v-mos transformation of NIH3T3 cells. Using mouse mammary tumor virus long terminal repeat sequences or hybrid promoters consisting of mouse mammary tumor virus and Moloney murine sarcoma virus long terminal repeat elements to express v-mos in C3H10T1/2 cells, we established cell lines representing different stages of morphological transformation in vitro. The threshold level for v-mos transformation was considerably lower than that for NIH3T3 cells, because no treatment with dexamethasone or primary selection other than transformation was necessary during standard transfection procedures. Using the cell lines mentioned we established an association of the level of v-mos expression with the transformation parameters examined, but not with p53 levels. Furthermore, the characterization of the different promoters showed (i) that the distal binding site confers hormone responsiveness to Moloney murine sarcoma virus promoter elements and (ii) that artifactual transcription initiation sites can be detected in mouse mammary tumor virus-Moloney murine sarcoma virus hybrid promoters which are, however, not regulated by the hormone. PMID- 3016523 TI - Protein phosphorylation in a tetradecanoyl phorbol acetate-nonproliferative variant of 3T3 cells. AB - The 3T3-TNR9 cell line is a variant of Swiss 3T3 cells which does not respond mitogenically to tumor promoters, but does respond mitogenically to epidermal growth factor, fibroblast growth factor, and serum. To elucidate differences between tumor promoters and polypeptide mitogens in the pathway(s) of mitogenesis which might be responsible for the nonresponsiveness of the 3T3-TNR9 cells, we have examined in these cells the early protein phosphorylation events known to be associated with mitogenesis in the parental 3T3 cells. We find that the 3T3-TNR9 cells display levels of tetradecanoyl phorbol acetate binding and of a calcium- and phospholipid-dependent protein kinase activity which are at least the equal of those seen in the parental 3T3 cells, implicating some postreceptor event in the nonmitogenic phenotype. In addition, we find that phosphorylation of the epidermal growth factor receptor and of 80-kDa and 22-kDa proteins, as well as the tyrosine phosphorylation of a 42-kDa protein, all proceed normally in the nonmitogenic variant, even though these phosphorylations must depend on the activation of different kinases. Thus, all these early phosphorylation reactions are intact in the 3T3-TNR9 cells. Although these phosphorylations may be necessary, they clearly are insufficient to trigger mitogenesis. PMID- 3016524 TI - Infection of immune mast cells by Harvey sarcoma virus: immortalization without loss of requirement for interleukin-3. AB - Cells from adult mouse spleens were cultured in WEHI-3 cell-conditioned medium, which contains the lymphokine interleukin-3 (IL-3). Under these conditions, cells grow well for 4 to 8 weeks; the cultures contain a variety of cell types for the first 1 to 2 weeks but are subsequently composed largely of immune mast cells. We found that infection of these cultures with Harvey sarcoma virus (HaSV) profoundly enhanced the growth potential of the cells, resulting in the reproducible isolation of long-term cell lines. These HaSV-infected cells appeared to be phenotypically identical to the immune mast cells found in uninfected cultures as determined by biochemical, immunological, and cytological tests. Although the cells expressed protein p21Ha-ras at levels similar to those in HaSV-transformed fibroblasts, they continued to require IL-3 for growth in vitro. Similar IL-3-dependent, long-term mast cell lines were also cultured from the enlarged spleens present in HaSV-infected mice. These results suggest that high-level expression of an activated Ha-ras oncogene enhances growth in these cells, perhaps by stimulating the progression of the cells into S, without affecting differentiation or altering the requirements for normal growth factor. PMID- 3016525 TI - Precise localization of m6A in Rous sarcoma virus RNA reveals clustering of methylation sites: implications for RNA processing. AB - N6-methyladenosine (m6A) residues are present as internal base modifications in most higher eucaryotic mRNAs; however, the biological function of this modification is not known. We describe a method for localizing and quantitating m6A within a large RNA molecule, the genomic RNA of Rous sarcoma virus. Specific fragments of 32P-labeled Rous sarcoma virus RNA were isolated by hybridization with complementary DNA restriction fragments spanning nucleotides 6185 to 8050. RNA was digested with RNase and finger-printed, and individual oligonucleotides were analyzed for the presence of m6A by paper electrophoresis and thin-layer chromatography. With this technique, seven sites of methylation in this region of the Rous sarcoma virus genome were localized at nucleotides 6394, 6447, 6507, 6718, 7414, 7424, and 8014. Further, m6A was observed at two additional sites whose nucleotide assignments remain ambiguous. A clustering of two or more m6A residues was seen at three positions within the RNA analyzed. Modification at certain sites was found to be heterogeneous, in that different molecules of RNA appeared to be methylated differently. Previous studies have determined that methylation occurs only in the sequences Gm6AC and Am6AC. We observed a high frequency of methylation at PuGm6ACU sequences. The possible involvement of m6A in RNA splicing events is discussed. PMID- 3016527 TI - Suppression of the hypomethylated Moloney leukemia virus genome in undifferentiated teratocarcinoma cells and inefficiency of transformation by a bacterial gene under control of the long terminal repeat. AB - The Moloney leukemia virus (M-MuLV) genome was introduced into undifferentiated teratocarcinoma cells by transfection of a plasmid with the virus genome linked to pSV2neo, which carries a bacterial drug resistance gene, neo, or by cotransfection with pSV2neo. In the resulting cells, the M-MuLV genome remained hypomethylated, but its expression was suppressed in cells in an undifferentiated state. The pattern of DNA methylation of the viral genome remained unchanged when the cells were induced to differentiate into epithelial tissues. However, spontaneous M-MuLV expression was detected with differentiation of the cells. To determine to what extent the viral long terminal repeat (LTR) was responsible for this suppression in undifferentiated cells, I constructed plasmids in which neo was placed under the control of the promoter sequence of the dihydrofolate reductase gene or the M-MuLV LTR, and compared the biological activities of the plasmids in Ltk- cells and in undifferentiated teratocarcinoma cells. In Ltk- cells, these plasmids were highly efficient in making the cells resistant to selection by G418. However, in undifferentiated teratocarcinoma cells, the M-MuLV LTR promoted neo gene expression at only 10% of the expected efficiency, as compared with the expression of the neo gene under the control of the simian virus to or dihydrofolate reductase promoter. Thus, the mechanisms of gene regulation are not the same in undifferentiated and differentiated teratocarcinoma cells. PMID- 3016526 TI - Novel method for identifying sequence-specific DNA-binding proteins. AB - We developed a general method for the enrichment and identification of sequence specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene. PMID- 3016528 TI - Hormonally mediated negative regulation of human pro-opiomelanocortin gene expression after transfection into mouse L cells. AB - We report results indicating that expression and hormonally controlled negative regulation of the human pro-opiomelanocortin (POMC) gene in mouse fibroblasts can be accomplished by the placement nearby of a simian virus 40 enhancer sequence. Expression resulting from correctly initiated transcription required the enhancer in cis both in cells stably transfected with the POMC gene and in a transient expression assay with constructs that fused that POMC promoter region to the protein-coding region of the herpes simplex virus thymidine kinase (TK) gene. Negative regulation of POMC transcription by glucocorticoids was demonstrated in transiently infected cells by assaying for TK activity encoded by the POMC-TK fusion constructs and by quantitative S1 nuclease mapping. The sequences responsible for such regulation were shown to be contained within a DNA segment that extends 670 base pairs upstream from the cap site for POMC mRNA. PMID- 3016530 TI - Influence of bromodeoxyuridine substitution of thymidine on sister-chromatid exchanges and chromosomal aberrations induced by restriction endonucleases. AB - Different levels of replacement of thymidine by 5-bromodeoxyuridine in mammalian DNA have been used to analyze restriction endonuclease-dependent induction of sister-chromatid exchanges and chromosomal aberrations. Data regarding enzyme action in whole cells and in isolated nuclei are presented and discussed. The results indicate a lack of correlation between enzyme effectiveness and the degree of 5-bromodeoxyuridine substitution in the target sequences, specific to the tested restriction endonucleases. PMID- 3016529 TI - Nuclear DNA content of cells in salivary gland tumours. PMID- 3016531 TI - Adverse effect of verapamil in a patient with the Lambert-Eaton syndrome. AB - A patient with the Lambert-Eaton syndrome (LES) and small cell lung cancer developed respiratory failure several hours after verapamil was given. Improvement in respiratory function did not occur when guanidine was given, but was delayed until verapamil was discontinued 3 days later. Although other factors may have contributed to the clinical deterioration of our patient, the temporal relationship to verapamil and the theoretical danger of calcium channel blockade lead us to believe that the drug should be used cautiously in LES. PMID- 3016532 TI - Passively transferred Lambert-Eaton syndrome in mice receiving purified IgG. AB - Purified IgG antibodies were prepared by ion-exchange chromatography from the plasma of a patient with nonneoplastic form of the Lambert-Eaton myasthenic syndrome (LES). The antibodies were injected into mice with daily doses of 0.15 10 mg for 20-22 days, following which the integrity of neuromuscular transmission was assessed in vitro in phrenic nerve-diaphragm muscle preparations. The injected animals manifested electrophysiologic features of human LES, which were characterized by: dose-dependent reduction in the quantal content of nerve-evoked endplate potentials, an abnormally small increase in the frequency of spontaneous miniature endplate potentials (MEPPs) with elevated [K+]o, and normal MEPP amplitude with no evidence of postjunctional deficiency. Crude immunoglobulins (Igs) from the same patient and two LES patients with associated malignancy similarly transferred the defects in quantal transmitter release. In contrast, animals receiving Igs from control subjects or from a patient with small-cell carcinoma of the lung manifested no functional impairment of neuromuscular transmission. Instead, the evoked release in these animals was significantly enhanced relative to that found in normal untreated mice. These results suggest that an IgG antibody produces the presynaptic impairment that is characteristic of LES and support the concept that LES with and without cancer has an autoimmune pathogenesis. PMID- 3016533 TI - Inhibitors of the mitochondrial cytochrome b-c1 complex inhibit the cyanide insensitive respiration of Trypanosoma brucei. AB - The cyanide-insensitive respiration of bloodstream trypomastigote forms of Trypanosoma brucei (75 +/- 8 nmol O2 min-1(mg protein)-1) is completely inhibited by the mitochondrial ubiquinone-like inhibitors 2-hydroxy-3-undecyl-1,4 naphthoquinone (UHNQ) and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). The Ki values for UHDBT (30 nM) and UHNQ (2 microM) are much lower than the reported Ki for salicylhydroxamic acid (SHAM) (5 microM), a widely used inhibitor of the cyanide-insensitive oxidase. UHNQ also stimulated the glycerol-3-phosphate dependent reduction of phenazine methosulfate, demonstrating that the site of UHNQ inhibition is on the terminal oxidase of the cyanide-insensitive respiration of T. brucei. These results suggest that a ubiquinone-like compound may act as an electron carrier between the two enzymatic components of the cyanide-insensitive glycerol-3-phosphate oxidase. PMID- 3016534 TI - Purification and characterisation of membrane-form variant surface glycoproteins of Trypanosoma brucei. AB - Membrane-form variant surface glycoprotein of Trypanosoma brucei can be prepared in the presence of para-chloromercuriphenylsulphonic acid. The membrane-bound enzyme that usually cleaves a lipid from this glycoprotein, thus producing the soluble variant surface glycoprotein, is inhibited by a range of sulphydryl reagents. The effect of such inhibitors, both on cell lysates and on semi purified enzyme, reveals that the enzyme may have a sulphydryl at or near its active site. Fatty acid analysis and isoelectric point measurements of membrane form and soluble form are presented. PMID- 3016535 TI - Glycerol, a metabolic end product of Trichomonas vaginalis and Tritrichomonas foetus. AB - Glycerol was demonstrated as an end product of anaerobic glucose metabolism in Trichomonas vaginalis and Tritrichomonas foetus, produced in addition to acetate, H2, CO2, and lactate or succinate. In T. vaginalis strain C-1, glycerol amounted to 16% of the fermentation products and was formed at an average rate of 38 nmol min-1 (mg protein)-1. Corresponding figures for T. foetus strain KV1 were 7% and 4.8 nmol min-1 (mg protein)-1. The amounts of glycerol detected compensated almost exactly for the deficits in fermentation products recognized earlier, thus complete redox balances can now be provided for both organisms. The metronidazole resistant T. foetus strain KV1-1MR-100 excreted only negligible amounts of glycerol and carried out an ethanol-CO2 fermentation. Aerobiosis hardly affected glycerol formation in T. vaginalis strains C-1 and NYH 286, but almost completely abolished it in T. foetus strain KV1. An NADP-dependent glycerol 3-phosphate dehydrogenase and a Mg2+-dependent glycerol 3-phosphatase were detected in the cytosol of both species. The phosphatase is distinct from the particle-bound nonspecific acid phosphatase. Glycerol kinase activity was not detected in either organism. Enhanced pCO2 did not affect the ratio of fermentation products in T. vaginalis strain C-1, but significantly increased the amount of succinate, and decreased the amounts of acetate, H2, and CO2, formed by T. foetus. PMID- 3016536 TI - Identification of visceral Leishmania species with cloned sequences of kinetoplast DNA. AB - Several efforts have been made in order to develop more precise and sensitive methods in the identification of Leishmania parasites. We report here the identification of cloned subfragments of minicircle kinetoplast DNA (kDNA) isolated from L. donovani, WR352, which show different taxonomic specificities. Analysis of these fragments demonstrates a significant sequence diversity within the kDNA minicircle. For example, one cloned fragment was found to be present in all visceral Leishmania species tested, but was not present in any of the cutaneous Leishmania species. Another cloned fragment was only found in the strain from which it had been derived, and was not present in any of the other strains tested. In similar experiments with the New World visceral leishmanias (L. chagasi, WR518) several different cloned kDNA fragments were found to react with all of isolates of the L. chagasi tested, but not with any cutaneous Leishmania species, either from the Old World or the New World. It is of interest to note that these cloned L. chagasi kDNA fragments reacted with isolates of African visceral Leishmania species but not with isolates from India. PMID- 3016538 TI - Impaired specific cellular response to HTLV-III before other immune defects in patients with HTLV-III infection. PMID- 3016537 TI - A second isolate of HTLV-II associated with atypical hairy-cell leukemia. PMID- 3016539 TI - HTLV-III seroconversion in a homosexual patient with common variable immunodeficiency. PMID- 3016540 TI - Possible nonspecific associations between malaria and HTLV-III/LAV. PMID- 3016541 TI - Prevalence of HTLV-III/LAV antibodies in patients with hemophilia and in their sexual partners in France. PMID- 3016542 TI - Nondonor HIV antibody testing in Minnesota. PMID- 3016544 TI - Gender dimorphism in brain. PMID- 3016545 TI - AIDS. US wins round in patent row. PMID- 3016543 TI - HIV infection with seroconversion after a superficial needlestick injury to the finger. PMID- 3016546 TI - Neurotransmission. Are there two functional classes of glutamate receptors? PMID- 3016547 TI - Biological chemistry. Long-range electron transfer. PMID- 3016548 TI - Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. AB - Methotrexate, a folate antagonist, blocks import into mitochondria of mouse dihydrofolate reductase fused to a mitochondrial presequence. Methotrexate does not mask the presequence, but stabilizes the dihydrofolate reductase moiety. It does not inhibit import of the authentic precursor from which the presequence is derived. This suggests that dihydrofolate reductase must at least partly unfold in order to be transported across mitochondrial membranes. PMID- 3016549 TI - Directional electron transfer in ruthenium-modified horse heart cytochrome c. AB - Cytochrome c can be modified by [(NH3)5RuII/III-] specifically at the imidazole moiety of histidine 33, and we have recently discussed the thermodynamics and kinetics of electron transfer within this modified protein. X-ray crystal structures of the oxidized and reduced forms of tuna cytochrome c indicate that the separation between the haem group of cytochrome c and the ruthenium label is 12-16 A. Internal electron transfer from the [(NH3)5RuII-] centre to the Fe(III) haem centre occurs with a rate constant k congruent to 53 s-1 (25 degrees C) (delta H = 3.5 kcal mol-1, delta S = -39 EU), as measured by pulse radiolysis. The measured unimolecular rate constant, k congruent to 53 s-1, is on the same timescale as a number of conformational changes that occur within the cytochrome c molecule. These results raise the question of whether electron transfer or protein conformational change is the rate limiting step in this process. We describe here an experiment that probes this intramolecular electron transfer step further. It involves reversing the direction of electron transfer by changing the redox potential of the ruthenium label. Electron transfer in the new ruthenium-cytochrome c derivative described here is from haem(II) to the Ru(III) label, whereas in (NH3)5Ru-cytochrome c the electron transfer is from Ru(II) to haem(III). Intramolecular electron transfer from haem(II) to Ru(III) in the new ruthenium-cytochrome c described here proceeds much slower (greater than 10(5) times) than the electron transfer from Ru(II) to haem(III) in the (NH3)5Ru cytochrome c. We therefore conclude that electron transfer in cytochrome c is directional, with the protein envelope presumably involved in this directionality. PMID- 3016550 TI - Synergism between immunoglobulin enhancers and promoters. AB - Enhancers are DNA sequences that stimulate transcription from eukaryotic promoters. This stimulatory effect can be exerted over large distances and from a position either 5' or 3' of a promoter. Enhancers have been found in the genomes of many viruses, and in some cellular genes such as those encoding immunoglobulin heavy chain and kappa light chain. An important feature of both viral and cellular enhancers is the ability of each enhancer to stimulate transcription from many promoters other than the one with which it is found associated. However, the question of whether cellular enhancers stimulate their 'own' promoter more efficiently than other promoters has apparently not been investigated. We show here that the kappa light-chain enhancer stimulates a kappa promoter about 20-fold more than it stimulates either the simian virus 40 (SV40) early promoter or a metallothionein (MT) promoter, two promoters that are very sensitive to other enhancers. Similarly, the heavy-chain enhancer stimulates a heavy-chain promoter much more than it stimulates the SV40 and MT promoters. This synergism between immunoglobulin enhancers and promoters might be due to the action of a protein that binds specifically to each of the regulatory elements. PMID- 3016551 TI - Expression of human adenosine deaminase in murine haematopoietic progenitor cells following retroviral transfer. AB - Adenosine deaminase (ADA) deficiency, an autosomal recessive inborn error of metabolism, leads to severe combined immune deficiency in man. This enzyme, although constitutively expressed in most tissues, is expressed at high level in immature T cells, and study of the pathophysiology of the disorder indicates that increased deoxyadenosine or altered methylation capacity have toxic effects on T cell maturation. Although bone marrow transplantation can correct the immune deficiency, this therapy is associated with graft-versus-host disease and incomplete immune restoration, and so our laboratory and others have sought to develop a method of gene replacement as a possible treatment for the disease. Moreover, characterization of the complementary DNA of the human ADA gene and some of its mutants makes it possible to design gene transfer strategies. We have now subcloned a human adenosine deaminase cDNA into the retrovirus shuttle vector pZIP-SV(B), and in this way have isolated a cell line, 4.2T, which produces high titres of replication-defective retrovirus which have been used to transfer the gene for human ADA to mouse bone marrow cells. Transfer and expression of the neomycin-resistance gene (neo) and the ADA gene in murine bone marrow colony forming units (CFU) was demonstrated by in vitro colony formation in the presence of the antibiotic G418 or 9-xylofuranosyladenine plus deoxycoformycin, respectively. Isoenzyme analysis also showed human ADA expression in the cultured mouse bone marrow. PMID- 3016552 TI - Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity. AB - Acquired immune deficiency syndrome (AIDS) is characterized by marked depletion of the T4+ helper subset of T cells. The aetiological agent of the disease, the human T-lymphotropic virus type III (HTLV-III)/lymphadenopathy-associated virus (LAV), specifically kills T4+ cells in vitro. Part of this specificity for the T4+ population residues in the relative efficiency with which HTLV-III infects these cells, as a result of a specific interaction between the T4 molecule and the virus envelope glycoprotein. In addition, the cytotoxic consequences of HTLV III replication are dependent on cell type, as certain lymphoid and myeloid cells can be productively infected without notable cytopathic effect. Here we investigate the basis for the specific cytotoxicity of the virus, and report that high-level expression of the HTLV-III envelope gene induces syncytia and concomitant cell death in T4+ cell lines but not in a B-lymphocyte line. Syncytium formation depends on the interaction of envelope-expressing cells with neighbouring cells bearing surface T4 molecules. These results explain, at least in part, the specific cytopathic effect of HTLV-III infections. PMID- 3016553 TI - A chromosomal rearrangement in a P. falciparum histidine-rich protein gene is associated with the knobless phenotype. AB - The significant morbidity and mortality associated with Plasmodium falciparum malaria results, in part, from the sequestration of parasitized erythrocytes in postcapillary venules, which may protect the parasite from splenic clearance and contribute to the pathogenesis of cerebral malaria. This sequestration has been linked to the expression of parasite-induced knob structures on the surface of the infected erythrocyte which mediate the cytoadherence phenomenon. While knobs are necessary for cytoadherence, they are not sufficient, requiring both parasite and host-encoded proteins. Spontaneous mutants of P. falciparum have been isolated from in vitro cultures which lack the ability to express knobs and fail to cytoadhere. A histidine-rich protein has been described which is associated with the knobby phenotype and may be a constituent of the knob. We now report the isolation of complementary DNA clones for a knob-associated histidine-rich protein (KAHRP) and demonstrate that in knobless mutants the gene for this protein has undergone a rearrangement, resulting in a deletion in the 3' coding sequence. Moreover, the chromosome to which the KAHRP gene maps is rearranged in these mutants, producing a telomeric location of the truncated gene. These observations explain the loss of expression of the messenger RNA and protein in such mutants and may explain the loss of the knob itself. The implications for the generation of spontaneous mutations in the parasite by this novel mechanism are discussed. PMID- 3016554 TI - Long-range restriction site mapping of mammalian genomic DNA. AB - Molecular analysis of many problems in genetics would be facilitated by the ability to construct restriction site maps of long stretches of genomic DNA and to directly place genes on these maps. Pulsed-field gradient gel electrophoresis allows measurement of the size of DNA fragments up to at least 2,000 kilobase pairs (kb) long and we have used this technique here to map sites for one class of infrequently cutting restriction enzyme over a total of 1,500 kb of mouse genomic DNA. The sites for these enzymes tend to be clustered in the genome. These clusters may correspond to the short stretches of C + G-rich unmethylated DNA often associated with mammalian genes. PMID- 3016555 TI - Phosphoinositol more than skin deep. PMID- 3016556 TI - The origin of Japanese HTLV-I. PMID- 3016557 TI - Graphical orientation procedure for the estimation of Kd and Bmax values of ligand binding to two receptor subpopulations. AB - Within a few minutes, the procedure described here provides to the investigator, performing binding studies, information about Kd and Bmax values of two possibly present receptor subpopulations. The procedure is based on the comparison of the experimental curve with values of ligand binding to only one receptor population at 4 defined levels of radioligand binding. The binding parameters are derived from comparison of 4 horizontal deviations at these levels with tabulated deviations. Kd and Bmax values can be further confirmed by comparison of the experimental data with the simulated binding curve obtained by entering the binding parameters found in the formula for ligand binding to two receptor subpopulations. The practical use of this procedure was demonstrated both for simulated and for experimental data; it was confirmed by comparison with binding parameters obtained by computer programs used for the evaluation of receptor heterogeneity. PMID- 3016558 TI - Inhibition of human colonic (Na+ + K+)-ATPase by arachidonic and linoleic acid. AB - The sodium pump, (Na+ + K+)-ATPase, which is involved in the transport of cations and water movement by the colonic mucosa, may be decreased in various diarrhoeal states. In this study, we have measured 3H-ouabain binding and (Na+ + K+)-ATPase activity in human colonic biopsy homogenates and the influence of various inflammatory and antiinflammatory compounds on these parameters. 3H-ouabain binds to one site of high affinity (KD 1.9 +/- 0.2 X 10(-9) mol/l) with a maximal binding capacity of 7.5 +/- 0.8 X 10(14) binding sites/g protein. Both arachidonic and linoleic acid inhibited (Na+ + K+)-ATPase activity (IC50 arachidonic acid: 7.5 X 10(-5) mol/l, linoleic acid: 6.5 X 10(-5) mol/l) and Mg2+ ATPase activity (IC50 arachidonic acid: 9 X 10(-5) mol/l, linoleic acid: 4 X 10( 5) mol/l). Arachidonic acid inhibited 3H-ouabain binding, (IC50 3.2 X 10(-5) mol/l). The following antiinflammatory compounds, at concentrations up to 1 X 10( 3) mol/l, did not influence ATPase activity directly nor reverse the arachidonic acid-induced inhibition: indomethacin (cyclooxygenase inhibitor), nordihydroguaiaretic acid (lipoxygenase inhibitor), sulphasalazine and its metabolites: 5-aminosalicylic acid, N-acetylaminosalicylic acid and sulphapyridine. These results indicate that human colonic (Na+ + K+)-ATPase is inhibited by the prostanoid precursors, arachidonic and linoleic acid. From a therapeutic point of view (effect on colonic (Na+ + K+)-ATPase and perhaps diarrhoea), the suppression of the production of these prostanoid precursors by drugs may, therefore, be beneficial in the treatment of inflammatory bowel disease. PMID- 3016559 TI - Neuronal efflux of noradrenaline induced by tris or lithium as substitutes for extracellular sodium. AB - Vasa deferentia of either untreated or reserpine (R) and/or pargyline (P) pretreated rats were incubated with 3H-noradrenaline and then washed with amine- and Ca2+-free solution until (after 100 min) the efflux of radioactivity largely originated from adrenergic nerve endings; COMT was inhibited by U-0521 (U). After 110 min of wash out, the sodium chloride in the wash-out solution was replaced by an equimolar concentration of either Tris-HCl or LiCl. This caused a desipramine sensitive (i.e., carrier-mediated) efflux of tritiated noradrenaline. The initial increase of the "low Na+"-induced efflux dependent on the experimental conditions: it was most pronounced when the axoplasmic concentration of noradrenaline was high (RPU) and relatively small when MAO and vesicular storage were intact (U). The effects of Li+ and Tris+ differed with regard to the time course of the efflux of tritium: under all three experimental conditions (RPU, PU, U), Tris+ caused the rate of efflux of tritium to increase gradually within the 30 min period of observation, while Li+ either had a "peak-effect" (RPU, PU) or a "plateau-effect" (U). Under "U-conditions" Tris+ caused a slowly increasing, pronounced increase with time of the efflux of both, 3H-noradrenaline and 3H DOPEG; whereas Li+ caused only a small and sustained increase of the efflux of 3H noradrenaline and a decrease in the efflux of 3H-DOPEG.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016560 TI - Rolipram, a stereospecific inhibitor of calmodulin-independent phosphodiesterase, causes beta-adrenoceptor subsensitivity in rat cerebral cortex. AB - Prolonged pretreatment of rats with the atypical antidepressant rolipram attenuates noradrenaline (NA) sensitivity of the cerebral cortical cAMP generating system. The development of this down-regulation is time (7 d treatment required) and dose dependent (EC50 = 0.35 mg/kg). Density of beta-adrenoceptor as measured by (-)-3H-dihydroalprenolol [(-)-3H-DHA] binding is also reduced by rolipram pretreatment. The effect of rolipram is absolutely stereospecific for the (-)-enantiomer (ED50 = 0.18 mg/kg). In addition, only with this isomer, a reduction in daily weight gain was found compared to sham treated controls. Presynaptic denervation using intracerebroventricular (i.c.v.) injections of 6 hydroxydopamine (6-OHDA) prior to or during rolipram treatment did not completely block the effect of a rolipram treatment on down-regulation of cerebral cortical beta-adrenoceptors. The data favor a pre- and postsynaptic action of rolipram different from all other antidepressants studied so far in this experimental setting. Rolipram is known as inhibitor of brain phosphodiesterase. Using partially purified calmodulin-independent phosphodiesterase from brain it is shown that exclusively the (-)-enantiomer of rolipram inhibits phosphodiesterase with an IC50 of 1.25 mumol/l whereas the (+)-isomer possesses little potency. Since a marked stereospecificity for the (-)-isomer of rolipram was displayed in all pharmacological parameters tested so far with (+)- and (-)-rolipram, it is suggested that stereospecific and isozyme specific inhibition of cAMP phosphodiesterase is, at least in part, related to the mechanism of action of the potential antidepressant drug rolipram and possibly of other antidepressants as well. PMID- 3016563 TI - Changing trends in small cell lung cancer. PMID- 3016561 TI - Functional characterization of substance P receptors in the rabbit ear artery. AB - Rabbit isolated ear arteries were perfused at a constant flow and stimulated with field pulses (5 Hz, 5 impulses). Different tachykinins and capsaicin depressed stimulation-induced vasoconstriction, substance P (SP) being the most potent inhibitor. The rank order of potency of the tachykinins was, SP approximately equal to physalaemin approximately equal to eledoisin greater than SP-methyl ester; that of SP and its C-terminal fragments, SP approximately equal to SP-(2 11) approximately equal to SP-(4-11) greater than SP-(6-11). SP-(1-9) was inactive. The SP antagonist (Arg5,D-Trp7,9,Nle11)SP-(5-11) 10 mumol/l shifted the concentration-response curve of SP to the right (pA2 = 5.43), whereas it did not reduce the action of capsaicin. Another SP antagonist (D-Pro4,D-Trp7,9,10)SP-(4 11) 10 mumol/l failed to affect the SP depression. Neither antagonist changed vasoconstriction by itself. Pretreatment of the arteries with a mixture of yohimbine, propranolol, atropine, diphenhydramine, burimamide, methysergide and indomethacin, all 1 mumol/l, did not influence the effect of SP or capsaicin. Only the inhibition by SP, but not that by capsaicin was abolished after mechanical destruction of the endothelium. SP, physalaemin and eledoisin, all 3 mumol/l, reduced vasoconstriction by noradrenaline or histamine; capsaicin 30 mumol/l depressed noradrenaline-induced vasoconstriction. In arteries preincubated with 3H-noradrenaline, electrical stimulation (1 Hz, 120 pulses) triggered an increase in the outflow of tritium and evoked vasoconstriction. SP 1 mumol/l did not change either basal or stimulation-evoked tritium outflow, whereas it reduced vasoconstriction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016564 TI - [Tumors of nerves in the hand]. PMID- 3016562 TI - Evaluation of LY163443, 1-[2-hydroxy-3-propyl-4-([4- (1H-tetrazol-5 ylmethyl)phenoxy]methyl) phenyl]ethanone, as a pharmacologic antagonist of leukotrienes D4 and E4. AB - LY163443,1-[2-hydroxy-3-propyl-4-([4- (1H-tetrazol-5-ylmethyl)phenoxy]- phenoxy]methyl)phenyl]ethanone, antagonized LTD4-induced contractions of guinea pig ileum, trachea, and lung parenchyma. Tracheal contractions to LTE4 were also inhibited by LY163443. The compound had minimal effect against ileal responses to LTC4 and parenchymal contractions to LTB4. Furthermore, LY163443 had little to no effect against contractions of isolated smooth muscles to histamine, bradykinin, PGF2 alpha, carbachol, serotonin or U46619. LY163443, given by oral administration to guinea pigs, blocked LTD4-induced increases in total pulmonary impedance (TPI). Similar responses elicited by histamine or U46619 were unaffected. Increases in TPI in response to i.v. administration of LTC4 were antagonized by LY163443 given by the same route. Ovalbumin challenge also increased TPI in guinea pigs previously sensitized against this antigen. In such animals, pretreated with pyrilamine, propranolol, and indomethacin, oral administration of LY163443 blocked the increase in TPI caused by ovalbumin. Additionally, LTD4 given intradermally to guinea pigs caused a vascular leakage which was suppressed by prior oral administration of LY163443. Finally, LY163443 relaxed isolated guinea pig trachea previously contracted with LTD4, histamine, or carbachol. Relaxation of tissues contracted by these latter two agonists suggested some inherent airway smooth muscle relaxant properties of the molecule. This was further demonstrated by showing some bronchodilator activity in an in vivo setting. Thus, this pharmacologic profile indicates that LY163443, or a member of the same chemical family, warrants consideration as a possible therapeutic agent in the treatment of asthma and in diseases characterized by an overproduction of LTD4 and LTE4. PMID- 3016565 TI - [Irritable bowel syndrome]. PMID- 3016566 TI - [Irritable bowel syndrome: an unmanageable diagnosis?]. PMID- 3016567 TI - [Melanotic progonoma of the upper jaw in a child]. PMID- 3016568 TI - [Role of serotonin in the development of the defensive reflex to food in the snail]. AB - 9-16 days after injection of 5,7-dihydroxytryptamine which elicits degeneration of serotonergic nerve cell terminals, pairing of food and electrical shock had no effect on responses of injected animals to food, while definite food-aversive reactions were observed in control animals. In neurophysiological experiments applications of serotonin in the chamber containing the nervous system was used as a reinforcing stimulus. The amplitude of synaptic responses to nerve stimulation increased significantly in preparations in which stimulation was paired with serotonin application. In neurons involved in defensive reactions after 3-7 pairings of a drop of juice to the chemoreceptive part of the skin with serotonin application a new spike response to food has appeared. Unpaired presentations of the same stimuli were not effective. It is concluded that serotonin is an important factor in formation of conditioned adverse reactions in the snail. PMID- 3016569 TI - [Postsynaptic reactions of neurons of the sensomotor cortex of the cat during evoked and self-sustained rhythmic activity of the "peak-wave" type]. AB - Intracellular correlates of evoked rhythmic cortical "spike-and-wave" potentials produced in sensorimotor cortex during 3/s stimulation of the thalamic relay nucleus (VPL) and of self-sustained "spike-and-wave" afterdischarges following 8 14/s stimulation of the same nucleus were studied in acute experiments on cats immobilized by myorelaxants. Intracellular recordings of pyramidal tract neurons revealed that different components of evoked "spike-and-wave" potentials, i. e. the spike-like negative wave and the long lasting negative wave, are postsynaptic in origin: the first is due to EPSPs with spike discharges, and the latter--to IPSPs of cortical neurons. Components of "spike-and-wave" afterdischarge mostly reflect the paroxysmal depolarizing shifts of the membrane potential of cortical neurons. After cessation of sustained "spike-and-wave" activity the long-lasting hyperpolarization accompanied by inhibition of spike discharges and subsequent recovery was observed in cortical neurons. It is presumed that the negative wave of the evoked "spike-and-wave" potential as well as slow negative potentials of direct cortical and primary responses reflect IPSPs of deeper parts of pyramidal tract neurons, while the waves of the sustained "spike-and-wave" afterdischarges are due to paroxysmal depolarizing shifts in cortical neurons. PMID- 3016570 TI - [Phenomenon of asynchrony of induced mediator release at the neuromuscular junction of the frog]. AB - "Hump-like" distortions of evoked end-plate currents were observed with the help of extracellular focal recordings in sartorius and cutaneous frog muscle preparations at 20 degrees C. These distortions resembled spontaneous or unitary evoked responses by their amplitude and time course. Statistical analysis allowed rejecting the simplified assumption that these "humps" were the result of spontaneous nervous signals superimposed on the evoked ones. The asynchronous release of single quanta forming the multiquantal response seemed to be a more plausible explanation. A distinct correlation between the distribution of synaptic delay values of unitary responses (at low quantal content) and observed asynchronous responses (at high quantal content) was found. A polymodal pattern of distribution of synaptic delays was shown in both cases. It is concluded that the presence of asynchronous responses and discrete character of variations in synaptic delays are an intrinsic property of the transmitter release mechanism. PMID- 3016571 TI - [Relation between temperature and processes of spontaneous quantum and non quantum mediator release from motor nerve endings of the mouse]. AB - The spontaneous quantal and nonquantal acetylcholine release was investigated at the temperature range from 10 to 35 degrees C in white mouse semidiaphragm. The quantal release was evaluated by calculation of miniature end-plate potentials frequency, while the nonquantal one--from the H-effect value. The spontaneous quantal release increased exponentially with the temperature growth. The temperature dependence of the nonquantal release showed two relative maxima: at 20 degrees and 35 degrees C. At 10 degrees C the nonquantal release was absent. The value of calculated effective energy of activation of the quantal release was 57.0 kJ/mol in the investigated temperature range. The effective energy of activation for the nonquantal release process in intervals 15-20 degrees C and 25 35 degrees C was 45.5 and 38.2 kJ/mol, relatively. It is suggested that the nonquantal release is rather due to active transport processes than to simple diffusion of acetylcholine molecules. PMID- 3016572 TI - [Modulation of pyramidal responses of the sensomotor cortex after combined stimulation of the lateral hypothalamus and the sensomotor cortex of the rat]. AB - Periodical pyramidal tract (PT) stimulation in freely moving rats has been shown to be accompanied by statistically significant increased functional activity of neuronal populations of the sensorimotor cortex (SMC) manifesting as potentiation of the primary positive phase of the pyramidal-cortical response (PCR). When periodical PT stimulation is combined with the lateral hypothalamus (LH) stimulation of the same periodicity, a statistically significant increase in the magnitude of potentiation of the PCR positive phase compared to that occurring without the LH activation is observed. When the periodical PT stimulation is combined with the LH and SMC stimulation of the same periodicity, the potentiation of the PSR positive phase increases significantly in comparison with the potentiation occurring without the SMC stimulation. PMID- 3016573 TI - [Mechanisms of development of long-periodicity oscillations in activity in nerve nets. Stochastically uniform nerve nets]. AB - The possibility of generation of long-term activity in the stochastic uniform networks consisting of neuron-like threshold elements was analyzed by the mathematical simulation model. It was shown that the long-term (hundreds of milliseconds) self-stopping activity in the networks with a positive feed-back between units can be generated. Termination of the activity was based on the following factors: 1) stochastic properties of network processes which determine the fluctuations of the activity level; 2) neuronal interaction leading either to synchronization of neuronal discharges and respectively to postactivation inhibitory processes, or to reconstruction of the microstructure of the neuronal network activity when weak-connected neurons are predominantly excited. Basing on the data obtained possible participation of the mechanism involved in formation of the long-term self-stopping activity of interneuronal networks of the spinal cord realizing different types of programmed rhythmical activity (generators) is discussed. PMID- 3016574 TI - [Mechanisms of the formation of long-periodicity oscillations in activity in nerve nets. Nets with pre- and postsynaptic inhibition]. AB - The role of presynaptic and postsynaptic processes in formation of the long-term (hundreds of milliseconds) activity of neuronal networks was analyzed by the mathematical simulation model. The long-term activity of networks with presynaptic inhibition was discontinued due to the depolarization of the neuronal terminals that achieved its critical level and to significant suppression of the effectiveness of synaptic interaction. The long-term activity of networks with postsynaptic inhibition was discontinued because of the activation of inhibitory neurons exerting strong hyperpolarizing effects on other neurons of the networks. Synchronization of neuronal discharges was important in achievement of the critical level by terminal depolarization or inhibitory postsynaptic processes that interrupted the network activity. Properties of neuronal networks with presynaptic and postsynaptic inhibition were compared with those of uniform neuronal networks (with a positive feedback between neurons only). It is concluded that introduction of the additional negative feedback circuits in a form of presynaptic or postsynaptic inhibition contributes to improvement of reliability and accuracy of the mechanism which terminates the network activity. PMID- 3016576 TI - Postnatal carcinogenic study of 1,2-dimethylhydrazine dihydrochloride in rats. AB - The outbred SPF rats of the Ripb: Wist stock were applied subcutaneously 1,2 dimethylhydrazine dihydrochloride (DMHD) at a concentration of 0.036% and 0.144% from the first day of age after the birth. In nine experimental groups DMHD was administered for first 5, 10, 15 and 20 days of age after the birth at total doses of 0.36 to 2.16 mg. With the highest applied dose, all animals died during the treatment, further deaths were recorded in groups in which DMHD was administered for first 5 to 10 days. The maximum length of survival was one year. The conclusion of the study was achieved only by the control animals and by 36.6% of males and 13.8% of females of the experimental group. From the 24th week of age, hepatic and renal tumors were detected in rats. In the liver hepatocellular carcinomas and benign hepatomas occurred, and were found in all groups which had been applied DMHD, furthermore cholangiomas, mostly in the groups which had been applied DMHD up to the age of 15 days and cavernomas in the groups which had been applied DMHD up to the age of 20 days. Practically in all rats which had been administered DMHD focal preneoplastic hyperplasia of liver cells was detected. In the kidney always a mixed malignant mesenchymal tumor was found in all animals of the groups treated up to age of 15 days. PMID- 3016575 TI - [Effect of strontium, barium and manganese ions on non-adrenergic inhibitory synaptic potentials and the action of ATP in the smooth muscle of the human intestine]. AB - The ionic mechanism of nonadrenergic inhibition in smooth muscles of human intestine was studied by the sucrose-gap method. The results obtained suggest that calcium-dependent potassium conductance which consists of apamin-sensitive and -insensitive components is involved in the action of nonadrenergic transmitter in smooth muscles of human intestine. PMID- 3016578 TI - The human tumour colony-forming assay using fresh specimens. PMID- 3016577 TI - Relationships of steroid hormone receptors, age and histological characteristics in human breast cancer. AB - A number of 178 human primary mammary carcinomas were evaluated for estradiol receptor (ER) content, patients' age, histologic type, tumor histological grade of differentiation and content of elastosis. For a subset of 78 cases progesterone receptor (PR) was also assayed. ER positivity was significantly elevated in patients over 50 years while PR positivity rate was increased in patients under 50; ER-positive-PR-positive (ER + PR +) cases were evenly distributed among age groups. Correlations of ER and PR with histological features revealed that: a) high levels of ER were found in lobular, cribriform and colloid carcinomas; b) ER+ cases but not PR+ tumors were more frequent in well differentiated tumors; c) ER+, PR+ and ER+ +PR+ tumors were strongly related to the presence of elastosis; d) contrasting with ER-PR+ tumors, ER+ PR+ cases were associated with features indicative of a favorable prognosis. It is concluded that the determination of ER and PR in mammary malignant tumors brings information regarding the evolution of breast cancer in human patients and the biology of malignant mammary cells. PMID- 3016579 TI - Effects of trimethyltin chloride on the LiCl dose-response function for conditioned taste aversions in rats. AB - Twenty-one days following intragastric administration of 6.0 mg/kg of TMT (TMT chloride base/kg) or equivolume distilled water, groups of rats were injected with various doses of LiCl (0, 0.15, 0.60 or 1.8 mEq/kg) following the consumption of a novel saccharin solution. Both groups subsequently avoided the consumption of saccharin, with the degree of the aversion directly related to the dose of LiCl. Further, there was no difference between the TMT- and non-TMT treated rats in the degree of aversion at any dose. These data suggest that the previously-reported effect of TMT on long-delay taste aversion learning was not likely due to changes in sensory responsiveness. PMID- 3016580 TI - Interaction of dimeric and monomeric enkephalins with NG108-15 hybrid cells. A kinetic analysis. AB - The binding of the enkephalin dimer [D-Ala2, Leu5-NH-CH2-]2 (DPE2) is characterized by its high affinity for receptors on NG108-15 hybrid cells, the affinity constant K = 4.7 X 10(9) M-1 is up to 8-fold that of monomers (0.6 to 1.0 X 10(9) M-1), and a maximal binding capacity equal to one half that of the monomers. Kinetic studies showed that DPE2 binds with a 2-fold higher rate, k1 = 6.3 X 10(7) M-1min-1, than monomers (2.4 to 3.8 X 10(7) M-1min-1), and dissociates at a slower rate than monomers. Dissociation of DPE2 was consistently bi- or multiphasic but increased about 12% only after 3 hr of dissociation in the presence of a large excess of unlabeled enkephalin. The dissociation kinetics of monomers varied with enkephalin and experimental conditions used. Consistent with the value for the maximal binding capacity, the kinetic studies are interpreted in support of the hypothesis that DPE2 binds by cross-linking two subunits of one receptor. PMID- 3016582 TI - Idiopathic Parkinson's disease and the Lewy body disorders. AB - A common clinical manifestation of idiopathic Lewy body disease is levodopa responsive idiopathic Parkinson's disease. Infrequently features such as dementia or autonomic failure predominate. The Lewy body is also reported; as an incidental finding in 7-10% of normal individuals mostly over the age of 60 as an incidental sporadic finding in Parkinson's syndrome from other causes, mostly over the age of sixty; in an additional group of degenerative disorders at a younger age, some with familial inheritance. The incidental finding of Lewy bodies can precede clinical Parkinson's disease. It is though they do not occur as an age-related feature, although this cannot be stated with certainty. Current evidence suggests that about 10% of the population may possess the pathological substratum for idiopathic Parkinson's disease. PMID- 3016583 TI - Human fetal adenohypophysis. Histologic and immunocytochemical analysis. AB - One hundred and forty human fetal pituitary glands were removed from fetuses at 7 40 weeks of gestation and studied by light microscopy and immunocytochemistry to localize adenohypophysial hormones. For immunocytology, the avidin-biotin peroxidase complex technique was more sensitive and identified hormones in younger fetuses than did the immunoperoxidase method. Adrenocorticotrophin, beta endorphin, and growth hormone were the first hormones detected; they were identified by intense cytoplasmic immunopositivity at 8 weeks of gestation. Between 10 and 20 weeks, many growth hormone containing cells were large and showed scattered, faint positivity; after 20 weeks, smaller cells with intense positivity predominated. alpha-Subunit of the glycoprotein hormones was identified at 9 weeks of development; beta-subunits of thyroid-stimulating hormone, follicle-stimulating hormone, and luteinizing hormone appeared by 12 weeks. Gonadotrophs differed in numbers related to fetal age and sex. From 15 to 25 weeks, glands of female fetuses contained more gonadotrophs than did those of males; after 25 weeks, there was no significant difference in total gonadotroph numbers. Throughout gestation, adenohypophyses of male fetuses had more luteinizing hormone containing cells than follicle-stimulating hormone containing cells; pituitaries of females had approximately the same numbers of follicle stimulating hormone containing and luteinizing hormone containing cells. Prolactin was identified in few small cells at 12 weeks; at term, prolactin containing cells were numerous, comparable to those seen in the hyperplasia of maternal glands in late gestation and during lactation. This comprehensive study indicates morphologic correlations with pituitary hormone extraction data and with the appearance of the various hormones in the fetal circulation. PMID- 3016581 TI - Differentiation of pre- and post-synaptic high affinity serotonin receptor binding sites using physico-chemical parameters and modifying agents. AB - The two 3H-labeled agonists [3H]8-hydroxy-2-(di-n-propylamino) tetralin ([3H]8-OH DPAT) and [3H]serotonin ([3H]5-HT) have been used to examine the effects of physico-chemical parameters and modulatory agents on the high affinity 5-HT receptor binding sites in various regions of the rat central nervous system. Sites labeled by [3H]8-OH-DPAT and [3H]5-HT were differentially sensitive to changes in incubation temperature and pH, such that the optimal interaction of [3H]8-OH-DPAT with specific sites in the striatum was at 30 degrees C and pH 7.4, whereas [3H]5-HT sites in the same region were most easily labeled at 2-23 degrees C and pH 8.2. Micromolar concentrations of Mn2+ enhanced [3H]5-HT binding but inhibited markedly [3H]8-OH-DPAT binding to striatal membranes. In contrast, both [3H]5-HT and [3H]8-OH-DPAT binding were increased by the cation in hippocampal membranes. Conversely, GTP reduced the binding of either ligand in the hippocampus but affected only [3H]5-HT binding in the striatum. Furthermore, N-ethylmaleimide inhibited equally [3H]5-HT and [3H]8-OH-DPAT binding to hippocampal membranes, but was markedly less potent against [3H]8-OH-DPAT binding to striatal membranes. These results led to the definition of assay conditions for studying separately [3H]8-OH-DPAT binding to "hippocampal-like" (HL) and "striatal-like" (SL) sites. [3H]8-OH-DPAT HL binding sites were particularly abundant in the hippocampus, septum and cerebral cortex, and exhibited pharmacological properties typical of the postsynaptic 5-HT1A subsites previously characterized with [3H]5-HT as the ligand. The regional distribution of [3H]8-OH DPAT SL binding sites was strikingly different from that of HL sites, but similar to that of serotoninergic terminals identified by their capacity to take up [3H]5 HT. The selective lesion by 5,7-dihydroxytryptamine of serotoninergic projections induced a marked loss of [3H]8-OH-DPAT SL binding sites in the striatum and the cerebral cortex, indicating that these sites were located presynaptically. In contrast, [3H]5-HT binding sites remained unchanged in lesioned rats, which confirmed further their exclusive postsynaptic location in brain. PMID- 3016584 TI - A rostral-caudal concentration gradient in cerebrospinal fluid adrenocorticotropin. AB - In two separate experiments, cerebrospinal fluid (CSF) adrenocorticotropin immunoreactive (ACTH-IR) concentrations in the rhesus monkey followed a significant rostral-caudal gradient. In the first study, CSF was sampled from an indwelling catheter in awake animals. The mean ACTH-IR concentration in the cisternal region was 12.3 pg/ml, as compared to 8.56 pg/ml in the lumbar region. In the second study, CSF was sampled in a different group of monkeys by percutaneous puncture at the cisterna magna and at L5-L6. In this study, the mean ACTH-IR concentration in samples collected from the cistern was also greater than the concentration from L5-L6. In addition, a significant correlation within subjects was found between samples collected from the two sites (r = 0.86). These results demonstrate that the site of CSF sampling is a variable in determining CSF ACTH-IR concentrations and suggest that lumbar CSF ACTH-IR concentrations in humans may be interpreted as indexes of ACTH changes at higher levels in the central nervous system. PMID- 3016585 TI - Diurnal variation in neuroendocrine response to stress in rats: plasma ACTH, beta endorphin, beta-LPH, corticosterone, prolactin and pituitary cyclic AMP responses. AB - The effects of restraint stress applied at different times of the day on levels of five stress-responsive plasma hormones (ACTH, beta-endorphin, beta-LPH, corticosterone and prolactin) and pituitary cyclic AMP levels were assessed. Different groups of rats were subjected to 15 min of restraint stress at 2-hour intervals over a 24-hour period. Rats were sacrificed immediately upon removal from their home cage (controls) or immediately following restraint (stressed). The time of day of stress exposure markedly affected the stress responses measured. Generally, responses to stress applied at the beginning of the dark cycle (18:00) were less than those seen following stress applied at the beginning of the light cycle (06:00). Stress at 06:00 increased levels of pituitary cyclic AMP 10-fold, while stress applied at 18:00 did not significantly increase pituitary cyclic AMP levels. In stressed rats, high correlations were seen among levels of hormones derived from the common precursor, proopiomelanocortin (ACTH, beta-endorphin, beta-LPH) and between these hormones and levels of pituitary cyclic AMP. These findings support the hypothesis that pituitary cyclic AMP is involved in the stress-induced release or synthesis of the pituitary hormones ACTH, beta-endorphin, and beta-LPH. PMID- 3016586 TI - Opioid ligand binding sites in the spinal cord of the guinea-pig. AB - The properties of opioid binding sites in membranes from the spinal cord of the guinea-pig were analyzed in experiments employing radiolabeled opioid ligands, selective or partially selective for mu, delta and kappa-type binding sites. Incubation was conducted at 37 degrees C in a quasi-physiological modified Krebs medium, containing sodium and magnesium. The types of binding sites were discriminated on the basis of their affinities for [3H-D-Ala2-MePhe4-Gly5 ol]enkephalin ([3H]DAGO), [3H-D-Ala2-D-Leu5]enkephalin, and [3H]ethylketocyclazocine and the relative potencies of the displacing ligands, DAGO, [D-Ser2-Leu5]enkephalyl-Thr and trans-3,4-dichloro-N-methyl-N-[2-(1 pyrrolidinyl)- cyclohexyl]benzeneacetamide methanesulfonate hydrate (U50488H), which are selective for mu, delta and kappa type binding sites respectively. In membranes from whole spinal cord, kappa type sites comprised about 60%, mu about 30% and delta about 10% of the total of mu, delta and kappa binding sites. Binding sites of the mu type were also found in the lumbo-sacral region of guinea pig spinal cord, in contrast to earlier reports of their absence from this tissue. Morphine showed a better than 500-fold selectivity for mu over kappa sites in spinal cord, while nalbuphine and (-)1-cyclopentyl-5-(1,2,3,4,5,6 hexahydro-8-hydroxy-3,6,11- trimethyl-2,6-methano-3-benzazocin-11-yl)3-pentanone methanesulfonate (WIN 44441-3) showed about a 10-fold selectivity for mu sites. The drug U50488H had about a 150-fold greater affinity for kappa than mu-type binding sites. PMID- 3016587 TI - Interaction between central alpha 1- and alpha 2-adrenoceptors on sympathetic tone in rats. AB - The interaction between piperoxan and alpha 2-agonists on sympathetic tone was studied in rats. The sympatho-inhibitory effect of alpha 2-agonists (clonidine, guanfacine, B-HT 933) was assessed by recording heart rate in normotensive bilaterally-vagotomized rats. Clonidine (3 micrograms/kg, i.c.v.) and B-HT 933 (100 micrograms/kg, i.c.v.) induced a bradycardia which was fully reversed by piperoxan (30 micrograms/kg, i.c.v.). However, in rats treated with guanfacine, piperoxan induced a partial recovery of the bradycardic effect. The injection of a small dose of the specific alpha 1-adrenoceptor blocking drug, AR-C 239 (10 micrograms/kg, i.c.v.) which, by itself did not modify heart rate, completely inhibited the reversal effect of piperoxan in rats treated with clonidine, B-HT 933 or guanfacine. In rat brainstem membranes, B-HT 933 was found to bind to both alpha 1- and alpha 2-adrenoceptors and was as potent as clonidine in competing for alpha 1-sites bound by [3H]prazosin. On the other hand, in bilaterally vagotomized rats, piperoxan (30 micrograms/kg, i.c.v.) induced an increase in blood pressure and heart rate which was inhibited by previous administration of AR-C 239 (10 micrograms/kg, i.c.v.). These data suggest that, by inhibiting central alpha 2-adrenoceptors, piperoxan unmasks central alpha 1-adrenoceptor stimulation by endogenous catecholamines leading to an increase in the sympathetic tone, but a full recovery in heart rate could be observed only with the mixed alpha 1- and alpha 2-adrenoceptor agonists, clonidine and B-HT 933. In addition, these data further indicate that alpha 1-adrenoceptors are implicated in a tonic control of the sympathetic nerve activity in normotensive rats. PMID- 3016588 TI - Evidence for transmission through sympathetic ganglia mediated by muscarinic receptors in anesthetized guinea pigs. AB - 3-Mercaptopropionic acid (3-MP), an inhibitor of the synthesis of GABA, acts in the central nervous system to increase arterial pressure (+50-60 mmHg) in anesthetized guinea pigs, apparently by sympathoadrenal activation. However, blockade of nicotinic receptors in autonomic ganglia with hexamethonium (10 or 20 mg/kg, i.v.) failed to effect any degree of sustained reversal of increases in blood pressure. Infusion of atropine methyl bromide (1 mg/kg, i.v.) likewise was without effect when administered alone, but completely reversed the hypertension induced by 3-MP when given after treatment with hexamethonium. These findings suggest that ganglionic transmission through either the "classical" nicotinic pathway or a muscarinic pathway is sufficient to sustain the sympathetically mediated pressor response elicited by 3-MP. PMID- 3016589 TI - Examination of the relationship between the uptake system for 5-hydroxytryptamine and the high-affinity [3H]imipramine binding site--I. Inhibition by drugs. AB - The relationship between the binding site for imipramine and the uptake system for 5-hydroxytryptamine was examined. This was determined from the interaction between various drugs (including tricyclic antidepressants) and the high affinity accumulation of [3H]5-hydroxytryptamine in cortical synaptosomes from the rat, and with the high affinity binding of [3H]imipramine to cortical membranes of the rat. Imipramine and clomipramine, but not desipramine, were potent inhibitors of both binding of [3H]imipramine and the uptake of [3H]5-hydroxytryptamine. However, ouabain, panuramine and 5-hydroxytryptamine itself, all inhibited the binding of [3H]imipramine only at concentrations greater than those required to inhibit the uptake of [3H]5-hydroxytryptamine. Kinetic analysis revealed that inhibitors of the uptake system for 5-hydroxytryptamine produced inhibition by different mechanisms, but this did not account for their differential potency against uptake and binding. It is concluded that the binding site for [3H]imipramine and the uptake site for 5-HT are not directly linked and that drugs may inhibit the uptake of 5-HT at sites other than the binding site for [3H]imipramine. PMID- 3016590 TI - Proconflict effect of GABA receptor complex antagonists. Reversal by diazepam. AB - The effect of drugs which down-regulate the function of GABA at the level of the GABA/benzodiazepine receptor complex was studied on the conflict test in the rat. The GABA receptor antagonist, bicuculline, and the blockers of the GABA-receptor coupled chloride channel, picrotoxin and pentylenetetrazol, produced a dose dependent proconflict effect. This effect occurred at dose levels which failed to affect unpunished behaviour. The most effective compounds were bicuculline and picrotoxin. The proconflict effect of these drugs was prevented by diazepam but not by the specific benzodiazepine antagonist, Ro15-1788. The data indicate that a diminished GABAergic activity at different subunits of the GABA receptor complex resulted in an enhancement of punishment-suppressed behaviour in rats. PMID- 3016591 TI - Reassessment of opioid binding sites in the rat brain. AB - Opioid binding sites have been characterized pharmacologically in membranes from different areas of the rat brain. Delta, mu and sites belonging to the kappa family (K1, K2, K3) have been detected. Delta sites were more abundant in cortex and striatum, mu sites in striatum and hypothalamus, while kappa binding site concentration was higher in deeper enkephalic structures (brainstem, cerebellum, hypothalamus) and the pituitary gland. A distinct distribution of each subtype of the kappa site was found: kappa 1 sites were higher in the spinal cord, kappa 2 sites in the brainstem and kappa 3 sites in cerebellum. The distribution of delta and kappa sites in the central nervous system was correlated with the distribution of proenkephalin-A derived peptides and precursors, suggesting that these peptides could be their endogenous ligands. PMID- 3016592 TI - Ultrastructural localization of cytochrome oxidase in human embryo brain cells. PMID- 3016593 TI - Improvement of retrieval in a system of conditioned reflexes elaborated in rats placed under the action of ACTH4-7 during ontogeny. PMID- 3016594 TI - Longterm habituation in neurons LPa3 and PPa3 of the snail. PMID- 3016595 TI - Functions of the hypophyseal-adrenocortical system after application of supraphysiological doses of corticosteroids in rabbits. PMID- 3016596 TI - Modification of electrical characteristics of bilayer lipid membranes by intramitochondrial thyroid hormone receptor. PMID- 3016598 TI - Altered autophosphorylation of adenosine 3',5'-phosphate-dependent protein kinase in the dunce memory mutant of Drosophila melanogaster. AB - The phosphorylation-dephosphorylation, in the presence of adenosine 5'-[gamma 32P]triphosphate, of a polypeptide of apparent molecular mass 53,000 has been compared in head homogenates of wild type and memory mutant dunceM11 strains of Drosophila melanogaster. In both strains, labelling of the 53 kilodalton protein required exogenous adenosine 3',5'-phosphate (cAMP), but in dunceM11 cAMP at higher concentration (above approximately 3 microM) caused the rapid disappearance of the label. This differential dephosphorylation can be attributed to the lack of a cAMP-specific phosphodiesterase isoenzyme in the mutant. Several lines of evidence indicate that the 53 kilodalton protein is identical with the regulatory subunit of cAMP-dependent protein kinase. The findings suggest that in the mutant's nerve cells the state of phosphorylation of the regulatory subunit of cAMP-dependent protein kinase is altered, which may contribute to the biochemical disorder leading to the memory deficit. PMID- 3016597 TI - The functional role of adrenocorticotropin in the postnatal ontogenesis of rats. PMID- 3016599 TI - Influence of stimulus length on directional bias of complex cells in cat striate cortex. AB - Visual cortical cells respond optimally to an oriented bar moving either in one unique direction or in directions 180 degrees apart. Length-dependence of this direction selectivity was investigated in the striate cortex of lightly anaesthetized cats. Approaching half of all complex cells showed some lability in their direction selectivity. The incidence was highest in standard and intermediate (length-summating) complex cells, less amongst special complex cells (those with only localized summation) and least amongst end-stopped cells, especially those of the special category. By contrast, direction selectivity of simple cells was length-independent. No correlation between a cell's overt selectivity (i.e. bidirectional, direction-biased or direction-selective) and its lability with bar length or polarity of contrast (light/dark) was evident. Moreover, since individual neurons amongst a population of complex cells could exhibit either increase, decrease, or no change of direction selectivity with length, it is unlikely that length per se can be coded by direction-selectivity. PMID- 3016600 TI - Excitatory neurotransmission within substantia nigra pars reticulata regulates threshold for seizures produced by pilocarpine in rats: effects of intranigral 2 amino-7-phosphonoheptanoate and N-methyl-D-aspartate. AB - Seizures produced by pilocarpine given i.p. to rats provide an animal model for studying the initiation, spread and generalisation of convulsive activity within the forebrain. Pilocarpine, 380 mg/kg, produces a sequence of behavioural and electroencephalographic alterations indicative of motor limbic seizures and status epilepticus, which is followed by widespread damage to the limbic forebrain resembling that occurring subsequent to prolonged intractable seizures. Microinjections of a selective antagonist at the N-methyl-D-aspartate receptor, (+/-)-2-amino-7-phosphonoheptanoate, into the substantia nigra pars reticulata, bilaterally, protects against the behavioural, electrographic and morphological features of seizures produced by pilocarpine, 380 mg/kg, with an ED50 of 0.0007 mumol (0.0004-0.0011). Microinjections of (+/-)-2-amino-7-phosphonoheptanoate, 0.005 or 0.01 mumol, into the substantia nigra pars compacta or into the dorsal part of mid-anterior striatum do not modify the electrographic and morphological sequelae of pilocarpine, 380 mg/kg. In rats pretreated with microinjections of N methyl-D-aspartate into the substantia nigra pars reticulata, a non-convulsive dose of pilocarpine, 100 mg/kg, results in recurrent motor limbic seizures and status epilepticus. The ED50 of N-methyl-D-aspartate for the generation of seizures after pilocarpine, 100 mg/kg, is 0.0014 mumol (0.001-0.0019). Electrographic monitoring shows a pattern and sequence of evolution of convulsant activity within the hippocampus and cortex similar to that produced with pilocarpine, 380 mg/kg, alone. Morphological examination of brains from rats treated with N-methyl-D-aspartate in the substantia nigra pars reticulata and subsequently given pilocarpine, 100 mg/kg, which underwent status epilepticus, reveals widespread damage to the amygdala, thalamus, olfactory cortex, substantia nigra, neocortex, and hippocampus. Microinjections of N-methyl-D-aspartate, 0.002 mumol, into either the substantia nigra pars compacta or dorsal striatum, bilaterally, do not augment seizures produced by pilocarpine, 100 mg/kg. The results indicate that the threshold for pilocarpine-induced seizures in rats is modulated by excitatory amino acid neurotransmission within the substantia nigra pars reticulata. PMID- 3016601 TI - Delayed up-regulation of alpha-adrenoceptor populations in particular regions of the rat brain after stimulation of the nucleus locus coeruleus. AB - We previously showed that electrical stimulation of the nucleus locus coeruleus resulted 4 weeks later in greatly improved performance in the acquisition and extinction of a food-reinforced operant task. To ascertain whether adrenergic receptors of particular brain regions were involved in this long term behavioral modification, we studied the characteristics of alpha 1, and alpha 2 and beta binding sites after stimulation of the locus coeruleus. In the first experiment these characteristics were studied, 4 weeks after treatment, in cortex, hippocampus, hypothalamus and the brainstem. Neither the number, nor the affinity of beta-receptors ([125I]iodocyanopindolol binding sites) was modified in any brain region. A significant increase in the number of alpha 1-receptors ([3H]prazosin binding sites) was observed in the cortex (62%). The number of alpha 2-receptors, ([3H]yohimbine binding sites), was significantly increased in cortex (99%), hippocampus (33%) and hypothalamus (113%). No significant alteration of the alpha 1, alpha 2 and beta-adrenoceptors was observed in the brainstem. To investigate the time course of these adrenoceptor changes, the characteristics of alpha 1, alpha 2 and beta-adrenoceptors were studied 2 weeks after stimulation using the same ligands and in the same brain regions. The only significant modifications observed were an increase of the alpha 2-adrenoceptors in the cortex (19.4%) and in the hypothalamus (54%). Furthermore, in both experiments, the increase in the number of alpha 1 and alpha 2-receptors was associated with a significant decrease in affinity. These results are discussed in relation to our previous behavioral and pharmacological findings. PMID- 3016602 TI - Nerve dependent regulation of neural cell adhesion molecule expression in skeletal muscle. AB - The expression of neural cell adhesion molecule was analysed by indirect immunofluorescence on adult mouse skeletal muscle subjected to a variety of experimental lesions. Adult mouse muscle does not express neural cell adhesion molecule at the sarcolemma. However, following denervation there is a rapid rise in neural cell adhesion molecule levels; this is initially in the cytoplasm of the myofibres but by 18 days there is intense reactivity at the sarcolemma. A nerve crush lesion was used to show that the increase in neural cell adhesion molecule levels following denervation is accompanied by a switch-off of neural cell adhesion molecule expression following reinnervation. Paralysis of skeletal muscle by botulinum toxin injection is sufficient to activate neural cell adhesion molecule expression. As paralysis of skeletal muscle by botulinum toxin is not accompanied by activation of muscle satellite cells or degeneration products of nerve or myelin, it suggests that the observed levels of neural cell adhesion molecule are synthesised by myofibres. As the expression of neural cell adhesion molecule in these lesions parallels the ability of skeletal muscle to accept innervation it is possible that neural cell adhesion molecule acts as a molecular cue at the sarcolemma in regulating synaptogenesis. PMID- 3016603 TI - Childhood encephalomyopathy with cytochrome c oxidase deficiency, ataxia, muscle wasting, and mental impairment. AB - The son of third cousins was normal until age 2 when he had difficulty walking. At age 8 there was limb weakness, ataxia, loss of tendon reflexes, dislalia, and he was mildly retarded. During fasting, urinary organic acid excretion was abnormally high. Cytochrome c oxidase activity in muscle was 7% of the normal mean. The enzyme in platelets was 16% of controls with a decreased cytochrome aa3 peak. These data suggest an autosomal recessive transmission of this variant of cytochrome c oxidase deficiency. PMID- 3016604 TI - Localization and quantification of beta-adrenergic receptors in human brain. AB - Little information is currently available on the localization of noradrenergic systems in the human CNS. We used quantitative autoradiography with [125I] iodopindolol to examine beta-adrenergic receptors in postmortem human brain. The concentration of beta-receptors was highest in all subfields of the hippocampus, followed by cerebellum, and then thalamic nuclei, basal ganglia, midbrain, and cerebral cortex. Low levels were found in white matter and hypothalamus. This distribution differed from the distribution of beta-receptors reported in membrane homogenates of human brain and also from the distribution of beta receptors in rat brain determined by autoradiography. The similarities and differences between the distribution of beta-receptors in the human and rat brains may have implications regarding the role of norepinephrine in the CNS of these two species. PMID- 3016605 TI - Primary amyloidosis with peripheral neuropathy and signs of motor neuron disease. AB - A 51-year-old man had primary amyloidosis, with typical amyloid neuropathy and signs of motor neuron disease, including widespread fasciculation in limb muscles, tongue atrophy and fasciculation, swallowing and chewing difficulty, symmetric hyperreflexia, and bilateral Hoffmann's signs. Fasciculations, fibrillations, and positive sharp waves were found in electromyography of all muscles tested. Motor nerve conduction velocities were moderately slow. Lambda chains were detected in serum and CSF. Amyloid was found in sural nerve biopsy. This combination of amyloid neuropathy and features of amyotrophic lateral sclerosis is related to the motor neuron disease of plasma cell dyscrasias. PMID- 3016607 TI - Present status and future role of surgery for malignant supratentorial gliomas. AB - Despite a voluminous literature it remains difficult to draw conclusions about the precise place of surgery in the management of patients with supratentorial malignant gliomas (anaplastic astrocytoma and glioblastoma multiforme). If the beneficial effects of radiotherapy are discounted, excisional surgery itself probably achieves no more than a survival of 4 to 6 months. The apparently beneficial effects of excisional surgery are related more to radiotherapy than to the surgery itself, beyond the immediate relief of raised intracranial pressure. So far in EORTC Brain Tumour Group studies there is no evidence of any significant beneficial effect from chemotherapy after excisional surgery. Whatever adjuvant methods of therapy are developed in the future, it will remain necessary to separate the effects of surgery from those of the adjuvant therapy. There will therefore be a continuing need for a basic human model whereby the effects of surgery can be clarified, and for that reason it will always be necessary to have a treatment arm without excisional surgery. A format is proposed taking into account favorable prognostic factors such as age and Karnofsky index, which should provide a basis for critical evaluation. The difficulties of adhering to the format are stressed. PMID- 3016608 TI - [Primary mucinous adenocarcinoma of Bahuin's valve]. PMID- 3016606 TI - Contemporary treatment of peripheral nerve and brachial plexus lesions. AB - The paper outlines modern microsurgical techniques utilized in the repair of injured peripheral motor and sensory neurons. The diagnostic evaluation and its timing, which depend on the level and the extent of the lesion, are proposed. The author stresses the need during the operation for close monitoring, which is a prerequisite of proper coaptation of the severed nerve structures. A technically perfect microsurgical repair provides optimal conditions for regeneration of the divided peripheral nerves and/or brachial plexus. The repair of avulsion injuries of the brachial plexus still poses many technical problems; the author proposes the use of intercostal nerves as new sources for grafts. Pain, which is one of the major problems occurring with peripheral nerve injuries, especially with lesions to the brachial plexus, is not dealt with in detail. The author maintains that the contemporary treatment of peripheral nerve injuries as a rule yields good results, while this is not yet true of the management of brachial plexus lesions. PMID- 3016609 TI - [Bran: horizons, virtues and limitations]. PMID- 3016610 TI - [Exogenous estrogens and the urinary tract]. PMID- 3016611 TI - Norepinephrine enhances stimulus-evoked calcium and potassium concentration changes in dentate granule cell layer. AB - Changes in extracellular Ca2+ and K+ concentrations were measured in the dentate gyrus with ion-selective/reference electrodes during high-frequency perforant path stimulation. Bath application of norepinephrine (NE, 50 microM) markedly enhanced both evoked decreases in extracellular calcium and increases in extracellular potassium concentration. These effects of NE were observed in the granule cell layer (stratum granulosum), but not 200 microns away in the dendritic layer (stratum moleculare). The beta-antagonist propranolol (1 microM) completely blocked the NE-induced enhancement of Ca2+ signals in the dentate. In contrast to the dentate, NE did not enhance evoked Ca2+ of K+ concentration changes in the CA1 pyramidal cell layer. These results indicate that NE markedly enhanced both Ca2+ and K+ fluxes, probably by a beta-receptor-mediated mechanism, in the dentate gyrus during high-frequency stimulation of a type able to elicit long-term potentiation (LTP). These increases may underly the action of NE in modulating LTP in the dentate gyrus. PMID- 3016612 TI - Increased neuropeptide Y (NPY) receptor binding in hippocampus and cortex of spontaneous hypertensive (SH) rats compared to normotensive (WKY) rats. AB - Specific 125I-NPY binding in various brain regions of spontaneous hypertensive (SH) rats and age-matched normotensive (WKY) rats was compared. SH rats exhibited significantly greater 125I-NPY binding than WKY rats in the hippocampus (43%) and cortex (18%), but not hypothalamus, midbrain, striatum or pons-medulla. Scatchard analysis indicated that the increased 125I-NPY binding in the hippocampus of SH rats represents a greater number of NPY binding sites. PMID- 3016613 TI - Presynaptic effects of cholecystokinin octapeptide on neuromuscular transmission in the frog. AB - Intracellular recordings were obtained from the frog sartorius muscle end-plate to investigate the effects of cholecystokinin octapeptide (CCK-8) on cholinergic transmission at the neuromuscular junction. A brief bath-application of CCK-8 (1 microM) produced a depression, followed by a long-lasting facilitation, of the amplitude and the quantal content of the end-plate potential (epp). CCK-8 had a biphasic effect, an initial depression followed by an augmentation of the frequency of the miniature epps. CCK-8 did not affect the sensitivity of the nicotinic receptor at the end-plate. These results suggest a significant role for CCK-8 in cholinergic transmission, possibly as a modulator of the evoked release of acetylcholine from motor nerve terminals. PMID- 3016614 TI - Effects of dibutyryl cyclic adenosine monophosphate and dibutyryl cyclic guanosine monophosphate on neuron activity of suprachiasmatic nucleus in rat hypothalamic slice preparation. AB - In order to elucidate the role of cyclic nucleotides in the suprachiasmatic nucleus, we examined the effect of dibutyryl cyclic AMP (c-AMP) and dibutyryl cyclic GMP (c-GMP) on single neuronal activity of the suprachiasmatic nucleus of the rat. A hypothalamic slice about 300 micron thick, including the suprachiasmatic nucleus, was cut coronally using a vibratome. By means of bath- as well as iontophoretic application, c-AMP and c-GMP caused mainly an inhibition and facilitation in suprachiasmatic neuronal activity, respectively. The present results suggest that c-AMP and c-GMP play an important role in regulation of suprachiasmatic neuronal activity as a second messenger and that these two nucleotides may function as a reciprocal messenger. PMID- 3016615 TI - Infection control: keeping current. PMID- 3016616 TI - Approaches to the study of drug interactions in behavioral pharmacology. AB - This paper discusses some of the approaches which have been used for analyzing drug interactions with an emphasis on applications for behavioral pharmacology. Two broad categories of drug interactions are defined: heterergic, when only one of the drugs is active in the behavioral measure employed, and homergic, when both of the interacting drugs have similar action. Two distinct models are presented for homergic drug interactions. The effect-addition model predicts that the combined action of two drugs is equal to the arithmetic sum of the individual effects. This is referred to as effect-additive and deviations from the predicted effects are described accordingly. The dose-addition model takes both dose and effect into account, and thus has a sounder theoretical basis. Leftward shifts in the dose-effect curves are described as equal to (dose-additive), or greater than (supra-additive) or less than (infra-additive) predicted on the basis of the relative potencies of the interacting drugs. Isobolographic methods facilitate data reduction and allow a graphic depiction of dose-addition analysis. A survey of the literature utilizing isobolographic techniques is presented. PMID- 3016617 TI - Constraints imposed on taste physiology by human taste reaction time data. AB - The speed with which an organism responds to stimulus events is reaction time (RT): the minimum time interval between stimulus arrival at a receptor organ, and an overt response by the organism. This time interval specifies maximum duration of all processes necessary for the RT sequence. Responses to any change in taste have RT less than 1 sec for suprathreshold concentrations. Therefore, constituent events at taste receptors, in the central nervous system (CNS), and at the response organ, must have sufficient durations less than 1 sec (Constraint 1). Taste stimulus durations of 50 msec, and therefore taste receptor events of approximately 50 msec, are sufficient for these responses (Constraint 2), as well as for taste quality identification responses (Constraint 3). Taste receptor latencies, neural conduction times, and RT response organ events are even briefer. Thus, 60% to 90% of human taste RT is CNS events. Taste receptor events remain crucial, but CNS processing is important, and apparently time limiting, in all human taste judgments. PMID- 3016618 TI - Mononucleosis-like-syndrome associated with acute AIDS retrovirus infection. AB - A 47-year-old man developed an acute infectious mononucleosis-like illness with a moderate lymphocytosis and numerous atypical lymphocytes in the peripheral blood. Seroconversion to the AIDS-associated virus occurred between 14 and 20 days following the onset of the acute illness. He was found to have reduction of the T4:T8 ratio, low mitogenic response to PHA and cutaneous anergy when tested at 25 and 136 days. These tests had returned to normal by 262 days. PMID- 3016619 TI - Hepatitis A infection in New Zealand children. AB - Antibody to hepatitis A virus (anti HAV), a marker of past infection, was assayed in 2000 sera collected as part of a national survey in 1978-79. The sera were obtained from children and young people aged 0-21 years, resident in all health districts of New Zealand. Anti HAV was detected in 307 sera, giving an overall prevalence of 15.4%. Prevalence increased steadily throughout childhood but more slowly during adolescence. There was no sex differential, the age-standardised rate/100 being 15.5 (95% confidence interval 13.2, 17.8) for males and 16.6 (14.4, 18.9) for females. However, the age-standardised rate for Maoris was 39.5 (33.2, 45.8) compared to 16.1 (12.8, 19.4) for Europeans, giving a risk ratio of 2.1. In addition, a marked north-south gradient in prevalence was demonstrated: the rate for children in the northern half of the North Island, when standardised for age and ethnicity, was 19.8 (16.8, 22.7) compared to 5.2 (3.2, 7.1) for South Island children, giving a risk ratio of 3.8. The higher prevalence of hepatitis A infection in Maori and northern North Island children mirrors our previously reported findings regarding markers of hepatitis B infection in this serum collection. PMID- 3016620 TI - AIDS and blood transfusion. PMID- 3016622 TI - An uncertain leukaemia. PMID- 3016621 TI - Once daily enalapril in general practice patients with mild to moderate essential hypertension. AB - Thirty-nine general practice patients with mild to moderate essential hypertension were treated with enalapril 10 to 40 mg once daily alone or in combination with hydrochlorothiazide 12.5 to 25 mg once daily for 20 weeks. Eighty-one percent of patients responded with a satisfactory reduction in supine diastolic blood pressure, and 58% became normotensive. No serious adverse clinical or laboratory effects were noted. Enalapril alone or in combination with low dose diuretic administered once daily was an effective alternative regimen for mild to moderate hypertension. PMID- 3016623 TI - Luteinizing hormone receptors in human ovarian follicles and corpora lutea during the menstrual cycle. AB - The binding of 125I-labeled human luteinizing hormone (hLH) to the 2000-g fraction of human ovarian follicles and corpora lutea during the entire menstrual cycle was examined. Specific high affinity, low capacity receptors for hLH were demonstrated in the 2000-g fraction of both follicles and corpora lutea. Specific binding of 125I-labeled hLH to follicular tissue increased from the early follicular phase to the ovulatory phase. Specific binding of 125I-labeled hLH to luteal tissue increased from the early luteal phase to the midluteal phase and decreased towards the late luteal phase. The results of the present study indicate that the increase and decrease in receptors for hLH during the menstrual cycle might play an important role in the regulation of the ovarian cycle. PMID- 3016624 TI - Human papillomavirus deoxyribonucleic acid in adenocarcinoma and adenosquamous carcinoma of the uterine cervix. AB - This report describes the detection of human papillomavirus type 16 or 18 deoxyribonucleic acid (DNA) in nine of 15 invasive tumors of the cervix, including three squamous carcinomas, four adenosquamous carcinomas, one glassy cell carcinoma, and one adenocarcinoma. The viral DNA was identified by Southern blotting and DNA hybridization. Human papillomaviruses may play an etiologic role in the development of at least some adenocarcinomas and adenosquamous carcinomas as well as most squamous tumors of the cervix. PMID- 3016625 TI - Human papillomavirus and lower genital neoplasia in renal transplant patients. AB - Lower genital cytopathology was evaluated in 105 immunosuppressed renal transplant recipients. Evidence of human papillomavirus infection was found in 17.5% and of lower genital neoplasia in 9.5%. The rate of the virus infection in the immunosuppressed was nine times greater than in a general population and 17 times greater than in a matched immunocompetent population. The rate of cervical neoplasia was 16 times greater than in a general population and nine times greater than in a matched immunocompetent population. In one-third of patients with human papillomavirus lesions and one-half of patients with neoplastic lesions, multiple lower genital sites were also involved. Of risk factors evaluated, only the number of sexual partners was associated with the development of human papillomavirus/lower genital neoplasia. PMID- 3016626 TI - Role of computed axial tomography of the chest in staging patients with nonmetastatic gestational trophoblastic disease. AB - Thirty-nine women with nonmetastatic gestational trophoblastic disease as determined by conventional staging studies were prospectively evaluated with computed axial tomography (CAT) of the lungs. Sixteen patients (41%) had pulmonary micrometastases detected by CAT, which were not detected by routine chest x-ray. Eight patients (20.5%) had indeterminate scans, and only 15 patients (38%) had negative scans. Eight of 16 patients (50%) with pulmonary micrometastases failed initial therapy with methotrexate-folinic acid rescue while one of eight (12.5%) patients in the indeterminate group and one of 15 (6.7%) patients in the true nonmetastatic group failed initial therapy (P less than .006). All patients who failed methotrexate-folinic acid rescue ultimately achieved prolonged remission with actinomycin D. Time to remission was significantly decreased in patients without evidence of pulmonary micrometastases (P = .03), but the total number of courses of chemotherapy was not significantly different (P = .06). No life-threatening toxicity occurred. Pulmonary micrometastases detected by CAT but not chest x-ray are predictive of an increased risk of methotrexate-folinic acid therapy failure. Computed axial tomography of the lungs identifies a group of patients at high risk for failure of methotrexate-folinic acid rescue, and, therefore, may be indicated for routine staging of patients with otherwise nonmetastatic gestational trophoblastic disease. PMID- 3016627 TI - Performance of sliding knots in monofilament and multifilament suture material. AB - Three different sliding knots were tested using five recently developed monofilament and multifilament suture materials. The resorbable materials were polyglactin-910 (Vicryl), polyglycolic-acid (Dexon-Plus), polyglyconate (Maxon), and polydioxanone (PDS), and the nonresorbable material was polypropylene (Prolene). For each type of sliding knot, three or five throws of suture were tested. Knot strength was determined by the loop holding capacity, which was defined as the strength at which the knot broke, or at which slippage in the knot amounted to more than 2 mm. When the three kinds of sliding knots were compared, identical sliding knots with identical throws around a single suture were found to be the most unreliable. Nonidentical and parallel sliding knots differed little with respect to knot reliability. Five-throw knots were generally stronger than three-throw knots. However, the effect of adding two extra throws to three throw sliding knots was only significant if monofilament suture material was used. Comparison of the different suture materials revealed major differences in knot holding ability. These findings indicate that knot strength is dependent on both the type of knot and the type of suture material, and surgeons should be cognizant of these variables. PMID- 3016628 TI - Virologic studies of HTLV-III/LAV in pregnancy: case report of a woman with AIDS. AB - The number of cases of acquired immunodeficiency syndrome (AIDS) in women is increasing. As of December 30, 1985, 1075 cases in women had been reported to the Centers for Disease Control; 81% of these cases occurred in women of childbearing age (15 to 45 years). The human T-lymphotropic virus type III/lymphadenopathy associated virus (HTLV-III/LAV) can be transmitted from mothers to their infants. Described is a woman with transfusion-acquired AIDS who was six weeks' pregnant at the time Pneumocystis carinii pneumonia was diagnosed. Despite the fact that HTLV III/LAV was isolated from her peripheral lymphocytes throughout pregnancy, transmission of the virus to her infant or husband does not appear to have occurred. PMID- 3016629 TI - Labial adhesions in a postpartum patient. AB - The occurrence of labial adhesions in a patient in the immediate postpartum period is described. The adhesions did not respond to topical estrogen therapy, and sharp separation was required. Factors related to possible etiology as well as management of this condition are discussed. PMID- 3016630 TI - Ovarian sex cord tumor with annular tubules: steroid profile. AB - The pre- and postoperative endocrine profile in a patient with an ovarian sex cord tumor with annular tubules resembles a granulosa cell tumor despite morphologic features of a Sertoli-Leydig cell tumor. Gonadotropin and prolactin levels were compatible with estrogen-progesterone synergy on hypothalamic pituitary function. PMID- 3016631 TI - Mixed germ cell tumor of the ovary with pure choriocarcinoma metastasis. AB - A case report of a 30-month disease-free survival in a ten-year-old girl with ovarian mixed germ cell tumor consisting of dysgerminoma, choriocarcinoma, and immature teratoma with pure choriocarcinoma paraaortic lymph node metastasis is presented. To prevent resistant cell colonies, the noncross-resistant combination of vinblastine-Adriamycin and vinblastine-actinomycin D-cytoxan were added to the initial four courses of vinblastine-cisplatin-bleomycin. There are no previously reported survivals in ovarian mixed germ cell tumors with pure choriocarcinoma metastasis. PMID- 3016632 TI - Safe disinfection of contact lenses after contamination with HTLV-III. AB - The human T-lymphotropic retrovirus type III (HTLV-III), the etiological agent of AIDS, has recently been detected in tears, cornea, and conjunctiva, raising the possibility of transmission of HTLV-III via contact lens trial sets used in routine fitting. We evaluated the ability of several contact lens cleaning solutions, with or without conditioning or disinfecting solutions, to disinfect contact lenses experimentally contaminated with HTLV-III. Following attempted disinfection, the lenses were cultured for residual HTLV-III on Hg cells for 28 days. Cultures without characteristic cytopathic effects, reverse transcriptase activity, and HTLV-III-specific antigen expression were considered negative. We found that all commercially available cleaning solutions tested were able to disinfect contact lenses exposed to HTLV-III. PMID- 3016633 TI - Ameliorating effects of thyroid hormone on rickets induced by a new rachitogenic diet. PMID- 3016634 TI - Management of children hospitalized for laryngotracheobronchitis. AB - During a 12-month period, 527 consecutive admissions for laryngotracheobronchitis (LTB) were reviewed to determine the epidemiology of hospitalized LTB patients and to better define patients who may benefit from therapy other than close observation. Viral cultures were obtained in 442 patients and were positive in 70%. Disease severity at the time of admission was unrelated to patient age or sex. Duration of hospitalization, however, was inversely related to age (p less than 0.001). Laboratory investigations were rarely abnormal or of therapeutic value. Patients who on admission had stridor without sternal and chest wall retractions recovered rapidly and spontaneously; they were frequently discharged within 48 hours and never required artificial airways. Children who had sternal and chest wall retractions on admission experienced longer hospitalizations, frequently received medical intervention such as aqueous mist therapy or racemic epinephrine, and had a 6% risk of requiring artificial airway support. This group of children should be studied selectively for the benefits of specific medical therapies and diagnostic evaluations. PMID- 3016635 TI - Kinetics of triiodothyronine action on Na-K-ATPase in single segments of rabbit nephron. AB - This study was initiated to define the dose- and time-dependence of triiodothyronine (T3) action on Na-K-ATPase in single microdissected nephron segments. For this purpose, the activity and the number of catalytic sites of Na K-ATPase, as determined by the specific binding of 3H-ouabain, were measured following a single injection of T3 to rabbits thyroidectomized since 8-12 days. Triiodothyronine restored both the activity and the number of catalytic sites of Na-K-ATPase in a dose-dependent manner in all nephron segments where the enzyme was decreased following thyroidectomy, i.e., the proximal and the collecting tubule. At a dose of 50 micrograms/kg bw, T3 restored Na-K-ATPase activity and 3H ouabain binding with the same kinetics. However, the kinetics depended on the nephron segments: in the proximal tubule, Na-K-ATPase stimulation occurred after a 12 h period of latency and was completed within 24 h whereas in the collecting tubule, the stimulation was biphasic with a first increase within the first 3 h and a second increase concomitantly to that observed in the proximal tubule. These results indicate that thyroid hormones regulate Na-K-ATPase activity by altering the number of catalytic sites of the enzyme. This control depends on two different mechanisms which differ by their time-dependence. PMID- 3016636 TI - Collagen metabolism of mouse skeletal muscle during the repair of exercise injuries. AB - The activities of prolyl 4-hydroxylase and beta-glucuronidase, the concentration of hydroxyproline as well as reticulin and collagen type III, IV and V stainings were followed in skeletal muscle during a 20-day period after a 9-h treadmill running in untrained and trained male mice, aged 4-6 months. The prolonged 9-h running of untrained mice temporarily increased prolyl 4-hydroxylase activity 2, 5 and 10 days after exercise, more prominently in the red than in the white part of quadriceps femoris-muscle, and in analogical manner as beta-glucuronidase activity in tibialis anterior-muscle. Twenty days after exercise these enzymatic activities were back to the control level. The hydroxyproline content of red muscle was increased for 10 and that of white muscle for 20 days after the exertion. Training for 45 days did not affect hydroxyproline content and prolyl 4 hydroxylase activity was at the control level after the training. A 9-h exercise increased prolyl 4-hydroxylase activity much less in trained muscle than in the untrained muscle and did not affect muscle collagen content. Histological observations showed fiber necrosis 2 days and signs of fiber regeneration 5 days after the exertion in untrained mice. Twenty days afterwards the regeneration was nearly completed. Reticulin staining was increased in injured muscle areas 10-20 days after the exertion. In immunohistochemical staining, antibodies to all studied collagen types (type III, IV and V) showed increased staining 5-20 days after the exertion in the areas of muscle injuries and regeneration.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016637 TI - ATPase activity in macula densa cells of the rabbit kidney. AB - Na-K- and Mg-activated ATPase activities were determined in maculae densae and glomeruli dissected from both superficial and juxtamedullary nephrons of normal rabbits, using an ultramicro method including a cycling reaction. Activities were expressed as Pi generated per macula densa or per glomerulus and normalized for tissue volume. Results indicate that the mean volume of superficial and juxtamedullary macula densa samples was not statistically different, while glomeruli from deep nephrons had sample volumes that were 29% larger than those from superficial nephrons (P less than 0.001). Correcting for volume both superficial and juxtamedullary macula densa samples had an Na-K-ATPase activity of 0.37 +/- 0.21 fmol X h-1 X (micron3)-1 X Mg-ATPase activity in both pools was also similar [0.41 +/- 0.07 and 0.52 +/- 0.1 fmol X h-1 X (micron3)-1]. Na-K ATPase activity in macula densa cells is estimated to be about 1/40th the activity of surrounding cortical thick ascending limb cells. Total glomerular ATPase per unit volume was significantly higher in glomeruli from superficial than from deep nephrons [0.41 +/- 0.04 vs. 0.28 +/- 0.04 fmol X h-1 X (micron3) 1, P less than 0.05]. There was no statistically significant activity of Na-K ATPase in either superficial or deep glomeruli. These results suggest that in contrast to previous reports, the macula densa contains Na-K-ATPase, but at a low level relative to surrounding tubular cells. Further, in normal rabbits, this activity is invariant in superficial and juxtamedullary samples. PMID- 3016638 TI - Cyclic GMP-dependent protein kinase relaxes skinned fibers from guinea pig taenia coli but not from chicken gizzard. AB - The effect of cGMP and cGMP-dependent protein kinase (cG-PK) on contraction and relaxation was studied in skinned smooth muscle fibers from guinea pig taenia coli and chicken gizzard. At a fixed [Ca2+] relaxation was significantly enhanced by activated cG-PK in fibers from guinea pig taenia coli, but not in those from chicken gizzard. The Ca2+-requirement for half maximal tension maintenance was shifted to the right. Relaxation was associated with a decline in phosphorylated myosin light chain-2 from 34% to 25%. Similarly to relaxation activated cG-PK inhibited tension development only in fibers from taenia coli. These results suggest that mammalian and chicken smooth muscle fibers respond differently to cG PK. PMID- 3016639 TI - [Adult T-cell lymphoma/leukemia associated with HTLV-I virus in Martinique: apropos of 2 cases]. AB - Two HTLV-I associated adult T cell leukemia cases were observed in patient from Martinique (French West Indies). These case are similar to the clinical entity, described by Takatsuki in 1977 in Japan and by Catovsky in Caribbean patients, characterized by a lymphadenopathy, skin lesions and visceral involvement, hypercalcemia, an aggressive course, and poor prognosis. The malignant cells with T4 phenotype and often suppressive function, were pleomorphic, mature, with prominent nuclear irregularities. Systematic research of HTLV-I virus or antibodies in patients with this clinical picture, to measure the influence of this virus in T cell lymphoproliferative diseases in France and in French West Indies is required. PMID- 3016640 TI - The human thyroglobulin gene is over 300 kb long and contains introns of up to 64 kb. AB - Thyroglobulin (Tg), the precursor of thyroid hormones, is a 660.000 Da dimeric glycoprotein synthesized exclusively in the thyroid gland. We have cloned the human thyroglobulin gene from cosmid and phage libraries and constructed a complete restriction map. The gene encodes an 8.7 kb mRNA, covers at least 300 kb DNA and contains at least 37 exons separated by large introns of up to 64 kb. A striking difference in structure between the 5' and 3' part of the gene suggests that it is composed of two evolutionarily different regions. The first 30 kb DNA encode 3 kb of the mRNA, yielding an exon:intron ratio of 1:10, whereas the remaining 270 kb encodes 5.7 kb of the mRNA with an exon:intron ratio of 1:47. In thyroid cells, the Tg gene is not rearranged and nuclear RNA homologous with sequences internal to the 64 kb intron is present, suggesting that the Tg gene is transcribed as a 300 kb RNA. PMID- 3016641 TI - Isolation of a deoxycytidylate methyl transferase capable of protecting DNA uniquely against cleavage by endonuclease R.Aqu I (isoschizomer of Ava I). AB - A sequence-specific modification methylase (M.AquI) was isolated and purified from Agmenellum quadruplicatum (Synechococcus PCC 7002). This enzyme uniquely methylates the deoxycytidylate residue in the sequence *CYCGRG indicated by the asterisk. It was shown to protect DNA against cleavage by restriction endonucleases AvaI, SmaI and XhoI, which recognize the sequences CYCGRG, CCCGGG, and CTCGAG, respectively. PMID- 3016642 TI - Floral tissue of Petunia hybrida (V30) expresses only one member of the chalcone synthase multigene family. AB - Twenty independent, petal-specific chalcone synthase (CHS) cDNA clones have been isolated from Petunia hybrida variety Violet 30 (V30). Sequence analysis shows that the largest of these clones contains the entire coding sequence. Using this clone in Southern blot analysis reveals the presence of multiple CHS gene copies in the genome of Petunia hybrida V30. Hybridization and sequence analysis of the CHS cDNA clones shows that they are all copied from a single mRNA species. This indicates the presence of only one transcriptionally active CHS gene in petals. Finally we report the identification, cloning and partial characterization of this gene. PMID- 3016643 TI - Isolation and computer-aided characterization of MmeI, a type II restriction endonuclease from Methylophilus methylotrophus. AB - A Type II restriction endonuclease, MmeI, has been purified from the obligate methylotroph, Methylophilus methylotrophus. The enzyme was shown to have the non palindromic recognition sequence 5'-T C C Pu A C (N)20-3', 3'-A G G Py T G (N)18 5' and to cleave (as indicated) on the 3' side, generating a two nucleotide 3' projection. Determination of the recognition sequence was achieved using two new computer programs; RECOG, which predicts recognition sequences from the pattern of restriction fragments obtained from DNAs of known sequence, and GELSIM, which generates graphical simulations of DNA band patterns obtained by gel electrophoresis of restriction digests of sequenced DNA molecules. PMID- 3016644 TI - Structural organization and nucleotide sequence of mouse c-myb oncogene: activation in ABPL tumors is due to viral integration in an intron which results in the deletion of the 5' coding sequences. AB - Bacteriophage libraries of mouse DNA were screened for sequences homologous to the v-myb oncogene and two overlapping clones containing the v-myb related region were isolated. Restriction enzyme mapping, heteroduplex analysis and nucleotide sequence analysis revealed the presence of nine exons. Six of these exons are homologous to the v-myb region while the other three exons are derived from the 5' region which is deleted in the viral oncogene. The sequences downstream to the sixth v-myb exon are not included in the 17 kbp of DNA sequences analyzed in this study. Comparison of the structure of the normal c-myb clone with its rearranged couterpart present in plasmacytoid lymphosarcomas revealed that the rearrangements occur in this locus as a result of viral integration. Present studies demonstrate that such a viral insertion interrupts the c-myb coding region at a region identical to that observed in the generation of the v-myb gene of avian myeloblastosis virus and results in the synthesis of mRNAs that lack the same 5' coding region. PMID- 3016645 TI - Structural organization of the Amy locus in seven strains of Drosophila melanogaster. AB - Restriction maps were made by Southern blot analysis of the Amy (alpha-amylase) region in 7 strains of D. melanogaster using endonucleases SalI, XhoI and EcoRI. These were compared to the map of lambda Dm65 which contains the cloned Amy region. Strains used produce either two amylase variants, a single variant, or no amylase, yet all 7 strains carry two Amy genes as inverted repeats at the Amy locus. This and the orientation of the repeats resembles the situation in lambda Dm65. Most restriction sites mapped are conserved but two strains contain a large insertion which differs in size and position between strains. A complex anomaly, probably an inversion, exists at the Amy locus in a null strain. Maps for our Amy1,3 strain and the lambda Dm65 clone are identical, the DNA of each having been derived from a Canton-S wild stock. Restriction and genetic maps of the Amy region were aligned and alleles assigned to the proximal and distal genes, Amy-p and Amy-d. PMID- 3016646 TI - Synthesis of two diastereoisomeric p-nitrophenyl phosphodiesters of 2',3' secouridine and their affinity for phosphodiesterases. AB - The synthesis of the p-nitrophenyl esters of the 5'- and 3'-phosphates of the nucleoside analogue 2',3'-secouridine are described. Unlike the corresponding diesters of thymidine, these two compounds are diastereoisomers. Their affinity for phosphodiesterases types I and II were investigated. Both analogues were hydrolysed very slowly by snake venom phosphodiesterase but their affinity for the enzyme was similar to that of the p-nitrophenyl ester of thymidine 5' monophosphate of which they were both competitive inhibitors with Ki approximately Km. Neither compound was hydrolysed by spleen phosphodiesterase but both competitively inhibited the p-nitrophenyl ester of thymidine 3' monophosphate, with Ki's slightly higher than the Km. Although for each enzyme the Ki of the correct analogue phosphodiester (i.e. the 5'-derivative for snake venom and the 3'-derivative for spleen) was the lower, the absolute specificity seen for the normal substrates had been lost. PMID- 3016647 TI - Nucleotide sequence and deduced amino acid sequence of Escherichia coli pyruvate oxidase, a lipid-activated flavoprotein. AB - The entire nucleotide sequence of the poxB (pyruvate oxidase) gene of Escherichia coli K-12 has been determined by the dideoxynucleotide (Sanger) sequencing of fragments of the gene cloned into a phage M13 vector. The gene is 1716 nucleotides in length and has an open reading frame which encodes a protein of Mr 62,018. This open reading frame was shown to encode pyruvate oxidase by alignment of the amino acid sequences deduced for the amino and carboxy termini and several internal segments of the mature protein with sequences obtained by amino acid sequence analysis. The deduced amino acid sequence of the oxidase was not unusually rich in hydrophobic sequences despite the peripheral membrane location and lipid binding properties of the protein. The codon usage of the oxidase gene was typical of a moderately expressed protein. The deduced amino acid sequence shares homology with the large subunits of the acetohydroxy acid synthase isozymes I, II, and III, encoded by the ilvB, ilvG, and ilvI genes of E. coli. PMID- 3016648 TI - The effect of preincubation of HeLa cell nuclei with ATP on the degradation of mononucleosomal DNA by micrococcal nuclease. AB - HeLa cell nuclei with DNA labeled with [3H] thymidine have been preincubated under varying conditions and then incubated with micrococcal nuclease. Aliquots, removed at increasing times, were analyzed for mononucleosomal size DNA and for acid-soluble DNA, the ratios were plotted and a slope was determined. Preincubation with ATP and a regenerating system increased the slope 2 fold. Optimum ATP concentrations were above 0.25 mM. The ATP effect was reversed by novobiocin. No inhibition of the ATP effect was observed with nalidixic acid, coumermycin, oxolinic acid, VM-26, aphidicolin, or 3 amino-benzamide. NAD or cAMP or cGMP had no effect with or without ATP. Other nucleoside triphosphates could substitute to varying degrees for ATP as could ATP analogues. Nuclei from log phase cells showed no ATP effect, but log phase cells, partially depleted of ATP by incubation with deoxyglucose, showed the effect. The effect was lost in nuclei on long-term storage. No evidence was found for differential degradation of core histones, histone H-1 or DNA, and there was no evidence of nucleosome sliding. PMID- 3016650 TI - Nucleotide sequence encoding the membrane protein of the IBV strain 6/82. PMID- 3016651 TI - Nucleotide sequence of a cDNA coding for the small subunit of human calcium dependent protease. PMID- 3016652 TI - Human aldolase B cDNA detects a Pvu II RFLP in healthy individuals. PMID- 3016649 TI - Specific cleavage of kinetoplast minicircle DNA from Leishmania tarentolae by mung bean nuclease and identification of several additional minicircle sequence classes. AB - Multiple sequence classes of kinetoplast minicircle DNA from Leishmania tarentolae were cleaved by mung bean nuclease in the presence of formamide, yielding unit length linear molecules which retained the anomalous electrophoretic mobility in acrylamide characteristic of minicircle DNA. No specific cleavage site sequence common to all minicircle sequence classes was apparent, although the main region of nuclease cleavage was localized approximately 350 bp from the unique SmaI restriction site of the conserved region found in all minicircle sequence classes. Covalent closure of the minicircle substrate was not a requirement for cleavage, as linearized network derived or cloned minicircles were also cleaved by mung bean nuclease at similar locations. The partial sequences of several new minicircle sequence classes released from the network by mung bean nuclease are also reported. PMID- 3016653 TI - RFLPs at the D19S19 locus of human chromosome 19 linked to myotonic dystrophy (DM). PMID- 3016654 TI - A frequent polymorphism of the complement component C4 gene. PMID- 3016655 TI - PvuII polymorphic site upstream to the human ApoCIII gene. PMID- 3016656 TI - A TaqI RFLP in Xq26-Xqter detected by pX45h [HGM8 no. DXS100h]. PMID- 3016657 TI - Gene activation properties of a mouse DNA sequence isolated by expression selection. AB - The MES-1 element was previously isolated from restricted total mouse cellular DNA by "expression selection"--the ability to reactivate expression of a test gene devoid of its 5' enhancer sequences. Mes-1 has been tested in long-term transformation and short-term CAT expression assays. In both assays MES-1 is active independent of orientation and at a distance when placed 5' to the test gene. The element is active with heterologous promoters and functions efficiently in both rat and mouse cells. MES-1 activates expression by increasing transcription from the test gene's own start (cap) site. Thus the expression selection technique can be used for the isolation of DNA sequences with enhancer like properties from total cellular DNA. PMID- 3016658 TI - Isolation of cDNA clones derived from a cellular gene transcriptionally induced by herpes simplex virus. AB - A small number of cellular proteins accumulate to high levels in cells infected with Herpes Simplex Virus (HSV) despite a generalised repression of most host cell bio-synthesis. An antibody to one such protein has been used to screen a lambda gt11 library and for polysome immunoprecipitation in order to isolate cDNA clones derived from the corresponding gene. The cDNA clones have been used in dot blot and nuclear run-off assays to show that HSV, like other DNA tumour viruses can transcriptionally induce a cellular gene. The mechanism of this effect which is dependent on viral protein synthesis and its possible significance in transformation by HSV are discussed. PMID- 3016659 TI - Primary structure of a proteinase inhibitor II gene from potato (Solanum tuberosum). AB - The isolation and characterization of a genomic clone encoding proteinase inhibitor II of potato (Solanum tuberosum) is described. The structure of this gene was determined by sequencing a genomic fragment of about 2 kb containing the entire RNA coding as well as about 900 nucleotides of the 5'-upstream and 250 nucleotides of the 3'-downstream region. The transcription start site was determined by RNase protection experiments. The comparison of the genomic sequence with cDNA sequences reveals the presence of one intron with a length of 117 nucleotides. The genomic clone contains an open reading frame of 462 nucleotides allowing for a protein of 154 amino acids. The proteinase inhibitor II gene displays typical features of eucaryotic genes. The sequence TATAAA is found 26 nucleotides upstream of the transcription initiation site and the sequence CAAAT at position--103. In the 3'-region the sequence AATAA is found 33 nucleotides in front of the poly-A addition site. PMID- 3016660 TI - Tripartite mitochondrial genome of spinach: physical structure, mitochondrial gene mapping, and locations of transposed chloroplast DNA sequences. AB - A complete physical map of the spinach mitochondrial genome has been established. The entire sequence content of 327 kilobase pairs (kb) is postulated to occur as a single circular molecule. Two directly repeated elements of approximately 6 kb, located on this "master chromosome", are proposed to participate in an intragenomic recombination event that reversibly generates two "subgenomic" circles of 93 kb and 234 kb. The positions of protein and ribosomal RNA-encoding genes, determined by heterologous filter hybridizations, are scattered throughout the genome, with duplicate 26S rRNA genes located partially or entirely within the 6 kb repeat elements. Filter hybridizations between spinach mitochondrial DNA and cloned segments of spinach chloroplast DNA reveal at least twelve dispersed regions of inter-organellar sequence homology. PMID- 3016661 TI - Part of the human ribosomal RNA locus stabilizes a plasmid in yeast. AB - Most yeast plasmids--particularly those containing chromosomal replicators (ARS)- are unstable and do not segregate equally to mother and daughter cells unless they contain centromeric sequences. We have screened a fraction of the human genome for sequences that stabilize YRp7, a plasmid containing ARS1. We selected a fraction which we hoped would be enriched in human centromeric sequences--the DNA attached to the nucleoskeleton. We obtained one human sequence that partially stabilized a yeast plasmid and, surprisingly, it contained sequences homologous to those coding for the 3' end of 18s rRNA, the transcribed spacer and 5' end of 28s rRNA. This sequence did not show any ARS activity nor did it increase the copy number of the plasmid and so probably improved partition of the plasmid between mother and daughter cells. It had no homology to yeast centromeres. PMID- 3016663 TI - Penicillin acylase from E. coli: unique gene-protein relation. AB - The nucleotide sequence of the gene (pac) coding for penicillin G acylase from E. coli ATCC 11105 was determined and correlated with the primary structure of the two constituent subunits of this enzyme. The pac gene open reading frame consists of four structural domains: Nucleotide positions 1-78 coding for a signal peptide, positions 79-705 coding for the alpha subunit, positions 706-867 coding for a spacer peptide, and positions 868-2538 coding for the beta subunit. Plasmids were constructed which direct the synthesis of a pac gene product lacking the signal peptide, and the synthesis of the alpha subunit or the beta subunit. The following results were obtained: The two dissimilar subunits are processing products of a single precursor polypeptide; the spacer peptide is removed during processing; the precursor polypeptide lacking the signal sequence is accumulated in the cytoplasm; it is not processed proteolytically in the cytoplasm and it does not display enzyme activity. Processing, therefore, requires translocation through the cytoplasmic membrane; processing follows a distinct sequential pathway in vitro. PMID- 3016662 TI - Mechanism of autonomous control of the Escherichia coli F plasmid: different complexes of the initiator/repressor protein are bound to its operator and to an F plasmid replication origin. AB - E protein, the 29 kd product of the F plasmid repE gene, plays both positive and negative roles in the autoregulation of F replication. We have cloned and expressed the repE gene in an inducible ATG-fusion vector and have detected specific binding of E protein to the repE operator and to four 19-base pair direct repeats (incB) within the F plasmid replication origin ori2. Binding of E protein at the repE operator occurs with higher affinity than at ori2(incB) and gives almost complete protection to at least 30 base pairs, whereas binding of E protein to the direct repeats in the ori2 region shows an alternating pattern of enhanced and reduced sensitivity to DNAase cleavage consistent with a protein induced folding of the DNA. These results provide direct biochemical support for a model of F plasmid replication in which the E protein serves both as an initiator of replication and as an autorepressor of its own synthesis. PMID- 3016664 TI - Site-directed mutagenesis of the binding site for ribosomal protein S8 within 16S ribosomal RNA from Escherichia coli. AB - Twelve specific alterations have been introduced into the binding site for ribosomal protein S8 in Escherichia coli 16S rRNA. Appropriate rDNA segments were first cloned into bacteriophage M13 vectors and subjected to bisulfite and oligonucleotide-directed mutagenesis in vitro. Subsequently, the mutagenized sequences were placed within the rrnB operon of plasmid pNO1301 and the mutant plasmids were used to transform E. coli recipients. The growth rates of cells containing the mutant plasmids were determined and compared with that of cells containing the wild-type plasmid. Only those mutations which occurred at highly conserved positions, or were expected to disrupt the secondary structure of the binding site, increased the doubling time appreciably. The most striking changes in growth rate resulted from mutations that altered a small internal loop within the S8 binding site. This structure is phylogenetically conserved in prokaryotic 16S rRNAs and may play a direct role in S8-16S rRNA recognition and interaction. PMID- 3016665 TI - Cloning and sequencing of Serratia protease gene. AB - The gene encoding an extracellular metalloproteinase from Serratia sp. E-15 has been cloned, and its complete nucleotide sequence determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature protein of the Serratia protease consists of 470 amino acids with a molecular weight of 50,632. The G+C content of the coding region for the mature protein is 58%; this high G+C content is due to a marked preference for G+C bases at the third position of the codons. The gene codes for a short pro-peptide preceding the mature protein. The Serratia protease gene was expressed in Escherichia coli and Serratia marcescens; the former produced the Serratia protease in the cells and the latter in the culture medium. Three zinc ligands and an active site of the Serratia protease were predicted by comparing the structure of the enzyme with those of thermolysin and Bacillus subtilis neutral protease. PMID- 3016666 TI - Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector. AB - Two bacterial antibiotic resistance genes, one coding for the neomycin phosphotransferase (NPT I) from Tn903, and the other coding for the chloramphenicol acetyltransferase from Tn9 were used as plant selectable markers. Both genes were introduced into the Nicotiana tabacum genome in a new plant expression vector, using the direct gene transfer method. The vector pDH51, used in these experiments contains a plant expression unit as a movable cassette, consisting of the strong cauliflower mosaic virus (CaMV) 35S RNA promoter and transcription terminator separated by a polylinker containing several unique restriction sites. PMID- 3016668 TI - Hydrocortisone modulates RA-induced growth inhibition of normal and transformed human embryonic lung fibroblasts. AB - All-trans retinoic acid (10(-5) M) added at seeding reduces the growth rate and saturation density of normal human embryonic lung fibroblasts of two lines (WI-38 and IMR-90) and similarly inhibits growth of SV40-transformed WI-38 cells (VA13A). The growth inhibitory effects of retinoic acid do not show serum dependency, and the viability of treated cells is 95-99% of controls. Old populations of WI-38 cells (cells at high population doubling levels) are more sensitive to the effects of retinoic acid than are young populations (cells at low population doubling levels), and population life span is reduced by continuous exposure to retinoic acid. When retinoic acid is combined with the glucocorticoid hydrocortisone, inhibition of VA13A cell growth is increased, whereas the retinoic acid-induced inhibition of normal cells is decreased. VA13A cells treated with retinoic acid alone, or in combination with hydrocortisone, exhibit a reversion to a more elongated, fibroblast-like appearance. This paper discusses the clinical implications of the relationship between retinoic acid and hydrocortisone. PMID- 3016667 TI - Nucleotide sequence of the intracisternal A-particle genome inserted 5' to the interleukin-3 gene of the leukemia cell line WEHI-3B. AB - The nucleotide sequence of the intracisternal A-particle genome IAP-IL3 is presented. This IAP element was found to have inserted upstream of the promoter of the interleukin-3 gene of the leukemia cell line WEHI-3B. IAP-IL3 is 5095 bp in length, with identical long terminal repeats (LTRs) of 337 bp. The LTRs show many of the conserved sequence elements identified in other retroviruses. Comparison with other available sequences of IAP genomes indicates that IAP-IL3 is a deleted type I element. It carries a deletion covering the 3' end of the putative IAP gag gene and extending into the 5' end of the putative IAP pol gene. IAP-IL3 has extensive sequence homology with an IgE-binding factor cDNA and evidence is presented indicating that it was derived from a member of the mouse IAP sequence family. Comparison between the pol region of IAP-IL3 and other retroviruses suggests that IAP-IL3 is most closely related to type B and type D retroviruses. PMID- 3016669 TI - Low-fat intake with falling fiber intake commensurate with rarity of noninfective bowel diseases in blacks in Soweto, Johannesburg, South Africa. AB - Among urban blacks in Johannesburg, South Africa, a measure of westernization of diet has occurred. Yet, the frequencies of most noninfective bowel diseases not only are low but also appear to have scarcely increased. To assess more adequately the current dietary pattern, a survey was undertaken. Results indicated a habitually low-fat intake, which supplied a mean of 24% of energy but also greatly decreased fiber intake (now about 14 g daily). Possibly, a meaningful rise in the incidence of bowel diseases requires, among other things, simultaneous rise in fat intake; alternatively, there are factors yet unknown in the dietary context of urban blacks (perhaps operating in their bowels) that are inhibiting rises in the diseases mentioned. PMID- 3016670 TI - Association of biogenic silica with disease. AB - Certain plants contain structures consisting of biogenic silica. This form of silica has been implicated as a causative agent in the high esophageal cancer areas of southern Africa, northeast Iran, and northern China. A spicule shape of biogenic silica is known to act as a tumor promoter in the mouse skin model system. The observation that fine biogenic silica fibers are found in leaves of sugarcane may pose a hazard for cane workers; also, the discovery that bracken contains silica fibers could have implications for browsing cattle. This paper reviews and discusses the involvement of biogenic silica in disease. PMID- 3016671 TI - [Changes of the lipid and protein profile in the obese child in diet therapy (with and without added fiber)]. AB - Considering the overcoming importance that cholesterol levels and lipoprotein pattern presents as atherosclerosis risk factor, the Authors have performed lipoprotein analysis in two groups of obese children (weight excess over 50% of the ideal weight). The patients were hospitalized and treated by dietetic therapy and moderate exercise for 15 days. In group 1 the diet was daily supplemented by fibres. In both groups a weight drop as well as reduction of total cholesterol and LDL cholesterol was observed, while serum triglycerides and VLDL cholesterol and HDL cholesterol remain substantially unchanged. PMID- 3016672 TI - [Poland syndrome. Clinical and structural considerations]. AB - The Authors reported 4 cases of Poland Syndrome. In one of these was possible to perform a skin and muscle biopsy. The clinical aspects of the patients, compared with those observed in the literature are reported. The disease is a malformative syndrome, and the histological features of skin and muscle appeared not structurally affected. PMID- 3016674 TI - [Therapy of infantile spasms (West syndrome) with sodium dipropylacetate]. AB - The authors report the results obtained in 42 patients affected by infantile spasms syndrome during treatment with Sodium Dipropylacetic acid. The subjects were divided into two groups according to the aetiology: idiopathic and secondary. In the first group the use of DPK as determined the disappearance of the seizures in 6 cases (40%), reduction of the crises beyond 50% in 7 cases (46.6%), while in 2 subjects (13.3%) the crises persisted. In the secondary group the crises ceased in 3 cases (11.1%), in 17 (62.9%) there were a reduction of the crises beyond 50%, no response to the drug was observed in 7 subjects (25.9%). In 10 patients the anticonvulsant treatment was progressively diminished and was substituted with hormonal treatment. The long term follow up (1-6 years) gives the following results: the seizures persisted in 2 cases (18.18%) among the idiopathic form and in 6 cases (28.57%) among the secondary group. Mental retardation was found in 4 subjects (36.36%) among the idiopathic group and in 12 patients (57.14%) among the secondary group. The authors shortly report the side effect of the hormonal treatment: they prefer the initiation of treatment of I.S. with anticonvulsant drug and suggest to resort to the ACTH when the initial treatment is unsuccessful. PMID- 3016673 TI - [Cortico-adrenal-genital syndrome. Diagnosis, therapy, follow-up]. AB - Recently new advances are achieved in the diagnosis of CAH, by discovering clinical forms different from the classical one: the late-onset and the cryptic CAH. While the diagnosis of classical form mainly depends on dosing 17-OH progesterone levels in all newborns, the detection of non-classical form requires measurement of adrenal steroids in basal conditions and after ACTH stimulation. The Authors recommend a careful follow-up of treated patients by frequent clinical and hormonal evaluations in order to prevent slightly unsuitable therapy that could compromise the achievement of adequate adult height. It is necessary to evaluate not only the daily total amount, but also the pattern of administration and which adrenal preparation is preferable. PMID- 3016675 TI - [Poland syndrome associated with Moebius syndrome]. AB - We present a case of a girl with Poland's Syndrome (absence of pectoral muscle and syndactyly) associated with Moebius Syndrome. More abnormalities, especially visceral, may be associated with it and therefore the clinician must know and identify them as soon as possible. We underline the importance of a precocious correction of the hand's abnormalities in order to avoid changes in the corporal pattern otherwise not correctible later. A retard in surgical treatment will produce a failure like in our patient. PMID- 3016676 TI - [Kaposi's herpetic eruption. Presentation of a clinical case treated with oral acyclovir]. AB - A case of Kaposi herpetic eruption in a one year old atopic child is reported. Oral Acyclovir treatment (100 mg, 5 times daily, for 5 days) was efficacious in arresting the hyperpyrexia, the systemic manifestations and the cutaneous lesions at 48 hours after the start of treatment. No toxicity was observed. Oral Acyclovir seems to be efficacious in primary herpetic infection, particularly in Kaposi herpetic eruption. PMID- 3016677 TI - Interaction of vasoactive intestinal peptide (VIP) with rat lymphoid cells. AB - Receptors for vasoactive intestinal peptide (VIP) have been characterized in rat lymphoid cells. The interaction of [125I] VIP with blood mononuclear cells was rapid, reversible, specific and saturable. At apparent equilibrium, the binding of [125I] VIP was competitively inhibited by native VIP in the 0.01-100 nM range concentration. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a Kd = 0.050 +/- 0.009 nM and a low binding capacity (2.60 +/- 0.28 fmol/10(6) cells), and a low-affinity class with a Kd = 142 +/- 80 nM and a high binding capacity (1966 +/- 330 fmol/10(6) cells). Secretin, glucagon, insulin and somatostatin did not show any effect at a concentration as high as 100 nM. With spleen lymphoid cells, stoichiometric studies were performed. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a Kd = 0.100 +/- 0.033 nM and a low binding capacity (4.60 +/- 1.07 fmol/10(6) cells), and low-affinity class with a Kd = 255 +/- 110 nM and high binding capacity (2915 +/- 1160 fmol/10(6) cells). With thymocytes, no binding was obtained under different conditions. PMID- 3016678 TI - Opiate receptor binding affinities of some D-amino acid substituted beta casomorphin analogs. AB - beta-Casomorphin-(5) and some analogs modified by the introduction of some D amino acids and D-pipecolic acid as well as by C-terminal amidation were tested for their affinities to mu- and delta-binding sites in rat brain membranes. The binding affinities of these compounds are compared with the known activities in the guinea pig ileum (GPI) and mouse vas deferens (MVD) test and their antinociceptive potencies in rats. The substitution of D-proline for proline in position 4 in beta-casomorphin-(5) and beta-casomorphin-(4)amide (morphiceptin) results in derivatives with very high mu-binding affinity and mu-selectivity. These affinities correspond to the respective analgesic potencies. Both binding to mu-receptors and analgesic potency are also enhanced by the introduction of D Phe in position 3. Testing D-Ala2 substituted derivatives with respect to their ability to compete for 3H-naloxone, we observed apparent differences between the pentapeptide amides (biphasic displacement curves) and the tetrapeptide amides (monophasic displacement curves). The substitution of L-Pro2 by D-pipecolic acid yields an analog with preferential delta-receptor affinity in the organ preparations (MVD) but preferential mu-receptor affinity in brain membranes. This finding suggests a possible difference between peripheral and central mu-binding sites. PMID- 3016679 TI - The effect of MSH/ACTH 4-10 on delayed response performance and post-test locomotor activity in rats. AB - Delayed response performance was measured in male, Long-Evans rats 1 hr after IP administration of various doses of MSH/ACTH 4-10 or control in a Hunter delayed reaction apparatus. Additional treatments consisting of naloxone 500 micrograms/kg (IP) and naloxone 500 micrograms/kg in conjunction with MSH/ACTH 4 10 95 micrograms/kg were also administered. Directly after delayed response performance was assessed, gross locomotor activity was determined. MSH/ACTH 4-10, at a dose of 95 micrograms/kg, significantly enhanced retention of a visual stimulus, while MSH/ACTH 4-10, at doses of 195 and 285 micrograms/kg, significantly impaired delayed response performance. Naloxone treatment resulted in significantly impaired delayed response performance when compared to control. However, naloxone plus MSH/ACTH 4-10 treatment failed to produce a significant difference from control in the delayed response performance paradigm. In post test locomotor activity determination, an apparent dose-response existed for MSH/ACTH 4-10 with the two highest doses (190 and 285 micrograms/kg) resulting in significantly increased locomotor activity. The observed delayed response performance data support theories implicating MSH/ACTH peptides in attentional processes involving visual stimuli. The fact that large doses of MSH/ACTH 4-10 disrupt delayed response performance while increasing post-test activity suggest that an optimum level of effect caused by the MSH/ACTH peptide exists in this paradigm. PMID- 3016680 TI - Episodic secretion of ACTH in rats. AB - While the circadian rhythm of pituitary adrenocorticotropin (ACTH) secretion has been well characterized, the ultradian rhythm has been less thoroughly investigated. To study the episodic nature of ACTH secretion, unrestrained, unanesthetized rats were bled continuously through indwelling jugular venous cannulae and blood sampled for up to 75 minutes at one-minute intervals beginning at 1100 hr (n=6) or 1730 hr (n=4). Sporadic low-amplitude micropulses were observed at both times of day. In addition, infrequent "superpulses" were observed in the evening. Analysis of pulse parameters revealed a significant (p less than 0.001) difference in pulse amplitude but no difference in pulse frequency of interpeak interval between morning and evening. As with other episodically secreted hormones, the threshold for pulse identification and the sampling interval were found to influence the observed pulse parameters. PMID- 3016681 TI - The effects of barbiturates upon the hemodynamic responses to intravenous methionine-enkephalin in dogs: modulation by the GABA complex. AB - In conscious animals, the intravenous administration of enkephalins increases heart rate (HR) and mean systemic arterial blood pressure (MAP); however, when given during barbiturate anesthesia, enkephalins reduce HR and MAP. We have investigated the potential role of the gamma-aminobutyric acid (GABA) complex (consisting of chloride-ion channel and binding sites for GABA, benzodiazepine, and barbiturate/picrotoxin) as the site of modulation of enkephalin responses by certain anesthetic agents in our chronically instrumented dog model. In our model, methionine-enkephalin (Met5-ENK) (35 micrograms/kg intravenously) increased HR and MAP, but following induction of general anesthesia with barbiturate (pentobarbital) or of sedation with benzodiazepine (diazepam), Met5 ENK produced vasodepressor responses despite differing levels of consciousness in the treated animals. Subsequent administration of picrotoxin restored pressor responses to Met5-ENK in the barbiturate-treated dogs, but not in those treated with benzodiazepine; picrotoxin did not alter the level of consciousness. Picrotoxin had no effect upon Met5-ENK responses in the conscious state. In contrast, alpha-chloralose, a convulsive anesthetic agent which does not appear to alter GABA complex activity, blunted but did not reverse pressor responses to Met5-ENK, despite causing a level of anesthesia similar to that produced by barbiturate. The observed pressor response to Met5-ENK during alpha-chloralose anesthesia was totally inhibited by naloxone, indicating that this response was still mediated by opiate receptors. Our data are compatible with modulation of enkephalin responses by GABA complex activity. Systemic enkephalins may generate afferent signals which may subsequently undergo GABA complex processing; the state of activation of the GABA complex may then determine whether systemic enkephalin signals are translated as vasopressor or vasodepressor responses. PMID- 3016682 TI - Autoradiographic visualization of CNS receptors for vasoactive intestinal peptide. AB - Receptors for VIP were characterized in the rat CNS. 125I-VIP bound with high affinity to rat brain slices. Binding was time dependent and specific. Pharmacology studies indicated that specific 125I-VIP binding was inhibited with high affinity by VIP and low affinity by secretin and PHI. Using in vitro autoradiographic techniques high grain densities were present in the dentate gyrus, pineal gland, supraoptic and suprachiasmatic nuclei, superficial gray layer of the superior colliculus and the area postrema. Moderate grain densities were present in the olfactory bulb and tubercle, cerebral cortex, nucleus accumbens, caudate putamen, interstitial nucleus of the stria terminalis, paraventricular thalamic nucleus, medial amygdaloid nucleus, subiculum and the medial geniculate nucleus. Grains were absent in the corpus callosum and controls treated with 1 microM unlabeled VIP. The discrete regional distribution of VIP receptors suggest that it may function as an important modulator of neural activity in the CNS. PMID- 3016683 TI - Brain CCK receptors: species differences in regional distribution and selectivity. AB - The binding of 125I-CCK-33 to its receptors prepared from cerebral cortex and cerebellum was studied in four species: mouse, rat, hamster, and guinea pig. Only the guinea pig showed significant binding to membranes from cerebellum and this binding was comparable to that observed for cerebral cortex. In all four species, the order of potency of unlabeled analogs to compete for the binding site was CCK 8 greater than CCK-33 greater than desulfated CCK-8 greater than CCK-4. While the affinity for CCK-8 and CCK-33 was similar in the various species, the relative affinity for desulfated CCK-8 and CCK-4 was less for hamster and guinea pig, indicating species differences in receptor specificity, as well as in regional localization. PMID- 3016686 TI - [Acquired immunodeficiency syndrome (AIDS)]. PMID- 3016684 TI - Eel calcitonin binding site distribution and antinociceptive activity in rats. AB - The distribution of binding site for [125I]-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing [125I]-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain. PMID- 3016685 TI - 125I-FK 33-824: a selective probe for radioautographic labeling of mu opioid receptors in the brain. AB - The selectivity of the Met-enkephalin analog FK 33-824 (FK) for mu opioid receptors has been, over the years, a matter of controversy. We report here pharmacological and radioautographic data demonstrating that at nanomolar concentrations. 125I-FK interacts exclusively with mu sites. (1) Specific binding of 125I-FK to rat striatal membranes is totally inhibited by mu- and/or delta preferring ligands according to monovalent, Michaelian kinetics, with a potency proportional to the affinity of competing drugs for mu receptors. (2) Unlabeled FK competes only at high concentration with the delta-selective ligand 3H-DPLPE and according to the same kinetics as the mu-selective agonist DAGO. (3) 125I-FK generates the same regional radioautographic labeling pattern as 3H-DAGO. We conclude that when used at nanomolar concentrations 125I-FK constitutes a selective probe for the radioautographic detection of mu opioid receptors at both light and electron microscopic levels. PMID- 3016687 TI - [Neurological symptoms in AIDS]. PMID- 3016688 TI - Water imbibition in the rat prostate during castration-induced regression. AB - The present study was conducted to examine changes in water concentration in the ventral prostate of adult rats at different intervals following castration. The prostatic dry weight was obtained by drying the fresh prostate at 70 degrees C for at least 110 hr and the prostatic water content was calculated from its wet and dry weight. Under normal conditions, the prostatic water concentration ranges from 81.9 to 82.7% in uncastrated rats. The prostatic water concentration started to increase at 8 hr postcastration (83.1%) but this increase was not statistically significant. By 16 hr postcastration, the prostatic water concentration (83.8%) became significantly higher than that of the uncastrated animals. In rats of day 1 to day 10 postcastration, the prostate contained significantly more water (84.4%-84.7%) than those in uncastrated animals. By day 21 postcastration, the prostatic water concentration (81.5%) was not significantly different from that of uncastrated rats. Unlike the prostate, the skeletal muscle did not significantly change the water concentration following castration. The 51Cr-EDTA space in the prostate was not significantly different in rats before and after castration. These results indicate that water imbibition in the rat prostate is associated with an active period of prostatic regression and that the change in the 51Cr-EDTA space during prostatic regression is not the major cause of water imbibition in the tissue. Therefore, the present findings suggest that castration-induced water imbibition in the rat prostate is caused by an increase in the intracellular water space. PMID- 3016689 TI - [AIDS: acquired immunodeficiency syndrome]. PMID- 3016690 TI - The effects of vitamin D3 on leg abnormalities in broilers. AB - Variable quantities of vitamin D3 (D3) ranging from 0 to 20,000 IU D3/kg of diet were incorporated in a corn-soybean meal basal diet and fed to male broiler chicks from day-old until 56 days of age. Four experiments were conducted to determine: 1) the requirement of D3 for growth and bone calcification of normal chicks, 2) the requirement of D3 for deficient chicks, and 3) if feeding up to 20,000 IU D3/kg of diet affects bone metabolism or increases the incidence of leg abnormalities. The parameters measured included: body weight, feed consumption, feed conversion, mortality rate, ionic and total serum calcium, kidney calcium, total blood phosphorus, tibial ash, tibial breaking strength, and tibial length. In addition, the type and the incidence of occurrence of skeletal abnormalities were recorded. The data indicate that feeding less than 200 IU D3/kg of diet produced significantly lower body weights, feed consumption and conversion values, serum ionic calcium, total serum calcium, tibial breaking strength, and percentage tibial ash values (P less than .05). For example, rachitic chicks fed 200 IU D3/kg of diet had significantly lower (P less than .05) levels of ionic calcium at 21 days than rachitic chicks fed 300, 400, or 1,500 IU D3/kg of diet. The optimal level of D3 for 0 to 56-day-old male broiler chicks, based on body weight and percentage tibial ash, is 400 IU D3/kg of diet. The vitamin D3 requirement for deficient chicks repleted with D3 appears to be between 300 to 400 IU D3/kg of diet. Feeding 1,500 to 20,000 IU D3/kg of diet does not appear to alter bone metabolism or increase the incidence of leg abnormalities. PMID- 3016691 TI - Utilization of sodium from sodium bicarbonate by broiler chicks. AB - Two experiments were performed with broiler chicks housed in battery brooders to ascertain their response to low levels of dietary sodium, supplied by either sodium chloride or sodium bicarbonate, and to compare the utilization of sodium from the materials. In each experiment, three replicate pens containing seven female day-old chicks were offered each treatment for a 21-day feeding period. Sodium chloride levels of 0, .05, .10, .15, or .20% were a portion of the dietary treatments. Additional groups received the same basal supplemented with .072, .144, or .216% sodium bicarbonate in the first experiment and .072 or .144% during the second, in order to provide sodium levels comparable to those resulting from .05, .10, or .15% sodium chloride. Group body weights, water consumption, feed consumption, and manure moisture served as evaluation criteria. As the level of sodium chloride was increased, body weights, feed and water intake, and manure moisture also increased. In neither experiment were there significant differences between the body weights of birds receiving sodium bicarbonate and those resulting from feeding comparable levels of sodium from sodium chloride. Average daily feed and sodium intake also did not differ statistically between groups receiving comparable sodium from either source. Water intake tended to be somewhat higher for sodium bicarbonate treatments and was significantly so for the .144% sodium bicarbonate treatment of the second experiment. Manure moisture was somewhat variable and did not differ statistically between sources at the same sodium level. It is concluded that the sodium from sodium bicarbonate was utilized in a manner comparable to that from sodium chloride. PMID- 3016692 TI - Efficacy of coarse spray administration of commercial intermediate infectious bursal disease vaccines. AB - The efficacy of two intermediate infectious bursal disease (IBD) vaccines (Clone Vac D-78 and S-706) for immunizing specific pathogen-free (SPF) White Leghorn chickens by coarse spray (CS) against subclinical IBD was compared. Both intermediate IBD vaccines were equally capable of immunizing day-old SPF chickens by CS and were safe as evidenced by the absence of morbidity, mortality, or severe gross and microscopic bursal pathology at 28 days of age. PMID- 3016693 TI - [Characterization of mucosal papillomas and carcinomas]. PMID- 3016694 TI - [Pagetoid skin infiltrates]. PMID- 3016696 TI - [Is the so-called sclerosing angioma of the lung a "pneumocytoma"? Immunohistochemical contribution to its histogenesis]. PMID- 3016697 TI - [Viral chemotherapy in 1986]. PMID- 3016698 TI - [Value of intraoperative ultrasonography and the extemporaneous assay of insulin during surgery of insulinoma]. PMID- 3016695 TI - [Risk of metastases with a peritoneo-caval shunt for malignant ascites]. PMID- 3016699 TI - [Hodgkin's disease in a male homosexual with LAV-positive serology]. PMID- 3016700 TI - [Clinical characteristics of Itsenko-Cushing syndrome with various morphological changes in the adrenal cortex]. AB - The frequency and nature of the main signs of Icenko-Cushing's syndrome were studied in 132 patients divided into 4 groups with relation to morphological changes in the adrenals. It was established that the peculiarities of morphological structure of the adrenals were not reflected in a clinical picture of disease. A more frequent development of some clinical manifestations of Icenko Cushing's syndrome was noted in the group of patients with a significantly higher mean level of cortisol in the blood plasma. The test with 8 mg of dexamethasone for suppression of 17-OCS excreted with urine was not strictly specific for any stage of morphological changes in the adrenal cortex. PMID- 3016702 TI - Tubulin: a factor necessary for the synthesis of both Sendai virus and vesicular stomatitis virus RNAs. AB - Tubulin acts as a positive transcription factor for in vitro RNA synthesis by two different negative-strand viruses: Sendai virus, a paramyxovirus; vesicular stomatitis virus (VSV), a rhabdovirus. A monoclonal antibody directed against beta-tubulin completely inhibited not only mRNA synthesis and RNA replication catalyzed in vitro by extracts of cells infected with either virus but also mRNA synthesis by detergent-disrupted purified virions. The synthesis of both a leader like RNA and the NP mRNA directed by detergent-disrupted purified Sendai virions was shown to be totally dependent on the addition of purified tubulin. The addition of purified tubulin, although not required, also stimulated mRNA synthesis directed by detergent-disrupted VSV virions 2- to 7-fold. Finally, there appears to be an association between tubulin and the L protein of VSV, since both monoclonal and polyclonal anti-tubulin antisera specifically immunoprecipitated not only tubulin but also the L protein of two different VSV serotypes from the soluble protein fraction of infected cells. PMID- 3016701 TI - Expression and site-specific mutagenesis of the poliovirus 3C protease in Escherichia coli. AB - We have engineered a segment of the poliovirus genome (nucleotides 5438-6061) that encodes the 183 amino acid residues of the 3C region and 25 residues of the 3D region of the viral polyprotein into an Escherichia coli expression vector. The 3C region is a virus-specific protease, which, when expressed in E. coli, is shown to be active and autocatalytic. In our system, three poliovirus-specific proteins are produced: a precursor polyprotein (3C-3D), an internal initiation product, and the mature protease (3C). Mutants in the 3C region have been constructed by oligonucleotide-directed mutagenesis and their effect on the proteolytic activity has been assayed by the in vivo production of the mature protease. The mutation of highly conserved residues (cysteine-47 or histidine 161) produced an inactive enzyme, while the mutation of a nonconserved residue (cysteine-153) had a negligible effect on the proteolytic activity. PMID- 3016703 TI - Stimulation of vesicular stomatitis virus in vitro RNA synthesis by microtubule associated proteins. AB - Microtubule-associated proteins purified from bovine brains stimulated the in vitro transcription and replication reactions of vesicular stomatitis virus. The products of these reactions were intact messenger or genome-sized RNA species. A preparation from HeLa cells containing tubulin and microtubule-associated proteins also stimulated vesicular stomatitis virus transcription in vitro. This observation is in accord with previous studies, which suggested that a host cell factor was involved with the function of the vesicular stomatitis virus RNA polymerase, and others that indicated that several animal viruses displayed an association with host cell cytoskeletal elements during their replication cycles. We show evidence in this report of a host cell protein that seems to have a functional role in interacting with the virion polymerase. PMID- 3016704 TI - Analysis of gamma delta resolvase mutants in vitro: evidence for an interaction between serine-10 of resolvase and site I of res. AB - The resolvase encoded by the transposon gamma delta mediates a site-specific recombination between the two copies of gamma delta in a cointegrate molecule to yield the final products of transposition. Several mutants of resolvase that lack recombinational activity have been isolated previously, and one of these, which has a serine-to-leucine change at position 10, we have characterized in vitro. We also have constructed a serine-to-cysteine change at position 10 by in vitro mutagenesis and have analyzed this mutant protein in vitro. We find that the cysteine-10 mutant is defective in recombinational activity but binds to the recombinational site, res, similarly to wild-type, as assayed by gel electrophoresis of the protein-DNA complexes. In contrast, the leucine-10 mutant has a specific defect in complex formation with site I, which contains the recombinational crossover point, although it can bind and provide the ancillary functions of resolvase at sites II and III. It has been shown that a phosphoserine linkage is made to the DNA during recombination; because serine-10 is absolutely conserved amongst the family of homologous site-specific recombination proteins, it is a good candidate for the active-site serine. The properties of these resolvase mutants with substitutions at position 10 are consistent with this hypothesis and suggest that serine-10 is in close contact with the DNA at site I. PMID- 3016705 TI - Erythrocyte homeostasis: antibody-mediated recognition of the senescent state by macrophages. AB - We tested the hypothesis that the accumulation of bound autologous antibody on a "senescent epitope" identifies aged erythrocytes for phagocytic removal by macrophages. Erythrocytes were collected from mice maintained on a hypertransfusion protocol designed to yield cells of defined age. The mouse erythrocytes were assayed for the presence of bound antibody by measuring their susceptibility to ingestion by macrophages from mouse peritoneal exudates and by flow cytofluorometry. Both assays disclosed that only the oldest mouse erythrocytes bore detectable levels of antibody. Flow cytofluorometric analysis revealed that the frequency distribution of IgG isotypes bound to the cells reflected their levels in normal serum. Finally, treatment with trypsin abolished the ability of the macrophages to ingest erythrocytes aged in vivo. These findings support the hypothesis that antibody mediates the clearance of senescent mouse erythrocytes from the circulation and demonstrate that the presence of a trypsin-sensitive recognition structure on macrophages is an essential requirement in this homeostatic process. PMID- 3016706 TI - Epidermal growth factor receptor is increased in multidrug-resistant Chinese hamster and mouse tumor cells. AB - Multidrug-resistant sublines of Chinese hamster lung and mouse tumor cells selected for high levels of resistance to vincristine or actinomycin D have increased numbers of epidermal growth factor (EGF) receptors compared to control cells. Evidence for this increase was found in six of six resistant cell lines with the use of receptor binding or immunoprecipitation techniques. Levels of 125I-labeled EGF binding to intact actinomycin D-resistant cells derived from DC 3F or CLM-7 Chinese hamster lines are increased 3- to 10-fold compared to controls. Scatchard analysis of these data suggests that increased binding is a result of increased receptor number rather than altered affinity of receptor for ligand. Affinity-labeling and immunoprecipitation studies confirmed the finding of increased receptor amount in resistant hamster and mouse cells. Multidrug resistant variants of DC-3F cells overproduce a plasma membrane glycoprotein (gp150-180) with several physicochemical properties that resemble those of EGF receptor. However, electrophoretic transfer blots with a polyclonal antibody to gp150-180 show that EGF receptor and gp150-180 are probably different molecules. Resistant cells described in this report manifest a normalized phenotype compared to transformed, tumorigenic, drug-sensitive parental cells. EGF receptor increase in resistant variants may be associated with this reverse transformation. PMID- 3016707 TI - Human J chain gene: chromosomal localization and associated restriction fragment length polymorphisms. AB - Gene clones encoding the human J chain, the protein that links immunoglobulin monomers, were recently described. Using probes from J chain clones we have now investigated the chromosomal location of this gene by analysis of somatic cell hybrids and by in situ chromosome hybridization. The gene is located on the long arm of chromosome 4 in band q21, the chromosomal band in which a consistent translocation with chromosome 11 has been observed in some acute leukemias. An additional human sequence that cross-hybridizes with some J chain gene probes is located on a different chromosome. Restriction fragment length polymorphisms deriving from length variations in a tandemly repeated region 5' to the J chain gene were detected; these should facilitate the analysis of genetic linkage between this gene and other markers on chromosome 4. PMID- 3016708 TI - Stable transformation of tobacco by electroporation: evidence for plasmid concatenation. AB - Electroporation (electric field-mediated DNA transfer) of tobacco protoplasts in the presence of the linearized plasmid pMON200 has led to the formation of transgenic plants. Defined electric shocks were delivered by capacitive discharges with readily available, low-cost electrical components. This transformation procedure is simple and efficient and may suggest a quick method for determining the appropriate electric fields for new cell systems. An optimal transformation frequency of 2.2 X 10(-4) (based on the number of cells subjected to the shock) was obtained with a single 2000-V/cm, 250-microseconds-duration capacitive discharge. Calli transformed to kanamycin resistance have been regenerated into whole plants. Southern blots of DNA from the transgenic plants demonstrate the integration of the selectable marker gene (neomycin phosphotransferase) at single or multiple genomic sites. In some cases, the plasmid appears to be integrated intact; in others, it is rearranged. The blots also provide evidence of plasmid recircularization and/or the formation of head to-head and head-to-tail concatemers in most of the plants analyzed. Although some plants apparently have multiple integration sites, analysis of progeny obtained by self-fertilization of the transgenic plants indicates that the kanamycin-resistance marker is inherited as a single dominant gene. PMID- 3016710 TI - Differentiation-dependent sensitivity of human B-cell-derived lines to major histocompatibility complex-restricted T-cell cytotoxicity. AB - Sets of Burkitt lymphoma lines and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) derived from the same individuals were compared for sensitivity to cytotoxic T-lymphocyte (CTL) clones. Major histocompatibility complex class I antigen-restricted CTL clones were generated by stimulating the lymphocytes of an EBV-seropositive individual with the autologous LCL. One clone (BK-20) lysed the autologous and allogeneic HLA-A11-expressing LCLs but not mitogen-induced B lymphoblasts. Thus the clone was selectively cytotoxic for LCLs. Allospecific CTL clones directed against the HLA-A11 antigen were generated from an EBV-seronegative individual. One clone (WP-36) was selectively cytotoxic for the appropriate allospecific LCL, whereas another clone (WP-21) lysed also T and B lymphoblasts. None of the four Burkitt lymphoma lines established in parallel with the CTL-sensitive LCLs were lysed. Two of the Burkitt lymphoma lines were EBV-negative, and EBV-positive sublines were derived from these by in vitro infection. One but not the other of the two convertants became sensitive to all three types of CTL clones. The CTL-sensitive converted line had also acquired some LCL characteristics: increased cell size, aggregation, and a shift in several of the B-cell-specific surface markers. The CTL-resistant convertant expressed EBV antigens but showed no phenotypic change. These findings suggest that the cellular phenotype plays a decisive role in the sensitivity of B-cell derived lines to the lytic effect of LCL-selective autologous and allogeneic CTLs. PMID- 3016709 TI - Detection of restriction fragment length polymorphisms at the centromeres of human chromosomes by using chromosome-specific alpha satellite DNA probes: implications for development of centromere-based genetic linkage maps. AB - We describe a general strategy for the detection of high-frequency restriction fragment length polymorphisms in the centromeric regions of human chromosomes by molecular analysis of alpha satellite DNA, a diverse family of tandemly repeated DNA located near the centromeres of all human chromosomes. To illustrate this strategy, cloned alpha satellite repeats isolated from two human chromosomes, 17 and X, have been used under high-stringency conditions that take advantage of the chromosome-specific organization of this divergent repeated DNA family. Multiple high-frequency restriction fragment length polymorphisms are described for the centromeric region of both chromosome 17 and X chromosome. Mendelian inheritance of the variants is demonstrated. The X-linked alpha satellite polymorphisms in particular are highly informative and constitute a virtually unique centromeric DNA marker for each X chromosome examined. Since the strategy we describe is a general one, the alpha satellite family of DNA should provide a rich source of molecular variation in the human genome and should contribute to the development of centromere-based genetic linkage maps of human chromosomes. PMID- 3016711 TI - A deletion in a rat major histocompatibility complex class I gene is linked to the absence of beta 2-microglobulin-containing serum molecules. AB - Class I major histocompatibility antigens are composed of a heavy chain that is noncovalently associated with beta 2-microglobulin (beta 2m). Most class I molecules are membrane bound, but mouse and rat cDNA clones and genes without a functional code for the transmembrane amino acids have been identified. The membrane-associated class I molecules are important in the control of cell mediated cytotoxicity, while the function of the soluble molecules remains unclear. Previous studies have shown that beta 2m circulates in rat serum in three different molecular weight classes. The first is free beta 2m (Mr, 12,000), the second is about Mr 70,000, and the third is roughly Mr 200,000. In an inbred subline of immunodeficient, diabetes-prone BioBreeding rats (BioBreeding/Hagedorn), previous work detected two restriction fragment polymorphisms in class I major histocompatibility complex genes, one of them a gene deletion on a 7-kilobase BamHI fragment and the other on a 2-kilobase BamHI fragment. In these rats we have found that the third serum beta 2m-binding size class is absent. Analysis of F1 and F2 individuals following cross-breeding between BioBreeding/Hagedorn rats and genetically related (nondiabetic) control BioBreeding w-subline rats demonstrated that the large-size serum peak of beta 2m was associated with the presence of the class I restriction fragments. PMID- 3016712 TI - Identification of a partial cDNA clone for the C3d/Epstein-Barr virus receptor of human B lymphocytes: homology with the receptor for fragments C3b and C4b of the third and fourth components of complement. AB - Human complement receptor type 2 (CR2) is the B-lymphocyte receptor both for the C3d fragment of the third component of complement and for the Epstein-Barr virus. Amino acid sequence analysis of tryptic peptides of CR2 revealed a strong degree of homology with the human C3b/C4b receptor, CR1. This homology suggested that CR1 gene sequences could be used to detect the CR2 sequences at conditions of low stringency hybridization. Upon screening a human tonsillar cDNA library with CR1 cDNA sequences, two clones were identified that hybridized at low, but not at high, stringency. Redundant oligonucleotides specific for CR2 sequences were synthesized and used to establish that the two cDNA clones weakly hybridizing with the CR1 cDNA contained CR2 sequences. One of these CR2 cDNA clones hybridized to oligonucleotides derived from two distinct CR2 tryptic peptides, whereas the other, smaller cDNA clone hybridized to oligonucleotides derived from only one of the CR2 peptides. Nucleotide sequence analysis of the CR2 cDNA confirmed that the site of oligonucleotide hybridization was identical to that predicted from the peptide sequence, including flanking sequences not included within the oligonucleotide probes. The CR2-specific cDNA sequences identified a poly(A)+ RNA species of 5 kilobases in RNA extracted from human B cells but did not hybridize to any RNA obtained from the CR2-negative T-cell line HSB-2, thus confirming the appropriate size and tissue-specific distribution for the CR2 mRNA. The striking peptide sequence homology between CR2 and CR1 and the cross hybridization of the CR2 cDNA with the CR1-specific sequences allow the placement of CR2 in a recently defined gene family of C3- and C4-binding proteins consisting of CR1, C4-binding protein, factor H, and now, CR2. PMID- 3016713 TI - Cholera toxin inhibits the T-cell antigen receptor-mediated increases in inositol trisphosphate and cytoplasmic free calcium. AB - The addition of monoclonal antibodies to the antigen receptor complex on the malignant human T-cell line Jurkat generates increases in inositol trisphosphate and in the concentration of cytoplasmic free calcium. Exposure of Jurkat cells to cholera toxin for 3 hr inhibited these receptor-mediated events and led to a selective, partial loss of the antigen receptor complex from the cellular surface. None of the effects of cholera toxin on the antigen receptor complex were mimicked by the B subunit of cholera toxin or by increasing intracellular cAMP levels with either forskolin or 8-bromo cAMP. These results suggest that a cholera toxin substrate can regulate signal transduction by the T-cell antigen receptor. PMID- 3016714 TI - Definitive identification of a member of the Epstein-Barr virus nuclear protein 3 family. AB - Some Epstein-Barr virus (EBV) immune human antisera are known to react with a 142 kDa protein, EBV-encoded nuclear antigen 3 (EBNA3), which, like EBNA1 and EBNA2, is likely to be involved in the establishment of latent infection or growth transformation. We have now constructed gene fusions between Escherichia coli lacZ and an EBV DNA open reading frame (BERF1; BamHI E fragment rightward open reading frame 1), which is transcribed into an mRNA in latently infected cells. Purified hybrid protein from one of these constructs, chosen because of its reactivity with EBNA3-positive human antisera, was used to affinity purify the specific antibody from human antiserum. This specific antibody was used to prove that EBNA3 is encoded, at least in part, by BERF1, and that EBNA3 is in the nucleus of each latently infected cell. In rodent cells, BERF1 encodes a 120- to 130-kDa protein, which translocates to the nucleus and is recognized by EBNA3 positive human antisera. Two other proteins similar in size to EBNA3 are detected in latently infected cells by EBV immune human antisera. Two EBV open reading frames related to BERF1 may encode these proteins. PMID- 3016716 TI - Inhibition of platelet function with synthetic peptides designed to be high affinity antagonists of fibrinogen binding to platelets. AB - We have constructed synthetic peptides modeled on the sequences of (i) Arg-Gly Asp, present in fibrinogen, fibronectin, and von Willebrand factor, and of (ii) the fibrinogen gamma chain (gamma 400-411) His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala Gly-Asp-Val. The concentration of each peptide that inhibits 50% of 125I-labeled fibrinogen binding to thrombin-stimulated platelets (IC50) was then determined. The IC50 for (gamma 400-411) was 48-180 microM at a fibrinogen concentration of 60 micrograms/ml. A substitution of arginine for alanine at position 9 decreased the IC50 to 14.5 microM. Arginine substitutions for all other residues on the amino-terminal side of the peptide Arg9-Gly-Asp-Val resulted in an IC50 of 0.4 0.8 microM, and the IC50 of the peptide Arg13-Gly-Asp-Val was 0.2-0.3 microM. This contrasts with an IC50 of 200 microM for Arg5-Gly-Asp-Val-Arg4 and an IC50 greater than 1 mM for the peptide Arg12. The inhibitory effect resulted primarily in a decreased affinity of fibrinogen binding to platelets, although the number of available binding sites had also decreased. Binding was completely inhibited. At concentrations between 10 and 18 microM, Arg9-Gly-Asp-Val blocked all ADP induced aggregation in citrated platelet-rich plasma. The peptide Tyr-His-His-Lys Arg-Lys-Arg-Lys-Gln-Arg-Gly-Asp-Val was labeled with 125I to quantitate its binding to thrombin-stimulated platelets; at saturation, 59,990 molecules were bound per cell (Kd = 3.8 X 10(-7) M). These modified synthetic peptides bind to platelets with the same affinity as does intact fibrinogen and inhibit platelet function. The increased affinity of these modified peptides is greater than 20 fold that of peptides comprised of only native sequences and is a prerequisite for the potential antithrombotic use of these agents. PMID- 3016715 TI - Characterization of two related Epstein-Barr virus-encoded membrane proteins that are differentially expressed in Burkitt lymphoma and in vitro-transformed cell lines. AB - Two related but differentially expressed potential membrane proteins of Epstein Barr virus are encoded by the same reading frame in the EcoRI D het region of the viral genome. Potential antigenic sites in the amino acid sequence of these proteins were selected by computer-aided prediction of the secondary structure and two oligopeptides corresponding to regions located in different parts of the proteins were synthesized chemically. Rabbit antisera to these peptides were used for immunoprecipitation of the native viral proteins from Epstein-Barr virus positive cell lines from various sources. Both predicted membrane proteins could be precipitated from cell lines that had been transformed in vitro with EBV or from cell lines derived from infectious mononucleosis patients. In cell lines established from Burkitt lymphoma, only the smaller polypeptide, which lacks 138 amino acids from the amino terminus, could be identified. Using the synthetic peptides as antigens in ELISA, we detected elevated antibody titers in sera from patients with infectious mononucleosis and nasopharyngeal carcinoma. PMID- 3016718 TI - Persistent expression of v-mos oncogene in transformed cells that revert to nonmalignancy after prolonged treatment with interferon. AB - BALB/c embryonic fibroblasts productively transformed by Moloney sarcoma virus and cultivated for over 600 generations in the presence of mouse alpha/beta interferon reverted to an apparently normal phenotype and were unable to produce tumors in nude mice. Nevertheless, the presence of an integrated Moloney sarcoma virus genome in the nonmalignant Moloney sarcoma virus-transformed interferon treated cell DNA could be shown by focus formation upon transfection and by hybridization with a v-mos probe. After digestion with various restriction endonucleases, similar hybridization patterns of v-mos sequences were obtained with DNAs from both reverted and transformed cells. However, additional integration sites and at least twice as many copies of the oncogene were found in the nonmalignant Moloney sarcoma virus-transformed interferon-treated cell DNA. Polyadenylylated RNA extracted from reverted and control cells contained two mos specific transcripts. Interestingly, the nonmalignant Moloney sarcoma virus transformed interferon-treated cells produced helper virus, but no detectable mos containing virions, suggesting that a posttranscriptional block in the v-mos gene expression had occurred in these cells. It should be stressed that, after up to 100 additional passages, cells cultured in the absence of interferon maintained their nontumorigenic character in spite of the persistent transcription of the mos oncogene. PMID- 3016719 TI - Mechanism of DNA polymerase I: exonuclease/polymerase activity switch and DNA sequence dependence of pyrophosphorolysis and misincorporation reactions. AB - Mechanistic features of several processes involved in the idling-turnover reaction catalyzed by the large (Klenow) fragment of Escherichia coli DNA polymerase I have been established. The exonuclease----polymerase activity switch involved in the excision/incorporation mode of idling-turnover occurs without an intervening dissociation of the enzyme from its DNA substrate. Comparative studies on the pyrophosphorolysis kinetics of related DNA substrates indicate a significant dependence of the reaction rate upon the DNA sequence within the duplex region upstream of the primer-template junction. Finally, a gel electrophoretic analysis of the products of the idling-turnover reaction has provided direct evidence for an alternative DNA sequence-dependent misincorporation/excision pathway. PMID- 3016717 TI - Genomic diversity of the acquired immunodeficiency syndrome retroviruses is reflected in alteration of its translational products. AB - We have isolated retroviruses from six acquired immunodeficiency syndrome (AIDS) and three lymphadenopathy syndrome (LAS) patients by cocultivation of patients' lymphocytes with phytohemagglutinin-stimulated normal T cells. In an effort to address the extent to which these viruses have identical genetic information or there is divergence in their genomic sequences, we have compared the nine retrovirus isolates by the following criteria: (i) antigenic cross-reactivity by highly specific and sensitive competition radioimmunoassay for the major internal antigen; (ii) restriction site mapping analysis; and (iii) immunoblot analysis and metabolic labeling of viral structural proteins and their analysis by polyacrylamide gel electrophoresis. The data indicate that individual retroviruses have significant restriction site polymorphism in their genome even though they were isolated from patients residing at one geographic location. Furthermore, this polymorphism is reflected in the variation of the apparent size of the gag and env gene-encoded structural proteins. The heterogeneity in AIDS retrovirus-encoded proteins may be due to either substitutions in the primary amino acid sequence of the protein or deletions or additions in the coding regions of proteins. The alterations in viral structural proteins among various AIDS retroviruses could have important implications in antigenic properties and/or pathogenicity in development of the disease, its detection, and ultimately its eradication. PMID- 3016720 TI - Cloning and characterization of a cDNA encoding the rat brain glucose-transporter protein. AB - Antibody raised against the human erythrocyte glucose transporter identified a recombinant lambda gt11 bacteriophage in a cDNA library prepared from immunoselected polysomal RNA from adult rat brain. The cDNA predicts a 492-amino acid protein that demonstrates 97.6% identity to the human hepatoma hexose carrier. The tissue distribution of the transporter mRNA is identical to that of immunologically identifiable protein and transport activity, except in liver in which high levels of transport are associated with little or no transporter mRNA or protein. As assayed by blot-hybridization analysis, mRNA from insulin responsive and nonresponsive tissues are indistinguishable. These data suggest that a genetically unrelated protein is responsible for hexose transport in normal liver. PMID- 3016721 TI - Insulin stimulates the generation from hepatic plasma membranes of modulators derived from an inositol glycolipid. AB - Insulin binding to plasma membrane receptors results in the generation of substances that acutely mimic the actions of the hormone on certain target enzymes. Two such substances, which modulate the activity of the high-affinity cAMP phosphodiesterase (EC 3.1.4.17), have been purified from hepatic plasma membranes. The two have similar properties and activities but can be resolved by ion-exchange chromatography and high-voltage electrophoresis. They exhibit a net negative charge, even at pH 1.9, and an apparent molecular weight of approximately 1400. The generation of these substances from membranes by insulin can be reproduced by addition of a phosphatidylinositol-specific phospholipase C purified from Staphylococcus aureus. This enzyme is known to selectively hydrolyze phosphatidylinositol and release from membranes several proteins that are covalently linked to phosphatidylinositol by a glycan anchor. Both enzyme modulating substances appear to be generated by the phosphodiesterase cleavage of a phosphatidylinositol-containing glycolipid precursor that has been characterized by thin-layer chromatography. Some of the chemical properties of these substances have been examined. They appear to be related complex carbohydrate-phosphate substances containing glucosamine and inositol. These findings suggest that insulin may activate a selective phospholipase activity that hydrolyzes a membrane phospholipid, releasing a carbohydrate-containing molecule that regulates cAMP phosphodiesterase and perhaps other insulin sensitive enzymes. PMID- 3016722 TI - Substitution of a serine residue for proline-87 reduces catalytic activity and increases susceptibility to proteolysis of Escherichia coli adenylate kinase. AB - Amino acid analysis, HPLC separation of trypsin digests, and sequence analysis showed that the thermosensitivity of the adenylate kinase (EC 2.7.4.3) from Escherichia coli K-12 strain CR341 T28 results from substitution of a serine residue for proline-87 in the wild-type enzyme. This mutation is accompanied by decreased affinity for nucleotide substrates and decreased catalysis. Circular dichroism spectroscopy showed a significant change of the secondary structure. This mainly corresponds to a reduction in alpha-helix content (39%) of mutant protein as compared to wild-type adenylate kinase (50%). Altered conformation of thermosensitive adenylate kinase was also manifested by an increase in susceptibility to proteolysis by trypsin. Ap5A and ATP, known to induce important conformational changes in eukaryotic adenylate kinase(s), protected the mutant enzyme against inactivation by trypsin. This seems to indicate that the "loosening" of the three-dimensional structure of E. coli adenylate kinase by proline----serine substitution is largely compensated for when an enzyme X ATP or enzyme X Ap5A complex is formed. PMID- 3016723 TI - Activating mutations of the c-Ha-ras protooncogene in chemically induced hepatomas of the male B6C3 F1 mouse. AB - Activated c-Ha-ras protooncogenes have recently been identified in the DNA of some spontaneous hepatic tumors found in 2-year-old B6C3 F1 mice. Activation of c Ha-ras has now been demonstrated in DNA from well-differentiated hepatomas initiated by a single dose of carcinogen given to male B6C3 F1 mice at 12 days of age. DNA from each of 25 hepatomas, induced by N-hydroxy-2-acetylaminofluorene, vinyl carbamate, or 1'-hydroxy-2',3'-dehydroestragole, containing transforming activity in the NIH 3T3 transfection assay. Southern analysis of NIH 3T3 cells transformed by DNA from 24 of these hepatomas revealed amplified and/or rear ranged restriction fragments homologous to a Ha-ras probe. The other tumor contained an activated Ki-ras gene. Immunoprecipitation and NaDodSO4/PAGE analysis of p21 ras proteins in NIH 3T3 transformants derived from a majority of the hepatomas suggested that the activating mutations were localized in the 61st codon of the c-Ha-ras gene. Creation of a new Xba I restriction site by an AT--- TA transversion at the second position of codon 61 was detected in DNA from primary tumors and NIH 3T3 cells transformed by DNA from 6 of 7 vinyl carbamate- and 5 of 10 1'-hydroxy-2',3'-dehydroestragole-induced hepatomas. Selective oligonucleotide hybridization demonstrated a CG----AT transversion at the first position of the 61st codon in NIH 3T3 transformants derived from 7 of 7 N-hydroxy 2-acetylaminofluorene-induced hepatomas. By the same criterion, an AT----GC transition at the second position of codon 61 was the activating mutation in 1 of 7 vinyl carbamate- and 5 of 10 1'-hydroxy-2',3'-dehydroestragole-induced tumors. Thus, c-Ha-ras activation is apparently an early event in B6C3 F1 mouse hepatocarcinogenesis that results directly from reaction of ultimate chemical carcinogens with this gene in vivo. PMID- 3016724 TI - Inhibin A-subunit cDNAs from porcine ovary and human placenta. AB - Inhibin, a gonadal protein that preferentially suppresses the secretion of pituitary follicle-stimulating hormone, has been isolated from porcine follicular fluid and characterized as a 32-kDa protein composed of 18-kDa and 14-kDa subunits. In the present work, oligonucleotide probes predicted from amino terminal inhibin amino acid sequences have been used to isolate, from a porcine ovarian lambda gt11 cDNA library, clones encoding the 18-kDa subunit, or A chain, of inhibin. DNA sequence analysis showed that the inhibin A chain is initially synthesized as a larger precursor protein and is predicted to be a glycopeptide. Inhibin A-chain mRNA is present specifically in the gonads, and its synthesis can be induced by treatment of the animal with gonadotropins. The porcine probe was used to isolate a human inhibin A-subunit cDNA from a placental cDNA library. The human precursor is highly homologous to its porcine counterpart and is predicted to generate an 18-kDa glycosylated inhibin A subunit. PMID- 3016725 TI - Role for topoisomerases in the release of DNA into the detergent-soluble fraction of eukaryotic cells. AB - Detergent-soluble DNA is the fraction (2-4%) of DNA that is released into the supernate upon mild detergent lysis. It is nonmitochondrial in origin. It labels efficiently with deoxy[3H]ribonucleosides and the labeling is prevented by inhibitors of polymerase alpha and ribonucleotide reductase. In previous publications we have characterized detergent-soluble DNA from splenocytes of immunologically activated mice. In this publication we show that incorporation of [3H]thymidine into detergent-soluble DNA is prevented by pretreatment with novobiocin, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), and teniposide (VM26), three inhibitors of type II topoisomerases. Camptothecin, an inhibitor of type I topoisomerases, also reduces incorporation of [3H]thymidine but only to 50% of control levels. In addition to affecting incorporation of [3H]thymidine, preincubation with the topoisomerase II inhibitors m-AMSA and VM26 alters the amount of DNA recovered in the detergent-soluble fraction. At low concentrations of m-AMSA the amount of detergent-soluble DNA increases somewhat, whereas at higher drug concentrations a marked decrease is observed. Treatment with VM26 results in diminished amounts of DNA being released into the detergent soluble fraction as well. However, maximal inhibition of detergent-soluble DNA release by VM26 requires the presence of camptothecin. Therefore, we suggest that topoisomerases play an important role in making a small part of lymphocyte chromatin detergent labile. Furthermore, these results are consistent with recent studies demonstrating a role for topoisomerases in yeast replication. Thus, the newly synthesized portion of detergent-soluble DNA may arise as DNA replication intermediates not yet stabilized into mature chromatin. PMID- 3016726 TI - Consecutive A X T pairs can adopt a left-handed DNA structure. AB - The capacity of six sequences with different numbers and orientations of A.T pairs flanked by alternating C.G pairs to adopt left-handed structures was evaluated in recombinant plasmids. A series of synthetic oligodeoxynucleotides were cloned into the BamHI site of pRW790, a small plasmid (approximately 2 kilobases) prepared especially for conformational studies of this type. Supercoil relaxation studies by two-dimensional gel electrophoresis on topoisomers of each plasmid revealed the energetics and structures of the left-handed helices. Also, the presence of supercoil-induced altered DNA conformations within the inserts of topoisomer populations of the plasmids was detected by reaction with S1 nuclease followed by restriction mapping of the cleavage sites. We conclude that consecutive T.A base pairs, whether alternating (TATA) or contiguous (TTTT), can adopt a left-handed conformation (presumably Z) when flanked by reasonably short runs of alternating (C-G)n (n = 3-5). Thus, these results substantially broaden the range of DNA sequences that can adopt left-handed Z conformations. PMID- 3016728 TI - Down-regulation of glucocorticoid receptor mRNA by glucocorticoid hormones and recognition by the receptor of a specific binding sequence within a receptor cDNA clone. AB - A cDNA clone for the rat glucocorticoid receptor (GR) was used to study mechanisms of GR mRNA regulation. Treatment of rat hepatoma culture cells with 0.5 microM dexamethasone caused a small, initial increase in the GR mRNA level after 6 hr as well as a 50% to 95% reduction of the GR mRNA level after 24 hr of incubation when studied by RNA blot hybridization. After 72 hr, the initial GR mRNA level was restored. The down-regulation of GR mRNA levels appears to be independent of protein synthesis, since it also was observed in the presence of cycloheximide. However, cycloheximide caused a 4-fold increase in intracellular levels of GR mRNA. Using an immunoprecipitation assay, we could demonstrate that the GR specifically interacts with a GR cDNA clone, which represents a 2.6 kilobase fragment of the 3' nontranslated region of the GR mRNA. Nuclease protection experiments indicate the presence of several internal GR-binding regions in the above fragment. PMID- 3016729 TI - Functional domains of rabbit thrombomodulin. AB - Thrombomodulin isolated from rabbit lung was separated by ion-exchange chromatography on DEAE-cellulose into a retarded (acidic) and a nonretarded (nonacidic) fraction. Both fractions contained the cofactor required for the activation of protein C. In addition, the acidic fraction (but not the nonacidic fraction) prevented the clotting of fibrinogen by thrombin ("direct" anticoagulant activity) and accelerated the inhibition of thrombin by antithrombin (effect corresponding to 2-10 international units of heparin per mg of protein). Both of these activities were readily neutralized by the synthetic polycation Polybrene, which did not appreciably affect protein C activation. They were also eliminated by digestion of thrombomodulin with bacterial heparinase, which, in addition, converted the acidic form of the protein C activation cofactor to a nonacidic form. Similar conversion observed during storage of thrombomodulin was attributed to endogenous proteinase activity. Density-gradient centrifugation of the acidic form of thrombomodulin in CsCl/4M guanidinium chloride failed to separate either of the direct or antithrombin-dependent anticoagulant activities from the protein C activation cofactor, which showed a buoyant density of 1.31-1.34 g/ml. The nonacidic cofactor had a lower density, 1.26-1.28 g/ml. Unreduced thrombomodulin yielded two major fractions of protein C activation cofactor on NaDodSO4/PAGE, with apparent Mr of approximately 68,000 and 57,000, respectively. The larger component contained essentially all of the direct and antithrombin-dependent anticoagulant activities. We propose that these activities as well as the negative charge and the higher buoyant density of the acidic, Mr 68,000 form of thrombomodulin are due to a heparin-like polysaccharide and, further, that this component can be separated from the major portion of the molecule, which contains the protein C activation site, through the action of a proteinase. PMID- 3016727 TI - Isolation and characterization of a human interleukin cDNA clone, homologous to mouse B-cell stimulatory factor 1, that expresses B-cell- and T-cell-stimulating activities. AB - A cDNA sequence coding for a human interleukin has been isolated from a concanavalin A-activated human T-cell cDNA library based on homology with a mouse interleukin cDNA that expresses B-cell stimulatory factor 1 (BSF-1) activity and T-cell- and mast-cell-stimulating activities. The human cDNA contains a single open reading frame encoding a protein of 153 amino acid residues including a putative signal peptide. Amino acid sequences of the mouse and human polypeptides, deduced from their cDNAs, share extensive homology with the exception of about 40 amino acid residues near the middle portion, which share little homology. Supernatant of COS-7 monkey cells transfected with the human cDNA clone stimulated proliferation of human helper T-cell clones and of anti-IgM activated human B cells, two properties of mouse BSF-1 on mouse cells. These results indicate that this human cDNA clone encodes a protein structurally and functionally homologous to mouse BSF-1. PMID- 3016730 TI - A large region (approximately equal to 95 kDa) of human factor VIII is dispensable for in vitro procoagulant activity. AB - Factor VIII (antihemophilic factor) is a high molecular weight plasma glycoprotein that participates in the blood clotting cascade. The recent cloning and sequence analysis of the cDNA encoding human factor VIII revealed an obvious domain structure for the protein, which can be represented as A1-A2-B-A3-C1-C2. We now report the DNA sequence analysis of porcine exons encoding the entire B domain and part of the A2 and A3 domains. We found an unusually high degree of porcine-human amino acid sequence divergence in the B region compared with the limited sequence available for other regions of the porcine factor VIII molecule. In addition to sequence divergence, there are numerous gaps in the porcine B domain totalling over 200 amino acids. Recombinant DNA techniques were used to effect the removal of large segments of DNA encoding the B domain from the full length human factor VIII cDNA. These constructs directed the synthesis of biologically active factor VIII when introduced into mammalian cells despite the deletion of up to 38% of the factor VIII molecule. PMID- 3016731 TI - Gene encoding the sigma 37 species of RNA polymerase sigma factor from Bacillus subtilis. AB - sigma 37 is a minor species of RNA polymerase sigma factor found in the Gram positive bacterium Bacillus subtilis. sigma 37 governs the transcription in vitro of genes that are turned on at an early stage in spore formation, as well as other genes that are switched on at the end of the exponential phase of growth but that are not under sporulation control. To study the role of sigma 37 in B. subtilis gene expression, we have cloned the gene for this minor species of sigma factor in Escherichia coli by using as a hybridization probe a synthetic oligonucleotide that was designed on the basis of the NH2-terminal amino acid sequence of sigma 37 protein. We determined the nucleotide sequence of the entire sigma 37 gene, which was found to encode a 262-amino acid residue polypeptide of 29.9 kDa. The predicted amino acid sequence of sigma 37 showed significant homology to that of other sigma proteins in a region that has been proposed to be the site of binding of these factors to core RNA polymerase. Genetic mapping experiments placed the gene for sigma 37, herein designated sigB, at 40 degrees on the genetic map of Piggot and Hoch [Piggot, P. & Hoch, J. A. (1985) Microbiol. Rev. 49, 158-179]. An insertion mutation was constructed in sigB and found not to impair growth or sporulation. PMID- 3016733 TI - Isolation of point mutations in bacteriophage Mu attachment regions cloned in a lambda::mini-Mu phage. AB - Twenty-one derivatives of a lambda::mini-Mu phage containing point mutations in the Mu attachment regions were isolated after mutD mutagenesis and selection for relief from Mu-specific replicative interference of lambda growth. DNA sequence analysis revealed that the single left-end mutant had suffered a T----C transition at position 1 of the Mu sequence, while the remaining 20 right-end mutants contained single base-pair insertions or deletions within the terminal 19 base pairs. A genetic assay showed that the right-end mutations revealed by sequencing were necessary for relief of the replicative inhibition of lambda growth. The properties of these mutants suggest that the terminal 2-base-pair and subterminal 8-base-pair inverted repeats are important for Mu-specific replicative transposition. PMID- 3016734 TI - Early postimplantation embryo lethality due to DNA rearrangements in a transgenic mouse strain. AB - Insertional mutagenesis in a transgenic mouse strain (HUGH/3) was caused by integration of plasmid DNA containing the human growth hormone gene and pBR322 plasmid sequences. From this study, which includes another instance of mutagenesis, and from other reports, it is apparent that insertional mutagenesis occurs fairly frequently during DNA integration in the mouse egg and that it is not specific for the exogenous DNA employed. The mutation in HUGH/3 is recessive and results in death of embryos homozygous for the donor sequences shortly after implantation, at the egg cylinder stage on days 4-5 of gestation. Restriction mapping of the insert and of the flanking DNA regions indicates that integration must have involved a series of complex events. Approximately five copies of plasmid sequences are arrayed in tandem but are interrupted at least twice by mouse cellular sequences. In addition, the mouse flanking DNA shows extensive rearrangements, probably including a deletion of at least 10 kilobases. The rearrangements may reflect an initially unstable DNA structure followed by attainment of a more stable conformation. PMID- 3016732 TI - Interpretation of dose-response curves for luteinizing hormone release by gonadotropin-releasing hormone, related peptides, and leukotriene C4 according to a hormone/receptor/effector model. AB - The dose-response curves for pituitary luteinizing hormone (LH) release in response to gonadotropin-releasing hormone and its agonists are unusually broad. It appears, however, that these ligands bind to a single class of receptors. It is shown that these dose-response data can be explained by either of two models in which ligand-receptor complexes stimulate LH secretion by interacting with either of two different effector systems or by interacting with a single effector system but forming monomeric and dimeric active effector complexes. A combination of these two basic models can account for the very broad, biphasic dose-response curve reported for LH release in response to leukotriene C4. PMID- 3016735 TI - Structure and expression of a tandem duplication of the Drosophila metallothionein gene. AB - A strain of cadmium-resistant Drosophila containing a chromosomal duplication of the metallothionein gene was isolated. This duplication is stably inherited in the absence of selective pressure, and larvae homozygous for it can produce approximately twice as much metallothionein RNA as wild-type larvae. The entire duplication was cloned within a 5.7-kilobase fragment; this fragment contained a direct, tandem repeat of 2.2 kilobases of DNA: 228 bases of 5' flanking DNA, the entire transcription unit, and 1.4 kilobases of 3' flanking sequences. The 3' region of the first repeated unit is joined to the 5' region of the second unit by a 6-base-pair segment we define as the novel joint. This joint forms part of a 10-base-pair inverted repeat of a segment within the 3' region of the first unit. Comparison of the sequences of the 5' and 3' boundaries revealed no extensive regions of similarity at a position corresponding to the novel joint, thus suggesting that a mechanism other than homologous recombination was involved in the origin of this duplication. PMID- 3016736 TI - Genes for the eight ribosomal proteins are clustered on the chloroplast genome of tobacco (Nicotiana tabacum): similarity to the S10 and spc operons of Escherichia coli. AB - The nucleotide sequence of a tobacco (Nicotiana tabacum) chloroplast gene cluster that encodes eight proteins homologous to Escherichia coli ribosomal proteins L23, L2, S19, L22, S3, L16, L14, and S8 has been determined. RNA gel blot hybridization revealed that all eight coding regions are expressed in the chloroplasts. The arrangement of the eight genes resembles that found in the E. coli S10 and spc operons. Among the eight genes, the L2 and L16 genes contain 666 and 1020-base-pair introns, respectively. These intron boundary sequences are consistent with the conserved boundary sequences of the chloroplast group III introns [Shinozaki, K., Deno, H., Sugita, M., Kuramitsu, S. & Sugiura, M. (1986) Mol. Gen. Genet. 202, 1-5]. PMID- 3016737 TI - Lethal osteogenesis imperfecta resulting from a single nucleotide change in one human pro alpha 1(I) collagen allele. AB - We have characterized a mutation in a pro alpha 1(I) procollagen gene (COL1A1) that results in lethal (type II) osteogenesis imperfecta. The mutation is a single base change that results in a cysteine-for-glycine substitution at position 988 of the triple-helical portion of half of the alpha 1(I) chains of type I collagen. The mutation thus disrupts the (Gly-Xaa-Yaa)n pattern necessary for triple-helix formation, where Xaa and Yaa are other amino acids. These experiments establish the minimal mutation in a type I collagen gene capable of producing lethal disease, and the lethality demonstrates a selective mechanism for the stringent maintenance of the collagen gene structure. PMID- 3016738 TI - Mutagenesis of the Ha-ras oncogene in mouse skin tumors induced by polycyclic aromatic hydrocarbons. AB - The importance of mutational activation of the Ha-ras protooncogene in polycyclic aromatic hydrocarbon-induced mouse skin tumors was investigated in a complete carcinogenesis model using repetitive applications of 7,12 dimethylbenz[a]anthracene (DMBA), or in an initiation-promotion model using a single application of dibenz[c,h]acridine (DB[c,h]ACR) or benzo[a]pyrene (B[a]BP) followed by chronic treatment with phorbol 12-myristate 13-acetate. DNA isolated from carcinomas induced by DMBA or DB[c,h]ACR, but not by B[a]P, efficiently transformed NIH 3T3 cells, and a high percentage of the transformed foci had an amplified Ha-ras gene. Restriction enzyme Southern blot analysis and DNA sequencing revealed that the amplified Ha-ras genes of the transformants had an A ---T transversion in the second position of the 61st codon. The same mutation was also detected in primary tumor DNA in a high percentage of the DMBA- or DB[c,h]ACR-induced carcinomas. Identification of the mutation in NIH 3T3 cells transformed with DNA from DB[c,h]ACR-induced benign skin papillomas suggests that it is an early event in skin carcinogenesis. Thus, mutation of the 61st codon of the Ha-ras-1 gene appears to be a critical step in the formation of mouse skin tumors induced in both of the two models tested. Our analyses also delineate two other classes of hydrocarbon-induced carcinomas--namely, tumors whose DNAs efficiently transform 3T3 cells but do not contain mutated ras genes and tumors whose DNAs do not transform 3T3 cells. PMID- 3016739 TI - Differences in sequences encoding the carboxyl-terminal domain of the epidermal growth factor receptor correlate with differences in the disease potential of viral erbB genes. AB - Eleven recently isolated erbB-transducing viruses as well as avian erythroblastosis virus (AEV)-R (ES4) and AEV-H have been characterized for the type of disease they cause, their ability to transform fibroblasts in culture, their ability to cause disease in pedigrees of chicken that differ in susceptibility to erbB-induced erythroblastosis, and the structure of their erbB genes. Differences in each of the biological parameters correlated with differences in erbB sequences encoding the C-terminal domain of the epidermal growth factor receptor (EGFR). Seven viruses were strain restricted in their ability to induce erythroblastosis and did not transform fibroblasts. These seven viruses contained v-erbB genes encoding the complete C terminus of the EGFR. AEV R and AEV-H were not pedigree restricted in their ability to induce erythroblastosis and could transform fibroblasts. These viruses contain v-erbB genes that lack codons for the immediate C terminus of the EGFR. Three viruses caused angiosarcoma and one caused fibrosarcoma. The angiosarcoma and fibrosarcoma-inducing viruses were not strain restricted and did not cause erythroblastosis. The v-erbB genes of each of these viruses contained extensive internal deletions or 3' truncations in sequences encoding the C-terminal domain of the EGFR. PMID- 3016740 TI - Introduction of plasmid DNA into the trypanosomatid protozoan Crithidia fasciculata. AB - Crithidia fasciculata cells were treated with a plasmid (pDK96) containing pBR322 sequences, a Leishmania tarentolae maxicircle autonomously replicating sequence, and the bacterial gene for aminoglycoside 3' phosphotransferase I inserted between the yeast alcohol dehydrogenase 1 promotor and terminator sequences. Resistant colonies were selected on agar plates containing paromomycin and screened for vector DNA by hybridization. Approximately 1% of the resistant colonies contained detectable vector DNA, which was present as extrachromosomal closed circular molecules ranging in copy number from 1 to 160 per cell. The plasmids could be recovered from Escherichia coli transformed to ampicillin resistance with Crithidia total cell DNA. Most of the recovered plasmids were a deleted product of pDK96, which lacked the maxicircle autonomously replicating sequence and contained a unique fragment of Crithidia nuclear DNA present at a low copy number in the wild-type genome. The plasmid DNA in resistant Crithidia was unstable even under selective conditions and was lost within 30 cell divisions. PMID- 3016741 TI - Homologous genes encode two distinct histidine-rich proteins in a cloned isolate of Plasmodium falciparum. AB - Two genes encoding distinct histidine-rich proteins in a Plasmodium falciparum clone exhibit high levels of homology, suggesting they have originated by duplication and divergence from a common ancestral sequence. Both genes have a similar interrupted structure and an exon that encodes closely related tandem repeats of very high histidine and alanine content. The most common repeat encoded by one gene, the hexapeptide Ala-His-His-Ala-Ala-Asp, differs in the sixth position from the most common repeat encoded by the other gene, the hexapeptide Ala-His-His-Ala-Ala-Asn. The divergence of the repeat domains is greater than that of the flanking regions, which exhibit 85-90% homology, including untranslated sequences. This suggests the tandem repeats are relatively labile elements within the genome that may provide the parasite with a means of rapid evolutionary change. PMID- 3016743 TI - Expression of type C-related endogenous retroviral sequences in human colon tumors and colon cancer cell lines. AB - Type C-related human endogenous retroviral sequences have been previously discovered and characterized [Martin, M. A., Bryan, T., Rasheed, S. & Khan, A.S. (1981) Proc. Natl. Acad. Sci. USA 78, 4892-4896]. We investigated the transcriptional pattern of these sequences to determine whether and to what extent their expression is altered in colon tumors and colon cancer cell lines as compared to normal colon mucosa (NCM). Of two long terminal repeat (LTR)-specific transcripts [3.6 and 2.1 kilobases (kb)], the 3.6-kb RNA was particularly abundant in NCM but strikingly decreased in most primary colon cancers tested. In NCM we identified three envelope gene (env)-related transcripts--namely, 3.0, 1.7, and 0.6 kb. Colon tumors appeared to express higher levels of these transcripts, especially the 1.7-kb species. In one case of dysplasia and in one benign tumor, this 1.7-kb transcript was clearly increased. We also examined the pattern of transcription in colon cancer cell lines HCT and Caco2. The LTR homologous 3.6-kb transcript, very abundant in NCM, was decreased in primary tumors and in HCT cells and virtually absent in Caco2 cells. The latter, however, appeared to produce the transcript when growing exponentially, indicating that Caco2 cultures provide an inducible system susceptible to in vitro manipulation. Both cell lines also contained higher amounts of the 1.7-kb env-related transcript. The decrease of the 3.6-kb RNA in colon tumors versus NCM may be the result of an altered pattern of differentiation, whereas the increase of the 1.7 kb RNA in tumors may represent an early marker of colon neoplasia. PMID- 3016742 TI - Immunoglobulin heavy chain locus of the rat: striking homology to mouse antibody genes. AB - DNA encoding the rat diversity segment (D), joining segment (JH), and constant (C) region mu, gamma 2a, gamma 1, gamma 2b, epsilon and alpha of the Ig heavy chain has been isolated from a cosmid library. Restriction mapping allowed us to identify two gene clusters: D-JH-C mu and C gamma 1-C gamma 2b-C epsilon-C alpha in addition to a single C gamma 2a gene. Analysis of genomic DNA by Southern blotting permitted identification of the C gamma 2c gene and led to the proposal of the following gene order for the rat Ig heavy chain locus: D-JH-C mu-C delta (C gamma 2c, C gamma 2a)-C gamma 1-C gamma 2b-C epsilon-C alpha. There is striking homology between the rat and mouse Ig heavy chain loci as regards gene order and distance between CH genes. Partial DNA sequencing confirms this homology and shows that exon sequences are more conserved than are intron sequences. One of the most conserved intron regions between rat and mouse is that spanning the Ig heavy chain enhancer (91% homology). However, the relationship between the different C gamma subclasses in rat differs from that in mouse. Comparison of the C gamma CH3 domains shows that the rat C gamma 2b gene is most homologous to mouse C gamma 2a/b, whereas the rat C gamma 1 and C gamma 2a genes, both very similar to each other, are most homologous to the mouse C gamma 1 gene. PMID- 3016744 TI - Patients with IgA nephropathy have circulating anti-basement membrane antibodies reacting with structures common to collagen I, II, and IV. AB - It has recently been shown that patients with IgA nephropathy have circulating IgA antibodies against extracts of human glomerular basement membrane. The present study extends those observations and demonstrates by using ELISA and immunoblotting techniques that patients with IgA nephropathy have circulating IgA antibodies against collagen IV alpha chains. The antigenicity of the alpha chains could be destroyed by digestion with collagenase, which indicates that the antigenic site(s) is located on the triple helical part of collagen IV. Furthermore, it was shown by inhibition tests that the IgA antibodies are directed against epitopes also present in collagen I and II isolated after pepsin digestion. PMID- 3016745 TI - Dopaminergic control of 125I-labeled neurotensin binding site density in corticolimbic structures of the rat brain. AB - In the rat brain, destruction of dopaminergic cell groups by injections of 6 hydroxydopamine into the ventral mesencephalic tegmentum results in large decreases in the number of neurotensin binding sites in the mesencephalon and the striatum. In contrast, these lesions produce an increase in the number of 125I labeled neurotensin binding sites in the lateral part of the prefrontal cortex despite a large decrease in cortical dopamine levels. Increases in the number of 125I-labeled neurotensin binding sites in this cortical area as well as in the entorhinal cortex, the nucleus accumbens, and the central part of the striatum were also obtained after chronic blockade of dopamine neurotransmission by a long acting neuroleptic pipotiazine palmitic ester. We propose that dopamine inputs regulate the density of postsynaptic neurotensin binding sites through cortical and subcortical dopamine receptors. Therefore, some of the clinical effects of neuroleptics in schizophrenic patients could be partly related to changes in neurotensin neurotransmission. PMID- 3016747 TI - Monocrotaline-induced cardiopulmonary damage in rats: amelioration by the angiotensin-converting enzyme inhibitor CL242817. AB - Pulmonary injury induced by the plant alkaloid monocrotaline is partially prevented by the angiotensin-converting enzyme (ACE) inhibitor captopril. CL242817 [(S-[R*,S*])-1-([3-acetylthio]-3-benzoyl-2-methyl-propionyl)- L-proline] is a new orally active ACE inhibitor under evaluation as an antihypertensive agent. To determine whether CL242817 also can modify monocrotaline-induced pulmonary injury, male rats were divided into four groups: control; CL242817 (60 mg/kg/day, po); monocrotaline (2.4 mg/kg/day, po); or monocrotaline plus CL242817, and were sacrificed after 6 weeks of continuous treatment. Rats receiving monocrotaline alone exhibited occlusive medial thickening of the pulmonary arteries, cardiomegaly, and right ventricular hypertrophy. Electron micrographs of monocrotaline-treated lung revealed degeneration of both endothelial and Type I epithelial cells, as well as marked interstitial hypercellularity and fibrosis. Hydroxyproline (collagen) content of monocrotaline treated lung also increased significantly, confirming the fibrosis observed in the electron micrographs. These structural changes were accompanied by decreased lung ACE and plasminogen activator (PLA) activities, indicative of pulmonary endothelial dysfunction. Concomitant CL242817 treatment ameliorated all anatomic manifestations of monocrotaline injury, particularly the right ventricular hypertrophy, pulmonary arterial occlusion, epithelial degeneration, and interstitial fibrosis. CL242817 also significantly prevented the monocrotaline induced increase in lung hydroxyproline content. In contrast, concomitant CL242817 did not significantly influence the suppressed lung ACE and PLA activities in monocrotaline-treated rats. CL242817 alone produced retarded weight gain, decreased heart weight relative to body weight, decreased lung hydroxyproline content and ACE activity, and increased serum ACE activity and plasma AII concentration. Thus CL242817 resembles captopril, both in its ability to ameliorate monocrotaline-induced pulmonary injury in rats, and in many of its side effects. PMID- 3016748 TI - Increased atrial natriuretic peptide (6-33) binding sites in the subfornical organ of water deprived and Brattleboro rats. AB - Binding sites for rat atrial natriuretic peptide (6-33) (ANP) were quantitated in the subfornical organ of chronically dehydrated homozygous Brattleboro rats unable to synthesize vasopressin; heterozygous Brattleboro rats, their controls, Long Evans rats and Long Evans rats after 4 days of water deprivation. Brain sections were incubated in the presence of 125I-ANP and the results analyzed by autoradiography coupled to computerized microdensitometry and comparison to 125I standards. Brattleboro rats and water deprived Long Evans rats presented a higher number of ANP binding sites than their normally hydrated controls. Our results suggest a role of ANP binding sites in the subfornical organ in the central regulation of fluid balance and vasopressin secretion. PMID- 3016749 TI - Determination of the total vitamin D3 content of cod-liver oil by high performance liquid chromatography. PMID- 3016746 TI - Control by cations of opioid binding in guinea pig brain membranes. AB - In membrane suspensions from guinea pig brain or cerebellum, NaCl, LiCl, NH4Cl, and KCl inhibit the equilibrium binding at 25 degrees C of the selective mu agonist [3H][2-D-alanine,4-methylphenylalanine,5-glycinol]enkephalin ([D Ala2,MePhe4,Gly-ol5]EK), the selective delta-agonist [3H][2-D-penicillamine,5-D penicillamine]enkephalin ([D-Pen2,D-Pen5]-EK), and the selective kappa-agonist [3H]dynorphin A-(1-9). Choline chloride inhibits mu- and kappa-binding but not delta-binding. The relative activities of these monovalent salts and the slopes of the dose-response curves are site-dependent. Binding at the kappa-binding site is also inhibited by CaCl2, MnCl2, and MgCl2. On the other hand, these divalent salts potentiate delta-binding, and MnCl2 and MgCl2 have both potentiating and inhibitory effects on mu-binding; CaCl2 inhibits but does not potentiate mu binding. Thus, the mechanisms by which monovalent cations inhibit opioid binding differ from those of divalent cations, and the mechanisms of action of both monovalent and divalent cations may differ at each site. When the antagonist [3H]naloxone, rather than the agonist [3H][D-Ala2,MePhe4,Gly-ol5]EK, is used to label the mu-binding site, the main effect of NaCl is to potentiate binding; a 22 fold higher concentration of LiCl is required to inhibit binding. The effects of NH4Cl, KCl, MnCl2, MgCl2, CaCl2, and choline chloride are little changed when [3H]naloxone is the ligand. PMID- 3016750 TI - Alteration of prostanoid metabolism in rats with magnesium deficiency. AB - Plasma and tissue concentrations of prostanoids PGE2, PGF2 alpha. 6-keto-PGF1 alpha (a stable metabolite of prostacyclin) and TXB2 (a stable metabolite of thromboxane A2) were measured in normal and magnesium-deficient rats. The mean values for prostanoids in plasma were significantly higher in magnesium-deficient rats than in normals (515 +/- 43 vs 296 +/- 31 pg/ml for 6-keto-PGF1 alpha, p less than 0.01, 3700 +/- 322 vs 346 +/- 33 pg/ml for TXB2, p less than 0.001 and 1234 +/- 132 vs 434 +/- 51 pg/ml for PGE2, p less than 0.001). Tissue levels of prostanoids were also significantly higher in magnesium-deficient rats as compared to normals. The increased synthesis of prostanoids is apparently linked to enhanced influx and translocation of Ca++ into the cells. If the adenylate cyclase is inhibited in magnesium deficiency, the lowered c-AMP will permit a high cyclooxygenase activity and a drastic increase in TXB2. It is possible that the changes in prostaglandin synthesis in magnesium deficiency are linked to the development of different diseases. PMID- 3016751 TI - Prostaglandin E1 (PGE1) stimulates in vitro renin release in the hibernating ground squirrel. AB - Using a renal cortical slice preparation obtained from hibernating ground squirrels, this study investigated whether three major intrarenal prostaglandins (PG) and other agents added at a 10(-5) M dose can affect renin release (RR), and if their effect on RR is correlated with changes in tissue cyclic AMP content (tcAMPc). Resting in vitro levels for RR and tcAMPc during hibernation were found to be comparable to those observed in non-hibernating (NH) mammals. Addition of PGE1 significantly stimulated RR while PGE2 and PGF2-alpha were ineffective. None of the three PG's tested altered resting levels of tcAMPC. When added by itself or in conjunction with any of the three PG's, the phosphodiesterase inhibitor theophylline did not modify resting levels of RR and tcAMPc, but it prevented the previous stimulatory effect of PGE1 alone on RR. Addition of lipid-soluble dcAMP, either alone or in conjunction with any of the three PG's, resulted in an increase in tcAMPc in all instances, no change in resting RR regarding PGE2 and PGF2-alpha, and again the prevention of the stimulatory effect of PGE1 alone on RR. These data suggest that: the resting activity of the renin-angiotensin system (RAS) and the adenylate cyclase-cAMP system (AC-cAMP) of the hibernating ground squirrel is comparable to that of NH species; in contrast, the sensitivity of the RAS of the hibernator to PG stimulation is less than that exhibited by the RAS of NH species when comparable in vitro concentrations of three major PG's are used; PGE1 stimulation of RR in the hibernator is independent of changes in tcAMPc; changes in tcAMPc may be inversely related to those in RR in the hibernator; and the known stimulatory effect of PG's on the renal cortical AC-cAMP of NH species is not seen in the hibernating ground squirrel at comparable PG doses. PMID- 3016752 TI - Differential rank order of potency for antagonism of LTC4- and LTD4-induced contractions of guinea pig trachea. AB - In the presence of 1-serine-borate complex (SB), employed to prevent LTC4 to LTD4 metabolism, desamino-2-nor-LTE, [4-hydroxy-5-[(2,2-carboxyethyl)thio]-6 nonadecenoic acid; DN-LTE1] equally antagonized the contractions elicited by LTC4 (-log KB = 5.8 +/- 0.2) and LTD4 (-log KB = 5.5 +/- 0.4) on the isolated guinea pig trachea. In contrast, FPL 55712 preferentially antagonized the LTD4-induced contractions in the presence of SB with a -log KB = 6.2 +/- 0.2, whereas only weak antagonism of the LTC4-induced contractions was observed (-log KB = 4.9 +/- 0.2). Thus, the rank order of potency for antagonizing the LTC4-induced contractions was DN-LTE1 greater than FPL 55712, whereas the rank order for antagonizing LTD4 was FPL 55712 greater than DN-LTE1. On tissues pretreated with 10 microM FPL 55712 without SB, 10 microM DN-LTE1 antagonized the contractions elicited by LTC4 but not those elicited by LTD4. Thus, based upon the differential rank order of antagonism of the LTC4- and LTD4-induced contractions by these LT antagonists under two different experimental conditions, these results imply the existence of multiple LT receptors in the guinea pig trachea. PMID- 3016754 TI - Leukotriene B4 and 20-OH-LTB4 in purulent peritoneal exudates demonstrated by GC MS. AB - Inflammatory peritoneal exudates from 11 patients with purulent peritonitis or non-perforative appendicitis were analyzed by gas chromatography mass spectrometry (GC-MS) for their content of 5S, 12R-dihydroxy-6,14-cis-8,10-trans eicosatetraenoic acid (LTB4) and its omega-oxidized catabolite, 5S,12R,20 trihydroxy-6,8,10,14-eicosatetraenoic acid (20-OH-LTB4). Eleven samples of peritoneal exudates were examined. LTB4 and 20-OH-LTB4 were demonstrated only in the samples from patients with purulent peritonitis. LTB4 was detected in 4 samples, and 20-OH-LTB4 was detected in 6 samples by GC-MS with selected ion monitoring. In addition, a nearly full spectrum corresponding to that of synthetic 20-OH-LTB4 was obtained with 3 samples. Neither LTB4 nor 20-OH-LTB4 were detected in samples from patients with non-perforative appendicitis. LTB4 or 20-OH-LTB4 may be related to the pathophysiological mechanism of purulent inflammations. PMID- 3016753 TI - Cell kinetic studies of PGD2 cytotoxicity on the in vitro growth of human neuroblastoma. AB - To determine the mechanism of the antineoplastic effect of PGD2, we studied the intracellular adenosine 3',5'-cyclic monophosphate (cAMP), cell growth and the kinetics of a human neuroblastoma (NCG line) in culture. Cells were maintained in RPMI 1640 with 10% FCS. cAMP level was determined for the cells, which were incubated for 10 min. with and without PGD2 (1 microgram/ml) in the presence of 100 microM papaverine. Growth inhibition was examined by counting viable cells after treatment with PGD2 (0-100 micrograms/ml) for 4 consecutive days starting at Day 4 after subculture. Effects on cell kinetics were examined for similarly treated cells by DNA cytofluorometry combined with 3H-thymidine autoradiography. In response to PGD2, the NCG line failed to increase its cAMP; however, cell growth was inhibited (IC50 13 micrograms/ml), accompanied by the marked decrease of S phase cells. The results indicate that PGD2 exerts its cytotoxic effect by a probable G1 block of the cell cycle and not through cAMP action. PMID- 3016755 TI - Lipoxygenase inhibitor and colchicine as anti-arthritic agents in the rat. AB - The non-steroidal anti-inflammatory agents do not have identical activities on the various pathways of arachidonic acid metabolism. The purpose of this study is to examine and compare the activities of colchicine, an anti-arthritic agent, indomethacin, a known prostaglandin synthesis inhibitor and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, in an experimental model of arthritis. Acute arthritis of the knee was induced in rats by injection of lipopolysaccharide (LPS) into the joints. Arthritis was characterized by an increase in joint diameter (18%), increased synovial weight (34%) and an increase in synovial prostaglandin E (PGE) production (56%). While administration of all of the agents examined abolished LPS-induced joint diameter and synovial weight increase, only indomethacin reduced increased PGE content. NDGA and colchicine had no inhibitory effect on LPS-induced PGE production, and moreover they actually stimulated PGE production when compared to control values. It is concluded that: Among the mediators of the inflammatory process are factors sensitive to colchicine and NDGA which are not PGs. Lipoxygenase products of arachidonic acid including leukotrienes may have an important role in inflammation. Leukotrienes and prostaglandins may act in concert in mediating the inflammatory process. PMID- 3016756 TI - Stimulation of brain lipase activity by polyamines. Comparison with the effect of ACTH. AB - Polyamines, as well as ACTH, strongly stimulate at pH 5.75 triacylglycerol lipase (TAGL) activity from rat brain. Whether the activating potency is expressed in terms of molar concentration or amount of positive charges, polyarginine, polylysine, spermine and spermidine exhibit, in this order, decreasing potencies. By contrast to other lipases, heparin (25 micrograms/ml) inhibits brain TAGL. Polyarginine, polylysine and spermine reverse the heparin-dependent inhibition and further stimulate TAGL activity above basal values; spermidine is much less potent. In the presence of heparin, ACTH has the greatest stimulating effect, being 1.6-fold and 3.3-fold more potent than polyarginine and polylysine, respectively. Taken together, the data suggest that polybasic effectors modify the interaction of TAGL with its substrate, resulting in increased levels of TAGL activity. In the presence of heparin, the enzyme charge density is mandatory for determining the stimulation process. Such cationic interactions appear to be specific of brain TAGL and should be considered in assessing any direct neuro hormonal role to ACTH or physiological polyamines in brain. PMID- 3016758 TI - Phencyclidine-induced stereotyped behaviors after injection of morphine and N allylnormetazocine (SKF 10,047) in rats. AB - We investigated the effects of N-allylnormetazocine (SKF 10,047) and morphine on the stereotyped behaviors induced by the intraperitoneal injection of phencyclidine (PCP). PCP-induced turning and backpedalling were significantly potentiated by pretreatment with SKF 10,047 (10 mg/kg) but sniffing and head weaving were not. On the other hand, pretreatment with morphine dose-dependently attenuated PCP-induced sniffing and head weaving, but not turning and backpedalling. These results suggest that PCP-induced stereotypy may be mediated by not only a sigma opioid receptor but also some other receptors. In addition, each component of PCP-induced stereotypy may be controlled by different opioid systems and/or neuronal systems. PMID- 3016757 TI - Foot shock induces time and region specific adrenergic receptor changes in rat brain. AB - Rats were subjected to 1 hr or 2 hr of electric foot shock for 1 day or 7 days and adrenergic receptor binding was evaluated in the hypothalamus, brainstem and cortex. beta-Adrenergic receptor density in the hypothalamus was dramatically reduced following 1 hr of shock. Following repeated shock, alpha 2-adrenergic receptors in the cortex and brainstem were observed to increase. Cortical alpha 2 adrenergic receptors were more sensitive to stress than the alpha 2-adrenergic receptors of the brainstem, alterations in the latter only reaching statistical significance following 7 days of shock and 24 hr of recovery. alpha 1- and beta adrenergic receptors in the brainstem and cortex were relatively resistant to stress induced changes. The significance of type of stress, duration of stress, and strain of rat for understanding the current data are discussed in the context of prior reports of stress induced receptor changes. PMID- 3016759 TI - beta-Carboline FG 7142-reduced aggression in male rats: reversed by the benzodiazepine receptor antagonist, Ro15-1788. AB - The beta-carboline FG 7142 decreases conspecific aggression in male hooded rats. The purpose of this study was to examine the effects of pretreatment with Ro15 1788 or chlordiazepoxide (CDP) in this paradigm. The six groups (n = 8) were saline, FG 7142 (5 mg/kg, immediate, IP), CDP (5 mg/kg, -10 min, IP), CDP (5 mg/kg, -10 min) plus FG 7142 (5 mg/kg, immediate), Ro15-1788 (10 mg/kg, -10 min, IP), and Ro15-1788 (10 mg/kg, -10 min) plus FG 7142 (5 mg/kg, immediate). Following injection of the more aggressive member of a pair of isolation-housed rats, the pair was observed in a living cage over four 6-min trials interpolated over a 40 min session. In the first 20 min after the injection FG 7142 decreased aggression, decreased the pinning of the other animal, and increased avoiding behavior. These effects were the opposite of those seen in the Ro15-1788-injected rats and Ro15-1788 pretreatment reversed the effects of FG 7142. CDP alone caused prolonged aggressive behavior but as a pretreatment only partially reversed the effects of FG 7142. PMID- 3016760 TI - Inescapable shock reduces [3H]Ro 5-4864 binding to "peripheral-type" benzodiazepine receptors in the rat. AB - [3H]Ro 5-4864 binding to "peripheral-type" benzodiazepine receptors was examined in brain and peripheral tissues of rats subjected to inescapable tailshocks. Two hours after a session of 80 (five-second) inescapable tailshocks, a significant reduction in [3H]Ro 5-4864 (10 mM) binding was observed in membranes from kidney (31%), cerebral cortex (29%), heart (19%) and pituitary (17%) compared to tissues from naive animals. In contrast, inescapable shock did not effect [3H]Ro 5-4864 binding to hippocampal, lung, or adrenal membranes. Scatchard analyses of [3H]Ro 5-4864 binding to renal membranes demonstrated that this session of tailshock reduced the density (Bmax) of "peripheral-type" benzodiazepine receptors without effecting the apparent affinity (Kd) of the radioligand for these sites. The effects of graded stress on [3H]Ro 5-4864 binding to cerebral cortex and kidney were investigated using 5, 20, or 80 (five-second) inescapable shocks. In cerebral cortical membranes, sessions of either 5 or 20 shocks did not affect, while 80 shocks reduced (29%) [3H]Ro 5-4864 (10 mM) binding. In renal membranes, 5 shocks significantly increased (35%), 80 shocks significantly decreased [3H]Ro 5-4864 (10mM) binding (31%). These findings demonstrate that the density of "peripheral-type" benzodiazepine receptors in both peripheral tissues and the central nervous system can be rapidly modulated by stress. PMID- 3016761 TI - Behavioral and neurochemical correlates of morphine and hypoxia interactions. AB - Decreased oxygen availability (hypoxia) impairs the synthesis of dopamine and serotonin in parallel with a decline in open-field behavior. If hypoxic-induced deficits in dopamine and serotonin metabolism are physiologically important, then stimulation of their synthesis may help reverse hypoxic-induced neurochemical and behavioral deficits. Acute morphine sulfate (50 mg/kg) increased dihydroxyphenylacetic acid/dopamine ratios (DOPAC/DA) (+20%), the conversion of [3H]tyrosine to [3H]dopamine (+73%) and open-field activity (+130%) in CD-1 male mice. However, morphine failed to significantly alter the incorporation of [3H]tryptophan to [3H]serotonin. Morphine antagonized the hypoxic-induced impairment of dopamine metabolism and locomotor activity. DOPAC/DA ratios of hypoxic animals that were treated with morphine were identical to controls, and conversion rates of [3H]tyrosine to [3H]dopamine were increased. Total distance in an automated activity monitor following the combination of morphine and hypoxia increased 79% compared to a 48% decrease with hypoxia alone. These results suggest that both hypoxia and morphine alter the dopaminergic system, but in opposite directions. These interactions may help to explain why morphine is able to ameliorate hypoxic-induced changes in behavior. PMID- 3016762 TI - Antagonism of morphine analgesia by intracerebroventricular naloxonazine. AB - Intravenous pretreatment with naloxonazine, an irreversible and selective antagonist of mu-1 sites for over 24 hr, reduces analgesia induced by morphine as well as a series of opiates and enkephalins. The present study evaluated whether intracerebroventricular (ICV) administration of naloxonazine produces similar long-term (24 hr) reductions in morphine analgesia on the tail-flick and jump tests. Naloxonazine failed to alter baseline tail-flick latencies or jump thresholds, but antagonized in a dose-dependent manner morphine analgesia for 24 hr. Naloxone had no effect at 24 hr. Morphine actions in the jump test were quite sensitive to doses of naloxonazine as low as 1 microgram/rat. Although tail-flick assays also revealed naloxonazine effects, far greater doses (30 micrograms/rat) were needed. Naloxonazine also shifted full morphine dose-response curves to the right. Again, naloxonazine antagonized morphine in the jump test more effectively than in the tail-flick assay. These data provide support for the involvement of the mu-1 opioid binding site in the central mediation of morphine analgesia and point out the differing sensitivities of two analgesiometric assay systems to naloxonazine. PMID- 3016763 TI - Tetrahydro-beta-carboline micro-injected into the hippocampus induces an anxiety like state in the rat. AB - Guide cannulae for bilateral micro-injection were implanted stereotaxically in the rat to rest just dorsal to the hippocampus. Following recovery, 1,2,3,4 tetrahydro-beta-carboline (THBC) hydrochloride in a concentration of 10 or 50 ng was infused bilaterally into the animal's hippocampus in a volume of 3.0 microliter. In the control condition, the artificial cerebrospinal fluid (CSF) vehicle was micro-injected into the hippocampus and a sham injection was made prior to the CSF or THBC infusion. The behavioral response of the rat was examined subsequently in an open-field chamber, in terms of the number of grid squares crossed, duration of grooming time and instances of freezing immobilization during the test interval of 7.5 min. Other behaviors recorded included the appearance of tail rigidity and the number of fecal boluses excreted. The intra-hippocampal infusion of the 10 ng dose of beta-carboline reduced the motor activity of the rat whereas the higher dose of THBC increased the duration of the freezing-immobilization. THBC failed to alter significantly the grooming activity of rats or their rate of defecation. Following repeated micro-injections of 50 ng of THBC, the duration of freezing-immobilization gradually decreased, but the response itself remained essentially intact. These results suggest that the well-known anxiogenic action of certain of the beta carboline class of aldehyde adducts may be mediated in part by neurons in the hippocampus, or the constituent pathways of this limbic system structure, or both. PMID- 3016764 TI - Acute effects of smoking marijuana on hormones, subjective effects and performance in male human subjects. AB - Four healthy male subjects smoked two marijuana cigarettes or one marijuana cigarette and one placebo cigarette, or two placebo cigarettes on separate days in a random order crossover design. Each marijuana cigarette contained 2.8% delta 9-tetrahydrocannabinol (THC). Plasma hormones and THC were measured before and after each smoking session. Plasma LH was significantly depressed and cortisol was significantly elevated after smoking marijuana. Nonsignificant depressions of prolactin, FSH, testosterone and free testosterone and elevation of GH also occurred. Concurrent measures of subjective effects via subscales of the Addiction Research Center Inventory, Single Dose Questionnaire and a Visual Analog Scale were generally elevated. Significant impairment on a psychomotor performance task paralleled elevations in subjective effects, hormone effects and peak THC determinations. Although all the hormone effects were within normal basal ranges, interactions between these systems, and their effects on behavior cannot be discounted. PMID- 3016765 TI - Inhibition of protein kinase activity by apomorphine. AB - The effect of apomorphine (1-20 microM) on protein kinase activity was studied in extracts from rat peritoneal mast cells and brain tissue. Apomorphine inhibited the cyclic AMP-dependent and the calcium-plus phosphatidylserine-dependent protein kinase activity with an IC50 between 1 and 6 microM, depending on the tissue and on the protein kinase involved. This effect might explain previous results on the apomorphine-induced inhibition of histamine release in rat peritoneal mast cells. PMID- 3016766 TI - The neuromuscular blocking activity of spectinomycin on the rat sciatic nerve gastrocnemius muscle preparation. AB - Spectinomycin displays a dose-dependent neuromuscular blocking activity in vivo. The neuromuscular blockade elicited by spectinomycin is potentiated by d tubocurarine. Neostigmine methylsulfate is unable to reverse the neuromuscular blocking activity of spectinomycin, whereas calcium chloride counteracts the neuromuscular blockade induced by this antibiotic. PMID- 3016767 TI - A cytosolic binding protein for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the uterus and deciduoma of rats. AB - The cytosol fraction of the uterus of proestrous rats and of deciduoma specifically binds 3H-2,3,7,8-tetrachlorodibenzo-p-dioxin (3H-TCDD). The 3H-TCDD binding protein has a sedimentation coefficient of 9 S in low ionic 10-40% sucrose density gradients. The binding of 3H-TCDD in the 9 S region is abolished by a 500-fold molar excess of unlabeled benzo[a]pyrene, 3-methylcholanthrene or by a 100-fold molar excess of unlabeled TCDD. Incubation of the binding protein with TCDD in amounts in excess of 500 nM causes aggregation of the TCDD binding protein. Neither estradiol, progesterone, testosterone, dihydrotestosterone nor cortisol competed with TCDD in binding to this 9 S protein. The decidual tissue contains two binding components for TCDD as shown by Scatchard analysis. One of the components has a high affinity for TCDD (Kd = 1.68 nM) and is saturable. The number of binding sites is about 75 fmol/mg protein. The TCDD binding protein eluted through a DEAE-cellulose column using a gradient of 0.25 M KCl. The binding of estradiol and progesterone to their respective receptors was not affected by TCDD or by other polycyclic aromatic hydrocarbons as shown by sucrose density gradients and by microtiter competition assays. These results suggest that TCDD acts by binding to its own receptor system in the target tissue and not by competing with estrogen or progesterone for binding to their receptors. The possible role of the receptor in teratogenesis is discussed. PMID- 3016768 TI - Regulation of active Na+-K+ transport in skeletal muscle. PMID- 3016770 TI - Beta-adrenergic receptors and their mode of coupling to adenylate cyclase. PMID- 3016769 TI - Functional properties of vertebrate olfactory receptor neurons. AB - The interaction of an odorant with the chemosensitive membrane of olfactory receptor neurons initiates a sequence of molecular and membrane events leading to sensory transduction, impulse initiation, and the transmission of sensory information to the brain. The main steps in this sequence are summarized in Figure 6. Several lines of evidence support the hypothesis that the initial molecular events and subsequent stages of transduction are mediated by odorant receptor sites and associated ion channels located in the membrane of the cilia and apical dendritic knob of the olfactory receptor neuron. Similarly, the membrane events associated with impulse initiation and propagation are mediated by voltage-gated channels located in the initial axonal segment and the axolemma. The ionic and electrical events associated with the proposed sequence have been characterized in general using a variety of experimental techniques. The identification, localization, and sequence of membrane events are consistent with the neurophysiological properties observed in specific regions of the bipolar receptor neuron. The influence of other cells in the primary olfactory pathway such as the sustentacular cells in the olfactory epithelium, the Schwann cells in the olfactory nerve, and the astrocytes in the olfactory nerve layer in the olfactory bulb on the physiological activity of the olfactory receptor neuron is an emerging area of research interests. The general principles derived from the experimental results described in this review provide only a framework that is both incomplete and of necessity somewhat speculative. As noted in the Introduction, the multidisciplinary study of the primary olfactory pathway is undergoing a renaissance of research interest. The application of modern biophysical, cell, and molecular biological techniques to the basic issues of odorant recognition and membrane excitability will clarify the speculations and lead to the establishment of new hypotheses. Three broad areas of research will benefit from such studies. First, the application of biophysical techniques will lead to a detailed characterization of the membrane properties and associated ion conductance mechanisms. Second, the isolation and biochemical characterization of intrinsic membrane and cytosolic proteins associated with odorant recognition, sensory transduction, and the subsequent electrical events will result from the utilization of cell and molecular biological techniques.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3016771 TI - Vasopressin excites ventromedial hypothalamic glucose-responsive neurons in vitro. AB - Effects of vasopressin (AVP), oxytocin (OXY), norepinephrine (NE), and glucose on the single-unit activity of hypothalamic ventromedial nucleus (VMN) in tissue slices were studied. While AVP was exclusively excitatory on 58% of the neurons, OXY could be excitatory or inhibitory and affected only 42% of the neurons. There was no correlation between the responses to these two peptides. Each of these two peptides could desensitize neuronal response to itself, but did not cross desensitize responses to each other. These results indicate that AVP and OXY do not act on the same population of VMN neurons through the same cellular mechanism. Furthermore, only the responses to AVP were correlated to responses to glucose and NE, two agents relevant to central regulation of feeding. This correlation with responses to feeding-relevant agents and the exclusively excitatory action on the VMN, which is involved in the regulation of feeding, suggest that AVP can play a role in the regulation of feeding, particularly the feeding induced by the injection of NE into the paraventricular nucleus, that is known to alter AVP release. PMID- 3016772 TI - Histamine in dorsal and ventral hippocampus. II. Effects of H1 and H2 histamine antagonists on exploratory behavior in male rats. AB - The effects on Hole-Board behavior of histamine (HA) microinjected into different parts of the hippocampus and the effects of pyrilamine (PYR, an H1-histamine antagonist), ranitidine (RAN, an H2-histamine antagonist) or alpha-fluoromethyl histidine (alpha-FMH, an irreversible inhibitor of the HA synthetizing enzyme) injected into the hippocampus on behavior were studied. Forty five nMol of HA were injected stereotaxically into the dorsal or ventral hippocampus. Five min later, Hole-board behavior was measured. It was observed that HA inhibited locomotion and rearing only in the rats injected into the ventral hippocampus. In other experiments, animals were microinjected into the ventral hippocampus with 135 nMol of PYR or RAN in 1 microliter of saline solution. Ten min later, they were microinjected with 45 nMol of HA. Hole-board exploratory activity was measured 5 min thereafter. Results showed that both PYR and RAN were effective in counteracting the inhibitory effect of HA on locomotor activity, but only RAN was able to block the inhibitory action of HA on rearing behavior. Head-dipping frequency was not affected by these treatments. In rats microinjected with 20 nMol of alpha-FMH, increased scores of locomotion were observed but the other behaviors (head-dipping frequency, grooming and rearing) were not affected. The present results support the hypothesis that HA in hippocampus may be exerting a regulatory role on behavior by interaction with H1 and H2 receptors. PMID- 3016773 TI - Rapidly and slowly adapting mechanoreceptors in the glans penis of the cat. AB - Sixty-one single mechanoreceptive fibers were surgically isolated from the dorsal nerve of the penis in anesthetized, mature, and sexually intact male cats. Impulse activity was recorded extracellularly. Receptive fields on the glans penis were stimulated with an accurate computer controlled mechanostimulator. Thirty-four units were categorized as rapidly adapting (RA) based on the absence of a response to sustained skin displacement. The remaining were slowly adapting (SA) units responding to sustained displacement. Most of the SA units were located in the distal smooth glans whereas RA units predominated in the proximal spiny glans. Displacement thresholds were significantly lower for RA units. All units encoded indentation velocity although the SA units were better suited to discriminate slowly moving stimuli. In addition, SA units encoded sustained displacement amplitude. Male cats with a denervated glans penis display disoriented mounting behavior disabling intromission. The presence of distally located SA mechanoreceptors suggests they may be the primary penile proprioceptors, mediating, along with the RA mechanoreceptors, successful completion of intromission. PMID- 3016774 TI - The curve-shift paradigm in self-stimulation. AB - Eleven rats were trained to press a lever in an operant chamber in order to earn rewarding trains of cathodal rectangular pulses of fixed intensity and variable frequency. The rate-frequency functions were examined under administration of two neuroleptics (pimozide and chlorpromazine) and three manipulations that interfered with bar pressing (muscular relaxation with methocarbamol, increased lever weight, and limitation of maximum response rates by an F1 reinforcement schedule). Chlorpromazine, and pimozide at low dosages produced a near parallel shift of the rate-frequency functions on the logarithmic axis of pulses, suggesting that these drugs decreased the reinforcing efficacy of the stimulation. The three conditions that interfered with bar-pressing decreased the asymptotic rates and produced small or moderate lateral shifts. Changes in the reinforcing efficacy of the stimulation following the above manipulations were inferred from the shift in the number of pulses required at zero and half-maximal performance (theta 0 and M50 indices, respectively). In the cases of the manipulations that interfered with bar-pressing, M50 indicated a larger artifactual change in the efficacy of the stimulation, compared to theta 0. This phenomenon was mainly due to the fact that the asymptote of the altered functions was shifted towards higher pulse numbers. PMID- 3016775 TI - Sleep in Macaca arctoides and the effects of prazosin. AB - Polygraphic (EEG, EOG, EMG) 12-hr recordings were made in 3 adult monkeys (Macaca arctoides) to document their normal sleep pattern and to examine the effects of prazosin, an alpha-1-adrenoceptor antagonist. In comparison to man, REM episodes appeared at shorter intervals (50 min) and they always ended in movement arousal or waking up. As in man the proportion of deep sleep decreased and the proportion of REM sleep increased towards morning. During eight hours after parenteral administration of prazosin, at doses without any noticeable behavioral side effects, the number of REM episodes and the amount of REM sleep was 30 to 80 percent greater than during the control nights. The changes were statistically nonsignificant. However, since the results were similar in all three monkeys, it can be suggested that prazosin had some effect on the cyclicity of sleep. Dose 1 mg/kg resulted in behavioral side effects and disturbed sleep. PMID- 3016776 TI - Short photoperiod stimulates brown adipose tissue growth and thermogenesis but not norepinephrine turnover in Syrian hamsters. AB - This experiment examined the effect of short photoperiod on food intake, body weight, carcass composition, and brown adipose tissue (BAT) activity in female Syrian hamsters (Mesocricetus auratus). BAT function was assessed by measuring sympathetic nervous system (SNS) activity in BAT (estimated by the rate of norepinephrine (NE) turnover), BAT mitochondrial content (estimated by cytochrome c oxidase activity), and BAT mitochondrial proton conductance (estimated by guanosine-5'-diphosphate (GDP) binding to isolated BAT mitochondria). Food intake and body weight were both increased in short photoperiod-housed hamsters (8-hr light, 16-hr dark; LD 8:16) when compared to those of the long photoperiod-housed controls (LD 16:8). The weight gain was entirely due to an increase in carcass lipid. Interscapular BAT (IBAT) pads from short photoperiod-housed hamsters were 53% heavier and contained comparably more protein and DNA. Short photoperiod produced a 118% increase in BAT cytochrome c oxidase activity and a 41% increase in specific mitochondrial GDP binding. Whether expressed per mg wet tissue or per pad, neither the endogenous concentration of NE nor its rate of turnover were changed by short photoperiod exposure. These results demonstrate a dissociation of BAT thermogenesis from SNS activity in BAT from short photoperiod-housed Syrian hamsters. PMID- 3016777 TI - Resident's scent: a critical factor in acute analgesic reaction to defeat experience in male mice. AB - Although it has recently been reported that defeated male mice evidence an acute non-opioid analgesia, little is currently known about the specific features of the defeat experience with which the analgesic reaction is associated. The present experiments not only confirm that defeat experience reliably induces acute antinociception in intruder mice, but show that a similar reaction also occurs as a consequence of exposure to an aggressive resident which does not attack during the brief test period, a well-characterized non-aggressive resident and the 'unoccupied' soiled home cage of an aggressive resident. Results also indicated that, with appropriate exposure duration, scent alone can give rise to a quantitatively similar analgesia to that observed in defeated mice. Furthermore, time-course comparisons and the absence of naloxone antagonism suggest that 'scent' and 'defeat' analgesias are mediated via a common non-opioid mechanism. Data are discussed in relation to the ecological significance of urinary odours in social communication in mice. PMID- 3016778 TI - Evidence for the involvement of the incC locus of broad host range plasmid RK2 in plasmid maintenance. AB - Plasmid pRK2501 is a deletion derivative of broad host range plasmid RK2 and encodes two trfB-regulated operons: the trfA operon which codes for both kilD, which interferes with plasmid maintenance if unregulated, and trfA whose protein product(s) is essential for replication from oriVRK2; and part of the trfB operon, containing both trfB/korA/korD, whose product negatively regulates transcription of both trfA and trfB operons, and incC, the product of which interferes in trans with inheritance of RK2 and certain of its derivatives. Plasmid pRK2501ts3 is a derivative with a point mutation in trfB, rendering plasmid maintenance temperature sensitive. Transcriptional fusions of the trfB operon and the galK gene demonstrate that this mutation derepresses trfB operon transcription at both 30 and 42 degrees C. The trfA operon is also derepressed by this mutation. Since the trfB gene product appears to be defective at both permissive and non-permissive temperatures the temperature sensitivity of pRK2501ts3 must be due to a secondary effect. In fact, it appears to arise from the inhibitory behavior of derepressed incC at the non-permissive temperature since a major class of "revertant" of pRK2501ts3 contains deletions inactivating incC and a reconstruction experiment demonstrates that such a deletion is sufficient for "reversion." Maxicell experiments show that at the non-permissive temperature the trfA operon polypeptide products are produced at much lower levels, an effect partly reversed by a deletion affecting incC. It is proposed that incC normally plays a role in maintenance of IncP plasmids by modulation of trfA operon expression. PMID- 3016779 TI - Location and cloning of the ultraviolet-sensitizing function from the chromosomally associated IncJ group plasmid, R391. AB - The IncJ plasmid R391, which specifies a uv-sensitizing function, has been shown to be associated with chromosomal DNA. Deletions originating from Tn10 insertion into the kanamycin-resistance determinant of plasmid R391 gave rise to uv resistant derivatives. This apparent linkage between the kanamycin-resistance determinant and the uv-sensitizing gene(s) was used to clone the uv-sensitizing function from plasmid R391 into pUR222. A recombinant plasmid containing both functions (KanR and Uvs+) was obtained. The uv-sensitizing function was mapped to a 4-kb EcoRI fragment. PMID- 3016780 TI - The presence of repeated DNA sequences and a partial restriction map of the pSym of Rhizobium fredii USDA193. AB - The large, 350-kb Sym (symbiotic) plasmid pRjaUSDA193 of Rhizobium fredii was examined to determine the frequency of repeated sequences present and to produce a physical and genetic map of a large region of the plasmid. A novel hybridization method, the Southern Cross, revealed that the plasmid pRjaUSDA193 contained many repeated sequences and assisted in restriction enzyme mapping of a 100-kb region containing nod genes. A cosmid clone bank was prepared with the broad-host-range cosmid pVK102. The restriction enzymes HindIII, HpaI, and KpnI were used to construct a physical map of overlapping clones. Labeled nod gene sequences were used to determine their location in the mapped region. PMID- 3016781 TI - Use of plasmid pTV1 in transposon mutagenesis and gene cloning in Bacillus amyloliquefaciens. AB - The plasmid pTV1, constructed in Bacillus subtilis as a tool for insertional mutagenesis by the transposon Tn917, has been transferred to Bacillus amyloliquefaciens by transduction with the phage PBS1. Insertional mutants containing Tn917 were observed in the new host. Southern blot analysis of such mutants indicated no preference for insertion sites. The copy numbers of pTV1 in B. amyloliquefaciens and B. subtilis were found to be 1.4 and 14, respectively; the plasmid is less stable against loss in B. amyloliquefaciens. The overall transposition rate in B. amyloliquefaciens is nevertheless comparable to that in B. subtilis and large numbers of mutants are readily obtained. The yield of auxotrophs was about 0.7% of all mutants, but the preponderance of glutamate auxotrophs seen in B. subtilis was not observed. A number of auxotrophs were identified as to nutritional requirements and those tested were found to be stable. Mutants deficient in extracellular proteases, amylase, and ribonuclease (barnase) were also found and the inactivated barnase gene has been cloned in Escherichia coli. It seems likely, therefore, that any B. amyloliquefaciens gene for which there is a functional test could be cloned by this technique. PMID- 3016782 TI - A common plasmid of Chlamydia trachomatis. AB - A 7.4-kb plasmid is a common and perhaps essential component of the Chlamydia trachomatis genome. This plasmid occurs as 10 copies per chlamydial chromosomal equivalent. It is unable to replicate in Escherichia coli. Complete plasmid genomes from eight serovars of C. trachomatis have been isolated in E. coli as cloned sequences ligated to pBR322. Restriction enzyme cleavage site mapping indicates that these plasmids are closely related. Homologous plasmid sequences have also been detected by DNA hybridization in all of the 200 clinically isolated strains of C. trachomatis which have been examined. DNA sequences homologous to the C. trachomatis plasmid were not found in eucaryotic DNA nor in a plasmid of similar size isolated from C. psittaci. C. trachomatis plasmid genes are expressed in vivo and the plasmid encoded gene products may play a role in the intracellular growth of this organism. Plasmid encoded genes were also expressed from the cloned C. trachomatis plasmid in E. coli minicells and using an E. coli S-30 in vitro transcription translation extract. PMID- 3016783 TI - Isolation and characterization of plasmids from Micromonospora zionensis and Micromonospora rosaria. AB - Three plasmids from Micromonospora species were isolated and characterized. Micromonospora zionensis NRRL5466 (a producer of sisomicin and G-52) carried a high-copy-number plasmid pMZ1 (9.9 kb). Micromonospora rosaria NRRL3718 (a producer of rosamicin) contained a large plasmid, pMR1 (53.5 kb), and a relatively small plasmid, pMR2 (11.0 kb). PMID- 3016784 TI - [Polyneuropathy and residual insulin secretion in diabetes mellitus type I]. AB - In 145 patients suffering from type-I-diabetes with or without signs or symptoms of polyneuropathy basal and glucose-glucagon-induced secretion of insulin was determined. Patients without remaining insulin secretion exhibited somewhat more often polyneuropathies, slowing of nerve conduction, or reduced respiratory heart arrhythmia. If diabetes lasts for more than 10 years, insulin secretion ist reduced to such a low level that its may not have any significant preventive capability with respect to polyneuropathy. PMID- 3016785 TI - [Correlation between the morphology of brain stem evoked auditory potentials of the brain stem and personality dimensions]. PMID- 3016787 TI - Clomipramine enhances prolactin and growth hormone responses to L-tryptophan. AB - The effects of the 5-hydroxytryptamine (5-HT) uptake inhibitor clomipramine on the prolactin (PRL) and growth hormone (GH) responses to L-tryptophan (LTP) were assessed in six normal subjects. Oral administration of 20 mg clomipramine 2 h prior to LTP infusion (50 mg/kg) significantly enhanced both endocrine responses, suggesting that the increases in plasma PRL and GH produced by LTP are mediated by 5-HT pathways. These findings, taken together with previous observations that 5-HT2 receptor antagonists do not attenuate the PRL response to LTP, suggest that the 5-HT-induced release of PRL in man may be mediated by 5-HT1 receptors. PMID- 3016786 TI - The antinociceptive effect of intracerebroventricularly administered prostaglandin D2 in the rat. AB - Intracerebroventricular administration of prostaglandin D2 (PGD2), the major PG in the rat brain, produced a dose-related anti-nociceptive effect in rats as assessed by the rat tail-hot wire, hot plate and phenylquinone-induced writhing techniques. The antinociceptive action of centrally-administered PGD2 was markedly attenuated after pretreatment of the rats with 5,6-dihydroxytryptamine, a selective neurotoxin for central serotonergic neurones, and p chlorophenylalanine, a specific inhibitor of serotonin synthesis, indicating that the PGD2 action is serotonin-mediated. Naloxone, an antagonist of endogenous opioid receptors, also antagonised the antinociceptive action of PGD2. Earlier reports from this laboratory have shown that the antinociceptive action of centrally-administered PGE1 in rats is a serotonin-mediated effect and is also antagonised by naloxone. It was further shown that PGD2, like PGE1, augments serotonin turnover in the rat brain. The present findings support the view that PGD2 shares some of the central actions of PGEs and, like the latter, may function as a neuromodulator in the central nervous system. PMID- 3016789 TI - Coolfont report: a PHS plan for prevention and control of AIDS and AIDS virus. PMID- 3016790 TI - Humoral hypercalcaemia of malignancy: metabolic and histomorphometric studies during surgical management of the primary tumour. AB - Several aspects of calcium metabolism were studied in five patients during the surgical exploration of malignant tumours associated with humorally-mediated hypercalcaemia. Before operation in all patients the renal tubular threshold for calcium reabsorption was raised and the threshold for renal tubular phosphate reabsorption depressed. On removal of the primary tumour in three cases, serum calcium returned to normal, renal calcium threshold fell, renal phosphate threshold rose, but urinary hydroxyproline excretion did not change. In two patients where the tumour proved inoperable, serum calcium remained elevated and no changes in renal calcium threshold or phosphate threshold occurred. Histomorphometry carried out on biopsy specimens from four patients showed normal bone resorption in three, and slightly increased resorption in one, without depression of osteoblastic bone formation. It is suggested that hypercalcaemia in these patients resulted mainly from an alteration in renal calcium threshold caused by a humoral substance released by tumour cells. Correction of hypercalcaemia on removal of the primary tumour was achieved rapidly and could be explained principally by a reduction in renal calcium threshold with increased loss of calcium into the urine. These data contrast with those of many previous studies which have emphasised the predominant role of accelerated osteoclastic bone resorption as the principal cause of hypercalcaemia in malignancy and suggest that a renal effect of the putative humoral agent may predominate in some cases. PMID- 3016791 TI - Effect of WR-2721 on fetal development in the rat. AB - The radioprotector WR-2721 has been shown to radioprotect all tissues studied except the central nervous system. However, it has not yet been used to radioprotect the fetus. In this study we determined that [14C]WR-2721 injected into pregnant rats quickly passed the placenta and was concentrated by the fetus. In addition, we evaluated the toxicity of WR-2721 (2.5-600 mg/kg) to pregnant rats and their fetuses during the period of major organogenesis at Days 9, 11, and 14 postconception. Pregnant animals were only slightly more (10%) sensitive (LD50, 580 mg/kg) to WR-2721 than nonpregnant cohort animals (LD50, 640 mg/kg). At concentrations of 50 mg/kg or less there was a small but statistically significant increase in fetal deaths, while at doses greater than 300 mg/kg a larger degree of fetal mortality occurred. Maximal fetal weight loss, to about 84% of control, was found at 500 mg/kg. No changes in head dimensions or gross malformations of the surviving fetuses were detected at any time or concentration. Of all the parameters measured in this study none demonstrated a predilection for any specific period of major organogenesis. The results of this study indicate that while WR-2721 demonstrates a dose-related embryotoxicity it is not teratogenic. PMID- 3016792 TI - [Evaluation of the cataractogenic effect of 9 GeV protons]. AB - A study was made of the regularities of formation of lenticular opacity in mice exposed to 9 GeV protons and 60Co-gamma-rays. The RBE coefficients, calculated by the nonparametric method, were found to depend upon dose and time after irradiation. It was shown that after small radiation doses (0.25 to 0.50 Gy) the RBE coefficients increased from 1 to 8 with increasing period of observation. With higher doses (up to 5.0 Gy) the RBE coefficient increase in time was less pronounced. PMID- 3016788 TI - Effects of trazodone treatment on alpha-2 adrenoceptor function in depressed patients. AB - Tricyclic antidepressant drugs reduce the sensitivity of alpha 2 adrenoceptors during long-term treatment. In the present study, the alpha 2 adrenergic agonist clonidine was administered to 11 depressed patients before and during treatment with the triazolopyridine antidepressant trazodone (TRZ). Clonidine's ability to decrease blood pressure (BP) and plasma levels of the norepinephrine metabolite 3 methoxy-4-hydroxyphenylethyleneglycol (MHPG), and to increase sedation and plasma growth hormone (GH), were measured. TRZ had little effect on these indices of pre and postsynaptic alpha 2 receptor function, suggesting that the antidepressant properties of TRZ are not related to changes in alpha 2 adrenoceptor sensitivity. PMID- 3016794 TI - Patterns of contrast enhancement of benign and malignant hepatic neoplasms during bolus dynamic and delayed CT. AB - Bolus dynamic and delayed computed tomographic (CT) scans of the liver were evaluated in 43 patients with 54 hepatic hemangiomas and 111 patients with primary or secondary malignant hepatic neoplasms. Twelve patterns of contrast enhancement were recognized during the bolus dynamic phase and delayed scanning. A "typical" CT pattern for hemangiomas (present in 29 of 54 hemangiomas [53.7%]) was established: (a) diminished attenuation prior to intravenous contrast medium administration (excluding lesions arising in a liver with diffuse fatty infiltration), (b) peripheral contrast enhancement during the bolus dynamic phase, and (c) complete isodense fill-in on delayed scan images. Using these criteria, we distinguished hemangiomas from malignant neoplasms in most patients. Only one of 63 (1.6%) malignant neoplasms manifested these typical CT criteria of hemangioma. There is an 86% chance that a lesion with the typical CT appearance of hemangioma is actually a hemangioma, even when found in a patient with a known nonhepatic primary neoplasm. PMID- 3016793 TI - [Characteristics of the carcinogenic effectiveness of 645 MeV protons and 60Co gamma-irradiation]. AB - The carcinogenic effect of 645 MeV protons and 60Co-gamma-rays (1, 2 and 4 Gy) on mongrel female rats was comparatively studied. The observations were made throughout the entire life span of the exposed animals. Revealed were a similar biological effectiveness of the two types of radiation and a common nature of the carcinogenic transformations. PMID- 3016795 TI - Comparative studies on morphogenesis of human, simian and bovine rotavirus in cell culture. AB - Regardless of different host cells and trypsin addition to inoculum, three rotavirus strains (human rotavirus strain Wa, SA 11 virus, bovine rotavirus) displayed the same features in adsorption, endocytosis at 30 min p.i. and the following steps of morphogenesis. The first progeny virus was regularly observed in the RER cisternae at 5 h p.i. It appears that virus yield enhancement by addition of trypsin was due to a greater number of cells having been infected. The infected single cell did not produce more virus particles. Crystalloid arrangement of provirus particles in viroplasm was only observed with the strain Wa virus. The strain Wa and bovine rotavirus produced intracytoplasmic and intranuclear tubes. SA 11 virus induced only intranuclear striated ribbons. PMID- 3016796 TI - The second messenger system in the stimulation of cellular growth. AB - A survey is given of recent results on ionic fluxes as fast events following proliferative stimulation of cells. From this, a hypothesis of interactions of ionic fluxes, pH and cAMP concentration shifts as a system of second messengers leading to cellular growth is developed. Own X-ray microanalytical results from hepatocytes in vivo of young and adult rats are shown to be consistent with that hypothesis. PMID- 3016798 TI - [AIDS--acquired immunodeficiency syndrome]. PMID- 3016797 TI - [Characterization of vasopressin receptors in cerebral endothelial cells]. AB - In target cells with receptors of the V2 type, vasopressin activates the adenylate cyclase and induces, finally, a clustering of intramembranous particles in the plasma membrane. Both characteristic effects, however, could not be observed at hippocampal capillaries of the rat. The presumed vasopressin receptors at cerebral capillaries that represent the blood-brain barrier are, therefore, probably not of the V2 type. PMID- 3016799 TI - [Coexistence of hereditary peripheral nerve hypersensitivity to pressure with other diseases of the peripheral nervous system]. PMID- 3016800 TI - Persistence of contaminant bacteria in dental laboratory pumice. PMID- 3016801 TI - Context specificity of latent inhibition in taste aversion learning. PMID- 3016802 TI - [Correction of the deficient mandibular alveolar crest with hydroxyapatite. Preliminary study]. PMID- 3016804 TI - [CT false negatives in supratentorial malignant astrocytomas]. PMID- 3016803 TI - [Adenoid cystic carcinoma: 3 clinical cases with different localizations in the ENT area]. PMID- 3016805 TI - [Cytomegalovirus: prevalence of antibodies in a control group]. PMID- 3016807 TI - [Pulmonary alveolar proteinosis. Case report]. PMID- 3016806 TI - [Results of digital subtraction angiography in children and adolescents]. AB - In 43 children and adolescents, DSA was carried out, in 29 cases by the intravenous and in 17 cases by the intra-arterial route. IV-DSA was usually performed by putting a cannula (14 to 18 G) into the antecubital vein (bolus injection of 5 to 40 ml. of contrast and 5 to 20 ml. saline at 2 to 12 ml./sec.). The indication for IV-DSA is the investigation of congenital cardio-vascular abnormalities (stenosis or atresia of the pulmonary artery, anomalies of the pulmonary veins or the aortic arch and following vascular surgery). In these cases the more invasive heart catheter examinations can be avoided. IA-DSA is performed with a catheter with a fine lumen (4 to 5 F), diluting the contrast with an equal amount of saline. The advantage of IA-DSA is that it is less invasive, uses less contrast and provides better contrast and detail. PMID- 3016809 TI - [Percutaneous catheter recanalization (angioplasty) of a subclavian occlusion with the subclavian steal syndrome]. PMID- 3016808 TI - [Congenital absence of the left proximal pulmonary artery and a high right-sided aortic arch]. PMID- 3016810 TI - Unusual evolution of chronic granulomatous disease with mediastinum localisation and diffusion into vertebrae, ribs and vertebral canal. PMID- 3016811 TI - Posterior mediastinal teratoma. PMID- 3016812 TI - Necrotic hepatic adenoma. PMID- 3016813 TI - [Periarterial soft tissue calcifications in the hand of patients undergoing dialysis for renal insufficiency]. PMID- 3016814 TI - [Intra-arterial DSA of the pelvic/leg vasculature using a 57 cm image intensifier]. AB - 100 consecutive intraarterial DSA's of the lower extremities obtained with a 57 cm image intensifier were evaluated retrospectively by 2 radiologists and 3 surgeons. The studies were considered diagnostic in 95% of the cases. The results, advantages and possible disadvantages are presented and compared to those associated with conventional film angiography and with i.v. DSA. PMID- 3016815 TI - Subtraction pitfalls in venous DSA of the renal arteries. AB - Venous DSA of the renal arteries was performed in 32 patients for suspected renovascular hypertension who were considered for percutaneous transluminal angioplasty (PTA). To trace the causes of contradictory or divergent findings in our material, v-DSA and conventional angiography prior to PTA were compared. Superimposition of dense bone structures on the origins of the renal arteries was the most common cause of different findings. In 22 arteries with superimposition, 11 were not the same (50%). In 42 arteries without superimposition 7 were not comparable (16.5%). The difference between both groups is statistically significant (chi square test: p less than 0.05). Other possible factors of influence such as motion artifacts, superimposition of vessels and calcifications in the region of the renal arteries, had no measurable effect. The importance and the origin of subtraction artifacts caused by dense bone structures are discussed. PMID- 3016816 TI - [Transvenous DSA of the extracranial cerebral arteries. Causes of reduced image quality based on a critical analysis of 1000 patients]. AB - In this study images of 1000 patients examined via transvenous DSA were analysed and the image quality scrutinised for each vascular region while determining the causes of image quality reduction. The results show that the diagnostic image quality was 98% (excellent/good 79%, fair: 19%) for a. carotis communis, 94% (69 and 25%, respectively) for carotid bifurcation, 84% (54%, 30%) for distal int. carotid arteries, 91% (65%, 26%) for vertebral arteries and 74% (45%, 29%) for basilar artery. Reasons for image quality reduction were artifacts due to movements by the patients, and were also due to vascular superposition. Reduced left ventricular function resulting in prolonged circulation time and dilution of contrast material was frequently combined with an advanced age of the patients (greater than 75 years). Our studies suggest that transvenous DSA is not useful in patients with significantly reduced cardiac output who do not cooperate properly and are of advanced age. It may, therefore, be omitted to avoid unnecessary stress and high cost. In such cases intraarterial DSA or conventional angiography is indicated. PMID- 3016817 TI - [Epidural empyema and orbital phlegmon. Computerized tomographic diagnosis of rare complications of sinusitis]. AB - Among 4019 computed tomograms of the head performed in 2 years we found 2 epidural empyemas combined with an ipsilateral orbital phlegmon and one periorbital abscess with an incipient orbital phlegmon. In all 3 patients they turned out to be intracranial or orbital complications of an adjacent paranasal sinusitis. The CT diagnosis was a guideline for further therapy and was verified by immediate surgery. The reliable diagnosis of orbital and intracranial space occupying lesions makes CT the radiological method of choice in case of suspicion of a complicated sinusitis. PMID- 3016819 TI - [Nuclear magnetic resonance tomographic diagnosis of joint changes. Value of chemical shift imaging]. AB - Chemical shift imaging permits separate demonstration of water and fat-bound protons in a selected plane. Compared with normal spin-echo sequences, specific information can be obtained in the investigation of internal joint structures. A pilot study was carried out on the possibility of showing normal and abnormal structures, using normal controls, and 16 patients with various joint abnormalities. PMID- 3016818 TI - [Nuclear magnetic resonance tomography of the temporomandibular joint. Comparison with computerized tomography and arthrography]. AB - 79 patients aged 6 to 66 years (9 men and 70 women) with abnormalities of the TMJs were examined by magnetic resonance tomography (132 joints) and the results were compared with CT (16 joints) and arthrography (39 joints). Magnetic resonance tomography showed forward luxation of the meniscus in 82 joints (62%). In 34 joints (26%) the meniscus spontaneously resumed normal position when the mouth was open, but in 48 joints (36%) the displacement was permanent. The accuracy of resonance tomography was equal to that of arthrography and superior to CT. It was particularly suitable for follow-up examination after surgery (23 cases) when invasive arthrography would be contraindicated or difficult. Because of the high resolution of the soft tissue components in the TMJ, resonance tomography should be able to diagnose inflammatory and degenerative changes in the meniscus and ligaments. PMID- 3016820 TI - [X-ray morphology and pathologic anatomy of osteoblastoma]. AB - Between 1974 and 1984, 24 patients with osteoblastomas were entered in the bone tumour registry of the Gerhard Domagk Institute of Pathology and Institute for Clinical Radiology of Westphalia. These cases are evaluated. Osteoblastoma is a benign primary bone tumour with a peak incidence between ten and 20 years and a sex ratio of 18 males to 4 females in our series. The site of predilection is the spine (59%) with long bones in second place (32%). The tumor is mostly situated in the medulla. Radiologically it usually appears as a well demarcated translucency with a narrow rim and marginal sclerosis. Variations in radiological appearance and the histology and treatment are described. A malignant variant, the "aggressive osteoblastoma", is discussed and one case is described. The classification of these cases is considered. PMID- 3016822 TI - [Malignant pleural mesothelioma. Value of 67Ga scintigraphy compared to computerized tomography]. AB - In 34 patients with suspected malignant pleural mesothelioma the results of computed tomography are compared with the findings of 67Ga-scintigraphy. The differential diagnosis of 14 pleural mesotheliomas, 7 pleural carcinoses, 10 inflammatory and 3 other pleural diseases is performed more accurately by CT than by scintigraphy. 67Ga uptake depends on the thickness of inflammatory as well as malignant lesions. Thus, numerous pleural processes that can be localised by CT escape scintigraphic detection. CT is indicated if there is clinical and radiological suspicion of pleural mesothelioma; in that case, there is hardly any indication for 67Ga scintigraphy. PMID- 3016821 TI - [Image analysis in hip sonography. Classification criteria]. AB - Based on 5000 ultrasound studies of infantile hips, Graf's classification and diagnostic criteria are reviewed with regard to their ability to discriminate between normal and pathological conditions. Additional criteria (delta angle and vector line) are presented, and the results compared. The proposed approach via visual analysis is explained as a flow chart. The consequences of the methodical limitations for use as a screening procedure are discussed. PMID- 3016823 TI - [Scintigraphic diagnosis of inflammatory small bowel stenoses in Crohn disease using 111In oxine-labeled leukocytes]. AB - 17 patients with known small bowel involvement in Crohn's disease (clinically active, n = 14; clinically inactive, n = 3) were examined within 8 days via barium enemas of the small bowel (Pansdorf's method or enteroclysis) and by 111In oxine labelled leucocytes. From 19 radiologically diagnosed small bowel stenoses 14 were classified as inflammatory and 5 as non-inflammatory. The leucocyte scan also showed 14 inflammatory stenoses. The not inflamed stenoses could not be diagnosed scintigraphically. The barium enemas of the small bowel and the leukocyte scans both correctly diagnosed the acute inflamed segments. The inability to show non-inflamed segments (n = 5) and to localise small bowel stenoses exactly is disadvantageous in the scan. The advantage of the leucocyte scan is a non-invasive examination without specific bowel preparation and the possibility to diagnose additionally inflamed large bowel segments (n = 4), fistulas and abscesses (n = 2). The leucocyte scan offers a useful expansion of the diagnostic tools in small bowel diseases, especially in radiological problems in patients with Crohn's disease. PMID- 3016824 TI - Pharyngo-oesophageal webs in dysphageal patients. A radiologic and clinical investigation in 1134 patients. AB - Among 1134 patients, cineradiologically examined because of dysphagia, 85 (7.5%) had webs in the pharyngo-oesophageal segment. Webs were more common in women (10%) compared to men (5%). Radiologic characteristics of the webs such as precise location, multiplicity, circumferential extension, thickness, accompanying streamline phenomenon and encroachment on the lumen, were compared to the presence of concomitant anaemia, thyroid disease, neoplasm, as well as the age and sex of the patients. Webs were regularly deeper in women compared to men. Patients with iron deficiency anaemia had thicker webs compared to patients without such anaemia. No other radiologic characteristics were found that could be used for distinguishing these potentially more significant webs from those in patients without such concomitant diseases. PMID- 3016825 TI - [Dynamic cavernosography. Radiologic diagnosis of venous induced erection disorders and erectile body diseases]. AB - In 30 to 50% erectile dysfunctions are due to vascular disorders. Roughly a third of these vasculogenic disturbances is based on venous disorders. These venous induced erectile failures may be objectivated and radiologically located via dynamic cavernosography, combined with a simultaneous recording of a cavernous pressure profile. Based on over 130 examinations it was possible to provide both an exact description of the normal venous drainage in normal potent men and to give a good idea of the different venous leakages in patients complaining of erectile dysfunctions. Congenital and acquired penile deviations along with Peyronie's disease may also be appropriate for dynamic cavernosography. PMID- 3016826 TI - [Comparison of the renal ultrasound-furosemide test with the isotope nephrogram. Possible clue to the retention phenomenon?]. AB - In 23 patients with essential hypertension renographic and sonographic examination of the kidneys was compared. No correlation could be observed for functional parameters and urinary excretion between the two different methods. Correlations have only been detected between renographic retentions and furosemide-conditioned swelling of the kidneys in sonography: patients with severe retention showed a statistically significant stronger response to furosemide. The increased retention in the renal pelvis could be responsible for alterations in sonographic findings. This study provides new data on the mechanisms of retention, whereby retention in the renal pelvis in some patients seems to be most probably caused by nephrosclerosis. PMID- 3016827 TI - [Intra-arterial digital subtraction angiography (i.a. DSA) in the preoperative diagnosis of abdominal masses]. AB - The diagnostic value of i.a. DSA, compared with conventional arteriography (n = 48), was proven in patients (n = 116) with abdominal tumours including intrahepatic masses. Arteries supplying the tumour, anomalous vessels and pathological malignant vascular abnormalities were visualised reliably in i.a. DSA despite reduced spatial resolution. An improved definition of hypervascular tumours was possible during the early arterial phase and of hypovascular or avascular masses during the late parenchymal phase of i.a. DSA. The dynamic evaluation of contrast flow is simpler and more reliable using the continuous mode in i.a. DSA than with the static imaging of a conventional film sequence. The advantages of i.a. DSA in preoperative vascular mapping and in detailed diagnosis of abdominal tumours and masses result in replacing conventional arteriography with very few exceptions. PMID- 3016828 TI - [Digital radiography. Experiences with the SP system]. AB - Digital radiography which would have to replace conventional radiography with equal or improved quality is the still missing link in the development of a fully digital imaging department of the future. Various attempts have been made to close this gap. This article describes the SP system, a large area detecting digital imaging system, originally designed by Fuji. Its components are the imaging plate, coated with a stimulable phosphor (SP), which is used like a conventional film-screen cassette, a laser-based image reader, the image processor and a laser-based image printer. After a six months' test in hospital routine, the advantages, shortcomings and possible improvements are discussed. PMID- 3016829 TI - [Computerized tomographic study of medieval coffins]. AB - Two medieval coffins containing neonates were examined by CT. The advantages of using computed tomography, compared with conventional x-ray techniques, for analysing various objects within a receptacle are demonstrated. PMID- 3016830 TI - [AIDS physiopathology and epidemiology of the infection with the LAV virus]. AB - LAV is the causative agent of AIDS. Its biological properties, assessed in vitro, fully account for the major immunological abnormalities which characterize the disease. LAV is a lentivirus which presents a selective tropism for T4+ lymphocytes, the very cells that are destroyed in AIDS. Its replication is associated with inhibition of lymphocyte proliferative capacity and with a cytopathic effect. In most instances, LAV infection is silent, for years at least, or it can result in various clinical syndromes among which AIDS is the most serious but the less frequent. Such a polymorphism can be explained by LAV biological properties and by the conjunction of numerous cofactors. PMID- 3016831 TI - [Transmission of AIDS virus by transfusion and blood products. Risks and preventive strategies]. AB - This article reviews some of the epidemiologic aspects of transmission of LAV through transfusion of blood and blood products in the light of data available until late December 1985 in France, Europe and the United States. As of December 1985, blood transfusion has been considered the etiologic factor in 2.58% of the 15,172 cases of AIDS reported in the USA and in 5.6% of 1,573 cases of AIDS registered in Europe. Whole blood, cellular blood components and plasma derivatives except Albumin and immunoglobulins have been incriminated in 1.75 to 2.22 of the above percentages. Hemophiliacs under long term therapy by factor VIII and factor IX concentrates (0.83 to 3.36 of the above percentages) represent a highly exposed group when one considers the small proportion of these individuals in the general population (1/10,000 inhabitants). Three preventive measures have been officially implemented in the French transfusion network: self refrainment and deferral of prospective donors belonging to risk factor groups, systematic screening of donated blood for the presence of anti-LAV antibodies and elimination of seropositive units, heat treatment of coagulation factor concentrates to achieve viral inactivation. Information and medical follow up of LAV-contaminated donors thus identified and of their partners represent an important issue among the current public health problems. PMID- 3016832 TI - [Metachronous ovarian metastasis of colorectal carcinoma (Krukenberg's tumor)]. PMID- 3016833 TI - [Nutrition and diabetes]. PMID- 3016834 TI - Atrial natriuretic factor: a new hormone of cardiac origin. PMID- 3016835 TI - Recent progress in the control of aldosterone secretion. PMID- 3016836 TI - Studies of the multiple molecular forms of bioactive parathyroid hormone and parathyroid hormone-like substances. PMID- 3016838 TI - Biochemical basis for improving chemotherapeutic regimens in liver malignancies. PMID- 3016839 TI - Concepts of liver resection for primary and secondary tumors. PMID- 3016837 TI - Nephrogenous cyclic AMP, adenylate cyclase-stimulating activity, and the humoral hypercalcemia of malignancy. PMID- 3016840 TI - Surgical treatment of primary liver cell carcinoma in China. PMID- 3016841 TI - Liver transplantation for liver tumors. PMID- 3016842 TI - Intraarterial chemotherapy for colorectal liver metastases and hepatomas using a totally implantable drug infusion pump. PMID- 3016844 TI - Radiolabeled antibody in the treatment of primary and metastatic liver malignancies. PMID- 3016843 TI - Combined use of drugs and radiation in the treatment of liver metastases. PMID- 3016846 TI - Restriction fragment length polymorphism of the human insulin gene region among type II diabetic Mexican-Americans and Tunisians. AB - The human insulin gene is flanked by a polymorphic locus that is located approximately 500 base pairs (bp) from the 5' end of the point where transcription begins (Bell et al. 1981; Bell et al, 1982). Its occurrence is due to an insertion-deletion region which gives rise to two major classes of alleles: those containing small insertions of 0-600 bp and those containing larger insertions of 1,600-2,200 bp (Owerbach and Nerup, 1982). Insertions of 600-1,600 bp are rare (Rotwein et al., 1983). The larger insertions have previously been reported to be associated with type 2 diabetes (Owerbach and Nerup, 1982). We have conducted studies on a Mexican-American population in Starr County, Texas (98% Mexican-American) and a Tunisian population in Tunis, Tunisia, to determine if the frequency distribution of these classes of insulin gene alleles are similar to the previously reported frequency distributions and if any of the classes of alleles are associated with type 2 diabetes in these populations. We conclude that none of the classes of insulin gene alleles are associated with type II diabetes among Mexican-Americans or Tunisians, and that the frequency distributions of the insulin gene alleles do not vary significantly between the Tunisians, Mexican-Americans, or the aggregate data resulting from combining the insulin gene frequencies of several of the populations described thus far (Bell et al., 1984). PMID- 3016845 TI - Stimulation of cAMP accumulation in rat aorta and diaphragm by fluorine containing compounds. AB - Evidence is presented that accumulation of cAMP in isolated rat thoracic aorta and diaphragm is stimulated by several fluorine containing compounds (NaF, Na2PO3F (MFP) and SnF2). Time course experiments with NaF showed that maximal stimulation of cAMP accumulation was observed within 2.5 min. NaF and MFP produced significant increases in cAMP accumulation at concentrations of 0.1 microM and SnF2 produced a significant increase at 0.01 microM. The sensitivity of these tissues to the fluorine compounds is similar to that previously reported (Curro and Mickunas, 1981) for the inhibition of norepinephrine induced contractions by fluorine compounds in isolated thoracic aorta. PMID- 3016847 TI - Chelation in metal intoxication. XX: Effect of pre-treatment with chelators on the distribution of mercury. AB - The effect of pre-treatment with sodium 2,3-dimercaptopropane 1-sulfonate (DMPS), sodium diethyldithiocarbamate (DDC) and calcium trisodium diethylenetriaminepentaacetate (DTPA) on the fate of injected mercuric chloride in rats was investigated. DMPS was the most effective chelator in reducing the uptake of Hg (203) in tissues and was the only chelator to enhance the urinary excretion of Hg (203). The results suggest affinity of Hg towards sulfur moiety and that the protective effects of pre-treatment are also shared by non-steroidal thiol chelators. PMID- 3016848 TI - Effects of chronic administration of a methionine-enkephalin analogue on the zona glomerulosa of the rat adrenal cortex. AB - The effects of D-ala2-met-enkephalinamide (DALA) on the zona glomerulosa of dexamethasone-ACTH-treated rats were investigated by coupled radioimmunologic and morphometric techniques. Short-term DALA administration provoked a significant increase in the aldosterone plasma level along with a notable lipid droplet depletion in zona glomerulosa cells. Long-term DALA treatment induced a striking hypertrophy of zona glomerulosa cells and a further rise in the blood concentration of aldosterone. These findings seem to indicate that DALA is involved not only in the acute enhancement of aldosterone output but also in the stimulation of the growth and steroidogenic capacity of rat adrenal zona glomerulosa. PMID- 3016849 TI - Foot-and-mouth disease vaccination: a multifactorial study of the influence of antigen dose and potentially competitive immunogens on the response of cattle of different ages. AB - Groups of 68 and 66 cattle aged 12 and 24 months respectively were each subdivided into 16 groups and inoculated with foot-and-mouth disease vaccines containing O1 Campos, A24 Cruzeiro and C3 Pando virus strains. The 140S antigen mass of the O1 and A24 valencies was varied while that of C3 was held constant. Multifactorial comparisons between the 21 day serum neutralising antibody titres showed that over most of the range there was a linear log dose response relationship. Doubling the antigen dose increased the serum antibody titres against both A24 Cruzeiro and O1 Campos by approximately 0.15 log10. The A24 antigen was about 30 times more immunogenic than the O1 with C3 intermediate between the two. At high antigen doses the responses flattened but the level at which this occurred depended on the immunogen administered. No difference could be demonstrated between the responses of 12- and 24-month-old cattle and there was no evidence of competitive inhibition or enhancement between the virus strains included in the vaccines. PMID- 3016850 TI - Electrophysiological evidence of peripheral nerve dysfunction in six dogs with botulism type C. AB - In six dogs with botulism type C electrophysiological examinations showed: fibrillation potentials and prolonged insertional activity; low amplitude of the evoked muscle action potential; decrease in amplitude of the compound muscle action potential with slow repetitive stimulation; slowing of motor and sensory velocities in the peripheral nerve; and restoration of velocity and amplitude corresponding to clinical improvement. These findings indicate peripheral nerve dysfunction which cannot be explained adequately by current knowledge of the action of botulinum toxin on cholinergic nerve endings. It is therefore suggested that botulinum toxin also interferes with peripheral nerve conduction. PMID- 3016851 TI - Serial inoculation of sheep with two bluetongue virus types. AB - Groups of sheep inoculated with bluetongue virus type 4 were challenged at various intervals after inoculation (from seven to 70 days) with bluetongue virus type 3. Examination of the clinical and serological response showed that animals were protected from challenge with a second bluetongue virus for up to 14 days after the inoculation of the first virus type. An adoptive transfer experiment in monozygotic sheep involving both antibody and T lymphocytes was carried out. Only partial protection was observed against heterologous virus challenge, indicating that although the T cell response has a cross-protective component, antibody is not involved. These observations indicate that current vaccination procedures should be reappraised, particularly in terms of revaccination with multiple bluetongue virus type. PMID- 3016853 TI - [Application of the passive hemagglutination test to the study of equine rhinopneumonitis. I. Serologic studies in rabbits inoculated with equine herpesvirus type 1]. PMID- 3016852 TI - Effects of naloxone on pituitary gonadotropins throughout sexual maturation in male and female rats. PMID- 3016854 TI - [Dietary fiber intake and fecal waste of nutrients in subjects living in areas with poor environmental sanitation]. PMID- 3016855 TI - [Mineral metabolism and secondary hyperparathyroidism in celiac disease]. PMID- 3016857 TI - [Resistance of gram-negative bacilli to 6 aminoglycosides]. PMID- 3016856 TI - [Primary adenocarcinoma of the vermiform appendix]. PMID- 3016858 TI - [Chemotherapy of pulmonary cancer]. PMID- 3016859 TI - [Thermal insulating properties of asbestos or kaowool as a casting mold lining]. PMID- 3016860 TI - [Emission tomography: its impact on the study of metabolism and on specific cerebral receptors in man]. PMID- 3016861 TI - [Treatment of asthma with high doses of beclomethasone]. PMID- 3016862 TI - [Procedures for the removal of superficial tooth stains]. PMID- 3016863 TI - [Regulation of bronchial motility]. PMID- 3016864 TI - [Medical and legislative aspects of air pollution]. PMID- 3016865 TI - Method to elaborate a national programme of short-course chemotherapy: experience of Romania. PMID- 3016866 TI - [Clinico-therapeutic considerations on the association of pulmonary tuberculosis and ulcerative disease]. PMID- 3016867 TI - [Immunostimulation with a lyophilized bacterial lysate (Bronchovaxom) in patients with chronic suppurative processes of the respiratory tract]. PMID- 3016869 TI - [T-lymphocyte subsets in tuberculous pleurisy]. PMID- 3016868 TI - [Causes responsible for delaying the detection of adult tuberculosis in the current stage of the anti-tuberculosis campaign]. PMID- 3016871 TI - [Evaluation of antitubercular chemotherapy under operational conditions]. PMID- 3016870 TI - [Determination of specific circulating antibodies in pulmonary tuberculosis using the ELISA immunoenzyme technic]. PMID- 3016872 TI - [Evaluation of integrated treatment of tuberculosis at 20 antituberculosis outpatient clinics]. PMID- 3016873 TI - [Methods for optimizing integrated antituberculosis control in the Cluj district]. PMID- 3016874 TI - [Short-term hospital treatment of uncooperative patients]. PMID- 3016875 TI - [Tuberculous pleurisy in adults; diagnostic and therapeutic aspects in 65 cases]. PMID- 3016876 TI - [Factors with a negative effect on the success of short-term antitubercular chemotherapy under operational conditions]. PMID- 3016877 TI - [Dynamic evaluation of integrated antitubercular actions in the Iasi district]. PMID- 3016878 TI - [Possibilities of preventing recurrences in pulmonary tuberculosis]. PMID- 3016879 TI - [Pathogenesis of articular chondrocalcinosis]. PMID- 3016880 TI - Nutrient and environmental growth factors for eight small-sized oral spirochetes. AB - The present investigation was carried out in order to obtain better information about the growth requirements for small-sized oral spirochetes containing two endoflagella from each cell-end. Eight strains of such spirochetes were isolated from subgingival plaque in patients suffering from advanced marginal periodontitis. The strains were maintained under anaerobic conditions in a fluid basal BHI medium with 15% inactivated rabbit serum, 0.07% Noble Agar and 5 micrograms/ml cocarboxylase. Firstly, the effect of trace amounts of oxygen in the atmosphere and pH in the medium on growth of the spirochete strains were examined. Secondly, the effect of different sera incorporated in the medium was examined, and thirdly, the effect of important growth factors in serum was studied by adding different serum components to the fluid basal medium instead of rabbit serum. Growth was always determined after 4 days' incubation at 35 degrees C, either by counting numbers of spirochete cells in a Petroff-Hauser counting chamber or by measuring the turbidity of the culture spectrophotometrically at 600 nm. There was no difference in growth by using an atmosphere containing 1% oxygen or an anaerobic atmosphere. It was found that serum (rabbit or human) was an essential growth component, and no single growth factor could replace rabbit serum. Only a long chain fatty acid mixture and an amino acid solution could, to a minor extent, stimulate growth compared to the basal medium without rabbit serum. Sodium bicarbonate inhibited growth of all strains. Finally, none of the strains fermented a series of low molecular weight carbohydrates, but all strains produced H2S and indole. PMID- 3016881 TI - Correlation between donor cytomegalovirus immunity and chronic graft-versus-host disease after allogeneic bone marrow transplantation. AB - Chronic graft-versus-host disease (GvHD) in bone marrow allograft recipients is frequently preceded by a cytomegalovirus (CMV) infection. To elucidate whether an immune reaction of transplanted donor cells against CMV was involved in the pathogenesis of chronic GvHD, the effect of donor pretransplant CMV immune status on chronic GvHD incidence was analyzed. In 43 bone marrow recipients at risk, the 2-yr cumulative chronic GvHD probability was 55% when the donor was immune, in contrast to 16.5% when the donor was non-immune (p less than 0.002). No correlation between recipient pretransplant CMV immunity and chronic GvHD was observed. Donor CMV immunity did not correlate with acute GvHD or posttranplant CMV infection and seemed to predispose for chronic GvHD regardless of donor and recipient age. However, the proportion of CMV immune donors increased with increasing donor and recipient age. This may account for the higher incidence of chronic GvHD in older patients. The present study suggests that chronic GvHD may be mediated by a reaction of immune donor cells against CMV infected recipient cells. PMID- 3016882 TI - Treatment of immune thrombocytopenic purpura in homosexual men. AB - Over the past 3 yr we have treated 6 homosexual men (age 22-55 yr) with immune thrombocytopenic purpura. 4 of the 6 have antibody to HTLV-III in their serum, 1 of these patients has the acquired immune deficiency syndrome (AIDS), 1 has AIDS related-complex (ARC), and a 3rd has persistent generalised lymphadenopathy (PGL). The platelet count at presentation was between 2 and 35 X 10(9)/l and in each case a bone marrow confirmed active platelet production. Antiplatelet antibodies were demonstrated in 3 of 4 patients tested. 3 of the 6 patients showed a partial response to prednisolone, 2 showed little or no response and the 6th showed a good response. 2 patients received high dose i.v. immunoglobulin - 1 had an excellent response prior to splenectomy, the other showed no response. 5 of the 6 patients had a splenectomy. 3 had a lasting remission (12-27 months after splenectomy), 1 of these has HTLV-III antibodies; 1 had a remission lasting 1 yr, followed by fluctuating thrombocytopenia (21-130 X 10(9)/l) and 1 showed no response. PMID- 3016883 TI - Asymptomatic virus shedding in men with genital herpes infection. AB - 13 men with a history of recurrent genital herpes simplex virus type 2 (HSV-2) infection were followed daily for 4 weeks with samples taken from the urethra for virus isolation. Asymptomatic virus shedding occurred in 5 men who had 1 single positive isolation each. Four of these urethral isolates were typed as HSV-1 and 1 as HSV-2. PMID- 3016884 TI - The importance of pre bone marrow transplantation serology in determining subsequent cytomegalovirus infection. An analysis of risk factors. AB - The yearly incidence of cytomegalovirus (CMV) infection among 73 consecutive bone marrow transplant (BMT) recipients was 68%. Recipients with negative CMV serology prior to transplantation had a yearly incidence of CMV infection of 35% compared to 87% in CMV seropositive patients (p = 0.0001). When the ages of donors and recipients were analysed as continuous variables, both recipients with a younger donor and young recipients had a lower incidence of CMV infection (p = 0.04; p = 0.05). White cell transfusions were significantly associated with an increased incidence of CMV infection (p = 0.03). If white cell transfusions were controlled for, lower marrow cell doses were significantly associated with an increased risk of CMV infection, compared to higher cell doses (p = 0.035). In multivariate analyses, the impact of negative recipient serology was so strong that the other analysed factors did not affect the prognosis for CMV infection, when taken together or separately. 14 patients had symptomatic CMV infection and 13 of those were seropositive prior to BMT. The one-year incidence of symptomatic CMV infection was 33%. None of 12 clinical factors analysed were significantly associated with symptomatic CMV infection. The CMV antibody titer level prior to BMT was not correlated to the risk for symptomatic CMV infection and/or death. The ability to respond with a significant titer rise after BMT was lowered for patients with interstitial pneumonitis compared to patients with other clinical symptoms of CMV infection. PMID- 3016885 TI - [Nongynecological occult cancers diagnosed in the course of their ovarian metastases. A clinical and anatomicopathological study of 8 cases]. AB - Malignant non-gynecological diseases manifested through ovary metastases are rare but not exceptional. In routine histological examinations of the ovary within the last 6 years we have found an unknown non-gynecological disease in 8 patients undergoing surgery in the gynecological clinic in Zurich. One lobular bilateral breast cancer, one pancreatic cancer, three stomach cancers and three lymphomas were diagnosed. The symptoms, treatment, prognosis and anatomo-pathological aspects are discussed. PMID- 3016886 TI - [Advances in the study of the receptors and surface antigens of hematopoietic stem cells]. PMID- 3016888 TI - [Mechanism of dimefline]. PMID- 3016889 TI - [Neurotoxicity of kainic acid and its uses]. PMID- 3016887 TI - [Advances in the study of the mechanism of anti-inflammatory agents]. PMID- 3016890 TI - [Advances in the subtype of opioid receptors]. PMID- 3016891 TI - Chicken heart ubiquinol: cytochrome c reductase--isolation by affinity chromatography and resolution of enzyme into subunits. AB - Chicken heart mitochondrial ubiquinol: cytochrome c reductase has been isolated as a monodisperse form by solubilization with Triton X-100 in low ionic strength and subsequent affinity chromatography. The enzyme complex contains less than 0.15 mumol ubiquinone and 12 mumol phospholipid per mumol enzyme. The spectrum of cytochrome b in the reduced exhibited two maximum alpha-absorption bands at 558 nm and 562 nm; a shoulder at 553 nm for c1. The purified enzyme complex consists of at least 9 subunits with apparent molecular weight of 47 K, 44 K, 36 K, 31 K, 25 K, 17 K, 15 K, 12 K and 9 K. The enzyme complex has been dissociated into subdomains of subunits in the presence of increasing concentration of salt and Triton X-100. Three subunits carrying the redox centers (cytochrome b, cytochrome c1 and Fe-S protein) have been further isolated. PMID- 3016894 TI - Metals and DNA: molecular left-handed complements. AB - Chiral metal complexes provide unique molecular probes for DNA. Chiral reagents that "recognize" different local structures along the DNA strand have been designed by a process in which the asymmetry in shape and size of the complex is matched to that of the DNA helical groove. As a result, the chiral metal complexes provide very sensitive probes for local helical structure, both left- and right-handed. Direct coordination of chiral complexes to the DNA bases adds an element of sequence selectivity to the probe design. With a suitable reactive metal center, reagents that target chemically specific sites along the strand may be developed. One such chiral reagent, which cleaves left-handed DNA sites with photoactivation, has been useful in mapping this distinct conformation and examining its biological role. The conformation-specific molecular cleaver, much like a DNA-binding enzyme, recognizes and reacts at discrete sites along the DNA strand. These site-specific chiral metal complexes provide exciting new tools for probing the local variations in DNA structure and its role in the regulation of gene expression. PMID- 3016893 TI - Fixing exposure limits for toxic chemicals in the UK--some case studies. AB - The former Director of the Health and Safety Executive in Britain describes the process by which limits are fixed for exposure of workers to toxic chemicals. Four examples of the process are analysed in detail, examining the controls introduced since 1975 for asbestos, man-made mineral fibres, styrene, and MbOCA. The toxicity assessments available to the decision makers are summarised, as are also the estimates of the additional costs to industry of stricter limits, and the possible economic effects on production and jobs. The author then draws some general conclusions based on British experience since 1975. These include: that decision taking was possible on the basis of uncertainty about the precise effects on humans of particular levels of exposure; that the decisions could be seen to be sensibly related to the toxicity assessments; and that the precise limit fixed was dependent as much on the costs and practicability of tighter limits as on the nature of the health effects. PMID- 3016892 TI - A new system for the synthesis of high levels of HBsAg sequence in Escherichia coli. AB - Using a system to study promoter activity, we have obtained a promoter fragment from E. coli chromosomal DNA. The HBsAg gene under the control of this promoter could be expressed in E. coli. The expression products are isolated and purified by means of a column of anti-HBs cross-linked to Sepharose 4 B. The pure products are characterized through the double diffusion method on 0.6% agarose and polyacrylamide-SDS gel electrophoresis. The synthesis of high levels of HBsAg sequences in E. coli is confirmed. PMID- 3016896 TI - Psychotomimesis mediated by kappa opiate receptors. AB - The kappa opioid agonists are analgesics that seem to be free of undesired morphine-like effects. Their dysphoric actions observed with the kappa agonist cyclazocine are thought to be mediated by an action at sigma-phencyclidine receptors. The benzomorphan kappa agonist MR 2033 is inactive at sigma phencyclidine receptors. In male subjects, the opiate-active (-)-isomer, but not the (+)-isomer, elicited dose-dependent dysphoric and psychotomimetic effects that were antagonized by naloxone. Thus, kappa opiate receptors seem to mediate psychotomimetic effects. In view of the euphorigenic properties of mu agonists, our results imply the existence of opposed opioid systems affecting emotional and perceptual experiences. PMID- 3016895 TI - Distinct pathways of viral spread in the host determined by reovirus S1 gene segment. AB - The genetic and molecular mechanisms that determine the capacity of a virus to utilize distinct pathways of spread in an infected host were examined by using reoviruses. Both reovirus type 1 and reovirus type 3 spread to the spinal cord following inoculation into the hindlimb or forelimb footpad of newborn mice. For type 3 this spread is through nerves and occurs via the microtubule-associated system of fast axonal transport. By contrast, type 1 spreads to the spinal cord through the bloodstream. With the use of reassortant viruses containing various combinations of double-stranded RNA segments (genes) derived from type 1 and type 3, the viral S1 double-stranded RNA segment was shown to be responsible for determining the capacity of reoviruses to spread to the central nervous system through these distinct pathways. PMID- 3016897 TI - Long-range electron transfer in heme proteins. AB - Kinetic experiments have conclusively shown that electron transfer can take place over large distances (greater than 10 angstroms) through protein interiors. Current research focuses on the elucidation of the factors that determine the rates of long-range electron-transfer reactions in modified proteins and protein complexes. Factors receiving experimental and theoretical attention include the donor-acceptor distance, changes in geometry of the donor and acceptor upon electron transfer, and the thermodynamic driving force. Recent experimental work on heme proteins indicates that the electron-transfer rate falls off exponentially with donor-acceptor distance at long range. The rate is greatly enhanced in proteins in which the structural changes accompanying electron transfer are very small. PMID- 3016899 TI - Immortalization of human T lymphocytes after transfection of Epstein-Barr virus DNA. AB - Epstein-Barr virus (EBV), a ubiquitous human herpesvirus, has the ability to transform human B lymphocytes. No other cell type has been experimentally transformed by EBV, either by intact virions or naked viral DNA and subgenomic fragments. Two immortalized human T-lymphoblastoid cell lines have now been established by transfecting cord blood lymphocytes with purified B95-8 viral DNA enclosed in fusogenic Sendai virus envelopes (RSVE) and then exposing the cells to EBV from a P3HR-1 cell subclone. One of these lines, which has been fully characterized, is termed HBD-1. This line is positive for EBV DNA and expresses surface OKT11, OKT4, and Tac receptors, but not M-1, mu immunoglobulin chains, EBV receptors, or B-1 surface markers. The cells contain fully rearranged T-cell receptor genes and germline immunoglobulin genes. The karyotype of the cells is normal, they do not require interleukin-2 for growth, and do not contain human T lymphotropic virus type I. However, the HBD-1 cells contain incomplete EBV genomes and express several EBV-determined antigens, including the early antigen type D, membrane antigens, but not EBV-determined nuclear antigen (EBNA). This association of the EBV genome with permanently growing hematopoietic cells of non B-cell lineage should prove useful in studies on the mechanism of EBV-mediated cell transformation. PMID- 3016898 TI - Insulin-stimulated hydrolysis of a novel glycolipid generates modulators of cAMP phosphodiesterase. AB - Insulin action may involve the intracellular generation of low molecular weight substances that modulate certain key enzymes. The production of two substances that regulate the activity of adenosine 3',5'-monophosphate phosphodiesterase was evaluated in cultured myocytes by incorporation of radiolabeled precursors. Insulin caused the rapid hydrolysis of a chemically undefined membrane glycolipid, resulting in the production of two related complex carbohydrates as well as diacylglycerol. Both the glycolipid precursor and the aqueous products were monitored by labeling with radioactive inositol and glucosamine. Depletion of the labeled precursor and the appearance of labeled water-soluble products and diacylglycerol occurred within 30 seconds after hormone treatment and was followed by rapid resynthesis of the precursor. The aqueous products that were radioactively labeled appeared chromatographically and electrophoretically identical to phosphodiesterase modulating activities produced by insulin from the same cells. The purified radiolabeled and bioactive substances had similar chemical properties. Hydrolysis of the glycolipid precursor and subsequent generation of products could be reproduced by incubation of extracted lipids with a phosphatidylinositol-specific phospholipase C. These studies suggest that insulin stimulates an endogenous, selective phospholipase C activity that hydrolyzes a novel glycolipid, resulting in the generation of a complex carbohydrate-phosphate substance containing inositol and glucosamine that may mediate some of the actions of the hormone. PMID- 3016900 TI - Calmodulin, cyclophilin, and cyclosporin A. PMID- 3016901 TI - PHS invites industry collaboration on AIDS vaccine. PMID- 3016902 TI - Direct polyclonal activation of human B lymphocytes by the acquired immune deficiency syndrome virus. AB - When B lymphocytes from normal human peripheral blood were incubated for 1 hour with the retrovirus that causes the acquired immune deficiency syndrome (AIDS), the B cells showed marked proliferation and differentiation. Proliferative responses to the virus peaked on day 4 and appeared to be independent of accessory cells. This finding was repeated with three separate viral isolates, one of which was from a patient from Zaire. The magnitude of the observed responses was comparable to that seen with standard polyclonal B-cell activators. This phenomenon may be at least partially responsible for the polyclonal B-cell activation seen in patients with AIDS. PMID- 3016903 TI - Detection of AIDS virus in macrophages in brain tissue from AIDS patients with encephalopathy. AB - One of the common neurological complications in patients with the acquired immune deficiency syndrome (AIDS) is a subacute encephalopathy with progressive dementia. By using the techniques of cocultivation for virus isolation, in situ hybridization, immunocytochemistry, and transmission electron microscopy, the identity of an important cell type that supports replication of the AIDS retrovirus in brain tissue was determined in two affected individuals. These cells were mononucleated and multinucleated macrophages that actively synthesized viral RNA and produced progeny virions in the brains of the patients. Infected brain macrophages may serve as a reservoir for virus and as a vehicle for viral dissemination in the infected host. PMID- 3016904 TI - Strategies for an AIDS vaccine. PMID- 3016906 TI - [A new use for enzymes]. PMID- 3016905 TI - Antiplatelet drugs in the management of patients with thrombotic disorders. PMID- 3016907 TI - [Effect of hypothalamic arcuate stimulation on the pain-evoked unit discharges of the thalamic parafascicular nucleus: a preliminary analysis]. PMID- 3016908 TI - Neurochemical mechanisms in the genetics of alcohol phenotypes. PMID- 3016909 TI - Cancer of the large bowel in children. AB - We present the morphologic and clinical features of large bowel carcinoma in 22 patients seen over 18 years at St. Jude Children's Research Hospital. We saw 18 cases of mucinous adenocarcinoma, three of well differentiated adenocarcinoma, and one case of poorly differentiated adenocarcinoma. The patients ranged in age from 9 to 19 years with a median age of 15. There were 12 female and ten male patients; 12 were black and ten were white. Eighteen patients had Dukes' stage C disease, with median survival of seven months and one patient alive 12 months after diagnosis. Three patients had Dukes's stage B disease, with two patients surviving 16 months each and one presently alive seven months after diagnosis. Only one patient had Dukes' stage A disease and is alive 121 months after diagnosis. Large bowel carcinoma in children is predominantly mucinous adenocarcinoma and occurs in the adolescent age group. Tumor distribution is fairly even throughout the large bowel, and all subtypes of the tumor are positive for carcinoembryonic antigen (CEA) on immunohistologic study. Active tumor and tumor regrowth are always accompanied by high serum levels of CEA. The tumor has no predilection for sex, but is significantly more frequent in blacks (P less than .05). Discovery of the tumor in an early stage improves the prognosis. PMID- 3016911 TI - The hepatitis B problem in the Philippines. PMID- 3016910 TI - Assessment of therapeutic plasmapheresis in demyelinating neurologic disorders. AB - Considerable controversy exists with regard to therapeutic efficacy of plasmapheresis in the immune-mediated demyelinating disorders of the peripheral and central nervous system: acute inflammatory polyneuropathy (Guillain-Barre syndrome), chronic inflammatory demyelinating polyneuropathy, and multiple sclerosis. In an effort to establish specific situations where plasmapheresis was of therapeutic value, we reviewed the experience at Southwestern Medical School, University of Texas Health Science Center at Dallas, and that published in the literature. In acute inflammatory demyelinating polyneuropathy (Guillain-Barre syndrome), plasmapheresis may prevent progression of the illness, and it significantly increases the rate of recovery. For patients with chronic inflammatory polyneuropathy, plasmapheresis has produced clinical improvement in 50% of corticosteroid-refractory patients. The use of plasmapheresis in patients with acute or chronic progressive multiple sclerosis still remains controversial. PMID- 3016912 TI - A rapid detection of herpes simplex virus by ELISA in asymptomatic pregnant women and neonates. AB - Herpes simplex virus (HSV) was detected by the enzyme-linked immunosorbent assay (ELISA). The assay system employed rabbit anti-HSV-2-coated microplates to detect HSV in clinical specimens and the same reagent labelled with peroxidase as a conjugate. The HSV type 2 obtained from vero cell culture and normal cell lysate (NCL) were used as positive and negative reference antigens respectively. HSV was detected in 40 (9.93%) of vaginal swabs obtained from 403 pregnant women just before the deliveries and in 39 (9.68%) fluid samples collected immediately after birth from the mouths of 403 newborns. HSV was detected in five pairs of mother newborn under investigation. There was no correlation between the incidence of HSV in mothers and newborns (p greater than 0.05). PMID- 3016913 TI - Gene mapping and physical arrangements of human chromatin in transformed, hybrid cells: fluorescent and autoradiographic in situ hybridization compared. AB - We compare a fluorescent in situ hybridization technique, using N-acetoxy-2 acetylaminofluorene (N-ACO-AAF) modified DNA adducts, with 3H-labeled DNA in situ hybridization for visualizing human transgenomes in HRAS1-selected, chromosome mediated gene transfer (CMGT), and mapping chromosomal SV40 in an SV40 transformed, human-mouse hybrid cell line. We demonstrate that individual HRAS1 CMGTs may contain multiple fragments of human chromatin. We deduce that the CMGT process can involve interstitial loss of mouse chromatin. We conclude that the N ACO-AAF technique gives finer resolution than 3H-labeled in situ hybridization. However, 3H-labeling is more sensitive and has allowed us to sublocalize SV40 in C121 to the region 7q31-35. PMID- 3016914 TI - Mapping human chromosomes in somatic cell hybrids using a low-copy-number repetitive sequence. AB - We have isolated a human-specific repetitive sequence with a copy number of several hundred (p11L26). Using Southern blots of EcoR1-digested DNA from somatic cell hybrids containing one or a few human chromosomes, band patterns specific for those chromosomes can be generated by hybridization with p11L26. Genomic copies of the sequence have also been mapped to subchromosomal regions using translocations. The probe offers a useful addition to the standard techniques for mapping human chromosomes in hybrid cell lines. PMID- 3016915 TI - Transfection of lymphoblastoid cells using DNA-loaded reconstituted Sendai virus envelopes: expression of transfected DNA and selection of transfected cells. AB - The American Burkitt's lymphoma cell line Loukes was cotransfected with cloned BamHI K fragment of EBV DNA and a vector pSV2neo. Reconstituted Sendai virus envelopes (RSVE) loaded with DNA were used for efficient gene transfer. Two cell lines have been obtained following culture in the presence of geneticin sulfate (G-418). Messenger RNA from both transfected DNAs was expressed during the whole period of observation, 42 days after transfection. This method provides a relatively simple and efficient means for selection of lymphoblastoid cells expressing a transfected gene. PMID- 3016917 TI - Ouabain-resistant mutants of Chinese hamster ovary cells are not directly affected in Na+, K+-ATPase. AB - The nature of biochemical lesions in a number of independent single-step ouabain resistant (OuaR) mutants of Chinese hamster ovary cells has been examined. The cellular uptake of 86Rb in the mutant cells was found to be resistant to inhibition by ouabain and correlated with the degree of resistance of the mutants to the drug. However, the plasma membrane Na+, K+-ATPase from all five of the OuaR mutants examined showed similar sensitivity to inhibition by ouabain as seen for the enzyme from the parental sensitive cells. These results provide evidence that, contrary to the general assumption, OuaR mutants of Chinese hamster ovary cells, which are widely used for genetic and quantitative mutagenesis studies, are not directly affected in the Na+, K+-ATPase. PMID- 3016916 TI - Comparison of methods for introducing vectors based on bovine papillomavirus-1 DNA into mammalian cells. AB - The intracellular structure of several vectors based on BPV-1 DNA has been analyzed following transfection into mouse C127 cells by the calcium phosphate method or, for the first time, by microinjection directly into the nucleus. It is shown that the method of introduction markedly affects the fate of a BPV-1 based vector. In general, microinjection appears to do little damage to DNA and is more likely to result in a vector replicating extrachromosomally as a monomeric structure of the same size as the input DNA. The method of selection for transformed cells, e.g., focus formation versus resistance to the neomycin analog G418, can also affect the intracellular state of the BPV-1 vector DNA. The nature of the recipient mammalian cell also influences whether a vector can replicate extrachromosomally or whether it integrates. BPV-1 based vectors, which replicated predominantly as multicopy intact extrachromosomal forms in mouse C127 cells, were always found to have integrated at low copy number in mouse LtAp20 cells. PMID- 3016918 TI - Site specificity of DNA methylation and expression of herpes simplex thymidine kinase gene. AB - Analysis of the methylation pattern of a single-copy herpes simplex virus thymidine kinase gene integrated into the genome of mouse L cells revealed that hypomethylation of five specific AvaI sites correlates with expression of the TK gene in all of the cell lines tested. Of these specific sites, one lies 5' to the coding region, one 3' to the coding region, and three lie within the coding region of the thymidine kinase gene. Analysis of methylation at a variety of other sites using other methylation-sensitive endonucleases revealed considerable variation in the methylation patterns, apparently unrelated to gene expression and subject to variation with time in culture. PMID- 3016919 TI - Mapping of human autosomal phosphoglycerate kinase sequence to chromosome 19. AB - In order to map human PGK sequences, DNA was prepared from 55 human-mouse somatic cell lines. The DNA was digested to completion with HindIII and Southern filters prepared. These filters were hybridized at high stringency conditions to a human PGK cDNA. Mouse and human X-linked and autosomal bands were distinguished and, in addition to known X-linked sequences, two autosomal PGK sequences were mapped: a 1-kb band to chromosome 19 and a 5-kb band to chromosome 6. The PGK cDNA probe was also hybridized to flow-sorted chromosomes confirming the presence of PGK sequences on the X chromosome and chromosomes 6 and 19. PMID- 3016921 TI - [Caries susceptibility and caries resistance induced by a cariogenic diet with trace elements]. PMID- 3016920 TI - Localization of multidrug resistance-associated DNA sequences to human chromosome 7. AB - Multidrug resistance in several human cell lines correlates with amplification or increased expression of two related DNA sequences, designated mdr1 and mdr2. These DNA sequences were used as probes for hybridization with DNA with a panel of human-mouse somatic cell hybrids and from individual human chromosomes separated by fluorescence-activated chromosome sorting. By these assays, both mdr1 and mdr2 sequences were localized to chromosome 7. PMID- 3016922 TI - Sindbis and West Nile virus infections in the Witwatersrand-Pretoria region. AB - From mid-December 1983 until mid-April 1984, there was an epidemic of Sindbis (SIN) virus infection in the Witwatersrand-Pretoria region in which hundreds of human cases were diagnosed clinically. Twenty-eight of these diagnoses were confirmed in the laboratory by seroconversion as being infections with SIN virus, and 5 cases of infection with West Nile (WN) virus were also found. Attempts to isolate virus from 66 patients, mainly from serum specimens, were unsuccessful. Infection rates for the mosquito vector Culex univittatus, collected at localities on the Witwatersrand in February and March, were mostly higher for both SIN and WN viruses than in previous years. The highest rates determined were 5.4 (SIN) and 9.6 (WN) per 1 000 mosquitoes. It is concluded that an epizootic of both viruses occurred which was manifested by a high level of viral activity in the feral Cx. univittatus-bird transmission cycle. Cx. univittatus efficiently transferred infection of SIN virus from this cycle to man to cause the epidemic of infection with that virus but it is unclear why there were apparently only a few cases of WN virus infection. PMID- 3016923 TI - [Malignant fibrohistiocytoma: a rare localization at the level of the glands]. PMID- 3016924 TI - The effects of alcohol abuse among the new chronically mentally ill. AB - In recent years, there has been a growing recognition of the high incidence of alcohol abuse among the new generation of chronically mentally ill. This article reports on a study that tracked a subgroup of the chronically mentally ill, those discharged from state psychiatric hospitals, through an entire community mental health aftercare system and its major auxiliary human service agencies. Those who were assessed by hospital discharge social workers as having a need for alcoholism services were found to be less likely to be referred for aftercare and to make contact with aftercare agencies post discharge; and for those with an alcoholism problem who do make contact, they generally received less service than those who did not have a need for alcoholism services. The professionals in both the mental health and alcoholism fields need to work together to better meet the needs of the chronically mentally ill with an alcohol problem. PMID- 3016925 TI - Occult lesions of the breast. AB - The biopsy of 148 clinically occult lesions of the breast found at mammographic examination yielded 21 instances of carcinoma (14.2 per cent). Five malignant lesions were lobular or in situ carcinoma, 16 were infiltrating ductal carcinomas and 11 were minimal carcinoma of the breast. Carcinomas found by mammographic examination were smaller and less likely to be associated with metastases to the axillary nodes than carcinomas of the breast diagnosed by palpation during the same time period. Biopsy of the breast was performed by several methods: 1, quandrantectomy with the patient under general anesthesia (the results of 29 biopsies yielded five instances of carcinoma, 17.2 per cent); 2, generous excision of breast tissue with the patient under general anesthesia and the lesion being localized by geometric co-ordinates based upon the mammograms (the results of 35 excisions yielded four instances of carcinoma, 11.4 per cent), and 3, mammogram-directed needle localization (the results of 84 procedures yielded 12 instances of carcinoma, 14.3 per cent). Because needle-directed biopsy causes less distortion of the breast, we prefer it to other biopsy techniques in the evaluation of occult lesions of the breast. PMID- 3016926 TI - beta-Endorphin in canine hemorrhagic shock. AB - Endogenous opiate peptides have been implicated in the cardiovascular depression of hemorrhagic shock. Beta-endorphin immunoreactivity was measured during hemorrhagic shock and the effects of beta-endorphin suppression during shock on cardiac hemodynamics and myocardial blood flow were studied. Fourteen dogs (group 1) were pretreated with 30 milligrams per kilogram of methylprednisolone (MP) prior to shock. Ten dogs (group 2) received saline solution and were the control group. All dogs were bled to a mean arterial blood pressure of 35 millimeters of mercury for two hours, then resuscitated with blood and lactated Ringer's solution. Beta-endorphin concentrations increased 600 per cent during shock in the control group. Dogs pretreated with MP had lower beta-endorphin concentrations (p less than 0.05) during shock than did dogs in the control group. The rate of left ventricular pressure rise (dp/dt) was significantly improved during shock (p less than 0.04) and after resuscitation (p less than 0.006) in dogs with suppressed beta-endorphin immunoreactivity. Blood flow from the coronary artery was higher during shock and after resuscitation (p less than 0.001) in the dogs in group 1. Our data indicate significant correlation between improved ventricular performance and decreased beta-endorphin levels in shock and after resuscitation. PMID- 3016927 TI - [Evaluation of gastric juice lactic dehydrogenase (LDH) in the prognosis and recurrence of gastric cancer]. PMID- 3016928 TI - [An observation of the histochemistry and ultrastructure of the borderline ovarian mucinous epithelial tumor]. PMID- 3016929 TI - [Preparation and characterization of 125I-HCG]. PMID- 3016930 TI - [Studies on a receptor radioassay system for chorionic gonadotropin receptors]. PMID- 3016931 TI - [An investigation on the CMV-CF antibody in a multitude of infants, children and adolescents]. PMID- 3016933 TI - A clinical study with brain brachytherapy for malignant gliomas. AB - A clinical trial with neutron brachytherapy for malignant glioma is now in progress at the University of Kentucky Medical Center. In the initial three year period, 32 patients entered the study. There were 24 males and eight females, and the peak incidence was seen in the seventh decade. All patients underwent surgical resection of tumor followed by Cf-252 implantation and external radiotherapy. The implantation procedure was well tolerated by the patients and demonstrated a short-term benefit in survival of patients who were able to complete planned radiotherapy. Older patients had less tolerance to radiotherapy. The evaluation of the beneficial effects of Cf-252 brachytherapy will require patients who are able to tolerate the whole course of therapy. PMID- 3016932 TI - [Results of percutaneous radiotherapy of mostly advanced cancers of the tongue]. AB - A retrospective study compiles the therapy results of 164 evaluable patients with lingual tumors who had been irradiated primarily or as part of a combined surgical-radio-oncologic treatment scheme at the Radiologic Hospital of the University of Munster. The five-year survival rate of all patients in all stages is 22%, and there were no statistically significant differences in the prognosis of carcinomas of the body of the tongue (22%) and carcinomas of the base of the tongue (25%). Our own experiences as well as the communications in literature show that the dose necessary, depending on the stage, is 55 to 60 Gy for small tumors (stages I and II), given within six weeks into the region of the primary tumor. In case of extended tumors (stages III and IV), 60 to 70 Gy have to be administered in seven to eight weeks. The split-course irradiation applied in our own cases produced a small rate of side effects without any demonstrable bad influence on the prognosis. For the irradiation of regional lymph nodes which has to be performed after neck dissection, too, 45 to 50 Gy should be given within four to five weeks. This dose should be increased by about 10 to 15 Gy in case of manifestations in extended regions. The excellent efficiency of intraoral electron irradiation is pointed out. PMID- 3016934 TI - The effect of interleukin-1 and interleukin-2 on the adrenergic control of hormone-sensitive lipase in the human adipocyte. AB - The effect of interleukin-1 and interleukin-2 on lipolysis and the adrenergic control of lipolysis was studied. Biopsy specimens of human adipose tissue were incubated in media containing 3H-palmitate and 14C-glucose, and the ratio of these isotopes was used to determine adipocyte lipolysis. Isoproterenol, clonidine, and theophylline were used in the media to stimulate the beta 1- and alpha 2-receptors and the subreceptor mechanism, respectively. Interleukin-1 had no effect on basal lipolysis, and at maximal receptor stimulation, it had no effect on the adrenergic receptor control of lipolysis. Interleukin-2 had no effect on basal lipolysis or on the beta-adrenergic receptor. Interleukin-2 significantly (p less than 0.02) decreased the alpha 2-inhibition of lipolysis by 68%. The effect of interleukin-2 on the alpha-receptor was demonstrated to be a significant 45% decrease (p less than 0.03) in the receptor responsiveness (a measure of the postreceptor mechanism) with no alteration in receptor sensitivity (a measure of receptor number). This data suggest that interleukin-2 stimulates lipolysis by decreasing the alpha 2-adrenergic inhibition of hormone-sensitive lipase. PMID- 3016935 TI - Perinatal cannabinoid exposure: effects on hepatic cytochrome P-450 and plasma protein levels in male mice. AB - Maternal exposure to the major psychoactive delta 9-tetrahydrocannabinol (THC), or to the nonpsychoactive cannabinol (CBN) or cannabidiol (CBD) on day 12 of gestation, or on day 1 postpartum, affected the concentrations of hepatic cytochromes P-450 in adult male offspring. Levels of P-450 were significantly increased in adult males prenatally exposed to cannabinoids, but were reduced after postnatal exposure. The response to exogenous testosterone was also differentially affected by perinatal cannabinoid exposure, with reduced plasma androgen in males prenatally exposed to THC, but increased levels of hormone in mice exposed postnatally to THC or CBN. There was a concomitant decrease in plasma albumin and increased gamma-globulin in adult males postnatally exposed to CBN. Beta-globulin levels were also significantly increased in adult males exposed to cannabichromene (CBC) on day 1 postpartum. Cannabinoid exposure during perinatal periods of development exert effects on hepatic function, plasma androgen levels, and on the immune system. These effects may reflect the ability of perinatal cannabinoid exposure to interfere with androgen-mediated processes of differentiation. PMID- 3016937 TI - AIDS: current status future problems. PMID- 3016936 TI - Experimental congenital disease with simian cytomegalovirus in rhesus monkeys. AB - Cytomegalovirus (CMV) infections occur worldwide and are responsible for severe damage to the child in from one to five newborns per 20,000 births. Animal models of congenital CMV infection resulting in disease have been developed in mice and guinea pigs. We report here the development of ventricular dilatation and leptomeningitis in rhesus monkeys, Macaca mulatta, following intrauterine infection with rhesus cytomegalovirus (RCMV). Central nervous system (CNS) lesions were associated with low cytomegalovirus fluorescent antibody titers in affected fetuses. In several infected animals, RCMV was isolated at necropsy from neural and nonneural tissues taken shortly after birth. This model allows investigators to study the pathogenesis and prevention of CNS changes following RCMV infection. PMID- 3016938 TI - Specific bronchial reactivity to toluene diisocyanate: relationship with baseline clinical findings. AB - One hundred and fourteen subjects with asthma induced by toluene diisocyanate were identified and the pattern of their bronchial responses to challenge with toluene diisocyanate was studied. An occupational type specific bronchial provocation test with toluene diisocyanate (10-25 parts per thousand million for 10-15 minutes) elicited an immediate response in 24, a late response in 50, and a dual response in 40 patients. Subjects with a dual response showed at diagnosis a longer duration of symptoms and a greater prevalence of airway obstruction; in these subjects FEV1 (percentage of predicted value) was lower than in subjects with immediate or late reactions to toluene diisocyanate. The percentage of current smokers and ex-smokers was significantly lower in subjects with a late response (26%) than in subjects with immediate or dual responses (56% and 57% respectively). In 27 of the 114 subjects a non-specific challenge test with methacholine was performed and subjects with dual responses showed greater non specific bronchial hyperresponsiveness than the other groups. These results suggest that a dual response to specific challenge in bronchial asthma related to toluene diisocyanate may be associated with more severe disease than other types of response, as assessed by duration of symptoms, baseline airway obstruction, and non-specific bronchial hyperresponsiveness. The high prevalence of non smokers and low prevalence of smokers in the group with a late response to specific challenge is so far unexplained. PMID- 3016940 TI - Xanthogranulomatous malignant fibrous histiocytoma arising from posterior mediastinum. PMID- 3016941 TI - Chemotherapy in non-small cell bronchial carcinoma. PMID- 3016939 TI - Primary oat cell carcinoma of the oesophagus. AB - Three cases of oat cell carcinoma of the oesophagus are presented and published reports reviewed. This is mainly a disease of older age with a 3:2 predominance of men. Of all published cases, 43 (47.3%) occurred in the middle third, 41 (45.1%) in the lower third, and four (4.4%) in the upper third. In one case it was multifocal and in two the location was not stated. Dysphagia was the most common symptom and was found in 82.5% of cases. Overall survival was 4.7 months. The longest survival in a patient treated by resection was 24 months and in a patient treated by chemotherapy 11 months. All but one of the patients had widely disseminated metastatic disease at death. It is concluded that surgery, possibly with adjuvant chemotherapy, holds out the best prospect for such patients. PMID- 3016942 TI - Potentiation of the antiplatelet action of adenosine in whole blood by dipyridamole or dilazep and the cAMP phosphodiesterase inhibitor, RA 233. AB - Adenosine (Ado, 10-50 microM), a potent inhibitor of ADP-induced human platelet aggregation in platelet-rich plasma (PRP), does not inhibit aggregation in whole blood. However, the Ado analogs, 2-fluoroadenosine, 2-chloroadenosine and 5'-N ethylcarboxamidoadenosine (NECA) which are resistant to deamination (2 fluoroadenosine) or deamination and phosphorylation (2-chloroadenosine and NECA), inhibit aggregation in whole blood with IC50 values of 12 microM, 2.3 microM and 0.26 microM, respectively. The inhibitory effect of NECA (200 nM) is potentiated by the platelet cAMP phosphodiesterase (PDE) inhibitor RA 233 (5 microM). Inhibition of the erythrocytic nucleoside transport system by dilazep (1 microM) or dipyridamole (10 microM), or blockade of Ado metabolism by 2'-deoxycoformycin (5 microM) plus 5-iodotubercidin (10 microM), evokes the antiaggregatory action of Ado in whole blood (IC50 congruent to 2 microM). RA 233 (5 microM) potentiates Ado-mediated inhibition about 10-fold when nucleoside transport or Ado metabolism is blocked. Ado (10 microM or 200 nM) is rapidly metabolized within 1 min in whole blood. When nucleoside transport is inhibited by dilazep or dipyridamole, or when Ado metabolism is blocked by 2'-deoxycoformycin and 5-iodotubercidin, 50 60% of the Ado remains in the plasma after 5 min. These results show that the failure of Ado to inhibit platelet aggregation in whole blood results from its rapid uptake and metabolism by erythrocytes. More importantly, these data emphasize the key role of nucleoside transport inhibition in the antiplatelet actions of dipyridamole and dilazep. In addition, superior therapeutic results may be obtained from the combination of blockade of nucleoside transport system with inhibition of platelet cAMP PDE. PMID- 3016943 TI - Thrombomodulin activity in commercial thromboplastin preparations. AB - Recently, it was reported that endothelial cells contain a membrane protein which serves as a cofactor for the activation of protein C by thrombin (thrombomodulin). Because many commercial thromboplastins are prepared from vessel-rich tissues/organs (lung, brain, placenta), it was hypothesized that some preparations may contain significant amounts of thrombomodulin; theoretically, thrombomodulin contaminations may enhance the prothrombin time. Among 8 different commercial thromboplastins tested two thromboplastins were found to contain significant amounts of thrombomodulin activity: Simplastin (64 U/ml) and Ortho (12.8 U/ml). However, we were unable to demonstrate that the presence of the thrombomodulin activity in these thromboplastins could affect the prothrombin time either by delaying fibrin formation (formation of thrombin-thrombomodulin complexes) or by inhibiting activated factor V (formation of activated protein C). PMID- 3016944 TI - Identification of receptors for fibrinogen and von Willebrand factor mediating aggregation in guinea pig platelets. AB - Monoclonal antibodies were prepared to guinea pig platelets and selected for their ability to inhibit ADP-induced platelet aggregation and ristocetin induced, Ca++-independent platelet agglutination. One antibody, PG-2, produced strong inhibition of aggregation induced by ADP, thrombin, collagen and arachidonic acid, while not inhibiting ristocetin-induced agglutination. A second antibody, PG-1, blocked ristocetin-induced agglutination, but did not inhibit aggregation induced by the previous agents. PG-2 blocked ADP-induced 125I-fibrinogen binding to washed guinea pig platelets by approximately 50%, but did not inhibit ristocetin-induced binding of 125I-vWF. Conversely, PG-1 selectively inhibited ristocetin-induced 125I-vWF binding, with the degree of inhibition inversely related to the ristocetin concentration. These studies suggest that in guinea pig platelets, fibrinogen and von Willebrand factor binding to different membrane sites are responsible for the aggregation response of stimulated platelets and the ristocetin-induced agglutination response respectively. These antibodies offer significant promise for the further development of a guinea pig animal model for studying platelet and megakaryocyte function. PMID- 3016945 TI - Synthesis by guinea pig megakaryocytes of platelet glycoprotein receptors for fibrinogen and von Willebrand factor. AB - In the preceding paper, we described two monoclonal antibodies, PG-1 and PG-2, that selectively blocked the binding of von Willebrand factor (PG-1) or of fibrinogen (PG-2) to guinea pig platelets. In this study we examine the structures and site of synthesis of these receptors. NP-40 lysates of radiolabeled guinea pig platelets were immunoprecipitated with monoclonal antibodies PG-1 or PG-2, and the precipitates analyzed by SDS-PAGE. PG-1 recognized a single polypeptide with reduced Mr of 143,000 daltons, while PG-2 precipitated two chains with reduced Mr of 121,000 and 93,000 daltons. Periodate [3H]borohydride labeling of platelets, in conjunction with two-dimensional SDS PAGE, showed that all three of the polypeptides are glycoproteins and that the 143,000 and 121,000 dalton chains are linked by disulfide bond(s) to smaller, approximately 25,000 dalton polypeptides. Guinea pig megakaryocytes synthesized polypeptides immunoprecipitable by PG-1 and PG-2, with molecular weights similar to polypeptides found associated with platelet membranes. These studies demonstrate that guinea pig platelets have functional receptors for fibrinogen and von Willebrand factor that are structurally homologous to human platelet glycoproteins Ib, IIb and IIIa, and that these glycoproteins are synthesized by megakaryocytes. PMID- 3016947 TI - Pharmacological and histological evidence for adrenergic innervation of the myoid cells in the rat seminiferous tubule. AB - Although the existence of seminiferous tubule contractions attributed to peritubular myoid cells is established, the control of the contractions is poorly understood. In this communication, it is shown that the rat seminiferous tubule responds to autonomic drugs and to stimulation of the perivascular nerve running along the spermatic vessels, by means of recording the intratubular pressure with a servo-null micropressure measuring device. Furthermore, the presence of nerve fibers close to the myoid cells is shown using a silver impregnation technique. Furthermore, the nature of the neurotransmitter contained in synaptic vesicles using 5-hydroxydopamine and L-DOPA as markers of adrenergic nerve elements with electron microscopy is shown in this report. It is concluded that there are adrenergic alpha- and beta-receptors and muscarinic receptors in myoid cells of the rat seminiferous tubule and that the contractions of seminiferous tubules are regulated by adrenergic nerve fibers. PMID- 3016946 TI - [Pathology and control of virus-induced enteritis in calves]. AB - Considerable economic loss can arise from virus-caused enteritis in calves, in the form of so-called infectious factor diseases, which often develop more seriously when bacterial organisms, such as E. coli become involved. Rota-, corona- and parvoviruses are of particular interest. These pathogens have a marked predilection for intestinal epithelium. Rotavirus destroys the epithelial cells of the upper parts of the villi. Coronavirus penetrates to the base of the small intestinal villi and the superficial and crypt colonic epithelium is frequently affected. Infection of the small intestinal crypt epithelium is characteristic of parvovirus; loss of epithelium at the villus tip is also observed. Damage of the mucosa results in a reduction in digestive and absorbing capacity. It is not possible to treat these virus strains specifically. Great importance is therefore attached to the vaccination of dams as immune prophylaxis. Consumption of sufficient colostrum and milk from vaccinated dams affords the calves good protection. The mechanism is based on the presence of milk antibodies in the calf's intestine which neutralise orally ingested pathogens. PMID- 3016948 TI - Increased forebrain beta-adrenergic ligand binding induced by trimethyltin. AB - Systemic injection of the organometal neurotoxin trimethyltin (TMT) into rats causes impairments in learning and memory. However, there is a discontinuity in dose-response functions, such that the deficit which emerges with a higher, acutely toxic dose, is qualitatively different from the impairment induced after lower doses. To investigate synaptic receptor changes associated with behavioral deficits, neurotransmitter-receptor ligand binding assays were done in forebrain areas of rats given TMT (3.6 or 7.5 mg/kg). Binding of the beta-adrenergic ligand, dihydroalprenolol in frontal cortex and amygdala/pyriform cortex was an inverted U-shaped function of TMT dose, with rats given the median dose exhibiting increased binding. The curvilinear dose-response functions in behavioral and biochemical assays suggest that altered forebrain noradrenergic neurotransmission could play a role in behavioral deficits. PMID- 3016949 TI - Effects of acrolein on macrophage functions in rats. AB - Male Sprague-Dawley rats were exposed to 0.1, 1.0 or 3.0 ppm acrolein or filtered air 6 h/day, 5 days/week for 3 weeks. Rats were tested one day following the last exposure and exhibited no change in pulmonary clearance of inhaled 35S-labeled Klebsiella pneumoniae at any acrolein concentration. Decreased numbers of peritoneal cells were obtained from exposed rats while the number of cells lavaged from the lungs was unchanged. Macrophages of acrolein-exposed rats had altered phagocytic and enzymatic patterns as compared to macrophages from animals breathing filtered air. However, these changes had no apparent effect on macrophage killing of inhaled bacteria and were therefore probably not indicative of extreme chemical toxicity. PMID- 3016950 TI - Exogenous steroids alter steroidogenesis in cultured Y-1 adrenal tumor cells by actions preceding cyclic AMP. AB - Using cultured Y-1 mouse adrenal tumor cells which produce 20 alpha-hydroxy-4 pregnen-3-one (20-DHP), it was found that 0.01 mM corticosterone and deoxycorticosterone increased basal and inhibited ACTH-induced 20-DHP production during consecutive 30 and 120 min incubations. Steroid effects were concentration dependent and reversible. Six other steroids tested did not stimulate 20-DHP production and varied in ability to inhibit ACTH-stimulated steroidogenesis. Experiments demonstrated that 20-DHP production following treatment with cholera toxin, N,0'-dibutyryl cyclic AMP (dbcAMP), or pregnenolone was not inhibited by exogenous steroids. Corticosterone (0.01 mM) increased basal and inhibited ACTH induced intracellular cyclic AMP (cAMP) production. Cytochalasin D, a microfilament perturbing agent, inhibited steroid-stimulated 20-DHP production, suggesting that ACTH and steroid stimulation mechanisms were similar. These findings taken together suggest that exogenous steroids can alter steroidogenesis by modifying plasma membrane adenylate cyclase activity. PMID- 3016951 TI - Extraction of corticosterone from cell homogenates and subcellular fractions of the rat adrenal cortex. III. ACTH-induced temporal subcellular redistributions of steroid precursors to corticosterone. AB - Cholesterol, pregnenolone, progesterone, 11-deoxycorticosterone (11-DOC) and corticosterone were quantitated in subcellular fractions isolated from in vivo adrenocorticotropin (ACTH)-stimulated rat adrenal zona fasciculata/reticularis. Six adrenal subcellular fractions separated by discontinuous sucrose gradient centrifugation (lipid, 0.125 M sucrose, cytosolic, microsomal, mitochondrial and nuclear) were extracted with alkaline ether/ethanol and assayed by high pressure liquid chromatography (HPLC). Lipid fractions contained the major cholesterol stores, while most pregnenolone and progesterone was found in lipid, microsomal and mitochondrial fractions. The 0.125 M sucrose and cytosol fractions together contained approximately 75% of the total 11-DOC and corticosterone. The five steroids were only present in small amounts in organelle fractions containing steroidogenic enzymes. Homogenate and lipid fraction cholesterol decreased between 10 and 15 min and again 30 min after ACTH injection. In the homogenate, lipid, microsomal and mitochondrial fractions, pregnenolone and progesterone were increased after ACTH injection; peak pregnenolone and progesterone concentrations were often measured in adrenal gland sucrose, cytosolic, microsomal and mitochondrial fractions 15 to 20 min after rats were injected with ACTH. Although ACTH increased 11-DOC and corticosterone in all but the mitochondrial and nuclear fractions, the sucrose, cytosolic and microsomal 11-DOC, and cytosolic corticosterone increased most dramatically. In many fractions, peak 11-DOC and corticosterone concentrations were most often observed between the 10 and 15 min periods and again at 30 min. PMID- 3016953 TI - Chronic intraoral herpes simplex infection in renal transplant recipients. PMID- 3016952 TI - Selection of high-affinity and high-specificity monoclonal antibodies for 1 alpha,25-dihydroxyvitamin D. AB - The preparation of high-affinity and high-specificity monoclonal antibodies to 1 alpha,25-dihydroxyvitamin D is described. Monoclonal antibodies were derived from Balb-c mice immunised with either 1 alpha-hydroxy-25,26,27-trinor-24 cholecalcioic acid or with 1 alpha-hydroxy-26,27-dinor-cholecalciferol-25-oxime, and spleen cells were hybridised with mouse myeloma cells. From six fusions nine monoclonal antibodies (MAb's) were selected from 676 antibody-secreting hybrids. Antibodies varied widely in their ability to bind 1 alpha,25-dihydroxyvitamin D3 (50% displacement of radioligand ranged from 25 - 900 pg); two had particularly useful characteristics for 1 alpha,25-dihydroxyvitamin D assay. MAb 5F2 has high affinity (Ka = 1.39 X 10(10) M-1) and does not discriminate between 1 alpha,25 dihydroxyvitamin D2 and D3, thus enabling the two forms to be measured together. MAb 1G7 is highly specific, having no cross-reactivity with 25-hydroxy-, 24,25 dihydroxy- or 25,26-dihydroxyvitamin D at concentrations found in normal human serum; this MAb has the potential to eliminate the lengthy extraction procedures involved in currently available assays for 1 alpha,25-dihydroxyvitamin D. PMID- 3016954 TI - [Properties of the enzymes of energy supply in the common frog under various physiological states. II. Changes in the heat resistance of adenosine triphosphatases and succinate dehydrogenase]. AB - The heat resistance of SDG, Na, K-ATPase and Mg-ATPase of the grass frog was determined in January, March and May, the number of animals examined being 30-40 in either experiment. It was found that the average level of the heat resistance of the enzymes studied shows significant, often differently directed changes, which depend on the physiological state of an organism. Negligible correlation between the thermal sensitivity of different enzymes of an organism during hibernation, completely disappear during the activity state. PMID- 3016955 TI - Capripox in the Yemen Arab Republic and the Sultanate of Oman. AB - Capripox was shown to be endemic in all the provinces of the Yemen Arab Republic and the Sultanate of Oman. Investigations into outbreaks of capripox indicated that some strains of capripox virus were infecting both sheep and goats and this was confirmed by inoculating experimental sheep and goats with isolates derived from field cases. The husbandry methods prevalent in the Middle East predispose to the rapid spread of capripox and annual vaccination of all sheep and goats must be considered as the only effective method of controlling the disease. PMID- 3016958 TI - Histologic-prognostic correlations in adenoid cystic carcinoma of major and minor salivary glands of the oral cavity. AB - Thirty-four cases of adenoid cystic carcinoma (ACC) of the major and minor salivary glands of the oral cavity, treated by wide surgical excision, were studied. The relationship between prognosis of the neoplasm and various morphologic factors were evaluated. Among the morphologic parameters previously studied, which according to various authors may be linked to the evolution of this tumor, we confirmed correlations for both histologic patterns and perineural invasion. In addition, the authors propose neoplastic growth type as a new parameter prognostically significant in ACC. In fact, none of the patients with the pushing type growth pattern died during the study period (p = 0.007). These same patients presented disease-free periods (mean 56 months, median 58 months) significantly longer than those with the infiltrating type growth pattern (mean 28 months, median 24 months). PMID- 3016956 TI - Infectious bursal disease immunisation failures in chickens in Nigeria. PMID- 3016957 TI - LAV/HTLV-III neutralizing antibodies in the sera of patients with AIDS, lymphadenopathy syndrome and asymptomatic seropositive individuals. AB - Serum samples which had previously been found positive for LAV/HTLV-III antibodies by the ELISA test and then confirmed by radioimmunoassay (Western blot) were tested for the presence of neutralizing antibodies. No neutralizing activity was found in the sera of a group of patients with the clinical diagnosis of AIDS. However in patients with LAS and other related pathologic conditions the percentage of sera positive for neutralizing antibodies was 27% and 55% respectively. At least 50% of the sera from seropositive asymptomatic individuals had neutralizing activity but with the exception of the haemophiliac group the mean titre was much lower than that of LAS patients. No relationship was found between the neutralizing titre and the antibody specificity detected by Western blot analysis for p41 and p120. PMID- 3016959 TI - [Ulcero-mutilating acropathies and spinal abnormalities. Apropos of 4 case reports]. PMID- 3016960 TI - [Effects of a dietary fiber, guar gum, on the control of diabetes]. PMID- 3016963 TI - [Catecholamine-sensitive adenylate cyclase activity and beta-adrenoreceptors of the myometrium in various functional states]. AB - Adenylate cyclase of the rabbit myometrium possesses different sensitivity to catecholamines in different functional states: in state of functional rest and labour the enzyme is activated by isoproterenol, in the state of pregnancy this activation is absent almost completely. The investigated functional states of myometrium are characterized by different density of beta-adrenoreceptors: density of beta-adrenoreceptors in the state of functional rest is 134 +/- 14 fmol/mg of protein, pregnancy -74 +/- 12 fmol/mg of protein, labour -80 +/- 11 fmol/mg. Dissociation constants of [3H]dihydroalprenolol for beta-adrenoreceptors of myometrium in states of functional rest, pregnancy and labour are 7.5 +/- 0.5 nM, 6.2 +/- 0.5 nM, 4.8 +/- 0.1 nM, respectively. PMID- 3016961 TI - [Ca2+-calmodulin-activated cyclic nucleotide phosphodiesterase from the rabbit myometrium]. AB - Two forms of soluble phosphodiesterase of cyclic nucleotides separating by DEAE cellulose ion-exchange chromatography and not only differing in physicochemical and catalytic parameters but also differently regulated by calmodulin are found in the doe myometrium. Calmodulin with 10(-7)-10(-5) M concentrations of Ca2+ promotes the two-fold activation of the 3':5'-AMP (but not of 3':5'-GMP) hydrolysis by the first form of phosphodiesterase. Trifluoperazine (10 microM) lowers the activating action of calmodulin. The second form of soluble phosphodiesterase is not sensitive to the action of both calmodulin and Ca2+. 3':5'-GMP (10 microM) inhibits the 3':5'-AMP hydrolysis by the first form of phosphodiesterase; calmodulin exerts no effect on this process. The data obtained testify to the possible participation of Ca2+ and calmodulin in Ca2+-calmodulin dependent phosphodiesterase regulation of the content of cyclic nucleotides (3':5'-AMP, in particular) in the doe myometrium. PMID- 3016962 TI - [Isolation and characteristics of the plasma membrane fraction from the swine myometrium]. AB - An accelerated method is developed for isolating a fraction of plasma membranes of pig myometrium using ultracentrifugation within the sucrose density gradient (15% and 30%). The membranes possessed the high activity of 5'-nucleotidase and Na+, K+-ATPase and the low activity of rhotenon-insensitive NADH-cytochrome c reductase. The vesicularized preparations of plasma membranes are able of ATP dependent accumulation of Ca2+ (7.5 +/- 0.3 nmol. 45Ca2+ per 1 mg of protein for 15 min). Phosphate increases the calcium accumulation in the presence of ATP and Mg2+. Ionophore A 23187 promotes a complete and rapid release of the previously active-accumulated calcium. The release of 45Ca2+ accumulated by the membrane fraction may be reached by introduction of 1 mM EGTA or DS-Na into the incubation medium, that evidences for the cation accumulation inside closed structures. Using concanavalin-A-sepharose 4B it is shown that 60% of membrane vesicles are turned inside out. The low saponine concentrations (0.0005%) which inhibit Ca2+ accumulation by plasma membranes but not by the endoplasmic reticulum inhibit this process by 60-70% in preparations of the isolated membrane fraction. The method has certain advantages over the previously applied methods used for isolating of plasma membrane fragments from smooth muscles. PMID- 3016964 TI - Influence of dissolved gases on chemical and biological effects of ultrasound. AB - The influence of dissolved gases (O2, Ar, N2O, and CO2) on the chemical and biological effects of 1.2 MHz continuous wave ultrasound was investigated. Spin trapping of OH and H radicals with 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) and observation of iodine liberation from a potassium iodide-starch solution were employed for monitoring the chemical effects, while lysing of mouse L5178Y cells was employed for monitoring the biological effects. The effectiveness of the dissolved gases in producing OH-DMPO adducts and H-DMPO adducts was O2 greater than Ar greater than N2O = CO2 approximately equal to O and Ar greater than O2 = N2O = CO2 approximately equal to O, respectively. A result similar to the yield of OH-DMPO was obtained from the liberation of iodine induced by ultrasound. In addition, the effectiveness of the dissolved gases in lysing mouse L5178Y cells by ultrasound was O2 = Ar greater than N2O = CO2 approximately equal to O. These results suggest that both dissolved N2O and CO2 gases in solution suppressed not only the chemical effect but also the biological effect. PMID- 3016965 TI - [Diagnostic parameters and their value in testicular tumors]. AB - Diagnosis of the retroperitoneum to deliniate metastases of nonseminomatous germinal cell testicular cancer, when combined with Computer tomography, Sonography and Lymphography has a high specificity. With regard to treatment, the differentiation between stage I and stage II is very important. In the so called high risk groups (MTU-Tumour, T3-Primary tumour) a stage I can actually be a stage II. Computer Tomography of the thorax should be made to deliniate a stage III tumour. PMID- 3016966 TI - [Nonbulky nonseminomas: N-staging value of image-producing study procedures with reference to possible surveillance management]. AB - In a retrospective study 72 patients with non-bulky nonseminomas were investigated in order to determine the value of computed tomography, sonography and lymphography in the detection of lymph node metastases. The diagnostic results were compared with the histological findings of the retroperitoneal lymph node dissection. In our evaluation computed tomography (n = 48) had an accuracy of 0.77, a sensitivity of 0.62 and a specificity of 0.89; sonography (n = 40) had an accuracy of 0.66, a sensitivity of 0.47 and a specificity of 0.81; lymphography (n = 54) had an accuracy of 0.56, a sensitivity of 0.59 and a specificity of 0.53. Our own results were compared with the results published elsewhere. PMID- 3016967 TI - Polyglycolic acid mesh in repair of renal injury. AB - To evaluate the use of a polyglycolic acid (PGA) mesh graft for partial nephrectomy, we replaced the upper pole of the left kidney in 12 New Zealand white rabbits with a free omental fat graft and the lower pole with PGA mesh. The mesh stopped the bleeding immediately during the operation. At forty-eight hours, the fibers were still intact, with an organized clot on the surface of the mesh. Between two weeks and two months, the fibers had begun to be digested by histiocytes, with formation of giant cells and fibroblastic proliferation with collagen deposition around the mesh. At three months, there was complete resorption of the PGA mesh and formation of a new fibrous capsule. There was no renal reaction and no difference between the PGA mesh and the omental fat graft. We believe that PGA mesh can be helpful in repairing injured kidneys by securing hemostasis and serving as a scaffold for the formation of a new capsule. PMID- 3016968 TI - [Immunoenzyme detection of enzootic bovine leukemia viruses (BLV) in cell cultures]. AB - There is a description of the method of immunoenzymic test used for detection of enzootic bovine leukosis virus (BLV) on the cells of foetal lamb spleen (FLS) permanently producing BLV and on the lymphocytes of infected animals. In both cases (in FLS cells and lymphocytes) the presence of BLV was demonstrated by the immunoperoxidase test. Using the defined serums from the animals after experimental and natural infection, BLV was detected by the above-mentioned test in FLS cells in all cases of the use of positive serums with anti-BLV antibodies. The specificity of the antibodies was also demonstrated in this way. The presence of BLV was analogically detected in the lymphocytes of the infected animals where the infection time was precisely defined. The method of immunoperoxidase test on lymphocytes is recommended in indicated cases for the demonstration of the presence of BLV in infected animals. PMID- 3016969 TI - [Changes in blood protein levels in piglets during development and during stress]. AB - The concentrations of total protein (TP), albumin and globulin in the blood serum were investigated in seven trials with 203 piglets weaned at the age of three to four weeks. The lowest values were recorded in the newborn piglets just before colostrum intake. Two days later the TP level was three times higher and that of globulin almost five times higher. Later the levels began to sink (more intensively in globulin) and the decrease lasted until the end of the second month of age. Albuminaemia increased from birth until the end of study (the beginning of the third month) without any greater influence of colostrum intake and at a rate reduced by weaning. The albumin-globulin quotient, the lowest on the second day of life, increased step by step. The better-growing piglets mostly had higher albuminaemia and their hypoglobulinaemia reached a normal level sooner after weaning as compared with the tail-enders. This suggests that nutrition, body growth and albumin and globulin synthesis are interdependent in piglets. An increase in serum corticosteroids induced by two days of starvation or two administrations of adrenocorticotropic hormone, caused an insignificant increase in the concentration of albumin. Two hours after ACTH administration, the high level of corticosteroids was not observed to be accompanied by a change in serum proteins. PMID- 3016970 TI - Isolation of very virulent pathotypes of Marek's disease virus from vaccinated chickens in Europe. AB - Two very virulent strains of Marek's disease virus were isolated from two separate farms in northern Italy that had experienced outbreaks of Marek's disease in vaccinated flocks. The isolates were similar to very virulent strains reported in the USA in terms of their enhanced pathogenicity, both for chickens vaccinated with the herpesvirus of turkeys, and for genetically resistant chickens. This first description of very virulent strains of Marek's disease virus from outside the USA suggests that at least some of the increasingly frequent disease outbreaks reported in Europe may be associated with such strains and that the adoption of bivalent or polyvalent vaccines containing, for example, attenuated Marek's disease virus plus herpesvirus of turkeys may be beneficial in the field. PMID- 3016971 TI - Aetiology of enzootic haematuria. AB - The precise aetiology of enzootic haematuria in cattle remains unknown. The involvement of bracken fern (Pteridium aquilinium) appears certain because of the close association between bracken fern infested farms and enzootic haematuria. Several toxic principles have been identified but the main carcinogenic element remains to be conclusively demonstrated. More recently, bovine papilloma virus has been implicated in the aetiology of enzootic haematuria. Its possible interaction with bracken fern carcinogen is discussed. PMID- 3016973 TI - Identification of bovine herpesvirus-1 polypeptides involved in serum neutralization. AB - Bovine herpesvirus (infectious bovine rhinotracheitis virus)-infected cell antigens were solubilized with Nonidet P-40. The crude antigen extract was separated by reaction with bovine hyperimmune serum in line immunoelectrophoresis; individual immunoprecipitates were used to immunize rabbits. Rabbit sera possessing serum neutralizing activity were analyzed by reaction with crude antigen extract in immunoprecipitation sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Four virus specified glycopeptides, with molecular weights of 69-75K, 77-81K, 82-92K and 108 115K, appeared to be involved in inducing serum neutralizing antibody. PMID- 3016972 TI - Changes in blood chemical components during experimentally induced Theileria annulata infections in cattle. AB - During the course of infection of calves with Theileria annulata levels of glucose, calcium, proteins, phosphorus, magnesium and potassium decreased and those of bilirubin, cholesterol and alkaline phosphatase increased with no changes in sodium and acid phosphatase levels. Several of these biochemical alterations have been explained in relation to the injury to the liver and other organs. PMID- 3016974 TI - Molecular pathogenesis of equine coital exanthema: temperature-sensitive function(s) in cells infected with equine herpesviruses. AB - Preliminary experiments have revealed that several laboratory and wild-type strains of the equine herpesvirus (EHV) triad were temperature-sensitive for growth when assayed at 39 degrees C. The efficiencies of plating (EOP) observed were 10(-2) for both EHV 1 and 2, and 1 X 10(-6) for EHV 3. The EOPs were determined by plaque assays which compared titrations at 34 degrees C and 39 degrees C on equine fetal dermal fibroblast cells. Growth yield experiments, assayed at 34 degrees C, reflected those EOP's, but did not indicate any difference in yields when infected cultures were incubated at 34 degrees C and 37 degrees C. Temperature shift experiments with EHV 3-infected cultures revealed that a temperature-sensitive function(s) responsible for the reduction in titer appeared to be a late function(s). All strains examined appeared to incorporate H3-thymidine into viral-density DNA at the non-permissive temperature of 39 degrees C. Electron microscopy of EHV 3-infected cell cultures, incubated continuously at the non-permissive temperature and examined at 18 h after infection, revealed structures consistent with the accumulation of nucleocapsids within the nucleus. The evidence presented is consistent with the hypothesis that in equine dermal cells infected with a plaque-purified wild-type strain of EHV 3 (1118LP), a function needed for the egress of nucleocapsids from the nucleus is absent at 39 degrees C. The significance of these findings relative to the pathogenicity of the disease (equine coital exanthema) caused by this virus is discussed. PMID- 3016975 TI - Effect of intratracheal challenge of fattening pigs previously immunised with an inactivated influenza H1N1 vaccine. AB - Intratracheal inoculation of a field isolate of influenza A H1N1 caused high fever, anorexia and dyspnoea in unvaccinated pigs. In a limited study, it was shown that animals vaccinated once with an inactivated influenza A H1N1 strain showed partial protection at challenge, indicated by mild or absent clinical signs and by the suppression of viral replication. There appeared to be a correlation between the hemagglutination-inhibition titers of the serum of vaccinated pigs and the degree of protection. Animals vaccinated with two spaced injections were completely protected at challenge. Viral replication was inhibited in their respiratory tract since no virus was isolated from animals at slaughter and no increase in antibody titer was observed in challenged vaccinates followed serologically. It was concluded that vaccination of swine against influenza with an inactivated vaccine can result in a protective immunity in the respiratory tract. The New Jersey vaccine strain could protect against swine influenza strains (H1N1) currently prevalent in several European countries. PMID- 3016976 TI - Evidence for an immunocompromising effect of bovine pestivirus on bovid herpesvirus 1 vaccination. AB - Five calves were given live intranasal vaccine against bovid herpesvirus 1 (BHV1) two days after intranasal inoculation of bovine pestivirus (BVDV). Another 5 were vaccinated in the absence of BVDV. Control unvaccinated groups were also maintained. All calves were challenged with virulent BHV1. The unvaccinated calves developed signs of infectious bovine rhinotracheitis (IBR) and both vaccinated groups showed a similar degree of clinical protection from IBR. Those given BVDV before vaccination shed up to 140 times more BHV1 (P less than 0.01) in the nasal mucus following challenge than those which had received BHV1 vaccine alone. The epidemiological significance of this is discussed. PMID- 3016977 TI - [Methods for synchronizing estrus and selecting recipient heifers in embryo transplantation]. AB - Threefold thorough gynecological investigations were carried out with a total of 108 heifers with normal cycle, which remained uninseminated--in their choice, treatment with hormonal preparations, and transplantation of embryos, reducing their number up to 76. With the first test group of 35 heifers estrus was synchronized with implants, containing 3 mg norgestomet each. On the 9th day these were removed, and the heifers were injected with 500 IU PMSG (Intervet) each. The animals of the second test group (73 in number) were injected with 7.5 mg prostaglandin F2-alfa at 11-day intervals. The conception rate was 31 per cent higher with heifer-recipients treated with norgestomet implants and serum gonadotropins. Death was established with 47 transplanted embryos (61.8 per cent). With 19 embryos (25 per cent) death set in on the 21st day, and with 28 ones (26.8 per cent)--within the period of the 60th days of pregnancy up to calving. In 17.10 per cent of the transplanted embryos migration was seen toward the opposite horn, on the side of which the ovary contained no yellow body. PMID- 3016978 TI - Detection of surface antigens defined by monoclonal antibodies in primary mucinous breast carcinomas. Relation to prognostic factors and recurrence-free survival. AB - Three monoclonal antibodies, 67D11, 115H10 and 115C2, raised against human milk fat globule membranes, have been applied to 207 primary mucinous breast carcinomas. The tumours reacted positively in 18% (67D11), 54% (115H10), and 20% (115C2) of the cases. The detected epitopes (MAM-3a (67D11), MAM-3b (115H10), and MAM-3c (115C2)) have formerly been shown to be markers of differentiation in infiltrating ductal carcinomas. In the present group of mucinous breast carcinomas, statistically significant correlation to high risk factors, such as occurrence of primary lymph node metastases, large tumour size, and local invasion of the tumour into overlying skin or deep fascie, were found. Furthermore, the antigen expression was less marked in pure mucinous carcinomas as compared to carcinomas also presenting with non-mucinous tumour areas. Thus, especially the antigen MAM-3b, is more frequently present in mixed tumours, in large tumours, in tumours with local invasion, and in tumours with primary lymph node metastases. However, no association could be demonstrated between expression of MAM-3b and recurrence-free survival. Mucinous carcinomas of the breast apparently differ from other carcinomas not only with respect to morphology, but also in their pattern of antigenic expression in relation to other prognostic factors. PMID- 3016979 TI - Morphology of a GHRH producing pancreatic islet cell tumour causing acromegaly. AB - A 54 year old woman suffered from acromegaly due to a pancreatic islet cell tumour producing GHRH. The tumour was demonstrated on CT scan. The diagnosis was established from elevated plasma levels of GHRH, GH and prolactin, and by the lack of signs of a pituitary adenoma in trans-sphenoidal surgery. Acromegaly was cured by tumour removal. Light microscopically, the tumour showed a medullary and microlobular pattern. The cells were large and often cuspidal. Small granules were found in semi-thin sections. Small aggregations of amyloid fibres were seen, mostly around capillaries. Immunocytochemistry revealed GHRH, NSE, neurotensin, serotonin, VIP and PP. S 100 was positive only in nerve fibres. Staining for GH, ACTH, calcitonin, alpha-HCG, beta-HCG, insulin, glucagon, gastrin, substance P, bombesin and somatostatin was negative. Ultrastructure showed oval partly lobulated nuclei with small nucleoli, moderate amounts of rough endoplasmic reticulum, many free ribosomes, some large Golgi fields and small numbers of secretory granules measuring 150 nm or, in a few cells, 650 nm. Only 4 other cases of pancreatic endocrine tumours causing acromegaly by ectopic GHRH secretion are described in the literature and these were similar to our case in many respects. PMID- 3016980 TI - The herpes simplex virus type 2 equivalent of the herpes simplex virus type 1 US7 gene and its flanking sequences. AB - Nucleotide sequencing studies (D. J. McGeoch, A. Dolan, S. Donald, and F. Rixon, 1985, J. Mol. Biol. 181, 1-14) have indicated that herpes simplex virus type 1 (HSV-1) has a coding sequence, referred to as US7, between the genes for the glycoproteins D and E (gD and gE). Northern blot analysis and nucleotide sequencing have been carried out to show that the type 2 virus (HSV-2) has an equivalent to the US7 gene. A comparison with the HSV-1 sequence has revealed some surprising similarities and differences. At the nucleotide level, HSV-2 has inserted a large sequence into the gE promoter, retained a large palindrome present in the coding sequence but not some tandem repeats, and deleted a region beside those repeats. At the amino acid level, the putative transmembrane sequence has been remarkably well conserved, and hydrophobic moment analysis indicates that it could be interacting with polar species within the plane of the membrane. Immediately after the deletion in the HSV-2 sequence, there is an N glycosylation signal, and HSV-2 has one more such signal than HSV-1. The longest conserved sequence at the nucleotide level codes for a region of polypeptide that is strongly predicted to fold into alpha-helix. Implications of these analyses to the structure and possible function of these molecules are discussed. PMID- 3016981 TI - The 3'-nucleotides of flavivirus genomic RNA form a conserved secondary structure. AB - The terminal noncoding regions of viral RNA genomes are presumed to contain signal sequences and sometimes also secondary structures involved in regulating viral RNA synthesis. Such signals would be expected to be highly conserved among related viruses. In order to identify replication signal features for flaviviruses we have compared the 3'-terminal nucleotide sequences of West Nile virus (WNV), Saint Louis encephalitis (SLE) virus, and yellow fever virus (YFV) genome RNAs. The existence of a stable 3'-terminal secondary structure was previously predicted by a cDNA sequence obtained from YFV genome RNA. We have confirmed the existence of this structure by direct RNA sequencing methods. Even though the size and shape of the 3'-terminal secondary structure is highly conserved, sequence conservation is restricted to the loop regions of the secondary structure and to 27 nucleotides immediately adjacent to the 5' side of the structure. The regions of conserved sequence represent likely signals for viral polymerase recognition and binding. However, the preservation of the configuration of the secondary structure by a means other than sequence conservation indicate that this structure is important for the survival of the virus. A WNV mutant, which replicates progeny genome RNA more efficiently than parental WNV, was found to have a 3'-genomic sequence identical to that of its parent virus. The sequence change conferring the phenotype of this mutant is therefore located in another region of the genome. PMID- 3016983 TI - Assembly factors in poliovirus morphogenesis. AB - Extracts of poliovirus-infected HeLa cells promoted the in vitro assembly of 14 S subunits into empty capsids antigenically indistinguishable from procapsids. When infected cells were treated with iodoacetamide, the extract lacked the assembly promoting activity. This activity was restored by the addition of heat-disrupted virions, but the empty capsids formed in this system were antigenically different from procapsids. This and other observations introduce a distinction between the "assembly promoting" and "antigenicity conferring" activities of infected-cell extracts. PMID- 3016982 TI - Characterization of polytropic MuLVs from three-week-old AKR/J mice. AB - An immunological focus assay using monoclonal antibodies on live adherent in vitro cell lines was employed to detect and isolate different types of murine leukemia viruses (MuLVs) from spleen and thymus cells of young (less than 1 month of age) AKR/J mice. In agreement with earlier studies, ecotropic viruses were detected from cells of both tissues in all mice tested, although only trace levels of ecotropic MuLV infectious centers were found with thymus cells from mice of this age. Polytropic MuLVs were not detected in mice less than 3 weeks of age; however, between the ages of 3 and 4 weeks, polytropic viruses were detectable in assays of spleen cells from 50% of the mice. No polytropic MuLVs were detected in assays of thymocytes from any mice of this age. Several polytropic MuLVs obtained from spleens of young mice were further characterized. All of the isolates were infectious for both mink and SC-1 (feral mouse) cells, and exhibited interference properties typical of polytropic MuLVs. However, none of the viruses induced obvious cytopathic effects (CPE) on mink cells. All of the viruses appeared antigenically similar with regard to their reactivities to a panel of 12 monoclonal antibodies directed at envelope antigens of polytropic MuLVs. RNase T1-resistant oligonucleotide analysis of a polytropic MuLV from a 26 day-old mouse indicated that its entire env gene was derived from nonecotropic sequences while the remainder of its genome was indistinguishable from the ecotropic parent. The isolate thus exhibited a genome structure typical of Class II polytropic MuLVs and is the first example of this type of MuLV isolated from AKR/J mice. Examination of polytropic MuLVs derived from the spleens and thymuses of 5- to 6-month-old mice indicated that only 2 of 10 isolates examined induced CPE on mink cells. Furthermore, most of the CPE-negative viruses isolated from spleen and thymus cells of these mice exhibited in vitro host ranges and antigenic reactivities similar to isolates from young mice, suggesting that this type of polytropic MuLV may originate in the spleen, subsequently spread to other tissues, and persist throughout the preleukemic period. The detection of polytropic viruses in a large proportion of very young mice is in contrast to previous studies which have not detected polytropic virus production in AKR mice less than 5 to 6 months of age.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3016984 TI - Point mutations in the U3 region of the long terminal repeat of Moloney murine leukemia virus determine disease specificity of the myeloproliferative sarcoma virus. AB - The myeloproliferative sarcoma virus (MPSV) is made up entirely of sequences derived from the Moloney murine leukemia virus (Mo-MuLV) and the cellular mos oncogene. As other members of the Moloney murine sarcoma virus (Mo-MuSV) family, MPSV transforms fibroblasts in vitro and causes sarcomas in vivo. In addition, however, MPSV also causes an acute myeloproliferative disease in adult mice. The mos oncogene is essential for its transforming capacity, but sequences specific to the long terminal repeat (LTR) U3 region of MPSV account for its expanded target specificity as compared to Mo-MuSV (C. Stocking, R. Kollek, U. Bergholz, and W. Ostertag, Proc. Natl. Acad. Sci. USA 82, 5746-5750 (1985)). The U3 region of the LTR of MPSV is, however, closely related to that of the Mo-MuLV, and it appeared likely that the difference between MPSV and Mo-MuSV was caused by a divergent evolution of Mo-MuSV LTRs. In this paper, we show that this is not the case. The few nucleotide differences in the LTR between Mo-MuLV and MPSV are crucial for the expanded host range of MPSV. Moreover, Mo-MuLV-related gag sequences retained in MPSV are not essential for the distinctive biological properties of MPSV. PMID- 3016985 TI - Expression of genes encoding vesicular stomatitis and Sindbis virus glycoproteins in yeast leads to formation of disulfide-linked oligomers. AB - Saccharomyces cerevisiae strains transformed with plasmids containing cDNAs coding for the glycoproteins of vesicular stomatitis or Sindbis viruses can be induced to produce large amounts of glycosylated virus glycoproteins. Studies reported here show that these proteins from high molecular weight disulfide linked oligomers in the yeast endoplasmic reticulum. Oligomers were also found for two genetically altered forms of VSV G; one of these was lacking the membrane anchor domain and the other had the cysteine in the cytoplasmic tail replaced with serine. These oligomers can be separated from the bulk of yeast proteins by brief high-speed centrifugation of yeast extracts prepared by boiling cells with 1% sodium dodecyl sulfate. Treatment with thiol-reducing agents converts the oligomers to soluble monomeric forms, and this procedure leads to a substantial purification of glycoproteins from bulk yeast protein. PMID- 3016987 TI - Transposition and replication of maxi-Mu derivatives of bacteriophage Mu. AB - The insertion of DNA fragments within the lac sequence of a MudI(Ap,lac) prophage resulted in the formation of a set of maxi-Mu genomes which were 39.8, 59, 85.6, and 88.2 kb long, respectively. The comparison of these maxi-Mu's with the 37.2 kb-long parental MudI(Ap,lac) indicated that the transposition frequency decreased as the length of the prophage increased. No replication of the two longest maxi-Mu's could be detected. The 59- and the 39.8-kb-long chimeric genomes were noted to replicate at approximately 1-2 and 30%, respectively, of the rate found with the MudI(Ap,lac) prophage. The length dependence of the transposition and replication could be explained by the impairment of an early step of the transposition/replication mechanism. PMID- 3016986 TI - Transcriptional mapping of early RNA from regions of the Shope fibroma and malignant rabbit fibroma virus genomes. AB - Malignant rabbit fibroma virus (MV) is a recombinant poxvirus derived from Shope fibroma virus (SFV) and rabbit myxoma virus (D. S. Strayer, E. Skaletsky, G. F. Cabirac, P. A. Sharp, L. B. Corbeil, S. Sell, and J. L. Leibowitz, 1983a, J. Immunol. 130, 399-404; W. Block, C. Upton, and G. McFadden, 1984, Virology 140, 113-124). We report here the transcriptional mapping of early RNAs transcribed from the SFV sequences within MV and from the corresponding regions in SFV. Hybridization analysis and S1 nuclease mapping of RNA using viral DNA probes were used to define 5' and 3' ends of the various transcripts. The RNAs described here are transcribed in one direction in a densely arranged head to tail fashion similar to that described for some vaccinia virus early transcriptional units. At late times of infection the early SFV RNAs are not detected whereas the early MV RNAs are present in minor amounts. The early SFV and MV transcripts range in size from 3170 to 425 nucleotides (nt) long. All of the longer transcripts are produced as a result of read through transcription. Three MV transcripts contain fused SFV and rabbit myxoma virus sequences due to transcription through the recombination junction region in the MV genome. Two other MV transcripts are transcribed from a unique initiation site near another recombination junction region resulting in RNAs that are composed of SFV sequences having unique 5' ends. PMID- 3016988 TI - Biochemical properties of a transforming nonkaryophilic T antigen of SV40. AB - We reconstructed into wt SV40 DNA a previously described deletion of the A gene, eliminating amino acids 110 through 152 of the large T (L. Fischer-Fantuzzi and C. Vesco (1985) Proc. Natl. Acad. Sci. USA 82, 1891-1895); the gene product of the new recombinant pACTSV2, like the previous product, has a cytoplasmic instead of a nuclear localization and efficiently transforms NIH3T3 cells. Three main functions of this nonkaryophilic large T (NKLT) were examined, and the results obtained were the following: the NKLT does not bind to the SV40 origin DNA under conditions where the normal large T shows specific binding; the NKLT has conserved the ability to form high molecular weight aggregates; the NKLT becomes phosphorylated in vivo at only two residues: serine 639 and threonine 701. This indicates that the NH2-terminal phosphorylation of the large T is unnecessary for established-cell transformation. In addition, this and previous evidence (K. H. Scheidtmann et al. (1984) J. Virol. 50, 636-640) suggest that the lack of phosphorylation in serines 106, 676, 677, and 679 may constitute a characteristic of the large T molecules with extranuclear localization. PMID- 3016989 TI - Cloning and fine mapping the DNA of equine herpesvirus type one defective interfering particles. AB - Equine herpesvirus type one (EHV-1) defective interfering (DI) particle DNA fragments were inserted into the XbaI site of the plasmid vector pACYC184. Five DI XbaI fragments, which ranged in molecular weight from 4.5 to 6.7 MDa, were selected for detailed analysis. Each DI DNA clone was labeled with 32P deoxynucleotides by nick translation and hybridized to genomic digests of EHV-1 standard (STD) DNA bound to nitrocellulose. All five clones were shown to hybridize to DNA sequences derived from the left terminus (0.0-0.04 map units) of the long (L) region and from the short (S) region inverted repeats (IRs, 0.79 0.86 and 0.93-1.00 map units) of the STD EHV-1 genome. Restriction enzyme mapping studies and Southern blot hybridizations employing cloned STD virus DNA fragments as probes revealed that these EHV-1 DI clones contain two major domains: (1) an L terminal region which maps to 0.01-0.04 map units and is highly conserved among all five clones, and (2) a region homologous to the IRs which appears to vary between individual clones. PMID- 3016991 TI - The properties and sequence of glycoprotein H of herpes simplex virus type 1. AB - The map position of the coding sequence of glycoprotein H of herpes simplex virus type 1 was determined by marker transfer studies in which DNA fragments cloned from a virus resistant to neutralisation by an anti-gH monoclonal antibody were used to transfer antibody resistance to wild type virus DNA following cotransfection. The gH coding sequence was mapped to the BglII "m" fragment of HSV-1 DNA (map coordinates 0.27-0.312), confirming the map position previously determined by intertypic recombinant analysis (Buckmaster et al., 1984). The complete nucleotide sequence of the BglII "m" fragment revealed two large open reading frames in addition to the thymidine kinase gene. The open reading frame lying immediately 3' of the thymidine kinase gene has a predicted translation product with the features of a large glycoprotein. This open reading frame translates to an amino acid sequence of 90,323 mol wt with a signal peptide, a membrane anchor sequence, a large external domain containing potential N glycosylation sites, and a charged C- terminal cytoplasmic domain. We suppose that this amino acid sequence corresponds to gH of HSV-1, and A. Davison (personal communication) has noted the existence of homologous glycoproteins predicted from the nucleotide sequences of Varicella-zoster virus and Epstein Barr virus. The properties of monoclonal antibody LP11, directed against gH show remarkable similarities to the properties for gD antibodies. LP11 efficiently neutralizes virus infectivity, blocks cell fusion by syncytial virus strains, and inhibits the formation of plaques when added to cell monolayers after infection. These similarities in antibody activity imply functional relatedness between gH and gD of herpes simplex virus. PMID- 3016990 TI - Molecular cloning of the Mason-Pfizer monkey virus genome: biological characterization of genome length clones and molecular comparisons to other retroviruses. AB - The molecular cloning of the DNA provirus of Mason-Pfizer monkey virus (M-PMV) is described. Fourteen independent clones of integrated M-PMV proviruses were isolated from a human embryo kidney cell line that had been previously derived from a single cell clone infected with M-PMV. Characterization of these clones for size of insert, restriction pattern of flanking DNA, and presence of repetitive DNA in the flanking sequences revealed that 10 of the isolates were identical while the four remaining clones were unique. Three independent clones of unintegrated M-PMV proviruses containing a single copy of the long terminal repeat (LTR) were cloned from acutely infected human embryo kidney cells, Transfection assays revealed that 13 of 14 integrated proviruses and 2 of 3 unintegrated proviruses were capable of producing infectious virus. One of the integrated provirus clones (clone 6A) produced consistently higher titers of virus than all of the other clones in all assays used and in two different cell lines, indicating that it contained a mutation that enhances virus replication. The virus recovered after transfection was shown to be capable of inducing cell fusion in nontransformed cell lines, confirming that this property is associated with M-PMV. One of the clones was hybridized under conditions of varying stringency, to molecular clones of type B, C, and D retroviruses. These studies revealed M-PMV to be most closely related to squirrel monkey retrovirus (D-type virus) and more distantly related to mouse mammary tumor virus (B-type virus). Hybridization was also detected with clones from the pol gene region of a family of human endogenous sequences. No homology was detected with Rous sarcoma virus or most mammalian C-type viruses tested. The exceptions were baboon endogenous virus and RD114 in which previously identified homology in the env gene was confirmed. These results suggest that the type D and type B viruses can be linked together in a group of viruses of similar ancestral origin analogous to that recently proposed for the human T-cell leukemia viruses and bovine leukemia virus. PMID- 3016992 TI - An SV40 mutant T antigen does not bind the SV40 viral origin. AB - F8dl is an SV40 deletion mutant that lacks over 60% of the coding sequences for large T antigen and yet is able to immortalize early passage rat cells, to transform established cell lines, and to cause tumors in animals. We report here on the further characterization of this mutant and show that (a) transformation by F8dl is protein mediated but does not require the action of the SV40 small t antigen; (b) the F8dl T antigens have, or are associated with, an ATPase activity; (c) the 34-kDa mutant T antigen of F8dl is localized in nuclei and cell membranes of F8dl transformants and binds to double-stranded DNA; (d) the 20-25 kDa forms of the mutant T antigen are cytoplasmic; and (e) the F8dl T antigens do not bind with high affinity to the SV40 origin of viral DNA replication. PMID- 3016993 TI - Analysis of the origin-specific nucleosome-free region in SV40 encapsidation intermediates. AB - The presence of a nucleosome-free region at the replication origin in encapsidation intermediates of SV40 was determined by measuring the sensitivity of the intermediates to digestion by restriction endonucleases which lie within (BglI and SphI) or outside of this region (KpnI, MspI, and EcoRI). As judged by hypersensitivity to digestion with BglI and SphI, the origin-specific nucleosome free region was found to be present in nonencapsidated and partially encapsidated SV40 chromosomes and previrions, but not virions. PMID- 3016994 TI - Transmission of (LTR, v-src, LTR) without recombination with a helper virus. AB - We have analyzed transmission of (LTR, v-src, LTR) cryptic structure integrated in the H-19 mammalian tumor cell line. From this cell line different isolates of transforming virus were rescued in heterokaryons produced by fusion with chicken fibroblasts infected by replication-competent avian leukosis virus RAV-1. One of them (F6) was used for the transformation of avian cells in the absence of the helper virus. In four transformed cell lines studied, the (LTR, v-src, LTR) structure was again integrated at a unique position in the cell DNA of each line. This indicated that the (LTR, v-src, LTR) structure is transmitted by the helper virus without recombination. This point has been further supported by the finding that a src-containing species corresponding in size to the nonpolyadenylated src mRNA is present in the RNA isolated from the rescued F6 transforming virus which might serve as template for the synthesis of (LTR, v-src, LTR) structure by the reverse transcriptase provided by RAV-1. PMID- 3016995 TI - Messenger RNA encoding the phosphoprotein (P) gene of human parainfluenza virus 3 is bicistronic. AB - The complete nucleotide sequence of the phosphoprotein (P) mRNA of human parainfluenza virus 3 (PIV-3) was derived from two cDNA clones spanning almost the entire P gene. The mRNA, excluding the poly(A) tail, is 2014 nucleotides long and is bicistronic. The first open reading frame (ORF) codes for the phosphoprotein (P) of mol wt 68,860. Seven nucleotides downstream from the first AUG codon, in a +1 reading frame, there is an additional ORF which can code for a polypeptide of mol wt 23,266. The latter protein appears to be similar to the C proteins found in cells infected with several paramyxoviruses. Comparison of the predicted amino acid sequence of the P and C proteins of PIV-3 with the corresponding Sendai virus proteins reveals considerable homology at the C terminal half. In contrast, the P and C proteins of PIV-3 share very little homology with the measles virus P and C proteins, respectively. PMID- 3016996 TI - The effects of hepes, bicarbonate and calcium on the cGMP content of vertebrate rod photoreceptors and the isolated electrophysiological effects of cGMP and calcium. AB - To evaluate the role of cGMP in vertebrate rod photoreceptors, two extracellular effectors of cGMP were used (calcium and buffer), and both cGMP content and electrophysiological responses were measured. In the dark when the cGMP content was reduced by 80%, electrophysiological effects mimicking a weak background light were observed. Continuous illumination, sufficient to suppress all electrophysiological responses, had only minor effects on cGMP. The data seem inconsistent with suggestions that steady-state levels of cGMP alone control the light responses of rod photoreceptors. The electrophysiological effects of extracellular calcium (under conditions of unaltered cGMP) were distinguishable from those of cGMP. PMID- 3016997 TI - [The effect of prolonged physical exercise on the binding capacity of beta adrenergic lymphocyte receptors and their response in essential hypertension]. PMID- 3016998 TI - [Cyclic nucleotides in patients with cancer and non-tumoral diseases of the gastrointestinal tract]. AB - The intracellular levels of adenosine-3,5-monophosphate and guanosine-3,5 monophosphate were compared in 76 patients with different patterns of stomach pathology and 98 cases of colonic diseases. A significant decrease in concentration of cyclic nucleotides in malignant tumors of the digestive tract was observed. The results point to essential functional changes taking place at cellular level in the grossly intact mucosa of the stomach and colon in tumor pathology. PMID- 3016999 TI - [Surgical treatment of lung cancer in elderly patients]. AB - The study was concerned with ann analysis of the immediate and long-term results of surgical treatment of 263 lung cancer patients aged 60 years and older. Postoperative lethality rates were as follows: lung resection-5.4%, pneumonectomy 8.9 and extensive and combined pneumonectomy-27.2%. 35.1% survived 5 years and longer. Extensive and combined pneumonectomy gave poor immediate and long-term results in the above group. X-ray, broncho- and mediastinoscopy offer means for evaluation of mediastinal involvement to avoid the said procedures. PMID- 3017000 TI - Effect of beta-propiolactone on blood group antibody detection. AB - A panel of 50 blood group antibodies covering a range of blood group antigens has been tested in the presence of 0.25% beta-propiolactone as a possible means of reducing infectivity of high-risk HTLV III/HBsAg samples. 11/50 (22%) antibodies could not be detected by the indirect antiglobulin test, and 6/40 (15%) were undetectable by the two-stage papain technique. PMID- 3017001 TI - Anti-HTLV-III screening of blood donors. AB - In a survey on 82,383 blood donor specimens from Hessen, West Germany, the observed percentage of repeatedly positive HTLV-III/LAV antibody ELISA results was 0.2%, but only 0.018% were confirmed by Western blot, immunofluorescence assay or radioimmunoprecipitation assay. These data are consistent with larger epidemiological data compiled from other German blood banks and suggest a high degree of nonspecific false-positive results; one of the causes for false positives are HLA-DR4 antibodies, which react in the HTLV-III/LAV test. Epidemiological data on 127 dialysis patients and 1,153 prison inmates are presented. PMID- 3017002 TI - The clinical importance of leukocyte depletion in regular erythrocyte transfusions. AB - Antilymphocyte, antigranulocyte and antiplatelet alloantibodies, T lymphocyte subsets, expression of HLA-DR antigens on T lymphocytes and NK cell function were determined in 11 homozygous beta-thalassemic children multitransfused ab initio with Erypur-filtered leukocyte-free red cell units (group A) and in 13 similar children multitransfused with standard packed red cell units (group B). No antibodies were found in group A patients, whereas 69% of group B patients were immunized. The two groups did not differ significantly with regard to the other test results. Considered together, thalassemia patients showed a percentage of T4+ cells and a NK cell function that were significantly lower than those found in a reference group of 16 healthy male blood donors. Thalassemics moreover showed a higher than normal percentage of T3+, T4+ and T8+ cells expressing HLA DR antigens. The results indicate that leukocyte-free red cells should be the treatment of choice for prospective recipients of multiple transfusions, since they are capable of preventing (or delaying) the production of alloantibodies against leukocytes and platelets. From the data of the present study, it does not seem that the transfusion of leukocyte-free red cells is capable of preventing the abnormalities of some immunological tests that occur in some multitransfused patients. Further investigations, however, are needed to draw conclusions on this problem. PMID- 3017003 TI - Transfusion-transmitted cytomegalovirus infections: significance and control. AB - Cytomegalovirus (CMV) is a herpes virus which can give rise to primary infections, reactivated infections, or reinfections in humans. Seroepidemiologic studies have shown CMV infection to be worldwide with the highest antibody prevalences detected in Third World countries; however, significant regional variations can be seen within a given country. Antibody prevalence varies directly with age and inversely according to socioeconomic status. Numerous prospective studies of blood transfusion recipients carried out since 1966 have shown marked differences in infection rates but relatively little associated disease. Infection rates were highest in seronegative recipients given large amounts of fresh blood. Recently published reports have shown substantially lower infection rates than earlier studies, a change likely to be due to the current practice of transfusing fewer units of older blood. CMV has not been found to play a significant role in the etiology of posttransfusion hepatitis. CMV infections have been found to be an important source of morbidity and mortality in immunocompromised patients. Several studies of transfused, premature infants have shown significant differences in infection rates and disease expression. Seronegative low-birth-weight infants receiving blood from seropositive donors are at greatest risk. Blood from CMV-seronegative donors substantially lowers the risk of infection. Receiving a kidney or heart from a CMV-seropositive donor appears to be a more salient risk factor than blood transfusion in renal and cardiac transplant patients who are also more likely to have symptomatic CMV infections. Leukocyte transfusions have been found to be a significant source of CMV infection and disease in bone marrow transplant patients.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017004 TI - Development of CMV antibody tests and their clinical evaluation. AB - An anti-CMV ELISA was developed in which monoclonal antibodies to immobilize the CMV antigens are used. The test was compared with the IHA test and there was 98.6% agreement. Using the CF test as a referee, sensitivity and specificity of the ELISA were calculated to be 98.6 and 99.0%, respectively. The seroconversion rate among Dutch donors was estimated from the age-related prevalence of anti-CMV antibodies to be about 0.4%. Specific anti-CMV IgM was searched for in a large donor population using an antibody-capture IgM ELISA; 1 of 600 donor sera was found positive. This makes it unlikely that screening for IgM anti-CMV will effectively prevent posttransfusion CMV infections. In order to meet the demand for simpler tests, a one-step ELISA was recently developed, based on the inhibition principle. Preliminary studies with this test indicate a high sensitivity and specificity. PMID- 3017005 TI - Application of robotics in blood banking. AB - To demonstrate the feasibility of using robots in blood banking applications several prototype systems were developed. Activities associated with sample testing and component preparation were examined. In one project, a general purpose laboratory robot (Zymate Laboratory Automation System, Zymark, Inc., Hopkinton, Mass.) was configured to prepare samples for microplate-based ABO/Rh testing. In a second project, this same robot was configured to carry out specific steps in evaluating bar-coded labels, as part of a quality control procedure. A fluid-handling robot (Sampler 505, Tecan AG, Hombrechtikon, Switzerland) was used to prepare the dilution of serum samples for the evaluation of an anti-HTLV-III test kit. It was then configured to aspirate, dilute and dispense samples for anti-HTLV-III and HBsAg testing. This robotic system is now in field trial. The use of large industrial robots for automating component production was also considered. The key element in this design was the development of a fixture that would hold the blood bag set during the balancing, centrifugation and expressing steps. However, a fixture which was capable of performing these operations and that was equivalent in size and adaptable to a standard centrifuge bucket could not be fabricated. PMID- 3017006 TI - HTLV-III antibody testing in three Danish blood banks. AB - The Organon Teknika Vironostika anti-HTLV-III/LAV test was evaluated in three Danish blood banks. The evaluation comprised in total 3,940 consecutive donors. In all three blood banks the tests were carried out exactly according to the manufacturer's instructions, using a low cut-off value defined as (4N + P)/5, where N and P are means of optical densities of known negative and positive samples. By this method the overall frequency of repeatably positive samples was 0.30%. When tested by Western blot, however, none of these samples were shown to contain specific antibodies against HTLV-III/LAV proteins. When testing different categories of patients, only sera containing HLA antibodies gave rise to false positive reactions. Finally, important differences in the results were observed regarding sample preparation, single or dual wavelength optical density readings, and the experience of the technical staff. PMID- 3017007 TI - [Morphological characteristics during replication of human HSV-2 (Wu)]. PMID- 3017008 TI - [Molecular epidemiological study of ADRV from 12 outbreaks]. PMID- 3017009 TI - [Morphological study of 4 strains of infectious bovine rhinotracheitis virus isolated in China]. PMID- 3017010 TI - [Dynamics of DNA synthesis in IBRV (infectious bovine rhinotracheitis virus) infected cells and the morphogenesis of the viruses]. PMID- 3017011 TI - [Translation in the wheat germ cell-free protein-synthesizing system of human rotavirus RNA]. PMID- 3017014 TI - [Early noninvasive detection of reperfusion and infarct by intravenous technetium 99m pyrophosphate scintigraphy following thrombolysis]. AB - To determine whether technetium-99-pyrophosphate accumulation immediately after intravenous thrombolysis can serve as a marker of reperfusion and infarct size, 17 patients with acute myocardial infarction were studied. Immediately after thrombolysis 10 mCi of technetium-99m pyrophosphate were injected intravenously. Coronary and left ventricular angiography were then performed in all patients, revealing patent coronary arteries in 13 patients. In all patients, 0.3 and 0.5 mCi of thallium-201 were injected into the right and left coronary artery, respectively, followed by planar scintigraphy. 6 patients with patent coronary arteries and a large thallium-201 defect had massive (more than one third of the cardiac silhouette) pyrophosphate accumulation (group A), whereas 7 patients with a small or no thallium-201 defect in the presence of a patent infarct artery had either focal or no pyrophosphate accumulation (group B). In contrast, 4 patients with an occluded infarct artery showed no acute pyrophosphate uptake despite a large thallium-201 defect (group C). Emission computed tomography confirmed the planar scintigraphic data in group A patients and revealed small thallium-201 defects and focal pyrophosphate accumulation in group B patients with negative planar scintigrams. Global and regional ejection fractions in the infarct area, measured from the acute and follow-up left ventricular angiograms, were higher in group A than in group B and C patients. It is concluded that early intravenous technetium-99m pyrophosphate scintigraphy in patients with acute myocardial infarction undergoing intravenous thrombolysis may serve as an indicator of reperfusion and infarct size. PMID- 3017013 TI - [Bronchial cancer from the viewpoint of 2 neighboring regional cancer registers]. AB - From follow-up examinations on 460 male and 67 female patients with bronchial cancer during the years 1981 and 1982 in the cantons of St. Gallen and Appenzell/Switzerland, as well as from the region of Vorarlberg/Austria various morphological and clinically registered parameters of importance to oncology were comparatively analysed. These regional clinical cancer registers have existed for many years and are recognized legitimately and clinically as an important partial aspect in diagnostic, therapy and prophylaxis of malignant tumours. PMID- 3017015 TI - [Senna: chemistry and pharmacology]. PMID- 3017012 TI - [Isolation of an endogenous guinea pig herpeslike virus from Hartley guinea pigs with latent herpes simplex virus type 2 infection]. PMID- 3017016 TI - Spin-labeling of influenza virus hemagglutinin permits analysis of the conformational change at low pH and its inhibition by antibody. AB - To study the conformational changes in the hemagglutinin (HA) molecule of A/seal/Mass/1/80 (H7N7) (Seal) influenza virus at low pH, a spin-labeling method was used. This method also permits study of antibody interaction with the HA. A synthetic nitroxide compound was used for spin-labeling of tyrosine residues of the isolated HA molecule. Electron spin resonance (ESR) spectra of the spin labeled HA at various pH values indicated that a conformational transition occurred under acidic conditions, and around pH 5.8 the HA molecule has maximal flexibility. Since virus-induced hemolysis occurs optimally at pH 5.8-5.9, the HA molecule in the maximally flexible conformation is considered to mediate membrane fusion. The ESR spectra of the antibody-bound HA at various pH values revealed that monoclonal antibodies to different regions on the molecule may inhibit the conformational change by different modes. One antibody inhibited the changes in the HA that resulted in flexibility, while the other did not. These results support the assumption that monoclonal antibodies, which failed to inhibit hemagglutination of the virus yet neutralized viral infectivity, inhibited the fusion step in the viral replication process by interfering with a low pH-induced conformational change in the HA molecule (Kida, H., Webster, R.G. and Yanagawa, R. (1983) Arch. Virol. 76, 91-99). PMID- 3017017 TI - Formation, characterization and interfering capacity of a small plaque mutant and of incomplete virus particles of infectious bursal disease virus. AB - Serial undiluted passages of infectious bursal disease virus in chick embryo cells were accompanied by a von Magnus type fluctuation of infectivity in viral harvests and a gradual decrease of plaque size. From the 9th undiluted passage on, the whole virus population consisted of small plaque-forming virus. The small plaque size remained constant when subsequent infections were carried out at low multiplicities. Small plaque virus interfered with the replication of large plaque standard virus. The small plaque/low yield mutation favoured the generation of defective particles which could be separated from complete particles by their lower densities in CsCl-gradients, where six fractions became visible and could be analyzed separately. Most of the defective virus particles had lost the larger of their two dsRNA segments and showed an aberrant protein composition. They had a very low residual infectivity and were also able to interfere with the replication of complete virus. PMID- 3017018 TI - Xanthogranulomatous pyelonephritis in children. Report of 7 cases and one associated with nephroblastoma. AB - 8 cases of XGP in children are reported. The presence of urolithiasis together with xanthogranulomatous lesion in 7 cases and of nephroblastoma in one may be of help in understanding the aetiopathogenesis. In all the cases the involved kidney did not function thus necessitating subcapsular nephrectomy via retroperitoneal approach. PMID- 3017019 TI - [After care of hand injuries]. PMID- 3017020 TI - [Adult multilocular cystic nephroma simulating a kidney carcinoma]. AB - The multilocular cystic nephroma is a rare benign renal tumour which has its origin in the metanephrogenic blastema. As to the frequency it occurred in adults and children approximately share and share alike. It is reported on a 54-year-old woman with a right-sided multilocular cystic nephroma, who was operated on under the clinical suspicion of a renal carcinoma. PMID- 3017022 TI - [Inoculation device for the intradermal administration of viral antigens]. PMID- 3017023 TI - [2-stage augmentation of the atrophic jaw with hydroxylapatite ceramic granules]. PMID- 3017024 TI - [Computerized tomographic analysis of the mandible augmented with hydroxylapatite ceramic granules]. PMID- 3017025 TI - [Hormonally active pancreatic tumors (apudomas). A review]. AB - Hormonally active pancreas tumours have ceased to be rare cases, and by means of up-to-date methods they are now more frequently diagnosed than they used to be in the past. An account, based on the authors' own experience, is given in this paper of diagnostic and surgical problems relating to the various hormone producing tumours of the pancreas. Particular reference is made to the treatment of malignant apudomata and to new concepts which have resulted from the introduction of H2 receptor blockers. PMID- 3017021 TI - [Comparative studies of calcium resorption modified by vitamin D2 and D3 in patients with chronic renal failure and dialysis patients]. AB - In 16 patients with a chronic renal insufficiency at the stage of the compensated retention and 13 test persons of the chronic haemodialysis programme determinations of the total calcium and of the ionized calcium fraction as well as investigation of the intestinal calcium absorption by means of the isotope 47Ca were performed. In these cases the influence of vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol) on these parameters of the calcium metabolism was compared. Under the treatment with vitamin D a normalization of the serum calcium levels concerning the total calcium concentration as well as with regard to the ionized calcium fraction was achieved; this effect took place parallel to the improvement of the intestinal calcium absorption. Vitamin D3, had a more favourable effect than vitamin D2. The necessary doses are below 40,000 U a day. PMID- 3017026 TI - [Value of preoperative study methods in midrectal cancer. Microscopy versus macroscopy]. AB - A retrospective analysis was made of data pre-operatively obtained from 110 patients who had undergone curative, palliative or exploratory surgery for midrectal carcinoma, between 1972 and 1983. Macroscopic tumour findings obtained from rectoscopy, in that context, were found to provide a clue to loco-regional spread (p = between 0.0001 and 0.0162). On the other hand, no information as to loco-regional spread proved to be recordable by histological typing of mid-rectum carcinomas, but for tubulo-papillary carcinoma (p = 0.0153). Grading could provide a clue as to possible lymph node involvement (p = 0.0205). A clearly differentiated prognosis could be usually made by macroscopic tumour appearance (p = 0.0001 to 0.0657). Histological typing was of no value for prognostication (p larger than 0.337), while the value of grading proved to be extremely low (p = 0.1819). Incidence of locally delimited recurrences could not be safely forecast at all, neither by macroscopic assessment (p larger than 0.119) nor by microscopy (p larger than 0.161). The information obtainable from macroscopic findings on both present phase and prognosis was generally greater than that recorded from histological processing of biopsy material (p = 0.0470) and should, therefore, by no means be neglected in pre-operative reconnaissance. PMID- 3017028 TI - [The half-life of glucocorticoid receptor in SMMC-7721 human hepatoma cell line]. PMID- 3017029 TI - [Cytoskeleton and the release of herpes virus]. PMID- 3017027 TI - [Neuroendocrine disorders in contusion of the brain in the adrenergic-corticoid stage of stress]. AB - The adrenergic-corticoid stage of stress in contusion of the brain is distinguished by a long duration and marked neuroendocrine changes, which must be taken into consideration in applying therapeutic measures. The duration of the neuroendocrine disorders in craniocerebral injury is determined by the severity of brain damage. PMID- 3017031 TI - Fine needle aspiration cytology of focal liver lesions. Results obtained with examination of both cytologic and histologic preparations. AB - The results of 197 consecutive fine needle aspirations (FNA) of focal liver lesions performed on 176 patients were reviewed, and the 176 single most diagnostic aspirates were analyzed in detail. The majority of specimens were obtained using a 20-gauge or 22-gauge needle with ultrasound guidance. An attempt was made to obtain both a cytologic and a tissue specimen from each aspirate. The overall accuracy of the procedure was 85%; the accuracies of the tissue and cytologic specimens were 67% and 73%, respectively. The combined procedure detected 81% of the documented malignant tumors; the tissue specimen detected 62%. Eleven tumors were identified only in the tissue specimen and 23 were identified only in the cytologic specimen. There were no false-positive diagnoses. Six of nine hepatocellular carcinomas were detected. These results show that FNA cytology is a safe, accurate, relatively noninvasive technique whose diagnostic yield may be improved by examination of both a histologic tissue and a cytologic preparation. PMID- 3017030 TI - [Algoneurodystrophy]. AB - The authors consider the extensive literature concerning a quite frequently observed clinical picture, that has been named in different way by each researcher. Algoneurodystrophy is the name used in this analysis. They make a review of the most important works appeared in literature. It points the contrasting aspects of the research made till now. Therefore, in authors' opinion, it's necessary for them to investigate with further studies various aspects of the disease (etiological, pathogenetic, clinical, therapeutic ones, etc.) associated with a prevention of the secondary and tertiary damage the earliest as possible. PMID- 3017032 TI - Fine needle aspiration cytology in a case of hepatoblastoma. AB - The cytologic features of a case of hepatoblastoma diagnosed by fine needle aspiration (FNA) in an 18-month-old female infant are described. A mass detected in the left lobe of the liver on routine examination was subjected to FNA. The distinctive cytologic findings included malignant cells in small clusters and in acinar arrangements. Some of the acinar structures had bile plugs. Fat vacuoles were present within the cytoplasm of some of the malignant cells. Immature hematopoietic cells were also present. PMID- 3017033 TI - Effects of angiotensin II infusion on the early morning surge of ACTH and o-CRH provoked ACTH secretion in normal man. AB - The question of whether elevated plasma angiotensin II (AII) levels modulate ACTH secretion in man still awaits a definite answer. We performed two sets of experiments pertinent to that problem: Seven healthy young males each received AII (5 ng/kg/min) and sham infusions on different days in a randomized sequence from 03.00 h to 06.00 h in the morning, while plasma ACTH and cortisol were measured every 20 min. Mean blood pressure rose by about 10 mmHg during AII infusion. Mean plasma ACTH levels were slightly higher with AII than with sham infusion in every single individual (P less than 0.05). Differences in a pre- and post-infusion period were significant. Plasma cortisol levels were almost identical with or without AII infusion. Nine healthy young males received AII (5 ng/kg/min) or sham infusions on different days from 16.30 h to 20.00 h in a randomized sequence and a 100 micrograms o-CRH injection at 17.00 h. Plasma ACTH and cortisol were measured every 15 or 30 min between 16.30 h and 20.00 h. Mean blood pressure rose by about 14 mmHg during AII infusion. The rapid increment and further change in plasma ACTH and cortisol was not significantly different between the AII and sham infusion studies. CONCLUSIONS: The dose of AII infused was probably just above the threshold of ACTH stimulation, although AII plasma levels obtained were probably far above the physiological range. On the adrenal level, a vasoconstrictor effect of AII may have prevented stimulation of cortisol. This may be different in states of sodium depletion with reduced vascular effects of AII.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017034 TI - Repetitive and continuous administration of human corticotropin releasing factor to human subjects. AB - ACTH secretion was studied in response to repetitive and continuous administration of human corticotropin releasing factor (CRF) in 14 healthy volunteers and 2 patients with secondary adrenal insufficiency. ACTH increases during repetitive CRF administration were within the same range in normal subjects independent of the intervals (60-180 min) between the CRF pulses (100 micrograms iv). When CRF was infused continuously (100 micrograms/h for 3 h) after an initial CRF bolus injection (100 micrograms iv), ACTH and cortisol remained elevated during the infusion at a nearly constant level (ACTH: 60 +/- 5 pg/ml; cortisol: 21.2 +/- 1 micrograms/dl; means +/- SE). A second CRF bolus injection at the end of the infusion did not lead to a significant further increase of ACTH and cortisol levels. This shows that there is no desensitisation or depletion of a ready releasable pool, as it is observed with other pituitary hormones after releasing hormone stimulation. Pulsatile administration of CRF in 2 patients with secondary adrenal insufficiency due to previous cortisol or glucocorticoid excess, respectively, revealed a blunted response to the first pulse which became normal after the following pulses. The latter could not be sustained until the next morning without CRF given overnight. These findings point to a hypothalamic defect being the cause of hypocortisolism after long-term cortisol suppression. PMID- 3017035 TI - Specific stimulatory effects of Graves' IgG on the release of triiodothyronine from the patients' own thyroids. AB - The present study was undertaken to investigate the release of thyroid hormones from cultured thyroid slices of untreated patients with Graves' disease in response to autologous IgG and IgG from other untreated patients with Graves' disease. Thyroid tissue (8-10 mg) was obtained by needle biopsy from 11 untreated patients with Graves' disease, and each biopsy was divided into 5 slices. Slices were then cultured in Ham's F-12 synthetic media for 7 days with IgG (1.3 mg/ml) obtained from the same patients, IgG obtained from other untreated patients, normal IgG, or bovine TSH (1.3 mIU/ml). Media were changed every day, and the concentrations of triiodothyronine (T3) in the media were measured by radioimmunoassay (RIA), and the concentrations of thyroidal cAMP were measured by radioimmunoassay on the last day of culture after incubation with 10 mM theophylline at 37 degrees C for 30 min. When thyroid slices were incubated with autologous IgG, the release of T3 increased from the 3rd day, and the increase was 3- to 10-fold above controls on the 5th day, and the production of thyroidal cAMP significantly increased 2- to 3-fold. However, when slices were incubated with IgG obtained from other untreated patients, the concentration of T3 in media and the production of thyroidal cAMP did not differ from that in controls. TSH increased the release of T3 from thyroid slices 2.5- to 10-fold above controls on the 5th day and the production of cAMP 4- to 5-fold. These results strongly suggest that thyroid hormone releasing IgG in patients with Graves' disease are highly specific for autologous thyroids.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017036 TI - Effects of phorbol esters on protein phosphorylation and free T3 release by mouse thyroid lobes. AB - Phorbols stimulated 32P incorporation into several proteins in mouse thyroid lobes. Phosphoproteins (34 K and 23 K) were detected by SDS-polyacrylamide gel electrophoresis and autoradiography. Among phorbols tested, 12-O-tetradecanoyl phorbol-13-acetate (TPA) was the strongest stimulator of protein phosphorylation. On the other hand, TPA at 50-200 ng/ml significantly stimulated free T3 release by mouse thyroid lobes. The stimulatory effect of TPA was not additive to that of TPA or dibutyryl cAMP. TSH stimulation of free T3 release by mouse thyroid lobes, however, was significantly inhibited by H-7 (an inhibitor of protein kinase C) at a concentration of 50 microM. These results suggest that protein kinase C may play some role in thyroid hormone secretion by phosphorylating the endogenous substrates. PMID- 3017037 TI - The 24-h cortisol secretory pattern in Cushing's syndrome. AB - The 24-h plasma cortisol profile was obtained at 20-min intervals in 18 patients with Cushing's syndrome (10 with Cushing's disease, 5 with adrenal adenoma, 2 with ectopic ACTH secretion and 1 of questionable aetiology). The mean cortisol level was maximum in the case of ectopic ACTH secretion. The coefficient of variation of cortisol levels was subnormal in all except 2 subjects. Periodogram calculations, providing a best-fit curve (B F C) for each profile, showed that the existence of a significant baseline variation is a frequent feature. In certain cases, it is compatible with the persistence of a true circadian rhythm (2 patients with Cushing's disease; 1 patient with adrenal adenoma). The alteration of plasma cortisol pulsatility is much more pronounced in patients with adrenal adenoma than in patients with Cushing's disease. This is consistent with the hypothesis of a predominantly tonic secretion blunting the episodic hormone release. In 9 patients with Cushing's disease, the plasma cortisol pattern was suggestive of a combination of episodic cortisol release under CRF control and of continuous cortisol secretion due to constant stimulation from an autonomous ACTH source. Two cases were possibly of hypothalamic origin, as suggested by the presence of enhanced cortisol pulsatility and of a normal circadian amplitude. The analysis of the 24-h profile of plasma cortisol in Cushing's syndrome contributes to our understanding of the physiopathological mechanisms underlying this disorder and may help the diagnosis of its aetiology. PMID- 3017038 TI - Male pseudohermaphroditism due to 17 beta-hydroxysteroid dehydrogenase deficiency: gender reassignment in early infancy. AB - Male pseudohermaphroditism due to 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) deficiency has a high prevalence within the Arab population of the Gaza strip and is characterised by marked virilization at puberty, leading in many cases to the spontaneous adoption of a male gender role. As a result of this, parents of 7 affected male infants (aged 1-10 months) born with female phenotype requested early gender reassignment. Diagnosis was suspected in 5 on the basis of a positive family history, but confirmed in all cases by the finding of low to normal testosterone levels (30-184 ng/dl) with high delta 4-androstenedione levels (188-808 ng/dl), after hCG. Treatment with im testosterone oenanthate (25 50 mg/dose) was given in one to three 3-months courses and penile size was increased into the normal range without evoking a significant increase in height velocity or skeletal maturation. Five patients underwent the first stage of male genitoplasty between 2 and 3 years of age. This consisted of bilateral orchidopexy, chordee release and penile lengthening - yielding finally an anatomically normal-sized and shaped penis. Androgen responsive male pseudohermaphroditism due to 17 beta-HSD deficiency or a similar defect and diagnosed in infancy should be treated as soon as possible with systemic testosterone before considering any sex change, and in preparation for male genitoplasty. Early gender reassignment according to genetic and gonadal sex is probably the management of choice for these cases since this may result in a normal adjustment to the male gender role, particularly after puberty. PMID- 3017039 TI - Clonidine and the sympatico-adrenal response to coronary artery by-pass surgery. AB - Clonidine was administered intravenously in an attempt to limit sympatico-adrenal activity and thereby reduce the incidence of arterial hypertension associated with coronary artery by-pass graft surgery (CABG). Forty patients scheduled for CABG were assigned to two groups. Twenty patients received clonidine 4 micrograms kg-1 before surgery, 2 micrograms kg-1 after cardiopulmonary by-pass and 1 microgram kg-1 when the skin was sutured. The other 20 patients served as controls. All patients were anesthetized with fentanyl, droperidol, nitrous oxide and alcuronium. During surgery 5 min after sternotomy, mean arterial pressure was 13 mmHg lower (P less than 0.01) in the clonidine group, while after operation the difference between the groups was negligible. Both during and after surgery the plasma catecholamine concentrations were significantly lower in the clonidine group (P less than 0.01). The greatest difference between the groups was seen 90 min after operation, when plasma noradrenaline and plasma adrenaline concentrations in the clonidine group were less than 1/3 of those in the control group (P less than 0.01). As judged by catecholamine concentrations clonidine was effective in attenuating sympatico-adrenal hyperactivity during and after surgery. Postoperative arterial hypertension was not reduced, however, and it is concluded that other factors besides sympatico-adrenal hyperactivity must be important. PMID- 3017040 TI - [The campaign against leprosy in an urban African environment: problems encountered at the level of case-finding and of monitoring of patients in Dakar]. AB - The authors first explain the main epidemiological parameters of leprosy in Dakar, their evolution and their differences with those of the rest of the country. The second part deals with case finding and reveals the essential importance of the voluntary detection which appears as rather early: 3.5% of second degree physical disabilities; 37% of monomacular lesions in the paucibacillary lesions. The third part explains the problems encountered on leprosy control with the study of a cohort of 241 patients: 64% were missing in 4 years and half of them during the first year. At the end of 4 to 6 years, only 19% of the patients had a regular attendance at treatment. The defects are significantly more frequent with the male patients, and with the people who have been residing in Dakar for less than two years. In the suggested solutions, the authors insist on the necessity to adopt short multidrug protocols and to make health education for patients so that they care about case finding with their contacts. PMID- 3017041 TI - Ultrastructure of cerebellar capillary hemangioblastoma. V. Large pinocytic vacuolar bodies (megalopinocytic vesicles) in endothelial cells. AB - Large pinocytic vacuolar bodies (megalopinocytic vesicles) containing electron dense granulo-fibrillary material, not previously described in micro-vascular endothelium of brain tumors, were observed in endothelial cells of all five cases of cerebellar hemangioblastoma studied ultrastructurally. They were present in 23% of a total of 132 capillary profiles studied. Some were prominent and aggregated to occupy a large portion of the endothelial cytoplasm. Unlike the ordinary pinocytic vesicles in endothelial cells, they were distributed predominantly in the vicinity of the nucleus and surrounded by abundant organelles. They were irregular and usually several times larger than macropinocytic vesicles. The larger vacuolar bodies were often surrounded by bundles of microfilaments which often anchored on their limiting membrane. They coexisted frequently with Weibel-Palade bodies and occasionally fused with them. Convergence of coated vesicles and micropinocytic vesicles with the vacuolar bodies was present. However, there was no direct contact between the vacuolar bodies and Golgi apparatus, rough and smooth endoplasmic reticulum and mitochondria. The vacuolar bodies were closely associated with pericytic foot processes. It was suggested that they were formed by invagination of the abluminal cytoplasmic membrane with engulfed extracellular material and migrated internally. Discharge of their contents into the vascular lumen and interendothelial space was observed. Some had a disrupted membrane with a suggestion of release of contents into the cytoplasmic matrix. Their function is unknown, but they may serve as a specific vehicle of transport or digestive mechanism in microvascular endothelium under certain pathophysiological conditions, such as neoplasm, to meet the increasing metabolic demands. PMID- 3017043 TI - Correlation of morphologic subtypes of liver cirrhosis with excess alcohol intake, HBV infections, age at death, and hepatocellular carcinoma. A study on 234 autopsy cases in Japan. AB - Two hundred thirty-four autopsy cases of liver cirrhosis were examined to correlate the tissue HBV markers and excess alcohol intake with the type of liver cirrhosis, and the incidence of hepatocellular carcinoma (HCC). The following four groups were classified as follows: (1) HBV marker-positive alcoholic group A, (2) HBV marker-negative alcoholic group B, (3) HBV marker-positive non alcoholic group C, and (4) HBV marker-negative non-alcoholic group D. Macronodular cirrhosis predominated in groups, A, C, and D, while in group B macronodular and micronodular cirrhosis were almost of the same frequency. The mean age at death of the patients with macronodular cirrhosis of HBV-positive alcoholic group A was similar to that of HBV-positive non-alcoholic group C but lower than that of HBV-negative alcoholic group B, suggesting a longer survival of alcoholics without HBV infection than that with HBV infection, when patients had macronodular cirrhosis at autopsy. In HBV-negative alcoholic group B, patients with macronodular cirrhosis had a higher mean age than those with micronodular cirrhosis, while in HBV-positive alcoholic group A, the mean age of patients with either cirrhosis was similar. This suggested that in the absence of HBV infection, macronodular cirrhosis in alcoholics may be related to the increased life span that allows a conversion of micronodular cirrhosis into macronodular one, and in HBV-positive alcoholics it may arise in relation to HBV infection. HCC was frequently associated with macronodular cirrhosis, regardless of the presence or absence of alcohol abuse or HBV infection, but rare in micronodular cirrhosis. PMID- 3017044 TI - [Four long-acting analgesic hydrazone derivatives of opiates which bind more firmly with opiate receptors]. PMID- 3017042 TI - Evidence of specific benzodiazepine binding to myometrial membrane preparations from human pregnant uterus. AB - The muscle relaxant effect of benzodiazepines (BDZ) is widely held to be mediated at central as well as at peripheral level. In this study we report that crude membrane preparations from the myometrium of pregnant women possess binding sites for [3H]-RO 5-4864, specific ligand for the peripheral type of BDZ receptor. Scatchard analysis shows a high affinity binding site (KD = 3.1 +/- 1.2 nM) and a class of low affinity binding sites (KD greater than 30 nM). Displacement experiments show that various BDZ are differently effective in inhibiting [3H]-RO 5-4864 binding: RO 5-4864 greater than diazepam greater than flunitrazepam greater than lorazepam greater than chlordiazepoxide. In conclusion, these data seem to demonstrate that human myometrium possesses specific binding sites of 'peripheral type' for BDZ. It may be suggested that these binding sites are involved in mediating the myometrial relaxant effect exerted by BDZ. Thus, peripherally active BDZ could be used as tocolytic agents, avoiding BDZ central effects. PMID- 3017045 TI - [Effects of anemodeanin A on DNA, RNA and protein of tumor cells in vitro and plasma cAMP in mice]. PMID- 3017046 TI - [Antidotal effect of sodium dimercaptosuccinate against acute poisoning by the monosodium salt of 2-dimethylamino-1,3-bisthiosulfo-propane]. PMID- 3017047 TI - Affinity of various compounds for benzodiazepine binding sites in rat brain, heart and kidneys in vitro. AB - Binding of several psychoactive, antiinflammatory, antihypertensive, and antiarrhythmic drugs to central and peripheral benzodiazepine (BZ) binding sites was studied in the brain, heart and kidneys of rats. Diazepam exhibited the highest affinity for all binding sites (Ki values at 0.01 microM level); another 1,4-BZ, oxazepam, had markedly lower affinity for peripheral binding sites (Ki 21 37 microM). Non-BZ compounds had low affinity for central BZ receptors; proquazone was the most potent (Ki 9.5 microM). The affinities of non-BZ compounds were higher for peripheral BZ binding sites. The Ki value for proquazone was approximately 0.1 microM; and many other antiinflammatory agents, and the vasodilators cyclandelate and nifedipine, produced Ki values in the micromolar level. beta-Blocking drugs, and several other antihypertensive and antiarrhythmic agents lacked affinity for both central and peripheral BZ binding sites. According to the results, the affinity for peripheral binding sites is independent of an affinity for central BZ receptors. Non-BZ compounds that bound to brain BZ receptors bound with equal affinity to both BZ1 and BZ2 subgroups of receptors. The compounds with affinity for peripheral BZ binding sites did not select between heart and kidneys, which suggests that these organs have similar binding sites. The role of the peripheral BZ binding sites has not yet been established. The findings of this study allow the selection of a more varied group of ligands to be used when investigating the physiological significance of these binding sites. PMID- 3017048 TI - Early abnormalities in myocardial cell function in experimental diabetes. PMID- 3017049 TI - Effect of muscle relaxants on the motor endplate of diabetic and glucocorticoid pretreated rats. AB - The susceptibility to d-tubocurarine, gallamine, pancuronium, succinylcholine, and decamethonium of the motor endplate innervated by the anterior tibial nerve was studied in alloxan diabetic rats and in rats pretreated with cortisone and dexamethasone. The sensitivity to various muscle relaxants of cholinergic receptors in the motor endplate of alloxan diabetic and glucocorticoid-treated rats was changed. Beside alterations in affinity, in some cases the kinetics of action were also altered as compared to controls. The phenomenon is suggested to be brought about by a modulator substance circulating in the blood of alloxan diabetic and glucocorticoid-treated rats. PMID- 3017050 TI - The hypothalamic and neurohypophysial vasopressor and oxytocic content as influenced by alpha-adrenergic blockade in stressed rats. AB - The hypothalamic and neurohypophysial vasopressor and oxytocic content as influenced by alpha-adrenergic blockade in stressed rats. Acta physiol. pol., 1985, 36 (3): 193-200. The effects of phenoxybenzamine (PBA; an alpha-adrenergic blocker) on hypothalamic and neurohypophysial vasopressin and oxytocin were investigated in stressed rats. Immobilization resulted in a decrease of both vasopressor and oxytocic activities in the hypothalamus and neurohypophysis, whereas in rats, exposed to cold the vasopressin and oxytocin content in the hypothalamo-neurohypophysial system was increased. Under treatment with PBA the vasopressin and oxytocin content in the neurohypophysis was diminished in stressed (both immobilized and cold-exposed) rats when compared to respective groups of untreated animals subjected to appropriate kind of stress. The response of the vasopressinergic and oxytocinergic neurones seems, therefore, to be dependent on the type of stress. The alpha-adrenergic transmission is probably in some way involved in the mechanisms of modified neurohypophysial function in stressed animals. PMID- 3017052 TI - Detection of type V collagen-degrading enzyme activity in human liver. AB - Type V collagen-degrading enzyme activity was detected as a metalloprotease acting at neutral pH in the human liver. Type V collagen extracted from human placenta and labeled with [1-14C] acetic anhydride was used as the substrate in the assay. Four major degradation products with relatively high molecular weights were observed upon polyacrylamide gel electrophoresis of the incubation mixture of type V collagen and liver homogenate. The significance of the measurement of this enzyme activity was discussed in relation to the clarification of the mechanism of liver fibrosis. PMID- 3017051 TI - Generation of active oxygen species by iron nitrilotriacetate (Fe-NTA). AB - Ferric nitrilotriacetate (Fe3+-NTA) solution showed maximum absorbance at pH 7.5. The iron was in ferric high-spin state and coordinated octahedrally with a relatively symmetric structure and also probably pentagonally. A spin trapping technique employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) yielded a DMPO spin adduct of unknown radical with three doublets (DMPO-Z) and a simple nitroxide radical (Y-NO.) in serum from rats injected intraperitoneally with Fe3+-NTA. When the Fe3+-NTA solution was diluted 500-fold with 50 mM NTA solution, DMPO-Z, Y-NO. and an additional signal, DMPO-OH were observed. The DMPO-Z signal was suppressed by a decrease in oxygen tension, alpha-tocopherol and 3-tert-butyl-4-hydroxy anisole (BHA). The DMPO-OH signal was suppressed in the presence of ethanol and catalase. Fe2+-NTA solution hardly produced DMPO spin adducts. The Fe3+-NTA solution produced a strong DMPO-OH signal in the presence of H2O2. Rose Bengal solution, a singlet oxygen generating system, produced the same DMPO adducts. Fe3+-NTA reacted with oxygen in solution. The oxygen was activated and might be similar to singlet molecular oxygen. In the presence of H2O2, the Fe3+-NTA solution generated a hydroxyl radical. Fe3+-NTA itself generated free radicals, but Fe2+-NTA did not. PMID- 3017053 TI - Urinary cyclic adenosine-monophosphate (cAMP) in children with familial hypophosphataemic rickets treated with vitamin D3 and phosphate given orally and by nocturnal intragastric infusions. PMID- 3017055 TI - Hemostatic factors and renin in Greenland Eskimos on a high eicosapentaenoic acid intake. Results of the Fifth UmanaK Expedition. AB - The Fifth UmanaK expedition compared the fatty acid composition of platelets, bleeding times before and after ingestion of acetylsalicylic acid, 24-hour urinary tetranorprostanedioate, creatinine and Na output, as well as plasma renin, serum electrolytes and antithrombin III in 20 Greenland Eskimos and 20 Danes. The results indicate that the prostaglandin production was not inhibited in the Eskimos, and that the antiaggregatory prostanoids predominate in Eskimos compared to Danes. Although blood pressure and 24-hour urinary Na output were similar, the plasma renin level was significantly higher in the Eskimos on a high eicosapentaenoic acid intake. PMID- 3017056 TI - Intracellular electrolytes in cardiac failure. AB - In congestive heart failure (CHF) there are several compensatory mechanisms operating which may influence electrolyte metabolism. The activation of the renin angiotensin-aldosterone system causes retention of sodium (Na) and losses of potassium (K) and magnesium (Mg). The secondary hyperaldosteronism may give rise to high intracellular Na and low intracellular K through a direct permeability effect on the cell membrane. The Mg deficiency may lead to a further increase of intracellular Na and decrease of intracellular K since Mg is a necessary ion for the function of the Na-K pump. In 297 patients with diuretic treated CHF we found that 42% had hypokalemia, 37% hypomagnesemia and 12% hyponatremia. We also found that 57% had excess muscle Na, 52% had depletion of muscle K and 43% had low muscle Mg. We have also shown that the low muscle K cannot be corrected by K supplementation when there is a concomitant Mg deficiency and that Mg infusions may change the disturbed relation between extra- and intracellular electrolytes towards normal. PMID- 3017054 TI - Indices of mineral metabolism in relation to blood pressure in a sample of a healthy population. AB - Indices of mineral metabolism in blood and urine were analysed in relation to blood pressure in 97 healthy subjects aged 16-82 years. In a multivariate analysis, after allowing for the effects of sex, body mass index (BMI) and age, there was an inverse relationship between plasma level of ionized calcium and mean blood pressure (MBP) (beta = -50.0 mmHg/mmol/l P-ionized calcium, p = 0.0005). In univariate analyses MBP also showed statistically significant inverse relationships with plasma ionized calcium, serum phosphate and renal threshold concentration of phosphate; positive relationships to MBP were found for fasting urinary excretion of calcium and cyclic adenosine monophosphate. However, when examined multivariately, only the relation between MBP and plasma ionized calcium persisted. This study supports previous findings of an inverse relationship between blood pressure and serum ionized calcium and extends the observations to the physiological range. It is further evident from this study that BMI and age should be taken into account in analyses of the relationship between blood pressure and mineral metabolism. PMID- 3017058 TI - Effects of ACE-inhibition on regional circulation in congestive heart failure. AB - During development of heart failure there are marked changes in the peripheral circulation. The renal and splanchnic circulations are reduced early. The cerebral circulation is maintained by autoregulation. Skeletal muscle blood flow is reduced in relation to the decrease of cardiac output. There are also important metabolic changes in these muscles with reduced mechanical efficiency. Treatment with arterial vasodilators increase cardiac output. However, most of this increase is usually shunted, probably in the splanchnic circulation. If any, only small increases in muscle blood flow have been reported. ACE-inhibition has been shown to have beneficial acute effects on the renal, cerebral and skeletal muscle blood flow with improved flow in these areas. This treatment seems to be the most beneficial vasodilatory alternative available so far. PMID- 3017057 TI - Positive inotropic drugs--digitalis. AB - Digitalis glycosides inhibit Na+K+-ATPase in the cells and have been used for scientific studies of cation transport over cell membranes. Furthermore, digitalis has a positive inotropic and antiarrhythmogenic effect. Specific binding sites for digitalis glycosides have been observed in erythrocytes, the myocardium and the central nervous system. Transcellular transport of digoxin has been found in the kidney, since digoxin is excreted by tubular secretion. Recent studies have discovered an endogenous digitalis-like substance both inhibiting Na K-ATPase and displacing digoxin from specific binding sites at the erythrocytes. The concentration of this component in plasma seems to be higher in hypertension than in normotension. Future studies will have to disclose other effects of this substance in order to evaluate whether it can be used as a drug in heart failure. PMID- 3017059 TI - Effects of ACE inhibition on renal regulation of salt and water. AB - CHF may activate the RAS by various mechanisms. Acute CHF is associated with high PRA, whereas chronic, stable disease is combined with normal values. The response to ACEI is affected by blood pressure, degree of activation of the RAS, salt balance and degree of possible renal failure. It may also be affected by concomitant diuretic or, e.g., digoxin therapy. ACEI improves RPF, GFR may remain normal or may increase, if it was previously impaired due to reduced RPF. Severe hypotension in combination with decreased autoregulatory capacity may decrease GFR. Generally, renal excretion of sodium and water increase. These changes in renal handling of salt and water are primarily caused by decreased AII. They are also augmented by inhibited sympathetic tone and thirst and decreased release of ADH and aldosterone. Increased synthesis of vasodilating and natriuretic PGs is probably also of some importance. Dilutional hyponatremia may be corrected by combined ACE inhibitor and furosemide treatment. Water and sodium excretion increase and sodium is redistributed from the intracellular space. Low serum sodium values increase and azotemia may be corrected, if ACE inhibitor doses are carefully titrated to avoid severe hypotension. These effects are ascribed mainly to a decrease of AII, thirst and ADH release. The effect of furosemide is improved since increased amounts of salt are delivered to the loop of Henle and access of furosemide to its site of action is facilitated by increased RPF. ACEI does not cause any obvious negative effects on renal handling of salt and water.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017060 TI - [HMSN type II with autosomal recessive transmission. Description of 2 cases with early onset]. PMID- 3017061 TI - [Eaton-Lambert syndrome. Clinical and electrophysiologic study of a case: effects of temperature on neuromuscular transmission]. PMID- 3017062 TI - Central- and peripheral-type benzodiazepine receptors. PMID- 3017063 TI - GABAergic transmission and anxiety. Proceedings of the 4th Capo Boi Conference on Neuroscience. Villasimius, Italy, June 1985. PMID- 3017064 TI - Regulation of the benzodiazepine receptor: in vivo and in vitro experiments. PMID- 3017065 TI - Structural analysis and localization of GABA/benzodiazepine/TBPS-receptor complex using monoclonal antibodies. PMID- 3017066 TI - Stress and GABAergic transmission: biochemical and behavioural studies. PMID- 3017067 TI - Endogenous benzodiazepine binding inhibitors from bovine cerebral cortex: isolation and purification. PMID- 3017068 TI - Isolation, purification, and immunochemical studies of benzodiazepine receptor(s) and its ligand(s) for mammalian brain. PMID- 3017070 TI - EEG interaction between delta 8-tetrahydrocannabinol and some sedative-anxiolytic drugs. Is the anxiogenic effect of Cannabis related to an action on the GABAergic system? PMID- 3017069 TI - Do the intrinsic actions of benzodiazepine receptor antagonists imply the existence of an endogenous ligand for benzodiazepine receptors? PMID- 3017071 TI - Structure and function of the barbiturate-modulated benzodiazepine/GABA receptor protein complex. PMID- 3017073 TI - Demonstration of GABA/barbiturate-receptor-mediated chloride transport in rat brain synaptoneurosomes: a functional assay of GABA receptor-effector coupling. PMID- 3017072 TI - Effect of benzodiazepine binding site ligands on [35S] t butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain: an autoradiographic study. PMID- 3017075 TI - Sonographic diagnosis of Klatskin tumors. AB - The sonograms of 14 patients with pathologically proven Klatskin tumors presenting between 1974 and 1985 were reviewed. All 14 patients demonstrated dilated intrahepatic bile ducts with a normal-sized extrahepatic biliary tree. In seven patients this was the only finding. The other seven patients demonstrated one or more additional abnormalities. These included an apparent intraductal mass at the confluence of the right and left intrahepatic ducts (four patients), enlarged portal lymph nodes (two patients), and hepatic metastases (one patient). With one exception, these additional findings were seen only in patients scanned after 1980, that is, using real-time sonography and up-to-date scanners. The presence of dilated intrahepatic ducts in a patient with a normal extrahepatic biliary tree should raise the possibility of Klatskin tumor. With high-resolution real-time sonography, further evidence suggestive of malignancy can be demonstrated in at least 50% of patients. PMID- 3017074 TI - Turnover of the benzodiazepine/gamma-aminobutyric acid receptor complex determined in situ. PMID- 3017076 TI - Cholangiocarcinoma as a late complication of choledochoenteric anastomoses. AB - Two cases of cholangiocarcinoma developing several years after choledochoenteric anastomoses are reported. It is speculated that these may represent heretofore unrecognized late complications of this procedure. The intermittent chronic obstruction with bile stasis and recurrent inflammation may be causative factors in the development of cholangiocarcinoma after choledochoenterostomy. PMID- 3017077 TI - Cholangiocarcinoma in a choledochal cyst: preoperative diagnosis. PMID- 3017078 TI - Angiography of small hepatocellular carcinomas: analysis of 105 resected tumors. AB - One hundred five hepatocellular carcinomas less than 5 cm in diameter were resected in 75 patients. The tumors were studied with respect to their detection rate by angiography and their angiographic features. Angiography identified 86 (82%) of the 105 lesions, missing 19 (18%). The findings included tumor vessels (70%) and tumor staining (76%). Pathologic analysis of the 19 undetected lesions showed that 74% of them were smaller than 2 cm in diameter and that they were well-differentiated carcinomas. Forty percent of 100 lesions were in the anterosuperior subsegment of the right lobe. PMID- 3017080 TI - Cutaneous and visceral hemangiomata in the Klippel-Trenaunay-Weber syndrome: antenatal sonographic detection. PMID- 3017079 TI - Accuracy of angiography in the diagnosis of small hepatocellular carcinoma. AB - Conventional hepatic arteriography combined with superselective infusion arteriography was carried out in 51 patients with hepatocellular carcinoma smaller than 5 cm, and angiograms of varying phases were analyzed. In cancers smaller than 40 mm, particularly in those smaller than 20 mm, so-called tumor stain in the capillary phase was the only abnormality seen in most but not all cases. Within a tumor stain, there were unstained areas in most cases and histologic examination in resected specimens showed them to be due to either necrosis, fibrosis, or fatty changes. Homogeneity and shape of the stain seemed to be related to growth speed and invasiveness of the cancer. Although overall diagnostic value of angiography for small hepatocellular carcinoma was high, super-superselective infusion hepatic arteriography produced nodular stains in 7 of 11 control cases of nonalcoholic cirrhosis without cancer, making difficult the differential diagnosis between stains due to tumors and those due to hyperplastic nodules of cirrhosis. PMID- 3017082 TI - Thallium-201/technetium-99m pyrophosphate overlap in patients with acute myocardial infarction after thrombolysis: prediction of depressed wall motion despite thallium uptake. AB - Intracoronary thallium-201/technetium-99m pyrophosphate planar scintigraphy was performed in 60 patients with acute myocardial infarction undergoing intracoronary thrombolysis to predict salvage of myocardium immediately after thrombolysis. In eight patients a significant overlap of new thallium uptake and technetium pyrophosphate accumulation was found after thrombolysis. Intravenous planar thallium scintigraphy revealed thallium uptake in the region of overlap in all patients; circumferential profile analysis showed no difference in the thallium scintigrams before and after technetium injections. Both findings indicate that overlap is not the result of scattering of technetium into the thallium window. Emission computed tomography revealed thallium/technetium pyrophosphate uptake in identical slices and regions. Regional wall motion in the area of overlap remained depressed in all patients, in contrast to patients with similar thallium uptake without overlap. These data suggest that thallium/technetium pyrophosphate overlap reflects the close proximity of viable and necrotic myocardial cells and predicts depressed wall motion after thrombolysis. PMID- 3017081 TI - Coronary vascular responses to hypoxia in the diabetic lamb: independence from adenosine and autonomic mechanisms. AB - We have previously found that the coronary dilator response to infused adenosine is attenuated in diabetic (alloxan) lambs. Adenosine responsiveness is restored by administration of insulin. The present studies tested the hypothesis that coronary flow changes with hypoxia, if mediated by adenosine, would also be modified. Studies were carried out in eight control and six diabetic lambs. The animals were anesthetized and prepared to maintain constant arterial pressure (reservoir), cardiac output (pump), and heart rate (paced). Atropine and practolol were given. Forced inspired oxygen was reduced in steps. Arterial and coronary sinus blood samples were analyzed for Po2, oxygen content, pH, hematocrit, and glucose. Myocardial oxygen delivery and uptake (MVO2) were calculated. Coronary flow increased identically in both control and diabetic animals as PaO2 was reduced below 60 Torr. Oxygen delivery and MVO2 fell equally in both groups. Acidosis potentiated hypoxic coronary flow changes. Alpha blockade (phentolamine) was without effect in control lambs but caused coronary flow to increase in diabetic lambs. Changes in coronary flow with hypoxia were unaffected, however. Insulin caused no change in the coronary dilator response to hypoxia in either control or diabetic lambs. It is concluded that coronary alpha tone is increased in diabetes but does not modify changes in coronary flow during hypoxia. As coronary flow responses to hypoxia were unaltered in diabetic lambs, and unaffected by insulin, adenosine may not be the primary mediator of coronary vascular dilatation. Potentiation of adenosine by tissue acidosis is apparently insufficient to explain these findings. The mechanism for coronary dilatation during hypoxia is unclear but may involve direct effects of reduced oxygenation of coronary vascular smooth muscle. PMID- 3017083 TI - Value of two-dimensional echocardiography, electrocardiography, and clinical signs in detecting right ventricular infarction. AB - Noninvasive tests for the diagnosis of right ventricular (RV) infarction--two dimensional echocardiography (2DE), ST elevation in V4R, and clinical parameters- were compared with equilibrium gated blood pool study (GBPS) in 50 patients after acute inferior myocardial infarction. Twenty-two of 50 patients had RV wall motion abnormalities on GBPS and 20 of 50 on 2DE. Sensitivity and specificity of 2DE was 82% and 93%, ST elevation in V4R was 50% and 71%, elevation of venous pressure was 77% and 85%, and a positive Kussmaul's sign was found in 59% and 89% for the detection of RV infarction compared to GBPS. Patients with RV infarction had higher peak creatine kinase levels and lower left ventricular ejection fractions than patients without RV infarction. Three patients died and all had significant left ventricular damage. At 20 weeks' follow-up, two thirds of the patients had no residual RV wall motion abnormalities, and all but two patients showed some recovery. PMID- 3017084 TI - Ventricular systolic and diastolic impairment during pacing-induced myocardial ischemia in coronary artery disease: simultaneous hemodynamic, electrocardiographic, and radionuclide angiographic evaluation. AB - This study examined the impairment in systolic and diastolic performance of both ventricles during pacing-induced myocardial ischemia in 12 men with coronary artery disease. Simultaneous hemodynamic, ECG, and radionuclide angiographic assessments were made: pre pacing (pre-P); intermediate pacing (P-1); maximum pacing (P-2); and immediately after pacing (post pacing (P-P). The prepacing measurements were made with the patient in the supine position and during leg elevation. Pacing produced a leftward and upward shift in the diastolic pressure volume relation, a progressive decrease in left ventricular (LV) end-diastolic volume (p less than 0.003) and right ventricular (RV) end-diastolic volume (p less than 0.01), concomitant with an increase in the pulmonary artery wedge pressure (p less than 0.004) and the right atrial pressure (p less than 0.04). The shift in the LV pressure-volume relation was associated with an initial increase (P-1), followed by a decrease (P-2) in the peak filling rate (p less than 0.001). Pacing also resulted in systolic dysfunction: abnormal LV ejection fraction responses in eight patients, LV regional wall motion abnormalities in eight patients, and abnormal RV ejection fraction responses in seven patients. Leg elevation resulted in a 7% increase in cardiac output, a 20% increase in RV end-diastolic volume, a 28% increase in right atrial pressure, a 29% increase in pulmonary artery wedge pressure, and a 10% increase in LV end-diastolic volume (p less than 0.05). Thus, the ischemic response to pacing results in systolic and diastolic LV and RV dysfunction, with the diastolic impairment being more frequent than the systolic impairment. PMID- 3017085 TI - Detection and localization of recent myocardial infarction by magnetic resonance imaging. AB - The potential of magnetic resonance imaging (MRI) to detect and localize acute myocardial infarction (AMI) in 27 patients a mean interval of 15 days after AMI was evaluated. Eighteen asymptomatic volunteers were also studied to determine the specificity of the observations. The diagnosis of AMI was established by conventional criteria; the infarct was localized by electrocardiography in all patients, technetium pyrophosphate scintigraphy in 19 and necropsy in 1 patient. MRI detected increased myocardial signal intensity in 88%, cavitary signal in 74% and regional wall thinning in 67% of the patients. At least 1 of these 3 features was seen in the area of the infarct in each patient. The sensitivity of these MRI observations was not influenced by location of the infarct or presence of Q waves. Asymptomatic volunteers also had increased myocardial signal in 83%, cavitary signal in 94% and wall thinning in 11% of cases. Some patients had these findings in myocardial segments not suspected of being involved by recent or remote AMI. It is concluded that AMI can be detected by MRI performed an average of 15 days after infarction. However, the hearts of normal volunteers and apparently normal myocardial segments of patients with AMI may have the MRI findings previously associated with AMI. Of these findings, wall thinning was the most predictive of and specific for AMI. PMID- 3017086 TI - Calcium-blocker withdrawal phenomenon: increase in affinity of alpha 2 adrenoceptors for agonist as a potential mechanism. AB - Precipitation of myocardial ischemia after withdrawal of calcium channel blocking drugs has been observed in some patients, but the mechanism of this phenomenon is not known. Among 15 patients with coronary artery disease who had been treated with nisoldipine, onset of severe unstable angina was observed in 2 and evolution of acute myocardial infarction in 1 patient after abrupt withdrawal of nisoldipine therapy. To determine a mechanism of myocardial ischemia after cessation of nisoldipine therapy, the status of alpha 2 adrenoceptors and platelet sensitivity to epinephrine was examined in 5 patients. After 6 weeks of therapy, there were no changes in the number of alpha 2 adrenoceptors (199 +/- 45 vs 188 +/- 35 fmol/mg protein, mean +/- standard error of the mean) or affinity for antagonist (dissociation constant 5.78 +/- 0.99 vs 4.70 +/- 0.87 nM), but there was a significant (p less than 0.01) increase in the affinity of alpha 2 adrenoceptors for agonist epinephrine. In vitro platelet sensitivity to epinephrine, but not ADP, also increased markedly. It is postulated that an increase in alpha 2-adrenoceptor affinity for agonist may relate to platelet hyperactivity, which may result in severe myocardial ischemia upon withdrawal of calcium blocking drugs in some patients. PMID- 3017088 TI - Gastrointestinal complications in renal transplant recipients. AB - In a 16-year study, 101 gastrointestinal (GI) lesions (16 fatal) developed in 580 renal transplant recipients seen in the authors' institution. Lesions were seen at all levels of the GI tract, but colonic lesions were the most common (42 patients) and were fatal in 8. Segmental ischemic colitis was the single most common morphologic diagnosis (14 patients). Seven of these patients had an unusual syndrome that clinically, at surgery, and on gross examination resembled inflammatory bowel disease. Lesions were segmental; involved bowel was thickened and erythematous with creeping peritoneal fat. Histologically, mucosa adjacent to the frank necrosis showed simplification and striking epithelial atypia. Specific identifiable viral infections caused 28% of the GI complications in this series. This incidence is higher than that in other reported series. Most of these infections can be diagnosed from endoscopically obtained material. These findings have therapeutic implications. PMID- 3017087 TI - Different pharmacological anatomy in the paraventricular hypothalamic nucleus, supraoptic nucleus, and suprachiasmatic nucleus of rats: quantitative autoradiography on angiotensin II receptor binding sites. AB - Angiotensin II (AII) and vasopressin (VP) play important roles in cardiovascular function. Using 125I-[Sar1,Ile8]-angiotensin II (125I-SI-AII), a potent AII antagonist, AII receptor binding sites were autoradiographically localized in three VP-producing areas of the hypothalamus and compared in hypertensive and normotensive rats. Within three major VP-producing areas, AII receptor binding was highest in the paraventricular hypothalamic nucleus and lowest in the supraoptic nucleus, suggesting that a differential AII regulation of separate VP systems exists in the brainstem. No statistical difference in 125I-SI-AII receptor binding was found between WKY and SHR rats in each of the three major VP producing nuclei studied. These results are consistent with a role of AII receptors in a subtle and complicated regulation of VP in cardiovascular function. PMID- 3017089 TI - Performance evaluation of the Abbott HTLV III EIA, a test for antibody to HTLV III in donor blood. AB - An enzyme immunoassay (EIA), designed to detect antibodies to human T-cell lymphotropic virus type III (HTLV III), was evaluated. The antibody test was found to be highly sensitive; serum from 221 of 223 (99.1%) patients with acquired immune deficiency syndrome (AIDS) was positive for antibodies to HTLV III. In addition, the antibody test was found to be highly specific; approximately 99.75% of 20,720 serum or plasma samples from blood donors were negative for antibody to HTLV III. In most cases, the Western Blot analysis agreed well with the EIA. Eighty-one of 82 (98.8%) EIA-positive samples from patients with AIDS were Western Blot positive. Of the EIA-positive blood donors, 21 of 36 (58%) were detected and confirmed by Western Blot analysis. A solid phase competitive EIA also was evaluated as an alternate procedure. The preliminary results indicate that this immunoassay has a high degree of sensitivity and specificity and could serve as an alternate procedure to detect antibody to HTLV III. PMID- 3017090 TI - Peroxidase activity in circulating mast cells in blast crisis of chronic granulocytic leukemia. Comparative studies with basophils and cutaneous mast cells. AB - Although the hematopoietic origin of mast cells is very probable, the cell from which they originate is still a matter of speculation. The description of "transitional basophil/mast cells" in myeloproliferative disorders has suggested a common origin for basophils and mast cells. In a case of mast cell transformation of chronic granulocytic leukemia, the authors have studied the morphology and peroxidase activity by three classical technics, of circulating mast cells and transitional "basophil/mast cells." These results were compared with those of blood and bone marrow basophils and those of cutaneous mast cells. In both mast cells and "transitional basophil/mast cells," peroxidase activity was revealed in the nuclear envelope, endoplasmic reticulum, and granules. This activity was detected in unfixed cells and in tannic acid-aldehyde-fixed cells but not in 1.25% glutaraldehyde-fixed cells, where the staining appeared only in the granules. The comparison of this activity with that of normal basophils and mast cells suggests that the proliferating cells in this case possess at the same time the peroxidase activity of basophils and mast cells. PMID- 3017091 TI - Pleural malignant mesothelioma following Wilms' tumor. AB - A 28-year-old woman had a left pleural malignant mesothelioma develop, which resulted in her death. At age four, she had undergone a left nephrectomy for Wilms' tumor and had received radiation therapy to the left renal fossa and to the right lung, the latter for a presumed diagnosis of metastatic tumor. Asbestos body counts of digested lung tissue were within the normal range. This is the fourth reported case of malignant mesothelioma following Wilms' tumor and is the first to provide quantitative analysis of asbestos in the lung. PMID- 3017092 TI - Long survival of diarrhea-associated hepatocarcinoma treated with Adriamycin and indomethacin. A case report. AB - The existence of tumors producing prostaglandins is well documented in the literature. At present, no case report of a prostaglandin-producing hepatocellular carcinoma has been published, to our knowledge. The authors report a patient with hepatocellular carcinoma associated with diarrhea mediated by prostaglandins, surviving 30 months after receiving treatment with indomethacin and Adriamycin. The authors will discuss the possible role played by indomethacin in the exceptional clinical course of the patient. PMID- 3017093 TI - Effects of diazepam on orthodontic tooth movement and alveolar bone cAMP levels in cats. AB - Cyclic AMP has been suggested as a possible intracellular mediator in bone remodeling during tooth movement. Accordingly, an increase in the level of this nucleotide should result in faster tooth movement. Breakdown of cAMP was inhibited by administration of diazepam in eight cats undergoing orthodontic tooth movement; another matched group of eight animals served as controls. Orthodontic appliances consisted of coil springs stretching between the right side maxillary and mandibular canines and third premolars. The data for tooth movement and cAMP concentrations were analyzed by repeated measures factorial analyses of variance. The results indicated that administration of diazepam increased the rate of tooth movement at P less than 0.0005 and, interestingly, although diazepam had no effect on undisturbed tissues, it lowered the cAMP levels in the periodontal tissues of orthodontically moved teeth at P less than 0.01. On the basis of these results, it was concluded that the concentration of cAMP did not correlate with bone remodeling in this model and perhaps should not be used as an index of periodontal-tissue response during orthodontic tooth movement. PMID- 3017095 TI - Corticotropin deficiency in Russell-Silver syndrome. PMID- 3017094 TI - Biochemical aspects of orthodontic tooth movement. I. Cyclic nucleotide and prostaglandin concentrations in tissues surrounding orthodontically treated teeth in vivo. AB - The objective of this study was to extract and assay cyclic nucleotides and prostaglandins from tissues surrounding orthodontically treated canines in cats. Seven groups of three to five female cats were treated by 80 g tipping force to one maxillary canine for 0 to 28 days. Tissue samples were removed from sites of compression and tension around treated teeth, and from the corresponding control sites. Cyclic nucleotides and prostaglandins were simultaneously extracted by a solvent system at 0 to -5 degrees C. A portion of the aqueous fraction was used for cAMP assay by a binding protein method; cGMP was purified by column chromatography and measured by radioimmunoassay. The solvent fraction was dried, reconstituted with assay buffer, and each of the prostaglandins measured by radioimmunoassay. Analysis of variance showed no significant differences between summary control and treated sites at each of the time periods studied. However, when interactions at secondary and tertiary levels were considered (such as tension and compression, position [apical-gingival sites] of tissue sample, and jaws), significant differences were found in PGE, cAMP, and PGF2 alpha values. These results demonstrate that alterations in the levels of each of these substances in tissues surrounding teeth may be brought about by long-term applications of orthodontic force in vivo. The method of tissue sampling, however, does not permit measurement of the levels of these substances in target cells alone, thus diluting the acute response that may have occurred in these cells. PMID- 3017096 TI - The dilemma of the multicystic dysplastic kidney. AB - Multicystic dysplastic kidney is the most frequent cause of an abdominal mass in the neonate, but controversy continues as to the optimal management of these lesions, since little is known about their natural history. Experience with two complicated cases and a review of reports of retained multicystic dysplastic kidneys suggest that such lesions pose a significant risk to their hosts. Malignancy, reversible hypertension, pain, and mass effect have been associated with retained lesions. Infection is another potential hazard that is frequently cited but poorly documented in the literature. In light of the currently low morbidity and mortality associated with operation and anesthesia in the neonatal period, resection appears to be the treatment of choice for the neonate with a multicystic dysplastic kidney. PMID- 3017097 TI - AIDS and gastroenterology. PMID- 3017098 TI - A submucosal antral mass caused by cytomegalovirus infection in a patient with acquired immunodeficiency syndrome. AB - A 29-yr-old homosexual man with acquired immunodeficiency syndrome presented with watery diarrhea and fever. Upper gastrointestinal endoscopy was performed to obtain duodenal aspirates and biopsies. A 4-cm submucosal mass in the gastric antrum was identified. Subsequent abdominal CT scan confirmed the presence of this antral mass. An attempt at CT guided needle biopsy was nondiagnostic. Because the mass possibly represented a Kaposi's sarcoma or lymphoma, exploratory laparotomy and open biopsy was performed. Examination of the biopsy specimen showed inflammatory debris with multiple intranuclear cytomegalovirus inclusions. This report describes a case of a submucosal antral mass caused by localized cytomegalovirus infection in a patient with acquired immunodeficiency syndrome. PMID- 3017099 TI - The Tecumseh Study of Illness. XIV. Occurrence of respiratory viruses, 1976-1981. AB - During 1976-1981, surveillance of respiratory infection in Tecumseh, Michigan included isolation of respiratory viruses using methods similar to those employed in 1965-1971. As in the earlier period, influenza viruses continued to be isolated annually, but not without some changes in regularity of appearance. Parainfluenzaviruses also exhibited some marked changes in patterns of occurrence, but years of activity of parainfluenza types 1 and 2 were similar to those reported in other studies. Respiratory syncytial viruses continued to be limited in appearance to relatively brief periods. Rates of isolation of parainfluenzaviruses and respiratory syncytial viruses demonstrated their importance in reinfections of older children and adults. This was confirmed by examination of characteristics of associated illnesses. PMID- 3017100 TI - Conjugated estrogen use and risk of endometrial cancer. AB - The relation between use of conjugated estrogens and the risk of uterine cancer was examined among 188 white women with newly diagnosed endometrial cancer and 428 controls hospitalized for nonmalignant conditions requiring surgery at the Boston Hospital for Women-Parkway Division, Boston, Massachusetts, in January 1970-June 1975. As in prior studies, the greatest increases in risk were associated with dosages of 0.625 mg or greater (relative risk (RR) = 3.8, 95% confidence interval (CI) = 2.2-6.6) and duration of use of 10 or more years (RR = 7.6). Risk was elevated whether or not use was cyclic. Cyclic use was associated with a higher risk (RR = 3.6, 95% CI = 2.2-6.6) than continuous use (RR = 2.4, 95% CI = 1.3-4.1), but the difference between these risk estimates was not statistically significant. Risk remained increased even among women who had discontinued use of conjugated estrogens five or more years previously (RR = 4.5). Cases who were previous users had less advanced lesions at diagnosis than had never users. The highest risk associated with use of conjugated estrogens was that for stage I, grade 1 disease with no myometrial invasion. However, increases in risk of more advanced disease were seen among long-term users. PMID- 3017101 TI - Case-control study of silicosis, silica exposure, and lung cancer in white South African gold miners. AB - A case-control study was undertaken to assess the association between lung cancer and silicosis or silica dust exposure in white South African gold miners. Cases and controls were identified from deaths reported to the Gold Miners Provident Fund for the period January, 1979-October, 1983. Two controls were matched to each case by year of birth (+/- 2 years) and by smoking (+/- 5 cigarettes or equivalents per day) assessed 10 years (+/- 2 years) prior to death. One hundred thirty-three matched triplets were identified. The results showed no overall association between lung cancer and radiological silicosis (OR = 1.08, p = 0.92). Autopsy data indicated no overall associations between lung cancer and silicosis of the lung parenchyma (OR = 1.49, p = 0.11), the pleura (OR = 0.72, p = 0.30), or the hilar glands (OR = 0.85, p = 0.72). A trend toward increased severity of silicosis of the parenchyma was evident; however, this was not statistically significant (p = 0.08). Odds ratios for lung cancer and silicosis were higher at lower levels of cumulative silica dust exposure (ORs = 2.43, 1.72, 1.35 and 0.62 for lung cancer and autopsy silicosis of the parenchyma for the lowest, second, third, and highest quartiles of dust exposure, respectively; all p greater than 0.05). Cases did not differ from controls for total silica dust exposure, length of exposure, weighted average intensity of exposure, or number of shifts at high dust (all p greater than 0.20). The data do not support the hypothesis of a carcinogenic role for silica dust and no statistically significant associations were found between lung cancer and silicosis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017102 TI - Asbestos dust exposure during brake repair. AB - About 10,000 tons of chrysotile per year are used in the Federal Republic of Germany for the production of friction materials. During brake repair an unknown number of approximately 300,000 mechanics in automobile service stations are exposed to asbestos dust. In a field study, asbestos fiber concentrations during brake repair were measured. Occupational histories and chest X-rays of brake service mechanics are being examined. Ninety dust measurements in 76 service stations were made by phase contrast microscopy and by scanning transmission electron microscopy. By electron microscopy, extremely fine chrysotile fibers with lengths less than 5 microns were identified in brake drum dust. Fibers with lengths greater than or equal to 5 microns constituted less than 1% of all chrysotile fibers counted in brake drum dust. Short-term asbestos dust exposures were measured by light microscopy in 101 personal samples during blowing out of brakes, and grinding and turning of brake linings. During blowing out of car brakes, as well as during grinding of brake linings, the product of fiber concentration with length greater than 5 microns and sampling time amounted to about 4-5 fibers/ml X min corresponding to a concentration of 10(6) fibers/m3 over 4-5 min. For trucks and buses higher amounts of 5-10 X 10(6) fibers/m3 X min were observed during these operations. From occupational histories of 210 vehicle mechanics, an average duration of employment of mean +/- s = 21 +/- 10 years and a mean cumulative fiber dose of mean +/- s = (0.54 +/- 1.1) X 10(6) fibers/m3 X years were calculated. PMID- 3017104 TI - Psychological performance in relation to central and peripheral nerve conduction in workers exposed to lead, zinc, and copper. AB - Psychological performance was examined in relation to central and peripheral nerve conduction by means of the Wechsler Adult Intelligence Scale test, short latency somatosensory-evoked potential (SSEP), and median nerve conduction velocity in 19 male gun-metal foundry workers exposed to lead, zinc, and copper. (Their blood lead concentrations--ie, 16-64 micrograms/dl with a mean of 42--and plasma zinc and copper concentrations were significantly higher than those of control subjects). In these workers, the score of picture completion (psychological performance) was significantly low; indicators of lead absorption, but no indicators of zinc and copper absorption, were significantly correlated with this score. The score of picture completion was significantly correlated with the N11-N13 latency of SSEP (conduction time in the spinobulbar region) in the workers; their N11-N13 latency, together with the N9 and N9-N11 latencies, was significantly prolonged and was significantly correlated with indicators of lead absorption. Furthermore, their maximal motor and sensory conduction velocities of the median nerve were significantly slowed. It is concluded that both psychological performance and central and peripheral nerve conduction may be impaired in lead-exposed workers with BPb's below approximately 60 micrograms/dl. PMID- 3017103 TI - Malignant mesothelioma caused by childhood exposure to long-fiber low aspect ratio tremolite. AB - A 41-year-old man was found to have a malignant mesothelioma of the pleura. During childhood in Corsica, he had been exposed at home to chrysotile ore from the Canari mine. Analysis of lung mineral content revealed background levels of chrysotile but an elevated level of tremolite and actinolite asbestos. The latter had a geometric mean length of 3.7 microns, a value considerably longer than we have found for tremolite and actinolite from Quebec chrysotile miners but roughly the same as the mean length of amosite and crocidolite in workers with occupational amphibole exposure. No tremolite or actinolite fibers of length greater than 8 microns microns and width less than 0.25 micron were observed. The mean aspect ratio of the tremolite and actinolite fibers was 7, a value similar to that found in chrysotile miners with mesothelioma but considerably less than the mean aspect ratio of amosite and crocidolite from those with occupational exposure. These data suggest that long-fiber tremolite is a potential mesothelial carcinogen in humans, and that fiber length is more important than fiber aspect ratio in this regard. PMID- 3017105 TI - Relation of human T lymphotropic virus type III antibodies to T lymphocyte subset abnormalities in hemophiliac patients. AB - The relationship of T lymphocyte subset abnormalities and the presence of antibodies to the human T lymphotropic virus type III (HTLV-III) was evaluated in 66 adult patients with hemophilia. Positive test results for antibodies to HTLV III were observed in 62 percent of patients with hemophilia A and 9 percent of patients with hemophilia B. Patients with HTLV-III antibodies had lower percentages and numbers of T helper (T4) cells and increased percentages of T suppressor (T8) cells compared with percents in patients without antibodies to HTLV-III. The mean T4/T8 ratio for antibody-negative patients was 1.45 compared with 0.65 for antibody-positive patients (p less than 0.0005). These abnormalities were more apparent among hemophiliac patients who received factor VIII concentrates. The strong association of lymphocyte subset abnormalities and seroreactivity to this virus suggests that infection by HTLV-III has occurred in this population rather than a passive exposure to viral antigens contained in factor concentrates. PMID- 3017107 TI - Renovascular hypertension: treatment with the oral angiotensin-converting enzyme inhibitor enalapril. AB - Sixteen patients with an established diagnosis of renovascular hypertension were entered in an open study of enalapril (MK421), an oral angiotensin-converting enzyme (ACE) inhibitor, for treatment of their hypertension. Initial blood pressure was 178.9 +/- 6.3/106.2 +/- 3.1 mm Hg during conventional therapy on a median of 3 different antihypertensive agents. All antihypertensive therapy was ceased and the patients admitted to hospital. Following introduction of enalapril, blood pressure fell to 161.5 +/- 6.9/90.6 +/- 4.1 mm Hg at 24 h (p less than 0.01 systolic and diastolic). Blood pressure control (diastolic blood pressure, phase V, less than 95 mm Hg) was achieved with monotherapy in 7 patients and in a further 5 patients with addition of a diuretic. Renal function was compromised in 4 patients, requiring cessation of enalapril in 2 instances. Enalapril is an oral ACE inhibitor useful in the treatment of renovascular hypertension. Close monitoring of renal function is necessary during the introduction of enalapril therapy in patients with renovascular hypertension. PMID- 3017106 TI - Male pseudohermaphroditism, partial androgen receptors defect, 11p13 deletion: indication of gene localization. AB - A partial androgen receptor defect was found in a boy with male pseudohermaphroditism and an 11p13 deletion. We hypothesize that a gene responsible for the function or structure of androgen receptors might be localized in the 11p13 band or in close proximity to it. PMID- 3017108 TI - New CPR guidelines: bicarb now a last resort. PMID- 3017110 TI - Bilateral breast cancer: the frequency of undiagnosed cancers. AB - The risk of developing cancer in the contralateral breast is five to seven times the risk of cancer occurrence for the normal female population. In patients in 365 consecutive operations, 15 frankly invasive cancers and four metastatic lesions were found in the contralateral breast as well as an additional 28 lesions in situ, totaling 13%. An additional 30% of the patients had severe dysplasia with an unknown potential for becoming cancerous. Only less than one third of the cancers in the contralateral breast were symptomatic (by palpation or mammography or both) but not recognized at the time of primary treatment. The majority of lesions in the contralateral breast are due to a second primary growth, and only a minority is metastatic. The results of 203 bilateral operations consisting of a modified subcutaneous mastectomy with axillary lymph node dissection and radiation are presented; this cosmetically acceptable procedure reduces the number of cancers in the contralateral breast to an incidence of 1% to 2%. PMID- 3017109 TI - Acquired immunodeficiency syndrome: epidemiology and significance for the obstetrician and gynecologist. AB - A retrovirus, variously named human T-lymphotropic virus type III or lymphadenopathy-associated virus, is now recognized as the etiologic agent of the acquired immunodeficiency syndrome. As of January, 1986, more than 16,000 cases of acquired immunodeficiency syndrome have been reported, and it is estimated that greater than or equal to 1 million Americans are now infected with human T cell lymphotropic virus/lymphadenopathy-associated virus. Most cases of overt acquired immunodeficiency syndrome still fall within well-defined "high-risk groups," that is, homosexual or bisexual men, intravenous drug users, recipients of transfused blood or blood components, and persons with hemophilia. Also at risk are heterosexual partners of persons who have overt acquired immunodeficiency syndrome or who are at risk for acquired immunodeficiency syndrome, as are children born to mothers who either have acquired immunodeficiency syndrome or are members of a "high-risk group." Transmission of the virus may occur via sexual contact, via parenteral exposure to contaminated blood products, or from an infected mother to her child. Of special concern to the practicing obstetrician are recent reports suggesting bidirectional heterosexual transmission, transmission via artificial insemination, and perinatal transmission. In addition, the isolation of human T-cell lymphotropic virus/lymphadenopathy-associated virus from human breast milk raises concerns regarding potential transmission by this route. PMID- 3017111 TI - Radioimmunoassay of free beta-subunit of human chorionic gonadotropin as a prognostic test for persistent trophoblastic disease in molar pregnancy. AB - A new radioimmunoassay system was established with a monoclonal antibody (1E5) that distinguishes the free beta-subunit of human chorionic gonadotropin in the presence of intact human chorionic gonadotropin, showing only 0.23% cross reactivity with the intact human chorionic gonadotropin molecule and virtually no cross-reactivity with other glycoprotein hormones or their beta-subunits. Serum samples, taken at initial diagnosis from nine patients with hydatidiform mole and spontaneous remission and 12 patients with subsequent progression to persistent trophoblastic disease, were assayed for free and total levels of the beta-subunit of human chorionic gonadotropin. The assay results were expressed as a ratio of nanograms of free beta-subunit per 1000 mIU of total beta-subunit. Eight of nine patients with mole and spontaneous remission had a ratio value less than 4 whereas 10 of 12 patients with subsequent persistent disease had a ratio value greater than 4. Statistical analysis with chi 2 showed a highly significant correlation of high ratios with eventual progressive disease (p = 0.0009). This study suggests that excessive production of the free beta-subunit of human chorionic gonadotropin may identify patients with a high likelihood of developing persistent trophoblastic disease. PMID- 3017112 TI - T-cell subsets and natural killer cell activity in patients with gestational trophoblastic neoplasia. AB - Lymphocyte counts, T-cell counts, B-cell counts, helper T-cell counts, and suppressor-cytotoxic T-cell counts were performed in 38 patients with gestational trophoblastic neoplasia and 38 normal control subjects. Natural killer cell activity was also assayed in 30 patients and 30 control subjects. The percentage and count of lymphocytes and the absolute counts (but not the percentages) of T cells, helper T cells, and suppressor-cytotoxic T cells in patients with gestational trophoblastic neoplasia were significantly lower than those of normal control subjects. In high-risk patients with gestational trophoblastic neoplasia, there was also a significant reduction of the helper T cell to suppressor cytotoxic T-cell ratio in comparison with that in normal control subjects. There was no significant difference in the B-cell counts or natural killer cell activity. The measurement of these parameters was not useful in predicting the response to chemotherapy. PMID- 3017113 TI - Tubuloreticular inclusions in placental chorionic villi of rhesus monkeys after maternal treatment with interferon. AB - Tubuloreticular inclusions were observed in placental chorionic villi of rhesus monkeys after pregnant female monkeys were injected intramuscularly with recombinant leukocyte A interferon (25 X 10(6) units/kg). They were identified in endothelial cells, fibroblasts, and Hofbauer cells of the chorionic villi, providing evidence that interferon crossed at least part of the maternal-fetal partition. Induction of tubuloreticular inclusions in these cells by exogenous interferon has not been previously reported. Tightly packed regular tubular arrangements appeared in endothelial and Hofbauer cells and loosely organized tubular arrays in fibroblasts. The maximum dimension of the tubuloreticular inclusions measured 3 micron and the diameter of the tubules was 20 nm. The tubuloreticular inclusions were continuous with, and surrounded by, smooth endoplasmic reticulum that was often connected to rough endoplasmic reticulum. The tubuloreticular inclusions were not detected in placental chorionic villi from monkeys not treated with interferon or from interferon-treated monkeys 30 days after cessation of treatment. These results indicate that the formation of tubuloreticular inclusions in rhesus monkey placentas was a transient response associated with elevated serum levels of interferon. PMID- 3017114 TI - Ocular features of the Hagberg-Santavuori syndrome. AB - The Hagberg-Santavuori syndrome, the infantile form of the lipopigment storage disorders (so-called neuronal ceroid-lipofuscinoses), is a rare autosomal recessive disease characterized by progressive mental and motor deterioration with an onset between 1 and 1 1/2 years of age. Visual impairment is usually evident early in the disease and hypopigmented retinal degeneration has been described. We studied two unrelated patients with the infantile Hagberg Santavuori form and found stellate posterior polar cataracts and retinal degeneration with hyperpigmented "bone spicules" in both. PMID- 3017116 TI - Myopericarditis and enhanced dystrophic cardiac calcification in murine cytomegalovirus infection. AB - In mice inoculated with murine cytomegalovirus (MCMV) an acute myopericarditis developed which varied from a focal lymphohistiocytic inflammation to intense inflammation with necrosis and cytomegalic inclusion-bearing cells. Sublethal doses caused focal transient nonspecific chronic inflammation, followed months later by an increased frequency and extent of dystrophic cardiac calcification. When such latently infected hearts were heterotopically transplanted into uninfected animals which were then immunosuppressed (IS), a fatal generalized CMV infection followed. Cytomegalic inclusion-bearing endothelial, fibroblastic, and myocardial cells were seen in the intense inflammation found in hearts taken from mice 4 days after lethal inoculation and transplanted into uninfected mice, which were then IS. These findings may be relevant to human cardiac transplantation because they show that MCMV regularly causes cardiac infection with both acute and chronic consequences; chronic injury may follow a morphologically nonspecific myopericarditis which might not be attributed to CMV infection. PMID- 3017115 TI - Contrasting features of T-lymphocyte-mediated diabetes in encephalomyocarditis virus-infected Balb/cBy and Balb/cCum mice. AB - Two closely related sublines of the Balb/c strain, Balb/cBy and Balb/cCum mice respond differently when inoculated with the diabetogenic M variant of the encephalomyocarditis (EMCM) virus. Although genetically similar, Balb/cBy mice develop severe hyperglycemia, whereas Balb/cCum animals exhibit only modest alterations in glucose tolerance. Virus concentrations in the pancreases of animals of both sublines are equivalent 3 days after inoculation and decrease rapidly to undetectable levels within 10 days, at a time when hyperglycemia in Balb/cBy mice peaks. These results support two conclusions: 1) direct virus induced injury to the beta cells probably is not responsible for hyperglycemia in Balb/c mice, and 2) virus replication in the pancreas does not predict diabetes susceptibility. Diabetes in Balb/cBy mice is immunologically mediated. These animals generate cytolytic T lymphocytes specific for beta cells during periods corresponding to glucose intolerance, and anti-thymocyte serum treatment of infected mice prevents the development of hyperglycemia. The pathogenesis of diabetes in Balb/cCum mice is not clear. Although cytolytic T cells appear concomitant with glucose intolerance, anti-thymocyte serum has not consistently prevented the development of the metabolic disease. PMID- 3017118 TI - Catabolism of intracellular protein: molecular aspects. AB - All living cells regulate the content and composition of their resident proteins, but the mechanisms by which this is accomplished are not understood. The process of protein degradation has an important role in determining steady state and fluctuations of protein concentrations in mammalian cells. This process may be regulated by innate properties of the protein substrates, by factors that interact or "brand" proteins for degradation or by the degradative machinery of the cell. For a specific protein, there appears to be a committed step, an irreversible event that leads to rapid and extensive degradation. That initial event may or may not involve 1) proteolysis, 2) a nonproteolytic covalent modification or branding event (e.g., oxidation, ubiquitin conjugation), 3) denaturation or unfolding of the protein, or 4) sequestration. The degradative machinery of cells may either recognize proteins committed to degradation or initiate degradation, but the process must be selective because there is great heterogeneity in the rates of degradation for different proteins of one cell. The degradative process certainly requires proteases, and it is probable that lysosomal and extralysosomal proteases are involved in the catabolism of cellular proteins. We review here briefly what is currently known about the factors that may determine the half-life of a protein in a mammalian cell, the role of the protein substrate and sequestration in the process, the proteolytic and nonproteolytic enzymes that may initiate the degradative process, and the regulation of extensive degradation of proteins in cells. PMID- 3017119 TI - Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells. AB - Aldosterone and insulin stimulate Na+ transport through mechanisms involving protein synthesis. Na+-K+-ATPase has been implicated in the action of both hormones. We examined the effect of aldosterone and insulin on Na+-K+-ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (ISC) in TB6C cells. Aldosterone increases Na+-K+-ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in ISC, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase ISC in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na+-K+-ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na+ entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na+-K+-ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on ISC. PMID- 3017117 TI - Retrovirus-induced osteopetrosis in mice. Ultrastructural evidence of early virus production in osteoblasts and osteocytes. AB - Newborn female strain NMRI mice were given injections of a mouse retrovirus (OA MuLV) known to induce osteopetrosis, osteoma, and lymphoma. Femur metaphyses and lumbar vertebrae were investigated ultrastructurally 3 d, 7 d and 28 d after infection. Budding, immature and mature virus was observed associated with osteoblasts and osteocytes, but not with osteoclasts or chondrocytes, 28 d after infection with the virus. No production of virus particles was observed in bone tissue in mock-treated controls. Thus, the primary target cell for OA virus in bone appears to belong to the osteoblastic/osteocytic cell lineage. PMID- 3017122 TI - Effects of theophylline and cAMP on Amphiuma small intestine. PMID- 3017120 TI - Prokaryotic adenylate cyclase toxin stimulates anterior pituitary cells in culture. AB - Bordetella pertussis synthesizes a variety of virulence factors including a calmodulin-dependent adenylate cyclase (AC) toxin. Treatment of anterior pituitary cells with this AC toxin resulted in an increase in cellular cAMP levels that was associated with accelerated exocytosis of growth hormone (GH), prolactin, adrenocorticotropic hormone (ACTH), and luteinizing hormone (LH). The kinetics of release of these hormones, however, were markedly different; GH and prolactin were rapidly released, while LH and ACTH secretion was more gradually elevated. Neither dopamine agonists nor somatostatin changed the ability of AC toxin to generate cAMP (up to 2 h). Low concentrations of AC toxin amplified the secretory response to hypophysiotrophic hormones. We conclude that bacterial AC toxin can rapidly elevate cAMP levels in anterior pituitary cells and that it is this response that explains the subsequent acceleration of hormone release. PMID- 3017121 TI - Convergence of sensory information from abdominal viscera in the rat brain stem. AB - This study was carried out to establish whether there was convergence of sensory information in the rat brain stem stimulated by physiological activation of gastric mechanoreceptors and hepatic glucoreceptors. Extracellular recordings were made from single neurons in the region of the dorsal vagal nucleus and nucleus of the solitary tract in the medulla. The responses of these neurons to gastric distension, hepatic portal vein perfusion of isotonic D-glucose, and hepatic portal vein infusion of isotonic saline were studied. Fifty-six neurons were studied; it was found that there was no significant difference in the proportion of neurons responding to gastric distension compared with the number responding to either form of hepatic stimulation. In 20 neurons (all 3 types of stimulation were tested on the same neuron), both excitation and inhibition were observed with both forms of visceral stimulation. Of the seven of these neurons that responded to hepatic portal vein infusion, four of them also had an input from gastric mechanoreceptors. Only three of the neurons that responded to hepatic stimulation showed a specific response to hepatic glucose perfusion; in the remainder a component of the response was due to the infusion of the volume itself. The results from these experiments have demonstrated an apparently weak functional synaptic projection carried by hepatic vagal afferents, particularly those responding to changes in portal glucose concentration, which may indicate a rather diffuse and nonspecific sensory system in the liver. These results have also demonstrated the convergence onto neurons in the brain stem of information from gastric and hepatic enteroceptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017124 TI - Basolateral membrane properties of juxtamedullary proximal tubule in newborn rabbit. AB - We have evaluated the functional and morphological changes that occur in juxtamedullary proximal convoluted tubules before and after the completion of nephrogenesis at approximately 3 wk of age. Tubules were dissected in connection with glomeruli in the deep juxtamedullary zone, crimped at both ends, and induced to swell in isotonic medium by sequential exposure to ouabain, reduced bath protein concentration, and collagenase. Basolateral membrane surface areas were measured morphometrically in paired tubules if possible. The swelling rates and basolateral areas were the same in tubules from 2-7- and 14-17-day-old rabbits (during nephrogenesis), had increased in 28-42-day-old rabbits (after nephrogenesis), and increased further in adults. Because the surface areas and swelling rates increased proportionally, the nephrons "matured" only after nephrogenesis was complete and then only in basolateral surface area. The intrinsic membrane properties of hydraulic conductivity and ion permeability apparently remained constant. PMID- 3017123 TI - Regulation of renal Na+-K+-ATPase in the rat: role of increased potassium transport. AB - The purpose of this study was to characterize the alterations in collecting tubule Na+-K+-ATPase activity produced by sustained increments in dietary potassium in the rat and to evaluate the role of aldosterone in their generation. In adrenal-intact animals, feeding a high-potassium diet (10-fold that of control) or administration of a high physiological dose of aldosterone (5 micrograms X 100 g-1 X day-1), which simulates the delivery rate of this hormone during potassium loading (both for 7 days), caused marked increments in Na+-K+ ATPase activity in the cortical collecting tubule (CCT) but had no effect on the enzyme in the inner stripe of the medullary collecting tubule (MCT). A significant increase in enzyme activity was also observed after smaller dietary potassium increments (2.5 and 5 times the control) and after 4 (but not 2) days of dietary potassium load. In adrenalectomized rats provided with physiological replacement doses of corticosterone and aldosterone (0.8 micrograms X 100 g-1 X day-1), Na+-K+-ATPase activity in both CCT and MCT was similar to that of adrenal intact controls but remained unchanged after 7 days on the potassium-enriched (10 fold) diet. In contrast, adrenalectomized animals receiving the high physiological dose of aldosterone displayed an increase in Na+-K+-ATPase activity of CCT comparable with that of adrenal-intact animals, whereas the enzyme activity in the MCT was unaffected. In conclusion, 1) following chronic potassium loading Na+-K+-ATPase activity increases significantly in the CCT with no change in its activity in the inner stripe of the MCT.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017125 TI - Extrarenal potassium adaptation: role of skeletal muscle. AB - Following the ingestion of a high-potassium-content diet for only a few days, the plasma potassium of rats rises only modestly in response to a previously lethal dose of potassium salts. This acquired tolerance, termed potassium adaptation, is principally the result of increased capacity to excrete potassium into the urine. However, a substantial portion of the acute potassium dose is not immediately excreted and is apparently translocated into cells. Previous studies have failed to show an increase in the content of potassium of a variety of tissues from such animals. Using 86Rb as a potassium analogue, we have shown that the skeletal muscle of potassium-adapted rats takes up significantly greater amounts of potassium in vivo in response to an acute challenge than does that of control animals. Furthermore, the same animals exhibit greater efflux of 86Rb following the termination of the acute infusion. We have also shown that the Na+-K+-ATPase activity and ouabain-binding capacity of skeletal muscle microsomes are increased by the process of potassium adaptation. We conclude that skeletal muscle is an important participant in potassium adaptation and acts to temporarily buffer acute increases in the extracellular concentration of potassium. PMID- 3017127 TI - Atrial peptides inhibit oxygen consumption in kidney medullary collecting duct cells. AB - Atrial natriuretic peptides (ANP) stimulate renal Na+ excretion by poorly understood mechanisms, perhaps involving direct inhibition of Na+ transport in the kidney medulla. To examine the effects of ANP on renal cells directly, we prepared highly purified cell suspensions derived from inner and outer medullary collecting duct and thick ascending limb of rabbit kidney and monitored ouabain sensitive oxygen consumption (QO2). Human ANP diminished QO2 by 27.4 +/- 1.6% (mean +/- SE) in inner medullary collecting duct cells but had no effect in cells derived from outer medullary collecting duct or thick ascending limb. The inhibitory effect of ANP was not additive with either amiloride or ouabain. ANP was without effect in the presence of amphotericin. These results indicate that ANP inhibited Na+ entry in inner medullary collecting duct cells. ANP-mediated inhibition of QO2 was dose dependent (Ki = 5.5 X 10(-10) M) and exhibited selectivity for peptide structure. These results suggest that atrial peptides enhance renal sodium excretion partly by direct inhibition of medullary collecting duct sodium transport. PMID- 3017126 TI - Immunodissection and culture of rabbit cortical collecting tubule cells. AB - A mouse monoclonal antibody designated IgG3(rct-30) has been prepared that reacts specifically with an antigen on the surface of all cells comprising the cortical and medullary rabbit renal collecting tubule including the arcades. Plastic culture dishes coated with IgG3(rct-30) were used to isolate collecting tubule cells from collagenase dispersions of rabbit renal cortical cells by immunoadsorption. Typically, 10(6) rabbit cortical collecting tubule (RCCT) cells were obtained from 5 g of renal cortex (2 kidneys). Initial purity was greater than 96% based on immunocytofluorescent staining with three different anti collecting tubule antibodies. Between 20 and 30% of the RCCT cells were reactive with peanut lectin suggesting that RCCT cells are a mixture of principal and intercalated cells. Approximately 10(7) RCCT cells were obtained after 4 to 5 days in primary culture. Moreover, RCCT cells continued to proliferate after passaging with a doubling time of approximately 32 h. RCCT cells passaged once and then cultured 4-5 days were found 1) to synthesize cAMP in response to arginine vasopressin (AVP), prostaglandin E2 (PGE2), isoproterenol, and parathyroid hormone, but not calcitonin, prostaglandin D2, or prostaglandin I, and 2) to release PGE2 in response to bradykinin but not arginine vasopressin or isoproterenol. Our results indicate that cultured RCCT cells retain many of the hormonal, histochemical, and morphological properties expected for a mixture of principal and intercalated rabbit cortical collecting tubule epithelia. RCCT cells should prove useful both for studying hormonal interactions in the cortical collecting tubule and as a starting population for isolating intercalated collecting tubule epithelia. PMID- 3017128 TI - Effects of 4-aminopyridine on rate-related depression of cardiac action potentials. AB - Canine cardiac Purkinje fibers and atrial trabeculae and rat and cat papillary muscles superfused with a hyperkalemic, hypoxic, and acidotic Tyrode solution were depolarized to membrane potentials (-70 to -60 mV) at which action potential amplitude declined as the coupling intervals of pacing stimuli were prolonged from 500 to 4,500 ms. The rate-related decline of action potential amplitude appeared to be due to time-dependent recovery of the early outward current rather than to a decrease in inward calcium current, since it was prevented by 4 aminopyridine (1.0 mM), but not by isoproterenol (1.0 microM), caffeine (5.0 mM), or CsCl (5-20 mM) and it was accompanied by an exponential increase of developed tension. Experiments using Purkinje fibers mounted in a single sucrose gap chamber demonstrated that the rate-related decline of action potential amplitude was maximal at membrane potentials between -70 and -40 mV and was negligible at less negative or more negative membrane potentials. These results may pertain to the mechanism for deceleration-dependent bundle branch block. PMID- 3017129 TI - Alpha-adrenergic receptors on rat ventricular myocytes: characteristics and linkage to cAMP metabolism. AB - When incubated with purified cardiomyocytes from adult rat ventricle, the alpha 1 antagonist [3H]prazosin binds to a single class of sites (8 X 10(4) per cell) with high affinity [dissociation constant (KD) = 82 pM]. Competition for [3H]prazosin binding by the alpha 2-selective antagonist yohimbine [inhibitor dissociation constant (KI) = 714 nM] and the nonselective alpha-antagonist phentolamine (KI = 168 nM) demonstrates that these receptors are of the alpha 1 subtype. In addition, incubation of myocyte membranes with [3H]yohimbine results in no measurable specific binding. Agonist competition for [3H]prazosin binding to membranes prepared from purified myocytes demonstrates the presence of two components of binding: 28% of alpha 1-receptors interact with norepinephrine with high affinity (KD = 36 nM), whereas the majority of receptors (72%) have a low affinity for agonist (KD = 2.2 microM). After addition of 10 microM GTP, norepinephrine competes for [3H]prazosin binding to a single class of sites with lower affinity (KD = 2.2 microM). Incubation of intact myocytes for 2 min with 1 microM norepinephrine leads to significantly less cyclic AMP (cAMP) accumulation (13.6 pmol/mg) than stimulation with either norepinephrine plus prazosin or isoproterenol (18 pmol/mg). Likewise, incubation of intact myocytes with 10(-6) M norepinephrine leads to significantly less activation of cAMP-dependent protein kinase (71 +/- 4%) than when myocytes are stimulated by both norepinephrine and the alpha 1-adrenergic antagonist, prazosin (95 +/- 1%), or the beta-adrenergic agonist, isoproterenol (100%).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017130 TI - Functional interdependence of discrete vagal projections to SA and AV nodes. AB - Dynamic modulation of chronotropic and dromotropic function was evaluated in anesthetized dogs before and after selective parasympathectomy of the atrioventricular (AV) node. Vagal and cardiac sympathetic efferent nerves were decentralized, and the distal cut ends of the cervical vagi were stimulated at frequencies from 1 to 25 Hz with step-wise voltage changes. To investigate parasympathetic modulation of AV conduction, stimulations were performed with and without atrial pacing. After determination of chronotropic and dromotropic responses, in the intact state, selective AV node parasympathectomy was performed as previously described [Am. J. Physiol. 248 (Heart Circ. Physiol. 17): H61-H68, 1985]. Stimulation protocols were then repeated. Control results in intact animals reveal a parallel and finely balanced vagal regulation of chronotropic and dromotropic responses over a wide range of frequency stimulations. Conversely, selective parasympathectomy of the AV node interrupts extrinsic vagal modulation of AV conduction without affecting parasympathetic control of chronotropic function. The dynamic balance of chronotropic and dromotropic function, modulated by extrinsic vagal input, reflects the parallel activation of functionally and anatomically distinct parasympathetic projections to the sinoatrial (SA) and AV nodal tissue. Thus present experiments allow a more detailed examination of vagal innervation of the SA and AV nodes, the importance of their balance through neuroregulation, and progress toward understanding the pathophysiology of disturbances to this balance. PMID- 3017131 TI - Cardiac function and chronotropic sensitivity to beta-adrenergic stimulation in sepsis. AB - An organism's cardiovascular response to sepsis is at least partly dependent on hormonal and neural modulation of myocardial function. We have investigated both intrinsic myocardial performance and one aspect of myocardial sensitivity to beta adrenergic stimulation in a model of sepsis in which animals, at the time studied, exhibited bacteremia, normal arterial blood pressure and cardiac output, elevated heart rate, and elevated plasma catecholamines. Intrinsic myocardial contractile function, studied with the isolated, perfused working heart preparation, was depressed over a range of preloads in septic animals, whereas heart rate was elevated. To determine whether hearts from septic animals could respond normally to beta-adrenergic stimulation, we studied chronotropic responses to isoproterenol in both Langendorff perfused hearts and in isolated right atria. In langendorff perfused hearts from septic animals, basal rates were significantly increased and lower concentrations of isoproterenol elicited greater increases in heart rate. In isolated right atria from septic animals, basal rates were also elevated and the EC50 for the chronotropic response to isoproterenol was significantly less than in atria from control animals. The maximal heart rate response to isoproterenol was not significantly different from control. These results indicate that in sepsis, despite apparently adequate in vivo cardiac performance, intrinsic myocardial function is depressed, but chronotropic sensitivity to beta-adrenergic stimulation is increased. PMID- 3017132 TI - A new technique for the study of vascular presynaptic receptors in freely moving rats. AB - A technique is described in which a silicon catheter is utilized for permanent catheterization of the portal vein, and curved platinum stimulation electrodes around this vein are used for the stimulation of the local nervous plexus. The method has a success ratio of over 70% during a period of more than 2 mo. Five animals were used in experiments over a period longer than 5 mo. Using this technique, we investigated the influence of variation of current density, pulse duration, and frequency of stimulation on the electrically evoked release of norepinephrine in portal blood. Enhancement of all stimulation parameters proportionally increased the evoked norepinephrine release. The alpha 2 adrenoceptor antagonist yohimbine was found to increase basal norepinephrine levels 4.43-fold and facilitated the evoked release additionally with a factor of 4.70. The results show that noradrenergic neurotransmission in the portal vein nervous plexus is controlled presynaptically by a strong local alpha 2-adrenergic autoinhibition. The method offers the means to explore locally the presynaptic modulation of endogenous neurotransmitter release in unrestrained unanesthetized animals. PMID- 3017133 TI - Protons, osmolytes, and fitness of internal milieu for protein function. AB - The composition of the intracellular milieu shows striking similarities among widely different species. Only certain values of intracellular pH, values that generally reflect alphastat regulation, and only narrow ranges of inorganic ion concentrations are found in the cytoplasm of the cells of most animals, plants, and microorganisms. In water-stressed organisms only a few types of low-molecular weight organic molecules (osmolytes) are accumulated. These highly conserved characteristics of the intracellular fluids reflect the need to maintain critical features of macromolecules within narrow ranges optimal for life. For proteins these features include maintaining adequate rates of catalysis, a high level of regulatory responsiveness, and a precise balance between stability and lability of structure (tertiary conformation, subunit assembly, and multiprotein complexes). The optimal values for these functional and structural features of proteins often lie near the midrange of possible values for these properties, and only under specific conditions of intracellular pH, ionic strength, and osmolyte composition are these optimal midrange values conserved. In dormant cells the departure of solution conditions from values that are optimal for protein function and structure may be instrumental in reducing or shutting down metabolic functions. Seen from a broad evolutionary perspective, the evolution of the intracellular milieu is an important complement to macromolecular evolution. In certain instances appropriate modifications of the internal milieu may reduce the need for adaptive amino acid replacements in proteins. PMID- 3017134 TI - Endogenous enkephalin modulation of dopamine neurons in ventral tegmental area. AB - Many lines of evidence support the possibility that the opioid pentapeptides Met- and Leu-enkephalin can modulate dopamine neurons in the ventral tegmental area (VTA). Thus microinjection of enkephalin analogues into the VTA of rats produces a dopamine-dependent increase in spontaneous motor activity and an increase in dopamine metabolism in certain mesolimbic dopamine terminal fields, such as the nucleus accumbens. To determine if these effects can be produced by endogenous enkephalins, an enkephalinase A inhibitor, thiorphan, was microinjected into the VTA to inhibit enkephalin metabolism. Thiorphan produced a dose-dependent (0.3 3.33 micrograms) increase in spontaneous motor activity that was blocked by pretreatment with the opioid antagonist naloxone (2.0 mg/kg ip) or the dopamine antagonist haloperidol (0.1 mg/kg ip). Thiorphan injection into the VTA increased dopamine metabolism in the nucleus accumbens, prefrontal cortex, and septum but not in the striatum. In all brain regions the increase in dopamine metabolism was blocked by pretreatment with naloxone. These data demonstrate that endogenous enkephalin in the VTA can increase the activity of A10 dopamine neurons, supporting a physiological role for enkephalin in mesolimbic and mesocortical dopamine-mediated behaviors. PMID- 3017135 TI - Characteristics of myocardial beta-adrenergic receptors during endotoxicosis in the rat. AB - The effects of in vivo endotoxin administration on beta-adrenergic receptors in rat ventricle membranes were studied using [3H]dihydroalprenolol as a radioligand. Nonlinear regression analysis of saturation binding indicated one site binding of antagonist in both control and endotoxic tissues. There was no change in maximum binding or dissociation constant of [3H]dihydroalprenolol at 0.5 or 3 h after endotoxin administration or when the rats were in the agonal stage of shock. Isoproterenol competition studies revealed that there was an increase in the slope of the curve from endotoxic tissues at the agonal stages (0.6 +/- 0.06 vs. 0.91 +/- 0.12, control and endotoxic, respectively, P less than 0.05) and that there was a decrease in affinity for isoproterenol binding. Control binding modeled to a two-state fit, whereas binding to endotoxin-exposed membranes modeled to one state of lower affinity. These data suggest that there is an alteration in receptor-adenylate cyclase coupling, which may account for an attenuation of agonist-stimulated cyclase activity. A modification in the beta adrenergic receptor may contribute to the decrease in myocardial performance during shock. PMID- 3017136 TI - Ultrastructural features of epithelial cell degeneration in rectal crypts of patients with AIDS. AB - Focal crypt epithelial cell degeneration (apoptosis) of the rectum is a characteristic pathologic feature in AIDS. The presence of apoptosis usually implies cell-mediated cytolysis, which would be an unexpected finding in this disease. We investigated the ultrastructural features of apoptosis in rectal biopsies from five AIDS patients (three homosexual males and two females with i.v. drug abuse), three heterosexual controls, and two homosexual male controls. Apoptosis was found in all AIDS patients and, to a lesser extent, in one heterosexual control. Ultrastructurally, vacuolization of several adjacent cells, leading to extrusion of cellular debris through the basal lamina and phagocytosis by macrophages, was seen. No intracellular or extracellular viral particles were detected in the regions containing apoptotic bodies, in epithelial cells near the crypt bases, in intraepithelial lymphocytes, or in macrophages. In summary, apoptosis in the rectal crypts of patients with AIDS has the same ultrastructural features as in other conditions, which suggests that its pathogenesis is related to immune rather than infectious factors. If this process occurs on a chronic basis in multiple cell types, it would promote general tissue depletion, which has been demonstrated to occur in AIDS. The presence of apoptosis in AIDS is not explained by current concepts of disease pathogenesis. PMID- 3017137 TI - Ectopic pituitary adenomas with normal anterior pituitary glands. AB - Two patients with ectopic pituitary adenomas and biopsy-proven normal anterior pituitaries are described. Both tumors were located in the sphenoid sinus. One tumor produced prolactin, and the other one was a plurihormonal adenoma that produced predominantly adrenocorticotropin and to a lesser extent thyroid stimulating hormone and alpha subunit. The patient with the plurihormonal tumor, who had Cushing's disease, was cured by surgery while the patient with the prolactinoma was treated by surgery and medical therapy. A review of these two cases and an additional nine cases from the literature of ectopic pituitary adenomas in patients with normal intrasellar anterior pituitaries indicate that these uncommon tumors are capable of secretory function and may be the only cause of excessive pituitary hormone production. PMID- 3017139 TI - [Morphofunctional and ultracytochemical characteristics of the epithelial cells of the endometrial glands in women during the menstrual cycle]. PMID- 3017138 TI - Glycogen-rich clear cell carcinomas of the breast. A clinicopathologic and ultrastructural study. AB - Ten cases of glycogen-rich clear cell carcinoma of the breast are described. Only two previous case reports have been published. These neoplasms are composed of clear cells with abundant glycogen. Ultrastructurally, two cases showed large quantities of non-membrane-bound glycogen and numerous empty glycogen lakes. neoplastic cells formed tight junctions, immature desmosomes, and occasionally had short microvilli. In nine cases the glycogen-rich carcinoma grew in a solid pattern only, while one case had both solid and papillary patterns. One case was associated with a signet-ring cell carcinoma. Seven of nine patients who underwent axillary dissections had nodal metastases. Five patients died with residual disease, and one is currently alive with local skin recurrence. These data suggest that glycogen-rich clear cell carcinoma is associated with frequent lymph node metastases and mortality. PMID- 3017141 TI - [Effects of substitution hormonal therapy on the hemostasis system in the pathologic climacteric]. PMID- 3017140 TI - [Serotonin and the hypophyseo-adrenal cortex system in patients with hyperprolactinemia]. PMID- 3017142 TI - Epstein-Barr virus DNA in lymphocytes of patients with the virus-associated hemophagocytic syndrome. AB - The virus-associated hemophagocytic syndrome (VAHS) is a histiocytic proliferative disorder with bone marrow and liver failure for which the connection with a specific virus is often tenuous. Epstein-Barr virus (EBV) is one candidate for the association, but serologic or culture confirmation may be lacking in a particular case. As a means of directly identifying the presence of EBV in patients' cells, molecular hybridization studies were carried out using a radioactively labeled viral DNA segment. DNA from mononuclear cells of two children with VAHS had specific hybridization to the EBV DNA probe. One of the patients had serologic evidence for EBV infection. Several immunologic deficiencies were found. VAHS may represent one of several hematologic and/or immunologic dysfunctions caused by EBV. PMID- 3017143 TI - Congenital mesoblastic nephroma with hypercalcemia. Pathogenetic role of prostaglandins. AB - A case of mesoblastic nephroma, hypercalcemia, and raised levels of prostaglandins in a 2-month-old female infant is reported. Plasma parathyroid hormone (PTH) was normal and urinary prostaglandins were raised. During surgery a prostaglandin arteriovenous gradient was demonstrated. A large quantity of PGE was extracted from the tumor by radioimmunoassay after incubation. Both blood calcium and urinary prostaglandins returned to normal after nephrectomy. These results show that this tumor produced prostaglandins, the mediators of hypercalcemia in this patient. PMID- 3017144 TI - Separation of acylcarnitines from biological samples using high-performance liquid chromatography. AB - A reverse-phase high-performance liquid chromatography technique to separate carnitine and acylcarnitines from a biological matrix is described. The method utilizes a step gradient to provide baseline resolution of acylcarnitines (individually or by class) for subsequent quantification using a sensitive radioenzymatic assay. The method requires minimal sample preparation and prevents any contamination among groups of acylcarnitines. This technique has been applied to liver tissues of rats obtained under a variety of conditions. These studies demonstrate the validity and utility of the HPLC method while confirming the applicability of the perchloric acid fractionation of acylcarnitines by functional class. The present HPLC method permits resolution of long-chain acylcarnitines in the presence of large excess concentrations of carnitine and short-chain acylcarnitines (coelution of unesterified carnitine with long-chain acylcarnitines less than or equal to 0.05%). Thus, the method will be of use in the study of acylcarnitines in biological systems over a broad spectrum of metabolic conditions. PMID- 3017145 TI - Highly sensitive immunoadsorption procedure for detection of low-abundance proteins. AB - A procedure that virtually eliminates nonspecific adsorption of radiolabeled proteins during immunoprecipitation was devised utilizing staphylococcal cells containing protein A (Staph A). Immunoprecipitates (antigen-antibody complexes) were solubilized from Staph A pellets into detergent micelles by incubation in a small volume of 1% sodium dodecyl sulfate (SDS) at 23 degrees C for 10 min. To allow re-formation of immunocomplexes and rebinding to new Staph A, the SDS solubilized material was diluted 20-fold in buffer containing 1% Triton X-100 and 0.5% sodium deoxycholate. Specific conductance measurements revealed that this solubilization and subsequent reimmunoadsorption of antibody-antigen complexes occur at SDS concentrations that are first above and then below its critical micelle concentration. This procedure lowered the nonspecific background from approximately 2250 parts per million (ppm) to less than 25 ppm with a final recovery of 30-50% depending on the antigen and antibody. Chaotropic agents such as 2 M urea, 0.2 M KOH, and 3.5 M MgCl2 (as well as combinations of urea and SDS) can substitute for 1% SDS, although the final recovery is somewhat lower. Fluorography of radiolabeled proteins obtained in this manner displays virtually undetectable background even for exposures as long as 2 months. These methods allowed the unambiguous detection of low-abundance antigens at a high level of sensitivity, for example, mouse mammary tumor virus protein products and epidermal growth factor receptor. PMID- 3017148 TI - Characterization of free radical-induced base damage in DNA at biologically relevant levels. AB - DNA damage induced by oxygen radicals, e.g., hydroxyl radicals generated in living cells either by cellular metabolism or external agents such as ionizing radiations, appears to play an important role in mutagenesis, carcinogenesis, and aging. Elucidation of the chemical nature of such DNA lesions at biologically significant quantities is required for the assessment of their biological consequences and repair. For this purpose, a sensitive method using gas chromatography-mass spectrometry with the selected-ion-monitoring technique (GC MS/SIM) was developed in the present work. DNA was exposed to hydroxyl radicals and hydrogen atoms produced by ionizing radiation in N2O-saturated aqueous solution. DNA samples were subsequently hydrolyzed with formic acid, trimethylsilylated, and analyzed by GC-MS/SIM. Characteristic ions from previously known mass spectra of DNA base products as their trimethylsilyl derivatives were recorded and the area counts of each ion were integrated. From these acquired data, a partial mass spectrum of each product was generated and then compared with those of authentic materials. This technique permitted the detection and characterization of a large number of free radical-induced based products of DNA, i.e., 5,6-dihydrothymine, 5-hydroxy-5,6-dihydrothymine, 5 hydroxymethyluracil, 5-hydroxyuracil, 5-hydroxycytosine, thymine glycol, 4,6 diamino-5-formamidopyrimidine, 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5 formamidopyrimidine, and 8-hydroxyguanine, simultaneously in a single sample after radiation doses from 0.1 to 10 Gy. Detectable amounts of the base products were found to be as low as approximately 10 fmol per injection.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017146 TI - Enhancement of immunoblot sensitivity by heating of hydrated filters. AB - Immunoblots of either dot or Western type were exposed to heat before reaction with antibody. Dramatic increases in immunoblot sensitivity were seen for certain antigen-antibody pairs after heating of either dry or hydrated nitrocellulose filters at or above 100 degrees C. Heating of filters in the hydrated state improved the linearity of immunodetection and produced the highest signal-to noise ratio. This treatment greatly increased immunoblot sensitivity with several peptide-generated antibodies, whereas decreased sensitivity was seen with antibodies against native proteins. Heating of hydrated filters after antigen immobilization is thus a potentially powerful way to increase the sensitivity of immunoblot analysis for antibodies that preferentially recognize epitopes in denatured proteins. PMID- 3017147 TI - Detection of transcription and translation in situ with biotinylated molecular probes in cells transfected with recombinant DNA plasmids. AB - The efficiency of transfection and subsequent expression of recombinant DNA plasmids in monolayers of CV-1 monkey kidney cells was analyzed by immunoperoxidase and in situ hybridization with biotin-nucleotide-labeled DNA molecular probes. Two recombinant plasmids were used for transfection. Both contained the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV) as the transcriptional promoter, but two different coding sequences were employed [bacterial chloramphenicol acetyltransferase (pRSVcat) and mouse casein alpha (pRSVcsn alpha)]. In our experiments up to 25% of the transfected cells were positive for pRSVcat expression by indirect immunoperoxidase assay with affinity purified, biotinylated anti-goat gamma-globulin after exposure to goat anti chloramphenicol acetyltransferase antibody. In duplicate cultures, where pRSVcat expression was monitored by in situ hybridization signal that was restricted to the cytoplasm in positive cells was identified as pRSVcat RNA by its sensitivity to alkali. Although transfection of CV-1 cells with pRSVcsn-alpha did not result in immunologically detectable alpha casein, greater than 14% of the cells possessed cytoplasmic RNA concentrations detectable by in situ hybridization. These observations provide comparative information on in situ hybridization and immunoperoxidase techniques. They further indicate that in situ hybridization can be used to evaluate the effectiveness of transfection with recombinant expression vectors. PMID- 3017149 TI - Specific binding of 125I-labeled human chorionic gonadotropin to gonadal tissue: comparison of limited-point saturation analyses to Scatchard analyses for determining binding capacities and factors affecting estimates of binding capacity. AB - Experiments were conducted to compare gonadotropin binding capacity calculated from limited-point saturation analyses to those obtained from Scatchard analyses, and to test the effects of membrane purity and source of gonadotropin receptors on determining the maximum percentage of radioiodinated hormone bound to receptors (maximum bindability). One- to four-point saturation analyses gave results comparable to results by Scatchard analyses when examining relative binding capacities of receptors. Crude testicular homogenates had lower estimates of maximum bindability of 125I-labeled human chorionic gonadotropin than more purified gonadotropin receptor preparations. Under similar preparation techniques, some gonadotropin receptor sources exhibited low maximum bindability. PMID- 3017150 TI - Affinity chromatographic procedure for the quantitative recovery of DNA fragments from agarose gels. AB - A simple and reliable method for the recovery of specific fragments of DNA from agarose gels is presented. The electroelution of the DNA onto the NENSORB cartridge matrix with the subsequent elution of the bound DNA by a methanol (50% v/v) wash has been shown to result in the quantitative recovery of the restriction fragment. Of importance is the fact that the DNA purified by this procedure is a viable substrate for further digestion by a second restriction endonuclease. The method does not require either phenol extraction or extensive desalting of the sample. PMID- 3017151 TI - Elimination of the effects of interferents on the quantitative determination of deuterium oxide in biological samples of infrared photometry. PMID- 3017152 TI - Epidural analgesic techniques in the management of cervical pain. AB - The injection of depot steroids into the cervical epidural space can maximize the conservative management of patients with cervical radiculopathy. We retrospectively studied 25 patients with cervical radiculopathy who received a total of 45 epidural injections of steroids. Sixty-four percent of the patients had a good or excellent response to cervical epidural steroid injection, whereas other conservative treatment modalities had not helped them. The patient's history and a description of the pain and the corresponding neurological abnormalities present were of value in the selection of patients who were most likely to respond favorably to epidural steroids, whereas laboratory studies were not as useful. Anesthesiologists, many already familiar with the use of epidural steroid injection in the treatment of low back pain, should add to their armamentarium the use of such techniques in the management of patients with acute and chronic cervical radiculopathy. PMID- 3017154 TI - A variant in the restriction endonuclease cleavage pattern of mitochondrial DNA in the domestic fowl, Gallus gallus domesticus. AB - The cleavage patterns of mitochondrial DNAs (mtDNAs) were investigated from 15 lines of domestic fowls, Gallus gallus domesticus, using 11 restriction endonucleases. The cleavage patterns with 10 restriction endonucleases were identical in all the lines. A variant was found in a line of White Leghorn in the pattern with MspI digestions. Cleavage patterns of the red jungle fowl, Gallus gallus gallus, were identical to the common patterns shown by the 14 lines of domestic fowls. PMID- 3017153 TI - [Phlebectasic edema caused by synovial sarcoma]. PMID- 3017155 TI - Genomic hybridization of bovine class II major histocompatibility genes: 1. Extensive polymorphism of DQ alpha and DQ beta genes. AB - Class II genes of the bovine major histocompatibility complex (MHC) were investigated by Southern blot analysis using human cDNA probes for DQ alpha, DQ beta, DR alpha and DR beta. The presence of a DQ-like and a DR-like subregion in cattle was clearly indicated. Highly polymorphic restriction fragment patterns were obtained when genomic DNA, digested with any one of the BamHI, EcoRI or PvuII restriction enzymes, was hybridized with the DQ alpha and the DQ beta probe. The polymorphisms were interpreted genetically by analysing five paternal half-sib families of the Swedish Red and White breed. The material comprised, besides the bulls, 28 offspring and their dams. The analysis resolved 9 and 12 allelic variants of DQ alpha and DQ beta respectively. Thus, this investigation establishes a method for routine typing of MHC class II gene polymorphism in cattle. The results were entirely consistent with close linkage of DQ alpha and DQ beta since no recombinant was found and since alleles at these loci occurred in complete linkage disequilibrium in the material investigated. Close linkage between DQ and the blood group locus M, which has previously been found to be closely linked to the serologically defined BoLA-A locus, was also indicated. In this study DNA was isolated from frozen semen samples of dead bulls, which shows that this type of analysis will be useful in genetic investigations in cattle breeds, where artificial insemination is practised. PMID- 3017156 TI - Exercise-induced "asthma" as a presentation of bronchial carcinoid. AB - Exercise-induced wheezing developed in a previously healthy 14-year-old boy. Chest radiographs revealed hyperlucency of the left lung. Bronchial tomography and bronchoscopy revealed a mass in the left mainstem bronchus, identified as a carcinoid tumor after surgical excision. The patient is now asymptomatic. Exercise-induced wheezing as the sole manifestation of this tumor has not been previously reported. PMID- 3017157 TI - Sites and mechanisms of basic narcotic receptor function based on current research. AB - Within the past decade, progress in opiate pharmacology has been phenomenal and has stimulated widespread interest. Several potent endogenous peptides with morphine-like activity were isolated and their structures were established. Opioid receptors in the brain were localized and characterized. Plausible hypotheses to explain the mechanism of action of the narcotic analgetics have been proposed. Antagonist-agonists that are potent analgesics with low addition liability were introduced for clinical application. Epidural administration of morphine and its surrogates to reduce side effects and prolong drug action is gaining increasing popularity. Extensive research is ongoing concerning the use of opioids in the treatment of shock and the mental states. Some of these advances will be discussed. PMID- 3017158 TI - Clinical evidence for different narcotic receptors and relevance for the clinician. AB - Analgesics such as morphine are toxic drugs that kill by producing respiratory depression. Further morphine-like drugs produce a high level of physical dependence and are highly reinforcing in some subjects. A systematic search, conducted over the last 50 years, for safer and less addicting analgesic has revealed that opioid analgesics act on different types of opioid receptors (mu, kappa, and sigma), and that they may function as mixed agonist-antagonists or as partial agonists. Thus some mixed agonists function as competitive antagonists at the mu receptor and as partial or strong agonists at the kappa or sigma receptor. When mixed agonists produce the same pharmacologic effects (eg, analgesia) by acting on different receptors, they invoke the principle of receptor dualisms. Drugs that produce agonist (analgesic) effects by acting on the kappa receptors are an order of magnitude safer than the mu agonists and produce a lesser degree of physical dependence than strong mu agonists. Thus safer, less addicting analgesics have been produced that act either as agonist-antagonists or by being partial agonists. PMID- 3017159 TI - Experimental amitriptyline intoxication: treatment of cardiac toxicity with sodium bicarbonate. AB - Overdose with amitriptyline and other tricyclic antidepressants can result in ventricular conduction abnormalities as well as severe ventricular arrhythmias. The arrhythmogenic effects of these compounds may be attributed to their direct local anesthetic actions in blocking sodium channels in cardiac membranes. Thus tricyclic-induced ventricular arrhythmias usually do not respond well to therapy with standard Class I antiarrhythmic drugs that also have the same direct local anesthetic action and may potentiate the adverse effects of tricyclic antidepressants. Cardiac toxicity was produced in dogs by the administration of amitriptyline, both orally and IV. At serum concentrations less than 2,000 ng/mL, sinus tachycardia occurred with widened QRS complexes. At higher concentrations, QRS duration became more markedly prolonged and was followed by ventricular tachyarrhythmias. Occurrence of ventricular tachyarrhythmias was associated with QRS durations of more than 0.11 second. Sodium bicarbonate (18 to 36 mEq) administered IV over either 30 seconds or two minutes rapidly converted ventricular tachycardia to normal sinus rhythm. Conversion was associated with abbreviation of the QRS complex and was accompanied by a rise in both systolic and diastolic pressures. The duration of sodium bicarbonate effect paralleled the duration of the changes in arterial pH and plasma bicarbonate concentrations. In vitro studies in cardiac Purkinje fibers suggested that reversal of amitriptyline induced cardiac membrane effects by sodium bicarbonate may be attributed not only to alkalinization but also to increased in extracellular sodium concentration, diminishing the local anesthetic action of amitriptyline and resulting in less sodium channel block.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017160 TI - Experimentally induced coronavirus infections in calves: viral replication in the respiratory and intestinal tracts. AB - Eleven 3- to 50-day-old colostrum-deprived gnotobiotic calves and seven 25- to 63 day-old colostrum-deprived conventional calves were allotted into 3 groups. Each group was inoculated with a fecal isolate of bovine coronavirus via different routes: orally/intranasally OR/IN, No. 1 through 8, group 1 calves; OR, No. 9 through 13, group 2 calves; IN, No. 14 through 18, group 3 calves. Nasal swab specimens and fecal specimens were collected daily and were examined for coronavirus antigen by use of direct immunofluorescent staining (nasal epithelial cells) or by use of immune electron microscopy (fecal specimens). All but 4 calves (No. 11, 13, 17, and 18) were euthanatized on postinoculation days (PID) 3 to 7. Calves 11 and 17 became severely dehydrated and died at PID 5. Calves 13 and 18 were evaluated for nasal and fecal shedding of coronavirus through PID 14. Distribution of coronavirus antigen in the respiratory and intestinal tracts of the 14 euthanatized calves was evaluated by use of direct immunofluorescent staining. All calves developed profuse diarrhea by PID 2 to 4; however, calves did not develop clinical signs of respiratory tract disease before euthanasia or death. Inoculated calves shed coronavirus in their feces as detected by use of immune electron microscopy. Infected nasal epithelial cells were detected in all but 2 orally inoculated calves (No. 9 and 10). Route of inoculation influenced the sequence of initial detection of coronavirus antigen from fecal specimens or nasal swab specimens.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017161 TI - Canine parvovirus: environmental effects on infectivity. AB - Effects of various environments on the infectivity of canine parvovirus-2 (CPV-2) were studied. When CPV-2 was subjected to several controlled indoor environments, the virus remained infective at approximate initial inoculation amount (median tissue culture infective dose [TCID50] = 10(5.5)/ml) for 12 months at temperatures less than -20 C, decreased to TCID50 of 10(2.3)/ml by 12 months at 4 C, and had a TCID50 of less than 10(1)/ml at room temperature (20 C) or higher in less than 2 months. The CPV-2 subjected to outdoor environments was not infective beyond 5 months, except that kept in areas protected from sunlight and drying conditions. The virus surviving in the outdoor environments was not infective for study dogs, whereas the virus maintained at less than 20 C was. PMID- 3017162 TI - An enzyme-linked immunosorbent assay for the detection of bovine antibodies to vesicular stomatitis virus. AB - An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect bovine antibody to vesicular stomatitis virus (VSV). Serum samples from cows experimentally infected with the New Jersey serotype of VSV (VSV-NJ) were assayed by the ELISA and serum-neutralization (SN) assay. The ELISA was as sensitive as the SN assay in detecting bovine antibody to VSV. The correlation between SN titers and ELISA values at absorbance at 405 nm was statistically significant. The ELISA was not specific for VSV-NJ, however, and could detect serum samples positive to the Indiana serotype of VSV that had SN titers of greater than or equal to 480. Nonspecific reactions were due to cross-reactive group-specific viral proteins that are shared by both serotypes. The cross-reactivity allows the use of a single rapid test in identifying both serotypes of VSV from the other exotic vesicular diseases, especially foot-and-mouth disease. The ELISA titers of serum samples positive for VSV-NJ were comparable with the corresponding SN titers of each sample. The sensitivity, rapidity, and ease of the ELISA system and the use of a single test in identifying both serotypes of VSV from the other exotic vesicular diseases make this ELISA suitable as a rapid diagnostic assay for VS. PMID- 3017164 TI - Inpatient alcoholism treatment. Who benefits? PMID- 3017163 TI - Transplantation of pancreatic islets in dogs. AB - Dispersed pancreatic islet tissue, prepared by collagenase digestion without separation of exocrine and endocrine components, was transplanted into the splenic pulp of 12 dogs made diabetic by total pancreatectomy. Four dogs (group 1) were given autotransplants, and all became euglycemic 4.5 +/- 1.5 days (mean +/- SE) after the transplantation was done. Three of these dogs remained euglycemic until splenectomized 60 days after transplantation was done. Four dogs (group 2) given allogeneic transplants from histocompatible littermates within the same group were administered cyclosporine (40 mg/kg of body weight/day; starting 2 days before transplantation was done until dogs were splenectomized), and 3 of these dogs became euglycemic 8.0 +/- 2.0 days after the transplant was done. Two of the 3 dogs that became euglycemic remained so until splenectomized 60 days after transplantation was done, and the 3rd was euglycemic until 31 days after transplantation. Four dogs (group 3) given allogeneic islet transplants from nonrelated histocompatible donors within the same group were given cyclosporine (40 mg/kg/day; as described for group 2), and none became euglycemic. PMID- 3017165 TI - Opportunities for the elimination of tuberculosis. PMID- 3017166 TI - Restriction fragment analysis of chromosomal DNA defines different strains of Mycobacterium tuberculosis. AB - As an initial step in gaining a better understanding of the important clinical properties that vary between strains of mycobacteria, we attempted to find molecular markers that would define different strains of Mycobacterium tuberculosis. We used restriction fragment analysis with the endonuclease MboI and hybridization with total M. tuberculosis DNA to examine DNA differences between 15 strains of M. tuberculosis. We were able to identify different strains using this method. In order to assess the sensitivity of this method in identifying different strains, we compared it with phage typing. The 2 methods appear to be similar in sensitivity and also to be complementary. There were 2 examples where restriction fragment analysis did not separate strains with different phage types. In addition, there were 2 examples where phage typing did not separate strains with different restriction patterns. Finally, there were 2 epidemiologically unrelated strains with the same restriction pattern and the same phage type. This method of restriction fragment analysis of chromosomal DNA is potentially useful for epidemiologic studies of tuberculosis. Additionally, by analyzing the genome of M. tuberculosis, molecular markers may well be defined that will be useful in discovering the pathogenesis of the clinical properties of M. tuberculosis, which previously have been poorly understood. PMID- 3017167 TI - Prevention of canine anaphylaxis with isoproterenol. AB - Sympathomimetic agents are used to reverse the cardiovascular and respiratory abnormalities that are associated with anaphylactic shock. In addition, in vitro studies have demonstrated that beta-adrenergic stimulation modulates IgE-mediated release of chemical mediators. To separately evaluate the ability of isoproterenol, a beta-adrenergic agonist, to prevent mediator release and anaphylactic shock, as well as to reverse the cardiopulmonary manifestations of systemic anaphylaxis, we administered a continuous infusion of isoproterenol before and during canine anaphylactic shock. An intravenous injection of Ascaris suum antigen was used to produce shock, which was characterized by a 50 +/- 15% (mean +/- SEM) decrease in mean arterial blood pressure, a 30 +/- 8% decrease in cardiac output, and a 122 +/- 95% increase in total pulmonary resistance. These changes were associated with significant increases in plasma histamine concentrations from 1.1 +/- 0.1 to 158.4 +/- 137 ng/ml. Administration of a continuous infusion of isoproterenol during anaphylactic shock did not significantly improve any of these physiologic abnormalities. However, when isoproterenol was administered prior to the injection of antigen, these physiologic changes were prevented and histamine release was inhibited in a significant proportion of animals. In contrast, beta-adrenergic stimulation did not prevent nonimmunologic shock and mediator release induced with compound 48/80. We conclude that the administration of a beta-adrenergic agonist prevented the cardiopulmonary manifestations of anaphylactic shock by inhibiting mediator release and that the physiologic effects of isoproterenol administered during systemic anaphylaxis were minimal. PMID- 3017168 TI - Usefulness of tumor markers in serum and lung lavage. PMID- 3017169 TI - Chronic hepatitis B in asymptomatic homosexual men with antibody to the human immunodeficiency virus. PMID- 3017170 TI - Transient antibody to human immunodeficiency virus. PMID- 3017171 TI - [Pharmacochemistry as seen through the history of benzodiazepines and their cellular binding sites (I)]. PMID- 3017172 TI - [Synthesis and pharmacology of some new GABA-ergics]. PMID- 3017173 TI - Residual carcinoma in bronchial resection line. AB - Microscopic residual tumour growth was found postoperatively in the bronchial resection lines in 44 out of a total of 1069 patients who underwent resection for pulmonary carcinoma. Bronchopleural fistula developed in six of these patients (13.6%). The incidence of bronchopleural fistula in the material overall was 4.2% (45 patients). Residual carcinoma therefore seemed to impair healing of the bronchial stump (p less than 0.01). Routine intraoperative frozen sections of the bronchial resection line can be recommended in order to reveal the residual carcinoma and thus lower the incidence of bronchopleural fistula. The five-year survival rate of these patients with microscopic residual carcinoma in their bronchial resection lines was similar to that in patients with the same TNM staging who had undergone resection for pulmonary carcinoma. Residual carcinoma in the bronchial resection line therefore did not worsen prognoses. The survival rate in our patients was better than survival rates reported for other series. Most of our patients had received postoperative radiotherapy. Previous reports relate to patients treated only surgically. Postoperative radiotherapy may therefore improve the prognosis in patients with residual tumour growth in their bronchial resection lines. PMID- 3017174 TI - Enalapril in chronic heart failure, a double-blind placebo-controlled study. AB - The efficacy of enalapril, a new angiotensin-converting enzyme inhibitor, was investigated in a double-blind placebo-controlled study in 24 patients with chronic heart failure. The patients belonging to NYHA functional class II-IV and treated with digoxin and diuretics were randomized to enalapril (12 patients) or placebo (12 patients) treatment for a 12-week period. Assessments were carried out at baseline and at 4 and 12 weeks during the treatment period. Complete data could be obtained on 10 patients receiving enalapril and on 11 patients receiving placebo. NYHA functional class improved by at least one class in 6 of 10 patients in the enalapril group, but only in 1 of 11 patients in the placebo group (chi 2 = 6.54; p less than 0.05). Duration of bicycle ergometer exercise increased significantly in the enalapril group from 8.8 +/- 3.4 to 11.3 +/- 4.2 (4 weeks) and to 11.2 +/- 3.6 min (12 weeks; p less than 0.05 for both), whereas it remained unchanged in the placebo group. Left ventricular ejection fraction by radionuclide ventriculography in the enalapril group increased significantly (baseline: 33.5 +/- 19.9%, 4 weeks: 40.0 +/- 20.0% (p less than 0.001), 12 weeks: 39.6 +/- 20.1% (p less than 0.01], whereas in the placebo group it did not change significantly from the baseline of 48.8 +/- 16.7%. The results indicate that enalapril induces a sustained relief of symptoms and improves exercise capacity in patients with heart failure. This subjective improvement appears to be accompanied by an increase in ejection fraction. PMID- 3017176 TI - [Symposium: Molecular biologic of peptide hormones]. PMID- 3017177 TI - [Symposium: Kinases and the transmission of information]. PMID- 3017179 TI - Lymphadenopathy/AIDS virus: genetic organization and relationship to animal lentiviruses. AB - This article presents data obtained by our group in the molecular characterization of the probable agent of the acquired immune deficiency syndrome (AIDS), the lymphadenopathy/AIDS virus (LAV). Molecular cloning and complete nucleotide sequencing of LAV allows a detailed comparison with other AIDS virus isolates, as well as other human and animal retroviruses. We have now molecular evidence that the AIDS virus is closely related to visna virus, prototype of the lentiviruses, whereas the other human retroviruses, i.e., human T-cell leukemia viruses type I and II (HTLV-I and II), are quite remote in the evolution. PMID- 3017178 TI - IgE immune complexes induce leukotriene B4 release from rat alveolar macrophages. PMID- 3017175 TI - [The heart, an endocrine gland]. AB - Two independent series of biomedical investigations have led to the discovery that the atria are a peptide-secreting endocrine gland. The first is mainly morphological and starts with the finding that mammalian atrial but not ventricular cardiocytes contain "dense bodies". These "dense bodies" later called "specific granules" were found to be different from lysosomes, to be made up of proteins and to incorporate both 3H-leucine and 3-H-fucose in a pattern typical of peptide-secreting endocrine cells. The finding that rat atrial granulation varied with the sodium and water balance led to the crucial observation that atrial extracts have natriuretic and diuretic effects. In less than 4 years, this new natriuretic hormone has been purified, sequenced and synthetized, and its cDNA and gene have been cloned. The ANF gene has been assigned to the distal short arm of chromosome 1 in band 1P36 while the mouse gene is localized in chromosome 4. The native and synthetic hormones exert identical wide ranging effects (possibly through particulate guanylate cyclase stimulation and adenylate cyclase inhibition) on the kidney, blood vessels, adrenal cortex and pituitary. Physiopathologic implications of the hormone in experimental hypertension, congestive heart failure and expansion of blood volume are already beginning to emerge. On the other hand, the search for natriuretic hormones or factors by studies of negative pressure breathing, atrial distention experiments, head-out water immersion, expansion of blood volume, Na+/K-ATPase inhibition and parabiosis experiments in Dahl rats has provided a general framework within which to interpret this new cardiac function. PMID- 3017180 TI - All three polyoma early genes are involved in transformation of rat embryo fibroblast cells. AB - The tumor progression concept was first defined by Foulds at the end of the nineteen fifties. His work about the evolution of mammary tumors led to the conclusion that a tumor is initiated as soon as a cell interacts with an oncogenic agent and that the tumor progresses by acquiring in a permanent and irreversible way, new properties which are characteristic of neoplasms. Molecular biology made possible the identification in transformed cells of molecular events such as chromosomal rearrangements, mutations, the loss of certain genes or integration of viral genes. Experimental models have been introduced in order to try to identify in vitro the successive steps leading to transformation and the corresponding cellular phenotypes. One of these models is the cellular transformation phenomenon induced by polyoma virus. In this model, it now seems clear that several steps and several genes are involved in the cellular transformation phenomenon. PMID- 3017181 TI - Are oncogenes involved in invasion and metastasis? AB - In carcinogenesis, the acquisition of invasiveness and metastatic capability are the key steps towards malignancy. Clinical and experimental data suggest that invasion and metastasis necessitate cellular functions other than growth and by implication the activation of separate cellular genes. We review here data about the role of known oncogenes in the acquisition of invasive and metastatic capabilities. Major importance is paid to the question whether actual techniques for DNA-transfection, selection, and testing of invasion and metastasis are valid for the study of the role of oncogenes in invasion and metastasis. We conclude that, so far, such a role of oncogenes is uncertain, because the majority of cell populations used as recipients in transfection experiments are able to acquire invasiveness and metastatic capability in an apparently spontaneous manner. PMID- 3017183 TI - Transformation of human embryo retinoblasts with simian virus 40, adenovirus and ras oncogenes. AB - Few human cell systems have been described in which a number of different genes induce transformation. The present investigation reports on our studies using primary human embryo retinoblasts as a model system to monitor transformation and the subsequent behaviour of individual transformants in terms of establishment, the frequency of immortalization and tumourigenic potential. SV40, Adenovirus E1 and E1A, and combinations of Adenovirus E1A and activated H-ras or N-ras were examined as transforming agents. Considerable differences were observed in the ability of these genes to transform human cells, to induce immortal lines and to produce cell lines with a tumourigenic phenotype. Activated ras genes were non transforming in this system and the degree of complementation with adenovirus E1As in transformation experiments was dependent on both the adenovirus serotype and the ras gene used. The development of tumourigenic cell lines required the expression of more than one oncogene and additional genetic events were required in some in some instances before immortal cell lines were obtained. These findings contribute to the concept that the development of cancer is a multi-step process. PMID- 3017184 TI - Preferential loss of large lumbar primary sensory neurons in carcinomatous sensory neuropathy. AB - Morphometric studies at autopsy of a patient with sensory neuropathy associated with small-cell lung carcinoma showed preferential loss of large-diameter sensory nerve cell bodies (fifth lumbar dorsal root ganglion), marked decrease of large myelinated fibers in the dorsal root and sural nerve, and almost total loss of myelinated fibers in the fasciculus gracilis. PMID- 3017185 TI - Chronic inflammatory demyelinating polyneuropathy of infancy: a corticosteroid responsive disorder. AB - We present the clinical, electrophysiological, and histopathological findings in 6 children with early-onset chronic inflammatory demyelinating neuropathy. The clinical features initially suggested a genetically determined disorder in each patient. Sural nerve biopsy showed changes of chronic demyelination with multifocal endoneurial edema and mononuclear cellular infiltrates. All children improved with corticosteroid therapy. PMID- 3017182 TI - N-ras and human cancer. AB - Activated N-ras genes have been detected in a variety of human tumours and tumour derived cell lines. It has been proposed that mutation or amplification of an N ras gene represents one of the key steps in the development of some human tumours. Here we discuss the various methods by which activated N-ras genes have been detected, the ways in which they have become activated, and their contributions to the transformed phenotype. PMID- 3017186 TI - Effects of ACTH on seizure susceptibility in the developing brain. AB - Adrenocorticotropic hormone (ACTH) has been frequently used as an anticonvulsant drug in some childhood seizure disorders. Despite its widespread use, few studies have evaluated the effects of ACTH on seizure susceptibility in the developing animal or the long-term consequences of ACTH treatment on brain development. In this study, ACTH was given either for 2 days or 14 days prior to kindling in 15-, 22-, and 30-day-old rats. Morphological changes in the brain were studied using routine light microscopy and dendrite branch counting following Golgi staining. Both acute and chronic ACTH treatment inhibited the rate of kindling in all three age groups. There were no differences in brain morphology between the controls and the ACTH-treated rats killed shortly after kindling. Rats treated with ACTH and killed as adults, however, had significantly more dendrite branches than did controls. In the immature brain, ACTH treatment reduces seizure susceptibility and has no long-term deleterious effects on neuronal growth. PMID- 3017188 TI - Primary central nervous system lymphoma related to Epstein-Barr virus in a patient with acquired immune deficiency syndrome. AB - The study of a patient suggested a relationship between Epstein-Barr virus infection and primary lymphoma of the central nervous system in the acquired immune deficiency syndrome. Deoxyribonucleic acid preparations from tumor tissue contained 30 to 100 copies of Epstein-Barr virus genome per cell when hybridized with a probe consisting of the Bam-HI K fragment of Epstein-Barr virus strain FF41. This hybridization study suggests that induction of this patient's central nervous system lymphoma was related to Epstein-Barr virus infection. PMID- 3017187 TI - Chronic demyelinating peripheral neuropathy in cerebrotendinous xanthomatosis. AB - Three siblings with chemically proved cerebrotendinous xanthomatosis presented with typical neurological manifestations of dementia and spinocerebellar disorder. Electrodiagnostic tests revealed demyelinating neuropathy in all three. Sural nerve biopsies showed loss of myelinated large fibers, marked Schwann cell proliferation, and onion bulb formation. Teased-fiber preparations confirmed the occurrence of segmental demyelination and remyelination. We suggest that demyelinating neuropathy is part of the neurological spectrum of cerebrotendinous xanthomatosis and should be considered in the differential diagnosis of a recessively inherited motor and sensory neuropathy. PMID- 3017189 TI - Biochemical interactions of tumor cells with the basement membrane. PMID- 3017190 TI - Complex transcriptional units: diversity in gene expression by alternative RNA processing. PMID- 3017192 TI - Mutants in glucose metabolism. PMID- 3017191 TI - Reactive oxygen intermediates in biochemistry. PMID- 3017193 TI - Extralysosomal protein degradation. PMID- 3017194 TI - Platelet activating factor: a biologically active phosphoglyceride. PMID- 3017195 TI - Arachidonic acid metabolism. PMID- 3017196 TI - Eukaryotic DNA replication. PMID- 3017197 TI - Neuropeptides: multiple molecular forms, metabolic pathways, and receptors. PMID- 3017198 TI - DNA polymorphism and human disease. PMID- 3017200 TI - [Formulation of the composition of a polymyxin ointment using a mathematical experimental design]. AB - Mathematical design of the experiment permitted detection of the action of independent factors of the component composition on antibacterial activity and rheological properties of polymyxin ointment. Optimal compositions of polymyxin ointment based on polyethylene oxide emulsion were scientifically grounded and recommended for further experimental investigation on animals. PMID- 3017199 TI - [The antibiotic galtamycin. The structure of galtamycinone]. AB - The structure of galtamycinone, a chromophore moiety of galtamycin, a new antitumor antibiotic was determined by spectral analysis, i.e. UV, NMR and mass spectrometry. Galtamycinone was shown to be 1,4,6-trioxy-10 [4 (e), 5 (e)-dioxy-6 (e)-methyl-tetrahydropyran-2(e)-yl]-8-methyl tetracendion-5,12. It represents a new type of anthracycline chromophores containing a C-glycoside bond in its molecule. PMID- 3017202 TI - Ability of monoclonal antibody to herpes simplex virus glycoprotein gB to promote healing of herpetic skin lesions in nude mice. AB - The effect of monoclonal antibody (MCA) to glycoprotein gB of herpes simplex virus (HSV) was studied in athymic nude mice inoculated with HSV intracutaneously in the midflank. HS1, the MCA used in the study, had a high neutralizing titer (1:2048) and had antibody-dependent cell-mediated cytotoxicity. HS1 was injected intraperitoneally at various intervals after HSV infection. HS1 injected 3 h after infection inhibited the development of skin lesions and most mice survived. Administration of HS1 at the time the local skin erosions appeared at the inoculated site (4-7 days after infection) was also effective, and in four of eight mice skin lesions completely healed. Furthermore, in three of four mice that survived, latent infections in the ganglia were also prevented as evidenced by the failure to detect HSV by co-cultivation with Vero cells. Administration of HS1 after the development of zosteriform skin lesions (5-9 days after infection) reduced virus in the ganglia and prolonged the survival time, though the disease was not completely arrested and all the mice died eventually. PMID- 3017201 TI - Thiosemicarbazones of 2-acetylpyridine, 2-acetylquinoline, 1-acetylisoquinoline, and related compounds as inhibitors of herpes simplex virus in vitro and in a cutaneous herpes guinea pig model. AB - A series of 111 thiosemicarbazones of 2-acetylpyridine, 2-acetylquinoline, 1 acetylisoquinoline, and related compounds were evaluated as inhibitors of herpes simplex virus in vitro and in a cutaneous herpes guinea pig model. All derivatives tested were potent inhibitors of virus replication with mean 50% inhibitory concentrations of 1.1 micrograms/ml for both type 1 and 2 herpes simplex virus. Inhibitory concentrations for cellular protein and DNA synthesis were considerably higher for many compounds resulting in in vitro therapeutic indices ranging from greater than 100 (highly selective) to less than 1 (negatively selective). All compounds were tested for dermal toxicity following topical administration of saturated solutions in 1,3-butanediol to the shaved, depilated skin of guinea pigs. Approximately 50% of the compounds produced slight to no dermal toxicity whereas the remaining compounds produced moderate to severe dermal toxicity. 28 compounds were evaluated in the cutaneous herpes guinea pig model against herpes simplex virus type 1. A number of N4-monosubstituted 2 acetylpyridine thiosemicarbazones produced highly significant reductions in days to healing and lesion score without producing untoward dermal toxicity. Structure activity relationships revealed that a reduction of the azomethine bond in the molecule (i.e., conversion of a thiosemicarbazone to a thiosemicarbazide) greatly diminished dermal toxicity apparently without producing a proportional decrease in antiviral activity. PMID- 3017203 TI - Synergism between anti-rhinovirus antivirals: various human interferons and a number of synthetic compounds. AB - DCF (dichloroflavan), enviroxime, chalcone Ro-09-0410 and HuIFN (Human interferon)-alpha 2, HuIFN-beta, HuIFN-beta X 401 and HuIFN-gamma, showed antiviral activity in vitro against RV2 (rhinovirus type 2) and RV9. Binary combinations of these drugs showed synergistic activity of which the combinations of HuIFN-gamma or HuIFN-alpha and enviroxime were of most interest. They were studied in more detail in tissue culture by virus yield experiments and in organ culture of human embryonic nasal epithelium and human embryonic tracheal culture in which there was a potent antiviral synergy. These results indicate that such combinations of drugs may be worthy of clinical study. PMID- 3017204 TI - Is fiber satiating? Effects of a high fiber preload on subsequent food intake of normal-weight and obese young men. AB - This study was designed to test whether a high fiber preload can suppress food intake at a subsequent test meal. Fifty male undergraduates, 31 of normal body weight and 19 at least 15 per cent overweight, participated as paid volunteers. So as not to inhibit spontaneous food intake, they were told that the investigators were studying the effects of the parasympathetic nervous system on cognitive processes and they were given a picture rating task to perform. The stated rationale for having the subjects eat the preload between two sets of pictures was to alter the state of parasympathetic activation. The preload consisted of two roast beef sandwich halves containing a total of 400 kcal and either 5.2 or 0.2 g of crude fiber in the bread. The preload was eaten by the subjects, their meal was intentionally interrupted and then they were allowed to finish eating 30-45 minutes later (test meal). Obese subjects ate significantly fewer sandwich halves after the high than after the low fiber preload. Obese subjects ate significantly more than non-obese subjects in the low fiber treatment so that the effect of the high fiber was to normalize their intake. Although both obese and normal-weight subjects reported feeling fuller after the high fiber preload, the non-obese subjects ate virtually the same amount in both treatments. Obese subjects reported liking the high fiber bread less well than the low fiber bread but additional statistical analyses showed an intake suppressing effect of fiber even after controlling for bread rating. These results support the hypothesis that fiber reduces caloric intake in obese people. PMID- 3017205 TI - Epileptogenic cerebral low-grade tumors: effect of interstitial stereotactic irradiation on seizures. AB - 15 patients with various types of surgically inaccessible epileptogenic cerebral neuroepithelial tumors were treated with stereotactic interstitial irradiation. Epilepsy was improved in all cases, being abolished in 6 and markedly reduced in another 7. The effects which became apparent soon after the radioisotope implant persisted. Possible mechanisms of the phenomenon are discussed. PMID- 3017206 TI - Central nervous system mechanisms for pain modulation. AB - Although a great deal has been learned about the neural basis for stimulation produced analgesia, it is evident that the 'analgesia systems' are much more complex than was initially thought. Part of the complexity derives from the fact that a number of different pathways, using several different neurotransmitters, can affect nociceptive transmission. Further complexity stems from evidence that nociceptive transmission can be modulated both at a spinal cord level and at higher levels of the nociceptive projection system, such as the thalamus. Hopefully, a greater understanding of the 'analgesia systems' will lead to explanations for a number of puzzling aspects of pain and perhaps to improved therapy. PMID- 3017207 TI - Deafferentation pain and stimulation of the thalamic sensory relay nucleus: clinical and experimental study. AB - This report summarizes our clinical experience in which the effects of both thalamic sensory relay nucleus (TSRN) and periaqueductal gray (PAG) stimulation were tested in the same series of patients with various forms of pain. The clinical data indicated that neurogenic pain due to deafferentation at the level of the peripheral nerves or the spinal cord was often controlled by TSRN stimulation but not by PAG stimulation. We also review the results of our experimental investigations in cats which were undertaken in an attempt to clarify the neurophysiologic basis of such differential clinical effects of TSRN and PAG stimulation. It appeared that abnormal hyperactivity within the trigeminal medullary dorsal horn following retrogasserian rhizotomy was far more frequently inhibited by TSRN stimulation than by PAG stimulation. PMID- 3017208 TI - An animal model for neuron-specific spinal cord lesions by the microinjection of N-methylaspartate, kainic acid, and quisqualic acid. AB - It has been shown that N-methylaspartate (NMA), kainic acid (KA), and quisqualic acid (QA) can produce preferential neuronal damage in various parts of the striatum, hippocampus, and thalamus with relative sparing of axons in transit. Thus far, the evidence that axons in transit escape destruction has been based largely on histological observations. To test the functional integrity of axons in passage, we made unilateral lesions with these agents in the cervical spinal cord of rats and compared the subsequent functional deficits with those seen after spinal cord hemisections. Observations were made in 14 rats. In each case, a laminectomy at the C6-C7 level was performed under general anesthesia. Animals receiving microinjections of KA, QA, or NMA showed motor and sensory deficits only in the ipsilateral forepaw and remained able to use the hindpaws normally. By contrast, animals undergoing spinal cord hemisection developed obvious motor deficits in the ipsilateral hindpaw in addition to the deficits in the forepaw. Histological observations of the spinal cords confirmed an extensive gray matter destruction with relative preservation of the long tracts in animals injected with KA, QA, and NMA. In addition, it was noted that spinal cord neurons appear relatively less sensitive to KA and more sensitive to QA than neurons in the thalamus, striatum, or hippocampus. The possible application of these findings for the production of dorsal root entry zone lesions will be discussed. PMID- 3017209 TI - Cerebral-evoked responses elicited by direct stimulation of the lateral spinothalamic tract in the human. AB - The authors recorded cerebral-evoked responses elicited by direct stimulation of the human lateral spinothalamic tract (LST) during percutaneous cordotomy to investigate central conduction of noxious stimuli. These responses consisted of four negative potentials, peak latency being 3.8 (N1), 8.4 (N2), 12.2 (N3) and 21.9 (N4) ms respectively. N1 showed wide distribution over the scalp and was considered to be of subcortical origin. N2-N4 were distributed in both the temporal and central area. The different distribution pattern of N2-N4 from conventional somatosensory-evoked potential suggested a different projection of LST from the medial lemniscus system. PMID- 3017210 TI - Cross-correlation analysis of thalamic neurons and EMG activity in parkinsonian tremor. AB - Bursting activity in cells cross-correlated with electromyographic (EMG) activity during parkinsonian tremor. Statistically significant evidence of cross correlation was found for 49% of cells located at the lesion target for relief of tremor. Statistically significant correlation was found for 90% of cells having tremor frequency power greater than twice 'average power' at nontremor frequencies. This population of cells may be involved in the generation of parkinsonian tremor. PMID- 3017211 TI - The role of oxidative processes in the cytotoxicity of substituted 1,4 naphthoquinones in isolated hepatocytes. AB - In order to clarify the role of oxidative processes in cytotoxicity we have studied the metabolism and toxicity of 2-methyl-1,4-naphthoquinone (menadione) and its 2,3 dimethyl (DMNQ) and 2,3 diethyl (DENQ) analogs in isolated rat hepatocytes. The two analogs, unlike menadione, cannot alkylate nucleophiles directly and were considerably less toxic than menadione. This decreased toxicity was consistent with the inability of DMNQ and DENQ to alkylate but we also found them to undergo lower rates of redox cycling in hepatocytes and a higher ratio of two electron as opposed to one electron reduction relative to menadione. Thus, facile analysis of the respective roles of alkylation and oxidation in cytotoxicity was not possible using these compounds. In hepatocytes pretreated with bischloroethyl-nitrosourea (BCNU) to inhibit glutathione reductase, all three naphthoquinones caused a potentiation of reduced glutathione (GSH) removal/oxidized glutathione (GSSG) generation and cytotoxicity relative to that observed in control cells. These data show that inhibition of hepatocyte glutathione reductase by BCNU results in enhanced naphthoquinone-induced oxidative challenge and subsequent cellular toxicity. That DMNQ and DENQ are cytotoxic, albeit at high concentrations, and that this cytotoxicity is potentiated by BCNU pretreatment suggest that oxidative processes alone can be a determinant of cytotoxicity. PMID- 3017212 TI - Three diadenosine 5',5''-P1,P4-tetraphosphate hydrolytic enzymes from Physarum polycephalum with differential effects by calcium: a specific dinucleoside polyphosphate pyrophosphohydrolase, a nucleotide pyrophosphatase, and a phosphodiesterase. AB - Two new enzymes that hydrolyze diadenosine tetraphosphate (Ap4A) have been isolated from the acellular slime mold Physarum polycephalum. Both enzymes are different from the Physarum Ap4A symmetrical pyrophosphohydrolase previously described on the basis of their substrate specificities, reaction products, molecular weights, and divalent cation requirements. One enzyme is a nucleotide pyrophosphatase that asymmetrically hydrolyzes Ap4A to AMP and ATP. This enzyme hydrolyzes several mono- and dinucleotides with the corresponding nucleotide monophosphate as one of the products. The percentage hydrolysis of NAD+, Ap4A, and Ap4G, each at 10 microM, was 100, 56, and 51, respectively. A divalent cation is required for activity, with Ca2+ yielding 20-30 times greater activity than Mg2+ or Mn2+. Values of Km for Ap4A and Vmax are similar to the corresponding values for Ap4A symmetrical pyrophosphohydrolase. The second enzyme is a phosphodiesterase I with broad substrate reactivity. This enzyme also asymmetrically hydrolyzes Ap4A, but it does not hydrolyze NAD+. Activity of the phosphodiesterase I is stimulated by divalent cations, with Ca2+ being 50-60 times more stimulatory than Mg2+ or Mn2+. The apparent molecular weights of the nucleotide pyrophosphatase and phosphodiesterase are 184,000 and 45,000, respectively. In contrast, the Ap4A pyrophosphohydrolase hydrolyzes Ap4A to ADP, is inhibited by Ca2+ and other divalent cations, and has an apparent molecular weight of 26,000 as previously reported. PMID- 3017213 TI - Partial purification of a nucleoside triphosphatase from the inner membrane of the chloroplast envelope of pea. AB - A Mg2+-NTPase has been partially purified from the inner membrane of the pea chloroplast envelope. Isolated envelope membranes were solubilized with Triton X 100 and fractionated by DEAE-Sephadex chromatography, followed by ultrafiltration and sucrose density gradient centrifugation. An approximate 35-fold increase in the specific activity of the vanadate and sodium fluoride sensitive NTPase was obtained. Analysis of the partially purified NTPase by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single 37-kDa polypeptide that appeared to be associated with the activity. In support of this identification, it is demonstrated that the 37-kDa polypeptide can be photolabeled with 8-azido ATP. PMID- 3017214 TI - Interaction of bovine renal mitochondrial cytochrome P-450 with antioxidants. AB - The antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), nordihydroguiaretic acid (NDGA), benzyl sulfoxide (BS), ferulic acid (FA), caffeic acid (CA), dimethyl sulfoxide (Me2SO), protocatechuic acid (PCA), and P 450 inhibitor metyrapone all acted to slow the previously noted loss of vitamin D3 1 alpha-and 24-hydroxylase activities in cultured bovine proximal tubule cells. The slowing of the loss of hydroxylase activities by antioxidants was increased by culturing cells in 5% O2 vs 19% O2. These same antioxidants also directly inhibited 1 alpha- and 24-hydroxylase activities. For a single antioxidant, or metyrapone, Ki's for inhibition of both hydroxylases were equal, ED50's for stabilization of both hydroxylase activities were equal, and Ki's and ED50's were not significantly different. These antioxidants prevented tert butylhydroperoxide (tert-BOOH)-mediated proximal tubule cell death at concentrations, i.e., 0.1 mM, which were effective in stabilizing hydroxylase activities. When added together, the antioxidants H2SeO3, uric acid, and trolox c gave slight stabilization of hydroxylase activities without inhibiting hydroxylase activities. Singly, these antioxidants did not stabilize or directly inhibit hydroxylase activities. This antioxidant combination augmented BHA- or BHT-mediated stabilization of both hydroxylase activities independent of any effects on inhibition. But the most potent antioxidants which acted to stabilize hydroxylase activities in culture also directly acted to inhibit hydroxylase activities. Antioxidant effects were additive for both inhibition and stabilization of hydroxylase activities. Stabilization of hydroxylase activities was dissociated from inhibition in the presence of maximal FA, CA, and BHA or FA, CA, and BHT combinations. Bovine renal mitochondrial cytochrome P-450 levels decreased in cultured bovine proximal tubule cells to nondetectable levels by 8 days in culture. When cultures were treated with BHA and BS, mitochondrial P-450 levels were almost twofold greater than in untreated controls. Percentage changes in mitochondrial P-450 levels closely paralleled percentage changes in hydroxylase activities elicited by antioxidant treatment regimes. Antioxidants which were effective inhibitors of hydroxylase activities in cultured bovine proximal tubule cells were also effective in inhibiting hydroxylase activities in isolated proximal tubule mitochondria, supplemented with a NADPH-generating source. Ki's for inhibition of hydroxylase activities were very similar in cultured cells and in isolated mitochondria.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3017215 TI - Phosphorylation- and ligand-induced conformational changes of rat liver fructose 1,6-bisphosphatase. AB - The effects of cyclic AMP-dependent phosphorylation on the structural properties of rat liver fructose-1,6-bisphosphatase were investigated by uv difference spectroscopy and circular dichroism. The incorporation of 4 mol of phosphate per mole of fructose-1,6-bisphosphatase induces a significant increase in the alpha helix content of the enzyme without affecting its spectrophotometric properties. The addition of fructose 1,6-bisphosphate or fructose 2,6-bisphosphate also affects the conformation of the enzyme. However, both the phosphorylated and the nonphosphorylated forms exhibit similar ligand-induced conformational changes. These results show that cyclic AMP-dependent phosphorylation of fructose-1,6 bisphosphatase induces a specific conformational change. They also suggest that this modification does not alter the interaction of the enzyme protein with fructose 1,6-bisphosphate and fructose 2,6-bisphosphate. PMID- 3017217 TI - Regulation of epidermal growth factor receptors by glucocorticoids during the cell cycle in HeLa S3 cells. AB - Glucocorticoids have been shown to increase epidermal growth factor (EGF) receptors in HeLa S3 cells via mechanisms dependent upon glucocorticoid receptors. We have now examined the basal and glucocorticoid-induced levels of epidermal growth factor (EGF) receptors in synchronized HeLa S3 cells and related these findings to glucocorticoid receptor nuclear binding, receptor activation, and several physiochemical properties of the glucocorticoid receptor. Quantitation of EGF receptor binding during the cell cycle indicates that no significant variation in EGF receptor number occurs during the cell cycle. Dexamethasone treatment of nonsynchronized HeLa S3 cells results in an approximately 131% increase in EGF receptor number within 48 h of treatment. Administration of glucocorticoids to cells synchronized at the late G1/S phase boundary of the cell cycle results in an approximately 80% increase in epidermal growth factor receptors 8-9 h after treatment. This hormone-induced response disappears as cells enter the G2/M and early G1 phases of the cell cycle. In contrast, hormone administration to synchronized cells during the G2/M phases is without effect after 8 or 9 h, but a response is evident when these cells reenter the late G1 phase. This inability of glucocorticoids to induce EGF receptor binding has been correlated with nuclear glucocorticoid receptor translocation at 37 degrees C in intact cells and activation of receptors in vitro to DNA binding proteins by warming. This reduction in nuclear receptor binding in intact cells and diminished in vitro activation of receptor are associated with the detection of a tightly binding glucocorticoid receptor form as analyzed by hydroxylapatite chromatography. These analyses suggest that the failure of glucocorticoids to induce EGF receptor binding during the G2/M and early G1 phases may be the result of decreased receptor activation which may result from a post-transcriptional modification of the glucocorticoid receptor. PMID- 3017216 TI - Release of iron from ferritin by cardiotoxic anthracycline antibiotics. AB - The use of the extremely effective anthracycline antitumor drugs adriamycin and daunomycin is limited by a severe, dose-dependent cardiomyopathy. Anthracycline induced toxicity has been proposed to involve iron-dependent oxidative damage to biological macromolecules yet little is known regarding the availability of physiologic iron. We now report that, in the presence of NADPH-cytochrome P-450 reductase, these drugs undergo redox cycling to generate superoxide which mediates a slow, reductive release of iron from ferritin, the major intracellular iron storage protein. Anaerobically, the semiquinone free radical forms of adriamycin and daunomycin catalyze a very rapid, extensive release of iron from ferritin. In contrast, diaziquone, an aziridinyl quinone antitumorigenic agent which is less cardiotoxic, is unable to release iron from ferritin. Thus, the present studies suggest that the cardiomyopathy observed with the anthracyclines, and perhaps their antineoplastic activity as well, may be related to their ability to delocalize tissue iron, thereby contributing to the formation of strong oxidants capable of damaging critical cellular constituents. PMID- 3017219 TI - Formation of the N'-methylnicotinamide adenine dinucleotide derivative of NAD in intact rat pituitary tumor GH3 and human promyelocytic leukemia HL-60 cells. AB - The NAD analog N'-methylnicotinamide adenine dinucleotide (N'AD) is formed in intact human promyelocytic leukemia HL-60 and in rat pituitary tumor GH3 cells during treatment of the cultured cells with the nicotinamide derivative N' methylnicotinamide (N'CH3NAm). N'AD formation is associated with the induced maturation of HL-60 cells and increased hormone production by GH3 cells during treatment with the nicotinamide derivative. N'AD is detected by HPLC analysis of cytoplasmic extracts as a peak which elutes near NAD. Four facts indicate that this compound is N'AD. First, a compound which elutes with identical time retention is produced by transglycosylation during reaction of NAD with pig brain NAD glycohydrolase in the presence of excess N'CH3NAm. Second, the putative N'AD is degraded by prolonged digestion with the NAD glycohydrolase to ADP-ribose. Third, N'AD formation is prevented by addition of nicotinamide along with N'CH3NAm to compete with binding of N'CH3NAm to the NAD glycohydrolase. Fourth, radioactive precursor labeling demonstrates that it contains adenosine, but it is not labeled by radioactive nicotinamide. The biological relevance of N'AD formation was evaluated. The appearance of N'AD precedes development of HL-60 maturation, and NAD levels increase, not decrease, as observed in other cell types, during treatment with N'CH3NAm. Therefore, we propose that N'AD, not the pyridine base itself, is the active species in inducing maturation. The results provide support of a role for NAD metabolism, probably ADP-ribosylation, in the regulation of HL-60 maturation and in hormone production by pituitary cells. PMID- 3017218 TI - The oxidation of arachidonic acid by the cyclooxygenase activity of purified prostaglandin H synthase: spin trapping of a carbon-centered free radical intermediate. AB - The ESR spin trapping technique was used to study the first detectable radical intermediate in the oxidation of arachidonic acid by purified prostaglandin H synthase. The holoenzyme and the apoenzyme, reconstituted with either hematin or Mn2+ protoporphyrin IX, were investigated. Depending on the different types of enzyme activity present, arachidonic acid was oxidized to at least two free radicals. One of these radicals is thought to be the first ESR detectable radical intermediate in the conversion of arachidonic acid to prostaglandin G2 and was detected previously in incubations of ram seminal vesicle microsomes, which are rich in prostaglandin H synthase. The ESR findings correlated with oxygen incorporation into arachidonic acid and prostaglandin formation, where the spin trap inhibits oxygen incorporation and prostaglandin formation by apparently competing with oxygen for the carbon-centered radical. Substitution of arachidonic acid by octadeuterated (5, 6, 8, 9, 11, 12, 14, 15)-arachidonic acid confirmed that the radical adduct contained arachidonic acid that is bound to the spin trap at one of these eight positions. An attempt was made to explain the apparent time lag between the metabolic activity observed in the oxygraph measurements and the appearance of the trapped radical signals. PMID- 3017220 TI - Characterization of polyisoprenyl phosphate phosphatase activity in rat liver. AB - The polyisoprenyl phosphate dephosphorylating activity of rat liver has been investigated with regard to substrate specificity, subcellular distribution, and transmembrane orientation. Total liver microsomes were employed as a source of enzymatic activity against a variety of 32P-labeled substrates. Susceptibility to dephosphorylation followed the order solanesyl phosphate greater than alpha-cis polyprenyl 19-phosphate = alpha-trans-polyprenyl 19-phosphate = dihydrosolanesyl phosphate greater than (S)-dolichyl 19-phosphate = (R)-dolichyl 19-phosphate = (R,S)-dolichyl 11-phosphate. There appeared to be no major effect of chain length from 11 to 20 isoprenes. Data obtained from inhibition studies using solanesyl [32P]phosphate as substrate were consistent with the substrate specificity studies and suggested that a single activity is responsible. With dolichyl [32P]phosphate as substrate, the phosphatase specific activity of the subcellular fractions prepared from rat liver was found to follow the sequence Golgi = smooth endoplasmic reticulum greater than plasma membrane greater than lysosomes = rough endoplasmic reticulum greater than nuclei greater than mitochondria. Transmembrane topography studies, using enzyme latency as a criterion, were consistent with an orientation of the active site facing the cytoplasm. PMID- 3017221 TI - Regulation of liver glycogen synthase phosphatase activity by ATP-Mg. AB - The kinetics of a synthase phosphatase reaction inhibited by ATP-Mg in a liver glycogen particle preparation were complex. In the presence of a physiological concentration of ATP-Mg, synthase phosphatase activity in the glycogen particle follows a biphasic course. Initially, the reaction was inhibited but later the reaction rate accelerated. The reaction was inhibited but the rate was constant in the presence of ATP-Mg with the addition of a physiological concentration of glucose 6-phosphate (Glc 6-P). Therefore, in most subsequent experiments Glc 6-P was added. The concentration of ATP-Mg at which 50% maximal inhibition (I0.5) occurred was approximately 0.1 mM in preparations obtained from rats given glucagon prior to being killed. In preparations from animals given glucose, the I0.5 was increased to 2.0 mM. The maximum inhibition was little changed in preparations from glucose- or glucagon-treated animals. Thus, administration of glucose in vivo reduced the sensitivity of the synthase phosphatase to ATP-Mg inhibition. Complexes of ATP with paramagnetic ions such as Co2+ and Mn2+ were less inhibitory than complexes with diamagnetic ions, including Ca2+ and Mg2+. Magnesium complexes of adenosine tetraphosphate and 5'-adenylimidodiphosphate also were inhibitory. Inhibition was independent of phosphorylase a and not a nonspecific, polyvalent anion effect. The best explanation for the distinctive effects of ATP-Mg in preparations from glucagon- and glucose-treated animals is that the respective treatments promote and stabilize different forms of synthase D or possibly synthase phosphatase with different affinities for ATP-Mg. These forms are interconvertible, as previously suggested, in studies employing EDTA (20). PMID- 3017222 TI - The effect of substrates and competitive inhibitors on the phosphatase-dependent activation of hepatic hydroxymethylglutaryl CoA reductase. AB - Previous studies have demonstrated that the in vitro activation of microsomal hepatic hydroxymethylglutaryl (HMG) CoA reductase by dephosphorylation is inhibited by HMG CoA or NADPH, the substrates of HMG CoA reductase (13). In the present study the effect of three competitive inhibitors of HMG CoA reductase on the activation of HMG CoA reductase was investigated. Adenosine-2'-monophospho-5' diphosphoribose, a competitive inhibitor for the NADPH binding site, blocked the phosphatase-mediated activation of HMG CoA reductase. By contrast, neither compactin nor mevinolin, competitive inhibitors for the HMG CoA binding site, altered the activation of HMG CoA reductase. Moreover, the HMG CoA-mediated inhibition of the activation of HMG CoA reductase was not blocked even by very high concentrations of either compactin or mevinolin. These observations suggest that HMG CoA can bind to two sites on HMG CoA reductase. One site of HMG CoA binding serves as a catalytic site and is competitively blocked by compactin or mevinolin, and the second binding site is an allosteric site to which only HMG CoA is capable of binding. The binding of HMG CoA to this second site inhibits the activation of HMG CoA reductase by phosphatases. PMID- 3017223 TI - Specific binding of 24R,25-dihydroxyvitamin D3 to chick intestinal mucosa: 24R,25 dihydroxyvitamin D3 is an allosteric effector of 1,25-dihydroxyvitamin D3 binding. AB - We have previously described a significant decrease in the positive cooperativity level and affinity of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] binding to its chick intestinal chromatin receptor induced in vitro by a physiological 10-fold molar excess of (24R)-25-dihydroxyvitamin D3 [24R,25(OH)2D3] [F. Wilhelm and A. W. Norman (1985) Biochem. Biophys. Res. Commun. 126, 496-501]. In this report, we have initiated a comparative study of the binding of 24R,25(OH)2[3H]D3 and 1,25(OH)2[3H]D3 to the the intestinal chromatin fraction obtained from vitamin D replete birds. 24R,25(OH)2[3H]D3 specific binding to this chromatin fraction was characterized by a dissociation constant (Kd) of 34.0 +/- 6.4 nM, a positive cooperativity level (nH) of 1.40 +/- 0.13, and a capacity (Bmax) of 47 +/- 8 fmol/mg protein. The very low relative competitive index (RCI) of 24R,25(OH)2D3 (0.11 +/- 0.03%) for the 1,25(OH)2D3 binding site/receptor, as well as the inability of 1,25(OH)2D3 to displace 24R,25(OH)2D3 from its binding site at a physiological molar ratio of 1:10, strongly suggest the independence of 24R,25(OH)2D3 and 1,25(OH)2D3 binding sites. Stereospecificity of the 24R,25(OH)2D3 binding sites was attested by the displacement of only 45 +/- 6% of 24R,25(OH)2D3 specific binding by equimolar concentrations of 24S,25(OH)2D3. Collectively these results suggest the existence of a binding domain/receptor for 24,25(OH)2D3 in the chick intestine which is independent of the 1,25(OH)2D3 receptor. PMID- 3017226 TI - [X-ray diagnosis of scirrhous carcinoma of the stomach]. AB - To find a scirrhous carcinoma in its early stage, X-ray examination should pay more effort to detect a small erosion or II c like depressed lesion located in the fundic gland mucosa. In addition, radiographic signs of a malignant relief and a localized resistance to the gastric expansion at double contrast technique are also important. Three typical cases are illustrated. PMID- 3017227 TI - [Surgical treatment of scirrhous carcinoma of the stomach]. AB - In surgical treatment of scirrhous gastric carcinoma, the important points are as follows: Extensive resection of the stomach should be performed. Left epigastric evisceration and extensive dissection of lymph nodes including the para-aortic nodes should be done for sufficient extirpation of the tissues around the stomach. Adjuvant therapy is recommended for intensive chemotherapy immediately after surgery and for postoperative long-term maintenance therapy. Development of a combined therapy which takes into account the characteristics of scirrhous gastric cancer is hoped for in the future. PMID- 3017225 TI - [Present diagnostic status of scirrhous type gastric cancer--with special reference to the endoscopic diagnosis]. AB - First, we presented an actual diagnostic situation in nowadays for gastric cancer of Borrmann 4, which is virtually the same as scirrhous gastric cancer. Among 12 patients treated by the author, all of whom were discovered late, only 3 underwent surgery. In fact, with inoperable cases in Borrmann 4, even those endoscopically found to show insufficient stretching of the gastric wall, thickening and tortuosity of folds, uneven gastric mucosa, redness and white coating, there may be negative in gastric biopsy. However, the significance of an endoscopic examination for diagnosis of scirrhous cancer is in obtaining proof of the carcinoma (especially when still operable) by gastric biopsy. Thus, one must strive not to overlook slight redness, white coating which means small erosions, but to go over gastric biopsy again and again. Next, with carcinoma presenting a leather bottle (linitis plastica type) of the stomach itself, the II c portion of the stomach consisted of fundic glands (undifferentiated carcinoma) shall become the primary focus supporting Nakamura's theory. One case of diffuse invasive cancer, mistakenly diagnosed as a II c case, and two cases of regional type, one similar to II c and the other a Borrmann 2 carcinoma of advanced carcinoma showing strongly fibrous scirrhous tendency toward infiltration, were jointly monitored. PMID- 3017224 TI - The identification and characterization of two cyclic nucleotide phosphodiesterases from bovine adrenal medulla. AB - Two soluble cyclic nucleotide phosphodiesterase activities, designated Peak I (Mr = 216,000) and Peak II (Mr = 230,000), have been isolated from bovine adrenal medulla by DEAE-cellulose chromatography. Peak I has Ca2+-independent, cGMP specific phosphodiesterase activity and Peak II has cGMP-stimulated cyclic nucleotide phosphodiesterase activity. Peak I hydrolyzes cGMP with hyperbolic kinetics and demonstrates a Km of 23 microM. Peak II hydrolyzes cGMP with hyperbolic kinetics but hydrolyzes cAMP with slightly sigmoidal kinetics and demonstrates Km values of 54 +/- 0.7 microM cGMP and 38 +/- 6 microM cAMP. Cyclic AMP and cGMP are competitive inhibitors of each other's hydrolysis, suggesting that these nucleotides may be hydrolyzed at the same catalytic site. Micromolar concentrations of cGMP cause a 5-fold stimulation of the hydrolysis of subsaturating concentrations of cAMP by the Peak II phosphodiesterase. Half maximal activation occurs at 0.5 microM cGMP and the result of activation is a decrease in the apparent Km for cAMP. Stimulation of the hydrolysis of subsaturating concentrations of cGMP by cAMP was also detected; however, cAMP is a less potent activator of the enzyme than cGMP. Cyclic AMP causes a 1.5-fold stimulation of cGMP hydrolysis and half-maximal activation occurs at 2.5 microM cAMP. PMID- 3017229 TI - [Endocrine therapy of scirrhous carcinoma of the stomach]. AB - Tissue localization of endogenous estrogens (estrone; E1, estradiol; E2, estriol; E3) progesterone was examined in scirrhous type of gastric cancer by the peroxidase-antiperoxidase (PAP) immunoperoxidase method. Twelve of 47 specimens showed positive estrogen staining, and 4 of 47 showed positive progesterone staining. The incidence of positive staining for these 4 substances was higher in male patients than in female patients. The number of specimens with positive E2 staining was the highest among E1, E2 and E3 staining. Prognosis of patients who had undergone curative surgery with positive estrogen staining were better than that of patients with negative estrogen staining. The existence of estrogen receptor (ER) and progesterone receptor (PgR) was also shown in 3 specimens and one specimen, respectively, from another 15 specimens with scirrhous type of gastric cancer by the dextran-coated charcoal assay. In a clinical test, Tamoxifen administered patients with scirrhous carcinoma following curative or noncurative gastrectomy showed better prognosis than non-Tamoxifen administered those. PMID- 3017228 TI - [Chemotherapy of Borrmann type 4 gastric cancer]. AB - In brief, the clinical course of patients with Borrmann type 4 gastric cancer is characterized by its marked tendency for peritoneal metastasis (P-factor). This paper dealt with the treatment of Borrmann type 4 gastric cancer from the viewpoint of the disease P-factor, and included the following topics; UFTM chemotherapy, pharmacokinetics of mitomycin C (i.v. vs i.p. use), case presentation, Port-A-catheter (implantable drug delivery system), suppression of collagen biosynthesis, prevention of the adverse effects of mitomycin C, neochemotherapy, and autopsy findings. PMID- 3017231 TI - [Analysis of the TNF receptor in KYM cells by affinity cross-linking]. AB - We investigated the identity of the TNF receptor on the KYM cell membrane by cross-linking 125I-TNF and the presumed receptor site with DSS, and subjecting the TNF-receptor complex to electrophoresis. Four specific bands were observed at 145 K, 50 K, 35 K, and 17 K, and those at 50 K, 35 K and 17 K being consistent with trimers, dimers and monomers of TNF, respectively. The 145 K band disappeared after addition of excess unlabelled TNF or anti-human recombinant TNF monoclonal antibody (IV3-E), which quenched the cytotoxic activity of TNF and inhibited the TNF binding to the receptor. The molecular weight of native TNF as estimated by gel filtration was 45 K and this observation showed that native TNF existed only as the TNF trimer. These results confirmed that 95K, i.e., the difference between 145 K and 50 K, is the molecular size of the TNF receptor. PMID- 3017230 TI - [Combination chemotherapy of cis-diamminedichloroplatinum (CDDP) and 5 fluorouracil (5-FU) in gastrointestinal tumors]. AB - Twenty-five patients with gastrointestinal tumors (stomach 13, colon 8, pancreas 2, liver 2) were treated with a combination chemotherapy regimen consisting of CDDP (30 mg/m2/day d 1, 2) and 5-FU (500 mg/m2/day d 1-3), repeated every 3 or 4 weeks. The patients comprised 14 males and 11 females with a median age of 50 years (range 24-69), and a median performance status of 80% (range 40-100%). Thirteen patients had had prior chemotherapy. Partial response was observed in 2 patients (colon and liver), which lasted for 2 months each, respectively. No objective response was observed in 11 patients evaluable for gastric cancer. Non hematological toxicities were nausea (92%), vomiting (56%), proteinuria (17%), transient elevation of BUN (8%), and hepatotoxicity (11%). Leukopenia and thrombocytopenia were observed in 71% and 25%, respectively. However, these toxicities were mild to moderate, and generally well tolerated. PMID- 3017233 TI - [Availability of the carbohydrate antigen (sialylated Lewis) as a tumor marker in pleural effusion]. PMID- 3017232 TI - [Analysis of TNF receptor by binding assay]. AB - The existence of a TNF receptor on TNF-sensitive tumor cells and on certain normal cells was elucidated by specific binding assay. A close correspondence (r = 0.855) was shown between the receptor number and the sensitivity of the tumor cells. However, for normal cells, despite the existence of TNF receptors, no cytotoxic effect was observed. Furthermore, certain normal diploid cells underwent proliferation as a result of TNF stimulation. It was therefore concluded that the existence of TNF receptor is essential but not sufficient in itself for TNF-induced cytotoxicity. PMID- 3017234 TI - Solitary Langerhans cell histiocytoma. AB - Biopsy of a solitary tumor of the buttock in a 3-month-old girl was diagnosed as a histiocytic proliferation suggestive of histiocytosis X. Electron microscopy showed Birbeck granules and dense myelinlike bodies within the cytoplasm of the tumor cells. An immunoperoxidase study, using a panel of monoclonal antibodies (OKT6, OKT4, and OKM1) and a polyclonal anti-S100 protein antibody, showed positive staining for OKT6 and OM1 and moderately positive staining for OKT4 and S100. After surgery to remove the tumor, visceral involvement could not be demonstrated during a 20-month follow-up. PMID- 3017236 TI - Suspected rotavirus encephalitis. AB - A 9 month old boy with suspected rotavirus encephalitis developed infantile spasms and delayed psychomotor development. Anti-rotavirus antibodies in cerebrospinal fluid, blood, and faeces were studied. PMID- 3017235 TI - Disturbed calcium and phosphate homeostasis during treatment with ACTH of infantile spasms. AB - Kidney histology of five infants who died during or immediately after treatment with adrenocorticotrophic hormone (ACTH) showed severe tubular and interstitial calcinosis. We therefore studied serum concentrations of calcium, inorganic phosphate, and parathormone, serum activities of alkaline phosphatase, and urinary excretion of calcium, inorganic phosphate, and cyclic adenosine monophosphate (cAMP) in 16 other children with infantile spasms before, during, and after 6 weeks of treatment with ACTH. During the treatment the following observations were made: hypocalcaemia developed in three infants; the mean daily urinary excretion of calcium in the group increased threefold and seven infants had hypercalciuria; the excretion of phosphate increased but its tubular reabsorption remained stable; and in most infants serum parathormone and urinary cAMP excretion increased, and in four infants they increased to supranormal concentrations. These biochemical changes were reversible in most infants. Radiographs suggested loss of bone mass by 3-4 weeks of treatment, with rapid recovery after treatment. We conclude that infants treated with ACTH for infantile spasms are at risk of suffering disturbance in calcium and phosphate homeostasis, which leads to nephrocalcinosis. PMID- 3017237 TI - Clinical features of acute gastroenteritis associated with rotavirus, enteric adenoviruses, and bacteria. AB - In a prospective one year study, comprising children with acute gastroenteritis admitted to hospital or treated as outpatients, the clinical and laboratory features of rotavirus diarrhoea (168 cases) were compared with those of enteric adenovirus (32 cases), bacterial (42), mixed (16), and non-specific (135) infections. The rotavirus disease was remarkably consistent, with a sudden onset of vomiting, a high frequency of fever and dehydration, and a mean duration of diarrhoea of 5.9 days. Outpatients excreting rotavirus had a similar but milder illness, mainly on account of less pronounced vomiting. The predominant symptom of enteric adenoviruses was long lasting diarrhoea (mean 10.8 days). Abdominal pain, bloody stools, prolonged diarrhoea (mean 14.1 days), leucocytosis, and a raised erythrocyte sedimentation rate strongly suggested a bacterial aetiology. Mixed infections caused longer lasting diarrhoea (mean 8.0 days) than rotavirus alone, but the severity of the illness was not increased. The clinical features of infection with unidentified pathogens most resembled those of bacterial infections. Respiratory symptoms were not significantly associated with any particular pathogen. Hypernatraemia and complications were uncommon. This study showed that the clinical features of gastroenteritis with rotavirus, enteric adenoviruses, and bacteria each exhibited patterns that could guide the experienced clinician to a presumptive diagnosis. PMID- 3017239 TI - Adrenocortical hyporesponsiveness after treatment with ACTH of infantile spasms. AB - The hypothalamic-pituitary-adrenocortical axis was studied in 10 infants before and during a six week period of treatment with adrenocorticotrophic hormone (ACTH) and three days and one and two weeks after its stopping. During the treatment 24 hour urinary cortisol excretion increased 20 to 350-fold (mean 100) above the basal value. Mean morning serum cortisol concentration, measured 24 hours after the preceding ACTH dose, did not increase. After the treatment mean urinary cortisol excretion was subnormal and mean morning serum cortisol concentration was below the pretreatment value. The mean serum cortisol response to a vasopressin test was reduced and shortened throughout the post-treatment observation period. The mean serum cortisol response to an intravenous ACTH test was not significantly different from the pretreatment response three days after treatment but was clearly reduced thereafter. At one and two weeks after treatment the basal concentrations of serum cortisol of one third of the patients and the post-ACTH concentrations of two thirds were subnormal. We conclude that in infants treatment with ACTH may cause adrenocortical hyporesponsiveness. PMID- 3017238 TI - Persistent protein losing enteropathy in post measles diarrhoea. AB - Faecal alpha 1 antitrypsin was measured in two groups of children with diarrhoea aged 6 months to 6 years during the acute and recovery stages of the illness. Group 1 comprised 19 children with a history of measles in the two weeks preceding admission to hospital. In this group there were six cases of Shigella species, six enterotoxigenic Escherichia coli, and five rotavirus, and two did not yield an aetiologic agent. Group 2 comprised 15 children with diarrhoea only. In this group there were five cases of Shigella species, five enterotoxigenic Escherichia coli, and five rotavirus. Children with rotavirus diarrhoea belonging to both groups showed a transient high faecal clearance of alpha 1 antitrypsin during the acute stage. Post measles cases of diarrhoea showed significantly higher faecal clearance of alpha 1 antitrypsin than group 2 subjects in both the acute and recovery stages. The faecal clearance of alpha 1 antitrypsin in both groups was significantly higher during the acute stage compared with the recovery stage. Highest faecal clearances of alpha 1 antitrypsin were observed in children with post measles shigellosis in the acute stage and they also had persistently raised concentrations, thus suggesting prolonged protein losing enteropathy. PMID- 3017240 TI - Hereditary multiple glomus tumours. AB - A 9 year old girl presenting with multiple glomus tumours is reported. Multiple glomus tumours are rare, often asymptomatic lesions, with a familial tendency and found in a more proximal location than their solitary counterparts. In our report four close relatives had similar lesions. Indications for surgical excision are discussed. PMID- 3017241 TI - The chemokinetic response of psoriatic and normal polymorphonuclear leukocytes to arachidonic acid lipoxygenase products. AB - Polymorphonuclear leukocytes (PMN) from ten patients with chronic stable plaque psoriasis, five of whom had more than 40% skin involvement and five with less than 20% involvement, responded in a dose-related fashion to stimulation with the arachidonic acid lipoxygenase products 5- and 12-hydroxyeicosatetraenoic acid (5- and 12-HETE) and leukotriene B4 (LTB4) in an in vitro chemokinesis assay. There was no significant difference in either the random migration or the chemokinetic response of psoriatic PMN to LTB4 when compared to the responses of PMN from a group of age- and sex-matched healthy controls. Psoriatic PMN migrated further in response to low doses of 5- and 12-HETE although the distance moved after maximal stimulation was no different to that observed in controls. No significant difference was observed in the responses of PMN obtained from patients with less than 20% skin involvement when compared to those with more extensive psoriasis. The small differences measured between the chemokinetic responses of psoriatic and control PMN to the lipoxygenase products tested are unlikely to be of pathogenetic significance. PMID- 3017242 TI - Evaluation of retinoids as inhibitors of [3H] all-trans retinoic acid binding to cellular retinoic acid-binding protein in rat skin and testes. AB - These experiments were designed to test the ability of certain analogs and metabolites of all-trans-retinoic acid (RA), 13-cis-retinoic acid, 4-hydroxy-all trans-retinoic acid, 4-keto-all-trans-retinoic acid, trimethylmethoxyphenol (TMMP) analog of retinoic acid, and TMMP analog of ethyl retinoate (etretinate) to compete for cellular retinoic acid-binding protein (CRABP) in skin and testes of rats. All retinoids, except etretinate, bind to CRABP in a competitive manner with a similar affinity (approximately 5 X 10(-9) M for skin and 3 X 10(-9) M for testes). In contrast, etretinate binds in a noncompetitive manner with a much lower affinity (7.7 X 10(-5) M for skin and 7.5 X 10(-5) M for testes). The values (microM) of IC50 for CRABP from rat skin are 0.43, 0.41, 0.95, 0.83, and 77.4 and those from rat testes are 0.59, 1.29, 2.25, 2.30, and 75.25 for all trans-RA, 13-cis-RA, 4-hydroxy-all-trans RA, 4-keto-all-trans-RA, TMMP analog of RA, and etretinate, respectively. Etretinate is a potent retinoid that is used in the treatment of psoriasis. The lack of quantitative correlation between IC50 and the biological activity of etretinate may be explained in that the active form of etretinate in the body may be the carboxylic acid form (TMMP analog of RA) which binds to CRABP with higher affinity. PMID- 3017243 TI - Human papillomavirus (HPV) DNA sequences demonstrated by in situ DNA hybridization in serial paraffin-embedded cervical biopsies. AB - An in situ DNA hybridization technique was used to identify various types of Human papillomavirus (HPV) in paraffin sections of serial punch biopsies taken from 64 patients having colposcopy because of abnormal smears. There women were in fact 64 consecutive patients out of 505 attending our clinic (at 6-month intervals) since 1981 for HPV infections. HPV 6 DNA sequences were found in 20%, HPV 11 in 17%, HPV 16 in 8% and HPV 18 in 5% of the 64 biopsies analysed with this method so far. More than 60% of HPV 6-positive lesions belong to HPV-NCIN (HPV lesion without concomitant CIN) or HPV-CIN I categories, as contrasted with HPV 16-positive lesions, 80% of which belong to HPV-CIN II and III categories. None of the HPV 16- or HPV 18-infected lesions regressed, as contrasted with 23% and 45% in those infected with HPV 6 and HPV 11, respectively (P less than 0.01). The rate of progression (38.4% and 45.5%, respectively) was markedly lower in HPV 6- and HPV 11 lesions as compared with that (80%) of HPV 16 lesions. The present results while supporting the concept on HPV 16 and HPV 18 as the high risk HPV types in cervical carcinogenesis also emphasize the applicability of the in situ DNA hybridization as a powerful tool in analysis of the specific HPV DNA sequences in routinely progressed biopsies of these lesions. PMID- 3017244 TI - Cytotoxic mechanisms in vitro against Epstein-Barr virus infected lymphoblastoid cell lines in rheumatoid arthritis. AB - Impaired regulation of latent infection with Epstein-Barr virus (EBV) may contribute to the pathogenesis of rheumatoid arthritis (RA) by allowing uncontrolled polyclonal B cell activation. The control of EBV infection in vitro is dependent on several cytotoxic lymphoid cell populations. The present report examines the suppression of early lymphoblastoid outgrowth by natural killer (NK) like cells and the ability to form cytotoxic T lymphocytes (CTLs) specific for EBV in vitro. The latter was measured by a regression assay of EBV induced lymphoblastoid transformation. In this assay the regression of B cell outgrowth at four and six weeks is due to the generation of CTLs specific for EBV. Patients with RA were defective in this ability to generate CTLs. Eight out of nine patients with RA had a geometric mean at the 50% regression end point equal to or greater than 20 X 10(5) cells/ml. In contrast, the geometric mean for all control donors was less than 4 X 10(5) cell/ml. NK activity was measured by a conventional 51Cr release assay with K562 targets. Patients with RA did not have significantly different activity from that of controls (RA patients, n = 4, 45.6 +/- 19.7% (means +/- SD) at 50:1, effector:target; normals, n = 5, 56.6 +/- 5.7%). No spontaneous NK activity was detected against allogeneic or autologous EBV infected B cell targets. When peripheral mononuclear cells from patients were incubated for six days with interleukin-2, lysis of EBV infected targets was seen. No difference in this activity was seen between RA and control studies. Overall, these studies show that patients with RA are defective in their ability to generate CTLs specific for EBV in vitro. PMID- 3017245 TI - Defective Epstein-Barr virus specific suppressor T cell function in progressive systemic sclerosis. AB - Several immunoregulatory defects of Epstein-Barr virus (EBV) induced B cell activation have been described in patients with rheumatoid arthritis (RA), suggesting that EBV may have a role in the pathogenesis of RA. We assessed EBV specific T cell regulation in 20 patients with progressive systemic sclerosis (PSS) and immune to EBV and in 10 control subjects also immune to EBV by comparing the secretion of IgM into supernatants of 16 day cultures of B cells alone and cocultures of B and autologous T cells. In control subjects autologous T cells mediated a significant decrease in the secretion of IgM by B cells at 12 and 16 days of culture. Analysis of individual responses showed the existence of two subgroups of patients with PSS: group I (10 patients) had a suppressor T cell function similar to that of controls; group II (10 patients) had a defective T cell function. Differences in the duration or severity of the disease, the slow acting therapeutic agents, and anti-inflammatory drugs could not account for these subdivisions. These results suggest that several immunoregulatory defects of EBV induced B cell activation exist in different connective tissue diseases. PMID- 3017247 TI - Closure of the urinary bladder with stainless steel and absorbable staples. AB - Stapling is widely accepted in the field of pulmonary and gastrointestinal surgery. However, this has not been the case in urinary tract surgery, presumably because of the possibility of stone formation on the staples. In addressing the issue, both stainless steel staples and absorbable staples (PolysorbTM) were evaluated in 104 linear stapled closures of dog bladders. The mucosa to mucosa closures were performed in bladders with sterile, acutely infected, and chronically infected urine. Staple lines were resected and examined both grossly and microscopically at periods from 1 week to 4 months after the closures. No animal suffered a clinical leak or abscess formation. Four steel closures developed exposed staples, one of which developed a small amount of crystal formation. Twenty-nine absorbable closures contained exposed staples in which two closures developed crystal formation. All closures were secure and healed without difficulty. It appears that closure of the bladder with stainless or absorbable staples is safe and effective. PMID- 3017246 TI - Patterns of radiographic abnormalities associated with basic calcium phosphate and calcium pyrophosphate dihydrate crystal deposition in the knee. AB - Radiographs and synovial fluids from 66 knees representing 59 patients with symptomatic osteoarthritis were evaluated to determine the pattern of radiographic abnormalities associated with basic calcium phosphate (BCP), calcium pyrophosphate dihydrate (CPPD), or both crystals together. Crystals were found in 71% of fluids. In general, CPPD crystals correlated with patient age, while BCP crystals correlated with joint degeneration. Synovial fluid BCP and CPPD crystals were found together more often than either alone. Joint compartment narrowing and osteophytes in three compartments are often associated with BCP crystals. PMID- 3017249 TI - Alterations of the functional properties of the parallel fibers in the cerebellum of the "scrapie mouse". AB - Alterations of the functional properties of the cerebellar parallel fibers have been investigated in scrapie mice by recording the compound action potential elicited by the superficial stimulation of the cerebellum. As soon as the 12th week following intracerebral inoculation, i.e. during the silent incubation period, there is a clear decrease of the response amplitude and of the recruiting properties. Depth profiles of the response indicate the progressive disappearance of the most superficial fibers. With respect to the remaining parallel fibers, the conduction velocity becomes progressively reduced, the duration of the refractory periods increases and the hyperexcitability and increased velocity characterizing the supernormal period are greatly reduced. Most of these electrophysiological alterations can be related to changes of the membrane properties and/or modifications of the metabolic processes ensuring the active ionic transport across the membrane. PMID- 3017248 TI - Novafil. A dynamic suture for wound closure. AB - Abdominal wound dehiscence was quantitatively studied in a rat model. Polybutester suture is a new monofilament nonabsorbable suture that has unique stress-strain properties that are potentially beneficial for abdominal wound closure. The abdominal volume at the moment of wound dehiscence was correlated with the extensibility of the suture material used for closure. Interrupted sutures of polybutester cut through the tissues at a mean abdominal volume of 212 +/- 3 ml. This volume was significantly (p less than or equal to 0.005) greater than the mean volumes reached with nylon (197 +/- 3 ml) or polyglycolic acid (187 +/- 4 ml). Closure of abdomens with continuous polybutester suture resulted in a mean rupture volume of 218 +/- 3 ml, which was significantly (p less than or equal to 0.005) greater than that achieved with the same suture employed as simple interrupted sutures (212 +/- 4 ml). The influence of width of tissue bite, suture size, and needle configuration was also evaluated. PMID- 3017250 TI - NAD kinase from Bacillus licheniformis: inhibition by NADP and other properties. AB - NAD kinase was purified 180-fold from Bacillus licheniformis to determine the role it plays in NADP turnover in this organism. The enzyme was found to have a pH optimum of 6.8 and an apparent Km for NAD of 2.7 mM. The ATP saturation curve was not hyperbolic; 5.5 mM ATP was required to reach half maximal activity. Both Mn2+ and Ca2+ could be substituted for Mg2+. Several compounds including nicotinic acid, nicotinamide, nicotinamide mononucleotide, quinolinic acid, NADPH, ADP, AMP and cyclic AMP did not affect NAD kinase activity. In contrast, the enzyme was inhibited by NADP at concentrations typically found in logarithmic cells of B. licheniformis. This inhibition was competitive with NAD and had a Ki of 0.13 mM. It is suggested that in vivo NAD kinase activity is highly dependent on the concentrations of NAD and ATP and the proportion of oxidized and reduced NADP. PMID- 3017251 TI - Accumulation of fructose 1,6-bisphosphate in mutant cells of mucoid Pseudomonas aeruginosa as an evidence of phosphofructokinase activity. AB - Phosphoglucose isomerase negative mutant of mucoid Pseudomonas aeruginosa accumulated relatively higher concentration of fructose 1,6-bisphosphate (Fru-1,6 P2) when mannitol induced cells were incubated with this sugar alcohol. Also the toluene-treated cells of fructose 1,6-bisphosphate aldolase negative mutant of this organism produced Fru-1,6-P2 from fructose 6-phosphate in presence of ATP, but not from 6-phosphogluconate. The results together suggested the presence of an ATP-dependent fructose 6-phosphate kinase (EC 2.7.1.11) in mucoid P. aeruginosa. PMID- 3017252 TI - [Leigh syndrome in the adult]. PMID- 3017253 TI - [Principles of hemoblastosis control in cattle using serological study methods]. PMID- 3017254 TI - [Results of leukosis control in the framework of a production experiment in the Gera district]. PMID- 3017256 TI - [Cultivation of enzootic bovine leukosis virus in cell lines]. PMID- 3017255 TI - [Results of enzootic bovine leukosis control in contaminated herds using serologic methods]. PMID- 3017257 TI - [Correlations between enzootic bovine leukosis virus and cellular immune system]. PMID- 3017258 TI - [Peculiarities of antibody dynamics in cattle with spontaneous bovine leukosis virus infection]. PMID- 3017259 TI - [Effects of colostrum antibodies on infectivity of bovine leukosis virus]. PMID- 3017260 TI - [Preliminary study on the presence in milk of antibodies against bovine leukemia virus]. PMID- 3017261 TI - [Experimental infection of lambs with enzootic bovine leukosis virus]. PMID- 3017262 TI - [Intrauterine infection and its significance in spreading of enzootic bovine leukosis as well as in taking appropriate measures of its control]. PMID- 3017263 TI - [Development of solid-phase enzyme immunoassay for the diagnosis of enzootic bovine leukosis]. PMID- 3017265 TI - [Role of saliva in transmission of bovine leukosis virus]. PMID- 3017264 TI - [Effects of different cultivation conditions on expression of bovine leukosis virus in permanently infected cell lines of fetal lamb kidneys]. PMID- 3017266 TI - [Occurrence of feline leukemia in Kosice]. PMID- 3017267 TI - [Immunological aspects of Marek's disease in poultry]. PMID- 3017268 TI - [Molecular modeling of the structure of barbiturate receptors]. PMID- 3017269 TI - Congenital microarteriovenous shunts. Angiographic and Doppler ultrasonographic identification. AB - We describe herein two cases of vascular malformations, one classified as hemangioma and the other as Klippel-Trenaunay syndrome. Clinical investigation in each case failed to demonstrate the presence of arteriovenous (AV) shunting. Arteriographic findings revealed only indirect evidence of AV shunting in each case. In contrast, systematic scanning with a Doppler ultrasonographic probe of the involved extremities provided evidence of AV shunting and pinpointed it in suspected arteriographic areas. Good correlation between the two methods was confirmed in the hemangioma case both preoperatively and intraoperatively. In the case of Klippel-Trenaunay syndrome, evidence of multiple AV shunts was obtained primarily with Doppler ultrasonography. In addition to arteriography, serial phlebography, when indicated, is also necessary for complete evaluation of concomitant venous malformations. The pathogenic mechanism of these vascular malformations was briefly reviewed, emphasizing AV shunting as a common link between the various anatomicoclinical forms. PMID- 3017270 TI - Utilization of NPN-supplements, other than urea, by ruminants. PMID- 3017272 TI - [Ultrastructural changes in the autonomic interneuronal synapse during activation in the presence of acetylcholinesterase suppression]. AB - Structural-functional reconstructions of the frog autonomic interneuronal synapsis have been studied at its activation with endogenic acetylcholine under conditions of acetylcholinesterase suppression. The investigation has been performed with preparations of isolated sympathetic trunk of Rana temporaria treated with armine (5 X 10(-6) M) and subjected to electrostimulation (5 imp/sec) up to a complete block of the synaptic transmission. Certain structural changes are revealed in the axo-somatic synapses, demonstrating an increased adhesive properties of the membranes, ("hatch-like" membranes, numerous submembranous aggregates, aggregates of the intercellular cleft and neuronal glial contacts). In the terminals changed according to the "light type", with poorly manifested changes, light synaptic vesicles loose their spheric form, their diameter decreases. In the boutons with more intensive changes, the vesicles gradually change into the mass of cluster-floccular material. In the boutons with intensively manifested disorders in the ultrastructure, a complete destruction of the light vesicles is observed. The great part of the ganglionic neuron bodies changes according the "dark type". In their neuroplasm a great amount of subsuperficial cisterns of the endoplasmic reticulum and formation of powerful fasciculi of microfilaments are noted to appear. PMID- 3017271 TI - The effects of treated straw meal on ileal and faecal digestibility of nutrients in pigs. AB - Four 40 kg castrated male pigs fitted with simple 'T' cannulas in the terminal ileum were given diets of varying crude fibre content in a change-over experiment with two periods. The basal diet was composed of wheat and fishmeal supplemented with minerals and vitamins. To this was added varying levels of partially hydrolysed straw meal to give crude fibre contents ranging from 40 to 132 g/kg. After adaptation to the particular levels of straw meal, faeces and ileal digesta were collected during successive 24 h periods. Nutrient digestibility values were determined by the chromic oxide ratio method. The addition of treated straw meal to the diet had little or no influence on the DM content of digesta or faeces. The excretion of N in faeces increased with increasing fibre intake but there was no effect on urine N excretion. The overall apparent digestibility of N was reduced from 89 to 79% as crude fibre intake increased from 40 to 132 g/kg but ileal apparent digestibility of N remained constant at about 68%, suggesting that the effect was mediated through hindgut bacteria. Increased fibre intake caused increased net secretion of Na in the small intestine and reduced the apparent absorption of P in the large intestine. PMID- 3017273 TI - [Protective action of serotonin on changes in the ultrastructure of the Retzius neuron induced by acetylcholine]. AB - A combined action of acetylcholine and serotonin is demonstrated to produce, in ultrastructure of the Retzius neuron of the leech, changes similar to those resulted from synaptic activation. Nevertheless, acetylcholine alone produces much deeper morphological shifts. A conclusion is made that serotonin not only retards impulse activity of the neuron, but it "slows down" development of rather great changes in its ultrastructure. PMID- 3017274 TI - Calcium carbonate precipitation in bicarbonate hemodialysis. AB - Calcium carbonate has been observed to precipitate in the fluid pathway of dialysate delivery systems dispensing bicarbonate-containing dialysates. Such precipitation can occlude the fluid pathway, leading to system malfunction and increased maintenance requirements. We show that commercial supplies of sodium bicarbonate are contaminated by trace amounts of sodium carbonate. This contamination may result in immediate precipitation of calcium carbonate on formulation of the dialysate, since bicarbonate-containing dialysates, as formulated, are metastable with respect to calcium carbonate. Sparging of the bicarbonate-containing concentrate with carbon dioxide converts any carbonate to bicarbonate, thus avoiding the formation of precipitates on addition of calcium ions. PMID- 3017275 TI - Accelerated cytomegalovirus retinitis secondary to immunosuppressive therapy. PMID- 3017276 TI - Angiotensin-converting enzyme (ACE) measurement in human serum using radioinhibitor ligand binding. AB - MK351A, a tyrosyl analogue of enalaprilic acid (MK422) is a potent inhibitor of angiotensin-converting enzyme (ACE). MK351A was radioiodinated with 125I and used to develop a radio inhibitor binding assay for human serum ACE. 125I MK351A associated rapidly and reversibly with human serum ACE (T 1/2 = 1/2 h). Bound 125I MK351A was displaced by an excess of cold MK351A, to give non-specific binding of less than 1%. Scatchard analysis of binding was linear (r = -0.99, n = 6, p less than 0.001), indicating a single class of binding site. ACE was estimated in serum from normal patients and patients with sarcoid by the radio inhibitor binding assay and by enzyme kinetic assay using Hip-His-Leu as substrate. The two methods for ACE estimation correlated closely (r = 0.87, n = 82, p less than 0.001). The radio inhibitor binding assay for human serum ACE is a simple, sensitive and specific assay which utilizes novel assay methodology. PMID- 3017277 TI - The production of hydroxyl radical by human neutrophils stimulated by arachidonic acid--measurements by ESR spectroscopy. AB - Human neutrophils incubated with sodium arachidonate generated hydroxyl radicals. The radical formed an adduct with the spin trap 5', 5-dimethyl-l-pyrroline-N oxide (DMPO) and was subsequently detected by electron spin resonance (ESR) spectroscopy. The ESR signal was inhibited by mannitol and superoxide dismutase but not by catalase. Removal of glucose from the reaction mixture or the presence of glucose metabolic inhibitors including 2-deoxy-D-glucose and 3-O-methyl-D glucose did not affect the production of hydroxyl radical by the neutrophils. The ESR signal was, however, inhibited by the lipoxygenase inhibitors nordihydroguaiaretic acid and N-ethylmaleimide. The involvement of lipoxygenase in the production of hydroxyl radical was demonstrated by the trapping of the radical with DMPO in a reaction mixture of soybean lipoxygenase and arachidonic acid (AA). These findings support our previous postulation that the metabolism of AA via the lipoxygenase pathway is a source of hydroxyl radical in stimulated neutrophils. PMID- 3017278 TI - Genetic analysis of Kunjin virus isolates using HaeIII and TaqI restriction digests of single-stranded cDNA to virion RNA. AB - The genotypic relatedness of 15 Kunjin (KUN) virus isolates from widely separated geographic regions in Australia was examined at the molecular level. HaeIII and TaqI restriction digest profiles of cDNA transcribed from virion RNA revealed a close genetic similarity between all isolates. We estimate that the nucleotide sequence divergence between any pair of KUN isolates is probably less than 1%. We conclude that a single KUN genetic type has existed in enzootic and epizootic areas of virus activity over an extended time span. The epidemiological implications of these results are discussed. PMID- 3017279 TI - An investigation of the potential of Aedes camptorhynchus (Thom.) as a vector of Ross River virus. AB - Aedes camptorhynchus (Thom.) collected on the mid-south coast of New South Wales during the winter of 1982 were highly susceptible to infection (ID50 = 10(2.4) VERO pfu/mosquito) when fed on rat tail skins containing blood and serial dilutions of the T48 strain of Ross River (RR) virus. After 2 d, when no virus was detectable, rapid proliferation allowed transmission from 5 d post ingestion. A maximum transmission rate occurred 9 d post-feeding when 4 of 4 infected mosquitoes transmitted virus. The susceptibility of Ae camptorhynchus to RR virus infection was compared with that of a laboratory colony of Ae aegypti (L.) (ID50 = 10(3.8) VERO pfu/mosquito). PMID- 3017280 TI - Vestibular neuronitis--serum and CSF virus antibody titer. AB - The cerebrospinal fluid (CSF) findings of patients with vestibular neuronitis were virologically evaluated and discussed in contrast to those of herpes zoster. CSF samples obtained from seven patients with vestibular neuronitis, aged 28 to 55 years, were examined. The results were as follows: The CSF protein level in the vestibular neuronitis showed the peculiar change; i.e. its level was normal at the onset period of vertigo, but it rose to abnormal levels mostly in the period of two weeks, while the cell count remained normal throughout all phases of our study. Herpes simplex virus (HSV) type 1 IgG antibody titers measured by indirect immunofluorescent antibody technique (IF) in paired sera rose in one of the seven cases of vestibular neuronitis, but the antibody titers of the same virus in the CSF were not detected. HSV type 1 IgG antibody titers measured by IF in the CSF were detected in two of seven cases of vestibular neuronitis, but not significant. The ratio of EB virus (EBV) capsid antigen IgG antibody titers in CSF to that in serum ranged from 1:160 to 1:80 in vestibular neuronitis. There was no direct available evidence that vestibular neuronitis caused a break in blood-CSF barrier, an increase in IgG synthesis in the central nervous system or active infection with HSV, varicella zoster virus (VZV), or EBV. In this paper, we summarized the recent information on studies of the CSF and a latent herpes virus infection in order to give perspective to the pathogenesis of vestibular neuronitis. PMID- 3017281 TI - Reproductive failure in pigs caused by encephalomyocarditis virus. PMID- 3017282 TI - The detection of antibody to avian infectious bronchitis virus by use of immunofluorescence with tissue sections of nephritic kidneys. PMID- 3017284 TI - DNA-protein cross-links: new insights into their formation and repair in irradiated mammalian cells. PMID- 3017283 TI - Cell kinetics of human epithelial ovarian cancers. AB - Tumor cellular proliferative activity was evaluated by 3H-thymidine labeling index (LI) determination in 73 lesions from 62 patients with ovarian cancers. The median LI value for the overall series was 5.8% and places this tumor type in a position of intermediate proliferative activity. In this case series, the relationship between proliferative activity and different morphologic and pathologic characteristics of the disease was analyzed. Similar median LI values were found for ascitic effusions and solid tumors and, among these, between primary tumors and metastases. No significant relation was observed between proliferative activity and histologic type, whereas a definite direct correlation was found between the kinetic variable and prognostic features such as pathologic stage and histologic grading. In fact median LI was higher for stage IV for stage I and for grade 3 than for grade 1 tumors. The strong association between tumor proliferative rate and conventional prognostic factors suggests that also on this tumor type the kinetic variable could play a role as a prognostic indicator and modulator of aggressiveness. PMID- 3017285 TI - DNA-drug binding and control of genetic information. PMID- 3017286 TI - Differential expression of SOS genes in an E. coli mutant producing unstable lexA protein enhances excision repair but inhibits mutagenesis. AB - The lexA41 mutant of E. coli is a UV-resistant derivative of another mutant, lexA3, which produces a repressor that is not cleaved following inducing treatments. lexA41 carried an additional mutation which changed amino acid 132 in the LexA protein from Ala to Thr. The resultant protein was unstable and was degraded both before and after an inducing treatment. This instability was greater at 42 degrees than at 30 degrees. The protein was more stable in Lon- mutants at both temperatures. lac operon fusions to most of the genes in the SOS regulon were used to show that the various damage-inducible genes were derepressed to different extents. uvrA, B, and D were almost fully derepressed. Consistant with this finding, the rate of removal of T4 endonuclease V-sensitive sites was more rapid in the UV-irradiated lexA41 mutant than in normal cells, suggesting a more active excision repair system. We propose that the instability of the LexA41 protein reduces the intracellular concentration of repressor to a level that allows a high level of excision repair. The additional observation that SOS mutagenesis was only weakly induced in a lexA41 uvrA- mutant implies that the mutant protein partially represses one or more genes whose products promote SOS mutagenesis. PMID- 3017287 TI - Electron spin resonance studies of the mechanism of radiation damage to DNA. PMID- 3017288 TI - Restoration of DNA repair in UV-sensitive Chinese hamster ovary cell by the denV gene from bacteriophage T4. PMID- 3017290 TI - Free radical mechanisms of DNA base damage. PMID- 3017289 TI - Activation of H-ras-1 oncogenes by chemical carcinogens. PMID- 3017291 TI - Mechanisms of spontaneous mutagenesis: clues from mutational specificity. PMID- 3017292 TI - Peroxyl radicals of nucleic acids and their components. PMID- 3017293 TI - Pathophysiology of superoxide radical as potential mediator of reperfusion injury in pig heart. AB - The role of oxygen-derived free radicals in myocardial reperfusion injury was studied using the isolated in situ pig heart model. The free radical scavengers, superoxide dismutase (SOD) and catalase, protected the ischemic pig heart subjected to one hour of normothermic regional ischemia followed by one hour of global hypothermic arrest and one hour normothermic reperfusion. A significant increase in thiobarbituric acid reactive material and oxidized glutathione appeared in the perfusate demonstrating free radical-mediated lipid peroxidation during reperfusion, and this was prevented by the addition of SOD plus catalase. The values of three important antioxidative enzymes, SOD, catalase, and glutathione peroxidase, showed reduced activities after 2 hours of ischemia. These values did not change significantly after 60 minutes of reperfusion following the 2 hours ischemic insult. The concentrations of high-energy phosphate compounds including creatine phosphate (CP), adenosine triphosphate (ATP), and total adenine nucleotide were reduced significantly during ischemia and reperfusion in hearts which were not protected by SOD and catalase. The plasma creatine phosphokinase levels were lowered appreciably as a result of SOD and catalase treatment. It may be concluded from these experiments that oxygen derived free radicals are present during reperfusion and SOD and catalase play a significant role in the protection of ischemic myocardium from reperfusion injury. PMID- 3017294 TI - Restriction endonuclease mapping of ribosomal RNA genes: sequence divergence and the origin of the tetraploid treefrog Hyla versicolor. AB - Hyla chrysoscelis (2n = 24) and H. versicolor (2n = 48) are a diploid-tetraploid species pair of treefrogs. Restriction endonuclease mapping of ribosomal RNA (rRNA) gene repeat units of diploids collected from eastern and western populations reveals no differences within rRNA gene coding regions but distinctive differences within the nontranscribed spacers. A minimum of two physical maps is required to construct an rRNA gene map for the tetraploid, whose repeat units appear to be a composite, with about 50% of the elements resembling the "western" diploid population and about 50% resembling the "eastern" population. These results imply that this population of the tetraploid species may have arisen from a genetically hybrid diploid. Alternatively, the dual level of sequence heterogeneity in H. versicolor may reflect some type of gene flow between the two species. The coding region of the rRNA genes in the tetraploid differs from that in either diploid in about 20% of all repeat units, as exemplified by a BamHI site located near the 5' terminus of the 28 S rRNA gene. If the 20% variant class of 28 S rRNA gene coding sequences is expressed, then there must be two structural classes of ribosomes; if only the 80% sequence class is expressed, then a genetic control mechanism must be capable of distinguishing between the two different sequence variants. It is postulated that the 20% variant sequence class may be correlated with a partial functional diploidization of rRNA genes in the tetraploid species. PMID- 3017295 TI - Pig mitochondrial DNA: polymorphism, restriction map orientation, and sequence data. AB - Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA) in pigs were analyzed using 18 enzymes which recognize six nucleotides and 1 four nucleotide-recognizing enzyme. Pigs including Taiwan native breeds and miniature strains maintained in Japan were examined in this study; four commercial breeds of pigs and Japanese wild boars have been investigated earlier [Watanabe, T., et al. (1985). Biochem. Genet. 23:105]. mtDNA polymorphisms were observed in the cleavage patterns of five restriction enzymes, Bg1II, EcoRV, ScaI, StuI, and TaqI. The results support the previous hypothesis that pigs must be derived from two different maternal origins, European and Asian wild boars, and that a breed, Large White, arises from both European and Asian pigs. Two HindIII cleavage fragments were cloned into the HindIII site of M13mp10 and were partially sequenced by the dideoxynucleotide-chain termination method. Furthermore, DraI and StuI cleavage sites were newly determined on the restriction endonuclease map. On the basis of these results, the restriction endonuclease cleavage map of pig mtDNA was rewritten. Comparing sequence data of pig mtDNA at 237 positions with those of cow, human, mouse, and rat mtDNA, the sequence difference, silent and replacement changes, and transitions and transversions among mammalian species were estimated. The relationships among them are discussed. PMID- 3017296 TI - Restriction endonuclease map variation in the Adh region in populations of Drosophila melanogaster. AB - Variation within a 12-kb region of the second chromosome of Drosophila melanogaster which includes the Adh transcriptional unit was studied in 59 isochromosomal lines drawn from two populations. A number of restriction-fragment length polymorphisms due to both restriction-site presence/absence and insertions/deletions were revealed and the proportion of polymorphic nucleotides was estimated as 0.017 in population "Chateau Tahbilk" and 0.025 in population "Groningen." The presence within population Groningen of two complementary haplotypes at a high frequency was noted and its possible origin is discussed. The distribution of insertion/deletion variants within the populations was consistent with an associated deleterious effect and significant clustering of such variants to a 1-kb region approximately 4 kb from the transcriptional unit was also revealed. Analysis of gametic disequilibrium showed that its intensity was not related to the physical distance separating the markers on the chromosome and that strong disequilibria may flank weaker disequilibria. Two alternative interpretations of these data are advanced based upon natural selection and the hitchhiking effect of a favorable mutation. Comparisons of gametic disequilibrium among the four populations now surveyed at this locus were made using model fitting by weighted least squares and several significant contrasts were revealed. PMID- 3017298 TI - The action of islet activating protein (pertussis toxin) on insulin's ability to inhibit adenylate cyclase and activate cyclic AMP phosphodiesterases in hepatocytes. AB - Treatment of hepatocytes with islet activating protein (pertussis toxin) from Bordetella pertussis blocked the ability of insulin to inhibit adenylate cyclase activity both in broken plasma membranes and in intact hepatocytes. Such treatment of intact hepatocytes with pertussis toxin did not prevent insulin from activating the peripheral plasma membrane cyclic AMP phosphodiesterase although it did inhibit the ability of insulin to activate the 'dense-vesicle' cyclic AMP phosphodiesterase. The ability of glucagon pretreatment of hepatocytes to block insulin's activation of the plasma membrane cyclic AMP phosphodiesterase was abolished in pertussis toxin-treated hepatocytes. It is suggested that the ability of insulin to manipulate cyclic AMP concentrations by inhibiting adenylate cyclase and activating the plasma membrane and 'dense-vesicle' cyclic AMP phosphodiesterases involves interactions with the guanine nucleotide regulatory protein system occurring in liver plasma membranes. PMID- 3017300 TI - Fractionation and quantification of calcium-dependent proteinase activity from small tissue samples. AB - Almost all mammalian tissues contain a cytoplasmic Ca2+-dependent proteolytic system consisting of two different proteinases (CDP I and CDP II) and an endogenous inhibitor specific for these proteinases. It was difficult to determine the relative activities of CDP I and CDP II directly and accurately without extensive purification, requiring relatively large amounts of tissue. We developed a simple technique based on Reactive Red-agarose affinity chromatography for quantitatively measuring CDP II activities in small (less than 100 mg) tissue samples. This technique is rapid, sensitive and highly reproducible. CDP II activities can be quantified in numerous tissue samples in a single day. Using this method, we analysed the components of the CDP system in various rat tissues and demonstrated quantitative differences in CDP II activities as well as relative differences in the amounts of CDP-inhibitor activity among the various tissues. This technique should prove useful in the efforts to define the currently unknown physiological function(s) of the Ca2+ dependent proteolytic system by allowing comparison of CDP activities in tissues under diverse conditions of protein metabolism. PMID- 3017299 TI - The presence of a Zn2+-dependent acid p-nitrophenyl phosphatase in bovine liver. Isolation and some properties. AB - The presence of a Zn2+-dependent acid p-nitrophenyl phosphatase (EC 3.1.3.2) in bovine liver was described. The enzyme was purified to apparent homogeneity and migrates as a single band during electrophoresis on polyacrylamide gel. The enzyme requires Zn2+ ions for catalytic activity, other bivalent cations have little or no effect. The enzyme, of Mr 118,000, optimum pH 6-6.2 and pI 7.4-7.5, was inhibited by EDTA, tartrate, adenine and ATP, but not by fluoride. The common phosphate esters are poor substrates for the enzyme, which hydrolyses preferentially p-nitrophenyl phosphate and o-carboxyphenyl phosphate. The Zn2+ dependent acid p-nitrophenyl phosphatase of bovine liver was different from the high-Mr acid phosphatases previously detected in mammalian tissues. PMID- 3017297 TI - Protein kinase activity of the insulin receptor. AB - The insulin receptor is an integral membrane glycoprotein (Mr approximately 300,000) composed of two alpha-subunits (Mr approximately 130,000) and two beta subunits (Mr approximately 95,000) linked by disulphide bonds. This oligomeric structure divides the receptor into two functional domains such that alpha subunits bind insulin and beta-subunits possess tyrosine kinase activity. The amino acid sequence deduced from cDNA of the single polypeptide chain precursor of human placental insulin receptor revealed that alpha- and beta-subunits consist of 735 and 620 residues, respectively. The alpha-subunit is hydrophilic, disulphide-bonded, glycosylated and probably extracellular. The beta-subunit consists of a short extracellular region which links the alpha-subunit through disulphide bridges, a hydrophobic transmembrane region and a longer cytoplasmic region which is structurally homologous with other tyrosine kinases like the src oncogene product and EGF receptor kinases. The cellular function of insulin receptors is dual: transmembrane signalling and endocytosis of hormone. The binding of insulin to its receptor on the cell membrane induces transfer of signal from extracellular to cytoplasmic receptor domains leading to activation of cell metabolism and growth. In addition, hormone-receptor complexes are internalized leading to intracellular proteolysis of insulin, whereas receptors are recycled to the membrane. These phenomena are kinetically well-characterized, but their molecular mechanisms remain obscure. Insulin receptor in different tissues and animal species are homologous in their structure and function, but show also significant differences regarding size of alpha-subunits, binding kinetics, insulin specificity and receptor-mediated degradation. We suggest that this heterogeneity of receptors may be linked to the diversity in insulin effects on metabolism and growth in various cell types. The purified insulin receptor phosphorylates its own beta-subunit and exogenous protein and peptide substrates on tyrosine residues, a reaction which is insulin-sensitive, Mn2+-dependent and specific for ATP. Tyrosine phosphorylation of the beta-subunit activates receptor kinase activity, and dephosphorylation with alkaline phosphatase deactivates the kinase. In intact cells or impure receptor preparations, a serine kinase is also activated by insulin. The cellular role of two kinase activities associated with the insulin receptor is not known, but we propose that the tyrosine- and serine specific kinases mediate insulin actions on metabolism and growth either through dual-signalling or sequential pathways.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3017302 TI - Human prostatic acid phosphatase has phosphotyrosyl protein phosphatase activity. AB - The major secreted isoenzyme of human prostatic acid phosphatase (PAcP) (EC 3.1.3.2), which catalyses p-nitrophenyl phosphate (PNPP) hydrolysis at acid pH values, was found to have phosphotyrosyl protein phosphatase activity since it dephosphorylated three different phosphotyrosine-containing protein substrates. Several lines of evidence are presented to show that the phosphotyrosyl phosphatase and PAcP are the same enzyme. A highly purified PAcP enzyme preparation which contains a single N-terminal peptide sequence was used to test for the phosphotyrosyl phosphatase activity. Both activities comigrated during gel filtration by high performance liquid chromatography. Phosphotyrosyl phosphatase activity and PNPP acid phosphatase activity exhibited similar sensitivities to different effectors. Both phosphatase activities showed the same thermal stability. Specific anti-PAcP antibody reacted to the same extent with both phosphatase activities. PNPP acid phosphatase activity was competitively inhibited by the phosphotyrosyl phosphatase substrate. To characterize further the phosphotyrosyl phosphatase activity, the Km values using different phosphoprotein substrates were determined. The apparent Km values for phosphorylated angiotensin II, anti-pp60src immunoglobulin G and casein were in the nM range for phosphotyrosine residues, which was about 50-fold lower than the Km for phosphoserine residues in casein. PMID- 3017301 TI - The inositol phospholipids: a stereochemical view of biological activity. PMID- 3017304 TI - Sulphation of proteins secreted by a human hepatoma-derived cell line. Sulphation of N-linked oligosaccharides on alpha 2HS-glycoprotein. AB - Several human glycoproteins, including alpha 1-antitrypsin, alpha 1-acid glycoprotein, transferrin, caeruloplasmin and alpha 2HS-glycoprotein, synthesized by the hepatoma-derived cell line HepG2 were observed to contain covalently linked sulphate. These proteins were estimated to contain about 0.1 mol of sulphate/mol of protein. The most abundant of the sulphated glycoproteins, alpha 2HS-glycoprotein, was analysed in detail. All of the sulphate on this protein was attached to N-linked oligosaccharides which contained sialic acid and resisted release by endoglycosidase H. Several independent analytical approaches established that approx. 10% of the molecules of alpha 2HS-glycoprotein contained sulphate. Our results suggest that a number of human plasma proteins contain small amounts of sulphate linked to oligosaccharides. PMID- 3017305 TI - Multi-frequency e.p.r. studies of a mercury-containing mixed-metal derivative of laccase. AB - Multi-frequency e.p.r. studies of a derivative of laccase containing one mercury atom and three of copper were carried out at -150 degrees C. The e.p.r. signal of the mercury derivative and fluoride-binding studies establish that a type-2-like copper centre is present. The signal suffers broadening, owing to g-strain, but at low frequencies (S-band) ligand hyperfine splitting can be resolved, and it can be explained in terms of coupling to three nitrogen atoms. The g values and the effect of solvent deuteration on the line width suggest that the fourth ligand in the equatorial plane is a water molecule. Simulations of the e.p.r. spectrum reveal that the site is slightly rhombic at -150 degrees C, a finding in accord with the proposed N3O donor set. Finally, it is emphasized that a structural reorganization of the type-2 copper site occurs with the binding of fluoride at low temperature. The reorganization may be linked to a conformational change which has previously been claimed to occur on cooling; however, this transition is not necessarily relevant to the temperature-dependence of fluoride binding. PMID- 3017303 TI - Homologous desensitization of beta-adrenergic receptors in lymphoma cells is not altered by the inactivation of Ni (Gi), the inhibitory guanine nucleotide regulatory protein. AB - We had previously demonstrated that the cyc- mutant of S49 wild-type lymphoma cells both desensitizes and undergoes a sequestration-internalization of the beta receptor in response to short-term treatment with adrenaline. The cyc- mutant of S49 wild-type lymphoma cells lacks the alpha s subunit of the stimulatory coupling protein Ns, but has fully functional Ni, the inhibitory component of the regulatory complex. This suggested that functional Ns was not required for desensitization. To examine the role of Ni in desensitization, both S49 wild-type and cyc- cells were treated with islet-activating protein under conditions that led to over 85% attenuation of Ni function in S49 wild-type cells and approx. 50% attenuation of Ni function in cyc- cells. This treatment had no effect on the adrenaline-induced desensitization of adenylate cyclase or the sequestration event measured by the apparent movement of beta-adrenergic receptors to a light vesicle fraction. Further, the desensitization event, which occurs before the sequestration event, observable only in intact cells, was also not altered by islet-activating-protein pretreatment of S49 wild-type cells. The data suggest that a functional Ni is not required for desensitization in the S49 lymphoma cells. PMID- 3017306 TI - Nucleotide sequence of the putative regulatory region of mouse lactate dehydrogenase-A gene. AB - The nucleotide sequence of approx. 3 kilobases including the regulatory region, a non-coding exon and the first protein-coding exon from mouse lactate dehydrogenase-A (LDH-A) gene has been determined. The putative initiation sites of transcription and translation were deduced by comparing the nucleotide sequence of mouse LDH-A gene with those of a mouse LDH-A processed pseudogene and the LDH-A full-length cDNAs from rat and human. The tentative TATA and CAAT boxes, and the hexanucleotides CCGCCC have been identified. The sequence of AAATCTTGCTCAA of mouse LDH-A gene has also been found to show striking homology to the cyclic AMP-responsive sequences of eukaryotic genes regulated by cyclic AMP. It has been reported previously that the protein-coding sequence of mouse LDH-A gene is interrupted by six introns and the 3' untranslated sequence of 485 nucleotides is not interrupted [Li, Tiano, Fukasawa, Yagi, Shimiza, Sharief, Nakashima & Pan (1985) Eur. J. Biochem. 149, 215-225]. An additional intron of 1653 base-pairs was found in the 5' untranslated sequence of 101 nucleotides at 24 nucleotides upstream to the translation start site. Thus, mouse LDH-A gene containing seven introns spans approx. 11 kilobases and its length of mature mRNA is 1582 nucleotides, excluding the poly(A) tail. PMID- 3017307 TI - Translational control of insulin biosynthesis. Evidence for regulation of elongation, initiation and signal-recognition-particle-mediated translational arrest by glucose. AB - The biosynthesis of insulin in the islets of Langerhans is strongly controlled at the translational level by glucose. We have used a variety of experimental approaches in efforts to dissect the mechanisms underlying the stimulatory effect of glucose. To assess its effects on rates of peptide-chain elongation, isolated rat islets were labelled with [3H]leucine at different glucose concentrations in the presence or absence of low concentrations of cycloheximide. Under these conditions, at glucose concentrations up to 5.6 mM, endogenous insulin mRNA did not become rate-limiting for the synthesis of insulin, whereas stimulation of non insulin protein synthesis was abolished by cycloheximide at all glucose concentrations, indicating either that insulin synthesis is selectively regulated at the level of elongation at glucose concentrations up to 5.6 mM, or that at these concentrations inactive insulin mRNA is transferred to an actively translating pool. Glucose-induced changes in the intracellular distribution of insulin mRNA in cultured islets were assessed by subcellular fractionation and blot-hybridization using insulin cDNA probes. At glucose concentrations above 3.3 mM, cytoplasmic insulin mRNA was increasingly transferred to fractions co sedimenting with ribosomes, and relatively more of the ribosome-associated insulin mRNA became membrane-associated, consistent with effects of glucose above 3.3 mM on both the initiation of insulin mRNA and SRP (signal recognition particle)-mediated transfer of cytosolic nascent preproinsulin to the endoplasmic reticulum. When freshly isolated islets were homogenized and incubated with 125I Tyr-tRNA, run-off incorporation of 125I into preproinsulin was increased by prior incubation of the islets at 16.7 mM-glucose. The addition of purified SRP receptor increased the run-off incorporation of [125I]iodotyrosine into preproinsulin, especially when the islets had been preincubated at 16.7 mM glucose. These findings taken together suggest that glucose may stimulate elongation rates of nascent preproinsulin at concentrations up to 5.6 mM, stimulates initiation of protein synthesis involving both insulin and non-insulin mRNA at concentrations above 3.3 mM, and increases the transfer of initiated insulin mRNA molecules from the cytoplasm to microsomal membranes by an SRP mediated mechanism that involves the modification of interactions between SRP and its receptor. PMID- 3017308 TI - A re-evaluation of evidence attributing the difference in cleavage rates of restriction endonuclease at different sites in the substrate to differences in Km values. AB - The only result supporting the hypothesis that the differences in restriction endonuclease cleavage rates at various target sites are caused by differences in Km values was reported by Forsblom, Rigler, Ehrenberg, Petterson & Philipson [(1976) Nucleic Acids Res. 3, 3255-3269]. The present work shows that the kinetic analysis in that paper is based on incorrect derivation and in fact provides no support for the hypothesis mentioned. PMID- 3017309 TI - Non-enzymic phosphorylation of polyphosphoinositides and phosphatidic acid is catalysed by bivalent metal ions. AB - Phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate and phosphatidic acid undergo non-enzymic phosphorylation by ATP in the presence of bivalent metal ions. The non-enzymic reaction is more rapid in a mixture of water, chloroform and methanol than in water alone. Chemical evidence indicates that the product formed from phosphatidylinositol 4-phosphate is the corresponding 4-pyrophosphate. This product shows an RF value very close to that of phosphatidylinositol 4,5-bisphosphate on t.l.c. with an acidic solvent commonly used to characterize and measure the latter; however, it can be separated readily with an alkaline solvent. Chemical evidence indicates that the products formed from phosphatidylinositol 4,5-bisphosphate and phosphatidic acid are also pyrophosphates. PMID- 3017310 TI - Target size of the adenosine Ri receptor. AB - The adenosine receptor of rat cerebral-cortical membranes was examined by radiation inactivation. In control membranes the receptor is distributed between high- and low-affinity states, that can be preferentially expressed by Mg2+ ions and guanine nucleotides respectively. Upon exposure to increasing doses of radiation, the high-affinity receptor decayed linearly as a function of radiation dose. This decay rate corresponded to a target size of 63,000 Da, when compared with the decay of the muscarinic cholinergic receptor that was also measured in these membranes. PMID- 3017311 TI - Preferential nucleosome placement on pBR322 restriction fragments. AB - Two restriction fragments of DNA containing the regulatory feature GTG/CAC were experimentally associated with core histones. The reconstituted DNA-histone complexes consisted of different forms of mononucleosomes. Lambda exonuclease and Fnu4HI were used to probe the structure of each distinct nucleoprotein complex. For each of the DNA fragments, one form of particle was produced that showed preferred placement of the core octamer on the DNA. The GTG/CAC base triplets may play some role in determining the final histone core positions in these reconstitutes. PMID- 3017313 TI - The effect of arabinose 1,5-bisphosphate on rat hepatic 6-phosphofructo-1-kinase and fructose-1,6-bisphosphatase. AB - The alpha- and beta-anomers of arabinose 1,5-bisphosphate and ribose 1,5 bisphosphate were tested as effectors of rat liver 6-phosphofructo-1-kinase and fructose-1,6-bisphosphatase. Both anomers of arabinose 1,5-bisphosphate activated the kinase and inhibited the bisphosphatase. The alpha-anomer was the more effective kinase activator while the beta-anomer was the more potent inhibitor of the bisphosphatase. Inhibition of the bisphosphatase by both anomers was competitive, and both potentiated allosteric inhibition by AMP. beta-Arabinose 1,5-bisphosphate was also more effective in decreasing fructose 2,6-bisphosphate binding to the enzyme. Neither anomer of ribose 1,5-bisphosphate affected 6 phosphofructo-1-kinase or fructose-1,6-bisphosphatase, indicating that the configuration of the C-2 (C-3 in Fru 2,6-P2) hydroxyl group is important for biological activity. These results are also consistent with arabinose 1,5 bisphosphate binding to the active site and thereby enhancing the interaction of AMP with the allosteric site. PMID- 3017312 TI - Hepatitis B virus transcripts in a human hepatoma cell line, Hep 3B. AB - Hep 3B, a human hepatoma cell line was examined for its RNA hybridizable to the hepatitis B virus sequence. Using probes that covered different regions of the hepatitis B virus genome, five species of RNA were observed of sizes 4.0, 3.3, 2.9, 2.6 and 2.2 kilobases. The RNAs covered surface antigen gene, pre-S and X regions. None of them had a core antigen sequence. RNA with a 4.0 kilobase size was the most abundant. Using S1 nuclease analysis, its 5' end of hepatitis B virus sequence was mapped at pre-S region and its 3' end of viral sequence was mapped at DR region. PMID- 3017314 TI - Detection of a potential transcription control sequence on the cauliflower mosaic virus genome by dinucleotide primed "in vitro" transcription. AB - The three sites of selective dinucleotide-primed "in vitro" transcription initiation on a cloned cauliflower mosaic virus DNA fragment have been localised by S1 nuclease mapping. Two of these sites lie within a region which has been shown to be essential for transcription complex formation on the viral sequences, one corresponding to a nuclease S1 hypersensitive site and the other to an imperfect repeat 100bp downstream. These sequences show striking homology with known transcription control sequences. These observations and the effect of the sequences on "in vitro" transcription raise the possibility that they may be involved in control of transcription of the viral genome. PMID- 3017315 TI - Structural studies on H+,K+-ATPase: determination of the NH2-terminal amino acid sequence and immunological cross-reactivity with Na+,K+-ATPase. AB - The NH2-terminal amino acid sequence of the 100 kilodalton subunit of porcine gastric H+,K+-ATPase has been determined to be YKAENYELYQVELGPGP. Although the NH2-terminal region of this protein is not similar to the same region of the lamb kidney Na+,K+-ATPase catalytic subunit, other regions of these ATPase proteins appear to be homologous. Both monoclonal and polyclonal antibodies raised to lamb kidney Na+,K+-ATPase and its alpha, but not beta, subunit cross-react with the 100 kilodalton protein of H+,K+-ATPase. PMID- 3017316 TI - Purification of cAMP-specific phosphodiesterase from rat heart by affinity chromatography on immobilized rolipram. AB - Affinity chromatography on a cAMP-specific phosphodiesterase inhibitor related to Rolipram, immobilized to AH Sepharose allowed to perform an efficient purification of the cAMP-specific phosphodiesterase isoenzyme from rat heart cytosol (102-fold purification with a 35% yield in a single step). This affinity chromatography involved a biospecific interaction since a 2 mM cAMP elution step at 30 degrees C was necessary for releasing the cAMP specific form tightly bound on the affinity gel. The cAMP eluate fraction exhibited a high specificity towards cAMP (cAMP/cGMP hydrolysis ratio 5-10), a marked sensitivity to Rolipram inhibition and could be resolved in two cAMP-specific, highly Rolipram-sensitive peaks of pI 6.7 and 4.8 by IEF on polyacrylamide gel plates. Protein stain of the IEF gel revealed a single band at pI 6.7. PMID- 3017317 TI - Diadenosine tetraphosphate activates cytosol 5'-nucleotidase. AB - The rate of hydrolysis of IMP (0.5 mM) by cytosol 5'-nucleotidase from Artemia embryos was increased up to 7-fold by concentrations of around 10 microM diadenosine tetraphosphate (Ap4A). Half maximal activation of the enzyme was accomplished with 5 microM Ap4A. The Km (S 0.5) values of the nucleotidase for IMP, GMP, AMP, XMP and CMP decreased about 10 fold in the presence of 10 microM Ap4A. Maximum velocity of the enzyme was not affected by Ap4A. ATP had been previously described as an activator of the enzyme. However, comparatively with Ap4A, concentrations of ATP two orders of magnitude higher are needed to elicit similar effects on the enzyme. Preliminary results indicate that Ap4A is also an activator of the cytosol 5'-nucleotidase from rat liver. PMID- 3017318 TI - Close linkage of human chromosomal pepsinogen A genes. AB - We have obtained a clone containing two pepsinogen A genes in a single insert by screening a recombinant cosmid library for human genomic DNA. Restriction endonuclease mappings of this cloned DNA showed that these two genes are very similar, but distinct in structure, and that they are closely linked to one another in the human chromosome DNA. The close arrangement of the genes with very similar structures could facilitate the homologous recombination or the unequal crossing-over which accounts for high frequency of haplotype variation in copy number of pepsinogen A genes as reported by Taggart et al. PMID- 3017319 TI - The yeast SRP gene: positive modulation by glucose of its transcriptional expression. AB - The yeast gene coding for a protein rich in serine residues (SRP) has been cloned. Its transcriptional expression is modulated positively by glucose as shown by Northern blots of mRNA prepared from cells grown in medium containing either glucose or raffinose or glycerol/ethanol as carbon sources. The positive elicitation of SRP mRNA by glucose was found to be associated with the promoter region of the SRP gene, using a plasmid with the lacZ gene fused with the SRP promoter region. The promoter region has been sequenced and disclosed all the characteristics of a strong yeast promoter. PMID- 3017321 TI - Role of calcium on TPA-induced secretion of ACTH and PGE2 by pituitary cells: effect of dexamethasone. AB - In pituitary cells in primary culture, ACTH and PGE2 secretions can be simultaneously stimulated by TPA, in the presence of Ca2+. However, whilst PGE2 secretion is under an absolute requirement for Ca2+, ACTH secretion is not. Both secretions are inhibited by dexamethasone but to various extents: PGE2 release is abolished in the presence of dexamethasone whilst only 35% of the TPA-induced ACTH release is sensitive to dexamethasone. Similar inhibitory effects are observed with mepacrine, a PLA2-inhibitor, suggesting that PLA2-activation could be related to these secretory process. Since PLA2-inhibition by dexamethasone is claimed to be mediated via lipocortin, these results suggest that a lipocortin like protein is present in pituitary cells and could be involved in the TPA induced secretions of PGE2 and ACTH. PMID- 3017320 TI - Purification of the insulin-like growth factor II (IGF-II) receptor from an IGF II-producing cell line, and generation of an antibody which both immunoprecipitates and blocks the type 2 IGF receptor. AB - 18,54-SF cells, which secrete rat insulin-like growth factor II (rIGF-II), have abundant type 2 IGF receptors. We have purified the type 2 receptor from these cells by solubilization of crude membranes in Triton X-100, followed by chromatography on agarose-immobilized rIGF-II. A partially purified receptor preparation, obtained by chromatography of solubilized membranes over wheat germ agglutinin, was used to immunize a rabbit. The antibody generated both immunoprecipitates the type 2 receptor, and specifically inhibits IGF-II binding to a variety of rat tissues, including 18,54-SF cells, BRL-3A cells and placenta. The presence of abundant type 2 receptors on an rIGF-II-secreting cell line is consistent with an autocrine role for IGF-II in select cells. PMID- 3017323 TI - Age-dependent changes in expression of alpha 1-adrenergic receptors in rat myocardium. AB - The expression of alpha 1-adrenergic receptors within ventricular myocardium of rats ranging in age from 21 days of fetal life to 24 months after birth was measured from [125I] 2-(beta hydroxy phenyl) ethylaminomethyl tetralone binding isotherms. No difference was observed in binding affinity between any of the age groups studied. The number of alpha 1-adrenergic receptors was found to be 60 120% higher in membranes from fetal or immature rats up to 25 days of age when compared with adult animals. The increased expression of alpha 1-adrenergic receptors in the developing heart relative to that observed in adult heart is consistent with the hypothesis that alpha 1-adrenergic receptor stimulation may modulate protein synthesis and growth in mammalian myocardium. PMID- 3017324 TI - Atrial natriuretic factor regulates steroidogenic responsiveness and cyclic nucleotide levels in mouse Leydig cells in vitro. AB - The effects of synthetic atrial natriuretic factor (ANF) on the regulation of mouse Leydig cell steroidogenesis have been studied in vitro. ANF in nanomolar concentration increased testosterone production by more than 30-fold over basal levels. Concomitantly, cyclic guanosine monophosphate levels were increased 35 fold; cyclic adenosine monophosphate levels fell minimally (15-20%). ANF at low concentration (1 X 10(-11) M) inhibited testosterone production by luteinizing hormone-stimulated cells, while at higher concentration (greater than 2 X 10(-9) M) ANF stimulated steroidogenesis beyond the level attained by luteinizing hormone alone. These results indicate that ANF can exert stimulatory effects on testosterone steroidogenesis in vitro, and that the mechanism may involve an intracellular messenger other than cyclic adenosine monophosphate. PMID- 3017322 TI - Absence of phosphorylation of retinoid-binding proteins by protein kinase C in vitro. AB - Cellular retinol-binding protein, cellular retinoic acid-binding protein, and fetal cellular retinol-binding protein were purified to homogeneity and each polypeptide had a molecular weight of 16,000. Their apoproteins were not phosphorylated under the same conditions. Their holoproteins did not inhibit the phosphorylation of histone III-S by protein kinase C. Each of these observations is contrary to the results reported by Cope et al. (Biochem. Biophys. Res. Commun., 120, 593-601, 1984). PMID- 3017325 TI - Regulation of atrial natriuretic peptide receptors in cultured vascular smooth muscle cells of rat. AB - To elucidate the regulation of vascular receptors for atrial natriuretic peptide (ANP), we have studied the binding capacity of 125I-labeled rat (r) ANP using cultured vascular smooth muscle cells from rat aorta. After preincubation with 3.2 X 10(-8) M rANP at 37 degrees C, the binding capacity decreased as a function of time; the maximal receptor loss (70-75%) occurred after 4 hrs and persisted for 24 hrs. Pretreatment with cycloheximide (20 micrograms/ml) and actinomycin D (2 micrograms/ml) similarly caused a dramatic reduction (approximately 80%) of the binding capacity after 24 hrs; the half-life (t1/2) of the receptor loss was approximately 7-8 hrs. Following removal of rANP, the "down-regulated" ANP receptors fully recovered in the presence of 10% fetal calf serum, but not in combination with either actinomycin D or cycloheximide. Concanavalin A dose dependently inhibited the binding. The binding capacity also decreased with time in the presence of tunicamycin (1 microgram/ml) with t1/2 of approximately 30 hrs. These data indicate that protein and carbohydrate moieties are essential for the functional integrity of the vascular receptor binding sites for ANP, and suggest that the recovery of the receptor loss by "down-regulation" requires concomitant RNA and protein synthesis. PMID- 3017326 TI - DNA fragments of 300 base pairs released from metaphase chromosomes by digestion with deoxyribonuclease I. AB - DNase I digestion of metaphase chromosomes, that have been extensively digested with Hae III, further released chromosomal DNA and proteins; 3.3% and 10.8% of the chromosomal DNA and proteins, respectively, remained insoluble. However, digestion of chromosomes first with DNase I followed by Hae III caused most of the proteins to remain in the insoluble fraction. DNase I released DNA fragments of 300 base pairs long which were not released by Hae III digestion. These DNA fragments may be protected by protein components from further fragmentation by DNase I. PMID- 3017328 TI - Role of methionine-1 in ubiquitin conformation and activity. AB - Methionine-1 of ubiquitin was oxidized to the sulfone without significant effect on biological activity or conformation at neutral pH. However, at low pH, the oxidized protein expanded to a more open conformation, similar in gel sieving properties to denatured ubiquitin but similar in secondary structure to native ubiquitin. This conformational transition was absent in the native protein. Interpretation of these results in the light of X-ray data suggests that ubiquitin contains two independently folded domains that are held together in part by a hydrogen bond between Met-1 and Lys-63 and which can be separated when this bond is broken. It is suggested that separation of these domains may occur upon ubiquitin conjugation. PMID- 3017330 TI - Isolation and initial characterization of a Bacillus subtilis mutant with novel protease secretion capability. AB - A bank of pTV32 (Tn 917 lacZ) - generated Bacillus subtilis mutants were examined on milk agar for the ability to produce proteases at 48 degrees C. A single mutant, BUL786, was isolated, which could hydrolyze casein after overnight incubation at 48 degrees C. This mutant secreted protease 10 fold more at 48 degrees C when compared to 37 degrees C, and part of the activity appears to be 48 degrees C-specific. At high temperatures, other strains of B. subtilis, including hyperprotease secretors, were unable to secrete protease to any significant degree. The BUL786 strain is missing the 97K major heat shock protein. Since a number of other proteins also appear to be secreted at 48 degrees C, this mutant may be a hypersecretor of exported proteins at temperatures greater than 45 degrees C. PMID- 3017329 TI - Calcium-induced alterations in the levels and subcellular distribution of proteolytic enzymes in human red blood cells. AB - Human red cells were treated with 100 microM Ca2+ and ionophore A 23187. This treatment induces remarkable changes in the activities of the two major proteolytic systems of red cells, i.e. Ca2+-dependent neutral proteinase and acid endopeptidases. Ca2+-dependent neutral proteinase undergoes intracellularly preliminary activation of the inactive proenzyme species, followed by eventual inactivation through self-proteolysis. Transient activation is shown by selective degradation of cytoskeletal proteins known to be targets of this enzyme system. Concomitantly, acid endopeptidase activity is substantially released from the membrane into the cytosol. Preliminary inactivation of the Ca2+-dependent neutral proteinase by exposure of Glucose 6-phosphate dehydrogenase-deficient red cells to auto-oxidizing divicine prevents alterations induced by Ca2+ loading on cytoskeletal membrane proteins, while leaving solubilization of acid endopeptidase activity unaffected. The two events, although dependent on Ca2+ loading, are therefore unrelated to each other. PMID- 3017327 TI - Atrial natriuretic factor receptors are negatively coupled to adenylate cyclase in cultured atrial and ventricular cardiocytes. AB - We have studied the effect of synthetic rat atrial natriuretic factor (ANF) on adenylate cyclase activity in cultured cardiocytes from atria (left and right) and ventricles from neonatal rats. ANF (Arg 101-Tyr 126) inhibited adenylate cyclase activity in a concentration dependent manner in cultured atrial (right and left atria) and ventricular cells. However the inhibition was greater in atrial cells as compared to ventricular cells. The maximal inhibition observed in ventricular cells was about 35% with an apparent Ki of about 10(-10) M, whereas about 55% inhibition with an apparent Ki between 5 X 10(-10) M and 65% inhibition with an apparent Ki of 10(-9) M were observed in right and left atrial cardiocytes respectively. The inhibitory effect of ANF was dependent on the presence of guanine nucleotides. Various hormones and agents such as isoproterenol, prostaglandins, adenosine, forskolin and sodium fluoride stimulated adenylate cyclase activities to various degrees in these atrial and ventricular cardiocytes. ANF inhibited the stimulatory responses of all these agonists, however the degree of inhibition varied for each agent. In addition ANF also inhibited cAMP levels in these cells. These data indicate that ANF receptors are present in cardiocytes and are negatively coupled to adenylate cyclase. PMID- 3017331 TI - The mitochondrial site of superoxide formation. AB - Ubiquinone and cytochrome b566 have both been postulated to cause mitochondrial O2 formation by autoxidation of their reduced forms. The present investigation was made to evaluate capabilities of the two candidates to transfer electrons to molecular oxygen out of sequence of the normal pathway of respiration. The results show that electron transfer from ubisemiquinone to oxygen depends on the availability of protons. In agreement with this finding autoxidation of redox cycling ubiquinone could not be observed due to its location in an aprotic environment of the mitochondrial membrane. However, O2 release from mitochondria was found to be related to the existence of low potential cytochrome b566. The transfer of this b type cytochrome to more positive values caused a concomitant decrease and finally inhibition of univalent electron transfer to oxygen out of sequence. Our findings suggest a role of cytochrome b 566 in mitochondrial O2 formation. A contribution by ubiquinone is unlikely as long as protons are deprived from penetrating into the domain where ubiquinone is operating. PMID- 3017332 TI - Pharmacological profiles of a potential LTB4-antagonist, SM-9064. AB - Antagonistic activity against leukotriene B4 (LTB4) and other pharmacological activities of SM-9064 were studied in vitro and in vivo. It inhibited the chemotaxis of rat polymorphonuclear leukocytes (PMNLs) induced by LTB4 (IC50 = 1.3 X 10(-7) M, not by other chemoattractants. Its agonistic activity was much less than that of LTB4. It suppressed the Arthus reaction-induced inflammation in mice with the dose of 5 mg/kg, i.p. or 10 mg/kg, p.o. These results suggest that SM-9064 is a candidate of LTB4 antagonists, which is effective in some type of inflammation. PMID- 3017334 TI - Autolytic activation of calcium-activated neutral protease. AB - Degradation of vimentin by native low calcium ion-requiring protease (mu CANP) was compared to that by autodigested mu CANP. On activation with 5 mM barium ions, a lag time was observed for the case of native mu CANP. This provides direct evidence that native mu CANP is inactive as a protease and must be autolyzed to be activated. Most of the protease activity can be accounted for by autodigested mu CANP with a 76 K polypeptide but another species with 50 K polypeptide may also be active. PMID- 3017333 TI - Requirement of free arachidonic acid for leukotriene B4 biosynthesis by 12 hydroperoxyeicosatetraenoic acid-stimulated neutrophils. AB - Stimulation of human neutrophils with 12-hydroperoxyeicosatetraenoic acid (12 HPETE) led to formation of 5S, 12S-dihydroxyeicosatetraenoic acid (DiHETE), but leukotriene B4 (LTB4) or 5-hydroxyeicosatetraenoic acid (5-HETE) was not detectable by reversed-phase high-performance liquid chromatography analysis. N formylmethionylleucylphenylalanine (FMLP) induced the additional synthesis of small amounts of LTB4 in 12-HPETE-stimulated neutrophils. The addition of arachidonic acid greatly increased the synthesis of LTB4 and 5-HETE by neutrophils incubated with 12-HPETE. In experiments using [1-14C]arachidonate labeled neutrophils, little radioactivity was released by 12-HPETE alone or by 12 HPETE plus FMLP, while several radiolabeled compounds, including LTB4 and 5-HETE, were released by A23187. These findings demonstrate that LTB4 biosynthesis by 12 HPETE-stimulated neutrophils requires free arachidonic acid which may be endogenous or exogenous. PMID- 3017335 TI - Selective oxidation of imidazole ring in histidine residues by the ascorbic acid copper ion system. AB - In connection with the physiological actions of active oxygen species on proteins, oxidative modification of histidine residues by the autoxidation of ascorbic acid was determined and the main oxidized compound was identified. Oxidation of imidazole ring by the ascorbic acid-copper ion system was considerably site-specific and assumed to be initiated by the addition of the hydroxyl radical (.0H) at C-2 position in the imidazole ring. PMID- 3017336 TI - 31P NMR spectra of rodent and avian fibroblasts transformed by Rous or Kirsten sarcoma viruses. AB - 31P NMR spectra of normal rodent and avian fibroblasts were compared to those of the same cells transformed either by the Rous sarcoma virus (RSV) or by the Kirsten sarcoma virus (Ki-MSV). Under physiological conditions, the spectra of living or perchloric acid extracted chicken embryo fibroblasts, rat cell line FR3T3 and mouse cell line C127 did not differ from those of their counterparts transformed by RSV or Ki-MSV. However, in the case of FR3T3 cells, on shifting from 37 degrees C to 20 degrees C, and particularly if PBS replaced serum growth medium, a different, though transitory, response of the transformed cells was detected. They then showed, within few minutes, a more rapid ATP depletion with accumulation of fructose 1,6-diphosphate (FDP), as compared to normal control cells. PMID- 3017337 TI - Re-evaluation of a method calculating cleavage rates at different sites of DNA from partial digestion of end-labelled molecule. AB - Nucleases cleave the same targets at different rates in different parts of DNA molecules. The cleavage rates can be calculated from the amounts of particular fragments occurring in the DNA partial digests, but these are very complex and difficult to separate. To simplify the analysis, the use of end-labelled DNA molecules was suggested by Lutter. It is shown in the present communication that the fundamental equation of Lutter's approach is erroneous. A correct equation is derived and recommended to be used for re-calculation of the results obtained previously. PMID- 3017338 TI - Phosphorylation of the calmodulin-dependent protein phosphatase by protein kinase C. AB - The calmodulin-dependent protein phosphatase was shown to be phosphorylated by the Ca2+, phospholipid-dependent protein kinase (protein kinase C). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 61 kDa catalytic subunit was phosphorylated. Phosphorylation by protein kinase C was stimulated up to 15-fold by addition of phosphatidyl-L-serine and between 0.5 to 1.0 mole of phosphate was incorporated per mole of phosphatase. It is possible that protein kinase C is involved in the regulation of the calmodulin-dependent protein phosphatase via this novel phosphorylation of the enzyme. PMID- 3017339 TI - Thermodynamics of proton transfer in carboxylic acid-retinal Schiff base hydrogen bonds with large proton polarizability. AB - During the photocycle of bacteriorhodopsin (BR) the chromophore, a retinal Schiff base, is deprotonated. Simultaneously an asp residue is protonated. These results suggest that this deprotonation occurs via a Schiff base - asp hydrogen bond. Therefore, we studied carboxylic acid - retinal Schiff base model systems in CCl4 using IR spectroscopy. The IR spectra show that double minimum proton potentials are present in the OH ... N in equilibrium with O- ... HN+ H-bonds formed and that the proton can easily be shifted in these bonds by local electrical fields. The thermodynamic data of H-bond formation and proton transfer within these H bonds are determined. On the basis of these data a hypothesis is developed with regard to the molecular mechanism of the deprotonation of the Schiff base of BR. PMID- 3017340 TI - Sequence and expression of a novel murine interferon alpha gene--homology with enhancer elements in the regulatory region of the gene. AB - A murine interferon gene (MuIFN alpha) has been isolated from a cosmid library. The sequence of a 1.2-kb HindIII-PstI fragment revealed a new MuIFN alpha gene which has not yet been described and which was termed MuIFN alpha 7. The coding sequence produced biologically active IFN when expressed in monkey cells under the control of an SV40 promoter. A comparison of the MuIFN alpha 7 gene with the known interferon genes in their coding and flanking sequences shows homologies between enhancer elements found in the 5' upstream region of the coding gene. The core element common to all known viral enhancers, GTGG(AAA/TTT)G is repeated four times in the MuIFN alpha 7 5'-flanking region, as in all known MuIFN alpha genes. PMID- 3017341 TI - Bovine cardiac mitochondrial ADP/ATP-carrier: two distinct mRNAs and an unusually short 3'-noncoding sequence. AB - cDNA clones for bovine cardiac mitochondrial ADP/ATP carrier have been isolated. They hybridize with two mRNAs that differ in size by about 300 nucleotides. The concentration ratio and total abundance of the two mRNAs varies among bovine tissues (heart, liver, kidney, and uterus). At least one of them has an unusually short 3'-noncoding sequence, which includes the two consensus cleavage/polyadenylation signal sequences CATTG and ATTAAA. The 3'-end shows complementarity to regions of human U4 small nuclear RNA. The predicted amino acid sequence confirms the reported sequence from Val207 to the C-terminal Val297, which has been proposed (residue 279-291) to contribute to a nucleotide binding site. PMID- 3017342 TI - Expression of the c-Ha-ras and c-myc genes in aflatoxin B1-induced hepatocellular carcinomas. AB - Expression and activation of several c-oncogenes in seven hepatocellular carcinomas from seven separate rats treated with aflatoxin B1 (AFB1) were examined by Northern and Southern blot analyses. Both c-Ha-ras and c-myc transcripts were elevated at high levels in all hepatomas. Moreover, in one of them, T2-1 hepatoma, the c-myc gene was amplified only in a tumor part of liver without significant rearrangement. N-ras specific transcripts were not elevated in these hepatomas. The present data suggest that the consistently increased expression or deregulation of the c-myc and c-Ha-ras genes may play an important role in the development of hepatomas induced by AFB1. PMID- 3017343 TI - A pertussis/choleratoxin-sensitive N protein may mediate chemoattractant receptor signal transduction. AB - Chemoattractant receptors on phagocytic leukocytes utilize a guanine nucleotide regulatory (N) protein to activate phospholipase C and subsequent biological responses. Since pertussis toxin inhibits activation of leukocytes by chemoattractants and ribosylates a ca. 40 kD protein in these cells it had generally been assumed that chemoattractant receptors are coupled to Ni. We now report that human polymorphonuclear leukocytes (PMNs), monocytes, and the myeloid HL-60 and U937 cell lines, but not erythrocytes or bovine brain contain a ca. 40 kD protein which is a substrate for ADP ribosylation by choleratoxin (CT). This N protein, termed Nc for chemotaxis-related N protein, comigrates with the ca. 40 kD PT substrate during one-dimensional gel electrophoresis. In vivo treatment of PMNs with PT or CT reduced high affinity binding of chemoattractants to membrane preparations from the cells, implying that chemoattractant receptors are coupled to an N protein which is a substrate for both PT and CT. We suggest that Nc rather than Ni couples chemoattractant receptors to phospholipase C. PMID- 3017344 TI - Supercoil-dependent recognition of specific DNA sites by chromosomal protein HMG 2. AB - The ability of the chromosomal high mobility group protein HMG 2 to recognize supercoil-dependent structures within the chicken adult beta-globin gene was investigated by examining its ability to protect such sites from digestion by S1 nuclease. Low molar ratios of HMG 2 were found to be sufficient for complete inhibition of S1 cleavage of a supercoiled plasmid containing the globin gene. Furthermore, HMG 2 protected an S1 cleavage site within the 5'-flanking region of the globin gene, in preference to a palindromic S1 site within the plasmid vector. PMID- 3017345 TI - Destabilization of target-sensitive immunoliposomes by antigen binding--a rapid assay for virus. AB - Interactions of antibody stabilized phosphatidylethanolamine (PE) immunoliposomes with Herpes Simplex virus (HSV) and virus infected cells were studied by detecting the immune-dependent lysis of liposomes. Employing PE immunoliposomes bearing anti-HSV glycoprotein D (gD) IgG, immune-specificity of these liposomes were documented by the sole ability of HSV and the HSV-infected L cells to induce immunoliposome lysis. In addition, inhibition of PE immunoliposome lysis by free anti-gD IgG, but not anti-HSV glycoprotein B IgG, indicated the target antigen specificity of these immunoliposomes. Based on these observations, alkaline phosphate encapsulated PE liposomes were used to directly detect HSV in fluid phase. This immunoliposome assay which does not require washing was shown to be very rapid and sensitive: 35pfu of HSV-1 in 5ul could be detected within 1.5hr. PMID- 3017346 TI - Efficient screening of recombinant DNA junctions afforded by probing with synthetic "bridge" oligonucleotides. AB - Procedures have been developed which simplify and expedite the screening of recombinant DNA constructions for those which only exhibit the desired DNA-DNA junctions. A synthetic DNA oligonucleotide designed to span (or "bridge") sequences around correct restriction enzyme junctions was used as a hybridization probe for the rapid identification of those sequences in several molecular cloning methodologies. It facilitated analyses of the products of random ligation reactions, as well as constructions harbored in bacteria and bacteriophage. "Bridge" probes, [32P]-end-labeled to very high specific activity, remained useful after several hybridizations and often circumvented lengthy restriction analyses. PMID- 3017347 TI - Characterization of human genomic DNA sequences homologous to the interleukin 2 cDNA. AB - Southern blot analysis of human placental DNA under low stringency hybridization conditions revealed several DNA fragments hybridizable to the human interleukin 2 (IL-2) cDNA. Four phage clones carrying these IL-2 cDNA-like sequences were isolated and their structures analyzed. A DNA fragment derived from one of the clones gave the strongest hybridization signal. Sequence analysis of this fragment revealed the presence of a cluster of three DNA segments, i.e. 20 base pairs (bp), 57 bp and 18 bp in length and having about 85%, 80% and 83% homology to three different parts of the coding region of human IL-2 cDNA, respectively. PMID- 3017348 TI - Transcriptional regulation of the transferrin receptor in differentiating HL-60 leukemic cells. AB - HL-60 promyelocytic leukemia cells were induced to differentiate along the granulocytic pathway by treatment with dimethylsulfoxide. A significant decrease in transferrin receptor specific mRNA was seen as early as 4 hr after exposure to inducer. Relative to untreated controls, transcript levels decreased 5-fold within 24 hr of exposure to dimethylsulfoxide and remained depressed throughout a 6-day treatment period. Reductions in receptor transcripts preceded the loss of surface receptor which in turn preceded the expression of the differentiated phenotype. The findings demonstrate that differentiation-associated regulation of the transferrin receptor can occur at the transcriptional level. PMID- 3017349 TI - Inactivation of mitochondrial adenosine triphosphatase from Trypanosoma cruzi by oxygen radicals. AB - Incubation of Trypanosoma cruzi mitochondrial ATPase (Fo-F1) with the xanthine oxidase system (XO), Fenton's reagent (Fe2+ + H2O2) and the ascorbate-Cu system, caused gradual loss of enzyme activity, which increased as a function of incubation time and rate of oxygen radical generation. The essential role of OH. radicals for ATPase inactivation was supported by a) the enzyme protection afforded by superoxide dismutase, catalase and mannitol, when using the XO system; b) the similar effect of mannitol and benzoate with Fenton's reagent; c) the similar effect of catalase, EDTA and histidine with the ascorbate-Cu system; d) the increased rate of ATPase inactivation by 1) the XO system supplemented with chelated iron, and 2) the ascorbate-Cu system supplemented with H2O2. Comparison of oxygen radical generators for their action on membrane-bound (Fo F1) and soluble F1 revealed that ascorbate-Cu was the most effective one, possibly because of its capability of producing OH. radicals that react preferentially with the enzyme at their formation site. PMID- 3017351 TI - A rapid and inexpensive method for preparing E. coli plasmid-DNA. AB - A simple, rapid and inexpensive scaled up miniprep procedure for preparing pure E. coli plasmid DNA is described. Bacterial cells were subjected to the boiling procedure and high molecular weight RNA was removed by LiCl-precipitation. Residual RNA and proteins were removed by subsequent treatment with RNase A and proteinase K/SDS respectively, followed by Sephadex G-50 and Sepharose 6B-Cl chromatography. The average yield from a 100 ml over-night bacterial suspension was 100 to 150 micrograms for pBR-322 DNA, and 250-500 micrograms for SP-6 derived recombinant plasmids. In addition, the described "scaled up" preparation does not require CsCl-ethidium bromide centrifugation; pure plasmid DNA can be prepared within 1 to 2 days. PMID- 3017350 TI - Confirmation that catalase is a glycoprotein. AB - Catalases which had been purified from the livers of mouse, rat and guinea pig were subjected to mild periodate oxidation followed by reduction with sodium boro[3H]hydride in order to test for the presence of sialic acid. A radioactively labelled moiety resulted, which behaved as a derivative of N-acetyl neuraminic acid during mild acid hydrolysis, neuraminidase treatment, ion exchange chromatography and paper chromatography. It is concluded that mammalian catalases are glycoproteins, and possess variable amounts of N-acetyl neuraminic acid in their carbohydrate moiety. PMID- 3017352 TI - Impaired uptake of D-glucose by tumoral insulin-producing cells. AB - At variance with the situation found in normal pancreatic islets, no equilibration of extracellular and intracellular D-glucose concentrations occurs in tumoral insulin-producing cells of the RINm5F line. This unexpected behaviour may account, in part at least, for the abnormal kinetics of glucose utilization in the tumoral cells and their poor secretory response to this hexose. PMID- 3017353 TI - Structure-activity relationships for N6-substituted adenosines at a brain A1 adenosine receptor with a comparison to an A2-adenosine receptor regulating coronary blood flow. AB - A series of 145 N6-substituted adenosines have been screened as inhibitors of the binding of [3H]cyclohexyladenosine to an A1-adenosine receptor in rat brain membranes and the results compared to the potencies of these analogs in increasing coronary blood flow via activation of an A2-adenosine receptor. The A1 receptor shows greater stereoselectivity in the N6 region of the receptor towards asymmetric aralkyl substituents, and shows greater bulk tolerance in the N6 region of the receptor such that it retains affinity for certain N6-tertiary alkyladenosines and N6-cycloalkyladenosines that are inactive at the coronary A2 receptor. At the A1 receptor, the most potent analogs have either aliphatic N6 substituents with four or more methylene residues or have an N6-halophenyl substituent. At the A2 receptor, the most potent analogs have an N6-phenethyl or similar heteroarylethyl substituent. Certain sets or series of analogs appear useful for identifying the subtypes of adenosine receptors involved in physiological functions. PMID- 3017354 TI - The effect of zinc in vivo and in vitro on the activities of angiotensin converting enzyme and kininase-I in the plasma of rats. AB - Two groups of rats were pair fed, for 18 days, diets containing either 2.6 (Zn deficient) or 100 mg Zn/kg (control diet). Plasma was assayed spectrophotometrically for the activity of kininase-I and angiotensin converting enzyme (ACE) in the presence of varying concentrations of added Zn2+. Zinc deficient rats had only 76% of the activity of both kininase-I and ACE compared with zinc supplemented control rats. There was a significant linear relationship between enzyme activity and concentration of zinc in plasma for both enzymes. When zinc was added to the enzyme incubation mixture for zinc deficient rats, the activity of ACE increased by 73% and that of kininase-I by 33%. This Zn2+ stimulated increase in enzyme activity was negatively correlated with the in vivo concentration of zinc in plasma, and a plateau in enzyme activity was seen at concentrations of plasma zinc that were commensurate with normal zinc status (over 14 mumol/1). The results demonstrate that the activities of both kininase-I and ACE are dependent on the concentration of zinc in vivo and in vitro, and suggest that information concerning the concentration of zinc in plasma and assay solutions be a prerequisite solutions be a prerequisite for the use of these enzymes in the clinical diagnosis of disease states. The results also showed that the activity of ACE and kininase-I in plasma could be used for the biochemical diagnosis of a suboptimal zinc status. PMID- 3017355 TI - Impaired regulation of beta 2-adrenergic receptor density in mononuclear cells during chronic renal failure. AB - Previous investigations have suggested that beta-adrenoceptor-mediated responses were decreased in uremia. To evaluate this phenomenon further, beta 2-receptor density in mononuclear cells, plasma catecholamines and plasma parathyroid hormone were studied in two groups of normotensive patients: group U, twenty-five chronic uremic patients with end-stage renal failure; group C, twenty-eight control subjects. Each group was divided into three age and sex-matched subgroups. Beta 2-receptor density was determined using (-)125 iodocyanopindolol. Despite a significant increase in plasma epinephrine in the group of uremic patients, there was a significant increase in beta 2-adrenoceptor density. On the other hand the uremic state did not influence (-)125 iodocyanopindolol binding affinity and plasma norepinephrine. Parathyroid hormone, as expected, was significantly elevated in all the uremic subgroups. It can be concluded that the uremic state is associated with an unexpected upregulation of beta 2-receptor density in mononuclear cells. The role of an endogenous beta-blocking substance is suggested. PMID- 3017357 TI - Suppression of MHV3 virus-activated macrophages by dieldrin. AB - Dieldrin (36 mg/kg body weight) administered intraperitoneally prolonged recovery from infection with mouse hepatitis virus 3 (MHV3) in the genetically-resistant A/J strain, affected the humoral anti-MHV3 IgG immune response, and inhibited the intrinsic antiviral activity of peritoneal macrophages upon in vitro rechallenge with the virus. Infection of untreated A/J animals and vehicle controls with MHV3 resulted in marked and reproducible activation of peritoneal macrophages, observed in vitro as resistance to MHV3-cytopathic effects 48 hr after rechallenge with the virus, whereas exposure to dieldrin resulted in apparent loss of the intrinsic capacity of cells to restrict replication of MHV3 and to protect them from cytolysis. In addition, in vitro treatment of MHV3 virus activated macrophages with dieldrin, mitomycin C and X-irradiation, inhibited the intrinsic capacity of cells to restrict MHV3 replication. This mechanism of cellular restriction of the virus by MHV3-activated macrophages from the resistant A/J strain appeared to be one of the sensitive targets for the suppressive action of dieldrin on host resistance, as no major changes in macrophage cellular parameters were observed in in vitro studies of cell viability, adherence to plastic, and superoxide anion generation; the increased cell yield in the peritoneal exudates during MHV3 virus infection was not affected by dieldrin exposure; and the attachment and uptake of [3H]MHV3 by virus activated macrophages was shown to be unchanged by dieldrin exposure. PMID- 3017356 TI - Prostaglandins and cannabis--XVI. Antagonism of delta 1-tetrahydrocannabinol action by its metabolites. AB - Prior exposure of cells in vitro to delta 1-tetrahydrocannabinol-7-oic acid (delta 1-THC-7-oic acid) reduced the degree of stimulation of prostaglandin synthesis incurred by subsequent treatment with delta 1-THC. The site of action of this inhibitory effect seemed to be on cyclooxygenase and not at the earlier step involving the phospholipase-mediated release of arachidonic acid. delta 1 THC-7-oic acid is a major metabolite of delta 1-THC and has no psychoactivity in humans. Our findings raise the possibility, however, that it may influence the in vivo activities of delta 1-THC by antagonizing its stimulatory action on cellular prostaglandin synthesis. PMID- 3017359 TI - Circadian rhythm in peripheral type benzodiazepine binding sites in human platelets. PMID- 3017358 TI - Role of polymorphonuclear leukocytes in connective tissue breakdown during the reverse passive Arthus reaction. AB - The reverse passive Arthus (RPA) reaction performed in the skin of rats was modified to allow for the determination of polymorphonuclear leukocyte (PMN) infiltration and hemorrhage, as well as changes in vascular permeability. After initiation of the RPA reaction, PMN infiltration, monitored by measurement of tissue myeloperoxidase (MPO, EC 1.11.1.7) content, increased dramatically with time. Depending on the experimental conditions used, PMN accumulation reached a maximum 2-10 hr after increased vascular permeability (125I-labeled albumin content) had peaked. Hemorrhage (59Fe-labeled erythrocyte accumulation) began to occur only after significant levels of PMN were reached and continued to increase proportionately to the level of PMN infiltration attained. Indomethacin administered 30 min prior to initiating the RPA reaction had no effect on vascular permeability increase but suppressed both PMN accumulation and hemorrhage development about 50%. When indomethacin was given 2 hr after the RPA reaction was begun, no effect on any of the RPA variables was noted. Dexamethasone suppressed the increase in vascular permeability (53%), PMN accumulation (78%), and hemorrhage (90%) when given 30 min prior to initiation of the reaction. Dexamethasone given 2 hr after initiating the RPA suppressed the entire reaction, but to a lesser extent. Catalase, as well as trasylol, alpha-1 antiproteinase and soybean trypsin inhibitor, inhibited PMN accumulation as well as hemorrhage when given intravenously at plus 2 hr. These results indicate that the damage to blood vessels during a severe RPA reaction is a direct consequence of PMN activity. PMID- 3017360 TI - Prevention of microsomal production of hydroxyl radicals, but not lipid peroxidation, by the glutathione-glutathione peroxidase system. AB - The glutathione-glutathione peroxidase system is an important defense against oxidative stress. The ability of this system to protect against iron-catalyzed microsomal production of hydroxyl radicals [oxidation of 4-methylmercapto-2-oxo butyrate (KMBA)] and lipid peroxidation was evaluated. When rat liver cytosol was added to microsomes, strong inhibition against KMBA oxidation was observed. No protection was found when the cytosol was boiled or dialyzed. In the latter case, the addition of 0.5 mM glutathione restored almost complete protection, whereas in the former case protection could be restored by the addition of both glutathione and glutathione peroxidase. Cysteine could not replace glutathione, nor could glutathione S-transferase replace glutathione peroxidase. The glutathione-glutathione peroxidase system was also very effective in decreasing production of hydroxyl radicals stimulated by the addition of menadione or paraquat to microsomes. In the absence of cytosol, the addition of glutathione plus glutathione peroxidase was also effective; however, 5 mM glutathione was necessary to protect against KMBA oxidation. The effective concentration of glutathione required for protection was lowered when glutathione reductase was added to the system, to regenerate reduced glutathione. These results indicate that low concentrations of glutathione in conjunction with glutathione peroxidase plus reductase can be very effective in preventing microsomal formation of hydroxyl radicals catalyzed by iron and other toxic compounds. Microsomal lipid peroxidation was decreased 40% by glutathione alone, and this decrease was potentiated in the presence of glutathione reductase. In contrast to KMBA oxidation, the combination of glutathione plus glutathione peroxidase was not any more effective than glutathione alone in preventing lipid peroxidation. The differences in sensitivities of microsomal lipid peroxidation and KMBA oxidation to glutathione peroxidase suggest that these two processes can be distinguished from each other, and that free H2O2 and hydroxyl radicals are involved in KMBA oxidation, but not lipid peroxidation. PMID- 3017363 TI - High potency congeners of isoproterenol. Binding to beta-adrenergic receptors, activation of adenylate cyclase and stimulation of intracellular cyclic AMP synthesis. AB - The potency of a p-toluide derivative and a p-trifluoromethyl derivative of isoproterenol on the beta1 receptor of turkey erythrocytes and on the beta2 receptor of S49 lymphoma cells was measured in comparison with isoproterenol. Binding assays showed that the p-trifluoromethyl derivative possessed an affinity for the receptor, which was two hundred times higher than that of isoproterenol, in the case of turkey erythrocytes, and sixty times higher in the case of the S49 cells. By measuring the Kact of adenylate cyclase, the activity of the p trifluoromethyl derivative was thirty to forty times higher than that of isoproterenol for the turkey erythrocyte membrane as well as for the S49 lysed cells. In stimulation of intracellular cyclic AMP accumulation, the Kact showed that the p-trifluoromethyl derivative was forty and twenty times more active than isoproterenol in turkey erythrocytes and in S49 cells, respectively. The p toluide derivative gave similar results. The superior affinity of the above isoproterenol congeners for both beta 1 and beta 2 adrenergic receptors makes these compounds excellent candidates for use as labeled agonist ligands in studies of beta receptors. The possibility is discussed that the relatively large substituent on the amino group of the catecholamine congeners might perhaps bind to lipids associated with the receptor. PMID- 3017361 TI - Characteristics of the active oxygen in covalent binding of the pesticide methoxychlor to hepatic microsomal proteins. AB - This study examined the characteristics of the active oxygen species involved in generation of the reactive intermediate of methoxychlor which covalently binds to liver microsomal proteins. The possibility that the active oxygen participating in the above reaction is the superoxide anion (O2-) or a species generated from O2- was examined with the help of superoxide dismutase (SOD) and with an SOD mimetic agent, CuDIPS [Cu2+(3,5-diisopropylsalicylic acid)2]. It was observed that, whereas CuDIPS inhibited covalent binding of methoxychlor metabolite(s), SOD did not. However, ZnDIPS [Zn2+(3,5-diisopropylsalicylic acid)2], which exhibits no SOD-mimetic activity, did not inhibit covalent binding. Furthermore, both CuDIPS and ZnDIPS had little or no effect on the formation of demethylated (polar) metabolites of methoxychlor, demonstrating that the inhibition of covalent binding by CuDIPS was not merely due to a general inhibition of the hepatic monooxygenase system. These findings suggested that O2- was involved in covalent binding, but was not accessible to SOD. Additional support for O2- involvement stems from the observation that alpha-tocopheryl acid succinate markedly inhibited covalent binding of methoxychlor. The possibility that hydrogen peroxide (H2O2) was involved in covalent binding of methoxychlor appears unlikely. Catalase had no effect on covalent binding when NADPH was the cofactor, and the use of H2O2 in place of NADPH did not yield covalent binding. Certain scavengers of hydroxyl radical (ethanol, t-butanol and benzoate) inhibited, and other known scavengers (DMSO and mannitol) did not inhibit, covalent binding. EDTA stimulated binding, desferal (desferrioxamine) exhibited no effect on binding, and diethylenetriaminepentaacetic acid (DETAPAC) inhibited binding. A possible explanation for this observation is that the Fe2+ needed for generation of X OH is much more easily obtained from Fe3+-EDTA than from Fe3+-desferal, which resists reduction. The inhibitory effect by DETAPAC may be due to chelation of another metal which is needed for the reaction. Lastly, certain scavengers of singlet oxygen inhibited covalent binding with little effect on the formation of polar metabolites of methoxychlor. In conclusion, these studies support the involvement of X OH and singlet oxygen, possibly derived from O2-, in the formation of the reactive methoxychlor intermediate.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3017362 TI - Effects of delta 9-tetrahydrocannabinol on glucagon receptor coupling to adenylate cyclase in rat liver plasma membranes. AB - Delta-Tetrahydrocannabinol (delta 9-THC), the principal psychoactive constituent of Cannabis sativa, was found to increase glucagon activation of liver plasma membrane adenylate cyclase. In the presence of 30 microM delta 9-THC, the EC50 for glucagon was decreased by 60% from 7.6nM to 3.1 nM. 11-OH-delta 9-THC, a psychoactive metabolite of delta 9-THC, also increased glucagon activation of adenylate cyclase while two cannabinoids without marihuana-like psychoactive potency, cannabinol and cannabidiol, did not. At 30 microM, delta 9-THC either slightly decreased or had no effect on the activation of adenylate cyclase by GTP, Gpp(NH)p, fluoride ion, forskolin or ATP alone. Delta 9-THC had no effect on the binding of [125I] glucagon to liver plasma membranes. Arrhenius plots demonstrated that delta 9-THC and 11-OH-delta 9-THC, but not CBD, decreased the activation energy above the break temperature. Therefore, delta 9-THC increased the coupling of the glucagon receptor to adenylate cyclase apparently by removing a constraint on receptor-Ns coupling. PMID- 3017364 TI - De novo analysis of receptor binding affinity data of beta-carbolines. PMID- 3017366 TI - Oxidation products are responsible for the resistance to the action of collagenase conferred on collagen by (+)-catechin. PMID- 3017365 TI - Regulatory effect of copper on rat adrenal cytochrome P-450 and steroid metabolism. AB - Accumulation of Cu2+ in rat adrenal glands produced a biphasic response in concentrations of the mitochondrial cytochrome P-450 and heme which were, in turn, reflected in abnormal steroid biosynthesis and output. In the mitochondria, 1 day after Cu2+ treatment, when the concentration of the metal ion was increased by 2- to 3-fold over the control value, a significant increase in cytochrome P 450-dependent steroid 11 beta-hydroxylase activity was observed. These effects were accompanied by a nearly 85% increase in concentrations of cytochrome P-450 and heme. In addition, the activity of delta-aminolevulinate synthetase was increased by 3-fold. In those animals lipid peroxidation, assessed by measuring concentrations of conjugated dienes, was reduced to approximately 50% of the control value. However, after 7 days of Cu2+ treatment (via a mini-osmotic pump), a significantly lowered rate of 11 beta-hydroxylase activity was noted, and the plasma concentration of corticosterone was also reduced significantly. Also, in the mitochondria, the concentrations of cytochrome P-450 and heme were decreased in comparison with the control values. These decreases were accompanied by elevated levels of the mitochondrial lipid peroxidation and a further increase in adrenal Cu2+ content (5-fold). At this time, delta-aminolevulinate synthetase activity remained elevated but to a lower extent than that observed after 1 day of Cu2+ treatment. In contrast to 11 beta-hydroxylase activity, the reduction in cytochrome P-450 content was not reflected in a decrease in the rate of cholesterol side-chain cleavage; rather this activity was increased in Cu2+ treated animals. Adrenal heme oxygenase activity was unaffected by either Cu2+ treatment, as was the specific content of cytochrome P-450 in the microsomal fraction. The present findings suggest that the Cu2+-mediated regulation of cytochrome P-450-dependent steroidogenic activity in the adrenal mitochondria is predominantly a reflection of the metal ion affecting heme biosynthesis and lipid peroxidation in this organ. Moreover, these actions appear to differentially affect the mitochondrial cytochrome P-450 species catalyzing different hydroxylation reactions in the adrenal steroidogenesis pathway. PMID- 3017367 TI - Structure-activity relationships of calmodulin antagonism by triphenylethylene antiestrogens. PMID- 3017368 TI - Phosphoribosylpyrophosphate synthetase superactivity. A study of five patients with catalytic defects in the enzyme. AB - Superactive phosphoribosylpyrophosphate (PRPP) synthetases were characterized in fibroblasts and erythrocytes from 5 unrelated men with gout and/or hyperuricemia and uric acid overproduction. The kinetic basis of enzyme superactivity in all patients was increased maximal reaction velocity. Affinities of the enzymes for substrates and activators and responsiveness to inhibitors were normal, and levels of immunoreactive enzyme in patient and control fibroblast and erythrocyte extracts were comparable. Enzymes purified to homogeneity from 2 patients confirmed the presence of isolated catalytic defects. Altered physical properties of certain of the superactive enzymes suggested the presence of several distinctive structural defects among the aberrant forms. Fibroblasts from each affected patient showed increased PRPP concentration and generation, as well as accelerated rates of all PRPP-requiring purine nucleotide synthetic pathways. These findings support the concept that enzyme superactivity results in uric acid overproduction as a consequence of increased rates of PRPP and purine nucleotide synthesis. Cultured cells from female relatives of 2 patients showed evidence for the heterozygous carrier state, as measured both by enzyme activities and by rates of PRPP and purine synthesis. The clinical phenotype in 4 patients was limited to early adult-onset gout and its consequences, whereas the fifth patient expressed a familial constellation of hyperuricemia, sensorineural deafness, ataxia, and renal insufficiency. The severity of the derangements in PRPP synthetase and in PRPP and purine synthesis in cells from the 5 patients, however, was comparable. The neurologic accompaniments of enzyme superactivity found in 1 family described here, and in 2 others described previously, thus may not necessarily be consequences of primary defects in PRPP synthetase. PMID- 3017370 TI - Enhancement of gamma-aminobutyric acid receptor binding by protopine-type alkaloids. AB - Protopine, cryptopine and allocryptopine were demonstrated to enhance 3H-gamma aminobutyric acid (3H-GABA) binding to rat brain synaptic membrane receptors. The above finding might be indicative of benzodiazepine-like activity of these alkaloids. PMID- 3017371 TI - Influence of topical corticosteroids on hormones in urine and plasma. AB - In the present study the effects on endogenous hormones of application of topical corticosteroids are investigated. Radioimmunological assays of the hormones cortisol, adrenocorticotrophic hormone (ACTH) and alpha-melanocyte stimulating hormone (alpha-MSH) before, during and after a large-area treatment with topical corticosteroids (30 g/d 0.025% fluocinolone acetonide ointment for 9 days) are performed in eight patients suffering from extensive inflammatory dermatoses (7 cases of eczema, 1 case of psoriasis). Cortisol was measured in plasma daily under treatment. In addition, gas chromatographic and mass spectrometric determinations of urocorticoids and uroandrogens are carried out in 24-h urine. A distinct, treatment-induced suppression of the endogenous steroid production was significant (-42.7%, p less than or equal to 0.05) within the first four days and diminished (-24.4%, p less than or equal to 0.10) after nine days' steroid therapy. The suppression of the plasma ACTH was also significant (-23.1%, p less than or equal to 0.05). The alpha-MSH level did not show any alterations. The measured urocorticoids showed an average fall of -38.9% (p less than or equal to 0.05). It is noteworthy that despite continued steroid treatment the marked initial suppression of the endogenous steroid production diminishes. It is possible that a therapy-induced "normalization" of the epidermis brings about a diminished steroid influx and thus a lower systemic load. Under the therapy conditions chosen here, the androgen metabolites in the urine did not show any significant fluctuations. PMID- 3017369 TI - Correlation between the presence of antibodies to the Epstein-Barr virus nuclear antigen type 2 and antibodies to the rheumatoid arthritis nuclear antigen in patients with rheumatoid arthritis. AB - The incidence of antibodies to Epstein-Barr nuclear antigen type 2 (EBNA-2) was determined in sera from rheumatoid arthritis (RA) patients and control subjects, by protein immunoblotting. Sixty-eight percent of the RA patients and 48% of the controls possessed anti-EBNA-2 antibodies. The titer of anti-rheumatoid arthritis nuclear antigen (RANA) in RA patient sera showed a stronger correlation with serum reactions to EBNA-2 than with reactions to EBNA-1. Our results indicate that the presence of EBNA-2 may make a major contribution to the RANA reaction. PMID- 3017372 TI - Modulation of the brain aversive system by GABAergic and serotonergic mechanisms. AB - Experiments performed in our laboratory, using electrical stimulation combined with microinjection of drugs in the dorsal midbrain central grey (CG) of the rat, evidenced that direct stimulation of GABA receptors with locally administered gamma-aminobutyric acid (GABA) or the GABAA receptor agonists 4,5,6,7 tetrahydroisoxazolo[5,4-c]pyridin-3-ol, isoguvacine and muscimol raised the aversive threshold, defined as the lowest electrical current intensity inducing flight or escape behaviour when applied to the dorsal CG. The GABAB receptor agonist baclofen was ineffective. Also, enhancement of endogenous GABA action through local injection of the benzodiazepines chlordiazepoxide and midazolam or of pentobarbital resulted in anti-aversive effects. Ro 15-1788 antagonized both chlordiazepoxide and midazolam, suggesting benzodiazepine receptor mediation. In contrast to pro-GABAergic drugs, microinjection of the GABA antagonists bicuculline and picrotoxin into the CG elicited flight behaviour, like the electrical stimulation. Similar experiments with drugs influencing serotonergic neurotransmission evidenced that intra-CG microinjection of serotonin (5-HT) or of the direct 5-HT receptor agonist 5-methoxy-N,N-dimethyltryptamine increased the aversive threshold. The anti-aversive effect of 5-HT was potentiated by the selective inhibitor of 5-HT neuronal uptake, zimelidine. Also, the latter drug increased the aversive threshold when given alone. The anti-aversive effect of 5 HT was antagonized by local pretreatment with either metergoline or ketanserin, the latter being a selective blocker of 5-HT2 receptors. In contrast to the GABA antagonists mentioned above, the 5-HT receptor blockers did not evoke aversive behaviour per se. Therefore, both GABAergic and serotonergic mechanisms are likely to play an inhibitory role in the dorsal CG integrating aversive behaviour.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017373 TI - Effects of basolateral amygdala lesions on taste aversions produced by lactose and lithium chloride in the rat. AB - In Experiment 1, intact rats were given either lactose or sucrose solutions. Although on first exposure they readily consumed lactose, its ingestion produced a conditioned taste avoidance which was partly extinguished by repeated sucrose exposure after lactose conditioning. In Experiment 2, rats with large bilateral electrolytic lesions of the basolateral amygdala and those with either sham or no operations were given two pairings of saline with LiCl injections (upper gastrointestinal tract discomfort) and in a separate condition access to high levels of lactose (lower gastrointestinal tract discomfort). Conditioned taste avoidances were measured both by two-bottle tests and by video recordings of the rats' orofacial and somatic responses. The lesions attenuated LiCl-induced taste aversion but not lactose-induced taste avoidance, results demonstrating that taste avoidance can occur without the basolateral amygdala. The results suggested that aversions based on distaste can be distinguished from avoidances based on danger, not only in terms of orofacial responses but also in terms of their neuroanatomical substrate. PMID- 3017375 TI - Focal brain stimulation attenuates morphine withdrawal behaviors. AB - Male albino rats were stereotaxically implanted with a bipolar stimulating electrode in the periaqueductal gray and also received a subcutaneous surgical implantation of a 72-mg morphine or a placebo pellet. Seventy-two hours following pellet implantation, naloxone-precipitated withdrawal was induced. Opiate withdrawal behaviors were observed and quantified. Animals then received focal brain stimulation for 30 min after which they were again observed for opiate withdrawal behaviors. The data suggest that focal brain stimulation attenuates morphine withdrawal behaviors, specifically the recessive behaviors associated with autonomic changes. These findings are consistent with those of other studies, from which it is speculated that more than one system is involved in the mediation of opioid dependence and analgesia. PMID- 3017374 TI - Oxytocin and vasopressin secretion in response to stimuli producing learned taste aversions in rats. AB - Administration of lithium chloride, copper sulfate, and apomorphine to rats each stimulated the secretion of oxytocin (OT) and, to a much lesser degree, arginine vasopressin. These agents are assumed to cause visceral illness in rats because of their effectiveness in promoting the acquisition of learned taste aversions. CuSO4 had a greater effect on plasma OT levels when administered ip rather than iv, whereas LiCl did not, results suggesting that LiCl probably stimulates OT secretion by central chemoreceptor activation whereas CuSO4 acts predominantly by local peritoneal irritation. A causal role for circulating OT in the acquisition of learned taste aversions was not found. These and other findings suggest that peripheral levels of OT may represent a quantifiable marker of visceral illness in rats and therefore might be useful in the interpretation of behavioral studies in which learned taste aversions are produced, provided that other stimuli of neurohypophyseal secretion are absent. PMID- 3017377 TI - Kinetics of the intracellular availability of heme after supplementing a heme deficient yeast mutant with 5-aminolevulinate. AB - The 5-aminolevulinic acid synthetase-deficient yeast mutant ole3 (Bard, M., Woods, R.A. & Haslam, J.M. (1974) Biochem. Biophys. Res. Commun. 56, 324-330), pregrown in a 5-aminolevulinic acid containing medium, can grow on glucose or galactose medium in the absence of heme for about 13 generations. When supplemented with 5-aminolevulinate before their 9th cell division, the cells can be induced to full respiratory competence. From the measurement of heme-dependent parameters (e.g. respiration, transcription of iso-1-cytochrome c mRNA and the post-translational proteolytic processing of the heme-free intermediate precursor of cytochrome c1 to its heme-containing mature form) it can be judged that heme is available in the cell about one hour after the addition of 5-aminolevulinate. The onset of respiration, however, does not occur to an appreciable extent before the 3rd hour of induction. The heme analogue deuteroporphyrin IX prevents respiratory adaptation but yet effects the transcription of heme-controlled genes. PMID- 3017376 TI - Chemical dependencies of learning in the rabbit olfactory bulb: acquisition of the transient spatial pattern change depends on norepinephrine. AB - Intracerebral cannulas were implanted in both olfactory bulbs of 6 rabbits. A surface electrode-array (8 X 8) was implanted epidurally on the lateral surface of the left bulb. Each rabbit was conditioned to respond to sniffing to an odor paired with cutaneous shock while receiving continuous intrabulbar infusion of either vehicle or propranolol (100 microM at 1 microliter/hr) in vehicle. After two training sessions to the original odor, a response to a new odor was conditioned under the influence of the alternate infusate. Electroencephalographic (EEG) activity was sampled on inspirations before and during odor presentations. During vehicle infusion a transient alteration in the pattern of activity was acquired that occurred during the second and third inspirations following presentation of the reinforced odor. The acquisition did not occur when propranolol was infused. No significant pattern changes occurred with unreinforced odors in either condition. There was no local anesthetic effect of the racemic mixture of propranolol found for any type of electric activity, including antidromic spike activity observed in an independent control group. Intrabulbar norepinephrine injection (100 microM, 10 microL) resulted in an amplitude increase of the bulbar 40-80-Hz EEG and a potentiation of the transient spatial pattern change to a novel odor, when compared with those observed during vehicle infusion. It is concluded that norepinephrine released under centrifugal control may act to prevent or delay habituation that otherwise occurs rapidly to unreinforced odors. PMID- 3017378 TI - The influence of forskolin on the metabolism of ecdysone and 20-hydroxyecdysone in isolated fat body of the blowfly, Calliphora vicina. AB - Rates of ecdysone and 20-hydroxyecdysone metabolism were measured by radio-assay in an in vitro system containing fat body isolated from blowfly larvae. The addition of forskolin which is known to stimulate artificially the intracellular adenylate cyclase system led to decreased rates of conversion of ecdysone into 20 hydroxyecdysone (= hormone activation) and of 20-hydroxyecdysone further to other metabolites (= hormone inactivation). The effect of forskolin was dose-dependent and reversible. Extracts prepared from larval brains were also tested. Some of them had the same effect as forskolin. It is concluded that the reactions leading from ecdysone to 20-hydroxyecdysone and further to hormonally inactive ecdysteroids are modulated by the intracellular level of cyclic nucleotides. We propose that a neurohormone is involved in the control of the reactions of the ecdysone metabolism. The observed new principle may contribute to the control of the titer of moulting hormone. PMID- 3017379 TI - Ernst Klenk Lecture, November 1985. Intracellular signalling through inositol trisphosphate and diacylglycerol. PMID- 3017380 TI - Natural antimicrobial systems and their potential in food preservation of the future. AB - The extensive literature on natural antimicrobial systems in animals, plants, and microorganisms is surveyed and particular systems are discussed, viz., the peroxidase systems in saliva and milk, singlet oxygen in the phagosome, cecropins and attacins in insects, complement, lysozyme and, to a limited extent, phytoalexins. The review draws attention to the cardinal role of targets on the cell envelopes of alien cells, especially bacteria, and emphasizes a possible approach to preservation based on the selection of specific agents for particular targets. The available evidence suggests that the perturbation of the homeostasis of all the organisms in the mixed flora of a food is unlikely to be achieved by one antimicrobial substance in isolation. Future studies need to consider therefore a tandem approach with two or more agents chosen because of their complementary action. Alternatively, natural system(s) and an established preservative method, either chemical or physical, warrant investigation. The extensive literature on the mechanisms whereby specialist pathogens overcome the defenses of plants and animals emphasizes the inherent dangers of selection leading to the persistent contamination of food processing areas with organisms tolerant of a particular antimicrobial system. PMID- 3017381 TI - Cloning, characterization, and tissue distribution of messenger RNA species expressed at moderate and scarce frequencies within the lactating guinea pig mammary gland. AB - In the lactating guinea pig mammary gland, the most abundant mRNA species encoding the major milk proteins, alpha-lactalbumin and caseins A, B, and C, have been extensively studied. Here we describe the isolation and characterization of cloned cDNA sequences representative of moderately abundant and scarce mammary gland mRNA species present at estimated concentrations of 1,400 (pgpO5), 540 (pgpKE6), 36 (pgpK1), and 2 (pgpJF4) copies per sequence per cell. RNA blotting showed these to represent mRNA species of 1,150, 1,900, 1,250, and 3,300 nucleotides in size, respectively. Hybrid selection cell-free synthesis showed that the mRNAs encoded proteins of Mr 33,000 (pgpO5), 58,000 (pgpKE6), and 36,000 (pgpK1). Studies on the tissue distribution of mammary gland mRNAs showed that the mRNA species of lower abundance, but not milk protein mRNAs, were expressed in other tissues but at concentrations differing from those in the mammary gland. None were expressed in all tissues, and so were not typical "housekeeping" proteins. We have used these cloned cDNA species to reinvestigate the apparent differential accumulation of moderately abundant poly(A)-containing mRNA species in polyadenylated and nonpolyadenylated cytoplasmic RNA populations of the mammary gland. Unlike previous observations, based on RNA excess hybridization using fractionated cDNA probes, the use of sequence-specific cloned cDNA probes showed that little intact mRNA was present in the nonpolyadenylated fraction. Thus previous observations were a reflection of the preferential accumulation of fragments of moderately abundant mRNA species, possibly a result of enhanced turnover. The significance of our results in terms of future investigations into factors which determine mRNA accumulation and tissue-specific expression is discussed. PMID- 3017382 TI - Human pleomorphic adenomas transplanted to nude mice. AB - Tissue from 13 human pleomorphic adenomas was transplanted to a total of 64 nude mice. Eight of the tumors were transplanted into a second passage of mice, 17 in all. In 39 mice of first passage, there was a definite increase in graft size. Microscopic examination showed no change in the histologic pattern from the donor tumor to the transplanted tissue. The heterochromatin pattern after staining with the DNA-specific fluorochrome D287/170 allowed distinction between human and murine cells and showed that both epithelial and mesenchymal cells were of human derivation. Autoradiographic studies with tritiated thymidine showed that both epithelial and mesenchymal tumor cells were labeled. Our results thus show that cell proliferation in the human pleomorphic adenoma takes place in epithelial areas as well as in mesenchymal areas. PMID- 3017384 TI - Pathologic quiz case 2. Paraganglioma (glomus jugulare). PMID- 3017383 TI - Glomus tumor of the facial canal. A case report. AB - Glomus tumors of the temporal bone are usually classified as either glomus tympanicum or glomus jugulare, depending on the site of origin. This report describes another anatomical site within the temporal bone from which glomus tumors may arise--the facial canal. PMID- 3017385 TI - Photoinactivation of acetylcholinesterase by erythrosin B and related compounds. AB - Acetylcholinesterase was rapidly inactivated when exposed to light in the presence of xanthene dyes. Photosensitizing efficiency paralleled the dye triplet state quantum yields, increasing in the order fluorescein less than eosin B less than eosin Y less than erythrosin B less than rose bengal. The observed first order rate constants of photoinactivation increased hyperbolically with dye concentration. Evidence for the formation of a dye-enzyme complex prior to inactivation was obtained from spectrophotometric and protein fluorescence quenching methods. The latter technique allowed estimates of the dye-enzyme dissociation constants for rose bengal (20 microM) and erythrosin B (30 microM). After photoinactivation, a portion of the dye became covalently bound to the enzyme. The photoinactivation reaction occurs in both aerobic (air saturated) and anaerobic (argon saturated) solution, with the rates of photoinactivation being about three to five times greater under the latter conditions. The aerobic reaction exhibits a large deuterium isotope enhancement effect and is largely (but not completely) quenched by 10(-2) M azide. The anaerobic reaction is unaffected by azide and exhibits only a small deuterium isotope effect. These results indicate that the photoinactivation reaction proceeds mainly by a type II (singlet oxygen mediated) pathway under aerobic conditions and by a type I (radical) pathway under anaerobic conditions. The enzyme was protected from inactivation by edrophonium, a competitive inhibitor, but not by d-tubocurarine, a peripheral-site ligand, indicating that destruction of a crucial residue at or near the catalytic site is an important component of the inactivation process. Extensive destruction of tryptophan undoubtedly occurs, at least under aerobic conditions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017386 TI - Rapid automated synthesis via diisopropyl phosphoramidite in situ activation. Chemical synthesis and cloning of a calmodulin gene. AB - A gene coding for a calmodulin was synthesized and cloned. The chemical synthesis of the gene, coding for 149 amino acids, was achieved by the enzymatic ligation of 61 chemically synthesized DNA fragments. The DNA fragments were synthesized using a solid support with a diisopropyl phosphoramidite intermediate and in situ activation. The automated standard cycle time was 10 min/addition. The synthesizer was designed and constructed from inexpensive, readily available parts and controlled by a Commodore 64 computer. The gene possesses 27 unique, regularly spaced, restriction endonuclease cleavage sites to facilitate gene mutagenesis. PMID- 3017387 TI - Regulation of rat liver phosphatidylethanolamine N-methyltransferase by cytosolic factors. Examination of a role for reversible protein phosphorylation. AB - The nature of cytosolic factors which modulate the activity of rat liver phosphatidylethanolamine (PE) methyltransferase was investigated. The combined additions of cytosol, Mg X ATP, and NaF to incubations with rat liver microsomes produced a 1.6-fold activation of the methyltransferase at pH 9.2 and a 1.3-fold stimulation at pH 7.0. Nonhydrolyzable 5'-adenylylimidodiphosphate could not substitute for ATP, although GTP could. The activation was time dependent, stable to reisolation of the microsomes by ultracentrifugation, and partially preventable by other cytosolic components. Despite these indications that PE methyltransferase might be a substrate for cytosolic protein kinases, cAMP and Ca2+-calmodulin exerted little influence on the activation reaction. Furthermore, microsomal PE methyltransferase activity was unaffected by purified preparations of cAMP-dependent protein kinase, calmodulin-dependent protein kinase, and casein kinase II, nor was methyltransferase activity influenced by the purified catalytic subunits of protein phosphatases 1 and 2A. Cytosol also contained inhibitors of PE methyltransferase which could overcome the Mg X ATP X NaF mediated activation of the enzyme, but were not affected by the thermostable phosphatase inhibitors 1 and 2. Part of this inhibitory activity (apparent molecular mass of 15 X 10(3) daltons) was insensitive to trypsin and chymotrypsin, stimulated by Mn2+, and partly inhibited by NaF. Therefore, regulation of methyltransferase by reversible phosphorylation, while still a tenable hypothesis, is apparently more complex than previously proposed. PMID- 3017388 TI - [Analysis of 240 cases of pleomorphic adenoma]. PMID- 3017389 TI - [Reconstruction of central nervous circuitries by axonal regeneration]. PMID- 3017390 TI - AIDS and intravenous drug abusers. PMID- 3017391 TI - Differential effects of atenolol and enalapril on memory during treatment for essential hypertension. AB - A randomized single-blind study was designed to compare the performance on memory tests requiring recall of information relevant to everyday life of two groups of hypertensive patients. One group of 13 patients were taking a beta-adrenoceptor blocker (atenolol) and the other group of 12 patients received the angiotensin converting enzyme inhibitor (enalapril). The results suggested that when compared with placebo the group of patients treated with enalapril showed no changes in memory function, whilst there was a mild, but consistent deficit in the group taking atenolol. PMID- 3017393 TI - Ageing and platelet alpha 2-adrenoceptors. PMID- 3017394 TI - The effect of a high fibre, low fat, low sodium diet on diabetics with intermittent claudication. PMID- 3017392 TI - The effect of moderate urine alkalinisation on low dose diethylcarbamazine therapy in patients with onchocerciasis. AB - Twenty-one patients with moderate to heavy infections with O. volvulus were treated with 25 mg of diethylcarbamazine (DEC) citrate twice daily for 10 days. In 11 patients the urine was made alkaline with sodium bicarbonate, 2 g, administered 6 hourly for three doses daily beginning 1 day before DEC was started and continued throughout the DEC therapy. Ten patients served as controls. The mean pre-dose plasma DEC concentration during treatment and the mean plasma DEC half-life were significantly higher in bicarbonate treated patients as compared to controls. Total urinary excretion of DEC was significantly less in the bicarbonate treated group than in controls. Mean overall total reaction was higher in bicarbonate-treated patients but the difference was not significant. The bicarbonate-treated group achieved a significantly greater reduction in skin microfilarial counts than the control group as assessed 1 week after completion of therapy, but there was little difference at 1 month. Microfilarial killing was associated with microfilarial mobilisation, alteration in peripheral leucocytes and elevation in serum aminotransferases in both groups. There was no effect of DEC on the number of adult worms recovered in nodules removed at the end of the therapy. This study indicates that moderate urinary alkalinisation alters the kinetics of DEC and the therapeutic response. However the severity of clinical reaction coupled with the inadequate level of microfilarial killing achieved make it unlikely that manipulation of urinary pH will be of practical value in onchocerciasis chemotherapy. PMID- 3017395 TI - Inadvertent immunosuppression as a health hazard. PMID- 3017396 TI - Expression of epidermal growth factor receptor (EGF-R) in human lung tumours. AB - Epidermal growth factor receptor (EGF-R) expression was assessed in 63 lung tumour samples with a monoclonal antibody (EGF-R1) by indirect immunoperoxidase staining on cryostat sections. All 15 small cell lung cancer samples were negative whereas over 80% of the 48 non small lung cancer stained positively. In 30 bronchial biopsies two monoclonal antibodies against the cytoplasmic part of the EGF-R were evaluated. These antibodies showed weaker staining than EGF-R1. No additional or enhanced staining as compared with EGF-R1 was observed, suggesting a lack of enhanced expression of a truncated EGF-R analogous to the v-erb-B oncogene product. Monoclonal antibodies against the EGF-R may be helpful diagnostically in differentiating small cell from non small cell lung cancer and may also be important in elucidating biological differences in primary lung cancer. PMID- 3017398 TI - Enhanced transplantability of human ovarian cancer lines in cyclophosphamide pretreated nude mice. PMID- 3017397 TI - Receptors for EGF and oestradiol and thymidinekinase activity in different histological subgroups of human mammary carcinomas. AB - The cellular content of receptors for epidermal growth factor (EGF) was measured in different histological subgroups of human mammary carcinomas. EGF receptors were detected in 36% of the ductal and all the medullary carcinomas. In contrast lobular and pure colloid tumours did not contain measurable amounts of the receptor. The receptor was found both among tumours with an euploid and aneuploid DNA pattern. The EGF receptor is thus found in carcinomas with a varying degree of differentiation as judged by the cellular DNA pattern. There was no correlation between the proliferative activity of the tumours as measured by thymidinekinase activity and the amount of EGF receptors in the tumour. Tumours with detectable EGF receptor often had low levels of oestrogen receptor. This finding could only partly be explained by the menstrual status of the patients. PMID- 3017399 TI - Antibody dependent cellular cytotoxicity against coronavirus 229E-infected cells. AB - An antibody dependent cellular cytotoxic (ADCC) reaction was elicited using human mixed lymphocyte cultures against coronavirus 229E-infected C-16 cells. The response was assayed in microtitre plates by radioactive chromium release. Optimal killing of target cells was achieved using cells labelled 22 h after infection. A prozone was present and the optimal dilution of sera was invariably 1:200. The majority (70%) of sera tested gave a positive ADCC response. These included all sera judged positive by ELISA, but also some which were judged negative. Two experiments using purified IgG from serum which was ADCC and ELISA positive suggested that the response is mediated by an IgG antibody and that the prozone is due to a separate serum component. PMID- 3017401 TI - Itraconazole therapy in pityriasis versicolor. AB - The efficacy of itraconazole was assessed in an open trial in 30 patients with disseminated lesions of pityriasis versicolor confirmed by direct microscopy. The patients were allocated randomly to one of two treatment regimens, 200 mg once daily for 5 days or 100 mg once daily for 10 days. On assessment 3 weeks after the end of the treatment, 25 patients were healed, two patients had mild residual lesions, two had considerable residual lesions and one patient had relapsed. One patient reported dyspepsia and one patient reported stomach ache. One patient had asymptomatic elevation of serum transaminase (GOT and GPT) but this had returned to normal 3 weeks after the end of therapy. PMID- 3017400 TI - Elastase, a marker for neutrophils in skin infiltrates. AB - The enzyme elastase (EC 3.4.21.37) has proved to be a convenient and extremely sensitive marker for the quantification of neutrophils in cutaneous infiltrates. Fluorometric assay using the synthetic substrate MeOSuc-Ala-Ala-Pro-Val-N methylcoumarin permitted the measurement of this enzyme in as few as five cells and was linear up to about 1000 cells per sample. The mean activity of lysates of human blood-derived neutrophils was 0.57 +/- 0.08 pmol of 7-amino-4-methyl coumarin released per hour per neutrophil. Extracts of normal human skin contained no measurable elastase activity but resulted in a slight inhibition of the neutrophil enzyme (mean 12%). Application to the in vivo situation has been demonstrated by the use of leukotriene B4 as chemotactic agent. A reproducible neutrophil infiltrate was found at a dose of 2 ng, well below the threshold for the appearance of microabscesses. PMID- 3017402 TI - Intracellular binding proteins for retinol and retinoic acid in early and term human placentas. AB - Specific binding of both [3H]retinol and [3H]retinoic acid was observed in the cytosol fraction from term placentas and specific binding of [3H]retinol but not [3H]retinoic acid was detected in the cytosol fraction from placentas of 8-12 week pregnancies. The elution volume of the bound radioactivity on Sephadex G-100 column chromatography was within the range expected for proteins of molecular weight 14 500, in agreement with the results of others for cellular retinol- and retinoic acid-binding proteins from other tissues. The role of these proteins in mediating the effects of vitamin A on growth and differentiation of the placenta and fetus has yet to be determined. PMID- 3017403 TI - Intraepithelial neoplasia arising in an artificial vagina. Case report. PMID- 3017404 TI - Eccrine spiradenoma of eyelid: case report. AB - A case report is presented of eccrine spiradenoma, a benign sweat gland tumour which, though not uncommon generally, is rare in the eyelid. The possibility of sweat gland tumour should be kept in mind in the diagnosis of eyelid tumours. PMID- 3017405 TI - Interaction of cytochrome c with mixed dimyristoylphosphatidylcholine dimyristoylphosphatidylserine bilayers: a deuterium nuclear magnetic resonance study. AB - Deuterium nuclear magnetic resonance (2H NMR) was used to study the interaction of cytochrome c (from horse heart) with bilayers of mixed dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylserine (DMPS). Three types of labeled lipids were used: chain-perdeuterated phosphatidylcholine (DMPC-d54), chain-perdeuterated phosphatidylserine (DMPS-d54), and phosphatidylserine labeled at the alpha-position of the head group (DMPS-d2). Liposomes containing equimolar mixtures of DMPC and DMPS were found to bind cytochrome c with a maximum ratio of about 1 mg of cytochrome c per 1 mg of DMPS. The 2H NMR spectra of equimolar mixtures of DMPC-d54-DMPS and DMPC-DMPS-d54 were examined with and without cytochrome c. No change of the NMR spectra of either DMPC or DMPS could be detected after protein addition, for temperatures both above and below the phospholipid phase transition region. On the other hand, in the liquid-crystalline state, the transverse relaxation time, T2e, was reduced by 30-40% after protein addition. Measurements of the spin-lattice relaxation time, T1, showed, under all circumstances, multiple components. For simplicity, we have examined the shape of the relaxation curves at short and long times. Addition of protein increased by 2-fold the value of the slow T1 component of DMPS-d54 but not that of DMPC-d54. Partially relaxed spectroscopy allowed us to assign this slow component (at least in part) to the methyl group and C2H2 groups near the methyl end of the chains, i.e., far from the binding sites of the extrinsic protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017407 TI - Characterization of the Ca2+-induced conformational changes in gelsolin and identification of interaction regions between actin and gelsolin. AB - Serum gelsolin, a Ca2+-dependent protein regulating the length of actin filaments, undergoes conformational changes upon binding Ca2+. These were detected and analyzed by several approaches including ultraviolet difference spectroscopy, circular dichroism studies, analytical ultracentrifugation, thiol group titration, and limited proteolytic digestions. The effect of Ca2+ binding on the UV absorption difference spectrum and the near-UV circular dichroism spectrum was consistent with changes in the environments of tyrosine and phenylalanine residues. In the presence of Ca2+, the S0(20),w value decreased from 5.3 to 4.7. This latter result implies a transformation to a more asymmetric molecular shape. Gelsolin contained only two accessible thiol groups per mole of protein, one of which was titratable in the native protein; it was more accessible to 5,5'-dithiobis(2-nitrobenzoic acid) in the absence than in the presence of Ca2+. The limited digestion of gelsolin from serum and bovine aorta smooth muscle by two different proteases, chymotrypsin and trypsin, proceeded much faster in the presence of Ca2+ than in its absence with the production of three main fragments of about 40K, 32K, and 21K. This fragment mixture was found still able to shorten F-actin in a Ca2+-dependent manner; this severing activity was expressed by the isolated 40K peptide. Gelsolin was cross-linked to F- and G actin by the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide (EDC), generating a covalent 130K binary complex (actin1-gelsolin1) followed by a covalent 180K ternary complex (actin2-gelsolin1).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017406 TI - Importance of the 10-13 region of glucagon for its receptor interactions and activation of adenylate cyclase. AB - The role of the Tyr10-Ser11-Lys12-Tyr13 region of glucagon in the binding interaction and activation of the glucagon receptor was investigated by means of the synthetic glucagon analogues [Phe13]glucagonamide, [Phe10]glucagonamide, [Phe10]glucagon, [Phe10,13]glucagon, [Pro11]glucagon, [Pro11,Gly12]glucagonamide, [Ala11]glucagon, and [Oac11-13]glucagonamide. These analogues were synthesized by solid-phase peptide synthesis on p-methylbenzhydrylamine or Merrifield resins with protected N alpha-tert-butyloxycarbonyl amino acids. Purification by dialysis, cation-exchange chromatography, gel filtration, and preparative reverse phase high-performance liquid chromatography (HPLC) gave products that proved homogeneous by thin-layer chromatography and HPLC and on analysis by amino acid analysis, by sequencing, and by alpha-chymotryptic peptide mapping with HPLC. Biological activities were examined by measurement of the stimulation of liver plasma membrane adenylate cyclase and by specific displacement of [125I]glucagon from glucagon receptors. The results of these studies indicate that while the biological "message" region of glucagon is located elsewhere, the 10-13 region has multiple roles in the glucagon-glucagon receptor interaction: this region provides functional groups for direct binding interaction with the receptor, and this region interacts with the receptor in such a way as to allow the "transduction message" portion of glucagon to interact and activate the receptor. PMID- 3017408 TI - Evidence that phosphorylase kinase exhibits phosphatidylinositol kinase activity. AB - Phosphorylase kinase phosphorylates the pure phospholipid phosphatidylinositol. Furthermore, it catalyzed phosphatidylinositol 4-phosphate formation using as substrate phosphatidylinositol that is associated with an isolated trypsin treated Ca2+-transport adenosinetriphosphatase (ATPase) preparation from skeletal muscle sarcoplasmic reticulum. On this basis a fast and easy assay was developed that allows one to follow the phosphatidylinositol kinase activity during a standard phosphorylase kinase preparation. Both activities are enriched in parallel approximately to the same degree. Neither chromatography on DEAE cellulose nor that on hydroxyapatite in the presence of 1 M KCl separates phosphatidylinositol kinase from phosphorylase kinase. The presence of a lipid kinase, phosphatidylinositol kinase, in phosphorylase kinase is not a general phenomenon; diacylglycerol kinase can be easily separated from phosphorylase kinase. Polyclonal anti-phosphorylase kinase antibodies as well as a monoclonal antibody directed specifically against the alpha subunit of phosphorylase kinase immunoprecipitate both phosphorylase kinase and phosphatidylinositol kinase. PMID- 3017409 TI - Binding of coagulation factor XI to washed human platelets. AB - The binding of human coagulation factor XI to washed human platelets was studied in the presence of zinc ions, calcium ions, and high molecular weight kininogen. Significant factor XI binding occurred at physiological levels of these metal ions when high molecular weight kininogen was present. Binding required platelet stimulation and was specific, reversible, and saturable. Scatchard analysis of the binding yielded approximately 1500 binding sites per platelet with an apparent dissociation constant of approximately 10 nM. Since the concentration of factor XI in plasma is about 25 nM, this suggests that in plasma factor XI binding sites on stimulated platelets might be saturated. Calcium ions and high molecular weight kininogen acted synergistically to enhance the ability of low concentrations of zinc ions to promote factor XI binding. The similarity between the concentrations of metal ions optimal for factor XI binding and those optimal for high molecular weight kininogen binding, as well as the ability of high molecular weight kininogen to modulate these metal ion effects, implies that factor XI and high molecular weight kininogen may form a complex on the platelet surface as they do in solution and on artificial negatively charged surfaces. PMID- 3017410 TI - Deglycosylated human follitropin: characterization and effects on adenosine cyclic 3',5'-phosphate production in porcine granulosa cells. AB - Chemical deglycosylation of gonadotropins with hydrogen fluoride (HF) has facilitated the investigation of the structure-function relationship of the individual peptide and oligosaccharide moieties in the mechanism of hormone action. These studies have dealt almost exclusively with lutropin or human choriogonadotropin. We report here the chemical characterization and biological properties of deglycosylated human follitropin (degly hFSH). Results indicate that deglycosylation of hFSH by HF removes 89% of the total carbohydrate without disruption of the peptide chain or significant loss of amino acid residues. However, a change in the conformation of the molecule was observed by measurement of the far-ultraviolet circular dichroic spectrum. The degly hFSH showed a 44% reduction in binding when tested in a FSH radioimmunoassay utilizing a polyclonal antibody. Binding of the degly hFSH to FSH-responsive tissues showed that the altered hormone bound with equal or better avidity than the intact hormone while the association constants were approximately the same for both preparations. The degly hFSH alone did not stimulate the FSH-stimulatable adenylyl cyclase (AC) activity of cellular homogenates of small follicle porcine granulosa cells. Furthermore, degly hFSH was a potent antagonist of hFSH-stimulatable AC activity when coincubated with intact hFSH. In intact granulosa cells, both the hFSH and the degly hFSH stimulated cAMP production and release by these cells. However, the degly hFSH was one-tenth as effective as the intact hormone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017412 TI - Bending and flexibility of kinetoplast DNA. AB - We have evaluated the extent of bending at an anomalous locus in DNA restriction fragments from the kinetoplast body of Leishmania tarentolae using transient electric dichroism to measure the rate of rotational diffusion of DNA fragments in solution. We compare the rate of rotational diffusion of two fragments identical in sequence except for circular permutation, which places the bend near the center in one case and near one end of the molecule in the other. Hydrodynamic theory was used to conclude that the observed 20% difference in rotational relaxation times is a consequence of an overall average bending angle of 84 +/- 6 degrees between the end segments of the fragment that contains the bending locus near its center. If it is assumed that bending results from structural dislocations at the junctions between oligo(dA).oligo(dT) tracts and adjacent segments of B DNA, a bend angle of 9 +/- 0.5 degrees at each junction is required to explain the observations. The extent of bending is little affected by ionic conditions and is weakly dependent on temperature. Comparison of one of the anomalous fragments with an electrophoretically normal control fragment leads to the conclusion that they differ measurably in apparent stiffness, consistent with a significantly increased persistence length or contour length in the kinetoplast fragments. PMID- 3017411 TI - Kinetic studies of Sendai virus-target membrane interactions: independent analysis of binding and fusion. AB - Fusion between Sendai virus and liposomes containing phosphatidylethanolamine (PE) and different mole fractions of ganglioside GD1a has been investigated. At different times after mixing the virus and liposomes, the mixture was diluted with a sucrose solution and centrifuged in an airfuge to separate the free and virus-associated liposomes. Since the HN protein of the virus was sensitive to the reducing reagent, inclusion of dithiothreitol in the sucrose solution dissociated the bound but not the fused liposomes. Thus, the kinetics of liposome virus binding and fusion could be independently measured. The validity of the assay was confirmed by electron microscopic observation of the virus-liposome mixtures. With trypsin-treated Sendai virus, in which the F glycoprotein of the virus had been selectively removed, only virus-liposome binding but not fusion was observed. The kinetic experiments were done under the condition of virus in large excess. Following a very fast initial binding phase, which was completed at the "zero time" of the measurement, the virus-liposome binding followed pseudo first-order kinetics. The subsequent fusion step was zero order. Judging from the discontinuity in the Arrhenius plots, both binding and fusion events were sensitive to the gel-liquid-crystalline phase transition of the target membrane. The binding rate constants had activation energies between 16 and 23 kcal/mol at temperatures above the transition. They were not sensitive to temperature change at temperatures below the transition. On the other hand, the fusion rate constants were not sensitive to temperature change above the transition, except for 6.3% GD1a liposomes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017413 TI - Use of substituted (benzylidineamino)guanidines in the study of guanidino group specific ADP-ribosyltransferase. AB - A number of substituted (benzylidineamino)guanidines with different substitutents in the benzene nucleus are synthesized by coupling substituted benzaldehydes with aminoguanidine, and these compounds are tested as substrates for cholera toxin catalyzed ADP-ribosylation. A spectrophotometric assay method for the measurement of ADP-ribosyltransferase activity is developed, making use of the absorption characteristics of some of these compounds and the difference in the ionic character of the free compounds and the ADP-ribosylated products. The kinetic parameters for the ADP-ribosylation of these compounds are evaluated. A correlation between log kcat or log (kcat/Km) and the Hammett substituent constant sigma is observed. This correlation suggests the importance of substrate electronic effects on the enzymatic reaction. The reactivity of these compounds as acceptors of ADP-ribosyl groups in the reaction catalyzed by cholera toxin increases with increasing electron-donating power of the substituents in the benzene function. The effect is primarily on the catalytic rate constant, kcat, not on the binding constant, Km. The results are consistent with an SN2 reaction mechanism in which the deprotonated guanidino group makes a nucleophilic attack on the C-1 carbon of the ribose moiety. PMID- 3017415 TI - Methionine-80-sulfoxide cytochrome c: preparation, purification and electron transfer capabilities. AB - In order to explore the electron-transferring properties of methionine-80 sulfoxide cytochrome c, the pure, chromatographically homogeneous methionine-80 sulfoxide cytochrome c was previously published procedure (Ivanetich, K.M., Bradshaw, J.J. and Kaminsky, L.S. (1976) Biochemistry 15, 1144-1153) was found to produce a mixture of products. In the pure derivative, visible spectroscopy indicates that the 695 nm band indicative of the Met-80-Fe coordination is missing, amino acid analysis indicates that only one methionine is modified to the sulfoxide, and the E0' is found to be 240 mV vs. N.H.E. For succinate cytochrome c reductase activity, the Km for modified cytochrome was about one ninth that of the native protein, while the maximum turnover number of the reductase with the modified protein was only about 54% of that with native protein. In contrast, the activity with cytochrome oxidase measured polarographically using ascorbate and TMPD under two different buffer/pH conditions, gave Km values that were very similar for both the native and modified cytochromes c, but the maximum turnover numbers of the oxidase with the modified protein were less than 40% of native in either buffer. It is concluded that the Met-80-sulfoxide cytochrome c in the reduced form is able to maintain substantially its heme crevice structure and thus maintain Km values similar to those of native protein. However, the low maximum turnover numbers for oxidase activity with the modified protein in the reduced state indicate that electron transfer itself has been significantly decreased, probably because the parity of acid/base and electrostatic interactions of Met-80 sulfur with the Fe in the two redox states has been disrupted. PMID- 3017414 TI - Rat hepatic (Na+, K+)-ATPase: alpha-subunit isolation by immunoaffinity chromatography and structural analysis by peptide mapping. AB - The catalytic alpha-subunit of rat hepatic (Na+, K+)-ATPase (EC 3.6.1.3) has been isolated by immunoaffinity chromatography from microsomes solubilized in n dodecyl octaethylene glycol monoether. The procedure employs an anticatalytic mouse monoclonal antibody ("9-A5") covalently linked to Sepharose 4B that specifically blocks phosphorylation of the sodium pump's alpha-subunit from [gamma-32P]ATP [Schenk, D. B., Hubert, J.J., & Leffert, H.L. (1984) J. Biol. Chem. 259, 14941-14951]. The hepatic subunit is virtually identical with purified rat, dog, and human renal alpha-subunits as judged by its apparent molecular weight after polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Mr 92K) and its two-dimensional tryptic and chymotryptic peptide maps on cellulose coated thin-layer plates. In contrast, the structures of authentic renal beta subunits from the three species differ significantly from each other as judged by their peptide maps; no detectable homologies are seen between their chymotryptic maps and those of putative hepatic "beta"-subunits (Mr 50K and 55K) eluted from 9 A5-Sepharose. Additional studies of ouabain-sensitive 86Rb+ uptake in primary cultures of adult rat hepatocytes reveal inhibition curves with single inflection points (ID50 = 0.1 mM ouabain) in the absence or presence of pump-stimulating peptides like insulin, glucagon, and epidermal growth factor. These findings indicate that rat hepatocytes express only one of two known structurally conserved forms of catalytic subunit (the renallike alpha form) and, if at all, structurally divergent forms of the sodium pump's beta-subunit. In addition, immunoaffinity chromatography with 9-A5-Sepharose facilitates the isolation of (Na+, K+)-ATPases from nonrenal tissues with low levels of sodium pumps. PMID- 3017416 TI - Interaction between blood components and a spin-labeled analogue of PAF-acether. AB - The interactions between a spin-labeled analogue of PAF-acether (designated as (0,2)PAF) and different human blood components (platelets, erythrocytes, and serum) have been studied. The rate of spin probe reduction by cytosol provided information about the internalization processes when the hydrolysis rate was also available. Although erythrocyte reactivity is lower than that of platelets, erythrocytes, because of their greater numbers, removed (0,2)PAF from whole blood faster than platelets. Lastly, erythrocytes may be more efficient traps for (0,2)PAF than serum acetylhydrolase. Criteria for extending these results to genuine PAF-acether are also discussed. PMID- 3017417 TI - Fusion of Sendai virus with negatively charged liposomes as studied by pyrene labelled phospholipid liposomes. AB - Sendai virus particles fuse with negatively charged liposomes but not with vesicles made of zwitterionic phospholipids. The liposome-virus fusion process was studied by dilution of the concentration-dependent excimer-forming fluorophore 2-pyrenyldodecanoylphosphatidylcholine contained in the liposomes by the viral lipids. The data were analyzed in the framework of a mass action kinetic model. This provided analytical solutions for the final levels of probe dilution and numerical solutions for the kinetics of the overall fusion process, in terms of rate constants for the liposome-virus adhesion, deadhesion and fusion. This analysis led to the following conclusions: At neutral pH and 37 degrees C, only 15% of the virus particles can fuse with the phospholipid vesicles, although all the virions may aggregate with the liposomes. The rate constants for aggregation, fusion and deadhesion are of the orders of magnitude of 10(7) M-1 X s-1, 10(-3) s-1 and 10(-2), s-1, respectively. The fraction of active virus increases with temperature. At acidic pH, both the fraction of 'fusable' virus and the rate of fusion increase markedly. The optimal pH for fusion is 3-4, where most of the virus particles are active. At higher pH values, an increasing fraction of the virus particles become inactive, probably due to ionization of viral glycoproteins, whereas at pH values below 3.0 the fusion is markedly reduced, most likely due to protonation of the negatively charged vesicles. While only 15% of the virions fuse with the liposomes at pH 7.4 and 37 degrees C, all the liposomes lose their content (Amselem, S., Loyter, A. Lichtenberg, D. and Barenholz, Y. (1985) Biochim. Biophys. Acta 820, 1-10). We therefore propose that release of entrapped solutes is due to liposome-virus aggregation, and not to fusion. Both trypsinization and heat inactivation of the virus particles inhibit not only the fusion process but also the release of carboxyfluorescein. This demonstrates the obligatory role of viral membrane proteins in liposome-virus aggregation. Reconstituted vesicles made of the viral lipid and the hemagglutinin/neuraminidase (HN) glycoprotein fuse with negatively charged liposomes similar to the intact virions. This suggests that the fusion of virions with negatively charged vesicles, unlike the fusion of the virus with biological membranes, requires only the HN and not the fusion glycoprotein. PMID- 3017418 TI - Control of red cell urea and water permeability by sulfhydryl reagents. AB - The binding constant for pCMBS (p-chloromercuribenzenesulfonate) inhibition of human red cell water transport has been determined to be 160 +/- 30 microM and that for urea transport inhibition to be 0.09 +/- 0.06 microM, indicating that there are separate sites for the two inhibition processes. The reaction kinetics show that both processes consist of a bimolecular association between pCMBS and the membrane site followed by a conformational change. Both processes are very slow and the on rate constant for the water inhibition process is about 10(5) times slower than usual for inhibitor binding to membrane transport proteins. pCMBS binding to the water transport inhibition site can be reversed by cysteine while that to the urea transport inhibition site can not be reversed. The specific stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamidostilbene-2,2' disulfonate) causes a significant change in the time-course of pCMBS inhibition of water transport, consistent with a linkage between anion exchange and water transport. Consideration of available sulfhydryl groups on band 3 suggests that the urea transport inhibition site is on band 3, but is not a sulfhydryl group, and that, if the water transport inhibition site is a sulfhydryl group, it is located on another protein complexed to band 3, possibly band 4.5. PMID- 3017419 TI - Effects of ethinyl estradiol on intestinal membrane structure and function in the rabbit. AB - Structural and functional properties of the small intestinal microvillus membrane were evaluated in the rabbit after administration of ethinyl estradiol, a synthetic estrogen with a demonstrated propensity to alter hepatic membrane lipid fluidity, and promote cholestasis. In the jejunum, no estrogen-induced changes in microvillus membrane total lipid, cholesterol or phospholipid content were observed. However, the ileal microvillus membrane in estradiol-treated animals demonstrates significant reductions vs. controls (per mg protein) in total lipid (0.55 milligrams vs. 0.89 milligrams) [corrected] and phospholipid (206.7 micrograms vs. 304.91 micrograms) (p less than 0.001) content, as well as modifications in specific phospholipid species. The increase in the ileal microvillus membrane cholesterol: phospholipid molar ratio (0.65 vs. 0.51, p less than 0.05) was associated with a significant decrease in membrane lipid fluidity reflected by an increase in fluorescence anisotropy measurements utilizing diphenyl hexatriene as the fluorophore (r at 25 degrees C = 0.306 vs. 0.282, p less than 0.05). Thermotropic lipid phase transitions, assessed by Arrhenius plots of both fluorescence data and ileal microvillus membrane p nitrophenylphosphatase activity demonstrate that phase changes occur between and 24 and 28 degrees C in both treated and untreated groups. Within the temperature range studied (40-10 degrees C) no differences from control were observed in microvillus membrane alkaline phosphatase activity following estrogen treatment. These data therefore indicate that ethinyl estradiol-induced effects on microvillus membrane lipid composition and physical properties occur predominantly in the ileum and appear to be related, in part, to specific alterations in the availability of phospholipid following estrogen treatment. PMID- 3017420 TI - Anchoring of phospholipase A2: the effect of anions and deuterated water, and the role of N-terminus region. AB - The effect of anions and deuterated water on the kinetics of action of pig pancreatic phospholipase A2 is examined to elaborate the role of ionic interactions in binding of the enzyme to the substrate interface. Anions and deuterated water have no significant effect on the hydrolysis of monomeric substrates. Hydrolysis of vesicles of DMPMe (ester) is completely inhibited in deuterated water. The shape of the reaction progress curve is altered in the presence of anions. The nature and magnitude of the effect of anions depends upon the nature of the substrate as well as of the anion. Substantial effects of anions on the reaction progress curve are observed even at concentrations below 0.1 M and the sequence of effectiveness for DMPMe vesicles is sulfate greater than chloride greater than thiocyanate. Apparently, anions in the aqueous phase bind to the enzyme, and thus compete with the anionic interface for binding to the enzyme. Binding of the enzyme to anionic groups on the interface results in activation and increased accessibility of the catalytic site possibly via hydrogen bonding network involving water molecule. In order to elaborate the role of the N-terminus region in interfacial anchoring, the action of several semisynthetic pancreatic phospholipase A2s is examined on vesicles of anionic and zwitterionic phospholipids. The first-order rate constant for the hydrolysis of DMPMe in the scooting mode by the various semisynthetic enzymes is in a narrow range: 0.7 +/- 0.15 per min for phospholipase A2 derived from pig pancreas and 0.8 +/- 0.4 per min for the enzymes derived from bovine pancreas. In all cases a maximum of about 4300 substrate molecules are hydrolyzed by each phospholipase A2 molecule. If anions are added at the end of the first-order reaction progress curve, a pseudo-zero-order reaction progress curve is observed due to an increased intervesicle exchange of the bound enzyme. These rates are found to be considerably different for different enzymes in which one or more amino acids in the N-terminus region have been substituted. Steady-state and fluorescence life time data for these enzymes in water, 2H2O and in the presence of lipids is also reported. The kinetic and binding results are interpreted to suggest that the N terminus region of phospholipase A2 along with some other cationic residues are involved in anchoring of phospholipase A2 to the interface, and the catalytically active enzyme in the interface is monomeric. PMID- 3017421 TI - Spin label study of local anesthetic-lipid membrane interactions. Phase separation of the uncharged form and bilayer micellization by the charged form of tetracaine. AB - The interaction between tetracaine and egg phosphatidylcholine (egg PC) multibilayers was examined. ESR spectra of an ester spin label indicate that at low uncharged anesthetic: lipid ratios, membrane organization decreases. At higher ratios, saturation and phase separation occur, as suggested by a second spectral component which appears when the water solubility of tetracaine is reached. However, experiments with the drug in the absence and in the presence of membranes, making use of a phospholipid spin label, suggest that the new phase does not consist of solid tetracaine alone. Location of the new phase in the membrane would require a change in partition coefficient, while its location outside would imply a mechanism whereby the anesthetic would come off the membrane as an aggregate containing spin probe and phospholipid. Charged tetracaine forms micelles which disrupt-unilamellar egg PC vesicles (Fernandez, M.S. (1981) Biochim. Biophys. Acta 646, 27-30). Micellar tetracaine added to bilayers containing a PC spin probe changes the spectrum from one typical of a bilayer into one typical of micelles, indicating the formation of a tetracaine egg PC mixed micelle. The effect is reversible upon dilution to concentrations below the critical micelle concentration of tetracaine. When membranes are prepared in the presence of a water-soluble spin label, TEMPOcholine, ascorbate destroys the signal of untrapped label; when mixed phospholipid-tetracaine are formed by addition of micellar tetracaine, this leads to a complete loss of the ESR signal. High drug concentrations are often used for anesthesia and could be related to morphological nerve damage caused by large doses of anesthetics. PMID- 3017422 TI - Effects of an ATP site affinity analog on some conformational and enzymatic properties of the canine kidney (Na+ + K+)-ATPase. AB - We have shown previously that the canine kidney Na+,K+ pump [Na+ + K+)-ATPase) reacts with the ATP affinity analog p-fluorosulfonylbenzoyladenosine (FSBA). At 20 degrees C, we find the time-course of this reaction to be that predicted for a first-order reaction accompanied by competing solvolysis of the reagent. The FSBA inactivated (Na+ + K+)-ATPase retains the ability to move between the E1 and E2 conformations that predominate in Na+ and K+ medium, respectively. Therefore, FSBA reaction with the enzyme does not interfere significantly with either its alkali metal cation binding or its conformational freedom. The ability of ATP to influence the enzyme's conformation by binding to the high-affinity nucleotide site is decreased, however, in proportion to the degree of inhibition of enzyme activity by FSBA. In addition, the ability of the enzyme to shift from the E1 to the E2 conformation through the (ATP + Na+)-dependent phosphorylation cycle is inhibited by FSBA treatment, as shown by the decreased ability of these substrates to stimulate the K+-dependent p-nitrophenylphosphatase activity. Both of these effects are consistent with specific reaction of FSBA with the ATP binding site of the enzyme. An additional effect of FSBA treatment is that it causes loss of p-nitrophenylphosphatase activity, but to a lesser extent than (Na+ + K+)-ATPase or Na+-ATPase activity. Binding of p-nitrophenylphosphate to the enzyme is apparently unaffected by FSBA treatment, since the Km for p nitrophenylphosphate is not changed. PMID- 3017424 TI - Tryptic and chymotryptic cleavage sites in sequence of alpha-subunit of (Na+ + K+)-ATPase from outer medulla of mammalian kidney. AB - Localization of selective proteolytic splits in alpha-subunit of (Na+ + K+) ATPase is important for understanding the mechanism of active Na+,K+-transport. Proteolytic fragments of alpha-subunit from pig kidney were purified by chromatography in NaDodSO4 on TSK 3000 SW columns. NH2-terminal amino acid sequences of fragments as determined in a gas phase sequenator were unambiguously located within the total sequence of alpha-subunit from sheep kidney (Shull, C.E., et al. (1985) Nature 316, 691-695) and pig kidney (Ovchinnikov, Y.A., et al. (1985) Proc. Acad. Sci. USSR 285, 1490-1495). The primary chymotryptic split in the E1-form is located between Leu-266 and Ala-267 while the tryptic cleavage site appears to be between Arg-262 and Ile-263 (Bond 3). Tryptic cleavage in the initial fast phase of inactivation of the E1-form is located between Lys-30 and Glu-31 (Bond 2). In the E2-form, primary tryptic cleavage is between Arg-438 and Ala-439 (Bond 1). Chymotryptic cleavage between Leu-266 and Ala-267 stabilizes the E1-form of the protein without affecting the sites for binding of cations or nucleotides. Titration of fluorescence responses demonstrates the importance of the NH2-terminal for E1-E2 transition. Protonation of His-13 facilitates transition from E1- to E2-forms of the protein. Removal of His-13 after cleavage of bond 2 can explain the increase in apparent affinity of the cleaved enzyme for Na+ and the shift in poise of E1-E2 equilibrium in direction of E1-forms. The NH2 terminal sequence in renal alpha-subunit is not conserved in alpha + from rat neurolemma or in alpha-subunit from Torpedo or brine shrimp. A regulatory function of the NH2-terminal part of the alpha-subunit may thus be a unique feature of the alpha-subunit in (Na+ + K+)-ATPase from mammalian kidney. PMID- 3017423 TI - Circular dichroism of the two major conformational states of mammalian (Na+ + K+) ATPase. AB - No alteration in the circular dichroic spectrum of fully active, membrane-bound (Na+ + K+)-ATPase is observed when the protein is cycled between the two major conformational states, E1 and E2. This finding is in agreement with the infrared study by Chetverin and Brazhnikov (J. Biol. Chem. 260 (1985) 7817) and demonstrates that any difference in secondary structure between the two conformers must be less than 2%. PMID- 3017426 TI - Presence of a low-affinity nucleotide binding site on the (K+ + H+)-ATPase phosphoenzyme. AB - The effects of Mg2+ and nucleotides on the dephosphorylation process of the (K+ + H+)-ATPase phosphoenzyme have been studied. Phosphorylation with [gamma-32P]ATP is stopped either by addition of non-radioactive ATP or by complexing of Mg2+ with EDTA. The dephosphorylation process is slow and monoexponential when dephosphorylation is initiated with ATP. When phosphorylation is stopped by complexing of Mg2+ the dephosphorylation process is fast and biexponential. The discrepancy could be explained by a nucleotide mediated inhibition of the dephosphorylation process. The I0.5 for ATP for this inhibition is 0.1 mM and that for ADP is 0.7 mM, suggesting that a low-affinity binding site is involved. When Mg2+ is present in millimolar concentrations in addition to the nucleotides the dephosphorylation process is enhanced. Evidence has been obtained that Mg2+ acts through lowering the affinity for ATP. In contrast to K+, Mg2+ does not stimulate dephosphorylation in the absence of nucleotides. Mg2+ and nucleotides show the same interaction in the dephosphorylation process of a phosphoenzyme generated from inorganic phosphate. These findings suggest the presence of a low affinity nucleotide binding site on the phosphoenzyme, as is found in the (Na+ + K+)-ATPase phosphoenzyme. This low-affinity binding site may function as a feed back mechanism in proton transport. PMID- 3017425 TI - Difference in phospholipid dependence between two isozymes of brain (Na+ + K+) ATPase. AB - The effect of phospholipase C on two isozymes (alpha (+) and alpha forms) of rat brain (Na+ + K+)-ATPase and the temperature-dependence of their activities were investigated. Phospholipase C from Clostridium welchii inhibited the activities of the enzymes treated with and without pyrithiamin or N-ethylmaleimide, a preferential inhibitor of the alpha (+) form, but the extent of the inhibition was higher in the control enzyme than in the treated enzymes. The treatment of the (Na+ + K+)-ATPase with phospholipase C altered a ratio between high- and low affinity components for ouabain inhibition. It also caused the similar change in a ratio between the alpha (+) and alpha forms of Na+-stimulated phosphorylation from [gamma-32P]ATP. These findings indicate that the alpha (+) form of rat brain (Na+ + K+)-ATPase is more sensitive to phospholipase C than the alpha form. Analysis of Arrhenius plots of the activities of the control and pyrithiamin treated enzymes showed that there was a difference between the two enzymes in a break point. We suggest that two isozymes of rat brain (Na+ + K+)-ATPase differ in the interaction with phospholipids or in the lipid-environment. PMID- 3017427 TI - Enrichment and biochemical characterization of boundary membrane contact sites from rat-liver mitochondria. AB - A subfraction of mitochondrial membranes was prepared from osmotically lysed rat liver mitochondria by density gradient centrifugation which contained the inner boundary membrane and the contact sites between this membrane and the outer membrane. The fraction was composed of inner and outer limiting membrane components as shown by the presence of specific marker enzymes, monoamine oxidase and glycerolphosphate oxidase. Surface proteolysis analysis, studies of cytochrome c permeability, and electron microscopy revealed the localization of the inner membrane component within a right-side-out outer membrane vesicle. Moreover, the outer membrane component in this fraction exhibited a higher capacity to bind hexokinase and had a higher specific activity of glutathione transferase than the pure outer membrane. In freeze-fracture analyses the fraction showed fracture plane deflections which may be specific for hydrophobic interactions between the two membranes. PMID- 3017428 TI - Demonstration and chemical modification of a specific phosphate binding site in the phosphate-starvation-inducible outer membrane porin protein P of Pseudomonas aeruginosa. AB - The interaction of phosphate ions with the Pseudomonas aeruginosa anion-specific protein P channel was probed. The single-channel conductance of protein P incorporated into planar lipid bilayer membranes in the presence of 0.3 M H2PO-4 was shown to be 6.0 pS, demonstrating that protein P channels allowed the permeation of phosphate. When large numbers of protein P channels were incorporated into lipid bilayer membranes in the presence of 40 mM Cl-, addition of small concentrations of phosphate resulted in reduction of macroscopic Cl- conductance in a dose- (and pH-) dependent fashion. This allowed calculation of an I50 value of e.g. 0.46 mM at pH 7.0, suggesting that the affinity of protein P for its normal substrate phosphate was at least 60-100-fold greater than the affinity of the channel for other ions such as chloride. Pyrophosphate and the phosphate analogue, arsenate, also inhibited macroscopic Cl- conductance through protein P with I50 values at pH 7.0 of 4.9 mM and 1.3 mM, respectively. To probe the nature of the phosphate binding site, the epsilon-amino groups of available lysine residues of protein P were chemically modified. Acetylation and carbamylation which produced uncharged, modified lysines destroyed both the anion (e.g. Cl-) binding site and the phosphate binding site as determined by single channel experiments and macroscopic conductance inhibition experiments respectively. Nevertheless, the modified proteins still retained their trimeric configuration and their ability to reconstitute single channels in lipid bilayer membranes. Methylation, which allowed retention of the charge on the modified lysine residues, increased the Kd of the channel for Cl- 33-fold and the I50 for phosphate inhibition of macroscopic Cl- conductance 2.5-4-fold. A molecular model for the phosphate binding site of the protein P channel is presented. PMID- 3017429 TI - Training increases the concentration of [3H]ouabain-binding sites in rat skeletal muscle. AB - Exercise is associated with a net loss of K+ from the working muscles and an increased plasma K+ concentration, indicating that the capacity for intracellular reaccumulation of K+ is exceeded. Training reduces the exercise-induced rise in plasma K+, and an increased plasma [K+] may interfere with physical performance. Since the clearing of K+ from the extracellular space depends on the capacity for active K+ uptake in skeletal muscle, the effects of training and inactivity on the total concentration of (Na+ + K+)-ATPase was determined. Following 6 weeks of swim training, the concentration of [3H]ouabain-binding sites in rat hindlimb muscles was up to 46% (P less than 0.001) higher than in those obtained from age matched controls. Whereas muscle Na+, K+ contents remained unchanged, the concentration of citrate synthase increased by up to 76% (P less than 0.001). Training induced no change in the [3H]ouabain-binding-site concentration in the diaphragm, but in the heart ventricles, the K+-dependent 3-O-methylfluorescein phosphatase activity increased by 20% (P less than 0.001). Muscle inactivity induced by denervation, plaster immobilisation or tenotomy reduced the [3H]ouabain-binding-site concentration by 20-30% (P less than 0.02-0.001) within 1 week. In conclusion, training leads to a significant and reversible rise in the concentration of (Na+ + K+)-ATPase in muscle cells. This may be of importance for the beneficial effects on physical performance by improving the maximum capacity for K+ clearance. PMID- 3017430 TI - A model for allosteric regulation of Na+/K+-transporting ATPase. PMID- 3017431 TI - The expression and genomic organization of randomly selected cloned Drosophila melanogaster genes. AB - A lambda recombinant DNA library containing Drosophila melanogaster nuclear DNA inserts was screened with cDNA made from oocyte and gastrula poly(A)+ RNA. 124 clones were isolated which represented sequences complementary to a distribution of abundancies of their RNAs. The clone set was then used as probes to identify those whose RNA abundancies changed during embryonic development. The vast majority of clones showed little difference during development. Four different clones were identified whose poly(A)+ RNAs were quantitatively regulated; two were oocyte-specific, and two were embryonic-specific. 44 clones were chosen for in situ hybridization to salivary gland polytene chromosomes. The location and distribution of their sites are described. A class of clones, identified by in situ hybridization to the nucleolus, is further described. These clones contain a scrambled array of ribosomal intervening sequences. PMID- 3017432 TI - Synthesis of epidermal growth factor (EGF) receptor in vitro using SP6 RNA polymerase-transcribed template mRNA. AB - The epidermal growth factor (EGF) receptor plays a key role in the control cellular proliferation, and its homology to the avian erythroblastosis virus erb B oncogene implicates its involvement in cellular transformation. The establishment of a correlation between the various structural domains of the EGF receptor and their functional counterparts would greatly advance our understanding of these processes. To this end, we have constructed an expression vector containing the SP6 viral promoter and an adjacent cDNA fragment encoding the full-length EGF receptor. Upon addition of SP6 RNA polymerase, this DNA is capable of generating large amounts of EGF receptor mRNA; this RNA can then be translated in vitro into immunoprecipitable EGF receptor protein. The translational efficiency of this EGF receptor RNA was found to be relatively low: approx. 100-fold lower than globin RNA synthesized using SP6 RNA polymerase. Use of these tools should now permit the synthesis and analysis of mutated EGF receptor protein in an effort to clarify the role of this receptor in growth control. PMID- 3017433 TI - Purification of mRNA coding for rat-liver fructose-1,6-bisphosphatase by polysome immunoabsorption. AB - The mRNA coding for rat liver fructose-1,6-bisphosphatase, which represents approx. 0.46% of total hepatic mRNA, has been purified to near homogeneity. Polysomes from rat liver were allowed to react with antibodies to rabbit anti fructose-1,6-bisphosphatase purified by affinity chromatography. The complex was immobilized on a protein A-Sepharose column. After the removal of unabsorbed polysomes, the specific mRNA was eluted and chromatographed on an oligo(dT) cellulose column. This method gave a 183-fold enrichment of the fructose-1,6 bisphosphatase mRNA to greater than 80% homogeneity as determined by electrophoreses of immunoprecipitated in vitro translation products on polyacrylamide slab gels in the presence of sodium dodecyl sulphate. PMID- 3017434 TI - Orientation of the beta subunit polypeptide of (Na+ + K+)ATPase in the cell membrane. AB - Although the animal cell (Na+ + K+)-ATPase is composed of two polypeptide subunits, alpha and beta, very little is known about the beta subunit. In order to obtain information about the structure of this polypeptide, the beta subunit has been investigated using proteolytic fragmentation, chemical modification of carbohydrate residues, and immunoblot analysis. The sialic acid moieties on the oligosaccharide groups on the beta subunit of (Na+ + K+)-ATPase were labeled with NaB3H4 after oxidation by sodium periodate, or the penultimate galactose residues on the oligosaccharides were similarly labeled after removal of sialic acid with neuraminidase and oxidation by galactose oxidase. All of the carbohydrate residues of the protein are located on regions of the beta subunit that are found on the non-cytoplasmic surface of the membrane. Cleavage of the galactose oxidase treated, NaB3H4-labeled beta subunit by chymotrypsin at an extracellular site produced labeled fragments of 40 and 18 kDa, indicating multiple glycosylation sites along the polypeptide. Neither the 40 kDa fragment nor the 18 kDa fragment was released from the membrane by chymotrypsin digestion alone, but after cleavage the 40 kDa fragment could be removed from the membrane by treatment with 0.1 M NaOH. This indicates that the 40 kDa fragment does not span the lipid bilayer. The 40 kDa fragment and the 18 kDa fragment are also linked by at least one disulfide bond. The 18 kDa fragment also contains all of the binding sites found on the (Na+ + K+)-ATPase for anti-beta subunit antibodies. Both the 40 kDa fragment and the 18 kDa fragment were also generated using papain or trypsin to cleave the beta subunit. These data indicate that the beta subunit of (Na+ + K+) ATPase contains multiple sites of glycosylation, that it inserts into the cell membrane near only one end of the polypeptide, and that one region of the polypeptide is particularly sensitive to proteolytic cleavage relative to the rest of the polypeptide. PMID- 3017435 TI - Characterization of the spleen green hemeprotein with magnetic and natural circular dichroism spectroscopy: positive evidence for a myeloperoxidase-type active site. AB - The green hemeprotein purified from bovine spleen has been characterized with magnetic and natural circular dichroism (MCD and CD) spectroscopy for the first time. The enzyme derivatives studied include the native high-spin ferric form and its high-spin chloride and low-spin cyanide and nitrite complexes, the ligand free high-spin ferrous form and its low-spin CO adduct, and Compounds II (ferryl iron species) and III (dioxygen adduct). All these enzyme states exhibit MCD spectra that are considerably different from the spectra of analogous complexes of normal heme iron. In particular, the following distinctions have been observed. The sign of the derivative-shaped MCD bands of the high-spin ferric and Compound II forms in the Soret (380-500 nm) region and of the ferrous low-spin and Compound III forms in both the Soret and visible (500-700 nm) regions are opposite to and, except for the high-spin ferric form, are less symmetric than those seen for normal heme iron systems. MCD intensities in the Soret region for the high-spin ferrous and low-spin ferric derivatives are noticeably smaller than those of normal heme proteins by a factor of up to ten. Prominent MCD bands are seen around 450 and 630 nm for the green hemeprotein derivatives; these features are considerably red-shifted (30-50 nm) relative to the analogous transitions observed for normal heme proteins. In contrast to the aforementioned spectral differences, the MCD and CD spectra of the spleen green hemeprotein derivatives are essentially identical to those previously reported for several derivatives of another spectroscopically anomalous heme-type enzyme, myeloperoxidase. This provides strong evidence that the two enzymes have identical prosthetic groups and endogenous axial ligands coordinated to the central iron. The novel MCD features of the green proteins, taken together with previously reported spectroscopic results, are most consistent with the presence of a chlorin-type prosthetic group in both proteins. In addition, the CD spectral similarities suggest that the two green proteins have nearly identical active-site environments. PMID- 3017436 TI - Transient resonance Raman spectroscopy shows unrelaxed heme following CO photodissociation from cytochrome-c peroxidase. AB - The 7 ns 436 nm pulses of an H2-shifted YAG laser have been used to photolyze the CO adduct of cytochrome-c peroxidase and produce the resonance Raman spectrum of the photoproduct. A 3 cm-1 downshift, relative to the spectrum of reduced enzyme, was observed for the porphyrin C-N breathing mode, v4. The downshift diminishes with decreasing CO /protein ratio, implying, in conjunction with a recent study of CO binding, that the unrelaxed heme is associated with adduct having a tilted, H-bonded FeCO unit. The downshift is eliminated when the phosphate buffer concentration is increased from 0.01 to 0.1 M. It is proposed that the heme relaxation under study involves a transition between two conformations, B and A, differing in the disposition of the distal residues, and having different v4 frequencies for unligated Fe(II) heme. Conformation B allows H-bonding to bound CO, and is favored at high CO and phosphate concentrations, while conformation A, which is unfavorable to CO H-bonding, is favored at low CO and phosphate concentrations. The recently reported absence of unrelaxed frequencies in the 7 ns photo-product of the CO adduct of horseradish peroxidase has been confirmed, and is attributed to lower stability for conformation B and a smaller A - B v4 difference. PMID- 3017437 TI - Tripolyphosphatase associated with S-adenosylmethionine synthetase isozymes from rat liver. AB - S-Adenosylmethionine (AdoMet) synthetase alpha and beta were purified to homogeneity, as judged by SDS-polyacrylamide gel electrophoresis from rat liver. When the purified enzymes were applied onto Sephacryl S-200, each synthetase was eluted together with a tripolyphosphatase. The activities of these isozymes in synthesizing AdoMet and in hydrolyzing tripolyphosphate decreased in parallel with increasing amounts of rabbit anti-(beta-form) IgG. The activity of the beta form isozyme was markedly stimulated by the addition of tripolyphosphate, whereas that of the alpha-form isozyme was inhibited. The tripolyphosphatase activity of both the alpha- and the beta-form was markedly stimulated by the addition of AdoMet. The tripolyphosphatases of each isozyme showed some other similar properties. PMID- 3017438 TI - Rapid affinity chromatographic purification of human lung and kidney angiotensin converting enzyme with the novel N-carboxyalkyl dipeptide inhibitor N-[1(S) carboxy-5-aminopentyl]glycylglycine. AB - Human angiotensin-converting enzyme has been purified, in a single chromatographic step, using a novel N-carboxyalkyl dipeptide CA-GlyGly (N-[1(S) carboxy-5-aminopentyl]glycylglycine) synthesised in our laboratory. CA-GlyGly is a weak competitive inhibitor, Ki = 0.18 mM, and its inhibitory profile is markedly pH-dependent. Human lung and kidney angiotensin-converting enzyme were solubilised with Triton X-100 and after ammonium sulphate fractionation the crude extract was applied to a column containing CA-GlyGly coupled to agarose via a 2.8 nm spacer group. Electrophoretically pure human angiotensin-converting enzyme could be eluted by raising the pH of the chromatography buffer from 7.50 to 9.50. The specific activity of human angiotensin-converting enzyme purified from lung was 104 units/mg, while that from kidney was 88 units/mg. Molecular weight for both enzymes was estimated to be 160,000. The Km with respect to hippuryl-L histidyl-L-leucine was 1.9 mM in the case of lung angiotensin-converting enzyme and 1.7 mM in that of kidney angiotensin-converting enzyme, while for the substrate angiotensin I Km values were 62 microM and 76 microM, respectively. Hydrolysis of either substrate was chloride-dependent and both enzymes were strongly inhibited by captopril. PMID- 3017440 TI - Differential kinetics and sensitivity to chloroquine of receptor-mediated insulin and prolactin endocytosis in liver parenchymal cells. AB - Systemically injected [125I]prolactin or [125I]insulin was accumulated and cleared from rat liver at different rates. Quantitative subcellular fractionation indicated a predominant accumulation of [125I]insulin in liver microsomes while [125I]prolactin was found in both the light-mitochondrial and microsomal fractions. The acidotropic agent chloroquine diminished the rate and extent of loss of each ligand from liver homogenates. In chloroquine treated rats, radiolabeled insulin accumulated in both the light-mitochondrial and the microsomal fractions. Subfraction of microsomes on discontinuous sucrose gradients revealed "early' endosomes in which ligand uptake was maximal at 2-5 min. In contrast, comparable subfraction of the of light mitochondrial fraction revealed "late' endosomes in which ligand uptake was maximal at 10-20 min. Chloroquine-treated rats showed a more marked enhancement of insulin compared to prolactin uptake in the "early' endosomes. It is suggested that "early' endosomes found in the Golgi-intermediate and -heavy fractions floated from parent microsomes may selectively degrade insulin but not prolactin. This could account for the apparently different kinetics of insulin and prolactin uptake into liver parenchyma. PMID- 3017439 TI - Detection of free radicals as a consequence of rat intestinal cellular drug metabolism. AB - Because the intestine is the first pass organ for orally administered drugs and because some of these drugs are known to undergo oxidative metabolism leading to the formation of free radicals, we investigated the potential for this to occur in cell suspensions of rat enterocytes. As part of our study, the effect of intracellularly produced superoxide on cellular metabolism was investigated. The drugs chosen were the quinone, menadione and the aromatic nitro-containing compound, nitrazepam. On incubation of both drugs with isolated enterocytes and the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), rapid appearance of an electron paramagnetic resonance (EPR) spectrum was recorded which was characteristic of hydroxyl radicals being spin trapped by DMPO giving 2,2 dimethyl-5-hydroxy-1-pyrrolidenyloxyl (DMPO-OH). Experiments were conducted which determined that the EPR spectrum of DMPO-OH resulted from the initial spin trapping of superoxide by DMPO to yield the corresponding nitroxide, 2,2-dimethyl 5-hydroxyl-1-pyrrolidenyloxyl (DMPO-OOH). Bioreduction of DMPO-OOH by glutathione peroxidase led to the rapid formation of DMPO-OH. We believe this enzymic pathway accounted for the EPR spectrum noted in incubations with either drug in the presence of the spin trap, DMPO. The incubation of enterocytes with both drugs did not mediate release of 51Cr nor lactate dehydrogenase. However, production of 14CO2 from [14C]glucose was severely inhibited (4-5-fold) in the presence of both drugs, while the incorporation of [14C]leucine into trichloroacetic acid precipitable protein was antagonized by menadione only. We conclude that superoxide can be demonstrated to arise as the result of enterocyte metabolism of menadione or nitrazepam. The consequence of oxidative metabolism of these drugs results in cellular dysfunction. PMID- 3017441 TI - Isolation and characterization of an inhibitor-sensitive and a polycation stimulated protein phosphatase from rat liver nuclei. AB - Two protein phosphatases were isolated from rat liver nuclei. The enzymes, solubilized from crude chromatin by 1 M NaCl, were resolved by column chromatography on Sephadex G-150, DEAE-Sepharose and heparin-Sepharose. The phosphorylase phosphatase activity of one of the enzymes (inhibitor-sensitive phosphatase) was inhibited by heat-stable phosphatase inhibitor proteins and also by histone H1. This phosphatase had a molecular weight of approx. 35,000 both before and after 4 M urea treatment. Its activity was specific for the beta subunit of phosphorylase kinase. Pretreatment with 0.1 mM ATP inhibited the enzyme only about 10%, and it did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as the catalytic subunit of phosphatase 1. The other phosphatase (polycation-stimulated phosphatase) was insensitive to inhibition by inhibitor 1, and it was stimulated 10-fold by low concentrations of histone H1 (A0.5 = 0.6 microM). This enzyme had a molecular weight of approx. 70,000 which was reduced to approx. 35,000 after treatment with 4 M urea. It dephosphorylated both the alpha- and beta-subunits of phosphorylase kinase. The enzyme was inhibited more than 90% by preincubation with 0.1 mM ATP and did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as phosphatase 2A. PMID- 3017442 TI - Increased activity of spermidine N1-acetyltransferase in the adrenal cortex of rats following administration of exogenous spermidine, carbamylcholine, 2 deoxyglucose and dopamine agonists. AB - The activity of N1-acetyltransferase was increased in the dissected adrenal cortex of the rat following a single administration of spermidine. The activity was maximal 6-8 h after the onset of treatment. The increase in enzyme activity was abolished when the rats were given cycloheximide 2 h after spermidine; this suggests that increased activity results from an augmentation in the synthesis of the enzyme. Adrenocortical spermidine N1-acetyltransferase was also induced by carbamylcholine, 2-deoxyglucose, apomorphine and piribedil, drugs that are known to cause induction of ornithine decarboxylase in that organ. Hypophysectomized rats showed reduced activity of spermidine N1-acetyltransferase when compared to sham-operated controls, and carbamylcholine no longer elicited an increase in enzyme activity in such animals. Adrenocortical spermidine N1-acetyltransferase activity of hypophysectomized rats is induced by corticotropin (ACTH). These results suggest a hormonal control over the activity of the enzyme in the adrenal cortex with ACTH acting as a mediator. PMID- 3017443 TI - Platelet-activating factor as a stimulus of exocytosis in human neutrophils. AB - The properties of platelet-activating factor (PAF-acether) 42-48ulus of exocytosis in human neutrophils have been re-investigated with particular attention to effects on cells that were not pretreated with cytochalasin B. Release of gelatinase, the most sensitive marker of exocytosis, was determined in addition to that of vitamin B-12-binding protein and beta-glucuronidase. Superoxide production was assayed as a measure of the respiratory burst. The effects of PAF-acether were compared to those of leukotriene B4 and N formylmethionylleucylphenylalanine (fMet-Leu-Phe). Our results show that PAF acether elicits marked secretion in untreated human neutrophils, and refute the prevalent view that cytochalasin B treatment is required for responsiveness. PAF acether induced abundant release of gelatinase, increasing on average from 20% at 10 nM to 35% at 1 microM. This release was very rapid, i.e., almost complete after 2 min. fMet-Leu-Phe induced the same maximum response already at 0.1 microM, but release was considerably slower. Leukotriene B4 was less potent with a maximum release of 20%. Exocytosis of gelatinase was always paralleled by liberation of smaller but significant amounts of vitamin B12-binding protein from the specific granules. In contrast to their effect on exocytosis, PAF-acether and leukotriene B4 were very weak stimuli of the respiratory burst when compared with fMet-Leu-Phe. PMID- 3017444 TI - Interaction of cyclic AMP and Ca2+ in the control of electrical coupling in heart fibers. AB - The influence of intracellular injection of cAMP on the electrical coupling of canine Purkinje cells was investigated. It was found that the nucleotide enhanced reversibly the cell-to-cell communication through an increase in junctional conductance. Dibutyryl cAMP (5 X 10(-4) M) plus theophylline (0.4 mM) decreased appreciably the intracellular longitudinal resistance (ri). The interactions of cAMP and Ca on the electrical coupling were also investigated. The nucleotide and Ca have opposite effects on the electrical coupling. In the presence of high [Ca2+]o solutions (6 mM), the intracellular injection of cAMP causes a transient increase in the coupling coefficient followed by an appreciable decrease in cell to-cell coupling. This reduction in intracellular communication was reversed by injecting EGTA into the same cell. The results of this study support the view that cAMP is a modulator of junctional conductance in cardiac muscle and that the compound interacts with Ca in the control of intracellular communication. PMID- 3017445 TI - Characterization of high-density lipoprotein binding and cholesterol efflux in cultured mouse adipose cells. AB - Binding of high-density lipoproteins to cultured mouse Ob1771 adipose cells was studied, using labeled human HDL3, mouse HDL and apolipoprotein AI- or AII containing liposomes. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 18, 42, 30 and 3.4 micrograms/ml, whereas the maximal binding capacities were found to be 160, 100, 90 and 21 ng/mg of cell protein. Apoprotein AI not inserted into liposomes did not bind. The binding of 125I-HDL3 was competitively inhibited by apolipoprotein AI-containing liposomes greater than mouse HDL greater than HDL3. The binding of 125I-labeled apolipoprotein AI- and 125I-labeled apolipoprotein AII-containing liposomes was competitively inhibited by HDL3, apolipoprotein AI- and apolipoprotein AII-containing liposomes. Dimyristoylphosphatidylcholine liposomes containing or not cholesterol did not interfere with the binding of labeled HDL3 or apolipoprotein-containing liposomes. Binding studies on crude membranes of Ob1771 adipose cells revealed the presence of intracellular binding sites for LDL and HDL3. Thus, adipose cells have specific binding sites for apolipoprotein E-free HDL and apolipoprotein AI (or AII) is the ligand for these binding sites. Long-term exposure of adipose cells to LDL cholesterol as a function of LDL concentration led to an accumulation of cellular unesterified cholesterol. This process was saturable and reversible as a function of time and concentration by exposure to HDL3 or apolipoprotein AI-containing liposomes, whereas apolipoprotein AII-containing liposomes did not promote any cholesterol efflux. Since long-term exposure of adipose cells to LDL and HDL3 did not affect the number of apolipoprotein B,E receptors and apolipoprotein E-free binding sites, respectively, it appears that adipose cells do not show efficient cholesterol homeostasis and thus could accumulate or mobilize unesterified cholesterol. PMID- 3017446 TI - Bradykinin-induced changes in myo-inositol 1,2-(cyclic)phosphate in rabbit papillary collecting tubule cells. AB - Rabbit renal papillary collecting tubule cells were harvested and grown in primary cultures. When labeled with myo-[2-3H]inositol and extracted under neutral conditions, a metabolite undetected under acidic extraction was observed on resolution by anion-exchange chromatography and which eluted under similar conditions with authentic DL-myo-inositol 1,2-(cyclic)phosphate; the mass spectrum of its pentakis(trimethylsilyl) derivative contains an identical ratio of selected ion fragments to the authentic standard. Bradykinin, demonstrated previously to increase labeling of free inositol polyphosphates, increases labeling of inositol 1,2 cyclic phosphate but over a time course subsequent to the formation of inositol trisphosphate. These observations are consistent with the model that bradykinin induces hydrolysis of phosphatidylinositol 4,5 bisphosphate which precedes hydrolysis of phosphatidylinositol in renal papillary collecting tubule cells. PMID- 3017447 TI - Cyclic AMP-dependent and -independent effects on tissue-type plasminogen activator activity in osteogenic sarcoma cells; evidence from phosphodiesterase inhibition and parathyroid hormone antagonists. AB - The plasminogen activator (PA) in clonal osteogenic sarcoma cells of rat origin (UMR 106-01 and UMR 106-06) and in osteoblast-rich rat calvarial cells has been characterized using specific antibodies to be tissue-type PA (tPA). An Mr value of 75,000 by SDS-polyacrylamide gel electrophoresis and fibrin autoradiography supports this characterization. There was also evidence for an Mr 105,000 component, which could be due to a proteinase-inhibitor complex. The mechanism of regulation of this tPA activity has been studied in the clonal osteogenic sarcoma cells. Parathyroid hormone (PTH) and prostaglandin E2, which increase cyclic AMP production in the sarcoma cells, also increased tPA activity. The sensitivity and magnitude of the tPA response to PTH and prostaglandin E2 were increased by simultaneous treatment with isobutylmethylxanthine (IBMX) at drug concentrations which had little effect themselves on tPA activity. In UMR 106-06 cells, which unlike UMR 106-01 cells show a cyclic AMP response to calcitonin, tPA activity was also increased in response to calcitonin, and the effect was enhanced by IBMX. 1,25-Dihydroxyvitamin D-3 also increased tPA activity in the cells, but this response was not modified by IBMX. Synthetic peptide antagonists of PTH responsive adenylate cyclase, [34Tyr]-hPTH (3-34) amide and [34Tyr]-hPTH (5-34) amide, inhibited the PTH-induced increase in tPA activity over the same concentration range at which they inhibited cyclic AMP production, but the antagonist peptides had no effect on the tPA responses to prostaglandin E2, calcitonin or 1,25-dihydroxyvitamin D-3. These data indicate that cyclic AMP mediates the actions of PTH, prostaglandin E2 and calcitonin in increasing tPA activity in the clonal osteogenic sarcoma cells. 1,25-Dihydroxyvitamin D-3, on the other hand, increases tPA activity through a mechanism independent of cyclic AMP. PMID- 3017449 TI - [Secondary active transport]. AB - Secondary active transport is defined as the transport of a solute in the direction of its increasing electrochemical potential coupled to the facilitated diffusion of a second solute (usually an ion) in the direction of its decreasing electrochemical potential. The coupling agents are membrane proteins (carriers), each of which catalyzes simultaneously the facilitated diffusion of the driving ion and the active transport of a given solute. The review starts with some considerations on the energetics followed by a presentation of the kinetics of secondary active transport. Examples of information which may be gained by such studies are discussed. In the second part, some examples of secondary transport are given; we also describe the characteristics of the corresponding carriers. The various transport systems presented are: the D-glucose/Na+ symport in brush border membranes, the lactose/H+ symport in E. coli, the Na+/H+ antiport, the different transport systems in the inner mitochondrial membrane. PMID- 3017448 TI - Glucocorticoid stimulation of choline-phosphate cytidylyltransferase activity in fetal rat lung: receptor-response relationships. AB - A number of previous studies using in vivo and cultured fetal lung models have shown that the activity of choline-phosphate cytidylyltransferase, the enzyme which catalyzes a rate-limiting reaction in de novo phosphatidylcholine synthesis, is increased by glucocorticoids and other hormones which accelerate fetal lung maturation. To examine the mechanism of this glucocorticoid action further, we examined the effect of dexamethasone on cytidylyltransferase activity in cultured fetal rat lung explants and related it to specific dexamethasone binding. Dexamethasone stimulated cytidylyltransferase activity in the homogenate, microsomal and 105,000 X g supernatant fractions. The hormone did not alter the subcellular distribution of the enzyme, however; the bulk of the activity was in the supernatant fraction in both the control and dexamethasone treated cultures. The dose-response curves for stimulation of cytidylyltransferase activity in the supernatant fraction and specific nuclear binding of dexamethasone were similar and both plateaued at approx. 20 nM. The EC50 for cytidylyltransferase stimulation was 6.6 nM and the Kd for dexamethasone binding was 6.8 nM. The relative potencies of various steroids for stimulating choline-phosphate cytidylyltransferase and for specific nuclear glucocorticoid binding were the same: dexamethasone greater than cortisol = corticosterone = dihydrocorticosterone greater than progesterone. The stimulation by dexamethasone of cytidylyltransferase activity and of choline incorporation into phosphatidylcholine were both abolished by actinomycin D. These data show that the stimulatory effect of dexamethasone on fetal rat lung choline-phosphate cytidylyltransferase activity is largely on the enzyme in the supernatant fraction and does not involve enzyme translocation to the microsomes as has been reported for cytidylyltransferase activation in some other systems. This effect of dexamethasone is a receptor-mediated process dependent on RNA and protein synthesis. PMID- 3017450 TI - [Energy-dependent translocation of amino-phospholipids in the erythrocyte membrane]. AB - In this review, we show how the stability of the asymmetric transverse distribution of phospholipids and the physiological role of the asymmetric distribution can be explained. Experiments with paramagnetic or fluorescent lipids enabled us to show that in fresh red blood cells, i.e. containing ATP, and in resealed ghosts containing ATP (1 mM) the amino derivatives (phosphatidylserine and phosphatidylethanolamine) are selectively transported from the outer monolayer to the inner monolayer of the membranes. On the other hand, phosphatidylcholine and sphingomyelin are not carried and diffuse spontaneously with a very long characteristic time. The ATP-dependent carrier mechanism can be inhibited by protein reacting groups (N-ethyl maleimide and ortho-vanadate), which very probably implies a transmembrane protein specific for amino phospholipids. The affinity for phosphatidylserine seems slightly higher than that for phosphatidylethanolamine. In addition we show the close parallel between the transverse distribution of phospholipids and cell shape. This leads us to suggest that the phospholipid translocation would be used to maintain the natural discoid shape of red blood cells. A possible generalisation of this mechanism to other cells and its implications for endocytosis are discussed. PMID- 3017451 TI - The vacuolar membrane of plant cells: a newcomer in the field of biological membranes. AB - The vacuole of plant cells is a large compartment whose functions (storage, regulation of cytoplasmic homeostasis) have been studied mainly using indirect methods. The recent development of procedures to isolate this organelle enables direct investigation of its properties and of those of its surrounding membrane, the tonoplast. Several problems are encountered when studying the tonoplast: the small quantities of membrane material recovered, the contamination by other membranes, and the lack of an unambiguous marker. This accounts for the scarcity of analytical data concerning this structure. The investigation of transport systems at the tonoplast was stimulated by the existence of a high pH gradient across this membrane and the accumulation of various solutes inside the vacuole (sucrose, organic acids, etc.). Up to now, the most studied system is the proton pumping ATPase. Its main characteristics are presented, together with a few data on other transport systems. PMID- 3017453 TI - [Molecular mechanisms activated during the emission of oxygen in photosynthesis]. AB - Photosynthetic oxygen evolution results from the dehydrogenation of water molecules by the chlorophyll-center cation of photosystem II. The primary charge separation occurs between the chlorophyll center and the primary acceptor. The positive charges are then transferred to the manganese cluster able to accumulate four positive equivalents. Protons are liberated during this process. The oxygen evolution requires the presence of Ca2+ and Cl-. The oxidation of two water molecules, and the transfer of the electrons to the plastoquinone pool, are done by a supra-molecular complex which utilizes photons as substrate, and can therefore transfer electrons against a large redox potential difference. PMID- 3017454 TI - Stimulation of 2',3'-cyclic nucleotide 3'-phosphodiesterase in human lymphocytes by Robinia pseudoacacia lectin. AB - 2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) activity was studied in human peripheral blood lymphocytes. Incubation of human lymphocytes with optimum mitogenic concentrations of Robinia pseudoacacia lectin produced an increase in CNP activity. This increase was not detected before 24 h of incubation. Furthermore, whereas unfractionated lymphocytes exhibited activation of CNP, this was not observed in B and T cell subpopulations. PMID- 3017455 TI - Cloning of yeast lysyl- and phenylalanyl-tRNA synthetase genes. AB - Cloning of yeast lysyl- and phenylalanyl-tRNA synthetase genes was accomplished by probing a lambda gt11 recombinant DNA expression library with antibodies directed against the purified enzymes. Several DNA clones encoding either the alpha or the beta subunit of phenylalanyl-tRNA synthetase were isolated. In each case, the inserted DNA was oriented in the same direction with respect to the lambda gt11 lacZ transcription unit, giving rise to the expression of hybrid proteins. The corresponding DNA fragments constitute suitable hybridization probes for the isolation of complete nucleotide sequences encoding the alpha and beta subunits of the enzyme. Recombinant DNA lambda gt11 clones encoding lysyl tRNA synthetase were also selected. One of these contained yeast DNA inserted with the opposite orientation with respect to lacZ. The lysogen corresponding to that recombinant DNA phage produced an active, native lysyl-tRNA synthetase. The 3.6 kbp DNA insert contained all the information necessary for the expression of yeast lysyl-tRNA synthetase in E. coli. PMID- 3017452 TI - [Molecular pharmacology of the catecholamine transporter of chromaffin granules from the bovine adrenal medulla]. AB - Tetrabenazine (TBZ) and reserpine are two inhibitors of the catecholamine uptake system of the chromaffin granule membrane. They are structural analogs of the substrates dopamine and serotonin and they inhibit the monoamine transporter, which catalyzes a H+/neutral amine antiport. [3H]Dihydrotetrabenazine ([3H]TBZOH) is bound by chromaffin granule membranes on one class of site (T sites, KD = 3 nM); [3H]reserpine is bound on T sites and a second class of site (R1 sites, KD = 0.7 nM). The two sites are involved in monoamine translocation. The substrates displace the ligands with different efficiency: noradrenaline (Km = 10 microM) displaces reserpine efficiently (EC50 = 30 microM), but TBZOH poorly (EC50 = 2000 microM); m-iodobenzylguanidine, which has recently been shown to be a substrate of the monoamine uptake system (Km = 5 microM), displaces TBZOH efficiently (EC50 = 25 microM), but reserpine inefficiently (EC50 = 300 microM). Since both substrates are translocated by the same transporter, this result confirms the existence of two sites with different properties. T sites are characterized by a linear relationship between the reciprocal of the dissociation constants of various drugs displacing [3H]TBZOH and their partition coefficient in octanol/H2O mixtures. This relationship, which indicates a hydrophobic environment of T sites, does not exist for R1 sites. T sites have been identified by covalent labeling with a derivative of TBZ coupled to an arylazido group. The labeled sites are borne by a 65,000 dalton protein. The kinetics of reserpine binding are accelerated in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017457 TI - Amplification and rearrangement of Ki-ras oncogene in human teratocarcinoma derived cell lines. AB - The structure of the ras gene family was analyzed in two human teratocarcinoma derived cell lines, Tera I and Tera II, by DNA restriction enzyme digestion and Southern blot. We report here a ten-fold amplification of the c-Ki-ras-2 gene in these cell lines, whereas no structural alterations seem to occur either in c-Ha ras-1 or N-ras. We also provide evidence indicating that no point mutation at codon 12, specifically recognized by Sac I, was detected. Moreover, DNA rearrangement, due to the loss of a Pvu II site located in the intervening sequences between the third and the fourth exon, has been found in both Tera I and Tera II. PMID- 3017456 TI - A novel RNA from early region 3 of adenovirus-2. AB - A novel RNA species was detected by Northern blotting among the polyadenylated nuclear transcripts from early region 3 of adenovirus-2. Preliminary mapping showed that it encompasses the E3 leader 1 and part of IVS 1. Primer extension experiments revealed that its 5' end (C) was the same as that of all E3 transcripts, and S1 nuclease mapping showed that its 3' end was a polyadenylation site (P1) known to be used in the late phase of infection. The body of this RNA is 618-619 nucleotides long and we propose to name it E3-CP1. Kinetic studies suggest that it is a primary transcript as are the E3 premessenger RNAs. PMID- 3017458 TI - Transformation of established murine fibroblasts with an activated cellular Harvey-ras oncogene or the polyoma virus middle T gene increases cell permissiveness to parvovirus minute-virus-of-mice. AB - Transformation of permanent rodent fibroblast cells by the polyoma virus middle T gene or the activated human Harvey-ras oncogene results in increased cellular permissiveness to the autonomous parvovirus minute-virus-of-mice. Parvoviral DNA amplification is restricted in the untransformed parental cell lines. Analysis of various parameters of the parvoviral life cycle shows that this block is partially overcome in the transformed lines. PMID- 3017459 TI - Molecular biology of liver regeneration. AB - Liver regeneration is a good system for studying cell proliferation in an in vivo, physiologically controlled situation. Various hepatotrophic factors, neuromediators, hormones and growth factors, presumably acting in synergy, seem necessary to induce the switch from quiescence to proliferation. As a consequence of this activation, a number of changes occurs in the hepatocyte: modifications of the plasma membrane proteins; metabolic changes such as variations in albumin and fibrinogen concentrations, and induction of the acute phase proteins; induction of several specific mRNAs; variations in cAMP concentrations, and consequently in the activity of protein kinases and several other enzymes; modifications in chromosomal proteins; induction of proteins involved in DNA replication. A model has been constructed which is more a basis for reflexion than a theoretical model. It takes into account the possible connections between the different molecular events cited above. It is hypothesized that DNA replication is at least partly uncoupled from mitosis, and that the initial events of the proliferative response may be triggered by nutritional elements. PMID- 3017460 TI - Self-stabilization of neuronal networks. I. The compensation algorithm for synaptogenesis. AB - Between the extreme views concerning ontogenesis (genetic vs. environmental determination), we use a moderate approach: a somehow pre-established neuronal model network reacts to activity deviations (reflecting input to be compensated), and stabilizes itself during a complex feed-back process. Morphogenesis is based on an algorithm formalizing the compensation theory of synaptogenesis (Wolff and Wagner 1983). This algorithm is applied to randomly connected McCulloch-Pitts networks that are able to maintain oscillations of their activity patterns over time. The algorithm can lead to networks which are morphogenetically stable but preserve self-maintained oscillations in activity. This is in contrast to most of the current models of synaptogenesis and synaptic modification based on Hebbian rules of plasticity. Hebbian networks are morphogenetically unstable without additional assumptions. The effects of compensation on structural and functional properties of the networks are described. It is concluded that the compensation theory of synaptogenesis can account for the development of morphogenetically stable neuronal networks out of randomly connected networks via selective stabilization and elimination of synapses. The logic of the compensation algorithm is based on experimental results. The present paper shows that the compensation theory can not only predict the behavior of synaptic populations (Wagner and Wolff, in preparation), but it can also describe the behavior of neurons interconnected in a network, with the resulting additional system properties. The neuronal interactions--leading to equilibrium in certain cases- are a self-organizing process in the sense that all decisions are performed on the individual cell level without knowing the overall network situation or goal. PMID- 3017461 TI - The sensitivity of the fetal rat adrenal gland to adrenocorticotropic hormone in vivo and in vitro. AB - Rat fetuses were treated with 1 IU ACTH on day 16 or 17 of gestation and autopsied on the next day. Other fetuses of the same litter were treated with saline alone. The adrenal glands of ACTH-treated fetuses were significantly heavier than those of saline-treated fetuses and of intact controls in every experimental period. These changes in the weight of fetal adrenal glands reflect histologically on changes in the size of adrenocortical cells which were enlarged in response to injected ACTH. Adrenal glands from rat fetuses of different ages were explanted to organ cultures for 2 days, in medium with or without ACTH added. The 13-day adrenal glands were not able to respond to ACTH, but the response appeared abruptly in the 14-day adrenal glands, judging from the increased cell size in histological sections. The overall results suggest that the fetal adrenal gland begins to respond to ACTH from day 14 of gestation. PMID- 3017462 TI - Lack of seasonal variation in platelet [3H]imipramine binding in humans. AB - The Bmax of [3H]imipramine (IMI) binding has been reported to be reduced in platelets of depressed untreated patients as compared with normal controls. However, it has also been suggested that this difference could be related to the failure to take into account seasonal variations in the binding parameters for [3H]IMI recognition sites in platelets. For this reason, [3H]IMI binding was studied throughout 1 year in platelet membranes from 11 control volunteers, with blood samples collected once a month. The Bmax and Kd values of [3H]IMI binding showed no significant variation throughout the 12-month period of the study. These results indicate that in the control population, the platelet [3H]IMI binding parameters remain stable, and that the decrease in Bmax observed in depressed untreated patients reflects a genuine difference, which may be considered to be a biological marker in depression. PMID- 3017463 TI - Sodium-potassium, magnesium, and calcium ATPase activities in erythrocyte membranes from manic-depressive patients responding to lithium. AB - Erythrocyte membrane Na+-K+ adenosine triphosphatase (ATPase), Mg2+ ATPase, and Ca2+ ATPase activities were compared among 23 euthymic manic-depressive patients responding to lithium therapy and 24 healthy controls. The two groups were similar in age, sex composition, body mass index, and community background. No significant differences were noted in mean ATPase activities between the two groups. However, plasma lithium concentration correlated positively with Na+-K+ ATPase and Mg2+ ATPase activities within the patient group, and six patients with plasma lithium levels in the range of 0.85-1.2 mM had Na+-K+ ATPase activities 62% greater than the control group mean. Possible biochemical mechanisms for the effects of lithium therapy on erythrocyte membrane functions are discussed. PMID- 3017464 TI - Gonadotropin-releasing hormone-induced alteration of bovine corpus luteum function. AB - Two experiments were conducted to evaluate effects of gonadotropin-releasing hormone (GnRH) on the function of the bovine corpus luteum during the estrous cycle. In Experiment 1, 10 beef heifers were assigned randomly into two groups; each heifer served as her own control. Heifers in Group I (n = 5) were injected i.v. with vehicle (saline) on Day 2 of the cycle (Day 0 = day of estrus) followed by an i.v. injection of 100 micrograms GnRH on Day 2 of the subsequent estrous cycle. Group II (n = 5) heifers were treated similarly except injections were given on Day 10 of the estrous cycle. All heifers were bled via the jugular vein at 15 min intervals beginning 30 min prior to injection and for 3 h after injection. Blood samples were also taken on alternate days after injection through Day 16 of the cycle. Gonadotropin-releasing hormone caused a significant release of luteinizing hormone (LH) on both treatment days with the peak occurring at 15 to 30 min postinjection. Treatment with GnRH on either Day 2 or 10 caused a reduction in serum progesterone levels on Days 12, 14 and 16 of the cycle (Group I, control 3.99, 3.97; 4.07 vs. treated 2.63, 3.45, 2.87; Group II, control 3.18, 3.82, 4.13 vs. treated 2.50, 2.82, 3.17 ng/ml, respectively; common SE = 0.24 p less than 0.03). Length of the estrous cycle did not differ between groups (Group I, control 20.7 vs. treated 20.9; Group II, control 20.7 vs. treated 21.1 days, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017465 TI - Stimulation of progesterone production by adrenocorticotropic hormone and prostaglandin E2 in rat luteal cells. AB - The effects of adrenocorticotropic hormone (ACTH), human chorionic gonadotropin (hCG) and prostaglandin E2 (PGE2) on the progesterone secretion of luteal cells from rats were studied. Corpora lutea were harvested on Day 6 of pseudopregnancy and digested by trypsin. Homogeneous suspensions of luteal cells were used for short-term incubation. ACTH, PGE2, and hCG were added to the medium and the changes in progesterone production were measured by radioimmunoassay (RIA). Furthermore, specific ACTH-binding sites of the luteal cell membrane were studied by Scatchard analysis. ACTH, PGE2 and hCG increased synthesis of progesterone, and the combination of hCG with ACTH or PGE2 further increased production of the hormone. The effect of ACTH could be prevented by indomethacin. These effect of ACTH seem to be connected with specific ACTH-binding sites of the luteal cell membrane and with increased production of PGE2. PMID- 3017467 TI - Axial heterogeneity and filtered-load dependence of proximal bicarbonate reabsorption. AB - A theoretical model was developed to examine the role of physical and chemical factors in the control of bicarbonate reabsorption in the renal proximal tubule. Included in the model were axial and radial variations in the concentrations of HCO3-, CO2 and related chemical species in the tubule lumen and epithelial cells. Relations between these concentrations and the solute fluxes across the brush border and basolateral membranes were also included, as were reaction rate and equilibrium expressions to describe the various buffering processes in the lumen and cells. The two most critical membrane parameters, the rate constant for H+ secretion at the brush border and the effective permeability of HCO3- at the basolateral membrane, were evaluated by comparing model predictions with available free-flow micropuncture data in the rat. It was found that the experimental observations could be explained only by decreasing one or both of these membrane parameters with axial position, suggesting a progressive decrease in HCO3- reabsorptive capacity along the tubule. For single nephron filtered loads of HCO3- up to about 1,400 pmol/min, absolute bicarbonate reabsorption was predicted to increase nearly in proportion to filtered load, whereas it was calculated to be relatively constant at higher filtered loads, irrespective of how filtered load was assumed to be varied. These predictions are in excellent agreement with most of the available micropuncture data in rats, as is the prediction that HCO3- reabsorption should change in parallel with CO2 partial pressure in the filtrate, at a given filtered load of HCO3-. Certain discrepancies between the model predictions and experimental observations are evident at very high filtered loads, and the implications of these are discussed in terms of possible adaptive responses of the tubule. PMID- 3017466 TI - Single-headed binding of a spin-labeled-HMM-ADP complex to F-actin. Saturation transfer electron paramagnetic resonance and sedimentation studies. AB - The interaction of actin and spin-labeled heavy meromyosin (MSL-HMM) was studied in the presence and absence of adenosine diphosphate or 5'-adenyl-yl imidodiphosphate (AMPPNP) to determine the contributions of single and double headed binding. The extent of single-headed binding to actin was deduced from a comparison of the fraction of immobilized heads (fi) with the fraction of bound molecules (fs) determined by saturation-transfer EPR (ST-EPR) and sedimentation, respectively. The ST-EPR measurements depend on the reduced motion of the spin label rigidly bound to the HMM heads upon the interaction of the latter with actin. During titration of acto-MSL-HMM with nucleotide, we measured changes in fi and fs brought about by dissociation of MSL-HMM from actin. On titration with ADP, fs changed very little, remaining above 0.8, while fi decreased to approximately 0.5 at 10mM ADP, a result consistent with extensive single-headed binding of MSL-HMM to actin. On titration with AMPPNP, single-headed binding was not detected; viz., fi and fs decreased in parallel. It was not necessary to postulate a nucleotide induced state of the bound heads, differing in motional properties from that of rigor heads, to account for the results. PMID- 3017469 TI - A specific model for the conformation of single-stranded polynucleotides in complex with the helix-destabilizing protein GP32 of bacteriophage T4. PMID- 3017468 TI - Dependency of delta pH-relaxation across vesicular membranes on the buffering power of bulk solutions and lipids. AB - The dependency of delta pH-relaxation kinetics across the membrane of sonicated small phospholipid vesicles on the concentration of internally entrapped buffer has been investigated by means of the pH-indicator dye pyranine. A very high contribution of lipid headgroups to the internal buffering power of the liposomes is observed, amounting to an equivalent phosphate buffer concentration of 110 mM. This localized two-dimensional proton/hydroxide ion reservoir must be considered in any determination of the H+/OH- permeability coefficient. Furthermore, it could have significance for energy-transduction across biological membranes. From the established linear relation between delta pH-relaxation rates and buffering power, net H+/OH- permeabilities of 3 X 10(-3) cm/s for soybean phospholipid (SBPL) and 1 X 10(-4) cm/s for diphytanoyl phosphatidylcholine (diphytanoyl PC) vesicles at pH 7.2 as well as buffering powers per lipid molecule of 6 X 10(-2) (pH-unit)-1 (SBPL) and 4 X 10(-2) (pH-unit)-1 (diphytanoyl PC) are calculated. In the case of diphytanoyl PC vesicles, delta pH-decay is accelerated by the presence of chloride ions. PMID- 3017472 TI - [Reversible accumulation of double- and single-strand breaks of DNA during transition of cells into the quiescent stage]. AB - The technique of neutral elution has been used to study DNA fragmentation in SV 40-transformed Hungarian hamster fibroblasts of 4/21 strain. Accumulation of DNA double-strand breaks was observed in growth-arrested cells. The breaks were repaired after the growth resumed. It is suggested that double-strand breaks are a sum of one-strand breaks. PMID- 3017471 TI - [Prevention of stress-induced disorders of myocardial Na, K-ATPase activity using adaptation to short-term stress]. AB - It was shown that adaptation to short-term emotional-painful stresses prevented both the decline in the activity and the increase in thermal denaturation rate of Na,K-ATPase in sarcolemmal vesicles isolated from the rat myocardium exposed to prolonged stress. The role of Na-pump damage in the disturbance of the electrical stability of the heart and the development of stress-induced arrhythmia is discussed. PMID- 3017470 TI - Retroviruses, immunosuppression and osteopetrosis. AB - Since decades, retroviruses are known to induce the formation of dense bones (osteopetrosis) in animals, either by increasing osteoblastic proliferation (ALVs viruses) or by decreasing osteoclastic bone resorption (FeLV virus). The latter is a good model of the inherited disease since neonatally infected cats die from wasting disease, like mutant op/op rats and children suffering from juvenile malignant osteopetrosis. These similarities have prompted this review of retrovirus-induced osteopetrosis in animals. We suggest the possibility that retroviruses might be involved, at least in part, in the induction of the human disease. PMID- 3017474 TI - [Analysis of the spectrum of ACTH-immunoreactive peptides in the brain of inbred mice]. AB - The ACTH-immunoreactive peptides (IP) distribution in the acid extract of brain and plasma fractions of C57BL/6, BALB/c and F1 (C57BL/6 X BALB/c) mice was investigated. IP were isolated by high-performance liquid chromatography. Interstrain differences in the IP spectrum were found in intact mice and mice after exposure to stress in the "open-field" test. PMID- 3017473 TI - [Effect ot thyroliberin on rat brain opiate receptors]. AB - The ability of thyroliberin to interact with opiate receptors of the rat midbrain and hypothalamus has been studied. It was shown by competitive displacement analysis that thyroliberin did not replace labeled opioid peptides in opiate receptor binding sites when added in vitro at concentrations of up to 10(-5) M. The specific binding of opioid peptides was increased by 10-20% in the presence of 10(-7)-10(-6) M thyroliberin. This effect was, probably, due to the rise in the affinity of high-affinity opiate receptors. At the same time the affinity of low-affinity binding sites was decreased. It is suggested that the antagonistic properties of thyroliberin are mediated by the modulation of the binding characteristics of enkephalin-low-affinity opiate receptors. PMID- 3017475 TI - PMN heterogeneity: long-term stability of fluorescent membrane potential responses to the chemoattractant N-formyl-methionyl-leucyl-phenylalanine in healthy adults and correlation with respiratory burst activity. AB - Polymorphonuclear neutrophils (PMNs) were isolated from 24 healthy adults 20 to 61 years of age and the proportion of cells that demonstrated depolarization responses to the synthetic chemotaxin N-formyl-methionyl-leucyl-phenylalanine (FMLP) were enumerated using the lipophilic fluorescent cyanine dye 3,3'-di pentyl-oxacarbocyanine [di-O-C(5)(3)] and flow cytometry. The membrane potential responses were correlated to the cells' respiratory burst capabilities as measured by nitroblue tetrazolium (NBT) reduction and/or superoxide production in response to FMLP; 40.2% +/- 15.1% (mean +/- SD) of cells depolarized to FMLP. The mean SE for duplicate determinations 1 hour apart was 3.8% (range 0.4% to 13.6%, n = 15). There was no correlation between the percentage of depolarizing PMNs and the yield of cells, the percentage of immature cells, or the circulating WBC count. There was no difference in the average age of men (34.9 +/- 9.9 years, n = 11) v women (33.8 +/- 8.5, n = 13) (mean +/- SD) studied, or in the percentage of depolarizing PMNs when men and women were compared (42.2 +/- 10.6% v 43.1 +/- 13.3%, respectively). However, there was a significant increase in the percentage of depolarizing PMNs with increasing age of women (r = .61, P less than .025) but not men (r = .03, P greater than .05). Analysis of variance revealed significantly greater person-to-person variability in the membrane potential response than in day-to-day variability in the same person (P less than .0005). The percentage of depolarizing PMNs in response to FMLP was significantly correlated with the percentage of NBT-positive cells from both purified PMNs and from whole blood (r = .849, P less than .0005, r = .857, P less than .05, respectively), and with the amount of superoxide produced, expressed as a percentage of that amount produced by cytochalasin B (cyto-B)-pretreated cells (r = .565, P less than .01). The data indicate that PMNs from healthy adults demonstrate a heterogeneous membrane potential response to the chemotaxin FMLP that correlates with the cells' oxidative responsiveness and that intersubject differences can be detected. In addition, the proportion of responsive PMNs increases with increasing age in women. PMID- 3017476 TI - Acute undifferentiated leukemia: implications for cellular origin and clonality suggested by analysis of surface markers and immunoglobulin gene rearrangement. AB - Although B cell leukemias and, recently, T cell leukemias can be identified both by surface marker and molecular analysis, there remains a population of acute undifferentiated leukemias (AUL) that cannot be allocated definitively to a single cell lineage. AUL was diagnosed in nine patients according to stringent criteria. We combined both immunologic and molecular approaches to analyze further the ambiguous origin of AUL cells. Southern blot analysis revealed rearranged Ig heavy-chain genes in seven patients and indicated a biclonal or oligoclonal leukemic cell population in three of them, including one case of AUL with translocation (4;11). Analysis of cell surface markers showed expression of at least one early B cell-associated antigen (BA-1, BA-2, B4, UL-38) in six of these seven patients, with coexpression of a myeloid antigen (VIM-2) in three patients. Leukemic cells of two other patients neither exhibited Ig chain gene rearrangements nor expressed B cell-associated antigens. T cell receptor beta chain genes showed germline configuration in all nine cases. Our results demonstrate heterogeneity among AUL patients based on molecular and surface marker analyses and suggest that most AUL blast cells are derived from a precursor cell that shares phenotypic and genotypic characteristics of early B cells with certain surface antigens of myeloid cells, in some cases of AUL more than one abnormal cell clone or subclone may exist, and the cellular origin, at least of some AULs exhibiting t(4;11), may be truly B cell lineage committed. PMID- 3017477 TI - Glycoprotein V is not the thrombin activation receptor on human blood platelets. AB - Thrombin activation of platelets involves two receptors: glycoprotein Ib (GPIb), which affects the kinetics of the response; and, as a strong candidate for the second, essential receptor, GPV, a hydrophobic, 82-kd glycoprotein with an isoelectric point (pI) of pH 5.85 to 6.55. Whole platelets were treated with endogenous platelets calcium-activated proteases, yielding a major fragment, GPV8, with molecular weight (mol wt) of 79 kilodaltons (kd). The fragment was purified by affinity chromatography on wheat germ agglutinin followed by ion exchange chromatography on DEAE-Sephacel using first a 0 to 0.7-mol/L and then a 0 to 0.3-mol/L NaCl gradient. A rabbit was immunized with the purified GPV8 for preparation of polyclonal antibodies. Crossed immunoelectrophoresis and two dimensional polyacrylamide gel electrophoresis (PAGE) electrophoretic blotting with the separate phases of a Triton X-114 phase partition of human platelets showed the characteristic pattern of GPV in the hydrophobic phase. During thrombin-induced platelet aggregation GPV is hydrolysed, releasing a fragment, GPVf1, to the supernatant. The fragment GPVf1 still contains a thrombin-binding site. Anti-GPV antibodies blocked GPV proteolysis, but did not inhibit platelet activation induced by thrombin. We conclude that proteolysis of GPV by thrombin is not essential for platelet activation. PMID- 3017478 TI - Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor. AB - We studied the effects(s) of two monoclonal antibodies, 6D1 and 10E5 (directed against platelet glycoprotein Ib [GPIb] and the GPIIb/IIIa complex, respectively), and purified human plasma fibrinogen on the binding of released platelet-von Willebrand factor (vWf) to the platelet surface. Neither of the monoclonal antibodies nor fibrinogen had any effect on the amount of platelet-vWf expressed on unstimulated platelets or on the amount expressed on platelets stimulated in the absence of extracellular Ca++. However, the antibody directed against GPIIb/IIIa inhibited 72% of the thrombin-induced increase in the platelet vWf bound to the platelet surface when platelets were stimulated in the presence of 5 mmol/L Ca++. The antibody against GPIb did not inhibit the surface expression of platelet-vWf on stimulated platelets in the presence of Ca++. Purified normal human fibrinogen inhibited the surface binding of platelet-vWf to thrombin-stimulated platelets to a degree similar to that observed with the monoclonal antibody directed against the GPIIb/IIIa complex. These data indicate that platelet-vWf released from platelets binds primarily to the GPIIb/IIIa complex at or near the plasma fibrinogen binding site. PMID- 3017480 TI - Hormones and hormone receptors in the etiology of breast cancer. AB - Many epidemiologic features of breast cancer suggest that endogenous hormones are of importance in the genesis of this disease. Endocrinologic studies based on total levels of various hormones in serum and urine have provided etiologic clues, but have failed to yield a clear understanding of the hormonal aberrations that promote the development of breast cancer. One reason for this is probably that neither urinary hormones, nor total levels of serum hormones accurately reflect biologically active levels to which the cells of origin are exposed. Levels of hormones unbound to serum sex binding globulin may be of more etiologic relevance, and future studies utilizing measurements of such hormone fractions may provide new information of etiologic importance. Some risk factors for breast cancer, especially age at birth of first child, may influence risk by altering the susceptibility of mammary ductal epithelial cells to estrogens or other endogenous hormones by altering the level of hormone receptor proteins in these cells. Additional studies to identify the determinants of hormone receptors in the breast are needed to test this hypothesis. PMID- 3017479 TI - Lactoferrin: affinity purification from human milk and polymorphonuclear neutrophils using monoclonal antibody (II 2C) to human lactoferrin, development of an immunoradiometric assay using II 2C, and myelopoietic regulation and receptor-binding characteristics. AB - Several investigators have now confirmed our original report demonstrating the myelopoietic suppressive activity of lactoferrin (LF) in vitro. In order to further clarify this activity, we used the recently produced and purified neutralizing antibody (II 2C) to LF to set up an immunoradiometric assay specific for LF and to affinity purify LF from lysates of peripheral blood polymorphonuclear neutrophils (PMN) obtained from healthy donors. Iron-saturated purified PMN LF was as active as iron-saturated affinity purified milk LF as a suppressor of the release of granulocyte-macrophage colony stimulating factors (GM-CSF) from mononuclear human peripheral blood leukocytes. The activities of both the PMN LF and milk LF were inactivated by preincubation with monoclonal anti-LF antibody (II 2C). In order to evaluate the methods of iron saturation of LF in vitro as measures of their functional activities, milk LF was iron saturated by four different methods, including ferric citrate, ferric ammonium sulphate, ferric chloride with nitriloacetate, and ferric chloride alone. The functional characteristics of all four preparations of LF saturated with iron in vitro were relatively equal and were more active than native LF. Resident mouse peritoneal macrophages separated into subpopulations of GM-CSF-producing cells by velocity sedimentation were evaluated for their LF-receptor binding capacity and for sensitivity to the suppression of GM-CSF release by LF. Iron saturated LF suppressed release of GM-CSF from only those fractions containing LF-receptor bearing cells, although not all fractions containing cells bearing receptors for LF responded to the suppressive activity of LF. These studies provide further evidence for the myelopoietic regulatory activity in vitro of PMN-derived LF, which is mediated through populations of mononuclear phagocytes having receptors for LF. PMID- 3017481 TI - Aminoglutethimide as second line therapy in advanced breast cancer. AB - Seventy-two women with advanced breast cancer were treated with aminoglutethimide and hydrocortisone (AG). All patients were previously treated with hormones and 59 patients had also received cytotoxic agents. A partial response to AG with a mean duration of 9 months (range: 4-26 months) was achieved in 24 patients (33%), 10 patients (14%) had stable disease, and 32 patients (44%) were progressing during AG therapy. Response to previous treatment seemed to be correlated to response to AG, as 47% of patients having responded to the first hormonal treatment also responded to AG, while only 16% of the progressors responded to AG. The number of previous treatments influenced neither the frequency nor duration of response to AG. The median survival after start of AG was 13 months, and 31% of the patients lived more than 2 years. Side-effects led to discontinuation of therapy in 9 patients (13%). Three patients developed reversible agranulocytosis. PMID- 3017482 TI - [Scanning electron microscopic demonstration of putrescine receptor sites on the surface of cancerous hepatocytes in culture]. PMID- 3017483 TI - Chelation in metal intoxication XXI: Chelation in lead intoxication during vitamin B complex deficiency. PMID- 3017485 TI - Genetic linkage. AB - Not all doctors would understand the statement "linkage is now established between a DNA marker and the locus for adult polycystic kidney disease at a recombination fraction of 5%". Yet over the next few years increasing numbers of DNA markers linked to single-gene disorders will become available and clinicians will need to identify patients who could benefit from this knowledge. PMID- 3017484 TI - Characterization and expression of collagen-like genes in Drosophila melanogaster. AB - We have used a cloned chicken collagen cDNA sequence to help identify hypothetic members of the collagen gene family from Drosophila melanogaster. Several experimental evidences have been obtained which indicate that the Drosophila genome contains numerous collagen-like sequences. We have characterized in more detail ten distinct DNA sequences that hybridized strongly to the heterologous collagen probe. By in situ hybridization we have shown that these sequences are dispersed throughout the Drosophila genome. Two of them are shown to originate from the previously described DCg 1 and DCg 2 collagen genes. In other respects, we show that in addition to DCg 1 and DCg 2, at least five putative collagen genes are expressed during the Drosophila lifetime. These genes are unique, and some of them are seen to be transcribed into different size classes of mRNAs. Additionally, the data presented so far demonstrate that the expression of these genes is regulated temporally and/or quantitatively during the Drosophila life cycle. PMID- 3017487 TI - Calcium channel inhibitors suppress the morphine-withdrawal syndrome in rats. AB - The effects of the Ca2+-channel blockers verapamil and nimodipine, on the behavioural signs of naloxone (1 mg kg-1)-induced abstinence syndrome in morphine dependent rats, were evaluated. The content of noradrenaline (NA) and of its metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) was measured, using high performance liquid chromatography and electrochemical detection or gas chromatography-mass spectrometry, in various brain regions of these animals. Possible interactions of nimodipine and verapamil with opioid receptors were evaluated by examining their ability to displace [3H]-naloxone binding to brain membranes. Verapamil (5, 10 and 50 mg kg-1) and nimodipine (1, 5 and 10 mg kg-1) dose-dependently reduced most of the signs of morphine abstinence. Naloxone precipitated abstinence decreased the NA content in the cortex, hippocampus, brainstem and cerebellum. In the same brain regions the content of MHPG increased, suggesting an increased release of the amine during morphine abstinence. Nimodipine (10 mg kg-1 i.v.) did not change the content of NA or MHPG in the cortex, hippocampus and brainstem. However, nimodipine pre-treatment markedly reduced the changes in NA and MHPG content induced by the abstinence syndrome. Neither verapamil nor nimodipine displaced [3H]-naloxone from its binding sites. These results suggest that Ca2+-channel blockers suppress the behavioural and neurochemical expressions of morphine abstinence by a mechanism that differs from those of opioids or alpha 2-adrenoceptor agonists. PMID- 3017486 TI - The effects of drugs acting at the GABAA-receptor/ionophore after chemical kindling with the benzodiazepine receptor ligand FG 7142. AB - Repeated administration of the beta-carboline benzodiazepine receptor ligand FG 7142 produces sensitization to its effects so that full seizures develop (chemical kindling); initially it is only pro-convulsant. The present study investigated alterations in the function of drugs which act at the different sites at the gamma-aminobutyric acid (GABA) benzodiazepine receptor complex, after repeated administration of FG 7142. In FG 7142 kindled mice decreased anticonvulsant and hypothermic effects of the GABA agonist muscimol were observed. The hypothermic effects of the GABA agonist progabide were reduced. In contrast a small increase in the hypothermic effect of pentobarbitone was seen. The convulsant effects of bicuculline and picrotoxin were unaltered when they were given intravenously but marginally increased when they were given by the intraperitoneal route. No changes were seen in the hypothermic effects of these drugs. No significant changes were seen in the convulsant or hypothermic effects of pentylenetetrazol. These results suggest that kindling with FG 7142 may alter GABA receptor function. PMID- 3017488 TI - Pharmacological characterization of the receptor involved in chemoexcitation induced by adenosine. AB - Experiments were performed on cats anaesthetized with pentobarbitone in which carotid body chemoreceptor activity was recorded from the peripheral end of a sectioned carotid nerve. Intracarotid (i.c.) injections of adenosine and its analogues, NECA (5'-N-ethylcarboxamidoadenosine), L-PIA(L-N6 phenylisopropyladenosine), and D-PIA(D-N6-phenylisopropyladenosine), caused dose related increases in chemosensory discharge. The rank order of potency as chemoreceptor stimulants was: NECA greater than adenosine greater than L-PIA greater than D-PIA. Infusion of theophylline antagonized the chemoexcitatory effects of NECA, and infusion of 8-phenyltheophylline (8-PT), which is a more potent adenosine antagonist with less activity as a phosphodiesterase inhibitor, reduced the chemoexcitation induced by adenosine. Infusion of 8-PT (10 micrograms min-1 i.c.), a dose which substantially reduced the effect of injected adenosine, also reduced the sensitivity of carotid chemoreceptors to hypoxia (10% O2 for 4 min). It is concluded that the adenosine receptors in the cat carotid body which mediate chemosensory excitation are xanthine-sensitive and appear to be of the A2 sub-type. Adenosine, released within the carotid body by physiological stimuli, may be involved in chemoexcitation. PMID- 3017489 TI - Autoradiographic localization of beta-adrenoceptors in pig lung using [125I] iodocyanopindolol. AB - The binding of the beta-adrenoceptor radioligand [125I]-iodocyanopindolol (I-CYP) has been studied in pig lung parenchyma and the distribution of binding sites visualised by light microscopic autoradiography. I-CYP binding was saturable (maximum binding capacity Bmax = 51 +/- 3 fmol mg-1 protein), involving sites with high affinity (dissociation constant KD = 73 +/- 10 pM). Specific I-CYP binding was displaceable both by beta-adrenoceptor agonists ((-)-isoprenaline greater than (-)-adrenaline greater than (+/-)-fenoterol greater than (-) noradrenaline greater than (+)-isoprenaline greater than (+/-)-RO363) and antagonists ((+/-)-propranolol greater than ICI-118551 greater than atenolol), indicating a predominance of beta 2-adrenoceptors. Further analysis showed that displacement data for the beta 1-selective antagonist atenolol and the beta 2 selective antagonist ICI-118551 were fitted best to a 2 binding site model and that both beta 1- and beta 2-adrenoceptors were present in pig lung in the ratio 28:72 respectively. Autoradiographic grains were localized over tissue and were most dense over alveolar walls greater than vascular endothelium greater than vascular smooth muscle greater than bronchial smooth muscle = bronchial epithelium. Atenolol (10(-5) M) caused a 31% reduction in specific grain density over alveolar wall tissue, while a 10 fold lower concentration of ICI-118551 (10( 6) M) caused a 50% decrease. These results are consistent with binding data in pig lung parenchyma demonstrating a mixed population of beta-adrenoceptors with a predominance of the beta 2 subtype. 6 It is possible that the previously described relaxant responses of the pig lung parenchyma strip to beta-agonists, mediated via beta 2-adrenoceptors, resulted from the sum of reactivities in airway and vascular smooth muscle together with relaxation of alveolar interstitial cells. PMID- 3017490 TI - Meptazinol has a similar agonist action on opioid receptors in field-stimulated mouse vas deferens and guinea-pig ileum. AB - The effects of the opioid receptor agonist RX783006 and of the opioid receptor partial agonist (+)-meptazinol have been examined on electrically induced twitch responses of the guinea-pig isolated ileum and of the mouse isolated vas deferens. Log10 concentration-tissue state curves were determined for (+) meptazinol and RX783006, alone, in combination and in the presence of naloxone (30 nM). Analysis of these log10 concentration-tissue state curves using the null equations derived and tested in the preceding paper indicates that the opioid agonist action of (+)-meptazinol on mouse vas deferens is quantitatively similar to that on guinea-pig ileum. The results also suggest that (+)-meptazinol acts as a functional antagonist on the guinea-pig ileum as well as on the mouse vas deferens. The potency of (+)-meptazinol relative to RX783006 has been measured by an indirect method which should eliminate any functional antagonistic action of (+)-meptazinol. This method gives a relative potency of (+)-meptazinol in both tissues which is three to six times greater than that measured directly on guinea pig ileum. This discrepancy may be due to experimental error but it may also indicate that direct measurements on guinea-pig ileum underestimate the agonist potency of this compound on opioid receptors. PMID- 3017491 TI - Chronic caffeine treatment reduces caffeine but not adenosine effects on cortical acetylcholine release. AB - The effects of both adenosine and caffeine on the release of acetylcholine (ACh) were investigated in slices of cerebral cortex taken from rats pretreated for 30 days with caffeine (100 mg kg-1 daily, dissolved in their drinking water) at rest and during electrical stimulation at frequencies of 0.2, 1 and 5 Hz. The effect of this treatment on adenosine binding sites was also investigated in cortical membranes using N-cyclohexyl-[3H]-adenosine ([3H]-CHA) as a ligand. The chronic caffeine treatment did not change animal growth patterns. Spontaneous exploratory activity appeared to be increased at the 3rd day but was unchanged at the 30th day when compared with controls. Caffeine-treatment increased the number of high affinity binding sites for [3H]-CHA by 64% over the control values. Low affinity binding site density and affinity constants were unaffected. Adenosine 30 microM added to the superfusion fluid decreased electrically stimulated ACh release both in rats drinking tap water and rats drinking caffeine. In rats drinking tap water, caffeine added to the superfusion fluid at a concentration of 50 microM enhanced ACh release, while at 0.5 mM it decreased ACh output from the slices. Both effects were abolished by pretreatment with caffeine in vivo. The results indicate that prolonged consumption of high doses of caffeine causes changes in the responsiveness of cholinergic neurones to caffeine. The change is not shared by adenosine, through whose recognition sites caffeine is believed to act. It is therefore possible that the adaptive changes following repeated caffeine administration involve either only the coupler-transducer mechanism activated by the antagonist, or effects unrelated to receptors. PMID- 3017492 TI - Electrophysiological studies in cultured mouse CNS neurones of the actions of an agonist and an inverse agonist at the benzodiazepine receptor. AB - The action of agents which bind with the benzodiazepine (BZ) receptor has been investigated by use of intracellular recordings from dissociated mouse neurones grown in tissue culture. The agents tested were midazolam (an agonist at the BZ receptor) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM-an inverse agonist at the BZ receptor). These were applied to the neurone under study by one of the following methods: iontophoresis; pressure application of known concentrations from blunt pipettes; directly in the perfusing medium. On only very few occasions did midazolam or DMCM have a direct effect on the membrane potential (EM) or conductance (GM) of the impaled neurone. For the neurones where direct effects were present, there was no consistent pattern of response. Neither substance affected the threshold for action potential generation. The effect of midazolam and DMCM on responses evoked by iontophoretic application of gamma-aminobutyric acid (GABA) was also investigated. Three parameters were used to quantify GABA responses: the depolarization (VGABA); the increase in GM (gGABA) measured with constant current pulses; using voltage clamp, the GABA current (IGABA). The GABA response should be quantified by a parameter which is linearly related to the number of GABA-operated channels which are conducting at any instant. VGABA does not fulfil this criterion. gGABA is an appropriate parameter, but is difficult to determine for large responses where the membrane is nearly short circuited. IGABA measured during voltage clamp fulfils this criterion. Midazolam (greater than 10(-6) M) reliably potentiated GABA responses with a parallel shift to the left of the dose-response curve. This is in agreement with biochemical studies where BZs increase the affinity of the GABA receptor for its ligand. The effect of DMCM on GABA responses was more variable. In the majority of cases GABA responses were reduced by DMCM. The threshold dose for this depression was usually around 10(-6) M, but was sometimes as low as 10(-8) M. Dose-response curves of IGABA or gGABA showed the inhibition to be of a non-competitive nature. The maximum inhibition achieved was around 70%. For a given neurone, and at doses which did not necessarily cause a reduction of the response to GABA, DMCM could antagonize the potentiating action of midazolam on GABA responses. A possible interpretation is that more than one BZ site per receptor complex must be occupied by a BZ agonist (or inverse agonist) before the functional changes for the complex as a whole can occur. Desensitization to GABA was increased by midazolam. PMID- 3017493 TI - The effects of an adenylate cyclase inhibitor on the electrophysiological correlates of neuromuscular transmission in the frog. AB - The presynaptic and postsynaptic effects of MDL 12,330A, an adenylate cyclase inhibitor in several biological tissues, were studied at motor endplates in frog cutaneous pectoris nerve-muscle preparations. This agent increased both spontaneous quantal acetylcholine (ACh) release and neurally-evoked ACh release approximately twofold during the first 20-40 min of application. The increased ACh release was accompanied by a profound irreversible depression in the amplitudes of the miniature endplate potentials (m.e.p.ps) and endplate potentials (e.p.ps). The response to iontophoretically-applied ACh was reduced in parallel with the amplitude of the spontaneous m.e.p.ps, indicating that the depression of synaptic transmission was postsynaptic in origin. Endplates were voltage-clamped to study the postsynaptic depression in more detail. It was observed that the peak endplate current (e.p.c.) was depressed without concomitant changes in: the kinetics of e.p.c. decay, the relationship between peak e.p.c. and membrane potential, the ACh equilibrium potential or the voltage sensitivity of the e.p.c. decay. This suggests that MDL 12,330A reduces the postsynaptic sensitivity to ACh by a voltage-dependent block of the cholinoceptor. The presynaptic enhancement and the postsynaptic depression of junctional transmission produced by MDL 12,330A are discussed in conjunction with current theories of the role of adenylate cyclase and cyclic nucleotides at nicotinic cholinergic synapses. PMID- 3017495 TI - Reversal by beta-funaltrexamine of the antinociceptive effect of opioid agonists in the rat. AB - The effect of the irreversible opioid receptor antagonist, beta-funaltrexamine (beta-FNA), on antinociception produced by mu- and kappa-receptor agonists was studied in the rat. beta-FNA, 20 to 80 mg kg-1, s.c., given 24 h before testing, produced a dose-related antagonism of the effects of morphine in the paw pressure, hotplate and tail-flick tests. Following the 80 mg kg-1 dose, the degree of antagonism of morphine was stable for up to 48 h after dosing, but was reduced by 5 days and had disappeared by 8 days. In the paw pressure test, beta FNA, 40 mg kg-1, s.c., antagonized the effects of fentanyl, buprenorphine, tifluadom, ethylketocyclazocine and proxorphan; it was without effect against the highly selective kappa-agonist, U-50,488. In light of these results, the possible opioid receptor selectivities of both the agonists and beta-FNA are reassessed. PMID- 3017494 TI - Novel interactions of cations with dihydropyridine calcium antagonist binding sites in brain. AB - The effects of monovalent (Na+, Li+, K+, Rb+) and divalent (Ca2+, Mg2+, Mn2+) cations on dihydropyridine calcium antagonist binding sites in brain and cardiac membranes were investigated using a low ionic strength buffer (5 mM Tris-HCl, pH 7.4), and the dihydropyridine, [3H]-nitrendipine. At 25 degrees C, the monovalent cations Na+, Li+, and K+ (100 mM) but not Rb+ significantly decreased the apparent dissociation constant (KD) but had no effect on the maximum binding site capacity (Bmax) of [3H]-nitrendipine in brain. The divalent cations Ca2+, Mg2+, and Mn2+ (2 mM) significantly increased the Bmax, but did not affect the KD of [3H]-nitrendipine. The effects of cations were concentration-dependent (EC50 monovalent cations 10-25 mM; EC50 divalent cations 50-200 microM) and demonstrated brain region selectivity. The effect of Ca2+, but not Mg2+ or Mn2+ on [3H]-nitrendipine binding was described by a two-site model. At 25 degrees C, neither mono- nor divalent cations altered the characteristics of [3H] nitrendipine binding to rat cardiac membranes. At 37 degrees C, Na+ (100 mM) but not K+ (100 mM) significantly increased the Bmax of [3H]-nitrendipine in rat brain membranes. Ca2+ (2 mM) significantly increased the Bmax of [3H] nitrendipine binding to rat brain membranes to a greater extent than at 25 degrees C. Both Na+ and K+ had no effect on [3H]-nitrendipine binding to cardiac membranes, while Ca2+ (2 mM) significantly decreased the KD of [3H]-nitrendipine. It is suggested that the selective effects of mono- and divalent cations on [3H] nitrendipine binding to rat brain and cardiac membranes may be associated with differences in the calcium current blocking activity of dihydropyridine calcium antagonists in brain and cardiac tissues. PMID- 3017496 TI - Classification of adenosine receptors mediating antinociception in the rat spinal cord. AB - Analogues of adenosine were injected intrathecally into rats implanted with chronic indwelling cannulae in order to determine a rank order of potency and hence characterize adenosine receptors involved in spinal antinociception. In the tail flick test L-N6-phenylisopropyl adenosine (L-PIA), cyclohexyladenosine (CHA) and 5'-N-ethylcarboxamide adenosine (NECA) produced dose-related antinociception which attained a plateau level. NECA and CHA also produced an additional distinct second phase of antinociception. D-N6-Phenylisopropyl adenosine (D-PIA) and 2 chloroadenosine (CADO) had very little antinociceptive activity in this test. The rank order of potency in producing the plateau effect was L-PIA greater than CHA greater than NECA greater than D-PIA = CADO, while that for the second phase of antinociception was NECA greater than-CHA. Pretreatment with both theophylline and 8-phenyltheophylline (8-PT) antagonized antinociception produced by CHA, with 8-PT being at least an order of magnitude more potent than theophylline. Both antagonists produced a significant hyperalgesia in the tail flick test. L-PIA and CHA also produced methylxanthine-sensitive antinociception in the hot plate test. These results suggest that activation of A1-receptors in the spinal cord can produce antinociception. Activation of A2-receptors may produce an additional effect, but the relative activity of CHA in this component of activity is unusual. PMID- 3017497 TI - Prostaglandin D2 interacts at thromboxane receptor-sites on guinea-pig platelets. AB - The anti-aggregatory prostanoid, prostaglandin D2 (PGD2) does not completely inhibit ADP-induced aggregation of guinea-pig platelets and thus produces a bell shaped dose-inhibition curve. The nature of this bell-shaped curve has now been investigated in guinea-pig platelet-rich plasma. Two selective thromboxane receptor antagonists, 13-aza-prostanoic acid (13-AZA; 16-64.4 microM) and BM 13.177 (5.9-29.8 microM), converted PGD2 to a full inhibitor of aggregation in a dose-related manner. The putative platelet PGD2 receptor antagonist, N-0164 (75 microM) also converted PGD2 to a full inhibitor of platelet aggregation. In contrast to 13-AZA and BM 13.177, higher concentrations of N-0164 (380 and 760 microM) caused a dose-related rightward shift of the PGD2 dose-inhibition curve. The thromboxane receptor antagonism of N-0164 was confirmed in studies in which the dose-aggregation curve to U-46619, a thromboxane mimetic, was competitively antagonized with a pA2 value of 4.67 and a slope of 1.13, comparable to that of 13-AZA. The results show that N-0164 acts as both a platelet PGD2 and thromboxane receptor antagonist in both human and guinea-pig platelet-rich plasma. The results further indicate that PGD2 can interact at thromboxane receptors in guinea-pig platelets. PMID- 3017498 TI - Should radiotherapy be used routinely in the management of benign parotid tumours? PMID- 3017499 TI - Failure to improve survival by improved diagnostic techniques in patients with malignant jaundice. AB - In a consecutive series of 118 patients with malignant obstructive jaundice at North Tees General Hospital only 70 patients were adjudged fit to undergo surgery. Tumour resection was possible in only four patients. Palliative bypass was performed in 45 patients while 21 patients had no procedure other than an exploratory laparotomy. In contrast to the accuracy of investigation the results of surgery have been poor with considerable mortality (42.8 per cent) and lack of long-term survivors. The results emphasize the role of non-surgical methods for relieving jaundice of malignant aetiology, especially in frail elderly patients and those who have metastases at the time of presentation. PMID- 3017500 TI - Hearing impairment in childhood. PMID- 3017502 TI - Immunocytochemical localization of pro-opiomelanocortin-derived peptides in the adult rat spinal cord. AB - A dispersed descending pro-opiomelanocortin (POMC) fiber system has been demonstrated by peroxidase-antiperoxidase (PAP) immunocytochemistry in the adult rat spinal cord. beta-endorphin, adrenocorticotrophic hormone (ACTH), alpha melanocyte-stimulating hormone (alpha-MSH) and 16K immunoreactive fibers exist in the spinal cord from cervical down to sacral level. Descending fibers running parallel in the dorsolateral and lateral funiculus send collaterals ventromedially or medially to terminate in the gray matter surrounding the central canal, where nociceptive neurons have recently been located, in addition to those nociceptive cells in the dorsal horn. After spinal transection at lower thoracic level, POMC peptide immunoreactivities disappeared below the lesion. Moreover, no POMC cell bodies were found in the spinal cord. Therefore, the descending fibers are most likely of supraspinal origin. PMID- 3017501 TI - Beneficial effects of angiotensin converting enzyme inhibition on renal function in patients with diabetic nephropathy. AB - The effects of angiotensin converting enzyme inhibition with captopril were investigated in patients with diabetic nephropathy and hypertension. After nine days' treatment with captopril glomerular filtration rate was unchanged in 13 patients, whereas renal plasma flow had increased from 265 to 302 ml/min/1.73 m2 body surface area (p less than 0.05) and the filtration fraction had decreased from 14.3 to 12.8% (p less than 0.025). During two years' treatment with captopril in 14 patients the mean arterial blood pressure had fallen by 5 mm Hg (p less than 0.005) and the deterioration in glomerular filtration rate had decreased from 10.3 to 2.4 ml/min/year (p less than 0.005). There was no correlation between the fall in blood pressure and the reduction in the deterioration of glomerular filtration rate. These findings suggest that the effects of angiotensin converting enzyme inhibition on renal haemodynamics protect renal function. Inhibitors of angiotensin converting enzyme should be considered for lowering blood pressure in patients with diabetic nephropathy. PMID- 3017503 TI - Autoradiographic localization of mu- and delta-opiate receptors in the forebrain of the rat. AB - The autoradiographic distributions of mu opiate receptors, labeled in vitro by [125I]D-Ala2-MePhe4-Met(o)5-ol-enkephalin (FK), and delta-opiate receptors, labeled by [3H]D-Ala2-D-Leu5-enkephalin (DADLE) in the presence of oxymorphone to block high affinity binding to the mu site, were examined and compared in the forebrain of the rat. The mu- and delta-receptors were differentially distributed in most structures. mu Binding sites were found in nearly all gray matter structures and showed heterogeneous patterns of density that were correlated with cytoarchitecture and neuronal connections. Laminar density profiles were seen in laminated structures such as olfactory bulb, cerebral cortex and hippocampus. Highest mu binding densities were in striatal patches and the habenular streak. delta Sites had distinct laminar patterns in the main olfactory bulb and cortex which differed from the mu patterns. The external plexiform layer of the main olfactory bulb had the greatest density of delta binding sites; cortex and striatum were also densely labeled. The septum, globus pallidus, preoptic area and hypothalamus were lightly labeled by both ligands. The magnocellular hypothalamic nuclei had negligible mu and delta labeling. The thalamus had dense mu but sparse delta sites. mu And delta binding sites were both present in the amygdala but had different distributions. Two fiber tracts--optic tract and fasciculus retroflexus--had FK labeling. In contrast, a portion of the corpus callosum was labeled by DADLE and not by FK. The results suggest an association of mu-opiate receptors with sensory, especially olfactory, and limbic projections in the forebrain, and delta-opiate receptors with intrinsic and commissural forebrain pathways. PMID- 3017506 TI - Myelin phagocytosis in Wallerian degeneration of peripheral nerves depends on silica-sensitive, bg/bg-negative and Fc-positive monocytes. AB - Previous experiments with nerves enclosed in millipore diffusion chambers had shown that myelin degradation during Wallerian degeneration depends on invasion by non-resident cells. The present study was aimed at a more precise identification of the invading cell population. Monoclonal antibody studies of degenerating nerves showed many cells with the Fc marker; cells having the Lyt-1, Lyt-2, Ia or Mac-1 markers were sparse or absent. Nerves transplanted into mice of the Chediak-Higashi bg/bg strain were invaded by cells lacking the bg/bg marker (giant lysosomes), while cotransplanted muscle tissue was invaded by cells with the bg/bg marker. Blocking monocytes with silica reduced both cell invasion and myelin degradation in degenerating nerves. These observations show that Wallerian degeneration of peripheral nerve fibers involves a subset of monocytes which are silica-sensitive and have Fc receptors but no bg/bg giant lysosomes. PMID- 3017505 TI - Correlational analysis of central noradrenergic neuronal activity and sympathetic tone in behaving cats. AB - The activity of the noradrenergic (NA) neurons of the feline locus coeruleus complex (LCx) were correlated with changes in peripheral sympathetic tone in behaving cats. LCx NA neurons exhibit stereotypical changes in discharge rate across behavioral states, falling virtually silent during paradoxical sleep (PS). Simultaneous recordings from LCx NA neurons and the cervical sympathetic trunk demonstrated a tonic reduction in nerve activity during PS as well. LCx NA neurons were also found to fall silent during induction of the scruff immobility reflex and significant pupillary miosis was seen during this behavior as well. These data support the hypothesis that NA neurons in the brain are a central analogue of the peripheral sympathetic system and demonstrate that the two systems operate in an integrated fashion in the behaving cat. PMID- 3017508 TI - Excitation of mouse motoneurones by GABA-mediated primary afferent depolarization. AB - Observations on the reflex properties of a mouse spinal cord preparation in vitro are reported. The findings show that the synaptically evoked, GABA-mediated, discharge of action potentials in primary afferent fibres, monitored as the dorsal root reflex, may lead to the excitation of motoneurones. Subthreshold, bicuculline-sensitive increases in motoneuronal excitability, followed by prolonged inhibition, may be seen in preparations in which the delayed reflex is not seen. Thus, primary afferent depolarization may both increase motoneuronal excitability and also cause presynaptic inhibition of afferent input. PMID- 3017507 TI - Alpha 2-adrenoceptor-GTP binding regulatory protein-adenylate cyclase system in cerebral cortical membranes of adult and senescent rats. AB - The characterization of [3H]clonidine binding and effects of GTP, forskolin, islet-activating protein (IAP) and cholera toxin on adenylate cyclase activity were investigated in cerebral cortical membranes from 70-day-old and 2-year-old rats. Neither Kd nor Bmax values in [3H]clonidine binding were changed between day 70 and year 2. The activation of adenylate cyclase by forskolin was significantly higher in senescent than in adult animals. The inhibitory effect of adrenaline, which was completely abolished by the pretreatment with IAP/NAD on forskolin/GTP-stimulated cyclase activity, was low in senescent rats compared to that in adult ones. The stimulatory effect of cholera toxin/NAD was also low at the senescent stage compared to that at the adult stage. It is suggested that ligand binding affinity and the density in alpha 2-adrenoceptors do not change between day 70 and year 2 but that GTP binding and/or coupling activity of inhibitory as well as stimulatory GTP binding regulatory protein to catalytic units decrease in synaptic membranes of 2-year-old compared to those of 70-day old rat brain. PMID- 3017504 TI - Mu receptors at discrete hypothalamic and brainstem sites mediate opioid peptide induced increases in central sympathetic outflow. AB - Synthetic human beta-endorphin, 7.25 nmol intracisternally, in conscious, freely moving, cannulated adult male rats increased plasma concentrations of the 3 catecholamines, epinephrine, norepinephrine and dopamine. Similarly administered equimolar morphine increased only plasma epinephrine concentration significantly. A 10-fold greater intracisternal dose of morphine significantly increased plasma concentrations of all 3 catecholamines. This effect was inhibited by prior intra arterial naloxone administration. Intracisternal administration of the selective mu receptor agonist [D-Ala2,NMe-Phe4,Gly-ol5]enkephalin (DAGO), 2.9 nmol, also increased plasma concentrations of the 3 catecholamines and, furthermore, these effects were significantly greater than those noted in response to equimolar beta endorphin. The greater potency of DAGO than beta-endorphin to increase catecholamine secretion suggests that this opioid peptide-induced effect is mediated at mu receptors. Administration of DAGO, 0.1 nmol, directly into either the hypothalamic paraventricular nucleus (PVN) or brainstem nucleus of the solitary tract (NTS) significantly increased plasma concentrations of all 3 catecholamines when compared with either saline-infused controls or animals administered DAGO into other brain areas. These catecholamine-stimulating effects of DAGO administered into either PVN or NTS were prevented by prior intra arterial naloxone administration. Heart rate, but not mean arterial blood pressure, increased in response to DAGO administration into the NTS while no significant cardiovascular changes were noted among the experimental groups in response to DAGO administered into the PVN. These data support a hypothesis that mu receptors at discrete and anatomically distant brain sites mediate opioid peptide-induced catecholamine secretion through activation of the central sympathetic outflow to the adrenal medulla and sympathetic nerve terminals. PMID- 3017509 TI - Interactions between septal and entorhinal inputs to the rat dentate gyrus: facilitation effects. AB - We examined facilitation effects between the medial septum and perforant path inputs to the dentate gyrus for the four possible combinations of paired-pulse activation. Facilitation effects occurred in all cases. The largest facilitation effects occurred when the septal pulse served as the conditioning pulse for the population spike subsequently evoked by a perforant path pulse. Using 3 pulses, we also examined the influence of septal activation on paired-pulse facilitation of the perforant path-granule cell population spike. A septal stimulation pulse, applied 6-10 ms prior to the onset of the population spike evoked by a perforant path conditioning pulse, did not affect the perforant path-dentate test response at any interpulse interval. If the septal pulse occurred immediately prior to population spike onset, however, there was a significantly greater depression of the test response from 70-3000 ms, but no effect at early intervals (20-50 ms). The effect of the septal pulse appears more consistent with a direct action of the septal terminals on granule cells than with an indirect action via the recurrent inhibitory interneurons. PMID- 3017510 TI - Dopamine decreases the calcium-activated afterhyperpolarization in hippocampal CA1 pyramidal cells. AB - The effect of dopamine (DA) on the calcium-activated potassium conductance underlying the slow afterhyperpolarization (AHP) which follows a train of action potentials in hippocampal pyramidal cells was studied utilizing the in vitro hippocampal slice preparation. Bath-applied DA (1-100 microM) significantly reduced the AHP in a reversible, dose-dependent manner. Neither the amount of current injected to elicit the AHP nor its initial amplitude had an effect on the reduction of the AHP by DA. DA did not depress calcium spikes, suggesting that the blockade of the AHP likely occurs at a step subsequent to the entry of calcium. Since DA's actions on the AHP closely mimicked those of norepinephrine, we examined the effect of beta-adrenergic antagonists on DA's actions. At concentrations which in other systems have been shown not to block DA stimulated adenylate cyclase, beta-adrenergic antagonists completely inhibited the reduction of the AHP by DA. In some cells DA also elicited small hyperpolarizations which were not blocked by application of dopamine receptor antagonists. These findings strongly suggest that a major electrophysiological action of DA in the hippocampus (i.e. blockade of the AHP) is due to its cross reactivity with beta adrenergic receptors and that rigid pharmacologic criteria must be used before attributing an action of DA unambiguously to its interaction with DA receptors. PMID- 3017513 TI - Wheat germ agglutinin inhibits nerve fiber growth and concanavalin A stimulates nerve fiber initiation in cultures of dorsal root ganglia neurons. AB - Treatment of dorsal root ganglion (DRG) neurons with the lectin wheat germ agglutinin (WGA; 25 micrograms/ml), which binds to N-acetylglucosamine containing glycoconjugates, inhibits nerve fiber growth in culture. DRG neurons treated with WGA have significantly reduced total output in nerve fiber length per neuron as well as reductions in the average length of the individual nerve fibers extended. The inhibition of nerve fiber growth by WGA is concentration-dependent, specific, reversible and not mimicked by treatment with several metabolic poisons. In contrast, treatment with the lectin concanavalin A (25 micrograms/ml), which binds to mannose-containing glycoconjugates, increases the number of nerve fibers produced per neuron. These results suggest that lectins which bind to distinct carbohydrate moieties can differentially regulate nerve fiber growth. PMID- 3017512 TI - Mice and rats are sensitized to the proconvulsant action of a benzodiazepine receptor inverse agonist (FG 7142) following a single dose of lorazepam. AB - Rats and mice were treated with lorazepam (1.0 mg/kg) or its vehicle. Six h later seizure threshold to i.v. pentylenetetrazole was determined following treatment with the benzodiazepine-receptor inverse agonist FG 7142, the antagonist Ro 15 1788 or a second dose of lorazepam. Although lorazepam alone was anticonvulsant 6 h after its administration, animals pretreated with lorazepam were more sensitive to the proconvulsant action of FG 7142 and less sensitive to the effects of a second treatment with lorazepam. PMID- 3017511 TI - Ineffectiveness of organic calcium channel blockers in antagonizing long-term potentiation. AB - Evidence has accumulated suggesting that the presence of calcium is critical for development of hippocampal long-term potentiation (LTP). However, there is a paucity of information about whether calcium's role in LTP is pre- or postsynaptic. In the present study, we examined the effectiveness of nitrendipine, verapamil, flunarizine and the benzodiazepine diazepam in: blocking voltage-dependent calcium channels; blocking synaptic transmission; and preventing development of LTP. Using the in vitro slice preparation, we obtained intracellular and extracellular recordings from guinea pig hippocampal CA1 pyramidal cells. At the cellular level, all 4 drugs were ineffective in blocking voltage-dependent calcium spikes (TTX resistant) and the calcium-dependent afterhyperpolarization. Verapamil and diazepam appeared to antagonize synaptic transmission, as reflected in smaller population spike amplitudes. Development of long-term potentiation was not affected by the presence of verapamil, flunarizine and diazepam. Nitrendipine appeared to reduce the percentage of slices exhibiting LTP; however, ethanol, the vehicle used to dissolve nitrendipine, was shown in separate experiments to reduce the percentage of slices exhibiting LTP. These results suggest that neither the organic calcium channel blockers--nitrendipine, verapamil, and flunarizine--nor micromolar concentrations of diazepam are potent blockers of extrasynaptic voltage-sensitive calcium channels in hippocampus. They thus cannot be used to demonstrate a specific pre- or postsynaptic calcium role in LTP. PMID- 3017514 TI - Opioid antagonist-induced modulation of cerebral and hippocampal development: histological and morphometric studies. AB - The role of endogenous opioid systems in preweaning cerebral and hippocampal development was explored in rats utilizing naltrexone, a potent opioid antagonist. Sprague-Dawley rats were given daily injections (s.c.) of either 1 or 50 mg/kg naltrexone to invoke a temporary or complete blockade, respectively, of opioid receptors throughout the first 3 weeks of postnatal life; animals injected with sterile water served as controls. At weaning (Day 21), macroscopic, morphometric, and histological assessments were undertaken. In general, 50 mg/kg naltrexone had a stimulatory action on brain development, whereas 1 mg/kg naltrexone had an inhibitory influence. In most cases, both males and females were affected comparably. Opioid antagonist action was especially directed at cellular and tissue differentiation, with marked changes in macroscopic and areal dimensions and histotypic organization observed in the cerebrum. A prominent effect on the cerebrum of the 1 mg/kg naltrexone group was a substantial increase in packing density of the neural cells, reflecting a reduced area for accommodating neural elements. Changes in the hippocampus were largely restricted to the 1 mg/kg group. However, the number of granule cells was increased in the dentate gyrus of the 50 mg/kg group, suggesting that opioid receptor blockade affects cell types undergoing postnatal proliferation. Cellular elements derived prior to naltrexone treatment (e.g., pyramidal neurons) were capable of being influenced in only differentiative capacity. Our results show that endogenous opioids are natural trophic factors in brain development and provide evidence for the crucial role of endogenous opioid-opioid receptor interaction in neuro ontogeny. PMID- 3017515 TI - Benzodiazepine binding sites: localization and characterization in the limbic system of the rat brain. AB - The distribution of benzodiazepine binding sites was analysed in limbic structures of rat brain by quantitative radioautography of brain sections incubated with 3H-flunitrazepam (3H-FLU). Quantitative estimation of the binding parameters was made in each range of postero-anterior sections taken. Distribution of 3H-FLU binding sites was found to be rather homogeneous in most of the structures examined but there were regional differences which resulted from variations in the densities of sites rather than in their affinities. A particular distribution pattern of 3H-FLU binding sites was observed in the cingulate cortex contrasting with the homogeneous postero-anterior distribution measured in other cortical areas in the same slices. A significantly greater density of sites was found in the anterior part of the structure as compared to the posterior part. This difference, which corresponds to a change in the density of sites without alteration of their apparent affinity and occurs at a precise anatomical level, is discussed with reference to the anatomical organization of this brain structure and to its possible functional implications. PMID- 3017516 TI - The effects of purified Mojave toxin on rat synaptic membrane (Ca+2 + Mg+2) ATPase and the dihydropyridine receptor. AB - Effects of purified Mojave toxin on rat synaptic membrane (Ca+2 + Mg+2)-ATPase and dihydropyridine receptor were determined. The toxin was observed to stimulate specifically (Ca+2 + Mg+2)-ATPase approximately two-fold with no effect on Mg+2 dependent ATPase activity. Examination of the effects of increasing amounts of purified Mojave toxin on binding of the calcium channel blocker, nitrendipine, indicated that the addition of 10 micrograms (4.5 X 10(-10) moles) of toxin resulted in greater than 90% inhibition of nitrendipine binding. Furthermore, binding studies revealed the toxin to have little affinity for the ligand indicating its interaction with calcium channel components. Since Mojave toxin has associated with it a phospholipase A2 activity, we investigated the effects of 4-bromophenacylbromide, a known inhibitor of phospholipase A2 activity in order to discern the possible effects of the purified toxin on synaptic membranes. At concentrations previously shown to be inhibitory of purified phospholipase A2 from cobra venom, both ATPase activity and nitrendipine binding of synaptic membranes were significantly inhibited. Thus we cannot rule out the possibility that the endogenous phospholipase activity of the purified toxin is responsible for its effects on the rat brain synaptic functions studied here. Binding studies conducted in the presence of verapamil and diltiazem indicated that the toxin interacts with allosteric sites responsible for regulation of the binding of nitrendipine. Although we have not tested the effects of Mojave toxin on other ion channels and/or receptors, results presented here suggest the potential usefulness of this toxin as a molecular probe of the calcium channel complex. PMID- 3017517 TI - [Characterization of lysosomal-type hyaluronidase in the culture medium of 2 cell lines derived from human hepatomas]. AB - A sensitive assay for hyaluronidase was developed using as a substrate, hyaluronic acid insolubilized on polystyrene microtest plates. Hyaluronic acid was measured exploiting the fact that it can bind immune complexes made up with hyaluronectin and alkaline phosphatase-conjugated anti-hyaluronectin antibodies. Hyaluronidase was detected in both cell line culture media. Optimum pH was between 3.25 and 3.75. Sodium chloride dependence was absolute, and the optimum concentration of sodium chloride was between 0.2 and 0.3 M. The activity was not affected by dialysis, and was suppressed by a 5 minute heating at 50 degrees C or by protease treatment. The molecular weight was 68 K as determined by gel permeation chromatography. The results are close to those reported for human lysosomal hyaluronidase. PMID- 3017519 TI - [Minirotavirus detected in the feces of infants with diarrhea in Nanjing]. PMID- 3017520 TI - [Advances in research on cardionatrin]. PMID- 3017518 TI - [Effects, in rats, of metapramine and carpipramine on the uptake of catecholamines and serotonin; relationship with 3H-imipramine binding]. AB - Carpipramine and metapramine inhibited 3H-imipramine binding both added in vitro and administered to the animal. They were inhibitors of NA uptake and they did not modified 5HT uptake. Only carpipramine affected DA release and uptake. PMID- 3017521 TI - NADP+ production using thermostable NAD+ kinase of corynebacterium flaccumfaciens AHU-1622. AB - A sonicate of Corynebacterium flaccumfaciens AHU-1622 had the highest NAD+ kinase activity (1.22 mU/mL culture broth) of the strains of bacteria we investigated. This enzyme was thermostable, with activity maintained at 50 degrees C for 1 h. This treatment inactivated phosphatase activity. Resting cells of the bacterium also had NAD+ kinase activity when treated at 60 degrees C for 30 min with 0.2% Triton X-100. NADP+ production was achieved using 8 mumol NAD+, 8 mumol ATP, 16 mumol MgCl2, 1.6 mumol NaN3, and 12 mU NAD+ kinase (0.1 g of permeabilized wet cells) in 2 mL of 0.1 M phosphate buffer, pH 7.5. The conversion ratio of NADP+ from NAD+ was 75% after 10 h of incubation at 50 degrees C, and the amount of accumulated NADP+ was 3 mumol/mL of reaction mixture. The NAD+ kinase activity of the permeabilized cells was stable and did not decrease after repeated use. PMID- 3017522 TI - Restriction endonuclease activities in the legionellae. AB - Fifteen legionellae isolates were studied for the presence of restriction endonucleases. At least three different specificities were found in nine of these isolates. One particular activity was present in two groups of isolates isolated from widely separated geographic areas. PMID- 3017523 TI - Immunoperoxidase plaque staining for the detection of pseudorabies virus. AB - A quantitative indirect immunoperoxidase plaque staining method was developed for the detection of pseudorabies virus infection in a pig kidney cell line (PK-15). The method is rapid and specific and foci of infection, represented by stained plaques, are easily counted by the unaided eye. Possible modification of this technique in a plaque reduction assay for the detection of antipseudorabies virus antibody is also discussed. PMID- 3017524 TI - Genomic variations and antigenic relationships among cytopathic rotavirus strains isolated in Quebec dairy herds. AB - Twelve isolates of bovine rotavirus, originating from eight dairy herds in Quebec known to have frequent epizootics of diarrhea in young calves in the last five years, were successfully propagated in cell cultures. The 12 isolates produced clear-cut plaques in BSC-1 cells and, except for one isolate, agglutinated human group "O" erythrocytes to an higher titer than bovine erythrocytes. Antisera to each isolate were produced in rabbits and used to study their antigenic relationships. All the isolates shared the group-specific immunofluorescent antigen and were antigenically related as demonstrated by the seroneutralization and hemagglutination-inhibition tests. However, the relationships to the Nebraska rotavirus was quite weak in cases of two Quebec isolates. When the genomes of the various isolates were compared by polyacrylamide gel electrophoresis, at least three different reproducible fractionation patterns could be identified. PMID- 3017525 TI - Distinct serotypes of porcine rotavirus associated with diarrhea in suckling piglets in southern Quebec. AB - Cytopathic rotavirus strains were isolated in cell cultures from the intestinal contents of diarrheic piglets on Quebec pig farms where repeated outbreaks of enteritis occurred. All the isolates shared the common group antigens of rotaviruses as revealed by immunofluorescence and counterimmunoelectrophoresis. A hemagglutinating activity was demonstrated with human group O, porcine and guinea pig erythrocytes. At least one of the isolates was clearly distinguished from the American prototype of porcine rotavirus (strain OSU) by neutralization and hemagglutination inhibition tests; a third serotype was also suspected. By polyacrylamide gel electrophoresis of RNA, it was not possible to differentiate these isolates. PMID- 3017527 TI - Arbovirus infections in several Ontario mammals, 1975-1980. AB - Serological studies for arboviruses were conducted on 725 animal sera collected in 22 Ontario townships between 1975 and 1980 including 44 coyote (Canis latrans), 277 red fox (Vulpes vulpes), 192 raccoon (Procyon lotor) and 212 striped skunk (Mephitis mephitis). Hemagglutination inhibition antibodies to two flaviviruses, namely St. Louis encephalitis and Powassan were found in 50% of coyote, 47% of skunk, 26% of fox and 10% of raccoon sera. Similarly, hemagglutination inhibition antibodies to a California serogroup virus, snowshoe hare, were found in 12% of fox, 7% of skunk, 7% of raccoon and 5% of coyote sera. No antibodies were detected to two alphavirus, namely eastern equine encephalitis and western equine encephalitis, antigens. This study affirms the endemic presence of Powassan and snowshoe hare virus and further delineates the scope of St. Louis encephalitis activity in Ontario. PMID- 3017526 TI - Induction of immunity against pneumonic pasteurellosis following experimental infection in calves. AB - Immunity against pneumonic pasteurellosis was studied in calves after recovery from experimental respiratory disease with Pasteurella haemolytica. Nine calves were exposed to aerosols of parainfluenza-3 virus and Pasteurella haemolytica A1 six days apart to produce respiratory disease. After recovery from the disease, these nine principal and four control calves were challenged with aerosols of bovine herpesvirus 1 and P. haemolytica A1 four days apart. With this viral bacterial challenge, the nine principal animals failed to develop clinical responses to this bacterial challenge and their lungs did not show the growth of P. haemolytica on cultures, whereas two of four control calves had elevated temperatures and developed necropurulent pneumonia with the isolation of P. haemolytica from the lungs. The principal calves had developed high levels of cytotoxin neutralizing antibodies in their sera following parainfluenza-3 virus P. haemolytica infection. This demonstrated that immunity against pneumonic pasteurellosis can be achieved, with a suggestion that further search for an effective vaccine for P. haemolytica is warranted. PMID- 3017528 TI - An epidemiological study of selected calf pathogens on Holstein dairy farms in southwestern Ontario. AB - Fecal samples from calves on 78 randomly selected Holstein dairy farms in southwestern Ontario were screened for Salmonella, Campylobacter jejuni/coli, enteropathogenic Escherichia coli, rotavirus and coronavirus. Based on the observed prevalence, 22% of farms had calves infected with Salmonella, 13% with Campylobacter jejuni/coli, 41% with enteropathogenic E. coli, 19% with rotavirus and 5% with coronavirus. These estimates can be modified, using a method developed by Mullen and Prost (1983) for the World Health Organization, to account for the nature of the laboratory test used. If the test is assumed to have no false positives (that is, if an organism is detected it must be there), then the observed prevalence estimates seen on this study may greatly underestimate the true prevalence of infected premises. The use of nipple feeders for calves was associated with an increased probability of farms having calves shedding detectable fecal levels of Salmonella, E. coli, or one of the two viruses. The use of group pens was associated with an increased odds of finding C. jejuni. Calves with diarrhea on these farms tended to have increased odds of shedding rotavirus, and E. coli with the K99 antigen. However, at the farm level, none of the organisms was associated with above median levels of morbidity. Farms positive for one or other of the viruses had increased odds of experiencing calf mortality relative to virus-negative farms, and farms positive for C. jejuni/coli had decreased odds of mortality.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017529 TI - The association between serological titers in infectious bovine rhinotracheitis virus, bovine virus diarrhea virus, parainfluenza-3 virus, respiratory syncytial virus and treatment for respiratory disease in Ontario feedlot calves. AB - A seroepidemiological study of the association between antibody titers to infectious bovine rhinotracheitis, parainfluenza-3, bovine virus diarrhea and bovine respiratory syncytial viruses, and treatment for bovine respiratory disease was conducted. A total of 322 calves from five different groups were bled on arrival, then one month later all cases (cattle treated for bovine respiratory disease) were rebled together with an equal number of controls (cattle not treated for any disease). Titers to these viruses varied significantly from group to group. Based on seroconversion, infectious bovine rhinotracheitis virus was active in 4.4%, bovine virus diarrhea virus in 24%, parainfluenza-3 virus in 69.5% and bovine respiratory syncytial virus in 71.3% of the cattle. Cattle with low titers to infectious bovine rhinotracheitis and/or bovine respiratory syncytial viruses on arrival, were at increased risk of subsequent treatment for bovine respiratory disease. Treated cattle also had significantly greater increases to parainfluenza-3 and/or bovine virus diarrhea viruses than control calves. Treatment rates varied considerably from group to group and were not strongly correlated with weight gain in the postarrival period. PMID- 3017530 TI - Antibody-mediated resistance against duck enteritis virus infection. AB - Vaccination of ducks with an apathogenic strain of duck enteritis virus resulted in protection against challenge with the virulent Lake Andes strain of duck enteritis virus by intramuscular inoculation or contact exposure. Antisera produced in the vaccinated ducks were able to transfer resistance against the challenge strain to recipient ducks. Antisera against duck enteritis virus were cytotoxic for duck enteritis virus-infected duck embryo fibroblasts in the presence of guinea pig complement. These in vivo and in vitro data suggest that the humoral immune mechanism plays a role in protecting ducks from duck enteritis virus infection. PMID- 3017531 TI - Development and evaluation of an indirect enzyme immunoassay for detection of porcine antibodies to pseudorabies virus. AB - An indirect enzyme immunoassay is described for detection of porcine serum antibody to pseudorabies virus. The analytical sensitivity of the enzyme immunoassay was found to be approximately 4.5 log 4 X 10 (5120 times) greater than the serum neutralization test, based on parallel end point titrations. The diagnostic sensitivity of the enzyme immunoassay was comparable or superior to that of the serum neutralization, based on the earliest detectable antibody after infection of swine with pseudorabies virus by intranasal or intrauterine routes or by contact with infected pigs. The enzyme immunoassay, at a screening dilution of 1:20, gave 100% agreement with ELISA results provided with a U.S. Department of Agriculture-Animal and Plant Health Inspection Service proficiency panel of 40 sera. One serum having demonstrable antibody by the enzyme immunoassay was seronegative by the serum neutralization test. PMID- 3017533 TI - Long-term survival in limited-stage small cell lung carcinoma. Experience in Rochester, New York from 1975 to 1981. AB - All patients with limited-stage small cell lung carcinoma (SCLC) diagnosed between January 1975 and 1981 in Rochester, New York, were collected. One hundred one patients were evaluable. By reviewing an entire community's experience with long follow-up, we were able to describe the response rates and survival in a large unselected population and compare them to results from concurrent cooperative group studies. Median survival for the entire group was 51 weeks, with only 18% alive at 130 weeks. There was no evidence for improvement in response or survival during the 6 years of study. Treatment results in the community as a whole were no different from that seen with cooperative group studies. A group who had initial surgery followed by adjuvant therapy had a significantly better survival and more long-term survivors than those not receiving surgery, but rare long-term survivors were seen with all treatment categories. Except for this small surgical subgroup, no other characteristics could be identified which were predictably associated with long-term disease-free survival. The overall poor survival of patient with localized SCLC suggests a need for the development of novel initial approaches to therapy. PMID- 3017532 TI - A prospective study of male sexual contacts of men with AIDS-related conditions (ARC) or AIDS: HTLV-III antibody, clinical, and immune function status at induction. PMID- 3017534 TI - Immunohistologic analysis of the phenomenon of spontaneous regression of numerous flat warts. AB - Among various tumors induced by human papilloma virus (HPV), flat warts are unique in that they show a systemic regression phenomenon after sudden occurrence of inflammation in all the tumors, leaving permanent immunity to flat warts in the host. When studied immunohistochemically, the presence of HPV antigen using papilloma virus genus-specific antiserum in 31 cases of regressing flat warts was not found; whereas it was demonstrated in the nuclei of upper epidermal cells of ordinary flat warts in 12 of 19 cases (63%). T-cell phenotype assessment in nine regressing flat warts using monoclonal antibodies showed that helper/inducer subsets constituted a major peritumoral dermal infiltrate with a moderate number of intermingling OKT6+ cells. In contrast, the tumoral epidermis was invaded by almost equal number of suppressor/cytotoxic T-cells and helper/inducer T-cells, where at least some keratinocytes also expressed HLA-DR antigen in addition to Langerhans cells. Most T-cells expressed HLA-DR antigen, a marker of activation, but only a small number of them were Tac antigen+, i.e., bearing interleukin 2 receptors. Leu 7+ natural killer cells were seldom found in the infiltrate. These data provide evidence that T-cell-mediated immune attack against tumor cells and not against intranuclear HPV antigen, induces the systemic spontaneous regression of numerous flat warts in humans. PMID- 3017535 TI - Determination of transferrin receptors on frozen sections of malignant B-cell lymphomas by immunofluorescence with a monoclonal antibody. AB - A series of 34 B-cell lymphomas was studied for the presence of transferrin receptors by immunofluorescence on frozen tissue sections with a monoclonal antibody. These lymphomas were classified by light microscopic study and furthermore investigated by immunohistochemical and morphometric methods. There were diffuse centrocytic, diffuse centroblastic, and follicular and diffuse centrocytic/centroblastic lymphomas. Significant differences in log mean nuclear area in these four histologic groups were found, as well as a significant correlation between log nuclear area and transferrin receptors as measured semiquantitatively by fluorescence intensity of the cell membranes. In addition, it was found that there is a strong correlation between the transferrin receptor activity and malignancy grading on histologic and morphometric basis. Therefore, the transferrin receptor determination on frozen sections appears to be a good method of malignancy grading. PMID- 3017536 TI - A variant of early gastric carcinoma. Histologic and histochemical studies of early signet ring cell carcinomas discovered beneath preserved surface epithelium. AB - Gastric carcinomas hidden beneath flat and intact mucosal surface epithelium are rarely discovered. Such a tumor in the early stage is at best diagnosed as an incidental finding, so that the diagnosis is probably always a surprise to the clinician. Six such cases of early gastric carcinoma were presented. Four were intramucosal lesions and the remaining two were invasive with submucosal extension. All the tumors are composed purely of signet-ring cells (diffuse-type by Lauren's classification). Histologic examination of the six cases revealed that certain features, which are not characteristically observed in ordinary signet-ring cell carcinomas, were commonly recognized. These included compact nests of uniform signet-ring cells sharing a common cytoplasmic wall, lack of desmoplastic stromal response, and intracytoplasmic mucin predominantly composed of neutral mucopolysaccharide. These six tumors are considered to be an incipient stage of signet-ring cell carcinoma. The findings also suggest a close histogenetic relationship between these tumors and the mucous neck cells in the basal region of gastric glands. The grossly unremarkable mucosal surface and histologically innocuous appearance associated with this form of tumor are emphasized for diagnosis. PMID- 3017537 TI - Long-term survival after brain metastasis from lung cancer. AB - A case is reported of prolonged survival after lobectomy for large cell undifferentiated carcinoma of the lung and resection of metastatic carcinoma of the brain. The patient had survived 11 years 5 months after lung resection and 10 years 4 months after excision of brain metastasis. A review of the reports of another 12 patients who survived 5 years or longer after craniotomy, shows that the surgical excision of a single metastatic lesion of the brain with or without postoperative irradiation offers the best hope for prolonged survival. PMID- 3017538 TI - Application of an in vitro antimetabolic assay to human germ cell testicular tumors for the preclinical evaluation of drug sensitivity. AB - An in vitro assay, which evaluates the effect of drugs on labeled nucleotide precursor incorporation 3H-thymidine and 3H-uridine after 3 hours of in vitro treatment, was applied to human germ cell testicular tumors. The assay was feasible on 78% of the 259 tumors, and the results were evaluable in 95% of these, which shows the good potential of its clinical application. In vitro response rates to conventional agents were comparable to clinical response rates reported in the literature for monochemotherapy regimens, thus demonstrating the accuracy of the assay in reproducing the sensitivity of the tumor type. The specificity of the assay in predicting drug sensitivity of individual tumors was investigated on 28 lesions from 24 patients who had residual disease after surgery. A significant correlation was found between in vitro and clinical sensitivity to the same drugs (P = 0.026), with an overall agreement of 92% when tumor metastases were tested in vitro. In contrast, no significant correlation, and a poor agreement (62%) was found when the primary tumor was tested. PMID- 3017539 TI - Modification of the hyperthermic response on neuroblastoma cells by cAMP and sodium butyrate. AB - The role of adenosine 3',5'-cyclic monophosphate (cAMP) and sodium butyrate in modifying the effect of heat on murine neuroblastoma cells (NBP2) in culture was evaluated on the criterion of survival. An elevation of cellular cAMP level by prostaglandin (PG) A2, a stimulator of adenylate cyclase, and 4-(3-butoxy-4 methoxybenzyl)-2-imidazolidinone (R020-1724), an inhibitor of cyclic nucleotide phosphodiesterase, only during heat treatment (43 degrees C and 40 degrees C) was sufficient to enhance the effect of heat. The extent of enhancement (additive versus synergistic) depended upon the cAMP stimulating agent and the experimental condition. When these agents were added after heat treatment for the entire observation period, they produced similar results. PGA2 and R020-1724 are known to increase the intracellular level of cAMP in these cells by three and fivefold, respectively; therefore, the effect of these agents in enhancing the heat response may be mediated by cAMP-dependent mechanisms. The presence of sodium butyrate during heat treatment alone was ineffective; however, when it was added before or after heat treatment for the entire observation period, the survival of heated cell was markedly reduced. PMID- 3017540 TI - Clinical and radiologic features of hepatocellular carcinoma originating in the caudate lobe. AB - Five patients with hepatocellular carcinoma in the caudate lobe were evaluated. Computed tomography (CT) scan and/or angiography clearly demonstrated multiple intrahepatic metastases in four (80%), and tumor thrombi in the portal vein in two (40%), and in the inferior vena cava in one. Even though there was no recognizable lung metastasis, metastases were found in the orbita in one patient, and in the ribs and thoracic vertebrae in two patients. Four patients died after a mean period of 5.5 months from the initial diagnosis. The mechanism for early invasion into the vessels and multiple intrahepatic metastases of hepatocellular carcinoma arising from the caudate lobe is discussed. PMID- 3017541 TI - Spontaneous remission of Kaposi's sarcoma in an HTLV-III-negative homosexual man. AB - Kaposi's sarcoma (KS) in homosexual men has been linked to the acquired immune deficiency syndrome (AIDS). We describe a 51-year-old homosexual man who developed extremity KS while taking corticosteroids. The KS resolved when the steroids were withdrawn. He did not have classically defined AIDS: no evidence of HTLV-III infection was found after serial ELISA and "western blot" analysis of the patient's serum nor after co-cultivation of his peripheral blood lymphocytes. This clinical observation is consistent with the hypothesis that AIDS and KS may have different etiologic agents. Corticosteroids should be used with caution in patients at risk for KS (including homosexual men) and may be complicated by the development of KS without HTLV-III-induced immunosuppression. PMID- 3017542 TI - Identification of a deoxyribonuclease activity in the nuclei of the cervical carcinoma HeLa. AB - A deoxyribonuclease activity has been identified in the nuclei of the human cervical carcinoma HeLa. This activity has the novel property of existing as a single strand specific endo- and exodeoxyribonuclease activity. These activities are ionic strength sensitive, with retardation of activity occurring at 50 mM NaCl and above, with the Mn++ ion preferred over the Mg++ ion as activating cation. This activity extensively damages DNA via its single strand nicking and gaping activity. The method used to solubilize this activity as well as the enzyme's characteristics are discussed. PMID- 3017543 TI - High performance liquid chromatography analysis of 1,25-dihydroxyvitamin D3 receptor in malignant cells. Correlation of effects on cell proliferation and receptor concentration. AB - Malignant cells were assayed for 1,25(OH)2D3 receptors and for the effects of 1,25(OH)2D3 on cell proliferation. The established lines studied were human promyelocytic leukemia (HL-60), T-cell lymphocytic leukemias (Molt-4, RPMI-8402, CEM), mouse leukemia (L1210), breast cancers (HT-39 and MCF-7) and a glioma (C-6) cultures. A TSK 3000 SW (0.75 X 60 cm) HPLC size exclusion column was used to characterize specific 1,25(OH)2D3 binding. We show for the first time that this column is capable of resolving the 3.2-3.5S 1,25(OH)2D3 mammalian receptor (Rs = 32 A) from the 5.5-6.0S form of the mammalian serum 25(OH)D3 transport receptor (Rs = 40 A). The molecular size of the 1,25(OH)2D3 receptors from these cancer cell lines was identical to that from rabbit intestine. HT-39, HL-60, MCF-7, Molt 4, C-6, RPMI-8402 and L1210 cells demonstrated specific 1,25(OH)2[3H]D3 binding (120, 90, 80, 45, 30 and 18 fmoles of sites/mg protein, respectively). Receptors were not detected in the CEM line. 1,25(OH)2D3 inhibited cell proliferation of HT 39, HL-60, MCF-7 and Molt-4 cells by 20% to 70%. In contrast, mouse leukemia (L1210) cells were stimulated to proliferate by this hormone. Proliferation of RPMI and CEM cells was not affected by 1,25(OH)2D3. We demonstrate that size exclusion HPLC of 1,25(OH)2D3 binding proteins from mammalian intestine and cancer cells provided a rapid method for identification of specific 1,25(OH)2D3 receptors. Furthermore, in the cells studied, the presence and concentration of 1,25(OH)2D3 receptors qualitatively predicted the potency of this hormone to alter cell proliferation. We believe this assay will be useful for rapid analysis of human tumor receptor concentrations. PMID- 3017544 TI - Translocation X;18 in synovial sarcoma. PMID- 3017545 TI - Distinct regions of the human cytomegalovirus genome are responsible for the immortalization and tumorigenicity of animal cells. AB - Dog embryo kidney cells were efficiently transformed by human cytomegalovirus (HCMV) particles or intact viral DNA. Negative results were obtained after transfection of the canine cells with recombinant plasmids carrying the HCMV Hind III-E subgenomic fragment or with limit Bgl II and Hind III digests of the viral genome. Immortalized dog cells with typical transformation properties appeared, however, after transfection with EcoR I fragments of the HCMV DNA. Distinct regions of the viral genome are probably responsible for the immortalization and the tumorigenicity of mammalian cells. PMID- 3017546 TI - Inhibition of mitochondrial protein synthesis leads to proliferation arrest in the G1-phase of the cell cycle. AB - Mitochondrial protein synthesis is specifically inhibited by low concentrations of tetracyclines. Prolonged inhibition of mitochondrial protein synthesis leads to a lack of oxidative ATP generating capacity, which results in proliferation arrest of normal and malignant cells of epithelial origin, as has been shown previously. The present study indicates that this holds true also for fibroblasts and sarcoma cells. It is shown that this proliferation arrest leads to accumulation of the growth-arrested cells in the G1-phase of the cell cycle. This offers several interesting possibilities to use tetracyclines in anticancer combination therapies. PMID- 3017548 TI - Hypnotic specificity of benzodiazepines. PMID- 3017547 TI - Superoxide radical potentiates invasive capacity of rat ascites hepatoma cells in vitro. AB - Effect of superoxide radical (O2-) produced extracellularly by hypoxanthine (HX) and xanthine oxidase (XO) on invasive capacity of rat ascites hepatoma cells was studied. Invasive capacity was estimated in vitro by counting the number of tumor cell colonies penetrated underneath cultured mesothelial cell monolayer. When the tumor cells had been treated with non-toxic doses of HX and XO, the formation of penetrated colonies increased with increasing concentrations of XO. This increment was completely inhibited by scavengers of active oxygen radicals, superoxide dismutase (SOD) in combination with catalase (CAT) added simultaneously at the time of HX-XO treatment. PMID- 3017549 TI - Brain mechanisms of synchronous sleep. PMID- 3017550 TI - Novel receptor specificity of selected benzodiazepines. PMID- 3017551 TI - Randomized comparisons of radiotherapy and carmustine versus procarbazine versus dacarbazine for the treatment of malignant gliomas following surgery: a Southwest Oncology Group Study. AB - Between 1977 and 1981, the Southwest Oncology Group entered 278 patients on a randomized study (SWOG 7703) to compare the effect of three different chemotherapeutic agents given in combination with radiotherapy (6000 rads over 7 weeks) following surgery for malignant gliomas. The chemotherapy regimens were: carmustine (BCNU)--80 mg/m2 iv daily X 3 every 6 weeks; procarbazine (PCB)--100 mg/m2 orally; or dacarbazine (DTIC)--175 mg/m2 iv daily X 5 every 4 weeks. Patients were stratified according to age, and degree of resection, with no differences identified between groups. The response rates (complete plus partial) for BCNU and DTIC were significantly better than for PCB [BCNU, 39%; PCB, 13%; and DTIC, 38% (P less than 0.01)]. The response duration and survival were somewhat better in patients treated with BCNU and DTIC, but compared to patients treated with PCB, the difference was not statistically significant. Median survival times were: BCNU, 45 weeks; PCB, 31 weeks; and DTIC, 49 weeks (P greater than 0.3). There were six toxic deaths with BCNU and four with PCB, most of which were due to infection associated with leukopenia. The high toxicity and minimal benefit of chemotherapy added to radiotherapy compared to historical results with radiotherapy alone suggest that combined treatment may not be indicated for some patients. PMID- 3017552 TI - Cancer in Bangladesh: a model for some problems and proposed solutions in the Third World. AB - Bangladesh typifies Third World cancer problems. Soluble problems include 1) assessment: establishment of national and hospital-based cancer registries for factual assessment of incidence and prevalence rates, which are different from those in the West and commonly include cancers of the head and neck or oropharynx, lymphoma, bronchus, esophagus, uterine corpus and cervix, penis, and hepatocellular carcinoma. These facts have consequences for 2) primary prevention. Three common cancers may be preventable--bronchus (smoking), head and neck (alcohol, tobacco and betel nut chewing, smoking), and hepatocellular carcinoma (mold contamination of unrefrigerated stored grains, hepatitis B vaccination). Four common cancers are often amenable to definitive treatment if only they are subjected to 3) early detection. These are cancers of the oropharynx (leukoplakia), cervix uteri (Pap smear), breast (self-exam) and hepatocellular carcinoma (serum alpha-fetoprotein levels). In addition, an analgesic plan at all levels of health care delivery might be instituted. These are practical, low-technology, and cheap and might result in the relief of much misery. PMID- 3017553 TI - Primary hepatocellular carcinoma: clinical, ultrasonic, and pathological patterns and correlations. AB - Twenty-seven patients with tissue diagnosis of hepatocellular carcinoma were reviewed retrospectively in an attempt to do a correlative study between clinical echographic patterns and the pathologic grading. The mean of the anterior hepatic linear projection (AHLP) was 33 cm; the surface was smooth in 26% and irregular in 30% and a single mass was detected in 44% of cases. Ultrasonically, three patterns were detected: 1) solitary mass in 66.6% of cases: hyperechogenicity in 22.2%, hypoechogenicity in 27.8%, and compound echogenicity in 50% of cases; 2) multiple masses in 25.9% of cases: hyperechogenicity in 71.4% and compound echogenicity in 28.6%; and 3) diffuse distortion of hepatic architecture with areas of heterogenous echopattern in 7.4% of cases. Pathological grading was carried out according to the generally accepted criteria, grades I in 29.6%, II in 33.3%, and III in 37.1% of cases. No correlation could be detected between the AHLP, patient age, sex, duration of complaint, or incidence of distant metastases and the pathologic grading. Also, there was no correlation between the duration of complaint and the AHLP. However, a significant relation was detected between the character of the hepatic surface and the pathologic grading; a smooth liver was associated with lower pathologic grading compared to either the surface with a single mass or to the irregular surface. On the other hand, no significant relation could be detected between lobar affection, ultrasonic pattern, or echogenicity and the pathologic grading. PMID- 3017554 TI - Herpes-related polypeptides from a human cervical carcinoma cell line. AB - Using antiserum against herpes simplex virus type 2 (HSV-2) infected cells, eight polypeptides with similar molecular weights could be immunoprecipitated from the nearly diploid, human cervical carcinoma cell line C4II and from HSV-2 transformed hamster and mouse cells. Only two of these polypeptides corresponded to those from HSV-2 infected cells, including the putative HSV-2 transformation related 35K protein. Partial proteolytic cleavage products of the immunoprecipitated 35K polypeptides from C4II and HSV transformed hamster and infected cells were indistinguishable; however, viral DNA and mRNA corresponding to the 35K polypeptide could not be detected in C4II or in transformed hamster or mouse cells by dot blot hybridization. A similar mechanism of transformation for the human cervical carcinoma cell line and HSV-2 transformed cells is proposed. PMID- 3017556 TI - Report of two cases of male breast cancer after prolonged estrogen treatment for prostatic carcinoma. AB - Reviewing our experience with 19 cases of male breast cancer, we found two patients with a 12-year history of estrogen therapy for prostatic carcinoma. One initially received diethylstilbene followed by estradiol, 80 mg monthly, and finally estramustin, 140 mg twice daily. The other patient had dienestrol, 2.5 mg daily, and later estradiol, 40 mg every 6 weeks. The possible causal relationship between prolonged estrogen administration and breast cancer in males is in contrast to available epidemiologic data. Therefore, the possibility of an association by chance cannot be ruled out. PMID- 3017555 TI - Human papillomavirus (HPV) infection of cervical lesions detected by immunohistochemistry and in situ hybridization. AB - Ten cases of cervical dysplasia, 13 cases of carcinoma in situ, nine cases of invasive carcinoma, and six cases of normal tissue were investigated for the presence of papillomavirus group-specific antigens and for viral DNA. Viral proteins were identified with genus-specific papillomavirus antibodies. Cloned HPV 6, 11, and 16 DNA labeled with 3H were used for in situ hybridization followed by autoradiographic detection of hybridized probes. In cervical dysplasias, five of ten samples were positive for HPV antigens, two of four cases contained HPV DNA related to type 16, and nine of thirteen cases of carcinoma in situ showed the presence of HPV antigens. In five of seven lesions of carcinoma in situ, HPV DNA was detected (HPV 6 three cases, HPV 16 two cases). For invasive carcinomas, HPV antigens were not observed in four cases. In the other five cases investigated, HPV antibodies were reactive with few keratinocytes located either at the surface or in keratinized tumour zones. HPV DNA related to type 6 was seen in three of six cases. Six cases of normal cervical mucosa served as controls. In these cases, HPV antigens (five samples) or HPV DNA (one specimen) were not detected. The study underlines the value of recombinant DNA techniques for screening of HPV infections in cervical cancer and precancer; 50-70% of cervical lesions contained HPV-related DNA. These data were comparable to the percentage of cervical dysplasia and carcinoma in situ positive for HPV antigens.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017558 TI - Plasma "cold-insoluble globulin" protects cytotoxic lymphocytes from ATP inhibition: 2. Immunization against viral cell surface antigen stimulates cytotoxic cells to lyse tumor cells. AB - The present studies examined the immunoregulatory factors that modulate T lymphocyte cytotoxic activity against tumor cells. At 30 days postvaccination with hepatitis B virus surface antigen(s) (HBsAg), peripheral blood lymphocytes (PBL) from HBsAb-seropositive healthy donors showed 8-10-fold increase in their cytotoxic activity against human hepatocellular carcinoma PLC/PRF/5 cells. However, there was no effect against human embryonic liver or lung fibroblasts (WI-35) cells. After cloning, these cytotoxic lymphocytes responded to HBsAg, phytohemagglutinin (PHA), and neuraminidase (VCN) with significant increases in cytotoxicity. These cloned, activated PBL also had no effect on human embryonic liver or lung fibroblasts. Exogenous 5'-triphosphate (ATP) inhibited the lymphocyte cytotoxic activities. The inhibition was ATP concentration-dependent. Also, colchicine, a tubuline depolymerizing agent, inhibited effector cell cytotoxic lymphocyte activity. Colchicine binds to the target cells without causing cell lysis. Preincubation of the cytotoxic lymphocytes with "cold insoluble globulin" (CIg) protected the cells from ATP and colchicine inhibition. PMID- 3017557 TI - Immunological reactivity of mucinous and serous ovarian adenocarcinomas. AB - Comparison of immunological reactivity of glycoprotein antigens extracted from individual cases of mucinous and serous ovarian adenocarcinomas was performed taking into account the immunological relationship with carcinoembryonic antigen (CEA), nonspecific cross-reacting antigen (NCA), alpha-1-antichymotrypsin, and alpha-1-acid glycoprotein. In all immunological tests, the specific immune sera against perchloric acid extracts of ovarian mucinous and serous cystadenocarcinomas and antisera against the reference antigens mentioned above were used. It was established that: 1) ovarian mucinous and serous adenocarcinomas are immunologically different and possess various tumor associated antigens, 2) ovarian mucinous adenocarcinomas contain considerable amounts of CEA and NCA, whereas serous type neoplasms show negligible amounts or lack of these antigens; and 3) in both types of tumors, alpha-1-antichymotrypsin and alpha-1-acid glycoprotein activities are found. Immunological data indicate that ovarian mucinous and serous adenocarcinomas derive from separate lineages of epithelium. PMID- 3017560 TI - Mast cells and synaptic transmission in sympathetic ganglia. PMID- 3017559 TI - Virus-inhibiting factor in primary and metastatic carcinoma of the liver. AB - Sera from patients with hepatoma, metastatic liver disease, cirrhosis of the liver, and benign diseases of the digestive tract have been tested for a virus inhibiting factor. Serum samples exerting complete inhibition of virus CPE in a dilution of at least 1:10 per 0.2 ml are considered positive, and those exerting 50% inhibition of CPE are regarded as weakly positive. An apparently positive antiviral activity was noted in the sera of patients with hepatoma (53.3%) and metastatic liver disease (46.6%). Patients with cirrhosis of the liver showed positivity in one of ten sera; no positive antiviral activity could be found in the sera of patients with benign diseases of the digestive tract. The percentage of weakly positive response was 33.3% and 13.3% for hepatoma and metastatic liver disease, respectively, and 10.0% for the benign diseases and 20.0% for the cirrhotic sera. The antiviral activity was more prominent in the embryonic foreskin fibroblasts and to a lesser degree in the bovine epithelial cells NBL-1. PMID- 3017562 TI - 17 beta-hydroxysteroid oxidoreductase activity in rat prostate after autosensitization to male accessory sexual glands. PMID- 3017561 TI - Immunoelectron microscopic and immunoblotting analyses of a retrovirus produced in a human lymphoblastoid cell line with a monoclonal antibody. PMID- 3017564 TI - [Antibodies against cytomegalovirus in various age groups in Wuhan District]. PMID- 3017563 TI - Intracellular pathways of endocytosed transferrin and non-specific tracers in epithelial cells lining the rete testis of the rat. AB - The uptake and pathway of different markers and ligands for fluid-phase, adsorptive and receptor mediated endocytosis were analyzed in the epithelial cells lining the rete testis after their infusion into the lumen of these anastomotic channels. At 2 min after injection, diferric transferrin bound to colloidal gold was seen attached to the apical plasma membrane and to the membrane of endocytic coated and uncoated pits and vesicles. The injection of transferrin-gold in the presence of a 100-fold excess of unconjugated diferric transferrin revealed no binding or internalization of transferrin-gold. Similarly, apotransferrin-gold was neither bound to the apical plasma membrane nor internalized by these cells. These results thus indicate the presence of specific binding sites for diferric transferrin. At 5 min, internalized diferric transferrin-gold reached endosomes. At 15 and 30 min, the endosomes were still labeled but at these time intervals the transferrin-gold also appeared in tubular elements connected to or associated with these bodies or seen in close proximity to the apical plasma membrane. At 60 and 90 min, most of the transferrin-gold was no longer present in these organelles and was seen only exceptionally in secondary lysosomes. These results thus suggest that the tubular elements may be involved in the recycling of transferrin back to the lumen of the rete testis. The coinjection of transferrin-gold and the fluid-phase marker native ferritin revealed that both proteins were often internalized in the same endocytic pit and vesicle and shared the same endosome.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017565 TI - [Maternal-neonatal paired studies on antibodies against human rotavirus (HRV)]. PMID- 3017566 TI - Negative control of DNA replication in composite SV40-bovine papilloma virus plasmids. AB - To identify DNA sequences that function in the control of DNA replication, we designed a hybrid replicon consisting of linked SV40 and BPV DNA sequences. In the composite SV40-BPV plasmid negative control encoded by BPV is dominant over the uncontrolled replication encoded by the positive factor, SV40 T antigen. Using a transient replication assay, we show that replication control requires three BPV elements. Two cis-acting sequences are closely linked to BPV replication origins. A third trans-acting element is encoded within the 5' part of the BPV E1 open reading frame (ORF) and is separable from the positive replication factor encoded within the 3' part of the same ORF. The controlled replication of SV-BPV composite replicons has enabled us to create permanent COS cell lines that stably maintain these plasmids as episomes. PMID- 3017567 TI - Repression of bovine papilloma virus replication is mediated by a virally encoded trans-acting factor. AB - Cells transformed with bovine papilloma virus type 1 mutants in the E6 or E6/7 genes are resistant to high-copy-number amplification of wild-type DNA after supertransfection. Transient and stable replication assays demonstrate this effect. If the supertransfected DNA has a mutation in a newly defined gene (M), this cellular immunity to high-copy-number replication is overcome, resulting in transient replication of the input DNA. In contrast, the resident plasmid does not participate in amplification and is maintained at a constant low copy number. Cotransformation of M- mutants and wild-type DNA into these cells leads to shutoff of replication of both genomes. Thus, M- mutants define a trans-acting negative modulator that regulates viral replication. This function is distinct from the positive factors required for replication. We propose a model that explains why the loss of E6 and E6/7 function leads to immunity of the infected cell. PMID- 3017568 TI - A fast-acting cytotoxin derived from Con A-activated porcine leukocytes. II. Mechanism of target cell lysis. AB - Serum-free culture supernatants of Con A-stimulated pig leukocytes contain cytotoxins (PCT) which have a fast-acting lytic effect on a variety of murine lymphomas. Mathematical analysis revealed that the cytodestructive reaction which leads to the immediate release of 51Cr from labeled target cells follows "single hit"/"first order" kinetics. The release of labeled compounds was found to be size dependent, such that low molecular isotope (86Rb) was released at faster rates than high molecular 51Cr-labeled complexes. The 51Cr release was strongly reduced by lowering the temperature to 4 degrees C. PCT-mediated target cell lysis could be competed by cold target cells and the factor could be absorbed by intact cells, plasma membranes and artificial liposomes. Neither the presence of EDTA or of various monosaccharides, nor exposure of target cells to trypsin, metabolic inhibitors, and cytoskeletal antagonists altered the target cells susceptibility to PCT-mediated lysis. Labeling of target cell DNA and subsequent exposure of these target cells to PCT revealed that target cell DNA is degraded into low-molecular-weight split products which is similar to that seen during T cell mediated target cell lysis. The analysis of the lytic event mediated by PCT thus revealed similarities to T-cell mediated target cell lysis. PCT was clearly distinct from the well-known cytolytic agents lymphotoxin (LT), tumor necrosis factor (TNF), or complement (C'). Taken together, the described characteristics make PCT a unique-acting cytolytic cytokine with anticancer activity and suggest further research concerning its mode of action and its possible therapeutic application. PMID- 3017569 TI - Intracellular epidermal interleukin 1-like factors in the human epidermoid carcinoma cell line A431. AB - Normal human epidermal cells produce, in primary culture, activities which stimulate the release of PGE2 and collagenase by dermal fibroblasts; this factor(s) might play an important role in epidermal-dermal interactions. Since these activities were mainly found in the cell lysates with only little being detected in the conditioned media, we investigated further the problem of cell associated versus released activity in the model of the human epidermoid carcinoma cell line A431. The activities were consistently found in the cell lysate and in the conditioned media only when the cells were leaky. No membrane associated activities were identified. Purification of the cytosolic activities were identified. Purification of the cytosolic activities yielded two differently charged species both with a MW of approximately 17K. The copurification of PGE2- and collagenase-stimulating activities with thymocyte comitogenic activity suggests a close physiochemical relation to IL-1. The activities described here might therefore correspond to the intracellular counterpart of epidermal IL-1 formerly described as epidermal cell-derived thymocyte activating factor (ETAF) and identified in the conditioned medium of cultured epidermal cells. These observations are of importance when studying the modulation of these activities. PMID- 3017570 TI - Purinergic modulation of T-lymphocyte activation: differential susceptibility of distinct activation steps and correlation with intracellular 3',5'-cyclic adenosine monophosphate accumulation. AB - The mechanism by which purinergic agonists modulate murine T-lymphocyte activation and proliferation was investigated. Adenosine and other compounds such as ATP and 2-chloroadenosine (ClAdo) were found to block T-cell mitogenesis induced by concanavalin A (Con A) in a dose-dependent fashion. The nonmetabolizable adenosine analog ClAdo was the most potent agent capable of inhibiting T-cell mitogenesis. Extracellular addition of the permeable cAMP analog dibutyryl cyclic AMP (dbcAMP) also led to a dose-dependent blockade of T cell mitogenesis, although with less efficiency when compared to ClAdo. Addition of IL-2-enriched fluids failed to reverse blockade of T-cell mitogenesis by ClAdo or dbcAMP. ClAdo blocked T-cell enlargement induced after 20 hr of culture with Con A. We analyzed the effect of micromolar concentrations of ClAdo on interleukin-2 (IL-2) production, expression of IL-2 receptors (7D4 and 3C7 surface antigens), and induction of IL-2 responsiveness after in vitro cultivation with Con A. ClAdo inhibited both IL-2 secretion and induction of IL-2 responsiveness up to control levels in the same dose range it inhibited T-cell mitogenesis. However, cell surface expression of IL-2 receptors was not affected. Short incubations of resting splenic T cells with ClAdo led to a dose-dependent accumulation of cyclic AMP in responding cells. This effect was markedly reduced by the purinergic antagonist 3-isobutyl-1-methylxanthine (IBMX) but was not prevented by the adenosine uptake blocker dipyridamole. ClAdo elicited cAMP accumulation in the same dose range it inhibited T-cell activation events. Extracellular administration of dbcAMP to splenic T cells stimulated by Con A mimicked the effects of ClAdo on T-cell activation parameters, as revealed by a dose-dependent blockade of both IL-2 secretion and IL-2 responsiveness induction, without affecting IL-2 receptor expression. Short incubations of Con A-activated T-cell blasts with ClAdo also led to a dose-dependent accumulation of cAMP. We then analyzed the effect of purines and dbcAMP on IL-2-mediated activated T-cell growth. Purines caused a dose-dependent inhibition of IL-2-mediated T-cell proliferation and ClAdo was the most potent purinergic agonist tested. The effect of ClAdo on Con A-induced T blasts was shifted to the right, if compared to earlier T-cell activation steps.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3017571 TI - Role of adenylate cyclase in human T-lymphocyte surface antigen capping. AB - Our recent studies indicated that capping of T3, T4 and T8 surface antigens on human T lymphocytes is augmented by interaction of adenosine with a purinergic receptor. We suggested that the T-cell capping process was mediated by an adenylate cyclase-coupled purinergic receptor that resulted in the generation of cAMP and occupancy of cAMP receptors. The present study was undertaken to examine whether activation of adenylate cyclase in the absence of purinergic stimulation is sufficient to regulate surface antigen capping. Treatment of T lymphocytes with forskolin or cholera toxin caused activation of adenylate cyclase and occupancy of intracellular types I and II regulatory subunits of protein kinase by cAMP, as demonstrated by photoaffinity labeling with [8-3H]N3-cAMP. Such treatment augmented the rate of capping of the T3, T4, and T8 antigens, which resulted in a significant decrement in the elapsed time to half-maximal capping of each antigen. These observations support the proposition that the normal T lymphocyte capping mechanism of both T3+, T4+ (inducer/helper) and T3+, T8+ (suppressor) subsets can be augmented by activation of adenylate cyclase. PMID- 3017572 TI - Modulation of Epstein-Barr virus-induced B-cell activation by concanavalin A. AB - Direct addition of the T-cell mitogen, concanavalin A (Con A), to cultures of Epstein-Barr virus (EBV)-stimulated peripheral blood mononuclear cells (PBMC) resulted in a dose-dependent inhibition of immunoglobulin M (IgM) secreted in the supernatant, as measured by an enzyme-linked immunosorbent assay. Furthermore, Con A inhibited IgM secretion of isolated T-depleted cells stimulated with EBV, and both the proliferation and IgM secretion of EBV-driven lymphoblastoid cell lines. T-Enriched cells, precultured for 48 hr with Con A, were also able to suppress the IgM response of fresh autologous PBMC stimulated with EBV. This suppression was radiation sensitive (2000 rad), a procedure which resulted in enhancement of the IgM secretion of the responder cells in two out of three experiments. Studies on the long-term effects of Con A showed that the early suppression of IgM secretion was transient and that the mitogen prevented the development of the cytotoxic T-cell response normally seen with lymphocytes from EBV-seropositive donors after 5 weeks of culture. Thus, Con A appears to modulate human lymphocyte responses to EBV by multiple mechanisms. PMID- 3017573 TI - Studies on the mechanism of specificity of human natural killer cells for tumor cells: correlation between target cell transferrin receptor expression and competitive activity. AB - Previous studies to determine the nature of the specificity of natural killer (NK) cells for leukemic cells indicated that functional transferrin (Tf) receptors may be one of the determinants recognized by NK cells. To further investigate these observations, the relationship between cellular Tf receptor expression and ability to compete with a control K562 cell preparation in a standard chromium release assay was studied. K562 cells were selected at different phases of growth by removing cells from tissue culture at 1, 3, and 5 days postfeeding. Under these conditions, K562 cells, respectively, displayed relatively high, medium, and low numbers of Tf receptors and corresponding competitive activity against a control K562 cell preparation. K562 cells were modified by either trypsin, heat, or sodium butyrate (differentiation inducer) pretreatment. An NK-resistant clone was also studied. There was a good correlation between Tf receptor expression and cold competitive activity of the above K562 cell preparations (r = 0.82, P less than 0.01). The different tumor target cell lines, K562, Molt-4, Raji, HL-60, and MeWo, which would be expected to express different ranges of specificity, did not show a significant correlation between Tf receptor expression and their cold competitive activities against Cr-51-labeled K562 cells. Rabbit reticulocytes which express high numbers of Tf receptors were tested for their ability to compete with K562 cells for NK cells. These cells were able to compete with K562 cells while mature rabbit red blood cells which do not express Tf receptors did not compete well. These findings support the contention that the Tf receptor may be involved in NK cell recognition of some tumor cells. PMID- 3017574 TI - Biochemical models of interferon-gamma-mediated macrophage activation: independent regulation of lymphocyte function associated antigen (LFA)-1 and I-A antigen on murine peritoneal macrophages. AB - IFN-gamma can induce the expression of both class II histocompatibility antigens (Ia) and the lymphocyte function associated (LFA)-1 antigen on murine peritoneal macrophages. We have examined the molecular changes which lead to altered expression of these two cell surface proteins to determine whether they are regulated by similar or independent mechanisms. While I-A antigen expression can be induced or enhanced by treatment of macrophages with either phorbol diesters and/or the Ca2+ ionophore A23187, these agents had no effect upon expression of LFA-1 under similar conditions. Macrophages from the A/J strain mouse exhibit a deficiency in their sensitivity to IFN-gamma which is seen in our studies as an inability of IFN-gamma to elevate I-A antigen expression. However, expression of I-A could be modulated in these cells by treatment with either phorbol diesters or A23187. In contrast, IFN-gamma could induce LFA-1 antigen on A/J derived macrophages and this was not affected by either phorbol or A23187. Thus these two antigens, despite coordinate expression in response to IFN-gamma in normal mouse strains, are clearly regulated independently. These results suggest that IFN gamma generates at least two independent molecular events in macrophages which ultimately modulate the expression of cell surface proteins important to the performance of activated functions. PMID- 3017575 TI - The role of transferrin in natural killer cell and IL-2-induced cytotoxic cell function. AB - The growth factor transferrin (Tf) enhanced natural killer (NK) cell cytotoxicity. This enhancement was due to direct effects on NK cell function, and Tf treatment of the K562 target cell had no effect on their sensitivity. NK cells were highly enriched in the low-density large granular lymphocyte population (LGL) by Percoll gradient centrifugation. Despite the direct effect of Tf on NK cells, the number of cells expressing receptors for Tf (TfR) in NK-enriched LGL was the same as the NK-cell-depleted high-density small lymphocyte population (SL). All populations, tested without stimulation, had very few TfR+ cells. Interleukin 2 (IL-2) could induce very high NK-like activity in the LGL but not in SL. Similarly, only LGL could be induced by IL-2 to express TfR. In serum-free cultures, only limited NK-like activity could be developed which was greatly enhanced by supplementing with Tf in the cultures. The importance of Tf in NK like development was confirmed by modulating the expression of TfR in IL-2 containing cultures with mouse monoclonal antibody OKT9 specific for TfR. OKT9 totally abrogated the induction of cytotoxic activity by IL-2 against K562 and NK resistant target. OKT9 inhibited the induction of cytotoxicity in both lymphocytes containing active NK cells and in those predepleted of active NK cells, indicating that the development of NK-like activity from both precursor populations requires Tf. The inhibition by OKT9 was only during the induction phase. The same antibody had no effect on the cytotoxicity of fresh NK cells or the mature IL-2-induced NK-like cells. Our data therefore do not support the hypothesis of TfR as the NK recognition structure. Instead, these results indicate that Tf is important for the development of NK and NK-like activities. PMID- 3017576 TI - Effect of interferon-gamma and 1 alpha,25-dihydroxyvitamin D3 on superoxide anion, prostaglandins E2, and mononuclear cell factor production by U937 cells. AB - Both interferon-gamma (IFN-gamma) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) induce changes in the human monocytic cell line U937 that may reflect cellular differentiation. The effects of recombinant IFN-gamma and 1 alpha,25(OH)2D3 on U937 cells with regard to the release of superoxide anion (O2 ), prostaglandin E2 (PGE2), and mononuclear cell factor (MCF) after stimulation with phorbol myristate acetate (PMA) were examined. PMA did not induce O2- production in untreated cells. A 3-day preincubation with IFN-gamma or 1 alpha,25(OH)2D3 resulted in a 5- to 10-fold increase in PMA-stimulated production of O2- as compared to cells preincubated in medium alone. The response was related to IFN-gamma and 1 alpha,25(OH)2D3 concentrations. In contrast, the PMA induced production of PGE2 and MCF does not require preincubation with either IFN gamma or 1 alpha,25(OH)2D3. These results suggest that O2- production and cytokine production (i.e., PGE2 and MCF) are modulated by different signals related to maturation processes. PMID- 3017578 TI - An acidic lymphokine distinct from interferon-gamma inhibits the replication of herpes simplex virus in human pulmonary macrophages. AB - Supernatants from concanavalin A-stimulated human peripheral blood mononuclear cells were fractionated by gel filtration and isoelectric focusing. A fraction with an isoelectric point of 2.2-3.3 containing macrophage migration inhibition factor activity inhibited the replication of herpes simplex virus type 1 in human pulmonary macrophages and U937 cells. This fraction did not inhibit the replication of herpes simplex virus in human fibroblasts. Moreover, the ability of this lymphokine fraction to inhibit viral growth in macrophages was not neutralized by antibody against interferon-gamma. These findings identify a macrophage specific antiviral lymphokine which is distinct biochemically and immunologically from interferon-gamma. PMID- 3017577 TI - Stimulatory effects of purified macrophage colony-stimulating factor on murine resident peritoneal macrophages. AB - A purified preparation of macrophage colony-stimulating factor (M-CSF) free of interferon and endotoxin activity was studied for its effects on resident murine peritoneal macrophages. M-CSF was found to induce profound morphologic alterations in resident macrophages. These changes included a marked increase in cell size, membrane ruffling, and cytoplasmic vacuolization. Further, after 72 hr of incubation with 1000 U/ml of M-CSF, there were significant increases in macrophage DNA synthesis as measured by autoradiography (P less than 0.001), and in macrophage monolayer protein content (P less than 0.01). None of these changes was seen in control macrophages or those exposed to recombinant interferon-gamma (IFN). Low activity levels of the ectoenzymes 5'-nucleotidase (5'NTD) and alkaline phosphodiesterase I (APD) have been associated with certain macrophage functions, particularly the expression of tumor cytotoxicity. Macrophage monolayers exposed to M-CSF demonstrated an unaltered level of 5'NTD activity from controls and a significantly increased level of APD activity (P less than 0.01) and did not demonstrate an increased ability to kill tumor cells, as measured by the 51Cr-release assay. On the other hand, IFN caused significant decreases in both 5'NTD (P less than 0.05) and APD (P less than 0.01) and also induced marked tumoricidal activity in macrophage monolayers. These results indicate that purified M-CSF induces highly specific alterations in the functional activity and morphologic appearance of resident macrophages and these changes are distinct from those induced by IFN. PMID- 3017579 TI - Immunoglobulin production in cultures of pokeweed mitogen stimulated human peripheral blood mononuclear cells requires interaction of interleukin 2 with the B cells. AB - Immunoglobulin (Ig) production in cultures of pokeweed mitogen (PWM)-stimulated human peripheral blood mononuclear cells (PBMC) is dependent on the presence of T cells. The latter can be replaced by a crude supernatant of PWM-stimulated PBMC, a conditioned medium containing interleukin 2 (IL-2) and presumably other helper factors for B cells. The present study examines the role of IL-2 among these helper factors. Anti-Tac, a monoclonal antibody that blocks the human IL-2 receptor, was found to suppress PWM-induced Ig production in cultures of human PBMC. The inhibition by anti-Tac was at least partly exerted at the level of B cells. Indeed, Ig production in cultures of T-cell-depleted PBMC or highly enriched B-cell preparations supplemented with PWM-induced conditioned medium was also inhibited by anti-Tac (82 +/- 13% inhibition of IgG and 83 +/- 12% inhibition of IgM production). When IL-2 was removed from the PWM-induced conditioned medium by absorption with an IL-2-dependent murine cytotoxic T-cell line, CTLL, the conditioned medium was unable to support PWM-induced Ig production by T-cell-depleted PBMC. On the other hand, recombinant IL-2 alone at a concentration similar to that of IL-2 in the PWM-induced conditioned medium was not able to support Ig production by PWM-stimulated B cells. We conclude that IL 2 acts on PWM-activated B cells in conjunction with other helper factors to induce Ig production and that anti-Tac inhibits PWM-induced Ig production by blocking IL-2 receptors on activated B cells. PMID- 3017580 TI - Demonstration of suppressor cells in coxsackievirus group B, type 3 infected female Balb/c mice which prevent myocarditis. AB - Coxsackievirus group B type 3 (CVB3) induces myocarditis in male Balb/c mice but produces little cardiac injury in females. Males develop cytolytic T lymphocytes (CTL) reactive to heart antigens which primarily cause the inflammation and cardiac injury observed in the disease. Infected female mice lack this CTL response because they rapidly produce suppressor cells inhibiting both cellular immunity and cardiac inflammation. Four lines of evidence demonstrate suppressor cells in females. First, females develop myocarditis when treated with low-dose cyclophosphamide under conditions known to preferentially eliminate suppressor cells but not other immune cells. Second, lymphocytes obtained from females at various times after infection prevent myocarditis when adoptively transferred into CVB3-infected males. Virus concentrations in the hearts of males receiving immune female cells and control males were equivalent. Thus protection did not result from accelerated virus elimination in recipient males. Third, CTL from CVB3 infected male mice could induce myocarditis in infected T-lymphocyte depleted but not in intact females suggesting the presence of an inhibitory T cell in the intact animals. Finally, male lymphocytes cultured on heart cell monolayers for 5 days generate significant cytolytic activity to myocyte targets. CTL generation could be inhibited by co-culture of the male cells with immune female lymphocytes. Nonimmune female cells were not inhibitory. PMID- 3017582 TI - Isolation, characterisation, growth and culture of endothelial cells from the rat aorta. AB - Rat aortic endothelial cells have been isolated by the explantation technique and grown in culture. They have been identified morphologically using standard staining techniques, biochemically by identification of angiotensin convertase and have been positively stained for Factor VIII-related antigen by immunofluorescence using both anti-human and anti-rat Factor VIII antibodies. The explantation technique is a successful alternative to enzyme digestion which is not applicable to rat aortic endothelial cells because of the nature of their attachment to the subendothelial layer. PMID- 3017581 TI - The relationship of abnormalities of cellular immunity to antibodies to HTLV-III in homosexual men. AB - A comprehensive evaluation of the cellular immune system (total T-cell, helper cell, suppressor cell, and natural killer cell numbers; in vitro interleukin-2 production, T-cell responses to mitogens and antigens, serum beta 2 microglobulin levels, and delayed hypersensitivity skin tests) was performed on 36 HTLV-III seronegative and 16 HTLV-III seropositive healthy homosexual men, 48 asymptomatic homosexual men with the chronic lymphadenopathy syndrome, 41 patients with AIDS, and 29 heterosexual controls without any known risk factors for AIDS. Our studies demonstrate that HTLV-III seronegative homosexual men have normal cellular immunity and are comparable to heterosexual controls. The abnormalities of lymphocyte subsets observed in HTLV-III seropositive healthy homosexual men are comparable to subjects with chronic lymphadenopathy. Assays of lymphocyte function, with the exception of delayed type hypersensitivity (DTH) skin tests, are similar in each group except patients with AIDS. Subjects with chronic lymphadenopathy were less responsive to DTH skin tests and HTLV-III seropositive healthy homosexuals were comparable to chronic lymphadenopathy subjects. We conclude that immunologic abnormalities in homosexual men are attributable to infection with HTLV-III. PMID- 3017583 TI - Opiate receptor mediated internalization of 125I-beta-endorphin in human polymorphonuclear leucocytes. AB - The early events in the interaction of (125I)-Tyr27-beta-endorphin with human polymorphonuclear leucocytes were investigated. Using ultrastructural autoradiography we found that the labeled peptide specifically bound to the plasma membrane and was internalized within two minutes of incubation at 37 degrees C. Both processes could be inhibited by unlabeled beta-endorphin or by the opiate antagonist diprenorphine. This finding was confirmed by radioreceptorassays. With longer incubation times the specific association of the labeled beta-endorphin with the cells decreased. About 10% of the tracer was degraded within 10 min of incubation as shown by gel chromatography. The morphological changes induced by 125I-beta-endorphin in the granulocytes were investigated under the microscope. The labeled peptide had the same biological effect as unlabeled beta-endorphin. PMID- 3017585 TI - Sensori-motor metronidazole neuropathy. PMID- 3017584 TI - Benzo[a]pyrene diol epoxide induces viral reactivation at concentrations that block DNA elongation in mammalian cells. AB - The survival of UV-irradiated Simian virus 40 (SV40) in CV-1P African green monkey kidney cells treated with (+/-)7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP-diol epoxide I) was studied. Enhanced survival of UV damaged SV40 was detected when CV-1P cells were treated with dose levels of BP-diol epoxide I corresponding to the exponential portion (0.33-1.11 microM) of a CV-1P cell survival curve. Dose levels of BP-diol epoxide I corresponding to the shoulder region (less than or equal to 0.16 microM) of a CV 1P survival curve did not induce viral reactivation. The shoulder region concentrations of BP-diol epoxide I selectively inhibited DNA initiation while the concentrations on the exponential portion of the curve preferentially inhibited DNA elongation. It was shown in a time course of enhanced viral survival at 0.66 microM BP-diol epoxide I that the reactivation response was fully induced by 24 h. In conclusion, the viral reactivation response was associated with concentrations of BP-diol epoxide I which induced lethal damage and preferentially inhibited DNA elongation. PMID- 3017586 TI - Synthesis and angiotensin converting enzyme inhibitory activity of 1,5 benzothiazepine and 1,5-benzoxazepine derivatives. II. PMID- 3017587 TI - Inhibition of adenosine 3',5'-cyclic monophosphate phosphodiesterase by components of Sophora flavescens Aiton. PMID- 3017589 TI - [Nursing care of patients with side effects of chemotherapy of choriocarcinoma and invasive mole]. PMID- 3017588 TI - Thermally controlled degradation of cytochrome c by cyclic peroxides: possible approach to the hyperthermic sensitizer. PMID- 3017590 TI - [Determination of the virus content in oral trivalent candy poliovaccine using monoclonal antibodies to poliovirus]. PMID- 3017591 TI - [Effect of Pyrola rotund folia on the blood flow and plasma cyclic nucleotide content in mice]. PMID- 3017592 TI - The proton pump/leak mechanism of unconsciousness. AB - There are few explanations which account for the manner in which the catastrophic physiological consequences of anaesthesia, cold narcosis or, for the matter, a short, sharp upper-cut, come about. Most studies terminate with the presentation of ever-better correlations between an end-point in a model system (dough consistency, rubber elasticity, bacterial, protozoal or animal mobility, liposome permeability, luciferase activity, etc.) and oil/water partition coefficients or with some arbitrary biological end-point. From what is currently known about the permeating pathways of non-electrolytes, ions and protons across membranes e.g. liposomes, the effect of anaesthetics on such pathways and the effect of temperature and pressure on both liposomes and whole animals, it is possible to develop a testable hypothesis. It is called the 'proton pump-leak' hypothesis and involves a number of linked biophysical and biochemical processes. It assumes that a living animal or plant is in a steady-state regarding all concentration gradients; passive leaks across membranes are balanced by temperature, pressure, and energy dependent ion/ion and/or proton/ion pumps (enzyme), working within an aqueous phase. Consciousness is dependent upon inter-neuronal communication via release of transmitter substances. Transmitter substances, characteristically either weak bases or weak acids e.g. catecholamines, accumulate passively in vesicles rich in acid-buffer, held to a low pH by the activity of H+/K+ energy driven pumps. Interference with this finely-balanced system either by changing the chemical potential of the hydrophobic (membrane) phase at NTP (with anaesthetics), or by changing the chemical potential of both hydrophobic and aqueous (pump) phases by hyperbaric, hypothermic, or anoxic conditions imposed (inevitably) on the whole animal, would result in the resetting of the steady state parameters. PMID- 3017594 TI - Percutaneous irradiation in cholangio-carcinoma. PMID- 3017593 TI - [A case of compression of the common peroneal nerve due to an intraneural mucoid pseudocyst]. AB - The Authors report a case of paralysis due to compression of the external common peroneal nerve by a mucoid pseudocyst. They emphasize the rareness of such lesion, as well as the importance of a prompt diagnosis and a conservative surgical treatment. PMID- 3017595 TI - WHO collaborative study on the use of monoclonal antibodies for the intratypic differentiation of poliovirus strains. AB - An international collaborative study was carried out to identify monoclonal antibodies that could reliably discriminate between wild polioviruses and strains derived from Sabin vaccine viruses. For poliovirus types 2 and 3, monoclonal antibodies were identified that reacted specifically with type 2 or type 3 strains which gave T1-oligonucleotide maps similar to or indistinguishable from that of Sabin vaccine virus, thus indicating their vaccine origin. These monoclonal antibodies failed to react with strains which gave T1 maps unrelated to that of Sabin vaccine virus. However for type 1, five of the six antibodies examined in the study reacted only with strains with a T1 map indistinguishable from that of type 1 Sabin vaccine virus. In contrast, other monoclonal antibodies against poliovirus types 1, 2 and 3 reacted broadly within a serotype. PMID- 3017598 TI - The transferrin receptor in hepatocyte nodules: binding properties, subcellular distribution and endocytosis. AB - Transferrin binding was found to be around 60-fold higher in hepatocyte nodules compared to normal liver, with no apparent differences in binding affinity or molecular weight of binding proteins, indicating that the increase in [125I]transferrin binding was the result of an increased number of binding sites with similar properties as in normal liver. The relative 'induction' of transferrin receptors was most marked in the total membrane fraction followed by membrane subfractions comprising endoplasmic reticulum, the Golgi complex and endocytic vesicles. Despite the increased number of transferrin receptors, the in vivo endocytosis of transferrin, measured as uptake of [125I]ferrotransferrin, was quantitatively similar in nodular cells compared to normal liver. The number of transferrin receptors in regenerating liver, 48 h after partial hepatectomy, was increased 20-fold over normal levels, but binding affinity, receptor structure and kinetics of transferrin uptake were normal. A slower than normal rate of 59Fe accumulation in hepatocyte nodules may suggest an alteration in the dissociation of iron from ferrotransferrin, thereby suggesting one mechanism relevant to the iron storage deficiency in nodular cells. PMID- 3017596 TI - Prospective randomized double-blind trial of nabilone versus domperidone in the treatment of cytotoxic-induced emesis. AB - A prospective randomized double-blind trial comparing the butyrophenone analogue domperidone (D) and the synthetic cannabinoid nabilone (N) in the treatment of cytotoxic-induced emesis was conducted in 38 patients receiving highly emetogenic chemotherapy regimens (70% containing cisplatin). Patients received 20 mg D or 1 mg N the night before chemotherapy and 8-hourly on each chemotherapy day for two consecutive cycles of treatment. Three of 19 patients randomized to N completed only one cycle because of disease progression or subjectively adverse effects. Four of 19 patients completed only one cycle of D because of lack of efficacy or chemotherapy toxicity. In all, 32 cycles of N and 33 cycles of D were evaluable for efficacy. The mean number of vomiting episodes in cycle 1 was 4.76 for N and 12.95 for D (P less than 0.02). The corresponding values for cycle 2 were 4.27 and 7.69 (P greater than 0.10), and for cycles 1 and 2 combined, 4.53 for N and 10.81 for D (P less than 0.01). Nausea and food intake scores did not differ significantly, although there was a trend towards less nausea and an increased food intake with N. Subjectively adverse effects were more frequent with N and included drowsiness, dizziness, dry mouth, and postural hypotension. N is superior to D for the control of cytotoxic-induced emesis. PMID- 3017597 TI - Relation between induction of ornithine decarboxylase and specific gene expression in rat liver in response to the tumor promoter agent clofibrate. AB - The time course of changes in a number of biochemical parameters in rat liver was studied during 10 days of clofibrate administration. Ornithine decarboxylase (ODC) and putrescine levels began to increase within hours of the first dose and reached maxima at about 36 h (40 and 10 times control levels, respectively) and then returned to normal levels by 48 h. This ODC induction by clofibrate is different from that seen in compensatory liver hyperplasia or diethylnitrosamine administration in that it was not accompanied by elevations in cAMP or increased activation of cytoplasmic cAMP-dependent protein kinases, type I or II. Messenger RNA levels, notably of the species coding for the enzymes of the peroxisomal beta oxidation pathway, increased in parallel with ODC and putrescine to reach a maximum also at 36 h. The enzymes of the peroxisomal beta-oxidation pathway, on the other hand, increased more gradually over time to reach a plateau at approximately 7 - 10 days. The magnitude of increase in mRNA (about 7-fold) was comparable to that of peroxisomal beta-oxidation as measured by cyanide insensitive palmitoyl-CoA-dependent NAD+ reductase activity; comparable increases in the specific content of enoyl-CoA hydratase: beta-hydroxyacyl-CoA dehydrogenase and of peroxisomal thiolase were observed, as determined by SDS electrophoresis. A gradual increase in long-chain acyl-CoA (1.5-fold) followed the increase in beta-oxidation, whereas a 2-fold increase in acid-soluble CoA (free CoA and short-chain acyl-CoA) was seen as early as 36 h. This sequence of changes is at variance with proposals that increased levels of long-chain acyl CoA mediate induction of peroxisomal beta-oxidation. PMID- 3017599 TI - Reduced number of gap junctions in rat hepatocarcinomas detected by monoclonal antibody. AB - A new rat monoclonal antibody was characterized which recognized the 26K protein in gap junctions of mouse, rat and human liver as shown by immunoblot, indirect immunofluorescence, and immunogold electron microscopy. This monoclonal antibody was used to investigate the abundance of gap junctions in chemically induced rat hepatocarcinomas. In comparison with livers of control animals we found in hepatocarcinomas an average decrease of 71% in the number of gap junctional immunofluorescent spots. A corresponding decrease of the total amount of the 26 K protein was detected by quantitative immunoblot. Changes in the proliferative state as well as in intercellular adhesion of hepatocarcinoma cells in contrast to normal hepatocytes might have contributed to cause this decrease of gap junctions in tumor tissue. Possibly the partial loss of gap junctions provided a selective advantage for those preneoplastic liver cells which developed into rapidly proliferating tumor cells. PMID- 3017600 TI - DNA strand breaks in human leukocytes induced by superoxide anion, hydrogen peroxide and tumor promoters are repaired slowly compared to breaks induced by ionizing radiation. AB - The repair kinetics and sensitivity to inhibitors of DNA strand breaks caused by superoxide anion, hydrogen peroxide, benzoyl peroxide and anthralin have been studied and compared with strand breaks caused by well-studied agents such as ionizing radiation and bleomycin. The latter two agents are generally believed to produce breaks indirectly by producing hydroxyl radicals, a very potent oxidizing species, which attack the phosphodiester backbone of DNA. As expected from earlier results, breaks induced by radiation and bleomycin rapidly disappear during post-treatment incubation as a consequence of the action of cellular DNA repair enzymes. Thus, strand breaks produced by hydroxyl radicals appear to be readily repaired in human leukocytes. By contrast, breaks caused by extracellular superoxide anion appeared not to be readily repaired, implying that some mechanism other than the generation of hydroxyl radicals in the vicinity of the DNA was involved. Inhibitors such as 3-aminobenzamide, cytosine arabinoside and adenine arabinoside affected the apparent rate of repair of radiation and methylmethane sulfonate-induced breaks but there was no indication that they affected superoxide anion-induced breaks. They partially inhibited repair of hydrogen peroxide-induced breaks. Breaks caused by benzoyl peroxide and anthralin were also apparently not repaired. We conclude that hydroxyl radicals are not likely the ultimate DNA strand-breaking species in cells exposed to extracellular superoxide anion and that the observed slow repair of DNA strand breaks may be significant in the mechanism of action of tumor-promoting agents. PMID- 3017601 TI - Nuclease P1-mediated enhancement of sensitivity of 32P-postlabeling test for structurally diverse DNA adducts. AB - Exceedingly sensitive procedures are required to detect the presence of covalent DNA adducts in humans exposed to environmental genotoxicants because of low levels of such derivatives (1 adduct in 10(8)-10(10) DNA nucleotides). A 32P postlabeling assay for detection and quantitation of carcinogen--DNA adducts with a sensitivity limit of 1 adduct in 10(7)-10(8) nucleotides has been described previously. In the standard procedure, DNA is enzymatically digested to 3' phosphorylated normal and adducted mononucleotides, which are quantitatively 32P labeled at their 5'-hydroxyl groups by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. 32P-labeled derivatives are resolved by t.l.c., detected by autoradiography and quantitated by counting. We now report that a minor modification of this procedure, entailing the postincubation of DNA digests with Penicillium citrinum nuclease P1 before 32P-labeling, enhanced the technique's sensitivity to 1 adduct in approximately 10(10) nucleotides for a 10 micrograms DNA sample. Nuclease P1 cleaves deoxyribonucleoside 3'-monophosphates of normal nucleotides to deoxyribonucleosides which do not serve as substrates for polynucleotide kinase, while most adducted nucleotides were found to be totally or partially resistant to the 3'-dephosphorylating action of nuclease P1. The additional enzymatic step enabled specific labeling of adducts in up to 20 micrograms of DNA with excess carrier-free [gamma-32P]ATP. The enzymatic digestion conditions were standardized to afford optimal adduct recovery. The new procedure was found to be simple, highly reproducible, and applicable to the detection and measurement of aromatic or bulky non-aromatic DNA adducts formed with such structurally diverse carcinogens as benzo[a]pyrene, 7,12-dimethyl benz[a]anthracene, dibenzo[c,g]carbazole, 4-aminobiphenyl, safrole and mitomycin C; most adducts were recovered quantitatively with a 500- to 1000-fold increase in 32P-count rates as compared with the standard procedure. PMID- 3017602 TI - Mechanism of cardiac dysfunction in hearts from endotoxin-treated rats. AB - Myocardial performance was assessed in isolated perfused working hearts 3 hr after in vivo endotoxin administration (LD50-6 hr) to rats. Hearts removed from endotoxin-treated rats developed approximately 70% of the peak systolic pressure and 50% of the cardiac output (45% of the aortic flow and 50% of the coronary flow) at 25 cm H2O left atrial filling pressure (LAFP) compared to hearts from vehicle-injected controls. Hearts from endotoxin-treated rats were also characterized by decreased mechanical responsiveness to isoproterenol challenge at a high LAFP. These data suggest that myocardial dysfunction associated with endotoxin shock persists in vitro under conditions in which flow returning to the heart is not a limiting factor. Cyclic AMP accumulation was reduced in myocytes from endotoxin rats in response to 1 microM isoproterenol (29% of control values) and 1 microM forskolin (35% of control values) in both the absence and presence of phosphodiesterase inhibition with 0.2 mM isobutylmethylxanthine. Adenylate cyclase activity in crude membrane preparations of ventricular tissue from endotoxin-treated rats was unresponsive to challenge by isoproterenol and was reduced to 65% of control values by 100 micron GppNHp and by 10 mM NaF. Maximal specific binding of (-)[3H]dihydroalprenolol was decreased in membranes from endotoxin (56 +/- 2 fmol/mg) compared to control (64 +/- 2 fmol/mg) rats (P less than 0.05) but with similar antagonist affinities. These results suggest a decreased adrenergic responsiveness in myocytes and ventricular membrane preparations from endotoxin rats that may be linked to the reduction in myocardial adrenergic responsiveness during endotoxemia. PMID- 3017603 TI - Stimulation of DNA synthesis in rat A10 vascular smooth muscle cells by threonine 59 insulin-like growth factor I. AB - The clonal smooth muscle cell line A10, derived from fetal rat aorta, binds 125I insulin-like growth factor I at a Type 1 insulin-like growth factor receptor. Threonine-59 insulin-like growth factor I, multiplication stimulating activity, and insulin inhibit the binding with IC50 = 10 nM, 84 nM, and 500 nM, respectively. Insulin in high concentrations (greater than 5 microM) completely inhibits 125I-insulin-like growth factor I binding to A10 cells. Threonine-59 insulin-like growth factor I and insulin stimulate [3H]thymidine incorporation into DNA in A10 cells that had been growth arrested by incubation in serum-free media (DMEM/0.1% BSA) for 24-36 hours. The stimulation produced by the peptides is 50-60% of the stimulation produced by 10% fetal calf serum. Low levels of serum (0.1 and 0.5%) also stimulate DNA synthesis, and the effects of Threonine 59 insulin-like growth factor I and low serum are additive. The ED50 for the effects of Threonine-59 insulin-like growth factor I, multiplication stimulating activity, and insulin are 6.8 +/- 0.3 nM, 36 +/- 2.5 nM, and 360 +/- 242 nM, respectively. Incubation of A10 cells for 24 hours with Threonine-59 insulin-like growth factor I or serum increases the protein content per culture dish by 85 +/- 21 and 183 +/- 26%, respectively (mean +/- SEM). Thus, both protein levels and DNA synthesis are increased by incubation with peptides. However, Threonine-59 insulin-like growth factor I does not increase the number of cells in serum starved cultures, although 10% fetal calf serum does.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017604 TI - Scintigraphic quantification of myocardial necrosis in patients after intravenous injection of myosin-specific antibody. AB - The Fab fragments of antimyosin antibodies, labeled with 99mTc, were used in the scintigraphic examination of 30 patients with myocardial infarction. The ability to detect necrosis and determine its extent from the antimyosin scan were compared with the results of quantitative regional wall motion analysis by contrast ventriculography at 10 to 14 days and 99mTc-pyrophosphate imaging. Antimyosin images recorded by planar and single photon-emission computed tomography (SPECT) delineated areas of myocardial necrosis in 27 of 30 patients (90%) compared with a 91% sensitivity of pyrophosphate in 21 of 23 patients. Infarct size was determined by both antimyosin and pyrophosphate SPECT images. Results by both techniques showed a significant correlation with computer-derived hypokinetic segment length (r = .79 for both, p = .002) and peak creatine kinase (r = .9 for both, p less than .01). Although sensitivity for and correlations with markers of necrosis were similar with both techniques, infarct size by pyrophosphate SPECT was 1.7 times larger than infarct size by antimyosin SPECT (p less than .01). Certain zones in the infarct area were differentially labeled; the nature and irreversibility of injury within these zones remains to be clarified. PMID- 3017605 TI - Effect of nedocromil sodium on histamine airway responsiveness in grass-pollen sensitive asthmatics during the pollen season. AB - We examined the effect of nedocromil sodium on histamine airway responsiveness in twelve grass-pollen sensitive patients during the 1984 pollen season. The study was a randomized double-blind crossover comparison of nedocromil sodium administered by a pressurized aerosol (4 mg b.d.) with placebo. Crossovers were made at 14-day intervals throughout 8 weeks of the grass pollen season. Histamine airway responsiveness was assessed twice before the pollen season and at the end of each 14-day treatment period. Results were expressed as the provocation concentration (PC) producing a 10% fall in FEV1 (PC10 FEV1) and a 40% fall in flow at 30% of the vital capacity (PC40 V30(P]. During the pollen season all patients developed hay fever and seven had symptoms of asthma. The observed lowest values of PC10 FEV1 and PC40 V30(P) during the placebo treatment periods were significantly lower than mean preseasonal values although not significantly lower than theoretical expected values. Geometric means PC10 FEV1 and PC40 V30(P) were significantly higher during nedocromil sodium treatment compared with placebo. These results indicate that nedocromil sodium has a small but statistically significant effect reducing histamine airway responsiveness in grass-pollen sensitive patients during the pollen season. PMID- 3017607 TI - An improved source of receptor for 1,25-dihydroxyvitamin D3 assay. PMID- 3017608 TI - Development of a cytochemical assay for plasma vasopressin: application to studies on water loading normal man. AB - A cytochemical assay has been developed to measure human plasma arginine vasopressin. It is based on the stimulation of Na+-K+, ATPase activity located in the outer medulla of the rat kidney, and is capable of detecting very low plasma arginine vasopressin concentrations, limit of detection 0.01 pmol/l. Specificity for vasopressin stimulation of the enzyme is conferred on the assay by the use of specific vasopressin antiserum. Index of precision of the assay is 0.21. Degradation of arginine vasopressin in plasma in inhibited by phenanthroline. Samples may be stored up to 8 weeks at -70 degrees C. Intra- and inter-assay coefficients of variation were 22% (n = 8) and 104% (n = 12), respectively. A sustained water load in eight healthy male adults caused a fall in plasma osmolality from a basal of 286.5 +/- 2.0 (mean +/- SEM) to 279.2 +/- 2.4 mmol/kg after the load (P less than 0.001), which was associated with a reduction in urine osmolality from 867 +/- 54 to 69 +/- 3 mmol/kg. Plasma immunoreactive arginine vasopressin fell from 1.3 +/- 0.3 pmol/l to become undetectable (less than 0.3 pmol/l), but plasma cytochemical arginine vasopressin decreased from 0.96 +/- 0.14 to 0.07 +/- 0.02 pmol/l. There was a curvilinear relationship between plasma osmolality and plasma cytochemical arginine vasopressin, which militated against the concept of an osmotic threshold for vasopressin release. PMID- 3017606 TI - Identification of lignans and phytoestrogens in urine of chimpanzees. AB - It was recently observed that the urinary excretion of animal lignans is low in postmenopausal breast cancer patients compared to normal omnivorous and vegetarian women. In addition, the mean excretion of the isoflavonic phytoestrogen equol tended to be lower. Because nonhuman primates appear to be remarkably resistant to the carcinogenic effect of estrogens, we investigated the possible occurrence of lignans and phytoestrogens in the urine of chimpanzees on their regular diet. Five major diphenols were isolated and identified by capillary gas chromatography and mass spectrometry by comparison with synthesized authentic reference compounds. Three of these compounds, the phytoestrogen equol and its precursor daidzein, the lignan enterolactone, were according to preliminary assays excreted in very large amounts. In addition, the lignan enterodiol and the daidzein metabolite O-desmethylangolensin were identified. It is concluded that the chimpanzee excretes both isoflavonic phytoestrogens and lignans in urine, apparently in high concentrations. It is suggested that these compounds may play a role in the maintenance of the resistance against carcinogenic effects of estrogens, which nonhuman primates possess, because both equol and enterolactone have been shown to have antiestrogenic properties in animals. However, much further work is necessary before the possible biological role of these compounds may be established. PMID- 3017609 TI - High activity of cyclic 3',5'-nucleotide phosphodiesterase in sera of patient with phaeochromocytoma. AB - Our previous observations that serum cyclic 3',5'-nucleotide phosphodiesterase activity varied in thyroid disorders and was positively correlated with thyroid function stimulated us to investigate the phosphodiesterase levels in sera of patients with pituitary and adrenal disorders, and the response to glucagon in normal subjects. Both serum cyclic AMP phosphodiesterase (cyclic AMP-PDE) and cyclic GMP phosphodiesterase (cyclic GMP-PDE) activities were measured at a low substrate concentration. Serum cyclic AMP-PDE activity was elevated in five patients with phaeochromocytoma and was not elevated in patients with Cushing's syndrome or acromegaly, compared to the level in normal subjects. Increased enzyme activities returned to normal after resection of the tumours. Intramuscular injection of glucagon to five healthy subjects elevated cyclic AMP levels and cyclic AMP-PDE activity in plasma. These results imply that the increased cyclic AMP level by the activation of cyclase may have induced cyclic AMP-PDE in the target organ and the soluble cyclic AMP-PDE may leak into blood vessels from target organs. PMID- 3017610 TI - Diarrhoea due to circulating agents. PMID- 3017612 TI - Linkage between the loci for cystic fibrosis and paraoxonase. AB - In a material of 22 Danish, 26 Canadian, 10 Australian, 5 English and 5 American families with at least 2 children affected with cystic fibrosis (CF) a combined positive LOD score of 3.46 was found for the relationship cystic fibrosis paraoxonase (PON) at recombination fraction theta = 0.07 in males and theta = 0.13 in females. Assuming a three allele model for PON the LOD score was 4.50 at the same recombination fractions. This confirms our earlier finding of an indication of CF-PON synteny. PMID- 3017611 TI - Indication against genetic localisation of the human transcobalamin II gene (TC2) on chromosome 16. AB - The genetic locus of human transcobalamin II (TC2) is not yet known. The mouse transcobalamin II gene has been assigned to mouse chromosome 11, linked to hemoglobin A. This fact suggested a similar linkage of transcobalamin II in man, assigning it thus to human chromosome 16. Our linkage investigation in a family material of more than 600 individuals demonstrated absence of linkage between transcobalamin II and phosphoglycolate phosphatase, which is very closely linked to hemoglobin A on chromosome 16. Additionally we confirmed absence of linkage with the chromosome 16 gene marker system haptoglobin. These two gene marker systems are located far from each other, and the total length of chromosome 16 is estimated only about 100 cM. Together with recent results of investigations in somatic mouse-man cell hybrids, we conclude that TC2 is not located on chromosome 16. Additionally we found absence of linkage between transcobalamin II and 6 phosphogluconate dehydrogenase, rhesus blood group (both on chromosome 1), GC (chromosome 4), Esterase D (chromosome 13) and AG; absence of close linkage with "debrisoquin polymorphism". PMID- 3017613 TI - Detection and exclusion of carriers of ornithine transcarbamylase deficiency by RFLP analysis. AB - By examining a restriction fragment length polymorphism (RFLP) detected by a gene specific DNA probe of ornithine transcarbamylase (OTC), we have been able to follow the segregation of the defective gene in a family with OTC deficiency. We have identified three sisters of the proband as carriers, and excluded a fourth as a carrier. PMID- 3017615 TI - Typing of families with classical phenylketonuria using three alleles of the Hindiii linked restriction fragment polymorphism, detectable with a phenylalanine hydroxylase cDNA probe. Family typing for PKU by linked HindIII RFLP. AB - A human phenylalanine hydroxylase cDNA clone was isolated from a human liver cDNA library. The size of the cDNA insert is approximately 2.4 kb and appears to be a near full length copy of a phenylalanine hydroxylase mRNA. This cDNA was used to probe for HindIII restriction enzyme polymorphisms in heterozygote typing of families with phenylketonuria. The use of three alleles of this polymorphism, as opposed to the two alleles as previously described, increases the informativity for typing - and therefore also for prenatal diagnosis - in certain families from 75% to 100%. PMID- 3017614 TI - DNA polymorphisms around the apo AI gene in normal and hyperlipidaemic individuals selected for a twin study. AB - We have investigated the allele frequencies, in a Norwegian population, of two restriction fragment length polymorphisms (RFLPs) in the apolipoprotein (apo) AI CIII-AIV gene region. The study group consisted of clinically well twins and their unrelated spouses. In the normotriglyceridaemic individuals tested, the frequency of the rare allele (S2) of the RFLP detected using the restriction enzyme Sst I was 0.17; for the RFLP detected with the enzyme Xmn I, the rare allele (X2) frequency was 0.06. The frequency of the S2 allele was lower in individuals with serum triglyceride levels above 1.7 mmol l-1, but this was not statistically significant. Conversely, the frequency of the X2 allele was higher in individuals with raised serum triglyceride levels, but similarly, did not reach statistical significance. Taken together with the data from our previous study on UK individuals, these results support the suggestion that inherited variations in this apolipoprotein gene cluster are involved in the determination of serum triglyceride levels. PMID- 3017616 TI - Antipsoriatic and antimetabolic agents as stimulators of the beta-adrenergic adenylate cyclase response of epidermis. PMID- 3017618 TI - Paget's disease of the breast presenting as a cerebellar metastasis. PMID- 3017617 TI - Eosinophil chemotactic factor derived from a malignant fibrous histiocytoma. PMID- 3017619 TI - Thyrotrophin (TSH) binding sites on Yersinia enterocolitica recognized by immunoglobulins from humans with Graves' disease. AB - Antibodies against the gram negative enteric bacterium Yersinia enterocolitica have been found in a high proportion of persons with autoimmune thyroid disorders, especially in those with Graves' disease or hyperthyroidism (Shenkman & Bottone, 1981). There is strong evidence that Graves' disease is caused by receptor autoantibodies which mimic the bioeffects of thyroid stimulating hormone (TSH) on the thyroid (Manley, Knight & Adams, 1982). Recently, saturable binding sites for TSH were demonstrated in Y. enterocolitica under non-physiological conditions (Weiss et al., 1983). We have characterized TSH binding sites on Y. enterocolitica under physiological conditions and studied their interaction with Graves' immunoglobulins (Ig's). Saturable and specific binding of receptor purified 125I-TSH to lysozyme/EDTA-treated Y. enterocolitica (serotype 03) was demonstrated under both non-physiological and physiological conditions. Scatchard binding plots were linear indicating a single class of binding site (Kd 1 X 10( 7) M, maximum of 30,000 binding sites per cell). In the presence of Graves' Ig's the binding of 125I-TSH to Y. enterocolitica was significantly inhibited. Graves' Ig's also precipitated a protein of relative molecular mass (Mr) 64,000 from Triton-solubilized, 125I-labelled Y. enterocolitica, similar in size to one of the proteins precipitated by Graves' Ig's from human thyroid membranes. These findings are consistent with the hypothesis that thyroid autoimmunity may be triggered by bacterial infection via a mechanism involving crossreactivity at the level of the TSH receptor. They also suggest that elements of mammalian endocrine systems are highly conserved and have a function in prokaryotes. PMID- 3017620 TI - Disturbance of the Epstein-Barr virus-host balance in rheumatoid arthritis patients: a quantitative study. AB - Rheumatoid arthritis (RA), seronegative spondyloarthropathy (SA) and osteoarthritis (OA) patients receiving no steroid or disease-modifying therapy have been monitored, along with healthy controls, for their prevailing level of Epstein-Barr virus (EBV) infection using four independent indices of the EBV-host balance, levels of virus shedding in throat washings as measured by a cord-blood transformation assay of improved sensitivity, frequency of virus-infected B cells in the circulating blood as measured by the rate of 'spontaneous' transformation in limiting dilution cultures, antibody titres to viral antigens, and virus specific cytotoxic T cell responsiveness as measured in the in vitro regression assay. All four parameters indicated significant disturbance of the virus-host balance accompanying RA, the range of values exhibited by RA patients as a group in each case extending beyond the normal control range in the direction of more active infection. However, observations with SA and OA patients suggested that such a disturbance may not be RA-specific. PMID- 3017622 TI - Natural killer cells in murine muscular dystrophy. IV. Characterization of Percoll fractionated splenic and thymic natural killer cells and natural killer sensitive thymocyte targets. AB - The Natural Killer (NK) activity in the thymus and NK-sensitive thymocyte targets of dystrophic mice was investigated. Dystrophic and normal mouse thymocytes or spleen cells were layered on discontinuous Percoll gradients (5 or 10% increments, respectively) between 40 and 70% and centrifuged at 1700 g for 30 min. All fractions were tested for either NK activity or used a 51Cr-labeled NK sensitive targets in a 6-hr 51Cr release assay. The density interface between the 50% (1.060 g/ml) and 60% (1.075 g/ml) Percoll fractions of either dystrophic or normal mouse spleen cells and the 40% (1.050 g/ml) and 50% (1.060 g/ml) Percoll fractions of either dystrophic or normal mouse thymocytes were found to contain the largest proportion of NK activity using YAC-1 lymphoma tumor cells as targets. In addition, the NK activity in dystrophic mouse spleen cells and thymocytes was significantly greater when compared with normal mouse controls. Target binding cell studies revealed that these Percoll fractions of dystrophic mouse spleen cells and thymocytes had greater numbers of conjugate-forming cells when compared with normal control groups. Cell depletion experiments using either anti-Thy 1.2, anti-asialo-GM 1 or anti-NK-1 plus complement treatment revealed that the cell responsible for NK activity in the 50% Percoll fraction interface of dystrophic mouse spleen cells was asialo-GM 1 positive. NK-1 positive, and partially Thy 1.2 positive. However, the cells displaying NK-activity in the thymus of normal or dystrophic mice were found to be highly Thy-1.2 positive and peanut agglutinin (PNA) negative. The density interface between the 60% (1.075 g/ml) and 65% (1.081 g/ml) Percoll fractions of either normal or dystrophic mouse thymocytes contained the largest proportion of NK-sensitive target cells. Interestingly, the 60% Percoll fraction of dystrophic mouse thymocyte targets was significantly more susceptible to NK-mediated lysis than that of the normal mouse thymocyte population. Cell depletion experiments revealed that the NK-sensitive thymocyte population was similar in both mice, that is, Thy-1.2 positive, cortisone sensitive, PNA positive, Dolichos biflorus (DBA) negative and asialo GM 1 negative. The results indicate that there are density differences between splenic and thymic NK cells. In addition, there are density and phenotypic differences between thymic NK cells and thymic NK-sensitive target cells. The findings support the hypothesis that there are different populations of NK cells. PMID- 3017621 TI - Abnormal B cell response to T cell-independent polyclonal B cell activators in haemophilia A. AB - In addition to T cell abnormalities, patients with haemophilia A show a separate B cell dysfunction. Characteristic are elevated spontaneous IgG (not IgM) levels in the patient's sera and in the culture supernatants of peripheral blood mononuclear cells. It is also observed that these cells fail to show a differentiation response to T cell-independent B cell activators. The B cell dysfunction correlates with the amount of factor VIII concentrates given prophylactically to patients with severe haemophilia. In contrast to the acquired immune deficiency syndrome (AIDS), the proliferation response to B cell mitogens is not affected. PMID- 3017623 TI - Interferon-mediated in vivo induction of beta 2-microglobulin on small-cell lung cancers and mid-gut carcinoids. AB - Selected neuroendocrine tumors, such as small cell lung cancer (SCLC), and neuroblastoma express markedly diminished class I major histocompatibility complex (MHC) antigens (HLA framework and beta 2-microglobulin, beta 2m). Another neuroendocrine tumor, mid-gut carcinoid, also expresses reduced beta 2m antigen as demonstrated herein. Antigen expression is greatly enhanced on SCLC cell lines by in vitro exposure to interferon (IFN). To determine whether IFN mediates similar effects in vivo, we examined by immunoperoxidase staining beta 2m expression in paraffin-embedded tumor tissue sections obtained from 4 SCLC and 7 mid-gut carcinoid patients before and after receiving partially purified human leukocyte IFN-alpha therapy. Before IFN treatment, 3/4 SCLC tumors and 5/7 mid gut carcinoids did not express beta 2m. By contrast, all tumors showed considerable expression of beta 2m after IFN therapy. Induction of class I antigens on tumor cells deficient in such expression may be one mechanism by which IFN exerts antitumor effects. We believe this is the first report of in vivo induction of class I MHC antigens in epithelial tumor cells in humans. PMID- 3017624 TI - [Behcet's disease with mononeuritis multiplex and histological studies of the tibial nerve]. PMID- 3017625 TI - Radionuclide brain perfusion studies in suspected brain death. AB - The radionuclide brain perfusion study (RPS) has been suggested as a method of confirming suspected brain death. The hospital records and RPSs of 34 patients referred were reviewed because brain death was suspected. In every case but one the RPS showed absent or drastically reduced cerebral blood flow. No patient survived more than five days, and 25 survived less than 24 hours. These findings are consistent with the results of previous reports of a total of 248 patients; only one of 248 survived and was discharged from the hospital after having had a positive RPS. The RPS is highly accurate in confirming brain death. PMID- 3017627 TI - Visualization of caput medusa in a patient with hepatoma. AB - A 70-year-old man presented with a history of alcohol abuse and three prior episodes of bleeding from esophageal varices. On physical examination, he had ascites and a bruit over the right lobe of the liver. A diagnosis of hepatoma was established by needle biopsy. Radionuclide studies demonstrated a photon deficient region in the liver by Tc-99m sulfur colloid, which picked up Ga-67 citrate. Angiography with Tc-99m labeled RBCs demonstrated an arterioportal fistula and a caput medusa. PMID- 3017626 TI - Differential diagnosis between Kaposi's sarcoma and pseudo-Kaposi's arteriovenous malformation by scintigraphy. AB - Kaposi's sarcoma and pseudo-Kaposi's arteriovenous malformation differ greatly in cause, evolution, prognosis, and treatment; yet their clinical macroscopic manifestations are similar. A simple and safe method of distinguishing between them is described. PMID- 3017628 TI - Contact highs and urinary cannabinoid excretion after passive exposure to marijuana smoke. AB - Five healthy men were passively exposed under pre- and postplacebo controlled conditions to sidestream smoke from four and 16 standard marijuana cigarettes (2.8% delta-9-tetrahydrocannabinol [delta-9-THC]) for 1 hour each day for 6 consecutive days. Subjective effects produced by the 16-cigarette exposure conditions were similar to those observed after active smoking of one 2.8% delta 9-THC marijuana cigarette. Effects after the four-cigarette condition were less pronounced. Concurrent physiologic measurements showed no clear trends or effects of smoke exposure for either condition. Daily mean plasma levels of delta-9-THC ranged from 2.4 to 7.4 ng/ml with an individual high of 18.8 ng/ml for the 16 cigarette condition. With the use of EMIT cannabinoid assays with 20 ng/ml (EMIT 20) and 100 ng/ml (EMIT 100) cutoffs, urines positive per subject under the four- and 16-cigarette passive exposure conditions were 4.6 +/- 2.2 and 35.2 +/- 3.8, respectively, for the EMIT 20 and 0.0 and 1.0 +/- 0.8, respectively, for the EMIT 100 assay. From the results of these studies, caution is clearly indicated for individuals who might be substantially exposed to heavy marijuana cigarette smoke environments and for those interpreting marijuana screening data. PMID- 3017629 TI - The influence of age on dorsal hand vein responsiveness to norepinephrine. AB - The influence of age on the responsiveness of dorsal hand vein alpha-receptors to local infusions of norepinephrine was investigated by the use of a novel technique, the linear variable differential transformer. Studies were conducted in two groups of healthy subjects, 26 elderly individuals (14 men and 12 women) 60 to 78 years old and 32 young individuals (24 men and eight women) 16 to 29 years old. There was wide interindividual variation in responsiveness to norepinephrine within both groups of subjects. The dose of norepinephrine required to produce 50% venoconstriction in the elderly ranged from 1.5 to 300 ng/min (geometric mean 24.0 ng/min). The dose required to produce 50% venoconstriction in younger individuals ranged from 1.6 to 360 ng/min (geometric mean 23.8 ng/min). These results suggest that there is no systematic influence of age on dorsal hand vein alpha-receptor responsiveness. A power calculation demonstrates a very small likelihood of a type II error. PMID- 3017630 TI - Human pharmacokinetics of the antiviral drug DHPG. AB - The pharmacokinetics of the antiviral drug 9-[2-hydroxy-1-(hydroxymethyl) ethoxymethyl]guanine (DHPG) were examined in six patients receiving 2.5 or 5.0 mg/kg every 8 or 12 hours for human cytomegalovirus (HCMV) pneumonitis or retinitis. Biexponential decay with a mean distribution t1/2 of 0.23 hours and terminal t1/2 of 2.53 hours was observed. Total clearance correlated well with and exceeded creatinine clearance by a factor of 2.4. Mean volume of the central compartment was 15.26 L/1.73 m2 and the volume of distribution at steady state was 32.8 L/1.73 m2. Peak (model predicted) and trough plasma concentrations were 4.75 to 6.20 micrograms/ml and less than 0.25 to 0.63 microgram/ml, respectively, in patients receiving 2.5 mg/kg. Peak concentrations are well above those needed to inhibit HCMV at the 50% level (ID50) and troughs are near this ID50. Cerebrospinal fluid concentrations of DHPG indicate a penetration of 24% to 67%. No accumulation of DHPG was apparent in these patients. However, dosage reduction is necessary in renal insufficiency. Neutropenia occurred in one patient. The plasma concentration profile of DHPG suggests potential beneficial activity against HCMV. PMID- 3017631 TI - The application of recombinant DNA methodology to the diagnosis of inherited disease. PMID- 3017632 TI - Pharmacologic modulation by prostaglandin E1 of superoxide anion production by human polymorphonuclear leukocytes. AB - Inhibition by prostaglandin E1 (PGE1) of superoxide anion (O2-.) production by isolated intact human neutrophils (PMNs) was investigated utilizing initial velocity enzyme kinetics. Lag time, linearity, rate, and extent of reaction were simultaneously examined. Dose-response data indicate progressive PGE1-induced suppression of O2-. synthesis by activated PMNs with a Ki value of 0.50 and 0.98 mumol/L for initial velocity and extent of reaction, respectively. There were no significant dose-related trends for either lag time or linearity for reactions with PGE1 concentrations less than 10(-6) mol/L; however, at concentrations of 10(-8) mol/L and greater, the length of reaction was progressively shortened. PGE1 inhibition of PMN-induced O2-. production does not involve PMN activation/desensitization since PGE1 itself cannot stimulate O2-. generation. Moreover, PGE1 does not function as a free-radical scavenger. These data indicate the clinical feasibility of utilizing PGE1 to titrate PMN-induced synthesis of active oxygen metabolites, in order to attenuate PMN-associated host autoinjury. PMID- 3017633 TI - Adrenocortical dysfunction in acute medical illness. AB - To further characterize the adrenocortical response to acute illness, we measured basal and adrenocorticotropic hormone (ACTH)-stimulated 11-deoxycortisol, androstenedione, and dehydroepiandrosterone sulfate (DHEAS). Acutely ill patients had higher ACTH-stimulated 11-deoxycortisol and androstenedione, and decreased basal and ACTH-stimulated androstenedione/cortisol and DHEAS/cortisol ratios. Our data suggest that a shift away from androgen synthesis toward the glucocorticoid pathway occurs in acute illness. PMID- 3017635 TI - Cryosurvival of herpes simplex virus-2 during cryopreservation of human spermatozoa. AB - Herpes simplex virus (HSV) is a sexually transmitted agent which has potential association with cervical cancer, as well as with high morbidity and mortality in perinatal infection. Its incidence is estimated just after gonorrhea and Chlamydia trachomatis infections. Cryobanking of human semen is accepted worldwide in therapeutic use for both husband and donor. Serious consequences could follow the clinical applications of cryobanked human semen in insemination, if some ejaculates contain HSV and the virus survives the processing of semen in cryopreservation. There is then the likelihood of infection of the female, and through her, the child, generally during parturition. PMID- 3017634 TI - Effects of butylated hydroxytoluene on membrane lipid fluidity and freeze-thaw survival in mammalian cells. AB - Butylated hydroxytoluene (BHT) increases the fluidity of membrane lipids in the hydrocarbon but not the polar regions, as measured by electron spin resonance spin label probes. BHT also sensitizes nucleated mammalian cells to freeze-thaw damage as measured by colony formation survival assays. Furthermore, the membranes of BHT-exposed cells are more susceptible to physical stress, as reflected by the BHT-induced sensitization to hypotonic stress. Since others have shown that BHT induces hexagonal phase lipids in lipid bilayers, this phenomenon may also influence the above survival results. PMID- 3017636 TI - Potentiation of sympathetic neurosecretion by forskolin and cyclic AMP in the rabbit iris-ciliary body. AB - Forskolin has been reported to stimulate cAMP formation and reduce intraocular pressure in rabbit and primate eyes. In view of recent evidence for the involvement of cAMP in modulation of transmitter release at adrenergic synapses, we have investigated the presynaptic effects of forskolin and other cAMP activators on field-stimulated secretion of 3H-norepinephrine (3H-NE) in the isolated, perfused rabbit iris-ciliary body. Forskolin (10(-7)-10(-5) M) was found to markedly enhance stimulation-evoked 3H-NE release without affecting basal (spontaneous) release. The response to forskolin was potentiated by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 0.5 mM) and was mimicked by the cell-permeant cyclic nucleotide analog 8-bromo-cAMP. 8-bromo-cGMP also produce a small enhancement of stimulus-evoked 3H-NE secretion, whereas IBMX alone had little effect on either stimulated or basal secretion. These results suggest that cAMP may play an important neuromodulatory role in regulation of norepinephrine release at intraocular synapses, and raise the possibility that the ocular hypotensive response to forskolin in rabbit eyes may be mediated, in part, by enhanced adrenergic neurosecretion. PMID- 3017637 TI - Tritiated thymidine labeling in carcinomas of the endocrine pancreas. PMID- 3017638 TI - Secretion of haemolysin by Escherichia coli. PMID- 3017639 TI - A case of silica granuloma. PMID- 3017640 TI - In vitro autoradiographic localization of angiotensin-converting enzyme in sarcoid lymph nodes. AB - Angiotensin-converting enzyme (ACE) was localized in sarcoid lymph nodes by an in vitro autoradiographic technique using a synthetic ACE inhibitor of high affinity, 125I-labelled 351A. The lymph nodes were from seven patients with active sarcoidosis who underwent mediastinoscopy and from six control subjects who had nodes resected at either mediastinoscopy or laparotomy. Angiotensin converting enzyme was localized in the epithelioid cells of sarcoid granulomata in markedly increased amounts compared with control nodes, where it was restricted to vessels and some histiocytes. In sarcoid lymph nodes, there was little ACE present in lymphocytes or fibrous tissue. Sarcoid nodes with considerable fibrosis had much less intense ACE activity than the nonfibrotic nodes. The specific activity of ACE measured by an enzymatic assay in both the control and sarcoid lymph nodes closely reflected the ACE activity demonstrated by autoradiography. Sarcoid lymph nodes with fibrosis had an ACE specific activity of half that of nonfibrotic nodes (p less than 0.05). There was a 15 fold increase in specific ACE activity in sarcoid nodes (p less than 0.05) compared to normal. Serum ACE was significantly higher in those sarcoid patients whose lymph nodes were not fibrosed compared with those with fibrosis (p less than 0.01). This technique offers many advantages over the use of polyclonal antibodies. The 351A is a highly specific ACE inhibitor, chemically defined and in limitless supply. This method enables the quantitation of results, and autoradiographs may be stored indefinitely for later comparison. PMID- 3017642 TI - [The lymphadenopathy associated virus]. PMID- 3017641 TI - Alveolitis of pulmonary asbestosis. Bronchoalveolar lavage studies in crocidolite and chrysotile-exposed individuals. AB - Bronchoalveolar lavage (BAL) findings in 27 individuals with crocidolite- or chrysotile-induced asbestosis were compared to BAL findings in 29 unexposed control subjects. Alveolitis, defined as an increase in the proportions and/or absolute numbers of inflammatory cells present in BAL fluid compared to values in control subjects, was present in 26 (96 percent) subjects with asbestosis. Most exhibited a neutrophil-eosinophil alveolitis, with neutrophil proportions increased to 7.4 +/- 0.7 percent and eosinophil proportions increased to 2.2 +/- 0.4 percent, compared to 2 +/- 0.5 percent and 0.4 +/- 0.01 percent, respectively, in control subjects (p less than 0.01 for both neutrophils and eosinophils). An increase in the total number of neutrophils and eosinophils per ml of lavage fluid was also seen (neutrophils 23 +/- 5 and eosinophils 13 +/- 4 per ml; p less than 0.05 compared to control subjects). Severity of the alveolitis, defined by the neutrophil or eosinophil proportions, was independent of a history of exposure to cigarette smoke. The pattern and severity of alveolitis in crocidolite- and chrysotile-induced asbestosis were similar. There was a significant correlation between duration of exposure to asbestos and neutrophil proportions (p less than 0.01). No significant difference in the severity of the alveolitis was observed between individuals with radiologic and physiologic evidence of asbestosis compared to those with asbestos exposure and crackles alone, suggesting that, in asbestosis as in other chronic interstitial lung diseases, radiologic and physiologic parameters do not reflect the severity of the alveolitis. This study demonstrates that a neutrophil-eosinophil alveolitis is present in individuals with crocidolite- and chrysotile-induced asbestosis, that this alveolitis is independent of cigarette smoking, and that the severity of the BAL changes is not reflected in radiologic and physiologic changes. PMID- 3017643 TI - [Cancer of the large intestine--a sequela of ureterosigmoidostomy]. AB - Carcinoma or polyps of the colon as a late complication of ureterosigmoidostomia (USS) have been reported with increasing frequency. Three patients are presented, two of them developed a signet-ring cell carcinoma after USS; one patient showed, 6 years after rediversion of the ureter, a carcinoma at the anastomotic site. Changes in the secretion patterns of sialo- and sulphomucines of the tumor and mucosa are histochemically demonstrated and, together with an appraisal of etiologic factors, discussed. We suggest a careful follow-up of patients with USS, particularly of those performed many years ago. PMID- 3017644 TI - [Correlation of HSV-2-induced early antigen with antibodies from cervical carcinoma patients]. PMID- 3017645 TI - [The determination and clinical significance of blood platelet aggregation function and factor VIII in patients with chronic liver disease]. PMID- 3017646 TI - Silica and oesophageal cancer. AB - The growth of animal cells in culture can be stimulated very powerfully when they are allowed to extend upon a solid surface. In normal fibroblasts, the maximum is reached either on a plane surface with an area of 2500 micron 2 or on a narrow fibre with a length of 250 micron. This growth-stimulating effect of fibres could help to explain how asbestos causes cancer. All asbestiform minerals are complex mixtures of different lengths, but siliceous macrohairs with a uniform length are borne by several species of the grass genus Phalaris. Some of these species are common contaminants of the bread eaten in a part of Iran where oesophageal cancer has an unexplained high incidence. A pure preparation of 200 micron silica fibres from one of these species is a powerful promoter of cancer in the skin of mice. Similar fibres from millet (Seteria italica) are associated with the same disease in China, and plant silica has long been known to be associated the it in South Africa. In addition, a rare thoracic tumour, which normally only occurs after exposure to asbestos, has been detected among sugarcane farmers in the United States and in India; fine silica fibres are liberated into the air during the harvest. PMID- 3017647 TI - Introduction to silicon chemistry and biochemistry. AB - An outline of the chemistry of silicon is given, stressing the reactions in water. In biological systems the metabolism of silicon is little known but much silica is deposited in a variety of amorphous forms. The differences between this biological silica and mineral silicas and silicates, which can be health hazards, are indicated. However both manufactured mineral phase and molecules containing silica can be valuable in medicine. PMID- 3017648 TI - Structural aspects of biogenic silica. AB - The objectives of this paper are to discuss the characterization of biogenic silica in terms of structural properties, and to elucidate the mechanisms of structural organization within biological systems. The scale of organization is a critical factor in the characterization of biosilicification processes, and order at the nanometre, micrometre and macroscopic levels is described. Molecular order is discussed in the light of high-resolution transmission electron microscopy and solid-state NMR results obtained from samples of biogenic silica. Microscopic organization is expressed in a range of structural motifs, e.g. gels, sheets, fibres, tubes and globular assemblies, and reflects the infinitely adaptive morphology of biogenic silica. Macroscopic structures such as curved rods, spicules, perforated plates, teeth and reticular frameworks can be assembled from these microscopic motifs. The mechanisms of structural organization involve spatial (scalar and vectorial) constraints, ordered particle aggregation and chemical regulation. The possible importance of organic surfaces is discussed. PMID- 3017649 TI - Silica in higher plants. AB - Opaline silica deposits are formed by many vascular (higher) plants. The capacity of these plants for silica absorption varies considerably according to genotype and environment. Plant communities exchange silica between soil and vegetation, especially in warmer climates. Silica deposition in epidermal cell walls offers mechanical and protective advantages. Biogenic silica particles from plants are also implicated in the causation of cancer. Recent techniques are reviewed which may aid in the identification of plant pathways for soluble silica movement to deposition sites and in the determination of ionic environments. Botanical investigations have focused on silicification of cell walls in relation to plant development, using scanning and transmission electron microscopy combined with X ray microanalysis. Silica deposition in macrohair walls of the lemma of canary grass (Phalaris) begins at inflorescence emergence and closely follows wall thickening. The structure of the deposited silica may be determined by specific organic polymers present at successive stages of wall development. Lowering of transpiration by enclosure of Phalaris inflorescences in plastic bags reduced silica deposition in macrohairs. Preliminary freeze-substitution studies have located silicon, as well as potassium and chloride, in the cell vacuole and wall deposition sites during initial silicification. PMID- 3017651 TI - [Serum angiotensin-converting enzyme, ceruloplasmin and lactic dehydrogenase in anthracosilicosis and anthracosilico-tuberculosis]. PMID- 3017650 TI - [Radionuclide cerebral angiographic observations on carotid transient ischemic attack]. PMID- 3017652 TI - [Clinical analysis of silicosis complicated by bronchogenic carcinoma]. PMID- 3017653 TI - Low frequency of 5'-flanking insertion of human insulin gene in Japanese non insulin-dependent diabetic subjects. AB - Polymorphism in the 5'-flanking region of the human insulin gene in 149 unrelated Japanese subjects [77 with non-insulin-dependent diabetes mellitus (NIDDM), 17 with insulin-dependent diabetes mellitus (IDDM), and 55 controls] was analyzed with Southern blot hybridization. We used the size of the hybridized fragments to classify the locus into three groups according to Bell's method (a short, class 1 allele averaging 570 base pairs; an intermediate-size, class 2 allele averaging 1320 base pairs, and a long, class 3 allele averaging 2470 base pairs in size). The allelic frequency of classes 2 and 3 in 298 alleles was 5.0% and in the 146 alleles of NIDDM, 7.8%. The value is lower than in Caucasians, American Blacks, and Pima Indians, and the results suggest that the 5'-flanking insertion is not a genetic marker in most NIDDM patients who are Japanese. However, the frequency of the 5'-flanking insertion in those who were not obese and had a family history of diabetes was higher than that of other NIDDM patients (P = .013), and the frequency in NIDDM patients with onset of diabetes at age less than or equal to 39 yr was lower than those whose onset was at age greater than or equal to 40 yr (P = .053). As NIDDM is a heterogeneous disorder, further analysis is needed. These results suggest that we cannot completely exclude the meaning of the insertion in NIDDM. On the other hand, the frequency in IDDM was 0%, lower than in NIDDM (P = .094). Because the number of subjects studied was small, this result is speculative. PMID- 3017654 TI - Calcium transport in pancreatic beta-cells: implications for glucose regulation of insulin release. PMID- 3017655 TI - Stimulus-secretion coupling in the pancreatic B-cell: the cholinergic pathway for insulin release. PMID- 3017656 TI - Structural organization and unusual codon usage in the DNA polymerase gene from herpes simplex virus type 1. AB - We have analyzed the protein and nucleic acid sequences of the DNA polymerase from herpes simplex virus type 1 (HSV-1) to provide insight into the expression and possible structure of this enzyme. Extensive similarity between the amino acid sequence and that of the Epstein Barr virus DNA polymerase is reported. We describe probable structural similarities between these proteins and the use of these similarities to define structural and functional domains within the polymerase. Analysis of base composition and codon usage reveals that several genes from HSV-1, including DNA polymerase, exhibit a strong preference for guanine or cytosine at the third codon position. This preference may result from the high guanine + cytosine content of the virus and produces a highly restricted codon usage, different from that of the host cell. Consequences of the unusual codon usage for viral expression include the potential for extensive mRNA secondary structure. PMID- 3017657 TI - Sequences upstream from the mouse c-mos oncogene may function as a transcription termination signal. AB - A region upstream from the mouse c-mos proto-oncogene, termed upstream mouse sequence (UMS), prevents expression of mos transforming activity. Previous studies suggested that the UMS prevented transcription readthrough. In this study, we constructed a recombinant DNA clone, pHTS3MS, with the UMS inserted downstream from both the mos gene and a truncated long terminal repeat containing only the U3 enhancer region. In this position UMS did not inhibit mos transforming activity. We examined cells transformed by pHTS3MS for RNA expression. S1 nuclease analysis showed that the UMS provides two polyadenylation signals to mos-containing RNA and nuclear run-on transcription showed that the primary transcripts terminate in UMS. In addition, using portions of the UMS, we found that a 360-bp fragment containing the UMS polyadenylation signals and sites inserted between the herpes simplex virus type 1 (HSV-1) thymidine kinase gene (tk) and its promoter inhibits tk transforming activity by 99% and prevents detectable expression of this construct in transient expression assays. Thus, the UMS must contain signals for polyadenylation and appears to function as a transcription terminator. PMID- 3017658 TI - Analysis of the organization and nucleotide sequence of the chromosomal gene for the beta-subunit of rat thyrotropin. AB - The gene for the beta-subunit of rat thyrotropin has been isolated from a library of rat DNA fragments cloned in bacteriophage lambda. The complete nucleotide sequence of the gene has been determined including a portion of 5'- and 3' flanking regions. The rat TSH-beta gene contains approximately 4879 nucleotides which ultimately lead to the production of a mRNA of about 554 nucleotides, exclusive of the 3' poly(A) tract. The first exon which represents only 5' untranslated sequences of the mRNA, is separated from the second exon by a very large, 3.9-kbp intervening sequence. The second and third exons are separated by a small, 377-bp intervening sequence. Southern blot analysis of total genomic DNA demonstrated that the rat genome contains sequences similar to the cloned gene, suggesting that no rearrangements occurred during the cloning process. PMID- 3017660 TI - [Correlation between nasopharyngeal carcinoma (NPC) and HLA in Hunan Province]. AB - 24 patients with NPC and 49 normal individuals in Hunan Province were typed for their HLA-A and -B antigens. The results showed that the antigen frequencies of HLA-A1 and HLA-Bw63 were significantly higher in NPC patient group than in the normal group (P = 0.0098 and P = 0.0028, respectively) and the antigen frequency of HLA-A11 was significantly lower in NPC patient group than in the normal group (P = 0.0075). In addition, the joint occurrence of A2 and B17 was increased in NPC patient group (P = 0.0350). The results suggested that HLA-A1 be a regional antigen associated with NPC in Hunan or Southern China, and that HLA-Bw63 be a new kind of antigen associated with NPC. PMID- 3017661 TI - [Immunohistochemical and cytochemical analysis of lymphocytes and plasma cells in nasopharyngeal carcinoma]. AB - Cytoplasmic IgA+, IgG+ and IgM+ in plasma cells, present in biopsy tissue of 68 patients with nasopharyngeal carcinoma (NPC) and 40 patients with chronic nasopharyngitis (CN), were studied by immunoperoxidase (PAP) technique. EB virus VCA-IgA serum antibody in all these patients was determined. At the same time, the activity of T lymphocytes of 41 patients (23 with NPC and 18 with CN) was investigated by alpha-naphthyl acetate esterase (ANAE) method. The number of T lymphocytes in NPC was far less than that in CN. This suggests that the impediment or deficiency in cellular immunity may promote the development and growth of tumor. The number of IgA+ plasma cells in NPC was obviously more than that in CN. As the increase in the level of VCA-IgA serum antibody in NPC patients corresponded to the increase in the number of IgA+ plasma cells in the tumor tissue, it was presumed that part of the IgA+ plasma cells might participate in the production and introduction of VCA-IgA antibody. We suggest that the examination of VCA-IgA serum antibody be a reliable screening test for NPC. No significant difference was found in the numbers of IgG+ plasma cells between NPC and CN. IgM+ plasma cells were rare in both. PMID- 3017659 TI - A method for constructing multiple tandem repeats of specific DNA fragments. AB - We describe a new method for constructing tandem repeats of DNA fragments that allows one to control the number of tandem copies appearing in a final recombinant DNA clone. The principle of the method is to prepare DNA fragments with predetermined cohesive ends and to ligate them together in such a way that unique multimers are generated. First, the fragment of interest is inserted into a vector with a multisite cloning region. Clones are picked with the fragment in both orientations, and the fragments are excised using different pairs of restriction enzymes. The resulting fragments with preprogrammed cohesive ends are mixed, ligated, and digested with one enzyme whose site appears in the cloning region of the vector. From this material multimeric fragments containing specific ends can be isolated and recloned into appropriate vectors. As an example of the method, we show the construction of clones containing either three or six tandem copies of the origin of DNA replication of the mouse polyoma virus. The prospect of using this technique to construct inverted repeat structures is discussed. As part of this work we have constructed a modified M13mp8 vector which contains a unique Xho I cloning site. PMID- 3017662 TI - [Clinical observation on the changes in plasma cyclic nucleotides in patients with leukemia before and after treatment and their relation to cell-mediated immunity]. AB - Level of plasma cyclic nucleotides was determined in 47 normal subjects and 63 patients with leukemia. Follow-up was carried out in 20 of these patients. The relation between the level of cyclic nucleotides and cell-mediated immunity was studied. THE RESULTS: There was no significant difference in cyclic adenosine monophosphate (cAMP) between the normal subjects and leukemic patients, whereas cyclic guanosine monophosphate (cGMP) level was markedly elevated and cAMP/cGMP ratio was obviously reduced in the untreated and unrelieved patients. There was no significant difference in the various types of leukemia. In patients with complete remission (CR) after treatment, their cyclic nucleotides were similar to those in normal subjects, but they remained abnormal in patients without clinical remission after treatment. In CR patients treated regularly for half a year, the cGMP level when first diagnosed was lower than that of patients who died soon or were unrelieved. Plasma cGMP level was negatively correlated to E-rosette forming cell (E-RFC) and cAMP/cGMP ratio was positively correlated to E-RFC. These results suggest that the plasma cGMP be used as additional parameter to monitor the treatment response and prognosis. Reduced E-RFC in leukemia may be related to the elevated cGMP and/or lowered cAMP/cGMP ratio. PMID- 3017663 TI - [Serum HSV-2 antibody in males from high and low incidence areas of cervical cancer]. AB - Serum HSV-2 antibody in male from high and low incidence areas of cervical cancer was assayed by enzyme-immuno-slide method. The results show that the geometric mean titer of the serum HSV-2 antibody in the male from high incidence area (413.5) is significantly higher than that from the low incidence area (210.4) (P less than 0.01). It indicates that the high cancer incidence might be related to the high HSV-2 infection rate in the spouses of the cervical cancer patients. PMID- 3017665 TI - [Bronchiolo-alveolar cell carcinoma (clear cell adenocarcinoma)--report of 6 cases]. AB - 6 cases of bronchiolo-alveolar cell carcinoma proven surgically and histologically were studied. Under the microscope, it was shown that the columnar tumor cells lined the inner alveolar walls and the bronchiolar epithelium revealed malignant changes. Diagnosis of bronchiolo-alveolar cell carcinoma was established. Under the electron microscope, in addition to its adenocarcinomatous features, tongue-like protrusions were present on the cell surface and abundant round electron-dense granules were accumulated above the nuclei. These were in accordance with the characteristics of clear cell adenocarcinoma. In 4 cases, lamella bodies were present in a few tumor cells. It suggests that the clear cell adenocarcinoma be able to synthesize round electron-dense granules and lamella bodies. PMID- 3017664 TI - [Malignant fibrous histiocytoma (MFH) of the maxilla--an analysis of 7 cases]. AB - Malignant fibrous histiocytoma is a rare tumor of the soft tissue or bone, especially if located in the head and neck. Up to the present, only 7 cases have been reported (6 in the mandible and only one in the maxilla). From Mar. 1958 to Mar. 1985, 7 patients with maxillary MFH, proved by pathology, were treated in our hospital. The X-ray manifestations were studied and analysed. The main X-ray characteristics are as follows: A large soft tissue mass in the maxilla, extending into the infratemporal fossa, pterygopalatina fossa, orbit, ethmoid sinuses, nasal cavity and cheek. Marked and extensive destruction of the maxilla with some amorphous reactive ossification. The bone appears like melting ice or ground glass. The value of computerized tomography in differential diagnosis and clinical staging of the maxillary malignant fibrous histiocytoma are discussed. PMID- 3017667 TI - [Cloning and expression of HTLV-III env gene fragment in Escherichia coli cells]. PMID- 3017666 TI - [Combination of radiation, chemotherapy and surgery in rectal cancer--an analysis of 128 patients]. AB - The results of 128 patients with rectal cancer treated by the combination of surgery, preoperative radiotherapy and chemotherapy from June 1975 to December 1979 are reported and compared with those by operation alone. The 5 year survival rates of the different groups were: 69.5% (16/23) for radical surgery plus preoperative radiotherapy and chemotherapy (RRCG), 56% (14/25) for radical surgery plus preoperative radiotherapy, 53.8% (7/13) for radical surgery plus chemotherapy and 40% (10/25) for radical surgery alone. The 5 year survival rate of RRCG was significantly higher than that of the radical surgery alone group (P less than 0.05). The authors believe that the combination of preoperative radiotherapy, chemotherapy plus radical surgery improves the 5 year survival rate and is worthy of further study. PMID- 3017668 TI - [Regulatory peptides consisting of less than 20 amino acid residues are statistically periodical]. PMID- 3017669 TI - [Effects of IPGJ on the cardiovascular system and cAMP and cGMP]. PMID- 3017670 TI - A review of the effects of repeated administration of selected phenylethylamines. AB - Several phenylethylamines are under consideration for international control. The effects of repeated administration of these compounds, including tolerance, physical dependence and central nervous system (CNS) toxicity, are reviewed here. The compounds can be divided into two major chemical groups: those with substituents on the ethylamine portion of the molecule and those with substituents on the phenyl ring. Although the effects of repeated administration have not been directly determined for most of the compounds, certain representative compounds of each chemical group have been examined in some detail. Prominent among the effects of repeated administration are CNS toxicity and tolerance development. Physical dependence has not been reported for any of these compounds. Future research with these compounds should emphasize the investigation of the CNS toxicity and the functional consequences of such effects for the organism. PMID- 3017672 TI - [Herpes zoster in early gastric carcinoma]. PMID- 3017671 TI - [Cytomegalovirus infection: pathogenesis, diagnosis and prevention]. PMID- 3017673 TI - [Intravenous thrombolysis treatment in fresh myocardial infarct. Success and aftercare]. PMID- 3017674 TI - [Oral hairy leukoplakia--early symptom of HTLV-III/LAV infection]. AB - Epstein-Barr virus was demonstrated electronmicroscopically in a leucoplakic area of the tongue of a man infected with HTLV-III/LAV. Oral "hairy" leucoplakia, diagnosed from the clinical findings, histology and by electronmicroscopy, in this patients is to be interpreted as the initial sign of an HTLV-III/LAV infection. PMID- 3017675 TI - Protein synthesis in astrocytes: 'spontaneous' and cyclic AMP-induced differentiation. AB - Primary cultures of mouse astrocytes have been used to study astroglial protein synthesis during 'in vitro' differentiation. Spontaneous age-related differentiation was compared to the effect of DBcAMP or forskolin, a drug which directly stimulates the adenylate cyclase and induces 'morphological differentiation' in these cells. Cell differentiation was followed in parallel by phase contrast microscopy and immunofluorescence techniques. Two antisera, one raised against GFA, the other against microtubule-associated protein 2 (MAP2) were used. Anti-GFA serum labelled the cells as early as 7 days in vitro. Anti MAP2 serum revealed a dense fibrous network at later stages of the culture, whereas the dividing astroblasts appeared poorly stained by this antibody. Both phase contrast microscopy and immunofluorescence techniques suggested that most of the cells spontaneously differentiate after 3 weeks of culture even in the absence of DBcAMP or forskolin. Forskolin, while accelerating differentiation after 7 days of culture, produced smaller cells than DBcAMP and had biphasic effects on cell morphology. Mono- and two-dimensional gel electrophoresis of the 35S-methionine labelled cells also showed that the major changes in protein synthetic activity occur spontaneously during the time course of the culture. Whatever the stage of the culture, DBcAMP or forskolin induced changes in the synthesis of only a few proteins. However, depending on the culture stage the proteins, which were positively or negatively controlled by these drugs, were not the same. PMID- 3017676 TI - Triiodothyronine receptors in developing mouse neuronal and glial cell cultures and in chick-cultured neurones and astrocytes. AB - The evolution of L-triiodothyronine (T3) receptors was studied in developing cultures of cells dissociated from cerebral hemispheres of 14-day-old mouse embryos, which present successive distinct periods of cell proliferation and/or maturation. These periods are characterized essentially as neuronal from 1 to 12 days in vitro (DIV) and glial between 12 and 60 DIV. Furthermore myelin-related membranes are produced in this culture system. Binding capacities of the T3 nuclear receptors increased from 1 to 6 DIV, when it reached a maximum (16 fmol/100 micrograms DNA). A similar increase of the DNA content of the cell was observed until 8 DIV. Thereafter a sharp fall of receptor concentration leading to a 5-fold decrease in the binding capacity occurred until day 15, a period at which neurones disappeared from the cultures. From 25 to 50 DIV (coinciding with the glial period), the concentration of receptor remained more or less constant (1-2 fmol/100 micrograms DNA). In parallel, the DNA content did not vary greatly between 30 and 50 DIV. Scatchard analysis revealed the presence of a single class of receptors at 6 and 20 DIV, representative of 'neuronal' and 'glial' periods, respectively. The equilibrium dissociation constant (Kd) of the nuclear receptor from cells at 6 DIV (2 X 10(-10) M) was similar to that found at 20 DIV. These results were confirmed using pure cultured neurones and astrocytes prepared from embryonic chick brain. The effect of T3 on the cellular gangliosides used as an index of neuronal cell maturation, and on cerebroside sulfotransferase (CST), an enzyme involved in the production of myelin sulfatides, was studied to determine a possible correlation between the binding capacity of the T3 nuclear receptor and the response of the cultured cells to thyroid hormone. Our data demonstrate that T3 had no significant effect either on the content of gangliosides or on their developmental pattern, while it increased the level of CST activity by 75% between 18 and 25 DIV. These results show that, although the concentration of T3 receptors per 100 micrograms DNA in glial cells was lower than that in neurones, it was nevertheless sufficient to elicit a response in oligodendrocytes. PMID- 3017677 TI - Non-traumatic quadriplegia and paraplegia in Dar es Salaam, Tanzania. PMID- 3017679 TI - [The use of porous hydroxylapatite ceramic in the filling of periodontal bone defects]. PMID- 3017678 TI - Post-kala-azar dermal leishmaniasis occurring long after cure of visceral leishmaniasis in Kenya. PMID- 3017680 TI - [Interaction between various muscle relaxants and our anticurare drugs]. PMID- 3017681 TI - [Analysis of the extension of epileptic discharges in the animal model]. AB - The study was aimed at the enlightenment of intracortical spreading mechanisms in focal epileptic seizures produced by local application of Acetylcholine. Experiments were made in rabbits. Epi- and intracortical seizure potentials were recorded simultaneously with special electrodes. The recorded signals were analysed using computer aided methods. The results show, that the spreading of the pathological events does not take place continuously but by successive generation of "active zones" independent of the original focus produced by Acetylcholine. In the epicortical recordings, the development of a new focus is indicated by a functional uncoupling between the superficial layers of the cortical area to be involved and the momentary active focus. This functional uncoupling is due to an activation of the middle cortical layers of this area by the focus. As a first sign, high frequent potentials can be observed in the middle cortical layers accompanied by an amplitude decrease in the superficial layers. In the further development high negative discharges appear in the middle cortical layers which lead to characteristic surface positive potentials. This phase indicates that a new focus has developed, i.e. the discharges are no longer triggered by the previous focus. These processes continue as long as inactive cortical areas are available. From the obtained results it can be assumed with high probability that in contrast to previous assumptions the generation of a new epileptic focus is not initiated via superficial axon collaterals but that other cortical connections must be responsible. Additionally, it has to be taken into account that extra- and/or intracellular changes of ion concentrations as a consequence of the massive synaptic bombardment may be responsible for the independent discharges in the focus. PMID- 3017682 TI - [Age changes in early somatosensory evoked potentials]. AB - There are characteristic age-related changes in the cervical and early cortical somatosensory potentials evoked by electrical stimulation of the median nerve. At an age of 40 to 50 years the latencies of the potential components and the transit times start increasing progressively. Moreover, there is an attenuation of the cervical and an enhancement of the cortical components with age. Considering the presumed neuronal basis of the bioelectric phenomena the changes are discussed in connection with aging processes of the spinal ganglion cells, cortical pyramidal cells and the locus coeruleus. PMID- 3017683 TI - [Function-oriented neurophysiologic diagnosis: long loop reflexes in spinal and cerebral lesions]. AB - Neurophysiological methods which allow to identify objectively lesions of the efferent cortico-spinal pathways in humans are with the exception of transcutaneous electrical stimulation of the motor cortex nor available. Long latency EMG responses from leg muscles are mediated by a "transcortical loop" and offer the possibility to detect in combination with sensory evoked potentials lesions both of the afferent proprioceptive and the efferent motor pathways. Subjects stood on a platform which was rotated toe-up (50 degrees/s, 4 degrees) around the axis of the ankle joint. This lead to two EMG responses of short- and medium latency in the stretched triceps surae muscle, and an EMG response of long latency (LL) from the antagonistic anterior tibial muscle. We investigated 135 patients with a spinal or cerebral lesion, detected by neuroradiological methods. We found a significant delay of LL in patients with spinal lesions and in patients with cerebral lesions on the affected side. A significant delay of LL could also be observed in patients with pure motor symptoms and normal latency of the cerebral potential (P 40) alone in the group of patients with spinal lesions revealed in about 45% a pathological result. The rate of pathological findings was about 65% when both methods (SEP and LL) were combined. The results indicate that the recording of LL is helpful to detect lesions of the efferent central pathways. In a follow-up study we investigated patients who underwent successful surgical treatment. We found a close correlation between the decrease in latencies of LL and the improvement of clinical signs. PMID- 3017684 TI - [AIDS--transmission by EMG needle electrodes?]. PMID- 3017685 TI - Evidence for a peripheral origin of the brainstem auditory evoked potential. PMID- 3017686 TI - Polyneuropathy in workers with long exposure to vinyl chloride. Electrophysiological study. PMID- 3017687 TI - Firing rate of low-threshold motor units after maximal voluntary contraction. PMID- 3017688 TI - The anterior pituitary-grafted rat: a valid model of chronic hyperprolactinemia. PMID- 3017689 TI - Receptor-mediated peptide transport through the blood-brain barrier. PMID- 3017690 TI - Benzodiazepine receptors and their relationship to the treatment of epilepsy. AB - Benzodiazepines (BDZ) interact with components of neuronal membranes to modify excitability in three different ways. Action at a high affinity central receptor (dissociation constant, KD, of 3 nM) linked to the GABAA recognition site enhances the inhibitory action of GABA by increasing the number of openings of Cl channels produced by a given concentration of GABA. This effect correlates with anticonvulsant activity as evaluated in the antipentylenetetrazol test in animals and with antimyoclonic activity in human beings. It also correlates with anxiolytic activity. Action at a lower affinity membrane site (KD 100 nM to 1 microM) limits repetitive firing as observed in isolated neurons (in a manner similar to the action of phenytoin or carbamazepine). This does not depend primarily on neurotransmitter mechanisms, but probably involves an increase in the population of sodium channels in the inactive state. Action at a lower affinity site (KD 45 microM) in presynaptic terminals decreases voltage sensitive Ca++ conductance and, by limiting Ca++ entry, decreases neurotransmitter release. The two lower affinity BDZ systems may be responsible for therapeutic action in status epilepticus and for sedative side-effects. The high affinity central benzodiazepine binding sites can be differentiated into BZ1 and BZ2 receptors by ligands (such as triazolopyridazines and Quazepam) that preferentially act on BZ1 sites. There are regional differences in the density of the two receptor subtypes, but these have not yet been correlated with specific actions of benzodiazepines. Differences between various 1,4- and 1,5-benzodiazepines in terms of therapeutic action in epilepsy and neurologic side-effects can probably be explained on the basis of variation in full or partial agonist action at the high affinity central receptor, or differing relative action at the high and low affinity receptors. PMID- 3017691 TI - Serum angiotensin-converting enzyme in diabetes mellitus: a negative report. AB - Serum angiotensin-converting enzyme (ACE) was measured by a direct photometric method in 78 normotensive diabetic patients and in 34 controls. For comparison, ACE was also assayed in 24 subjects by a radiometric procedure. We found no ACE elevations in diabetics and no significant difference in mean ACE levels between diabetics and normals. Within the diabetic group, enzyme levels were not affected by duration of disease, degree of metabolic control, or presence or not of microangiopathy. Only type I subjects had mean ACE significantly (p less than 0.05) higher than type II, very likely due to their younger age. Serum ACE data from photometric and radiometric methods significantly correlated. ACE measurement seems to be of scarce significance in diabetes mellitus. PMID- 3017692 TI - Characterization of the mutant N-acetylglucosaminylphosphotransferase in I-cell disease and pseudo-Hurler polydystrophy: complementation analysis and kinetic studies. AB - Complementation was examined among various types of I-cell disease and pseudo Hurler polydystrophy by monitoring N-acetylglucosaminylphosphotransferase activity in multinucleated cells produced by fusing pair combinations of cultured skin fibroblasts. Patients with the classical forms of these disorders (5 I-cell disease and 3 pseudo-Hurler polydystrophy cell lines) comprised one complementation group and 5 cell lines from patients with variant forms of pseudo Hurler polydystrophy comprised a distinct complementation group. In the first group, total or partial deficiency of the transferase activity was demonstrated with both natural (lysosomal enzymes) and artificial (alpha-methylmannoside) acceptor substrates with low Vmax but apparently normal Km values for the donor (UDP-GlcNAc) and acceptor (alpha-methylmannoside) substrates. The activity toward artificial substrate could be inhibited by adding exogenous lysosomal enzyme preparations to the reaction mixture. In the second group, the cells demonstrated deficiency of the transferase activity toward lysosomal enzyme acceptors but had normal activity toward alpha-methylmannoside acceptor and this activity could not be inhibited by the addition of exogenous lysosomal enzyme preparations. These findings suggest that N-acetylglucosaminylphosphotransferase is composed of at least two distinct subunits, a catalytic subunit which is absent or defective in the first complementation group, and a recognition subunit which is altered or deficient in the second group. PMID- 3017693 TI - Effect of triethyltin on enzyme activity in human adult and cord red cells. AB - Since organotin compounds represent an environmental health hazard, we determined the effect of triethyltin bromide (TTB) on red blood cell (RBC) enzyme activity. TTB produced a concentration-dependent inhibition of hexokinase and pyrimidine 5' nucleotidase for both adult and cord RBC. D-Glucose, but not ATP or MgCl2, prevented the hexokinase inhibition by TTB. Glucose-6-phosphate dehydrogenase, adenylate kinase, and hypoxanthine-guanine phosphoribosyltransferase were also inhibited by TTB. Cord RBC enzymes were more resistant to the effects of TTB than were adult RBC enzymes. Although TTB is a potent inhibitor of hexokinase, physiologic concentrations of glucose appear to protect the RBC during clinical tin intoxication. PMID- 3017694 TI - The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens. AB - The genetic transformation of plant cells by Agrobacterium tumefaciens is mediated by the genes of the Ti plasmid vir region. To determine the genetic and transcriptional organization of the vir region of pTiA6, vir plasmid clones were saturated with insertion mutations of a Tn3-lacZ transposon. This element is both an insertion mutagen and a reporter for the expression of the sequences into which it has inserted. One hundred and twenty-four vir::Tn3-lac insertions were analyzed for their mutagenic effect on Agrobacterium virulence, and for their expression of beta-galactosidase activity, the lacZ gene product, in vegetative bacteria and in bacteria cocultivated with plant cells. These data in conjunction with genetic complementation results show that the pTiA6 vir region contains six distinct vir complementation groups: virA, virB, virC, virD, virE and virG. Mutations in these loci eliminate (virA, virB, virD and virG) or significantly restrict (virC and virE) the ability of Agrobacterium to transform plant cells. Each of the vir loci corresponds to a single vir transcription unit: virA is constitutively expressed and non-inducible; virB, virC, virD and virE are expressed only upon activation by plant cells; and virG is both constitutively expressed and plant-inducible. The two largest vir operons, virB and virD, are probably polycistronic. The pTiA6 vir region also contains plant-inducible loci (pin) which are non-essential for virulence. PMID- 3017695 TI - Structure and expression of the chicken beta nerve growth factor gene. AB - The 3' exon of the chicken beta nerve growth factor (NGF) gene was isolated by the use of a murine cDNA probe. DNA sequence analysis of the clone suggests a mature chicken NGF protein of 118 amino acids, showing approximately 85% homology to mouse and human NGF. In addition to this conservation of the mature NGF, parts of the propeptide and the untranslated 3' end of the NGF gene are also highly homologous in chicken, human and mouse. Therefore, these sequences probably subserve important functions. Expression of NGF mRNA in various chicken tissues was examined by RNA blot analysis with a chicken NGF probe. A single mRNA of 1.3 kb was detected at high levels in heart and brain of 10-week-old roosters, and, at lower levels in spleen, liver and skeletal muscle. These data suggest a correlation between NGF expression and the density of sympathetic innervation in peripheral organs, in analogy with findings for mammalian tissues. In the adult avian brain, NGF mRNA is found at higher concentration in the optic tectum and cerebellum than in the cortex and hippocampus. This pattern of NGF expression differs from that previously described for the rat brain. During late stages of development (day 18), NGF mRNA was expressed both in heart and brain of embryos but at lower levels than in the adult. PMID- 3017696 TI - Reconstitution of beta 1-adrenoceptor-dependent adenylate cyclase from purified components. AB - In continuation of our efforts to reconstitute from purified components into lipid vesicles the signal transmission chain from beta 1-adrenoceptors to adenylate cyclase, we now report on the total reconstitution of the hormone dependent adenylate cyclase. In these reconstitution experiments we have employed the purified adenylate cyclase (C) from bovine brain and rabbit heart, the stimulatory GTP-binding protein (GS) purified from turkey erythrocytes and rabbit liver and the beta 1-adrenoceptor (R) from turkey erythrocytes. Several detergents were compared with respect to their suitability to allow reconstitution of subunits into phospholipid vesicles. While octyl polyoxyethylene (octyl-POE) was almost as potent as lauroyl-sucrose for preparation of vesicles containing GS.C, the latter detergent was clearly superior for vesicles enabling productive R.GS and R.GS.C coupling. The catalytic subunit from either bovine brain or rabbit heart was equally efficient in reconstitution. However, GS from turkey erythrocytes and rabbit liver revealed significant differences in RGS and RGS.C containing vesicles. While isoproterenol induced activation of GS by GTP gamma S was first order in both instances, kon with turkey GS was 0.12 min-1, whereas kon with rabbit liver GS was 0.6 min-1. Moreover, GTP gamma S activation of erythrocyte GS was significantly more dependent on the presence of hormone than that of liver GS, confirming observations made on the native membrane-bound system. Compared with stimulation by isoproterenol (GTP gamma S) (4-fold), stimulation by isoproterenol/GTP was modest (1.3- to 1.6-fold).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017697 TI - Evidence for dimer structure of proton-pumping cytochrome c oxidase, an analysis by radiation inactivation. AB - Cytochrome c oxidases, purified from bovine heart and the thermophilic bacterium PS3, were irradiated with a high-energy electron beam. The proton transport activities of both preparations and their electron transfer activities decreased as single exponential functions of the radiation dosage. Applying the target theory with alkaline phosphatase as an internal standard, the following functional molecular weights were obtained for cytochrome c oxidation and H+ pumping: 63-73 kd and 160-220 kd, respectively, for the bovine enzyme, and 80-100 kd and 190-230 kd for the PS3 enzyme. The results suggest that a dimer structure is necessary for H+ pumping, while a core part of monomer (presumably the largest two subunits, i.e. subunits I and II) is sufficient for cytochrome c oxidation. PMID- 3017698 TI - Temperature-sensitive mutants of MH2 avian leukemia virus that map in the v-mil and the v-myc oncogene respectively. AB - MH2 is an avian retrovirus that contains the v-mil and v-myc oncogenes. In vitro it transforms chick macrophages that are capable of proliferation in the absence of growth factor. Earlier work showed that v-myc induces macrophage transformation and that v-mil induces the production of chicken myelomonocytic growth factor (cMGF), thus generating an autocrine system. We describe the isolation of temperature-sensitive (ts) mutants of MH2 virus. As suggested by marker rescue experiments, one mutant bears a ts lesion in v-mil, whereas the other carries a mutation in v-myc. Ts v-mil MH2-transformed macrophages become factor-dependent at the non-permissive temperature (42 degrees C), while ts-v-myc MH2-transformed macrophages cease growing and acquire a more normal macrophage phenotype at 42 degrees C irrespective of the presence of cMGF. Both phenotypes can be reversed by backshift to the permissive temperature. These results suggest that the gene products of v-mil and v-myc function independently of each other and that v-mil is necessary for the maintenance of autocrine growth, whereas v myc is required to maintain the transformed phenotype. PMID- 3017699 TI - Transformation of quail embryo fibroblasts by a retrovirus carrying a normal human c-myc gene. AB - We have constructed avian retroviruses expressing the human c-myc oncogene. These viruses morphologically transformed primary quail embryo fibroblasts upon transfection and infection. Transformed cells produced viruses harboring a spliced c-myc gene and contained high levels of p64-67c-myc protein. One of these infectious viruses, vSX-AHM, was molecularly cloned and the nucleotide sequence of the spliced c-myc insert determined. No mutation was found within the c-myc coding sequence of this transforming clone when compared to the normal genomic progenitor. Thus, we concluded that no mutation within the human c-myc gene is required to induce primary avian embryo fibroblast transformation. PMID- 3017700 TI - The phenotypic characteristics of simian sarcoma virus-transformed human fibroblasts suggest that the v-sis gene product acts solely as a PDGF receptor agonist in cell transformation. AB - Previous studies have indicated that the oncogene v-sis of simian sarcoma virus (SSV) encodes a growth factor that is structurally and functionally similar to platelet-derived growth factor (PDGF). In the present investigation we have analysed the phenotypic characteristics of human foreskin fibroblasts transformed by SSV. It was found that the PDGF receptors were extensively down-regulated. This finding is consistent with a high, local, extracellular concentration of a PDGF-like factor, synthesized by the transformed cell. The receptors were up regulated by suramin, a drug that is known to dissociate PDGF and the v-sis product from the PDGF receptors. A cell-associated v-sis product of mol. wt 24,000 was identified by immunoprecipitation with PDGF antibodies; release of this component was induced by a high concentration of exogenous PDGF, indicating that a fraction of the product is associated with the PDGF receptors. SSV was not found to be an immortalizing virus; when serially passaged, SSV-transformed cells had essentially the same life-span as their non-transformed counterparts. Moreover, SSV did not induce growth in soft agar beyond the level afforded by exogenously added PDGF. Thus, the present study favors the notion that SSV transformation is mediated by a growth factor that mimics PDGF but has no further cellular effects. PMID- 3017701 TI - The transmembrane segment of the human transferrin receptor functions as a signal peptide. AB - The human transferrin receptor (TR) is a protein comprising 760 amino acid residues that spans the membrane once with its N terminus towards the cytoplasm. It is synthesized without a cleavable signal peptide. We have tested whether the signal responsible for its membrane insertion is present within its transmembrane peptide using a combined recombinant DNA/in vitro translation approach. The complete TR coding region was first reconstructed from overlapping TR cDNA clones and then engineered into an SP6-based transcription vector. In vitro transcription and subsequent translation in the presence of rough microsomes yielded TR molecules that were glycosylated and correctly inserted into the membrane. Two kinds of experiments demonstrated that the spanning region of the TR polypeptide contained the signal for translocation across the membrane of the rough endoplasmic reticulum. First, we deleted the spanning region of TR and showed that this deletion mutant could not be inserted. Second, we showed that two cytoplasmic proteins (the mouse dihydrofolate reductase and the chimpanzee alpha-globin) could be inserted into the microsomal membrane in the expected orientation when the TR transmembrane segment was added to their N termini. Thus, the spanning peptide was shown to be both necessary and sufficient for chain translocation. Further analyses demonstrated that the translocation event was dependent on the signal recognition particle. PMID- 3017702 TI - Reinitiation of translocation in the Semliki Forest virus structural polyprotein: identification of the signal for the E1 glycoprotein. AB - The biosynthesis of the Semliki Forest virus (SFV) structural proteins provides an interesting model system to study the reinitiation of translocation of membrane proteins into the endoplasmic reticulum membrane. The two transmembrane spike proteins, p62 and E1, are derived from a single polypeptide precursor. Once the first protein, p62, has been anchored and its cytoplasmic tail has been synthesized, translocation must be reinitiated to account for the insertion of the E1 protein. We have used deletion mutagenesis of the SFV cDNA to investigate the requirements for this event and map in detail the location of the signal. We have shown by deleting the region encoding the p62 signal and expressing the modified cDNA in COS cells that the p62 protein is not involved in the translocation of the E1 protein. The E1 signal was precisely mapped by progressive truncations of the 6 K peptide (located between p62 and E1 in the SFV polyprotein) and subsequent analysis in cell-free systems. A segment within the last 26 residues of the 6 K peptide was shown to be essential for translocation. This segment was then fused to the N-terminus of the chimpanzee alpha-globin and was shown to be sufficient for translocation. The E1 signal was cleaved efficiently even when attached to the alpha-globin protein. The activity of the signal was found to be SRP dependent in a wheat-germ cell-free system. We conclude that prior attachment of the ribosome to the membrane via the p62 signal peptide is not necessary for E1 translocation and that the reinitiation of translocation is mediated by an independent internal signal likely to be SRP dependent. PMID- 3017703 TI - Idiotypic selection of an antibody mutant with changed hapten binding specificity, resulting from a point mutation in position 50 of the heavy chain. AB - Somatic mutation occurs at a low rate in the rearranged antibody V region genes of the hybridoma line B1-8. delta 1 which expresses an antibody with specificity for the hapten 4-hydroxy-3-nitro-5-iodo-phenylacetyl (NIP). A mutant was selected which had lost a binding site-related idiotope but retained most of its other idiotypic determinants. The mutant had concomitantly lost NIP binding and acquired specificity for dinitrophenylated bovine serum albumin. It carried a single point mutation in position 50 of the heavy chain, resulting in the replacement of an arginine by a glycine. PMID- 3017705 TI - Normal T cell development is possible without 'functional' gamma chain genes. AB - The T cell-specific gamma gene family is organized into four V, J and C gene segments containing clusters (gamma 1, gamma 2, gamma 3, gamma 4) in germline DNA. We found that the V, J and C elements of gamma 2 are physically linked on a stretch of 6 kb of DNA while those of gamma 3 are found within a 15-kb region. Rearrangements take place only within the clusters, explaining the rigid rearrangement patterns seen in T lymphocytes. New V gamma, J gamma and C gamma gene segments were discovered and characterized allowing the better understanding of the potential germline diversity of the gamma gene family. No correlation with T cell function, i.e. cytolytic or helper, and the type of the productive gamma rearrangement could be established. In contrast we found that functional T cell clones have been able to mature without any functional gamma chain genes. PMID- 3017704 TI - Structure of the IgG-binding regions of streptococcal protein G. AB - The gene encoding the IgG-binding protein G from Streptococcus G148 was isolated by molecular cloning. A subclone containing a 1.5-kb insert gave a functional product in Escherichia coli. Protein analysis of affinity-purified polypeptides revealed two gene products, both smaller than protein G spontaneously released from streptococci, but with identical IgG-binding properties. The complete nucleotide sequence of the insert revealed a repeated structure probably evolved through duplications of fragments of different sizes. The deduced amino acid sequence revealed an open reading frame extending throughout the insert, terminating in a TAA stop codon. Analysis of the two gene products by N-terminal amino acid determination suggests that two different TTG codons are recognized in E. coli for initiation of translation to yield the two products. Based on these results several truncated gene constructions were expressed and analysed. The results suggest that the C-terminal part of streptococcal protein G consists of three IgG-binding domains followed by a region which anchors the protein to the cell surface. Structural and functional comparisons with streptococcal M protein and staphylococcal protein A have been made. PMID- 3017706 TI - Characterization of a human gene inducible by alpha- and beta-interferons and its expression in mouse cells. AB - An intact interferon-inducible gene has been isolated from a cosmid library of human genomic DNA. The gene (designated 6-16) encodes a mRNA of approximately 1 kb which is induced well by alpha- and beta- but poorly by gamma-interferons. Genomic and cDNA sequences indicate that the gene contains five exons, and that the mRNA encodes a hydrophobic polypeptide of 130 amino acids with a putative NH2 terminal signal sequence. The 5' end has been identified by primer extension. The corresponding genomic DNA contains a TATA box 20 nucleotides upstream of the putative transcription initiation site. After transfection of the human genomic cosmid into mouse Ltk- cells, human 6-16 mRNA is expressed in response to mouse alpha- and beta- but not gamma-interferons with the same kinetics and dose response as in the human cells. No such expression is observed in response to human interferons. It can be concluded that the human cosmid DNA contains all of the sequences necessary for alpha- and beta-interferon-induced gene expression and that the mechanisms governing such expression are conserved between murine and human cells. PMID- 3017707 TI - Transcriptional regulation of interferon-responsive genes is closely linked to interferon receptor occupancy. AB - We show that human glioblastoma cells, moving from exponential growth into a state of density-dependent growth arrest, demonstrate a 7-fold drop in the total number of alpha-IFN-receptors expressed per cell. This loss of receptor activity was not seen when cells were grown in the presence of anti-alpha-IFN-monoclonal antibody. The active binding sites expressed on the arrested cell population were of reduced affinity for IFN, relative to the high-affinity sites expressed on the growing cells, resulting in a 3-fold lower initial rate of IFN-binding to the arrested cells. We exploited this difference to investigate the relationship between IFN receptor binding and induced gene transcription. As determined by nuclear run-off assays, the transcriptional response of both the gene family 1-8 and 2-5A synthetase to IFN treatment also showed a 3-fold reduction in density arrested cells. Longer-term (0-8 h) induction and down-regulation of gene transcription in IFN-treated cells closely followed the binding to, and down regulation of, cell surface-localized IFN receptors. Furthermore, inhibition of the intracellular breakdown of IFN did not affect transcriptional responses to IFN. Thus transcription of these IFN-induced genes is closely linked to surface receptor occupancy and is most likely mediated by transmembrane signals alone. PMID- 3017708 TI - Mutations in conserved intron sequences affect multiple steps in the yeast splicing pathway, particularly assembly of the spliceosome. AB - Yeast introns contain three highly conserved sequences which are known to be required for splicing of pre-mRNA. Using in vitro mutagenesis, we have synthesized seven point mutations at five different sites in these signals in the yeast actin intron. The mutant introns were then inserted into each of three constructs, which allowed us to assess the consequences both in vivo and in vitro. In virtually every case, we found the efficiency of splicing to be significantly depressed; mature mRNA levels in vivo ranged from 0 to 47% of wild type. Surprisingly, the tightest mutations were not necessarily at the sites of nucleolytic cleavage and branch formation; these nucleotides are thus highly preferred, but are not absolutely necessary. Moreover, while particular nucleotides are specifically required for the final step in splicing, i.e. 3' cleavage and exon ligation, the predominant consequence of mutation within the conserved signals appears to be the inhibition of assembly of the splicing complex. PMID- 3017709 TI - Structural models for non-helical DNA. AB - Structural modelling techniques are employed to explore the energetic requirements for the transformation of classical B DNA into unwound yet double stranded DNA structures. Structural idealization using CORELS computer program of Sussman et al. followed by energy minimization using the EREF program of Levitt, leads to two regular non-helical models. In both models, the bases are conventionally paired and stacked, yet there is no net rotation between successive base pairs. One model, N1, has a 1-bp repeating unit; the second, N2, has a 2-bp repeating unit. The dihedral angles of the backbone all have values found either in the B or the Z form of DNA, except for the P-O5'-C5'-C4' angle, which is in the unprecedented g+ or g- domains. The energy difference found between the two N form models and B form DNA are 6.6 and 3.4 kcal/mol/nucleotide for N1 and N2 respectively. These relatively low energy differences encourage the idea that non-helical forms of DNA may contribute to the alternate DNA structures found in S1 nuclease sensitive and other regulatory regions of active genes. PMID- 3017712 TI - Calcium transport in membrane vesicles of Streptococcus cremoris. AB - Rightside-out membrane vesicles of Streptococcus cremoris were fused with proteoliposomes containing the light-driven proton pump bacteriorhodopsin by a low-pH fusion procedure reported earlier [Driessen, A.J.M., Hellingwerf, K.J. & Konings, W.N. (1985) Biochim. Biophys. Acta 808, 1-12]. In these fused membranes a proton motive force, interior positive and acid, can be generated in the light and this proton motive force can drive the uptake of Ca2+. Collapsing delta psi with a concomitant increase in delta pH stimulates Ca2+ uptake while dissipation of the delta pH results in a reduced rate of Ca2+ uptake. Also an artificially generated delta pH, interior acid, can drive Ca2+ uptake in S. cremoris membrane vesicles. Ca2+ uptake depends strongly on the presence of external phosphate while Ca2+-efflux-induced proton flux is independent of the presence of external phosphate. Ca2+ accumulation is abolished by the divalent cation ionophore A23187. Calcium extrusion from intact cells is accelerated by lactose. Collapse of the proton motive force by the uncoupler carbonylcyanide p trifluoromethoxyphenylhydrazone or inhibition of the membrane-bound ATPase by N,N'-dicyclohexylcarbodiimide strongly inhibits Ca2+ release. Further studies on Ca2+ efflux at different external pH values in the presence of either valinomycin or nigericin suggested that Ca2+ exit from intact cells is an electrogenic process. It is concluded that Ca2+ efflux in S. cremoris is mediated by a secondary transport system catalyzing exchange of calcium ions and protons. PMID- 3017710 TI - DNA conformation and chromatin organization of a d(CA/GT)30 sequence cloned in SV40 minichromosomes. AB - The alternating DNA sequence d(CA/GT)n is known to adopt the left-handed Z-form in negatively supercoiled DNA in vitro. This element represents a significant fraction of the highly repeated DNA sequences found in eukaryotic genomes. We have cloned an alternating d(CA/GT)30 sequence into SV40 minichromosomes at the unique HpaII restriction site which occurs in the transcriptional leader region of the viral late genes. By comparing the linkage differences of topoisomers obtained from viral DNA with or without the d(CA/GT)30 insert, at various stages of the lytic cycle, we conclude that this sequence does not predominate in the Z form in vivo. Furthermore, we find that the d(CA/GT)30 sequence is packaged into nucleosomal core particles and the region of the minichromosomes which contain the d(CA/GT)30 sequence is organized as nucleosomes. PMID- 3017711 TI - Infection with human T-lymphotropic virus type III and leukocyte interferon production in homosexual men. PMID- 3017713 TI - Membrane-bound hydroxylases in elicitor-treated bean cells. Rapid induction of the synthesis of prolyl hydroxylase and a putative cytochrome P-450. AB - Treatment of cell-suspension cultures of bean (Phaseolus vulgaris cv. Canadian Wonder) with an elicitor preparation heat-released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum resulted in rapid changes in the activities of two microsomal oxygenases, cinnamic acid 4-hydroxylase, involved in accumulation of wall-bound phenolics and phytoalexins, and proline 2 oxoglutarate dioxygenase (prolyl hydroxylase) involved in the post-translational modification of hydroxyproline-rich glycoproteins. An anti-(cytochrome P-450) monoclonal antibody, originally raised against rat cytochrome P-450 isoform c, has been shown to bind to bean microsomes and recognise in Western blots an Mr 48,000 polypeptide, which comigrates with a haeme-containing protein on SDS/polyacrylamide gel analysis and which has been tentatively identified as a cytochrome P-450 capable of the hydroxylation of cinnamic acid. A preparation of proline 2-oxoglutarate dioxygenase purified to homogeneity was used to immunise rabbits for the production of antiserum. An elicitor-induced polypeptide of Mr 65,000 was identified as prolyl hydroxylase while an antigenically related polypeptide of Mr 60,000 was also immunoprecipitated but not induced by elicitor treatment. Use of the two antibodies has demonstrated rapid transient increases in the synthesis of the Mr 48,000 and Mr 65,000 oxygenases in vivo and for mRNAs as measured in in vitro translations, particularly for the putative cytochrome P 450. These increases slightly precede corresponding changes in the synthesis of the soluble enzyme phenylalanine ammonia-lyase, in common with which these oxygenases probably share a mechanism of gene activation underlying the increased activities seen in response to elicitor treatment. PMID- 3017714 TI - Isolation and characterization of mutants from Schyzosaccharomyces pombe defective in glycerol catabolism. AB - Mutants unable to grow on glycerol were isolated from the fission yeast Schyzosaccharomyces pombe. Two types of mutants were obtained: one type was able to grow on dihydroxyacetone while the other one did not grow on this compound. The first type of mutants was defective in glycerol dehydrogenase while the second one was affected both in the glycerol dehydrogenase and in dihydroxyacetone kinase. It was found that the second type was defective in the derepression of several enzymes. The mutations were nuclear and monogenic and defined two complementation groups. Spontaneous revertants, able to grow on glycerol, were obtained from the first type of mutants. They have regained the glycerol dehydrogenase activity. The results presented provide genetic evidence for a pathway of glycerol catabolism in Sch. pombe involving dehydrogenation of glycerol as the first step followed by phosphorylation of the dihydroxyacetone formed. PMID- 3017715 TI - Tarantula hemocyanin mRNA. In vitro translation, cDNA cloning and nucleotide sequence corresponding to subunit e. AB - Following induction of hemopoiesis, poly(A)-rich RNA was prepared from the heart of the tarantula, Eurypelma californicum, and translated in rabbit reticulocyte lysates. In vitro translation products were immunoprecipitated with antiserum against whole dissociated Eurypelma hemocyanin. Analysis of the immunoprecipitate by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed a set of polypeptides comigrating with authentic Eurypelma hemocyanin. The mRNA was transcribed into cDNA, clones were constructed using the pUC9 vector and probed with a synthetic 17-mer oligonucleotide probe complementary to the amino acid sequence of the 'copper A' binding site of chelicerate hemocyanins. One clone, pHC4, contained a 1.62-kb cDNA insert, which was subcloned into phage M13. Sequence analysis by the dideoxynucleotide chain-termination method yielded a nucleotide sequence coding for 526 amino acids of Eurypelma hemocyanin subunit e. PMID- 3017716 TI - The effects of pressure on yeast cytochrome c peroxidase. AB - The effects of pressure on cytochrome c peroxidase [CcP(FeIII)], its cyano derivative (CcP X CN) and its enzyme-substrate complex (ES) have been studied. The effects of pressure on the binding of the substrate analog porphyrin cytochrome c (porphyrin c) to CcP X CN and ES have also been studied. High pressure causes CcP(FeIII) to undergo a high-spin to low-spin transition but has no detectable effect on either CcP X CN, which is already low spin, or on ES. The low-spin CcP(FeIII) structure at pressure is similar to the low-spin form at low temperature and the low-spin form of horseradish peroxidase at high pressure. delta V degree associated with the spin equilibrium is about 30 ml/mol and is independent of temperature. delta G degree is small, 4.7 kJ/mol at 0 degree C, while delta H degree is 14.2 kJ/mol at 1 bar (100 kPa). Pressure has no detectable effect on the binding equilibria of mixtures of CcP X CN plus porphyrin c or ES plus porphyrin c. This indicates that the interaction of CcP and porphyrin c results in little or no volume change; the same is true in the case of cytochrome c oxidase and porphyrin c. PMID- 3017717 TI - Interaction of vasoactive intestinal peptide (VIP) and N-terminally modified VIP analogs with rat pancreatic, hepatic and pituitary membranes. AB - Six vasoactive intestinal peptide (VIP) analogs inhibited [125I]iodo-VIP and [125I]iodo-helodermin binding to high-affinity VIP receptors in rat hepatic membranes. They also stimulated adenylate cyclase activity through these receptors, their decreasing order of potency being VIP greater than [D-Ala4]VIP greater than [D-Asp3]VIP greater than [D-Ser2]VIP greater than [D-His1]VIP greater than [D-Phe2]VIP greater than [D-Arg2]VIP, with the latter two peptides acting as partial agonists only. All VIP analogs tested on rat pancreatic membranes were able to stimulate adenylate cyclase, their order of potency being very similar to that observed on hepatic membranes. [D-Ser2]VIP, [D-His1]VIP, [D Arg2]VIP and [D-Phe2]VIP were partial agonists with an intrinsic activity of, respectively, 0.8, 0.7, 0.35 and 0.09 as compared to that of VIP = 1.0. [D Phe2]VIP competitively and selectively inhibited VIP-stimulated adenylate cyclase activity (Ki = 0.1 microM). On male rat anterior pituitary homogenates the order of potency of the peptides was VIP greater than [D-Ala4]VIP greater than [D Asp3]VIP greater than [D-Ser2]VIP greater than [D-His1]VIP. [D-Ser2]VIP and [D His1]VIP acted as partial agonists. Besides, [D-Phe2]VIP and [D-Arg2]VIP were inactive as well as unable to inhibit VIP-stimulated adenylate cyclase activity. These results indicated that (a) the efficacy of VIP receptor/effector coupling depended on the tissue tested; (b) the possibility exists to design a VIP antagonist by appropriate modification in the N-terminal moiety of the molecule. PMID- 3017718 TI - Proteins from rat liver cytosol which stimulate mRNA transport. Purification and interactions with the nuclear envelope mRNA translocation system. AB - Two polysome-associated proteins with particular affinities for poly(A) have been purified from rat liver. These proteins stimulate the efflux of mRNA from isolated nuclei in conditions under which such efflux closely stimulates mRNA transport in vivo, and they are therefore considered as mRNA-transport stimulatory proteins. Their interaction with the mRNA-translocation system in isolated nuclear envelopes has been studied. The results are generally consistent with the most recently proposed kinetic model of mRNA translocation. One protein, P58, has not been described previously. It inhibits the protein kinase that down regulates the NTPase, it enhances the NTPase activity in both the presence and the absence of poly(A) and it seems to increase poly(A) binding in unphosphorylated, but not in phosphorylated, envelopes. The other protein, P31, which probably corresponds to the 35,000-Mr factor described by Webb and his colleagues, enhances the binding of poly(A) to the mRNA-binding site in the envelope, thus stimulating the phosphoprotein phosphatase and, in consequence, the NTPase. The possible physiological significance of these two proteins is discussed. PMID- 3017719 TI - Conformational analysis of the octamer d(G-G-C-C-G-G-C-C) in aqueous solution. A one-dimensional and two-dimensional proton NMR study at 300 MHz and 500 MHz. AB - Proton NMR studies in H2O and 2H2O were carried out on the self-complementary DNA octamer d(G-G-C-C-G-G-C-C) and compared with similar studies on the dimethylated analogue d(G-G-m5C-m5C-G-G-C-C) [Sanderson, M. R., Mellema, J.-R., van der Marel, G. A., Wille, G., van Boom, J. H. & Altona, C. (1983) Nucleic Acids Res. 11, 3333 3346]. Base, H1', H2' and H2" resonances were assigned by means of two dimensional nuclear Overhauser spectroscopy (NOESY) and correlated spectroscopy (COSY) experiments. Chemical shift vs temperature profiles were used to analyze the temperature-dependent conformational behaviour and to extract thermodynamic parameters for the duplex-to-coil transition. Analysis of proton-proton couplings were used to discriminate between J1'2' and J1'2" and between the H2' and H2" resonances as well as to obtain conformational parameters of the sugar rings. From the NOESY and COSY experiments, the temperature profiles and the analysis of the coupling data it is concluded that: (a) the compound adopts a B-DNA-type helix in solution; (b) the sugar rings in the intact duplex display limited conformational freedom; (c) methylation of the cytosine bases at the C5 position has no measurable effect on the conformational behaviour of the octamer, nor on the conformation of the sugar rings; however, methylation does increase the stability of the duplex in aqueous solution under conditions of low salt concentration. PMID- 3017720 TI - [Attitude to the disease model and self-help groups as predictors of participation in after care and of therapeutic outcome in alcoholic patients]. AB - The study was conducted to determine the predictive value of attitudes towards the goals derived from the disease model of alcoholism (life-long acceptance of alcoholic status and endorsement of abstinence) as well as towards self-help groups. The criteria to be predicted were the intention to participate and actual participation in self-help groups as well as four indicators of therapeutic success. Only in cross-sectional analysis were significant correlations found. Longitudinally, there were no relevant relationships between the attitudes expressed by the patients at the end of inpatient treatment (t1) and subsequent participation in self-help groups or indicators of therapeutic success (t2: follow-up, 9 month after discharge). The discrepancy between cross-sectional and longitudinal analysis can be attributed to the fact that attitudes become increasingly less stable with time. This indicates that it is only possible to a limited extent to predict from the attitudes expressed during inpatient treatment how patients will actually behave in daily life outside the clinic. The consequences of these findings for therapy are discussed. PMID- 3017721 TI - Lactate dehydrogenase as a marker for testicular germ-cell tumours. AB - One hundred and eighty-four patients with testicular germ-cell tumours, 92 with seminoma and 92 with non-seminomatous germ-cell tumours (NSGCT) had total lactate dehydrogenase (LD) assay performed at the time of initial staging following orchidectomy. The proportion of patients with elevated plasma LD and the mean plasma LD increased with advancing stage and increasing tumour bulk for both seminoma and NSGCT. Of patients receiving cytotoxic chemotherapy, 87.5% with seminoma and 63% with NSGCT had elevated plasma LD with subsequent levels reflecting regression or progression of disease. Elevated plasma LD levels were seen in four of seven seminoma, and in 18 of 30 NSGCT patients relapsing after primary treatment. The LD assay provides useful information, and complements the routine measurement of alphafetoprotein (AFP) and beta human chorionic gonadotrophin (beta hCG) in the management of patients with testicular germ-cell tumours. PMID- 3017722 TI - Etoposide, cisplatin and doxorubicin in patients with small cell lung cancer: tumor response and long term survival. AB - One hundred and fourteen patients with small cell lung cancer received a combination of etoposide 80 mg/m2 IV days 1,2,3,15,16,17, cisplatin 20 mg/m2 IV days 1,2,3 and doxorubicin 40 mg/m2 IV day 1, repeated every 4 weeks. The observation time from the initiation of treatment is longer than 4 years for all patients. An 85% response rate (38% complete response) was obtained after 1-4 cycles in 105 evaluable patients (96 without prior antitumour treatments). The response rate was not influenced by initial performance status and minimally by disease extension, whereas the same prognostic factors correlated significantly with complete responses. For limited disease and WHO performance 0 all responses were complete. For extensive disease and performance 3 no complete response was obtained. Various chemotherapy regimens with or without radiotherapy were used during the maintenance phase. Forty five per cent of first relapses were in the lung or mediastinum and 18% in the nervous system (20% without and 7% with prophylactic cranial irradiation). The median survival was 13.4 months for limited and 8.5 months for extensive disease, and correlated with performance and response. Only 2 patients survived free of disease after 4.8 and 5.5 years. We conclude that the induction treatment as used here produces a high rate of partial and complete responses but a low rate of long survivors. PMID- 3017724 TI - Effect of age on the formation of cyclic nucleotides in guinea-pig tracheal smooth muscle in response to pharmacological agents. AB - The effects of isoproterenol, carbachol and other drugs on the cyclic AMP and cyclic GMP levels in tracheal smooth muscles of guinea-pigs of four different ages were investigated. Isoproterenol increased the cyclic AMP level several-fold in tracheal muscles from newborn (1 week) and young (4-7 weeks) guinea-pigs, but it caused less increase in the level in muscles from middle-aged (12 weeks) and old (20-24 weeks) guinea-pigs, although the basal cyclic AMP level at these ages was not significantly lower. The effects of prostaglandin E1 and cholera toxin in increasing the cyclic AMP level were also markedly less in muscle preparations from old guinea-pigs than in those from young ones. The increase in cyclic AMP levels caused by forskolin, an activator of adenylate cyclase did not decrease with age. Carbachol caused a 3- to 4-fold increase in the cyclic GMP level in muscle preparations from newborn and young guinea-pigs and more increase in the cyclic GMP level in preparations from middle-aged and old guinea-pigs. The increases in cyclic GMP level induced by high K+, histamine and sodium nitroprusside also increased with the age of the animals. These results suggest that the changes in the formation of cyclic AMP and cyclic GMP induced by various agents are due to changes at the post-receptor level. PMID- 3017723 TI - Enalapril reduces the catecholamine response to exercise in patients with heart failure. AB - Increased sympathetic activity occurs in congestive heart failure and may have deleterious effects. To examine the effects of angiotensin converting enzyme (ACE) inhibition on sympathetic activity in heart failure, the noradrenaline response to exercise was measured in 18 patients given enalapril or placebo in double-blind trial. The plasma noradrenaline response to graded exercise was significantly reduced after 4 weeks on active treatment but was unchanged by placebo treatment. The rate-pressure product at maximal exercise was significantly reduced after 4 weeks in the enalapril group but unchanged in the placebo group. These results suggest that ACE inhibition reduces sympathetic activity in patients with heart failure. PMID- 3017725 TI - Mechanism of the respiratory action of pentobarbital at the medullary and pontine levels. AB - A functional differentiation of the respiratory action of pentobarbital was made by applying the drug in cats to the ventral surface of the medulla and to the dorso-rostral surface of the pons. The involvement of a GABAergic mechanism was assessed by studying the interaction of bicuculline with pentobarbital at both levels of the brain-stem. In the medulla, pentobarbital (4 to 16 mg) caused an immediate and dose-dependent depression of tidal volume down to apnea with a minimal or no change in frequency. In the pons, the depression affected selectively the frequency (-45%) without modification in amplitude. Medullary structures were much more sensitive to the action of pentobarbital. Doses that induced apnea in the medulla did not attain 50% depression of the minute volume in the pons. Bicuculline (300 micrograms) completely antagonized the effect of pentobarbital in both regions, suggesting that a GABAergic mechanism may be involved in the respiratory action of pentobarbital. PMID- 3017726 TI - Distribution and coregulation of three peptide receptors in adrenals. AB - Receptors sites for angiotensin II and atrial natriuretic factor were concentrated in the zone glomerulosa of the adrenal cortex in the rat, mouse, hamster, rhesus monkey, guinea-pig and the cow. Angiotensin Ii receptor sites were also accumulated in adrenal medullary tissue of the rat, mouse and the hamster. With exception of the cow the zonae fasciculata and reticulata of the adrenal cortex were not labelled with the angiotensin II ligand, but showed a moderate density of atrial natriuretic factor receptor sites in all species except the rhesus monkey. In comparison, somatostatin receptors were even more heterogeneously distributed in all species mentioned above. In the rat, the increased growth of zona glomerulosa cells found after three weeks of sodium deprivation was accompanied by a similar increase in number of receptor sites for angiotensin II, atrial natriuretic factor and somatostatin. This shows that all three peptide receptors are regulated simultaneously by a single metabolic disturbance, suggesting that they might be localized on the same cell type in the adrenal cortex. PMID- 3017727 TI - Evidence from behavioural and in vitro receptor binding studies that the enkephalinergic system does not mediate acute ethanol effects. AB - The behavioural disturbances produced by acute exposure to ethanol have been related to changes in function of the opioid systems in the CNS. However, evidence in the literature is conflicting. The present report concerns the possible role of the enkephalinergic system in the mediation of acute ethanol effects. We used rats to study the ability of a selective opioid delta receptor antagonist (ICI 154129) to prevent the effect of ethanol on pain sensitivity, body temperature, sensorimotor performance and level of consciousness. Furthermore, in vitro receptor binding was measured to investigate whether or not ethanol, within a non-lethal concentration range, would change the binding parameters of the delta receptor ligand [3H][D-ala2, D-Leu5]enkephalin. ICI 154129 did not significantly influence the effects of ethanol in the behavioural experiments. Ethanol did not significantly change the binding parameters whether saturation or competition was measured in the receptor binding experiments. Thus, there was no evidence that the enkephalinergic system mediated the acute ethanol effects. PMID- 3017728 TI - Evidence for differing leukotriene receptors in gastric mucosa. AB - Exogenously administered LTD4 was found to increase pepsin secretion from and decrease the transgastric electrical potential difference across the stomachs of anesthetized cats. The former response was completely blocked by prior administration of a new and selective LTD4 antagonist, L-649,923, while the potential difference response was unaffected. This result, coupled with previous evidence of a difference in leukotriene potency order for the two responses, suggests the presence of functionally different leukotriene receptor subtypes in the gastric mucosa. PMID- 3017729 TI - Cannabidiol attenuates delta 9-tetrahydrocannabinol-like discriminative stimulus effects of cannabinol. AB - Interaction effects of cannabidiol (CBD) and cannabinol (CBN) were examined using a drug discrimination procedure in which rats had been trained to discriminate between 3 mg/kg of delta 9-tetrahydrocannabinol (delta 9-THC) and vehicle (2 ml/kg). The generalization effects of CBN to delta 9-THC were attenuated when CBN was administered together with CBD. The results were time- and dose-dependent. PMID- 3017730 TI - Angiotensin II receptor binding in the rat nucleus tractus solitarii is reduced after unilateral nodose ganglionectomy or vagotomy. PMID- 3017731 TI - Adrenal function in the Mongolian gerbil (Meriones unguiculatus): influence of confinement stress upon glucocorticosteroid, progesterone, dehydroepiandrosterone, testosterone and androstenedione plasma levels, adrenal content and in-vitro secretion. AB - The pattern of adrenal steroid secretion and the response to confinement stress were investigated in male Mongolian gerbils (Meriones unguiculatus). Steroid levels of glucocorticosteroids (GC), progesterone (P), dehydroepiandrosterone (DHEA), testosterone (T) and androstenedione (A) in plasma, adrenal tissue and superfusates of adrenals superfused in-vitro were measured by radioimmunoassay. The sensitivity of the assay systems and the low cross-reactivity of the antisera used allowed the determination of steroid levels in small samples (5-200 microliters), without prior chromatography. GC plasma levels were much higher than values of P, DHEA, T or A (176.7, 2.4, 3.3, 2.6 and 2.8 ng/ml, respectively). Confinement stress resulted in a significant increase of GC and DHEA plasma levels; similarly, adrenal content of GC, DHEA and P was markedly increased. In contrast, the applied stress factor had no significant effects on either plasma levels of P, T or A or on adrenal T or A content. Compared to plasma levels or adrenal content, amounts of steroids secreted from adrenals superfused in-vitro were very low (GC: 57.1, P: 2.1, DHEA: 23.0, T: 1.8, A: 3.0 pg/mg/min, respectively). Confinement stress significantly stimulated GC, P and DHEA secretion in-vitro but had no effects on T or A release. The secretion of GC, P, DHEA and T, but not of A was significantly increased by in-vitro stimulation with 0.01-10.0 mIU (1-24) ACTH. Interestingly, the amounts of GC and P, and of GC and DHEA secreted from incubated adrenal slices stimulated with (1 24) ACTH and from adrenals of controls and stressed gerbils superfused in-vitro were significantly correlated. By measuring steroid plasma levels and profiles of steroids secreted from adrenocortical and testicular tissue it now seems possible to characterize in more detail the effects of chronic intermittent stress upon the adrenalgonadal axis and the possible interrelationship between glucocorticosteroid and androgen secretion, especially in small laboratory animals from which only limited amounts of blood can be obtained repeatedly. PMID- 3017732 TI - ACTH secretory responsiveness of pituitary adrenotroph cell tumor to adrenocorticotropin-releasing factor in Cushing's disease and Nelson's syndrome. AB - ACTH-secretory responsiveness of the adrenotroph cell tumors to the exogenous CRF was evaluated in 4 cases of Cushing's disease and 3 cases of Nelson's syndrome. Significantly exaggerated ACTH release was evoked by the intravenous injection of CRF in all 3 cases of Nelson's syndrome, but in Cushing's disease there seemed to be an individual difference in the sensitivity of the pituitary to CRF, probably depending on the functional activity of hypothalamo-pituitary axis. PMID- 3017733 TI - The human insulin gene and diabetes mellitus. AB - The recombinant DNA technology is a useful tool to characterize the insulin gene and adjacent areas. A highly polymorphic region near the human insulin gene was detected and its possible relation to certain types of diabetes mellitus is discussed. In three cases the synthesis of a structurally abnormal insulin of lower biological activity leads to hyperglycemia. These mutant insulin gene sequences can be identified with the help of restriction endonuclease cleavage analysis. Insulin gene expression is regulated mainly at translational level. Elements near or within the insulin gene seem to be required for a sufficient and cell-specific expression. But the role of the polymorphic locus is not yet clear. First results on gene transplantation by administration of liposome entrapped insulin gene sequences are surprising, but at this time only of speculative value for medical research. PMID- 3017734 TI - Phorbol ester-induced surface transferrin receptor modulation. No correlation with decreased cell proliferation. AB - Both phorbol ester or diacylglycerol (DAG) reduce cell surface transferrin receptor (TFR) number in CEM cells (a human T-cell acute lymphoblastic leukemia line) and HL-60 cells (a human promyelocytic leukemia cell line). This effect occurs with a t1/2 of approx. 30 min and is mimicked by addition of phospholipase C to cell cultures. Although cell surface TFR number is reduced to 25-30% of the control level 5 h after phorbol ester administration, apparent cell proliferation (as measured by [3H]thymidine incorporation) remains unaffected. Although independent of extracellular calcium (EGTA is slightly enhancing), the phenomenon is completely blocked by 30-min pretreatment with the calcium channel blocker diltiazem. Dilitazem pretreatment, while preventing receptor redistribution, does not completely block the phorbol ester-induced increase in TFR phosphorylation thought to be associated with receptor redistribution. Thus, calcium channel blockade effectively dissociates the effects of tetradecanoylphorbol acetate (TPA) on TFR internalization and phosphorylation. Our results also demonstrate that phorbol ester-induced effects on the TFR can be mimicked by the endogenous stimulator of protein kinase C, DAG, whether added directly to cultures or produced by the cells in response to exogenous phospholipase C. Furthermore, the phenomenon of TFR redistribution here described is not associated with a decreased proliferative capacity. PMID- 3017736 TI - Converting Sendai virus into a specific fusogen whose cell target can be selected. AB - Covalent intermolecular hybrids of Fab anti-hemagglutinin-neuraminidase (HN) monoclonal antibody and avidin were prepared and characterized. These conjugates were used to block and redirect the fusion activity of Sendai virus (SV). After incubation of SV with Fab anti-HN: avidin conjugate on ice for 1-2 h, the SV fused only those P815 or BW5147 cells which were labeled with biotin-modified anti-cell surface immunoglobulin. The levels of cell-cell fusion obtained were at least as high as those achieved with unmodified SV and unlabeled P815 or BW5147 cells. These results demonstrate that it is possible to block the normal agglutinating activity of the HN molecules of SV and to introduce a new cell recognition feature without negating the fusogenic potential of the virus. Such an approach may be useful in harnessing the fusion activity of SV to a targeted delivery system for microinjection of macromolecules into selected cell populations. PMID- 3017735 TI - The effect of cAMP on tumour promoter responses mediated by C-kinase. AB - The phorbol ester tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induced several rapid changes in the HEL-37 mouse epidermal cell line. These included an alteration in cell morphology, inhibition of cell-cell communication, inhibition of epidermal growth factor (EGF) binding and a stimulation of phosphatidylcholine (PC) synthesis. The synthetic diacylglycerol sn 1-oleoyl-2 acetylglycerol (OAG) and sn 1,2-dioctanoylglycerol (diC8) caused similar changes, implying an involvement of the Ca2+- and phospholipid-dependent protein kinase (C kinase). Treatment of the cells with the cAMP-generating agents db-cAMP and isoproterenol together with the phosphodiesterase inhibitors aminophylline and isobutyl-methylxanthine (IBMX) prior to and during TPA, OAG or diC8 treatment protected the cells against the inhibition of both junctional communication and EGF binding. TPA-induced morphological changes and enhanced PC synthesis, however, were unaffected by elevated levels of intracellular cAMP. These experiments provide evidence for the existence of a dual regulatory system controlling some (but not all) tumour promoter effects. PMID- 3017737 TI - Tumor promoters and diacylglycerol activate the Na+/H+ antiporter of sea urchin eggs. AB - Various tumor promoters (TPA, lyngbyatoxin and aplysiatoxin) and diacylglycerol induced cytoplasmic alkalinization of sea urchin eggs independently of intracellular Ca2+ release. This response stimulated protein synthesis and was blocked by amiloride or a lack of extracellular Na+, procedures which inhibit the Na+/H+ antiporter. These results suggest that the antiporter which is responsible for cytoplasmic alkalinization in sea urchin eggs is activated directly or indirectly by protein kinase C in a Ca2+-independent manner. PMID- 3017738 TI - Cleavage of vimentin in dense cell cultures. Inhibition upon transformation by type 5 adenovirus. AB - The analysis on two-dimensional isoelectric focusing and SDS polyacrylamide gels (2D gels) of the Triton X-100 and high salt-insoluble fraction of fibroblast cell lines, certain epithelial cell lines and granulosa cells revealed various amounts of a vimetin cleavage product, with a more basic pI and with a MW (1,500-2,000) lower than that of intact vimentin. This cleavage product of vimentin which constituted as much as 30% of the total vimentin in an established rat embryo fibroblast cell line (CREF), was detected by a monoclonal antivimentin antibody in whole cell and Triton-insoluble extracts, and it has a phosphorylated variant which can be degraded to form the "staircase pattern" on 2D gels similarly to intact vimentin. This processing of vimentin occurred mainly in dense cell cultures and it could not be induced in sparse cell cultures by inhibiting DNA synthesis with ara C, or by arresting cell growth in medium containing 0.1% serum. Transformation of CREF cells with intact wild-type (H5wt) and host-range cold-sensitive mutants (H5hr1 or H5d1101) of type 5 adenovirus (Ad5), or transformation of CREF cells by Ca2+-mediated DNA transfection with the transforming E1a (0-4.5 map units) or E1a + E1b (0-11.5 map units) region of Ad5 inhibits the cleavage of vimentin in dense cultures only at temperatures which are permissive for expression of the transformed phenotype. The transformation of cells with bovine papilloma virus type 1, with T24 ras oncogene, or with RSV does not interfere with the cleavage of vimentin. The organization of the vimentin network in dense cultures, where the vimentin cleavage occurs, is very different from that of sparse untransformed and sparse or dense Ad5-transformed cells. The possibility that the acidic amino acid-rich C-terminus of vimentin is cleaved in dense cell cultures in conjunction with the reorganization of the vimentin network and the inhibition of this cleavage by transformation with Ad5, are discussed. PMID- 3017739 TI - A polyoma-derived plasmid vector maintained episomally in both E. coli and mouse hepatoma cells. AB - We describe a recombinant plasmid, pBBPY1, containing polyoma virus sequences which persists episomally in mouse hepatoma (MH) cells and can be shuttled between these cells and bacteria. This plasmid is composed of a subgenomic fragment of a polyoma virus mutant that includes two origins of replication; sequences of plasmid pML2; the xanthine-guanine phosphoribosyltransferase gene of Escherichia coli (Ecogpt) under the control of SV40 early-region promoter and RNA processing signals, providing a dominant selectable marker for mammalian transfection. MH cells from colonies growing in HAT medium (hypoxanthine, aminopterin and thymidine) were found to contain vector DNA molecules in an episomal state, the majority of them unrearranged. When HAT-selective pressure was applied for only 3 days, the resulting cells contained up to 50-100 copies of intact plasmid, i.e. 20-fold more than cells grown under standard selection conditions with continuous HAT-selective pressure. Contrary to standard conditions, transient selection does not alter the epithelial morphology nor ability of transfected hepatoma cells to produce albumin. PMID- 3017740 TI - Heterogeneity in distribution and content of p53 in SV40-transformed mouse fibroblasts. AB - The high level and intranuclear location of the cellular phosphoprotein p53 are usually regarded as invariant features of SV40-transformed fibroblast lines. During the development of improved methods for immunocytochemical detection of p53 using SVA31 E7 mouse fibroblasts, we have observed unexpected and marked variations in its distribution. Cells grown on plastic coverslips were fixed in acetone and the content and distribution of p53 and SV40 large T-antigen analysed by an indirect immunoperoxidase procedure using monoclonal antibodies PAb122 and PAb 248 for p53, and PAb416 for large T. First, we observed in all cultures an apparent reversal of intracellular compartmentalization, with strong cytoplasmic and absent nuclear/chromatin positivity in mitotic cells and young daughter cells. More importantly, for a short period, between 18-24 h after trypsinization and cell passage, we observed a marked overall reduction in detectable nuclear p53 content in all cells with both PAb122 and PAb248 antibodies. The first observation also held for SV40 large T-antigen, the second only for p53. These variations have important practical implications for the immunocytochemical analysis of cellular content and intracellular compartmentalization of p53. The biological implications of our findings are also discussed. PMID- 3017741 TI - Active function of membrane receptors for enveloped viruses. I. Specific requirement for liposome-associated sialoglycolipids, but not sialoglycoproteins, to allow lysis of phospholipid vesicles by reconstituted Sendai viral envelopes. AB - Phospholipid liposomes composed of phosphatidylcholine (PC) and cholesterol (chol), bearing the sialoglycoprotein glycophorin (GP), are able to effectively bind Sendai virus particles, but not to be lysed by them. Incorporation of gangliosides (gangl) into the above phospholipid vesicles (yielding liposomes composed of PC/chol/gangl/GP), although not increasing their ability to interact with Sendai virions, rendered them susceptible to the viral lytic activity. This was inferred from the ability of the virus to induce release of carboxyfluorescein (CF) upon interaction at 37 degrees C with liposomes composed of PC/chol/gangl/GP. Lysis of liposomes required the presence of the two viral envelope glycoproteins, namely the hemagglutinin/neuraminidase (HN) and the fusion (F) polypeptides, and was inhibited by phenylmethyl sulfonylfluoride (PMSF), dithiothreitol (DTT) and trypsin, showing that virus-induced lysis of PC/chol/gangl/GP liposomes reflects the fusogenic activity of the virus. Incubation of Sendai virus particles with liposomes containing the acidic phospholipid dicetylphosphate (DCP) but lacking sialic acid containing receptors, also resulted in release of the liposome content. Lysis of these liposomes was due to the activity of the viral HN glycoprotein, therefore not reflecting the natural viral fusogenic activity. Fluorescence dequenching studies, using fluorescently labeled reconstituted Sendai virus envelopes (RSVE), have shown that the viral envelopes are able to fuse with neutral, almost to the same extent, as with negatively charged liposomes. However, fusion with negatively charged liposomes, as opposed to fusion with neutral liposomes, was mediated by the viral HN glycoprotein and not by the viral fusion polypeptide. PMID- 3017742 TI - Subcellular distribution of the external and internal domains of the EGF receptor in A-431 cells. AB - Using specific antibodies directed against the external and internal domains of the epidermal growth factor (EGF) receptor, we have directly localized by the protein A gold technique at the electron microscopic level these receptor regions in A-431 epidermoid carcinoma cells. With all antibodies tested, 80-85% of the EGF receptors are found inside the cells, where they preferentially associate with lysosome-like structures, a tubulovesicular system, the rough endoplasmic reticulum and the nuclear envelope. The same distribution pattern is observed for antibodies directed against the external carbohydrate region of the receptor, an antibody against the protein core of the external segment of the receptor, and an antibody reacting with the internal kinase domain of the receptor, suggesting that both receptor segments are similarly distributed intracellularly. PMID- 3017743 TI - Action of retinoic acid on the diacylglycerol-induced ornithine decarboxylase activity, reduction in EGF binding and protein kinase C activation in rat tracheal epithelial 2C5 cells. AB - We have shown previously that in rat tracheal epithelial 2C5 cells the induction of ornithine decarboxylase (ODC) activity and the reduction in the binding of epidermal growth factor (EGF) by diacylglycerol is related to the activation of protein kinase C. In this paper we analyse the action of retinoic acid (RA) on these two parameters in order to determine whether RA acts on the level of protein kinase C. RA inhibits the induction of ODC activity by diacylglycerol (sn 1,2-dioctanoylglycerol) in a dose- and time-dependent manner. A biologically inactive analog of RA has no effect on this induction. RA does not affect the activation of protein kinase C by diacylglycerol in an in vitro assay. In contrast to the effect on ODC induction, RA does not counteract the reduction in EGF binding induced by diacylglycerol. These results are consistent with the concept that RA does not act at the level of protein kinase C and inhibits ODC induction during a stage following protein kinase C activation. PMID- 3017744 TI - Endemic pleural plaques. PMID- 3017745 TI - Carbonic anhydrase activity in the normal and injured peripheral nervous system of the rat. AB - The carbonic anhydrase reactivity of primary neurons and axons of the L4 and L5 lumbar levels was studied in rats before and after various surgical procedures including transection of the spinal cord, removal of dorsal root ganglia, and transection of ventral or dorsal roots or spinal nerves. In normal animals, carbonic anhydrase reactivity was confined to large and medium size neurons of the dorsal root ganglia, and was also present in a sizeable percentage of cells scattered throughout the thoracolumbar sympathetic chain and in the celiac ganglion. At root level, enzymatic staining could be detected in 48.7% of the dorsal root myelinated axons of most sizes, whereas in ventral roots, it was restricted to small myelinated axons, in a proportion much higher at the L4 than in the L5 level. Spinal motoneurons remained unlabeled, despite procedures aimed at increasing the somal concentration of carbonic anhydrase, such as ventral root ligation and blocking of the fast or slow axoplasmic transport using colchicine or iminodiproprionitrile. However, it is likely that reactive ventral root axons originate from neurons situated segmentally in the spinal cord, and do not constitute aberrant sensory fibers, as carbonic anhydrase activity remained unchanged in the L4 and L5 ventral roots after removal of the corresponding spinal ganglia, whereas it disappeared after damage to the spinal cord at the lumbar level, or at a site distal to a ventral root section. Enzymatic staining of neurons of the dorsal root ganglia was not modified by a dorsal rhizotomy, but showed a marked decrease after transection of the spinal nerve.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017746 TI - Character of (D-Phe-7) ACTH4-10-induced excessive grooming. AB - Rats receiving (D-Phe-7) ACTH4-10 exhibited excessive grooming during the first half of the hour-long observation period. This resulted in total grooming scores of about one-half of those produced by the longer ACTH fragments, ACTH1-24 or ACTH1-16 NH2. The excessive grooming induced by (D-Phe-7) ACTH4-10 was due to an enhancement of duration of grooming bouts and not to an increase in the frequency of occurrence. Furthermore, the neuropeptide-induced excessive grooming was eliminated by prior treatment with naloxone. PMID- 3017747 TI - Schistosoma mansoni: preparation, characterization of (Na+ + K+)ATPase from tegument and carcass. AB - The preparation and some biochemical properties of a (Na+ + K+)ATPase from male adult Schistosoma mansoni are described. After incubation in a membrane disruption medium, the tegument and carcass of the worms were separated and treated to obtain fractions enriched in (Na+ + K+)ATPase. The activity of the tegumental ouabain sensitive (Na+ + K+)ATPase at 37 C was 20.3 mumole Pi X mg-1 protein X hr-1 and represented 32% of the total ATPase activity. The (Na+ + K+)ATPase prepared from the carcass had a lower specific activity (3.7 mumole Pi X mg-1 protein X hr-1) but a higher relative activity (55%). Similar concentrations of Na+ and K+ activated the enzymes from both sources, and both enzymes were inhibited by similar concentrations of calcium. However, the enzyme from carcass was ten times more sensitive to ouabain than the enzyme from tegument. Comparison with results obtained on the (Na+ + K+)ATPase of human heart showed that the enzymes from the worms were more resistant to ouabain. The half maximal inhibitory concentration of dihydroouabain compared to that of ouabain was also different in the enzymes from human and worm. We conclude that (1) there exists at least one structural difference between the (Na+ + K+)ATPase of S. mansoni and that of the human host, and (2) it is useful to separately study the enzymes from tegument and carcass because they differ in sensitivity to cardiac glycosides. PMID- 3017748 TI - Synthesis of new 6H-indolo[2,3-b] [1,8]naphthyridines and their specific inhibition of benzodiazepine receptor. AB - Some 3-amino- and 3-hydroxy-8-halosubstituted 6H-indolo[2,3-b] [1,8]naphthyridines were synthesized and tested for their affinity for the benzodiazepine receptor in bovine cortical membranes. All prepared compounds were more active than the corresponding 8-unsubstituted derivatives. Moreover, among these compounds the 8-chloroindolonaphthyridines were clearly the most potent. PMID- 3017749 TI - Purification of an enzyme aggregate containing 3',5'-cyclic-nucleotide phosphodiesterase and nucleotidase. AB - Several steps of purification (octyl-Sepharose chromatography, Blue Sepharose 6B chromatography and sucrose density gradient centrifugation) led to a highly purified aggregate of the enzymes, 3',5'-cyclic-nucleotide phosphodiesterase (PDE) and nucleotidase. The purified enzyme aggregate showed an S value of 7.3 (SE +/- 0.3, n = 10). Further analysis by SDS-polyacrylamide gel electrophoresis (PAGE) revealed two proteins near 67 and 60 kDa. Dissociation of the 7.3 S enzyme aggregate showed a 3.6 S PDE form and a nucleotidase form at 4.2 S. Additionally, higher S value forms of the nucleotidase up to 17 S have been observed. Apparently, they had formed by self-association. SDS-PAGE of the 17 S nucleotidase form showed only one band at 67 kDa. This was taken as evidence for the homogeneity of the 17 S nucleotidase form and the self-association of the nucleotidase after dissociation from the 7.3 S enzyme aggregate. Furthermore, from this it could be concluded that the 67 kDa protein of the 7.3 S enzyme aggregate should be identified with the nucleotidase, and thus the 60 kDa band represents the PDE. PMID- 3017751 TI - Studies on the structure of the human C4b-binding protein gene. AB - Protein and cDNA sequence data have shown human C4b-binding protein to contain eight internally homologous repeat units, each approx. 61 amino acids in length. Repeat units conforming to the same consensus sequence have been found in other complement and non-complement proteins. Southern blot analysis together with isolation, characterisation and sequencing of genomic clones has allowed the study of intron/exon organisation in the human C4b-binding protein gene and the identification of a Bg/II restriction fragment length polymorphism. PMID- 3017750 TI - Deficiency of neutrophil membrane antigen detected by monoclonal antibody in rheumatoid arthritis. AB - Monoclonal antibodies (mAbs) against cell surface antigens and receptors are instrumental in defining specific membrane markers. mAbs GF 26.7.3 and MF 25.1 against human neutrophils modulated the activation mechanism of superoxide anion production induced by formyl-peptide and PMA in all subject. However, treatment with mAb MF 25.1 of neutrophils from patients with rheumatoid arthritis did not have any effect. This may suggest that the antigen which MF 25.1 binds is absent in rheumatoid conditions. This confirms our previous data showing that defective expression of membrane components is associated with neutrophil dysfunction. PMID- 3017752 TI - Analysis of the structures of the subunits of the cytochrome bc1 complex from beef heart mitochondria. AB - The interaction of the protein subunits of the bc1 complex from beef heart is analysed on the basis of protein chemical data and of secondary structure predictions suggesting a large number of amphipathic helices. Electrostatic interactions, i.e. helix-dipole interactions and ionic bonds, may play a major role in the stabilisation of the arrangement of the subunits within the multi protein complex, formation of subcomplexes and maintenance of the steric strain of cytochrome b. A model of the heme-carrying 'core' of cytochrome b, i.e. of helices II-V, is presented consisting of a twisted '4-alpha-helical' bundle held together by helix-dipole interactions and stabilised by the interaction with other protein subunits of the bc1 complex. PMID- 3017753 TI - Activation of a D-form of rabbit muscle glycogen synthase by Ca2+-activated protease. AB - Glycogen synthase isolated from rabbit skeletal muscle as a D-form (synthase D2) is activated by a rat liver cytosolic protein differing from any of the known protein phosphatases (D2 activase). Although reversible by phosphorylation by cyclic AMP-dependent protein kinase, the activation is a result of limited proteolysis by D2 activase, which has been identified as the Ca2+-activated protease. PMID- 3017754 TI - LLC-PK1 cell mutants in cAMP metabolism respond normally to phorbol esters. AB - Mutants of the pig kidney cell line, LLC-PK1, affected in cAMP metabolism, were examined for cAMP-dependent protein kinase (cAMP-PK) activity and for cAMP mediated induction of urokinase-type plasminogen activator (uPA). The FIB4 and FIB6 mutant cell lines possessed about 10% parental levels of cAMP-PK activity and concomitantly reduced uPA production (10-20% parental) in response to calcitonin, forskolin and 8-bromo cAMP. The FIB1, FIB2 and FIB5 mutant cell lines had about 70% parental levels of cAMP-PK and the synthesis of uPA was 40-60% parental. Thus, cAMP-mediated induction of uPA showed a dependence on the absolute levels of cAMP-PK. However, uPA synthesis in response to phorbol-12 myristate-13-acetate by all of the mutants was similar to parental, which indicates that enzyme induction mediated by phorbol esters does not involve cAMP or cAMP-PK. PMID- 3017755 TI - Specific binding of human growth hormone but not insulin-like growth factors by human adipocytes. AB - Binding of human GH (hGH) and insulin-like growth factors I and II (IGFI and II) to isolated human adipocytes from adult subjects was studied. Binding equilibrium for hGH at 24 degrees C was reached at 120 min and half-maximal specific binding at 6-8 ng/ml. Apparent Ka was 2.1 X 10(9) M-1 and Bmax 7.3 X 10(-11) M/10(6) cells. The human fat cell growth hormone receptor recognized neither bovine, ovine or rat GH nor human prolactin or placental lactogen. No specific receptors for human IGFII could be demonstrated. Thus, human adipocytes do not possess IGF receptors but have specific GH receptors which recognize hGH but not GH from lower species. PMID- 3017756 TI - Guanine nucleotides stimulate NADPH oxidase in membranes of human neutrophils. AB - In the chain of events by which chemotactic peptides stimulate NADPH oxidase catalyzed superoxide formation in human neutrophils, the involvements of a pertussis toxin-sensitive guanine nucleotide-binding protein (N-protein), mobilization of intracellular calcium and protein kinase C stimulation have been proposed. Superoxide formation was studied in membranes from human neutrophils; NADPH oxidase was stimulated by arachidonic acid in the presence of neutrophil cytosol. Fluoride and stable GTP analogues, such as GTP gamma S and GppNHp, which all activate N-proteins, enhanced NADPH oxidase activity up to 4-fold. GDP beta S inhibited the effect of GTP gamma S. These data suggest that NADPH oxidase is regulated by an N-protein, independent of an elevation of the cytoplasmic calcium concentration. PMID- 3017757 TI - Dolichol alters dynamic and static properties of mouse synaptosomal plasma membranes. AB - Dolichols are isoprenologues that are found in almost all tissues and whose biochemical function, aside from dolichol phosphate precursors, is not known. In addition, an understanding of the organizational and dynamic properties of dolichols in biological membranes has not been forthcoming. The purpose of the experiments reported here were to examine the effects of dolichol on the physical properties of mouse synaptic plasma membranes (SPM). Differential polarized phase fluorometry indicated that dolichol both fluidized and rigidified SPM. Membrane areas detected by diphenylhexatriene and trans-parinaric acid were selectively fluidized and rigidified, respectively. It also was found that the spin label, 5 doxyl stearic acid indicated that dolichol reduced membrane fluidity. These results report for the first time a structural effect of dolichol on a biological membrane. PMID- 3017758 TI - Identification of a rat liver cDNA and mRNA coding for the 28 kDa gap junction protein. AB - By screening of a rat liver cDNA library with complex and deoxyinosine containing oligonucleotide probes a cDNA clone was isolated and shown by sequencing to code for the amino-terminal half of the rat liver 28 kDa gap junction protein. The insert hybridized to a 1.9 kb species from rat and mouse liver poly(A)+ RNA in Northern blot analysis. In embryonic mouse hepatocytes the amount of the 1.9 kb mRNA increased 3-fold between 24 and 96 h in culture. This correlates with the previously described increase of the 28 kDa gap junction protein under these conditions. PMID- 3017759 TI - A surface NAD-glycohydrolase of human platelets may influence their aggregation. AB - Human platelets may hydrolyze externally added NAD+ yielding ADPR and nicotinamide. The extent of hydrolysis is significantly higher when the platelets are stimulated. The presence of external NAD+ strongly inhibits the aggregation induced by every agonist used. It seems that adenosine or ADPR itself generated by NAD+ hydrolysis may be responsible for the inhibition of aggregation. Evidence is given that some of the NAD+ hydrolysis product is taken up by stimulated platelets. PMID- 3017760 TI - A high resolution 1H NMR study of the solution structure of human epidermal growth factor. AB - 500 MHz 1H NMR studies of human epidermal growth factor are described. The backbone resonances of the 1-48 derivative of hEGF have been assigned using two dimensional techniques. Analysis of the type and magnitude of the observed sequential nuclear Overhauser effects and the NH-alpha CH spin-spin coupling constants allowed prediction of the secondary structure. Aspects of the tertiary structure are also identified. A pair of antiparallel beta-sheets involving residues 18-23 and 28-34 is a dominant feature of the solution structure. PMID- 3017761 TI - Cooperativity of the alpha beta-protomer structure in Na+,K+-ATPase functioning. A scanning microcalorimetry study. AB - Heat denaturation of the free and ligand-bound forms of purified Na+,K+-ATPase from pig kidney is studied with the scanning microcalorimetry technique. A single two-state transition is observed during denaturation of the free enzyme, the molar concentration of the cooperatively melting units being equal to the concentration of alpha beta-protomers (Mr approximately equal to 140 000). Upon interaction of the enzyme with phosphate, Mg2+, and strophanthidin, but not with Na+, the cooperativity of the protomer unfolding is lost, and the protein stabilization enthalpy becomes approximately equal to 230 kJ/mol higher. The data suggest that in a functionally active enzyme form, the alpha beta-protomers possess a rigid structure with tight association of their subunits and domains, this structural rigidity is essential for the Na+,K+-ATPase functioning and there is a unique non-active conformation of the enzyme which may play an important role in its in vivo regulation. PMID- 3017762 TI - A cytosine . cytosine base paired parallel DNA double helix with thymine . thymine bulges. AB - 500 MHz 1H NMR studies using 2D-NOESY indicate that the oligonucleotide d(CTCTCT) at low pH forms a parallel double helix with cytosine . cytosine base pairs and thymine . thymine bulges. This unusual structure may explain the hypersensitivity of S1 nuclease at low pH towards supercoiled plasmids containing d(CT)n inserts. PMID- 3017763 TI - Characterization of the distribution of potential short restriction fragments in nucleic acid sequence databases. Implications for an alternative to chemical synthesis of oligonucleotides. AB - We have searched the GenBank nucleic acid sequence database for potential short restriction fragments. All possible oligonucleotides up to length five are found at least once flanked by known restriction recognition patterns. Thus, searches in the database for a specific sequence corresponding to a desired oligonucleotide would often point to one or more sources of short, retrievable fragments containing that sequence. These results underscore the potential of nucleic acid sequence databases in planning experiments. PMID- 3017765 TI - Free radical metabolism of antiparasitic agents. AB - In recent years it has been apparent that many of the known antiparasitic drugs produce free radicals. Intracellular reduction followed by autooxidation yielding O.-2 and H2O2 has been suggested as the mode of action of nifurtimox on Trypanosoma cruzi and as the basis of its toxicity in mammals. On the other hand, free radical intermediates that do not generate oxygen-reduction products under physiological conditions have been found in the metabolic pathways of other antiparasitic nitro compounds (benznidazole, metronidazole, and other 5 nitroimidazoles) used in the treatment of diseases such as Chagas' disease, trichomoniasis, giardiasis, balantidiasis, amebiasis, and schistosomiasis. In these cases, as well as in the case of niridazole (used in the treatment of schistosomiasis), covalent binding or other interactions of the intermediates of nitroreduction with parasite macromolecules are possibly involved in their toxicity. Redox cycling of these compounds under aerobic conditions appears to be a detoxification reaction by inhibiting net reduction of the drugs. PMID- 3017764 TI - Complete amino acid sequence of the large subunit of the low-Ca2+-requiring form of human Ca2+-activated neutral protease (muCANP) deduced from its cDNA sequence. AB - The complete amino acid sequence of the large subunit (catalytic subunit) of human low-Ca2+-requiring-calcium-activated neutral protease (muCANP) was deduced from its cDNA base sequence. It is composed of 714 amino acid residues and its sequence is highly homologous to the chicken CANP sequence determined previously. Human muCANP, like chicken CANP, has a clear 4-domain structure, and their fundamental structures are essentially the same, although their Ca2+ sensitivities are significantly different. The role of each domain in the Ca2+ sensitivity and protease activity of CANP is discussed on the basis of sequence comparison. PMID- 3017766 TI - An electron spin resonance study of free radicals from catechol estrogens. AB - Electron spin resonance spectroscopy has been used to demonstrate production of semiquinone free radicals from the oxidation of the catechol estrogens 2- and 4 hydroxyestradiol and 2,6- and 4,6-dihydroxyestradiol. Radicals were generated by horseradish peroxidase/H2O2 or tyrosinase/O2, or by autoxidation, and were detected as their complexes with spin-stabilizing metal ions (Zn2+ and/or Mg2+). Radical production occurs via one- or two-electron oxidation of catechol estrogens, depending on the type of activating system. Autoxidation of catechol estrogens produces superoxide and H2O2 at physiological pH values. The present results also indicate a difference in the reactivity of quinones derived from 2- and 4-hydroxyestradiol. The toxicological significance of these reactions is discussed. PMID- 3017767 TI - Sonochemical free radical formation in aqueous solutions. AB - The phenomena of stable and transient acoustic cavitation in liquids exposed to ultrasound are briefly explained. The role of micronuclei, resonant bubble size, and rectified diffusion in the initiation of transient cavitation is reviewed. In aqueous solutions transient cavitation initially generates hydrogen atoms and hydroxyl radicals that may recombine to form hydrogen and H2O2 or may react with solutes in the gas phase, at the gas-liquid boundary, or in the bulk of the solution. The analogies and differences between sonochemistry and ionizing radiation chemistry are explored. The use of spin trapping and electron spin resonance to conclusively identify hydrogen atoms and hydroxyl radicals and to detect cavitation produced by continuous wave and by pulsed ultrasound is described in detail. PMID- 3017769 TI - Cholecalciferol and placental calcium transport. AB - Transplacental movement of calcium from mother to fetus is essential for normal fetal development. In most species, fetal plasma calcium levels are higher than maternal levels at term. The role of cholecalciferol metabolites, with specific emphasis on 1,25-dihydroxycholecalciferol (1,25(OH)2D), in placental calcium transport and maintenance of the fetomaternal gradient has been extensively investigated. In rats, there is not an absolute demand for 1,25(OH)2D for maintenance of fetal calcium homeostasis in utero, even though it is essential for maintenance of maternal plasma calcium levels. However, in sheep, the absence of 1,25(OH)2D results in disruption of both maternal and fetal calcium homeostasis. It is known that rat and human placentas contain specific cytosolic binding proteins for 1,25(OH)2D that are similar to the well-characterized intestinal receptor. Two calcium-binding proteins (CaBP) have been detected in rat and human placentas: a protein immunologically identical to the vitamin D dependent CaBP and a calcium-dependent ATPase. The levels of CaBP in rat placenta have been shown to increase in response to exogenously administered 1,25(OH)2D but cannot be obliterated with maternal vitamin D deficiency. No relationship has been shown between 1,25(OH)2D and placental Ca-ATPase in any species. Thus, the mechanism of action of 1,25(OH)2D in maintenance of the transplacental calcium gradient in sheep is unknown. In the pregnant rat (and perhaps human), 1,25(OH)2D is a critical factor in the maintenance of sufficient maternal calcium for transport to the fetus and may play a role in normal skeletal development of the neonate. PMID- 3017768 TI - Free radicals of acetaminophen: their subsequent reactions and toxicological significance. AB - The oxidation of acetaminophen to the corresponding phenoxyl free radical and N acetyl-p-benzoquinone imine by mammalian peroxidases is discussed. The acetaminophen free radical is very reactive--forming dimers, and, ultimately, melanin-like polymeric products. A model compound, leading to more stable metabolites, can be obtained by introduction of methyl groups next to the oxygen, to produce 3,5-dimethylacetaminophen. The electron spin resonance spectrum of this free radical could be completely analyzed. The phenoxyl radical of the dimethyl analog does not form polymers or bind with nucleophiles. N-Acetyl-p benzoquinone imine, a hepatic metabolite of acetaminophen, and its analog N acetyl-3,5-dimethyl-p-benzoquinone imine are metabolized by rat liver microsomes and NADPH to their corresponding p-aminophenoxyl free radicals. The p aminophenoxyl free radical formation could be suppressed by the deacetylase inhibitors sodium fluoride and paraoxon. Substitution of NADPH-cytochrome P-450 reductase for rat liver microsomes eliminates the deacetylase activity and results in the direct reduction of N-acetyl-3,5-dimethyl-p-benzoquinone imine to the 3,5-dimethylacetaminophen phenoxyl free radical. Neither the acetaminophen nor the 3,5-dimethylacetaminophen phenoxyl radical reduces oxygen to form superoxide or reacts with oxygen in any other detectable way. PMID- 3017772 TI - Eating to attain maximal health. PMID- 3017770 TI - Culture of human granulosa cells from an in vitro fertilization program: effects of extracellular matrix on morphology and cyclic adenosine 3',5' monophosphate production. AB - Human GCs obtained from patients undergoing an IVF procedure and treated with CC, hMG, and hCG, lack cAMP responsiveness to hCG stimulation in vitro. After 48 hours in culture, recovery of cAMP production was significantly higher in hCG stimulated cells grown on dishes coated with ECM than in cells grown on uncoated dishes. This difference correlated with a higher degree of morphologic differentiation of cells cultured on ECM. It is concluded that ECM provides superior culture conditions for the recovery of hormone-stimulated cAMP production and the maintenance of morphologic differentiation of preovulatory GCs from stimulated cycles. PMID- 3017771 TI - [Effect of 4-aminopyridine on synaptic transmission in the smooth muscle of the gastrointestinal tract of the guinea pig and man]. PMID- 3017773 TI - Immunoglobulin K chain genes in mouse hybridoma PTF-02 and parent myeloma. Insertion of hybridoma K gene into the plasmid pSV2-gpt. AB - The mouse myeloma line P3-X63-Ag8.653 currently used as the parent line for hybridoma construction contains only one (non-productive) gene for immunoglobulin K chains. The allelic gene is lost. In the mouse hybridoma PTF-02 two K genes can be found. One is identical with the gene of the myeloma parent line, the other originates in lymphocytes and is transcribed and translated in the K chain of the antibody secreted by the hybridoma. From the gene library of hybridoma PTF-02 in phage Charon 28 both K genes were isolated. Restriction endonuclease mapping and Southern blot hybridization demonstrated that the fragment comprising the lymphocyte gene was of the size (7.5 kb) sufficient to carry all exons, transcription and translation units. This gene was then recloned in the plasmid pBR322 and shuttle plasmid pSV2-gpt, which opens up possibilities for transfection of lymphoid cells and for study of the regulation of individual gene expression. PMID- 3017774 TI - The presence of retroviral particles in hybridoma cell lines. AB - The presence of very high numbers of type C and type A retroviral particles was repeatedly confirmed not only in myeloma cells NSI, Ag 8 and in thymoma cells BW5147, EL 4 but also in B and T cell hybridomas constructed from the myeloma and thymoma cells used. Retroviral particles were demonstrated by current electron microscopic and physicochemical methods. Biological tests for the induction of possible malignant or any pathological changes in the artificially infected sensitive cells during long-term cultivations in vitro or in vivo gave negative results. Nevertheless, on account of the hitherto unknown action of retroviruses one can suppose that monoclonal products from B and T cell hybridomas, particularly when used for practical purposes, should be purified because of their infectious nature. PMID- 3017775 TI - Effect of cytomegalovirus antigen on skin allograft survival in rabbits. AB - In experiments on rabbits it was found that the subcutaneous injection of cytomegalovirus (CMV) antigen into the draining lymphatic area of the skin allograft caused a decrease in graft survival. The difference of experimental versus control grafts was statistically significant. The finding supports some clinical data regarding the increased incidence of rejection episodes during CMV infection. PMID- 3017776 TI - [Molluscum contagiosum disclosing HTLV III infection]. AB - We report here a case of cutaneous molluscum contagiosum infection, which led to the diagnosis of HTL V III infection. Profuse mollusca contagiosa are one of the cutaneous lesions indicating the presence of AIDS at any stage. PMID- 3017778 TI - Cell surface receptors in development and immunity: a speculative review. PMID- 3017777 TI - Endocytosis and control of expression of transferrin receptor. PMID- 3017779 TI - Effect of melphalan on growth curves and cell cycle distribution of four human small cell carcinomas of the lung grown in nude mice. AB - Four human small cell carcinomas of the lung grown in nude mice were exposed to melphalan. Two of the tumors were derived from subpopulations isolated by in vitro cloning from the same tumor biopsy. The chemosensitivity of the tumors was determined by calculating the specific growth delay. Drug-induced changes in the cell cycle were detected by flow cytometric DNA analysis. The specific growth delay of the tumors was very different with the greatest differences between the two subpopulations originating from the same tumor. Melphalan induced a dose related S phase accumulation in three sensitive tumors, whereas no changes were seen in a resistant tumor. Furthermore, the amount of S phase accumulation reflected the sensitivity to melphalan. The results suggest that heterogeneity in chemosensitivity is an important reason for chemotherapy failures. PMID- 3017780 TI - Concanavalin A induced inhibition of 5'-nucleotidase from guinea pig skeletal muscle and bull seminal plasma: a comparative study. AB - Both purified and membrane-bound 5'-nucleotidases (EC 3.1,3.5) from guinea pig skeletal muscle and bull seminal plasma are inhibited by Concanavalin A (Con A). 5'-Nucleotidase purified from skeletal muscle is inhibited by Con A by an apparent uncompetitive process (K'i = 160 nM), while the lectin inhibits the particulate enzyme by an apparent non-competitive process (Ki = K'i = 50 nM). 5' Nucleotidase purified from bull seminal plasma is inhibited by Con A by an apparent non-competitive process (K'i = Ki = 270 nM), while the membrane-bound enzyme is subjected to a mixed type inhibition by the lectin (K'i greater than Ki; 30 and 14 nM, respectively). The enzyme purified from skeletal muscle exhibits a significant cooperativity in the interaction with Con A. The inhibition of bull seminal plasma particulate 5'-nucleotidase brought about by Con A is not completely reversed by addition of alpha-methyl-D-mannoside. PMID- 3017781 TI - Cyclic CMP phosphodiesterase: isolation, specificity and kinetic properties. AB - Cyclic CMP phosphodiesterase activity was demonstrated in rat liver, heart, brain, kidney, intestine, skeletal muscle, blood, testes, ovaries, spleen and lung; that present in the liver was purified to homogeneity by a sequential process of ammonium sulphate fractionation, gel filtration, two ion-exchange chromatographic steps, preparative electrophoresis and two affinity chromatographic stages with selection at each stage for maximum specificity. The final enzyme preparation was confirmed as a single protein by HPLC and isoelectric focussing; the total yield obtained was 1.5% and the final specific activity of 48.6 mumol cyclic CMP hydrolysed/min/mg reflected a 88,000 fold purification. The phosphodiesterase had a Mr of 2.8 X 10(4), pH optimum 7.2-7.4, isoelectric point between 4.2 and 4.4 and a Km of 9.0 mM cyclic CMP. This enzyme differs from a previously isolated cyclic CMP phosphodiesterase in its amino acid composition and specificity. The absolute specificity for 3',5'-cyclic CMP as substrate distinguishes this cyclic CMP phosphodiesterase from all other reported phosphodiesterases and shows it to be a novel enzyme. Its potential as a research tool and the significance of its occurrence are discussed. PMID- 3017782 TI - Short-term regulation of prolactin receptors in the liver, mammary gland and kidney of pregnant and lactating rats infused with ovine prolactin or human growth hormone. AB - Short-term regulation of prolactin (PRL) receptors was studied in ketamine anaesthetized 18-day pregnant or 7-day lactating female rats, by infusing them with various doses of oPRL or human growth hormone (hGH) for 0-3 h and measuring the binding of [125I]oPRL of [125I]hGH to the microsomal fractions prepared from the liver, mammary gland and kidneys of animals sacrificed at various states of infusion. Our main findings are: In pregnant rats, only 30% of liver receptors are unoccupied and infusion with 25 micrograms/h for 3 h of either oPRL of hGH decreased both free and total receptors by 22-30% while infusion with 250 micrograms/ml caused an additional decrease only in the free receptors. In the mammary gland and the kidney of pregnant rats, all receptors seem to be unoccupied; infusion with 25 micrograms/ml had none or a slight elevating effect on the number of both free and total receptors in the mammary gland but caused a significant 3-fold increase in the kidney; infusion with 250 micrograms/ml, however, resulted in a slight decrease in the mammary gland and a significant decrease in the kidney in both total and free receptors. In the liver of the lactating rats, there was no significant difference between the number of free and total receptors, but in mammary gland, specific binding to the total receptor was higher than to the free ones indicating partial occupancy; infusion with 25 micrograms/ml caused a significant decrease in free and total liver receptors without a remarkable change in the mammary gland and some decrease (by infusion with hGH only) in the kidney. In all cases, the changes in the specific binding resulted from the increase or decrease in receptor number and not from the change in receptor-hormone affinity. In almost all cases, infusion with oPRL or hGH yielded similar results. Infusion with both hormones did not affect the level of the endogenous rat prolactin. In conclusion, our results indicate the short-term regulation of PRL receptors by exogenous hormones is a complicated process which is affected by the level of the infused hormone, physiological state of the animal and may yield, simultaneously, different or even opposite changes in receptor number in various organs. PMID- 3017783 TI - Characterization of a prolactin-daunomycin ligand as a probe for drug targeting. AB - Conjugates of ovine prolactin and daunomycin were prepared for use as affinity labelled drug carriers in cancer cells carrying the prolactin receptor. The binding affinity of the conjugates to prolactin receptors in rat liver membrane preparations and in viable granulosa cells derived from estradiol- and pregnant mare serum gonadotropin (PMSG)-treated immature female rats was less than an order of magnitude lower than prolactin. The toxicity of the conjugate in cultured granulosa cells was dependent upon the concentration of the daunomycin present in the culture. The cytotoxic effect of the ligand was abolished by the addition of free prolactin or NH4Cl to the granulosa cell cultures. These conjugates may be useful probes in drug targeting against hormone-sensitive cancer. PMID- 3017784 TI - Discrimination between intact cell desensitization and agonist affinity changes. AB - The relationship between epinephrine-induced receptor-epinephrine affinity changes and intact cell adenylate cyclase activity was investigated using S49 lymphoma cells. It was demonstrated that 20 nM epinephrine caused desensitization (defined as a reduction in the rate of cAMP synthesis with time), but no significant change in receptor-epinephrine affinity as measured by competition with [125I]iodopindolol. On the other hand, treatment with 5 microM epinephrine caused no desensitization (as defined above), but did cause a very significant reduction in receptor-epinephrine affinity. It is suggested that there is a desensitization process which is distinct from the agonist affinity shift and which is expressed primarily as a change in the EC50 of the hormone-induced activation. The demonstration of extensive desensitization at low concentrations of hormone may have important physiological implications. PMID- 3017785 TI - Luteinizing hormone receptor induction in dispersed granulosa cells requires estrogen. AB - Studies on granulosa cell responses in vitro have routinely utilized cell preparations in which intercellular gap-junctions are maintained. The present study was conducted to determine if disruption of these junctions, prior to culture, would affect subsequent follicle-stimulating hormone (FSH)-stimulated luteinizing hormone (LH) receptor induction on these cells. Granulosa cells were expressed from ovaries of diethylstilbestrol (DES)-primed immature rats after a short incubation of the excised ovaries in culture medium alone or medium containing 6.8 mM EGTA. The latter procedure disrupts gap-junctions between granulosa cells thus providing a predominantly mono-dispersed cell suspension. The two cell preparations were cultured, separately, for 72 h in sterile polypropylene tubes in media containing either FSH or FSH plus various steroids (estradiol, testosterone, or 5 alpha-dihydrotestosterone (DHT)). LH receptor content of cells was determined at 72 h of culture. Both the cell yield and the proportion of viable cells obtained were enhanced by the EGTA pretreatment. LH receptors were induced in all FSH-containing cultures of non-dispersed granulosa cells. In the dispersed cell cultures, FSH alone failed to induce LH receptors. The inclusion of either estradiol or testosterone but not DHT with FSH, however, restored LH receptor induction to levels comparable to non-dispersed cultures. LH receptors were not induced in cultures of either cell preparation with steroids alone. Aromatase activity, however, was stimulated in both cell preparations by FSH alone. These results suggest that cell-cell communication may be necessary for LH receptor induction in granulosa cells and that estradiol (or an aromatizable androgen) can promote intercellular interactions if this communication has been disrupted. PMID- 3017786 TI - Cyclic AMP-dependent protein kinase-mediated desensitisation of adrenal tumour cells. AB - Y-1 mouse adrenal cortical tumour cells increase their production of steroids and cAMP while decreasing cell population growth in response to treatment with ACTH. With a fluorescent conjugate of the heat-stable protein kinase inhibitor, the time- and dose-dependent dissociation of cAMP-dependent protein kinase can be demonstrated following ACTH stimulation of Y-1 adrenal tumour cells, and the kinetics of its free catalytic subunits can be followed. After 60 min ACTH (6 X 10(-10) M) stimulation, a 4-fold rise in catalytic subunits was detected in the nucleus while a 2-fold increase was noted in the cytoplasm. When Y-1 cells had been previously treated with ACTH, they no longer secreted steroids to the same level in response to a subsequent exposure to ACTH. In addition to the altered steroidogenic response the cells become resistant to the effect of subsequent ACTH treatment on cell division and cAMP production as measured by protein kinase dissociation. Y-1 adrenal cells, that had been pretreated with ACTH, had an altered activation of protein kinase. Although there was an increase in cytoplasmic dissociation following subsequent ACTH stimulation of the pretreated cell, this increase was negligible when compared to that in the non-pretreated cultures. The nucleus of the ACTH-pretreated cell failed to significantly dissociate protein kinase following subsequent ACTH treatment. The data suggest that the phenomenon of desensitisation may be due to a decrease in dissociation of cAMP-dependent protein kinase, especially in the nucleus. PMID- 3017787 TI - Involvement of cyclic AMP-dependent protein kinase in prothoracicotropic hormone stimulated ecdysone synthesis. AB - Prothoracicotropic hormone (PTTH) is a brain neuropeptide that stimulates the prothoracic glands to synthesize ecdysone, an event that leads to insect molting. Both cyclic AMP (cAMP) and calcium have been implicated in PTTH action, with current evidence favoring cAMP as the messenger directly regulating ecdysone synthesis. To further define the role of cAMP in PTTH action, the activity of cAMP-dependent protein kinase (cAMP-PK) was examined in prothoracic glands from two developmental stages of the tobacco hornworm, Manduca sexta (day 3 fifth instar larvae and day 0 pupae). Prothoracic glands at each of these stages of development possess two forms of cAMP-PK which resemble the vertebrate type I and type II isozymes, with the latter being the predominant form (greater than 90%). Marked developmental differences exist in the degree of activation of soluble cAMP-PK following in vitro exposure of the prothoracic glands to PTTH. In larval glands, soluble cAMP-PK is activated within 3-10 min of initial exposure to doses of PTTH that stimulate ecdysone synthesis. By contrast, activation of soluble cAMP-PK in pupal glands occurs only when PTTH is administered in the presence of a phosphodiesterase inhibitor. Developmental differences in the activation of cAMP-PK by PTTH were qualitatively identical to previously observed differences in PTTH-stimulated accumulation of intracellular cAMP. The results suggest an involvement of soluble cAMP-PK in the response of day 3 fifth instar larval prothoracic glands to PTTH, but indicate a difference in the nature, intracellular location, or time course of activation, of hormone-sensitive protein kinase in day 0 pupal glands. PMID- 3017788 TI - Effects of streptozotocin-diabetes, fasting and adrenaline on phosphorylase phosphatase activities of rat skeletal muscle. AB - The distribution of the spontaneous and trypsin-stimulated phosphorylase phosphatase activities between glycogen particles and cytosol was examined in muscle extracts obtained from rats that had been fasted, made diabetic with streptozotocin or injected with adrenaline. In all conditions the particle-bound phosphatase activities decreased, glycogen was degraded and phosphorylase was released from the particles into the cytosol. However, in fasting and diabetes (but not after adrenaline) the combined glycogen particle + cytosolic phosphatase activities decreased, indicating that the activity lost from the particles was not simply shifted to the cytosol. Fasting and diabetes (but not adrenaline) also decreased the phosphatase-activating ability of the muscle extracts, which was, at least in part, attributable to the protein kinase FA. These data indicate the presence of at least two different mechanisms affecting the phosphatase system, one modified by fasting and diabetes, the other by adrenaline. PMID- 3017789 TI - Growth hormone receptors in cultured adipocytes: a model to study receptor regulation. AB - Acutely isolated rat adipocytes have been maintained in primary culture for several days and the effects of culture on the kinetics of 125I-human growth hormone (hGH) binding to adipocytes have been determined. A marked increase (500 1000%) in specific binding of 125I-hGH was observed over the first 3 days of culture--acutely isolated adipocytes (5.5 +/- 1.4%, mean +/- SE, n = 47) compared to 3-day cultured adipocytes (48 +/- 7%, mean +/- SE, n = 8). Specific binding of 125I-hGH to both acutely isolated and cultured adipocytes was dependent on incubation time and temperature (equilibrium being reached in 1 h at 37 degrees C and 2 h at 22 degrees C). Binding was reversible (t1/2 approximately 1.5 h). Scatchard analysis revealed linear plots and showed that the increase in binding during culture was due to an increase in the number of receptors per cell (approximately 20 000 to approximately 170 000) with little or no change in binding affinity (Ka approximately 1 X 10(9) M-1). Cycloheximide inhibited the increase in binding sites during culture suggesting a requirement for de novo protein synthesis. Addition of unlabelled hGH to the culture medium resulted in a marked down-regulation of the GH receptor by 2 days. The GH-induced decrease in receptor number was to due to receptor occupancy by exogenously added GH. The studies to date indicate that the cultured rat adipocyte should provide a useful model for a comprehensive study of the cellular mechanisms and dynamics of GH receptor regulation. PMID- 3017790 TI - Calcium-dependent actions of gonadotropin-releasing hormone agonist and luteinizing hormone upon cyclic AMP and progesterone production in rat ovarian granulosa cells. AB - The Ca2+ dependency of the direct stimulatory effect of the gonadotropin releasing hormone (GnRH) agonist analog [D-Ser(t-Bu)6]des-Gly10-GnRH-N-ethylamide (GnRHa) on progesterone production was investigated and compared to that of luteinizing hormone (LH) in rat granulosa cells from preovulatory follicles. Removal of extracellular Ca2+ by EGTA, or the use of the Ca2+ channel blockers verapamil and La3+, resulted in complete inhibition of GnRHa-induced progesterone production and a partial inhibition of LH-stimulated progesterone production (80, 80 and 50% inhibition respectively for EGTA, verapamil and La3+). Removal of extracellular Ca2+ increased the ED50 for LH-induced cAMP production by four-fold (from 80 to 330 ng/ml) and decreased maximal nucleotide formation by 44%. LH induced cAMP production was also inhibited partially by verapamil (35%) at 10(-4) M drug concentration. GnRHa had no effect on cAMP production in the presence or absence of Ca2+. GnRHa and LH were found to have maximal effects on progesterone production at about 0.5 mM of Ca2+ in the incubation medium. On the other hand the stimulatory effect of dibutyryl cAMP [Bu)2cAMP) on progesterone production showed little dependency on extracellular Ca2+. The calmodulin antagonist trifluoperazine (TFP) caused concentration-dependent inhibition of the stimulatory action of GnRHa and LH on progesterone production with IC50 values of 3 and 8 microM, respectively. The stimulatory effect of (Bu)2cAMP on progesterone synthesis was attenuated by verapamil and TFP. These results indicate that the direct stimulatory effect of GnRH on ovarian progesterone production is absolutely dependent on Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3017791 TI - Stimulatory effects of epidermal growth factor on steroidogenesis in Leydig cells. AB - We studied the effects of epidermal growth factor (EGF) on steroidogenesis in freshly prepared Percoll-purified Leydig cells from prepubertal and adult rats and mice, and in interstitial cells from immature rats cultured in the presence or absence of LH. It is demonstrated that EGF directly stimulates the output of C19-steroids (testosterone and androstenedione) as well as C21-steroids (progesterone, 17 alpha-hydroxyprogesterone and 20 alpha-hydroxypregn-4-en-3-one) in all systems studied. When combined with LH, EGF has little effect on freshly isolated cells but still stimulates steroidogenesis in cells cultured in the presence of LH. The strong inhibitory effects of EGF on androgen production that have been reported previously are only observed when cells that have lost part of their steroidogenic potential by prolonged culture in the absence of LH are acutely challenged with LH (or cholera toxin or dbcAMP) and EGF. Under the latter conditions EGF blocks the conversion of C21-steroids into C19-steroids. The stimulatory effects of EGF on androgen production are evident within the first hours of incubation and occur at ED50 values of 0.3 up to 2.5 ng/ml. They are not accompanied by any measurable change in the production of cAMP. The effects of EGF are compared to those of LHRH and those of SCF, a putative paracrine factor produced by Sertoli cells. Although there are many similarities between the effects of these three polypeptides on steroidogenesis in Leydig cells, our data indicate that they must act by different mechanisms. Moreover, SCF is the only one of these agonists that markedly stimulates androgen production in the presence of LH. PMID- 3017792 TI - Site and stage specific action of endogenous nuclease and micrococcal nuclease on histone genes of sea urchin embryos. AB - The early histone genes of sea urchin embryos are expressed exclusively during cleavage stages of embryogenesis. The chromatin containing these genes was examined by nuclease sensitivity. An endogenous nuclease active during cleavage, produces 1300-bp segments containing early histone genes. The cutting sites have been mapped; there are very sensitive sites close to the cap site for H1, H2A, H2B, and H4. Chromatin obtained from embryos of later stages, when the genes are not expressed, do not display this pattern of nuclease sensitivity. Micrococcal nuclease produces nucleosomes that contain histone genes when used with nuclei from later stages, but not with nuclei from cleavage stages. PMID- 3017793 TI - Identification and partial characterization of sperm receptor associated with the newly formed fertilization envelope from sea urchin eggs. AB - Prior studies from this laboratory have identified a proteoglycan-like component of high molecular weight from the surface of the egg of the sea urchin Strongylocentrotus purpuratus that serves as a receptor for sperm. In the present study, a glycoconjugate has been isolated from uncrosslinked fertilization envelopes prepared from eggs activated by treatment with ionophore. Based on its high molecular weight (greater than 5 X 10(6)) and its ability to inhibit fertilization by acrosome-reacted sperm, this glycoconjugate has the properties of the previously described sperm receptor. Components of the fertilization envelope of lower molecular weight (less than 10(6)) showed little or no ability to inhibit fertilization. PMID- 3017795 TI - Intercellular signaling is required for developmental gene expression in Myxococcus xanthus. AB - Certain developmental mutants of Myxococcus xanthus can be complemented (extracellularly) by wild-type cells. Insertions of Tn5 lac (a transposon which couples beta-galactosidase expression to exogenous promoters) into developmentally regulated genes were used to investigate extracellular complementation of the A group mutations. A- mutations reduced developmental beta galactosidase expression from 18 of 21 Tn5 lac insertions tested and that expression was restored to A- Tn5 lac cells by adding wild-type cells. The earliest A-dependent Tn5 lac normally expresses beta-galactosidase at 1.5 hr of development indicating a developmental block at 1-2 hr in A- mutants. A substance which can rescue the expression of this early Tn5 lac is released by wild-type (A+) but not by A- cells. This substance appears in a cell-free wash of wild-type cells or in starvation buffer conditioned by wild-type cells 1-2 hr after development is initiated. The conditioned starvation buffer also restores normal morphological development to an A- mutant. PMID- 3017794 TI - A global analysis of developmentally regulated genes in Myxococcus xanthus. AB - Tn5 lac is a transposon that fuses the transcription of lacZ to exogenous promoters. We generated 2374 Tn5 lac insertion-containing strains of Myxococcus xanthus, a soil bacterium that undergoes multicellular development which culminates in the formation of spores. Thirty-six strains were identified that specifically increase beta-galactosidase expression at some particular time during development and these expression times range from minutes after starvation initiates development to 24 hr, when sporulation begins. Different maximum levels of beta-galactosidase expression were also observed and the maximum for many strains that begin beta-galactosidase expression late in development was observed only if spores were disrupted. Seven of the 36 strains display mild to severe defects in aggregation and/or sporulation, as did an additional five strains whose beta-galactosidase expression was not developmentally regulated. Restriction maps of the DNA adjacent to the Tn5 lac insertions that are developmentally regulated and/or cause developmental defects show that most of the 41 insertions are in different regions of the Myxococcus genome. The developmentally regulated Tn5 lac insertions described here provide a set of at least 29 new developmental markers for Myxococcus. PMID- 3017796 TI - High frequency of class 3 allele in the human insulin gene in Japanese type 2 (non-insulin-dependent) diabetic patients with a family history of diabetes. AB - The restriction fragment length polymorphism in the 5' flanking region of the human insulin gene was studied in 155 nonobese Japanese subjects. The subjects consisted of 36 Type 2 (non-insulin-dependent) diabetic patients with a family history of diabetes mellitus, 42 Type 2 diabetic patients without a family history of diabetes, 42 Type 1 (insulin-dependent) diabetic patients, and 35 healthy volunteers who served as control subjects. It was demonstrated that, in Japanese healthy subjects and diabetic patients, the incidence of the insertion into 5' flanking region of the insulin gene was found to be significantly lower (p less than 0.05) than those in Caucasians and other races already investigated. Even though the class 3 gene allelic frequency in Type 2 diabetic patients without a family history of diabetes (0.060) was not higher than that in healthy subjects (0.014), in nonobese Type 2 diabetic patients with a family history of diabetes the allelic frequency of the inserted class 3 gene (0.111) was found to be significantly higher (p less than 0.02) than that in control subjects. These data suggest that the insulin gene polymorphism relates to the aetiology of diabetes mellitus. PMID- 3017797 TI - Studies on the insulin-antagonistic effect of catecholamines in normal man. Evidence for the importance of beta 2-receptors. AB - The insulin-antagonistic effect of adrenaline was studied in seven healthy subjects with the euglycaemic clamp technique using two insulin infusion rates (40 and 1200 mU X (m2)-1 min-1). The adrenergic receptor mediating the adrenaline effect was characterized by concomitant infusion of propranolol (beta 1 + beta 2 antagonist) or metoprolol (beta 1-antagonist). Each subject was studied four times (placebo, adrenaline, adrenaline + propranolol, adrenaline + metoprolol). Glucose turnover was measured with D(3-3H)-glucose. Similar plasma insulin levels were reached in all studies with the two insulin infusion rates (mean; placebo 51 +/- 3 and 7421 +/- 337 mU/l respectively). Glucose production was completely inhibited by the low insulin level during placebo infusion. Adrenaline antagonized this effect so that a significant glucose production was seen at the low but not at the high insulin level. Propranolol, but not metoprolol, reversed this insulin-antagonistic effect of adrenaline. Glucose utilization increased from 2.53 +/- 0.17 to 7.28 +/- 0.88 mg X kg-1 X min-1 during placebo when the insulin levels were increased from 4 +/- 0.3 to 51 +/- 3 mU/l. Increasing the insulin levels 150-fold to approximately 7500 mU/l only doubled the glucose utilization (14.68 +/- 1.14 mg X kg-1 X min-1). Adrenaline induced a pronounced inhibition of glucose utilization at both insulin levels (78% and 37% inhibition respectively). Propranolol, but not metoprolol, prevented this effect of adrenaline. Thus, physiological adrenaline levels exert a pronounced insulin antagonistic effect which is mediated by beta 2-receptor stimulation. The inhibitory effect on glucose uptake is maintained even at high insulin levels when hepatic glucose production is completely abolished. PMID- 3017798 TI - Bactericidal proteins and neutral proteases in diabetes neutrophils. AB - Reduced bacterial killing by polymorphonuclear leucocytes has been reported in patients with diabetes mellitus. Whether this is due to reduced content of bactericidal granular proteins has not been determined. We therefore immunochemically measured the content of myeloperoxidase, lactoferrin, lysozyme, cathepsin G and elastase in polymorphonuclear leucocytes from 50 insulin-treated diabetic patients. The peroxidase activity was also measured. Normal contents of myeloperoxidase and lactoferrin as well as normal peroxidase activity were found. The average contents of cathepsin G, elastase and lysozyme were 2.5, 3.2 and 2.6 micrograms/10(6) polymorphonuclear leucocytes, respectively, and thus 14, 45 and 18% higher than the contents of normal polymorphonuclear leucocytes. The results indicate that reduced intracellular killing of bacteria demonstrated in previous studies in diabetic patients does not appear to be related to a reduction in the content of bactericidal proteins. PMID- 3017800 TI - NH3 and propionate modulate the morphological response of aggregation-competent Dictyostelium discoideum to cAMP. AB - Two metabolites, NH3 and propionic acid, are known to act as morphogens during the development of Dictyostelium discoideum, specifically altering the course of morphogenesis and cytodifferentiation. They have also been shown to modulate the cAMP relay in this organism: NH3 by restricting intracellular accumulation, and propionate by inhibiting extracellular release. In the present study, we utilized the light-scattering properties of aggregation-competent cells in agitated suspension to demonstrate that the morphological responses of such cells to exogenous cAMP are also modulated by NH3 and propionate in a manner that has interesting implications for the overall control of morphogenetic movements in D. discoideum. Our experiments were conducted using a newly designed continuous-flow apparatus that represents a significant improvement in the technique. The apparatus is described in detail. PMID- 3017801 TI - [Viral infections and diabetic disease]. AB - Authors report recent data from medical literature on virus infections effect on diabetogenesis. Particularly they examine Rubella, Mumps, Coxsackie, Encephalomyocarditis and Herpesvirus infections which seem be more active on this process. Finally authors analyze the possible biological mechanism of viral diabetogenesis. PMID- 3017799 TI - Differentiation of TERA-2 human embryonal carcinoma cells into neurons and HCMV permissive cells. Induction by agents other than retinoic acid. AB - Retinoic acid induces the differentiation of NTERA-2 cl. D1 human embryonal carcinoma (EC) cells into neurons, cells permissive for the replication of human cytomegalovirus (HCMV), and other cell types that cannot as yet be classified but are distinguishable from the stem cells. We tested several additional agents for their ability to induce the differentiation of these EC cells. No differentiation was induced by butyrate, cyclic AMP, cytosine arabinoside, the tumor promoter 12 0-tetradecanoylphorbol 13-acetate (TPA), or the chemotherapeutic agent cis diaminedichloroplatinum, although morphological changes were detected at the highest concentrations of these agents that permitted cell survival. However, retinal, retinol, 5-bromouracil 2'deoxyribose (BUdR), 5-iodouracil 2'deoxyribose (IUdR), hexamethylene bisacetamide (HMBA), dimethylacetamide (DMA), and dimethylsulfoxide (DMSO) all induced some neuronal differentiation, but to a lesser extent than retinoic acid. Also, BUdR, IUdR, HMBA, and DMA induced the appearance of many cells permissive for the replication of HCMV. Differentiation was, in all cases, accompanied by the loss of SSEA-3, a globoseries glycolipid antigen characteristically expressed by human EC cells. However, another glycolipid antigen, A2B5, which appears in 60%-80% of differentiated cells 7 days following retinoic acid induction, was detected in less than 20% of the cells induced by the other agents studied. This implies that the HCMV-permissive cells induced by retinoic acid are not identical to those induced by BUdR, IUdR, and DMA. PMID- 3017802 TI - Cytomegalovirus colitis in acquired immune deficiency syndrome: radiologic spectrum. AB - Six cases of cytomegalovirus (CMV) colitis are described. The radiographic manifestations of this colitis are nonspecific and usually mimic the findings of ulcerative colitis with diffuse mucosal ulceration or granulomatous colitis with aphthous ulceration and skip areas. Terminal ileal involvement was noted in 1 patient. Nonspecific edema was present in 2 other cases. One patient demonstrated unusual cecal and ascending colonic nodularity due to pseudomembranes and, in another, large, flat discrete ulcerations were identified. Angiography, in 1 case, demonstrated marked hypervascularity and identified a site of hemorrhage in the ascending colon. With the radiographic identification of colitis in an immunocompromised patient, particularly a patient with acquired immunodeficiency syndrome (AIDS), CMV colitis must be strongly considered in the differential diagnosis. Endoscopic biopsy is the most effective method of establishing the diagnosis. PMID- 3017803 TI - Gastrointestinal hormones and gastrointestinal and pancreatic carcinomas. PMID- 3017804 TI - Effects of topical 5-aminosalicylic acid and prednisolone on prostaglandin E2 and leukotriene B4 levels determined by equilibrium in vivo dialysis of rectum in relapsing ulcerative colitis. AB - To determine the influence of inflammation and topical treatment with 5 aminosalicylic acid or prednisolone on arachidonic acid metabolism in vivo, we carried out a double-blind controlled study on the release of prostaglandin E2 and leukotriene B4 to the rectal lumen in 24 consecutive patients with proven distally located ulcerative colitis. Before and at days 15 and 29 a dialysis bag was placed in the emptied rectum for 4 h prior to assessing clinical, endoscopic, and histologic disease activity. A single enema was given daily at bedtime (1 g 5 aminosalicylic acid or 25 mg prednisolone) until complete remission or for a maximum of 4 wk. Clinical and endoscopic remission was obtained in 16 (7 on 5 aminosalicylic acid) and 11 (3 on 5-aminosalicylic acid) patients, respectively. Luminal concentrations of prostaglandin E2 and leukotriene B4 were positively correlated to disease activity and significantly decreased among the prednisolone treated patients. In both treatment groups a decrease toward normal levels occurred in patients responding to therapy. In retrospect, the pretreatment prostaglandin E2 and leukotriene B4 levels were significantly higher in patients not responding to therapy than in those improving during treatment. In conclusion, luminal prostaglandin E2 and leukotriene B4 levels may prove more useful predictors of the outcome of treatment in relapsing ulcerative colitis than clinical indices of disease activity. PMID- 3017806 TI - Regional assignments of three polymorphic DNA segments on human chromosome 15. AB - Hybridization of probe pDP151 (locus D15S2) to genomic human DNAs digested with EcoRI revealed allelic restriction fragments 9 and 11 kilobase-pairs (kb) in length. Hybridization of pDP151 to EcoRI-digested DNAs from 21 Chinese hamster X human hybrid cell clones containing different subsets of human chromosomes demonstrated cosegregation of the 9 and 11 kb EcoRI fragments with human chromosome 15. D15S2 and two other polymorphic loci previously mapped to chromosome 15--D15S1 and D15S6--were localized to specific regions on human chromosome 15. Eight Chinese hamster X human somatic cell hybrid clones derived from a human donor heterozygous for a balanced translocation between chromosomes 15 and 22 [t(15;22)(q14;q13.3); Oliver et al, Cytogenet Cell Genet 22:503-510, 1978] were studied. After digestion of human and hybrid DNAs with HindIII and Southern blotting, pDP151 (D15S2) and pMS1-14 (D15S1) hybridized to fragments of 4 and 4.5 kb, respectively. Further, pMS1-14 (D15S1) and p9-1a (D15S6) hybridized to EcoRI fragments of 3.5 and 3.2 kb. All fragments cosegregated with the der(22) derivative chromosome containing region 15q14----15qter. In situ hybridization of these probes to normal human chromosomes mapped the corresponding loci with greater precision: D15S1 to 15q15----15q21, D15S2 to 15q15----15q22, and D15S6 to 15q22----15q24. PMID- 3017805 TI - Transferrin receptors in the human gastrointestinal tract. Relationship to body iron stores. AB - Fluorescently labeled antibodies were used to identify transferrin receptors and mucosal transferrin in human gastrointestinal biopsy sections. Transferrin receptors were evident in the villous epithelium and the crypt areas of duodenum, ileum, and colon, predominantly in the basal-lateral area. In 7 subjects with low iron stores, the intensity of duodenal villous staining for receptor, on a scale of 0-4, was 2.1 +/- 0.3 (mean +/- SD). This value was significantly higher than the value in 13 subjects with normal iron stores (1.1 +/- 0.4). In 5 patients with hereditary hemochromatosis, duodenal transferrin receptor staining was not significantly different from that in the subjects with normal iron stores. Transferrin staining was found in the apical cytoplasm of epithelial cells in the duodenum, ileum, and colon, but observer assessment was not sufficiently reproducible to make a quantitative analysis. Our results suggest that iron deficiency is accompanied by an increase in transferrin receptors in duodenal absorptive cells, and the genetic lesion in hemochromatosis does not involve an increase in transferrin receptors in the intestinal mucosa compared with subjects with normal iron stores. PMID- 3017807 TI - Recombination between IS5 elements: requirement for homology and recombination functions. AB - Intermolecular recombination between two IS5 elements was measured, using bacteriophage lambda recombination vectors, and was compared to recombination between two copies of an SV40 segment cloned into the same vectors. Experiments were conducted in the presence and in the absence of RecA and Red functions, and with the recombining inserts in the same or in reversed orientation. Under all conditions, IS5 elements recombined in a manner similar to the SV40 inserts, indicating that IS-encoded functions did not confer measurable additional intermolecular recombination ability to IS5 in E. coli K-12. Bacteriophages containing reversed IS5 inserts, for which the 16 base pair (bp) termini are identical in 15 positions and which display 12 bp of uninterrupted homology, recombined at approximately the same low frequency under Rec+ and Rec- conditions, indicating that these short homologies were not good substrates for the Rec system. Bacteriophages having reversed inserts recombined better under Red+ than under Red- conditions, but the crossovers were located in nonhomologous regions flanking the element termini. This suggests that 12-bp homologies are not good substrates for the Red system. PMID- 3017808 TI - Geriatric psychiatry in the general hospital: the integration of services and training. AB - This article describes the development of a new psychiatric service for the elderly based in a university general hospital. The service aims to provide and coordinate comprehensive psychiatric care for a defined population and also to serve as a tertiary care unit with academic responsibilities. As part of the Department of Psychiatry, this specialized service is fully integrated into all the major clinical and teaching components of the department. As the demand for more geriatric psychiatry services and training increases over the next decade, innovative ways of responding to this need will have to be developed. This integrative model in a general hospital setting has enhanced the quality of health care delivery to the elderly at the same time as improving training and recruitment. PMID- 3017809 TI - [Restriction mapping of recombinant plasmids carrying the genes for arginine biosynthesis in Escherichia coli K-12]. AB - ArgA and argECBH genes of Escherichia coli K-12 were cloned on the pBR322 vector. Restriction maps of the recombinant plasmids were constructed. Deletion mutants of these recombinant plasmids, retaining the functional argA and argE genes, were obtained using different restriction enzymes. All of the recombinant derivatives have the replication properties of the pBR322 vector. PMID- 3017810 TI - [Isolation and characteristics of compound transposons Tn1000::Tn5]. AB - Two types of compound transposons were derived. In the first case, transposon Tn5 is inserted into the gene responsible for Tn1000 transposase synthesis. In the other, Tn5 is inserted into the region near the left end of Tn1000, where no functionally significant genes were found. It is known that translocation of the compound transposons does not depend on their size and takes place with the efficiency close to that characteristic of the intact Tn1000. Insertion of Tn5 into the gene coding for Tn1000 transposase results in sharp decrease in the efficiency of Tn1000 translocations. This effect, however, may be eliminated by introduction into the cell of the intact Tn1000. PMID- 3017811 TI - Tn951 derivatives designed for high-frequency plasmid-specific transposition and deletion mutagenesis. AB - We describe the construction of a system allowing high-frequency transposition and deletion mutagenesis with class-II transposons containing a kanamycin or a chloramphenicol-resistance marker. The system utilizes the transposition function of Tn3 and the resolution function of Tn951/Tn2501 which leads to an uncoupling of the resolution and repression functions. It consists of defective transposons inserted into conjugative, replication thermosensitive plasmids. The properties of the system are: easily selectable resistance markers, high transposition frequencies onto plasmids, low transposition frequencies onto the host chromosome, placement of the tnpA gene outside the transposons so that "second generation" transposition does not occur, possibility to transpose the whole system onto other plasmid vectors with different selection strategies, consecutive use of two transposons for deletion mutagenesis and restriction mapping. PMID- 3017812 TI - Structure of wheat gamma-gliadin genes. AB - We have cloned and sequenced two linked members of the wheat (Triticum aestivum) gliadin multigene family. One gene encodes a gamma-gliadin which is 292 amino acids (aa) long. S1 mapping indicates that this gene is transcriptionally active. The second gene, which is only marginally active by S1 mapping, is closely related to the first gene. Moreover, it is probably incapable of encoding a full length gamma-gliadin due to the presence of two premature in-frame stop codons. Neither gene contains introns. Nucleotide sequences of the two genes show the presence of characteristic repeats which have a derived aa consensus sequence Pro Gln-Gln-Pro-Gln-Gln-Pro-Phe-Pro-Gln. A comparison of the promoter regions of these gamma-gliadin genes with those of the alpha-gliadin genes shows the presence of a highly conserved region that could be involved in tissue specific and developmental regulation. PMID- 3017813 TI - An inducible eukaryotic host-vector expression system: amplification of genes under the control of the polyoma late promoter in a cell line producing a thermolabile large T antigen. AB - We have taken advantage of the inherent instability of integrated polyoma (Py) DNA sequences in the presence of a functional viral large T antigen (LT) to develop a eukaryotic host-vector system where copy number is controlled by temperature. A mouse cell line WOP32-4, that constitutively expresses a temperature sensitive (ts) LT, was transfected with plasmids containing the Py origin of DNA replication (ori) and either a neomycin-resistance gene (neo) or chloramphenicol acetyl transferase gene (cat) linked to the Py late promoter. Stable transformants were selected at 39 degrees C, the non-permissive temperature for the ts LT function. Upon shift to 33 degrees C, the resident Py sequences present in the WOP32-4 cells cannot excise due to an ori deletion. However, excision of the transfected plasmid molecules and subsequent extrachromosomal replication occur at high rates leading in some cases to the production of 1000-2000 copies per cell (average) of the plasmid. Proportional increases in either neo-specific mRNA or CAT activity were also observed. In situ hybridization for one cell line indicated that about 20% of temperature-shifted cells contained amplified plasmid DNA. PMID- 3017814 TI - A rapid and simple procedure for the preparation of homogeneously labeled DNA probes. AB - This report describes a novel technique for the preparation of homogeneously labeled DNA probes. The procedure uses a heteroduplex intermediate, which is directly prepared from a double-stranded plasmid, and therefore requires neither sub-cloning of the fragment of interest into a specialized vector nor the use of a synthetic primer. The technical advantages of the procedure are demonstrated by preparation of a probe able to determine the precise location and efficiency of utilization of the polyadenylation site used during transcription of the rat preproinsulin II gene in a transfected avian cell line. PMID- 3017815 TI - [Structural-metabolic and functional-behavioral indices of the postnatal toxicity of tetramethylthiuram disulfide in an experiment]. PMID- 3017817 TI - [Standards for ammofos in the atmosphere]. PMID- 3017816 TI - [Hazards of the redistribution of chemical and biological pollutants in an aqueous environment]. PMID- 3017818 TI - [Blastomogenic properties of erionite (acicular zeolite)]. PMID- 3017820 TI - [Role of prolactin and sex steroids in the pathophysiology of the mammary gland. IV. Various markers of tissue activity of the rabbit mammary gland after active immunization with prolactin]. PMID- 3017821 TI - Hormonal effects on collagenolytic activity in the isolated human ovarian follicular wall. AB - Tissue pieces from the wall (i.e. tunica albuginea with adjacent theca externa) of human follicles were incubated with and without various hormones and their potential influence upon the collagenolytic activity was evaluated. Following incubation the collagenase activity was determined in the incubation medium by measurement of the hydrolytic activity against the synthetic peptide 2,4 dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH. Stimulated collagenolytic activity was seen in the presence of relaxin and oxytocin whereas prostaglandin E2, prostaglandin F2 alpha, progesterone and 17 beta-estradiol were without effect. It is concluded that the stimulated collagenolytic activity induced by relaxin and oxytocin may be of importance for the degradation of collagen which occurs prior to follicular rupture. PMID- 3017819 TI - [Current aspects of the study of the combined effects of pesticides and fertilizers (review of the literature)]. PMID- 3017822 TI - Cytobiochemical studies of liver parenchyma in experimental histamine shock. PMID- 3017823 TI - [Viral conjunctivitis]. PMID- 3017824 TI - [Stimulation of the adrenal cortex in long-term steroid therapy. Increase of free cortisol in the urine following depot ACTH administration]. PMID- 3017825 TI - [Alzheimer's disease. Biological and pharmacotherapeutic aspects]. PMID- 3017826 TI - Some Epstein-Barr virus-transformed B lymphoblastoid cell lines have an altered structure around the mixed lymphocyte culture-inducing domain on DR molecules. PMID- 3017827 TI - Redox status in the control of pulmonary vascular tone. AB - There is mounting evidence that the tone, and possibly the structure, of the pulmonary vasculature is regulated by the redox status (GSH/GSSG, NADPH/NADP) of the pulmonary vascular smooth muscle cell. This hypothesis may explain some studies which have examined endogenous mediators and inhibitors of oxidative phosphorylation. An analogous model of redox regulation of cellular function and calcium flux is seen in the pancreatic beta-cell. The importance of redox status in the regulation of enzyme reactivity is well recognized. Sulfhydryl redox status may also be involved in the ability of the carotid body to detect changes in oxygen tension. It is likely that sulfhydryl redox status transduces the effect of changing oxygen tension for many physiologic control systems, including the pulmonary vasculature. PMID- 3017829 TI - Transcatheter partial splenic arterial embolization in patients with hypersplenism: a clinical evaluation as supporting therapy for hepatocellular carcinoma and liver cirrhosis. AB - Eleven cases with hypersplenism, one with liver cirrhosis and ten with hepatocellular carcinoma (HCC) associated with liver cirrhosis, underwent transcatheter partial splenic arterial embolization. In four of ten HCC cases, the spleen was accidentally infarcted during the procedure of transcatheter hepatic arterial embolization (TAE). The mean infarcted area of the spleen was 55.7%. An increase in the peripheral platelet count was particularly remarkable and continued over one year after the embolization. High fever and abdominal pain were observed in all cases. The fever was seen for 18.0 days and pain was noted for an average of 12.8 days after the embolization. Other adverse effects such as pleural effusion and ascites were transitorily observed. Splenic embolization is an effective supporting therapy for hypersplenism in patients with cirrhosis or HCC. PMID- 3017828 TI - Combination chemotherapy with c-DDP, cyclophosphamide, etoposide and prednisolone in small cell carcinoma of the lung. PMID- 3017830 TI - [Studies on new adenovirus types of subgenus D causing conjunctivitis--antigenic and restriction endonuclease analysis of adenovirus types 19 and 37]. AB - Neutralization test and DNA restriction endonuclease analysis were performed in strains of adenovirus types 19 (Ad-19) and 37 (Ad-37) isolated from patients with acute conjunctivitis in Sapporo, Kao-Hsiung (Taiwan) and Pusan (Korea) in the period of 1979 and 1984. Although Ad-19 and Ad-37 were differentiated, they were related to each other in neutralization test. There was no difference between prototype strain and isolates in Ad-19 and Ad-37 strains. By DNA restriction endonuclease analysis Ad-19 isolates were clearly distinct from Ad-19 prototype strain (AV-587) and Ad-37 strains. And it was suggested that these Ad-19 isolates from East Asia were as same as isolates from Europe named Ad-19a. Ad-37 isolated strains were as same as Ad-37 prototype strain (GW) by SmaI, SacI, SalI, BamHI, EcoRI, XhoI, HindIII restriction analysis. But some strains were distinct from prototype strain by only HindIII restriction analysis, and they were new subtype of Ad-37 which had never been reported. PMID- 3017832 TI - The effect of long term insulin treatment on kidney Na,K-ATPase activity in streptozotocin diabetic rats. PMID- 3017831 TI - The renal sensitivity for endogenous parathormone in patients with primary hyperparathyroidism, vitamin D deficiency and renal stones. AB - Baseline levels and increases in urinary cyclic AMP excretion (UcAMP) and immunoreactive parathormone (iPTH) were studied before and during infusion of EDTA in euparathyroid patients with renal stones (n=11), patients with primary hyperparathyroidism (PHP; n=14) and patients with vitamin D deficiency (n = 12). In all three groups, EDTA evoked a significant rise in iPTH and UcAMP. In patients with PHP and in those with vitamin D deficiency, there was a sufficiently close relationship between increments in iPTH (delta iPTH) and in UcAMP (delta UcAMP) (r = 0.90, P less than 0.001 and r = 0.67, P less than 0.02, respectively) to use this model to assess renal sensitivity for changes to endogenous PTH levels. We quantified sensitivity of the kidney for PTH, by calculating the ratio delta UcAMP/delta TPTH for the three studied groups. The ratio was comparable in patients with renal stones (16.7 +/- 10.3) and PHP (13.8 +/- 4.9, P greater than 0.10), but was significantly increased in patients with vitamin D deficiency (33.2 +/- 17.9; P less than 0.01 versus patients with renal stones and P less than 0.01 versus patients with PHP). Within the group of patients with PHP there was no correlation between baseline serum calcium concentrations and the ratio delta UcAMP/delta TPTH. It is concluded that in patients with vitamin D deficiency, renal sensitivity to PTH is increased compared with patients with PHP and euparathyroid patients with renal stones, perhaps an expression of a teleological useful adaptation of end organ sensitivity. PMID- 3017834 TI - Alpha-adrenergic receptors and cyclic AMP production in a group of schizophrenic patients. AB - Alpha 2-adrenergic receptor function was measured in platelets from chronic schizophrenic patients and normal controls. The number of alpha 2-receptors was greater in patients' platelets, and the prostaglandin E1 (PGE1)-stimulated cyclic AMP (cAMP) production lower, when compared with the normal controls. The changes measured may occur only in the platelet, but if central nervous system neurons share with platelets these changes, one might speculate that an increase in the number of alpha 2-receptors and a decrease in cAMP production may relate to the psychopathology of schizophrenia. PMID- 3017833 TI - Delta-sleep-inducing peptide (DSIP) inhibited CRF-induced ACTH secretion from rat anterior pituitary gland in vitro. AB - Delta-sleep-inducing peptide (DSIP, 10(-9) - 10(-7) M) significantly inhibited the CRF-induced ACTH release from rat anterior pituitary quarters in vitro. 10( 8) M DSIP showed the most prominent inhibition. DSIP (10(-8) M) also inhibited the CRF-activated cAMP levels in anterior pituitary tissue. DSIP did not influence basal ACTH or cAMP levels. Prostaglandin E2 (PGE2)-release from anterior pituitary quarters was not changed by DSIP. From these results, we conclude that DSIP inhibits CRF-induced ACTH release at the pituitary level through the inhibition of the cAMP system in corticotrophs. The involvement of PGE2 in this phenomenon is unlikely. PMID- 3017835 TI - PT rapidly moves from hospitals to other settings. PMID- 3017836 TI - Identification of carriers of mutant prealbumin gene associated with familial amyloidotic polyneuropathy type I by Southern blot procedures: study of six pedigrees in the Arao district of Japan. AB - Fifty-six Japanese individuals from six pedigrees with familial amyloidotic polyneuropathy (FAP), together with 2 individuals with symptomatic FAP from an unknown pedigree were analyzed, using the Southern blot procedures for the prealbumin gene structure. A human prealbumin cDNA was used as the probe. Altogether, these individuals included 20 with symptomatic FAP, 30 who were asymptomatic, and 8 disease-free spouses. Twenty individuals with symptomatic FAP were all heterozygous for the prealbumin genes, carrying one normal and one mutant gene. We confirmed the direct linkage between the mutant prealbumin gene and the Japanese type of FAP. Moreover, 10 of the 30 asymptomatic individuals from pedigrees with FAP were also heterozygous for the prealbumin gene. The number of asymptomatic individuals with the mutant prealbumin gene showed age related decreases, and none was over 40 years. A linkage between the mutant prealbumin gene and serum levels of the prealbumin variant was also evident. PMID- 3017837 TI - The gene for human apolipoprotein CI is located 4.3 kilobases away from the apolipoprotein E gene on chromosome 19. AB - We have isolated a cDNA clone for apolipoprotein CI and a genomic clone for apolipoprotein E, and by hybridisation and mapping experiments found the gene for apoCI to be located on the genomic apoE clone. The distance between the loci was 4.3 kb. PMID- 3017838 TI - Clustered GATA repeats (Bkm sequences) on the human Y chromosome. AB - Sixty eight individual clones of a human Y chromosome cosmid library were screened for the presence of GATA repeats, the major component of Bkm-related DNA sequences. Nine cosmid clones were found to cross-hybridize. The sequence organization of the repetitive base quadruplet GATA was analyzed using synthetic oligonucleotide probes. Subclones of GATA-positive cosmid clones were used for chromosomal localization of the Y-derived DNA sequences thus revealing male specificity or male-female homology. PMID- 3017839 TI - Bkm sequences are polymorphic in humans and are clustered in pericentric regions of various acrocentric chromosomes including the Y. AB - Probes of uncloned Bkm satellite DNA and a Drosophila clone 2 (8), consisting mainly of GATA repeats related to a major sequence component in Bkm, have been used to probe Southern blots of human male and female DNAs obtained from a Caucasian and an Australian aboriginal population and to human chromosomes in situ. Hybridization was observed to a distinct and an indistinct series of bands against a smeared background. The same distinct bands are identified in the DNA samples with both probes, but are most readily detected using the uncloned Bkm probe. Most restriction bands are common to both populations and some are polymorphic. However, certain bands appear to be characteristic of the Australian aboriginal samples. There are no distinct sex-linked patterns. However all of the small acrocentric human chromosomes, including the Y chromosome show hybridization to uncloned Bkm in situ. PMID- 3017840 TI - The isolation of genomic recombinants for the human apolipoprotein B gene and the mapping of three common DNA polymorphisms of the gene--a useful marker for human chromosome 2. AB - We have used four independently isolated cDNA probes for human apolipoprotein B (apo B), to isolate overlapping genomic recombinants for the 3' portion of the apo B gene. The cDNA clones and a unique fragment from the genomic recombinant have been used to identify the human apo B gene in DNA from a series of rodent X human somatic cell hybrids. Our results provide evidence for the assignment of this gene to the short arm of human chromosome 2 (p23-pter). We have used the cDNA probes to identify three common DNA polymorphisms. The first, detected with the restriction enzyme XbaI and our probe pAB4, has a rare allele frequency of 0.48. The other two polymorphisms are detected with the probe pAB3. The enzyme MspI detects at least three alleles, with frequencies of 0.67, 0.16 and 0.15, while that detected with the enzyme EcoRI has a rare allele frequency of 0.12. The relative position of these polymorphisms has been mapped using the genomic recombinants. Investigation of a small number of haplotypes indicates that there is linkage equilibrium between the polymorphisms, which have a total polymorphism information content (PIC) value of more than 0.8. These polymorphisms will provide useful markers for genetic studies on chromosome 2 and for the analysis of the involvement of variants of the apo B gene in the development of hyperlipidaemia. PMID- 3017841 TI - A routine method for the establishment of permanent growing lymphoblastoid cell lines. AB - Permanent lymphoblastoid cell lines are of great practical value in human clinical and experimental genetics. A detailed protocol for routine use is given for the establishment of lymphoblastoid lines from peripheral blood using Epstein Barr virus and the immunosuppressivum Cyclosporin A. In addition, the biologic basis of this transformation system is briefly summarized. PMID- 3017842 TI - Studies of a DNA marker (G8) genetically linked to Huntington disease in British families. AB - Close genetic linkage has been shown between the DNA sequence G8 (locus D4S10) and 16 British families with Huntington disease using the HindIII, EcoR1, Nci1, and Pst1 polymorphisms detected by G8, and by combining all the polymorphisms to give a combined haplotype. Two recombinants have been detected in these families giving a maximum lod score of 17.60 at a theta of 0.02. These results confirm the originally reported linkage between the loci and provide evidence against significant multilocus heterogeneity for Huntington disease. PMID- 3017843 TI - A polymorphic locus on the long arm of chromosome 20 defined by two probes from a single cosmid. AB - Two probes from the random human cosmid c1-37 detect restriction fragment length polymorphisms in humans. The loci revealed by these probes are in linkage equilibrium and constitute a compound polymorphic locus with a polymorphism information content of 0.54. A somatic cell hybrid panel has been used to map the probes to chromosome 20; in situ hybridization studies confirm this localization and indicate that the locus is on 20q13. This is the first polymorphic locus to be assigned to the long arm of chromosome 20. PMID- 3017844 TI - First trimester prenatal diagnosis of 21-hydroxylase deficiency by linkage analysis to HLA-DNA probes and by 17-hydroxyprogesterone determination. AB - The close genetic linkage between the gene for congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency and HLA genes allowed us to use the polymorphism of this system as a marker of the disease. HLA genotyping can be performed by using restriction enzyme fragments hybridized with specific probes instead of serologic methods. In seven pregnancies at risk for 21-OH deficiency, a first trimester prenatal diagnosis has been performed by determining the fetal genotype by linkage analysis of DNA from chorionic villi using HLA class I and class II probes. In four of these pregnancies, determination of 17-OH progesterone in first trimester amniotic fluid afforded a complementary approach to the diagnosis. PMID- 3017845 TI - The structural gene for the mitochondrial aldehyde dehydrogenase maps to human chromosome 12. AB - A cloned 850 bp cDNA fragment corresponding to the 3'-coding part of human ALDHI mRNA was used as a probe for the chromosomal assignment of the ALDHI gene. Southern blot analysis of human-rodent somatic cell hybrids indicates that the human ALDHI gene resides on chromosome 12. PMID- 3017846 TI - Deficiency of natural killer activity, but not of natural killer binding, in patients with lymphoadenopathy syndrome positive for antibodies to HTLV-III. AB - Blood lymphocytes (BL) of eleven patients with lymphoadenopathy syndrome (LAS) were studied for natural killer (NK) activity against the K562 cell line (using both the standard 51Cr release assay and the single-cell cytotoxicity assay on poly-L-lysine-coated coverslips) and for surface phenotype (employing OKT4, OKT8 and Leu7 monoclonal antibodies). A significant reduction in NK activity and in NK active cells was detected, while the percentage of target binding cells was not affected. Furthermore, the OKT4/OKT8 ratio was found to be inverted, and the Leu7+ subpopulation expanded. The patients had high titers of anti-HTLV-III antibodies. This study indicates that defective NK activity in LAS is secondary to an abnormality in the lytic event itself and not in target binding. PMID- 3017847 TI - [Polymorphonuclear neutrophils in dermatologic pathology]. PMID- 3017848 TI - [Scleromyxedema: a new therapeutic proposal]. PMID- 3017851 TI - A study of the HLA-D region in patients with classic Kaposi's sarcoma. AB - The HLA-D region in nine Sardinian patients with classic Kaposi's sarcoma was studied with two restriction enzymes, Eco RI and Eco RV, and two cDNA probes, DR beta and DQ beta. A total of 41 polymorphic restriction fragments were identified. One, an 11.5 kb Eco RV DQ beta fragment, was present in three of the patients but in none of the controls; a second, an 8.0 kb Eco RV DR beta fragment, was present in six patients and all the controls. No single fragment was identified which was significantly over or under-represented in either group. PMID- 3017852 TI - Role of hepatitis B virus in primary carcinoma of liver. PMID- 3017849 TI - Respiratory symptoms and pulmonary function in men exposed to toluene diisocyanate (TDI) in a foam factory. PMID- 3017850 TI - Chemotherapy of inoperable bronchogenic carcinoma. PMID- 3017853 TI - Atrial natriuretic factor and cyclic guanosine 3',5'-monophosphate in vascular smooth muscle. AB - To elucidate the molecular mechanism of the vascular action of atrial natriuretic factor (ANF), we investigated the effects of synthetic ANF and sodium nitroprusside on the levels of intracellular cyclic nucleotides and prostacyclin (measured as its stable metabolite 6-keto-prostaglandin F1 alpha) in cultured vascular smooth muscle cells from rat mesenteric artery and, in some experiments, from rat renal artery. Both ANF and sodium nitroprusside increased intracellular cyclic guanosine 3',5'-monophosphate (cGMP) levels in a dose-dependent manner but did not affect cyclic adenosine 3',5'-monophosphate levels or 6-keto prostaglandin F1 alpha synthesis. The stimulatory effect of ANF and sodium nitroprusside on cGMP levels were additive. Neither the deprivation of extracellular Ca2+ nor calcium entry blockers affected ANF-stimulated cGMP levels. Preincubation of ANF or sodium nitroprusside with kallikrein attenuated only the effect of ANF on cGMP levels. The effect of kallikrein was abolished by serine protease inhibitors. In contrast, the oxidant methylene blue inhibited the effect of sodium nitroprusside on cGMP levels, but not that of ANF. The stimulatory effect of ANF on cGMP levels was greater in cells from renal artery than in those from mesenteric artery. These results in cultured vascular smooth muscle cells further support the hypothesis that cGMP mediates the vasorelaxant action of ANF. PMID- 3017854 TI - Vascular responses to ouabain and norepinephrine in low and normal renin hypertension. AB - A circulating Na+, K+-ATPase inhibitor may cause arterial hypertension in patients with suppressed plasma renin activity, either directly or by sensitizing peripheral vessels to alpha-adrenergic stimulation. This hypothesis was tested by evaluating forearm arteriolar (plethysmographic technique) response to exogenous alpha-adrenergic stimulation by a 2-minute intra-arterial infusion of norepinephrine (0.1 microgram/dl tissue per minute) and to Na+, K+-ATPase inhibition by sequential 20-minute intra-arterial infusions of ouabain (0.36 and 0.72 microgram/dl tissue per minute). Two groups of hypertensive subjects with suppressed plasma renin activity, either essential or secondary to aldosterone excess, were compared with age-matched and sex-matched hypertensive subjects with normal plasma renin activity (n = 7 per group). No significant differences in forearm vascular response to norepinephrine were found among the three groups. Ouabain caused a highly significant, dose-related increment in forearm vascular resistance that was not accompanied by changes in the contralateral limb or systemic blood pressure. No significant interindividual differences in vascular responsiveness to ouabain were found. The individual increments in forearm vascular resistance during ouabain administration were unrelated to basal values or to plasma aldosterone, norepinephrine, or potassium concentrations. These data are not consistent with the hypothesis that suppressed basal Na+, K+-ATPase activity is primarily a characteristic of hypertensive patients with unresponsive plasma renin activity. Overall, these results cast doubts on the possibility of linking the development of human low renin hypertension to an endogenous Na+, K+ ATPase inhibitor. PMID- 3017855 TI - Polymorphonuclear leukocyte response to stimulation in vitro during pregnancy. AB - Oxidative metabolism of polymorphonuclear leukocytes (PMNLs) isolated from pregnant women in the third trimester and from controls were studied using zymosan-induced chemiluminescence (CL) and f-Met-Leu-Phe-stimulated superoxide (O2-) generation. CL was significantly increased during pregnancy, but a decrease was noted in cytochrome c reduction. Total cellular levels of beta-glucuronidase and lysozyme were diminished in PMNLs from pregnant subjects, with unaltered concentrations of cytosol lactate dehydrogenase. The capacity of PMNLs from pregnant women to degranulate did not differ from controls. It is suggested that during pregnancy, in vivo stimulation of PMNLs may occur to account for these changes. PMID- 3017856 TI - Activation-dependent redistribution of cellular components alters susceptibility of human neutrophils to cross-linking agents. AB - Contrary to resting cells, neutrophils stimulated with concanavalin A resist inhibition by bifunctional N-hydroxysuccinimide esters. Con A prestimulated, cross-linker-treated cells released superoxide upon restimulation with PMA but did not respond to chemotactic peptides. Although rates of PMA-elicited NADPH oxidase activity were lowered by the treatment, the activation parameters, namely lag times of the reaction, were not altered. The protection by Con A against blockade by cross-linkers developed concomitantly to the activation of NADPH oxidase and indicated redistribution of cross-linker-susceptible cellular components responsible for activation with PMA. The identity of these components is discussed. PMID- 3017857 TI - Converting enzyme activity of free airway cells. AB - The pulmonary circulation was previously shown to inactivate and even activate a number of autacoids. Further studies showed that these enzymatic activities were localized in vascular endothelial cells. The present results shows that guinea pig free airway cells (FACs) convert angiotensin I to angiotensin II and inactivate bradykinin, whereas they do not inactivate serotonin, prostaglandin E2, histamine, and noradrenaline. In the presence of a converting enzyme inhibitor (SQ14225), the angiotensin I was not converted into angiotensin II, but bradykinin was still inactivated, although the speed of inactivation was decreased. Furthermore, when FACs were separated as adherent and nonadherent cells, the cleavage of angiotensin I by adherent cells was similar to that of the total cell suspension. The inactivation of bradykinin was more pronounced in incubation with nonadherent than with the adherent cells. Using selected enzyme inhibitors, we also showed that the conversion of angiotensin by FAC is the result of specific kinase II activity most likely located in macrophages, whereas the bradykinin inactivation is the result of the cooperation of many enzymes including kinase I and II produced by various cell types. PMID- 3017858 TI - Effect of gold compounds on NADPH oxidase system of human neutrophils. AB - The inhibitory effects of gold compounds on the NADPH oxidase system of human polymorphonuclear leukocytes (PMNs) has been investigated. Auranofin (0.5-4.0 micrograms Au/ml) suppressed the rate of superoxide anion generation as well as the total yield in cells stimulated with phorbol myristate acetate and f-Met-Leu Phe. This implies that drug action may be occurring at the level of protein kinase C or steps subsequent to this in the signal transduction sequence. Sodium aurothiomalate (1-100 micrograms Au/ml) lacked such activity. Neither gold compound altered the ability of the granule-rich fraction of PMNs to produce oxy radicals whether this fraction was obtained from drug-treated cells or was treated after its isolation. Therefore, in order for auranofin to exhibit its inhibitory effects on the NADPH oxidase system, an intact cell membrane is necessary. PMID- 3017859 TI - Effects of gold compounds on function of phagocytic cells. Comparative inhibition of activated polymorphonuclear leukocytes and monocytes from rheumatoid arthritis and control subjects. AB - The effect of sodium aurothiomalate and auranofin on the generation of superoxide anions (O2-) by polymorphonuclear leukocytes (PMNLs) and adherent mononuclear phagocytic cells (AMNCs) has been investigated. Sodium aurothiomalate at final concentrations of 1, 10, and 100 micrograms Au/ml and auranofin ranging from 0.1 to 2.0 micrograms Au/ml were used in the reactions involving all cell types. Results have been compared between cells drawn from normal controls and patients with active rheumatoid disease. The effect of gold compounds on both cell types was assessed following activation by phorbol myristate acetate (1 X 10(-8) M) and N-formyl-methionyl-leucyl-phenylalanine (1 X 10(-4) M) using a cytochrome c reduction method. Sodium aurothiomalate at the maximum concentration modestly inhibited O2- generation by PMNLs but not AMNCs. Auranofin inhibits O2- generation by both cell types. Inhibition of cells from patients with rheumatoid arthritis was greater than that seen with cells from normal controls. PMID- 3017860 TI - Iodine deficiency disorders in India: review of control measures. PMID- 3017861 TI - [Risks of transmission of the AIDS virus: preventive approaches in dental medicine]. PMID- 3017862 TI - Purification and characterization of type II heat-labile enterotoxin of Escherichia coli. AB - Type II heat-labile enterotoxin (LT-II) of Escherichia coli has several biologic activities similar to cholera toxin (CT) and E. coli type I heat-labile enterotoxin (LT-I), but it is not neutralized by antiserum prepared against CT or LT-I. LT-II was purified from E. coli SA53 and from E. coli HB101(pCP3837), a strain that contains the cloned LT-II genes in a hybrid plasmid and produces up to 600 times more LT-II than does SA53. Purification involved sonic disruption of bacterial cells, ammonium sulfate fractionation, chromatography on Affi-Gel Blue, chromatofocusing, and gel filtration on Sephadex G-100. The LT-II purified to apparent homogeneity from HB101(pCP3837) had an isoelectric point of 6.8, induced increased vascular permeability in rabbit intracutaneous tests, caused rounding of cultured Y1 adrenal cells accompanied by increased intracellular cyclic AMP, and was 25 to 50 times more potent than CT or LT-I in the Y1 adrenal-cell assay. In contrast, purified LT-II did not cause secretion in ligated rabbit ileal segments at doses corresponding to CT controls that gave strongly positive reactions. LT-II was composed of two different polypeptides with MrS of 28,000 (A) and 11,800 (B); treatment of LT-II with trypsin cleaved the A polypeptide to fragments A1 (Mr, 21,000) and A2 (Mr, 7,000). The activity of LT-II was not blocked by ganglioside GM1 at concentrations that inactivated LT-I or CT. Antiserum against the LT-II from E. coli HB101(pCP3837) completely neutralized purified LT-II and the LT-II in crude extracts of SA53, but it did not neutralize purified LT-I or CT. PMID- 3017864 TI - Isolation and characterization of the sucrose 6-phosphate hydrolase gene from Streptococcus mutans. AB - The Streptococcus mutans GS-5 gene, scrB, coding for sucrose 6-phosphate hydrolase activity has been cloned into Escherichia coli utilizing the bacteriophage replacement vector lambda L47.1. DNA sequences containing the gene were initially subcloned into the moderate-copy-number plasmid vector pLG339 to yield active subclones. However, due to the instability of the resultant chimeric plasmids, the gene was subsequently subcloned into the low-copy-number vector pOU61 to yield the stable hybrid plasmid pMH613. Both plasmids contain a 6.6 kilobase EcoRI fragment from strain GS-5 and express both hydrolase and sucrase activities. The relative position of the gene in the insert has been determined after Tn5 mutagenesis and deletion analysis. The cloned enzyme was purified to near homogeneity after gel filtration and anion-exchange chromatography, chromatofocusing, and preparative polyacrylamide gel electrophoresis. The purified enzyme displayed a molecular mass of 58 kilodaltons, which is significantly higher than the 48-kilodalton enzyme previously purified from S. mutans GS-5. These results suggest that processing of the hydrolase occurs in S. mutans. PMID- 3017863 TI - Monoclonal antibodies against the M-protein and carbohydrate antigens of histoplasmin characterized by the enzyme-linked immunoelectrotransfer blot method. AB - Monoclonal antibodies (MAbs) of two different specificities were produced by immunizing mice with the semipurified M antigen of histoplasmin. One type, from clone CB4, was an immunoglobulin M that precipitated a polysaccharide present in histoplasmin and also formed immunoprecipitates with a cross-reactive polysaccharide present in extracts of Blastomyces dermatitidis and Coccidioides immitis. The second type of MAb, from clone EC2, was an immunoglobulin G that reacted in the enzyme-linked immunoelectrotransfer blot (EITB) assay with a doublet of proteins with an apparent molecular size of 70 to 75 kilodaltons. This molecule is proposed as the authentic M protein antigen that is recognized by M antibodies in sera from mice and rabbits immunized with Histoplasma capsulatum and from persons with histoplasmosis. The M factor also occurs in an abundant disulfide-bridged dimer which has a molecular size of 150 kilodaltons and is nonimmunoreactive under the conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PMID- 3017865 TI - Cloning of a Streptococcus mutans glucosyltransferase gene coding for insoluble glucan synthesis. AB - The gtfB gene coding for a glucosyltransferase (GTF) activity of Streptococcus mutans GS-5 was isolated on a 15.4-kilobase DNA fragment by using a lambda L47.1 gene library. The activity was catalyzed by gene products of 150 and 145 kilodaltons which reacted with antibodies directed against both soluble and insoluble glucan-synthesizing GTFs. The enzyme present in crude Escherichia coli extracts synthesized both soluble and insoluble glucans. The enzyme was partially purified from lysates of the lambda DS-76 clone and synthesized both types of glucans in a primer-independent fashion. In addition, the purified enzyme exhibited a pI of approximately 5.0. Southern blot analysis indicated that the cloned GTF gene represented a contiguous nucleotide sequence on the strain GS-5 chromosome. Furthermore, evidence for the existence of a distinct gene sharing partial homology with gtfB was also obtained. The gtfB gene was subcloned into plasmid pACYC184 into E. coli and exhibited GTF activity when carried on GS-5 inserts as small as 5 kilobases. The approximate location of the GTF promoter and the direction of gene transcription were also determined. The cloned enzyme was not secreted through the cytoplasmic membrane of E. coli, since most of the activity was found in the cytoplasm and, in lesser amounts, associated with the cytoplasmic membrane. The gtfB gene was insertionally inactivated by introducing a gene fragment coding for erythromycin resistance into the GTF coding region. After transformation of strain GS-5 with the altered gene, transformants defective in insoluble glucan synthesis were identified. These results indicate that the gtfB gene codes for a GTF involved in insoluble glucan synthesis in strain GS-5. PMID- 3017866 TI - Increase in progressive motility and improved morphology of human spermatozoa following their migration through Percoll gradients. AB - Human spermatozoa were separated on the basis of their motility in a discontinuous Percoll-gradient made up in tissue culture medium containing 10% (v/v) human serum (TCMS). Portions of ejaculates were placed on top of the gradients. After 3 h at 37 degrees C the bottom 1.5 ml was collected and the sperm washed free of the Percoll solution by centrifugation at 240 X g after dilution in TCMS. In this way the spermatozoa were separated from seminal fluid by means of the swimming rate of the sperm. When semen samples from normal men were used, total recovery of sperm after separated on a Percoll gradient was 21 +/- 2.3%. The progressive motility index increased by a factor of 15 +/- 1 when comparing separated samples with the same unseparated ejaculate, and the frequency of sperm with normal morphology increased from 60 to 85%. The improvements in these semen samples was attributable to the Percoll separation as the washing procedure itself was without effect. Using this method sperm of relatively unifirm motility and morphology can be collected. These may then be used for further biochemical and physiological studies. Improved sperm quality was also obtained when samples from patients with abnormal semen profiles were separated in this way, although the degree of improvement was much more variable than that obtained with semen from normal fertile men. This indicates that this method can be used in clinical practice in selected cases for the preparation of sperm for insemination or for in-vitro fertilization. PMID- 3017867 TI - Failure to induce cervical cancer in mice by long-term frequent vaginal exposure to live or inactivated herpes simplex viruses. AB - C57 mice aged 8-10 weeks in groups of 50 each received vaginal cotton pellets soaked in lysates of HEp-2 cells, either mock-infected or infected with herpes simplex virus I, herpes simplex virus 2, and highly attenuated recombinant viruses 5 times a week for 89 to 114 weeks. An untreated group was also included. The mock-infected and some of the infected cell lysates were exposed to ultraviolet light at a dose sufficient to inactivate virus. Smears of exfoliated vaginal cells collected once a month and histopathologic sections of genital organs removed at autopsy were coded and examined blind for the presence of abnormal cells indicative of malignant changes and cervical cancer, respectively. Sera collected before termination of the study were tested blind for the presence of antibody to infected cell lysates and to purified herpes simplex virus glycoprotein B. The results were as follows: Over 74% of 826 mice examined at autopsy contained tumors at non-genital sites. The tumors were randomly distributed among the various groups. Gross genital abnormalities were less common in untreated animals than in mice receiving vaginal implants. The fraction of mice which developed cervical cancer diagnosed by histopathologic examination was small (7.2%) and not significantly different among various groups. There was no correlation between the presence of abnormal exfoliated cells indicative of early invasive or invasive cancer lesions and the histopathologically proven diagnosis of micro-invasive or invasive cervical cancer. The incidence and levels of antibody were highest in animals exposed to live virus; some mice exposed to inactivated virus also developed weak or moderately high antibody levels. The presence of antibodies did not correlate with the presence of histopathologically proven cervical cancer. The results do not support the ability of herpes simplex viruses to cause genital neoplasia in mice. PMID- 3017869 TI - cAMP metabolism in B16 melanoma clones during the formation of experimental and spontaneous metastases. AB - The ability of B16 melanoma clones to form tumor colonies in the lung after i.v. injection (experimental metastases) correlates positively with their capacity to respond to activators of cyclic adenosine 3-, 5-monophosphate (cAMP) metabolism (such as melanocyte-stimulating hormone and forskolin). To investigate whether this relationship is causal, the cAMP responses of 4 B16 melanoma clones of differing colonizing potential have been examined in freshly established stock cell cultures, in pulmonary colonies in vivo and in cultures established from these lesions. In all cases, the cAMP responsiveness of the excised colonies (and cultures derived from them) mimicked the responsiveness of the cultures from which they arose. B16 clones exhibiting low cAMP responsiveness gave rise to few experimental metastases all of which were poorly responsive to activators of cAMP metabolism. Similarly, clones with high cAMP responsiveness formed multiple lung colonies which displayed a marked sensitivity to agents that stimulated cAMP production. Parallel experiments on the spontaneous metastatic behavior of the same clones revealed that the cAMP responsiveness of cells in the primary (intrafootpad) tumor and spontaneous metastatic lesions in the lung faithfully reflected the response profile of the original tumor-cell inoculum but no correlation was found between cAMP responsiveness and the capacity to form spontaneous metastases. These data suggest that cAMP-dependent events may influence the survival, arrest and organ colony formation by cells injected directly into the circulation but appear to be of little or no importance in determining the early event(s) involved in the evolution of spontaneous metastases prior to the entry of cells into the circulation. PMID- 3017868 TI - Analyses of transplanted murine tumors for HSV DNA sequences. AB - Meignier et al. (1986) report the results of exposure of C57BL/6NCr mice to vaginal plugs containing live or inactivated herpes simplex virus 1 or 2 (HSV-1 or HSV-2) or recombinant viruses 5 times a week for up to 114 weeks. Genital organs showing abnormalities were transplanted into nude mice. Of 33 transplants, 13 produced subcutaneous tumors in nude mice and 12 were subsequently transplanted into C57BL/6NCr mice. We report that the DNA extracted from coded tumor tissues of nude mice and from normal viscera of the same rodents did not hybridize with HSV-1 and HSV-2 DNA probes representing the viral genomic regions shown previously to be capable of morphologically transforming cells in culture. The sensitivity of the assays was such that we could detect 0.5 copies of the HSV sequences of complexity equal to or greater than 1 Kbp per cell DNA equivalent. To control for the sensitivity of the assays in the actual hybridizations, the tumor-cell DNA was also hybridized with a beta-globin mouse DNA probe. A striking feature of these control hybridizations was the detection of beta-globin polymorphism in some nude mouse tumors. The beta-globin polymorphism allowed us to conclude that the analyzed tissues contained significant amounts of the tumor cells occurring in the C57BL/6NCr mice. PMID- 3017870 TI - Epstein-Barr virus-related herpesvirus from a rhesus monkey (Macaca mulatta) with malignant lymphoma. AB - A herpesvirus (RhEBV) was isolated from a lymphoblastoid cell line (LCL) that became established from a malignant lymphoma in a rhesus monkey. The predominant cell marker in the LCL was that of B lymphocytes. RhEBV-induced viral capsid (VCA) and nuclear antigens (NA) in the LCL were serologically related to similar antigens known to be induced by human Epstein-Barr virus (EBV). RhEBV was of nonhuman primate origin and was clearly differentiated from EBV in the anti complement immunofluorescence reaction using human and non-human primate sera with antibodies to the NA induced by the respective viruses. While human sera reacted with NA induced by both EBV and RhEBV, monkey sera failed to recognize the NA induced by EBV. RhEBV-induced NA was present in nearly all the cells of a suspension prepared from the tumor tissue mass, but not in the monolayer fibroblasts derived from the tumor tissue or in the blood and lymph-node lymphocytes of clinically healthy animals. RhEBV induced in vitro transformation and establishment of LCLs from peripheral blood lymphocytes of normal rhesus and cynomolgus monkeys but not from those of 6 other non-human primate species tested. The LCLs, with predominant B-lymphocyte markers, established after treatment with RhEBV, all had evidence of the virus infection since nearly all cells in the culture expressed the virus-induced NA. PMID- 3017871 TI - Recombinant human interferon gamma suppresses HTLV-III replication in vitro. AB - Effect of human interferon gamma (rINF gamma) on HTLV-III replication was evaluated quantitatively via a novel infection system using HTLV-I-carrying MT-4 cells. Treatment of HTLV-III-infected MT-4 cells with different concentrations (I 1,000 U/ml) of rINF gamma, which did not affect the growth or viability of uninfected cells, significantly blocked the appearance of immunofluorescent antigens of HTLV-III and the virus-induced cytopathic effect in a dose-dependent manner. A plaque assay was applied to measure the exact amount of viral particles released from HTLV-III-infected MT-4 cultures either untreated or treated with rINF gamma after infection. The number of plaques per dish decreased with increasing drug concentrations. About 50% and 80% of HTLV-III replication were inhibited by the addition of 100 and 1,000 U/ml of rINF gamma, respectively. The effects of INF were observed by day 5 of incubation with the chemical. However, longer treatment of cells with rINF gamma permitted a gradual increase in viral replication. Re-addition of fresh INF into cultures did not change this pattern significantly. PMID- 3017873 TI - Characterization of (-)-(3H)-DHA binding to intact mouse lymphocytes: effect of experimental autoimmune orchitis on beta-adrenoceptor expression. AB - Autoimmune orchitis induced an increment in the beta-adrenoceptor populations in intact mouse lymphocytes, depending on their source. Characterization of (-)-(3H) DHA specific binding to intact normal cells indicate that thymic cells do not have the ability of binding a beta-adrenergic ligand. In contrast, spleen and lymph node cells showed a homogeneous population of beta-adrenergic receptors. ( )-(3H)-DHA binding was a rapid, reversible and stereospecific process. Competition experiments indicated the order of potency of the agonists and led to their definition as being of the beta 2-adrenoceptor subtype. Saturation assays and Scatchard analysis indicated a single class of binding sites (being free of allosteric or cooperative interaction) in both control and immune cells. However, spleen and lymph node cells from mice hyperimmunized with testicular preparations showed a marked increase in the number of beta-adrenoceptor sites with no changes in chi d. These results emphasize the use of lymphocytes possessing a homogeneous population of beta 2-adrenoceptors as an easily available model for monitoring beta-adrenoceptors in disease. PMID- 3017872 TI - The chronic cerebral effects of cannabis use. I. Methodological issues and neurological findings. AB - This paper examines the research evidence relating sustained use of marijuana to chronic cerebral impairment. Evidence from both American and cross-cultural studies is reviewed, with a particular emphasis on methodological problems in the research. The focus of this paper is on neurological findings while another paper focuses on neuropsychological findings. On the basis of available research, it was concluded that there is no evidence that marijuana produces gross structural cerebral changes and little evidence that it leads to functional impairment, although subtle impairment cannot be ruled out. PMID- 3017874 TI - Sympathoadrenergic regulation. PMID- 3017875 TI - Application of principles of metabolic control to the problem of metabolic limitations in sprinting, middle-distance, and marathon running. PMID- 3017877 TI - D-penicillamine inhibition of interleukin-1 production: a possible mechanism for its effect on synovial collagen synthesis? AB - Collagen production was investigated in cultured rabbit synovial fibroblasts exposed in vitro to D-penicillamine (D-Pen). The results show that these cells are rather insensitive to the drug since only a slight increase of the collagen amount secreted was observed for 48-h exposure to concentrations of 200-400 micrograms/ml. However, fibroblasts derived from the synovium of arthritic rabbits proved to be more susceptible to D-Pen, responding by a marked increase of collagen secretion even for concentrations of 50 micrograms/ml. This finding suggests that synovial fibroblasts of arthritic patients, probably stimulated by the inflammation process, could be target cells for the D-Pen action. The activities of 4-prolyl-hydroxylase (4-PH) and galactosylglucosyl-transferase (GGT) were assayed in the same cultures. A correlation has been found between the 4-PH activity and the collagen amount produced. In contrast, no alteration in the level of GGT on exposure to D-Pen was detected. Finally, D-Pen was shown to reduce in vitro the production of collagen-inhibiting factors by phytohaemagglutinin-stimulated mononuclear cells. This effect was associated with an inhibition of the release of monocyte cell factor (MCF/interleukin-1), suggesting that D-Pen could indirectly affect synovial collagen synthesis by interfering with interleukin-1 secretion. PMID- 3017876 TI - In vitro production of collagen by synovial fibroblasts from D-penicillamine treated arthritic rabbits. AB - In order to get further insight into the mechanism of D-penicillamine action on synovial tissue collagen synthesis, fibroblasts derived from drug-treated arthritic rabbits were cultured and labelled with radioactive proline. No evident correlation was found between the amount of newly synthesized collagen and the previous treatment of animals. In contrast, the prolyl-hydroxylase activity was reduced in cells from rabbits receiving D-penicillamine. This finding suggests that culture conditions may influence the collagen-synthesizing potentiality of the synovial fibroblasts without changing the level of enzyme activity. Therefore, the prolyl-hydroxylase activity could be considered here as a more reliable reflection of the in vivo situation. The ratio of type III to type I procollagens, as estimated by DEAE-cellulose chromatography, showed a rise in cultures from D-penicillamine-treated rabbits as compared to controls. This result indicates that long-term administration of the drug may alter the collagen composition of synovial tissue matrix in rheumatoid arthritis. The question remains, however, whether this alteration contributes to the beneficial effect of the drug. PMID- 3017878 TI - Polymorphonuclear leukocyte collagenase in carrageenin-induced inflammation: effect of nonsteroidal antiinflammatory drugs. AB - Experimental pleurisy induced by carrageenin in rats caused an accumulation of PMN-leukocytes in the pleural cavity and changes in the blood leukocyte population. During the course of the inflammation involved, up to 90-98% activation of latent collagenase was observed in blood PMN-leukocytes and in leukocytes obtained from pleural exudate. The NSAIDs tested for their effect on this process had significant inhibitory effects on both exudate and leukocyte accumulation as well as on the activation of latent collagenase. Timegadine, medosan and sulindac were most effective in preventing collagenase activation, felden and naproxen were less effective, and peroxinorm was without an influence, whereas indomethacin destroyed collagenase activity. These results suggest that the protective effects of NSAIDs against the activation of latent collagenase could be one of their therapeutic actions. PMID- 3017879 TI - A varicella-zoster immunization certification form for hospital employees. PMID- 3017880 TI - Adapting disease-specific isolation guidelines to a hospital information system. AB - The authors modified the Centers for Disease Control's guideline for disease specific isolation precautions to a hospital computerized information system. Entering a suspected diagnosis selected from the isolation option on computer terminals generated: a printout listing the isolation instructions, infective material(s), and persons who should avoid exposure; an order for the appropriate supplies; a patient charge based on the supplies required; and an option for stopping, changing, or listing the orders. In order to implement this system, both extensive in-service training for nurses and efforts to change ordering practices of physicians were necessary. Prevalence surveys before and after computerization were used to evaluate the new system. Combined surveys showed that isolation was ordered for only 21% of patients when indicated. Failure to isolate was identified as a significant problem. As a consequence, continuous surveillance and consultation of all infected patients were instituted, resulting in isolation orders for 81% when indicated. The computerized disease-specific system has resulted in better and more accurate use of isolation, probably due to in-service education and surveillance efforts. PMID- 3017881 TI - AIDS update: HTLV-III testing, immune globulins and employees with AIDS. PMID- 3017882 TI - Epstein-Barr virus as an etiological agent in the pathogenesis of lymphoproliferative and aproliferative diseases in immune deficient patients. PMID- 3017884 TI - Prevention of hepatocellular carcinoma by immunization against hepatitis B. PMID- 3017883 TI - Cancer cells, components of basement membranes, and proteolytic enzymes. PMID- 3017885 TI - Binding and activation of gonadotropin-releasing hormone receptors in pituitary and gonadal cells. PMID- 3017886 TI - Hormone dependence and independence of mammary tumors in mice. PMID- 3017887 TI - The gene for the major intrinsic protein (MIP) of the ocular lens is assigned to human chromosome 12cen-q14. AB - The gene for the human major intrinsic protein (MIP) of the ocular lens fiber membrane has been assigned to region cen-q14 of chromosome 12 through the use of somatic cell hybrids containing the whole chromosome and parts of human chromosome 12. PMID- 3017888 TI - The effect of stress on proton relaxation times (T1 and T2) of rat liver. AB - T1 of the liver has been shown to lengthen after sham operation or partial hepatectomy, reaching a maximum about 24 hours after surgery. Surgical stress, which includes tissue injury and repair, has been suggested as the source of the change. We subjected rats to three types of stress that do not include tissue damage and repair to determine the role of stress alone in the length of T1 of the liver. Prolonged swimming, ACTH injection, or fasting did not affect T1 times. Tissue damage and/or repair appear to have a major role in determining T1 in the liver. PMID- 3017890 TI - Transmission of acquired immune deficiency syndrome (AIDS) by a blood transfusion given in 1979 in Israel. AB - AIDS developed in a blood donor-recipient pair in Israel. Transmission of HTLV III/LAV probably occurred via a blood transfusion in November 1979. The donor was asymptomatic at the time of blood donation, but initial symptoms of AIDS-related complex developed in the donor 3 months later and in the recipient 6 months following the blood transfusion. PMID- 3017891 TI - Acquired immune deficiency syndrome (AIDS) HTLV III/LAV; the causal agent and modes of transmission. PMID- 3017892 TI - [Adenoid cystic sweat gland cancer]. AB - Two primary adenoid cystic carcinomas of the eccrine sweat glands of the skin are presented. This tumor, first described in recent years, represents the rarest form of carcinoma of the eccrine sweat gland. The tumor demonstrates characteristic histological features and clinically often exhibits slow progression and absence of lymph-node metastases. The most important differential diagnosis to be excluded is metastasis of an adenocarcinoma of an internal organ. PMID- 3017893 TI - Adenoid cystic salivary gland carcinoma: a clinicopathologic correlation. AB - Between 1960 and 1980, 71 cases of adenoid cystic carcinoma (ACC) were reviewed according to treatment modality and clinical course. Histologic review of pathologic slides was performed to classify the tumors into their predominant histologic pattern (tubular, cribriform, solid). The predominant histologic patterns of the tumors were equally divided between tubular and cribriform. Very few were classified as a solid pattern. In the patients receiving the same type of therapy (surgery and irradiation), the cribriform and tubular variants of ACC demonstrated no difference in the rate of distant metastases and overall survival. The cribriform variant demonstrated a significantly worse prognosis in terms of local recurrence rate. The patients who had a solid histologic pattern of ACC appeared to have an overall worse prognosis in terms of distant metastases and long-term survival. The long-term survival of patients with ACC may be related to the development of distant metastases despite local control. PMID- 3017894 TI - One-stage total mandibular reconstruction with rib, pectoralis major osteomyocutaneous flap. AB - One stage reconstruction of the mandible and lower third of the face with pectoralis major osteomyocutaneous flap incorporating the rib on a muscular pedicle in oral cancer case is described. The composite tissue provides the exact replacement of lost tissues. The technique described replaces adequate mucosa, soft tissue, and bone with minimum functional loss at the donor site. Two ends of the refashioned rib were anchored to masseteric muscle mass and zygoma. This has established functional rehabilitation of chewing, swallowing, and speech in a short period. PMID- 3017889 TI - Isolation and characterization of mRNA for cholinergic synaptic transmission. PMID- 3017895 TI - Malignant fibrous histiocytoma of the parotid gland associated with polycythemia. AB - Although malignant fibrous histiocytoma (MFH) is thought to be the most common soft tissue tumor of late adult life, it is extremely uncommon in the parotid gland. A case of MFH in the parotid is reported, associated with polycythemia, which remitted following surgical extirpation of the tumor. PMID- 3017896 TI - S-100 protein in salivary gland tumors: an immunohistochemical study of 129 cases. AB - Immunohistochemical distribution of S-100 protein was evaluated in 129 tumors from major and minor salivary glands. Also, two sensitive immunoperoxidase avidin biotin methods using either overnight incubation with primary antibody or pretreatment trypsin digestion and half-hour incubation were compared. Tumors with S-100 protein immunoreactivity were demonstrated in numerous benign and malignant histologic categories. Adenoid cystic carcinomas, carcinomas ex pleomorphic adenoma, clear cell carcinomas, and adenocarcinomas NOS showed inconsistent positive staining, whereas all monomorphic and pleomorphic adenomas and polymorphous low grade adenocarcinomas examined stained positively. No staining was observed in mucoepidermoid carcinomas or acinic cell carcinomas. Mesenchymal-like tumor cells with positive immunostaining were seen only in pleomorphic adenomas and trabecular-tubular adenomas. Equivalent results were found with both overnight and same-day digestion techniques. The consistent S-100 protein staining in some histologic tumor categories (pleomorphic and monomorphic adenoma and polymorphous low grade adenocarcinoma) compared to mucoepidermoid carcinoma that is devoid of S-100 protein immunoreactivity has application to some microscopic differential diagnostic situations. Inconsistent staining of adenoid cystic carcinomas and adenocarcinomas did not allow discrimination from other benign and malignant salivary gland tumors with similar histomorphology. PMID- 3017897 TI - [Cytomegaly as a postoperative complication]. PMID- 3017898 TI - Complication after embolization of internal iliac artery by gelatin sponge powder. PMID- 3017899 TI - Zonal analysis of metabolic profiles of articular-epiphyseal cartilage chondrocytes: a histochemical study. AB - Articular-epiphyseal cartilage from the femur of New Zealand rabbits was subjected to histochemistry for determination of the presence of metabolic enzymes along its zonal stratification. Glycolytic enzymes were strongly reactive in all of the zones. Krebs cycle enzymes, enzymes of the hexose monophosphate shunt and the respiratory chain enzymes showed a progressive increase in reactivity from the tangential zone through the top half of the epiphyseal zone. Indicators of lipid metabolism were fairly high in all regions of the cartilage. PMID- 3017900 TI - The effects of mucus on the binding of cationized ferritin by human and animal gastrointestinal epithelium. AB - Human gallbladder and gastric epithelial cells are normally covered with a layer of mucus. When specimens were exposed to cationized ferritin (CF) in vitro, they did not regularly bind nor internalise it. If the tissues were first exposed to the mucolytic agents cysteamine or pepsin, then the gallbladder epithelium readily bound CF and the gastric epithelium irregularly. The in vivo binding of CF by guinea pig gallbladder could be abolished by the induction of mucous hypersecretion by the antibiotic lincomycin. The removal of the mucus by mucolytic agents restored the binding of CF. The irregular binding of CF by gastric mucosa after the use of mucolytic agents suggests other factors may be at play. PMID- 3017901 TI - [Synovial sarcoma of the hypopharynx]. AB - A 22 year old man with a synovial sarcoma is reported. The first symptom was dysphagia for 5 months. He was treated by excision and post operative radiotherapy (60 Gy). The patient is without recurrence 38 months after surgery. A short review of diagnosis, histology, treatment and prognosis of synovial sarcoma follows. PMID- 3017902 TI - DR2+ haplotypes in insulin-dependent diabetes: analysis of DNA restriction fragment length polymorphisms. AB - Insulin-dependent diabetes (IDD) is strongly associated with certain HLA class II (Ia) antigens. The frequency of DR2 is significantly reduced in IDD; among DR2+ patients, the frequency of the subtype specificity Dw2 defined with homozygous typing cells (HTCs) is significantly reduced compared to DR2+ controls, and the specificity LD-MN2, which we have defined using primed lymphocyte typing reagents, is significantly increased. We have studied DNA restriction fragment length polymorphisms (RFLP) of DR2-LD-MN2+ individuals and homozygous typing cells carrying specificities antigenically related to LD-MN2. Using a number of different restriction enzymes, a characteristic pattern of fragments could be defined for DR2-LD-MN2 using both DQ beta and DR beta cDNA probes. This pattern was shared with some but not all of the antigenically related HTCs, and was distinct from that of DR2-Dw2. The RFLP pattern of DR2-LD-MN2 obtained with the DQ beta probe is identical, except for one band, to that of DR1-Dw1, suggesting that at least some part of the DQ region is identical in these two haplotypes. These results indicate that analysis of RFLP patterns can be used to help identify the genetic regions and, eventually, genes most important in the association of HLA and IDD. PMID- 3017903 TI - Effect of the radiosensitizer misonidazole and the radioprotector diethyldithiocarbamate on spontaneous metastasis formation of murine tumors. AB - The effect of treatment with the hypoxic cell radiosensitizer misonidazole (MISO) and the radioprotector diethyldithiocarbamate (DDC) on the formation of spontaneous lung metastases of four different spontaneously metastasizing murine tumors was investigated. The tumors were mammary carcinoma MCA-K, hepatocarcinoma HCA-1, and sarcomas SA-4020 and SA-NH. Multiple daily treatments with MISO significantly enhanced the incidence of metastases only in MCA-K. Because only MCA-K, but not the three remaining tumors, is immunogenic, the treatment with MISO may be associated with the promotion of metastasis primarily in the immunogenic tumors. Treatment of mice with DDC had no influence on metastatic spread. However, when given prior to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), DDC reduced BCNU-induced enhancement of HCA-1 metastases. PMID- 3017904 TI - Phase I trial of the hypoxic cell radiosensitizer SR-2508: the results of the five to six week drug schedule. AB - Sixty-five patients were entered on the long schedule of the Phase I trial of SR 2508. The planned total doses ranged from 30 to 40.8 g/m2 using various treatment schema including daily, split course, and every-other-day schedules. The individual dose size was 2 g/m2 for 56 patients and 1.7 g/m2 for nine. In contrast to misonidazole and desmethylmisonidazole, more SR-2508 can be administered as the duration of therapy is lengthened. All six patients on the 30 g/m2 step tolerated the drug without toxicity. This total dose was not achievable in the three week schedule. Additionally, a number of patients did not develop neuropathy at a cumulative dose of 40.8 g/m2. Although the analysis is not yet complete, a given patient's drug exposure as measured by their total AUC (mMxhr), defined as the area-under-the-curve of serum concentration of SR-2508 vs. time for a single dose times the number of doses given, is useful in predicting toxicity for that patient. The recommended starting schedule for the Phase II and III trials is 34 g/m2 over a 6 week period (2 g/m2 every other day). A total AUC of approximately 39 mMxhr should be tolerable. The drug regimen must be altered for patients who have a high AUC. Therefore, it is mandatory to have an accurate and rapid pharmacokinetic analysis for each patient. The clinical efficacy of the hypoxic cell sensitizers remains to be proven. However, using the guidelines derived from the Phase I trial, SR-2508 should be a relatively safe drug, producing minor or no toxicity. PMID- 3017905 TI - Binding of 3H-misonidazole to solid human tumors as a measure of tumor hypoxia. AB - Treatment-resistant, chronically hypoxic tumor cells have been assumed to exist in some solid human tumors, limiting their curability. To date, six patients with different types of tumors have been studied using radioactive labelled electron affinic compounds that bind to hypoxic cells. Although the gross clinical appearance of the tumors in all six patients was of a large and fixed mass which might on clinical grounds be expected to contain hypoxic cells, we have observed drug binding to hypoxic regions in only two, a rapidly growing small cell lung cancer (SCLC) and a malignant melanoma. The hypoxic fraction of the malignant melanoma was found to be 6% and the SCLC tumor approximately 10%. We have observed that areas of maximum adduct formation can be found in tumor cells immediately adjacent to blood vessels, suggesting that blood flow over the labelling interval was restricted. These preliminary studies suggest that sensitizer adduct formation in human tumor tissue may be a useful measure of tissue pO2 at the cellular level and that tumor hypoxia might be more related to the rate of tumor growth and histological grading than to tumor size. PMID- 3017907 TI - Diabetes insipidus associated with metastatic pancreatic carcinoma in a dog. AB - Diabetes insipidus was diagnosed in a dog with metastatic pancreatic carcinoma. Histologic examination of the pituitary gland revealed extensive invasion of the pars intermedia and neurohypophysis by metastatic tumor cells. PMID- 3017906 TI - Facial dermatosis in four dogs with hyperadrenocorticism. AB - In hyperadrenocorticism in the dog, cutaneous lesions may include alopecia, thin, hypotonic skin, hyperpigmentation, comedones, calcinosis cutis, secondary bacterial and fungal infections, and demodicosis. Skin lesions affecting only the face have not been reported in reviews of hyperadrenocorticism, nor has the disease been included in the differential diagnoses of facial dermatoses. This report involves 4 dogs with hyperadrenocorticism in which cutaneous signs were limited to the face. PMID- 3017908 TI - Modification of carboxyl-terminal region is the cause of activation of the src gene in avian sarcoma virus S2. AB - Viral oncogene product of avian sarcoma virus S2 was reported to have two alterations from proto-src product; a substitution of its extreme carboxyl terminus with a peptide of helper viral protein and a point mutation which altered the 501st amino acid from arginine to lysine. However, the following data suggest that lysine501 is more common in proto-src product than arginine501. Proto-src from two independent embryos which we analyzed encoded lysine for the 501st amino acid. Rous sarcoma virus and S1, another isolate which had transduced proto-src, also coded for lysine at the same position. Thus, in the case of S2, the oncogenic activation of the src gene appeared to be achieved with only an alteration at its carboxyl terminus and enhanced expression by long terminal repeats. PMID- 3017909 TI - Activated N-ras in a human rectal carcinoma cell line associated with clonal homozygosity in myb locus-restriction fragment polymorphism. AB - An N-ras transforming gene was detected in human rectal carcinoma-derived cells (7060) and molecularly cloned. The genetic lesion responsible for the transforming activity of the 7060 oncogene was localized to a single nucleotide transition from A to T in codon 61 of the predicted protein. This lesion in the second exon results in substitution of histidine for glutamine at this position. We also found an EcoRI restriction fragment length polymorphism, consisting of two alleles, of the human c-myb gene. The 7060-transformed epithelial cells showed the homozygous phenotype, while normal fibroblasts of the same patient showed the heterozygous phenotype. This suggests a relationship between the phenotypic change in the c-myb locus and the induction of the 7060 tumor. PMID- 3017910 TI - Superposition models of the discharge patterns of units in the lower auditory system. AB - Superposition of point processes has often been suggested as an abstract model for the generation of neural discharge patterns due to its simplicity (input spike trains are simply merged and then retransmitted by the model neuron). The properties of the superposition of two renewal processes are examined in relation to the properties of its components. A satisfactory condition is found so that the superposition of two renewal processes possesses negative serial dependence of intervent intervals. However, the imposition of a dead time of approximately the same duration as that of the components on their superposition process is shown to remove much of this dependence. Thus, the adequacy of the simple superposition of renewal processes as a model for discharge patterns from regions that are typified by negative serial dependence (such as the lateral superior olive) may be limited, but may suffice for the discharges of primary-like units. PMID- 3017911 TI - Searching for neural correlates of the hearing sensation fluctuation strength in the auditory cortex of squirrel monkeys. AB - Sounds with slow (less than 20 Hz) fluctuations may elicit the hearing sensation fluctuation strength. For AM tones, neural correlates of fluctuation strength were searched in the auditory cortex of unanesthetized squirrel monkeys. To enable a comparison of psychophysical and physiological data, the 'modulation' of the peristimulus time histogram was fitted by a sinusoidal function. The dependence of the amplitude of this function on modulation frequency, modulation depth and sound pressure level was often comparable to the dependence of fluctuation strength on the same stimulus parameters. In particular, as a function of modulation frequency, the neural data also show a bandpass characteristic at low modulation frequencies as was found for the hearing sensation fluctuation strength. PMID- 3017912 TI - Peroxide sensitivity of cold-shocked Salmonella typhimurium and Escherichia coli and its relationship to minimal medium recovery. AB - Cold-shocked Salmonella typhimurium displayed minimal medium recovery (MMR), viable counts on M9 minimal agar being much higher than those on tryptone soya yeast extract agar (TSYA). The addition of catalase to TSYA restored counts to the level found on M9 agar. Peroxide concentrations between 12 and 30 mumol/l were measured in TSYB but none was detected in M9 medium. Cold-shocked cells were sensitive to reagent hydrogen peroxide at a concentration similar to that found in TSYB. The minimal medium recovery phenomenon of cold-shocked cells is thus a manifestation of peroxide sensitivity. Changing the composition of growth media affected both cellular catalase activity and the magnitude of the MMR effect but the two properties were not directly related. Factors additional to cellular catalase activity must therefore affect susceptibility to peroxide following cold shock. Mutational loss of catalase, exonuclease III or recA-dependent DNA repair functions all increased the sensitivity of cold-shocked Escherichia coli to the inhibitory effects of peroxide present in rich medium. The peroxide resistant fraction of a cold-shocked population of Salm. typhimurium (i.e. those cells able to grow on TSYA) was more resistant to gamma radiation than the population as a whole. Cold shock thus sensitizes cells to more than one form of oxidative stress. Prior exposure of growing cells to 30 mumol/l hydrogen peroxide abolished their sensitivity to rich medium following cold shock implying that Salm. typhimurium contains an inducible system protecting against oxidative stress. PMID- 3017913 TI - Production of arachidonic acid metabolites by endothelial cells in hyperoxia. AB - This study investigated the response of bovine pulmonary artery endothelial cells to incubation in hyperoxia (95% O2-5% CO2). Changes in cell number and morphology, release of lactate dehydrogenase, and production of arachidonic acid metabolites were assessed during continuous exposure of confluent endothelial monolayers to air (air-5% CO2, "controls") or O2 (95% O2-5% CO2, "O2-exposed") for periods of 12-72 h. Control monolayer cell numbers remained constant (approximately 2,000,000 cells/flask), whereas the number of cells in O2-exposed monolayers decreased progressively to 30% of controls (P less than 0.01) by 72 h. As assessed by radioimmunoassay, both control and O2-exposed cells produced the prostacyclin metabolite, 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), and prostaglandin F2 alpha (PGF2 alpha), but no thromboxane metabolite (TxB2) was detected. The O2-exposed cells released significantly more 6-keto-PGF1 alpha and PGF2 alpha than control cells when apparent net production rates over the entire 72-h period were compared. In addition, both control and O2-exposed (48 h) endothelial monolayers released immunoreactive leukotriene B4 (LTB4) on stimulation with calcium ionophore (10 microM A23187). As with the cyclooxygenase products, O2-exposed cells released more immunoreactive LTB4 than did controls. Both cyclooxygenase and lipoxygenase metabolites of arachidonic acid are released by cultured endothelial cells during the development of O2 toxicity. PMID- 3017914 TI - Inconsistent differences between neutral detergent fiber and total dietary fiber values of fruits and vegetables. AB - A large number of fruits and vegetables were analyzed to determine levels of neutral detergent fiber (NDF) and total dietary fiber (TDF). The results showed that the TDF method is more precise than the NDF method. Further, the NDF values are significantly higher than the TDF values for apples and for miscellaneous fruits and vegetables, and significantly lower for stonefruit. For berryfruit, the differences between the NDF and TDF data are not significant. Also, a significant interaction between sample and method (except for berryfruit) indicated that, within groups, the (TDF-NDF) values vary so much that a "group difference" does not exist; consequently, figures from one method cannot be estimated from results of the other. PMID- 3017915 TI - Interaction between membrane proteins PBP3 and rodA is required for normal cell shape and division in Escherichia coli. AB - In Escherichia coli, the products of several genes are required for septation, and the products of several others are required for the maintenance of the rod shape of the cells. We show here that the combination of certain mutations in a division gene (ftsI) with a specific mutation in one of the shape genes (rodA) could produce cells with normal shape and division, although separately these mutations led to a loss of the capacity to divide (ftsI) or to form normal rod shaped cells (rodA). In contrast, combinations between other mutant alleles of these genes produced double mutants which had lost the capacity both to divide and to form rod-shaped cells. The mutual phenotypic correction observed within particular pairs of mutant genes suggests that the normal morphogenetic cycle of growth and division may require direct interaction between the two membrane proteins which are the products of these genes. PMID- 3017917 TI - Genetics of NAD metabolism in Salmonella typhimurium and cloning of the nadA and pnuC loci. AB - The nadA and pnuC loci of S. typhimurium were cloned and found to reside within a 2.2-kilobase region. Two-dimensional O'Farrell gel electrophoresis of the proteins produced after chloramphenicol amplification and subsequent release from chloramphenicol inhibition revealed NadA and PnuC to be 43,000- and 25,000 molecular-weight proteins, respectively. The data indicated that nadA and pnuC represent two distinct genes. PMID- 3017916 TI - Growth phase and ompR regulation of transcription of microcin B17 genes. AB - The synthesis of the peptide antibiotic microcin B17 was shown to occur as the cells entered the stationary phase of growth. This type of growth phase regulation is commonly observed in the production of a number of different bacterial products such as toxins and antibiotics. Microcin B17 synthesis is also dependent on the product of the ompR gene. To determine the role of transcription in this double regulation of microcin B17 production, operon fusions with Mu d1 (Ap lac) were constructed. Insertions were obtained in all four plasmid genes involved in production of microcin B17 (mcbA-D) and in the immunity region. Three classes of fusions were obtained. Fusions into mcbA, mcbB, and mcbC (first class) exhibited an increase in their transcription as the cells approached the stationary phase. These increases as well as basal levels of transcription were dependent on OmpR. Expression of fusions in mcbD and in the immunity region (second class) was also dependent on OmpR, but their expression remained constant throughout growth. One fusion in mcbC (third class) was obtained which was transcribed in the opposite direction than the others. It showed no growth phase regulation and no OmpR dependence. The implications of these results in terms of the transcriptional organization of the mbc genes are discussed. PMID- 3017918 TI - Serospecific antigens of Legionella pneumophila. AB - Serospecific antigens isolated by EDTA extraction from four serogroups of Legionella pneumophila were analyzed for their chemical composition, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The antigens were shown to be lipopolysaccharides and to differ from the lipopolysaccharides of other gram-negative bacteria. The serospecific antigens contained rhamnose, mannose, glucosamine, and two unidentified sugars together with 2-keto-3-deoxyoctonate, phosphate, and fatty acids. The fatty acid composition was predominantly branched-chain acids with smaller amounts of 3-hydroxymyristic acid. The antigens contain periodate sensitive groups; mannosyl residues were completely cleaved by periodate oxidation. Hydrolysis of the total lipopolysaccharide by acetic acid resulted in the separation of a lipid A-like material that cross-reacted with the antiserum to lipid A from Salmonella minnesota but did not comigrate with it on sodium dodecyl sulfate gels. None of the four antigens contained heptose. All of the antigen preparations showed endotoxicity when tested by the Limulus amebocyte lysate assay. The results of this study indicate that the serogroup-specific antigens of L. pneumophila are lipopolysaccharides containing an unusual lipid A and core structure and different from those of other gram-negative bacteria. PMID- 3017919 TI - Mutants of Escherichia coli defective for replicative transposition of bacteriophage Mu. AB - We isolated 142 Hir- (host inhibition of replication) mutants of an Escherichia coli K-12 Mu cts Kil- lysogen that survived heat induction and the killing effect of Mu replicative transposition. All the 86 mutations induced by insertion of Tn5 or a kanamycin-resistant derivative of Tn10 and approximately one-third of the spontaneous mutations were found by P1 transduction to be linked to either zdh 201::Tn10 or Tn10-1230, indicating their location in or near himA or hip, respectively. For a representative group of these mutations, complementation by a plasmid carrying the himA+ gene or by a lambda hip+ transducing phage confirmed their identification as himA or hip mutations, respectively. Some of the remaining spontaneously occurring mutations were located in gyrA or gyrB, the genes encoding DNA gyrase. Mutations in gyrA were identified by P1 linkage to zei::Tn10 and a Nalr gyrA allele; those in gyrB were defined by linkage to tna::Tn10 and to a gyrB(Ts) allele. In strains carrying these gyrA or gyrB mutations, pBR322 plasmid DNA exhibited altered levels of supercoiling. The extent of growth of Mu cts differed in the various gyrase mutants tested. Phage production in one gyrA mutant was severely reduced, but it was only delayed and slightly reduced in other gyrA and gyrB mutants. In contrast, growth of a Kil- Mu was greatly reduced in all gyrase mutant hosts tested. PMID- 3017920 TI - Characterization of Escherichia coli chemotaxis receptor mutants with null phenotypes. AB - Hydroxylamine mutagenesis was used to alter the tar gene that encodes the transmembrane Tar protein required for chemotaxis. Mutants defective in chemotaxis were selected, and the mutation was characterized by DNA sequencing. Two classes of mutations were found: nonsense and missense. The nonsense mutations were distributed throughout the gene, while the missense mutations were found to cluster in a region that includes 185 amino acids at the C-terminal end of the Tar protein. Partial characterization of mutant phenotypes suggested that some are completely defective in signaling while responding to attractants and repellents by differential methylation. Other mutants are undermethylated and constantly tumble, while yet another class of mutants is overmethylated and biased toward constant swimming with little or no tumbling. These mutants will be useful in experiments designed to understand the mechanism of chemotaxis. PMID- 3017921 TI - nifV-dependent, low-molecular-weight factor required for in vitro synthesis of iron-molybdenum cofactor of nitrogenase. AB - The molybdate- and ATP-dependent in vitro synthesis of the iron-molybdenum cofactor of nitrogenase requires a low-molecular-weight factor. The factor is present in extracts of nitrogen fixation-derepressed cultures of Klebsiella pneumoniae and Azotobacter vinelandii, but not in extracts of repressed cultures of these bacteria. Analysis of K. pneumoniae Nif mutants has indicated that the nifV gene product is the only nif protein (besides nifA) necessary for the synthesis and accumulation of the factor. The factor is stable to oxygen, temperatures below 120 degrees C, and extremely acidic and basic conditions. The activity of the factor was completely destroyed by dry ashing or digestion with sulfuric acid. The factor has been partially purified by filtration through an Amicon PM-10 DIAFLO membrane and chromatography on DEAE-cellulose, hydroxylapatite, silica gel, and Sephadex G-25. PMID- 3017922 TI - Immunological properties of monoclonal antibodies to human and rat prolyl 4 hydroxylase. AB - Monoclonal antibodies to human (8 clones) and rat (12 clones) prolyl 4 hydroxylase [EC 1.14.11.2] were prepared and characterized as regards subclass, subunit specificity, inhibition and crossreactivity. Among the antibodies to the human enzyme, four clones showed the IgG1 subclass, two IgA, one IgG2b, and one IgM. Four clones reacted with the alpha subunit of the enzyme, while the others reacted with the beta subunit. The enzymatic activity was inhibited by four clones. Five clones crossreacted with the rat enzyme. One clone inhibited the rat enzyme. Among the antibodies to the rat enzyme, seven clones showed the IgG1 subclass, four IgG2a and one IgG2b. Seven clones reacted with the alpha subunit, and four with the beta subunit. One reacted with neither subunit. The enzymatic activity was inhibited by seven clones. Seven clones crossreacted with the human enzyme. Three clones inhibited the human enzyme. PMID- 3017923 TI - Spontaneous induction of superoxide release and degranulation of neutrophils in isotonic potassium medium: the role of intracellular calcium. AB - When guinea pig peritoneal neutrophils were suspended in the isotonic medium of potassium, rubidium, and cesium ions at 37 degrees C, the cells released superoxide, while low activity was observed in the isotonic medium of sodium and lithium ions. The activity induced in the potassium medium was enhanced by potassium-ionophores, valinomycin, and gramicidin, and decreased by a potassium channel blocker, 4-aminopyridine. The superoxide-releasing activity was not affected by the presence or absence of extracellular calcium but was inhibited by an intracellular calcium antagonist-8-(N,N-diethylamino)-octyl-3,4,5 trimethoxybenzoate(TMB-8) with the half-inhibition concentration of 50 microM. The release of granular enzymes, lysozyme and beta-glucuronidase, was also induced in the isotonic potassium medium in the absence of extracellular calcium and inhibited by TMB-8. A remarkable elevation of the intracellular free calcium concentration in neutrophils, which was monitored by quin-2 fluorescence, was found when the cells were added to the potassium medium without calcium. The elevation was inhibited by the addition of TMB-8. These observations suggest that calcium mobilization from intracellular storage sites, not an influx of calcium from the extracellular medium, causes the release of superoxide and the granular enzymes in isotonic potassium medium. PMID- 3017924 TI - Characterization of ouabain-sensitive phosphatase activity in the absence of potassium ion in purified pig kidney Na,K-ATPase. AB - The ouabain-sensitive phosphatase activity of purified pig kidney Na,K-ATPase preparation in the absence of potassium ion ((-K)phosphatase) was examined precisely. During the preparation procedures, the (-K)3-O methylfluoresceinphosphatase ((-K)3-OMFPase) activity or the (-K)p nitrophenylphosphatase ((-K)pNPPase) activity appeared to be purified in parallel with the Na,K-ATPase activity. The (-K)phosphatase activity was competitively inhibited by ATP and by ADP, with the K1 values of 0.25 microM and 1.4 microM, respectively. These values are consistent with their Kd values for the high affinity ATP binding site of the Na,K-ATPase (Hegyvary, C. & Post, R.L. (1971) J. Biol. Chem. 246, 5234-5240). The substrate, pNPP, apparently competed with covalently bound fluorescein-5'-isothiocyanate (FITC), which is known to bind in the neighborhood of the high-affinity ATP binding site of the Na,K-ATPase, in both the (-K)phosphatase and the (+K)phosphatase reactions. The FITC-fluorescence intensity of FITC-labeled enzyme at the maximal steady-state activity of the ( K)phosphatase reaction was at a similar level to that of the E2 species. However, the FITC-labeled enzyme in the presence of only magnesium ion or only pNPP gave a fluorescence level similar to that of the E1 species. Oligomycin inhibited the ( K)phosphatase activity by at most 46%. On the basis of these results, it is strongly suggested that the (-K)phosphatase reaction is catalyzed at the high affinity ATP binding site of Na,K-ATPase, and the (-K)phosphatase reaction proceeds in a cyclic manner (E1----E2----E1). PMID- 3017925 TI - Purification and some properties of cytochrome c' from a strain of Achromobacter xylosoxidans. AB - Cytochrome c' was crystallized from Achromobacter xylosoxidans GIFU 543. The cytochrome was a basic protein and its molecular weight was 28,000. The pyridine ferrohemochrome showed absorption peaks at 415, 521, and 551 nm. The absorption spectra of the oxidized and reduced forms at neutral pH were almost the same as those of other cytochromes c' reported already. The reduced cytochrome c' reacted with CO and NO, and the NO complex showed a characteristic absorption spectrum. The midpoint redox potential of the hemoprotein was measured to be + 110 mV at pH 7.2. PMID- 3017927 TI - Activation of magnesium-dependent, neutral sphingomyelinase by phosphatidylserine. AB - The plasma membrane isolated from rat ascites hepatoma, AH 7974 cells was treated with 1% Triton X-100, which resulted in a more than 80% reduction in the phospholipid content of the plasma membrane. The delipidized plasma membrane showed only 18% of the activity of the magnesium-dependent, neutral sphingomyelinase in the untreated plasma membrane. On the addition of acidic phospholipids, especially phosphatidylserine, however, the enzyme activity in the delipidized membrane was markedly restored up to 77% of that in the untreated membrane. It was suggested that, considering the phospholipid composition of the AH 7974 plasma membrane (Koizumi, K. et al. (1977) Cell Struct. Func. 2, 145 153), phosphatidylserine may be a natural activator of neutral sphingomyelinase. PMID- 3017926 TI - Characterization of rat muscle fructose 1,6-bisphosphatase. AB - Fructose 1,6-bisphosphatase has been purified from rat muscle. Although the specific activity of the enzyme in the crude extract of rat muscle was extremely low, purification by the present procedure is highly reproducible. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The subunit molecular weight of the muscle enzyme was 37,500 in contrast to 43,000 in the case of the liver enzyme. Immunoreactivity of the muscle enzyme to anti muscle and anti-liver fructose 1,6-bisphosphatase sera was clearly distinct from that of the liver enzyme. All one-dimensional peptide mappings of the muscle enzyme with staphylococcal V8 protease, chymotrypsin, and papain showed different patterns from those of the liver enzyme. When incubated with subtilisin, the extent of activation of muscle fructose 1,6-bisphosphatase at pH 9.1 was smaller than that of the liver enzyme. The subtilisin digestion pattern of the muscle enzyme on SDS-polyacrylamide gel electrophoresis was distinct from that of the liver enzyme. The AMP-concentration giving 50% inhibition of the muscle enzyme was 0.54 microM, whereas that of the liver enzyme was 85 microM. The concentrations of fructose 2,6-bisphosphate that gave 50% inhibition of rat muscle and liver enzymes were 6.3 and 1.5 microM, respectively. Fructose 1,6 bisphosphatase protein was not detected in soleus muscle by immunoelectroblotting with anti-muscle fructose 1,6-bisphosphatase serum. PMID- 3017929 TI - Isolation and characterization of human heart cytochrome c oxidase. AB - Cytochrome c oxidase was isolated from human hearts and separated by SDS gel electrophoresis. The identity of polypeptide bands with known subunits was demonstrated by immunoblotting with monospecific antisera to rat liver cytochrome c oxidase subunits. The polarographically determined kinetics of cytochrome c oxidation were similar to those reported for the bovine heart enzyme. PMID- 3017928 TI - Cytochrome c oxidase from Paracoccus denitrificans in Triton X-100: aggregation state and kinetics. AB - Cytochrome c oxidase from Paracoccus denitrificans was homogeneously dispersed in Triton X-100. Using gel exclusion chromatography and sucrose gradient centrifugation analysis a molecular weight of the detergent-protein complex of 155,000 was determined. After subtraction of the bound detergent (111 mol/mol heme aa3) a molecular weight of 85,000 resulted, which agreed well with the model of a monomer containing two subunits. This monomer showed high cytochrome c oxidase activity when measured spectrophotometrically in the presence of Triton X 100 (Vmax = 85 s-1). The molecular activity, plotted according to Eadie-Hofstee, was monophasic as a function of the cytochrome c concentration. A Km of 3.6 X 10( 6) M was evaluated, similar to the Km observed in the presence of dodecyl maltoside [Nalecz et al. (1985). PMID- 3017931 TI - Stimulation of Escherichia coli adenylate cyclase activity by elongation factor Tu, a GTP-binding protein essential for protein synthesis. AB - A unique feature of eucaryotic adenylate cyclases is their interaction with GTP binding proteins that mediate hormonal responses. Until now, there has been no evidence for regulation of Escherichia coli adenylate cyclase by a GTP-binding protein. We describe here that the most abundant protein in E. coli, the GTP binding protein EF-Tu, which is important as an elongation factor in protein synthesis, also serves as a stimulator of adenylate cyclase activity. Homogeneous EF-Tu specifically increased the activity of purified adenylate cyclase as much as 70%; other E. coli GTP-binding proteins had no effect on enzyme activity. A study of the guanine nucleotide specificity for EF-Tu-mediated stimulation of adenylate cyclase activity suggested that the preferred activator is EF-Tu X GDP. To account for the GTP-specific stimulation of adenylate cyclase activity observed in intact cells, we propose that the nucleotide specificity for EF-Tu dependent activation of adenylate cyclase is governed by other factors in the cell. PMID- 3017930 TI - Stoichiometric conversion of oxygen to superoxide anion during the respiratory burst in neutrophils. Direct evidence by a new method for measurement of superoxide anion with diacetyldeuteroheme-substituted horseradish peroxidase. AB - Extracellular release of superoxide anion (O-2) and hydrogen peroxide (H2O2) during the respiratory burst of porcine and human neutrophils was studied by using diacetyldeuteroheme-substituted horseradish peroxidase as a trapping agent for these oxygen derivatives. The method permitted simultaneous measurement of oxygen consumption and formation of both O-2 and H2O2 in a single reaction mixture. When neutrophils were stimulated with phorbol myristate acetate in the presence of the heme-substituted peroxidase, a rapid accumulation of compound III, a complex of the enzyme with O-2, was observed accompanying an increase in oxygen consumption. During the process, amounts of compound III formed and oxygen consumed were stoichiometric, and no compound II, an indicator of H2O2 formation, was observed. These results establish that neutrophils stimulated with the phorbol ester produce exclusively O-2 as the primary oxygen metabolite and release it into the extracellular medium. When a limited amount of opsonized zymosan was used as the stimulus, compound III formation was also observed but it ceased at an early stage of oxygen consumption. When a sufficient amount of azide was included in the system, however, formation of compound II was noted in the later stage of oxygen consumption. The findings suggest that O-2, formed during phagocytosis, is converted to H2O2 within phagosomes and then diffuses out into the extracellular medium when its decomposition by catalase and/or peroxidases is blocked by azide. PMID- 3017932 TI - Inhibitory action of forskolin on adenylate cyclase activity and cyclic AMP generation. AB - In testicular Leydig cells, forskolin causes the expected stimulation of cAMP and testosterone production and potentiates gonadotropin-induced responses, when present in concentrations of 1-10 microM. In addition, when added at lower doses that did not affect cAMP generation and testosterone responses (100 nM), forskolin caused an increase in sensitivity to hormonal stimulation for all cAMP pools (extracellular, intracellular, and receptor-bound) and a 70% reduction in the ED50 for human chorionic gonadotropin (hCG) stimulation of testosterone production. Forskolin-induced increases in receptor-bound cAMP were less effective than those elicited by hCG in stimulating steroidogenesis. In contrast to the well-known stimulatory actions of forskolin, low doses of the diterpene (in the picomolar to nanomolar range) markedly inhibited the production of cAMP and testosterone. Such inhibitory actions of low-dose forskolin were prevented by preincubation of Leydig cells with pertussis toxin before addition of forskolin and/or hCG. Low concentrations of forskolin also inhibited adenylate cyclase activation by GTP and luteinizing hormone, and this effect was prevented by pretreatment of cell membranes with pertussis toxin. These studies have defined the stimulatory effects of forskolin on Leydig-cell cAMP pools, including potentiation of the hormonal increase in receptor-bound cyclic AMP by forskolin, and have provided additional evidence for the functional importance of cAMP compartmentalization during hormonal stimulation of steroidogenesis. We have also demonstrated a novel, high-affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation, an effect that appears to be mediated by the Ni guanine nucleotide regulatory subunit of adenylate cyclase. PMID- 3017934 TI - Spectroelectrochemical study of cytochrome c oxidase: pH and temperature dependences of the cytochrome potentials. Characterization of site-site interactions. AB - The cytochrome a and a3 sites in uninhibited, detergent-solubilized cytochrome c oxidase have been studied under a wide range of conditions using thin-layer spectroelectrochemistry. The observed absorbance changes at the alpha and Soret absorbance maxima have been used together to estimate the extents of reduction of cytochromes a and a3, using the absorbance properties of these cytochromes deduced from previous measurements employing ligand inhibition of cytochrome a3. The resulting Nernst plots, combined with the results of parallel studies on the carbon monoxide-inhibited enzyme (Ellis, W. R., Jr., Wang, H., Blair, D. F., Gray, H. B., and Chan, S. I. (1986) Biochemistry 25, 161-167; Wang, H., Blair, D. F., Ellis, W. R., Jr., Gray, H. B., and Chan, S. I. (1986) Biochemistry 25, 167 171), indicate that the cytochrome a site participates in anticooperative thermodynamic interactions which involve all three of the other metal sites in the protein. Using an analysis which resolves the intrinsic thermodynamic properties of the cytochromes from the effects of the intersite interactions, the pH, temperature, and ionic strength dependences of the cytochrome reduction potentials have been measured. The standard entropy of reduction of cytochrome a in the native enzyme is large and negative, in agreement with measurements on the carbon monoxide-inhibited enzyme. The reduction potential of cytochrome a is only moderately (less than -30 mV/pH unit) dependent upon pH, which implies that its reduction is linked to the uptake, on the average, of only about 0.5 protons at pH 7.0, and significantly less at the higher pH values relevant to the mitochondrial matrix. The thermodynamic properties of cytochrome a3 were found to be different in the two enzyme batches studied: in one batch, the cytochrome a3 reduction potential decreased steeply (about -56 mV/pH unit) with increasing pH, indicating stoichiometric (1 H+/e-) coupling of protonation to reduction. In the other batch, the cytochrome a3 potential was insensitive to pH below pH 7.5 and decreased at higher pH values in a manner suggesting coupling to an ionizable group with pKa near 7.8. The temperature dependence of the cytochrome a3 reduction potential indicates that its standard entropy of reduction is more positive than that of myoglobin, another high-spin metalloprotein heme, and significantly more positive than that of cytochrome a.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3017933 TI - Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4' nitroacetophenone. AB - The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P 450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P 450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH. PMID- 3017936 TI - Essential role of calcium influx in the adrenergic regulation of cAMP and cGMP in rat pinealocytes. AB - The role of Ca2+ in the adrenergic stimulation of pinealocyte cAMP and cGMP was investigated. In this tissue alpha 1-adrenoceptor activation, which by itself is without effect, potentiates beta 1-adrenergic stimulation of cAMP and cGMP 30- to 100-fold. The present results indicate that chelation of extracellular Ca2+ with EGTA or inhibition of Ca2+ influx with inorganic Ca2+ channel blockers (La3+, Co2+, Mn2+) markedly reduces the cyclic nucleotide response to norepinephrine, a mixed alpha 1- and beta-adrenergic agonist, but not to isoproterenol, a beta adrenergic agonist. In addition, the potentiating effects of alpha 1-adrenergic agonists were mimicked by agents which elevate cytosolic Ca2+, including K+ (EC50 = 2 X 10(-2) M), ouabain (EC50 = 2 X 10(-6) M), ionomycin (EC50 = 3 X 10(-6) M), and A23187 (EC50 = 2 X 10(-6) M); each potentiated the effects of beta-adrenergic stimulation but had no effect alone. Together these results indicate that an alpha 1-adrenoceptor-stimulated Ca2+ influx is essential for norepinephrine to increase pinealocyte cAMP and cGMP. PMID- 3017935 TI - Bovine brain-derived growth factor. Purification and characterization of its interaction with responsive cells. AB - A large-scale purification procedure for brain-derived growth factors, without chromatography in 0.1% trifluoracetic acid, has been developed. Two related brain derived growth factors have been purified to homogeneity from bovine brain using this procedure involving ammonium sulfate fractionation, followed by chromatography on CM-Sephadex C-50, sulfated Sephadex G-50, and heparin-Sepharose 4B. Brain-derived growth factor A (BDGF-A, molecular weight approximately 16,000) and brain-derived growth factor B (BDGF-B, molecular weight approximately 17,000) were eluted from heparin-Sepharose 4B at 1.2 and 1.6 M NaCl, respectively. Both BDGF-A and BDGF-B have a pI value of 5.7, have the same specific mitogenic activity, and react with mouse anti-BDGF-A antiserum. Both growth factors have a broad spectrum of mitogenic activity (vascular and aorta endothelial cells, chondrocytes, osteoblasts, epithelial cells, glial cells, fibroblasts, and smooth muscle cells), with a half-maximum effect at 10-20 pM. The binding of BDGF to bovine aorta endothelial cells and Swiss mouse 3T3 cells has been characterized. Binding of 125I-BDGF-A to these cells reached equilibrium in less than 15 min. Scatchard plot analysis of the binding of 125I-BDGF-A to endothelial cells and 3T3 cells showed a single class of high-affinity receptor with Kd values of 20 +/ 5 and 13 +/- 3 pM and receptor numbers/cell of 7,000 +/- 1,000 and 20,000 +/- 3,000, respectively. BDGF-B competed for 125I-BDGF-A binding in the same manner as unlabeled BDGF-A, suggesting that BDGF-A and BDGF-B bind to the same receptor. Basic pituitary fibroblast growth factor appeared to be a weak inhibitor, whereas platelet-derived growth factor and epidermal growth factor had no effect on 125I BDGF-A binding. Protamine, histone, polylysine, and polyarginine are potent inhibitors of the mitogenic activity of BDGF-A and BDGF-B and of binding of 125I BDGF-A to responsive cells. The half-time (t1/2) of internalization and degradation of cell surface-bound 125I-BDGF is 3 h. PMID- 3017937 TI - The effects of liposome-reconstituted apolipoproteins on the binding of rat intermediate density lipoproteins to rat liver membranes. AB - Rat intermediate density lipoproteins (IDL) bind specifically to high and low affinity binding sites on rat liver membranes. In a recent paper (Brissette, L., and Noel, S.-P. (1986) J. Biol. Chem. 261, 6847-6852), we have demonstrated that human low density lipoproteins and high density lipoproteins-3 can totally prevent the specific binding of rat IDL to the low affinity binding sites. The aim of the present studies was to determine the effects of apoA-I, apoC, and apoE, reconstituted into liposomes, on the binding of rat iodinated IDL to rat liver membranes. We found that a 50-, 100-, or 300-fold excess of liposome reconstituted apoE, apoC, or apoA-I, respectively, abolished the specific binding of IDL to the low affinity binding sites. Only apoE liposomes had an effect on the high affinity component; at a 100-fold excess no specific binding of IDL could be detected. Liposomes by themselves or associated with erythrocyte membrane proteins had virtually no effect on the binding of IDL. Taken together our results suggest that apoE is the only ligand that can compete efficiently for the sites that bind rat IDL with a high affinity. These sites may be the expression of both the remnant and the LDL receptors. The binding to the low affinity component probably represents weak interactions between IDL and "unspecified-lipoprotein binding sites," which can be entirely masked by human low density lipoproteins, high density lipoproteins-3, or liposome-reconstituted apoA-I, apoE, or apoC at appropriate concentrations. PMID- 3017938 TI - Properties of cyclic AMP-independent catabolite gene activator proteins of Escherichia coli. AB - The protein products of two crp alleles encoding mutationally altered catabolite gene activator proteins CAP and CAPc, which are functionally active in vivo in the absence of cAMP, were purified by an immunoaffinity purification procedure. These proteins bind cAMP with the same affinity as does the wild-type catabolite gene activator protein. From their susceptibility to the proteolytic enzyme subtilisin, we conclude that the two mutationally altered proteins adopt structural features adequate for biological activity and similar to the conformation that cAMP elicits or stabilizes in wild-type catabolite gene activator protein. We note, however, that their conformation is not unique and can be modulated by cAMP. The two altered proteins, CAP and CAPc, bind to the lactose promoter, giving rise to specific DNA-protein complexes in the absence of cAMP and promote initiation of specific lac messenger RNA synthesis. PMID- 3017939 TI - Identification of multiple binding sites for atrial natriuretic factor by affinity cross-linking in cultured endothelial cells. AB - In a previous study, we found that atriopeptin I was much weaker (EC50 greater than 500 nM) than atrial natriuretic factor (ANF-(8-33)) (EC50 = 0.3 nM) at increasing cyclic GMP in cultured endothelial cells. In this study, we used the cross-linking reagent disuccinimidyl suberate to investigate whether the differences in activity were due to the presence of multiple ANF receptors. When 98% of the ANF-binding sites on endothelial cells were occupied by tyrosine atriopeptin I after cross-linking, there was no difference in the concentration response curve to ANF-(8-33) with regard to cyclic GMP accumulation. In contrast, when 96% of the binding sites were occupied by cross-linked ANF-(8-33), a 60% decrease in the maximal cyclic GMP response was observed after the readdition of ANF-(8-33). These results suggest that ANF-(8-33) is binding to an additional site that atriopeptin I does not effectively bind. Affinity cross-linking of 125I ANF to intact endothelial cells resulted in the labeling of two sites of Mr approximately 66,000 and approximately 130,000. Approximately 94% of the 125I-ANF binding sites had an Mr approximately 66,000. Labeling of this site was inhibited by both tyrosine-atriopeptin I (KI = 0.9 nM) and ANF-(8-33) (KI = 0.09 nM). Although 0.1 microM tyrosine-atriopeptin (AP I) inhibited labeling of the 66,000 dalton site to nearly the same degree as ANF-(8-33), it produced only a 4-fold increase in cyclic GMP compared to a 400-fold increase with ANF-(8-33). These results suggest that the 66,000-dalton site is not coupled to guanylate cyclase and cyclic GMP formation. Tyrosine-AP I (KI greater than 10 nM) was much weaker at competing for the 130,000-dalton site than ANF-(8-33) (KI = 0.075 nM). Because the EC50 for cyclic GMP stimulation for tyrosine-AP I (greater than 100 nM) and ANF-(8-33) (0.4 nM) is closer to the KI values for the 130,000-dalton protein, this site probably mediates the marked stimulation of cyclic GMP. Our results demonstrate that endothelial cells contain two binding sites for ANF-(8-33) and suggest that only the less abundant site (Mr approximately 130,000) is the receptor coupled to the activation of guanylate cyclase. PMID- 3017940 TI - Mitochondrial DNA of two independent oligomycin-resistant Chinese hamster ovary cell lines contains a single nucleotide change in the ATPase 6 gene. AB - We have determined the nucleotide sequence of the URF A6L and ATPase 6 genes of the mitochondrial DNA of wild-type Chinese hamster ovary (CHO) cells and of two independently isolated, cytoplasmically inherited CHO mutant cell lines that are resistant to oligomycin, an inhibitor of the mitochondrial ATP synthase (ATPase) complex. Comparison of the nucleotide sequences of the mutants with that of their parental cell line revealed a single nucleotide difference, a G-to-A transition at nucleotide 433 of the ATPase 6 gene. This single base pair change predicts a nonconservative amino acid change, with a glutamic acid residue being replaced by a lysine residue at amino acid 145 of the ATPase 6 gene product in the mutants. This glutamic acid residue and several others in the surrounding amino acid sequence are conserved among all species examined to date. Analyses of several of the biochemical properties of the oligomycin-resistant CHO mutants indicate that the glutamic acid residue at position 145 of subunit 6 of the mitochondrial ATP synthase complex is important for the binding of oligomycin to the enzyme complex, but is not essential for proton translocation. PMID- 3017942 TI - Isolation of a cDNA clone encoding S-adenosylmethionine decarboxylase. Expression of the gene in mitogen-activated lymphocytes. AB - S-Adenosylmethionine decarboxylase was purified from bovine liver and digested with endopeptidase Lys-C; the resulting peptides were chromatographically separated. Peptides containing either methionine or tryptophan were subjected to sequence analysis. An oligonucleotide mixture of 48 sequences, which was 17 nucleotides in length, was synthesized based on one of these peptide sequences. This synthetic oligonucleotide mixture was labeled and used to screen a bovine cDNA library in phage lambda gt11. A clone was identified which contained a 1350 nucleotide insert. This insert contained nucleotide sequences coding for amino acid sequences of two of the peptides that were analyzed, thus proving that this cDNA clone codes for S-adenosylmethionine decarboxylase. A subcloned fragment from the coding region of the cDNA was used as a probe to analyze the expression of this gene in mitogen-activated lymphocytes. Northern blots revealed two message species of 2.4 and 3.6 kilobases in length. Both mRNAs were coordinately expressed and were present in polysomes. The levels of these mRNAs increased approximately 4-fold by 9 h after activation of the cells. The magnitude of the increase in these messages is to be compared with an 8- to 10-fold increase in the rate of synthesis of the protein. The apparent increase in translational efficiency of this message upon lymphocyte activation was confirmed by analyzing polysomes from these cells. In resting lymphocytes, the average size of polysomes containing mRNA coding for S-adenosylmethionine decarboxylase was 1.4 ribosomes per mRNA, and this value increased to 2.7 in stimulated cells. Thus, it appears that the increase in translational efficiency of this mRNA arises from an elevated rate of translational initiation, leading to more ribosomes per polysome encoding this particular message. This is not a general effect on the expression of all proteins, since there is no change in the translational efficiency of cytoplasmic actin upon activation of lymphocytes. PMID- 3017943 TI - Identification of NaK-ATPase inhibitors in human plasma as nonesterified fatty acids and lysophospholipids. AB - Elevated plasma levels of factors with cardiac glycoside-like activity have been implicated in the response to volume expansion in animals and in the pathogenesis of certain human diseases. We recently described four fractions (IR1, EI1, EI2, EI3) from normal human plasma that inhibit NaK-ATPase, displace ouabain from the enzyme, and exhibit digoxin-like immunoreactivity (Kelly, R. A., O'Hara, D. S., Canessa, M. L., Mitch, W. E., and Smith, T. W. (1985) J. Biol. Chem. 260, 11396 11405). In this report, we identify the active component of these plasma fractions as long-chain nonesterified fatty acids (NEFA) and lysophospholipids. These lipids were present in fractions EI1, EI2, and EI3 in quantities sufficient to account for all of the NaK-ATPase inhibitory activity. The digoxin-like immunoreactivity in fraction IR1 could be attributed to hydrocortisone and other endogenous steroids. To explore the nature of the lipid-NaK-ATPase interactions, we examined the effects of various ATP or sodium concentrations on the NaK-ATPase activity measured in the presence of NEFA. Varying sodium did not affect the inhibition of NaK-ATPase by linoleic acid. At less than 0.15 mM ATP, linoleic acid stimulated NaK-ATPase, but at higher ATP concentrations, the enzyme was progressively inhibited. In summary, NEFA and lysophospholipids, at levels similar to those occurring in human plasma, may account for all of the NaK-ATPase inhibitory activity observed in human plasma fractions. These lipids probably do not directly regulate NaK-ATPase in vivo under normal physiologic conditions, but may alter the sodium pump in disease states characterized by abnormalities in lipid metabolism or plasma protein binding. PMID- 3017941 TI - 4-Hydroxyphenylpyruvate dioxygenase is an iron-tyrosinate protein. AB - A resonance Raman investigation into the blue chromophore of 4 hydroxyphenylpyruvate dioxygenase, a non-heme iron enzyme from Pseudomonas P. J. 874, reveals the presence of enhanced vibrations characteristic of tyrosinate coordination to the iron center. The excitation profiles for these features show that they are associated with the 595 nm absorption feature. EPR studies of this enzyme indicate the presence of a high-spin ferric center in a rhombic environment, as evidenced by a signal at g = 4.3 with the correct intensity for the measured iron content. This enzyme thus belongs to the emerging class of iron tyrosinate proteins. PMID- 3017944 TI - Structure of sulfated glucuronyl glycolipids in the nervous system reacting with HNK-1 antibody and some IgM paraproteins in neuropathy. AB - Novel sulfated glucuronic acid-containing glycolipids have been identified in the nervous system. These glycolipids are highly antigenic and share antigenic determinants with several nervous system glycoproteins, such as neural cell adhesion molecules, myelin-associated glycoprotein, and ependymins. The structure of the major antigenic glycolipid from human peripheral nerve was determined by chemical and enzymatic degradation, incorporation studies, sugar analysis after permethylation, pertrimethylsilylation, and gas liquid chromatography-mass spectrometry techniques as well as fast atom bombardment-mass spectrometry of the native antigen. The following structure was established for the major antigenic glycolipid. sulfate-3-GlcA beta(1---3)Gal beta(1----4)GlcNAc beta(1----3)Gal beta(1----4)Glc beta(1----1)-ceramide. The major fatty acids in the ceramide were 18:0, 18:1, 24:0, and 24:1, with C18-sphingenine as the long chain base. PMID- 3017945 TI - A new DNA-dependent ATPase which stimulates yeast DNA polymerase I and has DNA unwinding activity. AB - Two forms of DNA-dependent ATPase activity were previously purified from the yeast Saccharomyces cerevisiae and characterized (Plevani, P., Badaracco, G., and Chang, L. M. S. (1980) J. Biol. Chem. 255, 4957-4963). Here, an additional DNA dependent ATPase (ATPase III) has been purified from S. cerevisiae to near homogeneity. This ATPase differs from those described previously in its chromatographic properties, molecular weight, reaction properties and immunological relatedness. Its molecular weight is about 63,000 in the presence of sodium dodecyl sulfate. It hydrolyzes ATP to ADP and orthophosphate in the presence of DNA as an effector. In addition, yeast DNA polymerase I, which is a true DNA replicase of yeast, is stimulated severalfold by this ATPase. Neither yeast DNA polymerase II nor prokaryotic DNA polymerases are stimulated. This stimulation is intrinsic to the ATPase activity, since both activities copurified in the last four steps of purification, showed the same heat stability and showed dependence on and hydrolysis of ATP. The ATPase III preparation also contains a DNA-unwinding (DNA helicase) activity, which unwinds double-stranded DNA in the presence of ATP. In the S. cerevisiae radiation-sensitive mutant rad3, no significant ATPase III activity could be detected, suggesting that the RAD3 gene, which codes for a different polypeptide, regulates the expression of ATPase III activity. PMID- 3017946 TI - Stoichiometric analysis of the specific interaction of the glucocorticoid receptor with DNA. AB - Purified preparations of activated glucocorticoid X receptor complex (GR) contain a Mr 94,000 hormone-binding polypeptide co-purifying together with a Mr 72,000 non-hormone-binding polypeptide (Wrange, O., Okret, S., Radojcic, M., Carlstedt Duke, J., and Gustafsson, J.-A. (1984) J. Biol. Chem. 259, 4534-4541). GR binds selectively to discrete regions of DNA in mouse mammary tumor virus (Payvar, F., DeFranco, D., Firestone, G.L., Edgar, B., Wrange, O., Okret, S., Gustafsson, J. A., and Yamamoto, K. R. (1983) Cell 35, 381-392). Such GR-binding DNA fragments were used to measure the stoichiometry of GR to DNA. Quantitative DNaseI protection "footprinting" analysis was used to ensure that saturation conditions for specific DNA-binding were achieved. Glycerol density gradient centrifugation was used to quantitate Mr 94,000 binding to specific and nonspecific DNA sites. One Mr 94,000 entity was bound per specific DNA site. A modified GR purification procedure resulted in increased amounts of Mr 72,000 polypeptide (1.6:1, 94,000:72,000 molar ratio), compared to previous GR preparations. Glycerol gradient centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the specific GR X DNA complex contained similar amounts of Mr 94,000 and Mr 72,000 polypeptide. It is as yet uncertain if the Mr 72,000 polypeptide is a functional subunit of GR or a co-purifying contaminant only. PMID- 3017947 TI - Identification of the ATP.Mg-dependent protein phosphatase activator (FA) as a myelin basic protein kinase in the brain. AB - The activating factor FA of the ATP.Mg-dependent protein phosphatase FcM was purified to near homogeneity from pig brain by a procedure involving chromatography on phosphocellulose, phosvitin-Sepharose 4B, and Blue Sepharose CL 6B. A specific myelin basic protein (MBP) kinase was found to co-purify with FA in a constant ratio throughout purification. It also proved impossible to separate the two activities on nondenaturing gel electrophoresis and 5-20% sucrose density gradient ultracentrifugation. Kinetic study indicated that MBP, presumably a substrate for FA, could compete with FcM for FA and thereby prevent the FA-mediated activation of the FcM activity. All the results taken together demonstrate that MBP kinase and FA are localized on the same protein. This, together with the data that FA, by activating the ATP.Mg-dependent phosphatase, promotes the dephosphorylation of [32P]MBP, phosphorylated by FA itself, suggests the evidence for a protein bearing two opposing activities involved in the regulation of brain functions. Moreover, since FA is tightly associated with the purified brain myelin membrane, the results further support the notion that FA may well be an endogenous protein kinase responsible for the cyclic phosphorylation-dephosphorylation of the central nervous system myelin. PMID- 3017948 TI - Interferons as gene activators. Cloning of the 5' terminus and the control segment of an interferon activated gene. AB - Treatment of cells with interferons induces various mRNAs and the corresponding proteins. We have described previously the isolation of a mouse cDNA clone (cDNA clone 202) which specifies an mRNA whose level is increased 20-fold in beta interferon-treated Ehrlich ascites tumor cells. The increase is a consequence of an increased rate of transcription. The mRNA encodes a 56,000-dalton protein. We report here the isolation of a genomic clone including the 5' terminus of the 202 gene with the interferon-responsive region. Experiments involving primer extension and protection from cleavage by S1 nuclease revealed the existence of multiple 5' termini of 202 mRNAs in Ehrlich ascites tumor and Ltk- cells. Treatment with beta-interferon increased the level of these 202 mRNAs with different 5' termini nonuniformly. A 0.8-kilobase DNA segment from the 202 gene (including its 5' flanking region and its 5'-terminal exon) was ligated to the chloramphenicol acetyltransferase gene, and the resulting construct was transfected into mouse Ltk- cells. Treatment of these cells with beta-interferon increased the expression of the chloramphenicol acetyltransferase gene 5-10-fold. Within the first, untranslated exon of the 202 gene, we found a 29-nucleotide long sequence that is partially homologous to sequences which occur upstream from interferon-inducible human HLA and metallothionein IIA genes (Friedman, R. L., and Stark, G. R. (1985) Nature 314, 637-639). PMID- 3017949 TI - Deletion and insertion mutants in the structural gene for ribosomal protein S1 from Escherichia coli. AB - Mutants have been constructed by deleting regions of the gene rpsA for ribosomal protein S1, which had been cloned in plasmid pACYC184. The mutant genes were analyzed for their ability to complement an S1 amber mutant containing a temperature-sensitive suppressor. Another series of mutants was constructed using the tac promoter plasmid pKK223-3, and the effect of the mutant proteins was analyzed in a strain wild type for rpsA. The gene products of all mutants were identified by the immunoblotting technique. Plasmids with a mutant rpsA gene which do not or only poorly complement the S1 amber mutation cause drastic growth reduction, whereas the overall protein synthesis is affected to different extents depending on the site of the deletion. Mutants which express S1 fragments comprising at least the NH2-terminal 100 amino acids stimulate or inhibit the synthesis of certain cellular proteins. The amount of chromosomal coded S1 was reduced by each mutant plasmid. Our data suggest that S1 has a general regulatory role during protein biosynthesis. PMID- 3017950 TI - Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae. Characterization of mutants in 34 complementation groups. AB - To identify nuclear functions required for cytochrome c oxidase biogenesis in yeast, recessive nuclear mutants that are deficient in cytochrome c oxidase were characterized. In complementation studies, 55 independently isolated mutants were placed into 34 complementation groups. Analysis of the content of cytochrome c oxidase subunits in each mutant permitted the definition of three phenotypic classes. One class contains three complementation groups whose strains carry mutations in the COX4, COX5a, or COX9 genes. These genes encode subunits IV, Va, and VIIa of cytochrome c oxidase, respectively. Mutations in each of these structural genes appear to affect the levels of the other eight subunits, albeit in different ways. A second class contains nuclear mutants that are defective in synthesis of a specific mitochondrial-encoded cytochrome c oxidase subunit (I, II, or III) or in both cytochrome c oxidase subunit I and apocytochrome b. These mutants fall into 17 complementation groups. The third class is represented by mutants in 14 complementation groups. These strains contain near normal amounts of all cytochrome c oxidase subunits examined and therefore are likely to be defective at some step in holoenzyme assembly. The large number of complementation groups represented by the second and third phenotypic classes suggest that both the expression of the structural genes encoding the nine polypeptide subunits of cytochrome c oxidase and the assembly of these subunits into a functional holoenzyme require the products of many nuclear genes. PMID- 3017951 TI - Escherichia coli topoisomerase I can segregate replicating pBR322 daughter DNA molecules in vitro. AB - The reconstituted pBR322 DNA replication system has been used to identify a mechanism for the processing and segregation of daughter DNA molecules by Escherichia coli topoisomerase I (Topo I) during the terminal stages of DNA replication. At low concentrations of Topo I (sufficient to confer specificity to the replication system for DNA templates containing a ColE1-type origin of DNA replication), the major products of the replication reaction were: multigenome length, linear, double-stranded DNA molecules (an aberrant product); multiply interlinked, catenated, supercoiled DNA dimers; and a last Cairns-type replication intermediate. Thirty- to fifty-fold higher concentrations of Topo I led to the appearance of form II and form I pBR322 DNA as the only synthetic products. A model was developed in which Topo I, bound to a single-stranded gap on the parental H strand DNA just upstream of the origin of DNA replication, catalyzed the decatenation of the intermolecular linkages between the two daughter DNA molecules that were generated by primosome-catalyzed unwinding of the residual nonreplicated parental duplex DNA in the last Cairns-type intermediate. At low concentrations of Topo I, however, the intermolecular linkages persisted and, within the context of this replication system, were not removed by DNA gyrase. In support of this model it was demonstrated that: there was a single-stranded gap between the nonreplicated parental duplex region and the 5' end of the nascent leading-strand DNA; the number of intermolecular linkages in the catenated supercoiled DNA dimers was inversely related to the concentration of Topo I; the supercoiled DNA dimers did not serve as a precursor of the final form I DNA product; and maturation of the last Cairns-type replication intermediate to form I DNA was not affected by the presence of coumermycin, a potent inhibitor of the activities of DNA gyrase. PMID- 3017952 TI - Ca2+ regulates the inositol tris/tetrakisphosphate pathway in intact and broken preparations of insulin-secreting RINm5F cells. AB - The two-step isomerization of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) to Ins 1,3,4-P3 via the intermediate inositol 1,3,4,5-tetrakisphosphate (Ins-P4) was studied in intact RINm5F cells and in subcellular fractions. Muscarinic stimulation with carbamylcholine leads to a rapid (2 s) rise in both Ins-1,4,5-P3 and Ins-P4, whereas Ins-1,3,4-P3 was produced only after a lag of at least 5 s. In cells with depleted Ca2+ stores, the rise in Ins-1,4,5-P3 was nearly tripled, and that of Ins-1,3,4-P3 markedly diminished as compared to control cells. Raising the free Ca2+ concentration from 10(-7) to 10(-5) M increased inositol 1,4,5-triphosphate-3-kinase activity in cytosolic fractions by 2 1/2-fold (EC50 for Ca2+ approximately 0.8 microM) but had no effect on the activity of inositol 1,4,5-triphosphate-5-phosphomonoesterase. At 10(-7) M Ca2+ these two enzymes displayed comparable activity when assayed at concentrations of Ins-1,4,5-P3 occurring in stimulated cells; however, at 10(-5) M Ca2+, kinase activity predominates. These results suggest that Ins-1,4,5-P3 counter-regulates its own levels through the activity of inositol 1,4,5-trisphosphate 3-kinase and that the increase in [Ca2+]i may account for the transience of the rise in Ins-1,4,5-P3 seen during muscarinic stimulation of RINm5F cells. PMID- 3017953 TI - Inhibition of peptide amidation by disulfiram and diethyldithiocarbamate. AB - Peptidylglycine alpha-amidating monooxygenase is a copper- and ascorbate dependent enzyme that converts peptides with COOH-terminal glycine residues into the corresponding alpha-amidated product peptides. The relatively selective copper chelator N,N-diethyldithiocarbamate (DDC) and its disulfide dimer, disulfiram (Antabuse), were used to determine whether the availability of copper affects the production of two alpha-amidated pro-ACTH/endorphin-derived peptides, alpha-melanotropin (alpha MSH) and joining peptide. When mouse pituitary corticotropic tumor cells (AtT-20) were grown in medium containing micromolar concentrations of disulfiram or DDC, alpha-amidation of newly synthesized joining peptide was specifically inhibited in a dose-dependent manner. In rats injected twice with disulfiram or DDC, the ability of the intermediate pituitary to alpha amidate newly synthesized alpha MSH and joining peptide was inhibited in a dose dependent manner; at disulfiram doses equivalent to those used in alcohol abuse therapy (4 mg/kg/day), only about 10% of the newly synthesized peptides were correctly alpha-amidated. Chronic treatment of rats with DDC or disulfiram produced a dose-dependent increase in the pituitary content of glycine-extended alpha MSH and joining peptide; the total amount of pro-ACTH/endorphin-related material was unaltered. After 11 days of treatment with 4 mg/kg/day disulfiram, about one-third of the pituitary alpha MSH and joining peptide were present in the glycine-extended rather than the alpha-amidated form; pituitary extracts normally contain almost entirely alpha-amidated peptides. PMID- 3017954 TI - Regulation of membrane-associated and cytosolic phospholipase C activities in human platelets by guanosine triphosphate. AB - The effects of guanine nucleotides, thrombin, and platelet cytosol (100,000 X g supernatant) on the hydrolysis of polyphosphoinositides by phospholipase C was examined in isolated platelet membranes labeled with [3H]inositol. Guanosine 5' (3-O-thio)triphosphate (GTP gamma S) (10 microM) caused a 2-fold stimulation of polyphosphoinositide hydrolysis, compared to background. GTP gamma S (10 microM) plus thrombin (1 unit/ml) stimulated the release of inositol triphosphate, inositol diphosphate, and inositol phosphate 500, 300, and 250%, respectively, compared to GTP gamma S alone. Cytosol prepared from unlabeled platelets slightly increased the release of inositol phosphates from [3H]inositol-labeled membranes. Addition of cytosol plus GTP gamma S (10 microM), however, resulted in a 300% enhancement of the release of inositol phosphates compared to membranes incubated with thrombin and GTP gamma S. The stimulatory effects of cytosol and GTP gamma S on polyphosphoinositide hydrolysis were also observed when membranes were replaced by sonicated lipid vesicles prepared from a total platelet lipid extract. These data suggest that PIP2 hydrolysis in platelets is catalyzed by a soluble phospholipase C which is regulated by a GTP-binding regulatory protein. PMID- 3017955 TI - Kinetic studies on the processing of human beta-lipotropin by bovine pituitary intermediate lobe pro-opiomelanocortin-converting enzyme. AB - The kinetics of the previously reported paired basic residue-specific pro opiomelanocortin-converting enzyme from bovine pituitary intermediate lobe secretory vesicles (Loh, Y. P., Parish, D.C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205) were studied using human 125I-beta-lipotropin as substrate. The enzyme, at a concentration of 20 ng/100 microliters cleaved human beta lipotropin to yield gamma-lipotropin, a beta-melanotropin linked to beta endorphin intermediate and beta-endorphin, whereas at an enzyme concentration of 40 ng/100 microliters, the substrate was completely cleaved to yield beta endorphin and beta-melanotropin. These products were identified by their immunological properties and size on sodium dodecyl sulfate-polyacrylamide gels. The 125I-beta-endorphin product was further shown by high pressure liquid chromatography to contain two forms; the major form co-eluted with 125I-Arg0-beta h-endorphin and the minor form with 125I-beta h-endorphin. No COOH-terminal shortened forms of beta-endorphin were detected. The products formed indicate cleavages at two of the three pairs of basic residues of human beta-lipotropin, at Lys37-Lys38 and Lys57-Arg58, but not at Lys86-Lys87. The cleavage at Lys57 Arg58 occurred primarily in between these basic residues. The Km values for the cleavage of the Lys37-Lys38 and Lys57-Arg58 pairs were 1.9 and 2.5 microM, respectively. The Vmax values for the cleavage of the Lys37-Lys38 and Lys58-Arg58 pairs were 4.8 nmol/micrograms of enzyme/h and 9.1 nmol/micrograms of enzyme/h, respectively. PMID- 3017956 TI - Loss of thyroidal inducibility of Na,K-ATPase with neoplastic transformation in tissue culture. AB - Thyroidal induction of the plasma membrane Na,K-ATPase is a characteristic of mammalian tissues that exhibit a thermogenic response to this hormone. To facilitate analysis of the pathways mediating this response, we defined the conditions needed for reproducible thyroidal induction of this enzyme, as well as mitochondrial cytochrome c oxidase, in established cell lines in tissue cultures. In confluent monolayers of nontransformed mouse embryo fibroblasts (C3H/10T1/2), triiodothyronine modulated Na,K-ATPase and cytochrome c oxidase activities in a concentration- and time-dependent manner. Similar increases in Na,K-ATPase activity were obtained in other rodent embryo cells (SWISS/3T3 and NIH/3T3) and in human fibroblasts (WI-38). In contrast, neoplastic transformation of all of these cell lines resulted in loss of inducibility of Na,K-ATPase by thyroid hormone, regardless of the initiating mechanism (i.e. spontaneous, x-ray, chemicals, viruses). PMID- 3017957 TI - The protein substrate binding site of the ubiquitin-protein ligase system. AB - In order to gain insight into the mechanisms that determine the selectivity of the ubiquitin proteolytic pathway, the protein substrate binding site of the ubiquitin-protein ligase system was identified and examined. Previous studies had shown that the ligase system consists of three components: a ubiquitin-activating enzyme (E1), ubiquitin-carrier protein (E2), and a third enzyme, E3, the mode of action of which has not been defined. E3 from rabbit reticulocytes was further purified by a combination of affinity chromatography, hydrophobic chromatography, and gel filtration procedures. A 180-kDa protein was identified as the subunit of E3. Two independent methods indicate that E3 has the protein binding site of the ubiquitin ligase system. These are the chemical cross-linking of 125I-labeled proteins to the E3 subunit and the functional conversion of enzyme-bound labeled proteins to ubiquitin conjugates in pulse-chase experiments. The trapping of E3 bound protein for labeled product formation was allowed by the slow dissociation of E3 X protein complex. The specificity of binding of different proteins to E3, examined by both methods, showed a direct correlation with their susceptibility to degradation by the ubiquitin system. Proteins with free alpha-NH2 groups, which are good substrates, bind better to E3 than corresponding proteins with blocked NH2 termini, which are not substrates. Oxidation of methionine residues to sulfoxide derivatives greatly increases the susceptibility of some proteins to ligation with ubiquitin, with a corresponding increase in their binding to E3. However, a protein derivative which was subjected to both amino group modification and oxidation binds strongly to the enzyme, even though it cannot be ligated to ubiquitin. It thus seems that the substrate binding site of E3 participates in determining the specificity of proteins that enter the ubiquitin pathway of protein degradation. PMID- 3017958 TI - Structure, expression, and evolution of a heat shock gene locus in Caenorhabditis elegans that is flanked by repetitive elements. AB - A locus containing two hsp16 genes in Caenorhabditis elegans has been characterized by DNA sequencing. Each gene encodes a 16-kDa polypeptide which is expressed following heat induction. The two genes, designated hsp16-2 and hsp16 41, are arranged in divergent orientations, and each contains a single intron of 46 and 58 base pairs, respectively. Although both gene transcripts are spliced efficiently in vivo, hsp16-41 corresponds to a previously isolated cDNA which contains an unspliced intron sequence. The 5'-noncoding regions of both genes contain TATA boxes preceded 18 or 19 nucleotides upstream by a heat shock regulatory sequence. The 3'-noncoding regions contain polyadenylation signals (AATAAA) either downstream (hsp16-2) or immediately adjacent (hsp16-41) to a sequence capable of forming a hairpin. This pair of hsp16 genes is flanked by three copies of an approximately 200-bp dispersed repetitive element (two copies on one side and a single one on the other side of the locus) which occurs in at least 70 copies throughout the C. elegans genome, and has been designated CeRep 16. Together with data described previously (Russnak, R. H., and Candido, E. P. M. (1985) Mol. Cell. Biol. 5, 1268-1278), the results presented here define a family of four distinct, related small heat shock protein genes. These are arranged in divergently transcribed pairs at two loci. The hsp16-48/41 genes code for one class of HSP16, 143-amino acid residues long, while the hsp16-1/2 genes encode the other class, which is 2 amino acid residues longer. Thus each locus codes for the two major types of HSP16. The two loci differ in a number of respects, including the presence of a tandem inverted duplication of two heat shock protein genes at one locus, and of repetitive elements at the other. Sequence comparisons allow us to propose a scheme for the evolution of the four genes and reveal conserved features of noncoding regions which may be involved in the regulation of their transcription, RNA processing, or translation. Using locus-specific hybridization probes, we have found that the genes at locus hsp16 2/41 are expressed at levels approximately 20-40-fold higher than those at locus hsp16-1/48. PMID- 3017959 TI - Hydroxyl/bile acid exchange. A new mechanism for the uphill transport of cholate by basolateral liver plasma membrane vesicles. AB - In order to characterize the driving forces for the concentrative uptake of unconjugated bile acids by the hepatocyte, the effects of pH gradients on the uptake of [3H]cholate by rat basolateral liver plasma membrane vesicles were studied. In the presence of an outwardly directed hydroxyl gradient (pH 6.0 outside and pH 7.5 inside the vesicle), cholate uptake was markedly stimulated and the bile acid was transiently accumulated at a concentration 1.5- to 2-fold higher than at equilibrium ("overshoot"). In the absence of a pH gradient (pH 6.0 or 7.5 both inside and outside the vesicle), uptake was relatively slower and no overshoot was seen. Reductions in the magnitude of the transmembrane pH gradient were associated with slower initial uptake rates and smaller overshoots. Cholate uptake under pH gradient conditions was inhibited by furosemide and bumetanide but not by 4, 4'-diisothiocyano-2,2'-disulfonic stilbene (SITS), 4-acetamido-4' isothiocyanostilbene-2,2'-disulfonic acid (DIDS), or probenecid. In the absence of a pH gradient, an inside-positive valinomycin-induced K+ diffusion potential caused a slight increase in cholate uptake which was insensitive to furosemide. Moreover, in the presence of an outwardly directed hydroxyl gradient, uphill cholate transport was observed even under voltage clamped conditions. These findings suggest that pH gradient-driven cholate uptake was not due to associated electrical potentials. Despite an identical pKa to that of cholate, an outwardly directed hydroxyl gradient did not drive uphill transport of three other unconjugated bile acids (deoxycholate, chenodeoxycholate, ursodeoxycholate), suggesting that a non-ionic diffusion mechanism cannot account for uphill cholate transport. In canalicular vesicles, although cholate uptake was relatively faster in the presence of a pH gradient than in the absence of a gradient, peak uptake was only slightly above that found at equilibrium under voltage clamped conditions. These findings suggest a specific carrier on the basolateral membrane of the hepatocyte which mediates hydroxyl/cholate exchange (or H+-cholate co transport). A model for uphill cholate transport is discussed in which the Na+ pump would ultimately drive Na+/H+ exchange which in turn would drive hydroxyl/cholate exchange. PMID- 3017960 TI - Autoproteolysis of the small subunit of calcium-dependent protease II activates and regulates protease activity. AB - Calcium-dependent protease II (CDP-II) from bovine heart is a heterodimer with subunit molecular weights of 80,000 and 26,000. Previous studies have demonstrated that the protease requires 350 microM Ca2+ for half-maximal activity and that the large subunit contains both the catalytic and Ca2+ binding functions of the enzyme. The function of the small subunit has been unclear. We have examined the effect of Ca2+ on structural and catalytic properties of CDP-II in the presence and absence of substrate proteins. When incubated with Ca2+ in the absence of substrate, CDP-II undergoes a series of autoproteolytic cleavages that sequentially reduce the small subunit's molecular weight from 26,000 to 24,000 to 22,000 to 17,000. During this time there is no detectable change in the 80-kDa subunit, which remains associated with the autolyzed small subunit. The rate of autoproteolysis is dependent on temperature and on the concentration of Ca2+ (half-maximal rate at approximately 600 microM Ca2+). The first cleavage appears to be unimolecular because its rate is unaffected by CDP-II concentration or by the presence of exogenous protein substrates. Subsequent cleavages result in the formation of the 80-kDa/17-kDa heterodimer and appear to occur by bimolecular reactions; rates of these reactions were slowed by decreasing CDP-II concentrations and by the presence of protein substrates. Autoproteolysis of the small subunit has two distinct functional consequences, each of which is associated with different forms of the autolyzed protease. Our results indicate that the 80-kDa/26-kDa form of CDP-II represents an inactive proenzyme and that the initial Ca2+-dependent cleavage of the 26-kDa subunit results in activation of the protease. The activated enzyme hydrolyzes protein substrates with a Ca2+ concentration requirement of 350 microM for half-maximal rates. The further autoproteolysis, which results in the formation of the 80-kDa/17-kDa heterodimer, serves to reduce the Ca2+ concentration requirement for protease activity by 25 fold. Thus, these results provide evidence for specific roles of the small subunit in the regulation of CDP-II activity. PMID- 3017961 TI - Structure of proteoheparan sulfates from fibroblasts. Confluent and proliferating fibroblasts produce at least three types of proteoheparan sulfates with functionally different core proteins. AB - [3H]Leucine- and [35S]sulfate-labeled proteoheparan sulfates were isolated from postconfluent or proliferating cultures of human skin fibroblasts. Cell layers were solubilized by Triton X-100, and transferrin-binding macromolecules were isolated by affinity chromatography. Proteoglycans with no affinity for transferrin were purified by using ion-exchange and gel permeation chromatography. Postconfluent cells synthesize a proteoheparan sulfate of Mr 350,000 (as determined by gel permeation chromatography) which has affinity for transferrin as well as for octyl-Sepharose. Its core protein (Mr 180,000) consists of two disulfide-bonded polypeptides of Mr 90,000. This species was not detected in cultures of proliferating cells. Proliferating and confluent cells also synthesize other forms of proteoheparan sulfates (Mr 200,000-400,000) which have no affinity for transferrin. However, most of them have affinity for octyl Sepharose. The core protein of proteoheparan sulfates made by proliferating cells has Mr 50,000. A smaller form (Mr 250,000) of this proteoglycan was solubilized by Triton X-100, whereas a larger form (Mr 400,000) remained associated with the pericellular matrix. A third type of proteoheparan sulfate (Mr 200,000) without affinity for transferrin nor octyl-Sepharose was associated with postconfluent cell layers but not with proliferating ones. Its core protein has Mr 35,000. Heparan sulfate oligosaccharides (Mr 6,000 or higher) were found in proliferating cells but not in postconfluent ones. PMID- 3017963 TI - Differences in the kinetic properties of BamHI endonuclease and methylase with linear DNA substrates. AB - BamHI endonuclease and methylase were found to exhibit a kinetic preference for linear pBR322 DNA substrates containing the recognition site in a central location. The Km values for substrates having the recognition site in a terminal location were approximately 3-fold greater than those with a centrally located site. This phenomenon may be partially due to facilitated transfer of the enzymes to the recognition site over nonspecific flanking sequences. The exploitation of facilitated transfer by these enzymes has been inferred from studies demonstrating kinetic preferences for longer DNA substrates. The reaction rates of the endonuclease were 9-fold greater with full-length pBR322 DNA than with a 74-base pair derivative. The methylase exhibits a kinetic preference for longer substrates but only under conditions of comparatively higher DNA concentrations. In addition, the methylase has the property of increasing long chain preference with increasing salt concentrations up to 120 mM. Increasing salt concentrations decreased the endonuclease's preference for longer substrates. Nonspecific inhibition studies revealed qualitative and quantitative differences between the two enzymes under catalytic conditions. These studies suggest that BamHI endonuclease and methylase interact with nonspecific DNA in different ways. PMID- 3017962 TI - The regulation of intracellular pH in monkey kidney epithelial cells (BSC-1). Roles of Na+/H+ antiport, Na+-HCO3(-)-(NaCO3-) symport, and Cl-/HCO3- exchange. AB - Using the pH-sensitive absorbance of 5 (and 6)-carboxy-4',5'- dimethylfluorescein, we investigated the regulation of cytoplasmic pH (pHi) in monkey kidney epithelial cells (BSC-1). In the absence of HCO3-, pHi is 7.15 +/- 0.1, which is not significantly different from pHi in 28 mM HCO3-, 5% CO2 (7.21 +/- 0.07). After an acid load, the cells regulate pHi in the absence of HCO3- by a Na+ (or Li+)-dependent, amiloride-inhibitable mechanism (indicative of Na+/H+ antiport). In 28 mM HCO3-, while still dependent on Na+, this regulation is only blocked in part by 1 mM amiloride. A partial block is also observed with 4,4' diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (1 mM). With cells pretreated with DIDS, 1 mM amiloride nearly totally inhibits this regulation. Cl- had no effect on pHi regulation in the acidic range. In HCO3(-)-free saline, Na+ removal leads to an amiloride-insensitive acidification, which is dependent on Ca2+. In 28 mM HCO3-, Na+ (and Ca2+) removal led to a pronounced reversible and DIDS sensitive acidification. When HCO3- was lowered from 46 to 10 mM at constant pCO2 (5%), pHi dropped by a DIDS-sensitive mechanism. Identical changes in pHo (7.6 to 6.9) in the nominal absence of HCO3- led to smaller changes of pHi. In the presence but not in the absence of HCO3-, removal of Cl- led to a DIDS-sensitive alkalinization. This was also observed in the nominal absence of Na+, which leads to a sustained acidification. It is concluded that in nominally bicarbonate-free saline, the amiloride-sensitive Na+/H+ antiport is the predominant mechanism of pHi regulation at acidic pHi, while being relatively inactive at physiological values of pHi. In bicarbonate saline, two other mechanisms effect pHi regulation: a DIDS-sensitive Na+-HCO3- symport, which contributes to cytoplasmic alkalinization, and a DIDS-sensitive Cl-/HCO3- exchange, which is apparently independent of Na+. PMID- 3017965 TI - The nucleotide sequence of the serA gene of Escherichia coli and the amino acid sequence of the encoded protein, D-3-phosphoglycerate dehydrogenase. AB - The nucleotide sequence of serA, the structural gene of Escherichia coli which codes for D-3-phosphoglycerate dehydrogenase, has been determined. The structural gene contains 1233 nucleotides which code for the 409 amino acids of the subunit of the tetrameric enzyme, as well as the initiator methionine, which is cleaved from the mature protein, and the termination codon. The majority of the primary structure of the enzyme has been confirmed by automated Edman degradation of peptide fragments produced by a variety of cleavage agents. Comparison of the amino acid sequence of phosphoglycerate dehydrogenase with other NAD-dependent oxidoreductases reveals less than 20% homology, although conservation of certain specific residues in the coenzyme binding domain appears to be evident. PMID- 3017964 TI - Differential and common recognition of the catalytic sites of the cGMP-dependent and cAMP-dependent protein kinases by inhibitory peptides derived from the heat stable inhibitor protein. AB - Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein of cAMP-dependent protein kinase (Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., and Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992) were tested as inhibitors of cGMP-dependent protein kinase. The peptides themselves were not substrates. cGMP-dependent protein kinase activity was assayed using histone H2B and two synthetic peptide substrates. Consistent with previous observations of other peptide inhibitors of this enzyme (Glass, D. B. (1983) Biochem. J. 213, 159-164), the inhibitory peptides had no effect on the phosphorylation of histone H2B, but they competitively inhibited cGMP-dependent phosphorylation of the two peptide substrates. The parent inhibitor peptide, PKI(5-24)amide, and a series of analogs had Ki (or IC50) values for cGMP-dependent protein kinase in the range of 15-190 microM. In contrast to their effects on the cAMP-dependent protein kinase, the inhibitory peptides were substantially less potent with cGMP-dependent protein kinase, and potency was reduced by the presence of the NH2-terminal residues (residues 5-13). We conclude that the two protein kinases share a recognition of the basic amino acid cluster within the pseudosubstrate region of the peptide, but that the cGMP-dependent protein kinase does not recognize additional NH2 terminal determinants that make the inhibitor protein extremely potent toward the cAMP-dependent enzyme. Even- when tested at high concentrations and with peptide substrates, the native inhibitor protein did not inhibit cGMP-dependent protein kinase under assay conditions in which the peptides derived from it were inhibitory. Thus, the native inhibitor protein appears to have structural features which block interaction with the cGMP-dependent enzyme and enhance its selectivity for cAMP-dependent protein kinase. PMID- 3017966 TI - Three partial reactions of ribulose-bisphosphate carboxylase require both large and small subunits. AB - Three partial reactions of ribulose-bisphosphate carboxylase/oxygenase were measured in the presence and absence of small subunits using the enzyme from the cyanobacterium, Synechococcus ACMM 323, whose small subunits may be reversibly dissociated from its octameric, large-subunit core. These partial reactions were: the exchange of the proton at C-3 of the substrate, ribulose 1,5-bisphosphate, with the medium which is indicative of C-2, C-3 enolization; the hydrolysis of the 6-carbon reaction intermediate, 3-keto-2-carboxy-D-arabinitol 1,5 bisphosphate, to two molecules of 3-phosphoglycerate; and the decarboxylation of the 6-carbon intermediate, which is catalyzed only by the deactivated, divalent metal-ion-free carboxylase. None of these partial reactions was catalyzed by the small-subunit-depleted, large-subunit octamer to an extent greater than that expected from the residual small subunit content (about 3%), implying that small subunits are required for all three reactions. Clearly, the small subunit's influence is not restricted to any single stage of the catalytic sequence. Under conditions where it was possible to demonstrate tight binding of the reaction intermediate analog, 2-carboxy-D-arabinitol 1,5-bisphosphate, to the large subunit octamer, no binding of the 6-carbon intermediate could be detected. We suggest that either the tight-binding form of the 6-carbon intermediate is the hydrated gem-diol, not the ketone, or the large subunits by themselves intrinsically possess a trace of catalytic activity which discharges any bound intermediate before it can be measured. PMID- 3017968 TI - Studies on the transverse tubule membrane Mg-ATPase. Lectin-induced alterations of kinetic behavior. AB - Transverse tubule (TT) membrane vesicles contain a very active Mg-ATPase (EC 3.6.1.3). Concanavalin A (ConA) and other lectins were found to activate the TT Mg-ATPase from chicken skeletal muscle up to 25-fold yielding specific activities greater than 800 mumol/h/mg. The sarcoplasmic reticulum Ca-ATPase and the sarcolemma Na,K-ATPase were unaffected by ConA. 125I-Labeled lectin binding to the TT membrane Mr 102,000 glycoprotein supports the contention that this protein is identical with or is intimately associated with the TT Mg-ATPase. The ATPase exhibited non-Michaelis-Menton kinetics with both apparent negative cooperativity (n = 0.723; S0.5, Mg-ATP = 14 microM) and substrate inhibition (Ki, Mg-ATP = 10.2 mM), both of which were eliminated in the presence of ConA. Under the same conditions, ConA also abolished the unusual temperature dependence and potent Triton X-100 inhibition. The similarities in ConA suppression of both Triton and substrate inhibition suggest that these ligands may be interacting through a non catalytic site and that Triton is serving as a nucleotide-mimetic agent. The unique kinetic responses are consistent with a homotropic substrate modifier mechanism wherein the enzyme can be viewed as possessing a single catalytic and a single regulatory site on a single polypeptide chain. It is proposed that ConA interferes either with ligand interaction at a putative regulatory site or blocks communication between a regulatory site and the catalytic site. The possible nature of the regulatory site and its modulation by a ConA-like, endogenous, skeletal muscle lectin and their combined role in excitation-contraction coupling is discussed. PMID- 3017967 TI - Desensitization of the beta-adrenergic receptor-coupled adenylate cyclase in cultured mammalian cells. Receptor sequestration versus receptor function. AB - Human A431 and rat glioma C6 cells exposed to isoproterenol underwent a time- and dose-dependent loss of isoproterenol-stimulated adenylate cyclase activity. Desensitization was accompanied by sequestration of beta-adrenergic receptors, which became less accessible to the hydrophilic antagonist 3H-labeled 4-(3-tert butylamino-2-hydroxypropoxy)benzimidazole-2-one hydrochloride ([3H]CGP-12177) and redistributed from the heavier density plasma membrane fraction to a lighter density membrane fraction. Prior treatment of the cells with concanavalin A or phenylarsine oxide blocked sequestration of the receptors but not desensitization of the agonist-stimulated adenylate cyclase. The membranes from such pretreated cells were exposed to alkali to inactivate adenylate cyclase, and the receptors were transferred to a foreign adenylate cyclase by membrane fusion with polyethylene glycol. beta receptors from desensitized cells exhibited a reduced ability to maximally stimulate the foreign adenylate cyclase, but remained accessible to [3H]CGP-12177 in the fused membranes. When isoproterenol-treated cells were washed free of agonist, there was a time-dependent recovery of agonist responsiveness and [3H]CGP-12177-binding sites. Using the fusion technique, the receptors recovered their functional activity in the resensitized cells. In concanavalin A-treated cells, desensitization and resensitization appeared to occur in the absence of receptor sequestration. Finally, membranes from desensitized cells pretreated with concanavalin A were fused with polyethylene glycol and assayed for agonist-stimulated adenylate cyclase. There was no reversal of the desensitized state. Thus, the primary, essential step in the desensitization process is a reduction in functional activity of the beta adrenergic receptor. In contrast, sequestration of the receptors is not a prerequisite, but a secondary event during desensitization. PMID- 3017969 TI - Identification of a gastrin binding protein in porcine gastric mucosal membranes by covalent cross-linking with iodinated gastrin. AB - A gastrin binding protein (GBP) has been identified in detergent extracts of porcine gastric mucosal membranes by covalent cross-linking to 125I [Nle15]gastrin with disuccinimidyl suberate. The apparent molecular weight of the cross-linked complex (80,000) is uneffected by reduction suggesting that the GBP is not composed of disulfide-bonded subunits. Subtraction of the molecular weight of 125I-gastrin indicates that the molecular weight of the GBP is 78,000. A similar molecular weight has been observed previously for the gastrin receptor (74,000) on intact canine parietal cells and plasma membranes therefrom, and for the receptor for the related hormone cholecystokinin (76,000-85,000) on pancreatic acinar membranes under reducing conditions. The similarity in molecular weight between the gastrin receptor and the solubilized GBP suggests that the latter protein is probably the gastrin receptor. However, the concentration (2 microM) of [Nle15]gastrin required for 50% inhibition of cross linking of gastrin to the GBP solubilized in 0.1% Triton X-100 is 200-fold greater than the value (10 nM) observed for the gastrin receptor on isolated canine gastric parietal cells. A lower concentration (0.3 microM) of [Nle15]gastrin was required to inhibit cross-linking in a milder detergent (0.4% digitonin, 0.08% cholate). Thus, the reduced affinity for gastrin of the putative solubilized form of the gastrin receptor appears to be a result of detergent extraction. PMID- 3017970 TI - Purification of a three-subunit ubiquinol-cytochrome c oxidoreductase complex from Paracoccus denitrificans. AB - A ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex has been purified from the plasma membrane of aerobically grown Paracoccus denitrificans by extraction with dodecyl maltoside and ion exchange chromatography of the extract. The purified complex contains two spectrally and thermodynamically distinct b cytochromes, cytochrome c1, and a Rieske-type iron-sulfur protein. Optical spectra indicate absorption peaks at 553 nm for cytochrome c1 and at 560 and 566 nm for the high and low potential hemes of cytochrome b. The spectrum of cytochrome b560 is shifted to longer wavelength by antimycin. The Paracoccus bc1 complex consists of only three polypeptide subunits. On the basis of their relative electrophoretic mobilities, these have apparent molecular masses of 62, 39, and 20 kDa. The 62- and 39-kDa subunits have been identified as cytochromes c1 and b, respectively. The 20-kDa subunit is assumed to be the Rieske-type iron sulfur protein on the basis of its molecular weight and the presence of an EPR detectable signal typical of this iron-sulfur protein in the three-subunit complex. The Paracoccus bc1 complex catalyzes reduction of cytochrome c by ubiquinol with a turnover of 470 s-1. This activity is inhibited by antimycin, myxothiazol, stigmatellin, and hydroxyquinone analogues of ubiquinone, all of which inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain. The electron transfer functions of the Paracoccus complex thus appear to be similar, and possibly identical, to those of the bc1 complex of eukaryotic mitochondria. The Paracoccus bc1 complex has the simplest subunit composition and one of the highest turnover numbers of any bc1 complex isolated from any species to date. These properties suggest that the structural requirements for electron transfer from ubiquinol to cytochrome c are met by a small number of peptides and that the "extra" peptides occurring in the mitochondrial bc1 complexes serve some other function(s), possibly in biogenesis or insertion of the complex into that organelle. PMID- 3017971 TI - A 66-base pair insert bridges the deletion responsible for a mouse model of beta thalassemia. AB - The breakpoints of the deletion responsible for the Hbb(th-1) mouse model of beta thalassemia have been isolated. A 3709 (+/- 2)-base pair (bp) region, including the entire beta major globin gene and 2 kilobases of 5' flanking region, is deleted. A novel 66 (+/- 2)-bp sequence, ending in a stretch of 25 dA:dT base pairs, was found to bridge the deletion. A region of the normal murine genome, containing the first 43 bp of the deletion-associated insert (DAI), but lacking the 25-bp dA:dT sequence, was isolated. All normal mice tested contain this DAI like element and several inbred strains contain an additional DAI-like element. The sequence spanning the Hbb(th-1) deletion may be a reverse transcript of this region. PMID- 3017972 TI - Identification of distinct receptors for two hemopoietic growth factors (granulocyte colony-stimulating factor and multipotential colony-stimulating factor) by chemical cross-linking. AB - Granulocyte colony-stimulating factor (G-CSF) and multipotential colony stimulating factor (multi-CSF or interleukin 3) are two members of a family of hemopoietic growth and differentiation factors. Using biologically active radioiodinated derivatives and chemical cross-linking (predominantly with the homobifunctional reagent disuccinimidyl suberate) followed by gel electrophoresis and autoradiography, receptors for these two factors have been identified. The G CSF receptor was identified as a single subunit protein of Mr approximately 150,000 while two molecular species able to specifically cross-link to 125I-multi CSF were identified of Mr approximately 75,000 and 60,000. For both CSFs specificity of formation of cross-linked species was demonstrated by showing that the homologous unlabeled CSF (but not other CSFs) competed for formation of the complexes with the appropriate dose-response relation, by showing that saturation occurred over the appropriate range of 125I-CSF concentration and by showing that the cellular specificity of CSF binding paralleled that for cross-linked complex formation. The formation of cross-linked complexes was dependent on the concentration and type of chemical cross-linker, especially for cross-linking of 125I-multi-CSF. Based on a number of criteria it is suggested that the two species cross-linked to 125I-multi-CSF do not represent receptors of different affinity but, rather, two noncovalently associated subunits of a receptor complex. PMID- 3017973 TI - Purification and properties of the groES morphogenetic protein of Escherichia coli. AB - The morphogenesis of lambda proheads is governed by the products of at least four bacteriophage-coded genes (B, C, E and Nu3) and two host-coded genes (groES (mopB) and groEL (mopA)). Earlier genetic experiments indicated that the phenotypes of some of the groES- mutations could be suppressed by mutations in the groEL gene, suggesting an interaction between the two groE proteins in vivo (Tilly, K., and Georgopoulos, C. P. (1982) J. Bacteriol. 149, 1082-1088). The Mr 15,000 groES protein was overproduced and purified to homogeneity by monitoring its presence after polyacrylamide gel electrophoresis. Both gel filtration on an AcA34 sizing column and glycerol gradient centrifugation indicate that the groES protein possesses an oligomeric structure of Mr 80,000. In agreement, electron microscopic pictures of the purified groES protein show that it possesses a symmetrical ring-like structure. The sequence of the first five amino acids and the overall composition of the purified protein match those predicted by the nucleotide sequence of the groES gene. The following results implicate a physical association between the groES and groEL proteins in vitro. The groES protein inhibits the weak ATPase activity of the groEL protein, with a maximal effect seen at a 1:1 molar ratio; the two proteins cosediment during glycerol gradient centrifugation in the presence of ATP and Mg2+; and the groES protein binds specifically to a groEL-affinity column. These results help explain why mutations in either of the groE genes exhibit similar phenotypes with respect to both lambda and bacterial growth. PMID- 3017975 TI - The complete nucleotide sequence of the structural gene TOP2 of yeast DNA topoisomerase II. AB - The nucleotide sequence of the gene TOP2 encoding DNA topoisomerase II of the yeast Saccharomyces cerevisiae has been determined. The entire coding sequence of the gene lies within an open reading frame, and there are 1429 amino acids in the single polypeptide protein if translation is assumed to start at the first ATG in this frame. The calculated subunit and molecular mass of the enzyme is 164,000 and 328,000 daltons, respectively. The transcriptional starts of the gene and a polyadenylation site of the message have also been mapped. PMID- 3017974 TI - Electrical potential accelerates the E1P(Na)----E2P conformational transition of (Na,K)-ATPase in reconstituted vesicles. AB - We have used renal (Na,K)-ATPase, covalently labeled with fluorescein, and phospholipid vesicles reconstituted with labeled enzyme, to detect conformational transitions induced by acetyl phosphate in the presence of Mg2+ and Na+ ions. Equilibrium fluorescence measurements show quenching of the fluorescein fluorescence, which is thought to reflect conversion of the initial E1 form to the phosphorylated E2P form. These fluorescence changes occur on inside-out oriented pumps. The rates of acetyl phosphate-induced fluorescence changes have been measured using a stopped-flow fluorimeter. The rate of fluorescence quenching (1.5-3 s-1) is a measure of the rate of the E1P(Na)----E2P transition. The quenching is preceded by a fast fluorescence increase (12.3 +/- 4 s-1) associated with phosphorylation of E1 to E1P(Na), shown clearly in experiments with enzyme treated with oligomycin. Oligomycin greatly reduces the rate of the fluorescence quenching (0.044 +/- 0.01 s-1). Using potassium-loaded vesicles treated with valinomycin or lithium-loaded vesicles treated with Li+ ionophore N,N'-diheptyl-N,N'-didiethyl ether, 5,5-dimethyl-3,7-dioxanonanediamide in order to induce electrical diffusion potentials, negative inside, the rates of the fluorescence quenching are accelerated by up to 4-fold. The experiments demonstrate that the conformational transition E1P(Na)----E2P, associated with transport of 3 Na+ ions, is a voltage-sensitive reaction, carrying a net positive charge. This confirms a prediction based on transport experiments. In experiments with fluorescein-labeled (Na,K)-ATPase, the use of acetyl phosphate rather than ATP, which does not bind, provides a valuable tool to detect fluorescence signals accompanying steps in the turnover cycle. PMID- 3017976 TI - Iron mediates paraquat toxicity in Escherichia coli. AB - The role of iron ions in paraquat toxicity was studied in bacterial system. We show that addition of ferrous iron led to an enhancement of the bacterial killing, whereas addition of chelating agents, such as nitrilotriacetate and desferrioxamine, markedly reduced, up to a total abolishment, the toxic effects. The calculated rates of bacterial killing are proportional to both paraquat and iron concentrations, and conform to the rate equation: dN/dt = -k[paraquat] [Fe2+]. The killing constant for iron, k, is 24-fold smaller than the corresponding value for copper. Mannitol, an OH. scavenger, has a partial protective effect: 15-35% at concentrations range of 1-50 mM, respectively. Histidine, on the other hand, provided a more efficient protection that may be due to a combination of various effects. Induction of endogenous superoxide dismutase and catalase provided partial protection (about 25%). These findings, together with an earlier study on the role of copper in paraquat toxicity (Kohen, R., and Chevion, M. (1985) Free Rad. Res. Commun. 1, 79-88) indicate that transition metals play a central catalytic role in the production of the deleterious effects of paraquat, probably by redox cycling and producing OH. via the site-specific Fenton reaction. PMID- 3017978 TI - Gonadotropin-releasing hormone activates a rapid Ca2+-independent phosphodiester hydrolysis of polyphosphoinositides in pituitary gonadotrophs. AB - Addition of gonadotropin releasing hormone (GnRH) to pituitary cells prelabeled with [32P]Pi or with myo-[2-3H]inositol, resulted in a rapid decrease in the level of [32P]phosphatidylinositol 4,5-bisphosphate (approximately 10 s), and in [32P]phosphatidylinositol 4-phosphate (approximately 1 min), followed by increased labeling of [32P]phosphatidylinositol and [32P]phosphatidic acid (1 min). GnRH stimulated the appearance of [3H]myo-inositol 1,4,5-trisphosphate (10 s), [3H]myo-inositol 1,4-bisphosphate (15 s), and [3H]myo-inositol 1-phosphate (1 min) in the presence of Li+ (10 mM). Li+ alone stimulated the accumulation of [3H]myo-inositol 1-phosphate and [3H]myo-inositol 1,4-bisphosphate but not [3H]myo-inositol 1,4,5-trisphosphate, but had no effect on luteinizing hormone release. The effect of GnRH on inositol phosphates (Ins-P) production was dose related (ED50 = 1-5 nM), and was blocked by a potent antagonist [D-pGlu,pClPhe,D Trp]GnRH. Elevation of cytosolic free Ca2+ levels ([Ca2+]i), by ionomycin and A23187 from intracellular or extracellular Ca2+ pools, respectively, had no significant effect on [3H]Ins-P production. GnRH-induced [3H]Ins-P production was not dependent on extracellular Ca2+ and was noticed also after extracellular or intracellular Ca2+ mobilization by A23187 or ionomycin, respectively. The effect of GnRH on [3H]Ins-P accumulation was not affected by prior treatment of the cells with the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate or with islet-activating protein pertussis toxin. These results indicate that GnRH stimulates a rapid phosphodiester hydrolysis of polyphosphoinositides. The stimulatory effect is not mediated via an islet-activating protein-substrate, is not dependent on elevation of [Ca2+]i, neither is it negatively regulated by 12-O tetradecanoylphorbol-13-acetate which activates Ca2+/phospholipid-dependent protein C kinase. The results are consistent with the hypothesis that GnRH induced phosphoinositide turnover is responsible for Ca2+ mobilization followed by gonadotropin release. PMID- 3017977 TI - Reconstitution of human epidermal growth factor receptors and its deletion mutants in cultured hamster cells. AB - DNA sequences encoding the human epidermal growth factor (EGF) receptor and various EGF-receptor deletion mutants were transfected into chinese hamster ovary (CHO) cells devoid of endogenous EGF receptors. A functional human EGF-receptor is expressed on the surface of heterologous CHO cells with the following properties: it exhibits typical high affinity (10%; Kd = 3 X 10(-10) M) and low affinity (90%; Kd = 3 X 10(-9) M) binding sites for 125I-EGF; it is expressed as a polypeptide of 170,000 molecular weight with intrinsic protein tyrosine kinase activity. EGF stimulates the kinase activity leading to self-phosphorylation and to phosphorylation of exogenous substrate; 125I-EGF is rapidly internalized into the CHO cells by receptor mediated endocytosis and; EGF stimulates DNA synthesis in the cells expressing the human EGF-receptor. Deletion of 63 amino acids from the C-terminal end of EGF-receptor, which removes two autophosphorylation sites, abolishes the high affinity state of the receptor. Nevertheless, this receptor mutant is able to undergo endocytosis and to respond mitogenically to EGF to a similar extent as the "wild type" receptor. Further deletions from the cytoplasmic domain give rise to low affinity endocytosis-defective receptor mutants. Finally, deletion of the transmembrane domain of the human receptor yields an EGF-receptor ligand binding domain which is secreted from the cells. PMID- 3017979 TI - Attenuation of sn-1,2-diacylglycerol second messengers. Metabolism of exogenous diacylglycerols by human platelets. AB - The metabolism of exogenous [3H]diacylglycerols by intact human platelets was studied in order to examine: the metabolic fate of these second messengers in an intact cell, the effect of diacylglycerol kinase and diacylglycerol lipase inhibitors on this metabolism, the effect of agonist stimulation on metabolism, and the dependence of metabolism on diacylglycerol chain length. When 2.5 microM [3H]dioctanoylglycerol (diC8) was added to 10(9) platelets it was rapidly metabolized; 80% was converted to various products in 2.5 min. Initially, 40% was recovered as 3H-labeled phospholipid (predominantly phosphatidic acid) reflecting the action of diacylglycerol kinase, 20% was recovered as [3H]glycerol due to the action of diacylglycerol and monoacylglycerol lipases, and small amounts were recovered as triacylglycerol and monoacylglycerol. Thrombin stimulation of platelets did not affect the rate or pathway of metabolism. Pretreatment of platelets with the diacylglycerol kinase inhibitors, diC8ethyleneglycol or 1 monooleoylglycerol, inhibited 3H-labeled phospholipid production 47% and 75%, respectively, and resulted in a longer lived diC8 signal. The diacylglycerol lipase inhibitor, RHC 80267, inhibited the production of water-soluble metabolites 75%. Despite inhibition of the lipase, the overall metabolism of exogenous [3H]diC8 occurred at a similar rate as in control platelets due to an increased flux towards phospholipid. The ability of exogenous diacylglycerols to be metabolized by diacylglycerol kinase correlated well with their ability to activate protein kinase C in platelets. [3H]Dibutyroylglycerol, didodecanoylglycerol, and ditetradecanoylglycerol, were not metabolized by this route. These diacylglycerols were still metabolized via the lipase pathway. The results indicate that platelets possess potent attenuation systems to defend against the accumulation of diacylglycerol second messengers, and that the primary metabolic fate of cell-permeable, exogenous diacylglycerols is conversion to phosphatidic acid. PMID- 3017980 TI - Regulation of magnesium but not calcium transport by phorbol ester. AB - Phorbol esters in the presence of Ca2+ apparently mimic diacylglycerol in activating protein kinase C. Resulting phosphorylations alter multiple cellular processes including inhibition of the action of Ca2+-mobilizing agonists. In contrast to this inhibition of Ca2+ mobilization, addition of 4 beta-phorbol 12 myristate 13-acetate (PMA) to murine S49 lymphoma cells stimulated Mg2+ influx severalfold without any detectable alteration of Mg2+ efflux or of Ca2+ influx or efflux. Stimulation of Mg2+ influx did not require extracellular Ca2+, was half maximal at 10 nM PMA, and was characterized by a marked increase in the Vmax of Mg2+ influx without change in the Ka for Mg2+. Stimulation of Mg2+ influx was not mimicked by 4 alpha-phorbol didecanoate, which does not activate protein kinase C and was not the result of Na+/H+ exchange activity. The effect of PMA on Mg2+ influx was inhibited by the beta-adrenergic agonist (-)-isoproterenol, which we have previously shown to inhibit Mg2+ influx by a non-cyclic AMP-dependent mechanism (Maguire, M. E., and Erdos, J. J. (1980) J. Biol. Chem. 255, 1030 1035). Forskolin, a direct activator of adenylate cyclase, also inhibited PMA stimulation of Mg2+ influx, suggesting the presence of both cyclic AMP-dependent and -independent influences on Mg2+ influx. We have also previously demonstrated that Mg2+ influx occurs solely into a small subcytoplasmic pool (Grubbs, R. D., Collins, S. D., and Maguire, M. E. (1984) J. Biol. Chem. 259, 12184-12192). PMA did not alter this compartmentation; rather, it almost doubled the content of this cytosolic Mg2+ pool. These data indicate that, in addition to phorbol ester modulation of intracellular Ca2+ mobilization, substantial changes in Mg2+ flux and content occur. They further demonstrate that Mg2+ influx is regulated by a variety of stimuli. It seems likely that such alterations in Mg2+ flux and content would have physiological consequences. PMID- 3017981 TI - The functional domains of coagulation factor VIII:C. AB - A lack of factor VIII:C, manifested as a bleeding disorder due to the absence of clot formation, is known as hemophilia A, an X chromosome-linked inherited disease afflicting 1-2 males/10,000. To determine the minimum functional domain(s) essential for factor VIII:C activity, we have expressed the amino terminal (92-kDa) and carboxyl-terminal (80-kDa) proteolytic cleavage products as individual, secreted polypeptides in monkey cells without the 909-residue central region. We have found that neither terminal domain alone is able to promote coagulation in factor VIII:C-deficient plasma. However, when the 92- and 80-kDa peptides are co-expressed, clotting activity is readily detected. Thus, these two chains alone constitute an active or activatable complex. The central domain is required neither for activity nor for the assembly of an active complex from two chains expressed in trans. These results suggest that a truncated derivative of factor VIII:C may be useful in coagulation therapy. PMID- 3017983 TI - Termination sequences in the control region of the Ad2-specific VARNA2 gene. AB - Both VARNA genes in the adenovirus genome are transcribed by host DNA-dependent RNA polymerase III. Nevertheless, late in infection the mass ratio of VARNA2 to VARNA1 in adenovirus-infected human KB or HeLa cells is about 1:40. This difference is most likely due to differences in promoter strength which may be attributed to sequence differences in the control regions of the two genes. To investigate this possibility, the two genes were cloned into separate plasmid molecules and their transcription efficiencies were compared in vitro. The VARNA2 gene was transcribed in vitro at least 50 times less efficiently than the VARNA1 gene. The competing strengths of the two genes were similar, which suggests that the B block sequence in the VARNA2 gene may be fully functional for sequestering factors. Therefore, the major difference must reside in the region upstream of the B block. By analyzing the hybrid RNAs transcribed from a hybrid gene and 27 fused genes in which only the control region of the VARNA1 gene was fused to various lengths of the 5'-flanking and the coding regions of the VARNA2 gene, one major and one minor termination site, which caused premature termination, were revealed downstream of the A block sequence and in the 5'-flanking region of the VARNA2 gene, respectively. The presence of termination sequences, a suboptimal interblock spacing, and an altered A block sequence in the control region may result in a weaker promoter in the VARNA2 gene. PMID- 3017982 TI - Inhibition of the oxidative burst in human neutrophils by sphingoid long-chain bases. Role of protein kinase C in activation of the burst. AB - The neutrophil oxidative burst is characterized by increased cellular O2 consumption due to the activation of a membrane-associated superoxide-generating NADPH-oxidase. The response is triggered by a variety of stimuli, including opsonized zymosan, formylmethionylleucinephenylalanine (FMLP), arachidonate, short-chain diacylglycerols, and phorbol myristate acetate (PMA). We herein demonstrate that incubation of cells with sphinganine or sphingosine blocks or reverses activation by these agonists. The inhibition is reversible, does not affect cell viability, and does not affect another complex cell function, phagocytosis. Inhibitory concentrations of sphinganine did not significantly affect cytoplasmic calcium levels or FMLP-generated calcium transients. Structural requirements for inhibition of the oxidative burst include a long aliphatic chain and an amino-containing head-group, and there is modest specificity for the native (erythro) isomer of sphinganine. Inhibition involves stimulus-induced activation mechanisms rather than a direct effect on the NADPH oxidase, since sphinganine did not inhibit NADPH-dependent superoxide generation in isolated membranes containing the active enzyme. Activation by FMLP, diacylglycerol, PMA, opsonized zymosan, and arachidonate was blocked by the same concentrations of sphinganine, indicating that these agonists share a common inhibited step. Three lines of evidence indicate that this step involves protein kinase C. First, in a micelle system and in platelets, long-chain bases are inhibitors of this enzyme (Hannun, Y., Loomis, C., Merrill, A., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609). Second, sphinganine blocks PMA stimulated incorporation of 32PO4 into neutrophil proteins. Third, sphinganine inhibits the binding of [3H]phorbol dibutyrate to its cellular receptor, known to be protein kinase C. We suggest that long-chain bases function as physiologic modulators of cellular regulatory pathways involving protein kinase C. PMID- 3017984 TI - Molecular heterogeneity in the infantile and juvenile forms of Sandhoff disease (O-variant GM2 gangliosidosis). AB - There are two major beta-hexosaminidase, EC 3.2.1.52, isozymes in normal human tissues. They exist as active dimers of alpha- and/or beta-subunits. A defect of their beta-subunit results in Sandhoff disease (O-variant GM2 gangliosidosis), an inherited, clinically heterogeneous, lysosomal storage disease. The status of the HEXB gene, pre beta-polypeptide chain mRNA, and residual beta-hexosaminidase activities were examined in a clinically and ethnically diverse collection of 16 fibroblast cell lines from patients with Sandhoff disease. Differentiation of the two major clinical types, infantile and juvenile onset, could be made by the determination of the activity of the residual beta-hexosaminidase eluting in the same pH range as hexosaminidase A. All the juvenile lines were found to have normal or reduced levels of pre beta-chain mRNA and no gross abnormalities in the HEXB gene. Of the 11 infantile type cell lines examined, four were found to contain no detectable pre beta-chain mRNA. Two cell lines in this group contained partial gene deletions localized to the 5' end of the HEXB gene. One of these cell lines has previously been assigned to the single complementation group in Sandhoff disease, conclusively demonstrating that the primary gene defect in the majority of Sandhoff cases is in the HEXB gene itself. These data suggest that each clinical group is made up of a collection of different HEXB mutations. PMID- 3017985 TI - Organization, nucleotide sequence, and transcription of the chicken HSP70 gene. AB - We have isolated a gene encoding a 70,000-Da heat shock protein (HSP70) from a chicken genomic library. The recombinant clone C411 was isolated by hybridization to a radioactively labeled plasmid containing Drosophila HSP70 gene sequences. The identity was established by hybrid selected translation using a subcloned restriction fragment, pC1.8, and total cytoplasmic RNA from chicken cells. The selected mRNA translated in vitro a product which co-migrates with in vivo synthesized chicken HSP70 by SDS-polyacrylamide gel electrophoresis. By Northern blot analysis of poly(A)+ mRNA from heat-shocked cells, we detected a 2.6 kilobase pair mRNA homologous to pC1.8. The nucleotide sequences within pC1.8 and a flanking 2.0-kilobase pair HindIII fragment contain a single contiguous open reading frame of 1905 nucleotides that corresponds to a protein of predicted size 70,000 daltons. Upstream of the initiator methionine codon, ATG, lie sequences homologous to the canonical transcription elements TATA, CCAAT, and SP1 binding site, and two overlapping heat shock elements. The order and spacing of these sequences share many features in common with the promoter for the human HSP70 gene. PMID- 3017986 TI - The catalytic bimodality of mammalian phosphoglycerate mutase. AB - Rabbit muscle phosphoglycerate mutase, presumed to manifest an absolute cofactor requirement for activity, has been found to express catalysis (3 +/- 1% of optimum) in the absence of added D-glycerate-2,3-P2. Isotope experiments indicate that this catalysis proceeds through a binary phosphoryl enzyme-glycerate intermediate which dissociates into free enzyme and monophosphoglycerate. 32P Labeled phosphoglycerate mutase is formed by reaction with either D-32P-glycerate 3-P or D-U32P-glycerate-2,3-P2. In each case, the acid lability and alkali stability of the covalent adduct, phosphoenzyme, is consistent with a phosphohistidyl residue having been formed within the active site. D-[U 14C]Glycerate reacts with phosphoenzyme to generate D-[U-14C]monophosphoglycerate which, in turn, can react further with phosphoenzyme to yield D-[U-14C]glycerate 2,3-P2. The pH profile for the cofactor-independent activity exhibits an optimum at 6.0 as opposed to 7.0 when D-glycerate-2,3-P2 is present in the reaction medium. Bisubstrate kinetics (pH 7.0, 23 degrees C) with D-glycerate-3-P concentration as the variable, yields a family of reciprocal plots which is in accord with a modified ping-pong mechanism when D-glycerate-2,3-P2 concentrations are greater than 10(-1) Km (where Km = 0.33 microM). Progressively diminishing concentrations (much less than Km) of D-glycerate-2,3-P2 produce curvilinear reciprocal plots that approach linearity as a limit in accordance with single substrate kinetics. PMID- 3017987 TI - Appendix II. The catalytic and stability properties of phosphorylated mammalian phosphoglycerate mutase. AB - Because of the critical nature of the experiments described, Sigma phosphoglycerate mutase was carefully examined for the possible occurrence of phosphorylated enzyme, using isotope-labeling techniques. Also, the influence of purposely formed phosphoryl enzyme on mutase catalysis in the absence of added 2,3-DPG was determined. PMID- 3017988 TI - Structure of the lc and nmpC outer membrane porin protein genes of lambdoid bacteriophage. AB - The lc gene of the lambdoid bacteriophage PA-2 and the nmpC gene located on a defective lambdoid prophage in the 12-min region of the Escherichia coli K12 chromosome have been sequenced. The porin proteins encoded by these two genes were almost identical, with only 4 of the 365 residues of the precursor forms of the proteins being different. The Lc and NmpC proteins were strongly homologous to the OmpC, ompF, and PhoE proteins, with greater than 56% of the residues identical in each case. Sequencing of the region flanking the lc gene allowed precise positioning of this gene with respect to the rightward cos site of the phage and to sequences which are homologous between PA-2 and lambda. In wild-type strains of E. coli K12, the nmpC gene is not expressed and contains an IS5 insertion near the 3' end of the coding region. This insertion deletes 18 residues from the COOH terminus of NmpC protein and adds 8 residues from an open reading frame extending into IS5 sequence. Expression of this form of the gene in an expression vector plasmid demonstrated that this altered protein is still capable of being translocated to the outer membrane. Plasmid expression experiments using lc-nmpC hybrid genes show that it is the presence of the IS5 insertion which prevents expression of the porin in wild-type E. coli K12. In the nmpC mutant which expresses the protein, there has been a precise excision of the IS5 which regenerates a COOH terminus of NmpC protein which is identical to that of the Lc protein. Blot hybridization detected no mRNA transcripts from the wild type nmpC gene, although transcripts were readily detected from the lc gene in PA 2 lysogens and from the nmpC mutant which has excised the IS5. This indicates that IS5 affects the production or stability of transcripts from the adjacent nmpC gene. PMID- 3017989 TI - Arachidonic acid metabolism by canine tracheal epithelial cells. Product formation and relationship to chloride secretion. AB - Canine tracheal epithelial cells freshly isolated from mongrel dog trachea were used to study relationships between arachidonic acid metabolism and chloride ion movement. High performance liquid chromatography (HPLC) analysis of the cell incubation media after the addition of A23187 showed the presence of prostaglandin H synthase and lipoxygenase-derived metabolites. The major prostaglandin H synthase metabolite identified by HPLC, gas chromatography, and mass spectrometry was prostaglandin (PG) D2. The major lipoxygenase metabolites were leukotriene (LT) C4 and LTB4. LTB4 was identified by HPLC, UV spectroscopy, and gas chromatography. Straight phase HPLC of the methyl esters indicated only a minor formation of LTB4 isomers. LTC4 was identified by HPLC, UV spectroscopy, and conversion to LTD4 by gamma-glutamyl transpeptidase. Analysis by radioimmunoassays indicated approximately 1-2 ng of LTB4 and peptide LT formed by 10(6) cells after A23187 stimulation. The addition of ionophore A23187 caused a rapid release of arachidonic acid metabolites which was completed within 5 min of stimulation. Cl- secretion was measured in parallel studies of excised tracheas in Ussing chambers. Cl- secretion occurred at 2-3 min after the addition of ionophore, and the most rapid change occurred with the highest PGD2 concentrations. Indomethacin produced a concentration-dependent inhibition of PGD2 formation and Cl- movement. The addition of PGE2, PGD2, and PGH2 effectively stimulated Cl- secretion. LTC4 also stimulated Cl- secretion, but the stimulation was inhibited by indomethacin. These results indicate that canine tracheal epithelial cells metabolize arachidonic acid via both prostaglandin H synthase and lipoxygenase enzymes. It appears that endogenous PGD2 formation is the important variable controlling the Cl- ion movement in canine trachea. PMID- 3017990 TI - Monoclonal antibodies to Fc receptors for IgG on human mononuclear phagocytes. Antibody characterization and induction of superoxide production in a monocyte cell line. AB - We have utilized monoclonal antibodies against the two IgG Fc receptors (p40 and p72) of U937 cells to stimulate the release of superoxide. The monoclonal antibody (mAb) specific for p40 (IV3) has been described elsewhere. A murine IgG1 mAb specific for the high affinity p72 Fc receptor (designated mAb FcR32 or simply mAb 32) bound to the same p72 precipitated by Sepharose-human IgG as shown by preclearing experiments and by identical isoelectric focussing patterns. Binding of mAb 32 to p72 was independent of the Fc region of the antibody since Fab' fragments of mAb 32 affinity adsorbed p72. The binding of both mAb 32 and human IgG1 to the intact U937 cell was not reciprocally inhibitory, indicating that mAb 32 does not interfere with the ligand binding site of p72. mAb 32 bound to human monocytes, U937, and HL60 cells, but not to granulocytes or lymphocytes. U937 cells cultured in gamma-interferon and 1,25-dihydroxycholecalciferol generated superoxide when incubated with mAb 32 or IV3 followed by cross-linking with F(ab')2 anti-murine Ig. Incubation with mAb 32 or IV3 alone or with 3 of 5 other anti-U937 mAbs cross-linked with anti-murine Ig did not result in superoxide generation. Immune complex-mediated superoxide production was inhibited 80% by IgG, but not by mAb 32 or IV3. PMID- 3017991 TI - Isolation and characterization of a high molecular weight stable pink form of uteroferrin from uterine secretions and allantoic fluid of pigs. AB - A pink, high molecular weight form of uteroferrin (Uf) has been isolated from uterine secretions and allantoic fluid of pigs. This protein fraction (denoted FIII) which is relatively stable under physiological conditions of pH, ionic strength, and temperature has a molecular weight of about 80,000, a value approximately twice that of purple Uf (Mr approximately 35,000) isolated from a separate fraction (FIV) by gel filtration. The visible absorption spectrum, EPR signal, and acid phosphatase activity of Uf in FIII are almost identical to those of FIV Uf after the latter has been reduced by 2-mercaptoethanol. However, unlike reduced FIV Uf, the pink, high molecular form does not revert to purple, nor does it show loss of EPR signal and phosphatase activity in the presence of oxygen. In addition, it does not become purple at orthophosphate concentrations which inhibit Uf acid phosphatase activity. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate has shown that FIII consists of approximately equal amounts of Uf polypeptides (Mr = 35,000 and 37,000) and a group of three polypeptides (Mr = 40,000, 46,000, and 50,000) antigenically unrelated to Uf. The latter share a common epitope not found on Uf and are probably differentially processed forms of the same protein. FIII can be dissociated by pH conditions below 5.0, by exposure to antibodies raised against Uf or the associated polypeptides, and by sodium dodecyl sulfate at 100 degrees C. The polypeptides in FIII are not therefore linked by disulfide bonds. Treatment with dimethyl suberimidate, however, results in a cross-linked complex (Mr approximately 82,000) consisting of Uf and the associated polypeptides. It is concluded that this high Mr form of Uf is a heterodimer of fully activated Uf and a second polypeptide of unknown function. PMID- 3017992 TI - Regulation of kinase and intermolecular bonding in intact and truncated epidermal growth factor receptor. AB - Tyrosine kinase activity of the epidermal growth factor (EGF) receptor can be regulated by its state of association. Studies done with the purified receptor solubilized in Triton X-100 indicate that dimer formation results in negative regulation of kinase, whereas successive binding of EGF and ATP shift the association equilibrium toward the catalytically active monomeric form. The promotion of the monomeric state by ATP can be mimicked by various nonphosphorylating analogs indicating that nucleotide binding rather than autophosphorylation is responsible for stabilizing the monomeric receptor form. Truncated receptor forms, lacking either the external EGF-binding domain or the internal kinase (ATP-binding) domain, are unable to form stable dimers. These results suggest that both intra- and extracellular domains of the receptor act to stabilize the kinase-regulatory dimer. PMID- 3017993 TI - The secY protein can act post-translationally to promote bacterial protein export. AB - Conditionally lethal Escherichia coli mutants in secY (prlA) show defective export of proteins to the periplasm and outer membrane. It has been proposed that this gene and other sec genes must act on pro-OmpA at an early stage of protein synthesis in order to allow later translocation to occur. We have described a temperature-sensitive mutation in which the secYts function is impaired at the nonpermissive temperature (Ito, K. (1984) Mol. Gen. Genet. 197, 204-208). A plasmid bearing the wild-type secY gene under the control of the lactose operon (Shiba, K., Ito, K., Yura, T., and Cerretti, D. P. (1984) EMBO J. 3, 631-635) has been introduced into this mutant strain. We now report that the in vivo chase of pulse-labeled full length pro-OmpA to mature OmpA is accelerated by inducing the synthesis of the wild-type secY protein at the end of the period of pulse labeling. We have also assayed the requirements for secY function for in vitro protein translocation. Membranes derived from secY ts cells which were incubated at 42 degrees C were inactive in vitro in the post-translational uptake and processing of pro-OmpA. Thus, the secY protein can act post-translationally, enhancing the translocation of completed pro-OmpA polypeptide chains across the plasma membrane. PMID- 3017995 TI - Monocyte-conditioned medium, interleukin-1, and tumor necrosis factor stimulate the acute phase response in human hepatoma cells in vitro. AB - Human hepatoma cells mimic the acute phase response after treatment with monocyte conditioned medium. Levels of secreted fibrinogen, alpha-1 acid glycoprotein, C reactive protein, haptoglobin, and the third component of complement were elevated compared with control levels after 48 h of incubation with conditioned supernatant medium from an enriched fraction of normal peripheral monocytes. Albumin levels declined and alpha-1 antitrypsin remained unchanged. Levels of specific mRNA were measured by hybridization to slot blots and Northern blots and changed in correspondence with protein alterations. Interleukin-1 and tumor necrosis factor stimulated the third component of complement, but did not elevate any other member of the acute phase group and were therefore only partially active in this system. The identification of an in vitro model of the human acute phase response will permit analysis of the molecular basis for coordinate regulation of this group of facultative genes. PMID- 3017996 TI - Amino-terminal deletion mutants of the Rous sarcoma virus glycoprotein do not block signal peptide cleavage but can block intracellular transport. AB - Protein sequence requirements for cleavage of the signal peptide from the Rous sarcoma virus glycoprotein have been investigated through the use of deletion mutagenesis. The phenotypes of these mutants have been characterized by expression of the cloned, mutated env genes in CV-1 cells using a late replacement SV40 vector. The deletion mutations were generated by Ba131 digestion at the XhoI site located near the 5' end of the coding sequence for the structural protein gp85, which is found at the amino terminus of the precursor glycoprotein, Pr95. The results of experiments with three mutants (X1, X2, and X3) are presented. Mutant X1 has a 14 amino acid deletion encompassing amino acids 4-17 of gp85, which results in the loss of one potential glycosylation site. In mutants X2 and X3 the amino terminal nine and six amino acids, respectively, of gp85 are deleted. During the biosynthesis of all three mutant polypeptides, the signal peptide is efficiently and accurately cleaved from the nascent protein, even though in mutants X2 and X3 the cleavage site itself has been altered. In these mutants the alanine/aspartic acid cleavage site has been mutated to alanine/asparagine and alanine/glutamine, respectively. These results are consistent with the concept that sequences C-terminal to the signal peptidase site are unimportant in defining the site of cleavage in eucaryotes. Mutants X2 and X3 behave like wild-type with respect to protein glycosylation, palmitic acid addition, cleavage to gp85 and gp37, and expression on the cell surface. Mutant X1, on the other hand, is defective in intracellular transport. Although it is translocated across the rough endoplasmic reticulum and core-glycosylated, its transport appears to be blocked at an early Golgi compartment. No terminal glycosylation of the protein, cleavage of the precursor protein to the mature products, or expression on the cell surface is observed. The deletion in X1 thus appears to destroy signals required for export to the cell surface. PMID- 3017994 TI - Reorganization of polymerized actin: a possible trigger for induction of procollagenase in fibroblasts cultured in and on collagen gels. AB - Changes in cell shape are postulated to modulate gene expression during differentiation of a number of cell types, including rabbit synovial fibroblasts, which are inducible for expression of the zymogen form of the metalloendopeptidase, collagenase. In the work presented here, fibroblasts cultured on and within hydrated collagen gels were allowed to contract by release of the gels from the sides of the culture dish. Within 24 h of cell release, synthesis and secretion of procollagenase was initiated in the absence of any chemical manipulation. Fibroblasts grown in and on collagen also responded to 12 O-tetradecanoylphorbol-13-acetate and cytochalasin B with morphologic change and induced procollagenase. However, colchicine, which altered morphology to varying degrees in cells on plastic, on collagen, and within collagen gels, did not induce procollagenase expression. In all cases, the enzyme was induced only after reorganization of polymerized actin, rather than after a change in cellular morphology per se. As a first approach to identifying other aspects of the stimulated phenotype that could affect collagen turnover, the expression of collagen and endogenous metalloproteinase inhibitors in relation to procollagenase secretion was investigated. Collagen secretion by fibroblasts decreased when procollagenase secretion was induced by the pharmacologic agents, but not when cells were stimulated by contraction on or within collagen gels. The expression of two endogenous inhibitors was not coordinately regulated with induction of procollagenase. Therefore, the extracellular matrix and the cellular actin cytoskeleton may transduce signals that modulate the tissue remodeling phenotype of fibroblasts. PMID- 3017997 TI - Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT20 cells. AB - We have studied by electron microscopy and immunocytochemistry the formation of secretory granules containing adrenocorticotropic hormone (ACTH) in murine pituitary cells of the AtT20 line. The first compartment in which condensed secretory protein appears is a complex reticular network at the extreme trans side of the Golgi stacks beyond the TPPase-positive cisternae. Condensed secretory protein accumulates in dilated regions of this trans Golgi network. Examination of en face and serial sections revealed that "condensing vacuoles" are in fact dilations of the trans Golgi network and not detached vacuoles. Only after presumptive secretory granules have reached an advanced stage of morphological maturation do they detach from the trans Golgi network. Frequently both the dilations of the trans Golgi network containing condensing secretory protein and the detached immature granules in the peri-Golgi region have surface coats which were identified as clathrin by immunocytochemistry. Moreover both are the site of budding (or fusion) of coated vesicles, some of which contain condensed secretory protein. The mature granules below the plasma membrane do not, however, have surface coats. Immunoperoxidase labeling with an antiserum specific for ACTH and its precursor polypeptide confirmed that many of the coated vesicles associated with the trans Golgi network contain ACTH. The involvement of the trans Golgi network and coated vesicles in the formation of secretory granules is discussed. PMID- 3017998 TI - Role of intracellular calcium and protein kinase C in the endocytosis of transferrin and insulin by HL60 cells. AB - The role of the cytosolic free calcium concentration ([Ca2+]i) and of protein kinase C on the internalization of transferrin and insulin in the human promyelocytic cell line HL60 was investigated. [Ca2+]i was selectively monitored and manipulated by the use of the fluorescent Ca2+ indicator and buffer quin2, while receptor-ligand internalization was studied directly by quantitative electron microscope autoradiography. Decreasing the [Ca2+]i up to 10-fold below resting level had no effect on the internalization of transferrin or insulin. Similarly, a 10-fold elevation of the [Ca2+]i using the calcium ionophore ionomycin caused little or no change in the endocytosis of the two ligands. In contrast, activation of protein kinase C by phorbol myristate acetate markedly stimulated the internalization of both occupied and unoccupied transferrin receptors, even in cells with very low [Ca2+]i. The insulin receptor was found to behave differently in response to phorbol myristate acetate, however, in that only the occupied receptors were stimulated to internalize. We conclude that the [Ca2+]i plays only a minor role in regulating receptor-mediated endocytosis, whereas protein kinase C can selectively modulate receptor internalization depending on receptor type and occupancy. PMID- 3018000 TI - Stress induces an increased hexose uptake in cultured cells. AB - Temperature-sensitive mutants have revealed a region of the herpes simplex virus 1 genome that affects both the uptake of hexose and the synthesis of heat shock proteins. Other inducers of heat-shock proteins, namely heat shock itself and arsenite, likewise induce an increased uptake of hexose. The increased uptake, like that induced by insulin, is insensitive to the presence of actinomycin D or cycloheximide. It is concluded that an increased hexose uptake, reflecting an activation or relocation of existing hexose transport protein, is a general biochemical response of stressed cells. PMID- 3017999 TI - Myoblast fusion is regulated by a prostanoid of the one series independently of a rise in cyclic AMP. AB - The role of prostanoids in the regulation of chick myoblast differentiation has been investigated. At 3 X 10(-6) M, indomethacin and chloroquine specifically inhibit cell fusion. They do not affect cell proliferation, alignment, or the expression of two muscle-specific proteins, namely, the acetylcholine receptor and the muscle-specific form of creatine phosphokinase. The results demonstrate that it is indomethacin's activity as an inhibitor of prostaglandin synthesis at the cyclooxygenase step that causes the block of cell fusion, whereas chloroquine probably acts at the earlier step of phospholipase A. Prostaglandin E1 (PGE1), but not prostaglandin E2 (PGE2), rapidly reverses the inhibition of fusion imposed by indomethacin or chloroquine. The dose response of the myoblasts to PGE1 is a bell-shaped curve with a 100% reversal of fusion at approximately 10( 9) M. Eicosatrienoate and linoleate reverse the inhibition of fusion with similar kinetics, whereas arachidonate is completely ineffective. The ability of PGE1 and eicosatrienoate but not PGE2 and arachidonate to restore fusion to control levels implies that fusion is specifically regulated by a prostanoid of the one series. The reversal of the fusion-block by linoleate further suggests that this fatty acid provides the necessary source of eicosatrienoate in the myoblast plasma membrane. At 10(-8) M and above, PGE1 and PGE2 stimulate adenylate cyclase and depress control fusion as does 10(-5) M isoproterenol. The beta-adrenergic blocker propranolol abolishes both isoproterenol's inhibition of myoblast fusion and its activation of adenylate cyclase. The similar depressions imposed on cell fusion by 10(-8)-10(-6) M prostanoid and 10(-5) M isoproterenol suggest that in both cases the depressive effects are mediated by cyclic AMP. It is concluded that a prostanoid of the one series regulates fusion by a cyclic AMP-independent mechanisms. PMID- 3018002 TI - Purified HDL-apolipoproteins, A-I and C-III, substitute for HDL in promoting the growth of SV40-transformed REF52 cells in serum-free medium. AB - The lipid-free apolipoproteins of human high density lipoprotein (HDL) have been assayed for their ability to substitute for native HDL in promoting the growth of a SV40-transformed REF52 cell line in serum-free medium. Total HDL apolipoproteins (apoHDL) were found to mimic almost exactly the growth promoting effects of whole HDL. The apoHDL-associated growth promoting activity eluted from a Sephacryl S-200 column in two separate fractions coinciding with the protein peaks of apolipoprotein A-I and the C group of apolipoproteins. These two fractions, designated S-II and S-IV, respectively, acted additively in promoting WT1A cell growth when tested at saturating concentrations. The active component in the S-II fraction maximally stimulated WT1A cell growth at 40-60 micrograms/ml and was identified as apolipoprotein A-1 by NaDodSO4 polyacrylamide gel electrophoresis and affinity chromatography on anti-(apoA-I). The active component in the S-IV fraction was maximally active at 1-2 micrograms/ml and was identified as apolipoprotein C-III by DEAE ion exchange high pressure liquid chromatography and polyacrylamide gel electrophoresis (at pH 8.3) in 6 M urea. These results indicate that the growth promoting effect of HDL on WT1A cells is mediated via the HDL-apolipoproteins, A-I and C-III, and that the mechanism responsible does not necessarily involve their participation in the uptake (or utilization) of HDL-associated lipids. PMID- 3018001 TI - Prominent glutamine oxidation activity in mitochondria of avian transplantable hepatoma induced by MC-29 virus. AB - Well coupled mitochondria were isolated from transplantable chicken hepatoma induced by MC-29 virus. The mitochondrial phosphate-dependent and phosphate independent glutaminase activities were increased compared with those from normal chicken liver. Glutamate dehydrogenase was undetectable in the tumor mitochondria. Oxypolarographic tests showed the following: glutamine oxidation was prominent in the tumor mitochondria and was mediated through an NAD-linked reaction, while mitochondria from the liver showed a feeble glutamine oxidation; glutamine oxidation by tumor mitochondria was inhibited either by aminooxyacetate, inhibitor of transaminases, or prior incubation of mitochondria with DON (6-diazo-5-oxonorleucine), which inhibited mitochondrial glutaminases. Bromofuroate, inhibitor of glutamate dehydrogenase, had little or no effect; and glutamate oxidation was also inhibited by aminooxyacetate, while it was not affected by DON. These findings clearly show a high glutamate oxidation activity in the hepatoma and indicate that the product of glutamine hydrolysis, glutamate, is catabolized via transamination in the mitochondria to supply ATP. PMID- 3018003 TI - Intracellular location of the Ah receptor. AB - The subcellular distribution of the Ah receptor from the mouse hepatoma line, Hepa-1, was investigated following cytochalasin B treatment and cell enucleation. Probing the resultant cytoplast and nucleoplast fractions with radiolabelled tetrachlorodibenzo-p-dioxin (TCDD) revealed the presence of a specifically bound peak of receptor only in the cytoplast fraction. However, the quantity of receptor recovered in these experiments was only 10-12% of the expected value. We therefore undertook an investigation to determine the fate of the Ah receptor in the presence of cytochalasin B. Incubation of Hepa-1 cells with this compound resulted in a rapid loss or inactivation of cytosolic binding activity with a concomitant decrease in the amount of receptor partitioned into the nucleus at all time periods examined. Control experiments indicated that cytochalasin B did not compete with TCDD for binding to the Ah receptor and furthermore, that its mechanism of action could not be attributed to a non-specific effect on all cytosolic proteins. The results obtained are discussed in relation to the proposed models for induction by the estrogen and glucocorticoid binding receptors. PMID- 3018004 TI - Reversible inactivation of the Ah receptor associated with changes in intracellular ATP levels. AB - We have previously demonstrated that incubation of Hepa-1 cells in the presence of cytochalasin B (CB) results in a time- and temperature-dependent loss of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding activity (Gudas et al., 1986). We show here that this loss of binding activity is probably attributable to a CB induced inhibition of glucose transport, as incubation of cells in the presence of glycolytic inhibitors or in glucose free medium caused a similar effect. All conditions leading to loss of binding also caused a marked reduction in cellular ATP concentration, suggesting that ATP (or perhaps another energy-dependent molecule) is required for maintaining the receptor in the active state. Inactivation of the Ah receptor occurred in the cytosol but not when it had translocated to the nucleus. Reactivation of receptor binding activity occurred readily in vivo and did not require de novo protein synthesis. However, attempts to restore receptor binding activity in vitro were not successful. To our knowledge this is the first reported evidence indicating that the TCDD binding Ah receptor can exist in both an inactive and an active form, with the amount present in the active ligand binding form being coupled to the energy state of the cells. PMID- 3018005 TI - Persistent infection with a nontransforming RNA virus leads to impaired growth factor receptors and response. AB - The potential role of viral persistence with nontransforming viruses on cellular growth and cellular function has received little attention. We found that when infected with type 3 reovirus (five plaque-forming units (PFU/cell), balb/C 3T3 cells (a mouse embryo fibroblast cell line) undergo a limited lytic phase. The surviving cells, about 90% of the original cells, appear morphologically normal by light microscopy and exhibit normal growth patterns in serum-supplemented medium but are persistently infected by electron microscopy. These persistently infected cells shed infectious virus in the culture medium (1.6-60 X 10(6) PFU per 10(6) cells per 24 h). In comparison to control uninfected 3T3 cells, the persistently infected cells exhibit a 70-90% decrease in receptor number for epidermal growth factor (EGF). This occurs without production of any EGF-like material and is associated with a parallel decrease in EGF-stimulated DNA synthesis. By contrast, insulin receptors are increased in number three-fold and insulin and serum stimulated DNA synthesis are comparable to control uninfected cells. These results suggest that persistent infection with a nontransforming virus may lead to major alteration in control of cell growth by specific growth factors. PMID- 3018006 TI - Down-modulation of receptors for granulocyte colony-stimulating factor on human neutrophils by granulocyte-activating agents. AB - Purified human blood neutrophils were able to bind radioiodinated murine granulocyte-colony-stimulating factor (G-CSF) in a specific manner. This factor has previously been shown to stimulate functional activities of human and murine neutrophilic granulocytes and to be functionally analogous to human-derived CSF beta. The binding of 125I G-CSF to human neutrophils was competed for equally by unlabeled G-CSF and CSF beta but not by other CSF's. Saturation analysis indicated that human neutrophils displayed about 700-1,500 receptors for G CSF/CSF beta per cell. Three other agents (N-formyl-methionine-leucine phenylalanine, bacterial lipopolysaccharide, and human CSF alpha) known to activate neutrophils did not compete directly for G-CSF binding sites but, in preincubation experiments at 37 degrees C, were able to down-modulate the expression of G-CSF receptors on human neutrophils in a dose- and time-dependent manner. This effect was specific since the same agents have been shown elsewhere to up-regulate the expression of other granulocyte surface antigens and other agents were much less effective at down-modulating G-CSF receptors. Since the granulocyte-activating agents increase the sensitivity of human neutrophils to G CSF/CSF beta and mimic some of the actions of G-CSF on neutrophils, it is suggested that G-CSF receptor down-modulation might be a mechanism whereby these agents activate G-CSF receptors and thereby exert some of their effects. PMID- 3018007 TI - Transforming activity of human nasopharyngeal carcinoma DNA. AB - NIH 3T3 cells were transfected with the DNAs from biopsy specimens of human nasopharyngeal carcinoma (NPC, EBV DNA positive) using calcium phosphate precipitation method. The malignant, transformed foci of NIH 3T3 cells have been observed and cloned. The hybridization of transfectant DNA digested by EcoRI with total human leukocyte DNA as probe was performed. The strong signal of smear comparing with NIH 3T3 DNA as control was observed. It was implied that the putative human transforming sequences had been integrated into transformed cells. Employing soft agar culture, the transformed cells can grow and form cell colonies. Following transfer, the foci were able to grow and adhere to a glass wall. These cells were easily agglutinated by con A. The cloned foci have been inoculated into nude mice with the formation of highly malignant sarcomas. In preliminary experiments for characterizing the transforming sequences, Ha-ras and Blym 1 were found in transfectants derived from one of the NPC DNA samples. It is implicated that these two oncogenes might be responsible for the acquisition of malignant phenotypic character of some human NPC. The further identification of oncogenes in NPC is currently in progress. PMID- 3018008 TI - A pilot study on universal immunization of newborn infants in an area of hepatitis B virus and primary hepatocellular carcinoma prevalence with a low dose of hepatitis B vaccine. AB - Hepatitis B virus (HBV) had been considered as the main causative factor of primary hepatocellular carcinoma and universal immunization of newborns was recommended as the major approach to control hepatitis and hepatoma in areas of prevalence. As the initial phase of the first vaccination program for such a purpose, a pilot study was done from September 1983 to May 1984 in a high incidence rural area of China. In an area of 214,343 inhabitants, 1,703 newborns (99% of all births) were vaccinated. Ninety-seven percent of all vaccinees were followed up at 1 year. The vaccine used was Hep-B Vax, given intramuscularly at 0, 1, and 6 months after birth. Four immunization regimes were used: 5-micrograms or 2.5-micrograms doses with or without hepatitis B immune globulin (HBIG) added in the case of carriers children. These groups were defined by drawing lots at community level. A matched control was selected on a voluntary basis. Each group consisted of 400 infants. Vaccination was proven to be very safe and well accepted by the public. The prevalence of HBV infection in the area was further demonstrated by the high HBsAg-positive rate measured: 14.2% of the 1,180 mothers (3.9% were also e-antigen positive), 7.6% and 10.1% of the unvaccinated children at 6.5 months and 1 year of age, respectively. It was shown that vaccination with a 5-micrograms or 2.5-micrograms dose significantly lowered the HBsAg positives to a level close to 1.5% versus 10% in the control group at 1 year. An 85% protection was thus achieved. A 5-micrograms dose plus HBIG did not show additional benefit. A 2.5-micrograms dose plus HBIG gave less protection, and anti-HB levels were also significantly lower than in other groups. Among the 12 failures found in the 5-micrograms and 2.5-micrograms groups, 11 were born to HBsAg-positive mothers, nine of whom also had e-antigen. Available data showed that 29% of children born by e-antigen-positive and 2.7% of children born by e antigen-negative carriers had the risk of becoming carriers during the first year of life following vaccination. The present study demonstrated the feasibility and rationale of conducting universal immunization of newborns in endemic rural area for controlling hepatitis and hepatoma. The significance of the possible use of the vaccination at lower dose had also been stressed. PMID- 3018010 TI - Cell signalling through phospholipid metabolism. PMID- 3018009 TI - Nerve injury complications. Management of neurogenic pain syndromes. AB - Postural brachial plexus compression neuropathy occurs more frequently than usually realized. The cause is multifactorial with certain predisposing anatomic and congenital factors that are not uncommon. An inciting event is required to cause a clinically significant syndrome. The event can be a specific traumatic episode or cumulative trauma leading to an adoption of a guarding posture, which results in a self-perpetuating cycle and brachial plexus nerve compression. The diagnosis and management may be complicated by concurrent vascular compression, concurrent reflex sympathetic dystrophy, and associated inflammatory musculotendinous conditions. Diagnosis relies on the appreciation of a peculiar symptom complex of pain and paresthesias. The important clinical signs are a supraclavicular Tinel's sign and a positive stress abduction test. Treatment includes exercises, patient education, and behavior modification. However, misdiagnosis can lead to inappropriate treatment, such as unnecessary carpal and cubital tunnel releases. Operative treatment is reserved for those severe cases that are resistant to extended and intense physical therapy. The preferred surgical technique involves an anterior, supraclavicular approach allowing for complete visualization and release of intrinsic and extrinsic nerve compression. Awareness is the key to making the diagnosis, and successful treatment requires a multidisciplinary approach. It is generally accepted that injuries to peripheral nerves result in serious losses of function. Paresthesias and motor weakness cause immediate functional limitation, and place the hand at risk for further injury. The system has little regenerative capacity, and the chance for recovery is poor even under the best circumstances. Therefore the treatment of acute nerve injuries can be difficult and frustrating.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018011 TI - Oncogenes. PMID- 3018012 TI - [Spontaneous rupture of a hepatocarcinoma. Diagnostic and therapeutic problems]. PMID- 3018014 TI - Hypoglycemia: a systematic approach to specific diagnosis. PMID- 3018013 TI - AIDS: what is now known. III. Clinical aspects. PMID- 3018016 TI - Evaluation of a new assay for antibodies to LAV/HTLV III. AB - The sensitivity, specificity and reproducibility of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to LAV/HTLV III produced by Genetic Systems was assessed with the identical panel of sera used in previous evaluations of anti-HTLV III ELISAs. The results from this study show that the Genetic Systems anti-LAV/HTLV III ELISA proved to be of equivalent sensitivity and to have higher specificity than assays currently used in Australia for screening purposes while maintaining high levels of intra- and inter-laboratory reproducibility. PMID- 3018015 TI - Factors contributing to intrinsic loading capacity in silica-based packing materials for preparative anion-exchange protein chromatography. AB - Properties of the matrix and stationary phase which affect the intrinsic loading capacity of silica-based packing materials for preparative anion-exchange chromatography of proteins were investigated. Polyethyleneimine-coated controlled porosity glass beads ranging from 100 to 2000 A in pore diameter were used to evaluate the effects of pore diameter and surface area. Protein binding was found to depend on accessible, rather than total, support surface area. Consequently, wide-pore, high surface area media provide maximum intrinsic loading capacity. Increasing the number of positively charged sites on the stationary phase by increased coating or by quaternization of amines increases hemoglobin-binding capacity. PMID- 3018017 TI - Routine detection of human rotavirus by latex agglutination: comparison with enzyme-linked immunosorbent assay, electron microscopy and polyacrylamide gel electrophoresis. AB - A commercially available latex agglutination test, RotaScreen (Mercia Diagnostics Ltd., West Byfleet, Surrey, U.K.) was evaluated for the detection of human rotaviruses in stool specimens. The results obtained were compared with those from 3 other routine assay systems used in this laboratory: enzyme-linked immunosorbent assay (ELISA), electron microscopy (EM) and polyacrylamide gel electrophoresis (PAGE) of viral ribonucleic acid. 400 stool samples were examined by the 4 assay systems under routine conditions. RotaScreen latex agglutination was found to be more sensitive than EM and PAGE, and highly specific for rotavirus antigens. PMID- 3018018 TI - Detection of herpes simplex virus and adenovirus DNA by dot blot hybridization using in vitro synthesized RNA transcripts. AB - A new diagnostic assay was developed to detect herpes simplex virus (HSV) and adenovirus DNA in clinical specimens using in vitro synthesized radioactively labelled RNA transcripts from virus-specific DNA fragments cloned in transcription vector pSP 64/65. RNA probes derived from HSV-I-Eco-RI-G-DNA fragment show a sensitivity of less than 3 pg of whole plasmid DNA and hybridize only with DNA of HSV I and II, but not with other viral or cellular DNA. The analysis of 15 clinical specimens showed concordance with virus isolation, except for two culture-negative samples of cerebrospinal fluid of patients with suspected HSV encephalitis, which was confirmed by serology as well as by hybridization. Using RNA transcripts from adenovirus-2-Hind-III-D-DNA fragment, we attained a sensitivity of less than 3 pg of whole plasmid DNA. This probe detected different types of adenovirus, but failed to hybridize to other viral, bacterial or cellular DNA. Compared with the cell culture method this assay did not show any false-positive or false-negative results in 16 different clinical specimens. The technique is sensitive, specific and useful for screening clinical specimens and may be helpful in confirming the diagnosis of HSV encephalitis. PMID- 3018020 TI - The persistent infection of a canine thymus cell line by equine infectious anaemia virus and preliminary data on the production of viral antigens. AB - Equine infectious anaemia virus (EIAV) was adapted to the Cf2Th cell line, a heterologous malignant line from canine thymus. A persistent infection was monitored for 100 serial passages by demonstrating the presence of virus and viral antigens at each 10th passage by electron-microscopy, immunodiffusion and immunofluorescence. Chromosome analysis of EIAV-infected cells indicated they had a karyotype resembling the control cells of similar passage history. Virus infected cells, grown in roller cultures for 65 days without subculturing, continuously produced viral antigens into supernatant fluids which were harvested every 3-4 days. Antigen peaks were observed at approximately 12-day intervals. Immunoprecipitin lines of identity were demonstrated between ether-extracted antigens from virus-infected canine cell line and known positive EIAV antigen extracted from infected equine spleen and a commercial source. Replication of a non-oncogenic retrovirus (EIAV) resulted in the continuous release of viral antigens from a persistently infected and infinite cell line. PMID- 3018019 TI - An improved method for detection of ganglionic latency in herpes simplex virus type-1 infected guinea pigs. AB - A simple method was developed which increased the sensitivity and reliability of detecting latent herpes simplex virus type-1 (HSV-1) in trigeminal ganglia of guinea pigs. Animals were infected with the Shealey strain of HSV-1 immediately following scarification of the cornea and maintained for 30-40 days to ensure that true latency was established. Viral latency was defined as the appearance of infectious virus in ganglia only upon cultivation in vitro. Thus, ganglia from similarly infected animals, homogenized immediately upon removal, did not contain infectious virus. Excised ganglia were incubated intact in high glucose medium and yielded maximal positive results (90-100%) by the twelfth day of incubation. This method was compared with the standard cocultivation technique in which minced fragments of ganglionic tissue were explanted onto Vero cell cultures. Cocultivation yielded a considerably lower latency rate and was more variable (29 57%) than the whole ganglion culture method. PMID- 3018021 TI - Immunoprecipitation of Epstein-Barr virus EBNA1 protein using human polyclonal serum. AB - We have developed a method that permits the use of human polyclonal serum to immunoprecipitate BamH1-K EBNA(EBNA1) from EBV transformed cell lines and from cells transfected with an expression vector containing the Bam K region of EBV. Serum from healthy seropositive donors is preabsorbed once with lysate of EBV negative Burkitt lymphoma cells, then fractionated by gel filtration. The main IgG fraction is then used for the immunoprecipitations. Immunoprecipitated material is visualized by immunoblotting using the same serum. Two proteins with apparent molecular weights of 74 and 62 kD are specifically precipitated from extracts of B95-8 cells. Several proteins are immunoprecipitated from cells transfected with the Bam K containing vector, the apparent molecular weights of the 4 major bands are 74, 68, 62 and 57 kD. Labelling of transfected cells with [3H]glycine and [32P]orthophosphate shows that the 74 and 62 kD proteins can be labelled with both isotopes. PMID- 3018022 TI - An improved radioimmunoassay method for the detection of IgG antibodies against cytomegalovirus. AB - The non-specific binding seen with human sera in a radioimmunoassay for the detection of IgG antibodies specific for CMV can be reduced greatly by using a murine monoclonal antibody as a radiolabelled detecting antibody. Such non specific binding formerly obtained with a polyclonal detecting antibody was due to the binding of the polyclonal reagent to factors on the solid phase other than IgG molecules. PMID- 3018023 TI - Adrenal steroid responses to continuous intravenous adrenocorticotropin infusion compared to bolus injection in normal volunteers. AB - We compared the adrenal steroid responses after synthetic ACTH-(1-24) (Cosyntropin) administration given by either continuous iv infusion or bolus injection in 11 normal women and 6 normal men. Each subject received 250 micrograms Cosyntropin as a bolus iv injection on 1 occasion and as a continuous 2-h iv infusion on another occasion, in random order. There was a 1-week interval between the studies. We measured the plasma levels of cortisol, 11-deoxycortisol, 17-hydroxyprogesterone, progesterone, pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, delta 5-androstenediol, androstenedione, and testosterone by RIA 15 and 0 min before and 30, 45, 60, and 120 min after administering ACTH. The steroid concentrations and their increments, ratios, or areas above baseline did not differ significantly between the bolus injection and the continuous infusion. Thus, at the dose of 250 micrograms, a bolus ACTH injection stimulates adrenal steroid secretion as effectively as a 2-h continuous ACTH infusion. PMID- 3018024 TI - Adrenal steroid secretion in girls with pseudoprecocious puberty due to autonomous ovarian cysts. AB - To evaluate the role of adrenal steroids in pseudoprecocious puberty due to large ovarian follicular cysts, we studied the serum 17-hydroxyprogesterone response to a combination of dexamethasone suppression followed by iv ACTH administration in two girls and compared the results to those in girls with premature thelarche, normal prepubertal girls, and a girl with true precocious puberty. Although basal serum 17-hydroxyprogesterone levels were normal in all subjects, there was incomplete suppression of 17-hydroxyprogesterone with dexamethasone in the two girls with pseudoprecocious puberty and large ovarian cysts. The 17 hydroxyprogesterone response to ACTH was much greater in these girls (360 and 540 ng/dl) than in the girls with other types of precocious puberty (mean +/- SD, 71 +/- 15 ng/dl) or in normal prepubertal girls (80 +/- 20 ng/dl). The girls with large ovarian cysts had decreased gonadotropin responses to GnRH, which were reversed subsequent to removal of the cyst. Removal of the ovarian cysts also restored the dexamethasone suppressibility of serum 17-hydroxyprogesterone and abolished the progression of pubertal development. However, 17 hydroxyprogesterone responses to ACTH were still elevated (160 and 350 ng/dl). Preoperatively, both girls had increased levels of dehydroepiandrosterone sulfate, 16-hydroxydehydroepiandrosterone sulfate, and another unidentified steroid sulfate. These steroid sulfates were also found in the cyst fluid from the one patient from whom the fluid was obtained. These results suggest that steroid production by the adrenal gland may stimulate the development of small ovarian cysts (which may be present in normal prepubertal girls) into large ovarian cysts capable of causing gonadotropin-independent precocious puberty. PMID- 3018025 TI - Evidence for the presence of specific receptors for N-formyl chemotactic peptides on human spermatozoa. AB - Synthetic N-formylated peptides are potent chemoattractants for human spermatozoa in vitro. The specific structure-activity relations for eliciting a chemotactic response and the ability of the antagonist tertbutoxycarbonyl-phenylalanyl-leucyl phenylalanyl-leucyl- phenylalanine (Boc-Phe-Leu-Phe-Leu-Phe) to inhibit the chemotaxis induced by these peptides strongly suggest the presence of receptors on human spermatozoa. The following studies were performed to identify specific binding sites on human spermatozoa by using [35S]-N-formyl-methionyl-leucyl phenylalanine [( 35S]f-Met-Leu-Phe), a potent chemotactic peptide. Binding of the [35S]formyl-peptide to human spermatozoa was rapid (t1/2, 8 min) and reversible. Binding isotherms of the saturation experiments revealed a single class of high affinity, low capacity binding sites (equilibrium dissociation constant, 17.7 nM; maximal binding, 109 fmol/2 X 10(6) cells) and an average number of 60,000 receptors per cells. The biological potencies of a series of formyl peptides as chemoattractants correlated closely with their relative abilities to compete with [35S]f-Met-Leu-Phe for specific binding to human spermatozoa. These data fulfill the major criteria for demonstration of specific receptors for chemotactic peptides on human spermatozoa. It is likely that these receptor sites initiate the chemotactic response of human spermatozoa to N-formyl peptides. PMID- 3018026 TI - In vivo regulation of beta-adrenergic receptors on human mononuclear leukocytes: assessment of receptor number, location, and function after posture change, exercise, and isoproterenol infusion. AB - We studied the regulation of beta-adrenergic receptors in human mononuclear leukocytes (MNL). Total receptor number was determined as specific binding at 4 C of [3H] dihydroalprenolol or [125I]iodopindolol, and redistributed receptors were defined as those binding sites to which the hydrophilic antagonist CGP-12177 did not have access. Receptor function was assessed as cAMP accumulation stimulated by isoproterenol. In in vitro experiments, high concentrations of isoproterenol desensitized receptor function and promoted redistribution of about 80% of the receptors away from the cell surface. However, three in vivo protocols (upright posture for 3 h, moderate exercise, and infusion of isoproterenol for 30 min) redistributed few beta-adrenergic receptors on MNL. The 30-min isoproterenol infusion did not alter later cAMP accumulation, but posture change and exercise increased isoproterenol-stimulated cAMP accumulation in intact MNL. Infusion of isoproterenol for 120 min redistributed 9 +/- 2% (+/- SEM) of the receptors and decreased isoproterenol-stimulated cAMP accumulation by 19 +/- 6%. Isoproterenol stimulated adenylate cyclase activity in membranes isolated from MNL previously was found to be decreased with upright posture, and we confirmed these findings in assays that did not include exogenous GTP, but instead relied upon guanine nucleotides retained in the membrane preparation. However, when excess GTP was included, isoproterenol-stimulated adenylate cyclase activity in MNL membranes was not altered by posture change. We conclude that substantial receptor redistribution of beta-receptors on MNL does not readily occur in physiological situations. PMID- 3018027 TI - Relative sensitivity and responsivity of serum cortisol and two adrenal androgens to alpha-adrenocorticotropin-(1-24) in normal and obese, nonhirsute, eumenorrheic women. AB - The alpha ACTH-(1-24) threshold dose and the response slope were determined for cortisol (F), delta 4-androstenedione (A), and dehydroepiandrosterone (DHEA) in 10 normal and 16 obese eumenorrheic nonhirsute women matched for age. Each woman received 1 mg dexamethasone at 2300 h and again at 0700 h the next morning. At 0700 h, a continuous alpha ACTH-(1-24) infusion was begun at an initial dose of 30 ng/1.5 m2 body surface area X hr. The ACTH infusion rate was doubled every hour for 5 consecutive h to a maximum dose of 480 ng/1.5 m2 X h. Blood samples were collected for steroid assays before the infusion and at the end of each hour. The ACTH threshold dose was defined as the dose that produced a steroid response significantly above the basal level. The ACTH threshold dose for serum F and DHEA stimulation was not different between the groups, but the threshold dose for A was significantly lower in the obese women. Basal and stimulated serum DHEA to F ratios were significantly higher in the obese women. In both groups, the mean F response slope was significantly higher than that for DHEA, which in turn, was significantly higher than that for A. The mean DHEA response slope was significantly greater in the obese women. The F and A response slopes were not different between the groups. We conclude that the relative responsivity of the steroids to ACTH was the same in both groups: F greater than DHEA greater than A; in the obese women, the ACTH threshold dose for F stimulation was lower (greater sensitivity) than for DHEA or A stimulation; and in the obese women, the ACTH threshold dose for A was significantly lower (increased sensitivity) and the slope of the DHEA response to ACTH was steeper (greater responsivity) than in normal women. PMID- 3018028 TI - Familial disorder with increased number of insulin receptors: a new category of insulin receptor abnormality. AB - We found a family in which 6 of 13 family members had extremely high insulin binding to their erythrocytes. The specific binding values of these patients were 3- to 4-fold higher (22.8-28.6% of added [125I]insulin) than normal (mean +/- SD, 7.1 +/- 0.8%; r = 44). Scatchard analysis revealed that in each patient increased insulin binding was due to increased binding capacity, with little change in affinity. In all patients, reticulocyte counts were within the normal range. From the pedigree analysis, the pattern of inheritance was considered to be autosomal dominant. The propositus was more extensively studied. His mononuclear leukocytes and Triton X-100-solubilized preparations of erythrocyte ghosts also had high (approximately 3-fold) insulin-binding capacity compared to that of normal subjects. In contrast, the number of ouabain-binding sites and kinetics of sugar transport were normal. From these findings, we propose a new type of inherited abnormality of the insulin receptor. PMID- 3018029 TI - Calcium, calmodulin, and cyclic adenosine monophosphate modulate prostaglandin E2 release from isolated human gastric mucosal cells. AB - We studied prostaglandin E2 (PGE2) release from isolated cells of the human gastric mucosa. Mucosal cells were enzymatically isolated from biopsy specimens of human fundic mucosa. The results from these crude cell preparations were compared to those obtained in fractions with enriched (65-80%) or depleted parietal cell content (3-7%) which were prepared from gastric mucosa obtained at surgery. PGE2 release in the enriched parietal cell fractions exceeded that from crude or parietal cell depleted preparations 3- and 13-fold, respectively. However, despite this quantitative difference, all preparations responded similarly to the test agents. Newly synthesized PGE2 was not stored intracellularly but was released into the incubation medium. Release increased linearly for 30 min. Addition of the calcium ionophore A23187 enhanced PGE2 release 4- to 5-fold. The effect of A23187 required the presence of extracellular Ca2+ (10(-3) mol/liter). Assuming that A23187 alters Ca2+ flux in gastric cells as it does in other cell systems our data indicate that increased Ca2+ influx enhances PGE2 release. Since calmodulin is of importance for intracellular Ca2+ action, the calmodulin antagonists trifluoperazine and W7 were tested. Both antagonists inhibited PGE2 release by 65-85%, trifluoperazine being slightly more effective. Activation of the adenylate cyclase system by forskolin or direct addition of (Bu)2cAMP, a stable cAMP-analog, also inhibited PGE2 release. We conclude that PGE2 is released from parietal and from nonparietal cells of the human gastric mucosa, although the major quantity is released from the light density fraction that is enriched in parietal cells. In parietal and nonparietal cells Ca2+ is of importance in the regulation of gastric mucosal PGE2 release and calmodulin seems to mediate this intracellular action of Ca2+. cAMP inhibits PGE2 release from gastric cells. PMID- 3018030 TI - Decreased alpha 2-adrenergic receptors on platelet membranes from diabetic patients with autonomic neuropathy and orthostatic hypotension. AB - Platelet adrenergic receptors were studied in normal subjects and diabetic patients with autonomic neuropathy to determine the relationship between adrenoreceptor status and orthostatic hypotension. The binding of [3H]clonidine and [3H]yohimbine to platelet membranes was measured in diabetic patients with autonomic neuropathy and orthostatic hypotension (n = 12) and without orthostatic hypotension (n = 11), diabetic patients without autonomic neuropathy (n = 12), and normal subjects (n = 9). Mean basal and standing plasma norepinephrine levels were not different in the four groups, and there was no relationship between orthostasis and norepinephrine responses. The diabetic patients with orthostatic hypotension had a significantly greater fall in mean blood pressure [31 +/- 2.8 (+/- SE) mm Hg] than any of the other three groups. Diabetic patients with diabetic autonomic neuropathy and orthostatic hypotension had a 30-40% decrease in number of platelet alpha 2-adrenergic receptors, as demonstrated by [3H]clonidine and [3H]yohimbine binding. The maximum number of binding sites for clonidine was 34 +/- 2.8 (+/- SE) fmol/mg protein in normal subjects, 27.4 +/- 3.4 in diabetic patients with neuropathy, 26 +/- 2.5 in diabetic patients with autonomic neuropathy without orthostatic hypotension, and 20.4 +/- 3.8 fmol/mg protein in diabetic patients with autonomic neuropathy with orthostatic hypotension (P less than 0.001). The maximum number of binding sites for yohimbine was 112 +/- 12.6 in normal subjects, 127 +/- 10 in diabetic patients without orthostatic hypotension, and 87 +/- 12.4 fmol/mg protein in patients with diabetic autonomic neuropathy with orthostatic hypotension (P less than 0.001). Reduced platelet alpha 2-receptors are associated with postural hypotension in diabetic autonomic neuropathy. If applicable to the postjunctional alpha 2 adrenergic receptor on sympathetic neurons, reduced vascular responses to changes in posture would be expected despite normal or enhanced norepinephrine secretion. PMID- 3018031 TI - The role of calcium in steroidogenesis in fetal zone cells of the human fetal adrenal gland. AB - The human fetal adrenal gland is composed primarily of fetal zone (FZ) cells, which have a high rate of steroidogenesis. The purpose of this study was to examine the role of calcium in the regulation of steroidogenesis by FZ cells. Dispersed FZ cells were incubated in Krebs-Ringers medium at 37 C for 3 h in the presence of ACTH, (Bu)2cAMP, or forskolin in addition to various drugs. The medium contents of dehydroepiandrosterone sulfate (DS), cortisol (F), and cAMP were quantified by RIA. After the addition of ACTH (10(-10)-10(-5) M), DS and cAMP secretion increased. The addition of EGTA to the medium inhibited ACTH- and forskolin-stimulated DS, F, and cAMP secretion by 50% as well as (Bu)2cAMP stimulated steroidogenesis. The addition of calcium (10(-5)-10(-2) M) had only a slight effect on the secretion of DS or F in the absence of ACTH or (Bu)2cAMP. In the presence of ACTH and (Bu)2cAMP, however, increasing amounts of calcium resulted in a 2- to 3-fold increase in the rates of DS and F secretion. The addition of either A23187, a calcium ionophore, or verapamil, a calcium channel blocker, inhibited ACTH-stimulated DS and F secretion by 90%. The rate of cAMP formation was greater after ACTH plus verapamil treatment than after ACTH treatment alone, whereas A23187 inhibited ACTH-stimulated cAMP secretion to basal levels. Both A23187 and verapamil inhibited ACTH- and cAMP-stimulated pregnenolone secretion. The metabolism of 22R-hydroxycholesterol to pregnenolone was inhibited by A23187 and verapamil. In conclusion, our results suggest that extracellular calcium is important for activation of the human adrenal FZ cell adenylate cyclase system, while intracellular calcium plays a multifaceted role in controlling steroid production. PMID- 3018032 TI - Preservation of dopaminergic and alpha-adrenergic function in children with growth hormone neurosecretory dysfunction. AB - The integrity of dopaminergic and alpha-adrenergic neurotransmitter regulation of GH secretion was examined in children with decreased GH secretion. Children with GH neurosecretory dysfunction (GHND; n = 16) those with classical GH deficiency (n = 9), and short but otherwise normal children (n = 12) underwent 24 h GH studies (blood sampling every 20 min for 24 h) and provocative tests using arginine, insulin hypoglycemia, L-dopa (dopaminergic) and clonidine (alpha adrenergic), and GH-releasing hormone (GHRH). GHND was defined as children with height in the first percentile or below, growth velocity of 4 cm/yr or less, low plasma somatomedin-C for age, delayed skeletal age by 2 or more yr, peak serum GH responses to any one (or more) provocative test of 10 ng/ml or more, and mean 24 h GH concentration below 3 ng/ml. GHND and GH-deficient children had reduced endogenous GH secretion, expressed as mean serum 24-h GH concentration [1.6 +/- 0.1 (+/- SEM) and 2.1 +/- 0.1 vs. 6.1 +/- 0.5 ng/ml (GH-deficient and GHND vs. normal, respectively); P less than 0.01]. the mean peak serum GH levels after arginine [8.2 +/- 2.0 vs. 20.8 +/- 6.6 ng/ml (GHND vs. normal); P less than 0.05] and insulin [9.3 +/- 1.0 vs. 16.2 +/- 1.7 ng/ml (GHND vs. normal); P less than 0.01) were lower in GHND children. The mean peak responses after L-dopa [13.4 +/- 3.4 vs. 14.6 +/- 4.7 ng/ml (GHND vs. normal); P = NS] and clonidine [19.0 +/- 2.2 vs. 23.3 +/- 3.8 ng/ml (GHND vs. normal); P = NS] were preserved in GHND children. In GH-deficient children, mean peak serum GH concentrations after all four provocative tests were low (arginine, 2.7 +/- 0.8; insulin, 2.6 +/- 0.8; L dopa, 3.0 +/- 0.9; clonidine, 3.4 +/- 1.0 ng/ml; all P less than 0.01 vs. normal). The mean peak serum GH concentration after GHRH was blunted in GH deficient children (9.1 +/- 1.7 ng/ml) compared to those in GHND (32.9 +/- 8.5 ng/ml) and normal (43.2 +/- 6.4 ng/ml) children (P less than 0.01). The area under the GH curve after GHRH stimulation was greater for normal than GHND children (P less than 0.05). These data demonstrate preservation of dopaminergic and alpha-adrenergic neurotransmitter pathways in GHND children. They further suggest a defect in the release of pituitary GH secondary to an abnormality in alternative neurotransmitter pathways resulting in decreased GHRH and/or increased somatostatin secretion. PMID- 3018033 TI - Changes in insulin-like growth factors I and II and their binding protein after a single intramuscular injection of growth hormone. AB - The concentrations of insulin-like growth factors I and II (IGF-I and IGF-II) and their binding proteins in serum were measured in 10 GH-deficient patients before and after a single 6-IU injection of GH. Serum IGF-I concentrations were initially low, increased significantly by 8 and 24 h, and decreased to pretreatment levels 48 and 72 h after GH administration. Serum IGF-II concentrations also were low initially and did not increase by 8 and 24 h, but were, however, significantly higher 48 and 72 h after GH administration. In GH deficient patients before GH administration, binding of IGF-I or IGF-II to serum proteins was restricted primarily to proteins of 50K mol wt. Little or no binding to proteins of 150,000 mol wt was found. By 8 and 24 h after GH injection, IGF-I, but not IGF-II, bound primarily to a protein(s) of 150K mol wt, as in normal subjects. IGF-II remained bound to a 50K mol wt protein. By 48 and 72 h after administering GH, however, the binding pattern was reversed, and IGF-II, but not IGF-I, bound predominantly to a protein(s) of 150K mol wt. Our data demonstrate both a temporal dissociation in the responses of IGF-I and IGF-II to GH and a similar temporal dissociation in the binding of IGF-I and IGF-II to the large mol wt (150K) binding protein. This dissociation, particularly the latter, may provide a means for better characterization of protein fractions in binding IGF, particularly in terms of specificity. PMID- 3018034 TI - Somatomedin-C binding to cultured human fibroblasts is dependent on donor age and culture density. AB - Cultured fibroblasts have been used extensively to study age-related changes in the cellular response to serum stimulation. Since somatomedin-C (Sm-C) is an important growth factor in serum, we determined if there were age-related changes in Sm-C fibroblast receptor number or affinity and if culture density influenced these changes. Skin fibroblasts were obtained from six normal donors in three separate age groups and tested for their capacity to bind Sm-C. Sparse cultures (10-15K cells/well) derived from fetal donors had an affinity for Sm-C that was 4.7-fold greater than that of cultures derived from elderly (74-96 yr old) donors [9.4 +/- 0.2 (+/- SD) compared to 2.0 +/- 0.2 X 10(10) M-1]). When grown to high density (60-100K cells/well), the affinity of the fetal cultures was significantly reduced to 2.2 +/- 0.2 X 10(10) M-1 (P less than 0.001) and was not significantly different from the affinity of high density elderly donor cultures (2.3 +/- 0.4 X 10(10) M-1). Intermediate age donors (3-14 yr old) also had a significant reduction in receptor affinity with increasing density. Fetal donor cultures showed no density-dependent changes in receptor number. Fetal donor cells at low density had 5.2 +/- 1.0 X 10(4) receptors/cell compared to 5.9 +/- 0.6 X 10(4) receptors/cell in the high density cultures. In contrast, cells derived from donors aged 3-14 yr had 12.0 +/- 1.6 X 10(4) receptors/cell at 15K cells/well and 5.1 +/- 0.6 X 10(4) at 80K cells/well (P less than 0.05). Cultures from elderly donors had significantly greater mean receptor numbers per cell compared to fetal donor cells at four of five densities tested and had a significantly lower receptor number per cell with increasing culture density 25.2 +/- 1.2 X 10(4) (10-15K cells/well) compared to 5.2 +/- 0.2 X 10(4) (60-100K) cells/well. Thus, increasing donor age at low culture density was associated with an increase in receptor number per cell and a decrease in receptor affinity. At high culture densities, these differences were not detected. These changes in Sm C receptor number and affinity at low density could lead to donor age-related changes in the cellular response to Sm-C. PMID- 3018035 TI - Chronic active Epstein-Barr virus infection in patients with Chediak-Higashi syndrome. AB - The results of clinical and Epstein-Barr virus (EBV) serological studies on nine Chediak-Higashi syndrome (CHS) patients are reported. Persistently elevated antibodies to the viral capsid antigen (VCA) and the restricted component of the early antigen complex (EA-R) developed in six patients who experienced primary EBV infection which either remained silent or were accompanied by clinical signs of infectious mononucleosis (IM). Hepatosplenomegaly and moderate lymphadenopathy, both clinical signs of the accelerated phase, remained detectable in the six patients for a long period of time after seroconversion. The clinical, serological, and histopathological observations are suggestive of a nonmalignant lymphoproliferative disease and consistent with an immunodeficiency to EBV. The abnormal serological responses to EBV in CHS are therefore considered manifestations of a chronic active EBV infection which may result in lethal lymphoproliferation. The three as yet seronegative CHS patients revealed no signs of the accelerated lymphoproliferative phase of the syndrome. PMID- 3018036 TI - Oral succession of gram-negative bacilli in myelosuppressed cancer patients. AB - Aerobic and facultative gram-negative bacilli (GNB) have been reported to increase on various body surfaces in the seriously ill and debilitated patient. This study examined quantitative aspects of GNB succession at five oral sites in cancer patients before and during myelosuppressive chemotherapy. GNB concentrations increased sharply during chemotherapy at 25 to 50% of the oral sites in both acute nonlymphocytic leukemia and small-cell lung carcinoma patients. Most sites did not exhibit shifts of GNB to levels higher than 0.1% of the cultivable flora. When shifts occurred, all sites sampled in the mouth were usually affected and GNB usually represented more than 10% of the cultivable flora. Low levels of indigenous microflora were observed in most sites exhibiting GNB shifts. None of the subjects harboring high levels of GNB developed the symptoms of acute infection which are commonly observed in myelosuppressed patients. Although Pseudomonas aeruginosa and Klebsiella pneumoniae were recovered from some sites, most GNB were nonpathogenic species of Pseudomonas; Pseudomonas pickettii was the most frequently recovered. PMID- 3018037 TI - Phenotypic comparison of Pseudomonas aeruginosa strains isolated from a variety of clinical sites. AB - Pseudomonas aeruginosa elaborates a number of extracellular products which have been shown to play a role in the pathogenesis of disease caused by this organism. In this study, we showed that the host environment markedly affects the levels of exoproducts produced. We compared the phenotypes of a number of P. aeruginosa strains obtained from a variety of clinical sources, including burn wounds, skin wounds, urine, cystic fibrosis sputum, acute pneumonia sputum, and blood. The clinical isolates were examined quantitatively for levels of total protease, elastase, phospholipase C, exotoxin A, and exoenzyme S produced in vitro under defined conditions. The exoproduct levels varied significantly, depending on the site of isolation. Elevated levels of elastase were demonstrated in strains isolated from acute lung infections, phospholipase C levels were elevated in urinary tract and blood isolates, exotoxin A levels were elevated in blood isolates, and exoenzyme S levels were increased in acute pneumonia isolates. Isolates from cystic fibrosis sputum produced low amounts of virtually all of the tested exoproducts, particularly as compared with sputum isolates from acute P. aeruginosa lung infections. PMID- 3018038 TI - Alternative cell line for virus isolation. AB - A human lung carcinoma cell line (A549) was compared with various other cell lines to determine susceptibility to viral growth. In the first phase of the study, A549 cells were compared with human embryonic kidney (HEK) and cynomolgus monkey kidney (CMK) cells for isolation of upper-respiratory disease viruses by using 1,248 throat swab specimens from basic-combat trainees. Of the 552 virus isolates, 507 were adenoviruses, 41 were polioviruses, and 4 were herpes simplex viruses (HSV). Of the isolates, 518 (93.8%) were isolated in A549 cells, 480 (87.0%) were isolated in HEK cells, and 262 (47.5%) were isolated in CMK cells (P less than 0.001). In the second phase of the study, A549 cells were compared with a human diploid fibroblast cell strain (MRC-5) and Vero monkey kidney (VMK) cells for the isolation of HSV from 1,157 specimens submitted for culture. Of the 227 HSV isolates, 210 (92.5%) were isolated in A549 cells, 202 (89.0%) were isolated in VMK cells (P greater than 0.1 for A549 versus VMK cells), and 167 (73.6%) were isolated in MRC-5 cells (P less than 0.001 for A549 versus MRC-5 cells). These results suggest that A549 cells are more susceptible to adenovirus infection and at least as susceptible to HSV infection compared with the other cell cultures evaluated. Detracting factors for the use of A549 cells were a slight loss of sensitivity to adenovirus at passage 120 and a concurrent change in the morphology of the cells. The A549 cell line proved to be an efficient, practical, and economical alternative cell system for the isolation of adenovirus and HSV in particular. Initial indications are that other clinically significant viruses may be grown in A549 cells; however, additional studies need to be performed. PMID- 3018039 TI - Cytotoxic and cytotonic factors produced by Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis. AB - Complete toxigenicity studies were performed on 341 strains of Campylobacter spp., including 23 nonhuman isolates. Toxin profiles based on both cytotonic and cytotoxic factors were determined after analyzing responses in Vero, HeLa, CHO and Y-1 cells. Suckling mouse assays were consistently negative for all culture filtrates tested. Toxin-producing strains were frequently encountered among both the human and nonhuman strains of Campylobacter jejuni, C. coli, and C. laridis investigated. Strains isolated from outbreaks demonstrated parallels in serotype, biotype, and toxigenicity profile, although no clear association could be demonstrated. Biphasic culture conditions conducive to the production of both toxic factors were delineated for the propagation of test Campylobacter strains. Cytotonic effects of Campylobacter culture filtrates were determined in Vero and CHO cells, and cyclic AMP accumulation in cells exposed to these culture filtrates was compared with that in cells exposed to reference toxigenic strains of Vibrio cholerae and Escherichia coli. Partial neutralization of C. jejuni enterotoxin was demonstrated by using antitoxins to cholera toxin and E. coli heat-labile enterotoxin. No neutralization of C. jejuni cytotoxin could be achieved by using antitoxins to either Clostridium difficile cytotoxin or E. coli Verotoxin (0157:H7). PMID- 3018040 TI - Enzyme-linked immunosorbent assay spin amplification technique for herpes simplex virus antigen detection. AB - A comparative study of herpes simplex virus diagnosis by standard cell culture and a new hybrid test (enzyme-linked immunosorbent assay spin amplification technique) was done on 300 specimens. The new test was found to be equally sensitive and specific, much less expensive to perform, and to report all results in 48 h. PMID- 3018042 TI - Polymorphism of the human complement C4 and steroid 21-hydroxylase genes. Restriction fragment length polymorphisms revealing structural deletions, homoduplications, and size variants. AB - Several autoimmune disorders as well as congenital adrenal hyperplasia (CAH) are either associated or closely linked with genetic variants of the fourth component of complement (C4A and C4B) and the enzyme steroid 21-hydroxylase (21-OH). These proteins are encoded by genes that are located downstream from the genes for complement proteins, C2 and factor B (BF) between HLA-B and -DR in the major histocompatibility complex (MHC). Previous studies of variants and null alleles were based on electrophoretic mobility of C4 protein and linkage with disease phenotypes. These data did not permit analysis of the basis for the observed null alleles and duplicated variants. We studied this region of the MHC in 126 haplotypes for a structural analysis of the four adjacent loci, C4A, 21-OHA, C4B, and 21-OHB. About half of the C4 genes typed as C4 null are deleted and several unrecognized homoduplicated C4 alleles were detected. Hence the frequencies of different C4 structural variants must be recalculated based on a direct analysis of the genes. Analysis of the C4/21-OH genes of patients with the classical (salt wasting) form of CAH showed that some involve a deletion of the C4B and 21-OHB genes; whereas for two only the 21-OHB gene is deleted, i.e., the C4B gene is present. Together, these data provide a better understanding of the mechanisms generating and importance of deleted C4 and 21-OH null alleles in human disease. PMID- 3018043 TI - Very late activation antigens on rheumatoid synovial fluid T lymphocytes. Association with stages of T cell activation. AB - Lymphocytes from the synovial fluid of eight out of eight rheumatoid arthritis (RA) patients had elevated very late activation antigen-1 (VLA-1) expression (10 36% positive cells), whereas peripheral blood lymphocytes (PBL) from RA patients and healthy controls had low VLA-1 expression (0-6% positive cells). During 1-2 wk of in vitro culture, VLA-1 increased on synovial fluid cells but remained low on PBL. In comparison, the interleukin 2 receptor (IL-2 R) was less prominent than VLA-1 on fresh synovial fluid cells, did not increase on cultured synovial fluid T cells, but did increase greatly on cultured PBL. The mitogen PHA reversed or prevented the appearance of VLA-1+, IL-2 R- synovial fluid cells during in vitro culture, thus giving IL-2 R+, VLA-1- cells. These results emphasize that VLA-1+ SF cells are different from resting cells or IL-2 R+ activated PBL T cells, and VLA-1 on synovial fluid T cells may be incompatible with mitogen stimulation. In addition, the VLA-2 heterodimer (165,000/130,000 relative molecular mass [Mr]) was regulated opposite to the VLA-1 heterodimer (130,000/210,000 Mr) on synovial lymphocytes, and thus the VLA-1/VLA-2 ratio is another indicator of the stage of T cell activation. PMID- 3018041 TI - The unique endocrine milieu of the fetus. AB - Table II summarizes in tabular form the major features of the fetal endocrine milieu discussed in the foregoing pages. The mammalian fetus develops in an environment where respiration, alimentation, and excretory functions are provided by the placenta. Fetal tissue metabolism is oriented largely to anabolism; body temperature is modulated by maternal metabolism, and fetal tissue thermogenesis is maintained at a basal level. Tissue and organ growth appear to be regulated by growth factors which probably function by autocrine or paracrine mechanisms during most of gestation (72, 146-148). In this milieu conventional endocrine control systems are largely redundant, and other transient systems more appropriate to the intrauterine environment have evolved. We have developed some insights into these systems, but much more information is necessary before we can truly understand this fascinating environment. PMID- 3018045 TI - Receptor recognition of maleyl-albumin induces chemotaxis in human monocytes. AB - We demonstrate here that the exceptionally active maleyl-albumin receptor of human monocytes functions in vitro as a chemoattractant receptor. Chemotaxis of human monocytes occurs at an effective median dose of 3-4 microM maleyl-albumin, a concentration representing 1% of the total albumin in the adult human. Computerized analyses by LIGAND of the saturable binding of maleyl-albumin to human monocytes reveal two classes of binding sites, described by dissociation constants of 37 nM and 5.3 microM with maximal binding of 1.6 and 23 pmol maleyl albumin/mg cellular protein, respectively. Chemotaxis of human monocytes thus occurs at concentrations of maleyl-albumin promoting binding to the lower affinity sites. We propose that conformational isomers of albumin that are chemotactic may form in vivo and that albumin, in addition to receptor independent plasma transport functions, may also play an important role in the receptor-mediated recruitment and accumulation of phagocytic cells at sites of inflammation and injury. PMID- 3018047 TI - Suspected adverse methylphenidate-imipramine interactions in children. AB - Two cases are presented which illustrate the dangers inherent in utilizing a polypharmacy approach in treating children with psychotropic medication. In each case, severe adverse effects, including cognitive and mood deterioration, were experienced by the child when treated with a combination of methylphenidate and imipramine. Possible mechanisms at the neurotransmitter level are described as postulated determinants for this pharmacological interaction. PMID- 3018048 TI - Are we making progress in the drug treatment of lung cancer? PMID- 3018044 TI - Role of plasma gelsolin and the vitamin D-binding protein in clearing actin from the circulation. AB - We determined the plasma kinetics of both actin and complexes of actin with the two high affinity actin-binding proteins of plasma, gelsolin, and vitamin D binding protein (DBP). Actin is cleared rapidly from the plasma by the liver (half-disappearance time, 0.5 h). Using radiolabeled actin-binding proteins, we found that actin accelerated the clearance of both plasma gelsolin and the vitamin D-binding protein. In separate experiments we found that DBP-actin complexes were cleared more quickly than gelsolin-actin complexes, at a rate comparable to the clearance of actin from the blood. A low affinity interaction (dissociation constant, 2.9 X 10(-4) M) between actin and fibronectin was found, suggesting that little actin will bind to fibronectin in plasma. We conclude that while plasma gelsolin and DBP may both clear actin from the circulation, DBP appears to play a more important role. By so doing, DBP may conserve the filament severing activity of plasma gelsolin. PMID- 3018046 TI - Atrial natriuretic peptide transcription, secretion, and glomerular receptor activity during mineralocorticoid escape in the rat. AB - The mechanisms that mediate renal "escape" from the sodium-retaining effects of mineralocorticoids are incompletely understood. This study was undertaken to determine whether atrial natriuretic peptide (ANP) may play a role in the escape phenomenon. Immunoreactive ANP in rat plasma increased 2.5-fold above baseline values at 12 and 24 h after a single depot injection of desoxycorticosterone acetate in oil and returned to baseline thereafter. In addition, specific pre-pro ANP messenger RNA content in rat atria was significantly elevated as early as 12 h after mineralocorticoid administration and remained elevated at 24, 48, and 72 h, indicating a prompt and sustained increase in ANP biosynthesis. Renal glomerular ANP receptor density was down-regulated appropriately with rising plasma ANP levels, and receptor affinity was unchanged. Thus, mineralocorticoid administration in the rat is a powerful stimulus for ANP release and for atrial myocyte ANP synthesis, which suggests a potential role for this hormone in overriding mineralocorticoid-induced renal sodium retention. PMID- 3018049 TI - Intraatrial extension of hepatocellular carcinoma detected with ultrasound. PMID- 3018050 TI - Severe cutaneous reactions to captopril and enalapril; histological study and comparison with early mycosis fungoides. AB - A severe non-dose related skin eruption attributable to treatment with captopril was recently reported: this is distinct from the dose related rashes that have been widely described. Ultrastructural and immunohistochemical studies were carried out to determine in detail the histological features of this eruption: the histological appearances were similar to those found in early mycosis fungoides, so that this disease was erroneously diagnosed in one case. Unlike most other complications resulting from treatment with Captopril, an indistinguishable rash can result from treatment with enalapril, a newer angiotensin converting enzyme inhibitor. PMID- 3018052 TI - Atypical keloids after dermabrasion of patients taking isotretinoin. AB - Six patients underwent dermabrasion while on or having recently completed isotretinoin (Accutane) therapy. All patients developed keloids in atypical locations; the keloids eventually responded to topical or intralesional steroid therapy. Retinoids have a modulatory effect on connective tissue metabolism, including suppression of collagenase, which may enhance keloid formation. Dermabrasion should be delayed in those patients taking or recently on isotretinoin therapy. PMID- 3018051 TI - Human papilloma virus in condyloma acuminata of the anus. PMID- 3018053 TI - Extramammary Paget's disease. PMID- 3018054 TI - Computed tomography of monkey brain tumors. AB - Thirty-five Japanese monkeys were inoculated intracerebrally with chick embryo fibroblasts that were producing Schmidt-Ruppin strain of Rous sarcoma virus. Tumors were induced in 54.3% (19/35). Computed tomography detected tumors in 10 symptomatic animals with an average latency of 32.6 (15-43) days. At autopsy, the brains were sectioned into 5 mm slices, coplanar to the CT image. Various CT features of high- and low-density area correlated well with the histopathological findings, such as tumor, hemorrhage, necrosis, and peritumoral edema. Contrast enhanced CT detected 10 tumors greater than 4 mm in diameter, and there was +/- 2 mm potential error in determining tumor size. Follow-up CT revealed growth of tumors in four animals and stabilization of tumor in two animals. Large brain size, 90-110 g in adult monkeys, and availability of induced tumors offer an excellent brain tumor model for CT studies. PMID- 3018055 TI - Effects of sodium bicarbonate, magnesium oxide, and a commercial buffer mixture in early lactation cows fed hay crop silage. AB - Sixteen early lactation Holstein cows fed 70% concentrate: 30% hay crop silage were used to determine effects of .7% sodium bicarbonate, .7% sodium bicarbonate plus .28% magnesium oxide, or 1.8% commercial buffer mixture (total ration dry basis). This mixture contained a variety of buffers, alkalis, and other compounds known to affect milk production or composition in some circumstances. Buffers did not affect dry matter intake, milk yield, or milk composition but decreased efficiency of milk production. Ruminal fluid pH was not affected, but fecal pH and digestibilities of dry matter, organic matter, energy, acid detergent fiber, and cellulose were increased by the mixed buffers compared with sodium bicarbonate alone. Total ruminal volatile fatty acid concentration was reduced by buffers. Compared with sodium bicarbonate alone, mixed buffers increased ruminal ammonia concentration, acetate proportion, and acetate:propionate ratio and decreased proportions of propionate and butyrate. Valerate was reduced by all three buffers. Ruminal volume and liquid dilution rate were unaffected, but buffers increased total fluid outflow from the rumen. Higher amounts of buffers or alkalis may be necessary to offset low rumen pH and affect production with hay crop silage-based diets. PMID- 3018056 TI - Laser treatment of hereditary multiple glomus tumors. AB - Familial multiple glomus tumors are rare. A family is reported here with the disease documented in three generations. While this type of glomus tumor is not classically associated with pain, all members of this family who exhibited multiple glomangioma were symptomatic. Treatment with both the argon laser and carbon dioxide laser was successful in relieving symptoms and improving the appearance of only the more superficial glomus tumors. PMID- 3018057 TI - Toxicity of erythrosin B to the house fly (Diptera: Muscidae) PMID- 3018060 TI - Nutritional implications and dietary management postproctocolectomy and ileal reservoir construction. PMID- 3018059 TI - [Disordered regulation of collagen production in the skin fibroblasts of systemic scleroderma patients]. PMID- 3018061 TI - Update: acquired immune deficiency syndrome. PMID- 3018058 TI - Exposure of Culicoides variipennis (Diptera: Ceratopogonidae) to hair clippings to evaluate insecticide-impregnated ear tags in cattle. PMID- 3018062 TI - Muconaldehyde formation from 14C-benzene in a hydroxyl radical generating system. AB - It has recently been proposed that muconaldehyde, a six carbon, alpha, beta unsaturated dialdehyde, may be a hematotoxic metabolite of benzene. The present studies indicate that trans, trans-muconaldehyde is formed from benzene in vitro in a hydroxyl radical (.OH) generating system containing ascorbate, ferrous sulfate and EDTA in phosphate buffer, pH 6.7. Muconaldehyde formed from benzene in the .OH generating system was identified by trapping it with thiobarbituric acid (TBA), which results in the formation of an adduct with a 495 nm absorption maximum and a 510 nm fluorescence emission maximum. These maxima were identical to those observed after reacting authentic trans, trans-muconaldehyde with TBA. This finding was supported by thin layer chromatography and solid phase extraction studies. In those studies benzene-derived muconaldehyde cochromatographed with the muconaldehyde/TBA standard. Analyses of the products from the .OH generating system using high performance liquid chromatography (HPLC) confirm that trans, trans-muconaldehyde is a product of benzene ring fission. Regardless of whether or not TBA was used for trapping, samples from the .OH system incubated with benzene contained a peak which cochromatographed with the muconaldehyde standard. The radioactivity profile of fractions collected during HPLC analysis demonstrates 14C-benzene to be the source of the trans, trans-muconaldehyde. The role of hydroxyl radicals in the formation of muconaldehyde was investigated by using dimethyl sulfoxide, mannitol, and ethanol as .OH scavengers. These scavengers, at concentrations of 10 and 100 mM, were found to cause a dose-dependent decrease in the formation of muconaldehyde.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018064 TI - Superoxide radical scavenging ability of centrophenoxine and its salt dependence in vitro. AB - The superoxide radical scavenging ability of centrophenoxine (CPH) and its components (dimethylaminoethanol = DMAE, p-chlorophenoxyacetic acid = PCPA) was studied in vitro using the method of pyrogallol autoxidation, cytochrome c reduction and photoxidation of o-dianisidine in salt-free assay media and in the presence of increasing NaCl or KCl concentrations. The CPH proved to be a superoxide radical scavenger in all three systems used, however, the rate constant for this reaction was rather low (1.7 X 10(2) M-1 s-1). This scavenging ability decreased linearly with increasing ionic strength. DMAE and PCPA behaved in a somewhat contradictory manner. The former proved to be a weak superoxide radical generating compound being partially sensitive to the ionic strength. The latter showed either superoxide radical scavenging or generating effects in various assays depending on the actual salt concentrations of the media. On the basis of the results one has to assume that the superoxide radical scavenger ability of CPH may hardly be responsible for the in vivo effects of this compound, therefore, its OH. radical scavenger reactions the rate constant of which is about 10(9) M-1 s-1 may be of much greater importance. PMID- 3018063 TI - Superoxide anion is generated from cellular metabolites by solar radiation and its components. AB - Several endogenous cellular constituents were tested for their ability to produce superoxide anion (O2-) from ground-state molecular oxygen upon irradiation by solar radiation. The pyridine cofactors NADPH and NADH, riboflavin, and the nucleosides 2-thiouracil and 4-thiouridine were found to sensitize the transmission of photon energy from solar radiation and monochromatic radiation (290, 334, 365, and 405 nm) to oxygen, resulting in O2- formation, as detected by superoxide dismutase-inhibitable cytochrome c reduction. Quantum yields for the production of O2- indicate that NADPH is the most efficient and riboflavin the least efficient of the compounds tested. These data indicate that endogenous compounds may participate in the production of O2- by solar radiation and imply that O2- may play a role in sunlight-induced erythema and dermal carcinogenesis. PMID- 3018065 TI - Hydrogen peroxide causes dimethylthiourea consumption while hydroxyl radical causes dimethyl sulfoxide consumption in vitro. AB - Addition of increasing concentrations of hydrogen peroxide (H2O2) caused progressive decreases in dimethylthiourea (DMTU) concentrations which were inhibitable by simultaneous addition of catalase, but not the superoxide anion (O2-.) scavenger, superoxide dismutase (SOD), or hydroxyl radical (.OH) scavengers, such as mannitol, sodium benzoate or dimethyl sulfoxide (DMSO). In parallel, addition of increasing concentrations of H2O2 with FE++/EDTA (but not H2O2 alone) caused decreases in DMSO concentrations which were inhibitable by simultaneous addition of .OH scavengers but not SOD or catalase. Addition of DMTU, but not DMSO, also decreased H2O2 concentrations in vitro. The results indicate the relative scavenging specificities of DMTU and DMSO for H2O2 and .OH, respectively. The findings also suggest that measurement of DMTU or DMSO consumption could help assess the contribution of O2 metabolites in biological systems. PMID- 3018066 TI - Peroxidation of liposomes promoted by human polymorphonuclear leucocytes. AB - Human polymorphonuclear leucocytes were found to promote peroxidation of phospholipid liposomes upon stimulation by phorbol myristate acetate. Peroxidation required the presence of either pyrophosphate-chelated or ADP chelated iron, whereas iron chelated to EDTA or ATP had no effect. Peroxidation was also catalyzed by ferritin, but not by transferrin. Superoxide dismutase abolished the peroxidation, whereas catalase and apparently also the hydroxyl radical scavenger dimethyl sulphoxide were inactive, indicating that the peroxidation was mediated by superoxide radicals but not by hydrogen peroxide or hydroxyl radicals. Xanthine oxidase-promoted peroxidation was studied for comparison and showed similar characteristics except that transferrin catalyzed the peroxidation. Peroxidation of membrane lipids may be a mechanism whereby granulocytes cause tissue damage in inflammation. The drugs paracetamol, gentisic acid and 5-aminosalicylic acid inhibited lipid peroxidation, probably through their ability to react with the superoxide anion. PMID- 3018067 TI - Platelet angiotensin receptors in young and old humans. AB - Platelets from healthy human volunteers were studied for angiotensin II (AII) binding sites. Platelets were isolated from whole blood by differential centrifugation, and binding sites were analyzed by Scatchard plots of radioactive ligand binding data. The number of platelet AII receptor sites was significantly higher in human beings of advanced age compared with younger persons. The affinity of receptor sites was not different in young and old participants. The increased number of binding sites bore no relationship to salt intake as documented by history, plasma renin activity, or blood pressure. A significant portion of the increase in platelet AII receptor sites in older adults in this study is related directly to the age of the individuals. PMID- 3018069 TI - Recurrent ileal mucinous adenocarcinoma in an ileostomy. AB - We report an adenocarcinoma arising in an ileostomy after colectomy for ulcerative colitis and discuss the treatment of a localized recurrence. PMID- 3018068 TI - The effect of sodium bicarbonate versus aluminum-magnesium hydroxide on postprandial gastric acid in duodenal ulcer patients. AB - When ingested 1 hour after a meal, conventional liquid antacids have a buffering effect of approximately 2 hours, while in the fasting state their effect is brief, lasting less than 1 hour. We tested the hypothesis that equal doses of antacid, one water soluble (sodium bicarbonate) and the other water insoluble (aluminum hydroxide plus magnesium hydroxide, MaaloxR), would have similar durations of postprandial buffering if the water soluble antacid regenerates the particulate protein buffer of the meal that leaves the stomach more slowly than liquids. Tests were conducted in random order on three separate days in 10 patients with duodenal ulcer. The effects of 30 ml of 2.39 M sodium bicarbonate (6.17 g, about 1 teaspoonful), the aluminum-magnesium antacid, each equivalent to 71.7 mmol of in vitro buffer, and water as a control on pH, hydrogen ion activity, and titratable acidity were compared. Thirty milliliters of each was swallowed 1 and 3 hours after ingestion of a standard solid plus liquid. Compared to the water control each dose of sodium bicarbonate significantly increased intragastric pH and decreased hydrogen ion activity and titratable acidity for only 1 hour. Each dose of the aluminum-magnesium antacid significantly buffered intragastric contents for 2 hours. These findings indicate that sodium bicarbonate transiently buffers postprandial intragastric contents. Therefore, sodium bicarbonate fails to reconstitute the protein buffer of the meal effectively, and the observations suggest that it leaves the stomach rapidly with the liquid phase of the meal. However, the water insoluble, aluminum-magnesium antacid has a longer duration of buffering, probably because it leaves the stomach more slowly, largely with the solid portion of the meal. PMID- 3018070 TI - Lactic acidosis associated with cirrhosis, hepatoma, hemoperitoneum, and hyperphosphatemia. AB - A patient with cirrhosis developed hemoperitoneum, lactic acidosis, and hyperphosphatemia in the absence of shock. At post-mortem examination occult multicentric hepatocellular carcinoma eroding the liver capsule was present. The case emphasizes the central role of the liver in lactic acidosis, indicates that hemoperitoneum may precipitate this complication, and documents the association of lactic acidosis and hyperphosphatemia. PMID- 3018071 TI - Precipitating antibody development in chicks experimentally infected with herpes virus of turkey and Marek's disease virus. PMID- 3018072 TI - Effect of simulated poor specimen handling on ELISA for rotavirus detection. PMID- 3018073 TI - Haemodynamic rebound phenomena after abrupt cessation of propranolol therapy in portal hypertensive rats. AB - The haemodynamic effect of sudden termination of propranolol therapy was studied in sham-operated and portal hypertensive rats. All animals were injected with propranolol (20 mg/kg/day) or saline i.p. for 10 days, then had an isoproterenol infusion test performed 48 h or 72 h after cessation of injections. The dose of isoproterenol required to increase the heart rate by 50 beats/min (CD50), was significantly lower in both sham-operated and portal hypertensive rats at 48 h after propranolol withdrawal. Maximum chronotropic response (Rmax), was significantly higher only in portal hypertensive rats at 48 h after propranolol withdrawal. These results show the existence of a transient beta-adrenergic hypersensitivity state following propranolol withdrawal in normal and portal hypertensive rats. PMID- 3018074 TI - Resolution of the clinical features of tyrosinemia following orthotopic liver transplantation for hepatoma. AB - The clinical history before transplantation and subsequent clinical and biochemical course of 3 children and one adult with hereditary tyrosinemia treated by orthotopic hepatic transplantation is described. All four patients are now free of their previous dietary restrictions and appear to be cured of both their metabolic disease and their hepatic neoplasm. PMID- 3018076 TI - Radiation therapy and non-small cell lung cancer: current perspectives. PMID- 3018075 TI - Hepatitis B virus DNA in mononuclear blood cells. A frequent event in hepatitis B surface antigen-positive and -negative patients with acute and chronic liver disease. AB - We have investigated 38 hepatitis B surface antigen (HBsAg)-positive and 34 negative patients with acute and chronic liver disease for the presence of hepatitis B virus (HBV) DNA in peripheral mononuclear blood cells. Among the HBsAg-positive subjects HBV DNA was detected in the mononuclear cells of asymptomatic HBV carriers (2/6), patients with acute hepatitis (8/8), chronic active hepatitis (18/21), and with hepatocellular carcinoma (2/3); the viral DNA sequences were also identified in the mononuclear cells of patients with HBsAg negative acute hepatitis (2/3), chronic active hepatitis (5/15) and hepatocellular carcinoma (5/16), some of these showing no evidence of HBV by conventional serological markers. By contrast HBV DNA was not detected after resolution of the acute viral infection. For 7 patients different mononuclear cell-enriched subpopulations were assayed and the viral DNA was observed in T lymphocytes (both OKT4+ and OKT8+ enriched subsets) and/or in B enriched lymphocytes; the restriction DNA patterns showed in some patients a genetic organisation of the viral DNA similar to those observed in the liver (including free monomeric and oligomeric HBV DNA and results consistent with integrated viral sequences); however, no HBV DNA replicative forms were detected. These results show that the hepatitis B virus infection of mononuclear blood cells (including lymphoid cells) is a frequent event at all stages of the viral infection which might be related to immunological abnormalities observed in HBV carriers; in addition the mononuclear blood cells analysis may provide an insight to the liver cells status. PMID- 3018077 TI - Suppressive effect of human natural killer cells on Epstein-Barr virus-induced immunoglobulin synthesis. AB - The suppressive effect of human natural killer (NK) cells on Epstein-Barr virus (EBV)-induced immunoglobulin (Ig) synthesis by autologous B cells was investigated. By Percoll discontinuous density gradient centrifugation, low density fractions enriched for NK cells were isolated from human peripheral blood lymphocytes. These NK-enriched fractions were added to purified autologous B cells in the presence of EBV, were cultivated for 8 days, and were examined for their suppressive effect on Ig synthesis by an enzyme-linked immunosorbent assay. The fractions markedly suppressed both IgM and IgG synthesis induced by EBV. It was possible to reduce the suppressive effect of NK-enriched cells by complement dependent lysis of NK cells and Leu-11, but not by OKT3 monoclonal antibody, indicating that NK cells may be responsible for the suppression of Ig synthesis. Upon close examination of interferon (IFN) activity, it was revealed that the co cultures of NK-enriched cells and EBV-infected B cells generated production of IFN-alpha, which might be produced by NK cells in response to EBV-stimulated B cells. Addition of anti-IFN-alpha but not anti-IFN-gamma serum almost completely abrogated the suppressive effect of NK-enriched cells on Ig synthesis, indicating that IFN-alpha produced are required for the NK cell-mediated suppression of Ig synthesis. However, addition of IFN-alpha into purified B cells showed no direct suppressive effect on EBV-induced Ig synthesis by B cells in the absence of NK cells. Nevertheless, NK cells when previously incubated with IFN-alpha and added to B cells showed a suppressor activity on Ig synthesis to a level higher than that of untreated NK controls. These results strongly suggest the possibility that NK cells display an interaction with EBV-infected B cells and produce IFN alpha, which in turn activates NK cells. These activated NK cells suppress the Ig synthesis by B cells, which undergo transformation induced by EBV. PMID- 3018078 TI - Glycoprotein C of herpes simplex virus 1 is an inhibitor of the complement cascade. AB - Mammalian cells in culture express membrane receptors for C3b when infected with HSV-1. C3b binding is mediated by glycoprotein C (gC), a virus-specified membrane glycoprotein. In view of the inhibitory functions of other C3b-binding proteins, we studied the capacity of gC to modulate complement activation. Glycoprotein C was purified from HSV-1-infected cells by immunoaffinity chromatography. Glycoprotein C, but not a control viral glycoprotein, demonstrated dose-dependent acceleration of decay of C3bBb sites. In addition, gC produced a dose-dependent, time-independent depression of the overall hemolytic efficiency of C3bBb sites. Inhibition of C5b6-initiated reactive lysis of cells bearing C3b, but not cells bearing antibody alone, by gC suggests that the second effect represents interference with the C3b-C5/5b interaction. This hypothesis is supported by the failure of gC to inhibit reactive lysis when added after C5b67 insertion into target cells. Glycoprotein C does not accelerate C14b2a decay, nor does it impair classical pathway hemolytic efficiency when excess C5 is present. By limiting available C5/5b, some gC inhibition of C3b-C5/5b interactions can be unmasked in the classical pathway system. Glycoprotein C is devoid of factor I co-factor activity. HSV-1 gC is a modulator of complement activation, especially via the alternative pathway, and may represent a novel viral mechanism for evading host defense processes. PMID- 3018079 TI - Involvement of natural killer cells in coxsackievirus B3-induced murine myocarditis. AB - The role of natural killer cells in the temporal development of coxsackievirus B3 induced myocarditis in adolescent CD-1 male mice was examined. Inoculation of purified CVB3m induced maximum NK cell activity in the splenic populations at 3 days postinoculation (p.i.) as assessed by lysis of YAC-1 cells; maximum virus titers in heart tissues were also found at day 3 p.i. Mice depleted of NK cells after injection of anti-asialo GM1 antiserum i.v. had decreased NK cell activity, increased CVB3m titers in heart tissues, and exacerbated myocarditis. Although lesion number was not increased in heart tissues of the latter mice, lesions in these mice exhibited increased myocyte degeneration and dystrophic calcification above that found in lesions of mice inoculated with CVB3m only. No alteration in interferon titers were observed in CVB3m-infected mice treated with anti-asialo GM1 antiserum as compared with normal CVB3m-infected mice. Measurements of splenic NK cell activity in mice inoculated with doses of 10(2) to 10(8) PFU of CVB3m per mouse or UV-irradiated virus suggest that replication of CVB3m is required for NK cell activation. An amyocarditic variant of CVB3m (ts5R) was shown to replicate in heart tissues and to elicit NK cell activity comparable to that elicited by CVB3m. Therefore, the data suggest that NK cell activation depends on virus replication and that these cells provide some protection against CVB3m-induced myocarditis by limiting virus replication in heart tissues. PMID- 3018081 TI - Leukocyte inhibitory factor (LIF) potentiates neutrophil responses to formyl methionyl-leucyl-phenylalanine. AB - The ability of purified (80,000-fold) human leukocyte inhibitory factor (LIF) to modulate several formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe)-induced neutrophil functions was evaluated. Although not affecting directed migration itself, at low concentrations (1/2 to 2 U/ml), LIF was demonstrated to potentiate chemotaxis induced by f-met-leu-phe (40.3% +/- 8.1) and to reduce the concentration of f-met-leu-phe necessary for maximal chemotaxis (10(-8) to 10(-9) M). Similarly, LIF did not directly induce the respiratory burst, but potentiated both superoxide generation (151.6% +/- 77) and hydrogen peroxide production (54.9% +/- 15.5) in the presence of f-met-leu-phe (10(-7) M). LIF was also shown to induce degranulation of neutrophil-specific granules in a dose-dependent manner. Neutrophil-specific granules have been shown to contain an intracellular pool of receptors for f-met-leu-phe, and on degranulation provide the surface membrane with a fresh source of receptors. Our data suggested that LIF potentiation of neutrophil stimulation by f-met-leu-phe might be mediated, at least in part, by increasing the number of available membrane receptors as a result of its ability to induce degranulation. Radioligand receptor analysis using f-met-leu-[3H] phe was performed, and LIF was shown to mediate an increase in receptors for f-met-leu-phe from an average of 18,600 on untreated cells to 27,000 after pretreatment with LIF. This increase in receptors could "sensitize" the neutrophils for f-met-leu-phe and possibly explain the potentiation of neutrophil stimulation observed in the presence of the ligand. LIF was also found to have a more generalized effect on the transduction of neutrophil activation stimuli, mediating a 35.8% increase in superoxide production after exposure to calcium ionophore. The data do not permit a determination as to whether the increase in receptor number is responsible for the potentiation of f-met-leu-phe mediated function, or whether this occurs secondary to the more generalized effect on neutrophil stimulation transduction. PMID- 3018080 TI - Tumor-specific idiotype vaccines. I. Generation and characterization of internal image tumor antigen. AB - The concept of idiotype vaccines against tumor-associated antigens (TAA) was tested in the DBA/2 L1210 lymphoma subline, L1210/GZL. Monoclonal antibodies against a TAA that cross-reacts with the envelope glycoprotein gp52 of the mammary tumor virus were used to make hybridoma anti-idiotype antibodies (Ab2). In this report we describe the characterization of monoclonal anti-idiotypic antibodies against the combining site of 11C1 (Ab1), which recognizes a shared determinant of gp52 of mouse mammary tumor virus (MMTV) and the TAA of L1210/GZL. Hybridomas expressing the internal image of gp52 were screened by an idiotype inhibition assay. Mice sensitized with radiated L1210/GZL cells produced specific delayed type hypersensitivity (DTH) against the Ab2 hybridoma. Five Ab2 hybridomas were selected and were used to immunize DBA/2 mice. Such immunized animals showed specific DTH reaction against a challenge with the L1210/GZL tumor cells. Similar results were obtained in mice immunized with purified Ab2. Fluorescence-activated cell sorter analysis demonstrated that fluorescence staining of L1210/GZL cells by 11C1 can be completely inhibited with preabsorption on Ab2 hybridoma cells. Mice immunized with 2F10 and 3A4 coupled to keyhole limpet hemocyanin (KLH) contained antibodies binding to MMTV. But only in mice immunized with 2F10-KLH was significant inhibition of L1210/GZL tumor growth observed. Collectively, these results indicate that certain anti-idiotypic antibodies can mimic the MMTV gp52 antigen, as well as the gp52-like epitope expressed on the L1210/GZL tumor cells. These properties of anti-idiotypic antibodies mimicking TAA could be exploited for making idiotype vaccines against tumors. PMID- 3018082 TI - Characterization of the plasma membrane bound GTPase from rabbit neutrophils. I. Evidence for an Ni-like protein coupled to the formyl peptide, C5a, and leukotriene B4 chemotaxis receptors. AB - We have characterized the GTPase activity of the Ni-like guanine-nucleotide binding regulatory protein in rabbit neutrophil plasma membranes. The low Km (3.64 +/- 0.87 X 10(-7) M) GTPase copurified with the formyl peptide receptor in the plasma membrane fraction obtained by discontinuous sucrose density gradient centrifugation. The Vmax (23.9 +/- 2.91 pmol/mg/min) and Km of the unstimulated enzyme were similar to those reported for Ni in other cell types. The activity of the unstimulated enzyme was both magnesium and sodium dependent and linear over the first 4 min of the assay. The chemoattractants, formyl-methionyl-leucyl phenylalanine (fMLP), C5a, and leukotriene B4 (LTB4) stimulated the GTPase in purified neutrophil plasma membrane preparations, whereas other secretagogues, such as A23187 and PMA, were without effect. Lineweaver-Burk analysis showed an fMLP-induced increase in Vmax (31.94 +/- 4.80 pmol/mg/min) (33.1 +/- 9.5%) but not in Km. The dose-response curve for fMLP stimulation showed an ED50 of 4.1 +/- 1.0 X 10(-8) M and an overall 22.2 +/- 3.1% maximal stimulation. C5a (30 micrograms/ml) increased the activity of the GTPase 21.3 +/- 5.7% and 10(-7) M LTB4 produced a 32.2 +/- 5.4% increase. Activated pertussis toxin treatment of neutrophil plasma membranes inhibited by 72.5 +/- 14.3% the stimulation of GTPase activity induced by fMLP; however, activated cholera toxin had no effect on the inhibition of fMLP stimulation, suggesting a direct role for an Ni-like protein in the coupling process. In contrast to the lack of inhibition of fMLP stimulation by activated cholera toxin treatment of plasma membranes, both pertussis toxin and to a lesser extent cholera toxin treatment reduced fMLP, C5a, and LTB4 stimulation of the GTPase in sonicates prepared from pretreated whole cells. Pertussis toxin inhibited fMLP stimulation of the GTPase by 75 +/- 7%, C5a stimulation was inhibited by 83 +/- 13%, and LTB4 stimulation was inhibited completely. Sonicates prepared from neutrophils treated similarly with cholera toxin showed a smaller inhibition of GTPase activity (50 +/- 4% and 14 +/- 9% for fMLP and LTB4, respectively) with the exception of C5a, where CT inhibition (81 +/- 32%) equaled pertussis toxin inhibition. Similarly, pertussis toxin completely inhibited the release of the granule enzyme N-acetyl-glucosaminidase by all three chemoattractants, whereas cholera toxin, except with C5a stimulation, had little or no effect.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3018085 TI - Purification of the B lymphocyte receptor for the C3d fragment of complement and the Epstein-Barr virus by monoclonal antibody affinity chromatography, and assessment of its functional capacities. AB - The human C3d receptor (complement receptor type 2, CR2), that also serves as the B lymphocyte receptor for the Epstein-Barr virus, was purified from detergent lysates from the B lymphoblastoid cell lines, SB and Raji, by monoclonal antibody affinity chromatography using the anti-CR2 monoclonal antibody, HB-5. Relative to the concentration of cellular protein and receptor that was initially solubilized by detergent, the procedure provided a 37,000-fold purification with a 40-50% recovery of CR2. The purified receptor presented a single Coomassie blue-stained band when analyzed by SDS-PAGE, and it retained its function of binding to C3 Sepharose. The N-terminus of CR2 was blocked. The amino acid composition was significantly similar to that of the C3b/C4b receptor, factor H and C4 binding protein, suggesting that CR2 may be a member of this newly defined protein family. However, CR2 did not exhibit the regulatory functions of these proteins, namely, the decay dissociation of the classical or alternative pathway C3 convertases and serving as a cofactor for the cleavage of C3b. PMID- 3018084 TI - Epstein-Barr virus immortalization of normal cells of B cell lineage with nonproductive, rearranged immunoglobulin genes. AB - Most continuous cell lines derived by EBV immortalization of peripheral blood cells are composed of phenotypically mature B lymphocytes, and secrete Ig. Occasionally, EBV-immortalized cell lines have failed to secrete Ig. Expansion and characterization of one of these EBV-induced cell lines, VDS-O, showed that in addition to a lack of Ig secretion, surface and intracytoplasmic Ig were absent. Cell surface phenotyping revealed that VDS-O belongs to the B cell lineage, because it expresses the B cell restricted antigens B1 and B4, while it lacks T cell and monocyte-associated determinants. Analysis of the Ig gene organization in VDS-O revealed that the Ig genes are rearranged for both heavy (gamma) and light (kappa) chains. However, the expected gamma-heavy chain and/or kappa-light chain RNA species were not detected. These findings demonstrate the existence in normal peripheral blood of cells of B cell lineage susceptible to EBV immortalization that have Ig genes that are rearranged but are nonproductive. PMID- 3018083 TI - Lipopolysaccharide modulates receptors for leukotriene B4, C5a, and formyl methionyl-leucyl-phenylalanine on rabbit polymorphonuclear leukocytes. AB - Peripheral blood polymorphonuclear leukocytes (PMNL) isolated from rabbits after an i.v. injection of endotoxin exhibited decreased chemotactic migration in response to leukotriene B4 (LTB4) and C5a, but not N-formyl-methionyl-leucyl phenylalanine (fMLP), after endotoxin treatment. The binding of radiolabeled LTB4, fMLP, and C5a to isolated PMNL was assessed in order to determine whether altered receptor expression could account for the observed functional changes. Control PMNL expressed binding sites for fMLP, LTB4, and C5a similar to those previously characterized from human PMNL. Control PMNL expressed a single class of 14,600 +/- 2700 receptors for fMLP with a mean dissociation constant (Kd) of 2.0 +/- 0.6 nM at 0 degrees C, whereas two subclasses of binding sites were expressed for LTB4: 10,300 +/- 6800 high-affinity and 85,600 +/- 53,000 low affinity binding sites per PMNL with mean Kd for LTB4 of 0.75 +/- 0.43 nM and 70 +/- 58 nM (mean +/- SD, n = 5), respectively. Control PMNL bound [125I]-C5a in a dose-dependent and saturable manner at 24 degrees C. At saturating concentrations of C5a, PMNL obtained from control rabbits bound 270,000 +/- 50,000 molecules of [125I]-C5a with half-maximal binding occurring at [125I]-C5a concentrations of 5.5 +/- 1.9 nM. The binding of LTB4 and C5a to PMNL obtained 24 hr after an i.v. injection of endotoxin was markedly decreased compared with control PMNL. PMNL from endotoxin-treated rabbits exhibited 68% fewer high-affinity binding sites per PMNL for LTB4 and a 51% decrease in the amount of [125I]-C5a bound at saturating concentrations compared with control PMNL. There was no significant change in the Kd of the high-affinity binding sites for LTB4, no change in the Kd and number of the low-affinity binding sites for LTB4, and a small decrease in the apparent Kd for C5a to 3.3 +/- 1.1 nM. Even though the pretreatment with i.v. endotoxin did not alter chemotactic or degranulation responses elicited by fMLP, the endotoxin pretreatment induced an eightfold increase in the receptor density without altering the Kd for fMLP. Decreased receptor expression could account in large part for the decreased chemotactic responsiveness towards C5a and LTB4 induced by LPS. The finding that a substantial increase in receptors for fMLP need not be accompanied by a comparable functional change suggests that decreased efficiency in receptor coupling to intracellular biochemical events may also result from i.v. endotoxin. PMID- 3018086 TI - Skin levels of arachidonic acid-derived inflammatory mediators and histamine in atopic dermatitis and psoriasis. AB - Since the biochemical events leading to cutaneous inflammation in atopic dermatitis and psoriasis are unknown, we studied the levels of arachidonic acid derived mediators of inflammation as well as histamine in the suction blister fluid obtained from lesional and nonlesional skin of patients with these dermatoses. Mediator levels were determined radioimmunologically. Skin from healthy controls and uninvolved skin from patients contained very low or unmeasurable levels of the 5-lipoxygenase metabolite of arachidonic acid, leukotriene (LT) B4. In contrast, higher levels of LTB4-like immunoreactivity were detected in suction blister fluid from lesional atopic dermatitis skin, and even higher concentrations occurred in psoriasis lesions. LTB4-like immunoreactivity from atopic dermatitis suction blister fluid cochromatographed on reverse-phase high-pressure liquid chromatography with authentic LTB4, thus excluding cross-reaction of the LTB4-antibody with arachidonic acid or monohydroxyeicosatetraenoic acids. In contrast, suction blister concentrations of the cyclooxygenase metabolite of arachidonic acid prostaglandin (PG) E2 showed no significant differences between lesional and nonlesional patient skin and healthy control skin. PGD2 determined as a stable metabolite could not be detected in these samples. Histamine concentrations in lesional skin were within normal range. The elevated levels of the potent proinflammatory and immunomodulating mediator LTB4 could be involved in the pathogenesis of cutaneous inflammation in atopic dermatitis and psoriasis. In addition, they might explain the therapeutic efficiency of glucocorticosteroids, which among other actions inhibit the release of arachidonic acid from phospholipid stores by blocking the enzyme phospholipase A2. However, the specificity of disease expression in atopic dermatitis and psoriasis must be due to factors other than cutaneous LTB4 elevation. PMID- 3018087 TI - ATP-induced cell contraction with epidermolysis bullosa dystrophica recessive and normal dermal fibroblasts. AB - Human dermal fibroblasts cultured on glass coverslips and permeabilized by glycerol can be induced to undergo cell shrinkage by the addition of ATP in buffer containing calcium and magnesium. They reduce in size by 72% in 10 min. ATP-induced cell contraction is linked to an aggregation of cytoplasmic filaments as demonstrated by rhodamine-phalloidin F-actin staining. Before the addition of ATP, glycerinated cells have parallel arrangements of staining cytoplasmic filaments. Afterward, dermal fibroblasts from patients with epidermolysis bullosa dystrophica recessive (EBdr) show only a 10-20% reduction in cell size, and little F-actin aggregation staining can be demonstrated. Epidermolysis bullosa dystrophica recessive fibroblasts have been reported to produce excess prostaglandin E2 (PGE2) and cAMP. The preincubation of normal dermal fibroblasts for 24-30 h with 10 micrograms/ml PGE2, 10 micrograms/ml cholera toxin, or 1 mM dibutyl cAMP will reduce ATP-induced cell contraction to less than 20%. Treated cells showed little disruption of cytoplasmic F-actin. Epidermolysis bullosa dystrophica recessive fibroblasts preincubated with the cyclooxygenase inhibitor indomethacin at 10 micrograms/ml restored cell contraction to 74%. These treated cells also show aggregation of F-actin filaments. The process of ATP-induced cell contraction can be altered by the intracellular concentrations of cAMP, the levels of which are elevated in the fibroblasts in EBdr patients. A mechanism for cAMP inhibition of cell contraction is discussed. PMID- 3018089 TI - Seasonal rhythm of the plasma level of alpha-melanocyte stimulating hormone. AB - The present results indicate the presence of a seasonal rhythm of immunoreactive alpha-melanocyte stimulating hormone (alpha-MSH) in 20- to 40-year-old subjects of skin type I (light color of skin and eyes, red hair, no tanning after sun exposure) and skin type II (light color of skin, eyes, and hair, rare tanning) with raised levels of alpha-MSH in summer and low levels in winter. With increasing age of the investigated subjects, the seasonal rhythm seems to be lost. In subjects with skin type III (light skin, brown eyes and black hair, strong pigmentation after sun exposure) alpha-MSH shows only insignificant variations over the whole year. A seasonal rhythm of ACTH could not be demonstrated. A diurnal rhythm could be seen for ACTH, but not for alpha-MSH. To summarize, one can suppose that the seasonal rhythm of alpha-MSH is controlled by a varying UV exposure of the integument which is different over the whole year. PMID- 3018088 TI - Altered [125I]epidermal growth factor binding and receptor distribution in psoriasis. AB - Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that [125I]EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers. PMID- 3018090 TI - Cytochemical and biochemical localization of lipase and sphingomyelinase activity in mammalian epidermis. AB - Despite a wealth of new information on epidermal lipids and their role in permeability barrier function and desquamation, little is known about the location of the enzymes that regulate their catabolism. In this study we have localized lipase (triacylglycerol hydrolase) and sphingomyelinase in the outer epidermis simultaneously by cytochemical and cell fractionation techniques. Aldehyde-fixed tissues (100-microns slices) incubated in either Tween 85 or triolein plus taurocholate/calcium chloride-containing buffer, pH 7.2 or 4.5, were then exposed to lead to form insoluble soaps, and processed for electron microscopy. Simultaneously, cell homogenates and isolated lamellar body fractions were incubated with methylumbelliferyl oleate under similar conditions, with released, free methylumbelliferone serving as an index of lipase activity. On electron microscopy and cell fractionation, both lipase and sphingomyelinase were localized primarily to intercellular domains in the stratum corneum. In the stratum granulosum lipases were found, both ultrastructurally and biochemically, in lamellar bodies and ultrastructurally in both the perinuclear cistern and mitochondria. In summary, these studies: by demonstrating lipid-catabolic enzymes in the intercellular domains of the stratum corneum, lend further support to the 2-compartment model of the stratum corneum; provide new information about the location of lipid-catabolic enzymes in differentiating epidermis; and provide insights about how lipids are processed during permeability barrier formation and desquamation. PMID- 3018091 TI - Human monkeypox: a study of 2,510 contacts of 214 patients. AB - A study of 2,510 contacts of 214 patients with human monkeypox was conducted in Zaire from 1980 to 1984. Among the contacts of 130 primary cases of human monkeypox, a further 22 co-primary and 62 secondary cases were detected, and an additional fourteen people who had no evidence of clinical disease had positive serological results. A majority of the clinical and subclinical cases of monkeypox occurred in children less than 10 years of age. Immunity in vaccinated persons now appears to be waning because 16 overt cases occurred in contacts who had been vaccinated. The overall attack rate for contacts without a vaccination scar (7.2%) differed significantly from the attack rate for those who had been vaccinated in the past (0.9%). The attack rate for household contacts was significantly higher than that for other contacts, among both unvaccinated (four times higher) and vaccinated (seven times higher) household contacts. Many unvaccinated contacts living in the same household as the index case under conditions of maximum exposure, however, escaped not only the disease but also infection. PMID- 3018092 TI - Association of HTLV-III with Epstein-Barr virus infection and abnormalities of T lymphocytes in homosexual men. AB - Homosexual men were studied for associations among human T-lymphotropic virus type III (HTLV-III) infection, Epstein-Barr virus (EBV) infection, and T cell abnormalities. The presence of IgG antibody to EBV capsid antigen and antibody to EBV early antigen was significantly associated with augmented counts of suppressor T cells in healthy HTLV-III-seronegative men. HTLV-III-seropositive asymptomatic subjects had significantly enhanced titers of antibody to EBV and lower ratios of helper to suppressor T cells compared with HTLV-III-seronegative homosexual men. Of three men who seroconverted to HTLV-III, two had a greater than fourfold increase in titer of IgG antibody to EBV capsid antigen after seroconversion. These results suggest that the interaction of HTLV-III and EBV and their immunologic perturbations are significant in the natural history of this retrovirus infection in homosexual men. PMID- 3018094 TI - Deficient responses of pulmonary macrophages from healthy smokers to antiviral lymphokines in vitro. AB - The antiviral function of pulmonary macrophages obtained by broncholavage of healthy smokers and nonsmokers was studied. Compared with nonsmokers' cells, smokers' macrophages produced significantly more virus during in vitro infection with herpes simplex virus type 1 (HSV-1). Exposure of macrophages to either antiviral macrophage-activating factor or interferon-gamma for 20 hr before infection resulted in diminished production of HSV-1 by both types of macrophages. However, in contrast to smokers' cells, exposure of nonsmokers' macrophages to these antiviral lymphokines totally prevented viral replication. This difference could not be attributed to diminished adsorption of virus to smokers' macrophages or to an increased proportion of extracellular to intracellular virus in smokers' cell cultures. The effect of smoking on viral infectivity did not appear to be mediated by secretion of a soluble factor by the macrophage because incubation of nonsmokers' cells with supernatant from smokers' cell cultures did not affect the growth of HSV-1. PMID- 3018096 TI - Hospital-associated epidemic of pharyngitis and conjunctivitis caused by adenovirus (21/H21 + 35). PMID- 3018097 TI - Transformation-related growth factors and their receptors. AB - Cellular transformation may be accomplished in vitro and in vivo through the concerted action of growth factors and oncogenes. This association has demonstrated that malignant growth results from aberrations in growth factor signal transduction pathways that normally operate to control proliferation. Activation of genes that code for growth factors and/or their receptors provides tumor cells with potential mechanisms to maintain their proliferative state. Tumor cells have been shown to produce endogenous substances that augment their growth (autocrine stimulation), as well as responding to exogenous substances (paracrine stimulation). With solid tumor cells these responses have been shown to involve aberrant expression of growth factor and/or receptor genes. The study of the interrelationship of these various growth regulatory molecules is important not only in the identification of gene products essential to cellular proliferation, but also in providing clues as to what forces are driving tumor cell growth. PMID- 3018095 TI - The time from infection with human immunodeficiency virus (HIV) to the onset of AIDS. PMID- 3018093 TI - Use of bronchoalveolar lavage to diagnose acute diffuse pneumonia in the immunocompromised host. AB - We compared the diagnostic information obtained by bronchoscopy and needle aspiration of the lung with information obtained concurrently by open-lung biopsy in 15 marrow transplant recipients. Bronchoscopy included a wash, brush, and bronchoalveolar lavage. Laboratory evaluation included standard histological and cytological methods, specific immunofluorescence with monoclonal antibodies, and DNA hybridization to detect cytomegalovirus (CMV). Bronchoscopy permitted diagnosis of five of six patients with CMV pneumonia, whereas needle aspiration alone permitted diagnosis of only one of six. Bronchoalveolar lavage alone provided the diagnosis in four of six patients. Bronchoscopy also identified the bacterial component of a combined infection and identified one case of primary lung hemorrhage. Immunofluorescent staining and hybridization studies each diagnosed one case of CMV pneumonia not identified by standard techniques. The diagnostic sensitivity of the bronchoscopy was 89%, as compared with 17% for needle aspiration, and there were no complications. PMID- 3018098 TI - Early and late interstitial pneumonia following human bone marrow transplantation. AB - Interstitial pneumonia is a major determinant of early and late morbidity and mortality following bone marrow transplantation. Among 952 patients receiving allogeneic marrow grafts in Seattle, 35% developed interstitial pneumonia within 100 days of transplant. Development of early cytomegalovirus (CMV) or idiopathic interstitial pneumonia was infrequent in patients with aplastic anemia prepared only with cyclophosphamide. Use of total body irradiation (TBI) in the transplant preparation, increasing patient age, pretransplant seropositivity for CMV antibody and post-transplant development of graft-versus-host disease (GVHD) all increased the risk of CMV pneumonia. Late interstitial pneumonia was studied in patients with chronic GVHD. Among 198 patients with extensive chronic GVHD, 31 episodes of interstitial pneumonia (seven idiopathic, six CMV, six pneumocystis, five miscellaneous and four unknown causes, and three varicella-zoster) were observed 3-24 months after transplant. In untreated patients with chronic GVHD, 15% developed late interstitial pneumonia. Patients with chronic GVHD who received prednisone +/- azathioprine as immunosuppressive therapy and trimethoprim sulfamethoxazole for infection prophylaxis had an 8% incidence of interstitial pneumonia. Patients with chronic GVHD given immunosuppressive treatment without trimethoprim sulfamethoxazole prophylaxis had a 28% incidence of interstitial pneumonia. Trimethoprim sulfamethoxazole significantly reduced the incidence of late interstitial pneumonia in patients with chronic GVHD (p = 0.001). PMID- 3018099 TI - The biology of human cytomegalovirus infection after bone marrow transplantation. AB - Human cytomegalovirus (HCMV) infection remains the most common infectious cause of morbidity after bone marrow transplantation (BMT). In a prospective study of 127 BMT recipients who received blood cultures for HCMV between days 28 to 105 after marrow grafting, HCMV viremia occurred in 68 patients (53.4%). Twenty patients (15.7%) had one or two positive cultures, and 48 (37.7%) had greater than or equal to three positive cultures. Fifty-nine patients (46.4%) had no viremia. HCMV-associated interstitial pneumonia (HCMV-IP) occurred in one-third of the viremic patients. Quantitative measurements of infectious HCMV or of HCMV DNA in lung tissue were made to determine whether HCMV replication correlated with clinical disease. Using DNA probes, viral DNA was measured by dot-blot hybridization, and this correlated with infectious HCMV. However, neither HCMV DNA nor HCMV viral titer correlated with time from the onset of pneumonia to death. The hypothesis is presented that HCMV-IP is caused by immunologic events induced after HCMV infection. In this model HCMV alterations in recipient cell surfaces induce donor alloreactivity to minor histocompatibility differences and lead to the subsequent pneumonitis which we term HCMV-IP. This model suggests that prevention of HCMV-IP will require early use of antiviral therapy or late use of immune response modification. PMID- 3018101 TI - Prophylactic use of immunoglobulin in bone marrow transplantation. PMID- 3018100 TI - Therapeutic use of newer antiviral agents. PMID- 3018102 TI - Experience with sandoglobulin in bone marrow transplantation. PMID- 3018103 TI - Induction of xanthine oxidase and heme oxygenase and depression of liver drug metabolism by interferon: a study with different recombinant interferons. AB - Induction of xanthine oxidase in mouse liver by interferon (IFN) was studied with three different recombinant human leukocyte IFN molecules: IFLrA, IFLrD and the hybrid IFLrA/D(Bgl II). The ability of different IFN species to induce xanthine oxidase correlated with their ability to depress liver cytochrome P-450-dependent drug metabolism, supporting the hypothesis that reactive oxygen metabolites generated by xanthine oxidase might be responsible for this impairment of liver function by IFN. The antioxidant N-acetylcysteine protected in vivo against the depression of liver drug metabolism by IFLrA/D. IFLrA/D was also found to induce liver microsomal heme oxygenase, an effect that was probably secondary to the observed depression of cytochrome P-450. PMID- 3018104 TI - Recurrent intraoral herpes simplex virus infection. AB - A 48-year-old female had primary herpetic gingivostomatitis, followed by recurrent intraoral herpes simplex virus (HSV) disease; HSV isolates were obtained from the swabs of primary and recurrent lesions; restriction endonuclease cleavage analysis of the viral DNAs extracted from Vero cells infected with the HSV isolates according to the method of Hirt was carried out. The viral DNAs were cleaved by restriction endonucleases such as BamHI, KpnI and SalI and resolved by agarose gel electrophoresis, followed by staining with ethidium bromide. Consequently, their cleavage patterns were very similar to one another and were identified as HSV type 1. From these findings, it can be concluded that primary and recurrent lesions of this case are caused by the same virus. PMID- 3018105 TI - Pleomorphic adenoma of the buccal gland in a child. AB - The rare case of a 9-year-old Japanese girl with pleomorphic adenoma of the buccal mucosa is described. We review the literature of pleomorphic adenoma in children. PMID- 3018106 TI - [Pathological and clinical observations on precancerous lesions of the vulva]. AB - The vulvar epithelial lesions which have been subjected to histological examinations in Nagasaki University Hospital (1965-1985) and its 5 affiliated hospitals (1975-1985) included 133 cases in vulvar dystrophies, 72 in other benign lesions, 18 in squamous cell carcinoma in situ, 10 in vulvar Paget's disease, and 59 in invasive carcinoma. These lesions were studied pathologically and endocrinologically, and the pathogenesis of vulvar carcinoma and whose clinical problems were discussed. Serum hormone level in lichen sclerosus In a lichen sclerosus group, serum testosterone level was higher, as compared to that of a coetaneous group in the control, and androstenedione remained normal whereas 5 alpha-dihydrotestosterone (DHT) was decreased significantly. This led us to apply a 2% testosterone propionate ointment to the local skin, and which resulted in an apparent increase in DHT; the greater the increase multiple, the more the significance in clinical efficacy; suggesting that application of testosterone ointment may lead to the activation of 5 alpha-reductase and subsequently to an increase in DHT by which clinical symptoms also were alleviated. Papillomavirus antigen A test was carried out, for each lesion to search human papillomavirus (HPV) antigen, using avidin-bioton peroxidase complex (ABC) method with monoclonal antibody against papillomavirus. The results obtained revealed that antigens were negative on all in the case with lichen sclerosus and in a hyperplastic dystrophy case not associated with atypia, but cases judged as being positive included 9 of 14 with atypia and 5 of 6 with carcinoma in situ.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018107 TI - Construction of genomic libraries of mycobacterial origin: identification of recombinants encoding mycobacterial-specific proteins. AB - A complete genomic library from Mycobacterium vaccae (2785 recombinants) and a partial genomic library of M. leprae and BCG (300 and 1750 clones, respectively) were constructed in the plasmid pBR322. Bam HI was selected as the restriction endonuclease for obtaining DNA cleavage products. Evidence was obtained for limited expression of the cloned mycobacterial DNA inserts in Escherichia coli. A recombinant has been identified which codes for antigen immunoreactive with rabbit anti-M. leprae antibody but not with anti-H37Rv antibody. PMID- 3018108 TI - [High resolution electron microscopy study of crystallized fluorapatite using a silica-hydro gel method]. PMID- 3018109 TI - [A case of isolated adrenocorticotropic hormone deficiency with primary empty sella]. PMID- 3018110 TI - [A case of partial hypopituitarism with the normal size of empty sella following normal pregnancy and delivery--deficiency of adrenocorticotropic hormone and prolactin]. PMID- 3018112 TI - Neonatal cytomegalovirus infection. PMID- 3018111 TI - Platelet dysfunction in uremia. II. Correction by arachidonic acid of the impaired exposure of fibrinogen receptors by adenosine diphosphate or collagen. AB - Previous studies showed that platelets from patients with uremia have a marked decrease in their aggregation response to adenosine diphosphate (ADP) and collagen as single agents or as a pair. It is known that small amounts of arachidonic acid can enhance the sensitivity of platelets to concentrations of ADP or collagen that do not cause aggregation when used singly. Stimulation of platelets by certain agonists induces the formation of fibrinogen receptors on the platelet surface. The binding of fibrinogen that follows is essential for platelet aggregation. The platelet membrane glycoprotein IIb-IIIa complex appears to be the site of the fibrinogen receptor. Therefore, we investigated the binding of iodine 125-labeled fibrinogen to uremic platelets exposed to ADP, collagen, or arachidonic acid as single agents and as pairs. When aggregation and binding were studied in response to ADP, collagen, or the combination of ADP with collagen, uremic platelets had reduced aggregation and bound abnormally low amounts of fibrinogen. In contrast, platelets from patients with uremia bound as much 125I fibrinogen and aggregated as well as controls when ADP or collagen were used in combination with low concentrations of arachidonic acid. Studies with a monoclonal antibody (B 79.7) suggested that the number of glycoprotein IIb-IIa molecules is the same in uremic and normal platelets. We conclude that uremia impairs the exposure of fibrinogen receptors on platelets in response to ADP or collagen without affecting the glycoprotein IIb-IIa complex quantitatively. Correction by arachidonic acid of the impaired aggregation and exposure of fibrinogen receptors by ADP or collagen suggests that abnormal release of endogenous arachidonic acid plays a role in the dysfunction of platelets in uremia. PMID- 3018113 TI - Computerized axial tomography of the nose, paranasal sinuses, nasopharynx and pterygoid regions. PMID- 3018114 TI - Monocyte-derived growth factors for human tumor clonogenic cells. AB - Human peripheral blood monocytes secrete a soluble factor that enhances the ability of human epithelial tumor cell lines to clone in soft agar. Monocytes increased colony growth in a concentration-dependent manner, although inhibition of cell growth was observed in the presence of high concentrations of monocytes. Addition of indomethacin to monocytes did not abrogate this inhibition. Exposure of monocytes to endotoxin increased their ability to secrete stimulatory factors. Nonadherent lymphocytes were unable to support colony growth. Conditioned media from unstimulated monocytes also increased colony growth. Growth-promoting activity was detected in the media within the first 24 hr of culture, reaching a peak at 72 hr. Activity was not observed in monocyte lysates. Monocytes released this activity when cultured in the presence of both serum-free and serum containing media. The activity was nondialyzable, relatively heat stable, and failed to adhere to CM-Sephadex. The demonstration of a monocyte-derived factor that enhances growth of epithelial tumor colonies supports findings indicating that inflammatory products may enhance tumor cell growth in vitro. PMID- 3018115 TI - Host genetic control of retrovirus-induced leukemogenesis in the mouse: direct genetic and epistatic effects. PMID- 3018116 TI - On the divergence of members of a transposable element family. AB - Statistical properties of the amount of divergence of members of a transposable element family are studied. The analysis is based on the model proposed by Langley et al. describing the evolution of a family of selectively neutral transposable elements in a finite haploid population of size 2N. By considering the time back to the most recent common ancestor of two copies, both the probability of identity and the moments of the number of sites that differ between two sampled copies are obtained. Our analytic results are consistent with the numerical results of Ohta for a similar model. The effects of gene conversion are also examined. In agreement with Slatkin, we find that gene conversion has a minimal effect on the probability of identity providing that the rate of deletion is sufficiently large. PMID- 3018117 TI - An ultrastructural analysis of the inclusion body in the type II pneumocyte processed by rapid freezing followed by freeze-substitution--an autoradiographic study. PMID- 3018118 TI - Reduced affinity of intestinal receptors for 1,25-dihydroxycholecalciferol in phosphorus-deficient chicks. AB - The binding of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) to its intestinal receptor was studied in chicks fed a phosphorus (P)-deficient diet. The equilibrium association constant (Ka) was determined by Scatchard analysis. Association (Kass) and dissociation (Kdis) rate constants were determined in experiments on hormone uptake and release respectively. The Ka, determined at 4, 12 and 19 degrees C, decreased progressively during P deficiency, due to the decrease in Kass, but Kdis was not affected. During prolonged P deficiency the concentration of binding sites (Nmax) also decreased. Duodenal calcium-binding protein (CaBP) increased during 10 days of P deficiency and then decreased. The long-term decrease in receptor affinity and Nmax may account for the observed reduction in receptor occupancy and the decrease in the high level of intestinal CaBP stimulated during early P deficiency. The resulting decrease in Ca absorption may minimize the hypercalcaemia induced by the deficiency. PMID- 3018119 TI - Effect of human alpha-atrial natriuretic polypeptide on adrenocortical function in man. AB - To determine the effects of atrial natriuretic polypeptide (ANP) on plasma levels of ACTH, cortisol, aldosterone and dehydroepiandrosterone (DHEA), synthetic human alpha-ANP (h alpha-ANP) was infused i.v. into eight normotensive, disease-free volunteers, at a dose and duration previously found to be sufficient to produce apparent cardiovascular and renal effects. The mean basal concentration of plasma ACTH determined by radioimmunoassay was 18.2 +/- 3.1 ng/l. Plasma ACTH concentrations tended to be decreased during the infusion in all subjects. However, the change in plasma ACTH concentrations during infusion of h alpha-ANP was essentially the same as that during the infusion of saline. The mean plasma cortisol concentration was significantly suppressed from 25 to 40 min after the end of synthetic h alpha-ANP infusion. At 90 min after infusion, the mean plasma level of cortisol reverted to the pretreatment level. There was a non-significant increase in plasma renin activity following the infusion. The mean plasma aldosterone concentration was reduced by 15% (P less than 0.05) during the infusion and returned to preinfusion levels 10 min after termination of the infusion, after which the mean plasma concentration declined to the level seen during infusion. Administration of h alpha-ANP had no significant influence on plasma DHEA concentrations, but there was a tendency to decrease during the infusion. Our data suggest that synthetic h alpha-ANP inhibits adrenocortical steroidogenesis in man. PMID- 3018120 TI - Evidence for the presence of the pituitary insulin secretagogue beta-cell trophin in human plasma. AB - It has been demonstrated that the insulin secretagogue beta-cell-trophin, ACTH(22 39), is present in human plasma. The hormone, separated from plasma by affinity chromatography on a corticotrophin-like intermediate-lobe peptide antibody column, behaves similarly to synthetic beta-cell-trophin on a gel filtration column and on reverse-phase high-performance liquid chromatography. Sufficient amounts of the hormone were isolated from the plasma of two patients with Nelson's syndrome to demonstrate its biological activity on the perfused rat pancreas. PMID- 3018121 TI - Cooperation between cytotoxic and helper T lymphocytes in protection against lethal Sendai virus infection. Protection by T cells is MHC-restricted and MHC regulated; a model for MHC-disease associations. AB - The in vivo importance of class I MHC regulation of the Tc response to a natural pathogenic agent of high virulence was studied on the basis of our previous demonstration of a major difference in the capacity to generate a Sendai virus specific Tc response between C57BL/6 (B6, H-2b) mice and H-2Kb mutant B6.C-H-2bm1 (bm 1) mice. These two mouse strains differ from each other only in three amino acids in the crucial H-2Kb restriction element for this response. bm 1 mice, in contrast to B6 mice, are Tc nonresponders against this virus, but show Sendai specific T cell proliferation, antibody production, and DTH reactions, as well as NK cell activity, equal to those of B6 mice. B6, Sendai Tc-deficient bm 1 and T cell-deficient B6 nu/nu mice differ from each other in susceptibility to lethal pneumonia induced by i.n. inoculation of virulent Sendai virus. The lethal dose (LD50) in B6 mice averaged 152 TCID50, in bm 1 mice, 14 TCID50 and in B6 nu/nu mice 0.5 TCID50. The importance of Tc was also shown by the complete protection of B6 nu/nu mice against infection with a lethal virus dose by i.v. injection of a Sendai virus-specific, IL-2-dependent and H-2Kb-restricted B6 Tc clone. In vivo protection by this Tc clone was H-2Kb-restricted. Apart from Tc, an important role for virus-specific Th cells is evident from the difference in susceptibility between bm 1 and B6 nu/nu mice. This conclusion was supported by the demonstration that the mean survival time of B6 nu/nu and bm 1 nu/nu mice could be significantly prolonged, in an I-Ab-restricted manner, by the injection of in vitro-propagated, Sendai-specific B6 or bm 1 Th clones after a lethal dose of Sendai virus, and by the demonstration that inoculation of these Th clones provided help to virus-specific Tc by means of IL-2 production. Strikingly, Th and Tc cooperate in anti-Sendai virus immunity, since permanent survival of lethally infected nu/nu mice was only achieved by inoculation of a mixture of Tc and Th clones or a mixture of a Tc clone and rIL-2. This study provides a unique model for the study of MHC-disease associations. PMID- 3018122 TI - Herpesvirus saimiri strain 11 immortalizes a restricted marmoset T8 lymphocyte subpopulation in vitro. AB - Herpesvirus saimiri induces a fatal lymphoproliferative syndrome in a variety of New World primate species. We now show that cell lines derived from PBL of the common marmoset by in vitro-immortalization with H. saimiri strain 11 represent a remarkably restricted lymphocyte population. These cell lines have NK cell function, phenotypically express both suppressor/cytotoxic (T8) and NK cell (NKH1)-associated antigens, and express a T cell receptor. This subpopulation of lymphocytes is a very minor population of cells in the peripheral blood of common marmosets (less than or equal to 3%). The specificity in the interaction between H. saimiri strain 11 and a subpopulation of common marmoset lymphocytes represents an example of a restricted viral lymphotropism and may have important implications for the disease induced by this virus in New World monkeys. PMID- 3018123 TI - Extended left hepatectomy for interlobar hepatoblastoma--a case report. PMID- 3018124 TI - The complete DNA sequence of varicella-zoster virus. AB - The entire DNA sequence of varicella-zoster virus (VZV) was determined using the M13-dideoxynucleotide technology. The genome is variable in size, but the sequence which was obtained comprises 124884 bp. Analysis of the sequence indicated that the genome contains 70 genes distributed about equally between the two DNA strands. The genes are organized compactly, but regions of overlap between protein-coding regions are not extensive. Many of the genes are arranged in 3'-coterminal families, and at least one is spliced. The discerned organization of VZV genes and that deduced for herpes simplex virus type 1 (HSV 1) from published transcript mapping data indicate that these two members of the Alphaherpesvirinae are very similar in gene layout. Comparisons of the predicted amino acid sequences of VZV proteins with those available for HSV-1 proteins generally suggest evolution from an ancestral genome, and allow the functions of several VZV genes to be deduced, although limited regions where the genomes differ in functional organization were also identified. PMID- 3018125 TI - Analysis of varicella-zoster virus DNAs of clinical isolates by endonuclease HpaI. AB - The DNAs of 20 strains of varicella-zoster virus (VZV) isolated from epidemiologically unrelated individuals, and of 15 strains isolated from vesicles of vaccinees with varicella or zoster after vaccination, were compared by restriction enzyme cleavage using HpaI. Differences were found in the sizes of the HpaI-F, -G and -K fragments of the wild strains. The gel migration patterns of the HpaI-F and -G fragments, but not of the HpaI-K fragment, were polymorphic in the different strains isolated from the vaccinees. The effects of serial passages in vitro and in humans on the genome stability of VZV were investigated by HpaI analysis. The DNA profiles of the HpaI-K fragments from six isolates recovered from room-mates infected in a single outbreak were identical, but the mobilities of their HpaI-F and -G fragments varied. The DNA profiles of the Oka vaccine virus after 10 and 85 passages in human embryo cells differed only in the HpaI-F fragment. The profiles of these fragments in DNA derived from two isolates obtained at different times from a vaccinee with varicella followed by zoster were compared with those of the Oka (parental) and Oka (vaccine) strains, and identical results were obtained for the two viruses. In addition, the same DNA profiles of HpaI fragments were obtained from three sequential isolates from one person and also from two isolates from another with varicella and zoster. Thus, it was concluded that: three variable fragments (HpaI-K, -F and -G) were not changed in the DNAs of isolates derived from the same patient; HpaI-K was stable both on passage in vitro and after human transmission in the case of the same outbreak, but was different among all wild-type strains isolated in epidemiologically unrelated outbreaks; HpaI-F was very unstable both on passage in vitro and in human infections by either vaccine or wild-type strains; HpaI-G was not influenced by passage in vitro but varied among wild-type strains. Using physical maps of VZV DNA established by others, three variable regions on the viral genome were identified. One was located near the 0.16 coordinate, which is covered by HpaI-K (variable region I, VRI). Another was represented by HpaI-F (VRII), the most unstable fragment, and mapped at about the 0.35 coordinate. The third was VRIII near the right terminus, covered by HpaI-G. PMID- 3018126 TI - The effect of triterpenoid compounds on uninfected and herpes simplex virus infected cells in culture. II. DNA and protein synthesis, polypeptide processing and transport. AB - The effect of the triterpenoid drugs carbenoxolone sodium (CBX) and cicloxolone sodium (CCX) on DNA and protein synthesis in uninfected and herpes simplex virus (HSV)-infected BHK-21 or Flow 2002 cells has been studied. No consistent effect of 500 microM-CBX or 300 microM-CCX on HSV DNA synthesis was identified. With 300 microM-CCX, some inhibition of cellular DNA synthesis was observed, but this was relatively slight. The restriction enzyme digest profiles of HSV DNA from drug treated cells appeared normal. The synthesis of several HSV-specified polypeptides was much reduced by treatment with CBX or CCX and the transport of certain proteins between the nuclear and cytoplasmic compartments of infected cells was also affected. CBX or CCX treatment strongly inhibited post translational glycosylation and sulphation of both host- and HSV-specified proteins, but the phosphorylation of only a few proteins appeared affected. The drugs induced quantitative changes in the synthesis of some BHK cell polypeptides, but these were not considered important. CCX treatment of Flow 2002 cells, however, induced the synthesis of several new polypeptides, some of which had the same apparent molecular weights as identified Flow 2002 cell stress proteins. When treated with concentrations greater than 50 microM-CCX, the plasma membranes of both uninfected and HSV-infected cells became increasingly leaky. The SDS-PAGE polypeptide profile of purified virus particles made in BHK cells treated with 300 microM-CCX differed markedly from that synthesized under drug free conditions. The greatly reduced amount of infectious virus made in cells treated with 300 microM-CCX was more thermolabile at 42 degrees C than virus produced in the absence of the drug. Our results indicate that the antiviral activity of the triterpenoid drugs is non-specific and operates by interfering with or changing the normal function of cell membranes so that cells, although retaining their viability, can no longer assemble the virus components efficiently into infectious particles. As a consequence, the population of virus particles made is of inferior quality. While we have no evidence that there is also a specific anti-HSV effect, this possibility cannot yet be ruled out. PMID- 3018127 TI - Zosteriform spread of herpes simplex virus type 2 genital infection in the guinea pig. AB - A model of herpes simplex virus type 2 (HSV-2) infection was developed in the guinea-pig that permits investigation of the role of neural spread of virus in the pathogenesis of genital skin disease. After HSV-2 inoculation of an abraded area lateral to the external genital skin, virus was first detected in the ipsilateral (to the inoculation site) peripheral nerves or genital skin on day 2 or 3 post-inoculation followed by detection in the ipsilateral dorsal root ganglia and spinal cord on day 3 or 4. Virus also spread to the contralateral dorsal root ganglia, contralateral peripheral nerves and contralateral genital skin on day 4 or 5 although lesions were only rarely detected on the contralateral side. Genital skin lesions first developed on day 5 and were followed by the development of lesions along the medial aspect of the ipsilateral hindlimb. Virus was first detected in the vaginal vault on day 4 or 5. The development of lesions appeared to be unrelated to vaginal virus replication but was associated with the recovery of virus from peripheral nerves. Recurrent genital skin lesions were seen more commonly on the inoculated side than the contralateral side. Since the development of skin lesions appeared to result from virus emerging from nerve endings, an event similar to the terminal event that is believed to be involved in the development of recurrent lesions, we now have a model which may be useful in further exploring the pathogenesis of herpetic disease. PMID- 3018128 TI - Genetically determined difference in the antiviral action of alpha/beta interferon in cells from mice resistant or susceptible to herpes simplex virus type 2. AB - Resistance of mice to infection with herpes simplex virus type 2 (HSV-2) is genetically determined. Embryonic cells from susceptible BALB/c and resistant C57BL/6 mice were equally sensitive to infection with HSV-2 as judged by plaque area, plaquing efficiency, endpoint titration and virus yield. Cells from C57BL/6 mice showed a higher sensitivity than cells from BALB/c mice to the protective action of two preparations of alpha/beta interferon against challenge with HSV-2. This was evident both from c.p.e. inhibition and yield reduction experiments. The difference in sensitivity was dependent on virus dose and was greatest (up to 50 fold) with low virus doses. An analysis of the genetics of the alpha/beta interferon sensitivity in cells from embryos of parental mice and embryos derived from reciprocal matings between HSV-2-resistant and -susceptible mice suggested that interferon sensitivity is inherited as a co-dominant autosomal trait. The induction of the interferon-induced enzyme 2'-5'-oligoadenylate synthetase was also different in cells from the two mouse strains, since significant levels were only detected in cells from C57BL/6 mice. It is suggested that differential interferon sensitivity of cells from HSV-2-resistant and -susceptible mice might be a factor of importance for the course of the infection. PMID- 3018130 TI - Nucleotide sequence analysis of the haemagglutinin-neuraminidase gene of Newcastle disease virus. AB - The nucleotide sequence of the haemagglutinin-neuraminidase (HN) gene of Newcastle disease virus (NDV) has been determined. The HN gene is 2031 nucleotides long, approximately 13.5% of the viral genome. The nucleotide sequence contains a single long open reading frame which would encode a protein of 577 amino acids, with a mol. wt. of 63,149. This is in good agreement with estimates of the molecular weight of the unglycosylated HN protein. Analysis of the amino acid sequence reveals six potential glycosylation sites and shows the major hydrophobic region to be close to the N terminus. This provides evidence for the N-terminal attachment of HN to the viral membrane. The hydrophilic nature of the extreme N-terminal amino acids suggests the absence of a cleaved signal sequence. Analysis of the long non-coding region at the 3' end of the mRNA encoded by the HN gene of NDV suggests a possible explanation for the origin of HN0 in extremely avirulent strains of NDV. There are regions of high homology between the deduced amino acid sequence of the NDV HN glycoprotein and the HN glycoproteins of two other paramyxoviruses, Sendai virus and simian virus 5 (SV5). An alignment of the HN amino acid sequences of these viruses shows 32% of amino acid residues are conserved between NDV and SV5, and 23% between NDV and Sendai virus. In contrast, only very limited homology is found between NDV HN and the influenza virus glycoproteins. PMID- 3018129 TI - The expression of human papillomavirus type 18 E6 protein in bacteria and the production of anti-E6 antibodies. AB - Human papillomavirus type 18 (HPV-18) has recently been closely linked with human malignant cervical lesions. The early region of the genome of the related bovine papillomavirus (BPV) has been shown to be important for the production of the transformed phenotype. BPV E6 has been shown to be a transforming protein. We report the primary structure of the HPV-18 E6 open reading frame and its predicted amino acid sequence. Both E6 protein and E6-beta-galactosidase fusion protein were synthesized in bacteria. Antisera were raised against the E6-beta galactosidase fusion protein and against an E6 N-terminal peptide which was 14 amino acids long. We show that these antisera reacted on Western blots against E6 synthesized in bacteria. The HPV E6 antigen and antibodies described here will be useful in understanding HPV expression and its association with human malignancies and may also be diagnostically useful. PMID- 3018131 TI - Immunological relationships between mumps virus and parainfluenza viruses studied with monoclonal antibodies. AB - The immunological relationships between mumps virus and parainfluenza viruses were investigated with 74, 78 and 80 previously developed monoclonal antibodies directed against five major structural proteins of mumps virus, Sendai virus (a murine parainfluenza type 1 virus) and parainfluenza type 3 virus. These monoclonal antibodies were reacted with the three viruses, with parainfluenza type 2 virus and with Newcastle disease virus (NDV) in ELISA and immunofluorescence (IF) tests. In addition, immunoprecipitation tests with [35S]methionine-labelled extracellular virions were carried out with cross reacting monoclonal antibodies. None of all 232 monoclonal antibodies against the three viruses cross-reacted with either parainfluenza type 2 virus or NDV in ELISA and IF tests. In the collection of 74 mumps virus monoclonal antibodies, three directed against the nucleocapsid (NP) protein, polymerase protein, and fusion protein cross-reacted with Sendai virus. Two Sendai virus monoclonal antibodies directed against two different epitopes of the haemagglutinin neuraminidase (HN) protein cross-reacted with parainfluenza type 3 virus. Six other Sendai virus monoclonal antibodies directed against four different epitopes of the HN protein and one directed against the NP protein cross-reacted with mumps virus. Eight out of 80 monoclonal antibodies directed against parainfluenza type 3 virus cross-reacted with Sendai virus. One was directed against the HN protein, four were directed against a minimum of two epitopes of the matrix protein and three were directed against three different epitopes of the NP protein. The different cross-reactions found show that Sendai virus is antigenically related to both mumps virus and parainfluenza type 3 virus. In contrast, no antigenic relationship could be demonstrated between mumps virus and parainfluenza type 3 virus. PMID- 3018132 TI - Detection and characterization of infectious DNA intermediates of a primary foamy virus. AB - DNA from human T-lymphoid (Molt-4) and hamster kidney (BHK-21) cells infected with the T-lymphotropic simian foamy virus LK-3 was shown to be infectious, when assayed by transfection of BHK-21 cells. The proviral genome was further characterized by blot hybridization to a specific cDNA probe, which had been prepared by reverse transcription in vitro using viral RNA and RNA-dependent DNA polymerase present in cytoplasmic extracts of infected BHK-21 cells. This probe hybridized to a DNA species of 14 kbp in extracts from LK-3-infected diploid human fibroblasts, Molt-4 and BHK-21 cells, whereas no hybridization occurred with DNA from the respective uninfected controls. No integrated proviral DNA could be demonstrated, and the 14 kbp DNA was shown not to represent circular DNA. The patterns of restriction endonuclease and S1 nuclease fragments indicated a unique configuration of linear double-stranded DNA containing a single-stranded section separating two subunits one of which may be sufficient to transmit LK-3 by transfection with DNA. PMID- 3018134 TI - Delta-9-tetrahydrocannabinol enhances release of herpes simplex virus type 2. AB - This study was undertaken to determine the effect of micromolar concentrations of delta-9-tetrahydrocannabinol (Delta-9-THC) on herpes simplex virus type 2 (HSV-2) replication in vitro. Virus-infected Vero cells pretreated for 24 h with 10(-5) M or 10(-6) M-Delta-9-THC yielded 100-fold increases in infectious extracellular virus. Transmission electron microscopy of drug-treated cells revealed plasma membrane dissolution, distension of the smooth and rough endoplasmic reticulum, and the appearance of macrovacuoles in the cytoplasm containing aggregates of virus. These results suggest that Delta-9-THC enhances the release of HSV-2 by perturbing cellular membranes in virus-infected cells. PMID- 3018133 TI - Detection of human papillomavirus type 16 DNA and evidence for integration into the cell DNA in cervical dysplasia. AB - The presence of human papillomavirus (HPV) type 16 DNA in biopsies from precancerous lesions and from early lesions of human cervical cancer, and the integration of virus DNA into host cell DNA were analysed by dot blot and Southern blot hybridizations. HPV 16 DNA was detected in 23% of mild dysplasias, 32% of moderate dysplasias, 55% of severe dysplasias and 62% of carcinomas in situ by dot blot hybridization. Digestion of the DNA with restriction enzymes PstI and BamHI followed by Southern blot analysis revealed the presence of some typical restriction fragments of HPV 16 DNA in most virus-positive samples. In addition, we detected submolar fragments which might represent virus-cell junction sequences in 86% of dysplasias, suggesting that the integration of HPV 16 DNA could occur in the precancerous stage. PMID- 3018135 TI - Reduced temperature can block different glycoproteins at different steps during transport to the plasma membrane. AB - Reduced temperature has been shown to block the cell surface expression of Sendai virus haemagglutinin-neuraminidase (HN) and fusion (F0) glycoproteins at different steps of their intracellular transport. At 20 degrees C, HN was confined to the rough endoplasmic reticulum or cis Golgi compartment, while F0 acquired complete resistance to digestion by endo-beta-N-acetylglucosaminidase-H and therefore was blocked at a more distal location in the pathway of cell surface expression. The significance of these results for different pathways of transport to the cell surface is discussed. PMID- 3018136 TI - In vitro stimulation of presensitized mouse spleen cells with poliovirus type 1, Mahoney, and enhancement of poliovirus-specific hybridomas. AB - In vivo immunization of BALB/c mice with poliovirus type 1, strain Mahoney, or with its purified polypeptides resulted in 0.2 to 0.5 antigen-specific hybridoma microcultures per 10(6) spleen cells. Stimulation of spleen cells from mice immunized with poliovirus or with its polypeptides in vitro with poliovirus 6 days prior to fusion with the myeloma cells led to a six- to 20-fold increase in the number of positive microcultures, i.e. after stimulation the yield of poliovirus-specific hybridomas was up to 3.8 antigen-specific microcultures per 10(6) spleen cells. The in vitro stimulation of spleen cells primed in vivo was demonstrated by the detection of poliovirus-specific antibody-producing cells 6 days after in vitro cultivation in the presence of poliovirus as antigen. Only spleen cells stimulated under these conditions in vitro gave rise to specific antibody-producing cells and yielded antigen-specific hybridomas after somatic hybridization. PMID- 3018137 TI - Characterization of monoclonal antibodies against human rotavirus hemagglutinin. AB - Three monoclonal antibodies capable of specifically inhibiting hemagglutination of human rotavirus were produced. Their hemagglutination inhibition (HI) activity was specific to the homologous strain (KUN) used for immunization. The monoclonal antibodies with HI activity were highly effective in neutralizing the infectivity of the KUN strain. These antibodies reacted with Vp80, and 80,000 molecular weight (MW) protein present in the viral outer shell. It was confirmed by immunoblotting assay with the monoclonal antibodies that the antigenic site of human rotavirus hemagglutinin (HA) resides on Vp80 and on its smaller trypsin cleavage products Vp30 (MW 30,000) and Vp24 (MW 24,000). Immunofluorescence studies using the antibodies revealed that the HA antigen of the KUN strain developed at the final stage of virus maturation. PMID- 3018139 TI - The association of rhinoviruses with lower respiratory tract disease in hospitalized patients. AB - An analysis of 32 hospitalized infants and children from whom rhinoviruses were isolated in our diagnostic laboratories in 1982-83 suggests that these agents are associated with lower respiratory tract disease with focal findings in susceptible patients. In 23 cases, an acute lower respiratory disease was the cause for admission, while nine patients were cultured after new respiratory symptoms developed during hospitalization. Presenting signs and symptoms included cough (23), fever (19), rhinorrhea (19), respiratory distress (14), and decreased feeding (15). Seventeen of 25 chest x-rays showed new focal abnormalities. Twenty five patients had a significant underlying disease including seven with malignancies, six with respiratory tract abnormalities, and four with congenital heart lesions. Six of the remaining seven patients were less than 2 1/2 months of age. In no cases were significant bacterial or fungal pathogens isolated; two did have concomitant viral isolates. Rhinoviruses in the appropriate clinical setting are associated with significant pulmonary disease. PMID- 3018138 TI - Testing for antibodies to AIDS-associated retrovirus (HTLV-III/LAV) by indirect fixed cell immunofluorescence: specificity, sensitivity, and applications. AB - Seropositivity to the AIDS-associated retrovirus, HTLV-III/LAV, has profound implications. Simple and reliable tests are needed to detect such antibodies. A rapid, sensitive indirect immunofluorescence assay (IFA) on acetone-fixed virus producing CEM/LAV-N1 cells was adapted for detection of human antibodies to HTLV III/LAV. Specific and nonspecific patterns of of immunofluorescent reactivity were easily distinguished, and results paralleled those obtained by Western blotting and radioimmunoprecipitation (RIP), indicating that there is no need to confirm IFA positivity. In contrast, the commercial enzyme-linked immunosorbent assay (ELISA) was less reliable. False positives occurred with sera from seven hemophiliacs that were negative on Western blots, and false-negative reactions were observed on two occasions. These involved low-titer AIDS-patients' sera that were positive on Western blots, and from one of which virus was successfully isolated. Our results emphasize the requirement for confirmatory assays when the ELISA test is used for primary screening of sera for antibodies to HTLV-III/LAV. The IFA method is especially well-suited to quantitative analysis of serum antibody levels. Our data suggest that serum antibody titers rise as disease progression occurs, ultimately falling as severe complications ensue. It is suggested that in laboratories where the demand for HTLV-III/LAV antibody testing is not excessive (1,000-2,000 sera/month), IFA could serve as the only serological assay for both screening and epidemiological purposes. PMID- 3018140 TI - Protein kinase in nondiabetogenic coxsackievirus B4. AB - Alkali-dissociated, purified preparations of prototype coxsackievirus B4 release a protein kinase that catalyzes the incorporation of gamma-phosphate from 32P labeled ATP into three virus capsid proteins (VP1, VP3, VP4), several additional proteins of the particle, and exogenous acceptor proteins. Using protamine sulfate as an acceptor protein, we detected nearly 20-fold more enzyme activity in membrane-bound virions (MBV) than in virions of the virus. The activity in the MBV is cyclic nucleotide-independent, divalent cation-dependent, and has a pH optimum of 8.0. Phosphoserine is labeled with 32P. The enzyme activity sediments at about 5S and is separated into at least two peaks of heterogeneous proteins by ion-exchange chromatography. The patterns of phosphorylation by these enzyme peaks are somewhat similar. Coxsackievirus-associated protein kinase appears to be located internally in the virus and may be host-cell-coded. The enzyme appears to be lacking in a variant of the virus that produced diabetes in mice. PMID- 3018141 TI - An immunoglobulin G capture assay (GACRIA) for anti-HTLV III/LAV and its use as a confirmatory test. AB - An immunoglobulin G (IgG) antibody capture assay (GACRIA) methodologically distinct from other assays for the detection of antibodies to human T lymphotropic virus type III/lymphadenopathy-associated virus (anti-HTLV III/LAV) was developed and used to test 1,055 serum samples previously screened by a competitive assay (COMPRIA). Eight hundred and twenty-three (78%) sera were positive and 129 (12.2%) negative in both assays. The coefficient of correlation between the two assays was 0.87, and COMPRIA appeared more sensitive than GACRIA. On retesting 103 sera that gave initially conflicting results, 81 discrepancies were resolved, 77 because of a change in the COMPRIA result. The 22 persistently discrepant samples gave an equivocal or positive result by COMPRIA and were negative by GACRIA. Thirteen of these 22 were positive in a commercial ELISA (ELAVIA). Twenty of them were also tested by Western blot; all were reactive with at least two HTLV III/LAV protein bands. The persistently discrepant samples were of four types: laboratory control sera (n = 2); naturally occurring weakly reactive samples (n = 5); samples that were anti-HTLV III/LAV IgG negative but IgM positive (n = 5; all five individuals from whom these samples were drawn were symptomatic); samples from one laboratory that were probably accidentally contaminated with anti-HTLV III/LAV-positive serum (n = 10). It was concluded that GACRIA for anti-HTLV III/LAV is specific and adequately sensitive and, in conjunction with other assays, is a useful confirmatory test whose format could readily be changed to ELISA. PMID- 3018142 TI - Activation of alpha 2-adrenoreceptors enhances haloperidol-induced suppression of operant behavior. AB - Inhibition of catecholamine synthesis by alpha-methyl paratyrosine (alpha-MT) was previously shown to potentiate the behavioral suppression caused by dopamine receptor antagonists. This effect of alpha-MT is in all probability due to inhibition of the compensatory increase in dopamine turnover induced by the dopamine receptor antagonists. In the present study we investigated the effect of the alpha 2-adrenoreceptor agonist clonidine on the haloperidol-induced suppression of food-reinforced lever-pressing behavior (fixed ratio 40:1) in rats. Small behaviorally inactive doses of clonidine were found, in analogy with alpha-MT, to enhance the haloperidol-induced suppression of the lever-pressing behavior. The haloperidol-induced increase in dopamine synthesis (measured as the accumulation of DOPA after inhibition of aromatic amino acid decarboxylate) was antagonized by clonidine in the striatum as well as in the dopamine rich limbic regions. Prazosin, a selective alpha 1-adrenoreceptor antagonist had no effect on the clonidine induced behavioral changes. Idazoxane, a selective alpha 2 adrenoreceptor antagonist, counteracted both the behavioral and biochemical effects of clonidine, indicating that these effects of clonidine are mediated via its action on alpha 2-adrenoreceptors. The present findings provide support for the notion that alpha 2-adrenoreceptors may participate in the regulation of nigro-striatal as well as meso-limbic dopaminergic activity. It is suggested that alpha 2-adrenoreceptor agents, especially in combination with classical antipsychotics, might be of therapeutic value in the treatment of disorders associated with abnormal dopaminergic activity. PMID- 3018143 TI - Effect on hypothalamic self-stimulation of the novel beta-carbolines ZK 93 426 (a benzodiazepine receptor antagonist) and ZK 91 296 (a putative partial agonist). AB - Low doses (300 micrograms/kg-1.0 mg/kg) of the novel beta-carboline, ZK 91 296, a putative agonist at the benzodiazepine receptor, produced a significant increase in the rate of variable-interval self-stimulation responding, similar to that found with typical benzodiazepines. This effect was blocked by simultaneous administration of the specific benzodiazepine-receptor antagonists Ro 15-1788 (2.0 mg/kg), and ZK 93 426 (10 mg/kg). Neither antagonist, ZK 93 426 (100 micrograms/kg-10 mg/kg) or Ro 15-1788 (2.0 mg/kg), had any effect on self stimulation when given alone. Unlike all benzodiazepine-receptor agonists previously tested, higher doses of ZK 91 296 did not depress self-stimulation response rates, even at a dose-level 100 times greater than the maximally stimulant dose. It is uncertain why ZK 91 296 lacks depressant effects: available evidence does not conclusively favour any single current explanation, but is consistent with it acting as a "partial" agonist. PMID- 3018144 TI - Involvement of postsynaptic alpha 1- and alpha 2-adrenoceptors in the flexor reflex activity in the spinal rats. AB - Clonidine (0.2 mg/kg i.v.) and St 587 (1 mg/kg i.v.), selective agonists of alpha 2- and alpha 1-adrenoceptors, respectively, increased the flexor reflex amplitude in the spinal rat. Yohimbine and rauwolscine, alpha 2-adrenoceptor antagonists, inhibited dose-dependently the effect of clonidine but not of St 587. However, prazosin and clozapine, alpha 1-adrenoceptor antagonists, diminished the action of both agonists in a dose-dependent manner. After central chemosympathectomy caused by 6-OH-DA or DSP-4 the response to clonidine did not differ from that in the sham-operated animals. In 6-OH-DA treated rats yohimbine (1 mg/kg i.v.) antagonized the effect of clonidine to the same extent as it did in unlesioned animals. It is concluded that the results are functional evidence that alpha 1- and alpha 2-adrenoceptors are present in the rat spinal cord, and stimulation of each receptor increases flexor reflex activity. PMID- 3018145 TI - Normal patterns of melatonin levels in the pineal gland and body fluids of humans and experimental animals. AB - The normal 24 hours patterns of melatonin were described in experimental animals and man. The rhythms are generated by neuronal activity originating in the suprachiasmatic nuclei of the hypothalamus; the activity of these nuclei are synchronized by the prevailing light:dark environment as perceived by the retinas. As a consequence of this synchronization, pineal melatonin levels are always high at night and low during the day, irrespective of whether the animal being studied is nocturnally or diurnally active. The rhythmic production of melatonin in the pineal gland is interrupted by the exposure of animals, or man, to light during the normal dark period. Several factors may change the 24 hours pattern of melatonin production. Because daylength changes throughout the year there are seasonal effects on the melatonin rhythm. Also, age is a major factor in determining the ability of the pineal gland to metabolize serotonin to melatonin. In advanced age pineal melatonin production is severely limited in both the human and in experimental animals. A variety of endocrine manipulations have been tested in terms of their impact on the ability of the pineal to produce melatonin. The majority of these have been rather innocuous although hypophysectomy does significantly curtail the synthesis of melatonin. To date, melatonin cycles have been usually described in rodents maintained under controlled environmental conditions in the laboratory; it can only be assumed that these rhythms are similar in animals in their natural habitat. PMID- 3018146 TI - Pineal calcification: its mechanism and significance. AB - On the basis of conventional transmission electron microscopy and ultracytochemical reactions for demonstration of calcium, for glucose-6 phosphatase, and for Ca2+-ATPase, intracellular and extracellular mineralization foci in the superficial pineal gland of the Mongolian gerbil (Meriones unguiculatus) have been described. The initial intracellular calcification sites occur in the cytoplasmic matrix, vacuoles, mitochondria and the endoplasmic reticulum of large clear pinealocytes. These loci, and particularly those within the cytoplasmic matrix, transform into acervuli by a further addition of hydroxyapatite crystals. The cells gradually degenerate, die, break down, and the acervuli reach the extracellular space. It has been suggested that the reason for a rise in intracellular calcium levels could be the incapacity of Ca2+-ATPase to eliminate this cation from the cell, so that the hypercalcemic intracellular milieu becomes favourable for the initial crystallization. The primary extracellular mineralization sites occur in the calcium-rich flocculent material. The mineralization process in the gerbil pineal gland is interpreted as a histophysiological phenomenon intimately related to the metabolic activity of the pineal gland. PMID- 3018148 TI - Effects of octopamine on miniature excitatory junction potentials from developing and adult moth muscle. AB - Intracellular recordings of excitatory junction potentials (EJPs) and miniature EJPs (MEJPs) were made from the dorsal longitudinal muscle of Manduca sexta to determine the sites of action of octopamine. MEJPs increased in amplitude and frequency as the moth developed during the 3 days before eclosion. DL-Octopamine (5 X 10(-6) M) increased the amplitude of excitatory junction potentials in both immature moths (one day before eclosion) and adults. Octopamine (10(-5) M) also increased the amplitude and frequency of MEJPs from immature animals (one and two days before eclosion) but had the opposite effect on adults and pharate adults ready to eclose. Treatment with octopamine (10(-5) M) resulted in a decrease in input resistance and a hyperpolarization in both immature and adult muscle fibers. The results suggest that octopamine acts both presynaptically and postsynaptically but that the increase in the amplitude of the evoked response is due primarily to influences on presynaptic processes. PMID- 3018147 TI - Puberty in the rat: modulation by melatonin and light. AB - Although a role has been found for melatonin in species which have a seasonal reproductive cycle, very little is known on the role of melatonin in species such as the rat where seasonal cycles are a minor component of reproduction. But the rat is a photosensitive species, because it responds to changes in the lighting environment. Females do not become quiescent but they can have irregular estrous cycles, another way of controlling population dynamics. In this species, exogenous melatonin can exert an antigonadotropic action, providing conditions which apply to other species are also respected for the rat: melatonin must be given at the right time of the day, 9 to 12 hours after the onset of light, and at a given period of life, before the onset of puberty. Melatonin probably acts on the pattern of GnRH pulsatile secretion and the subsequent alterations of the hypothalamic-gonadal axis differ according to the sex of the animal. Endogenous melatonin rhythms are modified by the lighting environment, and results obtained with exogenous melatonin suggest that they could be one of the factors controlling timing of sexual maturation. PMID- 3018150 TI - Serotonin immunoreactivity of neurons in the gastropod Aplysia californica. AB - Serotonergic neurons and axons were mapped in the central ganglia of Aplysia californica using antiserotonin antibody on intact ganglia and on serial sections. Immunoreactive axons and processes were present in all ganglia and nerves, and distinct somata were detected in all ganglia except the buccal and pleural ganglia. The cells stained included known serotonergic neurons: the giant cerebral neurons and the RB cells of the abdominal ganglion. The area of the abdominal ganglion where interneurons are located which produce facilitation during the gill withdrawal reflex was carefully examined for antiserotonin immunoreactive neurons. None were found, but two bilaterally symmetric pairs of immunoreactive axons were identified which descend from the contralateral cerebral or pedal ganglion to abdominal ganglion. Because of the continuous proximity of this pair of axons, they could be recognized and traced into the abdominal ganglion neuropil in each preparation. If serotonin is a facilitating transmitter in the abdominal ganglion, these and other antiserotonin immunoreactive axons in the pleuroabdominal connectives may be implicated in this facilitation. PMID- 3018149 TI - Effects of octopamine and forskolin on excitatory junction potentials of developing and adult moth muscle. AB - Intracellular recordings were made from the dorsal longitudinal muscle of Manduca sexta to determine the effects of development and octopamine on the excitatory junction potential (EJP) produced in response to electrical stimulation of the motor nerve. Observations were made on pharate moths during the last 3 days before eclosion and on adults. In saline, the highest values for EJP amplitude and maximum rate of rise and for resting membrane potential are reached on the nineteenth day of the pupal period, the day the animal ecloses; adult values are slightly lower. In animals of all ages tested, DL-octopamine (5 X 10(-6) M) increases EJP amplitude and maximum rate of rise. Increases in amplitude are greater in animals at stage day 17 and 18 than in animals at stage day 19 and adult. Octopamine has no effect on EJP rise time (onset to peak) or recovery time (peak of EJP to 70% recovery). Octopamine causes a hyperpolarization of about 6 mV. The results show that developmental changes in synapse properties are paralleled only in part by changes induced by octopamine. Both development and octopamine increase EJP amplitude and maximum rate of rise, and neither alter rise time. EJP recovery time changes with development but not in response to octopamine. Forskolin (10(-4) M) mimics the effects of octopamine on day 17 animals. EJP amplitude and maximum rate of rise are increased by forskolin, and rise time and recovery time are unaffected. Forskolin, like octopamine, causes a 6 mV hyperpolarization of the muscle fiber. These results suggest that octopaminergic modulation at the Manduca sexta dorsal longitudinal neuromuscular junction may be mediated by changes in intracellular levels of cyclic AMP. PMID- 3018152 TI - Hemes and hemeproteins, 2: The pH-dependent equilibria of microperoxidase-8 and characterization of the coordination sphere of Fe(III). AB - Titration of the monomeric heme octapeptide from horse heart cytochrome c, microperoxidase-8 (MP-8) from pH 1 to pH 13 in 20% (v/v) methanol-water solutions, mu = 0.1, at 25 degrees C shows three reversible concentration independent pKs (4.43 +/- 0.09; 8.90 +/- 0.03; 10.48 +/- 0.09) which are ascribed to successive proton loss from the conjugate acid of His (and its coordination to Fe(III)), bound H2O, and from bound His to form an imidazolate complex, respectively. The equilibrium constant for coordination of imidazole between pH 5.5 and 7.0 is independent of pH (logK = 4.45) which proves that His-18 is coordinated to Fe(III) in aqueous solution. PMID- 3018151 TI - Hemes and hemoproteins. 1: Preparation and analysis of the heme-containing octapeptide (microperoxidase-8) and identification of the monomeric form in aqueous solution. AB - The heme-octapeptide from cytochrome c, Microperoxidase-8 (MP-8), was prepared by peptic and tryptic digestion of horse heart cytochrome c and purified by gel permeation chromatography in about 50% yield. Conditions for the identification of MP-8 by TLC and analysis by HPLC are described. Study of the concentration dependence of the absorption spectrum showed that at concentrations of less than or equal to 2.5 X 10(-5) M in aqueous solution at pH 7, 25 degrees C and mu = 0.1, MP-8 exists as an equilibrium mixture of monomers and dimers with KD = 1.17 +/- 0.02 X 10(5) M-1, decreasing to 1.21 +/- 0.02 X 10(4) M-1 and 2.16 +/- 0.21 X 10(3) M-1 in 20% and 50% (v/v) methanol:water mixtures, respectively. Comparison of the Soret region spectrum of monomeric MP-8 with other hemoproteins suggests that it is six-coordinate in aqueous solution with water and His as axial ligands. PMID- 3018153 TI - Characterization of the A1 adenosine receptor-adenylate cyclase system of cerebral cortex using an agonist photoaffinity ligand. AB - Endogenous adenosine acting via A1 adenosine receptors is capable of inhibiting adenylate cyclase activity and neurotransmitter release in the brain. In this report, we describe the synthesis and attributes of a new series of A1 adenosine receptor agonists. One of these, [125I]N6-2-(4-amino-3-iodophenyl)ethyladenosine, can be used as a radioligand and another, [125I]N6-2-(4-azido-3 iodophenyl)ethyladenosine, as a photoaffinity probe. The unlabeled ligand, N6-2 (4-aminophenyl)ethyladenosine, and its iodinated product are full agonists, inhibiting cyclic AMP production in rat cerebral cortex membranes to the same extent as the prototypic A1 agonist N6-R-1-phenyl-2-propyladenosine. These new ligands are not substrates for adenosine deaminase. The new photoaffinity azide described here labels an Mr 38,000 protein that displays all the pharmacological characteristics expected of the A1 adenosine receptor. This is the same molecular weight protein previously described using a cross-linking radioligand. This new azide compound demonstrates a 15-fold higher efficiency of incorporation, making it the photoaffinity probe of choice for tissues containing low concentrations of A1 adenosine receptors. PMID- 3018154 TI - Uptake and metabolism of [3H]adenosine by Aplysia ganglia and by individual neurons. AB - [3H]Adenosine was taken up and metabolized by isolated ganglia of the marine mollusc Aplysia californica. After 2 h, most of the radioactivity was recovered as metabolites, including ATP, ADP, and AMP, as well as the deaminated products, inosine, hypoxanthine, and uric acid. Little remained in the form of adenosine. These pathways were not uniformly distributed among various tissue elements. In most individual neurons, inosine and its breakdown products were the principal metabolites of [3H]adenosine, whereas ATP and other nucleotides predominated in the connective tissue sheath. Endogenous levels of ATP, ADP, AMP, and adenosine in ganglia, sheath, and individual neurons were also determined using a fluorimetric-HPLC assay. The concentrations of the nucleotides were quite uniform in sheath and among the individual neurons assayed (1-5 pmol/microgram of protein); however, concentrations of adenosine were considerably higher in neurons than in the sheath. PMID- 3018155 TI - Calcium-activated neutral proteinase of human brain: subunit structure and enzymatic properties of multiple molecular forms. AB - Calcium-activated neutral proteinase (CANP) was purified 2,625-fold from postmortem human cerebral cortex by a procedure involving chromatography on diethylaminoethyl (DEAE)-cellulose, phenyl-Sepharose, Ultrogel AcA-44, and DEAE Biogel A. The major active form of CANP exhibited a molecular weight of 94-100 kilodaltons (Kd) by gel filtration on Sephacryl 300 and consisted of 78-Kd and 27 Kd subunits. Two-dimensional gel electrophoresis resolved the small subunit into two molecular species with different isoelectric points. CANP degraded most human cytoskeletal proteins but was particularly active toward fodrin and the neurofilament protein subunits (145 Kd greater than 200 Kd greater than 70 Kd). The enzyme required 175 microM Ca2+ for half-maximal activation and 2 mM Ca2+ for optimal activity toward [methyl-14C]azocasein. Other divalent metal ions were poor activators of the enzyme, and some, including copper, lead, and zinc, strongly inhibited the enzyme. Aluminum, a neurotoxic ion that induces neurofilament accumulations in mammalian brain, inhibited the enzyme 47% at 1 mM and 100% at 5 mM. A second CANP form lacking the 27-Kd subunit was partially resolved from the 100-Kd heterodimer during DEAE-Biogel A chromatography. The 78 Kd monomer exhibited the same specific activity, calcium ion requirement, pH optimum, and specificity for cytoskeletal proteins as the 100-Kd heterodimer, suggesting that the 27-Kd subunit is not essential for the major catalytic properties of the enzyme. The rapid autolysis of the 27-Kd subunit to a 18-Kd intermediate when CANP is exposed to calcium may explain differences between our results and previous reports, which describe brain mCANP in other species as a 76 80-Kd monomer or a heterodimer containing 76-80-Kd and 17-20-Kd subunits. The similarity of the 100-Kd human brain CANP to CANPs in nonneural tissues indicates that the heterodimeric form is relatively conserved among various tissues and species. PMID- 3018156 TI - Biochemical and immunocytochemical evidence for a deficiency of normal interfascicular oligodendroglia in the CNS of the dysmyelinating mutant (md) rat. AB - Carbonic anhydrase was assayed and carbonic anhydrase and 5'-nucleotidase were localized in the CNS of myelin-deficient mutant rats and normal littermates. The carbonic anhydrase specific activities were reduced by 61% and 29% in the mutants' forebrains and cerebella, respectively, and the total carbonic anhydrase activity in the spinal cords was reduced by 35%. Immunostained cells were found in gray matter from both normal and mutant rats, but, in the mutants, there was a marked deficiency of interfascicular oligodendrocytes in the regions that are normally occupied by white matter. It is suggested that a developmental study could indicate the step(s) at which normal differentiation of interfascicular oligodendroglia is blocked in this mutant. PMID- 3018157 TI - Characterization of brain calpains. AB - A new, simple one-step procedure [Karlsson et al. Biochem. J. 231, 201-204 (1985)] for the separation of calpains I and II was used prior to the characterization of these enzymes from rabbit brain, using alkali-denatured casein as the substrate. Enzyme activity was dependent on Ca2+ ions and free-SH groups and was maximal around pH 7.4. Incubation of calpains I and II with Ca2+ in the absence of substrate led to a rapid loss of enzyme activity. Enzyme activity was linear at room temperature and millimolar Ca2+ concentrations. However, when incubation of calpain I was performed with micromolar Ca2+ concentrations at room temperature proteolytic activity exhibited a lag period of approximately 10 min. This activation period was not as evident with calpain II. PMID- 3018159 TI - Modulatory effects of thyroxine treatment on central and peripheral benzodiazepine receptors in the rat. AB - The effects of 10 days of D-thyroxine (T4) treatment on central benzodiazepine (BZ) receptors in the brain and on peripheral-type BZ binding sites in the heart, kidney, and testis of rats were studied. The experimental hyperthyroidism resulted in an increase in the density of cortical central BZ receptors, without any alteration of the affinity of the receptors to [3H]flunitrazepam. The increase in cortical central BZ receptors was also accompanied by the up regulation of peripheral BZ binding sites in the heart, kidney, and testis. The affinity of the peripheral BZ binding sites for the ligand [3H]PK 11195 was not affected by T4 treatment in any of these three organs. The increase in the density of brain cortical central BZ receptors was less prominent than the increase in the peripheral BZ binding sites. The modulatory effect of T4 treatment on central and peripheral BZ receptors might be attributed to the direct interaction of the thyroid hormone at these sites or might reflect a physiological compensatory adaptation mechanism to thyrotoxicosis associated with hypermetabolism, anxiety, and stress. PMID- 3018158 TI - Effect of phospholipases on chronic opiate action in neuroblastoma X glioma NG108 15 hybrid cells. AB - Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with opiate agonist resulted in loss of the acute opiate inhibition of adenylate cyclase activity with a concomitant increase in the enzymatic activity observable on addition of the antagonist naloxone. The role of membrane lipids in the cellular expression of these chronic opiate effects was investigated by the hydrolysis of phospholipids with various lipases. Treatment with phospholipase C from Clostridium welchii produced an enzyme concentration-dependent decrease of prostaglandin E1-stimulated adenylate cyclase activity in control or etorphine treated (1 microM for 4 h) hybrid cells. In addition, incubation of hybrid cells with phospholipase C concentrations of greater than or equal to 0.5 U/ml completely abolished the compensatory increase in adenylate cyclase activity after chronic opiate treatment. This attenuation of the increase in adenylate cyclase activity by phospholipase C could be prevented by inclusion of phosphatidylcholine but not of phosphatidic acid during the enzymatic incubations. The specificity of the phospholipids involved in expression of the chronic opiate effect could be demonstrated further by the absence of effect exhibited by phospholipase C from Bacillus cereus and phospholipase D. Hydrolysis of the acyl side chains of phospholipids with phospholipase A2 did not alter the chronic opiate effect after removal of lysophosphatides with bovine serum albumin. Because the guanylylimidodiphosphate- and NaF-sensitive adenylate cyclase activities were not affected by these phospholipase treatments, the expression of the compensatory increase in adenylate cyclase activity is mediated via an increase in the coupling between hormonal receptor and adenylate cyclase with the participation of the polar head groups of the phospholipids and not the hydrophobic side chains. PMID- 3018160 TI - Alpha 1-adrenergic receptor-mediated downregulation of angiotensin II receptors in neuronal cultures. AB - Previous evidence has suggested that brain catecholamine levels are important in the regulation of central angiotensin II receptors. In the present study, the effects of norepinephrine and 3,4-dihydroxyphenylethylamine (dopamine) on angiotensin II receptor regulation in neuronal cultures from rat hypothalamus and brainstem have been examined. Both catecholamines elicit significant decreases in [125I]angiotensin II-specific binding to neuronal cultures prepared from normotensive rats, effects that are dose dependent and that are maximal within 4 8 h of preincubation. Saturation and Scatchard analyses revealed that the norepinephrine-induced decrease in the binding is due to a decrease in the number of angiotensin II receptors in neuronal cultures, with little effect on the receptor affinity. Norepinephrine has no significant actions on [125I]angiotensin II binding in cultures prepared from spontaneously hypertensive rats. The downregulation of angiotensin II receptors by norepinephrine or dopamine is blocked by alpha 1-adrenergic and not by other adrenergic antagonists, a result suggesting that this effect is initiated at the cell surface involving alpha 1 adrenergic receptors. This is further supported by our data indicating a parallel downregulation of specific alpha 1-adrenergic receptors elicited by norepinephrine. In summary, these results show that norepinephrine and dopamine are able to alter the regulation of neuronal angiotensin II receptors by acting at alpha 1-adrenergic receptors, which is a novel finding. PMID- 3018161 TI - Pitfalls in the assay of cyclic AMP-dependent protein kinase activity in microsacs from Torpedo marmorata. AB - Endogenous phosphorylation of the nicotinic acetylcholine receptor (nAChR) in microsacs from Torpedo marmorata was found to be affected by several reagents commonly used in the preparation of cyclic AMP (cAMP)-dependent protein kinases and in its activity determination. The presence of a Na+,K+-ATPase inhibitor is essential to avoid a rapid depletion of ATP, even when a membrane fraction highly enriched in the nAChR is used. The presence of the thiol reducing agent dithiothreitol was found to abolish the cAMP dependence of nAChR phosphorylation, whereas the less potent reagent 2-mercaptoethanol did not affect the assay. Concentrations in the millimolar range of the chelators EDTA and EGTA were found to inhibit nAChR phosphorylation effectively. This inhibition was not due to a withdrawal of Ca2+ by the chelators, but rather to a reversible inhibition by the Mg2+ complexes. These observations may explain some of the discrepancies found in the literature concerning endogenous and exogenous nAChR phosphorylation. PMID- 3018162 TI - Effects of prenatal phenobarbital on benzodiazepine receptor development. AB - Phenobarbital (PB) was administered to pregnant mice during days 9-21 of gestation. Forebrain and cerebellar [3H]flunitrazepam ([3H]FLU) binding was assayed in the offspring at birth and at 21 days of age. Prenatal treatment produced a decrease in the number (Bmax) of [3H]FLU receptors in both the forebrain and cerebellum at birth. A small decrease in the [3H]FLU dissociation constant (KD) values in the forebrain was also detected at birth, but no changes were seen in the [3H]FLU KD values in the cerebellum. No changes were observed in forebrain and cerebellar [3H]FLU Bmax or KD values at 21 days of age, indicating that the effects of prenatal exposure to PB on [3H]FLU binding are eliminated during the postnatal development of the forebrain and cerebellum. The receptor affinity for the triazolopyridazine CL 218,872, which distinguishes the type I and type II benzodiazepine (BDZ) receptors, was not altered by prenatal PB treatment. The coupling of the BDZ receptor to the gamma-aminobutyric acid and pentobarbital binding sites was unaffected by exposure to PB in utero. PMID- 3018163 TI - Solubilization of the benzodiazepine/gamma-aminobutyric acid receptor complex: comparison of the detergents octylglucopyranoside and 3-[(3-cholamidopropyl) dimethylammonio]1-propanesulfonate (CHAPS). AB - Octylglucopyranoside (OCTG) was three times more efficient than 3-[(3 cholamidopropyl)-dimethylammonio]1-propanesulfonate (CHAPS) in solubilizing the benzodiazepine (BDZ)/gamma-aminobutyric acid (GABA) receptor complex from rat cerebellar synaptic membranes. OCTG-solubilized receptor preparations had ligand binding characteristics that were significantly different from those of the CHAPS solubilized receptors. The inclusion of phospholipids in the solubilization media improved the binding characteristics of both soluble receptor preparations and appeared absolutely necessary for the maintenance of chloride facilitation of flunitrazepam (FNZ) binding to OCTG-solubilized receptors. FNZ and ethyl-beta carboline-3-carboxylate bound to OCTG-solubilized preparations with equilibrium dissociation constants of 2.2 nM and 1.6 nM, respectively, and chloride (150 mM) and GABA (100 microM) + chloride facilitated the binding of FNZ by 15% and 55%, respectively; these ligand binding characteristics are similar to those of membrane-located BDZ receptors. Cartazolate, a pyrazolopyridine that facilitated the binding of FNZ to membrane-located and CHAPS-solubilized receptors, did not facilitate FNZ binding to OCTG-solubilized receptors. These results are discussed in terms of an interaction between the membrane lipid phosphatidylserine (PS) and cartazolate; PS appears to have the capacity to inhibit the effects of cartazolate on FNZ binding. Storage of the soluble receptor preparations for 24 h at 4 degrees C resulted in the loss of several characteristic BDZ receptor binding properties. Incorporation of the OCTG-solubilized receptor complex into liposomes prevented these losses but this procedure did not protect the CHAPS solubilized receptors. We conclude that OCTG may have some advantages over CHAPS as the detergent of choice for the solubilization and reconstitution in liposomes of a functional BDZ/GABA receptor-chloride ionophore complex. PMID- 3018164 TI - Alpha 1-adrenergic receptors in neuronal cultures from rat brain: increased expression in the spontaneously hypertensive rat. AB - Neuronal cells in primary culture from 1-day-old brains of normotensive, Wistar Kyoto strain (WKY) and spontaneously hypertensive (SH) rats have been utilized to study the expression of alpha 1-adrenergic receptors. Binding of a selective alpha 1 antagonist, [125I]2-[beta-(4-hydroxy-3-iodophenyl)-ethylaminomethyl] tetralone ([125I]HEAT) to neuronal membranes prepared from primary brain cultures of WKY and SH rats was 75-80% specific, rapid, and time-dependent although the binding was 1.5-2 times higher in neuronal membranes from SH rat brain cultures. Kinetic analysis of the association and dissociation data demonstrated no significant differences between rat strains. Competition-inhibition experiments provided IC50 values for various antagonists and agonists in the following order: prazosin less than phentolamine less than yohimbine less than phenylephrine less than norepinephrine less than propranolol, suggesting that [125I]HEAT bound selectively to alpha 1-adrenergic receptors. Scatchard analysis of the binding data provided straight lines for both strains of rats, indicating the presence of a homogeneous population of binding sites. It also showed that the increase in the binding in neuronal cells from SH rat brains over those from normotensive WKY controls was a result of an increase in the number of alpha 1-adrenergic receptors. Incubation of neuronal cultures from both strains of rats with phenylephrine, an alpha 1-adrenergic agonist, caused a time- and dose-dependent decrease in the binding of [125I]HEAT. This decrease was due to a decrease in the number of alpha 1-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018165 TI - Neurotensin stimulates inositol phospholipid metabolism and calcium mobilization in murine neuroblastoma clone N1E-115. AB - Murine neuroblastoma cells (clone N1E-115) possess neurotensin receptors that mediate cyclic GMP synthesis. Because of the hypothesized relationship between phospholipid metabolism, intracellular Ca2+, and cyclic GMP synthesis, we determined with these cells the effects of neurotensin on 32P labeling of phospholipids, release of inositol phosphates, and intracellular Ca2+ (as determined with the use of Quin-2, a fluorescent probe sensitive to free Ca2+ levels). Neurotensin stimulated incorporation of 32P into phospholipids, especially phosphatidylinositol and phosphatidate. Neurotensin also stimulated the release of [3H]inositol phosphates with an EC50 of about 1 nM. Mean basal Ca2+ concentration in these cells was 134 nM and this level was increased in a rapid and dose-dependent manner by neurotensin, with an EC50 of 4 nM. Since the EC50 for neurotensin in stimulating cyclic GMP synthesis is 1.5 nM and the KD for binding of [3H]neurotensin at 0 degrees C is 11 nM, all these different effects appear to be shared proximal consequences of neurotensin receptor activation. PMID- 3018166 TI - Biochemical characterization of alpha-adrenergic receptors in human brain and changes in Alzheimer-type dementia. AB - Using ligand binding techniques, we studied alpha-adrenergic receptors in brains obtained at autopsy from seven histologically normal controls and seven patients with histopathologically verified Alzheimer-type dementia (ATD). Binding of the alpha-adrenergic antagonists [3H]prazosin and [3H]yohimbine to membranes of human brains exhibited characteristics compatible with alpha 1- and alpha 2-adrenergic receptors, respectively. Binding of both ligands was saturable and reversible, with dissociation constants of 0.15 nM for [3H]prazosin and 5.5 nM for [3H]yohimbine. [3H]Prazosin binding was highest in the hippocampus and frontal cortex and lowest in the caudate and putamen in the control brains. [3H]Yohimbine binding was highest in the nucleus basalis of Meynert (NbM) and frontal cortex and lowest in the caudate and cerebellar hemisphere in the control brains. Compared with values for the controls, [3H]prazosin binding sites were significantly reduced in number in the hippocampus and cerebellar hemisphere, and [3H]yohimbine binding sites were significantly reduced in number in the NbM in the ATD brains. These results suggest that alpha 1- and alpha 2-adrenergic receptors are present in the human brain and that there are significant changes in numbers of both receptors in selected regions in patients with ATD. PMID- 3018167 TI - On the concept of third occipital headache. AB - One of the putative causes of headache is osteoarthritis of the C2-3 zygapophysial joint. A technique for blocking the third occipital nerve which innervates this joint was devised and used as a screening procedure for headache mediated by this nerve. Seven out of ten consecutive patients presenting with suspected cervical headache were found to suffer pain mediated by the third occipital nerve and stemming from a C2-3 zygapophysial joint. Because third occipital headache may be indistinguishable clinically from tension or other forms of headache, third occipital nerve blocks are advocated as means of establishing this largely unrecognised diagnosis. PMID- 3018168 TI - Slowed motor conduction in lumbosacral nerve roots in cauda equina lesions: a new diagnostic technique. AB - New techniques have been developed for the electrophysiological assessment of patients with suspected cauda equina lesions using transcutaneous spinal stimulation (500-1500 V: time constant 50 microseconds) to measure motor latencies to the external and sphincter and puborectalis muscles from L1 and L4 vertebral levels. These latencies represent motor conduction in the S3 and S4 motor roots of the cauda equina between these levels. Similarly motor latencies can be recorded from spinal stimulation to the anterior tibial muscles (L4 and L5 motor roots). Transrectal stimulation of the pudendal nerves is used to measure the pudendal nerve terminal motor latency. In 32 control subjects, matched for age and sex, mean motor latencies from L1 and L4 spinal stimulation were 5.5 +/- 0.4 ms and 4.4 +/- 0.4 ms (mean + SD). In the 10 patients with cauda equina disease including ependymoma, spinal stenosis, arachnoiditis and trauma, these latencies were 7.2 +/- 0.8 ms and 4.6 +/- 0.9 ms, a significant increase in the L1 latency. The L1/L4 latency ratios to the puborectalis muscle were 1.36 +/- 0.09 in control subjects and 1.72 +/- 0.13 in cauda equina patients. Pudendal nerve terminal motor latencies were normal in eight of the 10 patients with cauda equina disease. The single fibre EMG fibre density in the external and sphincter muscle (normal, 1.5 +/- 0.16) was increased in patients with cauda equina lesions (1.73 +/- 0.28), but was increased more than two standard deviations from the mean only in three patients. This increase in fibre density was not of diagnostic value since it was also found in two of the four patients with low back pain. Slowing of motor conduction in the cauda equina is thus a useful indication of damage to these intraspinal motor roots. These investigations can be used in the selection of patients for myelography, and to follow progress in patients managed conservatively. PMID- 3018169 TI - The relationship of peripheral trauma and pain to dystonia. PMID- 3018170 TI - Possible peripheral neuropathy at high altitude. PMID- 3018172 TI - Late responses as aids to diagnosis in peripheral neuropathy. PMID- 3018171 TI - Sensorimotor neuropathy and cisplatin and adriamycin toxicity. PMID- 3018173 TI - Mechanism of phagocytosis by Schwann cells. AB - 33B rat Schwannoma cell line is known to exhibit phagocytic properties analogous to those of normal Schwann cells. The mechanism of phagocytosis by this cell line was investigated by studying the effect of known modulators of phagocytosis on the uptake of latex particles by these cells. Treatments which block energy production of the host cell, such as incubation at 4 degrees C and treatment with sodium azide, markedly inhibited the phagocytosis of latex particles by these cells. Phagocytosis was dose-dependently, and completely, inhibited by cytochalasin B, demonstrating an important role of microfilaments. Colchicine produced a minor inhibition of phagocytosis only at the highest concentration (10(-3) M) tested, suggesting that intact microtubules are not crucial for latex phagocytosis. Dibutyryl cyclic AMP was without any effect on the phagocytosis. Thus, latex phagocytosis by rat Schwannoma cells is an active, energy-dependent process requiring intact microfilaments with only a minor dependence on microtubules. PMID- 3018174 TI - Progressive axonopathy: an inherited neuropathy of boxer dogs. Quantitative and morphometric analysis of the peripheral nerve lesion. AB - Previous studies have described and illustrated the lesions in the peripheral nerves in progressive axonopathy, an inherited neuropathy of Boxer dogs. The present paper assesses these changes using quantitative techniques. Cervical and lumbar nerve roots and tibial, phrenic and medial cutaneous radial nerves have been studied in affected and age-matched normal dogs aged 2 months to 3 years. The dorsal and ventral nerve roots, and to a lesser extent the proximal nerves, contain a proportion of swollen myelinated axons whereas in the middle and distal nerves the larger diameter fibres fail to develop to their expected maximum calibre. The unmyelinated axons remain the same size as those in normal dogs. Myelin sheath changes, with attenuation or loss of the sheath and/or remyelination, become increasingly prevalent through the course of the disease, always maintaining a proximal to distal decrease in their frequency. Quantification indicates that, particularly in the ventral roots, many axons have disproportionately thin sheaths with shortened internodes. Axonal degeneration and regeneration increase in frequency in the distal nerves as the disease progresses. The cervical ventral roots prove an exception in that they contain large numbers of regenerating clusters at most stages. It is suggested that in progressive axonopathy an axonal transport failure may occur in the roots leading to the axonal swellings, as a result of which a developmental hypoplasia occurs in the more distal, larger diameter fibres. The prominent, but unevenly distributed, myelin sheath changes indicate a severe disturbance in axon-sheath cell inter-relationships. PMID- 3018175 TI - The role of 2-chloroprocaine and sodium bisulfite in rat sciatic nerve edema. AB - In order to evaluate the possible mechanisms of local anesthetic toxicity, the rat sciatic nerve was exposed to various solutions including Nesacaine (containing the antioxidant sodium bisulfite), 2-chloroprocaine in the Nesacaine vehicle (0.2% sodium chloride), 0.2% sodium bisulfite in 0.2% sodium chloride, or 0.2% sodium chloride alone. All solutions were pH balanced between 2.9 and 3.2. Forty-eight hours (h) following extraneural administration of 1 ml volumes, significant edema was produced by all solutions containing 3% 2-chloroprocaine, but not with 0.2% bisulfite in sodium chloride or with sodium chloride alone. Intrafascicular administration of five to ten microliter volumes of these solutions produced edema at 48 h in all cases, but the highest levels were observed with Nesacaine and the lowest levels with 0.2% bisulfite. The results of this study implicate the local anesthetic 2-chloroprocaine in the production of nerve edema, which is inconsistent with other reports that the toxicity of Nesacaine-CE can be attributed to the antioxidant bisulfite. PMID- 3018176 TI - Membrane alterations in cerebral cortex when using PIPES buffer. AB - When used for vascular perfusion of brain, 0.1 M PIPES-buffered 3% glutaraldehyde resulted in the formation of expanded, vesicle-filled cell processes limited by multiple membrane layers. These structures, termed multivesicular myelin figures and interpreted as artefacts, were most common in layer 2 of the cerebral cortex. When cacodylate or phosphate buffer was used instead of PIPES buffer in the primary fixative, such structures were not seen. The use of a more concentrated initial aldehyde fixative, PIPES-buffered, markedly reduced the size and numbers of these artefacts when compared to PIPES-buffered 3% glutaraldehyde only. Slowing the initial perfusion rate increased the size and frequency of occurrence of multivesicular myelin figures with PIPES buffer when compared to optimum perfusions. Prolonged initial exposure to PIPES buffer by using it to wash out the blood and then perfusing with fixative 5 min later did not increase the number or size of multivesicular myelin figures but did reduce the multivesicular nature of the artefacts. We suggest that the non-toxic nature of PIPES buffer allowed the formation of these membranous artefacts, while phosphate and cacodylate interfered with the cellular activity during the process of fixation. PMID- 3018177 TI - Cytochemical identification of cerebral glycogen and glucose-6-phosphatase activity under normal and experimental conditions. II. Choroid plexus and ependymal epithelia, endothelia and pericytes. AB - Intracellular glycogen and glucose-6-phosphatase (G6Pase) activity were identified cytochemically within epithelia of the choroid plexus and ependyma of the cerebral ventricles including the median eminence and area postrema, the cerebral endothelium and pericytes from control, salt-stressed and fasted adult mice. Identification of glycogen was obtained by employing osmium tetroxide potassium ferrocyanide and the periodic acid-thiocarbohydrazide-silver protein technique as ultrastructural contrast stains. A lead-capture method was used to localize G6Pase activity with glucose-6-phosphate or mannose-6-phosphate as substrate. Cerebral G6Pase functions predominantly as a phosphohydrolase to convert glucose-6-phosphate to glucose. Some glucose-6-phosphate in vivo may be derived from the breakdown of glycogen stores. Within the sampled cell types, presumptive glycogen appeared as electron-dense, isodiametric particles scattered throughout the cytoplasm. Reaction product for G6Pase activity was localized consistently within the lumen of the nuclear envelope and endoplasmic reticulum and frequently within an outer saccule of the Golgi complex under normal conditions. Choroid plexus epithelia from stressed mice exhibited a qualitative increase in cytoplasmic glycogen and a decrease in G6Pase activity; the other cell types did not express demonstrable alterations in glycogen concentration and G6Pase activity. The results indicate that glycogen and G6Pase activity are prevalent within non-neural cells of the adult mammalian CNS. Glucose utilization in the choroid plexus epithelium may be altered by stressful conditions that influence the functional activity of this cell. PMID- 3018178 TI - Incidence of peripheral neuropathy and cerebellar ataxia in chronic alcoholics. AB - A total of 78 chronic alcoholics were examined neurologically as well as by electroneurography, myography and posturography. Clinical signs of peripheral neuropathy were detected in 45% of these patients, with electromyographic and neurographic abnormality in 67% and 55% respectively. Clinical signs of cerebellar ataxia were found in 33% of our patients, whereas posturographic measurements of increased sway were recorded in 69%. The posturographic characteristics of cerebellar anterior lobe atrophy were observed in two-thirds of the latter patients. The severity of cerebellar-ataxia did not correlate with the degree of neuropathy. This lack of correlation is interpreted as an indication of different pathogenetic mechanisms acting on peripheral nerves and cerebellum. PMID- 3018179 TI - False positive-false negative CT scan in late epileptic seizure: a meningioma glioblastoma association. AB - A false-negative finding on initial CT is reported in a case of supratentorial glioma. This observation was peculiar because the first CT revealed a meningioma which might initially have been related to the clinical symptoms. The term false positive-false negative CT is proposed. The reasons for such CT failures are discussed. The accuracy of clues as to the localization of the glioma provided by EEG is emphasized. PMID- 3018181 TI - Staging and the evaluation of prognosis for patients with small-cell carcinoma of the lung. PMID- 3018180 TI - Appearance of Guillain-Barre syndrome in patients during corticosteroid treatment. AB - Three patients developed acute Guillain-Barre syndrome while on steroid treatment. The first patient suffered from ulcerative colitis and developed Guillain-Barre syndrome when the steroid dosage was being tapered. Another patient with long-standing multiple sclerosis received steroids during relapse and the Guillain-Barre syndrome appeared while treatment was reduced. The third patient, with aqueductal stenosis and ventriculoatrial shunt, who received steroids when malfunction of the shunt was suspected, developed Guillain-Barre syndrome while steroids were being tapered. Based on the putative immune pathogenesis of inflammatory demyelinating polyneuropathy, the occurrence of the syndrome in these patients could have been due to a selective effect of low-dose steroids on a specific, maybe suppressor lymphocyte subpopulation. PMID- 3018182 TI - Long-term disease-free survival in small-cell carcinoma of the lung: a study of clinical determinants. AB - The influence of treatment and of pretreatment patient characteristics on the probability of long-term disease-free survival in small-cell lung cancer (SCLC) was investigated in a consecutive series of 874 patients. The patients were included in six controlled treatment trials from 1973 to 1981, using different combinations of chemotherapy with or without irradiation. All patients underwent pretreatment staging, including bronchoscopy, peritoneoscopy with liver biopsy, and bone marrow examination. The same procedures were repeated in patients without overt signs of disease 18 months from initiation of treatment, and patients without evidence of SCLC were regarded as long-term survivors. Seventy two patients were disease-free at restaging, corresponding to 13% of 443 patients with limited-stage disease and 3% of 431 patients with extensive-stage disease. The possible relationship between different pretreatment variables and the probability of 18 months' disease-free survival was investigated by multiple regression analysis. Disease extent was the most important determinant of long term survival. Being a woman was a positive factor and hypouricemia had negative influence on the long-term results, while features such as performance status and serum lactate dehydrogenase (LDH) did not have significant influence in the regression model. Differences between the efficacy of the applied treatment regimens were less in limited disease than they were in extensive disease, in which six-agent regimens of alternating chemotherapy was significantly better than treatment with three- or four-agent regimens. Accordingly, disease extent seems to be the most pivotal determinant of long-term survival in SCLC, but influence of the patient's sex and serum urate concentration should also be considered. PMID- 3018183 TI - Treatment of small-cell lung cancer on protocol: potential bias of results. AB - During a 27-month period, 215 new cases of lung cancer were diagnosed at five McGill University hospitals. Only 44 patients (20%) so diagnosed were treated on available chemotherapy protocols. Six categories were used to define reasons for nonparticipation. The most important were medical reasons (MRs), 46%; non-medical reasons (NMRs), 20%; and physician preference (PP), 16%. The three remaining categories, representing 18% of exclusions, were death before diagnosis (DBD), surgical treatment (S), and a miscellaneous group (M). Median survival of patients on and off protocol was 10 and 7 months, respectively. Patients with limited disease treated off protocol for NMR and those treated with surgery did better than patients on protocol. Patients with extensive disease not enrolled because of MR did worse, and those excluded because of PP did better than patients treated on protocol. The implication of these findings for other cancer studies is that analysis of chemotherapy trials often represents treatment results in a small proportion of all patients with a given neoplasm. As such, caution must be exercised when extrapolating results to the group as a whole. We suggest that complete demographic data, including proportion of patients participating and reasons for exclusion, be included in all chemotherapeutic trial reports. PMID- 3018184 TI - Synchronous occurrence of glioblastoma multiforme in a husband and wife. AB - Glioblastomas developed within two years of each other in an otherwise unrelated married couple in their fifties. There was a daughter who died of Hodgkin's disease but no other unusual incidence of cancer in either siblings, parents or other children. No clear etiology of risk factors for brain tumor were identified. The development of such conjugal tumors, although apparently rare, raises important etiologic questions. PMID- 3018185 TI - Pituitary adenomas in patients under 20 years old. A clinicopathological study of 12 cases. AB - The clinical manifestations, pathological features and follow-up data on 12 cases of pituitary adenoma in patient less than 20 years old were evaluated. This group represented 8.5% of our 142 cases of pituitary adenoma from all age groups during the period of study. There were five males and seven females whose ages ranged from 13 to 19 years at diagnosis. Immunocytochemical staining demonstrated the presence of prolactin in 10 tumors, ACTH in one tumor and the remaining neoplasm was negative for the five major pituitary hormones (prolactin, hGH, ACTH, TSH, gonadotrophin). The results of the immunocytochemistry correlated appropriately with the clinical manifestations. Extension beyond the sella turcica at presentation was a common feature as evidenced by the high incidence of visual defects (75%). A complete excision was accomplished in only two patients. The aggressive behavior of these tumors was demonstrated by a recurrence rate of 50% and only a single long-term cure. Early detection and therapy, if possible, are essential for the successful management of pituitary adenomas in younger patients as implied by our study and other reports in the literature. PMID- 3018186 TI - Tooth pulp input to the shell region of nucleus ventralis posteromedialis of the cat thalamus. AB - A population of neurons in the somatosensory part of the nucleus ventralis posteromedialis (VPM proper) that responded to electrical stimulation of the tooth pulp were studied in cats under urethan-chloralose anesthesia. Two classes of units responsive to electrical stimulation of the contralateral canine tooth pulp were identified. One class was responsive only to tooth pulp stimulation and these units were designated as tooth pulp specific (TPS) units. The other class of units responded to mechanical stimulation of the contralateral trigeminal integument in addition to tooth pulp stimulation. Their receptive field characteristics identified them as wide dynamic range (WDR) units responsive to tooth pulp stimulation. Both classes of units were located in the shell region of the caudal VPM proper; TPS units were coexistent with trigeminal nociceptive specific (NS) units and were found in the dorsomedial as well as ventromedial parts of the NS zone. WDR units responsive to electrical stimulation of the tooth pulp were located in the dorsomedial as well as ventromedial parts of WDR zone, a narrow band, approximately 300 micron wide, just in front of the NS zone. Tooth pulp units in the dorsomedial shell region of the VPM proper responded to the maxillary canine tooth pulp, whereas those in the ventromedial shell region responded to the mandibular canine tooth pulp. Some tooth pulp units in these two regions were responsive to stimulation of both maxillary and mandibular canine teeth. Both TPS and WDR units were antidromically excited by electrical stimulation of the SI area of the somatosensory cortex. Cooling the dorsolateral surface of the caudal medulla oblongata reversibly blocked tooth pulp evoked responses of TPS and WDR units. Trigeminal tractotomy just above the level of the obex irreversibly abolished tooth pulp-evoked responses of TPS and WDR units. These findings suggested that TPS neurons in the marginal layer of the trigeminal subnucleus caudalis and WDR neurons in the lateral part of the subnucleus reticularis dorsalis relay afferent impulses derived from the tooth pulp to the shell region of the VPM proper. PMID- 3018187 TI - Characterization of ameboid microglia isolated from developing mammalian brain. AB - Ameboid microglia are isolated from the cerebral tissue of neonatal rat by selective cell adhesion to plastic. Histochemical markers show that the microglial preparations are homogeneous (95 +/- 3%) and represent a 10% yield from starting cultures. Isolated ameboid microglia contain nonspecific esterase activity, the macrophage surface antigens MAC-1 and MAC-3, and acetylated low density lipoprotein receptors. Ameboid cells have functional properties similar to those of macrophages, including the ability to engulf 5 micron latex beads, to secrete Interleukin-1 (IL-1) and to release superoxide anion. Unlike monocytes and adherent spleen cells, ameboid microglia do not show peroxidase activity by histochemical stain. Unlike resident peritoneal macrophages, ameboid microglia proliferate in vitro. Scanning electron microscopy shows that ameboid cells have short, spinous processes that can be distinguished from the ruffled surfaces of body macrophages. Our observations suggest that ameboid microglia are a distinct class of mononuclear phagocytic cells. Retinoic acid and dimethyl sulfoxide, agents known to accelerate differentiation in vitro, stimulate ameboid cells to develop thin processes several hundred microns in length. These "process-bearing" microglia eventually lose the capacity to engulf latex beads and to proliferate. They also show reductions in nonspecific esterase activity and in the binding of acetylated low-density lipoprotein. We suggest that in vitro ameboid microglia differentiate into nonphagocytic cells similar to ramified microglia found in normal adult brain. The isolation techniques described here provide the opportunity to study the composition and function of different microglial subpopulations during the development of the CNS. PMID- 3018188 TI - Ontogeny of substance P receptor binding sites in rat brain. AB - The ontogeny of substance P (SP) receptor binding sites in rat brain has been studied using both membrane binding assays and in vitro receptor autoradiography. The density of SP binding sites is maximal 1 d before birth and decreases thereafter to reach adult values 14 d after birth. During the early postnatal period, the distribution of SP binding sites undergoes major modifications. For example, very high densities of SP binding sites are present in most brain stem nuclei from 1 to 14 d after birth, while it is not the case in adults. In the striatum, SP receptors are distributed in a "patchy" manner early after birth, while it is much more homogeneous in the adult. This demonstrates that SP receptors undergo major redistributions during postnatal development. The very high density of SP binding sites present in the brain at its early stages of development may indicate that SP could be an important factor involved in the early organization of the CNS. PMID- 3018189 TI - Blockade of D-2 dopamine receptors strongly enhances the potency of enkephalins to inhibit dopamine-sensitive adenylate cyclase in rat neostriatum: involvement of delta- and mu-opioid receptors. AB - The interactions between dopamine receptors and opioid receptors coupled to adenylate cyclase in rat neostriatum were investigated. cAMP efflux from neostriatal slices induced by simultaneous activation of (stimulatory) D-1 and (inhibitory) D-2 dopamine receptors with 30 microM dopamine was inhibited by the preferential delta-opioid receptor agonist [D-Ala2-D-Leu5] enkephalin (DADLE) and the mu-opioid receptor agonist morphine with an EC50 of 100 and 800 nM, respectively. On selective D-1 receptor activation (i.e., with D-2 receptors blocked by 10 microM (-)sulpiride), the EC50 of DADLE was strongly reduced to 3 nM, whereas that of morphine was unaffected. When D-1 and D-2 receptors were activated simultaneously, the inhibitory effects of DADLE (0.3 microM) and morphine (3 microM) on cAMP efflux were antagonized equally well by naloxone, a mu-opioid receptor antagonist. In contrast, on selective D-1 receptor activation, naloxone was about 20 times more potent in antagonizing the inhibitory effect of morphine than DADLE. Moreover, the delta-opioid receptor antagonist ICI 174864 (0.75 microM) did not affect the inhibitory effect of morphine but antagonized that of DADLE, provided that D-2 receptors were blocked. The highly selective delta-opioid receptor agonist [D-Pen2-D-Pen5] enkephalin (DPDPE) inhibited dopamine-stimulated cAMP efflux only when D-2 receptors were blocked. Similar results were obtained when the agonists SKF 38393 and LY 141865 were used to activate D-1 and D-2 receptors, respectively. These data indicate that blockade of D-2 receptors in the neostriatum elicits the coupling of delta-opioid receptors to dopamine-sensitive adenylate cyclase, thereby making it considerably more sensitive to inhibition by the enkephalins. PMID- 3018190 TI - Differential down-regulation of D1-stimulated adenylate cyclase activity in rat forebrain after in vivo amphetamine treatments. AB - Amphetamine has complex behavioral actions in the rat that depend upon the release of dopamine in striatal and mesolimbic brain regions. To explore a possible role of the dopamine-sensitive cAMP second-messenger system in mediating these effects, we examined the effects of in vivo amphetamine treatments on the D1 receptor-coupled adenylate cyclase system in membranes from striatal and mesolimbic rat brain regions. The results show that amphetamine produces a regional, dose- and time-dependent down-regulation of adenylate cyclase activity. Intermediate and high doses of amphetamine (2.5 and 7.5 mg/kg, respectively), but not a low dose (1.0 mg/kg), resulted in a decrease in the apparent Vmax and/or an increase in the apparent Ka for the selective D1 partial agonist, SKF38393, in striatal membranes 30 min after amphetamine treatment. Treatment of rats with 7.5 mg/kg amphetamine for 30 and 60 min, but not 10 min, similarly resulted in a down regulation of D1-mediated adenylate cyclase activity in striatal membranes. In contrast, in mesolimbic tissues, no amphetamine treatment at any time resulted in an alteration of SKF38393-stimulated adenylate cyclase activity relative to saline controls. The results of behavioral analyses also showed that animals exhibiting intense stereotypies had significantly lower striatal apparent Vmax values than did those animals engaged in moderate or no behavioral activity at the time of decapitation. These findings demonstrate that amphetamine treatments result in a down-regulation of striatal, but not mesolimbic, dopamine-sensitive adenylate cyclase activity that parallels the intense, stereotyped behaviors characteristic of dopaminergic activation in the striatum. PMID- 3018191 TI - Distribution of neuronal receptors for nerve growth factor in the rat. AB - To survey the distribution of neuronal receptors for NGF, sections of the rat brain, spinal cord, and peripheral ganglia were incubated in vitro with radioiodinated NGF and examined by autoradiography. NGF binds selectively with high affinity to most sympathetic neurons and many primary sensory neurons together with their intraspinal or intramedullary axons. In autoradiographs of the brain, labeled neuronal perikarya are seen in the basal forebrain, the caudate-putamen, the medulla oblongata, the ventral cochlear nucleus, and the dorsal nucleus of the lateral lemniscus. The distribution of neurons binding NGF resembles the distribution of cholinergic neurons in the forebrain, but these 2 systems overlap very little in the brain stem. In extracts of the brain or spinal cord enriched for plasma membranes, avid binding sites are regionally manifest with properties similar to those of fetal peripheral neurons. The localization of neurons expressing the high-affinity receptor for NGF defies simple correlation with neurotransmitter function or embryogenesis. PMID- 3018192 TI - Dual peroxidase and colloidal gold-labeling study of angiotensin converting enzyme and angiotensin-like immunoreactivity in the rat subfornical organ. AB - The cellular relationships between angiotensin converting enzyme (ACE) (EC 3.4.14.1) and angiotensin-like immunoreactivity (AGLI) were examined in the subfornical organ (SFO). Brains from adult rats were fixed by vascular perfusion with 3.75% acrolein and 2% paraformaldehyde. The region containing the SFO was then sectioned on a vibrating microtome. Partially permeabilized sections were immunocytochemically labeled using the peroxidase-antiperoxidase (PAP) or combined PAP and immunogold methods. Goat antiserum to ACE was localized to both non-neuronal and neuronal cells within the SFO. Intense peroxidase immunoreactivity for ACE was associated with the ventricular and basal surface of ependymal cells, the luminal surface of the vascular endothelium, portions of glial membranes exposed to extracellular spaces, and membranous organelles within neuronal processes. Two antisera raised in rabbits against angiotensin II showed peroxidase immunoreactivity within the extracellular spaces and throughout the cytoplasm of numerous axon terminals and a few perikarya and dendrites in the SFO. Axon terminals and dendrites also showed aggregates of AGLI in smooth membranes and vesicles near the plasmalemma. Gold labeling for AGLI was evident in only 6% of the axon terminals and in a smaller number of dendrites containing peroxidase immunoreactivity for ACE. The low incidence of terminals containing both markers appeared to at least partially reflect limited penetration of the 10 nm gold particles. These results provide the first ultrastructural evidence that ACE is associated with the plasmalemma and membranous organelles strategically located for interaction with precursors of angiotensin II or other peptides within the cerebrospinal fluid, extracellular spaces and neurons of the SFO. PMID- 3018193 TI - Cytosolic calcium elevation and cGMP production induced by serotonin in a clonal cell of glial origin. AB - It has been shown recently that astroglial cells of the mammalian CNS possess receptors for neurotransmitters. In order to analyze what sequences of cellular events occur upon activation of these glial receptors, we utilized a 5-HT receptor in a rat clonal cell of glial origin as a model system. When the C6BU-1 glioma cells were exposed to 5-HT, the cytosolic Ca2+ concentration ([Ca2+]i) was elevated and the cellular content of cGMP was increased in a dose-dependent manner. 5-HT receptor antagonists and a Ca2+ entry blocker suppressed the increases in both [Ca2+]i and cGMP. The magnitude of the cGMP increment depended on the environmental Ca2+ concentration and was totally blocked by Ca2+ depletion. Application of a Ca2+ ionophore increased [Ca2+]i and cGMP. There was a tendency for extremely high [Ca2+]i to suppress the cGMP increment. On the contrary, membrane-permeable cyclic nucleotide analogs failed to increase [Ca2+]i. These results suggest that the following sequence of events occurs in 5 HT-induced C6BU-1 cells: activation of 5-HT receptors, Ca2+ influx, a rise in [Ca2+]i, activation of guanylate cyclase, and, finally, activation of cyclic nucleotide phosphodiesterase. PMID- 3018195 TI - Adenosine stimulates glycogenolysis in mouse cerebral cortex: a possible coupling mechanism between neuronal activity and energy metabolism. AB - Adenosine promotes a concentration-dependent hydrolysis of 3H-glycogen newly synthesized from 3H-glucose by mouse cerebral cortical slices. The EC50 for this effect is 7 microM. Theophylline antagonizes the glycogenolysis induced by adenosine with an EC50 of 80 microM. The rank-order of potencies of adenosine agonists is adenosine 5'-cyclopropyl-carboxamide greater than 2-chloroadenosine much greater than N6-cyclohexyladenosine = adenosine, suggesting that adenosine promotes glycogenolysis via receptors of the A2 type. This contention is substantiated by the weak stereospecificity observed for the glycogenolytic action of D- and L-(phenylisopropyl)-adenosine. The glycogenolysis elicited by adenosine at 10 and 100 microM is inhibited by ouabain at 10 microM, a concentration of the cardiac glycoside not significantly affecting 3H-glycogen levels per se. Interestingly, the previously demonstrated glycogenolytic action of vasoactive intestinal peptide (Magistretti et al., 1981, 1984) and of norepinephrine (Quach et al., 1978) is also antagonized by ouabain. These results demonstrate the existence of a metabolic action of adenosine, which is sensitive to ouabain and appears to be mediated by A2 receptors. The concentrations at which adenosine promotes glycogenolysis are of the same order of magnitude as those reached extracellularly by the nucleoside during neuronal depolarization (Pull and McIlwain, 1972). This set of observations therefore supports the notion that adenosine plays a modulatory role in the coupling between neuronal activity and energy metabolism in the CNS. PMID- 3018194 TI - Nerve growth factor receptors on chick embryo sympathetic ganglion cells: binding characteristics and development. AB - NGF is essential for the development and maintenance of sympathetic and certain sensory neurons. The NGF receptors on the surface of sympathetic ganglion cells from chick embryos were characterized; they consist of high-affinity receptors with a dissociation constant of about 10(-11) M, and low-affinity receptors with a dissociation constant of about 10(-9) M. There are more than 10 times as many low-affinity as high-affinity receptors per cell. The heterogeneity of NGF binding is not due to negatively cooperative interactions among the receptors. The high- and low-affinity components of NGF binding defined at steady state correspond to slowly and rapidly dissociating components of bound NGF seen in kinetic experiments. In addition, a very slowly dissociating component of bound NGF was observed; this component was a small fraction of binding at low concentrations of NGF but increased to 20-60% of bound NGF at the highest NGF concentrations examined. This very slowly dissociating component of bound NGF accounts for several peculiarities in the binding data not accounted for by steady-state binding of NGF to its high- and low-affinity receptors. Developmental studies showed that both high- and low-affinity NGF receptors were present on chick embryo sympathetic ganglion cells from 6.5 to 20 d in ovo. No significant differences in the numbers or affinities of the receptors were seen with cells from ganglia at 9, 11, or 15 d of development. Cultured non-neuronal cells from sympathetic ganglia had only low-affinity NGF receptors. PMID- 3018196 TI - Detection and characterization of beta-adrenergic receptors and adenylate cyclase in coated vesicles isolated from bovine brain. AB - To assess whether internalization of beta-adrenergic receptor occurs in the CNS, we have isolated clathrin-coated vesicles from bovine forebrain and examined them for the presence of beta-adrenergic receptor binding and adenylate cyclase activities. A coated vesicle enriched preparation isolated by successive D2O Ficoll density gradient centrifugations was applied to a glass bead permeation column to achieve further purification. Two major peaks of protein were eluted from the column and monitored by electron microscopy and SDS-PAGE. Peak II contained almost exclusively coated vesicles (98%), whereas peak I, which appeared in the void volume, contained larger smooth vesicles and few coated vesicles. beta-Adrenergic receptor binding to peaks I and II was measured with 125I-cyanopindolol (CYP) as ligand in Sepharose 4B column assays. 125I-CYP was found to bind specifically and saturably to both peaks I and II with a Bmax of 28 +/- 4 and 32 +/- 3 fmol/mg protein, respectively. 3H-CGP 12177, a hydrophilic beta-adrenergic receptor ligand, did not label receptors present in peak II, but it specifically bound to synaptic plasma membranes (SPM) prepared from bovine hippocampus and, to a lesser extent, to peak I. These results suggest that receptors present in coated vesicles are cryptic in nature. In the displacement of 125I-CYP binding by (-)-isoproterenol, addition of 50 microM GppNHp caused a significant "right shift" with SPM and peak I but not the peak II preparation. Adenylate cyclase activities could also be detected in both peaks I and II (specific activities, 21 +/- 0.6 and 24 +/- 0.5 pmol cAMP/mg protein/min, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018197 TI - Increased presynaptic ATP levels coupled to synaptic activity at the crayfish neuromuscular junction. AB - Levels of ATP and related adenylates were measured in the terminal region of efferent nerves in the crayfish opener muscle using the luciferin-luciferase method. Following 1 min of stimulation at 50 Hz, the average (+/- SE) ATP content rose from 13.4 (+/- 1.5) to 19.0 (+/- 2.1) nmol/mg dry weight. The amounts of ADP, AMP, and the phosphagen phosphoarginine did not change significantly. Thus, the increased ATP was not derived from any of these potential sources. The increase was found to depend on synaptic activation, however, for its magnitude was directly related to the concentration of extracellular Ca2+, and it was blocked when CoCl2, verapamil, ruthenium red, or gamma-methylglutamate and picrotoxin were added to the bath. Addition of ATP to the bath solution also increased nerve ATP levels. Based upon measurements of sucrose distribution, only 50% of this increase was in the extracellular water space. The remainder of the ATP had either entered the nerve, become adsorbed extracellularly, or both. Addition of 2-deoxy-D-glucose and gamma-32P-ATP to the bath resulted in the formation of 32P-2-deoxy-D-glucose-6-P by the nerve. This suggests that a fraction of the extracellular ATP does enter the neuron chemically intact. To determine whether exogenous ATP is the source of the increased ATP measured in the nerve following stimulation, the bath was assayed for ATP. Stimulation did cause ATP levels to increase significantly; however, the maximum concentration was 3 orders of magnitude lower than that required to increase ATP levels in resting nerve.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018198 TI - Dual-component synaptic potentials in the lamprey mediated by excitatory amino acid receptors. AB - The synaptic mechanisms underlying amino acid-mediated excitation in the lamprey spinal cord have been investigated. Fine stimulating electrodes were used to stimulate single axons in the spinal cord and evoke unitary EPSPs in lamprey motoneurons and one type of premotor interneuron, the CC interneuron. Three types of EPSP, distinguished by their time course and sensitivity to amino acid antagonists, were seen. Fast EPSPs had a fast rise time (mean, 6.5 msec) and a short half-decay time (mean, 22.5 msec). Slow EPSPs lasted at least 200 msec, had a slow rise time (mean, 28 msec), and a long half-decay time (mean, 109 msec). The third type of unitary potential, called "mixed" EPSP, also lasted at least 200 msec, had a fast rise time (mean, 12 msec), and a long half-decay time (mean, 105 msec). Lamprey neurons were found to possess 3 types of excitatory amino acid receptor: N-methyl-D-aspartate (NMDA), kainate, and quisqualate receptors. 2 Amino-5-phosphonovaleric acid (APV) or Mg2+ blocked the depolarizations caused by N-methyl-D,L-aspartate (NMA) but not those of kainate or quisqualate. Cis-2, 3 piperidine dicarboxylic acid (PDA) blocked the depolarizations caused by NMA and kainate but not those of quisqualate. Fast EPSPs were unaffected by the bath application of APV or Mg2+ but were greatly reduced by PDA, suggesting that these EPSPs were mediated by non-NMDA, possibly kainate receptors. Both APV and Mg2+ blocked the slow EPSPs, suggesting that they were mediated by NMDA receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018200 TI - Hyperthyroidism caused by a toxic intrathoracic goiter with a normal-sized cervical thyroid gland. AB - The rare presentation of hyperthyroidism caused by an intrathoracic goiter with a normal-sized cervical thyroid gland is described. The toxic intrathoracic goiter demonstrated avid uptake of [131I] and [99mTc]pertechnetate, with comparatively faint isotopic accumulation seen in the cervical thyroid. A chest roentgenogram and radioisotope scan should be mandatory in cases of hyperthyroidism having no cervical thyroid enlargement to explore the possibility of a toxic intrathoracic goiter. PMID- 3018199 TI - Excitatory synaptic drive for swimming mediated by amino acid receptors in the lamprey. AB - In order to investigate the properties and pharmacology of the excitatory synaptic drive received by motoneurons during swimming in the lamprey, propriospinal excitatory interneurons were activated as a population by the regional application of N-methyl-D,L-aspartate (NMA) to either the 6-8 rostral most or the 6-8 caudal-most segments of lengths of isolated spinal cord. This caused a rhythmic motor output to be generated in these regions. Synaptic potentials that were phase-locked to, and dependent on, the rhythmic motor activity of the segments exposed to the agonist could be recorded in motoneurons lying outside the activated regions. The synaptic drive to motoneurons located rostral or caudal to the activated regions was studied. Motoneurons received both descending and ascending synaptic input, which consisted of alternating excitatory and inhibitory phases. The inhibition could be reversed by chloride injection and blocked by strychnine, leaving an oscillating excitatory phase. The descending excitatory drive could extend 1-9 segments from the active region, while the ascending excitatory drive was recorded only in motoneurons that were 1 3 segments rostral to the active region. Both types of drive occurred in phase with the ipsilateral ventral root discharge: The peak depolarization of the descending drive occurred at the same point in the swimming cycle as that of the depolarizing phase seen during fictive swimming, while that of the ascending drive occurred significantly later. Both ascending and descending drives were partially reduced in amplitude by 2-amino-5-phosphonovaleric acid or Mg2+. The blocking action of Mg2+ was, in both cases, voltage dependent. Cis-2,3-piperidine dicarboxylic acid or kynurenic acid caused a much greater reduction in the amplitude of the oscillations. These results suggest that a major part of the excitatory drive for swimming in lamprey motoneurons is generated by populations of propriospinal interneurons with relatively long descending and/or short ascending axons, which fire rhythmically during swimming and release an amino acid transmitter that excites motoneurons through N-methyl-D-aspartate (NMDA) and non-NMDA receptors. This information will allow these important neurons to be identified in future experiments. PMID- 3018201 TI - Severe systemic reaction to diphosphonate bone imaging agents: skin testing to predict allergic response and a safe alternative agent. AB - We describe a severe systemic reaction which occurred in a patient on two occasions after i.v. injection of chemically related diphosphonate bone imaging agents. Skin testing showed reactivity to multiple commercially available diphosphonate compounds but no significant response to pyrophosphates. A subsequent pyrophosphate bone scan resulted in no adverse reaction. Severe systemic reactions to diphosphonates can occur, skin testing may prove useful in evaluating allergic reactions, and pyrophosphates appear to be a safe alternative agent in patients proven or suspected allergic to diphosphonates. PMID- 3018202 TI - Use of factor analysis in the evaluation of left to right cardiac shunts. AB - We have compared two methods of data processing for the quantitation of left-to right cardiac shunts using first-pass radionuclide angiography. These two methods are used for curve generation in the deconvolution analysis. The standard method involves manual definition of regions of interest. A newer method--factor analysis--provides automatic curve generation and is therefore more operator independent. Both techniques yield curves of the venous input, lung, and background. The venous input curve is deconvolved by the lung curve, and the resultant unit impulse response is fitted by the gamma variate method to quantitate the left-to-right shunt fraction. Both techniques--factor analysis and regions of interest (ROIs)--separated the shunt patients (n = 16) from the control subjects (n = 20) with a p less than 0.001. There was less interobserver variability with the curves obtained by factor analysis than with those obtained by regions of interest. The coefficient of correlation of factor analysis results with oximetry, r, was 0.90. High sensitivity and specificity, each 94%, was achieved with curves generated by factor analysis. Time vs. activity curves generated by ROIs can achieve high sensitivity and low specificity, or vice versa, depending on the cutoff level defined for separation of the left-to-right shunt patients from the control group. PMID- 3018203 TI - Diagnosis in the Cushing's syndrome revisited. PMID- 3018205 TI - Calcium, metals and nervous system in the elderly. PMID- 3018206 TI - An x-ray fluorescence technique to measure in situ the heavy metal burdens of persons exposed to these elements in the workplace. AB - Assays of hair and body fluid concentrations may be valuable measurements of acute exposure to a heavy metal, but they do not provide insight into the total heavy metal intake when the intake is low and chronic. The use of an x-ray fluorescence technique (XRF) enables measurement of the long-term retention of various heavy metals in select tissues in vivo. XRF was used to measure the mercury content of head and bone tissue in 298 dentists with long-term exposure to mercury-containing amalgams. It was also used to evaluate the lead burden of persons suspected of having elevated lead exposure at the workplace, and to assay the lead levels in urban and rural children. These studies indicated that the x ray fluorescence method of assaying heavy metals in vivo is noninvasive, safe, rapid, and sensitive to levels of many heavy metals that accumulate in human tissues. PMID- 3018207 TI - The "three-piece" osteotomy and interpositional bone graft for augmentation of the atrophic mandible. AB - A study of 20 patients who underwent augmentation of an atrophic mandible by a "three-piece" osteotomy and interpositional bone graft technique is presented. The results show a reduced rate of bone resorption in the posterior regions and a reduced incidence of sensory nerve disturbances in comparison with previous visor/sandwich techniques. PMID- 3018204 TI - Effect of preventing coprophagy in the rat on neutral detergent fiber digestibility and apparent calcium absorption. AB - The effect of coprophagy on apparent neutral detergent fiber (NDF) digestibility, and calcium absorption was evaluated by housing rats in four types of cages: regular, metabolic, and two anticoprophagy cages: a short linear tube cage which allowed the rat forward and backward movement of about one-half a body length and a long tube cage assembled to form a square which allowed the rat to move in one direction through the tube but not turn around. Three-day weight gains of animals in the regular or metabolism cages were greater than those of rats housed in either anticoprophagy cage. In contrast, average food intake did not differ among the four housing conditions. Wet, but not dry, fecal weights were greater in the two groups of rats in the anticoprophagy cages. Neither fecal NDF (% dry wt) nor apparent NDF digestibility was affected by housing conditions. Apparent calcium absorption was decreased by the anticoprophagy housing. The differences in body weight and apparent calcium absorption suggest that prevention of coprophagy in the rats produces significant changes in the efficiency of food and nutrient utilization. The failure to detect differences in NDF digestibility indicates that coprophagy has little impact on the study of fiber digestibility. PMID- 3018208 TI - Malignant fibrous histiocytoma of the tongue: report of a case and ultrastructural observations. AB - A 42-year-old female presented with a 2.5 cm symptomless swelling on the left lateral surface of the tongue. Histologically, there were large, often pleomorphic, fibroblast-like and histiocyte-like cells, multinucleate giant cells and cells of variable morphology. Many of the cells had abundant vesicular cytoplasm. There were also aggregations of neutrophilic or eosinophilic leukocytes, or both. The cells in some areas formed a fascicular or storiform pattern, or both. Ultrastructurally, the vesicular appearance was due mostly to markedly dilated endoplasmic reticulum, but many of the neoplastic cells contained some lipid. There were some myofibroblasts and a few xanthofibroblasts. The cells of variable morphology had many of the ultrastructural features of the fibroblast and histiocyte-like cells. There were also some small undifferentiated cells with minimal cytoplasm. These ultrastructural features confirmed the histological diagnosis of malignant fibrous histiocytoma. PMID- 3018209 TI - Detection of papillomavirus DNA in oral papillomas and carcinomas: application of in situ hybridization with biotinylated HPV 16 probes. AB - Four oral papillomas and 7 carcinomas were studied for the presence of HPV DNA by means of in situ hybridization. Hybridization was carried out with a HPV 16 probe labelled with biotin under different conditions (nick translation/photobiotin). Subsequently, a modified biotin-avidin-alkaline phosphatase procedure was used to visualize virus infected cells. Four/4 papillomas and 4/7 carcinomas were seen to contain HPV harbouring cells. Positive cells were located at intermediate and superficial cell layers in papillomas and in keratinized zones in carcinomas. Analogous results were found with nick translated and photobiotinylated probes. DNA-biotinylation in conjunction with the biotin-avidin-alkaline phosphatase detection system proved to be a sensitive, convenient and rapid modification of in situ hybridization. PMID- 3018212 TI - A diffusion chamber technique for testing of antifungal drugs against Sporothrix schenckii in vivo. AB - Growth of the yeast form of Sporothrix schenckii (ATCC 14804) was determined in diffusion chambers with 0.45 and 3.0 micron pore size over a period of 24 to 192 h after subcutaneous implantation into mice. Numbers of S. schenckii in 0.45 micron chambers increased significantly by 192 h when inocula of 10(3) and 10(5) colony forming units were implanted. In chambers with a pore size of 3.0 microns, only a slight decrease of fungal growth occurred, although host cells readily passed the filter membrane and phagocytosed yeast-form cells. The activities of amphotericin B, ketoconazole, itraconazole, ICI 153.066, vibunazole and potassium iodide against S. schenckii in implanted chambers were determined in terms of their effects on S. schenckii. ICI 153.066, ketoconazole, itraconazole and amphotericin B significantly reduced the numbers of reisolated S. schenckii in both types of chambers. There was a slight activity with vibunazole but none with potassium iodide. PMID- 3018213 TI - Pulmonary sequestration and congenital cystic adenomatoid malformation in an infant. AB - Pulmonary sequestration and congenital cystic adenomatoid malformation (CCAM) are individually well known but infrequent congenital malformations of the lung. We report a rare case of pulmonary sequestration and CCAM occurring concurrently in the same infant. PMID- 3018211 TI - Effects on Trypanosoma brucei differentiating bloodstream trypomastigotes and established procyclic trypomastigotes when grown in the presence of respiratory inhibitors. PMID- 3018210 TI - Circulating thymulin and thymosin-alpha 1 activity in pediatric acquired immune deficiency syndrome: in vivo and in vitro studies. AB - Twenty-five children with acquired immune deficiency syndrome (AIDS) or AIDS related complex had a characteristic pattern of T cell deficiency. Abnormally low plasma thymulin levels preceded the development of peripheral blood T cell abnormalities. In contrast to patients with congenital T cell deficiencies, our patients had elevated serum levels of thymosin-alpha 1. Treatment with thymosin fraction 5 in three children with AIDS resulted in only transient clinical and immunologic improvement. PMID- 3018215 TI - [Interferon sensitivity of various cell lines]. AB - An economic assay to determine the yield of interferon is necessary for the induction, production and purification of interferon. Using the inhibition of cytopathic effect of vesicular stomatitis virus in a microtitre system, we compared the susceptibility of different cell lines against an internal IFN alpha standard obtained from Sendai virus-induced human leukocytes. Human fibroblasts carrying trisomy G 21 and bovine MDBK cells showed the highest sensitivity followed by normal human fibroblasts. Human RH kidney cells exhibited a susceptibility similar to that of human WISH amnion cells frequently used by others. The human amnion cell line FL and monkey Vero kidney cells, as described here, were unsuitable for the determination of IFN yields. PMID- 3018214 TI - Extravascular clot formation and platelet activation on variously treated root surfaces. AB - The platelet attachment and activation potential of a variety of treated, human root surfaces was studied employing an established in vitro method for determining thrombogenicity. In addition, scanning electron microscopic observations were made of the morphological appearance of blood components in initial clot formation on root surfaces containing periodontal ligament (PDL). For the platelet study, the crowns were removed from freshly extracted teeth with and without periodontal disease (PD) and the roots were sectioned. The following surface conditions were created: (1) PDL present, (2) PD, (3) PD planed, (4) PD planed and demineralized, (5) Condition 4 treated with collagenase. All conditions were incubated with Indium-1 ll-labeled platelets with and without the addition of prostacyclin, an inhibitor of platelet activation. It was observed that the greatest number of attached platelets were found in Condition 1, that platelet activation was significantly enhanced in Condition 4 and this effect was reversed by Condition 5. The scanning electron microscopic observations were made on freshly extracted teeth with an intact PDL in which the roots were sectioned and either reinserted immediately into the extraction site or incubated in platelet-rich plasma. It was seen that platelets were involved early in clotting and activated by this surface. This study suggests the possible use of platelet attachment and activation as an indicator for root surface thrombogenicity and perhaps of the fibrous attachment potential. PMID- 3018216 TI - Tolerance to the luteinizing hormone and prolactin suppressive effects of delta-9 tetrahydrocannabinol develops during chronic prepubertal treatment of female rats. AB - To determine if prepubertal female rats develop tolerance to the hormone suppressive action of delta-9-tetrahydrocannabinol (THC), we tested the ability of THC (10 mg/kg i.p.) to block luteinizing hormone (LH) and prolactin (PRL) surges acutely at first proestrus in animals that were treated repetitively with THC before puberty. Rats were pretreated with 10 mg THC/kg b.wt. twice daily beginning at 27 days of age and the blocking efficacy of THC treatment at first proestrus was compared to that in animals that were previously untreated or were pretreated only with drug vehicle. Vaginae were opened at 30 days of age to permit identification of first proestrus by vaginal smears, and serial blood samples for LH and PRL radioimmunoassays were collected before and after treatment at proestrus. In previously untreated or vehicle-pretreated animals, THC treatment at 1300 hr on the day of first proestrus inhibited the afternoon surges of LH and PRL, whereas hormone surges occurred in control animals injected with only vehicle at 1300 hr proestrus (P less than .05). In contrast, animals pretreated with THC exhibited marked LH and PRL surges whether treated at 1300 hr proestrus with THC or vehicle. Among animals receiving only vehicle at proestrus, there was no difference in serum LH concentrations or in timing of the LH surge between the group pretreated with THC and the group pretreated with vehicle. These results are consistent with the hypothesis that ovulation eventually occurs in the pubertal rat treated chronically with THC because tolerance develops to its gonadotropin suppressive effects. PMID- 3018217 TI - Action at the mu receptor is sufficient to explain the supraspinal analgesic effect of opiates. AB - Mu, delta and kappa opiate receptors have been implicated in the production of analgesia. In order to study the relative contributions of these receptor types to supraspinally mediated analgesia, apparent pA2 values and rank order potencies were determined for i.c.v. injected highly selective opioid agonists in the mouse using a thermal nociceptive test. Drugs used included the prototypical mu agonist morphine, putative mu agonists [D-Ala2, N-methyl-Phe4, Gly5-ol] enkephalin and BAM 22P, putative delta agonists [D-Pen2, D-Pen5] enkephalin, [D-Thr2, Thr6, Leu5] enkephalin and [D-Ser2, Leu5, Thr6] enkephalin and the putative kappa agonists [trans-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide methane sulfonate hydrate] and Dynorphin A (1-13). We were unable to demonstrate significant analgesic potencies for i.c.v. injected Dynorphin A (1-13) or BAM 22P in the absence of marked behavioral abnormalities. The rank order potency of the remaining compounds studied was found to be: [D Ala2, N-methyl-Phe4, Gly5-ol] enkephalin greater than [D-Thr2, Thr6, Leu5] enkephalin greater than [D-Ser2, Leu5, Thr6] enkephalin greater than Morphine greater than [D-Pen2, D-Pen5] enkephalin greater than [trans-3, 4-dichloro-N methyl-N[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methane sulfonate hydrate]. Apparent pA2 values of morphine, [D-Ala2, N-methyl-Phe4, Gly5-ol] enkephalin and [D-Pen2, D-Pen5] enkephalin in naloxone antagonism trials did not differ significantly. These results indicate that although both mu- and delta selective ligands produce potent analgesia, a single receptor (mu) is sufficient to explain the supraspinal effects of opiate drugs. PMID- 3018218 TI - Comparison of endothelium-dependent relaxation in bovine intrapulmonary artery and vein by acetylcholine and A23187. AB - This study was conducted to compare the influence of endothelium on mechanical responses of bovine intrapulmonary artery and vein to acetylcholine (ACh) and A23187 and to determine if a relationship exists between the responses and cyclic GMP (cGMP) accumulation. ACh and A23187 induced relaxation in artery and A23187 induced relaxation in vein. The relaxant responses in both vessels were abolished by rubbing the intimal surfaces, indicating that the relaxant responses depended upon the presence of a functionally intact endothelium. cGMP accumulation was temporally associated with both ACh- and A23187-induced endothelium-dependent relaxation. Both the relaxant responses and the accompanying cGMP accumulations were abolished or reduced markedly by intimal rubbing or pretreatment with methylene blue. Atropine abolished relaxation and cGMP accumulation induced by ACh in artery, but not relaxation and cGMP accumulation induced by A23187. Whereas indomethacin did not affect either ACh- or A23187-induced relaxation in artery, it slightly, but significantly, reduced A23187-induced relaxation in vein. In contrast to its effect in artery, ACh only induced contractile responses in vein and did not alter cGMP levels, whether or not functional venous endothelium was present. However, ACh did relax veins when arterial endothelium was present in crossover experiments using a modified sandwich technique. At concentrations which induced endothelium-dependent relaxation in artery, ACh similarly induced no, or only minimal, contraction in both artery and vein in which endothelium was functionally destroyed. These findings demonstrate that bovine intrapulmonary artery and vein exhibit endothelium-dependent relaxation in response to A23187, and similar to ACh-induced relaxation in the artery, A23187 induced relaxation is associated closely with accumulation of cGMP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018219 TI - Metaphit, an acylating ligand for phencyclidine receptors: characterization of in vivo actions in the rat. AB - Metaphit, which acylates phencyclidine (PCP) receptors in vitro, was shown to acylate PCP receptors and antagonize the behavioral and electrophysiological effects of PCP in vivo. Metaphit (2 mumol/rat) administered i.c.v. produced PCP like stereotyped behavior and ataxia in 10 to 20% of rats. At a lower dose, Metaphit (1 mumol/rat) antagonized the ability of PCP to induce stereotyped behavior and ataxia for 3 and 4 days, respectively. The Metaphit-induced antagonism of PCP induction of stereotyped behavior and ataxia was dose-dependent and specific as Metaphit did not antagonize induction of stereotyped behavior by amphetamine. Further evidence for a specific PCP receptor mechanism was the finding that PCP pretreatment blocked the effects of subsequent Metaphit administration. Metaphit also antagonized PCP-induction of stereotyped behavior, but not ataxia, after i.v. administration. Doses of Metaphit that produced long term antagonism of the behavioral effects of PCP also produced a significant decrease in the maximum binding, but not Kd, of the binding of the PCP analog, [3H]-1-(2-thienyl)cyclohexyl]piperidine, in Metaphit-pretreated rats. The binding of [3H]etorphine and [3H]spiroperidol was not altered significantly by pretreating rats with Metaphit. (-)-Cyclazocine and (+)-SKF 10,047 induced stereotyped behavior and ataxia that was not antagonized by Metaphit pretreatment. In electrophysiological experiments, Metaphit, like PCP, initially depressed the firing of caudate neurons as does PCP, but then irreversibly inhibited PCP-induced depression of caudate neurons. These results suggest that metaphit antagonized the effects of PCP by selectively acylating PCP receptors and that (-)-cyclazocine- and (+)-SKF 10,047-induced behavioral effects are not mediated primarily by PCP receptors. PMID- 3018220 TI - Bone resorption induced by parathyroid hormone and dibutyryl cyclic AMP: role of carbonic anhydrase. AB - The role of carbonic anhydrase in bone resorption induced by parathyroid hormone (PTH) and dibutyryl cyclic AMP (DBcAMP) was studied using an in vitro neonatal mouse half-calvarial culture system. Both PTH (16.7 nM) and DBcAMP (0.3 mM) were effective in stimulating bone resorption, as assessed by measuring changes in media calcium concentrations. Bones treated with PTH or DBcAMP for 96 hr contained significantly greater carbonic anhydrase activity than control bones [PTH Treated/Control (T/C) = 2.44; DBcAMP T/C = 2.34]. Both PTH and DBcAMP significantly enhanced calvarial acid phosphatase activity relative to control values [PTH T/C = 1.48; DBcAMP T/C = 1.30]. Neither PTH nor DBcAMP significantly altered calvarial alkaline phosphatase activity. Bone resorption induced by PTH and DBcAMP was inhibited by the carbonic anhydrase inhibitor acetazolamide, but not by the acetazolamide analog CL 13,850 (N-t-butylacetazolamide), which does not inhibit carbonic anhydrase. These results support the concepts that PTH induced bone resorption requires the action of osteoclastic carbonic anhydrase and that the action of PTH on bone is mediated, in part, by the action of cyclic AMP. PMID- 3018221 TI - Autonomic mechanisms for morphine and amphetamine mydriasis in the rat. AB - Morphine sulfate and amphetamine sulfate dilate the pupil of the conscious, unrestrained rat. To examine the mechanisms underlying amphetamine and morphine mydriasis, the pupil diameter of freely moving rats was measured from photographs taken before and after these drugs were administered s.c., i.c.v. or by instillation into the eye. Some rats received monoamine antagonists, sympathetic denervation, or adrenalectomy before drug administration. The results indicate that both amphetamine and morphine mydriasis are primarily due to centrally mediated inhibition of parasympathetic outflow, although for both drugs there may be a sympathetic component. Pharmacological evidence indicates that the mydriatic effect of a low dose of amphetamine is mediated by actions on both alpha-1 and alpha-2 adrenergic receptors, whereas only alpha-2 adrenergic receptors are involved in morphine mydriasis. PMID- 3018222 TI - Putative roles for lysophospholipids as mediators and lipoxygenase-mediated metabolites of arachidonic acid as potentiators of stimulus-secretion coupling: dual mechanisms of p-hydroxymercuribenzoic acid-induced insulin release. AB - This paper explores the mechanism whereby insulin (I) secretion is stimulated by p-hydroxymercuribenzoic acid (PHMB), an agent which inhibits the esterification of arachidonic acid (AA) into phospholipids in intact rat islets. An effect of PHMB on I release could be seen even at substimulatory glucose concentrations (0 1.7 mM) and was resistant to blockade of energy flux using antimycin A, or of glucose metabolism using mannoheptulose. It was, however, inhibited by Ni++, Co++, La , replacement of chloride in the buffer by the impermeant anion isethionate or reduced ambient temperature (16 degrees C), but not by extracellular Ca++ depletion or 8-(N,N-diethylamino) octyl 3,4,5-trimethoxy benzoate hydrochloride (a putative stabilizer of intracellular Ca++ stores); thus PHMB's effect may require the translocation of membrane-associated Ca++ stores, leading to exocytotic hormone release. Although PHMB increases the accumulation of lipoxygenase-derived metabolites of AA, I secretion at 1.7 mM glucose unexpectedly was resistant to cyclooxygenase or lipoxygenase inhibition and could not be reproduced by exogenous AA (0.18 through 262 microM). However, it could be mimicked closely by exogenous lysophosphatidylcholine, which also shared with PHMB an identical profile of reversibility and pharmacologic inhibitability. Lysophospholipid (lyso-PL)-induced I release could not be attributed to detergent effects because, for example, it occurred in the absence of significant 51Cr release. The lyso-PL effect demonstrated structural specificity (lysophosphatidyl ethanolamine and lysophosphatidylserine being essentially inactive) and was specific for lyso-PLs as neither phosphatidylcholine itself nor glycerophosphorylcholine (the deacylation product of lysophosphatidylcholine) had any effect. In contrast to the effects of lyso-PLs, the energy-dependent effects of glucose (16.7 mM) or the amino acid alpha-ketoisocaproic acid (15 mM) on I release were abrogated by inhibitors of phospholipases or lipoxygenase. This effect of phospholipase inhibition could be circumvented by exogenous lyso-PLs. We conclude that lyso-PLs (generated by energy-dependent phospholipid deacylation or by inhibition of reacylation) may be true mediators of I release, whereas the role of concomitantly generated oxygenation products of AA is restricted to the modulation of stimulated release. PMID- 3018223 TI - Effects of chronic SCH23390 treatment on the biochemical and behavioral properties of D1 and D2 dopamine receptors: potentiated behavioral responses to a D2 dopamine agonist after selective D1 dopamine receptor upregulation. AB - Chronic treatment of rats with SCH23390 (0.5 mg/kg/day s.c.), a D1 dopamine receptor antagonist, for 21 days resulted in an increase in D1 dopamine receptors but produced no change in D2 dopamine receptors. During habituation to locomotor activity cages the rats treated chronically with SCH23390 showed significantly higher locomotor activity than controls treated chronically with saline. When injected with the selective D1 dopamine receptor agonist SKF38393 (3 mg/kg), rats treated chronically with SCH23390 showed significantly greater stereotypy and locomotor activity responses. Surprisingly, rats treated chronically with SCH23390 also showed significantly higher locomotor activity and stereotypy responses when treated with the selective D2 dopamine receptor agonist, quinpirole (LY171555) (0.3 mg/kg). These results indicate that a selective increase in D1 receptors may not be necessary, but is sufficient, to lead to an enhanced behavioral response to either selective D1 or D2 dopamine receptor agonists. If, indeed, an enhanced stereotypy and locomotor activity response to dopaminergic agonists in rats after a brief chronic treatment with a neuroleptic drug is predictive of tardive dyskinesia potential in the clinical setting, these results can suggest that SCH23390 may also induce tardive dyskinesia in humans. Adenylate cyclase activity stimulated by guanine nucleotides, forskolin or dopamine was enhanced after chronic treatment with SCH23390. However, dopamine stimulated adenylate cyclase activity was not potentiated detectably by the increase in receptor number over the more general increase in guanine nucleotide stimulated cyclic AMP production. Additionally, no change was observed in dopamine competition for [3H]SCH23390 binding, with dopamine's RH/RL ratio remaining unchanged.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018224 TI - Downregulation of [3H]Ro5-4864 binding sites after exposure to peripheral-type benzodiazepines in vitro. AB - Peripheral-type benzodiazepine (BZD) binding sites undergo a rapid and pronounced downregulation after exposure to these compounds in vitro. Friend erythroleukemia cells were incubated with micromolar concentrations of BZD after which they were washed thoroughly and the binding of the specific peripheral-type BZD radioligand [3H]Ro5-4864 was determined. Exposure to the peripheral-type BZD Ro7-3351 decreased the number of [3H]Ro5-4864 binding sites from 324 to 41 fmol/10(6) cells with no change in affinity. Downregulation appears to require active cellular processes because it is blocked when exposure to BZD is at 4 degrees C rather than at 37 degrees C. Furthermore, whereas [3H]Ro5-4864 binding is decreased substantially in membrane preparations made from downregulated cells, it is not altered when membrane preparations from control cells are exposed to BZD. The time course of downregulation is quite rapid, as it occurs within minutes. In contrast, the return of sites requires days and there is a close relationship between return of sites and growth of new cells. The ability of BZDs to downregulate correlates more closely with affinity for the peripheral-type site than with biological activity. The ability to undergo downregulation is characteristic of receptors and its occurrence suggests that peripheral-type BZD binding sites are functional receptors. PMID- 3018226 TI - Short-term desensitization of muscarinic cholinergic receptors in mouse neuroblastoma cells: selective loss of agonist low-affinity and pirenzepine high affinity binding sites. AB - The effects of brief incubation with carbamylcholine on subsequent binding of [3H]N-methylscopolamine were investigated in mouse neuroblastoma cells (clone N1E 115). This treatment demonstrated that the muscarinic receptors in this neuronal clone can be divided into two types; one which is readily susceptible to regulation by receptor agonists, whereas the other is resistant in this regard. In control cells, both pirenzepine and carbamylcholine interacted with high- and low-affinity subsets of muscarinic receptors. Computer-assisted analysis of the competition between pirenzepine and carbamylcholine with [3H]N-methylscopolamine showed that the receptor sites remaining upon desensitization are composed mainly of pirenzepine low-affinity and agonist high-affinity binding sites. Furthermore, there was an excellent correlation between the ability of various muscarinic receptor agonists to induce a decrease in consequent [3H]N-methylscopolamine binding and their efficacy in stimulating cyclic GMP synthesis in these cells. Thus, only the agonists that are known to recognize the receptor's low-affinity conformation in order to elicit increases in cyclic GMP levels were capable of diminishing ligand binding. Taken together, our present results suggest that the receptor population that is sensitive to regulation by agonists includes both the pirenzepine high-affinity and the agonist low-affinity receptor binding states. In addition, the sensitivity of these receptor subsets to rapid regulation by agonists further implicates their involvement in desensitization of muscarinic receptor-mediated cyclic GMP formation. PMID- 3018225 TI - Beta adrenoceptor control of the microvascular reserve in rabbit myocardium. AB - This study was performed to determine if the unperfused microvascular reserve in the rabbit heart can be controlled by beta adrenoceptors. Anesthetized, open chest rabbits (N = 54) were subjected to saline i.v., 0.7 mg/kg of atenolol i.v., 1 mg/kg of practolol, 0.1 microgram/kg of salbutamol, 1 microgram/kg of salbutamol or 0.7 mg/kg of atenolol + 1 microgram/kg of salbutamol treatments. In half these animals coronary flows were determined before and after treatment using radioactive microspheres. The others were given 100 mg/kg of fluorescein isothiocyanate-dextran and the hearts were removed and analyzed for perfused and total microvascular morphology. The fluorescence marked the perfused microvessels and the slides were stained to show all vessels. Control group blood flow was 213 +/- 25 ml/min/100 g. Flows decreased 25% with atenolol and practolol whereas no change was seen with salbutamol or atenolol + salbutamol. Total capillary volume fraction ranged from 0.15 to 0.20 mm3/mm3 with 60% of this perfused in controls. Atenolol significantly reduced this to 47% whereas practolol (90%), high-dose salbutamol (97%) and salbutamol + atenolol (99%) resulted in a significant mobilization. Low-dose salbutamol resulted in a mobilization of capillaries to a degree intermediate (79%) between controls and high-dose salbutamol. Total arteriolar volume fraction ranged from 0.002 to 0.004 mm3/mm3 and in controls 65% of this was perfused. This percentage was reduced with atenolol and significantly increased with all other treatments except practolol. Thus, blockade of beta-1 adrenoceptors results in decreases in the percentage of perfused microvessels whereas stimulation of beta-2 adrenoceptors results in an increase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018228 TI - Human psychopharmacology of ketocyclazocine as compared with cyclazocine, morphine and placebo. AB - The activity of ketocyclazocine, a putative kappa opioid receptor agonist, was studied and compared with that of morphine, a mu opioid receptor agonist, and cyclazocine, a putative kappa and sigma opioid receptor agonist, vs. placebo in 10 drug abusers. The measures included vital signs and pupil measurements, established and new observer- and subject-completed psychopharmacologic questionnaires and several methods of drug discrimination. The results indicate that ketocyclazocine is different from morphine-like agonists in that it produces only minimal miosis and lacks euphoriant action. It causes a dysphoric state and was clearly discriminated from morphine. The dysphoria and pattern of responses was similar to cyclazocine though ketocyclazocine was discriminated from cyclazocine. This is consistent with the concept that morphine and ketocyclazocine have separate modes of primary activity. The similarity between ketocyclazocine and cyclazocine obscures the assignment of particular drug effects to activity at the kappa receptor. PMID- 3018227 TI - Hypotensive and renal vasodilator effects of carbocyclic adenosine (aristeromycin) in anesthetized spontaneously hypertensive rats. AB - Systemic arterial pressure and renal blood flow were measured in pentobarbital anesthetized spontaneously hypertensive rats to assess the influence and mechanism of action of metabolically stable adenosine analogs on renal hemodynamics. (-)-Aristeromycin (carbocyclic adenosine; CA), a model carbocyclic nucleoside, was characterized with respect to adenosine receptor pharmacology by comparison to the effects elicited by the prototypic adenosine analogs 5'-N ethylcarboxamide adenosine (NECA; an adenosine A1 and A2 receptor agonist) and N6 cyclohexyl adenosine (an adenosine A1 agonist). Intravenous bolus injections of CA and NECA caused dose-dependent hypotension and renal vasodilatation. Although CA and NECA were equally efficacious hypotensive agents, NECA was approximately 100-fold more potent than CA. CA was a more efficacious renal vasodilator than NECA. In contrast, at doses which had minimal effects on systemic arterial pressure, N6-cyclohexyl adenosine decreased renal blood flow. The hypotensive and renovascular effects of the adenosine analogs but not those of a control vasodilator, methacholine, were attenuated by i.v. administration of the xanthines aminophylline and 8-phenyltheophylline; thus, the effects of the nucleosides on renal blood flow in vivo appear to be attributable in part to activation of adenosine receptors. The profile of cardiovascular effects caused by CA suggests that this agent acts primarily as an adenosine A2 receptor agonist. PMID- 3018229 TI - In vivo and in vitro effects of WR-2721 in experimental hypercalcemia in the rat. AB - The effects of WR-2721 [S-2-(3-aminopropylaminoethyl)phosphorothioic acid] in two in vivo and in vitro models of experimental hypercalcemia in the rat were examined. Chronic WR-2721 administration by osmotic minipump (250 mg/kg/24 hr) reduced serum calcium from 12.0 +/- 0.1 to 9.5 +/- 1.0 mg/dl (P less than .01) in rats receiving 1,25-(OH)2 Vitamin D3. Control rats receiving Vitamin D without WR 2721 had a rise in serum calcium to 13.4 +/- 0.2 mg/dl over the same 5-day period. In an experimental form of humoral hypercalcemia of malignancy, the Walker carcinosarcoma tumor-implanted rat, WR-2721 reduced serum calcium from 13.6 +/- 0.3 to 8.4 +/- 0.6 mg/dl by 5 to 6 days (P less than .001). In vitro bone resorption assays utilizing fetal rat long bones in organ culture showed complementary results. WR-2721 (10(-4) M) blocked bone resorption (assayed as percentage of 45Ca release) induced by both conditioned medium derived from cell lines of Walker carcinosarcoma (7.6 +/- 1.4 vs. 24.0 +/- 1.8%, P less than .01) and by addition of 1,25-(OH)2 Vitamin D3 (10(-8) M) (9.8 +/- 0.8 vs. 17.3 +/- 1.0%, P less than .01). These results suggest that WR-2721 may be effective in controlling clinical hypercalcemia due to excess bone resorption. PMID- 3018230 TI - Behavioral effects of opioid peptides selective for mu or delta receptors. I. Morphine-like discriminative stimulus effects. AB - The morphine-like discriminative stimulus effects of opioid peptides with selectivity for the mu- or delta-opioid receptors were examined in rats trained to discriminate 3.0 mg/kg of morphine (s.c.) from saline in a two-choice discrete trial avoidance paradigm. The mu-selective peptides D-Ala2-NMePhe4 Gly5(ol)enkephalin, FK 33,824 and morphiceptin, the delta-selective peptides D Ala2-D-Leu5enkephalin and metkephamid and beta-endorphin (mu- and delta selective) produced morphine-like stimulus effects after administration into the lateral ventricle. Generalization with the morphine cue was dose-dependent and occurred over a wide range of doses (0.01-30 micrograms), depending upon peptide. On a molar basis, the order of relative potency of the peptides as morphine-like discriminative stimuli was: D-Ala2-NMePhe4-Gly5(ol)enkephalin = FK 33,824 greater than beta-endorphin greater than D-Ala2-D-Leu5enkephalin = metkephamid greater than morphiceptin. The discriminative effects of D-Ala2-NMePhe4 Gly5(ol)enkephalin, D-Ala2-D-Leu5enkephalin and beta-endorphin were antagonized by low doses of s.c. naltrexone (0.01-1.0 mg/kg). Furthermore, the stimulus effects of s.c. morphine were antagonized by 24-hr pretreatment of rats with the irreversible mu-antagonist beta-funaltrexamine (5.0 micrograms i.c.v.). Based upon the order of relative potency of the peptides and the relative potency for antagonism of their discriminative effects by naltrexone and beta-funaltrexamine, mu-opioid receptors in the brain appear to be an important element in the genesis of morphine-like discriminative effects by opioid peptides. PMID- 3018231 TI - Behavioral effects of opioid peptides selective for mu or delta receptors. II. Locomotor activity in nondependent and morphine-dependent rats. AB - The i.c.v. administration of opioid peptides having selectivity for the mu receptor (D-Ala2-NMePhe4-Gly5(ol)enkephalin and FK 33,824) produced effects on the locomotor activity of nondependent and morphine-dependent rats that differed both quantitatively and qualitatively from those effects produced by peptides having selectivity for the delta receptor (D-Ala2-D-Leu5enkephalin and metkephamid) and beta-endorphin, which has similar affinity for both receptors. Peptides selective for the mu receptor: had a biphasic effect on locomotor activity of nondependent rats, inducing an increase at low doses and an initial decrease followed by a later increase at higher doses and had an enhanced stimulant effect on locomotor activity with tolerance to the depressant effect in morphine-dependent rats. Peptides selective for the delta receptor and beta endorphin: induced only a dose-related increase in the locomotor activity of nondependent rats and had effects on the locomotor activity of morphine-dependent rats that did not differ substantially from those in nondependent rats. Naltrexone (0.1 mg/kg s.c.) and beta-funaltrexamine (5.0 micrograms/rat i.c.v.), an irreversible antagonist, each blocked to a comparable extent the effects of D Ala2-NMePhe4-Gly5(ol)enkephalin and DAla2-D-Leu5enkephalin on the locomotor activity of nondependent rats. Thus, effects of opioid peptides that act predominantly at mu or delta receptors on locomotor activity cannot be differentiated in nondependent rats by antagonists but can be differentiated in morphine-dependent rats. These results suggest that the depressant and stimulant effects of opioid peptides on locomotor activity are mediated by distinct neuronal sites. PMID- 3018232 TI - Effects of the renin-angiotensin system on the reflex response of the adrenal medulla to hypotension in the dog. AB - We have studied the influence of the renin-angiotensin system on the control of catecholamine release from innervated and denervated adrenal glands of anaesthetized dogs. Captopril reduced the resting release of catecholamines and inhibited release evoked either by lowering carotid sinus pressure or by stimulating the peripheral end of the cut splanchnic nerve. Both responses were restored by exogenous angiotensin II, and the reflex response could also be restored by corticotrophin. Cycloheximide, in the presence of captopril, further reduced the resting release of catecholamines and prevented the restoration of the reflex response by angiotension II. Plasma renin activity did not rise during baroreceptor tests lasting 10 min, but catecholamine release was evoked from the first minute. We conclude that the response of the adrenal medulla to sympathetic activity requires a minimum circulating concentration of angiotensin II. It is severely impaired by inhibition of the renin-angiotensin system but function can be restored either by exogenous angiotensin II or by corticotrophin. PMID- 3018233 TI - Excitatory synaptic interactions between CA3 neurones in the guinea-pig hippocampus. AB - Excitatory synaptic interactions between CA3 neurones in slices from guinea-pig hippocampus were examined. Recurrent excitatory post-synaptic potentials (e.p.s.p.s) were evoked by action potentials in a single presynaptic neurone or by the antidromic activation of part of the CA3 pyramidal cell population. The peak amplitude of unitary e.p.s.p.s was 1-2 mV at potentials between -64 and -70 mV. Their time to peak was 7-12 ms and the initial phase of their decay was slower than that of a somatically injected voltage pulse. Recurrent e.p.s.p.s were often followed by a small (0.3 mV) hyperpolarization, or undershoot. Recurrent e.p.s.p.s were compared with e.p.s.p.s evoked by stimulating mossy fibres, which terminate proximally on apical dendrites of CA3 pyramidal cells. They were of slower time course and reversed at a more positive potential than mossy fibre e.p.s.p.s. Some synaptic terminals made by recurrent axon collaterals apparently terminate at distant locations on apical dendrites. The decay of both recurrent e.p.s.p.s and dendritic voltage pulses was prolonged by membrane depolarization within a 10-15 mV subthreshold potential range. Voltage-dependent inward currents activated by the synaptic depolarization may contribute to the slow initial decay of these synaptic events. The undershoot did not occur when transmission of a unitary e.p.s.p. failed and was of slower time course than the hyperpolarization due to an inhibitory post-synaptic potential (i.p.s.p.). It was suppressed by intracellular application of K+ channel blockers and probably reflects an intrinsic outward current activated as a consequence of the synaptic depolarization. Considerable temporal summation of synaptic potentials occurred when recurrent synapses were activated twice at an interval of 5-10 ms, typical of the spontaneous burst firing pattern of CA3 neurones. The mean facilitation of a second e.p.s.p. at this interval was about 0.6. The efficacy of a third and subsequent e.p.s.p.s at similar interval was reduced. Presynaptic bursts of three to five action potentials evoked summed e.p.s.p.s of amplitude 2-4 mV, with time to peak 20-40 ms and decaying phase of similar duration. Their rising phase was relatively smooth and summed events were succeeded by an undershoot. Presynaptic bursts could cause a post-synaptic neurone to discharge. PMID- 3018235 TI - [Time series analysis of the neuronal impulse signal and its physiological significance]. PMID- 3018234 TI - Comparative development of end-plate currents in two muscles of Xenopus laevis. AB - The development of miniature end-plate currents (m.e.p.c.s) was studied in the superior oblique and interhyoideus muscles of Xenopus laevis. An analysis of m.e.p.c. decays shows that each muscle possesses its own characteristic programme of end-plate current development. In the superior oblique, the exponential decay constants of m.e.p.c.s were initially about 3 ms; they declined within half a day to 1 ms and remained at that value for six weeks. They then gradually became longer, reaching a mean value of 1.7 ms at late metamorphosis. In the interhyoideus, m.e.p.c. decay constants were initially about 6 ms. They declined in less than one day to a mean value of 2.6 ms and remained there for the following seven weeks. Upon completion of metamorphosis, the decay constants underwent a further decrease to about 1 ms. In both muscles, the changes in m.e.p.c. decays were correlated with developmental changes in muscle contraction speeds, as measured by maximum twitch frequencies. The above changes in end-plate currents in the superior oblique and interhyoideus muscles are discussed in terms of the development of acetylcholine receptor channel gating and acetylcholinesterase activity. PMID- 3018236 TI - Particles versus solid forms of hydroxyapatite as a treatment modality to preserve residual alveolar ridges. AB - In two separate but related studies, different forms of hydroxyapatite were implanted into the extraction sockets of human teeth to delay alveolar resorption and to form the background for a comparison of the treatment modalities. The significant differences in the treatment modalities and the postoperative sequelae seem to merit this report. The implantation of the particles appears to be clinically, a more expedient procedure than the implantation of cones. The time required to select an appropriate-sized cone, modify the cone as needed to achieve a snug fit into the extraction socket, and seat the cone deeply enough in the extraction socket to assure at least 2 mm of bone above the top of the cone implant was significantly greater than the time required to fit and pack particles into an extraction socket. None of the postimplantation problems encountered with cones was encountered in using the particle implants. The postimplantation problems encountered with cones included submucosal prominence, erosion through the mucosa (dehiscence), migration, loss of the implant, or surgical maintenance or resubmergence. Data from these two studies suggest that the implantation of particles into the extraction sockets of human teeth to delay alveolar ridge resorption is a more prudent, forgiving, considerate, problem free, and predictable procedure than the implantation of cones. PMID- 3018237 TI - Using soft vinyl stents to facilitate augmentation of maxillary anterior atrophic ridges with hydroxyapatite. AB - A technique has been described for fabrication of a surgical template made of soft vinyl mouthguard material. Use of this stent at the time of surgery can be of great value to the oral surgeon and the prosthodontist. The risk of excessive augmentation and migration of the particulate hydroxyapatite to an undesired area will be minimized if the stent is used as a matrix. Uniform results in size and form of the ridge can be obtained from the surgical procedure when the stent is used. The denture-bearing area of the maxillary anterior ridge will improve significantly, enabling the dentist to make a maxillary complete denture. PMID- 3018239 TI - Pancreatic glucagonoma with lymph node metastases: disease-free survival six years after resection. PMID- 3018238 TI - Demonstration of various acid hydrolases and preliminary characterization of acid phosphatase in Naegleria fowleri. AB - Extracts of the pathogenic ameba Naegleria fowleri, prepared by freeze-thawing and sonication, were analyzed for their content of various hydrolytic enzymes that have acid pH optima. The organism is rich in acid phosphatase activity as well as a variety of glycosidases which include beta-glucosidase, beta galactosidase, beta-fucosidase, alpha-mannosidase, hexosaminidase, arylsulfatase A, and beta-glucuronidase. The crude extract contained only negligible levels of sphingomyelinase, neuraminidase, or arylsulfatase B. All of the hydrolases exhibited higher activity at pH 5.5 than at 7.0, indicating that they are truly "acid" hydrolases. In general, after centrifugation (100,000 g, 1 h), except for arylsulfatase B, more than half of the activity of each of the various hydrolases was recovered in the supernatant fraction. The acid phosphatase in the high-speed supernatant was purified 45-fold (32% yield) by chromatography on QAE-Sephadex and Sephadex G-200 and shown to have the following properties: pH optima, 5.5; Km (4-methylumbelliferyl phosphate), 0.60 mM; molecular weight (estimated by gel filtration chromatography), 92,000; inhibited by heteropolymolybdate complexes but not by L(+) sodium tartrate (0.5 mM) or sodium fluoride (0.5 mM). In addition, unlike the tartrate-resistant acid phosphatase of Leishmania donovani, the major acid phosphatase of N. fowleri is less than 5% as effective in inhibiting superoxide anion production by f-Met-Leu-Phe-stimulated human neutrophils. The finding of high levels of a number of acid hydrolases in Naegleria fowleri raises several questions that merit further study: Do the hydrolases perform a housekeeping function in this single cell eukaryote or do they play some role in the pathogenic process that ensues when the organism infects a suitable host? PMID- 3018240 TI - Parvovirus infection in a family with wheeze in an adult. AB - In a family outbreak of human parvovirus infection, a 59-year-old doctor had a prodromal illness with wheeze. This was followed by a distinct acute episode with itch and arthropathy and a two-month phase characterized by intermittent cough and wheeze. PMID- 3018241 TI - 3,4-Dihydro-2-phenyl-2H-pyrano[2,3-b]pyridines with potent antirhinovirus activity. AB - A general synthesis to the title compounds 1, substituted in the 6-position and on the phenyl ring, is outlined. Eighteen analogues were compared with respect to in vitro activity against rhinovirus types 1A, 9, and 64. Compounds 1c and 1h, the 6-bromo- and 6-(methylsulfonyl)-3',4'-dichlorophenyl analogues, afforded median MIC50 values against 23 rhinovirus serotypes of 0.05 and 0.13 micrograms/mL, respectively. Mice dosed orally with 200 mg/kg of 1c or 1h exhibited serum levels well in excess of each compound's MIC50, indicating that some analogues have the potential to be orally effective drugs. PMID- 3018242 TI - Synthesis and opioid antagonist potencies of naltrexamine bivalent ligands with conformationally restricted spacers. AB - Bivalent ligands 1-4 with naltrexamine pharmacophores and spacers of different lengths containing a fumaryl moiety were synthesized and evaluated for mu and kappa opioid antagonist activity on the electrically stimulated guinea pig ileal longitudinal muscle (GPI). The fumaryl moiety was incorporated into the spacer in order to determine the effect of conformational restriction of the spacer on the relationship between spacer length and opioid antagonist potency. While it was found that the fumaryl and succinyl series (11) possessed a very similar structure-potency profile with respect to antagonism at mu opioid receptors, the interaction of these two series at kappa receptors differed substantially from one another. This difference was manifested by the longer spacer requirement for peak kappa antagonist potency in the fumaryl relative to the succinyl series. It is concluded that the conformational restriction imposed by the fumaryl group in a short spacer (n = 0) prevents effective interaction of both pharmacophores with vicinal recognition sites of the kappa receptor system; as the spacer is lengthened (n = 2) and becomes more flexible, the simultaneous occupation of vicinal recognition sites occurs with greater facility. PMID- 3018243 TI - Influence of alkyl chain ramification on estradiol receptor binding affinity and intrinsic activity of 1,2-dialkylated 1,2-bis(4- or 3-hydroxyphenyl)ethane estrogens and antiestrogens. AB - The influence of a symmetrical introduction of CH3 substituents in the alpha or beta positions of the 1,2-dialkyl-1,2-bis(4-hydroxyphenyl)ethane estrogens hexestrol (ethyl, HES) and octestrol (n-propyl, OCES) [isopropyl (1), tert-butyl (2), sec-butyl (3), isobutyl (4)] and the 1,2-dialkyl-1,2-bis(3 hydroxyphenyl)ethane antiestrogens metahexestrol (ethyl, MetaHES) and metaoctestrol (n-propyl, MetaOCES) [isopropyl (5), tert-butyl (6), sec-butyl (7), isobutyl (8)] on estradiol receptor (E2R) binding affinity and intrinsic activity is described. The synthesis of compounds 1-8 was accomplished by reductive coupling of (a) the corresponding alpha-alkylbenzyl alcohols with TiCl3/LiAlH4 and separation of the meso diastereoisomers (compounds 2-4, 7, and 8), (b) alpha tert-butyl-3-methoxybenzyl chloride with CoCl2/EtMgCl and isolation of the meso configurated isomer (6), and (c) the isopropyl phenyl ketones with TiCl4/Zn and subsequent hydrogenation of the corresponding cis-hex-3-enes (compounds 1 and 5). The binding affinity of 1-8 to the calf uterine E2R was measured relative to that of [3H]E2 by a competitive binding assay. Compounds 1 and 5 showed relative binding affinity (RBA) values exceeding those of HES and MetaHES, respectively. All other derivatives showed RBA values smaller than the corresponding parent compounds. The intrinsic activity was monitored in terms of uterotrophic and antiuterotrophic activity. It is striking that the introduction of a CH3 group in the alpha positions of MetaHES and MetaOCES led to compounds with full intrinsic activity (5-7), i.e., estrogens without antiestrogenic properties. No correlation between E2R binding affinity and intrinsic activity was found. PMID- 3018244 TI - N6-substituted N-alkyladenosine-5'-uronamides: bifunctional ligands having recognition groups for A1 and A2 adenosine receptors. AB - The coronary vasoactivity of N-ethyl-1'-deoxy-1'-(6-amino-9H-purin-9-yl)-beta-D ribofuranuronamide (NECA, 1) is over 2 orders of magnitude greater than that of adenosine, and the vasoactivity of certain N6-substituted adenosines is as much as 1 order of magnitude greater. Such results suggest that a combination of appropriate modifications at N6 and C-5' might additively augment the agonist potency of adenosine. At low temperatures 1-deoxy-1-(6-chloro-9H-purin-9-yl) 2',3'-O-isopropylidene- beta-D-ribofuranosyl chloride (5), obtained in three steps from inosine, reacts with amines to yield uronamides. The subsequent reaction of such uronamides with amines at elevated temperatures displaces the purine 6-chloro group to yield, after deblocking, N-alkyl(or aryl)-N6-alk(ar)yl adenosine-5'-uronamides. At the coronary artery A2 receptor the potency of N6 modified analogues of 1 is similar to that of the N6-substituted adenosine, rather than equal to or greater than 1. As agonists in the A2 receptor-mediated stimulation of adenylate cyclase in plasma membranes of PC12 pheochromocytoma cells or human platelets, N6-substituted analogues of 1 are intermediate between the high potency of 1 and the lower potency of the N6-substituted adenosines. At the A1 receptor of rat brain the potency of an N6-substituted analogue of 1 is often greater than that of the corresponding N6-substituted adenosine. At all four receptors, replacing the ethyl group of N-ethyl-N6-3-pentyladenosine-5' uronamide by larger alkyl groups reduces potency; amides of secondary amines are inactive or have only marginal activity. Analogues of 1 containing a chiral center in the N6 substituent retain the stereoselectivity characteristic of each of the four receptors. Thus, at either A1 or A2 adenosine receptors, adenosine analogues interact with both the N6 and the C-5' receptor regions. However, the effects of N6 and C-5' modifications on potency are less than additive, evidence that the interaction of a substituent with its receptor region influences the interaction of other substituents with their respective receptor regions. PMID- 3018245 TI - Synthesis of anthraquinonyl glucosaminosides and studies on the influence of aglycone hydroxyl substitution on superoxide generation, DNA binding, and antimicrobial properties. AB - A series of anthraquinonyl glucosaminosides (10a-e) were synthesized by Koenigs Knorr glycosidation of the corresponding aglycones (11a-e) with bromo sugar 12 followed by saponification. These glycosides were intended to serve as models to study the role played by the hydroxyl substituents on the aglycone portion of the antitumor anthracycline antibiotics. Superoxide generation as measured in rat heart sarcosomes was found to increase with the addition of successive hydroxyl groups to the anthraquinone nucleus. The 1,8-dihydroxy pattern was determined to generate significantly less superoxide than the 1,4-dihydroxy pattern. Hydroxyl substitution was also observed to stabilize the complex formed between the anthraquinones and DNA and was required for antibacterial activity against a number of Gram-positive organisms. PMID- 3018246 TI - Carbocyclic analogues of 5-halocytosine nucleosides. AB - Carbocyclic analogues of 5-halocytosine nucleosides were prepared by direct halogenation of the carbocyclic analogues of cytidine, 2'-deoxycytidine, 3' deoxycytidine, or ara-C. The 5-chloro and 5-bromo derivatives of the cytidine (carbodine) and of the 2'-deoxycytidine analogues and the 5-iodo derivatives of all four of the cytosine nucleoside analogues were prepared. All of the C-5 halocytosine nucleosides, as well as the parent C-cytosine nucleosides, were tested against a strain of herpes simplex virus type 1 (HSV-1) that induces thymidine kinase in host cells. Carbodine, 5-bromocarbodine, C-2'-deoxycytidine, C-5-bromo-2'-deoxycytidine, the four C-5-iodocytosine nucleosides, and C-ara-C inhibited replication of this strain of HSV-1 in cultured cells. Most of these compounds were tested also against the type 2 virus (HSV-2) in vitro and were active. The greatest activity observed was exerted by C-5-iodo-2'-deoxycytidine in inhibiting replication of HSV-1 in L929 cells. In tests against these DNA viruses, carbodine, a ribofuranoside analogue that had been shown previously to be highly active against human influenza A virus in vitro, was the most active compound against HSV-2 and one of the most active compounds against HSV-1 in Vero cells. 5-Bromocarbodine was active against influenza virus, but it was less active than carbodine. PMID- 3018247 TI - Cystic fibrosis carrier detection using a linked gene probe. AB - Cloned DNA markers which are closely linked to the gene defect causing cystic fibrosis have recently been described. These markers are sufficiently informative for carrier detection in 80% of families where there is a living cystic fibrosis child and unaffected sibs. The tightly linked DNA marker pJ3.11 was used in this study to identify carriers in six families and exclude carrier status in two subjects. Risk calculations for recessive diseases using linked DNA probes may be complex, but useful information for counselling can be obtained in this way. PMID- 3018249 TI - Poland syndrome associated with 'morning glory' syndrome (coloboma of the optic disc). AB - A 12 year old girl with the Poland syndrome and the 'morning glory' syndrome is described. The patient presented with absence of the left pectoralis major muscle, hypoplasia of the left arm, symbrachydactyly, and ipsilateral coloboma of the optic disc. This is the first report of the association of these two congenital anomalies. PMID- 3018250 TI - Distribution and genetic location of Tn7 in trimethoprim-resistant Escherichia coli. AB - A series of 178 strains of Escherichia coli, highly resistant to trimethoprim, isolated from hospital patients and patients in the community between 1979 and 1983, was examined for the presence of Tn7 on a plasmid or on the chromosome only, by transposition to RP4 and restriction endonuclease digestion with Hind III. Of the isolates, 57% carried Tn7. Comparison of hospital isolates from 1979 to 1980 and 1982 showed that although the proportions that carried Tn7 were similar (63% and 57%) there had been a significant change in the genetic location of the transposon. The proportion of plasmid-mediated Tn7 had fallen from 62% to 30% with a corresponding rise in Tn7 located exclusively on the chromosome from 38% to 70%. This change may be the result of continuing transposition of Tn7 from plasmids to the bacterial chromosome followed by plasmid loss. The consequent reduction in the mobility of trimethoprim-resistance genes may in turn lead to changes in the incidence of resistance. PMID- 3018248 TI - Application of three intragenic DNA polymorphisms for carrier detection in haemophilia B. AB - In the west of Scotland use of a single intragenic restriction fragment length polymorphism (F9(VIII)/TaqI) allowed definitive genetic counselling for 45% of females at risk of being carriers for haemophilia B. Two further intragenic RFLPs, F9(VIII)/XmnI) and F9(VIII)/DdeI, have been applied to this population and by using all three polymorphisms the carrier status could be determined in 68% of females at risk. Linkage disequilibrium was apparent between these three RFLPs, and in the west of Scotland the single most clinically useful polymorphism was F9(VIII)/TaqI followed by F9(VIII)/DdeI and then F9(VIII)/XmnI. Overall, prenatal diagnosis by DNA analysis could be offered to 31 of 37 (84%) carriers (obligate and detected) in these families. PMID- 3018251 TI - Coagglutination reactions between Candida albicans and oral bacteria. AB - An agglutination assay for detecting intermicrobial adherence between the cells of Candida albicans and various oral bacteria is described. Strains of Streptococcus sanguis, S. salivarius, S. mutans, S. mitis, Fusobacterium nucleatum and Actinomyces viscosus all coagglutinated with C. albicans. No interaction could be demonstrated between the cells of Bacteroides melaninogenicus and those of C. albicans. Preliminary investigations of these interactions suggest that binding of F. nucleatum and A. viscosus to C. albicans is mediated by bacterial proteins, possibly lectins. Other mechanisms must account for the binding of oral streptococci to C. albicans. The possible implications of these findings in relation to oral mucosal colonisation and oral candidal clearance are discussed. PMID- 3018252 TI - Factors affecting growth of Legionella pneumophila in liquid media. AB - The growth in liquid media of Legionella pneumophila serogroups 1-6 was monitored turbidimetrically and factors affecting growth rate were studied. The presence of inhibitors, use of detoxifying agents and the method of broth preparation each had significant effects on cultivation. Cysteine was essential for growth; the optimal concentration was 100 micrograms/ml, but supplemental iron had no demonstrable effect. PMID- 3018253 TI - Antibodies against herpes simplex virus in a group of healthy humans without reactions to herpes simplex virus antigens in blasttransformation assays. AB - 22 healthy individuals with different reactions in humoral and cell-mediated immunity to herpes simplex virus (HSV), as found in complement-fixation, neutralization, and blasttransformation assays have been further investigated using antibody-dependent cell-mediated immunity (ADCC) as a serological test. Sera from all individuals were absorbed on HSV-infected, varicella zoster virus (VZV)-infected or uninfected cell monolayers, before they were used in ADCC with HSV-infected cells, as target. The results with absorbed sera were compared with the results from unabsorbed sera. Fresh uninactivated sera have also been used in some of the investigations. By these experiments a group of individuals was found, who were HSV-positive in ADCC, but negative in all other, above-mentioned tests. Because of these reactions they are proposed to be HSV-positives without a latent infection. PMID- 3018254 TI - Rosette inhibitory factor (RIF) augments cyclic AMP generation by helper T lymphocytes. AB - The effect of Rosette Inhibitory Factor (RIF) on cyclic AMP generation by peripheral blood mononuclear cells (PBMC) and lymphocyte subpopulations (OKT4+ or helper T cells, OKT8+ or suppressor T cells, B lymphocytes) was analyzed. The results were compared to those observed with isoproterenol, an agent which is known to inhibit erythrocyte rosette (ER) formation and increase cyclic AMP levels in T cells. RIF elevated cyclic AMP levels in PBMC, an effect which was observed to be confined to helper T cells. Isoproterenol also elevated levels of cyclic AMP in PBMC but non-selectively augmented levels of the cyclic nucleotide in all lymphocyte subpopulations. Whereas isoproterenol increased cyclic AMP levels within 30 min, RIF did so after a lag period of 4 hr, a delay which is necessary for inhibition of ER formation. These studies reaffirm the selective effect of RIF on helper T cells. We conclude that inhibition of ER formation and possibly other immunoregulatory effects associated with RIF may be mediated via modulation of the adenyl cyclase-cyclic nucleotide network. PMID- 3018255 TI - Adrenergic regulation of ion transport by primary cultures of canine tracheal epithelium: cellular electrophysiology. AB - We examined the effect of adrenergic agents on the cellular electrical properties of primary cultures of canine tracheal epithelium. Both isoproterenol and epinephrine stimulated Cl secretion, as evidenced by an increase in transepithelial voltage and a fall in transepithelial resistance. Moreover, both agents appear to increase the conductance of apical and basolateral membranes. However, the pattern of response was different. Isoproterenol initially depolarized apical voltage psi a and decreased the fractional resistance of the apical membrane fR. These changes are consistent with an initial increase in apical Cl conductance. In contrast, epinephrine acutely hyperpolarized psi a and increased fR, changes consistent with an initial increase in basolateral K conductance. Following the acute effect of epinephrine, psi a depolarized and fR decreased to values not significantly different from those observed with isoproterenol. The acute increase in basolateral K conductance produced by epinephrine appeared to result from stimulation of alpha adrenergic receptors because it was reproduced by addition of the alpha agonist phenylephrine, and blocked by the alpha antagonist phentolamine. The ability of prazosin but not yohimbine to block the acute epinephrine-induced increase in K permeability indicates the presence of alpha 1 adrenergic receptors. The acute alpha adrenergic-induced increase in basolateral K conductance may be mediated by an increase in cell Ca because the response was mimicked by addition of the Ca ionophore A23187. In contrast, the response to isoproterenol was similar to that observed with addition of 8-bromo-cAMP and theophylline. These results indicate that both beta and alpha adrenergic agents mediate the ion transport processes in canine tracheal epithelium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018257 TI - Role of the Na+/H+ antiport in the regulation of the internal pH of Ehrlich ascites tumor cells in culture. AB - Ehrlich ascites tumor cells contain a Na+ uptake system, which is activated by internal protons and is inhibited by amiloride with an IC50 of 25 microM and by dimethylamiloride with an IC50 of 0.6 microM at 1 mM external Na+. Decrease of external Na+ or addition of amiloride is followed by a decrease of internal pH. Taken together, these findings suggest the presence of an operative Na+/H+ antiport system, which is involved in the regulation of internal pH. We cannot find a significant contribution of a proton pump activated by glycolysis to the pH gradient. At an external pH between 7.0 and 7.6, quiescent cells are more alkaline than exponentially growing cells (0.1 to 0.17 units). Accordingly, an increase of the affinity of the Na+/H+ antiport for internal protons in quiescent cells is demonstrated by the following findings: The internal pH, at which the half-maximal activation of the amiloride-sensitive Na+ uptake occurs, is shifted from 6.85 to 7.1 at 1 mM external Na+. The threshold value of external pH, below which a pronounced effect of amiloride on steady-state internal pH is observed, is shifted from 7.0 in growing to 7.5 in quiescent cells at physiological Na+ concentrations. Therefore, we conclude that quiescent Ehrlich ascites tumor cells raise their internal pH by increasing the affinity of their Na+/H+ antiporter to internal protons. The Na+/H+ antiport cannot be activated further by addition of serum growth factors to quiescent cells. All experiments were performed at bicarbonate concentrations in the medium which do not exceed 0.5 mM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018256 TI - Characterization of the Band 3 substrate site in human red cell ghosts by NDS TEMPO, a disulfonatostilbene spin probe: the function of protons in NDS-TEMPO and substrate-anion binding in relation to anion transport. AB - NDS-TEMPO is a specific disulfonatostilbene spin label for the Band 3 substrate site (K.F. Schnell, W. Elbe, J. Kasbauer & E. Kaufmann, Biochim. Biophys. Acta 732:266-275, 1983). The pH dependence of NDS-TEMPO binding and of chloride and sulfate binding was studied in resealed human erythrocyte ghosts. pH was varied from 6.0 to 9.0. The ESR spectra from NDS-TEMPO-labeled red cell ghosts exhibited a strong immobilization of membrane-bound NDS-TEMPO. Changes of pH had no effect upon the mobility of membrane-bound NDS-TEMPO. A mutual competition between NDS TEMPO binding and the binding of the substrate-anions, chloride and sulfate, was observed throughout the entire pH range. The maximal number of NDS-TEMPO binding sites per cell was in the range of 9.0 X 10(5) to 1.10 X 10(6) and was found to be insusceptible to changes of pH. The NDS-TEMPO/substrate-site and the chloride/substrate-site dissociation constants amounted to 1.25 microM and to 17 mM and were independent of pH from pH 6.0 to 8.0, while the sulfate/substrate site dissociation constant displayed a strong pH dependency with a maximum of approximately 50 mM at about pH 7.0. The NDS-TEMPO inhibition constants from the chloride and the sulfate flux experiments were 0.5 microM (0 degree C) and 1.8 microM (25 degrees C), respectively, and are in close accordance with the NDS TEMPO/substrate-site dissociation constants. Our studies provide strong evidence for the assumption that NDS-TEMPO binds in fact to the substrate site of Band 3. They show that the strong pH dependence of the chloride and of the sulfate transport cannot result from the pH dependency of substrate-anion binding, but point to the participation of ionizable regulator sites in transport catalysis. These regulator sites seem to be positioned outside the substrate site of the Band 3 transport domain. PMID- 3018259 TI - Mechanisms of spontaneous mutagenesis: an analysis of the spectrum of spontaneous mutation in the Escherichia coli lacI gene. AB - We have obtained via DNA sequence analysis a spectrum of 174 spontaneous mutations occurring in the lac I gene of Escherichia coli. The spectrum comprised base substitution, frameshift, deletion, duplication and insertion mutations, of which the relative contributions to spontaneous mutation could be estimated. Two thirds of all lacI mutations occurred in the frameshift hotspot site. An analysis of the local DNA sequence suggested that the intensity of this hotspot may depend on structural features of the DNA that extend beyond those permitted by the repeated tetramer at this site. Deletions comprised the largest non-hotspot class (37%). They could be divided into two subclasses, depending on whether they included the lac operator sequence; the latter was found to be a preferred site for deletion endpoints. Most of the deletions internal to the lacI gene were associated with the presence of directly or invertedly repeated sequences capable of accounting for their endpoints. Base substitutions comprised 34% of the non hotspot events. Unlike the base substitution spectrum obtained via nonsense mutations, G . C----A . T transitions do not predominate. A new base substitution hotspot was discovered at position +6 in the lac operator; its intensity may reflect specific features of the operator DNA. IS1 insertion mutations contributed 12% of the non-hotspot mutations and occurred dispersed throughout the gene in both orientations. Since the lacI gene is not A + T-rich, the contribution of IS1 insertion to spontaneous mutation in general might be underestimated. Single-base frameshift mutations were found only infrequently. In general, they did not occur in runs of a common base. Instead, their occurrence seemed based on the "perfection" of direct or inverted repeats in the local DNA sequence. Three (tandem) duplication events were recovered. No repeated sequences were found that might have determined their endpoints. PMID- 3018258 TI - Kinetic studies on the stimulation of Na+-H+ exchange activity in renal brush border membranes isolated from thyroid hormone-treated rats. AB - Na+-H+ exchange activity in renal brush border membrane vesicles isolated from hyperthyroid rats was increased. When examined as a function of [Na+], treatment altered the initial rate of Na+ uptake by increasing Vm (hyperthyroid, 18.9 +/- 1.1 nmol Na+ X mg-1 X 2 sec-1; normal, 8.9 +/- 0.3 nmol Na+ X mg-1 X 2 sec-1), and not the apparent affinity KNa+ (hyperthyroid, 7.3 +/- 1.7 mM; normal, 6.5 +/- 0.9 mM). When examined as a function of [H+] and at a subsaturating [Na+] (1 mM), hyperthyroidism resulted in the proportional increase in Na+ uptake at every intravesicular pH measured. A positive cooperative effect on Na+ uptake was found with increased intravesicular acidity in vesicles from both normal and hyperthyroid rats. When the data were analyzed by the Hill equation, it was found that hyperthyroidism did not change the n (hyperthyroid, 1.2 +/- 0.06; normal, 1.2 +/- 0.07) or the [H+]0.5 (hyperthyroid, 0.39 +/- 0.08 microM; normal, 0.44 +/ 0.07 microM) but increased the apparent Vm (hyperthyroid, 1.68 +/- 0.14 nmol Na+ X mg-1 X 2 sec-1; normal 0.96 +/- 0.10 nmol Na+ X mg-1 X 2 sec-1). The uptake of Na+ in exchange for H+ in membrane vesicles from normal and hyperthyroid animals was not influenced by membrane potential. H+ translocation or debinding was rate limiting for Na+-H+ exchange since Na+-Na+ exchange activity was greater than Na+ H+ exchange activity. Hyperthyroidism caused a proportional increase and hypothyroidism caused a proportional decrease in Na+-Na+ and Na+-H+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018260 TI - Insertions of palindromic DNA sequences into the J-F intercistronic region of bacteriophage phi X174 interfere with normal phage growth. AB - The effect of hairpin (cruciform) size on the regulation of gene expression was investigated by cloning a series of palindromic sequences into the non-essential J-F intercistronic region of the bacteriophage phi X174 ins6 genome. Genetic stability of the insert sequence and its effect on the growth efficiency of the phage was used as an initial measure of the biological consequence of hairpin insertions. Multimers of increasing size of the BamHI linker sequence C-C-G-G-A-T C-C-G-G were inserted into the PvuII site of the parental strain ins6. The largest hairpin that could be constructed and maintained in the phi X174 genome had a stem length of 22 base-pairs and a loop size of four nucleotides (linker tetramer). However, this structure proved to be disadvantageous to the phage and was rapidly deleted from its genome. Trimer inserts were more stable, but were eventually deleted also. Monomer and dimer inserts, though genetically stable, decreased the growth efficiency of the phage as judged by competitive growth experiments and measurements of burst size. The physical formation of these hairpins was shown by restriction digests of single-stranded DNA with BamHI and HpaII. We argue that these secondary structures form in vivo, at least in the single-stranded genome and the polycistronic mRNAs, and were responsible for the observed growth defects. PMID- 3018261 TI - Mini-F E protein: the carboxy-terminal end is essential for E gene repression and mini-F copy number control. AB - Mini-F is a segment of the conjugative plasmid F consisting of two origins of replication flanked by regulatory regions, which ensure a normal control of replication and partitioning. Adjacent to the ori-2 origin is a complex coding region that consists of the E gene overlapped by three open reading frames with the coding potential for 9000 Mr polypeptides here designated 9 kd-1, 9 kd-2 and 9 kd-3. In this paper, we show that open reading frame 9 kd-3 is preceded by active promoter and Shine-Dalgarno sequences. The E coding region specifies: an initiator of replication, which acts at the ori-2 site; a function that negatively regulates the expression of the E gene; and a function involved in mini-F copy number control. To assign one of these functions to one of the overlapping coding sequence, we have isolated, characterized and sequenced mutations mapping in the E coding region. In this paper, we analyse two mutations (cop5 and pla25) that abolish the repression of the E gene. As these mutations affect the primary structure of protein E itself but not the 9 kd polypeptides, we conclude that protein E takes part in the negative regulation of its own synthesis. In addition, the localization of the cop5 and pla25 mutations indicates that the carboxy-terminal end of the E protein is involved in the autorepression function. The cop5 mutation causes an eightfold increase of the mini-F copy number. The pla25 mutation leads to the inability of the derived mini F plasmid to give rise to plasmid-harbouring bacteria. The ways in which the cop5 and pla25 mutations may lead to such phenotypes are discussed in relation to the different functions mapping in the E coding sequence. PMID- 3018262 TI - Location and analysis of byssal structural proteins of Mytilus edulis. AB - The acellular attachment organ (byssus) of the marine mussel Mytilus edulis L. is composed of threads that emanate from the body of the mussel to adhesive discs that anchor the threads to rocks, sand and other mussels. Three proteins have been purified by immunohistological methods and located to specific regions of the byssus. A collagenous protein with subunit molecular weights of 53,000, 55,000 and 65,000 is found in the matrix of the elastic thread region. Its 73,000 MW precursor was extracted from foot glands in the area proximal to the animal body and was identified by immune cross-reactivity. A cystine-rich, acidic protein was found in all regions of the byssus associated with a third protein, the polyphenolic protein. The L-dopa-containing polyphenolic protein appears in the cortex of the entire thread and adhesive plaque and at the substrate-plaque interface. Antiserum to this protein stains spherical vesicles in the phenol gland of the foot. Using immuno-electrophoretic methods, the polyphenolic protein and the cystine-rich protein were shown to form high molecular weight aggregates with aging of the byssus. PMID- 3018264 TI - Heterogeneous distribution of beta adrenoceptors in the dog left ventricle. PMID- 3018263 TI - The link between myocardial contraction and relaxation: the effects of calcium antagonists. AB - In isolated rat hearts perfused at constant coronary flow and heart rate the amount of shortening in their major axis (L), the developed tension (T) and their time derivatives (L and T) were measured after different interventions. Relative changes of maximal velocities of contraction (+L, +T) and relaxation (-L, -T) were assessed by the ratio between both velocities (+L/-L, +T/-T). After the interventions, cAMP intracellular levels and cAMP dependent protein kinase activity were measured. The negative inotropic effect of verapamil (10(-7) M) and nifedipine (5 X 10(-7) M) was accompanied by a relatively greater decrease in maximal velocity of relaxation. Consequently the ratio +L/-L increased. Verapamil increased +L/-L from 1.27 +/- 0.07 to 1.57 +/- 0.08 (P less than 0.001). Nifedipine increased +L/-L from 1.25 +/- 0.05 to 1.77 +/- 0.12 (P less than 0.001). Whereas nifedipine increased intracellular cAMP levels from 0.403 +/- 0.036 pmol/mg wet weight to 0.534 +/- 0.047 (P less than 0.05), verapamil did not alter them. Neither verapamil nor nifedipine affected cAMP protein kinase activity. Increasing Ca2+ in the perfusate did not change the ratio +L/-L. A decrease in extracellular Ca2+, on the other hand, produced a greater decrease in -L than in +L, so that the ratio +L/-L increased from 1.27 +/- 0.05 to 1.49 +/- 0.10 (P less than 0.05). No changes were detected in cAMP levels or its protein kinase activity. Similar results were obtained when +T/-T was analyzed. To offset the negative inotropic effect caused by calcium antagonists, either increased extracellular Ca2+ or isoproterenol can be used.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018265 TI - Roussel award for cardiology. The mechanism of nucleotide induced calcium translocation across sarcoplasmic reticulum membranes: evidence for a non translocated intermediate pool of calcium. AB - Our previous data suggested that, in cardiac muscle sarcoplasmic reticulum fragments, GTP hydrolysis occurs by an alternative enzyme cycle of the Ca2+ ATPase which is insensitive to (Ca2+) and does not involve an acyl phosphate intermediate. Despite this, GTP induces the incorporation of calcium into a membrane pool that is not translocated to the vesicular lumen. The present study suggests that this GTP-induced intermediate calcium pool is identical to a modulable component of the calcium translocation process in that: it has an identical pH sensitivity; the initial incorporation of calcium in response to GTP eliminates the initial rapid burst and lag component of the typical ATP-induced calcium uptake curve when ATP is added during GTP-induced calcium accumulation. Instead, the addition of ATP during GTP-induced calcium accumulation results in the prompt onset of the linear phase of calcium translocation; GTP-induced calcium accumulation directly affects the pH sensitivity of subsequent ATP induced calcium accumulation. We suggest that the intermediate calcium pool is in series with calcium translocation and is the site of the pH sensitivity observed in calcium flux. PMID- 3018266 TI - GDP activates rabbit heart adenylate cyclase, but does not support stimulation by isoproterenol: a re-appraisal of the control mechanism. AB - The effect of GDP on rabbit heart adenylate cyclase has been determined under conditions where only 0.08% to 0.26% of an added 100 microM was converted to GTP in the course of the assay. At concentrations of 100 microM, GDP stimulated basal cyclase activity to the same extent as GTP and guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). Isoproterenol increased activity in the presence of GTP or guanylyl imidodiphosphate (Gpp(NH)p), but not in the presence of GDP or GDP beta S. It is suggested that the hydrolysis of GTP to GDP is the "turn-off" mechanism for beta receptor stimulation of cardiac adenylate cyclase, but not for stimulation by GTP alone. The effects of GDP and GDP beta S are readily removed by washing, implying that their binding to Ns (the guanine nucleotide binding protein) is weak. GDP beta S initially competes with Gpp(NH)p, reducing Gpp(NH)p-stimulated activity. As stimulation of cyclase activity by Gpp(NH)p develops, in the course of 30 min, Gpp(NH)p becomes no longer displaceable by GDP beta S. Isoproterenol does not release 3H-Gpp(NH)p or reduce Gpp(NH)p-stimulated activity, once the nucleotide has become tightly bound. Nor does isoproterenol change the relative affinities of GDP beta S and Gpp(NH)p when these analogs are given together. There is, therefore, no evidence that isoproterenol acts by releasing tightly bound GDP from Ns, or that it 'unlocks' the guanine nucleotide binding site in the myocardial sarcolemma. In this, the cardiac adenylate cyclase system differs from the avian erythrocyte system. The action of isoproterenol is best explained by an increased dissociation of alpha(GTP) and beta,gamma-subunits of the Ns protein. PMID- 3018268 TI - Subcellular distribution of cardiac 5'-nucleotidase: alteration of microsomal pool in hypertrophied pig heart. AB - The subcellular distribution of the 5'-nucleotidase activity was investigated in normal and hypertrophied pig hearts; normal rat hearts were used for comparison. The left ventricular hypertrophy was induced in pigs by banding the supravalvular aorta for 4, 8 and 12 weeks. By employing different procedures for the isolation of cardiac membranes, a major catalytic site for 5'-nucleotidase was found to be located at sarcolemma in rat heart and microsomes (sarcoplasmic reticulum) in pig heart. A progressive decrease in the homogenate and microsomal 5'-nucleotidase activity occurred upon the development of myocardial hypertrophy in pigs. This reduction in microsomal 5'-nucleotidase activity was characterized by a depression in both apparent Vmax and Km values. These results indicate that a primary 5'-nucleotidase pool is present in the intracellular compartment of the pig heart and is altered during the development of hypertrophy. PMID- 3018267 TI - Muscarinic receptors in mammalian myocardium: effects of atrial and ventricular receptors on phosphatidylinositol metabolism and adenylate cyclase. AB - Cat myocardium was used to investigate muscarinic receptor function in atria and ventricles. Carbachol and oxotremorine were used to determine agonist binding to the muscarinic receptors. It was found that carbachol was bound with almost the same characteristics in atria (KdH 1 microM; KdL 150 microM) as in ventricles (KdH 3 microM; KdL 150 microM). However, in the presence of guanylylimidodiphosphate Gpp(NH)p a difference was apparent so that the ventricular curve was shifted to a majority of low affinity sites whereas in atria two affinity sites remained even if the guanine nucleotide concentration was increased to 10 mM. Oxotremorine was bound with almost equal affinity in both atria and ventricles. The addition of Gpp(NH)p left all the receptors in the low affinity state irrespective of receptor localisation. The two agonists were also used to determine inhibition of adenylate cyclase activity. It was shown that the magnitude of adenylate cyclase inhibition was more pronounced in ventricles than in atria whether it was induced by oxotremorine or carbachol. When the effects of muscarinic agonists were determined on phosphatidylinositol metabolism it was shown that carbachol mediated a greater effect in atria than in ventricles. Almost no effect was seen with oxotremorine on phosphatidylinositol breakdown. Pirenzipine binding showed the presence of M1 receptors both in atria and ventricles. On the basis of diversity of muscarinic agonists on function and receptor occupancy it is suggested that heterogeneity exists for muscarinic receptors in both atria and ventricles. PMID- 3018269 TI - Sequence and evolution of rhesus monkey alphoid DNA. AB - Analysis of rhesus monkey alphoid DNA suggests that it arose by tandem duplication of an ancestral monomer unit followed by independent variation within two adjacent monomers (one becoming more divergent than the other) before their amplification as a dimer unit to produce tandem arrays. The rhesus monkey alphoid DNA is a tandemly repeated, 343-bp dimer; the consensus dimer is over 98% homologous to the alphoid dimers reported for baboon and bonnet monkey, 81% homologous to the African green monkey alpha monomer, and less than 70% homologous to the more divergent human alphoid DNAs. The consensus dimer consists of two wings (I and II, 172 and 171 bp, respectively) that are only 70% homologous to each other, but share seven regions of exact homology. These same regions are highly conserved among the consensus sequences of the other cercopithecid alphoid DNAs. The three alpha-protein binding sites reported for African green monkey alpha DNA by F. Strauss and A. Varshavsky (Cell 37: 889-901, 1984) occur in wings I and II, but with one site altered in wing I. Two cloned dimer segments are 98% homologous to the consensus, each containing 8 single-base pair differences within the 343-bp segment. Surprisingly, 37% of these differences occur in regions that are evolutionarily conserved in the alphoid consensus sequences, including the alpha-protein binding sites. Sequence variation in this highly repetitive DNA family may produce unique nucleosomal architectures for different members of an alphoid array. These unique architectures may modulate the evolution of these repetitive DNAs and may produce unique centromeric characteristics in primate chromosomes. PMID- 3018270 TI - The distribution of P-element sequences in Drosophila: the willistoni and saltans species groups. AB - This report describes the distribution of P-element sequences among members of the closely related willistoni and saltans species groups of the subgenus Sophophora. Gel-blotting analyses showed that many, but not all, species from each of these groups possess sequences with homology to the P transposable element of Drosophila melanogaster, a sophophoran species belonging to the melanogaster species group. Furthermore, P-homologous fragments are present in lower numbers in willistoni- and saltans-group species than in D. melanogaster P strains, and, in some species of those two groups, exhibit species-characteristic hybridization patterns. On the basis of these results, it is proposed that P elements have had a long evolutionary history in the willistoni and saltans lineages. PMID- 3018271 TI - Drosophila virilis histone gene clusters lacking H1 coding segments. AB - Approximately 30-40% of Drosophila virilis DNA complementary to cloned Drosophila histone genes is reduced to 3.4-kilobase-pair (kbp) segments by Bgl I or Bgl II digestion. The core histone genes of a 3.4-kbp Bgl II segment cloned in the plasmid pDv3/3.4 have the same order as the D. melanogaster core histone genes in the plasmid cDm500: H2B H3 H4 H2A. Nonetheless, pDv3/3.4 and cDm500 have different histone gene configurations: In pDv3/3.4, the region between the H2B and H3 genes contains 0.35 kbp and cannot encode histone H1; in cDm500, the region contains 2.0 kbp and encodes histone H1. The lack of an H1 gene between the H2B and H3 genes in 30-40% of D. virilis histone gene clusters suggests that changes in histone gene arrays have occurred during the evolution of Drosophila. The ancestors of modern Drosophila may have possessed multiple varieties of histone gene clusters, which were subsequently lost differentially in the virilis and melanogaster lineages. Alternatively, they may have possessed a single variety, which was rearranged during evolution. The H1 genes of D. virilis and D. melanogaster did not cross-hybridize in vitro under conditions that maintain stable duplexes between DNAs that are 75% homologous. Consequently, D. virilis H1 genes could not be visualized by hybridization to an H1-specific probe and thus remain unidentified. Our observations suggest that the coding segments in the H1 genes of D. virilis and D. melanogaster are greater than 25% divergent.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018272 TI - Serum enzymes of collagen synthesis and type III procollagen aminopropeptide in Nigerian patients with sickle cell disease. AB - Serum immunoreactive prolyl hydroxylase protein (S-IRPH), galactosylhydroxylysyl glucosyltransferase activity (S-GGT), and the aminoterminal propeptide of type III procollagen (S-Pro(III)-N-P) were measured in 20 patients with sickle cell disease and the values were compared with those in 20 apparently healthy Nigerians. The means for the two enzymes and S-Pro(III)-N-P were significantly elevated in the sickle cell disease patients. Significant correlations were found between the values of the two enzymes and the protein (S-Pro(III)-N-P) within the sickle cell disease patients. The data confirm that collagen formation is found in the bone, liver, or other organs of patients with this disease. The measurement of S-GGT and S-Pro(III)-N-P in prospective studies might be helpful in predicting general and hepatic fibrogenesis in sickle cell disease. PMID- 3018273 TI - Chromium effects on chondrocytic differentiation in vitro. AB - A tissue culture study was conducted on the effects of chromium on chondrocytic differentiation. Mesenchymal cells from stage 22-24 chick limb buds were dispersed and cultured as micromasses, where they differentiated into chondrocytes. Addition of chromium(VI) to the cultures indicated that the production of proteoglycans (as detected by Alcian blue staining) was more sensitive to chromium's effects than was cell proliferation. Whereas Alcian blue nodule formation was inhibited by 1 microM Cr(VI), cell proliferation (as detected by cell counts) was not. Chromium (VI) was added to cultures at daily intervals, and these studies indicated that the interval of d 1-2 was the most sensitive period. ADP-ribose transferase activity in these cultures was measured; the pattern of enzyme activity in control cultures was high 1 and 24 h after the start of culture, decreased abruptly between 24 and 48 h, and then decreased more gradually. In the presence of Cr(VI), elevated ADP-ribose transferase levels were maintained throughout the culture period. We suggest that, in presence of 1.0 microM chromium(VI) or higher concentrations, the balance of events favors nucleolytic action rather than repair of damage. PMID- 3018274 TI - Baclofen overdose with cardiac conduction abnormalities: case report and review of the literature. AB - Cardiac conduction abnormalities and hypertension developed in a patient who ingested approximately 500 mg of baclofen (Lioresal). The patient also exhibited the more common features of baclofen overdose including coma, respiratory depression, hypotonia, and hyporeflexia. A review of the literature and a discussion of the interesting manifestations and treatment of baclofen overdose are included. PMID- 3018275 TI - Gestational trophoblastic disease with coexistent normal fetus: evaluation by ultrasound-guided chorionic villus sampling. PMID- 3018276 TI - Exogenous mouse mammary tumor virus proviral DNA isolated from a kidney adenocarcinoma cell line contains alterations in the U3 region of the long terminal repeat. AB - Mouse mammary tumor virus (MMTV) is a B-type retrovirus which induces predominantly mammary carcinomas after a relatively long latency period. To date, very little is known about the reasons for the strict tissue specificity of MMTV. The BALB/cf/Cd strain of mice, which was infected with milk-borne MMTV (C3H), shows a high incidence of kidney adenocarcinomas, and our data suggest that MMTV might be involved in the formation of these tumors. Newly integrated exogenous MMTV proviruses were found in the genome of transplanted tumor cells as well as in the DNA of a cell line derived from one tumor, but not in normal cells of BALB/cf/Cd mice. The MMTV DNA in these tumor cells was transcribed and viral RNA synthesis was strongly stimulated by glucocorticoid hormones. Viral structural polypeptides, comparable in size and antigenicity to MMTV polypeptides of infected mammary tumor cells were synthesized and processed normally in the cell line and were organized correctly into intracytoplasmic particles. Heteroduplex analysis of the molecularly cloned MMTV proviral DNAs of kidney and mammary tumor origin revealed a high degree of homology in the gag, pol, and env genes. A striking difference, however, was observed in the U3 region of the two LTRs that might relate to the different tissue specificity of the two viruses. PMID- 3018277 TI - Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen. AB - The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into a retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of psi 2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10(6) cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture. PMID- 3018278 TI - Organization of the thymidylate synthase gene of herpesvirus saimiri. AB - Herpesvirus saimiri codes, unlike most other herpesviruses, for a thymidylate synthase (TS). The TS gene of herpesvirus saimiri is unusual in structure and regulation of expression. It is transcribed into a nonspliced mRNA of 2,190 nucleotides. The single open reading frame of the viral TS gene, instructing a polypeptide of 33.5 kilodaltons, has extensive sequence homology with the corresponding TS coding sequences of human cells and of various procaryotes; the putative polypeptide derived from the nucleotide sequence of the herpesvirus saimiri TS gene is 70% identical with the human enzyme. The untranslated regions of the herpesvirus saimiri TS gene do not share homology with the other characterized eucaryotic or bacterial TS genes. The 5' untranslated sequence has 22 ATG triplets shortly followed by stop codons. The herpesvirus saimiri TS gene, which may be weakly transcribed during immediate early and early times of virus replication, is maximally expressed at the late phase. Various parameters suggest that the TS gene has been acquired in virus evolution by an ancestral herpesvirus from the cellular genome. PMID- 3018279 TI - Translation and processing of mouse hepatitis virus virion RNA in a cell-free system. AB - The first event after infection with mouse hepatitis virus strain A59 (MHV-A59) is presumed to be the synthesis of an RNA-dependent RNA polymerase from the input genomic RNA. The synthesis and processing of this putative polymerase protein was studied in a cell-free translation system utilizing 60S RNA from MHV-A59 virions. The polypeptide products of this reaction included two major species of 220 and 28 kilodaltons. Kinetics experiments indicated that both p220 and p28 appeared after 60 min of incubation and that protein p28 was synthesized initially as the N-terminal portion of a larger precursor protein. When the cell-free translation products were labeled with N-formyl[35S]methionyl-tRNAi, p28 was the predominant radioactive product, confirming its N-terminal location within a precursor protein. Translation in the presence of the protease inhibitors leupeptin and ZnCl2 resulted in the disappearance of p28 and p220 and the appearance of a new protein, p250. This product, which approached the maximal size predicted for a protein synthesized from genomic RNA, was not routinely detected in the absence of inhibitors even under conditions which optimized the translation reaction for elongation of proteins. Subsequent chelation of ZnCl2 resulted in the partial cleavage of the precursor protein and the reappearance of p28. One-dimensional peptide mapping with Staphylococcus aureus V-8 protease confirmed the precursor product relationship of p250 and p28. The results show that MHV virion RNA, like many other viral RNAs, is translated into a large polyprotein, which is cleaved soon after synthesis into smaller, presumably functional proteins. This is in marked contrast to the synthesis of other MHV proteins, in which minimal proteolytic processing occurs. PMID- 3018280 TI - Detection of a genome-linked protein (VPg) of hepatitis A virus and its comparison with other picornaviral VPgs. AB - The nucleotide sequence corresponding to the P3 region of the hepatitis A virus (HAV) polyprotein genome was determined from cloned cDNA and translated into an amino acid sequence. Comparison of the amino acid sequences of the genome-linked proteins (VPgs) of other picornaviruses with the predicted amino acid sequence of HAV was used to locate the primary structure of a putative VPg within the genome of HAV. The sequence of HAV VPg, like those of other picornaviral VPg molecules, contains a tyrosine residue as a potential binding site for HAV RNA in position 3 from its N terminus. The potential cleavage sites to generate VPg from a putative HAV polyprotein are between glutamic acid and glycine at the N terminus and glutamic acid and serine or glutamine and serine at the C terminus. A synthetic peptide corresponding to 10 amino acids of the predicted C terminus of HAV VPg induced anti-peptide antibodies in rabbits when it was conjugated to thyroglobulin as a carrier. These antibodies were specific for the peptide and precipitated VPg, linked to HAV RNA, from purified HAV and from lysates of HAV infected cells. The precipitation reaction was blocked by the synthetic peptide (free in solution or coupled to carrier proteins) and prevented by pretreatment of VPg RNA with protease. Thus, our predicted amino acid sequence is colinear with the nucleotide sequence of the VPg gene in the HAV genome. From our results we concluded that HAV has the typical organization of picornavirus genes in this part of its genome. Similarity among hydrophobicity patterns of amino acid sequences of different picornaviral VPgs was revealed in hydropathy plots. Thus, the VPg of HAV appears to be closely related to VPg1 and VPg2 of foot-and-mouth disease virus. In contrast, HAV VPg has a unique isoelectric point (pI = 7.15) among the picornavirus VPgs. PMID- 3018281 TI - Promiscuous trans activation of gene expression by an Epstein-Barr virus-encoded early nuclear protein. AB - We identified an Epstein-Barr virus (EBV) gene product which functions in transient-expression assays as a nonspecific trans activator. In Vero cells, cotransfection of the BglII J DNA fragment of EBV together with recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene gave up to a 100-fold increased expression of CAT activity over that in cells transfected with the recombinant CAT constructs alone. The BglII J fragment acted promiscuously, in that increased CAT synthesis was observed regardless of whether the promoter sequences driving the CAT gene were of EBV, simian virus 40, adenovirus, or herpes simplex virus origin. Cleavage of cloned BglII-J plasmid DNA before transfection revealed that activation was dependent upon the presence of an intact BMLF1 open reading frame. This was confirmed with subclones of BglII J and with hybrid promoter-open reading frame constructs. This region of the genome is also present in the rearranged P3HR-1-defective DNA species, and defective DNA clones containing these sequences produced a similar activation of CAT expression in cotransfection experiments. The heterogeneous 45-60-kilodalton polypeptide product of BMLF1 may play an important regulatory role in expression of lytic-cycle proteins in EBV-infected lymphocytes. PMID- 3018282 TI - Identification and mapping of Epstein-Barr virus early antigens and demonstration of a viral gene activator that functions in trans. AB - The BamHI M DNA fragment of the Epstein-Barr virus (EBV) genome was inserted in two orientations into a simian virus 40-based expression vector, and the EBV specific proteins produced in COS-7 monkey cells were examined. In one orientation, termed BamHI-M rightward reading frame 1 (BMRF1), a set of phosphoproteins ranging in size from 47,000 to 54,000 daltons was synthesized. These proteins reacted with monoclonal and polyclonal antisera, defining them as components of the EBV early antigen diffuse set of proteins (EA-D). The BamHI M DNA fragment in the opposite orientation, termed BamHI-M leftward reading frame 1 (BMLF1), directed the synthesis of a nuclear antigen detected by antibodies in serum from a patient with nasopharyngeal carcinoma. The BMLF1 antigen was not detected by monoclonal or polyclonal antibodies directed against the EA-D complex. A series of deletion mutants were constructed in the BamHI M DNA fragment, and the EA-D complex and BMLF1 antigen were mapped to discrete open reading frames in this DNA fragment. A test for several possible functions of these antigens showed that the BMLF1 antigen had the ability to activate or enhance, in trans, the level of expression of a gene under the control of the adenovirus early region 3 promoter or the simian virus 40 early promoter in the absence of its cis-acting enhancer. These experiments demonstrate a new gene function, encoded by EBV, that may be important in the positive regulation of viral or cellular genes. PMID- 3018283 TI - Site-directed mutagenesis of the gag-myc gene of avian myelocytomatosis virus 29: biological activity and intracellular localization of structurally altered proteins. AB - Transfection of chicken embryo cells with pMC29, a plasmid vector containing the sequences for the acute transforming virus MC29, and a cloned transformation defective helper virus, p delta Mst, resulted in morphological transformation, the synthesis of P110gag-myc (the product of the gag-myc oncogene), and the production of infectious virus. MC29 mutants bearing site-directed deletions within the gag-specific sequences or within the middle portion of the myc sequences efficiently induced transformation of chicken embryo cells in culture. However, variants containing deletions of sequences in the amino-terminal half or carboxy-terminal portion of the myc gene were defective for transformation. The gag-myc proteins encoded by these variants efficiently localized to the cell nucleus. Premature termination mutants were isolated which encoded gag-myc proteins lacking the carboxy-terminal 185 residues; these truncated proteins localized to both the nucleus and the cytoplasm. Deletion of as few as 11 residues within the middle of the myc-specific sequences (residues Ile-239 to Glu 249) significantly reduced the efficiency of chicken hematopoietic cell transformation. PMID- 3018285 TI - Mammalian cell transformation by a murine retrovirus vector containing the avian erythroblastosis virus erbB gene. AB - A recombinant murine retrovirus vector containing the v-erbB gene of avian erythroblastosis virus was constructed to investigate v-erbB as a transforming gene for mammalian cells. A restriction fragment containing the v-erbB sequences from a molecular clone of avian erythroblastosis virus was inserted into a Moloney murine leukemia virus vector. The construct, designated MuLV/erbB, transformed NIH 3T3 cells at a high efficiency in the DNA transfection assay. Individual MuLV/erbB transfectants grew in soft agar and were tumorigenic. The transfectants contained v-erbB DNA sequences, expressed v-erbB-specific transcripts, and synthesized v-erbB-related glycoproteins. The majority of transfectants produced two major v-erbB gene products of 58 and 66 kilodaltons. However, some transfectants produced much smaller v-erbB-specific proteins. Tunicamycin experiments revealed that the size heterogeneity observed between different transfectants was not due to variations in glycoprotein processing, implying that, in some cases, alterations in the MuLV/erbB genome occurred during the transfection process. These findings indicate that expression of the complete v-erbB gene product is not required for transformation of NIH 3T3 cells. A transmissible murine v-erbB (M-erbB) virus was generated by infection of nonproducer transfectants with amphotrophic murine leukemia virus. Transmission of the rescued M-erbB virus was confirmed by DNA, RNA, and protein analyses. The introduction of a transforming v-erbB gene into mammalian cells by virus infection provides a means of analyzing the mechanism by which this epidermal growth factor receptor-related gene alters the growth and differentiation of cells from various lineages. PMID- 3018284 TI - Use of lambda gt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins. AB - A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambda gt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambda gt11 vector, the cloned proteins were expressed in Escherichia coli as beta-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of [14C]glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX; gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved. PMID- 3018286 TI - A new acute transforming feline retrovirus with fms homology specifies a C terminally truncated version of the c-fms protein that is different from SM feline sarcoma virus v-fms protein. AB - The HZ5-feline sarcoma virus (FeSV) is a new acute transforming feline retrovirus which was isolated from a multicentric fibrosarcoma of a domestic cat. The HZ5 FeSV transforms fibroblasts in vitro and is replication defective. A biologically active integrated HZ5-FeSV provirus was molecularly cloned from cellular DNA of HZ5-FeSV-infected FRE-3A rat cells. The HZ5-FeSV has oncogene homology with the fms sequences of the SM-FeSV. The genome organization of the 8.6-kilobase HZ5 FeSV provirus is 5' delta gag-fms-delta pol-delta env 3'. The HZ5-and SM-FeSVs display indistinguishable in vitro transformation characteristics, and the structures of the gag-fms transforming genes in the two viruses are very similar. In the HZ5-FeSV and the SM-FeSV, identical c-fms and feline leukemia virus p10 sequences form the 5' gag-fms junction. With regard to v-fms the two viruses are homologous up to 11 amino acids before the C terminus of the SM-FeSV v-fms protein. In HZ5-FeSV a segment of 362 nucleotides then follows before the 3' recombination site with feline leukemia virus pol. The new 3' v-fms sequence encodes 27 amino acids before reaching a TGA termination signal. The relationship of this sequence with the recently characterized human c-fms sequence has been examined. The 3' HZ5-FeSV v-fms sequence is homologous with 3' c-fms sequences. A frameshift mutation (11-base-pair deletion) was found in the C-terminal fms coding sequence of the HZ5-FeSV. As a result, the HZ5-FeSV v-fms protein is predicted to be a C-terminally truncated version of c-fms. This frameshift mutation may determine the oncogenic properties of v-fms in the HZ5-FeSV. PMID- 3018288 TI - Positive and negative autoregulation of the adeno-associated virus type 2 genome. AB - The defective human parvovirus, adeno-associated virus (AAV), requires multiple functions provided by a coinfecting helper virus for viral replication. In addition, it has recently been shown that at least one AAV gene is also required for AAV DNA replication. In this paper, we investigate the autoregulation of the AAV genome by analyzing the expression of mutant AAV genomes upon transfection into adenovirus-infected human cells. Evidence is presented which indicates that the AAV genome regulates its own gene expression in at least two ways. First, either the AAV p5 gene or both the p5 and p19 genes appear to encode a trans activator of AAV transcription. Frameshift mutations within the p5 or p19 gene severely inhibited the synthesis and accumulation of all AAV transcripts. The defective accumulation of transcripts could be complemented in trans, in a manner independent of DNA replication, by cotransfection with a capsid deletion mutant. Second, evidence is presented which suggests that the p5 and p19 genes contain negative cis-active regulatory elements. Deletion of sequences within the p5 and p19 genes enhanced the accumulation of the p5 transcript in cis upon complementation with an AAV capsid deletion mutant, whereas certain deletions enhanced p40 RNA accumulation in the absence of trans activation by the p5 gene. PMID- 3018287 TI - Molecular analysis and pathogenesis of the feline aplastic anemia retrovirus, feline leukemia virus C-Sarma. AB - We describe the molecular cloning of an anemogenic feline leukemia virus (FeLV), FeLV-C-Sarma, from the productively infected human rhabdomyosarcoma cell line RD(FeLV-C-S). Molecularly cloned FeLV-C-S proviral DNA yielded infectious virus (mcFeLV-C-S) after transfection of mammalian cells, and virus interference studies using transfection-derived virus demonstrated that our clone encodes FeLV belonging to the C subgroup. mcFeLV-C-S did not induce viremia in eight 8-week old outbred specific-pathogen-free (SPF) cats. It did, however, induce viremia and a rapid, fatal aplastic anemia due to profound suppression of erythroid stem cell growth in 9 of 10 inoculated newborn, SPF cats within 3 to 8 weeks (21 to 58 days) postinoculation. Thus, the genome of mcFeLV-C-S encodes the determinants responsible for the genetically dominant induction of irreversible erythroid aplasia in outbred cats. A potential clue to the pathogenic determinants of this virus comes from previous work indicating that all FeLV isolates belonging to the C subgroup, an envelop-gene-determined property, and only those belonging to the C subgroup, are potent, consistent inducers of aplastic anemia in cats. To approach the molecular mechanism underlying the induction of this disease, we first determined the nucleotide sequence of the envelope genes and 3' long terminal repeat of FeLV-C-S and compared it with that of FeLV-B-Gardner-Arnstein (mcFeLV-B-GA), a subgroup-B feline leukemia virus that consistently induces a different disease, myelodysplastic anemia, in neonatal SPF cats. Our analysis revealed that the p15E genes and long terminal repeats of the two FeLV strains are highly homologous, whereas there are major differences in the gp70 proteins, including five regions of significant amino acid differences and apparent sequence substitution. Some of these changes are also reflected in predicted glycosylation sites; the gp70 protein of FeLV-B-GA has 11 potential glycosylation sites, only 8 of which are present in FeLV-C-S. PMID- 3018289 TI - Cell receptors for the mammalian reovirus: reovirus-specific T-cell hybridomas can become persistently infected and undergo autoimmune stimulation. AB - We have previously described the development of virus-specific helper T cell hybridomas which recognize structural determinants shared by type 1 and type 3 reoviruses that have been exposed to UV radiation. We have found that T-cell hybridomas become persistently infected with live type 3 reovirus used for the immunization. Persistently infected T-hybridoma cells were found to spontaneously produce interleukin 2 (IL-2). To analyze the mechanism of induction of IL-2 secretion of persistently infected T-cell hybridomas, we exposed T-cell hybridomas specific for UV-treated virus to replicating type 3 reovirus. The T cell hybridomas became infected but did not produce IL-2 unless simultaneously exposed to syngeneic I-A+ antigen-presenting cells. In this situation, the persistently infected T-cell hybridomas produced IL-2 without being reexposed to virus. This process was not a consequence of nonspecific IL-2 gene activation, which occurs in cells persistently infected with reovirus, because reovirus infection did not activate IL-2 secretion in T-cell hybridomas with other antigenic specificities. Reovirus exposure also resulted in persistent infection of certain antigen-presenting B-cell tumor lines. The persistently infected B cell tumor lines could stimulate reovirus-specific helper T cells but not T-cell hybridomas of other specificities. The data support the thesis that persistent infection of reovirus-specific T cells creates a mechanism in which the virus released from these cells is processed and then reexpressed by I-A+ antigen presenting cells. The IA antigen and reovirus structures on the antigen presenting cells then restimulate the T cells through their specific receptors, resulting in IL-2 synthesis and release. These observations may be relevant to mechanisms of autoimmunity induced by virus. PMID- 3018290 TI - Regulation of polyomavirus late promoter activity by viral early proteins. AB - To assess the effect of the polyomavirus (Py) early proteins, the large T (LT), middle T (MT), and small T (ST) antigens, on gene expression from the Py late promoter, replication-defective plasmid constructs with the bacterial chloramphenicol acetyltransferase (cat) gene linked to this promoter were cotransfected into mouse or rat cells with plasmids capable of producing either LT, MT, or all three early proteins. When target CAT plasmids contained a truncated early region and thus had the coding potential for MT and ST, base-line CAT activities were low, whereas cotransfection with an LT plasmid resulted in up to 70-fold stimulation of CAT activity that was also reflected in similar increases in the level of steady-state mRNA. Studies with target plasmids with deletions within the Py regulatory region indicated that at least the major LT binding site C and a functional enhancer region were both required for maximal stimulation of CAT activity. However, although enhancer deletions totally suppressed the ability of target plasmids to be trans activated, a consistent two to fourfold stimulation of CAT activity by LT was still observed with a plasmid in which all three major LT-binding sites were deleted. Of four mutant LTs incapable of binding Py DNA but retaining immortalization potential, only one showed a low but significant trans-activating ability. When the early coding region was completely eliminated from the target plasmid, base-line CAT activity was increased 10-fold. LT failed to stimulate CAT activity to the same levels observed with target plasmid containing the truncated early region, but this limited response could be enhanced by supplying, in addition, MT and ST. Our results suggest that LT trans activation may involve the formation of a complex of transcriptional factors which interacts with the enhancer, an interaction that is facilitated both by the binding of LT to the Py regulatory region and by the presence of MT or ST or both, and that a significant portion of LT stimulation of late gene expression is a result of the removal of the competing early transcriptional unit via autoregulation. In addition, our results suggest that LT trans activation involves a second indirect component acting independently of LT binding and that the immortalization and trans activation functions of LT can be dissociated. PMID- 3018291 TI - Pathogenicity of herpes simplex virus mutants containing drug resistance mutations in the viral DNA polymerase gene. AB - Three herpes simplex virus mutants that contain drug resistance mutations in the DNA polymerase gene exhibited no significant reduction in replication in the ears of mice compared with the wild type after inoculation at that site but were attenuated for pathogenicity after intracerebral inoculation. Cataracts were common sequelae in mice that survived mutant infections. PMID- 3018293 TI - A recombinant murine retrovirus for simian virus 40 large T cDNA transforms mouse fibroblasts to anchorage-independent growth. AB - A recombinant murine retrovirus containing the intact cDNA sequence for the simian virus 40 (SV40) large T antigen (T) was constructed by using the pZIPNeo SV(X)1 vector. Psi 2 packaging cells were then transfected, and G418-resistant clones were used to generate helper-free viral stocks. NIH 3T3 mouse fibroblasts infected by the recombinant T cDNA retrovirus were selected fro G418 resistance. Such cultures synthesized authentic SV40 T and were transformed to anchorage independent growth at high efficiency. Therefore, this vector has allowed the study of the transformation properties of T under conditions of neutral drug selection and in the absence of SV40 small t antigen. PMID- 3018294 TI - Coxsackievirus B3 infection alters plasma membrane of neonatal skin fibroblasts. AB - Replication of coxsackievirus B3 occurred for days in cultures of murine neonatal skin fibroblasts in the absence of cytopathology and resulted in alteration of the plasma membrane. Dual immunofluorescence studies showed that the lectin Ulex europaeus agglutinin I bound only to cells producing viral capsid antigens. Cultures of coxsackievirus B3-inoculated murine neonatal skin fibroblasts showed maximum binding of this lectin at 72 h postinoculation. These data show that in a nonlytic infection a picornavirus can alter the surface of an infected cell. PMID- 3018292 TI - Replication of human cytomegalovirus in human peripheral blood T cells. AB - The replication of human cytomegalovirus (HCMV) strain AD169 was studied in human peripheral blood granulocytes, monocytes-macrophages, B lymphocytes, and T lymphocytes. Progeny virus was produced in some T-cell cultures stimulated in the allogeneic mixed lymphocyte reaction and was regularly obtained when stimulated T cells were grown in the presence of interleukin 2. Replication of HCMV in these cultures was documented by increases in titer, expression of early and late antigen as assessed by indirect immunofluorescence and Western blot, and viral DNA synthesis as determined by dot-blot assays. Approximately 0.05% of cells in virus-producing cultures formed infectious centers, indicating that only a subset of cells takes part in active virus replication. In double-immunofluorescence experiments this subset was found to consist primarily of the T3+ and T8+ phenotype. By infection of preparatively separated T4+ and T8+ T lymphocytes, however, it could be shown that both T-cell subsets were susceptible to HCMV infection as indicated by increases in titer and by DNA kinetics. We conclude from these data that the T lymphocyte might be a target for HCMV in vitro, which is in accordance with in vivo findings in HCMV-infected patients. PMID- 3018295 TI - Modulation of humoral response to a 12-amino-acid site on the poliovirus virion. AB - Most monoclonal antibodies to poliovirus 3 but not poliovirus 1 require a single 12-amino-acid sequence in virion protein VP1 for neutralization (site 1). None of the available monoclonal antibodies requiring this site bound virions after tryptic cleavage of site 1. This result allowed the amount of site 1-specific antibodies to be determined in an antiserum by comparing its reactivity with virus and trypsin-cleaved virus. Antisera to poliovirus 3 Sabin strain (PS3) but not poliovirus 1 Sabin showed site 1 immunodominance, consistent with the frequency of isolation of site 1-specific monoclonal antibodies to these viruses. Cleavage of site 1 prior to immunization dramatically reduced the immunogenicity of this site in PS3. However, the antiserum against trypsin-cleaved PS3 still had a high neutralization titer, demonstrating that sites other than site 1 can elicit a neutralizing response to PS3. Other antisera to PS3 showed significant variability in the response to site 1, indicating that other factors, such as the genetic background of inbred mouse strains, the species immunized, and the immunization protocol, also affect immunodominance. In particular, a serum from a human infant recently immunized with oral trivalent vaccine had little response to site 1. PMID- 3018296 TI - Reovirus guanylyltransferase is L2 gene product lambda 2. AB - Reovirus guanylyltransferase, studied as a covalent enzyme-GMP intermediate, was used to guanylate appropriate acceptor molecules in vitro to produce authentic cap structures. Guanylyltransferase activity was associated with lambda 2, the 140-kilodalton product of the L2 gene segment of reovirus serotypes 1 and 3. PMID- 3018297 TI - Identification of coding regions for various Epstein-Barr virus-specific antigens by gene transfer and serology. AB - Baby hamster kidney cells were transfected with BamHI fragments of Epstein-Barr virus (EBV) DNA (B95-8 strain) cloned into the pLTR vector containing retroviral enhancer and promoter sequences. Seventeen fragments (BamHI-A, -B, -C, -D, -E, G, -K, -L, -M, -O, -P, -Q, -R, -U, -V, -X, and -Z) expressed antigenically distinct EBV-specific products recognized by EBV-immune human sera. PMID- 3018298 TI - Analysis of 15 different genome types of adenovirus type 7 isolated on five continents. AB - A total of 15 different genome types of adenovirus type 7 (Ad7), i.e., Ad7p, Ad7p1, Ad7a, Ad7a1 to Ad7a5, Ad7b, Ad7c, Ad7d, Ad7d1, Ad7e, Ad7f, and Ad7g, were identified among 40 selected strains isolated in Europe, Asia, North America, South America, and Australia by using restriction endonucleases BamHI, BclI, BglI, BglII, BstEII, EcoRI, HindIII, HpaI, SalI, SmaI, XbaI, and XhoI. Eight of them, Ad7p1, Ad7a1 to Ad7a5, Ad7d1, and Ad7g, are newly discovered. All 15 genome types could be distinguished by the four restriction endonucleases BamHI, BclI, BglI, and XbaI. At least four restriction sites differed between Ad7d and Ad7g. Pairwise analyses of comigrating DNA restriction fragments of all 15 Ad7 genome types were performed and presented in a schematic fashion. According to the degree of comigration of DNA restriction fragments, the 15 genome types could be divided into three clusters. Ad7b was the dominant genome type in different parts of the world and may have evolved in China into Ad7d and further to Ad7d1. PMID- 3018299 TI - Effect of interferon on replication of herpes simplex virus types 1 and 2 in human macrophages. AB - Macrophages derived from human peripheral blood and cultured for 1 week were permissive for the replication of herpes simplex virus (HSV) types 1 and 2. Low titers of interferon (IFN) were produced after virus infection. The yield of infectious virions was reduced by pretreatment of cells with natural and recombinant IFN-alpha and natural IFN-beta. Recombinant and natural IFN-gamma exhibited very low antiviral activity. Treatment of cells with IFN-gamma mixed with IFN-alpha or with IFN-beta did not result in a synergistic inhibition of virus yield. We studied the synthesis of HSV type 1- and HSV type 2-coded proteins in macrophages treated with IFN-beta. Induction of the HSV beta-protein DNA polymerase was strongly inhibited in IFN-treated cells in a dose-dependent manner. As shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, other beta- and gamma-proteins of HSV were inhibited as well. Immunofluorescence studies revealed a strong inhibition of the expression of immediate early alpha protein ICP4. The results indicate that IFN acts early during the viral replication cycle to inhibit the synthesis of HSV alpha- and beta-proteins. PMID- 3018301 TI - Generation and characterization of a recombinant Moloney murine leukemia virus containing the v-myc oncogene of avian MC29 virus: in vitro transformation and in vivo pathogenesis. AB - A new retrovirus consisting of the v-myc oncogene sequences of avian MC29 virus inserted into the genome of Moloney murine leukemia virus (M-MuLV) was generated. This was accomplished by constructing a recombinant DNA clone containing the desired organization, introducing the recombinant DNA into mouse NIH 3T3 cells, and superinfecting the cells with replication-competent M-MuLV. The construction was designed so that an M-MuLV gag-myc fusion protein would be produced. The resulting virus, M-MuLV(myc), morphologically transformed uninfected NIH 3T3 cells. Stocks of M-MuLV(myc)-M-MuLV were infected into secondary mouse embryo cultures. M-MuLV(myc) induced striking growth and proliferation of hematopoietic cells. These cells were of the myeloid lineage by morphology, phagocytic properties, and surface staining with Mac-1 and Mac-2 monoclonal antibodies. They resembled mature macrophages, although they displayed minor properties of immaturity. The myeloid cells were transformed in comparison with uninfected myeloid cells since they were less adherent and had unlimited proliferative capacity and reduced growth factor requirements. The transformed myeloid cells with proliferative potential were actually myeloid progenitors which apparently underwent terminal differentiation to macrophages. It was possible to derive a permanent line of factor-independent macrophages from M-MuLV(myc)-transformed myeloid cells. M-MuLV(myc) also immortalized and morphologically transformed mouse embryo fibroblasts. These in vitro properties closely resembled the biological activity of MC29 virus in avian cells and suggested that the nature of the v-myc oncogene was an important determinant in transformation specificity. Neonatal NIH Swiss mice inoculated intraperitoneally with M-MuLV(myc)-M-MuLV only developed lymphoblastic lymphoma characteristic of the M-MuLV helper alone, and no acute fibrosarcomas or myeloid tumors resulted. In light of the strong myeloid transformation observed in vitro, the absence of acute in vivo myeloid disease was noteworthy. Interestingly, when a derivative of M-MuLV(myc) carried by a nonpathogenic amphotropic MuLV helper was inoculated, T lymphomas developed with long latency. Molecular hybridization confirmed that these tumors contained M MuLV(myc). PMID- 3018304 TI - Leads from the MMWR. Enterovirus surveillance--United States, 1986. PMID- 3018303 TI - Leads from the MMWR. Recommendations for preventing transmission of infection with human T-lymphotropic virus type III/lymphadenopathy-associated virus during invasive procedures. PMID- 3018302 TI - Topoisomerase II and other DNA-delay and DNA-arrest mutations impair bacteriophage T4 DNA packaging in vivo and in vitro. AB - A survey of DNA packaging in vivo and in vitro during infections caused by T4 DNA delay and DNA-arrest amber mutants revealed a common DNA packaging-deficient phenotype. Electron microscopy revealed high proportions of proheads partially filled with DNA in vivo, indicating normal initiation but incomplete encapsidation. In contrast, exogenous mature T4 DNA was packaged in vitro by several early-gene mutant extracts. Detailed analysis of gene ts39 mutants (subunit of topoisomerase II) showed that in vivo packaging is defective, yet expression of late proteins appeared normal and the concatemeric DNA was not abnormally short or nicked. Although g39 amber mutant extracts packaged DNA in vitro, two of three ts39 mutant extracts prevented encapsidation of the exogenous DNA. The temperature-sensitive (ts) gp39 in a mutant topoisomerase II complex may have interfered with packaging in vivo and in vitro by interacting with DNA in an anomalous fashion, rendering it unfit for encapsidation. These results support the hypothesis that T4 DNA packaging is sensitive to DNA structure and discriminates against encapsidation of some types of defective DNA. PMID- 3018300 TI - Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells. AB - An in vitro poliovirus RNA-synthesizing system derived from a crude membrane fraction of infected HeLa cells was used to analyze the mechanism of initiation of poliovirus plus-strand RNA synthesis. This system contains an activity that synthesizes the nucleotidyl proteins VPg-pU and VPg-pUpU. These molecules represent the 5'-terminal structure of nascent RNA molecules and of virion RNA. The membranous replication complex is also capable of synthesizing nucleotidyl proteins containing nine or more of the poliovirus 5'-proximal nucleotides as assayed by the formation of the RNase T1-resistant oligonucleotide VPg pUUAAAACAGp or by fingerprint analysis of the in vitro-synthesized RNA. Incubation of preformed VPg-pUpU with unlabeled nucleoside triphosphates resulted in the formation of VPg-pUUAAAACAGp. This reaction, which appeared to be an elongation of VPg-pUpU, was stimulated by the addition of a soluble fraction (S 10) obtained from uninfected HeLa cells. Preformed VPg-pU could be chased into VPg-pUpU in the presence of UTP. Our data are consistent with a model that VPg-pU can function as a primer for poliovirus plus-strand RNA synthesis in the membranous replication complex and that the elongation reaction may be stimulated by a host cellular factor. PMID- 3018306 TI - Sites of rhinovirus recovery after point inoculation of the upper airway. AB - Spread of rhinovirus infection in the nose was studied after point inoculation of 25 microL of rhinovirus suspension either under the right inferior turbinate via the test duct (26 volunteers) or onto the posterior nasopharyngeal wall (through the mouth under direct vision) (six volunteers). Epithelial brush samples obtained daily from the anterior and posterior portions of the inferior turbinate in both nasal cavities and the nasopharynx were cultured for rhinovirus. Point inoculation of rhinovirus produced a high infection rate, but the entire nasal lining was not infected at the time of peak symptoms. Virus was usually first detected at the nasopharyngeal site, with subsequent spread of infection anteriorly to one or both inferior turbinates. Spread to the left turbinate was not detected in seven of 17 volunteers during the first five days after right eye inoculation. Virus recovery from the mucosa declined by day 16 and ceased by day 21. PMID- 3018305 TI - Effects of exposure to factor concentrates containing donations from identified AIDS patients. A matched cohort study. AB - We compared recipients of eight lots of factors VIII and IX voluntarily withdrawn from distribution because one donor was known to have subsequently developed the acquired immunodeficiency syndrome with a nonexposed cohort matched by age, sex, and factor use. The factor VIII recipient cohorts did not differ in prevalence of antibody to human immunodeficiency virus (HIV) (exposed, 75%; nonexposed, 86%), T cell subset numbers (median: exposed, 619 T-helper cells per cubic millimeter; nonexposed, 659 T-helper cells per cubic millimeter), T-helper to T-suppressor ratios, or immunoglobulin levels. Exposed individuals had higher levels of immune complexes by C1q binding and staphylococcal binding assays and lower responses to phytohemagglutinin and concanavalin A. However, only the staphylococcal binding assay values were outside the normal range for our laboratory. Factor IX recipient cohorts did not differ in HIV antibody prevalence (exposed, 30%; nonexposed, 40%) or any immune tests. Although exposed and nonexposed individuals did not differ from each other in a clinically meaningful fashion at initial testing, both the exposed and nonexposed cohorts had high rates of HIV seroprevalence. Market withdrawals were clearly insufficient means of limiting the spread of HIV in hemophilic patients; however, the currently available methods of donor screening and viral inactivation of blood products will prevent continued exposure within this population. PMID- 3018308 TI - Consensus conference. The impact of routine HTLV-III antibody testing of blood and plasma donors on public health. PMID- 3018307 TI - HIV antibody screening. An ethical framework for evaluating proposed programs. AB - We believe that the greatest hope for stopping the spread of HIV infection lies in the voluntary cooperation of those at higher risk--their willingness to undergo testing and to alter their personal behavior and goals in the interests of the community. But we can expect this voluntary cooperation--in some cases, sacrifice--only if the legitimate interests of these groups and individuals in being protected from discrimination are heeded by legislators, professionals, and the public. Yet voluntary testing is not enough. We must proceed with vigorous research and educational efforts to eliminate both the scourge of AIDS and the social havoc that has accompanied it. PMID- 3018309 TI - Lung as a metabolic organ. PMID- 3018310 TI - [Transmission of pain sensation via the right phrenic nerve during cholecystectomy]. PMID- 3018311 TI - [Effects of edrophonium on neuromuscular transmission studied with isolated arm method under general anesthesia]. PMID- 3018312 TI - [Requirement of cyclic 3',5'-monophosphate in the production of a permeability increasing factor by Vibrio cholerae]. PMID- 3018314 TI - [Eating habits and bowel transit time]. AB - Bowel transit times of Chokai (Akita Pref.) and Ohasama (Iwate Pref.) peoples were 34.1 and 43.9 hours, respectively, and the difference between the two populations was 0.05 less than p less than 0.10. There was no significant difference in weight of stools passed per day, and they were 191.6 g, and 174.2 g, respectively. Stool weight per excretion of Chokai people was 196.1 g and that of Ohasama people was 167.9 g, and the difference between the two was p less than 0.05. Significant negative correlation between the stool weight and the bowel transit time was observed among Chokai people, but it was not significant among Ohasama people. Dietary fiber was not analysed, but from the view point of intake frequency of each food item or food group, there were no clear differences in eating habits which affect directly to the differences of bowel habits of the two populations. PMID- 3018313 TI - [Dietary factors to induce and suppress colon cancer development]. AB - It has been clear that the promoting stimulus from bile acids and other colonic constituents play an important role in colon cancer induction in man and in animal models, although essential for the initiation of this cancer probably is relatively week. The level and concentration of bile acids in the colon largely depend on the amount of dietary fat and fiber. High fat diet increases fecal bile acids, and fiber increases the fecal volume and may have a protecting effect through dilution of the bile acids in presenting to the colonic mucosa. It is suggested in animal models that changes of the enterohepatic circulation of bile acids following surgery such as cholecystectomy and right-sided hemicolectomy could provide another cocarcinogenic stimulus or anticarcinogenic mechanism in the development of this cancer. PMID- 3018315 TI - [Adenoid cystic carcinoma of the head and neck--a clinical study of 27 cases]. AB - Adenoid cystic carcinoma (ACC) is frequently seen in the salivary glands, but may occur at other sites in the head and neck. We report 27 patients with ACC treated at the Department of Otorhinolaryngology, Mie University School of Medicine, for 15 years. The survival rate dropped from 78% at five years to 36% at 10 years. This suggests that the long-term prognosis of ACC is poor. According to Batsakis, we classified ACC according to histopathology and emphasize that the solid cellular pattern had the worst prognosis. PMID- 3018316 TI - [A resectable case of juvenile hepatocellular carcinoma in an asymptomatic HBV carrier]. AB - A 28-year-old man was admitted to our hospital in October 1983 as he was found to have HBs-antigenemia at the time of blood donation. Since he had no symptoms and showed normal liver function, HBeAg(-), HBeAb (+) and HBcAb 87% (200x), he was diagnosed as an asymptomatic HBV-carrier. Unexpectedly, high serum AFP (13,500 ng/ml) was detected. Subsequently, computed tomography, angiography and ultrasonography demonstrated a tumor, 4.5 X 4.0 cm in diameter, in the left lobe of the liver. On Feb. 24, 1984, the tumor was radically resected. The postoperative course was excellent. This case suggested the relationship between hepatoma and asymptomatic HBV carriers and the importance of screening hepatoma patients to detect asymptomatic carriers. PMID- 3018317 TI - [Adult T cell leukemia accompanied by marked swelling of the left thigh]. AB - A patient with adult T cell leukemia (ATL) whose chief complaint was marked swelling of left thigh is reported. A 72-year-old woman was diagnosed as having ATL based on a high titer of anti-ATLA antibody and abnormal lymphocytes with convoluted nuclei. Computed tomography revealed swelling of her left thigh and a large low-attenuation mass in its muscles. Aspirate from her thigh contained many abnormal cells. Though chemotherapy reduced the swelling for two weeks, she had a relapse and died. This is the first case of ATL having such an onset reported in Japan. PMID- 3018318 TI - [An autopsy case of adult T-cell leukemia with acute hepatic failure]. AB - An autopsy case of ATL with acute hepatic failure is reported. A 39-year-old man born in Miyazaki Prefecture was admitted because of jaundice and general malaise of about 10 days' duration. Palpation of the abdomen revealed moderate hepatomegaly: the patient had clear consciousness. The white cell count was 62,500/mm3, with 34% abnormal lymphocytes having lobulated or convoluted nuclei. Marked liver dysfunction (TB 30.5 mg/dl; GOT 2,740 U; GPT 1,031 U; LDH 3,833 U) was noted. He developed hepatic coma and died of acute hepatic failure on the third hospital day. At autopsy, the enlarged liver weighed 2,000 g and was diffusely infiltrated by ATL cells. PMID- 3018321 TI - [Pathophysiology of pre-ATL (preleukemic state of adult T cell leukemia)]. PMID- 3018319 TI - [Inflammatory fibrous histiocytoma of the retroperitoneum]. AB - A 64-year-old man with the chief complaint of abdominal fullness was hospitalized on the suspicion of pancreatic tumor. The tumor was thought to be a pancreatic or retroperitoneal tumor, based on examinations of ultrasonography and CT scan of the upper abdomen, angiography of abdominal arteries, and ERCP. Laparotomy revealed that the tumor was inoperable inflammatory fibrous histiocytoma. Combined anti-cancer chemotherapy with doxorubicin, cyclophosphamide, and vincristine induced partial response. The duration of response was six months, and the patient died of tumor growth 18 months after the initial diagnosis. PMID- 3018320 TI - [Smoldering adult T-cell leukemia--HTLV-I has also an etiologic role in the diseases other than ATL]. PMID- 3018322 TI - [Immunologic characterization of lymphoid tumor cells from adult T-cell leukemia (ATL) and peripheral T-cell lymphoma (PTCL) in an ATL-endemic area]. PMID- 3018323 TI - [Treatment of adult T-cell leukemia]. PMID- 3018325 TI - [Physiopathology of thrombosis: protein C and thrombomodulin]. PMID- 3018324 TI - [Successful treatment with 2'-deoxycoformycin in a case of adult T-cell leukemia]. PMID- 3018326 TI - [Physiopathology of thrombosis: plasma fatty acids]. PMID- 3018327 TI - [Trends in the study of genetic engineering in cardiovascular diseases]. PMID- 3018328 TI - [Use of monoclonal antibodies in cancer therapy]. PMID- 3018329 TI - [Red cell enzyme anomalies and hemolytic anemia]. PMID- 3018331 TI - [A case of Paget's disease-like metastatic skin carcinoma involving the glans penis originating from bladder carcinoma]. PMID- 3018333 TI - [A case of multiple hormone producing tumor of the pancreas presenting WDHA syndrome]. PMID- 3018332 TI - [Reversal of portal blood flow in a case of primary hepatocellular carcinoma demonstrated by the ultrasonic pulse Doppler method during a short clinical course]. PMID- 3018330 TI - [Malignant fibrous histiocytoma of the pancreas]. PMID- 3018334 TI - [Experimental nephritis induced by Coxsackie B4 virus in mice--transient mesangial proliferation associated with acute viremia]. PMID- 3018335 TI - Activation of alveolar macrophages in pulmonary sarcoidosis: lack of correlation with serum angiotensin-converting enzyme activity. AB - To identify the activation of alveolar macrophages as a direct measure of disease activity in pulmonary sarcoidosis and to evaluate the possible relationship with serum angiotensin-converting enzyme (ACE) levels, we studied the morphology and function of alveolar macrophages obtained by bronchoalveolar lavage from patients with active disease (n = 12) and inactive disease (n = 5), and from normal controls (n = 11). At the time of lavage, 6 of 12 patients with active disease showed an elevation of serum ACE levels. When alveolar macrophages were cultured, more cells from active disease adhered to glass, spread out, and were positive for NBT reduction (p less than 0.001). Lysosomal enzyme (beta-galactosidase) activity was higher only in patients with active disease (p less than 0.001). In active disease, there were no significant differences in morphology or function of alveolar macrophages between higher and normal serum ACE levels. Thus, alveolar macrophages were activated in active pulmonary sarcoidosis and no correlation was found with serum ACE levels. These results suggest that serum ACE levels do not accurately reflect the activity of disease in the lung. PMID- 3018336 TI - Werner's syndrome associated with cholangiocarcinoma. AB - A 38-year-old Japanese man had cholangiocarcinoma in association with typical features of Werner's syndrome. The replicative capacity of fibroblasts in culture was characteristically reduced, and the in vitro natural killer cell activity was deficient. He died of massive G-I tract bleeding 8 months after admission. PMID- 3018337 TI - Chronic polymyositis: presence of coxsackievirus A9 antigen in muscle. AB - We report a case of chronic recurrent polymyositis associated with increasing antibody titers of coxsackievirus A9 in serum during clinical exacerbations. Muscle biopsy specimens showed pathologic changes consistent with chronic myositis, including perivascular mononuclear cell infiltration and hyalinization of muscle fibers with cytoplasmic vacuolations. The specific fluorescence was observed in the muscle fibers stained with antiserum for coxsackievirus A9. These findings indicate that this viral subtype as the etiologic agent in this case and virus plays a pathogenic role in some cases of chronic polymyositis. PMID- 3018339 TI - [Clinical and experimental studies on increases of immunoglobulins in patients with pneumoconiosis]. PMID- 3018338 TI - Bradykinin-induced cyclic AMP accumulation in mouse fibrosarcoma independent of prostaglandin E2 formation. AB - The relationship between bradykinin (BK)-induced prostaglandin E2 (PGE2) and cyclic AMP syntheses in mouse fibrosarcoma cells (HSDM1C1) was investigated. Maximal BK-induced increases in cyclic AMP preceded increases in PGE2 production. PGE2 synthesis reached maximum at a much lower concentration of BK than cyclic AMP synthesis. Indomethacin completely inhibited BK-induced PGE2 production, but did not influence the cyclic AMP levels. Arachidonic acid in the medium induced PGE2 production in large quantities, but increased cyclic AMP accumulation only slightly. A high PGE2 concentration increased cyclic AMP levels only slightly. Theophylline increased basal and BK-mediated cyclic AMP levels, but did not affect PGE2 production at all. These results indicate that BK-evoked PGE2 and cyclic AMP syntheses in HSDM1C1 are not dependent upon each other. PMID- 3018341 TI - [In vitro cultivation of human testicular germ cell tumors. (I). Establishment of three cell lines derived from embryonal carcinomas]. PMID- 3018342 TI - Role of fat, animal protein, and dietary fiber in breast cancer etiology: a case control study. AB - A case-control study of 818 breast cancer (BC) patients and 2 matched control groups, surgical controls (SCs) and neighborhood controls (NCs), was undertaken in Israel between 1975 and 1978. The interview schedule included a detailed dietary history based on the frequency of consumption of 250 food items, which were grouped according to their principal nutrient component. The average frequency of consumption of each food item in each nutrient group was computed. Medical, demographic, hormonal, and parity histories were also obtained. Risks associated with fat, animal protein, and fiber consumption were evaluated. Two types of analysis were performed [in 2 age groups (less than 50 yr and greater than or equal to 50 yr)], using the conditional logistic method: evaluating the risk attributable to nutrition only and controlling for nondietary confounding factors as well. When no adjustment for nondietary confounding factors was made, the risk increased with fat intake in both age groups [one-tailed P-value for linear trend = .08 and .07 in age less than 50 and .01 and .10 for the greater than or equal to 50 age category for the BC case (BCC)-SC and BCC-NC comparisons, respectively]. Increased fiber intake decreased the risk in the younger age group (one-tailed P-value for linear trend = .06 and .07 for the BCC-SC and BCC-NC comparisons, respectively), while in the 50-or-over age category the trend was inconsistent. The risk associated with animal protein was much less clear. For women in the highest quartiles of fat and animal protein intake and the lowest quartiles of fiber intake, risk was about twice as high as that for women in the lowest quartiles of fat and animal protein intake and in the highest quartile of fiber intake (one-tailed P-value for linear trend = .04 and .08 for age less than 50 and .08 and .09 for the age category greater than or equal to 50 BCC-SC and BCC-NC comparisons, respectively). When hormonal and demographic confounding factors were controlled for, this pattern persisted but it remained significant for 1 control only. Power increased when cases were analyzed against both controls simultaneously (one-tailed P-value for linear trend = .10 for age less than 50 and .02 for age greater than or equal to 50). Thus a higher fat-animal protein and lower fiber diet is associated with increased cancer risk, but this relationship needs to be studied further. PMID- 3018340 TI - [Preoperative cytodiagnosis of peripheral lung cancer]. PMID- 3018343 TI - Virus-induced pancreatic cancer in guinea fowl: a morphologic study. AB - The morphologic lesions in the pancreas of 350 guinea fowls infected with avian osteopetrosis virus strain Pts-56 were studied. Focal unspecific changes in the acinar tissue, including reduction of zymogen content, diminution of cytoplasm, and dilatation of acinar lumens accompanied with substitution of the affected acini by tubular structures lined with centroacinar-like cells, developed 2 months from post infection (p.i.) in about 30% of the birds. Adenomatous growths of ductules with mucin-producing or mucin-nonproducing epithelium were observed independently or parallelly to these changes after 3 months p.i. Pancreatic carcinomas arose as early as 4 months p.i. The number and size of the neoplastic foci increased with time, and after the 6th month neoplastic foci were established in almost all infected guinea fowls. Neoplastic proliferations of type A and D endocrine cells were identified in the carcinoma foci of separate cases. Other associated neoplasms were bone tumors and papillomatous growths of the duodenal mucosa. PMID- 3018344 TI - Immune response to avian leukosis virus determined by the cell-mediated focus reduction (CEMFOR) assay. AB - A cell-mediated focus-reduction (CEMFOR) assay was used to determine the sequential development of cell-mediated immunity to avian oncornaviruses and the nature of the cells participating in this immune reaction. Peripheral blood leukocytes of Leghorn chickens that had regressed Rous sarcoma virus-induced tumors or were immune to avian leukosis virus had CEMFOR activity. The response was biphasic early in the infection. Peripheral blood leukocytes from nonimmune chickens or viremic, immunologically tolerant chickens did not have CEMFOR activity. Sera from leukosis virus-immune chickens blocked CEMFOR activity. The early CEMFOR response was mediated by T-cells. The late response was mediated by an adherent cell population possibly augmented by the presence of T-cells. PMID- 3018345 TI - In vivo immunologic selection of proviral gene deletion variants from a nonproducer clone. AB - The mechanism by which tumors recur at sites of injection of retrovirus-infected fibrosarcoma cell lines was investigated. Previously, it was established that tumor recurrences reflect outgrowth of rare cells that lack viral antigens and are susceptible to superinfection with the homologous retrovirus. In the present study clones isolated from a retrovirus-infected cell line were evaluated as precursors for tumor recurrence. Under conditions of in vivo immunologic selection, a clone that contained a single abbreviated copy of the provirus formed variants that lacked the proviral gene. Tumor variants lacking the proviral gene grew progressively in both nonimmune and virus-immune male Sewall Wright strain 2 guinea pigs. Tumor recurrence could be prevented by superinfection of the virus-infected fibrosarcoma cell line or by superinfection of the precursor for tumor recurrence. Cell lines infected with retroviruses varied in frequency of tumor recurrence formation. This model may be useful in analyzing gene deletion as a mechanism of tumor escape from host immunologic attack. PMID- 3018346 TI - Cytomegalovirus infections: a review. PMID- 3018347 TI - Sodium transport in red blood cells from dialyzed uremic patients. AB - Studies on red blood cell (RBC) sodium (Na) transport in chronic renal failure have described abnormalities in the ouabain-sensitive Na, K pump. We now report Na transport in RBC using cation flux methodology, measuring both the ouabain sensitive Na, K pump and the ouabain-insensitive Na, K cotransport (CoT) and Na, lithium (Li) countertransport (CTT) in 28 subjects on hemodialysis, eight subjects on chronic ambulatory peritoneal dialysis (CAPD) and 29 control subjects. Intracellular cation content and passive permeability of Na were also examined. Mean Na efflux through the ouabain-sensitive Na, K pump was not reduced in dialysis patients when compared to normal subjects, whether measured in fresh cells (1.41 +/- 0.05 vs. 1.30 +/- 0.03 mmole/liter RBC/hr; P less than 0.05) or in Na-loaded cells (7.10 +/- 0.24 vs. 6.90 +/- 0.22; NS). There was, however, a marked and uniform suppression of the CoT pathway in Na-loaded cells from dialysis patients versus controls (0.14 +/- 0.02 vs. 0.41 +/- 0.05 mmole/liter RBC/hr; P less than 0.001). Mean CTT activity, as measured by Li efflux, was not different between dialysis and normal subjects. Uremic and normal RBC had similar intracellular Na or K content as well as passive permeability for either ion. This indicates that intracellular cationic homeostasis is maintained, perhaps secondary to balanced changes in cationic flux activity through these transport pathways. PMID- 3018348 TI - Mineralocorticoid-stimulated renal acidification: the critical role of dietary sodium. AB - Recent in vitro studies of isolated distal nephron segments have demonstrated that mineralocorticoid hormone stimulates H+ secretion by both Na+-dependent and Na+-independent mechanisms, and the Na+-independent acidification mechanism has a greater capacity. These in vitro data suggest that mineralocorticoid administration in vivo might increase renal acid excretion when an augmentation in distal Na+ reabsorption is precluded by rigid restriction of dietary Na+; under these circumstances, virtually all Na+ delivered to the distal nephron is reabsorbed in the basal state. In the present studies, prolonged (12 days) administration of DOC (15 mg/day) was undertaken in both Na+-fed and rigidly Na+ restricted dogs with chronic HCl acidosis. Na+-fed animals responded to DOC administration with a large increment in net acid excretion and complete correction of metabolic acidosis. Marked hypokalemia and significant kaliuresis also occurred. Na+-restricted dogs experienced no changes in renal acid excretion, systemic acid-base equilibrium, plasma [K+] or K+ balance. These results suggest that both renal H+ and K+ excretory responses to prolonged mineralocorticoid hormone administration in vivo are critically dependent on the availability for reabsorption of surplus Na+ within the distal nephron; this requirement is met when the diet, and hence the final urine, contains Na+ but cannot be satisfied when dietary Na+ is rigidly restricted. PMID- 3018349 TI - [Differential diagnostic potentials of 2-stage hepatic scanography]. PMID- 3018350 TI - [Ultrasonic diagnosis of gallbladder cancer]. PMID- 3018353 TI - [Differential diagnosis of viral hepatitis]. PMID- 3018351 TI - Demonstration of spermatozoa in the lung of an AIDS patient. AB - Using the DNA-binding fluorochrome, DAPI, spermatozoa and Pneumocystis carinii were easily demonstrated in postmortem lung sections of an AIDS patient. The possible practical significance of these findings is discussed. PMID- 3018352 TI - HTLV-III antibodies in hemodialysis patients--a consequence of blood transfusions? AB - An investigation for HTLV-III antibodies in chronic hemodialysis patients revealed in four out of 276 patients a positive result using the ELISA and western blot techniques. All HTLV-III positive patients had received blood transfusions. As it has been shown that a needle stick could transmit the HTLV III, it is suggested that hemodialysis patients who have received frequent blood transfusions should be screened. PMID- 3018354 TI - [Role of viral infection in chronic nonspecific lung diseases]. PMID- 3018355 TI - A case of hemophilia A with seroconversion to human T-lymphotropic virus III. PMID- 3018356 TI - [Congress for continuing education in oncology for nursing personnel]. PMID- 3018359 TI - Expression of monocyte-specific oncogenes c-fos and c-fms in HL60 cells treated with vitamin D3 analogs correlates with inhibition of DNA synthesis and reduced calmodulin concentration. AB - Monocytic differentiation of cultured leukemic cells HL60 can be induced by a variety of compounds including analogs of vitamin D3. We studied the expression of oncogenes c-fos, c-fms, c-fes, c-myb, c-myc, and c-Ha-ras during this process, and attempted to relate these and a conventional marker of monocytic differentiation, the nonspecific esterase activity, to changes in cytosol levels of the Ca2+-binding proteins. The analogs of vitamin D3, 1 alpha,25 dihydroxycholecalciferol, 1 alpha,25-dihydroxy-26,27-hexafluorocholecalciferol, and 1 alpha,25-dihydroxy-24R-fluorocholecalciferol, reduced the total calcium binding activity and the cellular levels of calmodulin, but calbindin D28k could not be detected in HL 60 cells. A correlation was evident between the potency of these compounds as inducers of differentiation demonstrated by nonspecific esterase activity, the degree of reduction in calmodulin concentration, the rate of the inhibition of DNA synthesis and of expression of c-myc and c-myb genes, and the induction of the expression of oncogenes c-fos and c-fms. These experiments indicate that the events which underlie monocytic differentiation of HL 60 cells can be observed to occur in discrete steps which include an inhibition of DNA synthesis, a reduction of cellular levels of calmodulin, and altered expression of several oncogenes. PMID- 3018358 TI - Lymphoid cell lines as a model system for the study of Wolman's disease: enzymatic, metabolic and ultrastructural investigations. AB - Epstein-Barr virus (EBV) transformed lymphoid cell lines (LCL) were established from blood lymphocytes of a patient affected with Wolman's disease (WD) and from her parents. These LCL showed a severe deficiency in acid lipase activity using every substrate in comparison to LCL from normal subjects, in which acid lipase activity was similar to that in blood lymphocytes. In the LCL from Wolman's disease a major accumulation of neutral lipids was observed, mainly cholesteryl esters, CE (amount around 7 times higher than in normal cells and ratio of esterified/free cholesterol increased by 10 times) and to a lesser extent triglycerides, TG (amount increased by 1.5 times). Electron microscopy showed the storage vacuoles of neutral lipids quite characteristic of this lysosomal disease. The reported data demonstrated the validity of transformed LCL as a cellular model system in culture for experimental studies of Wolman's disease and for investigating the lysosomal metabolism of neutral lipids. PMID- 3018360 TI - Herpes simplex virus suppression of human endothelial matrix protein synthesis is independent of viral protein synthesis. AB - Protein synthesis was determined in cultures of human umbilical cord vein endothelial cells (EC) infected with either herpes simplex type 1 (HSV-1) or type 2 (HSV-2). Monolayers were infected for 1 hour with either 5 or 20 infectious virus particles per EC (multiplicity of infection of 5 or 20). At different times after infection, infected and noninfected cultures were pulsed with either 5 mu Ci/ml of [14C]proline or 25 mu Ci/ml of [35S]methionine for 1 or 2 hours. Autoradiograms of sodium dodecyl sulfate-gel electrophoresis showed suppression of matrix protein synthesis by 4 hours postinfection which became almost complete at 6 hours. Multiplicity of infection of 20 was more effective than multiplicity of infection of 5 at suppressing matrix protein synthesis at 4 or 6 hours postinfection. Infection of EC with ultraviolet light-inactivated virus resulted in the shutoff of host-cell as well as virus protein synthesis. Similar results were observed when monolayers of EC were infected with intact virus in the presence of 2 micrograms/ml of actinomycin-D, an inhibitor of RNA synthesis. On the other hand, uninfected EC in the presence of actinomycin-D continued to synthesize protein from pre-existing mRNA. The time of shutoff of synthesis of specific matrix proteins varied with the protein i.e., shut-off of type IV collagen occurred first, followed by that of fibronectin, and then of thrombospondin. The data suggest that the degree of suppression of synthesis of EC matrix proteins after infection with HSV-1 or HSV-2 is dependent on the dose of the virus inoculum; it occurs at the translational level and is not dependent on new viral protein synthesis. PMID- 3018361 TI - Tetrahydrocannabinol stability in whole blood: plastic versus glass containers. AB - The stability of tetrahydrocannabinol (THC) in whole blood during storage was investigated. Two types of containers were used: plastic tubes (polystyrene) and glass vials. Freshly drawn blood was spiked with 10 pmol THC/mL and stored for four days at room temperature (20 degrees to 25 degrees C). The material was kept for four weeks at -20 degrees C. Blood samples from 16 persons apprehended by the police on suspicion of cannabis smoking were collected into the two types of containers and stored frozen (-20 degrees C) until analysis. THC was quantified by capillary column gas chromatography/electron impact mass spectrometry (GC/EI MS) combined with selected ion monitoring (SIM). Deuterium-labeled THC (THC-d3) was used as internal standard (IS) and was added to the samples immediately prior to extraction. The THC level remained unchanged in glass vials. Samples from the plastic containers had lost 60 to 100% of their THC during storage. In 13 authentic samples, the THC level in blood from the plastic containers varied 0 to 87% from the level in the same blood stored in glass vials. The IS was undetectable in some of these samples. PMID- 3018357 TI - Disorders of the pyruvate dehydrogenase complex. AB - Pyruvate dehydrogenase deficiency may be a non-specific consequence of many different neurological degenerative disorders. There are also serious methodological problems in estimating the activity of this enzyme complex. PMID- 3018362 TI - Potentials of wide-bore fused silica capillary columns for substance identification by means of retention indices. AB - A wide-bore capillary column with a methylsilicone stationary phase has been evaluated with regard to its ability to identify substances by means of their retention indices. Column efficiency was compared with a normal packed column and a medium-bore capillary column, and the impact of carrier gas flow and load capacity were investigated. It was shown that under defined conditions retention indices on the wide-bore capillary column were comparable to those available in a data bank obtained on packed columns. PMID- 3018364 TI - Epidermal growth factor receptors in parathyroid tumors. AB - Hyperparathyroidism is caused by parathyroid adenomas, hyperplastic parathyroid glands, or rarely parathyroid carcinoma. Membrane receptors to epidermal growth factor (EGF), a growth-stimulating polypeptide, have been shown in other endocrine tissues such as thyroid, breast, and ovary, but not in parathyroid glands. Therefore we studied abnormal parathyroid glands from fourteen patients for the presence of EGF receptors. The binding of radioiodine-labeled EGF to the crude membrane fractions was studied using competitive inhibition with unlabeled EGF. In ten patients with solitary parathyroid adenomas, seven adenomas had no EGF binding, three had low affinity EGF binding with dissociation constants (Kd) of 28 to 148 nM and maximal specific binding (Bmax) of 285 to 1944 fmole/mg protein. In two patients with multiple adenomas, a high affinity EGF binding with Kd of 0.28 to 2.8 nM and Bmax of 6.7 to 43 fmole/mg protein was found. In one patient with hyperplastic parathyroid glands secondary to renal failure, a high affinity EGF binding with Kd of 1.7 nM and Bmax of 18 fmole/mg protein was found. In one patient with persistent hyperparathyroidism following a successful renal transplant (tertiary hyperparathyroidism), a low affinity EGF binding with Kd of 25 nM and Bmax of 219 fmole/mg protein was found. The binding of EGF did not correlate with the preoperative serum calcium or PTH levels. Thus, hyperplastic parathyroid glands (either primary or secondary) have high affinity EGF receptors whereas solitary parathyroid adenomas do not. PMID- 3018365 TI - Vasoactive intestinal peptide augmentation of cholecystokinin-8-stimulated pepsinogen secretion: evidence for dual modulation of chief cell function. AB - Pepsinogen (PPG) secretion from chief cells (CC) is dually modulated by adenosine 3':5'-monophosphate (cAMP) and calcium second messenger systems. Vasoactive intestinal peptide (VIP) stimulates cellular function by elevating intracellular levels of cAMP. In contrast, cholecystokinin-8 (CCK-8) acts by producing a rise in intracellular calcium concentration. Consequently, it was the purpose of this study to test whether VIP (acting through cAMP-mediated systems) could augment CCK-8 (acting through calcium-dependent systems)-stimulated PPG secretion. Collagenase dispersed rabbit isolated gastric glands (IGG) were incubated alone (unstimulated) or with secretagogues for 30 min. VIP in graded doses of 10(-11) to 10(-7) M was used alone or in combination with CCK-8 (10(-9) M). PPG levels were determined using an assay based on pepsin hydrolysis of [14C]methemoglobin. Results are expressed as percentage of total pepsinogen (within the IGG) secreted above unstimulated levels. VIP alone (10(-11) to 10(-7)) or CCK-8 alone (10(-9)) did not significantly stimulate PPG secretion (P greater than 0.05). The combination of CCK-8 (10(-9) M) plus VIP (10(-7) M) significantly stimulated PPG secretion above unstimulated levels (P less than 0.05). Thus, the combination of VIP and CCK-8 produced greater PPG secretion than either secretagogue alone. These data support the hypothesis that secretagogues acting through either cAMP or calcium-mediated systems contribute to the regulation of PPG secretion from CC and that the two second messenger systems act in concert achieving at least additive effects. PMID- 3018366 TI - Paradoxical effect of trifluoperazine, a calmodulin antagonist, on pepsinogen secretion. AB - Pepsinogen secretion (PS) is modulated at the intracellular level by both cAMP and calcium ion. Cholecystokinin octapeptide (CCK-8), a potent stimulus for PS, is believed to act through calcium. The most extensively studied pathway for calcium-mediated modulation involves the formation of calcium/calmodulin complexes, leading to activation of calmodulin. We have therefore examined the hypothesis that an inhibitor of calmodulin might inhibit PS stimulated by CCK-8. The phenothiazine derivative trifluoperazine (TFP) was chosen as a calmodulin antagonist. We measured in vitro secretion of pepsinogen by isolated gastric glands as a function of TFP concentration 10(-6) M-5 X 10(-4) M), in the presence and absence of a maximal concentration of CCK-8 (10(-7) M). Cellular viability was determined by measurement of release of the enzyme lactate dehydrogenase (LDH) into the medium. TFP did not significantly inhibit PS stimulation by CCK-8 at any concentration (P greater than 0.05). At 10(-4) M, TFP actually augmented PS stimulation by CCK-8 (P less than 0.05). TFP alone significantly stimulated PS (P less than 0.05) at 5 X 10(-5) M and above. TFP did not raise cAMP levels at any concentration tested (P less than 0.05), in contrast to the adenylate cyclase activator forskolin, 10(-5) M, which caused a 6- to 37-fold increase (P less than 0.05). TFP, 2 X 10(-4) did not increase LDH levels significantly (P less than 0.05). Thus a calmodulin inhibitor, TFP, paradoxically stimulates PS. This stimulatory effect of TFP is not cAMP-dependent and is not accompanied by a nonspecific release of LDH into the medium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018367 TI - Hepatic blood flow measurement with inert gas clearance. AB - Inert gas clearance has been used for 20 years to measure hepatic blood flow. Injection of a saline solution of 85Kr or 133Xe is usually made via the PV, and the resulting hepatic clearance is monitored with a Geiger-Muller tube, scintillation crystal, or gamma camera. Complex slow components in 133Xe clearance curves, once believed to indicate a correspondingly complex hepatic microcirculation, are now considered to be caused by nonhepatic radioactivity. Normal liver is therefore believed to receive a homogeneous perfusion throughout the depth of tissue in any given region. HA blood and PV blood are normally completely mixed in the hepatic sinusoids. Macroscopic variations in tissue perfusion may exist in different lobes of the liver in both animals and man. The technique expresses flow in units of milliliters per minute per 100 g. Accurate and acceptably reproducible results have been obtained after PV injection of isotope; fast component analysis of 133Xe clearance is most appropriate, while beta detection of 85Kr yields a simple monoexponential curve. Normal hepatic blood flow in dogs and in man is 100-130 ml min-1 100 g-1. Employing sites of isotope administration other than the PV produces inaccurate results unless appropriate corrections are made. Accuracy of flow measurement is critically dependent on a knowledge of the partition coefficient of the gas used. Liver disease per se does not affect measurement accuracy, and many practical features make the technique an attractive tool for the measurement of hepatic hemodynamics in man. Nevertheless, it is essential that the investigator be aware of certain limitations of the method, and carefully apply current concepts of clearance curve analysis and interpretation, in order to derive maximum advantage. PMID- 3018368 TI - Arthritis and an endocrine abnormality. PMID- 3018363 TI - Superoxide generation during cardiopulmonary bypass: is there a role for vitamin E? AB - The cytotoxic metabolites of oxygen [superoxide (O-2), hydrogen peroxide (H2O2), and hydroxyl (OH.)] have been demonstrated to be involved in the peroxidation of membrane lipids consequently altering membrane composition, morphology, and function. Of all the lines of defense adopted by living organisms against toxic oxygen free radicals, vitamin E is most effective in the prevention of membrane damage. Cardiopulmonary bypass (CPB) has been shown to activate complement and cause sequestration of leukocytes which can recruit, adhere, and stimulate release of cytotoxic oxygen radicals. A prospective study of 30 patients evaluated the effects of CPB with and without an exogenous free radical scavenger (Group I, N = 20, control) and (Group II, N = 10, vitamin E) on H2O2 (a marker of oxygen free radicals) malonaldehyde (a marker of lipid peroxidation), transpulmonary leukosequestration, and plasma levels of vitamins E and C. Group I showed a progressive increase in H2O2 during CPB from 65 +/- 6 to 130 +/- 11 micron/ml (P less than 0.0001); plasma vitamin E decreased from 15 +/- 3 to 6 +/- 1 mg/liter (P less than 0.0001) while vitamin C increased from 1.6 +/- .3 to 2.3 +/- .3 mg/dl (P less than 0.0001). Group II showed no significant increase in H2O2 (from 78 +/- 8 to 93 +/- 5 microns/ml) during CPB and a significant reduction in H2O2 levels compared to Group I (P less than 0.001); plasma vitamins E and C did not change significantly in Group II.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018369 TI - Effect of vinblastine, a potent antimicrotubular agent on steroid secretion by perifused frog adrenal glands. AB - The role of microtubules in adrenal steroidogenesis was examined in vitro, using frog interrenal tissue. Adrenal dice from Rana ridibunda were perifused with amphibian culture medium and the effect of various antimicrotubular drugs was studied. The amounts of corticosterone and aldosterone released in the effluent perifusate were radioimmunoassayed using specific antisera. Administration of colchicine, nocodazole, and vinblastine (10(-5) M) did not affect spontaneous secretion of corticosterone and aldosterone. These results indicated that, in contrast to microfilaments which play an important role in spontaneous steroidogenesis, the microtubular system is not required for basal corticosteroid secretion. However, vinblastine (10(-5) M) was responsible for a marked decrease in ACTH-induced stimulation of corticosterone and aldosterone production. Conversely, vinblastine did not significantly alter the response of interrenal tissue to dibutyryl cAMP, forskolin and NaF, indicating that the microtubules are involved in an early step of ACTH action, namely at the level of the receptor subunit. PMID- 3018370 TI - Desensitization of Sertoli cell-enriched cultures by FSH, L-isoproterenol and glucagon. Influence on subsequent stimulation of 17 beta-estradiol production. AB - Sertoli cell-enriched cultures derived from 19-day old rats were exposed to FSH, L-isoproterenol, glucagon and dbcAMP for 24 up to 96 h. The influence of primary stimulation with these agonists on the response of the cells to subsequent stimulation with the homologous or heterologous agonists was investigated. Particular attention was paid to the response of the aromatase system defined as the ability of the cells to convert testosterone into 17 beta-estradiol. The responsiveness of this system was compared with the responsiveness of two other systems: adenylate cyclase and phosphodiesterase. It could be demonstrated that preincubation with the mentioned agonists results in a decreased responsiveness (maximal response) and a decreased sensitivity (ED50) upon re-stimulation with the homologous agonists. Preincubation with FSH also provokes partial desensitization for glucagon and L-isoproterenol. This heterologous desensitization can be mimicked by dbcAMP. The mentioned desensitization reactions are accompanied by a marked decrease in the accumulation of cAMP in the medium. Whereas desensitization of the adenylate cyclase occurs rapidly, desensitization of the aromatase response requires protracted stimulation for 3-4 days. In contrast with the adenylate cyclase and the aromatase system, the responsiveness of phosphodiesterase to FSH, L-isoproterenol, glucagon and dbcAMP is not affected by repeated stimulation for 96 h. It is concluded that hormonal desensitization affects both early (cAMP) and late (aromatase) responses of the Sertoli cell. However, some responses, such as phosphodiesterase activity seem to escape the desensitization process. PMID- 3018371 TI - Regulatory factors of glucocorticoid binding in early and term rat placenta. AB - We have measured by an exchange procedure the binding of [3H]dexamethasone in cytosol of early (10-13 days) and late (19-22 days) placentas from pregnant rats. Binding was 3-fold higher in late placentas both in the presence of Na2MoO4. We then studied some possible regulatory factors in order to explain differences in binding at both gestational ages. The activity of enzymes compromising the phosphorylation (acid and alkaline phosphatases) or stability (protease) of the receptor were normal or lower in early as opposed to late placenta, discarding these enzymes as leading regulatory factors. Cyclic nucleotides were also studied, in view that they regulate steroid binding in uterus and placenta. Both basal and epinephrine-stimulated production of cAMP were higher in early placenta. cAMP (but not cGMP) inhibited [3H]dexamethasone binding by reducing the number of sites without changing the Kd. Moreover, addition of epinephrine in concentrations that maximally stimulated cAMP, inhibited subsequent binding of [3H]dexamethasone in cytosol. We suggest that cAMP may be a modulator of glucocorticoid binding at the early stages of placental development. The significance of this mechanism may be understood in terms of the opposing effects of cAMP and glucocorticoids on placental progesterone production. PMID- 3018372 TI - Involvement of cycloheximide-sensitive mediators in the steroidogenic action of adrenocorticotropin and angiotensin II. AB - The involvement of short-lived proteins in the steroidogenic action of corticotropic peptides has been investigated in vitro by means of a perifusion technique using frog adrenal glands. Graded concentrations of cycloheximide (10( 7) M to 10(-5) M) led to a dose-related inhibition of corticosterone and aldosterone production. The perifusion model gives detailed information on the kinetics of the inhibitory effect of cycloheximide. This effect was rapidly observed (the lag period was about 15 min), maximum inhibition being obtained 25 min after the end of administration of the protein synthesis inhibitor. Whatever the concentration of cycloheximide, corticosteroid output returned to basal values 2 h after the onset of cycloheximide infusion. Stimulation of steroidogenesis by ACTH and angiotensin II was totally inhibited by cycloheximide (10(-6) M) indicating that the synthesis of a labile protein was required for the adrenal response to corticotropic peptides. In addition, the stimulatory effect of cAMP and PGE1, which are considered to be the second messengers of ACTH and angiotensin II in amphibian interrenal gland, was blocked by cycloheximide. Taken together, these data suggest that a labile protein is involved in an early step of corticosteroid biosynthesis in the frog. PMID- 3018373 TI - Temporal effects of progesterone domination on estrogen and oxytocin receptors in hamster uterus. AB - The purpose of this study was to determine whether progesterone (P)-induced down regulation of estrogen receptors (Re) and oxytocin receptors (ROT) changes with the time of P exposure. Ovariectomized hamsters were given s.c. Silastic implants of estradiol (E2) and P for 4, 8 and 16 days. Cytosol and nuclear Re were measured at low temperature with the pyridoxal phosphate exchange assay, and ROT was assayed in the membrane fraction by [3H] oxytocin binding. Nuclear Re and ROT were down regulated throughout the 16-day P exposure period, but cytosol Re (and total Re) increased progressively from 4 to 16 days indicating that the down regulation of cytosol Re escapes P control with time. This conclusion was supported by P withdrawal studies in which P implants were removed for 6 or 12 h. P withdrawal resulted in equivalent recovery responses of nuclear Re and ROT after 4, 8 and 16 days of P exposure. Although cytosol Re recovery to P withdrawal occurred at 4 and 8 days, no response was obtained after 16 days of P exposure. Uterine weight increased during steroid treatment, and morphometric analysis of the P-dominated uterus revealed significant increases in the cross sectional area of the endometrium and myometrium with time of P exposure. Cytological examination of the uterus showed prominent secretory changes in the epithelial compartment on day 16 with accumulation of secretion in the uterine lumen. These results demonstrate that P can chronically down regulate nuclear Re and ROT. However, the control of cytosol Re varies with the time of P exposure, and cytosol Re levels become refractory to P domination by 16 days. The present observations indicate that the escape of cytosol Re from P control may be associated with the proliferation of one of more uterine cell populations such as glandular and luminal epithelial cells. PMID- 3018374 TI - Regioselective reaction of thiols with catechol estrogens and estrogen-O quinones. AB - Incubations of [3H]estradiol and [3H]2-hydroxyestradiol (2-OHE2) with rat liver microsomes and mushroom tyrosinase were carried out in the presence of glutathione and 2-mercaptoethanol. A ratio of about 3.5:1 for the C-4 and C-1 thioether conjugates of 2-OHE2 was observed. Chemical reaction of estradiol-2, 3 O-quinone with various thiols showed that alkyl and phenyl thiols gave about a 1:1 ratio of C-4 to C-1 thioethers. However, reaction of the O-quinone with 4 nitrothiophenol gave a C-4/C-1 ratio of 0.25 while 4-bromothiophenol gave a C-4/C 1 ratio of 4.0. These studies suggest that the regioselectivity of the reaction of thiols with estrogen catechols and O-quinones may be dependent on the nature of the thiol compounds and less on steric hindrance. PMID- 3018375 TI - 3 beta-hydroxysteroid isomerase dehydrogenase in guinea-pig kidney: possible involvement in 11-deoxycorticosterone formation in situ. AB - 3 beta-Hydroxysteroid isomerase dehydrogenase, capable of acting on C21- and C19 3 beta-hydroxy-5-ene-steroids has been found in guinea-pig kidney at equivalent levels to those in guinea pig testes. Of the 3 beta-hydroxy-5-ene-steroids present in guinea pig serum, 21-hydroxypregnenolone occurs in highest concentration (17 nM) followed by pregnenolone (10 nM), whereas 17 alpha-hydroxy pregnenolone and dehydroepiandrosterone occur in very low concentrations (less than 0.5 nM). Furthermore, the concentration of 21-hydroxypregnenolone relative to 11-deoxycorticosterone (the mineralocorticoid of the guinea pig), is 10:1 (Nishikawa and Strott, Steroids 41 (1983) 105-120). The apparent Km value for 21 hydroxypregnenolone, for the reaction yielding 11-deoxycorticosterone as catalysed by guinea pig kidney microsomes, was 85 nM and the Vmax 33 pmol/min per mg protein. Pregnenolone was a competitive inhibitor (apparent Ki = 5 microM) in the above reaction. A sex difference in the level of the enzyme in the kidney was found (activity in the female was one-third of that in the male) which may indicate that the enzyme is under partial androgen control. 3 beta-Hydroxysteroid isomerase dehydrogenase activity was also detected in guinea pig liver and again it was lower in the female. Whilst the exact role of 3 beta-hydroxysteroid isomerase dehydrogenase in guinea-pig kidney remains uncertain, the data suggest that it may utilise blood-borne 21-hydroxypregnenolone, the later then playing the role of a prohormone. PMID- 3018377 TI - Normal development of cardiac beta adrenoceptors in mice exposed to ethanol in utero. AB - Abnormalities of cardiac physiology and anatomy may occur in children with the fetal alcohol syndrome. To understand the basis of these abnormalities, research has been performed which shows that in mice exposed to ethanol from gestation day 8 to birth via a liquid diet regime with pairfed controls there are ultrastructural changes in the cardiac myocytes. To determine if the exposure to ethanol also affects the development of the cardiac noradrenergic system, which in turn could cause developmental abnormalities, beta adrenoreceptor binding was characterized by Scatchard analyses of concentration dependent binding curves in newborn mice exposed to ethanol in utero. Although body and heart weight were lower in newborns from both the pairfed and ethanol groups compared to a normally fed group, no differences in the densities of the beta adrenoceptors or in the Kd values for binding were seen due to the ethanol or the liquid diet regime. Therefore, at least one component of the development of the cardiac sympathetic system was not altered by ethanol exposure or a reduced caloric intake. PMID- 3018376 TI - Ecological relevance of memory tests and the prediction of relapse in alcoholics. AB - In an inpatient alcoholism rehabilitation program, 56 men were administered two 10-min memory tests: the Product Recall Test (PRT), designed to assess memory for familiar stimuli (assumed to be relatively high in ecological relevance), and the Memory-for-Designs Test (MFD), a test of memory for novel patterns of stimuli (assumed to be relatively low in ecological relevance). Approximately 74% of subjects who recalled less than or equal to half the items of the PRT relapsed at 3 months compared to only 33% of the subjects who recalled more than half the items. Performance on the MFD was not related to relapse rate. PRT performance was almost as predictive of relapse at 3 months as aftercare attendance, and combining both of these variables further improved predictability. The results suggest that the familiarity of the stimuli employed in memory tests may be important in tapping cognitive deficits of alcoholics that place these subjects at increased risk for relapse. The implication of these findings for the time effective identification of early relapsers from alcoholism treatment programs are discussed. PMID- 3018378 TI - Binding of (3H)-piretanide to a specific receptor of renal medulla. AB - We had previously demonstrated that (3H)-piretanide binds to a receptor located on medullary membranes of canine kidney. Here we show that binding is specific for a particular group of loop diuretics. Sulfonamide diuretics of the benzoic acid family, other substituted sulfonamides, and phenoxy-acetic acid derivates displace (3H)-piretanide from its receptor. Loop diuretics that do not act at the luminal tubular membrane do not displace piretanide, nor do diuretics with a different site and mode of action (thiazides; inhibitors of the Na+/H+ antiporter (amiloride), of Na+K+ ATPase (ouabain), or of carbonic anhydrase (acetazolamide). We demonstrate that no interference occurs between the piretanide receptor and membrane bound receptors of several neurotransmitters. PMID- 3018379 TI - Energy storage during DNA girase activity. AB - In this work, we develop a minimal two-cycle model for the action of DNA girase. One of the cycles describes the ATP dependent chemicomechanical transduction performed by the enzyme. The other cycle describes the relaxing activity exhibited by girase on supercoiled DNA in the absence of substrates. Supercoiling of DNA is described as a random walk on a topological index. The mechanical energy storage in DNA is manifested in the fact that in general, the kinetic constants for the cycles do not satisfy Wegscheider relations. Finally, a connection is established between the degree of DNA supercoiling and the hydrolysis of ATP. PMID- 3018380 TI - The pH in the neighborhood of membranes generating a protonmotive force. AB - The chemiosmotic mechanism provides a way whereby energy inherent in a chemical combustion process is extracted and transduced: first into the energy of electron X volts of the electron redox system and second into proton X volts as protons are forced to leave the interior of the cell, creating an electro-chemical protonic potential (the protonmotive force). Here we consider the distribution of potential and pH across the membrane and the phases bathing the membrane in more detail. The distribution of hydrogen ions parallel to the surface is also described. It is shown that the voltage and pH gradients due to the proton extrusion occur near to the membrane (approximately 2 nm). This implies that the pH is much lower immediately outside the membrane than in the cytoplasm or in usual neutral growth or isotonic media. It provides a link between the points of view of Mitchell and Williams. It requires that literature models for the role of the protonmotive force in the maintenance of wall thickness in Gram-positive organisms, the adhesion of microbes to surfaces, and the transport of auxin in plants be modified. PMID- 3018381 TI - Effect of ginsenosides Rg1, Rc and Rb2 on hormone-induced lipolysis and lipogenesis in rat epididymal fat cells. AB - Ginsenosides Rb2, Rc and Rg1 suppressed corticotropin-induced, dibutyryl cyclic AMP-induced and epinephrine-induced lipolysis with the relative potencies Rb2 greater than Rc greater than Rg1. The inhibition of corticotropin-induced lipolysis by ginsenoside Rg1 could not be overcome by increasing the dose of the lipolytic hormone while that of ginsenosides Rc and Rb2 was nearly abolished by corticotropin at a dose of 40 nM which by itself produced maximal lipolysis. Dibutyryl cyclic AMP-stimulated lipolysis was also inhibited. Only ginsenoside Rb2 suppressed glucagon-induced lipolysis. All three ginsenosides did not inhibit basal lipolysis or basal incorporation of D-[3-3H]glucose into lipids. Insulin stimulated lipogenesis was diminished by ginsenosides Rg1 and Rc but not by ginsenoside Rb2. PMID- 3018382 TI - Protective metabolic effects of propranolol during total myocardial ischemia. AB - Clinical trials have shown an increase in survival in patients treated with beta blockers after infarction. In addition, the majority of patients undergoing myocardial revascularization are also treated preoperatively with beta blockers. It is commonly thought that beta blockers exert their protective effect primarily by decreasing heart rate and subsequent myocardial work. The present study was designed to determine whether beta blockade has any primary protective metabolic effects on globally ischemic myocardium. Thirty-four anesthetized dogs underwent total myocardial ischemia at 37 degrees C. High-energy nucleotide and lactate levels in left ventricular tissue samples were determined at control and at 15 minute intervals as well as at the onset of ischemic contracture in 24 dogs. Seventeen dogs were treated with propranolol before ischemia. The time to ischemic contracture in control dogs was 63.3 +/- 1.4 minutes compared with 75.9 +/- 2.2 minutes in the propranolol-treated group (p less than 0.01). In addition to significantly delaying the onset of ischemic contracture, propranolol also decreased the rate of anaerobic glycolysis during ischemia. Ischemic contracture occurred in the control group with an average adenosine triphosphate level of 1.26 +/- 0.08 mumol compared to 0.91 +/- 0.08 mumol/gm wet weight for the beta blocked group (p less than 0.0025). These are the first data suggesting that the protective effects of beta blockade may be related to a beneficial effect on ischemic myocardial metabolism allowing myocardium to survive with lower levels of adenosine triphosphate. PMID- 3018383 TI - Steroid-responsive bronchiolitis after human heart-lung transplantation. AB - This report describes the clinical course of a patient who developed obliterative bronchiolitis after viral infection on three separate occasions. Long-term follow up is given. It is suggested that the syndrome of late pulmonary deterioration after transplantation may be steroid responsive if treatment is initiated early in the natural history of the syndrome. In addition, it is suggested that increased emphasis should be placed on the documentation of viral infection in transplant recipients to define a possible interaction between infection and rejection. PMID- 3018384 TI - B cells from leukemic patients are relatively resistant to in-vitro EBV transformation. AB - Samples of peripheral blood mononuclear cells (PBMC) from normal donors or from leukemic patients were used to obtain Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL). Whereas the rate of transformation was around 85% with normal donors, it was only around 20% with leukemic patients. However no explanation for this resistance of B cells from leukemic patients to in-vitro EBV transformation could be found. Indeed no correlation exists between this EBV resistance and the age, sex, diagnosis or chemotherapeutic regimen. Furthermore no correlation is apparent between the percentage of B2+ cells, which should have EBV receptors, and the successful EBV transformation. PMID- 3018386 TI - Lymphoblastoid B cell lines produce an interleukin-1-like activity that can be serologically distinct from macrophage interleukin-1. AB - Antigen presentation by macrophages is accompanied by the production of interleukin-1 (IL-1) for successful T cell triggering. We have described EBV infected B cell lines that served as antigen presenting cells (APC) and now report the ability of these cells and their supernates, as well as EBV negative B cell lines, to support IL-2 production by a T cell line JM in the presence of the monoclonal antibody that recognizes the CD3 complex. Both EBV-transformed B cell lines and EBV-negative lines, and their supernates, exhibited IL-1-like activity in two different IL-1 dependent assays. These IL-1-like molecules were released constitutively from five of six EBV positive lines and three of five non-infected B cell lines. The activity from both, one virally infected and one non-infected B cell line, eluted from Sephadex G-75 in two peaks at 15-18K and 30-35K and could not be neutralized by antibody specific for macrophage IL-1. Supernates having IL 1-like molecules also contained a higher molecular weight inhibitor of proliferation. These data indicate that: both EBV-positive and EBV-negative B cell lines are capable of elaborating the IL-1-like activity; and the IL-1-like activity from these cells can be serologically distinct from macrophage IL-1. This suggests the presence of a family of molecules with IL-1 activity that derive from different cell types. PMID- 3018385 TI - Differences in the plasma membrane glycoproteins of cultured myeloblastoid and promyelocytic human leukemia (HL60) cells. AB - Since the mechanisms that control synthesis of surface and internal granule membranes are closely-related within the Golgi apparatus, we have compared the plasma membrane proteins and glycolipids of cells of the human promyelocytic line HL-60 with those of its agranular myeloblastoid variant (HL60-D), and of other human myeloid lines (KG-1a, KG-1 and ML-2). Proteolytic degradation by granule enzymes altered the protein profiles unless multiple inhibitors were included in the cell suspension before lysis and during subsequent handling of the extracts. Polyacrylamide gel electrophoretic profiles of the proteins accessible to lactoperoxidase-catalyzed 125I-labeling or to periodate [3H]-borohydride labeling, as well as those of the glycoproteins bound to and eluted from immobilized concanavalin A, showed distinct patterns. The apparent molecular weights of the two major sialylated glycoproteins were larger in cell lines with a greater content of azurophilic granules. Also, the blastic line incorporated less fucose into glycolipid and contained less complex gangliosides and neutral glycolipids than did the parent. These data demonstrate that, within the limits of this culture model, cells capable of cytoplasmic granule production express a different constellation of surface components. PMID- 3018387 TI - Acute effect of smoking on superoxide production by pulmonary alveolar macrophages. PMID- 3018389 TI - The roles of diet and exercise in the management of patients with insulin dependent diabetes mellitus. AB - Current dietary recommendations for patients with diabetes are similar to those for the US population in general, including a moderate intake of sucrose. Awareness of the carbohydrate content of meals will allow adjustments of the dose of meal-related short-acting insulin when the size of the meal is altered; thus, meal planning can be flexible without sacrificing glycemic control. Although exercise may have potential benefits for patients with diabetes, it is also associated with greater risk in these persons than in their nondiabetic counterparts and frequently complicates the management of their disease. If there are no contraindications to regular, vigorous exercise and the patient desires to participate, careful self-monitoring of blood glucose, combined with snacks and insulin dose adjustment, should enable the intensively treated patient to exercise safely. PMID- 3018388 TI - Airway responses to inhaled ouabain in subjects with and without asthma. AB - Challenges with ouabain and histamine were performed a week apart in 10 patients with asthma and 5 normal subjects. Concentrations were increased cumulatively until specific airway conductance decreased by 30% or the maximal concentration of 1.0% was reached. At low concentrations, ouabain induced bronchodilatation in six patients who had asthma. Bronchodilatation gradually decreased with increasing concentrations and was followed by bronchoconstriction in two patients with asthma who had high airway sensitivity to histamine. Ouabain caused only bronchoconstriction in three patients with severe asthma. The normal subjects showed mild bronchodilatation or no response to ouabain. Several possible biochemical mechanisms may be responsible for the bronchodilatory response to low doses of ouabain, such as stimulation of adenylate cyclase or (Na+,K+)-adenosine triphosphatase. The absence of a bronchodilatory response to ouabain in patients with severe asthma suggests an impairment in the activity of these enzymes. PMID- 3018390 TI - Non-Hodgkin's lymphoma associated with hypercalcemia and increased activity of serum angiotensin-converting enzyme. AB - We describe two patients with diffuse non-Hodgkin's lymphoma, hypercalcemia, and increased activity of serum angiotensin-converting enzyme. A mechanism similar to that operative in sarcoidosis is speculated to have caused the hypercalcemia. A lymphokine elaborated by the malignant lymphoma may cause activated macrophages to produce 1,25-dihydroxyvitamin D3. PMID- 3018391 TI - Staging and treatment of Wilms' tumors. PMID- 3018392 TI - Effects of neuropeptide Y (NPY) at the sympathetic neuroeffector junction. Can pre- and postjunctional receptors be distinguished? AB - Neuropeptide Y (NPY) is widely distributed in central and peripheral neurons. In sympathetic postganglionic neurons, NPY coexists with noradrenaline. NPY and its structural relative peptide YY (PYY) appear to exert three principally different effects at the sympathetic neuroeffector junction. Firstly, NPY has a direct postjunctional effect; this effect is manifested as a vasoconstriction when studied on the guinea pig iliac vein. Secondly, NPY has an indirect postjunctional effect in that it potentiates the response to various vasoconstrictors; this was studied on the rabbit femoral artery and vein, using noradrenaline and histamine, respectively, as vasoconstrictors. Thirdly, NPY acts prejunctionally in that it suppresses the release of noradrenaline from sympathetic nerve terminals; this was studied in the rat vas deferens. The aim of the investigation was to examine whether the three effects of NPY were mediated by the same type of receptor. For this purpose, we examined the effects of a series of NPY-related peptides, namely NPY, PYY, desamido-NPY, and five C terminal fragments (NPY 19-36, NPY 24-36, PYY 13-36, PYY 24-36 and PYY 27-36). NPY and PYY were active in all three assay systems. The C-terminal amide appears to be crucial for maintaining the biological activity, since desamido-NPY was inactive in the three test systems. Interestingly, PYY 13-36 was almost as active as NPY and PYY in suppressing the electrically evoked contractions of the vas deferens; PYY 13-36 was inactive in the two other test systems. None of the shorter fragments had any biological activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018393 TI - The effect of 5-fluorouracil on rat adrenal function. AB - This study was undertaken to determine the effects of 5-fluorouracil on corticosteroidogenesis and adrenal size in the rat. The rats were injected intraperitoneally with 5-FU (12.5 mg kg-1 or 25 mg kg-1), normal saline or actinomycin D (0.02 mg kg-1), daily for 10 days. High-dose 5-FU induces adrenal hyperplasia, in association with mild impairment of corticosteroidogenesis (manifest by lower corticosterone and higher ACTH levels). The response to low dose 5-FU and actinomycin D is of lesser adrenal hyperplasia, relative to high dose 5-FU (P less than 0.05) and elevated corticosterone levels. There may be a dose-related effect on the suppression of corticosteroidogenesis in the rat by 5 FU. PMID- 3018394 TI - Two cases with objective complete response to neo-adjuvant chemotherapy of breast carcinoma. AB - This report describes two patients in whom primary treatment with chemotherapy completely eliminated palpable, and in one case, histological evidence of previously cytologically verified breast carcinoma. PMID- 3018396 TI - Etoposide: a pharmacokinetic profile including an assessment of bioavailability. AB - The pharmacokinetics of intravenous etoposide (50-150 mg m-2) have been studied in 17 patients. Bioavailability studies on either the capsule or intravenous (i.v.) formulation were performed in 13 patients, 7 of whom received both oral formulations given in the dose range 50-250 mg m-2. After i.v. administration the mean +/- SD half-lives were t1/2 alpha 0.62 +/- 1.01 h and t1/2 beta 6.04 +/- 2.5 h. The bioavailability of etoposide was extremely variable: for the capsule it was 38 +/- 14% (range 10-55) and for the i.v. formulation it was 53 +/- 25% (range 31-88). The i.v. formulation was not significantly better than the capsule. The results confirm the low and variable bioavailability of oral etoposide. PMID- 3018395 TI - Predictive value of T-cell subset derangements in lung cancer. AB - The proportions of T-lymphocytes, T-lymphocyte subsets, NK cells, DR determinant and interleukin-2 receptor-bearing T-lymphocytes were enumerated in 39 patients with lung cancer prior to any chemotherapy. T-lymphocytes, suppressor/cytotoxic T cells and interleukin-2 receptor-bearing T-cells were found to be significantly higher in patients responding than in those not responding to chemotherapy. Such mononuclear cell subset analysis by monoclonal antibodies might be additional information to consider before undertaking treatment. PMID- 3018397 TI - [Immunohistochemical localization of S-100 protein in granular cell myoblastoma]. AB - Three cases of granular cell myoblastoma have been studied in order to determine the presence and distribution of the S-100 specific protein in the neoplastic cells, using immunocytochemical staining techniques, through the modified avidin biotin method. Positive immunostaining was observed in the three cases studied. The comparative study of various control cases histogenetically originating from neuroectoderm (melanoma) and specifically from Schwann cells, as also the presence of strongly positive staining in Schwann cells of peripheral nerve fibres situated inside and outside the tumor, support the concept of the neurogenic origin of this interesting tumor. PMID- 3018399 TI - [Eosinophilic adenoma of the nose and ethmoid bone simulating a malignant tumor]. AB - A 82-year old woman patient was first diagnosed to have a dacryocystitis and was treated at the Department of Ophthalmology. Clinical examination and x-ray of the paranasal sinuses including tomographs led to the suspicion of a malignant, infiltratively growing tumour in the region of the left cheek and of the left ethmoidal cells. After endonasal removal of the tumour that obstructed the left nasal cavity and ethmoidal cells, histological diagnosis revealed an eosinophilic adenoma. Dacryocystitis had been caused secondarily by the impairment of lacrymal flow. Now, one year later, the patient is free from any recurrence and clinical or subjective signs and symptoms. PMID- 3018398 TI - [Significance of computerized tomography in middle ear diagnosis]. AB - Due to the important improvement represented by the latest computed tomography (CT) technology in respect of detail and excellent contrast resolution normal and pathological structures of the inner and middle ear can be visualised. The clinical and diagnostic value of CT in comparison to standard x-rays and tomography is prospectively evaluated on the basis 74 patient examinations. Today CT is unsurpassed in the diagnosis of cholesteatomas and glomus tumours and ranks first among all imaging modalities. Chronic inflammatory diseases and deformities of the middle ear can be evaluated in most cases on the basis of standard x-rays and tomography. The importance of CT is primarily complementary, except in preoperative cases. Fractures should be visualised first via standard x-rays. In cases of questionable complications (e.g. tympanic or intracranial haematomas), CT should be the next step. Conventional tomograms are not necessary. PMID- 3018400 TI - [Angiofibrolipoma of the tonsil]. AB - Benign tumors of the pharynx are very rare. The authors report on the remarkable case of a tonsillar angiofibrolipoma. PMID- 3018401 TI - [Neuroradiologic and surgical treatment of a recurrent angiofibroma supplied by the internal carotid artery]. AB - If blood supply to the brain hemisphere is disturbed following closure of internal homolateral carotid artery tumors of the skull base with involvement of this artery should not be operated on radically. The authors describe the electrophysiological monitoring of cortical evoked somato-sensory potentials. If there is no alteration of the evoked potentials after preliminary reversible blockade of the internal carotid artery this vessel can be definitely closed using a detachable balloon. Thereafter the whole tumor including the carotid artery can be removed. The authors describe a case of juvenile angiofibroma operated on in this way. The combined interventional-neuroradiological and surgical management widens the range of skull base surgery. PMID- 3018402 TI - [Principles of cholesterol and bile acid metabolism in the liver and small intestine (I)]. PMID- 3018403 TI - New role for heparan sulfate: regulator of leukotriene generation in mouse E-mast cells. AB - In this work, bovine heparan sulfate, pig mucosa heparin and squid chondroitin sulfate-E glycosaminoglycans (GAGs) were compared as to their affect on the synthesis of leukotrienes C4 (LTC4) and B4 (LTB4) as well as on the production of prostaglandin D2 (PGD2) in cultured mouse E-mast cells (E-MC). The maximum percent increase in LTC4 generation in cells treated with 0.2 micrograms heparan sulfate was 52 +/- 3% (mean +/- S.E., n = 5). Whereas 0.5 micrograms of the GAG increased the production of LTB4 by 50% Ten micrograms of heparin slightly increased the LTC4 production (33%) whereas lower doses were found to be ineffective. No significant increase in LTC4 production was demonstrated when the IgE sensitized E-MC were treated with chondroitin sulfate-E GAG prior to the antigen challenge. Neither one of the three GAGs, at the various doses used, affected the antigen induced exocytosis of beta-hexosaminidase from the IgE sensitized E-MC. After 15 min preincubation with heparan sulfate, antigen induced release of PGD2 from 1 X 10(6) sensitized cells was inhibited from 5.4 ng +/- 0.1 ng into 3.5 ng +/- 0.1 ng at 0.5 micrograms/ml GAG. All of the three types of GAGs used were uneffective when the E-MC were activated by calcium ionophore A23187. PMID- 3018404 TI - Glucocorticoid suppression of the sympathetic nervous system and adrenal medulla. AB - Studies were performed to evaluate the effects of glucocorticoids on the activity of the sympathetic nervous system and adrenal medulla. Plasma concentrations of norepinephrine and epinephrine were measured in rats in which endogenous glucocorticoids were removed by bilateral adrenalectomy and in rats to which exogenous glucocorticoids were administered. In intact rats, dexamethasone (2.5, 25 or 250 micrograms) pretreatment suppressed ether vapor-induced elevations of norepinephrine and epinephrine concentrations in plasma. Corticosterone (3 mg/kg), similar to dexamethasone, attenuated the elevation of plasma concentrations of norepinephrine and epinephrine in rats exposed to ether vapor. Glucocorticoids did not alter the elevation of plasma catecholamines stimulated by intracerebroventricular injections of corticotropin-releasing factor or calcitonin gene-related peptide, thus demonstrating functional integrity of the sympathetic nervous system and adrenal medulla. Adrenalectomy resulted in elevation of basal plasma norepinephrine levels and accentuation of ether vapor induced elevations of plasma norepinephrine concentrations in rats. Dexamethasone (25 ug) administration blunted the effects of adrenalectomy on both basal and ether vapor-stimulated levels of plasma norepinephrine. It is concluded that glucocorticoids acting at as yet undefined sites may be involved in the regulation of sympathetic nervous system and adrenal medullary function. PMID- 3018406 TI - Kappa receptor mediated opioid dependence in rhesus monkeys. AB - The kappa receptor-selective agonist U-50, 488 was administered chronically to rhesus monkeys. Tolerance developed to the overt behavioral effects of U-50,488 without cross-tolerance to morphine. Withdrawal behaviors produced by deprivation, naloxone or quadazocine administration in U-50, 488-dependent monkeys consisted of hyperactivity, excessive grooming, and yawning. The syndrome was suppressed in a dose-related manner by a kappa agonist, ethylketazocine, but not by doses of morphine that suppressed its own withdrawal. The mu-selective antagonist, beta-funaltrexamine, at doses which are active in morphine-dependent monkeys, did not precipitate withdrawal in U50, 488-dependent monkeys. Dependence, which is the result of activity at the kappa receptor, was distinct from morphine dependence. PMID- 3018405 TI - Stability of alpha-1 adrenoceptors in surgically excised human brain. AB - Alpha-1 adrenoceptor sites were measured in membranes prepared from nonepileptic superficial cortex following temporal lobectomy for lesions in deep medial structures. There was no significant change in receptor density (Bmax) or affinity (Kd) when paired samples were either frozen immediately or kept at room temperature for 24 hours before freezing and storage at -70 degrees C. Regional variability in the Bmax or Kd of alpha-1 adrenoceptor binding was not observed in serial samples from lateral temporal cortex. We previously reported a localized decrease in alpha-1 adrenoceptors in epileptic foci when compared to adjacent nonepileptic tissue obtained from the same patient. As nonepileptic control tissue from an adjacent gyrus is frequently not available in the same specimen, the stability of alpha-1 adrenoceptors justifies the use of postmortem brain for comparative studies. PMID- 3018407 TI - Effect of atriopeptin III on renin release in vitro. AB - The ability of atriopeptin III (AP) to directly inhibit renal renin release has not been resolved. This issue was examined in a series of experiments performed in a system of rat renal cortical slices (dry weight 1.91 mg) in which the goal was to explore the effects of AP on renin release induced by cyclic AMP (cAMP) coupled stimuli or by agents which are believed to decrease intracellular calcium (Cai). Concentration response relationships were initially established for all test agents. The cAMP stimuli utilized were isoproterenol (10(-5) M), forskolin (10(-5) M), and dibutyryl cAMP (3 X 10(-4) M); each of these agents produced a significant increase in renin release in the system (with isoproterenol a 59% increase, with forskolin 37%, and with dibutyryl cAMP 52%). The addition of AP (2.09 X 10(-8) M, a minimum inhibitory concentration derived from preliminary studies) significantly blunted these increases; in the case of the dibutyryl cAMP stimulated renin release, the inhibition was partial as a significant 25% increase in renin occurred in the presence of AP. The addition of the calcium channel blocking agent diltiazem (10(-4) M) resulted in a significant increase in renin release (364 to 567 ng X mg-1, p less than .05) which was not blocked by the addition of AP. Similarly, TMB-8 (0.6 X 10(-4) M), another agent thought to lower Cai, also resulted in increased renin release (455 to 810 ng X mg-1), p less than .01) which was also unaffected by the addition of the AP. In summary, these results show that AP is capable of partially inhibiting renin release in vitro, particularly renin release coupled to cAMP action. In contrast, renin release induced by a decline in Cai appears to be unaffected by the addition of AP. PMID- 3018408 TI - Pharmacological profile of adenosine A2 receptor in PC12 cells. AB - The PC12 cell line, a clone isolated from a pheochromocytoma tumor of rat adrenal medulla, was shown to exclusively contain stimulatory adenosine (A2) receptors linked to adenylate cyclase (AC). AC was stimulated 6-7 fold by several agonists with a rank order of potency of 5'-N-Ethyl carboxamidoadenosine (NECA) greater than 2-Chloroadenosine (2-CADO) greater than (R)-N-Phenylisopropyladenosine (R-( )-PIA) greater than N6-Cyclopentyladenosine (CPA) greater than N6 Cyclohexyladenosine (CHA) greater than S-(+)-PIA. AC activity was antagonized by a variety of adenosine receptor antagonists with a potency order of 1,3,-Dipropyl 8-(2-amino-4-chlorophenyl)xanthine (PACPX) greater than 1,3,-Diethyl-8 phenylxanthine (DPX) greater than 8-Phenyltheophylline greater than 3-Isobutyl-1 methylxanthine (IBMX) greater than 8-(p-sulfophenyl)theophylline (PST) greater than 7-(beta-chloroethyl)theophylline greater than theophylline = enprofylline = caffeine. Under conditions known to favour receptor-mediated Ni-coupled inhibition of AC, R-(-)-PIA failed to inhibit both basal and forskolin stimulated AC activity in PC12 cells, confirming the absence of an A1 mediated response. On the other hand, adenosine agonists inhibited AC activity in rat cortical membranes with a rank order of potency of CPA greater than R-(-)-PIA greater than CHA greater than NECA greater than S-(+)-PIA greater than 2-CADO. These findings suggest that PC12 cells are functionally deficient in an A1 receptor linked AC response but are efficiently coupled to A2 stimulatory receptors. The cells should prove useful for further study of A2 adenosine receptors and to establish selectivity profiles of compounds acting at both A1 and A2 receptors. PMID- 3018409 TI - Stimulation of Na+-Ca2+ exchange in heart sarcolemma by insulin. AB - Insulin was found to stimulate Na+-dependent Ca2+ uptake in dog heart sarcolemma in a concentration dependent manner (0.001 to 1 milliunits/ml). Maximal stimulation (160 to 170%) was seen at 0.1 to 1 milliunits/ml of insulin. Unlike Na+-dependent Ca2+ uptake, ATP-dependent Ca2+ uptake was unaltered by 1 microunit/ml of insulin. However, high concentrations of insulin (0.01 to 1 milliunits/ml) significantly increased the ATP-dependent Ca2+ uptake activity of heart sarcolemma; maximal increase (60%) was observed at 1 milliunit/ml of insulin. The Na+ K+-ATPase activity did not change upon incubating sarcolemma with insulin. The membrane preparation exhibited specific insulin binding characteristics. The Scatchard plot analysis of the data indicated two binding sites for insulin; the association constants for the high and low affinity sites were 2 X 10(9) M-1 and 4.4 X 10(8) M-1, respectively. These results support the view regarding the presence of insulin receptors in the heart cell membrane and indicate a dramatic effect of insulin on the sarcolemmal Ca2+ transport systems. PMID- 3018411 TI - Amiloride potentiates atrial natriuretic factor inhibitory action by increasing receptor binding in bovine adrenal zona glomerulosa. AB - The interaction of atrial natriuretic factor (ANF) with the diuretic amiloride was studied in bovine adrenal zona glomerulosa. Amiloride enhances 2 to 3-fold high affinity binding of [125I] ANF to zona glomerulosa membrane receptor with an ED50 of 10 microM. This effect is due to a recruitement of high affinity receptor sites and to an increase of their affinity from a Kd of 23 to 8 pM. This enhancing effect is almost equipotently elicited by guanabenz, while clonidine is 20-fold less potent and arginine is inactive. ATP reduces by 30 to 50% [125I] ANF binding with an IC50 of 50 microM. Amiloride and ATP opposite effects on [125I] ANF binding are mutually competitive. Low concentrations of amiloride (less than 100 microM) potentiate the inhibitory effect of ANF in hormone-stimulated steroid secretion with a 3-fold decrease in ANF IC50 at 10 microM amiloride. Higher concentrations of amiloride (greater than 100 microM) directly inhibit aldosterone secretion with an IC50 of 500 microM and a maximum of 80 to 100% reversal of stimulation by various secretagogues. These results indicate that amiloride synergistically potentiates ANF inhibitory action by altering ANF receptor binding properties. They also suggest a role for sodium transport and for phosphorylation-dephosphorylation mechanisms in the mode of action of ANF. PMID- 3018410 TI - The benzodiazepine receptor ligand, methyl beta-carboline-3-carboxylate, is both sedative and proconvulsant in chicks. AB - Certain pharmacological properties of methyl beta-carboline-3-carboxylate (beta CCM), a benzodiazepine receptor ligand, have been investigated in chicks. Although beta-CCM has been established previously as an "inverse agonist" of benzodiazepine receptors in rodents, having effects opposite to those of benzodiazepines in a variety of tests, in chicks this compound had a different pharmacological profile. Firstly, in contrast to the overt convulsant action of beta-CCM in other species, beta-CCM (0.05-40 mg/kg) did not produce convulsions by itself in chicks, but it was only proconvulsant. Secondly and most surprisingly, beta-CCM, like diazepam, produced in chicks a sedation which could be blocked by the benzodiazepine receptor antagonist Ro 15-1788. Thus it appears that beta-CCM can function both as an agonist and as an inverse agonist in this animal. PMID- 3018412 TI - Adrenocorticotropin administration increases testosterone secretion in adult male rats. AB - It appears that the effect of acute administration of pituitary-adrenal hormones on the pituitary-gonadal axis is species-dependent. However, no information is available with regard to the effect of acute adrenocorticotropin (ACTH) administration on testosterone secretion in rats. The present data indicate that acute ACTH administration can increase serum testosterone levels without modifying luteinizing hormone (LH) levels. Since this rise was not observed in castrated rats, it must be assumed that increased serum testosterone was of gonadal origin. The action of ACTH on testosterone secretion was likely an indirect one since there is no evidence at present for a direct, short-term action of the pituitary-adrenal axis on Leydig cell function. PMID- 3018413 TI - Starvation-induced changes in rat brain corticotropin-releasing factor (CRF) and pituitary-adrenocortical response. AB - Starvation-induced changes in CRF concentration in major brain regions and abnormalities in the pituitary-adrenal axis were examined in rats using rat CRF radioimmunoassay. The CRF concentrations in the hypothalamus and cerebellum were significantly reduced in the completely starved rats, while those in the midbrain, thalamus and neurointermediate lobe of the pituitary were significantly increased in the semi-starved or completely starved rats. No significant changes in the CRF concentrations were found in the pons, medulla oblongata and cerebral cortex. In the completely starved rats, the serum ACTH level was significantly reduced, whereas the serum corticosterone level was markedly elevated. These observations suggest that starvation may stimulate the CRF-ACTH-corticosterone system and that not only hypothalamic CRF but also extrahypothalamic CRF may be discretely related to feeding behavior or starvation. The reduced serum ACTH level in starved rats may be ascribed to the negative feedback effect of the elevated serum corticosterone. PMID- 3018415 TI - Cerebral cortical GABA and benzodiazepine binding sites in genetically seizure prone rats. AB - Adult male and female genetically seizure-prone rats were assessed for sound induced seizures. Heterozygous control groups were compared with mild seizure (designated GEPR 3) and severe seizure animals (GEPR 9). Groups of animals were killed and crude synaptosome fractions (P2) prepared from freshly dissected cerebral cortices. Binding sites for gamma-aminobutyric acid (GABA) were assessed by [3H]-muscimol in the absence or presence of excess GABA and/or pentobarbital. Binding sites for benzodiazepines were assessed by [3H]-flunitrazepam in the presence or absence of clonazepam. Compared to controls, GEPR 3 animals had a modest increase and GEPR 9 animals a larger increase in Bmax for both high and low affinity GABA sites, with no change in Kd. Chloride-dependent, barbiturate enhanced GABA binding (increased Bmax) was observed in all conditions and groups. Likewise benzodiazepine binding (Bmax) increased slightly in GEPR 9 animals. There were no observed changes in binding sites for a survey of biogenic amines. Seizure-prone animals appear to have compensatory denervation-like supersensitivity for their most prominent inhibitory receptor, which may or may not be linked to the seizure event. PMID- 3018414 TI - Abnormalities in the central cholinergic transmitter system of the genetically epilepsy-prone rat. AB - Seizure-experienced Genetically Epilepsy-prone Rats (GEPRs) have increased acetylcholine content and choline acetyltransferase activity in the thalamus and striatum. These cholinergic differences are accompanied by a slight but statistically significant reduction in acetylcholinesterase activity in the midbrain. In addition, no abnormalities were found in the numbers of specific 3H QNB binding sites in the striatum, hippocampus, inferior colliculi or cortex. Other work has shown no difference in muscarinic receptor function as measured by carbachol-stimulated inositol-1-phosphate formation. These data suggest a possible presynaptic defect in the striatal and thalamic cholinergic system which may play some role in the seizure-prone state of the GEPR. However, caution must be used in interpreting these cholinergic derangements since more recent findings show no differences in thalamic acetylcholine content in seizure-naive GEPRs. Thus, the original cholinergic abnormalities detected in the seizure-experienced GEPR may be an enduring response to seizure activity. PMID- 3018416 TI - Ethanol induced conformational changes of the peptide ligands for the opioid receptors and their relevance to receptor interaction. AB - The FT-IR (Fourier Transform Infrared) Spectrum of [Met 5]-enkephalinamide in aqueous solution shows the presence of both the beta-turn and beta-sheet conformations. The beta-turn and beta-sheet conformations of enkephalins have been proposed to play a role in receptor selectivity. Addition of ethanol alters these secondary structural features and hence the effect of ethanol on ligand receptor interaction may be mediated primarily through conformational changes of the ligand rather than those of the receptor. PMID- 3018417 TI - Transferrin receptor on rat Kupffer cells in primary culture. AB - Kupffer cells may play a role in the turnover of iron in acute viral hepatitis. The transferrin receptor of rat Kupffer cells in primary culture was therefore investigated in this study. Daily specific bindings on 125I-diferric transferrin (Tf) to rat Kupffer cells in primary culture from day 3 to day 6 of culture were 1.64 +/- 0.08%, 4.16 +/- 0.05%, 4.34 +/- 0.07% and 2.63 +/- 0.07%, respectively. The specificity of the Tf binding sites was examined by competition studies showing that galactose (30 mmol x l-1) and ovalbumin (90 mumol x l-1) did not compete for the binding sites, but human lactoferrin (50 mumol x l-1) competed for the binding sites by about 30%. The affinity and capacity of Tf receptor on rat Kupffer cells in 5-day culture were analyzed according to the method of Scatchard. A single class of 125I-diferric Tf binding sites with an affinity constant of 1.65 x 10(7) l x l-1) and a capacity of 6.86 x 10(6) sites/cell was found. After zymosan (500 micrograms/ml) preincubation for 30 min, the binding capacity increased about 1.7-fold, and this increase depended upon the increase of the affinity of Tf receptor. These data suggest that Kupffer cells in the activated state accelerate the removal of elevated serum iron. PMID- 3018418 TI - [Dynamic radionuclide ventriculography in the diagnosis of heart and lung diseases]. AB - The authors have provided a comparative analysis of various modifications of radionuclide ventriculography and assessment of their potentialities for clinical use. A total of 120 patients with chronic nonspecific lung diseases, coronary heart disease and rheumatic heart disease were examined. It was established that the most accurate information to assess myocardial contractility was obtained by a method of the determination of the ejection fraction by the "activity-time" curve with a fixed zone of interest, and the universal patient's position was the left front oblique projection. The right ventricular ejection fraction was determined in parallel with the study of the left ventricular ejection fraction characterizing myocardial contractility in cardiovascular pathology. The study of the former extended significantly the potentialities of radionuclide ventriculography, allowed the assessment of the hemodynamic state of the lesser circulation and compensatory potentialities of the right ventricle of the heart in cardiopulmonary pathology. PMID- 3018420 TI - [Effect of dynamic and isometric exercise on systemic and regional left ventricular contractility in myocardial infarct based on radionuclide ventriculographic data]. PMID- 3018419 TI - [Cardio-scintigraphy (using 99 mTc-pyrophosphate) in the evaluation of the results of complete or partial myocardial revascularization]. PMID- 3018421 TI - [Current problems of radionuclide studies in cardiology]. PMID- 3018422 TI - [Radionuclide methods applicable to the evaluation of the functional state of the extremities in transosseous osteosynthesis]. PMID- 3018423 TI - In vivo opioid receptor occupation in the rat brain following exercise. AB - The effects of prolonged swim-stress (2 h and 1 h) upon brain opioid receptor binding of tritiated [3H]diprenorphine were investigated in male Sprague-Dawley rats. This was accomplished by injecting the label intravenously immediately following the swim, then allowing 20 min for tracer washout from non-specific binding sites, sacrificing the animal, dissecting the brain into several discrete areas (medulla-pons, mid-brain, mesolimbic, caudate, thalamus, and hypothalamus), and subsequently preparing homogenates from each brain area. Data were obtained from scintillation counting of the homogenates. A separate support experiment measured circulating beta-endorphin endorphin like immunoreactivity immediately following 2 h of swim-stress. Blood-borne beta-endorphin levels were significantly enhanced by the swim. Additionally, [3H]diprenorphine binding was insignificantly elevated following the 1-h swim and significantly greater in 5 of 6 brain areas examined subsequent to the 2-h swim. Greater availability of opioid receptors to allow enhanced binding of [3H]diprenorphine may have been caused by decreased competition for available receptors from endogenously produced peptides or possibly by alterations in receptor-binding characteristics. These proposed explanations await further investigation. As a result of these studies, we conclude: exercise-induced enhancement of peripheral beta-endorphin probably does not have a supraspinal action; and prolonged swim-stress apparently alters opioid receptor occupancy in the rat brain, and this effect may be dependent upon exercise duration. PMID- 3018424 TI - Adrenocortical function of female endurance runners and joggers. AB - The aim of the present study was to investigate the long-term effects of endurance exercise on the function of the adrenal cortex of 18 female runners, 12 control subjects, and 13 joggers and their 11 control subjects by measuring the serum concentrations of cortisol and dehydroepiandrosterone sulfate and the responses of cortisol to intravenous ACTH injection. All of the participants were studied over one menstrual cycle, during a light training period in the autumn and a hard training period in the spring. There were no significant between-group differences in the concentrations of cortisol in the autumn or the spring. However, the mean spring vs autumn concentrations of cortisol were significantly increased in runners during the follicular and luteal phases of the menstrual cycle. The concentrations of cortisol in the ACTH response test were also increased at 30 and 60 min in runners and all the time in joggers in spring, in relation to the respective values in the autumn. The absolute and relative rises of cortisol in response to ACTH did not differ between the groups, but the relative spring vs autumn increase of cortisol was significantly higher at 60 min in the runners and lower at 30 min in the control subjects of the joggers. There were no differences in the serum concentrations of dehydroepiandrosterone sulfate between the groups, or between spring and autumn values in any group. In conclusion, chronic endurance exercise per se did not appear to alter the function of the adrenal cortex, while an undefined spring-associated factor, possibly the high luminosity, appeared to induce an increase in cortisol secretion in female runners. PMID- 3018425 TI - Post-traumatic hypopituitarism. Six cases and a review of the literature. AB - The typical patient with post-traumatic hypopituitarism is a young adult male presenting months to years after an automobile accident, following which he was unconscious for several days. He will probably have sustained a fracture of the base of the skull and on recovery is likely to have permanent visual or other neurological sequelae. Temporary or permanent diabetes insipidus may have occurred. The features of panhypopituitarism such as weight loss, fatigue, faintness, loss of libido, and impotence may have been ascribed to depression or the "postconcussion syndrome" and often inappropriate treatment and rehabilitation advised. The striking feature on review of the literature is that the pathological consequences of head injury to the pituitary and hypothalamus have been well described, while only 47 cases of traumatic hypopituitarism have been reported. The most likely reason for this disparity is that head injury of sufficient severity to cause hypothalamic and pituitary damage commonly led to death. More patients now survive, owing to the availability of intensive care; accordingly, most cases have been reported in the last 15 years. However, several patients are described in whom the initiating head injury was not associated with a skull fracture or followed by coma. We recommend that patients with major head injury (defined by post-traumatic amnesia greater than 24 hours), and in particular those with fractures of the base of the skull or diabetes insipidus should be closely monitored for symptoms and signs of endocrine dysfunction and appropriate dynamic pituitary-function tests performed. PMID- 3018426 TI - Nuclear thyroid hormone receptors in cultured human fibroblasts: improved method of isolation, partial characterization, and interaction with chromatin. AB - In order to characterize the nuclear thyroid hormone receptors in human tissue, an improved method for isolation of nuclei from cultured human fibroblasts was developed. This method provided nuclei with a protein/DNA ratio of 2.8 and recovery of 42%. The purity of nuclei was verified by phase contrast and electron microscopy, which showed normal appearance of chromatin structure. Nuclear binding assay was performed by incubation of whole cells at 37 degrees C or isolated nuclei at 22 degrees C with L-triiodothyronine (T3). In both cases, an affinity constant (Ka) of 2.0-3.0 X 10(10) M-1 and an average binding capacity of 41 femtomoles of T3/100 micrograms DNA (3,100 binding sites/nucleus) were obtained. During incubation of the nuclei, 13% to 16% of receptors that had an identical Ka was released into the medium. Salt extraction recovered 85% to 90% of the receptors, which had a Ka of 4.5 X 10(10) M-1 and the capacity of 0.13 pmol of T3/mg protein. The Ka fo. L-thyroxine (T4) was seven to 18 times lower than that for T3, but the capacity was the same in isolated nuclei, receptors released during incubation of nuclei, and in salt-extracted receptors. Of the iodothyronines examined, affinity for triiodothyroacetic acid was the highest, followed by L-T3, D-T3, L-T4. Isokinetic glycerol gradient analysis revealed that salt-extracted receptors had a sedimentation coefficient of 3.4 S, whereas micrococcal nuclease digested receptors showed two major (6.0 to 6.5 and 12.5 S), and two minor (17 and 19 S) peaks. These results were virtually identical to those obtained with rat liver nuclei analyzed in parallel studies.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018427 TI - Amino-terminal structure of spoOA protein and sequence homology with spoOF and spoOB proteins. AB - The previously reported nucleotide sequence of the spoOA coding region of Bacillus subtilis suggested that the protein is initiated with either of two possible initiation codons, ATG and GTG, 84 base pairs apart. To determine which codon is utilized as an initiator in B. subtilis, we constructed a fusion gene in which the promoter and NH2-terminal region of the spoOA gene was connected to the chloramphenicol acetyltransferase gene (cat gene). After introduction of the plasmid carrying the spoOA-cat fusion gene into B. subtilis cells, the fusion protein was purified by affinity chromatography. The sequence of NH2-terminal amino acids of the fusion protein was determined and the result established that the GTG codon is utilized as an initiator in B. subtilis. Comparison of the amino acid sequences revealed a marked homology between the spoOA (NH2-terminal half) and spoOF proteins. A less striking but significant homology was also found between the spoOA (COOH-terminal half) and spoOB proteins. This suggests the presence of a common functional domain structure for these proteins that are supposed to play key regulatory roles in sporulation. PMID- 3018428 TI - Nucleotide sequence of the Escherichia coli hisD gene and of the Escherichia coli and Salmonella typhimurium hisIE region. AB - In this paper we report the nucleotide sequence of the hisD gene of Escherichia coli and of the his IE region of both E. coli and Salmonella typhimurium. The hisD gene codes for a bifunctional enzyme, L-histidinol:NAD+ oxidoreductase, of 434 amino acids with a molecular mass of 46,199 daltons. We established that the hisIE region of both S. typhimurium and E. coli is composed of a single gene and not, as previously believed, of two separate genes. The derived amino acid sequence indicates that the hisIE gene codes for a bifunctional protein of 203 amino acids with an approximate molecular mass of 22,700 daltons. We also determined the nucleotide sequence of a deletion mutant in S. typhimurium which abolishes the hisF and hisI functions but retains the hisE function. We deduced that the mutant produces a chimeric protein fusing the aminoterminal region of the upstream hisF gene to the carboxyl-terminal domain of the hisIE gene which encodes for the hisE function. In view of these results the structural and functional organization of the histidine operon in enteric bacteria needs to be revised. The operon is composed of only 8 genes and the pathway leading to the biosynthesis of the amino acid requires 11 enzymatic steps. PMID- 3018429 TI - Expression of biosynthetic genes from Pseudomonas aeruginosa and Escherichia coli in the heterologous host. AB - We examine the expression of constitutive or repressible, monocistronic genes from Pseudomonas aeruginosa and Escherichia coli after their transfer to the heterologous host. To this end, chromosomal DNA from P. aeruginosa was cloned into the mobilizable broad-host-range vector pKT240; recombinant plasmids carrying the argA, argF, or proC genes were identified by complementation of the corresponding auxotrophic mutations. The isofunctional E. coli genes and the E. coli proB gene were subcloned into pKT240 from existing recombinant plasmids. The enzyme expression specified by the Pseudomonas genes in E. coli, calculated per gene copy, ranged from 0.3%-5% of the levels observed in Pseudomonas. Fusion of the P. aeruginosa proC gene to the E. coli consensus tac promoter resulted in very high proC enzyme production in E. coli, indicating that, at least in this case, the expression barrier is essentially at the level of transcriptional initiation. The E. coli argA and argF enzymes, which are controlled by repression in their native host, were synthesized constitutively in P. aeruginosa at 5% of the levels measured in E. coli under derepressed conditions. The constitutive E. coli proB and proC genes were expressed at high levels (ca. 50%) in the heterologous host. These results support the idea that P. aeruginosa may be a more permissive host than E. coli for the heterologous expression of genes from gram-negative bacteria. PMID- 3018430 TI - Heterologous expression and regulation of the lysA genes of Pseudomonas aeruginosa and Escherichia coli. AB - The Pseudomonas aeruginosa lysA gene encoding diaminopimelate decarboxylase (DAP decarboxylase) was cloned into a broad host range vector. This gene complemented a lys mutation at the lys-12 locus of P. aeruginosa and a lysA defect in Escherichia coli. The P. aeruginosa DAP-decarboxylase was synthesized constitutively in P. aeruginosa as well as in E. coli, where the Pseudomonas lysA gene was poorly expressed. By contrast, the E. coli lysA gene was expressed well in P. aeruginosa and subject to lysine regulation when the E. coli LysR activator protein was provided. This indicates that the mechanism of transcriptional activation for the E. coli lysA gene is effective in the heterologous host. PMID- 3018431 TI - Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator. AB - Several versatile, multi-copy, promoter-probe plasmid vectors have been constructed that replicate in a wide range of Streptomyces species. Transcriptional activity is detected by the expression of a promoter-less aminoglycoside phosphotransferase gene (neo) derived from the transposon Tn5; expression of this gene confers kanamycin and neomycin resistance on Streptomyces lividans. An efficient transcriptional terminator from E. coli phage fd has been inserted upstream of the neo coding region to prevent significant transcriptional read-through from vector promoters. A translational stop codon situated downstream from the site(s) used for cloning and preceding and in frame with the ATG start codon of the neo gene ensures the detection of transcriptional, rather than translational, fusions. Relative promoter strengths can be determined by gradient plate assays of kanamycin resistance, by measuring the amount of aminoglycoside phosphotransferase produced or by estimating neo mRNA synthesised. The high copy number of the vectors facilitates the rapid isolation and characterisation of promoter-active fragments and convenient restriction sites are available for DNA sequencing and S1 mapping of cloned inserts. Some derivatives contain a polylinker that facilitates the insertion, excision and analysis of cloned fragments and which enhances the use of these plasmids as general cloning vectors. PMID- 3018432 TI - Polar mobilization of the Escherichia coli chromosome by the ColE1 transfer origin. AB - Mobilization of the plasmid ColE1 from cells containing a conjugative plasmid (such as F) requires the synthesis of ColE1 mob proteins, and the presence, in cis, of bom (basis of mobility), a region of ColE1 containing the origin of transfer (oriT). The process of ColE1 transfer is thought to resemble that of the conjugative plasmid F, although the plasmids share little sequence homology. In F, conjugation is preceded by a strand-specific nicking event at oriT. The nicked strand is then conducted to the recipient with the 5' end leading. This is believed also to occur with ColE1, but direct biochemical confirmation has been precluded by its small size (6.65 kb). To test this hypothesis genetically, a novel method, using a lambda dv-based vector, has been devised to site specifically integrate bom (or any other cloned sequence) into the chromosome of Escherichia coli. When provided with suitable mobilizing plasmids, such strains were found to transfer the chromosome in a polar way. From these data, the orientation of transfer of ColE1 was deduced and shown to be analogous to F. PMID- 3018433 TI - Controlled gene expression utilising lambda phage regulatory signals in a cyanobacterium host. AB - This study presents plasmid systems that utilize regulatory signals of bacteriophage Lambda to accomplish regulated expression of cloned genes in an A. nidulans R2 derivative strain. An operator-promoter region and the temperature sensitive repressor gene cI857 of bacteriophage Lambda were employed. Linked to a cyanobacterial replicon, the plasmid vectors efficiently transformed Anacystis and were stably maintained within this host. The cat structural gene, encoding chloramphenicol acetyltransferase, was used to demonstrate that expression can be regulated by temperature shift. We have identified in extracts from the vector bearing Anacystis, a protein similar in size and immunology to the Lambda repressor. The systems described should allow controlled expression of adventitious genes in the cyanobacterial host. PMID- 3018435 TI - Heterologous insertion of transforming DNA and generation of new deletions associated with transformation in Aspergillus nidulans. AB - The analysis of four transformants for the proline catabolism (prn) gene cluster of Aspergillus nidulans is reported. Using a combination of traditional genetic methodology and Southern hybridisation we have shown that in two cases multiple copies of the transforming plasmid have been integrated into linkage groups other than VII, which contains the prn cluster. In the other two cases integration of the plasmid has probably occurred homologously. The phenotype of these transformants is broadly consistent with increased copy number resulting in increased expression. Genetic manipulation of these transformants using the sexual or parasexual cycles has shown that recombination events during and possibly also subsequent to integration of the transforming DNA can generate new mutational lesions, in particular, deletions. PMID- 3018434 TI - IS21 insertion in the trfA replication control gene of chromosomally integrated plasmid RP1: a property of stable Pseudomonas aeruginosa Hfr strains. AB - Broad host range IncP-1 plasmids are able to integrate into the chromosome of gram-negative bacteria. Strains carrying an integrated plasmid can be obtained when the markers of a temperature-sensitive (ts) plasmid derivative are selected at non-permissive temperature; in this way Hfr (high frequency) donor strains can be formed. The integrated plasmids, however, tend to be unstable in the absence of continuous selective pressure. In order to obtain stable Hfr donor strains of Pseudomonas aeruginosa PAO, we constructed a derivative of an RP1 (ts) plasmid, pME134, which was defective in the resolvase gene (tnpR) of transposon Tn801. Chromosomal integration of pME134 was selected in a recombination-deficient (rec 102) PAO strain at 43 degrees C. Plasmid integration occurred at different sites resulting in a useful set of Hfr strains that transferred chromosomal markers unidirectionally. The tnpR and rec-102 mutations prevented plasmid excision from the chromosome. In several (but not all) Hfr strains that grew well and retained the integrated plasmid at temperatures below 43 degrees C, the insertion element IS21 of RP1 was found to be inserted into the trfA locus (specifying an essential trans-acting replication function) of the integrated plasmid. One such Hfr strain was rendered rec+; from its chromosome the pME134::IS21 plasmid (= pME14) was excised and transferred by conjugation to Escherichia coli where pME14 could replicate autonomously only when a helper plasmid provided the trfA+ function in trans. Thus, it appears that trfA inactivation favours the stability of chromosomally integrated RP1 in P. aeruginosa. PMID- 3018436 TI - Nucleotide sequence of the lig gene and primary structure of DNA ligase of Escherichia coli. AB - The DNA ligase of Escherichia coli catalyses the NAD-dependent formation of phosphodiester linkages between 5'-phosphoryl and 3'-hydroxyl groups in DNA. It is essential for DNA replication and repair of damaged DNA strands. We determined the nucleotide sequence of the lig gene of Escherichia coli coding for DNA ligase and flanking regions. The coding frame of the gene was confirmed by the amino acid composition and the amino- and carboxyl-terminal amino acid sequences of the purified ligase. The ligase consists of 671 amino acid residues with a molecular weight of 73,690. PMID- 3018437 TI - Regions associated with the stable maintenance of plasmid pSC101 and its tetracycline resistance. AB - Two regions tentatively called unsA and unsR were identified on pSC101. One, unsA, corresponds to less than 650 bp of the N-terminal in the tetracycline resistance structural gene and seems to inhibit stable maintenance of pSC101. The other, unsR, is defined within the 1 kb XhoI-EcoRI region located upstream of the tetracycline resistance structural gene and is a regulatory gene clearly distinct from tetR (Unger et al. 1984); it serves as a suppressor of the unsA function. PMID- 3018438 TI - Lytic activity localized to membrane-spanning region of phi X174 E protein. AB - Lytic activity of the phi X174 E (lysis) protein had previously been localized to the amino terminal 51 amino acids (a.a.) of the molecule (Blasi and Lubitz 1985). This E gene lytic activity has here been further localized to the amino terminal 29 a.a., a region of the protein which is thought to just span the cell membrane (Young and Young 1982). phi X174 E gene fusions to both the lacZ gene and the chloramphenicol acetyl transferase (CAT) gene resulted in fusion proteins with lytic activity. Fusion to a third protein, trpE, did not result in lytic activity. These results support a model of oligomerization of the phi X174 E protein for lytic activity since both beta-galactosidase and CAT exist as tetramers in their native state. A difference in the composition of the charged amino acids at the cytoplasmic boundary between the various fusion proteins could also account for these results, since these amino acids may play a role in proper anchoring of the E protein in the cell membrane. In a spontaneous E gene mutant, which introduces a proline residue at position 9 of the E protein, lytic activity of the E protein was decreased, but not abolished. The presence of the helix breaking proline at this position may interfere with insertion of the lysis protein into the cell membrane, leading to the decreased functional activity of the protein. PMID- 3018439 TI - Conservation of primary structure in the hisI gene of the archaebacterium, Methanococcus vannielii, the eubacterium Escherichia coli, and the eucaryote Saccharomyces cerevisiae. AB - A 2.7 kilobase pair (Kb) fragment of DNA, which complements mutations in the hisI locus of Escherichia coli, has been cloned and sequenced from the genome of the methanogenic archaebacterium Methanococcus vannielii. The cloned DNA directs the synthesis of three polypeptides, with molecular weights of 71,000, 29,000 and 15,600 in minicells of E. coli. Subcloning and mutagenesis demonstrates that hisI complementation results from the activity of the 15,600 molecular weight polypeptide. The primary structure of this archaebacterial gene and its gene product have been compared with the functionally equivalent gene and protein from the eubacterium E. coli (hisI) (Chiariotti et al. 1986) and from the eucaryote Saccharomyces cerevisiae (his4A) (Donahue et al. 1982). The DNA sequences of the archaebacterial and eubacterial genes are 40% homologous, the archaebacterial and eucaryotic DNA sequences are 47% homologous and, as previously reported (Bruni et al. 1986) the eubacterial and eucaryotic DNA sequences are 45% homologous. In E. coli the hisI locus is part of a bifunctional gene (hisI/E) within the single his operon. In S. cerevisiae the his4A locus is part of a multifunctional gene (his4) which encodes a protein with at least four enzymatic activities. The his genes of S. cerevisiae do not form an operon and are not physically linked. The M. vannielii hisI gene does not appear to be part of a multifunctional DNA sequence and, although it does appear to be within an operon, the open reading frames (ORFs) 5' and 3' to the M. vannielii hisI gene are not related to any published his sequences.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018440 TI - Sequence of the promoter and 5' coding region of pepN, and the amino-terminus of peptidase N from Escherichia coli K-12. AB - The pepN gene of Escherichia coli K-12 has been cloned onto a multi-copy plasmid and shown to encode a polypeptide which co-migrates with purified peptidase N. Transformed strains have been shown to contain up to a one hundred fold increase in the amount of peptidase N. We isolated the peptidase N protein and determined the sequence of its first 15 amino acids. By restriction mapping, we identified and subcloned the 5' region of the pepN gene and then determined its nucleotide sequence. Comparison of the actual amino acid sequence with that predicted from the extended open reading frame found in the DNA sequence indicated that peptidase N is not synthesized as a pre-protein precursor. The presumed region preceding the open reading frame contained nucleotide sequence having homology to the procaryotic promoter consensus sequences for the -35 and the -10 regions and the ribosome binding site. PMID- 3018441 TI - Studies on lipase directed export of Escherichia coli beta-lactamase in Staphylococcus carnosus. AB - The lipase (lip) gene of Staphylococcus hyicus was used to study the expression of the Escherichia coli beta-lactamase (bla) gene in S. carnosus. The bla gene, devoid of its promotor and most of the signal sequence, was fused to the lip structural gene at various positions. A set of 11 secretion vectors (pLL beta 1 to pLL beta 11) was isolated and analysed. All secretion vectors caused beta lactamase production and activity in S. carnosus. However, the amount of hybrid proteins secreted was influenced by the length of the NH2-terminal lipase portion. An increased concentration, comparable to that of the native lipase, of secreted lipase/beta-lactamase hybrid proteins was only found when the lipase portion of the construct comprised more than 101 amino acids of the NH2-terminal region of the lipase preprotein; the proposed lipase signal peptide is 36 amino acids long. If the hybrid proteins constructed contained 101 or less amino acids of the NH2-terminal lipase preprotein, only low amounts of secreted hybrid proteins were detectable and a significant portion of the hybrid proteins and beta-lactamase activity was found in the cellular fraction. The results indicate that the lipase possesses adjacent to the signal peptide a peptide domain that is essential for the secretion of the lipase/beta-lactamase hybrid proteins. PMID- 3018442 TI - Feedback regulation of RNA polymerase subunit synthesis after the conditional overproduction of RNA polymerase in Escherichia coli. AB - The beta and beta' subunits of RNA polymerase are thought to be controlled by a translational feedback mechanism regulated by the concentration of RNA polymerase holoenzyme. To study this regulation in vivo, an inducible RNA polymerase overproduction system was developed. This system utilizes plasmids from two incompatibility groups that carry RNA polymerase subunit genes under lac promoter/operator control. When the structural genes encoding the components of core RNA polymerase (alpha, beta and beta') or holoenzyme (alpha, beta, beta' and sigma 70) are present on the plasmids, induction of the lac promoter results in a two fold increase in the concentration of functional RNA polymerase. The induction of RNA polymerase overproduction is characterized by an initial large burst of beta beta' synthesis followed by a gradual decrease as the concentration of RNA polymerase increases. Overproduction of RNA polymerase in a strain carrying an electrophoretic mobility mutation in the rpoB gene results in the specific repression of beta beta' synthesis off the chromosome. These results indicate that RNA polymerase feedback regulation controls beta beta' synthesis in vivo. PMID- 3018443 TI - Conditionally lethal nusAts mutation of Escherichia coli reduces transcription termination but does not affect antitermination of bacteriophage lambda. AB - Termination of transcription at bacteriophage lambda terminators as well as at the Escherichia coli trp a attenuator was examined in the conditionally lethal mutant (nusAts11) defective in the NusA protein of E. coli. Experiments using terminator-assay lambda vectors revealed that the efficiency of termination at both rho-dependent (lambda tL1) and rho-independent (lambda tL2 and trp a) terminators decreases in the mutant. The mutation does not block lambda phage growth at either permissive or nonpermissive temperatures, nor does it affect the lambda Q protein antitermination activity at the t6s terminator. These results indicate that NusA is required for transcription termination, and that lambda N and Q-mediated antitermination may not require the NusA protein function in the nusAts11 mutant. PMID- 3018445 TI - Identification of Tn4430, a transposon of Bacillus thuringiensis functional in Escherichia coli. AB - The mobile genetic element Tn4430, originating from the gram-positive bacterium, Bacillus thuringiensis, and previously described as the Th-sequence, is the first transposon isolated from the genus Bacillus. In the present work a gene (APH-III) conferring resistance to kanamycin was inserted into this 4.2 kb transposon. Transposition experiments showed that Tn4430 omega APH-III could transpose in the gram-negative host Escherichia coli when its insertion functions were supplied by an intact copy of Tn4430. By transposing Tn4430 omega APH-III directly onto pBR322, it was possible to determine the nucleotide sequence of the terminal inverted repeats of Tn4430 and of the target DNA site. Identical 38 bp in inverted orientation are situated at each end of the transposon and there is a direct duplication of 5 bp at the insertion site. Thus, it is clear that Tn4430 is closely related to the transposons belonging to the Tn3 family (class II elements). PMID- 3018444 TI - Amplification of the amyE-tmrB region on the chromosome in tunicamycin-resistant cells of Bacillus subtilis. AB - In a class of tunicamycin-resistant mutants (tmrA7) of Bacillus subtilis, the production of extracellular alpha-amylase is increased by about five fold. The tmrA7 characteristics (tunicamycin resistance and hyperproduction of extracellular alpha-amylase) can be transferred to recipient cells by transformation. In the transformants and the original tmrA7 mutant, typical amplification of the region from 4 kb upstream of the amyE gene to the tmrB gene on the chromosome was detected. The repeating unit, 16 kb in size, repeats tandemly about five and ten times in the mutant and transformants, respectively, and the alpha-amylase production is proportional to the copy number of the amyE gene. Simultaneous amplification of the tmrB gene, which is responsible for tunicamycin resistance in the multicopy state, and the alpha-amylase structural gene (amyE) seems to be the cause of the pleiotropy of the tmrA7 mutation. PMID- 3018447 TI - Role of the RecF gene product in UV mutagenesis of lambda phage. AB - E. coli recF mutants have a greatly reduced capacity for Weigle mutagenesis of ultraviolet light-irradiated lambda phage. A recF 332::Tn3 mutation was introduced into an E. coli recA441 lexA51 strain which constitutively expresses SOS functions. Weigle mutagenesis of phage lambda could occur in the resulting strain in the absence of host cell irradiation, and was increased when the recA441 (tif) allele was activated by increased temperature and excess adenine. The inability of recF strains to support Weigle mutagenesis can therefore be ascribed to a defect in expression of SOS functions after irradiation. PMID- 3018446 TI - Mistranslation during phenylalanine starvation. AB - Starvation for phenylalanine led to leucine misincorporation frequencies of 0.1 and 0.6 at UUC codons in the argI transcript of Escherichia coli, but no detectable misincorporation at a UUU codon. Under similar starvation conditions the relative synthesis of full sized MS2 coat protein, encoded by the RNA virus or a DNA copy, is greatly reduced, preventing analysis of the protein. This reduction in amount is unaffected by a rpsL mutation. PMID- 3018448 TI - Spontaneous amplification of yeast CEN ARS plasmids. AB - Transformation of Saccharomyces cerevisiae with several yeast CEN4 ARS1 plasmids containing the his3-delta 4 allele (as well as the URA3 and TRP1 markers) yielded His+ transformants at 0.1%-50% the frequency of Ura+ Trp+ transformants. Additional His+ derivatives arose on continuous growth of transformants originally scored as His- Ura+ Trp+. In all cases, the His+ phenotype was not due to plasmid or host mutations but invariably correlated with an up to 12-fold increase in plasmid copy number. On removal of selective pressure, the His+ phenotype was lost more readily than the Ura+ Trp+ markers, with a corresponding decrease in plasmid copy number. Also, the amplification did not decrease the mitotic loss rate of the Ura+ Trp+ markers. These results indicate that CEN ARS plasmids can be spontaneously amplified to higher levels than previously observed. However, when amplified, apparently not all copies exhibit the characteristic stability of CEN ARS plasmids. PMID- 3018451 TI - Isolation of cytophatic strains of rotavirus from pigs. AB - Twenty-two rotavirus isolates were recovered from piglets suffering from diarrhea. The isolates readily propagated in MA-104 cell cultures where they induced typical cytopathic effects (CPE) and possessed the physicochemicals properties of the members of rotavirus genus, Family Reoviridae. The electron microscopy study, conducted in MA-104 infected cells, revealed virus particles which possessed the peculiar morphology of rotavirus. The isolates were neutralized by a reference porcine rotavirus antiserum: all were devoid of hemagglutinating activity for red cells of swine, cattle, sheep, rabbit, chicken and guinea pig. PMID- 3018450 TI - Preliminary characterization of a fast-growing strain of human hepatitis A virus. AB - A fast-growing strain of Human Hepatitis A (HHA) virus was selected by progressively shortening by the time between serial passages of the isolate in the simian cell line Frp/3. The virus so selected infected 90-95% of the cells in 7-10 days, and developed a strong cytopathic effect (CPE). The synthesis of HHA specific antigens and the CPE were neutralized simultaneously by standard anti HHA sera. This fast-growing strain has the characteristics of a picornavirus. PMID- 3018449 TI - Study of receptors for vesicular stomatitis virus in vertebrate and invertebrate cells. AB - Early interactions between vesicular stomatitis virus (VSV) and susceptible cells were examined in cell lines of mammalian (HeLa), bird (CER), piscine (EPC) and arthropod (Aedes albopictus) origin showing different permissiveness to VSV growth. The chemical nature of receptors was investigated either by modification of cell surfaces with different enzymes or by competition for VSV binding between extracted membrane components and whole cells. Results obtained indicate that in all cell models, membrane lipid components show receptor activity whereas glycid groups participate to the in virus binding to a different extent. PMID- 3018452 TI - Partial characterization of two temperature-sensitive mutants of junin virus. AB - Two temperature sensitive (ts) mutants of Junin virus, a member of Arenaviridae, have been partially characterized. Both mutants, named ts-32 and ts-40, had a relative plating efficiency 40 degrees C/34 degrees C lower than 10(-3) an exhibited a leak yield below 10(-4). Standard growth curves showed that at 34 degrees C the viral mutants multiplied slower than wt virus and at high multiplicity did not display autointerference. No differences in thermolability were observed between wt and ts mutants. By contrast, when the pathogenic properties of the mutants were investigated they were significantly attenuated for mice. At the restrictive temperature both mutants were unable to synthesize viral-specific polypeptides, while at the permissive temperature the pattern was similar to wt virus. Shift-up and down experiments suggested that ts defect is expressed between 2 and 4 hours post-infection. It is concluded that ts-32 and ts 40 are early function mutants. The possible nature of their defect is discussed. PMID- 3018453 TI - Dissemination of a gentamicin resistance plasmid in the microbial population of hospital patients. AB - The dissemination of a gentamicin resistant plasmid, originally found in strains of Klebsiella and termed pk181, into the microbial population of patients of the Orvieto Hospital was studied during 1982. Five hundred and seventy-four strains of Gram-negative bacilli were examined, transferable gentamicin resistance being revealed in five different bacterial species. The resistance was shown to be encoded by 81-megadalton plasmids in Escherichia coli and Enterobacter cloacae, and by 93-megadalton plasmids in Serratia marcescens and Pseudomonas spp. Restriction endonuclease digestion of plasmid DNA showed that the fragment patterns of the 81-megadalton plasmids from E. coli and enterobacter cloacae were identical to one another and to the pattern of plasmid pk181. The fragment patterns of the 93-megadalton plasmids from Serratia and Pseudomonas, on the contrary, differed substantially from those of the 81-megadalton plasmids. PMID- 3018454 TI - Hypothesis: the gingival tissue as a reservoir for herpes simplex virus. AB - The reservoir site of Herpes simplex virus (HSV), from the primary infection until reactivation and in between recurrences was found to be in the dorsal roots of the trigeminal ganglion or in the sensory root ganglion. However, the triggering of viral genome in the ganglion does not exclusively explain the recurrences and appearance of skin lesions. Other sites cannot, therefore, be excluded, and virus may well be occult also in extraneural tissues. Herpes virus antigens were found by us in the sulcular epithelium of approximately 60% of patients with clinically healthy gingivae; we therefore hypothesised that the epithelial cells might act as the preferential site for latent HSV. In order to prove this assumption, viral genome should be traced in cells of oral tissues, and efforts should be made to rescue virus. PMID- 3018455 TI - Relationship between antibody titres against human cytomegalovirus detected by enzyme immunoassay and titres to individual viral polypeptides studied by immunoblotting. AB - Several sera with different IgG and IgM titres measured by enzyme immunoassay were studied in serial dilutions for their reactivity with human Cytomegalovirus structural polypeptides separated in SDS-PAGE and electrotransferred to nitrocellulose. The different immunogenicity of the viral proteins is documented and a hypothetical evolution of the humoral immune response to individual polypeptides is presented. PMID- 3018456 TI - Resistance of HSV-1 growth to aphidicolin in two aphidicolin resistant cell lines. AB - The growth of herpes simplex virus type 1 (HSV-1) was examined in two cell lines resistant to aphidicolin by inhibition of infectious progeny production in the presence of the drug. The results reported show that the virus yield, which was severely inhibited by the drug in sensitive cells, was only slightly inhibited in the resistant cell lines. This suggests that the metabolic alteration known to confer resistance to the cell line (that is the altered deoxyribonucleoside triphosphate pool) may also allow viral growth to proceed in the presence of aphidicolin, and that therefore HSV replication at least partially depends upon the host replicative apparatus. PMID- 3018457 TI - Serological differences between human T-lymphotropic virus type-I (HTLV-I) and HTLV-III/LAV. AB - Sera from each of five preselected groups of patients with acquired immune deficiency syndrome (AIDS), AIDS-related complex (ARC), hemophilia, adult T-cell leukemia (ATL), and healthy controls were examined for antibodies to human T-cell leukemia (T-lymphotropic) virus type-I (HTLV-I) and HTLV-III by indirect immunofluorescence (IF) and radioimmunoprecipitation (RIP) methods. All sera from five patients with AIDS, ARC, and hemophilia reacted at titers from 1 : 512 to 1 : 5,120 with fixed H9/HTLV-III cells by IF but not with fixed MT-1 cells carrying HTLV-I. Similarly, sera from patients with AIDS, ARC, and hemophilia precipitated HTLV-III-specific polypeptides of 120K, 46K, and 24K. In contrast, sera from five patients with ATL did not react with fixed H9/HTLV-III cells, but reacted with fixed MT-1 cells. Moreover, HTLV-I-specific polypeptides of 68K, 28K, and 24K were precipitated with sera from ATL-patients but not with anti-HTLV-III-positive sera. Recently, we infected HTLV-I-carrying MT-4 cells with HTLV-III and provoked strong cytopathic effects. This system enabled testing for neutralizing antibodies to HTLV-III. Neutralizing titers to HTLV-III of five anti-HTLV-III positive sera ranged from 1 : 720 to 1 : 9,000. In contrast, all five seronegative controls showed no or only low reactivity to HTLV-III envelope (1 : 80 and 100). However, three out of five anti-HTLV-I-positive sera exhibited weak cross-reactivities with HTLV-III. The reactivities were expressed as less than 1 : 160, except for one case (1 : 720). They were considered to be nonspecific since they were negative for HTLV-III antibodies in the radioimmunoprecipitation studies. PMID- 3018458 TI - Budding process and fine structure of lymphadenopathy-associated virus (LAV). AB - The budding process and fine structure of lymphadenopathy-associated virus (LAV), were studied by indirect immunofluorescence (IF) and electron microscopy (EM). By IF, LAV antigen was seen to be distributed focally within infected CCRF-CEM cells. Consistent with this finding, electron micrographs showed that LAV particles occurred in a focally aggregated state in a restricted area of the surface of the infected cells. LAV particles possessed bar-shaped, dense and central or eccentric cores. In addition, two or more cores were occasionally observed in one virus particle, or the cores were sometimes absent when thin sections were examined. The envelope of the virus particles had an irregular structure, although LAV particles were approximately spherical. PMID- 3018459 TI - Synergistic inhibitory effect of acyclovir and human native beta-interferon on the growth of herpes simplex virus type 2 in human embryo fibroblast cell cultures. PMID- 3018462 TI - Human papilloma virus and cervical cancer. PMID- 3018460 TI - [Cyclic adenosine-3',5-monophosphate in Streptomyces antibioticus and its possible role in regulating oleandomycin biosynthesis and the growth of the culture]. AB - When glucose is substituted for sucrose in the fermentation medium for Streptomyces antibioticus, the pH of the cultural broth becomes more acidic, the rate of protein synthesis in the mycelium rises, and the rate of oleandomycin synthesis decreases abruptly. The dynamics of cAMP (cyclic monophosphate) accumulation was studied in the process of biosynthesis by the culture in different media. Most of the synthesized cAMP (80-90%) was shown to be excreted into the medium. Glucose stimulates cAMP synthesis and excretion from the mycelium by a factor of 1.5-3. No distinct correlation was found between cAMP content in S. antibioticus cells and the level of oleandomycin biosynthesis. A correlation between changes in the concentration of exocellular cAMP and protein synthesis in the mycelium suggests that the excreted cAMP may be involved in regulating the growth of the culture producing the antibiotic. PMID- 3018461 TI - The pathogenesis of cystic fibrosis: a proposal for calmodulin as the basic biochemical defect. AB - In cystic fibrosis (CF) there are two major defects which lead to most clinical manifestations of the disease. These are the electrolyte sweat defect and the abnormality of mucous secretions. Both may be satisfactorily explained by increased intracellular Ca++. In Na+ reabsorbing cells, such as exocrine sweat glands, increased Ca++ inhibits transepithelial Na+ transport. In mucus-secreting cells, high Ca++ levels may lead to physiochemical changes in secreted mucus. Increased intracellular Ca++ levels are hypothesized to be caused either by a defective Ca++ efflux cellular mechanism, or by increased intracellular binding of Ca++. PMID- 3018463 TI - HIV antibodies in intravenous drug abusers. PMID- 3018464 TI - Transcobalamin I as a "marker" for fibrolamellar hepatoma. AB - A 12 year old girl with a localised fibrolamellar hepatoma had a raised serum unsaturated vitamin B12 binding capacity (UBBC) and transcobalamin I (TCI) prior to complete resection and chemotherapy. Regular clinical and radiological follow up detected no recurrence of her disease, but the UBBC and TCI slowly rose. Local recurrence and pulmonary metastases became detectable 2 1/2 years after diagnosis, 18 months after the UBBC and TCI level became elevated. Measurement of UBBC and TCI can help in the early detection of recurrence long before there is clinical or radiological evidence of recurrent fibrolamellar hepatoma. PMID- 3018465 TI - [Detection of the Koutango virus (Flavivirus, Togaviridae) in Somalia]. PMID- 3018466 TI - [Results of studying foci of arbovirus infections in the southern Maritime Territory]. PMID- 3018467 TI - Functional studies in brain and heart with positron emission tomography. AB - Positron Emission Tomography (PET) has evolved in the last years into a powerful research technique for the study of the physiology and pathophysiology of the human brain in vivo. These procedures now need no longer be viewed only as research studies. They are ready to be applied clinically on a wide scale. This article will give a short overview of the technical and methodological background and outline the clinical research applications by using the most developed tracers in the field: glucose and its analogs, oxygen, fatty acids, various perfusion markers and receptors, all labelled with positron emitting isotopes. They allow the quantitative measurement of local tissue functions in an essentially non-invasive way. PMID- 3018468 TI - [Granular-cell myoblastoma. A clinical case]. PMID- 3018469 TI - Human T-lymphotropic virus type III/lymphadenopathy-associated virus: agent summary statement. PMID- 3018471 TI - Immunization of children infected with human T-lymphotropic virus type III/ lymphadenopathy-associated virus. PMID- 3018470 TI - Chikungunya fever among U.S. Peace Corps volunteers--Republic of the Philippines. PMID- 3018472 TI - Acquired immunodeficiency syndrome (AIDS) in western Palm Beach County, Florida. PMID- 3018473 TI - [Measurement of the portal blood flow in man by continuous local thermodilution method: II. Portal hemodynamics before and after hepatectomy]. AB - We found that measurements of portal blood flow by continuous thermodilution were highly reproducible even after hepatectomy. Our subjects numbered 59 in all: In these patients having diseases of the liver and biliary tract, we studied portal hemodynamics during percutaneous transhepatic portography. Of these, 37 underwent hepatectomy. We chose 19 subjects from this group, and measured again both portal venous flow and portal venous pressure many times, continuing for 14 more days. In all 19 patients checked after hepatectomy, portal hemodynamics became hypodynamic, and this change was greater when the amount of liver resected was large. In 18 of these patients, hemodynamics started to improve after the 7th postoperative day. Changes in hemodynamics were not significantly different in patients with or without cirrhosis. In one patient who died of hepatic failure, the portal hypodynamic state did not improve. With this exception, in patients with major resections, portal venous flow per liver volume had increased after surgery and continued to increase. This was not true for patients with minor resections. Portal hemodynamics are important in the functioning and regeneration of the remaining liver, and it is necessary to understand and medically correct portal hemodynamics before and after hepatectomy. PMID- 3018474 TI - [Clinical study on serum 5'-nucleotide phosphodiesterase isoenzyme-V as a predictor of liver metastases in patients with gastric and colorectal cancers]. AB - By separating 5'-nucleotide phosphodiesterase isoenzyme-V (5'-NPD-V) as a fast moving isoenzyme by polyacrylamide electrophoresis, the determination of serum 5' NPD-V was performed in 302 preoperative patients with gastric and colorectal cancers to assess the clinical usefulness for suspecting liver metastases. Serum levels of CEA, alpha-fetoprotein and tumor markers were simultaneously measured. Angiography, CT scan and echo were also performed preoperatively. The normal values of serum 5'-NPD-V in 67 healthy subjects except heavy smokers were less than 3.0mm. 5'NPD-V values determined in patients with and without liver metastases were as follows: In gastric cancer 1.5 +/- 2.0mm and 8.6 +/- 9.0mm, and in colorectal cancer 2.2 +/- 3.3mm and 5.8 +/- 5.3mm, respectively, indicating a significant difference (p less than 0.05). The sensitivity of 5'-NPD V in gastric cancer was 0.682, the specificity was 0.892, the predictability was 0.518, and the accuracy was 0.862. The results in colorectal cancer were 0.600, 0.958, 0.805 and 0.800, respectively. Serum 5'-NPD-V value was elevated progressively in accordance with extent of liver involvement. When assessed by 5' NPD-V and CEA, 80.9% of patients with liver metastases proved to be correctly diagnosed. The results suggest that 5'-NPD-V is clinically a useful marker in that the isoenzyme provides the rationale for the further detection of tumor localization in the liver. PMID- 3018475 TI - [A case of gastric perforation due to invasive liver cell carcinoma to the stomach]. AB - A 70 year old man complaining of abdominal pain was admitted to our hospital and was suspected to have a gastric cancer with perforation by barium meal X-ray and by endoscopy. Ten days before, admission X-ray examination revealed the free air in the abdominal cavity but this patient didn't complain of any sign of peritonitis. He underwent left upper abdominal evisceration through upper median incision and median sternophrenicotomy. Resected specimen of the stomach had giant ulcer which looked like the gastric cancer. The pathological diagnosis of this lesion was liver cell carcinoma, Edmondson's grade 4, metastasis from the liver with lymph nodes and pancreatic involvement. PMID- 3018476 TI - Receptor reserve in the calcium-dependent cyclic AMP response of astrocytoma cells to muscarinic receptor stimulation: demonstration by agonist-induced desensitization, receptor inactivation, and phorbol ester treatment. AB - Activation of muscarinic cholinergic receptors on 1321N1 human astrocytoma cells results in attenuation of cyclic AMP accumulation, apparently as a consequence of increases in phosphoinositide hydrolysis, Ca2+ mobilization, and the activation of a Ca2+ calmodulin-regulated phosphodiesterase. Preincubation of these cells with carbachol for 30-90 min resulted in a 15-30-fold shift to the right of the concentration effect curves for carbachol or methacholine for attenuation of cyclic AMP accumulation, with no change occurring in the maximal effect observed with either agonist. In contrast, preincubation with carbachol for 30-90 min resulted in an essentially complete loss of effects on cyclic AMP accumulation of the muscarinic receptor agonists oxotremorine, arecoline, and bethanechol. In contrast to carbachol and methacholine, oxotremorine, arecoline, and pilocarpine were much less effective inducers of desensitization. Inactivation of muscarinic receptors with propylbenzylcholine mustard, or preincubation of cells with 4 beta phorbol 12 beta-myristate 13 alpha-acetate, which has been shown to markedly decrease the phosphoinositide response of 1321N1 cells to cholinergic agonists, produced differential effects on the cyclic AMP response to carbachol and oxotremorine that were similar to those observed after preincubation with carbachol. The data can be explained by the presence of "reserve" in the series of steps that result in muscarinic receptor-mediated activation of phosphodiesterase. That is, it is proposed that carbachol and methacholine mobilize much more Ca2+ than is necessary for maximal activation of phosphodiesterase, whereas oxotremorine and several other muscarinic receptor agonists do not. PMID- 3018477 TI - Muscarinic responses and binding in a murine neuroblastoma clone (N1E-115): cyclic GMP formation is mediated by a low affinity agonist-receptor conformation and cyclic AMP reduction is mediated by a high affinity agonist-receptor conformation. AB - Murine neuroblastoma cells (clone N1E-115) possess two subtypes of the muscarinic receptor each of which separately mediates a cyclic nucleotide response. The formation of cyclic GMP is postulated to involve a low affinity agonist-receptor conformation, whereas the reduction of prostaglandin E1-stimulated cyclic AMP formation appears to involve a high affinity conformation. Further evidence supporting this hypothesis was obtained in experiments measuring the equilibrium dissociation constants for the full agonist carbachol by the method of partial receptor inactivation. Quinuclidinyl benzilate (QNB) was employed to occlude muscarinic receptors; measurements with [3H] QNB ensured that the amount of QNB appearing in the assay after washout had only a minimal effect on the determination of the equilibrium dissociation constants. Carbachol mediated cyclic GMP formation with an equilibrium dissociation constant (KD) of 325 microM and cyclic AMP reductions with a KD value of 13 microM. These KD values are similar to but somewhat higher than those determined by direct binding at 15 degrees, and they are strong evidence in support of the view that a low affinity conformation mediates cyclic GMP formation, whereas a high affinity conformation mediates cyclic AMP reductions. PMID- 3018478 TI - Characterization of the binding of [3H]-(+/-)-L-364,718: a new potent, nonpeptide cholecystokinin antagonist radioligand selective for peripheral receptors. AB - [3H]-(+/-)-L-364,718 a new, potent and selective nonpeptide peripheral cholecystokinin (CCK) antagonist bound saturably and reversibly to rat pancreatic membranes. The radioligand recognized a single class of binding sites with a high affinity (Kd = 0.23 nM). The binding of [3H]-(+/-)-L-364,718 was stereospecific in that the more biologically active (-)-enantiomer demonstrated greater potency than the (+)-enantiomer. The rank order of potency of various CCK agonists and antagonists in displacing [3H]-(+/-)-L-364,718 correlated with their ability to displace [125I]CCK-8 and their known pharmacological activities in peripheral tissues. However, the absolute potencies of agonists were greater in displacing [125I]CCK-8 than [3H]-(+/-)-L-364,718. As described for other physiologically relevant receptor systems, the potency for displacement of [3H]-(+/-)-L-364,718 binding by CCK agonists, but not antagonists, was reduced by guanosine 5'-(beta, gamma-imido)triphosphate and NaCl and enhanced by MgCl2. [3H]-(+/-)-L-364,718 also demonstrated specific binding to bovine gall bladder tissue but not guinea pig brain or gastric glands, consistent with its selectivity as a peripheral CCK antagonist. [3H]-(+/-)-L-364,718 binding to pancreatic membranes was not affected by various pharmacological agents known to interact with other common peptide and nonpeptide receptor systems. These data indicate that [3H]-(+/-)-L-364,718 represents a new potent nonpeptide antagonist radioligand for the study of peripheral CCK receptors which may allow differentiation of agonist and antagonist interactions. PMID- 3018479 TI - Modulation of the chloride ionophore by benzodiazepine receptor ligands: influence of gamma-aminobutyric acid and ligand efficacy. AB - t-Butylbicyclophosphorothionate (TBPS) produces dose-dependent enhancement of [3H]propyl beta-carboline-3-carboxylate ([3H]PCC, 40 pM) binding to the benzodiazepine1 (BZ1) receptor subtype in hippocampus. Furthermore, TBPS enhancement of [3H]PCC binding was antagonized by micromolar concentrations of gamma-aminobutyric acid (GABA) in a way reversible by bicuculline. BZ receptor ligands that are "GABA positive" (i.e., enhance GABA neurotransmission) allosterically inhibited [35S]TBPS binding, whereas "GABA-negative" ligands (i.e., inhibit GABA neurotransmission) produced the opposite effect. The efficacy of the ligands as modulators of [35S]TBPS binding was consistent with their reported in vivo pharmacology. The effects of positive and negative ligands on [35S]TBPS binding were modulated by micromolar concentrations of GABA. Examination of the kinetics of [35S]TBPS binding suggested the presence of slowly and rapidly dissociating components. The GABA-positive clonazepam stabilized the rapidly dissociating component of [35S]TBPS binding, whereas methyl beta carboline-3-carboxylate had a similar effect on the slowly dissociating component. It is speculated that the slowly dissociating component of [35S]TBPS binding is associated with a closed chloride channel, whereas the opposite is proposed for the rapidly dissociating component. The differential effects of GABA positive versus GABA-negative ligands on [35S]TBPS binding and the modulatory effect of GABA provide further evidence to suggest that [35S]TBPS labels a site near the chloride ionophore linked to the GABA-BZ receptor complex. PMID- 3018480 TI - Leukotriene B4 induces formation of inositol phosphates in rat peritoneal polymorphonuclear leukocytes. AB - Leukotriene B4 (LTB4) induced rapid breakdown of prelabeled inositol phospholipids in rat peritoneal polymorphonuclear leukocytes (PMNs). Formation of [3H]inositol triphosphate ([3H]IP3) was rapid, with a peak of 250-300% of the control level, after 5-15 sec of exposure to LTB4. Accumulation of [3H]inositol bisphosphate was rapid, peaking after 30 sec of treatment. Accumulation of [3H]inositol monophosphate was also rapid in the presence of LiCl. The kinetics of [3H]IP3, [3H]inositol bisphosphate, and [3H]inositol monophosphate accumulation suggest that LTB4 may interact with receptors in PMNs and activate phospholipase C which in turn induces hydrolysis of inositol-phospholipids. The agonist activities of several LTB4 analogs were employed to investigate the structure-activity relationships of LTB4 receptor-mediated activation of phosphatidylinositol hydrolysis. Increases in [3H]IP3 formation were dependent upon the concentration of LTB4 and the agonist analogs. The rank order potency of these analogs was equivalent to that of the pharmacological activity of LTB4 agonists in the PMN chemotaxis assay. Furthermore, the islet activation protein isolated from Bordetella pertussis inhibited LTB4-induced [3H]IP3 formation. The tumor-promoting phorbol myristate acetate also inhibited LTB4-induced [3H]IP3 formation. The LTB4 receptors on a partially purified PMN membrane were characterized. LTB4 binding to the receptors was stereoselective and specific. The binding affinity (Kd) of [3H] LTB4 to the receptors was 1.3 +/- 0.2 nM. The maximum density of binding was 5.5 +/- 1.8 pmol/mg of protein. The rank order potency of binding affinities of several LTB4 analogs was equivalent to that of the induction of IP3 response induced by LTB4 and analogs. These results suggest that LTB4 may interact with receptors in rat PMNs, activate G protein-regulated phospholipase C, and induce [3H]IP3 formation. PMID- 3018481 TI - Association of a spin-labeled local anesthetic with the allosterically coupled noncompetitive inhibitor site on the acetylcholine receptor. AB - Radioligand binding and ESR were used to study the association of a spin-labeled local anesthetic, 2-[N,N-dimethyl-N-(2,2,6,6-tetramethylpiperidinooxyl)] ethyl-4 hexyloxybenzoate iodide (C6SLMel), with acetylcholine receptor-enriched membranes from Torpedo californica. In the presence of carbamylcholine, we found that C6SLMel competitively inhibits [3H]phencyclidine binding with high affinity (KD = 8.7 X 10(-7) M for C6SLMel), whereas in the presence of alpha-toxin or in the absence of agonist, C6SLMel binds with lower affinity (KD = 2 X 10(-5) M). At concentrations lower than 1 X 10(-5) M, C6SLMel does not bind to the agonist site but enhances [3H]acetylcholine binding. These findings show that C6SLMel binds to the allosterically coupled noncompetitive inhibitor site which is regulated by agonist binding. In addition, C6SLMel preferentially associates with the desensitized receptor state known to exhibit high affinities for agonists and local anesthetics. ESR measurements of C6SLMel bound to receptor membranes in the absence of agonist show moderately immobilized spectra. Addition of carbamylcholine results in the appearance of a strongly immobilized component. Prior exposure to alpha-toxin blocks the carbamylcholine-induced, strongly immobilized component in the ESR spectrum. Furthermore, in the presence of carbamylcholine, back-titration of bound C6SLMel with phencyclidine decreases the highly immobilized component at concentrations consistent with the KD for phencyclidine. These findings indicate that C6SLMel detects conformational changes between the resting and desensitized acetylcholine receptor states that occur at the noncompetitive inhibitor binding site. The strongly mobilized component is not affected by ferricyanide addition, suggesting that the binding site is in a region not readily accessible to anion collision from the aqueous phase. PMID- 3018482 TI - Effects of sodium valproate on mitochondrial membranes: electron paramagnetic resonance and transmembrane protein movement studies. AB - Sodium valproate (VPA), the salt of a branched short-chain fatty acid, is a major antiepileptic whose mode of action, as yet unclear, may involve effects on the organization of membranes. VPA was either injected into rats whose liver and kidney mitochondria were then isolated, or was preincubated with isolated mitochondria. First, liver and kidney mitochondria were studied with paramagnetic probes. The electron paramagnetic resonance spectra of proteins of VPA-treated mitochondria spin-labeled with 4-maleimido-2,2,6,6-tetramethyl-1-pyrrolidinoxyl showed that the ratio of weakly immobilized to strongly immobilized SH groups was reduced with respect to control mitochondria, more so in liver than in kidney mitochondria of VPA-injected rats, and more so in kidney than in liver mitochondria for VPA-incubated mitochondria. Spectra of mitochondrial lipids spin labeled with 5-doxyl stearic methyl ester showed that VPA had no significant effect on order parameters S. Second, the transmembrane movement of aspartate aminotransferase was studied by incubating liver mitochondria in a sucrose succinate medium and then fractionating them. The translocation of aspartate aminotransferase from mitoplasts, vesicles formed of inner membrane and matrix, to the intermembrane fluid, was significantly higher in VPA-treated than in control mitochondria. Thus, VPA, at concentrations in the range of those used therapeutically, interacted with membranes by modifying the structural organization of the internal mitochondrial membrane, essentially the membrane protein conformation. PMID- 3018483 TI - Effects of nitroprusside on the bradykinin responsiveness of human fibroblasts. AB - The effects of agents that cause vasodilatation and hypotension, such as endogenously produced bradykinin (BK) or the drug nitroprusside (NP), appear to result from effects on cyclic nucleotides (cGMP, cAMP) and arachidonate metabolism. Cultured human fibroblasts, which possess B2 BK receptors and respond to NP with an increase in cGMP, were used to study the interaction of these agents at the molecular level. Addition of BK or NP to cultured human fibroblasts caused a rapid increase in cGMP. The effect of NP was usually maximal within 30 sec, after which cGMP content declined. The increase in cGMP produced by BK reached a maximum at approximately 1 min and then fell; the rise with NP was more than 10 times that with BK. At 30 sec, cGMP content with NP plus BK was less than with NP alone. At later times, however, effects of BK and NP were slightly more than additive and maximal cGMP levels were reached at 90 sec. BK increased prostaglandin production by the fibroblasts; it is believed that the kinin induced elevation in cAMP content is secondary to increased prostaglandin formation. NP caused a small, early increase in cAMP without significant effect on prostaglandin I2 (PGI2); after 2.5 min, effects on PGI2 and cAMP were greater with BK and NP than with BK alone. To study further the roles of arachidonate metabolites in the fibroblast response to BK and NP, the cyclooxygenase inhibitor, indomethacin, and the combined lipoxygenase and cyclooxygenase inhibitor, 5,8,11,14-eicosatetraynoic acid (ETYA), were added to fibroblasts prior to BK or NP. Increases in cAMP or PGI2 with BK or BK plus NP were blocked by indomethacin or ETYA. These effects of BK or BK plus NP on cAMP thus appear to be mediated through cyclooxygenase products of arachidonate metabolism. Indomethacin and ETYA did not affect cGMP in the presence of BK plus NP but enhanced NP-stimulated cGMP accumulation by 40-50%; effects of NP on cGMP may be independent of or perhaps inhibited by cyclooxygenase derivatives. Cellular responses to BK plus NP differed quantitatively and temporally from the sum of effects of BK and NP alone. Through interactions of this type, in vivo responses to drugs like NP may be influenced by levels of BK or similar endogenous mediators. PMID- 3018484 TI - Changes in the levels of three different classes of histone mRNA during murine erythroleukemia cell differentiation. AB - We used a gene-specific S1 nuclease assay to study the changes in steady-state mRNA levels of several core histone variants during the differentiation of murine erythroleukemia cells. These studies allowed us to distinguish three distinct expression classes of histone genes. The expression of the major replication dependent class of histone genes was tightly linked to DNA synthesis. The concentrations of these transcripts decreased rapidly as cell division slowed during the process of differentiation. In contrast, the replication-independent H3.3 transcript levels were constitutively maintained throughout differentiation and were unaffected by inhibitors of DNA or protein synthesis. We also identified among the cloned histone genes used as probes a third expression class, the partially replication-dependent variants. Expression of these transcripts became transiently uncoupled from the reduced rate of DNA synthesis accompanying the early stages of differentiation. We show that their synthesis is sensitive to the DNA synthesis inhibitor hydroxyurea but that selective uncoupling from DNA synthesis of these histone mRNAs occurs at a specific stage of differentiation. We present several hypotheses to explain how this might be accomplished. The expression characteristics of the mRNAs studied coincided with those of the proteins for which they code, indicating that changes in the relative levels of the different variants is mediated at least in part by changes in mRNA levels. PMID- 3018485 TI - Evolution of the dispersed SUC gene family of Saccharomyces by rearrangements of chromosome telomeres. AB - The SUC gene family of Saccharomyces contains six structural genes for invertase (SUC1 through SUC5 and SUC7) which are located on different chromosomes. Most yeast strains do not carry all six SUC genes and instead carry natural negative (suc0) alleles at some or all SUC loci. We determined the physical structures of SUC and suc0 loci. Except for SUC2, which is an unusual member of the family, all of the SUC genes are located very close to telomeres and are flanked by homologous sequences. On the centromere-proximal side of the gene, the conserved region contains X sequences, which are sequences found adjacent to telomeres (C. S. M. Chan and B.-K. Tye, Cell 33:563-573, 1983). On the other side of the gene, the homology includes about 4 kilobases of flanking sequence and then extends into a Y' element, which is an element often found distal to the X sequence at telomeres (Chan and Tye, Cell 33:563-573, 1983). Thus, these SUC genes and flanking sequences are embedded in telomere-adjacent sequences. Chromosomes carrying suc0 alleles (except suc20) lack SUC structural genes and portions of the conserved flanking sequences. The results indicate that the dispersal of SUC genes to different chromosomes occurred by rearrangements of chromosome telomeres. PMID- 3018486 TI - Poliovirus mutant that does not selectively inhibit host cell protein synthesis. AB - A poliovirus type I (Mahoney strain) mutant was obtained by inserting three base pairs into an infectious cDNA clone. The extra amino acid encoded by the insertion was in the amino-terminal (protein 8) portion of the P2 segment of the polyprotein. The mutant virus makes small plaques on HeLa and monkey kidney (CV 1) cells at all temperatures. It lost the ability to mediate the selective inhibition of host cell translation which ordinarily occurs in the first few hours after infection. As an apparent consequence, the mutant synthesizes far less protein than does wild-type virus. In mutant-infected CV-1 cells enough protein was produced to permit a normal course of RNA replication, but the yield of progeny virus was very low. In mutant-infected HeLa cells there was a premature cessation of both cellular and viral protein synthesis followed by a premature halt of viral RNA synthesis. This nonspecific translational inhibition was distinguishable from wild-type-mediated inhibition and did not appear to be part of an interferon or heat shock response. Because the mutant is recessive, our results imply that (at least in HeLa cells) wild-type poliovirus not only actively inhibits translation of cellular mRNAs, but also avoids early inhibition of its own protein synthesis. Cleavage of the cap-binding complex protein P220, which has been associated with the selective inhibition of capped mRNA translation, did not occur in mutant-infected cells. This result supports the hypothesis that cleavage of P220 plays an important role in normal poliovirus mediated translational inhibition. PMID- 3018487 TI - Sequence-dependent DNA replication in preimplantation mouse embryos. AB - Circular, double-stranded DNA molecules were injected into nuclei of mouse oocytes and one- or two-cell embryos to determine whether specific sequences were required to replicate DNA during mouse development. Although all of the injected DNAs were stable, replication of plasmid pML-1 DNA was not detected unless it contained either polyomavirus (PyV) or simian virus 40 (SV40) DNA sequences. Replication occurred in embryos, but not in oocytes. PyV DNA, either alone or recombined with pML-1, underwent multiple rounds of replication to produce superhelical and relaxed circular monomers after injection into one- or two-cell embryos. SV40 DNA also replicated, but only 3% as well as PyV DNA. Coinjection of PyV DNA with either pML-1 or SV40 had no effect on the replicating properties of the three DNAs. These results are consistent with a requirement for specific cis acting sequences to replicate DNA in mammalian embryos, in contrast to sequence independent replication of DNA injected into Xenopus eggs. Furthermore, PyV DNA replication in mouse embryos required PyV large T-antigen and either the alpha beta-core or beta-core configuration of the PyV origin of replication. Although the alpha-core configuration replicated in differentiated mouse cells, it failed to replicate in mouse embryos, demonstrating cell-specific activation of an origin of replication. Replication or expression of PyV DNA interfered with normal embryonic development. These results reveal that mouse embryos are permissive for PyV DNA replication, in contrast to the absence of PyV DNA replication and gene expression in mouse embryonal carcinoma cells. PMID- 3018489 TI - Point mutations implicate repeated sequences as essential elements of the CYC7 negative upstream site in Saccharomyces cerevisiae. AB - The transcription of the CYC7 gene of Saccharomyces cerevisiae, encoding the iso 2-cytochrome c protein, is controlled by two upstream regulatory elements, a positive element and a negative element. The nature of the DNA sequences in the negative element were investigated in a two-part approach. The first involved the construction of a CYC7-galK fusion gene which placed the coding sequence of the Escherichia coli galactokinase gene under the regulation of the CYC7 upstream sequences. This fusion allowed the quantitation by galactokinase enzyme assays of the effects on gene expression of a variety of previously isolated deletion mutations within the negative site. The results suggested that the negative site contained three related sequences. This hypothesis was tested in the second part of these studies, the selection of point mutations within the region of the negative site which led to increased CYC7 expression. Point mutations were introduced by a technique which induced mutations within a localized region at high efficiency. All but one of the mutations involved more than a single base pair change. The mutations followed the pattern that multiple base-pair changes occurred in one repeat or single base-pair changes occurred in two repeats, with the exception of one mutant, which had a single base-pair change in one repeat. This pattern of mutations and the base pairs that were altered strongly supported the hypothesis that the repeats are integral elements of the negative site. PMID- 3018488 TI - Transcription of mouse rDNA and associated formation of the nucleolus organizer region after gene transfer and amplification in Chinese hamster cells. AB - Mouse rDNA can initiate transcription by using only Chinese hamster cell components, and this is associated with nucleolus organizer activity. To demonstrate this, we transferred a 3.2-kilobase segment of mouse rDNA containing the promoter, the transcription initiation site, and part of the external transcribed spacer to dihydrofolate reductase-deficient Chinese hamster cells by cotransformation with an abbreviated mouse dhfr gene. Stepwise selection for methotrexate resistance produced sublines in which the mouse rDNA was usually coamplified with the donor dhfr DNA and occupied the same site or sites in the hamster genome, as shown by in situ hybridization. Transcription from mouse rDNA was demonstrated in two such lines, and S1 protection mapping indicated faithful initiation of the transcript. In some cells from both lines, the chromosome segments containing amplified mouse rDNA showed multiple silver-staining regions (i.e., active nucleolus organizers). Although the transferred mouse rDNA was able to use the rDNA transcriptional machinery of the Chinese hamster, the level of transcription was much lower than expected from the rDNA copy number, and a large fraction of each amplified region showed no silver staining. Since the absence of silver staining is generally correlated with the absence of transcription, many copies of the amplified mouse rDNA may have been in a chromatin conformation in which they could not be transcribed. This was not associated with the extensive methylation seen in other amplified, inactive rDNA sequences. PMID- 3018490 TI - Definition of essential sequences and functional equivalence of elements downstream of the adenovirus E2A and the early simian virus 40 polyadenylation sites. AB - In addition to the highly conserved AATAAA sequence, there is a requirement for specific sequences downstream of polyadenylic acid [poly(A)] cleavage sites to generate correct mRNA 3' termini. Previous experiments demonstrated that 35 nucleotides downstream of the E2A poly(A) site were sufficient but 20 nucleotides were not. The construction and assay of bidirectional deletion mutants in the adenovirus E2A poly(A) site indicates that there may be redundant multiple sequence elements that affect poly(A) site usage. Sequences between the poly(A) site and 31 nucleotides downstream were not essential for efficient cleavage. Further deletion downstream (3' to +31) abolished efficient cleavage in certain constructions but not all. Between +20 and +38 the sequence T(A/G)TTTTT was duplicated. Function was retained when one copy of the sequence was present, suggesting that this sequence represents an essential element. There may also be additional sequences distal to +43 that can function. To establish common features of poly(A) sites, we also analyzed the early simian virus 40 (SV40) poly(A) site for essential sequences. An SV40 poly(A) site deletion that retained 18 nucleotides downstream of the cleavage site was fully functional while one that retained 5 nucleotides downstream was not, thus defining sequences required for cleavage. Comparison of the SV40 sequences with those from E2A did not reveal significant homologies. Nevertheless, normal cleavage and polyadenylation could be restored at the early SV40 poly(A) site by the addition of downstream sequences from the adenovirus E2A poly(A) site to the SV40 +5 mutant. The same sequences that were required in the E2A site for efficient cleavage also restored activity to the SV40 poly(A) site. PMID- 3018491 TI - Glucocorticoid receptor binding and activation of a heterologous promoter by dexamethasone by the first intron of the human growth hormone gene. AB - In this study DNA-binding and gene transfer experiments were performed to examine a potential glucocorticoid regulatory element (GRE) in the human growth hormone gene. As assayed by nitrocellulose filter binding, only two regions of the human growth hormone gene, the 5'-flanking sequences and a fragment containing part of the first intron, were retained preferentially by purified glucocorticoid receptor complexes. The relative binding by the transcribed sequences was three times greater than the relative binding by the 5'-flanking sequences, but less than the relative binding by a fragment containing the human metallothionein-IIA gene GRE. The intron, but not the 5'-flanking sequences, generated a "footprint" when the receptor complex was used to protect the segments against exonuclease III digestion; the protected sequence spanned nucleotides +86 to +115 in the first intron and contained a structure homologous in 14 of 16 nucleotides to a 16 nucleotide consensus GRE. The hexanucleotide 5'-TGTCCT-3', thought to be important for GRE activity, not only was found in this sequence and in the 5' flanking region, but also was present twice in the 3' end of the gene that did not show specific receptor binding. The latter results suggest that the hexanucleotide alone is not sufficient to generate specific receptor binding tight enough to be assayed in this way. To test the biological activity of the intron binding site, a fragment containing these sequences was fused 5' to the human metallothionein-IIA gene promoter depleted of its GRE and linked to the structural sequences of the herpes simplex virus thymidine kinase (TK) gene. When this hybrid gene was transfected into Rat 2 TK- cells, its expression was induced threefold by the glucocorticoid dexamethasone, as assessed by transfection efficiency and RNA blotting analyses. Expression of the same gene without the human growth hormone gene segment was not affected by the steroid, whereas the wild-type human metallothionein-IIA gene promoter containing its GRE responded to the hormone by a sixfold increase in thymidine kinase mRNA. These results indicate that the human growth hormone gene contains a structure within its first intron that can function as a GRE. PMID- 3018492 TI - The proto-oncogene c-ets is preferentially expressed in lymphoid cells. AB - The transforming sequences of the avian acute leukemia virus, E26, contain two distinct oncogenes, v-mybE and v-ets, fused together. By using a probe containing v-ets sequences, polyadenylated transcripts of the c-ets proto-oncogene were detected in avian tissues; they included a major 7.0-kilobase and a minor 2.0 kilobase species. These c-ets mRNAs were detected at high levels only in lymphoid organs and in avian T and B lymphoid cell lines. A similar pattern of c-ets transcription was observed in human hematopoietic cell lines, with transcripts detected in lymphoid B and T cells but not in erythroid or myeloid cells. The E26 oncogene was inserted into an inducible expression vector, and a 90-kilodalton protein (bp90) was produced in bacteria. Rabbit antisera raised to purified bp90 precipitated P135gag-mybE-ets, the v-mybE-ets polyprotein expressed in E26 transformed cells, and also reacted with p50v-mybA, the transforming protein of the avian myeloblastosis virus. Antiserum to bp90 was absorbed with a bacterially synthesized v-mybA protein to remove anti-myb activity. The absorbed anti-bp90 serum retained the ability to immunoprecipitate P135gag-mybE-ets from E26 transformed cells and specifically reacted with a 56-kilodalton polypeptide (p56) detected in chicken lymphoid organs and in T and B lymphocytes of both avian and human origin. The data suggest that p56 is a translational product of the c-ets proto-oncogene and imply that p56 may be involved in regulating the growth of lymphoid cells. PMID- 3018493 TI - Quantitative assessment of actin transcript number in eggs, embryos, and tube feet of the sea star Pisaster ochraceus. AB - Actin coding sequence cDNA probes were used to quantitate the number of transcripts in RNA from eggs, embryos, and tube feet of the sea star Pisaster ochraceus. Transcript concentrations were measured in both total RNA and in poly(A)+ RNA by titration and hybridization kinetic methods. Surprisingly, the actin transcript number in sea star eggs is two orders of magnitude greater than in sea urchin eggs. There are at least 2.9 X 10(5) actin transcripts per sea star egg, 1.2 X 10(5) per 48-h gastrula and 1.9 X 10(5) per 72-h gastrula. The number of actin transcripts per unit mass of extracted tube foot RNA is lower than in developmental stages. The relative abundance and size of actin transcripts was determined by Northern and dot blot analyses using probes containing actin coding DNA or 3'-untranslated-region sequences. The actin transcript in eggs and embryos is 2,300 nucleotides (nt) long and originates from the Cy (cytoplasmic) gene class. In tube feet, the most abundant actin transcript is 2,200 nt long and originates from the M (muscle) gene class. Tube feet also contain, at lower abundance, 2,300-nt transcripts of the Cy gene type expressed in eggs and embryos. PMID- 3018494 TI - Regulation of the human c-myc gene: 5' noncoding sequences do not affect translation. AB - The influence of untranslated 5' sequences on c-myc expression was compared by measuring the translational efficiencies of mRNAs which contain leaders derived from exon 1 or intron 1 of the human c-myc gene. Expression plasmids were constructed and introduced into COS cells, and the levels of c-myc mRNA and protein were examined. Our results show that mRNAs transcribed from constructs containing exon 1 or intron 1, which have different folding potential, are translated with approximately equal efficiencies. This suggests that the translation of c-myc mRNA is not controlled by secondary structure alone. In addition, we observed that transcripts in which exon 1 was deleted are not translated more efficiently, but are present at a higher steady-state level. Thus, this example provides evidence for possible control at the transcriptional level. Finally, since the c-myc product was produced in each of our test systems, the results suggest that this protein does not regulate its own transcription or translation via a specific interaction with c-myc exon 1 alone. PMID- 3018496 TI - Cloning of hexokinase structural genes from Saccharomyces cerevisiae mutants with regulatory mutations responsible for glucose repression. AB - The regulatory hexokinase PII mutants isolated previously (K.-D. Entian and K.-U. Frohlich, J. Bacteriol. 158:29-35, 1984) were characterized further. These mutants were defective in glucose repression. The mutation was thought to be in the hexokinase PII structural gene, but it did not affect the catalytic activity of the enzyme. Hence, a regulatory domain for glucose repression was postulated. For further understanding of this regulatory system, the mutationally altered hexokinase PII proteins were isolated from five mutants obtained independently and characterized by their catalytic constants and bisubstrate kinetics. None of these characteristics differed from those of the wild type, so the catalytic center of the mutant enzymes remained unchanged. The only noticeable difference observed was that the in vivo modified form of hexokinase PII, PIIM, which has been described recently (K.-D. Entian and E. Kopetzki, Eur. J. Biochem. 146:657 662, 1985), was absent from one of these mutants. It is possible that the PIIM modification is directly connected with the triggering of glucose repression. To establish with certainty that the mutation is located in the hexokinase PII structural gene, the genes of these mutants were isolated after transforming a hexokinaseless mutant strain and selecting for concomitant complementation of the nuclear function. Unlike hexokinase PII wild-type transformants, glucose repression was not restored in the hexokinase PII mutant transformants. In addition mating experiments with these transformants followed by tetrad analysis of sporulated diploids gave clear evidence of allelism to the hexokinase PII structural gene. PMID- 3018495 TI - Nuclear compartmentalization of the v-myb oncogene product. AB - Nuclei obtained from chicken leukemic myeloblasts transformed by avian myeloblastosis virus were fractionated into various subnuclear compartments, which were then analyzed by specific immunoprecipitation for the presence of the leukemogenic product, p48v-myb, of the viral oncogene. In cells labeled for 30 or 60 min with L-[35S]methionine and in unlabeled exponentially dividing leukemic cells analyzed by Western blotting, p48v-myb was detected within the nucleoplasm (29 +/- 9% [standard deviation] of the total), chromatin (7 +/- 4%), and lamina nuclear matrix (64 +/- 9%). Also, in myeloblasts analyzed by immunofluorescence during mitosis, p48v-myb appeared to be dispersed through the cell like the lamina-nuclear matrix complex. Strong attachment to the nuclear matrix-lamina complex suggests that p48v-myb may be involved in DNA replication or transcription or both. PMID- 3018497 TI - Simian virus 40 minichromosomes contain torsionally strained DNA molecules. AB - Sundin and Varshavsky (J. Mol. Biol. 132:535-546, 1979) found that nearly two thirds of simian virus 40 (SV40) minichromosomes obtained from nuclei of SV40 infected cells become singly nicked or cleaved across both strands after digestion with staphylococcal nuclease at 0 degrees C. The same treatment of SV40 DNA causes complete digestion rather than the limited cleavages produced in minichromosomal DNA. We have explored this novel behavior of the minichromosome and found that the nuclease sensitivity is dependent upon the topology of the DNA. Thus, if minichromosomes are pretreated with wheat germ DNA topoisomerase I, the minichromosomal DNA is completely resistant to subsequent digestion with staphylococcal nuclease at 0 degrees C. If the minichromosome-associated topoisomerase is removed, virtually all of the minichromosomes are cleaved to nicked or linear structures by the nuclease treatment. The cleavage sites are nonrandomly located; instead they occur at discrete loci throughout the SV40 genome. SV40 minichromosomal DNA is also cleaved to nicked circles and full length linear fragments after treatment with the single strand-specific endonuclease S1; this cleavage is also inhibited by pretreatment with topoisomerase I. Thus, it may be that the nuclease sensitivity of minichromosomes is due to the transient or permanent unwinding of discrete regions of their DNA. Direct comparisons of the extent of negative supercoiling of native and topoisomerase-treated SV40 minichromosomes revealed that approximately two superhelical turns were removed by the topoisomerase treatment. The loss of these extra negative supercoils from the DNA probably accounts for the resistance of the topoisomerase-treated minichromosomes to the staphylococcal and S1 nucleases. These findings suggest that the DNA in SV40 intranuclear minichromosomes is torsionally strained. The functional significance of this finding is discussed. PMID- 3018498 TI - Characterization of the myosin light-chain-2 gene of Drosophila melanogaster. AB - Recombinant DNA clones encoding the Drosophila melanogaster homolog of the vertebrate myosin light-chain-2 (MLC-2) gene have been isolated. This single-copy gene maps to the chromosomal locus 99E. The nucleotide sequence was determined for a 3.4-kilobase genomic fragment containing the gene and for two MLC-2 cDNA clones generated from late pupal mRNA. Comparison of these sequences shows that the gene contains two introns, the positions of which are conserved in the corresponding rat sequence. Extension of a primer homologous to the mRNA reveals two start sites for transcription 12 nucleotides apart. The sequence TATA is not present ahead of the mRNA cap site. There are two major sites of poly(A) addition separated by 356 nucleotides. The protein sequence derived from translation of the cDNA sequence shows a high degree of homology with that for the DTNB myosin light chain (MLC-2) of chicken. A lower degree of sequence homology was seen in comparisons with other evolutionarily related calcium-binding proteins. RNA blots show high levels of expression of several transcripts during the developmental time stages when muscle is being produced. In vitro translation of hybrid selected RNA produces two polypeptides which comigrate on two-dimensional gels with proteins from Drosophila actomyosin, although the cDNA sequence reveals only one 26-kilodalton primary translation product. PMID- 3018500 TI - Regulation of cellular morphology by the Rous sarcoma virus src gene: analysis of fusiform mutants. AB - We have been interested in how Rous sarcoma virus (RSV) influences transformed cell morphology and compared the molecular properties of chicken embryo cells (CEC) infected with mutants of RSV that induce the fusiform transformed cell morphology with those of CEC infected by wild-type RSV, which induces the more normal round transformed cell morphology. We looked for properties shared by all fusiform mutant-infected cells, because these may be responsible for maintaining the fusiform morphology. Five different fusiform mutants, two wild-type RSVs, and one wild-type back revertant of a fusiform mutant were studied. In the fusiform mutant-infected cells, the localization and myristylation of pp60src were determined and the extent of expression of the extracellular matrix protein fibronectin was examined at both the mRNA and protein levels. The phosphorylation of vinculin on tyrosine also was examined in the same CEC. Within all fusiform mutant-transformed CEC, pp60src was dramatically absent from the adhesion plaque sites normally seen in cells transformed with wild-type RSV, and these transformed CEC all expressed more fibronectin mRNA and protein in the extracellular matrix than did the wild-type RSV-transformed CEC. The absence of pp60src from the adhesion plaques was not due to lack of myristylation of the src protein, and tyrosine phosphorylation of vinculin was not related to fibronectin expression. These results suggest that the inverse relationship between pp60src in the adhesion plaques and fibronectin expression in the extracellular matrix may be interconnected phenomena and could be related to the maintenance of the fusiform transformed morphology. PMID- 3018501 TI - Chromosomal organization of chicken histone genes: preferred associations and inverted duplications. AB - We present a detailed picture of the disposition of core and H1 histone genes in the chicken genome. Forty-two genes were located within four nonoverlapping regions totalling approximately 175 kilobases and covered by three cosmid clones and a number of lambda clones. The genes for the tissue-specific H5 histone and other variant histones were not found in these regions. The longest continuous region mapped was 67 kilobases and contained 21 histone genes in five dissimilar clusters. No long-range repeat was evident, but there were preferred associations, such as H1 genes with paired, divergently transcribed H2A-H2B genes and H3-H4 associations. However, there were exceptions, and even when associations such as H1-H2A-H2B we maintained, the order of those genes within a cluster may not have been. Another feature was the presence of three (unrelated) clusters in which genes were symmetrically ordered around central H3 genes; in one such cluster, the boundaries of a duplicated H2A-H4 gene pair contained related repeat sequences. Despite the dispersed nature of chicken histone genes, the number of each type was approximately equal, being represented as follows: 6 H1, 10 H2A, 8 H2B, 10 H3, and 8 H4. PMID- 3018499 TI - A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface. AB - We investigated the role of glycosylation in intracellular transport and cell surface expression of the vesicular stomatitis virus glycoprotein (G) in cells expressing G protein from cloned cDNA. The individual contributions of the two asparagine-linked glycans of G protein to cell surface expression were assessed by site-directed mutagenesis of the coding sequence to eliminate one or the other or both of the glycosylation sites. One oligosaccharide at either position was sufficient for cell surface expression of G protein in transfected cells, and the rates of oligosaccharide processing were similar to the rate observed for wild type protein. However, the nonglycosylated G protein synthesized when both glycosylation sites were eliminated did not reach the cell surface. This protein did appear to reach a Golgi-like region, as determined by indirect immunofluorescence microscopy, however, and was modified with palmitic acid. It was also apparently not subject to increased proteolytic breakdown. PMID- 3018502 TI - Alternative model for chromatin organization of the Saccharomyces cerevisiae chromosomal DNA plasmid TRP1 RI circle (YARp1). AB - TRP1 RI circle (now designated YARp1, yeast acentric ring plasmid 1) is a 1,453 base-pair artificial plasmid composed exclusively of Saccharomyces cerevisiae chromosomal DNA. It contains both the TRP1 gene and ARS1 (a DNA sequence that permits extrachromosomal maintenance of recombinant plasmids). This high-copy number, relatively stable plasmid was shown to be organized into nucleosomes comparable to typical yeast chromatin, containing a possible maximum of nine nucleosomes per circle. Therefore, YARp1 can be used to examine the structure of chromatin of both a chromosomally derived replicator and a functional gene. By mapping regions of micrococcal nuclease cleavage in chromatin versus purified DNA, we located the positions of protected regions on the circle with reference to six unique restriction sites. Measurements made on patterns of early digestion products indicated that a region of approximately 300 base pairs in the vicinity of ARS1 was strongly resistant to micrococcal nuclease. The remainder of the plasmid appeared to be associated with five positioned nucleosomes and two nonnucleosomal, partially protected regions on the bulk of the molecules. After similar extents of digestion, naked DNA did not exhibit an equivalent pattern, although some hypersensitive cleavage sites matched sites found in the chromatin. These results are consistent with the interpretation that the protected domains are aligned with respect to a specific site or sites on the small circular chromatin. PMID- 3018503 TI - Biologically active mutants with deletions in the v-mos oncogene assayed with retroviral vectors. AB - We have constructed retroviral expression vectors by manipulation of the Moloney murine leukemia virus genome such that an exogenous DNA sequence may be inserted and subsequently expressed when introduced into mammalian cells. A series of N terminal deletions of the v-mos oncogene was constructed and assayed for biological activity with these retroviral expression vectors. The results of the deletion analysis demonstrate that the region of p37mos coding region upstream of the third methionine codon is dispensable with respect to transformation. However, deletion mutants of v-mos which allow initiation of translation at the fourth methionine codon have lost the biological activity of the parental v-mos gene. Furthermore, experiments were also carried out to define the C-terminal limit of the active region of p37mos by the construction of premature termination mutants by the insertion of a termination oligonucleotide. Insertion of the oligonucleotide just 69 base pairs upstream from the wild-type termination site abolished the focus-forming ability of v-mos. Thus, we have shown the N-terminal limit of the active region of p37mos to be between the third and fourth methionines, while the C-terminal limit is within the last 23 amino acids of the protein. PMID- 3018504 TI - Molecular cloning and structural analysis of murine thymidine kinase genomic and cDNA sequences. AB - Two functional cytosolic thymidine kinase (tk) cDNA clones were isolated from a mouse L-cell library. An RNA blot analysis indicated that one of these clones contains a nearly full-length tk sequence and that LTK- cells contain little or no TK message. The nucleotide sequences of both clones were determined, and the functional mouse tk cDNA contains 1,156 base pairs. An analysis of the sequence implied that there is an untranslated 32-nucleotide region at the 5' end of the mRNA, followed by an open reading frame of 699 nucleotides. The 3' untranslated region is 422 nucleotides long. Thus, the gene codes for a protein containing 233 amino acids, with a molecular weight of 25,873. A comparison of the coding sequences of the mouse tk cDNA with the human and chicken tk genes revealed about 86 and 70% homology, respectively. We also isolated the tk gene from a mouse C57BL/10J cosmid library. The structural organization was determined by restriction mapping, Southern blotting, and heteroduplex analysis of the cloned sequences, in combination with a mouse tk cDNA. The tk gene spans approximately 11 kilobases and contains at least five introns. Southern blot analysis revealed that this gene is deleted in mouse LTK- cells, consistent with the inability of these cells to synthesize TK message. This analysis also showed that tk-related sequences are present in the genomes of several mouse strains, as well as in LTK- cells. These segments may represent pseudogenes. PMID- 3018505 TI - Myosin light-chain 1 and 3 gene has two structurally distinct and differentially regulated promoters evolving at different rates. AB - DNA fragments located 10 kilobases apart in the genome and containing, respectively, the first myosin light chain 1 (MLC1f) and the first myosin light chain 3 (MLC3f) specific exon of the rat myosin light chain 1 and 3 gene, together with several hundred base pairs of upstream flanking sequences, have been shown in runoff in vitro transcription assays to direct initiation of transcription at the cap sites of MLC1f and MLC3f mRNAs used in vivo. These results establish the presence of two separate, functional promoters within that gene. A comparison of the nucleotide sequence of the rat MLC1f/3f gene with the corresponding sequences from mouse and chicken shows that: the MLC1f promoter regions have been highly conserved up to position -150 from the cap site while the MLC3f promoter regions display a very poor degree of homology and even the absence or poor conservation of typical eucaryotic promoter elements such as TATA and CAT boxes; the exon/intron structure of this gene has been completely conserved in the three species; and corresponding exons, except for the regions encoding most of the 5' and 3' untranslated sequences, show greater than 75% homology while corresponding introns are similar in size but considerably divergent in sequence. The above findings indicate that the overall structure of the MLC1f/3f genes has been maintained between avian and mammalian species and that these genes contain two functional and widely spaced promoters. The fact that the structures of the alkali light chain gene from Drosophila melanogaster and of other related genes of the troponin C supergene family resemble a MLC3f gene without an upstream promoter and first exon strongly suggests that the present-day MLC1f/3f genes of higher vertebrates arose from a primordial alkali light chain gene through the addition of a far-upstream MLC1f-specific promoter and first exon. The two promoters have evolved at different rates, with the MLC1f promoter being more conserved than the MLC3f promoter. This discrepant evolutionary rate might reflect different mechanisms of promoter activation for the transcription of MLC1f and MLC3f RNA. PMID- 3018507 TI - Phosphatidylinositol kinase activities in normal and Rous sarcoma virus transformed cells. AB - The phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and diacylglycerol kinase activities in the plasma membrane-rich fraction of chicken embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus increased when the cells were shifted from the nonpermissive temperature, 41 degrees C, to the permissive temperature, 35 degrees C. Temperature shift from 35 to 41 degrees C decreased the lipid kinase activities in the membrane vesicles. These changes accompanied the changes observed in pp60v-src protein kinase activity. Thermal inactivation at 41 degrees C did not appreciably reduce PI and PIP kinase activities in membrane vesicles prepared from uninfected or Rous sarcoma virus-transformed cells, whereas pp60v-src protein kinase activity in the membrane vesicles was rapidly inactivated under the same conditions. These data suggest that pp60v-src may indirectly enhance PI and PIP phosphorylation but not directly contribute to this pathway. PMID- 3018508 TI - Burkitt lymphoma cell line carrying a variant translocation creates new DNA at the breakpoint and violates the hierarchy of immunoglobulin gene rearrangement. AB - The Burkitt lymphoma cell line KK124, which contains a reciprocal t(8;22) translocation, was shown to have rearranged in a region 3' to the c-myc proto oncogene on chromosome 8 and 5' to the lambda constant region on chromosome 22. The breakpoint was cloned and sequenced, revealing that c-myc and a portion of its 3' region abutted a complete lambda variable gene that had undergone V-J recombination. Since this cell line expresses kappa light chain, this lambda rearrangement violates the previously proposed hierarchy of immunoglobulin gene rearrangement. A novel duplication of normal chromosome 8 sequences was also found at the breakpoint. The first exon of c-myc and its flanking sequence from the translocated allele was sequenced and compared with a normal counterpart. Extensive mutation was found within the first exon in contrast to its 3' and 5' flanking regions. S1 nuclease analysis revealed that it was the translocated c myc being expressed and that there was a promoter shift from P2 to P1. The detailed structural analysis of this cell line provides clues concerning mechanisms of chromosomal translocation and c-myc deregulation in Burkitt lymphomas. PMID- 3018506 TI - ATTAAA as well as downstream sequences are required for RNA 3'-end formation in the E3 complex transcription unit of adenovirus. AB - We mapped the location of the E3A RNA 3' end site in the E3 transcription unit of adenovirus 2. The procedure used was nuclease-gel analysis with 32P-labeled RNA probes. The poly(A) addition sites were microheterogeneous and were located approximately 17 to 29 nucleotides downstream from an ATTAAA sequence. To identify the sequences that make up the E3A RNA 3' end signal, we constructed five viable virus mutants with deletions in or near the E3A RNA 3' end site. The mutants were analyzed for E3A RNA 3' end formation in vivo. No effect was observed from a 47-base-pair (bp) deletion (dl716) or a 72-bp deletion (dl714) located 22 and 19 nucleotides, respectively, upstream of the ATTAAA. In contrast, E3A RNA 3' end formation was abolished by a 554-bp deletion (dl708) that removes both the ATTAAA and the poly(A) addition sites, a 124-bp deletion (dl713) that removes the ATTAAA but leaves the poly(A) addition sites, and a 65-bp deletion (dl719) that leaves the ATTAAA but removes the poly(A) addition sites. These results indicate that the ATTAAA, as well as downstream sequences, including the poly(A) addition sites, are required for E3A RNA 3' end formation. PMID- 3018509 TI - New host cell system for regulated simian virus 40 DNA replication. AB - Transformed monkey cell lines (CMT and BMT) that inducible express simian virus 40 (SV40) T antigen from the metallothionein promoter have been isolated and characterized. Immunoprecipitation of pulse-labeled T antigen demonstrates a 5- to 12-fold increase in the rate of synthesis on addition of heavy-metal inducers to the culture medium. Radioimmunoassay of cell extracts indicates the accumulation of three- to fourfold more total T antigen after 2 days of induction by comparison with uninduced controls. A direct correlation was found between the level of T-antigen synthesis and the extent of SV40 DNA replication in inducible cells. Inducible BMT cells expressing a low basal level of T antigen were efficiently transformed by a vector carrying the neomycin resistance marker and an SV40 origin of replication. These vector sequences were maintained in an episomal form in most G418-resistant cell lines examined and persisted even in the absence of biochemical selection. Extensive rearrangements were observed only if the vector contained bacterial plasmid sequences. Expression of a protein product under the control of the SV40 late promoter in such vectors was increased after heavy-metal-dependent amplification of the template. These results demonstrate the ability of BMT cells to maintain a cloned eucaryotic gene in an amplifiable episomal state. PMID- 3018510 TI - Extrachromosomal replication of shuttle vectors in Dictyostelium discoideum. AB - We cloned a 12.3-kilobase (kb) endogenous plasmid, Ddp1, found in several wild type and laboratory strains of Dictyostelium discoideum into pBR322. The cloned plasmids have been used to cotransform D. discoideum cells with B10S, a transformation vector carrying a gene fusion conferring resistance to G418. Whereas B10S DNA alone appears to integrate in a tandem array, the cloned Ddp1 plasmids replicate extrachromosomally and are stably maintained in the absence of selection with an average copy number of 50 to 100 copies per cell. The Ddp1 derived plasmids can be directly recovered by transforming Escherichia coli with bulk nuclear DNA from these cells. Preliminary deletion analysis indicates that not all regions of Ddp1 are necessary for stable replication in D. discoideum. Several recombinant vectors which replicate extrachromosomally in D. discoideum were also isolated. One contains the Act6-neor gene fusion from B10S recombined into one of the cloned derivatives of Ddp1 and can be used to directly transform D. discoideum amoebae, selecting for G418 resistance. Another recombinant is only 5.6 kb and resulted from a deletion of a 16.6-kb cloned Ddp1 hybrid plasmid. An analysis of the vector DNAs present in clones derived from single D. discoideum transformants is also described. PMID- 3018511 TI - Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells. AB - We describe the construction of an adeno-associated virus (AAV) vector in which the coding sequence of the procaryotic gene neo is expressed under the control of the major AAV promoter p40. This AAV-neo vector allowed stable expression of neo as a dominant selective marker in mammalian cells by selection of cells which were resistant to the antibiotic geneticin (G418). When the vector was introduced into human (293 or HeLa) cell lines by a DNA transfection procedure, stable geneticin-resistant colonies were obtained. When the vector was first packaged into AAV particles and then introduced into cells via particle infection, geneticin-resistant cells were obtained at higher frequencies than those obtained by DNA transfection. In geneticin-resistant cells the AAV-neo vector was integrated at low copy number and could be rescued by subsequent infection with wild-type AAV and the helper adenovirus or, in some cases, by infection with adenovirus alone. The rescued AAV-neo vector could then be recovered as amplified unintegrated DNA from a Hirt lysate. These results demonstrate that AAV can be used as a transducing viral vector for stable integration and expression of a foreign gene in mammalian cells. The high frequency of integration and the ability to rescue the integrated vector suggest that this vector system may be useful for selecting genes from cDNA libraries. This vector may also be useful for introduction of genes into cells which are refractory to transfection in procedures such as those involving the use of CaPO4 or DEAE-dextran. PMID- 3018512 TI - Organization, primary structure, and evolution of histone H2A and H2B genes of the fission yeast Schizosaccharomyces pombe. AB - The histone H2A and H2B genes of the fission yeast Schizosaccharomyces pombe were cloned and sequenced. Southern blot and sequence analyses showed that, unlike other eucaryotes, Saccharomyces cerevisiae included, S. pombe has unequal numbers of these genes, containing two histone H2A genes (H2A-alpha and -beta) and only one H2B gene (H2B-alpha) per haploid genome. H2A- and H2B-alpha are adjacent to each other and are divergently transcribed. H2A-beta has no other histone gene in close proximity. Preceding both H2A-alpha and -beta is a highly conserved 19-base pair sequence (5'-CATCAC/AAACCCTAACCCTG-3'). The H2A DNA sequences encode two histone H2A subtypes differing in amino acid sequence (three residues) and size (H2A-alpha, 131 residues; H2A-beta, 130 residues). H2B-alpha codes for a 125 amino-acid protein. Sequence evolution is extensive between S. pombe and S. cerevisiae and displays unique patterns of divergence. Certain N-terminal sequences normally divergent between eucaryotes are conserved between the two yeasts. In contrast, the normally conserved hydrophobic core of H2A is as divergent between the yeasts as between S. pombe and calf. PMID- 3018515 TI - Transformation-defective mutant of avian myeloblastosis virus that is temperature sensitive for production of transforming protein p45v-myb. AB - We have characterized a mutant of avian myeloblastosis virus (strain GA907/7) that shows a reduced capacity to transform myelomonocytic cells at the nonpermissive temperature. Myeloblasts transformed by this mutant suffer a substantial decrease in the amount of the transforming protein p45v-myb when shifted from the permissive to the nonpermissive temperature. We presume that the 5- to 10-fold decrease in the amount of p45v-myb causes the loss of the transformed phenotype. The decrease is due to a reduction in the level of v-myb mRNA. Mutant GA907/7 thus provides genetic evidence that p45v-myb is the transforming protein of avian myeloblastosis virus and apparently represents an unusual defect in the production or stability of mRNA. PMID- 3018513 TI - DNA-mediated transfer and expression of a human DNA repair gene that demethylates O6-methylguanine. AB - Human liver DNA was transfected into CHO cells (mex-) along with pSV2gpt and colonies were selected first for resistance to mycophenolic acid and then to chloroethylnitrosourea. Transformants were obtained that contained approximately 10,000 molecules of O6-alkylguanine alkyltransferase (mex+) per cell. Their genome contained at least three copies of the human Alu sequence. PMID- 3018514 TI - An adenovirus 2-coded tumor antigen located on the endoplasmic reticulum and nuclear envelope is required for growth of transformed cells in Ca2+-deficient media. AB - Rat embryo cell lines containing the adenovirus 2 E1a region together with normal or mutant forms of the N-terminal half of the E1b region (HindIII G fragment) were generated by using a dominant selection marker, neo. Biochemically transformed cells containing a nonmutated HindIII G fragment proliferated more rapidly in Ca2+-deficient media, whereas cells containing a specific deletion within the E1b-encoded, 175-amino-acid (175R) (19-kilodalton) T-antigen gene and nontransformed cells grew at a slower rate. Furthermore, transformed cells that did not express the 175R T antigen and untransformed cells could not replicate their DNA efficiently in low-Ca2+ medium. Our results suggest that Ca2+ ions may provide an important stimulus for cell proliferation in adenovirus-transformed cells through a mechanism that involves the functions of the 175R T antigen. PMID- 3018517 TI - Type 1 human T-cell leukemia virus small envelope protein expressed in mouse cells by using a bovine papilloma virus-derived shuttle vector. AB - In an attempt to express the small (transmembrane) envelope protein p21e of type 1 human T-cell leukemia (lymphotrophic) virus (HTLV-1) exclusive of other viral gene products, we have constructed a recombinant plasmid clone (pMBE-1) in a bovine papillomavirus-derived mammalian expression vector. Mouse C127 cells transfected with the pMBE-1 plasmid expressed the introduced HTLV-1 viral gene(s) as demonstrated by Northern blot and indirect immunofluorescence with natural human antisera. The transfected mouse cells were injected into BALB/c mice, and a monoclonal antibody was recovered which specifically recognizes a 21-kilodalton protein present in HTLV-1 virions, indicating that the pMBE-1 plasmid encodes the small envelope protein. PMID- 3018516 TI - The bovine papillomavirus distal "enhancer" is not cis essential for transformation or for plasmid maintenance. AB - We constructed a mutant of bovine papillomavirus type 1 (BPV-1) DNA that lacked a transcriptional enhancer located 3' to the polyadenylation site of the early viral RNAs expressed in transformed cells. This mutant DNA, when separated from the procaryotic sequences, transforms mouse cells with an efficiency comparable to that of the full BPV-1 genome, and it exists as a stable multicopy plasmid in transformed cells. The BPV-1 distal enhancer suppresses the effects of a cis inhibitory element in pML2 sequences but is not essential for the expression of the viral genes involved in cellular transformation or plasmid maintenance. PMID- 3018518 TI - Behavior of a Drosophila melanogaster transposable element in Saccharomyces cerevisiae. AB - The Drosophila melanogaster transposable element 412 is transiently unstable in Saccharomyces cerevisiae when present on a freely replicating plasmid. The 412 element undergoes recombination to form two circular molecules, a 412 deletion plasmid and, presumably, a 412 circle. The 412 deletion plasmid contains a single long terminal repeat which most likely is the result of homologous recombination within the long terminal repeats. This recombination occurs at or shortly after transformation and is independent of both the RAD52 gene product and the Flp gene of 2 micron DNA. PMID- 3018520 TI - Deletions extending from a single Ty1 element in Saccharomyces cerevisiae. AB - Chromosomal rearrangements associated with one Ty1 element in the iso-1 cytochrome c (CYC1) region of Saccharomyces cerevisiae yeast cells were examined. Most of the rearrangements were deletions of the three linked genes, CYC1, OSM1, and RAD7, and resulted from recombination involving the single Ty1 element and a solo delta in the same orientation. These deletions differed by the number of Ty1 elements (zero, one, or two) remaining after deletion and by restriction site heterogeneities associated with these elements. A single Ty1 element remained at the deletion junction point much more frequently than no Ty1. Apparently the Ty1 associated delta element nearer to the solo delta was involved more often in recombination than the more distal Ty1-associated delta element. The restriction site data implicate gene conversion and suggest that site-specific recombination within the deltas, if occurring, is not the only mechanism of delta-delta recombination. Three other rearrangements bore deletions which began at the end of the Ty1 element and extended into regions not bearing Ty1 or delta sequences. Two of these deletions eliminated 7 kilobases of DNA, although they differed by an associated reciprocal translocation. The third involved a deletion of 14.7 kilobases of DNA associated with an overlapping inversion. PMID- 3018519 TI - Nucleotide sequence and expression in vitro of cDNA derived from mRNA of int-1, a provirally activated mouse mammary oncogene. AB - The mouse int-1 gene is a putative mammary oncogene discovered as a target for transcriptionally activating proviral insertion mutations in mammary carcinomas induced by the mouse mammary tumor virus in C3H mice. We have isolated molecular clones of full- or nearly full-length cDNA transcribed from int-1 RNA (2.6 kilobases) in a virus-induced mammary tumor. Comparison of the nucleotide sequence of the cDNA clones with that of the int-1 gene (A. van Ooyen and R. Nusse, Cell 39:233-240, 1984) shows the following. The coding region of the int-1 gene is composed of four exons. The splice donor and acceptor sites conform to consensus; however, at least two closely spaced polyadenylation sites are used, and the transcriptional initiation site remains ambiguous. The major open reading frame is preceded by an open frame 10 codons in length. The mRNA encodes a 41 kilodalton protein with several striking features--a strongly hydrophobic amino terminus, a cysteine-rich carboxy terminus, and four potential glycosylation sites. There are no differences in nucleotide sequence between the known exons of the normal and a provirally activated allele. The length of the deduced open reading frame was further confirmed by in vitro translation of RNA transcribed from the cDNA clones with SP6 RNA polymerase. PMID- 3018521 TI - Characterization of the human c-fms gene product and its expression in cells of the monocyte-macrophage lineage. AB - The McDonough strain of feline sarcoma virus contains an oncogene called v-fms whose ultimate protein product (gp140v-fms) resembles a cell surface growth factor receptor. To identify and characterize the protein product of the proto oncogene c-fms, antisera were prepared to the viral fms sequences and used to detect specific cross-reacting sequences in human choriocarcinoma cells (BeWo) known to express c-fms mRNA. Both tumor-bearing rat sera and a rabbit antiserum prepared to a segment of v-fms expressed in Escherichia coli detected a 140 kilodalton (kDa) glycoprotein in the BeWo cells. Tryptic fingerprint analysis of [35S]methionine-labeled proteins indicated that the viral fms proteins and the 140-kDa BeWo cell protein were highly related. This 140-kDa glycoprotein contained an associated tyrosine kinase activity in vitro and was labeled principally on serine after 32Pi metabolic labeling. These results suggest that the 140-kDa protein in BeWo cells is the protein product of the human c-fms proto oncogene. This conclusion is supported by the finding that a similar protein is detectable only in other human cells that express c-fms mRNA. These other human cells include adherent monocytes and the cell line ML-1, which can be induced to differentiate along the monocyte-macrophage pathway. This is in agreement with current thought that the c-fms proto-oncogene product functions as the CSF-1 receptor specific to this pathway. PMID- 3018523 TI - Enhancer-dependent expression of the rat preproinsulin gene in bovine papillomavirus type 1 vectors. AB - The effect of position in a bovine papillomavirus type 1 (BPV-1) vector on foreign gene expression was assessed with the rat preproinsulin (rI1) gene. The rI1 gene was inserted at each of the BPV-1/pML2d junctions in either transcriptional orientation in derivatives of the pdBPV-1(142-6) vector which consists of the BamHI linear genome of BPV-1 DNA cloned into pML2d. Transformed lines of C127 cells were established and assayed for rI1 gene expression. Cells containing the rI1 gene at the 3' end of the BPV-1 transforming region expressed rat proinsulin, whereas cells with the gene at the 5' end of the nontransforming region did not. Variability in the plasmid copy number or in the extent of DNA rearrangement could not account for this difference. We conclude that the expression of the rat preproinsulin gene (which is normally tissue specific for pancreatic islet cells) in C127 cells depends on the transcriptional activation afforded by viral enhancer sequences located at the 3' end of the transforming region. Intervening BPV-1 or pML2d sequences appear to block this enhancer mediated gene activation. In agreement with enhancer-dependent activation, a rat preproinsulin gene located in a blocked position (i.e., not adjacent to the BPV-1 enhancer) could be activated by the insertion of a DNA fragment containing the simian virus 40, Moloney murine sarcoma virus, or BPV-1 enhancer element adjacent to the rI1 gene. Thus, a gene which is normally not expressed in a particular cell may be activated when placed adjacent to a viral enhancer in a BPV-1 vector. PMID- 3018522 TI - Transformation by the v-fms oncogene product: role of glycosylational processing and cell surface expression. AB - The effect of glycosylational-processing inhibitors on the synthesis, cell surface expression, endocytosis, and transforming function of the v-fms oncogene protein (gp140fms) was examined in McDonough feline sarcoma virus-transformed Fischer rat embryo (SM-FRE) cells. Swainsonine (SW), a mannosidase II inhibitor, blocked complete processing, but an abnormal v-fms protein containing hybrid carbohydrate structures was expressed on the cell surface. SW-treated SM-FRE cells retained the transformed phenotype. In contrast, two glucosidase I inhibitors (castanospermine [CA] and N-methyl-1-deoxynojirimycin [MdN]) blocked carbohydrate remodeling at an early stage within the endoplasmic reticulum and prevented cell surface expression of v-fms proteins. CA-treated SM-FRE cells reverted to the normal phenotype. Neither SW, CA, nor MdN affected either endocytosis or the tyrosine kinase activity associated with the v-fms gene product in vitro. These results demonstrate the necessity of carbohydrate processing for cell surface expression of the v-fms gene product and illustrate the unique ability to modulate the transformed state of SM-FRE cells with the glycosylational-processing inhibitors CA and MdN. PMID- 3018525 TI - DNA-mediated transformation of Chlamydomonas reinhardi cells: use of aminoglycoside 3'-phosphotransferase as a selectable marker. AB - Using a modified vector, we developed a method for DNA-mediated transformation of Chlamydomonas reinhardi with increased efficiency. The vector contained the yeast 2 microns origin of replication as a heterologous replicon. The aminoglycoside 3' phosphotransferase (APH) gene linked to the simian virus 40 early promoter was used as an antibiotic selectable marker. The C. reinhardi transformants were resistant to 12 micrograms of G418 or 150 micrograms of kanamycin per ml. A quick blot mRNA analysis demonstrated the presence of RNase-sensitive transcripts from the APH gene in the transformants, suggesting that the acquisition of antibiotic resistance was due to the expression of the APH gene. Southern blot analysis revealed the presence of free plasmid DNA in the transformant. The transforming vector was recovered by transforming recipient bacteria with the total DNA extracted from the C. reinhardi transformant. PMID- 3018524 TI - Isolation of a simian virus 40 T-antigen-positive, transformation-resistant cell line by indirect selection. AB - In an attempt to identify cellular genes that might be involved in simian virus 40 (SV40) transformation, we have set out to isolate cells which express T antigen but are not transformed. SV40 DNA and the herpes simplex virus thymidine kinase gene were cotransfected into tk- 3T3 fibroblasts. Of 72 colonies screened that were resistant to hypoxanthine-aminopterin-thymidine, 57 were T antigen positive as judged by immunofluorescence. One of these lines, A27, had a normal growth phenotype in monolayer overgrowth and soft agar assays. It contained intact SV40 sequences that could be rescued by fusion to permissive cells. This rescued virus was fully capable of transforming nonpermissive cells to the same extent as did wild-type virus. The A27 cells, however, were not transformable by infection with SV40 or by transfection of SV40 DNA. It is likely that these cells were altered in a cellular function required for the establishment of the transformed state. PMID- 3018526 TI - Activation of Ha-ras p21 by substitution, deletion, and insertion mutations. AB - The transforming activity of naturally arising ras oncogenes results from point mutations that affect residue 12 or 61 of the encoded 21-kilodalton protein (p21). By use of site-directed mutagenesis, we showed that deletions and insertions of amino acid residues in the region of residue 12 are also effective in conferring oncogenic activity on p21. Common to these various alterations is the disruption that they create in this domain of the protein, which we propose results in the inactivation of a normal function of the protein. PMID- 3018527 TI - Inhibition of cell growth by monoclonal anti-transferrin receptor antibodies. AB - Five anti-murine transferrin receptor monoclonal antibodies have been characterized with respect to immunoglobulin class, effects on binding of transferrin, and effects on AKR1 lymphoma cell growth in vitro. The immunoglobulin M (IgM) antibodies, but not the IgG antibodies, prevent cell growth. We suggest that the profound effects of the IgM antibodies on cell growth are probably due to extensive cross-linking of cell surface receptors. In support of this, we are able to mimic the growth-inhibiting effects of the IgM antibodies by adding antiimmunoglobulin to an IgG antibody. By flow microfluorimetry, we show that an IgG antibody by itself induces up to a 10-fold downward regulation in the cell surface transferrin receptor, which is accompanied by accelerated receptor degradation. A similar downward regulation is seen in mutant cells resistant to growth inhibition by an IgM antibody, when grown in the selecting antibody. Wild-type cells grown in the presence of IgM antibody do not show receptor downward regulation. Inhibitory effects of antibody plus antiimmuoglobulin on mutant cells are also consistent with extensive cross linking causing inhibition of growth. PMID- 3018528 TI - A putative origin of replication of plasmids derived from Epstein-Barr virus is composed of two cis-acting components. AB - A genetic element of Epstein-Barr virus, oriP, when present on recombinant plasmids allows those plasmids to replicate and to be maintained in cells that express the Epstein-Barr virus-encoded nuclear antigen EBNA-1. Here we define the DNA sequences required for oriP activity. Two noncontiguous regions of oriP are required in cis for activity. One consists of approximately 20 tandem, imperfect copies of a 30-base-pair (bp) sequence. The other required region, approximately 1,000 bp away, is at most 114 bp in length and contains a 65-bp region of dyad symmetry. When present together on a plasmid, these two components supported plasmid replication even when the distance between them was varied or their relative orientation was altered, or both. When present alone on a plasmid that expresses a selectable marker, the family of 30-bp repeats efficiently conferred a transient drug-resistant phenotype in human 143 cells that is dependent on the presence of EBNA-1. This result leads us to suggest that EBNA-1 interacts with the 30-bp repeated sequence to activate oriP. To test whether the 30-bp repeats might cause the increased transient expression of drug resistance by enhancing transcription, the family of 30-bp repeats was tested for the ability to activate the simian virus 40 early promoter present in plasmid pA10CAT2 (Laimins, et al., Proc. Natl. Acad. Sci. U.S.A. 79:6453-6457). In this assay, the 30-bp repeats could activate the simian virus 40 early promoter in Raji cells, an EBNA-positive Burkitt's lymphoma cell line, but not detectably an EBNA-positive 143 cells in which oriP also functions. PMID- 3018529 TI - Transformation of cultured Drosophila melanogaster cells with a dominant selectable marker. AB - We have developed a method for the stable and efficient introduction of foreign DNA into Drosophila melanogaster tissue culture cells. A plasmid vector was constructed that carries the bacterial neomycin resistance gene under the transcriptional control of the copia transposable element long terminal repeat promoter. After calcium phosphate-DNA transfection, this vector rendered D. melanogaster cells resistant to the aminoglycoside G-418, a derivative of gentamicin. The vector DNA appeared to be integrated in long tandem arrays of 10 to 20 copies per cell and was stable for many generations in the absence of selection. To test the usefulness of this system for introducing nonselected DNA into D. melanogaster cells, a gene fusion between the P transposable element and the hsp70 promoter was inserted into the copia-neomycin resistance plasmid. After transfection and establishment of a G-418-resistant cell line, the hsp-P fusion gene was found to be efficiently transcribed after heat shock. PMID- 3018530 TI - A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome c. AB - The CYC1-239-O mutation in the yeast Saccharomyces cerevisiae produces a -His-Leu replacement of the normal -Ala-Gly- sequence at amino acid positions 5 and 6, which lie within a dispensable region of iso-1-cytochrome c; this mutation can accommodate the formation of a hairpin structure at the corresponding site in the mRNA. The amount of the altered protein was diminished to 20% of the wild-type level, whereas the amount of the mRNA remained normal. However, in contrast to the normal CYC1+ mRNA that is associated mainly with four to seven ribosomes, the bulk of the CYC1-239-O mRNA is associated with one to four ribosomes. These results suggest that the stable secondary structure within the translated region of the CYC1 mRNA diminishes translation by inhibiting elongation. PMID- 3018531 TI - Analysis of the mouse dhfr promoter region: existence of a divergently transcribed gene. AB - The use of murine dihydrofolate reductase (dhfr) gene amplification mutants enabled us to identify important structural and functional features of the dhfr promoter region. We found another transcription unit, at least 14 kilobases in size, which initiates within 130 base pairs of the major dhfr transcript and is transcribed divergently. The 5' ends of both transcripts were analyzed and found to have multiple initiation sites. The major dhfr transcript and the divergent transcript appear to share the same promoter region; the longer transcripts of the dhfr gene overlap with the divergent transcripts and use a different promoter region. The divergent transcript appears to code for a protein; an homologous sequence to its first exon is found in the corresponding location near the human dhfr gene. PMID- 3018532 TI - Analysis of an activatable promoter: sequences in the simian virus 40 late promoter required for T-antigen-mediated trans activation. AB - The late promoter of simian virus 40 (SV40) is activated in trans by the viral early gene product, T antigen. We inserted the wild-type late-promoter region, and deletion mutants of it, into chloramphenicol acetyltransferase transient expression vectors to identify promoter sequences which are active in the presence of T antigen. We defined two promoter activities. One activity was mediated by a promoter element within simian virus 40 nucleotides 200 to 270. The activity of this element was detectable only in the presence of an intact, functioning origin of replication and accounted for 25 to 35% of the wild-type late-promoter activity in the presence of T antigen. The other activity was mediated by an element located within a 33-base-pair sequence (simian virus nucleotides 168 to 200) which spans the junction of the 72-base-pair repeats. This element functioned in the absence of both the origin of replication and the T-antigen-binding sites and appeared to be responsible for trans-activated gene expression. When inserted into an essentially promoterless plasmid, the 33-base pair element functioned in an orientation-dependent manner. Under wild-type conditions in the presence of T antigen, the activity of this element accounted for 65 to 75% of the late-promoter activity. The roles of the 33-base-pair element and T antigen in trans-activation are discussed. PMID- 3018534 TI - In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene. AB - The human p53 gene was cloned and characterized by using a battery of p53 DNA clones. A series of human cDNA clones of various sizes and relative localizations to the mRNA molecule were isolated by using the human p53-H14 (2.35-kilobase) cDNA probe which we previously cloned. One such isolate, clone p53-H7 (2.65 kilobases), spans the entire human mature p53 mRNA molecule. Construction of the human cDNA clones in the pSP65 RNA transcription vector facilitated the generation of p53 transcripts by the SP6 bacteriophage RNA polymerase. The p53 specific RNA transcripts obtained without further processing were translated into p53 proteins in a cell-free system. By using this rapid in vitro transcription translation assay, we found that whereas clone p53-H7 (2.65 kilobases) coded for a mature-sized p53 protein, a shorter cDNA clone, p53-H13 (1.8 kilobases), dictated the synthesis of a smaller-sized p53 protein (45 kilodaltons). The p53 proteins synthesized in vitro immunoprecipitated efficiently with human-specific anti-p53 antibodies. Genomic analysis of human DNA revealed the presence of a single p53 gene residing within two EcoRI fragments. Heteroduplex analysis between the full-length cDNA clone p53-H7 and the cloned p53 gene indicated the presence of seven major exons. PMID- 3018533 TI - The level of expression of adenovirus type 2 transforming genes governs sensitivity to nonspecific immune cytolysis and other phenotypic properties of adenovirus 2-simian virus 40-transformed cell hybrids. AB - Syrian hamster embryo cells transformed by adenovirus type 2 (Ad2) or simian virus 40 (SV40) differ markedly in morphology, tumorigenicity, and susceptibility to in vitro lysis by nonspecific cytotoxic cells. Hybrid cells formed by fusing Ad2- and SV40-transformed Syrian hamster embryo cells may express only SV40 T antigens or both SV40 and Ad2 T antigens. Hybrids that express only SV40 T antigens are indistinguishable from the nonhybrid SV40-transformed phenotype, whereas hybrid cells that express T antigens from both viruses closely resemble the nonhybrid parental Ad2-transformed phenotype. Because these hybrid cells have been useful in the study of neoplastic transformation, we determined the amount of viral antigens that they accumulate in an attempt to correlate the level of expression of the transforming viral genes with some of their phenotypic properties. Hybrid cells that expressed proteins from both viruses showed reduced levels of SV40 T antigens compared with those of hybrid cells that did not express Ad2 T antigens. We also found that the production of several cellular proteins that influence cytomorphology was inhibited in hybrid and nonhybrid cells that expressed Ad2 T antigens, and the repression of these cellular proteins correlated with a change in cytomorphology from fibroblastic to spherical. Finally, we showed that the susceptibility of our hybrid cells to in vitro lysis by natural killer cells and activated macrophages, two putative host effector cells involved in defense against neoplasia, correlated closely with the level of expression of a 58,000-dalton Ad2 protein. The results reported here, together with the results of previous studies, indicate that the oncogenic potential of hybrid cells that express both Ad2 and SV40 antigens is extremely sensitive to Ad2 expression, whereas other phenotypic properties depend on Ad2 expression in a dose-dependent manner. PMID- 3018535 TI - Synthesis of predominantly unspliced cytoplasmic RNAs by chimeric herpes simplex virus type 1 thymidine kinase-human beta-globin genes. AB - The herpes simplex virus type 1 thymidine kinase (tk) gene lacks introns and produces stable mRNA in the absence of splicing. We have prepared a hybrid gene by placing the first exon, first intron (first intervening sequence, designated IVS1), and most of the second exon of the normal human beta-globin gene into the 3' untranslated region of the tk gene. Although this hybrid gene contains all globin sequences presumed necessary for the splicing of IVS1, predominantly, unspliced stable cytoplasmic RNA is produced in both long- and short-term expression assays. Moreover, stable unspliced cytoplasmic RNA is detected whether the intron is situated in a sense or an antisense orientation. Efficient splicing of IVS1 is obtained either by deleting the majority of tk coding sequences or by relocating the globin sequences from the 3' to the 5' untranslated region of the tk gene. PMID- 3018537 TI - Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants. AB - The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest. PMID- 3018536 TI - Suppressors of Saccharomyces cerevisiae his3 promoter mutations lacking the upstream element. AB - Transcription of the Saccharomyces cerevisiae his3 gene requires an upstream promoter element and a TATA element. A strain containing his3-delta 13, an allele which deletes the upstream promoter element but contains the TATA box and intact structural gene, fails to express the gene and consequently is unable to grow in medium lacking histidine. In this paper we characterize His+ revertants of his3 delta 13 which are due to unlinked suppressor mutations. Recessive suppressors in three different ope genes allow his3-delta 13 to be expressed at wild-type levels. In all cases, the suppression is due to increased his3 transcription. However, unlike the wild-type his3 gene, whose transcripts are initiated about equally from two different sites (+1 and +12), transcription due to the ope mutations is initiated only from the +12 site, ope-mediated transcription is regulated in a novel manner; it is observed in minimal medium, but not in rich broth. Although ope mutations restore wild-type levels of transcription, his3 chromatin structure, as assayed by micrococcal nuclease sensitivity of the TATA box, resembles that found in the his3-delta 13 parent rather than in the wild type strain. This provides further evidence that TATA box sensitivity is not correlated with transcriptional activation. ope mutations are pleiotropic in that cells have a crunchy colony morphology and lyse at 37 degrees C in conditions of normal osmolarity. ope mutations are allele specific because they fail to suppress five other his3 promoter mutations. We discuss implications concerning upstream promoter elements and propose some models for ope suppression. PMID- 3018538 TI - Promoter domains required for expression of plasmid-borne copies of the herpes simplex virus thymidine kinase gene in virus-infected mouse fibroblasts and microinjected frog oocytes. AB - A transient expression assay was used to measure the relative template activities of mutated tk genes in mouse L cells induced in trans by herpes simplex virus (HSV). In this assay, expression of the wild-type HSV type 1 tk gene is induced at least 200-fold by the superinfecting virus. Genetic lesions that were assayed include 5' deletions, clustered base substitutions, single base substitutions, intrapromoter inversions, and intrapromoter recombinants with the HSV type 2 tk gene. Roughly half of the mutations that were tested were found to weaken tk expression efficiency, and the remaining mutations did not alter expression. The spatial distribution of mutations that reduce expression efficiency in trans induced mouse fibroblasts facilitated the construction of a map of promoter domains. The most gene-proximal promoter domain is located between 16 and 32 base pairs (bp) upstream of the tk mRNA cap site and contains a TATA homology. Two more distally located promoter domains were mapped to discrete locations upstream from the TATA homology. One of these distal domains is located between 47 and 79 bp upstream from the mRNA cap site, and the other is located between 84 and 105 bp upstream from the tk gene. The boundaries of these three promoter domains, with one exception, coincided with the set of domains delineated previously in a frog oocyte microinjection assay. The concordant behavior of tk promoter mutants in microinjected frog oocytes and trans-induced mouse fibroblasts leads us to propose that recognition and activation of the HSV tk promoter is mediated by cellular transcription factors that are common to frogs and mice. PMID- 3018539 TI - Delineation of transcriptional control signals within the Moloney murine sarcoma virus long terminal repeat. AB - We identified three distinct elements within the Moloney murine sarcoma virus long terminal repeat that control transcription. The phenotypes of unidirectional deletion mutants of the long terminal repeat were assayed in microinjected frog oocytes and in transfected mouse fibroblasts. Steady-state levels of RNA bearing the same 5' terminus as the authentic Moloney murine sarcoma viral transcripts were measured by primer extension in assays that included a pseudo-wild-type internal reference. Mutant phenotypes define the boundaries of three functional elements. A region between 21 and 31 base pairs upstream from the mRNA cap site contains AT-rich sequences that function to establish the transcription start site. A second control element, termed the distal signal, lies between 31 and 84 base pairs upstream of the mRNA cap site. A CAT box consensus sequence is located at the 5' boundary of the distal signal. Additional components of the distal signal include a hexanucleotide sequence that is repeated four times. The distal signal augments transcription efficiency in oocytes but contributes only weakly to long terminal repeat-mediated expression in mouse fibroblasts. A third transcriptional control element lies between 156 and 364 base pairs upstream of the mRNA cap site. This element includes the 75-base-pair repeats previously identified as the Moloney murine sarcoma virus enhancer. In contrast to the distal signal, the Moloney murine sarcoma virus enhancer is crucial for significant expression in mouse fibroblasts but does not contribute to transcriptional expression in frog oocytes. PMID- 3018540 TI - Alternate utilization of two regulatory domains within the Moloney murine sarcoma virus long terminal repeat. AB - The Moloney murine sarcoma virus long terminal repeat (LTR) harbors two distinct positive activators of transcription, namely, a distal signal and an enhancer. In this report we demonstrate that infection by herpes simplex virus (HSV) can markedly affect the utilization of these two Moloney murine sarcoma virus transcription signals. We investigated the HSV-mediated trans-acting effects with two goals in mind: first, to gain insight into LTR function, and second, to probe the mechanisms used by HSV to establish its own transcription cascade. In mock infected cells, LTR-mediated expression was heavily dependent on the Moloney murine sarcoma virus enhancer but was effectively distal signal independent. HSV infection mobilized the use of the LTR distal signal and concomitantly alleviated enhancer dependence. Indeed, enhancer function may actually be inhibited by HSV trans-acting factors. These results suggest that the two positive control signals of the Moloney murine sarcoma virus LTR facilitate transcriptional activation by two different pathways. We further observed that the identity of the structural gene driven by the LRT, as well as the state of integration of a transfected template, can exert a substantial effect on the response of a template to HSV infection. According to these findings, we propose a tentative model to account for the initial temporal shift of the HSV transcriptional cascade. PMID- 3018541 TI - Expression of human beta-globin genes in transgenic mice: effects of a flanking metallothionein-human growth hormone fusion gene. AB - In an attempt to place a human beta-globin gene in an open chromatin domain regardless of its site of integration in the mouse genome, we microinjected into fertilized mouse eggs a construct in which the human beta-globin gene and a mouse metallothionein-human growth hormone fusion gene were juxtaposed and oriented in opposite directions. Mice that developed from injected eggs and that grew larger than normal were analyzed for human beta-globin mRNA. The globin genes were not expressed in erythroid tissue but were expressed with the same tissue specificity as metallothionein-human growth hormone. These results suggest that sequences which control metallothionein-human growth hormone gene expression are capable of stimulating the expression of a flanking gene in an orientation-independent and tissue-specific manner. As a control for this experiment, we deleted the metallothionein-human growth hormone transcription unit and noted that the human beta-globin gene then was expressed at high levels with erythroid tissue specificity. PMID- 3018542 TI - Clonal variants of PC12 pheochromocytoma cells with defects in cAMP-dependent protein kinases induce ornithine decarboxylase in response to nerve growth factor but not to adenosine agonists. AB - We have isolated and partially characterized three mutants of the pheochromocytoma line PC12 by using dibutyryl cyclic AMP (cAMP) as a selective agent. Each of these variants, A126-1B2, A208-4, and A208-7, was resistant to both dibutyryl cAMP and cholera toxin when cell growth was measured. In comparison to wild-type PC12 cells, each of these mutants was deficient in the ability to induce ornithine decarboxylase (ODC) in response to agents that act via a cAMP-dependent pathway. In contrast, each of these mutants induced ODC in response to nerve growth factor. To understand the nature of the mutations, the cAMP-dependent protein kinases of the wild type and of each of these mutants were studied by measuring both histone kinase activity and 8-N3-[32P]cAMP labeling. Wild-type PC12 cells contained both cAMP-dependent protein kinase type I (cAMP PKI) and cAMP-dependent protein kinase type II (cAMP-PKII). Regulatory subunits were detected in both soluble and particulate fractions. The mutant A126-1B2 contained near wild-type PC12 levels of cAMP-PKI but greatly reduced levels of cAMP-PKII. Furthermore, when compared with wild-type PC12 cells, this cell line had an altered distribution in ion-exchange chromatography of regulatory subunits of cAMP-PKI and cAMP-PKII. The mutant A208-4 demonstrated wild-type-level binding of 8-N3-[32P]cAMP to both type I and type II regulatory subunits, but only half the wild-type level of type II catalytic activity. The mutant A208-7 had type I and type II catalytic activities equivalent to those in wild-type cells. However, the regulatory subunit of cAMP-PKI occurring in A208-7 demonstrated decreased levels of binding 8-N3-[32P]cAMP in comparison with the wild type. Furthermore, all mutants were defective in their abilities to bind 8-N3-[32P]cAMP to the type II regulatory protein in the particulate fraction. Thus, cAMP-PK was altered in each of these mutants. We conclude that both cAMP-PKI and cAMP-PKII are apparently required to induce ODC in response to increases in cAMP. Finally, since all three mutants induced ODC in response to nerve growth factor, the nerve growth factor-dependent induction of OCD was not mediated by an increase in cAMP that led to an activation of cAMP-PK. These mutants will be useful in the elucidation of the many functions controlled by cAMP and nerve growth factor. PMID- 3018544 TI - In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster. AB - We describe a method for examining the in vivo distribution of a protein on specific eucaryotic DNA sequences. In this method, proteins are cross-linked to DNA in intact cells, and the protein-DNA adducts are isolated by immunoprecipitation with antiserum against the protein. Characterization of the DNA cross-linked to the precipitated protein identifies the sequences with which the protein is associated in vivo. Here, we applied these methods to detect RNA polymerase II-DNA interactions in heat-shocked and untreated Drosophila melanogaster Schneider line 2 cells. The level of RNA polymerase II associated with several heat shock genes increased dramatically in response to heat shock, whereas the level associated with the copia genes decreased, indicating that both induction of heat shock gene expression and repression of the copia gene expression by heat shock occur at the transcriptional level. Low levels of RNA polymerase II were present on DNA outside of the transcription units, and for at least two genes, hsp83 and hsp26, RNA polymerase II initiated binding near the transcription start site. Moreover, for hsp70, the density of RNA polymerase II on sequences downstream of the polyadenylate addition site was much lower than that observed on the gene internal sequences. Examination of the amount of specific restriction fragments cross-linked to RNA polymerase II provides a means of detecting RNA polymerase II on individual members of multigene families. This analysis shows that RNA polymerase II is associated with only one of the two cytoplasmic actin genes. PMID- 3018545 TI - Regulation of simian virus 40 gene expression in Xenopus laevis oocytes. AB - Expression of the simian virus 40 (SV40) early and late regions was examined in Xenopus laevis oocytes microinjected with viral DNA. In contrast to the situation in monkey cells, both late-strand-specific (L-strand) RNA and early-strand specific (E-strand) RNA could be detected as early as 2 h after injection. At all time points tested thereafter, L-strand RNA was synthesized in excess over E strand RNA. Significantly greater quantities of L-strand, relative to E-strand, RNA were detected over a 100-fold range of DNA concentrations injected. Analysis of the subcellular distribution of [35S]methionine-labeled viral proteins revealed that while the majority of the VP-1 and all detectable small t antigen were found in the oocyte cytoplasm, most of the large T antigen was located in the oocyte nucleus. The presence of the large T antigen in the nucleus led us to investigate whether this viral product influences the relative synthesis of late or early RNA in the oocyte as it does in infected monkey cells. Microinjection of either mutant C6 SV40 DNA, which encodes a large T antigen unable to bind specifically to viral regulatory sequences, or deleted viral DNA lacking part of the large T antigen coding sequences yielded ratios of L-strand to E-strand RNA that were similar to those observed with wild-type SV40 DNA. Taken together, these observations suggest that the regulation of SV40 RNA synthesis in X. laevis oocytes occurs by a fundamentally different mechanism than that observed in infected monkey cells. This notion was further supported by the observation that the major 5' ends of L-strand RNA synthesized in oocytes were different from those detected in infected cells. Furthermore, only a subset of those L-strand RNAs were polyadenylated. PMID- 3018543 TI - Activation of immediate-early, early, and late promoters by temperature-sensitive and wild-type forms of herpes simplex virus type 1 protein ICP4. AB - To better define the activities on herpes simplex virus type 1 gene expression of temperature-sensitive and wild-type forms of the transcriptional regulatory protein ICP4, regulatory sequences from immediate-early, early, and late herpes simplex virus genes were fused to the gene for chloramphenicol acetyltransferase (CAT). These constructs were used in trans induction and cotransfection experiments with wild-type and temperature-sensitive mutant alleles of ICP4. The ICP4 genes used in this study were cloned from the KOS strain (wild type) and two phenotypically distinct temperature-sensitive ICP4 mutants, tsB32 and tsL14 (DeLuca et al., J. Virol. 52:767-776, 1984), both alone and in conjunction with three other immediate-early genes. The latter series of plasmids was used to assess the influence of additional immediate-early gene products on gene expression in the presence of a given ICP4 allele. The results of this study demonstrate that the phenotypes of these ICP4 mutants observed in cell culture at the nonpermissive temperature were determined in part by activities associated with the mutant ICP4 polypeptides and that these activities differed from those of wild-type ICP4. Low levels of wild-type ICP4 had a marginal but reproducible stimulatory effect on immediate-early CAT gene expression, especially the pIE4/5CAT chimera. This effect was diminished with increasing quantities of ICP4, suggesting an inhibitory role for the wild-type form of the protein. The ICP4 mutants had a strong stimulatory effect on immediate-early CAT expression, consistent with their phenotypes at 39 degrees C. The mutant forms of the ICP4 polypeptide differed in their ability to induce CAT activity from an early chimeric gene. Thus, the tsL14 form of ICP4 was effective in early gene induction (i.e., ptkCAT was induced), whereas the ICP4 derived from tsB32 was slightly inhibitory. Cotransfection of tsB32 ICP4 simultaneously with other immediate early genes resulted in a marginal increase in ptkCAT induction. This induction was enhanced when the gene for ICP4 was inactivated by restriction enzyme cleavage, substantiating the inhibitory effect of the tsB32 form of ICP4. The two mutant ICP4 genes (tsB32 and tsL14) were unable to trans-activate either of the late CAT constructs (p5CAT and pL42CAT) tested. Cotransfecting tsL14 ICP4 with the other immediate-early genes resulted in activation of p5CAT but not pL42CAT. Taken together, these studies demonstrate that (i) low levels of wild-type ICP4 have stimulatory effect on immediate-early promoters and that higher concentrations of wild-type ICP4 have an inhibitory effect on these promoters, (ii) isolated mutant form of ICP4 exhibit activities that reflect the phenotypes of the mutants from which they were isolated, and (iii) immediate-early gene products other than ICP4 are involved in determining the distinct phenotypes of the two mutants at 39 degrees Celsius. PMID- 3018547 TI - Reproducible and variable genomic rearrangements occur in the developing somatic nucleus of the ciliate Tetrahymena thermophila. AB - We analyzed the extent, reproducibility, and developmental control of genomic rearrangements in the somatic macronucleus of the ciliate Tetrahymena thermophila. To exclude differences caused by genetic polymorphisms, we constructed whole-genome homozygotes, and we compared the homozygous progeny derived from single macronuclear differentiation events. This strategy enabled us to identify a novel form of variable rearrangement and to confirm previous findings that rearranged sequences occur at a high frequency in the Tetrahymena genome. Rearrangements studied here were deletions of both unique and interchromosomally dispersed repetitive DNA sequences involving DNA rejoining of internal, nontelomeric regions of macronuclear DNAs. We showed that although rearrangements of some sequence classes are reproducible among independently developed macronuclei, other specific sequence classes are variably rearranged in macronuclear development. The variable somatic genomes so produced may be the source of phenotypically variant cell lines. PMID- 3018546 TI - Recombination hot spot in the human beta-globin gene cluster: meiotic recombination of human DNA fragments in Saccharomyces cerevisiae. AB - We describe a novel system for the analysis of sequence-specific meiotic recombination in Saccharomyces cerevisiae. A comparison of three adjacent restriction fragments from the human beta-globin locus revealed that one of them, previously hypothesized to contain a relative hot spot for genetic recombination, engages in reciprocal exchange during yeast meiosis significantly more frequently than either of the other two fragments. Removal of the longest of four potential Z-DNA-forming regions from this fragment does not affect the high frequency of genetic recombination. PMID- 3018548 TI - Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells. AB - Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules. PMID- 3018549 TI - Common regulatory elements control gene expression from polyoma early and late promoters in cells transformed by chimeric plasmids. AB - In a previous report we showed that transcripts initiating from the late promoter of integrated polyoma plasmids could be detected at significant levels when neomycin resistance (neo) coding sequences were linked to this promoter. In this report we used chimeric plasmids that contain either a limited portion of the polyoma genome or deletions within the polyoma noncoding regulatory region to determine the sequence requirements for late promoter activity in this system. We observed no absolute requirement for either the polyoma early coding region or the origin of DNA replication for Neo-r colony formation. We were therefore able to independently assess the effects of deletions in the polyoma enhancer region on gene activity in both the early and late directions. We measured the ability of cells transfected with plasmids containing deletions in this region to form colonies in either semisolid or G418-containing medium under nonreplicative conditions. Our results indicate that either the PvuII 4 fragment, which contains the simian virus 40 core enhancer sequence, or a region from nucleotides 5099 to 5142, which contains the adenovirus type 5 E1A core enhancer sequence, can be deleted without significantly affecting gene expression in either direction. However, a deletion of nucleotides 5099 to 5172 reduced activities to similar extents in both directions, and a plasmid containing a larger deletion of nucleotides 5055 to 5182 showed a further reduction in activity. Although having no effect by itself, a second origin region deletion of nucleotides 5246 to 127 when present in these mutant backgrounds caused either a further reduction or elimination, respectively, of both G418 and agar colony-forming ability, suggesting the presence of an additional common regulatory element within this region. A comparison of 5' ends of neo transcripts present in cells transformed by these plasmids suggested that the reduction in activity was due to deletion of regulatory rather than structural elements of the late promoter. Our results indicate that the noncoding region of polyoma contains multiple complementing regulatory elements that control the level of both early and late gene expression. PMID- 3018550 TI - Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells. AB - Cultured animal cells rearrange foreign DNA very efficiently by homologous recombination. The individual steps that constitute the mechanism(s) of homologous recombination in transfected DNA are as yet undefined. In this study, we examined the topological requirements by using the genome of simian virus 40 (SV40) as a probe. By assaying homologous recombination between defective SV40 genomes after transfection into CV1 monkey cells, we showed that linear molecules are preferred substrates for homologous exchanges, exchanges are distributed around the SV40 genome, and the frequency of exchange is not diminished significantly by the presence of short stretches of non-SV40 DNA at the ends. These observations are considered in relation to current models of homologous recombination in mammalian cells, and a new model is proposed. The function of somatic cell recombination is discussed. PMID- 3018553 TI - A molecular mechanism for the activation of the first component of complement by immune complexes. AB - The proposed activation mechanism is based upon several key concepts, including the "S"-structure for the folding of the C1r2C1s2 tetramer among the C1q arms [Poon, et al., J. molec. Biol. 168, 563-577 (1983)]; the locations of the catalytic domains on the tetramer and the resulting functional relevance of the "S"-structure [Colomb et al., Phil. Trans. R. Soc. B306, 282-292 (1984)]; the structure of C1-inhibitor [Odermatt et al., FEBS Lett. 131, 283-289 (1981)]; and the control of C1 activation by C1-inhibitor [Ziccardi, J. Immun. 128, 2505-2508 (1982)]. The proposed activation mechanism has four main features: steric exclusion of C1-inhibitor from C1 when it binds to an immune complex; signal generation through multivalent binding of the C1q heads to an irregularly arranged cluster of antibody Fc regions, and signal transmission through the movement of the stiff C1q arms about their semi-flexible joints, causing distortion of the symmetrical cone of C1q arms; induction of rapid activation by a shift in equilibrium favoring the autocatalytic conformation of C1r2C1s2; and release of the activated C1s from the C1q arms, so that the ends of the tetramer are free for interaction with C4 and C2 and C1-inhibitor, and the C1q subcomponent becomes more flexible, allowing access of C1-inhibitor to C1r. PMID- 3018551 TI - Identification of sequences in the herpes simplex virus thymidine kinase gene required for efficient processing and polyadenylation. AB - The herpes simplex virus (HSV) type 1 thymidine kinase gene (tk) was resected from its 3' end with BAL 31 exonuclease. Two sets of plasmids were isolated that lacked information distal to the two copies of the hexanucleotide 5'-AATAAA-3' located at the 3' end of the HSV tk gene. The presence of a simian virus 40 origin of DNA replication in each plasmid facilitated analysis of patterns of transcription in transfected Cos-1 monkey cells. Transcription analyses were performed with an S1 nuclease protection assay. Efficient processing and polyadenylation at the normal site still occurred when all sequences more than 44 or 46 base pairs (bp) downstream from the first AATAAA were removed (pTK311R/SV010 and pTK209R/SV010). Removal of an additional 7 bp (pTK312R/SV010) decreased the amount of tk mRNA processed at that normal site, and tk mRNA polyadenylated at a cryptic site within pBR322 sequences began to appear. The normal processing and polyadenylation site was not used at all when an additional 12 bp was removed (pTK314R/SV010); the small amount of tk mRNA produced was processed and polyadenylated at the cryptic pBR322 site. The region of the tk gene critical for efficient processing and polyadenylation of tk mRNA is located 20 to 38 bp downstream from the first AATAAA, distal to the polyadenylation site, and as RNA can form a stem-loop structure containing AAUAAA. Similar G + T-rich elements were located in DNA fragments which substitute efficiently for the HSV tk processing and polyadenylation signal and were not found in AATAAA-containing DNA fragments which substitute inefficiently for the HSV tk signal. PMID- 3018554 TI - [Postnatal development of the sympathoadrenergic system in premature and newborn infants]. AB - Autonomic regulatory mechanisms and some metabolic functions are predominantly influenced by the sympathetic nervous system. Premature and mature newborns show a high variability and a low adaptability of the sympathetic nervous system. In order to investigate whether sympathetic systems are completely developed at birth or underly a postnatal maturation process we have determined plasma levels of adrenaline and noradrenaline as well as the density and affinity of alpha- and beta-adrenoceptors on thrombocytes and lymphocytes in pre- and mature newborns and adults. Catecholamines were determined by means of a radioenzymatic method, number and affinity of adrenoceptors by use of the radioactive labelled antagonists 3-H-Yohimbine and 125-I-Cyano-Pindolol. Beta-adrenoceptor responsiveness was assessed by measurement of cyclic AMP in lymphocytes before and after stimulation of beta-adrenoceptors by isoprenaline. A linear relationship occurred between the gestational age and the number of adrenoceptors on lymphocytes, whereas the alpha-adrenoceptors on thrombocytes showed no age dependency. The basal content of cyclic AMP and the accumulation in response to beta-adrenoceptor stimulation by isoprenaline was significantly lower in newborns than in adults. Since noradrenaline and adrenaline plasma levels were not significantly different in newborns and adults it is assumed that the low density of beta-adrenoceptors in premature and mature newborns is due to a postnatal maturation and not to a "down regulation" by circulating catecholamines. Our results suggest that an immaturity of beta-adrenoceptors is involved in the poorly developed adaptive control in newborns. PMID- 3018552 TI - Polyomavirus mutation that confers a cell-specific cis advantage for viral DNA replication. AB - The structural and biological properties of a polyomavirus mutant selected in Friend erythroleukemic cells were investigated. The growth efficiency of this mutant (PyFL78) was compared with that of the parental PyA2 strain by a growth competition assay in Friend erythroleukemic and 3T3 (or 3T6) cell lines. The results reveal that PyFL78 displays a cis-acting growth advantage over the PyA2 parental strain in Friend erythroleukemic cells but not in 3T3 or 3T6 cells. This cell-specific cis advantage is shown to be due to modifications within the polyomavirus noncoding regulatory region. PMID- 3018555 TI - Mutagenic and cytotoxic effects of oxygen free radicals generated by methylviologen (paraquat) on Escherichia coli with different DNA-repair capacities. AB - Investigations were carried out to examine the mutagenic and cytotoxic effects of oxygen free radicals on E. coli. E. coli B strains with different DNA-repair capacities were exposed to methyl viologen, commonly called paraquat (1,1' dimethyl-4,4'-bipyridinium dichloride, MV), which has been shown to act as an intracellular generator of superoxide radicals. The results obtained were as follows: The cytotoxicity of MV in E. coli was dioxygen-dependent and due to the extent of intracellular generation of superoxide radicals. Cells containing higher levels of superoxide dismutase were more resistant to the toxic effect of MV. The cytotoxicity of MV was greater in DNA repair-deficient E. coli, Bs-1(exrA uvrB), NG30(recA) and R15(polA), than in DNA-repair-proficient strains (B/r and H/r30) and Hs30 (uvrB). MV was found to be mutagenic to E. coli H/r30 and Hs30 under aerobic conditions. The mutation frequencies to streptomycin resistance and to arginine prototrophy increased with the dose of MV in both strains. However, E. coli NG30 was unmutable by MV. The mutation induction did not occur under anaerobic conditions. The expression of the umu operon in E. coli was induced by MV under aerobic conditions. From these results, it was concluded that superoxide radicals intracellularly generated by MV include DNA damage, which causes cytotoxicity and mutation induction in E. coli, and that DNA damage induced by oxygen radicals is repairable by at least recA, polA and exrA(lexA) gene controlled mechanisms. PMID- 3018556 TI - Introduction, rescue and expression of plasmid genes in mammalian cells and Escherichia coli. AB - A shuttle-vector system is described for the study of mutational specificity in mammalian cells. Using a plasmid (pGKTK) carrying the E. coli galactokinase gene (gk) and the herpes simplex virus thymidine kinase gene (tk), we demonstrate the introduction of a foreign gene into the chromosome of a mammalian cell (TK- mouse fibroblasts) and its efficient rescue back into E. coli. This system makes use of two genes, each of which can expressed in both E. coli and mammalian cells, thereby permitting one marker to be the mutational target and the other to maintain stable integration in the host. In addition, expression of both genes in bacteria makes it possible to deletion map mutants to facilitate their sequencing. In the case of a putative single-copy transformant (T8), about half of the rescued plasmids are identical in size and restriction pattern to the original plasmid. Each of these expressed the tk gene, indicating the fidelity of the rescue system. PMID- 3018557 TI - X-rays mutate human lymphoblast cells at genetic loci that should respond only to point mutagens. AB - We have demonstrated that X-rays induce mutations at 4 of 5 genetic loci. 2 of these loci, which code for a mRNA synthesis factor (resistance to 5,6 dichlororibofuranosylbenzimidazole) and tubulin (resistance to podophyllotoxin), are "small-marker" loci, in that they theoretically respond only to mutations which eliminate a toxin-binding site while leaving the major function of the protein intact. Thus mutations induced by X-rays in these two loci are most likely due to base-pair substitution-type alterations. X-Rays did not induce mutations in the Na+/K+ ATPase (resistance to ouabain), another small-marker locus. Two other loci, hypoxanthine guanine phosphoribosyl transferase (resistance to 6-thioguanine) and thymidine kinase (resistance to trifluorothymidine), are "whole-gene" targets in that they theoretically respond to a wide variety of mutagenic changes. X-Rays induced dose-dependent increases in mutant fraction at both of these loci. Ethyl methanesulfonate (EMS), an agent thought to produce mutations primarily through a base-pair substitution mechanism, induced mutations at all genetic loci tested. The pattern of mutations at the small-marker loci induced by EMS was different than that induced by X rays, suggesting that the specificities of the mutagens and/or of the loci are different. PMID- 3018558 TI - Differentiation of Schistosoma haematobium from related species using cloned ribosomal RNA gene probes. AB - The ribosomal RNA (rRNA) gene units of Schistosoma mansoni (lateral spined eggs) and six species of schistosomes with terminal spined eggs (S. haematobium, S. curassoni, S. bovis, S. intercalatum, S. margrebowiei and S. mattheei) have been studied. The schistosome rRNA gene unit consists of a regular interspersion of the two genes encoding the large and small rRNA units with two spacers. The large spacer is not transcribed while the small spacer is part of the transcription unit. Variation in the rRNA gene unit of the species studied is demonstrated and takes three forms: First, variation in DNA sequence leads to both reduced homology in the spacer regions between species and loss or gain of restriction sites. Second, variation in the length of the transcribed spacer is demonstrated and DNA insertions of 0.2 kilobases (kb) and 0.1 kb are observed in S. mattheei and S. margrebowiei, respectively. Third, the length of the non-transcribed spacer region varies between species. S. haematobium has a 0.5 kb deletion in this region, while that of S. margrebowiei contains varying numbers of a 0.4 kb DNA insert. These interspecific variations have been shown to be conserved within species. Analysis of the rRNA genes by DNA hybridisation techniques therefore serves as a means of species identification, whereby it is possible to differentiate S. haematobium from other schistosome species with terminal spined eggs. Similarly, S. margrebowiei and S. mattheei may be clearly distinguished, although no major variation has been detected between S. curassoni, S. bovis and S. intercalatum. All these species differ from S. mansoni by the absence of certain restriction sites in the non-transcribed spacer. PMID- 3018560 TI - Maternal antibodies against fetal cardiac antigens in congenital complete heart block. AB - An immunologic basis for congenital heart block has been proposed previously. To investigate the association between congenital heart block and maternal antibodies capable of crossing the placenta, we used immunofluorescence to examine serum samples from 41 mothers and 8 affected children, together with serum from controls, for antibodies to fetal cardiac tissue. Twenty-one mothers (51 percent) had IgG antibody reactive with fetal heart tissue, as compared with only 9 of 94 controls (10 percent; P less than 0.001). Three of 8 affected babies, but none of 50 healthy babies, had similar antibodies. The antibodies reacted with all myocardial tissue and were not directed specifically to the conduction system. They also reacted with other fetal tissues and could be distinguished from nuclear and smooth-muscle autoantibodies. We also observed a higher occurrence of antibodies to cytomegalovirus, but not to Epstein-Barr virus, in these mothers. Autopsy specimens from babies with congenital heart block examined by immunoperoxidase staining showed deposition of immunoglobulin and complement components in all cardiac tissues. These findings strengthen the case implicating immune reactivity related to maternal antibody in the development of some but not all cases of congenital heart block. PMID- 3018559 TI - Hydrolysis of phosphoproteins and inositol phosphates by cell surface phosphatase of Leishmania donovani. AB - Leishmania donovani promastigotes contain intense tartrate-resistant cell surface acid phosphatase (ACP1) which blocks superoxide anion production by activated human neutrophils [A.T. Remaley et al. (1984) J. Biol. Chem, 259, 11173-11175]. An extensively purified preparation of ACP1 dephosphorylates several phosphoproteins which are phosphorylated at serine residues; these include: pyruvate kinase (Km 1.6 microM; Vmax 71.4 U (mg protein)-1), phosphorylase kinase (Km 0.076 microM; Vmax 5.4 U (mg protein)-1) and histones (Km 4.86 microM; Vmax 2.2 U (mg protein)-1). However, the specific activity of the leishmanial phosphatase on these phosphoproteins is very low as compared to other phosphoprotein phosphatases. The phosphatase activity of ACP1 was also low on phosphohistone phosphorylated at tyrosine residues. Phosphatidylinositol-4,5 diphosphate (PIP2) and inositoltriphosphate (IP3) were also tested as ACP1 substrates. PIP2 was hydrolyzed rapidly by ACP1. The rate of hydrolysis of PIP2 was higher at pH 6.8 (Km 2.35 microM; Vmax 107 X 10(3) U (mg protein)-1) than at pH 5.5 (Km 4.16 microM; Vmax 71 X 10(3) U (mg protein)-1). 32P-labeled IP3 was also a substrate for ACP1; the hydrolysis products consisted of a mixture of inositoldiphosphate and inositolmonophosphate. ACP1 and ten other phosphatases were tested for their ability to dephosphorylate proteins and to inhibit O2- production by stimulated human neutrophils. There was no correlation between the protein phosphatase activity of the acid- and alkaline phosphatases and their ability to block neutrophil O2- production. The results indicate that ACP1 probably blocks the production of reduced oxygen intermediates by a mechanism that does not involve dephosphorylation of phosphoproteins; however, the possibility that the parasite's phosphatase affects phagocyte metabolism by degrading PIP2 or IP3 should be considered. PMID- 3018561 TI - Conjugated estrogens for the management of bleeding associated with renal failure. AB - Bleeding is a major complication of uremia. Both cryoprecipitate and desmopressin effectively shorten the prolonged bleeding time and favorably influence clinical bleeding, but the former carries the risk of transmitting blood-borne infectious diseases, and both cryoprecipitate and desmopressin have a short duration of action. Preliminary evidence has suggested that estrogens may be useful, and we therefore performed a randomized, double-blind, crossover trial comparing the effect of conjugated estrogens with that of placebo on hemorrhagic tendencies and the bleeding time in six patients with uremia who were on maintenance hemodialysis. Five daily infusions of placebo or conjugated estrogens were administered at the beginning of one-month trial periods. Estrogen shortened the bleeding time in all six patients. The effect was detectable six hours after the first infusion, reached its maximum in all patients between days 5 and 7, and lasted for 14 days. By day 16 after the last infusion, the bleeding time had returned to base line in four of the six patients. No side effects were noted during or after estrogen infusion. Estrogens did not influence the circulating level of von Willebrand factor or change its multimeric structure. Moreover, the defective platelet aggregation and thromboxane formation observed in the patients were not corrected by estrogens. We conclude that conjugated estrogens are an adequate alternative to cryoprecipitate or desmopressin for the treatment of bleeding associated with renal failure, especially when a longer duration of action is needed and immediate onset of the effect is not essential. The mechanism of action of estrogens remains to be clarified. PMID- 3018562 TI - CDC's definition of AIDS. PMID- 3018563 TI - Defective regulation of Epstein-Barr virus infection in patients with AIDS or AIDS-related disorders. PMID- 3018564 TI - Reversibility of severe hypothyroidism with supplementary iodine in patients with endemic cretinism. AB - The reversibility of thyroid dysfunction in children with endemic cretinism treated with supplemental iodine is unknown. To study this question we conducted a five-month follow-up of 51 patients with cretinism (age 14 and below), who were randomly assigned to treatment (0.5 ml of intramuscular iodized oil) and control groups. The geometric mean initial serum level of thyrotropin (223 microU per milliliter; SD, 97 to 513) and the mean (+/- SD) initial serum level of thyroxine (1.0 +/- 1.2 micrograms per deciliter) indicated that all patients had severe hypothyroidism. Within one month after receiving the iodized oil, 13 of 14 of the younger patients (less than 4 years) and 1 of 9 of the older patients (4 to 14 years; P less than 0.001) had thyrotropin values below 20 microU per milliliter. Five months after treatment, the levels of thyrotropin had decreased and those of thyroxine had increased in all children, but greater changes occurred in the 13 younger patients than in the 14 older patients. The mean levels of thyrotropin were 2 microU per milliliter (SD, 0.6 to 6) vs. 38 microU per milliliter (SD, 11 to 132; P less than 0.001), and the mean (+/- SD) levels of thyroxine were 13.1 +/- 2.8 vs. 8.1 +/- 4.6 micrograms per deciliter (P less than 0.001). In the untreated group, 3 of the 9 younger patients and none of the 15 older patients recovered normal thyroid function within five months. We conclude that iodine supplementation restored a biochemically euthyroid state in all younger children with cretinism but only some of the older children. In addition, some younger patients became euthyroid without iodine supplementation. PMID- 3018567 TI - Platelet and vascular function during fish-oil administration. PMID- 3018568 TI - Case 52-1985: oat-cell carcinoma of the lung. PMID- 3018566 TI - Comparison of captopril and enalapril in patients with severe chronic heart failure. AB - To evaluate the concept that long duration of action is an advantageous property of angiotensin-converting enzyme inhibitors in the treatment of severe heart failure, we randomly assigned 42 patients to therapy with either a short-acting inhibitor (captopril, 150 mg daily) or a long-acting inhibitor (enalapril, 40 mg daily) for one to three months while concomitant therapy with digoxin and diuretics was kept constant. The treatment groups had similar hemodynamic and clinical characteristics at base-line evaluation and similar initial responses to converting-enzyme inhibition. During long-term therapy, captopril and enalapril produced similar decreases in systemic blood pressure, but the hypotensive effects of enalapril were more prolonged and persistent than those of captopril. Consequently, although the patients in both groups improved hemodynamically and clinically during the study, serious symptomatic hypotension (syncope and near syncope) was seen primarily among those treated with enalapril. Sustained hypotension also probably accounted for the decline in creatinine clearance (P less than 0.05) and the notable retention of potassium (P less than 0.05) observed in the patients treated with enalapril but not in those treated with captopril. We conclude that when large, fixed doses of converting-enzyme inhibitors are used in the treatment of patients with severe chronic heart failure, long-acting agents may produce prolonged hypotensive effects that may compromise cerebral and renal function, and thus they may have disadvantages in such cases, as compared with short-acting agents. PMID- 3018565 TI - Failure of antepartum maternal cultures to predict the infant's risk of exposure to herpes simplex virus at delivery. AB - In 414 pregnant women with a history of recurrent genital herpes simplex infection, we studied the correlation between asymptomatic viral shedding in late pregnancy and at the time of delivery. Antepartum cultures for asymptomatic reactivation of herpes simplex virus were positive in 17 of the 414 women (4.1 percent). None of these women had positive cultures at the time of delivery. Cultures of specimens obtained at delivery from 5 of 354 asymptomatic mother infant pairs (1.4 percent) were positive for asymptomatic excretion of herpes simplex virus. None of these women had had antepartum cultures that documented asymptomatic excretion of herpes simplex virus, despite the fact that culturing was repeatedly performed during the four weeks before delivery. Asymptomatic shedding of herpes simplex virus occurred with the same frequency at delivery, whether or not any episodes of symptomatic recurrence were noted during the pregnancy (1.4 vs. 1.3 percent). We conclude that antepartum maternal cultures do not predict the infant's risk of exposure to herpes simplex virus at delivery. PMID- 3018569 TI - A theory on the nature of physiologic opiate dependence: a formal statement. PMID- 3018571 TI - Electrophilic affinity ligands for the phencyclidine (PCP) receptor. PMID- 3018572 TI - Studies of kappa agonist. PMID- 3018570 TI - Central infusion of rats with agents selective for different types of opioid receptor. PMID- 3018574 TI - A stage model of HTLV-III LAV infection in intravenous drug users. PMID- 3018573 TI - The central and peripheral effects of delta-9-tetrahydrocannabinol on gastrointestinal transit in mice. PMID- 3018575 TI - Marijuana effects and behavioral contingencies. PMID- 3018576 TI - Pharmacology of benzodiazepine antagonists. PMID- 3018577 TI - Evaluation of new compounds for opioid activity (1985). PMID- 3018578 TI - Transfer of radioiodide to milk and its inhibition. PMID- 3018579 TI - Bovine papillomavirus genome elicits skin tumours in transgenic mice. AB - Transmission of the bovine papillomavirus-1 (BPV-1) genome through the mouse germ line results in the heritable formation of fibropapillomas of the skin, a tissue specific phenotype analogous to that observed in natural BPV-1 infection of cattle. Oncogenesis is slow, with tumours first arising at 8-9 months of age, usually in areas prone to wounding. Extrachromosomal BPV-1 DNA is detected in all tumours, whereas normal tissues show only integrated DNA. PMID- 3018581 TI - Tissue-specific expression of the human tropomyosin gene involved in the generation of the trk oncogene. AB - The trk oncogene is a human transforming gene generated by the fusion of tropomyosin gene sequence to a truncated tyrosine kinase receptor gene. We have now characterized the normal tropomyosin gene from which the trk oncogene is derived. At least two different transcripts are expressed by this gene using a tissue-specific alternative messenger RNA splicing mechanism: a 2.5-kilobase (kb) mRNA encoding a 248-amino-acid tropomyosin in human fibroblasts and a 1.3-kb mRNA encoding a 285-amino-acid tropomyosin in human skeletal muscle. The rearrangement which generates the trk oncogene preserves most of the tropomyosin-coding sequences of the normal gene, including exons alternatively spliced in muscle and non-muscle tissue. We therefore expect the trk oncogene to show a tissue-specific pattern of transforming activity. Correct expression of the trk oncogene can occur only in non-muscle tissues. In muscle tissue the oncogene would almost certainly be inactive, as splicing according to the alternative muscle pattern aborts synthesis of the tyrosine kinase domain. PMID- 3018582 TI - Membrane biophysics. Surface conduction of protons. PMID- 3018580 TI - Olfactory dysfunction in humans with deficient guanine nucleotide-binding protein. AB - The guanine nucleotide-binding stimulatory protein (Gs) couples hormone-receptor interaction to the activation of adenylate cyclase and the generation of cyclic AMP. Studies using frog neuroepithelium indicate that the sense of smell is mediated by a Gs-adenylate cyclase system, and this prompted us to test olfaction in the only known model of Gs deficiency in the animal kingdom, Gs-deficient (type 1a) pseudohypoparathyroidism (PHP), which occurs in humans. Such patients are resistant to the cAMP-mediated actions of several hormones. (Although Henkin has reported disturbances in the sense of smell in six patients with PHP, currently available biochemical measurements such as the cAMP response to parathyroid hormone (PTH) and determination of Gs activity were not reported and olfactory testing was limited.) In the present study, we found that all Gs deficient patients had impaired olfaction when compared with PHP patients who had normal Gs activity (type 1b PHP, in which patients are resistant only to the action of PTH in the kidney). This is the first evidence of human olfactory impairment which can be related to Gs deficiency and suggests that Gs-deficient PHP patients may be resistant to cAMP-mediated actions in other non-endocrine systems. PMID- 3018583 TI - Gene regulation by proteins acting nearby and at a distance. AB - Transcription of genes can be controlled by regulatory proteins that bind to sites on the DNA either nearby or at a considerable distance. Recent experiments suggest a unified view of these apparently disparate types of gene regulation. PMID- 3018584 TI - Tight linkage between a splicing mutation and a specific DNA haplotype in phenylketonuria. AB - The first phenylketonuria mutation identified in the human phenylalanine hydroxylase gene is a single base substitution (GT----AT) in the canonical 5' splice donor site of intron 12. Direct hybridization analysis using specific oligonucleotide probes demonstrates that the mutation is tightly associated with a specific restriction fragment-length polymorphism haplotype among mutant alleles. The splicing mutation is the most prevalent phenylketonuria allele among Caucasians, and the results suggest the possibility of detecting carriers of the genetic trait who have no family history of phenylketonuria. PMID- 3018585 TI - Two glucagon transducing systems. PMID- 3018587 TI - Bovine chromogranin A sequence and distribution of its messenger RNA in endocrine tissues. AB - Chromogranin A is contained in storage vesicles of chromaffin cells of the adrenal medulla and released with catecholamines when the splanchnic nerve is stimulated. Chromogranin A is similar to secretory protein I (SP-I), a major secreted protein of the parathyroid. Chromogranin A/SP-I immunoreactivity is abundant in endocrine cells that secrete peptide hormones from storage vesicles. Chromogranins may act in neuroendocrine secretion by binding intravesicular calcium. Serum levels of chromogranin are raised in hypertension and endocrine neoplasia. We report here the isolation and sequencing of a cDNA encoding bovine chromogranin A, providing the first complete primary structure of a chromogranin protein. Chromogranin A is a highly acidic protein with an apparent relative molecular mass (Mr) of 75,000 on SDS-PAGE, but an actual Mr of 48,000. Adrenal medulla, brain, pituitary and parathyroid are all sites of synthesis of chromogranin A. The primary structure of chromogranin A, and the presence of chromogranin mRNA in the parathyroid, indicate that chromogranin A and SP-I are identical. PMID- 3018586 TI - Activation of two signal-transduction systems in hepatocytes by glucagon. AB - The ability of glucagon to stimulate glycogen breakdown in liver played a key part in the classic identification of cyclic AMP and hormonally stimulated adenylate cyclase. But several observations indicate that glucagon can exert effects independent of elevating intracellular cAMP concentrations. These effects are probably mediated by an elevation of the intracellular concentration of free Ca2+ although the mechanism by which this occurs is unknown. We show here that glucagon, at the low concentrations found physiologically, causes both a breakdown of inositol phospholipids and the production of inositol phosphates. Indeed, we show that the glucagon analogue, (1-N-alpha-trinitrophenylhistidine,12 homoarginine)glucagon (TH-glucagon), which does not activate adenylate cyclase or cause any increase in cAMP in hepatocytes yet can fully stimulate glycogenolysis, gluconeogenesis and urea synthesis, stimulates the production of inositol phosphates. This stimulation of inositol phospholipid metabolism by low concentrations of glucagon provides a mechanism whereby glucagon can exert cAMP independent actions on target cells. We suggest that hepatocytes possess two distinct receptors for glucagon, a GR-1 receptor coupled to stimulate inositol phospholipid breakdown and a GR-2 receptor coupled to stimulate adenylate cyclase activity. PMID- 3018588 TI - Transposon-dependent mutant phenotypes at the Notch locus of Drosophila. AB - Many mutations at complex genetic loci in the fruitfly Drosophila melanogaster are associated with insertions of transposable elements. At the Notch locus, members of one class of insertion-associated mutations, termed glossy-like, produce a recessive viable, smooth-eye phenotype with mottled pigmentation. Members of a second class, facet, produce a recessive viable, rough-eye phenotype with homogeneous pigmentation. Both classes of mutations fail to complement Notch lethal mutations, so they behave as Notch alleles. Here we report that each glossy-like mutation is associated with an insertion of the same transposable element (flea). Each flea insertion occurs in the same orientation, but at different locations within intervening sequences of the Notch locus. In contrast, each facet mutation is associated with insertion of a unique, non-flea, transposable element. Insertions producing a facet phenotype and insertions causing a glossy-like phenotype can break Notch intervening sequences at precisely the same location. This suggests that the type of insertion element rather than its position within an affected gene is the primary determinant of the phenotype observed. PMID- 3018589 TI - AIDS virus and scrapie agent share protein. PMID- 3018590 TI - Elimination of action potentials blocks the structural development of retinogeniculate synapses. AB - Although the influence of electrical activity on neural development has been studied extensively, experiments have only recently focused on the role of activity in the development of the mammalian central nervous system (CNS). Using tetrodotoxin (TTX) to abolish sodium-mediated action potentials, studies on the visual system show that impulse activity is essential both for the normal development of neuronal size and responsivity in the lateral geniculate nucleus (LGN), and for the eye-specific segregation of geniculo-cortical axons. There have been no anatomical studies to investigate the influence of action potentials on CNS synaptic development. We report here the first direct evidence that elimination of action potentials in the mammalian CNS blocks the growth of developing axon terminals and the formation of normal adult synaptic patterns. Our results show that when TTX is used to eliminate retinal ganglion-cell action potentials in the cat from birth to 8 weeks, the connections made by ganglion cell axons with LGN neurones, retinogeniculate synapses, remain almost identical morphologically to those in the newborn kitten. PMID- 3018591 TI - Normal p21N-ras couples bombesin and other growth factor receptors to inositol phosphate production. AB - Many receptors, in response to ligand activation, trigger inositol phospholipid breakdown, which leads to rapid intracellular responses. The sustained activation of this pathway is believed to be at least one of the factors involved in the stimulation of cell growth and there has been much speculation that certain oncogenes use this pathway to effect uncontrolled cellular proliferation. It has been suggested, by analogy with the receptor-mediated control of adenylate cyclase, that the receptor stimulation of inositol phospholipid metabolism is mediated through a guanine nucleotide regulatory protein (G-protein) called Gp (or Np). Although such a species has not been identified, there is now strong experimental evidence that this process is mediated by a G-protein distinct from the stimulatory and inhibitory G-proteins (Gs and Gi, respectively). The ras genes code for a plasma membrane protein, p21, whose only known biochemical property is a high-affinity GTPase activity. We show here that the expression of normal p21N-ras in NIH 3T3 fibroblasts leads to the coupling of certain growth factor receptors to stimulated inositol phosphate production. We propose that the N-ras proto-oncogene encodes a protein which couples the receptors for certain growth factors to the stimulation of phospholipase C. Thus, N-ras p21 may be the putative Gp or a functionally related protein. PMID- 3018592 TI - N-ethylmaleimide (NEM) diminishes alpha 2-adrenoceptor mediated effects on noradrenaline release. AB - The effect of N-ethylmaleimide (NEM), which has been shown to abolish rather selectively inhibition of adenylate cyclase, on the alpha 2-adrenoceptor-mediated modulation of noradrenaline release was studied. Slices of the rabbit hippocampus were loaded with 3H-noradrenaline, superfused continuously and stimulated twice electrically. NEM (30 mumol/l) applied for 30 min enhanced both basal and stimulation-evoked tritium overflow significantly. Occupation of the receptor by the alpha 2-adrenoceptor agonist clonidine prior to and during NEM treatment did not protect the alpha 2-adrenoceptor-mediated autoinhibitory feedback system from being affected by NEM. Preincubation of the hippocampal slices with NEM was without any influence on 3H-noradrenaline uptake. The inhibitory effect of clonidine on 3H-noradrenaline release was attenuated in a non-competitive manner. In addition, the facilitatory effect of the alpha 2-adrenoceptor antagonist yohimbine on the stimulus-evoked tritium overflow was reduced. The facilitation of the evoked noradrenaline release by yohimbine or yohimbine or yohimbine and NEM converged with increasing concentrations of yohimbine, suggesting that yohimbine and NEM were acting at the same signal-transduction system. These results are compatible with the idea that NEM, by alkylating the Ni-unit of a presynaptically located adenylate cyclase, prevents the alpha 2-adrenoceptor mediated modulation of noradrenaline release. PMID- 3018593 TI - Changes in central alpha-adrenoceptors and noradrenaline content after high sodium intake in Sabra salt-sensitive and salt-resistant rats. AB - Several studies have suggested a correlation between sodium accumulation and the development of hypertension. However, the mechanisms whereby sodium is able to increase blood pressure remain unclear. In the present study, alpha-adrenoceptors and noradrenaline contents have been studied in the cerebral cortex, hypothalamus and medulla oblongata in the Sabra rat strain in order to define their role in the resistance or sensitivity to sodium-induced hypertension. Alpha-Adrenoceptors were defined using the selective ligands 3H-prazosin and 3H-rauwolscine for alpha 1- and alpha 2-adrenoceptors, respectively. Under normal sodium diet, alpha 2 adrenoceptor density was higher in cerebral cortex and lower in hypothalamus and medulla oblongata of SBN (salt-resistant) compared to SBH (salt-sensitive) rats. Five weeks of high sodium intake induced a decrease in alpha 2-adrenoceptor density in cerebral cortex and an increase in hypothalamus only in SBN rats. These changes abolished the differences between SBH and SBN rats observed with a normal sodium diet. No changes in density and affinity of alpha 2-adrenoceptors were observed in medulla oblongata of SBN and SBH rats. Density and affinity of alpha 1-adrenoceptors were similar in SBN and SBH rats in all the tissues studied and they were unaffected by the high sodium diet. Noradrenaline contents in cerebral cortex, hypothalamus and medulla oblongata were also similar in the two rat substrains under normal sodium diet, but high sodium intake induced a decrease cerebrocortical noradrenaline content only in SBN rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018595 TI - Cardiac sarcolemmal purity is essential for the verification of adenylate cyclase inhibition via A1-adenosine receptors. AB - Inhibition of cardiac adenylate cyclase by adenosine receptor agonists was reinvestigated in a more homogeneous sarcolemmal vesicular preparation than used in a previous study. Microsomal particles obtained by differential centrifugation were further fractionated on a shallow density gradient of Percoll. Two populations of plasma membrane vesicles were partially resolved. Identical peaks were identified for adenylate cyclase activity and [3H]ouabain binding, whereas 5'-nucleotidase activity and beta-adrenoceptor binding displayed an additional peak at higher density, where angiotensin converting enzyme, a marker for endothelial plasma membranes, was at maximal activity. Significant inhibition by N6-cyclohexyladenosine (CHA), as measured in each fractionation step following homogenization, was observed only at the activity peak of adenylate cyclase. Moreover, analysis of the degree and rank order of potency of several adenosine analogs was indicative for interaction with A1-adenosine receptors. Accordingly, the peak in adenosine receptor binding, using (-)[125I]iodo-N6-hydroxyphenyl isopropyladenosine as the radioligand, coincided with CHA-inhibitable adenylate cyclase activity. By contrast, adenylate cyclase was slightly stimulated by CHA in the higher density range, an action suggested to be mediated via A2-adenosine receptors, which recently have been demonstrated to exist on guinea-pig coronary endothelium. It is concluded that the full extent of adenosine receptor-mediated adenylate cyclase inhibition in the heart is only to be demonstrated if contamination of the sarcolemmal preparation with endothelial membrane components is kept to a minimum. PMID- 3018594 TI - Effect of prostaglandin E2 on ACTH and beta-endorphin release from rat adenohypophysis in vitro after secretagogues which can mimic various first or second messengers. AB - The purpose of the present study was to further characterize the inhibition by prostaglandin E2 (PGE2) of adrenocorticotropin (ACTH) and beta-endorphin release from rat anterior pituitary fragments in vitro. Peptide hormone release was induced by vasopressin, which initiates secretion via cell surface receptors, or by secretagogues which can mimic various post-receptor mechanisms and the effect of PGE2 was examined. Concentration-response curves of the effect of vasopressin on the release of beta-endorphin-like (beta-End-IR) and ACTH-like immunoreactivity (ACTH-IR) were constructed in the absence or presence of a fixed concentration of PGE2. The concentration-response curve of vasopressin was shifted to the right about 8-fold by PGE2 (1 mumol/l) without altering the maximum effect. PGE2 (60 nmol/l-1 mumol/l) markedly reduced beta-End-IR release induced by 8-bromoadenosine-3',5'-cyclic-monophosphate (8Br-cAMP) (1 mmol/l). Omission of Ca2+ from the incubation medium did not prevent PGE2-induced inhibition of 8Br-cAMP-evoked secretion. 4 beta-Phorbol, 12 beta-myristate, 13 alpha-acetate (PMA) stimulated beta-End-IR and ACTH-IR release in a concentration dependent manner. This effect was not blocked by indometacin or eicosatetraynoic acid. PG E2 (greater than 100 nmol/l) reduced PMA (100 nmol/l)-elicited secretion by about 50%. PG E2 (1 mumol/l) almost halved beta-End-IR release caused by K+ (30 mmol/l). After pre-incubation in Ca2+-free medium, re-introduction of Ca2+ (1.3 mmol/l) elicited beta-End-IR release. This response was abolished by PG E2 (1 mumol/l). The addition of Ba2+ (10 mmol/l) to a Ca2+-free medium markedly enhanced beta-End-IR release.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018597 TI - [Methods for laboratory studies of LAV/HTLV III infection and reliability of the results]. PMID- 3018596 TI - [AIDS in the Netherlands; anno 1986]. PMID- 3018598 TI - [Sensory neurography, visual and somatosensory evoked potentials (VEP and SEP) in lead-exposed children]. PMID- 3018599 TI - [Visual evoked potentials and peripheral nerve conduction velocity in Friedreich ataxia]. PMID- 3018600 TI - Effect of sodium bicarbonate preloading on ischemic renal failure. AB - Rats pretreated with sodium bicarbonate were functionally protected from the damage of bilateral renal artery occlusion. The rise in serum creatinine (day 1 minus day 0) during the first 24 h after ischemia was 2.88 +/- 0.28 mg% in the bicarbonate-loaded animals versus 3.90 +/- 0.26 mg% in their matched controls (p less than or equal to 0.01). Pretreatment with acetazolamide produced a similar alkaline urine as the bicarbonate loading (pH 8.3 vs. 7.0 in controls) and a similar degree of protection (delta creatinine 2.85 +/- 0.41 vs. 4.23 +/- 0.26 mg%; p less than or equal to 0.01). A direct effect of sodium loading was excluded by comparing NH4HCO3 with NaHCO3 loading and observing no difference in delta creatinine levels after ischemia (3.39 +/- 0.69 vs. 3.20 +/- 0.61 mg%). These data indicate that NaHCO3 protects in this model of acute renal failure and further suggest that the mechanism of protection is not related to either systemic alkalosis or sodium loading. PMID- 3018601 TI - Factors affecting renal vein renin ratio in renal artery stenosis. Secretion of inactive renin. AB - All four factors which theoretically may affect the renal vein renin ratio in unilateral renal artery stenosis--increased renin secretion and diminished renal plasma flow on the stenotic side; suppressed renin secretion and renin extraction on the contralateral side--have been assessed. In a series of patients with unilateral renal artery stenosis, the renal vein ratio of active renin was more closely related to the reduction of renal plasma flow than to renin secretion rate on the affected side. On the contralateral side renin secretion was suppressed while angiotensin II was extracted. During long-term treatment with the converting enzyme inhibitor enalapril, peripheral plasma angiotensin II was lowered, while active renin concentration was markedly elevated, both in arterial plasma and in renal venous plasma of the stenotic kidney; the contralateral kidney became a net extractor of active renin. Thus, all 4 factors which theoretically affect the renal vein renin ratio can operate clinically. Both before and during enalapril, the affected kidney secreted inactive renin. PMID- 3018602 TI - A comparison of enalapril plus hydrochlorothiazide with standard triple therapy in renovascular hypertension. AB - This prospective, double-blind, multicenter study compared enalapril plus hydrochlorothiazide with standard triple therapy (STT; hydrochlorothiazide, timolol, and hydralazine) with regard to safety, tolerability, antihypertensive efficacy, and effect on renal function in 75 patients with documented renovascular hypertension. Both groups showed a significant mean decrease in systolic and diastolic blood pressure during the double-blind study, with the enalapril group showing a mean 12 mm greater decrease in systolic blood pressure as compared to STT (less than 0.05). Effective treatment of diastolic hypertension was noted in 96% of the enalapril group as compared to 82% on STT (p less than 0.05). STT failure was seen exclusively in patients with bilateral renal artery stenosis of high grade and frequently in association with impaired renal function. cPAH, a measure of effective renal plasma flow, showed a significant increase in the enalapril group, as compared to the STT (p less than 0.05). In contrast, there was a bimodal response of CIn (GFR): 80% of patients in the enalapril group showed no significant change while 20% (10 patients) showed a mean decrease of 28% along with a 12% increase in CPAH (p less than 0.01). No acute renal failure or toxic side effects were noted in the enalapril group. Enalapril plus hydrochlorothiazide is very effective in treating renovascular hypertension and is without significant toxic side effects. The self-limited increase in serum creatinine seen in 20% of renovascular hypertensive patients receiving enalapril and hydrochlorothiazide may identify a subset of patients with unilateral or bilateral high grade renal artery stenosis who should be treated with angioplasty or operative intervention. PMID- 3018604 TI - Low levels of calpain activity in Chiroptera brain: implications for mechanisms of aging. AB - Calcium-dependent neutral proteases ("calpains") have been implicated in degenerative processes in muscles and neurons, suggesting that they might also play a role in age-related brain pathologies and perhaps in brain aging itself. Because Chiroptera exhibit an unusual maximum life span, relative to other mammalian orders, we investigated the activity of these enzymes in the brain of two species of bats. As in other mammals, brain calpain degrades many proteins associated with the cell cytoskeleton. However, enzyme activity is 5-7 fold lower in bat's brain than in a similar-sized mammal, such as the mouse. Moreover, the maximal life span of bats predicted from the equation relating calpain activity and maximal life span across a wide range of mammals is close to the observed values. These results strengthen the hypothesis that calpain activity is somehow linked to the rate at which brains age. PMID- 3018603 TI - Renal function and hemodynamics during treatment with enalapril in primary hypertension. AB - Thirty-nine hypertensive patients were entered into a randomized, double-blind protocol to assess the effects of enalapril (10-20 mg b.i.d.), or combined enalapril (10-20 mg b.i.d.) and hydrochlorothiazide (25-50 mg b.i.d.) therapy on renal function and hemodynamics. Enalapril, either alone or in combination with hydrochlorothiazide, effectively controlled blood pressure. In patients with an initial inulin clearance less than or equal to 80 ml/min/1.73 m2, inulin and p aminohippurate clearances were markedly improved toward normal following either drug therapy. The filtration fraction was either unchanged (monotherapy) or increased (combination therapy), suggesting a direct glomerular effect of angiotensin-converting enzyme inhibition. Renal vascular resistance was decreased in all patients. These observations suggest that enalapril, either alone or in combination with a diuretic, has the potential to reverse renal function abnormalities encountered in the hypertensive state. PMID- 3018605 TI - Partial characterization of an endogenous factor which modulates the effect of catecholamines on synaptosomal Na+, K+-ATPase. AB - We have previously presented evidence for the existence of a brain soluble factor which mediates the stimulation of synaptosomal ATPases by catecholamines. The stimulation of synaptosomal ATPases by dopamine plus brain soluble fraction was not modified if the soluble fraction was heated for 5 min at 95 degrees C. One day after preparation, the soluble factor inhibited the Na+, K+-ATPase, but not the Mg2+-ATPase activity, and subsequent addition of noradrenaline stimulated the ATPases activities. The inhibitory effect of a 24 h soluble fraction disappeared if the soluble fraction was dialyzed; in this case, noradrenaline did not activate the enzyme activities. Gel filtration in Sephadex G-50 permitted separating a subfraction which inhibited ATPase activity (peak II) from another which stimulated ATPase activity (peak I). Peak I stimulated both Na+, K+, and Mg2+ ATPases. Peak II inhibited only Na+, K+-ATPase, and when stored acidified, it mediated ATPases stimulation by noradrenaline. PMID- 3018607 TI - [Clinico-pathological studies of CSF dissemination of glioblastoma and medulloblastoma]. AB - Clinico-pathological findings of CSF dissemination which was diagnosed on CT scan, were studied on 13 cases of glioblastoma and 9 cases of medulloblastoma. The type of CSF dissemination and the prognosis of patients were both different between glioblastoma and medulloblastoma. In the former, the dissemination was predominantly in ventricular walls and in the latter, in basal cisterns. The mean survival time after the diagnosis of dissemination is 6 months of glioblastoma as compared with 13 months of medulloblastoma. The Pathological studies show that subependymal and/or subpial infiltration of tumor cells, and thickness of arachnoid membrane by marked mesodermal reaction were demonstrated in cases of glioblastoma. On the contrary, tumor cells of medulloblastoma grow markedly in the subarachnoid space and/or on the ependymal layers. From these pathological findings of CSF dissemination, it will be resulted that the prognosis of glioblastoma is much more poor that of medulloblastoma. PMID- 3018608 TI - Induction of luteinizing hormone release by electrochemical stimulation of the medial preoptic area in delta 9-tetrahydrocannabinol-blocked proestrous rats. AB - The predominant psychoactive constituent of marijuana, delta 9 tetrahydrocannabinol (THC), blocks the preovulatory luteinizing hormone (LH) surge and ovulation in rats treated with THC (10 mg/kg body weight) during the early afternoon of proestrus. When THC-blocked proestrous rats were subjected to unilateral electrochemical stimulation (100 microA anodal DC for 45 s) in the medial preoptic area (mPOA), serum LH was significantly elevated at 30, 60 and 90 min after stimulation in comparison with LH levels measured in sham-stimulated control animals at those times. The induced LH release was sufficient to elicit ovulatory responses comparable to the spontaneous ovulations observed in control rats treated only with the drug vehicle. These results are consistent with the hypothesis that THC inhibits gonadotropin secretion by action within the central nervous system, but demonstrate that the central inhibitory effect of THC does not prevent the release of LH-releasing hormone (LHRH) when the LHRH neurosecretory units are activated by brain stimulation. Thus, the antiovulatory effect of THC appears to result from an inhibition of LH secretion which does not involve the direct blockade of LHRH release. PMID- 3018609 TI - Effects of 6-hydroxydopamine on the hypothalamo-pituitary-adrenocortical axis. AB - Female rats were treated with two intraventricular injections each of 350 micrograms 6-hydroxydopamine (6-OHDA) and used for experiment 4 or 14 days later. The response to laparotomy, as assessed by subsequent in vitro corticosterone release, was unaffected by the drug, but that to the smaller stress of a skin cut was significantly reduced. Both fast and delayed feedback responses to corticosterone administration were still evident in 6-OHDA-treated animals. When determined 14 days after treatment, hypothalamic concentrations of epinephrine (E) and norepinephrine (NE) were reduced by 46 and 84%, respectively. There was no significant change in content of immunoreactive corticotropin-releasing factor (CRF-41). The acetylcholine-stimulated release of CRF bioactivity from control hypothalami incubated in vitro was significantly inhibited by E or NE, with E being at least 10 times more potent on a molar basis. This effect of NE was enhanced in hypothalami removed from 6-OHDA-treated rats, complete inhibition of acetylcholine-stimulated release of CRF being produced by 0.6 nM NE, as opposed to 6.0 nM for untreated controls. At the level of the anterior pituitary gland, tissue content of adrenocorticotropin (ACTH) was unaffected by treatment, but that of luteinizing hormone (LH) in the same tissues was significantly increased. The corticotrophic response of fragments of the gland incubated or perifused in vitro to hypothalamic extract, CRF-41, arginine vasopressin or E was reduced. In contrast, the response of the tissue to gonadotropin-releasing hormone (GnRH) added in vitro was not significantly affected.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018610 TI - GnRH release from the mediobasal hypothalamus: in vitro inhibition by corticotropin-releasing factor. AB - The effects of corticotropin-releasing factor (CRF) on GnRH release from the adult male rat mediobasal hypothalamus (MBH) and median eminence (ME) were studied in an in vitro incubation system. CRF induced a dose-related inhibition of GnRH release from both the MBH and the isolated ME, and this inhibition was blocked by treatment with CRF receptor antagonist. Moreover, GHRF, a hypothalamic peptide similar in size to CRF, did not alter the ME release of GnRH. These results demonstrate that CRF can inhibit in vitro release of GnRH by a CRF receptor-mediated mechanism at the level of the neurosecretory terminals in the ME. Thus, increased hypothalamic CRF secretion associated with stress and adrenalectomy may inhibit hypothalamic GnRH release and produce the suppression of pituitary luteinizing hormone secretion which occurs under these conditions. PMID- 3018606 TI - Light and dark adaptation influences GABA receptor sites in the chick retina. AB - The aim of the present study was to investigate the effect of environmental conditions such as light-and-dark-adaptation on the plasticity of GABA receptor sites in the chick retina. In chicks exposed to light for 5 hr (light-adapted), specific [3H]GABA binding was increased by 35% in comparison to the binding found in chicks maintained in darkness (dark-adapted). Conversely, in the retina of chicks exposed to darkness for 5 hr, specific [3H]GABA binding was decreased by 28% with respect to that found in chicks kept in the light. Scatchard analysis of the binding data revealed that the affinity of GABA for its receptor binding site was higher in the retinas of light-adapted chicks than in those of dark-adapted chicks (Kd values of 19.20 +/- 1.23 and 27.20 +/- 1.47 nM, respectively). On the contrary, the maximal number of binding sites (Bmax) remained unchanged in light- and dark-adapted chicks (5.2 +/- 0.10 and 5.3 +/- 0.15 pmol/mg protein, respectively). These results suggest the involvement of GABA receptors in the regulation of visual function. PMID- 3018611 TI - Catechol estrogen formation by the CNS: regional distribution of estrogen-2/4 hydroxylase activity in rat brain. AB - Estrogen-2/4-hydroxylase (E-2/4-H) activity was measured by a direct product isolation assay in punch biopsy specimens obtained from nine nuclear regions from forebrain of adult male rats. Tritiated catechol estrogens were isolated from incubations of tissues with [6,7-3H]estradiol from all regions studied. The amount of 4-hydroxyestradiol (4-OH-E2) formed equaled or exceeded that of 2 hydroxyestradiol (2-OH-E2). There were significant regional differences in the amounts of catechol estrogen produced. The difference was nearly 8-fold between the arcuate-median eminence (ARC-ME) and the medial preoptic nucleus (POM), regions with the highest and lowest specific activities, respectively (37.7 +/- 6.2 vs. 5.1 +/- 0.7 pmol/mg protein/10 min 2-OH-E2, mean + SEM, n = 6). The supraoptic nucleus was the site of second highest concentrations of E-2/4-H activity (20.3 pmol 2-OH-E2/mg protein/10 min). Estrogen-2/4-H activity in the paraventricular (PVN) and periventricular (PERI) nuclear regions, though only about half that in the SON, was significantly greater than in the remaining brain areas (nucleus interstitialis striae terminalis, caudate, anterior hypothalamic and medial preoptic nuclei and cortex. The ARC-ME, the region with the highest E 2/4-H activity is where the dopaminergic neurones and terminals from the Gn-RH neurons are concentrated. The functions regulated by these two classes of neurones, the secretion of prolactin and gonadotrophins, respectively, have been the subject of most of the previous studies aimed at establishing the role of catechol estrogen formation in the hypothalamus.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018612 TI - Action potentials and frequency-dependent secretion in the mouse neurohypophysis. AB - The frequency-dependence of secretion of arginine vasopressin (AVP) from the mouse neural lobe in vitro was studied and found to be comparable to that reported for the rat neural lobe in vitro. For a stimulus train of 600 pulses, the secretion of AVP per pulse (i.e., facilitation) increased to a maximum at 20 Hz. Compound intracellular action potentials were recorded from the mouse neural lobe using optical recording methods and potentiometric dyes. These extrinsic optical signals reflect the true time courses of transmembrane potential changes (e.g., action potentials), and the action potentials recorded from mouse neural lobes had a duration of 5 ms; at half-maximum peak height. Optical recordings during repetitive stimulation showed that significant spike broadening occurred in each subsequent spike at 10 and 16 Hz stimulation. These data are consistent with a spike broadening hypothesis of frequency-dependent facilitation in the neural lobe. However, 4-aminopyridine, a drug which causes spike broadening in neural tissues by blocking potassium channels, did not produce an increase in secretion of AVP per stimulus from the mouse neural lobe. PMID- 3018614 TI - Involvement of central vasopressin receptors in the control of blood pressure. AB - Intraventricular perfusion with a hypertonic sodium chloride solution elicits increases in cerebrospinal fluid vasopressin and blood pressure and a decrease in heart rate. The central peptide response was greatly reduced in the hypertensive rat. Central pretreatment with the vasopressin (V1) antagonist completely abolished the pressor response to hypertonic sodium chloride in the normotensive animal. Results suggest that a central vasopressin receptor may play a role in the control of blood pressure. PMID- 3018615 TI - Comparison of benzodiazepine receptor binding in membranes from human or rat brain. AB - Specific high affinity binding of [3H]flunitrazepam to membranes from human brain was stimulated by gamma-aminobutyric acid (GABA), pentobarbital, 1-ethyl-4 (isopropylidene-hydrazino)-1H-pyrazolo[3,4b]pyridine-5-carboxy lic acid ethyl ester hydrochloride (SQ 20009) and avermectin B1a and was unaffected by 2 microM 4'-chlorodiazepam (Ro 5-4864) indicating that [3H]flunitrazepam in human brain as well as in rat brain predominantly binds to benzodiazepine receptors specific to brain, which was associated with a GABA receptor and several modulatory binding sites for drugs. The potency of several selective and non-selective ligands for benzodiazepine receptors for inhibition of the binding of [3H]flunitrazepam was compared in membranes from human or rat brain cerebellum, hippocampus and cerebral cortex. It was demonstrated that all these compounds, derived from different chemical structures, had a remarkably similar potency for inhibition of the binding of [3H]flunitrazepam in the corresponding regions of the human or rat brain. However, irreversible labelling of benzodiazepine binding sites with [3H]flunitrazepam and subsequent SDS-polyacrylamide gel electrophoresis and fluorography revealed more photolabelled protein bands in human than in rat cerebellum and hippocampus. The results seem to indicate that, although the pharmacological properties of reversible binding of [3H]flunitrazepam are remarkably similar in membranes from rat or human brain, the molecular heterogeneity of benzodiazepine binding sites is even greater in human than in rat brain. PMID- 3018613 TI - Central nervous system control of pituitary vasopressin receptors: evidence for involvement of multiple factors. AB - The regulation of pituitary vasopressin (VP) receptor concentration was investigated in rats with antero-lateral cuts (ALC) placed around the hypothalamus, as well as in Brattleboro homozygotes (HO) that genetically suffer from a lack of AVP. Hypothalamic ALCs caused a reduction in (3H)-AVP binding, while counteracting the dramatic fall in binding that normally occurs after adrenalectomy. Surprisingly, in HO rats, long-term adrenalectomy did cause pituitary AVP receptor number to decrease to an extent similar to that seen in normal rats. However, the receptor disappeared twice as rapidly in heterozygote controls than in HO animals, with calculated half-lives of 1.1 and 2.0 days, respectively. In HO, chronic administration of VP reduced receptor concentration by about 80%, while the same dose of oxytocin (OT) produced only a 20-30% reduction. Whereas dexamethasone injections did reverse the depressing effect of adrenalectomy on pituitary AVP receptors, they failed to enhance binding in sham operated controls, treated or not with VP; thereby suggesting a central site of action of the steroid. In contrast, in rats with hypothalamic ALCs (i.e. with the pituitary lacking central control), corticosterone implants did antagonize the reduction in receptor density caused by adrenalectomy. We conclude that the pituitary AVP receptor system lies mainly under control of the central nervous system, through a mechanism of action that not only seems to imply AVP and OT, but probably also some other hypothalamic factor(s). Glucocorticoids appear to exert a dual effect, acting indirectly through negative feedback control of neuropeptide release and, possibly, also directly on the pituitary to regulate binding sites. PMID- 3018616 TI - Purinergic modulation of the seizure threshold for pentylenetetrazol in the rat. AB - The effects of various metabolically-stable analogs of adenosine on the threshold for seizures in rats was determined by measuring the dose of pentylenetetrazol (PTZ), infused through a tail vein, required to elicit a myoclonic jerk. The adenosine receptor agonists, 2-chloroadenosine (2-ClAdo), cyclohexyladenosine (CHA) and L- and D-phenylisopropyladenosine (L- and D-PIA), all produced dose dependent elevations of the seizure threshold for pentylenetetrazol in rats. L Phenylisopropyl-adenosine was the most potent analog of adenosine tested with a dose as small as 5 micrograms/kg (i.v.) producing a 23% increase in seizure threshold for pentylenetetrazol. The rank order of the potency of adenosine agonists in increasing the seizure threshold was L-PIA greater than 2-ClAdo greater than CHA greater than D-PIA, with L-PIA being 79 times more potent than D PIA. In contrast to these effects, the adenosine receptor antagonist, theophylline, elicited a proconvulsant effect in doses from 15 to 60 mg/kg (i.p.). The effect of theophylline in reducing seizure threshold for pentylenetetrazol peaked at 30 mg/kg, a dose which reduced the seizure threshold by approx. 27%. Support for the involvement of recognition sites for adenosine in the observed modulation of seizure threshold was provided by the antagonism of the elevation of the seizure threshold for pentylenetetrazol induced by 2-ClAdo, by pretreatment with theophylline (5 mg/kg, i.v.). These findings provide support for the idea that endogenous adenosine may function as a regulator of seizure susceptibility. PMID- 3018617 TI - 2-Amino-6-trifluoromethoxy benzothiazole, a possible antagonist of excitatory amino acid neurotransmission--I. Anticonvulsant properties. AB - 2-Amino-6-trifluoromethoxy benzothiazole (PK 26124) prevented convulsions induced in rodents by maximal electroshock, inhibitors of the synthesis of gamma aminobutyric acid (GABA) and ouabain, but was inactive against seizures provoked by GABA antagonists, unlike diazepam, chlordiazepoxide, phenobarbital and valproic acid. 2-Amino-6-trifluoromethoxy benzothiazole prevented seizures induced by sound stimuli in DBA/2 mice (ED50 = 0.66; 2.1 and 4.1 mg/kg, i.p. according to the seizure component), postural seizures in El mice (ED50 = 7.5 mg, i.p.) and seizures induced by photic stimulation in the baboon, Papio papio, at 4 and 8 mg/kg (i.v.). This spectrum of anticonvulsant activity closely resembles that reported previously for dicarboxylic amino acid antagonists. Indeed, PK 26124 prevented seizures induced by L-glutamate (ED50 = 8.5 mg/kg, i.p.) or by kainate (ED50 = 9.25 mg/kg, i.p.) and tremors induced by harmaline (ED50 = 2.5 mg/kg, i.p.) In these tests diazepam was inactive (L-glutamate) or as potent as PK 26124 (kainate, harmaline), whereas it was 10-20 times more potent than PK 26124 against seizures induced by inhibitors of the synthesis of GABA. Together, these data suggest that PK 26124 possesses antagonistic properties of excitatory dicarboxylic amino acids, which may contribute to its anticonvulsant action. PMID- 3018619 TI - Specific [3H]neurotensin binding to rat spinal cord membranes. AB - The binding of [3H]neurotensin to membranes prepared from rat spinal cord has been studied in vitro. Scatchard analysis of saturation binding data indicated that [3H]neurotensin binds with high affinity (Kd = 6.3 nM) to a single, saturable population of binding sites (Bmax = 12.4 pmol/g tissue). Neurotensin1 13 (IC50 = 5.9 nM) and neurotensin8-13 (IC50 = 3.7 nM) were potent inhibitors of [3H]neurotensin binding whereas neurotensin1-8 was virtually inactive at concentrations up to 10(-5) M. Sodium chloride (150 mM) significantly inhibited binding, while potassium chloride (5 mM), magnesium chloride (10 mM), manganese chloride (1 mM) and GMP-PNP (0.1 mM) were without effect. The characteristics of the binding of [3H]neurotensin obtained in this study are consistent with this ligand binding to a physiologic neurotensin receptor in rat spinal cord membranes. PMID- 3018618 TI - Effect of ascorbate on the toxicity of morphine in mice. AB - The effects of ascorbic acid on the toxicity of morphine in mice were investigated. An intraperitoneal dose of sodium ascorbate (1 G/kg) injected 10 min prior to morphine (500 mg/kg, i.p.) was found to provide significant protection against mortality due to respiratory depression, while having no effect on the lethality of the pentobarbital. Pretreatment with ascorbate had no effect on the distribution of morphine in brain tissue, nor did it alter the pH of the plasma. Administration of ascorbate in vivo also produced no inactivation of binding to opioid receptors. It is postulated that ascorbate antagonizes the lethality of morphine by selectively affecting neuronal activity. PMID- 3018621 TI - The effects of the GABA-mimetic drugs, progabide and baclofen, on the biochemistry and function of 5-hydroxytryptamine and noradrenaline. AB - Administration to mice of a single dose of (+/-)-baclofen (5 mg/kg) or progabide (100 mg/kg) significantly inhibited the head-twitch response mediated by 5 hydroxytryptamine (5-HT2) receptors 30 min (but not 3 hr) later, when the response was produced by injection of carbidopa (25 mg/kg) plus 5 hydroxytryptophan (5-HTP; 100 mg/kg). No change was seen in the head-twitch response when induced at this time by 5-methoxy-N,N-dimethyltryptamine (5-MeODMT; 5 mg/kg). Inhibition of the head-twitch response after injection of 5-HTP was produced by pretreatment with (-)-baclofen, but not (+)-baclofen; injection of (+)-baclofen with the (-)-baclofen did not alter the attenuation of the behaviour produced by the active isomer. Twenty-four hours after the last injection of progabide, given repeatedly (100 mg/kg injected 5 times over 10 days) specific binding of [3H]ketanserin in the frontal cortex was enhanced and the head-twitch response to both 5-HTP and 5-MeODMT was markedly increased. The sedation response mediated by alpha 2-adrenoceptors, which followed the injection of clonidine (0.25 mg/kg) was attenuated. Repeated administration of baclofen (10 mg/kg per day in drinking water) also increased the number of 5-HT2 receptors in the frontal cortex (16%) and enhanced the head-twitch behaviour after injection 5-HTP or 5-MeODMT. Clonidine-induced sedation, number of beta-adrenoceptors in the cortex and apomorphine-induced locomotor activity were all unchanged.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018620 TI - Inhibitory effect of taurine on 4-aminopyridine-stimulated release of labelled dopamine from striatal synaptosomes. AB - 4-Aminopyridine (4-AP) stimulated the release of [3H]dopamine from striatal synaptosomes in the rat. At a concentration of 200 microM, 4-aminopyridine increased the spontaneous efflux of dopamine by 170%. The effect of 4 aminopyridine was calcium-dependent, being abolished when calcium was omitted from the incubation medium. Taurine, at a concentration of 25 mM, decreased the stimulatory effect of 4-aminopyridine from 170 to 49%, in the presence of 2.5 mM calcium. When the concentration of calcium in the superfusion medium was reduced to 0.1 mM, taurine had a complete inhibitory effect on the release of [3H]dopamine stimulated by 4-aminopyridine. The effect of taurine was dose dependent. Glycine had no effect on the release of [3H]dopamine stimulated by 4 aminopyridine, either in the presence of absence of calcium, whereas gamma aminobutyric acid (GABA) showed a slight inhibitory effect in both conditions. The results suggest that taurine antagonizes the release of [3H]dopamine induced by 4-aminopyridine through an effect mediated by calcium. PMID- 3018622 TI - Baclofen activates two distinct receptors in the rat spinal cord and guinea pig ileum. AB - Following intrathecal injection, pretreatment with both D-baclofen and 5 aminovaleric acid (5-AV) inhibited the antinociceptive effect of L-baclofen, but homotaurine (3-aminopropane sulphonic acid, APS) was inactive as an antagonist (rank order D-baclofen greater than 5-AV greater than APS = 0). In an established GABAB system, the electrically stimulated guinea pig longitudinal muscle myenteric plexus preparation, APS and 5-AV but not D-baclofen reduced the inhibitory effect of L-baclofen (APS = 5-AV greater than D-baclofen = 0). Receptors with which baclofen interacts in the spinal cord to produce antinociception differ from GABAB receptors with respect to the rank order of potency of antagonists as well as close structural analogs, and these criteria could be used for characterization of such receptors. PMID- 3018623 TI - Glioblastoma occurring after radiation therapy for meningioma: case report and review of literature. AB - A 32-year-old man developed an intracranial glioblastoma multiforme 10 years after irradiation for an incompletely resected convexity meningioma. The association of these two tumors is exceedingly rare. Therefore, we propose that this is a case of radiation-induced glioma and review the evidence supporting this view. PMID- 3018624 TI - Invasiveness in primary intracranial tumors: Part 1. An experimental model using cloned SV40 virus-produced hamster brain tumors. AB - This report presents an experimental model for study of the cellular and molecular biology of invasiveness in tumors. It uses SV40 virus for the production of primary intracranial tumors that are invasive for normal brain and vary markedly and predictably in this invasiveness. Cell cultures of dissociated 1- to 2-day-old Syrian hamster cerebral cortex (Cx), brain stem (Bs), cerebellar hemisphere (Cbh), and cerebellar vermis (Cbv) were transformed with SV40 virus and inoculated intracerebrally into newborn hamsters. All 368 animals that developed intracranial tumors were killed, and tumor was taken for histological and immunofluorescence studies, assessment of extent of invasiveness, and preparation of cell cultures from which cells were cloned by dilution plating or growth in soft agar. A few hamsters were perfused with glutaraldehyde for studies of tumor ultrastructure. All cloned and uncloned tumor cells were reinoculated to produce second- and third-passage tumors. Characteristic differences in morphology and growth rate were observed between normal astrocytes derived from each brain region, and these phenotypic differences were retained after virus transformation and tumor production. Cloned and uncloned Cx cell-derived tumors of second and third passage diffusely invaded adjacent normal brain, although those of first passage invaded only slightly. Except for extracerebral spread, these tumors resembled human astrocytic series tumors. Bs and some Cbh cell derived tumors were also astrocytic but more undifferentiated and only slightly invasive; Cbv and other Cbh cell-derived tumors were sarcomatous and only extended along perivascular spaces or were not invasive at all. The tumor cells contained glial fibrillary acidic protein and SV40 T-antigen. These results suggest that astrocytes from different brain regions vary in genomic stability and support the theory that differences in invasiveness reflect the development of heterogeneity and subsequent selection of more aggressive subpopulations of cells. PMID- 3018626 TI - Motor neuron disease associated with cancer. PMID- 3018625 TI - Familial long thoracic nerve palsy: a manifestation of brachial plexus neuropathy. AB - Long thoracic nerve palsy causes weakness of the serratus anterior muscle and winging of the scapula. It is usually traumatic in origin. Isolated long thoracic nerve palsy has not been recognized as the major manifestation of familial brachial plexus neuropathy, but I have studied the syndrome in four members of three generations of one family. One individual suffered an episode of facial paresis. The inheritance pattern was autosomal dominant. PMID- 3018627 TI - Influence of cyanide intoxication on the composition of myelin lipids in rats fed during development on a diet containing various amounts of lipids. PMID- 3018628 TI - Case of diagnosis. AIDS. PMID- 3018629 TI - [The endplate effects of atracurium. Clinical study]. PMID- 3018630 TI - [Breast thermography in the follow-up of substitutive hormonal therapy in menopause]. PMID- 3018632 TI - Pyridazinyl-GABA derivatives as GABA and glycine antagonists in the spinal cord of the cat. AB - Of two arylaminopyridazine derivatives of gamma-aminobutyric acid (GABA) tested as antagonists of the inhibitory actions of glycine and GABA in the spinal cord of pentobarbitone-anaesthetized cats, one - SR95531 - was sufficiently selective to be of use in microelectrophoretic investigations of GABA-mediated synaptic transmission. PMID- 3018631 TI - Cytomegalovirus antibody status in couples. AB - Sera from fifty couples were assayed for the presence of antibody to Cytomegalovirus. In twenty of the couples only one partner was seropositive. If serological status accurately distinguishes between infected and uninfected individuals, these data indicate that conjugal transmission of Cytomegalovirus is inefficient. PMID- 3018633 TI - Aniracetam augments, and midazolam inhibits, the long-term potentiation in guinea pig hippocampal slices. AB - The effects of aniracetam, a nootropic drug, and midazolam, which produces amnesia, on the long-term potentiation (LTP) of population spikes was investigated using hippocampal slices (CA3 area) from the guinea pig. Aniracetam at concentrations of 10(-7) and 10(-8) M, but not at 10(-6) M, significantly augmented LTP. On the other hand, midazolam (10(-9) M) significantly suppressed LTP. The suppressive effect was antagonized by Ro 15-1788 (10(-8) M). Both drugs did not affect the population spikes in the absence of tetanic stimulation at those concentrations. It was suggested that in vitro application of LTP is a feasible model system for evaluating the nootropic activity of drugs. PMID- 3018634 TI - A revised method for generation of unitary postsynaptic potentials for quantal analysis in the hippocampus. AB - For quantal analysis of excitatory postsynaptic potentials (EPSPs) in hippocampal CA3 neurons, a method was devised to evoke trains of unitary EPSPs at intended intervals and to control the number of unitary EPSPs composing each train at will. The amplitudes of the first EPSPs in EPSP trains evoked with this method were measured. From the mean and variance of the EPSP amplitudes, the mean number of quanta (m) released by a single impulse and the mean amplitude of EPSPs induced by individual quanta (q) were estimated in a standard solution and in a high calcium solution. PMID- 3018635 TI - Localization of nerve growth factor receptors in cholinergic neurons of the human basal forebrain. AB - Nerve growth factor (NGF) receptors were visualized in the basal human forebrain using an immunohistochemical procedure with a monoclonal antibody previously shown to recognize human melanoma cell NGF receptors. The receptors were found to be exclusively located in the medical septal nucleus, the diagonal band of Broca, and the nucleus basalis. This location coincided with that of cell bodies of ascending cholinergic neurons of the basal forebrain. In addition, NGF receptor positive cells were costained for acetylcholinesterase. These findings indicate that cholinergic neurons of the basal forebrain but none of the other neurons located in this area express receptors for NGF. Results suggest that NGF acts as a trophic factor for cholinergic neurons in the human brain in a similar way as has been established in recent years for the rat brain. PMID- 3018636 TI - Long-term potentiation in guinea pig hippocampal slices monitored by optical recording of neuronal activity. AB - Pre- and postsynaptic potential changes evoked by stimulation of the Schaffer collateral-commissural input to CA1 pyramidal neurons were optically recorded in guinea pig hippocampal slices after staining the preparation with a suitable voltage-sensitive fluorescent dye. Brief tetanic stimulation induced long-term potentiation of synaptic transmission as monitored both optically and electrically. The results demonstrate that non-invasive optical techniques can be used to study long-term changes in spatial neuronal interactions, possibly involved in learning and other higher functions of the nervous system. PMID- 3018637 TI - Prevention of amygdala kindling with an inhibitor of gamma-aminobutyric acid uptake. AB - A novel, specific inhibitor of gamma-aminobutyric acid (GABA) uptake, SKF 89976-A (N-[4,4-diphenyl-3-butenyl]-nipecotic acid), was administered daily (15 mg/kg, i.p.) 30 min prior to amygdala stimulation in adult female rats. Whereas control rats developed full kindled seizures after 9.4 +/- 1.2 amygdala stimulations. SKF 89976-A-treated rats had not progressed beyond an early stage of kindled seizures by the termination of drug treatment after 22 episodes of daily amygdala stimulation. Following cessation of SKF 89976-A treatment, full kindled seizures developed after 4.1 +/- 0.9 additional amygdala stimuli. The data suggest that SKF 89976-A can inhibit generalization of amygdala-kindled seizures, possibly by enhancement of central GABAergic activity. PMID- 3018638 TI - Non-coordinate localization of corticotroph-neurophysin and beta-endorphin in the anterior pituitary gland of the rat. AB - Anti-neurophysin serum was applied in the immunohistochemical technique to anterior pituitary tissues obtained from normal and chronically dehydrated rats and also from rats with chronic diabetes insipidus (Brattleboro strain). In all cases there was a positive staining in the corticotrophs, which also stained for either beta-endorphin (beta-END) or adrenocorticotrophin hormone (ACTH). It was concluded that corticotroph-neurophysin may be synthesized independently of either ACTH or beta-END. PMID- 3018639 TI - Update on pediatric oncology. PMID- 3018640 TI - [3H]glutamate binding sites in the rat pituitary. AB - A significant activity of [3H]glutamate binding was detected in homogenate particulate preparations obtained from the rat pituitary, in addition to central structures including the cerebral cortex. In contrast to the cerebral binding, the pituitary binding was significantly inhibited by Na+ ions. It was also found that neurohypophysis possessed more than a two-fold higher binding activity than that found in adenohypophysis. These results suggest a possible significance of glutamate in rodent pituitary. PMID- 3018641 TI - The inhibitory feedback pathway from the forelimb to C3-C4 propriospinal neurones investigated with natural stimulation. AB - Light mechanical stimulation of the skin and passive joint movements in the forelimb gave effective activation of interneurones located medially in the C3-C4 segments. Such interneurones may be inhibitory to C3-C4 propriospinal neurones (PNs) and recording from PNs revealed that the stimuli which activated the interneurones evoked inhibition in the PNs. It is postulated that a movement commanded via the C3-C4 PNs evoke impulses in forelimb afferents which by negative feedback control transmission in the C3-C4 PNs and thus govern the execution of the movements. PMID- 3018642 TI - Fish oils and coronary heart disease. PMID- 3018643 TI - Use of the deoxyuridine suppression test to evaluate vitamin B12 and folate status. AB - The deoxyuridine suppression test (DUST), performed on bone marrow cells, or peripheral blood lymphocytes, provides a rapid, dynamic assessment of vitamin B12 and folate status. The principles of this test are described and the use of the DUST in haematological practice at Wellington Hospital is reviewed. The advantages of the test are the speed of obtaining results, and an accurate assessment of the patient's condition when other haematological tests may be misleading. PMID- 3018644 TI - Guidelines for breast feeding following maternal radiopharmaceutical administration. PMID- 3018645 TI - [Experiences with the follow-up patient care at the Berlin-Steglitz health department]. PMID- 3018646 TI - Before you test a patient for AIDS.... Ohio Department of Health recommendations for clinical management of individuals with HTLV-3 infection. PMID- 3018648 TI - [EPICO in the treatment of small cell bronchial cancer. 3. Intermediate analysis]. AB - 61 patients with untreated small cell lung cancer were treated with a combination of epirubicin 70 mg/m2, cyclophosphamide 1,000 mg/m2 and oncovin 2 mg every 3 weeks. The mean age of patients was 57 years. 51 patients were evaluable. 30 patients were classified to the stage limited disease, 21 patients to extensive disease. 9 complete and 23 partial remissions were achieved (remission rate 64%). The overall survival was 14 months, the mean survival of responders 16 months. 32 patients were alive at the end of the study. Performance status and extent of disease influenced significantly the result of treatment. The cytostatic activity of EPICO is comparable to three other drug combinations. The benefit of EPICO might be the lower cumulative toxicity of epirubicin and therefore enabling a longer duration of treatment. PMID- 3018647 TI - Treatment of AIDS-related Kaposi's sarcoma with recombinant gamma-interferon. AB - Four patients with the acquired immunodeficiency syndrome and Kaposi's sarcoma were treated with subcutaneous human recombinant gamma-interferon at a dosage of 200 micrograms = 2 X 10(6) U twice daily for at least 28 days. While the dosage of interferon was well tolerated, progressive disease was observed in all four patients studied. These results might be due to the selection of the patients presenting advanced stages (3 out of 4) with recurrent disease after previous chemotherapy. PMID- 3018649 TI - Epirubicin in advanced gastrointestinal (GI) cancer. AB - A review of the activity of epirubicin in advanced gastrointestinal tumors is presented. In gastric cancer epirubicin has comparable activity to its parent compound doxorubicin with less toxicity. Epirubicin seems particularly active in pancreatic cancer as shown by the results of two EORTC Gastrointestinal Group studies. In advanced colorectal cancer results of different studies are conflicting and the drug may have limited activity in rectosigmoid cancer. PMID- 3018650 TI - Experimental chloroquine retinopathy. AB - Chloroquine retinopathy was produced experimentally in the eye of the albino corydoras (one of the tropical fish) by daily administration of chloroquine (0.1 mg per os). The enucleated eyes were examined from the 14th day to 3 months after the beginning of drug administration under light and electron microscopy. The first change of retina was the appearance of membraneous cytoplasmic body (MCB) in the cytoplasm of ganglion, amacrine, bipolar and horizontal cells. MCB might be degenerated lysosome. They showed lamellar figures or crystalline lattice-like structures. Secondarily, these MCB appeared in the inner segments of photoreceptor cells. The outer segments of rod cells disappeared, and then those of cone cells. Although photoreceptor cells were diminished in number in advanced degeneration, the cells of inner nuclear layer and ganglion cells were maintained in number. The presence of MCB dose not mean death of cells. The retinal pigment epithelial cells contained MCB in its cytoplasm only in severe degenerative cases, and did not show other remarkable changes. MCB also appeared in the cytoplasm of pericytes of retinal vessels. Chloroquine is considered to damage directly photoreceptor cells most severely. PMID- 3018652 TI - Intraoral solitary glomus tumor (glomangioma): case report and literature review. AB - The glomus tumor, or glomangioma, is a benign neoplasm arising from the normal glomus. Glomus tumors of the oral cavity are rare, with only ten cases reported in the literature. We report the light and electron microscopic features of an additional case of glomus tumor of the lip which occurred as a solitary, painless, submucosal mass in a 51-year-old woman. Clinical, diagnostic, and histogenetic aspects are discussed. PMID- 3018651 TI - Visor osteotomy augmentation of the mandible with posterior onlay bone graft or with hydroxylapatite: a comparative study. AB - A frequent and severe ridge atrophy, the anterior spheroid basal bone with posterior bilateral concavities of the mandible, has been defined. Two groups of sixteen patients who had residual ridge augmentation of the mandible with two different techniques were studied. The visor osteotomy with posterior hydroxylapatite was superior to the visor with posterior onlay bone graft in terms of ridge form, bone resorption, patient satisfaction, and neurosensory disturbances. Nevertheless, there remained some problems of anterior resorption and backward rotation of the visor segment for both techniques and of posterior resorption with the partial onlay bone graft. PMID- 3018653 TI - Nonsquamous tumors of the oral cavity. AB - The clinical presentations of various nonsquamous tumors of the oral cavity are reviewed, along with their gross pathology, histology, and treatment. Survival rates are presented for those neoplasms that occur frequently enough to allow meaningful analysis of various modes of therapy. PMID- 3018654 TI - Personality characteristics of patients with chronic pain in comparison with normal controls and depressed patients. AB - The Karolinska Scales of Personality (KSP) were used for the description of self reported personality characteristics in pain patients. The patients were a selected group, suffering from chronic non-malignant pain mainly of neurological origin, and had in all cases an established somatic diagnosis. In comparison with normal controls, the pain patients differed only with respect to two KSP scales, showing significantly more negative childhood experiences and less Inhibition of Aggression than the normals. In contrast, there were several and pronounced differences between the pain patients and two psychiatric groups with depressive disorders, one with and one without pain. Thus this group of chronic pain patients did not show the personality characteristics predisposing for depressive disorders. Furthermore, the KSP personality factors were found to be related to variables describing consequences of the pain, but the power of the KSP to predict the outcome of the pain treatment appeared to be limited. The KSP appears to have several advantages in comparison, e.g., with the Minnesota Multiphasic Personality Inventory, the most frequently used method for the assessment of chronic pain patients. Unlike the MMPI, the KSP reflects aspects of personality and is not primarily a measure of psychopathology. In addition, the KSP is more up-to-date and its completion is less time-consuming. The usefulness of the KSP for the assessment of chronic pain patients deserves therefore further examination. PMID- 3018656 TI - Technetium 99m pertechnetate thyroid scintigraphy: congenital hypothyroid screening. AB - Technetium 99m pertechnetate thyroid scans were performed on 57 infants referred for evaluation of suspected congenital hypothyroidism. Thyroid anatomy may be characterized by four general types, based on the scintigraphic findings: (1) normal size and location; (2) ectopic location; (3) no detectable thyroid activity; (4) normal location with increased size or uptake. There are diverse etiologies of congenital hypothyroidism. Correlation of thyroid scintigraphy with blood T4 and TSH levels allows specific etiological diagnosis in the majority of cases of congenital hypothyroidism. PMID- 3018655 TI - Delta viral hepatitis. Histopathology and course. AB - Delta viral hepatitis is important to recognize on hepatic tissue as its presence may explain severe or fulminant acute viral hepatitis, "relapse" of acute viral hepatitis, fulminant acute viral hepatitis in a chronic hepatitis B carrier, exacerbation of previously silent chronic active hepatitis, and the change in course of persistent viral hepatitis to progressive chronic active hepatitis. Histologic clues to support delta infection are primarily the severity of hepatocellular necrosis and inflammatory response. Specific immunologic procedures are required for diagnosis and include serum testing (antigen, antibody for IgG and IgM types) and antigen detection in tissue by immunofluorescence or immunoperoxidase staining. This agent is currently important as a cause of hepatitis in IV drug users, male homosexual patients, and recipients of blood products. Further studies are needed with careful accurate classification of hepatitis B patients in order to recognize the pattern of spread, severity, and outcome of delta hepatitis in other populations. PMID- 3018657 TI - High resolution ultrasonography of the parotid gland in children. AB - High resolution ultrasonography has revolutionized imaging of the superficial parotid gland. The fine morphology of the parotid gland can be exquisitely visulized. Anatomical landmarks such as the mastoid tip, the sternocleidomastoid muscle, the styloid process, the posterior facial vein, the internal jugular vein, and the external carotid artery can easily be identified in relationship to the parotid. The course of the facial nerve can be inferred by visualization of these landmarks. Common masses within the parotid in children such as hemangiomas, mixed parotid tumors, and lymphadenopathy can be demonstrated. Parotid masses are uncommon in children but can be easily evaluated by high resolution ultrasonography. PMID- 3018658 TI - Effect of the synthetic inhibitor tosylamino-phenylethyl-chloromethylketone on chemotactic peptide receptor activation and superoxide production in human neutrophils. AB - It was previously shown that inhibitors such as tosylamido-phenylethyl chloromethylketone (TPCK) inhibit superoxide production by human neutrophils. These studies suggested that a chymotrypsin-like protease inhibited by TPCK was involved in the activation of the neutrophils oxidative system. In this study, we attempted to define the step in cellular activation and/or cell function inhibited by TPCK. TPCK 10(-5) M did not inhibit the following early events thought to be involved in the activation of oxidase. 1) f-met-leu-phe-induced activation of phospholipase C assessed by the production of inositol-tris phosphate (IP3), 2) f-met-leu-phe-induced membrane potential changes, 3) f-met leu-phe-induced increase in free cytosolic calcium, and 4) phorbol-myristate acetate-induced protein phosphorylation in 32P labeled neutrophils. We also showed that TPCK 10(-5) M inhibited bactericidal activity of neutrophils on Staphylococcus aureus, whereas it did not inhibit the ingestion rate of endotoxin coated Oil red O particles. We conclude that 1) TPCK at the concentration of 10( 5) M inhibits superoxide production but not ingestion of Oil red O particles and 2) TPCK inhibits superoxide production at a step distal from calcium mobilization and protein phosphorylation. Radiolabeled TPCK may therefore be a useful tool to study, whether a protease is involved in the activation of the oxidative system distal to second messenger generation. PMID- 3018659 TI - Alanine enhances jejunal sodium absorption in the presence of glucose: studies in piglet viral diarrhea. AB - We measured the response of jejunal sodium (Na) absorption to neutral amino acid (L-alanine) and to dipeptides (L-alanyl-L-alanine, glycylsarcosine) in normal piglets and in piglets with acute viral diarrhea after experimental infection with transmissible gastroenteritis (TGE) virus. In the TGE jejunum villi were blunted, crypts were deepened, and the epithelium was composed of relatively undifferentiated cells with reduced disaccharidase, decreased sodium-potassium stimulated ATPase, and elevated thymidine kinase activities. The response of Na absorption to a maximal concentration of L-alanine (20 mM) or D-glucose (30 mM) was significantly blunted in TGE jejunum in Ussing chambers. However, the addition of L-alanine together with D-glucose caused a significantly greater increment of Na absorption than either L-alanine or D-glucose alone in control and TGE tissue. The effect of Na absorption of the dipeptide L-alanyl-L-alanine (10 mM), which was rapidly hydrolyzed by control and TGE mucosa, was similar to that of L-alanine (20 mM), while glycylsarcosine, a poorly hydrolyzed dipeptide, did not change net Na absorption in the jejunum. Our data support the concept of separate carrier systems for neutral amino acid and hexose in the crypt-type intestinal epithelium characterizing viral enteritis. We speculate that a sodium cotransporting amino acid, if added to oral glucose-electrolyte solutions, could benefit oral rehydration therapy in acute viral diarrhea; neither of the dipeptides tested here can be expected to enhance absorption to any greater extent than its constituent amino acids. PMID- 3018661 TI - Virilizing adrenocortical tumors in childhood. PMID- 3018660 TI - Immune response to herpes simplex virus in patients with recurrent herpes labialis. II. Relationship between interferon production and cytotoxic responses. AB - The relationship between the development of cytotoxic cellular immune response to herpes simplex virus type I (HSV-1)-infected autologous cells and the production of interferon (IFN) was studied using in vitro secondary sensitization of peripheral blood leukocytes in subjects with recurrent herpes labialis (RHL) and in normal controls without any history of recurrent herpes labialis. There was a significant discordance between optimal HSV-1 antigen dose required for induction of peak cytotoxic responses and for maximal activity of IFN. Moderate IFN activity (6-100 U/ml) was demonstrated in all HSV-1 antigen-stimulated peripheral blood leukocytes collected from subjects during both acute and convalescent phase of RHL. However, only 50% of seropositive controls and no seronegative controls exhibited detectable IFN activity, when stimulated with HSV-1 antigen, although such in vitro stimulation resulted in maximal virus-specific cell-mediated cytotoxicity. A correlation of virus specific cytotoxic activity to HSV-1 and IFN production (r = 0.38, p less than 0.05) was less marked than that of cytotoxic activity to K562 (natural killer-sensitive target cells) and IFN titer (r = 0.48, p less than 0.01). Furthermore significant reverse correlations between cytotoxicity against HSV-1-infected autologous cells and a titer of gamma-IFN was observed in samples with high cytotoxic activity. These observations suggest that gamma-IFN produced by HSV immune T cell may also act as an autoregulatory factor against the production of cytotoxic cellular activity against HSV-1-infected autologous cells. PMID- 3018662 TI - [Bronchiolo-alveolar lung cancer in a 10-year-old child with paraneoplastic systemic lupus erythematosus]. PMID- 3018663 TI - Influence of potassium depletion on potassium conductance in proximal tubules of frog kidney. AB - In order to test for the contribution of intracellular potassium activity to the link of sodium/potassium-ATPase activity and potassium conductance, studies with conventional and potassium selective microelectrodes were performed on proximal tubules of the isolated perfused frog kidney. The peritubular transference number for potassium (tk), i.e., the contribution of peritubular slope potassium conductance to the slope conductance of the cell membranes (luminal and peritubular), was estimated from the influence of peritubular potassium concentration on the potential difference across the peritubular cell membrane (PDpt). During control conditions, PDpt is -65 +/- 1 mV, intracellular potassium activity (Ki) 57 +/- 2 mmol/l and tk 0.41 +/- 0.05. The resistance in parallel of the luminal and peritubular cell membranes (Rm) is 44 +/- 4 k omega cm, the resistance of the cellular cable (Rc) 137 +/- 13 M omega/cm. When the cells are exposed 10 min to potassium free perfusates (series I), PDpt increases by -28 +/- 3 mV within 2 min and then decreases gradually to approach the control value within 10 min. Ki decreases by 22 +/- 3 mmol/l and Rc increases by 35 +/- 10%. After a transient decrease, Rm increases by 36 +/- 9%. Readdition of peritubular potassium leads to a transient increase of PDpt, a gradual decrease of Rm and Rc as well as a gradual increase of Ki. tk recovers only slowly to approach 65 +/- 8% of control value within 3 and 79 +/- 10% within 6 min.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018665 TI - [Splenic lesions following transcatheter arterial embolization of malignant liver tumors]. PMID- 3018664 TI - Quick isolation of rat medullary thick ascending limbs. Enzymatic and metabolic characterization. AB - This paper describes a rapid and simple method for isolation of medullary thick ascending limbs (MTAL) from rat kidney. The technique takes advantage of the fact that MTAL represents a high fraction of the inner stripe (IS) tissue in the outer medulla, and that this nephron segment is more resistant than others to mechanical and enzymatic disruption. Special attention was given in the design of each step of the isolation procedure in order to improve purity and yield of the preparation. Major steps are the following: careful dissection of the IS; cutting IS tissue into small pieces of regular size (approximately equal to 1 mm3); mild and brief enzymatic hydrolysis in a 65 U/ml collagenase solution; separation of long MTAL segments from other tubule fragments and cells, and washing of the collagenase solution, on a nylon sieve (100 microns opening). This technique does not require lengthy centrifugations and provides about 6 mg fresh tissue (= 1 mg protein) from two rat kidneys in 2 h. Light microscopy and transmission electron microscopy show a good purity (at least 95%) and good preservation of TAL ultrastructural morphology. Adenylate cyclase responsiveness to arginine vasopressin (AVP), glucagon (GLU) and salmon calcitonin (SCT) of the MTAL suspension is similar to that reported for single microdissected rat MTAL. Viability of the MTALs was demonstrated by the ability to accumulate cyclic AMP in presence of AVP, GLU, SCT and forskolin. Normal oxygen consumption was 45.1 +/ 2.4 (SEM) microliter . mg protein-1 . h-1 (n = 8).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018666 TI - [Myeloid splenomegaly and LAV/HTLVIII virus infection]. PMID- 3018667 TI - Restriction endonuclease activity induced by NC-1A virus infection of a Chlorella like green alga. AB - A type II restriction endonuclease, CviBI, was isolated from a eukaryotic, Chlorella-like green alga infected with the dsDNA containing virus NC-1A. The enzyme recognizes the sequence GANTC and cleaves DNA between the G and A. Methylation of deoxyadenosine in the GANTC sequence probably inhibits enzyme activity. In vitro CviBI cleaves host nuclear DNA but not viral DNA. A survey of 18 other viruses which infect the same Chlorella sp. revealed that infection with 5 of these viruses also induced a restriction endonuclease which cleaves DNA into the same size fragments as CviBI. PMID- 3018668 TI - The ribosomal spacer in Xenopus laevis is transcribed as part of the primary ribosomal RNA. AB - S1 mapping of Xenopus laevis ribosomal RNA transcripts, both in oocyte microinjection experiments and in vivo, shows that all but 212 bp of the so called "non-transcribed" spacer (NTS) of the ribosomal DNA repeat is transcribed as part of the primary ribosomal transcript. The 40S pre-ribosomal RNA (pre-rRNA) is therefore a processing intermediate. The primary ribosomal transcript co terminates with the previously described spacer transcripts [Moss], at a site 213 bp upstream of the 40S pre-rRNA initiation site. This mode of transcription suggests a simple mechanism for the recently proposed phenomenon of "readthrough enhancement", [Moss et al, Moss], where readthrough transcription from an upstream gene may enhance transcription of a gene immediately downstream in the tandem ribosomal repeat. PMID- 3018669 TI - Association of the herpes simplex virus regulatory protein ICP4 with specific nucleotide sequences in DNA. AB - We report that the herpes simplex virus (HSV) transcription regulatory protein designated ICP4 is a component of a stable complex between protein and specific nucleotide sequences in double-stranded DNA formed by addition of exogenous DNA to either a crude extract obtained from HSV-1 infected cells or a partially purified preparation of native ICP4. DNA sites which are bound directly or indirectly to ICP4 have been designated ICP4/protein binding sites. Three independent ICP4/protein binding sites have been identified by DNAse footprinting; two are in the vector pBR322 and one is located approximately 100 nucleotides upstream from the HSV glycoprotein D mRNA cap site. Comparison of the nucleotide sequences in these three sites reveals several regions of homology. We propose that the sequence 5'-ATCGTCNNNNYCGRC-3' (N = any base; Y = pyrimidine; R = purine) forms an essential component of the ICP4/protein binding site. PMID- 3018670 TI - Molecular mechanism of introduction of the hidden break into the 28S rRNA of insects: implication based on structural studies. AB - We determined the structure around the gap region between 28S alpha and 28S beta rRNA of Bombyx mori, a lepidopteran insect, to know the introduction mechanism of the hidden break, an interruption of the phosphodiester bond specific to the 28S rRNA of protostomes. Sequence analysis and S1 nuclease mapping suggested that a stretch of 30 nucleotides is excised from the mid region of the 28S rRNA to generate the hidden break. The length of excluded stretch was very various among three insects so far studied. However, the gap responsible for the hidden break was located in a fixed position in the 28S rRNA irrespective of insect species. It was suggested that extremely AU-rich sequence including the specific UAAU tract forming a loop can be a signal for the introduction of the hidden break. The same signal seemed also involved in splitting the dipteran 5.8S rRNA. PMID- 3018671 TI - The use of single-stranded DNA and RNase H to promote quantitative 'hybrid arrest of translation' of mRNA/DNA hybrids in reticulocyte lysate cell-free translations. AB - Single-stranded cDNA clones complementary to the 5' end of TMV RNA have been used to explore the conditions necessary for efficient 'hybrid arrest of translation' in the reticulocyte lysate. It is shown that incubations of 20 minutes at 60 degrees in 0.1 M KCl are sufficient to give almost complete arrest of translation using a clone complementary to the 5'-non-coding region and first 171 coding nucleotides of TMV RNA. However, hybrids with DNA complementary to regions of the mRNA downstream of the first AUG gave variable and in some cases almost no arrest of translation in the reticulocyte lysate unless they were first digested with RNase H. A simple and rapid method for giving complete and highly specific arrest of translation of particular mRNAs in complex mixtures has been developed using both cDNA clones and synthetic oligodeoxynucleotides in conjunction with RNase H digestion. Evidence is presented that suggests that 'hybrid arrest of translation' in the wheat-germ cell-free system is primarily due to the action of RNase H. When a reticulocyte lysate was doped with 20 U/ml of RNase H, its ability to translate unannealed mRNA was unaffected but it translated DNA/RNA hybrids extremely poorly. PMID- 3018672 TI - Nucleotide sequence of the Escherichia coli replication gene dnaZX. AB - The Escherichia coli 2.2 kilobase dnaZX region contains one 1929 nucleotide reading frame which directs the synthesis of two protein products involved in DNA polymerization. The larger consists of 643 amino acids in a deduced 71,114 dalton chain which could be the tau subunit of DNA polymerase III. The smaller, the DNA polymerase III gamma subunit, is encoded by the same reading frame as the larger. The dnaZX sequence contains a region homologous to ATP binding sites, suggesting that these replication factors are adenine nucleotide binding proteins. PMID- 3018673 TI - Sequence of 2,617 nucleotides from the 3' end of Newcastle disease virus genome RNA and the predicted amino acid sequence of viral NP protein. AB - DNA fragments complementary to the Newcastle disease virus genome (strain D26) were cloned and sequenced. The sequence of 2,617 nucleotides from the 3' end of the genome was determined and an open reading frame (OP-1) consisting of 1,467 nucleotides, most likely encoding NP protein, was found in this region. This was followed by a second unfinished open reading frame (OP-2) of at least 729 nucleotides which continued beyond the 2,617th nucleotide. Another relatively short (312 nucleotides long) open reading frame (OP-2') was found overlapping with OP-2, but its significance is still unclear. The amino acid sequence deduced from the nucleotide sequence of OP-1 showed a moderate homology to that of the NP protein of Sendai virus in the central portion of the peptide. The leader sequence of 53 nucleotides was also identified. The 5' end of mRNAs synthesized in the infected cells was analyzed and found to be m7GpppA, suggesting that the transcription of viral mRNAs starts with A, but not with G residue. PMID- 3018674 TI - Oligonucleotide duplexes containing inosine, 7-deazainosine, tubercidin, nebularine and 7-deazanebularine as substrates for restriction endonucleases HindII, SalI and TaqI. AB - Synthetic hexadecanucleotide duplexes containing a single purine nucleotide analogue in the recognition sites of the restriction endonucleases HindII, SalI and TaqI were used to investigate the restriction site determinants required by these enzymes for sequence recognition and phosphodiester bond cleavage. The enzymes were, in general, unaffected by changes introduced into the minor groove of the helix. SalI was found to be inhibited by the major groove modifications introduced into the fourth position of its recognition sequence GTCGAC. HindII and TaqI were, by contrast, able to cleave the sites containing the analogues at this position. TaqI and, to a lesser extent, HindII could also be shown to tolerate "mismatch analogues" at this site. PMID- 3018675 TI - Light regulation of a SSRubisco-nos chimaeric gene: photoregulatory control sequences from a C3 plant function in cells of a CAM plant. AB - A SSRubisco-nos chimaeric gene has been constructed, in an oncogenic Ti-plasmid vector. A 900bp soybean SSRubisco upstream fragment, carrying CAAT and TATA boxes and transcription initiation point, was fused to the nos coding region, the fusion site being within the 5'-untranslated region. When this chimaeric construct was transferred to Kalanchoe cells, nos expression was shown to be light-regulated. Thus DNA sequences responsible for light-dark control of gene expression are wholly or partly contained within the 900bp soybean SSRubisco upstream region. Moreover, this is the first demonstration that photoregulatory elements in a gene derived from a C3 plant, function in cells of a plant exhibiting the CAM trait. PMID- 3018676 TI - Nucleotide sequence of the yeast cell division cycle start genes CDC28, CDC36, CDC37, and CDC39, and a structural analysis of the predicted products. AB - The nucleotide sequences of the yeast cell division cycle start genes CDC36, CDC37, and CDC39 are presented. An open reading frame corresponding in size and mapped position to the mRNA for each gene was revealed. These sequences, as well as that of the CDC28 gene, were analyzed for the presence of consensus sequences postulated to be transcriptional or translational signals, or to be involved in mRNA processing. In addition, the predicted protein products of the four genes were subjected to a number of structural and statistical analyses including codon usage bias analysis, secondary structure analysis and hydropathicity analysis. PMID- 3018678 TI - Three RFLPs for the insulin receptor gene INSR: EcoRI, Pst I, Hind III. PMID- 3018677 TI - Nucleotide sequence of an insertion sequence (IS) element identified in the T-DNA region of a spontaneous variant of the Ti-plasmid pTiT37. AB - We have identified and determined the nucleotide sequence of an IS element (IS136) of Agrobacterium tumefaciens. This is the first IS element isolated and sequenced from a nopaline type Ti-plasmid. Our IS element has 32/30 bp inverted repeats with 6 mismatches, is 1,313 bp long and generates 9 bp direct repeats upon integration. IS136 has 3 main open reading frames (ORF's). Only ORF1 (159 codons) is preceded by sequences that are proposed to serve functional roles in transcriptional and translational initiation. No DNA sequence homology was found between IS136 and IS66, an IS element isolated from an octopine type Ti-plasmid. PMID- 3018679 TI - PvuII polymorphic sites in the human insulin receptor gene INSR. PMID- 3018680 TI - Human renin (REN) gene locus: BglII, RsaI and TaqI RFLPs. PMID- 3018681 TI - RFLP for a gene-related sequence of alpha 1-antitrypsin (AAT). PMID- 3018682 TI - An anonymous single copy genomic clone (M8) (D2S13) on chromosome 2 identifies a moderately frequent RFLP. PMID- 3018684 TI - A new RFLP for D18S3(B74) an anonymous genomic clone localized to 18p113. PMID- 3018683 TI - An anonymous human single copy genomic clone (D8S5) (TL11) on chromosome 8 identifies a moderately frequent RFLP. PMID- 3018685 TI - [Simultaneous 201Tl/99mTC seven-pinhole tomography in acute myocardial infarct]. AB - Combined infarction scintigraphy with 201Tl-chloride and 99mTc-pyrophosphate (PPi) by simultaneous seven-pinhole tomography was investigated with a phantom as well as in patients. No artificial defects occurred when the collimator was centered correctly in axial position, but a very high standard of image uniformity and linearity of the gamma camera was required. Artefacts by overlying activity from the skeleton or cardiac blood pool were not observed. All 11 controls showed normal results. Despite a poor depth resolution due to limitations of the system even small areas of partially damaged myocardium could be recognized and correlated three-dimensionally. Of 24 patients with proven myocardial infarction, in 16 both a positive (99mTc-PPi) and a negative (201Tl) image was obtained in congruence with the necrosis. 8 patients (33%) showed discordant results providing however additional information on the nature and extent of the necrosis. 4 out of 6 non-transmural infarctions seen by tomography had been suspected clinically. PMID- 3018686 TI - Mental health aftercare. Where is nursing? AB - The author reviews nursing's entrance into mental health aftercare and relates it to the problems that have ensued since the 50s. The role of the community mental health nurse is explored and compared with concepts of aftercare and perceptions of prominent psychiatric/mental health nursing authors. The realities of community mental health nursing in the '80s are contrasted with the thoughts and the ideas of others endeavoring to make contributions to the needs of discharged psychiatric patients. The author reminds us that aftercare begins before the patient leaves a psychiatric facility and stresses that nurses are a natural bridge between the hospital and the community. PMID- 3018689 TI - VIP receptor activity during HT29-18 cell differentiation and rat intestinal development. AB - In undifferentiated clonal HT29-18 cells, VIP produced an important (17-fold) and persistent increase in cyclic AMP generation for 10 min. This activation measured at 37 degrees C in the absence of IBMX was transient for 2 min and was considerably reduced (4-fold increase) in enterocyte-like cells. Retrodifferentiation of HT29-18 cells (after substitution of galactose for glucose) resulted in a partial reversion of VIP receptor activity. Cyclic AMP levels reached a peak value at 1 min but remained elevated over basal values for 10 min. Similarly, maturation of intestinal mucosae in 19-day-old rat fetuses and 5 day-old rats was accompanied by a remarkable reduction of VIP receptor activity. Cyclic AMP phosphodiesterase, adenylate cyclase activities and VIP receptor desensitization might explain this phenomenon. In contrast, the potency of VIP and the pharmacological properties of the VIP receptor are unchanged. We therefore assume that VIP receptor activity is an indicator of intestinal cell differentiation and we suggest that this neuropeptide may be a regulator of the normal and tumoral development of the intestinal mucosae in man and rat. PMID- 3018688 TI - Comparative efficacy of seven synthetic glucagon analogs, modified in position 1, 2 and/or 12, on liver and heart adenylate cyclase from rat. AB - Crude fresh membranes from rat liver and membranes from rat heart obtained according to Snyder and Drummond were tested for adenylate cyclase activation by glucagon (Gn) and seven glucagon analogs including (Ala2)-, (Arg12)-, (Des-His1, Arg12), (Phe1, Arg12)-, (N-Ac-His1, Arg12)-, (1-Me-His1, Arg12)-, and (3-Me-His1, Arg12)-glucagon. (Des-His1, Arg12)-glucagon acted as a competitive antagonist in heart membranes and as a partial agonist in liver membranes. Results obtained with analogs where His1 was modified suggest that the size of the imidazole ring and the charge of its nitrogen 1, but not the charge of the free amino group of histidine, played a major role in biological activity. When comparing functional glucagon receptors in liver and heart membranes, it appears that the first receptors were more sensitive to the hormone and more efficiently coupled to adenylate cyclase. PMID- 3018690 TI - Functional receptors for VIP, GIP, glucagon-29 and -37 in the HGT-1 human gastric cancer cell line. AB - Three separate sets of receptors sensitive to VIP, GIP and pancreatic/entero glucagons, have been characterized in HGT-1 cells. The order of relative potencies of VIP receptor agonists was VIP greater than rh GRF-43, rh GRF-29 greater than PHI greater than hp GRF-40, secretin. G-37 was about 4 times less potent than G-29 in HGT-1 cells (G-29 greater than G-37), whereas it was about 20 times more potent than G-29 in rat fundic glands (G-37 greater than G-29). Adenylate cyclase in HGT-1 cells was stimulated by VIP, G-29, G-37 and GIP, over a concentration from 3.16 X 10(-9) to 3.16 X 10(-7) M GIP. The experimental data: (1) support the enterogastrone activity of GIP, via adenylate cyclase activation and somatostatin release by gastric D cells; (2) demonstrate that HGT-1 cells originating from a human fundic tumor are sensitive to the glucagon-like peptides G-29 and -37, as rat fundic glands; (3) indicate that the pharmacological properties of the VIP receptor in this human gastric cell line are similar to those characterized in normal human gastric glands. PMID- 3018687 TI - Effector mechanisms of peptides of the VIP family. AB - The present review is focused on the exocrine pancreas and liver where the only known effector mechanism of VIP is the activation of adenylate cyclase in plasma membranes. A two-state model of activation-deactivation of the enzyme visualizes the participation of VIP receptors and Ns, the guanyl nucleotide stimulatory protein of adenylate cyclase. In the rat pancreas, VIP and GRF receptors are indistinguishable and disulfide bridges influence their functional integrity. The antagonism of VIP and somatostatin perhaps requires, at the adenylate cyclase level, the contribution of Ni, the guanyl nucleotide inhibitory protein. The potentiation of VIP by various stimulants acting on Ca2+ movements may rely on later events, e.g., on a concerted activation of protein kinases. When comparing quantitatively peptide binding to receptors with adenylate cyclase activation, cyclic AMP levels and amylase secretion, a tool is at hand to tailor synthetic agonists and antagonists of VIP, with appropriate changes in the N-terminal moiety of the peptide (a good agonist allows efficient coupling of receptors to the adenylate cyclase system). Apart from stimulus-secretion coupling, VIP may influence protein synthesis in the rat pancreas, through the phosphorylation of ribosomal protein S6, and may alter the activity of the endoplasmic reticulum via the phosphorylation of Mr = 21 kDa and Mr = 25 kDa proteins. In rat liver membranes, high affinity VIP receptors are specifically labelled with 125I helodermin and are coupled to adenylate cyclase (at variance with low affinity VIP receptors). These receptors are highly responsive to divalent cations and to guanyl nucleotides. PMID- 3018691 TI - Characterization of the vasoactive intestinal peptide (VIP) binding sites: a biochemical and an immunological approach. AB - The initial event of VIP action is its interaction with a specific receptor at the surface of a target cell. The understanding of the fine mechanism of action of VIP requires the characterization and of course the purification of the receptor. The understanding of the mechanism which regulates the number of receptor sites at the cell surface, if it occurs, is also a way to characterize the properties of VIP receptor. In this paper we report a number of data which represent several attempts to characterize VIP receptor in a human colonic adenocarcinoma cell (HT 29 cells). We have characterized a monoclonal antibody which partially inhibits 125I-VIP binding to HT 29 cells. We have specifically cross-linked, on intact HT 29 cells, a major polypeptide of Mr-64,000 with 125I VIP using DTSP or DSS as cross-linking reagents. This polypeptide behaves like a high affinity binding site for VIP. We have demonstrated that VIP is rapidly internalized in HT 29 cells (in less than 10 minutes) and that simultaneously VIP receptors were no more detectable on the cell surface by cross-linking experiments. This suggests that VIP is internalized together with its receptor. PMID- 3018692 TI - Solubilization of active receptors for VIP from guinea pig lung. AB - The zwitterionic detergent 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (CHAPS) was used to solubilize functional receptors for vasoactive intestinal peptide (VIP) from guinea pig lung. The binding of [125I]-VIP to the soluble receptors was time-dependent, reversible and saturable. Trypsin destroyed the activity of the receptors, indicating their proteinic nature. Dithiothreitol reduced the binding in a dose-dependent manner, suggesting the importance of disulfide bond(s) in receptor-binding activity. Binding of [125I]-VIP to the receptors was reversible and was competitively inhibited by unlabeled VIP, with 50% inhibition obtained at 305 nM VIP. PMID- 3018693 TI - Receptors for vasoactive intestinal peptide on isolated human sweat glands. AB - Human sweat gland function is predominantly under cholinergic control. Sweat secretion may also be regulated by Vasoactive Intestinal Peptide (VIP), which coexists with acetylcholine in nerves to sweat gland acini and ducts. We have examined the expression of VIP receptors on isolated human eccrine sweat glands. Two classes of specific binding sites for VIP were demonstrated: one with high affinity (Kd = 0.58-2.4 nM), and another with low affinity (Kd = 175-288 nM). The data suggest a physiological regulatory role for VIP in sweat gland function. PMID- 3018696 TI - VIP binding sites on synaptosomes from rat cerebral cortex: structure-binding relationship. AB - The structural requirements for VIP interaction with receptors on synaptosomes from rat cerebral cortex was investigated by the ability of VIP and VIP fragments, secretin analogues and fragments, peptides of the VIP/secretin family and several other regulatory peptides to inhibit specific 125I-VIP binding. Only large VIP fragments interacted with the VIP receptors with potencies relative to VIP ranging from 0.9-0.006%. The rank order of inhibition was: VIP 7-27 greater than VIP 11-28 greater than VIP 1-22-NH2 greater than VIP 16-28. Shorter fragments: VIP 18-28; VIP 18-28-NH2; VIP 19-28; VIP 21-28; VIP 22-28; VIP 1-18; VIP 1-18-NH2; VIP 1-10-NH2; VIP 1-6; VIP 16-20 and VIP 16-19 had no effect. Secretin fragments and analogues inhibited 125I-VIP binding with potencies of 2.2 0.01% relative to VIP in the order; secretin greater than (Ala4, Val5) secretin greater than (D-Ala4) secretin greater than (D-Phe6) secretin greater than secretin 5-27 greater than secretin 14-27. Other peptides of the VIP/secretin family inhibited 125I-VIP binding with potencies of 200-1%; avian VIP greater than porcine VIP greater than PHI = secretin greater than human GRF, whereas glucagon and GIP showed no inhibition. Among twenty-five other regulatory peptides only avian PP and somatostatin were inhibitors with relative potencies of 0.02% and 0.03%, respectively. In conclusion it may be emphasized that the intact VIP molecule is essential for VIP interaction with its receptors in the rat brain cortex.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018694 TI - Secretin receptor activity in rat gastric glands. Binding studies, cAMP generation and pharmacology. AB - We measured 125I-secretin binding to membranes prepared from rat fundic glands and compared the abilities of natural and synthetic secretin (SN) analogs to inhibit 125I-secretin binding and to activate the cAMP generating system in glandular and subcellular preparations from the fundus and antrum. The natural peptides structurally related to porcine secretin (pSN) included: chicken secretin (cSN), vasoactive intestinal peptide (VIP), porcine peptide with N terminal histidine and C-terminal isoleucine amide (PHI), helodermin, growth hormone releasing factors isolated from the rat hypothalamus (rhGRF-43, rhGRF-29) or from a human pancreatic tumour (hpGRF-40). These peptides inhibited the binding of 125I-secretin to rat fundic membranes: pSN greater than cSN greater than PHI, VIP and activated the cAMP generating system in fundic glands, according to the following order of potency; pSN greater than cSN greater than PHI, VIP greater than rhGRF-29 greater than rhGRF-43. Porcine peptide with N terminal tyrosine and C-terminal tyrosine (PYY), GIP, SOM and hpGRF-40 were inactive. Structural requirements for secretin receptor activity were evaluated with four synthetic secretin analogs corresponding to porcine secretin substituted at the N-terminal end by sequence portion of VIP, GIP, GLU and SOM: Ala4-Val5-SN(VIP-SN); Tyr1-Ala2-Glu3-SN (GIP-SN); Gln3-SN (GLU-SN) and Phe1-Phe1 Trp3-Lys4-SN (SOM-SN). The relative potencies of the analogs in fundic and antral preparations were: pSN greater than VIP-SN greater than VIP, GIP-SN greater than GLU-SN greater than SOM-SN for 125I-secretin displacement and cAMP production (glandular cAMP generation and adenylate cyclase activation).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018695 TI - Actions of VIP, hGRF, PHI and secretin: comparative studies in cerebral cortex and adenohypophysis. AB - We have examined the effects of hGRF on cyclic AMP and glycogen levels in mouse cerebral cortical slices. hGRF-44-NH2 and hGRF-28-OH did not stimulate cyclic AMP formation nor glycogenolysis and did not antagonize the stimulatory effects of VIP on cyclic AMP formation and glycogenolysis. These observations indicate that despite the structural homologies with VIP, hGRF does not interact with VIP receptors coupled to adenylate cyclase in mouse cerebral cortex. This is in contrast with observations in other tissues and species, such as rat and human intestinal epithelial membranes and rat pancreas. We have also compared the effects of hGRF, VIP, PHI and secretin on Growth Hormone (GH) release and cyclic AMP levels in anterior pituitary cells in vitro. VIP and PHI, but not secretin, promote at a high concentration (10(-6) M) a small but significant release of GH. This GH release is accompanied by increases in cyclic AMP levels. The concentration of VIP and PHI required to elicit these effects is high and suggests that VIP and PHI act as low affinity pharmacological analogs of hGRF on hGRF pituitary receptors. PMID- 3018697 TI - Vasoactive intestinal peptide actions on cyclic AMP levels in cultured striatal neurons. AB - The actions of vasoactive intestinal peptide (VIP) on intracellular cyclic AMP, in primary cultures of striatal neurons, were examined. VIP stimulated cyclic AMP formation five-fold over basal levels in neurons after 6 days in vitro (DIV); half maximal activation (EC50) was obtained with 10 nM of the peptide. VIP stimulation was both more potent and effective than those due to adrenocorticotropin (ACTH), dopamine (DA) or serotonin (5-HT). VIP efficacy was augmented to 15-20-fold in the presence of 0.1 microM forskolin, which had virtually no effect on cyclic AMP production alone; VIP potency was unaffected. At saturating concentrations of VIP (0.1-1.0 microM), no other agonist can further activate cyclic AMP production. Under these conditions, the interaction with opiate, DA D2 and 5-HT1 receptors, whose activation results in the inhibition of cyclic AMP production, was shown. During the differentiation of striatal neurons, VIP stimulation of cyclic AMP over basal levels, in the presence of 0.1 microM forskolin, decreases progressively from 30-fold after 3 DIV to 11-fold after 10-13 DIV. PMID- 3018698 TI - Circadian cycles in VIP content and VIP stimulation of cyclic AMP accumulation in the rat pineal gland. AB - VIP content in the rat pineal gland and cyclic AMP accumulation in response to VIP in the daily light and dark cycle were examined. VIP content in the pineal varied significantly during the day and night; the content decreased during exposure to light and was lowest at the onset of darkness, 6 p.m. (mean +/- SE, 23 +/- 5 pg/pineal), and increased during the night and was highest at the onset of light, 6 a.m. (72 +/- 12 pg/pineal). Response of cyclic AMP accumulation to VIP varied with a periodicity inversely related to the daily light and dark cycle of VIP content; cyclic AMP accumulation in response to 10(-7) M VIP increased in proportion to periods of exposure to light and peaked at 6 p.m., and decreased with the onset of darkness. PMID- 3018699 TI - Ectopic G-29 and G-37 glucagon secretion by hypercalcemic infantile renal tumors. AB - Four hypercalcemic infantile renal tumors were shown to secrete glucagon-like peptides. These unusual tumors were histologically classified as rhabdoid tumors of the kidney (3 cases) and a cellular mesoblastic nephroma (1 case). Elevated G 29 and G-37 glucagon levels were detected in the plasma and tumor extracts as well as in the supernatants of cultured tumor explants. Three of these tumors were heterotransplanted into the nude mice and serially passaged from a mouse to another. The glucagon level decreased in the transplanted tumor extracts with the number of passage. PMID- 3018700 TI - VIP regulation of a human pancreatic cancer cell line: Capan-1. AB - VIP and secretin control the secretory function of the normal pancreas. We analysed their regulatory functions in a human pancreatic cancer cell line: Capan 1. Saturation binding experiments with 125I-VIP showed the existence of one class of binding sites of very high affinity: KD 6.4 +/- 3.0 X 10(-11) M and a low Bmax: 12 fmoles/10(6) cells, in both intact cells and membrane preparations. This site has not yet been described in normal or tumorous digestive cells. Competition binding experiments let us characterize two more binding sites, KD: 2.1 +/- 0.7 X 10(-9) M and 5.0 +/- 0.6 X 10(-8) M and the corresponding Bmax: 120 and 500 fmoles/10(6) cells. These sites are similar to those found on cells of the digestive tract. Competition binding experiments gave the following IC50: 3.0 +/- 0.9 X 10(-9) M for VIP; 2 +/- 0.6 X 10(-6) M for PHI; and 1 +/- 0.7 X 10(-5) M for secretin. VIP elicited a cAMP rise, the half maximal response being obtained at 1.2 X 10(-10) M. Secretin induced a cAMP response but only for concentrations higher than 10(-8) M. VIP receptors were found to be modulated by two factors: cell ageing and cell density. Cells chronically treated with VIP showed a slight decrease of their proliferation; insulin exerted an opposite effect. It is concluded that at the difference of normal pancreatic cells, the present cell line lacks secretin-preferring receptors and acquires some of the properties of intestinal cells. PMID- 3018701 TI - VIP interaction with intestinal epithelial cells and liver plasma membranes from hepatectomized rats. AB - Specific binding of VIP and VIP-stimulated cyclic AMP accumulation were studied in jejuno-ileal (villous and crypt) and colonic epithelial cells three days after partial (70%) hepatectomy in the rat. The binding capacity (but not the affinity) of VIP receptors, and the efficiency (but not the potency) of VIP on cyclic AMP stimulation increased after hepatectomy (as compared to sham-operated rats). On the other hand, the affinity (but not the binding capacity) of VIP receptors in plasma membranes of the regenerating liver was decreased as compared to the control condition. The possibility that these findings may be related to an involvement of VIP in hepatic growth regulation deserves further investigation. PMID- 3018702 TI - Conformation of peptides of the secretin-VIP-glucagon family in solution. AB - The significance of a well defined molecular architecture in hormone receptor interaction and the methods available for the study of preferred conformations are discussed. The conformational freedom in glucagon is a major obstacle in the determination of its biologically relevant geometry. In the secretin molecule intramolecular forces generate a folded, partially helical conformation. In respect of long range cooperative interactions resulting in a compact molecule with secondary-tertiary structure secretin is similar to globular proteins. In VIP some characteristics of secretin and also of glucagon can be recognized. Further progress in conformation analysis can be expected from the study of rigid, cyclic analogs in which the biological activities of the parent hormones are retained or even enhanced. Such analogs have well defined conformations without external stabilization from membrane mimicking lipids. Therefore, they provide information on the biologically relevant geometry of the hormones and contribute also to our knowledge of receptor sites. PMID- 3018703 TI - Comparative structural requirements of thirty GRF analogs for interaction with GRF- and VIP receptors and coupling to adenylate cyclase in rat adenopituitary, liver and pancreas. AB - The ability of 30 synthetic GRF(1-29)-NH2 analogs to stimulate adenylate cyclase activity was investigated in membranes from rat adenopituitary, rat liver and rat pancreas. In adenopituitary membranes, GRF and GRF analogs interacted with specific GRF receptors, whereas in liver and pancreatic membranes, they interacted with VIP receptors. The C-terminal moiety of GRF was responsible for GRF receptor recognition as the hybrid analog (His1, D-Ala2)-GRF(1-9), VIP(10-28) stimulated pituitary adenylate cyclase through the occupancy of VIP receptors only. When GRF or VIP receptors were occupied by GRF analogs, the N-terminal part of the ligand appeared critical for adenylate cyclase activation. This was established by testing 30 GRF analogs mono-, bi- or tri-substituted in positions 1 to 10. Major observations included: (a) the characterization of (N-Ac-Tyr1, D Arg2)-GRF(1-29)-NH2 as an antagonist of GRF-stimulated pituitary adenylate cyclase; (b) the discovery of the (N-Ac-Tyr1, D-Phe2)-, (His1, D-Ala2, D-Ser3, NLeu27)-, and (His1, D-Ala2, D-Thr7, NLeu27)-derivatives of GRF(1-29)-NH2 as specific antagonists of VIP receptors in rat pancreatic membranes; (c) the importance of the free NH2 function of amino acid residue 1 for pancreatic adenylate cyclase activation, and (d) the decreased efficiency of iodinated (Tyr1)-GRF(1-29)-NH2 as opposed to the non iodinated form, in all systems tested. PMID- 3018704 TI - Identification of two pro-VIP forms in a human neuroblastoma cell line. AB - The immunoreactivity of VIP and PHI standards, immobilized on a nitrocellulose membrane, was first assayed with various detection procedures. For VIP, the double bridge peroxidase-antiperoxidase (PAP) method was the most sensitive procedure, giving a detection limit of 0.1-0.3 pmol per mm2 with the 4 rabbit anti-VIP antisera tested. By contrast, the detection limit of immobilized PHI was 100 times higher with the 4 rabbit anti-PHI/PHM antisera tested presumably because major antigenic sites were masked in the immobilized peptide. With this information at hand, the VIP and PHI immunoreactivity of human neuroblastoma NB OK-1 cells was tested after extraction, SDS-PAGE, electrotransfer, and PAP immunodetection. Two faint immunoreactive bands corresponding to two pro-VIP forms with an Mr of, respectively, 19 kDa and 18 kDa, were detected in undifferentiated cells. These distinct bands increased progressively and markedly during differentiation in the presence of dibutyryl cyclic AMP. In addition, two intermediary VIP forms of lower Mr (11 kDa and 6 kDa) and 3 kDa VIP itself were also present after 2 days of differentiation. The 19 kDa and 18 kDa pro-VIP forms were detected with a sensitivity several times higher than that of VIP and their staining was specific for VIP epitopes. By contrast, when using 4 rabbit anti PHI/PHM antisera, we observed essentially the strong unspecific staining of a 17 kDa polypeptide. VIP immunoreactivity was also visualized by immunocytochemistry in neuroblastoma cells cultured on glass coverslips and fixed in situ. Specific VIP staining using the PAP method was present in 10 percent of the cells in the undifferentiated state.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3018705 TI - Structural requirements for gastric inhibitory polypeptide (GIP) receptor binding and stimulation of insulin release. AB - The effect of bovine GIP 1-42 and several of its fragments in competing with the binding of 125I-GIP to beta-cell plasma membranes from transplantable hamster insulinoma, and in stimulating insulin release from the isolated perfused rat pancreas, was investigated. Our results, in association with the results of previous studies, indicate that the sequence 17-38 is necessary for receptor binding and biological activity of GIP. By contrast, the N-terminal portion of GIP can be removed without seriously impairing the activity of the molecule. PMID- 3018706 TI - Interaction of synthetic N- and C-terminal fragments of helodermin with rat liver VIP receptors. AB - Synthetic helodermin was more potent than natural helodermin (purified from the venom of Gila monster) in activating adenylate cyclase in rat liver membranes. Two possible reasons for this discrepancy were discussed. Comparing adenylate cyclase activation to the binding of 125I-natural helodermin and 125I-VIP in the presence of six synthetic helodermin fragments (1-27, 7-35, 13-35, 17-35, 18-35 and 22-35), we conclude that the effects of both synthetic and natural helodermin were mediated through an interaction with VIP receptors. The C-terminal (28-35) extension of these peptides favored VIP receptor recognition. Their N-terminal extremity was not necessary for binding and for ensuing adenylate cyclase activation, illustrating again the atypical coupling of VIP receptors with the effector enzymatic system, in rat liver membranes. PMID- 3018707 TI - The receptors of the VIP family peptides (VIP, secretin, GRF, PHI, PHM, GIP, glucagon and oxyntomodulin). Specificities and identity. AB - A model is proposed for the receptors of the VIP family peptides including a ligand and a cellular domain. Specificities of the receptors are due to different ligand binding sites. Three subgroups of the family can be distinguished accordingly: glucagon and oxyntomodulin; GIP; VIP, secretin r and hGRF, PHI and PHM. In the same species, the expression of these different sites is cell specific resulting in a stoichiometry of the ligand-receptor interaction which is compatible with physiological regulation of cell function. Specificities of the interaction as studied by native and synthetic analogs is supported both by restricted sequences of amino acids (such as that including the N-terminal histidine residue), and membrane-induced configuration of the ligand. Identity of the receptors is related to their interactions with subunits of the adenylate cyclase system. Arguments are put forward indicating that the alpha subunit of the guanyl regulatory protein is a reasonable candidate for directly transducing to the adenylyl cyclase the information contained in the activated ligand-binding site subunits. Evidence of functional and molecular heterogeneity of the recognizing site and of the alpha subunits leads to the supposition that some types of specific complementarity is retained at this level of interaction, further enhancing the possibility of species and cell differences. On the other hand, the identities found in other sequences of the alpha and ras oncogene products extend to the receptor of the VIP family peptides a pattern of organization which is similar to that recently described for the insulin family of receptors. The role of ligand specific receptor mediated regulation in homologous or heterologous desensitization is reviewed in brief for the peptides of the VIP family as well as the appearance of the specific receptor during the ontogenesis or the cell differentiation. The co-distribution of plasma membrane receptors from other families further adds to the cell specificity resulting for each differentiated cell in unique patterns of recognizing site. Some examples of receptor-receptor interaction are given, indicating that the integration of the different signals by cells might occur at an early step through the transmembranair domain of the receptor. PMID- 3018709 TI - Hepatocellular carcinoma presenting as a solitary metastasis to the scapula. Case report and review of the literature. AB - Although primary hepatocellular carcinoma is uncommon, metastasis to the upper extremity as a presenting symptom is even more rare. Recent case reports and autopsy surveys document that extrahepatic spread of this carcinoma occurs in 30% to 78% of patients, who usually are without regional symptoms involving bone. Although metastatic spread to the lungs and lymph nodes occurs more commonly, the incidence of bone metastases has increased according to previous reports and is now estimated from 2% to 13%. This case report concerns widely disseminated hepatocellular carcinoma presenting initially without any other systemic signs except for shoulder pain and induration. Due to the aggressive nature of this tumor, early detection is crucial. Early diagnosis may offer the only real hope for establishing effective treatment and improving the chances for long-term survival. PMID- 3018708 TI - Adenosine triphosphatase activity of crystalline inclusions in alveolar soft part sarcoma. An ultrahistochemical study of a case. AB - A case of alveolar soft part sarcoma was studied by light and electron microscopy and by electron microscopic enzyme histochemistry of adenosine triphosphatase (ATPase) and 5'nucleotidase(5'Nase). The tumor showed distinct alveolar pattern and diastase resistant PAS positive crystalline inclusions were found in the cytoplasm. Ultrastructurally, characteristic rhomboid crystals and dense granules were observed and they were positive for Mg++- and Ca++-ATPases but negative for 5'Nase. Tumor cell membrane also showed positive activity of ATPase in addition to 5'Nase. These results would support the myogenous derivation of alveolar soft part sarcoma. PMID- 3018710 TI - Cytomegaloviral infection in children. Recent advances in clinical management. PMID- 3018711 TI - Glomus tumour of the hallux: diagnosis by Doppler-shift ultrasound and digital subtraction angiography. AB - A case is presented of a glomangioma with typical history and clinical findings, proven by operation and histology. Unique radiographic features are demonstrated including visualization of the tumour on a soft tissue radiograph and associated hyperaemic bone changes, continuous wave Doppler results indicating hyperaemia and an arterio-venous malformation, and the clear demonstration of the tumour in both frontal and lateral views was possible by intra-arterial digital subtraction angiography (DSA) under local anaesthesia. Fibrous dysplasia of a femur was an incidental finding. PMID- 3018712 TI - Evaluation of the immunogenicity, stability, pathogenicity, and immunodepressive potential of four commercial live infectious bursal disease vaccines. AB - The study was divided into three experiments. In each, day-old specific pathogen free (SPF) White Leghorns were injected subcutaneously with one of the following commercial live infectious bursal disease (IBD) vaccines: Burcell, Bursine, Clone Vac D-78, and S-706. In the first experiment, the immunogenicity of each vaccine was determined by challenge with virulent IBDV at 25 days of age. All four vaccines were equally efficacious in preventing clinical IBDV infection. The second experiment determined the stability and pathogenicity of the four vaccines by successive back passage of each virus in SPF chicks for 4 consecutive weeks. Although all vaccines were capable of spreading to contact controls, only the two intermediate vaccines (D-78 and S-706) produced slightly atrophied bursae and moderate microscopic bursal lesions. However, both vaccines were stable, because neither reverted back to increase virulence nor resulted in morbidity or mortality associated with virulent IBDV after successive passage. In the third experiment, the immunodepressive potential of each vaccine virus was determined by examining the ability of IBD-vaccinated birds to respond to Newcastle disease (ND) virus vaccination. None of the four vaccines was found to be immunodepressive, as all IBD vaccinated birds responded to NDV vaccination. PMID- 3018713 TI - Diseases of the salivary glands. PMID- 3018714 TI - ["Paradent" use in various diseases of the oral mucosa]. PMID- 3018715 TI - [Transbronchial lymph node puncture using the fiber bronchoscope in pulmonary neoplasms]. PMID- 3018716 TI - Purification and characterization of pyridoxal kinase from human erythrocytes. AB - Pyridoxal kinase has been purified 50,000-fold from human erythrocytes. The purification procedure included dextran-induced aggregation of red blood cells, ammonium sulphate fractionation of the haemolysate, DEAE-cellulose chromatography, hydroxyapatite chromatography. Sephadex G-100 gel filtration and omega-aminooctyl agarose chromatography. The enzyme preparation migrated as a single protein and activity band on analytical gel electrophoresis. Determination of the Michaelis constants for pyridoxal, pyridoxine and pyridoxamine using a new assay gave comparable values of 33 microM, 16 microM and 6.2 microM respectively. Various amines were shown as competitive inhibitors of pyridoxal kinase with respect to ATP. The inhibition order was: N-dansyl-1,8-diaminooctane greater than 1,8-diaminooctane greater than 1,6-diaminohexane greater than 1,4-diaminobutane greater than gamma-aminobutyric acid, whereas octane, hexane and butane were not inhibitors. Results suggest that the amino groups on the above inhibitors are essential for competitive inhibition at saturating concentrations of pyridoxal. It was also observed that increasing the chain length of the hydrophobic backbone of these competitive inhibitors can facilitate its action. PMID- 3018717 TI - The role of ACTH in placental steroidogenesis. AB - We have studied the possible function of the placental adrenocorticotrophic hormone-(ACTH-) like substance (PALS) in placental steroidogenesis by measuring oestradiol (E2) and progesterone (P4) in term human placental explants incubated with commercially available porcine ACTH I-39. There was a dose-dependent increase in the E2 and P4 released into the medium at 24 h as compared to controls. At 48 h, no significant effect was noted. Propranolol (10(-5) M) did not block the effect of ACTH on P4 release. The data suggest that ACTH may have a regulatory role on placental steroidogenesis. The possible mechanisms of action of PALS on the placenta and the adrenal are discussed, and the role of PALS in the maintenance of pregnancy and in maternal response to stress is suggested. PMID- 3018718 TI - Use of multiple radioligands to characterize adenosine receptors in human placenta. AB - To identify and characterize fully the adenosine receptor of human placenta, particulate preparations of placental parenchyma were examined using several adenosine receptor ligands, [3H]2-chloradenosine ([3H]2-Cl-ADO), [3H]N6 ethylcarboxamideadenosine ([3H]NECA), and [3H]N6-(L-phenylisopropyl)-adenosine ([3H]PIA). Each of the three ligands bound to the particulate preparations with unique binding characteristics. Binding of [3H]PIA characterized a high-affinity low-capacity receptor while the [3H]NECA displayed lower-affinity, higher capacity binding. Saturation isotherms for [3H]2-Cl-ADO appeared intermediate in terms of affinity and binding capacity. Saturation isotherms of binding data also disclosed displaceable non-receptor binding at high labelled ligand concentrations. Competitive binding studies supported the observation that PIA and NECA bind to sites of high and low affinity, respectively. However, the multicomponent competition curves for [3H]2-Cl-ADO binding suggest binding to the receptors with differential affinity. These studies confirm the presence of adenosine receptors in the human placenta and suggest that these receptors are of both the high-and low-affinity subtypes. PMID- 3018719 TI - Isolation and characterization of trophoblast from murine placenta. AB - A discontinuous density gradient centrifugation method, devised to isolate enriched populations of trophoblast from murine definitive placentae, is described. It is concluded that the isolated adherent cells are trophoblast on the basis of the following characteristics: they are fetally derived, as determined by their donor glucose phosphate isomerase phenotype in embryo transfer experiments; epithelial cells, as shown by the presence of cytokeratin filaments and the absence of vimentin; negative for the stage-specific embryonic antigen-I (SSEA-I); and capable of progesterone secretion. Initially, they grew as individual polygonal cells, tending to form tight confluent monolayers with poorly defined intercellular boundaries. They were mono- or binucleate and increased their nuclear size with time. After two days, giant cells appeared to be formed from binucleated cells by nuclear fusion, and multinucleated cells appeared forming syncytia. Some of these cells also seemed to form giant cells. A low percentage (1 to 10 per cent) of contaminating cells, mainly macrophage-like cells, was observed. The isolated cells were a mixture of alkaline phosphatase- (AP-)positive and AP-negative cells, with some of the latter having phagocytic capacity. All were Fc receptor-negative. The possible identity of these cells in relation to trophoblast in the intact placenta is discussed. This method of isolating and characterizing trophoblast cells from the definitive mouse placenta will be a useful tool for studying the biology and immunology of trophoblast. PMID- 3018720 TI - [Spasmophilia or panic attack?]. AB - For many years, the symptoms grouped under the label "spasmophilia" have been differently evaluated in France by psychiatrists, who ascribe them to hysteria or anxiety, and by endocrinologists and general practitioners for whom they are all due to neuromuscular hyperexcitability, the cause of which must be sought in the biochemistry of calcium. Recent studies on anxiety should reduce the gap between these discordant views. The concept of anxiety itself has changed owing to the discriminant effects of psychotropic drugs and to a classification based on precise diagnostic criteria without theoretical presuppositions. Since anxiety can be biochemically induced, the hypotheses put forward by "spasmophilologists", such as disorders of calcium metabolism or hyperventilation, can now be tested in the laboratory. PMID- 3018721 TI - Interaction between proteins localized in membranes. AB - We present a conceptual framework for evaluating the effect on the self association of proteins in membranes due to the presence of other proteins at high concentrations (excluded volume effect) and the high concentration and preoriented state of the reactive species. We have calculated the magnitude of such effects using plausible values for the concentrations of proteins in membranes, for the degree to which proteins may tilt and move vertically, and for their dimensions. Compared to the association of monomers tumbling freely in an experimentally realistic volume, we calculate that these factors alone can increase the likelihood of forming dimers 10(6)-fold and of forming trimers and higher oligomers many orders of magnitude greater. We discuss the implications of our calculations for experimental manipulations of membrane proteins, for biosynthetic assembly of multisubunit membrane proteins and formation of membrane lesions by assemblies of exogenous proteins, and for the activation of cellular events induced by the interaction of membrane receptors with themselves or with other membrane proteins. PMID- 3018722 TI - In vitro transcription of human mitochondrial DNA: accurate termination requires a region of DNA sequence that can function bidirectionally. AB - Mammalian mitochondrial genomes have a presumptive transcription termination site at the 16S rRNA-tRNALeu gene boundary. We have developed an in vitro system from human KB cells that terminates transcription at this gene boundary. By S1 nuclease protection, the 3' ends of terminated transcripts were mapped 3 and 4 base pairs upstream of the 16S rRNA-tRNALeu gene boundary, in agreement with in vivo data. By high-resolution sizing of transcripts, the 3' end was mapped 7 +/- 1 base pairs downstream from the gene boundary. Termination occurs with equal efficacy from transcriptional initiation at the heavy- or light-strand promoter. All template nucleotide sequence information necessary for termination appears to be located near the termination site itself. An unexpected observation is that the termination region functions bidirectionally. PMID- 3018723 TI - Biologically active precursor for transforming growth factor type alpha, released by retrovirally transformed cells. AB - Retrovirus-transformed rat cells that actively express transforming growth factor type alpha (TGF-alpha) release into the medium a soluble protein of 17-19 kDa that has been identified as a precursor for TGF-alpha. The identification of this protein as a TGF-alpha precursor is based on its recognition by specific antibodies and by the ability of elastase to convert this protein into a 6-kDa polypeptide with the properties of mature TGF-alpha. This TGF-alpha precursor binds to epidermal growth factor/TGF-alpha receptors and activates the receptor associated tyrosine kinase activity in intact cells. The biological potency of this precursor is not markedly increased by conversion into mature TGF-alpha in vitro. These studies demonstrate the ability of transformed cells to release a TGF-alpha precursor capable of strong mitogenic action in vivo. PMID- 3018724 TI - A DNA binding protein specific for an origin of replication of herpes simplex virus type 1. AB - We have identified a protein that binds specifically to an origin of replication (oris) of the herpes simplex virus type 1 genome. The oris binding protein, detectable only in nuclear extracts of infected cells, shows the same time course of appearance as the herpesvirus-induced DNA polymerase and the DNA binding protein ICP8. The partially purified oris binding protein generates a DNase I "footprint" that spans 18- of the 90-base-pair minimal oris sequence. The oris binding protein may, therefore, be analogous to other origin-specific binding proteins that are required for the initiation of viral and chromosomal DNA replication. PMID- 3018725 TI - Photoaffinity labeling of the gonadotropin receptor with native, asialo, and deglycosylated choriogonadotropin. AB - Human choriogonadotropin (hCG) is a heterodimeric hormone composed of an alpha and a beta subunit. hCG and its asialo (ashCG) and deglycosylated (dghCG) forms vary in their ability to stimulate hormone responsive adenylate cyclase. ashCG is a partial agonist, and dghCG is an antagonist. Photoactivatable moieties were coupled to hCG, ashCG, and dghCG, and the derivatives were radioiodinated. Competitive binding studies indicate that all of the derivatives had a similar affinity for the gonadotropin receptor on porcine granulosa cell membranes. Radiolabeled derivatives were used to photoaffinity label the gonadotropin receptor. Radiolabeled complexes were separated by NaDodSO4/PAGE. All of the derivatives produced similar autoradiographic patterns, except that dghCG produced an additional 48-kDa complex. To investigate the structure of the complexes further, peptide mapping of proteolytic digests was used. All, except for the 48-kDa complex, generated similar peptide maps indicating a relationship between those complexes in which the smaller components are part of the larger. The 48-kDa complex contained both subunits of 40-kDa dghCG. Therefore, this complex is expected to contain an additional component of 8 kDa. The complex was generated whether the hormone-receptor complex was photoaffinity labeled on cells, on isolated membranes, or after solubilizing in detergent. Formation was blocked by excess hCG and did not occur in the absence of UV irradiation. We conclude that the hCG derivatives are able to photoaffinity label the hCG receptor but that the dghCG derivative can photoaffinity label an additional component that was not observed when derivatives of hCG or ashCG were used to label the receptor. PMID- 3018726 TI - Species-specific in vitro synthesis of DNA containing the polyoma virus origin of replication. AB - In vitro replication of DNA containing the polyoma (Py) virus origin of replication has been carried out with cell-free extracts prepared from mouse FM3A cells. The in vitro system required the Py virus-encoded large tumor (T) antigen, DNA containing the Py virus origin of replication, ATP, and an ATP-regenerating system. The replication reaction was inhibited by aphidicolin, suggesting the involvement of DNA polymerase alpha in this system. Simian virus 40 (SV40) T antigen could not substitute for the Py T antigen. Cell extracts prepared from HeLa cells, a source that replicates SV40 DNA in the presence of SV40 T antigen, replicated Py DNA poorly. The addition of purified DNA polymerase alpha-primase complex isolated from FM3A cells enabled HeLa cell extracts to replicate Py DNA with the same efficiency as FM3A cell extracts. Complementary experiments have shown that FM3A cell extracts do not support SV40 DNA replication unless supplemented with DNA polymerase alpha-primase complex from HeLa cells [Murakami, Y., Wobbe, C.R., Weissbach, L., Dean, F.B. & Hurwitz, J. (1986) Proc. Natl. Acad. Sci. USA 83, 2869-2873]. These results indicate that the host-cell source of the DNA polymerase alpha-primase complex plays an important role in discriminating between SV40 T antigen- and Py T antigen-dependent replication of their homologous DNA in vitro. This may explain the host-range specificity of these viruses in vivo. PMID- 3018727 TI - Functional expression of rat cytochrome c in Saccharomyces cerevisiae. AB - To determine whether a mammalian cytochrome c could efficiently replace iso-1 cytochrome c, which is encoded by the yeast CYC1 gene, the coding sequence of RC9 (a nondefective processed gene from rat) was cloned in both single- and multiple copy expression vectors under the direction of the yeast alcohol dehydrogenase 1 (ADC1) promoter. Upon transformation of a CYC1 deletion strain, the multiple-copy construct restored a wild-type growth rate on lactate medium; such growth normally requires derepressed amounts of iso-1-cytochrome c. These transformants expressed a level of hybrid ADC1/RC9 mRNA approximately 5- to 10-fold greater than the amount of message from the endogenous ADC1 gene and produced a steady state level of rat cytochrome c equivalent to that of the wild-type yeast protein. A requirement for the vector was evidenced by its absence in all transformants that lost the lactate growth phenotype after propagation in nonselective medium. In contrast, the level of vector-specific message in single copy was equivalent to that of the endogenous ADC1 mRNA, but transformants exhibited no significant growth on lactate. Constructions having a small deletion or a mammalian intron within the rat cytochrome c coding region failed to support lactate-dependent growth, indicating that complementation depends upon proper translation of the correct rat coding sequence. Therefore, the rat polypeptide, when expressed at normal physiological levels, is recognized by the yeast machinery involved in the multiple steps required for the processing and transport of an active cytochrome c as well as its functional interaction with the respiratory apparatus. PMID- 3018728 TI - Beta-agonist- and prostaglandin E1-induced translocation of the beta-adrenergic receptor kinase: evidence that the kinase may act on multiple adenylate cyclase coupled receptors. AB - beta-Adrenergic receptor kinase (beta-AR kinase) is a cytosolic enzyme that phosphorylates the beta-adrenergic receptor only when it is occupied by an agonist [Benovic, J. Strasser, R. H., Caron, M. G. & Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. USA 83, 2797-2801.] It may be crucially involved in the processes that lead to homologous or agonist-specific desensitization of the receptor. Stimulation of DDT1MF-2 hamster smooth muscle cells or S49 mouse lymphoma cells with a beta-agonist leads to translocation of 80-90% of the beta AR kinase activity from the cytosol to the plasma membrane. The translocation process is quite rapid, is concurrent with receptor phosphorylation, and precedes receptor desensitization and sequestration. It is also transient, since much of the activity returns to the cytosol as the receptors become sequestered. Stimulation of beta-AR kinase translocation is a receptor-mediated event, since the beta-antagonist propranolol blocks the effect of agonist. In the kin- mutant of the S49 cells (lacks cAMP-dependent protein kinase), prostaglandin E1, which provokes homologous desensitization of its own receptor, is at least as effective as isoproterenol in promoting beta-AR kinase translocation to the plasma membrane. However, in the DDT1MF-2 cells, which contain alpha 1-adrenergic receptors coupled to phosphatidylinositol turnover, the alpha 1-agonist phenylephrine is ineffective. These results suggest that the first step in homologous desensitization of the beta-adrenergic receptor may be an agonist promoted translocation of beta-AR kinase from cytosol to plasma membrane and that beta-AR kinase may represent a more general adenylate cyclase-coupled receptor kinase that participates in regulating the function of many such receptors. PMID- 3018729 TI - A synthetic approach to structure-function relationships in the murine epidermal growth factor molecule. AB - Murine epidermal growth factor, a 53-amino acid peptide that is mitogenic for a number of cell types, has been synthesized by the solid-phase method. The synthetic peptide is identical to the natural material in amino acid composition, chromatographic behavior, receptor binding, and stimulation of DNA synthesis. Fragments of the EGF molecule corresponding to residues 42-53, 32-53, and 15-53 were constructed as well as the methionine sulfoxide derivative of EGF, [Met(O)21]EGF-(1-53), and a polymeric form of EGF. [Met(O)21]EGF-(1-53) was slightly less active than EGF in receptor binding and stimulation of DNA synthesis. Polymeric EGF was 1/100th as active as EGF, while EGF-(15-53) was less potent than EGF-(1-53) by a factor of 10(4). EGF-(32-53) was even less active and EGF-(43-53) was inactive. PMID- 3018731 TI - Inositol cyclic triphosphate [inositol 1,2-(cyclic)-4,5-triphosphate] is formed upon thrombin stimulation of human platelets. AB - Cleavage of polyphosphoinositides in vitro by phospholipase C results in formation of both cyclic and noncyclic inositol phosphates. We have now isolated the cyclic product of phosphatidylinositol 4,5-bisphosphate cleavage, inositol 1,2(cyclic)-4,5-triphosphate [cIns(1:2,4,5)P3], from thrombin-treated platelets. We found 0.2-0.4 nmol of cIns-(1:2,4,5)P3 per 10(9) platelets at 10 sec after thrombin; none was found in unstimulated platelets or in platelets 10 min after thrombin addition. We conclude that cIns(1:2,4,5)P3 is a major product of polyphosphoinositide metabolism in thrombin-stimulated platelets. PMID- 3018730 TI - Isolation and characterization of a complementary DNA specific for human aromatase-system cytochrome P-450 mRNA. AB - A cloned complementary DNA sequence has been isolated from a human placental cDNA library in the bacteriophage expression vector lambda gt11 after screening with polyclonal antibodies against human placental aromatase-system cytochrome P-450 (P-450Arom). A single recombinant clone, lambda hAROM1, was characterized by its ability to generate a beta-galactosidase fusion protein that reacted independently with polyclonal antibodies raised against beta-galactosidase and cytochrome P-450Arom and with monoclonal antibodies specific for cytochrome P 450Arom. The cDNA insert, which was found to be 1.8 kilobases in length, was radiolabeled and used to analyze poly(A)+ RNA isolated from human placenta and total RNA isolated from human adipose stromal cells cultured in the absence or presence of regulatory factors. The radiolabeled cDNA hybridized to several size species of mRNA in both placental and adipose stromal cell RNA fractions. Changes in the levels of adipose stromal cell RNA that hybridized to the cDNA insert were associated with comparable changes in the levels of translatable cytochrome P 450Arom mRNA and aromatase system activity. These findings are indicative that lambda hAROM1 contains DNA sequences complementary to human cytochrome P-450Arom mRNA and are suggestive that regulatory factors affect aromatase activity by altering the transcriptional activity of the cytochrome P-450Arom gene. PMID- 3018732 TI - Efficient reversion of simian sarcoma virus-transformation and inhibition of growth factor-induced mitogenesis by suramin. AB - Simian sarcoma virus, an acutely transforming primate retrovirus with capacity to induce gliomas and sarcomas in experimental animals, has acquired its transforming properties by transducing the cellular gene sequences that encode one of the constituent chains of platelet-derived growth factor. Suramin, a drug used in the treatment of trypanosomiasis and onchocerciasis, has previously been reported to inhibit the interaction of platelet-derived growth factor with its cell surface receptor. We show here that suramin efficiently reverts the simian sarcoma virus-induced transformed phenotype in human and rat fibroblasts and propose that this is due to neutralization of an externalized v-sis product. Moreover, we show that suramin inhibits the action of a broad spectrum of growth factors. PMID- 3018733 TI - Intracellular potassium depletion in IM-9 lymphocytes suppresses the slowly dissociating component of human growth hormone binding and the down-regulation of its receptors but does not affect insulin receptors. AB - We have investigated whether the slowly dissociating component of insulin and human growth hormone (hGH) binding and the homologous down-regulation of their receptors in IM-9 cultured human lymphocytes are due to distinct conformations of the receptor or, rather, to a redistribution within the cell. To do so, we used intracellular K+ depletion, which has been shown to inhibit reversibly coated-pit formation and ligand internalization in some cell lines. IM-9 cells incubated in K+-free buffer after a hypotonic shock rapidly lost their K+, which was stabilized at +/- 50% of control by incubation in K+-free binding assay buffer. In K+-depleted cells, the hGH dissociation kinetics became monoexponential and, in contrast with control cells, compatible with the equilibrium constant derived from saturation and association data using a simple model. The loss of hGH receptors during competition studies was abolished. The down-regulation by unlabeled hGH was decreased by 80%. In contrast, insulin receptor kinetics remained unchanged (non-first-order) in the K+-depleted cells; the negative cooperativity and the down-regulation (60%) were identical to those of control cells. Quantitative electron microscopic autoradiography showed a decrease of +/- 50% in the fraction of 125I-labeled hGH internalized. The number of visible coated pits in the membrane was reduced by 80%. Thus, in IM-9 cells, association with coated pits and endocytosis appear to play a major role in the kinetics of hGH binding and in the down-regulation of its receptors, but not in insulin receptor binding kinetics and down-regulation. PMID- 3018735 TI - Fibrinogen mitogenic effect on hemopoietic cell lines: control via receptor modulation. AB - The possibility that the mitogenic effect of fibrinogen, a major plasma protein (3 mg/ml), is mediated by specific membrane receptors was studied. Specific binding analysis showed that fibrinogen receptors are present only on hemopoietic cell lines that respond to its mitogenic effect. The mitogenic fibrinogen receptor is not recognized by antibodies specific for the platelet fibrinogen receptor or is not competitively blocked by synthetic peptides containing the Arg Gly-Asp sequence, which is common to fibronectin, fibrinogen, vitronectin, and other cell-attachment proteins. The lymphoma-derived pre-B-cells (Raji) have 149,000 receptors, whereas the lymphoma-derived T cells (JM), which are 3 times smaller, have 54,000 receptors. These receptors have a Kd of 2 X 10(-7) M. They are inducible by stimuli specific for the cell lineage: activators of the breakdown of phosphatidylinositol phosphates, such as platelet activating factor for Raji cells, and adenylate cyclase agonists and cAMP analogues for JM cells. The stimuli have no mitogenic effect in the absence of fibrinogen; they do not change the Kd. Each stimulus increases the number of fibrinogen receptors in a dose-dependent manner, which correlates strongly (r = -0.98, n = 5) with an increased growth rate of cells in the presence of fibrinogen. This correlation concludes that the mitogenic effect of fibrinogen is controlled via receptor modulation. PMID- 3018734 TI - Phosphorylation of the fibronectin receptor complex in cells transformed by oncogenes that encode tyrosine kinases. AB - The fibronectin (FN) receptor in avian cells has been characterized previously as a complex of three membrane glycoproteins of about Mr 160,000, Mr 140,000, and Mr 120,000 (simply termed protein band 1, band 2, and band 3, respectively). Monoclonal antibodies to the band 3 protein of the complex prevent FN and laminin binding both in vivo and in vitro and enable the detection of the receptor proteins in the plasma membrane and in adhesion plaques. Association of the FN receptor proteins with the adhesion-plaque protein talin also has been reported. We now find that the band 2 and band 3 proteins in the complex are phosphorylated in Rous sarcoma virus-transformed chicken cells but not in normal chicken cells. Phosphorylation occurs predominantly on tyrosine and is accompanied by a reorganization of the receptor complex in the membrane of the transformed cells. Whereas normal cells contain the FN receptor in focal contacts and cellular processes between cells, v-src-transformed cells exhibit a more diffuse distribution of this receptor. In addition to the viral v-src oncogene, cells transformed by other avian oncogenes that also encode tyrosine kinases (v-fps, v erbB, and v-yes) also express the receptor complex proteins in the phosphorylated state regardless of whether the transforming protein is detectable in adhesion plaques. These results suggest that the altered FN and laminin receptor proteins may contribute to the transformed phenotype, but their significance and role in the transformed state remain to be established. PMID- 3018736 TI - Characterization of mutations induced by 2-(N-acetoxy-N-acetyl)aminofluorene in the dihydrofolate reductase gene of cultured hamster cells. AB - To determine the types of alterations in gene structure that are induced by the carcinogen 2-(N-acetoxy-N-acetyl)aminofluorene, we used this compound to generate mutations at the dihydrofolate reductase (DHFR) locus (DHFR) in Chinese hamster ovary cells. Twenty-nine independent enzyme-deficient mutants were isolated. A profile of the 26-kilobase (kb)-long gene was obtained by Southern blot analysis of the mutant and parental DNAs digested with BstEII/Kpn I. Hybridization to a mixed probe of 10 DHFR genomic and cDNA fragments revealed 12 bands that scan 34 kb. Twenty-one DHFR- clones (72%) contained small mutations (changes less than 100 base pairs in size). Large or small deletions involving various parts of the gene occurred in eight of the mutants (28%). A large deletion (greater than 35 kb) with 5' and 3' breakpoints mapping to approximately the same location was noted in four mutants. One mutant has undergone a deletion of 550-900 bp that eliminated the first coding exon. Concomitantly, a chromosomal event (either translocation, insertion, or inversion) has separated the 5' flank from the body of the gene. In another mutant, four deletions have occurred at the DHFR 5' end and internally. Restriction fragment length polymorphism analysis of the mutant DNAs with exon-specific probes localized three mutations. One mutant has lost a Taq I (TCGA) site, and another has lost a Sac I (GAGCTC) site. In a third, a GC-- -TA transversion has created a BstEII (GGTNACC) site. Finally, we used HPLC to determine the ratio of acetylated (12%) to deacetylated (88%) 2-aminofluorene adducts formed in the parental cells. A correlation between the mutational specificities and the conformational changes induced by the two types of DNA adducts is discussed. PMID- 3018737 TI - Cis-acting sequences responsible for the transcriptional activation of human T cell leukemia virus type I constitute a conditional enhancer. AB - Transcription from the long terminal repeat promoter of human T-cell leukemia virus type I is activated in the presence of a trans-activator protein, TA-I, encoded in the 3' part of the genome. A series of long terminal repeat mutants and hybrid promoter constructs have been studied in a transient expression assay for their ability to be activated in the presence of the trans-activator protein. The sequences responsible for trans-activation have properties similar to those of transcription enhancer elements. They act relatively independent of position and orientation and activate both the homologous as well as heterologous promoters only in the presence of the trans-activator protein. Therefore, the trans-activator protein of human T-cell leukemia virus type I acts via an inducible enhancement mechanism. PMID- 3018739 TI - Antitumor effects of an immunotoxin made with Pseudomonas exotoxin in a nude mouse model of human ovarian cancer. AB - An immunotoxin composed of Pseudomonas toxin coupled to an antibody to the human transferrin receptor was evaluated for its effect on ovarian cancer. In the tumor model employed, 60 million human ovarian cancer cells were injected into the peritoneal cavity of an immunodeficient nude mouse. By day 5, cancer cells were implanted and growing in small clusters throughout the peritoneal cavity. On days 5-8, 0.3-2 micrograms of immunotoxin was injected into the peritoneal cavity. Control mice died with malignant ascites at 34-58 days after the implantation of tumor cells, whereas immunotoxin-treated mice lived to 100 days or longer. Irrelevant immunotoxins or antibody alone had no antitumor activity. These findings suggest that intraperitoneal injection of immunotoxins may have a role in the treatment of ovarian cancer. PMID- 3018738 TI - Interferon gamma and granulocyte/macrophage colony-stimulating factor inhibit growth and induce antigens characteristic of myeloid differentiation in small cell lung cancer cell lines. AB - The expression of several macrophage and hemopoietic cell surface markers recently described on small-cell lung cancer (SCLC) cell lines was studied by use of flow cytometry. The antigens Leu-M3, Leu-7, and HLA-DR were examined for their modulation by human interferon gamma and granulocyte/macrophage colony stimulating factor (GM-CSF). Both of these lymphokines generally induced enhanced expression of hemopoietic markers in several SCLC lines. A differential response to these two hormones was observed, in that qualitative and quantitative differences in marker modulation among the tested cell lines were apparent. In addition to regulating the antigenic phenotype of these cells, both interferon gamma and GM-CSF had antiproliferative effects on SCLC lines as determined by [3H]thymidine incorporation and clonal growth in agar. These results suggest that interferon gamma and GM-CSF promote a differentiation process in SCLC cell lines that has characteristics in common with myeloid differentiation. These findings support the theory that SCLC tumors are hemopoietic cells that arise from macrophages or their precursors and suggest new therapeutic modalities for the treatment of lung cancer. PMID- 3018740 TI - Effects of reagent and cell-generated hydrogen peroxide on the properties of low density lipoprotein. AB - Low density lipoprotein (LDL) isolated from human plasma anticoagulated with EDTA (EDTA/LDL) was 4-fold more resistant to oxidation by reagent H2O2, as assayed by the thiobarbituric acid (TBA) assay, than LDL prepared from plasma anticoagulated with citrate (CDP/LDL). The LDLs required 1-3 mM H2O2 for maximal oxidation by this assay, and ED50S were 1.7 X 10(-3) M for EDTA/LDL and 4.5 X 10(-4) M for CDP/LDL. Oxidation was enhanced 2.3-fold by Cu2+ ions. Rabbit endothelial cell line monolayers released two orders of magnitude less H2O2 than was required to oxidize LDL and failed to induce TBA reactivity in either EDTA/LDL or CDP/LDL after a 24-hr coincubation. However, this LDL was subsequently degraded by mouse macrophages more rapidly than untreated LDL. Freshly isolated human monocytes (2 X 10(6) cells per ml), with or without phorbol myristate acetate (100 ng/ml) to trigger the respiratory burst, did not oxidize LDL in the TBA assay, despite producing large amounts of reactive oxygen intermediates. EDTA/LDL, CDP/LDL, and acetoacetylated LDL failed to trigger H2O2 release from human monocytes or macrophages. These results separate oxidation of LDL as measured by TBA assay from the modification of LDL by rabbit aortic endothelial cell line that leads to its subsequent enhanced degradation by macrophages. PMID- 3018741 TI - An Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA5) partly encoded by the transformation-associated Bam WYH region of EBV DNA: preferential expression in lymphoblastoid cell lines. AB - Four peptides were synthesized on the basis of amino acid sequences deduced from a highly spliced transcript encoded by the Bam W, Y, and H fragments of the Epstein-Barr virus (EBV) genome [Bodescot, M., Chambraud, J. B., Farrell, P. J. & Perricaudet, M. (1984) EMBO J. 3, 1913-1917]. Rabbit antisera against three of the four peptides identified a nuclear polypeptide that varied between 22 and 70 kDa in molecular size. Four of 20 EBV-positive human sera contained antibodies against this polypeptide. Since this is the fifth EBV-determined nuclear antigen (EBNA) discovered in growth-transformed cells, it is designated EBNA5. The antigen was detected in virus nonproducer lines (less than 0.01% EBV early antigen expression) and is thus not dependent on the viral cycle. It was differentially expressed depending on the origin of the lines. All 10 lymphoblastoid cell lines tested expressed EBNA5, but it could not be detected in 10 of 11 EBV-carrying Burkitt lymphoma lines. Infection of tonsillar lymphocytes with the B95-8 strain of EBV induced six EBNA5-specific polypeptides that varied between 41 and 70 kDa in molecular size with regular increments of 6 kDa. This may be due to the fact that the EBNA5 coding sequence includes the Bam W internal repeat. Parallel infection of the EBV-negative Burkitt lymphoma line Ramos with the same viral substrain did not induce detectable levels of EBNA5, nor was this antigen present in permanently EBV-converted Ramos sublines. These findings imply that the expression of the viral genome varies among B cells having different phenotypes. PMID- 3018742 TI - Immunological identification of the major platelet low-Km cAMP phosphodiesterase: probable target for anti-thrombotic agents. AB - Immunoblot and enzyme-activity analyses, using specific immunological probes, indicated that more than 80% of the total low-Km cAMP phosphodiesterase activity present in bovine and human platelets resided in a single phosphodiesterase isozyme. In the presence of protease inhibitors, the platelet enzyme has an apparent subunit size of 110 kDa and appears immunologically and structurally indistinguishable from a recently purified bovine heart isozyme. When protease inhibitors were absent during homogenization and centrifugation, this platelet phosphodiesterase was susceptible to sequential proteolysis forming 80-kDa and 60 kDa peptides. As a previous report on the purification of the platelet low-Km cAMP phosphodiesterase described a 61-kDa protein, our data would suggest that this was a proteolytic fragment. Moreover, in our study a 40-70% increase in catalytic activity was associated with proteolysis. Further similarities between the platelet and heart phosphodiesterases were demonstrated by pharmacological studies that showed identical inhibitor profiles for both enzymes. Several known phosphodiesterase inhibitor compounds that have been found useful in inhibiting platelet aggregation also inhibited the platelet low-Km cAMP phosphodiesterase with potencies very similar to their antithrombotic effects. Cilostamide, Ro 15 2041, milrinone, papaverine, isobutylmethylxanthine, and theophylline inhibited the 110-kDa platelet enzyme with IC50 values of 0.04, 0.13, 0.46, 1.4, 2.6, and 110 microM, respectively. PMID- 3018743 TI - Synthesis of the complete trans-activation gene product of human T-lymphotropic virus type III in Escherichia coli: demonstration of immunogenicity in vivo and expression in vitro. AB - Human T-lymphotropic virus type III (HTLV-III) contains a gene (tat-III) the product of which activates the expression of viral genes in trans. We have expressed in Escherichia coli the complete tat-III-encoded protein as well as a truncated form that lacks three amino acids from the amino terminus. These proteins are recognized by sera of many, but not all, infected individuals including patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex, as well as asymptomatic seropositive persons. Seropositivity for the tat III protein does not appear to correlate with the clinical stage of HTLV-III related disease. Antibodies raised in rabbits against the E. coli-produced protein detect the native protein (apparent molecular mass, 14.5 kDa) in a virus producing cell line. A second protein (26 kDa), of unknown origin but viral related, is also specifically recognized by the immune serum. PMID- 3018744 TI - Reaction of DNA with chemically or enzymatically activated mitomycin C: isolation and structure of the major covalent adduct. AB - The antitumor antibiotic mitomycin C is shown to form a covalent complex with calf thymus DNA under anaerobic conditions in the presence of either NADPH cytochrome c reductase/NADPH, xanthine oxidase/NADH, or the chemical reducing system H2/PtO2. Digestion of the complex with DNase I/snake venom diesterase/alkaline phosphatase yields a single mitomycin deoxyguanosine adduct as the major DNA alkylation product, identified as N2-(2'' beta,7'' diaminomitosen-1'' alpha-yl) 2'-deoxyguanosine (Structure 2). Two minor adducts, 2-5% each of the total adduct pool, are isolated and identified as the 1'' beta stereoisomer of 2 (Structure 3), and 10''-decarbamoyl-2 (Structure 7). The same results were obtained with M13 DNA and poly(dG-dC).poly(dG-dC); however, in the latter case, a minor adduct apparently possessing two deoxyguanosine and one mitomycin unit is isolated. Digestion of the covalent mitomycin-calf thymus DNA complex with nuclease P1 yields four dinucleotide adducts, all of which consist of 2 linked at its 3' end to each of the four possible 5' nucleotides (A, T, G, and C). Upon treatment of each dinucleotide adduct with snake venom diesterase/alkaline phosphatase, 2 is released along with the corresponding free nucleoside. In apparent conflict with the present results, previous reports from another laboratory have indicated that modification of calf thymus DNA by mitomycin C under conditions identical to those described here result in the isolation of three mitomycin C mononucleotide adducts possessing linkages of the drug to N2 and O6 of guanine and N6 of adenine. Evidence is shown suggesting that the latter adducts are actually three of the above four dinucleotide derivatives of 2 obtained independently by us and, thus, all of them in fact possess an identical N2-mitosenylguanine adduct moiety. Model-building studies indicate an excellent fit of the guanine N2-linked drug molecule inside the minor groove of B DNA with no appreciable distortion of the DNA structure. PMID- 3018746 TI - The avian beta-adrenergic receptor: primary structure and membrane topology. AB - Partial amino acid sequence information allowed the isolation of cDNA clones encoding the turkey erythrocyte beta-adrenergic receptor. Antisera raised against synthetic peptides encoded by the cDNA crossreacted with the purified receptor and appropriate tryptic fragments, confirming the identity of the cDNA. The receptor is composed of 483 amino acids and has a molecular mass of 54 kDa. Its sequence suggests that it is arranged predominantly in seven membrane-spanning sequences and a long cytoplasmic carboxyl-terminal domain. The extracellular amino-terminal domain contains a consensus sequence for N-glycosylation. The beta adrenergic receptor displays overall structural similarity and weak sequence homology with rhodopsin. Because both proteins act by regulating GTP-binding proteins, a compact structure based on seven membrane-spanning regions may be a general model for receptors that act on G proteins. PMID- 3018745 TI - Purified human platelet-derived growth factor receptor has ligand-stimulated tyrosine kinase activity. AB - The platelet-derived growth factor receptor (PDGF-R), a 180-kDa single-chain polypeptide, was purified from membranes of the human osteogenic sarcoma cell line MG-63. Purification was achieved by treatment of membranes with PDGF and ATP, followed by solubilization with nonionic detergent and successive chromatography on solid-phase anti-phosphotyrosine monoclonal antibody and DEAE cellulose. The PDGF-R, which was estimated to be 50-80% pure by NaDodSO4/polyacrylamide gel electrophoresis of 32P-labeled preparations, was free of contaminating epidermal growth factor receptor and had no detectable phosphatase activity. It specifically bound 125I-labeled PDGF, a reaction quantified by binding of the ligand-PDGF-R complex to the anti-phosphotyrosine antibody. The purified receptor displayed PDGF-stimulatable tyrosine kinase activity, assayed by autophosphorylation of PDGF-R at tyrosine residues and by phosphorylation of angiotensin II. The Km for ATP in the autophosphorylation reaction was 7.5 microM. Addition of PDGF did not change the Km but increased the Vmax 1.7-fold. PMID- 3018748 TI - Growth hormone-releasing factor stimulates proliferation of somatotrophs in vitro. AB - The mitogenic effect of the hypothalamic peptides growth hormone-releasing factor (GRF) and somatostatin on cultured growth hormone (GH)-producing cells (somatotrophs) was studied. Using autoradiographic detection of [3H]thymidine uptake and immunocytochemical identification of GH-producing cells, we show that 5 nM GRF causes a 20-fold increase in the percentage of somatotrophs labeled with [3H]thymidine. The total number of somatotrophs in GRF-treated cultures was increased by 60%. Somatostatin had no measurable effect on the labeling index by itself, but it partly inhibited the GRF-induced increase in both the labeling index and the total number of cells. Forskolin caused an increase in both the percentage of somatotrophs with a [3H]thymidine-labeled nucleus and the somatotroph number similar to that caused by GRF. GH secretion as well as cellular GH content in the GRF- or forskolin-treated cells increased with culture time over the entire period, whereas secretion and content of GH gradually decreased in control or somatostatin-treated cultures during the entire culture period. These data suggest that GRF and somatostatin regulate the mitotic activity of GH-producing cells and that the effect of GRF is possibly mediated by cyclic AMP. PMID- 3018747 TI - Anaerobic regulation of nitrogen-fixation genes in Rhodopseudomonas capsulata. AB - A Rhodopseudomonas capsulata nifH::lacZ gene fusion was used to isolate constitutive mutants of R. capsulata, unable to repress nif gene transcription anaerobically with every fixed-nitrogen source tested. When these nifc strains were grown aerobically, nif gene transcription was repressed. These results indicate that the regulation of nif gene transcription by fixed nitrogen is different from the regulation by oxygen. Under anaerobic conditions, nif gene transcription in both R. capsulata and Klebsiella pneumoniae is specifically prevented by inhibitors of DNA gyrase [DNA topoisomerase type II (ATP hydrolyzing), EC 5.99.1.3]. A recent study has shown that anaerobically grown Salmonella typhimurium have high DNA gyrase activity, whereas aerobically grown cells have high DNA topoisomerase type I (EC 5.99.1.2) activity and DNA that is more relaxed [Yamamoto, N. & Droffner, M. L. (1985) Proc. Natl. Acad. Sci. USA 82, 2077-2081]. In view of these results, we suggest that the control of nif gene transcription in response to oxygen is determined by the action of DNA gyrase and DNA topoisomerase I. Thus, although nitrogen control of nif gene expression requires the products of regulatory genes for which constitutive mutations can be isolated, oxygen appears instead to prevent the adoption of a DNA conformation necessary, directly or indirectly, for nif gene transcription. PMID- 3018750 TI - Significant potential secondary structures in the Epstein-Barr virus genome. AB - This paper identifies all statistically significant dyad symmetry combinations in the Epstein-Barr virus genome. The distribution of long dyad symmetry pairings emphasizes two regions, the 5' third of the 3.1-kilobase-pair (kbp) repeat and the oriP region, the latter essential for Epstein-Barr virus replication during latency. A 600-base-pair (bp) stretch in the 3.1-kbp repeat can establish an extended hairpin loop of stem length in excess of 208 bp of predominantly G + C stacking. Moreover, the 3.1-kbp repeat has the potential to form a wide variety of secondary structures based on juxtapositions of sizable palindromes, close dyad symmetry pairings, and direct repeats. The 3.1-kbp repeat presents several features that portend it as an important control region. The oriP region contains an abundance of statistically significant dyad symmetry combinations that strongly correlate with the "21 X 30 bp" tandem repeat units and four truncated copies of this repeat unit 1 kbp downstream. Each of the units centers on the same approximately 30-bp palindrome. Contrasts in the content and the secondary structure formations associated with the 3.1-kbp repeat units versus those of the oriP region are discussed in relation to viral or cellular function. PMID- 3018749 TI - Recombinant murine retroviruses containing avian v-myc induce a wide spectrum of neoplasms in newborn mice. AB - NFS/N mice were infected within 48 hr of birth with pseudotypes of recombinant murine leukemia viruses containing avian v-myc developed T-cell, pre-B-cell, and B-cell lymphomas and epithelial tumors including pancreatic and mammary adenocarcinomas. Primary hematopoietic and epithelial tumors and continuous in vitro cell lines derived from some of these tumors, established in the absence of added growth factors, exhibited clonal integrations of v-myc and expressed v-myc RNA. These results show that deregulated expression of the myc oncogene in mammalian cells can initiate a wide variety of neoplasms. PMID- 3018751 TI - Efficient introduction of plasmid DNA into human hemopoietic cells by encapsidation in simian virus 40 pseudovirions. AB - Introduction of DNA into human hemopoietic cells is required for the study of regulatory mechanisms operating in these cells, as well as for possible procedures of gene therapy. However, with hemopoietic cells the conventional technique of calcium phosphate precipitation is inefficient. The pathway of encapsidation of plasmid DNA as simian virus 40 (SV40) pseudovirions for the introduction of new genetic material was therefore investigated. Encapsidation was achieved in COS (monkey kidney) cells, which express SV40 large tumor (T) antigen constitutively. The vector, pSO, was introduced to the COS cells by DNA transfection. It carried the SV40 origin of replication (ori), to facilitate replication of the plasmid in the COS cells. The SV40 capsid proteins were supplied in trans by a helper SV40 virus. The bacterial chloramphenicol acetyltransferase gene cat was used as a model for gene transmission. After encapsidation, the pseudovirions were used in infection of the human erythroleukemic cell line K562 and of normal human bone marrow cells. The results demonstrate that the cat gene can be transmitted with high efficiency. Over 40% of the infected K562 cells and 30% of the infected bone marrow cells were observed to contain plasmid DNA 48 hr after infection. Moreover, the results suggest that the efficiency of gene transmission by this vector can be improved and so may approach the theoretical 100%. PMID- 3018752 TI - Analysis of mutations in the transmembrane region of the aspartate chemoreceptor in Escherichia coli. AB - Site-specific mutagenesis was used to replace an alanine with a lysine residue and to create a deletion of seven amino acids into the first transmembrane region (TMI region) of the aspartate chemoreceptor in Escherichia coli. The mutations resulted in the loss of aspartate chemotaxis on tryptone motility plates. However, both mutant proteins were able to associate with the membrane and to bind aspartate. They were both refractory to methylation or to modification of the C-terminal region of the protein by the cheB gene product. These results suggested that the integrity of the TMI domain of the protein was required to maintain the function of the cytoplasmic portion of the receptor. The Lys-19 mutant retained the ability to generate a repellent response. Analysis of suppressor mutations of the Lys-19 mutation suggested that formation of an ion pair or specific changes in a 40 amino acid stretch in the cytoplasmic region of the protein (from amino acid 264 to amino acid 303) could suppress the effects of the Lys-19 mutation. The TMI region of the protein may be involved in transmembrane transmission of signals from the periplasmic portion of the cell to the cytoplasmic portion of the Tar protein. PMID- 3018753 TI - Prospect for prevention of human immunodeficiency virus infection: purified 120 kDa envelope glycoprotein induces neutralizing antibody. AB - This study initiates an effort to develop a safe vaccine against the acquired immunodeficiency syndrome (AIDS) that is caused by infection with a retrovirus designated human immunodeficiency virus (HIV) [formerly human T-cell lymphotropic virus type III (HTLV-III)]. Other retrovirus models have shown that purified external glycoprotein subunits are immunogenic. The external envelope glycoprotein of HIV (gp120) has a molecular size of 120 kDa, is responsible for virus infectivity, and induces strong antibody response in humans. Purified HIV virus preparations contain relatively little gp120 so HIV-infected cells were used as the antigen source. The gp120 was localized on cell membranes and was solubilized with low levels of nonionic detergent. The glycoprotein was further purified by immunoaffinity chromatography over a resin prepared from IgGs isolated from patients. Homogeneity was achieved following extensive dialysis and polyacrylamide gel electrophoresis. The gp120 isolated from infected cells was shown to be structurally identical by peptide maps to virion gp120 and the amino terminal amino acid sequence confirmed that the molecule was specified by the HIV genome. Goat, horse, and rhesus monkey (Macaca mulatta) immune sera to gp120 precipitated the homologous antigen and neutralized the in vitro infectivity of HIV. The induction of neutralizing antibody indicates that a gp120 subunit vaccine against HIV is theoretically possible. PMID- 3018754 TI - Conservation of amino acid sequence of VP8 and cleavage region of 84-kDa outer capsid protein among rotaviruses recovered from asymptomatic neonatal infection. AB - Within the past few years, rotavirus strains were recovered from four discrete prolonged outbreaks of infection in newborn nurseries in which affected infants failed to develop significant symptoms. The virus strains recovered from each outbreak belonged to a different human rotavirus serotype and thus each of the four human rotavirus serotypes was associated with asymptomatic infection of neonates. Marked conservation of sequence was observed among the fourth genes of the nursery rotavirus strains in a previous study using RNA X RNA hybridization, while a different conserved set of fourth gene sequences was identified among virulent human rotaviruses representing the four known serotypes. In the present study, this sequence dimorphism was further evaluated by comparing the sequence of the region of the fourth gene of virulent and asymptomatic human rotaviruses that codes for the VP8 protein, downstream cleavage sites, and the NH2 terminus of VP5. The corresponding sequences of a simian rotavirus were also determined. The fourth segment (+) strand RNA has a 5' conserved nontranslated sequence of nine nucleotides and encodes a VP8 protein of 240 amino acids in human rotavirus strains and 241 amino acids in simian rotavirus strains. Human and simian rotaviruses exhibit many similarities in this region of their genome, including identical NH2-terminal amino acid sequences, conservation of arginine at the two trypsin cleavage sites, and the position of a cysteine residue. Alignment of amino acid sequences of the VP8 protein, the downstream cleavage region, and the NH2 terminus of VP5 of asymptomatic and virulent human rotavirus strains indicates a high degree of homology (96% or more) among the asymptomatic viruses (serotypes 1, 2, 3, and 4), while homology between asymptomatic strains and virulent viruses is considerably less (68-72%). A high degree of conservation of amino acid sequence (92-97%) is also observed among three of the virulent strains (serotypes 1, 3, and 4). At 48 positions in the protein sequence of VP8, the cleavage region, and the NH2 terminus of VP5, an amino acid is conserved among asymptomatic rotaviruses, while a different amino acid is conserved among virulent rotaviruses. Notably, three of these differences are located within the cleavage region between VP8 and VP5. These findings suggest that the fourth genes of virulent and asymptomatic human rotavirus strains represent two lines of divergent evolution from a common ancestor. Also, it is possible that this sequence dimorphism may be responsible in part for the difference in virulence between these two groups of human rotaviruses. PMID- 3018755 TI - Cellular localization of human immunodeficiency virus infection within the brains of acquired immune deficiency syndrome patients. AB - Dysfunction of the central nervous system (CNS) is a prominent feature of the acquired immune deficiency syndrome (AIDS). Many of these patients have a subacute encephalitis consistent with a viral infection of the CNS. We studied the brains of 12 AIDS patients using in situ hybridization to identify human immunodeficiency virus [HIV, referred to by others as human T-cell lymphotropic virus type III (HTLV-III), lymphadenopathy-associated virus (LAV), AIDS associated retrovirus (ARV)] nucleic acid sequences and immunocytochemistry to identify viral and cellular proteins. Nine patients had significant HIV infection in the CNS. In all examined brains, the white matter was more severely involved than the grey matter. In most cases the infection was restricted to capillary endothelial cells, mononuclear inflammatory cells, and giant cells. In a single case with severe CNS involvement, a low-level infection was seen in some astrocytes and neurons. These results suggest that CNS dysfunction is due to indirect effects rather than neuronal or glial infection. PMID- 3018756 TI - Role of calcium in regulating the cyclic GMP cascade of phototransduction in retinal rods. AB - Both cGMP and Ca2+ appear to be involved in the process of phototransduction in vertebrate rods, but their precise roles have been the subject of debate. To investigate the role of Ca2+ we have artificially increased the calcium buffering capacity of the rod by using a patch pipet to incorporate the calcium buffer 1,2 bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) into the rod cytoplasm. In the presence of buffer the Na+-Ca2+ exchange current became greatly slowed, suggesting that the cytoplasmic calcium concentration (Cai) had indeed been buffered substantially. Although the presence of buffer had negligible effect on the rising phase of the light response, it profoundly altered the later behavior. Responses to brief flashes became prolonged and exhibited an overshoot, apparently because the shut-off process was modified. The normal acceleration of time-to-peak with brighter flashes (an early sign of light adaptation) disappeared. Responses to steady adapting illumination took much longer than normal to settle to a steady level, although the final level represented a similar fractional suppression of current. With superimposed test flashes the presence of such adapting illumination caused a more rapid recovery, whereas the presence of calcium buffer slowed the recovery. The results are consistent with the idea that the rapid drop in Cai, which has recently been shown to accompany the light response, is involved in terminating the light response, and that Cai is thereby involved in setting the operating point and sensitivity of phototransduction. From comparison with other work we infer that Cai appears to act, at least in part, by means of control of cGMP phosphodiesterase activity. PMID- 3018758 TI - Structure of evolving populations of Saccharomyces cerevisiae: adaptive changes are frequently associated with sequence alterations involving mobile elements belonging to the Ty family. AB - Haploid a and diploid a/alpha and a/a populations of Saccharomyces cerevisiae evolving in laboratory environments for up to 300 generations were analyzed for sequence rearrangements associated with the Ty family of transposable elements. In contrast to results with Escherichia coli, evolving populations of yeast exhibit a high frequency of sequence rearrangements associated with mobile genetic elements. In particular, adaptive shifts in these populations are often associated with such sequence rearrangements. The results are most compatible with the explanation that there is direct selection for some of the sequence rearrangements. In addition, the pattern of changes suggests that the structure of evolving microorganism populations may be more complex than expected. PMID- 3018759 TI - Key hormonal and metabolic control systems modifying nutrient use. PMID- 3018760 TI - The physiological bases of nutrient responses; growth and fattening. PMID- 3018757 TI - Involvement of brain opiate receptors in the immune-suppressive effect of morphine. AB - We previously reported that a single systemic injection of a high dose of morphine (greater than or equal to 20 mg/kg) transiently suppresses splenic natural killer cell cytotoxicity in rats. The present study examined the possibility that the immune-suppressive effect of morphine is mediated by opiate receptors in the brain. Supporting this hypothesis, we found that morphine (20 or 40 micrograms) injected into the lateral ventricle suppressed natural killer cell activity to the same degree as a systemic dose higher by three orders of magnitude. This effect was blocked by an opiate antagonist, naltrexone. Natural killer cell activity was unaffected by systemic administration of N-methyl morphine, a morphine analogue that does not cross the blood-brain barrier. These data implicate opiate receptors in the brain in morphine-induced suppression of natural killer cell cytotoxicity. PMID- 3018761 TI - Dynamic models: their use in understanding and predicting nutrient response. PMID- 3018762 TI - Corticotropin-releasing activity in the rat gastric antrum. AB - Rat gastric antrum, duodenum, pancreas, and spleen were extracted in acetic acid, treated with acetone, and purified on a C-18 cartridge. These extracts, in a dose equivalent to one respective organ, were examined for CRF bioactivity in vitro using rat half pituitaries, with gastric antrum extract showing a significant CRF activity. The antrum extract showed a dose-related CRF activity in vitro using rat pituitary cell culture, and the dose-response curve appeared to be parallel with that of synthetic rat hypothalamic CRF. Subsequent ion-exchange chromatography on a SP-Sephadex column showed that antrum CRF coeluted with basic materials (SP-III fraction), while rat hypothalamic CRF coeluted with weakly basic materials (SP-II fraction). The SP-III fraction was further purified by gel filtration on Sephadex G-50. CRF activity was eluted in two areas: large mol wt fraction (10,000-15,000) and small mol wt fraction (1500-2000). Hypothalamic CRF was eluted between them. The CRF activities of the two fractions were completely abolished by trypsin digestion, suggesting a peptide nature. The large molecular weight fraction exhibited a steeper dose-response curve than the hypothalamic CRF in vitro using cell culture, and the response to a dose equivalent to two antra exceeded the maximum response exhibited by the hypothalamic CRF. However, the fraction failed to increase serum corticosterone when injected in pharmacologically blocked rats. On the other hand, the small molecular weight fraction showed a lesser CRF activity and a similar dose-response curve to that of the hypothalamic CRF as tested in vitro. This fraction significantly stimulated corticosterone secretion in vivo as well. The small molecular weight activity did not appear to be due to other peptides or amines which have been reported as causing ACTH release. Although the physiological roles of the small molecular weight antrum CRF are unknown, it is possible that this CRF plays a role during stress as a tissue CRF. PMID- 3018763 TI - Estrogens and Chlamydia trachomatis. AB - Isolates of Chlamydia trachomatis were inoculated in nonreplicating McCoy cells and incubated for 48 hr with various concentrations of hormones. Only the estrogens, particularly 17-beta-estradiol, had an affect on the subsequent infection of the McCoy cells by the Chlamydia. Exposure to 2-4 ng/ml (10(-8) M) estradiol during inoculation and incubation was associated with no change in the initial binding of Chlamydia to McCoy cells, but significantly more (about twofold) Chlamydia inclusions in the McCoy cells after 48 hr incubation. This effect was dose dependent, could be blocked with anti-estrogens, and also occurred with replicating McCoy cells. Localization studies suggest that this effect on the infectivity of Chlamydia trachomatis is dependent upon initial interactions of estrogens with McCoy cells. Both light microscopy and electron microscopy of the McCoy (fibroblast) cells showed no morphological changes after exposure to the estrogens under the incubation conditions employed in these studies. Estrogens may modify host susceptibility to Chlamydia infections in a manner independent of morphological changes in mammalian cells. PMID- 3018764 TI - Rapid induction of thymic lymphomas by isopropyl methanesulfonate: a preliminary report. AB - The direct-acting SN1 alkylating agent isopropyl methanesulfonate (IMS) was carcinogenic by subcutaneous injection in female Hsd:(ICR)BR mice, causing thymic lymphoid neoplasms within 7 months in at least 20 of 32 treated mice. No such neoplasms were observed in mice treated with the direct-acting SN2 methyl homolog, methyl methanesulfonate (MMS). Both the IMS-treated mice and the MMS treated mice initially received 20 mumole of the respective compounds by sc injection once weekly; however, because of toxic effects the dose of IMS was reduced to 10 mumole per injection on the 63rd day and further reduced to 5 mumole per injection on the 120th day, after which this dose was maintained until the 202nd day when the last surviving IMS-treated mouse became moribund and was sacrificed. In 2 of the MMS-treated mice, 93% of which were alive at 288 days, tumors were observed at the site of injection, one being a papilloma and the other a subcutaneous sarcoma. IMS has not previously been implicated as a carcinogen, to our knowledge. Its induction of thymic lymphomas may conceivably be related to its ability to alkylate exocyclic oxygen atoms in the DNA of hemopoietic cells. PMID- 3018765 TI - Role of volume and prostaglandin synthesis in the suppression of renin by saline in the rat. AB - To evaluate the contribution of plasma volume expansion per se on acute inhibition of renin release by sodium chloride infusion, renin responses to comparable plasma volume expansion with intravenous infusions of sodium chloride, sodium bicarbonate, or albumin were studied in separate groups of sodium chloride depleted rats. In addition, urinary prostaglandin E2 (PGE2) excretion rate was compared in the saline- and sodium bicarbonate-infused animals to evaluate the relationship between acute changes in renin release and intrarenal PGE2 synthesis. All three groups were plasma volume-expanded by approximately 55%. Plasma renin activity (PRA) decreased in response to saline (12.3 +/- 1.0 to 6.7 +/- 0.7 ng AI/ml/hr; P less than 0.01) whereas PRA did not change with sodium bicarbonate (11.3 +/- 1.4 to 10.2 +/- 1.5) or albumin (9.9 +/- 0.7 to 8.2 +/- 1.0). The rate of PGE2 excretion was not changed by either saline (72.2 +/- 13.1 to 72.3 +/- 18.7 pg/min) or sodium bicarbonate infusion (70.7 +/- 8.8 to 64.9 +/- 7.0). These results support the hypothesis that acute suppression of PRA by infusion of saline is not dependent upon volume expansion per se. In confirmation of earlier observations, inhibition of renin release by sodium chloride was related to chloride. Finally, the results suggest that the renal tubular mechanism for inhibition of renin release by sodium chloride is not related to overall changes in renal PGE2 synthesis in the rat. PMID- 3018766 TI - Genetic-dependent alterations in adrenal stress response and adrenocortical cell function of the domestic fowl (Gallus domesticus). AB - Strain-dependent differences in adrenocortical function were investigated in male White Leghorn domestic fowl. Adrenocortical function of Cornell K strain (K) (genetic control), autosomal dwarf strain (ADW), and sex-linked recessive dwarf strain (SLD) was evaluated in vivo by measuring plasma corticosterone and in vitro by measuring acute (2 hr) corticosterone production by enriched adrenocortical cell populations. Regardless of strain, there was an age-dependent decrease (27-57%) in plasma corticosterone from 1 to 12 weeks of age. However, there was a tendency for plasma corticosterone values of ADW and SLD to be, respectively, greater and less than that of K. In addition, at 12 weeks of age, plasma corticosterone responses of ADW and SLD to transient heat stress (50 degrees C, 30 min) were, respectively, 22.8% greater and 15.9% less than that of K. Strain differences in adrenal weight and relative adrenal weight (mg% body wt) were also apparent. At 12 weeks of age, adrenal weights of ADW and SLD were, respectively, 33 and 42% less than that of K, whereas relative adrenal weights were, respectively, 27.6% greater and 22.4% less than that of K. In addition there were strain-dependent differences in adrenocortical function at the cellular level. Although there were no consistent strain differences in basal and maximal corticosterone production by cells, there were strain differences in cellular sensitivity to ACTH and pregnenolone. On an equal cell concentration basis, the half-maximal steroidogenic concentrations (ED50 values or effective doses for 50% maximal effect) of ACTH for ADW and SLD adrenocortical cells were, respectively, 0.23 and 2.07 times the ED50 value for K cells. In addition, the ED50 value of pregnenolone for ADW cells was 0.46 times that for K and SLD cells. Since ED50 values are a measure of cellular sensitivity (the greater the ED50 value the lesser the cellular sensitivity), the order of sensitivity to ACTH was ADW greater than K greater than SLD and the order of sensitivity to pregnenolone was ADW greater than K = SLD. However, there were no strain differences in ED50 values of 8-bromo-cyclic AMP. These data suggest that strain differences in plasma corticosterone response to stress are, in part, due to differences in relative adrenal weight and differences in adrenocortical cell function. PMID- 3018767 TI - Changing concepts in mutation research. PMID- 3018768 TI - The use of animal virus or shuttle vectors to characterize mutagenesis in mammalian cells. PMID- 3018769 TI - Molecular analysis of mutations induced by deoxyribonucleotide pool imbalances in Chinese hamster ovary cells. PMID- 3018770 TI - The human T-cell leukemia/lymphotropic viruses: recent developments. PMID- 3018771 TI - Cytogenetic manifestations of alteration of gene expression and their relevance to cancer. PMID- 3018772 TI - Development of biochemical markers sensitive to ecogenetic variation and their use in assessing risk from genotoxic exposures. PMID- 3018773 TI - Genetic toxicology problems and perspectives: a case study and overview of Egyptian environment. PMID- 3018775 TI - Tests for chemical-induced germline transposition in Drosophila melanogaster. PMID- 3018774 TI - Prospects for DNA methods to measure human heritable mutation rates. PMID- 3018776 TI - Sexing of mammalian sperm. PMID- 3018777 TI - Expression of MTV sequences introduced into the mouse germline. PMID- 3018778 TI - Developmental regulation of transcript splicing in Drosophila melanogaster. PMID- 3018779 TI - c-Abl is differentially expressed during mouse spermatogenesis. PMID- 3018780 TI - The presence of cytoplasmic retinoic acid binding proteins in amphibian tissues and their possible role in limb regeneration. PMID- 3018781 TI - Responsiveness of dental epithelial and mesenchymal cells to epidermal growth factor. PMID- 3018782 TI - Cyclic AMP shortens mitotic phase of sea urchin egg cleavage cycle. PMID- 3018784 TI - Extracellular cAMP in Dictyostelium discoideum tissue. PMID- 3018783 TI - Introduction of genes into mouse embryonic stem cells. PMID- 3018785 TI - Adhesion and migration of avian neural crest cells: an evaluation of the role of several extracellular matrix components. PMID- 3018786 TI - Mechanisms of extracellular matrix turnover in normal and neoplastic cells. PMID- 3018788 TI - Calcium stimulation of embryonic palate mesenchymal prostaglandin synthesis. PMID- 3018789 TI - Sperm-egg interaction and fusion in fertilization. PMID- 3018790 TI - Brain iron-deficiency causes reduced learning capacity in rats. AB - Rats made nutritionally iron-deficient (ID) showed a significant deficit in water maze learning compared with normal rats. The deficit was substantially greater the longer the rats stayed on the ID diet. The deficit in learning was established prior to any significant decrease in hemoglobin (Hb) level in the blood. Three weeks after the ID rats were placed on a control diet, the Hb level was restored to normal value, but the cognitive deficit remained. Previous studies showed that the behavioral effects of ID are mediated by a decrease in the functional activity of the dopaminergic system. The ID effects on learning and memory may be related to the irreversible diminished dopaminergic neurotransmission that results from ID. PMID- 3018787 TI - Role of purpurin in neural retina histogenesis. PMID- 3018791 TI - Differential effects of amphetamine and related compounds on locomotor activity and metabolic rate in mice. AB - Locomotor activity was measured by photobeam interruptions, and metabolic rate by the production of CO2 (as minute volume expired CO2, or VECO2) in mice. d Amphetamine (0.3 to 10 mg/kg IP) increased locomotor activity in a dose-dependent manner while suppressing VECO2 over the same 72-min test period, compared to saline-injected controls. This phenomenon of divergent effects on locomotor activity and metabolic rate required central stimulation, as neither ammonium sulfate nor p-hydroxyamphetamine suppressed VECO2. Oxygen consumption was also suppressed by d-amphetamine, indicating that the suppression of VECO2 involved more than a change in respiratory quotient. When baseline activity rates were increased with running wheels, VECO2 and activity were both suppressed by d amphetamine; VECO2 was suppressed by d-amphetamine more in exercising mice than in sedentary mice. Anorexigenic agents phenmetrazine, aminoxaphen, and fenfluramine, when administered in doses equimolar to maximally effective doses of d-amphetamine, did not consistently affect activity or VECO2. Evidence for mediation of the VECO2 response by corticosterone and endogenous opioid peptides was negative. Further work, with other mediators of the stress response, or with more complete dose-effect studies with anorexigenic compounds, may be necessary to explicate the mechanism of this counter-intuitive divergence of two measures of activity in mice. PMID- 3018793 TI - The possible role of benzodiazepine receptors in morphine analgesia. AB - Diazepam within its therapeutic dose range, was shown to have no effect on nociception, but was shown to antagonize the analgesic action of morphine. This antagonism was found to be statistically significant at 0.5 mg/kg diazepam. To elucidate the mechanism of this inhibitory action of diazepam against morphine analgesia, Ro 15-1788, the specific antagonist of benzodiazepine receptors was used. As a result, Ro 15-1788 was found to partially reverse the inhibitory action of diazepam against morphine analgesia. This overall interaction between the supramolecular GABA receptor complex and morphine is discussed. PMID- 3018794 TI - REM sleep deprivation induces a decrease in norepinephrine-stimulated 3H-cyclic AMP accumulation in slices from rat brain. AB - Beta adrenergic sites in rat brain are reduced after repeated treatment with antidepressant drugs, with REM sleep deprivation (REMSd) having the same effect. This paper reports the effects of REMSd in the production of 3H-cyclic AMP in frontal cortical slices by NE challenge. Data presented in this paper report a marked decrease in 3H-cyclic AMP synthesis after REMSd, which is in accordance with previous results showing adrenergic receptor down-regulation following REMSd. Results are discussed in view of possible interaction with dopaminergic systems and depression management. PMID- 3018792 TI - Reinforcing properties of cocaine in the medical prefrontal cortex: primary action on presynaptic dopaminergic terminals. AB - The presynaptic mechanisms involved in the initiation of cocaine reinforcement were investigated using neurotoxin lesions. Rats were trained to intracranially self-administer cocaine (50 to 90 pmol) into the medial prefrontal cortex and after dose-effect analyses were completed, each rat received a unilateral 6 hydroxydopamine lesion (4 micrograms in 0.2 microliter) at the self administration site. The lesion selectively decreased dopamine content in the medial prefrontal cortex (-45%) and decreased cocaine-maintained responding to vehicle levels. Lever-pressing could be reinstated by substituting dopamine (300 pmol) but not serotonin for cocaine. Dopamine self-administration was attenuated by including equimolar concentrations of the D2 dopaminergic antagonist sulpiride in the injectate. These results suggest that the initiation of reinforcing neuronal activity in the medial prefrontal cortex appears to result in part through the direct interaction of cocaine with presynaptic reuptake sites associated with dopaminergic nerve endings. The resulting increased synaptic concentration of the neurotransmitter may then interact with postsynaptic D2 binding sites to activate neuronal systems involved in the mediation of this reinforcement. PMID- 3018795 TI - Mechanisms of lateralized hyperactivity following focal brain injury in the rat. AB - Recent work in this lab which explores the differential behavioral and neurochemical changes following left versus right cerebral damage is reviewed and neural mechanisms which may account for this asymmetry are proposed. Damage to the right frontoparietal cortex in rats, caused by either ischemia or suction, produces hyperactivity for as long as a month after surgery. These lesions also produce decreases in norepinephrine (NE) levels in both ipsilateral and contralateral cortex and in the locus coeruleus. Similar lesions in the left cortex, however, do not produce these behavioral or biochemical changes. Similar lateralized responses have also been produced by intracortical injections of NE neurotoxins, by cortical island lesions, by destroying cortical efferents, and by producing lesions in the nucleus accumbens, which receives a cortical input. These lateralized responses suggest that the neural mechanisms that mediate this phenomenon include both cortical and subcortical components. It is proposed that the neuroanatomical asymmetry is in either accumbal efferents or their postsynaptic connections. PMID- 3018796 TI - Effects of chronic electrode implantation on dopaminergic neurons in vivo. AB - The present study evaluated the effects of chronic electrode implantation on stimulus-dependent increases of the dopamine (DA) metabolite dihydroxyphenylacetic acid (DOPAC) in relationship to a well characterized in vivo model which used electrical stimulation from acute electrode placements in the nigro-striatal pathway. Five days after bipolar electrodes were implanted into the nigro-striatal pathway, non-contingent electrical stimulation (100 microA, 25 Hz, 1.5 msec duration, 20 min session) did not change DA or DOPAC concentrations in the caudate nucleus, nucleus accumbens, or olfactory tubercles, whereas the same stimulation from acute electrode placements causes significant ipsilateral increases in caudate DOPAC. Although DOPAC concentrations did not change when these chronically implanted electrodes were stimulated, similar chronic electrode placement supported intracranial self-stimulation (ICSS). In order to examine the effects of self-stimulation on DOPAC concentrations, five ICSS test groups were established for comparison: implanted only, trained only, minimum response rate, 50% maximum response rate and maximum response rate. Following a 50 min test session, a comparison of either DA, or DOPAC concentrations across the different ICSS conditions revealed no change for the caudate nucleus, nucleus accumbens or olfactory tubercles. Likewise, there was no change between the stimulated and unstimulated sides within each ICSS group. When a comparison was made between implanted only and maximal ICSS response rate groups for changes in DA or DOPAC concentrations in the frontal cortex, no differences were found. Apparently, chronic electrode implantation abolished the ability to electrically stimulate nigro-striatal dopaminergic neurons under non contingent conditions, and the relationship between dopaminergic neurons and ICSS appears to be indirect in nature. PMID- 3018797 TI - Cannabidiol-caused depression of spinal motoneuron responses in cats. AB - Intracellular recording techniques were used on spinal motoneurons in the cat in order to define the synaptic pharmacology of cannabidiol (CBD). The cannabinoid produces only depression of electrophysiological responses of the motoneurons: For instance, the drug decreases the amplitude of excitatory postsynaptic potentials (EPSPs); this reduction does not appear to be the result of a change in the afferent input. In addition, CBD raises the firing threshold and decreases the amplitude of motoneuron action potentials; the effects on action potentials are related to changes in postsynaptic membrane conductances, probably involving at least sodium conductance. The spinal motoneuron effects provide potential electrophysiological mechanisms for CBD's central depressant actions. PMID- 3018798 TI - Influence of environment on GABA receptors in muricidal rats. AB - The influence of environment (isolation) on GABA receptor numbers ( [3H]-muscimol binding sites) and affinities was investigated in specific limbic areas known to be involved with the development of muricide. Olfactory bulbs of isolated rats were found to have identical numbers of [3H]-muscimol binding sites whether or not they were muricidal. However, in the olfactory bulbs of the aggregated animals a greater than two-fold increase was found in numbers of [3H]-muscimol binding sites in those rats that were muricidal. In the amygdala muricidal rats had a 32-34% increase in [3H]-muscimol binding sites over non-muricidal rats regardless of environment. In the septum non-muricidal rats had fewer [3H] muscimol binding sites than muricidal rats and although the trend was evident, statistical vigor was seen only in the aggregated animals. Neither muricide nor isolation significantly influenced [3H]-muscimol binding numbers in the hypothalamus. GABA Ki values were examined in all brain regions and found to be the same in the isolated and aggregated animals whether or not they were muricidal. We concluded that environment appears to influence apparent GABA receptor numbers in the olfactory bulbs and septum whereas muricidal behavior correlates well with an increase in apparent number of GABA receptors in the amygdala. GABA receptors in the hypothalamus were not influenced by either environment or aggression. PMID- 3018799 TI - Upregulation of brain benzodiazepine receptors by electroconvulsive shocks. AB - Locomotor activity and footshock-induced aggressive behaviour in rat was decreased in a dose-dependent manner by diazepam. These effects were significantly potentiated by chronic electroconvulsive shock (ECS). Specific 3H diazepam binding showed an upregulation of benzodiazepine receptors in the rat frontal cortex by ECS. This upregulation can explain the decreased therapeutic benefit observed in patients receiving both benzodiazepines and electroconvulsive shocks. PMID- 3018800 TI - Structure-activity relationships in cannabinoids. PMID- 3018801 TI - Morphologically different biopsy specimens of the human gastric mucosa. I. The use of enzymatic cell isolation for quantitative determination of parietal cells. AB - Single biopsies of human gastric mucosa from controls and different groups of patients were used for enzymatic cell isolation by pronase and collagenase and subsequent count of parietal and nonparietal cells. This procedure was tested in regard to its validity and delivered the following cell numbers. Total gastric cells/mg wet weight gastric mucosa: normal gastric mucosa [controls (C), n = 95] 31,500 +/- (SEM) 1,490, chronic superficial gastritis (GI; n = 49) 36,300 +/- 2,770, chronic gastritis with beginning atrophy (GII; n = 36) 44,100 +/- 3,050 (p less than 0.025), chronic atrophic gastritis (GIII; n = 12) 40,100 +/- 5,760, duodenal ulcer (DU; n = 26) 29,340 +/- 2,280, gastric ulcer (GU; n = 23) 37,090 +/- 3,000, gastric resection according to Billroth I (BI; n = 7) 57,480 +/- 12,360 (p less than 0.005) and Billroth II (BII; n = 12) 52,560 +/- 6,730 (p less than 0.005). Parietal cells/mg wet weight gastric mucosa: 1,910 +/- 490 (C), 1,980 +/- 140 (GI), 1,700 +/- 200 (GII), 1,170 +/- 220 (GIII, p less than 0.025), 2,580 +/- 240 (DU, p less than 0.05), 1,690 +/- 150 (GU), 1,500 +/- 250 (BI), 1,360 +/- 320 (BII). Parietal cell concentration (density) did not differ in males and females and did not change with age. The method delivers relevant cell numbers, is suitable to detect qualitative differences and can be used for the interpretation of biochemical studies. PMID- 3018802 TI - Calcium entry blocking activity of dilazep in dog coronary artery. AB - Calcium entry blocking activities of adenosine and its potentiating compounds (dipyridamole, lidoflazine and dilazep) were studied in potassium (100 mmol/l) depolarized, dog, large coronary artery strips, in comparison to nifedipine, verapamil and diltiazem. Apparent pA2 values were calculated by using concentration-response curves for calcium before and 30 min after the addition of each dilator drug. The order of potency (using both pA2 and IC50 values) for the calcium entry blocking effect was: nifedipine greater than verapamil greater than diltiazem greater than lidoflazine greater than dilazep. Dipyridamole and adenosine had negligible calcium entry blocking activities (about 10,000 times less potent than verapamil). The calcium entry blocking activity of verapamil (using pA2 values) was 39.8 times less potent than nifedipine, and 3.6, 21.4 and 97.7 times more potent than diltiazem, lidoflazine and dilazep, respectively. The maximum relaxations induced by adenosine (3.7 X 10(-4) mol/l) and dipyridamole (5 X 10(-5) mol/l) were less than 20% that of 3 X 10(-4) mol/l papaverine. However, the other test drugs caused 80-90% relaxation under similar conditions. The relaxing effect of adenosine was inhibited by 8-phenyltheophylline (adenosine receptor antagonist) and potentiated by EHNA (an adenosine deaminase inhibitor), while dilazep-induced relaxation was not affected by these drugs. These findings suggest that the calcium entry blocking effect of dilazep in dog, large coronary artery strips is not mediated through adenosine. PMID- 3018804 TI - Spinal cord injury community follow-up. Role of the physical therapist. AB - This study was conducted over a 14-month period to assess the physical therapy intervention needs of 201 patients who attended monthly spinal cord injury (SCI) outpatient clinics conducted in three outlying local communities of northern California. Methods to identify and provide appropriate physical therapy services for the patients were explored. An experienced physical therapist from a regional SCI center and other clinic staff members screened the patients and identified 66 patients (33%) who needed physical therapy services, including evaluation (82%), patient education (62%), and referrals to appropriate local health care professionals or equipment vendors (52%). Losses of joint range of motion, changes in sitting posture as a result of increased muscle tone or contracture, and malaligned or misfitting lower extremity orthoses were identified as problem areas not commonly recognized in routine follow-up examinations. Recommendations based on our study findings included the use of a screening form for physical therapy needs at each clinic, improved patient education about the role of the physical therapist as a resource person during follow-up care, coverage for each of the three clinics on a biannual basis, and continued study of the mechanisms used by other SCI centers to fulfill the outpatient needs of their patients. Physical therapy involvement in SCI follow-up services can maximize efficient use of our health care resources and provide early identification and management of specific postdischarge needs. PMID- 3018803 TI - The role of beta-lactamase inhibitors in chemotherapy. PMID- 3018805 TI - Photoaddition of chlorpromazine to guanosine-5'-monophosphate. PMID- 3018806 TI - Porphyrin photosensitization of proteins in cell membranes as studied by spin labelling and by quantification of DTNB-reactive SH-groups. PMID- 3018807 TI - The site-specific inhibition of Bgl I cleavage by psoralen photoadducts. PMID- 3018808 TI - Free radical production by chlorpromazine sulfoxide, an ESR spin-trapping and flash photolysis study. PMID- 3018809 TI - Behavioral effects of intraventricular dibutyryl cyclic AMP in domestic fowl. AB - Intraventricular administration of dibutyryl cyclic AMP (dbcAMP) to domestic fowl induced behaviors within 60 seconds which persisted for 7-120 minutes. Stereotyped head movements and increases in preening were observed at the lowest dose (50 nmol), while at higher doses (150 and 225 nmol) head movements were interspersed with escape behavior, increases in locomotor activity, salivation and a loss of coordination. Administration also elicited vocalizations, mainly laying and type 1 warning calls. These calls contained many abnormal elements, possibly caused by relaxation of the syringeal musculature. The rate of calling was influenced by testosterone, being greater in hens and capons than in roosters or capons implanted with testosterone propionate. Caponization also intensified escape behavior. No behaviors were induced by administration of the hydrolysis product of dbcAMP, butyric acid. These behavioral effects of dbcAMP are similar to those reported to occur during electrical stimulation of loci in the avian brain. PMID- 3018811 TI - Effects of ergot alkaloids on stress-induced analgesia. AB - Repeated, thirty minute anticipation of unavoidable painful stimulation causes endorphin-induced analgesia in rats. This type of stress-induced analgesia (SIA) develops rapidly during the first minutes of the exposure to anticipation stress. SIA can be demonstrated during the whole period of anticipation stress. Ergot drugs (DH--ergotoxine, lisuride, trans-9,10 dihydrolisuride) administered 30 min before the onset of anticipation stress, blocked completely this form of SIA. On the other hand, no effect of ergot alkaloids in the tail-flick latency, as measured under resting conditions, was observed. Possible interactions of ergot alkaloids with opiate receptors as an important mechanism by which ergot drugs affect SIA are discussed. PMID- 3018810 TI - Influences of feeding and drinking on circadian rhythms of opioid peptides in plasma, hypothalamus and pituitary gland in rats. AB - Changes of plasma, hypothalamic and pituitary immunoreactive beta-endorphin (IR beta-end), methionine-enkephalin (IR-Met-enk) and ACTH (IR-ACTH) were studied under various conditions of feeding and watering in rats. When rats were fed from 17:00 to 09:00 hr and water was given ad lib, plasma IR-beta-end and IR-ACTH had parallel circadian rhythms with a peak before feeding and drinking. In the hypothalamus, IR-beta-end and IR-Met-enk showed parallel circadian rhythms with a decrease before these behaviors. When rats were fed from 09:00 to 17:00 hr, the peaks of plasma IR-beta-end and IR-ACTH shifted to one hour before the onset of feeding and drinking. When feeding and watering were restricted to 17:00-09:00 hr and 09:00-12:00 hr respectively, plasma IR-beta-end and IR-ACTH exhibited parallel circadian rhythms with two separate peaks at one hour before drinking and feeding, respectively. In the hypothalamus, IR-beta-end, IR-Met-enk and IR ACTH showed parallel circadian rhythms with a decrease before feeding but not before drinking. When rats were fed from 17:00 to 20:00 hr, plasma IR-beta-end increased and neurohypophysial IR-beta-end and IR-Met-enk decreased at 16:00 hr, one hour before feeding. It was observed that locomotor activities increased at the time of transition from light to dark and at one hour before the onset of feeding and drinking. The present results suggest that endogenous opioid peptides may have some physiological roles in feeding and drinking behaviors. PMID- 3018813 TI - Cardio-pulmonary arrest--asystole: a review of the medications used to restart the heart. PMID- 3018812 TI - Development of resistance of the outer membrane of brain and liver mitochondria. AB - The outer membrane resistance of brain and liver mitochondria to the action of digitonin was studied in 5- and 60-day-old rats. Disintegration of the outer membrane was evaluated by means of cytochrome oxidase activity. The mitochondria were isolated by the Ficoll gradient method. Brain mitochondria were found to be significantly more resistant to digitonin than liver mitochondria. It was also demonstrated that the resistance of the outer membrane of both brain and liver mitochondria rose approximately 1.3- to 1.4-fold during ontogenesis. PMID- 3018815 TI - The effect of ginsenosides on adrenocorticotropin secretion in primary culture of rat pituitary cells. PMID- 3018814 TI - Restriction maps and homologies of the three plasmids of Agrobacterium rhizogenes strain A4. AB - Agrobacterium rhizogenes strain A4 is a virulent agropine-type strain possessing three plasmids: plasmid a (pArA4a, 180 kb) is not necessary for plant transformation, plasmid b (250 kb) is the root-inducing plasmid (pRiA4), and plasmid c (pArA4c) is a cointegrate of pArA4a and pRiA4. The total plasmid DNA (pArA4) of strain A4 was cloned in the cosmid pHSG262 and the library obtained was used to establish BamHI maps of the three plasmids. The plasmids a and Ri have an apparently identical region and a partly homologous region, and are different in the remaining regions including their origins of replication. Another agropine-type A. rhizogenes strain, HRI, bears only one plasmid, which is the Ri plasmid (pRiHRI). pRiHRI and pRiA4 present the same restriction maps for a great part, but are different in a region of 48 kb; however, this region of pRiHRI is found unmodified in pArA4a and may have a role in the virulence of the bacteria. The comparison between the restriction maps of the plasmids of strain A4 leads us to propose that the recombination event leading to pArA4c formation occurs within the identical regions of pArA4a and pRiA4. In addition, the comparison with the already established map of pRiHRI suggests that strain HRI could have been derived from a recombination event between the two homologous regions of pArA4c with subsequent loss of the smaller plasmid. PMID- 3018816 TI - [Alcoholics in detoxification treatment: provision of further measures for rehabilitation]. AB - Detoxified alcoholics in psychiatric hospitals of a West German large city are investigated with regard to the question who of them get recommendations for further rehabilitation: to take part in a self help group or therapy. Patient documentations of two years are analysed. Results show that few detoxified alcoholics get advice for further rehabilitation. These persons are socially more integrated than those without advice. PMID- 3018817 TI - Alpha 2-adrenoceptor levels on platelets of patients with major depressive disorders. AB - 3H-p-Aminoclonidine binding to platelets of patients with primary, unipolar major depressive disorder was compared to that of a normal healthy control population. The variances of platelet alpha 2-adrenoceptor Kd and Bmax values in patients were significantly greater than in the control group. No significant difference could be demonstrated between the Kd values of the two different groups, but the Bmax value of the depressed group was significantly lower than that of controls. We propose that an abnormal platelet alpha 2-adrenoceptor density may be used as a biological marker for major depressive disorder. PMID- 3018819 TI - Platelet 3H-imipramine binding in bipolar patients. AB - Platelet 3H-imipramine binding was investigated in 31 control subjects and 19 hospitalized bipolar patients, either in the hypomanic or the depressed phase of illness. The mean Bmax value in the bipolar depressed patients did not differ significantly from that in the control subjects or the hypomanic patients. Differences in timing of the assay after blood collection, membrane preparation, protein content used in the assay, or binding of radioactive ligand to the equipment do not appear to explain the discrepancy between these results and previous findings. PMID- 3018818 TI - Imipramine binding sites on platelets of patients with major depressive disorder. AB - 3H-Imipramine binding to platelets of patients with primary, unipolar major depressive disorder was investigated and compared to that of a normal, healthy control population. No significant differences could be demonstrated between either the Kd or the Bmax values of the two groups. A negative correlation was observed between imipramine Bmax values and the Hamilton anxiety ratings of the depressed patients. Patients who displayed psychomotor retardation tended to have lower platelet imipramine Bmax values than patients with psychomotor agitation. It is suggested that platelet imipramine Bmax values may be a biological marker for subtypes of depression. PMID- 3018820 TI - Vasopressin and oxytocin: hypothalamic modulators of the stress response: a review. AB - ACTH secretion is primarily controlled by hypothalamic secretion of corticotropin releasing factor (CRF) into pituitary portal blood. However arginine vasopressin (AVP) and oxytocin (OT) can modulate the actions of CRF and at times may be important mediators of stress-induced ACTH secretion. The relative contributions of CRF, AVP, and OT to the control of ACTH secretion vary with different types of stress. In general, AVP stimulates ACTH secretion in all species studied. OT also stimulates ACTH release in rats but is inhibitory in primates. The involvement of AVP and OT in the control of ACTH secretion may have important implications for physiological and pathological conditions associated with activation of the hypothalamo--hypophysial--adrenal cortical axis. PMID- 3018821 TI - Cortisol and corticosterone response after syn-corticotropin in relationship to dexamethasone suppressibility of cortisol. AB - The current study was designed to investigate whether glucocorticoid output after syn-ACTH stimulation is different in depression associated with dexamethasone suppression test (DST) nonsuppression from the euthymic state and DST suppression. We gave 28 depressives a DST and an adrenocortical challenge with synthetic ACTH. Fourteen patients were nonsuppressors on the DST. After successful drug treatment, the subjects were reinvestigated by both tests; all DSTs revealed plasma cortisol concentrations below the criterion value of 50 ng/ml. Cortisol and corticosterone responses after syn-ACTH tended to be higher during depression. After clinical remission, higher cortisol and corticosterone responses occurred in those patients who were DST nonsuppressors during depression. This finding suggests that patients who suffer from a depression which is linked to an abnormal pituitary--adrenocortical regulation develop an enhanced sensitivity of the adrenal cortex to ACTH. PMID- 3018822 TI - Central and peripheral cholinesterase inhibition: effects on anterior pituitary and sympathomimetic function. AB - Ten physically healthy inpatients of mixed diagnosis received, in a randomized, counterbalanced double-blind paradigm, physostigmine (22 micrograms/kg) and neostigmine (11 micrograms/kg). Infusions were separated by at least 2 days. The differential effects of physostigmine and neostigmine on plasma concentrations of cortisol, prolactin, growth hormone, ACTH, beta-endorphin/beta-lipotropin-like immunoreactivity, dopamine, norepinephrine, and epinephrine are reported. Administration of physostigmine, unlike that of neostigmine, was associated with statistically significant increases in plasma concentrations of cortisol, prolactin, ACTH, beta-endorphin/beta-lipotropin-like immunoreactivity, and epinephrine, presumably via central mechanisms. In a separate study, 15 subjects, mostly depressed inpatients, were pretreated with methscopolamine (0.75 mg) on one day and scopolamine (0.5 mg) on another day, at least 2 days apart, in a randomized, counterbalanced double blind paradigm and subsequently on each day received physostigmine (22 micrograms/kg). Scopolamine significantly attenuated the physostigmine-associated increase in plasma concentrations of cortisol, growth hormone, prolactin, ACTH, and dopamine compared to methscopolamine, and a close-to-significant attenuation of epinephrine as well. These results provide further evidence that physostigmine's effects on plasma concentrations of pituitary hormones and epinephrine occur via central mechanisms and are muscarinically mediated. PMID- 3018824 TI - Differential effects of ethyl (R,S)-nipecotate on the behaviors of highly and minimally aggressive female golden hamsters. AB - The GABA uptake inhibitor ethyl (R,S)-nipecotate produces a dose-dependent suppression of aggression in highly aggressive hamsters but not in minimally aggressive ones. This suppression occurs at doses below those producing peripheral cholinergic effects; at the highest dose used it persists after these effects have dissipated. Doses sufficient to suppress aggression have no significant effect on grooming, locomotor activity and other behaviors but do affect sunflower seed acceptance. The differential effects of the drug on highly and minimally aggressive animals may indicate that their differences in aggression are due to differences in endogenous GABAergic activity. These results, together with previous evidence for parallel circadian variation in GABA uptake and aggressive behavior, suggest that GABA uptake may be an important endogenous regulator of aggression. PMID- 3018823 TI - The influence of central noradrenergic function on 5-HT2-mediated head-twitch responses in mice: possible implications for the actions of antidepressant drugs. AB - This study has investigated the influence of central noradrenergic function on 5 HT2-mediated head-twitch responses in mice. Central injection of low doses of the alpha 1-adrenoceptor agonists phenylephrine or methoxamine, or peripheral administration of the antagonist prazosin had no effect on the head-twitches induced by 5-methoxy-N,N-dimethyltryptamine (5-MeODMT). High doses of both alpha 1-adrenoceptor agonists and antagonists markedly inhibited this response. Head twitches induced by 5-MeODMT were potently inhibited by low doses of the alpha 2 adrenoceptor agonist clonidine, and potentiated by the antagonists idazoxan and yohimbine. Clonidine also potently inhibited this response when produced by 5 hydroxytryptophan (5-HTP) and carbidopa. The action of the beta-adrenoceptor agonist clenbuterol on head-twitches was paradoxical, this drug enhancing the responses to precursor loading (5-HTP/carbidopa) but inhibiting those induced by direct agonists (5-MeODMT, quipazine). Lesioning noradrenergic neurons by central injection of 6-hydroxydopamine (6-OHDA) or peripheral administration of DSP-4 resulted in enhanced head-twitch behaviour. 6-Hydroxydopamine lesioning did not alter the inhibition of head-twitch responses by clonidine but prevented their enhancement following withdrawal from repeated desmethylimipramine (DMI) administration. It is therefore suggested that head-twitch behaviour may be under tonic control by a population of alpha 2-adrenoceptors which are not on presynaptic noradrenergic terminals, but are postsynaptic and located "down stream" of the 5-HT2 receptor. In addition, the enhancement of this behaviour produced by withdrawal from repeated DMI administration probably also resulted from alterations in central noradrenergic function. PMID- 3018825 TI - Benzodiazepine-induced hyperphagia: stereospecificity and antagonism by pyrazoloquinolines, CGS 9895 and CGS 9896. AB - Non-deprived male rats were familiarized with a highly palatable diet until baseline consumption in a 30-min daily access period had stabilised. Stereospecificity of the hyperphagic effect of benzodiazepine receptor agonists was demonstrated using two enantiomers, the (S)-enantiomer being Ro11-3128 (methylclonazepam) and the (R)-enantiomer, Ro11-3624. The benzodiazepine receptor antagonists, Ro15-1788 and CGS 8216, reversed the hyperphagic effect of Ro11 3128. These data confirm the mediation of the hyperphagic effect of benzodiazepines by specific receptors. In further experiments, the effects of the pyrazoloquinolines CGS 9895 and CGS 9896 were examined both alone and also in combination with clonazepam. In doses of 1.25-10.0 mg/kg, neither CGS 9895 nor CGS 9896, when given alone, had a significant effect on the consumption of the palatable diet. Both, however, dose-dependently antagonised the hyperphagic effect of clonazepam. In a test of palatable food consumption, therefore, both compounds can be characterised as benzodiazepine antagonists. PMID- 3018826 TI - Benzodiazepine receptor-mediated effect of CGS 8216 on milk consumption in the non-deprived rat. AB - The pyrazoloquinoline CGS 8216, which binds with high affinity to central benzodiazepine recognition sites, produced a highly significant reduction in the consumption of familiar, sweetened milk by non-deprived male rats, when administered in a dose of 20 mg/kg, IP. The anorectic effect was present during the first 5 min period of a 20-min drinking test, and remained in evidence throughout the remainder of the test. The benzodiazepine receptor antagonist Ro15 1788, administered 15 min before the consumption test, produced a dose-related (10-40 mg/kg, IP) reversal of the anorectic effect of CGS 8216, during the first 10 min of the test. Injection of Ro15-1788 alone had no significant effect on milk ingestion. This experiment shows that the reduction in the consumption of a palatable liquid food by CGS 8216 can be attributed to an action at benzodiazepine receptors. The result is consistent with the characterization of CGS 8216 as a weak benzodiazepine receptor inverse agonist. PMID- 3018829 TI - [Hydroxylapatite reconstruction--progress compared to earlier methods of restorative alveoloplasty]. PMID- 3018827 TI - Possible psychophysiologic mechanisms in premature labor. PMID- 3018828 TI - [Repair of periodontal bone pockets with hydroxylapatite]. PMID- 3018830 TI - [HTLV III/LAV (AIDS)-infection protection]. PMID- 3018831 TI - Equilibrium dialysis studies of the binding of radioprotector compounds to DNA. AB - Radioprotection by WR-2721, S-2-(3-aminopropylamino)ethyl phosphorothioate, is thought to involve its corresponding thiol (WR-1065) or symmetrical disulfide (WR 33278). It has been suggested that these metabolites concentrate close to the DNA target molecule; to test this hypothesis we have measured their in vitro binding to DNA. The binding of WR-33278 (0.05-0.4 mM) to calf thymus DNA (6 mM, with respect to DNA phosphate) was determined at 50, 100, and 150 mM KCl in 1 mM Tris, pH 7, by equilibrium dialysis. The binding of WR-1065 (0.5-8mM) was determined at 25, 50, and 100 mM KCl, under similar conditions, but with 2 mM EDTA and 3 mM dithiothreitol (DTT) added to the dialysis buffer to prevent thiol oxidation. Drug levels were quantitated by HPLC after fluorescent labeling with monobromobimane; disulfide samples were reduced with DTT prior to analysis. Dissociation constants (Kd = [Free Drug] [DNA site]/ [bound drug] ) under these conditions were found to vary with ionic strength, being in the range of 0.02 +/- 0.01 to 0.18 +/- 0.06 mM for WR-33278 and 0.43 +/- 0.24 to 3.5 +2/- 1.5 mM for WR 1065. WR-2721, glutathione, cysteine, and DTT showed no detectable binding to DNA in 25 mM KCl. However, cysteamine and cystamine did bind to DNA, with unbound drug to bound drug ratios of 8 +/- 2 and 0.6 +/- 0.1, respectively, at total drug concentrations of 1 mM. Cystamine and WR-1065 bound to DNA with comparable affinity under similar conditions. These results indicate that binding of WR 33278 and WR-1065 by DNA phosphate are probably significant in the mechanism of radioprotection by WR-2721. PMID- 3018832 TI - [Mediator function of molecular factors--lipoxygenase enzyme systems during exposure to ionizing radiation]. AB - This paper, which is a review of the national literature on the mediatory function of lipoxygenase systems affected by ionizing radiation, reports data on the functional role of lipoxygenases, their regulator--mediate contribution to radiation response of plants and animals. The latest data are submitted concerning the biological regulatory systems, eicosanoids--leukotrienes, formed under the effect of lipoxygenases which catalyse oxidation of polyunsaturated arachidonic acid (20:4). The structure and function of leukotrienes are described for these are necessary in studying the biochemical functions of lipoxygenases after ionizing irradiation. Emphasis is made on biologically active leukotrienes which take part in biological processes involved in inflammatory and hypersensitive reactions under the effect of radiation. Possible involvement of the lipoxygenase systems in metabolism regulation, its resistance and activation in a living body during development of radiopathological processes are discussed. The modelling concepts are considered for perspective radiation research aimed at biotechnological utilisation of lipoxygenases. Some regularities of mediatory function of lipoxygenase systems have been found. An assumption has been made that lipoxygenases and leukotrienes play an important role in the life of irradiated cells and tissues. PMID- 3018833 TI - [Optimization of the composition of the radioprotective complex APAETP+mexamine and analysis of its action]. AB - The method of mathematical theory of experiment was used to find optimum variants of the radioprotective complex APAETP + mexamine. The character of their pharmacological interaction, depending on their dose ratio, was determined. It is suggested that it is conditioned by the specific role of different mechanisms involved in the radioprotective effect. PMID- 3018834 TI - [The effect of small doses of ionizing radiation on the cAMP system in the thyroid gland of chick embryos]. AB - Adenylate cyclase activity and cyclic adenosine monophosphate (cAMP) content were studied in the thyroid gland of intact and irradiated (0.029 Gy given before incubation) embryos and chickens. The enzyme activity was stimulated and the nucleotide content increased on the 18th day of the embryo development. It is suggested that the observed stimulation of the cAMP system is associated with the increased secretion of the thyrotropic hormone which controls the functional activity of the thyroid gland. PMID- 3018835 TI - [Modification by mesoporphyrin-IX derivatives of Chinese hamster cell damage induced by high energy helium ions and protons]. AB - The damaging effect of high-energy helium ions (4 GeV), protons (9 GeV), and standard radiation was different as studied on Chinese hamster cell cultures (clone 431). A possibility of increasing the survival of cells by the pre- and post-irradiation treatment with a mesoporphyrin-IX derivative was demonstrated. It is suggested that the radioprotective effect of the modifier is associated with the decreased level of aberrant mitoses. PMID- 3018836 TI - [Estimation of the contribution of the dose rate to the effect of whole body uniform irradiation with 1000 MeV protons]. AB - Some biochemical indices of peripheral blood and spleen and the survival rate of albino rats after whole-body uniform irradiation with 1000 MeV protons delivered in two ways have been studied. The data obtained indicate that the dose rate contributes to the biological effect of impulse proton radiation. PMID- 3018837 TI - [Measurement procedure for further radiologic analysis of bone. A review of the quantitative radiology of the skeleton]. AB - The reasons for using measurements of bone density for further analysis of changes in metabolic bone diseases are pointed out. Besides the easy methods of visual comparison of X-ray images and "microradioscopy", the methods of quantitative radiology are discussed. The X-ray morphometry method is presented, including the new results of measurement of various bones of the skeleton. Structural analysis of spongy and compact bone tissue from the X-ray image using opto electronic image transformation is demonstrated. The techniques of X-ray photodensitometry and isotope-densitometry are explained. The importance of a reproducible calibration standard and the special value of reference systems containing a known concentration of hydroxyapatite are shown. Special information is given regarding the present status of the development of the new technique of quantitative computer tomography (QCT) or computer tomometry with the aid of a solid reference system. This new reference system is constructed from various concentrations of hydroxyapatite in "solid water" (a polyethylene mixture) and is demonstrated in detail. The results of comparing measurements of the 2nd lumbar vertebral body are shown. Finally, the clinical importance of measurements of the "apatite value" of vertebral trabecular bone tissue and of various trabecular areas of the skeleton is emphasized. PMID- 3018838 TI - Topographic histochemistry of the cerebellum. 5'-nucleotidase, acetylcholinesterase, immunology of FAL. PMID- 3018839 TI - Immunobiology of human papillomavirus infection. PMID- 3018840 TI - Aleutian disease: a persistent parvovirus infection of mink with a maximal but ineffective host humoral immune response. PMID- 3018841 TI - Herpesvirus latency and recurrence. PMID- 3018842 TI - An illustrated guide to the structure of the human cytomegalovirus genome and a review of transcription data. PMID- 3018844 TI - [5'-nucleotidase and its clinical significance in diseases of the liver and biliary tract]. PMID- 3018843 TI - [Release of plasma membrane-bound enzymes by phosphatidylinositol-specific phospholipase C]. PMID- 3018845 TI - 5-Hydroxytryptamine and (+)-lysergic acid diethylamide displace [3H]thyrotropin releasing hormone at its binding sites in the rat limbic forebrain. AB - Binding of [3H]thyrotropin-releasing hormone (TRH) to its receptors in the rat limbic forebrain was partially displaced by 5-hydroxytryptamine (5-HT, ligand for 5-HT1 receptors) and (+)-lysergic acid diethylamide ((+)-LSD, ligand for 5-HT1 and 5-HT2 receptors) at nanomolar concentrations. Spiperone (ligand for 5-HT2 receptors) displaced [3H]TRH in a dose-dependent manner at micromolar concentrations. These results suggest that some TRH receptors are related to 5 HT1 receptors, probably adjoining them on the membrane. This type of TRH receptor is shown to be among the high-affinity receptors which we reported previously. The significance of the receptor-coexistence is such that TRH facilitates serotonergic transmission by increasing the density of 5-HT1 receptors. This finding seems to support a pharmacological observation of other investigators that TRH potentiates 5-HT-induced hyperactivity in mice, probably by affecting postsynaptic 5-HT receptors. PMID- 3018846 TI - Effects of peripherally injected pituitary peptides on recognition memory in pigeons. AB - The effects of peripheral injection of arginine vasopressin (AVP), oxytocin (OT), arginine vasotocin (AVT), adrenocorticotrophic hormone (ACTH4-10) and alpha melanocyte-stimulating hormone (alpha-MSH) were studied, using pigeon subjects and a pair comparison task, with and without intervening delays between stimuli presentation. The highest dose (20 micrograms/kg) of AVT impaired performance by disrupting input, but not storage. General cognitive ability was unimpaired, as were perceptual mechanisms. None of the other peptides affected recognition memory, in that forgetting curves were unchanged when compared with control. The results are discussed in terms of species-specific roles for these peptides. PMID- 3018847 TI - [Seroepidemiology of the HTLV-III virus in a high-risk Panamanian population. Preliminary report]. PMID- 3018848 TI - [Magneto-pharmaceutic contrast changes in nuclear magnetic resonance tomography]. AB - Changes in contrast due to paramagnetic substances depend on many factors, such as the properties and concentration of the substance, the external pulse and time parameters and the inherent relaxation time of the tissues. The physical background for contrast enhancement by paramagnetic substances is reviewed and the various groups of magneto-pharmaceuticals which have been used experimentally and clinically are described. The effect on signal intensity due to changes in dose, pulse sequence, repetition and echo times have been simulated and measured on phantoms and the effect of various tissue relaxation times on contrast has been investigated. PMID- 3018849 TI - [Subjective evaluation and quantitative gray-scale analysis in the sonographic diagnosis of diffuse changes in the liver parenchyma]. AB - Two investigators were asked to evaluate independently the echogenicity, coarsening and inhomogeneity of the hepatic echo pattern in a semi-quantitative manner; they had no knowledge of the diagnosis and used the same apparatus. The three subjective criteria were largely independent from the observer. Approximately three-quarters of all patients with slightly increased echogenicity showed a subjective appearance of coarsening; the impression of inhomogeneity increased significantly with increasing echogenicity. This is of importance in the sonographic evaluation of cirrhosis of the liver and in the diagnosis of diffuse liver metastases in a fatty liver. Grey scale analysis within a region of interest showed satisfactory correlation between the measured mean grey scale and echogenicity. Standard deviation within the grey scale was redundant, and there was no correlation between the other subjective characteristics and quantitatively measurable values. PMID- 3018850 TI - [Real-time sonography in extranodal lymphoma]. AB - Sonography is the primary imaging method for staging malignant lymphomas. The accuracy of sonography in diagnosing hepatic and splenic involvement was examined in 132 histologically proven cases. Focal involvement was differentiated from diffuse lymphoma. The value of sonography is discussed with reference to cases showing involvement of the gastro-intestinal tract, kidneys, suprarenals, pancreas, testes, breast, thyroid, parotid and muscle. PMID- 3018851 TI - [CT-cisternography of the basal cisterns. A roentgen anatomic study]. AB - Air cisternograms at post mortem and positive contrast cisternograms on patients were performed in order to study intracisternal structures, particularly cranial nerves, as seen on CT. Air and contrast CT cisternograms showed excellent demonstration of the second, third, fifth, sixth, seventh and eighth cranial nerves. The ninth and tenth cranial nerves could not always be separated from each other and demonstration of the first, fourth, eleventh and twelfth cranial nerves was often not possible or was unsatisfactory. With a knowledge of the normal anatomy and of important surrounding structures, the individual cranial nerves are easily identified. The anthropologic baseline appears highly suitable for CT examination of the basal cisterns. The complementary coronal projection is also very valuable. PMID- 3018852 TI - [Magnetic resonance tomography of brain tumors--comparison of the results using the multi-echo technic and gadolinium-DTPA]. AB - In thirty-seven MR examinations of intracranial tumours equivalent sections were obtained in a multi-echo technique before and after intravenous injection of 0.1 mmol. gadolinium DTPA/kg body weight. From this comparison the following preliminary conclusions have been drawn concerning the demonstration of the tumour, its delineation and type: Contrast administration does not unequivocally improve the sensitivity. In 55% of the cases, differentiation between tumour and oedema respectively normal brain tissue was easier after Gd-DTPA. Diffusely infiltrating gliomas remain a problem, since their extent is uncertain with or without contrast medium. The structure of the tumour can already be adequately characterized by the multi-echo technique. In order to diagnose the type of tumour, the criteria which apply to Gd-DTPA are similar to those used for iodine containing contrast media in CT. PMID- 3018853 TI - [NMR-tomography of cerebral midline tumors and cervical cord processes in children]. AB - Tumours of the brain, mesencephalon and cervical spinal cord in 10 children were examined by MRI. The most useful anatomical information was supplied by the sagittal midplane tomogram. Due to the prolongation of T2-times in brain tumours T2-weighted images were appropriate for detecting pathological structures even without space-occupying signs. Questions of operability and therapeutic effectiveness were reliably answered. PMID- 3018854 TI - [Results of a comparative study of MR, CT and sonography of patients with primary hyperparathyroidism]. AB - Twenty-three patients with primary hyperparathyroidism were examined by MR, CT and sonography in order to localise the parathyroid adenoma. In twenty patients, surgery was performed and the findings confirmed histologically. Fifteen patients had an adenoma in a parathyroid and in three, the adenoma was in the mediastinum. In one patient there was hyperplasia of all parathyroids. Accuracy of MR was 98%, of CT 98% and sonography 94% in previously unoperated patients and is therefore similar for these methods. This is also true for previously operated patients (four) where MR was 88% accurate, CT 83% and sonography 91%. In sixteen patients without previous operation, MR had the highest sensitivity with 79%. In previously operated patients, the sensitivity of MR and sonography was equal, with 67%, and CT was similar with 66%, MR was able to differentiate adenomas by the specific measurements of T1 and T2. PMID- 3018855 TI - [MR of ischemic brain diseases. A comparison with CT, PET (18-fluorodeoxyglucose) and angiographic results]. AB - The results of MRI and CT in 55 patients with brain infarcts were compared; in 26 of these cases an additional PET examination was obtained in order to study the regional glucose utilisation. MRI was superior to CT, demonstrating 11% more of the infarcts, particularly during the first 24 hours, in small lesions confined to the grey or subcortical white matter and in infratentorial ischemic lesion. On the other hand, only CT was able to show fresh hemorrhage, although MRI was the method of choice to demonstrate old blood collections. To characterise the follow up of an infarct, CT and MRI were similar, except the marginal contrast enhancement sometimes demonstrated by CT studies between the 2nd and 4th week after stroke event. PET was inferior to show details because of its poorer spatial resolution, but anyhow had a high sensitivity and provided additional informations concerning secondary inactivations of brain areas not directly damaged. Additionally PET was able to demonstrate areas of anaerobic glycolysis and lesions of diminished glucose utilisation in TIAs. Small areas of gliosis in the white matter of the cerebral hemispheres were frequently found in patients with cerebro-vascular diseases; they were best shown by MRI, but do not correlate with the extent of vascular stenoses or occlusions, shown by angiography. PMID- 3018857 TI - [Analysis of the NMR signal for biological information in spinal diagnosis]. AB - Discs of 23 patients and volunteers were examined for the purpose of obtaining T1 and T2-relaxation times. First of all, the signal characteristics of the surface coil were evaluated. The relaxation times of 100 discs were then measured by using standard spin echo sequences. There was a correlation between age and T2 values for healthy discs (R = -0.744). This was not found in case of degenerated discs. The mean value of T1 for healthy discs was found to be 825 ms. The results show a possibility of using relaxation measurements in routine diagnosis for the early identification of disc degeneration. PMID- 3018856 TI - [Single photon emission computed tomography of patients with benign and malignant spinal diseases]. AB - SPECT has been used in a comparative study with planar bone scintigraphy in 54 patients. Spatial resolution of the method is sufficient to localise the vertebral bodies, the spinous processes, the intervertebral, costotransverse and costovertebral joints. In all patients it was possible to relate the areas of increased uptake to specific anatomical sites of the spine known to be affected in the different conditions. A detailed localisation is rarely possible using planar scintigraphy alone, due to the complex osseous anatomy of the spine. In some patients lesions could be seen only with SPECT. SPECT is an invaluable supplement to planar scintigraphy of the spine. PMID- 3018858 TI - [Imaging of cruciate ligament injuries using MR tomography]. AB - The cruciate ligaments of the knee joint can be demonstrated by magnetic resonance tomography, without using invasive methods. High resolution coils and section thickness of less than 5 mm are necessary. Sixty-six patients and volunteers were examined; the results show that the normal cruciate ligaments present as homogeneous zones of low signal intensity within the surrounding fat. Injuries to the cruciate ligaments interrupt the image of the ligaments, change their anatomical position or produce a signal of intermediate intensity. Eighteen operations were carried out on fifteen patients with cruciate ligament injuries and there was good agreement between the MR and the operative findings. Sixteen patients had had previous cruciate surgery and in these there was only partial correlation with the clinical findings. PMID- 3018859 TI - [Nuclear magnetic resonance tomography in the diagnosis of muscular diseases]. AB - Forty-nine patients with various systemic muscle diseases were examined by MR using a 1 Tesla magnet and the appearances of different conditions are analysed. Emphasis was placed on the analysis of patients with progressive muscular dystrophies, myositis, myotonia dystrophica and other muscle diseases. The investigation was begun in March 1984 and was continued until September 1985. Certain characteristic patterns of selectively involved muscles could be recognised. The pattern corresponds to our present understanding of the early phases of muscle diseases, whether inflammatory or due to fatty degeneration. The T1 and T2 relaxation times in various patients were quantified and changes in the normal pattern were analysed. Attention is drawn to the value of MR when carrying out a biopsy and for treatment of muscle diseases. PMID- 3018860 TI - [Adult respiratory distress syndrome. Radiologic and clinical chemical findings]. AB - Our present-day knowledge concerning the clinico-chemical and radiological findings in adult respiratory distress syndrome are described. Three typical case histories have been selected to illustrate this condition; they were due to multiple trauma or sepsis. It is stressed that radiology is in a key position for making the diagnosis and for observing the course of the illness. PMID- 3018862 TI - A new transhepatic endoprosthesis to prevent dislodgement. AB - A new transhepatic endoprosthesis, a so-called screw endoprosthesis with a spiral shaped elevation to prevent dislodgement, is presented. The elevation is created by a silver wire wound around the endoprosthesis. Further, the wire may be used as a marker for radiotherapy or for taking biopsies, and it makes it easier to control the position of the endoprosthesis. The screw endoprosthesis was used in 22 patients, including 9 patients with high strictures in which dislodgement is known to be frequent, and 2 patients in whom previously inserted ordinary endoprostheses had been dislodged. The insertion of the screw endoprosthesis was easy. The complication rate was similar to that of ordinary transhepatic endoprosthesis. No cases of dislodgement were observed. PMID- 3018861 TI - [Digital subtraction angiography of cancerous changes in the ear, nose and throat area]. AB - Intravenous and intra-arterial digital subtraction angiography is able to demonstrate hypervascular tumours in the ear, nose and throat territory. Direct puncture of a venous bypass used for intra-arterial chemotherapy, within the external carotid artery territory, is devoid of risk and can be used for assessing the accessibility of the tumour. Lesions of low vascularity can only be recognised by DSA on the basis of vessel displacement. These lesions are not a primary indication for examination by digital angiography. PMID- 3018863 TI - [Radiation exposure of patients and operators during interventional radiology]. AB - Surface doses received by patients and operators were measured during 30 interventional radiological procedures (ten percutaneous transhepatic biliary drainages, ten percutaneous nephrostomies, ten percutaneous transluminal angioplasties). In addition, organ doses to the patient were determined using an Alderson-Rando phantom. These served as a basis for calculating the so-called somatic dose indices. It was found that the somatic radiation risk to the patient is relatively small despite prolonged periods of fluoroscopy. However, exposure of the hands and lenses of the operator could easily reach the limits thought acceptable while carrying out these procedures with additional angiography. PMID- 3018864 TI - [Energy dependence of Hounsfield numbers]. AB - The linear absorption co-efficient of various tissues, as determined by Phelps, was converted into Hounsfield units. This demonstrated their close energy dependence; the possibility of tissue characterization and differentiation by CT is pointed out. PMID- 3018865 TI - [1.5 tesla nuclear magnetic resonance tomography of a patient with metallic osteosynthesis material]. PMID- 3018866 TI - [Detection of liver involvement in hereditary hemorrhagic telangiectasia (Rendu Osler-Weber disease) using imaging procedures]. PMID- 3018867 TI - Three cases of pigmented villonodular synovitis of the knee. Ultrasound and computed tomographic findings. PMID- 3018868 TI - Retroperitoneal Castleman's tumour of plasma-cell type. PMID- 3018870 TI - [New puncture aid in angiography (puncture doppler sonde)]. PMID- 3018869 TI - [Malignant fibrous histiocytoma of the CNS]. PMID- 3018871 TI - [Blue tongue, a new disease of sheep in La Reunion (Indian Ocean)]. PMID- 3018872 TI - [Experimentation, in wool-producing sheep, on the innocuity and efficacy of a modified poxvirus, isolated from a Mauritanian sheep with nodular sheep pox]. PMID- 3018873 TI - [Anatomoclinical correlations in primary hepatic carcinoma: a study of 130 cases from the geographic area of Valencia]. PMID- 3018874 TI - [Endogenous opioids in respiratory inhibition by laryngeal stimulation]. AB - The reflex effect elicited by mechanical stimulation of the glottis has been studied in dogs. The three components of the response--sudden apnea, constriction of the glottis and bradycardia--were modified by naloxone, although not in the same degree. These results suggest the existence of opioid interneurons between laryngeal afferent fibers and central inspiratory neurons. PMID- 3018875 TI - [Evaluation of free and bound fractions resulting from the interaction of prolactin with its specific receptors]. AB - Binding of either "cold" or 125I-PRL to their specific receptors (fraction after centrifugation at 15,000 and 100,000 X g) obtained from late pregnant rat liver, pre- and post-dissociation with MgCl2, has been studied. Binding was higher with cold hormone (delta 21.63%) than with 125I-PRL. Similarly, binding to the 100,000 X g fraction was also higher than to the 15,000 X g one. Dissociation by MgCl2 improved binding to the 100,000 X g fraction (delta 17.27%), while reduced the 15,000 X g fraction binding (delta 11.71%), underlying the impurity of the latter fraction. Control studies with rLH, rFSH, hACTH, insulin, glucagon and hGH evidenced the specificity of the preparation to bind lactogenic hormones. Binding increases with PRL and receptor concentration, reaching equilibrium between bound PRL/unbound PRL. An amount of PRL unable to bind to the receptor is always present. Even with high receptor concentrations (3,500 micrograms/0.1 ml) there is still about 25% of unbound PRL. When reincubating this previously unbound PRL with a fresh receptor preparation identical to the one used in the first incubation, a similar proportion of bound PRL/unbound PRL is obtained. These results suggest the existence of a heterogeneity in the receptor preparation. PMID- 3018876 TI - Renin granules isolated from rat kidney cortex by continuous colloidal silica (Percoll) density gradient centrifugation. AB - Renin granules were isolated from rat kidney cortex by a continuous polyvinyl pyrrolidone-coated colloidal silica (Percoll) density gradient centrifugation. A major peak of renin activity was found at a density of 1.12-1.13 g/ml, and the specific activity of renin in the peak fraction was increased by approximately 70 fold, as compared with that in the kidney cortex homogenate. On the other hand, activities of other reference enzymes, such as succinate dehydrogenase, acid phosphatase and glucose-6-phosphatase, were not detectable in the peak fraction. When the extract of the peak fraction was applied to a pepstatin column, trypsin activated renin could not be detected in the breakthrough fractions. These results indicate that renin granules of the rat kidney cortex contain only active renin. PMID- 3018877 TI - Comparative effects of parenteral and oral administration of selected dithiocarbamates on body burdens and organ distribution of cadmium in mice. AB - Diethyldithiocarbamate (DDTC), N-methyl-N-dithiocarboxyglucamine (MDCG), and 4 carboxamidopiperidine-N-dithiocarboxylate (CAP-N-DTC) were compared at equimolar doses administered by the ip and po routes for effectiveness in mobilizing metallothionein (MT)-bound cadmium (Cd) using 109Cd in mice. DDTC was highly effective by both routes in lowering hepatic, renal, and splenic Cd levels, but enhanced redistribution of Cd to testes and brain. While MDCG and CAP-N-DTC were much less active by the oral route, each effected a significant reduction of renal Cd levels, and MDCG reduced the testicular Cd burden. Neither analog lowered hepatic Cd levels impressively when given po. PMID- 3018879 TI - Heterochromatin and germ line-restricted DNA. PMID- 3018878 TI - The differentiation of germ and somatic cell lines in nematodes. PMID- 3018880 TI - Failure of argon laser to halt cytomegalovirus retinitis. AB - This report describes the experience with Argon laser used in an attempt to slow the advance of CMV retinitis in two eyes of two patients with AIDS, who were not able to be treated with antiviral chemotherapy. The treatment was performed to create a retinal scar to attempt to slow cell-to-cell spread of the virus through retinal tissue. In both patients, treatment was not successful in preventing spread of the CMV infection. Foci of retinitis, beyond the treated areas, became apparent within 2 weeks of treatment. Autopsy histopathology confirmed the diagnosis of CMV retinitis in both patients and necrosis due to CMV retinitis was seen in laser treated retina, and on both sides of laser treated retina. PMID- 3018881 TI - Macrophage recognition of self from nonself: implications for the interaction of macrophages with neoplastic cells. PMID- 3018883 TI - [AIDS etiology and epidemiology]. PMID- 3018884 TI - [The clinical symptoms of HTLV-III infections]. PMID- 3018882 TI - Leporine acquired immune deficiency disease. AB - We have identified a recombinant leporipoxvirus that produces disseminated fibromas and a severe combined immune deficiency disease of sudden onset. The virus is recombinant between the SFV and the MV. MV was identified as a trace contaminant in stocks of SFV (Patuxent strain). Rabbits inoculated with the original uncloned stock of SFV prepared in vitro develop local tumors that subsequently regress. However, tumor extracts prepared from these animals, when injected into a second group of rabbits, produced MV syndrome. Rabbits with MV syndrome develop severe, usually lethal, Pasteurella or Bordetella infections and have disseminated fibroxanthosarcomas more similar to those produced by myxoma virus. The virus that induces this syndrome has been isolated by two cycles of plaque purification. This virus is indistinguishable from SFV using cross neutralization and electron microscopy. Analyses of restriction enzyme digests of MV and plaque-purified SFV show them to be quite dissimilar and indicate that MV is recombinant between SFV and myxoma virus. This recombinational event resulted in approximately 5.5 kb of myxoma virus DNA within each of the inverted terminal repeats being replaced by a similar amount of DNA derived from the corresponding region of the SFV genome. Thus, MV contains approximately 149 kb of myxoma sequences and 11 kb of SFV sequences. Immunofluorescent studies of spleen and lymph nodes from MV-infected rabbits demonstrate that viral antigens are present predominantly in the sinusoidal lining cells in lymph nodes and in phagocytes in the splenic cords. This contrasts with the distribution of antigen observed in myxoma virus-infected rabbits where myxoma-specific antigens are present in large amounts in hyperplastic epithelium overlying tumors, particularly in the nasal mucosa and in spleen and lymph node cells. MV-infected rabbits essentially lose their lymphocyte proliferative response to T and B cell mitogens and are unable to initiate an antibody response to SRBC, as determined by a modified Jerne plaque assay. In vitro MV severely depresses the mitogen responses of normal B and T lymphocytes after two days of culture. Lymphoid cells and lysates of lymphoid cells from MV-infected rabbits will suppress mitogen- and antigen induced responses in vitro. MV can grow in lymphocytes, but replication of MV is less efficient in lymphocytes than in RK-13 cells. Thus, MV produces a disseminated viral infection, systemic myxofibromas, and a severe combined immune deficiency in rabbits. The molecular and immunologic basis for these effects is now under study. PMID- 3018885 TI - [HTLV-III infection serologic diagnosis]. PMID- 3018886 TI - [Papilloma virus infection and the clinical significance]. PMID- 3018887 TI - [Oral papillomavirus infections]. PMID- 3018889 TI - Hepatitis in day care centers: epidemiology and prevention. AB - Hepatitis A is a significant health problem in day care centers, causing outbreaks that average 12 cases in size and three months in duration. These outbreaks have three characteristic features: children have mild or asymptomatic infections; adults (primarily parents) are the major group with clinical hepatitis; and persons having contact with one- or two-year-old children run the highest risk of infection. Outbreaks are commonest in centers that are large, have long operating hours, and enroll children younger than the age of two years (i.e., those in diapers). The presence of such children is necessary for the rapid spread of the disease. Nationally, outbreaks occur primarily in areas with many infant/toddler centers, which often form the focus for epidemics. Prevention relies on hygiene, especially hand washing. Disease control depends on early detection of outbreaks and aggressive use of immunoglobulin. The spread of hepatitis B has not been documented in day care centers; however, when a child carrying hepatitis B virus enrolls in a center, a low risk of transmission may exist and precautions are recommended, with a focus on personal hygiene. PMID- 3018888 TI - Current concepts of vascular endothelial and smooth muscle cell communication. PMID- 3018890 TI - Varicella in day care centers. AB - Spread of varicella in day care is controlled by excluding children at the first signs of illness. Exclusion is generally ineffective. Minimally ill children might be permitted to attend day care. This approach may lead to exposure of those in high-risk groups, i.e., adults or immunocompromised children. Morbidity is greater in adults, but susceptibility among day care workers is probably low. Immunocompromised children can be vaccinated or given varicella-zoster immune globulin (VZIG) after exposure. Questions about the risks of varicella-zoster vaccination (V-Z) concern the production of latent infection and subsequent zoster and the effect on the epidemiology of infection, i.e., its possible delay until adulthood. Zoster has been found not to be more frequent in immunized than in nonimmunized leukemic children, and normal vaccinees retain good antibody titers five years after vaccination and have titers similar to individuals who have had varicella 10 years after vaccination. Vaccine efficacy is excellent, but its desirability will be determined after resolution of questions concerning the long-term impact of its use. PMID- 3018891 TI - Development of vaccines against hepatitis A and hepatitis B. AB - Hepatitis A and hepatitis B are viral infections of the liver. Hepatitis A is spread by the fecal-oral route--i.e., by ingestion of virus shed in the stool of acutely infected individuals. The virus is transmitted from person to person or (in outbreaks) via contaminated food or water. Population groups at increased risk of acquiring hepatitis A include children and staff in day care centers. Hepatitis B is spread by blood and other body fluids from acutely infected individuals or chronically infected carriers. Infection occurs when virus contained in these fluids enters the body through mucosal surfaces or breaks in the skin. A vaccine against hepatitis B has been developed. It consists of noninfectious hepatitis B surface antigen purified from the plasma of chronic carriers. The three sequential inactivation treatments used in manufacture of the vaccine kill hepatitis B virus and other infectious agents that may be present in human plasma, including the human T cell-lymphotropic virus that causes the acquired immunodeficiency syndrome. The vaccine is well tolerated, highly immunogenic, and highly effective in preventing hepatitis B. Both live attenuated and killed vaccines against hepatitis A are also being investigated. A live attenuated vaccine is preferred and seems feasible on the basis of initial studies in animals and volunteers. PMID- 3018893 TI - Infection with human T-lymphotropic virus type III/lymphadenopathy-associated virus: considerations for transmission in the child day care setting. AB - Many public health and social issues relate to infection with the human T lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV)-the virus that causes the acquired immunodeficiency syndrome. One such issue is the care and education of infected children, both in schools and in day care facilities. To date, transmission of HTLV-III/LAV in either the school or the day care setting has not been documented. However, because the virus has been isolated from a variety of body fluids, contact with such fluids from an infected child poses at least a theoretical risk of transmission. Past experience with transmission of hepatitis B virus provides a possible model to aid in understanding the epidemiology of HTLV-III/LAV infection. The risk of transmission of HTLV-III/LAV in day care settings is not known, and infection with this virus carries serious public health and clinical implications. PMID- 3018894 TI - Assessment of the presence of cytomegalovirus-neutralizing antibody by a plaque reduction assay. AB - Evaluation of the in vitro neutralizing activity of 30 immunoglobulin G (IgG) preparations and seven batches of pooled plasma against human cytomegalovirus (CMV) was performed by plaque-reduction assays. There were definite differences detected by these methods that could permit the selection of preparations with highest titers for clinical trials. The addition of guinea pig complement to the plaque-reduction assay enhanced inhibition of CMV; thus, guinea pig complement should be used in assays of IgG preparations. There was no relationship between the ELISA-derived optical density values of the IgG samples and the titers determined by plaque neutralization. These data suggest that the results of plaque-reduction neutralization assays provide important information about preparations of antibody that may be selected for patient administration. The preparations selected for their high titers by neutralization assays differ significantly from those identified by another method. PMID- 3018892 TI - Group day care and cytomegaloviral infections of mothers and children. AB - Cytomegalovirus (CMV), the leading cause of congenital viral infection, occurs commonly among children in group day care. Urinary or salivary excretion of CMV was found frequently among children in three centers serving mostly middle-income white families. Although there was center-to-center variation, CMV excretion was uncommon among infants under one year of age; peak rates of viral shedding, ranging from 44% to 100%, were noted for two-year-olds. A longitudinal study at a single center indicated that children usually acquired CMV during their second year of life and usually shed virus for two years or longer. The high prevalence of silent CMV infection among the children in day care argues against the exclusion of any child known to have CMV infection; such children have sometimes been excluded because of the potential risk of CMV transmission to pregnant workers. PMID- 3018895 TI - Passive immunotherapy for encephalitis caused by herpes simplex virus. AB - Passive administration of antiviral antibody has been assessed in animal models as a potential form of treatment for infections due to herpes simplex virus (HSV). Previous investigative work has demonstrated the beneficial effect of immune serum administered either before or after HSV infection of mice. In some studies the protective effect of antibody was abrogated in the absence of T lymphocytes, an observation suggesting that cellular effector mechanisms may be necessary for antibody efficacy. In the present study of a mouse model of encephalitis caused by HSV type 2, a 0.5-ml dose of human immunoglobulin (1,250 mg of immunoglobulin G/kg of body weight) prepared at pH 4.25 for intravenous administration reduced mortality and prolonged survival when administered by intraperitoneal injection 24 hr before or up to 8 hr after intranasal viral challenge. The serum titer of HSV-neutralizing antibody and the response to therapy were dose dependent. Clinical trials of passive immunotherapy for severe HSV infections may be warranted. PMID- 3018897 TI - [Development and recovery of neuronal networks in the child's brain studied by nodal analysis]. PMID- 3018896 TI - In vitro and in vivo antibody-dependent cellular cytotoxicity of intravenous immunoglobulin G preparations against herpes simplex virus. PMID- 3018898 TI - [Diagnostic possibilities and therapeutic results in non-deficiency rickets]. PMID- 3018899 TI - [Staghorn calculi with hyperexcretion of uric acid: clinical treatment]. PMID- 3018900 TI - [New evaluation of 3 immunoenzyme assay kits for the detection of anti-LAV antibodies]. PMID- 3018901 TI - [Bacillus thuringiensis--an agent for the biological control of Culicidae]. PMID- 3018903 TI - [Infectious pneumonia in immunocompromised patients]. PMID- 3018902 TI - [The treatment of bronchopulmonary cancer]. PMID- 3018904 TI - [Adult respiratory distress syndrome (certainties and hypotheses)]. PMID- 3018906 TI - [Round table on "Diagnosis of diffuse fibrotic interstitial pneumopathies"]. PMID- 3018905 TI - [Effect of selenium on the fibrogenic and immune mechanisms in experimental silicosis]. PMID- 3018907 TI - [Present status of the detection and diagnosis of bronchopulmonary cancer]. PMID- 3018908 TI - Superoxide generation by synovial fluid neutrophils enhanced by immune complexes and suppressed by rheumatoid factor in synovial fluid. AB - Synovial fluid (SF) from patients with rheumatoid arthritis (RA) enhanced superoxide generation by neutrophils isolated from RA SF, in contrast to SF from patients with osteoarthritis. These superoxide generation-enhancing substances may be intermediate-sized immune complexes and a complement C5-derived fragment. Rheumatoid factor (RF) isolated from RA SF suppressed superoxide generation enhancing activity of aggregated IgG. Therefore, biologically active RF may block the interaction of the immune complexes with neutrophils accumulating in RA SF, and protect the joint tissue from the effects of oxygen radicals or proteases. PMID- 3018910 TI - Delay in the absolute latency of auditory brainstem response (ABR) component P1 in acute inflammatory demyelinating disease. AB - A patient with an acute inflammatory demyelinating disease and bilateral involvement of the peripheral (i.e. extracranial) portion of the VIII n. is described. The diagnosis of VIII n. involvement was made on the basis of grossly prolonged Auditory Brainstem Response (ABR) component P1 latencies which resolved to normal latencies in one year. It is suggested that involvement of the peripheral myelin is not uncommon in central demyelinating disease even in the absence of auditory system complaints. PMID- 3018909 TI - The set of growth factors stimulatory for a transformed rat cell of line NRK-49F depends on the identity of the transforming virus. AB - Re-cloned rat fibroblast line NRK-49F was transformed by human adenovirus type 5 which had been grown in human cells of the KB carcinoma line. Cell subclones were isolated from seven independent transformation events, and seven untransformed cell subclones were isolated in parallel from control cultures which had been inoculated with lysate of uninfected KB cells. The adenovirus-transformed cells differed from the control untransformed fibroblasts by being typically small and cuboidal, showing multilayered growth, producing colonies in soft-agar medium, and growing to higher saturation density. Adenovirus-transformed subclones showed no infective virus and no consistent difference from control subclones in karyotype (mode = 41). Like the original re-cloned NRK-49F cells, all fourteen subclones required epidermal growth factor, fibronectin, insulin, and retinoic acid for optimal growth in serum-free culture. Thus, transformation by HA5 did not alter the required set, in contrast to the earlier finding that transformation of this same re-cloned line by polyoma virus eliminated the requirements for insulin and retinoic acid. PMID- 3018911 TI - The central auditory conduction at term date and three months after birth. III. Middle latency responses (MLRs). AB - Middle latency responses (MLRs) were obtained in 25 healthy newborns with a follow-up recording at 3 months in all but three. A four-channel recording provided topographic information. Linkage of the ear references reduced the myogenic contamination of the recordings. P0 and Na proved to be the most consistent components in the sinusoidal MLR wave sequence. Topographic differences suggest a generation of P0 and Na contralateral to stimulation. A significant latency decrease was found for P0 and na between term and 3 months. The most important latency and amplitude changes may occur before term date and immediately thereafter. The MLRs are the link between the auditory brainstem responses (ABRs) and the auditory cortical responses (ACRs), and it may be possible to use them for recordings in newborn infants. They provide information about the generation of specific components as well as regarding the auditory afference in the stimulus propagation between the brainstem and the cortical auditory areas. PMID- 3018912 TI - The central auditory conduction at term date and three months after birth. IV. Auditory cortical responses. AB - Auditory cortical responses (ACRs) were recorded in 25 healthy mature newborns with a follow-up at 3 months. The waveform, latencies and amplitudes for the ACRs for this time period are studied. Topographic differences between the central and central-temporal derivations and changes between term and 3 months are found with respect to latencies and amplitudes for different peaks and troughs. The ACRs, obtained as a part of a protocol covering the entire auditory afference, offer consistent parameters, which can be used to study developmental neurophysiological properties of audition and which are potential diagnostic tools for the detection of deviant sensory or mental development. PMID- 3018913 TI - Nuclear thyroxine binding in human mononuclear blood cells from hyperthyroid and hypothyroid patients before and after treatment. AB - Nuclear binding of [125I]T4 in human mononuclear blood cells was examined in six hyperthyroid and six hypothyroid patients before and after treatment. In hypothyroid patients the nuclear T4 binding was initially increased and subsequently normalized as the patients became euthyroid suggesting a homeostatic counter-regulation. The thyroid state did not affect the nuclear T4 binding in hyperthyroid patients. Treatment with methimazol however increased the nuclear T4 binding, suggesting either a direct effect of methimazol on the hormone-receptor interaction or that the patients had become slightly hypothyroid during the treatment. The TSH was shown not to affect nuclear T4 binding. The thyroid state of the patients did not significantly affect the nuclear accumulation of the T3 produced intracellularly by deiodination of T4. PMID- 3018915 TI - Nuclear uptake of 1,25-dihydroxy[3H]-cholecalciferol in peripheral blood monocytes. AB - The active vitamin D metabolite, 1,25-dihydroxycholecalciferol induces differentiation of monocytes into macrophages. The pharmacological induction of differentiation of primitive, rapidly proliferating cell lines into more mature cells with lower proliferative potential is a new dimension in the treatment of myeloproliferative disorders, which may prove to be an important alternative to more traditional regimens. Furthermore, the cell primarily engaged in bone resorption--the osteoclast--represents another differentiated form of mononuclear phagocytes, and 1,25-dihydroxycholecalciferol increases the number of osteoclasts. Since the cellular action of 1,25-dihydroxycholecalciferol is exerted mainly through its binding to nuclear receptors, a detailed knowledge of ligand-receptor interactions is mandatory for future work in this area. In order to investigate the interaction between 1,25-dihydroxycholecalciferol and its receptor in mononuclear cells, the nuclear uptake of the hormone was studied using a whole cell assay. The nuclear uptake of 1,25-dihydroxy[3H]cholecalciferol in human monocytes at physiological temperature and pH was saturable, specific, and fully reversible. When eight normal individuals were investigated, the maximal binding capacity (Bmax) was 0.4-8.4 fmol/10(6) cells and the dissociation constants (Kd) were 0.12-0.45 nmol/l. The characterization of the nuclear uptake of 1,25-dihydroxy[3H]cholecalciferol in intact human monocytes shows that it is mediated by binding of the ligand to a specific nuclear receptor. The binding to the nuclear receptor is the result of the passage of ligand across the cytoplasmic membrane and of the cytoplasmic transport of ligand. In contrast to conventional receptor assays in hypertonic cellular extracts, this system provides information on the role of the cytoplasmic membrane in relation to the nuclear uptake of 1,25-dihydroxycholecalciferol, which may be closer to in vivo cellular conditions. PMID- 3018914 TI - Binding and degradation of 125I-gastrin by plasma membranes from homogenized rat gastric mucosa. AB - Binding of 125I-gastrin to the 270-30,000 g fraction from homogenized rat oxyntic mucosa was studied. 'Specific' binding was calculated by subtracting the binding at excess cold gastrin from the binding with labelled gastrin (250 pM) only. At 30 degrees C specific binding rose rapidly to a short-lived maximum before falling gradually, whereas at 15 degrees C and 0 degree C specific binding rose gradually to a higher plateau level. The reduced binding at 30 degrees C could be caused by degradation of either the tracer or the binding site or by a combination of these two events. Degradation of 125I-gastrin was evaluated by trichloroacetic acid (TCA) precipitation, fast protein liquid chromatography, and binding to a gastrin antibody (immunoreactivity). The effect of incubation on the binding site was evaluated by preincubation of the homogenate fraction before adding gastrin. In separate studies, the proteolytic activity of the homogenate fraction was studied by TCA precipitation of radioactive casein. Different enzyme inhibitors tested were virtually ineffective in preventing gastrin and casein degradation. Only lowering the incubation temperature to 15 degrees C or lower could prevent this degradation. The reduced and transient binding of 125I-gastrin at 30 degrees C most probably reflects tracer degradation. Accordingly, the gastrin binding experiments were performed at 15 degrees C. Only homogenates from the oxyntic area of the stomach bound 125I-gastrin specifically and with a Kd of 0.8 nM (Scatchard analysis). However, micromolar concentrations of unlabelled gastrin were required to inhibit half maximal binding of the tracer. The tracer binding was unaffected by secretin, slightly reduced by a CCK-9 analogue, and more markedly reduced by pentagastrin. PMID- 3018916 TI - Early defect of phagocytic cell function in subjects at risk for acquired immunodeficiency syndrome. AB - We studied the functions of peripheral blood monocytes and polymorphonuclear cells in 15 apparently healthy homosexual men, eight homosexual or bisexual subjects with unexplained generalized lymphadenopathies (pre-AIDS), four homosexual men with acquired immunodeficiency syndrome (AIDS), and 15 heterosexual men. In comparison with normal controls, the homosexual groups studied presented a decreased monocyte candidacidal activity for Candida pseudotropicalis that gradually deteriorates as the clinical symptoms progress towards AIDS. The monocyte phagocytic function was retained. Although the phagocytic and candidacidal activities of the polymorphonuclear cells did not differ from those of the normal controls, the candidacidal activity in some of the cases studied was unusually enhanced, indicating that the cells were in an activated state. In addition, only two of nine sera tested from asymptomatic homosexual males were positive for antibodies to HTLV-III/LAV, while six out of eight pre-AIDS and both of the two AIDS patients tested had antibodies to AIDS associated retrovirus. We suggest that in AIDS the phagocytic system is already involved, together with B and T lymphocyte abnormalities, during the early events of the syndrome, even without the detection of AIDS-associated retrovirus antibodies. PMID- 3018917 TI - Production of specific antibodies by cerebrospinal fluid lymphocytes in patients with herpes zoster, mumps meningitis and herpes simplex virus encephalitis. AB - We applied a new method consisting of short-term culture (18 h) of lymphocytes from cerebrospinal fluid (CSF-L) and peripheral blood (PBL) in viral antigen coated ELISA plates and subsequent measurement of IgG and IgM antibodies bound to antigen. Utilizing mumps virus, herpes simplex virus (HSV), varicella zoster virus (VZV), and measles virus as antigens, we demonstrated production by CSF-L of antibodies against the aetiological agent only in all patients with mumps meningitis and HSV encephalitis and also in all patients with herpes zoster without central nervous system (CNS) symptoms. This might be considered as direct evidence that specific antibodies are produced within the CNS in inflammatory nervous system diseases. CSF-L usually produced higher amounts of antibodies than the corresponding number of PBL. In comparison with concentrations of free antibodies determined in parallel, our method had higher specificity and sensitivity and gave more precise information about the antibody response in infections of the nervous system. PMID- 3018918 TI - Induction of immunoglobulin secretion and DNA synthesis in human lymphocytes in vitro by cytomegalovirus preparations. AB - Immunoglobulin secretion as evidenced by plaque-forming cells (PFC) in an indirect haemolysis-in-gel assay, and DNA synthesis were induced in human blood lymphocytes by the following preparations of cytomegalovirus (CMV): Nucleocapside Nc-CMV antigen, membrane M-CMV antigen, crude C-CMV preparations and CMV incubated adherent cells. Peak stimulations occurred around day 6 in culture. Nc CMV and M-CMV only stimulated PFC and DNA synthesis in lymphocytes from CMV seropositive individuals. C-CMV also stimulated lymphocytes from CMV seronegative individuals but gave better responses in lymphocytes from CMV seropositive individuals. CMV-incubated adherent cells occasionally stimulated lymphocytes from CMV seronegative individuals but always in CMV seropositive blood donors. Nc , M-, and C-CMV gave high numbers of PFC in B cells enriched by sheep erythrocyte sedimentation and in co-cultures of enriched T and B cells. Almost no PFC were induced in enriched T cells. DNA synthesis induced by all the three antigenic CMV preparations increased after removal of adherent cells. Strong DNA synthesis was induced in enriched T cells compared to almost none in enriched B cells. It is concluded that some pure CMV preparations act as antigens and more crude preparations induce polyclonal activation. Different CMV preparations may be used to diagnose CMV, to study immune reactivity against CMV and as a model for CMV infections in vitro. PMID- 3018919 TI - Blood monocyte and neutrophil functions in the acquired immune deficiency syndrome. AB - The acquired immune deficiency syndrome (AIDS) is characterized by infections with microorganisms against which phagocytes (especially monocytes/macrophages) play an important role. Therefore, various functions of blood monocytes and neutrophils were tested in 16 patients with AIDS. Neutrophil chemotactic responses towards casein and N-formyl-methionyl-L-leucyl-L-phenylalanine were depressed in patients with a short duration of disease (n = 9), whereas they were normal in those with a longer duration (n = 5), P less than 0.05. Neutrophil superoxide anion release was normal. In contrast, we found no evidence of an altered monocyte activity in the patients, since chemotactic responsiveness, phagocytosis of opsonized Candida albicans, and superoxide anion release were all normal. These findings suggest that the depressed neutrophil chemotaxis may play an important role in the high incidence of opportunistic infections in AIDS. Furthermore, it appears that in AIDS the immune deficiency does not extend to peripheral blood monocytes, but it does not exclude the possibility that the function of tissue macrophages is abnormal. PMID- 3018920 TI - Monoclonal antibodies specific for Sendai virus. II. Production of monoclonal anti-idiotypic antibodies. AB - The monoclonal antibodies A and B specific for the HN molecule of Sendai virus were used to induce polyclonal and monoclonal anti-idiotypic antibodies. No response was observed in allogeneic Lewis rats, low responses in syngeneic LOU rats, and high responses in allogeneic BN rats and xenogeneic Balb/c mice. The monoclonals A and B share a similar or identical idiotype, since polyclonal anti idiotypic antisera to antibody A cross-reacted completely with antibody B and vice versa. The same was found with the three monoclonal anti-idiotypes 1, 2, and 3 elicited in a BN rat or in Balb/c mice. None of the polyclonal or monoclonal anti-idiotypes reacted with other monoclonal antibodies specific for Sendai virus, even when these recognized the same epitope as antibodies A and B. The monoclonal anti-idiotypic antibodies could be used to induce anti-Sendai antibodies in BN rats. PMID- 3018921 TI - [Reactivation of a latent bovine herpes mamillitis virus (BHV-2) infection in an animal with questionable IBR serology]. PMID- 3018922 TI - Identification of human N-ras as the common oncogene in NIH 3T3 cells transformed with DNAs from human primary hepatic cancer and hepatoma 7402 line. AB - DNA was extracted from NIH 3T3 cells transformed with DNAs from human primary hepatic cancer (PHC) and Hepatoma 7402 cell line. The transformant DNA was analyzed by Southern transfer and hybridization with 32P-labeled probes of various oncogenes. The EcoRI 7.2 and 9.0 kb bands characteristic of human N-ras gene were identified in transformed NIH 3T3 cells derived both from PHC and 7402 DNA. The BamHI 6.6 kb band characteristic of human c-Ha-ras I was present only in 7402 transformants, but not in PHC transformants. Using 35S-methionine incorporation, immunoprecipitation with anti-p21 monoclonal antibodies, SDS-PAGE and autoradiography, it was demonstrated that p21 synthesis was remarkably enhanced in 7402 cells as well as in transformed cells derived from both 7402 and PHC DNA. Taking the data together, it strongly implies that N-ras is one of the transforming genes for human liver cancer. PMID- 3018923 TI - Expression of N-ras gene in human primary hepatocarcinoma. AB - Poly(A)+ RNA was isolated from 9 specimens of human primary hepatic carcinoma, 1 non-tumorous liver tissue adjacent to cancer and 1 normal liver tissue samples. The Oligo-dT cellulose-purified poly(A)+ RNAs were subjected to formaldehyde agarose gel electrophoresis, Northern transfer and hybridization with various oncogene probes. Two RNA species, 5.6 kb and 2.2 kb were identified by N-ras gene hybridization in 6 out of 9 mRNA samples from primary hepatic carcinoma specimen. N-ras specific mRNA was not detectable in mRNA samples from normal human liver and tumor surrounding cirrhotic tissue. No detectable hybridization of mRNA from hepatoma and normal liver with Ki-ras or Ha-ras was observed. As human N-ras gene has been identified in DNA of mouse transfectants transformed with PHC DNA, it strongly suggests that N-ras gene might be responsible for the transforming activity of part of cases of human liver cancer. PMID- 3018924 TI - The site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating. AB - WIN 51711 and WIN 52084 are structurally related, antiviral compounds that inhibit the replication of rhino (common cold) viruses and related picornaviruses. They prevent the pH-mediated uncoating of the viral RNA. The compounds consist of a 3-methylisoxazole group that inserts itself into the hydrophobic interior of the VP1 beta-barrel, a connecting seven-membered aliphatic chain, and a 4-oxazolinylphenoxy group (OP) that covers the entrance to an ion channel in the floor of the "canyon." Viral disassembly may be inhibited by preventing the collapse of the VP1 hydrophobic pocket or by blocking the flow of ions into the virus interior. PMID- 3018925 TI - Tandem duplication of D-loop and ribosomal RNA sequences in lizard mitochondrial DNA. AB - Some Cnemidophorus exsanguis have mitochondrial DNA's (mtDNA's) that are 22.2 kilobases (kb) in size, whereas most have mtDNA's of 17.4 kb. Restriction site mapping, DNA transfer hybridization experiments, and electron microscopy show that the size increment stems from the tandem duplication of a 4.8-kb region that includes regulatory sequences and transfer and ribosomal RNA genes. This observation is notable in that sequences outside of the control region are involved in major length variation. Besides revealing a novel form of mtDNA evolution in animals, these duplications provide a useful system for investigating the molecular and evolutionary biology of animal mtDNA. PMID- 3018926 TI - A genetic approach to promoter recognition during trans induction of viral gene expression. AB - Viral infection of mammalian cells entails the regulated induction of viral gene expression. The induction of many viral genes, including the herpes simplex virus gene encoding thymidine kinase (tk), depends on viral regulatory proteins that act in trans. Because recognition of the tk promoter by cellular transcription factors is well understood, its trans induction by viral regulatory proteins may serve as a useful model for the regulation of eukaryotic gene expression. A comprehensive set of mutations was therefore introduced into the chromosome of herpes simplex virus at the tk promoter to directly analyze the effects of promoter mutations on tk transcription. The promoter domains required for efficient tk expression under conditions of trans induction corresponded to those important for recognition by cellular transcription factors. Thus, trans induction of tk expression may be catalyzed initially by the interaction of viral regulatory proteins with cellular transcription factors. PMID- 3018927 TI - New rule proposed for protein degradation. PMID- 3018928 TI - Early signals in the mitogenic response. AB - Polypeptide growth factors, regulatory peptides, and a variety of pharmacological agents acting alone or synergistically induce mitogenesis in cultured fibroblasts. The early signals in the membrane, cytosol, and nucleus promoted by these extracellular factors, together with their mitogenic effectiveness, are integrated in a unified hypothesis for the regulation of fibroblast growth. PMID- 3018929 TI - Molecular analysis of the hotspot of recombination in the murine major histocompatibility complex. AB - Biological and serological assays have been used to define four subregions for the I region of the major histocompatibility complex (MHC) in the order I-A, I-B, I-J, and I-E. The I-J subregion presumably encodes the I-J polypeptide of the elusive T-cell suppressor factors. Restriction enzyme site polymorphisms and DNA sequence analyses of the I region from four recombinant mouse strains were used to localize the putative I-B and I-J subregions to a 1.0-kilobase (kb) region within the E beta gene. Sequencing this region from E beta clones derived from the two mouse strains: B10.A(3R), I-Jb and B10.A(5R), I-Jk initially used to define the I-J subregion revealed that these regions are identical, hence the distinct I-Jb and I-Jk molecules cannot be encoded by this DNA. In addition, the DNA sequence data also refute the earlier mapping of the I-B subregion. Analysis of the DNA sequences of three parental and four I region recombinants reveals that the recombinant events in three of the recombinant strains occurred within a 1-kb region of DNA, supporting the proposition that a hotspot for recombination exists in the I region. The only striking feature of this hotspot is a tetramer repeat (AGGC)n that shows 80 percent homology to the minisatellite sequence which may facilitate recombination in human chromosomes. PMID- 3018930 TI - In vivo half-life of a protein is a function of its amino-terminal residue. AB - When a chimeric gene encoding a ubiquitin-beta-galactosidase fusion protein is expressed in the yeast Saccharomyces cerevisiae, ubiquitin is cleaved off the nascent fusion protein, yielding a deubiquitinated beta-galactosidase (beta gal). With one exception, this cleavage takes place regardless of the nature of the amino acid residue of beta gal at the ubiquitin-beta gal junction, thereby making it possible to expose different residues at the amino-termini of the otherwise identical beta gal proteins. The beta gal proteins thus designed have strikingly different half-lives in vivo, from more than 20 hours to less than 3 minutes, depending on the nature of the amino acid at the amino-terminus of beta gal. The set of individual amino acids can thus be ordered with respect to the half-lives that they confer on beta gal when present at its amino-terminus (the "N-end rule"). The currently known amino-terminal residues in long-lived, noncompartmentalized intracellular proteins from both prokaryotes and eukaryotes belong exclusively to the stabilizing class as predicted by the N-end rule. The function of the previously described posttranslational addition of single amino acids to protein amino-termini may also be accounted for by the N-end rule. Thus the recognition of an amino-terminal residue in a protein may mediate both the metabolic stability of the protein and the potential for regulation of its stability. PMID- 3018931 TI - Nuclear and mitochondrial DNA comparisons reveal extreme rate variation in the molecular clock. AB - The discovery that the rate of evolution of vertebrate mitochondrial DNA is rapid, compared to the rate for vertebrate nuclear DNA, has resulted in its widespread use in evolutionary studies. Comparison of mitochondrial and nuclear DNA divergences among echinoid and vertebrate taxa of similar ages indicates that the rapid rate of vertebrate mitochondrial DNA evolution is, in part, an artifact of a widely divergent rate of nuclear DNA evolution. This disparity in relative rates of mitochondrial and nuclear DNA divergence suggests that the controls and constraints under which the mitochondrial and nuclear genomes operate are evolving independently, and provides evidence that is independent of fossil dating for a robust rejection of a generalized molecular clock hypothesis of DNA evolution. PMID- 3018932 TI - Physiological variation in alpha-adrenoceptor-mediated arterial sensitivity: relation to agonist affinity. AB - Vascular smooth muscle from different arteries of the rabbit varies in sensitivity to norepinephrine, even when factors known to contribute to this variation are excluded. Sensitivity to norepinephrine mediated through the alpha adrenoceptor is linearly related to the agonist dissociation constant, but is not significantly related to receptor reserve. These results suggest that agonist affinity is the primary determinant of sensitivity to norepinephrine, at least in these arteries, and that this is a locally regulated characteristic which may account for regional sensitivity changes. PMID- 3018933 TI - Brominating oxidants generated by human eosinophils. AB - Eosinophils are white blood cells that in humans are found in association with helminthic infections and various inflammatory disease processes. These cells contain a unique lysosomal peroxidase that oxidizes halides to generate highly reactive and toxic hypohalous acids. Although chloride is found in vivo at concentrations at least 1000-fold greater than those of other halides, human eosinophils did not preferentially oxidize chloride under physiologic conditions. Instead, eosinophils used bromide, a halide with a hitherto unknown function in humans, to generate a halogenating oxidant with characteristics similar, if not identical, to those of hypobromous acid. These results indicate that physiological concentrations of bromide arm human eosinophils with the ability to generate and release an unusual oxidant capable of destroying a wide range of prokaryotic and eukaryotic targets. PMID- 3018934 TI - Infectious agents in the pathogenesis of rheumatoid arthritis. PMID- 3018935 TI - Hepatic encephalopathy and cerebral edema. PMID- 3018936 TI - Clinical experience with charcoal and resin hemoperfusion. PMID- 3018937 TI - [Histopathological studies in the evaluation of hydroxyapatite root canal cements]. PMID- 3018938 TI - [Pulp response to a spherical sub-microfilled resin and its effectiveness in pulp protection with a dentin-adhesive lining material]. PMID- 3018939 TI - [A case of recurrent pleomorphic adenoma with a rare histological pattern. Immunohistochemical identification of tumor cells using the PAP method]. PMID- 3018940 TI - [A histological study of hydroxyapatite implantation in experimental bone defects in rats]. PMID- 3018941 TI - Use of monoclonal antibodies in trophoblastic neoplasia. PMID- 3018942 TI - AIDS--the facts. PMID- 3018943 TI - Large brain metastasis not shown by computerized tomography. AB - We have described a patient with metastatic small cell carcinoma of the lung in whom cranial CT and contrast-enhanced CT failed to show metastases. When the patient died four days later, two sizable intracerebral metastatic foci were found at autopsy. Reviewing previously reported causes of nonvisualization of lesions, we have speculated on reasons for the lack of CT findings in this case. PMID- 3018944 TI - Occult gastric cancer manifested by progressive shortness of breath in a young adult. AB - We have reported a case of occult, diffuse gastric cancer in a young adult with progressive shortness of breath and bilateral pulmonary interstitial infiltrates. Progressive shortness of breath may be the first or only manifestation of occult gastric cancer caused by either lymphangitic carcinomatosis or microscopic tumor emboli to the lungs. Widespread recognition of this syndrome, a high index of suspicion, and prompt lung biopsy are necessary to make the correct diagnosis. With progress in chemotherapy for malignant diseases, early diagnosis and specific treatment may improve the prognosis of this condition. PMID- 3018945 TI - Anesthesia-induced pulmonary macroatelectasis: a cause of false-positive computerized tomography. AB - In three pediatric patients having CT of the chest done under general anesthesia, we encountered multiple areas of macroatelectasis in the lung. All patients had malignant disease, and the chest findings could have been interpreted as metastatic spread to the lung. To prevent this critical false-positive diagnosis, we believe general anesthesia for CT of the chest should be given with controlled rather than spontaneous ventilation. PMID- 3018946 TI - Evaluation of hydroxylapatite graft materials in canine cervical spine fusions. AB - The efficacy of ceramic hydroxylapatite implant materials as graft materials for cervical spine fusion was evaluated in canines. Bioresorbable and non bioresorbable systems were evaluated at time periods ranging from 1 to 24 weeks. Implant interbody position and progression of fusion were evaluated radiographically and histologically. Implant fracture and extrusion into adjacent soft tissues occurred in nine of 23 cases. Implant fracture occurred in many of the remaining 14 cases, however, the implant materials remained within the interspace. Implant fracture occurred with both implant systems. Radiographically little evidence of fusion was observed at less than 6 weeks, however by 12 weeks evidence of fusion was noted and was confirmed histologically. No difference in fusion rate or degree of fusion was observed between the two implant systems. PMID- 3018947 TI - [Lupus mice and arteritis]. PMID- 3018948 TI - Tetanus antibodies as a marker of potential efficacy of killed polio immunization. AB - The use of antibodies to tetanus toxin as a marker of efficacy of parenteral immunization was examined in a randomized sample of 1212 sera representative of the total black infant population of the RSA between 24 and 35 months. All but one of these sera had protective levels of antibodies measured by enzyme-linked immunosorbent assay. In contrast, in a previous study only 59-80% had tritypic immunity to polio and 70-84% mono- or bitypic immunity. Thus, this serological marker was found to be unreliable and some possibilities for this remarkably high level of antitetanus antibodies are considered. PMID- 3018949 TI - The simplified endoscopic Congo Red test for completeness of vagotomy. PMID- 3018951 TI - Human papillomavirus and cervical neoplasia. PMID- 3018950 TI - Human papillomavirus and cervical disease in a Mexican-American community. PMID- 3018952 TI - CDC recommendations for preventing transmission of AIDS during invasive procedures. PMID- 3018953 TI - Distribution of transposable elements in prokaryotes. AB - We consider models for the distribution of the number of elements per host genome for families of transposable elements (TEs). The hosts are assumed to be prokaryotes. These models assume a constant rate of infection of uninfected hosts by TEs, replicative transposition within each host, and a reduction of the fitness of a host dependent on the number of TEs it contains. No provision was made for the deletion of individual TEs within a host or for recombination, since both are relatively rare events in prokaryotes. These models mostly assume that the TE performs no function for the host, and that the reduction in fitness with increased copy number is due to effects such as the impairment of beneficial genes by transposition or homologous recombination. We also consider a model in which the TEs can convey a selective advantage to the host. The equilibrium distributions of copy number are determined for these models, and are of a variety of classical types. Relevant parameters of the models are estimated using data on the distribution of insertion sequences in natural isolates of Escherichia coli. PMID- 3018954 TI - Effects of a thromboxane synthetase inhibitor and a cAMP phosphodiesterase inhibitor, singly and in combination, on platelet behaviour. AB - The effects of dazoxiben, a thromboxane synthetase inhibitor, and AH-P 719, a cAMP phosphodiesterase inhibitor, on arachidonic acid (AA)-induced platelet behaviour were determined. The levels of cAMP present in platelet-rich plasma (PRP) after stimulating the platelets with AA in the absence and presence of the agents were also measured. AH-P 719, as well as dazoxiben, was more effective as an inhibitor of AA-induced platelet behaviour in PRP from some individuals than in PRP from others, and the effectiveness with which it inhibited platelet behaviour paralleled that of dazoxiben. A combination of both agents was more effective than either agent alone. Both AH-P 719 and dazoxiben increased the level of cAMP in AA-stimulated platelets but again they were more effective in PRP from some individuals than others. A combination of AH-P 719 and dazoxiben always resulted in higher levels of cAMP than either agent alone. These results imply that cAMP is involved in determining the effects of thromboxane synthetase inhibitors on platelet behaviour, and indicate that the anti-thrombotic potential of a combination of a thromboxane synthetase inhibitor and a cAMP phosphodiesterase inhibitor may be greater than that of the individual agents. PMID- 3018957 TI - Thrombomodulin inhibits the activation of factor XIII by thrombin. PMID- 3018955 TI - An IgM lupus anticoagulant that neutralizes the enhancing effect of phospholipid on purified endothelial thrombomodulin activity--a mechanism for thrombosis. AB - An anticoagulant activity was isolated from the plasma of a patient with a strong lupus-like anticoagulant using gel filtration by high performance liquid chromatography. IgM were detected in this anticoagulant fraction which exhibited specificity towards 50% phosphatidylcholine - 50% phosphatidylserine vesicles and cardiolipin. These phospholipids were able to produce an apparent 3-fold enhancement of purified human protein C activation by human alpha-thrombin in the presence of purified human placenta thrombomodulin. In the absence of phospholipid, the anticoagulant fraction had no effect on thrombomodulin activity. The anticoagulant fraction could neutralize the enhancement of thrombomodulin activity by phospholipid in a dose-dependent manner. This study suggests that the neutralization of phospholipid might result in a reduced activation of protein C which could be responsible for the occurrence of thrombotic complications in a proportion of patients with lupus anticoagulants. PMID- 3018956 TI - Further studies on aggregation of platelet-type von Willebrand's disease platelets by human von Willebrand factor. AB - Platelet-type von Willebrand's disease (vWD) is a bleeding disorder characterized by a heightened interaction between platelets and von Willebrand factor (vWF) as the result of an intrinsic platelet abnormality (probably in GPIb). Platelet aggregability was nearly normal in response to thrombin, wheat germ agglutinin and Ricinus communis agglutinin in this disorder. Unmodified platelets showed no aggregation upon the addition of peanut agglutinin. Partially purified human vWF induced little aggregation of washed patient platelets, but the aggregation was greatly enhanced in the presence of plasma devoid of vWF. Monoclonal antibodies directed against GPIb and GPIIb/IIIa as well as EDTA completely inhibited vWF induced aggregation. These results indicate that human vWF induces aggregation of platelet-type vWD platelets in the presence of divalent cations and some plasma cofactor(s), and that both GPIb and GPIIb/IIIa are involved in this aggregation. PMID- 3018958 TI - The use of Epstein-Barr virus transformation for the establishment of class II positive cord blood cells for use in analysis of maternal sera. AB - The use of Epstein-Barr virus transformed cord blood cells to detect maternal antibodies to neonatal class II DR and DQ antigens and to allotype the reacting cells is described. PMID- 3018959 TI - Identification of two subtypes of HLA-DR2 by Southern blot analysis. AB - We studied the polymorphism of HLA-DR2 by Southern blot analysis. Genomic DNA from 12 DR2 positive unrelated individuals was digested separately with five restriction endonucleases, Bam HI, Eco RI, Eco RV, Hind III and Pvu II. Hybridizing restriction fragments were visualized using radiolabeled beta-chain cDNA probes homologous to the DR and DQ subregions of the HLA. The results in the present study demonstrate that the two subtypes of DR2, DR2.1 and DR2.2, as defined by serological and cellular (PLT) techniques can be identified by Southern blot analysis. These two subtypes were identifiable after cleaving genomic DNA with Bam HI and EcoRV. DR2.1 correlated with restriction endonuclease fragments at 2.8 kb (DR beta) and 6.6 kb (DQ beta) after Bam HI digest, while DR2.2 correlated with a 2.9 kb (DR beta) and a 2.6 kb (DQ beta) restriction fragment after Eco RV digest. In addition, inheritance of the restriction fragment lengths defining DR2.1 and DR2.2 were observed in two families. In contrast, no differences between DR2.1 and DR2.2 were observed with the restriction enzymes Eco RI, Hind III and Pvu II. PMID- 3018960 TI - Positive selection of activated T cells of the T8 (CD8) sub-type by immunomagnetic separation. AB - By conjugating a monoclonal IgM antibody of CD8-specificity to magnetite containing polymer particles, we have developed a rapid and simple one-step procedure for positive selection of T8 cells. The method ensured good yield and viability of pure T8 cells. The positively selected T8-fraction from MLC activated T cells retained their cytolytic capacity and gave a moderate proliferative response. Most of the proliferative response occurred in the negatively selected T4 population which showed only a marginal cytotoxic activity against PHA blasts as target cells. Transferrin receptor expression, as measured in a direct radiobinding assay, could also be demonstrated both among pure T4 and T8 cells upon MLC-activation. PMID- 3018961 TI - DNATYPE--a personal computer program for HLA-DR typing based upon restriction fragment length polymorphisms. AB - The use of specific cDNA probes and selected combinations of restriction endonucleases makes it possible to characterize specific antigens by the molecular weights of the hybridized restriction enzyme digests of DNA. We have developed a simple program suitable for personal computers which utilizes such results to deduce HLA-DR phenotypes of any nucleated cell from which DNA may be extracted. PMID- 3018963 TI - Immunohistochemical and electron microscopic demonstration of human papillomavirus in dysplasia of the uterine cervix. AB - Twenty-five cases of dysplasia of the uterine cervix were studied for the presence of human papillomavirus (HPV) by means of immunohistochemical and electron microscopic techniques. Serial sections of the same histological specimen were examined in each case. HPV was detected in 14 cases by both immunohistochemistry and electron microscopy, while 10 cases were negative with both methods. In only one case, there was a discrepancy in the results derived from these two methods. It was concluded that the relation between HPV infection and cervical dysplasia was confirmed and that immunohistochemical and electron microscopic methods led almost to the same result in detecting HPV in cervical dysplasia. PMID- 3018962 TI - High frequency of HLA-DR5 in Greek patients with haemophilia A and haemophilia B. AB - Sixty unrelated Greek patients with haemophilia (46 with haemophilia A and 14 with haemophilia B) were typed for HLA-A, B and DR antigens. A highly significant increase in the frequency of HLA-DR5 was observed in both groups of patients (58.6% vs 30.0%, chi 2 = 10.47, pc less than 0.03, RR = 3.31 for haemophilia A and 78.5% vs 30.0%, chi 2 = 12.32, pc less than 0.007, RR = 8.5 for haemophilia B). An increased frequency of HLA-B13 was also observed in patients with haemophilia A (15.2% vs 5.7%, chi 2 = 5.74, pc less than 0.4, RR = 2.9). Thirty of the 60 patients (50.0%) were positive for LAV/HTLVIII antibodies. HLA-DR5 was equally distributed in patients with and without these antibodies (63.3% and 63.3%, respectively). The presence of DR5 did not correlate with the severity of haemophilia A or B. These results may suggest an influence of gene(s) on chromosome 6 in haemophilia A and haemophilia B and no effect of HLA antigens in the susceptibility to LAV/ HTLVIII infection among haemophiliac patients. PMID- 3018965 TI - Ultrastructural alterations in rats adrenal cortical cells produced by 4 aminopyrazolopyrimidine and ACTH administration. AB - The correlation between adrenal cortical steroid metabolism and ultrastructural alterations was studied in rat adrenal glands stimulated by 4 aminopyrazolopyrimidine (4-APP) and ACTH. Ultrastructural alterations were focused on the outer fasciculata (Outer zone) and inner fasciculata-reticularis (Inner zone) of the rat adrenal cortical cells. In 4-APP administered rats, the most striking ultrastructural alteration was noted in the characteristic tubular profiles of smooth endoplasmic reticulum, mainly of the Inner zone. In ACTH administered rats, proliferation of fine tubulo-vesicular profiles of smooth endoplasmic reticulum was frequently seen in intermitochondrial space in both the Outer and the Inner zones. On the other hand, by 4-APP plus ACTH administration, the most striking ultrastructural changes were detected in mitochondria, mainly of the Inner zone. These mitochondria had a highly electron dense matrix and protrusions or blebs of mitochondrial outer membrane were frequently observed. In contrast, no remarkable changes were detected in the Outer zone of the cortex, except a slight enlargement of the mitochondria. On the bases of our findings, it is strongly suggested that the Inner zone of the cortex after 4-APP plus ACTH administration, at least in part, probably corresponds to highly activated steroid synthesis and it is further, speculated that steroidogenesis in the adrenal cortical cells after 4-APP administration is partially impaired in mitochondrial 11 beta-hydroxylase system. PMID- 3018964 TI - Effects of neonatal methylmercury exposure on adrenergic receptor binding sites in peripheral tissues of the developing rat. AB - Neonatal exposure to methylmercury produces changes in patterns of tissue growth and function, in part, due to alterations in adrenergic neuronal input. To explore the mechanisms by which these changes come about, newborn rats were exposed to methylmercury (1 or 2.5 mg/kg per day) throughout the preweaning stage and the ontogeny of adrenergic receptor binding sites evaluated in liver, kidney, heart and lung, using [3H]prazosin (alpha 1-receptors), [3H]rauwolscine (alpha 2 receptors) and [125I]pindolol (beta-receptors). In the kidney, methylmercury caused decreases in beta- and alpha 1-receptor binding and increases in alpha 2 binding; previous work has shown that beta-receptor-mediated responses are generally enhanced in methylmercury-exposed pups, and the down-regulation of beta receptor binding thus probably represents a compensatory action secondary to alterations in post-receptor coupling mechanisms. The effects of methylmercury on hepatic adrenergic receptors were different from those seen in the kidney, with substantial elevations in beta- and alpha 1-receptor binding apparent in the preweaning stage; this agrees also with the differences in effects of the mercurial on trophic reactivity and growth in the 2 tissues. Despite the fact that methylmercury causes activation of neonatal cardiac sympathetic nerves, beta receptor binding sites in the heart were unaffected by methylmercury exposure; the failure to down-regulate cardiac postsynaptic receptors in the face of increased nerve activity again represents an anomaly of synaptic regulatory function. These results indicate that methylmercury alters adrenergic responsiveness, in part, through actions on the development of receptor binding sites, and further, that the organ-specificity and receptor subtype-selectivity are consistent with subsequent effects of the organomercurial on adrenergic participation in target organ growth; however, changes in receptor binding alone do not account for all of the effects of methylmercury on synaptic activity or trophic responses. PMID- 3018966 TI - [Use of collalysine++ phonophoresis in the combined treatment of hypertrophic and keloid scars of the face and neck]. PMID- 3018968 TI - Selenium dioxide oxidation of vitamin D3 acetate to a dimer of 1-oxotransvitamin D3 acetate. AB - The treatment of vitamin D3 acetate with selenium dioxide and t-butyl hydroperoxide leads to a mixture from which a Diels-Alder dimer of 1 oxotransvitamin D3 acetate was isolated. PMID- 3018967 TI - Possible abnormalities of steroid secretion in children with Smith-Lemli-Opitz syndrome and their parents. AB - In early infancy, two unrelated children with Smith-Lemli-Opitz syndrome were found to have elevated levels of androgen sulfates. When the steroid conjugates in the serum of normal infants were hydrolyzed and chromatographed on Sephadex LH 20, 4 androgen containing peaks (I, II, III, IV) were found. In the serum from these two infants with Smith-Lemli-Opitz syndrome, Peaks I and III were increased, but Peaks II and IV were absent. The parents of the two children, and of three additional unrelated children with Smith-Lemli-Opitz syndrome, had exaggerated 17-hydroxyprogesterone responses to an intravenous bolus of ACTH. These findings suggest that a defect in steroid metabolism may be linked to the Smith-Lemli-Opitz syndrome. PMID- 3018969 TI - Accessibility of thymic cortical lymphocytes to particles translocated from the peritoneal cavity to parathymic lymph nodes. AB - In an attempt to evaluate the relative effectiveness of barriers protecting thymic cortical lymphocytes from antigens or other macromolecules, mice were given an intraperitoneal (i.p.) or intravenous (i.v.) injection of polyvinylpyrrolidone (PVP)-coated silica particles (Percoll), measuring approximately 17 nm in diameter. Pathways of translocation of this material were studied by transmission electron microscopy. Particles were rapidly cleared from the peritoneal cavity, and large quantities appeared in parathymic lymph nodes. From there, small amounts of Percoll reached lymphatics surrounding the thymus and traversed the thymic capsule, preferentially in the area close to the lymph nodes, to enter the cortical parenchyma within less than 2 h. Considerable numbers of cortical thymocytes contained endocytosed particles. At later time intervals after both i.p. and i.v. injection. Percoll was identified within cytoplasmic vesicles of macrophages scattered along cortical capillaries. However, particles given i.v. were never found in intercellular spaces or in the cytoplasm of thymocytes of the thymic cortex. Thus, the lymphoid parenchyma of the thymic cortex is accessible to small particles carried through the capsule with the flux of interstitial fluid. We conclude that the capsule-thymus barrier is less efficient than the blood-thymus barrier. PMID- 3018970 TI - Post-encephalitic epilepsy and arbovirus infections in an isolated rainforest area of central Liberia. AB - Among a population of 4.436 Bassa, Kpelle and Mano people in the Gbawein and Wroughbarh Clan region of Grand Bassa Country, Liberia, 123 cases of epilepsy could be documented. In 38% of these cases infections involving the central nervous system precipitated the onset of seizures. Sera from 67 epilepsy patients, 50 direct healthy relatives and 22 geographically matched controls were tested for antibodies to 16 arboviruses of the Togaviridae and Bunyaviridae known to occur in Africa. Antibodies to arboviruses were found in 16.5% of the epilepsy patients, 36% of the mostly older family members, and in 22% of the controls. Males and females were equally affected as were the different clans and language groups. Although meningoencephalitis with sequelae, like seizures, are known to result from arbovirus infections, no evidence for a correlation between epilepsy in this are of Central Liberia and previous arbovirus infection could be established. PMID- 3018971 TI - A study of seasonal trace element intakes and hair trace element concentrations in selected households from the Wosera, Papua New Guinea. AB - The trace element status of nine households from the Wosera subdistrict of Papua New Guinea was assessed. Individual weighed three-day dietary intakes were carried out in April during the hungry season when yams were not eaten and in July when yams were available. Representative staple food items analyzed 'as eaten' for energy, protein, Ca, Mn, Cu and Zn and mean daily energy and nutrient intakes calculated. Hair samples were also collected in July, washed, and analyzed for Zn, Cu and Mn by INAA. Seasonal differences in energy, protein, Ca, Fe and Mn intakes were related to changes in yam consumption. The Zn status of the male (M) and female (F) adults and children (Ch) was sub-optimal based on low median hair Zn levels (microgram/g): -F = 78, n = 8; M = 108, n = 8; Ch2-4yrs = 79, n = 8; Ch5-10yrs = 94, n = 7 and inadequate intakes of readily available Zn (mean + SD): - April M + F = 7.0 + 1.6 mg/day; July M + F = 8.2 + 1.5 mg/day; less than three percent Zn from animal products. In contrast their Mn status, as indicated by elevated median hair Mn levels (microgram/g) (F = 25.1; M = 26.3; Ch2-4yrs = 21.5; Ch5-10yrs = 15.2) was high, attributed to elevated Mn intakes (April M + F = 11.0 + 2.2 mg/day; July M + F = 7.5 + 1.4 mg/day). Median hair Cu levels (microgram/g) (F = 7.6; M = 7.5; Ch2-4yrs = 8.6; Ch5-10yrs = 7.8) and mean Cu intakes (April M + F = 2.2 + 0.4 mg/day; July M + F = 3.0 + 0.6 mg/day) were within the range noted for persons consuming predominantly plant-based diets. PMID- 3018973 TI - [Calcium pyrophosphate dihydrate deposition disease. Clinical picture, diagnosis and treatment]. PMID- 3018972 TI - [Glioblastoma multiforme. Prognosis and treatment]. PMID- 3018974 TI - Expression of the human fes cellular oncogene in renal cell tumors. AB - Renal cell tumors were screened for expression of the cellular oncogenes c-abl, c fes, c-fms, c-myc, c-ras, and c-sis in dot blot hybridization analysis. Expression of c-ras and c-myc was clearly detectable in most of the 15 tumors that were studied. The c-fes oncogene appeared to be expressed in only two of them. Comparative Southern blot analysis of molecularly cloned human c-fes DNA and genomic DNA of the 15 renal tumors revealed no major genetic differences. Northern blot analysis of poly(A)-selected RNA from the fes-positive tumors with the complete viral v-fes oncogene of the Gardner-Arnstein strain of feline sarcoma virus as a molecular probe revealed hybridization of RNA species of 3.0 and 4.5 kb, respectively. The 3.0 kb c-fes transcript has also been reported in RNA from patients suffering from acute myelogenous leukemia. The 4.5 kb transcript, however, has not been described before and represents either a c-fes related splicing intermediate or, more likely, a completely processed transcript. The results of this study could imply that human c-fes coding sequences are more extensive than was previously assumed. PMID- 3018976 TI - Retroperitoneal germinoma. PMID- 3018975 TI - Magnetic resonance imaging of Wilms tumor. AB - Magnetic resonance imaging (MRI) in 4 children with Wilms tumor suggests the usefulness of this newer imaging modality in evaluating the organ of origin and defining the extent of Wilms tumor. Coronal T1-weighted images were the most useful pulsing sequence for evaluating these children. PMID- 3018977 TI - [Malignant chemodectomas of the neck]. PMID- 3018978 TI - [Sialogenic tumors of the palate]. PMID- 3018979 TI - [Non-invasive methods of examination in the diagnosis of aneurysms]. AB - Radionuclide angiography and ultrasonic echolocation were used for the examination of 38 patients with aneurysms. Isotope angiography was fulfilled in all the 38 patients, ultrasonography was used in 20 patients. Radionuclide angiography and ultrasonography are thought to be sufficiently effective methods of diagnosis of vessel aneurysms. PMID- 3018980 TI - [Treatment of soft tissue sarcomas of the leg]. AB - An analysis of data of 548 patients with different diseases of soft tissues of lower extremities after using a complex of diagnostic measures (clinical, radionuclide data, thermography, puncture, biopsy) has shown that the number of erroneous conclusions may be minimized to 2%. Modern advances of plastic surgery can widen indications for preserving operations in malignant tumors of soft tissues of lower extremities without prejudice to the main principle of oncology concerning its fascial character. Combined treatment can also improve the results. PMID- 3018981 TI - A scanning and transmission electron microscopic study of rotavirus-induced intestinal lesions in neonatal gnotobiotic dogs. AB - Neonatal gnotobiotic dogs orally inoculated with canine rotavirus had ultrastructural changes limited to the jejunal and ileal regions of the small intestine. Early scanning electron microscopic findings consisted of swollen villus epithelial cells, denuded foci on intestinal villi, and slight to moderate villus atrophy. Later changes were slight villus atrophy with no denuded intestinal villi. Transmission electron microscopic changes in villus epithelial cells from 12 to 48 hours post-inoculation included: rotavirus particles associated with intracytoplasmic vacuoles near the terminal web and apical tubules; viral particles in dilated cisternae of rough endoplasmic reticulum; and moderate numbers of necrotic cells having no microvilli, swollen mitochondria, membrane-bound lipid-like material in the cytoplasm, clumped chromatin around the periphery of the nucleus, and disruption of the cytoplasmic membrane. In jejunum and ileum at 72 to 154 hours post-inoculation, there were fewer necrotic villus epithelial cells and fewer virus particles. In addition, the ultrastructural morphology of the majority of the villus epithelial cells was similar to crypt epithelium. These studies showed that rotavirus infected the villus epithelial cells with subsequent propagation of the rotavirus and destruction of villus epithelial cells. PMID- 3018982 TI - Neuropathology of experimental 3-nitro-4-hydroxyphenylarsonic acid toxicosis in pigs. AB - Twenty pigs were fed a diet containing 187.5 mg kg-1 of 3-nitro-4 hydroxyphenylarsonic acid (3-nitro). Ten pigs were euthanized at intervals up to 29 days, 3-nitro was withdrawn from the diet of the remaining pigs on day 30, and these animals were subsequently euthanized at intervals up to 49 days after commencement of the experiment. A nervous syndrome characterized by clonic convulsive episodes inducible by exercise, developed at day 11. Paraparesis was apparent at day 22 progressing to paraplegia by day 33 (3 days after cessation of 3-nitro feeding). Histopathologic examination revealed myelin and axonal degeneration in the white matter of the spinal cord coincident with the onset of nervous signs. Marchi-positive degeneration was present in the dorsal funiculus at cervical level at day 22. Lesions intensified with increasing duration of toxicosis and while degenerate fibers were seen in all funiculi, there was preferential involvement of the fasciculi gracilis and cuneatus, the peripheral regions of the ventral and lateral funiculi, and a discrete area of the dorsal region of the lateral funiculus. Peripheral and optic neuropathies were evident from day 32 but were always mild and focal. The experiment establishes 3-nitro as a central-peripheral neurotoxicant of pigs. PMID- 3018983 TI - Histopathological and hematological findings in myeloid leukemia induced by a new feline leukemia virus isolate. AB - Myeloid leukemia was induced by a new feline leukemia virus isolate FeLV-AB/GM-1 in a high proportion of cats. The latency period was short. Three to 5 weeks after infection early changes were detectable in the bone marrow, and cats developed leukemia 5 to 8 weeks after infection. The results of the present histological and cytological studies suggested that there were two stages in the development of leukemia. The first stage appeared to be equivalent to the syndrome of bone marrow dysplasia or preleukemia which, however, converted rapidly to leukemia. Cytopenia(s) were the main hematological findings in all preleukemic and leukemic cats. White blood cell counts were low or normal, but the number of leukemic and abnormal cells increased in the peripheral blood with the progression of the disease. This reliable model system lends itself to further studies to elucidate the pathogenesis of myeloproliferative disorders. PMID- 3018984 TI - Immunohistochemical detection of canine adenovirus in paraffin sections of liver. AB - An avidin-biotin immunoperoxidase procedure was optimized for detection of canine adenoviral antigens in formalin-fixed, paraffin-embedded liver. Long-term stability of viral antigen was shown by successful demonstration of virus in liver tissue preserved up to six years from dogs with infectious canine hepatitis. This immunohistochemical stain was applied to sections from livers with a wide range of inflammatory lesions. Examination of sections from 53 dogs yielded five livers with small amounts of adenovirus. An additional virus positive liver was identified from a dog with no hepatic inflammation. Although a cause and effect relationship remains to be determined, these findings suggest a possible connection between canine adenovirus and spontaneous chronic hepatitis. PMID- 3018985 TI - Pi granules and related intracytoplasmic inclusions in equine Schwann cells. PMID- 3018986 TI - Functional pituitary acidophil adenoma in a cat with diabetes mellitus and acromegalic features. PMID- 3018987 TI - Isolation of bluetongue virus from sheep in India. PMID- 3018988 TI - Porcine influenza outbreak. PMID- 3018990 TI - Kangaroo gait. PMID- 3018989 TI - Evaluation of a combined rotavirus and enterotoxigenic Escherichia coli vaccine in cattle. AB - A vaccine of rotavirus and K99 antigen from enterotoxigenic Escherichia coli was emulsified in oil adjuvant and administered intramuscularly to pregnant cows. Calves born to and reared on vaccinated dams were protected against experimental rotavirus infection at five days old when compared with calves from unvaccinated control cows. Field trials of the vaccine were carried out in 40 commercial herds, in which half the cows in each herd were selected at random for vaccination and half were left unvaccinated. In 31 herds (2641 cows) there was no significant diarrhoea problem (less than 10 per cent morbidity); these herds were excluded from further analysis. The nine remaining herds did experience a calf diarrhoea problem of greater than 10 per cent morbidity, but on four farms the disease was associated with cryptosporidiosis and on a fifth no enteropathogens were detected; these five farms (461 cows) were also excluded from further analysis. Of the remaining four herds, two beef suckler herds (105 cows) had concurrent rotavirus and cryptosporidial infections, and vaccination was associated with a decreased excretion of rotavirus but not with a decreased incidence of diarrhoea. In the other two dairy herds (68 cows) with prevaccination rotavirus problems, there was a significantly decreased incidence of diarrhoea in calves born to vaccinated cows. No natural field challenge of enterotoxigenic E coli was encountered on any of the trial farms. PMID- 3018991 TI - The effects of ACTH, prednisolone and Escherichia coli endotoxin on some clinical haematological and blood biochemical parameters in dwarf goats. AB - ACTH (microgram kg-1 i.v.) and prednisolone (1 microgram-1 i.v.) caused a moderate but statistically significant inhibition of rumen contractions, whereas no effects on heart rate and body temperature were observed. Both hormones induced hyperglycaemia and leucocytosis, characterised by moderate lymphopenia and a profound increase in the number of circulating neutrophils. A significant decrease in plasma iron and increase in plasma zinc concentrations were observed. After 3 daily i.m. injections of ACTH (10 micrograms-1 day-1) decreases were seen in both serum Alkaline phosphatase (ALP) activity and plasma trace metal concentrations; heart rate was significantly higher. Intraveneous injection of E. coli endotoxin (0.1 microgram kg-1) caused shivering, fever, inhibition of rumen contractions, changes in heart rate, lymphopenia, neutropenia followed by neutrophilic leucocytosis, hypoferraemia, hypozincaemia, hypoglycaemia and a decline in serum ALP activity. ACTH, given i.m. for 3 days, reduced the febrile responses to E. coli endotoxin, modified the changes in heart rate, intensified the inhibition of rumen contractions, and induced a more marked decrease in the number of circulating neutrophils. ACTH pretreatment did not affect the endotoxin induced decrease in blood glucose concentrations nor the drop in plasma zinc and iron values. These results suggest that glucocorticosteroids are not primarily involved in the fall in plasma iron and zinc concentrations during E. coli endotoxin-induced fever, the effects of endotoxin released glucocorticosteroids on white blood cells and blood glucose are masked by some other effect(s) of endotoxin, and in dwarf goats, ACTH has antipyretic properties without influencing normal body temperature. This effect is probably not dependent on adrenal cortical activity. PMID- 3018993 TI - Isolation of a porcine respiratory, non-enteric coronavirus related to transmissible gastroenteritis. AB - A porcine respiratory, non-enteric virus which is related to the coronavirus transmissible gastroenteritis virus (TGEV) has been isolated in pigs and in cell culture. The isolate was designated TLM 83. It has become very widespread and enzootic among the swine population in Belgium and in other swine raising countries. It causes an infection of the lungs and appears to spread by aerogenic route. It does not replicate in the enteric tract. The experimental infection in conventional and gnotobiotic pigs in isolation remains subclinical. The infection, either experimental or in the field, results in the formation of antibodies which neutralise the classical enteric TGEV. Based on this relationship, this virus is assumed to be a new TGEV-related porcine respiratory coronavirus or TGEV itself which has totally lost its tropism for the enteric tract. PMID- 3018992 TI - Clinical application of neuromuscular electrophysiology in the dog: a review. AB - A review of the literature concerning the application of electromyography and electroneurography in canine neurology is presented. Measurements of amplitude and duration of motor unit potentials in normal dogs varied largely in the various reports. It was therefore concluded that these measurements are of limited clinical value. The results of motor and sensory nerve conduction studies in normal dogs are summarised. The differences between the methods used are discussed. It is concluded that variations in reported normal values are due mainly to differences in method. The application of electromyography and electroneurography in neuromuscular disorders in the dog is systematically presented, based upon the reported diagnoses. The considerable number of first descriptions of newly-recognised and described neuromuscular diseases appears to be related to the introduction of neuromuscular electrophysiology into veterinary medicine. PMID- 3018994 TI - The effect of parainfluenza virus type 3 and Pasteurella haemolytica on oxygen dependent and oxygen-independent bactericidal mechanisms of ovine pulmonary phagocytic cells. AB - Groups of caesarean-derived, colostrum-deprived lambs were inoculated by the intratracheal route with Pasteurella haemolytica, either alone or 4 or 6 days after the inoculation of parainfluenza virus type 3 (PI3). Other groups were inoculated with PI3 followed by veal infusion broth, or with uninfected cell culture fluid followed by veal infusion broth (controls). All lambs were killed 24 h after the second inoculation. Pulmonary phagocytic cells were recovered by lavage and separated into alveolar macrophage (AM) and neutrophil fractions by density gradient centrifugation. Bacterial proliferation was detected in the lungs of all five lambs inoculated with P. haemolytica 6 days after PI3 but in only one of five inoculated with P. haemolytica 4 days after PI3 and one of five inoculated with P. haemolytica alone. The number of phagocytic cells recovered from the lungs was highest in animals inoculated with P. haemolytica 6 days after PI3 and, overall, a greater number of both AM and neutrophils was recovered from the lungs of animals where bacterial proliferation occurred (greater than 10(5.0) P. haemolytica 100 g-1 lung) than from those that controlled the bacterial infection. Oxygen-dependent bactericidal activity of AM and neutrophils was measured by chemiluminescence. Infection with PI3 and P. haemolytica increased the chemiluminescence responses. The highest responses were recorded from lambs inoculated with P. haemolytica 6 days after PI3, the group where pulmonary clearance was poorest. Overall, responses were higher in lambs in which bacterial proliferation occurred than in those that controlled the infection. On the other hand, oxygen-independent bactericidal activity, measured by the direct effects of neutrophil lysates on Escherichia coli, was lowest in lambs inoculated with P. haemolytica 6 days after PI3 and was lower in lambs where bacterial proliferation occurred. PMID- 3018995 TI - Comparison by plaque assay of bovine rotavirus and a bovine enterovirus-like agent. AB - Enterovirus-like particles from feces of calves are a frequent source of contamination of bovine rotavirus isolates. A study of plaque formation using BSC 1 cells indicated differences in behaviour of the viruses which could be used for differentiation the purification. The enterovirus-like particles produced well defined plaques earlier and reached their optimal size much more rapidly than did the rotavirus. Furthermore, plaques produced by bovine enterovirus-like particles were significantly larger than those of bovine rotavirus. The viral cytopathic effects on the cells within the plaques were also characteristic for each virus. PMID- 3018996 TI - [The role of contrast sphenography in the x-ray diagnosis of inflammatory diseases of the sphenoidal sinuses]. PMID- 3018998 TI - The synthesis and transport of SV40 structural proteins. AB - The kinetics of the synthesis and transport of viral structural proteins, Vp1 and Vp3, and of actin in SV40 infected TC7 cells were studied. The newly synthesized proteins were found in the NP-40-soluble (Sol) fraction of the cell cytoplasm. The majority of newly synthesized viral structural proteins, destined for the cell nucleus for virion assembly, were transported to the cell nucleus (Nuc) between 10 and 30 min after synthesis, whereas the majority of newly synthesized actin remained in the Sol fraction of the cytoplasm, suggesting that some specific mechanism exists for selecting the proper sites for transport. The synthesis and transport of both Vp1 and Vp3 throughout infected cells were similar. However, there is a difference in the transport properties of these two proteins. Once Vp1 was synthesized, the mature Vp1 was transported to both the cytoskeletal (Csk) and the Nuc fractions in the absence of further protein synthesis, whereas the movement of Vp3 from the Sol to the Csk, but not to the Nuc fraction, was partially inhibited in the absence of protein synthesis. Modification of Vp1 occurred in the cell cytoplasm before transport to the cell nucleus. Its modification pattern suggests that the Csk is the site for the modification of Vp1. The efficiency of viral protein transport to the cell nucleus was diminished after 47 hr of infection. This trend was preceded by a decrease in the ability to incorporate label into actin 12 hr earlier in infection. Thus, some marking event appears to have occurred prior to the actual decrease in transport efficiency and the integrity of the cytoarchitecture appears to be important for viral protein transport. PMID- 3018999 TI - Conservation of the c-myc coding sequence in transduced feline v-myc genes. AB - We have cloned the normal feline c-myc locus and determined the nucleotide sequence of all three exons. The feline c-myc gene shows close homology to other mammalian c-myc genes, particularly human c-myc. The feline and human sequences are colinear within the open reading frame for the putative c-myc product but show insertions and deletions relative to each other outside this domain. We have also analyzed a cloned FeLV provirus, CT4, which contains the host-derived myc gene. In this provirus the v-myc sequences are located at the 3' end of the pol gene, replacing pol and env sequences. Nucleotide sequence analysis of CT4 shows an open reading frame for a v-myc gene product which may be expressed without fusion to any viral protein sequences. This contrasts with another FeLV v-myc (LC), in which myc and gag sequences were found to be fused. Unlike previously identified avian v-myc genes, the feline v-myc genes contain exon 1-derived sequences, but these have been truncated or internally deleted. The FeLV CT4 v myc sequence shows very few coding changes relative to c-myc and the FeLV LC v myc coding sequence is unchanged relative to c-myc apart from fusion to gag. These results are discussed in relation to the mechanism of transduction and activation of myc by FeLV. PMID- 3018997 TI - [Use of NMR tomography in clinical practice (a review of the literature)]. PMID- 3019000 TI - Physical characterization of the genome of feline herpesvirus-1. AB - The physical structure of the genome of feline herpesvirus-1, a major upper respiratory tract pathogen of cats, was studied. Purified FHV-1 DNA was analyzed by restriction endonuclease and gamma 5' exonuclease digestion, blot hybridization, and electron microscopy. To facilitate further studies, nine bacteriophage clones were isolated which contained 85% of the viral genome as SalI inserts, and DNA from these clones was used in blot hybridization experiments and as substrates for restriction digest analysis. Data from these studies permitted construction of a SalI and partial HindII and EcoRI restriction maps of the viral genome. FHV-1 DNA is approximately 134 kb in size and is composed of a long (L) and a short (S) segment. The long segment (U1) is 104 kb in size and is composed of unique DNA. The adjacent S segment is approximately 30 kb in size and contains a central portion of unique DNA (Us) which is approximately 8 kb in size. The Us region is bounded by inverted repeat sequences which are 11 kb in size. Therefore, the physical structure of the FHV-1 genome is similar to the genomes of other alpha-herpesviruses. PMID- 3019002 TI - The effect of cerulenin on the synthesis of the precursor gag polyprotein in defective murine leukemia and sarcoma virus producing cell lines. AB - The effect of cerulenin, an inhibitor of de novo fatty acid (and cholesterol) biosynthesis, on the synthesis of the precursor gag polyprotein, Pr65gag in a defective murine leukemia virus (334C) producing murine cell line (3JE) and a defective murine sarcoma virus (Gazdar) producing hamster cell line (HTG-2) was examined. In contrast to Moloney murine leukemia virus (M-MuLV) producing cell lines (MJD-54, clone 2) the amount of the Pr65gag remaining in the presence of cerulenin (20 micrograms/ml) was greatly reduced in both defective virus-infected cells. This effect appears specific for the Pr65gag polyprotein, since the env precursor polyprotein Pr80env was normally synthesized and remained undegraded in cerulenin-treated 3JE-infected cells. Thin-section electron micrographs showed an increased accumulation of virion particles in vesicles of treated HTG-2 cells. PMID- 3019001 TI - Characterization of Fischer rat embryo (CREF) cells transformed by bovine papillomavirus type 1. AB - Transformation of an established Fischer rat embryo (CREF) cell line by bovine papillomavirus type 1 (BPV-1), in contrast to transformation by type 5 adenovirus or the T24 (Ha-ras) oncogene, resulted in transformants which did not exhibit a major increase in saturation density or a decrease in 125I-epidermal growth factor binding. BPV-1-transformed CREF clones did, however, grow in agar suspension culture and were tumorigenic in both nude mice and Fischer rats. The majority of transformed clones contained multiple extrachromosomal copies of BPV DNA. One transformed CREF clone contained integrated BPV-1 DNA which underwent sequence rearrangements following tumor formation in a Fischer rat and reestablishment in cell culture. BPV copy number varied in subclones of transformants isolated from the same monolayer focus, in agar-derived subclones of the same transformed focus, in tumors and in tumor-derived BPV-transformed CREF subclones. The degree of expression of specific transformation-related phenotypes, i.e., saturation density, growth in agar, and tumorigenicity, was not correlated with BPV copy number in transformed clones. Analysis of the biological properties of tumor-derived BPV-1-transformed CREF subclones indicated that certain transformants developed a stable increase in expression of transformation related properties, a process termed "progression." TPA did not enhance the frequency of BPV-1 transformation or BPV DNA copy number in transformed CREF cells. The present study demonstrates that the CREF- transformation system can be utilized to study the molecular basis of BPV-1 transformation and should prove useful in studying the role of specific BPV-1 transforming proteins in regulating expression of the transformed phenotype and the mechanism by which transformed cells undergo progression of the transformed state. PMID- 3019003 TI - Nucleotide sequence of the CMII v-myc allele. AB - The entire nucleotide sequence of the transduced v-myc allele in the genome of avian oncogenic retrovirus CMII was determined. The CMII v-myc and the chicken c myc alleles differ in their shared coding sequences by a single nucleotide substitution causing a glutamic acid/alanine exchange in the predicted sequences of the corresponding protein products. This mutation has not been found in the transduced v-myc alleles of avian oncogenic retroviruses MC29, MH2, and OK10. We conclude that no specific, if any, missense mutation of the c-myc coding sequence is necessary for oncogenic activation upon transduction of the cellular gene. PMID- 3019004 TI - Conservation in rotaviruses of the protein region containing the two sites associated with trypsin enhancement of infectivity. AB - The primary structure of the region in the outer layer protein VP3, containing the two sites associated with trypsin enhancement of infectivity of rotavirus was found to be greatly conserved in cultivable human rotavirus serotypes 1 (Wa), 2 (DS1), and 3 (P) and in four human rotaviruses directly purified from feces. Significant differences with this conserved sequence were found in human rotavirus serotype 4 (ST3), isolated from an asymptomatic neonate, and in seven animal rotaviruses. However, the two trypsin cleavage sites were conserved in every rotavirus VP3 sequence analyzed. PMID- 3019005 TI - Spleen specific expression of an MMTV related transcript associated with the Mtv 6 locus in BALB/c mice. AB - We have detected an MMTV related transcript which is expressed in a spleen specific manner in BALB/cHeA mice. Using a recombinant inbred series between BALB/cHeA and STS/A mice (C X S RI series) we have identified RNA associated with the Mtv-3 locus of the STS/A strain. This transcript initiates at the same site in the MMTV LTR as already reported for Mtv-2 and Mtv-8. The novel spleen specific MMTV transcript in the BALB/cHeA strain has a different structure as compared to the transcripts associated with the Mtv-2, Mtv-3, or Mtv-8 loci. We have tentatively identified the Mtv-6 locus as the source of these unique transcripts. PMID- 3019006 TI - Sequence of the Sendai virus L gene: open reading frames upstream of the main coding region suggest that the gene may be polycistronic. AB - The sequence of the L gene of Sendai virus, encompassing 6799 nucleotides, has been determined, completing the primary sequence of the entire virus genome. An open reading frame beginning at position 569 codes for a basic protein of 2048 amino acids with an estimated Mr of 231,608. No nucleotide sequence similarities with the analogous L gene of vesicular stomatitis virus were observed. However, comparison of the deduced amino acid sequences of both proteins revealed a conserved 18 amino acid sequence that may have functional significance. Two additional overlapping reading frames which precede the L protein sequence could encode proteins with MrS of 6474 and 14,026, suggesting that the gene is polycistronic. PMID- 3019007 TI - A transforming plasmid from HSV-2 transformed cells contains rat DNA homologous to the HSV-1 and HSV-2 genomes. AB - Morphologically transformed rat (3Y1) cell lines were established following transfection with HSV-2 mtrII DNA sequences (0.585 to 0.601 map units). The mtrII sequences were cloned in plasmids containing the neor gene. Cells resistant to the antibiotic G-418 were passed into soft agarose, and clonal lines were established from individual colonies. The DNAs from two cell lines examined by Southern blot hybridization were shown to contain the original transfected viral DNA sequences in a fashion consistent with a multiple and complex pattern of integration. From one cell line, an approximately 20-kbp plasmid was isolated after transformation of bacteria with Hirt supernatant DNA. This plasmid was capable of rapidly transforming rat cells at a greater than 1000-fold higher frequency than the mtrII DNA. This plasmid consists mainly of unique sequence rat DNA with two copies of the HSV-2 mtrII region DNA (HSV-2 genomic map unit location of ca. 0.595) present at sites distant from each other. The rat DNA in the rescued plasmid is homologous to the putative focus-forming sequences present in the HSV-2 mtrIII (0.53 to 0.58 map units) and the colinear HSV-1 DNA. The genomic copy of these rat sequences in four HSV-2 mtrII transformed cell lines appears to have undergone rearrangement. These data provide evidence that the HSV 2 mtrII sequences are involved in transformation, and that the HSV-2 mtrII region may affect transformation by rearranging the cellular sequences that are homologous to mtrIII. PMID- 3019008 TI - Geographical prevalence of two types of Epstein-Barr virus. AB - The Jijoye EBV strain is characterized by a substitution of 1.8 kb in the C terminal part of the EBNA 2 gene compared to B95-8 or M-ABA virus. This made it possible to construct hybridization probes specific for M-ABA (type A) and Jijoye viruses (type B), which have been used to type the EBV genomes in 38 spontaneously established cell lines. Type A is more prevalent being found in 31 of 38 cases; type B virus was found in five cell lines (Jijoye, LY 67, QIMR-GOR, BL 16, and BL 29); and two cell lines, Daudi and EB-3, contained neither the M ABA- nor the Jijoye-specific sequences. EBV type B appears to be less ubiquitous, since all type B isolates, including AG 876 virus, originated from Central Africa, La Reunion, and New Guinea. All the other cell lines, carrying EBV type A, were established from patients from Central Africa (4), North Africa (7), New Guinea (1), and Asia (6) and from white individuals (13). The restricted geographical localization of EBV type B in parts of the southern hemisphere and its similarity to herpesvirus papio (T. Dambaugh, K. Hennessy, L. Chamnankit, and E. Kieff (1984) Proc. Natl. Acad. Sci. USA 81, 7632-7636) could suggest that such viruses may have evolved by recombination of EBV with a related Old World monkey virus, alternatively, evolution of virus variants within the human species also being conceivable. PMID- 3019009 TI - Dynamic and nonspecific dispersal of human T-cell leukemia/lymphoma virus type-I integration in cultured lymphoma cells. AB - The progression of HTLV-I proviral integration over a 3-year period of in vitro culture was examined in two human lymphoma lines, Hut 102 and MJ. Using specific HTLV-I molecular clones and a Southern analysis at different cell passages, Hut 102 increased from 2 to 19 integrated proviral integrations while MJ increased to at least 25 different integrations by passage 43. During the progress of increased superinfection and novel integration in vitro some of the previous proviral integrations were lost from the cultures. The 19 integrations of late passage Hut 102 cells were shown to be dispersed to 19 different human chromosomes by analysis of 34 distinct rodent X Hut 102 somatic cell hybrids which segregated human chromosomes (and included proviral integrations) in different combinations. The two primary integrations in Hut 102 were located on human chromosomes 4 and 20, respectively. A similar pattern of nonspecific integration was observed in somatic cell hybrid analysis of the 25 proviral integrations of MJ. The dynamic infection-reintegration process in vitro revealed in these studies may confuse experimental verification of potential cis acting functions of HTLV-I in the as yet poorly understood mechanism of neoplastic transformation. PMID- 3019011 TI - [Acquired immunodeficiency syndrome]. PMID- 3019012 TI - [Complications after surgical treatment of polymorphic adenomas of the parotid gland]. PMID- 3019010 TI - Site-specific rearrangements in the long terminal repeat of extra mouse mammary tumor proviruses in murine T-cell leukemias. AB - Extra MMTV proviruses in T-cell leukemias of GR and C57/BL10 mice contain alterations in their long terminal repeat (LTR) sequence. The four different leukemias studied contain different deletions, but common hallmarks were observed around the recombination sites. At the 5' end of the deletions we observed a common nonamer sequence, AGACAGGTG, in two leukemias and an almost identical sequence, AGAGCAGGTG, in two other leukemias. At the 3' end of the deletions we invariably found a common stretch of five nucleotides, TTAAA. Three of the four leukemias showed nonconserved crossover sites. The deletions in two leukemias were replaced with neighboring sequences, generating direct repeats. The MMTV LTR characteristic open reading frame and glucocorticoid response element were altered in all four rearranged MMTV LTRs. These results demonstrate site specific rearrangements in the LTR of extra MMTV proviruses in T-cell leukemias and suggest that these rearrangements might permit expression of MMTV in a new target cell. PMID- 3019013 TI - [Insulin and ACTH test in patients in the initial stages of essential hypertension]. PMID- 3019014 TI - [Differential diagnosis of paresthesias in alcoholic polyneuropathy and multiple sclerosis]. PMID- 3019015 TI - [Use of electroanalgesia and percutaneous electrostimulation in the treatment of pain syndromes]. PMID- 3019017 TI - Effects of prednisolone on beta-adrenergic desensitization of normal peripheral lymphocytes: an in vitro model for steroid-controlled tachyphylaxis. AB - In vitro culture of human peripheral blood lymphocytes with the beta-adrenergic catecholamine isoproterenol for 24 hours, induced homologous beta-adrenergic desensitization, i.e. a large decrease in the number of beta-adrenergic binding sites and loss of the adenylate cyclase response to isoproterenol, without altering the effectiveness of prostaglandin E1 to stimulate the enzyme. Lymphocyte cultures pulsed for 24 hours with isoproterenol, washed free of the agent and cultured in hormone-free medium for 48 hours still showed marked suppression of the beta-adrenergic adenylate cyclase response and a lack of beta receptors. When prednisolone was added to the isoproterenol-depleted resuspension medium of desensitized lymphocytes, however, the beta-adrenergic system recovered fully within 48 hours. Treatment of desensitized lymphocytes with prednisolone in the continuous presence of isoproterenol failed to restore beta-adrenergic responsiveness of the cells. The results are discussed with respect to the reconstituting effect of prednisolone on beta-adrenergic responsiveness in bronchial asthma after therapy induced tachyphylaxis. PMID- 3019016 TI - [Angiotensin converting enzyme: its levels and its effect on blood pressure regulation]. PMID- 3019018 TI - [Activation of the macrophage/lymphocyte system in AIDS, ARC and AIDS risk groups]. AB - Elevated neopterin levels are indicative of activation of the cellular immune system. Studies on excretion of neopterin in patients with AIDS and ARC, as well as in members of risk groups have demonstrated repeated or permanent stimulation of the immune system. This stimulation can be observed in all risk groups independent of LAV/HTLV-III infection. Additionally, in vitro replication of LAV/HTLV-III has been observed to be quantitatively greatest in activated CD4+ lymphocytes. Hence, we conclude that activation of the cellular immune system represents the central cofactor for progressive LAV/HTLV-III infection. These findings seem to contrast with most reports in the literature to date, but observations of other authors corroborate them. As consequence of these studies, therapeutic regimens using immunostimulatory strategies should be prevented in AIDS and ARC patients. Immunosuppressive treatment should be considered. PMID- 3019019 TI - [HTLV-III serology, epidemiology and clinical aspects of imprisoned i.v. drug dependent males in Austria]. AB - Sera of 71 intravenous drug abusers imprisoned in Austria were tested for HTLV III antibodies and the results correlated with the case histories and available clinical data. 12 sera, i.e. 17%, were found to be positive. Seronegative and seropositive drug addicts showed no notable difference regarding the average duration of previous drug abuse or the incidence of sexually-transmitted diseases. Among HTLV-III seropositive drug addicts there was found to be a significantly higher incidence of frequent needle-sharing and history of previous i.v. drug abuse in Amsterdam. 6 of 12 HTLV-III seropositive drug addicts had developed the lymphadenopathy syndrome (LAS); chronic liver disease and positive hepatitis B serology were equally frequent in HTLV-III seronegative persons, HTLV III seropositive persons without symptoms and LAS patients. PMID- 3019020 TI - Diazoxide in the management of patients with insulinoma. PMID- 3019021 TI - Evaluation of image-diagnosing methods of enlarged parathyroid glands in chronic renal failure. PMID- 3019023 TI - Treatment of cancer of the lung. PMID- 3019022 TI - 99mTc(V)-dimercaptosuccinic acid scintigraphy for medullary thyroid carcinoma. PMID- 3019025 TI - Comparative study of kinin destroying activity of blood enzymes in respiratory sarcoidosis patients. AB - A total of 192 patients with active sarcoidosis of the stages I and II were examined. The state of the kinin system of the blood was studied on the basis of the values of the rate of kininogenesis and kinin-destroying activity of the blood. The rate of kininogenesis was assessed by the content of kininogen and prekallikrein in the blood and by kallikrein activity. Antikinin potential was measured by the values of the activity of carboxypeptidase N (KI), angiotensin converting enzyme (ACE) and total kininase activity (TKA) of the blood. KI and ACE were estimated by the rate of hydrolysis of synthetic substrates, TKA was estimated biologically by inactivation of the native substrate bradykinin. In sarcoidosis patients activation of the kinin system is detected which at early stages of the disease is accompanied by stimulation and later by depression of TKA. No correlation is found between values of KI, ACE and TKA. Possible mechanisms of these disturbances detected are discussed. PMID- 3019024 TI - N-sulphoconjugation of alicyclic, alkyl- and aryl-amines in vivo and in vitro. AB - Radioactive 35S in 3'-phosphoadenosine 5'-phosphosulphate was incorporated into alicyclic, alkyl- and aryl- amines in the presence of hepatic 105 000 g supernatants of female rats. 4-Phenylpiperazine, 4-phenyl-1,2,3,6 tetrahydropyridine (PTHP), 1,2,3,4-tetrahydroisoquinoline, N-methylbenzylamine, desmethylzotepine and desmethylzimeldine showed the highest conjugation with 35SO3 among the amines tested. Incorporation of 35SO3 into alicyclic and alkyl amines was higher at pH 10.0 than at pH 7.4 but the incorporation into arylamines was the opposite. A greater amount of 35SO3 was incorporated into the secondary alkylamines than the corresponding primary amines. Radioactive reaction products were identified as N-sulphoconjugates of amines by comparison on t.l.c. with synthetic authentic compounds. Reaction products of desmethylimipramine (DMI) and PTHP in vitro were isolated as their sulphoconjugates, identified by comparison of field desorption mass spectra, u.v. spectra, retention time on h.p.l.c. and RF values on t.l.c. with synthetic standards. DMI N-sulphonate and PTHP N-sulphonate were detected in the body of female rats treated orally with DMI and PTHP, respectively. These results indicate that N-sulphoconjugation is a common metabolic pathway of alicyclic, alkyl- and aryl-amines in vivo and in vitro. PMID- 3019026 TI - [Embryonal carcinoma of the lung--a contribution to the differential diagnosis of pulmonary coin lesions]. AB - The unusual case of an embryonal cell carcinoma of the lung mimicking pulmonary coin lesions on chest radiography is reported. The differential diagnostic problems and theories of genesis of the tumor are discussed. PMID- 3019027 TI - [Acquired immunologic deficiency syndrome (AIDS) and other sexually transmissible diseases with gastrointestinal manifestations]. AB - Various kinds of gastrointestinal symptoms are seen in connection with AIDS. Furthermore, because of special sexual practices, other sexually transmitted diseases are found, especially in the oral cavity, the oesophagus and the anorectum. Whereas in most of the cases infections can be treated successfully under a strict drug regimen, the treatment of malignant lymphomas or Kaposi's sarcoma is problematic. When the digestive tract is affected by the latter diseases, treatment is normally restricted to symptomatic measures. In the future, gastroenterologists will be faced with the above mentioned problems more frequently. PMID- 3019028 TI - [AIDS--Crohn disease as a diagnostic error. Case report]. AB - A twenty six year old homosexual male with the diseases Hepatitis B, Lues, Gonorrhoea, ascending myelitis, lymphadenitis got additionally a chronic inflammatory bowel disease, which first was discussed as Crohn's disease. Two "attacks" were "successfully" treated with Methylprednisolone. The patient died twelve month later in the follow of a pneumocystitis carinii pneumonia. The cause of the symptoms of the chronic-inflammatory bowel disease was a cytomegalovirus infection with acquired immune deficiency-syndrome. PMID- 3019029 TI - [Multiple generalized glomus tumors. Morphologic and biochemical findings]. AB - A 54 year-old woman developed approximately 150 generalized glomus tumors since she had been 23 years old. Painful tumors showed more glomus cells than those being not painful. Furthermore, the patient suffered from persisting congenital capillary hemangioma (strawberry mark). Histological examination revealed that glomus cells were located in the vessel walls. The large amount of Dopa found in the glomus tumor tissue supports the assumption that it may be innervated by adrenergic efferent nerves. PMID- 3019030 TI - [Psoriasis in AIDS--remission with high dosage co-trimoxazole therapy]. PMID- 3019032 TI - [Functional reorganization of the chemosensitivity of central neurons]. PMID- 3019031 TI - Retention and egg production of Microphallus pygmaeus in mice: the influence of the adrenal cortex. AB - Fewer adult Microphallus pygmaeus are recovered from the small intestine of adrenalectomized male mice than from sham-operated counterparts. Preinfection intraperitoneal injection of 2 IU or 4 IU ACTH daily for 7 days increases the initial primary worm burden in male mice. Preinfection intramuscular injection of 325 micrograms corticosterone daily for 7 days increases the initial worm burden and alters the subsequent rate of decline and duration of the primary infection. However, egg production is unaffected. The changes in parasite numbers are attributed to the immunosuppressive action of corticosterone. PMID- 3019033 TI - [Elements of theoretical neurophysiology]. PMID- 3019034 TI - [Assessment of indices of hemodynamics and hormonal background as criteria for the effectiveness of different variants of prolonged epidural analgesia postoperatively]. PMID- 3019035 TI - [A physical map of the DNA of streptococcal bacteriophage CI]. PMID- 3019036 TI - [Neutrophil chemotaxis and its role in the pathogenesis of dermatoses]. PMID- 3019037 TI - Genome organisation of the FBR-osteosarcoma virus complex: identification of a subgenomic fos-specific message. AB - The FBR murine virus complex together with the FBJ murine virus complex are known to be bone tumor inducers in newborn mice. Both transforming viruses have transduced c-proto-fos-derived sequences in their genome. FBR-MuSV was molecularly cloned as a biologically active 10-kbp EcoRI fragment from non productively transformed rat embryo fibroblasts into Charon phage 4A (lambda MOL503) and subsequently subcloned in plasmid pBR322 (pMOL503). Its natural associated helper FBR-MuLV, excized as an internal 8.2-kbp PstI proviral DNA fragment from chronically infected NIH/3T3 cells, was cloned into the unique PstI site of pBR322. Comparative analysis of the restriction maps of FBR-MuSV and FBR MuLV together with the electron microscopic analysis of heteroduplex DNA molecules formed between both molecular clones suggested that FBR-MuLV is the parental virus of FBR-MuSV. fos- and fox-specific DNA hybridisation probes identified a genomic sized 3.3-kb mRNA and a subgenomic 2.2-kb messenger RNA. Using a 5'-gag hybridisation probe, only the genomic 3.3-kb RNA molecule was detected, demonstrating that a donor splice site is present upstream of the gag sequences and used to generate the fos-specific 2.2-kb subgenomic mRNA. PMID- 3019038 TI - Polypeptides encoded by varicella-zoster virus unique short sequences. AB - The SalI-I and K DNA fragments which lie within the unique short sequences (Us) and contain a portion of the inverted repeat sequences (IRs/TRs) of varicella zoster virus (VZV) DNA were cloned in an in vitro transcription vector system (pGEM-2). RNA was transcribed from both strands, translated in vitro and analyzed by SDS-PAGE. The results showed that SalI-I and K each codes for three primary translation products. Polypeptides with Mrs of 19,000 (19K), 47K, 93K/90K are encoded by SalI-I and polypeptides of 12K, 19K, and 50K are encoded by SalI-K. These results are consistent with the predicted genetic expression of the VZV SalI-I and SalI-K DNA fragments. PMID- 3019039 TI - [Participation of GABA in mediating the effects of ethanol in rats with differing susceptibility to the development of experimental alcoholism]. AB - The influence of ethanol and of GABA receptors blocker bicuculline on recovery cycles of primary response of the sensorimotor cortex was studied in rats with strong and weak inclination to development of experimental alcoholism. It is found that in rats of the first group, inhibition in the cerebral cortex was weakened in comparison with the rats of the second group. Ethanol in non-narcotic doses intensified the inhibitory processes and its effects could be prevented or suppressed by bicuculline. The conclusion is made about GABA participation in mediation of ethanol effects on inhibitory processes in the cerebral cortex. PMID- 3019040 TI - Epstein-Barr virus and malaria in relation to Burkitt's lymphoma in Papua New Guinea. PMID- 3019041 TI - Adenocarcinomas of the small intestine in golden hamsters. PMID- 3019043 TI - [There will exist no interdependence between bovine leukosis and infectious bovine rhinotracheitis]. PMID- 3019042 TI - [Disinfection of sewage sludge by aerobic thermophilic treatment]. AB - Thermal stabilization of sewage sludge, performed in a pilot plant under aerobic conditions, resulted in microbiological and virological decontamination. A minimum temperature of 50 degrees C was necessary to inactivate coliform bacteria, E. coli and Salmonella sp. The rate of inactivation of these bacteria and enteric viruses was dependent on the concentration of microbes in the raw sludge, the temperature and the detention time of the sludge. At a temperature of 56 +/- 1 degree C, which was achieved by autogeneous heating of the processed sludge, pathogens were usually inactivated after two hours, but the time required for inactivation was shorter when the temperature was higher. PMID- 3019044 TI - [Detection of antibodies to Orthopoxvirus cameli in the sera of East African dromedaries with ELISA]. PMID- 3019045 TI - Isolation and identification of a poxvirus from a domestic cat and a human contact case. PMID- 3019046 TI - Reduction of rotavirus-, coronavirus- and E. coli-associated calf-diarrheas in a large-size dairy herd by means of dam vaccination with a triple-vaccine. PMID- 3019047 TI - [Components of the outer membrane of Yersinia pseudotuberculosis and their role in the pathogenesis of pseudotuberculosis]. AB - The pore-forming protein, lipopolysaccharide-protein complex and lipopolysaccharide of Y. pseudotuberculosis outer membrane have been shown to participate in the penetration of the bacteria into the cells of the macroorganism, to produce a toxic effect on these cells and to enhance the ingesting activity of macrophages in small doses, while suppressing it in large doses. When introduced parenterally, protein induces a more pronounced clinical picture of specific reactive hepatitis in experimental animals and greater changes in their kidneys than the lipopolysaccharide--protein complex. PMID- 3019048 TI - [West Nile fever (West Nile encephalitis)]. PMID- 3019049 TI - Influence of hormone treatment applied or begun in different phases of the cell cycle on hormonal imprinting in Tetrahymena. AB - Primary exposure to a hormone (hormonal imprinting) alters--in the case of the Tetrahymena increases--cellular response to re-exposure(s) to the same hormone. The intensity of hormonal imprinting depends on the phase of the cell cycle in which the primary exposure has taken place. The effect of imprinting was greater on the cells exposed to the hormone in phase G1 than on those exposed in phase S or G2. The response pattern of the progeny generations corresponded to that of the primarily exposed (imprinted) ancestor cell, irrespective of their own pre exposure in phase G1, G2 or S of their cycle. PMID- 3019050 TI - Influence of low and high temperature on diiodotyrosine imprinting in Tetrahymena. AB - Hormonal imprinting takes place at the primary interaction between target cell and hormone, and alters cellular response to the hormone for lifetime (at the unicellular level in many subsequent generations). Imprinting induced in Tetrahymena cells by diiodotyrosine at the optimum temperature of 25 degrees C took effect on re-exposure to the hormone at 25 degrees C and 15 degrees C, but failed to take effect if the cells were first exposed to the hormone at 15 degrees C or 32 degrees C. PMID- 3019051 TI - Superoxide anion, superoxide dismutase and lipid peroxidation in the murine macrophage cell line C4M phi. AB - A remarkable increase in the production of superoxide radicals and SOD activity was measured in suspension of the murine macrophage cell line C4M phi treated with Lentinan (4-10 X 10(3) micrograms/5 X 10(6) cells). In activated macrophages the decrease of lipid peroxidation could be interpreted as a consequence of enhanced SOD activity. PMID- 3019052 TI - Growth hormone-releasing factor does not stimulate phosphoinositides breakdown in primary cultures of rat and human pituitary cells. AB - It has been reported that rat growth hormone releasing factor (rat GRF-43), similarly to the two human GRFs (GRF-40 and 44) stimulates adenylate cyclase activity in pituitary cells. Controversial findings have been presented by two different groups on the action of GRF on phosphoinositides (PI) metabolism, a phenomenon linked to Ca-- mediated intracellular mechanisms. In the work to be reported, we evaluated the accumulation of inositol phosphates induced by GRF exposure in primary cultures of rat and human pituitary cells. Addition of rat GRF-43 to rat pituitary cells at doses up to 1 microM had no effect on inositol phosphates accumulation, while already at a dose as low as 0.05 nM it increased growth hormone secretion in the incubation medium significantly. In the same cell system, TRH, a known activator of PI breakdown, significantly increased [3H]inositol phosphates. In primary cultures of human somatotrophs from acromegalic subjects as in rats, addition of hpGRF-40 and also of TRH did not elicit any modification in the accumulation of [3H]inositol phosphates. Consistent with in vivo findings, both peptides induced a significant release of GH in the medium. Our results show that the GH releasing effect of GRF does not involve the hydrolysis of phosphatidylinositol in normal rat as well as in tumoral human somatotrophs. In addition it appears that the anomalous response of TRH on adenomatous cells from acromegalic patients is differently mediated in respect to the action of the tripeptide on normal lactotrophs and thyrotrophs. PMID- 3019053 TI - The relative importance of glucocorticoids and mineralocorticoids in the development of adrenal regeneration hypertension in rats. AB - The adrenocortical tissue which regenerates after adrenal enucleation, and contralateral uninephrectomy and adrenalectomy, resembles histologically zona fasciculata tissue which normally synthesises glucocorticoids. However, increases in blood pressure after enucleation (adrenal regeneration hypertension-ARH] were preceded by a rise in exchangeable body sodium similar to that found with mineralocorticoid-induced hypertension (e.g. DOC/salt rat model). Glucocorticoid involvement in ARH rats was tested, firstly by infusing dexamethasone into control and ARH rats to see whether ACTH suppression would lower blood pressure by reducing adrenocortical activity and, secondly, by infusing dexamethasone into rats with intact adrenals to see whether conditions for ARH (i.e. uninephrectomy and/or saline consumption) pre-disposed rats to the hypertensinogenic properties of glucocorticoids. Low-dose dexamethasone infusions (10 micrograms/day for 5 days) in ARH rats did not affect blood pressure but in control animals caused a significant (P less than 0.01) increase from 128 +/- 3 to 151 +/- 5 mmHg. Corticosterone, 18-hydroxycorticosterone and deoxycorticosterone plasma concentrations were suppressed in both groups by dexamethasone treatment; plasma renin concentrations were lower in ARH rats than in controls. Uninephrectomy or 1% NaCl as drinking fluid did not affect the blood pressure rise induced by sc infusion of 10 micrograms dexamethasone/day for 14 days in rats with intact adrenals. The temporal relationship between blood pressure changes and exchangeable body sodium in ARH rats resembles that in mineralocorticoid-induced hypertension. Glucocorticoid, unlike mineralocorticoid, induced hypertension is not affected by a reduction in renal mass or increased sodium intake. PMID- 3019054 TI - Effects of progesterone induced postmaturity on CBG and pituitary-adrenal activities in the rat foetus. AB - CBG and pituitary-adrenal activities were investigated in intact rat foetuses, in newborns spontaneously delivered by vaginal way and in postmature foetuses from mothers with delayed parturition caused by daily progesterone injection from day 20 of gestation. The postmature foetuses had lower body weights and higher adrenal weights on day 22, 23 and 24 of gestation than newborns of the same conceptional age. The corticosterone binding capacity of the plasma as well as the binding capacity of CBG for corticosterone decreased in intact foetuses for the last 3 days of gestation and stayed very low in pups from day 0 to day 8 postpartum. These parameters decreased more slowly in postmature foetuses; however, the differences between the latter and intact foetuses or newborns were not statistically significant. Similar evolution occurred in intact pregnant and suckling females as well as in females with prolonged gestation. The fall in CBG activity in normal rat pups and the subsequent rise in free steroids could explain a sharp decrease in plasma ACTH levels as well as the drop in adrenal and plasma corticosterone concentration. In foetuses with prolonged gestation, the same phenomenon did not occur. Stress conditions produced by maintaining growing foetuses in utero and the development of severe jaundice maintained high ACTH levels. In contrast, the fall in adrenal and plasma corticosterone concentrations in spite of the high level of circulating ACTH could be mainly due to the progesterone inhibition of the steroidogenic activity of the foetal adrenals. PMID- 3019055 TI - Effects of forskolin, luteinizing hormone and prostaglandin F2 alpha on isolated rat corpora lutea. AB - The diterpene forskolin increased in a dose-dependent way cyclic AMP (cAMP) accumulation in isolated corpora lutea from immature rats injected with an ovulatory dose of pregnant mare's serum gonadotropin (PMSG). The cAMP increase was significant already after 1 min of incubation with forskolin, and cAMP continued to rise to a maximum at about 30-60 min, with a clear decrease after 240 min of incubation. The forskolin effect was more pronounced in very young corpora lutea (1-day-old) than in older corpora lutea. There was a clear discrepancy between the marked effect on cAMP accumulation by forskolin and the steroidogenic response when compared with the corresponding effects of LH. Forskolin also increased progesterone production, but this effect was marginal compared with that of LH. Prostaglandin F2 alpha (PGF2 alpha) did not influence the forskolin stimulated cAMP increase. PGF2 alpha has previously been shown to inhibit the stimulatory effect of LH on cAMP formation in this type of corpora lutea. The fact that PGF2 alpha did not inhibit the forskolin stimulated cAMP production indicates that PGF2 alpha does not act directly on the adenylate cyclase. PMID- 3019056 TI - Affinity to lectin, biological and immunological characteristics of human chorionic gonadotropins from pregnant women and trophoblastic tumour patients. AB - To compare the affinity to lectin of human chorionic gonadotropins (hCG) from pregnant women and trophoblastic tumour patients, a small amount of urine was fractionated by a lentil lectin (LcH) affinity chromatography. The LcH-bound fractions were eluted with 2% mannoside solution, and each fraction was assayed for hCG activities by radioimmunoassay. In pregnant women, more than 90% of hCG immunoactivity in urine was bound to the LcH column and eluted from it, whereas 8 to 26% and 37 to 51% of the activity were not adsorbed to the affinity column and were recovered in the LcH-unbound fraction in the patients with hydatidiform mole and choriocarcinoma, respectively. These results suggest that LcH affinity chromatography of urinary hCG contributes to differential diagnosis between pregnancy and trophoblastic tumours. To characterize the properties of hCG from the urine of choriocarcinoma patients, with or without the LcH affinity, hCG activities in both LcH-unbound and LcH-bound fractions were measured by in vivo and in vitro bioassays and each of the activities was compared with the activities measured by radioimmunoassay. The results showed that the LcH-unbound fraction contained hCG molecules defecting to induce the biological activity of hCG in vivo. PMID- 3019058 TI - Dose and time effects of treatment with low doses of a LRH agonist on testicular axis and accessory sex organs in rats. AB - LRH and its agonists have been shown to exert both stimulatory and inhibitory effects on testicular function. In the present study, the dose and length of treatment were tested to determine the appearance of the stimulatory and inhibitory effects of LRH agonist on testicular axis including the three levels. Two doses of an agonist of LRH, 40 and 100 ng/100 g body weight (buserelin, 'agonist'), were administered daily for 1 to 15 days to adult male rats. Control rats received the vehicle only. On day 1, 2, 4, 8 and 15 of treatment, the pituitary, testicular and peripheral levels (weight of accessory sex organs and androgen receptors in ventral prostate) were tested 6 h after the last injection. For the 15 days of treatment with both doses, a stimulatory effect of the 'agonist' was observed on LH and FSH release. A short exposure (1-2 days) to the low dose of the 'agonist' had a stimulatory effect on the density of LH/hCG testicular receptors (326 +/- 49 vs control 185 +/- 21 fmol/mg protein, mean +/- SEM), on the weights of seminal vesicles and ventral prostate and exposure to both doses led to high plasma testosterone levels (13.8 +/- 0.5 and 13.7 +/- 0.7 ng/ml, respectively, vs control 2.6 +/- 0.3 ng/ml), and to an increased density of nuclear androgen receptors in the ventral prostate (142 +/- 9 and 144 +/- 15 fmol/mg protein respectively vs control 97 +/- 12 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019057 TI - Preparation and properties of human chorionic gonadotropin antagonist for biological studies: antifertility effects in the female rat. AB - We compared the effects of treatment of clinical grade hCG powders with anhydrous HF (hydrogen fluoride) or TFMS (trifluoromethane sulfonic acid). HF treatment yielded stable product (DG-hCG), which had desirable antagonistic activity in mouse Leydig cells. Binding of HF-hCG to ovarian granulosa cell FSH receptors was less than 5% as compared to purified ovine FSH. As LH/hCG receptor specificity was not significantly compromised crude hCG could be directly used in obtaining large amounts of the antagonist. The effects of antagonist (DG-hCG) and agonists crude hCG and purified hCG were evaluated in pregnant rats. When administered between days 1 to 5 of pregnancy, crude DG-hCG inhibited serum progesterone and oestradiol levels and implantation. The effect was dose-dependent. However, both crude hCG and purified hCG elevated progesterone level and partially inhibited implantation (up to about 40%). In the post-implantation period (days 8-11) crude DG-hCG treatment induced abortion due to a decrease in circulating progesterone and oestradiol levels. The agonists, crude hCG and purified hCG, on the other hand, elevated both steroid levels in serum and induced partial termination of pregnancy (up to 50%). During the second half of pregnancy, when luteotropic support of LH becomes unnecessary in the rat, crude DG-hCG (antagonist) had no effect on parturition. However, crude or purified hCG caused delay in parturition by sustaining high level of progesterone in circulation. Our data demonstrate that the antifertility effects of crude DG-hCG are more potent and consistent than the administration of either crude or purified hCG in the pregnant rat. PMID- 3019059 TI - Neural functions of TRH. PMID- 3019060 TI - CRF: its regulation of ACTH and pro-opiomelanocortin peptide release and its extra hypothalamic occurrence. AB - CRF-41 and vasopressin are the major part of the hypothalamic complex responsible for the release of ACTH. The potentiated response of the corticotroph to mixtures of the peptides and their co-localization in the same neurosecretory vesicles would indicate their obligatory co-secretion in many stress situations. The occurrence of CRF at peripheral sites especially in the placenta and in the plasma of women in their third trimester of pregnancy is suggestive of further roles for this peptide although care should be exercised in interpreting simple immunohistochemical staining in certain tissues. When ACTH is released from the pituitary several other peptides which form part of its precursor are co released. The effect of these peptides on the adrenal gland and the way their activity is expressed has led to a post-secretional proteolytic control mechanism being proposed. PMID- 3019061 TI - CRF and catecholamines; their place in the central and peripheral regulation of the stress response. PMID- 3019062 TI - LHRH, the role of LHRH (agonists) in the regulation of gonadal function. AB - Studies which have contributed to the present knowledge about peripheral effects of LHRH agonists are reviewed and compared with studies on the mechanism of effects of LHRH on the pituitary gland. It is concluded that LHRH or LHRH agonists can directly stimulate or inhibit the activity of gonadal cells from rats but not from man. Effects of LHRH on other human or rat peripheral tissues have been observed. LHRH- or LHRH-like molecules can be isolated from rat testes, but there is not enough evidence to consider LHRH- or LHRH-like peptides as general and important paracrine factors for regulation of gonadal function. On the other hand studies with LHRH agonists have stimulated investigations on cell cell interactions in the gonads and data have been collected which show that peptides which are less or non-related to LHRH can modulate functional properties of gonadal cells via pathways not employed by gonadotropins. PMID- 3019063 TI - Platelet-activating factor--a powerful lipid autacoid possibly involved in microangiopathy. AB - Microvascular injury includes diffuse endothelial and periendothelial damage and increased leukocyte/platelet-endothelium interactions including cell adhesion and liberation of powerful autacoids, growth and coagulation factors, all capable of causing vessel wall injury. Platelet-activating factor (PAF) is an ether-lipid mediator of inflammation, produced by and active on both platelets and endothelial cells (EC) at nanomolar concentrations. Its structure, biosynthesis and origin as well as some regulatory properties of the related enzymes are considered. The in vivo effects of PAF, as well as its action on selected cell types, such as platelets, neutrophils and EC, are briefly reviewed. PAF production may explain the intervention of platelets and neutrophils as well as increased vascular permeability in kidney, lung and retinal diseases. Pharmacological modulation of PAF production and activity is discussed with particular attention to the inhibitory effect of calcium dobesilate, a drug used in human diabetic retinopathy, on PAF production from the human endothelial cell line EA926. PMID- 3019064 TI - Evolution of localization of the reactions of adenosine triphosphatase (Mg++-ATP ase), 5'nucleotidase (5'nt), alkaline phosphatase (AP), and acid phosphatase (AcP) in developing rat testis. I. Physiological conditions. AB - The experiments were performed upon the rats aged 1, 4, 7, 15, 30, 45, 60, 90 d, and 1,5 a. The behavior of the following reactions was described: for adenosine triphosphatase stimulated by Mg++(Mg++-ATP-ase), for 5'nucleotidase (5'Nt), for alkaline phosphatase (AP), for acid phosphatase (AcP). The first 3 are markers of the transport enzymes in cells, and the 4th is a marker of lytic processes. It was estimated on the basis of the examined reactions that a full metabolic maturity of the gonad was revealed since the 45th d of post-fetal life. PMID- 3019065 TI - Blood coagulation-fibrinolytic system and endothelial cells. AB - Endothelial cells, under normal conditions, possess antithrombotic nature, whereas during damage, various components of the cell may initiate blood coagulation. These functions are greatly influenced and controlled by thrombin and plasmin through their direct actions or their formation in the blood coagulation-fibrinolytic system. PMID- 3019066 TI - Pyrimidine salvage enzymes in human tonsil lymphocytes: II. Purification and properties of deoxycytidine kinase. AB - Human tonsillar lymphocytes incorporate about 7-10 times more [3H]-deoxy thymidine than [3H]-deoxycytidine at the same extracellular nucleoside concentration, and both incorporations could be inhibited by 10(-5) M arabinosyl cytosine to 99%. On the other hand, the crude extract of these cells had about 10 times more deoxycytidine kinase than deoxythymidine kinase specific activity, as it was reported previously as well. Therefore, the differences in the labelling of these cells by [3H]-deoxycytidine and [3H]-thymidine cannot be simple due to the different levels of phosphorylating enzymes in the cells. The deoxycytidine kinase of human tonsillar lymphocytes was purified 38.6-fold by DEAE-Sephadex chromatography, and some kinetic and regulatory properties of the purified enzyme were investigated. Deoxycytidine kinase was eluted as a single peak from DEAE Sephadex column and also from Sephadex G-100 column indicating a molecular weight of about 60 000; no isoenzymes could be detected. During the purification procedure an increase of the enzyme activity was observed suggesting the inhibition of the enzyme in the cells. An apparent Km of 13 microM for deoxycytidine and Ki of 55 microM for arabinosyl-cytosine were found for the tonsillar deoxycytidine kinase. The enzyme was strongly inhibited by dCTP, but not by the other deoxyribonucleoside triphosphates. PMID- 3019067 TI - Regulation of beta-adrenergic sensitivity by guanine nucleotides in rat brain. AB - Binding of beta-adrenergic receptor by labelled dihydroalprenolol was inhibited in the presence of GTP or GppNHp in rat brain particulate fractions. The inhibition was suspended when the particulate fraction was pretreated at 30 degrees C for 20 minutes and washed 3 times. It is concluded that the inhibition was due to endogenous bound catecholamines. PMID- 3019068 TI - Interaction of immunosuppressive agents and human adenovirus infection in mice. AB - It was examined whether sensitivity of mice to human adenovirus infections is affected by immunosuppressive agents. Mice treated with the lymphotropic cytostatic dianhydrodulcitol (DAD) or antilymphocytic serum (ALS) did not become susceptible to human adenovirus type 12 infection, as the virus did not replicate -not even in its component forms--in the animals. On the other hand, the effect of both DAD and ALS on the lymphoid organs of mice was intensified by human adenovirus infection. In tissue cultures the reproduction of adenovirus was facilitated by DAD. PMID- 3019069 TI - Studies on enterotoxin of Shigella dysenteriae type 1. II. Systemic effects in rabbits of Shigella dysenteriae 1 enterotoxin. AB - The enterotoxin of Shigella dysenteriae type 1 is an acid and heat-labile protein. It induces a gut dilatory response and increases the levels of blood glucose, serum alkaline phosphatase and serum acid phosphatase in rabbits. PMID- 3019070 TI - Benign monoclonal IgAK gammopathy associated with polyneuropathy and dysautonomia. AB - The first case of benign IgAK monoclonal gammopathy associated with peripheral neuropathy is described. Dysautonomia is an unusual, yet prominent, manifestation of neuropathy in this patient. Electrodiagnostic testing and nerve biopsy were compatible with demyelination and axonal loss. Myelin sheath, perineural, and endoneural interstitial tissue fixation of anti-IgA and anti-kappa light chains was demonstrated by direct immunofluorescence microscopy. Absorption studies utilizing human peripheral nerve myelin resulted in complete removal of the paraprotein band. Analytic procedures with myelin-associated glycoprotein and gangliosides, however, were negative. Based on these findings, an alternative etiology for this neuropathy is hypothesized. PMID- 3019071 TI - Spinal cord syndromes in the acquired immune deficiency syndrome. AB - Two patients with AIDS developed paraparesis. Neuropathological post mortem examination in one revealed cytomegalovirus polyradiculopathy, and in the second, vacuolar myelopathy which occurred in association with brain lesions resembling Marchiafava-Bignami Syndrome. PMID- 3019073 TI - Clinical and epidemiological features of keratoconus genetic and external factors in the pathogenesis of the disease. AB - Clinical and epidemiological features of keratoconus (KC) were studied in a series comprising all the 212 KC patients treated at Oulu University Central Hospital from 1964 to 1984. Altogether 294 keratoconus patients and relatives were examined ophthalmologically by the author. The prevalence rate of KC needing ophthalmic care was estimated to be 0.03% (75/260,000). The annual incidence was 0.0015% (75/260,000 per 20 years) and remained the same throughout the period studied. 62.7% (133) of the patients were male and 37.3% (79) female. 73% were aged 24 years or younger at the onset of symptoms, the mean age of the males at the first examination being 26.5 +/- 8.2 years, and that of the females 30.6 +/- 13.7 years. Corneal transplantation was carried out on 65 of the 144 patients coming from the area served by Oulu University Central Hospital. Familial KC was found in 19 of the 101 families investigated in the north of Finland (19%) and in 5 of the 58 from the south (9%). The higher frequency of familial KC in the north is probably due to the more pronounced effect of gene pooling in the larger families (mean family size 4.9 persons as compared with 3.5 in the south). The inheritance was found to be attributable to a dominant autosomal mode in 24 out of 28 multiple-case families (85%), the disease being inherited from the mother in 15 cases and the father in 9. Data on the order of birth of keratoconic children were obtained from 159 families. 169 out of a total of 688 children were affected (25%). If families with only one child were excluded, then 47 of the 149 first children (32%) and 44 of the 149 second children (30%) had KC. Thus the disease is characterized by incomplete penetrance and variable expressivity. 122 HLA-A,B,C antigen typings were performed in 18 multiple-case families and the HLA genotypes expressed as haplotypes. In 15 families with more than one child affected, 27 keratoconic children were noted to share the mutual haplotype with the affected parent, whereas 3 had inherited the mutual haplotype from the healthy parent (p less than 0.001). The HLA haplotype could thus serve as a marker for KC inside the family. Connective tissue symptoms and abnormalities were seen in 31 out of 46 KC patients (67%) and in 60 out of 122 first-degree relatives from the town of Oulu and its surroundings (49%).(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3019072 TI - Autonomic function tests in healthy controls and in terminal uraemia. AB - Healthy individuals were tested with a battery of autonomic function tests in order to make up a normal data base for a variety of parasympathetic and sympathetic effector organ tests. 16 non-diabetic uraemic patients were also tested. Pathological findings were common, especially in the reflexes of parasympathetic nerves. PMID- 3019074 TI - Oral rotavirus vaccination in breast- and bottle-fed infants aged 6 to 12 months. AB - Oral bovine rotavirus vaccine RIT 4237 was tested at two dose levels in 217 children between 6 and 12 months of age who received either breast milk or bottle milk (cow's milk or infant formula) before vaccination. Of the children 65% were initially seronegative for rotavirus ELISA IgG and IgM antibody. The full vaccine dose (10(8.3) 50% tissue culture infective doses, TCID50) induced seroconversion rates 81% and 86% and booster response rates 69% and 64% in breast and bottle-fed children, respectively. There was no difference in the vaccine response of infants receiving cow's milk or infant formula either. It is concluded that the RIT 4237 rotavirus vaccine at the dose level 10(8.3) TCID50 gives a satisfactory response in both breast- and bottle-fed children in this age group, but multiple vaccinations may be needed for maximal efficacy. PMID- 3019075 TI - Serum steroids and success of corticotropin therapy in infantile spasms. AB - Serum levels of 8 steroids and urinary cortisol excretion were determined in 10 infants before and during corticotropin therapy for infantile spasms. High serum dehydroepiandrosterone-androstenedione concentration ratio distinguished the 6 infants with good therapeutic response from the 4 with poor response (p = 0.001). No such distinction was obtained directly by any of the serum steroid levels, 24 hour urinary cortisol, or serum pregnenolone-progesterone concentration ratio. This suggests that the therapeutic effect of corticotropin may be mediated by steroid factors other than cortisol. Inhibition of the 3 beta-hydroxysteroid dehydrogenase system in zona reticularis might be beneficial during the corticotropin therapy. PMID- 3019076 TI - Incipient germ cell tumor in Sertoli-cell-only syndrome testis, accompanied with retroperitoneal teratocarcinoma and widespread metastases. AB - Incipient germ cell tumor in Sertoli-cell-only syndrome testis was examined in an autopsy case of retroperitoneal teratocarcinoma with widespread metastases. Although both testes of a 28-year-old man had clinically been small and free from tumor mass to palpation, histopathological examinations revealed a malignancy in the right testis with the appearance of Sertoli-cell-only syndrome. The left testis showed solely the histology of Sertoli-cell-only syndrome. The testicular malignancy consisted of undifferentiated, atypical germ cells mainly confined within approximately one-tenth of seminiferous tubules, and only one small cartilage nodule. Some tubules showed intratubular growth pattern suggestive of seminoma. A few syncytiotrophoblast-like giant cells occurred in the tubules. These findings seem to furnish substantial evidence to the concept that atypical germ cells are the origin of testicular germ cell tumors of different types. PMID- 3019077 TI - Subcellular characterization of the transferrin-transferrin receptor and iron accumulating system of established human erythroid and monoblastoid tumour cell lines. AB - The organelles in two human tumour cell lines in culture - U-937 and K-562 - involved in the receptor mediated endocytosis (RME) of transferrin (Tf) were studied after isolation from homogenates by density gradient separation. They were also studied by electron microscopy after labelling of living cells with Tf coated colloidal gold particles. Three different Tf containing fractions with densities 1.038 (plasma membrane), 1.040 (light endosomes) and 1.051 g/ml (heavy endosomes) were identified. Ultrastructural studies of the distribution of gold label indicated that Tf was present in structures normally involved in RME of other ligands, probably including the recently identified "compartment of uncoupling of receptor-ligand complex" (CURL). The endocytosed iron was found to be rapidly transferred into the cytosol, as shown by density gradient centrifugation. Isoelectric focusing analysis showed that the iron mainly became bound in ferritin. In the hemoglobin synthesizing cell line K-562, however, iron was also inserted into hemoglobin. The finding that heavy and light endosomes process Tf suggests that Tf follows the same route as other ligands, including the epidermal growth factor and low density lipoprotein, in a presumed prelysosomal pathway, despite the fact that Tf does not dissociate from its receptor. Our findings are thus consistent with the notion that an endosome system similar to that in other types of RME is responsible for the cleavage and separation of iron from the carrier Tf. PMID- 3019078 TI - Modulation of cholinergic transmission by opiate peptide and FMRFamide on identified neurons of Helix pomatia L. (Gastropoda, Mollusca). AB - Identified neurons regulating visceral functions in Helix pomatia reacted selectively to FMRFamide peptide applied to the membrane, some becoming depolarized others hyperpolarized. The effect of morphine and leu-enkephalin was similar to that of FMRFamide. The ACh-induced depolarization was decreased by FMRFamide, morphine, leu-enkephalin and naloxone on the neuron LPa5, while this modulatory effect was not observed on other neurons involved in the regulation of visceral functions. The opiate peptides and FMRFamide may play a modulatory role in molluscan CNS. PMID- 3019079 TI - Effect of prolonged restraint on glycogen content and activities of four enzymes of carbohydrate metabolism in the liver of rats. AB - Triphasic changes in glycogen content and activities of four enzymes of carbohydrate metabolism (glucose-6-phosphatase, phosphoenolpyruvate carboxykinase, hexokinase, and glucose-6-phosphate dehydrogenase) were studied in the liver of male Wistar rats exposed to 1, 5, 10, 15, 20, 30, 45, 60, 70 and 90 day movement restrain in pencil cases. It was assumed that these three phases corresponded to the alarm, resistance and exhaustion stages of Selye's general adaptation syndrome. In hypokinetic rats, however, a transition of the alarm reaction to the resistance stage was registered later, and hepatic glycogen accumulation was reduced in comparison with the standard pattern observed in chronic stress. PMID- 3019080 TI - Effect of specific opioid-receptor antagonist naloxone on rats with hereditary hypothalamic diabetes insipidus (Brattleboro strain) during acute hemorrhagic shock. AB - The development of an acute hemorrhagic shock in rats with hereditary diabetes insipidus (DI), lacking vasopressin, is very dramatic, compared to rats of the parent strain Long Evans (LE). After removal of 50% of the circulating blood for LE, and 30% for DI, the mortality for both LE and DI groups was 50%, the shock index being 0.049 and 0.028, respectively. Infusion of either vasopressin (107 mU/100 g) or naloxone (0.2 mg/100 g) in DI rats, prevented the progression of hemorrhagic shock into irreversible stage, and augmented survival up to 66% and 57%, respectively. The specific opioid antagonist naloxone exerted a therapeutic effect on rats with hemorrhagic shock by antagonism of opioid receptors, without influencing ACTH and aldosterone secretion in DI rats. This is another evidence for the role of beta-endorphin in the pathogenesis of hemorrhagic shock. PMID- 3019081 TI - The hypothalamic and neurohypophysial vasopressin and oxytocin content under various states of adrenergic transmission in dehydrated and subsequently rehydrated rats. AB - Rats dehydrated for 8 days and subsequently rehydrated were given intracerebroventricularly (i.c.v.) methoxamine hydrochloride (MX) or dihydroergotamine methanosulphonate (DHE), each in a daily dose of 10 micrograms dissolved in 10 microliter of 0.9% sodium chloride. A single dose of MX injected to normally hydrated animals increased the release of hypothalamic and neurohypophysial vasopressin but did not affect significantly the oxytocic activity in the hypothalamus as well as in the neurohypophysis. Under conditions of dehydration MX did not influence the hypothalamic vasopressin content but it stimulated the neurohypophysial vasopressin depletion. On the contrary, MX distinctly inhibited the decrease of hypothalamic and neurohypophysial oxytocin content in dehydrated animals. In rehydrated animals MX restrained some what the renewal of hypothalamic vasopressin and oxytocin storage but intensified this process in the neurohypophysis. A single dose of DHE decreased the vasopressin content in the hypothalamus as well as the oxytocin content both in the hypothalamus and neurohypophysis. Under conditions of dehydration DHE stimulated the depletion of hypothalamic vasopressin and oxytocin. On the contrary, DHE strongly inhibited the depletion of oxytocin in the neurohypophysis of dehydrated rats. DHE restrained the renewal of hypothalamic vasopressin and oxytocin stores as well as intensified this process in the neurohypophysis of subsequently rehydrated rats. PMID- 3019082 TI - Oxygen, ischemia and inflammation. PMID- 3019083 TI - Effects of chloride ion substitution on the frequency dependence and alpha autoinhibition of [3H]noradrenaline secretion in guinea-pig vas deferens. AB - Guinea-pig isolated vas deferens, in which the stores of noradrenaline (NA) had been labelled by preincubation with [3H]NA, was used to study the effects of substitution of chloride with other anions, on transmitter secretion evoked by electrical stimulation of postganglionic sympathetic nerves. Chloride was omitted from the Tyrode's solution and substituted with acetate, nitrate, methylsulphate, sulphate or HEPES. All substituents increased both the spontaneous and the stimulus-evoked fractional secretion of [3H]NA under control conditions. The effect was essentially the same at all frequencies of stimulation (1-8 Hz). In the presence of the alpha-adrenoceptor blocker phentolamine, which enhanced the secretion of [3H]NA in all media, the secretion was not further enhanced by chloride substitution. Chloride substitution reduced the depressing effect of exogenous NA on the secretory response. In conclusion, chloride ions may be required for full expression of alpha-adrenoceptor-mediated inhibition of [3H]NA secretion, but do not markedly influence facilitation by increased frequency of stimulation. PMID- 3019084 TI - Presynaptic inhibition of acetylcholine release. AB - High potassium (51 mM) has been shown to evoke release of acetylcholine ([3H]ACh and endogenous ACh) from cholinergic nerves in rat bronchial smooth muscle. The release of [3H]ACh was reduced by 85% when the Ca2+ concentration was changed from 2 to 0.1 mM. The veratridine-induced release was completely inhibited by tetrodotoxin, but tetrodotoxin did not reduce the potassium-evoked release. The muscarinic agonist, oxotremorine, reduced the potassium stimulated release of [3H]ACh, without affecting the basal release. In contrast, scopolamine substantially potentiated the potassium-evoked release. Adenosine had a dual effect in the rat bronchi. Adenosine inhibited the potassium-evoked release of [3H]ACh and this presynaptic effect of adenosine was antagonized by 8 phenyltheophylline. Adenosine also induced contraction of the bronchial smooth muscle and there was potentiation by adenosine of the ACh-induced contraction. The results indicate that cholinergic nerve terminals in the rat bronchi possess muscarinic receptors which inhibit the release of ACh. Adenosine may have analogous effects, e.g. presynaptic inhibition of transmitter release in addition to postsynaptic enhancement of bronchial smooth muscle contraction. PMID- 3019085 TI - Adenosine potentiates the effects of LH and isoproterenol on luteal cells but not on isolated intact corpora lutea. AB - Adenosine potentiated the stimulatory effect of luteinizing hormone (LH) in a dose-dependent way on the production of cyclic AMP (cAMP) in isolated cells from heavily luteinized rat ovaries and from individual rat corpora lutea of various ages (2- and 5-day-old). Such an effect has earlier been reported only for cells from heavily luteinized ovaries (Behrman et al. 1983). A similar potentiating effect of adenosine was now seen on catecholamine-stimulated cAMP production in isolated cells from 2-day-old corpora lutea. Adenosine did not, however, potentiate LH- or catecholamine-stimulated cAMP production in isolated intact corpora lutea. PMID- 3019086 TI - Increased beta I-adrenoceptor density after 6-hydroxydopamine pretreatment in rat colon and lung. AB - Estimation of beta-adrenoceptor-binding sites with 125I-(-)-pindolol in rat colon show a proportion of 30% beta I-adrenoceptors and 70% beta 2-adrenoceptors. Studies on the isolated colon strip have revealed a neuronal beta-adrenoceptor involved in the inhibitory response of colon motility to beta-adrenoceptor stimulation. In order to further characterize the beta-adrenoceptors in the colon, acute and chronic treatments with 6-hydroxydopamine were made. Both acute pretreatment of rats with 6-hydroxydopamine for 8 and 24 h (one intravenous injection) and chronic treatment for 3 days (implanted osmotic mini-pumps), reduced the noradrenaline tissue content by 90%, and successively increased the beta-adrenoceptor-binding sites from 14.2 to 21.7 fmol mg-I P-I in colon and from 158 to 240 fmol mg-I P-I in lung membranes. Displacement of the radiolabelled ligand by the selective beta-adrenoceptor antagonists, pafenolol and ICI 118.551 showed that the density of beta I-adrenoceptor binding sites was more than doubled, whereas the density of beta 2-adrenoceptor-binding sites was only marginally increased by chronic treatment with 6-hydroxydopamine. Thus sympathetic denervation by 6-hydroxydopamine treatment produced a selective increase in beta I-adrenoceptors in the rat colon. These results may indicate that stimulation of beta I-adrenoceptors in both colon and lung have a neuronal linkage. PMID- 3019087 TI - Generation and transmission of ovine ureteral contractions, with special reference to prostaglandins. AB - The pattern of spontaneous rhythmic contractions was studied in isolated preparations from calyx, pelvic, middle and distal parts of the sheep ureter. The frequency of contractions was highest in intrarenal specimens (13.3 +/- 0.8 contractions min-I). The regional difference in contractions is consistent with proximal dominant pacemaker cells. In isolated rings, indomethacin (10(-5) M) inhibited and finally stopped rhythmic motility. After stoppage prostaglandin PGF2 alpha (10(-6)-10(-5) M) promptly re-established contractions in a manner characteristic of each specimen, apparently according to the pre-existent dominant pacemaker (frequency and pattern). This was demonstrated in a frequency analysis which showed a highly significant correlation of pacemaker frequencies before and after indomethacin. Experiments using dual recordings from both ends of a longitudinal preparation (tandem mode) showed that co-ordinated contractile waves travelling from one end to the other could be initiated with PGF2 alpha. These results have been interpreted to indicate that prostaglandins in ureteral smooth muscle play a predominant role in co-ordinating intercellular impulse transmission for which gap junctions could be responsible. The presence of such structures, as clearly demonstrated by an ultrastructural study, lends support to this hypothesis. PMID- 3019088 TI - Effects of some aminoquinolines on spontaneous quantal acetylcholine release. PMID- 3019089 TI - Serum and cerebrospinal fluid antibodies to cytomegalovirus in schizophrenia. AB - Antibodies to cytomegalovirus (CMV) were determined in the serum and cerebrospinal fluid (CSF) by complement-fixing (CF), enzyme immunoassay (EIA), and enhanced virus neutralization test (EVNT), in acute unmedicated schizophrenic patients and neurological controls. An elevated level of CF antibody was observed in three serum specimens from the schizophrenic patients and in one control specimen. No CF antibody was present in the CSF samples of the two patient groups tested. By EIA none of the serum or CSF specimens was positive for IgM antibody to CMV. By EVNT, 17% of the schizophrenic patients exhibited a CSF/serum ratio greater than 2 SD, whereas the corresponding figure for the control group was 4% (P greater than 0.05). The role of CMV in the etiopathogenesis of schizophrenia is discussed in the light of the present and previous negative findings. PMID- 3019090 TI - Alcohol withdrawal and opiate withdrawal--similarities and differences. PMID- 3019091 TI - Replication and its regulation of ColE2 and ColE3. PMID- 3019092 TI - DNA replication of the resistance plasmid R100 and its control. PMID- 3019093 TI - Illegitimate recombination: role of type II DNA topoisomerase. PMID- 3019094 TI - Escherichia coli proteins involved in regulation of transcription termination: function, structure, and expression of the nusA and nusB genes. PMID- 3019095 TI - Negative control of transcription by cAMP and cAMP receptor protein in Escherichia coli. PMID- 3019096 TI - Structure and regulation of mammalian ribosomal RNA gene. PMID- 3019098 TI - Evolution and molecular architecture of copia-like transposons in Drosophila. PMID- 3019097 TI - Genetic control of protein secretion and localization. PMID- 3019099 TI - Mechanisms of stable plasmid inheritance. PMID- 3019100 TI - Compartmentation of hormone action in adult mammalian cardiomyocytes. PMID- 3019101 TI - A role for mitochondria in myocardial adenosine production. AB - The results from the previous work (Bukoski et al, 1983) and those presented here indicate that mitochondria from rat heart are capable of producing adenosine in concert with a sarcolemmal 5'-nucleotidase. In addition, and perhaps more importantly, these data also indicate that mitochondria can produce adenosine in the presence of inhibition of sarcolemmal 5'-nucleotidase. Given the current understanding of the regulation of mitochondrial respiration and thus oxygen consumption, mitochondria may be uniquely situated to sense increases in myocardial oxygen demand and respond to this with a feedback signal (adenosine) which can return the system to a state of balance. PMID- 3019102 TI - Energy compartmentation and active transport in proximal kidney tubules. PMID- 3019103 TI - Glycerol kinase deficiency: compartmental considerations regarding pathogenesis and clinical heterogeneity. PMID- 3019104 TI - Microcompartmentation at the mitochondrial surface: its function in metabolic regulation. PMID- 3019105 TI - Hormonal regulation of creatine kinase BB. PMID- 3019106 TI - Ambisense RNA genomes of arenaviruses and phleboviruses. PMID- 3019108 TI - Causes of polyneuropathy in the elderly. AB - Seventy-four patients over 65 years with electrophysiologically confirmed polyneuropathies were reviewed. Common causes were diabetes (27%), neoplasms (13%) and the Guillain-Barre syndrome (11%). In only 28% was no cause identified. PMID- 3019107 TI - Regulation of protein synthesis in virus-infected animal cells. PMID- 3019109 TI - Data to the intracellular effects of prostacyclin in rat gastric mucosa. AB - Using specific radioimmunoassay it seems that Prostacyclin has a strong effect on the cyclic nucleotide-content of the rat gastric antral mucosa. Shortly (1 min) after an intragastric application of 100 micrograms/kg Prostacyclin the cAMP and the cGMP-content showed a significant decrease. Investigating the antral and fundic mucosal calmodulin-levels, it seems that 15-30 mins after Prostacyclin application both tend to increase. It seems very probable that the primary intracellular effect of Prostacyclin in the gastric mucosa is directed to the so called 'second messenger system' but later Prostacyclin activates the calmodulin system too. PMID- 3019110 TI - In vitro and in vivo effects of piroxicam on rat polymorphonuclear responsiveness after induction of two acute non-specific inflammatory reactions. AB - The effect of piroxicam on rat polymorphonuclear leucocytes (PMN) has been studied in vitro and in vivo after the induction of two acute, non specific inflammatory reactions (pleurisies induced by calcium pyrophosphate crystals (CaPP) or isologous serum). An inhibition of chemotaxis by piroxicam has been demonstrated by two techniques, the filter and agarose assays in vivo and in vitro. An inhibition of random cell migration has been observed only at the higher drug concentration using agarose assay with CaPP-elicited cells. Piroxicam also inhibited superoxide anion generation and O2 consumption of CaPP- and serum elicited cells. These findings suggest that piroxicam may have a direct effect on PMN responses and that this activity could, at least in part, contribute to its anti-inflammatory properties. PMID- 3019111 TI - Feline polymorphonuclear leukocytes respond chemotactically to leukotriene B4 and activated serum but not to F-Met-Leu-Phe. AB - The chemotactic response of feline polymorphonuclear leukocytes (PMNs) to three types of chemoattractants was studied. Feline PMNs responded to leukotriene B4 as well as to agarose-activated autologous and homologous serum. However, no response was obtained to N-formylmethionylleucylphenylalanine (FMLP), and four similar peptides that activate the FMLP receptor (N formylnorleucylleucylphenylalanine, N-formylmethionylphenylalanine, methionylleucylphenylalanine, and pepstatin). Thus, feline PMNs are similar to equine, porcine, bovine and canine PMNs which also do not respond chemotactically to these peptides. PMID- 3019112 TI - Prostaglandins, pain, and inflammation. AB - The prevention of hyperalgesia by inhibition of prostaglandin synthesis is the most plausible hypothesis on the mechanism of NSAID action. This review also discusses other possibilities of controlling inflammatory hyperalgesia, including the development of peripherally acting opiates. These are obtained by reduction of the opiates' lipophilic properties. The drugs' undesired central effects are thus eliminated, while their peripheral analgesic action is preserved. Moreover, it appears that the adrenergic system is also involved in inflammatory hyperalgesia. This implies that beta-receptor blockers might be useful in eliminating the adrenergic component of inflammatory pain. Metamizol, in contrast to the NSAIDs, is effective even in manifest prostaglandin or sympathetic hyperalgesia. PMID- 3019113 TI - Intraosseous glomus tumor: report of a case and review of the literature. PMID- 3019114 TI - Technetium-99m pyrophosphate imaging in acute renal failure associated with nontraumatic rhabdomyolysis. AB - Technetium-99m pyrophosphate (Tc-PYP) imaging was performed in five patients with acute renal failure associated with nontraumatic rhabdomyolysis. Four patients had phencyclidine intoxication and one had viral pneumonia. During the acute phase, marked uptake of pyrophosphate was seen in all patients in several muscle groups, but always in the thigh adductors. The results show that phencyclidine intoxication can result in diffuse muscle uptake of Tc-PYP without overt evidence of muscle injury. Tc-PYP imaging may provide a clue to the cause of acute renal failure in patients with suspected rhabdomyolysis in whom elevations of serum creatine phosphokinase concentrations are equivocal. PMID- 3019115 TI - Psychiatric symptoms and brain tumor. AB - Psychiatric symptoms may be the only clue to the presence of a brain tumor. Careful physical, neurologic and psychiatric examinations will reveal the diagnosis. Affective and schizophrenia-like psychoses are related to dysfunctions of the right and left hemispheres, respectively. Lesions of the temporal lobes commonly cause depression. Psychotropic medications may improve symptoms in the presence of tumor. There is no clinical method of localizing or excluding a brain tumor by its psychiatric manifestations. PMID- 3019116 TI - Exercise radionuclide angiography in patients with mitral stenosis: value of right ventricular response. AB - We observed 26 patients with mitral stenosis and 19 normal volunteers with exercise gated radionuclide angiography. Although no differences were seen between normal subjects and patients with mitral stenosis at rest in left (LV) and right (RV) ventricular ejection fraction, significant differences were found for exercise change in ejection fraction for both ventricles, exercise time, exercise workload, and the percent change in LV end-diastolic, LV stroke, and RV end-systolic counts (ESC). Because nearly all of the normals (18/19) had a decrease in RVESC, patients with stenosis were divided into two groups according to whether RVESC increased or decreased. Significant differences were found between these two groups for age, New York Heart Association class, prevalence of atrial fibrillation, echocardiographic mitral valve area, and prognosis, that is, number undergoing catheterization and surgery. We conclude that exercise radionuclide angiography does yield information that has significant clinical and prognostic value in patients with mitral stenosis. PMID- 3019117 TI - Evaluation of air-purifying respirators for protection against toluene diisocyanate vapors. AB - Two brands of air-purifying organic vapor cartridges (Willson and Survivair) and a disposable respirator (3M) were evaluated for protection against toluene diisocyanate (TDI) vapors. The respirators/cartridges were tested by generating dynamic atmospheres of TDI at concentrations of 0.2 and 1.5 ppm or greater, which are substantially above the currently accepted exposure limits. The TDI atmospheres were generated by controlled and continuous evaporation and dilution techniques. The relative humidity of the final TDI atmosphere was maintained at 50%. For the testing of Survivair and Willson respirators, one cartridge was mounted on a stainless steel plate and placed inside an exposure chamber through which air was drawn unidirectionally at 32 L/min. Periodically, the air before and after the cartridge was monitored for TDI. In the case of the disposable, valveless 3M respirator, a breathing pump was used to simulate the breathing through the respirator. The TDI atmosphere was respired through the respirator at 28.8 L/min (24 cycles/min at 1.2 L/cycle). As before, the concentration of TDI was measured periodically before and after the respirator. There was no significant breakthrough (less than 0.5%) of TDI in any of the respirators tested for 40 hr at 0.2 ppm and for 20 hr at 1.5 ppm or higher concentration of TDI. The detection limits of the post-respirator TDI measurements ranged from 0.4 to 0.02% of the pre-respirator concentration. It is important to note that, at the present time, because the odor threshold for TDI is higher than the ceiling exposure limit (poor warning property), NIOSH and most of the respirator manufacturers do not recommend the use of air-purifying respirators in isocyanate containing environments. PMID- 3019118 TI - Prolonged hemodynamic effect of a slow-release nitroglycerin ointment. AB - Nitroglycerin (NTG) ointment has been shown to be effective in the treatment of angina pectoris and congestive heart failure. Its duration of action is usually 4 to 6 hours. This study presents data that show that a new slow-release NTG ointment produces hemodynamic improvement over at least 24 hours. Twenty patients with coronary artery disease were tested with serial gated equilibrium radionuclide ventriculography before and at various stages of continuous, once-a day use of slow-release NTG ointment and 4 days after cessation of therapy. NTG ointment significantly (p less than 0.005) decreased left ventricular end diastolic and end-systolic volumes both at rest (23% and 33%) and during handgrip exercise (22% and 32%) when examined after continuous usage of at least 24 hours. Ejection fraction increased 21% at rest, from 0.42 +/- 0.15 to 0.51 +/- -0.18, p less than 0.0005). The ratio of peak systolic pressure to end-systolic volume increased 85% at rest (p less than 0.05) and 54% during exercise (p less than 0.01). All values had returned to baseline 4 days after cessation of treatment. Thus, slow-release NTG ointment may be useful in the treatment of angina pectoris and congestive heart failure on a once-a-day basis. PMID- 3019120 TI - Relationship between changes in the histologic subtype of small cell carcinoma of the lung and the response to chemotherapy. AB - It has been documented that changes in the histopathologic subtype of small cell carcinoma of the lung (SCCL) may occur after chemotherapy. The significance of such changes with respect to response to treatment has not yet been studied. In a retrospective review of 25 patients, we correlated their response from chemotherapy with morphologic changes seen in subsequent histologic material. Eleven patients responded to therapy and 14 failed to respond. A difference in original histologic subtype was found in 10 (71%) of the nonresponders and in only two (18%) of the responders. The difference was statistically significant (p less than 0.05). We conclude that patients with "pure" SCCL in the initial biopsy specimen who fail to respond to chemotherapy are likely to have mixed or combined histologic subtypes in subsequent tissue specimens, although we cannot preclude their pre-existence. An attempt to search for different histologic subtypes is warranted in patients who do not respond to chemotherapy regimens considered to be efficacious in SCCL. PMID- 3019119 TI - Localization of the site of ventricular premature complexes by radionuclide angiographic phase imaging. AB - To investigate whether gated radionuclide angiographic phase imaging is useful for visually displaying the origin of ventricular premature complexes (VPCs), 82 patients were studied by gating only VPCs. The VPC "origin" by the scintigraphic method was defined as the area of earliest phase and was compared with that predicted by 12-lead electrocardiographic criteria in all patients and to invasive electrophysiologic mapping in 10. Separating the right ventricle into 3 and the left ventricle into 4 segments, the phase imaging method and the electrocardiographic criteria agreed as to ventricle of VPC origin in 69 patients (84%) and segment of origin within each ventricle in 46 (56%). When baseline ventricular wall motion was analyzed, the 2 methods agreed to the ventricle of VPC origin in 31 of 33 patients (94%) with normal wall motion, 20 of 23 (87%) with segmental wall motion abnormalities and 19 of 26 (73%) with diffuse wall motion abnormalities. Agreement between the 2 methods as to specific segmental localization of the arrhythmia focus was noted in 21 of 33 patients (64%) with normal wall motion, 11 of 23 (48%) with segmental wall motion abnormalities and 12 of 26 (46%) with diffuse hypocontractility. In the 10 patients with endocardial mapping studies, the phase imaging technique confirmed the segment of VPC origin in all 10; the electrocardiographic method was accurate in 8. Thus, gated radionuclide angiographic phase imaging methods may be of value in noninvasively defining the origin of spontaneous VPCs. The visual format allows ready interpretation of the arrhythmia origin, and there may be an advantage to this approach over electrocardiographic morphometric criteria. PMID- 3019121 TI - The resolution of phenotypic ambiguity in a case of human leukemia using cytogenetics and immunoglobulin gene rearrangement. AB - A 28-year-old woman presented with acute leukemia. Surface antigen analysis showed monoclonal kappa immunoglobulin distribution. Morphology was consistent with either acute lymphoblastic leukemia or acute promyelocytic leukemia, hypogranular variant. This dilemma was resolved by examining the leukemic blasts for immunoglobulin gene rearrangement. None were found, ruling-out involvement of the B-cell lineage in this malignancy. Cytogenetic analysis showed a clonal abnormality diagnostic of acute promyelocytic leukemia. Immunoglobulin gene rearrangement in cytogenetics are invaluable in the care of patients with acute leukemia and can resolve ambiguities that result from phenotypic analysis. PMID- 3019122 TI - Absence of mononuclear phagocyte antigens in malignant fibrous histiocytoma. AB - Nine malignant fibrous histiocytomas (MFHs) were tested for the presence of macrophage-associated antigens by an alkaline phosphatase-anti-alkaline phosphatase immunohistochemical method. One myxoid and eight pleomorphic storiform tumors were analyzed with eight antibodies reacting with separate antigens previously found on cells of mononuclear phagocyte lineage. Malignant cells did not react with any of the antibodies, while benign histiocytes within tumor were consistently labeled with all of the antibodies tested. While the malignant cells of MFH previously have been found to share some functional and morphologic properties with histiocytes, they do not possess the surface and cytoplasmic antigens that have been found on various macrophage populations. The malignant cells of MFH are fibroblastic or poorly differentiated mesenchymal cells rather than neoplastic histiocytes. PMID- 3019123 TI - Reactivity of serologic tests for the detection of antibody specific to cytomegalovirus. II. Latex agglutination and enzyme-linked immunosorbent assay. AB - Two hundred seven sera were assayed for antibody-specific for cytomegalovirus (CMV) by two enzyme immunoassays (EIAs; Bioenzabody, Litton Bionetics, and Abbott CMV Total Antibody EIA, Abbott Laboratories) and latex agglutination (LA; CMVScan, Hynson, Westcott and Dunning). The overall accuracy of the LA, Litton EIA, and Abbott EIA was 95.8%, 86.2%, and 88.6%, respectively. Although the Abbott EIA had a sensitivity of 98.8%, the specificity was only 35.5%. The positive predictive values of the LA, Litton EIA, and Abbott EIA were 99.4%, 95.9%, and 88.9%, respectively, while the negative predictive values of each of these tests were 81.1%, 56.2%, and 84.6%, respectively. PMID- 3019124 TI - Evaluation of three commercial screening tests for AIDS virus antibodies. AB - Three commercial enzyme-linked immunosorbent assays (ELISA) for acquired immune deficiency syndrome (AIDS) virus antibodies were evaluated using serum that had been characterized by an ELISA and western blot procedure developed at the University of California at Davis (UCD). Each of the commercial tests was more specific than the UCD ELISA, but the UCD ELISA was more sensitive in the detection of sera that lacked reactivity by western blot to the envelope glycoprotein (gp-41). The HTLV-III Bio-EnzaBead (Litton Bionetics, Charleston, SC) was less sensitive and specific than the Abbott HTLV-III EIA (Abbott Laboratories, North Chicago, IL) or the Virgo HTLV-III ELISA (Electro-Nucleonics Inc., Columbia, MD). Overall, 22.9% (57 of 250) and 51.0% of sera that were repeatedly (X2) positive by commercial screening kits tested at blood donor centers and clinical laboratories, respectively, were confirmed by western blot. These results indicate that the screening assays for AIDS virus antibodies are not equal in performance and that positive screening test results must be confirmed by a more specific test like western blot before results are released. PMID- 3019125 TI - Reye's syndrome. Salicylate metabolism, viral antibody levels, and other factors in surviving patients and unaffected family members. AB - To investigate possible etiologic factors for Reye's syndrome (RS), five survivors and their unaffected family members were studied. This study showed low salicylate levels among the patients with RS compared with siblings and parents when challenged with three doses of aspirin. Thus, the patients with RS, three to ten years after having had RS, exhibited normal or increased ability to metabolize aspirin. We found significantly higher antibody levels to influenza A and varicella among the patients with RS, further supporting the importance of these viral infections in the etiology of the syndrome. PMID- 3019126 TI - Acute Chlamydia trachomatis respiratory infection in childhood. Serologic evidence. AB - Serum samples from 184 infants and children whose blood was drawn during a clinic visit were tested for antibody to Chlamydia trachomatis, Epstein-Barr virus, and cytomegalovirus. Lifetime illness history was obtained from clinic records. Fifteen percent had anti-C trachomatis IgM antibody. Anti-C trachomatis IgM without IgG was significantly associated with upper respiratory tract syndromes within the 14 days prior to phlebotomy in 6- to 10-year-old patients. This association was not due to polyclonal activation from Epstein-Barr virus infection. A definitive study of chlamydial illness in children rather than infants appears to be indicated. PMID- 3019127 TI - Influence of progressive salt restriction on urinary bicarbonate wasting in uremic acidosis. AB - In steady state, the acidosis in the majority of 17 uremic patients was characterized by a persistent bicarbonaturia (FEHCO3 ranging between 0% and 17.65%). An NH4Cl loading test in 17 patients revealed two distinct groups: group A (n = 11) with complete disappearance of the urinary bicarbonate loss and a mean UpH of 5.39 +/- 0.10 at a PHCO3 level of 13.3 +/- 0.5 mEq/L; and group B (n = 6) with urinary acidification disturbances with a persistent FEHCO3 ranging between 1.06% and 3.15% and a mean UpH of 6.53 +/- 0.06 at a PHCO3 level of 13.5 +/- 0.7 mEq/L. Between the two groups, there were no differences in CCr, plasma Na, K, Cl, Ca, PO4, PCO2, and aldosterone levels. Calculation of the THCO3/TNa reabsorption ratio over a wide range of PHCO3 levels revealed no differences between the two groups. The mean levels of circulating PTH were significantly higher in group B compared with group A (40.1 +/- 10.8 mU/dL v 19.3 +/- 4.4 mU/dL; P less than .05), and the spontaneous steady-state FENa was more pronounced in group B than in group A (12.1% +/- 1.5% v 4.9% +/- 0.7%; P less than .05). Four patients from group B with a well-documented salt-losing nephropathy (FENa ranging from 10.20% to 15.10%) were submitted to a progressive dietary salt restriction over several weeks. At this stage, the four patients no longer had bicarbonaturia, and the urinary pH decreased to levels between 5.15 and 5.65 during NH4Cl-induced acidosis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019129 TI - DNA contamination in commercial restriction endonucleases. PMID- 3019128 TI - DNA "fingerprints" and segregation analysis of multiple markers in human pedigrees. AB - Tandem-repetitive DNA hybridization probes based on a putative human recombination signal detect multiple polymorphic minisatellite fragments in human DNA. The genetic complexity of the resulting individual-specific DNA "fingerprints" was investigated by studying a large sibship affected by neurofibromatosis and a more extensive pedigree segregating for two different hemoglobinopathies. The segregation of up to 41 different heterozygous DNA fragments from each parent could be analyzed in a single sibship, using two different repeat probes. Most of these variable DNA fragments could not be paired as alleles, to an extent which suggests that the DNA fingerprints are together derived from approximately 60 heterozygous loci (approximately 120 variable fragments), only a proportion of which can be scored in a given individual. Two or three of the DNA fragments detected by one probe showed tight linkage and may be derived from long minisatellite(s) that are cleaved to produce more than one polymorphic DNA fragment. Excluding allelic and linked DNA fragments, almost all remaining scorable fragments segregated independently, allowing up to 34 unlinked loci to be examined simultaneously. These loci are scattered over most or all of the human autosomes. Minisatellite probes are therefore suitable for rapid marker generation and can be applied to linkage analysis in human pedigrees. PMID- 3019130 TI - Sample-size considerations and strategies for linkage analysis in autosomal recessive disorders. AB - The opportunity raised by recombinant DNA technology to develop a linkage marker panel that spans the human genome requires cost-efficient strategies for its optimal utilization. Questions arise as to whether it is more cost-effective to convert a dimorphic restriction enzyme marker system into a highly polymorphic system or, instead, to increase the number of families studied, simply using the available marker alleles. The choice is highly dependent on the population available for study, and, therefore, an examination of the informational content of the various family structures is important to obtain the most informative data. To guide such decisions, we have developed tables of the average sample number of families required to detect linkage for autosomal recessive disorders under single backcross and under "fully informative" matings. The latter cross consists of a marker locus with highly polymorphic codominant alleles such that the parental marker genotypes can be uniquely distinguished. The sampling scheme considers families with unaffected parents of known mating types ascertained via affected offspring, for sibship sizes ranging from two to four and various numbers of affected individuals. The sample-size tables, calculated for various values of the recombination fractions and lod scores, may serve as a guide to a more efficient application of the restriction fragment length polymorphism technology to sequential linkage analysis. PMID- 3019131 TI - Linkage disequilibria between pairs of loci within a highly polymorphic region of chromosome 2Q. AB - A recently described region on chromosome 2q contains seven restriction fragment length polymorphisms (RFLPs) revealed by single-copy probes isolated from a 20 kilobase (kb) segment of a single cosmid insert. Analysis of six of these loci demonstrates modest amounts of linkage disequilibrium. This reflects the presence of a substantial number of different haplotypes in this chromosome region and indicates that the region could be used as one highly polymorphic locus. No consistent relationship is found between the amount of linkage disequilibrium and the physical distance between pairs of loci. For seven of the 10 pairs of diallelic loci studied, the observed disequilibrium can be attributed primarily to the absence of the minor haplotype from the population. These results suggest that, for small regions of the genome, factors such as mutation, genetic drift, and population admixture may have effects that outweight those of recombination. In addition, results are reviewed which show that estimates of linkage disequilibrium coefficients for tightly linked loci are very imprecise. Thus, the inference of gene order from linkage disequilibrium values must be regarded with caution. PMID- 3019132 TI - Determining the mode of inheritance of RFLP-associated diseases using the affected sib-pair method. AB - It is determined that the classical HLA sib-pair method can be used for the detection of linkage and determination of mode of inheritance of a disease when applied to non-HLA data, where attention is restricted to that subset of all parental matings that can produce affected sibs where unequivocal determination of haplotype sharing by descent values can be obtained. With the increasing use of restriction fragment length polymorphisms (RFLPs) to detect disease genes, it is predicted that this method will be applicable to many diseases. PMID- 3019134 TI - Subclinical cervico-spino-bulbar effects of lead: a study of short-latency somatosensory evoked potentials in workers exposed to lead, zinc, and copper. AB - Subclinical central and peripheral nervous system dysfunction among lead-exposed workers was studied by measuring short-latency somatosensory evoked potentials (SSEP) and maximal motor and sensory nerve conduction velocities (MCV and SCV) following stimulation of the median nerve at the wrist. The examinations were conducted in 20 gun-metal foundry workers exposed to lead, zinc, copper, and tin, with blood lead (BPb) concentrations of 16 to 64 micrograms/dl (mean, 42 micrograms/dl). The interpeak latency of SSEP in the cervico-spino-bulbar region [N9(Erb)-N13 latency] was significantly prolonged, and the MCV and SCV in the forearm were significantly slowed. Multiple regression analysis revealed that the yield of urinary lead following challenge with calcium disodium ethylenediamine tetraacetate (CaEDTA) and packed red blood cell volume were the major factors associated with the prolongation of SSEP latency in the cervico-spino-bulbar region. Similarly, the interpeak latency in the upper central nervous system (N13 N20 latency) was inversely related to the zinc concentration in erythrocytes; latency up to the Erb's point [N9(Erb) latency], which reflects conduction time in a long pathway of the sensory median nerve, was inversely related to urinary zinc level; the MCV and SCV in the palm were positively related to erythrocyte zinc concentration and plasma copper concentration, respectively. These findings suggest that the subclinical neurophysiological effects of lead occur not only in peripheral nerves but also in the central nervous system. It appears that zinc antagonizes the central and peripheral neurologic dysfunction caused by lead; similarly, copper antagonizes the peripheral sensory nerve dysfunction. PMID- 3019133 TI - Processing of types I and III procollagen in Ehlers-Danlos syndrome type VII. AB - The processing of types I and III procollagen was studied in skin fibroblast cultures from type VII A and B of the Ehlers-Danlos syndrome [EDS] and age matched controls. Synthesis of collagenous proteins was significantly increased in EDS type VII B, and the activities of prolyl-4-hydroxylase and galactosylhydroxylysyl glucosyltransferase were slightly increased in these cell lines, reflecting increased biosynthesis of collagen. The synthesis of collagenous proteins was close to normal in EDS type VII A cells. The synthesis of type III procollagen per cell was increased, as also was the ratio of immunoreactive type III procollagen to total collagen production. The activity of type I procollagen amino-terminal proteinase was decreased in skin fibroblasts of type VII A and normal in those of type VII B relative to cell protein or DNA. Type III amino-terminal proteinase activity was of a level found in normal cells when expressed relative to the protein or DNA, and the release of type III amino terminal propeptides was nevertheless not disturbed in these EDS type VII cell cultures. The results show that only the conversion of type I procollagen is defective in EDS type VII, and no general defect in procollagen processing can be found in EDS type VII as has been suggested in the case of dermatosparaxis, a connective tissue disorder in animals caused by disturbed procollagen conversion. PMID- 3019135 TI - Leukotrienes and other lipoxygenase products of arachidonic acid synthesized in the kidney. AB - Lipoxygenase products are synthesized in the kidney. Rabbit medulla and murine and human glomeruli produce 12- and 15-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE). Minor amounts of leukotrienes are formed under normal conditions, but it is likely that the resident renal cells are capable of synthesizing these metabolites. Rat glomeruli and papillae possess the enzymes necessary to process leukotriene C4 into leukotrienes D4 and E4. However, the enzyme activity of the papillae is masked due to the presence of an inhibitor detected in the 10,000 g supernate of the papillary homogenate. 12-HETE synthesis is markedly increased in glomeruli from rats with nephrotoxic serum nephritis and leukotriene B4 synthesis in glomeruli from rats with cationic bovine gamma-globulin-induced glomerulonephritis. In vivo consequences of the association between the resident glomerular cells and the bone marrow-derived cells have been studied in vitro in co-incubation experiments. Glomeruli release factors that stimulate the cyclo oxygenase and lipoxygenase pathways in macrophages. Co-incubation of glomeruli, platelets, and polymorphonuclear leukocytes results in the formation of 12,20 diHETE and an excess of 12-HETE. Lipoxygenase products, regardless of their origin, modify the renal functions. Leukotriene C4 binds specifically to rat glomeruli and human cultured glomerular epithelial cells. Leukotrienes C4 or D4 administered in vivo cause renal vasoconstriction and a decline in the glomerular filtration rate. In vitro, these two sulfidopeptide leukotrienes promote epithelial cell proliferation and produce mesangial cell contraction. The lipoxygenase pathway is also implicated in the attachment of macrophages to glomeruli and in the oxidative burst of glomerular mesangial cells during phagocytosis. The future use of specific inhibitors of the synthesis or antagonists of the lipoxygenase products, particularly the leukotrienes, should provide a tool for evaluating the role of these metabolites in renal diseases. PMID- 3019136 TI - Effects of acute angiotensin converting enzyme inhibition on renal blood flow in patients with stable congestive heart failure. AB - Renal blood flow was serially measured as the left renal vein blood flow using the continuous thermodilution technique during acute angiotensin converting enzyme inhibition in 20 patients with stable congestive heart failure. Eleven patients received captopril orally, and the remaining nine patients received enalaprilat intravenously. During the control period, left renal vein blood flow and cardiac output did not correlate closely (r = 0.57). Following administration of captopril or enalaprilat, stroke volume index increased from 20 +/- 7 to 25 +/ 8 ml/M2 (p less than 0.001), while pulmonary capillary wedge pressure decreased from 26 +/- 8 to 19 +/- 8 mm Hg (p less than 0.001). Left renal vein blood flow increased in all patients despite a consistent reduction in systemic arterial pressure. At peak effect, left renal vein blood flow increased from 295 +/- 86 to 443 +/- 122 ml/min (p less than 0.001), while mean systemic arterial pressure fell from 81 +/- 13 to 71 +/- 14 mm Hg (p less than 0.001). Thus, in patients with stable congestive heart failure, acute angiotensin converting enzyme inhibition, although decreasing substantially systemic arterial pressure, consistently enhances renal blood flow. PMID- 3019137 TI - Activation of cyclic AMP phosphodiesterase by phorbol and protein kinase C pathway. AB - Insulin (INS) stimulates, and diabetes inhibits, low Km cAMP phosphodiesterase (PDE). This mechanism, at least in part, accounts for the lowering of cyclic AMP levels in plasma and tissue of diabetic patients and animals. Phorbol, a tumor promoting agent known to act through protein kinase C and calcium translocation, exhibits a powerful effect stimulating PDE in rat adipose tissue. Nifedipine, a calcium channel blocker, inhibits insulin, but not phorbol stimulated PDE. These data demonstrate new effects of inositide diacylglycerol-Ca++ pathway components on PDE and suggest some common pathways of activation of low Km cAMP PDE through insulin and phorbol esters. PMID- 3019139 TI - Renal hypophosphatemic osteomalacia unmasked by hyperthyroidism. AB - A case of renal hypophosphatemic osteomalacia (RHO) that was unmasked by hyperthyroidism is presented. The patient presented at age 64 with pathologic leg fractures. There was no family history of osteomalacia or rickets. Initial evaluation revealed hyperthyroidism, which was treated with radioactive iodine. Despite control of thyroid function, the patient had recurrent pathologic fractures. Further evaluation revealed histologically proven osteomalacia and the biochemical findings of RHO: elevated serum alkaline phosphatase, decreased serum phosphate and tubular resorption of phosphate, and normal serum calcium, parathyroid hormone, and vitamin D levels. Other causes of osteomalacia were excluded. Treatment with phosphate and calcitriol reversed the osteomalacia. This case demonstrates that hyperthyroidism, and possibly other illnesses that affect vitamin D or bone metabolism, may unmask metabolic bone disease and that physicians should be alert for the subtle clinical and biochemical indicators of unrecognized metabolic bone disease in adults. PMID- 3019138 TI - Immunomodulation by bronchial lavage cells in normal individuals and patients with bronchogenic carcinoma. AB - The ability of bronchoalveolar lavage cells to facilitate lymphoproliferation to mitogen in a system which allows assessment of pulmonary alveolar macrophage accessory cell function was investigated. Bronchoalveolar lavage cells were obtained from healthy non-smokers and smokers and from patients undergoing diagnostic bronchoscopy. Lavaged cells were cultured with monocyte-depleted homologous blood lymphocytes obtained from healthy, young volunteers and stimulated with suboptimal (2 micrograms/ml) or optimal (20 micrograms/ml) concentrations of phytohemagglutinin. Mitogen responses of lymphocytes in all groups were related to the number of lavage cells added, increasing with 1:100 and 1:10 bronchoalveolar lavage cell to lymphocyte ratios and decreasing with 1:2 and 1:1 ratios. Lymphoproliferative responses observed in smoker and nonsmoker cultures were not different. In contrast, maximal proliferative responses of cell cultures from patients with epidermoid and small cell carcinoma were decreased compared with cultures from patients with adenocarcinoma or controls. These data show that pulmonary bronchial lavage cells from smokers and nonsmokers provide similar dose related augmentation and suppression of lymphocyte mitogenic responses. Furthermore, accessory cell function of lavage cell populations is normal in patients with adenocarcinoma, but depressed in patients with epidermoid or small cell carcinoma. PMID- 3019140 TI - Small cell lung cancer: a problem of tumor heterogeneity. AB - Small cell undifferentiated carcinoma represents a subtype of lung cancer that possesses biologic and clinical characteristics that make it significantly distinct from other forms. A major impact on the natural history of this disease has been accomplished during the past 15 years, including the potential for cure by non-surgical treatment modalities. Further progress in the management of this disorder has been impaired by a number of factors that appear to be inherent to the biology of the tumor and its clinical features. Analysis of initial clinical trials and more detailed examination of this tumor in vitro have permitted the elucidation of many barriers to curative outcome presently being evaluated at the laboratory and clinical levels. These include clear biologic and morphologic heterogeneity; problems with chemotherapy responsiveness including drug resistance; the potential for combining chemotherapy and radiation modalities; the re-examination of the role of surgical intervention in selected patients; and the need to deal with central nervous system dissemination of tumor cells. Further advances in this disease will be dependent on the successful integration of laboratory and clinical disciplines. PMID- 3019143 TI - Immunohistochemical localization of human chorionic gonadotropin, placental lactogen, and pregnancy-specific globulin in early placentation trophoblast: implications for evaluating trophoblastic differentiation in germ cell neoplasms. PMID- 3019141 TI - Extramammary Paget's disease of the vulva and anus: use of intraoperative frozen section margins. AB - Thirteen consecutive cases of vulvar Paget's disease treated by us between 1975 and 1984 underwent pathologic retrospective review. In the first group of eight patients having Paget's disease not involving the anal mucosa, the extent of disease was completely defined by frozen-section margins. Additional intraoperative resections were necessary in five of the eight. None had residual involvement on permanent sections and none had recurrences in 3 to 8 years of follow-up, although two died of unrelated causes. A second group of two patients early in the series had frozen sections to define some but not all of the margins. Both had positive perineal margins on permanent sections. One has required two subsequent revisions after 3 years of follow-up and the other has been free of disease during 9 years of follow-up. A third group of three patients all had anal mucosal involvement and all had associated mucinous adenocarcinoma of the rectum. Frozen sections actually discovered the presence of one of the three carcinomas. After appropriate radical operations, all three are alive and free of disease between 2 and 9 years of follow-up. It is concluded: patients with vulvar Paget's disease can reduce the need for subsequent operations with the use of frozen sections to define surgical margins; anal mucosal involvement of Paget's disease represents an ominous sign of underlying carcinoma. PMID- 3019142 TI - Treatment of cornual pregnancy with methotrexate: case report. PMID- 3019144 TI - Confirmation of HTLV-III virus in cornea. PMID- 3019145 TI - Subcellular distribution of calmodulin and its binding proteins within the rat submandibular gland. AB - The involvement of calmodulin (CaM) in rat submandibular gland mucin secretion was investigated in vitro using a dispersed cell preparation. The CaM antagonist trifluoperazine (TFP) inhibited mucin secretion in response to both isoproterenol and dibutyryladenosine 3',5'-cyclic monophosphate. The inhibitory concentrations of TFP were between 10 and 100 microM. One millimolar TFP was toxic to submandibular cells and resulted in decreased levels of cellular ATP and a significant release of lactate dehydrogenase. Determination of CaM levels via radioimmunoassay within various subcellular fractions indicated that the majority of the CaM within the rat submandibular cell was located within the cytoplasm. CaM binding proteins were also identified within these subcellular fractions utilizing a gel overlay procedure. The two major, specific CaM binding proteins present within rat submandibular cells were a 59-kDa cytosolic protein and a 47 kDa membrane-associated protein. PMID- 3019146 TI - Evidence for physiological regulation of myometrial gap junction permeability. AB - We have investigated whether direct intercellular communication between uterine smooth muscle cells of delivering rats is influenced by intracellular adenosine 3',5'-cyclic monophosphate concentration ([cAMPi]) and agents relevant to the hormonal control of pregnancy and parturition. The rate of diffusion of phosphorylated 2-[3H]deoxy-D-glucose (2-DG) in strips of longitudinal myometrium from rats in labor, indicated by the apparent diffusion coefficient for this molecule, was observed to be significantly reduced in tissues with elevated [cAMPi] (after treatment with dibutyryl cAMP, 8-bromo cAMP, forskolin, and theophylline) in the absence of any change in the area of gap junctions (GJs). Similarly, several agonists that elevate [cAMPi] in this tissue (e.g., isoproterenol, relaxin, carbacyclin, prostaglandin E2) also reduced 2-DG diffusion. These data suggest that the permeability of GJs in uterine smooth muscle may be regulated by [cAMPi] and physiologically relevant agonists. Control of GJ permeability may be important for the physiological regulation of intercellular communication and the extent of synchronous contractile activity in the uterine wall during pregnancy and parturition. PMID- 3019147 TI - Velocity of shortening and myosin isozymes in two types of rabbit fast-twitch muscle fibers. AB - The fast-twitch tibialis anterior muscle of the rabbit was stimulated (10 Hz, 8 h/day for 7 wk) to cause complete transformation of the fibers from type IIb to type IIa. The velocity of unloaded shortening of permeabilized single fiber segments dissected from control and chronically stimulated tibialis anterior muscles was measured by the slack test at 20 degrees C. The myosin isozymes in these segments were separated on pyrophosphate-containing polyacrylamide gels. Peptide mapping of the myosin chain was performed on the myosin bands cut from the gels. The velocity of unloaded shortening of the IIb fibers was significantly higher (2.50 +/- 0.09 fiber length/s; n = 6) than that of the IIa fibers (1.33 +/ 0.08 fiber lengths/s; n = 6). The two groups of fibers differed with respect to their alkali light chain complement, as assessed by nondenaturing gel analyses, and with respect to their myosin heavy chain complement, as demonstrated by peptide mapping. Thus two groups of fast-twitch muscle fibers that contain distinguishable myosin isozyme contents differ in their velocities of unloaded shortening by a factor of two. PMID- 3019148 TI - Vasopressin binding sites in toad bladder: studies with the photoaffinity analogue [Phe(p-N3)3]AVP. AB - Toad bladders were exposed to [Phe(p-N3)3]AVP (N3-AVP), an analogue of vasopressin with a photoreactive p-azido group in position three, in the presence and absence of ultraviolet (UV) light. Bladders exposed to the analogue in the presence of UV light showed an increase in membrane permeability to water, which persisted in spite of repeated and prolonged washout of analogue. In contrast, the hydroosmotic response induced by the analogue in the absence of UV light was readily reversed on washout. Aliquots of a broken epithelial cell preparation, derived from bladders that had been exposed to the analogue in the presence of UV light, bound less tritium-labeled vasopressin ([3H]AVP) than control aliquots that had been exposed to the analogue in the absence of UV irradiation or irradiated in the absence of the analogue. Membrane preparations that had not been photolabeled had specific binding sites for [3H]AVP in excess of 1,800 fmol/mg protein without evidence of saturation at a [3H]AVP concentration of 250 nM. Conversely, photolabeled membranes were saturated at a [3H]AVP concentration of 100 nM. The present studies demonstrate that a high proportion of [3H]AVP binding sites can be covalently labeled with N3-AVP and that at least some of these N3-AVP-bound sites are functional in triggering an increase in membrane permeability to water. PMID- 3019149 TI - Studies on final differentiation of rat renal proximal tubular cells in culture. AB - The ontogeny of effective Na and K permeability has been studied in renal epithelial cells isolated from the outermost superficial cortex from adult and young (10-15 days) rats. The cells were cultured for 2-4 days and exhibited phloridzin-inhibitable alpha-methylglucoside uptake, characteristic of renal proximal tubular cells (RPTC). Intracellular concentrations of K, Na, Cl, and P and kinetics of changes in intracellular ionic content after inhibition of Na-K ATPase with 1 mM ouabain (or by incubation in low-K medium) were measured in individual cells using electron probe analysis. Intracellular concentrations of K, Na, Cl, and P were equivalent in young and adult rat RPTC. Adult rat and young rat cells preincubated in K-free medium rapidly recovered normal intracellular K and Na contents when returned to 5.5 mM K medium. The recovery was almost immediately blocked by ouabain. Effective permeabilities measured as half time of K efflux and Na influx after ouabain inhibition of Na-K-ATPase were higher in adult than in young RPTC cultured for less than 4 days. Effective K and Na permeabilities decreased significantly with increasing time in culture in adult but not in young rat RPTC. Among young rat RPTC, half times of Na and K fluxes were significantly correlated to age. Effective K and Na permeabilities were lower in both young and adult rat RPTC that had been serum deprived for 24 h than in cells that had been continuously cultured in serum. In cells cultured for 3 days and serum deprived for 1 day, the addition of serum significantly increased K and Na permeability both in young and adult RPTC, but the effect was more pronounced in young RPTC where permeability reached the same high values as in adult RPTC continuously cultured in serum. In conclusion, effective Na and K permeabilities and serum activation of "permeability units" change during ontogeny. These ontogenic changes might be blunted after a few days in culture due to dedifferentiation of adult rat RPTC. PMID- 3019150 TI - Isolation of smooth muscle cells from swine carotid artery by digestion with papain. AB - A new method is described for the preparation of viable, elongated smooth muscle cells from the swine carotid artery. Cells were prepared by papain digestion of pressurized arteries in calcium-free solution. After digestion, the arteries were everted, and fine strips were teased from the intimal surface of the media in calcium-free solution, releasing single cells. Viability was assessed by exclusion of trypan blue and by appearance under phase-contrast microscopy. By these criteria, approximately 20% of the isolated cells were viable. The most distinguishing and unexpected characteristic of these cells was their length. Mean length of the relaxed viable cells was 240.4 +/- 47.4 microns (SD, n = 76), which is much longer than previously reported for arterial smooth muscle cells. Calcium (1.6 mM) caused most of the viable cells to contract slightly, and the mean cell length in calcium was 194.4 +/- 57.7 microns. Cells in 1.6 mM calcium contracted substantially in response to 10 microM histamine or the calcium ionophore A23187 (10 microM), demonstrating that histamine receptors and the contractile apparatus were still functional. PMID- 3019151 TI - Chronic DOCA treatment increases Ca absorption: role of hypercalciuria and vitamin D. AB - To examine the effects of mineralocorticoidism on calcium (Ca) absorption and to define the mechanism, rats received a high-salt diet and injections of vehicle or deoxycorticosterone acetate (DOCA). Net (44.2 vs. 31.4 mg/day) and percent Ca absorption (28.1 vs. 20.1%) was increased after 5 days of DOCA. This was associated with increased duodenal 45Ca uptake. Thus despite the hypercalciuria, Ca balance was similar. Although the hypercalciuria persisted chronically, the gut effects were sustained, which maintained normal ionized Ca, bone Ca, and Ca balance. Urinary cyclic adenosine monophosphate was elevated by DOCA. Compared with appropriate controls, neither DOCA alone nor polydipsia (elicited by dextrose) produced similar magnitudes of hypercalciuria as DOCA plus high-salt diet. These maneuvers also failed to increase Ca absorption. Neutralization of the metabolic alkalosis neither attenuated the DOCA-induced hypercalciuria nor abolished the Ca hyperabsorption. In vitamin D-deprived rats, the hypercalciuria but not the intestinal effects of DOCA were reproduced. Serum 1,25 dihydroxyvitamin D3 levels were increased during chronic DOCA treatment (224 vs. 139 pg/ml). These data best fit the hypothesis that increased Ca absorption is secondary to the calciuric effects of DOCA and high-salt diet and is mediated via the increased parathyroid hormone and 1,25-dihydroxyvitamin D3 activities. PMID- 3019152 TI - Beta-adrenergic contribution to glucagon-induced glucose production and insulin secretion in uremia. AB - Spontaneous or propranolol-induced hypoglycemia can occur in uremic humans. We studied glucose kinetics (using [3-3H]glucose) in five uremic humans 24 h after hemodialysis and in seven normal controls. The effect of glucagon infusion at rates of 3, 6, 12, and 18 ng X kg-1 X min-1 at 60-min intervals was compared with either saline or beta-adrenergic blockade (propranolol infusion). In uremics, plasma glucose increased by 20-25% and by 40-50% at the 3 and 6 ng X kg-1 X min-1 glucagon doses, respectively, with no further increases at higher infusion rates. Glucose production increased transiently and in tandem with glucose uptake at each glucagon increment (P less than 0.0001). During beta-adrenergic blockade, the effect of glucagon in stimulating glucose production was blunted by 14-24% at the 6-18 ng X kg-1 X min-1 doses (P less than 0.05). During saline infusion, plasma insulin concentrations increased progressively to peak levels fourfold above basal at the 18 ng X kg-1 X min-1 dose. This increase in plasma insulin was virtually abolished by concomitant beta-adrenergic blockade (P = 0.0002). In contrast to uremic subjects, normal controls exhibited lesser degrees of hyperglycemia and hyperinsulinemia at all glucagon infusion rates. Propranolol infusion had no effect on the increments in glucose production and uptake nor on the plasma insulin response. These results suggest that in uremic humans propranolol independently reduces the hepatic response to glucagon and the insulin secretory response to hyperglycemia and/or hyperglucagonemia. These observations provide a possible mechanism for the adrenergic regulation of glucose homeostasis in uremia. PMID- 3019153 TI - Placental lactogen and GH receptors in sheep liver: striking differences in ontogeny and function. AB - To determine whether changes in the relative biological potencies of ovine placental lactogen (oPL) and ovine growth hormone (oGH) during development derive from ontogenetic changes in the binding of these hormones to hepatic receptors, we have compared the binding of 125I-oPL and 125I-oGH to hepatic membranes from fetal lambs and pregnant sheep at mid- and late gestation and from postnatal sheep at 1 day to 7 mo of age. Specific high-affinity 125I-oPL binding sites in ovine fetal liver were detected as early as day 70 of gestation (term = 145 days), and the number of fetal 125I-oPL binding sites increased progressively throughout the latter half of gestation, reaching a maximum (11.2 fmol/mg protein) at 3-7 days before parturition. The potency of oPL (Kd 0.27 nM) in competing for 125I-oPL binding sites was 90 and 1,300 times greater than that of oGH and ovine prolactin, respectively. Although the number of fetal 125I-oPL binding sites increased throughout pregnancy, there was little or no specific binding of 125I-oGH noted in the fetus. Treatment of fetal liver membranes with 4 M MgCl2 did not enhance the subsequent specific binding of 125I-oGH, suggesting that the low specific binding of oGH did not result from occupation of hepatic receptors by endogenous circulating oPL or oGH. In contrast, MgCL2 treatment markedly increased the apparent number of fetal 125I-oPL binding sites, suggesting that oPL receptors in fetal liver are partly saturated in vivo by oPL.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019154 TI - Sodium-proton exchange in colon brush-border membranes. AB - Apical membrane vesicles were prepared from proximal and distal segments of the large intestine of the rat by a method based on morphological criteria and were used to determine 22Na uptake. In both preparations an outwardly directed proton gradient stimulated 22Na uptake. In proximal colon a decrease in vesicular volume induced by an increased media osmolarity led to diminished 22Na uptake at 90 min; a significant (50-60%) portion of uptake represented binding. Initial uptake was linear for 10 s and extrapolated through zero, indicating minimal extravesicular binding. Initial uptake was a saturable function of medium Na concentration. In both preparations initial influx of 0.1 mM NaCl was inhibited by amiloride (0.1 1.0 mM), 15 mM NaCl, 15 mM LiCl, and 15 mM NH4Cl. From the characteristics of the initial 22Na influx we conclude that the apical membrane from colonocytes of proximal and distal segments contains a Na-H exchange with properties similar to those described in other epithelia. PMID- 3019155 TI - Preferential hepatic uptake of iron from rat asialotransferrin: possible engagement of two receptors. AB - Hepatic iron uptake from and degradation of rat asialotransferrin prepared from the least anionic (major) component of rat transferrin were studied in intact rats. In experiments lasting 60-90 min, rat asialotransferrin delivered a three to four times larger fraction of the Fe dose to the liver than rat transferrin. Variations in the concentration of endogenous circulating rat 2Fe-transferrin by up to 300% failed to affect the enhanced hepatic delivery of Fe from rat asialotransferrin. However, pretreating the animals with a large dose of asialomucin, or fully sialylated human transferrin, or a combination of both did affect the delivery. In all cases, rat asialotransferrin delivered Fe to the liver at rates comparable with those seen with rat transferrin. The reason for the efficacy of human transferrin was clarified in competitive binding studies on rat hepatocytes and reticulocytes, which showed that human transferrin possessed an approximately sevenfold higher affinity for rat transferrin receptors than the homologous protein. These findings suggest that the enhanced hepatic uptake of Fe from rat asialotransferrin is mediated by simultaneous binding of the ligand both through its glycan and transferrin receptor affinity site. Pretreatment with asialomucin and human transferrin had no suppressing effect on basal hepatic delivery of iron from rat 2Fe-transferrin. The data suggest that deposition of a significant fraction of Fe in rat liver from rat transferrin is likely to take place by a mechanism not involving transferrin receptors. Desialylation shortened the metabolic half-life of rat transferrin from 33 to 24 h.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019156 TI - Adrenergic agents stimulate and cholinergic agents inhibit H+ secretion by amphibian jejunum. AB - In vitro segments of Amphiuma jejunum secrete H+ spontaneously. This study explored the effect of cholinergic and adrenergic agents on H+ secretion. Segments of mucosa were short-circuited and exposed on their mucosal surface to HCO3- -buffered medium while the pH of the unbuffered serosal medium was held by the pH-stat technique. Methacholine added to the serosal medium nearly abolished the spontaneous short-circuit current (Isc) and serosal alkalinization (JHCO3-) with an EC50 of 3.7 X 10(-7) M. Subsequent addition of norepinephrine (NE) to the serosal medium caused a dose-dependent increase in Isc and JHCO3. For three catecholamines the order of potency was epinephrine greater than NE greater than isoproterenol. The spontaneous Isc was significantly reduced (P less than 0.05) by the gastric H+-K+-ATPase inhibitor omeprazole, while the NE-induced Isc was unaltered by the inhibitor. Replacement of medium Na+ with choline abolished the response to NE. The NE-induced Isc was also reduced by methacholine. Acetazolamide inhibited the spontaneous and NE-induced Isc and JHCO3. In summary, cholinergic and adrenergic agents have opposing effects on intestinal H+-HCO3- transport. Jejunal acid secretion may be controlled in part by these antagonistic influences. Adrenergically activated acid secretion occurs by a different mechanism than spontaneous acid secretion. PMID- 3019157 TI - Response of rat jejunum to changes in sodium and volume balance. AB - Initial studies determined whether a renal factor facilitates decreased jejunal absorption following volume expansion of the anesthetized rat. Volume expansion (VE) decreased jejunal absorption to the same extent in both normal and nephrectomized animals. Furthermore, VE of a donor animal failed to alter jejunal absorption in a recipient following cross circulation. Thus, a hormonal factor is not implicated in the jejunal response to VE. Additional experiments demonstrated that rats ingesting a high-Na diet exhibited levels of jejunal absorption lower than animals fed a normal-Na diet. High-Na animals were not volume expanded. Plasma aldosterone concentrations and plasma renin activity were reduced in high Na animals. Bilateral adrenalectomy-nephrectomy inhibited jejunal absorption. However, neither bilateral adrenalectomy nor bilateral nephrectomy alone inhibited jejunal absorption. Furthermore, inhibition of angiotensin formation in adrenalectomized animals failed to alter jejunal absorption. Decreased jejunal absorption in high-Na animals is not due to volume expansion or to inhibition of the renin-angiotensin-aldosterone axis. PMID- 3019158 TI - Phosphate transport across renal proximal tubular cell membranes. AB - The transport of phosphate across the plasma membrane of the renal proximal tubular epithelial cell is thought to take place through the activities of specific transporters located in the membrane. The activities of these transporters are essential to effect the reabsorption of phosphate present in glomerular ultrafiltrate. In addition, their activities are thought to be important for the maintenance of metabolic functions in the proximal tubular cell. Studies utilizing proximal tubular brush-border and basolateral membranes isolated from mammalian kidney have provided significant insights into the mechanisms by which phosphate transport across the brush-border and basolateral membranes of the intact proximal tubular cell occurs and is modulated. In this editorial review, the results of many of these studies are summarized. Particular emphasis is placed on studies that utilized isolated membranes to determine the mechanism by which the phosphaturic action of parathyroid hormone is mediated in the renal proximal tubule. On the basis of studies conducted in isolated membranes and in more physiologically intact preparations, models are constructed to integrate the role of the brush-border and basolateral membranes in the transport of phosphate into and out of the renal proximal tubular cell. PMID- 3019160 TI - Prostaglandin E2 and cyclic AMP response to vasopressin in renal medullary tubular cells. AB - Rat renal medullary tubular cells, prepared by collagenase dispersion and hypotonic lysis, were introduced in Teflon chambers for superfusion. Prostaglandin (PG) E2 and cyclic adenosine 5'-monophosphate (cAMP) production was measured in the effluent. Arginine vasopressin (AVP) but not 1-deamino-8-D arginine vasopressin (dDAVP) (10(-10)-10(-6) M), induced a dose-dependent increase in PGE2 synthesis, whereas AVP and dDAVP produced a similar dose dependent increase in cAMP synthesis. The Ca2+ ionophore A23187 (10(-6) M) stimulated PGE2 synthesis but not cAMP production. In contrast, forskolin (10(-5) M) stimulated cAMP synthesis without affecting PGE2 generation. The pressor antagonists dEt2AVP and d(CH2)5Tyr(Me)AVP (10(-5) M) completely inhibited the PGE2 response to 10(-8) M AVP, whereas d(CH2)5-D-LeuVAVP (10(-6) M), a mixed pressor-antidiuretic antagonist, inhibited incompletely. dEt2AVP did not significantly affect cAMP synthesis in response to 10(-8) M AVP, whereas d(CH2)5 D-LeuVAVP, and unexpectedly also d(CH2)5Tyr(Me)AVP, were inhibitory. dPTyr(Me)AVP (10(-7) M), a pressor antagonist, had an unexpectedly high cAMP-stimulating capacity. In Ca2+-free media containing EGTA, the PGE2 response to AVP and A23187 was inhibited. Nifedipine (10(-6) M) did not significantly inhibit the AVP stimulated PGE2 production. Thus AVP-stimulated PGE2 synthesis is linked to a V1 receptor in renal medullary tubular cells and occurs independently to the activation of adenylate cyclase through a V2-receptor. PMID- 3019159 TI - Electrophysiological studies on primary cultures of proximal tubule cells. AB - Primary confluent monolayers were grown from proximal tubule fragments of rabbit kidneys. The fragments were obtained by gradient centrifugation and seeded on an ad hoc dish whose bottom was a permeable and transparent collagen membrane. The culture medium was a mixture of 50% Ham's F-12 and 50% Dulbecco's modified Eagle's medium supplemented with insulin, transferrin, ethanolamine, sodium selenite, and amino acids. The monolayers were studied at 6-14 days after seeding. Transmission electron microscopy revealed cuboidal cells 8.5-10.5 microns high, with a 1.5 to 2.5-microns apical brush border, abundant mitochondria, vacuoles, lysosomes, and irregular basal interdigitating processes. Cyclic AMP synthesis was stimulated by parathyroid hormone and was insensitive to vasopressin and isoproterenol. Electrophysiological studies performed with the same physiological salt solution on both sides revealed a transepithelial voltage of -2.6 +/- 0.6 mV (n = 10) and a basolateral membrane voltage of -51.0 +/- 4.5 mV (n = 13), both referred to the basal solution. The transepithelial electrical resistance was 7 +/- 2 omega X cm2. The apical membrane depolarized on addition of glucose to the apical side and hyperpolarized on removal of glucose. Changes in apical membrane voltage on addition of varying glucose concentrations (at [Na] = 135 mM, 37 degrees C) demonstrate the presence of a glucose transport system with an apparent Km of 3.54 +/- 0.54 and a Vmax of 7.2 +/- 0.4 mV. Thus this preparation exhibits morphological and electrophysiological characteristics of proximal tubule cells; these studies demonstrate the feasibility of the use of intracellular microelectrode techniques to study the transport properties of cultured epithelia. PMID- 3019161 TI - Regulation of renal Na-K-ATPase in the rat: effect of uninephrectomy. AB - This study has examined the temporal profile and the segmental localization along the rat nephron of the increase in Na-K-ATPase produced by uninephrectomy, and the role of the adrenal gland in the generation of the increase in enzyme activity. In adrenal-intact rats, an increase in Na-K-ATPase activity in the cortical collecting tubule (CCT) was observed at 1 wk (140 +/- 13% of sham, P less than 0.05) and sustained at 2 wk (140 +/- 8% of sham, P less than 0.05). In contrast, the enhancement of enzyme activity in the proximal convoluted tubule (PCT) was transient (at 1 wk: 164 +/- 20% of sham, P less than 0.05; and at 2 wk: 97 +/- 9% of sham, P greater than 0.5). No changes in Na-K-ATPase activity were observed in the other nephron segments studied: pars recta, medullary thick ascending limb, cortical thick ascending limb, distal convoluted tubule, and medullary collecting tubule. In adrenalectomized rats, CCT enzyme activity was lower than in adrenal-intact rats (761 +/- 84 vs. 1,984 +/- 276 pmol X mm-1 X h 1, P less than 0.001) and was not altered by uninephrectomy (849 +/- 91 pmol X mm 1 X h-1, NS). We conclude that the increase in Na-K-ATPase activity following uninephrectomy is restricted to two segments of the nephron and follows a distinctive pattern in each. In the PCT a transient enhancement in enzyme activity is observed, whereas in the CCT the increase in Na-K-ATPase is sustained and requires the presence of an intact adrenal gland. PMID- 3019163 TI - Renin-angiotensin system, converting-enzyme inhibition and kidney function in aging female rats. AB - The age-related changes in the renin-angiotensin system were investigated in normotensive 3-, 10-, 20-, and 30-mo-old female Wistar rats. Plasma renin concentration and immunofluorescent renal renin index remained constant from 3 to 10 mo, then decreased as the animals become older, whereas plasma concentration of renin substrate diminished slightly between 10 and 20 mo and plasma converting enzyme activity was not modified with age. Acute inhibition of converting-enzyme activity by intravenous administration of 0.1 mg/100 g body wt S 9780 (the diacid form of S 9490) was followed by a 7- to 8-mmHg decrease in arterial pressure and a concomitant 10-12% increase in inulin and p-aminohippuric acid (PAH) clearance in the 10- and 20-mo-old rats. On the other hand, neither glomerular filtration rate, PAH clearance nor arterial blood pressure were affected by converting enzyme inhibition in the 30 mo-old rats. These results indicate that the activity of the renin-angiotensin system is progressively reduced with age and suggest that angiotensin II does not play an important role in the maintenance of blood pressure and kidney hemodynamics in normotensive female senescent rats. PMID- 3019162 TI - Responses of ACTH, epinephrine, norepinephrine, and cardiovascular system to hemorrhage. AB - Hemorrhages of various magnitudes were performed on conscious rats, and arterial pressure, heart rate, and plasma levels of adrenocorticotropin hormone (ACTH), epinephrine, and norepinephrine were measured. Eight rats were prepared with chronic femoral arterial cannulas and received a 10, 15, or 20 ml/kg X 3 min hemorrhage in random order on day 4, 7, or 10 after surgery. Mean arterial blood pressure, heart rate, and plasma ACTH, epinephrine, and norepinephrine concentrations were determined before and 20 min after hemorrhage. Arterial blood pressure decreased significantly immediately after each hemorrhage and slowly recovered over the next 20 min. Heart rate did not change during the 10 ml/kg X 3 min hemorrhage but decreased significantly after 15 and 20 ml/kg X 3 min hemorrhages. Plasma ACTH and epinephrine levels increased significantly 20 min after the 15 and 20 ml/kg X 3 min hemorrhages but not after 10 ml/kg X 3 min hemorrhage. Norepinephrine increased significantly 20 min after the 20 ml/kg X 3 min hemorrhage but not after the 10 or 15 ml/kg X 3 min hemorrhage. There was no significant effect of time and repeated hemorrhages on resting levels of plasma ACTH, epinephrine, norepinephrine, osmolality, or proteins. Since hemorrhage leads to a fall in arterial pressure and a subsequent rise in plasma ACTH, the relationship between plasma ACTH and mean arterial blood pressure during hemorrhage was examined in both conscious and acutely prepared pentobarbital sodium-anesthetized rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019165 TI - Malignant granular cell tumor? PMID- 3019164 TI - Control of renin secretion by Ca2+ and cyclic AMP through two parallel mechanisms. AB - The cellular mechanism of action of cyclic AMP (cAMP) mediating the beta adrenergic stimulation of renin secretion was studied, with special reference to its interactions with the inhibitory pathway of renin secretion by Ca2+ calmodulin. Forskolin, a potent stimulator of adenyl cyclase that bypasses the hormone-receptor interactions, stimulated renin secretion in vitro from rabbit renal cortical slices in a concentration-dependent manner. Renin secretion stimulated by submaximal concentration of forskolin was partly or completely antagonized or blocked by raising intracellular Ca2+ concentration by incubating slices in a high-K+ depolarizing medium, but renin secretion stimulated by the maximal effective concentration of forskolin was not inhibited by Ca2+. In addition, the maximal effective concentration of forskolin (10(-5) M) increased renin secretion by a fixed amount independent of medium (by inference, intracellular) Ca2+ concentration in the range of 10(-8) to 10(-6) M in a high-K+ medium. Furthermore, the degree of stimulation of renin secretion by forskolin was greater with greater removal of the inhibitory effect of Ca2+ calmodulin pathway on renin secretion with use of potent calmodulin antagonists, suggesting that the stimulatory effect of cAMP on renin secretion may be maximal in the absence of the inhibitory influence of Ca2+. These results are consistent with the hypothesis that cAMP (by inference, the beta-adrenergic stimulus) stimulates renin secretion through a cellular mechanism independent of that through the Ca2+ -calmodulin pathway. PMID- 3019166 TI - [Hormonal balance during labor. I. Changes in ACTH, cortisol, progesterone and estradiol levels]. PMID- 3019167 TI - [Lasers in obstetrical and gynecological practice. The nature of laser radiation]. PMID- 3019168 TI - [Current viewpoints in the anti-D prevention of rhesus alloimmunization in the pregnant woman and of hemolytic disease of the newborn infant (a review of the literature)]. PMID- 3019169 TI - Impact of AIDS on Alabama hospital care. PMID- 3019170 TI - AIDS in Alabama. The first 1,000 days. PMID- 3019171 TI - Testing for HTLV-III/LAV antibody. PMID- 3019172 TI - AIDS: the national perspective. PMID- 3019173 TI - Herpes genitalis. PMID- 3019174 TI - Pituitary-adrenal responses to morphine and footshock stress are enhanced following prenatal alcohol exposure. AB - The effect of prenatal ethanol exposure on pituitary-adrenal function in adult offspring was assessed by measuring corticosterone (CS) levels in plasma following exposure to two forms of footshock stress, intermittent and continuous, and after administration of 20 mg/kg of morphine. The footshock experiments were conducted at two time points in the circadian pituitary-adrenal cycle. Prenatally ethanol-exposed (E) rats had higher levels of CS than pair-fed and normal controls following intermittent footshock when tested at the crest of the CS circadian rhythm. However, this difference was not present when intermittent footshock was presented at the trough of the circadian cycle. At either time of day, there were no differences among the prenatal treatment groups in the basal condition or following continuous footshock. In addition, E rats had significantly higher levels of plasma CS than controls following morphine. Plasma adrenocorticotropin (ACTH) levels were measured after intermittent footshock at the crest of the circadian rhythm and were significantly higher in E rats than in controls. These results extend our previous reports of enhanced activation of the hypothalamo-pituitary-adrenal axis in response to other stressors as well as to ethanol in adult rats exposed to ethanol in utero. They also confirm that E rats are differentially hyperresponsive only to intermittent footshock stress, not to continuous footshock, as we had found to be the case when the analgesia induced by these two stressors was the dependent measure. PMID- 3019175 TI - [Myasthenia gravis as an anesthesia risk]. AB - Myasthenics must be considered as surgical risk patients. It is imperative to know the exact pathophysiology of the disease pattern with its three types of crisis including their treatment in order to perform safe anaesthesia and to reduce the rate of perioperative complications. In the preoperative phase we must consider a few specific angles besides the routine manipulations: Treatment with cholinesterase inhibitors as practised in myasthenics is continued unchanged or with only slightly reduced dosage up to the day of the operation. If necessary, oral administration may be changed to intramuscular or intravenous application. Premedication is carried out as far as possible without any drugs contraindicated in myasthenics. The patient may get regional or full anaesthesia, the latter always via intubation. We prefer inhalation anaesthetics because they are easily monitored. Neuroleptanalgesia, however, is also possible. One must accept the somewhat higher risk of postoperative respiratory insufficiency since in most cases subsequent artificial respiration must be performed anyway. Relaxation is effected, if at all necessary, via a non-depolarising muscle relaxant in low dosage (one-half to one-tenth of normal dosage). Measurement and monitoring of neuromuscular transmission via the nerve stimulator is mandatory. Succinylcholine is used only in case of vital indication (half of the normal dose). After surgery the patient is transferred to the intensive care ward in intubated position, extubation being performed only after spontaneous breathing has been safely assured. In postoperative analgetic treatment the opiate antagonist pentazocine (Fortral) showed the best results as far as our experience goes. With careful monitoring, however, it is also possible to employ other highly effective analgesics.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019176 TI - [Protective effect of lidocaine in maintaining the function of peripheral nerves]. AB - The conduction preserving effect of lidocaine was investigated in sheathed vagus nerves of the rabbit. The nerves were preincubated for one hour in solutions containing either 5 (group I, n = 12) or 20 mmol/l glucose (group II, n = 12). Subsequently, the nerves were deprived of glucose, one half of each group being exposed to 0.1 mmol/l (ca. 0.0025 g/dl) lidocaine hydrochloride. Lidocaine prolonged significantly the 50% and 100% extinction times of compound action potentials of both myelinated A and unmyelinated C axons: In group I, conduction was blocked within 120 +/- 11 and 123 +/- 9 minutes (mean +/- SEM) respectively, whereas in group II conduction block occurred within 174 +/- 16 resp. 183 +/- 17 minutes. In contrast, A and C compound action potentials of nerves incubated without lidocaine were extinguished within 69 +/- 5 and 78 +/- 6 minutes, respectively (group I, p less than 0.001) or 106 +/- 9 minutes (group II, p less than 0.005). The results suggest that administration of subblocking concentrations of lidocaine by standard Bier block technique may increase the margin of safety during operations employing a pneumatic tourniquet, especially if the blood flow to the nerves is impaired by vascular diseases or local anaesthetics containing adrenaline. PMID- 3019177 TI - Tumours of the glomus jugulare. Case report and anaesthetic management for the combined two stage operation. AB - Two cases are reported using a two stage procedure for the excision of glomus jugulare tumours. The anaesthetic problems which this may produce are discussed, and recommendations are made regarding the anaesthetic conduct for this procedure. A two stage, combined procedure is suggested to reduce anaesthetic morbidity. PMID- 3019178 TI - Management of a glomus jugulare tumour with internal carotid artery involvement. AB - A previously healthy 40-year-old female presented for surgical resection of a large glomus jugulare tumour with extensive involvement of the carotid siphon and intracranial extension. Conduct of anaesthesia with specific reference to cerebral protection is discussed. A combination of induced hypothermia, barbiturate therapy, normotension, normocarbia and prior clamping of the distal internal carotid artery was chosen. The role of barbiturates as a therapeutic intervention is debated. PMID- 3019179 TI - Determination of cinnamic acid and its analogues by electrophoresis in a fused silica capillary tube. PMID- 3019181 TI - Outbreak of fatal adenoviral type 7a respiratory disease in a children's long term care inpatient facility. PMID- 3019180 TI - Studies on the male antifertility agent gossypol acetic acid. VII. Effect of motility stimulated factors on the revival of human spermatozoal motility after gossypol treatment in vitro. AB - Human spermatozoa were incubated with gossypol acetic acid (100 micrograms/1 X 10(6) spermatozoa/ml) at 37 degrees C for 30 min. The drug treatment inhibited the spermatozoal motility significantly. Washing of the spermatozoa, after gossypol treatment, did not effect their motility. A partial revival in the motility of the spermatozoa was observed when gossypol treated spermatozoa were incubated, after washing, with motility stimulating factors, e.g. theophylline, dibutyryl-cAMP and Kallikrein. PMID- 3019183 TI - [Familial hypophosphatemia and rickets caused by calcium deficiency]. PMID- 3019182 TI - Experimental model of anaphylaxis-induced beta-adrenergic blockade in the airways. AB - The influence of an anaphylactic challenge on the relaxing effects of isoproterenol, prostaglandin E1 (PGE1), forskolin, and aminophylline was examined using an isolated tracheal muscle preparation from ovalbumin (OA)-sensitized guinea pigs. As compared with histamine (4 X 10(-5) M) challenge, an anaphylactic challenge with OA (5 micrograms/mL) caused a rightward shift of the concentration response curves for isoproterenol, PGE1, and forskolin but not for aminophylline. Their 50% effective concentrations were increased by about 1.7- to 2.8-fold of the histamine challenged preparations. These results indicate that anaphylactic challenge of the sensitized guinea pig tracheal muscle diminishes relaxing effects of isoproterenol, PGE1, and forskolin but not aminophylline. Possible mechanisms of these subsensitivities may involve both impaired coupling mechanism between receptor and adenylate cyclase and decreased adenylate cyclase activity, rather than their receptor numbers or affinity. PMID- 3019184 TI - [Structure and properties of animal and bacterial collagenases]. AB - Everywhere is found collagen, collagenases are also found. The author reviews the properties of these collagenases: structure, biosynthesis, activity, inhibition. The therapeutical applications are also discussed. They seem promising since a collagenase I, Achromase can be industrially produced and has been shown interesting both in laboratory studies and in clinical trials. PMID- 3019185 TI - [Peptide inhibitors of a bacterial collagenase]. AB - The properties of the cleavage site of collagen by collagenases remain discussed. The authors report their studies on the active site of the collagenase of a bacteria, achromobacter, based on two types of methods. The first type uses synthetic substrates, the second one enzymatic inhibitors. The results of both methods are reviewed. The inhibitors seem more sensitive than substrates to study the relation between structure and activity of the enzyme. PMID- 3019186 TI - [Possible role of glutathione peroxidase in the regulation of collagenase activity]. AB - Glutathione peroxidase (Se-GPx) is a selenoenzyme which catalyzes the reduction of hydroperoxides by glutathione (GSH), in most mammalian cells. Several Slow acting drugs that are used in the treatment of rheumatoid arthritis, including D Penicillamine, alpha-mercaptopropionylglycine and gold salts, are specific inhibitors of Se-GPx. In situation of oxidant stress, Se-GPx activity is a major source of glutathione disulfide (GSSG), an essential activator of leucocyte collagenase. Hence the possibility that the enzymatic reduction of hydroperoxides produced during chronic inflammation would play an important role in the destruction of joint tissue of arthritic patients. Inhibition of a protective system such as Se-GPx may therefore be involved in the mechanism of action of D Penicillamine and gold salts, but it could also explain some of their undesirable or toxic effects. Confirmation of this hypothesis would open the way to new pharmacological strategies. PMID- 3019187 TI - Efficacy of an orally administered modified-live porcine-origin rotavirus vaccine against postweaning diarrhea in pigs. AB - A commercially available, porcine-origin rotavirus vaccine was evaluated for efficacy against postweaning diarrhea due to rotavirus infection in pigs. Weight gains were compared at 5 intervals after weaning. Visual scoring was used to evaluate fecal consistency. Rectal swab specimens were cultured for hemolytic E coli and evaluated for rotaviral antigen by use of enzyme-linked immunosorbent assay. Milk from dams and sera from pigs and dams were evaluated for rotavirus neutralizing antibodies by use of a plaque-reduction test. Significant differences between vaccinates and controls were not found in the determinants evaluated. Selected rotavirus-positive fecal and rectal swab specimens were examined for double-stranded (ds) RNA by use of direct electropherotyping, and the results were compared with the dsRNA pattern of rotavirus propagated from the vaccine. Only electropherotypes typical of field strain virus were found in the fecal and rectal swab specimens evaluated. Sera from guinea pigs and from a gnotobiotic pig immunized against the field strain rotavirus neutralized Ohio State University strain rotavirus (homologous to the vaccine rotavirus strain), but did not neutralize the Gottfried strain of rotavirus. This indicated that, although the dsRNA electropherotypes of the field and vaccine strains of the virus were different, the serotypes were similar, if not identical. PMID- 3019188 TI - Immunologic comparison of the proteins of pseudorabies (Aujeszky's disease) virus and bovine herpesvirus-1. AB - The immunologic relationship between bovine herpesvirus-1 and pseudorabies virus was examined by 80% serum cross-neutralization test, enzyme-linked immunosorbent assays, and Western immunoblotting procedures. Immunogenic cross reactivity between the 2 viruses was observed with both the serum-neutralization test and the enzyme-linked immunosorbent assay. A probing of viral Western immunoblots with rabbit hyperimmune antiserum showed that there were a number of viral specific cross-reactive proteins between bovine herpesvirus-1 and pseudorabies virus. PMID- 3019190 TI - Interrelationships among liposoluble vitamins in ruminants. AB - Plasma concentrations of vitamins A and E were examined in sheep, and a transitory decrease was observed after a single massive dose of vitamin D3 (5 X 10(6) IU) was administered orally or parenterally. Administration of a large dose of vitamin E to sheep decreased plasma retinol concentrations within 72 hours, but thereafter, the plasma retinol concentrations returned to near baseline values. Oral administration of a single pharmacologic dose of dl-alpha-tocopherol (5 g) to sheep caused a slow increase of this vitamin in the blood plasma. In cattle, a single IM administration of 3 liposoluble vitamins (A, D3, and E) at acceptable concentrations had no detectable influence on plasma alpha-tocopherol concentrations with the sampling intervals used. Plasma concentrations of alpha tocopherol in these cattle showed a marked seasonal pattern; the concentrations increased from January to a peak in July, with a subsequent decrease in the fall. Also reported are estimates of inter- and intraindividual variation in plasma liposoluble vitamin concentrations. PMID- 3019189 TI - First isolation of calicivirus from reptiles and amphibians. AB - Calicivirus isolations were made from 4 poikilothermic species in a zoologic collection. Viruses were recovered from 8 asymptomatic Aruba Island rattlesnakes (Crotalus unicolor; rectal swab samples) and from 8 symptomatic animals (4 Aruba Island rattlesnakes, 2 Bell's horned frogs [Ceratophrys orata], 1 rock rattlesnake [C lepidus], and 1 eyelash viper [Bothrops schlegeli] tissue samples obtained at necropsy). On the basis of cross-neutralization test results, the 16 isolates were antigenically indistinguishable and were considered to represent a unique calicivirus serotype, tentatively designated reptilian calicivirus Crotalus type 1. These isolations could not be associated causally with any specific disease entity either in naturally infected poikilotherms or in experimentally infected snakes and pigs. PMID- 3019191 TI - Multiple bluetongue virus-cloned genetic probes: application to diagnostics and bluetongue virus genetic relationships. AB - A shotgun-cloning method incorporating all 10 bluetongue virus genome segments can simultaneously produce complete and partial copies of any of the genome segments. We report here 4 different cloned probes derived from 3 genome segments and individually defined by different hybridization recognition capabilities. One probe hybridized strongly with all 5 United States prototype strains of the 5 different bluetongue virus (BTV) serotypes existing in the United States and, as such, is a strong candidate for a broad BTV diagnostic probe in the United States. Another probe derived from genome segment 2 of BTV-17 hybridized only with the BTV-17 prototypic serotype, thereby demonstrating serospecific hybridization diagnostic potential. The implications for diagnostic and genetic relationship studies on BTV, using various genetic probes, are discussed. PMID- 3019192 TI - Investigation of possible vaccine-induced epizootics of infectious bovine rhinotracheitis, using restriction endonuclease analysis of viral DNA. AB - Viral DNA was extracted from each of 14 modified-live (ML) bovine herpesvirus 1 vaccines, representing all of the ML infectious bovine rhinotracheitis virus (IBRV) vaccines licensed by the US Department of Agriculture for use in cattle. Restriction endonucleases Pst I and Bgl II were used to establish restriction enzyme patterns for the vaccinal viruses. Viral DNA from isolates obtained from 6 field samples of IBRV (1 from Colorado, 1 from West Virginia, 3 from Wisconsin, 1 from South Dakota) were digested with restriction endonucleases, and patterns were compared to evaluate the role of vaccinal virus in these field epizootics of infectious bovine rhinotracheitis. Animals from which field samples were obtained had been vaccinated with ML IBRV vaccine before the epizootic of infectious bovine rhinotracheitis occurred in the herds. In 2 of the 6 field samples, DNA restriction endonuclease analyses patterns from the isolates were indistinguishable from the pattern for the vaccinal viruses used. In the remaining 4 field samples, DNA restriction endonuclease analyses patterns of the IBRV from isolates were different from those of the vaccinal viruses. PMID- 3019193 TI - Density-gradient sedimentation of feline bone marrow cells with polyvinyl pyrrolidone-coated silica gel. AB - Feline bone marrow cells were separated based on their density distribution in a continuous polyvinyl-pyrrolidone-coated silica gel density gradient. Morphologic identification of cytocentrifuged preparations at specific densities revealed a progressive increase in density with maturation of the cells within the granulocytic and erythroid series. Segmented eosinophils peaked at 1.084 g/ml, monocytes at 1.07 g/ml, and lymphocytes spanned the density of 1.068 to 1.084 g/ml. Bone marrow samples from 6 healthy specific-pathogen-free cats were separated by the continuous polyvinyl-pyrrolidone-coated silica gel gradient and were studied for progenitor colony formation in methylcellulose. Erythroid colony formation was greatest at a density of 1.084 g/ml and also appeared in cells from 4 cats at a lighter density of 1.016 to 1.05 g/ml. Colony formation in the granulocytic series revealed progenitors throughout the gradient with enrichment at 1.055 g/ml. PMID- 3019194 TI - Respiratory clearance of 99mTc-DTPA and pulmonary involvement in sarcoidosis. AB - To investigate the relationships between the respiratory epithelial clearance of micronic aerosolized 99mTc-DTPA (RC-DTPA) and pulmonary function, serum angiotensin-converting enzyme (SACE), and lymphocytic alveolitis in patients with sarcoidosis, RC-DTPA was measured in 49 nonsmokers with pulmonary sarcoidosis and 38 normal nonsmokers. Pulmonary involvement was evaluated on chest roentgenograms (type O = normal, type I = hilar adenopathies, type II = hilar adenopathies associated with parenchymal shadows, type III = parenchymal shadows without adenopathy) and by pulmonary function tests. Serum angiotensin-converting enzyme was determined, and a bronchoalveolar lavage was performed for alveolar lymphocyte differential counting (Ly%). RC-DTPA was increased (greater than or equal to 1.96%/min) in 12 of 31 patients with type II or III involvement but was normal in all 18 patients with type O or I involvement (p = 0.002). Patients with increased RC-DTPA had low FVC, TLC, FEV1, and resting Pao2 (p less than 0.05); resting and exercise AaPo2 were increased (p less than 0.05), but RC-DTPA correlated negatively with FEV1 (p less than 0.01), Pao2 at rest (p less than 0.005), and DLCO (p less than 0.05) and positively with resting and exercise AaPO2 (p less than 0.01). In patients with increased RC-DTPA (42 +/- 17%), Ly% did not differ from Ly% in patients with normal RC-DTPA (34 +/- 16%). SACE was increased in patients with increased RC-DTPA (56 +/- 26 U/ml versus 38 +/- 16 U/ml; p = 0.007) and correlated positively with RC-DTPA (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019195 TI - Tumor killing by human alveolar macrophages and blood monocytes. Decreased cytotoxicity of human alveolar macrophages. AB - Tumor killing by human alveolar macrophages (AM) might be an important mechanism of pulmonary defense against neoplastic disease. We compared AM and blood monocytes (Mo) for the ability to kill 2 neoplastic targets, A549 human lung adenocarcinoma cells and P815 mastocytoma cells. Blood monocytes were able to kill both targets, whereas AM killed neither. Tumor killing by Mo was spontaneous and was not increased by incubation with lipopolysaccharide. Because the P815 target is highly sensitive to lysis by hydrogen peroxide (H2O2), it afforded the opportunity to compare AM and Mo for the ability to kill tumors by the production of toxic oxygen compounds. Comparable amounts of superoxide anion were produced by AM and Mo after stimulation with phorbol myristate acetate. However, luminol enhanced chemiluminescence of AM was far less than that of Mo, suggesting that AM could not utilize the myeloperoxidase-H2O2-halide ion system for tumor killing. The addition of exogenous peroxidase to cultures of AM and P815 cells enabled AM to kill this tumor cell. Our results suggest that as Mo mature into AM, their ability to kill tumor cells declines and that AM may be unable to kill H2O2 sensitive tumors because of a loss of myeloperoxidase during maturation. PMID- 3019196 TI - Small cell lung cancer. AB - Lung cancer is the predominant fatal neoplasm of our time, and SCLC, which accounts for about 25% of all lung cancer, if untreated results in death in about 3 months. Currently employed aggressive combination chemotherapy has allowed a 4- to 5-fold improvement in median survival over untreated patients. Ten to 20% of patients with limited disease can be expected to have a long-term (2-yr) survival. The majority of patients, however, have extensive disease. For these patients the median survival is about 7 months. Less than 2% survive 2 yr. During the last 10 yr, experience in the treatment of thousands of patients has been reported. These trials, using a large variety of drug combinations, doses, and schedules as well as multiple modalities including radiotherapy, surgery, and bone marrow transplantation, demonstrate that a plateau has been reached with our present therapeutic approach. The development of new effective therapeutic strategies as well as prevention of SCLC require a better basic understanding of the cellular pathophysiology of the disease. A consistent chromosomal abnormality has been associated with SCLC. This may provide new insight into predisposition and pathogenesis of SCLC. How this chromosomal abnormality relates to loss of control of cell growth is under intense investigation. Similarly, during the past 3 yr, the identification of growth regulatory oncogenes has greatly improved our understanding of malignancy. The discovery that metastatic cells escape immune surveillance has led to attempts at modulating antigenic expression. The modulation of cellular antigenic expression may facilitate the destruction of tumor cells by host defense mechanisms. The understanding of the genetic basis of drug resistance may lead to approaches that prevent or delay resistance. This century has witnessed the emergence of SCLC as an important fatal neoplasm. It has also been during this time that another, formerly dominant pulmonary condition, tuberculosis, has been controlled. The reduction of tuberculosis was accomplished by a combination of scientific understanding, beginning with the discovery of Koch's bacillus, and public health measures. Perhaps a similar parallel for SCLC as well as other forms of cancer will be written. Basic cellular investigations with the new tools of molecular biology as well as measures to control exposure to predisposing environmental factors such as component of cigarette smoke may one day lead to control of SCLC. PMID- 3019197 TI - The diagnostic challenge of metastatic linitis plastica. Two cases and a consideration of the problem. AB - Linitis plastica of the stomach is usually difficult to diagnose preoperatively and can extend locally and metastasize widely before recognition. This insidious manner of spread may cause unusual presentations in organs distant from the stomach. The authors recently treated two patients who were initially found to have radiologic abnormalities of the colon and bladder, respectively. One patient was surgically explored with a view towards the resection of a primary colonic tumor, the other to determine the nature of a bladder tumor. Intraoperative histologic interpretation of frozen tissue samples from the gastric wall in each case radically altered the surgical approach and the prognosis. The clinical presentations illustrate that it may be difficult to obtain preoperative diagnostic tissue samples in such cases and that it is important for the surgeon to inspect and palpate the stomach wall during all abdominal explorations for cancer. PMID- 3019199 TI - Is bone marrow examination in small-cell lung cancer really necessary? AB - Of 403 patients with small-cell lung cancer, we identified by aspiration, biopsy, or both 67 with bone marrow involvement and found the two procedures to be complementary in detecting marrow involvement. The mean surface area of the positive biopsy specimens was significantly greater than that of a randomly selected group of negative biopsy specimens, suggesting that the larger the specimen, the greater the chance of detecting tumour. Patients with marrow involvement had only a slightly worse prognosis compared with other patients who had extensive disease. Only 7 of the 403 patients (1.7%) had extensive disease based on marrow involvement alone. Because bone marrow examination rarely changes the stage of cancer in noninvasively assessed patients, and has no impact on the tolerance of chemotherapy and only a small effect on length of survival, we do not recommend this procedure in the routine staging of small-cell lung cancer. PMID- 3019198 TI - [Early pseudo-deficiency or Prader's hypocalcemic familial type-I rickets]. PMID- 3019200 TI - Transfusion-acquired human immunodeficiency virus infection among immunocompromised persons. AB - Transmission of the human immunodeficiency virus (HIV) was studied in a group of patients with cancer who received transfusion of blood components harvested from a single, asymptomatic, seropositive donor. Of ten living recipients, nine had antibodies to the virus in fresh or cryopreserved sera at a median of 384 days (range, 237 to 686) after transfusion. In three patients, an enzyme-linked immunosorbent assay was negative at the same time that Western blot and radioimmunoprecipitation techniques showed seropositivity. Cultures for HIV obtained at a median of 615 days (range, 322 to 714) after transfusion were positive in seven of nine seropositive recipients. Six seropositive recipients have developed immunologic and clinical sequelae of HIV infection at a median of 286 days (range, 56 to 745) after transfusion. The sera of the two patients without clinical sequelae neutralized HIV in an in-vitro assay, whereas the seven other seropositive patients lacked such neutralizing antibodies. Our study characterizes the clinical, serologic, virologic, and immunologic manifestations of HIV infection in immunocompromised persons. PMID- 3019202 TI - Recommendations for providing dialysis treatment to patients infected with human T-lymphotropic virus type III/lymphadenopathy-associated virus. Centers for Disease Control, Department of Health and Human Services. AB - Maintenance dialysis can be continued in patients with end stage renal disease who have manifestations of human T-lymphotropic virus type III/lymphadenopathy associated virus (HTLV-III/LAV) [now known as the human immunodeficiency virus], including the acquired immunodeficiency syndrome, or who are positive for antibody to HTLV-III/LAV. These patients can be dialyzed in hospital-based or free-standing dialysis units using conventional infection-control precautions. Transmission of HTLV-III/LAV is adequately prevented by standard blood and body fluid precautions, and disinfection and sterilization procedures. PMID- 3019201 TI - Resolution of a contraceptive-steroid-induced hepatic adenoma with subsequent evolution into hepatocellular carcinoma. PMID- 3019204 TI - Testing for human immunodeficiency virus. PMID- 3019205 TI - Secretin, cholecystokinin, and vasoactive intestinal polypeptide in pancreatic cholera. PMID- 3019206 TI - The role of epidemiology in helping CDC improve public health. PMID- 3019203 TI - The human interleukin-2 receptor: normal and abnormal expression in T cells and in leukemias induced by the human T-lymphotropic retroviruses. AB - The human receptor for interleukin-2 (T-cell growth factor) plays a critical role in the growth of T cells and is required for full expression of the normal immune response. Through hybridoma and recombinant DNA techniques, the interleukin-2 receptor protein has been biochemically characterized and purified; full-length copies of its complementary DNA have been molecularly cloned, sequenced, and expressed in eukaryotic cells; and the receptor gene has been characterized. Transient expression of the interleukin-2 receptor gene occurs during normal T cell activation, and high- and low-affinity forms of the membrane receptor exist. A naturally occurring, soluble receptor has also been isolated, and its levels in serum correlate with the activity of various diseases. Deregulation of interleukin-2 receptor expression occurs in T-cell leukemias produced by the human T-lymphotropic retroviruses types I and II (HTLV-I and -II) and has been causally linked to the action of the trans-activator (taf) gene of these viruses. Monoclonal antibodies specific for the interleukin-2 receptor are being evaluated in the treatment of HTLV-I-induced leukemias and other conditions involving the inappropriate function of activated T cells. PMID- 3019208 TI - [Ecology and epidemiology of transmissible viruses of sandflies in Italy]. PMID- 3019209 TI - CAMPEDS--a dust sampling and assessment system for use in hard rock mines. PMID- 3019207 TI - [A synchronizer DNA; the kDNA of trypanosomes]. PMID- 3019210 TI - [Surgical approach in benign tumors of the infraspheno-temporal fossa by cervico transoral access]. AB - Surgical treatment in three patients with benign tumors of the infrasphenotemporal fossa was through a transoral-cervical approach, a procedure possessing particular advantages when compared with the anterior an external approaches. PMID- 3019211 TI - [Preventive treatment with oral acyclovir of recurrent erythema multiforme, associated with herpes infection or not. Preliminary study]. PMID- 3019212 TI - [Efficacy and tolerability of enalapril compared to altizide combined with spironolactone in patients with moderate arterial hypertension]. AB - The aim of this study was to compare the efficacy and safety of enalapril and a combination of a thiazide diuretic (altizide) and a diuretic that reduce potassium excretion (spironolactone), in patients presenting moderate essential hypertension. The four-month, double-blind study of two parallel groups involved 20 patients (12 males, 8 females) of mean age 54 years. All patients first received placebo for 15 days. Subsequently, 10 patients received 20 mg enalapril per day and 10 patients 15 mg altizide combined with 25 mg spironolactone in the same tablet. Blood pressure was measured every five minutes for 30 minutes with an automatic apparatus (Dynamap 845), before administration of placebo, before treatment and one, two and three months after treatment. There was no significant change of pulse rate with enalapril or with the diuretic combination. Both treatments produced an identical significant drop in systolic pressure. On the other hand enalapril seemed more efficacious in reducing diastolic pressure at the fourth month: respective pressures 98.2 +/- 13.3 mmHg and 81.0 +/- 8.5 mmHg (p less than 0.005) with enalapril, 98.6 +/- 7.4 mmHg and 86.0 +/- 6.1 mmHg (p less than 0.001) with the diuretic association (17% versus 12%, p less than 0.05). Safety was good with both medicinal preparations and the few side-effects noted did not necessitate withdrawal of treatment. PMID- 3019213 TI - Hepatocellular carcinoma--a clinical study. AB - Hepatocellular carcinoma afflicts mainly Chinese Singaporeans 75/77 (97.4%), of which 71/77 (92.2%) of the patients are males. It is rare below the 3rd decade of life (1.3%), with the peak incidence occurring in the 5th to 7th decade of life (68.5%). Common presenting features are hepatomegaly (85.7%), jaundice (63.6%), and right hypochondrial pain (51.9%). Liver function tests were abnormal in 98.7%. Alpha-foetoprotein were positive in 61/77 (79.2%) of patients. Hepatitis B surface Antigen were positive in 43.75 (57.3%) of patients. Radiology and ultrasound studies demonstrated that 70.1% had lesions involving both lobes at diagnosis. Only 4/77 (5.1%) had surgical resections of the tumour. 50/65 (76.9%) died within six months of diagnosis, 11/65 (16.9%) survived for one year, 1/65 (1.5%) for 1 1/2 years, 1.65 (1.5%) for 2 years and 2/65 (3.0%) for more than 2 1/2 years; the longest survivor is still alive, at 4 1/2 years after diagnosis. PMID- 3019214 TI - Treatment of irresectible hepatocellular carcinoma with intrahepatic arterial lipoidal mixed with adriamycin and mitomycin C. AB - Four patients with advanced hepatocellular carcinoma, but with stage I functional disease, were treated with intrahepatic arterial lipoidal mixed with small doses of Adriamycin (20 mg) and Mitomycin C (10 mg). Regression was seen in 3 out of the 4 patients. In 2 patients, there was substantial regression of tumour clinically, radiologically and biochemically. The treatment was tolerable without marrow depression or deterioration of liver function. Mild fever (37 degrees C) was seen in 2 and epigastric pain in 1. This form of treatment opens up scope for further improvement in the management of irresectable hepatocellular carcinoma. PMID- 3019215 TI - 4'-Epidoxorubicin (Epirubicin) as a single agent in advanced primary hepatocellular carcinoma--a preliminary experience. AB - 41Epidoxorubicin (Epirubicin) is a new anthracycline analogue which has activity in a variety of cancers. Advanced primary hepatocellular carcinoma is a rapidly fatal neoplasm. Studies with doxorubicin (Adriamycin) in Africa and Asia have shown a response rate around 30-50% with modes gains in median survivals in patients who responded. We report here our preliminary experience with epirubicin in advanced primary hepatocellular carcinoma. We treated 13 patients with advanced primary hepatocellular carcinoma with epirubicin using a dose of schedule of 60 mg/m +/- at the beginning and gradually escalated to 90 mg/m +/- as tolerated. All patients had no prior chemotherapy. Median age was 51 years with range of 31 to 70 years. Cardiotoxicity was serially monitored with radionuclide multigated left ventriculogram. Objective responses were observed in 3 (23%) patients of the 13 evaluable patients. The median duration of these responders was 30 weeks (range 20 to 42). The overall median survival was 11 weeks. Two patients had cardiotoxicity as measured by a significant fall in left ventricular ejection fraction. The toxicities are much less than doxorubicin and better tolerated. We feel that epirubicin has definite activity in primary hepatocellular carcinoma even in patients with advanced disease. PMID- 3019216 TI - [Effect of the ingestion of dietary fiber on the digestibility of lipids in a sorghum-consuming African population]. AB - Digestibility measurements were carried out on 12 men. Their habitual diet, deficient in animal products, is based on sorghum meals which supply between 2.4 and 4.2 g of crude fiber per 100 g of dry matter. Over three 11-day periods, the subjects received 3 successive diets (A, B and C) which supplied respectively 3.3, 4.8 and 5.4 g of crude fiber per 100 g of dry matter. Reduced lipids digestibility was noted, even for diet A which was the poorest in fiber content. No difference was observed between diet A and diet B (92.3 and 91.7% respectively). The apparent digestibility of lipids dropped to 86.1 with diet C. True digestibility of lipids is hidden by poorly digestible dietary lipids. Lipid losses increased more rapidly than nitrogen losses with increasing of fiber content in diets. On these regimens, there were no significant changes in concentrations of fecal fat. Concentration of fecal nitrogen decreases for diets B and C. PMID- 3019217 TI - Competition of n-3 and n-6 polyunsaturated fatty acids in the isolated perfused rat heart. AB - When perfused with exogenous arachidonic acid (AA) or eicosapentaenoic acid (EPA), the rat heart incorporated these fatty acids into phospholipids, chiefly as phosphatidylcholine. The pattern of fatty acid incorporation at any given concentration of fatty acid in the perfusate was not different between n-3 and n 6 polyenoates. When rat hearts were perfused with the same amounts but different mixtures of EPA and AA, the incorporation of EPA showed a marked increase proportional to the EPA/AA ratio present in the perfusate. Results indicated that cardiac muscle phospholipids incorporate n-3 and n-6 polyenoates equally effectively and hence enrichment of n-3 polyenoate can displace AA in the cardiac phospholipid pool. PMID- 3019218 TI - Effects of DN-9693, a new metastasis inhibitor, on murine hematogenous metastasis. AB - 1,5-Dihydro-7-(1-piperidinyl)-imidazo[2,1-b]quinazolin-2(3H)-one dihydrochloride hydrate (DN-9693), a new c-AMP: phosphodiesterase inhibitor was examined for its inhibitory effects on platelet aggregation induced by metastasizing tumor cells and on blood-borne metastases of these tumors. 1-3 microM of DN-9693 completely inhibited platelet aggregation induced by B16 melanoma subline BL6 and Lewis lung carcinoma (3LL) cells. Platelets prepared from mice intravenously or orally administered with DN-9693 failed to aggregate after the addition of BL6 cells. Intravenous injection of DN-9693 was effective in protecting the mice inoculated with 1 X 10(6) BL6 cells against acute pulmonary embolic death. Either intravenous or oral administration of DN-9693 (1-10 mg/kg) sufficiently suppressed thrombus formation and subsequent pulmonary metastasis caused by intravenously inoculated BL6 or 3LL cells. Spontaneous pulmonary metastasis of 3LL was also inhibited by DN-9693. Continuous administration of DN-9693 during and after surgical excision of the primary tumors was the most effective treatment against the development of pulmonary metastases of 3LL. PMID- 3019220 TI - In vitro heterogeneity in human gliomas. Are all transformed cells of glial origin? AB - Primary cultures from 30 human gliomas (26 high grade, 3 low grade, and one oligoastrocytoma) were studied to examine the mixture of cells that make up the outgrowth, and their interactions. GFAP + ve Fibronectin - ve cells had a predominantly process forming (PF) morphology and formed complex networks around explants. Fibronectin + ve GFAP-ve cells had a flattened adherent (FA) morphology, tended to increase in time in culture, in 11 tumours showed a transformed pattern of growth, and may derive from perivascular mesenchyme. Neoplastic glial cells in vivo, or in vitro, may influence the behaviour and nature of other cells in tumour tissue which may be of significance in tumour behaviour and progression. PMID- 3019219 TI - Clinical relevance of oncogene expression in head and neck tumours. AB - A study was undertaken to determine whether the variation in the increased expression of three oncogenes (Ha-ras, Ki-ras and myc) could be correlated with various clinicopathological parameters of squamous cell carcinoma (SCC) of the head and neck region. No correlation was found with sex, age, site of primary tumour, level of differentiation of the tumour, previous X-ray treatment, or fate. The one exception was myc expression with TNM staging of SCC. A significant increase was found for myc expression in TNM Stage III and IV as compared to the combined stages of I and II. Elevated expression of Ha-ras, Ki-ras and myc oncogenes was found in pleomorphic salivary adenoma (PSA), but at a lower level than SCC. It is proposed that in a percentage of cases the elevated expression of these oncogenes in PSA corresponds to a relatively early event in the multistep process of carcinogenesis. PMID- 3019221 TI - The avian and mammalian ets genes: molecular characterization, chromosome mapping, and implication in human leukemia. AB - The mammalian homologs of the ets-region from the transforming gene of avian erythroblastosis virus, E26, consist of two distinct domains located on different chromosomes. Using somatic cell hybrid panels, the mammalian homologs of the 5' v ets-domain, ets-1, were mapped to chromosome 11 in man, to chromosome 9 in mouse, and to chromosome D1 in cat. The mammalian homologs of the 3'v-ets domain, ets-2, were similarly mapped to human chromosome 21, to mouse chromosome 16, and to feline chromosome C2. We conclude that the ets sequence shared by the virus, chicken, and man is likely to contain at least two functionally dissociable domains, identifiable as ets-1 and ets-2. The human ets- locus is transcriptionally active and encodes a single mRNA of 6.8 kb, while the second locus, human ets-2 encodes three mRNAs of 4.7, 3.2 and 2.7 kb. By contrast, the chicken homolog, having a contiguous ets-1 and ets-2 sequence, is primarily expressed in normal chicken cells as a single 7.5 kb mRNA. Because chromosome translocations have been associated with different human hematopoietic malignancies, we have used our human probes to study specific translocations occurring in acute leukemias. The human ets- gene was found to translocate from chromosome 11 to 4 in t(4;11) (q21;23) and the human ets-2 gene was found to translocate from chromosome 21 to 8 in t(8;21) (q22;q22). Significantly, both translocations were associated with an expression of ets genes which differed from that found in normal diploid lymphoid cells. PMID- 3019222 TI - Nuclear thyroid hormone receptors in human cancer tissues. AB - Saturable, high affinity binding sites for 3,5,3' triiodothyronine (T3) were identified in nuclei isolated from human tumors of various origins (breast cancers, other epitheliomas, sarcomas, tumors of the central nervous system). Nuclear T3 receptors were present in all samples of primary breast cancer (n = 93; average Cmax = 215 fmol/mg DNA) and in metastatic tissues originating from breast tumors. A significantly lower T3 binding capacity was found in non-tumor tissues, obtained from breast sites distal to the tumor (n = 30; average Cmax = 133 fmol/mg DNA; paired t-test: p less than 0.01). Specific nuclear T3 receptors were also present in other epitheliomas (n = 8; average Cmax = 432 fmol/mg DNA), sarcomas (n = 4; average Cmax = 297 fmol/mg DNA) and cerebral tumors (n = 13; average Cmax = 364 fmol/mg DNA. In 93 cases of breast cancer, a negative relationship was found between the nuclear T3 receptor level and the involvement of axillary lymph nodes (Pearson chi square value: p = 0.017). Except a possible relationship between the T3 receptor and the progesterone receptor concentrations, no significant correlation was observed between the nuclear T3 binding capacity in breast cancer samples and other clinical and biochemical parameters: age, tumour stage, histopathological grade, serum concentrations of thyroid hormones, TSH, CEA (carcinoembryonic antigen) and prolactin, cytoplasmic estrogen receptors. The presence of high affinity T3-binding sites in human tumor nuclei indicates that the thyroid hormones may play a role, at the cellular level, on the development of certain human cancers. PMID- 3019223 TI - Prolactin binding in benign and malignant mammary tissue of female dogs. AB - Prolactin receptor (PRL-R) concentrations were determined in membrane preparations of canine mammary tumours and of non-affected mammary tissues by a radioreceptor-assay using ovine prolactin (oPRL) both for 125I-labelling and for displacement. Receptor levels greater than or equal to 3 fmol/mg membrane protein were considered positive. Histologically non-affected samples of mammary tissue from 6 dogs were PRL-R positive (12-195 fmol/mg protein). These levels were positively correlated with epithelium content (based on surface area in microscopic sections; r = 0.943, P less than 0.02). In tumour samples where pre existing mammary epithelium (PME) was present (3 non-malignant and 6 malignant tumour samples; PME content 5-10%), the cut-off limit for PRL-R positivity was increased to 50 fmol/mg protein to forestall false positives due to non-affected tissue. If no PME was present the general limit of 3 fmol/mg protein was maintained. All 18 non-malignant tumours showed PRL-R (18-162 fmol/mg protein). The PRL-R levels were positively correlated with levels of oestrogen-(ER; r = 0.735, P less than 0.002) and progesterone receptors (PgR; r = 0.556, P less than 0.02) as measured by a multi-concentration dextran-coated charcoal method. ER and PgR levels were also proportional (r = 0.660, P less than 0.01). In 6 dogs bearing primary cancers with 5-10% PME, 1 out of a total of 6 tumours was PRL-R positive. In 9 dogs bearing primary or locally recurrent cancers without PME, significant PRL-R levels were measured in 2 out of a total of 10 tumours. Three metastatic sites in 2 other dogs were PRL-R positive. In 2 dogs (1 with a PRL-R negative local recurrence) the metastatic lesions were PRL-R negative. Thus 5 dogs of a total of 18, had PRL-R positive mammary cancers (3-377 fmol/mg protein). Unlike in non-malignant lesions the ER, PgR, and PRL-R levels were not related. In mammary cancer the presence of PRL-R was less common (P less than 0.001), and the ultimate levels less high (P less than 0.001) than in non malignant tumours. In comparative studies using pooled membrane preparations from benign mammary tissues, oPRL was far more effective than canine prolactin (cPRL) in displacing 125I-oPRL; canine growth hormone (cGH) in this respect was ineffective. It is concluded that non-malignant mammary tissue in the dog generally is PRL-R positive; only some mammary cancers retain the PRL receptors. PMID- 3019224 TI - Regression of rat mammary tumors associated with suppressed growth hormone. AB - Serum growth hormone (GH) was suppressed in female rats bearing mammary tumors induced by 7, 12, dimethylbenz(a)anthracene (DMBA) or N-nitrosomethylurea(NMU). Serum GH was suppressed due to treatment with a human GH analog produced by the plerocercoid stage of the tapeworm Spirometra mansonoides. Rats treated with plerocercoid growth factor (PGF) via plerocercoid infection had accelerated growth rates despite marked reductions in GH levels. Approximately two-thirds of the mammary tumors induced by either DMBA or NMU regressed during three weeks of exposure to PGF while most of the control tumors continued to grow. The data support an important regulatory role for GH in growth of mammary tumors in rats. PMID- 3019225 TI - Receptors for epidermal growth factor in human mammary carcinomas and their metastases. AB - The tumor cell content of the receptor for epidermal growth factor (EGF-R) and estrogen receptor (ER) was analyzed in 36 primary breast carcinomas and their metastatic deposits. Twenty-five percent of the primary tumors and 48 percent of the metastases exhibited measurable levels of the receptor. The frequency of EGF R positivity was the same for cutaneous and lymph node recurrences. No discrepancy was observed with respect to ER content between primary tumors and their metastases. PMID- 3019226 TI - Purification and biological activities of an angiogenesis factor from human placenta. AB - Several crude angiogenesis preparations, as well as a purified angiogenesis factor from human placenta, were tested for their ability to stimulate the production of plasminogen activator (PA) and collagenase activities, motility, chemotaxis, DNA synthesis and clonal growth in cultured endothelial cells. Treatment of capillary endothelial cells resulted in a stimulation of all the above activities, whereas endothelial cells derived from large vessels did not respond. These cellular activities are postulated to be responsible for capillary growth in vivo. PMID- 3019227 TI - Inhibition of antibody synthesis and expression of T-cell membrane markers by serum factors of patients with small cell carcinoma of the lung. AB - Serum of patients with small cell lung cancer as well as serum fractions recovered by column chromatography inhibit the in vitro antibody synthesis in a PWM driven lymphocyte culture system and the capacity of T-lymphocytes to form rosettes with sheep erythrocytes. Utilizing an in vitro antibody synthesis system recomposed of previously separated B-cells and T-helper cells, we could show that the inhibiting high-molecular weight factors (between 40,000 to 60,000 dalton) from the patients' serum are suppressing antibody synthesis by inactivation of T helper cell function. The mechanisms of inhibition are still unclear. The factors inhibiting E-rosette formation which are in the molecular weight range of 10,000 dalton and lower are not the same as the factors suppressing the antibody production. PMID- 3019228 TI - Parkinson's disease: nigral receptor changes support peptidergic role in nigrostriatal modulation. AB - Autoradiographic studies reveal densities of binding to somatostatin, neurotensin, mu-opiate, and benzodiazepine receptors in substantia nigra specimens from neurologically normal human brains. Binding to nigral angiotensin converting enzyme is also dense, whereas more modest densities of kappa-opiate, dopamine, and serotonin receptors are noted. In nigral specimens taken from patients with idiopathic Parkinson's disease, substantial reductions in somatostatin, neurotensin, mu-opiate and kappa-opiate receptors contrast with more modest reductions in dopamine and benzodiazepine I receptor subtypes. Angiotensin converting enzyme, serotonin, and benzodiazepine II binding are virtually unaltered. These results underscore the likelihood of strong peptidergic influences on normal and pathologically altered human nigrostriatal circuitry. PMID- 3019229 TI - Peripheral neuropathy associated with mitochondrial myopathy. AB - Twenty patients with mitochondrial myopathy were investigated for the presence of peripheral neuropathy. There were clinical features of a mild sensorimotor neuropathy in 5 patients (25%) and nerve conduction studies were abnormal in 10 patients (50%). Electrophysiological studies of the whole group showed significant impairment of motor and sensory conduction, compared with controls. Sural nerve biopsy and morphometric studies were performed on 4 patients with clinical neuropathy. There was a reduction in density of myelinated fibers and electron microscope features of axonal degeneration affecting myelinated and unmyelinated fibers. Abnormal mitochondria containing paracrystalline inclusions were seen in the Schwann cell cytoplasm of two nerves. PMID- 3019230 TI - Reduced phospholipase D activity in brain tissue samples from Alzheimer's disease patients. AB - Biochemical examinations of brain tissue samples obtained from patients with Alzheimer's disease have revealed a decreased quantity of the neurotransmitter acetylcholine and reduced activity of choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine formation. It has been suggested that the choline moiety of lecithin, a substitute ubiquitously present in membranes of mammalian cells, could be mobilized for acetylcholine formation. The activity of phospholipase D, an enzyme which releases choline directly from lecithin, was measured in homogenates of Alzheimer brain tissue and found to be reduced by 63%. ChAT activity was reduced by 58% and 5'-nucleotidase activity was reduced by 27% in these homogenates. PMID- 3019231 TI - Glial bundle formation in spinal roots following experimental neuronopathy. AB - Spinal motor neurons and dorsal root ganglion cells were caused to degenerate by axoplasmic transport of toxic lectins via the sciatic nerve. Within a few months, loss of axons and glial bundle formation were seen in the proximal portions of the L4-6 ventral and dorsal roots. The glial bundles observed were structurally the same as those described in humans, but they were occasionally also invested with Schwann cell cytoplasmic processes, the cell membrane of which was directly apposed to that of astrocytes. Basal lamina surrounding the outer surface of the bundles was shared by these two as a continuous sheet. This unique complex of astroglial and Schwann cell processes, completely covered by basal lamina, may be a newly formed interface between the central and peripheral nervous systems and thus may severe as a guide and potential pathway for regenerating neurites. PMID- 3019232 TI - Prevention of rhinovirus and poliovirus uncoating by WIN 51711, a new antiviral drug. AB - WIN 51711, a potent new antipicornavirus drug, has been shown to inhibit an early event in the replication cycle of human poliovirus type 2 and human rhinovirus type 2. WIN 51711 was not virucidal and had no measurable effect on the adsorption of [3H]uridine-labeled virions to cells. When virion penetration of the plasma membrane was determined through loss of sensitivity to neutralizing antisera, WIN 51711 had no effect on poliovirus penetration, but inhibited rhinovirus penetration by 40%. In the presence of WIN 51711, exposure of neutral red-encapsidated virus-infected cells to light at 3 h postinfection resulted in a 3-log reduction in the number of infectious centers, indicating that WIN 51711 maintained the viral RNA in the encapsidated state after penetrating the cell membrane. The inhibition of uncoating by WIN 51711 in the neutral red assay was found to be concentration dependent, with a concentration of 0.03 micrograms/ml resulting in a 90% inhibition of uncoating. Sucrose gradient sedimentation of lysates from whole cells infected with [3H]uridine-labeled poliovirus showed that poliovirions remained intact in the presence of WIN 51711, but were uncoated in the absence of drug. WIN 51711 also prevented thermal inactivation of poliovirus infectivity, indicating a direct stabilizing effect of this compound on virion capsid conformation. PMID- 3019233 TI - Synergy between RU 28965 (roxithromycin) and human neutrophils for bactericidal activity in vitro. AB - The in vitro effects of RU 28965 (roxithromycin), a new semisynthetic macrolide, on human neutrophil activity were compared with those of erythromycin. RU 28965, at a concentration as low as 0.1 microgram/ml, significantly enhanced the phagocytosis and killing of Staphylococcus aureus by neutrophils. Erythromycin displayed a less stimulating effect in a dose-dependent manner. Phagocytosis of Klebsiella pneumoniae was also increased after incubation of neutrophils with RU 28965, but killing was not altered. Neutrophil chemotaxis, myeloperoxidase activity, and O2 consumption were unchanged in the presence of RU 28965. PMID- 3019234 TI - Inhibition of electron transfer and uncoupling effects by emodin and emodinanthrone in Escherichia coli. AB - The anthraquinones emodin (1,3,delta-trihydroxy-6-methylanthraquinone) and emodinanthrone (1,3,8-trihydroxy-6-methylanthrone) inhibited respiration-driven solute transport at micromolar concentrations in membrane vesicles of Escherichia coli. This inhibition was enhanced by Ca ions. The inhibitory action on solute transport is caused by inhibition of electron flow in the respiratory chain, most likely at the level between ubiquinone and cytochrome b, and by dissipation of the proton motive force. The uncoupling action was confirmed by studies on the proton motive force in beef heart cytochrome oxidase proteoliposomes. These two effects on energy transduction in cytoplasmic membranes explain the antibiotic properties of emodin and emodinanthrone. PMID- 3019236 TI - Antirhinovirus compound 44,081 R.P. inhibits virus uncoating. AB - 44,081 R.P., or 2-[(1,5,10,10a-tetrahydro-3H-thiazolo[3,4-b]isoquinolin- 3 ylidine)amino]-4-thiazole acetic acid, is a compound which selectively inhibits rhinovirus in cell cultures. The compound, which was unable to inactivate infectivity of virions, appeared to act prior to RNA and protein synthesis without affecting adsorption or penetration of virus in MRC5 cells. In contrast, it protected intracellular [5-3H]uridine-labeled virions against the effects of RNase treatment, indicating that inhibition of virus uncoating is the mode of action of 44 081 R.P. PMID- 3019237 TI - Analysis by using DNA probes of the OXA-1 beta-lactamase gene and its transposon. AB - From recombinant clone pTY27, encoding an OXA-1 beta-lactamase gene, we performed subcloning experiments to more precisely delimit the gene. We describe the use as probes of six different restriction fragments known from subcloning experiments to be within the structural gene or part of the transposable element, Tn2603, flanking the OXA-1 determinant. We showed that the OXA-1 structural gene is slightly related to the OXA-2 determinant and also that sequences within Tn2603 are common to all the OXA- and PSE-producing strains tested. For epidemiological purposes, we began nucleotide sequencing of the OXA-1 determinant, and from preliminary sequence data we synthesized an oligonucleotide 15 bases in length, corresponding to a sequence within the OXA-1 gene. This oligonucleotide was found to be specific for the OXA-1 determinant, because it hybridized only to bacteria producing that beta-lactamase. PMID- 3019235 TI - Synergistic inhibition of human T-cell lymphotropic virus type III replication in vitro by phosphonoformate and recombinant alpha-A interferon. AB - Phosphonoformate and recombinant alpha-A interferon synergistically inhibited the replication of human T-cell lymphotropic virus type III in cultured peripheral blood lymphocytes. T-cell proliferative capability was maintained by this combination, and toxicity was minimal. PMID- 3019238 TI - Antiherpetic effects of a human alpha interferon analog, IFN-alpha Con1, in hamsters. AB - The efficacy of a novel consensus form of human alpha interferon designated IFN alpha Con1 was evaluated against herpesvirus infections in vitro and in vivo. At comparable antiviral concentrations, natural lymphoblastoid IFN, IFN-alpha Con1, the molecular subtype IFN-alpha, and the hybrid IFN-alpha AD(Bgl) obtained by recombinant DNA methods conferred similar protection against herpes simplex virus type 1 and type 2 (HSV-2) infections of human cells in vitro. Whereas 7 X 10(5) U of IFN-alpha AD(Bgl) administered in 7 intraperitoneal (i.p.) doses between -4 and 96 h relative to infection protected 90% of mice from a lethal HSV-2 infection, a similar treatment regimen with IFN-alpha Con1 conferred no protection. Systemic HSV-2 infection of hamsters was rapidly lethal, but a single i.p. treatment with 10(6) U of either IFN-alpha Con1 or IFN-alpha AD(Bgl) was highly effective and protected 90 and 75% of animals, respectively, when given 6 h before infection; treatment with IFN-alpha Con1 protected 45% of animals when administered 10 h after infection. In addition, IFN-alpha Con1 was highly protective against acute cervicovaginal HSV-2 infection of hamsters when administered either in a single i.p. dose of 10(6) U at -6 or 10 h relative to infection or in multiple i.p. doses of 10(6) U between -6 and 120 h relative to infection. Protection was manifested by a delay in the onset of and a reduction in duration of infection, a reduction in the number of positive cervicovaginal infections, and an increase in the survival rate. PMID- 3019239 TI - Effect of glutaraldehyde on the activity of some DNA restriction endonucleases. AB - The effect of the bifunctional crosslinking reagent glutaraldehyde on the activity of the restriction enzymes Bam HI, Hind III, EcoRI, and Tthlll I was investigated. The four enzymes exhibited differential sensitivity to inactivation. Tthlll I was the most sensitive, with activity losses occurring at levels of 0.0025% and above. Hind III was the most stable of the four and remained fully active at concentrations as high as 0.075%. Addition of BSA to incubation mixtures generally had a stabilizing effect. Implications of these results for the design of glutaraldehyde-based methods for the immobilization of restriction endonucleases are discussed. PMID- 3019240 TI - Cyclic AMP activation of a triglyceride lipase in broken cell preparations of rat heart. AB - The effect of CAM [cyclic AMP, Mg-ATP, and 3-isobutyl, 1-methylxanthine (MIX)] on triacylglycerol (TG) lipase activity in extracts from heparin-perfused rat heart was determined. TG lipase activity in homogenate, 10,000g supernatant, 105,000g supernatant, ammonium sulfate supernatant, and the eluate from heparin-Sepharose was increased between 62 and 151% when incubated with a combination of 0.3 mM cyclic AMP, 5 mM MgCl2, and 2 mM ATP. The addition of Mg-ATP + cyclic AMP caused a greater activation of TG lipase in the various fractions than did Mg-ATP + MIX or cyclic AMP + MIX. These results suggest that activation may be mediated by the classical cyclic AMP-protein kinase cascade. Control and CAM-stimulated activities were increased by heparin and inhibited by NaCl and protamine sulfate. In the absence of serum in the assay, the CAM system caused a relatively greater stimulation of lipolytic activity in each fraction compared to when serum was present in the assay. However, the absolute values were 6.1 to 16.3-fold greater with serum in the assay than without serum. In a similar manner, TG lipase activity was stimulated by CAM between 1.75 and 4.26-fold at pH 7.4, and only between 1.62 and 2.51-fold at pH 8.1. However, the absolute values at pH 8.1 were 6.77 to 31.83-fold greater than those seen at pH 7.4. These data demonstrate, for the first time, the cyclic AMP activation of a TG lipase above basal levels in cell-free fractions of rat heart. It is intriguing to speculate that the intracellular fraction of lipoprotein lipase may play a role in the hormonal regulation of cardiac TG lipolysis. PMID- 3019241 TI - Analysis of the peroxidatic mode of action of catalase. AB - Catalase is an enzyme which can function either in the catabolism of hydrogen peroxide or in the peroxidatic oxidation of small substrates such as ethanol, methanol, or elemental mercury (Hg0). It has been reported that native catalase can peroxidatically oxidize larger organic molecules (e.g. L-dopa) and that catalase maintained at alkaline pH for various lengths of time demonstrates an increase in peroxidase activity using guaiacol as substrate. We have shown, by using two distinct methods of H2O2 introduction for measuring peroxidase activity, that native catalase shows no peroxidatic activity toward these larger organic molecules. We have also shown, through the use of these peroxidase assays and by enzyme absorption spectra, that the peroxidase activity attributed to catalase maintained at alkaline pH is a catalytic but not enzymatic activity associated with a hematin group attached to a denatured catalase monomer. Possible mechanisms for the catalytic and peroxidatic modes of action of catalase involving hydride-ion transfer are discussed. PMID- 3019242 TI - Tracheal carbohydrate antigens identified by monoclonal antibodies. AB - In a previous study we described a family of monoclonal antibodies directed against tracheal antigens having a variety of cellular and subcellular distributions. In the present study, we have extended our findings on four representative antibodies to determine the periodate sensitivity, glycosidase sensitivity, and apparent molecular weight of the corresponding antigens. Since mild periodate oxidation selectively cleaves carbohydrate moiety leaving amino acids intact, loss of antigenicity following this treatment suggests the involvement of sugar residues in the antigenic determinant. This can be confirmed by testing the sensitivity of the antigens to specific glycosidases. By enzyme linked immunosorbent assay (ELISA), all four antibodies were found to have highest affinity for void volume components isolated by Bio-Gel A15m chromatography of the total tracheal secretion. Further analysis of this void volume material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions followed by immunoblot analysis revealed that all antigens were carried by high-molecular-weight species (greater than 200,000) which were periodate-Schiff positive but reacted poorly with Coomassie blue. In parallel experiments using immunofluorescence and ELISA, antibody binding was compared under control conditions and following periodate treatment of antigens under varying intensities (10 mM IO4-, 10 min, 4 degrees C; 50 mM IO4-, 1 h, 4 degrees C; 100 mM IO4-, 12 h, 20 degrees C). Similar results were obtained with the two methods, indicating a partial loss of antigenicity for one of the four antigens following the mildest periodate treatment, and total loss of antigenicity for all four antigens following each of the two prolonged treatments. All four antigens showed marked sensitivity to digestion with mixed exoglycosidases and three antigens were also susceptible to endo-beta galactosidase digestion. Antigenicity was not decreased during incubation with chondroitinase ABC, heparitinase, or heparinase. Immunofluorescence analysis of tracheal tissue sections showed that the four antibodies recognized determinants in different locations, including gland and goblet cell cytoplasmic granules and the apical epithelial membrane. The characteristic immunofluorescence patterns of all antibodies were abolished by periodate incubation of the tracheal sections. Thus, the four antibodies appear to recognize carbohydrate antigens carried by high-molecular-weight glycoproteins, each with different cellular origins. PMID- 3019243 TI - Characterization and affinity crosslinking of receptors for tumor necrosis factor on human cells. AB - Receptors for tumor necrosis factor (TNF) were characterized in the U-937 human histiocytic lymphoma cell line with the aid of highly purified recombinant human TNF, radiolabeled with 125I. Saturation binding to specific cell surface receptors occurred with less than 15% nonspecific binding. Analysis of the equilibrium binding data obtained at 4 degrees C revealed a single class of noninteracting binding sites. The mean number of binding sites per cell was calculated to be 12,000, and the apparent dissociation constant (Kd) was 2 X 10( 10) M. Crosslinking of 125I-TNF to the cell surface receptor with disuccinimidyl suberate, followed by NaDodSO4-polyacrylamide gel electrophoresis of the cell lysate, revealed a TNF-receptor complex with a molecular weight of approximately 100,000. Binding to concanavalin A-Sepharose suggested that the TNF receptor is a glycoprotein. PMID- 3019244 TI - Effects of metal ions on sphingomyelinase activity of Bacillus cereus. AB - Some divalent metal ions were examined for their effects on sphingomyelinase activity of Bacillus cereus. The enzyme activity toward mixed micelles of sphingomyelin and Triton X-100 proved to be stimulated by Co2+ and Mn2+, as well as by Mg2+. Km's for Co2+ and Mn2+ were 7.4 and 1.7 microM, respectively, being smaller than the Km for Mg2+ (38 microM). Sr2+ proved to be a competitive inhibitor against Mg2+, with a Ki value of 1 mM. Zn2+ completely abolished the enzyme activity at concentrations above 0.5 mM. The concentration of Zn2+ causing 50% inhibition of the enzyme activity was 2.5 microM. Inhibition by Zn2+ was not restored by increasing concentrations of Mg2+ when the concentration of Zn2+ was above 10 microM. Ba2+ was without effect. When sphingomyelinase was incubated with unsealed ghosts of bovine erythrocytes at 37 degrees C, the enzyme was significantly adsorbed onto the membrane in the presence of Mn2+, Co2+, Sr2+ or Ba2+. Incubation with intact or Pronase-treated erythrocytes caused enzyme adsorption only in the presence of Mn2+. In the course of incubation, the enzyme was first adsorbed on the membranes of intact bovine erythrocytes in the presence of Mn2+; then sphingomyelin breakdown proceeded with ensuing desorption of adsorbed enzyme. Hot-cold hemolysis occurred in parallel with sphingomyelin breakdown. In this case, the hydrolysis of membranous sphingomyelin as well as the initial enzyme adsorption took place in the following order: unsealed ghosts greater than Pronase-treated erythrocytes greater than intact erythrocytes. PMID- 3019245 TI - [The organization of metabolism : subcellular localization of glycolytic enzymes]. AB - The subject of cellular metabolic organization (with focus on carbohydrate metabolism) is reviewed. The existence of a "soluble" phase in the cell is considered unlikely. A note of caution regarding metabolic compartmentation as shown by isotope studies is presented. Emphasis is given to the description of experiments purporting to show the influence of protein crowding, interactions between glycolytic enzymes, and reversible association to structural proteins or to organelles. The transient nature of those associations is considered to be particularly relevant to the organization and regulation of glycolysis. The proposed role of isozymes as structural determinants of metabolic compartmentation is stressed. A few studies based on immunocytochemical observations of glycolytic enzymes are briefly described. A list of questions whose answers are badly needed is presented. PMID- 3019246 TI - Fructose 2,6-bisphosphate in the control of metabolism. PMID- 3019247 TI - Activation of fructose-1,6-bisphosphatases by monovalent cations and its relationship with a fructose-2,6-bisphosphate allosteric site. AB - The effects of potassium ions on pig kidney fructose-1,6-bisphosphatase activity have been studied. At low (non-inhibitory) concentrations of fructose-1,6 bisphosphate K+ shows an inhibitory effect and the apparent Km for fructose-1,6 bisphosphate increases as the concentration of monovalent cation increases. The inhibition by high substrate concentrations is decreased by addition of the potassium ions. Modification of a highly reactive cysteine residue with cyanate or N-ethylmaleimide results in the loss of activation of the enzyme by K+. Significant protection to the loss of potassium activation and substrate inhibition is afforded by the presence of low concentrations of fructose-2,6 bisphosphate or inhibitory levels of fructose-1,6-bisphosphate. Non-inhibitory concentrations of the substrate give partial protection against the loss of monovalent cation activation. The inhibitor AMP markedly increases the reactivity of the cysteine residue. The carbamoylated enzyme is not inhibited by excess of Mg2+ as compared to native enzyme. The results suggest that K+ decreases the affinity of the enzyme for fructose-1,6-bisphosphate at both the catalytic site and an allosteric site for fructose-2,6-bisphosphate. Furthermore, they lead to the proposal that monovalent cations activation could be due to the removal of both Mg2+ and substrate inhibitions. PMID- 3019248 TI - [Concurrence of multiple and integrated mechanisms in the modulation of enzyme activities: significance for the regulation of metabolic fluxes]. AB - The activity of some enzymes in a given metabolic pathway is modulated through multiple mechanisms, which operate in a simultaneous and coherent way to produce either stimulation or inhibition. The operation of these mechanisms is illustrated with several enzymes involved in glucose metabolism, by choosing examples from the presentations at the Symposium. Thus the reciprocal interactions of the regulatory mechanisms acting upon hexokinase D ('glucokinase'), phosphofructokinase, fructose 1,6-bisphosphatase and pyruvate kinase were discussed, as well as their relationships with the induction of enzyme conformational changes. In addition, the effects of covalent interconversions on glutamine synthetase activity were briefly analyzed. An outstanding feature exhibited by all these enzymes is the display of a great number of elasticity coefficients, which are differential quotients measuring the dependence of enzymatic activity on each variable that modulates it. A general assumption is that these enzymes make an important contribution to the control of the metabolic flux in which they participate. The flux control, however, appears to be shared in different degrees by all the components of the system, and may be quantified through the differential quotient denominated control coefficient. Some of the problems that emerge in any attempt to estimate these coefficients in the living cells are discussed. The problems derive partly from the complex subcellular structure, the formation of functional compartments resulting from reversible association of the enzymes, one to another and to different cellular components, and the actual state of cell water. These problems make that the results obtained with purified and highly diluted enzymes in most enzymological studies should not be extrapolated directly to what happens in vivo, without a careful evaluation of each particular case. The regulatory role of enzyme activity of fructose 2,6-bisphosphate and its eventual participation as an intermediary in the hormonal control of glycolytic and gluconeogenic fluxes are emphasized. The regulation of yeast fructose 1,6-bisphosphate activity is discussed in relation to the eventual role of allosteric modulators and covalents interconversions as signals for the initiation of intracellular degradation of the enzyme during catabolic inactivation. PMID- 3019249 TI - [The protocol of informing cancer patients of their condition]. AB - The seven points we must consider in informing cancer patients of their condition are as follows: It is necessary that informing the patient has one or more of the following purposes: Patients have a right to know the truth. Any patient who desires to know the nature of his own disease has a right to be told. To avoid legal action. In cases where information is uncertain, which may cause difficulty for the patient. To avoid the burden of fabricating an untrue explanation. To help the patient bear radical treatment such as surgery, radiation and/or chemotherapy. To allow the patient to put an end to his/her life's work or finalize problems of property. To help the patient see the need to change his/her way of life. Are the conditions sufficient or not? Almost all these conditions must be clear. Is there any constructive purpose in informing the patient? Does the patient have a rational and receptive character? Is the patient in a mentally stable condition? Is these personal experience of death in the patient's family. Does the patient have a sure faith, thoughts and/or views of life and death? Is there enough cooperative work among the medical team? Is there enough cooperative work among the patient's family? Who tells the truth to the patient? Is there a need to inform the patient in stages? At what stage of the disease is the patient told? Where is the patient told? Comprehensive care must be given after informing the patient of his/her condition. PMID- 3019250 TI - [Pathological variability and cancer treatment in lung cancer]. AB - It is well known that the biological behavior of lung cancer varies according to histological type and growth pattern. Therefore, more precise analysis of various morphologic features affecting prognosis are needed based on tumor histology. Lung cancer is mainly classified into 4 major histological types; squamous cell carcinoma, small cell carcinoma, adenocarcinoma and large cell carcinoma. Recently lung cancer has been subclassified into small cell carcinoma and non small cell carcinoma on the basis of responsiveness to chemotherapy and radiation therapy. In small cell carcinoma, combination of chemotherapy and radiation therapy is the main modality for treatment, and the most important prognostic factor is LD and ED stage classification. Even so, a new histological classification is proposed according to the responsiveness to chemotherapy and radiation therapy. On the other hand, surgical therapy is the first choice for the treatment of non-small cell lung cancer and the most important prognostic factor is TNM and related stage classification. In squamous cell carcinoma, moreover, extended resection is sometimes tried because of its local invasiveness. Early lung cancer of hilar type is mostly squamous cell carcinoma and highly curative by resection. The biological behavior of adenocarcinoma is the most variable among lung cancers. In the histopathological study of surgically resected cases, moreover, histological differentiation, nuclear atypia of tumor cells, mitotic frequency, grades of scarring associated with a tumor, and degrees of infiltration of T-zone histiocytes is closely related to prognosis. Scoring of these factors may help a clinician to reach a decision on the necessity and type of postoperative adjuvant chemotherapy, particularly in cases of Stage I adenocarcinoma. Most cases of large cell carcinoma including giant cell carcinoma ultrastructurally reveal features of differentiation toward adenocarcinoma and/or squamous cell carcinoma. Giant cell carcinoma shows the most unfavorable prognosis because of its rapid growth. However, among operable cases of giant cell carcinoma, some long-term survivors do exist. Evaluating these forms of biological behavior according to tumor histology at the time of treatment, it is easier to decide whether or not adjuvant therapy is necessary. PMID- 3019251 TI - [Intra-arterial administration of epirubicin in the treatment of non-resectable hepatocellular carcinoma. Epirubicin Study Group for Hepatocellular Carcinoma]. AB - A group study was conducted to investigate the effect of intra-hepatic arterial administration of epirubicin in the treatment of non-resectable hepatocellular carcinoma(HCC). Sixty-four patients were entered into the study. There were 51 males and 13 females, with an age range from 32 to 79 years, the average being 59.1 years. Fifty-four patients had associated cirrhosis of the liver. Epirubicin at a dose of 60-90 mg/m2 was infused as a bolus into the hepatic artery 1 to 4 times (average 1.8) at an interval of 3 weeks to 3 months. Tumor size was properly evaluated in 53 patients. There were 1 CR, 7 PR, 7 MR, 27 NC, and 11 PD. Thus, the response rate (CR + PR) was 15.1%. Seventeen patients are still alive 305 to 720 days (mean 505 days) after the initial treatment. The most common side effects of this drug were bone marrow suppression, gastrointestinal complaints, and alopecia. Cardiac toxicity was negligible at the doses used in this study. A retrospective comparison of the present results with those for patients treated by intra-arterial administration of doxorubicin demonstrated that epirubicin is more effective than doxorubicin in terms of survival rate. The current study indicates that epirubicin may be a promising alternative in the treatment of HCC. The appropriate dosage of the drug and the interval for infusion are to be elucidated. Also, the combination of epirubicin with other cytostatic drugs is open for study. PMID- 3019252 TI - [A phase II study of epirubicin in advanced lung cancer]. AB - A phase II study of epirubicin (EPI) was performed on 65 eligible lung cancer patients. EPI was administered intravenously at the dosage of 60 mg/m2 for non small cell lung cancer (NSCLC) and 75 mg/m2 for small cell lung cancer (SCLC) at the interval of three to four weeks. The response rate of 57 measurable patients was 7.0% (predicted true response rate was 1.9-17.0%). In SCLC, the overall response rate was as low as 8.3% (2/24), but a better response rate, 20% (2/10), was observed in patients given no prior therapy. In NSCLC, the response rate was 6.1% (2/33) as a whole and 12.5% (2/16) in patients given no prior therapy. We were able to conclude that EPI was a moderately active agent in SCLC and marginally effective in NSCLC. Although the dose limiting factor was leukopenia, its incidence and degree seemed low as compared with adriamycin (ADM). As for alopecia or GI tract side effects, those associated with EPI appeared less acute than those produced by ADM. No cardiac toxicities were observed, partly because of the low cumulative maximum dose of 311 mg/m2 used in our study. PMID- 3019253 TI - [A case of hepatoma with a remarkable response to gamma-interferon administration]. AB - A 59-year-old male was admitted to our hospital with complaints of general fatigue, abdominal distension and edema in the legs in February, 1985. Laboratory findings were as follows: GOT 152 IU/l, GPT 129 IU/l, LDH 555 IU/l, ALP 1147 IU/l, gamma-GTP 413 IU/l, T.-Bil 2.1 mg/dl and AFP 422.6 ng/ml. Multiple SOLs were recognized in both lobes of the liver by abdominal CT scan and echography. Interferon-gamma (gamma-IFN: KW-2202; Kyowa Hakko Co.) therapy was started in March from an initial dose of 1 X 10(6) units and was increased up to 4 X 10(6) units, 2 X 10(6) units being administered as a maintenance therapy for 12 weeks. The tumors became remarkably smaller in size, AFP was decreased to 38.8 ng/ml, and PR was obtained. The only side effect was temporary fever. The patient was subsequently followed without gamma-IFN at an outpatient clinic for about 100 days, but finally died due to rupture of esophageal varices and hepatic failure. PMID- 3019254 TI - [A randomized comparison of radiotherapy with CAP and CVP in non-small cell lung cancer]. PMID- 3019255 TI - Monoclonal antibodies to the beta-adrenergic receptor: modulation of catecholamine-sensitive adenylate cyclase by the antibody. AB - We have developed two types of hybridomas producing monoclonal antibodies to the turkey erythrocyte beta 1-adrenergic receptor in order to study the beta adrenergic-cAMP system of epidermis. Splenic cells from BALB/c mice immunized with partially purified turkey erythrocyte beta 1-receptors were fused with mouse myeloma cell line SP2/0-Ag14. Five hybridomas of 17 positive cells producing antibodies which could precipitate soluble turkey erythrocyte beta 1-receptors were cloned by the limiting dilution method. The antibodies cross-reacted with beta 1- and beta 2-adrenergic receptors and stained epidermal basal cells with immunocytochemical techniques. Neither type of antibody interfered with the antagonist binding, i.e., all antibodies bound to sites other than the ligand binding site on the surface. One type of antibody inhibited epinephrine stimulated adenylate cyclase activity in our "leaky" epidermal cell system. The data suggest that the antibody interferes with the coupling of the receptor to the regulatory protein. PMID- 3019256 TI - DNA polymerase activity in the n.hexadecane-induced hyperkeratotic epidermis. AB - DNA polymerase activity was determined in hyperkeratotic epidermis of the guinea pig by topical application of n.hexadecane. Epidermal cells were separated by Percoll gradient centrifugation. DNA polymerase alpha activity was higher in the basal cells, but polymerase beta activity was higher in granular cells than in cells from the other layers. The hyperkeratosis was accompanied by an increase in polymerase alpha activity in both the squamous and basal cells. However, polymerase beta activity decreased in the granular cells and was distributed almost uniformly across the epidermis. The distribution pattern of polymerase activities in the hyperkeratotic epidermis was not simply an enhancement of the pattern in normal epidermis. PMID- 3019258 TI - The effect of sublethal cyanide exposure on plasma vitellogenin levels in rainbow trout (Salmo gairdneri) during early vitellogenesis. PMID- 3019257 TI - Effect of selective enzymatic digestions on skin biopsies from pseudoxanthoma elasticum: an ultrastructural study. AB - Skin biopsies from patients with pseudoxanthoma elasticum (PXE) were studied by electron microscopy either before or after selective digestions with collagenase, elastase, trypsin, hyaluronidase, chondroitinase AC and ABC, with the aim of identifying an eventual organic component associated with mineralization within the elastin fibers and the chemical nature of the enormous aggregates of filaments very often associated with, but distinct from mineralized elastin fibers. The results obtained, on both embedded thin sections and fresh tissue fragments, showed that elastin fibers, whether mineralized or not, were sensitive only to elastase, and they did not contain significant amounts of materials different from elastin that could be accounted for by ion precipitation; the aggregates of microfilaments in strict connection with altered elastin fibers were mostly sensitive to elastase and hyaluronidase, were partially removed by trypsin and chondroitinase, and were not modified by collagenase, which seems to indicate that the microfilaments consist mainly of abnormally aggregated elastin molecules together with low sulfated proteoglycans. It may be concluded that PXE is a complex genetic disorder of the connective tissue, and that mineralization of elastin is only one of the alterations of the extracellular matrix. PMID- 3019259 TI - [Multilocular cystic nephroma. Apropos of a case]. PMID- 3019261 TI - Studies on epinephrine-induced lung edema in the rat. I. Selective alpha 1 adrenoceptor involvement. AB - Phentolamine (Phe) prevents the induction by epinephrine (E: 1800 nmol/kg, i.v.) of lung edema (LE) in urethane-anesthetized and bivagotomized rats in a dose related manner (from 246 to 3933 nmol/kg, i.v.). Since Phe blocks and E activates both alpha 1- and alpha 2-adrenoceptors, the evidence does not allow us to link LE to selective (alpha 1 or alpha 2) or non-selective (alpha 1 + alpha 2) alpha adrenoceptor activation. Accordingly, we tried to find out whether: phenylephrine (PE), a selective alpha 1-adrenoceptor agonist, and B-HT 920, a selective alpha 2 adrenoceptor agonist, would cause LE; prazosin (Praz), a selective alpha 1 adrenoceptor antagonist, and yohimbine (Yoh), a selective alpha 2-adrenoceptor antagonist, would protect rats against LE caused by E or PE. We found that: 1) PE (from 736 to 5892 nmol/kg, i.v.), but not B-HT 920 (from 190 to 12200 nmol/kg, i.v.), caused LE, while both drugs increased arterial blood pressure; 2) Praz prevented induction of LE, whether by E (1800 nmol/kg, i.v.) or by PE (5892 nmol/kg, i.v.), in a dose-related manner (from 15 to 119 nmol/kg, i.v.). In contrast, Yoh was ineffective at doses up to 7675 nmol/kg, i.v. We conclude, therefore, that E-induced LE in urethane-anesthetized and bivagotomized rats strictly depends on alpha 1-adrenoceptor activation. The outcome of E-induced LE is usually rat death, the incidence of which depends on the dose. Since alpha 1 adrenoceptor agonists rank in the same order of potency for the induction of death as for that of LE, and since Phe and Praz protect from death at LE preventing doses, there seems to be some link between LE and death, even though protection against death is obtained with doses of antagonists lower than those abolishing induction of LE. Finally, alpha 1-agonists cause maximum arterial hypertension at all doses used, irrespective of induction of LE and of protective pretreatment against LE. PMID- 3019262 TI - Pharmacologic evidence that high dose chronic amitriptyline and desipramine down regulate alpha 2-receptor-mediated hypothermia in the rat. AB - The effects of a three week course of treatment of amitriptyline (AMI) and desipramine (DMI) (3.2 and 10 mg/kg, s.c., b.i.d.) were studied on the hypothermic response to clonidine (0.1 mg/kg, s.c.) in male adult albino rats. Single doses of clonidine in normal unmedicated rats produced a marked (about 2 degrees C) fall in body temperature. In contrast, 0.9% NaCl control injections had no significant effect. The clonidine-induced hypothermia was prevented by yohimbine pretreatment (3.2 mg/kg, i.p., 1/2 hr before), suggesting an alpha 2 mediated response. On days 8 and 14 of chronic tricyclic antidepressant (TCA) treatment, the hypothermic response was prolonged. Four days after the abrupt withdrawal of 20 days of TCA treatment (10 mg/kg, s.c., b.i.d.), the clonidine induced hypothermia response was attenuated consistent with down-regulation of alpha 2-receptors. This latter effect was not observed with a dose of 3.2 mg/kg, s.c., b.i.d. of either TCA. PMID- 3019263 TI - Prejunctional opioid receptors in the pulmonary artery of the rabbit. AB - The possible existence of prejunctional opioid receptors at postganglionic sympathetic vasoconstrictor axons was studied in superfused spiral strips of the main pulmonary artery of the rabbit. Ethylketocyclazocine 0.1 and 1 mumol/l and dynorphin-(1-13) 0.1 mumol/l but not morphine 0.01-1 mumol/l, (D-Ala2, NMePhe4, Gly-ol5)-enkephalin 0.001-1 mumol/l, Leu5-enkephalin 0.1-5 mumol/l and (D-Pen2, L Pen5)-enkephalin 0.01-1 mumol/l reduced contractions of the artery strips elicited by electrical field stimulation at 0.5 or 1 Hz. The effect of ethylketocyclazocine was antagonized by naloxone. Ethylketocyclazocine 1 mumol/l did not change contractions elicited by exogenous noradrenaline (5-10 nmol/l). In artery strips preincubated with 3H-noradrenaline, ethylketocyclazocine 1 mumol/l diminished both the overflow of tritium and the contraction evoked by field stimulation at 1 Hz. All inhibitory effects were small, amounting maximally to about 20%. The results indicate that the postganglionic sympathetic axons of the artery possess prejunctional opioid receptors, activation of which leads to inhibition of the release of noradrenaline and, in consequence, inhibition of neurogenic vasoconstriction. It seems likely that the receptors are, at least predominantly, of the kappa-type. PMID- 3019260 TI - A systematic study of host defense processes in badly injured patients. AB - A prospective study of factors predisposing to infection in badly injured patients has disclosed: the dominant roles of two specific parameters: monocyte antigen presenting capacity, and opsonic capacity of diluted serum; the potential value of further assessment of: the predictive value of plots of activated T cells/total T-cells versus monocyte antigen presenting capacity, the apparent protective effect of the ability to sharply increase specific IgM in response to infection, and the apparent protective effect of cytomegalovirus (CMV) infection in the first 28 days after injury against major bacterial infection; the lack of value of analysis of other T- and B-cell subsets in such patients; and the need to clarify CMV and transfusion status with respect to interpretation of such data. The specific role of variable transfusion and of specific serum immunoglobulins will require further and more discriminating study. PMID- 3019264 TI - Therapy-related leukemia and myelodysplasia in small-cell lung cancer. Report of a case and results of morphologic, cytogenetic, and bone marrow culture studies in long-term survivors. AB - The bone marrow of 11 patients with small-cell lung cancer, who survived more than two years following combined-modality therapy, was subjected to morphologic, cytogenetic, and bone marrow culture studies. One patient, after a prodrome of anemia and thrombocytopenia, developed acute leukemia 60 months after the start of chemotherapy. Four months before frank leukemia developed, bone marrow culture studies showed a marked inability to form colonies. Cytogenetic studies demonstrated an abnormal clone of cells that included the deletion of the long arm of chromosome 5. No morphologic abnormalities were noted in the bone marrow of any other long-term survivor; however, the mean corpuscular volume of peripheral red blood cells was greater than normal in three of four patients who remain alive and disease free. In one of these patients marrow culture studies also failed to grow colonies. The other patients showed a decreased ability to form multilineage colonies and colonies of the granulocyte-macrophage lineage in vitro compared with a control population. All patients showed some degree of aneuploidy on cytogenetic analysis; in two cases approximately 50% of cells were aneuploid. However, no clonal abnormality was detected in any patient. Follow-up for the development of secondary acute leukemia and other long-term complications continues in these patients. PMID- 3019266 TI - [Facial dystonias. Review of a series of 21 cases]. PMID- 3019265 TI - Properties of the purified APS-kinase from Escherichia coli and Saccharomyces cerevisiae. AB - Adenylylsulphate kinase (EC 2.7.1.25, ATP:adenylylsulphate 3'-phosphotransferase) has been isolated from Escherichia coli and from Saccharomyces cerevisiae. As major steps of purification, affinity chromatography on Sepharose CL 6B ("blue" or "red") and chromatofocusing on polybuffer PBE 94tm were employed. The proteins were obtained in nearly homogeneous state after five chromatographic steps. The isolated enzymes from both sources appeared predominantly to exist as dimers. Upon reduction of the protein with dithiothreitol, it disintegrated into assumingly identical smaller subunits (E. coli rom Mr 90-85,000 to 45-40,000 and S. cerevisiae from 52-49,500 to 28-29,500). Both forms, dimer and monomer were found catalytically active. Preincubation of the isolated enzyme from either source in the presence of thioredoxin plus DTT, reduced glutathione or DTT increased the activity significantly. Treatment of the enzyme with SH-blocking reagents inactivated the enzyme irreversibly as compared to the inactivation caused by oxidants (2,6-dichlorophenol-indophenol, ferricyanide or oxydized glutathione). This oxidant induced inactivation was less pronounced for the fungal enzyme than for the bacterial protein. The enzyme from E. coli required thioredoxin in order to alleviate the GSSG-induced inactivation. PMID- 3019268 TI - Increased serotonin2 and beta-adrenergic receptor binding in the frontal cortices of suicide victims. AB - A statistically significant 28% increase in the mean (+/- SD) number of serotonin2 receptors (127.8 +/- 13.4 vs 99.6 +/- 11.1 fmol/mg of protein) and a 73% increase in beta-adrenergic receptor binding (14.5 +/- 1.5 vs 8.4 +/- 1.5 fmol/mg) was found in the frontal cortices of violent suicide victims compared with matched controls. No significant differences were found in the number of serotonin1 binding sites (109.5 +/- 13.4 vs 99.9 +/- 8.8 fmol/mg). We have previously reported a reduced density of presynaptic tritiated imipramine binding sites on serotonergic nerve terminals in the frontal cortices of suicide victims. These data support the hypothesis that suicide completed by violent methods is associated with reduced presynaptic serotonergic activity that has generated compensatory upregulation of the postsynaptic serotonin2 receptor sites. The increase observed in beta-adrenergic binding suggests that there may also be a concomitant reduction in presynaptic noradrenergic activity associated with suicide. If antidepressant pharmacotherapies specifically downregulate cortical beta-adrenergic and/or serotonin2 receptors in depressed subjects, as has been demonstrated in animal studies, and since these effects would be in the opposite direction of the receptor changes found in suicide victims, they may account for the therapeutic action of antidepressants on suicidal behavior and depressive disorders. PMID- 3019267 TI - Electroconvulsive shock therapy and maximum binding of platelet tritiated imipramine binding in depression. AB - We studied the tritiated imipramine binding values in platelets from 12 hospitalized untreated patients with endogenous depression and found a significant decrease in maximum binding (Bmax) values compared with a control population of the same age and sex. There were no changes in the equilibrium dissociation affinity constant values between the untreated depressives and the control population. After at least six sessions of electroconvulsive therapy and at the time when a significant clinical improvement of depression was confirmed, the Bmax value of tritiated imipramine binding in platelets was slightly increased but was still significantly below that of the control values. However, when six of these patients were reexamined after 12 to 18 months, at a time when they were euthymic, the Bmax of tritiated imipramine binding in platelets was found in the same range as the values of the control population. Our results indicate that clinical improvement precedes the changes in Bmax of tritiated imipramine binding in platelets from depressed patients. The tritiated imipramine binding in platelets is a useful biologic marker in affective disorders. Furthermore, our results suggest that tritiated imipramine binding in platelets may be a state-dependent biologic marker in depression. PMID- 3019270 TI - Imaging of neurotransmitter receptors in the living human brain. PMID- 3019269 TI - Prostaglandin receptor sensitivity in psychiatric disorders. AB - The cyclic adenosine monophosphate (cAMP) responses to prostaglandin E1 (PGE1) in platelets and leukocytes from drug-free schizophrenic patients, depressive patients, and normal controls have been compared. Both schizophrenic and depressive patients had a significantly lower platelet cAMP response to PGE1 than controls. The platelet cAMP response to PGE1 did not discriminate among exacerbated, remitted, and poor-prognosis schizophrenic patients, or between exacerbated and remitted depressive patients. The cAMP response to PGE1 was negatively correlated with global symptom severity in actively ill schizophrenic patients, but was not correlated with symptom severity in exacerbated depressive patients. The leukocyte cAMP response to PGE1 did not differ among normal controls, schizophrenic patients, and depressive patients. These data indicate that a diminished platelet cAMP response to PGE1 may be a marker common to both schizophrenia and depression but that this effect does not extend to a cAMP linked PGE1 receptor on another blood cell type. PMID- 3019271 TI - Alpha 2-adrenergic receptor function in depression. The cortisol response to yohimbine. AB - There is evidence that the abnormalities in hypothalamic-pituitary-adrenal (HPA) axis function observed in patients with depression may be related to changes in central neurotransmitter receptor function. To evaluate this possibility further, the alpha 2-adrenergic receptor antagonist yohimbine hydrochloride, which increases brain norepinephrine turnover, was administered to 40 patients with DSM III major depression (18 melancholic, 22 nonmelancholic) and 16 healthy controls. Plasma free 3-methoxy-4-hydroxyphenylglycol (MHPG) level was measured as an index of noradrenergic function, and plasma cortisol level was used to assess the HPA response. Baseline cortisol levels were elevated in melancholic depressed patients, but not in nonmelancholic patients, when compared with healthy controls. The cortisol response to yohimbine was significantly greater in depressed patients than in controls, despite similar MHPG responses between groups. Since there is evidence that stimulation of postsynaptic alpha 2 adrenergic receptors inhibits HPA axis function, the abnormally increased cortisol response to the alpha 2-antagonist yohimbine suggests a relative subsensitivity of postsynaptic alpha 2-adrenergic receptors in depression. PMID- 3019272 TI - High resolution image analysis of cell nuclei in tissue sections of primary and metastatic carcinomas. AB - The present study examines whether certain histological tumour types can be differentiated on account of their nuclear image with the aid of automated image analysis. For karyometric investigations three tumour types (adenocarcinomas, squamous cell carcinomas and mammary carcinomas) were chosen, which occur frequently as occult primary tumours. From each type ten primary tumours with their corresponding lymph node metastases were examined. 100 cell nuclei were measured from each case using 4 micron thick paraffin sections stained with gallocyanin-chromalum. For each cell nucleus 21 contour and texture features were determined. Through the application of linear classifiers 41 out of 52 cases (25 primary tumours, 27 metastases) of these three tumour types were correctly classified. Eight cases could not be classified with certainty and only three cases were wrongly classified. In addition, within the group of adenocarcinomas differences due to localisation were detected which allow us to draw conclusions on the seat of the primary tumour. PMID- 3019273 TI - [Signet-ring-cell lymphoma. Light and electron microscopic study of gastric involvement]. AB - The authors report on a malignant Non-Hodgkin lymphoma of the stomach of a 73 year-old male. Histologically, it was revealed to be a follicularly growing, obviously secondary centroblastic lymphoma. Crystalline cytoplasmic inclusions in numerous centrocytes have led to displacement of the nucleus to the cell margin and thus to the picture of a signet-ring-cell lymphoma. Electronmicroscopically, there were found big crystalline electron-dense deposits in the rough endoplasmic reticulum. The finding was compared with literature data, and the histological differential diagnosis is discussed. PMID- 3019274 TI - Malignant fibrous histiocytoma arising in the liver. PMID- 3019275 TI - Evaluation of an automated hematology system (Technicon H-1). AB - We evaluated the Technicon H-1 hematology system, a new third-generation blood cell analyzer that performs a complete blood cell count, including red blood cell (RBC) morphometric studies and a full white blood cell differential count. Linearity and precision were acceptable over the entire clinical range. We compared the H-1 with the Technicon H-6000 and the Coulter S Plus IV. Close correlation was observed for all equivalent parameters except the mean corpuscular hemoglobin concentration (MCHC), the mean platelet volume, and the basophil count. Poor correlation for the MCHC reflects the novel methodology of the H-1 for measuring RBC size and, independently, RBC hemoglobin content. The H 1 MCHC correlates with hypochromia as assessed morphologically, while the MCHC of the H-6000 or S Plus IV does not. Further technological innovations include measurement of the mean neutrophil peroxidase content and nuclear lobularity. PMID- 3019277 TI - The effect of various dietary fibres on tissue concentration and chemical form of mercury after methylmercury exposure in mice. AB - The whole-body retention of mercury after exposure of BALB/c mice to methylmercury was measured in animals fed fibre-free, 5% pectin, 5% cellulose or 5, 15 or 30% wheat bran diets. The rate of elimination of mercury was dependent on the diet fed, with dietary bran increasing the rate of elimination. The incorporation of 15 or 30% bran in the diet of the mice decreased the total mercury concentration in the brain, blood and small intestine, although the effects were significant only in those animals on 30% bran diet. The fibres had little effect on mercury levels in other tissues. The proportion of mercury found in the mercuric form was significantly greater in liver, kidneys and gut of mice fed bran. The results suggest that dietary bran may reduce the levels of mercury in the brain after methylmercury exposure and may therefore reduce the neurotoxic effects of the organomercurial. We suggest that wheat bran exerts its effects on mercury retention and brain level via a modification of the metabolic activity of the gut microflora. PMID- 3019276 TI - Effect of suloctidil on rat liver. AB - The effect of suloctidil (120 mg/kg body weight PO for 3 weeks) on rat liver was investigated using biochemical and morphological methods: enzymatic activities characteristic of the main cellular compartments were used as biochemical markers of hepatocyte function and morphometry was applied to investigate morphological changes. No sign of hepatotoxicity was evidenced after suloctidil treatment (liver weight; cytochrome c oxidase; glucose 6-phosphatase; NADPH-cytochrome c reductase; D-amino acid oxidase; urate oxidase; fatty acid oxidation; peroxisomal number, volume and size distribution). Suloctidil increased catalase activity by 22% without morphologically detectable changes in the peroxisomes. After suloctidil treatment, slightly increased mitochondrial volume fraction and slightly decreased mitochondrial number were noted without significant changes in cytochrome c oxidase. Clofibrate, at the same dose, increased NADPH-cytochrome c reductase, catalase, acylCoA oxidase, mitochondrial and peroxisomal number and volume fraction, and decreased urate oxidase activity. PMID- 3019278 TI - Comparison of the primate alphaherpesviruses. I. Characterization of two herpesviruses from spider monkeys and squirrel monkeys and viral polypeptides synthesized in infected cells. AB - Biological and biochemical properties of two neurotropic herpesviruses of New World monkeys--Herpesvirus saimiri type 1 (HVS-1) and Herpesvirus ateles type 1 (HVA-1)--were examined and compared. HVS-1 and HVA-1 both exhibited a time course of replication similar to another primate herpesvirus, SA 8. Both viruses grew rapidly and high titers of infectious virus were readily produced. HVS-1 and HVA 1 were also able to replicate efficiently in cell lines derived from a number of primate and non-primate species. Analysis of proteins synthesized in infected cells revealed the presence of over 30 virus-specific proteins ranging from less than 30,000 to over 200,000 daltons apparent molecular weight. Both viruses specified synthesis of a major capsid polypeptide of 148,000 daltons. Pulse labeling of cells during infection demonstrated temporal differences in the kinetics of synthesis of individual viral proteins and post-translational modification of a number of viral polypeptides. Glycosylated polypeptides synthesized in HVS-1 and HVA-1 infected cells were identified which ranged from approximately 49,000 to 120,000 daltons. Structural polypeptides of HVA-1 and HVS 1 virions were identified by SDS-PAGE analysis of purified virions. Taken together with clinical data on the diseases caused by these viruses, these studies indicate that HVS-1 and HVA-1 appear similar in many respects to both the human herpes simplex viruses and alphaherpesviruses of other primates. PMID- 3019279 TI - Further investigation on the mode of entry of human rotavirus into cells. AB - Entry of the KUN strain of human rotavirus into MA 104 cells was studied by electron microscopy. Double-shelled rotavirus particles attached to the cell membrane, and in the presence of trypsin their nucleic acids were expelled from the virus core into the cytoplasm through radial spaces between the capsomeres and the cell membrane pores formed after their attachment. This mechanism was considered to be analogous to those of phages. PMID- 3019280 TI - Latent infection of human ovarian teratocarcinoma cells with human cytomegalovirus. Brief report. AB - Persistent infection with human cytomegalovirus (HCMV) can be established in cultures of human ovarian teratocarcinoma (PA1) cells, and maintained for more than 200 days. Infected cultures maintained at 34 degrees C (PA1CMV34) and 37 degrees C (PA1CMV37) entered crisis and subsequently displayed massive cytopathic effects (CPE), whereas infected cultures maintained at 32 degrees C (PA1CMV32) and 39 degrees C (PA1CMV39) continued to release small amounts of infectious virus until 240 or 151 days post-infection (p.i.) respectively. PA1CMV32 cultures shifted to 37 degrees C at 258 days p.i. resumed synthesis of infectious virus which resulted in cell destruction, indicating that latent infection with HCMV was maintained in PA1 cells at 32 degrees C. In contrast, PA1CMV39 cells did not produce infectious virus even when cultured at 37 degrees C for more than 100 days after the temperature shift. PMID- 3019281 TI - Effect of concanavalin A and succinyl concanavalin A on cytomegalovirus replication in fibroblasts. AB - In order to investigate inhibition of viral replication, human embryonic fibroblasts infected with cytomegalovirus (CMV) were treated with 0 to 25 micrograms/ml concanavalin A (Con A) and 0 to 150 micrograms/ml succinylated Con A (S-Con A). Alterations in cellular morphology occurred by day 2 post infection (p.i.) in cultures treated with 10 micrograms/ml Con A and 25 micrograms/ml S-Con A. With increasing concentrations of Con A and S-Con A there was decreased virus production from day 4 p.i. to day 10 p.i. Increasing levels of Con A and S-Con A also reduced the number of cells in culture. When virus titres were corrected to take cell number into account, decreased CMV titres in Con A and S-Con A treated cells appeared mainly to reflect decreased cell numbers. In support of this finding, in comparison with untreated CMV-infected fibroblasts, viral DNA synthesis was reduced and acid phosphatase levels were increased in CMV-infected cells treated with Con A or S-Con A. PMID- 3019282 TI - [Accumulation of 99mTc-pyrophosphate and structural changes in cardiac muscle during pharmacologic correction of stress injuries of the heart]. AB - It has been established that natrium oxybutirate, prolactin, inderal and ionol affecting different links of pathogenetic chain of stress injury, efficiently prevent structural damage to cardiomyocytes and the accumulation of 99mTc pyrophosphate in the myocardium. PMID- 3019283 TI - [Electron microscopic characteristics of oncocytoma of the lung, small intestine and adrenal gland]. AB - Electron microscopic characteristic of 3 oncocytomas (of lung, small intestine, adrenal) is presented. All the tumours had a similar ultrastructure with a large number of mitochondria in their cytoplasm. The electron microscopic study confirmed the diagnosis of oncocytoma in 2 cases. In one case the diagnosis of oncocytoma was made on the basis of the electron microscopic examination. PMID- 3019284 TI - [Muciparous cricoid apudoma of the breast]. AB - Four cases of muciparous cricoid-cell breast tumours with their cells containing endocrine granules are presented. The granules were detected with argyrophile reaction. Problems of histogenesis of the neoplasms are briefly discussed. They are defined as muciparous apudomas of the breast. PMID- 3019285 TI - [Diagnosis of epithelioid sarcoma]. AB - Epithelioid sarcoma is a rare soft tissue tumour. Small-nodular structure, necrotic foci in the centre of tumour nodes, moderate lymphocytic infiltration are characteristic histologic features of this tumour. Majority of tumour cells have the appearance of epithelioid histiocytes. Some tumour cells are found in small cavities resembling hyalin cartilage lacunes. Signs of a synovial, fibroblastic and histiocytic differentiation are observed ultrastructurally in the epithelioid sarcoma cells On the basis of the literature analysis and their own ultrastructural data the authors conclude that there should be a relation of this tumor with pluripotential stem cells of mesenchyma surrounding distal part of the chondral bone germ. PMID- 3019286 TI - Immunohistochemical localization of transferrin receptors in junctional and sulcular epithelium of human gingiva. AB - Transferrin receptors were found on the apical cells of the junctional epithelium and on the basal and adjacent suprabasal cells of the sulcular epithelium, consistent with the rapidly renewing character of these tissues. High density of receptors was found in the coronal portion of the junctional epithelium and on the sulcular cells adjacent to the tooth surface. This may indicate the proliferative potential of such cells rather than their actual division. An inert substratum, stimulating the tooth surface in vitro caused positive expression of the transferrin receptor, but not so extensively as in vivo. Thus factors not present in culture may normally activate the distinct non-differentiating junctional and sulcular cells to express the receptor. PMID- 3019288 TI - Orbital and choroidal metastases from carcinoma of the male breast. AB - Simultaneous uveal and orbital spread of breast carcinoma occurred in a male patient. We believe that this case is unusual because of the rarity of ocular metastases from carcinoma of the breast in men and the extremely long interval (ten years) between detection of the primary tumor and appearance of metastatic disease. PMID- 3019287 TI - The effects of calmodulin antagonists on prostaglandin E2-induced responses in rat calvarial bone cells. AB - Osteoclastic (OC) and osteoblastic (OB) cells were isolated by sequential collagenase digestions of new-born rat calvaria. Prostaglandin E2(PGE2) did not alter total calmodulin levels after a 5 or 60 min incubation. The calmodulin antagonists, trifluoperazine (TFP) at 10-50 microM and W-7 (50 microM) inhibited PGE2-induced increases in calcium uptake by OC cells, but had no effect on control OC or OB calcium levels. W-5 (50 microM), a chlorine-deficient analogue of W-7 with weak anti-calmodulin activity, had no effect. Compound 48/80 (100-500 micrograms/ml), a highly effective calmodulin antagonist in other systems, had no effect on PGE2-induced calcium levels or control calcium uptake. There was inhibition of PGE2-induced increases in cyclic AMP by compound 48/80 (100 micrograms/ml) in both OC and OB cells but no effect on control levels. TFP at 50 microM inhibited both control and PGE2-induced increases in cyclic AMP but at 10 microM it lessened only the hormone-induced effect. W-7 (100 microM) inhibited PGE2-induced increases in OC and OB cyclic AMP but had no effect on control levels; W-5 (50 microM) had no effect on either of these. Dibutyryl cyclic AMP had no effect on control calcium uptake, PGE2-induced increases or W-7 inhibition of the PGE-2 effect on calcium uptake. The calmodulin antagonists, at doses which had affected only PGE2-induced increases in calcium uptake and/or cyclic AMP production, had no effect on leucine uptake by OC or OB cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019289 TI - Effect of iron chelation on severity of ocular inflammation in an animal model. AB - Metabolites of oxygen-free radicals generated by polymorphonuclear leukocytes and macrophages are believed to inflict the initial tissue damage in acute inflammations. Of the various oxygen products, hydroxyl radicals are known to be potent toxic agents, and their generation depends largely on the presence of free iron. Treatment of experimental uveitis in Lewis rats with an iron chelator, deferoxamine mesylate, resulted in marked reduction in choroidal inflammation and suppression of retinal damage. These findings suggest that in experimental uveitis the severity of ocular inflammation and tissue damage may be mediated by the iron-catalyzed generation of hydroxyl radicals, and deferoxamine may thus serve as an anti-inflammatory agent. PMID- 3019290 TI - Chronic renal failure. PMID- 3019291 TI - An association between encephalomyocarditis virus infection and reproductive failure in pigs. AB - An outbreak of reproductive failure, characterised by mummified foetuses and stillbirths, was investigated in an intensive piggery. Six foetuses that died towards the end of gestation had multifocal myocardial necrosis and encephalomyocarditis virus was recovered from 4 of these foetuses but not from 6 mummified foetuses. There was also a significant increase in failure of conception or early embryonic deaths in sows mated at the same time as sows which produced affected litters. PMID- 3019292 TI - Serological identification of an Australian adenovirus isolate from sheep. PMID- 3019293 TI - Generation of human anti-rubella monoclonal antibodies from human hybridomas constructed with antigen-specific Epstein-Barr virus transformed cell lines. AB - Human monoclonal antibodies (humab) directed against viral antigens were developed by combining immortalization of human primary B lymphocytes by Epstein Barr virus (EBV) infection with consecutive fusion of selected immortalized lymphoblasts with an established, human lymphoblastoid cell line. Using EBV infection a high rate of immortalized B lymphoblastoid cells was obtained which unfortunately could not be cloned because at least 10 cells per microtiter well had to be seeded to get cells growing. However, fusion of these immortalized lymphoblastoid cells which had been selected for antiviral humab production with an established cell line resulted in hybridomas which could easily be cloned. Among the antiviral humab producing hybridomas thus generated, were three which produced anti-rubella humab. Rubella virus consists of three structural proteins, the core protein C and the two envelope proteins E1 and E2. By the western blot technique we were able to show that two humab reacted with the core protein and the third humab with the envelope protein E1. From the hybridomas grown in stationary cultures, highly purified humab preparations were obtained by subjecting the concentrated culture supernatant to immuno-affinity chromatography. PMID- 3019296 TI - [Role of cytomegaloviruses (CMV) in blood transfusions--virologic and immunologic aspects]. PMID- 3019295 TI - Hemodynamic effects of dibutyryl cyclic GMP in anesthetized dogs. AB - Dibutyryl cyclic GMP was given intravenously to anesthetized dogs with doses of 0.0625, 0.125, 0.25, 0.5 and 1.0 mg/kg, respectively. It induced decreases in blood pressure, heart rate, max LV dp/dt and total peripheral resistance with increase of stroke volume in a dose-dependent manner. Although these effects began to appear 30 sec after the administration in each dose, the duration of the effect was observed 50 sec after the administration. Effects of 1 mg/kg dibutyryl cyclic GMP on mean blood pressure and max LV dp/dt were almost equal to those of 3 micrograms/kg acetylcholine, but dibutyryl cyclic GMP was revealed to have a slower onset and longer acting effects than acetylcholine. The administration of atropine did not block the effect of dibutyryl cyclic GMP but blocked those of acetylcholine. These results indicate that dibutyryl cyclic GMP does not exert these effects through muscarinic receptors in spite of its acetylcholine-like effects. PMID- 3019294 TI - Effects of potassium conductance inhibitors on spontaneous diastolic depolarization and abnormal automaticity in human atrial fibers. AB - The capability of generating spontaneous diastolic depolarization and automaticity was investigated in vitro by means of standard microelectrode techniques in 50 human atrial preparations. Samples were classified within two groups: group 1 was composed of 12 well-polarized preparations exhibiting action potentials that were fast responses (mean maximum diastolic potential: -75.5 mV and Vmax greater than 100 V/s); group 2 was composed of 38 partially-depolarized samples (mean maximum diastolic potential: -50.3 mV and Vmax less than 10 V/s) and was further divided into two subgroups. Subgroup 2A consisted of 20 spontaneously beating preparations and subgroup 2B consisted of 18 non-automatic partially-depolarized specimens. Highly-polarized fibers from group 1, although exhibiting a slight diastolic depolarization which was almost entirely suppressed by 2 mM caesium, never presented spontaneous activity under our experimental conditions. 90% of automatic fibers from subgroup 2A were sampled from dilated atria. In automatic preparations, diastolic depolarization was usually separated into two phases: an initial phase, also present in non-automatic fibers, and a late phase. Changes in the initial phase were not accompanied by concomitant changes in the spontaneous rate. Abnormal automaticity was clearly related to the late diastolic phase (absent in non-automatic fibers), the generation of which appeared to be a specific property of automatic fibers. The use of K conductance inhibitors (caesium, 4-aminopyridine, barium, low K solutions) provided indirect evidence that neither delayed outward ix current nor if type inward current are principally responsible for abnormal automaticity. PMID- 3019297 TI - [Granulocyte functions in stored blood]. PMID- 3019298 TI - [Iatrogenic disorders of granulocyte function]. PMID- 3019299 TI - [The problem of post-transfusion hepatitis]. PMID- 3019300 TI - [Practical significance of the AIDS problem in blood donation services]. PMID- 3019301 TI - [Rational microanalysis technic in the detection of antibodies to HTLV-III, CMV and EBV with indirect immunofluorescence]. PMID- 3019302 TI - [Endogenous opiates: 1. Enkephalins and their role in the regulation of gonadotropin secretion]. PMID- 3019303 TI - DNA restriction-fragment variation in the gene family encoding high molecular weight (HMW) glutenin subunits of wheat. AB - Restriction enzyme digests of DNA from nullisomic-tetrasomic and intervarietal chromosome substitution lines of wheat were probed with a high molecular weight (HMW) glutenin cDNA. Three restriction endonucleases were used to investigate restriction-fragment differences among five wheat varieties. The results suggest that the hybridizing fragments contain single gene copies and permit the identification of the subunit encoded by each gene. Restriction-fragment variation associated with previously established allelic differences between varieties was observed. Also, there is a clear relationship between the electrophoretic mobility of a HMW subunit and the length of the central repetitive section of the gene encoding it. These results are discussed with reference to the evolution of the HMW glutenin gene family and the uses of restriction-fragment variation in plant breeding and genetics. PMID- 3019304 TI - Effect of cyclic AMP-dependent hormones and Ca2+-mobilizing hormones on the Ca2+ influx and polyphosphoinositide metabolism in isolated rat hepatocytes. AB - The effect of the interaction between the Ca2+-mobilizing hormone adrenaline, used as alpha-adrenergic agonist, and cyclic AMP-dependent hormones, including beta-adrenergic agonists and glucagon, on the initial 45Ca2+ uptake rate and polyphosphoinositide metabolism were investigated in isolated rat hepatocytes. Each hormone alone increased the initial 45Ca2+ uptake rate. When adrenaline was added without inhibitor, it induced a rise in the initial 45Ca2+ uptake rate larger than the sum of the rises elicited by its alpha and beta components singly. Similarly, when adrenaline was used as an alpha-agonist and added together with glucagon, it enhanced the initial 45Ca2+ uptake rate synergistically. Kinetic analysis of the initial 45Ca2+ uptake rate measured at different Ca2+ concentrations suggested that the increased influx elicited by the combination of adrenaline as alpha-adrenergic agonist and glucagon reflects an activation of the rate of Ca2+ transport via a homogeneous population of Ca2+ channels or carriers. Dose-response curves for the alpha-adrenergic action of adrenaline or glucagon applied in the presence of increasing doses of glucagon or adrenaline showed that each hormone increases the maximal response to the other without affecting its ED50. Measurement of polyphosphoinositide hydrolysis and of the inositol phosphates formed in the presence of adrenaline or vasopressin and/or glucagon showed that Ca2+-mobilizing hormones and glucagon had no synergistic effects on inositol 1,4,5-trisphosphate production. It is therefore proposed that the synergistic action of glucagon and Ca2+-mobilizing hormones on Ca2+ influx occurs at a step that takes place close to the Ca2+ channels or carriers themselves. The Ca2+ gating involved might be mainly controlled by two products, one of them arising from the polyphosphoinositide metabolism, and the other from the increase in internal cyclic AMP. PMID- 3019305 TI - A flow-dialysis method for obtaining relative measures of association constants in calmodulin-metal-ion systems. AB - A flow-dialysis apparatus suitable for the study of high-affinity metal-binding proteins has been utilized to study calmodulin-metal exchange as a measure of relative calmodulin-metal association constants. Calmodulin labelled with radioactive 153Gd was dialysed against buffer containing various competing metal ions. The rate of label exchange was monitored by a gamma-ray scintillation detector. Competing metals used were Ca2+ and Cd2+, and the lanthanides Gd3+, Eu3+, La3+ and Lu3+. All exchange processes were first-order, and two categories of metal were found: Ca2+ and Cd2+ in one, the lanthanides comprising the other. In addition calmodulin-metal complexes with radioactive 109Cd and 45Ca released the bound label without any competing metal being added to the buffer. The kinetics of this metal loss can be described by two consecutive first-order processes, and the fraction of label associated with each rate can be determined. Studies of phosphodiesterase activation by calmodulin show Cd2+ and calmodulin to cause 80% of the maximum activation found when Ca2+ and calmodulin are used. PMID- 3019306 TI - alpha-Melanocyte-stimulating-hormone precursors in the pig pituitary. AB - The occurrence of intermediates from the processing of ACTH-(1-39) [adrenocorticotropic hormone-(1-39)] to alpha-melanocyte-stimulating hormone was investigated in normal pig pituitaries by the use of sensitive and specific radioimmunoassays for ACTH-(1-13), ACTH-(1-14), ACTH-(1-13)-NH2 and ACTH-(1-39). Fractionation by reverse-phase h.p.l.c. revealed ACTH(1-17) and their acetylated analogues. The intermediate lobe contained NO-diacetyl-ACTH-(1-13)-NH2, N-acetyl ACTH-(1-13)-NH2 and ACTH-(1-13)-NH2. In addition, the corresponding ACTH-(1-14) peptides (the glycine-extended precursor of the amidated peptides) were detected in lower amounts in both the intermediate lobe and the anterior lobe. ACTH-(1 17), ACTH-(1-13) and their acetylated analogues could not be detected in the anterior lobe or the intermediate lobe. The results suggest that an endopeptidase initially cleaves ACTH-(1-39) at the Lys-16-Arg-17 bond. ACTH-(1-16) is then processed by a pituitary carboxypeptidase to ACTH-(1-14) and ACTH-(17-39) by the aminopeptidase to ACTH-(18-39). PMID- 3019307 TI - Turnover of phosphomonoester groups and compartmentation of polyphosphoinositides in human erythrocytes. AB - The turnover of phosphomonoester groups of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was investigated in human erythrocytes by short-term labelling with [32P]Pi. The procedure applied ensured a quantitative extraction of erythrocyte polyphosphoinositides as well as their reliable separation for the determinations of pool sizes and specific radioactivities. The pool sizes of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 are 25, 11 and 44 nmol/ml of cells respectively. Under steady-state conditions, the phosphorylation fluxes from [gamma-32P]ATP into PtdIns4P and PtdIns(4,5)P2 are in the ranges 14 22 and 46-94 nmol X h-1 X ml of cells-1 respectively. Only 25-60% of total PtdIns4P and 6-10% of total PtdIns(4,5)P2 take part in the rapid tracer exchange, i.e. are compartmentalized. In isolated erythrocyte ghosts, the turnover of PtdIns4P approximately corresponds to that in intact erythrocytes, although any compartmentation can be excluded in this preparation. Under the conditions of incubation employed, the turnover of PtdIns(4,5)P2 is more than one order of magnitude smaller in isolated ghosts than that obtained for intact erythrocytes. PMID- 3019308 TI - Regulation of rat heart cytosol 5'-nucleotidase by adenylate energy charge. AB - A 5'-nucleotidase (EC 3.1.3.5) was highly purified from the soluble fraction of rat heart. The preparation appeared homogeneous by the criterion of polyacrylamide-gel electrophoresis. The enzyme was activated by ATP and ADP, and inhibited by Pi. When AMP was used as substrate, the velocity/substrate concentration plot was sigmoidal. ATP or ADP changed the plot to hyperbolic and decreased S0.5. Pi increased both the sigmoidicity of the plot and S0.5. When IMP was used as substrate, the velocity/substrate plot was hyperbolic. ATP or ADP decreased Km and increased V. Pi changed the plot to sigmoidal and increased S0.5. Within the range of adenylate energy charge observed in surviving mammalian cells (0.7-0.9), the rate of AMP-hydrolysing activity catalysed by the 5' nucleotidase increased sharply with decreasing energy charge. The highest activity was observed at an energy-charge value of about 0.6. The response was also observed in the presence of Pi. No change in IMP-hydrolysing activity was observed in the physiological range of adenylate energy charge, but in the presence of Pi the activity gradually increased with increasing energy charge. These results suggest the possibility that this enzyme participates in production of adenosine, a vasodilator, during hypoxia and in removal of IMP, which accumulates during the hypoxia, in the heart. PMID- 3019309 TI - Thyrotropin cross-links to the thyrotropin receptor through both the alpha and beta subunits. AB - We have recently shown that the beta subunit of thyrotropin (TSH) can be cross linked to the TSH receptor [Buckland, Strickland, Pierce & Rees Smith (1985) Endocrinology (Baltimore) 116, 2122-2124; Buckland, Strickland & Rees Smith (1985) Biochem. Soc. Trans. 13, 942-943]. We failed, however, to cross-link the alpha subunit to the receptor, leaving the role of this subunit in the TSH-TSH receptor interaction uncertain. We now report the successful cross-linking of the TSH alpha subunit to the receptor by the use of two different cross-linking reagents. Our studies suggest therefore that both subunits of TSH form part of the hormone's receptor-binding site. PMID- 3019310 TI - Molecular size of N-acetylglucosaminylphosphotransferase and alpha-N acetylglucosaminyl phosphodiesterase as determined in situ in Golgi membranes by radiation inactivation. AB - The radiation inactivation method was used to determine the molecular size of the two enzymes that participate in the synthesis of the phosphomannosyl recognition marker of lysosomal proteins. The determinations were carried out in situ, in Golgi membranes isolated from normal human placenta and cultured skin fibroblasts. A molecular size of 228 +/- 29 kDa was found for placental N acetylglucosaminyl-phosphotransferase, and 129 +/- 11 kDa for placental alpha-N acetylglucosaminyl phosphodiesterase. The values for the fibroblast enzymes were about 20% higher, 283 +/- 27 kDa and 156 +/- 14 kDa for the transferase and phosphodiesterase respectively. Triton X-100 had no effect on the molecular size of these enzymes. PMID- 3019311 TI - Nucleotide and derived amino acid sequences of a cDNA coding for pre-uteroglobin from the lung of the hare (Lepus capensis). AB - An almost full-length cDNA coding for pre-uteroglobin from hare lung was cloned and sequenced. The derived amino acid sequence indicated that hare pre uteroglobin contained 91 amino acids, including a signal peptide of 21 residues. Comparison of the nucleotide sequence of hare pre-uteroglobin cDNA with that previously reported for the rabbit gene indicated five silent point substitutions and six others leading to amino acid changes in the coding region. The untranslated regions of both pre-uteroglobin mRNAs were very similar. The amino acid changes observed are discussed in relation to the different progesterone binding abilities of both homologous proteins. PMID- 3019312 TI - Evidence suggesting that a novel guanine nucleotide regulatory protein couples receptors to phospholipase C in exocrine pancreas. AB - The initial response of many cells to 'Ca2+-mobilizing' agonists is phospholipase C-mediated hydrolysis of phosphatidylinositol bisphosphate to inositol trisphosphate (IP3) and diacylglycerol. It has been suggested, by analogy with receptor regulation of adenylate cyclase, that 'Ca2+-mobilizing' receptors may interact with a guanine nucleotide-binding protein (G protein) to regulate phospholipase C activity. Here we report increased accumulation of IP3 in response to caerulein or carbachol in electrically permeabilized rat pancreatic acinar cells. The stable analogues of GTP (guanosine 5'-[gamma-thio]trisphosphate and guanosine 5'-[beta, gamma-imido]triphosphate) stimulate IP3 accumulation and potentiate the effects of caerulein and carbachol. This synergism demonstrates an interaction between receptors, a G protein and phospholipase C. These responses are unaffected by pretreatment of the cells with pertussis or cholera toxins under conditions that produce substantial covalent modification of Gi and Gs, the proteins that couple receptors to adenylate cyclase. We therefore conclude that the G protein that couples receptors to phospholipase C in exocrine pancreas is probably neither Gi nor Gs; instead, we propose that a different G protein mediates this effect. PMID- 3019313 TI - The oxidation-state-dependent ATP-binding site of cytochrome c. A possible physiological significance. AB - Cytochrome c binds certain physiological anions that are known to modulate the biological properties of the protein, although it is not known whether this effect is fortuitous or has physiological significance. We have examined the ability of the protein and its semisynthetic analogues to associate with certain of these anions, e.g. ATP, ADP, Pi and citrate. Our results show that specific residues or clusters of residues on the surface of horse heart cytochrome c are involved in the recognition sites for these anions. We also observed that binding at one site is linked to the oxidation state of the protein. PMID- 3019314 TI - Conformational changes in the bilirubin-human serum albumin complex at extreme alkaline pH. AB - Light-absorption, c.d. and fluorescence of the bilirubin-albumin complex were investigated at extreme alkaline pH. Above pH 11.1 albumin binds the bilirubin molecule, twisted oppositely to the configuration at more neutral pH. On the basis of light-absorption it is shown that two alkaline transitions occur. The first alkaline transition takes place at pH between 11.3 and 11.8, co-operatively dissociating at least six protons. The second alkaline transition takes place at pH between 11.8 and 12.0. It probably implies a reversible unfolding of the albumin molecule, increasing the distance between tryptophan-214 and bilirubin, and partly exposing the liganded bilirubin to the solvent. PMID- 3019315 TI - Disulphide reduction alters the immunoreactivity and increases the affinity of insulin-like growth-factor-I receptors in human placenta. AB - We previously identified two forms of the insulin-like growth-factor-I (IGF-I) receptor in human placenta: a lower-affinity form reactive with an autoantiserum (B-2) to the insulin receptor and a higher-affinity non-immunoreactive form [Jonas & Harrison (1985) J. Biol. Chem. 260, 2288-2294]. Evidence is now presented that the lower-affinity immunoreactive forms are convertible into higher-affinity non-immunoreactive forms via reduction of receptor disulphide bonds. Treatment of placental membranes with increasing concentrations of dithiothreitol (DTT): (1) converted native Mr-290 000 heterotetrameric IGF-I receptors into Mr-180 000 dimers (determined by chemical cross-linking of 125I IGF-I with disuccinimidyl suberate); (2) increased 125I-IGF-I binding, owing to an increase in receptor affinity; and (3) abolished the reactivity of Triton solubilized IGF-I receptors with antiserum B-2 and transformed the curvilinear plot of IGF-I binding to a linear form. In isolated complexes between receptor and B-2 antibody, DTT increased 125I-IGF-I binding and released a single class of higher affinity IGF-I receptors of Mr 180,000. Thus DTT-treated IGF-I receptors have similar properties to the higher-affinity non-immunoreactive forms of the native receptor, except that reduced dimeric forms are not detected by cross linking of 125I-IGF-I to native membranes. Cleavage of the inter-dimeric disulphide bonds is therefore not a prerequisite for higher-affinity binding or loss of immunoreactivity. These observations suggest that the thiol redox state of the IGF-I receptor in vivo is an important determinant of receptor conformation and therefore of the biological responses to IGF-I. PMID- 3019316 TI - Extraction, purification, identification and metabolism of 3',5'-cyclic UMP, 3',5'-cyclic IMP and 3',5'-cyclic dTMP from rat tissues. AB - The large-scale extraction and partial purification of endogenous 3',5'-cyclic UMP, 3',5'-cyclic IMP and 3',5'-cyclic dTMP are described. Rat liver, kidney, heart, spleen and lung tissues were subjected to a sequential purification procedure involving freeze-clamping, perchlorate extraction, alumina and Sephadex ion-exchange chromatography and preparative electrophoresis. The samples thus obtained co-chromatographed with authentic cyclic UMP, cyclic IMP and cyclic dTMP on t.l.c. and h.p.l.c. and the u.v. spectra of the extracted samples were identical with those of the standards. Fast atom bombardment of the three cyclic nucleotide standards yielded mass spectra containing a molecular protonated ion in each case; mass-analysed ion kinetic-energy spectrometry ('m.i.k.e.s') of these ions produced a spectrum unique to the parent cyclic nucleotide. The extracted putative cyclic UMP, cyclic IMP and cyclic dTMP each produced a m.i.k.e.s. identical with that obtained with the corresponding cyclic nucleotide standard. Rat liver, heart, kidney, brain, intestine, spleen, testis and lung protein preparations were each found capable of the synthesis of cyclic UMP, cyclic IMP and cyclic dTMP from the corresponding nucleoside triphosphate, of the hydrolysis of these cyclic nucleotides and of their binding, with the exception that cyclic dTMP was not synthesized by the kidney preparation. PMID- 3019318 TI - Detection of radical species in haematin-catalysed retinoic acid 5,6-epoxidation by using h.p.l.c.-e.p.r. spectrometry. AB - E.p.r. signals were detected in an all-trans-retinoic acid/haematin incubation mixture by using an e.p.r. spin-trapping technique. The spin adducts are presumably attributable to some intermediates in haematin-catalysed retinoic acid 5,6-epoxidation, since addition of nitrosobenzene to the reaction mixture dose dependently inhibited the epoxidation. Analysing the reaction mixture by h.p.l.c. e.p.r. spectrometry resulted in the detection of three peaks (III-1, III-2, IV) ascribable to the radical species. Two (peaks III-1 and -2) of the three peaks, which appeared 10 min after the reaction had started, seem to be attributable to the radical species directly participating in the epoxidation. The radicals trapped by nitrosobenzene do not appear to be derived from active oxygen, since none of these peaks were detected in a similar h.p.l.c. analysis of O2- and OH. generating systems. They are presumably derived from retinoic acid. This view is also supported by the following results: none of these peaks were detected in the h.p.l.c. elution profile of the reaction mixture when retinoic acid was absent; peaks III-1 and 2 were detected even under anaerobic conditions, and their peak heights were unchanged under aerobic conditions. PMID- 3019319 TI - Substrate specificity and regulation of the maize (Zea mays) leaf ADP: protein phosphotransferase catalysing phosphorylation/inactivation of pyruvate, orthophosphate dikinase. AB - The protein substrate specificity of the maize (Zea mays) leaf ADP: protein phosphotransferase (regulatory protein, RP) was studied in terms of its relative ability to inactivate/phosphorylate pyruvate, orthophosphate dikinase from Zea mays and the non-sulphur purple photosynthetic bacterium Rhodospirillum rubrum. The dimeric bacterial dikinase was inactivated by the maize leaf RP via phosphorylation, with a stoichiometry of approximately 1 mol of phosphate incorporated/mol of 92.7-kDa protomer. Inactivation required both ADP and ATP, with ADP being the specific donor for regulatory phosphorylation. The requirements for inactivation/phosphorylation in this heterologous system were identical with those previously established for the tetrameric maize leaf dikinase. The ADP-dependent maize leaf RP did not phosphorylate alternative protein substrates such as casein or phosvitin, and its activity was not affected by cyclic nucleotides, Ca2+ or calmodulin. The regulation of the maize leaf ADP: protein phosphotransferase was studied in terms of changes in adenylate energy charge and pyruvate concentration. The change in adenylate energy charge necessary to substantially inhibit phosphorylation of maize leaf dikinase was not suggestive of it being a physiological modulator of phosphotransferase activity. Pyruvate was a potent competitive inhibitor of regulatory phosphorylation (Ki = 80 microM), consistent with its interaction with the catalytic phosphorylated intermediate of dikinase, the true protein substrate for ADP-dependent phosphorylation/inactivation. PMID- 3019317 TI - The membrane topography of ecto-5'-nucleotidase in rat hepatocytes. AB - The transmembrane topography of the rat hepatocyte ectoenzyme 5'-nucleotidase was studied by the use of glycoprotein labelling and limited-proteolysis techniques. Comparison, by one-dimensional peptide mapping, of enzyme iodinated from outside the cell with that iodinated in the solubilized state showed that no additional iodination sites were revealed on solubilization. Incubation of newly synthesized enzyme in a microsomal membrane fraction with proteinase showed that the entire molecule of 5'-nucleotidase was protected from proteolysis. These data suggest that little, if any, of the 5'-nucleotidase molecule is present on the cytoplasmic side of the plasma membrane. No evidence was found for a previously proposed interaction between 5'-nucleotidase and actin, although the ability of preparations of 5'-nucleotidase to prevent inhibition of deoxyribonuclease I by actin was explained by minute traces of ATPase activity. Comparison of peptide maps of enzyme labelled by iodination or by methods specific for carbohydrate showed that in both cases predominantly one section of the molecule was labelled. It is proposed that the enzyme is a short-stalked integral membrane protein without a cytoplasmic domain in which about one-third of the molecule forms the accessible molecular surface. PMID- 3019320 TI - Effect of parathyroid hormone on phospholipid metabolism in osteoblast-like rat osteogenic sarcoma cells. AB - Previous results have shown that 1,25-dihydroxycholecalciferol [1,25(OH)2D3] enhances the synthesis of phosphatidylserine (PS) and suppresses the synthesis of phosphatidylethanolamine (PE) in osteoblast-like rat osteogenic sarcoma UMR 106 cells [Matsumoto, Kawanobe, Morita & Ogata (1985) J. Biol. Chem. 260, 13704 13709]. In the present study, the effect of parathyroid hormone (PTH) on phospholipid metabolism is examined by using these cells. Treatment of UMR 106 cells with human PTH-(1-34)-peptide suppresses the synthesis of phosphatidylethanolamine in a dose- and time-dependent manner without affecting the synthesis of PS. The maximal effect on PE synthesis is obtained with 2.4 nM human PTH-(1-34)-peptide when the cells are treated for 48 h or longer. In addition, when human PTH-(1-34)-peptide is added together with the maximal dose of 1,25(OH)2D3, there is a further decline in PE synthesis, whereas the stimulation of PS synthesis by 1,25(OH)2D3 is not altered. Because methylation of PE is suggested to affect hormone receptor-adenylate cyclase coupling, the observed change in PE metabolism by PTH and 1,25(OH)2D3 may be, at least in part, involved in the development of desensitization phenomenon to PTH in these cells. PMID- 3019321 TI - The susceptibility towards proteolysis of intermediates during the renaturation of yeast phosphoglycerate mutase. AB - The renaturation of the tetrameric enzyme phosphoglycerate mutase from baker's yeast after denaturation in guanidinium chloride was studied. Three proteinases (trypsin, chymotrypsin and thermolysin) cause extensive loss of activity of samples taken during the early stages of refolding. As judged by SDS/polyacrylamide-gel electrophoresis, the proteinases cause substantial degradation of the polypeptide chain with no evidence for large quantities of fragments of Mr greater than 6500. These data suggest that the early intermediates in the refolding, especially the folded monomer, possess a number of sites that are susceptible to proteolysis. PMID- 3019322 TI - The chemoattractant des-Arg74-C5a regulates the expression of its own receptor on a monocyte-like cell line. AB - The interaction of the chemoattractant des-Arg74-C5a (C5a des Arg) with its receptor on a human monocyte-like cell line, U-937, was examined. The data obtained suggest that C5a des Arg receptor expression is regulated by the extracellular concentration of C5a des Arg itself. PMID- 3019323 TI - ATP production and consumption of rabbit reticulocytes increase in an amino-acid enriched medium. AB - The ATP production and the main processes of ATP consumption, globin synthesis, Na+, K+-ATPase, and proteolysis of rabbit reticulocytes in two different media (1. complete medium with amino acids and glucose and 2. glucose containing electrolyte medium) were measured. The ATP formation in the complete medium amounts to 157 mmol/l X h which is 28% higher than in the incomplete medium. The increased ATP formation rate is due to increased ATP consumption by the more than doubled protein synthesis in the complete medium, whereas proteolysis and Na+,K+ ATPase are nearly unchanged. The increase in the glucose consumption in the complete medium is not fully accounted for by the increased oxidative degradation of glucose in the citric acid cycle and by lactate accumulation. PMID- 3019324 TI - Influence of postnatal hypoxia on 32P-labeling of polyphosphoinositides and phosphatidic acid in striatum synaptosomes from rat brain. AB - Striatum synaptosomes prepared from adult rats which had been exposed to postnatal hypoxia incorporate 32P-phosphate into phosphatidylinositol-4,5 trisphosphate (PI-4,5-P2) with decreased rate. 32P-incorporation amounted to 57% of the control for PI-4,5-P2 labeling and was slightly diminished for phosphatidic acid and PI-4-P. Exposure to hypoxia of adult rats did not affect inositol phospholipid labeling. The inhibitory effect of dopamine on 32P phosphate incorporation was reduced only after postnatal hypoxia. 32P incorporation rates and the dopamine inhibitory effect were not influenced by external calcium. A working hypothesis is suggested for the dopamine action on specific receptors which may be linked to the polyphosphoinositide metabolism and membrane calcium release. The long lasting effects of an early postnatal hypoxia on 32P-incorporation rates into polyphosphoinositides and phosphatidic acid could reflect the role of the proposed dopamine receptor interaction. PMID- 3019325 TI - Developmental changes of glycosidase activities in rat renal cortex. AB - The activities of beta-glucosidase as well as of alpha- and beta-galactosidase increase in the renal cortex of the rat during the early postnatal development. However, while the two galactosidases reach adult values around the end of the second week of life, beta-glucosidase activity exhibits a second steep increase after the third post-uterine week. This rise in activity correlates with the functional maturation of kidney and is confined nearly exclusively to the cytosolic isozyme. The effects of taurocholate, Triton X-100, and dexamethasone on beta-glucosidase isozymes were studied. The results suggest that taurocholate should be included in the assay medium for the measurement of the beta glucocerebrosidase activity in the diagnosis of Gaucher's disease. For the determination of the total beta-glucosidase activity in samples containing mainly the lysosomal isozyme (perinatal kidney, isolated lysosomes) the use of Triton X 100 is recommended. PMID- 3019326 TI - Inhibition of fructose 1,6-bisphosphatase from pig liver by fructose 2,6 bisphosphate. AB - The inhibition of pig liver fructose 1,6-bisphosphatase by fructose 2,6 bisphosphate has been investigated over a wide range of substrate concentration by measuring the release of labelled inorganic phosphate from [1-32P]fructose 1,6 bisphosphate. The activity of the enzyme can be inhibited completely by fructose 2,6-bisphosphate. The inhibiting effect is most pronounced at low substrate concentrations. The results have been analyzed in terms of a mathematical model assuming a competitive interaction of fructose 1,6-bisphosphate and fructose 2,6 bisphosphate at the catalytic site as well as a synergistic cooperation of the two hexose bisphosphates at an inhibiting allosteric site of the enzyme. PMID- 3019327 TI - Probing of mRNA binding sites involved in interactions with rat liver ribosomes using poly(U) spin labeled at the ribose moiety. AB - The interaction of rat liver ribosomes with poly(U), spin labeled (SL) at the 2'OH groups of ribose residues by N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1 oxyl)-imidazole, has been studied by electron spin resonance (ESR) spectroscopy. The ESR spectra demonstrate that SL-poly(U) with a modification of 1 spin label per 20 ribose residues binds to 80S ribosomes as well as to 40S subunits in a 1:1 stoichiometry at 12 mM MgCl2. The same result is found with highly modified poly(U) bearing 1 SL per 4 ribose residues. Addition of excessive amounts of unmodified poly(U) displaces bound SL-poly(U) from the ribosome which points to a competition for the same binding site at the ribosome. The biological activity of SL-poly(U) was tested with regard to trigger 80S ribosomes for binding of Phe tRNAPhe and for poly(Phe) synthesis. SL-poly(U) bearing 1 SL group per 20 ribose residues directs the binding of only 50% of the amount of Phe-tRNAPhe bound to ribosomes in the presence of unmodified poly(U). When SL-poly(U) bearing 1 SL group per 4 ribose residues is used, this value drops to 25%. Poly(Phe) synthesis is even more impaired: In the presence of poly(U) bearing 1 SL-group per 20 ribose residues only about 30% of the amount of poly(Phe) coded by unmodified poly(U) are synthesized and in the presence of SL-poly(U) bearing 1 SL group per 4 ribose residues poly(Phe) synthesis is completely abolished. The results suggest that modification of the ribose moiety has only a relatively small influence on the binding of mRNA to ribosomes but causes substantial impairment of the mRNA function, whereas, as shown earlier (Ebert et al., Acta Biol. Med. Germ. 41, 431, 1982), modification of the base moiety of poly(U) (in a proportion of 1 SL per 30 bases) does not influence coding efficiency for poly(Phe) synthesis. PMID- 3019328 TI - Insulin-like growth factor I (IGF I) receptor autophosphorylation and kinase activity. Effect of a human polyclonal antibody (pIgG) AB - IGF I receptor is a tyrosine kinase capable of phosphorylating the receptor itself and other substrates. A high degree of homology does exist in tyrosine kinase domain among receptors for several polypeptide growth factor receptors and this enzymic activity has been indicated as a possible mediator of biological action. Nevertheless growth factor receptors possess peculiar specificities both in their functions and tissue distribution. A human polyclonal IgG (pIgG), previously characterized as anti insulin receptor antibody, able to inhibit insulin receptor kinase activity, was used to further investigate subunit homologies and differences in antigenicity and functional regulation between IGF I and insulin receptors, IGF I receptor tyrosine kinase was stimulated by a IGF I analog (aIGF I), produced by DNA recombinant technology, pIgG was able to inhibit IGF I receptor kinase activity, thus revealing antigenic homologies between the kinase domains of insulin and IGF I receptors. However the more pronounced inhibition of IGF I receptor-compared with insulin receptor kinase activity by pIgG suggests the existence of different regulatory mechanisms. PMID- 3019329 TI - Differential effect of sodium butyrate on cyclic AMP phosphodiesterase activities in butyrate sensitive and resistant mastocytoma cells. AB - The effect of sodium butyrate on the intracellular cyclic AMP levels and the activities of cyclic AMP-regulating enzymes were examined in two types of mastocytoma p-815 cells in culture: one type (S cell) was sensitive and the other (R cell) was resistant to the induction of differentiation by sodium butyrate. In the presence of sodium butyrate, adenylate cyclase activity increased in both S and R cells to the same degree, whereas the level of cyclic AMP was elevated only in S cells. Cyclic AMP phosphodiesterase activity increased in R cells but not in S cells. Cyclic AMP phosphodiesterase activities of two cell populations differed in their response to sodium butyrate and they seem to have an important role in regulating cellular level of cyclic AMP that might be an important factor in controlling cell differentiation. PMID- 3019330 TI - Inhibition of natural killer cell activity of human lymphocytes by eicosapentaenoic acid. AB - The effect of eicosapentaenoic acid (EPA) on natural killer (NK) cell activity of human lymphocytes was examined. The addition of an emulsion of trieicosapentaenoyl-glycerol (EPA-TG) emulsified with purified phosphatidylcholine from krill to a cytotoxicity assay system resulted in a marked depression of NK activity. The inhibition was proportional to the concentration of EPA-TG emulsion, and was observed as early as the first one hour of incubation at various effector to target cell ratios. Pretreatment of effector cells with EPA-TG emulsion resulted in significant suppression of their NK activity. Inhibition of cytotoxicity was not due to direct toxicity to effector cells or decreased target cell binding. These results indicate that EPA is a potent inhibitor of NK activity in vitro. PMID- 3019331 TI - Failure of cAMP to replace ACTH in over-riding the melanosome aggregation effect of epinephrine. PMID- 3019333 TI - pH measurements by 31p NMR in bacterial suspensions using phenyl phosphonate as a probe. AB - Phenylphosphonate was used as an internal reference for 31p NMR measurements of E. coli cytoplasmic pH. Phenylphosphonate diffused into the cells allowing determination of pH, independent of magnetic susceptibility differences between intercellular and extracellular pools. Phenylphosphonate was used to measure pH changes during succinate uptake and metabolism, as well as during uncoupling of the transmembrane pH gradient by pentachlorophenol. PMID- 3019332 TI - Activation of NADPH-oxidase by arachidonic acid involves phospholipase A2 in intact human neutrophils but not in the cell-free system. AB - The mechanism involved in the stimulation of NADPH-oxidase by arachidonic acid (AA) in intact human neutrophils was studied and compared with that involved in a cell-free system. [3H]-AA was released from pre-labeled cells upon AA stimulation, and phospholipase A2 inhibitors reduced in parallel the release of [3H]-AA and superoxide. Cyclooxygenase, lipoxygenase or protein kinase inhibitors failed to affect either response. In a cell-free system, no release of [3H]-AA was observed after AA addition, whereas NADPH-oxidase was activated; the generation of superoxide was not inhibited by phospholipase inhibitors and was not initiated by adding phospholipase A2 to the preparation. Thus AA stimulates NADPH-oxidase through a phospholipase A2 mediated pathway in intact cells, but activates the oxidase independent of phospholipase A2 in a broken cell system, suggesting distinctive mechanisms of activation for each system. PMID- 3019334 TI - Levels of topoisomerase II and DNA polymerase alpha are regulated independently in developing neuronal nuclei. AB - A possible correlation between activity levels of topoisomerase II and DNA polymerase alpha was studied in neuronal nuclei from developing rat brain. A high level of canonical topoisomerase II activity was detected in neuronal nuclei throughout the development even at a late stage (28 days after birth) when the activity of DNA polymerase alpha decreased to less than 2% of the fetal level. Thus, in contrast to other systems, topoisomerase II does not change in parallel with DNA polymerase alpha during neuronal development. Our results suggest that topoisomerase II is required to maintain some fundamental processes in differentiated cells, including transcription. PMID- 3019335 TI - Analogs of thyrotropin-releasing hormone: hypotheses relating receptor binding to net excitation of spinal lower motor neurons. AB - Experimentally and clinically, treatment with high-doses of TRH produces a net excitation of spinal lower motor neurons (LMNs) that is subsequently reduced or completely lost through continuous or repeated exposure to the peptide. This is operationally termed "autorefractoriness" (AR). We have performed biochemical and in vivo pharmacologic experiments to investigate the mechanism(s) of AR. Biochemically, we classified TRH and several analogs into three groups based on their binding by spinal-cord TRH-receptors (TRH-Rs): high-affinity, (low nanomolar range; MeTRH, TRH); intermediate-affinity (mid-nanomolar range; MK-771, RX77368) or low-affinity (micromolar range; DN-1417, PNP). When tested in vivo for LMN excitatory activity in cordotomized (T8) rats, TRH and MK-771 produced rapid-onset excitation followed AR. In contrast, sustained excitation with much less AR was produced by the low affinity analog DN-1417. Based on these results, we have formulated two receptor-based hypotheses to explain AR: a) rapid TRH-R desensitization (conversion to an inactive form) by high- but not low-affinity TRH-analogs; and b) a slower down-regulation (cellular internalization) of the agonist-receptor complex, most evident with high-affinity agonists. Thus, low rather than high-affinity TRH-analogs may be superior to TRH for providing sustained LMN excitation (increase of strength) in motor neuron degenerative disorders. PMID- 3019336 TI - Transferrin receptor cycling by human lymphoid cells: lack of effect from inhibition of microtubule assembly. AB - Flow cytometry was used to follow the uptake and release of fluorescein conjugated ferro-transferrin by CCRF-CEM human T - leukaemia cells on an individual cell basis. Pretreatment with the stathmokinetic agent vincristine for 5 hr had no effects on the cycling of transferrin, although microscopic examination of the cells showed a substantial proportion to be arrested in metaphase with cytoplasmic dispersal of tubulin, indicating inhibition of microtubule assembly. Tubulin is therefore unlikely to play a major role in the transferrin cycle of exponentially-growing cells, despite earlier reports that the cytoskeleton is involved in receptor clustering and down regulation. PMID- 3019337 TI - EPR studies show that all lanthanides do not have the same order of binding to calmodulin. AB - Calmodulin, spin labeled at Tyr-99, has been titrated with the lanthanides La3+, Nd3+, Eu3+, Tb3+, Er3+ and Lu3+ as well as Ca2+ and Cd2+. The titration was monitored by EPR and changes in mobility of the spin label, due to binding into the labeled site and protein conformational change, were observed. Comparison of these titration curves with theoretical binding curves for the various calmodulin metal species, show that different lanthanides have different high affinity sites. Three basic categories were observed, with Lu3+ and Er3+ behaving like Ca2+, Eu3+ and Tb3+ binding in the opposite order from Ca2+, and La3+ and Nd3+ different from either Ca2+ or Tb3+. PMID- 3019338 TI - Characterization of affinity-purified type I insulin-like growth factor receptor from human placenta. AB - The binding affinities of type I IGF receptor, purified to near homogeneity from human placental membranes, were characterized. For this receptor preparation, free of type II IGF receptor and essentially free of insulin receptor, dissociation constants of Kd = 0.05 nM for IGF I and of Kd = 0.2 nM for IGF II (linear Scatchard plots) were determined. Competitive binding studies indicated a cross-reactivity of approximately 40% for IGF II to the type I IGF receptor. PMID- 3019339 TI - Cell-free synthesis of ubiquinone-binding protein of mitochondrial cytochrome bc1 complex. AB - In vitro synthesis of the ubiquinone-binding protein of mitochondrial cytochrome bc1 complex has been studied. In vitro translation of rat liver poly(A)+ RNA followed by immunoprecipitation with antibody directed against bovine ubiquinone binding protein yielded a polypeptide with a molecular weight that was the same as that of the mature protein. It was also found that this polypeptide was imported into mitochondria and became resistant to trypsin treatment, and that the import required the energized state of the inner membrane. PMID- 3019340 TI - [3H]muscimol photolabels the gamma-aminobutyric acid receptor binding site on a peptide subunit distinct from that labeled with benzodiazepines. AB - Affinity column-purified GABA-benzodiazepine receptor protein from bovine brain was photoaffinity labeled with both [3H]flunitrazepam and [3H]muscimol. Gel electrophoresis in sodium dodecyl sulfate revealed that the benzodiazepine binding site labeled with [3H]flunitrazepam was primarily associated with a major peptide subunit revealed by protein staining with Mr = 52 kiloDaltons, with minor labeling of a second peptide of Mr = 57 kiloDaltons, corresponding to a second major stained band. Covalent incorporation of [3H]muscimol was limited to the 57 kiloDalton band, with no labeling of the 52 kiloDalton peptide, showing that the GABA binding site is carried by a subunit distinct from that carrying the benzodiazepine binding site. PMID- 3019341 TI - Identification of a monoclonal antibody which can distinguish between two distinct species of the type I receptor for insulin-like growth factor. AB - A monoclonal antibody was identified which equally inhibits 125I-labeled insulin and insulin-like growth factor I (IGF-I) binding to their respective receptors in human IM-9 lymphoid cells and solubilized placenta receptor preparations. In contrast, this monoclonal antibody inhibits insulin but not IGF-I binding to human hepatoma (HepG2) cells, fibroblasts and muscle cells. These results indicate that there are two distinct species of the type I insulin-like growth factor receptor (which we have named type IA and type IB) and suggest that this monoclonal antibody may be useful in determining whether different biological effects are mediated through these two receptors. PMID- 3019342 TI - Decreased level of calpain inhibitor activity in red blood cells from Milan hypertensive rats. AB - In mature red cells of rats from Milan Normal (MNS) and Hypertensive Strains (MHS), the soluble Ca2+-dependent neutral proteinase (calpain) is present in similar amounts as the form requiring 0.1-0.2 mM Ca2+ for maximum catalytic activity. The amount of the endogenous calpain inhibitor, however, differs greatly in the red cells of the two strains. In red cells from hypertensive rats the activity of the inhibitor is 10 times less with a ratio of inhibitor to calpain activity (unit/unit) of 0.2; compared to red cells from normal rats, in which this ratio is approximately 2. This is the first demonstration of the existence, in a mammalian cell, of such a low ratio of calpain to inhibitor and implies the occurrence of a potentially "unregulated" intracellular soluble proteinase. This abnormal condition may be responsible for some of the structural and metabolic changes reported in rats of the genetically determined MHS strain. PMID- 3019343 TI - Synergistic action of adenosine and fMet-Leu-Phe in raising cAMP content of purified human monocytes. AB - The content of cAMP was measured in monocytes treated with fMet-Leu-Phe and adenosine, either singly or in combination. Adenosine caused a small and variable rise in cAMP, which was considerably less than that caused by fMet-Leu-Phe. The rise induced by peptide plus adenosine was twice the sum of the increases caused by each agent alone. An inhibitor of phosphodiesterase also enhanced the adenosine-induced rise in cAMP. The data suggest that the increase in cAMP by adenosine-induced cyclase activation is limited by the activity of phosphodiesterase, and that the latter can be inhibited by fMet-Leu-Phe. PMID- 3019344 TI - Evidence that thyrotropin-releasing hormone-induced increases in GTPase activity and phosphoinositide metabolism in GH3 cells are mediated by a guanine nucleotide binding protein other than Gs or Gi. AB - Thyrotropin-releasing hormone (TRH), vasoactive intestinal polypeptide (VIP) and acetylcholine stimulated high affinity GTPase activity in GH3 cell membrane preparations. The effects of acetylcholine and VIP were blocked by pretreatment of cultured cells with pertussis toxin and cholera toxin respectively. Such pretreatment, which causes covalent modification of the guanine nucleotide binding proteins (G-proteins) of adenylate cyclase, did not, however, block the effects of TRH on GTPase activity or phosphoinositide breakdown. These data suggest that TRH receptors interact with a G-protein discrete from those associated with regulation of adenylate cyclase activity. PMID- 3019345 TI - Occurrence of alpha 2-adrenergic effects on adenylate cyclase activity and (3H) clonidine specific binding in brown adipose tissue from foetal rats. AB - Noradrenaline maximally stimulates adenylate cyclase activity in brown adipocytes from foetal rats by 400%. Isoproterenol maximally stimulates the adenylate cyclase activity by 600%. The differences observed in the dose-response curves of adenylate cyclase activity to isoproterenol or noradrenaline were prevented in the presence of clonidine (a alpha 2-agonist) or yohimbine (alpha 2-antagonist) respectively. (3H)-clonidine binds specifically to brown fat membranes saturable (from 1.75 to 20 nM). Scatchard analysis revealed a Bmax of 22 fmol/mg and a KD of 10 nM. PMID- 3019346 TI - Phosphatidylinositol 4,5-bisphosphate formation in rabbit skeletal and heart muscle membranes. AB - Incubation of rabbit skeletal muscle microsomes or isolated triads with gamma 32P ATP/Mg2+ in the absence and in the presence of added phosphatidylinositol resulted in the formation of phosphatidylinositol 4-phosphate catalyzed by phosphatidylinositol kinase. When phosphatidylinositol 4-phosphate was added as exogenous substrate, phosphatidylinositol 4,5-bisphosphate was also formed demonstrating the presence of a membrane bound phosphatidylinositol 4-phosphate kinase. Triads were broken mechanically in a French press and separated on a continuous sucrose gradient. Incubation of these fractions with gamma 32P ATP/Mg2+ resulted in a rapid labeling of phospholipid in a membrane fraction banding between transverse tubules and the terminal cisternae. Partial triad breakage and triad reformation experiments indicated that this phosphatidylinositol kinase was associated with T-tubules. When exogenous phosphatidylinositol 4-phosphate was employed as substrate phosphatidylinositol 4,5-bisphosphate and phosphatidic acid were formed, indicating the presence of all the enzymes of the polyphosphoinositide signaling system in this special membrane fraction. In contrast, heart muscle microsomes or plasma membranes can catalyze this reaction sequence from endogenous formed phosphatidylinositol 4 phosphate. PMID- 3019348 TI - The nucleotide sequence of the mitochondrial DNA genome of an abundant petite mutant of Saccharomyces cerevisiae carrying the ori1 replication origin. AB - We have determined the 903 bp nucleotide sequence of the mitochondrial DNA genome of a Saccharomyces cerevisiae petite mutant BB5. This petite, containing the 265 nucleotide ori1 region, is representative of a class of petites arising at exceptionally high frequency within the population of spontaneous petites derived from a particular mit- strain Mb12. The DNA sequences of both the ori1 region and the flanking intergenic regions have been compared to those of the corresponding regions of mtDNA in a previously reported petite strain, a1/1R/1 of Bernardi's laboratory, that has a similar (880 bp) repeat unit. The BB5 petite genome carries a canonical ori1 sequence that is identical in both petite mtDNAs, but the flanking intergenic sequences show significant differences between the two petite strains. The divergence is considered to arise from differences in the sequences flanking ori1 in the respective parent strains. PMID- 3019347 TI - Fasting increases fat cell adenylate cyclase sensitivity to stimulatory agonists through enhanced ability of the stimulatory regulatory component Ns to dissociate. AB - In rat fat cell membranes, a 72-hour fasting fails to alter the adenylate cyclase stimulatory responses to Mn2+, forskolin and cholera toxin and the cholera toxin catalyzed [alpha-32P] ADP ribose incorporation into the Mr = 42,000 and 46,000/48,000 alpha s peptides of Ns. In contrast, dose-response curves for GTP stimulation of basal and isoproterenol-stimulated adenylate cyclase display higher maximal responses in fasted rats under conditions restraining (2 mM Mg2+) but not promoting (10 mM Mg2+) the dissociation of Ns. Moreover, at 10 mM Mg2+, the sensitivity of isoproterenol-stimulated adenylate cyclase to GTP is clearly increased in fasted rats. Finally, fasting reduces by 40% the lag-phase of adenylate cyclase activation by Gpp(NH)p. Taken together, these results are consistent with the hypothesis that the permissive effect of fasting on the fat cell adenylate cyclase response to stimulatory agonists is related to increased ability of Ns and the ternary H.R.Ns. complex to dissociate which is likely due to enhanced Ns affinity for guanine nucleotides. PMID- 3019349 TI - Peroxidase activity in the mouse uterus, placenta and fetus during pregnancy. AB - Changes in peroxidase activity during pregnancy were examined in CD-1 mice. Peroxidase activity was measured with guaiacol as the substrate in uterine extracts of nonpregnant mice and in uterine, placental, and fetal extracts of pregnant mice on days 9, 12, 14, 16, and 18 of gestation. Uterine peroxidase activity in nonpregnant mice was high, but declined logarithmically to only 0.2% by day 18 of pregnancy. In contrast to this decline, a concomitant 50-fold logarithmic increase in fetal peroxidase activity was observed between day 12 and 18. Activity in placental extracts did not change significantly throughout the gestational period examined. These results suggest that membrane bound peroxidase in mouse uterus and fetus undergoes major shifts during pregnancy. PMID- 3019350 TI - Factors affecting Ca2+ transport in mouse islet and kidney mitochondria. AB - The transport of Ca2+ in islet and kidney mitochondria respiring on succinate was inhibited by atractylate and fluorocitrate, and stimulated by pyruvate, isocitrate, alpha-ketoglutarate, dibutyryl cAMP, oligomycin and bongkrekate, and by in vivo administration of glucagon, glyceraldehyde or glucose. The kidney [beta-hydroxybutyrate]/[acetoacetate] ratio was increased in glyceraldehyde treated mice. The data suggest a relationship, which might be influenced by cAMP, between activity of pyruvate, isocitrate and alpha-ketoglutarate dehydrogenases and transport of Ca2+ in islet and kidney mitochondria. A contributory role of reductive carboxylation for Ca2+ uptake, and a role of citrate for Ca2+ retention are discussed. PMID- 3019351 TI - Ionic signal transduction in growth factor action. AB - Growth factors are polypeptides which exert their mitogenic action through binding to specific high-affinity receptor molecules on the cell surface of target cells. This interaction leads to the rapid activation of a receptor-linked signal transduction system, involving the stimulation of an intrinsic receptor tyrosine phosphokinase activity, the breakdown of inositol lipids, and the production of ionic signals. In this contribution we have analysed the nature and origin of the ionic signals, and we have applied monoclonal antibodies against the receptor for epidermal growth factor (EGF), as well as tumour-promoting phorbol esters, to dissociate the early cellular responses to growth factors. Evidence is presented that the ionic signals are coupled to the breakdown of inositol lipids. The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) would lead to the production of 1,2-diacylglycerol (DG) and inositol triphosphate (IP3). DG production results in the stimulation of protein kinase C, which causes the activation of Na+/H+ exchange by increasing its affinity for cytoplasmic H+. Consequently, a rise in cytoplasmic pH is observed. This response can be mimicked by the tumour promoter TPA, which can replace DG in activating the protein kinase C. Independently, IP3 production leads to the rapid mobilization of Ca2+ from intracellular stores. Monoclonal antibodies against the EGF receptor differed in their ability to evoke EGF-like responses upon binding to the EGF receptor. Of the three anti-EGF receptor IgGs tested, one was directed against the EGF-binding domain (2E9), and the others (2D11 and 2G5) were directed against sugar moieties not involved in EGF binding. Receptor tyrosine kinase activity could be stimulated by 2E9 as well as 2D11, but not by 2G5. Only 2D11 induced morphological changes similar to EGF. None of the antibodies was able to trigger the production of ionic signals, which implies probably that antibody binding to the receptor, even when they bind to the EGF binding domain, is an insufficient stimulus for the breakdown of inositol lipids. Most importantly, these monoclonal antibodies were also not able to induce DNA synthesis in quiescent human fibroblasts, not even after cross-linking of the EGF receptors by a second antibody. It may thus be concluded that the stimulation of the intrinsic receptor tyrosine phosphokinase activity can be dissociated from other early responses, and that none of the identified early responses is a sufficient trigger for the mitogenic action of growth factors. PMID- 3019352 TI - Regulation of cell-to-cell communication by phosphorylation. AB - The cyclic AMP-activated protein kinase I, a serine- and threonine phosphorylating enzyme, regulates cell-to-cell communication. Its deficiency in mutant cells is associated with deficiency of communication. The communication defect is corrected by introduction of the catalytic subunit of the enzyme into the mutant cells. Activation of the enzyme by cyclic AMP in normal cells causes an increase of communication, namely an increase of junctional permeability associated with an increase in the number of membrane particles of gap junction. This upregulation of cell-to-cell membrane channels constitutes a basic mechanism whereby cell communities set their degree of communication. The mechanism is normally put into motion by adenylate cyclase-activating hormones. The mechanism is counteracted by tyrosine-phosphorylating protein kinase (src protein), which downregulates junctional permeability, a fast and reversible effect on the channels, independent of the action of the kinase on the cytoskeleton. The two T proteins coded by the SV-40 genome cause a similar channel downregulation. PMID- 3019353 TI - Studies on several pyrrolo[2,3-d]pyrimidine analogues of adenosine which lack significant agonist activity at A1 and A2 receptors but have potent pharmacological activity in vivo. AB - 5'-Deoxy-5-iodotubercidin was previously reported to cause potent muscle relaxation and hypothermia when injected i.p. into mice. In normotensive rats, i.v. injection reduced blood pressure and heart rate. 5-Iodotubercidin possessed the same in vivo activities whereas tubercidin was pharmacologically almost inactive. None of these compounds interacted significantly with Al adenosine receptors, as determined by their ability to displace 3H-N6 phenylisopropyladenosine or 3H-5'-N-ethylcarboxamidoadenosine bound to rat brain membranes. Furthermore these compounds were much weaker than adenosine as agonists of adenosine-stimulated adenylate cyclase in guinea-pig brain slices (A2 receptors). A previous report showed that 5'-deoxy-5-iodotubercidin and 5 iodotubercidin were very potent inhibitors of adenosine kinase from rat or guinea pig brain and were potent inhibitors of 3H-adenosine uptake into brain slices; relative to the halogenated derivatives, tubercidin was quite weak as an inhibitor of adenosine kinase and of adenosine uptake. We therefore propose that a significant part of the in vivo activity of the two halogenated tubercidin analogues may not be due to a direct agonist action at A1 and/or A2 adenosine sites (as proposed for a number of other metabolically-stable analogues of adenosine) but may result from an inhibition of reuptake of endogenously-released adenosine; the increased extracellular levels of adenosine resulting from this action could then interact directly with membrane receptors. Consistent with this, low concentrations of 5'-deoxy-5-iodotubercidin were shown to significantly potentiate the effects of exogenous adenosine on blood pressure and heart rate in anaesthetized rats and on adenosine-stimulated cAMP generation in guinea-pig brain slices. None of these compounds interacted with central benzodiazepine receptors. The cardiovascular and behavioural effects of 5'-deoxy-5 iodotubercidin and 5-iodotubercidin were blocked by theophylline; results from the cardiovascular studies suggest there may be different adenosine receptors in heart and blood vessels. PMID- 3019354 TI - Differential effects of mepacrine, chloroquine and hydroxychloroquine on superoxide anion generation, phospholipid methylation and arachidonic acid release by human blood monocytes. AB - The 4-aminoquinolines chloroquine (CQ) and hydroxychloroquine (HCQ), and previously the 9-aminoacridine mepacrine (quinacrine) (MP), have been widely used in the treatment of inflammatory disorders such as rheumatoid arthritis and systemic lupus erythematosus. Their effects are believed to be mediated through phagocytic cells but the precise biochemical basis remains uncertain. We have investigated the effects of these drugs on monocyte superoxide anion (SO) generation elicited by 5 different stimuli-opsonised zymosan (STZ), FMLP, A23187, TPA and fluoride--and sought correlations with effects on two processes which have been linked with monocyte activation, namely arachidonate (AA) release and transmethylation of phosphatidyl ethanolamine (PE) to phosphatidylcholine (PC). In all experiments conditions were adjusted to achieve leucocyte concentrations of drug comparable to those found during in vivo therapy. Neither CQ nor HCQ had any marked effect on SO release induced by TPA, A23187 or fluoride ion, excluding a significant effect on protein kinase C (PKC), calmodulin-dependent kinase(s) or the membrane-bound, superoxide generating NADP(H) oxidase. In contrast MP inhibited the response to TPA and A23187. Each drug also had different effects on surface receptor-dependent responses; thus HCQ inhibited FMLP- but not STZ induced SO release, whereas CQ and MP inhibited the response to both stimuli. Each drug also displayed different effects on AA release and phospholipid (PL) methylation; MP and HCQ, but not CQ, inhibited STZ-stimulated AA release while MP and CQ but not HCQ inhibited basal rates of PL-methylation in mononuclear cells (MNC). However, only MP inhibited PL-methylation in an enriched monocyte population. We conclude that despite their close structural similarity, MP, CQ and HCQ each have different metabolic effects and their actions cannot simply be attributed to inhibition of lysosomal functions. Other possible mechanisms of action are discussed. The selective effects of each drug also provide further evidence for multiple pathways of monocyte activation. PMID- 3019355 TI - Role of redox cycling and lipid peroxidation in bipyridyl herbicide cytotoxicity. Studies with a compromised isolated hepatocyte model system. AB - The role of active oxygen species and lipid peroxidation in the toxic effects of diquat, paraquat and other bipyridyl herbicides remains controversial. In vitro studies have shown that these compounds are potent generators of active oxygen species by redox cycling and that they stimulate lipid peroxidation. In vivo studies have failed, however, to show clear evidence of lipid peroxidation resulting from toxic exposures to these compounds. We have directly compared the abilities of three bipyridyl herbicides, diquat (DQ), paraquat (PQ) and benzyl viologen (BV), to generate superoxide anion radical (O2-.) in rat liver microsomes and H2O2 in hepatocytes and correlated this with their relative toxicities to a compromised isolated hepatocyte system. DQ was the most potent generator of O2-. and H2O2, being slightly more potent than BV and much better than PQ. This ability of the bipyridyls to generate active oxygen was positively correlated with the ability to induce toxicity in hepatocytes pretreated with 1,3 bis-(2-chloroethyl)-1-nitrosourea (BCNU) to inhibit their glutathione reductase activity, i.e. DQ greater than BV greater than PQ. DQ caused a rapid depletion of cellular GSH and a concomitant increase in GSSG in this system. Toxicity, measured as loss of plasma membrane integrity, was pronounced after only 30-60 min of incubation and was accompanied by a significant increase in lipid peroxidation. The onset of lipid peroxidation could not be separated temporally from the expression of toxicity. However, the total inhibition of lipid peroxidation by the antioxidants Trolox C, promethazine and N,N'-diphenyl-p phenylenediamine only delayed toxicity, indicating that, even though lipid peroxidation may play some role in enhancing bipyridyl herbicide toxicity, it is not essential for the toxicity to manifest itself. PMID- 3019356 TI - Effect of proteases and phospholipases on [3H]yohimbine binding to human platelet membranes. AB - Trypsin and chymotrypsin inactivated specific [3H]yohimbine binding sites in the partially purified human platelet membranes in a concentration- and time dependent fashion. The maximal inactivation (70-80% of control) was incomplete regardless of the concentrations of the proteases used or the incubation time. Scatchard analysis of the binding data showed that the total number of binding sites was reduced, but the affinity of the receptor to the ligand remained unaffected. Pretreatment of the membranes with unlabeled yohimbine or epinephrine produced a 20-30% increase in the specific [3H]yohimbine binding; however, this treatment offered only a slight protection (10-15%) against trypsin-induced inactivation of [3H]yohimbine binding. Pretreatment with phospholipase A2 produced a complete inhibition, while pretreatment with phospholipase C resulted in only a partial (70-80% of control) reduction in [3H]yohimbine binding. The inhibitory effects were not reversed when the specific binding of [3H]yohimbine was carried out with membranes treated with phospholipases and subsequently washed with defatted bovine serum albumin, suggesting that products released from phospholipolysis were not involved in the inhibition of [3H]yohimbine binding. These results suggest that the integrity of the receptor proteins and phospholipids is necessary for the specific binding of the ligand to the alpha 2 adrenoreceptor proteins of the human platelet membranes. PMID- 3019357 TI - Inositol phospholipid metabolism and platelet function. PMID- 3019358 TI - Evidence that interleukin-1 induction of synovial cell plasminogen activator is mediated via prostaglandin E2 and cAMP. AB - Addition of the cyclooxygenase inhibitor indomethacin to human synovial cells in culture, at concentrations which completely block prostaglandin E2 (PGE2) synthesis, reversibly inhibited the interleukin-1 (IL-1) stimulation of cell associated and extracellular plasminogen activator (PA) production. Results of mixing experiments suggested that the inhibition by indomethacin was not due to stimulation of production and/or activation of a PA inhibitor, but reflected inhibition of PA synthesis. Simultaneous addition of PGE2 or dibutyryl cAMP prevented the inhibition by indomethacin. Addition of the phosphodiesterase inhibitor, theophylline, the adenylate cyclase stimulator, forskolin, or dibutyryl cAMP caused an enhancement of the IL-1 induction of synovial cell PA. These results suggest that the IL-1 induction of synovial cell PA occurs via generation of endogenous PGE2 and cAMP. PMID- 3019359 TI - Neutral proteases in human osteoarthritic synovium. AB - The activities of neutral collagenolytic enzymes (CE) and neutral proteoglycan degrading enzymes (PE) in the synovial membranes of osteoarthritis (OA) patients were determined. The total neutral metallo-CE activity showed a significantly higher level of activity when the membranes of OA patients were compared with those of controls. The severely and moderately inflamed synovia had significantly more enzyme activity than did either mildly inflamed or control synovia. Steroids reduced the total metallo-CE activity. In specimens with severe inflammation, the active form of the neutral metallo-CE was significantly elevated over that found in controls. The serine-CE activity was also significantly elevated in OA synovia with severe inflammation and synovial hypertrophy. The total and active neutral metallo-PE was significantly elevated in synovial membranes of OA patients with severe inflammation. Moreover, the serine-PE showed much more activity in OA patients than in controls. The enzyme activity remained at a significantly high level in the OA synovium, regardless of the presence or absence of macroscopic synovial hypertrophy or the histologic grading of the synovium (mild, moderate, severe). Our data indicate that, in OA, an increased level of neutral proteases in the synovia could be involved in the local tissue destruction of the periarticular structures. Because of the very high level of serine proteases, their diffusion may render plausible a degradative action on the cartilage surface. PMID- 3019361 TI - Factors in addition to the direct cytopathic action of HTLV-3/LAV contribute to the loss of T4 cells characteristic of AIDS. PMID- 3019360 TI - Influence of cholesterol feeding on the production of eicosanoids, tissue plasminogen activator and superoxide anion (O2-) by rabbit blood monocytes. AB - The metabolism in vitro of exogenous and endogenous arachidonic acid was studied in circulating blood monocytes obtained from control (control group) and cholesterol (0.5%)-fed (cholesterol group) rabbits. The production of superoxide anion (O2-), tissue plasminogen activator (t-PA) and adherence of monocytes were assessed in both groups of animals. The amounts of cyclooxygenase and lipoxygenase products derived from exogenously added [1-14C]AA were not significantly different in monocytes collected from both groups of animals. However, the amounts of PGD2, TXB2 and PGE2 formed from endogenous substrate were decreased significantly in monocytes obtained from the cholesterol group compared to those from the control group. The production of immunoreactive LTB4 was not suppressed significantly in monocytes collected from the cholesterol group. The production of O2- and t-PA was suppressed significantly in monocytes obtained from the cholesterol group and these cells adhered onto glass surfaces more efficiently than control cells. Since the formation of prostanoids from endogenous but not exogenous substrate is reduced, an effect of cholesterol on the liberation of AA from phospholipid pools is implicated. PMID- 3019362 TI - Cytomegalovirus viremia increases with progressive immune deficiency in patients infected with HTLV-III. AB - Cytomegalovirus (CMV) viremia was studied in 15 homosexual patients [two healthy, nine with the acquired immune deficiency syndrome (AIDS), five with the AIDS related complex (ARC)], in one patient with transfusion-related AIDS, and in four individuals with cancer or connective tissue disorders. While CMV viremia was absent in the latter patients, in the healthy homosexuals, and in three stable ARC patients, it was present in clinically-deteriorating ARC patients and in all nine AIDS patients. Some CMV isolates caused limited proliferation of fibroblasts in the culture medium. The rapidity and ease of CMV isolation from leukocytes of patients at different stages of infection with human T-lymphocytotropic virus type III (HTLV-III) indicate that the CMV titer in leukocytes increases as immune deficiency worsens. In ARC patients CMV viremia may be predictive of clinical complications in the next 3 months. In AIDS patients CMV viremia at high titer was an unfavorable prognostic sign. PMID- 3019364 TI - [Malignant hyperthermia following halothane anesthesia for more than 3 hours]. PMID- 3019363 TI - AIDS statistics and the risk for minorities. AB - Analysis of the AIDS case data for the United States reported to the Centers for Disease Control reveals quite different risk group profiles for minorities and for whites. A much higher percentage of minority male AIDS cases are heterosexual, the ratio of bisexual to homosexual cases is two to three times higher, and IV needle use is a greater risk factor in minority populations. Among women and children with AIDS, 75% and 82% respectively are minorities. Whites at risk for AIDS tend to be concentrated in the gay community but blacks at risk for AIDS are dispersed throughout the entire community. As a result, AIDS education programs aimed a gay populations do not reach most of the minority populations at risk. AIDS prevention programs need to take this fact into account. PMID- 3019365 TI - The role of corticocortical projections in self-stimulation of the prelimbic and sulcal prefrontal cortex in rats. AB - Four experiments were performed to assess the nature of the contribution of the corticocortical projections between the prelimbic and sulcal divisions of the rat prefrontal cortex to self-stimulation (SS) of these sites. The first experiment showed that transection of these projections by parasagittal knife cuts or bilateral electrolytic lesions of the prelimbic cortex had no effect on SS of the sulcal cortex. The second experiment demonstrated that SS of the prelimbic cortex could be obtained after transection of the corticocortical projection path. The third experiment demonstrated that the deficit in prelimbic SS, seen to follow such bilateral transections, is a function of the amount of exposure to the stimulation given to the animals after the lesion. The fourth experiment showed that the stimulation-dependent process underlying the acquisition of prelimbic and sulcal SS could be dissociated by the knife cuts. The discussion focused on the implications of these findings for an account of prefrontal self-stimulation behavior. PMID- 3019367 TI - Characterization of the porcine ACTH receptor with the aid of a monoclonal antibody. AB - Monoclonal antibodies were raised against the ACTH receptor by immunization of BALB1 c mice with porcine adrenal cortex cell membranes. Competition and binding experiments confirmed that one of these antibodies, IV/14/9, reacted with the ACTH receptor in competition with ACTH and caused a definite ACTH-like effect. This antibody was used to characterize the hormone receptor of the porcine adrenal cortex. The number of antibody binding sites per cell was calculated from a Scatchard plot to be 124,100 +/- 10,000. The curve was linear indicating the existence of a single class of receptors. The finding that a high concentration of IV/14/9 totally suppressed maximally ACTH-induced steroidogenesis confirms the view that only a single class of ACTH receptors exists. The antibody IV/14/9 neither reacted with intact porcine thymus cells nor with normal porcine lymphocytes but was bound to the mouse adrenal tumor cell line Y1 and to normal rat adrenal cortex cells with a low affinity. Two dimensional electrophoresis of lysates of porcine adrenal cortex cells and subsequent blotting of the proteins on nitrocellulose, using IV/14/9 as a primary and anti-mouse Ig as a secondary reagent, permitted the detection of three forms of the receptor, two of which had an identical apparent molecular mass of about 85,000 Da (isoelectric points: 6.14 and 6.27). The third was somewhat larger (94,000 Da and pI = 6.21). PMID- 3019366 TI - Ethanol-induced hypothermia in rats: possible involvement of opiate kappa receptors. AB - The effect of the opiate antagonists naloxone and MR2266 on ethanol-induced hypothermia and changes in Ca2+-stimulated Mg2+-ATPase activity in brain regions were investigated in the present study. Administration of different doses of ethanol (0.5-2 g/kg, IP) produced a dose-dependent hypothermia. Ca2+/Mg2+-ATPase activity in the hypothalamus was stimulated at 30 min and 2 hr after ethanol (2 g/kg, IP) treatment. In cortex, enzyme activity was inhibited by ethanol at 30 min with no change seen at 2 hr. Naloxone (7.5 mg/kg, SC) at a dose which did not affect body temperature or enzyme activity, partially inhibited ethanol-induced hypothermia and enzyme activity at the earliest time (30 min) but not at 2 hours. The opiate Kappa antagonist MR2266 (5 mg/kg, SC), however, significantly protected against ethanol hypothermia and enzyme activation measured at 30-120 min. This evidence suggests that ethanol-induced hypothermia and subsequent activation changes of Ca2+/Mg2+-ATPase in the hypothalamus may be regulated by opiate Kappa receptors, and that Ca2+ ions play an important role in mediating the effects of ethanol. PMID- 3019369 TI - Juvenile angiofibromas. Behavior and treatment of extensive and residual tumors. AB - In a 15-year period, 40 juvenile angiofibromas were treated surgically (lateral or extended rhinotomy) at the Mayo Clinic, Rochester, Minn. Tumors were staged according to size and extension by using the Sessions classification. Stage III tumors involved the middle fossa or cavernous sinus or both structures, with or without extension into the orbit. Seven patients had stage I, ten had stage IIA, and six had stage IIB disease. Five tumors extended to the skull base (stage IIC). There were 12 cases that included intracranial extension (stage III), none of which involved the sella. There were eight cases with residual tumor: four (stages IIB [one], IIC [one], and III [two]) were not treated again, and four (stage III) were treated with various methods, including operation, embolization, and irradiation. No patient died. No complications or morbidity was experienced by the four patients who were not treated for residual tumor. PMID- 3019368 TI - Surgical treatment of recurrent pleomorphic adenoma of the parotid gland. AB - Recurrent pleomorphic adenomas of the parotid gland warrant consideration because of the potential for facial nerve injury occurring with surgical treatment and the risk of malignant conversion. Forty-eight cases of recurrent pleomorphic adenoma treated at the University of Michigan, Ann Arbor, between 1935 and 1975 were retrospectively analyzed. The results of surgical procedures for recurrence were determined with respect to tumor control and resultant facial nerve function. Malignant conversion developed in three (6%) of 48 cases. The results of this study underscore the importance of adequate surgical excision of initial recurrences as well as primary tumors to prevent tumor recidivism. Tumor control rates and facial nerve preservation are enhanced with formal parotidectomy for recurrent tumor when feasible. In cases in which facial nerve identification and dissection is not possible, en bloc total parotidectomy offers effective, though not absolute, control of extensive recurrence. PMID- 3019370 TI - Purification and characterization of catalase HPII from Escherichia coli K12. AB - Catalase (hydroperoxidase II or HPII) of Escherichia coli K12 has been purified using a protocol that also allows the purification of the second catalase HPI in large amounts. The purified HPII was found to have equal amounts of two subunits with molecular weights of 90,000 and 92,000. Only a single 92,000 subunit was present in the immunoprecipitate created when HPII antiserum was added directly to a crude extract, suggesting that proteolysis was responsible for the smaller subunit. The apparent native molecular weight was determined to be 532,000, suggesting a hexamer structure for the enzyme, an unusual structure for a catalase. HPII was very stable, remaining maximally active over the pH range 4-11 and retaining activity even in a solution of 0.1% sodium dodecyl sulfate and 7 M urea. The heme cofactor associated with HPII was also unusual for a catalase, in resembling heme d (a2) both spectrally and in terms of solubility. On the basis of heme-associated iron, six heme groups were associated with each molecule of enzyme or one per subunit. PMID- 3019371 TI - Effects of triorganotin-mediated anion-hydroxide exchange upon reconstituted cytochrome c oxidase proteoliposomes. AB - Proteoliposomes containing cytochrome c oxidase and an internally trapped fluorescent pH probe (pyranine) were used to monitor respiration-dependent internal alkalinization and membrane potential formation. A maximum steady-state pH gradient of about 0.4 pH unit (vesicle interior alkaline) was obtained during active respiration in presence of reducing substrates and cytochrome c. This pH gradient was abolished by the triorganotin compounds tripropyl-, tributyl-, and triphenyl-tin chloride. At the same time, the membrane potential, measured by carbocyanine dye uptake, was slightly increased in value. Valinomycin, which abolishes the membrane potential, restores the value of delta pH at low trialkyltin concentrations. The organotin compounds acted as electroneutral ionophores which exchanged intravesicular OH- ions with external SCN-, I-, and CI ions, but not NO3- or SO4(2-) ions. Abolition of delta pH is accompanied by an increase in respiration rate, but full respiratory stimulation only occurs when both delta psi and delta pH are abolished by addition of both triorganotin and valinomycin. The triorganotin-valinomycin combination leads to active KCl accumulation by the respiring proteoliposome, and it is necessary to postulate an electrically neutral KCl efflux process to explain the continued steady respiration of the proteoliposomes in the presence of this ionophore combination. PMID- 3019372 TI - [Long-term prognosis of infantile spasms after standard ACTH treatment in Japan]. PMID- 3019373 TI - Cannabis psychosis. PMID- 3019375 TI - The pharmacodynamics and dose-response relationships of the angiotensin converting enzyme inhibitor, cilazapril, in essential hypertension. AB - In a double-blind, placebo controlled, crossover study 12 patients with essential hypertension received single doses of 5, 10 and 20 mg of cilazapril, a new angiotensin converting enzyme (ACE) inhibitor. All doses similarly and significantly (P less than 0.05) reduced supine and erect blood pressure without increasing heart rate. The hypotensive effect was evident within 1 h, maintained for up to 8 h, with a maximal effect at 6 h. There was no discernible effect on blood pressure at 24 h after dosing. Plasma ACE activity was markedly inhibited to the same extent after all doses, with a peak inhibition of 94-96% at 2-3 h. At 24 h residual inhibition of ACE was 49-54%. Plasma renin activity increased in a dose-dependent manner with a peak at 6 h, and returned to baseline at 24 h. No correlation was found between the reduction in blood pressure and plasma renin activity, either at baseline or following cilazapril. There were no significant changes in plasma noradrenaline and the responses to upright posture and to dynamic exercise were preserved. There was no evidence of impaired exercise performance. Cilazapril is a potent ACE inhibitor with a rapid onset and a prolonged duration of action. These results suggest that peak ACE inhibition is achieved by 5 mg and that lower doses may be useful in clinical practice. PMID- 3019374 TI - Calcium antagonists and their mode of action: an historical overview. AB - The Ca2+ antagonists are a novel group of drugs useful in management of a variety of cardiac disorders. They differ from one another in terms of their chemistry, tissue specificity and selectivity. As a group, however, they share the common property of slowing Ca2+ entry through voltage-activated, ion-selective channels. Some of them exhibit other properties, including that of interfering with Na+ transport. At least one of them, diltiazem, has an intracellular action. Specific high and low affinity binding sites have been identified for two of the major groups of Ca2+-antagonists, with the binding sites for verapamil and its derivatives being distinct from those which can be occupied by the dihydropyridines. The number (Bmax) and affinity (KD) of these binding sites changes under certain pathological conditions--including a reduction in ischaemia and in spontaneous hypertension, an increase in the latter, at present, only demonstrated for the dihydropyridine binding sites. The sensitivity of a particular tissue to these drugs will depend upon a number of factors including the number of binding sites that are present, the contribution made by the Ca2+ entering through the voltage-activated channels to the functioning of the tissue, and properties which are peculiar to a particular type of Ca2+ antagonist, for example, whether, as in the case of verapamil, they exhibit use-dependence. PMID- 3019376 TI - Burkitt-like lymphoma in an English child: characterisation of tumour biopsy cells and of the derived tumour cell line. AB - An eight year old English boy presented with an abdominal undifferentiated 'Burkitt-like' lymphoma. Lymphoma cells from ascitic fluid were cultured on a human embryo fibroblast feeder layer and, after a short lag period, a cell line (DH-BL) was established which, like the original tumour, was both negative for the Epstein-Barr nuclear antigen (EBNA) and expressed a monoclonal pattern of surface immunoglobulin (alpha lambda). DH-BL also possessed the Burkitt-related 8:14 chromosome translocation in all metaphases analysed; no other chromosomal abnormalities were present. The cell surface phenotype of the original biopsy cells and the cultured tumour cells in early passage were investigated using a panel of monoclonal antibodies to B lineage-associated antigens. These antibodies had recently been used to characterise African 'endemic' Burkitt's lymphoma (BL) biopsy cells and their derived cell lines. The cell surface phenotype of this English EBNA negative Burkitt-like lymphoma biopsy was indistinguishable from that previously shown by biopsies of EBNA positive endemic BLs. It therefore appears that both the endemic and sporadic forms of BL, as illustrated by this case, may be derived from the same subset of progenitor cells. PMID- 3019378 TI - Potentiation of the generation of reactive oxidants by human phagocytes during exposure to benoxaprofen and ultraviolet radiation in vitro. AB - The effects of ultraviolet (UV) radiation on the spontaneous membrane-associated oxidative metabolism of human polymorphonuclear leukocytes (PMNL) and mononuclear leukocytes (MNL), co-incubated in the presence and absence of the non-steroidal, anti-inflammatory drug (NSAID) benoxaprofen at various concentrations, were investigated in vitro. Assays of superoxide generation and luminol-enhanced chemiluminescence (CL) were used to detect the production of reactive oxidants by PMNL and MNL. Benoxaprofen in the absence of UV radiation caused a dose-related activation of superoxide production and CL by PMNL. Exposure of PMNL to UV radiation in the absence of benoxaprofen caused a slight increase in superoxide generation and an unsustained slight increase in CL within 10 s. Exposure of both MNL and PMNL to UV radiation in the presence of benoxaprofen had a marked synergistic effect on both superoxide generation and CL. These effects were also achieved with UVA irradiation but were not detected when adherent cell depleted MNL or sodium fluoride (NaF) (10-2 M) pulsed PMNL, or PMNL from children with chronic granulomatous disease, were used. The pro-oxidative effects of benoxaprofen and UV radiation alone and in combination are dependent on intact phagocyte membrane-associated oxidative metabolism. It is postulated that the pro oxidative interactions which occur between human phagocytes, benoxaprofen and ultraviolet radiation cause the dermatological side-effects of benoxaprofen. PMID- 3019377 TI - Mast cells and matrix degradation at sites of tumour invasion in rat mammary adenocarcinoma. AB - Significant numbers of mast cells have been demonstrated histologically around the periphery of the invasive rat mammary adenocarcinoma 13672NF. The number of mast cells at microfoci along the tumour:host tissue junction was significantly greater than that found in normal mammary tissues, and few mast cells were detected within the tumour itself. Mast cell degranulation, often associated with disruption and lysis of the connective tissue matrix, was a common feature in later stages of tumour proliferation. When soluble products derived from purified rat peritoneal mast cells were added to monolayer cultures of rat stromal fibroblasts or tumour cells they stimulated a significant increase in total collagenase production, and the mast cell products were also capable of activating the latent collagenases thus produced. Histological examination indicated that degradation of local collagenous matrix was a common feature of mast cell degranulation, an observation possibly explained by the release of mast cell enzymes and/or the potential of this cell to modulate the expression of collagenolytic activity by surrounding cells. These observations suggest that, at least in some tumours, mast cells contribute to the connective tissue breakdown commonly associated with tumour invasiveness and metastatic spread. PMID- 3019379 TI - Ionophore A23187 stimulates arachidonic acid release and cyclic GMP accumulation in pig epidermis. AB - We studied the effect of Ionophore A23187 (A23187) on phospholipid degradation and the accumulation of cyclic GMP in pig epidermis. A23187 stimulated the release of AA, probably partially through the activation of phospholipase A2. A23187 also stimulated the accumulation of epidermal cyclic GMP, but the activity of cyclic GMP-dependent phosphodiesterases was not altered. Mepacrine, an inhibitor of phospholipase A2, inhibited the A23187-stimulated accumulation of cyclic GMP. The results suggest that A23187 stimulates the accumulation of epidermal cyclic GMP by a calcium-dependent process which also requires products of phospholipid degradation. PMID- 3019380 TI - Healthy HTLV-I carriers in Japan: the haematological and immunological characteristics. AB - The haematological and immunological characteristics of 34 healthy anti-HTLV-I antibody-positive individuals (HTLV-I carriers) in southwestern Japan were examined. No significant difference was noted between carriers and the controls in counts of RBC, WBC and the absolute number of lymphocytes. The serum IgG in the carriers was higher than that of the controls. The percentages of OKT4, OKT8, OKIa1 and B1-positive cells were found to be normal in the peripheral blood of the carriers, whereas the percentages of OKT11 and anti-Tac-positive cells were significantly higher in the carriers than in the controls. A correlation was observed between the percentages of anti-Tac-positive cells and the titres of anti-HTLV-I antibody in the carriers. After a 72 h incubation of peripheral blood lymphocytes with medium alone, the percentage of anti-Tac-positive cells tended to decrease in the controls, but to increase in carriers, with the appearance of large blastoid cells resembling blastic transformed lymphocytes cultured with mitogen. Tac and Ia antigens were markedly expressed on these large blastoid cells. PMID- 3019381 TI - Serum angiotensin converting enzyme, ceruloplasmin, and lactic dehydrogenase in anthracosilicosis and anthracosilicotuberculosis. PMID- 3019383 TI - Characterization of phenylalanine hydroxylase. AB - Iron can be bound to phenylalanine hydroxylase (PAH) in two environments. The assignment of the electron paramagnetic resonance spectrum of PAH to two, overlapping high-spin ferric signals is confirmed by computer simulation. Both environments are shown to be populated in the crude enzyme. Reconstitution of the apoenzyme demonstrated that the two iron environments are not interconvertible. Oxygen consumption during PAH reduction by tetrahydropterin in the absence of phenylalanine but not in its presence explains the different reduction stoichiometries (tetrahydropterin:enzyme) that have been observed. PMID- 3019382 TI - Herpes simplex virus type 1 persistence and latency in cultured rabbit corneal epithelial cells, keratocytes, and endothelial cells. AB - Cell cultures of rabbit corneal epithelium, keratocytes, and endothelium were used to determine the lytic cycle of herpes simplex virus type 1. Viral growth was fastest in epithelial cells. A novel HSV-1 in-vitro latency system was established in the three distinct cell types. Cell cultures were inoculated at low multiplicities of infection with HSV-1. Temperature manipulation alone was used to induce and reactivate latent HSV-1 infections. The presence of cellular stress proteins was demonstrated at supraoptimal temperatures. All cell types were capable of maintaining latent viral infections under these conditions. Viral persistence was present in 20% of epithelial cell cultures at supraoptimal temperatures, but not in keratocyte cultures or endothelial cell cultures. PMID- 3019384 TI - A spectral study of cobalt(II)-substituted Bacillus cereus phospholipase C. AB - The coordination sphere of both the structural and catalytic zinc ions of Bacillus cereus phospholipase C has been probed by substitution of cobalt(II) for zinc and investigation of the resultant derivatives by a variety of spectroscopic techniques. The electronic absorption, circular dichroic, magnetic circular dichroic, and electron paramagnetic resonance spectra were found to be strikingly similar when cobalt(II) was substituted into either site and are consistent with a distorted octahedral environment for the metal ion in both sites. Octahedral coordination appears comparatively rare in zinc metalloenzymes but has been suggested for glyoxalase I [Sellin, S., Eriksson, L. E. G., Aronsson, A.-C., & Mannervik, B. (1983) J. Biol. Chem. 258, 2091-2093; Garcia-Iniguez, L., Powers, L., Chance, B., Sellin, S., Mannervik, B., & Mildvan, A. S. (1984) Biochemistry 23, 685-689], transcarboxylase [Fung, C.-H., Mildvan, A. S., & Leigh, J. S. (1974) Biochemistry 13, 1160-1169], and the regulatory binding site of Aeromonas aminopeptidase [Prescott, J. M., Wagner, F. W., Holmquist, B., & Vallee, B. L. (1985) Biochemistry 24, 5350-5356]. Phospholipase C is so far unique in having two such sites. PMID- 3019386 TI - Nucleotide sequence binding preferences of nogalamycin investigated by DNase I footprinting. AB - Four DNA restriction fragments, designated tyrT, pTyr2, pUC13, and Xbs1, have been used as substrates for footprinting studies with DNase I in the presence of the anthracycline antibiotic nogalamycin. With each fragment a distinct pattern of antibiotic-protected binding sites is observed, but no concensus sequence emerges from the data. All sites are located in regions of alternating purine pyrimidine sequence, most commonly associated with the dinucleotide steps TpG (CpA) and GpT (ApC), suggesting that the preferred binding sites may contain all four nucleotides and/or that peculiarities of the dynamics of DNA conformation at alternating sequences may be critical for nogalamycin binding. Some concentration dependence of footprinting patterns is evident, in contrast to previous studies with a variety of sequence-specific ligands. Enhanced susceptibility to attack by DNase I is commonly observed at sequences flanking strong antibiotic-binding sites. Nogalamycin selectively inhibits cleavage of DNA at certain guanine containing sequences by the G-specific photosensitized reaction with methylene blue. Comparison of these effects with its action on the G-specific reaction with dimethyl sulfate suggests that the amino sugar moiety of nogalamycin may be preferentially located in the minor helical groove at some binding sites but in the major groove at others. PMID- 3019385 TI - Iron-containing metallocenes as active site directed inhibitors of the proteinase that cleaves the NH2-terminal propeptides from type I procollagen. AB - Derivatives of ferrocene (dicyclopentadienyliron) (Fc) were examined as active site directed inhibitors of type I procollagen N-proteinase, the enzyme that cleaves the NH2-terminal propeptides from type I procollagen. The compounds were shown here to be reversible, competitive inhibitors of the enzyme. The effectiveness of the Fc inhibitors varied with modification of the cyclopentadienyl (cp) rings. The monocarboxylic acid (I) and the 1,1' dicarboxylic acid (II) derivatives of Fc inhibited 50% of the enzymic activity (I50) at concentrations of 1.0 and 0.5 mM, respectively. The Ki values were 0.3 mM for both I and II. Derivatization of the carbonyl alpha to the cp ring of compound I (FcCOCH2CH2COOH, III) increased the inhibitory activity (I50 = 0.100 mM; Ki = 0.065 mM). Removal of the carbonyl alpha to the cp ring of III did not improve inhibitory activity: FcCH2CH2COOH, I50 = 2 mM; FcCH = CHCOOH, I50 = 1.5 mM. The active inhibitory species apparently contained iron in the 3+ valence state since two ferrocenium derivatives were very effective inhibitors: ferrocenium tetrachloroferrate, IV (I50 = 0.030 mM; Ki = 0.004 mM), and carboxyferrocenium hexafluorophosphate, V (I50 less than 0.1 mM; Ki less than 0.05 mM). In addition, reduction of III with ascorbic acid abolished its inhibitory activity. Compounds I and III stabilized the enzyme to heat denaturation in the absence of exogenous calcium; compound IV did not stabilize the enzyme. Further observations indicated that Fc derivatives were specific inhibitors of procollagen N-proteinase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019387 TI - Covalent modification of the inhibitor binding site(s) of Escherichia coli ADP glucose synthetase: specific incorporation of the photoaffinity analogue 8 azidoadenosine 5'-monophosphate. AB - The photoaffinity agent 8-azidoadenosine 5'-monophosphate (8-N3AMP) is an inhibitor site specific probe of the Escherichia coli ADP-glucose synthetase (ADPG synthetase). In the absence of light, 8-N3AMP exhibits the typical reversible allosteric kinetics of the physiological inhibitor AMP. In the presence of light (254 nm), the analogue specifically and covalently modifies the enzyme, and photoincorporation is linearly related to loss of catalytic activity up to at least 65% inactivation. The substrate ADPG provides nearly 100% protection from 8-N3AMP photoinactivation, while the substrate ATP provides approximately 50% protection and the inhibitor AMP, approximately 30% protection. These three adenylate allosteric effectors of E. coli ADPG synthetase also protect it from photoincorporation of 8-N3AMP. A structural overlap of the inhibitor and substrate binding sites is proposed which explains the protection data in light of the known binding and kinetic properties of this tetrameric enzyme. PMID- 3019388 TI - Alteration of intramolecular disulfides in insulin receptor/kinase by insulin and dithiothreitol: insulin potentiates the apparent dithiothreitol-dependent subunit reduction of insulin receptor. AB - Dithiothreitol (DTT) was observed to increase both beta-subunit autophosphorylation and exogenous substrate phosphorylation of the insulin receptor in the absence of insulin. The natural protein reducing agent thioredoxin was also observed to increase the insulin receptor beta-subunit autophosphorylation. The activation of the insulin receptor/kinase by both DTT and thioredoxin was found to be additive with that of insulin. Further, the increase in the insulin receptor beta-subunit autophosphorylation in the presence of DTT and insulin was demonstrated to be due to an increase in the initial rate of autophosphorylation without alteration in the extent of phosphorylation. Similarly, the increase in the exogenous substrate phosphorylation was due to an increase in the Vmax of phosphorylation without significant effect on the apparent Km of substrate binding. In the presence of relatively low concentrations of DTT, insulin was found to potentiate the apparent insulin receptor subunit reduction of the native alpha 2 beta 2 heterotetrameric complex into alpha beta heterodimers, when observed by silver staining of sodium dodecyl sulfate-polyacrylamide gels. N-[3H]Ethylmaleimide ([3H]NEM) labeling in the absence of DTT pretreatment demonstrated that only the beta subunit had accessible sulfhydryl group(s). However, treatment of insulin receptors with DTT increased the amount of [3H]NEM labeling in the beta subunit as well as exposing sites on the alpha subunit. Further, incubation of the insulin receptors with the combination of DTT and insulin also demonstrated the apparent insulin-potentiated subunit reduction without any increase in the total amount of [3H]NEM labeling.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019389 TI - Apolipoprotein C-III/sphingomyelin recombinants: formation, isolation, and characterization. AB - The association of apolipoprotein C-III (apoC-III) from human very low density lipoprotein with sphingomyelin from egg yolk (EYSM) has been studied at the transition temperature (Tc) of the phospholipid. Upon incubation of aliquots of the apoprotein with increasing amounts of sphingomyelin, the alpha-helical content of the apoprotein increased from 20% in the absence of EYSM to a limiting value of 67% at a protein:lipid molar ratio of 1:200. The tryptophan fluorescence spectrum of the apoprotein exhibited a gradual blue shift from 356 nm in the absence of EYSM to 348 nm when the protein:lipid ratio in the complex had reached 1:50. Gel filtration chromatography of complexes formed by incubating the apoprotein and phospholipid at differing apoC-III:EYSM ratios demonstrated a disintegration of sphingomyelin vesicles into particles of decreasing size with increasing proportion of protein. This effect was confirmed by sedimentation velocity experiments in which the observed sedimentation coefficient of EYSM decreased from 14.0 S (for vesicles) to a limiting value of 7.0 S when the apoprotein:phospholipid ratio reached 1:50 in the complex. Electron micrographs of negatively stained EYSM vesicles showed spherical particles of 380-A diameter. Addition of apoC-III led to the formation of disk-shaped structures whose diameter decreased to a limiting value of 204 +/- 34 A at a protein:lipid ratio of 1:50. In contrast, the disk thickness was relatively constant at 51 +/- 2 A for all isolated complexes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019390 TI - Human lysosomal sphingomyelinase: substrate efficacy of apolipoprotein/sphingomyelin complexes. AB - Human apolipoprotein stimulation of sphingomyelin (SM) hydrolysis by sphingomyelinase from human skin fibroblasts has been studied. Apolipoproteins A I, A-II, B, C-I, and E do not enhance sphingomyelin hydrolysis above control levels. In contrast, apoC-II stimulates sphingomyelin hydrolysis by approximately 2.5-fold. ApoC-III, the most potent apoprotein activator, stimulates hydrolysis by 3-4-fold. ApoC-III stimulation is not significantly different for the three different isoforms which carry 0, 1, or 2 sialic acid residues. The amino terminal half of this apoprotein, C-III(1-40), which does not bind to phospholipid surfaces, does not activate sphingomyelinase. In contrast, the carboxyl-terminal half, C-III(41-79), which strongly binds to phospholipid surfaces, stimulates sphingomyelin hydrolysis to the same level as that produced by the intact, full-length apoprotein. Incubation of sphingomyelin vesicles with increasing proportions of apoC-III results in the formation of complexes of increasing apoC-III:SM ratio and decreasing radius. The hydrolysis of sphingomyelin in the 1:50 (mol/mol) complex was more than 2-fold greater than that of the 1:200 (mol/mol) complex. The rate of hydrolysis of egg yolk sphingomyelin in the 1:50 complex was maximal [0.9 mumol h-1 (mg of protein)-1] at the gel----liquid-crystalline phase transition temperature (Tm) of the complex (40 degrees C). The rate of hydrolysis fell markedly at either higher or lower temperature. Determination of the apparent Km and Vmax values below, at, and above Tm indicated that the temperature dependence of sphingomyelin hydrolysis was attributable primarily to changes in Vmax.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019392 TI - Binding of N-acetylgalactosamine-specific lectins to spin-labeled galactosamine derivatives. AB - Legume seed lectins specific for N-acetyl-alpha-D-galactosaminyl end groups from Amphicarpaea bracteata, lima bean, Griffonia simplicifolia, Dolichos biflorus, and soybean were compared with respect to binding of several spin-labeled derivatives of D-galactosamine by electron spin resonance and precipitin inhibition analysis. Spin-label II [methyl 2-[[(2,2,5,5-tetramethyl-1 oxopyrrolidin-3-yl) carbonyl]amino]-2-deoxy-alpha-D-galactopyranoside], spin label III [1-(methyl 2-deoxy-alpha-D-galactopyranosid-2-yl)-3-(2,2,6, 6 tetramethyl-1-oxypiperidin-4-yl)-2-thiourea], and spin-label IV [1-[4-[[(methyl 2 deoxy-alpha-D-galactopyranosid-2-yl)amino]carbonyl]phenyl]-3-(2, 2,6-tetramethyl 1-oxypiperidin-4-yl)-2-thiourea] contain 2-N-(oxypiperidinyl) or 2-N (oxypyrrolidinyl) substituents varying in length and polarity of the linker arm between the glycoside and nitroxide ring. Spin-labels II and III were found to bind very weakly to all the lectins tested (Kd greater than or equal to 1.0 mM). Spin-label IV, containing a planar, nonpolar 2-N-phenyl group, was bound very strongly (Kd = 0.1-0.4 mM) and was moderately immobilized (2T parallel = 48-56 G) by all lectins except that from D. biflorus. Notably, the affinity of spin-label IV to lima bean lectin was 18-fold greater than that for methyl N-acetyl-alpha galactosaminide. These results suggest that when the bulky oxypiperidinyl moiety lies in a position close to the sugar ring, it interferes with binding; in the cases where a phenyl group spacer exists, the aromatic ring in some cases actually enhances binding.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019391 TI - Raman and infrared spectra of cytochrome c peroxidase-carbon monoxide adducts in alternative conformational states. AB - Resonance Raman (RR) spectra are reported for CO-bound cytochrome c peroxidase (CCP). At low pH, two forms are observed: form II, with nu Fe-C = 530 cm-1 and delta FeCO = 585 cm-1, and form I, with nu Fe-C = 495 cm-1 and no detectable delta FeCO. They appear to have coincident nu CO infrared bands, at 1922 cm-1. These low-pH forms, similar to those observed for horseradish peroxidase (HRP), are attributed to tilted, H-bonded CO and perpendicular CO, respectively. The frequencies differ between the two proteins, a weaker H bond to CO being indicated for CCP. As with HRP, the equilibrium between forms I and II is shifted toward the latter at increasing CO concentrations, suggesting that secondary binding of CO perturbs the distal residues. At high pH [8.4, tris(hydroxymethyl)aminomethane buffer] the form II fraction converts to another form, II', with nu FeC = 503 cm-1, delta FeCO = 575 cm-1, and nu CO = 1948 cm-1; a tilted, non-H-bonded geometry is suggested. If phosphate buffer is used, however, form II (H bonded) persists at pH 8.4. This result establishes a role for phosphate in stabilizing the H-bonded form of the enzyme; it is suggested that phosphate binds near the distal imidazole and substantially increases its pKa. The conformational state is also influenced by aging. Fresh protein contains purely high spin FeIII heme, as monitored by the high-frequency RR spectrum, and yields form II almost exclusively at elevated CO concentrations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019393 TI - Demonstration of a collisional interaction of ubiquinol with the ubiquinol cytochrome c2 oxidoreductase complex in chromatophores from Rhodobacter sphaeroides. AB - Ubiquinone-10 can be extracted from lyophilized chromatophores of Rhodobacter sphaeroides (previously called Rhodopseudomonas sphaeroides) without significant losses in other components of the electron-transfer chain or irreversible damages in the membrane structure. The pool of ubiquinone can be restored with exogenous UQ-10 to sizes larger than the ones in unextracted membranes. The decrease in the pool size has marked effects on the kinetics of reduction of cytochrome b-561 induced by a single flash of light and measured in the presence of antimycin. The initial rate of reduction, which in unextracted preparations increases on reduction of the suspension over the Eh range between 170 and 100 mV (pH 7), is also stimulated in partially UQ-depleted membranes, although at more negative Eh's. When the UQ pool is completely extracted the rate of cytochrome (Cyt) b-561 reduction is low and unaffected by the redox potential. In membranes enriched in UQ-10 above the physiological level the titration curve of the rate of Cyt b-561 reduction is displaced to Eh values more positive than in controls. This effect is saturated when the size of the UQ pool is about 2-3 times larger than the native one. The reduction of Cyt b-561 always occurs a short time after the flash is fired; also the duration of this lag is dependent on Eh and on the size of the UQ pool. A decrease or an increase in the pool size causes a displacement of the titration curve of the lag to more negative or to more positive Eh's, respectively. Similarly, the lag becomes Eh independent and markedly longer than in controls when the pool is completely extracted. These results demonstrate that the rate of turnover of the ubiquinol oxidizing site in the b-c1 complex depends on the actual concentration of ubiquinol present in the membrane and that ubiquinol from the pool is oxidized at this site with a collisional mechanism. Kinetic analysis of the data indicates that this reaction obeys a Michaelis Menten type equation, with a Km of 3-5 ubiquinol molecules per reaction center. PMID- 3019394 TI - Rapid redox equilibrium between the mitochondrial Q pool and cytochrome b during triphasic reduction of cytochrome b by succinate. AB - The reliability of monitoring the redox reactions of cytochrome b using the different wavelengths employed by different authors has been reexamined. It was found that 562-575 nm is suitable in succinate: cytochrome c reductase but not in mitochondria, in which case 562-540 nm is a better pair. Direct optical measurements of the redox reaction kinetics of the mitochondrial Q pool using a commercial dual-wavelength spectrophotometer are possible when succinate is used as the electron donor. Using the correct wavelength pair, and with malonate to slow down the electron input, the reduction course of cytochrome b was still triphasic but a plateau or a turn replaced the oxidation phase previously reported by several authors. At the same time, the reduction course of the Q pool was also triphasic, and in perfect match with that of cytochrome b. Destruction of the Rieske iron-sulfur cluster by British anti-Lewisite (BAL) + O2 treatment or prereduction of the high-potential components made the reduction of both Q and b monophasic. The plot of log (Q/QH2) against log (b3+/b2+) gave a straight line with an n value of 1.7 for cytochrome b at pH 7.4. This n value rose to 2.0 at pH 6.5 and dropped to 1.4 at pH 8.5. On the other hand, the mid-point potential of cytochrome b relative to that of the Q pool remained essentially unchanged between pH 6.5 and 8.4. BAL treatment had a small effect on the midpoint potential of cytochrome b relative to that of the Q pool and had no effect on the n value. Addition of quinone homologues and analogues extended the plateau phase in the reduction of cytochrome b, but exogenous quinones did not equilibrate rapidly with cytochrome b. It was concluded that the appearance of the plateau between the two reduction phases of Q and b is caused by the rapid delivery of electrons to the high-potential components of the respiratory chain as envisaged in the Q cycle; the unexpected n value for cytochrome b suggests a concerted reduction by QH2 of two species of cytochromes b-562. PMID- 3019395 TI - The effect of ring substituents on the mechanism of interaction of exogenous quinones with the mitochondrial respiratory chain. AB - In uncoupled pig-heart mitochondria the rate of the reduction of duroquinone by succinate in the presence of cyanide is inhibited by about 50% by antimycin. This inhibition approaches completion when myxothiazol is also added or British anti Lewisite-treated (BAL-treated) mitochondria are used. If mitochondria are replaced by isolated succinate:cytochrome c oxidoreductase, the inhibition by antimycin alone is complete. The reduction of a plastoquinone homologue with an isoprenoid side chain (plastoquinone-2) is strongly inhibited by antimycin with either mitochondria or succinate:cytochrome c reductase. The reduction by succinate of plastoquinone analogues with an n-alkyl side chain in the presence of mitochondria is inhibited neither by antimycin nor by myxothiazol, but is sensitive to the combined use of these two inhibitors. On the other hand, the reduction of the ubiquinone homologues Q2, Q4, Q6 and Q10 and an analogue, 2,3 dimethoxyl-5-n-decyl-6-methyl-1,4-benzoquinone, is not sensitive to any inhibitor of QH2:cytochrome c reductase tested or their combined use, either in normal or BAL-treated mitochondria or in isolated succinate:cytochrome c reductase. It is concluded that quinones with a ubiquinone ring can be reduced directly by succinate:Q reductase, whereas those with a plastoquinone ring can not. Reduction of the latter compounds requires participation of either center i or center o (Mitchell, P. (1975) FEBS Lett. 56, 1-6) or both, of QH2:cytochrome c oxidoreductase. It is proposed that a saturated side chain promotes, while an isoprenoid side chain prevents reduction of these compounds at center o. PMID- 3019396 TI - Conformational alterations within the glycocalyx of erythrocyte membranes studied by spin labelling. AB - The structure of the glycocalyx of the membrane of human erythrocytes and spectrin-depleted vesicles was studied under various conditions by two spin labelling approaches: covalently labelling sialic acid residues of the glycocalyx and incorporation of a charged hydrophobic spin probe, CAT 16, being sensitive to alterations on the membrane surface into the lipid phase. Although cell electrophoretic measurements which were performed, additionally, indicated an erection of the glycocalyx upon decreasing the ionic strength of the suspension medium a more restricted mobility of spin-labelled sialic acid residues was found, in this case probably due to electrostatic interactions. The enhanced mobility of the spin probe CAT 16 at low ionic strength as well as in the case of neuraminidase-treated cells could be caused by reduced steric and electrostatic interaction with glycoproteins and glycolipids. La3+ adsorption and virus attachment on the human erythrocyte membrane were accompanied with a reduced mobility of sugar headgroups of the surface coat. No indication of cluster formation or lateral segregation of glycophorin molecules was found upon virus binding. After denaturation of the spectrin cytoskeleton of intact erythrocytes, increased mobility of spin-labelled sialic acid residues was observed. PMID- 3019397 TI - Leucine transport in relation to the activities of Na+-H+ antiporter and Na+/K+ pump stimulated by serum and a tumor promoter. AB - Fetal calf serum and 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the rate of leucine uptake by Chang liver cells in Na+-containing medium. Addition of monensin to the incubation medium also increased the leucine uptake. All these agents were capable of raising the cytoplasmic pH, which was blocked by a prior addition of amiloride or removing Na+ from assay medium, suggesting activation of Na+-H+ exchange across the cell membrane by fetal calf serum and TPA. The stimulation of leucine uptake by monensin and fetal calf serum was blocked completely or incompletely by addition of ouabain or amiloride. The basal and fetal-calf-serum- or TPA-stimulated leucine uptake was extensively depressed by the presence of an excess of 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid in the incubation medium. Based on these results it is proposed that the transport of leucine by the system L is stimulated by fetal calf serum and TPA with a high concentration of Na+ outside the cells as a result of alkalinization of the cytoplasm and coordinated activation of (Na+ + K+)-ATPase by these stimulators to maintain the transmembrane Na+ gradient and also hyperpolarize the cell membrane. PMID- 3019398 TI - Isolation of human neutrophil plasma membranes employing neutrophil cytoplasts and changes in the cell-surface proteins upon cell activation. AB - A plasma membrane fraction, highly enriched in 5'-nucleotidase activity, was prepared from human neutrophils by disruption of previously formed neutrophil cytoplasts (enucleated neutrophils), which were devoid of intracellular organelles. This plasma membrane fraction shows an extremely low contamination by specific and azurophilic granule markers as compared to previous reported preparations. Nevertheless, a novel tertiary granule (Mollinedo, F. and Schneider, D.L. (1984) J. Biol. Chem. 259, 7143-7150), unlike specific and azurophilic granules, fuses partially with the cell surface under the experimental conditions used for cytoplast preparation. Comparison between the external cell-surface proteins in resting neutrophils and neutrophil cytoplasts by lactoperoxidase-catalyzed iodination showed some differences both in deletion and in addition of proteins. In resting cells, iodine was incorporated into at least 13 proteins ranging in size from over 200 to 30 kDa. A 140 kDa polypeptide, representing the major labeled surface component in resting neutrophils, was absent from cytoplasts. Furthermore, high-molecular-weight proteins (110 and over 160 kDa were more exposed to iodination after cytoplast preparation. Activation of human neutrophils by N-formylmethionylleucylphenylalanine induced some alterations in the pattern of labeled cell-surface proteins, which correlated to a certain degree with those observed during cytoplast preparation. PMID- 3019399 TI - Properties of the porin of Haemophilus influenzae type b in planar lipid bilayer membranes. AB - The major outer membrane protein (40 kDa) of the bacterium Haemophilus influenzae type b is a porin which forms transmembrane permeability channels. It has an exclusion limit for oligosaccharides of about 1.4 kDa. When this protein was added to the aqueous phase which was bathing a planar lipid bilayer, it caused the conductance of the membrane to increase by several orders of magnitude. At low protein concentrations (2-10 pM), the conductance of the membrane increased in a stepwise fashion with an average single-channel conductance of 1.1 nS in 1 M KCl. Single-channel experiments were performed with a variety of different salts. The conductance of single channels was proportional to the specific conductance of the aqueous solution which was bathing the membrane. Current through the pores was proportional to the applied voltage, indicating that these pores are not voltage-controlled. The 40 kDa porin was very slightly cation-selective: the pores were about 1.6-times more permeable to potassium ions than to chloride ions. These properties of the 40 kDa porin are those of large water-filled channels and are characteristic of most bacterial porins. The single-channel conductance of the porin is, however, much smaller than might be expected from its exclusion limit. A model is proposed which could explain the differences in apparent pore size. PMID- 3019400 TI - Mechanism of inhibition of mitochondrial enzymatic complex I-III by adriamycin derivatives. AB - We demonstrate here that complex I-III of bovine heart mitochondrial membrane is inhibited by adriamycin derivatives. This inhibition is a cardiolipin-dependent process. This lipid, specific to the inner mitochondrial membrane, has been shown previously to interact specifically with adriamycin in model membranes (Goormaghtigh, E., Chatelain, P., Caspers, J. and Ruysschaert, J.-M. (1980) Biochim. Biophys. Acta 597, 1-14) and in mitochondrial membranes (Cheneval, D., Muller, M., Toni, R., Ruetz, S. and Carafoli, E. (1985) J. Biol. Chem. 260, 13003 13007). The differential scanning calorimetry data indicate that, in multilamellar liposomes, the formation of antibiotic-cardiolipin complexes induces a clustering of cardiolipin molecules. Conformational analysis of the antibiotic-cardiolipin complexes suggests that plane-plane interactions between the antibiotics aromatic moieties stabilize this complex formation. Possible mechanisms of inactivation of complex I-III by adriamycin are proposed. PMID- 3019401 TI - Unsuitability of the 86Rb+ uptake method for estimation of (Na+ + K+)-ATPase activity in innervated tissues. AB - (Na+ + K+)-ATPase activity was estimated by 86Rb+ uptake in dog saphenous vein to determine the validity of the technique in tissues that have a sympathetic innervation. When saphenous vein rings were incubated at 37 degrees C in Krebs' solution containing 86Rb+, the cardenolide acetylstrophanthidin caused a concentration-dependent inhibition of Rb+ uptake. The threshold for inhibition was approx. 10 nM acetylstrophanthidin and the maximum effect was obtained at 9 microM. In the upper part of this concentration range (greater than 1 microM) acetylstrophanthidin released noradrenaline from the sympathetic nerve terminals associated with the tissue. In this upper part of the acetylstrophanthidin concentration range the alpha-adrenoceptor antagonist phentolamine (8 microM) reduced, by up to 25%, the degree of 86Rb+ uptake inhibition caused by the cardenolide. In other experiments, saphenous vein strips were loaded with 86Rb+ and perifused with Krebs' solution containing acetylstrophanthidin. At concentrations which release noradrenaline, acetylstrophanthidin increased the efflux of 86Rb+. Phentolamine (8 microM) prevented the acetylstrophanthidin evoked efflux of the isotope as did prior in vitro denervation of 86Rb+ loaded strips with 6-hydroxydopamine. Exogenous noradrenaline (1-100 microM) added to the perifusing fluid also caused an efflux of 86Rb+ that was attenuated by phentolamine. The data indicate for dog saphenous vein that with low concentrations of acetylstrophanthidin the extent of 86Rb+ accumulation might accurately reflect prevailing (Na+ + K+)-ATPase activity. At higher concentrations of acetylstrophanthidin, however, noradrenaline is released from the nerve endings and causes 86Rb+ efflux from the smooth muscle cells consequent upon alpha-adrenoceptor activation. Since this efflux reduces the extent of Rb+ accumulation, measurement of the latter does not adequately reflect uptake mediated by the activity of (Na+ + K+)-ATPase. This is significant because in most applications of the 86Rb+ uptake method it is the estimate of Rb+ accumulation made in the presence of a high concentration of cardenolide that forms the basis of all subsequent calculations with respect to (Na+ + K+)-ATPase activity. PMID- 3019402 TI - Nucleotide specificity of the E2K----E1K transition in (Na+ + K+)-ATPase as probed with tryptic inactivation and fragmentation. AB - The nucleotide specificity for the E2K----E1K conformational transition in (Na+ + K+)-ATPase as the key step for overall hydrolytic activity and coupled cation transport has been investigated. Use has been made of tryptic inactivation, which is biexponential in time for the enzyme in the presence of Na+ with or without nucleotides (E1 conformation) and monoexponential in the presence of K+ (E2 conformation). ATP, AdoPP[NH]P and CTP in order of decreasing effectivity induce the biphasic tryptic inactivation pattern in the presence of K+. Their order of effectivity is inversely related to the rate constant of the second (slow) phase of inactivation. In the presence of K+ and either ITP or GTP tryptic inactivation remains monoexponential, indicating that these nucleotides cannot drive the E2K-- -E1K transition. Tryptic inactivation has been compared with tryptic fragmentation of the alpha-subunit (apparent mol. wt. 94 kDa) of (Na+ + K+) ATPase. In the E1 conformation (Na+ present) a 71 kDa fragment is formed during the second phase of inactivation. In the E2 conformation (K+ present) the alpha subunit is split to fragments of 41 and 52 kDa. In the presence of K+ and ATP, ADP, AdoPP[NH]P or CTP the 71 kDa fragment is formed in amounts which decrease in the order ATP approximately equal to ADP greater than AdoPP[NH]P greater than CTP. In the presence of K+ and AMP, ITP or GTP the 71 kDa fragment is absent and only the E2 fragments are formed. From these and literature data we arrive at a specificity order for the E2K----E1K transition of ATP greater than ADP greater than AdoPP[NH]P greater than CTP greater than ITP = GTP = AMP. The same order holds for K+ transport in the K+-K+ exchange and for overall hydrolytic activity (Na+ + K+ present) with the natural nucleoside triphosphates as substrates. This marks the E2K----E1K transition as the step in the reaction mechanism that determines nucleotide specificity for (Na+ + K+)-activated hydrolysis and coupled cation transport. PMID- 3019403 TI - Purification and characterization of the beta 2-adrenergic receptor from calf lung. AB - Improved methods for the solubilization and purification of the mammalian beta 2 adrenergic receptor have allowed this protein to be characterized further. In the present study, the beta 2-adrenergic receptor has been solubilized from calf lung membranes using a 0.4% digitonin/0.08% cholate-Tris buffer with multiple proteinase inhibitors. This solubilization buffer produced 60-75% solubilization of the receptor, which retained complete ligand-binding activity as determined by Scatchard analysis. Subsequent receptor purification employed a modified acebutolol-agarose affinity resin. The eluate from the affinity resin was then purified further by HPLC-gel exclusion chromatography on a Spherogel TSK-3000 column. The receptor, detected by [3H]dihydroalprenolol or [125I]iodocyanopindolol binding, eluted with a retention time identical to that of IgG (Stokes radius 49 A). Autoradiography following SDS-PAGE of the purified iodinated receptor clearly demonstrated two distinct bands: a major band of 67 kDa and a minor band of 53 kDa. With the addition of leupeptin to the proteinase inhibitor regimen, the 53-kDa band became less apparent. Two-dimensional gel electrophoresis indicated that the 67-kDa peptide behaved as a predominantly single species with a pI of 6.0 +/- 0.2. The purified receptor protein recognized adrenergic ligands with a specificity identical to that of the membrane-bound beta 2-adrenergic receptor. PMID- 3019404 TI - Direct determination by 2H-NMR of the ionization state of phospholipids and of a local anaesthetic at the membrane surface. AB - Protonation and deprotonation of the primary amino group of phosphatidylethanolamine, and of the lipid phosphate groups of phosphatidylethanolamine with phosphatidylcholine, have been observed directly and isothermally in equimolar mixed fluid bilayers of the two phospholipids. In addition, the acid-base titration of the secondary amino group of the local anaesthetic, tetracaine, whilst partitioned into the bilayers, has also been determined. Here we show how studies by deuterium nuclear magnetic resonance of non-perturbing deuterons, specifically placed at the membrane polar-apolar interface, can give direct information about the electrostatics at a membrane surface. PMID- 3019405 TI - Changes in sinusoidal plasma membrane enzyme activities during the pre replicative phase of liver regeneration. AB - Changes in a range of plasma membrane enzyme activities during the early period of liver regeneration are thought to be related to the initiation of DNA synthesis and the triggering of cellular activation. The sinusoidal plasma membrane was isolated from control and partially hepatectomized animals at various intervals during the pre-replicative phase. The specific activities of 5' nucleotidase, (Na+ + K+)-ATPase, Ca2+-ATPase, Mg2+-ATPase showed that after partial hepatectomy changes in the enzyme activities at the sinusoidal plasma membrane region occur. These changes are probably related to the remodeling of the cell-surface that occurs before the division of hepatocytes. PMID- 3019406 TI - Transport characteristics of system A in the rat exocrine pancreatic epithelium analyzed using the specific non-metabolized amino acid analogue alpha methylaminoisobutyric acid. AB - The selectivity and kinetics of system A amino acid transport in the rat exocrine pancreatic epithelium were characterized using the specific analogue alpha methylaminoisobutyric acid. Unidirectional influx of alpha-methylaminoisobutyric acid was measured in isolated perfused pancreata by rapid dual tracer dilution. In cross-inhibition experiments DL-methylalanine, L-serine, L-cysteine, glycine, L-phenylalanine and L-glutamine were effective inhibitors of influx, whereas L glutamate and L-lysine were less effective. In the presence of sodium alpha methylaminoisobutyric acid influx was saturable with an apparent Kt = 1.7 +/- 0.2 mM and Vmax = 0.49 +/- 0.03 mumol/min per g (mean +/- S.E., n = 6). Influx of alpha-methylaminoisobutyric acid at 50 microM and 100 microM concentrations was significantly inhibited as the perfusate sodium concentration was gradually decreased from 156 mM to 26 mM by isoosmolar choline replacement. Estimated Kt values for sodium at these two methylaminoisobutyric acid concentrations approximated 200 mM. System A activity in the basolateral membrane of the exocrine pancreatic epithelium exhibits a high transport affinity, a wide tolerance for different amino acids and a dependency upon the extracellular sodium concentration. PMID- 3019408 TI - Changes in conformation of spin-labeled calmodulin by phospholipids. AB - Spin-labeled calmodulin was synthesized and the effects of phospholipids on its conformation were examined by ESR spectroscopy. Phosphatidylserine (0.1-1.0 mM) increased the signal intensity of the ESR spectrum of spin-labeled calmodulin and decreased the apparent rotational correlation time in the presence of 0.1 mM CaCl2. This change was reversed by addition of excess calcium, and in the absence of calcium phosphatidylserine did not change the spectrum, suggesting that the change in spin-labeled calmodulin brought about by phosphatidylserine was not induced by a hydrophobic interaction of the two, but by inhibition of the binding of calcium to calmodulin. L-Serine and O-phospho-L-serine had no effect on the ESR signals of spin-labeled calmodulin. The effects of various other phospholipids were also examined. Their inhibitory activities were in the order phosphatidic acid greater than phosphatidylserine greater than phosphatidylglycerol = phosphatidylinositol; phosphatidylethanolamine and phosphatidylcholine had no effect on the spectra. The effects of these phospholipids were dependent on their binding activities toward calcium. Furthermore, phosphatidic acid and phosphatidylserine at 1 mM reduced the activity of calmodulin-dependent phosphodiesterase by 16.4 and 8.7%, respectively. These findings indicate that spin-labeled calmodulin did not interact with the phospholipids by a hydrophobic interaction, but that calcium binding to spin-labeled calmodulin interfered with phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol, and some of these phospholipids inactivated calmodulin. Thus the activity of calmodulin may be regulated in part by some phospholipids. PMID- 3019407 TI - A low-calcium-requiring calcium-activated neutral proteinase from human placenta. AB - A low-calcium-requiring calcium-activated neutral proteinase (mu CANP) has been purified to homogeneity from human placenta. The purification procedure includes chromatography on DEAE-cellulose, Ultrogel AcA-22 and DEAE-Sephadex in succession. The purified mu CANP is a thiol proteinase and requires calcium for activity. Half-maximal activation occurs at 40 microM calcium. It is a heterodimer with subunits of 74 kDa and 32 kDa. (The placental mCANP has subunits of 70 kDa and 32 kDa.) Mn2+ or Sr2+, in combination with Ca2+, activates the enzyme synergistically. The presence of both mCANP and mu CANP in equal proportion in human placenta is reported for the first time. This will facilitate a comparative study of these two forms of human calcium-activated neutral proteinase, especially their physiological structural and functional interrelationship. Maximal activation of the autolysed mCANP occurs at a calcium concentration much higher than that for mu CANP; and this autolysed mCANP does not cross-react with antiserum against mu CANP, suggesting that the two forms of proteinase are independent species. PMID- 3019409 TI - Leukotriene synthesis by human gastrointestinal tissues. AB - The prostaglandin and leukotriene synthesizing capacity of human gastrointestinal tissues obtained at surgery was investigated using radioimmunoassay for prostaglandin E2, leukotriene B4 and sulfidopeptide leukotrienes. The leukotriene immunoassay data were validated by high-pressure liquid chromatography (HPLC). During incubation at 37 degrees C, fragments of human gastric, jejuno-ileal and colonic mucosa released considerably larger amounts of prostaglandin E2 than of leukotriene B4 and sulfidopeptide leukotrienes. Gastrointestinal smooth muscle tissues released even larger amounts of prostaglandin E2, but smaller amounts of leukotrienes than the corresponding mucosal tissues. Adenocarcinoma tissue released larger amounts of leukotriene B4, sulfidopeptide leukotrienes and prostaglandin E2 than normal colonic mucosa. Ionophore A23187 (5 micrograms/ml) did not stimulate release of prostaglandin E2 from any of the tissues investigated, but enhanced release of leukotriene B4 and sulfidopeptide leukotrienes. HPLC analysis demonstrated that immunoreactive leukotriene B4 co chromatographed almost exclusively with standard leukotriene B4, while immunoreactive sulfidopeptide leukotrienes consisted of a mixture of leukotrienes C4, D4 and E4. Leukotriene synthesis by human gastrointestinal tissues was inhibited by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) and the dual enzyme inhibitor BW755C (3-amino-1-(trifluoromethylphenyl)-2-pyrazoline hydrochloride). Synthesis of prostaglandin E2 was inhibited by the cyclooxygenase inhibitor indomethacin as well as by BW755C. Incubation of gastrointestinal tissues in the presence of glutathione decreased the amounts of leukotrienes D4 and E4, while release of leukotriene C4 was simultaneously increased. On the other hand, incubation of tritiated leukotriene C4 with incubation media from human gastric or colonic mucosa resulted in conversion of the substrate to [3H]leukotriene D4 and [3H]leukotriene E4. The results indicate the capacity of human gastrointestinal tissues to synthesize the 5-lipoxygenase-derived products of arachidonate metabolism, leukotriene B4 and sulfidopeptide leukotrienes, in addition to larger amounts of prostaglandin E2. Furthermore, considerable activities of the sulfidopeptide leukotriene-metabolizing enzymes gamma-glutamyl transpeptidase and dipeptidase were detected in human gastrointestinal tissues. These enzymes might play an important role in biological inactivation and/or change of biological profile of sulfidopeptide leukotrienes generated in the human gastrointestinal tract. PMID- 3019410 TI - Oxygen concentration-dependent metabolism of leukotriene B4 by hepatocyte monolayers. AB - Leukotriene B4 was found to be metabolized by rat hepatocyte monolayers at a rate that was linear with increasing substrate concentration from 74 to 740 nM leukotriene B4. The rates of metabolism were dependent on the O2 concentration and were 315, 213, 80, and 36 pmol leukotriene B4 per min per nmol cytochrome P 450 at 20% (212 microM), 4% (42.5 microM), 2% (21.2 microM), and 1% (10.6 microM) O2, respectively. The metabolic rate was not linear with respect to O2 concentration; however, half maximal rate occurred at 4% O2, and O2 concentration found in the pericentral region of normally oxygenated liver. These results suggest that in vivo conditions of hypoxia or ischemia that lead to blood O2 concentrations less than 4% may drastically decrease hepatic clearance of leukotriene B4. PMID- 3019411 TI - Isolation and characterization of multiple forms of Mg2+-dependent phosphatidate phosphohydrolase from rat adipose cytosol. AB - In the present studies, we have made several unique observations. First, we have shown that cytosolic phosphatidate phosphohydrolase from adipose tissue subjected to butyl-agarose chromatography was resolved into four different components. These components, designated as passthrough (PT), D150, D250 and E, were present in the proportions of 51:7:24:16, respectively, in the rat adipose cytosol. Comparison of the properties of these components revealed some similar properties, and also several differences. These components showed the same pH optimum, required Mg2+ for activity and were inhibited by N-ethylmaleimide, indicating a requirement of active sulfhydryl groups for activity. These components differed from one another with respect to hydrophobicity, sedimentation behavior, Stokes diameter, Km values, thermolability and susceptibility to proteinase treatment. Second, we have shown that each component of this system was associated with lipids which were found to be essential for the catalytic activity. Perturbation of this association by organic solvent or by adding excess amounts of exogenous lipids resulted in the loss of enzyme activity. Finally, we analyzed lipid composition of individual components. These studies suggest that the multi-component system of Mg2+-dependent phosphatidate phosphohydrolase may be a part of the cytomembrane network. PMID- 3019412 TI - Quantitative role of different embryonic tissues in mevalonate metabolism by sterol and nonsterol pathways. Relationship with enzyme activities of cholesterogenesis. AB - 3-Hydroxy-3-methylglutaryl-CoA reductase, mevalonate kinase, mevalonate-5 phosphate kinase and mevalonate-5-pyrophosphate decarboxylase activities have been determined in brain, liver, intestine and kidneys from 19-day-old chick embryo. Levels of brain reductase and decarboxylase were clearly higher than those found in the other tissues assayed. However, only small differences were observed in the activity of both kinases among the different tissues. Mevalonate metabolism by sterol and nonsterol pathways has been investigated in chick embryo at the same developmental stage. Mevalonate incorporation into total nonsaponifiable lipids was maximal in liver, followed by intestine, brain and kidneys. The shunt pathway of mevalonate not leading to sterols was negligible in both brain and liver, while a clear CO2 production was observed in intestine and kidneys. Sterols running in TLC as lanosterol and cholesterol were the major sterols formed from mevalonate by brain and kidney slices, while squalene and squalene oxide(s) were found to be mainly synthesized by liver slices. Minor differences in the percentage of different sterols were observed in chick embryo intestine. The importance of free and esterified cholesterol accumulation in the different tissues on the inhibition of cholesterogenic activity is discussed. PMID- 3019413 TI - The role of apolipoproteins of HDL in the selective uptake of cholesteryl linoleyl ether by cultured rat and bovine adrenal cells. AB - Rat adrenal cells in culture were used to study the uptake of cholesteryl linoleyl ether [( 3H]cholesteryl linoleyl ether), a nonhydrolyzable analog of cholesteryl ester. When [3H]cholesteryl linoleyl ether was added in the form of liposomes, its uptake was enhanced by adrenocorticotropin (ACTH) and by addition of milk lipoprotein lipase and interfered by heparin. When the adrenal cells were incubated with homologous [3H]cholesteryl linoleyl ether-HDL, ACTH treatment also resulted in an increase in [3H]cholesteryl linoleyl ether uptake. The uptake of [3H]cholesteryl linoleyl ether was in excess of the uptake and metabolism of 125I labeled HDL protein and was not sensitive to heparin. Unlabeled HDL or delipidated HDL reduced very markedly the uptake of [3H]cholesteryl linoleyl ether, while addition of phosphatidylcholine liposomes had little effect. Attempts were made to deplete and enrich the adrenal cells in cholesterol and, while depletion resulted in a decrease in [3H]cholesteryl linoleyl ether-HDL uptake, enrichment of cells with cholesterol had no effect. Among the individual apolipoproteins tested, apolipoprotein A-I and the C apolipoproteins reduced [3H]cholesteryl linoleyl ether uptake, while apolipoprotein E was not effective. Since the labeled ligand studied was a lipid, these effects could not be due to an exchange of apolipoproteins, but indicated competition for binding sites. Preferential uptake of human [3H]cholesteryl linoleyl ether-HDL3 by bovine adrenal cells was found when compared to the uptake and metabolism of 125I labeled HDL. The present results suggest that the preferential uptake of HDL cholesteryl ester (as studied with [3H]cholesteryl linoleyl ether) requires an interaction between the apolipoproteins of HDL and cell surface components. PMID- 3019414 TI - Gangliosides and neutral glycosphingolipids of normal tissue and oat cell carcinoma of human lung. AB - Concentration and composition of gangliosides and neutral glycosphingolipids of adult human lung, and lung small cell carcinoma were studied. The structures of the glycolipids were determined by quantitative component determination, enzymic degradation, permethylation and fast atom bombardment mass spectrometry. Adult human lung contained mainly gangliosides with lactosylceramide as the basic core, GM3, GD3 and GT3, and approx. equal proportions (10%) of gangliosides of the gangliotetraosyl- and lactotetraosylceramide series. 18 gangliosides with different carbohydrate moieties were identified: four of them were only found in the tumor tissue. The adult human lung contained 85 nmol (77-120) gangliosides and 140 nmol neutral glycosphingolipids per g wet weight. Globoside was the major neutral glycolipid and there were only minor amounts of glycolipids of the lactotetraose series. In small cell carcinoma tissue the concentration of neutral glycosphingolipids was approximately twice as high than in normal lung tissue, and there was a markedly larger concentration of both lactosylceramide and glycolipids of the lactotetraose series and fucose derivatives of these. The concentration of gangliosides varied between 202 and 415 nmol per g wet weight. Compared to normal lung tissue, the tumor tissue had a lower proportion of GD3, and a higher proportion of complex gangliosides, and they contained five tumor associated gangliosides: Fuc-GM1, Fuc-GD1b, 3'-LM1, Fuc-3'-LM1 and 6'-nLM1. PMID- 3019415 TI - Elongation of C20 polyunsaturated fatty acids by human skin fibroblasts. AB - Human skin fibroblasts actively elongate a portion of incorporated C20 polyunsaturated fatty acids to their respective C22 derivatives. As much as 40% of incorporated [14C]eicosapentaenoate is elongated within 8 h and 85% by 48 h. Elongation of [14C]arachidonate is initially less than half that of [14C]eicosapentaenoate and plateaus at 20-30% of incorporated 14C-labeled fatty acid. The elongation of 5,8,11-[14C]eicosatrienoate is intermediate between that of 20:4(n-6) and 20:5(n-3). Docosatetraenoate is not an effective inhibitor of the elongation of arachidonate, thus suggesting that the observed plateau is not due to product inhibition. When concentrations of exogenous fatty acids are increased, these cells elongate substantial quantities of C20 polyunsaturated fatty acids; elongation of eicosapentaenoate is consistently more extensive than that of arachidonate. Eicosapentaenoate is also an effective inhibitor of the elongation of [14C]arachidonate. Increases in exogenous arachidonate up to 10 microM result in an increase in elongation of [14C]arachidonate both in absolute quantities and as a percentage of that incorporated; the arachidonate thus acts as a positive modulator of its own elongation. Increased eicosapentaenoate also enhances the elongation of [14C]eicosapentaenoate, but only at lower concentrations (0.02-0.15 microM). The factors which regulate the elongation of C20 polyunsaturated fatty acids in human skin fibroblasts serve to permit extensive elongation of eicosapentaenoate while retaining incorporated arachidonate primarily in its C20 form. PMID- 3019416 TI - Effect of dimethylaminoethylphosphonate on phospholipid metabolism in Tetrahymena. AB - Dimethylaminoethylphosphonate (DMAEP) was incorporated into the phospholipids of the ciliate protozoan Tetrahymena thermophila at the expense of both phosphatidylethanolamine and phosphatidylcholine, but it had no effect on the levels of the 2-aminoethylphosphonolipid. The newly formed DMAEP-lipid accounted for almost 50% of the phospholipids of the organism. The DMAEP was incorporated into the phospholipids using both the ethanolaminephosphotransferase and cholinephosphotransferase pathways. The DMAEP-lipid was not methylated to the trimethyl derivative, confirming the lack of methylation of phosphonolipids by Tetrahymena. PMID- 3019417 TI - Deacylation of penicilloylated penicillin-binding proteins from Micrococcus luteus: kinetic study of thiol-promoted release of the bound penicilloyl moiety. AB - The stability of covalent complexes obtained by labelling penicillin-binding proteins 1-6 from Micrococcus luteus with a radioactive derivative of ampicillin has been examined in the presence of thiols. When the incubation medium contained only 1 mM 2-mercaptoethanol, the complexes were almost unaffected for at least 1 h. If 20 mM dithiothreitol was also included in the medium, the amount of bound radioactivity decreased throughout the incubation period. The breakdown of the complexes derived from penicillin-binding proteins 4 and 5 proceeded very slowly, following an apparent first-order kinetics, whereas the kinetics of deacylation of other penicillin-binding proteins exhibited a biphasic pattern with an initial fast phase followed by a slow one, each of which could be approximated by an apparent first-order reaction. This behavior is explained adequately by a two step mechanism: the penicilloylated penicillin-binding proteins are first deacylated in a reversible exchange with the added thiol, giving rise to an intermediate thioester; once formed, this intermediate is hydrolysed irreversibly. A simple graphical method has been devised to deduce rate constants from the time course of the reaction. Theoretical curves have been constructed, and they fit experimental data satisfactorily. The results point out that added thiols may effectively interfere with the quantitation of penicillin-binding proteins; therefore, the stability of penicilloylated penicillin-binding proteins should be checked carefully when these protecting agents are included in membrane extracts or incubation media. PMID- 3019418 TI - Hormonal control of collagenase inhibitor production in rabbit uterine cervical fibroblast-like cells. AB - Rabbit uterine cervical fibroblast-like cells maintained in fetal calf serum-free medium were found to biosynthesize and secrete a collagenase inhibitor into the culture medium. All the properties of this inhibitor were similar to those that have been described so far for the tissue inhibitor of metalloproteinases. Both progesterone and 17 beta-estradiol significantly increased the level of collagenase inhibitor without the proliferation of cells. These data suggest that both progesterone and estradiol regulate collagenolysis in the uterus bifunctionally by acceleration of the inhibitor production in addition to their known inhibitory actions towards collagenase biosynthesis. PMID- 3019419 TI - Catabolic properties of aglycofibrinogen synthesized by tunicamycin-treated human hepatoma (HepG2) cells and rabbit hepatocytes. AB - Human hepatoma cell (HepG2) or rabbit hepatocyte monolayers were incubated with [35S]methionine in presence or absence of tunicamycin, a potent inhibitor of asparagine-linked glycosylation. The 35S-labeled nonglycosylated and control fibrinogens purified from the media were used to evaluate the influence of the oligosaccharide on the catabolic properties of this glycoprotein. Plasmin, pronase, cathepsin D or cathepsin B each degraded the nonglycosylated and control fibrinogens similarly, as evidenced by the release of trichloroacetic acid soluble radioactivity and by SDS-polyacrylamide gel electrophoresis and autoradiography of plasmic digests. Nonglycosylated and control fibrin clots also showed no differences in susceptibility to plasmic digestion. The two forms of fibrinogen demonstrated the same plasma half-life in rabbits. These data indicate that the oligosaccharide does not influence the proteolytic stability or the in vivo plasma survival of fibrinogen, and suggest that other biochemical determinants may influence the catabolic properties of this molecule. PMID- 3019420 TI - Muscle adenylate kinase in Duchenne muscular dystrophy. AB - On the basis of electrophoretic and enzyme inhibition studies it was postulated that an aberrant adenylate kinase occurs in muscle and serum of patients with Duchenne muscular dystrophy (Schirmer, R.H. and Thuma, E. (1972) Biochim. Biophys. Acta 268, 92-97; Hamada, M. et al. (1981) Biochim. Biophys. Acta 660, 227-237; Hamada et al. (1985) J. Biol. Chem. 260, 11595-11602). On the basis of the following results we conclude that Duchenne muscular dystrophy patients do not possess an unusual adenylate kinase isoenzyme. In muscle biopsies from five Duchenne patients, the electrophoretic mobility of adenylate kinase and the inhibition of the enzyme by P1, P5-di(adenosine-5')pentaphosphate (Ap5A) was normal. Because of the high SH-group content of the extracts from Duchenne muscle, high concentrations of Ellman's reagent were needed to inhibit adenylate kinase activity in these samples. In Duchenne plasma the adenylate kinase activity was elevated. Like in muscle specimens, the DTNB inhibition curves were shifted to higher reagent concentrations; this was due to a high SH-group content of Duchenne plasma when compared with normal plasma. With respect to inhibition by Ap5A and electrophoretic mobility, Duchenne adenylate kinase in Duchenne plasma behaved like normal muscle adenylate kinase in normal plasma. It was noted that normal muscle adenylate kinase changes its electrophoretic behaviour when mixed with normal or Duchenne plasma. This finding had been considered previously as evidence for the presence of an aberrant adenylate kinase in Duchenne plasma. PMID- 3019421 TI - Localization of leukotriene D4-metabolizing metalloenzyme on the cell surface of human neutrophils. AB - The localization of leukotriene D4-metabolizing enzyme on the cell surface was examined using human neutrophils. Intact neutrophils rapidly converted leukotriene D4 to leukotriene E4. However, when neutrophils were modified chemically by diazotized sulfanilic acid, a poorly permeant reagent which inactivates cell surface enzymes selectively, the leukotriene D4-metabolizing activity of neutrophils decreased significantly without any inhibition of the cell viability or marker enzymes of cytosol, granules, microsome and mitochondria. The leukotriene D4-metabolizing enzyme activity of the membrane fraction was inhibited by modification to the same extent as that of Mg2+ dependent ATPase, a cell-surface marker enzyme. Among various enzyme inhibitors examined, a metal chelator, o-phenanthroline, strongly suppressed the leukotriene D4-metabolizing activity of intact neutrophils and the o-phenanthroline inactivated enzyme activity was fully reactivated by Co2+, Mn2+ and Zn2+. These results would suggest that some metalloenzyme located on the cell surface is involved in the conversion of leukotriene D4 to leukotriene E4 by neutrophils. PMID- 3019422 TI - Identification of phosphorylethanolamine in 31P-NMR spectra of human peripheral blood lymphocytes. AB - The 31P-NMR spectrum of intact human peripheral blood lymphocytes contains a large unidentified peak in the phosphomonoester region. The pH dependency of the 31P-NMR chemical shift of this peak in perchloric acid extracts of peripheral blood lymphocytes was recorded. It was compared to the pH dependency of the chemical shift of phosphorylethanolamine, phosphorylcholine, and ribose 5 phosphate in model solutions. An excellent agreement was found between the behavior of phosphorylethanolamine and the unidentified peak. To further substantiate this assignment phosphorylethanolamine was added to extracts and the pH titrations were repeated. The added phosphorylethanolamine gave exactly the same chemical shift as the unidentified peak and no difference was observed with pH titrations. The concentration of phosphorylethanolamine in human peripheral blood lymphocytes was estimated by 31P NMR to be 2.4 mumol/10(9) cells (range 0.9 4.3/10(9) cells, n = 4). PMID- 3019423 TI - Alkylacetylglycerophosphocholine stimulates Na+-Ca2+ exchange, protein phosphorylation and polyphosphoinositide turnover in rat ileal plasmalemmal vesicles. AB - The novel ether phospholipid, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), isometrically contracted helically cut rat ileal smooth muscle strips in a dose- and time-dependent manner. Utilizing an enriched plasma membrane vesicular preparation from rat ileal longitudinal smooth muscle, AGEPC specifically stimulated Na+-Ca2+ exchange in a dose- and time-dependent manner. Concomitant with the AGEPC stimulation of Na+-dependent Ca2+ influx in plasma membrane vesicles is an enhanced turnover of the polyphosphoinositides, an elevated concentration of phosphatidic acid and also an enhanced phosphorylation of an Mr 40,000 plasmalemmal protein. The mechanisms by which AGEPC may regulate ileal plasmalemmal Ca2+ flux and contractility are considered. PMID- 3019424 TI - The assay of the activity of protein kinase C with the synthetic oligopeptide substrate designed for histone kinase II. AB - The activity of histone kinase II was determined on the basis of its ability to phosphorylate the nonapeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide designed previously as a specific substrate for this enzyme. Histone kinase II was purified from calf thymus extract by DEAE-cellulose chromatography followed by hydroxylapatite chromatography and high-performance liquid chromatography on a Protein Analysis column (I-125). The Mr value of histone kinase II estimated by the latter method was 50,000-55,000, but several observations indicated that histone kinase II was a product of a proteolytic process. Since the substrate specificity determinants for histone kinase II known from our previous investigations are very similar to those for protein kinase C, it was presumable that histone kinase II was the proteolytic fragment of protein kinase C. Therefore, the nonapeptide was tested as a substrate for protein kinase C prepared from rabbit brain extract by DEAE-cellulose chromatography. The activity of histone kinase II was also detected in brain extract. Histone kinase II was eluted from the DEAE-cellulose in the known position of the proteolytic fragment of protein kinase C. The nonapeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide proved to be a better substrate than H1 histone for the detection of the activity of protein kinase C because it was not phosphorylated by the cAMP-dependent protein kinase and the Vmax of protein kinase C was about one order of magnitude higher with the peptide than with H1 histone. The apparent Km of protein kinase C for the peptide was identical with that of histone kinase II (0.2 mM). PMID- 3019425 TI - Effects of age and androgens upon functional vasoactive intestinal peptide receptors in rat prostatic epithelial cells. AB - The specific binding of vasoactive intestinal peptide (VIP) and the stimulatory effect of VIP upon cyclic AMP accumulation in isolated epithelial cells of rat ventral prostate were age dependent. The number of VIP receptors decreased but the efficiency of VIP on cyclic AMP accumulation increased in prostatic epithelium when considering the periods 35-65 days and 3-6 months. Since these features could be related to the known age-related decrease of androgen and androgen-receptor levels, we studied the effect of testosterone and its 5 alpha reduced metabolite dihydrotestosterone upon both steps of VIP action. The two steroid hormones exerted a non-competitive inhibition on VIP-induced cyclic AMP accumulation but did not modify VIP binding to its specific receptors. This modulatory effect of androgens might involve their interaction with specific sites on the cell membrane leading to modifications of membrane activities including adenylate cyclase, as has been suggested by an increasing number of recent reports. PMID- 3019426 TI - [The role of structural factors in radiolytic damage of human hemoglobin]. AB - A significant difference was discovered in low temperature (77 K) gamma radiolytic behavior of 20% aqueous solutions of human oxyhemoglobin and partly denaturated methemoglobin. In the latter case twice as high yield of the sum of free radicals and OH radicals was observed, as well as presence in the ESR spectrum of a narrow singlet line at g 2.00 (absent for irradiated solutions of oxyhemoglobin) ascribed to the stabilized electron. PMID- 3019427 TI - [Changes in NMR-relaxation of plasma protons in animals during experimental carcinogenesis]. AB - Studies were carried out of changes of NMR-relaxation of water protons bound to blood plasma proteins of linear animals in the course of induced cancerogenesis and total reaction of the organism to damage. It has been shown that the ratio between the times of spin-lattice and spin-spin relaxation can serve as a criterion of the development of induced cancerogenesis at histologically determined stage of its development. PMID- 3019428 TI - [Redox reactions of flavins and coenzyme Q-10 in the heart during experimental ischemia and reperfusion]. AB - Free radical EPR-signal intensity of the rat heart tissue decreases during ischemia and increases at the initial steps of reperfusion, relaxation characteristics of the signal being unchanged. These data imply that free radical content of the heart decreases during ischemia and increases upon reperfusion. PMID- 3019429 TI - [Determining the motility of glycoprotein oligosaccharides from lymphocyte membranes using the spin label method]. AB - Splenocyte glycoproteins solubilized by papain were purified on lectyl-lectin Sepharose 4B. Glycoproteins eluted from lectin were spin-labeled at carbohydrates. Quantitative evaluation of ESR spectra of spin-labeled glycoproteins pointed to strong restriction of reorientation of the spin label bound to oligosaccharides. Calculated correlation time of relaxing volume tagged with the spin label was equal to the molecular weight about 6000-7000 dalton. This value suggests the existence of flexibility of the glycoproteins studied. PMID- 3019431 TI - Molecular mechanisms of photosensitization. AB - The first part of this article is devoted to basic concepts of photosensitization and to the primary photophysical and photochemistry processes involved in the reaction. The electronic configuration of molecular oxygen in its ground or activated states, which intervene in numerous photosensitized reactions, is reviewed. Finally, the main photosensitized reactions are reviewed and classified into three different groups: reactions due to radicals (type I), reactions due to singlet oxygen (type II) and those which do not involve oxygen (type III). PMID- 3019430 TI - [Effect of the phase state of lipids on their interaction with cobra cytotoxin]. AB - Interaction between cytotoxin of the Central Asia cobra venom and dimiristoylphosphatidylcholine bilayer depending on its phase state was studied by ESR with spin label. A conclusion can be drawn that the efficiency of cytotoxin effect on the membranes depends on their phase state. Cytotoxin molecules are incorporated into myophile region of the bilayer, only if the latter is in the liquid crystal state. The interaction between cytotoxins and lipids of the bilayer in a gel state is in the main conditioned by electrostatic forces. PMID- 3019432 TI - [Activated radical species of oxygen]. AB - Oxygen free radicals are often formed during photosensitization processes. Kinetic and thermodynamical characteristics are briefly described for OH and O2-. radicals. PMID- 3019434 TI - Drug-induced photosensitivity: phototoxic and photoallergic reactions--a few molecular aspects. AB - Drug-induced photosensitivity involves mainly phototoxic and photoallergic reactions. The main features of phototoxic and photoallergic reactions are presented and some molecular aspects involved in the mechanisms leading to an adverse skin response are illustrated with examples. PMID- 3019433 TI - Biomolecular photoalterations mediated by phenothiazine derivatives. AB - This survey focuses on recent developments in the field of the ultraviolet photochemistry and photobiology of phenothiazine derivatives. One of the major alterations introduced by this kind of photosensitized reaction is a covalent addition of the photosensitizer or one of its photoproducts onto the macromolecular target. This reaction has been observed with soluble and membrane proteins, lipids and DNA. In the latter case, the addition occurs at the level of guanine residues and leads to inhibition of DNA replication. PMID- 3019435 TI - Photosensitized UVA light induction of the SOS response in Escherichia coli. AB - Several of the factors controlling the extent of the ultraviolet light (and particularly of UVA 320 nm less than lambda less than 380 nm) induced SOS response in E. coli have been studied using a sfiA::lacZ fusion. The decreased 254 nm induced sfiA expression level triggered by a UVA-induced growth delay (Caldeira de Araujo A. & Favre A. (1986) Embo J., 5, 175-179), is closely mimicked by a transient chloramphenicol protein synthesis inhibition. In a nuvA mutant strain (lacking the growth delay effect), UVA light triggers a 30-40% lower SOS response at temperatures higher than 20 degrees C when illumination is performed under anaerobic conditions: endogenous oxygen-mediated photosensitized reactions appear to contribute to the SOS response. In contrast to the temperature independence of the sfiA induction levels obtained after 254 nm irradiation, the UVA induced response is 30-60% lower when the temperature (T) increases from a value lower than 10 degrees C to a value higher than 20 degrees C. This indicates that detoxifying enzymes play a role at T greater than 20 degrees C. Also the in vitro photooxydation of NADH to give NAD+ is described and its possible role in endogenous photosensibilizations discussed. To explain the contrasted mutagenic efficiencies of UVA light treatment when applied to cells in buffer at high fluences, and to growing cells at low fluence rates, we propose that intrinsically the UVA-induced DNA damages are able to trigger the SOS response (cyclobutyl pyrimidine dimers and some O2-dependent lesions) but also constitute premutagenic sites (some lesions leading to alkali-labile DNA breaks).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019436 TI - Hematoporphyrin and hematoporphyrin-derivative photosensitization of mitochondria. AB - Laser micro-irradiation experiments show that mitochondria are profoundly affected when cells are irradiated with Hp as the photosensitizer. Functional as well as enzymatic studies on isolated mitochondria show that coupling between respiration and oxidative phosphorylation, Ca2+ transport and respiration are successively lost under irradiation in the presence of either Hp or HpD. ATP driven Ca2+ uptake, which is not impaired under anoxic conditions with HpD alone, is impaired in the absence of oxygen by the synergistic action of HpD and nitroimidazoles. PMID- 3019437 TI - Aspects of psoralen phototumorigenesis with emphasis on the possible role of tumour promotion. AB - 8-Methoxypsoralen in combination with UVA radiation (PUVA) is carcinogenic in mice and probably so in man. PUVA is genotoxic and so has tumour initiation potential. Some evidence suggests that PUVA has other biological effects which may be equated with tumour promotion. Thus, the use of a two-stage model, similar to that of chemical carcinogenesis, may be a useful experimental approach for the further understanding of PUVA carcinogenesis. PMID- 3019438 TI - 5'-Nucleotidase activity in adult and fetal rabbit lungs. AB - 5'-Nucleotidase was assayed in the membrane fractions isolated from rabbit lung homogenate. The enzyme activity was measured from the rate of hydrolysis of [U 14C]cytidine 5'-monophosphate (CMP). The optimal pH of the rate of hydrolysis is around 8.0 and the enzyme activity is stimulated by Mg2+. The apparent Km and Vmax of the enzyme in the microsomal fraction for CMP were 3.33 mM and 1.43 mumol/min/mg protein, respectively. During lung development the enzyme activity increased moderately at late gestational ages and reached its maximum level in adults (p less than 0.001). The developing profile of 5'-nucleotidase activity is similar to the developing pattern of the CMP content in rabbit lungs. PMID- 3019439 TI - The effects of psychotropic drugs on synthesis of DNA and the infectivity of herpes simplex virus. PMID- 3019440 TI - Aberrant parenting and delayed offspring development in rats exposed to lithium. AB - Natural lithium (Li) salts, including those used routinely in manic depressive illness, consist of two stable nonradioactive isotopes: lithium-7 (Li-7) (92.6%) and lithium-6 (Li-6) (7.4%). Female rats (3 months old) were treated with either Li-7 chloride or Li-6 chloride or were untreated prior to and during gestation and lactation. Birth weights were lower for Li-treated animals than for normal pups. Maternal behavior of all Li-treated mothers was altered. Li-7 mothers ignored their pups and nursed them infrequently. Li-6 mothers groomed and nursed their pups more often than normal mothers. All pups showed delays in development, especially in the maturation of depth perception. Although Li-6-treated dams were over-protective mothers, their offspring showed longer developmental delays than those of Li-7-treated offspring. PMID- 3019441 TI - Arginine vasopressin stimulation and imipramine. PMID- 3019442 TI - Experimental application of polyvinyl alcohol-silica for small artificial vessels. AB - Polyvinyl alcohol-silica (PVA-SiO2) composite and heparinized PVA-SiO2 were examined in vitro and in vivo as materials to coat artificial vessels to be used for the replacement of small arteries. PVA-SiO2 was observed to prolong coagulation time and on heparinized PVA-SiO2 surfaces no blood coagulation was noticed after a period of two days using the Lee-White and plasma recalcification methods. After placing non-coated and coated surfaces in contact with blood components in vitro and in vivo, the degree of blood component adhesion was greater in non-coated woven Dacron than in PVA-SiO2 coated Dacron. The degree of adhesion was even less in heparinized PVA-SiO2 coated Dacron. Furthermore, artificial vessels made of these 3 types of materials were used to replace parts of the canine abdominal aorta and were removed one and a half years later. Patency rates were as follows: non-coated 2/7, PVA-SiO2-coated 4/7, heparinized PVA-SiO2-coated 8/12. The inner surfaces of these prostheses were observed with light microscopy and scanning electron microscopy. Intima formation was thinner on the PVA-SiO2 composite surfaces than on the control surfaces. Heparin acted as a local anticoagulant and PVA-SiO2 limited intima formation. This report showed that PVA-SiO2 composite coated surfaces can be effective for small artery replacement due to good tissue affinity and anticoagulability. PMID- 3019443 TI - Direct measurement of the initial proton extrusion to oxygen uptake ratio accompanying succinate oxidation by rat liver mitochondria. AB - The problem of obtaining very early ratios for the H+/O stoichiometry accompanying succinate oxidation by rat liver mitochondria was attacked using new techniques for direct measurement rather than extrapolations based on data obtained after mixing and the recovery of the electrode from initial injection of O2. Respiration was quickly initiated in a thoroughly mixed O2-containing suspension of mitochondria under a CO atmosphere by photolysis of the CO cytochrome c oxidase complex-. Fast responding O2 and pH electrodes were used to collect data every 10 ms. The response time for each electrode was experimentally measured in each experiment and suitable corrections for electrode relaxations were made. With uncorrected data obtained after 0.8 s, the extrapolation back to zero time on the basis of single-exponential curve fitting confirmed values close to 8.0 as previously reported (Costa et al., 1984). The data directly obtained, however, indicate an initial burst in H+/O ratio that peaked to values of approximately 20 to 30 prior to 50 ms and which was no longer evident after 0.3 s. Newer information and considerations that place all extrapolation methods in question are discussed. PMID- 3019444 TI - Large transient nonproton ion movements in purple membrane suspensions are abolished by solubilization in Triton X-100. AB - Light-induced release/uptake of both protons and other ions cause transient changes in conductivity in suspensions of purple membrane (PM) fragments (Marinetti, Tim, and David Mauzerall, 1983, Proc. Natl. Acad. Sci. USA, 80:178 180). We find that the release/uptake of nonproton ions with quantum yield greater than 1 is observed at most pHs and ionic strengths. Only at both low pH and low ionic strength is the conductivity transient mostly due to protons. Our hypothesis is that during the photocycle, changes occur in the PM's dense surface charge distribution that result in changes in the number of counterions bound or condensed at the membrane surface. To test this, the PM structure was perturbed with the nonionic detergent Triton X-100. Immediately after addition, Triton does not abolish the nonproton ion movements; in fact at low detergent concentrations (0.02% vol/vol) the signal amplitudes increased considerably. However, when PM is completely solubilized into monomers in Triton, the conductivity transients are due to protons alone, though at lower quantum yield compared with native PM. These results suggest that changes in the surface charge distribution in native PM's photocycle could contribute to proton transfer between the aqueous phase and bR itself. PMID- 3019445 TI - Lateral diffusion of lipid probes in the surface membrane of human platelets. An electron-electron double resonance (ELDOR) study. AB - Electron-electron double resonance (ELDOR) techniques employing [14N], [15N] 16 Doxylstearate spin-label pairs have been used to measure the lateral diffusion constant, D, of lipids in the surface membrane of intact human blood platelets. For freshly prepared platelets, D is 1.0 X 10(-8) cm2/s at 37 degrees C and for platelets stored for 3 d at room temperature under accepted routine blood bank conditions, D is 2.6 X 10(-8) cm2/s at 37 degrees C. This is the first time that D in the surface membrane of platelets is reported. The marked increase in D for stored platelets may be attributed at least partly to loss of cholesterol during storage, suggesting a correlation between lipid lateral diffusion and cholesterol levels in cell membranes. PMID- 3019447 TI - [Effect of buspirone and diazepam on cGMP level in the rat cerebellum]. AB - Diazepam at a dose of 5 and 10 mg/kg significantly decreases (by 50 and 60%, respectively) cGMP content 30 min following intraperitoneal injection to rats. Buspirone, at a dose of 2.5-25 mg/kg produced a nonsignificant (up to 18%) and at a dose of 50 mg/kg a statistically significant (p less than 0.05) 30% decrease in cerebellum cGMP content. Taking into account the identical anxiolytic effects of diazepam and buspirone, it is suggested that pharmacological effects of buspirone are not linked to the activation of GABA-ergic system. PMID- 3019446 TI - [Electroencephalographic analysis of inter-central relations in hypothalamo reticulo-limbic brain structures during treatment with corticosteroids and neuropeptides]. AB - Intercentral relations between hypothalamus, limbic system and reticular formation were studied in rabbits and rats under systemic and central action of DSIP, ACTH, corticosteroids and stress (aggressive-defensive behaviour). The results obtained demonstrate changes in the adrenal cortex resulting from stress inducing adrenocortical hormone content. The increase was achieved by the rise in ACTH level resulting in corticosteroid level elevation (endogenous elevation aggressive behaviour) and by corticosteroid injections (exogenous elevation). Correlation analysis of structural interrelations after ACTH and corticosteroid injections demonstrated an increased correlation between hypothalamo-reticular limbic structures. DSIP was shown to have an opposite effect. Correlation analysis revealed the potentials for the formation of new functional interrelations between hypothalamo-reticular-limbic structure in the motivation of aggression (stress) and the levels of corticosterone and DSIP. DSIP action depends on the initial corticosteroid blood level and is more marked in stress inducing concentrations. PMID- 3019448 TI - [Effect of brain acid extract products in inbred mice on 3H-diazepam reception]. AB - The influence of brain acid extract products, isolated by high-performance liquid chromatography on H3-diazepam binding was investigated in synaptosomal membranes of C57BL/6 and BALB/c mice. Fractions with stimulatory and inhibitory activity were isolated. Quantitative and qualitative differences in the effects and structure of ACTH-immunoreactive peptide fractions under study were established. PMID- 3019449 TI - [Characteristics of opiate receptors in the brain and spinal cord of morphine tolerant mice]. AB - The in vitro studies of 3H-morphine binding to synaptosomal brain and spinal cord membranes and the in vivo detection of pA2 values were carried out in mice. Both morphine-tolerant and intact animals were used. Morphine-tolerant mice showed no changes in specific binding and naloxone pA2 values. Desensitization of neural tissue is most likely to result from variation in translation from opiate receptors to subreceptor effector systems. PMID- 3019450 TI - [The need for the correct choice of free ligand concentration in determining parameters of benzodiazepine binding]. AB - In the studies of 3H-diazepam binding to the rat brain membranes it has been shown that insufficiently high concentrations of free ligand might lead to incorrect determination of Bmax. Thus, free ligand concentration in the range of 0.5 to 16 nM (the most often used ones) and low receptor-protein concentrations (0.08 to 0.12 mg/sample) were far from being saturating and therefore could not be applied for the analysis in Scatchard coordinates. In this case Bmax value would be considerably below the true Bmax value. It has been concluded that for the determination of Bmax of 3H-benzodiazepine binding the range of concentrations used should be at least 0.25 to 64 nM. PMID- 3019451 TI - [Early manifestations and mechanism of the neurotoxic action of organophosphorus pesticides]. AB - The inhibition of neurotoxic esterase activity in chicken brain has been studied in vitro and in vivo. Aphos exposure, causing chicken paralysis, has demonstrated that the initial stage of delayed neurotoxicity was significant esterase activity inhibition (by 60-80%) within 3-24 hours after the pesticide administration. The inhibition of cholinesterase activity occurred both in the blood and sciatic nerve. The delayed conduction through peripheral nerves caused by demyelination has been revealed in the latent period (before the clinical signs of intoxication). PMID- 3019452 TI - Ca2+ and phospholipid-dependent protein kinase (protein kinase C) activity is not necessarily required for secretion by human neutrophils. AB - Ca2+-dependent and phospholipid-dependent protein kinase (PKC) is a receptor for and is activated by phorbol esters. This enzyme is reportedly involved in the mechanism of superoxide anion (O2-) production and the release of intracellular granule contents from human neutrophils. As previously reported by others, we found that greater than 75% of the total cellular PKC activity existed in a soluble form in untreated neutrophils and that this activity was enhanced in a dose-dependent manner by phorbol 12-myristate 13-acetate (PMA) and by phorbol 12,13-dibutyrate (PDBu). Furthermore, mezerein, an analogue of PMA that is thought to be a competitive inhibitor, did not activate PKC, and on the contrary, inhibited PMA-stimulated activity in a dose-dependent manner. Pretreatment of intact neutrophils with PMA or PDBu caused the "translocation" of PKC activity to the insoluble cell fraction; PKC translocation was not detected after mezerein stimulation at any of the tested concentrations. Neither did mezerein cause an increase in intracellular Ca2+, as monitored by Quin 2 fluorescence. Both phorbol esters and mezerein stimulated intact neutrophils to generate O2- and release lysosomal enzymes into the extracellular medium. Finally sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated key differences in the patterns of endogenous phosphoproteins of neutrophils stimulated with phorbol as compared with mezerein. We therefore suggest that PKC activation may not be the only pathway required to elicit neutrophil responses. PMID- 3019453 TI - Purification and functional evaluation of mature neutrophils from human bone marrow. AB - Human myeloid maturation proceeds within the bone marrow and results in a mature neutrophil that is released into the peripheral circulation. Previous reports have indicated that neutrophils from bone marrow demonstrate decreased adherence, impaired phagocytosis, and decreased nitroblue tetrazolium dye reduction when stimulated. Due to lack of a suitable method for isolating purified bone marrow neutrophils, these studies have been performed by microscopic techniques. We now report a method for isolating 1 X 10(8) neutrophils [bands plus polymorphonuclear leukocytes (PMNs)] from 10 mL of bone marrow aspirate sample. By means of a discontinuous Percoll-gradient centrifugation through densities of 1.085, 1.095, and 1.10 g/mL a leukocyte-rich suspension of bone marrow can be separated into three leukocyte layers. By combining the lower two leukocyte layers (M2/3), a population of neutrophils consisting of 26% bands and 63% PMNs is seen. When compared with peripheral blood PMNs, these bone marrow neutrophils had a lower alkaline phosphatase activity, decreased ingestion of Oil Red O-coated particles, impaired superoxide release on stimulation with the chemotactic peptide Fmet-leu phe (FMLP) or the tumor promotor phorbol myristate acetate (PMA), and less activity of the NADPH-dependent oxidase. These results indicate that morphologically mature neutrophilic cells within the bone marrow exist in a still functionally immature state. PMID- 3019454 TI - Variant chronic granulomatous disease: modulation of the neutrophil defect by severe infection. AB - The present studies document the cellular and biochemical processes involved in granulocyte O2- production in three patients from two kindreds with variant chronic granulomatous disease (CGD). Rates of O2- production were 9% to 30% of normal, depending on the individual tested and the stimulus; the two brothers from one family responded to each stimulus with rates very similar to each other. Kinetic analysis of NADPH-dependent O2- production in subcellular fractions revealed all three to have NADPH oxidases with both diminished substrate affinity for NADPH (high Kmapp) and decreased maximal velocities of O2- production. Their granulocytes had normal lag times for activation of the respiratory burst but abnormal rates of stimulus-induced membrane depolarization. Cytochrome b was not found in granulocytes or subcellular fractions despite the use of a spectrophotometric assay sensitive enough to detect the cytochrome if its content were proportional to the residual rate of O2- generation. A striking finding in one patient from each kindred was a threefold to tenfold decrease in the rate of O2- production accompanying serious infection. The residual O2(-)-generating activity of CGD variants helps to explain their relative freedom from the recurrent infections of the classic disease. However, the marked decrease described in the present study indicates the potential for a vicious cycle in which an infection, once established, leads to increasing impairment of host defense. PMID- 3019455 TI - Malignant fibrous histiocytoma of the bladder. PMID- 3019456 TI - Comparison of harvesting methods for islet transplantation. AB - One problem preventing islet transplantation becoming a clinical reality in the treatment of insulin-dependent diabetes is the unsatisfactory, low yield processes for harvesting islets. This study was designed to find the most effective harvesting process and to examine the feasibility of obtaining more than one graft from a single pancreas. After confirming that normoglycaemia at one month could be achieved in dogs using 50 per cent of an autograft prepared by a previously established method, novel techniques were assessed using 33 per cent of the tissue obtained. An isolation process involving perfusion of the duct by enzymes allowed normoglycaemia using only one-third of the graft preparation and these animals exhibited significantly improved glucose handling and insulin output when compared with recipients of grafts prepared by the established method. A significant improvement in harvesting technique has, therefore, been demonstrated and this will exploit the potential of multiple donor grafts in islet transplantation and the method may be worthy of trial in the human gland. PMID- 3019457 TI - Iron and the outcome of infection. PMID- 3019458 TI - AIDS virus antibody in polytransfused dialysis patients vaccinated against hepatitis B. PMID- 3019459 TI - Industrial exposure to hydrogen cyanide: implications for treatment. PMID- 3019460 TI - Childhood gastroenteritis: a population study. AB - A prospective study of gastroenteritis based on a population was carried out for 12 months on over 7000 children in general practice. The incidence of gastroenteritis was highest in the first year (127.7 children affected per 1000) and second year (90.8) of life, and gastroenteritis was rare after six years of age. Children from urban areas had gastroenteritis more commonly than children from semirural areas. A potential pathogen was isolated from half of the specimens: 78% were viruses, and rotavirus was identified most often. PMID- 3019461 TI - AIDS, cotton wool spots, and cytomegalovirus retinitis. PMID- 3019462 TI - Cold-restraint stress reduces [3H]etorphine binding to rat brain membranes: influence of acute and chronic morphine and naloxone. AB - [3H]Etorphine binding was characterized in rat brain homogenates depleted of endogenous opioids from animals acutely and chronically treated with morphine or naloxone and either unstressed or subjected to a 3-h restraint period in the cold. There was significant reduction in the number of high-affinity opiate binding sites in brain tissue from stressed as compared to unstressed animals. Despite the fact that the opiate drug regimens used produce marked behavioral and physiological effects, stress-induced opiate receptor depletion was not influenced by the drugs or by withdrawal. The various drug treatments also failed to produce significant changes in opiate receptor site densities or affinities in either stressed or unstressed animals. We propose that persistent activation of opiate receptors by endogenous opioids released during restraint stress leads to receptor 'down-regulation'. PMID- 3019463 TI - Quantitative autoradiographic localization of neuropeptide Y receptors in the rat lower brainstem. AB - Using quantitative autoradiography, we have characterized the binding of 125I Bolton-Hunter coupled neuropeptide Y ([125I]NPY) and observed the localization of 125I-NPY receptors in the rat lower brainstem. [125I]NPY bound to the receptors in a specific, saturable and reversible manner with high affinity. The binding was blocked only by unlabeled NPY but not by NPY-related peptides i.e. peptide YY, pancreatic polypeptide (avian and human), nor by neurotensin. [125I]NPY receptors were revealed to be coupled to the guanosine triphosphate (GTP)-binding protein. Regional distribution study showed that [125I]NPY has a distinctive pattern of distribution in the rat lower brainstem, being particularly concentrated in the area postrema and the medial subnucleus of the nucleus tractus solitarii. These results suggest that such NPY receptors have an important role in cardiovascular regulation. PMID- 3019464 TI - Serotonin-1C sites in the choroid plexus are not linked in a stimulatory or inhibitory way to adenylate cyclase. AB - The association of the serotonin recognition sites in the pig choroid plexus (5 HT-1C sites) with an adenylate cyclase was examined. The interaction of serotonin and mianserin with [3H]mesulergine binding was not affected by the stable GTP analogue GppNHp. The binding of [3H]serotonin to choroid plexus membranes was also unaffected by GppNHp while a dose-dependent decrease was observed in pig cortical and hippocampal membranes. The porcine choroid plexus contained a forskolin- and histamine-sensitive adenylate cyclase. Serotonin, however, was ineffective in this preparation. While forskolin-stimulated adenylate cyclase in the rat hippocampus was inhibited by serotonin forskolin-stimulated adenylate cyclase in the choroid plexus was insensitive to serotonin. These results indicate that the serotonin recognition sites in the choroid plexus are not linked in a stimulatory or inhibitory way to an adenylate cyclase, in contrast with other 5-HT-1 receptor subtypes. PMID- 3019465 TI - Histochemical demonstration of vibrissae-representing patchy patterns of cytochrome oxidase activity within the trigeminal sensory nuclei in the cat. AB - Cytochrome oxidase histochemistry revealed patchy patterns of the enzyme activity in transverse sections through the caudal part of the ventral subnucleus of the principal sensory trigeminal nucleus, interpolar spinal trigeminal nucleus, and layer IV of the caudal spinal trigeminal nucleus in the cat. By the transganglionic transport method of horseradish peroxidase, the patterns were indicated to replicate the spatial array of the facial vibrissae. PMID- 3019466 TI - Dopaminergic involvement in the estrogen-induced suppression of frequency of pulsatile luteinizing hormone secretion in the ovariectomized rat. AB - This experiment examined whether various catecholaminergic synthesis inhibitors and receptor blockers affect the inhibitory effect on the frequency of pulsatile luteinizing hormone (LH) secretion induced by local application of estradiol benzoate (EB) into the preoptic suprachiasmatic area (POSC) of ovariectomized rats. Ovariectomized rats were pretreated with either alpha-methyl-p-tyrosine (AMPT; tyrosine hydroxylase inhibitor), AMPT plus threo-dihydroxyphenylserine (DOPS; norepinephrine precursor), diethyldithiocarbamate (DDC; dopamine-beta hydroxylase inhibitor), pimozide (dopaminergic receptor blocker), phenoxybenzamine (alpha-adrenergic receptor blocker), propranolol (beta adrenergic receptor blocker), or their vehicles. EB implantation into the POSC reduced the frequency of existing pulsatile LH secretion in vehicle-treated rats. Pretreatment of the rat with AMPT, AMPT plus DOPS, or pimozide did not affect the basal pulsatile LH secretion but eliminated the suppressive effect of EB implantation on the LH pulse frequency. In rats pretreated with DDC or phenoxybenzamine, basal pulsatile LH secretion was significantly inhibited, and EB implantation did not lower serum LH further in these rats. Propranolol had no obvious effect on either basal pulsatile LH secretion or EB-induced suppression of LH pulse frequency. These findings suggest that, in addition to the alpha adrenergic involvement in maintaining the basal pulsatile LH secretion, the intact dopaminergic system is required for the suppression of the frequency of LH pulses induced by estrogen. PMID- 3019467 TI - Opioid receptor alterations in a genetic model of generalized epilepsy. AB - Autoradiography was used to examine opioid receptor binding in the Mongolian gerbil, a genetic model of the epilepsies. Coronal brain sections of seizure resistant (SR) and seizure-sensitive (SS) (both pre- and post-seizure conditions) gerbils were labeled with [3H]dihydromorphine. SS (pre-seizure) gerbils demonstrated overall greater brain opioid binding when compared to SR animals. The periaqueductal gray, substantia nigra and medial geniculate body were specific areas in SS (pre-seizure) gerbils which demonstrated highly significant increases in opioid binding compared to SR animals (% increase vs SR were 98%, 91.3% and 42.9%, respectively). Scatchard analysis demonstrated that the increase in opioid binding was due to an increase in the total number of receptors without a significant change in receptor affinity (i.e. periaqueductal gray area: total number of binding sites was 12.7 (SR) and 18.0 fmol/mg tissue (SS pre-seizure), while Kd values were 4.0 (SR) and 4.0 mM (SS pre-seizure). Opioid binding was also increased in the SS (post-seizure) animals when compared to SR animals, especially in the substantia nigra. However, when compared to SS (pre-seizure) gerbils, there was a general but not significant, decrease in opioid binding in SS post-seizure gerbils. The increased opioid binding in the SS (pre-seizure) gerbil compared to SR gerbils could reflect an up-regulation due to a deficit in endogenous ligand (e.g. a deficit in synthesis or decreased release) which could underlie the seizure diathesis in the gerbil. PMID- 3019468 TI - [3H]muscimol binding of GABA receptors in the visual cortex of normal and monocularly deprived cats. AB - In vitro receptor binding techniques were used to compare the total number, affinity and regional distribution of GABA receptors in visual cortex, as revealed by [3H]muscimol binding, in 5 normal and 5 monocularly deprived (MD) cats. Analysis of saturation kinetics and pharmacological specificity indicated that binding was to a single site having the characteristics of the GABAA receptor. No differences were found between normal and MD cats in either number or affinity of receptors. Within visual cortex, there were laminar differences in the density of binding, but no evidence for a lateral (columnar) organization. Label was densest in the superficial layers (I-IV), lowest in layer V and intermediate in layer VI. This pattern of label varied with incubation parameters with layer IV showing the densest label when high concentrations of [3H]muscimol and short rinse times were used. There were no differences between normal and MD cats in regional distribution of receptors under any incubation condition. PMID- 3019469 TI - The suppression of hippocampal potentials by the benzodiazepine antagonist Ro 15 1788 may be mediated by purines. AB - The benzodiazepine antagonist Ro 15-1788 has been reported to suppress paired pulse inhibition in the hippocampal slice. It is now shown that the depression of orthodromic synaptic transmission by Ro 15-1788 can be prevented by the adenosine antagonist 8-phenyl-theophylline, or by adenosine deaminase. Since Ro 15-1788 has previously been shown to inhibit adenosine-uptake into rat brain tissue, it is suggested that this property, leading to an accumulation of extracellular adenosine, may underlie its effects on synaptic transmission. PMID- 3019471 TI - Glial cells influence membrane-associated enzyme activity at the blood-brain barrier. AB - Glial cells have been shown to influence several cerebral endothelial cell properties in vitro. A situation similar to the endothelial-astrocyte relationship existing at the blood-brain barrier (BBB) can be produced by growing cultured cerebral endothelium on one side of a filter and glial cells on the other in an enclosed double chamber. In this setting membrane-associated reaction product on the cerebral endothelial cell for both Na+,K+-ATPase and non-specific alkaline phosphatase was markedly increased when the endothelial cells were co cultured with glial cells. In addition, the distribution of reaction product on the cerebral endothelial cell membrane was similar to that reported in vivo. These observations support a glial influence on enzyme activity at the BBB. PMID- 3019470 TI - Chronic dietary lithium induces increased levels of myo-inositol-1-phosphatase activity in rat cerebral cortex homogenates. AB - The monovalent lithium ion inhibits the enzyme myo-inositol-1-phosphatase at concentrations comparable to those which are useful in the treatment of manic depressive illness. However, dialyzed cortical homogenates from rats which have been fed diets containing lithium carbonate demonstrate increased myo-inositol-1 phosphate phosphatase activity. Over a 4-week period, there is an approximate doubling of the lithium-sensitive myo-inositol-1-phosphatase activity in the homogenate. PMID- 3019472 TI - Heterogeneous distribution of benzodiazepine receptors in the human striatum: a quantitative autoradiographic study comparing the pattern of receptor labelling with the distribution of acetylcholinesterase staining. AB - The distribution of benzodiazepine receptors in the human striatum was studied by quantitative autoradiography following in vitro labelling of cryostat sections with [3H]flunitrazepam, and the pattern of receptor-labelling was compared to the distribution of acetylcholinesterase (AChE) staining in adjacent sections. A heterogeneous pattern of benzodiazepine receptors was found in all regions of the striatum. The highest densities of receptors were seen in the ventral striatum (nucleus accumbens and olfactory tubercle), where very dense receptor patches aligned with both AChE-poor and AChE-rich regions. The dorsal striatum (caudate nucleus and putamen) contained lower concentrations of benzodiazepine receptors, but dense receptor patches were still evident (especially in the caudate nucleus) and these aligned with AChE-poor striosomes. PMID- 3019473 TI - Kynurenic acid distinguishes kainate and quisqualate receptors in the vertebrate retina. AB - Excitatory amino acid receptors (EAARs) underlie major synaptic pathways in the brain, retina and spinal cord. Several subclasses of EAARs have been proposed, based on pharmacological studies using a variety of agonists and antagonists. Kynurenic acid (Kyn), a metabolite of tryptophan, has been recently proposed as a potent EAAR antagonist. In this report, we show that Kyn can be used to separate two distinct classes of EAAR in the vertebrate retina: it blocks kainic acid (KA) responses but has minimal effects on responses mediated by quisqualate (QQ). At concentrations which block the KA responses, Kyn also blocks the light-evoked synaptic responses of all types of third-order neurons in the retina. These results suggest that KA receptors are the major receptor subtypes which underlie synaptic transmission and that QQ receptors are minimally utilized by light activated pathways under the conditions of our experiments. PMID- 3019474 TI - Benzodiazepine receptors in rat brain are altered by adrenalectomy. AB - The effects of adrenalectomy on benzodiazepine receptors in discrete regions of rat brain were examined using [3H]flunitrazepam as binding ligand. The concentration of benzodiazepine receptors was significantly increased by 25, 50 and 71% in hippocampus, striatum and hypothalamus, respectively, after adrenalectomy. In contrast, adrenalectomy did not affect the concentration of benzodiazepine receptors in cerebral cortex, olfactory bulb and cerebellum. No significant differences in the apparent binding affinity (Kd) values were seen following adrenalectomy in any brain region examined. The adrenalectomy-induced increases in [3H]flunitrazepam binding sites were completely reversed by glucocorticoid replacement with dexamethasone. These results demonstrate that adrenalectomy is capable of selectively modulating benzodiazepine receptors in brain regions presumably involved with glucocorticoid negative feedback. The data further suggest additional mechanisms by which endogenous hypothalamic-pituitary adrenocortical hormones may affect 'anxiety' levels. PMID- 3019475 TI - The arcuate nucleus: a site for gamma-aminobutyric acid regulation of prolactin secretion. AB - The effects on prolactin (Prl) secretion following microinfusion of muscimol or N methyl-aspartate (NMA) into the medial basal hypothalamus (MBH) of male rats was determined. A highly significant increase in Prl occurred following microinfusion of muscimol (500 pmol) into the arcuate nucleus. Microinfusion of muscimol into nearby sites, such as the ventromedial nucleus and dorsal medial nucleus, was not effective in altering Prl secretion. Similarly, microinfusions of artificial cerebrospinal fluid were not effective. NMA (50 pmol), a dose found to affect hormone secretion in other CNS areas, did not alter Prl secretion when infused into the MBH. The present study was able to discriminate between CNS areas by using small volumes (250 nl) and small quantities of the amino acid agonists. This data indicates that GABAergic systems in the arcuate nucleus control Prl release from the pituitary, and that one possible mechanism is via the tuberoinfundibular dopamine system. PMID- 3019477 TI - Nigroamygdaloid dopamine neurons: nigral modulation of their activity. AB - In order to study the mechanisms regulating the dopaminergic nigroamygdaloid cells, the release of dopamine was observed in the central nucleus of the amygdaloid complex. Halothane anesthetized rats were implanted, according to the experiment, with one or two push-pull cannulae in the central nuclei of the amygdala (ACE), the substantia nigra (SN) and/or the caudate nucleus (CN). Canulae were supplied with artificial cerebrospinal fluid (CSF) containing tritiated tyrosine, and labeled dopamine [3H]DA was evaluated in successive superfusate fractions. Electrical stimulation of the medial forebrain bundle with an implanted bipolar electrode induced an increase of the [3H]DA release in the ipsi- and contralateral ACE. Electrical stimulation of the SN produced only a very delayed effect in the ipsilateral ACE but an immediate and large increase of [3H]DA release in the contralateral structure. Superfusion of unlabeled DA and alpha-methyl-p-tyrosine in the SN remained ineffective on the [3H]DA release in the ipsilateral ACE. In this structure the release of [3H]DA was, however, decreased by nigral superfusion with gamma-amino-butyric acid (GABA). D-(+) Amphetamine (1 microM), when superfused in the CN, induced a large enhancement of the [3H]DA release in the ipsilateral ACE simultaneously with the local increase of [3H]DA release. The results presented here are in agreement with the previous studies concerning the anatomical organization of the dopaminergic nigroamygdaloid pathway. The DA cell bodies located in the SN appear insensitive to a local action of DA, perhaps due to a lack of autoreceptors. They are, however, powerfully inhibited by GABA and the relation observed between the [3H]DA release in the CN and ACE support the hypothesis that the SN can act as a relay between the extrapyramidal and limbic systems. PMID- 3019476 TI - Corticotropin releasing factor receptor-mediated stimulation of adenylate cyclase activity in the rat brain. AB - Corticotropin releasing factor (CRF)-stimulated adenylate cyclase activity and receptor binding were examined in rat brain homogenates using a potent synthetic CRF analog--[D-Tyr3,D-Pro4,Nle18,21,alpha-helical]CRF3-41 (alpha-hel CRF3-41). Binding of alpha-hel CRF3-41 in the rat brain was saturable, reversible, of high affinity and exhibited relevant peptide specificity. This analog also stimulated adenylate cyclase activity of various brain regions; the greatest magnitude of stimulation was in the cerebral cortex followed by the septum, cerebellum and thalamus. Adenylate cyclase stimulation in the cerebral cortex was concentration dependent with an ED50 of 2.5 +/- 0.4 nM for alpha-hel CRF3-41 and an ED50 of 16 +/- 2 nM for ovine and rat CRF. Maximal stimulation was comparable for all peptides. Agonist-stimulated adenylate cyclase activity was competitively blocked by the CRF antagonists. The inactive CRF analog, ovine CRF1-39, at concentrations less than 1 microM, did not significantly stimulate adenylate cyclase. Adrenalectomy, which has been reported to modulate CRF receptor number and CRF stimulated adenylate cyclase activity in the anterior pituitary, had no effect on CRF receptor binding or CRF-stimulated adenylate cyclase activity in the cerebral cortex. These results suggest that, as in the anterior pituitary, at least some of the physiological responses mediated by CRF receptors in the brain utilize the cyclic nucleotide regulatory pathway as a post-receptor mechanism. PMID- 3019478 TI - N-methyl-D-aspartate receptors coexist with kainate and quisqualate receptors on single isolated catfish horizontal cells. AB - Horizontal cells enzymatically isolated from catfish retina were exposed to the putative neurotransmitters aspartate (Asp) or N-methyl-D-aspartate (NMDA). Under voltage clamp conditions, inward currents were recorded when the holding potential was more negative than zero and outward currents were recorded when the membrane potential was more positive than zero. The current voltage curve was highly non-linear in the range of membrane potential between -30 and -100 mV. This non-linearity was largely removed in zero magnesium solution. 2-Amino phosphonovaleric acid selectively blocked Asp and NMDA responses. These response characteristics are consistent with the presence of NMDA receptors in these cells. PMID- 3019479 TI - Spinal opiate modulation of cardiovascular reflexes in the exercising dog. AB - The possible role of spinal opiate receptors on hind limb muscle afferents, participating in arterial blood pressure and heart rate responses to ischemic exercise, were investigated following lumbosacral intrathecal injection of morphine and naloxone in chronically instrumented dogs. Morphine markedly attenuated these responses. Naloxone had no effect alone but blocked the morphine responses. Thus, spinal opiate receptors may modulate ascending afferent information mediating cardiovascular reflexes from ischemic muscle. PMID- 3019480 TI - Reversal of organophosphate-induced muscle block by neomycin. AB - The organophosphate, diisopropyl fluorophosphate, and the aminoglycoside antibiotic, neomycin, both independently block neuromuscular transmission. At the neuromuscular junction, neomycin reduces the presynaptic release of acetylcholine, whereas diisopropyl fluorophosphate irreversibly inhibits acetylcholinesterase, thereby increasing the acetylcholine concentration. In the rat diaphragm preparation, the block in neuromuscular transmission caused by diisopropyl fluorophosphate could be reversed by adding neomycin. PMID- 3019482 TI - Opiate receptors in rat pituitary are confined to the neural lobe and are exclusively kappa. AB - The distribution and density of opiate receptor subtypes in rat pituitary were examined by quantitative autoradiography of tritiated ligand-binding to slide mounted sections under conditions optimized to label mu, delta, or kappa opiate receptors. Mu and delta receptor-binding was virtually undetectable in the pituitary. Kappa receptor-binding was confined to the neural lobe where it was densest in the external rim. Autoradiographic silver grains in emulsion-coated, Nissl-stained sections were preferentially located between cells, suggesting kappa receptor localization on nerve terminals and/or processes of pituicytes. PMID- 3019481 TI - Delta opioid receptors in human neuroblastoma cell lines. AB - Opioid receptor sites were detectable in 4 out of 9 human neuroblastoma cell lines tested, in the human retinoblastoma line Y79 NHT C10 and in the mouse neuroblastoma line Neuro 2A. All of these cell lines expressed delta sites, while only one coexpressed mu sites (SK-N-SH). Together with delta sites previously found in rodent neuroblastoma lines, these results suggest that the expression of delta sites is under less stringent control than that of mu and chi sites. A large number of delta sites (greater than 10,000 sites per cell) is expressed in IMR-32 and NMB neuroblastoma lines. Agonist binding was sensitive to Na+ and guanine nucleotides. The delta sites in IMR-32 and NMB cells were further characterized with delta selective ligands and [3H]DADL tracer. Their delta binding affinities were identical to those of the mu and delta cell line SK-N-SH; therefore the presence of mu sites does not appear to affect the binding behavior of the delta sites by any potential interaction among the binding proteins. Further, close correlations were found when comparing ligand binding in the human neuroblastoma cell lines with those of mouse neuroblastoma cells and rodent brain, an indication that the delta receptor is highly preserved among different species. PMID- 3019483 TI - Neonatal peptides affect developing rats: beta-endorphin alters nociception and opiate receptors, corticotropin-releasing factor alters corticosterone. AB - The dose-response relationship of neonatal (days 1-7) administration of beta endorphin (BE) and corticotropin-releasing factor (CRF) on body weight, eye opening, response to thermal pain, and concentrations of plasma and adrenal corticosterone were measured in developing rat pups. At the highest dose (50 micrograms/pup), neonatal CRF reduced body weight, while 10 and 50 micrograms/pup accelerated eye opening. In addition, a reduction in concentration of plasma and adrenal corticosterone was correlated with the dose of neonatal CRF, whereas adrenal weights were not altered. BE produced none of these effects, but 1, 10 and 50 micrograms/pup on days 1-7 significantly reduced baseline latencies in a novel water-bath tail-flick test on day 9. These same animals showed reduced numbers of brain opiate receptors on day 14. The results indicate that peptide administration during the sensitive neonatal period can alter the development of physiological processes that will later be influenced by the peptide. PMID- 3019484 TI - 5'-Nucleotidase of cerebellar molecular layer: reduction in Purkinje cell deficient mutant mice. AB - The molecular layer of the cerebellar cortex contains high levels of the enzyme 5'-nucleotidase (EC 3.1.3.5), localized predominantly to Bergmann glia. In the present study, histochemical methods have been employed to examine the distribution of cerebellar 5'-nucleotidase in mice of two separate mutant strains in which Purkinje cells are eliminated selectively. In homozygous 'Purkinje cell degeneration' (pcd) and 'nervous' (nr) mice, molecular layer 5'-nucleotidase is greatly reduced and residual enzyme activity in colocalized with surviving Purkinje cells. These results suggest strongly that the expression of 5' nucleotidase activity by Bergmann glia is under the inductive influence of their associated Purkinje cells. PMID- 3019485 TI - Separation of components of hippocampal field potentials by digital filtering on a PC-based microcomputer. AB - A versatile digital filtering system is described which enables separation of wave components of evoked potentials based on their frequencies. The utility of this technique is exemplified by application of digital filtering to hippocampal field potentials. Separation of population spikes from lower frequency synaptic potentials provides less ambiguous interpretation of the compound slope of the field potential during the first 5 milliseconds of the response. The entire procedure of stimulation, data acquisition, and digital filtering was implemented on a PC-compatible computer. PMID- 3019486 TI - Projections of the mesencephalic locomotor region in the rat. AB - Retrograde labeling and electrophysiological techniques were used to determine the organization of efferent projections of the mesencephalic locomotor region (MLR) in the rat. Injections of horseradish peroxidase (HRP) or fluorescent dyes were made into suspected terminal zones of the MLR. After allowing for retrograde transport, the same animals underwent precollicular transection under anesthesia. The MLR was identified physiologically by the induction of controlled locomotion on a treadmill at low current (less than 50 microA) levels. Stimulation sites were marked and the brain processed accordingly. Retrogradely labeled cells in proximity (less than 0.5 mm) to locomotion-inducing stimulation sites were considered to be putative MLR neurons and were plotted according to stereotactic coordinates. These studies revealed the intrinsic organization of MLR neurons projecting to various brainstem sites. This organization may be related to the different types of transmitter systems involved in mediating MLR function. PMID- 3019488 TI - [Leukotriene C4 binding sites in the mouse forebrain: autoradiographic demonstration]. AB - The presence of binding sites for leukotriene C4 (LTC4) is demonstrated in the mouse forebrain, by using autoradiography of sections incubated with tritiated LTC4. The binding of LTC4 is inhibited by an excess of cold LTC4, but unaffected by the presence of a large excess of LTD4, which differs from LTC4 by the absence of a glutamic acid residue. The density of binding sites is minimal on fiber bundles and on choroid plexuses, maximal at the level of granule cell-rich structures such as the dentate gyrus and entorhinal area, and high in the cerebral cortex, thalamic relay nuclei and the caudoputamen. These data suggest that leukotrienes and their receptors might play a role as regulators of central neural activity, a hypothesis which was recently proposed by Lindgren et al. PMID- 3019487 TI - beta-Funaltrexamine (beta-FNA) and the regulation of body and brain development in rats. AB - The effects of beta-FNA, a highly selective and irreversible mu opioid receptor antagonist, in altering body and brain development in preweaning rats were determined. Animals given beta-FNA did not differ from controls in body weights, brain and cerebellar weights, macroscopic dimensions of the brain, the area of the cerebellum, or in organ weight. The dosage of beta-FNA utilized (5 mg/kg) blocked morphine-induced analgesia (2 mg/kg morphine sulfate, SC) for each injection period (i.e., 48 hr). In contrast to beta-FNA treatment, rats given naltrexone (50 mg/kg SC) in a regimen which completely blocked the opioid receptor throughout ontogeny exhibited marked increases in somatic and neurobiological growth. These results suggest that, in and by themselves, mu receptors selectively antagonized by beta-FNA do not play an important role in regulating development. PMID- 3019489 TI - [A case of overlapping reading frames in Escherichia coli]. AB - The two promoters and the first 65 nucleotides of a gene related to the rrnB operon are localized in the coding sequence of the btuB gene. PMID- 3019490 TI - [Circulating lupus-type anticoagulant, a risk factor for thrombosis by inhibition of protein C activation]. AB - The lupus anticoagulant is a risk factor of thrombosis. The non thrombogenic endothelial surface could be a target for the lupus anticoagulant. We have investigated the effect of purified immunoglobulins G of five patients with LA on the thrombomodulin activity of cultured human endothelial cells from umbilical cord vein. The rate of activation of purified protein C (PC) (30 nM) by the endothelial cells in the presence of thrombin (0.1 U/dish) has been measured by hydrolysis of substrate S 2366. Activated PC has been 7.37 +/- 0.78 pmoles X ml-1 X h-1 in the presence of buffer and 7.2 +/- 0.78 pmoles X ml-1 X h-1 in the presence of control IgG (2 mg/dish). Heat aggregated IgG did not induce any significant change. Patient's IgG lowered significantly the rate of PC activation (4.86 +/- 1.04 pmoles X ml-1 X h-1, p less than 0.001). Fab fragment from two of these patient's IgG displayed the same inhibition. Moreover neutralization of this effect was obtained by addition of phospholipids (70% phosphatidylcholine, 30% phosphatidylserine) in excess to patient's IgG. Activation of PC has been also performed using purified rabbit thrombomodulin and a similar inhibition by patient's IgG was found. These results seem to indicate that antibodies present in the IgG fractions containing LA could be directed against phospholipids associated to thrombomodulin activity. Reduction of PC activation if present in the patients with LA could play a role in the occurrence of thrombosis. PMID- 3019491 TI - [Effects of insulin and somatomedin C on the function of cultured Leydig cells]. AB - Porcine cultured Leydig cells (LC) lose hCG receptors and hCG responsiveness (cAMP and testosterone) when they are cultured for three days in a defined medium without insulin or somatomedin C (Sm-C) (Insulin-like growth factor I). In the presence of insulin (50 ng/ml) or of Sm-C (10 ng/ml) the loss of the hCG receptor number and the decreased cAMP response to hCG were prevented, but the steroidogenic response to hCG was only partially prevented. This parameter became normal when cells were pretreated with either Sm-C (10 ng/ml) plus insulin (50 ng/ml) or with insulin alone at high concentrations (5 micrograms/ml). These results indicate that both Sm-C and insulin acting through their own receptors increase Leydig cell steroidogenic capacity by increasing hCG receptor number and improving some step beyond cAMP formation. PMID- 3019493 TI - Neural control of vasomotor tone of large coronary arteries. AB - In man, the occurrence of constrictions of large coronary arteries accompanied by transient myocardial ischemia is now well established. However, the role of neural factors involved in such coronary artery spasms is still a matter of conjecture. A consistent reduction (9 +/- 2%) of the diameter of the large coronary arteries can be obtained in the conscious dog with alpha-adrenergic receptor stimulation with methoxamine in spite of the concomitant pressor rise (65 +/- 5%). Smaller reductions in coronary diameter can be obtained with electrical efferent sympathetic stimulation in anesthetized dogs. The diameter of a conduit artery such as the aorta can be reduced (5%) by reflex increases in sympathetic efferent activity: therefore it is not unlikely that similar neural influences might be exerted on the coronary tree as well. In normal life, stressful situations, such as emotion or exercise, will be accompanied by a drastic increase in sympathetic drive to the heart, together with a marked increase in coronary flow. The latter will induce an endothelial mediated vasodilation; however the net effect on coronary size of these two potentially opposite mechanisms is as yet unexplored. In the laboratory, intracoronary bradykinin and regional myocardial ischemia initiate a reflex increase in sympathetic activity to the heart; in the clinics acute myocardial ischemia can be accompanied by signs of sympathetic overactivity. The extent to which such increases in sympathetic activity, could play a role in the control of coronary tone and hence in the pathophysiology of coronary artery disease, is still under investigation. PMID- 3019492 TI - Effects of ascorbic acid on alkaline phosphatase activity and hormone responsiveness in the osteoblastic osteosarcoma cell line UMR-106. AB - L-ascorbic acid at physiological concentrations (10 micrograms/ml) increased alkaline phosphatase activity in the osteoblastlike rat osteosarcoma cell line, UMR-106. The increase was dose-dependent and detectable at 6 hours after the addition of 100 micrograms/ml ascorbic acid to the medium. Treatment of the cells with 100 micrograms/ml ascorbic acid potentiated the response of cAMP to both PTH and PGE1, while cell growth was inhibited. Furthermore, the number of colonies formed by the cells grown in the soft agar was significantly reduced by increasing concentrations of ascorbic acid. These results indicate that ascorbic acid might play some role in the differentiation of osteoblasts. PMID- 3019495 TI - Herpes simplex keratitis: a clinico-pathologic case report. AB - A case of herpes simplex keratitis in which it was difficult to make a conclusive diagnosis by routine histochemistry is presented. Electron microscopy, however, revealed virus particles in the stromal cells. PMID- 3019494 TI - Genetic mapping of the 5S rRNA gene cluster of the nematode Caenorhabditis elegans. AB - We have identified a restriction fragment length difference (RFLD) affecting the genomic sequences immediately flanking the 5S rRNA gene cluster in the Bristol and Bergerac strains of the nematode Caenorhabditis elegans. We have used this RFLD as a molecular marker to follow the segregation of the 5S rRNA gene cluster through a series of two- and three-factor interstrain crosses. Our results show that the 5S rRNA gene cluster maps between unc-76 and dpy-21 on the right arm of linkage group V. This genetic localization provides a linkage group V "landmark" with which to localize other cloned sequences by in situ hybridization. PMID- 3019496 TI - Dysfunctional uterine bleeding. AB - Dysfunctional uterine bleeding is defined as abnormal uterine bleeding in the absence of organic disease. It is the result of anovulation or the abnormal local production of prostaglandins, and in each case the primary fault is inappropriate hormone formation. There are two approaches to diagnosis. The traditional one is primarily concerned with the exclusion of cancer of the endometrium; this concern results in the frequent resort to uterine curettage. The second approach is to limit curettage to patients whose symptoms are not ameliorated by medical therapy. The aim of medical treatment is either to produce secretory change in the endometrium or to decrease the formation of uterine prostaglandins. Intermittent progesterone treatment is used to cause secretory changes in the endometrium. Decreased production of the prostaglandins is achieved indirectly by causing atrophy of the endometrium or directly through the use of prostaglandin synthetase inhibitors. Surgery in the form of dilatation and curettage has no long-term therapeutic effect; hysterectomy is definitive therapy. PMID- 3019497 TI - Reduction of intimal hyperplasia in canine autologous vein grafts with cod-liver oil and dipyridamole. AB - To determine the effects of cod-liver oil and a combination of cod-liver oil and dipyridamole on vein-graft intimal hyperplasia, 76 segments of undistended jugular vein were interposed between bilaterally divided femoral arteries in 38 mongrel dogs who received a 2% cholesterol diet. Ten control animals received the diet alone, 8 received cod-liver oil containing 1.8 g of eicosapentaenoic acid daily 1 week before and for 6 weeks after operation, and 20 dogs received 1.8 g of eicosapentaenoic acid and 75 mg of dipyridamole daily 1 week before and for 6 weeks after operation. A similar and significant (p less than 0.01) increase in serum cholesterol was observed in all three groups. Prothrombin, partial thromboplastin and clotting times and the platelet count were unchanged in the controls and in those receiving cod-liver oil. Clotting time increased in the animals receiving a combination of cod-liver oil and dipyridamole (p less than 0.001). Measurements (406 +/- 27) of intimal thickness were made from each graft. Intimal thickness was 3.7 +/- 0.1 micron before implantation and increased to 78 +/- 8 micron after in the controls. Cod-liver oil limited the increase in intimal thickening, to 24 +/- 3 micron (p less than 0.001); cod-liver oil and dipyridamole further reduced the increase in intimal thickening, to 17 +/- 1.4 micron (p less than 0.001). The data indicate that a combination of cod-liver oil and dipyridamole is more effective than cod-liver oil alone in reducing canine vein-graft intimal hyperplasia (p less than 0.03). PMID- 3019498 TI - Eradication of bovine leukemia virus infection in commercial dairy herds using the agar gel immunodiffusion test. AB - Demands for bovine leukemia virus test negative breeding cattle and for semen from bovine leukemia virus test negative bulls by several countries have encouraged the eradication of bovine leukemia virus infection from selected herds in Canada. This project was undertaken to evaluate the suitability of the agar gel immunodiffusion test, standardized to detect anti-bovine leukemia virus glycoprotein antibodies, for eradication of bovine leukemia virus from commercial dairy herds. Of nine participating herds, the prevalence rate of bovine leukemia virus infection was low (less than 10%) in three, medium (11-30%) in four and high (greater than 30%) in two. The herds were tested by the agar gel immunodiffusion test, reactors were removed and the herds were then retested at regular intervals. The results indicate that it is possible to eliminate bovine leukemia virus infection from the herds after two to three cycles of agar gel immunodiffusion tests and prompt removal of the reactors. PMID- 3019500 TI - Attempts to establish congenital bluetongue virus infections in calves. AB - Three bovine fetuses were inoculated in utero with approximately 10(3) plaque forming units of type 11 bluetongue virus. The gestational ages of the fetuses at the time of inoculation were 106, 113 and 122 days. They were spontaneously aborted 104, 65 and 109 days later, respectively, and the first and third of these fetuses were recovered. There was no grossly normal cerebral tissue, the meninges formed fluid filled sacs, and the cerebellums were reduced in size. Bluetongue virus was not isolated from the fetuses but the older one had neutralizing antibody. The three dams developed neutralizing antibody to bluetongue virus. The present work supports the observation by others that early fetal infections with bluetongue virus normally result in severe central nervous system damage and not in clinically normal, persistently infected calves. PMID- 3019499 TI - Prompt arousal from fentanyl-droperidol-pentobarbital anesthesia in dogs: a preliminary study. AB - Groups of fentanyl-droperidol-pentobarbital-anesthetized dogs (n = 6 dogs/group) were given IV saline solution (control group), graded doses of naloxone (0.01, 0.1, 1.0, 10.0 mg/kg) or fixed doses of 4-aminopyridine (0.5 mg/kg), yohimbine (0.4 mg/kg), or doxapram (5.0 mg/kg) alone or in combination with a fixed dose of naloxone (1.0 mg/kg). The purpose was to determine which drug or drug combination would produce arousal most quickly without producing obvious undesirable side effects. Control group mean arousal time, mean walk time and mean duration of postarousal sedation were 66.1 minutes, 112.4 minutes and 5.6 hours, respectively. Naloxone (1.0 mg/kg) decreased mean arousal time to 10.8 minutes without significantly decreasing mean walk time or mean duration of postarousal sedation. The combination of naloxone + doxapram decreased mean arousal time and mean walk time to 1.0 minute and 57.1 minutes, respectively, without decreasing mean duration of postarousal sedation. In all groups, emergence from anesthesia was smooth. Relapses or undesirable side effects were not observed. Naloxone + doxapram is superior to naloxone alone for arousal of fentanyl-droperidol pentobarbital-anesthetized dogs. PMID- 3019501 TI - Possible sex differences in the susceptibility of calves to rotavirus infection. AB - The agar gel precipitation test was used to detect rotavirus antibodies in the serum of 562 calves and bovine rotavirus antigen in the feces of 347 calves. Significantly more females had rotavirus antibodies in the serum (P less than 0.01) and rotavirus antigen in the feces (P less than 0.1) than did male calves. Female buffalo calves were also found to be more susceptible than male buffalo calves to rotavirus infection. PMID- 3019502 TI - Rates of death from testicular cancer in Ontario in 1964-82: analysis by major histologic subgroups. AB - In 1977 the rate of death from testicular cancer in Ontario began to decline following a long period of relative stability. This coincided with the addition of cisplatin to the chemotherapeutic regimens used in the treatment of disseminated germ-cell testicular cancer. A study was carried out to determine whether the decline has been similar for the two major histologic subgroups, seminoma and nonseminoma. By means of Ontario Cancer Registry data, histologic type was determined for all testicular cancers causing death in Ontario residents between 1964 and 1982, and death rates were calculated for seminoma and nonseminoma germ-cell testicular cancer. The rates of death from nonseminoma exceeded those from seminoma for the entire study period. Although the death rates for both subgroups declined by about 50% after 1976, the reduction in the overall rate of death from testicular cancer is primarily attributable to the decline in the rate for nonseminoma germ-cell testicular cancer. The increased overall rate of death from testicular cancer (all types) between 1980 and 1984 is cause for concern and indicates the need for continued monitoring of the trends in rates of death from this disease. PMID- 3019503 TI - A 10-year experience with combined modality therapy for stage III small cell lung carcinoma. AB - During the past 10 years, 240 patients with Stage III small cell lung carcinoma (SCLC) were treated with one of five chemotherapy programs plus thoracic irradiation. In addition, prophylactic cranial irradiation was administered concurrently with thoracic irradiation to 194 patients receiving CAML-HC, VCAM, or MOCA. Seventy-two patients had disease confined to the chest (Stage IIIM0), 30 patients had disease in the chest plus ipsilateral supraclavicular nodal involvement (Stage IIIM0SCN+), and 138 patients had distant metastatic disease (Stage IIIM1); the median survivals were 15.2 months, 12.6 months, and 8.4 months, respectively. The overall complete response rate was 30% and the overall response rate (complete and partial) was 76%. The overall response rates by stage were 86% for Stage IIIM0, 90% for Stage IIIM0SCN+, and 67% for Stage IIIM1. Eight patients (3%) were alive and free of disease at 24 months. Due to continued disease relapse in this group (four of eight patients), long-term survivors should not be identified for a minimum of 3.5 years from the time of initial therapy. Prophylactic cranial irradiation (PCI) effectively reduced the incidence of central nervous system (CNS) relapse in patients with a complete response to therapy (44% relapse without PCI versus 13% relapse with PCI, P less than 0.01). More effective chemotherapy is required for the successful treatment and improved long-term survival of patients with SCLC. PMID- 3019504 TI - Five-year survival of patients with oral cancer and its association with antibody to herpes simplex virus. AB - Levels of antibody to herpes simplex virus type 1 (HSV-1) were measured in 70 patients with untreated squamous cell carcinoma of the mouth. After treatment the actuarial survival was determined at quarterly intervals for 5 years and was found to be associated with the pretreatment level of antibody to the virus. Patients with levels of IgM antibody to HSV-1 which were above the median level had a 5-year survival of only 56% whereas those with levels below the median had a higher survival of 72%. Patients with no detectable IgM antibody to HSV-1 had a 5-year survival of 81%. The reverse was seen with IgG antibody to HSV-1. Patients with higher than the median level of IgG antibody had a 5-year survival of 73%, whereas those with IgG antibody below the median had a 5-year survival of 56%. No relationship was seen between survival and levels of IgA antibody to HSV-1, or between survival and antibody of any class to cytomegalovirus. The data are consistent with the reported association between oral cancer and HSV-1. PMID- 3019505 TI - Cystosarcoma phyllodes. A clinicopathologic study of 26 cases. AB - Twenty-six cases of cystosarcoma phyllodes diagnosed at M. D. Anderson Hospital were reviewed. The following criteria were evaluated for possible correlation with local recurrence, uncontrolled local recurrence, metastasis, and tumor death: tumor size, stromal overgrowth, tumor necrosis, mitotic rate, stromal cellularity, nuclear size and pleomorphism, the presence of specialized stroma, and initial therapy. Of the 26 tumors, seven caused death. Five patients developed metastatic spread, and all of them died of tumor. Five patients had local recurrence, which was uncontrolled in three (two patients died with uncontrolled recurrence alone, and one with uncontrolled recurrence and metastasis). Stromal overgrowth was present in eight cases. Six of the seven patients who died of tumor had stromal overgrowth, including all five with metastasis. Correlation of stromal overgrowth with metastatic spread and tumor death was significant at P levels of 0.0014 and 0.02, respectively. It is concluded that stromal overgrowth is a significant histologic indicator of malignant behavior in cystosarcoma phyllodes. PMID- 3019506 TI - Impaired primary, but not secondary, immune response in breast cancer patients under adjuvant chemotherapy. AB - The antibody response after vaccination against tick-borne encephalitis (TBE) was studied in patients with breast cancer. Although sex- and age-matched control persons produced high titers of anti-TBE antibodies 2 to 4 weeks after the second of two consecutive vaccinations, patients with breast cancer who were first vaccinated after the start of adjuvant chemotherapy consisting of cyclophosphamide, methotrexate and 5-fluorouracil (CMF) failed to do so. The lack of anti-TBE antibody production was found not only in patients under CMF chemotherapy, but also in those primarily vaccinated 6 to 12 months after the termination of CMF treatment. Patients with breast cancer who had been vaccinated either before or after the onset of disease, but before the initiation of chemotherapy, were shown to have developed significant anti-TBE antibody titers which persisted throughout the course of adjuvant treatment and could be boostered by revaccination during the course of CMF administration. The authors conclude that patients with breast cancer undergoing adjuvant chemotherapy experience a serious and prolonged defect in primary antibody production, whereas secondary immune responses remain unimpaired. PMID- 3019507 TI - Severe impairment of antioxidant system in human hepatoma. AB - Catalase (CAT), glutathione-peroxidase (GSH-Px) activity and reduced glutathione content (GSH) were measured in patients who had hepatocellular carcinoma, and values compared with those of normal liver and liver adjacent to neoplastic tissue. The results showed a remarkable reduction of CAT in tumor and corresponding tumor-free tissue (P less than 0.001 and P less than 0.02, respectively). All neoplastic samples had a significant lower activity of CAT than the corresponding adjacent tumor-free tissue (P less than 0.05). The GSH-Px activity of tumor tissue also was lower than normal (P less than 0.001) but similar to that of adjacent tissue. No correlation was noted between the two enzyme activities. Glutathione content was extremely low in tumor (P less than 0.001) and even in tumor-free tissue (P less than 0.05) when compared with normal liver. In all cases the content of GSH in neoplastic tissue was lower than that of the corresponding tumor-free tissue (P less than 0.05). Whereas in normal liver the activity of GSH-Px was positively correlated with the content of GSH, in the neoplastic tissue such a relationship disappeared. All these findings suggest that the antioxidant system of hepatocellular carcinoma cell is severely impaired. PMID- 3019508 TI - Influence of chemotherapy on superoxide anion-generating activity of polymorphonuclear leukocytes in patients with lung cancer. AB - The superoxide anion (O2-) generating activity of polymorphonuclear leukocytes (PMN) in peripheral venous blood obtained from 15 patients with lung cancer was measured weekly after drug treatment. The amount of O2- generated by the cells before chemotherapy was 5.74 +/- 0.42 nmol/minute/10(6) cells (mean +/- SE). The value decreased gradually and reached a nadir of 1.98 +/- 0.24 at 2 weeks after chemotherapy, at which time PMN count also reached a nadir of 1359 +/- 251/microliters (mean +/- SE). The decreased O2- generating activity of the PMN recovered gradually thereafter and reached the level seen before the chemotherapy at 4 weeks. Expression of Fc receptors on the plasma membrane in PMN was measured after chemotherapy by the binding of iodine 125 (125I)-immune complexes to the cells. Almost 40% decrease of the binding was observed in the cells obtained at 2 weeks after chemotherapy, compared with that in the cells before chemotherapy. These results indicate that PMN present in peripheral blood at 2 weeks after chemotherapy are not fully matured, in terms of O2- generating activity and the expression of Fc receptors. PMID- 3019509 TI - Placental-like alkaline phosphatase. Re-evaluation of the tumor marker with exclusion of smokers. AB - This report demonstrates that smoking is a major factor of nonspecific elevation of the tumor marker placental-like alkaline phosphatase (PLAP). In 98 healthy nonsmokers the mean of the enzyme activity was determined as 0.068 U/L (range, +/ 2 SD 0-0.144 U/L) compared to a mean of 0.378 U/L (range, +/- 2 SD 0-1.02 U/L) in 65 smokers. In view of this finding the usefulness of PLAP as a tumor marker was re-evaluated in 286 patients with various neoplasms and a negative smoking history. Of these patients, 23% and 50% had elevated values for PLAP and carcinoembryonic antigen, respectively. When compared to the range of PLAP in normal smokers only 4.1% of the patients showed elevated values. An increased incidence of elevated PLAP was found in patients with tumors of the lung, pancreas, stomach, colon/rectum, ovaries, and in 2 of 3 seminomas. It was concluded from the data that PLAP is a useful tumor marker for selected neoplasms provided its use is confined to nonsmokers. PMID- 3019510 TI - Thrombocytosis in patients with malignant pleural mesothelioma. AB - Thrombocytosis above 40.0 X 10(4)/mm3 occurred in five of six (83%) patients with malignant mesothelioma. In contrast, the incidence of thrombocytosis was 7.5% in patients with lung adenocarcinoma, 12.5% in squamous cell carcinoma, 5.1% in small cell carcinoma, and 41.7% in large cell carcinoma, respectively. The platelet count in large cell carcinoma was significantly higher than that in other cell types of lung cancer; however, the platelet count in malignant mesothelioma was much higher than that in large cell carcinoma. These results show that the incidence of thrombocytosis seems to be high in malignant mesothelioma, although the mechanism is thus far unknown. PMID- 3019511 TI - The effects on resting energy expenditure of different tumor types. AB - The presence of a malignant tumor is said to influence the resting energy expenditure (REE) of the host. This study assesses the hypothesis that different tumor types exert differing effects on REE. REE was measured using indirect calorimetry in 84 cancer patients. Fifty-one patients had colorectal cancer, 22 had gastric cancer, and 11 had non-small cell bronchial cancer. For each tumor type, REE correlated significantly with body weight and lean body mass (LBM). The slope of the regression line for the bronchial cancer patients was significantly different from the colorectal (P less than 0.02) and gastric (P less than 0.02) cancer patients when REE was related to LBM. When related to body weight, the slope of the line of the bronchial cancer patients was significantly different from that of the gastric cancer patients only (P less than 0.05). The bronchial cancer patients had a significantly higher REE than the colorectal (P less than 0.005) and gastric (P less than 0.02) cancer patients when REE was expressed in terms of body weight. However, when corrected for LBM, no significant differences in REE were found between the groups. The presence of hepatic metastases did not influence REE in any of the tumor groups. Differing relationships between REE and indices of body size have been detected in differing tumor types. Consequently, the use of heterogeneous cancer groups may be inappropriate in studies of REE and cancer. PMID- 3019512 TI - Bone marrow involvement in anaplastic small cell lung cancer. Diagnosis, hematologic features, and prognostic implications. AB - The hematologic changes and prognostic implications of bone marrow involvement in small cell lung cancer (SCLC) were examined in 133 patients undergoing staging procedures including bone marrow aspiration and trephine biopsy. Bone marrow involvement was found to be present in 27 of 133 (20%) at diagnosis. In most instances bone marrow involvement was associated with the presence of other metastases but in five patients (4%) the bone marrow was the sole site of metastasis. Bone marrow biopsy proved superior (14%) to bone marrow aspiration (5%) in detecting marrow infiltration. Peripheral blood hematologic changes were infrequent even in patients with positive bone marrow biopsy results. Although patients with bone marrow involvement had a shorter median survival (9 weeks) than bone marrow-negative patients (33 weeks) the adverse effect on survival appears to have been mainly due to the presence of concomitant metastases at other sites. Intensive chemotherapy was tolerated to the same degree by bone marrow-positive and bone marrow-negative patients. PMID- 3019513 TI - Diet, nutrition, and cancer. The role of fiber. AB - The mechanisms by which dietary fiber could inhibit development of colon cancer include effects on fecal weight and transit time, adsorption of bile acids, dilution of colonic contents, production of short chain fatty acids (products of fiber fermentation), inhibition of dehydroxylation of bile acids, and regulation of energy intake. Review of the literature suggests that effects on fecal weight and transit time and adsorption of bile acids are not likely mechanisms. Since concentration of bile acids is lower in feces of less susceptible populations, dilution of colonic contents may contribute to fiber effects. High colonic pH is associated with promotion of cancer and production of short chain fatty acids would counteract this effect. Animals maintained on calorie-restricted diets exhibit fewer spontaneous or experimentally induced tumors. Regulation of energy intake by fiber may contribute towards reduction of colon cancer incidence in man especially when caloric content is low from infancy. PMID- 3019514 TI - A clinical and histopathologic analysis of the results of conservation surgery and radiation therapy in stage I and II breast carcinoma. AB - One hundred eighty women with clinical Stage I or II operable breast carcinoma were treated by radiotherapy following local tumor excision at Yale-New Haven Hospital through 1980. With a median follow-up time of 6.9 years, the actuarial 5 year overall and disease-free survival rates were 82% and 78%, respectively. The 5-year actuarial breast-recurrence-free survival rate was 92%. Several clinical histopathologic features and treatment parameters were assessed for their significance as predictors of local breast failure or distant relapse. Cox lifetable regression analysis showed that patients with clinical Stage II carcinomas had significantly worse overall and relapse-free survival rates, but clinical stage alone had no effect on the rate of breast recurrence. Furthermore, a decrease in overall and disease-free survival was evident when necrosis was present in the tumor or when patients had an infiltrating lobular carcinoma. Breast recurrence-free survival was also influenced adversely by the presence of these two tumor features, especially when either tumor necrosis or infiltrating lobular carcinoma was found in conjunction with clinical Stage II lesions. Other histologic features such as grade, vascular invasion, perineural invasion, or the presence of an intraductal component of carcinoma did not affect outcome, nor did the treatment techniques employed appear to have a differential effect. PMID- 3019515 TI - Constitutional interstitial deletion of 11p11 and pericentric inversion of chromosome 9 in a patient with Wiedemann-Beckwith syndrome and hepatoblastoma. AB - A constitutional interstitial deletion on the short arm of chromosome #11 and an inversion of the heterochromatin of chromosome #9 were detected in a 1.5-year-old boy with Wiedemann-Beckwith syndrome (WBS) and hepatoblastoma. Of 37 malignant and nine benign neoplasms reported in approximately 250 cases with complete and incomplete forms of WBS, this is the fourth patient with hepatoblastoma. To date, 28 cases of WBS have been cytogenetically investigated with banding techniques. Constitutional anomalies have been found in only nine cases: Various anomalies resulting in a common triplication of the 11p15 region in six cases, reciprocal translocations t(11;22) and t(X;1) and an inversion of chromosome #2 in the three remaining cases. Triplication 11p15 was only present in one of four cases with a tumor. The breakpoints of the unique del(11)(p11.1p11.2) present in our case are proximal to those of del(11p13-11p14) and dup(11p15) observed thus far in both the aniridia-Wilms' tumor association and in WBS. Inversion of chromosome #9--one of the heterochromatin variants associated with elevated chromosomal instability, increased congenital abnormalities, and cancer proneness--may have been causally connected with a genetic imbalance resulting in the de novo deletion of 11p11. Therefore, we suggest that in these high-risk groups, C-banding studies should be performed together with high resolution chromosome analysis in order to also reveal the incidence and significance of C-band variants in individuals with such cancer prone syndromes. PMID- 3019516 TI - Effect of nickel sulfate on cellular proliferation and Epstein-Barr virus antigen expression in lymphoblastoid cell lines. AB - Nickel is found in high levels in the environment of the high-risk areas for nasopharyngeal carcinoma (NPC) of China. Epstein-Barr virus (EBV) is associated with NPC and the interaction of nickel and EBV may be a contributive cofactor to the development of NPC. The study of the in vitro effect of nickel sulfate on cell proliferation and EBV-antigen expression demonstrated that nickel increases cell proliferation of some EBV-positive lymphoblastoid cell lines and increases early antigen expression of Raji cells. Nickel exerted variable effects on viral capsid antigen (VCA): increasing VCA-positive cells in B95-8 cells while decreasing VCA in P3HR-1 cells. It is proposed that the uptake of nickel in NPC high-risk areas could be one of the factors responsible for cancer development in the nasopharynx in China. PMID- 3019517 TI - Augmentation of cytotoxicity of chemotherapy by human alpha-interferons in human non-small cell lung cancer xenografts. AB - Three human non-small lung cancer xenograft lines were used to study the activity of combinations of cytotoxic drugs with human alpha-interferons (IFNs). Statistically significant potentiation of cis-platinum (CDDP) and cyclophosphamide (CY) given weekly in a low dose was seen when human lymphoblastoid interferon (IFN-alpha nl) (2 X 10(5) mu/mouse/day) was administered simultaneously. The median tumor doubling times for CDDP in the three tumors (35, 22, and 29 days) increased to 52, 51, and 41 days when IFN alpha nl was added. A similar though less marked effect was seen with CY (median doubling time increased from 21.5, 19.5, and 27 days to 32, 27, and 35 days with the addition of IFN-alpha nl). IFN-alpha nl alone at this dosage was shown to have some cytotoxic activity. Similar potentiation of CDDP and ifosfamide was seen in two tumors when human recombinant alpha-2 interferon was added at a lower dose (2 X 10(4) mu/mouse/day). Median doubling times for CDDP increased from 17 and 14 days to 27 and 18.5 days with the addition of human recombinant alpha-2 interferon, whereas for ifosfamide they increased from 11.5 and 14 days to 15 and 16 days. Human recombinant alpha-2 interferon in this dose had no effect as a single agent. PMID- 3019518 TI - Mechanism of action of the novel anticancer agent 6-fluoro-2-(2'-fluoro-1,1' biphenyl-4-yl)-3-methyl-4-quinolinecarbo xylic acid sodium salt (NSC 368390): inhibition of de novo pyrimidine nucleotide biosynthesis. AB - Exposure of cultured clone A human colon tumor cells to 25 to 75 microM of NSC 368390 [6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinolinecarbox yli c acid sodium salt, DuP 785] for 48 to 72 h resulted in a 99.9% cell kill as determined by clonogenic assay. Cells exposed to NSC 368390 became depleted in intracellular pools of uridine 5'-triphosphate and cytidine 5'-triphosphate. Both uridine 5'-triphosphate and cytidine 5'-triphosphate were decreased to 50% of levels in control cells at 3 h and were undetectable at 15 h after addition of 25 microM of NSC 368390 to the cultures. Similar effects were observed in L1210 leukemia cells. Addition of 0.1 mM of uridine or cytidine restored intracellular pools of uridine 5'-triphosphate and cytidine 5'-triphosphate to control levels and rescued clone A cells from NSC 368390 cytotoxicity. Addition of uridine circumvented NSC 368390 cytotoxicity in L1210 cells, but addition of cytidine did not. This result is consistent with the fact that L1210 cells lack cytidine deaminase and thus cannot form uridine or its anabolites from cytidine. These results indicated that NSC 368390 inhibits a step in the de novo biosynthetic pathway leading to uridine 5'-monophosphate. Therefore, the effects of NSC 368390 on the six enzymes that comprise the de novo pathway leading to the formation of uridine 5'-monophosphate were examined. The results showed that NSC 368390 was a potent inhibitor of dihydroorotate dehydrogenase, the fourth enzyme in the pathway; thus, this study demonstrates that NSC 368390 exerts its tumoricidal effect by inhibiting a step in de novo pyrimidine biosynthesis resulting in the depletion of critical precursors for RNA and DNA synthesis. PMID- 3019519 TI - Demonstration of Epstein-Barr virus-specific DNA polymerase in chemically induced Raji cells and its antibody in serum from patients with nasopharyngeal carcinoma. AB - Epstein-Barr virus (EBV) has been found to be associated with nasopharyngeal carcinoma (NPC), and antibodies with high frequency and titer to EBV proteins have been found in sera from NPC patients. Raji cells, an EBV genome-carrying nonproducer cell line, treated with 12-O-tetradecanoylphorbol-13-acetate and n butyrate induced a unique EBV DNA polymerase which has properties similar to the EBV DNA polymerase induced by 12-O-tetradecanoylphorbol-13-acetate in P3HR-1 cells, an EBV producer cell line. The possible presence of antibodies to this EBV DNA polymerase in NPC patient serum was examined. The mean number of EBV DNA polymerase units neutralized was 380 +/- 168 units/ml serum (mean +/- SD) in 48 sera from patients with NPC, whereas that in the sera from 52 healthy donors was 62 +/- 56 units/ml (p less than 0.01). The EBV DNA polymerase antibody was found to be associated with the immunoglobulin G but not the immunoglobulin A fraction, and its titer was not correlated with the titers against EBV DNase or virus capsid antigen-immunoglobulin A. Whether the EBV DNA polymerase antibody is against the EBV DNA polymerase core protein or its stimulating protein is still being investigated. This study demonstrated the high frequency and high titer of antibody against EBV DNA polymerase in serum from NPC patients and suggested the potential of utilizing this antibody titer to complement other methods for the early diagnosis or prognosis of NPC. PMID- 3019520 TI - Detection of a polyoma virus-induced tumor-associated membrane antigen in mouse cells by the macrophage migration inhibition test. AB - Soluble membrane fractions derived from polyoma tumor cells trigger lymphocytes, derived from polyoma-immunized animals, but not from nonimmunized controls, to release the lymphokine, macrophage migration-inhibitory factor. The reaction can be blocked by sera from polyoma-bearing animals. Absorption of these sera with polyoma cells, but not with nonpolyoma cell lines, abrogates this activity. These findings suggest that there is a polyoma virus-induced membrane component that can induce polyoma-specific macrophage migration inhibition. PMID- 3019521 TI - Biosynthesis and expression of the disialoganglioside GD2, a relevant target antigen on small cell lung carcinoma for monoclonal antibody-mediated cytolysis. AB - Monoclonal antibodies (MAbs) 126 (immunoglobulin M) and 14.18 (immunoglobulin G3) react strongly with the cell surface of small cell carcinoma of the lungs (SCCL) and are unreactive with most normal tissues and other neoplasms with the notable exception of tumors derived from cells of neural crest origin. These MAbs react specifically with the oligosaccharide portion of the disialoganglioside GD2. Analysis of total gangliosides from cultured cell lines derived from SCCL indicates that GD2 is a predominant ganglioside. A comparison of the reactivities of MAbs against GD2 with those directed against gangliosides GM2 and GD3, each differing from GD2 by a single sugar residue, clearly indicates that GD2 is preferentially expressed by cultured cells derived from SCCL. Membranes isolated from these cells exhibit GD2 synthetase activity which specifically converts the precursor GD3 to GD2 in the presence of uridine diphosphate-N-acetyl galactosamine as the glycosyl donor. We present evidence that in SCCL, GD2 serves as a relevant target antigen for monoclonal antibody-mediated cytolysis. Specifically, we demonstrate that MAb 14.18 (immunoglobulin G3), can lyse small cell carcinoma of the lung targets by either complement- or antibody-dependent cellular cytotoxicity. PMID- 3019522 TI - Roles of cytomegalovirus and Chlamydia trachomatis in the induction of cervical neoplasia in the mouse. AB - Cytomegalovirus and Chlamydia trachomatis are prevalent sexually transmissible pathogens. They produce persistent infections of the cervix and have been associated with cervical neoplasia. Cytomegalovirus has also been shown to induce transformation of cells in culture. Because of the high prevalence of genital infections with these pathogens and evidence that they may have oncogenic effects on the cervix, cytomegalovirus (strain AD-169) and C. trachomatis (serovar LGV-2) were tested for oncogenicity in a mouse model in which induction of cervical neoplasia by repeated exposure to inactivated herpes simplex viruses has been demonstrated previously. Cotton tampons, saturated with UV-inactivated cytomegalovirus, C. trachomatis, or corresponding control fluids, were inserted into the vaginas of virgin C57 mice 3 times a week. Smears of vaginal aspirates for cytological examination were obtained every 5 weeks. After 75-90 weeks of exposure, the mice were sacrificed and serial sections of their reproductive tracts were examined. Cervical dysplasia was detected by histological examination in 51% and cervical carcinoma in 10% of mice exposed to cytomegalovirus. In control mice, in contrast, dysplasia developed in 3% and carcinoma in none. The progression from normal cervical epithelium to dysplasia to carcinoma observed with cytomegalovirus exposure was similar to that observed previously in this model after exposure of mice to herpes simplex virus types 1 and 2. The frequencies of cervical abnormalities in mice exposed to C. trachomatis or corresponding control fluid were low, and differences between the two groups were not statistically significant. These data indicate that strain AD-169 of cytomegalovirus is oncogenic for the mouse cervix and suggest that the LGV-2 serovar of C. trachomatis is not. PMID- 3019523 TI - Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. AB - The lack of highly exploitable biochemical differences between normal tissues and some tumors can theoretically be circumvented by a strategy utilizing gene insertion prophylactically to create tissue mosaicism for drug sensitivity, thereby ensuring that any tumor arising clonally will differ from part of the normal cell population. Elements of the strategy were tested with neoplastic BALB/c murine cell lines bearing the herpes thymidine kinase gene. Exposure to the herpes thymidine kinase-specific substrate 9-([2-hydroxy-1 (hydroxymethyl)ethoxy]methyl)guanine ablated the clonogenic potential of the cells in vitro, and administration of this drug to BALB/c mice bearing tumors produced by the cell lines uniformly induced complete regression of the tumors. The observed responses to therapy imply that the strategy may prove valuable when the genetic technology needed for its human implementation becomes available. PMID- 3019524 TI - Recurrent chromosome changes at 3p21 in Epstein-Barr virus-transformed cells derived from human prolymphocytic leukemia. AB - Cytogenetic studies of Epstein-Barr virus-transformed lymphoblastoid cells obtained from a patient with prolymphocytic leukemia revealed the cells to contain recurrent chromosome aberrations involving band 3p21. The in vivo leukemic cells from which the cell lines originated contained a der(3)t(3;17?)(p21;q11?) and a der(13)t(13;3)(q34;p21), as well as numerical (-Y, -8, and -17) and other structural [8p- and der(8)t(8;?)(q13?;?)] changes. The cells in established culture showed additional chromosome aberrations involving band 3p21 of the previously normal and abnormal chromosomes 3. After 200 days of culture, the cells contained a new translocation, i.e., t(3;14)(p21;q32). The cells showed further chromosomal breakage and reunion at band 3p21 at later passages. The affected chromosomal band (3p21) is close to one of the constitutive fragile sites, i.e., 3p14.2. Northern blot analysis of the messenger RNA of the cultured cells did not show an increased or altered expression of the c-raf-1 protooncogene (located at 3p25) when compared with the mRNA of Epstein Barr virus-transformed lymphoblastoid cell lines from normal subjects with a normal karyotype. Also, the established cells did not show DNA rearrangements or RNA alterations of the c-myc gene. PMID- 3019526 TI - Significance of 1,25-dihydroxyvitamin D3 receptor in primary breast cancers. AB - Specific high affinity receptors for 1,25-dihydroxyvitamin D3 are present in several human breast cancer cell lines, and this hormone can regulate the replication of these cells. These receptors are also present in primary breast carcinomas. The present study has resulted from the follow-up for up to 68 mo of 263 women, who had had 1,25-dihydroxyvitamin D receptor (1,25-DR) levels measured in their primary tumors. Survival data on 191 women were correlated with the levels of 1,25-DR and other steroid hormone receptors, menopausal status, and age by life table analysis. Survival was not affected by 1,25-DR level in either absolute terms or relative levels. However, the late development of lymph node metastases in eight of 47 individuals was correlated with the 1,25-DR level (P = 0.05). There was no correlation between 1,25-DR or other hormone receptor levels and the development of hypercalcemia or bone metastases in the small number of individuals so affected. As we had observed previously, there was no correlation between the level of 1,25-DR and that of the other steroid hormones. These data show that the presence of 1,25-DR in primary breast cancers is independent of other prognostic indicators and, inasmuch as it correlated with late lymph node metastasis, may be an adverse prognostic indicator. PMID- 3019525 TI - Induction of an unusual type of shared phosphorylation in human and avian cells by tumor-promoting phorbol esters or transformation. AB - Alterations in patterns of protein phosphorylation after exposure to phorbol esters were compared in chicken embryo fibroblasts and KD cells (a human fibroblast line) by two-dimensional gel electrophoresis. A substantial increase in phosphorylation was observed of a major, markedly acidic protein of pI = 4.5 in two-dimensional gels of each cell type. However, the apparent molecular weights of these phosphoproteins differed substantially in the two species with a molecular weight of 67,000 in chicken fibroblasts and one of 80,000 in human KD cells. Both phosphoproteins, termed 67K and 80K respectively, contained phosphoserine and a small amount of phosphothreonine, but no detectable level of phosphotyrosine. Tryptic and chymotryptic phosphopeptide maps of 67K were nearly identical to those of 80K. These results indicate that they are related molecules, even though considerably different in apparent molecular weight, and that the induction of phosphorylation of these closely related, major, acidic phosphoproteins by phorbol esters is conserved from avian to human cells. In chicken embryo fibroblasts infected by a temperature-sensitive mutant of Rous sarcoma virus, phosphorylation of 67K was found to be elevated at 36 degrees C (transformed phenotype) compared to 41.5 degrees C (normal). Although the function of these closely related 67K and 80K phosphoproteins is unknown, the elevated level of phosphorylation could be involved in some aspects of transformation, and the increase is mimicked by treatment with phorbol esters. PMID- 3019527 TI - Effects of differing purified cellulose, pectin, and hemicellulose fiber diets on fecal enzymes in 1,2-dimethylhydrazine-induced rat colon carcinogenesis. AB - The fecal microflora enzymes, beta-glucuronidase and beta-glucosidase, as well as fecal bacterial counts, were examined during colon carcinogenesis in rats administered parenteral 1,2-dimethylhydrazine and fed nutritionally equivalent diets free of fiber or containing one of three single sources of dietary fiber (cellulose, hemicellulose, and pectin). Whereas pectin-fed animals had increased fecal beta-glucuronidase activities, those fed cellulose and hemicellulose, two fibers protective in dimethylhydrazine colon neoplasia, had decreased activities. Although fecal bacterial counts were not significantly changed, similar differential changes in fecal beta-glucosidase activity were noted: cellulose but not pectin or hemicellulose feeding was associated with reduced activity. Although cellulose fiber may cause differing physiological effects resulting in a reduction in colonic neoplasia development in this experimental animal model, decreased bacterial metabolic enzyme activation of carcinogens or cocarcinogens may lead to diminished exposure of colonic cells to exogenous or endogenous mutagens. PMID- 3019528 TI - Role of reactive oxygen metabolites in crocidolite asbestos toxicity to mouse macrophages. AB - Crocidolite asbestos is toxic to macrophages in vitro. We hypothesize that this toxicity is mediated by the generation of reactive oxygen metabolites. Elicited mouse peritoneal macrophages were found to release reactive oxygen metabolites upon incubation with crocidolite asbestos in vitro. Crocidolite toxicity to both primary cultures of mouse peritoneal macrophages and P388D1 cells, a mouse macrophage-like cell line, could be prevented by a hypoxic environment or by addition of the reactive oxygen metabolite scavengers, superoxide dismutase and catalase. In addition, if crocidolite fibers were presoaked with the iron chelator deferoxamine, no macrophage death occurred. In an attempt to mimic crocidolite-induced cytotoxicity, P388D1 cells or primary elicited macrophages were exposed to the nontoxic mineral particle titanium dioxide in the presence and absence of ferric chloride. Titanium dioxide was only lethal when ferric chloride was added. This toxicity was prevented by superoxide dismutase, catalase, or deferoxamine. These results suggest that crocidolite-induced injury to macrophages depends on the formation of reactive oxygen metabolites. Iron present in crocidolite fibers may catalyze the production of hydroxyl radical from superoxide anion and hydrogen peroxide generated during phagocytosis. These highly reactive hydroxyl radicals are postulated to mediate lethal cell injury. PMID- 3019529 TI - Inhibition of phorbol ester stimulated interleukin 2 production by copper(II) complexes. AB - Superoxide dismutase mimetic copper(II) complexes, such as copper(II)(3,5 diisopropylsalicylate)2 (CuDIPS), inhibit phorbol ester stimulated tumor promotion in mouse skin. Therefore, CuDIPS was tested as a potential inhibitor of another effect of phorbol esters, induction of interleukin 2 (IL2) synthesis, in the mouse thymoma cell line EL4. CuDIPS inhibited phorbol ester induced IL2 production in a concentration dependent manner with a 50% inhibitory concentration of about 10 microM. However, the ligand 3,5-diisopropylsalicylic acid also inhibited the induction of IL2 by phorbol esters (50% inhibitory concentration, 15 microM). Since the superoxide dismutase mimetic activity of CuDIPS is not stable in the presence of ethylenediaminetetraacetic acid, the effects of CuDIPS could be due to the free ligand and not to the intact metallocomplex. Consequently, a series of extremely stable copper(II) macrocyclic compounds was synthesized, and the reduction potential, superoxide dismutase mimetic activity, and ability to inhibit phorbol ester induced IL2 production were determined for each. Of the copper(II) macrocyclic complexes studied, only the most potent superoxide dismutase mimetic compound was found to inhibit phorbol ester induced IL2 production. Copper(II) complexes had to be added no later than 4 following phorbol ester administration to be effective inhibitors of the IL response, suggesting that these compounds act subsequent to the binding of phorbol esters but prior to the transcription of IL2 messenger RNA. Adherence of EL4 cells to substrate in response to phorbol esters was unaffected by copper(II) compounds. In summary, copper(II) compounds with appropriate reduction potentials can act within a defined time period to inhibit some, but not all, of the effects of phorbol esters on EL4 cells. PMID- 3019530 TI - Induction of anchorage-independent growth of JB6 mouse epidermal cells by 1 alpha,25-dihydroxyvitamin D3. AB - 1 alpha,25-Dihydroxyvitamin D3 [1 alpha,25(OH)2D3], a hormonally active form of vitamin D3, was shown previously to enhance chemically induced transformation of BALB 3T3 cells and Syrian hamster embryo cells. This report demonstrates that 1 alpha,25(OH)2D3, like phorbol ester tumor promoters, induces anchorage independent growth of mouse JB6 epidermal cells. When plated on agar plates containing 1 alpha,25(OH)2D3 at concentrations higher than 0.05 ng/ml or 0.12 nM, JB6 cells formed colonies on the surface of agar plates dose dependently. This anchorage-independent growth was further confirmed by stimulation of DNA synthesis after liquefying the agar layer with NaI. A phorbol-ester resistant variant of JB6 cells was also resistant to 1 alpha,25(OH)2D3 in terms of induction of anchorage independency. Induction of anchorage-independent growth was specific for 1 alpha,25(OH)2D3: other derivatives of vitamin D3 also induced colony formation on agar plates but only at a higher concentration (500 ng/ml) and to much less extent than did 1 alpha,25(OH)2D3. JB6 cells were found to contain a receptor specific for 1 alpha,25(OH)2D3 with a Kd of 55.7 pM and Nmax of 102.5 fmol/mg protein, suggesting a receptor-mediated mechanism of the induction. The clone that was resistant to 1 alpha,25(OH)2D3 also contained the receptor. DNA-cellulose chromatography showed that a 1 alpha,25(OH)2D3-receptor complex interacted with DNA. In contrast to 1 alpha,25(OH)2D3, retinoic acid did not induce anchorage-independent growth of JB6 cells, but it inhibited the induction by 1 alpha,25(OH)2D3 when applied with it. PMID- 3019531 TI - Predominant role for DNA damage in etoposide-induced cytotoxicity and cell cycle perturbation in human SV40-transformed fibroblasts. AB - The cytotoxic and cell kinetic effects of the epipodophyllotoxin 4,6 demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyr anoside) (VP-16) in cultured mammalian cells are thought to relate to the induction of DNA damage, specifically DNA strand interruptions. In an effort to explore this relationship in human cells we have identified a VP-16-hypersensitive human cell system, namely an SV40-transformed fibroblast line (AT5BIVA) originally derived from an ataxia telangiectasia (AT) patient. Evidence is presented that enhanced VP-16 sensitivity may be a consistent in vitro feature at AT derived cells. However, the intrinsic sensitivity (DNA strand breaks per lethal hit quantitated by nucleoid sedimentation) was the same for AT5BIVA and a corresponding normal control, indicating that the AT cell line accumulated more drug-induced DNA damage during short-term VP-16 exposures. It is suggested that AT cells may have abnormal topoisomerase II activity. The cell cycle responses of normal and AT cells to VP-16 exposure were complex, with the generation of parasynchronous S phase populations and the accumulation of cells in G2. Differences in cell killing or DNA strand breakage between normal and AT cells could only be correlated with the magnitude and kinetics of the G2 retention phenomenon. In short, there are several similarities in the action of ionizing radiation and VP 16. We suggest that the sensitivity of cellular DNA to VP-16-induced DNA damage and the kinetics of the G2 delay may be useful parameters for predicting the survival probability of drug-treated human tumor populations. PMID- 3019532 TI - Enhanced release of hydrogen peroxide and metabolites of arachidonic acid by macrophages from SENCAR mice following stimulation with phorbol esters. AB - Chronic inflammation has long been associated with carcinogenesis. Phorbol esters which are potent promoters of tumors in mouse skin are also potent inflammatory agents in skin and cause inflammatory cells to release large quantities of reactive oxygen intermediates and oxidized lipid products. SENCAR mice have been bred for their sensitivity to the promotion of tumors by phorbol esters and C57BL/6 mice have been shown to be resistant. We quantified the release of H2O2 and metabolites of arachidonic acid by macrophages obtained from SENCAR and C57BL/6 mice, following exposure to phorbol esters and other stimulants. The basal level for secretion of H2O2 in resident peritoneal macrophages was negligible in cells from both strains. Conversely, inflammatory macrophages from SENCAR mice, elicited by the injection of sterile casein, secreted 4 times more H2O2 than the corresponding cells from C57BL/6 mice. Furthermore, cells from SENCAR mice required less than one-third the amount of phorbol ester to obtain 50% of the maximal response than that required by cells from C57BL/6 mice. This difference was less when zymosan was used as a stimulant. Both resident and inflammatory macrophages from SENCAR mice released more metabolites of arachidonic acid than cells from C57BL/6 mice when exposed to phorbol esters, but macrophages from C57BL/6 mice released more metabolites when stimulated with zymosan. Few differences in the pattern of released metabolites were noted between the strains of mice. There were large differences in the relative amounts of individual metabolites released when different stimulants were used. The enhanced response to phorbol esters of chronic inflammatory cells from SENCAR mice correlates with the enhanced sensitivity to the promotion of tumors by phorbol esters in these animals. PMID- 3019533 TI - Modulation by normal serum factors of Kirsten murine sarcoma virus-induced transformation in adult rat cells infected in early passage. AB - Adult rat adrenal cells, infected with Kirsten murine sarcoma virus in early passage, transform consistently in 10% fetal bovine serum (FBS)-supplemented medium. Substitution of 3% horse serum (HS) for FBS reverses early foci and delays transformation. The influence of the serum on DNA synthesis, anchorage dependence, tumorigenicity, and subcellular Mr 21,000 transforming protein (p21) distribution was followed from infection in passage 1 to complete transformation. In FBS, increased expression of p21 preceded other evidence of transformation. Subsequently, p21-positive cells transformed morphologically, but initially their growth parallelled that of coexisting untransformed cells. Foci formed at passages 5 to 10, and the cells became anchorage independent and tumorigenic at passages 10 to 20. As transformation in FBS progressed, p21 relocated from a diffuse distribution to sites of retraction from substrata and then to ruffles and lamellae on cellular processes. Early in transformation, HS-medium reduced proliferation of morphologically normal and morphologically transformed p21 positive cells. This effect was counteracted by the addition of FBS or the Mr 50,000 to 100,000 fraction of FBS. Fully transformed, tumorigenic cells grew rapidly in both sera but, if transferred from FBS to HS, became more anchorage and density dependent, and p21 relocated from cell processes to the cell bodies. In immortal lines, the substitution of HS for FBS accelerated rather than delayed the progression of Kirsten murine sarcoma virus-induced transformation. These results show that Kirsten murine sarcoma virus-induced transformation of adult presenescent cells is controlled by physiological factors to which immortal cells appear refractory. The changes in subcellular distribution of p21 during transformation parallel the expression of some, but not other, transformation parameters and suggest a possible association of p21 with increased membrane activity. PMID- 3019535 TI - Clonal origin of human hepatoma determined by integration of hepatitis B virus DNA. AB - The hepatitis B virus genome is integrated in cellular DNA of human hepatocellular carcinoma from hepatitis B surface antigen-positive patients. Using this phenomenon, we determined the clonal origin of hepatocellular carcinoma from the integration mode of hepatitis B virus DNA. The molecular size and the number of restriction fragments of integrated hepatitis B virus DNA in several parts of tumors in the same liver and in metastatic tumors were compared by Southern blot analysis. Of 14 cases of hepatoma, 13 cases were monoclonal; in one case, a different clone of hepatoma was found in one part of the tumor. In three of 13 cases of monoclonal hepatoma, metastatic tumors in lymph nodes and the lung were also examined and found to be the same clone as the liver tumors. These results indicate that hepatocellular carcinomas were usually generated from a single tumor cell even though tumor cells spread in the liver and invaded other organs for a long time. Development of different clones of tumor was apparently unusual but was observed in one case of hepatocellular carcinoma. PMID- 3019534 TI - Growth-dependent expression of human Mr 53,000 tumor antigen messenger RNA in normal and neoplastic cells. AB - We have investigated the expression of Mr 53,000 protein (p53) in total RNA isolated from human peripheral blood mononuclear cells stimulated by phytohemagglutinin, in serum-stimulated human diploid fibroblasts, and in normal and tumor cells of human epithelial colon tissue. We have found that the expression of p53 messenger RNA is growth regulated in human cells following kinetics similar to that previously shown in mouse 3T3 cells, and is increased in the large majority of colon adenocarcinomas in comparison to adjacent normal mucosa and adenoma. This increased expression of p53 is accompanied by a nearly proportional increase in the expression of histone H3. As the expression of histone H3 is restricted to the S phase of the cell cycle and therefore measures the growth fraction of a given population, we suggest that the increased expression of p53 observed in the large majority of colon tumors simply reflects the increased number of cycling cells frequently found in a neoplastic tissue. At variance with these findings a true overexpression of p53 was detected in one SV40-transformed human fibroblasts cell line. PMID- 3019537 TI - Morphological, biological, biochemical, and karyotypic characteristics of human pancreatic ductal adenocarcinoma Capan-2 in tissue culture and the nude mouse. AB - Human pancreatic ductal adenocarcinoma Capan-2, derived from a 56-yr-old male Caucasian, has been studied in both tissue culture and the nude mouse. In tissue culture, tumor cells showed epithelial-like features, whereas in the nude mouse, the tumor grew as a well-differentiated adenocarcinoma, resembling histopathologically the original neoplasm. Ultrastructurally, the neoplastic cells showed characteristics of ductal epithelium. The allozyme phenotypic profile of Tumor Capan-2 was determined in eight genetically determined loci, and chromosome studies showed a hypotetraploid pattern with a number of morphological and numerical changes. Carcinoembryonic antigen was produced in trace amounts, and lactate dehydrogenase was represented only by Isoenzyme 5, regardless of environmental conditions. The characteristics of Capan-2 tumor make it a valuable addition to the small number of available pancreatic tumor lines in studies aiming at clarifying certain aspects of the biology of this type of malignancy. PMID- 3019536 TI - Reductive activation of diaziquone and possible involvement of free radicals and the hydroquinone dianion. AB - To analyze the mode of action of diaziquone [AZQ] on DNA, we examined the activity of two AZQ analogues and N,N',N"-triethylenethiophosphoramide on three forms [supercoiled (Form I), open circular (Form II), and linear (Form III)] of PM-2 DNA. The AZQ analogues contained chlorine atoms which substituted either the carbethoxyamino groups or the aziridine groups of the parent compound. N,N',N" triethylenethiophosphoramide is a triaziridine compound containing pentavalent phosphorus instead of a quinone group. We found that only when reduced with sodium borohydride did AZQ change the topology of the three forms of PM-2 DNA by introducing mainly single strand breaks. The AZQ analogue containing only aziridines (RQ2) was active in both its oxidized and its reduced forms, while the analogue containing only the carbethoxyamino groups (RQ14) or N,N',N" triethylenethiophosphoramide were not active in either form. Under similar experimental conditions, Adriamycin alone altered the electrophoretic mobility of PM-2 DNA, while borohydride reduced Adriamycin did not. By using electron spin resonance spectroscopy, we showed that dihydrodiaziquone (AZQH2) oxidizes to the semiquinone in the presence of oxygen. Although AZQH2 was active against DNA, it was not active against cellular DNA synthesis as measured by [3H]thymidine incorporation into exponentially growing HEp-2 cells. However, AZQ alone prevented [3H]thymidine incorporation into HEp-2 cells. We found that HEp-2 cells have the ability to reduce AZQ to its free radical anion, but AZQH2 does not autoxidize to the free radical in the presence of cells. The reductive ability of HEp-2 cells may be responsible in part for preventing the oxidation of AZQH2 to the free radical. We found that under our conditions (1-h incubations) the aziridines are essential for the activity of aziridinyl quinones against PM-2 DNA and that in the case of AZQ the hydroquinone is also required. PMID- 3019538 TI - Effects of the bifunctional antitumor intercalator ditercalinium on DNA in mouse leukemia L1210 cells and DNA topoisomerase II. AB - Ditercalinium, a 7H-pyridocarbazole dimer (bisintercalator) belongs to a new class of antineoplastic intercalating agents. To investigate its mechanism of cytotoxicity, the effects of ditercalinium on DNA were assessed using normal (L1210) and drug-resistant (L1210/PyDi1) mouse leukemia cells. Alkaline elution assays demonstrated that ditercalinium produced no DNA strand breaks, DNA-protein cross-links, or DNA-DNA cross-links, eliminating these effects as cytotoxic lesions. This result sets ditercalinium apart from other intercalating agents with respect to its interaction with DNA. Nucleoids (histone-depleted chromatin) from ditercalinium-treated L1210 cells were considerably more compact than those from untreated cells, as determined by sedimentation in neutral sucrose gradients. In contrast, nucleoids from ditercalinium-treated L1210/PyDi1 (resistant) cells were similar in compactness to those from control cells. Thus, ditercalinium altered chromatin structure in vivo. The effect of the bisintercalator on purified DNA topoisomerase II, an intracellular target of monointercalators, was measured in vitro. Ditercalinium (5 X 10(-7) M) completely inhibited both the formation of covalent complexes between this enzyme and simian virus 40 DNA and the enzyme-induced DNA cleavage. In addition, ditercalinium induced DNA catenation in the presence of topoisomerase II and adenosine triphosphate. Thus, the cytotoxicity of ditercalinium may derive from a mechanism that, although involving topoisomerase II, is manifested by condensation of DNA rather than by the induction of protein-associated DNA strand breaks. PMID- 3019539 TI - Induction of anchorage-independent growth by epidermal growth factor and altered sensitivity to type beta transforming growth factor in partially transformed rat kidney cells. AB - A partially transformed cell line (NRK-PT14) was isolated from normal rat kidney (NRK) cells. Like NRK cells, NRK-PT14 cells required epidermal growth factor for anchorage-independent growth, but lost the additional requirement for exogenous type beta transforming growth factor (TGF-beta). Compared to NRK cells, NRK-PT14 cells did not secrete elevated levels of TGF-beta, but exhibited an altered response to this growth factor. Monolayer growth of NRK cells in a serum-free medium was inhibited by TGF-beta, whereas growth of NRK-PT14 cells was stimulated by TGF-beta. In addition, TGF-beta stimulated epidermal growth factor binding to high affinity sites in NRK cells, but decreased epidermal growth factor binding to NRK-PT14 cells during growth of the cells in serum-free medium. These qualitative changes in the response to TGF-beta may be representative of an intermediate stage in the spontaneous transformation of NRK cells. PMID- 3019540 TI - A nonviral, virus strain-specific antigen expressed on rat cells transformed by avian sarcoma virus. AB - Five of six rat sarcomas, induced by the Schmidt-Ruppin (SR) strain of avian tumor virus, expressed a Mr 60,000 tumor cell surface antigen (TSA), immunoprecipitable from non-ionic detergent extracts. Expression of the antigen was exclusive to rat cells transformed by the SR virus strain. Moreover, expression of TSA appeared restricted by cell type. The five TSA-positive SR transformed rat cell lines tested were apparently of fibroblastic origin, but cultured rat cerebral endothelial cells (RCE-T1), transformed by SR virus, showed no expression of TSA. However, the antigen emerged on cultured tumors obtained after histoincompatible transplantation of these cells into newborn rats of another strain (tumor digest cells). Investigation of TSA for immunological relationship to viral structural antigens and the src gene product indicated that the TSA is distinct from any of these and more probably derives from a virus directed alteration in a host molecule. PMID- 3019541 TI - Expression of melanoma-associated antigens by normal and neurofibroma Schwann cells. AB - The cell surface antigen distribution on traumatic neuroma Schwann cells and neurofibroma Schwann-like cells was characterized using monoclonal antibodies that define melanoma-associated antigens. Immunofluorescence staining of cultured cells, immunoprecipitation of radioiodinated antigens from cells placed in short term cultures, and immunoperoxidase staining of frozen tissue sections revealed most of the melanoma-associated antigens tested on traumatic neuroma and neurofibroma Schwann cells and on fetal and adult femoral nerve. The cross reactivity of the antibodies with neural cells may reflect the common neural crest embryological origin of Schwann cells and melanocytes. Cell sorter analysis of neurofibroma cells using a monoclonal antibody directed against the melanoma nerve growth factor receptor resulted in cell cultures highly enriched for Schwann-like cells which may bear the genetic defect responsible for neurofibromatosis. The antigen detected by this monoclonal antibody is the neurofibroma nerve growth factor receptor and the antibody was a potent inhibitor of nerve growth factor binding to neurofibroma cells. PMID- 3019543 TI - Urinary transforming growth factors in neoplasia: separation of 125I-labeled transforming growth factor-alpha from epidermal growth factor in human urine. AB - Purified human epidermal growth factor (hEGF) from urine promotes anchorage independent cell growth in soft agar medium. This growth is enhanced by transforming growth factor-beta (TGF-beta), and is specifically inhibited by hEGF antiserum. Transforming growth factors of the alpha type (TGF-alpha), potentially present in normal human urine or urine from tumor-bearing patients, also promote anchorage-independent cell growth and compete with EGF for membrane receptor binding. Consequently, TGF-alpha cannot be distinguished from urinary hEGF by these two functional assays. Therefore, a technique for separation of TGF-alpha and related peptides from urinary EGF based on biochemical characteristics would be useful. Radioiodination of characterized growth factors [mouse EGF (mEGF), hEGF, and rat TGF-alpha (rTGF-alpha)], which were then separately added to human urine, was used to evaluate a resolution scheme that separates TGF-alpha from the high level of background hEGF present in human urine. Commercially available methyl bonded microparticulate silica efficiently adsorbed the 125I-labeled mEGF, 125I-labeled hEGF, and 125I-labeled rTGF-alpha that were added to 24-h human urine samples. Fractional elution with acetonitrile (MeCN) of the adsorbed silica released approximately 70 to 80% of the 125I-labeled mEGF and 125I-labeled hEGF between 25 and 30% MeCN, and over 80% of the 125I-labeled rTGF-alpha between 15 and 25% MeCN, with retention after dialysis of less than 0.2 and 1.7% of the original urinary protein, respectively. Consequently, a single-step enrichment of about 400-fold for mEGF and hEGF, and 50-fold for rTGF-alpha were achieved rapidly. Subsequent Bio-Gel P-10 chromatography indicated that 125I-labeled mEGF and 125I-labeled hEGF eluted later than would be predicted on the basis of their reported molecular weight of approximately 6000, whereas 125I-labeled rTGF-alpha eluted from Bio-Gel P-10 at an approximate molecular weight of 8000 to 9000. 125I labeled rTGF-alpha bound to carboxymethyl cellulose and eluted at a less acidic pH than did hEGF. On reverse phase high performance liquid chromatography using a linear gradient of 18 to 35% MeCN over 120 min, 125I-labeled rTGF-alpha was comparatively hydrophilic, eluting at 22% MeCN in contrast to the more hydrophobic 125I-labeled hEGF, which eluted at 27% MeCN. These observed biochemical differences between hEGF and TGF-alpha provide a rationale for resolution of these and perhaps other related putative transforming growth factors from bulk urine of tumor-bearing patients. PMID- 3019544 TI - Re: Growth of cell lines and clinical specimens of human non-small cell lung cancer in a serum-free defined medium. PMID- 3019542 TI - Establishment and characterization of a cell line from a human gliosarcoma. AB - A human gliosarcoma culture was characterized from the time of inception to the time of establishment of the cell line (SF-539 BT). Immunohistochemical analysis of the original tumor showed 2 distinct regions of cells. The gliomatous regions were identified by immunostains for glial fibrillary acidic protein and the sarcomatous regions by immunostains for laminin, collagen type IV, procollagen type III, and fibronectin. In early-passage culture, both types of cells maintained their characteristic immunohistochemical profiles; however, after the fourth subcultivation in monolayer culture, no cells expressing glial fibrillary acidic protein could be identified. All cells had become morphologically uniform and expressed laminin, collagen type IV, procollagen type III, and fibronectin only. The immunostaining profile of clones grown in soft agar was similar to that of cells in monolayer culture. At establishment, SF-539 BT has a saturation density of 1.3 X 10(6) cells/25 sq cm, a doubling time of 32 h, and a plating efficiency of 22% in monolayer culture. The tumor cell line is resistant to 1,3 bis(2-chloroethyl)-1-nitrosourea, has an abnormal karyotype, grows anchorage independently, and forms a tumor that most closely resembles a spindle cell sarcoma in athymic mice. Its ultrastructure in monolayer culture consists of large cells with an expanded rough endoplasmic reticulum and abundant multivesicular bodies; in athymic mice, extracellular collagen fiber formation is prominent. DEAE-cellulose chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis of cultures labeled with [3H] proline demonstrated interstitial collagen formation. We conclude that the cell line at establishment is a collagen-producing spindle cell sarcoma that resembles the sarcomatous regions of the original mixed tumor. Further cell separation and characterization studies are needed to determine the pathogenesis of mixed tumors such as gliosarcoma. PMID- 3019545 TI - Automated fluorescent analysis for drug-induced cytotoxicity assays. AB - The human tumor clonogenic assay has been reported to predict for sensitivity of human tumors to a variety of drugs. However, this assay requires large numbers of viable cells, is time-consuming, and takes at least 2 weeks before results are available. To circumvent these problems, Weisenthal developed a microscope-based dye exclusion assay. Because this method is also time-consuming and subject to observer error, we have developed an automated method of quantitating drug cytotoxicity using a flow cytometric cell sorter (FCM). After incubation of drug exposed tumor cells, acetaldehyde-fixed duck red blood cells (DRBC) are added. Dead tumor cells and the fixed DRBC are stained by the fluorescent dye propidium iodide, which penetrates dead cell membranes. A two-parameter analysis (cell size as measured by narrow angle light scatter vs propidium iodide fluorescence) enables determination of the live tumor cell:DRBC ratio. There was a strong correlation between the FCM method and manual counting (r = 0.958 for cell lines, r = 0.831 for fresh leukemic cells, P less than 0.0001 in both cases). We conclude that the automatized FCM method gives compatible results to the manual dye exclusion assay and increases efficiency. PMID- 3019547 TI - Beta adrenoceptor density and adenylate cyclase response in right atrial and left ventricular myocardium of patients with mitral valve disease. AB - Beta adrenoceptor density, measured by radioligand binding techniques, is reportedly much higher in atrial than in ventricular myocardium of patients with mitral valve disease. In the present study adenylate cyclase activity, both basal and in response to beta adrenergic agonists and sodium fluoride, in biopsy preparations from these same patients was significantly lower in atrial than in ventricular tissue when stimulated with either isoproterenol, noradrenaline, isoproterenol combined with terbutaline, or sodium fluoride. Terbutaline stimulated and basal adenylate cyclase activity was not significantly different in the two cardiac regions. The ratios of receptor density to isoproterenol stimulated and to sodium fluoride stimulated adenylate cyclase activity were 4-5 times higher in atrial than in ventricular biopsy specimens. Thus ventricular beta receptors, although present in comparatively low concentrations, are coupled to considerably more catalytic moieties of the receptor-adenylate cyclase complex than their atrial counterparts. The reason for this is probably a relative lack of coupling proteins (N components) in atrial tissue. A weak positive correlation between receptor density and isoproterenol stimulated adenylate cyclase activity was found in atrial but not in ventricular tissue. This may indicate individual variation in receptor-adenylate cyclase coupling. Furthermore, no correlation was found between atrial and ventricular values for any variable. One reason for this may be the different haemodynamic stresses in the two cardiac chambers. PMID- 3019546 TI - Cisplatin and etoposide before definitive radiation therapy for inoperable squamous carcinoma, adenocarcinoma, and large cell carcinoma of the lung: a phase I-II study of the Radiation Therapy Oncology Group. AB - A trial of "neoadjuvant" cisplatin-etoposide and radiation therapy was conducted by the Radiation Therapy Oncology Group for non-small cell carcinoma of the lung limited to the thorax. Thirty evaluable patients were studied: two achieved complete response and four achieved partial response after chemotherapy. All patients underwent radiation therapy as planned, with no unusual acute reactions. Sixteen patients had local failure, and 13 had distant metastasis. Twenty-seven patients are dead, two are alive with cancer, and one is clinically free of cancer at 145 weeks. Five of six patients who survived greater than or equal to 2 years after treatment had adenocarcinomas. There was no unexpected late toxicity. This combination of chemotherapy and radiotherapy is unlikely to improve results in the treatment of inoperable non-small cell carcinoma of the lung over those with radiotherapy alone. PMID- 3019548 TI - Protective effects of the calmodulin antagonist bepridil on ischaemia induced in the rat myocardium. AB - The effects of a block of the slow calcium channel by bepridil and its antagonising effects on calmodulin were determined in the ischaemic rat myocardium. When isolated rat hearts were treated with a cardioplegic solution containing bepridil or verapamil there was a significant diminution in ischaemic damage in the myocardium. Bepridil was more effective than verapamil in protecting against this ischaemic damage. Bepridil effectively inhibited the activation of calmodulin dependent enzymes, such as calcium calmodulin dependent cyclic nucleotide phosphodiesterase and myosin light chain kinase. The inhibitory effect of verapamil on these enzymes was weaker than that of bepridil. Since bepridil protects the myocardium and minimises ischaemia induced damage these events might be related to a calmodulin antagonistic action as well as a calcium channel blocking action. PMID- 3019549 TI - [The effect of triiodothyronine on the beta adrenergic receptor system in human lymphocytes]. PMID- 3019550 TI - [Receptors and second messengers of hormone effects from a slightly different aspect]. PMID- 3019552 TI - Major transcript of the frameshifted coxII gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA. AB - The mitochondrial cytochrome oxidase (cox) subunit II gene from trypanosomes contains a frameshift at amino acid 170. This gene is highly conserved in different trypanosome species, suggesting that it is functional. Sequence determination of coxII transcripts of T. brucei and C. fasciculata reveals four extra, reading frame-restoring nucleotides at the frameshift position that are not encoded in the DNA. Southern blot analysis of DNA of both trypanosome species failed to show the existence of a second version of the coxII gene. We conclude, therefore, that the extra nucleotides are added during or after transcription of the frameshift gene by an RNA-editing process. PMID- 3019551 TI - Ontogenesis of corticotropes and lactotropes in situ in the pituitary gland of the hamster. An immunohistochemical study. AB - The development of corticotropes and lactotropes was investigated in the golden Syrian hamster using an anti-porcine ACTH antiserum and a homologous anti-hamster PRL antiserum. Oval corticotropes were first visible in the ventral region of the pars distalis at 13 days of gestation. By the end of gestation, corticotropes were found throughout the pars distalis and in the pars intermedia. Corticotropes in the pars distalis of postnatal hamsters were either round or irregularly shaped, often appearing in clusters. Throughout development, corticotropes often appeared to be surrounding other cells. Scarce, very small lactotropes were first observed in the pars distalis of hamsters on the first postnatal day. The number of these cells, which were either round or polyhedral, increased dramatically between 4 and 20 days of postnatal life. These observations indicate that the sequence of appearance of corticotropes and lactotropes in the hamster is similar to that in other species and that lactotropes are confined to the pars distalis of postnatal hamsters. PMID- 3019553 TI - Resistance to antimycin A in yeast by amplification of ADH4 on a linear, 42 kb palindromic plasmid. AB - A yeast strain lacking a functional copy of ADH1 has been isolated that is resistant to antimycin A because of the presence of multiple copies of a nuclear gene, ADH4. The amplified copies of ADH4 exist on linear molecules 42 kb in length, which can be separated from chromosomal DNA by orthogonal-field alternation gel electrophoresis. These amplified molecules are palindromes that reanneal rapidly after denaturation to form linear, snap-back molecules 21 kb in length. The amplified ADH4 sequences are bounded by telomere-homologous sequences. The chromosomal copy of ADH4 is the most distal marker on the left arm of chromosome VII, and the amplified ADH4-containing molecules appear to contain two copies of the region extending from ADH4 to the telomere. PMID- 3019554 TI - A cis-acting element within the 5' leader of a cytomegalovirus beta transcript determines kinetic class. AB - During cytomegalovirus (CMV) infection, gene expression is regulated by transcriptional and posttranscriptional events. Although recent studies have established that posttranscriptional controls are important determinants of gene expression in several eukaryotic systems, the precise signals and mechanisms have not been clearly identified. We present evidence for a cis-acting signal, contained within the 5' leader region of a CMV beta (or early) gene, that acts posttranscriptionally in the regulation of gene expression. Addition of this signal to an alpha (or immediate early) gene construct converted expression of the indicator protein to the beta class, even though the gene remained under alpha transcriptional control. Deletion of a portion of the beta gene leader sequence (nucleotides +62 to +142) reverted expression to the alpha class. This cis-dominant signal appears to act by blocking expression posttranscriptionally until a viral function activates full gene expression at the appropriate time in infection. PMID- 3019555 TI - The telomeres of the linear mitochondrial DNA of Tetrahymena thermophila consist of 53 bp tandem repeats. AB - We have cloned and sequenced the telomeric DNA of the linear mitochondrial DNA (mtDNA) of T. thermophila BVII. The mtDNA telomeres consist of a 53 bp sequence tandemly repeated from 4 to 30 times, with most molecules having 15 +/- 4 repetitions. The previously recognized terminal heterogeneity of the mtDNA is completely accounted for by the variability in the number of repeats. The 53 bp repeat does not resemble known telomeric DNA in sequence, repeat size, or number of repetitions. The termini occur at heterogeneous positions within the 53 bp repeat. The junction of the telomeric repeat with the internal DNA is at a different position within the telomeric repeat on each end of the mtDNA. We propose a model for the maintenance of the mtDNA ends involving unequal homologous recombination. PMID- 3019556 TI - Self-assembly of purified polyomavirus capsid protein VP1. AB - The polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in E. coli, was isolated as oligomers resembling the dissociated capsomeres derived from viral capsids. Image analysis of low-dose electron micrographs demonstrates that these VP1 oligomers are exclusively pentamers. The purified VP1 pentamers associated to form capsid-like assemblies and polymorphic aggregates at high ionic strength. The capsid-like assemblies were stabilized at low ionic strength by the addition of calcium. Self-assembly of the unmodified, recombinant DNA-generated VP1 implies that the posttranslational charge modifications of VP1 and the minor virion protein components, VP2 and VP3, are not essential for capsid formation. The nonequivalently related subunits of the penta- and hexavalent capsomeres therefore must spontaneously switch their bonding specificity during assembly. PMID- 3019557 TI - Oligomerization is essential for transport of vesicular stomatitis viral glycoprotein to the cell surface. AB - Using ts045, a temperature sensitive strain of Vesicular stomatitis virus, we show that oligomerization of G protein is a prerequisite for its transport from RER to the Golgi apparatus and for its subsequent maturation. While wild-type G forms an oligomer in the RER, ts045 G synthesized at the nonpermissive temperature does not. When the permissive temperature is reinstated, ts045 G forms an oligomer and moves to the Golgi. The state of oligomerization was determined by chemical cross-linking and by the ability of a microinjected monoclonal antibody specific for the carboxy-terminal five amino acids of the cytoplasmic tail of G to cause patching of G in intracellular membranes. We conclude that formation of an oligomer of G protein, probably a trimer, is necessary for G protein maturation. PMID- 3019559 TI - A retrovirus vector expressing the putative mammary oncogene int-1 causes partial transformation of a mammary epithelial cell line. AB - In mammary tumors induced by the mouse mammary tumor virus (MMTV), the int-1 gene is frequently activated by adjacent proviral insertions and is thereby strongly implicated in tumorigenesis. To seek a direct biological effect of int-1 that would validate its proposed role as an oncogene, we constructed a retrovirus vector containing the gene and examined its effects on tissue culture cells. Expression of int-1 in a mammary epithelial cell line caused striking morphological changes, unrestricted growth at high cell density, and focus formation on a monolayer, although the cells were not tumorigenic in vivo. This partial transformation induced by int-1 was not observed in cells infected by an otherwise identical virus bearing a frameshift mutation in the gene. These findings strongly support the hypothesis that int-1 plays a functional role in MMTV-induced mammary tumorigenesis. PMID- 3019558 TI - Protein kinase C phosphorylates human platelet inositol trisphosphate 5' phosphomonoesterase, increasing the phosphatase activity. AB - Phosphoinositide breakdown in response to thrombin stimulation of human platelets results in the formation of the calcium-mobilizing messenger molecules inositol 1,4,5-trisphosphate and inositol 1,2-cyclic-4,5-trisphosphate and of diglyceride, which activates protein kinase C. We find that protein kinase C phosphorylates and thereby increases the activity of inositol 1,4,5-trisphosphate 5' phosphomonoesterase, a phosphatase that hydrolyzes these molecules to inert compounds. The 5'-phosphomonoesterase phosphorylated using [gamma-32P]ATP comigrates on SDS-polyacrylamide gels with a protein (40 kd) phosphorylated rapidly in response to thrombin stimulation of 32PO4-labeled platelets. Peptide maps of proteolytic digests of these two phosphorylated proteins indicate that they are the same. We propose that platelet Ca2+ mobilization is regulated by protein kinase C phosphorylation of the inositol 1,4,5-trisphosphate 5' phosphomonoesterase. These results explain the observation that phorbol ester treatment of intact human platelets results in decreased levels of inositol trisphosphate and decreased Ca2+ mobilization upon subsequent thrombin addition. PMID- 3019560 TI - The interaction of recombination proteins with supercoiled DNA: defining the role of supercoiling in lambda integrative recombination. AB - Lambda integrative recombination depends on supercoiling of the phage attachment site, attP. Using dimethylsulfate protection and indirect end-labeling, the interaction of the recombination proteins Int and IHF with supercoiled and linear attP has been studied. Supercoiling enhances the binding of Int to attP, but not if a truncated attP site is employed or if IHF is omitted. We reason that the altered affinity reflects the formation of a higher-order nucleoprotein structure, an "attP intasome," that involves Int and IHF assembly of both arms of attP into a wrapped configuration. The good correlation between the degree and sign of supercoiling needed to promote recombination and that needed for the "attP intasome" indicates that the primary role of supercoiling is to drive the formation of the wrapped structure. PMID- 3019561 TI - Most of the yeast genomic sequences are not essential for cell growth and division. AB - To determine the fraction of the yeast Saccharomyces cerevisiae genome that is required for normal cell growth and division, we constructed diploid strains that were heterozygous for random single disruptions. We monitored the effects of approximately 200 independent disruptions by sporulating the diploids and examining the phenotype of the resulting haploid strains. We found that only 12% of the disruptions were haploid-lethal, 14% resulted in slow growth, and an additional 4% were associated with some other new phenotype (such as an auxotrophic requirement). No obvious new phenotype was detected for 70% of the disruptions. PMID- 3019563 TI - Regulation of AIDS virus expression. PMID- 3019562 TI - Genetic basis of the complementary DpnI and DpnII restriction systems of S. pneumoniae: an intercellular cassette mechanism. AB - Cells of S. pneumoniae contain either DpnI, a restriction endonuclease that cleaves only the methylated DNA sequence 5'-GmeATC-3', or DpnII, which cleaves the same sequence when not methylated. A chromosomal DNA segment containing DpnII genes was cloned in S. pneumoniae. Nucleotide sequencing of this segment revealed genes encoding the methylase and endonuclease and a third protein of unknown function. When the plasmid was introduced into DpnI cells, recombination during chromosomal facilitation of its establishment substituted genes encoding the DpnI endonuclease and another protein in place of the DpnII genes. DNA hybridization and sequencing showed that the DpnI and DpnII segments share homology on either side but not between themselves or with other regions of the chromosome. Thus, the complementary restriction systems are found on nonhomologous and mutually exclusive cassettes that can be inserted into a particular point in the chromosome of S. pneumoniae on the basis of neighboring homology. PMID- 3019564 TI - Demonstration of virus-specific transcriptional activator(s) in cells infected with HTLV-III by an in vitro cell-free system. AB - Human T cell lymphotropic viruses (HTLV's) differ from most other retroviruses based on the presence of trans-regulatory genes for virus expression. In this study, using an in vitro cell-free transcription system, we demonstrate that nuclear extracts obtained from cells infected with HTLV-III (human immunodeficiency virus, HIV) contain a factor(s) that stimulates transcription specifically from the HTLV-III promoter. Present studies indicate that this activation is mainly involved in the initiation of transcription from the HTLV III promoter. PMID- 3019565 TI - Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA. AB - Closed-circular HBV DNA was introduced into cells of the established human hepatoma culture HepG2. The culture medium of one of 40 single-cell clones contained HBV surface antigen (HBsAg), core-related antigens (HBc/eAg), and HBV DNA sequences. HBV DNA and DNA polymerase activity were detected in particles resembling both nucleocapsids and complete virions (Dane particles). Intracellular integrated and extrachromosomal HBV DNA sequences were detected. Relaxed-circular and single-stranded forms of viral DNA were identified as likely replicative intermediates of the HBV genome. In conclusion, in vitro production of Dane-like particles by transformed human hepatocytes has been achieved. This model should be valuable as a cell culture system for studying virus replication and virus-host cell interactions. PMID- 3019567 TI - Regulation of rat natural killing. II. Inhibition of cytolysis and activation by inhibitors of lipoxygenase: possible role of leukotrienes. AB - Rat splenic natural killer (NK) cell activity against 51Cr-labeled YAC-1 or TMT 081 tumor cells can be augmented by culturing at 37 degrees C for 18 hr. Inhibitors of the lipoxygenase pathway of arachidonic acid metabolism, NDGA, alpha-phenanthroline, quercetin, ETYA, BW755C, esculetin, and timegadine, inhibited this NK activation and also inhibited NK cytotoxicity when added directly to the NK assay. However, there was a partial loss of sensitivity of activated NK cells to suppression by NDGA, BW755C, and esculetin. Indomethacin failed to reverse the inhibition of NK activation caused by NDGA. However, LTB4 and LTC4 (0.01 microgram/ml) were able to reverse the inhibitory effect of NDGA on NK activation. Furthermore, spleen cells cultured for 18 hr synthesized detectable amount of LTC4 in their supernatants. NDGA inhibited the LTC4 synthesis in a dose-dependent manner. These data therefore suggest that leukotrienes are responsible for NK activation, and lipoxygenase activity is essential for NK cytolytic activity. PMID- 3019566 TI - Regulation of respiratory burst in murine peritoneal macrophages: differential sensitivity to phorbol diesters by macrophages in different states of functional activation. AB - Activation of macrophages either in vivo or in vitro can modulate the capacity to generate and secrete reactive oxygen intermediates including H2O2 and O2-. Thus, the cellular and biochemical components requisite for execution of the respiratory burst must be regulated during the activation process. In the present report, we have examined murine peritoneal macrophages in different stages of activation for their sensitivity to stimulants of respiratory burst known to activate protein kinase c (i.e., phorbol dibutyrate or diacylglycerol). The results demonstrated that more highly activated macrophages showed, in addition to greater magnitude of H2O2 or O2- production, a two- to fourfold greater sensitivity to these stimuli. While more active macrophages also exhibited a higher rate of H2O2 secretion, the time at which secretion was measured did not account for or modulate the heightened sensitivity. The increased sensitivity to stimulation was dependent upon the stage of activation and not on the agent used to elicit the macrophages. Increased sensitivity of the more active macrophage populations was also seen when physiologic stimuli (i.e., insoluble immune complexes or unopsonized zymosan) were used. These findings indicate that macrophage activation for H2O2 secretion modulates the sensitivity to stimulation such that more H2O2 is produced in a shorter time and at a lower concentration of stimulus, thereby heightening the inflammatory response in several independent ways. Because all the stimuli employed in the present study have in common the ability to activate protein kinase c (either directly or indirectly), the data also suggest that this form of macrophage activation may involve, at least in part, modulation of the stimulus-response coupling mechanisms which utilize this enzyme. PMID- 3019568 TI - Specific transfer factor protects mice against lethal challenge with herpes simplex virus. AB - Bovine transfer factor (TFd) specific to herpes simplex virus (HSV)1 or to HSV2 was prepared by immunizing calves with the corresponding virus. The TFd preparations were then injected into Swiss mice in an attempt to protect them against a subsequent lethal challenge with HSV1 or HSV2 virus. It was thus shown that injection of anti-HSV TFd protects the mice against the corresponding HSV virus, whereas the injection of a nonspecific TFd (anti-CMV) fails to protect against a challenge with HSV1. Furthermore, a dose-response effect was observed, since potent TFd preparations were ineffective when they were used at one-fifth of the original concentration. It seems, therefore, that animal models may be used to assay the potency of TFd preparations specific for herpes viruses. PMID- 3019569 TI - Recombinant interleukin-2-induced polyclonal proliferation of in vitro unstimulated human peripheral blood lymphocytes. AB - Human peripheral blood mononuclear cells as well as T-cell-enriched or T-cell depleted populations were found to proliferate in response to recombinant interleukin-2 (IL-2) in vitro in the absence of a lectin preactivation signal. This proliferative response was detected at Day 2, peaked at Day 5, and was dependent on the concentration of IL-2 used. At the initiation of culture, these cells did not appear to be activated as determined by the expression of Tac antigens. In cultures of unfractionated T-cell-enriched suspensions, high concentrations of IL-2 resulted in preferential expansion of the OKT8+ population, although both OKT4+ and OKT8+ cells proliferated in response to IL-2 when cultured alone. These studies demonstrate that human lymphocytes obtained by standard fractionation procedures from peripheral blood are capable of proliferation in response to IL-2 without in vitro preactivation signals given by the addition of mitogens or antigens to cultures. These findings suggest that in vivo IL-2, in the absence of other exogenous stimuli, may directly influence immune responses and thus may have a potential role as a clinical immunopharmacologic agent. PMID- 3019570 TI - Differential recognition of tumor-derived and in vitro Epstein-Barr virus transformed B-cell lines by fetal calf serum-specific T4-positive cytotoxic T lymphocyte clones. AB - Two interleukin-2 (IL-2)-dependent cytotoxic T-cell clones were obtained by limiting dilution from a lymphocyte culture stimulated in vitro with the autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) in the presence of fetal calf serum (FCS). Both clones uniformly had a T3+, T4+, Dr+ phenotype and lysed autologous B blasts, the autologous LCL, and allogeneic B cell lines sharing major histocompatibility complex (MHC) class II antigens. The cytotoxic function was triggered by FCS-derived components. There was no killing if the sensitive targets were cultured in serum-free medium or in medium supplemented with human serum. Sensitivity to lysis could be restored by exposing the targets to FCS for at least 6 hr at 37 degrees C. Monoclonal antibodies directed to T-cell-specific surface antigens and MHC class II antigens inhibited lysis with different efficiencies depending on the target cell origin. Killing of Burkitt's lymphoma (BL)-derived cell lines was blocked more easily than killing of LCLs. LCLs but not BL lines induced proliferation of the T-cell clones in the absence of exogenous IL-2. The differences were not related to quantitative variations in the expression of MHC class II antigens, indicating that BL lines differ from LCLs in other cell membrane properties that may influence antigen presentation. The results suggest that the affinity of effector/target binding, which is probably influenced by the concentration of antigenic determinants expressed on the target cell membrane, determines whether proliferative responses or cytotoxicity are induced in the antigen-recognizing T cells. PMID- 3019571 TI - Interleukin-1 production and antigen presentation by normal human peritoneal macrophages. AB - Human peritoneal macrophages from healthy females have been investigated for their capability to produce interleukin-1 (IL-1), their expression of HLA-DR and DQ, and for their antigen-presenting capacity in concanavalin A, tetanus toxoid (TT), and autologous T-cell proliferative responses. Fifteen out of thirty macrophage populations produced IL-1 but the activity was 1/5 to 1/10 that of peripheral blood mononuclear cells stimulated under similar conditions. High levels of HLA-DR were expressed on all macrophages while lower and more variable levels of DQ were found. All macrophages induced mitogen-dependent T-cell proliferation while the ability to induce a proliferative response to TT was variable, 12/23 tests were positive. In five samples stimulatory capacity of macrophages in the absence of TT was as strong as in the presence of the stimulus, suggesting that in vivo processed immunogen could be responsible for the proliferative response. The surface density of HLA-D-region-determined antigens was not indicative of the macrophages' ability to induce antigen specific proliferation. IL-1 production, however, correlated with this function. Antigen presentation was not confined exclusively to peritoneal populations consisting of recently immigrant monocyte-like cells, nor were all young macrophages able to present antigen. This may reflect on the diversion of macrophage function by the local environment. PMID- 3019574 TI - The interleukin-2 receptor on malignant cells: a target for diagnosis and therapy. AB - Interleukin-2 (IL-2) is a lymphokine synthesized by T cells following activation. Resting T cells do not express IL-2 receptors, but receptors are rapidly expressed on T cells following interaction of the antigen-specific T-cell receptor complex with appropriately processed and presented antigens. Anti-Tac, a monoclonal antibody that recognizes the IL-2 receptor, has been used to purify the receptor. The receptor is a 55-kDa glycoprotein comprised of 251 amino acids including a single 19-amino transmembrane domain and a short intracytoplasmic domain composed of 13 amino acids at the carboxy terminus. Normal resting T cells and most leukemic T-cell populations examined did not express IL-2 receptors; however, the leukemic cells of all patients with human T-cell lymphotrophic virus (HTLV-I)-associated adult T-cell leukemia (ATL) expressed the Tac antigen. In HTLV-I-infected cells, the 42-kDa long open reading frame (tat) protein encoded in part by the tat region of HTLV-I may act as a transacting activator that induces transcription of the IL-2-receptor gene, thus providing an explanation for the constant association of HTLV-I infection of lymphoid cells and IL-2 receptor expression. The constant display of large numbers of IL-2 receptors which may be aberrant in the ATL cells may play a role in the uncontrolled growth of these leukemic T cells. Patients with the Tac-positive ATL are being treated with both unmodified and toxin-conjugated forms of anti-Tac monoclonal antibody directed toward this growth factor receptor. PMID- 3019573 TI - Reversal of murine alveolar macrophage-mediated suppression of plaque-forming cell response by sodium periodate. AB - Murine alveolar macrophages (AM) have been shown to suppress the in vitro plaque forming cell (PFC) response of spleen cells previously primed with sheep erythrocytes (SRBC) in a dose-dependent manner. Mild oxidation of cell membranes on viable AM with sodium periodate resulted in total abrogation of AM-mediated suppression of the PFC response, while periodate treatment of spleen cells resulted only in partial reduction of the suppression. Pretreatment of AM with sodium periodate followed by addition of the aldehyde blocking agent, hydroxylamine, resulted in restoration of the PFC-suppressing activity of AM. Periodate treatment of AM also resulted in significantly increased macrophage-T cell binding and cluster formation. These observations suggest that the generation of aldehyde moieties on AM membrane sialoglycoconjugates promotes positive macrophage-lymphocyte interactions, resulting in abrogation of AM mediated suppression of the PFC response. PMID- 3019572 TI - Presentation of Candida albicans and purified protein derivative soluble antigens by Epstein-Barr virus-transformed human lymphoblastoid B-cell lines. AB - Cells other than the macrophage can function as antigen-presenting cells (APCs). These class II-bearing accessory cells include dendritic cells, epidermal Langerhans cells, B cells, murine B-cell tumors, and human Epstein-Barr virus transformed lymphoblastoid cell lines (EBV-LCL). We investigated the ability of EBV-LCL to present two soluble antigens, Candida albicans and purified protein derivative of tuberculin (PPD). The EBV-LCL derived from B cells of two different individuals can present both antigens to bulk cultures of autologous antigen primed peripheral blood lymphocytes. The responses of PPD-reactive T-cell clones were weaker to PPD when presented by EBV-LCL than by PBL-APCs, with some clones responding only to PPD presented by PBL-APCs. This suggests that EBV-LCL are not equivalent to PBL monocytes in APC function, and that expression of class II major histocompatibility complex antigen is not sufficient in enabling antigen presenting capability. PMID- 3019575 TI - Function of T cells from elderly humans: reductions of membrane events and proliferative responses mediated via T3 determinants and diminished elaboration of soluble T-cell factors for B-cell growth. AB - The abilities of T cells from young and elderly humans to cap membrane T3 determinants, proliferate in response to anti-OKT3 antibody, and elaborate soluble factors required for the growth of B-cell colonies were investigated. The results showed that T cells from approximately 50 to 80% of elderly subjects had reductions in ligand-induced T3-mediated membrane events and proliferative responses in addition to decreased elaboration of soluble B-cell growth factors. Thus, these previously unrecognized abnormalities in T cells might contribute to the decline of immunocompetence observed among some elderly humans. PMID- 3019578 TI - Production of cytotoxin to a tumor cell in co-cultures of macrophage cell line with bovine leukemia virus producing cell line. AB - J 774 cells (macrophage cell line) were co-cultured with fetal lamb kidney cells which permanently produce Bovine leukemia virus (BLV-FLK cells) and after co culture, the cells removed by scraping were subcultured once every for 2-4 days. Strong cytotoxicity to a cell (C8 cells; a clone of F81 cell line which contains mouse sarcoma virus genome) was found in culture of cells from generations 11 after the passages of co-cultures. The activity to the cells was continuously found in the fluid without any other addition of stimulants. The above results show that J 774 cells were activated by the co-culture with BLV-FLK cells and produced the cytotoxic factor which revealed strong cytotoxicity to a C8 cells. PMID- 3019577 TI - Putative second messengers affect cell coupling in the seminiferous tubules. AB - Junctional transfer of ions in monolayer primary cultures of Sertoli cells and in intact seminiferous tubules from 20 day-old rats has been investigated by using electrophysiological techniques. The electrotonic coupling of both intact tubule and monolayer culture was inhibited by the presence of dibutyryl cyclic AMP (10( 4) M) in the medium. In contrast, 1-oleoyl-2-acetylglycerol (10(-5) M) and 12-O tetradecanoyl-phorbol-13-acetate (10(-7) M) decreased the junctional resistivity and increased the extent of the coupling in immature tubules. The significance of this modulation of the cell coupling in the seminiferous epithelium is discussed. PMID- 3019580 TI - [Enzyme immunoanalysis of antibodies against type A hepatitis viruses]. PMID- 3019576 TI - Leukotriene B4 causes proliferation of interleukin 2-dependent T cells in the presence of suboptimal levels of interleukin 2. AB - We have recently found that endogenous leukotriene B4 (LTB4) production is a necessary component of mitogen-stimulated T-cell proliferation. In this report, we address the relationship between LTB4 and interleukin 2 (IL-2) in stimulating proliferation of IL-2 responsive T cells. We employed an IL-2 responsive T-cell line, HT-2 and also human peripheral blood T cells rendered IL-2 responsive by culture with phytohemagglutinin (PHA). In the presence of concentrations of IL-2 that resulted in suboptimal stimulation of HT-2 cells (5-20% of maximum proliferation), LTB4 at 10(-8) or 10(-10) M produced a several-fold increase in [3H]thymidine incorporation, resulting in levels of [3H]thymidine incorporation that were comparable to those produced by HT-2 cells stimulated with optimal concentrations of IL-2. Similar results were obtained with the peripheral blood T cells. Leukotriene C4, prostaglandin E2 (PGE2), and arachidonic acid did not share with LTB4 the ability to stimulate proliferation of HT-2 cells. Hydrocortisone and nordihydroguairetic acid (NDGA), drugs which block endogenous LTB4 production in cell cultures inhibited [3H]thymidine incorporation of HT-2 cells stimulated by suboptimal but not optimal levels of IL-2. This inhibition could be overcome by the readdition of LTB4 to the cultures. Thus, LTB4 in physiologic concentrations (10(-10) M) can substitute for IL-2 in stimulating proliferation of IL-2 responsive T cells, provided that some IL-2 is present in the culture. Endogenous LTB4 may act synergistically with endogenous IL-2 in promoting T-cell proliferation. PMID- 3019581 TI - [The present status of findings in human papillomavirus diseases of the uterine cervix]. PMID- 3019579 TI - Effects of inhibitors of arachidonic acid metabolism on intercellular adhesion of SV40-3T3 cells. AB - Mepacrine, an inhibitor of arachidonic acid mobilisation, and NDGA, a lipoxygenase inhibitor, were found to impair the aggregation of SV40-3T3 cells but the effects could not be unequivocally dissociated from non-specific actions of the drugs. No effect on aggregation was observed even after prolonged exposure of the cells to the cyclooxygenase inhibitors aspirin and indomethacin. These results argue against a possible regulatory role for endogenous AA metabolites in intercellular adhesion of SV40-3T3 cells. PMID- 3019582 TI - Effects of thyroidectomy on the Ah receptor and enzyme inducibility by 2,3,7,8 tetrachlorodibenzo-p-dioxin in the rat liver. AB - Thyroidectomy of rats confers some protection, by an unknown mechanism, from the weight loss, immunotoxicity, and mortality induced by 2,3,7,8-tetrachlorodibenzo p-dioxin (TCDD). Since at least some of the many effects of TCDD appear to be mediated by the Ah receptor, perhaps the thyroid plays a role in regulation of this receptor, thereby modifying the toxicity of TCDD. We tested this hypothesis by comparing TCDD-binding characteristics of the receptor and hepatic enzyme inducibility by TCDD (a receptor-mediated response) in thyroidectomized (ThX) and euthyroid rats. There were no significant differences in levels of TCDD binding in vitro in hepatic cytosol, in receptor affinity, nor in the molecular size of the TCDD-bound receptor in untreated ThX rats compared to controls fed ad libitum or pair-fed. Total hepatic cytochrome P-450 (P-450) levels and NADPH-menadione oxidoreductase (NMOR) activity were unaffected by thyroid status, whereas 7 ethoxycoumarin O-deethylase (ECOD) activity was approx. 50% lower in ThX animals than in ad libitum or pair-fed controls. At 3 and 10 days after TCDD administration (10 micrograms/kg, i.p.), P-450 concentrations and NMOR and ECOD activities were induced by approximately the same proportions in ThX and pair-fed intact rats; however, the absolute levels of the induced activities were lower in ThX than in pair-fed controls. It was concluded that hypothyroidism does not regulate Ah receptor concentration or function in the liver. Therefore, the modulation of TCDD toxicity by hypothyroidism appears not to involve changes in the hepatic Ah receptor. PMID- 3019584 TI - [First-trimester diagnosis of beta-thalassemia]. PMID- 3019583 TI - The binding of CC-1065 to thymidine and deoxyadenosine oligonucleotides and to poly(dA).poly(dT). AB - In this work, we report on the binding of the novel antitumor agent CC-1065 to poly(dA).poly(dT) and to mixtures of dA and dT oligomers as determined by electronic absorption and circular dichroism (CD) methods. In addition, the DNA binding properties of CC-1065 and its binding mechanism are compared to those of netropsin. CC-1065 binds to the polymer by at least three mechanisms to produce one irreversibly and two reversibly bound species. One reversibly bound species is moderately stable, but in time (days), it converts to the irreversibly bound species. Both of these species bind within the minor groove of the polymer and exhibit intense CC-1065 induced CD spectra. The other reversibly bound species does not acquire an induced CD. CC-1065 forces B-form duplex formation between mixtures of single strand dA and dT oligomers and binds irreversibly to the duplexes without showing the presence of an intermediate, reversibly bound species. The induced CD increases with increasing length of the oligomer, from the 5-mer (barely detectable CD) to the 14-mer (intense CD). The 7-, 10- and 14 mer mixtures bind about 1, between 1 and 2, and between 2 and 3 CC-1065 molecules, respectively. Computer graphic models of the CC-1065-DNA complex show that the covalent adduct of CC-1065 and unreacted CC-1065 can attain the same close van der Waals contacts between adenine C2 hydrogens and antibiotic CH groups that were observed in the crystal structure of the netropsin-DNA complex. These contacts may account for the dA-dT base pair binding specificity of CC-1065 and for the stability of the reversibly bound CC-1065 species. PMID- 3019585 TI - Phorbol diacetate inhibits superoxide anion radical production and tumor promotion by mezerein. AB - The ability of the non-promoter phorbol diacetate (PDA) to modulate superoxide anion radical production by the complete tumor promoter phorbol myristate acetate (PMA) or the second stage promoter mezerein was assessed. Superoxide anion radical production was measured by the superoxide dismutase inhibitable reduction of nitroblue tetrazolium (NBT) to a blue intracellular formazan precipitate. These studies demonstrated that superoxide anion radical production by murine peritoneal exudate cells (PEC) stimulated by i.p. injection with mezerein (100 ng) is inhibited in a dose-dependent manner by co-administration with PDA (1-1000 ng). There was no effect on the number of formazan-positive PEC when PDA was co administered with PMA. In a two-stage tumor promotion bioassay in female SENCAR mice initiated with 25.6 micrograms dimethylbenz[a]anthracene (DMBA) followed by first stage promotion with PMA (4X, 2 micrograms), co-administration of mezerein (2 micrograms) with 2 micrograms or 20 micrograms PDA reduced the number of papillomas after 14 weeks by 38% and 44%, respectively, compared with mezerein treatment alone. PDA (20 micrograms) when co-administered with mezerein (2 micrograms) does not inhibit mezerein induced hyperplasia in mouse skin. These results suggest a correlation between the ability of PDA to inhibit both superoxide anion radical production and tumor promotion by mezerein. PMID- 3019586 TI - Preservation of glomerular filtration rate in human heart failure by activation of the renin-angiotensin system. AB - When renal perfusion pressure is reduced in experimentally induced low-output states, glomerular filtration rate is preserved by angiotensin II-mediated efferent arteriolar vasoconstriction, but available evidence in man suggests that angiotensin II supports renal function only to the extent that it preserves systemic blood pressure. We performed simultaneous assessments of cardiac and renal function in 56 patients with severe chronic heart failure before and after 1 to 3 months of converting-enzyme inhibition. Among the 29 patients with a pretreatment renal perfusion pressure under 70 mm Hg, patients with preserved renal function (creatinine clearance greater than 50 ml/min/1.73 m2) had markedly elevated values for plasma renin activity (11.8 +/- 3.8 ng/ml/hr) and showed a significant decline in creatinine clearance after converting-enzyme inhibition (61.1 +/- 3.0 to 45.9 +/- 5.3 ml/min/1.73 m2; p less than .05). In contrast, although similar with respect to all pretreatment demographic, hemodynamic, and clinical variables, patients with a creatinine clearance under 50 ml/min/1.73 m2 had low values for plasma renin activity (3.4 +/- 0.8 ng/ml/hr) and, despite similar drug-induced decreases in systemic blood pressure, showed no change in creatinine clearance after therapy with captopril or enalapril (32.6 +/- 2.5 to 41.4 +/- 3.8 ml/min/1.73 m2). Changes in creatinine clearance varied linearly and inversely with pretreatment values for plasma renin activity (r = - .64, p less than .001); converting-enzyme inhibition effectively abolished the pretreatment difference in renal function seen in the high- and low-renin subgroups. In the 27 patients with a renal perfusion pressure of 70 mm Hg or greater, creatinine clearance did not vary significantly with plasma renin activity and was not altered by therapy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019587 TI - A study of lipid effects on the digoxin immunoassay and on the binding to and activity of Na+/K+-ATPase. AB - The present study has examined the cross-reactivity of fatty acids and mono- and di-glycerides in the fluorescence polarization immunoassay for digoxin. The ability of these compounds to inhibit 86Rb uptake by the erythrocyte as well as their ability to displace 3H-ouabain from membrane-bound dog kidney ATP-ase was also assessed. Some unsaturated fatty acids (palmitoleic, palmitelaidic, oleic, linoleic, linolelaidic, linolenic, gamma-linolenic and arachidonic) were found to cross-react significantly in the digoxin immunoassay and to inhibit 3H-ouabain binding to membrane-bound Na+/K+-ATPase. Of the monoglycerides studied mono-11 eicosenoin was found to cross-react in the digoxin immunoassay, inhibit red cell 86Rb uptake and displace 3H-ouabain from its receptor, membrane-bound Na+/K+ ATPase. Two other monoglycerides, 1-monolinoleoyl and 1-monolinolenoyl glycerides, were able to displace 3H-ouabain from membrane-bound Na+/K+-ATPase, but had no effect on the digoxin immunoassay or on red cell 86Rb uptake. PMID- 3019588 TI - Erythrocyte membrane alterations and plasma lipids in patients with chylomicronemia and in Tangier disease. AB - The relationship between erythrocyte membrane structural and functional alterations and plasma lipids was studied in three patients with chylomicronemia due to either lipoprotein lipase (LPL) deficiency, apo C-II deficiency (in an individual who also suffers from thalassemia minor) or coexistent diabetes mellitus (and decreased LPL activity) and in a patient with Tangier disease. All of the patients' erythrocytes had significantly elevated phosphatidyl-choline (PC): sphingomyelin (Sph) ratios (most marked in the patient with Tangier disease). Major differences were observed in the PC: Sph ratios of erythrocytes and plasma. The pattern of changes in erythrocyte membrane enzyme activities differed despite similarities in the lipid composition of the erythrocytes. The changes in osmotic fragility (OF) were inversely related to the membrane cholesterol:phospholipid ratio. An even stronger negative correlation was found between OF at the lowest NaCl concentrations and the activities of both Na+,K+- and Mg++-ATPases. The ratio of total: surface sulfhydryl titres also correlated significantly with OF. PMID- 3019589 TI - The chemistry of aluminum as related to biology and medicine. AB - The increasing number of roles discovered for Al3+ in physiological processes demands an understanding of how Al3+ interacts with compounds in biological systems. Al3+ is expected to complex with oxygen donor ligands, especially phosphates, and it does so in soils, in the gastrointestinal tract, and in cells. The stability of Al3+ complexes has generally been misjudged because of lack of recognition that free, aqueous Al3+ is not the dominant form in neutral solutions and that the solubility of Al(OH)3 limits the free Al3+ at the plasma pH 7.4 to less than 10(-11) mol/L. In the presence of inorganic phosphate, the permitted free Al3+ is decreased further, through formation of insoluble aluminum phosphate. This precipitate facilitates the elimination of Al3+ from the body. In contrast, citrate solubilizes Al3+, and an appreciable fraction occurs as a neutral complex that may pass through membranes and provide a vehicle for Al3+ absorption into the body. In the blood plasma the most likely small-molecule complex is that with citrate, while the only competitive protein complex is that with transferrin, a protein built to transport Fe3+ but whose sites are only 30% occupied. PMID- 3019590 TI - Biochemical alterations and complications in diabetes. PMID- 3019591 TI - Vasopressor and vasodepressor hormone-systems in adolescent hypertension. AB - We investigated the activity of the renin-angiotensin-aldosterone-system, the secretion of catecholamines and the kallikrein-kinin-system in 126 adolescents randomly selected from a large study of 1342 young people examined in an epidemiological survey conducted in Cologne in 1975, 1976 and 1980. 73 of them with arterial blood pressures below 145/90 mm Hg were called "normotensives" (systolic blood pressure 127.2 +/- 1.0 mm Hg, diastolic bp 79.7 +/- 0.8 mm Hg). They were compared with 53 "hypertensives" (systolic blood pressure 147.2 +/- 1.6 mm Hg, diastolic bp 93.7 +/- 1.1 mm Hg). Urinary catecholamines were significantly higher in the hypertensives (155.0 +/- 13.3 micrograms/d) compared to the normotensives (100.7 +/- 5.3 micrograms/d) (p less than 0.001) whereas plasma levels of adrenaline and noradrenaline were similar. Serum aldosterone levels and plasma-renin-concentrations were not different between the two groups. Angiotensin-converting-enzyme-activity was slightly higher in the hypertensive group (107.1 +/- 3.5 U/l versus 98.0 +/- 2.6 U/l, p less than 0.001). Urinary kallikrein excretion was found to be modestly lower in hypertensives compared to normotensives (0.40 +/- 0.05 versus 0.55 +/- 0.06 mU/mg creatinine). Urinary excretion of sodium and potassium, blood levels of glucose, uric acid, cholesterol and triglycerides were similar in both groups. The results of the present study suggest an increased sympathetic activity in the early stage of hypertension in adolescents. PMID- 3019592 TI - Captopril as an aid to diagnosis in childhood hypertension. PMID- 3019593 TI - The determination of mineralocorticoid receptors in human mononuclear leukocytes from patients with mineralocorticoid excess: physiological and pathological implications. AB - The affinity and the capacity of mineralocorticoid receptors (MR) in human mononuclear leukocytes (HML) were determined in 9 patients with Conn's syndrome (PA) and in 3 patients with pseudohypoaldosteronism (PHA). The number of binding sites per cell was 136 +/- 39 (mean +/- SD) in PA. One case with PHA had no MR, and of the other 2 patients, one had 50 and the other 55 receptors per cell. The capacity of normal controls ranged from 200 to 400 receptors per cell (n = 20). We conclude that the etiology of PHA is due to a lack of MR in the target tissues and that a down regulation of MR may exist in PA. PMID- 3019594 TI - Cardiovascular effects of the converting enzyme inhibitor ramipril (HOE 498) in anesthetized dogs with acute ischemic left ventricular failure. AB - The non-sulfhydryl converting enzyme inhibitor Ramipril (HOE 498) is characterized by long lasting antihypertensive activity in man. To examine its cardiovascular potential in heart failure, ramipril was administered during acute ischemic left ventricular failure in pentobarbital anesthetized dogs, induced by repeated injections of plastic microspheres into the left main coronary artery. Repeated embolizations produced stable left ventricular (LV) pump failure characterized by LV enddiastolic pressure of 22 mmHg, reductions in LV dp/dt max and cardiac output. Blood pressure and heart rate were not changed while total peripheral resistance increased. After a stabilization period ramipril was administered in two doses at 30 or 100 micrograms/kg as an intravenous bolus followed by continuous infusion of 3 or 10 micrograms/kg/min for 150 min. Ramipril in the lower dose decreased LV enddiastolic pressure by 8 mmHg, mean pulmonary artery pressure by 4 mmHg, systemic blood pressure by 40 mmHg and total peripheral resistance by 1280 dyn x sec x cm-5. LV dp/dt max, heart rate and cardiac output remained unchanged during ramipril administration. More pronounced effects were obtained with the higher dose. In conclusion, the improvements of hemodynamics produced by ramipril during acute ischemic left ventricular failure in anesthetized dogs are best explained by a reduction in both preload and afterload. PMID- 3019595 TI - Saralasin blocks the effect of angiotensin II and extracellular fluid saline expansion on the Na-K-ATPase inhibitor release in rats. AB - A low molecular weight substance which behaves like ouabain as inhibitor of brain membrane Na-K-ATPase and 3H-ouabain binding was found in plasma after saline expansion of extracellular fluid or angiotensin II infusion into the third brain ventricle in the rat. Intracerebroventricular infusion of angiotensin II antagonist, saralasin, blocks the increase of the Na-K-ATPase inhibitor produced by infusion of angiotensin II into the third ventricle or extracellular fluid saline expansion. PMID- 3019596 TI - Number of "high genes" involved in determining the activity of paraoxonase. AB - The genetics of paraoxonase activity is further analysed on the basis of a Danish family material (Eiberg & Mohr 1981), namely a random sample of the investigated two mating types. The starting point is the earlier assumption that the segregation into high and low activity is due to alleles on a single locus with the frequency 0.726 of low genes (Eiberg & Mohr 1981, Nielsen et al. 1986). It is shown that a hypothesis of two "high genes", h1 and h2 on the same locus, with allele frequencies 0.117 and 0.157, both high genes being dominant over low genes and h2 dominant over h1, may explain the observed pattern of segregation. The hypothesis would imply average activities of genotype 1h1 of 960 microM PNP/1, of 1h2 1385 microM PNP/1, of h1h1 1202 microM PNP/1, and of h1h2 and h2h2 2048 microM PNP/1. It cannot be excluded that there are more than two "high genes". It is further shown that more than one "low gene" must be involved. PMID- 3019597 TI - Plasma-free eicosapentaenoic acid/arachidonic acid ratio: a possible new coronary risk factor. AB - Seventy-six effort angina patients who had typical angina on exertion documented by treadmill stress test with evidence of ischemic ST-segment depression and 78 healthy volunteers in urban Japan were investigated in this study. Plasma free fatty acids (FFA) in both groups were determined using high-performance liquid chromatography. The relationships between the total cholesterol, high-density lipoprotein (HDL), triglycerides in plasma, and the genesis of coronary heart disease were also examined. The ratio (0.08 +/- 0.08) of eicosapentaenoic acid (EPA)/arachidonic acid (AA) in plasma FFA was significantly lower in effort angina patients than that (0.15 +/- 0.12) in healthy volunteers. The lower ratio was due to significantly lower levels of EPA in the patients than in normals. In 42% of angina patients, the ratio is below 0.03. In all age subgroups except the age 30-39 subgroup, the ratio of EPA/AA was significantly lower in patients than in normals, when divided into four subgroups by using a 10-year age interval. Though the total cholesterol and triglycerides were not significantly different between the two groups, HDL was significantly lower and total cholesterol/HDL ratio was significantly higher in effort angina patients than in healthy volunteers. However, there was no correlation between EPA/AA ratio and HDL in individuals in either group. From these results, it could be concluded that lower EPA/AA ratio is a new coronary risk indicator other than HDL. PMID- 3019599 TI - Herpes simplex virus and human papilloma virus in the development of cervical carcinoma. PMID- 3019598 TI - Bile salts as endogenous digitalis like factors. AB - Digitalis-like factors were assayed by radioimmunoassay of digoxin in 6 bile samples obtained from patients at autopsy and in plasma from three patients with combined hepatic and acute renal failure. None of the patients received digoxin. Digitalis like factor values in bile samples were 23 to 85 nmol digoxin equivalents/1. Bile salt concentrations ranged from 38-104 mmol/l in the bile and 28-184 mumol/l in the plasma of these subjects. Bile, plasma digitalis like factor extracts and bile salt standards (0.1-3 mM) showed concentration dependent displacement of [125I]-digoxin from digoxin antibody, inhibition of hog brain Na,K-ATPase and displacement of [3H]-ouabain from Na,K-ATPase. The concentration displacement curves suggest that bile salts could account for 50-79% of the total digitalis like factors in the six bile samples and 2-7% in the plasma of the three patients. High performance liquid chromatographic fractionation of a bile sample showed digitalis like factor peaks co-eluating with standards of tauro- and glycocholate, tauro- and glycochenodeoxycholate and tauro- and glycodeoxycholate. These bile salt peaks accounted for 78% of the total digitalis like factors in all high performance liquid chromatographic peaks in bile, but only 7% of the total digitalis like factor activity in all high performance liquid chromatographic peaks in an extract of plasma from one of the patients with hepatic and renal failure. The bile salts appear to be examples of endogenous digitalis like compounds which do not act by simple competitive ligand binding to antidigoxin antibody and Na,K-ATPase. They make an important contribution to digitalis like factor activity in bile, but not in plasma. PMID- 3019600 TI - Maturation of a primitive neuroectodermal brain tumor? A case study with some remarks on the classification and nomenclature of 'primitive' CNS tumors. AB - A brain tumor in a 4-year-old child is described. The neoplasm was partly cystic and showed an a-typical multi-differentiated aspect. Microscopically the neoplasm had a clear-cut 'malignant' morphology. This tumor represents possibly a partly maturated primitive neuroectodermal brain tumor. The term PNET is briefly discussed in relation to the clinical implications. PMID- 3019601 TI - Rat ventral prostate cell lysis at 2 degrees C. AB - In order to physically separate epithelial from nonepithelial cells, well-minced rat ventral prostate at 2 degrees C was passed through a tissue sieve, and the disrupted tissue suspended and washed several times before centrifugation on a Ficoll gradient. While limited separation of single prostate epithelial and nonepithelial cells and small aggregates could be achieved, the yield of intact undamaged cells was low, and many nuclei contaminated the 2 major cell fractions obtained from the gradient. During subsequent experiments, it became apparent that most single cells released from prostates minced at 2 degrees C rapidly lysed, yielding cytoplasmic debris and cell nuclei. Yet the morphology of red and white blood cells, examined by light microscopy, was unaffected, suggesting a soluble factor was not responsible. These results indicated that mechanical dissociation of rat ventral prostate at 2 degrees C with release of single cells was accompanied by a powerful prostate cell-associated lytic 'event', affecting both epithelial and connective tissue cells, without destruction of cell nuclei or accompanying red and white blood cells. Some properties of this process are described in this report. PMID- 3019602 TI - High fibre, low sodium and low fat diet in white and black type 2 diabetics with mild hypertension. AB - White and black mildly hypertensive Type 2 diabetics were given an intended diet high in unrefined carbohydrate (50% daily energy) and fibre (40-45 g/day) while low in fat (25% daily energy) and sodium (60-80 mmol/day) for 3 months. Both white and black diabetic hypertensive patient groups demonstrated a significant reduction of systolic (p less than 0.01 and p less than 0.001 respectively) and diastolic blood pressure (p less than 0.001 and p less than 0.01 respectively). This was accompanied by a significant reduction of daily urine sodium excretion, urine sodium: potassium ratio, weight and glycosylated haemoglobin in both groups. Only whites had a significant reduction of serum triglyceride level (p less than 0.05) although blacks showed similar trends. Compliance to the dietary regimen, assessed by a trends. Compliance to the dietary regimen, assessed by a scoring method appeared comparable in both groups. These data suggest this modified dietary regimen might be considered an attractive alternative to conventional antihypertensive drug therapy in mildly hypertensive Type 2 diabetic black as well as white patients. PMID- 3019604 TI - Superoxide anion production and degranulation of rat neutrophils in response to acetaldehyde-altered liver cell membranes. AB - Rat liver membrane vesicles were exposed to acetaldehyde, with or without reduction of the resultant adducts formed. Superoxide anion production and degranulation of rat neutrophils, upon stimulation with the liver membrane vesicles, were measured by cytochrome c reduction before and after the addition of superoxide dismutase, and beta-glucuronidase release respectively. Preincubation with acetaldehyde significantly enhanced superoxide anion production by both the reduced and non-reduced membrane samples (1.7-fold and 4.4 fold, respectively). Preincubation with acetaldehyde significantly enhanced degranulation (1.5-fold) of neutrophils in response to the non-reduced membranes only. The reductive process itself caused a marked increase (2.4-fold) in the ability of the membrane vesicles to stimulate degranulation. Cytochalasin B, an inhibitor of phagocytosis, did not reduce degranulation, implying that it occurred as a consequence of cell surface stimulation. Neutrophil superoxide anion production and lysosomal enzyme release in response to acetaldehyde-altered liver cell membranes could be an important mechanism of hepatocyte injury in alcoholic liver disease. PMID- 3019603 TI - Humoral hypercalcaemia of malignancy: report of two further patients with biochemical studies on tumour extracts. AB - Tumour extracts from two patients with humoral hypercalcaemia of malignancy contained material which stimulated adenylate cyclase in chick renal membranes and in rat osteosarcoma cells. Adenylate cyclase-stimulating activity in each system was inhibited by a specific parathyroid hormone (PTH) antagonist. Studies in two HPLC systems suggested that the adenylate cyclase-stimulating factors extracted from these tumours differed from each other and from synthetic human parathyroid hormone 1-34. The presence of similar PTH-like adenylate cyclase stimulating material(s) in oncogenic osteomalacia suggests that adenylate cyclase stimulating factor(s) may not be the direct or the sole cause of hypercalcaemia. PMID- 3019605 TI - Assessment of serologic markers for Epstein-Barr virus. AB - Antibody responses to early antigen (EA) and viral capsid antigen (VCA) were analyzed in 48 proven cases of Epstein-Barr Virus infection and in 48 age- and sex-matched healthy controls to establish optimal cutoff values for diagnosing EBV infection. Predictive values were determined for individual EA and VCA antibody titers and for EA to VCA antibody ratios and the optimal dilution cutoff values for positivity of EA (1:20), VCA (1:640), and EA to VCA (0:031) were selected. When evaluated on a subset of 10 VCA IgM positive cases and 35 negative controls, the three selected cutoff values identified as infections nine of 10, four of 10, and 10 of 10 of the cases and one of 35, none of 35, and one of 35 of the controls, respectively. When evaluated individually on 22 cases of suspected EBV infection who were heterophile antibody-negative and presented with symptoms compatible with EBV infection, an equal number of VCA IgM-positive and negative cases were identified as EBV infections. Overall, the cutoffs EA, VCA, and ratio identified 19 of 22 (86.4%), 14 of 22 (63.6%), and 18 of 22 (81.8%), respectively, and all cases could be identified using combinations of these values. Although these serologic values may be used with some accuracy, until more definitive markers are described a combination of heterophile responses, lymphocyte analysis, clinical symptoms, and serologic cutoff values should be used to assess the role of EBV in patient evaluation. PMID- 3019606 TI - Evaluation of microsomes isolated from fresh and frozen myotomal tissue of Atlantic cod (Gadus morhua). AB - Microsomes were isolated from fresh and frozen myotomal tissue of Atlantic cod by two procedures. Electron microscopy revealed one method to yield microsomes containing greater quantities of myofibrillar debris than the other and this was reflected as reduced 5'-nucleotidase, acid phosphatase and succinate dehydrogenase marker enzyme activity. Overnight freezing of myotomal tissue did not affect the marker enzyme activity of microsomes isolated by either procedure. Morphological changes were observed among microsomes prepared from myotomal tissue retained for 8 weeks at -30 degrees C. Accordingly, 5'-nucleotidase was marginally elevated and acid phosphatase and succinate dehydrogenase activity reduced in comparison to fresh microsome preparations. PMID- 3019607 TI - Studies on purine turnover in the camel (Camelus dromedarius) and zebu (Bos indicus). AB - Significantly higher hypoxanthine over uric acid ratios were found in camel plasma and urine, with respect to those of zebu. Enzyme levels of purine catabolism were markedly lower in camel than in zebu liver. Oxidation of hypoxanthine appears to be the limiting step of purine metabolism in camel liver. Any hepatic hypoxanthine appears to be actively converted into IMP in camel liver, rather than oxidized to uric acid. PMID- 3019608 TI - Dilated cardiomyopathy: emerging role of endomyocardial biopsy. AB - The development of safe techniques of endomyocardial biopsy has led to a significant increase in our understanding of the etiology and pathogenesis of dilated cardiomyopathy. The scope of patients for whom this technique is absolutely clinically indicated, however, remains quite narrow and should be restricted to those centers with active cardiac transplant programs, large oncology practices, or those involved with active research into the etiology and treatment of patients with heart muscle disease. The most exciting concept to emerge from the use of EMB is the role of myocarditis in the development of dilated cardiomyopathy. It can accurately be stated that a subset of patients with the clinical presentation of dilated cardiomyopathy may in fact have histologic evidence of myocarditis. Although there is a suspicion that immunosuppressive therapy may be helpful, a randomized trial of large numbers of patients is necessary before definitive conclusions may be drawn. If immunosuppressive therapy proves to be efficacious in active myocarditis, then one could argue that all patients with heart failure of unknown cause with no evidence of valvular or coronary artery disease should undergo endomyocardial biopsy as part of the routine diagnostic workup. However, this recommendation must be considered premature. Since routine histologic findings are nonspecific in dilated cardiomyopathy, biochemical, pharmacologic, and cell culture techniques may provide more definitive information regarding the functional state of the heart muscle. In conclusion, endomyocardial biopsy is rapidly emerging as a useful diagnostic tool in the evaluation of patients with heart failure of unknown cause. PMID- 3019609 TI - Mechanisms of in situ nick translation of chromosomes using restriction endonucleases. AB - In situ nick translation of mammalian chromosomes by restriction endonuclease treatment to nick the chromosomal DNA, and 'translation' in the presence of DNA polymerase I and biotinylated dUTP, results in a distinct banding pattern. Further experiments have elucidated the mechanisms producing these bands. The hypothesis is presented that differences in the local conformation of the DNA protein complex, rather than the DNA sequence itself, lead to the nick translation bands. The different DNase I sensitivity along the chromosomes suggests that the bands, which were clearly evident, reflect morphological units closely related to biological functions. PMID- 3019610 TI - Probable occurrence of brain basic protein factor I, an activator of phosphoprotein phosphatases. AB - Basic protein factor I (BPFI was purified to homogeneity from bovine brain by boiling and trichloroacetic acid-precipitation of tissue homogenate, followed by DEAE-cellulose, Sephadex G-150, Affi-Gel-phenothiazine, and Bio-Gel P-6DG chromatographic procedures. The preparation appeared as a single protein band in the SDS-polyacrylamide gel electrophoresis with a minimal Mr of 13,200. The factor was a basic protein as indicated by an estimated isoelectric point of pH 8.3 and a high content of amino acids including arginine, histidine, lysine and others. In the absence of Mn2+, the factor stimulated the phosphoprotein phosphatases (PPase) from rabbit brains. Unlike histones or protamine, the factor was a poor substrate for megamodulin-dependent protein kinase. In addition, the factor did not interact significantly with E. coli megamodulin. PMID- 3019611 TI - Chronic asthma due to toluene diisocyanate. AB - Twelve subjects were studied with inhalation challenge testing to toluene diisocyanate (TDI) because of suspected TDI asthma based on a consistent clinical and occupational history. In seven cases, TDI asthma was documented by a positive inhalation challenge to low levels of TDI. Six of the seven TDI reactors had persistent respiratory symptoms and required daily treatment even though they had been removed from isocyanate exposure for intervals as long as 12 years (mean 4.5 years). These six TDI reactors had either dual (four cases) or late bronchospasm (two cases) to less than 20 ppb TDI, and all had a positive methacholine or cold air challenge prior to study. The one TDI reactor with a negative methacholine challenge had a positive (immediate) bronchospastic response to a TDI challenge performed one week after removal from isocyanate exposure. Five workers had a negative TDI challenge, two of whom had persistent respiratory symptoms and positive methacholine challenges at the time of TDI inhalation testing. We conclude that respiratory symptoms may persist following long-term removal from occupational exposure to TDI and are associated with nonspecific bronchial hyperreactivity. The TDI sensitivity may also persist for a long time even in the absence of any additional occupational exposure. Long-term prospective studies of symptomatic isocyanate workers are needed to fully define the extent of this problem. PMID- 3019612 TI - Airway inflammation and airway hyperresponsiveness. PMID- 3019613 TI - Crystalluria and ciprofloxacin, influence of urinary pH and hydration. AB - Ciprofloxacin is one of the newer 4-quinolones. It combines a high antibacterial activity and a broad spectrum with favourable pharmacokinetic properties. The present study was designed to detect the influence of urinary pH and fluid consumption on crystalluria. Six healthy volunteers aged 25-41 years, 3 of each sex, participated in the study. Single doses of 1,000 and 500 mg of ciprofloxacin were given orally. The urinary pH was varied by giving each subject three different diets: a regular diet, a diet supplemented by ammonium chloride to acidify urine, and a diet supplemented by sodium bicarbonate to obtain alkaline urine. The urine volume and pH were measured and microscopically examined at 37 degrees C immediately after voiding. After the very high dose of 1,000 mg ciprofloxacin the regular diet regimen led to crystalluria in only one subject. Even with this high dose, but with the acidifying regimen, no crystals were observed in any one of the volunteers. When bicarbonate was supplemented 5 to 6 volunteers presented crystals in 22 of the 36 urine samples. 21 of the crystalluric urine samples showed a pH greater than or equal to 7.3. After the usual 500-mg dose and regular diet no crystals were observed at all; only in 3 subjects who received bicarbonate supplement crystals have been seen. In the urine of two subjects crystals emerged 'ex vivo' after some hours of storage at both 37 degrees C and room temperature; these results show the importance of sediment observation at 37 degrees C immediately after voiding to differentiate between real and 'ex vivo' crystalluria. Results of different examinations permit the conclusion that the crystals contain mostly unchanged ciprofloxacin as major component and magnesium as characteristic element. Participation of the metabolite 2 in the crystal formation cannot be excluded. No significant change was observed in blood counts and blood chemistry of any subject. Urinalysis showed no modifications except the eventual presence of the typical drug-related crystals. Hematuria never occurred. PMID- 3019615 TI - [The roentgenologic manifestations of primary malignant fibrous histiocytoma (MFH) of the bone]. PMID- 3019616 TI - [Malignant mixed tumor of the salivary glands: a clinicopathological analysis of 15 cases]. PMID- 3019614 TI - Heterogeneity of [3H]ethyl beta-carboline-3-carboxylate binding sites and [3H]gamma-aminobutyric acid binding sites. AB - Binding properties of [3H]flunitrazepam ([3H]FNZ), [3H]ethyl beta-carboline-3 carboxylate ([3H]beta CCE), [3H]muscimol ([3H]MUSC) and [3H]gamma-aminobutyric acid ([3H]GABA) to bovine cortical membranes and to their Triton extracts were studied. GABA, 1 X 10(-5) M, stimulated [3H]FNZ binding of frozen, thawed and washed membranes by an increase in affinity without alteration of the maximal number of binding sites, and this GABA stimulated [3H]FNZ binding can be inhibited by bicuculline methobromide. Freeze, thaw and wash with Triton X-100 removed the low affinity [3H]MUSC binding sites. The ratios of [3H]FNZ binding sites to [3H]beta CCE binding sites and [3H]MUSC binding sites to [3H]GABA binding sites were always found to be about 1:2 in both membranes and soluble extracts. The fact that [3H]beta CCE, after displaced by clonazepam, can be further displaced by unlabelled beta CCE from its binding sites and that a portion of [3H]beta CCE binding sites can be survived from FNZ-photolysis implied that there are at least two subclasses of beta CCE binding sites, one is sensitive to beta CCE only and the other is sensitive to both beta CCE and benzodiazepines (BZs). [3H]GABA, after displaced by MUSC, can be further displaced by unlabelled GABA from its binding sites. The results also support that there are two subclasses of BZ-related GABA binding sites, one is sensitive to GABA only and the other is sensitive to both GABA and MUSC. Furthermore, the decay rates of [3H]beta CCE and [3H]GABA binding activities exposed to various degree of electron bombardment are identical, which is evident that these two binding sites are believed to be functional associated in a macromolecular complex with molecular mass about 220,000 Mr. PMID- 3019617 TI - [Na, K+-ATP-ase activity of erythrocyte membranes in progressive muscular dystrophy]. PMID- 3019619 TI - [Experimental study of multiple organ failure its relation to cAMP and ATP]. PMID- 3019618 TI - [Surgical treatment of thymic tumor and myasthenia gravis]. PMID- 3019621 TI - [Action of monoclonal antibody against a hepatocellular carcinoma cell line (PLC/PRF/5)]. AB - Monoclonal antibody (A9-84) against a hepatocellular carcinoma cell line (PLC/PRF 5) was produced by somatic cell fusion. The hybridoma clones were screened by a rapid solid-phase enzyme-linked binding assay. The target cells were cultured in 96-well Linbro plate and fixed by methanol for screening. The specificity of the antibody was studied by enzyme-linked binding assay and immunofluorescence methods. It shows that A9-84 do not respond to 8 different human cancer cell lines (4 liver cancer, 1 esophageal cancer, 1 stomach cancer, 1 multiple myeloma and 1 lymphoblast cell line) and the peripheral mononuclear cells of 91 normal subjects. A9-84 is the subtype of IgG3. It is capable of inhibiting the growth of cultured PLC/PRF/5 cells with or without complement. PMID- 3019622 TI - [Carcinoid of ectopic pancreas tissue--islet cell tumor]. AB - A case of islet cell tumor--carcinoid of the ectopic pancreas is reported. The patient had been operated six times because of recurrent abdominal tumor mass and intermittent hypoglycemic coma. The first five operations were done with the diagnosis of mesotheliosarcoma by pathology. The last operation was done in our hospital with the final diagnosis of carcinoid--islet cell tumor. In addition to the light microscopic view, several diagnostic features were observed as follows: Under electron microscopy, membrane bound secretive granules were present in the tumor cytoplasm. Neither brush-like microvilli nor lumen formation of the mesothelioma were found. By immunohistochemistry, positive granules to immunoperoxidase stain of insulin were observed in some tumor cytoplasm. By laboratory test, levels of blood 5-hydroxytryptamine and urinary 5 hydroxyindolacetic acid were elevated. Sometimes it is difficult to differentiate carcinoid from islet cell tumor only by morphology, because both are derived from the same origin during the embryonic development which is probably related to the endocrine system from the embryonic foregut. The latter arises from the neuro ectoderm and belongs to APUD system. PMID- 3019620 TI - Construction, analysis, and application to 46,XY gonadal dysgenesis of a recombinant phage DNA library from flow-sorted human Y chromosomes. AB - The analysis of a recombinant human Y-enriched Hind III total digest phage library prepared from the DNA of flow sorted human Y chromosomes is described. Out of 43 phage inserts from the library thus far mapped, 25 revealed hybridization with Y chromosomal DNA. These inserts may be divided into five groups according to their degree of Y specific hybridization: inserts that hybridize with one single copy or slightly repeated Y-specific DNA sequence, Y specific repeated sequences of various restriction fragment lengths, Y chromosomal DNA sequence(s) shared by a sequence on the X and/or on autosomes, Y specific DNA sequences in addition to multiple X and/or autosomal sequences, or Y specific repeated DNA in addition to multiple X and/or autosomal sequences. Application of probes from this library for diagnostic purposes is shown in two 46,XY patients with gonadal dysgenesis and small deletions of the Y short arm. PMID- 3019624 TI - [Infantile acro-localized papulovesicular syndrome]. AB - Papular acrodermatitis (Gianotti-Crosti syndrome) was seen in a six-year-old girl. The disease was marked by the characteristic triad of a papular-vesicular rash, lymphadenopathy and liver damage. Serological findings suggest an infection with Epstein-Barr virus as the causative factor. In such cases hepatitis-B induced papular eruptive acrodermatitis should be considered in differential diagnosis. PMID- 3019623 TI - Prevalence of viral warts on the hands of retail butchers. AB - The prevalence of viral warts on the hands was found to be the same in a group of 113 workers in butcher's shops as in a control group of 115 mechanics. This low prevalence is discussed and statistically compared to previous studies in the literature. PMID- 3019625 TI - [Adrenomyeloneuropathy, a rare cause of primary adrenal cortex insufficiency]. AB - Inherited via the X chromosome, adrenomyeloneuropathy is a rare cause of primary adrenocortical insufficiency. Neurological signs are of central and peripheral demyelinization, while endocrinologically it is characterized by Addison's disease and primary testicular insufficiency. In two patients with this condition the metabolic defect in the breakdown of long-chain fatty acids was confirmed by an increased hexakosan (C 26) blood level. One patient had an isolated failure of the zona fasciculata; in the other there was clinically manifest complete adrenocortical insufficiency. Both patients had incipient hypogonadism. In the second case, neurological symptoms preceded the endocrinological ones, while in the first both the family history and the adrenocortical insufficiency led to the diagnosis. In peripheral neuropathy in a young male, attention should always be given to signs of incipient adrenocortical insufficiency. PMID- 3019627 TI - [IBR/IPV serodiagnosis with ELISA methods in blood, single milk and bulk milk samples; control measures for maintaining normal cattle herds; eradication measures with reference to vaccination]. PMID- 3019626 TI - [Concentrating of leukosis antibodies in bulk milk (using a dialysis system) for study in an ELISA system]. PMID- 3019628 TI - [Morphological changes caused by adrenalectomy in rabbits]. PMID- 3019629 TI - [Toxicity of the combination preparation nivalin-P]. PMID- 3019630 TI - [Antimetastatic action of bacterial endotoxins and changes in the activity of the enzymes of purine metabolism in macrophages]. AB - The antimetastatic action of bacterial endotoxins (BET), E. coli 0111:B4, B in particular, as well as their influence on the adenosine deaminase and 5' nucleotidase activity were studied in peritoneal macrophages of mice bearing lung Lewis carcinoma. BET inhibition of lung metastasis growth was found to be due to such changes in macrophage adenosine metabolism, that testifies to the rise of their functional activity. The change in the level of macrophage purine metabolism can be considered as an important evidence of the effectiveness of the drugs capable to inhibit the metastasis growth. PMID- 3019631 TI - Presence of luteinizing hormone releasing hormone (LHRH) in milk. AB - Evidence is presented a luteinizing hormone-releasing hormone (LHRH)-like substance exists in milk of the rat and that this substance is transferred to the offspring during suckling. This milk "LHRH" generates an inhibition curve parallel to authentic LHRH in the LHRH radioimmunoassay (RIA) and coelutes, to a significant extent, with LHRH upon Sephadex G-25 chromatography. An increase in LHRH-like immunoreactivity (LHRH-LI) can be detected in both the stomach and plasma of infantile rats during suckling. Available ovarian LHRH receptors increase in the pups after separation of the mother and return to basal values following suckling. This change is prevented by i. v. administration of an LHRH antiserum. Milk LHRH stimulates gonadotropin release in vitro and inhibits both gonadotropin-induced steroid secretion from granulosa cells in culture and the binding of LHRH to ovarian membranes. PMID- 3019632 TI - Activation of the rat kidney mineralocorticoid receptor. AB - Activation of the rat kidney mineralocorticoid receptor was investigated using diethylaminoethyl (DEAE)-cellulose, DNA-cellulose, and gel permeation chromatography. Specific labeling of the mineralocorticoid receptor was achieved by labeling with [3H]aldosterone in the presence of the pure glucocorticoid RU28362. The specificity of labeling was confirmed by the lack of immunoreactivity of [3H]aldosterone-labeled material with the monoclonal antiglucocorticoid receptor antibody BURG-1. The unactivated aldosterone mineralocorticoid receptor complex did not bind to DNA-cellulose, was eluted from DEAE-cellulose at relatively high salt (195 mM KCl) concentration, and had an apparent Stokes radius when chromatographed on Sephacryl S300 of 6.3 nm. After activation, the aldosterone-mineralocorticoid receptor complex had increased affinity for DNA-cellulose and decreased affinity for DEAE-cellulose and appeared as a smaller complex when chromatographed on Sephacryl S300. These changes were blocked by sodium molybdate. Our results indicate that activation of the rat kidney mineralocorticoid receptor is analogous to activation of the glucocorticoid receptor and suggest that activation of the mineralocorticoid receptor involves dissociation of a multimeric receptor form. PMID- 3019633 TI - Role of arachidonic acid in the regulation of adrenocorticotropin release from rat anterior pituitary cell cultures. AB - In addition to cAMP-dependent mechanisms, stimulation of pituitary ACTH secretion by various stimuli, including CRF, may involve phospholipid and arachidonic acid turnover. To determine the role of phospholipase A2 activation in corticotroph function, we studied the effect of exogenous arachidonic acid, phospholipase A2, and the phospholipase A2 activator melittin on ACTH release in cultured rat anterior pituitary cells. Incubation with 1-100 micron arachidonic acid, 0.01-1 micron melittin, 0.1-10 U/ml phospholipase A2, and 0.01-10 nM CRF caused dose dependent increases in ACTH release to 8.1 +/- 1.1- (+/- SE), 16.2 +/- 0.9-, 13.6 +/- 1.2-, and 2.9 +/- 0.3-fold; respectively. The participation of the major pathways of arachidonic acid metabolism in the control of ACTH release was analyzed in cells treated with nordihydroguaiaretic acid, a lipoxygenase inhibitor; indomethacin, a cycloxygenase inhibitor; and 5,8,11,14 eicosatetraynoic acid, an inhibitor of both pathways. The effects of arachidonic acid, melittin, and CRF were partially blocked by 10 micron nordihydroguaiaretic acid and 5,8,11,14-eicosatetraynoic acid, but were significantly enhanced by 10 micron indomethacin. These results suggest that arachidonic acid is mainly metabolized through the lipoxygenase pathway to a stimulatory metabolite and, to a lesser extent, through the cycloxygenase pathway to an inhibitory metabolite. Arachidonic acid release from anterior pituitary cells labeled with [3H]arachidonic was analyzed during cell column perifusion and stimulation by CRF and other secretagogues. Two-minute pulses of CRF (10 nM), vasopressin (10 nM) and phorbol 12-myristate 13-acetate (100 nM) caused immediate 1.5- to 2-fold increases in [3H]arachidonic acid release, and melittin (100 nM) caused a 5-fold increase in [3H]arachidonic acid release. The ability of both exogenously added and endogenously generated arachidonic acid to stimulate ACTH secretion, together with the stimulation of arachidonic acid release by ACTH secretagogues and the attenuation of stimulated ACTH release by lipoxygenase blockers, indicate that lipoxygenase products of arachidonic acid metabolism participate in the control of ACTH secretion. PMID- 3019634 TI - Effects of N-ethylmaleimide on gonadotropin and beta-adrenergic receptor function coupled to rabbit luteal adenylyl cyclase. AB - The effects of the sulfhydryl-reactive alkylating agent N-ethylmaleimide (NEM) on the rabbit luteal adenylyl cyclase system were studied. Treatment of luteal membranes with NEM revealed three activities with differing sensitivities to NEM treatment. When luteal membranes were treated with NEM on ice for 30 min, it was found that NaF-stimulated adenylyl cyclase activity in cholate extracts of these membranes was most sensitive to this treatment. Half-maximal inhibition was obtained at 0.09 mM NEM. The activity of the stimulatory guanine nucleotide- and Mg-binding regulatory component (Ns), as assessed by functional reconstitution of NaF-stimulated adenylyl cyclase activity into membranes from the cyc-variant of the S49 mouse lymphoma, was less sensitive to this treatment, with half-maximal inhibition occurring at 0.69 mM NEM. In contrast, high affinity gonadotropin and beta-adrenergic binding, as assessed by competitive displacement of [125I]iodo hCG by bovine LH and (-)3-[125I]iodocyanopindolol by isoproterenol, was unaffected by NEM concentrations up to 50 mM when membranes were treated on ice. However, when membranes were treated with NEM at 25 C for 30 min, high affinity gonadotropin and beta-adrenergic binding demonstrated similar sensitivities to NEM treatment, such that 50 mM NEM completely inhibited high affinity binding to both receptors. Under either of the conditions described above, neither the number of receptors nor the affinities of the labeled probes for their receptors were altered by NEM treatment. Thus, there appears to be at least three NEM sensitive sites necessary for the functioning of the rabbit luteal adenylyl cyclase system, one associated with the catalytic component, one on Ns which interacts with the catalytic component, and one involved in high affinity agonist binding. Furthermore, it appears that formation of the high affinity binding state is regulated similarly for gonadotropin and beta-adrenergic receptors. PMID- 3019635 TI - The dynamics of steroid and adenosine 3',5'-cyclic monophosphate output in perifused interstitial cell aggregates derived from prepubertal rat testes. AB - The characteristics of the steroidogenic response of reaggregated rat interstitial cells were examined in a perifusion system. Interstitial cells were isolated from 19-day-old rat testes by digestion with collagenase. The cells were cultured for 3 days as monolayers and were resuspended by brief treatment with trypsin. Constant gyratory shaking of the dispersed cells resulted in the formation of round and compact aggregates of 70-140 microns. The functional characteristics of these aggregates were examined by studying the output of cAMP, C19 steroids (testosterone and androstenedione), and C21 steroids (progesterone, 17 alpha-hydroxyprogesterone, 20 alpha-hydroxypregn-4-en-3-one) in a perfusion system. It is demonstrated that reaggregated interstitial cells maintain their responsiveness to LH, LHRH, and Leydig cell stimulatory factor(s) produced by Sertoli cells for at least 12 days. When exposed to low concentrations of LH (1 ng/ml), either in a continuous or in a pulsatile fashion, perifused aggregates maintain a constant output of steroids for more than 20 h. Under these conditions, LH-dependent differentiation of the steroidogenic machinery can be observed in vitro. In fact, although the sum of the measured steroids remains constant, C21 steroids progressively decrease whereas C19 steroid output increases during perifusion. When perifused with high concentrations of LH (10 ng/ml), desensitization becomes the predominant phenomenon. It is demonstrated that the steroid output of reaggregated interstitial cells considerably exceeds that of similarly treated cells maintained as monolayers. Moreover, perifusion of aggregates results in a 6-fold increase in steroid output as compared to static incubation and in a selective increase in androgen output. It is concluded that prepubertal interstitial cells allowed to reaggregate in suspension culture form functional multicellular structures. Perifusion of these aggregates is a useful tool in the study of the dynamics of the regulation of steroidogenesis. PMID- 3019636 TI - Central administration of corticotropin-releasing factor modulates oxytocin secretion in the rat. AB - Recently, it has been reported that oxytocin (OT), classically known for its function during parturition and lactation, is secreted in response to stressful stimuli in male rats. In these and in the present report it was found that swimming stress, restraint stress, ether stress, and footshock stress elevate OT secretion without affecting arginine-vasopressin (AVP) secretion. In the present studies, we investigated the possible modulation of OT secretion by CRF which is known to be released during stress. Male and female rats received intraventricular (icv) injections of 0.75 nmol (5 micrograms) rat CRF and were killed 5 min after the treatment. CRF significantly elevated OT secretion in male and female rats 3.4- and 4-fold, respectively. Plasma AVP levels were not affected by the treatment. The effect of CRF on OT release was structure specific since rat CRF, ovine CRF, and sauvagine were equipotent releasers of OT while an inactive analog to CRF, ovine CRF did not change plasma OT levels. In another set of experiments rats were pretreated with either CRF-antiserum (0.5 ml iv) or dexamethasone (20 micrograms/rat ip) and then injected with icv CRF. Both CRF antiserum and dexamethasone blocked the rise in ACTH release after icv CRF completely but did not influence the OT response. This suggests CRF may be acting centrally but not at the level of the neurohypophysis to change OT secretion. Since parvocellular but not magnocellular neurons of the paraventricular nucleus have been demonstrated to be steroid sensitive in immunohistochemical studies, we suggest CRF may act directly or indirectly upon magnocellular neurons to increase OT release. Intravenous administration of 0.75 nmol CRF increased both OT and AVP levels in peripheral blood. The magnitude of this increase was similar (2- to 4 fold stimulation) to responses after icv administration of CRF. Intravenous administration of CRF results in hypotension and may therefore cause a baroreceptor mediated release of AVP and OT. From the above evidence we conclude: physical and mental stresses which do not result in changes in blood volume or osmolality evoke an increase in OT secretion while AVP secretion remains unchanged; CRF administered icv mimics OT responses observed after ether stress or footshock stress; CRF may play a role in regulating stress-induced OT secretion in the rat. PMID- 3019637 TI - Binding studies with rat myometrial and mammary gland membranes on effects of manganese on relative affinities of receptors for oxytocin analogs. AB - Mg2+ increases the potency of oxytocin (OT) analogs in stimulating uterine contractions. Generally, the enhancing effects of Mg2+ are inversely related to the potency of the peptide. To determine the site of metal ion action, we measured the effects of Mn2+, another potentiating metal ion, on the ability of a series of peptides to inhibit the binding of [3H]OT to receptor sites on both uterine myometrial and mammary gland plasma membranes. The analogs used in this study were derivatives of 7-glycine oxytocin, which is about 10 times more active when the Mg2+ concentration in the uterine smooth muscle bath is increased from 0 to 0.5 mM. We found a generally good correlation between the ability of the analogs to inhibit [3H]OT binding to both receptor systems and their biological potencies. An increase in Mn2+ concentration from 1 to 10 mM enhanced the affinity of uterine membranes for the analogs, in inverse proportion to their potencies. This selective enhancement occurred regardless of the structural modification of peptide. These results suggest that the metal ion effect occurs at the receptor level and is not a property of the peptide per se. In contrast to the uterus, the affinities of mammary gland receptors for two low potency analogs were unaffected by increased Mn2+ concentration. The mechanisms of the metal ion effect are not entirely understood, but it appears that Mn2+ allows the conformation of the myometrial receptor to adapt to less well-fitting ligands. Although the metal ion effects on mammary gland receptors are more difficult to interpret, it is clear that uterine and mammary gland receptors are different with respect to the mechanisms of interaction with peptides. PMID- 3019638 TI - Tissue and gene specific hypomethylation of the human parathyroid hormone gene: association with parathyroid hormone gene expression in parathyroid glands. AB - An association between decreased cytosine methylation at specific sites adjacent to or within eukaryotic genes and increased gene expression has been described. To determine if tissues secreting PTH show hypomethylation within the vicinity of the PTH gene, we compared the degree of cytosine methylation in DNA from parathyroid glands and control tissues (leukocytes, anterior pituitary, posterior pituitary, and placenta). We digested DNA with HpaII (which cleaves only unmethylated CCGG sequences) and MspI [which cleaves CCGG and C 5-methyl cytosine GG], hybridized the DNA fragments to a PTH complementary DNA probe, and scanned autoradiograms of Southern blots. After MspI digestion all tissues yielded equivalent amounts of a single hybridizing fragment of 6.7 kilobases. The degree of hypomethylation at sites within and flanking the PTH gene was determined as the ratio of the amount of hybridizing fragments obtained by methylation sensitive digestion (HpaII) relative to methylation-insensitive digestion (MspI). DNA from all parathyroid glands showed significantly greater hypomethylation of the PTH gene than did DNA from control tissues that did not express the PTH gene. Despite variability in the levels of secretory activity of the different parathyroid glands, we found no significant differences in the degree of hypomethylation of the PTH gene. In contrast to the PTH gene studies, hypomethylation was not seen using GH probe on the same blots. Our findings thus suggest that tissue and gene specific hypomethylation of the PTH gene is associated with expression of the gene. PMID- 3019639 TI - Characterization of in vitro dopamine synthesis in the median eminence of rats with haloperidol-induced hyperprolactinemia and bromocriptine-induced hypoprolactinemia. AB - In order to investigate the mechanism of PRL action on dopamine synthesis in tuberoinfundibular dopaminergic (TIDA) neurons, in vitro dopamine synthesis in the median eminence of hypothalamic slices was compared between hyperprolactinemic and hypoprolactinemic rats, Hyper- and hypoprolactinemia were induced in ovariectomized rats by repetitive injections of the dopamine antagonist haloperidol (Halo) and the dopamine agonist bromocriptine (Bromo), respectively. In vitro dopamine synthesis in TIDA neurons was estimated by measuring 3,4-dihydroxyphenylalanine (DOPA) accumulated in the median eminence after incubation of hypothalamic slices with a DOPA decarboxylase inhibitor. Treatment with Halo or Bromo produced increases or decreases, respectively, in the concentration of PRL in serum and in in vivo DOPA accumulation in the median eminence, as compared with vehicle treatment. The basal rate of in vitro DOPA accumulation in the median eminence was increased in Halo-treated rats and was decreased in Bromo-treated rats. The increase in basal DOPA accumulation after Halo treatment was inhibited by Ca2+ removal from medium or tetrodotoxin addition. A CA2+ -dependent increase in DOPA accumulation in the median eminence by depolarization was greater in Halo-treated rats than in Bromo-treated rats. This difference in DOPA accumulation was due to the changes in PRL secretion after Halo and Bromo treatments, since hypophysectomy abolished it. Incubation of hypothalamic slices in Na+-free media to increase the intracellular concentration of Ca2+ through inhibition of Na+-Ca2+ exchange caused an increase in DOPA accumulation. The rate of DOPA accumulation in Na+-free media was increased in Halo-treated rats and was decreased in Bromo-treated rats. On the other hand, neither Halo nor Bromo treatment altered the increase in DOPA accumulation induced by (Bu)2cAMP or forskolin. These results support the view that PRL stimulates dopamine synthesis in TIDA neurons by mechanisms which include an increase in the firing rate of TIDA neurons and increased depolarization-induced synthesis due to an enhanced response of the component that regulates dopamine synthesis to intracellular Ca2+. PMID- 3019640 TI - Adenylate cyclase inhibition is not involved in the adrenal steroidogenic response to angiotensin II. AB - Angiotensin II (AII) receptors in adrenal glomerulosa cells are coupled to adenylate cyclase inhibition. We investigated the importance of cyclase inhibition in adrenal steroidogenesis by treating adrenal glomerulosa cells with the toxin of Bordetella pertussis (20 ng/ml) for 3 and 18 h. This treatment prevented inhibition of forskolin-stimulated adenylate cyclase by AII. However, the aldosterone response to AII was not altered by toxin treatment. These results strongly suggest that adenylate cyclase inhibition is not directly involved in mediating the adrenal actions of AII. In addition, ACTH-induced steroidogenesis also was unaffected by toxin treatment demonstrating that cyclase inhibition is not involved in suppressing steroidogenesis via the cAMP pathway. PMID- 3019641 TI - Mechanism of action of dopamine on the in vitro release of gonadotropin-releasing hormone. AB - Controversy exists on whether dopamine (DA) stimulates or inhibits GnRH secretion and whether its effects are mediated via alpha-adrenergic receptors or dopaminergic receptors. As a means to examine this conflict, we have utilized an in vitro superfusion system to study the effects of DA, norepinephrine (NE), phentolamine (alpha-antagonist), pimozide (DA antagonist), and two DA agonists (apomorphine and bromocryptine) on GnRH release from isolated mediobasal hypothalami from adult male rats. In this dynamic system, graded concentrations of both NE and DA (2.0 nM to 2.0 microM) led to a dose-dependent increase in GnRH output during the 10 min interval that followed each pulse dose of NE (P less than 0.02) or DA (P less than 0.05). The DA-induced GnRH release was reproducible, consistent, and significant over five successive pulses (20 microM) at 30-min intervals (P less than 0.02). Coinfusion of phentolamine (20 microM) prevented the DA (20 microM) induced release of GnRH (P less than 0.03), but pimozide (20 microM) had no significant effect on DA-induced GnRH release (P greater than 0.3). The two DA agonists, apomorphine and bromocryptine, at doses up to 2.0 microM and 200 nM, respectively, had no significant effect on GnRH release. To determine whether DA was causing a direct stimulation of alpha adrenergic receptors or being enzymatically converted to NE which could then stimulate alpha-receptors to induce GnRH release, rats were injected with sodium diethyldithiocarbamate (DDC) (550 mg/kg BW) ip, 1 h before death. DDC blocks the enzymatic conversion of DA to NE, and this was reflected by a 37% decrease in hypothalamic NE efflux during the superfusion. However, pulses of DA, even in the presence of DDC, were associated with a marked dose-dependent increase in hypothalamic NE efflux, and DDC failed to prevent the subsequent stimulation of GnRH release. We conclude that the apparent DA-induced release of GnRH is most probably attributable to DA-induced release of hypothalamic NE which, in turn, acts through alpha-adrenergic receptors on peptidergic neurons to stimulate GnRH release. PMID- 3019642 TI - Plasma catecholamines do not participate in pituitary-adrenal activation by immobilization stress in rats with transection of nerve fibers to the median eminence. AB - The effect of immobilization stress was studied in rats in which the CRF and arginine vasopressin-containing innervation of the median eminence was destroyed by an anterolateral cut (ALC) around the medial basal hypothalamus. One week after surgery, the rats with ALC were subjected to immobilization and they showed a normal rise in plasma corticosterone, a smaller than normal rise of plasma ACTH, and an increased response of plasma epinephrine and norepinephrine. When in rats with an ALC the response of plasma catecholamines was prevented by guanethidine pretreatment and adrenal enucleation the small rise in plasma ACTH was unchanged during immobilization. In addition, the plasma corticosterone and ACTH rises during immobilization in the rats with ALC were not influenced by prior treatment with phentolamine (2.5 mg/kg ip) or propranolol (2.5 mg/kg ip). These findings suggest that the large rises in plasma epinephrine and norepinephrine levels during immobilization do not contribute to changes in plasma ACTH or corticosterone levels when hypothalamic regulation via CRF and/or arginine vasopressin is interrupted by ALC. PMID- 3019643 TI - Pretranslational and posttranslational mechanisms for regulating beta-endorphin adrenocorticotropin of the anterior pituitary lobe. AB - Stress-induced activation of secretion of ACTH and beta-endorphin (beta-END) from anterior lobe corticotrophs leads to both short term and longer term perturbation of the system. Immediately following an acute stress session, the rate of translation of the ACTH/beta-END precursor proopiomelanocortin appears accelerated by 50% and the rate of conversion of the precursor into products is doubled. These changes appear to take place at the translational and posttranslational level and reflect a better use of the preformed messenger RNA which compensates for the stress-induced peptide depletion. When the animal is subjected daily to the stress session, longer term mechanisms appear to emerge. The ACTH/beta-END stores in the gland are increased, apparently owing to an increase in transcription, as reflected by a small but significant increase in proopiomelanocortin messenger RNA. The posttranslational processing is no longer accelerated after further stress. This longer term mechanism appears to be pretranslational and to supplant the posttranslational mechanisms observed after acute stress. These two levels of control may represent different points in the regulation of this critical peptide system. PMID- 3019644 TI - Guanyl nucleotide regulation of human chorionic gonadotropin binding to rat luteal tissue. AB - Guanyl nucleotides are known to have a dual effect on most hormone-sensitive adenylate cyclase systems, regulating activation of the adenylate cyclase enzyme and binding of hormone to receptor. In the ovary, guanyl nucleotides have been shown to be required for hCG stimulation of luteal adenylate cyclase, but no effect on binding has been observed. Evidence has been obtained which suggests that guanyl nucleotide regulation of hCG binding is masked in most luteal membrane preparations by the presence of a large excess of unregulated receptor. A highly purified urea-washed membrane fraction has been prepared. Binding of hCG to this preparation was decreased (approximately 45%) in the presence of 5' guanylylimidodiphosphate (GMPPnP). The maximal effect of GMPPnP occurred at 10 microM, with the half-maximal effect at 0.2 microM. These levels compare favorably with the GTP concentrations required for hCG stimulation of luteal adenylate cyclase. Analysis of equilibrium binding experiments showed that GMPPnP acted by reducing the number of binding sites by 45%. However, kinetic experiments suggested that this effect was due to a significant decrease in the affinity of a fraction of the hCG-binding sites. Association of hCG and its receptor was unaffected by the presence of GMPPnP (100 microM), whereas the dissociation of 40-50% of bound hormone was significantly accelerated (30-fold) by its presence. The guanyl nucleotide effect required the presence of MG+2; other divalent cations (Ca+2, Mn+2, and Co+2 could not be substituted. The ratio of beta-adrenergic to hCG-binding sites in the urea-washed heavy membrane preparation was elevated, suggesting that a sizable fraction of uncoupled hCG receptor had been removed. The results show that hCG binding to its luteal receptor is modulated by guanyl nucleotide and suggest that the modulation only occurs in those receptors that are directly coupled to the adenylate cyclase enzyme. PMID- 3019645 TI - Increased pituitary sensitivity to glucocorticoid feedback during the stress nonresponsive period in the neonatal rat. AB - Neonatal rats show a diminished response to stress [the stress-nonresponsive period (SNRP)] from day 2-3 until day 14 of age; the physiological bases for the SNRP are unknown. We examined whether enhanced sensitivity of the brain or pituitary to the inhibitory feedback effects of circulating glucocorticoids (GC) contributes to the SNRP. Age-related changes in the ability of corticosterone (CORT) and dexamethasone (DEX) to inhibit the ACTH secretion induced by urethane or CRF were studied. We also examined the ACTH response to ether stress or CRF in intact or 24 h-adrenalectomized 5-day-old rats. Plasma ACTH did not increase in intact rats after ether stress (basal: 64.6 +/- 9.1 pg/ml vs. stressed: 66.8 +/- 8.9 pg/ml; P greater than 0.05), whereas small elevations occurred after CRF challenge (184.6 +/- 40 pg/ml; P less than or equal to 0.01). Five-day-old adrenalectomized rats, which had elevated basal ACTH concentrations, increased ACTH secretion after exposure to ether or CRF. Thus, negative feedback appears to mediate critically the SNRP. Furthermore, sensitivity to such feedback was enhanced during the SNRP since the capacity of CORT to inhibit urethane-induced ACTH secretion in vivo declined with age; 1 mg/kg BW was the minimal dose that inhibited ACTH secretion at day 10, whereas at day 18, the threshold for a similar inhibition was 5 mg/kg BW. In contrast, at both ages, a dose of 10 micrograms/kg BW DEX inhibited ACTH release. In vitro dose response studies in whole pituitaries further demonstrated the enhanced pituitary sensitivity to GC feedback during the SNRP since the IC50 for CORT inhibition of CRF-induced ACTH release increased from days 3-5 to days 22-23. A similar, although not statistically significant trend was observed for DEX inhibition. Thus, neonatal rats exhibit an enhanced pituitary sensitivity to GC during the SNRP and removal of this inhibition allows ACTH secretion in response to ether stress. PMID- 3019646 TI - Adenosine 3',5'-monophosphate-binding capacity in small and large ovine luteal cells. AB - The photoaffinity analog [32P]8-azidoadenosine cAMP ([32P]8-N3cAMP) was used to study available cAMP-binding sites on cAMP-dependent protein kinases in homogenates of ovine small (12-22 microns) and large (greater than 22 microns) luteal cells. Binding of the analogs to specific proteins was detected by autoradiography after sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and was quantitated by liquid scintillation counting. In homogenates of untreated small and large cells, only the type I isoenzyme of the regulatory subunit (R1) of protein kinase had a measurable number of available cAMP-binding sites. Small luteal cells were incubated for 2 h with increasing concentrations of ovine LH (oLH), cholera toxin, or forskolin, and media were collected for quantification of cAMP and progesterone. Cells were harvested and homogenized, and intracellular cAMP content and photoincorporation of [32P]8 N3cAMP into RI were measured. Treatment of small luteal cells with oLH, cholera toxin, and forskolin resulted in dose-dependent increases in cAMP in both the cells and the incubation media and in the progesterone content of the media. These increases were accompanied by a dose-dependent decrease in photoincorporation of [32P]8-N3cAMP into RI of small cells, which was correlated (P less than 0.05) with the increase in progesterone secretion. A time-dependent decrease (P less than 0.05) in photoincorporation in small cells incubated with oLH (100 ng/ml), cholera toxin (1000 ng/ml), or forskolin (5 microM) preceded an increase (P less than 0.05) in progesterone secretion by these cells. Large ovine luteal cells incubated with increasing concentrations of cholera toxin or forskolin demonstrated a dose-dependent decrease in photoincorporation of [32P]8 N3cAMP into RI. The time-dependent decrease (P less than 0.05) in photoincorporation in large cells incubated with cholera toxin (1000 ng/ml) or forskolin (5 microM) was not followed by enhanced progesterone secretion. These observations are consistent with a role for cAMP involvement in progesterone secretion by ovine small luteal cells and suggest a lack of cAMP involvement in progesterone synthesis by large cells. PMID- 3019647 TI - Spontaneous diabetic BB rat: studies of cyclic adenosine 3',5'-monophosphate phosphodiesterase and calmodulin. AB - Uncontrolled diabetes in man is associated with increased plasma and tissue levels of cAMP and decreased cAMP phosphodiesterase (PDE) activity. Spontaneously diabetic BB rats (SDR) were used in these experiments. Specific tissues (i.e. liver and epididymal fat) were studied without therapeutic insulin. Another group of normal animals were rendered diabetic by streptozotocin (STZ) and killed without benefit of insulin therapy. Calmodulin (CM), a small molecular weight protein essential for activation of specific cAMP PDE was assayed. STZ diabetes is associated with a decrease (58%) in CM biological activity and in immunoreactive CM in fat (69%) and liver (13%) tissues. Similarly, SDR rats and the nondiabetic genetic controls (NDR) demonstrate decreased CM bioactivity in fat (76% and 56%, respectively) and decreased CM immunoreactivity in liver (68% and 74%, respectively) compared to normal control rats. In addition, maximum velocity (Vmax) of the low Michaelis-Menten constant (Km) cAMP PDE is decreased in SDR animals, as compared to controls in both fat (42%) and liver (39%) tissues. Similar data are presented for NDR animals. STZ diabetes is also associated with a reduction in Vmax of the low Km cAMP PDE in both liver (70%) and fat (70%) tissues. These changes found in the NDR animals suggests that the diabetic defect may be under dual regulation: genetic and environmental. PMID- 3019648 TI - The effects of furosemide on adenosine 3':5'-cyclic monophosphate, guanosine 3':5'-cyclic monophosphate and corticosterone production stimulated by adrenocorticotropin in monolayer cultured rat adrenal cells. AB - Furosemide has been reported to have a suppressive effect on ADH-, PTH- and adrenaline-stimulated adenosine 3':5'-cyclic monophosphate (cAMP) production, but the effect on adrenocorticotropin (ACTH) action has not yet been elucidated. In the present study, therefore, the effects of furosemide on cAMP and also on guanosine 3':5'-cyclic monophosphate (cGMP) and corticosterone, stimulated by ACTH in monolayer cultured rat adrenal cells, were investigated. The intra- and extracellular cAMP stimulated by ACTH was dose-dependently suppressed by furosemide within the concentration range of 10(-3) M to 3 X 10(-3) M, and the suppressive effect of the drug was accompanied with decreased corticosterone production. However, non-stimulated basal corticosterone production was not influenced by the drug even at 3 X 10(-3) M. A similar suppressive effect of dibutyryl cAMP-stimulated corticosterone production by 3 X 10(-3) M furosemide was observed. The intracellular cAMP bound to its binding protein in sonicated adrenal cell extract was also suppressed in a very similar dose-dependent manner to total cAMP. However, though the effect on corticosterone production was also observed when the calcium concentration in the loading medium was changed, the magnitude of the effectiveness (percent of control) was relatively constant at each calcium concentration, suggesting that furosemide may not affect the site(s) at which calcium acts. Intracellular cGMP, on the other hand, was increased by 10(-3) M to 3 X 10(-3) M of furosemide, suggesting an intensifying effect of furosemide on guanylate cyclase activity. Dibutyryl cGMP-stimulated corticosterone production was also increased at the same concentration range. These results indicated that furosemide may act not only on adenylate cyclase but also on the additional step(s) to suppress the resultant corticosterone production. In contrast to the effects of furosemide on such cAMP-mediated processes, this drug treatment appeared to enhance cGMP-mediated corticosterone production. PMID- 3019649 TI - Effects of an LHRH agonist on endocrine function, LHRH receptor and LH/hCG receptor in the pituitary-gonadal axis of male rats. AB - The effects of an LHRH agonist (LHRHa), [D-Ser (tBu)]6 des-Gly-NH210) ethylamide, on endocrine function and the LHRH and LH/hCG receptors in the pituitary-gonadal axis were examined. The LHRHa was injected at 100 ng/100 g body weight into male rats once a day for 4 weeks and its effects were observed until 2 weeks after the end of treatment. Due to LHRHa treatment, the plasma LH concentration began to increase on day 3, reached a peak on day 7, and then decreased, although it remained above the control level during the treatment. The pituitary LH content decreased on day 1, reached a minimum (about 40% of the control) between days 3 and 7, and then was maintained at 60% of the control level until week 4. In contrast, the pituitary LHRH receptor concentration increased only on day 3, and the association constant (Ka) remained unchanged during the observation period. The testis weight and plasma testosterone concentration began to decrease on day 3, reached the minimum on day 7 and remained at this level until week 4, and their levels were not completely restored to normal 2 weeks after cessation of treatment. The testicular LH/hCG receptor concentration was decreased on day 1, and markedly decreased to 10-15% of the control value between day 7 and week 4, but the Ka value was slightly increased during the treatment. However, these values had completely recovered 2 weeks after the cessation of treatment. The testicular LHRH receptor concentration increased between days 1 and 7, returned to the control level in weeks 2 and 4, and then decreased 2 weeks after cessation of treatment. Its Ka value was reduced in weeks 2 and 4. These data suggest that the inhibitory effect of LHRHa on the gonad in male rats is not due to reduced pituitary LH release, but to changes in the number and Ka values of gonadal receptors for LH/hCG and LHRH. PMID- 3019650 TI - A bioassay for thyroid stimulating immunoglobulins of patients with Graves' disease using porcine thyroid monolayer cells. AB - A bioassay for thyroid stimulating immunoglobulins (TSI) of patients with Graves' disease was developed by porcine thyroid monolayer cells. Thyroid cells were prepared by dispersion using collagenase and trypsin. Aliquots of the cell suspension (2 X 10(6) cells/1.5 ml/dish) in Ham's F-12 medium (pH 7.2) containing 10% calf serum and 1.5 mM Hepes were seeded and cultured in air at 36 C. On day 6 of culture, cells were incubated with test samples (IgG or bTSH) in 1 ml of serum free, 0.5 mM IMX-included fresh medium for an additional time, and cAMP in the cells was measured by radioimmunoassay. Intracellular cAMP was increased within 5 minutes after the addition of bTSH and the maximal increase was observed after 30 min. Responses of cAMP were in a dose-related manner up to 10 mU/ml of bTSH. With the addition of IgG from untreated Graves' patients, dose-related increases in cAMP were also observed up to 10 mg/ml IgG and the maximal response was seen at 2 hours incubation. Thyroid stimulating activity in IgG's from normal subjects and patients with Graves' disease was tested with a dose of 10 mg/ml and 2 hours incubation and the activity was expressed as a percent of the control (incubated in the same experiment without IgG). One hundred forty one of 145 untreated patients showed higher activity (228 +/- 51.8%, mean +/- SD; 127-393%, range) than normal subjects (103 +/- 13.3%, mean +/- SD, n = 24; 80-129%, range). Sequential changes in TSI activity in 27 patients after initiating thionamide drugs were studied for 24 months. Initially all 27 patients showed positive TSI and 6 months later 15 remained positive. At 6 months after that, 10 of 23, 4 of 16, and 2 of 6 followed patients showed positive TSI. These results indicate that this bioassay is clinically useful for detecting TSI. PMID- 3019651 TI - Effects of dibutyryladenosine 3',5'-monophosphate on steroid biosynthesis in humans. AB - The effects of dibutyryladenosine 3', 5'-monophosphate (DBcAMP), a nucleotide analogue, on blood pressure, serum electrolytes and plasma corticoid concentrations were investigated in 10 normotensive healthy subjects who received a constant diet containing 5-8 g sodium chloride in hospital. The systolic blood pressure did not change after infusion of 0.25 or 0.33 mg/kg/min of DBcAMP for 20 min. On the other hand, the diastolic blood pressure was significantly decreased after the infusion of DBcAMP. The levels of serum sodium and potassium were significantly decreased after the infusion of DBcAMP. After infusion of 0.25 mg/kg/min of DBcAMP for 20 min, the changes in plasma levels of 6 corticoids [progesterone, deoxycorticosterone (DOC), 18-hydroxy-deoxycorticosterone (18-OH DOC), corticosterone, cortisol and dehydroepiandrosterone sulfate (DHEA-S] revealed no significant changes. After infusion of 0.33 mg/kg/min of DBcAMP for 20 min, the plasma levels of cortisol, corticosterone and 18-OH-DOC were significantly increased and the changes in plasma levels of aldosterone showed a tendency to increase but this was not significant. The plasma levels of DOC and DHEA-S were not appreciably changed, while the plasma levels of progesterone were significantly decreased after the infusion of 0.33 mg/kg/min of DBcAMP. It is speculated therefore that DBcAMP may act to enhance the activity of the sodium potassium pump and to promote steroid biosynthesis dose-dependently in humans. PMID- 3019653 TI - Inhibition of TSH binding to chicken thyroid by some cases of LATS (long acting thyroid stimulator). AB - TSH receptor antibody (TRAb) activity using chicken thyroid receptor (c-TRAb) and porcine thyroid receptor (p-TRAb) was determined by the incubation of 125I-bovine TSH with each receptor. Both c-TRAb and p-TRAb activity in LATS positive and negative Graves' sera were compared. 15 out of 39 LATS positive sera and 4 out of 46 LATS negative sera had positive c-TRAb activity. On the other hand, all LATS positive sera and 33 out of 46 LATS negative sera had positive p-TRAb activity. No relationship between c-TRAb and p-TRAb activity was observed, and there was also no correlation between c-TRAb and LATS activity. Changes in c-TRAb, p-TRAb and LATS activity in the clinical course of patients with Graves' disease were examined. These activities were parallel in some cases, but in others they were not. A weak c-TRAb activity was observed in 4 out of 29 Hashimoto's disease, but all cases with thyroid cancer and subacute thyroiditis showed no activity. Sera with positive c-TRAb activity did not stimulate chicken thyroid in chick bioassay. These results suggest that some cases of TRAb in Graves' disease (mainly LATS) inhibit TSH binding to chicken thyroid receptor (non-mammalian species) in the same way as mammalian thyroid, but may not have any stimulatory action on thyroid hormone synthesis. It is interesting to note that TRAb including LATS have the similar effect on TSH receptor even in nonmammalian species. PMID- 3019654 TI - The homonuclear Overhauser effect in H2O solution of low-spin hemeproteins. Assignment of protons in the heme cavity of sperm whale myoglobin. AB - Proton-proton Overhauser effects were observed in 1H2O solutions of sperm whale metcyano myoglobin. Dipolar connectivities involving hyperfine-shifted exchangeable protons such as the proximal and distal histidine ring NH's allowed us to categorize signals as arising from residues located on one side of the heme plane or on the other. With these connectivities, as well as spin-lattice relaxation times, spectral assignments were reached that were used to derive structural and dynamic information about the heme environment. Thus, it was shown that the distal histidine residue does not titrate down to pH approximately 4.1 and that the beta CH2 of the proximal histidine side chain tumbles with the same correlation time as the protein. Some other applications and limitations are presented. PMID- 3019652 TI - Relationship between potency of blocking type thyrotropin-binding inhibitor immunoglobulin in three women with primary myxedema and thyroid function of their neonates. AB - Three neonates born to three mothers with primary myxedema who have thyrotropin binding inhibitor immunoglobulin (TBII) were continually examined after birth. One neonate showed a high TSH level in mass-screening for congenital hypothyroidism and developed transient hypothyroidism. Her TBII disappeared at 114 days of age, and she remained euthyroid after discontinuation of thyroxin replacement at 146 days of age. The other two neonates were euthyroid, though they had positive TBII. In three mothers, the doses of IgGs that inhibited 125I TSH binding to the level of 50% were compared. The potency of IgG from the mother whose neonate developed hypothyroidism was stronger than that of IgG from the other two mothers. And the elevation of cAMP induced by bovine TSH in suspension culture with porcine thyroid follicles was significantly reduced in the presence of IgG from the three mothers when compared with normal IgG. The thyroid stimulation blocking activity was more potent in the mother whose neonate developed hypothyroidism than in the other two mothers. This study suggests that the thyroid function of neonates born to primary myxedema with blocking type TBII is influenced by the potency of TSH-binding inhibitor and thyroid-stimulation blocking activity of the mother. PMID- 3019656 TI - Effect of chrysotile asbestos fibers on germ cells of mice. AB - An Indian form of chrysotile asbestos procured from a local asbestos factory (Hyderabad) was tested for its toxic effects on spermatocytes and sperm of mice. Swiss albino male mice were fed orally with chrysotile asbestos suspended in water. The concentration tested was 20 mg/kg/day. Chronic oral administration of chrysotile failed to induce chromosomal aberrations and abnormal sperms in mice. PMID- 3019655 TI - Morphology of tracheal and bronchial epithelium and type II cells of the peripheral lung of the guinea pig after inhalation of toluene diisocyanate vapors. AB - Toluene diisocyanate (TDI), a polymerizing agent used in production of plastics, can cause airways disease in some exposed individuals. Using guinea pigs as a model, the response of the airways and the type II cells of the peripheral lung was monitored morphologically and morphometrically after exposure to TDI vapors at 30 ppb, 260 ppb, and 3100 ppb. The two low doses of TDI caused little change in airways epithelium. There was no gross inflammatory cell infiltrate, however, surface infoldings and intracellular ciliated cysts increased in numbers. Animals exposed to 3100 ppb TDI 4 h/day for 5 days, sustained considerable damage to the epithelium, and stratified nonkeratinizing cells lined the airways until one week after exposure. Polymorphonuclear leukocytes were present in the early period after exposure. Increased numbers of eosinophils were present between one and two weeks following exposure. Mitoses in the epithelium were common during recovery. In the peripheral lung, though a modest subjective increase in the number of type II cells was seen after 3100 ppb TDI, the volume density of type II cells, and organellar components (lamellar bodies, mitochondria, cisternal bodies) did not change significantly after any exposure level of TDI. PMID- 3019657 TI - Subacute effects of nitrogen dioxide on membrane constituents of lung, liver, and kidney of rats. AB - Male Wistar rats were exposed to 0.4, 1.2, and 4.0 ppm nitrogen dioxide (NO2) for up to 14 weeks to examine subacute effects of NO2 on membrane constituents of lung, liver, and kidney. In the lung, cytochrome P-450 decreased to 59% (P less than 0.01) and 57% (P less than 0.01) of the control values after 1 and 10 weeks of exposure to 4.0 ppm NO2, respectively, and remained at control levels at other exposure periods. The activity of succinate-cytochrome c reductase also decreased to 75% (P less than 0.01) of the control values after 2, 4, and 14 weeks of exposure to 4.0 ppm NO2, respectively. Exposures to 0.4 and 1.2 ppm NO2 resulted in similar patterns of alterations in these enzymes. In the liver, cytochrome P 450 decreased to 72% (P less than 0.01), 70% (P less than 0.05), and 73% (P less than 0.05) of the control values after 1, 5, and 8 weeks of exposure to 4.0 ppm NO2, respectively, and remained at control levels at other exposure periods. The activity of NADPH-cytochrome P-450 reductase also decreased in a fashion similar to cytochrome P-450. Exposures to 0.4 and 1.2 ppm NO2 resulted in similar patterns of alterations in these enzymes. In addition, cytochrome b5 showed a reduced value between 5 and 12 weeks of exposures to 1.2 and 4.0 ppm NO2 and then recovered. In the kidney, all components of the microsomal electron-transport systems increased during 12-week exposures to 1.2 and 4.0 ppm NO2. These results show that subacute exposures to 0.4-4.0 ppm NO2 caused a periodic reduction in microsomal cytochrome P-450 and mitochondrial succinate-cytochrome c reductase in the lung and in components of the microsomal electron-transport systems in the liver, whereas exposures to 1.2 and 4.0 ppm NO2 resulted in induction of the microsomal electron-transport systems in the kidney. PMID- 3019658 TI - Fiber contamination of vermiculites: a potential occupational and environmental health hazard. AB - Vermiculite ores from Montana, Virginia, and South Africa have been analyzed for the presence of amphibole contamination. Fibrous actinolite was found in unexpanded Montana vermiculite ore at a maximum concentration of 2.0%. The fibers persisted in the expanded ore at a maximum concentration of 0.6%. Actinolite was also found in the Virginia vermiculite ore but at a lower concentration and mostly as cleavage fragments with low length-to-width ratios. South African ore contained rare anthophyllite fibers also with low length-to-width ratios. Vermiculite ores have the potential for amphibole contamination and can represent potential health hazards without proper occupational and environmental control measures. PMID- 3019659 TI - Studies on surface properties of asbestos. I. Active sites on surface of chrysotile and amphiboles. AB - The study of the acid-base properties of asbestos reveals a striking analogy between amphiboles and chrysotile. The high sites' density with basic character, evidenced by use of various probe molecules, is very similar for the two asbestos types (chrysotile and crocidolite) and on the same order as the density encountered in some catalysts. Discussion of the surface structure reveals that it is the noncrypting lateral surfaces which possess an electron donor character while the surfaces of the extremities of the fibers with crypting character possess a null or very weakly positive charge. PMID- 3019660 TI - Studies on surface properties of asbestos. III. Interactions between asbestos and polynuclear aromatic hydrocarbons. AB - The purpose of this study is to reveal the nature of the physicochemical interactions between asbestos and polynuclear aromatic hydrocarbons (PAH) in an organic liquid medium, and to assist in the understanding of synergistic effects between asbestos and PAH in bronchopulmonary cancers. The adsorption curves of three PAH (phenanthrene, fluorene, dimethyl-7,12-benzanthracene) on chrysotile and crocidolite are multistep isotherms and show the formation of bidimensional condensed layers (2D) of PAH. This phenomenon is observed with solids having a dominant basic character (asbestos, magnesia) but is not detected with acidic solids (alumina, silica-alumina). The elimination of water and dissolved gases (O2, CO2) in the liquid medium increased the affinity of asbestos for PAH. The coadsorption CO2-phenanthrene on the substrate decreased the adsorbed quantities of solute but did not inhibit the formation of layers (2D). The adsorption is weaker on leached chrysotile, than on original chrysotile; the amorphous silica coating the fibers has no affinity for PAH; the adsorption is only due to some active sites present on the surface of residual chrysotile which is accessible to phenanthrene. The formation of layers (2D) is due to strong interactions between PAH having an induced or permanent dipole moment and the active electron donor sites present on the mineral surface. The equilibrium equation between the adsorbed layer and the PAH in solution is established by reference to theoretical studies, and the results allowed us to classify the charge density of the mineral surface. The interactions between PAH and asbestos allowed us to explain the differences introduced in the kinetics of PAH uptake toward the cells when PAH is preadsorbed on asbestos. This fact could, in part, explain the synergistic effects observed in carcinogenesis. PMID- 3019661 TI - Studies on surface properties of asbestos. IV. Catalytic role of asbestos in fluorene oxidation. AB - To determine whether asbestos is a basic catalyst, catalytic oxidation of fluorene to fluorenone in a heterogeneous system was tested. It was shown that oxidation was quantitatively possible on the surface of all basic minerals, such as asbestos (chrysotile and crocidolite) and magnesia, but was not possible with acidic mineral materials such as silica. The effects of different factors are discussed. PMID- 3019662 TI - Neonatal developmental pattern of superoxide dismutase and aniline hydroxylase in rat lung. AB - The developmental biology of superoxide dismutase and aniline hydroxylase was followed in rat lungs from prenatal stage to 3 months old. Total superoxide dismutase activity as determined by spectrophotometry as well as electrophoresis was high in the prenatal rat lung, decreased in the first 24 hr postpartum, increased within 7 days, and then decreased gradually to adult levels. On polyacrylamide gel electrophoresis only two isozymic forms of superoxide dismutase were located as achromatic zones in the fetal lung. In the adult rat lung, there were three molecular forms of superoxide dismutase, two in the postmitochondrial supernatant and one in the mitochondrial fraction. Unlike superoxide dismutase, aniline hydroxylase was detectable only after 5 days of age and the activity exhibited a gradual increase afterward up to 1 month of age. The developmental pattern of superoxide dismutase and aniline hydroxylase activities in lung may be significant in understanding the mechanism of body defenses and their regulatory modulations in response to toxic air pollutants and environmental stress. PMID- 3019663 TI - Regulation of lung fibroblast proliferation and protein synthesis by bronchiolar lavage in experimental silicosis. AB - Male Sprague-Dawley rats were subjected to a single, intratracheal instillation of 30 mg Min-U-Sil silica in sterile saline and were sacrificed 3, 7, or 14 days following instillation. Control animals were instilled with sterile saline only. Silica instillation produced an inflammatory reaction followed by histological changes characteristic of lung fibrosis. Thickened alveolar septa associated with inflammatory cells transforming into large multifocal fibrotic nodules were detected in silica-exposed animals. Increased numbers of bronchoalveolar cells (principally macrophages), elevated levels of protein (principally serum albumin), and lysozyme, proteolytic (trypsin-like), and myeloperoxidase activities were detected in lavage fluids obtained from animals instilled with silica. These factors (except for lysozyme activity) were elevated above control levels from 3 to 7 days postinstillation and declined to near control levels by Day 14. The rate of DNA, collagen, and noncollagen protein synthesis was significantly elevated in lung tissue minces from silica-treated rats 3 and 7 days after instillation. Elevated levels of total protein, and lung collagen in particular, were observed 9 weeks after insult. Lavage fluid from silica instilled rats stimulates DNA synthesis in cultures of proliferating and quiescent rat lung fibroblasts. Lavage fluid from silica-instilled rats also stimulates lung fibroblasts to increase collagen and noncollagen protein synthesis. PMID- 3019664 TI - The mouse immunoglobulin heavy-chain enhancer: effect on transcription in vitro and binding of proteins present in HeLa and lymphoid B cell extracts. AB - The ability of the mouse immunoglobulin heavy chain gene (IgH) enhancer to stimulate in vitro transcription from the adenovirus-2 major late promoter (Ad2MLP) has been investigated. In agreement with the in vivo cell-type specificity of this enhancer, a stimulation can be observed in BJA-B lymphoid B cell, but not in HeLa cell, extracts. Under identical conditions, the Simian virus 40 (SV40) enhancer stimulates transcription in both extracts to approximately the same extent. In addition we have found that a sequence, previously shown to inhibit transcription in vitro in HeLa cell extracts, is also inhibitory in B cell extracts. DNase I footprint and DMS-methylation protection experiments indicate that each cell type contains proteins which bind to specific sequences of the IgH enhancer. The relationship between the binding of these proteins and the preferential activity of the IgH enhancer in B cells is discussed. PMID- 3019665 TI - Full-length von Willebrand factor (vWF) cDNA encodes a highly repetitive protein considerably larger than the mature vWF subunit. AB - Full-length human von Willebrand factor (vWF) cDNA was assembled from partial, overlapping vWF cDNAs. This cDNA construct includes a coding sequence of 8439 nucleotides which encode a single-chain precursor of 2813 amino-acid residues, representing a putative signal peptide, a prosequence and mature vWF of 22, 741 and 2050 amino acids, respectively. This represents the longest coding sequence determined to date. In-vitro expression of full-length vWF cDNA revealed the synthesis of a polypeptide with a mol. wt corresponding with that of the unglycosylated precursor. The precursor is a highly repetitive protein which consists of two duplicated (B, C), a triplicated (A), a quadruplicated (D) and a partly duplicated domain (D'), in the following order: H-D1-D2-D'-D3-A1-A2-A3-D4 B1-B2-C1-C2-OH. Both the prosequence, composed of two D domains (D1, D2), and mature vWF harbor an arg-gly-asp ('R-G-D') sequence which has been implicated in cell-attachment functions. It is argued that the pro-sequence is equivalent to von Willebrand Antigen II (vW AgII). PMID- 3019666 TI - Molecular characterisation of a hypervariable region downstream of the human alpha-globin gene cluster. AB - We have characterised an unusual, highly polymorphic region of DNA located 8-kb downstream of the human alpha-globin gene complex. This hypervariable region (alpha-globin 3' HVR) is composed of an array of 17-bp tandem repeats, the number of which differs considerably (70-450) from one allele to another. The sequence of the 17-bp repeats is highly conserved within and between alleles. Furthermore, this sequence identifies a core oligonucleotide [5'-GNGGGG(N)ACAG-3'] that is common to three previously characterised hypervariable regions. At reduced stringency, a probe to the 3' HVR detects a new family of multiallelic loci that will be of value in the study of human genetics. PMID- 3019667 TI - Studies on the expression of an H-2K/human growth hormone fusion gene in giant transgenic mice. AB - Transgenic mice carrying the H-2K/human growth hormone (hGH) fusion gene were produced by microinjecting into the pronucleus of fertilized eggs DNA molecules containing 2 kb of the 5' flanking sequences (including promoter) of the class I H-2Kb gene joined to the coding sequences of the hGH gene. Thirteen transgenic mice were obtained which all contained detectable levels of hGH hormone in their blood. Nine grew larger than their control litter-mates. Endogenous H-2Kb and exogenous hGH mRNA levels were analysed by S1 nuclease digestion experiments. hGH transcripts were found in all the tissues examined and the pattern of expression paralleled that of endogenous H-2K gene expression, being high in liver and lymphoid organs and low in muscle and brain. Thus 2 kb of the 5' promoter/regulatory region of the H-2K gene are sufficient to ensure regulated expression of hGH in transgenic mice. This promoter may therefore be of use to target the expression of different exogenous genes in most tissues of transgenic mice and to study the biological role of the corresponding proteins in different cellular environments. PMID- 3019668 TI - Isolation of the human gene for bone gla protein utilizing mouse and rat cDNA clones. AB - cDNAs which encode bone gla protein (BGP), an abundant gamma-carboxylated protein of bone, have been cloned from rat and mouse osteosarcoma cell lines. DNA sequence analysis indicates that the cDNAs code for both the 50 (rat) or 46 (mouse) amino acids of the mature proteins and a 49 amino acid leader peptide. The leader peptide of each BGP includes the expected hydrophobic signal sequence and an apparent pro sequence. Although there is no homology between the mature forms of BGP and the gamma-carboxylated clotting factors, we note that there is some homology between their leader peptides. These cDNAs have been used to examine the modulation of BGP mRNA levels by osteoblastic cells in response to hormones. The cDNAs have also allowed isolation of the human BGP gene; analysis of this gene indicates the presence of four exons. Comparison of the exon structure of the BGP gene and the Factor IX (a gamma-carboxylated clotting factor) gene suggests that the exons encoding the part of the leader peptides presumably directing gamma-carboxylation arose from a common ancestral sequence. PMID- 3019669 TI - The budding mechanism of spikeless vesicular stomatitis virus particles. AB - Virus particles, lacking the spike G-glycoproteins, are produced during infection of Vero cells with the vesicular stomatitis virus mutant ts045 at the restrictive temperature 39.5 degrees C. At this temperature the mutated G proteins are blocked in their intracellular transport in the endoplasmic reticulum. We have studied the role of the G proteins in the formation of these spikeless virus particles. The results showed that the spikeless particles contain a full complement of membrane anchors, derived from the carboxy-terminal end of the G protein. Our observations suggest that virus particles are formed at the restrictive temperature with G protein which is later cleaved to produce spikeless particles. We suggest that this is due to a leak of G protein to the cell surface at 39.5 degrees C where budding then takes place, presumably driven by a G protein C-terminal tail--nucleocapsid interaction. PMID- 3019670 TI - Structural and functional dissection of an MHC class I antigen-binding adenovirus glycoprotein. AB - The early transmembrane glycoprotein E19 of adenovirus-2 binds to class I antigens of the major histocompatibility complex (MHC). The association is initiated in the endoplasmic reticulum of infected cells and abrogates the intracellular transport of the class I molecules. To examine which parts of the E19 molecule are responsible for the association with the class I antigens and which parts confine the protein to the endoplasmic reticulum we have constructed a series of mutated E19 genes, which have been expressed in an improved mammalian expression vector. By various manipulations the membrane anchoring and the cytoplasmic domains were removed from the protein. The biosynthesis of the mutant protein was examined. All mutant proteins were secreted from the cells suggesting that the transmembrane and/or cytoplasmic portions of the E19 molecule are responsible for its confinement to the endoplasmic reticulum. The ability to associate with class I antigens was retained by the lumenal domain of the E19 protein. PMID- 3019671 TI - Analysis of RNA cleavage at the adenovirus-2 L3 polyadenylation site. AB - Processing at the L3 polyadenylation site of human adenovirus-2 involves endonucleolytic cleavage generating the 3' terminal sequence -UAOH to which adenosine residues are added. This dinucleotide is 19 nucleotides downstream of the AAUAAA polyadenylation signal. The ATP analog cordycepin triphosphate (3' dATP) inhibits poly(A) synthesis, but precursor RNA is processed to give a product terminating in -UAAH. Addition of only one adenosine analog demonstrates that the initial poly(A) tract is synthesized by polymerization of single residues rather than by ligation of preformed poly(A). Cleavage is not coupled to polyadenylation since incubation with an ATP analog containing a non-hydrolyzable alpha--beta bond generates a product with a 3' terminus coincident with the UAOH) addition site. Addition of this accurately processed RNA to a nuclear extract results in efficient polyadenylation, suggesting that downstream sequences are not required for synthesis of the poly(A) tract. Finally, processing at the L3 poly(A) site may involve both endonucleolytic and exonucleolytic activities. PMID- 3019672 TI - DNA helicase activity of SV40 large tumor antigen. AB - Large tumor antigen (T antigen) was extracted from SV40-infected African Green Monkey cells and purified to homogeneity by immunoaffinity chromatography. The purified T antigen preparations unwind DNA duplices of greater than 120 bp in a reaction which is dependent on magnesium ions and ATP hydrolysis. Based on these and other properties of the reaction we classify this newly discovered enzymatic activity as a eukaryotic DNA helicase. The helicase and the known ATPase function of T antigen cosediment with the mono- or dimeric 4-6 S form of T antigen, but not with higher T antigen aggregates. The helicase activity seems to be an intrinsic function of SV40 T antigen. First, several different T antigen-specific monoclonal antibodies interfere with the DNA unwinding activity; monoclonals which are known to reduce the T antigen-specific ATPase most strongly inhibited the helicase reaction. Second, mutant T antigens with impaired ATPase function also showed a reduced DNA unwinding activity. PMID- 3019674 TI - Functional analysis of the 5' flanking sequence of a vaccinia virus late gene. AB - A series of mutations, including 5' and 3' deletions, as well as insertions were introduced into the 5' flanking nucleotide sequence of a vaccinia virus late gene. This DNA has been shown previously to contain all the necessary elements for correct regulation of the gene most probably transcribed by the viral RNA polymerase. To facilitate the assays, the mutated DNA was fused to the chloramphenicol acetyltransferase gene and inserted into the genome of live vaccinia virus. The effects of the mutations on expression of the chimeric gene were studied by both enzyme assays and nuclease S1 analysis. The results showed that 5' deletions up to about 15 bp from the putative initiation site of transcription still yielded high levels of gene expression. All mutations, however, that deleted the authentic late mRNA start site, abolished promoter activity. PMID- 3019673 TI - Differential regulation of papilloma virus early gene expression in transformed fibroblasts and carcinoma cell lines. AB - Treatment of bovine papilloma virus (BPV) 1-transformed mouse fibroblasts with cycloheximide led to a 10-fold increase in the amount of viral transcripts, after as little as 1 h of protein synthesis inhibition. Northern blots revealed no qualitative changes in the RNA pattern. Nuclear run-on experiments showed about a 7-fold increase in specific transcriptional activity after cycloheximide treatment. The half-life of BPV1 mRNA was twice as long as in untreated controls. These results indicate that both RNA synthesis and degradation of viral RNA are controlled by labile proteins. Cycloheximide stimulation turned out to be independent of the BPV1 E2 gene activity which enhances viral transcription. Cycloheximide treatment had no effect on the amount of human papilloma virus (HPV) 18 transcripts in cervical carcinoma derived HeLa and C4-1 cells. Transcription of HPV16 in the cervical carcinoma line SiHa was likewise unaffected. The differential regulation of transcription in transformed fibroblasts and cancer-derived cells, and the significance for malignant conversion are discussed. PMID- 3019676 TI - A new homeo-box is present in overlapping cosmid clones which define the mouse Hox-1 locus. AB - A new murine homeo-box (Hox1-3) has been isolated and studied with respect to its structure and transcriptional pattern. This homeo-box is part of a gene which is specifically regulated during mouse prenatal development and expressed in a restricted number of teratocarcinoma tumours as well as in adult testis. Hox1-3 is shown to be a member of a cluster of at least four homeo-boxes lying within a 75-kb segment of DNA on mouse chromosome 6. The structure of the whole cluster, the Hox-1 locus is presented. PMID- 3019677 TI - GATC sequence and mismatch repair in Escherichia coli. AB - The Escherichia coli mismatch repair system greatly improves DNA replication fidelity by repairing single mispaired and unpaired bases in newly synthesized DNA strands. Transient undermethylation of the GATC sequences makes the newly synthesized strands susceptible to mismatch repair enzymes. The role of unmethylated GATC sequences in mismatch repair was tested in transfection experiments with heteroduplex DNA of phage phi 174 without any GATC sequence or with two GATC sequences, containing in addition either a G:T mismatch (Eam+/Eam3) or a G:A mismatch (Bam+/Bam16). It appears that only DNA containing GATC sequences is subject to efficient mismatch repair dependent on E. coli mutH, mutL, mutS and mutU genes; however, also in the absence of GATC sequence some mut dependent mismatch repair can be observed. These observations suggest that the mismatch repair enzymes recognize both the mismatch and the unmethylated GATC sequence in DNA over long distances. The presence of GATC sequence(s) in the substrate appears to be required for full mismatch repair activity and not only for its strand specificity according to the GATC methylation state. PMID- 3019675 TI - Identification of an Epstein-Barr virus-coded thymidine kinase. AB - We have demonstrated the presence of an Epstein-Barr virus (EBV)-coded thymidine kinase (TK) by producing biochemically transformed, TK-positive mammalian cell lines using either microinjection of whole EBV virions or calcium phosphate mediated transfection of the SalI-B restriction endonuclease fragment of EBV DNA. Analysis of these cell lines showed that: (i) EBV DNA was present in the cell lines, (ii) sequences from the SalI-B restriction endonuclease fragment of EBV were expressed, (iii) a TK activity was present and (iv) a protein with antigenic cross-reactivity with the herpes simplex virus (HSV) TK was produced. The identity of the EBV TK gene was determined by demonstrating that a recombinant plasmid, which expressed the protein product of the BXLF1 open reading frame as a fusion protein, could complement TK- strains of E. coli. A comparison of the predicted amino acid sequences of the TK proteins of EBV and HSV-1 revealed significant regions of homology. PMID- 3019678 TI - DNA-protein recognition: demonstration of three genetically separated operator elements that are required for repression of the Escherichia coli deoCABD promoters by the DeoR repressor. AB - The sequences required for full repression of the Escherichia coli deoP1 and P2 promoters by the deoR repressor (DeoR) have been analyzed in vivo. Using recombinant techniques, we have constructed a set of deo-lacZ fusions which contain different parts of the sequences involved in the regulation of deo expression on low copy number fusion vectors. Since these vectors are present in only one copy per chromosome at temperatures below 37 degrees C, this vector system allows very accurate studies of gene control signals. Our results show that three DeoR operator sites exist in the deoP1-P2 regulatory region. Two of these loci overlap the initiation sites for deoP1 (O1) and deoP2 (O2) transcription located 599 bp apart, whereas the third site (OE) is present approximately 270 bp upstream of P1. DeoR repression of both P1 and P2 transcription is weak on promoter fragments which only contain one operator site (O1 or O2). Enhanced repression by deoR is observed on promoter fragments containing two operator sites. However, all three sites are needed for full repression. These findings are discussed with respect to upstream and downstream control regions of eukaryotic genes. PMID- 3019679 TI - Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of the E. coli relB operon. AB - The parB region of plasmid R1 encodes two genes, hok and sok, which are required for the plasmid-stabilizing activity exerted by parB. The hok gene encodes a potent cell-killing factor, and it is regulated by the sok gene product such that cells losing a parB-carrying plasmid during cell division are rapidly killed. Coinciding with death of the host cell, a characteristic change in morphology is observed. Here we show that the killing factor encoded by the hok gene is a membrane-associated polypeptide of 52 amino acids. A gene located in the Escherichia coli relB operon, designated relF, is shown to be homologous to the hok gene. The relF gene codes for a polypeptide of 51 amino acids, which is 40% homologous to the hok gene product. Induced overexpression of the hok and relF gene products results in the same phenomena: loss of cell membrane potential, arrest of respiration, death of the host cell and change in cell morphology. The parB region and the relB genes were cloned into unstably inherited oriC minichromosomes. Whereas the parB region also conferred a high degree of genetic stability to an oriC minichromosome, the relB operon (with relF) did not; therefore the latter does not appear to 'stabilize' its replicon (the chromosome). The function of the relF gene is not known. PMID- 3019681 TI - Occurrence of antigenically distinct rotaviruses in infants in Bulgaria. PMID- 3019680 TI - Identification of the stable free radical tyrosine residue in ribonucleotide reductase. AB - The small subunit of iron-dependent ribonucleotide reductases contains a stable organic free radical, which is essential for enzyme activity and which is localized to a tyrosine residue. Tyrosine-122 in the B2 subunit of Escherichia coli ribonucleotide reductase has been changed into a phenylalanine. The mutation was introduced with oligonucleotide-directed mutagenesis in an M13 recombinant and verified by DNA sequencing. Purified native and mutant B2 protein were found to have the same size, iron content and iron-related absorption spectrum. The sole difference observed is that the mutant protein lacks tyrosyl radical and enzymatic activity. These results identify Tyr122 of E. coli protein B2 as the tyrosyl radical residue. An expression vector was constructed for manipulation and expression of ribonucleotide reductase subunits. It contains the entire nrd operon with its own promoter in a 2.3-kb fragment from pBR322. Both the B1 and the B2 subunits were expressed at a 25-35 times higher level as compared to the host strain. PMID- 3019682 TI - Comparison of cytopathic effect and an enzyme-linked immunosorbent assay using monoclonal antibodies for typing herpes simplex virus isolates. PMID- 3019683 TI - Structure and drug inducibility of the human cytochrome P-450c gene. AB - A human genomic clone (lambda hP-450mc-1), highly homologous to the rat cytochrome P-450c gene, was isolated and analyzed for the complete nucleotide sequence. The gene structure coincides with that of a recently reported human gene isolated from genomic DNA of a human breast carcinoma cell line, MCF-7 [Jaiswal, A.K., Gonzalez, F.J. & Nebert, D.W. (1985) Nucleic Acids Res. 13, 4503 4520] with notable exceptions in the first intron: a 320-base-pair fragment is inserted and a 650-base-pair fragment is deleted in the gene examined in the present study. The 320-base-pair insert appears to contain a moderately repetitive sequence (approx. 140 copies) in the human genome. The 650-base-pair fragment, present in intron 1 of the reported sequence, is dislocated in the lambda hP-450mc-1 to about 10(4) base pairs upstream from the putative transcription initiation site. The results of Southern blot analysis using human total DNA were compatible with the gene structure of lambda hP-450mc-1. A fusion gene, which was constructed by ligating the 5' flanking region of the gene to the structural gene for prokaryotic chloramphenicol acetyltransferase (CAT), inducibly expressed the CAT activity in mouse Hepa-1 cells in response to administered methylcholanthrene, indicating that the isolated human gene is indeed of methylcholanthrene inducibility. PMID- 3019684 TI - Overproduction, isolation and determination of the amino-terminal sequence of the SecY protein, a membrane protein involved in protein export in Escherichia coli. AB - The gene product of secY (prlA) is an integral membrane protein with an essential role in protein export in Escherichia coli. When the protein was overproduced, using a plasmid, it was degraded rapidly in the cell. The lon or the htpR mutation did not slow down this degradation, but low-temperature growth conditions (30 degrees C) did so appreciably. On the other hand, the copy number of the pUC8-based plasmid was higher at higher temperatures. Thus, the plasmid was first amplified at 42 degrees C and the protein was then accumulated at 30 degrees C. The SecY protein was isolated in sodium dodecyl sulfate (SDS) denatured form from the membranes of the overproducing cells, using SDS-SDS two dimensional gel electrophoresis. Its NH2-terminal sequence confirmed the secY reading frame and the translation initiation site assigned previously. The SecY protein does not undergo NH2-terminal processing except for the removal of the initiator methionine. PMID- 3019685 TI - Cross-linking of bromodeoxyuridine-substituted oligonucleotides to the EcoRI and EcoRV restriction endonucleases. AB - We have synthesized several self-complementary oligodeoxynucleotides which contain bromodeoxyuridine in various positions within and outside of the recognition sequence for the EcoRI and EcoRV restriction endonucleases. These oligodeoxynucleotides are cleaved in the presence of Mg2+ by their respective enzyme. Upon irradiation by long-wavelength ultraviolet light and in the absence of Mg2+ they are cross-linked in low yield to their enzymes, forming 1:1 and 1:2 (oligodeoxynucleotide:enzyme subunit) adducts. Cross-linking occurs with both specific and non-specific complexes. With EcoRI the site of cross-linking was determined to be at or close to Met-137, i.e. in a region of the molecule implicated by other studies from our laboratory [Scholtissek et al. (1986) J. Biol. Chem. 261, 2228-2234] in the binding and cleavage of the substrate. PMID- 3019686 TI - Binding of low-density lipoprotein and chylomicron remnants to the hepatic low density lipoprotein receptor of dogs, rats and rabbits demonstrated by ligand blotting. Failure to detect a distinct chylomicron-remnant-binding protein by ligand blotting. AB - In this paper, human low-density lipoprotein (LDL), rat chylomicron remnants and very-low-density lipoproteins of beta-mobility from cholesterol-fed rabbits (beta VLDL) have been shown to bind strongly to a protein present in solubilised liver membranes of rats, rabbits and dogs by ligand blotting with biotin-modified lipoproteins. This binding protein was identified as the LDL-receptor on several criteria. First, binding of the lipoproteins to the receptor was saturable and Ca2+-dependent; secondly, the apparent relative molecular mass of the binding protein (ranging from 128,000 in the rabbit, 145,000 in the rat to 147,000 in the dog) was similar to that of the purified bovine LDL receptor. Finally, binding activity was greatly increased in the livers of rats treated with oestrogen in pharmacological doses and absent from the liver of Watanabe heritable hyperlipidaemic (WHHL) rabbits that have a genetic defect in the LDL receptor. Some binding was also observed to a high-molecular-mass protein present in solubilised liver membranes of rats and rabbits, which, in rabbits at least, shared antigenic determinants with rabbit apoB and was not likely to be related to the LDL receptor as it was present in equal amounts in normal and WHHL rabbits. No evidence was obtained for a specific chylomicron remnant binding protein, distinct from the LDL receptor, whose activity could be detected in solubilised liver membranes by ligand blotting although a variety of solubilisation and fractionation conditions were employed. PMID- 3019687 TI - Cloning and sequencing of the gene encoding cytochrome c3 from Desulfovibrio vulgaris (Hildenborough). AB - The gene encoding the redox protein cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) has been cloned using two synthetic oligonucleotides (one 17-mer and one 18-mer), designed to recognize the structural gene. Plasmid pCYC3 was derived from the clone and contains a 7.5 X 10(3)-base EcoRI-HindIII insert of D. vulgaris DNA in pUC9. A 674-base-pair fragment of this insert was sequenced with the dideoxy-chain-termination procedure and found to contain the entire structural gene encoding cytochrome c3 bracketed by apparent Escherichia coli consensus sequences for initiation and termination of transcription. The amino acid sequence of 107 residues, derived from protein sequencing [Trousil, E. B. and Campbell, L. L. (1974) J. Biol. Chem. 249, 386-393], is confirmed by the nucleic acid sequence, which shows in addition that it is preceded by a hydrophobic, positively charged signal sequence of 21 residues. This amino terminal extension functions in the export of cytochrome c3, which is thought to reside in the periplasm of D. vulgaris. PMID- 3019688 TI - Purification and properties of phosphofructokinase 2/fructose 2,6-bisphosphatase from chicken liver and from pigeon muscle. AB - Phosphofructokinase 2 and fructose 2,6-bisphosphatase extracted from either chicken liver or pigeon muscle co-purified up to homogeneity. The two homogeneous proteins were found to be dimers of relative molecular mass (Mr) close to 110,000 with subunits of Mr 54,000 for the chicken liver enzyme and 53,000 for the pigeon muscle enzyme. The latter also contained a minor constituent of Mr 54,000. Incubation of the chicken liver enzyme with the catalytic subunit of cyclic-AMP dependent protein kinase in the presence of [gamma-32P]ATP resulted in the incorporation of about 0.8 mol phosphate/mol enzyme. Under similar conditions, the pigeon muscle enzyme was phosphorylated to an extent of only 0.05 mol phosphate/mol enzyme and all the incorporated phosphate was found in the minor 54,000-Mr constituent. The maximal activity of the native avian liver phosphofructokinase 2 was little affected by changes of pH between 6 and 10. Its phosphorylation by cyclic-AMP-dependent protein kinase resulted in a more than 90% inactivation at pH values below 7.5 and in no or little change in activity at pH 10. Intermediary values of inactivation were observed at pH values between 8 and 10. Muscle phosphofructokinase 2 had little activity at pH below 7 and was maximally active at pH 10. Its partial phosphorylation resulted in a further 25% decrease of its already low activity measured at pH 7.1 and in a negligible inactivation at pH 8.5. Phosphoenolpyruvate and citrate inhibited phosphofructokinase 2 from both origins non-competitively. The muscle enzyme and the phosphorylated liver enzyme displayed much more affinity for these inhibitors than the native liver enzyme. Fructose 2,6-bisphosphatase from both sources had about the same specific activity but only the chicken liver enzyme was activated about twofold upon incubation with ATP and cyclic-AMP-dependent protein kinase. All enzyme forms were inhibited by fructose 6-phosphate and this inhibition was released by inorganic phosphate and by glycerol 3-phosphate. Both liver and muscle fructose 2,6-bisphosphatases formed a 32P-labeled enzyme intermediate when incubated in the presence of fructose 2,6-[2-32P]bisphosphate. PMID- 3019689 TI - Reaction of phosphofructokinase 2/fructose 2,6-bisphosphatase with monoclonal antibodies. A proof of the bifunctionality of the enzyme. AB - Monoclonal antibodies were derived from mice immunized against homogeneous chicken liver phosphofructokinase 2/fructose 2,6-bisphosphatase. Of 112 clones, 30 were found to secrete antibodies that specifically reacted with the antigen in enzyme-linked immunoabsorbant assay (ELISA) while 17, which were ELISA-negative, produced antibodies that affected the enzymic activity of the antigen. Four clones were subcloned and used for an extensive investigation of the reaction of the corresponding antibodies with the supposedly bifunctional enzyme. A definite proof of the bifunctionality of the enzyme was obtained from the two following observations. First, the two activities were similarly retained by the four antibodies that had been coupled to Sepharose. Second, one of the antibodies inhibited both activities with the same efficiency. Furthermore, the antigen antibody reaction led to the formation of aggregates with an apparent molecular mass of several megadaltons, showing that the two subunits of the antigen reacted with the same antibody and were therefore identical. The four monoclonal antibodies affected the activity of phosphofructokinase 2. This effect was seen as an up to 17-fold activation as well as an up to 85% inhibition. Only one of the four antibodies (antibody 10) had inhibitory effects on fructose 2,6 bisphosphatase, an effect which was in part explained by a decrease in the rate of formation of the intermediary phosphoenzyme. All the effects described above were obtained on both the chicken liver and the pigeon muscle enzymes but with lower doses of antibody in the case of the former enzyme. Antibody 10 was also shown to react with mouse liver phosphofructokinase 2/fructose 2,6 bisphosphatase, and with phosphofructokinase 2 from chicken brain, heart and testis and from frog skeletal muscle and liver. None of the four antibodies cross reacted with phosphofructokinase 2 from Saccharomyces cerevisiae or from spinach leaves. PMID- 3019690 TI - Fusion of rat erythrocytes by membrane-mobility agent A2C depends on membrane proteolysis by a cytoplasmic calpain. AB - The membrane-mobility agent 2-(2-methoxyethoxy)ethyl-cis-8-(2 octylcyclopropyl)octanoate (A2C) promotes fusion of rat, but not of human, erythrocytes. The difference in fusibility was shown to be correlated with membrane proteolysis, a process induced by Ca2+ in the rat erythrocytes or hemolysate-loaded ghosts, but not in the human cell. Membrane proteolysis is necessary but not sufficient for fusion. Fusion requires both Ca2+ and A2C [Kosower, N. S., Glaser, T. and Kosower, E. M. (1983) Proc. Natl Acad. Sci USA 80, 7542-7546]. Membrane proteolysis (Ca2+-dependent) and fusion (Ca2+ and A2C dependent) requires a Ca2+-activated cytoplasmic thiol protease, as shown by the following observations. In intact rat erythrocytes, proteolysis and fusion are prevented by thiol alkylation and by inhibitors of Ca2+-dependent thiol proteases. Inhibitors to other proteases have no effect. Erythrocyte ghosts undergo proteolysis and fusion only when loaded with non-inhibited hemolysate, irrespective of membrane status (native or alkylated membrane). A partially purified cytosolic enzyme, identified as calpain I, promotes proteolysis in rat erythrocyte ghosts. A2C induces fusion only in such calpain-treated ghosts. PMID- 3019691 TI - Cooperative effect of thyroid and glucocorticoid hormones on the induction of hepatic phosphoenolpyruvate carboxykinase in vivo and in cultured hepatocytes. AB - The present study investigates the effect and interaction of glucocorticoid and thyroid hormones on the induction of phosphoenolpyruvate carboxykinase (PEPck) mRNA and enzyme protein under in vivo conditions and in serum-free cultured hepatocytes from hypothyroid rats. In hypothyroid/adrenalectomized rats T3 significantly enhanced the cAMP induced PEPck mRNA activity within 3-6 h. This effect was further enhanced by the presence of glucocorticoids. The half-life of PEPck mRNA, as determined after administration of cordycepin, was not affected by hypothyroidism or hyperthyroidism (t 1/2 approximately equal to 45 min), but considerably prolonged by the absence of glucocorticoid hormones (t 1/2 less than 80 min). In hepatocytes in culture Bt2cAMP (0.2 mM) provoked an increase in translatable PEPck mRNA within 2 h incubation time. Preincubation with either T3 (0.1 microM) or dexamethasone (0.1 microM) for 4 h significantly enhanced the cAMP response on PEPck mRNA. Addition of both, T3 plus dexamethasone further enhanced this Bt2cAMP-mediated effect. By measurement of PEPck synthesis corresponding findings were observed. It is concluded that glucocorticoid and thyroid hormones predominantly enhance the cAMP-provoked induction of hepatic PEPck mRNA and, consequently, of PEPck synthesis. Their effect is rapid, significant and additive, indicating an independent action. While glucocorticoids, in addition, accelerate PEPck mRNA degradation, the PEPck mRNA decay rate is similar in the presence and absence of thyroid hormones. PMID- 3019692 TI - The interactions of cytochrome c and porphyrin cytochrome c with cytochrome c oxidase. The resting, reduced and pulsed enzymes. AB - Cytochrome c oxidase forms tight binding complexes with the cytochrome c analog, porphyrin cytochrome c. The behaviour of the reduced and pulsed forms of the oxidase with porphyrin cytochrome c have been followed as functions of ionic strength; this behaviour has been compared with that of the resting oxidase [Kornblatt, Hui Bon Hoa and English (1984) Biochemistry 23, 5906-5911]. All forms of the cytochrome oxidase studied bind one porphyrin cytochrome c per 'functional' cytochrome oxidase (two heme a); it appears as though porphyrin cytochrome c and cytochrome c compete for the same site on the oxidase. The resting enzyme binds cytochrome c 8 times more strongly than porphyrin cytochrome c; the reduced enzyme, in contrast, binds the two with almost equal affinity. In all three cases, resting, pulsed and reduced, the heme-to-porphyrin distance is estimated to be about 3 nm. The tight-binding complexes formed between cytochrome oxidase and porphyrin cytochrome c can be dissociated by salt. Debye-Huckel analysis of salt titrations indicate that the resting enzyme and the reduced enzyme are similar in that the product of the interaction charges on the two proteins is about -14. The product of the charges for the pulsed enzyme is -25, indicating that on average another positive and negative charge take part in the interaction of the two proteins. While there is one tight binding site for cytochrome c per two heme a, cytochrome c is able to 'communicate' with four heme a. In the absence of cytochrome c, electron transfer from tetramethylphenylenediamine to the oxidase to oxygen results in the conversion of the resting form to the 'oxygenated'; in the presence of cytochrome c, the same electron transfer results in the appearance of the 'pulsed' form. Cytochrome c titrations of the enzyme show that a ratio of only one cytochrome c to four heme a is sufficient to convert all the oxidase to the 'pulsed' form. Porphyrin cytochrome c, like cytochrome c, catalyzes the same conversion with the same stoichiometry. The binding data and salt effects indicate that major structural alterations occur in the oxidase as it is converted from the resting to the partially reduced and subsequently to the pulsed form. PMID- 3019693 TI - Characterization of inverted repeated sequences in Ascaris nuclear DNA. AB - The inverted repeated sequences of the chromatin-eliminating nematode Ascaris lumbricoides var. suum have been examined by electron microscopy and by hydroxyapatite chromatography, both in the germ-line and in the somatic DNA. 38% of the inverted repeats of the germ-line DNA analysed in the electron microscope have a single-stranded loop, in comparison to about 50% of looped structures in the somatic DNA. The loops are on average 2.3 X 10(3) base pairs (bp) long. The rest of the foldback DNA consists of simple hairpins. The average length of looped and unlooped inverted repeats is of the order of 300-400 bp in the germ line and in the somatic DNA. The content of S1-resistant foldback duplexes isolated by hydroxyapatite chromatography amounts to 1.3% in spermatids, with an average length of 350 bp, and to 1.1% in intestinal or larval cell nuclei, with a length of about 320 bp. We estimate by two different methods that there exist approximately 12500 inverted repeats per haploid germ-line genome and approximately 8000 in the haploid somatic genome. A statistical analysis of the data indicates that the great majority of the foldback sequences are randomly distributed in the Ascaris genome, with a spacing of about (40-80) X 10(3) bp, both in the germ-line and in the somatic DNA. PMID- 3019694 TI - Differential inhibition of prostaglandin and superoxide production by dexamethasone in primary cultures of rat Kupffer cells. AB - Dexamethasone inhibited the stimulus-induced prostaglandin E2 formation by rat Kupffer cells in primary culture, e.g. after treatment with zymosan, phorbol ester, calcium ionophore A23187, platelet-activating factor or lipopolysaccharide. Prostaglandin E2 production from added free arachidonic acid was not influenced by the hormone. The time course, as well as the partial inhibition of the hormone effect by actinomycin D and cycloheximide, point to the hormone-induced formation of a protein which regulates phospholipase A2. The hormone did not affect the phagocytotic activity of the Kupffer cells. The quantity of [3H]arachidonic acid incorporated into phospholipids was also not altered by dexamethasone. After stimulation with zymosan, [3H]arachidonic acid was liberated from phosphatidylcholine only. Superoxide generation by rat Kupffer cells was induced by zymosan, phorbol ester and, to a much smaller extent, by platelet-activating factor. A23187 and lipopolysaccharide were without effect. In contrast to prostaglandin formation, the generation of superoxide was not influenced by dexamethasone. These results indicate that in cultured rat Kupffer cells prostaglandin formation and superoxide generation are independently triggered processes. PMID- 3019695 TI - Sulfite oxidase from chicken liver. The role of imidazole and carboxyl groups for the reaction with cytochrome c. AB - Oxidation of sulfite to sulfate by sulfite oxidase is inhibited when the enzyme is treated with reagents known to modify imidazole and carboxyl groups. Modification inhibits the oxidation of sulfite by the physiological electron acceptor cytochrome c, but not by the artificial acceptor ferricyanide. This indicates interference with reaction steps that follow the oxidation of sulfite by the enzyme's molybdenum cofactor. Reaction with diethylpyrocarbonate modifies ten histidines per enzyme monomer. Loss of activity is concomitant to the modification of only a single histidine residue. Inactivation takes place at the same rate in free sulfite oxidase and in the sulfite-oxidase--cytochrome-c complex. Blocking of carboxyl groups with water-soluble carbodiimides inactivates the enzyme. But none of the enzyme's carboxyl groups seems to be essential in the sense that its modification fully abolishes activity. The pattern of inactivation by chemical modification of sulfite oxidase is quite similar to that observed previously for cytochrome c peroxidase from yeast [Bosshard, H. R., Banziger, J., Hasler, T. and Poulos, T. L. (1984) J. Biol. Chem. 259, 5683-5690; Bechtold, R. and Bosshard, H. R. (1985) J. Biol. Chem. 260, 5191-5200]. The two enzymes have very different structures yet share cytochrome c as a common substrate of which they recognize the same electron-transfer domain around the exposed heme edge. PMID- 3019696 TI - Effect of tropomyosin on the interactions of actin with actin-binding proteins isolated from pig platelets. AB - Several non-muscle tropomyosins have been reported to lack the ability to polymerize in a head-to-tail manner [Dabrowska, R. et al. (1983) J. Muscle Res. Cell Motil. 1, 83-92; Cote, G.P. (1983) Mol. Cell. Biochem. 57, 127-146]. Unlike rabbit skeletal muscle tropomyosin, these proteins could therefore not protect the F-actin microfilaments neither from disassembly or from cross-linking by the other actin-associating factors. However, we have provided evidence that, in vitro, pig platelet tropomyosin, although shorter in molecular length, exhibits the same properties as the muscle protein: it self-associates and forms a 1:6 complex with platelet filamentous actin under physiological conditions [Pruliere et al. (1984) J. Muscle Res. Cell Motil. 6, 126]. In this paper, we examine the effects of several other actin-binding proteins on the microfilaments saturated with this non-muscle tropomyosin. Since contractile proteins often vary with the cell type and may require different conditions for their interactions, we have developed a procedure which allows the parallel purification of actin-binding protein (ABP), vinculin, alpha-actinin, gelsolin as well as actin and tropomyosin from the same batch of cells. Thus, using an homogeneous system, we show by viscometry, sedimentation and densitometry, and by electron microscopy, that pig platelet tropomyosin can protect the structure of the microfilaments from the action of the modulating factors to the same extent as rabbit skeletal muscle alpha-tropomyosin. Our data suggest that interaction of ABP, vinculin or alpha actinin can occur only with the ends of the filaments when F-actin is saturated with tropomyosin, while cross-linking takes place by interactions with sites localized along the entire length of F-actin in the absence of tropomyosin. Moreover, the presence of tropomyosin on F-actin leads to the total inhibition of gelsolin severing activity, although it did not prevent the binding of gelsolin to the F-actin--tropomyosin complex. This suggests that pig platelet as well as skeletal muscle tropomyosins have the ability to increase the strength of the interaction between actin monomers within the filament. This also suggests that the binding sites of gelsolin along the filaments are not localized in the groove of the F-actin helix. PMID- 3019697 TI - The aromatic 1H-NMR spectrum of plasminogen kringle 4. A comparative study of human, porcine and bovine homologs. AB - The isolated kringle 4 domain of human plasminogen has been compared with homologous structures from bovine and porcine sources, both free and in the presence of the ligand 6-aminohexanoic acid, by two-dimensional 1H-NMR spectroscopies at 300 MHz and 600 MHz. The chemical-shift-correlated, spin-echo correlated, and double-quantum-correlated aromatic spectra of the three proteins reveal that the globular conformation of the fourth kringle is closely maintained throughout the set of homologs. Direct comparison shows that the three conserved Trp residues (at sites 25, 62 and 72) which exhibit highly non-degenerate subspectra, find themselves in similar intramolecular environments. In particular, proton Overhauser experiments reveal that the close steric interaction between the Trp-II (Trp62 or Trp25) indole group and the aromatic ring at site 74 (Tyr74 or Phe74) is strictly preserved. This feature forces the kringle inner loop, closed by the Cys51-Cys75 link, to fold back onto itself so as to place the site 74 residue proximal to the Cys22-Cys63 bridge. Single residue substitutions enable unambiguous assignments of His-I to His3, Tyr-III to Tyr41 and Tyr-IV to Tyr74. From this direct evidence, comparison with the kringle 1 spectrum, and the previously reported chemical modification of Tyr-II (Tyr50) [Trexler M., Banyai L., Patthy L., Pluck N. D. & Williams R. J. P. (1985) Eur. J. Biochem. 152, 439-446], Tyr-I and Tyr-V (the latter, an immobile ring on the 600 MHz time scale) could be assigned to Tyr2 and Tyr9, respectively. Since Trp-III has previously been assigned to Trp72 at the lysine-binding site, the present study completes the assignment of 10 out of 12 aromatic spin systems in the kringle 4 1H-NMR spectrum; the only ambiguity which remains concerns the Trp-I and Trp-II indole spin systems, which are totally identified but as yet only tentatively assigned to Trp25 and Trp62, respectively. PMID- 3019698 TI - Calcium and polyphosphoinositides: their distribution in relation to the membrane changes occurring in the head of boar spermatozoa. AB - Ejaculated boar spermatozoa, previously incubated in a rigorously Ca++-free medium, were exposed to Ca++ for different incubation times and processed for the detection of Ca++ localization by a pyroantimonate technique. The distribution of polyphosphoinositides, anionic phospholipids natural constituents of membrane known to bind Ca++, was investigated using a specific cytochemical probe, i.e., neomycin conjugated with horseradish peroxidase. The in situ localizations thus obtained revealed: short exposure to Ca++ ions (10 min) evocated a Ca++-induced release of calcium from the nonmitochondrial intracellular store, i.e., the outer acrosomal membrane; a more prolonged exposure (20 min) triggered the occurrence of fusional and exocytotic events, that appeared to be morphologically related to the acrosome reaction; the outer acrosomal membrane, which is the fusigenic sperm membrane, was the elective site of the neomycin/peroxidase labeling. When assayed for the presence of a phospholipase C-like activity, the detergent extract obtained from boar spermatozoa exhibited substantial amount of p-nitrophenyl phosphorylcholine hydrolyzing activity. The results, on the whole, allow us to suggest a relationship between Ca++ and polyphosphoinositides turnover in the events triggering the acrosome reaction, the exocytotic process peculiar to mammalian spermatozoa. PMID- 3019699 TI - Water permeability in human erythrocytes: identification of membrane proteins involved in water transport. AB - The water permeability of human erythrocytes has been monitored by nuclear magnetic resonance (NMR) before and after treatment of the cells with various sulfhydryl reagents. Preincubation of the cells with N-ethylmaleimide (NEM), a non-inhibitory sulfhydryl reagent, results in a faster and more sensitive inhibition of water exchange by mercurials. The inhibition of water exchange by p chloromercuribenzene sulfonate (PCMBS) was maximal at a binding of approximately 10 nmol PCMBS per mg protein when non-specific sulfhydryl groups are blocked by NEM. Inhibition by PCMBS has been correlated with the binding of 203Hg to erythrocyte membrane proteins. A significant binding of label to band 3 and the polypeptides in band 4.5 occurs, with approximately 1 mol of mercurial bound per mol of protein. Inhibition of water transport by sulfhydryl reagents does not induce major morphological changes in the cells as assessed by freeze-fracture and scanning electron microscopy. PMID- 3019700 TI - Cytoskeletal disruption during human cytomegalovirus infection of human lung fibroblasts. AB - Human cytomegalovirus (HCMV) infection causes a rapid, progressive disruption of the host cell cytoskeleton that correlates with actin depolymerization. Whole mount (3D) electron microscopy was used to analyze the cytoskeleton of uninfected and HCMV-infected human lung fibroblast cells. Within 2 min of HCMV infection, localized areas of cytoskeletal disruption were observed. Disruption extended throughout the cytoplasm during the ensuing 45 to 90 min of infection and resulted in generalized cytoskeletal disorganization. Actin depolymerization occurred, as indicated by an increase in DNase I inhibition and alteration in the fluorescence pattern with rhodamine-conjugated phalloidin. Thus, actin appears to be the primary cytoskeletal target involved during HCMV infection. Fractionation of the virus seed inoculum showed that development of DNase I inhibitory activity in infected cells was associated only with the virus-containing fractions. Cytochalasin B treatment at early times of HCMV infection stimulated progeny virus production. This study demonstrates that rapid cytoskeletal disruption occurs during early periods of HCMV infection and indicates that actin depolymerization facilitates viral infectivity. PMID- 3019701 TI - Increased alpha 2- but similar beta-adrenergic receptor activities in subcutaneous gluteal adipocytes from females compared with males. AB - alpha 2 and beta-adrenergic binding and action were studied in subcutaneous adipocytes from the gluteal region in females and males. The beta-selective antagonist [3H]dihydroalprenolol and the alpha 2-selective antagonist [3H]yohimbine were used to identify the beta- and the alpha 2-receptor, respectively. The biological effects mediated by these receptors were determined by measuring the glycerol release induced by isoproterenol (beta-receptor agonist) and by clonidine (alpha 2-receptor agonist). The study consisted of health volunteers (eighteen females and fifteen males). Compared to men the alpha 2-receptor binding was increased by 73% (P less than 0.01) in adipocytes from females. From Scatchard analysis it was determined that the increased binding was due to an increased receptor number (438 v. 262 fmol mg-1 protein, P less than 0.001) with unaltered Kd (1.18 v. 1.35 nmol l-1, P greater than 0.05). This increased alpha 2-receptor binding in female and adipocytes was associated with a significantly increased sensitivity (P less than 0.05) and maximal antilipolytic effect of clonidine (P less than 0.05). The beta-receptor binding was similar in adipocytes from females and males. However, isoproterenol was significantly more lipolytic in female adipocytes (P less than 0.01). Since the combination of theophylline and adenosine deaminase also was significantly more lipolytic in female adipocytes the enhanced effect of isoproterenol then seemed to be due to mechanisms not directly related to the hormone-beta-receptor binding. Finally, the mixed beta- and alpha 2-receptor agonist, adrenaline showed biphasic effects on lipolysis, with stimulatory effect at low concentrations (less than 500 nmol l 1) and pronounced inhibitory effect at higher concentrations (greater than 1 mumol l-1).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019702 TI - Studies on the antihypertensive effect of single doses of the angiotensin converting enzyme inhibitor ramipril (HOE 498) in man. AB - The time course of the blood pressure lowering effect and the dose-response relationship of the new angiotensin converting enzyme inhibitor ramipril (HOE 498) were studied in 8 patients, with essential hypertension. As compared with placebo, a single oral dose of 2.5 mg ramipril lowered systolic and diastolic blood pressure. The antihypertensive action of single oral doses of 5, 7.5 and 10 mg ramipril was more pronounced. No change in heart rate occurred. Angiotensin converting enzyme activity was suppressed after all doses of ramipril studied. Plasma renin activity increased after 2.5 mg and 5 mg ramipril. Plasma aldosterone was not affected by 2.5 mg, but it fell after 5 mg ramipril. Thus, ramipril produced prolonged inhibition (more than 12 hours) of angiotensin converting enzyme activity and lowered blood pressure in patients with essential hypertension. PMID- 3019703 TI - Effect of acarbose on carbohydrate tolerance during administration of a fibre free formula diet on healthy subjects. AB - The influence of the disaccharidase inhibitor acarbose on carbohydrate tolerance was investigated in healthy subjects during substitution of fibre-free formula diets for normal food. Two separate experiments showed that acarbose was highly efficient in retarding and diminishing the postprandial rise in blood glucose and serum insulin when administered with these diets for 10 to 14 days. Acarbose decreased the area under the postprandial curves of blood glucose from 1.836 to 504 mg/dl X min in Study 1, and from 587 to -302 mg/dl X min in Study 2. The area under the serum insulin curves was reduced from 5.022 to 1.440 microU/ml X min in Study 1, and from 7.990 to 918 microU/ml X min in Study 2. In addition, acarbose greatly diminished the interindividual variation in postprandial serum insulin concentration. Its efficacy in reducing the glycaemic response to a test meal in both experiments was found to depend on the time of initiation of therapy; in contrast, the serum insulin response was only time-dependent in Study 2. Use of fibre-free diets to standardise experimental conditions proved to be a valuable tool in investigating these details. PMID- 3019704 TI - T cell help in human antigen-specific antibody responses can be replaced by interleukin 2. AB - Recombinant IL 2, and immunosorbent/high performance liquid chromatography purified interleukin 2 (IL 2) obtained from the human T cell leukemic line Jurkat, but not interferon-alpha or -gamma, were able to substitute for T cells in specific antibody responses to influenza virus by T cell-depleted (E-) human peripheral blood mononuclear cells, and resulted in antibody formation equivalent to that obtained in the presence of T cells. The antibody response was shown to be antigen specific by using two non-cross-reacting strains of influenza virus (A/X31 and B/HK). IL 2 in this assay therefore functions as a T cell-replacing factor. Less than 1% of T (UCHT1+) cells were present in the E- preparations, and this number did not increase during the 7-day culture with antigen and IL 2. Because the frequency of T helper cells for X31 is known to be less than 5 X 10( 5), this low number of contaminating cells excluded indirect action of IL 2 through antigen-specific T helper cells. Three to four times less IL 2 was required for antibody production by E- cells than was needed for optimal proliferation by an IL 2-dependent T cell line. Moreover, the concentration of anti-Tac required for 50% inhibition of the IL 2-induced antibody response was 50 times less than required for 50% inhibition of IL 2-dependent proliferation by the T cell line. But when T cells were added back to the E- cells, the anti-Tac inhibition curve shifted back to that obtained with the T cell line. In cell labeling experiments, Leu 11+ cells but not HNK1+ cells were increased in E- cells cultured with antigen and IL 2. This increase in Leu 11+ cells was abolished by prior passage of the E- cells through Sephadex G-10 columns without affecting the IL 2-induced antibody response. From these experiments we conclude that IL 2 can replace T cells in specific antibody responses, and that the IL 2 effect is not mediated indirectly through T cells or large granular lymphocytes. PMID- 3019705 TI - IgE receptors on human lymphocytes. III. Expression of IgE receptors on mitogen stimulated human mononuclear cells. AB - Human peripheral blood mononuclear cells (PBMC) were tested for the expression of Fc epsilon-receptor (Fc epsilon R) after stimulation with various mitogens in the absence of IgE. Fc epsilon R were found on virtually all the cells from 19 Epstein-Barr virus-transformed B cell lines including those derived from cord blood, from one agamma-globulinemic patient and VDS-0 pre-B cells. Hence, the data clearly indicate that Fc epsilon R may be expressed on very immature B cells. PBMC cultures stimulated with either pokeweed mitogen (PWM), phytohemagglutinin (PHA) or concanavalin A displayed an early increase of their content in Fc epsilon R-bearing cells followed by a decrease to levels below those of control cultures. After fractionation of the PWM-stimulated cultures into T and B cell-enriched preparations, most of the Fc epsilon R+ cells were in the in the B cell fractions and the same low levels of Fc epsilon R+ cells were found in the T cell fractions isolated from the PWM-stimulated and from the control cultures. Double-labeling experiments, employing biotinylated F(ab')2 monoclonal antibody to FcR and either fluorescein isothiocyanate-conjugated B1 or Mo2 monoclonal antibodies, indicated that PWM mainly exerted its effect on B cells and on monocytes. This effect was T cell dependent and it was mediated by soluble factors of T cell origin. At the peak of the PHA or concanavalin A response, most of the Fc epsilon R-bearing cells were found in the B cell fraction but the T cells isolated from mitogen-stimulated cultures contained significant more Fc epsilon R+ cells than those from the control cultures, suggesting that T cell mitogens had increased the expression of Fc epsilon R on some T cells. This view was supported by the finding of a higher proportion of Fc epsilon R+ cells in PHA-stimulated than in control cultures of highly purified T cells with a maximum response at the end of the culture period. Double-labeling experiments at the peak (day 2) of the peripheral blood mononuclear cell response indicated that the expression of Fc epsilon R was increased on B cells (B1+) and on monocytes (Mo2+). By using the same approach at the peak of the T cell response (day 7), it was found that T cells isolated from PHA-stimulated cultures expressed more Fc epsilon R than those isolated from control cultures.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3019706 TI - Interaction of periodate-oxidized target cells and cytolytic T lymphocytes: a model system of "polyclonal MHC recognition". AB - In oxidation-dependent cytotoxicity (ODCC), cytolytic T lymphocytes (CTL) non specifically recognize, bind to and lyse oxidized target cells (O-TC) but the precise mechanism whereby CTL react with O-TC is far from clear (Berke, G., Immunol. Rev. 1983. 72:5). Here we present evidence that CTL/O-TC interactions are blocked by aldehyde-reactive reagents such as hydroxylamine, adipic acid dihydrazide and thiocarbohydrazide and that preformed CTL/O-TC conjugates dissociate upon reduction with NaBH4, suggesting that active aldehyde groups of O TC rather than intercellular Schiff bases are involved in the recognition and lysis of O-TC by CTL in ODCC. The aldehydes are bound to trypsin-sensitive, non-H 2 glycoproteins that appear to be different and unique in the three different target cell lines so far examined (EL4, L1210, R1.1). In view of these and previous findings we would like to suggest that in ODCC, active aldehydes react with adjacent major histocompatibility complex and perhaps other cell-surface molecules to create a multitude of modified conformations, responsible for the "polyclonal" (nonspecific) MHC recognition and lysis of O-TC by CTL, as well as for an altered pattern of H-2 antibody binding to O-TC. PMID- 3019707 TI - Biosynthesis of the subcomponents C1q, C1r and C1s of the first component of complement (C1) by guinea pig hepatocyte primary cultures. AB - Thus far, the synthesis of C1q by liver cells has not been demonstrated. To investigate this possibility, viable hepatocytes were isolated from the liver of guinea pigs and primary cultures were established. The cells (10(6) cells/ml) were cultured under serum-free conditions for 8 days and the culture medium was changed every 24 h. The few contaminating Kupffer cells were lysed by preincubating the cell cultures with a monoclonal (22C4-8) antibody directed against a nonpolymorphic Ia determinant and preabsorbed rabbit serum. The hemolytic activity of C1 and its subcomponents C1q and C1r/C1s was tested in the supernatants. Guinea pig hepatocyte primary cultures synthesize and secrete up to 3 X 10(3) effective C1q molecules/cell/24 h and 34 X 10(3) effective C1r/C1s molecules/cell/24 h. The synthesis of C1q and C1r/C1s could be reversibly inhibited by cycloheximide (50 micrograms/ml). Furthermore, to demonstrate de novo synthesis of the C1q subcomponent, endogeneous labeling with 3H-proline (or 14C-proline) was performed. The immunoprecipitated C1q from cellular lysates and culture medium was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Compared to biosynthetically labeled guinea pig C1q from peritoneal macrophages, three corresponding bands (30, 28 and 24 kDa, respectively) were detectable in the fluorograph. The data show that guinea pig hepatocytes are able to synthesize C1 subcomponents, whereby the synthesis of C1q and C1r/C1s occurs independently. PMID- 3019708 TI - Epstein-Barr virus-induced IgE production in limiting dilution cultures of normal human B cells. AB - The induction of in vitro IgE production in human B cells from normal, nonatopic donors has been difficult and somewhat controversial. We report that IgE production is consistently observed in limiting dilution cultures of in vitro Epstein-Barr virus (EBV)-infected normal human B lymphocytes. The frequency of IgE-committed, EBV-responsive cells ranged from 1/810 to 1/10 000 B lymphocytes, and it was similar in peripheral (blood, tonsil) and central (bone marrow) tissue sites. Poisson distribution analysis of these limiting dilution cultures suggested that IgE-committed B cells comprise 0.1-1% of all EBV-responsive B lymphocytes. PMID- 3019710 TI - Lithium ions have a potent and selective inhibitory effect on cyclic GMP formation stimulated by neurotensin, angiotensin II and bradykinin. AB - The effect of lithium ion (Li+) on receptor-mediated synthesis of cyclic GMP, a putative second messenger, was examined using intact murine neuroblastoma cells (clone N1E-115). Lithium chloride potently inhibited cyclic GMP formation stimulated by the neuropeptides, neurotensin, angiotensin II and bradykinin in an identical concentration-dependent (IC50 s of around 12 mM), saturable and reversible manner. In the presence of veratridine, an alkaloid which by stimulating sodium channels can increase Li+ entry into the cells, Li+ inhibited neurotensin-stimulated cyclic GMP formation more potently (IC50 = 7 mM). No effect of Li+ was observed on phosphodiesterase (EC 3.1.4.17) activity. These results suggest that Li+ may interfere with the function of these receptors through its inhibitory effect at a common site in the pathway of receptor mediated cyclic GMP formation. PMID- 3019709 TI - Further evidence for a role of delta-opiate receptors in the presynaptic regulation of newly synthesized dopamine release. AB - The effects of the specific delta-agonist of opiate receptors, DTLET (Tyr-D-Thr Gly-Phe-Leu-Thr), the specific mu-agonist DAGO (Tyr-D-Ala-Gly-(Me)Phe-Gly-ol) and of kelatorphan (N-((2R)-3-(hydroxyaminocarbonyl-2-benzyl-1-oxopropyl)-L-alanine), a potent inhibitor of the enkephalin-degrading enzymes, on the spontaneous release of [3H]dopamine ([3H]DA) synthesized from [3H]tyrosine were examined in rat striatal slices. DTLET (10(-7) M, 10(-6) M) and kelatorphan (5 X 10(-6) M) enhanced markedly the release of newly synthesized [3H]DA, while DAGO (10(-6) M) was inactive. The stimulatory effects of DTLET (10(-7) M) and kelatorphan (5 X 10(-6) M) were prevented in the presence of naloxone (3 X 10(-6) M; 10(-4) M respectively) or ICI 154,129 (10(-5) M), a selective antagonist of delta-opiate receptors. While DTLET (10(-7) M) stimulated the 30 mM potassium-evoked release of newly synthesized [3H]DA, it did not affect the potassium-evoked release of [3H]DA previously synthesized in tissues. A higher concentration of DTLET (10(-6) M) was required in the latter case. In contrast to the release observed with striatal slices, DTLET (10(-7) M), 10(-6) M) or DAGO (10(-6) M) did not affect the spontaneous release of newly synthesized [3H]DA from nucleus accumbens slices. In addition, DTLET (10(-6) M) was without effect on the potassium-evoked release of newly synthesized [3H]DA in this structure. The present results confirmed that delta-opiate receptors are involved in the presynaptic regulation of [3H]DA release.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019711 TI - Further evidence that a beta-adrenergic mechanism regulates water intake: role of the subfornical organ. AB - The daily water intake was reduced in rats by subcutaneous administration of propranolol. The fluid intake after 24 h of dehydration in nephrectomized animals was decreased by propranolol. In intact rats diazepam did not modify the postdehydration water intake. Propranolol injected into subfornical organ decreased water consumption after 24 h of fluid deprivation but did not change the dipsogenic response to carbachol given in the same structure. These results suggest that a beta-adrenoceptor mechanism participates in the regulation of the daily water intake. After a stimulus such as dehydration, beta-blockade modifies water consumption by a non-renal mechanism. This mechanism involves neither a general depressant nervous activity nor a local anesthetic action and could be located in a periventricular structure, the subfornical organ. PMID- 3019712 TI - Alpha-adrenoceptor responsiveness in the aged rat. AB - The effects of ageing on vascular alpha 1- and alpha 2-adrenoceptors were examined using anaesthetised and pithed young (3-7 months) and old (21-24 months) Sprague-Dawley rats. In pithed animals, the pressor and cardioinhibitory effects of the alpha 2-adrenoceptor agonist xylazine were significantly reduced in old animals (8- and 6-fold shift), but the pressor effects of the alpha 1 adrenoceptor agonist amidephrine were not significantly altered by ageing. In anaesthetised rats, the pressor response to the mixed alpha-adrenoceptor agonist noradrenaline (NA) was not significantly altered, and the pressor potency of the alpha 1-adrenoceptor agonist amidephrine was only slightly reduced (1.7-fold shift) in old animals. In the presence of cocaine (1 mg kg-1) the pressor potency of NA was markedly reduced (13-fold shift) in old animals. In the presence of prazosin (1 mg kg-1) to eliminate alpha 1-adrenoceptor-mediated responses, the pressor potency of NA was markedly reduced in old as animals as compared to young animals (16-fold shift). The neuronal uptake blocker cocaine (1 mg kg-1) significantly potentiated the pressor response to NA only in young. In summary, we have found a reduced alpha 2-adrenoceptor-mediated pressor and cardioinhibitory responsiveness and a reduced neuronal uptake of NA in old animals with little change in alpha 1-adrenoceptor-mediated vascular responsiveness. PMID- 3019714 TI - Effect of repeated electroconvulsive shocks on isoprenaline-induced changes of the endogenous inhibitor of cAMP-dependent protein kinase in rat brain. AB - The effect of repeated electroconvulsive shocks (ECS) on the responsiveness to isoprenaline of an endogenous, specific inhibitor of cAMP-dependent protein kinase (type I inhibitor) was tested in rat hippocampus and brainstem. Isoprenaline produced a dose-dependent decrease in the type I inhibitor activity in these brain structures. Neither the basal activity nor the isoprenaline induced changes of type I inhibitor activity were affected by a single ECS. However, a series of 11 ECS markedly reduced the response of the type I inhibitor activity to isoprenaline. The action of isoprenaline was completely blocked by propranolol and markedly enhanced by aminophylline. The results indicate that the subsensitivity of beta-adrenoceptors is accompanied by reduced responsiveness of type I inhibitor activity to isoprenaline. PMID- 3019713 TI - Neuromedin N: high affinity interaction with brain neurotensin receptors and rapid inactivation by brain synaptic peptidases. AB - Neuromedin N (NN) is a novel neurotensin (NT)-like hexapeptide recently isolated from porcine spinal cord. NN competitively inhibited the binding of monoiodinated [Trp11]NT to rat brain synaptic membranes with a 19-fold lower potency than NT. In the presence of 1 mM 1,10-phenanthroline or 10 microM bestatin, the potency of NN relative to NT was increased about 5-fold. NN was readily degraded by rat brain synaptic membranes, and NN-(2-6) was the major degradation product. NN-(2 6) did not bind to NT receptors at concentrations up to 1 microM whether or not peptidase inhibitors were present in the binding assay. The rate of degradation by synaptic membranes was nearly 2.5 times higher for NN than for NT. NN degradation by membranes was totally prevented by 1,10-phenanthroline and markedly inhibited by bestatin. The presence of NN in the central nervous system, its high potency to interact with brain NT receptors and its rapid inactivation by brain synaptic peptidases make it a potential neurotransmitter candidate acting at the NT receptor. PMID- 3019715 TI - 'Peripheral' and 'central' type benzodiazepine receptors in Maudsley rats. AB - In this study the binding to 'central' and 'peripheral' benzodiazepine receptors in various brain areas in MR/N and MNR/N rat strains was investigated. The specific ligands [3H]BCCE and [3H]RO 5-4864 were used for binding to 'central' and 'peripheral' benzodiazepine receptors, respectively. Neither [3H]BCCE nor [3H]RO 5-4864 binding was changed in MNR/N strain in comparison to MR/N rat strain in cerebral cortex, cerebellum, hippocampus and medulla-pons. Scatchard analysis of [3H]BCCE or [3H]RO 5-4864 binding to cerebellar membranes also revealed no changes in either affinity or density in both types of benzodiazepine receptors. Autoradiographic analysis of brain regions using microdensitometry shows no differences in [3H]flunitrazepam binding between the MR/N and MNR/N strains. Our results, therefore, fail to confirm a previous report showing alterations in benzodiazepine receptors in Maudsley rats. PMID- 3019717 TI - Light microscopic autoradiographic localisation of [3H]glycine and [3H]strychnine binding sites in rat brain. AB - Receptor autoradiography has been employed to determine the distribution of strychnine-insensitive glycine binding sites in rat brain using [3H]glycine as a ligand. The location was significantly different from and more widespread than glycine sensitive [3H]strychnine binding sites. Highest binding densities were observed in hippocampus, cortex, subiculum and amygdala followed by striatum, cerebellum and olfactory areas. Characterisation of the binding indicated that it was saturable, of high affinity, stereoselective and displaced by structurally related amino acids. The results support the existence of two glycine receptor subtypes: strychnine-sensitive and strychnine-insensitive. PMID- 3019716 TI - Baclofen suppresses bursting activity induced in hippocampal slices by differing convulsant treatments. AB - Epileptiform activity was induced in area CA3 of hippocampal slices by superfusion of medium containing 50 microM bicuculline and 3.5 mM K, 50 microM bicuculline and 5 mM K, 50 nM kainic acid and 3.5 mM K, or 7 mM K. Burst potentials were recorded at rates between 5 and 44/min, depending on the convulsant treatment. Baclofen reduced the frequency of burst firing in all slices tested in a dose-dependent manner, with little change in the morphology of individual bursts. Thus baclofen primarily affected the initiation of epileptiform discharges. IC50 values varied between 27 and 500 nM and were positively correlated with the rate of bursting. These experiments indicate that baclofen, at concentrations present in the CSF of patients treated for spasticity, has an anticonvulsant-like effect in the hippocampal formation and suggest that its mode of action is to reduce the excitability of pyramidal cells. PMID- 3019718 TI - In vivo binding of putative delta-selective opioid antagonists to central opiate receptors. AB - The in vivo opiate receptor binding of three putative delta-selective opioid antagonists, the irreversible oripavine ligand NIH 10236 and two enkephalin analogs, ICI 154,129 and ICI 174,866, was studied in rat brain. Following i.c.v. injection, potent and apparently irreversible opiate receptor binding was observed for NIH 10236 with similar affinity for mu, delta and kappa sites. However, opiate receptor binding was not evident either after s.c. injection of NIH 10236 or ICI 154,129, or following i.c.v. administration of ICI 154,129 or ICI 174,864. The receptor sites that mediate the pharmacological effects of these drugs should be reevaluated. PMID- 3019719 TI - Platelet high affinity adrenoceptor binding sites labelled with the agonist [3H]UK-14,304, in depressed patients and matched controls. AB - Increased numbers of platelet high affinity alpha 2-adrenoceptors binding sites have been reported in depressed patients using the agonist radioligand [3H]clonidine, whereas no differences from controls have been found using antagonist radioligands. We have measured platelet high affinity alpha 2 adrenoceptors in 13 depressed patients and 14 well-matched controls using a new selective agonist radioligand, [3H]UK-14,304. Unlike previous studies using [3H]clonidine, we find no differences in Bmax or KD of the high affinity alpha 2 adrenoceptor binding sites between the 2 groups. PMID- 3019720 TI - The motor effect of the capsaicin-sensitive inhibitory innervation of the rat ureter. AB - Neurokinins activate a series of tetrodotoxin (TTX)-insensitive rhythmic contractions of the rat isolated ureter. Field stimulation or capsaicin (1-3 microM) produced a transient inhibition of neurokinin-activated ureteral motility in preparations from control but not from capsaicin-pretreated (50 mg/kg s.c.) rats. The inhibitory action of field stimulation but not that of capsaicin was prevented by TTX. It is concluded that a capsaicin-sensitive inhibitory innervation exists in the rat ureter. PMID- 3019721 TI - Stereospecific binding of the antidepressant rolipram to brain protein structures. AB - The characteristics for the binding of the selective cAMP phosphodiesterase inhibitor and antidepressant agent rolipram to brain and peripheral organs were investigated. (+/-)-[3H]Rolipram equilibrium binding and Scatchard analysis revealed saturable, reversible, stereospecific, Mg2+-dependent and heat-sensitive binding with an apparent Hill number of 1. Binding was detected both to membrane bound and soluble sites, with dissociation constants Kd of 1.2 and 2.4 nM, respectively, and binding site concentrations (Bmax) of 19.3 and 23.6 pmol/g rat forebrain. The (-)-enantiomer of rolipram was ca. 20 times more effective than the (+)-enantiomer in displacing (+/-)-[3H]rolipram from membranes. Rolipram bound to brain tissue of all mammalian species tested including man, while tissue from bird and fish showed less binding. Organs other than brain exhibited only negligible binding. Only specific cAMP phosphodiesterase inhibitors (ICI 63.197, Ro 20-1724) were potent competitors, while rolipram itself was inactive in a variety of receptor binding assays of neuroactive ligands. The kinetics of (-) [3H]rolipram binding to the particulate fraction revealed a complex association and dissociation behaviour. The nature of the rolipram binding protein(s) is not clear, but the low affinity binding site evident from binding kinetics may represent a rolipram-sensitive phosphodiesterase isoenzyme also common to some peripheral organs, while the high affinity binding site(s) may be related to PDE isoenzymes more confined to the central nervous system. PMID- 3019722 TI - Vascular alpha-adrenoceptor blockade by E. coli endotoxin in the rat. AB - It has been previously shown that Escherichia coli endotoxin (ENDO) reduces the pressor effect of phenylephrine. This work attempted to define precisely the involvement of vascular alpha-adrenoceptors in this action. The dose-response curves of three alpha-agonists noradrenaline, St 587 and B-HT 933 in pithed rats were shifted rightwards by pretreatment (i.v. injection of 10 and 100 micrograms/kg) with ENDO. ENDO 10 and 30 micrograms shifted the dose-response curve of noradrenaline in perfused rat kidney. It can be suggested that ENDO has a potent blocking action on vascular alpha-adrenoceptors without evident discrimination between alpha 1 and alpha 2 subtypes. PMID- 3019723 TI - Modulation of peripheral benzodiazepine binding sites following chronic estradiol treatment. AB - A 10 day estradiol treatment of rats resulted in a marked reduction (43%) in peripheral benzodiazepine (BZ) binding sites in the testis and an up-regulation (27%) of these sites in the kidney. No significant alterations in brain central BZ receptors and heart peripheral binding sites were observed. The down regulation of BZ binding sites in the testis may be related to or accompany estrogen-induced testicular atrophy. The physiological and pharmacological implications of the alterations obtained in the kidney are as yet unclear. PMID- 3019724 TI - Electrophysiologic effects of prazosin during acute myocardial ischemia. AB - Prazosin, 100 micrograms/kg, had no effect on baseline refractoriness or intraventricular conduction in anesthetized dogs. During 1 h of coronary artery occlusion followed by reperfusion, prazosin significantly blunted the shortening of the ventricular effective refractory periods within ischemic myocardial region relative to vehicle-treated animals. Prazosin treatment also prevented the delayed conduction of paced ventricular complexes entering and exiting the ischemic zone. These effects may be associated with the blockade of alpha 1 adrenoceptor activation during the acute phase of myocardial ischemia. PMID- 3019725 TI - Phospholipase A2 products induce pulmonary beta-adrenoceptor deviations. PMID- 3019726 TI - Differences between central and peripheral rat alpha-adrenoceptors revealed using binuclear ligands. AB - We have used two homologous series of binuclear ligands, diacridines and diquinolines, and the radioligand receptor assay to compare the topology of alpha 1- and alpha 2-adrenoceptors in rat cerebral cortex and kidney membranes. While the chain length-dependence of affinity of the diacridines, as well as that of the diquinolines, for the alpha 1-adrenoceptors of these central and peripheral tissues are similar, we find marked differences in affinity profiles for interaction with central and peripheral alpha 2-adrenoceptors. In the context of our previously proposed model for the binding of diacridines and diquinolines to alpha-adrenoceptors the results suggest that the surface features of central and peripheral alpha 2-adrenoceptors differ in the area surrounding the noradrenaline binding site. This difference may prove to be of therapeutic relevance. PMID- 3019727 TI - Interference of BN 52021 (ginkgolide B) with the bronchopulmonary effects of PAF acether in the guinea-pig. AB - The interaction between the ginkgolide BN 52021 and the effects of PAF-acether on the bronchopulmonary system of the guinea-pig was studied. In pentobarbitone or ethyl carbamate-anaesthetized animals, BN 52021 (1 mg/kg i.v. or 10 mg/kg p.o.) inhibited bronchoconstriction, the hematocrit increase and the accompanying thrombopenia and leukopenia induced by PAF-acether (33-100 ng/kg) and failed to block the bronchoconstriction produced by collagen, arachidonic acid and the tripeptide formyl-Met-Leu-Phe (FMLP). BN 52021, 3 mg/kg, reduced the bronchoconstriction induced by aerosolized PAF-acether. BN 52021, 300 microM, also inhibited the superoxide production by PAF-acether-stimulated alveolar macrophages and failed to reduce the same effects when triggered by FMLP (0.01-1 microM). BN 52021 blocked the formation of thromboxane-triggered by PAF-acether (100 ng) injected into perfused lung, under conditions where the effects of arachidonic acid where not modified. Finally, pretreatment of parenchyma lung strips with BN 52021 (100 microM) partially inhibited the contraction induced by PAF-acether (0.1 microM) and suppressed the accompanying release of thromboxane. BN 52021 is a selective antagonist of the effects of PAF-acether on the bronchopulmonary system and on circulating blood cells of the guinea-pig. PMID- 3019728 TI - Chronic antidepressant treatment reduces central beta-adrenoceptor sensitivity in a behavioural test. AB - The ability of beta-adrenoceptor agonists to potentiate the head-twitch response to 5-hydroxytryptophan (5-HTP) in mice was used to assess the in vivo sensitivity of beta-adrenoceptors 48 h after cessation of acute or chronic administration of desmethylimipramine (DMI) or iprindole (IPD). Neither acute nor chronic antidepressant administration significantly altered the head-twitch response to 5 HTP alone. Forty-eight hours after withdrawal from chronic but not acute pretreatment with DMI or IPD the potentiating effects of dobutamine, prenalterol and salbutamol were significantly decreased. This is consistent with a reduction in beta-adrenoceptor density and suggests that the 'spare' beta-adrenoceptor pool is small enough for this to result in functional subsensitivity. Implications of these findings are discussed. PMID- 3019729 TI - Increased gastric cholinergic activity evoked by 5-hydroxy-L-tryptophan in the rat. AB - In gastrointestinal tissues such as rat stomach, exogenous 5-hydroxytryptamine (5HT) has little or no ability to affect nerve activity. However, endogenous 5HT might act differently, and this was investigated by stimulating 5HT synthesis using 5-hydroxy-L-tryptophan (5HTP). In longitudinal strips of rat forestomach, 5HTP (50 and 500 microM) increased cholinergically mediated contractions evoked by electrical field stimulation, probably by facilitating acetylcholine release; contractions evoked by exogenous acetylcholine were unaffected by 5HTP. The ability of 5HTP to increase electrically evoked contractions was long-lasting, required the presence of pyridoxal (a monoamine decarboxylase cofactor) and was reduced by the decarboxylase inhibitor carbidopa, but not by 6-hydroxydopamine. In the presence of paroxetine and nialamide, the 5HTP-induced increase in cholinergically mediated contractions was short-lasting. In anaesthetised rats 5HTP caused stimulation of gastric motility, which was blocked or reduced by atropine. These findings suggest that in the rat 5HTP stimulates gastric cholinergic activity, by increasing the concentration of 5HT at sites which normally synthesise 5HT. PMID- 3019731 TI - A re-appraisal of the mode of action of 5-HT in inhibiting GABA-induced cholinergic contractions in the guinea-pig ileum. AB - In the guinea-pig ileum, pretreatment with 5-hydroxytryptamine (5-HT) (5 microM) for 4-5 min inhibited both 5-HT- and gamma-aminobutyric acid (GABA)-induced cholinergic contractions without consistently altering those induced by electrical field stimulation. Cisapride (1 micron) antagonized 5-HT-induced cholinergic contractions but left those induced by GABA or twitch responses unchanged. These results indicate that the 5-HT action in inhibiting GABA-induced cholinergic responses may arise at the interneuronal level, thus suggesting that GABA may also indirectly activate cholinergic terminal neurons. These findings rule out the possibility of 5-HT acting as an intermediate transmitter in this type of response. PMID- 3019730 TI - An electrophysiological analysis of the effect of reactive blue 2, a putative P2 purinoceptor antagonist, on inhibitory junction potentials of rat caecum. AB - The electrophysiological effects of the putative P2-purinoceptor antagonist reactive blue 2 (RB2) were investigated in strips of circular muscle of rat caecum with the sucrose gap technique. RB2 (0.1-1 mM) antagonized in a concentration-dependent manner, the amplitude, rate of rise and speed of onset of the inhibitory junction potentials elicited either with single pulse or with train of field stimulation at 10 Hz. A fully effective concentration of RB2 (0.5 mM) decreased the membrane response to high strength hyperpolarizing constant current pulses, thus indicating an increase in membrane conductance. At this concentration RB2 inhibited the hyperpolarization induced by the stable ATP analogue alpha,beta-methylene ATP, but did not significantly inhibit noradrenaline-induced hyperpolarization. RB2 (0.5 mM) also abolished the ability of carbachol to elicit spikes, while the carbachol-induced depolarization and the amplitude of accompanying mechanical responses were largely unaffected. The possible mechanisms responsible for the RB2-induced effects are discussed. PMID- 3019732 TI - Calcium or magnesium concentration affects the severity of organophosphate induced neuromuscular block. AB - When the rat diaphragm muscle was treated with dusopropyl fluorophosphate, an organophosphate inhibitor of acetylcholinesterase, a decrement in the compound action potential and in the force of contraction was observed. It was found that the decrement produced by 30 Hz nerve stimulation for 1 s could be reversed by lowering the calcium concentration or increasing the magnesium concentration. PMID- 3019734 TI - (+)-3-PPP antagonizes the discriminative stimulus effects of (+)-N allylnormetazocine. AB - The functional significance of the reported affinity of (+)-3-PPP and (+)-N allylnormetazocine (NANM) for the same binding site in rat brain membranes was assessed by studying (+)-3-PPP as a agonist and antagonist of (+)-NANM in rats trained to discriminate 5.0 mg/kg (+)-NANM from saline. Over a wide dose range, (+)-3-PPP was able to block the discriminative stimulus effects of (+)-NANM, with complete antagonism at 1.0 mg/kg i.p. Since (+)-NANM is a prototype sigma-opioid agonist, (+)-3-PPP is a good candidate for being a competitive sigma antagonist. PMID- 3019733 TI - Autoradiographic localization of alpha-adrenoceptors, muscarinic acetylcholine receptors and opiate receptors in the heart. AB - Using in vitro autoradiography the distribution of [3H]rauwolscine, [3H]prazosin and [3H]quinuclidinyl benzilate binding sites has been demonstrated in cardiac tissue taken from the cat and rat. A similar distribution of both alpha 1- and alpha 2-adrenoceptor sites was seen but the distribution of muscarinic acetylcholine sites was markedly different. alpha-Adrenoceptors were present predominantly in ventricular muscle whereas muscarinic acetylcholine receptors exhibited a greater density in atrial tissue compared to ventricular muscle. Opiate receptors were absent from cardiac tissue. PMID- 3019735 TI - Autoradiographic mapping of substance P receptors in lung. PMID- 3019737 TI - Oestrogen modulates dopamine-controlled behaviours in the male ferret. AB - Oestradiol (100 micrograms/kg) was administered to male ferrets on three successive days. Two and three days after the last oestradiol injection the effects of apomorphine (0.5 mg/kg) on stereotyped, predatory and scent marking behaviour were intensified. Thus, three days of oestradiol administration produced a supersensitive dopaminergic system two days after the last oestradiol injection. However, no initial antidopaminergic effect of oestradiol could be demonstrated one day after the last oestradiol injection. PMID- 3019738 TI - GABA-serotonergic interactions in the regulation of central sympathetic pathways. AB - Methysergide, a serotonin antagonist, and clonidine, an alpha 2-adrenoceptor agonist, lowered arterial blood pressure, heart rate and inferior cardiac sympathetic nerve activity in the anesthetized cat. Picrotoxin, a GABA antagonist, caused a reduction in the inhibition of sympathetic activity produced by methysergide. In contrast, picrotoxin did not effect the reduction of sympathetic nervous discharge resulting from administration of clonidine. These observations suggest that an interaction exists between GABAergic and serotonergic neurons which regulates central sympathetic pathways, possibly by a tonic GABAergic inhibition of serotonergic sympathoexcitatory pathways. PMID- 3019736 TI - Muscle relaxant action of phenobarbitone in genetically spastic rats: an electromyographic study. AB - The muscle relaxant effect of phenobarbitone was studied in genetically spastic rats which exhibit spontaneous tonic activity in the electromyogram (EMG) of the gastrocnemius muscle. Phenobarbitone, 10-30 mg/kg i.p., reduced the tonic activity in the EMG of the gastrocnemius muscle of such rats in a dose- and time dependent manner. The GABA antagonists bicuculline, 2 mg/kg i.p., and picrotoxin, 2 and 3 mg/kg i.p., reduced the muscle relaxant effect of phenobarbitone, 20 and 30 mg/kg. The benzodiazepine receptor antagonists, Ro 15-1788, 5 mg/kg, and CGS 8216, 5 mg/kg (doses which do not affect tonic activity in the EMG), failed to alter the depressant effect of phenobarbitone 30 mg/kg, in the EMG. Beta Carboline-3-carboxylic acid methylester (beta-CCM), 2 mg/kg i.p., while not affecting the tonic activity in the EMG, reversed the depressant effect of phenobarbitone, 30 mg/kg. Both Ro 15-1788, 5 mg/kg, and CGS 8216, 5 mg/kg, prevented the reversal of the depressant action of phenobarbitone, 30 mg/kg, produced by beta-CCM, 2 mg/kg. The results indicate that the muscle relaxant action of phenobarbitone in genetically spastic rats is mediated via GABA-related mechanisms and add further support to the hypothesis that both Ro 15-1788 and CGS 8216 are specific antagonists at benzodiazepine receptors, devoid of intrinsic activity at moderate doses. The results also suggest that reversal of the muscle relaxant action of phenobarbitone by beta-CCM is mediated via a GABA/benzodiazepine receptor/chloride ionophore complex. PMID- 3019739 TI - Elevation of cyclic AMP by prostacyclin is accompanied by relaxation of bovine coronary arteries and contraction of rabbit aortic rings. AB - The role of cyclic AMP (cAMP) in the control of vascular smooth muscle tension was examined by comparing the effects of prostacyclin (PGI2) on tension and cAMP levels in helical strips of bovine coronary arteries and in rabbit aortic rings, both denuded of endothelium. In bovine coronary arteries, PGI2 elevated cAMP levels and relaxed the muscles. The PGI2-induced cAMP elevation preceded the relaxation and both parameters were altered in a dose-dependent manner by increasing concentrations of PGI2 (0.3, 3 and 30 microM). These results are consistent with a role for cAMP as a mediator of vascular smooth muscle relaxation. Cyclic AMP levels were also elevated by PGI2 in a concentration- and time-dependent manner in rabbit aortic rings. However, in direct contrast to the results in the bovine coronary arteries, PGI2-induced elevation of cAMP in the aortic rings was accompanied by contraction rather than relaxation. Isoproterenol, a drug which is generally believed to relax smooth muscles by virtue of its ability to increase tissue levels of cAMP, relaxed PGI2-contracted aortic rings with no further elevation of cAMP beyond that caused by the PGI2 alone. These results demonstrate that cAMP elevation and relaxation of vascular smooth muscle are not always well correlated. It is possible that some form of functional compartmentalization of cAMP or cAMP-dependent protein kinase exists in these tissues. PMID- 3019740 TI - Sensitivity of the PGF2 alpha-versus carbachol-contracted trachea to relaxation by salbutamol, forskolin and prenalterol. AB - A comparison has been made of the abilities of salbutamol, forskolin and prenalterol to relax guinea-pig tracheal rings contracted equivalently with either prostaglandin F2 alpha (PGF2 alpha) or carbachol. In the absence of spontaneous tension, 10(-6) M PGF2 alpha and 4 X 10(-7) M carbachol induced equivalent contractions of tracheal rings. Tracheal contractions induced by PGF2 alpha were more sensitive to the relaxant effects of salbutamol, forskolin or prenalterol than were contractions induced by carbachol. Each tracheal relaxant had a lower IC50 value for inhibiting PGF2 alpha-induced contractions, and prenalterol (a beta-adrenoceptor partial agonist) induced more complete relaxation of PGF2 alpha- than carbachol-induced tracheal tension. It is concluded that the tracheal relaxant activity of these compounds is in part dependent upon the contractile agent which induces tension, and that the variable sensitivity of different isolated tracheal muscle preparations to the actions of tracheal relaxants may result from differences in the contracting stimulus being antagonized in each of these models. PMID- 3019741 TI - Contradictory evidence concerning the "universal" role of growth hormone in the regulation of the enzyme activities of hepatic steroid metabolism. AB - Recent publications suggest that the sexual dimorphism observed in the activities of enzymes involved in drug and steroid metabolism in rat liver are due to sex specific differences in the rate of growth hormone release. In this paper we set out to demonstrate that this hypothesis cannot be generalized, but has its limitations. Prepuberal hypophysectomy led to the expected "masculinization" of the activities of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSDH), microsomal 3 alpha-HSDH and microsomal 5 alpha-reductase which could be reversed by continuous infusion of human growth hormone (hGH). However, one activity did not conform to this pattern: cytoplasmic 17 beta-HSDH activity reacted to hypophysectomy with a "feminization" and was completely unaffected by hGH infusion. Moreover, microsomal 3 alpha-HSDH in hypophysectomized rats was "feminized" as efficiently by infusion of ovine prolactin (oPRL) as by hGH. Ablation of the pituitary caused loss of measurable cytoplasmic receptor oestrogen concentrations. The inability of either hypophyseal hormone to cause consistent and significant elevation of oestrogen receptor concentrations is probably due to the early age at which the animals were hypophysectomized. PMID- 3019742 TI - Glycogenolytic effect of cortisol previous to its interaction with the cell nucleus in mouse liver. AB - Activities of hepatic glycogen synthase a and glycogen phosphorylase a have been studied in mouse liver at different times after an acute intraperitoneal administration of hydrocortisone. It has been observed an increase of glycogen synthase a activity and a decrease of glycogen phosphorylase a between 2 and 3.5 hours after cortisol injection. An early effect, previous to the synthase activation has been discovered. Cortisol caused an increase of glycogen phosphorylase a activity in mice 45 min after injection. This early effect of cortisol is independent of protein synthesis and it does not imply an increase in cAMP levels. PMID- 3019743 TI - Effect of prolonged administration of CRF on plasma concentrations of ACTH in patients with Addison's disease. AB - To investigate the possibility that the prolonged administration of corticotropin releasing factor (CRF) might suppress rather than enhance pituitary ACTH secretion 24-hour infusions (50 micrograms/h) of ovine CRF were performed in 6 patients with adrenocortical insufficiency. CRF induced a heterogeneous behaviour of plasma ACTH concentrations in these hypercorticotropinemic patients but both a sustained increase and a suppression of ACTH was clearly absent at the end of the 24 hour infusion period. Thus, continuous administration of CRF does not appear to be a promising way to control abundance of plasma ACTH concentrations in patients with Addison's disease. PMID- 3019744 TI - The role of cAMP and prostaglandins in gastric acid secretion after pentagastrin administration. AB - The role of cAMP and prostaglandins as specific intracellular effectors of gastrin action at the level of the parietal cells has not been sufficiently clarified. For this reason we studied the responses of the parietal cells to stimulation with pentagastrin (6 micrograms/kg i.m.) during theophylline infusion (which causes an increase in the intracellular cAMP) and during acetylsalicylic acid infusion (which inhibits the prostaglandin synthesis) in 28 healthy volunteers. Both theophylline and acetylsalicylic acid provoked a significant increase of gastric acid secretion after pentagastrin. Our results suggest that: 1. an increase in intracellular cAMP may be the basis of the stimulatory effect of gastrin on gastric acid secretion 2. a decrease in the synthesis of prostaglandins may lead to a greater gastric acid response after pentagastrin. PMID- 3019745 TI - Clones defective in metabolic cooperation selected from a pluripotent feeder dependent mouse embryonal carcinoma cell line. AB - Starting from embryonal carcinoma (e.c.) cells capable of extensive differentiation in culture, the technique of thioguanine 'kiss of death' has been used to select four independent metabolic cooperation-defective variants. The communication ability of these variant cell lines has been quantified by autoradiographic measurement of the transfer of uridine nucleotides, and also by an assay of the extent of junction-mediated rescue from ouabain toxicity by resistant fibroblasts. The cell lines which are defective in ability to transfer nucleotides, as measured by the uridine nucleotide transfer assay, are also defective in their ability to differentiate into endoderm and to form the cavitated 'embryoid bodies' which are produced by the parental cell line when grown in suspension culture. However, it is not clear whether this is related to the defects in metabolic cooperation, since clones which had been subjected to the same selective conditions but which cooperate normally have also lost some of the capacity to undergo this differentiation. Endoderm differentiation was classified into two categories, one being visceral endoderm and the other, primary plus parietal endoderm, on the basis of morphology, immunocytochemical staining for alpha-fetoprotein, and basement membrane formation. With the exception of correlations arising from variations between experiments and differences between cell lines, there is no statistical association between these two categories of differentiation. The formation of cavities was observed only in embryoid bodies with endoderm differentiation: the present of either category was a sufficient condition for cavitation to occur. PMID- 3019746 TI - Inhibition of transcription does not affect the total amount of ubiquitinated histone 2A in chromatin. AB - Using a polyclonal anti-ubiquitin antibody in Western blotting experiments, we detected three antibody-binding components in a HeLa cell extract: ubiquitin, a ubiquitin-histone 2A conjugate (uH2A) and a 17 kD protein, probably corresponding to an additional ubiquitin conjugate. Since ubiquitination of histone 2A (H2A) has been invoked in the transcription process, the amount of uH2A was studied after inhibition of ribosomal RNA (rRNA) synthesis with actinomycin D and of heterogeneous nuclear RNA (hnRNA) synthesis with 5,6-dichloro-1-beta-D ribofuranosylbenzimidazole (DRB). The amount of uH2A did not change, suggesting that the overall level of ubiquitination of histone 2A is not directly coupled to on-going transcription of either rRNA or hnRNA. Since the uH2A content of protein coding genes constitutes a considerable portion of total chromatin uH2A, it seems also likely that there is no major change in the degree of ubiquitination on the templates of the protein-coding genes themselves upon cessation of transcription. It is proposed that the pattern of ubiquitination of histone 2A is established on a long-term basis and that it is related to the overall organization and distribution of the chromatin material in the interphase nucleus. PMID- 3019747 TI - Chondrocyte activation in response to factor(s) produced by a continuous line of lapine synovial fibroblasts. AB - We have established a permanent line of lapine synovial fibroblasts called HIG 82. Upon appropriate stimulation, these cells mimicked primary cultures of lapine synovial cells in producing substances which activated primary cultures of lapine articular chondrocytes. Activated chondrocytes secreted prostaglandin E2 (PGE2) and latent neutral collagenase, gelatinase, and caseinase, but not acid hydrolases, into their culture media. PGE2 itself did not activate the chondrocytes. Heating the crude, synovial-conditioned media at 70 degrees C for 30 min reduced their activating activity by 49.3 +/- 20.5% (n = 7). Production of PGE2 by chondrocytes was maximal during the first day of exposure to synovial conditioned media, whereas the production of neutral proteinases peaked during the second day. All the chondrocyte-stimulating activity was present in a fraction of Mr 10,000-25,000. Unlike the crude conditioned medium, this partially purified material retained full activity following heating to 70 degrees C for 30 min. These data indicate that synovial fibroblasts (type B synoviocytes) are a source of chondrocyte activator(s) and that neutral, but not acid, proteinases may be involved in extracellular proteolysis which leads to the resorption of the cartilaginous matrix seen in bioassays of catabolin. PMID- 3019748 TI - Human bone marrow stromal cell colonies: response to hydrocortisone and dependence on platelet-derived growth factor. AB - Long-term bone marrow cultures are dependent on the formation in vitro of an adherent cell layer that supports hematopoiesis. We have grown bone-marrow adherent cells, termed stromal colony-forming units, or CFU-ST, as isolated adherent colonies, and examined some of their growth requirements. Bone marrow mononuclear cells separated from aspirates by density centrifugation and cultured in medium supplemented with fetal calf serum or human plasma gave rise to adherent colonies (CFU-ST). An average of 23.4 +/- 2.1 (mean +/- SEM, n = 19) CFU ST were produced by 10(5) bone marrow mononuclear cells. CFU-ST could not be cultured from similarly prepared peripheral blood mononuclear cells. The colonies were composed of spindle cells, flat cells, and fat-containing cells, with all three types often present in the same colony, suggesting derivation from a common progenitor. Cells were negative for nonspecific esterase and factor VIII antigen. Hydrocortisone added to the cultures at concentrations of 10(-7) M induced the formation of adipose cells in the center of one-third to one-half of the colonies but did not affect CFU-ST number. Human platelet-poor plasma and platelet-rich plasma were substituted for fetal calf serum in the medium. When all determinations for four experiments were averaged, platelet-rich plasma gave 17.8 +/- 1.2 (mean +/- SEM, n = 16) colonies, whereas platelet-poor plasma gave only 0.2 +/- 0.1 colonies (n = 15). When purified platelet-derived growth factor (PDGF) was added to platelet-poor plasma, growth of CFU-ST was enhanced, and a dose-response relationship was found between size of colonies and concentration of added PDGF. Granulocyte-macrophage colony stimulating factor added to cultures had no effect on the growth of CFU-ST. PMID- 3019749 TI - GABAergic neurotransmission within the reticular part of the substantia nigra (SNR): role for switching motor patterns and performance of movements. AB - In order to investigate the role of GABAergic neurotransmission within the reticular part of substantia nigra (SNR) in the switching of motor patterns and the performance of movements, cats trained to walk on the running belt of a treadmill at constant speed were subjected to three different tests: a food dispenser test measuring the animals' capacity to switch motor patterns in order to get access to food during walking; an obstacle test measuring the animals' capacity to switch motor patterns in reaction to incoming obstacles; EMG recording of two representative antagonistic muscles of the hindlimb during walking on the treadmill. Local injection of a moderate dose of the GABA antagonist picrotoxin (PTX; 250-500 ng/0.5 microliter) into the SNR disrupted the animals' capacity to switch motor patterns in the food dispenser test, but not in the obstacle test. These animals displayed normal EMG patterns during walking. Higher doses of intranigral injections of PTX, however, impaired the execution of movements per se as detected by an increased number of 'faults' in the obstacle test and pathological EMG patterns during walking. These experiments support the view that the SNR plays a distinct role for switching motor patterns; the SNR is involved in the control of movements per se; the degree of motor disorder depends on the degree of pathology within this brain structure. PMID- 3019750 TI - The role of the anterior intralaminar nuclei and N-methyl D-aspartate receptors in the generation of spontaneous bursts in rat neocortical neurones. AB - The nature of spontaneous unitary activity of rat neocortex was investigated during slow wave sleep and urethane anaesthesia. Neurones in layer IV and V locations fired in a burst-pause pattern at a low burst repetition rate (0.5-4 per second) during both stage 3/4 sleep and urethane anaesthesia. Occasionally an alternative mode of firing (spindle clusters), associated with focal spindle wave activity, was also found to occur in both states. Using dual microelectrode implants it was found that the onset times of bursts (but not spindle clusters), coincided in the same and opposing cortices, whether in functionally similar or disparate areas. The highest probability was that burst onsets occurred simultaneously (resolution = 2.56 ms, interquartile range = 40 ms). Spontaneous unitary activity was investigated in the thalamus for temporal correlation with spontaneous unitary activity in neocortex under urethane anaesthesia. Neurones of the anterior intralaminar group (aIL) consistently fired in a burst-pause pattern such that each aIL burst showed a strong tendency to precede a cortical burst. Unilateral electrical stimulation of the aIL nuclei evoked widespread bilateral entrainment of cortical bursts. In contrast stimulation of VPl, or cutaneous sites, evoked only short duration spike responses together with burst abolition in the appropriate restricted Sml area. Ionophoresis of NMDA (N-Methyl D Aspartate) onto Sm1 neurones increased the probability of cortical burst responses to aIL stimulation in addition to decreasing the latency by 20-40 ms (n = 11). Ionophoresis of 2APV (2-amino 5-phosphono valeric acid) caused simultaneous abolition of spontaneous cortical bursts and bursts evoked by aIL stimulation. Short latency responses to cutaneous and VPl stimulation were unaffected by ionophoresis of 2APV sufficient to cause burst elimination, suggesting that this pathway does not operate via a 2APV sensitive receptor mechanism. Anatomical features of the aIL nuclei and their overall cortical projection pattern are discussed in relationship to these findings. The activation of cortical NMDA/APV sensitive receptors by aIL afferents in the "spontaneous" generation of bursts in cortical cells is discussed. PMID- 3019752 TI - Integration in descending motor pathways controlling the forelimb in the cat. 14. Differential projection to fast and slow motoneurones from excitatory C3-C4 propriospinal neurones. AB - The projection of C3-C4 propriospinal neurones (PNs) to alpha-motoneurones of forelimb muscles has been analysed with the aid of antidromic stimulation of the ascending branch of the PNs to the lateral reticular nucleus (LRN). A single stimulus of 500 microA applied in the caudo-dorsal part of the LRN evoked a maximal or greater than 90% maximal monosynaptic EPSP in the motoneurones. Systematic mapping of EPSPs evoked by stimulation of 500 microA in and around the LRN revealed that at this strength there was hardly any co-activation of a medial system (Peterson et al. 1979) which evoked small monosynaptic EPSPs with shorter latency and faster time course. The LRN EPSP amplitude was positively correlated with the homonymous group Ia EPSP amplitude, the input resistance and the afterhyperpolarization (AHP) duration. It is therefore postulated that the LRN EPSP amplitude is correlated with motor unit type (Burke 1967, 1968; Burke et al. 1973) with the largest EPSPs in slow (S), the smallest in fast, fatiguable (FF) and possibly intermediate sized in fast, fatigue resistant (FR) units. There was only a small difference in latency of the LRN EPSP in fast and slow motoneurones, while the time course was considerably slower in the latter. It is suggested that slow motoneurones receive projection both from fast and slowly conducting PNs but fast motoneurones mainly from fast PNs. Comparison of the disynaptic pyramidal EPSPs and the LRN EPSPs revealed a positive correlation, but the amplitude ratio pyramidal EPSP: LRN EPSP was smaller in slow than in fast motoneurones. A negative correlation was found between this amplitude ratio and the latency of the disynaptic pyramidal EPSP. It is suggested that this correlation reflects the excitability level in the PNs and that low excitability is due to inhibition of the PNs. PMID- 3019753 TI - Integration in descending motor pathways controlling the forelimb in the cat. 15. Comparison of the projection from excitatory C3-C4 propriospinal neurones to different species of forelimb motoneurones. AB - In the preceding report (Alstermark and Sasaki 1986) it was shown that a stimulus of 500 microA applied in the lateral reticular nucleus (LRN) evokes a maximal or near monosynaptic EPSP (LRN EPSP) in forelimb motoneurones. This EPSP which is assumed to be selectively mediated by C3-C4 propriospinal neurones (PNs), was used to estimate the strength of the excitatory projection from C3-C4 PNs. A systematic comparison was made of the size and time course of the maximal LRN EPSP in various species of forelimb alpha-motoneurones innervating shoulder, elbow, wrist and digit muscles. The LRN EPSP was evoked in all investigated species of forelimb motoneurones. When either the peak amplitude or the underlying area of the LRN EPSP was compared, a three-fold range was found with some tendency for the size to vary in the order of wrist greater than shoulder approximately equal to elbow greater than digit greater than intrinsic paw motor nuclei. Generally, a positive correlation was found in each motor nucleus between the peak amplitude of the LRN EPSP versus the monosynaptic homonymous group Ia EPSP, input resistance and afterhyperpolarization duration respectively (cf. Alstermark and Sasaki 1986). It is therefore postulated, that the LRN EPSP peak amplitude is correlated with motor unit type. Comparison of the time course of the LRN EPSPs was made by measuring the time-to-peak (T-t-p) and half-width (H w). The finding in the preceding report that the T-t-p and H-w is longer in slow than in fast motoneurones was confirmed and extended to all the investigated motor nuclei. The hypothesis that both fast slow motoneurones receive projection from a group of fast C3-C4 PNs, while slow motoneurones receive an additional projection from a group with lower conduction velocity, can therefore be applied to all forelimb motor nuclei. In addition, it is proposed that some slow shoulder, wrist and digit motoneurones receive projection from a special subpopulation of C3-C4 PNs with very slow conduction velocity. PMID- 3019751 TI - Monosynaptic connexions of low threshold muscle afferents with hindlimb motoneurones in the turtle spinal cord. AB - Excitatory postsynaptic potentials (EPSPs) were recorded intracellularly from hindlimb motoneurones of the anaesthetized fresh water turtle. The EPSPs were evoked from low threshold muscle afferents and the amplitudes saturated for stimuli less than two times the nerve threshold. The segmental latencies of these EPSPs, measured from the initial positive peak of the triphasic cord dorsum potential to the onset of the EPSP, ranged from 1.5 to 3.1 ms. The intraspinal conduction time of afferents was estimated by recording afferent volleys in the grey matter along the vertical course of intraspinal afferent fibres. The synaptic delay was estimated by subtracting the latency of the afferent volley at the deepest region of the dorsal horn from the segmental latency of the EPSP (in the range from 1.6 to 2.1 ms) recorded in the same microelectrode track. The average value was 0.99 ms (range: 0.9-1.1 ms), which was close to the known synaptic delay of cold-blooded animals. Therefore, the EPSPs in this range of segmental latencies were regarded as monosynaptic. Taking account of the intraspinal afferent conduction time (0.8 ms on average) and another synaptic delay, the latency for disynaptic transmission would be 2.8 ms or more. Thus, EPSPs having segmental latencies of 1.5-3.1 ms were suggested to be almost all monosynaptic in nature, at least under the present conditions of deep anaesthesia. On the basis of the above criteria for the monosynaptic nature of EPSPs, the pattern of convergence of monosynaptic excitatory inputs from various muscle afferents was investigated. Monosynaptic EPSPs were induced from the homonymous muscle nerve and the nerve innervating the synergist at the same joint. The heteronymous EPSPs were also found between muscles within each group of the anterodorsal musculature and the posteroventral musculature. No monosynaptic connexions were found between anterodorsal and posteroventral muscles except between the muscles innervated by the peroneal and the tibial nerve. PMID- 3019754 TI - Histogenesis and tissue reconstruction of mixed mesodermal tumor. AB - The clonal cell lines designated as HIRS-BMS and HIRS-BMA were established from HIRS-BM (multipotent primitive cells which differentiated into rhabdomyosarcoma and adenocarcinoma cells) by a single cell plating method. The HIRS-BMS was a rhabdomyosarcoma cell line composed of elongated fibrous cells which contained myoglobin. The HIRS-BMA was an adenocarcinoma cell line composed of round cells. The tissue reconstruction of these cell lines was studied in vitro (rotation culture system) and in vivo (transplantation into hamster cheek pouch). The HIRS BMS cells produced rhabdomyosarcoma, both in vitro and in vivo, and the HIRS-BMA produced poorly differentiated adenocarcinoma, in vitro and in vivo. The mixture of HIRS-BMS and HIRS-BMA produced a mixed mesodermal tumor resembling the original tumor. These results support the theory of a combined tumor as a cause of mixed mesodermal tumor. PMID- 3019755 TI - Determination of erythrocyte pyrimidine 5'-nucleotidase activity by 31P nuclear magnetic resonance: comparison of normal controls and multiple sclerosis patients. AB - A technique to assay erythrocyte pyrimidine 5'-nucleotidase activity in situ using 31P nuclear magnetic resonance spectroscopy is presented. The assay is chemically specific, simple and applicable to untreated lysates. A comparison of enzyme levels in normal controls and in multiple sclerosis patients employing the assay yielded no significant differences between both groups. Difficulties encountered in the quantitative analysis of the assay using 1H-NMR spectroscopy are briefly discussed. PMID- 3019757 TI - Effects of 18-hydroxydeoxycorticosterone on central nervous system excitability. AB - The effects of 18-hydroxydeoxycorticosterone (18-OH-DOC) on central nervous system excitability were studied in adrenalectomized rats. Sixty-four evoked potentials (EP) recorded from the pontine reticular formation were averaged before and after the injection of vehicle and hormone. 750 micrograms of 18-OH DOC dissolved in 0.5 ml of a 4:1 saline Cremophor-EL solution were injected i.v. A decrease of 55.7 +/- 6.1% in the amplitude of the EPs was observed with the hormone 16.3 min +/- 2.7 (SE) after injection. Amplitude values returned to baseline levels 38 min +/- 6.8 (SE) after injection. The secretion of 18-OH-DOC is greatly increased by ACTH and might modulate central nervous system function. PMID- 3019756 TI - The in vitro characterization of the inhibition of mouse brain protein kinase-C by retinoids and their receptors. AB - The mechanism of the in vitro inhibition of Ca2+-, phosphatidylserine-dependent protein kinase C (PK-C)2 by the purified holo (ligand-saturated) forms of cellular retinol-binding protein (cRBP) and cellular retinoic acid-binding protein (cRABP) was studied. We report here that the PK-C-inhibitory action of holo-cRBP and holo-cRABP is due to their respective ligands, all-trans-retinol and all-trans-retinoic acid; the reduced phosphorylation of the holo-retinoid binding proteins and brain cytosolic proteins is not the result of a retinoid induced soluble phosphatase or protease activity; retinoids reduce PK-C affinity for calcium and phosphatidylserine in vitro; and the structure-function activity of the retinoids and the specific interaction of these compounds with their binding proteins are important in blocking the activity of PK-C. These observations suggest that the inhibitory effect of retinoids on plasma membrane associated PK-C activity pays a significant role in defining the early epigenetic aspects of PK-C-dependent tumor promotion and may be a physiological mechanism by which retinoids induce terminal differentiation in cell types that do not express soluble retinoid-binding proteins. PMID- 3019759 TI - [Effect of dimetpramid and metoclopramide on the rate of catecholamine turnover in the subcortical-stem structures of the rat brain]. AB - Dimetpramide, a new antiemetic drug (manufactured in the USSR) from the group of substituted benzamides, produced, due to blockade of catecholaminergic receptors, an increase in dopamine and noradrenaline turnover rates in the subcortical brainstem regions responsible for the behavioural activity when administered at considerably higher doses than those of metoclopramide, a conventional drug of similar type. It is suggested that the probability of the occurrence of undesirable side effects of dimetpramide in clinical practice (extrapyramidal and sedative effects) would be much less than with metoclopramide. PMID- 3019758 TI - Possible target of Abelson virus phosphokinase in cell transformation. AB - By fusing interphase cells to cells undergoing mitosis, the interphase chromosomes can be visualized. When analyzed in this way, chromosomes of normal mouse cells show characteristic undercondensed centromeric regions. We have found that the centromeric regions of chromosomes from Abelson virus-transformed cells are fully condensed. Abelson virus transforms mouse cells by introducing into them a virally encoded phosphokinase that is expressed constitutively. Thus, we propose that the condensation of centromeric chromatin is a result of overphosphorylation by the Abelson virus phosphokinase, and that the centromeric region is the relevant target of overphosphorylation in transformed cell growth. PMID- 3019761 TI - [Pharmacological activity of cytochrome C]. AB - Cytochrome C (a single dose range of 1-200 mg/kg, intraperitoneally) does not possess the antihypoxic activity (altitude chamber, methemoglobinemia, circulatory hypoxia), decreases (in a dose of 50 mg/kg) diuresis in rats modifying diversely (depending on the dose used) the hypnotic effects of general anesthetics and the convulsant activity of corazol and arecoline. PMID- 3019760 TI - [Effect of amizil on the carbohydrase activity in intact animals and animals subjected to stress exposure]. AB - Effect of a central cholinolytic drug amizyl on the activity of some digestive enzymes involved in carbohydrate hydrolysis was studied in experiments on control rats and animals exposed to immobilization stress. The experiments showed that amizyl administration modifies the activity of carbohydrases that could affect disintegration and assimilation of carbohydrases. Preliminary administration of the drug was shown to prevent substantially the changes in the enzyme activity occurring during stress situation. PMID- 3019762 TI - [Relationship between the cyclic nucleotide content of blood plasma and the clinical course of chronic alcoholism]. AB - In patients with chronic alcoholism out of abstinence the plasma cAMP concentration was shown to decrease and that of cGMP to increase, cAMP/cGMP ratio was 1.8 in contrast to control value of 4.2. During abstinence the plasma cAMP level was higher and cAMP/cGMP ratio in patients with abstinence was 3.1. In patients with acute hallucinosis the plasma cAMP level was lower but during delirium, on the contrary, as well as the plasma cGMP level, higher than in control. The treatment of alcoholic patients relieved clinical symptoms of abstinence but the plasma cyclic nucleotide levels didn't change significantly. In most patients after cessation of delirium the plasma cAMP level was significantly lower as compared with pretreatment level. PMID- 3019763 TI - Calpain-calpastatin and fusion. Fusibility of erythrocytes is determined by a protease-protease inhibitor [calpain-calpastatin] balance. AB - Rat erythrocytes fuse when treated with the membrane mobility agent, 2-(2 methoxyethoxy)ethyl-cis-8-(2-octylcyclopropyl)octanoate (A2C) and Ca2+, whereas human cells do not. Membrane proteolysis promoted by calpain is required for rat cell fusion [(1986) Eur. J. Biochem., in press]. Human calpain induced a selective proteolysis in both the human and rat erythrocyte ghosts (mainly band 4.1 in the human, band 4.1 and band 3 in the rat cell) and rendered them fusible. Calpastatin (calpain inhibitor) prevented A2C-induced fusion in both ghosts, via inhibition of proteolysis. The human erythrocyte has excess calpastatin and resists A2C-promoted fusion. A regulatory role of calpastatin in membrane fusion is thus indicated. PMID- 3019764 TI - Studies on inorganic pyrophosphatase using imidodiphosphate as a substrate. AB - Baker's yeast inorganic pyrophosphatase has been found to catalyze Mg2+-dependent hydrolysis of imidodiphosphate yielding phosphate and amidophosphate. The reaction proceeds linearly in the presteady state. The catalytic constant is maximal at pH 9.0 and equals 0.5 min-1. Kinetic titrations of the enzyme with imidodiphosphate and Mg2+ have provided direct evidence for the involvement of three Mg2+ per active site in the transition state of the pyrophosphatase reaction. PMID- 3019765 TI - Absence of structural homology between sup1 and sup2 genes of yeast Saccharomyces cerevisiae and identification of their transcripts. AB - The results of Southern blotting demonstrate that sup2 is a unique gene in Saccharomyces cerevisiae that does not possess homologous sequences in the yeast genome. The direct hybridization of DNA fragments, containing cloned sup1 and sup2 genes, did not reveal any structural homology between these two genes. By Northern blotting analysis the sizes of the transcripts were determined to be 1.6 kb for sup1 gene and 2.5 and 1.4kb for sup2 gene. Experiments with RNA isolated from yeast mutant with impaired splicing demonstrated that sup1 and sup2 genes do not contain introns. PMID- 3019767 TI - A gene in Paracoccus for subunit III of cytochrome oxidase. AB - The region of Paracoccus denitrificans chromosome where the genes coding for cytochrome oxidase (cytochrome aa3) subunits are located has been cloned. DNA sequencing revealed an open reading frame that codes for a protein homologous to the subunit III of the eukaryotic, mitochondrial enzyme. This subunit is absent from the isolated Paracoccus oxidase. It now seems that it is part of the native enzyme in the bacterial cytoplasmic membrane. This may explain the observed discrepancies in the function of the isolated enzyme. PMID- 3019768 TI - Use of fluoride ion as a probe for the guanine nucleotide-binding protein involved in the phosphoinositide-dependent neutrophil transduction pathway. AB - Fluoride activation of neutrophils was found to be associated with phosphoinositide turnover, as monitored by the time-dependent accumulation of inositol phosphates. Unlike phosphoinositide turnover induced by the chemotactic peptide, formylmethionylleucylphenylalanine, that induced by fluoride was not inhibited by pretreatment with pertussis toxin. The translocation of protein kinase C activity from the cytosolic to the membrane compartment was also observed in fluoride-stimulated cells. We have proposed that the mode of action of this halide ion involves interaction with a GTP-binding protein which serves as an intermediary unit between the receptors for inflammatory stimuli and the phosphoinositide-specific phosphodiesterase. PMID- 3019769 TI - Altered protein kinase activities of lymphoid cells transformed by Abelson and Moloney leukemia viruses. AB - Five different types of protein kinase activities have been evaluated in cell lines from murine lymphomas induced by Abelson leukemia virus (A-MuLV), whose oncogene codes for a tyrosine protein kinase. Such activities were compared with those of normal cells and of cells transformed by Moloney leukemia virus (M MuLV), lacking oncogene sequences in its genome. While cAMP-dependent protein kinase and casein kinase-1 do not undergo significant changes, casein kinase-2 rises in both A-MuLV and M-MuLV infected lymphocytes, becoming largely associated with the particulate fraction of transformed cells. Protein kinase-C on the other hand is unchanged in M-MuLV transformed cells but it undergoes a 2-3-fold increment in both soluble and particulate fractions of A-MuLV transformed lymphocytes, which also display high tyrosine protein kinase activity. PMID- 3019766 TI - The modulation of cytochrome c electron self-exchange by site-specific chemical modification and anion binding. AB - The site-specific chemical modification of horse heart cytochrome c at Lys-13 and -72 using 4-chloro-3,5-dinitrobenzoic acid (CDNB) increases the electron self exchange rate of the protein. In the presence of 0.24 M cacodylate (pH* 7.0) the electron self-exchange rate constants, kex, measured by a 1H NMR saturation transfer method at 300 K, are 600, 6 X 10(3) and 6 X 10(4) M-1 X s-1 for native, CDNP-K13 and CDNP-K72 cytochromes c respectively. Repulsive electrostatic interactions, which inhibit cytochrome c electron self-exchange, are differentially affected by modification. Measurements of 1H NMR line broadening observed with partially oxidised samples of native cytochrome c show that ATP and the redox inert multivalent anion Co(CN)3-6 catalyse electron self-exchange. At saturation a limiting value of approximately 1.4 X 10(5) M-1 X s-1 is observed for both anions. PMID- 3019770 TI - Activation of the Na+/K+-ATPase by interleukin-2. AB - Activated B61.SF.1 and CTLL-2 T lymphocyte clones which are strictly dependent on interleukin-2 (IL-2) for growth were used to study the activation of Na+/K+ ATPase. 50% of [3H]thymidine maximal incorporation was obtained when the extracellular concentration of Na+ or K+ was reduced to 50 or 2 mM, respectively. 'Quiescent' CTL clones stimulated with IL-2 showed an increase of 48-380% in ouabain-sensitive 86Rb uptake. Furthermore, this stimulation was completely inhibited by a monoclonal antibody PC.61 directed at the IL-2 receptor. The activation of the pump was dependent on the dose of IL-2, took place at the same doses of IL-2 that were required to stimulate cell proliferation and was linear for at least 30 min. PMID- 3019771 TI - Organization of hydroxyapatite crystals within collagen fibrils. AB - Transmission electron micrographs of individual mineralized collagen fibrils show that hydroxyapatite crystals are located mainly within the fibrils at the level of the gap regions. The plate-shaped crystals are observed to be more or less uniformly stacked across the fibril diameter. We therefore suggest that the crystals are primarily located in 'grooves' created by contiguous adjacent gaps. The proposal is consistent with the observed crystal distribution in the fibril and with their average widths, which are almost 10-times greater than an individual gap diameter. PMID- 3019773 TI - Complete recovery of the phosphoenzyme-forming activity of nucleoside-diphosphate kinases after reconstitution of their subunits. AB - Two distinct subunits [alpha-subunit (Mr 21 000, pI 7.6) and beta-subunit (Mr 19 000, pI 6.5)] of nucleoside-diphosphate (NDP) kinases highly purified from HeLa S3 cells can be separated by FPLC using a Mono P column in the presence of 6 M urea and 1% pharmalyte (pH range between 5.0 and 8.0). Comparatively high [32P]phosphate incorporation was detected when these two subunit fractions were reconstituted in vitro. Available evidence suggests that these two enzyme subunits are necessary for the formation of phosphoenzyme, which functions as an intermediate in NDP kinase action. PMID- 3019772 TI - Protein kinase C regulates leukotriene B4 receptors in human neutrophils. AB - Three protein kinase C (PKC) activators, viz. phorbol myristate acetate, mezerein, and rac-1-O-myristoyl-2-acetylglycerol, inhibited human neutrophil binding of [3H] leukotriene B4 (LTB4) by reducing the number of high-affinity receptors available to the arachidonic acid metabolite. The inhibitory effect occurred in whole cells and cytoplasts but not in isolated membranes; it appeared to involve the activation of PKC rather than direct competition for binding sites. PKC may govern cellular responsiveness by regulating the receptor-linked bioactions of endogenous mediators like LTB4. PMID- 3019774 TI - Inositol phosphate production following alpha 1-adrenergic, muscarinic or electrical stimulation in isolated rat heart. AB - A possible participation of polyphosphoinositide metabolism in the excitation contraction coupling in heart was investigated. Isolated rat ventricles prelabelled with myo-[2-3H]inositol were stimulated by conditions that increase mechanical activity. Both noradrenaline and carbachol increased the basal level of IP3, IP2 and IP by the activation of alpha 1-adrenergic and muscarinic receptors, respectively. Electrical stimulation accelerated inositol lipid degradation by phospholipase C thus enhancing the IP3 level as compared to quiescent ventricles. It is proposed that IP3 may be involved in excitation contraction coupling in cardiac tissue. PMID- 3019775 TI - ADP stimulates IP3 formation in human platelets. AB - Aspirinated human platelets labeled with 32PO4 showed a 1.7-fold increase in [32P]IP3 when stimulated with ADP. ADP-stimulated mobilization of internal Ca2+ and phosphorylation of myosin were enhanced in the presence of extracellular Ca2+ but the increase in IP3 was not significantly affected by external Ca2+. The Ca2+ ionophore, ionomycin, elevated internal Ca2+ and induced myosin phosphorylation without a detectable change in IP3. These results indicate that the mechanism of ADP stimulation of human platelets is similar to that of other platelet agonists and supports the theory that IP3 functions to liberate internal Ca2+. PMID- 3019776 TI - Protein kinase C activators modulate differentiated thyroid function in vitro. AB - Exposure of porcine thyroid cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) leads to inhibition of differentiated thyroid function. We investigated whether this effect is mediated via protein kinase C activation. TPA, phorbol 12,13-didecanoate and phorbol 12,13-dibutyrate inhibited TSH stimulated iodine organification in porcine thyroid cells by 98, 96 and 45%, respectively. Non-tumour promoting phorbol esters had no effect. The diacylglycerol analogue, sn-1,2-dioctanoylglycerol had similar but quantitatively less activity than TPA. Dibutyryl cAMP could not reverse any inhibition noted. Under conditions that caused significant inhibition of differentiated function, TPA caused translocation of thyroidal protein kinase C from the cytosol to its membrane-bound form. These data provide evidence that the mechanism of phorbol action on thyroid function in vitro includes activation of protein kinase C. PMID- 3019778 TI - In situ ductal carcinoma of the breast: implications of disease pattern and treatment. AB - Ninety-seven patients with in situ ductal carcinoma (DCIS) of the breast have been reviewed. The commonest presenting feature was a breast lump, and residual carcinoma was found in the mastectomy specimen in 63% of patients. Furthermore, 13 cases had evidence of infiltrating carcinoma when the mastectomy specimen was examined. Thus excision alone would have left residual in situ or infiltrating carcinoma in two-thirds of the cases. A wide excision, by removing local residual disease, would still have left multifocal disease in one-third of cases. Studies need to be conducted to determine whether conservative treatment of DCIS can yield results which are as good as those following total mastectomy. PMID- 3019779 TI - Wait-and-see policy in clinical stage I non-seminomatous germ cell tumors of the testis. AB - Thirty-three patients with a non-seminomatous germ cell tumor of the testis in clinical stage I were treated only by orchidectomy. The very careful follow-up- with tumor marker assays every 3 weeks, chest X-rays every 6 weeks and CT-scans of the lungs and retroperitoneum every 3 months--revealed metastases in 7 of the patients (21%). All these relapses were diagnosed within 6 months of the orchidectomy. Para-aortic node metastases were found in 5 of the 7 patients, with additional inguinal node metastases in 1 and additional lung metastases in 1; 2 patients had only lung metastases. Six of the 7 patients with a relapse were given chemotherapy (PVB); 1 patient refused chemotherapy. In view of residual disease a surgical excision was performed; it revealed necrosis as well as mature teratoma. All 33 patients are still alive, the post-orchidectomy follow-up period being 12-38 months. PMID- 3019780 TI - Prolonged survival without treatment in a patient with primary hepatocellular carcinoma. AB - The proportion of primary hepatocellular carcinomas which are technically operable with hope of cure is small. Recommended therapy ranges from aggressive therapy to a nihilistic approach. There is little data available on the natural history of untreated tumours and we report such a case. A review of the literature regarding survival with primary hepatocellular carcinoma shows that the prognosis of patients with this tumour may vary considerably. Such variability has important implications for those involved in the design and interpretation of trials of treatment for hepatocellular cancer. PMID- 3019777 TI - Identification of three protein kinases which phosphorylate threonyl-tRNA synthetase from rat liver. AB - Threonyl-tRNA synthetase is phosphorylated in Chinese hamster ovary cells labeled with 32Pi [(1984) J. Biol. Chem. 259, 11160-11161]. Phosphorylation of the purified synthetase from rat liver has been examined with five different protein kinases. Three of the enzymes phosphorylate the synthetase, protease activated kinase I, the cAMP-dependent protein kinase, and the Ca2+, phospholipid-dependent protein kinase. Phosphorylation occurs exclusively on seryl residues. Two dimensional phosphopeptide maps of tryptic digests of the phosphorylated synthetase are distinct with each protein kinase. These data suggest that multiple phosphorylation of the synthetase may occur in vivo. PMID- 3019781 TI - Eponyms in oncology. Max Wilms (1867-1918). PMID- 3019782 TI - Phosphorylated derivatives of myo-inositol. AB - The structures of myo-inositol and its isomers are reviewed, together with those of the phosphoinositides, and of the inositol phosphates resulting from phosphodiesteratic cleavage. Inositol trisphosphate and related derivatives are considered with regard to their second messenger function. Strategies for possible disruption of enzymatic steps leading to the formation and degradation of these substances are discussed. PMID- 3019783 TI - Formation and biological action of inositol 1,4,5-trisphosphate. AB - A wide variety of receptors appear to be coupled to a phospholipase C (EC 3.1.4.3) that hydrolyzes inositol lipids. This reaction is believed to provide a link between receptor activation and cellular Ca2+ mobilization. The mechanisms by which this occurs are believed to involve inositol 1,4,5-trisphosphate (1,4,5 IP3), which signals release of Ca2+ from the endoplasmic reticulum. In rat parotid acinar cells made permeable with saponin, 1,4,5-IP3 induced rapid release of sequestered Ca2+. In intact parotid cells, the concentration-response relationship for methacholine-induced IP3 formation was similar to the relationship for muscarinic receptor occupancy by methacholine. About 10-fold lower concentrations of methacholine were sufficient to increase cytosolic [Ca2+] and to activate secretion, indicating an excess IP3 forming capacity for the muscarinic receptor. The mechanisms for the coupling of receptors to IP3 formation were studied in pancreatic acinar cells made permeable electrically. In this preparation, nonhydrolyzable derivatives of GTP potentiated agonist-induced IP3 production, which suggests the involvement of a guanine nucleotide-dependent regulatory protein. The effects of agonists and guanine nucleotides were not altered by pretreating the acinar cells with cholera or pertussis toxins, which indicated that the regulatory protein linking receptors to IP3 formation is distinct from the ones involved in the regulation of adenylate cyclase. PMID- 3019785 TI - Phosphoinositide kinase activity and transformation. AB - We have used the DNA tumor virus polyoma as a model system to examine whether the phosphatidylinositol (PI) turnover pathway is a critical target for transforming gene products. Polyoma-infected cells show elevated levels of polyphosphoinositides and polyphosphoinositols, and a PI kinase activity is associated with middle T antigen, a transforming gene product of polyoma virus. In anti-T immunoprecipitates from polyoma-infected or -transformed cells, comparisons of wild-type and polyoma mutants defective for transformation show a strong correlation between middle T-associated PI kinase activity and transforming ability. Middle T has previously been found to associate at the plasma membrane with pp60 c-src and to activate it as a tyrosine kinase. c-src itself does not appear to phosphorylate PI; however, the middle T/pp60 c-src tyrosine kinase activity may be important for activation of PI kinase. Ammonium orthovanadate, a tyrosine phosphatase inhibitor, elevates the middle T/pp60 c-src associated PI kinase activity. We propose that middle T/pp60 c-src activates a PI kinase and modulates PI turnover in vivo by tyrosine phosphorylation. PMID- 3019784 TI - Effects of lithium on phosphoinositide metabolism in vivo. AB - All of the known pathways for metabolizing the phospholipase C (EC 3.1.4.10) products of phosphoinositide metabolism eventually lead to myo-inositol monophosphates and products that are hydrolyzed by myo-inositol 1-phosphatase (EC 3.1.3.25). That enzyme is inhibited by lithium (Ki about 1 mM). In animals treated with LiCl, elevations of myo-inositol 1-phosphate (1-IP) occur in brain that appear to result from endogenous neural activity for they are diminished by the anesthetics halothane and pentobarbital. Lithium is thus a useful tool for assessing endogenous in vivo cerebral phosphoinositide metabolism. The 1-IP elevation is also useful for revealing in vivo central nervous system (CNS) receptor activity that is stimulated by endogenous or exogenous processes such as the effects of centrally acting drugs and of seizures. Stimulation of the CNS in the presence of lithium causes myo-inositol to be sequestered in 1-IP in proportion to the amount of stimulation. Thus if the inositol level falls sufficiently resynthesis of the phosphoinositides may be compromised and receptor response to stimuli may be reduced. Evidence for such an occurrence would support the theory that this is one mechanism by which lithium acts in the therapy of manic illness. We extended our efforts to identify such a lowering of phosphoinositide levels to mice where cerebral metabolism can be halted more rapidly than in rats. However, the only change detected was a small elevation in phosphatidylinositol 4-phosphate. We were successful, however, in causing all of the phosphoinositides to be reduced in rat cerebral cortex by pilocarpine stimulation after lithium treatment, a procedure that causes seizures. The same procedure causes the largest reduction in cortical myo-inositol levels that we have observed, and thus may represent the point where the inositol decrement is sufficient to interfere with resynthesis of the lipids. PMID- 3019787 TI - A child with 45,X/46,X,del(Y)(q12) identified with a Y-specific probe. PMID- 3019786 TI - Ethiodol inhibits phagocytosis by pelvic peritoneal macrophages. PMID- 3019788 TI - [Role of ACTH fragments in regulating electrogenesis and electrotonic synaptic interaction of pond snail neurons]. AB - The hypothesis of neuropeptides involvement in intercellular interaction was checked on the neurons VD1 and RPD2 connected with electrotonic synapse with two way transmission in nerve ganglia of the pond snail. The preparation was perfused with natural and synthetic fragments of ACTH (2 X 10(-7) M). In perfusion with ACTH4-10 solution, synapse became rectified whereas in ACTH4-7 and ACTH5-10 solutions it obtained partially rectified properties. After exposure to ACTH4-7- Pro--Gly--Pro, synapse obtained rectifying properties with one--way increase in conductivity following temporary two-way increase of transmission efficiency. With the use of Pro--Gly--Pro--ACTH4-7--Pro--Gly--Pro, inhibition of the two--way conductivity occurred. Neuropeptides seem to modulate synaptic transmission. Impulse priority depends on the initial level of the cell MPs and is purposefully modulated by the peptides under test. PMID- 3019789 TI - [Acetylcholine activation of the sodium pump in frog muscle]. AB - The effect of acetylcholine, ouabain, atropine and d-tubocurarine on the activity of K+, Na+-pump of the frog muscles was studied. Acetylcholine enhanced the activity of K+, Na+-pump, ouabain and atropine preventing the enhancement. D tubocurarine was ineffective in blocking the ionic pump. PMID- 3019790 TI - [Paradoxical reactions of a neuromuscular preparation (on the 100th anniversary of their discovery by N.E. Vvedenskii)]. AB - The discovery of the intensity of repetitive stimulation pessimum and of the paradoxical response to stregthening of stimuli in a parabiotic nerve, is presented in historical aspect. Both phenomena are shown to be based on the mutual electrical delay in propagated APs when the number of synchronously activated nerve fibers is increased. The delay may cause a propagation block through these nerve regions where the safety factor is diminished. PMID- 3019791 TI - [Electrophysiologic manifestations of the effect of monoaminergic systems on the cerebral cortex]. AB - Various forms of electropgraphic display of monoaminergic neurotransmitter system effect upon the cerebral cortex are discussed. Comparison of theoretical and experimental results suggests a modulating role of serotoninergic and noradrenergic systems. PMID- 3019792 TI - [Processes facilitating conduction in the vesical plexus of the cat]. AB - In the cat isolated bladder plexus, cholinergic transmission from parasympathetic "entry" was shown to be less obvious at low-frequency stimulation and much more effective (up to 300-1800%) at a high-frequency one (1-20/sec). During posttetanic potentiation (up to 5 min) it is possible to reach the 100% saturation of ganglion motor pool. The phenomena of facilitation were shown to be conducted through presynaptic mechanisms. The facilitation processes are more obvious in the bladder plexus than in sympathetic ganglia and allow to filtrate low and amplify high frequencies of activation which is particularly significant during urine storage and evacuation. PMID- 3019793 TI - [Relation between neuronal uptake and beta-adrenoreactivity of the rat heart]. AB - A positive correlation exists between the activity of neuronal uptake and the heart beta-adrenoreactivity. The amount of catecholamines in the myocardium is unrelated to this interconnection. PMID- 3019794 TI - [Contractile function and antioxidative system of the myocardium of the intact rabbit during hyperbaric oxygenation]. AB - Daily exposures of rabbits to the HBO (2 ata, 1 hr) enhanced activity of glutathione-peroxidase for all 28 days of exposure. Other parameters of the antioxidative defence and the contractile function remained unchanged. The 2.5 ata oxygenation sharply reduced the activity of antioxidative enzymes, the antioxidative activity of lipids, and the tissue resistance against the induced peroxide oxidation of lipids. The heart contractile function was obviously worsened. Sites of necrosis appeared in the myocardium tissue. Increase in oxygenation seems to lead to changes of adaptation of the antioxidative system, but the exhaustion of the latter's power reserves potentiates the toxic effect of hyperoxia. PMID- 3019795 TI - [Desensitization of alpha-adrenoreceptors of the small intestine of the rat]. AB - A study performed on the isolated section of the rat small intestine has shown that at a slow increase in the concentration of the specific alpha-adrenomimetic phenylephrine in the incubation medium 1.0 X 10(-8) M/sec) adrenoreceptors pass into a state of desensitization without excitation, and no reaction takes place. At 2.5 X 10(-8)-10 X 10(-8) M/sec the reaction develops depending on the rate of phenylephrine introduction and is stabilized at 5.5-5.8 X 10(-6) M. Apparently, under these conditions an equilibrium is set up between activation and desensitization of adrenoreceptors. The role of desensitization of alpha adrenoreceptors in the regulation of adrenergic reaction is discussed. PMID- 3019796 TI - [Abrikosov tumor--a clinical surprise diagnosis]. PMID- 3019797 TI - Stimulation of low Km cyclic AMP phosphodiesterase by sulphydryl modification. AB - Low concentrations of the organic mercurials, p-chloromercuribenzoate or p chloromercuriphenylsulphonate activate the particulate low Km phosphodiesterase from adipose tissue and liver. Higher concentrations are inhibitory. Enzyme which has been activated by treatment of adipocytes with insulin, is not activated by the organic mercurials although inhibition by higher concentrations is seen. Enzyme from non-insulin treated adipocytes is activated and solubilised by mild trypsin treatment. Enzyme activated by either insulin treatment, or by p chloromercuribenzoate is not further activated by trypsin, but it is solubilised. PMID- 3019798 TI - Endocrine-dependent regulation of tetrahydrobiopterin levels and guanosine triphosphate cyclohydrolase activity. AB - The role of endocrine organs in the regulation of tetrahydrobiopterin (BH4) levels and guanosine triphosphate cyclohydrolase (GTP-CH) activity was studied in the spleen, bone marrow and brain of rats and mice. Following hypophysectomy, BH4 levels and GTP-CH activity were significantly decreased in both spleen and bone marrow. Fourteen days after hypophysectomy GTP-CH activity and BH4 levels were approximately 25% of control levels in both tissues. In contrast, BH4 levels and GTP-CH activity in brain were not significantly different from control values. The decrease in GTP-CH activity and BH4 levels in spleen and marrow could not be reversed by high doses of ACTH or by a pituitary extract. Removal of the thyroid gland resulted in significant decreases in BH4 levels and GTP-CH activity in spleen; marrow and brain levels were not affected. BH4 levels in spleens of thyroidectomized rats returned to control values following treatment with either triiodothyronine or thyroxine. Adrenalectomy and castration had no effect on biopterin metabolism in bone marrow, spleen or brain. Tissue levels of BH4 and GTP-CH were also studied in mutant mouse strains having mutations in either pituitary or thyroid functions in order to examine further the role of these tissues in the regulation of the biosynthesis of this cofactor. The results of this study indicate that factors secreted from the pituitary are important in the regulation of BH4 levels and GTP-CH activity in spleen and bone marrow and that the thyroid gland also plays a role in regulation in the spleen. Levels of BH4 and GTP-CH in the brain, if regulated, appear to be independent of the endocrine tissues studied. PMID- 3019799 TI - Retinoic acid-binding protein in the axolotl: distribution in mature tissues and time of appearance during limb regeneration. AB - Analysis of cytoplasmic protein preparations from axolotl tissues revealed the presence of a cytoplasmic retinoic acid-binding protein (CRABP), of approximate molecular weight 17K. This protein was found to be present at various concentrations in skin, muscle, and limb tissue preparations, but not in liver and serum preparations. The distribution and molecular weight of this protein agrees with that reported in mammalian studies. The level of CRABP in cone stage blastemas was found to be significantly higher than that found in nonregenerating whole limb preparations. The level falls gradually, to approach normal, towards the completion of regeneration. Such an increase, at the start of regeneration, was not altered by 4 days pretreatment with 36 mg/liter all-trans-retinoic acid, a sufficient dose to produce pattern effects. Competition experiments confirmed that the all-trans and 13-cis isomers of retinoic acid bind to CRABP with similar high efficiencies, and that the arotinoid, Ro 13-6298, exhibits only a fraction of this binding activity. Retinol, retinol palmitate, and retinol acetate were unable to compete with [3H]retinoic acid for binding to CRABP. The results presented here are discussed in terms of their possible value to understanding pattern specification in the regenerating urodele limb. PMID- 3019800 TI - Changes during differentiation in requirements for cAMP for expression of cell type-specific mRNAs in the cellular slime mold, Dictyostelium discoideum. AB - A number of genes encoding developmentally regulated mRNAs in the cellular slime mold, Dictyostelium discoideum, have been described. Many of these are regulated by cAMP. Analysis of the earliest time at which elevated levels of cAMP can induce the expression of these mRNAs reveals a more complex pattern of regulation in which genes change in their ability to be induced in response to cAMP with developmental stage. A prestalk mRNA (C1/D11) previously thought not be regulated by elevated levels of cAMP is inducible by cAMP between aggregation and loose mound stage; later in development its expression becomes independent of elevated cAMP. The early prespore genes (prespore class I) also show two modes of regulation; early in development they are induced independently of continuous elevated levels of cAMP, while later in development their expression is dependent upon elevated cAMP. The period during development when the prestalk genes are cAMP inducible precedes by 2 hr the first time at which either the early prespore class I or late prespore class II mRNAs are inducible by continuous elevated levels of cAMP. Previous analysis of these mRNAs has been carried out using Dictyostelium cells grown axenically. In this report we have studied the developmental expression of these mRNAs in cells grown on bacteria. A substantial shutoff of the class I prestalk and early prespore (class I) mRNAs not seen in axenically grown cells is observed when bacterially grown cells are plated for development. Less than 10% of the maximal level of these mRNAs remains in the cells at the time of mature spore and stalk differentiation. Additionally, in the bacterially grown cells two distinct patterns of developmental regulation are observed for mRNAs which in axenically growing cells appear to be constitutively expressed throughout growth and development. PMID- 3019801 TI - Cyclic AMP and NH3/NH4+ both regulate cell-type-specific mRNA accumulation in the cellular slime mold, Dictyostelium discoideum. AB - Dictyostelium discoideum cells plated for development until aggregation stage, and then dissociated into media containing glucose, albumin, and cAMP will form into clumps and undergo prespore and prestalk differentiation. Differentiation in this in vitro system is dependent on three components: cAMP, multicellularity, and the acquisition of "differentiation competence" which the cells acquire in a period between interphase and aggregation stage when plated on Millipore filters. We have used this system to explore aspects of the multicellular environment which are involved in regulation the accumulation of the different prespore- and prestalk-specific messenger RNAs. Two classes of prespore messenger RNA, as well as a prestalk-specific messenger RNA, all require the acquisition of differentiation competence in order to be expressed in response to cAMP. Additionally, all of these messenger RNAs require agglomerate formation for maximal expression. The addition of 33 mM ammonium sulfate (NH4)2SO4, however, can entirely replace the requirement for agglomerate formation for expression of the prestalk-specific messenger RNA, and can partially substitute for agglomerate formation in inducing the expression of both classes of prespore-specific messenger RNAs. In this system, cAMP is essential for the initial induction of expression of all three classes of messenger RNAs. In this system, cAMP is essential for the initial induction of expression of all three classes of messenger RNAs while agglomerate formation or elevated NH3/NH+4 is essential only for the maintenance of the elevated levels of the messenger RNAs. PMID- 3019802 TI - A transient expression assay for tissue-specific gene expression of alcohol dehydrogenase in Drosophila. AB - The regulation of expression of the alcohol dehydrogenase gene of Drosophila was examined by injecting plasmids containing the gene directly into preblastoderm embryos and subsequently staining for alcohol dehydrogenase activity in somatic cells of larvae and adults. The alcohol dehydrogenase genes introduced in this manner were expressed normally in both adults and larvae; i.e., alcohol dehydrogenase activity was found exclusively in tissues where it would normally be expressed. Activity was found in some cells in more than 90% of all surviving third instar larvae, but not all cells which would normally express the enzyme were positive, presumably due to the random distribution of the injected DNA to the cells of the embryo. Regulated expression was not dependent on the vector used: tissue-specific expression was obtained from alcohol dehydrogenase genes inserted in the P-element vector, Carnegie-4; in pBR322; in pUC18; or in bacteriophage lambda. The bulk of the injected DNA was not integrated into the chromosome and appeared to persist throughout development as supercoiled and nicked circles. Using the procedure and in vitro mutagenesis, we were able to show that the alcohol dehydrogenase gene was expressed in a normal tissue specific manner in larvae if there were 777 nucleotides of upstream information present. PMID- 3019803 TI - Alleviation of infantile amnesia in rats by internal and external contextual cues. AB - A previous study [Richardson, R., Riccio, D.C., and Jonke, T. (1983). Alleviation of infantile amnesia in rats by means of a pharmacological contextual state. Devel. Psychobiol., 16:511-518] found that ontogenetic forgetting ("infantile amnesia") in rats was attenuated by a "contextual matching" manipulation: Subjects trained in a distinct pharmacological state and tested in that state retained learned fear better than saline controls. To determine whether improved retention could be obtained with salient but nonpharmacological contexts, two experiments were conducted employing fear conditioning in preweanling rats. In Experiment 1, an internal context (illness induced by lithium chloride) present at training and testing reduced infantile forgetting. In Experiment 2, matching an exteroceptive context (home nest shavings) at training and testing was also found to be sufficient to alleviate infantile amnesia. In both experiments, retention was not improved in controls exposed to the context at training only or testing only, indicating that the contextual effect is not on acquisition or retrieval processes per se. These findings provide indirect support for views that emphasize the role of contextual cues in retrieval (Spear, 1978). In addition, they indicate that contextual matching is not limited to a state dependent drug, but may include a wide range of "distinctive" stimuli. PMID- 3019805 TI - Novel gene activated in rat insulinomas. AB - Insulinomas can be induced in experimental animals by the combined administration of diabetogenic agents with polyadenosine diphosphate (polyADP)-ribose synthetase inhibitors. A complementary DNA (cDNA) library that was constructed from streptozocin-nicotinamide-induced rat insulinomas has been found to contain a novel gene encoding a basic protein of 145 amino acids. The gene was expressed in alloxan-nicotinamide-induced insulinomas as well as in streptozocin-nicotinamide induced insulinomas but not in normal pancreatic islets or in regenerating islets. This indicates that the activation of the gene designated rig, i.e., rat insulinoma gene, may be a general feature of pancreatic beta-cell transformation. PMID- 3019804 TI - Decreased myo-inositol content and Na+-K+-ATPase activity in superior cervical ganglion of STZ-diabetic rat and prevention by aldose reductase inhibition. AB - Decreased sciatic nerve myo-inositol content and Na+-K+-ATPase activity have been associated with slowing of motor nerve conduction in the acutely diabetic rat and have been invoked as possible pathogenic factors in diabetic peripheral neuropathy. Aldose reductase inhibitors prevent these abnormalities in peripheral nerves of the streptozocin (STZ)-diabetic rat. Whether an analogous biochemical abnormality occurs in the autonomic nervous system and plays a role in the development of diabetic autonomic dysfunction is unknown. Therefore we examined the effect of 8 wk of untreated STZ diabetes and administration of 20 mg X kg-1 X day-1 of the aldose reductase inhibitor sorbinil on myo-inositol level and Na+-K+ ATPase activity in rat superior cervical ganglia. Both myo-inositol concentration and Na+-K+-ATPase activity were reduced in ganglia from untreated STZ-diabetic rats, and sorbinil administration prevented these abnormalities. Thus, a sorbinil responsive metabolic defect involving myotional abnormalities in the somatic and autonomic nervous systems in diabetes. PMID- 3019807 TI - Beta-adrenergic receptors and adenylate cyclase activity in diabetic rat fat cells. AB - Our study describes the effect of norepinephrine on adenylate cyclase activity in adipocytes from control and diabetic rats. The results show that diabetes induced an increase in both basal and norepinephrine-stimulated adenylate cyclase activity. This higher activity was not suppressed when the animals were treated for 2 days with the beta-blocking agent propranolol. On the other hand, adipocytes from control animals treated with propranolol showed a higher adenylate cyclase activity (basal and in response to norepinephrine). beta Adrenergic receptors were examined in adipocytes from control and diabetic rats with and without treatment with propranolol. The results show a higher beta receptor density in adipocytes from diabetic animals. When the animals were treated with propranolol, the beta-blocker induced a higher receptor density in adipocytes from control animals without affecting the already increased receptor density in diabetic preparations. The data suggest that adenylate cyclase activity in response to norepinephrine in adipose tissue is increased during at least a certain period of the diabetic state. This increase in adenylate cyclase activity is accompanied with an increase in beta-receptor density, but in contrast to control animals, this receptor density is not further increased with propranolol treatment. PMID- 3019806 TI - Expression of class I MHC proteins on RIN-m5F cells is increased by interferon gamma and lymphokine-conditioned medium. AB - To examine whether products of the immune system interact with the pancreatic beta-cell, rat insulinoma cells (RIN-m5F line) were cultured in the presence of conditioned medium from concanavalin A-activated mouse spleen cells (CAS medium). Indirect immunofluorescence and flow cytometry revealed that after culture in CAS medium, RIN-m5F cells had an 8- to 10-fold increase in class I major histocompatibility complex (MHC) proteins, whereas class II MHC proteins remained undetectable, and the level of insulin and/or insulin-like growth factor 1 receptors was unchanged. The stimulation of class I MHC expression on RIN-m5F cells by CAS medium could be mimicked by recombinant interferon-gamma. Analysis of 125I-surface-labeled cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that in the presence of CAS medium, there was a major increase in the expression of proteins of 48,000, 32,000, and 12,000 Mr and a minor increase in proteins of 17,000 and 9,000 Mr. Precipitation with monoclonal antibody identified the 48,000- and 12,000-Mr proteins as the class I MHC protein and beta 2-microglobulin, respectively. The ability of lymphokine-conditioned medium to increase the expression of RIN-m5F cell surface proteins, including the class I MHC proteins, provides a potential mechanism for enhancing the immune-mediated destruction of the beta-cell. PMID- 3019808 TI - Cyclic AMP phosphodiesterase in diabetes. Effect of glyburide. AB - Low-Michaelis constant cAMP phosphodiesterase (PDE; EC3.1.4.C) activity is inhibited in tissues of rats with type I ketosis-prone diabetes and is restored to normal by insulin treatment. To determine whether the oral hypoglycemic agent glyburide affected tissue cAMP PDE activity in non-insulin-dependent oral agent treatable diabetes, cAMP PDE activity was measured in the liver and fat of animals rendered diabetic by low-dose streptozocin (STZ-DM) and treated for 3 wk with oral glyburide (360 micrograms/kg). The results were compared with PDE activity in the liver and fat of untreated STZ-DM and normal control rats. At the time of death, low-Km cAMP PDE activity [as maximum velocity (Vmax)] in STZ-DM rats was decreased to 66% of control values in the liver and to 65% in fat (P less than .001). PDE activity was restored toward normal by glyburide treatment: 91% in the liver (P less than .01) and 80% in fat (P less than .05). Calmodulin and calmodulin-like activity (PDE-activator activity) in the liver and fat was decreased in diabetes and restored toward normal after glyburide treatment (P less than .05). These data demonstrate that oral agents as well as insulin can restore the activity of cAMP PDE in the low-dose STZ-DM model, which is in some ways similar to type II diabetes. PMID- 3019810 TI - Comparison of the therapeutic effect of wheat bran, mebeverine and placebo in patients with the irritable bowel syndrome. AB - There is no generally accepted treatment in the irritable bowel syndrome (IBS), probably because of a lack of convincing therapeutic trials. In the present study, 120 outpatients with IBS participated in a prospective randomized therapeutic trial. According to a double-blind design, 40 patients received 400 mg/day mebeverine and 40 patients received a placebo. In an open branch of the trial, 40 patients were treated with 15 g/day wheat bran. The effects of treatment on symptoms were noted after 4, 8, 12 and 16 weeks of therapy. A significantly superior symptomatic effect of bran in comparison to mebeverine and placebo was demonstrated after 12 weeks, but could not be confirmed at the end of the study. There was no significant difference between the symptomatic effect of mebeverine and placebo. The compliance with the therapy was about 80% for 4 weeks, but dropped to about 50% at the end of the trial. This points to a particular difficulty in the management of patients with IBS. The results of this trial suggest that bran and mebeverine are no ideal therapy for patients with IBS but they support the therapeutic use of bran in patients with IBS. PMID- 3019809 TI - Glutathione redox state is not the link between polyol pathway activity and myo inositol-related Na+-K+-ATPase defect in experimental diabetic neuropathy. AB - Decreased glutathione levels in the ocular lens have been invoked as a possible cause for the decreased lenticular Na+-K+-ATPase in diabetes because both are corrected by aldose reductase inhibitors, and the Na+-K+-ATPase is known to be susceptible to oxidation inactivation. Because an analogous Na+-K+-ATPase defect that is prevented by aldose reductase inhibitors has been described in diabetic peripheral nerve, we examined the effect of streptozocin (STZ) diabetes and aldose reductase inhibition on reduced (GSH) and oxidized (GSSG) glutathione levels in crude homogenates of rat sciatic nerve. Neither GSSG nor GSH levels were altered by 2 or 8 wk of untreated diabetes or by aldose reductase inhibition. Because the defect in Na+-K+-ATPase is fully expressed by 4 wk of STZ diabetes, we conclude that altered glutathione redox state plays no detectable role in the pathogenesis of this defect in diabetic peripheral nerve. PMID- 3019812 TI - Marihuana-induced embryotoxicity in the rabbit. AB - Few teratogenic studies in animals have been performed simulating marihuana smoking in man. An inhalation marihuana teratology study was conducted in albino rabbits utilizing a modified automatic smoking machine originally developed for rats and mice. Appropriate numbers of dams were exposed to 4 puffs (0.14 mg/kg), 8 puffs (0.72 mg/kg), or 16 puffs (1.44 mg/kg) once daily during gestation Days 6 to 18, and sacrificed on Day 28. Control dams were exposed to 12 puffs of placebo cigarettes or sham-treated for a similar duration in the absence of any smoke. Consistency of smoke was monitored by cigarette weights, total particulate matter, concentrations of carbon monoxide (CO), and tetrahydrocannibinol (THC) in smoke, carboxyhemoglobin levels, and plasma THC levels. Except for a transient decrease in dam respiration rates, other gross toxic signs were absent. Reproductive parameters of mothers were generally normal except for a dose related embryotoxicity predominantly associated with early resorptions. Despite twice the number of embryo/fetal deaths, there were no marihuana soft tissue or skeletal defects. A correlation between dam demises and CO levels among placebo exposed animals was related to greater quantities of CO being generated during placebo combustion. It has been shown in the rabbit that marihuana is embryotoxic and not a teratogen at plasma THC levels found in human females. PMID- 3019811 TI - Increased platelet thromboxane receptor sensitivity in diabetic patients with proliferative retinopathy. AB - Platelet aggregation to collagen in 12 Type 1 (insulin-dependent) diabetic patients with background retinopathy and 12 Type 1 diabetic patients with proliferative retinopathy was compared with an age- and sex-matched control group. An analogue of prostaglandin H2, 11,9 epoxymethano-prostaglandin H2, which directly stimulates thromboxane receptors, and EP 092, which is a competitive thromboxane A2 receptor antagonist, were used to investigate changes at platelet thromboxane receptor level in these groups. The concentration of collagen (EC50) required to give 50% of maximum aggregation did not differ between the two diabetic groups and the control group. However, platelets from the proliferative retinopathy group were significantly more sensitive to the thromboxane mimetic (11,9 epoxymethano-prostaglandin H2) (p less than 0.005) than the background retinopathy and control groups. This change may be a factor in the development of proliferative retinopathy. PMID- 3019813 TI - Technical grade but not recrystallized alpha-naphthylthiourea potentiates superoxide release by rat neutrophils stimulated in vitro by phorbol myristate acetate. AB - alpha-Naphthylthiourea (ANTU) causes pulmonary edema and pleural effusion in rats. It has been suggested that ANTU pneumotoxicity may be mediated by blood neutrophils (PMNs) via the release of reactive oxygen species. Accordingly, we tested the effect of technical grade ANTU (tANTU) on the ability of rat peritoneal PMNs to release superoxide (O2-). tANTU did not itself stimulate O2- production by PMNs, but it increased the O2- released in response to PMN stimulation by phorbol myristate acetate (PMA). This effect was dependent upon the amount of tANTU added. In PMNs activated in vitro by a submaximal PMA stimulus, addition of 20 micrograms/ml tANTU doubled superoxide release. When tANTU was recrystallized from ethanol, the purified ANTU was not effective in potentiating the effect of PMA on PMNs. This suggests that an impurity in technical grade ANTU is capable of increasing O2- release by stimulated PMNs. tANTU and recrystallized ANTU caused similar pneumotoxicity in rats in vivo, suggesting that the unidentified impurity does not markedly influence the biologic effects of ANTU. PMID- 3019814 TI - [Pulmonary carcinomatosis: a rare cause of acute cor pulmonale]. AB - Five cases of neoplastic pulmonary embolism are reported in whom the clinical presentation was consistent with acute cor pulmonale. Perfusory lung scintigraphy was negative in all the cases. Four patients died within 7 days, one after 30 days from starting of symptoms. At autopsy in all the cases neoplastic diffuse embolization of pulmonary arteries was seen with or without thrombosis. In two cases lymphatic carcinosis was also evident. In the literature the majority of cases are reported to have a subacute clinical course as compared to the acute clinical evolution of our series. We suggest to keep in mind the diagnostic hypothesis of vascular pulmonary carcinosis in the cases of acute cor pulmonale with negative perfusory lung scintigraphy. PMID- 3019815 TI - [When cells communicate: journey through Disse's space]. PMID- 3019816 TI - Autoradiographic localization of mu- and delta-type opioid receptors in the gastrointestinal tract of the rat and guinea pig. AB - The distribution of delta- and mu-type opioid binding sites in the gastrointestinal tract of the rat and guinea pig was studied by autoradiography after in vitro incubation of tissue slices with 3H-D-Ala2,D-Leu5-enkephalin, and 3H-naloxone or 3H-dihydromorphine to locate delta- and mu-type opioid receptors, respectively. In the gastric fundus, both mu- and delta-type binding sites were found to occur associated with the circular muscle, muscularis mucosae, and submucosal plexus, whereas in the corpus and antrum, binding was located primarily in the submucosal plexus, deep muscular plexus, and mucosa. Some mu type opioid receptor sites were present in the myenteric plexus. A dense distribution of both mu- and delta-type binding sites was observed throughout the mucosa of the duodenum and ileum of the rat. In guinea pig ileal tissue, however, only mu-type binding could be demonstrated, occurring in the submucosal plexus and diffusely over the muscle layers. Endogenous opioid peptides, acting at these receptors sites, might be involved in the control of gastrointestinal motility, endocrine and exocrine secretions, as well as intestinal fluid and electrolyte transport. PMID- 3019817 TI - Variations of gastric transmucosal potential difference and lesion formation during hemorrhagic shock in the rat. AB - We measured transmucosal potential difference (PD) of the stomach in anesthetized rats before, during, and after hemorrhagic shock, and investigated the effects of various drugs on the PD and gastric lesion during this period. After hemorrhagic shock, there was a decrease of PD and an increase of luminal pH in the saline perfused stomach, the degree of these changes being dependent on a fall in the arterial blood pressure. The graded reduction of PD in response to hemorrhagic shock was similarly observed in the acid-perfused stomach as in the saline perfused one. However, gastric lesions developed only in the former, and a significant correlation was found between the lesion index and the fall in blood pressure, the reduction in PD, or the concentration of HCl as the perfusate. Subcutaneously administered propantheline bromide (30 mg/kg) or cimetidine (100 mg/kg) had no effect on gastric lesion and PD reduction caused by hemorrhagic shock. These lesions were significantly inhibited by 16,16-dimethyl prostaglandin E2 (10 micrograms/kg) or sulindac (100 mg/kg), a scavenger of OH., and aggravated by indomethacin (1 mg/kg), with less effect on the PD reduction. Intravenous infusion of NaHCO3 (0.5 M) also significantly prevented the lesion with a concomitant suppression of the PD reduction in response to hemorrhagic shock, but these effects were significantly reversed by pretreatment of the animals with acetazolamide (50 mg/kg). These results indicate that during hemorrhagic shock the PD may largely reflect the impairment of mucosal blood flow and may be used as an indicator of mucosal vulnerability to acid, gastric lesions develop only in the presence of exogenous acid, and production of prostaglandins and superoxide radicals may be involved in the pathogenesis of gastric lesions. PMID- 3019818 TI - A new method to assay des-gamma-carboxyprothrombin. Results obtained in 75 cases of hepatocellular carcinoma. AB - A new method for assaying the activity of des-gamma-carboxyprothrombin (DCP), using staphylocoagulase on undiluted adsorbed plasma, is described. The thrombin coagulase formed is measured on a chromogenic substrate, and the results are expressed in milliunits per milliliter of increment of the optical density following the release of p-nitroaniline. Levels of DCP in 96 normal subjects were under 10 mU/ml (mean, 3.58 mU/ml). Of 70 nonhepatectomized patients with hepatocellular carcinomas, 74% had increased DCP levels of between 20 and 420 mU/ml (most of the values were between 20 and 100 mU/ml). Des-carboxyprothrombin and alpha-fetoprotein measurements gave complementary information, one marker or the other being positive in 87% of hepatocellular carcinoma. Fourteen of 15 patients with metastatic carcinoma of the liver had normal DCP levels, as did 95 patients with liver cirrhosis and 13 patients with chronic hepatitis. When the level of "total factor II" is below 40%, it is recommended that a second determination of DCP be performed 5 days after the injection of vitamin K, to exclude any vitamin K deficiency (in the case of hepatocellular carcinoma the DCP level will remain elevated). The DCP assay appears more sensitive and more specific than the alpha-fetoprotein assay for the diagnosis of hepatocellular carcinoma; furthermore, both tests are complementary. PMID- 3019819 TI - Levobunolol for the long-term treatment of glaucoma. AB - Systemically, levobunolol is as effective as propranolol for cardiovascular indications, with a greater potency and greater duration of action. Ocularly, levobunolol is as effective and as safe as topical timolol for the long-term treatment of elevated IOP. The utility of topical levobunolol as an additional, effective beta-blocker for the treatment of glaucoma will be determined by additional research and use by ophthalmologists in countries where levobunolol is approved. PMID- 3019820 TI - The effects of hemicholinium-3 (Hc-3) during tetanic (TP) and posttetanic potentiation (PTP) on sympathetic ganglion. AB - An electrophysiological analysis has been made of the synaptic plasticity in the eighth sympathetic ganglion of the toad. The mean amplitude of Compound Action Potential (CAP) recorded extracellularly was taken as a measure of the synaptic transmission. Repetitive stimulation of the preganglionic axonal fibers at 50 Hz for 40 seconds in the presence of Hc-3 led to facilitatory and depressant actions on ganglionic synaptic transmission. Hc-3 in the presence of elevated extracellular Ca2+ concentration (5.4 mM) increased Tetanic Potentiation (TP) and Posttetanic Potentiation (PTP) on eighth sympathetic ganglion. Hc-3 plus 3,4 diaminopyridine (3,4-DAP) increased synaptic transmission, TP, and PTP from postganglionic CAP responses to supramaximal preganglionic stimulation. PMID- 3019821 TI - Opiate-like and adrenocorticotrophin-like materials in equine pancreas. AB - Equine pancreatic acetone powder was extracted with an acetone-water-HCl mixture. An acid acetone powder resulted by adding a copious volume of acetone to the extract. The powder was subjected to salt fractionation, gel filtration and chromatography on CM-cellulose. Steroidogenic activity, ACTH-like immunoreactivity and opiate receptor binding activity were distributed among the CM-cellulose chromatographic fractions derived from material unretarded as well as from material retarded on Sephadex G-25. The data indicates a separation of steroidogenic and opiate receptor binding activities, and the presence of opiate like molecules with different affinities of binding to mu and delta opiate receptors. PMID- 3019822 TI - Postnatal development of responsiveness to adrenergic agents of longitudinal and circular smooth muscles from cat terminal ileum. AB - The changes in the responsiveness of the isolated longitudinal and circular smooth muscles from cat terminal ileum to adrenergic agents during the postnatal period were studied by the effect of noradrenaline (0.01-100 mumol) on the smooth muscle tone and phasic contractions. Cumulatively applied noradrenaline (0.01-10 mumol) decreased the longitudinal muscle tone without age-dependent differences via inhibitory alpha 1-adrenoceptors. Noradrenaline decreased the phasic contraction amplitude of longitudinal muscle via inhibitory alpha 1 adrenoceptors. Cumulatively applied noradrenaline (0.01-10 mumol) exerted an age dependent effect on the circular muscle tone via stimulatory alpha 1 adrenoceptors. alpha 1-adrenoceptors continue to develop functionally at 60 days postnatal. Noradrenaline decreased the phasic contraction amplitude of the circular muscle and exerted a stimulant effect on the tone which suggested an existence of two alpha 1-adrenoceptor subtypes. PMID- 3019823 TI - Modulation of receptor mediated leukotriene release in the perfused heart. AB - The chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) induces peptide leukotriene release at concentrations of 20-25 pmol/ml 3 min after the start of FMLP infusion. FMLP-induced leukotriene release in rabbit hearts is not blocked by the leukotriene receptor antagonist FPL-55712 at concentrations that totally antagonize the hemodynamic effects of exogenously infused peptide leukotrienes. Moreover, propyl gallate, a lipoxygenase inhibitor, does not block FMLP-induced leukotriene release. However, the chemotactic peptide antagonist (Boc-Phe-Leu-Phe-Leu-Phe-OH) totally antagonized FMLP-induced leukotriene release suggesting that the release is via a different mechanism, possibly a receptor mediated event. PMID- 3019824 TI - Effects of alpha beta methylene ATP on membrane potential, neuromuscular transmission and smooth muscle contraction in the rat tail artery. AB - alpha beta methylene ATP (meATP, 400 nM-1 microM) caused depolarization and constriction of the smooth muscle of the rat tail artery, and block of the excitatory junction potential (e.j.p.). A similar depolarization caused by 25 mM K did not block the e.j.p. Contractions caused by nerve-released noradrenaline acting on alpha-adrenoceptors were potentiated by meATP but this was probably due to the depolarization as 25 mM K had a similar effect. MeATP blocked e.j.p.s recorded in the presence of 3 microM tetrodotoxin, suggesting that meATP was not blocking e.j.p.s by a local anaesthetic action. PMID- 3019825 TI - The role of dopamine in salivation in the rat parotid gland. AB - The ability of dopamine to evoke secretion from the rat parotid gland has been investigated. The actions of dopamine were investigated alone and during carbachol mediated secretion. Dopamine increased flow rate and protein secretion in both experimental models. The increase in protein secretion is mediated via beta 1-adrenoceptors whereas the increase in flow rate may be due to activation of specific dopamine receptor. PMID- 3019826 TI - Enhanced relaxation response of canine coronary artery to isoproterenol and salbutamol after removal of endothelial cells. AB - The importance of the endothelium for isoproterenol- and salbutamol-induced relaxation of canine coronary artery was examined. The relaxation effect of isoproterenol and salbutamol was significantly enhanced (P less than 0.05) upon removal of the coronary artery endothelium. In contrast, the relaxation effect of acetylcholine was completely abolished. These results indicate that canine coronary endothelium modulates beta-adrenoceptor-mediated relaxation, and that the role of the endothelium in agonist-induced relaxation of vascular smooth muscle appears to be heterogeneous. PMID- 3019827 TI - Up-regulation of brain [3H]diazepam binding sites in chronic caffeine treated rats. AB - Brain [3H]diazepam and [3H]L-phenylisopropyladenosine binding sites in caffeine treated (75 mg/kg/day, i.p. 12 days) and caffeine withdrawn (30 days) rats were examined. Treatment with caffeine (75 mg/kg/day) for 12 days increases the Bmax (maximum binding capacity) for [3H]diazepam binding by 30.9% whereas the same treatment increases the Bmax for [3H]L-PIA binding by 165%. The Bmax for [3H]diazepam binding sites returns to slightly below control levels but [3H]L-PIA binding sites remain elevated after 30 days of caffeine withdrawal. These findings suggest that the up-regulation of [3H]diazepam binding sites seen in caffeine treated rats may not be adequately explained by a direct antagonism of caffeine on benzodiazepine receptors. Other modes of interaction therefore must be considered. PMID- 3019828 TI - [Cancer of the colon in childhood. Report of 2 cases]. PMID- 3019829 TI - Cloning and template activity of the origins of replication of phage phi 29 DNA. AB - A 73-bp fragment from the left end of phi 29 DNA and a 269-bp fragment from the right end have been cloned in plasmids pPLc28 and pKK223-3, respectively, after removal of the terminal protein p3 by treatment with piperidine. In addition, the 73- and 269-bp fragments were cloned together in plasmid pKK223-3 in such a way that the two termini of phi 29 DNA were joined. Treatment of the latter recombinant plasmid with AhaIII releases several fragments, two of which contain the phi 29 DNA terminal sequences at the DNA end. These two fragments initiated replication specifically at the ends of the DNA giving rise to the formation of the p3-dAMP complex. The activity was about 15% of that obtained with phi 29 DNA protein p3. All remaining recombinant plasmids were essentially inactive when tested as templates either in circular form or after cutting in such a way that placed the origin of phi 29 DNA replication close but not at the DNA end. PMID- 3019830 TI - Initiation of phage phi 29 DNA replication by mutants with deletions at the carboxyl end of the terminal protein. AB - Series of deletions at the C end of p3, the phage phi 29 DNA terminal protein (TP), have been constructed and characterized. Measurements of the activity of those deletion mutants in the formation of the p3-dAMP initiation complex in vitro indicate the need of an intact C-end for the normal TP primer function in DNA replication. It appears that the region at the C-end between aa 240 and 262 of p3, or part of it, might be essential for the normal TP function. PMID- 3019831 TI - Cloning of Micrococcus luteus 3-methyladenine-DNA glycosylase genes in Escherichia coli. AB - The 3-methyladenine-DNA glycosylase (m3ADG) excises 3-methyladenine (m3A) residues formed in DNA after treatment with alkylating agents. In Escherichia coli, the repair of this type of damage depends on the products of the genes tagA and/or alkA, which code for m3ADG I (20 kDa) and II (30 kDa), respectively. The tagA- and alkA--single mutants are sensitive to alkylating agents, the double mutant much more so. We have cloned two genes of Micrococcus luteus that can partly substitute the function of the E. coli tagA- and alkA- genes. An M. luteus genome bank was made by shotgun cloning of EcoRI + BamHI-digested DNA into pBR322. Two hybrid plasmids were identified that confer methylmethane sulfonate (MMS) resistance to the tagA- ada+ mutant and a capacity to reactivate MMS treated bacteriophage lambda. Each hybrid plasmid directed the synthesis of 21 kDa m3ADG in E. coli tagA- ada-, which were not inhibited by 4 mM m3A. However, the restriction maps of the two cloned genes were different, and they showed no sequence homology as judged by the lack of cross hybridization. PMID- 3019832 TI - Molecular cloning of sequences encoding the human heat-shock proteins and their expression during hyperthermia. AB - Plasmids containing cDNA copies of mRNAs induced in HeLa cells by heat shock have been isolated and characterized. In vitro translation of RNAs selected by hybridization to plasmid DNAs identified sequences representing the three major classes (89, 70 and 27-kDa) of heat-shock proteins (hsp) and a 60-kDa minor hsp. Plasmids with inserts specific for the 27, 60, and 70-kDa hsp each hybridize with a single discrete size class of heat-inducible mRNA. Plasmids specific for the 89 kDa protein, however, hybridize with either a 2.7- or 2.95-kb mRNA species. Both mRNAs are coordinately induced during heat shock. We show that the characteristic pattern of induction and repression of each class of hsp during sustained hyperthermia is the result of changes in the steady state level of each mRNA. PMID- 3019833 TI - Nucleotide sequence and transcription of a human glycine tRNAGCC gene and nearby pseudogene. AB - A bacteriophage lambda clone containing a 15.4-kb human DNA fragment was isolated and found to contain a glycine tRNA gene and, 758 bp away, a pseudogene, both with an anticodon of GCC. The nucleotide (nt) sequence of a 1362-bp segment of this clone, encompassing the gene, pseudogene, and their flanking regions, was determined. The gene and pseudogene have an identical sequence of eight nt (5' CAGCTGGA-3') in their 5'-flanking regions immediately preceding the coding regions, as well as characteristic transcription termination sites of five consecutive T nt in the 3'-flanking regions. Neither of these genes has intervening sequences. Only one of the two genes was efficiently transcribed in vitro by RNA polymerase III in a HeLa cell-free system. During the course of transcription, primary transcripts of one gene were processed to yield mature sized products. In contrast, the level of transcription of the second gene was significantly less than that of the first, and no mature-sized products could be detected. The nt sequence of the inefficiently transcribed gene has two base substitutions compared to the sequence of the efficiently transcribed gene, and the DNA sequence predicted from the human placental tRNAGlyGCC sequence. One of these nt substitutions is a C to T transition in the TTCG sequence within the B block of the characteristic internal split promoter sequence. The precursor product relationships of the tRNA transcripts were established by comparing the RNase T1 and RNase A fingerprints of the precursors and products. PMID- 3019834 TI - Nucleotide sequence of the split tRNAleu(UAA) gene from Sorghum bicolor chloroplasts. AB - The nucleotide (nt) sequence of the split tRNAleu(UAA) gene and 328 nt of its flanking regions from sorghum chloroplasts (cp) has been determined. This gene is located in the BamHI-6 fragment in a map position very similar to that of maize. The exon of sorghum tRNAleu gene has an identical nt sequence to its counterpart in maize. Although the 450 nt of intron in sorghum is 8 nt shorter than that of maize, the nt sequence between them shows 97% homology. Like maize and broad bean, the intron from sorghum cp tRNAleu gene could be folded into a secondary structure which is similar to the postulated structure of the intron from the auto-spliceable rRNA precursor of Tetrahymena. Both introns from sorghum and maize contain open reading frames (ORFs) which are conserved at the N terminus. The putative AUG initiation codon for both ORFs is located in the stem region of a 12-bp secondary structure of highly A + T-rich sequences. PMID- 3019835 TI - Three classes of homologous Bacillus thuringiensis crystal-protein genes. AB - Four homologous genes encoding insecticidal crystal proteins from Bacillus thuringiensis have been cloned in Escherichia coli. Differences in lengths of HindIII restriction fragments containing the 5' ends of the genes allowed the identification of three classes termed the '4.5- and 5.3- and 6.6-kb-class genes'. A survey of 24 strains from subspecies kurstaki and thuringiensis revealed strains containing one, two, or three of these classes of crystal protein genes. The 4.5- and 6.6-kb-class genes encode polypeptides of Mr 133,500 and 133,330, respectively, while the two 5.3-kb-class genes encode a ca. 130-kDa polypeptide. The polypeptide composition of crystals from the B. thuringiensis strains agreed with the composition predicted from the content of the three classes of genes. The representative genes from each class were isolated from B. thuringiensis plasmids of different sizes. An analysis of plasmid DNA flanking three of these genes revealed a complex pattern of two different inverted repeat (IR) sequences, IR2150 and IR1750. Four variant forms of IR1750 were found. The IR elements were located near deletions and rearrangements adjacent to crystal protein genes and may account for the diversity of plasmids carrying crystal protein genes in other subspecies of B. thuringiensis. PMID- 3019836 TI - High-level expression of the Epstein-Barr virus EBNA1 protein in CV1 cells and human lymphoid cells using a SV40 late replacement vector. AB - To construct a recombinant plasmid designed to yield large amounts of the Epstein Barr virus (EBV) nuclear antigen, EBNA1, the EBV BamHI-K fragment (B95-8 strain) was inserted into an expression vector composed of SV40 and pBR322 DNA. The vector replicates in both Escherichia coli and eukaryotic cells. Introduction of such a BamHI-K-containing vector into CV1 monkey cells (using DEAE-dextran, glycerol and chloroquine diphosphate) gave high yields of the correct size EBNA1 protein in 40-50% of the transfected cells. Maximal amounts of EBNA1 could be extracted from the cells at 65-72 h post transfection. Using a quantitative ELISA assay, it was estimated that transfected cells express 500-1000 times more EBNA1 than lymphoid cells, latently infected with EBV. A monoclonal antibody directed against EBNA1 immunoprecipitated two proteins of 74 and 62 kDa from transfected cells. These same two proteins were detected in immunoprecipitation and immunoblot experiments using human EBV-positive polyclonal serum, although this serum also detected several other protein products in transfected cells. In vivo labelling of transfected cells with [32P]orthophosphate showed that the 74- and 62-kDa proteins are modified by phosphorylation. The same vector construction was also used to transfect an EBV-negative human lymphoblastoid cell line (Ramos). Expression of the EBNA1 protein was obtained in up to 20% of the cells. PMID- 3019837 TI - Sequence analysis of the pyr-4 (orotidine 5'-P decarboxylase) gene of Neurospora crassa. AB - The pyr-4 gene of Neurospora crassa encodes orotidine-5' -phosphate decarboxylase, which catalyses the sixth step in the pyrimidine biosynthetic pathway. The complete nucleotide sequence of a 1.8-kb genomic fragment containing the pyr-4 gene has been determined. Using transposon mutagenesis, the coding region has been identified, and the amino acid (aa) sequence deduced. Comparison of the pyr-4 aa sequence with URA3, the equivalent gene of Saccharomyces cerevisiae, showed extensive blocks of homology, with non-homologous sequences between these blocks being generally much longer in Neurospora than in yeast. Computer-predicted protein secondary structure of pyr-4 and URA3 was conserved within equivalent blocks. Upstream sequences of pyr-4 were compared with other sequenced Neurospora genes and possible promoter sequences identified. PMID- 3019838 TI - The sequence and mom-transactivation function of the C gene of bacteriophage Mu. AB - The mom gene of bacteriophage Mu encodes a DNA modification function. The gene is regulated on the transcriptional level by Dam-specific methylation and a trans acting Mu function, and on a post-transcriptional level by the product of gene com. The gene encoding the transactivator has been cloned and mapped. By complementation analysis the activation function (also designated Dad) was shown to be the product of gene C. Transactivation of the mom promoter was shown in the following assay: the mom promoter and N-terminal part of com were fused in frame to lacZ. Cells containing such fusion plasmids were infected with M13 clones expressing C in the presence of IPTG and XGal. Successful transactivation results in the formation of blue plaques. Moreover, we have determined the sequence of gene C and found that it has a coding capacity of 140 amino acids. The promoter for C (pc) is likely to be located at least 0.5 kb upstream from the gene. A transcription terminator is found directly downstream from the C-coding region. PMID- 3019839 TI - The fos gene product undergoes extensive post-translational modification in eukaryotic but not in prokaryotic cells. AB - To investigate the properties of the fos oncogene, we have constructed bacterial and yeast vectors which express the entire fos-coded protein (Fos) and two C terminal deletion products. In Escherichia coli, Fos proteins were expressed from the phage lambda pL promotor under the control of the temperature-sensitive lambda repressor. In vitro transcription/translation studies indicate that these vectors produce Fos proteins of the expected sizes. However, in vivo, Fos protein accumulation is observed only in hosts with the Lon- phenotype. In Saccharomyces cerevisiae, the fos gene was expressed from the PHO5 promoter which is induced under low-phosphate conditions. In contrast to the situation in E. coli, in which the heterologous proteins appeared as single major products when subjected to sodium dodecyl sulfate - polyacrylamide gel electrophoresis, the Fos proteins in S. cerevisiae displayed extensive Mr heterogeneity. Pulse-chase analyses indicated that this heterogeneity was a consequence of extensive post translational modification. These modifications occurred to an equivalent extent on the products coded by the fos gene with C-terminal deletions and thus appear not to be controlled by the missing domain. PMID- 3019840 TI - Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. AB - A single-copy integration vector was used for the in vitro construction of translational fusions to the lacZ gene of Escherichia coli. Insertion of a single copy of the lacZ fusion into the B. subtilis chromosome leads to an easily detected Amy- phenotype. A trpE-lacZ fusion was constructed in which the trp promoter directs hybrid beta-galactosidase (beta Gal) synthesis. The level of beta Gal in a wild-type strain carrying the trpE-lacZ fusion in the chromosome is regulated by exogenous tryptophan, while a 5-methyltryptophan-resistant mutant constitutively synthesizes betaGal. A trpF-lacZ fusion was constructed and used to determine the effect of a frameshift mutation in the trpE gene on expression of the trpF-lacZ fusion. The frameshift mutation in trpE led to a three-fold reduction in the levels of the trpF-lacZ fusion. The levels of the betaGal activity of these integrated lacZ fusions appear to provide a quantitative measure of the expression of B. subtilis genes under single-copy conditions. PMID- 3019841 TI - The nodI gene product of Rhizobium leguminosarum is closely related to ATP binding bacterial transport proteins; nucleotide sequence analysis of the nodI and nodJ genes. AB - The nucleotide sequence of a 2-kb fragment immediately downstream of the nodABC genes of the Rhizobium leguminosarum symbiotic plasmid pRL1JI has been determined. Genes corresponding to the two open reading frames identified are named nodI and nodJ. Tn 5 insertions into these genes result in a "nodulation delayed" phenotype. The predicted amino acid sequence of the nodI gene shows considerable homology to inner-membrane-located gene products involved in active transport systems in Escherichia coli and Salmonella typhimurium. The predicted product of the nodJ gene is very hydrophobic, suggesting that it may be an integral membrane protein. PMID- 3019842 TI - [Catechol estrogens]. PMID- 3019844 TI - Postoperative extended-field irradiation in patients with pelvic and/or common iliac node metastases from cervical carcinoma stages IB to IIB. AB - Radical hysterectomy with pelvic and common iliac lymphadenectomy was done for 207 Stage IB (148), IIA (19), and IIB (40) cervical carcinomas. Pelvic nodal involvement was limited in 30 (14.5%) cases, whereas common iliac nodes were involved in 16 (7.7%) cases. Common iliac node metastases were significantly increased, when the number of positive pelvic nodes increased from 2 to 3 or 4 or more (21.4% to 73.3%, P less than 0.05), when the tumor invaded deeper than 20 mm (3.7% to 22.2%, P less than 0.001), and when the tumor extended into parametrial tissues (4.8% to 14.8%, P less than 0.05). Postoperative extended-field irradiation was administered to 40 patients with nodal metastases. The 3-year disease-free rates were 85% in 24 patients with positive pelvic nodes, and 51% in 16 patients with common iliac node metastases; 70% in total. These results indicate that postoperative extended-field irradiation is essential for those patients with nodal metastases from locally resectable cervical carcinomas. PMID- 3019843 TI - Pharmacokinetics of mitomycin-C in plasma and tumor tissue of cervical cancer patients and in selected tissues of female rats. AB - Mitomycin-C (MMC) is an alkylating agent which has shown significant activity in gynecologic cancers, both in vivo and in vitro. We determined the delivery of MMC to target tissue by comparing plasma and tumor tissue concentrations of MMC as measured by high-performance liquid chromatography (HPLC) in five patients with cervical cancer. In a companion study, we measured MMC concentrations in plasma and selected tumors of female rats given an equivalent dose. In patients, the mean terminal half-life and total body clearance rates of MMC were 40 min and 275 ml/min/m2, respectively. The mean cervical tumor to plasma concentration of MMC was 1.26 +/- 0.34 (mean +/- SE, n = 4). In female rats, the terminal half-life and total body clearance rates of MMC were 28.4 min and 270 ml/min/m2, respectively. Tissue concentrations of MMC in rats were lower than plasma concentrations measured at corresponding times. The highest concentrations were found in lung and uterus (including cervix) with lower concentrations in ovary and liver. The mean half-life for elimination of MMC from tissues of rats was 20.3 +/- 2.8 min (mean +/- SE, n = 6). Based on similar pharmacokinetic parameters in rats and patients, the rat appears to be a suitable model for the disposition of MMC in human patients. The near equivalent drug concentrations found in tumor and plasma of patients suggests that the in vitro tests conducted at concentrations based on plasma level may be relevant to cervical tumor tissue, as well. PMID- 3019846 TI - Multiple myeloma following reactivated Epstein-Barr virus infection in a renal transplant recipient. PMID- 3019845 TI - Peripheral neuropathy and ototoxicity of dichlorodiamineplatinum: instrumental evaluation. Preliminary results. AB - Clinical use of cis-platinum in the treatment of many human tumors is increasing. Since side effects could represent a limitation of its use, we evaluated neurotoxic effects of this compound in a group of 23 patients on antiblastic treatment for gynecological neoplasms. Evaluation was performed by clinical neurological examination, neurophysiological data (electromyography, maximal motor conduction velocity, and sensory conduction velocity) and potentials. Seven patients reported subjective symptoms of neurological involvement. In 2 cases there was a change in the electromyographic pattern. The otolaryngological examination showed a change in the hearing threshold in 3 patients and in 1 case the fatigue test was positive. Neurotoxic effects and hearing damage was not related to the treatment schedule. PMID- 3019847 TI - Improved procedure for cytochemical demonstration of myeloperoxidase (MPO) activity on blood and bone marrow smears: preliminary study on blood neutrophils. PMID- 3019848 TI - Basis for selection of anticoagulant drugs for therapeutic trials in human malignancy. AB - Evidence indicates that progression of the Lewis lung carcinoma in mice and small cell carcinoma of the lung in humans is retarded by warfarin administration. This suggests that vitamin K-dependent pathways are of importance in the pathogenesis of these tumors. Available data were reviewed for these tumor types in an attempt to explore mechanisms and to gain insights that might guide the selection of other coagulation-reactive drugs for testing in future controlled clinical trials in small cell carcinoma of the lung. While many differences exist between the Lewis lung tumor and small cell carcinoma of the lung, both are rapidly growing malignancies of pulmonary origin that metastasize early to kill the host after a short time. Both are favorably influenced by combination chemotherapy and radiation therapy as well as anticoagulant treatment. Peripheral blood changes indicative of disseminated intravascular coagulation occur in each of these tumor types, and tumor cells from both are capable of interacting with the coagulation mechanism. While many details concerning the host-tumor interaction remain to be elucidated, the considerable and diverse information available for these tumor types provides a secure base for future investigation. It is postulated that certain drugs in addition to warfarin might reasonably be studied in controlled clinical trials of small cell carcinoma of the lung and that drugs other than warfarin might be effective for tumor types that are not responsive to this agent. PMID- 3019849 TI - [Eaton Lambert syndrome--an autoimmune disease]. PMID- 3019850 TI - [Differential diagnosis, pathogenesis and therapy of alcoholic polyneuropathy]. AB - The most common forms of polyneuropathies are the alcoholic and diabetic polyneuropathies. They each constitute 1/3 of all polyneuropathies. The first symptoms shown by the alcoholic polyneuropathy are symmetric sensory disturbances with loss of tendon reflexes and of vibration sense in the peripheral segments of the lower extremities. At the beginning one almost always finds pressure pain in the calves. Important differential clues in diagnosis compared to the diabetic neuropathy, are the age at which the disease begins, the degree to which the autonomic nerve fibres and the cranial nerves are affected, as well as the form of manifestation. Pathogenetically, a direct toxic alcohol effect can above all be suspected in accordance with the typical electrodiagnostic findings with a neurogenic pattern in the EMG in the case of normal or slightly diminished conduction velocity, and in agreement with the morphological finding of an axonal degeneration in most of the biopsies. Possibly, in a small number of cases a vitamin deficiency or a malabsorption can play a causal role. The prognosis is good by complete abstinence from alcohol. PMID- 3019851 TI - [Motor grammar of the spinal cord--status and revision]. AB - Modern neurophysiological research, mainly in the second third of this century, has led to a set of widely accepted findings on the basic processes and rules of interactions between bodily periphery and the spinal level, which may be called a kind of elementary "grammar" of the spinal cord. As far as these apply to motor control functions, they are expressed here in condensed form as ten "paragraphs", which until recently belonged to the conventional standard teaching of neurophysiology. Each paragraph is then critically reviewed, supplemented and revised, as far as necessary, based on research progress in the last thirty years. The review deals with: Alpha motoneurones and their motor units; the muscle spindle with its sensory and motor structures, and its efferent controls by gamma and beta neurones; the spinal connections and effects of the afferents from primary and secondary spindle endings; properties and spinal effects of the Golgi tendon organs; new aspects of the flexor reflex afferents (FRA), of recurrent Renshaw feedback, and of presynaptic inhibition. The last paragraph stresses the importance of supraspinal controls of the spinal moto- and interneurones. PMID- 3019852 TI - Gastrointestinal carcinoma--are there age-related differences in tumor behavior? AB - Among the gastric cancers, the percentage of diffuse carcinomas decreases with increasing age, while the incidence of the intestinal type increases markedly (up to 50 years 29.6%, older than 70 years 61.5%). Accordingly, in patients older than 70, gastric carcinomas limited to the antrum are seen significantly more frequently (32.5%) than in younger patients (21.1%). In patients aged 70 years or less, the rectum is more frequently affected than the colon (ratio 1.41:1), than is the case in older patients (1.17:1). With respect to stage distribution and the pTNM classification, colorectal carcinomas and gastric carcinomas show only small age-related differences. The prognosis of both carcinomas is independent of age when the survival rates are corrected for age, post-operative mortality is excluded, and only patients with identical stages are compared. In patients older than 70, cancer is not less aggressive, but, as a result of the limitations of therapeutic possibilities, and greater side effects of treatment, is more dangerous than in younger patients. PMID- 3019853 TI - [Mass survey of hepatocellular cancer]. PMID- 3019854 TI - [Cytogenetic study of endometrial carcinoma]. AB - Cytogenetic studies were performed on endometrial specimens of four patients with hyperplasia. Six with adenocarcinoma and one with mixed mesodermal tumor. All 65 cells obtained from hyperplasia specimens excluding one cell, had a normal female karyotype. However, cells from five adenocarcinoma specimens had chromosomal abnormalities, though a specimen of a well differentiated adenocarcinoma showed a normal karyotype. A few numerical abnormalities which were clonal in origin, were noted in one case each. Three kinds of structural abnormalities involving chromosomes 1 were identified as clonal in origin; del (1) (p21), t. dic(1; 16) (p21; q24), and i (lq). Since carcinoma cells had two chromosomes 1 of normal morphology, the presence of the marker chromosome led to the partial trisomy or tetrasomy of the long arm of a chromosome 1, being characteristic of cells from adenocarcinoma of the endometrium. A successively transplantable tumor has been produced from a poorly differentiated carcinoma cells with the t. dic (1; 16) (p21; q24) marker chromosome. Histological and chromosomal examinations were performed in cells from the passage 1, 4 and 5 tumors in order to explore the role of this marker played in the formation of the endometrial carcinoma. Though the degree of differentiation status of tumor cells had been changed during transplantations, the t. dic (1; 16) (p21; q24) marker were observed consistently among these cells. This suggested that the rearrangement of chromosome 1 was not produced as a result of genetic instability due to tumor progression, but rather specifically associated with the endometrial carcinogenesis. None of karyotypic changes excluding the marker and the tetraploid was responsible for these phenotypic changes. PMID- 3019855 TI - [Studies on Epstein-Barr virus (EBV) infection and reactivity of peripheral B lymphocytes to EBV in patients with ataxia telangiectasia]. AB - Ataxia telangiectasia (AT) is a primary immunodeficiency syndrome characterized by oculocutaneous telangiectasia, ataxia, recurrent infection and development of malignancies. Epstein-Barr virus (EBV) is a B-cell lymphocytotropic virus which causes infectious mononucleosis and is also highly associated with Burkitt's lymphoma, nasopharyngeal carcinoma and lymphoproliferative disorders in immunodeficient patients. 10 Japanese patients with AT were studied concerning the status of EBV infection by specific EBV serology, and reactivity of peripheral lymphocytes to EBV. All the AT patients had high EBV antibody titers of IgG to viral capsid antigen (VCA) and early antigen (EA), while low titers of IgG to EBV-associated nuclear antigen (EBNA), compared with age and sex matched healthy controls. However, significant differences were not apparent with antibodies to several other viruses between the AT patients and controls. These antibody characteristics were thought to be that an activated EBV infection occurred in AT patients. Then the lymphocytes were exposed to B95-8 strain EBV. There was no significant differences in EBNA induction frequency at 24 hours prior to cellular DNA synthesis, between the AT and controls. EBV-specific T cell killer function was very low as judged with the days of establishment of lymphoblastoid cells expressing EBNA on all cells after EBV exposure, when compared with the lymphocytes from controls. These AT lymphoblastoid cells easily expressed EA and VCA by cultivation at lower temperature of 33 degrees C, 12-0 tetradecanoyl-phorbol-13-acetate treatment, 60Co irradiation and by P3HR-1 strain EBV infection. Malignant transformation with high colony forming efficiency in soft agarose and tumor formation in nude mice easily occurred with some of AT lymphoblastoid cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019856 TI - Internalization and degradation of glucagon by human mononuclear cells. AB - The processing of glucagon by circulating human mononuclear cells was examined. Glucagon bound to the membrane with a turnover time of 4.4 minutes per site after 15 minutes of incubation and 8 minutes per site after 90 minutes. The amount of intact intracellular hormone increased by 3-fold by 90 minutes suggesting a slowing of intracellular processing with prolonged incubation. Excess unlabelled insulin also slowed the processing of glucagon at the degradative step with no effect on binding or internalization of glucagon. Subcellular fractionation of the cells showed that most hormone accumulated in the 500xg pellet and in the 100,000xg supernatant. N-ethylmaleimide blocked intracellular glucagon degradation suggesting a role for intracellular sulfhydryl-dependent enzymes. Kinetic analysis of the dissociation of glucagon revealed a second order process with K values of 2.2 X 10(-2) fm-1 min-1 and 1.4 X 10(-2) fm-1 min-1 for dissociation from membranes and from membranes + intracellular sites, respectively. T 1/2 values were 6 min. for membrane dissociation and 9 min for membranes + cells. These findings suggest that glucagon interaction with mononuclear cells has characteristics similar to other receptor bound ligands including internalization processing and metabolism. PMID- 3019858 TI - Adrenal and testicular function in 14 patients with adrenoleukodystrophy or adrenomyeloneuropathy. AB - Testicular and adrenal function were evaluated in 12 patients with adrenoleukodystrophy and 2 patients with adrenomyeloneuropathy. Although only 5 subjects had clinical symptoms suggesting adrenal insufficiency, an additional 5 showed laboratory evidence of reduced adrenal reserve. 9 of the 14 patients developed neurological deficits prior to the onset of clinical or biological adrenal insufficiency. In the remaining 5 patients, adrenal insufficiency antedated the appearance of neurological symptoms; 2 of these 5 patients had only laboratory evidence of hypoadrenocorticism, and 3 had both clinical and laboratory abnormalities. None of the prepubertal patients had detectable signs of testicular insufficiency, while 3 of the 7 pubertal/adult patients had elevated serum LH or FSH levels. This mild testicular deficiency was seen only in association with clinical adrenal insufficiency and significant neurological impairment. PMID- 3019857 TI - Response to low-dose pulsatile cortisol in Addison's disease with suspected corticotropinoma. AB - A 65 year old woman with long-standing Addison's disease treated with oral glucocorticoid and mineralocorticoid replacement had persistently high ACTH levels, inadequate suppression of ACTH on low-dose dexamethasone, sellar enlargement, and pigmentation, and thus resembled patients alleged to develop corticotropinomas while on oral replacement for adrenal insufficiency. Since animal studies suggested that rapid rises of corticosteroids within the physiologic range can inhibit ACTH release, we administered brief infusions of cortisol every three hours with total daily dose equal to her chronic dose. Prompt suppression of ACTH and immunoreactive beta-endorphin occurred during each cortisol dose profiled, suggesting a role for ultradian cortisol fluctuations in tonic inhibition of ACTH secretion in humans, and a possible therapeutic benefit of mimicking ultradian cortisol rhythms during replacement therapy. PMID- 3019859 TI - Dissociation and enrichment of rat atrial myocytes containing atrial natriuretic factor (ANF). AB - Methodologies developed for the dissociation and subsequent enrichment of muscle and nonmuscle cells from atrial myocardium were used to evaluate the contribution of these cell populations to the natriuretic, diuretic and vasoactive properties of crude atrial tissue extracts. Suspensions of single cells, which contained approximately 34% myocytes, were prepared from atrial tissue blocks with a collagenase-trypsin digestion followed by gentle mechanical disruption. Differential centrifugation and unit gravity sedimentation techniques were employed to enrich the 'muscle' and 'nonmuscle' cell suspensions to a purity of approximately 91 and 95%, respectively. Cell extracts were bioassayed for natriuretic activity in saline-expanded, pentobarbital-anesthetized, female rats. Extracts obtained from 'initial' and 'muscle' cell suspensions significantly enhanced sodium and chloride excretion as well as urine flow while extracts from 'nonmuscle' cell suspensions had no effect on renal function. Sodium excretion was dose-dependent and increased linearly with increasing numbers of extracted and infused myocytes. This simple two-step centrifugation and sedimentation protocol can be utilized to obtain enriched atrial myocyte populations for subsequent physiologic and biochemical studies. PMID- 3019860 TI - Alcohol and cancer. PMID- 3019861 TI - The unfolding GABA story. PMID- 3019862 TI - The isolation of functionally heterogeneous hepatocytes of the proximal and distal half of the liver acinus in the rat. AB - The objective of this study was to isolate hepatocytes of the proximal half (Zones 1 and 2) or distal half (Zones 2 and 3) of the liver acinus. The zonal origin of the isolated hepatocytes was recognized by: the presence in hepatocytes of a fluorescent marker, acridine orange, selectively delivered to either the proximal or the distal half of the acinus by in situ perfusion prior to cell isolation and the measurement of the induction of cytochrome P-450 by phenobarbital, an induction known to occur predominantly in the distal half of the acinus. Following the selective labeling of the acinus with acridine orange, livers were perfused with collagenase in either the portal to hepatic vein direction (anterograde) or in the retrograde direction. Hepatocytes isolated by either an anterograde or a retrograde perfusion were separated by centrifugation in a Percoll density gradient. This procedure isolated populations of proximal or distal hepatocytes, respectively, which were intact and 90% fluorescent. In an effort of assessing the heterogeneity of the separated proximal and distal hepatocytes, each population was further fractionated by centrifugal elutriation. This resulted in the arbitrary separation of proximal or distal hepatocytes into five fractions. Total cytochrome P-450 was determined spectrophotometrically in each of the fractions isolated from controls and after 3 days of the in vivo administration of phenobarbital. On the basis of the pattern of fluorescence in isolated hepatocytes and on the cytochrome P-450 inductive response to phenobarbital administration, it is proposed that: the anterograde or retrograde perfusion of the liver with collagenase separated hepatocytes predominantly of the proximal or distal half of the liver acinus, respectively and that hepatocytes of the distal half of the liver acinus responded to phenobarbital administration with the highest level of cytochrome P-450 induction, indicating that the isolated hepatocytes conserved the functional heterogeneity observed in vivo. PMID- 3019863 TI - Cellular oncogenes and the pathogenesis of human cancer. PMID- 3019864 TI - The erB-related growth factor receptors. PMID- 3019865 TI - Histologic features associated with long-term survival in breast cancer. AB - Three basic cytologic variants were identified among cases of infiltrating ductal carcinoma of the breast (NOS). These variants were found to have different distributions among patients surviving 25 years after mastectomy, compared with matched short-lived control subjects. Multivariate analyses indicated that recognition of these cytologic variants enhanced the prognostic value of tumor grade and blood vessel invasion. Patients with tumors designated here as histologic type C, which others have called invasive cribriform carcinoma, appear to have a higher probability of long-term survival than patients with other histologic subtypes. Since they account for approximately 20 per cent of 25-year survivors, they should be distinguished from patients with other types of infiltrating ductal carcinoma (NOS). PMID- 3019866 TI - Glomerulonephritis in congenital cytomegalic inclusion disease. AB - Except for renal transplant recipients, glomerulonephritis has only very rarely been associated with renal cytomegalovirus (CMV) infection. The kidneys of five infants with congenital cytomegalic inclusion disease, including renal infection, were examined at autopsy. Two of the infants had glomerulonephritis. The younger, a 4-month-old female, had diffuse proliferative and necrotizing glomerulonephritis; virus was present in nuclei and cytoplasm of glomerular endothelial cells and, possibly, in leukocytes as well. There were no electron dense deposits. The other infant, a 5-month-old male, had diffuse mesangial and focal segmental proliferative and sclerosing glomerulonephritis; electron-dense mesangial deposits were seen ultrastructurally. Three additional infants (a newborn male, a 2-day-old male, a 6-week-old female), all with CMV in tubules and one with a single glomerular inclusion, had only rare glomerular abnormalities, i.e., mesangial proliferation in less than 10 per cent of glomeruli (one infant) and segmental sclerosis in less than 1 per cent of glomeruli (all three infants). Thus, congenital renal CMV infection was associated with proliferative glomerulonephritis in the two infants who survived the longest. The three with shorter survival times had only minor glomerular alterations. PMID- 3019867 TI - Aniridia and Wilms' tumor in a child constitutionally mosaic for 11p-;12q+: a new chromosomal change also present in Wilms' tumor cells of the blastema type. AB - A child with congenital aniridia was assessed closely, by repeated abdominal ultrasound examinations, beginning at birth. The Wilms' tumor subsequently discovered and removed was analyzed karyotypically and found to have some cells with a terminal deletion of chromosome 11; in other cells this deletion was associated with a duplication in the long arm of chromosome 12. These findings were identical to those observed in the patient's peripheral blood mononuclear cells. This case further substantiates the association between changes in chromosome 11 and Wilms' tumor and demonstrates how chromosomal abnormalities in early infancy may lead to the development of Wilms' tumor. PMID- 3019868 TI - Liposarcoma of the breast: a clinicopathologic study of 20 cases. AB - The clinical and pathologic features of 13 patients with pure liposarcomas of the breast and seven patients with liposarcomas that had arisen in cystosarcomas are reviewed. Metastases occurred in three of the 13 patients with pure mammary liposarcomas, and two women died of tumor. One of the patients with a liposarcoma that had arisen in a cystosarcoma had a local recurrence one year after diagnosis but was subsequently lost to follow-up study. Fourteen of the 20 patients for whom follow-up information was available for as long as 14 years had no evidence of recurrence. Features associated with the development of recurrence, based on the cases studied here and previous reports, included the pleomorphic liposarcoma pattern and an infiltrative margin. Features associated with tumor-free survival included the well-differentiated liposarcoma pattern, male gender (two patients), and a circumscribed microscopic tumor margin. There were no axillary lymph node metastases. Follow-up data indicate that complete surgical excision of tumor with tumor-free margins is necessary, but total mastectomy and removal of the axillary tail are not required unless these procedures are needed for complete excision. PMID- 3019870 TI - Different inducibility and possible significance of several concomitant "fragile sites" in two brothers. AB - Concomitance of four fragile sites (at 16p13, 16q22, 16q23, Yq12) in the lymphocyte cultures of two brothers is reported. The expression of each of these fragile sites was enhanced (or induced) by different culture conditions. Some of the inducing conditions are already known and others are reported here for the first time. The meaning of the fragile sites is discussed and a possible viral etiology suggested. Concomitance of some of them may be a potential causal factor for deletions, translocations, or inversions. PMID- 3019871 TI - A partnership between the community, state and federal government: rhetoric or reality. PMID- 3019869 TI - Repeated DNA sequences in the distal long arm of the human X chromosome. AB - Two DNA probes from within a single large insert from a recombinant phage-DNA library that was constructed from flow-sorted chromosomes enriched for the human X chromosome were shown to hybridize with repeated X-specific and autosomal DNA sequences. The X-chromosomal repeated sequences were assigned to the distal long arm of the X chromosome by both hybrid mapping and in situ hybridization. Fine mapping places these repeats in a region of Xq28 between DX13 (DXS15, in distal Xq28) and factor VIII (F8C, in proximal Xq28). The location of the X-specific repeats makes them potentially useful for future investigations of diseases mapping to the distal long arm of the X chromosome, such as the fragile X syndrome. PMID- 3019872 TI - Double heterozygote leftward/rightward deletion type alpha-thalassaemia in Saudi Arabs. AB - Restriction endonucleases have been used for the investigation of deletion type alpha-thalassaemias in the Saudi population. Using Bgl II digestion, we diagnosed 2 cases with 15.8- and 7.0-kb alpha-globin gene fragments. The 15.8-kb Bgl II fragment is obtained in cases with rightward deletion, while the 7.0-kb fragment occurs in cases with leftward deletion. The 2 cases reported in this paper are heterozygous leftward/rightward deletion cases. The Bam HI digestion results and haematological parameter values are reported. This is the first report of heterozygous leftward/rightward deletion in the Saudi population. PMID- 3019873 TI - Phosphoglycolate phosphatase polymorphism: gene frequencies in three Italian samples. AB - The electrophoretic polymorphism of the PGP locus has been studied in about 1,700 Italians. The sample consisted of individuals from Viareggio (North-Central Italy), Rome (Central Italy) and Cagliari (Sardinia, Southern Italy). Comparison among the three groups showed a high degree of heterogeneity. The Sardinian sample was well differentiated from the other two concerning the frequencies of both the PGP3 and of PGP2 alleles. The frequency of the PGP1 allele varied from 0.900 (Viareggio) to 0.987 (Cagliari). The gene frequencies, together with those available for other European populations were plotted against the latitudes of the different localities sampled and fitted to a North-South cline. PMID- 3019876 TI - [Malignant eccrine poroma]. PMID- 3019875 TI - A rat model of purine nucleoside phosphorylase deficiency. AB - Purine nucleoside phosphorylase (NP; EC 2.4.2.1) deficiency is associated with selective T-cell dysfunction and normal B-cell immunity. In order to create an in vivo model of this immune deficiency, we administered 8-aminoguanosine to rats. This water-soluble nucleoside was rapidly converted by NP to the more potent inhibitor 8-aminoguanine, which has a Ki of 0.19 microM. The accumulation of inosine in plasma showed that administration of 8-aminoguanosine was effectively inhibiting NP activity. The administration of 8-aminoguanosine with deoxyguanosine produced increased levels of dGTP only in thymus cells, and increased levels of GTP in cells from thymus, spleen and lymph node and in red cells. This correlated with assays of deoxyguanosine kinase, which showed significantly higher activity in thymus cells than in cells from spleen and lymph node. The intraperitoneal injection of 8-aminoguanosine alone or with deoxyguanosine for 8 consecutive days caused significant decreases in the number of thymus cells (P less than 0.001) and in lymph node and spleen lymphocytes (P less than 0.01). These data showed that the administration of 8-aminoguanosine to rats provided an animal model of NP deficiency that will allow studies of the specific regulation of T-cell function. PMID- 3019874 TI - Induction of rat secretory IgA antibodies against cholera toxin by a synthetic peptide. AB - There is accumulating evidence concerning the possible importance of secretory IgA antibodies in defence mechanisms against infections of the gastrointestinal tract, including cholera. Intestinal IgA antibodies are also thought to play a major role in protection against the diarrhoeogenic effects of cholera toxin. We therefore attempted to induce secretory IgA antibodies towards a reactive synthetic peptide from the cholera toxin B subunit sequence. We report that rat biliary secretory IgA antibodies against the CTP3 peptide (residues 50-64 of the B subunit) were obtained by three intra-Peyer's patch immunizations, at 2-week intervals, with CTP3 conjugated to tetanus toxoid in complete Freund's adjuvant. Purified secretory IgA fractions from bile of such immunized rats reacted with the carrier toxoid, but also with the CTP3 peptide, and with the native cholera toxin, they also partially neutralized its biological activity, as assayed by inhibition of in vitro cholera toxin-induced cAMP production in mouse thymocytes. PMID- 3019877 TI - Surface markers of NK cells in peripheral blood of patients with cirrhosis and hepatocellular carcinoma. AB - The surface markers and NK activity of blood lymphocytes in 38 normal controls, 57 cirrhotic patients and 22 cirrhotics with hepatocellular carcinoma were studied by the flow cytofluorometry using monoclonal antibodies. A reduction of OKM1+ cells was remarkable in cirrhotics, and a further decrease in HNK-1+ cells as well as OKM1+ cells was observed in cirrhotics with hepatocellular carcinoma, accompanied by depression of NK activity. We postulate that the decreased NK activity in these patients may result from the disturbed maturation of NK cells. PMID- 3019878 TI - Age-related changes in beta-adrenoceptors of lymphocytes. AB - The density of adrenoceptors (Bmax) is greater on B than on T splenocytes. It decreases more or less rapidly on membranes of both populations, as animals age. The exception, we have observed in this respect, is an increase in Bmax on B cells of SJL mice, between the 6th and 25th week of life. PMID- 3019879 TI - Animal models for retrovirus-induced immunodeficiency disease. PMID- 3019880 TI - Studies on in vitro production of Paul-Bunnell antibodies. AB - Two cell lines secreting Paul-Bunnell antibodies were established by means of Epstein-Barr virus-induced immortalization of B lymphocytes from patients with infectious mononucleosis. Progressive loss of antibody production, most probably due to a shutdown of antibody secretion by individual cells was, however, observed. The first of the established cell lines lost its secretion ability after 10 weeks, and the second after 33 weeks of culture. Further improvements of the technique are necessary to stabilize the antibody secretion. PMID- 3019881 TI - Nucleotide sequence of cDNA and derived amino acid sequence of rabbit complement component C3 alpha-chain. AB - The nucleotide sequence coding for 726 amino acid residues of the alpha-chain of rabbit C3 was determined from a cDNA clone. Subfragments of the cDNA produced by restriction endonucleases were inserted into the bacteriophage M13 and sequenced using the dideoxynucleotide technique. The derived amino acid sequence was compared with those of human and mouse C3, which have been previously reported [by De Brujn, M.H.L. and Fey, G.H. (1985) Proc. Natl. Acad. sci. USA 82, 708, and Westel, R.A. et al. (1984) J. Biol. chem. 259, 13857, respectively]. There was 79% or 78% homology in amino acid sequence between rabbit and human or mouse C3, respectively. All of the cysteinyl residues were conserved among the three molecules, and the sequence around the thioester site was also highly conserved. Several regions having low homology were found: one of them was the small fragment released by factor I cleavage. PMID- 3019882 TI - Neonatal rotavirus infection in Pondicherry. PMID- 3019883 TI - Immunological status of patients of primary hepatic carcinoma. PMID- 3019884 TI - Monoclonal antibodies against Japanese encephalitis virus. PMID- 3019885 TI - Hepatocellular carcinoma and cirrhosis of liver. PMID- 3019886 TI - Pleomorphic adenoma of Krause's gland in lower lid. PMID- 3019887 TI - In vitro secretion of immunoreactive tonin from dispersed rat submandibular gland cells. AB - Dispersed cells from the submandibular gland of the male rat were prepared by collagenase treatment to study the mechanism by which immunoreactive tonin is secreted in vitro. Norepinephrine, epinephrine, and phenylephrine stimulated tonin release, an effect that was inhibited by phentolamine but not by propranolol, whereas isoproterenol, carbachol, histamine, and serotonin did not stimulate tonin release. The stimulatory effect elicited by alpha-adrenergic agonists was inhibited by both removal of Ca2+ from the medium and addition of diltiazem and nifedipine, both selective calcium channel blockers. The divalent cation ionophore A23187 stimulated tonin release in the presence of Ca2+, but not in the presence of Mg2+. Dibutyryl cyclic adenosine 3',5'-monophosphate, methylisobutylxanthine, angiotensin II, and vasoactive intestinal peptide had no effect on tonin release. The apparent molecular size of immunoreactive tonin released into the medium under basal and norepinephrine-stimulated conditions was similar to that of standard tonin by gel exclusion chromatography. These data suggest that the in vitro secretion of immunoreactive tonin from rat submandibular gland is initiated by activation of alpha-adrenergic receptors and apparently involves a mechanism dependent not on cyclic adenosine 3',5' monophosphate, but on the influx of extracellular Ca2+. PMID- 3019889 TI - Organization of genes encoding two outer membrane proteins of the Lyme disease agent Borrelia burgdorferi within a single transcriptional unit. AB - OspA and OspB are major outer membrane proteins and antigens of the Lyme disease spirochete, Borrelia burgdorferi. We examined the organization of ospA and ospB, the genes encoding these proteins. The location and direction of transcription of each osp gene was determined by subcloning, deletion analysis, and transposon Tn5 mutagenesis. Transposon Tn5 insertions within the ospA gene abrogated expression of ospB, suggesting that these genes are transcribed from a common promoter. Northern blot analysis of mRNA from B. burgdorferi with two DNA probes individually specific for ospA or ospB identified a 2.2-kilobase transcript that hybridized with each probe. These studies indicate that the two osp genes of B. burgdorferi constitute a single transcriptional unit. PMID- 3019890 TI - Copurification of Leptospira interrogans serovar pomona hemolysin and sphingomyelinase C. AB - The hemolytic and sphingomyelinase C activities of supernatants of cultures of Leptospira interrogans serovar pomona tended to copurify when isoelectric fractionation was carried out. Both activities focused primarily at pH 8.1. Considered in conjunction with other circumstantial evidence, the results led to the conclusion that sphingomyelinase C is responsible for hemolysis. PMID- 3019888 TI - Interference with granulocyte function by Staphylococcus epidermidis slime. AB - The interaction of Staphylococcus epidermidis slime with human neutrophils (PMN) was examined by using isolated slime and allowing bacteria to elaborate slime and other extracellular products in situ. S. epidermidis slime was found to contain a chemoattractant. Incubation of PMN with 50 micrograms or more of slime per ml inhibited subsequent chemotaxis of the PMN to n-formyl-methionyl-leucyl phenylalanine by 27% and to zymosan-activated serum by 44 to 67% with increasing slime concentrations. S. epidermidis slime stimulated little degranulation of untreated PMN. After pretreatment of PMN with 5 micrograms of cytochalasin b per ml, slime predominantly induced release of specific granule contents (33.8% lactoferrin release by 250 micrograms of slime per ml versus 10% myeloperoxidase release by 250 micrograms of slime per ml). By a surface phagocytosis assay, PMN uptake of radiolabeled S. epidermidis which were incubated for 18 h on a plastic surface for slime expression was less than that for S. epidermidis adhered to the plastic for 2 h or grown in unsupplemented nutrient broth. These results suggest that S. epidermidis slime interaction with PMN may be potentially detrimental to host defense and may contribute to the ability of this organism to persist on surfaces of foreign bodies in the vascular or central nervous system. PMID- 3019891 TI - Isolation, characterization, and mapping of Tn5 insertions into the 140 megadalton invasion plasmid defective in the mouse Sereny test in Shigella flexneri 2a. AB - Using Shigella flexneri 2a YSH6000, we isolated 304 independent Tn5 insertion mutants in the 230-kilobase invasion plasmid, pMYSH6000. The site of each Tn5 insertion was assigned to 23 SalI fragments on the previously made SalI cleavage map of pMYSH6000. Among the 304 insertions, 150 were negative in expression of four phenotypes examined (mouse Sereny test [Ser], invasion into epithelial cells [Inv], Congo red binding [Pcr], and inhibition of bacterial growth [Igr] ): 12 were Ser- Inv+ Pcr+ Igr+, and 142 were positive in all four phenotypes. Tn5 insertions in the avirulent mutants were distributed in two separate SalI fragments, F and G, and in four contiguous SalI fragments, B, P, H, and D. Fragment G contains a novel class of determinant(s) which is required only for Ser+ but not for Inv+, Pcr+, and Igr+. Fragment F contains the previously characterized virF locus. B, P, H, and D each contained both virulent and avirulent Tn5 insertions. This indicates that more than two gene clusters exist within this region. Both are required for expression of all four virulence phenotypes. PMID- 3019892 TI - Cloning and expression in Escherichia coli of the Streptococcus pneumoniae gene encoding pneumolysin. AB - A gene bank of Sau3A1-generated Streptococcus pneumoniae DNA fragments was constructed in Escherichia coli K-12 by cloning into the BamHI site of the cosmid vector pHC79. Clones expressing the pneumolysin determinant were selected by testing for hemolytic activity which could be inhibited by antibody to purified pneumolysin and by cholesterol. Restriction analysis of pneumolysin-positive recombinant cosmid DNA indicated that the coding sequence for the toxin was located within a 2.9-kilobase-pair (kbp) ClaI DNA fragment. This fragment, which included 0.35 kbp of vector pHC79 DNA, was subcloned into the plasmid pBR322. E. coli cells harboring this recombinant plasmid (designated pJCP20) produced approximately one-third of the amount of pneumolysin found in the donor S. pneumoniae strain. Plasmid pJCP20 was stably maintained in E. coli and resulted in the accumulation of active pneumolysin in the cytoplasm. Western blot analysis showed that E. coli harboring pJCP20 produced two forms of the toxin with molecular weights of 54,000 and 52,000. The lower-molecular-weight form was indistinguishable from native pneumolysin. Subcloning the 2.9-kbp DNA fragment into the expression vector pEV31 allowed the determination of the direction of transcription of the pneumolysin gene. The pneumolysin-coding sequence (approximately 1.5 kbp) has been localized to within a 1.75-kbp segment of pneumococcal DNA. PMID- 3019893 TI - Molecular organization and expression of the gtfA gene of Streptococcus mutans LM7. AB - The Streptococcus mutans LM7 gene gtfA was cloned in Escherichia coli along with flanking regions of the chromosome as a fragment representing 10.3 kilobases (kb) of streptococcal DNA. Restriction endonuclease mapping revealed that the cloned DNA consisted of four EcoRI fragments with gtfA sucrase activity localized to one fragment, EcoRI-B (2.4 kb). Subsequent analysis with E. coli minicells indicated that three polypeptides were encoded on the 10.3-kb insert (55 [GtfA], 45, and 35 kilodaltons). Neither the 45- nor 35-kilodalton polypeptide exhibited any detectable sucrase activity. The approximate positions and directions of transcription of the two larger proteins were determined from minicell protein profiles displaying truncated versions of these polypeptides. The restriction endonuclease data for the cloned gtfA gene were used to develop a strategy for insertional inactivation of this locus in vivo. An internal HincII fragment of the gtfA gene was removed and replaced with a DNA fragment containing a tetracycline resistance determinant. This new recombinant plasmid was linearized and then transformed into S. mutans GS5 and S. mutans V403 where it was incapable of replication. It was predicted that Tcr colonies would result from double crossover recombinational events involving homologous regions flanking the gtfA gene. This was verified by Southern DNA hybridization analyses. The inactivation of the gtfA gene in both S. mutans GS5 and S. mutans V403 resulted in a decrease of water-soluble exopolysaccharide but no detectable changes in the amounts of water-insoluble polymers. PMID- 3019894 TI - [Asymptomatic excretors of rotavirus]. AB - Asymptomatic Carriers of Rotavirus. Samples of faeces from healthy, mostly adult persons, which were sent to us according to legal requirements to permit food handling, were additionally tested for the presence of rotaviruses. 1.6% of this group proved to be positive at the time of sample collection. Contrary to the typical winter peak of diseased patients, the virus carriership was evenly distributed throughout the year in healthy persons. The results are discussed especially with regard to epidemiological points of view. PMID- 3019895 TI - Congenital varicella syndrome. AB - The authors are reporting a typical case of congenital varicella syndrome following maternal varicella during the 17th week of pregnancy. At birth, the newborn showed necrotic bullae on the skin that healed later with characteristic scars. Other typical anomalies, i.e. hypoplastic limbs with muscular atrophy and clubfoot, intrauterine atrophy, dysphagia and anisocoria were also found. In view of the risk of serious malformations following intrauterine varicella infection attempts should be made to prevent varicella zoster virus infection during pregnancy. PMID- 3019896 TI - Acute biphasic hepatitis A: are different viruses involved? PMID- 3019898 TI - Somatic rearrangement of the c-myc oncogene in primary human diffuse large-cell lymphoma. AB - Chromosome translocations involving 8q24, the band to which c-myc has been mapped (Dalla-Favera et al., 1982), are a uniform finding in Burkitt's lymphoma (Bernheim et al., 1981). However, in only a minority of the tumors is the rearrangement of the c-myc locus sufficiently close to the gene to be detected with currently available probes (Dalla-Favera et al., 1983). Approximately 25% of diffuse large-cell lymphomas have also been reported to have translocations involving 8q24 (Mitelman, 1985), but there have been no reports of c-myc rearrangements in this form of non-Hodgkin's lymphoma. We have examined the structure of the c-myc locus in primary tumor tissue of 10 cases of diffuse large cell lymphoma. In one patient, Southern blot analysis revealed additional c-myc fragments in the tumor DNA but not in the germ-line DNA. Southern blot analysis using probes from both the heavy- and light-chain immunoglobin loci showed that the myc rearrangement was unlikely to involve the immunoglobulin loci in this patient. PMID- 3019897 TI - [Inhibition of gyrase by 4-quinolones: effect on the structure of DNA]. AB - DNA gyrase is an essential enzyme in bacterial metabolism. Its function is the introduction of negative supercoils into DNA. These favour the unwinding of certain parts of DNA und thus are likely to facilitate processes like replication and transcription. Inhibition of DNA gyrase by quinolones and possible effects on the structure of DNA are reviewed. PMID- 3019900 TI - STLV-I antibodies in feral populations of East African vervet monkeys (Cercopithecus aethiops). AB - Serum samples from feral populations of African green monkeys (Cercopithecus aethiops) were screened for antibodies to the simian T-lymphotropic virus, type I (STLV-I). Blood samples had been collected from 336 monkeys in 4 regions of central and southern Kenya in 1978 and 1979, from 114 monkeys in central Ethiopia in 1973, and from 85 monkeys from the Kampala region of Uganda in 1966. A total of 178/535 monkeys (33%) were seropositive (STLV-I+). Only 4/114 monkeys (4%) from Ethiopia were seropositive compared to 25/85 Ugandan monkeys (29%) and 149/336 Kenyan monkeys (44%). Epidemiological analysis of the Kenyan monkeys showed that 37% of the males and 54% of the females were STLV-I+, and that there was a progressive increase in the proportion of STLV-I+ monkeys of both sexes with age, rising from an average of 16% in infants (less than 9 months) to an average of 69% in adults (greater than 42 months). The proportion of STLV-I+ monkeys was higher among females in each age category. Seropositivity for antibodies to STLV-I had no apparent effect on the health of monkeys, and no association with the occurrence of Hepatocystis parasitemia was seen in this species. The analysis of data from infants of STLV-I+ mothers showed that seroconversion had occurred in 1 of 3 cases, suggesting that vertical transmission of the STLV-I virus is not an inevitable consequence for infants of seropositive mothers. PMID- 3019901 TI - Identification of a cell-surface glycoprotein mediating cell adhesion in EBV immortalized normal B cells. AB - Phorbol ester treatment markedly enhanced aggregation of Epstein-Barr virus (EBV) immortalized normal B cells. Monoclonal antibody (MAb) 60.3, recognizing a leukocyte common antigen, completely inhibited intracellular adhesion, whereas MAbs reacting with leukocyte common antigen T200, C3b receptor, T-cell-associated antigen TA-I, C3d (EBV) receptor, brain-granulocyte/T-lymphocyte antigen, transferrin receptor, surface immunoglobulin, Class-I or Class-II transplantation antigens or a B-cell-specific antigen (BI) showed no inhibitory effect. Both the spontaneous and phorbol-ester-enhanced cell aggregations were similarly inhibited by Fab fragments made from antibody 60.3. Phorbol esters also enhance binding between EBV-immortalized normal B cells and autologous or allogeneic blood mononuclear cells depleted of B lymphocytes. This process, which is independent of immunity to EBV, was similarly blocked by the antibody fragments. Antibody 60.3 precipitated 3 non-covalently associated surface glycopolypeptides with apparent MW of 90, 130 and 160/kDa from EBV-infected B cells. Although dissociation of a similar protein complex from granulocytes by sodium dodecyl sulfate treatment clearly indicated the presence of the epitope on the smallest component, the same treatment did not dissociate the protein complex from EBV immortalized normal B cells. It is thus concluded that the 90-kDa glycopolypeptide, either alone or associated with the larger glycopolypeptides, mediates cell adhesion of the B cells. PMID- 3019899 TI - Reversible inhibition of lymphokine-activated killer cell activity by lipoxygenase-pathway inhibitors. AB - Natural killer (NK) cells and lymphokine-activated killer (LAK) cells are anomalous cytotoxic cells which are potentially important in host defense against cancer. Several studies have demonstrated that natural killer (NK) cell activity can be suppressed by chemical inhibitors of the lipoxygenase pathway through inhibition of the production of leukotriene B4 (LTB4). The present study investigated the effects of the lipoxygenase inhibitors BW755C and nordihydroguaiaretic acid (NDGA) on NK and LAK cell activity. NK cell function of fresh peripheral blood mononuclear cells (PBMC) was determined via a standard chromium release assay employing K562 as the tumor target. The LAK cell activity of PBMC which had been stimulated with 10 IU of interleukin-2 for 72 hr was determined against the NK-resistant cell line Daudi. Both BW755C and NDGA inhibited NK and LAK cell function at a variety of concentrations. Indomethacin, a prostaglandin synthesis inhibitor, did not bring about an appreciable diminution in NK or LAK cell activity. Inhibition of NK and LAK cell activities by BW755C and NDGA could be reversed by washing the effector cell suspensions prior to the cytotoxic assay or by adding LTB4 (10(-11)-10(-8) M) directly to the effector:target suspensions. These data indicate that certain arachidonic acid oxidation products of the lipoxygenase pathway are essential for the function of LAK cells. PMID- 3019902 TI - Epstein-Barr virus-related ultrastructural modifications of plasma membrane during B-cell transformation. AB - Ultrastructural modifications are described in the plasma membrane of in vitro established human B cells. By the freeze-fracture technique, intramembrane particles (IMPs) are quantified in B lymphocytes following Epstein-Barr virus (EBV) transformation in vitro, and in B-lymphoma (Burkitt-type) cells, either positive or negative for EBV genome. Analysis shows an overall increase in IMP density as compared to normal controls. Differences are observed between the protoplasmic and exoplasmic faces of fractured membranes as well as among in vitro transformed and clearly neoplastic cells. Results indicate that conformational changes in IMP distribution parallel neoplastic evolution of transformed cells. PMID- 3019903 TI - Steroid therapy in Bell's palsy. AB - The effect of steroid therapy was studied on twenty-eight cases of Bell's palsy. Complete and partial recovery was obtained in twenty-five cases. Delay in starting the therapy with steroids seems to be a major factor responsible for the failures. PMID- 3019904 TI - Stimulatory effects of muramyl dipeptide and its butyl ester derivative on the proliferation and activation of macrophages in vitro. AB - Muramyl dipeptide (MDP) and the butyl ester derivative, N-acetylmuramyl-L-alanyl D-glutamine-alpha-n-butyl ester (MDP[Gln]OnBu), were shown to induce the in vitro proliferation of oil-induced guinea pig peritoneal exudate cells (PEC). Both agents induced 10-20 fold increases in tritiated thymidine incorporation in PEC cultures. The maximal effects occurred in 72 h cultures stimulated with either 0.1 microgram MDP or 10 micrograms MDP[Gln]OnBu. The mitogenic effects of MDP appeared to be mediated by a macrophage product detected in the supernatants of MDP-stimulated cultures. Supernatants of MDP- or MDP[Gln]OnBu-stimulated PEC cultures were also inhibitory to normal fibroblast growth and cytotoxic to L929 tumor cells. These results indicated that these agents may stimulate macrophages by modulating secretory functions. In addition, either peptidoglycan was capable of activating bactericidal activity in in vitro macrophage cultures. Initial studies of possible mechanisms of action revealed an early increase in the level of cyclic GMP. The possible role of cyclic GMP in mediating the stimulation of macrophage secretory processes is discussed. PMID- 3019905 TI - Increased interleukin-1 and modulation of interleukin-2 production by murine macrophages and lymphocytes treated with LF 1695. AB - Previous results have demonstrated that lectin-induced T cell proliferation was potentiated or suppressed by LF 1695, a synthetic immunomodulator, depending on the dose used. Therefore the activity of this compound was investigated on murine IL-1 and IL-2 production. Adherent peritoneal cells, incubated with LF 1695, could secrete high levels of IL-1 with only a slight elevation in intracellular IL-1. This effect apparent at 5 and 10 micrograms/ml was linked to a transient state of activation. At low doses, LF 1695 increased IL-2 production by Con A stimulated spleen cells. A decrease was found at higher doses only when cells were preincubated 20 h with the compound. In murine macrophages stimulated either by A 23187 or LPS PGE2 synthesis was inhibited by LF 1695 even at low doses. However, supernatant LTB4 level was increased in LF 1695-treated culture with a time-dependent effect. Therefore modulation of lectin-induced T cell proliferation by LF 1695 may be IL-2 production-mediated. Inhibition of the cyclooxygenase and stimulation of the lipoxygenase pathway of arachidonic acid metabolism may be responsible for this pattern of activity. PMID- 3019906 TI - Pagetoid Bowen's disease on the breast. PMID- 3019908 TI - Primitive neuroectodermal tumor of the endometrium: report of two cases, one with electron microscopic observations. AB - Two primary primitive neuroectodermal uterine malignant tumors are reported. One initially had the appearance of neuroblastoma but after radiotherapy showed both glial and ganglionic differentiation. The second neoplasm exhibited medulloepithelial canals, as well as glial, neuroblastic, and focal neuronal differentiation. Both patients died with widespread metastatic disease after both radiotherapy and chemotherapy. PMID- 3019907 TI - Evaluation of silica gel cartridges coated in situ with acidified 2,4 dinitrophenylhydrazine for sampling aldehydes and ketones in air. AB - A procedure for coating in situ silica gel in prepacked cartridges with 2,4 dinitrophenylhydrazine (DNPH) acidified with hydrochloric acid is described. The coated cartridge was compared with a validated DNPH impinger method for sampling organic carbonyl compounds (aldehydes and ketones) in diluted automotive exhaust emissions and in ambient air for subsequent analysis of the DNPH derivatives by high performance liquid chromatography. Qualitative and quantitative data are presented that show that the two sampling devices are equivalent. The coated cartridge is ideal for long-term sampling of carbonyls at sub to low parts-per billion level in ambient air or for short-term sampling of carbonyls at low ppb to parts-per-million level in diluted automotive exhaust emissions. An unknown degradation product of acrolein has been tentatively identified as x-acrolein. The disappearance of acrolein in the analytical sample matrix correlates quantitatively almost on a mole for mole basis with the growth of x-acrolein. The sum of the concentration of acrolein and x-acrolein appears to be invariant with time. PMID- 3019909 TI - Brown adipose tissue in patients with phaeochromocytoma. AB - Intra-abdominal adipose tissue was obtained at laparotomy from three subjects with high circulating noradrenaline concentrations in the presence of phaeochromocytoma. Light and electron microscopy confirmed typical brown adipose tissue, both adjacent to, and in one case distant from, the tumour. Biochemically, in terms of high cytochrome-C oxidase activity, mitochondrial GDP binding, GDP-inhibitable uncoupled mitochondrial respiration, and specific concentration of uncoupling protein (mean 31 +/- 7 micrograms/mg mitochondrial protein) the tissue possessed all the unique features of thermogenically active brown adipose tissue. These findings are contrasted with low results obtained from a case with Cushing's disease, and the significantly lower results (mean 2.5 +/- 1.8 micrograms/mg) in a group of control adults (P less than 0.02). In the presence of high circulating noradrenaline concentrations, the intra-abdominal fat of human adults, including the omental fat, which is brown adipose tissue in infancy, becomes reactivated and may be contributing to the weight loss which is typically seen with phaeochromocytoma. Human adult brown adipose tissue thus has the biochemical potential for the thermogenic activity required in order to contribute to the regulation of energy balance and body weight. PMID- 3019910 TI - Intermittent protein-sparing fasting with abdominal belting. AB - In an attempt to minimize the weight regain that often follows effective weight reduction, a programme has been evaluated which involved the use of a nylon waist cord which could be readily tightened but not lengthened, during a regime of alternating periods of a liquid diet and a high-fibre natural diet combined with behavioural modification. Forty subjects completed the initial cycle of a liquid regime with an overall weight loss of 7.7 kg during the treatment period. Thirty six of these subjects have been followed up for a mean of 12 months after the completion of the treatment programme. Five subjects are difficult to categorize having worn the cord for some but not all of this time. Fourteen have continued to wear the waist cord throughout and have achieved a further mean weight loss of 4.8 kg. This differs significantly from the 17 subjects who cut off the cord and who have regained a mean of 6.6 kg. The results suggest that an adjustable waist cord is a valuable aid to achieving successful and permanent weight reduction in some subjects. PMID- 3019911 TI - Purification and characterization of rabbit muscle acylphosphatase in the thiol ( SH) form. AB - Modifications in the muscle acylphosphatase purification procedure enabled us to isolate the enzyme with its sole cysteine in the -SH form; this enzyme form is the most abundant in vivo. Our data demonstrates that the enzyme forms purified by previously reported procedures can be easily derived from a reaction of the SH enzyme with oxidized glutathione. Probably most, or even all, of these enzyme forms are artifacts due to the purification. The SH-acylphosphatase shows kinetic parameters similar to those reported for the mixed disulfide with glutathione and S-S dimer, except for the specific activity value, which is about twice as much, and the Km, which is reduced. PMID- 3019912 TI - Chemical properties of water-soluble porphyrins. 5. Reactions of some manganese (III) porphyrins with the superoxide and other reducing radicals. AB - Solution properties of three manganese porphyrins, in monomeric form, were investigated. These were the 'picket-fence-like' porphyrin Mn(III) alpha,alpha,alpha,beta- tetra-ortho(N-methylisonicotinamidophenyl)porphyrin (Mn(III)PFP) and two 'planar unhindered' porphyrins, the Mn(III)TMPyP (tetrakis (4-N-methylpyridyl)porphyrin) and Mn(III)TAP (tetra(4-N,N,N trimethylanilinium)porphyrin). The porphyrin properties studied were: the absorption spectra in their manganic and manganous forms; acid/base properties of the aquo complexes; the effect of potential axial ligands (up to a concentration of 0.1 mol dm-3) and their one electron reduction potentials. Knowing these properties, the reaction of the Mn(III) porphyrins with the superoxide radical and other reducing radicals were studied using the pulse radiolysis technique. The second-order reaction rate constant of O2- with the Mn(III) porphyrins, which governs the catalytic efficiency of the metalloporphyrins upon the disproportionation of the superoxide radical, was 5.1 X 10(7) to 4.0 X 10(5) dm3 mol-1 s-1, depending on the pH and the nature of the metalloporphyrin. These values are at least one order of magnitude lower than found for Fe(III)TMPyP. One electron reduction of the three Mn(III) porphyrins by eaq-, CO2-, CH2OH and (CH3)2COH had similar second-order rate constants (10(9)-10(10) dm3 mol-1 s-1). That for (CH3)2(CH2)COH was about 10(5) dm3 mol-1 s-1. Reduction in all cases produced the corresponding Mn(II) porphyrin and no intermediate was found. The oxidation reaction of the Mn(II) porphyrins by O2- was approximately two orders of magnitude faster when compared to the reduction of Mn(III) porphyrins with the same radical. Since the reactivities of O2- towards the three manganese (III) compounds follow their reduction potentials, it is suggested that these reactions are governed by an outer-sphere mechanism. This suggestion is corroborated by the finding that water molecules acting as axial ligands, in these aqueous solution systems, are not replaced by another potential ligand when the latter is in the concentration range of 100 mM or less. PMID- 3019913 TI - Physical mechanism for inactivation of metallo-enzymes by characteristic X-rays. AB - Measurements have been made of the inactivation of the metallo-enzyme dihydro oratic dehydrogenase in solution by characteristic X-rays at energies above and below the K absorption edge of the constituent iron atom. From the dose-survival curves and knowledge of the equilibrium electron spectrum generated by the X-ray 'field', inactivation cross-sections are deduced and expressed in terms of intrinsic efficiencies for the various proposed direct and indirect mechanisms of inactivation. It is concluded that the inactivation is caused by direct X-ray interaction in an area equivalent to about 30 per cent of the mean geometrical cross-section of the molecule, and is independent of whether the target is wet or dry. The contribution from Auger electron cascades, Coulomb charges etc. initiated by the inner-shell vacancy in the metal atom is negligible--possibly due to saturation effects. It seems that the presence of the metal atom simply serves to enhance the overall interaction probability with the molecule in a manner consistent with expectations from the photon absorption coefficients. No anomalously large damage is detected. These conclusions are supported by comparison with published results for other metallo-enzymes and bromine-loaded bacteria. PMID- 3019914 TI - Isolation, cultivation and haemadsorption of buffalo pox virus on BHK-21 cell line from Dhule epidemic (Western India). AB - An epidemic of buffalo pox infection in buffaloes, white cattle, horses, goats and human beings in the Dhule district of Maharashtra State (India) during December, 1975 to March, 1976 is reported. Buffalo pox virus from this epidemic has been propagated in BHK-21 cell line directly from the skin scab materials collected from those animals in phosphate glycerine buffer. A specific cytopathic effect (CPE) produced in these cell lines was differentiated from the non specific CPE by the demonstration of the characteristic 'Rosette' or 'Sun flower' like haemadsorption with 0.4% fowl erythrocytes. The CPE started appearing as early as 18 hours and the detachment and peeling off the cells from the glass surface were noted on the 4th day onwards. Eosinophilic inclusion bodies with giant cells (multi-nucleated) formation and fusion of the cells were also observed with a few stellate cells. PMID- 3019915 TI - Susceptibility to varicella-zoster virus among adults at high risk for exposure. AB - The adult health care provider who is susceptible to varicella zoster virus (VZV) represents a risk to her or himself and to patients. Nineteen percent of employees at this Children's Hospital had no or uncertain prior experience with VZV, and of these, 28% were found to be VZV susceptible, representing 5% of the total population of 2,730 hospital employees. During the 12 months of study, six of the potential 135 to 137 VZV-susceptible individuals acquired varicella. VZV susceptible health care providers should be aware of their potential to both acquire and transmit VZV. PMID- 3019916 TI - Is primary cytomegalovirus infection an occupational hazard for obstetric nurses? A serological study. AB - The results are reported of a 4-year prospective study of the incidence of primary cytomegalovirus (CMV) infection in the nursing staff of a specialist obstetric hospital. The absence of seroconversion found in personnel attending patients with confirmed CMV-infection justifies reassuring staff members in "high risk" areas of the adequacy of the methods used to combat cross-infection. On the other hand, a low rate of seroconversion (1.2% per annum) in the staff who nurse normal mothers and "rooming-in" babies emphasizes the need for the rigorous observance of hygienic precautions by all personnel in all areas. The results of this Australian investigation are discussed in relation to the northern hemisphere experience of CMV-seroconversion in pediatric nurses. PMID- 3019917 TI - [Clinical diagnosis: Cushing's disease. Laboratory diagnosis: normal findings. A contribution on the rhythmic activity of endocrine adenomas]. PMID- 3019918 TI - HSV-1 recovery from ocular tissues after viral inoculation into the superior cervical ganglion. AB - New Zealand albino rabbits were inoculated in the right superior cervical ganglion with 25 microliter of herpes simplex virus type 1 (HSV-1) (McKrae strain; 10(3) or 10(5) PFU/ml). Positive tear film swabs were detected at least once in 28/32 (88%) of ipsilateral eyes and 6/32 (19%) of contralateral eyes beginning on postinoculation (PI) day 2-6. The average HSV-1 titer in the tear film was 4.0 X 10(3) PFU in ipsilateral eyes and 2.7 X 10(3) PFU in contralateral eyes, determined from eye washes after inoculation of 25 PFU of HSV-1. In selected rabbits, the aqueous humor was positive for virus on PI days 3, 4, 5, 6, and 8. the aqueous humor in ipsilateral eyes showed positive results in 9/11 (82%) of the eyes tapped on PI 3, 13/18 (72%) on PI 4, 5/11 (45%) on PI 5, 1/6 (17%) on PI 6, and 1/2 (50%) on PI 8. No virus was detected in aqueous humor tappings in any contralateral eyes (0/65). Conjunctivitis and iritis (iris hyperemia) appeared in all ipsilateral eyes beginning as early as PI day 1. Conjunctivitis occurred in 1/21 (4.8%) of contralateral eyes. Cells and flare appeared in 18/21 (86%) of ipsilateral eyes and 2/21 (9.5%) of contralateral eyes. Hyphema was noted in 3/21 (14%) of ipsilateral eyes. Of the eyes with iritis, 12/21 (57%) developed corneal edema. Corneal dendritic ulcers were observed in 4/21 (19%) of ipsilateral eyes and 2/21 (9.5%) of contralateral eyes. No ocular fundus changes were seen in any contralateral or ipsilateral eyes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3019919 TI - The accumulation of myoinositol and rubidium ions in galactose-exposed rat lens. AB - When rat lens is incubated in 30 mM galactose overnight, the extent of accumulation of rubidium ions (Rb) and myoinositol (MI) are affected, as well as the Na-K ATPase activity. Rb accumulation and Na-K ATPase activity are only slightly affected compared to the dramatic drop in MI accumulation. These changes are completely abolished by sorbinil, which blocks polyol formation, or by rendering the galactose medium hypertonic to offset the osmotic effect of polyol formation. On the other hand, the addition of excess MI to the galactose medium had no effect on correcting these changes. The results obtained are consistent with the polyol-osmotic theory of sugar cataract formation. PMID- 3019920 TI - Peripheral neuropathy in Cockayne syndrome. AB - Two siblings with Cockayne syndrome are reported. In one case a sural nerve biopsy showed a demyelinating peripheral neuropathy with occasional inclusions in Schwann cells made up of electron dense finely granular material intermingled with vacuoles or lamellar structures. The significance, if any, of this accumulated material remains unclear. The presence, in addition, of small finely lamellar intra-axonal osmiophilic bodies suggests an associated axonal involvement. PMID- 3019922 TI - A new radioiodine exchange labeling technique. AB - A new technique of radioiodine exchange labeling is presented. The method was developed with respect to the short physical half lives of 123I and 121I, the altered activity concentrations and varying impurities, which hamper the previously introduced labeling methods. The described "one step reaction" is applicable to a series of organic compounds with different physicochemical and physiological behaviour as well as to all radioisotopes of iodine. PMID- 3019921 TI - Inhibition and reversal of endotoxin-, aggregated IgG- and paf-induced hypotension in the rat by SRI 63-072, a paf receptor antagonist. AB - Platelet activating factor (paf) given intravenously produces systemic hypotension in the rat. Similar effects can be induced using endotoxin or heat aggregated IgG challenges, which are thought to involve endogenous paf release. Extending this concept, we have examined the ability of the paf antagonist SRI 63 072 to inhibit or reverse systemic hypotension induced with paf, heat-aggregated IgG or endotoxin 0111-B4 in rats. At 100 ng kg-1 paf, there occurred a 38.6 +/- 5.1% decrease in carotid mean arterial pressure (MAP) followed by a 3.2 +/- 0.7 min recovery period (RP) to return to normal pressure values. The ED50 of SRI 63 072 was 0.16 mg kg-1 i.v. (MAP) and 0.25 mg kg-1 (RP) when given 1-5 min before the paf challenge. Endotoxin (15 mg kg-1 i.v.) produced a hypotensive response (54 +/- 8% decrease in MAP) and a corresponding 80% decrease in mesenteric artery blood flow. When given 2-8 min after endotoxin, 1.0 mg kg-1 i.v. SRI 63-072 totally restored blood pressure and artery blood flow. SRI 63-072 similarly reversed heat-aggregated IgG (10 mg kg-1) induced reduction of MAP, with an ED50 of 0.05 mg kg-1 i.v. The observations that SRI 63-072 can inhibit or reverse systemic vascular effects produced from paf and other provocators of endogenous paf release strongly implicates paf as a common final mediator of hypotension and shock. As SRI 63-072 is a competitive receptor antagonist, the hypotensive effects of these provocators appear to be mediated by vascular receptors for paf. PMID- 3019923 TI - The separation of 99mTc(Sn)EHDP complexes by HPLC and GPC. AB - For the characterization of the multi-component bone-scan agent 99mTc(Sn)EHDP we have analysed the complex mixture with reversed phase ion pair chromatography (IPC) and soft gel permeation chromatography (GPC). With IPC five major complexes were found within a separation-time of 40 min. To avoid decomposition of the complexes during separation, the concentrations of EHDP and the reductant Sn(II) in the eluent had to be identical to the EHDP and Sn(II) concentrations used for the preparation of the complexes. To investigate the stability of the complexes we applied separation by IPC, followed by re-analysis of the fractions within 1 h and after 21-25 h. It appears that there is a state of equilibrium between the five complexes. Within 1 h after isolation the separated complexes were still more than 90% in their original form while after 20 h considerable amounts of the other complexes are found. The two complexes with the largest retention time with IPC are the most stable ones. When the total mixture was re-analysed after 26 h all five components appeared to be still present, but the relative amount of the most stable component had increased. Using GPC for the separation of the complex mixture, we found four major peaks within a separation time of 14 h. The elution orders of the complexes with the two separation methods are opposite. PMID- 3019925 TI - Aqualuminescence of alkaline luminol in the presence of fluorescein. AB - The light yield from both luminol and fluorescein is studied at a fixed concentration of luminol and by varying the concentration of fluorescein in 3 X 10(-2) M basic solution by direct chemical reaction as well as by aqualuminescence technique. The emission spectra of chemiluminescence and aqualuminescence of the alkaline luminol-fluorescein mixture are recorded on a Fuoss spectrograph. A 4-fold increase in the aqualuminescence intensity of luminol has been observed in the presence of fluorescein as compared to that of pure luminol. The results are explained on the basis of reactions of colour centres with the activators. PMID- 3019926 TI - Dosimetry concepts and measurements in food irradiation processing. AB - The associations between the dosimetry concepts, Minimum absorbed dose (D min), maximum absorbed dose (D max), and average dose and median dose are investigated for the case of a large cobalt-60 plaque source irradiating homogeneous bulk product in a two-pass, two-sided irradiation. It is assumed that to a first approximation the intensity of radiation decreases exponentially with the depth, t, in the product. A series of mathematical relationships is derived for the average dose, the maximum and minimum dose, the median dose [defined as (D max/D min)/2], and the uniformity ratio (defined as U.R. = (D max/D min). The relationships are derived in terms of a constant D0 (the dose on the surface of the product in the pass close to the source) and the relaxation length (mu t) of the radiation in the product. Since the uniformity ratio and other dose parameters can be calculated for certain chosen values of mu t, the individual values of mu (the energy absorption coefficient) and t do not need to be known. By dividing the dose range from D min to D max into 10 equal fractions, the amount of product irradiated to each of the fractions is calculated, and it is shown that, independent of the value of U.R., about a third of the product receives a dose in the first fraction above D min. It is also shown that for a given median dose, the average dose decreases as U.R. increases. The calculated dose relationships are confirmed by measurements in homogeneous dummy product, using the lyoluminescence of glutamine to measure dose. The implications of these results for the regulation of the food irradiation process and for the design of irradiation facilities are discussed. PMID- 3019924 TI - Production of no-carrier added 67Cu. AB - Copper-67 has been produced at the BLIP by 68Zn(p, 2p) and 70Zn(p, alpha) reactions with 193 MeV protons and at the HFBR using the 67Zn(n, p) route. The effective cross-sections of natZn(p, 2pxn)61,64,67Cu reactions were measured at 200 MeV and compared with predicted values obtained from semi-empirical formulae given by Silberberg and Tsao. From these cross-sections, the fraction of 64Cu in the final 67Cu preparations was estimated and compared with the experimental values. A procedure has been developed consisting of electrodeposition and ion exchange for isolation of no-carrier-added radiocopper from a Zn target. The method is adaptable for remote hot cell operation. The overall radiochemical yield of 67Cu and the separation factors from a Zn target and other radionuclides were evaluated. The saturation yield of 67Cu produced by the fast neutron 67Zn(n, p) reaction and the corresponding cross-section were remeasured. Experiments have been initiated to attach 67Cu to monoclonal antibodies for immunotherapy applications. PMID- 3019927 TI - The 17th Japan Conference on Radiation and Radioisotopes. September 2-4, 1985, Sankei Kaikan, Tokyo. Abstracts. PMID- 3019928 TI - A system for in vivo measurement of bone calcium by local neutron activation of the hand. AB - A 252Cf neutron-irradiation facility designed specifically for clinical in vivo measurement of calcium in the hand is described. Results of preliminary measurements are presented. Hand phantoms were exposed to the neutron beam for 10 min and the induced 49Ca activities were counted for 10 min after an elapsed time of 2 min. The results indicate that with a 2 X 100 micrograms 252Cf neutron source and two 20.3 X 12.7 cm NaI crystals, the counts per gram of bone mineral mass changes by about 4% for each 100 cm3 change in the overall volume (soft tissue plus bone) of the hand. For hands of equal volumes the counts per gram are expected to be almost independent of the bone volume. With an absorbed dose equivalent of 150 mSv (15 rem), the sensitivity is about 200 counts per 10 min per gram Ca. The statistical reproducibility of the results is better than 3% for the average value of 11 g Ca in the normal hand. PMID- 3019929 TI - A device for small scale experimental cell irradiators using 90Sr applicators. PMID- 3019930 TI - Preparation of [99mTc(DMPE)2Cl2]+ for myocardial imaging by using Fe-(DMPE) complexes. AB - The preparation of [99mTc(1,2-bis(dimethylphosphino)ethane)2Cl2]+ ([99mTc(DMPE)2Cl2]+) for imaging the myocardium is investigated. Starting from Fe(III)- or Fe(II)-DMPE and 99mTcO4- different preparation variants are compared. In these reactions either ascorbic acid or DTPA serves as an agent for complexing iron. A simple procedure using lyophilized initial components is represented. Its application yields [99mTc(DMPE)2Cl2]+ with a radiochemical purity of more than 95%. Organ distribution studies performed in rats emphasize the high myocardial accumulation of this preparation. PMID- 3019931 TI - omega-Iodophenyl fatty acids: a convenient method of radioiodination. AB - A solid-phase radioiodination technique for omega-iodophenyl fatty acids using ammonium sulfate is described. The radioiodinations are regioselective, high in yield (95%), short in reaction time (1 h) and capable of yielding high specific activity products although at lower yields. Purification is exceptionally simple: a single passage through an ion exchange column to remove unreacted I- is all that is required. Syntheses of several omega-iodophenyl fatty acids are also described. PMID- 3019933 TI - NCA 16 alpha-[18F]fluoroestradiol-17 beta: the effect of reaction vessel on fluorine-18 resolubilization, product yield, and effective specific activity. AB - Although the reported synthesis of the title compound resulted in a high radiochemical yield (43% based on resolubilized 18F), the effective specific activity at EOS was low (166 Ci/mmol). Reduction in the amount of carrier fluoride in the target water improved the effective specific activity of the product, but with a concommitant decrease in the resolubilized yield of the fluoroestradiol (12.7%). A re-examination of the labeling parameters was performed to determine the conditions that would increase the yield of the fluoroestrogen and maintain a high effective activity for the product. Since the amount of resolubilized 18F in THF is important in obtaining high specific activity compounds in this type of synthesis, several types of vessels were investigated to determine their effect on the evaporation of the [18O]H2O target water and subsequent resolubilization of 18F into THF. Of the vessels (Pt crucible, borosilicate glass, siliconized borosilicate glass, Vacutainer), the Vacutainer afforded the highest resolubilization of 18F into THF (90%), resulting in an improved resolubilized yield for the fluoroestradiol (28%) and an increased effective specific activity at EOS for the product (1600-3939 Ci/mmol). PMID- 3019932 TI - Studies on the interaction between SeO2 and sulfur compounds and distribution of Rb, Zn, Co, Fe and Hg in mice by instrumental neutron activation analysis. AB - Content of Se, Rb, Zn, Co, Fe and Hg in liver, kidneys, spleen, brain and blood of SAS/4 mice were determined after i.p. injection with SeO2, gluthathione, cysteine, cysteamine or methionine. Instrumental neutron activation analysis (INAA) was applied as the analytical method. Se was incorporated in all the examined organs, the the efficiency of the incorporation depended upon the sulfur compounds injected. Injection with above compounds affects the contents of the other elements in all mice organs. PMID- 3019934 TI - Technetium-sulfur colloid. AB - The chemistry of the technetium-sulfur colloid produced by the reaction of sodium thiosulfate with acid was investigated. A commercial kit was duplicated, and analyses of elemental sulfur, bisulfite and residual thiosulfate were carried out. The colloidal dispersions were filtered through Nuclepore graded membranes, and the percentages of sulfur and of 99mTc in the various filtrates were determined. In all cases--with varying acid, thiosulfate and time of incubation- there was a rough agreement between the two percentages for particles 0.4 micron in diameter or more. However, for small particles (less than 0.1 micron) there was virtually no sulfur, but there was an appreciable percentage of technetium. It was concluded that the technetium sulfide nuclei formed first, and that the supersaturated sulfur deposited in part on them and in part on its own nuclei. It was found that raising the pH of the preparation to weakly alkaline values and reheating the solution dissolved most of the deposited sulfur by the reaction with sulfite to form thiosulfate, leaving much smaller, virtually sulfur-free technetium sulfide particles. Such a preparation was found to be as efficient as the technetium-antimony sulfide colloid for lymphograms in dogs. Potassium trithionate, K2S3O6, used in place of sodium thiosulfate, produced small Tc-S colloid particles with less sulfur than the conventional thiosulfate-acid system. PMID- 3019935 TI - Reactor production and detection of radiolabeled cis-platinum. AB - Small quantities of radiolabeled cis-platinum were prepared by two methods. The thermal neutron irradiation of potassium tetrachloroplatinite followed by radiochemical synthesis was compared to the direct irradiation of the cis platinum itself. Both methods produced usable quantities of tracer, but the direct method proved most practical. Radiochemical purification and analysis data are also presented. Liquid scintillation and NaI(Tl) counting techniques were evaluated. Counting the numerous x-rays produced from several platinum radioisotopes in a well-type NaI(Tl) detector proved most convenient for routine use. It is concluded that these methods provide a useful route for the preparation, analysis and determination of measurable quantities of radiolabeled cis-platinum for small scale applications. PMID- 3019936 TI - Determination of free fatty acids in irradiated tuna loins on storage. PMID- 3019937 TI - Preparation of 99mTc-tin-phosphate polyvinyl pyrollidone stabilized colloid and distribution in bone marrow. AB - Technetium-99m-Sn-phosphate colloid was prepared in the presence of polyvinylpyrollidone(PVP), molecular weight 44,000, for bone marrow imaging. Size of the colloidal particles, as determined by coulter counter, microphotographic and electron microscopic studies was 15-35 nm. The colloid preparation was checked for the presence of any soluble components by Sephadex G-25 chromatography. Scintigraphy in rabbits showed a high concentration of the colloid in the bone marrow. Tissue distribution studies in rabbits showed 26.7% of the injected dose at 1 h post-injection in the bone marrow collected from femoral shaft and head. Although liver and spleen showed considerable levels of activity, kidneys and compact bone did not show any uptake. The colloid cleared from the blood exponentionally with a first phase showing a relatively fast clearance while in the second phase it disappeared more slowly. Uptake of the colloid in human bone marrow was close to that in the rabbit and thus clinical evaluation is warranted. PMID- 3019938 TI - Synthesis of 15-(p-iodophenyl) pentadecanoic acid labelled with carbon-14. AB - 15-(p-Iodophenyl) pentadecanoic acid labelled with carbon-14 in the carboxyl group is obtained with good yields in five steps from [14C]NaCN, starting from 14 bromo 1-phenyl tetradecane 2. Specific radioactivity: 0.019 mCi/mg, 8.45 mCi/mmol; radiochemical yield: 74.6%. PMID- 3019940 TI - Licencing of medical x-ray equipment in 1899: background and procedure. AB - The full text of probably the first worldwide licencing procedure for x-ray equipment has now become available. The document is dated 27 December 1898 and is a formal request from one authority to another to look into what was going on in an institute which called itself Institut fur Rontgenuntersuchungen. In response to this, a committee considering both formal and scientific aspects of licencing was founded. This paper shows in brief the scientific background on the detrimental radiation effects available at the turn of the century, and reports on the decisions made by the authorities. PMID- 3019939 TI - A 122Xe-122I generator for remote radio-iodinations. AB - A 122Xe-122I generator system is described that produces 122I extraction efficiencies of approximately 60%. Radiocontaminants were less than 0.1% at the time of 122I removal following a 10 min ingrowth period. The chemical form of 122I was identified as [122I]iodide, and the [122I]iodide was remotely incorporated into radiopharmaceuticals for PET studies with an overall efficiency of as much as 40%. PMID- 3019941 TI - Rapid reductive-carboxylation of secondary amines, one pot synthesis of N'-(4-11C methyl)imipramine. AB - A new rapid high yield synthesis of radiolabeled N'-(4-11C-methyl)imipramine has been developed using a reductive-carboxylation approach, in which 11CO2 is reacted with either N'-trimethylsilyldesimipramine or N'-lithium derivative of desimipramine, followed by lithium aluminum hydride reduction, to give no carrier added or carrier added 11C-labeled imipramine respectively. The final product is characterized by chromatographic and spectroscopic methods. PMID- 3019942 TI - [18F]fluoride from a small cyclotron for the routine synthesis of [18F]2-fluoro-2 deoxy-D-glucose. AB - [18F]Fluoride was produced via the 18O (p, n) 18F reaction using a low volume stainless steel 18O-water target in a small cyclotron. The average 18F production rate for 60 runs was about 720 microCi/microA-min. A typical bombardment time of 20 min produced 120 mCi of 18F-fluoride. [18F]2-Fluoro-2-deoxy-D-glucose ([18F]2FDG) was prepared using the procedure developed by Tewson [J. Nucl. Med. 24, 718 (1983)] involving a protected cyclic sulfate glucose derivative as precursor. The routine synthesis and quality testing of [18F]2FDG required 1 h and, typically, yields of 30-35 mCi of greater than 95% HPLC-pure product were obtained. PMID- 3019943 TI - Stereospecific approach to the synthesis of [18F]2-deoxy-2-fluoro-D-mannose. AB - The reaction of methyl 4,6-O-benzylidene-3-O-benzyl-2-O-trifluoromethanesulfonyl beta-D- glucopyranoside in acetonitrile at 75 degrees C for 30 min with [18F]tetra-n-butylammonium fluoride, followed by silica gel column chromatographic purification, gave the corresponding [18F]methyl 4,6-O benzylidene-3-O-benzyl-2-fluoro-beta-D-mannopyranoside with complete regio- and stereoselectivity (42% radiochemical yield). Hydrolysis of the radiolabeled fluoromannopyranoside intermediate with either 6 N HCl or 50% methanesulfonic acid for 30 min at 120 degrees C, followed by purification by column chromatography (ion retardation resin and neutral alumina), gave pure [18F]2 deoxy-2-fluoro-D-mannose ([18F]2-FDM) with an overall radiochemical yield (from [18F]fluoride ion) of 34%. Extension of this methodology to the no carrier added (nca) synthesis under phase transfer conditions (Kryptofix 222/K 18F/acetonitrile) gave nca [18F]2-FDM in a radiochemical yield of 75%. Purity and identity of the fluorinated products were confirmed by 1H and 19F NMR spectroscopy. The synthetic procedure described here permits for the first time the routine preparation of large amounts of [18F]2-FDM for tomographic studies. PMID- 3019944 TI - An improved synthesis of (3-N-[11C]methyl)spiperone. AB - An improved radiochemical synthesis of (3-N-[11C]methyl) spiperone is reported. The radiotracer was prepared by N-alkylation of spiperone in dimethylformamide in the presence of equimolar aqueous tetrabutylammonium hydroxide and was purified by semi-preparative reversed phase high performance liquid chromatography (C-18 HPLC). The average radiochemical yield was approximately 21% end-of-synthesis (EOS) with an average specific activity of over 2750 mCi/mumol EOS. The total time for synthesis and specific activity determination was approximately 21 min. PMID- 3019945 TI - Measurements of 99Tc in biological samples: problems with the evaluation of radiochemical yield. AB - Measurements of 99Tc in vegetables have been made. The radiochemical yield is measured by tracing the process with a known amount of 99mTc. Differences in chemical behaviour of the problem 99Tc relative to the tracer 99mTc have been found under certain conditions and the way to overcome them is reported. PMID- 3019946 TI - Synthesis of nitrogen-13 labeled alkylamines via amination of organoboranes. AB - Organoboranes react with nitrogen-13 labeled ammonia to produce alkylamines in moderate yield. When 13N labeled ammonia was bubbled into a tetrahydrofuran solution containing 0.5M tridecylborane, 1-[13N]aminodecane was formed in 25-30 min from the end of bombardment (EOB) in 40-60% overall yield. 1-[13N]aminooctane and 1-[13N]aminohexane were also synthesized from appropriate organoboranes in similar yield. PMID- 3019947 TI - Carbon-11 labeled dialkylketones: synthesis of 9-[11C]heptadecan-9-one. AB - 9-[11C]heptadecan-9-one was synthesized from di-n-octylthexylborane via cyanidation with K11CN. The rearrangement of the organoborane intermediate followed by alkaline oxidation produced the title compound in 55-60 min from the end of bombardment (EOB) in 50-70% overall yield. The reaction sequence is applicable for the synthesis of various dialkyl ketones. PMID- 3019948 TI - The effect of DMSO treatment on the composition of 99mTc(Sn)EHDP. AB - To find an explanation for the reported positive effect of a dimethyl sulfoxide (DMSO) treatment on the performance of the bone scan agent technetium-(tin) ethane-1-hydroxy-1, 1-diphosphonate, we compared the composition of the agent, prepared with and without treatment with DMSO by using high performance ion-pair chromatography (IPC). The preparation obtained with the DMSO treatment appeared to contain a larger fraction of large and highly charged polynuclear complexes than the preparation without the DMSO treatment. According to experiments by other investigators the smaller 99mTc(Sn)EHDP complexes (early eluting components in IPC) give lower bone/blood ratios than the larger ones. The results presented in this paper show that the explanation for the effect of the DMSO treatment may be that the Sn-EHDP complexes which are removed by extraction with DMSO, give relatively small 99mTc(Sn)EHDP complexes. Without these small complexes a superior bone scan agent is obtained. From experiments in which an excess of 99TcO4 over Sn(II) was used, it was concluded that at least one of the technetium complexes contains Sn(IV). On the other hand, the absence of Sn from a late eluting technetium complex was proven. PMID- 3019950 TI - Search for 99mTc labeled DTS bifunctional radiopharmaceutical: role of functional groups in myocardial accumulation. AB - Development of 99mTc-bifunctional radiopharmaceutical (BR) is attracting the interest of various research groups. In the present paper, various molecules containing a neutral 99mTc-dithiosemicarbazone (DTS) structure as the technetium chelating site, along with various functional groups (amino, carboxyl or isobutyl group with diverse charge) are tested for their chemical or biological functions. The study on the effect of those functional groups is carried out in vitro and in vivo. The validity of introducing an amino group along with the technetium chelating site DTS for myocardial accumulation is discussed. PMID- 3019949 TI - [Biochemistry of amino acid derivatives of [103Ru] ruthenocene. Comparison with 131I hippuran]. AB - The potential radiopharmaceuticals: ruthenocenoyl alanine, ruthenocenoyl methionine, 1'-methyl-ruthenocenoyl glycine and its esters were labelled with 103Ru starting from the analogous ferrocene compounds. In a series of tests in mice and rats these substances were compared with hippuran and ruppuran (= ruthernocenoyl glycine, a ruthenocene-amino acid analogue of hippuran). The organ distribution of these compounds was measured at various times after injection. Kidney concentrations of 1'-methyl-ruthenocenoyl glycine and its esters were found to be extremely high, followed by a rapid excretion. In contrast with these compounds, ruthenocenoyl methionine indicated a significantly greater affinity for liver than for kidney, but not for pancreas. Ruthenocenoyl alanine exhibits a high affinity for tumor cells. The advantages of 97Ru labelled radiopharmaceuticals compared with 99mTc or 123I/131I labelled compounds are discussed. PMID- 3019951 TI - Effect of gamma radiation on the metamorphic stages of Dermestes maculatus DeGeer (Coleoptera: Dermestidae). AB - The effects of gamma-radiation on all stages of the hide beetle Dermestes maculatus, DeGeer were studied. Eggs of D. maculatus were more susceptible to gamma radiation than other stages. Egg radiosensitivity decreased with increasing embryonic development. An absorbed dose of 200 Gy killed the 1st, 6th and 7th instar larvae, but the 4th and 5th instar larvae were more resistant. The developmental period increased in treated larvae. Pupae (24 h) treated with 150 Gy failed to eclose, but eclosion was not affected in older pupae. Adults from female pupae irradiated at 72 h with 150 Gy were infertile, but male pupae required more than 200 Gy for sterilization. The average number of eggs per female decreased with increasing doses when either the male or female of the pair was irradiated as puape or adults. Adult males were sterile after irradiation 300 Gy and adult females treated with the same dose failed to lay eggs. Newly emerged irradiated adults or female adults from irradiated 72-h-old pupae recovered some fertility after treatment with doses as high as 150 Gy. Adult males from irradiated 72-h-old pupae were treated at doses of 50 and 100 Gy showed a higher reproductive capacity at 60 days than at 15 days post-treatment. A dose between 200 and 300 Gy was necessary to provide complete sterility of 24-h-old adults. PMID- 3019952 TI - The synthesis of the neuropeptide Met-enkephalin and two metabolic fragments labelled with 11C in the methionine methyl group. AB - Starting from the corresponding N-benzyloxycarbonyl S-benzyl homocysteine peptide benzyl esters, Met-enkephalin and two metabolites, Gly-Phe-Met and Phe-Met, have been labelled with 11C for application in positron emission tomography in vivo. All labelling experiments were accomplished in high radiochemical yields within 30-40 min from start of the [11C]methyl iodide synthesis. Alkylations with this reagent were performed in liquid ammonia, using sodium to generate the free peptides with their reactive sulphide anions, essentially as previously described for [methyl-11C]methionine. The products were purified by liquid chromatography (LC) to a radiochemical purity of 98% or better. PMID- 3019953 TI - Synthesis and identification of [76As]arsenic trichloride. AB - Radiosynthesis of arsenic trichloride (AsCl3) was achieved using 76As as the radionuclide of choice for preliminary investigations. The synthesis required neutron activation of arsenic trioxide (As2O3) followed by reaction with sulfur monochloride (S2Cl2) under a dry inert atmosphere. The synthesized 76AsCl3 was purified by distillation or partially purified by treatment with dry acetonitrile followed by filtration. Identification was accomplished by thin layer chromatography (TLC) and reversed phase high performance liquid chromatography (HPLC). 76AsCl3 was used for synthesis of a dihydrophenarsazine derivative. PMID- 3019954 TI - Response to RIT 4237 oral rotavirus vaccine in breast-fed and formula-fed infants. AB - Twenty-six full-term newborns (15 males and 11 females) were followed-up from birth to 5 months of age. During the first month of life, all of them were breast fed. Thereafter those infants whose mothers produced enough milk continued breast feeding (n = 16) while the remaining (n = 10) changed to an adapted milk formula supplying approximately 2 g/kg/day of protein and 100 Kcal/kg/day. At 4 months of life, all infants were vaccinated with one oral dose of RIT 4237 rotavirus vaccine of bovine origin. Before and one month after the vaccination, total protein immunoglobulin and IgM type antibodies against rotavirus were evaluated in serum. Growth, weight, length, head circumference and nutritional serum parameters were comparable in both groups of infants as well as the immune response to the RIT 4237 vaccine. Moreover, the "take" of RIT 4237 oral rotavirus vaccine was not lowered by the concomitant administration of human breast-milk which is known to contain rotavirus antibodies. Therefore, breast-feeding is probably not a contraindication for vaccination with RIT 4237, which is most important in developing countries where rotavirus infection is common in young infants and results in acute diarrhoea often leading to death. PMID- 3019955 TI - Food in the aetiology of cancer. AB - During recent years much evidence has accumulated indicating that diet and nutrition may be important in the aetiology of human cancer. This paper discusses some of the components of diet that have been implicated as both causative and protective agents. Total calorie intake and overnutrition have been associated with breast and uterine cancers, high fat intake with cancer of the breast and large bowel and nitrates with gastric cancer. High fibre intakes are suggested to protect against colo-rectal cancer, and vitamin A, selenium and vitamin E have been inversely associated with various cancers. PMID- 3019956 TI - Immunocytochemical localization of 17 beta-hydroxysteroid dehydrogenase in porcine testis. AB - The immunocytochemical localization of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) in porcine testes was examined by applying an indirect immunofluorescence method using an antiporcine testicular 17 beta-HSD antibody. Only the Leydig cells located in the interstitial tissue exhibited a positive immunoreaction for 17 beta-HSD: the germ cells and Sertoli cells located in the seminiferous tubules were entirely negative. These results suggest that, in porcine testis, the biosynthesis of testicular testosterone, the final step of which is the conversion of androstenedione to testosterone, takes place in the Leydig cells. PMID- 3019958 TI - Is adenocarcinoma/large cell carcinoma the most radiocurable type of cancer of the lung? AB - Radiation therapy is widely considered the primary treatment for inoperable "non small" cell carcinoma of the lung. In clinical investigations, distinction has been infrequent among the histopathologic subtypes of non-small cell carcinoma. Studies have shown significant differences between squamous cell carcinoma and adenocarcinoma/large cell carcinoma; adenocarcinoma/large cell carcinoma has a greater propensity for extrathoracic dissemination, especially to the brain, and it is less curable by resection when regional lymph node metastases are present. No differences have been documented between adenocarcinoma and large cell carcinoma. A retrospective study was undertaken to determine the results of definitive radiation therapy by histopathologic subtype of non-small cell carcinoma of the lung. Between July 1977 and April 1983, 134 patients with non small cell carcinoma of the lung underwent definitive radiation therapy with curative intent. All patients had performance status scores of 80 to 100 (Karnofsky), and received minimum total doses within the tumor of 60 Gy in 6 to 7 weeks, five fractions per week. The median period of observation was 63 months. Ninety patients had squamous cell carcinoma; 44 had adenocarcinoma/large cell carcinoma. The two groups of patients were comparable in respect to age and Stage; there were significantly more women with adenocarcinoma/large cell carcinoma (27%) than with squamous cell carcinoma (13%). The median survival for patients with squamous cell carcinoma was 11.5 months; the 2 and 4 year survival rates were 21 and 7%, respectively. The median survival for patients with adenocarcinoma/large cell carcinoma was 18 months; 2 and 4 year survival rates were 38 and 23%, respectively. Comparison of the overall survival experience did not show a significant difference between the two cell types (p = .12 using Gehan's generalized Wilcoxon test). However, comparison of the proportion of patients with adenocarcinoma/large cell carcinoma surviving 18 months (50%) was significantly higher (p = .02) than that with squamous cell carcinoma (30%). A small body of data from the literature also suggests a better long-term prognosis for adenocarcinoma/large cell carcinoma. This observation requires confirmation from large trials with histopathologic review. If it is confirmed, there are important implications for therapeutic strategies in future clinical investigations of inoperable carcinoma of the lung. PMID- 3019957 TI - Ultrahistochemical localization of Na+-K+ ATPase, Ca2+-ATPase and alkaline phosphatase activity in a calcium-transporting epithelium of a crustacean during moulting. AB - Periodical changes in Na+-K+-ATPase, Ca2+-ATPase and non-specific alkaline phosphatase activity were observed using cytochemical techniques in the posterior caeca of the crustacean amphipod, Orchestia cavimana, during the moult cycle. These changes were considered in relation to the calcium transport mechanisms in the posterior caecal epithelium. For both ATPases as well as alkaline phosphatase, the specific reaction products were most intense during the pre exuvial period, i.e. when calcium is slowly transported against a concentration gradient: the localization of Na+-K+-ATPase activity in microvilli and the upper extracellular channels strongly supports the hypothesis that this enzyme is involved in an indirect, sodium-dependent mechanism for the transport of calcium. The detection of Ca2+-ATPase activity in microvilli would seem to indicate that this enzyme plays a role in the direct, active extrusion of Ca2+ at this level. Although the role of alkaline phosphatase in the transport of calcium remains unclear, the histochemical detection of this enzymatic activity throughout the apical part of the caecal epithelium suggests that this enzyme may be involved in calcium secretion. In post-exuvial period, we found only weak specific reaction products, thus indicating a reduced active calcium transport as these ions are rapidly reabsorbed down the concentration gradient. PMID- 3019959 TI - The use of positron emission tomography in pion radiotherapy. AB - The radioactive debris produced by pion radiotherapy can be imaged by the technique of Positron Emission Tomography (PET) as a method of non-invasive in situ verification of the pion treatment. This paper presents the first visualization of the pion stopping distribution within a tumor in a human brain using PET. Together with the tissue functional information provided by the standard PET scans using radiopharmaceuticals, the combination of pion with PET technique can provide a much better form of radiotherapy than the use of conventional radiation in both treatment planning and verification. PMID- 3019960 TI - Drug resistance in human lung cancer cell lines: cross-resistance studies and effects of the calcium transport blocker, verapamil. AB - We have developed multi-drug resistant variants of three human lung cancer cell lines by growth in increasing concentrations of adriamycin (ADM). Each of the lines shows reduced ADM content after acute drug exposure when compared with the corresponding parent line. The resistance to ADM may in each case be partially overcome by the concurrent use of 6.6 microM verapamil (VRP) (a calcium transport blocker). In small cell line NCI-H69 the growth inhibitory effect of VRP alone is greater in the resistant variant than in the parent line. Little or no change in the ADM sensitivity of parent cells is brought about by concentrations of VRP up to 15-20 microM. In the resistant line, a dose-related effect of VRP is seen with little effect below 1.0 microM and a gradual loss of resistance at higher doses. ADM-resistant cells of line NCI-H69 show only a small degree of resistance to two anthracyclines (aclacinomycin A and Ro 31-1215). This can be further reduced when these drugs are combined with verapamil. PMID- 3019961 TI - A novel interaction of diethyldithiocarbamate with the glutathione/glutathione peroxidase system. AB - Diethyldithiocarbamate (DDC) exhibits a variety of pharmacologic activities, including both radioprotective and sensitizing properties. Since the glutathione/glutathione peroxidase system may be a significant factor in determining radiation sensitivity, the potential mechanisms of action of DDC in relation to this system were examined in vitro. The interaction of DDC with reduced glutathione (GSH) was tested using a simple system based on the reduction of cytochrome c. When DDC (0.005 mM) was incubated with GSH (0.5 mM), the reduction of cytochrome c was eightfold greater than that expected from an additive effect of DDC and GSH. GSH could be replaced by oxidized glutathione and glutathione reductase. Cytochrome c reduced by DDC was oxidized by mitochondria. The interaction of DDC with both the hexosemonophosphate shunt pathway and the mitochondrial respiratory chain suggests the possibility of linking these two pathways through DDC. Oxidation of DDC by peroxide and reversal by GSH indicated that the drug can engage in a cyclic reaction with peroxide and GSH. This was confirmed when DDC was used in the assay system for glutathione peroxidase (GSHPx) without GSHPx. DDC at a concentration of 0.25 mM was more active than 0.01 unit of pure GSHPx in eliminating peroxide, and much more active than the other sulfhydryl compounds tested. These studies indicate that DDC can supplement GSHPx activity or substitute for it in detoxifying peroxides, and suggests a unique role in the chemical modification of radiation sensitivity. PMID- 3019962 TI - Radiobiological evaluation of a newly synthesized cysteamine derivative. AB - A new cysteamine-based compound, I109 (N-glycylglycyl-S acetylcysteamine trifluoroacetate) was tested on both normal tissues and tumors to evaluate its radioprotective potential. I109, which is three times less toxic than WR-2721, was injected at a dose approximately equal to half the LD50(30 days). The Protection Factor (PF = gamma ray dose ratio) after whole body irradiation was 1.3-1.4 for intestinal death and 1.4-1.5 for hemopoietic death when intervals of 40 or 20 min elapsed between injection and the end of irradiation. A crypt cell assay was done for both I109 and WR-2721; PFs were 1.1 and 1.4, respectively. I109 was then tested on five solid tumors. For each cell line (4 human, 1 murine) one dose of radiation was delivered. Surviving fraction ratios with and without drug ranged between 2.4 and 14.1 when an interval of 20 min elapsed between injection and the end of irradiation. The degree of radioprotection proved time dependent for EMT6 and HRT18; radioprotection afforded by WR-2721 on these tumors is either similar or greater than radioprotection afforded by I109 depending on the time interval between injection and irradiation. PMID- 3019963 TI - Radioprotection in rat spinal cord with WR-2721 following cerebral lateral intraventricular injection. AB - The capacity of WR-2721 to provide radioprotection in central nervous system (CNS) tissue was assessed in F-344 rats irradiated with Cs-137 to the cervical spinal cord 45 min following injection of either 0.33 mg (0.60 X LD50) of WR-2721 or carrier solution in the right lateral cerebral ventricle. The radiation dose groups were 20, 26, 32, or 38 Gy; the dose rate was 1.48 Gy/min. Following irradiation, the time in weeks to forelimb and hindlimb paralysis was measured and statistical significance was assessed by means of the log rank sum test. The median times in weeks to forelimb paralysis in control vs. WR-2721-treated rats were, respectively, 20 vs. 22 at 38 Gy, 19 vs. 31 at 32 Gy (p less than 0.01), 23 vs. 28 at 26 Gy (p less than 0.01), and 49 vs. 60 at 20 Gy (p less than 0.01). The median times to hindlimb paralysis in control vs. WR-2721-treated rats were respectively, 20 vs. 29 at 38 Gy (p less than 0.001), 20 vs. 35 at 32 Gy (p less than 0.01), 23 vs. 34 at 26 Gy (p less than 0.001), and 58 vs. 65 at 20 Gy (p less than 0.01). From these results, we calculated the DMF for forelimb paralysis to be 1.3 and for hindlimb paralysis, 1.6. Histological studies from selected spinal cords from symptomatic killed rats showed petechial hemorrhages, rare microvascular thrombi, and scattered microinfarcts in both gray and white matter. In the white matter columns, there were scattered microfoci of demyelination. The histological findings did not differ between the control and WR-2721-treated groups, but were worse in the higher dose groups. These data indicate that WR 2721 has the capacity to be radioprotective in CNS tissues, when it is administered by a route that bypasses the blood-brain barrier. PMID- 3019964 TI - Radioprotection against cataract formation by WR-77913 in gamma-irradiated rats. AB - Protection by WR-77913 against radiation-induced cataract formation in rats was observed following intraperitoneal (i.p.) administration of drug (1160 mg/kg) 15 30 min before exposure to 15.3 Gy of Cs-137 whole head irradiation. Control groups included irradiated, non-protected animals, and sham-irradiated aging controls. Protection was documented photographically and by analysis of eye lens constituents. All non-protected irradiated animals developed dense cataracts throughout the lens between 90-120 days post-irradiation, while WR-77913 protected animals developed minimal lens opacification through 200 days post irradiation. No opacification in aging controls was seen. Lens protein analysis by Lowry assay and size exclusion HPLC showed radioprotected and aging control animals were similar in protein content, distribution of total and soluble protein, and degree of lens hydration. This contrasted significantly with cataractous lenses of non-protected animals. In cataractous lenses, the soluble protein concentration in the 25-43 K dalton range was approximately 10% of that found in radioprotected or aging control lenses. Hydration was substantially higher in cataractous lens. These results indicate that WR-77913 protects against lens opacification, protein insolubilization, and hydration in lenses of irradiated animals. Biodistribution studies with [S-35]-WR-77913 showed ocular uptake of drug within 15 minutes after i.p. injection, which remained relatively constant through 60 min. The relative order of drug concentration for individual eye components was: globe greater than total eye approximately equal to humor greater than lens. Although the mechanism of radioprotection observed remains to be elucidated, WR-77913 clearly prevents radiation-induced cataracts in rats. The potentially significant clinical use for this radioprotective compound is being investigated further. PMID- 3019966 TI - Modification of WR-2721 toxicity and radioprotection by an inhibitor of alkaline phosphatase. AB - We used levamisole, an inhibitor of alkaline phosphatase, to study the role of that enzyme in mediating the metabolic activation, toxicity, and radioprotection of WR-2721 in intact mice. We found the toxicity of WR-2721 was slightly decreased by prior subcutaneous (SQ) injection of 40 mg/kg of levamisole. In studying the effect of levamisole on WR-2721 radioprotection, we found that intraperitoneal (i.p.) injection of levamisole had little or no effect on radioprotection of the gastrointestinal and the hematopoietic systems. Even this small reduction of protection was due in part to the toxicity of levamisole as demonstrated when levamisole was injected following, rather than before, WR-2721 radiation treatment. To determine whether levamisole inhibited the activation (i.e., dephosphorylation) of WR-2721 to WR-1065, we assayed WR-1065 in the jejunum using an HPLC electrochemical assay. SQ injection of 75 mg/kg levamisole 10 min prior to WR-2721 reduced the WR-1065 observed 10 min after WR-2721 administration by 37%. In conclusion, levamisole appears to be too toxic and non specific to be useful in studying and regulating the metabolism, toxicity and radioprotection of WR-2721. PMID- 3019965 TI - Comparative biodistribution and radioprotection studies with three radioprotective drugs in mouse tumors. AB - The organ level biodistribution and tumor radioprotective properties of three drugs have been compared: WR-2721 (NSC 296961), WR-3689 (NSC 327729), and WR 77913 (NSC 318809). The three drugs have similar distribution patterns in normal mouse tissues. At 30 minutes after intraperitoneal injection, highest levels of 35S from radiolabeled protector are found in kidney and submandibular salivary gland, with lowest levels in brain and moderately low values in tumor and skin. Three of four tumors examined take up less WR-3689 than the other two protectors. For the three protectors, the dose modifying factors for the RIF-1 tumor irradiated in vivo and assayed in vitro are 1.5-1.7, but do not vary as predicted by differential uptake of drug into this neoplasm. In RIF-1, WR-3689 is taken up most avidly, but the three drugs tend to be equally protective. PMID- 3019967 TI - A method for the combined measurement of ethiofos and WR-1065 in plasma: application to pharmacokinetic experiments with ethiofos and its metabolites. AB - An analytical method for the combined measurement of ethiofos (WR-2721) and a major metabolite (WR-1065) in plasma is described. Plasma samples were subjected to conditions which quantitatively converted both ethiofos and bound WR-1065 to free WR-1065 which was subsequently separated by HPLC and detected electrochemically using established procedures. Although bound WR-1065 in plasma is thought to exist mainly in the form of mixed disulfides, the symmetrical disulfide, WR-33278, also was quantitatively converted to the free thiol form. Standard curves were linear over the range 0.10 to 25 micrograms/mL (0.75 to 186 mumol/L). Mean precision over the range was 5.4% (coefficient of variation, CV) and recoveries of various mixtures of ethiofos, WR-1065 and WR-33278 averaged 102% (CV = 6.6%). This analytical procedure and others specific for ethiofos, free WR-1065 and WR-33278 were applied to dosing experiments in which the parent drug and its major metabolites were variously administered to beagle dogs and rhesus monkeys. Following i.v. administration of ethiofos (120-150 mg per kg body weight) to monkeys, plasma concentrations of unchanged drug ranged from 477 micrograms/mL (2.23 mM) down to the minimum detectable limit of the analytical procedure (0.05 micrograms/mL, 0.23 microM) 2-3 hours postinfusion. Clearances averaged 43.5 +/- 13.4 (SD) mL min-1 kg-1 and half-lives observed in the 20-60 minute postinfusion period were 8-15 min. PMID- 3019968 TI - Human pharmacokinetics of WR-2721. AB - The pharmacokinetic properties of WR-2721 were investigated in 13 cancer patients given a 150 mg/M2 intravenous bolus dose of the drug. An average plasma clearance value of 2.17 L/min was obtained. Very little of the drug or the two metabolites, WR-1065 and WR-33278, were excreted in urine obtained after the blood collection schedule. Plasma concentrations of WR-2721 decreased by 94% within 6 minutes of drug administration. The mean value of 6.44 L obtained for the steady-state volume of distribution indicates that the extravascular space occupied by the drug is small. These observations suggest that in human cancer patients, WR-2721 is rapidly taken up by tissues and converted to metabolites. PMID- 3019969 TI - Phase I/II trials of WR-2721 and cis-platinum. AB - Renal dysfunction is a well-known dose-limiting toxicity of cis-platinum. Previous studies demonstrated a 32% incidence of nephrotoxicity following a 100 mg/M2 single dose of cis-platinum with mannitol diuresis. WR-2721 is an aminothiol which in the animal model improves renal tolerance to cis-platinum by factors of 1.3 to 1.7. Phase I trials were initiated to establish the toxicity when WR-2721 was given prior to escalating doses of cis-platinum. Fifty-two patients received 161 courses of WR-2721 prior to cis-platinum (60-150 mg/M2) with mannitol and hydration. Nephrotoxicity, measured by twice weekly serum creatinines and monthly creatinine clearances, occurred in only 10/97 (10%) courses with 120 mg/M2 of cis-platinum. No patient experienced a creatinine elevation after 135 mg/M2 of cis-platinum. With 150 mg/M2 of cis-platinum, 6/17 (35%) courses were associated with transient nephrotoxicity. Five patients who developed transient creatinine elevations following 150 gm/M2 of cis-platinum were subsequently retreated with WR-2721 and cis-platinum (100 mg/M2) without nephrotoxicity. Bone marrow suppression was mild and infrequent. Mild to moderate peripheral neuropathies were noted in seven patients following cumulative cis platinum doses ranging from 460-1160 mg/M2. Objective partial responses were observed in 26/45 (58%) patients with measurable or evaluable disease. Thus, there is no evidence that WR-2721 protects against the antitumor efficacy of cis platinum in man. Compared to retrospective series, our data suggest that WR-2721 may provide some protection against platinum-induced nephrotoxicity and neurotoxicity. Controlled trials will be required to define the potential clinical benefit of WR-2721 and cis-platinum. PMID- 3019970 TI - Toxicity and biodistribution of the radioprotectors, WR2721, WR77913, and WR3689, in the CNS following intraventricular or intracisternal administration. AB - The radioprotective capacity of the phosphorothioate compounds, WR2721, WR77913, and WR3689, in the CNS is being evaluated following injection of the drugs into the lateral cerebral ventricle or the cisterna magna of F-344 rats. This approach circumvents the blood-brain barrier and permits an assessment of the CNS toxicity and regional distribution of these compounds. Following intraventricular injection in 150-200 gm female rats, the LD50 doses for WR2721, WR77913, and WR3689 were respectively 0.60 +/- 0.07 mg (S.E.), 2.36 +/- 0.13 mg, and 3.56 +/- 0.26 mg. Following intracisternal injection the LD50 doses were 0.71 +/- 0.18 mg, 4.12 +/- 1.09 mg and 3.03 +/- 0.68 mg, respectively. WR 2721 produced lethargy, unsteady gait, and dishevelment but these signs all resolved completely within 1 3 days in survivors. In addition to these signs, WR77913 and WR3689 produced severe convulsions. At high doses, following intraventricular administration, all three drugs were associated with cerebral and diencephalic periventricular necrosis and ipsilateral necrosis of the lateral hippocampus. Biodistribution studies were performed with [S-35]-labeled derivatives of the drugs and tissue sampling. The three drugs demonstrated similar patterns. Forty-five minutes following either the intraventricular or intracisternal route of drug delivery the highest drug concentrations were in the brainstem, cerebellum, and cervical cord. Additional studies with autoradiography revealed that intraventricular injection was associated with high drug uptake in the cerebral white matter, the periventricular diencephalon, and the periaqueductal mesencephalon. The biodistribution and toxicity data together suggest that the drugs can be ranked, WR3689 greater than WR77913 greater than WR2721, according to the level of drug thiol that can be achieved in the CNS tissues with intraventricular or intracisternal injection. Tissue levels achievable with WR2721 following these two routes of administration are as high as levels others have reported as radioprotective in rodent skin and gut. PMID- 3019971 TI - Final comments on amended insulin-glucose ratio. PMID- 3019972 TI - Evaluation of canine hyperadrenocorticism, using computed tomography. AB - Abdominal computed tomography was performed in 9 dogs with hyperadrenocorticism and in 2 healthy dogs. Both adrenal glands were identified in all dogs. Computed tomography allowed accurate identification of the sites of adrenal gland dysfunction, when interpreted in combination with a biochemical diagnosis of canine hyperadrenocorticism. This accuracy permitted the retroperitoneal approach to be used for all adrenalectomies. Use of contrast medium (although not essential) was helpful in the computed tomographic identification of blood vessels, kidneys, and other abdominal organs. PMID- 3019973 TI - Pituitary-dependent hyperadrenocorticism in a cat. AB - Pituitary-dependent hyperadrenocorticism was diagnosed in a 9-year-old, male castrated cat that had polyuria, polyphagia, pendulous abdomen, truncal hair loss, congestive heart failure, and insulin-resistant diabetes mellitus. Results of pituitary-adrenal function testing revealed inadequate serum cortisol suppression following dexamethasone administration, exaggerated serum cortisol responses after exogenous ACTH stimulation, and high plasma ACTH concentrations. The pathologic findings of bilateral adrenocortical hyperplasia and a pituitary adenoma that immunostained well for ACTH-related peptides confirmed pituitary dependent hyperadrenocorticism. PMID- 3019974 TI - Dietary fiber in the Japanese diet as investigated in connection with colon cancer risk. AB - The low risk of colon cancer among the Japanese suggests a high intake of dietary fiber. Composite diets for 1959, 1970, and 1979 were prepared using food consumption data from the National Nutrition Survey in Japan and analyzed for non starch polysaccharides (NSP) at the Dunn Clinical Nutrition Centre in Cambridge. The results showed that average intake of NSP by Japanese in the above years did not exceed 13 g per day, which is as low as the corresponding intake by the Scandinavians and the British, whose risk of colon cancer is known to be high. PMID- 3019975 TI - Chromosome instability in cultured skin fibroblasts from patients with familial polyposis coli and Peutz-Jeghers' syndrome. AB - Detailed chromosome studies on cultured skin fibroblasts from patients with familial polyposis coli (FPC) and Peutz-Jeghers' syndrome (PJS) showed an increased incidence of chromosome aberrations at early passage levels, as compared with the age-comparable controls. The occurrence of certain clones of karyotypically abnormal cells was recognized in both groups of patients. There was no clear evidence for the emergence of such clones in the controls within the range of the number of cells observed. Our observations may suggest that the increased chromosome instability in the FPC and PJS patients is due to a genetic predisposition rather than an age-related expression of a normal phenomenon. The present studies, however, have not revealed any specific break or exchange point, although several sites were involved in three or more rearrangements. PMID- 3019976 TI - Cisplatin and vindesine combination chemotherapy for non-small cell lung cancer: a randomized trial comparing two dosages of cisplatin. AB - Forty-five patients with advanced non-small cell lung cancer were randomly allocated to receive vindesine (3 mg/m2 every week) plus either high-dose cisplatin (120 mg/m2 every 4 weeks) or low-dose cisplatin (80 mg/m2 every 3 weeks). All patients were previously untreated. The response rate for the high dose regimen of cisplatin was 39% (9/23) and that for the low-dose regimen of cisplatin was 33% (7/21); the difference was not statistically significant. Only one patient treated with high-dose cisplatin achieved complete response, lasting 6.5 months. The median duration of response was 5.6 months (range, 2.7-7.7) in the high-dose cisplatin group and 6.8 months (range, 1.9-8.9) in the low-dose cisplatin group. The median survival times for the 23 patients treated with the high-dose regimen of cisplatin and for the 21 patients treated with the low-dose regimen of cisplatin were 9.0 and 10.8 months, respectively. Significantly more azotemia occurred in the high-dose cisplatin group than in the low-dose cisplatin group (P less than 0.05). Combination chemotherapy with cisplatin and vindesine showed significant antitumor activity in patients with non-small cell lung cancer. However, the high-dose regimen of cisplatin did not result in a significantly better response rate or survival advantage, and was associated with greater toxicity. PMID- 3019977 TI - A case report of synchronous small cell lung cancer and gastric cancer successfully treated with carboplatin. AB - A 53-year-old man complained of anorexia and abdominal distention of one month's duration. The chest X-ray demonstrated a mass in the left lung with hilar and mediastinal adenopathy and a lytic lesion in the right fourth rib. A transbronchoscopic biopsy of the mass revealed oat cell carcinoma (WHO classification). The endoscopic evaluation also revealed a gastric lesion (IIc type). Biopsy of this lesion indicated signet ring cell gastric cancer. An abdominal CT scan demonstrated multiple liver metastases. Based on these findings, the patient was diagnosed as having synchronous lung and gastric primaries, with liver and bone metastasis from lung cancer. Carboplatin (CBDCA) was administered by intravenous drip infusion of 450 mg/m2. After a second treatment with CBDCA about 3 weeks later, the patient achieved a partial response at the primary site of lung cancer as well as at the liver and bone metastases. In addition, repeat endoscopy of the stomach demonstrated a complete regression. A biopsy specimen taken by gastroscopy was negative for cancer cells. Subsequent chemotherapy for small cell lung cancer was administered with cyclophosphamide, adriamycin, and vincristine, and to date there is no evidence of recurrence. Further studies on CBDCA treatment of small cell lung cancer and gastric cancer are needed to establish the efficacy of this drug against these two histologically different cancers. PMID- 3019978 TI - In vitro evaluation of SF-2103A, a novel carbapenem antibiotic, as a beta lactamase inhibitor. AB - SF-2103A, a new carbapenem antibiotic, exhibited a broad antibacterial spectrum and a potent inhibitory activity against a wide range of beta-lactamases, in particular, against cephalosporinases, with lower I50 values than those displayed by sulbactam and clavulanic acid. Using a fixed combination and checkerboard titration, in vitro synergy against the majority of the beta-lactamase-producing strains tested was demonstrated between SF-2103A and various beta-lactam antibiotics, especially cefotaxime, ceftizoxime, and cefoperazone. The synergistic effect of SF-2103A was more pronounced than that of sulbactam. The in vitro synergy was also confirmed by bactericidal and bacteriolytic activities and morphological effects. PMID- 3019979 TI - Effect of sodium bicarbonate and sodium bentonite on digestion, solid and liquid flow, and ruminal fermentation characteristics of forage sorghum silage-based diets fed to steers. AB - Six ruminally cannulated steers, five Holsteins and one Hereford (250 to 295 kg), were fed 84% forage sorghum silage plus 16% supplement or 50% forage sorghum silage plus concentrate and supplement diets containing either no addition (controls), 1% sodium bicarbonate (NaHCO3) or 2% sodium bentonite in a 2 X 3 factorial arrangement of treatments in a 6 X 6 Latin-square experiment with 3-wk periods. Sodium bicarbonate increased dry matter (DM) intake when concentrate was included, but neither compound affected intake of the 84% silage diet. Bentonite lowered DM, neutral detergent fiber (NDF), and acid detergent fiber (ADF) digestibilities, but NDF disappearance from nylon bags was unchanged. Ruminal pH, osmolality and L(+) and D(-) lactate were not affected by treatment. Both NaHCO3 and bentonite tended to lower ruminal NH3-N concentrations. Bentonite lowered the molar proportion of isobutyrate in ruminal fluid relative to controls, but proportions of other volatile fatty acids (VFA) and total VFA concentrations were unchanged. Neither NaHCO3 nor bentonite affected ruminal liquid or solid volumes, dilution rate constants or ruminal outflow rates. Markers overestimated volumes, but correction with measured volumes did not change interpretation of treatment effects. It was concluded that control diets had sufficiently high baseline values of pH, dilution rate and acetate proportion to preclude changes induced by either compound, especially at 1 or 2% of DM intake. An effect on palatibility through neutralization of silage acids may have been responsible for the intake response to NaHCO3. PMID- 3019980 TI - Identification of individual aminoglycoside-inactivating enzymes in a mixture by HPLC determination of reaction products. AB - A method is described for the detection and identification of two aminoglycoside acetylating (AAC(2') or AAC(3) or AAC(6')) or -phosphorylating (APH(2'') or APH(3')) enzymes when these are present in the same crude enzyme extract. Crude enzyme extracts were prepared from reference strains of bacteria producing AAC(2'), AAC(3), AAC(6'), APH(2'') or APH(3') enzymes and mixed to give the following paired enzyme combinations: AAC(2') + AAC(3), AAC(2') + AAC(6'), AAC(3) + AAC(6') and APH(2") + APH(3'). These enzyme mixtures were examined by the radioenzymatic profile method for the identification of aminoglycoside-modifying enzymes and their reaction products with butirosin, lividomycin and kanamycin (AAC) or lividomycin and kanamycin (APH) characterized by high performance liquid chromatography (HPLC). Only two of the eight enzymes present were identified by the radioenzymatic method, but all eight were correctly identified by the HPLC method. PMID- 3019981 TI - In-vitro effect of itraconazole, ketoconazole and amphotericin B on the phagocytic and candidacidal function of human neutrophils. AB - Neither itraconazole nor ketoconazole had significant effects on the phagocytosis or killing of Candida albicans blastospores by human neutrophil polymorphonuclear (PMN) leucocytes, whether following simultaneous addition of drug with the blastospores, prior treatment of the PMN cells with drug, or prior treatment of the blastospores with drug. Following simultaneous addition, amphotericin B caused significant impairment of phagocytosis in tests with PMN cells from one of two donors, but had no effect on subsequent killing of ingested blastospores. Prior treatment of PMN cells or blastospores with amphotericin B had no effect on phagocytosis or killing. In tests in which blastospores of an azole-sensitive and an azole-resistant strain of C. albicans were added to PMN cells, fewer than 25% of ingested blastospores of either strain had initiated germ tube formation after 6 h. Addition of ketoconazole had no effect on the proportion of ingested blastospores of either strain which germinated. Ketoconazole had no effect on germ tube elongation of ingested blastospores of the azole-resistant strain, but caused marked inhibition of germ tube elongation of ingested blastospores of the azole-sensitive strain. After 24 h, the azole-resistant strain had produced abundant mycelium and few phagocytic cells remained intact. With the azole sensitive strain, PMN cells were still evident at 24 h, with ingested germ-tube forming blastospores, most of which were dead. PMID- 3019982 TI - Membrane-disorganizing property of polymyxin B nonapeptide. AB - The possibility of improving the antibacterial activities of drugs normally excluded by Gram-negative bacteria with polymyxin B nonapeptide (PMBN) has been explored. In vitro, PMBN rendered clindamycin, erythromycin, novobiocin, rifampicin and vancomycin very active against a number of Gram-negative enteric bacteria. The drug also sensitized the previously resistant bacterial strains to human, mouse or guinea pig serum. However, parenterally administered PMBN failed to influence bacterial growth in chambers implanted into mice and guinea pigs. It was also ineffective in experimental septicaemia at a dose of up to 200 mg/kg or when combined with antibiotics with which it interacted synergistically in vitro. PMID- 3019983 TI - Aminoglycoside-phosphotransferases APH(3')-IV and APH(3") synthesized by a strain of Campylobacter coli. AB - Campylobacter coli strain 981 of animal origin was resistant to erythromycin, tetracycline, streptomycin, kanamycin, ribostamycin, neomycin, paromomycin, lividomycin, and butirosin. Resistance to aminoglycosides of strain 981 was mediated by phosphotransferases APH (3') type-IV and APH (3"). C. coli 981 harboured three plasmids of 24, 34, and 40 Megadaltons respectively. None of these plasmids were transferable to Escherichia coli K-12 by conjugation. PMID- 3019985 TI - Effects of cefotaxime and cefodizime on human granulocyte functions in vitro. AB - In vitro, cefotaxime and cefodizime enhanced significantly the bactericidal activity of human neutrophils against Staphylococcus aureus P 209 A, but not phagocytosis. The increase was about 150% for cefotaxime and 400% for cefodizime at concentrations as low as 1 mg/l. Furthermore, by two different techniques (NBT and cytochrome C reduction tests) cefotaxime but not cefodizime significantly enhanced superoxide anion production by zymosan-stimulated neutrophils. Other neutrophil functions (chemotaxis and myeloperoxidase-mediated iodination of proteins) were not significantly altered by either antibiotic, even at concentrations as high as 1000 mg/l. PMID- 3019984 TI - Comparative activities of the beta-lactamase inhibitors YTR 830, clavulanate and sulbactam combined with extended-spectrum penicillins against ticarcillin resistant Enterobacteriaceae and pseudomonads. AB - The in-vitro synergistic activity of YTR 830, a new beta-lactamase inhibitor, combined with four extended-spectrum penicillins (ticarcillin, piperacillin, mezlocillin and apalcillin) against ticarcillin-resistant clinical isolates of Gram-negative enteric bacilli was compared with that of clavulanate and sulbactam. Synergy testing was performed with fixed concentrations of beta lactamase inhibitors (8 mg/l) combined with doubling dilutions of beta-lactams in microdilution trays. Synergy was defined as a four-fold or greater decrease of beta-lactam MIC in the combination compared with the beta-lactam alone. For 79 ticarcillin-resistant Enterobacteriaceae, ticarcillin-YTR 830 and ticarcillin clavulanate were synergistic against 90% of strains; for ticarcillin-sulbactam, 70% showed synergy. The synergistic activity of all three inhibitors was similar against strains resistant only to ticarcillin; for strains resistant to all four extended-spectrum penicillins, the activity of ticarcillin with YTR 830 and clavulanate was similar (synergy against 79% of strains) and superior to ticarcillin-sulbactam (synergy against 39% of strains). YTR 830 was more active than clavulanate against Serratia, Citrobacter, Proteus and Providencia spp. Piperacillin, mezlocillin and apalcillin susceptible strains, with MICs of 8-16 mg/l, showed synergy with inhibitors against 37-87% of strains. Amongst pseudomonads, no synergy was demonstrated against Pseudomonas aeruginosa; ticarcillin produced synergy with the inhibitors against Ps. maltophilia, while piperacillin-YTR 830 and apalcillin-YTR 830 were synergistic against Ps. cepacia. YTR 830 appears to have comparable in-vitro activity to that of clavulanate, and further development of this compound is warranted. PMID- 3019986 TI - Treatment of cryptococcal meningitis in mice with fluconazole. AB - Fluconazole is a recently developed triazole with activity in vitro against Cryptococcus neoformans, water solubility, and excellent oral absorption. We compared fluconazole in murine cryptococcosis with ketoconazole and amphotericin B. Fluconazole was highly effective in suppressing cryptococcosis in mice challenged by the intravenous and intranasal routes, and was comparable with the other two drugs in its protective capacity. However, fluconazole was superior to ketoconazole and comparable with amphotericin B after intracerebral challenge. Fluconazole may warrant clinical evaluation in cryptococcosis. PMID- 3019987 TI - Synergy of amoxycillin combined with clavulanate and YTR 830 in experimental infections in mice. AB - YTR 830, a new beta-lactamase inhibitor, is synergistic with amoxycillin in vitro against a number of beta-lactamase-producing organisms. The combination of amoxycillin-YTR 830 was compared to amoxycillin-clavulanate in the treatment of experimental Staphylococcus aureus, Citrobacter freundii and Proteus mirabilis infections in mice. Both combinations were synergistic with amoxycillin against all three test organisms. The amoxycillin-clavulanate combination was superior against S. aureus and C. freundii while amoxycillin-YTR 830 was more effective against P. mirabilis. The difference in efficacy between the two drug combinations appears to relate to the degree of protection afforded the animals by the beta-lactamase inhibitor alone. YTR 830 is a promising new agent and should undergo further investigation. PMID- 3019988 TI - Effects of alveolar pressure on lung angiotensin-converting enzyme function in vivo. AB - Angiotensin-converting enzyme lines the luminal surface of pulmonary capillary endothelial cells. The metabolism of its synthetic substrate, 3H-benzoyl-L phenylalanyl-L-alanyl-L-proline ([3H]BPAP) has been used as an indicator of pulmonary microvascular function. Because the flow-volume status of the pulmonary capillaries is dependent on intra-alveolar pressure, we have studied the effects of airway pressure on endothelial plasmalemmal angiotensin-converting enzyme function in rabbit lungs in vivo. Static inflation of the lungs to a pressure of 0 or 5 Torr did not change percent transpulmonary metabolism and Amax/Km ratio (defined as E X Kcat/Km and thus, under normal conditions, an indirect measure of perfused endothelial luminal surface area) compared with control measurements during conventional mechanical ventilation. When the inflation pressure was increased to 10 Torr, percent metabolism of [3H]BPAP remained unaltered but Amax/Km decreased to 60% of the control value. This decrease was in close relation to the decrease in pulmonary blood flow. Addition of 5 cmH2O positive end-expiratory pressure (PEEP) to the mechanical ventilation also decreased Amax/Km values and pulmonary blood flow but did not influence percent metabolism of [3H]BPAP. These results suggest that the detected alterations in apparent enzyme kinetics were more likely due to hemodynamic changes than to alterations in angiotensin-converting enzyme function. Thus high static alveolar pressures as well as PEEP probably reduced the fraction of perfused microvessels as reflected in changes in Amax/Km ratios. This information should prove useful in interpreting the response of pulmonary endothelial enzymes to injury. PMID- 3019989 TI - Fatigue of isolated rat diaphragm: role of impaired neuromuscular transmission. AB - We compared the contributions of impaired neuromuscular transmission (transmission fatigue) and impaired muscle contractility (contractile fatigue) to fatigue of the isolated rat diaphragm. To make this comparison, we measured the differences in active tension elicited by direct muscle stimulation and by indirect (phrenic nerve) stimulation before and after fatigue induced by indirect supramaximal stimulation at varying frequencies and durations. Transmission fatigue was observed after all experimental protocols. Although significant contractile fatigue was not demonstrated after brief periods of low-frequency stimulation (6 min, 15 Hz, 25% duty cycle), it was present after longer or higher frequency stimulation. We repeated the direct stimulation in the presence of neuromuscular blockade with 6 microM d-tubocurarine to demonstrate that a reduced response to stimulation of intramuscular branches of the phrenic nerve during direct stimulation was not responsible for the apparent contractile fatigue. Since we found significant decreases in the response to direct stimulation even after neuromuscular blockade, we could verify the presence of contractile fatigue. We conclude that both contractile and transmission fatigue can occur in the isolated rat diaphragm and that transmission fatigue is a much more important factor after brief periods of fatiguing contractions. PMID- 3019990 TI - Diffusion-related differences in elimination of inert gases from the lung. AB - Partial pressures of intravenously infused acetylene, Freon 22, and isoflurane (gases with similar solubilities in blood but differing molecular weights) were compared in arterial and mixed venous blood and mixed expired gas of 13 anesthetized mongrel dogs to determine whether gas molecular weight influenced gas exchange. Analysis of covariance was used to account for the variables of ventilation-perfusion ratio, partition coefficient, and experimental run before individual gas effects were sought. A gas effect difference was observed such that the arterial fractional retention of isoflurane (mol wt 184.5) would be 12% higher than that of acetylene (mol wt 26) if the two gases had identical partition coefficients. This effect was neither significantly increased by positive end-expiratory pressure nor decreased by high-frequency oscillatory ventilation. To test whether the individual gas effect was greater with gases with disparate erythrocyte and plasma partition coefficients, the exchange of ethyl iodide (erythrocyte-to-plasma solubility ratio 8.1) and diethyl ether (solubility ratio 0.95) was compared in five dogs. A larger difference between the elimination of the two gases was observed than predicted from the differences in molecular weight. The observed individual gas effect appears to be diffusion related, influenced both by the molecular weight of a gas and its erythrocyte plasma partition coefficient ratio. PMID- 3019991 TI - Increased yield of mouse mammary tumor virus (MMTV) by cultivation of monolayer derived mammary tumor cells in suspension. AB - The MJY-alpha epithelial-like mammary tumor cell line was adapted for cultivation in suspension using a shaker culture technique. Replication of suspension (MJY beta) cells was more sensitive than monolayer cells to decreases in the concentration of serum in the medium. Comparison of amino acid incorporation and lactate production rates revealed additional differences between monolayer and suspension cultures. In addition, growth in suspension resulted in 10- to 400 fold increases in mouse mammary tumor virus (MMTV) production by the mammary tumor cells. Increases in MMTV yield were detected within 48 h of culture initiation and MMTV production remained elevated throughout 20 cell passages in suspension. Exposure of MJY-beta cells to 14 microM hydrocortisone further increased MMTV yield two- to five-fold. The MJY-beta suspension cultures demonstrated that these epithelial-like cells do not require attachment to a solid substrate for replication or for MMTV production. Loss of structural polarization associated with growth as a monolayer resulted in stimulation of MMTV production greater than and independent of steroid exposure. PMID- 3019992 TI - Effect of high fibre intake on serum vitamin A levels. PMID- 3019993 TI - Prolonged sodium stibogluconate therapy in Indian kala-azar. PMID- 3019994 TI - Detection of homology to the beta bacteriophage integration site in a wide variety of Corynebacterium spp. AB - In toxigenic conversion of Corynebacterium diphtheriae C7, beta bacteriophage DNA integrates into either of two chromosomal attachment sites, attB1 or attB2. These attB sites share a 96-base-pair sequence with the attP sites of beta-related phages. The distribution of attB-related sites in other species of Corynebacterium was assessed by hybridization with a DNA probe containing both attB sites of the C7 strain and a second DNA probe containing the attP site of a beta-related phage. All but one of the 15 C. diphtheriae strains tested, regardless of origin or colonial type, contained at least two BamHI fragments that hybridized strongly to both of these probes under conditions of high stringency. Strains of C. ulcerans and C. pseudotuberculosis, species in which conversion to toxinogeny has also been demonstrated, also had one or two hybridizing BamHI fragments. The functionality of these sites as integration sites was demonstrated by isolating lysogens of all three species following single infection with one or more beta-related phages. As predicted, following lysogenization one of the DNA fragments that had exhibited homology with the attB1-attB2 probe was replaced by two hybridizing fragments. Other species of Corynebacterium, including pathogens and nonpathogens from animals, plant pathogens, and soil isolates also carried at least one BamHI fragment that hybridized with the attB1-attB2 and attP probes. The data indicate that sequences homologous to the beta phage integration sites in C. diphtheriae have been conserved in members of the genus Corynebacterium. PMID- 3019995 TI - Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204. AB - A Pseudomonas putida strain designated RE204, able to utilize isopropylbenzene as the sole carbon and energy source, was isolated. Tn5 transposon mutagenesis by means of the suicide transposon donor plasmid pLG221 yielded mutant derivatives defective in isopropylbenzene metabolism. These were characterized by the identification of the products which they accumulated when grown in the presence of isopropylbenzene and by the assay of enzyme activities in cell extracts. Based on the results obtained, the following metabolic pathway is proposed: isopropylbenzene----2,3-dihydro -2,3-dihydroxyisopropylbenzene----3 isopropylcatechol----2 -hydroxy-6-oxo-7-methylocta-2,4-dienoate----isobutyrate + 2-oxopent-4-enoate----amphibolic intermediates. Plasmid DNA was isolated from strain RE204 and mutant derivatives and characterized by restriction enzyme cleavage analysis. Isopropylbenzene-negative isolates carried a Tn5 insert within a 15-kilobase region of a 105-kilobase plasmid designated pRE4. DNA fragments of pRE4 carrying genes encoding isopropylbenzene catabolic enzymes were cloned in Escherichia coli with various plasmid vectors; clones were identified by (i) selection for Tn5-encoded kanamycin resistance in the case of Tn5 mutant plasmids, (ii) screening for isopropylbenzene dioxygenase-catalyzed oxidation of indole to indigo, and (iii) use of a Tn5-carrying restriction fragment, derived from a pRE4::Tn5 mutant plasmid, as a probe for clones carrying wild-type restriction fragments. These clones were subsequently used to generate a transposon insertion and restriction enzyme cleavage map of the isopropylbenzene metabolic region of pRE4. PMID- 3019996 TI - Control of sensitivity to inactivation by H2O2 and broad-spectrum near-UV radiation by the Escherichia coli katF locus. AB - Mutations in the Escherichia coli katF gene (hydroperoxidase II) result in sensitivity to inactivation by H2O2 and broad-spectrum near-UV (NUV; 300 to 400 nm) radiation. Another mutation, nur, originally described as conferring sensitivity to inactivation by broad-spectrum and monochromatic NUV, also confers sensitivity to inactivation by H2O2. Genetic analysis via transduction suggests that the nur mutation allele of the katF locus. As previously reported for broad spectrum and monochromatic NUV wavelengths, the sensitivity of a particular strain to H2O2 inactivation is also independent of the recA and uvrA alleles. Extracts of nur and katF strains lack catalase (hydroperoxidase II) as revealed by polyacrylamide gels stained for such activity, which is consistent with the genetic results. PMID- 3019997 TI - Metastable regulation of type 1 piliation in Escherichia coli and isolation and characterization of a phenotypically stable mutant. AB - Type 1 piliation in Escherichia coli exhibits phase variation due to the inversion of a small, ca. 300-base-pair, element that regulates pilA (fimA), the gene that encodes the structural subunit of pili (Abraham et al., Proc. Natl. Acad. Sci. USA 82:5724-5727, 1985). We have used the inversion as an assay to characterize a stably piliated mutant. The mutant strain did not exhibit the pilA ON and pilA OFF colonial variants characteristic of the wild type; rather, every clone produced a level of pilA expression intermediate between ON and OFF wild type populations. The mutant phenotype was conferred by a lesion at a previously undescribed locus between hemA and trpA, which we have termed pilG. Examination of the pilA promoter region in four pilG mutant populations indicated that the phenotypic stability conferred by the pilG mutation was not due to an inability to carry out the inversion. Rather, all pilG mutant populations consisted of approximately equal mixtures of ON and OFF individuals. We suggest that pilG mutants may undergo such rapid switching of the pilA promoter that populations exhibit an intermediate level of pilA expression and phenotypic stability. PMID- 3019998 TI - Location of the right boundary of the virulence region on Agrobacterium tumefaciens plasmid pTiC58 and a host-specifying gene next to the boundary. AB - The right boundary of the virulence (Vir) region of the nopaline plasmid pTiC58 of Agrobacterium tumefaciens was determined by transposon insertion, cartridge emplacement, and deletion mutagenesis. Genetic complementation with mutant and wild-type alleles led to the identification of the virE locus at the right boundary, which was located about 6 kilobases from the left border of the segment of DNA that is transferred into the plant genome. virE is 2.0 kilobases long and encodes at least one protein of 69 kilodaltons. Various mutations in virE resulted in different truncated lengths of the 69-kilodalton protein. As this protein was increasingly truncated from the carboxy terminus, the host range of A. tumefaciens and the frequency of tumor formation diminished concomitantly. Thus, as one of its functions, the 69-kilodalton protein of virE is probably involved in some aspect of the host range specificity of A. tumefaciens and in infection efficiency. PMID- 3019999 TI - Effects of DNA gyrase inhibitors in Escherichia coli topoisomerase I mutants. AB - Relaxation of titratable supercoils in bacterial nucleoids was measured following treatment of topA mutants with coumermycin or oxolinic acid, inhibitors of DNA gyrase. Relaxation occurred after treatment of the mutants with either inhibitor. We detected no significant difference in relaxation between topA- and topA+ strains treated with coumermycin. This finding, together with previous observations, supports the idea that relaxation caused by coumermycin probably arises from the relaxing activity of gyrase itself. The source of DNA relaxation caused by oxolinic acid was not identified. Nucleoid supercoiling can be increased by adding oxolinic acid to a strain that carries three topoisomerase mutations: delta topA, gyrB225, and gyrA (Nalr) (S. H. Manes, G. J. Pruss, and K. Drlica, J. Bacteriol. 155:420-423, 1983). We found that this increase in supercoiling requires partial sensitivity to the drug and at the delta topA and gyrA mutations. Full resistance to oxolinic acid in the presence of the delta topA, gyrB225, and gyrA mutations was conferred by an additional mutation that maps at or near gyrB. PMID- 3020000 TI - Purification and characterization of an inorganic pyrophosphatase from the extreme thermophile Thermus aquaticus. AB - An inorganic pyrophosphatase was purified over 600-fold to homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme is a tetramer of Mr = 84,000, has a sedimentation coefficient of 5.8S, a Stokes radius of 3.5 nm, and an isoelectric point of 5.7. Like the enzyme of Escherichia coli, the pyrophosphatase appears to be made constitutively. The pH and temperature optima are 8.3 and 80 degrees C, respectively. The Km for PPi is 0.6 mM. A divalent cation is essential, with Mg2+ preferred. The enzyme uses only PPi as a substrate. PMID- 3020001 TI - Mini-mu bacteriophage with plasmid replicons for in vivo cloning and lac gene fusing. AB - New mini-Mu transposons with plasmid replicons were constructed with additional features for in vivo DNA cloning and lac gene fusing in Escherichia coli. These mini-Mu replicons can be used to clone DNA by growing them with a complementing Mu bacteriophage and by using the resulting lysate to transduce Mu-lysogenic cells. These mini-Mu phage have selectable genes for resistance to kanamycin, chloramphenicol, and spectinomycin-streptomycin, and replicons from the high-copy number plasmids pMB1 and P15A and the low-copy, broad-host-range plasmid pSa. The most efficient of these elements can be used to clone genes 100 times more frequently than with the previously described mini-Mu replicon Mu dII4042, such that complete gene banks can be made with as little as 1 microliter of a lysate containing 10(6) helper phage. The 39-kilobase-pair Mu headful DNA packaging mechanism limits the size of the clones formed. The smallest of the mini-Mu elements is only 7.9 kilobase pairs long, allowing the cloning of DNA fragments of up to 31.1 kilobase pairs, and the largest of them is 21.7 kilobase pairs, requiring that clones carry insertions of less than 17.3 kilobase pairs. Elements have been constructed to form both transcriptional and translational types of lac gene fusions to promoters present in the cloned fragment. Two of these elements also contain the origin-of-transfer sequence oriT from the plasmid RK2, so that clones obtained with these mini-Mu bacteriophage can be efficiently mobilized by conjugation. PMID- 3020002 TI - Origin of small beta-lactamase-specifying plasmids in Haemophilus species and Neisseria gonorrhoeae. AB - Fifty-nine percent of unselected strains of Haemophilus parainfluenzae were found to carry small, phenotypically cryptic plasmid DNA species. Using filter blot hybridization, we found several plasmids which were homologous to the small beta lactamase-specifying plasmids pJB1 and pFA7, which were originally isolated from Haemophilus ducreyi and Neisseria gonorrhoeae, respectively. Detailed filter hybridization studies combined with electron microscope heteroduplex analysis suggested that three cryptic plasmids are completely homologous to the non-TnA sequences of pJB1. One cryptic plasmid was found to be highly homologous to pJB603, a small beta-lactamase plasmid previously found in two isolates of H. influenzae. A second group of plasmids were found to carry sequences homologous to pJB1 and other sequences homologous to pJB603. These results strongly suggest that small beta-lactamase plasmids found in Haemophilus species and N. gonorrhoeae may have arisen by insertion of the transposable beta-lactamase specifying element TnA into small, phenotypically cryptic replicons resident in H. parainfluenzae. Attempts to reproduce such a recombination event in the laboratory were not successful. PMID- 3020003 TI - Effects of anaerobic regulatory mutations and catabolite repression on regulation of hydrogen metabolism and hydrogenase isoenzyme composition in Salmonella typhimurium. AB - Hydrogen metabolism in Salmonella typhimurium is differentially regulated by mutations in the two anaerobic regulatory pathways, defined by the fnr (oxrA) and oxrC genes, and is controlled by catabolite repression. The synthesis of the individual hydrogenase isoenzymes is also specifically influenced by fnr and oxrC mutations and by catabolite repression in a manner entirely consistent with the proposed role for each isoenzyme in hydrogen metabolism. Synthesis of hydrogenase isoenzyme 2 was found to be fnr dependent and oxrC independent, consistent with a role in respiration-linked hydrogen uptake which was shown to be similarly regulated. Also in keeping with such a respiratory role was the finding that both hydrogen uptake and the expression of isoenzyme 2 are under catabolite repression. In contrast, formate hydrogenlyase-dependent hydrogen evolution, characteristic of fermentative growth, was reduced in oxrC strains but not in fnr strains. Hydrogenase 3 activity was similarly regulated, consistent with a role in hydrogen evolution. Unlike the expression of hydrogenases 2 and 3, hydrogenase 1 expression was both fnr and oxrC dependent. Hydrogen uptake during fermentative growth was also both fnr and oxrC dependent. This provided good evidence for a distinction between hydrogen uptake during fermentation- and respiration dependent growth and for a hydrogen-recycling process. The pattern of anaerobic control of hydrogenase activities illustrated the functional diversity of the isoenzymes and, in addition, the physiological distinction between the two anaerobic regulatory pathways, anaerobic respiratory genes being fnr dependent and enzymes required during fermentative growth being oxrC dependent. PMID- 3020004 TI - Spontaneous deletion of a 20-kilobase DNA segment carrying genes specifying isopropylbenzene metabolism in Pseudomonas putida RE204. AB - The genes encoding isopropylbenzene metabolism in Pseudomonas putida RE204 are readily lost in two ways: by loss (curing) of plasmid pRE4 which specifies the catabolic pathway and by deletion from pRE4 of an approximately 20-kilobase segment of DNA carrying the catabolic genes. The presence of DNA sequences at the ends of the catabolic gene region sharing homology with one another suggests that the deletions result from recombination events between these homologous sequences. PMID- 3020005 TI - Requirement of the Escherichia coli dnaA gene product for plasmid F maintenance. AB - There are DnaA protein-binding sites in at least one F origin of replication, and only potentially leaky dnaA(Ts) mutations had ever been used in previous studies indicating that F replication was independent of the dnaA gene product. Here we show that an Escherichia coli dnaA::Tn10 host which does not make a dnaA gene product cannot sustain autonomous or integrated F plasmid maintenance. PMID- 3020006 TI - Development of a gene cloning system for the hydrogen-producing marine photosynthetic bacterium Rhodopseudomonas sp. AB - Seventy-six strains of marine photosynthetic bacteria were analyzed by agarose gel electrophoresis for plasmid DNA content. Among these strains, 12 carried two to four different plasmids with sizes ranging from 3.1 to 11.0 megadaltons. The marine photosynthetic bacterium Rhodopseudomonas sp. NKPB002106 had two plasmids, pRD06S and pRD06L. The smaller plasmid, pRD06S, had a molecular weight of 3.8 megadaltons and was cut at a single site by restriction endonucleases SalI, SmaI, PstI, XhoI, and BglII. Moreover, the marine photosynthetic bacterium Rhodopseudomonas sp. NKPB002106 containing plasmid pRD06 had a satisfactory growth rate (doubling time, 7.5 h), a hydrogen-producing rate of 0.96 mumol/mg (dry weight) of cells per h, and nitrogen fixation capability. Plasmid pRD06S, however, had neither drug resistance nor heavy-metal resistance, and its copy number was less than 10. Therefore, a recombinant plasmid consisting of pRD06S and Escherichia coli cloning vector pUC13 was constructed and cloned in E. coli. The recombinant plasmid was transformed into Rhodopseudomonas sp. NKPB002106. As a result, Rhodopseudomonas sp. NKPB002106 developed ampicillin resistance. Thus, a shuttle vector for gene transfer was constructed for marine photosynthetic bacteria. PMID- 3020007 TI - Cloning and expression of Mycobacterium bovis BCG DNA in "Streptomyces lividans". AB - The ability of "Streptomyces lividans" to use the expression signals of genes from Mycobacterium bovis BCG was tested in vivo by using gene fusions. Random DNA fragments from M. bovis BCG were inserted into promoter-probe plasmids in Escherichia coli and in "S. lividans." Comparison with promoter activity detected with random DNA fragments from the respective hosts suggested that "S. lividans" efficiently utilizes a high proportion of mycobacterial promoters, whereas a smaller fraction are expressed, and expressed more weakly, in E. coli. M. bovis BCG DNA fragments were also inserted into the specially constructed translational fusion vector (pIJ688) in "S. lividans." pIJ688 contains the kanamycin phosphotransferase gene (neo) from transposon Tn5, truncated at its amino terminus, as the indicator. The results suggested that "S. lividans" uses M. bovis BCG translational signals almost as efficiently as its own signals. Moreover, several hybrid proteins with an M. bovis BCG-derived amino terminus seemed to be reasonably stable in "S. lividans." These experiments indicate that "S. lividans" may be a suitable host for the expression of Mycobacterium leprae and Mycobacterium tuberculosis genes from their own signals. This is a precondition for the expression of entire biosynthetic pathways, which could be valuable in the production of diagnostic and therapeutic agents. The vectors may also have wider applications for the analysis of gene expression in Streptomyces. PMID- 3020008 TI - Association of transformation of xenosomes from nonkiller to killer with extrachromosomal DNA. AB - Extrachromosomal DNA in the form of covalently closed circular DNA molecules was isolated from killer and nonkiller xenosomes, bacterial endosymbionts of the marine protozoan Parauronema acutum. Restriction endonuclease digests of these molecules derived from 12 isolates revealed consistent, readily identifiable, differences in the pattern of fragments of the killer as compared with those present in the nonkiller. Transformation of the nonkiller to killer by infection is also accompanied by a change from the nonkiller to killer pattern. Based on analysis of fragments resulting from restriction endonuclease digests, two circular duplex DNA molecules, each 63 kilobase pairs (kbp) in length, were identified in the 263-20 nonkiller stock and mapped. The maps revealed that each possesses a single BamHI site and multiple BglI, BstIIE, PstI, and SalI sites. A distinguishing feature of these maps is that the two molecules share a region about 17 kbp in length in which multiple restriction sites are in register with each other. Allowing for a 0.5-kbp insertion or deletion and the introduction or removal of only a few restriction sites, an additional stretch extending approximately 31 kbp beyond this sequence could also be considered to be homologous. The structure of the killer plasmid appears to be more complex, and we have been unable, as yet, to construct physical maps for this DNA. We postulate that the killer plasmid DNA is composed of three, perhaps four, circular 63-kbp duplexes, at least one which contains a single BamHI site and another which contains two BamHI sites. The remaining molecules may represent copies of either or both of the other two, modified to contain additional restriction sites. Transformation from the nonkiller to the killer is visualized as the insertion of restriction sites at various points along parent nonkiller plasmid DNA molecules. The mechanism by which these sites are introduced is unknown. PMID- 3020009 TI - Enhancement of ECT benefit by yohimbine. AB - Three patients with major depression who were pretreated with yohimbine 10 mg p.o. showed a dramatic response without major adverse effects following two electroconvulsive treatments (ECT). The hypothesis that the alpha 2-adrenergic antagonist yohimbine accelerates ECT-induced postsynaptic beta-adrenergic receptor down-regulation is discussed. PMID- 3020010 TI - Epstein-Barr virus infection and depression. PMID- 3020011 TI - Update on neuroreceptor mechanisms and their implication for the pharmacotherapy of affective disorders. AB - Unlike "acceptors" (binding sites), receptors for neurohormones display the dual function of recognition and biologic response via coupling to effector systems that generate second messengers or to ion channels that modify the passage of specific ions. The demonstration that membrane receptors for norepinephrine are coupled in a stimulatory (beta 1, beta 2) or inhibitory (alpha 2) way via nucleotide regulatory proteins to adenylate cyclase, thus increasing or decreasing the formation of the second messenger cyclic AMP, is discussed. Serotonin receptors are linked to phosphatidylinositol hydrolysis generating two second messengers, diacylglycerol and inositol triphosphate. Pharmacologically, serotonin (5-HT) receptors that are linked to phosphatidylinositol hydrolysis are of the 5-HT2 type in the cerebral cortex and of the 5-HT1c type in the choroid plexus. The final common pathway of signal transduction appears to be the protein kinase-mediated phosphorylation of cellular proteins. Glucocorticoid receptors have been found to be located in the cytoplasma or nuclei of aminergic cell bodies and may exert their effects by modifying the genomic expression of the respective neurons. The two aminergic receptor systems are biomolecularly linked, with glucocorticoids exerting a modulatory role. The implications of central receptor research for the pharmacotherapy and the pathophysiology of affective disorders are reviewed. PMID- 3020012 TI - Isomerization and denaturation of homologous cytochromes c: correlation between local and gross conformational changes. AB - To elucidate the relationship between local and gross conformational changes, three types of conformational changes, i.e., alkaline isomerization, ligand replacement, and guanidine hydrochloride (GuHCl) denaturation, of a set of homologous and modified cytochromes c were investigated by spectroscopic methods. Cytochromes c examined include the horse, tuna, Candida, monomeric Saccharomyces, and disulfide-linked dimeric Saccharomyces proteins. Correlations were found between the apparent pK (pKa) for the isomerization and the equilibrium constants for the binding of imidazole and azide to the heme iron: the lower the pKa value, the higher the binding constant. This is explained by the fact that the isomerization and ligand replacement involve similar conformational changes localized around the sixth coordination position of the heme. A good correlation was also observed between the susceptibilities to local and gross conformational changes: the lower the pKa value for the isomerization, the lower the GuHCl concentration for the midpoint of the denaturation. Thermodynamic analysis suggests that this correlation is not due to the involvement of similar conformational changes in the two processes. Cooperative stabilization of the tertiary structure is proposed to interpret this correlation. PMID- 3020013 TI - Isolation and characterization of monoclonal antibodies against calcium-activated neutral protease with low calcium sensitivity. AB - Fifteen hybridomas secreting antibodies against calcium-activated neutral protease (CANP), especially those for rabbit muscle mCANP with low calcium sensitivity, have been produced by the cell fusion technique. Eight of the monoclonal antibodies belong to the class IgG1, one to the class IgG2a, and six to the class IgG2b. The antibodies from these clones were characterized with regard to their relative binding affinities to the large subunits (80K) and the small subunits (30K) of mCANP as well as mu CANP, which is another type of CANP with high calcium sensitivity. Fourteen antibodies bound only to the 80K subunit of mCANP and one antibody bound to the 80K subunit of both mCANP and mu CANP. These antibodies recognized rat mCANP but not chicken CANP, with the exception of one antibody. Examination of the effects of these antibodies on the enzyme activity of mCANP showed that six antibodies partially inhibited the enzyme activity and the others were noninhibitory. These monoclonal antibodies should be useful for analyzing the fine structure of CANPs and the mechanism of the activation of mCANP, and also for determining the intracellular localization of mCANP. PMID- 3020014 TI - Structure and expression of the mouse prealbumin gene. AB - We cloned a genomic DNA fragment which covers the entire sequence of the mouse prealbumin gene and then studied the structure. The coding regions are separated into four exons by three introns, and these numbers, the sizes of the exons and the relative sites of the exon-intron junctions are all in complete agreement with those determined for the human gene. The sequences of four exons can be aligned perfectly with that of the previously determined mouse prealbumin cDNA. In addition to the exon regions, we found two highly conserved DNA regions between the mouse and human prealbumin genes, one in the 5'-flanking region of the gene and the other in the 3' end region of the first intron. These DNA regions contain several consensus glucocorticoid receptor-binding site sequences, and the latter also contains an enhancer sequence present in the immunoglobulin kappa-chain joining-constant kappa intron. RNA hybridizing to the mouse prealbumin cDNA was detected in the extracts from liver, brain, and kidney, but was not detected in testes, spleen, or heart. Little change was caused in the level of prealbumin mRNA in the liver by administration of dexamethasone to mice. PMID- 3020015 TI - The gene crtI mediates the conversion of phytoene into colored carotenoids in Rhodopseudomonas capsulata. AB - Carotenoids are membrane pigments present in all photosynthetic organisms, providing essential photoprotective functions. The first carotenoid formed in the pathway is phytoene, a colorless compound which is then converted into colored carotenoids by a series of dehydrogenation reactions. In the photosynthetic bacterium Rhodopseudomonas capsulata mutations that affect carotenoid biosynthesis before colored carotenoids are formed have a "blue-green" phenotype as opposed to the "red" of wild type cells. We have extracted carotenoids from several blue-green mutants and found that two strains (BPY69 and BPY102) accumulate phytoene and no colored carotenoids. These mutants failed to dehydrogenate phytoene in an in vitro assay. However, dehydrogenation of this compound can be achieved in vitro by adding a cell-free extract from another blue green mutant blocked earlier in the pathway. Genetic complementation and deletion mapping indicate that the gene crtI is responsible for the conversion of phytoene into colored carotenoids in these mutants. PMID- 3020016 TI - Truncated atrial natriuretic peptide analogs. Comparison between receptor binding and stimulation of cyclic GMP accumulation in cultured vascular smooth muscle cells. AB - A series of truncated atrial natriuretic peptide analogs were examined as a means of defining the structural requirements for receptor occupancy and stimulation of cyclic GMP accumulation in bovine aortic smooth muscle cells. It was determined that deletion of amino acids from the carboxyl and/or amino termini of the peptides diminished their ability to increase cyclic GMP levels. Deletion of amino acids from the carboxyl terminus had the greatest effect, and atrial natriuretic peptide analogs lacking the carboxyl-terminal phenylalanyl-arginyl tyrosine tripeptide were 100-1000-fold less active than parent compounds in stimulating intracellular cyclic GMP accumulation. In marked contrast to the cyclic GMP effects, deletion of amino- and/or carboxyl-terminal amino acids had only minor effects on the affinity of the peptides for specific smooth muscle cell-associated receptors. Peptide analogs lacking the phenylalanyl-arginyl tyrosine tripeptide bound to receptors with an affinity only 1.1-5-fold weaker than the parent compounds. Thus, there was no correlation between apparent receptor binding affinity of atrial natriuretic peptide analogs and potency of these same peptides for stimulating intracellular cyclic GMP accumulation. Furthermore, analogs that bound to receptors and failed to elicit significant cyclic GMP responses did not antagonize or modulate increases in cyclic GMP induced by parent compounds. These data are most consistent with the existence of multiple subpopulations of atrial natriuretic peptide receptors on aortic smooth muscle cells. PMID- 3020018 TI - Alpha and beta forms of cytochrome c oxidase observed in rat heart myocytes by low temperature Fourier transform infrared spectroscopy. AB - Carbon monoxide bound to myoglobin and cytochrome c oxidase in separated adult rat heart myocytes has been observed with Fourier transform IR spectroscopy at low temperatures. CO complexes of these two proteins can be spectrally separated through temperature manipulation of the relaxation of the photolyzed systems. Photolyzed carboxymyoglobin relaxes very rapidly above 80 K, whereas the CO photolyzed from cytochrome a3 associates with CuB and relaxes very slowly below 140 K. Cytochrome c oxidase is found to be present in two major molecular forms which we designate alpha and beta. Each form contains an a3Fe and its associated CuB which we observe by their CO complexes. The predominant FeCO band, the alpha form of cytochrome oxidase, is similar to that previously seen in beef heart mitochondria, but with a slightly larger activation enthalpy, delta H = 46 kJ/mol. At least one of the beta forms is similar, but two have not been observed in beef heart mitochondria. Upon photolysis of alpha-FeCO, the alpha-CuCO species is formed. This band splits into two at low temperature. Up to half of the FeCO band area of the intact myocytes is distributed among three or more minor species (beta forms). The beta-FeCO bands all appear to be associated with only one beta CuCO band which does not split at low temperature. After photo-dissociation of CO, the beta forms relax considerably faster than the alpha form, achieving 50% recombination in 10% of the time required for the alpha form. In a tissue slice from an opossum heart exposed to CO, we observed alpha and beta forms of cytochrome oxidase very similar to those in the rat heart myocytes. The cause of the differences between the alpha and beta forms of the enzyme is unknown, but their possible role in the control of respiration is discussed. Carboxymyoglobin contained within intact rat heart myocytes was very similar to sperm whale carboxymyoglobin, but with a much smaller amount of the lower frequency minor component. PMID- 3020017 TI - Changes in cGMP concentration correlate with some, but not all, aspects of the light-regulated conductance of frog rod photoreceptors. AB - Cyclic GMP has been implicated in controlling the light-regulated conductance of rod photoreceptors of the vertebrate retina. However, there is little direct evidence correlating changes in cGMP concentration with the light-regulated permeability mechanism in living cells. A preparation of intact frog rod outer segments suspended in a Ringer's medium containing low Ca2+ has been used to demonstrate that initial changes in total cellular cGMP concentration parallel changes in the light-regulated membrane current over a wide range of light intensities. At light intensities bleaching from 160 to 5.6 X 10(6) rhodopsin molecules/rod/s, decreases in the response latency for the cGMP kinetics parallel decreases in the latent period of the electrical response. Further, changes in the rate of the cGMP decrease parallel the rate of membrane current suppression as the light intensity is varied. Up to 10(5) cGMP molecules are hydrolyzed per photolyzed rhodopsin, consistent with in vitro studies showing that each bleached rhodopsin can activate over 100 phosphodiesterase molecules. Addition of the Ca2+ ionophore, A23187, does not affect the initial kinetics of the cGMP decrease or of the electrical response, excluding a direct role for Ca2+ in the initial events of phototransduction. These results are consistent with cGMP being the intracellular messenger that links rhodopsin isomerization with changes in membrane permeability upon illumination. It is unlikely, however, that light induced changes in total cGMP concentration are the sole regulators of membrane current. This is suggested by several observations: at bright light intensities, the subsecond light-induced cGMP decrease is essentially complete prior to complete suppression of membrane current; maximal light-induced decreases in cGMP concentration occur at all light intensities tested, whereas the extent of membrane current suppression varies over the same range of light intensities; changing the external Ca2+ concentration from 1 mM to 10 nM in the dark causes an increase in membrane current that is significantly more rapid than corresponding changes in cGMP concentration. Thus, light-induced changes in total cellular cGMP concentration correlate with some, but not all, aspects of the visual excitation process in vertebrate photoreceptors. PMID- 3020019 TI - Regulation of uridine kinase. Evidence for a regulatory site. AB - Uridine kinase from mouse Ehrlich ascites tumor cells may exist at 4 degrees C in multiple aggregation states that only slowly equilibrate with one another. Increasing the temperature leads to dissociation, and the appearance of a single predominant species: at 22 degrees C the enzyme exists as a tetramer. There is also a break in the dependence of enzyme activity on temperature as measured in an Arrhenius plot. The feedback inhibitors CTP and UTP cause the enzyme to dissociate to the monomer, whereas the substrate ATP reverses this process. Kinetic studies show that the monomer has little or no activity. Studies of the reaction mechanism show that binding of substrates is ordered, leading to a ternary complex, and release of products is ordered: uridine is the first substrate bound, ADP the first product released. Except for the inhibitors UTP and CTP, all other nucleoside triphosphates, whether purine or pyrimidine, or containing ribose or deoxyribose, act as phosphate donor. Especially interesting are the opposite effects of CTP and dCTP on uridine kinase: unlike CTP, dCTP does not dissociate the enzyme and is competent as a phosphate donor. We propose that the various effects of different ligands are best explained by the existence of a regulatory site (with more stringent specificity than the catalytic site) that controls dissociation of uridine kinase to the inactive monomer. PMID- 3020020 TI - Redox-cycled oxidase. One of the reaction products of reduced cytochrome c, cytochrome c oxidase, and dioxygen. AB - Pulsed and oxygenated forms of cytochrome c oxidase are believed to be variants of the oxidized enzyme. They were produced as a consequence of one or more reduction-oxidation cycles of the resting form and are characterized by an increase of the alpha band intensity and a red-shift of the Soret absorption band to 428 nm. The rate of decay of these species back to the resting enzyme varies appreciably and appears to depend on the nature of the reductant and/or oxidant used in their preparation. Here we report that if resting oxidase is incubated with either reduced or oxidized cytochrome c and then exposed to dioxygen, an activated form is rapidly produced which appears to be more oxidized than the starting material. This finding suggest some degree of partial reduction of the resting enzyme, but this by itself cannot explain the extent of activation. Our results further question the significance of the optical spectral "signature" of the oxygenated (Okunuki, K., and Sekuzu, I. (1954) Seitaino Kagaka 5, 265-272), pulsed (Antonini, E., Brunori, M., Colosimo, A., Greenwood, C., and Wilson, M. T. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3128-3132), and "420 nm" species (Kumar, C., Naqui, A., and Chance, B. (1984) J. Biol. Chem. 259, 2073-2076, 11668-11671), which are thought to be activated forms of oxidized cytochrome c oxidase. PMID- 3020021 TI - Solubilization and purification of rat pituitary gonadotropin-releasing hormone receptor. AB - Gonadotropin-releasing hormone (GnRH) receptors were solubilized from rat pituitary membrane preparations in an active form by using the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid). The solubilized receptor exhibits high affinity, saturability, and specificity. The soluble supernatant retained 100% of the original binding activity when stored at 4 or -20 degrees C in the presence of 10% glycerol. The receptors were resolved into two components on the basis of chromatography on wheat germ agglutinin-agarose. Homogeneous receptor preparation was obtained by two cycles of affinity chromatography on immobilized avidin column coupled to [biotinyl-D Lys6]GnRH. The overall recovery of the purified receptor was 4-10% of the initial activity in the CHAPS extract, and the calculated purification -fold was approximately 10,000 to 15,000. Analysis of iodinated purified GnRH receptors by autoradiography indicated the presence of two bands, Mr = 59,000 and 57,000. This was confirmed by photoaffinity labeling of the partially purified receptors and suggests that both components can specifically bind the hormone. PMID- 3020022 TI - Reductive release of iron from ferritin by cation free radicals of paraquat and other bipyridyls. AB - NADPH-cytochrome P-450 reductase-catalyzed reduction of paraquat promoted the release of iron from ferritin. Aerobically, iron release was inhibited approximately 60% by superoxide dismutase, whereas xanthine oxidase-dependent iron release was inhibited nearly 100%. This suggests that both superoxide and the paraquat cation radical can catalyze the release of iron from ferritin. Accordingly, under anaerobic conditions, the paraquat radical mediated a very rapid, complete release of iron from ferritin. Similarly, the cation free radicals of the closely related chemicals, diquat and benzyl viologen, also promoted iron release. ESR studies demonstrated that electron transfer from the paraquat cation radical to ferritin accounts for the reductive release of iron. The ferritin structure was not altered by exposure to the paraquat radical and also retained its ability to re-incorporate iron. These studies indicate that release of iron from ferritin may be a common feature contributing to free radical-mediated toxicities. PMID- 3020023 TI - Catecholamine activation of the membrane transport of long chain fatty acids in adipocytes is mediated by cyclic AMP and protein kinase. AB - Membrane transport of long chain fatty acids in the isolated adipocyte can be stimulated 5-10-fold by epinephrine (Abumrad, N. A., Perry, P. R., and Whitesell, R. R. (1985) J. Biol. Chem. 260, 9969-9971). This study shows that isoproterenol and norepinephrine are more potent than epinephrine in activating the transport process. The stimulatory effect on transport is mediated by beta-receptor interaction and cAMP. This was shown by the following. alpha-Receptor agonists and antagonists were ineffective; methylisobutylxanthine at low concentration (3 microM) potentiated the effect of a suboptimal dose (0.01 microgram/ml) of epinephrine and was stimulatory at high concentration (100 microM) in the absence of epinephrine; and cAMP analogs were very effective activators. Involvement of the cAMP-dependent protein kinase was indicated by two lines of evidence. 1) Combinations of cAMP analogs which are specific for sites 1 and 2 of the protein kinase, respectively, had synergistic effects on fatty acid transport. Combinations of analogs specific for the same site were only additive in their effects. This is similar to the pattern of protein kinase activation in vitro and to that of lipolysis activation in the intact adipocyte (Beebe, S. J., Holloway, R., Rannels, S. R., and Corbin, J. D. (1984) J. Biol. Chem. 259, 3539-3547). 2) Treatment of cells with various metabolic poisons abolished the stimulatory effect of norepinephrine. The response of fatty acid transport to catecholamines showed multiple parallels with that documented for lipolysis except that it was much more rapid. This suggested that the transport process was a regulatory step in fatty acid mobilization. This interpretation is supported by the observation that basal Vmax for transport is much too slow to accommodate the rate of fatty acid release which is observed following stimulation of intact cells with adrenergic hormones. PMID- 3020024 TI - The mechanism of the search for homology promoted by recA protein. Facilitated diffusion within nucleoprotein networks. AB - recA protein promotes the homologous pairing of single strands with duplex DNA by polymerizing on the single strands to make presynaptic nucleoprotein filaments which are polyvalent with respect to duplex DNA and which consequently form large networks or coaggregates when duplex DNA is added. Previous work has shown that efficient homologous pairing occurs within these networks. In the experiments described here, we observed that the length of the duplex DNA determined the stability of coaggregates, their steady state level, and the yield of joint molecules. Correspondingly, heterologous duplex DNA when preincubated with presynaptic filaments excluded subsequently added homologous duplex DNA from coaggregates and inhibited homologous pairing; the extents of exclusion and inhibition were determined by the length of the heterologous duplex DNA. On the other hand, long heterologous duplex DNA when added together with short homologous duplex DNA was capable of stimulating the absorption of the homologous molecules into coaggregates and increasing the rate of homologous pairing. In reactions involving short duplex molecules, polyamines exerted comparable effects on coaggregation and homologous pairing. We conclude that coaggregates are instrumental in homologous pairing, that they constitute distinct domains that are responsible for the processive or first order character of the pairing reaction, and that they act by concentrating DNA and facilitating diffusion. PMID- 3020025 TI - Deletion of exon encoding cysteine-rich repeat of low density lipoprotein receptor alters its binding specificity in a subject with familial hypercholesterolemia. AB - The proposed ligand binding domain of the low density lipoprotein (LDL) receptor consists of a 40-amino acid cysteine-rich unit that is repeated with some variation seven times. We describe here a mutant allele at the LDL receptor locus in which one of the seven repeats has been deleted. This mutation was found in a patient with the clinical syndrome of homozygous familial hypercholesterolemia. By molecular cloning, we show that the deletion arose by homologous recombination between repetitive Alu sequences in intron 4 and intron 5 of the gene. The deletion removes exon 5, which normally encodes the sixth repeat of the ligand binding domain. In the resultant mRNA, exon 4 is spliced to exon 6, preserving the reading frame. This mRNA produces a shortened protein that reaches the cell surface and reacts with anti-receptor antibodies but does not bind LDL, which contains apoprotein B-100 as its major protein component. Surprisingly, the deleted protein retains the ability to bind and internalize beta-migrating very low density lipoprotein, a lipoprotein that contains apoprotein E as well as apoprotein B-100. These data support the hypothesis that the seven repeated sequences in the receptor constitute the LDL binding domain. The data further indicate that the sixth repeat is required for binding of LDL, but not beta migrating very low density lipoprotein, and that deletion of a single cysteine rich repeat can alter the binding specificity of the LDL receptor. PMID- 3020026 TI - Regulation of intracellular pH in human platelets. Effects of thrombin, A23187, and ionomycin and evidence for activation of Na+/H+ exchange and its inhibition by amiloride analogs. AB - Intracellular pH (pHi) of human platelets was measured with the fluorescent dye 2',7'-bis(carboxyethyl)5,6-carboxyfluorescein under various conditions. Stimulation by thrombin at 23 degrees C caused a biphasic change in pHi (initial pHi 7.09); a rapid fall of 0.01-0.04 units (correlated with the rise of [Ca2+]i measured with quin2) followed after 10-15 s by a sustained rise of 0.1-0.15 units pHi. The fall of pHi and [Ca2+]i mobilization was reduced by early (5 s) addition of hirudin, but the later elevated pHi was not reversed by hirudin added after 30 s, although this strips thrombin from receptors and rapidly returns [Ca2+]i to basal levels. In Na+-free medium, or in presence of the Na+/H+ antiport inhibitors, 5-(N,N-dimethyl)amiloride (DMA) or 5-(N-ethyl-N-isopropyl)amiloride (EIPA), thrombin caused a greater fall of pHi (0.22-0.26 units) that was sustained. DMA or EIPA could also reverse the alkalinization response to thrombin. Ca2+ ionophores (ionomycin, A23187) decreased platelet pHi by 0.02-0.15 units, but without an increase of pHi comparable to that following thrombin; DMA and EIPA enhanced the fall of pHi (0.14-0.33 units). Cytoplasmic acidification produced by nigericin (K+/H+ ionophore) was followed by return towards normal that was abolished by Na+/H+ antiport inhibitors. The phorbol diester phorbol 12 myristate 13-acetate had little effect on resting pHi but increased the rate of recovery 2-3-fold after cytoplasmic acidification by nigericin, ionomycin, or sodium propionate. These results indicate that elevation of [Ca2+]i by thrombin enhances H+ production, but the subsequent alkalinization is independent of receptor occupancy or elevated [Ca2+]i and stimulation of the Na+/H+ antiporter by thrombin probably involves some mechanism apart from regulation by H+ and protein kinase C. PMID- 3020027 TI - Identification of N-[p-(2-benzimidazolyl)phenyl]maleimide-modified residue participating in dynamic fluorescence changes accompanying Na+,K+-dependent ATP hydrolysis. AB - Na+,K+-ATPase from pig kidney was specifically modified with a sulfhydryl fluorescent reagent, N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM), by pretreatment of N-ethylmaleimide. The preparation thus obtained retained 100% of initial Na+,K+-ATPase activity and contained 1 BIPM residue/alpha-chain, and it showed almost 2-fold larger fluorescence changes accompanying ATP hydrolysis than the previous preparations which retained 60% of initial activity and contained 3 4 BIPM residues/alpha-chain (Taniguchi, K., Suzuki, K., and Iida, S. (1982) J. Biol. Chem. 257, 10659-10667). Extensive trypsin (Sigma type I) treatment of the new preparation produced mainly two different fluorescent peptide peaks in both ion-exchange and reverse-phase chromatography. Amino acid sequence analysis of both peptides showed that they had the same common sequence, Ser-Tyr-X-Pro-Gly Met-Gly-Val, except that the larger one contained Ala-Leu next to the Val residue. From the comparison of the amino acid sequence deduced from cDNA from sheep kidney (Shull, G. E., Schwartz, A., and Lingrel, J. B. (1985) Nature 316, 691-695), X was shown to correspond to Cys-964 of the alpha-chain in Na+,K+ ATPase. The data suggest that the microenvironment of the BIPM residue covalently bound to the sulfhydryl group of Cys-964 changes accompanying sequential appearance of reaction intermediates of Na+,K+-ATPase. PMID- 3020028 TI - Linkage, evolution, and expression of the rat apolipoprotein A-I, C-III, and A-IV genes. AB - The genes coding for three of the proteins of the lipid transport system, apolipoproteins A-I (apoA-I), C-III (apoC-III), and A-IV (apoA-IV), are closely linked and tandemly organized as a multigene family in the human genome. The evolution of this multigene family was studied by cloning and extensive restriction mapping analysis of an approximately 20-kilobase genomic DNA fragment containing the rat apoA-I gene. Low stringency hybridization blotting analysis of this DNA fragment using human apoC-III and apoA-IV cDNA probes revealed that the apoA-I, apoC-III, and apoA-IV genes are also closely linked and tandemly organized in the rat genome. Complete characterization of the rat apoA-I, apoC III, and apoA-IV genes showed that their relative location, size, direction of transcription, and intron-exon organization are remarkably similar to those of the corresponding human genes. The relative steady state apoA-I, apoC-III, and apoA-IV mRNA levels in various rat tissues were determined by quantitative dot blot hybridization of tissue total RNA using the corresponding gene probes. Adult liver and intestine, but not colon, brain, spleen, muscle, heart, lung, and kidney, contain apoA-I, apoC-III, and apoA-IV mRNAs. Fetal liver and intestine contain apoA-I but not apoC-III or apoA-IV mRNAs. During neonatal development the liver contains apoA-I and apoC-III but not apoA-IV while the intestine contains apoA-I, apoC-III, and substantial amounts of apoA-IV mRNAs. In adulthood and during aging both liver and intestine contain all three apoA-I, apoC-III, and apoA-IV mRNAs. These results indicate that the apolipoprotein A-I/C-III/A-IV multigene family was established before mammalian radiation and suggest that these genes are similarly organized in the genomes of all mammals. In addition, these results indicate that expression of the rat apoA-I, apoC-III, and apoA-IV genes is liver- and intestine-specific and regulated by fetal-, neonatal-, and aging-related factors. PMID- 3020029 TI - Acute ACTH regulation of adrenal corticosteroid biosynthesis. Rapid accumulation of a phosphoprotein. AB - Two-dimensional gel electrophoresis was used to monitor proteins synthesized in unstimulated control and in adrenocorticotropic hormone (ACTH)- or cAMP stimulated rat adrenal cells. Four proteins, which have similar proteolytic peptide maps, have been identified. The two found primarily in unstimulated cells are referred to as pb and pa, where pb is the protein with more basic isoelectric point. Similarly, proteins ib and ia were detected only in stimulated cells. The synthesis of pb occurs only in unstimulated cells and that of ib only in stimulated cells. Protein ib accumulates with the same lag time, rate, and stimulant dose response as the increase in steroid hormone synthesis. Pulse-chase studies showed that protein ib is not produced from pb by a post-translational modification. Proteins pb and ib thus seem identical with proteins p and i previously identified in rat adrenal cortex and corpus luteum (Krueger, R.J., and Orme-Johnson, N. R. (1983) J. Biol. Chem. 258, 10159-10167, and Pon, L.A., and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 6594-6599). The acidic forms, pa and ia, appear after a longer lag time and are produced at a slower rate than the basic forms. Pulse-chase studies showed that the disappearance of the basic form of each protein occurs concurrently with the appearance of the corresponding acidic form. Addition of [32P]orthophosphate to stimulated adrenal cells allowed direct demonstration that proteins ib and ia are phosphorylated. Moreover, alkaline phosphatase treatment of [35S]methionine-labeled, cAMP-stimulated adrenal cells caused a large decrease in the amounts of ib and ia and the appearance of proteins with the same two-dimensional electrophoretic mobilities as pb and pa. These observations suggest that protein ib may mediate stimulation of steroidogenesis, be produced by an ACTH- or cAMP-dependent, cotranslational phosphorylation of protein pb, and be lost by a cycloheximide-insensitive, post translational conversion to ia. PMID- 3020030 TI - Phosphorylation of nerve growth factor receptor proteins in sympathetic neurons and PC12 cells. In vitro phosphorylation by the cAMP-independent protein kinase FA/GSK-3. AB - We have examined phosphorylation of nerve growth factor (NGF) receptor in cultured sympathetic neurons and PC12 cells. Dissociated rat superior cervical ganglion neurons or PC12 cells were incubated with 32Pi to label cellular phosphoproteins. Membrane proteins were solubilized, and NGF receptor proteins were immunoprecipitated with the monoclonal antibody 192-IgG. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that NGF receptor components of Mr = 80,000 and Mr = 210,000 were phosphorylated. Phosphorylation of neither species was affected by treating the cells with NGF or phorbol 12-myristate 13-acetate. When the 80,000-Da protein was subjected to complete trypsin proteolysis and then analyzed by reverse phase liquid chromatography, two 32P-labeled peptides were resolved. The more hydrophobic peptide accounted for most of the 32P and contained only phosphoserine; the other peptide contained phosphoserine and phosphothreonine. No phosphotyrosine was detected in the receptor proteins. When receptor molecules from nonlabeled PC12 cells were immunoprecipitated and then incubated in vitro with [gamma-32P]ATP and the cAMP-independent protein kinase FA/GSK-3, phosphorylation occurred predominantly on serine and to a lesser extent on threonine. However, the immunoprecipitated receptor proteins neither autophosphorylated nor were they detectably phosphorylated by cAMP-dependent protein kinase, casein kinase II, or protein kinase C (the Ca2+/phospholipid-dependent enzyme). We conclude that binding units of the NGF receptor are phosphorylated constitutively in at least two sites in intact cells and that they can be phosphorylated by FA/GSK-3 in vitro. PMID- 3020031 TI - Intracellular modifications of human apolipoprotein E. AB - We have used pulse-chase techniques to study the synthesis, intracellular modification, and secretion of human apolipoprotein E by cultures of HepG2 cells and peripheral blood human monocyte macrophages. We have found that modified apoE isoproteins are detectable intracellularly after 14 min of pulse, and their relative concentration increases linearly over a 2-h pulse-chase period. At the same time, the relative concentration of unmodified apoE decreases. All the major modified apoE isoproteins appear simultaneously, and they correspond to the sialo apoE forms apooEs2, apoEs4, and apoEs6. ApoE secretion is first detected after a 30-min pulse. Secreted apoE consists of 92% of the same sialated apoE forms observed intracellularly. ApoE sialation and secretion were not affected by treatment of the HepG2 cultures with tunicamycin, whereas monensin inhibited both the intracellular sialation and secretion of this protein. These findings suggest that apoE is secreted in the form of three major isoproteins generated by intracellular modification of this protein with one or more O-linked oligosaccharide chains containing sialic acid. PMID- 3020032 TI - Cloning and expression of an intron-deleted phage T4 td gene. AB - The 1017-bp intron within the cloned phage T4 td gene was deleted by oligonucleotide-directed mutagenesis. Induction of thymidylate synthase activity and mature td mRNA from this intronless construct (pKTd delta I) was compared both in vivo and in vitro with expression from plasmids bearing td genes in which the introns contain either no change (pKTd2), an XbaI linker inserted about 200 nucleotides from the 3'-end (pKTdX-1), or a deletion of two-thirds of the central portion (pKTd delta 1-3). Slightly more synthase accumulated in cells carrying pKTd delta I as compared to the other td genes when induction was performed at 30, 37, or 42 degrees C. Dramatically different results were observed in vitro, where enzyme activity synthesized from pKTd delta I DNA appeared earlier and reached severalfold higher levels than with pKTd2 DNA. In addition, thymidylate synthase expression from pKTdX-1 was impaired relative to pKTd2, while pKTd delta 1-3 accumulated enzyme at levels intermediate between those of pKTd2 and pKTd delta I. Under both in vivo and in vitro conditions, increasing levels of mature td mRNA preceded and paralleled those in enzyme activity for all four plasmids, demonstrating comparable translation of the mRNAs produced. From these results it would appear that the splicing of td RNA is much more efficient in vivo than in vitro, suggesting that other cellular components may facilitate in vivo processing of this intron-containing transcript. PMID- 3020033 TI - Polyanionic-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p dioxin. A comparison with the glucocorticoid receptor. AB - The interaction of the rat hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p dioxin (dioxin) with immobilized heparin (heparin-Sepharose) or DNA (DNA cellulose) has been compared to the polyanionic-binding properties of the rat hepatic glucocorticoid receptor. Both the nonoccupied and in vitro occupied forms of the receptors interacted with heparin-Sepharose but with varying strength, as determined by ligand binding assays or an enzyme-linked immunosorbent assay based on a monoclonal antibody against the steroid- and DNA-binding Mr approximately 94,000 glucocorticoid receptor protein. In the absence of ligand, both the dioxin and glucocorticoid receptors eluted from heparin-Sepharose at 0.1-0.2 M KCl, in contrast to the in vitro occupied receptor forms which eluted at 0.3-0.4 M KCl. Following elution of the in vitro occupied dioxin receptor from heparin Sepharose, it was efficiently retained on DNA-cellulose and eluted at an ionic strength of approximately 0.2 M KCl. In the presence of 20 mM sodium molybdate which is known to inhibit the activation of steroid hormone receptors to a DNA binding form, both the dioxin and glucocorticoid receptors eluted at 0.1-0.2 M KCl from heparin-Sepharose. In analogy to what has previously been shown for the glucocorticoid receptor, sodium molybdate stabilized a large dioxin-receptor complex with a sedimentation coefficient, S20,w, of 9-10 S, a Stokes radius of approximately 7.5 nm, and a calculated Mr of 290,000-310,000. Limited proteolysis of both the dioxin and glucocorticoid receptors with trypsin which is known to eliminate the DNA-binding property of both receptor forms also resulted in a decreased strength in the interaction of both in vitro occupied receptors with heparin-Sepharose (elution at 0.1-0.2 M KCl). In line with these data, calf thymus DNA in solution competed for receptor binding to heparin-Sepharose. In conclusion, the chromatographic properties of the dioxin receptor on heparin Sepharose are indistinguishable from those of the glucocorticoid receptor, and both receptors appear to be structurally and functionally closely related proteins. PMID- 3020034 TI - Isolation and characterization of homogeneous acetate kinase from Salmonella typhimurium and Escherichia coli. AB - Acetate kinase from Salmonella typhimurium and Escherichia coli was purified to electrophoretic homogeneity. The amino acid compositions of both proteins were similar, and the apparent molecular weights were the same, about 40,000 for the putative monomers. The native proteins gave higher molecular weights, suggesting that the enzymes may be oligomers, perhaps with two polypeptide subunits. Steady state kinetic studies were performed with the enzymes isolated from both organisms and the kinetic constants were determined. The Km values were 0.07 and 7 mM for ATP and acetate, respectively. In contrast to earlier studies using less pure preparations, the homogeneous enzymes from both strains were active only with acetate but not with propionate or butyrate. The enzyme activity was cold labile, and the length of reactivation time in the presence of Mg X ATP and acetate was dependent on protein concentration, suggesting that the monomer may not be catalytically active. The enzyme was phosphorylated with [gamma-32P]ATP and the phosphoprotein was isolated. Phosphoacetate kinase was capable of transferring the phosphate group to either ADP or acetate. The accompanying paper (Fox, D. K., Meadow, N. D., and Roseman, S. (1986) J. Biol. Chem. 261, 13498 13503) shows that the phosphoryl group of phosphoacetate kinase can also be reversibly transferred to Enzyme I of the phosphoenolpyruvate:glycose phosphotransferase system. PMID- 3020035 TI - Phosphate transfer between acetate kinase and enzyme I of the bacterial phosphotransferase system. AB - Interactions between homogeneous acetate kinase and proteins of the phosphoenolpyruvate:glucose phosphotransferase system (PTS) were studied. The phosphorylation of D-glucose was followed spectrophotometrically using a coupled assay system, and acetate kinase and GTP were found to substitute for phosphoenolpyruvate provided that each of the PTS proteins was present in the mixture. To further define the phosphoryl transfer reaction pathway, the system was simplified to include only the homogeneous, soluble PTS proteins. 32P was transferred from [gamma-32P]ATP to the protein IIIGlc, but this transfer reaction required acetate kinase, and the PTS proteins Enzyme I and HPr. These results suggested that acetate kinase interacts with the first protein in the PTS sequence, Enzyme I. Acetate kinase was therefore incubated with [32P] phospho Enzyme I, and a direct transfer of the phosphoryl group was observed without the addition of any other protein. These results show that there is a reversible transfer of the phosphoryl group between Enzyme I and acetate kinase. The possible role of this interaction in regulating sugar uptake by the Krebs cycle is discussed. PMID- 3020037 TI - On the fidelity of DNA synthesis. Pyrophosphate-induced misincorporation allows detection of two proofreading mechanisms. AB - The effect of pyrophosphate on the fidelity of in vitro DNA synthesis has been examined. Pyrophosphate enhances misincorporation by Escherichia coli DNA polymerase I in copying phi X174 DNA. The increased misincorporation is directly proportional to the extent of inhibition of the rate of polymerization. In contrast, pyrophosphate is not detectably mutagenic with avian myeloblastosis virus DNA polymerase or DNA polymerases alpha and beta from animal cells, which lack associated proofreading activities. This suggests that increased misincorporation by pyrophosphate is not due to an increase in misinsertions by DNA polymerase, but rather due to inhibition of proofreading by pyrophosphate. However, the pyrophosphate-induced infidelity has a different specificity from, and is not competitive with, two experimental markers of 3'----5' exonuclease proofreading; i.e. the effects of the next nucleotide or the addition of deoxynucleoside monophosphates. These distinctive features suggest a second mode of proofreading susceptible to inhibition by pyrophosphate. This concept is discussed in relation to models for proofreading described in the literature. PMID- 3020036 TI - An EPR and electron nuclear double resonance investigation of carbon monoxide binding to hydrogenase I (bidirectional) from Clostridium pasteurianum W5. AB - Previous Mossbauer and electron nuclear double resonance (ENDOR) studies of oxidized hydrogenase I (bidirectional) from Clostridium pasteurianum W5 demonstrated that this enzyme contains two diamagnetic [4Fe-4S]2+ clusters and an iron-sulfur center of unknown structure and composition that is characterized by its novel Mossbauer and ENDOR properties. In the present study we combine ENDOR and EPR measurements to show that the novel cluster contains 3-4 iron atoms. In addition, we have used EPR and ENDOR spectroscopies to investigate the effect of binding the competitive inhibitor carbon monoxide to oxidized hydrogenase I, using 13C-labeled CO and enzyme isotopically enriched in 57Fe. Treatment of oxidized enzyme with CO causes the g-tensor of the paramagnetic center to change from rhombic to axial symmetry. The observation of a 13C signal by ENDOR spectroscopy and analysis of the EPR broadening show that a single CO covalently binds to the paramagnetic center. The 13C hyperfine coupling constant (Ac approximately equal to 21 MHz) is within the range observed for inorganic iron carbonyl clusters. The observation of 57Fe ENDOR signals from two types of iron site ([A1c] approximately 30-34 MHz; [A2c] approximately 6 MHz) and resolved 57Fe hyperfine interactions in the EPR spectrum from two nuclei characterized by [A1c] confirm that the iron-sulfur cluster remains intact upon CO coordination, but show that CO binding greatly changes the 57Fe hyperfine coupling constants. PMID- 3020039 TI - Modulation of receptors and cytotoxic response of tumor necrosis factor-alpha by various lectins. AB - The effects of various lectins on the interaction of the human cervical carcinoma cell line ME-180 with recombinant human tumor necrosis factor-alpha (rTNF-alpha) was investigated. rTNF-alpha is known to have cytotoxic effects on this tumor cell line and has been reported to interact with these cells through a single class of specific high affinity receptors (Kd = 0.45 nM; approximately 1790 binding sites/cell). Exposure of cells to concanavalin A (ConA) causes an approximately 2-fold increase in rTNF-alpha receptors without any significant change in their affinity constant (Kd = 0.36 nM; approximately 3662 binding sites/cell). This increase in receptor number is dependent on temperature, the time of exposure and dose of ConA, and does not require the synthesis of new proteins. In spite of an increased binding of rTNF-alpha to cells, the cell killing induced by rTNF-alpha is totally blocked by ConA. Cells are also protected by this lectin from the synergistic cytotoxic effects of rTNF-alpha and recombinant human interferon-gamma. Furthermore, it was also found that ConA decreases the rate of internalization and dramatically inhibits the release and degradation of rTNF-alpha by the cells. These results, overall, demonstrate that ConA increases total number of binding sites for rTNF-alpha but blocks the transduction of the signal for the cytotoxic response. PMID- 3020038 TI - Design of biologically active peptides with non-peptidic structural elements. Biological and physical properties of a synthetic analogue of beta-endorphin with unnatural amino acids in the region 6-12. AB - Modelling studies with beta-endorphin have clearly demonstrated that an amphiphilic secondary structural segment is a salient feature of the biologically active conformation of this 31-residue opioid peptide hormone. Here, we have initiated the synthesis of peptide models using unnatural building blocks by designing a beta-endorphin analogue (peptide 6) in which the hydrophilic linker region between the NH2-terminal enkephalin (residues 1-5) and the COOH-terminal helix (residues 10-28, sequence identical to that of peptide 3 in region 13-31, Fig. 1) consists of four units of gamma-amino-gamma-hydroxymethylbutyric acid connected by isopeptidic linkages. Peptide 6 has physical properties similar to that of peptide 3, as shown by surface monolayer and circular dichroism studies. The binding affinities of the two peptides to delta- and mu-receptors are also similar. In rat vas deferens assays, the present model is equipotent to peptide 3. The most striking result of all is the potent analgesic activity displayed by peptide 6 when injected intracerebroventricularly into mice. The potencies of peptides 6 and 3 are comparable in these assays. These studies clearly illustrate that one can use unusual building blocks to construct structural regions of synthetic analogues and still preserve the biological activity of peptide hormones. PMID- 3020040 TI - Receptor-cytoskeleton interactions and membrane traffic may regulate chemoattractant-induced superoxide production in human granulocytes. AB - When dihydrocytochalasin (dhCB) was added either prior to or after CHO-Met-Leu Phe (fMLP), the rate and duration of superoxide production in human granulocytes stimulated by fMLP was augmented. This effect was maximal when dhCB was added before fMLP, increasing the rate 1.5-3-fold. The effect of dhCB was progressively diminished for later additions and was undetectable after 6-10 min. The effects of dhCB could be blocked by the additional presence of 10 microM t-Boc-Phe-Leu Phe-Leu-Phe (where t-Boc is t-butoxycarbonyl) indicating a requirement for receptor occupancy. In the presence of dhCB, the reversible binding of fML[3H]P was elevated and the formation of slowly dissociating surface complexes of occupied receptor and cytoskeleton was inhibited. Myeloperoxidase and lactoferrin release from fMLP-stimulated cells was induced by dhCB but was only partially correlated with the potentiating effects of dhCB on superoxide production and receptor expression. To circumvent the complicating effects of degranulation on the analysis of the functional consequences of receptor-cytoskeletal associations, cells were also preincubated with 100 nM fMLP at 15 degrees C. Under these conditions, the majority of the surface receptors became irreversibly occupied and coisolated with the cytoskeletal fraction of the cell. Subsequent exposure of the cells to fMLP at 37 degrees C resulted in no superoxide production. This desensitization was blocked by dhCB which also inhibited coisolation of the ligand and cytoskeletons. Conversion of receptor to a slowly dissociating state may represent its trapping in an inactive form and would provide a role for receptor-cytoskeleton interactions in the termination of the granulocyte response to chemoattractants. The inhibition of such receptor "sequestration" and the induction of new receptor expression could, therefore, partially account for dhCB-induced potentiation of the fMLP response. PMID- 3020041 TI - Partial agonism in the glucagon receptor system is a consequence of the two-state rat hepatic receptor. AB - We have previously demonstrated that the glucagon receptor binds hormone to form a low affinity complex which, by a time- and temperature-dependent mechanism, is converted to a high affinity complex (Horwitz, E.M., Jenkins, W.T., Hoosein, N.M., and Gurd, R.S. (1985) J. Biol. Chem. 260, 9307-9315). In this report we have investigated the effects of agonist concentration, potency, and intrinsic activity on the characteristics of the two, interconvertible states of the glucagon receptor. As the glucagon concentration is increased from 0.02 to 0.50 nM, the initial velocity of binding increases. The conversion of a low affinity to a high affinity complex is the rate-limiting step in the overall binding reaction and approaches its maximal velocity as the hormone concentration exceeds 0.20 nM. At equilibrium, 87-90% of the hormone-receptor complexes are in the high affinity state at all hormone concentrations examined. [S-methyl-Met27]glucagon, a full agonist with reduced potency, binds to the two-state system in a manner analogous to that of native glucagon. The binding of N alpha-biotinyl-N epsilon acetimidoglucagon, a partial agonist with reduced potency, effects a two-state system where the high affinity state accounts for only 35% of the total hormone receptor complexes at equilibrium. We conclude that the formation of the high affinity complex is the rate-limiting step involved in glucagon binding; reduction in binding potency with full agonism is due to a reduction in the affinity of the ligand for the unoccupied receptor and not to an alteration of the interconversion of the two states, and decreased intrinsic activity is due to a quantitative decrease in conversion of the low to high affinity state. PMID- 3020042 TI - Nonequivalence in the electronic structure of the prosthetic groups between two alpha-subunits within deoxycobalthemoglobin as determined by single-crystal EPR spectroscopy. AB - An artificial hybrid hemoglobin, alpha(Co)2 beta(Fe)2, the alpha- and beta subunits of which contain cobaltous and ferrous protoporphyrins IX, respectively, and its complementary hybrid, alpha(Fe)2 beta(Co)2, were prepared from human hemoglobin, crystallized in the deoxy state, and examined by electron paramagnetic resonance (EPR) spectroscopy. The orientations of the porphyrin normals in these deoxy Fe-Co hybrid hemoglobins in terms of the g parallel signals, were closely coincident with those of the heme normals of deoxyhemoglobin determined by x-ray crystallography. Two sets of axially symmetric EPR signals were found in the alpha(Co)-subunits, whereas only one set was observed in the beta(Co)-subunits. Nonequivalence in the electronic structures of the prosthetic groups between the two alpha(Co)-subunits, designated alpha I and alpha II, within deoxy-alpha(Co)2 beta(Fe)2 hybrid hemoglobin was correlated to these two distinct EPR signals. The interaction between the epsilon-nitrogen of the imidazole ring of the proximal histidine and the cobaltous ion in deoxy-alpha I(Co)-subunit is different from that in the deoxy-alpha II(Co)-subunit. The absence of a strict molecular dyad axis in the deoxy-alpha(Co)2 beta(Fe)2 hybrid hemoglobin suggests that the affinity state of the alpha(Co)-subunits may be partially switched to the R-state having a higher affinity for oxygen. Upon partial ligation of carbon monoxide to the beta(Fe) subunits, the line width of the g parallel and perpendicular signals of the alpha II(Co)-subunit was found to become somewhat narrower without disruption of the crystal structure. This suggests that there may be very close contacts between the alpha- and beta-subunits of different hemoglobin molecules which appear to be responsible for stabilizing the deoxy crystal structure after partial ligation in the crystalline state. PMID- 3020043 TI - Loss of sigma-factor of RNA polymerase of Xanthomonas campestris pv. oryzae during phage Xp10 infection. AB - Ten min after infection of Xanthomonas campestris pv. oryzae by phage Xp10, a sharp decrease in the activity of the host RNA polymerase was observed. Host RNA polymerase from phage-infected and uninfected cells was purified, and their properties were compared. The enzyme from uninfected cells contained four polypeptides with Mr = 155,000, 155,000, 93,000, and 37,000, respectively, and assembled with a stoichiometry of alpha 2 beta beta' sigma. The enzyme from infected cells lacked the sigma-subunit. The enzyme from uninfected cells utilized Xp10 DNA and poly[d(A-T)] as templates, the enzyme from phage-infected cells failed to transcribe Xp10 DNA, but retained the ability to transcribe poly(A-T). The regions of the Xp10 genome transcribed by the two enzymes were also investigated. The enzyme from uninfected cells transcribed the leftmost 25 30% of the Xp10 genome. The enzyme from phage-infected cells also transcribed the same region, but the enzyme activity was very low. Other properties such as (a) the response to RNA polymerase inhibitors, (b) the effect of N-ethylmaleimide, (c) the requirement of Mg2+ and Mn2+, and (d) the optimum temperature and pH of the two enzymes were very similar. PMID- 3020044 TI - Specific binding of [3H]dihydrokadsurenone to rabbit platelet membranes and its inhibition by the receptor agonists and antagonists of platelet-activating factor. AB - Kadsurenone inhibits specifically and competitively the specific binding of 3H labeled platelet-activating factor ([3H]PAF) to rabbit platelet membranes. Since the 5-propyl analog of kadsurenone (dihydrokadsurenone) retains roughly the same potency as kadsurenone, [3H]dihydrokadsurenone was therefore synthesized through tritiation of kadsurenone. Specific binding of [3H]dihydrokadsurenone in rabbit platelet membranes is saturable. Scatchard analysis of binding data reveals the presence of a single class of binding sites with an equilibrium dissociation constant (KD) of 16.81 ( +/- 0.57) nM. The total number (Bmax) of detectable binding sites is 2.27 ( +/- 0.09) pmol/mg protein. Both C16- and C18-PAF fully displace the specific binding of (3H]dihydrokadsurenone (5 nM) with an identical ED50 of 3.6 X 10(-9) M. Dihydrokadsurenone and kadsurenone also displace the specific binding with roughly the same potency (ED50 = 4.4 X 10(-8) M). Several other PAF analogs and PAF receptor antagonists tested show relative potencies roughly similar to those found in the [3H]PAF-specific binding assay. Other pharmacological agents with no PAF antagonistic activities did not inhibit the specific binding of [3H]dihydrokadsurenone. These results agree with our previous conclusion that kadsurenone is a specific and competitive receptor antagonist and strongly suggest that PAF and the PAF receptor antagonists tested may interact at a common binding site in the PAF receptor. PMID- 3020045 TI - Organization and transcription of the gluconate operon, gnt, of Bacillus subtilis. AB - The gluconate (gnt) operon of Bacillus subtilis has been cloned and sequenced. Analysis of the sequence (5482 base pairs) revealed four open reading frames, each of which was preceded by a Shine-Dalgarno sequence. These four frames were designated from the 5'-end as gntR, gntK, gntP, and gntZ. The gntR and gntK genes overlapped by 5 bases. The gntK and gntP gene products (consisting of 513 and 448 amino acids) were identified as gluconate kinase and permease, respectively, by means of insertional inactivation and deletion analysis of these genes subcloned in plasmid pC194. The functions of the gntR and gntZ gene products (243 and 468 amino acids) are presently unknown. S1 nuclease mapping and subcloning in a promoter probe vector (pPL603B) provided evidence that the gnt operon was transcribed as a polycistronic mRNA. Besides the gnt promoter about 40 base pairs upstream of the gntR gene, we detected two overlapping internal promoters between the gntP and gntZ genes. The gnt transcripts terminate about 45 base pairs downstream of the gntZ gene. PMID- 3020046 TI - The eye lens has an active ubiquitin-protein conjugation system. AB - Using exogenous 125I-ubiquitin, ubiquitin-lens protein conjugation was observed with supernatants of cultured rabbit lens epithelial cells and lens cortex tissue. Conjugation was ATP-dependent with the greatest variety and amount of conjugates larger than 150 kDa. In vivo production of ubiquitin-protein conjugates in cultured rabbit and beef lens epithelial cells and rabbit lens tissues of different developmental age was established using immunological detection. There were limited similarities between conjugates found in youngest as opposed to oldest tissue. Cultured rabbit cells contained 27 pmol/mg free ubiquitin and 18 pmol/mg conjugated ubiquitin. Levels of free ubiquitin in lens tissue epithelium, cortex, and core were 36, 5, and 5 pmol/mg, respectively. There were only 2 pmol/mg conjugated ubiquitin in each of these tissues. Hydrolysis of 125I-ubiquitin was catalyzed by supernatants of cultured lens cells, beef and human lens tissues, and reticulocytes. Degradation was greatest in epithelial tissues, and least in core. This corroborates studies which show that proteolytic capabilities are attenuated in older tissue. Decreased initiation of proteolysis by ubiquitination as well as diminished proteolysis in older lens tissue may be related to the accumulation of damaged proteins in aging lens tissue. PMID- 3020047 TI - Immunoreactivity and ouabain-dependent phosphorylation of (Na+ + K+) adenosinetriphosphatase catalytic subunit doublets. AB - Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 6% polyacrylamide was used to resolve the 100-kDa catalytic (alpha subunit) polypeptide of (Na+ + K+)-adenosinetriphosphatase from various tissues. The catalytic subunit was identified on immunoblots with antisera against mouse brain catalytic subunit and lamb kidney holoenzyme. Immunoblots and Coomassie Blue-stained companion gels showed double species of the 100-kDa subunit in sucrose gradient fractions of mouse brain and kidney, bovine grey and white matter, purified lamb kidney and duck salt gland holoenzyme, electroplax microsomes, and NaI-extracted microsomes of goldfish and rat brain. The apparent molecular mass differences between the two species in each tissue all ranged between 5 and 8 kDa. Both forms in rat brain and lamb kidney enzyme contain common epitopes reactive with antibodies immunoaffinity-purified on either species from mouse brain. In addition, ouabain dependent acid-stable inorganic phosphate incorporation was tested with mouse brain, lamb kidney, and electroplax enzyme. Ouabain-dependent phosphorylation was demonstrated in both species in lamb kidney and electroplax and in the larger of the two forms in mouse brain. These results suggest that double species of the phosphorylatable subunit are present generally in epithelial as well as excitable tissues and in fish and avian as well as mammalian species. Work is needed to elucidate their qualitative and quantitative characteristics in different tissues. PMID- 3020048 TI - Structure and expression of the rat apolipoprotein E gene. AB - The rat apolipoprotein E gene was isolated from a genomic library by screening with a cDNA probe. The nucleotide sequence of the gene plus a 5' flanking region of 623 nucleotides and a 3' flanking region of 234 nucleotides was determined. The gene contained three introns, which were located in the 5' nontranslated region and in the regions coding for the signal peptide and the mature protein. A "TATA box" sequence TATAATT was found beginning at nucleotide -32. Two copies of the sequences GGGCGG or CCGCCC, potential binding sites for the transcription protein factor Sp 1, were found adjacent to the TATA box, beginning at nucleotides -52 and -72. The 5' proximal flanking region, up to about 140 nucleotides, was found to be highly conserved (82% homology) in the gene for rat and human. This region was GC-rich (68% G + C) and contained self-complementary sequences with the potential to form hairpin loop structures, suggesting a regulatory role of this region in gene transcription. Two open reading frames with promoter sequences were found, one located in the first and the other in the second intron. Expression of rat apolipoprotein E mRNA in murine L cells transfected with the cloned gene was detected at levels comparable to that in rat liver. Rat apolipoprotein E expressed in transfected cells was secreted from the cells into the medium. PMID- 3020049 TI - A calcium-dependent 35-kilodalton substrate for epidermal growth factor receptor/kinase isolated from normal tissue. AB - We have previously reported the isolation of a 35-kDa protein from A-431 cells that, in the presence of Ca2+, can serve as a substrate for the epidermal growth factor (EGF) receptor/tyrosine kinase (Fava, R.A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). We now report the detection of an antigenically related 35 kDa protein in a number, but not all, of rat, pig, and human tissues. These antigenically related proteins also can serve as substrates for the EGF receptor/kinase in the presence of Ca2+. All of these proteins share the property of reversible, Ca2+-dependent binding to the particulate fraction (presumably membranes) of cell homogenates. We have isolated the 35-kDa substrate from porcine lung and have demonstrated that it is a Ca2+-binding protein. The amino terminal sequence and the site of tyrosine phosphorylation therein have been determined. The positions of the acidic amino acid residues amino-terminal to the tyrosine phosphorylation site bear a distinct resemblance to the sequence in the homologous region of a number of other substrates for tyrosine kinases. Based on available data, the 35-kDa protein clearly differs from the protein I complex derived from intestinal mucosa and thought to be related to the proteins isolated herein (Gerke, V., and Weber, K. (1985) J. Biol. Chem. 260, 1688-1695). Finally, we report a striking sequence homology between the porcine 35-kDa described herein and human lipocortin, a phospholipase A2 inhibitor. PMID- 3020050 TI - Rat glutathione S-transferases supergene family. Characterization of an anionic Yb subunit cDNA clone. AB - High multiplicity of GSH S-transferases (GST) with overlapping substrate specificities may be essential to their multiple roles in xenobiotics metabolism, drug biotransformation, and protection against peroxidative damage. Subunit composition analysis of rat liver GSH S-transferases indicated that heterodimer associations were not random, limiting the generation of GST isozyme multiplicity. We have analyzed a Yb subunit cDNA clone, pGTR187, that may correspond to an anionic Yb subunit sequence. Comparison with other GSH S transferase cDNA sequences and blot hybridization results indicates that the multiple Yb subunits are encoded by a multigene family. This Yb subunit sequence has very limited homology to Ya and Yc subunit cDNAs, but slightly more sequence homology to the Yp subunit cDNA. More consistent sequence homology is found at the amino acid level with 28% conservation throughout the coding sequences. These results and results published from other laboratories clearly indicate that rat GSH S-transferases are products of at least four different gene families that constitute a supergene family. Conceptually, the supergene family may encode GSH S-transferases of very different structures that are essential to metabolize a multitude of xenobiotics in addition to serving other physiologically important functions. PMID- 3020052 TI - The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing. AB - The complete nucleotide sequences of rat M1- and M2-type pyruvate kinase mRNAs were determined by sequencing the cDNAs and by analyses of S1 nuclease mapping and primer extension. The sequences have an identical molecular size of about 2220 nucleotides excluding a poly(A) tail and include 1593-nucleotide coding region. Their nucleotide sequences are identical except for 160-nucleotide sequences within the coding regions. The amino acid sequences of the M1- and M2 type subunits deduced from the cDNA sequences differ by only 45 residues within domain C, which constitutes the main region responsible for intersubunit contact. The sequence of this region of the M2-type shows higher homology than that of the M1-type with the corresponding sequence of the L-type. Since the M2- and L-types are allosteric enzymes, unlike to the M1-type, the residues common to the M2- and L-types, but not the M1-type may be important for mediating the allosteric properties. Genomic clones encoding both M1- and M2-type isozyme mRNAs were isolated. By partial sequence analysis of a clone lambda MPK37 four exons were identified, of which two adjacent exons coded the M1- and M2-specific sequences, respectively. The two remaining exons present downstream coded amino acids common to the two isozymes. Thus, we conclude that the M1- and M2-type isozymes of pyruvate kinase are produced from the same gene probably by alternative RNA splicing. PMID- 3020051 TI - Pulse-chase studies of the synthesis and intracellular transport of apolipoprotein B-100 in Hep G2 cells. AB - The synthesis and secretion of apolipoprotein B-100 (apoB-100) have been studied in a human hepatoma cell line, the Hep G2 cells. The time needed for the synthesis of apoB-100 was estimated to be 14 min, which corresponds to a translation rate of approximately 6 amino acids/s. ApoB-100 was compared with albumin and alpha 2-macroglobulin as to the distribution between the membrane and the luminal content in the endoplasmic reticulum (ER) and the Golgi apparatus. The results suggested that apoB-100 approximately followed the distribution of these secretory proteins in the Golgi, while the ratios between the percent membrane-bound apoB-100 and percent membrane-bound albumin or alpha 2 macroglobulin were 3-4:1 in the ER. This may suggest that apoB-100 occurs in a membrane-associated form in ER prior to the integration in the lipoproteins. Pulse-chase studies combined with subcellular fractionation was used to investigate the kinetics for the intracellular transfer of apoB-100. A 3-min pulse of [35S]methionine was followed by an increase in apoB-100 radioactivity in the ER during the first 10-15 min of chase. The following 10-15 min of chase were characterized by linear decrease in apoB-100 radioactivity with a decay rate of approximately 6%/min. The residence kinetics for apoB-100 in the ER differed from that of transferrin and probably also from that of albumin. By comparing the time for the pulse maximum in ER with that in the denser Golgi fractions the time needed for the transfer between ER and Golgi could be estimated to be 10 min. The time needed for the secretion of newly synthesized apoB-100 was estimated to be 30 min. This indicates that the transfer of the protein through the Golgi apparatus to the extracellular space requires 20 min. PMID- 3020053 TI - Perturbation of mitochondrial composition in muscle by iron deficiency. Implications regarding regulation of mitochondrial assembly. AB - It has been reported that the mitochondrial cytochromes and citrate cycle enzymes occur in constant proportions to each other and increase or decrease roughly in parallel in response to various stimuli. The purpose of this study was to determine whether this proportionality is an obligatory consequence of the way in which mitochondria are assembled. Severe iron deficiency was used to bring about decreases of the iron-containing constituents of the mitochondrial respiratory chain in skeletal muscle. Cytochrome c concentration and cytochrome oxidase activity were decreased approximately 50%, while succinate dehydrogenase and NADH dehydrogenase activities were decreased by 78% in iron-deficient muscle. On electron microscopic examination, mitochondria in iron-deficient muscles had relatively sparse numbers of cristae. The iron deficiency had little or no effect on the levels of a range of mitochondrial matrix enzymes, including citrate synthase, isocitrate dehydrogenase, fumarase, aspartate aminotransferase, 3 hydroxyacyl-CoA dehydrogenase, 3-ketoacid-CoA transferase, and acetoacetyl-CoA thiolase. These results show that the usual constant proportions between the constituents of the mitochondrial respiratory chain and matrix enzymes are not obligatory; they provide evidence that mitochondrial matrix enzymes and respiratory chain constituents can be incorporated into mitochondria independently and that the ratios between them can vary within wide limits. PMID- 3020054 TI - Characterization of 5'-flanking region of heart myosin light chain 2A gene. Structural and functional evidence for promoter activity. AB - Two recombinant clones, lambda LC5 and lambda LC13, encompassing the entire regulatory myosin light chain 2 (MLC2A) gene of chicken heart muscle were isolated. Of these, lambda LC5 which contains a large 5'-flanking sequence of about 7.0 kb, was characterized by a partial nucleotide sequence analysis. A TATA like sequence (TATTTTTA) and a CAAT-box (CAAAAGT) are located at positions -32 and -59, respectively, which most likely constitute the functional promoter region in the gene. Based on primer extension reaction with a synthetic 20-mer corresponding to the 5'-leader sequence and total poly(A+) RNA, the probable transcription initiation site in the gene was located. The gene promoter activity was demonstrated following transient expression of recombinant genomes containing the chicken upstream sequence fused to the bacterial chloramphenicol acetyltransferase (CAT) or to the rat preproinsulin II genes. The extracts from a Quail fibroblast cell line (QT35) transfected with the construct (pLCo5.2iCat) containing the putative chicken promoter, and the CAT gene promoted the formation of 3'-acetate chloramphenicol. Another construct (pBC12LC5.2f) contains the rat preproinsulin II gene placed under the control of chicken promoter and a simian virus 40 origin of replication. Transfection of COS cell line with pBC12LC5.2f DNA resulted in an efficient expression of rat preproinsulin mRNA initiating from the chicken promoter. The transfection assay also allowed detection of chicken MLC2A gene transcripts by S1-nuclease protection of end-labeled DNA probes. A comparison of the MLC2A upstream gene sequence with those available for skeletal myosin light chains revealed no common sequence elements, suggesting that cardiac MLC2A gene promoter region has diverged considerably from its counterparts in skeletal muscle. PMID- 3020055 TI - Inactivation of poliovirus with beta-propiolactone. AB - The recovery of poliovirus D-antigen after virus inactivation was studied for two inactivating agents (beta-propiolactone and formalin) using the three poliovirus types (Sabin types 1, 2 and 3). With beta-propiolactone (BPL), D-antigen recoveries were high (88, 88 and 60%, respectively) but were significantly less when formalin was used (22, 15 and 25%). beta-Propiolactone inactivated virus was purified, combined with Freund's adjuvant and used to hyperimmunize rabbits. High titres (50 000-200 000) of specific neutralizing antibody were obtained. PMID- 3020056 TI - Standardization of U.S. Reference Rh0 (D) Immune Globulin by quantitative automated hemagglutination. AB - An automated hemagglutination procedure was used to assess the relative potency of US Reference Rh0 (D) Immune Globulin, Lot 3, with respect to the International Reference Preparation, WHO Anti-D immunoglobulin, Lot 68/419. A value of 300 international units (IU) of anti-D per ampoule has been assigned to Lot 68/419. In 25 assays, the mean value for Lot 3 was 820 IU anti-D per milliliter when tested in parallel with Lot 68/419. PMID- 3020057 TI - The antibody response of seronegative infants to inactivated poliovirus vaccine of enhanced potency. AB - The immunogenic efficacy of inactivated poliovirus vaccine of enhanced potency (IPV-E), containing 40, 8 and 32 D-antigen units of types 1, 2 and 3, respectively, was evaluated in tritypic seronegative infants. Eighty infants aged six to 45 weeks, with no antibody detectable at a 1 : 4 dilution, were given two doses of a quadruple vaccine containing diphtheria-pertussis-tetanus (DPT) vaccine and IPV-E at intervals of four weeks (37 infants, group 1) or eight weeks (43 infants, group 2) between doses. All infants of group 2 responded with antibody to the three types of polioviruses. In group 1, all responded to types 1 and 3 antigens but only 36 responded to type 2. Antibody titres were higher in infants immunized at eight week than at four week intervals. Thus, two doses of IPV-E, especially when given eight weeks apart, are sufficient for primary immunization against poliomyelitis. If DPT vaccine of enhanced potency is combined with IPV-E, two doses of such a quadruple vaccine may be sufficient for primary immunization against four diseases; this possibility deserves evaluation. PMID- 3020058 TI - A rapid and automated colorimetric assay for evaluating the sensitivity of herpes simplex strains to antiviral drugs. AB - A rapid and sensitive colorimetric assay has been developed to evaluate the sensitivity of herpes simplex viruses (HSV) to antiviral agents. The chessboard titration of viruses and antiviral drugs and the automatic reading and analysis of the data allows objective and accurate results to be rapidly obtained. Virus sensitivity was expressed as an ED50 value which was the concentration of drug (micrograms/ml) reducing viral cpe by 50%. The ED50 values of antiviral drugs [acetylguanosine (ACV), idoxuridine (IDU), deoxycytidine (IDC) and bromovinyl deoxyuridine] for several HSV reference strains were determined and the method was then applied to clinical specimens. The selection of ACV and IDU resistant mutants was performed on a cloned sensitive HSV 1 ocular strain. We observed cross-resistance between ACV and IDU for the mutants isolated. The resistance to thymidine-kinase-dependent antiviral agents varied in inverse ratio to the thymidine kinase activity induced by HSV strains. PMID- 3020059 TI - The biological performance of calcium phosphate ceramics in an infected implantation site: II. Biological evaluation of hydroxyapatite during short-term infection. AB - Macroporous hydroxyapatite was implanted submucosally in the rat middle ear and studied after intratympanic injection of a Staphylococcus aureus suspension. The middle ear infection was induced 1 week after the implantation, and the effects of infection on the middle ear and the implant material were evaluated after 1, 3, 7, and 14 days by light and electron microscopy. The findings in the infected middle ear with an implant corresponded well with those described for the infected middle ear cavity without an implant. The reactions of the tissue over the implant were similar to those of the original mucosa of the middle ear. Bone was deposited on the implant and in its pores in relatively large quantities. Biodegradation, due at least partially to phagocytic activity of macrophages and multinucleated cells, was more prominent than previously found. This higher degree of biodegradation may be attributed to the use of the mucosal implantation technique, because this was the only point of divergence with respect to material or methods from earlier work reported by our group. The present results, together with those published earlier, suggest that this material has promising features for use as a bone substitute in reconstructive middle ear surgery. Definitive conclusions on biological performance and biofunctionality will, however, have to await long term clinical trials. PMID- 3020060 TI - The biological performance of calcium phosphate ceramics in an infected implantation site: I. Biological performance of hydroxyapatite during Staphylococcus aureus infection. AB - In the present study the biological performance of macroporous and dense hydroxyapatite after implantation in the rat middle ear was evaluated during an induced Staphylococcus aureus middle ear infection. The course of the infection was similar to that in the absence of an implant. Hydroxyapatite was frequently integrated with fibrous ingrowths in the middle ear lumen, originating solely from the infection. Good epithelial covering of the implant with all types of epithelial cells of importance for middle ear defence, was found. Increase of the exudate in the pores due to the infection was relatively small, and most of the exudate was restricted to pores on the implant surface. The bony tissue in the pores was not influenced significantly by the induced infection. Degradation of hydroxyapatite was consistent with earlier results obtained in the non-infected middle ear. The results obtained so far suggest that hydroxyapatite is highly suitable for middle ear implantation. PMID- 3020061 TI - Superior vena caval syndrome in primary mediastinal germ cell tumors. AB - Upon examination, nine of 45 patients (20%) with primary mediastinal germ cell tumors were found to have superior vena caval syndrome. All patients were men (average age, 27). Superior vena caval syndrome was found with all mediastinal germ cell tumor cell types except mature teratoma. Primary mediastinal germ cell tumors should be strongly considered in the diagnosis of young men with a mediastinal mass and superior vena caval syndrome. PMID- 3020062 TI - Collagenase production by smooth muscle: correlation of immunoreactive with functional enzyme in the myometrium. AB - A monospecific antibody to rat uterine collagenase has been produced and employed to study the cell of origin and the time course of production of this enzyme in the involuting rat uterus. The specificity of the anti-collagenase antibody was confirmed by immunoprecipitation, Western analysis, and by its ability to inhibit the activity of collagenase. Parallel measurements of functional enzyme, both latent and active, bound to tissue collagen were also made in nonpregnant, gravid, and postpartum rat uteri. Immunohistochemical staining of collagenase in sections of rat uterus showed the enzyme to be present in the perinuclear region of the smooth muscle cells only of the involuting myometrium. No detectable collagenase was present in the prepartum or nonpregnant uterus. Identity of the smooth muscle cells was confirmed using an anti-smooth muscle actin antibody. In addition, the cultured uterine cells from which the immunizing antigen was obtained were also identified as smooth muscle cells. Specificity of the tissue staining was confirmed by the ability of pure rat uterine collagenase to block the reaction of the antibody with the tissue. These observations indicate that smooth muscle cells are capable of producing collagenase and are consistent with the hypothesis that this enzyme presides over the massive collagen degradation seen in postpartum uterine involution. Furthermore, measurement of collagenase bound to uterine collagen revealed that collagenase activity could be detected only at the time that the cells could be seen to be producing the enzyme by immunolocalization. These findings support the concept that collagenase is produced only as needed and not stored, either intra- or extra-cellularly. PMID- 3020063 TI - Effects of transformation on the expression of laminin and fibronectin by neural cells. AB - We have studied laminin and fibronectin expression in a collection of rat cerebellar neural cell lines transformed with a mutant of Rous sarcoma virus which is temperature sensitive for transformation. We show that regardless of their neuronal or glial properties the cell lines produce both laminin and fibronectin. Laminin is expressed in similar amounts in cell lines grown at either the permissive or nonpermissive temperature for transformation, while fibronectin is generally expressed at higher levels in cells kept at the nonpermissive temperature. To provide further evidence that neural cells can produce laminin and fibronectin, double label immunofluorescence studies were conducted on primary cerebellar cultures. Both laminin and fibronectin were found to be present in the primary culture, and laminin was found to be associated with a subpopulation of astrocytes. PMID- 3020065 TI - Na+-dependent amino acid transport is a major factor determining the rate of (Na+,K+)-ATPase mediated cation transport in intact HeLa cells. AB - Little is known concerning the effects of Na+-coupled solute transport on (Na+,K+)-ATPase mediated cation pumping in the intact cell. We investigated the effect of amino acid transport and growth factor addition on the short term regulation of (Na+,K+)-ATPase cation transport in HeLa cells. The level of pump activity in the presence of amino acids or growth factors was compared to the level measured in phosphate buffered saline. These rates were further related to the maximal pump capacity, operationally defined as ouabain inhibitable 86Rb+ influx in the presence of 15 microM monensin. Of the growth factors tested, only insulin was found to moderately (22%) increase (Na+,K+)-ATPase cation transport. The major determinant of pump activity was found to be the transport of amino acids. Minimal essential medium (MEM) amino acids increased ouabain inhibitable 86Rb+ influx to a level close to that obtained with monensin, indicating that the (Na+,K+)-ATPase is operating near maximal capacity during amino acid transport. This situation may apply to tissue culture conditions and consequently measurements of (Na+,K+)-ATPase activity in buffer solutions alone may yield little information about cation pumping under culture conditions. This finding applies especially to cells having high rates of amino acid transport. Furthermore, rates of amino acid transport may be directly or indirectly involved in the long-term regulation of the number of (Na+,K+)-ATPase molecules in the plasma membrane. PMID- 3020064 TI - Prostaglandin E2 synthesis in the inner medullary collecting duct of the rat: implications for vasopressin-dependent cyclic AMP formation. AB - The effects of osmolality on prostaglandin E2 (PGE2) biosynthetic capacity and the interaction between endogenous PGE2 synthesis and vasopressin (AVP)-dependent cyclic AMP generation were examined in papillary collecting ducts (PCD) microdissected from collagenase-digested rat kidneys. Increasing medium osmolality with NaCl:urea (1:2 molar ratio) progressively increased PGE2 synthesis in PCD up to 1,500 mOsm. Addition of NaCl:urea or NaCl alone were equally effective in stimulating PGE2 biosynthetic capacity in PCD. In contrast, addition of urea alone had a much smaller stimulatory effect on PGE2 synthesis. Inhibition of endogenous PGE2 synthesis with naproxen (10(-5)M) suppressed AVP dependent cAMP formation in PCD when incubated in 300 mOsm medium but had no effect when incubated in 1,500 mOsm medium. Addition of 2.5 X 10(-5) M PGE2 also suppressed AVP-dependent cAMP formation in PCD only when incubated in 300 mOsm medium. The present study suggests that the PCD is a site of active PGE2 synthesis that is modulated by osmolality. Our results do not support the concept that endogenous PGE2 antagonizes vasopressin action via inhibition of AVP dependent cAMP formation. PMID- 3020067 TI - Liver autophagy in the influenza B virus model of Reye's syndrome in mice. AB - Biochemical evidence is presented for the autophagic destruction of liver mitochondria in the influenza B virus model of Reye's syndrome in mice. Separation of lysosomes and autophagic vacuoles from mitochondria was accomplished by prior treatment of the mice with Triton WR-1339, resulting in uptake of detergent by these organelles (tritosomes), reducing their densities. The organelles were banded in a discontinuous sucrose gradient. Total protein in the heavy tritosomal fraction increased from 1-2% in controls to 7-8% in virus treated animals. Ornithine carbamoyl transferase (OCTase), a mitochondrial marker, increased from 2-3% (controls) to 11-15% (virus-treated), and glucose-6 phosphatase, a marker for endoplasmic reticulum, increased from 1-2% (controls) to 8-10% (virus-treated). beta-Galactosidase, a soluble enzyme in the lysosome, and OCTase also increase in the cell extract fraction following virus treatment, indicating that there was turnover of heavy lysosomal contents. PMID- 3020066 TI - Solubilization and assay of a colony-stimulating factor receptor from murine macrophages. AB - The colony-stimulating factor, CSF-1, selectively stimulates the survival, proliferation, and differentiation of mononuclear phagocytes. The solubilization, assay, and characteristics of the CSF-1 receptor from the J774.2 murine macrophage cell line are described. The recovery of cell-surface receptor in the postnuclear supernatant membrane fraction of hypotonically disrupted cells was 76%. Recovery of the ligand binding activity of the receptor after solubilization of this fraction with 1% Triton X-100 was approximately 150%. The binding of 125I CSF-1 to intact cells and membrane preparations was consistent with the existence of a single class of high-affinity receptor sites. In contrast, the equilibrium binding of 125I-CSF-1 to the solubilized postnuclear fraction indicated the existence of two distinct classes of binding site (apparent Kds 0.15 nM and 10 nM). A rapid assay was developed for the high-affinity sites, which were shown to be associated with the CSF-1 receptor. The function of the low-affinity sites, which have not been demonstrated on intact cells or cell membranes and which are 13 times more abundant than the high-affinity sites, is unknown. The solubilized high-affinity receptor-CSF-1 complex was stable on storage at 0 degrees C and -70 degrees C but dissociated at 37 degrees C. Dissociation also occurred at 0 degrees C in buffers of low pH (4.0) or high ionic strength (0.7 M NaCl). PMID- 3020068 TI - Antipeptide antibodies directed against the carboxy-terminal region of SV40 structural proteins VP2 and VP3. AB - Rabbits were immunized with a synthetic heptapeptide of the sequence Arg-Asn-Arg Ser-Ser-Arg-Ser corresponding to the carboxy-terminal region of the SV40 viral proteins VP2 and VP3. The raised antibodies recognize the viral proteins in enzyme-linked immunosorbent (ELISA) and Western blot assay. Specificity of the antibodies were confirmed by competition experiments. The antibodies recognize VP2 and VP3 in infected cells by immunofluorescence and in subcellular fractions by ELISA. No interaction with virions was observed. PMID- 3020069 TI - Amplification of the N-myc oncogene in an adenocarcinoma of the lung. AB - c-myc oncogene is the most extensively studied member of the myc gene family, which now consists of three characterized members, namely the c-myc, N-myc, and L myc genes. Deregulation owing to amplification and/or rearrangements of the c-myc gene have been described in a variety of human malignancies. Several neuroblastomas have amplifications of the N-myc genes. The c-myc, N-myc, or L-myc oncogenes are also found amplified in different cell lines from small cell carcinomas of the lung. In this study, we have examined the c-myc, N-myc, and c erbB oncogenes in 34 clinical and autopsy tumor specimens representing various histopathological types of human lung cancer, including nine small cell lung cancers. A 30-fold amplification of the N-myc gene was found in a tumor histopathologically and histochemically verified as a typical adenocarcinoma. No amplifications of the c-myc or c-erbB oncogenes were seen in any of the tumors. In the DNA of one small cell carcinoma, an extra c-myc and N-myc cross hybridizing restriction fragment was observed, possibly owing to an amplification of a yet uncharacterized myc-related gene. PMID- 3020070 TI - Effects of a stable enkephalin analogue, (D-Met2,Pro5)-enkephalinamide, and naloxone on cortical blood flow and cerebral blood volume in experimental brain ischemia in anesthetized cats. AB - The effects of intracarotid injection of the stable enkephalin analogue (D Met2,Pro5)-enkephalinamide (ENK) and intravenous administration of naloxone on the cerebrocortical blood flow (dye dilution method) and cerebral blood volume (CBV) (photoelectric method) were investigated during unilateral brain ischemia in anesthetized cats. Both parameters were measured simultaneously in the intact and ischemic (middle cerebral artery occluded) hemispheres. An intracarotid injection of ENK 0.5 mg/kg induced a significant increase in cortical vascular resistance and a -87% decrease in cerebrocortical blood flow from 25 +/- 3 to 4 +/- 3 ml/100 g/min, without CBV alteration in the ischemic hemisphere. Naloxone (1 mg/kg i.v.), on the other hand, induced a marked two-fold increase in cerebrocortical blood flow and a significant elevation of CBV from 5.9 +/- 0.5 to 7.4 +/- 0.7 vol% in the ischemic hemisphere. No change in cerebrocortical blood flow or CBV was observed in the intact hemisphere either after ENK or after naloxone administration. Arterial blood gases and hematocrit remained unchanged. On the basis of the present findings, we conclude that besides other factors, endogenous opioid mechanisms may also participate in ischemic cerebrovascular reactions and the cerebral circulatory effects of naloxone probably reflect its opiate receptor blocking property and not simply its other non-opiate-related actions. PMID- 3020071 TI - Arthropathies associated with calcium-containing crystals. AB - Monosodium urate crystals are clearly related to acute attacks of gout and to the hard tissue destruction of chronic tophaceous gout. Fortunately, the acute attacks are readily treated with anti-inflammatory drugs, and destructive changes due to tophi may be prevented or reversed, at least in part, by the intelligent control of serum urate levels. Control of gout is one of the premier success stories of modern medicine. In contrast, the number of patients who have arthritis associated with crystals that contain calcium appears to be rising- perhaps a function of better recognition, perhaps related to the aging of the population. CPPD and BCP crystals can be associated with acute or subacute inflammation, but as in acute gout, it is easily controlled with anti inflammatory drugs or by local injections of corticosteroids. A direct relationship of BCP and CPPD crystals to the associated destructive arthropathies has been hypothesized and is supported by clinical observations, animal studies, and in vivo experiments. Unlike gout, which is usually associated with a systemic metabolic abnormality (i.e., hyperuricemia), calcium crystals deposition seem to be a localized phenomenon, although numerous local sites in several joints are often involved in a given patient. Tissue degeneration in gout clearly follows (tophaceous) crystal deposition. Calcium crystal deposition may follow, rather than precede, destructive joint changes. Alternatively, both destructive changes and crystal deposition may derive independently from a common, still obscure, biochemical abnormality of joint tissues. P. A. Dieppe and colleagues believe that calcium crystal deposition follows either primary or secondary tissue degeneration but that the crystals exert a positive feedback effect (amplification loop) that accelerates degeneration. Each of those formulations of a pathogenetic role for crystals may be true in a given case, analogous to the etiology of primary and secondary forms of hyperuricemia and to sodium urate crystal deposition coexistent with osteoarthritis (tophus formation in Heberden's nodes). Conclusive proof of a significant role for BCP or CPPD crystals in the pathogenesis of human joint tissue damage depends on interrupting the postulated disease mechanism and showing that this prevents joint deterioration and leads to significant repair of existing damage. Our current position is somewhat analogous to that of our colleagues who had to contend with management of gouty arthritis before the advent of effective drugs for control of hyperuricemia. PMID- 3020072 TI - Sensitive assay for serum angiotensin-converting enzyme and separation of angiotensin analogues by high-performance liquid chromatography with fluorescence detection. AB - A high-performance liquid chromatographic method is described for the assay of angiotensin-converting enzyme in human serum and for the separation of angiotensins and their analogues after pre-column fluorescence derivatization with benzoin. Angiotensin II, formed enzymatically from angiotensin I, is converted into a fluorescent derivative which is then separated isocratically from the substrate and biological substances in the enzyme reaction mixture on a reversed-phase column (TSK gel ODS-120T). The lower limit of detection for angiotensin II is 0.66 pmol per enzyme assay tube. The method is simple and sensitive, and requires as little as 5 microliter of human serum. Angiotensin analogues can also be separated and quantified by the chromatographic technique, and thus this method permits the use of the analogues of angiotensin I as substrates. PMID- 3020073 TI - Separation of phospholipids using a diethylaminoethyl-silica gel column and thin layer chromatography. AB - Phospholipids derived from egg yolk are readily separated by DEAE-silica gel column chromatography using stepwise gradient elution. The overall recovery of phospholipids from the column is 85-95% at a loading capacity of 120 mg of lipids per 10 g of DEAE-silica gel. The complete separation of eight phospholipids (phosphatidylcholine, sphingomyelin, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine and lysophosphatidylserine; 5 micrograms each) is also achieved by one-dimensional DEAE-silica gel thin-layer chromatography with the solvent system chloroform-methanol-water-pyridine-58% ammonia solution (130:55:8:4:4, v/v). PMID- 3020074 TI - Ectopic adrenocorticotropin syndrome caused by lung cancer that responded to corticotropin-releasing hormone. AB - ACTH responses to corticotropin-releasing hormone (CRH) were studied in three patients with the ectopic ACTH syndrome caused by lung cancer. Plasma ACTH responded to synthetic CRH in two of three patients. Tumor tissues obtained from these two patients contained CRH and ACTH. In one patient, tumor ACTH secretion was stimulated by CRH in vitro. Tumor CRH was immunologically, chromatographically, and biologically similar to hypothalamic CRH. In addition, multiple forms of immunoreactive beta-endorphin were present in plasma and the tumor extracts. From these results, we conclude that some patients with the ectopic ACTH syndrome have tumors that produce both ACTH and CRH and that CRH can stimulate ACTH secretion by such tumors. Other patients with the ectopic ACTH syndrome do not have ACTH responses to CRH. Therefore, procedures other than CRH testing are needed to differentiate patients with Cushing's syndrome due to ectopic ACTH/CRH production from those with Cushing's disease, since the latter also usually have ACTH responses to CRH. PMID- 3020075 TI - Stimulation of creatine kinase activity by calcium-regulating hormones in explants of human amnion, decidua, and placenta. AB - We have used stimulation of the activity of the brain type creatine kinase (CK) isoenzyme as a response marker to examine the effects of vitamin D metabolites, PTH, and calcitonin in cultured explants of placenta, decidua, and amnion from normal human deliveries. We found a biological response to PTH in placenta and amnion and to vitamin D metabolites in all three tissues. In the amnion, CK activity increased 2.3-fold after 24 h of incubation in 2.5 nM 1,25 dihydroxyvitamin D3 [1,25-(OH)2D3], 3.8-fold when incubated with 12.5 nM 24,25 dihydroxyvitamin D3 [24,25-(OH)2D3] and 2.7-fold when incubated with 10 U/ml bovine PTH. In the decidua, 24,25-(OH)2D3, but not 1,25-(OH)2D3 or bPTH caused a 1.7-fold increase in CK activity. In contrast, the placenta responded to 1,25 (OH)2D3 with a 1.6-fold increase in CK activity and to bPTH, with a 1.7-fold increase but did not respond to 24,25-(OH)2D3. Bovine calcitonin (100 ng/ml) had no effect on CK activity in any of the three tissues. Nearly all CK in both the unstimulated and stimulated explants was the brain type isoenzyme. CK activity increased significantly between 1 and 4 h after hormonal treatment in all experiments. The enzyme activity rose steeply with dose and reached a significant increase, and usually a plateau, at hormone concentrations considered to be physiological in vivo. [3H]Thymidine incorporation into DNA increased in parallel to stimulation of CK activity in all experiments, except that PTH did not increase DNA synthesis in the placenta. PTH did cause an increase in cAMP production in explants of amnion (1.5-fold) and placenta (2.6-fold). PMID- 3020077 TI - Effects of luteinizing hormone and dibutyryl adenosine 3',5'-monophosphate in cultured granulosa cells from polycystic ovaries. AB - The present study was undertaken to assess the ability of granulosa cells from subjects with normal and polycystic ovaries (PCO) to secrete progesterone throughout a 10-day culture period. LH levels in serum and follicular fluid from PCO patients were significantly (P less than 0.001) higher than those in normal subjects. In the absence of LH, progesterone secretion by granulosa cells cultured from PCO follicles did not differ significantly from that of cells from normal early and midfollicular phase follicles. Granulosa cells cultured from follicles from normal subjects in the early and midfollicular phases responded to LH (100 ng/ml) with an 8- to 20-fold increase in progesterone production. In contrast, LH increased progesterone production to a much lesser extent (up to 4 fold) in cells from the ovaries of patients with PCO. Progesterone secretion by granulosa cells from normal ovaries in response to LH diminished as intrafollicular endogenous progesterone and LH levels increased. Cells from PCO follicles cultured with (Bu)2cAMP (100 micrograms/ml) secreted progesterone in quantities comparable to those secreted by (Bu)2cAMP-stimulated normal ovaries in the early and midfollicular phases. These data demonstrate the discrepancy between the ability of granulosa cells from PCO and normal follicles to secrete progesterone in response to stimulation by LH and (Bu)2cAMP. These results suggest that in women with PCO, the persistent elevation of follicular LH may lead to impaired progesterone production in response to exogenous LH. PMID- 3020076 TI - The effect of exogenous hyperinsulinemia on proinsulin secretion in normal man, obese subjects, and patients with insulinoma. AB - To examine possible feedback inhibition of insulin on proinsulin secretion, we measured serum proinsulin levels before and after 120 min of euglycemic hyperinsulinemia (90-100 mU/liter) in 11 normal and 7 obese hyperinsulinemic subjects and 6 patients with beta-cell adenoma (n = 4), carcinoma, or hyperplasia. Baseline proinsulin levels accounted for 19%, 14%, and 56% of the total immunoreactive insulin in the 3 groups, respectively. Compared to normal subjects, baseline proinsulin levels were elevated (P less than 0.02) by 4- and 6 fold in obese subjects and patients with autonomous insulin secretion, respectively, but there was an overlap between the groups. In both normal and obese subjects, hyperinsulinemia suppressed proinsulin secretion by 45-50% (P less than 0.02), whereas no response occurred in the patients. Thus, the 120 min values were clearly different in the patients and the normal or obese subjects. After removal of the adenoma in 4 patients, baseline proinsulin levels and the response to hyperinsulinemia were normalized, but they remained elevated after a partial pancreatectomy or tumor removal in the patients with beta-cell hyperplasia or carcinoma. Thus, proinsulin secretion is under negative feedback control of insulin in both normal man and hyperinsulinemic obese subjects. In patients with insulinoma or beta-cell hyperplasia, this control is lost. PMID- 3020078 TI - High levels of corticotropin-releasing hormone immunoactivity in maternal and fetal plasma during pregnancy. AB - Corticotropin-releasing hormone immunoactivity (CRHi) was measured in the plasma of 31 pregnant women and 6 nonpregnant women as well as in the umbilical cord plasma of 40 term fetuses. CRHi was not detectable (less than 44 pg/ml) in the plasma of 6 nonpregnant women or in 6 women in the first trimester of pregnancy. Mean plasma CRHi rose progressively to 58 +/- 18 and 270 +/- 68 pg/ml during the second and third trimesters, respectively, and again became undetectable within 24 h after delivery. Mean CRHi in 40 umbilical cord plasma samples was 136 +/- 16 pg/ml. Gel filtration of both fetal and maternal plasma showed that the majority of the CRHi eluted in the same position as synthetic human CRH. There was no significant correlation between CRHi and either beta-endorphin or ACTH in umbilical cord plasma, suggesting that this CRHi may not be primarily responsible for the release of beta-endorphin and ACTH into fetal plasma at delivery. A close correlation (r = 0.82) was found between simultaneously obtained maternal and umbilical cord plasma CRHi in 10 maternal-fetal pairs, supporting a common source for this peptide in maternal and fetal circulation. A placental source for fetal and maternal CRHi was suggested by the finding of a higher CRHi concentration in the umbilical vein than in the umbilical artery and by the disappearance of this peptide from maternal plasma after delivery. We conclude that a large amount of CRHi is secreted by the placenta into both the maternal and fetal circulation during pregnancy and suggest that this may be an important modulator of the maternal and fetal hypothalamic-pituitary-adrenal axis during gestation. PMID- 3020079 TI - Short term administration of potassium perchlorate restores euthyroidism in amiodarone iodine-induced hypothyroidism. AB - We studied the effect of potassium perchlorate (KClO4) in patients with hypothyroidism due to amiodarone. The short term administration of KClO4 to six such patients led to prompt restoration of euthyroidism, while the three untreated patients remained hypothyroid for 2-6 months. Since KClO4 inhibits thyroid iodide transport, thereby blocking further entrance of iodide into the thyroid and decreasing intrathyroidal iodide content, amiodarone-associated hypothyroidism is probably secondary to the inhibitory effect of excess intrathyroidal iodine on thyroid hormone synthesis. PMID- 3020080 TI - Normal activity of nucleoside triphosphate pyrophosphatase in alkaline phosphatase-deficient fibroblasts from patients with infantile hypophosphatasia. AB - Inorganic pyrophosphate (PPi) influences the formation of bone mineral. In the rare inherited disease hypophosphatasia, abnormal extracellular metabolism of PPi occurs together with defective skeletal mineralization. The primary biochemical defect in this condition is a deficiency of the bone/liver/kidney (tissue nonspecific) isoenzyme of alkaline phosphatase (AP), an enzyme that catalyzes the extracellular breakdown of PPi. Fibroblast lines derived from patients with hypophosphatasia manifest the deficiency of AP activity that occurs in vivo and thus are a suitable model for this condition. Using these cells from patients with the severe (infantile) form of the disease, we examined aspects of PPi metabolism in hypophosphatasia, in particular the formation of PPi from ATP by ecto-nucleoside triphosphate (NTP) pyrophosphatase. This enzyme is believed to catalyze the extracellular generation of PPi in vivo. We found that normal fibroblasts possess ecto-NTP pyrophosphatase and that infantile hypophosphatasia cell lines have normal activity and cellular distribution of this enzyme compared with cell lines derived from age-matched normal subjects. This suggests that extracellular generation of PPi is normal in hypophosphatasia. The results also provide further evidence that ecto-NTP pyrophosphatase and AP are distinct entities and that hypophosphatasia does not involve a general loss of enzyme activities from cell surfaces. PMID- 3020081 TI - In vitro exposure of preimplantation bovine embryos to vesicular stomatitis virus. AB - Vesicular stomatitis virus New Jersey serotype (VSV-NJ) adhered to 14 of 20 zonae pellucidae intact (ZP-I) bovine embryos exposed in vitro. The VSV-NJ-exposed ZP-I bovine embryos were washed by a single- or multiple-pipette procedure. The multiple-pipette washing procedure was more efficient in removing unattached virus than the single-pipette procedure, but neither washing procedure was effective in consistently removing attached virus from ZP-I embryos. The virus plaque assay with Vero-MARU cells was more sensitive than was the suckling mouse intracerebral inoculation procedure for detection of VSV-NJ from the sonic extracts of bovine embryos. A maximum of 15 infective VSV-NJ particles were detected adhering to one virus-exposed, washed ZP-I bovine embryo. PMID- 3020083 TI - Immunofluorescence for detection of antibodies against human cytomegalovirus induced membrane antigens. AB - This paper describes an improved method for the in vitro detection of antibodies specifically directed against human cytomegalovirus (CMV)-induced membrane antigens present on the surface of CMV-infected fibroblasts (CMV-MA). Viable cells were found to be essential for specific visualization of CMV-MA staining. The addition of divalent cations (2.6 mM Ca2+ and 2.2 mM Mg2+) and glucose (180 mM) to the incubation and washing buffers improved the viability and morphology of the cells and increased the cell yield at the end of the assay. Clustering of antigen-antibody complexes on the surface of viable CMV-infected cells was prevented by low-temperature incubation (0 to 4 degrees C) rather than by the addition of agents which act on the metabolism of the cell. No interaction with the CMV-induced Fc receptor was observed at 0 degrees C with either human sera or murine monoclonal antibodies. The specificity of the CMV-MA reaction was confirmed by using monoclonal antibodies to CMV nuclear, cytoplasmic, and membrane-associated antigens. Furthermore, a microplate modification of the membrane fluorescence test is described which is suitable for multisample screening purposes. This method can be applied to the determination of anti-CMV MA antibody titers in human sera and to the screening of hybridoma supernatants for the presence of antibodies with specificity for CMV-MA. PMID- 3020082 TI - Comparison between children treated at home and those requiring hospital admission for rotavirus and other enteric pathogens associated with acute diarrhea in Melbourne, Australia. AB - The etiology of acute diarrhea in children less than 42 months of age attending one pediatric hospital in Melbourne, Australia, was studied during a 7-month period encompassing the winter of 1984. Pathogens identified in 157 children treated as outpatients with mild disease were compared with those in 232 children hospitalized with severe disease. The pathogens (and frequencies among outpatients and inpatients, respectively) detected were rotaviruses (32.5 and 50.9%), enteric adenoviruses (8.9 and 7.4%), Campylobacter jejuni (7.2 and 1.3%), and Salmonella sp. (4.0 and 1.7%). Electropherotypes of rotavirus strains from outpatients and inpatients were compared. Two strains predominated during the 7 months of this study and were observed with equal frequency from outpatients and inpatients. Rotaviruses of the same electropherotype caused a wide spectrum of disease, with symptoms ranging from mild to severe, life-threatening diarrhea. The similarity of etiological agents identified from children with mild and severe forms of acute diarrhea suggests that the etiology of community enteric illness can be reasonably inferred from the etiology of inpatient disease in children in the same geographic area. During the winter epidemic period, the severity of symptoms associated with rotavirus infection in young children is likely to be determined by the inherent susceptibility of the host rather than by genetic differences in the strains of infecting rotaviruses. PMID- 3020084 TI - Identification of Campylobacter pyloridis isolates by restriction endonuclease DNA analysis. AB - Campylobacter pyloridis isolates recovered from gastric biopsy specimens of 16 patients were examined by restriction endonuclease DNA analysis with HindIII. For 8 of these 16 patients two different isolates were compared to study the persistence of the colonizing strains and the stability of their DNA digest patterns during a period of 2 years (two patients), the identity or nonidentity of different colony types within one culture (two patients), and the nature of the relapses after apparently successful antibacterial therapy (four patients). The isolates from the 16 patients all produced different DNA digest patterns. Comparison of the two different isolates recovered from the same patients showed that these isolates were identical in all eight cases. Laboratory subculturing of a C. pyloridis strain (10 times) did not change its DNA digest pattern. These results indicate the stability of the DNA digest patterns and a marked variability of these patterns among isolates from different patients. Using restriction endonuclease DNA analysis, we found the persistence in the stomach of the same C. pyloridis strain during a period of 2 years and the identity of different colony types within one culture. The relapses after apparently successful antibacterial treatment could be attributed to recrudescence rather than reinfection. Restriction endonuclease DNA analysis is a sensitive and useful method for identifying C. pyloridis isolates. PMID- 3020085 TI - Enzyme-linked immunosorbent assays for Snow Mountain and Norwalk agents of viral gastroenteritis. AB - Enzyme-linked immunosorbent assays (ELISAs) for antigen detection and blocking ELISAs for serum antibody rises were developed for the Snow Mountain and Norwalk agents of viral gastroenteritis. The ELISAs were as sensitive as the existing radioimmunoassays and were specific for the Snow Mountain or Norwalk agent. The blocking ELISAs detected the same number of significant rises in antibodies to these agents as did the existing blocking radioimmunoassays. PMID- 3020087 TI - Viral isolation versus immune staining of infected cell cultures for the laboratory diagnosis of herpes simplex virus infections. AB - The Difco Laboratories Cellmatics kit, an immune staining method for the detection of herpes simplex virus after amplification in culture, was found to be 94.6% (138 of 146) sensitive and 100% (302 of 302) specific. However, conventional culturing detected 10 (6.4%) additional viruses that were not herpes simplex. For a full-service diagnostic laboratory, viral isolation is important. PMID- 3020086 TI - Reduction of nonspecific fluorescence in respiratory specimens by pretreatment with N-acetylcysteine. AB - Treatment of nasopharyngeal specimens with 0.25% N-acetylcysteine for 20 min at room temperature effectively reduced the nonspecific fluorescence encountered during direct examination of the specimens by immunofluorescence. The treatment did not affect specific antigen staining or the viability of several common respiratory viruses. PMID- 3020088 TI - Autoantibodies to Alzheimer and normal brain structures from virus-transformed lymphocytes. AB - B-Lymphocytes from two patients with Alzheimer's disease and one healthy subject were transformed into lymphoblastoid cells by exposure to Epstein-Barr virus. In culture, more than 50% of these cells secreted sufficient IgM or IgG antibody (mainly IgM) to allow immunohistochemical screening against cryostat sections of normal and Alzheimer temporal cortex. More than 30% of the IgM antibodies from each subject recognised brain components, namely: neurons, astrocytes, nuclei, nucleoli, and Alzheimer plaques and neurofibrillary tangles. This methodology represents a major addition to the procedures currently available for the generation of antibodies towards normal and pathological structures in human brain. PMID- 3020089 TI - Chronic caffeine administration exacerbates renovascular, but not genetic, hypertension in rats. AB - The purpose of this study was to determine whether or not caffeine would exacerbate renovascular hypertension. Therefore, we examined the effects of chronic caffeine administration on arterial blood pressure in rats subjected to either unilateral renal artery clipping (2K-1C rats) or sham-operation. Animals in each group were randomly assigned to receive either 0.1% caffeine in their drinking water or normal drinking water, and systolic blood pressure was monitored for 6 wk. Caffeine markedly exacerbated the severity of hypertension in 2K-1C rats and caused histological changes consistent with malignant hypertension. 6 wk after surgery, systolic blood pressure, plasma renin activity, and creatinine clearance in control 2K-1C rats were 169 +/- 5 mmHg (mean +/- SEM), 4.4 +/- 0.5 ng AI X ml-1 X h-1, and 2.9 +/- 0.2 ml/min, respectively; as compared with 219 +/- 4 mmHg, 31.8 +/- 7.8 ng AI X ml-1 X h-1, and 1.4 +/- 0.3 ml/min, respectively, in 2K-1C rats receiving caffeine (all values were significantly different compared with control 2K-1C). Chronic caffeine administration did not alter systolic blood pressure, plasma renin activity, or creatinine clearance in sham-operated rats or spontaneously hypertensive rats. Chronic treatment with enalapril (a converting enzyme inhibitor) prevented the development of hypertension in control 2K-1C rats and caffeine-treated 2K-1C rats; however, withdrawal of enalapril precipitated a rapid rise in systolic blood pressure in caffeine-treated 2K-1C rats, but not in control 2K-1C rats. These experiments indicate that caffeine specifically exacerbates experimental renovascular hypertension and might worsen the hypertensive process in patients with renovascular hypertension. PMID- 3020090 TI - Prostaglandin E2 and collagenase production by fibroblasts and synovial cells is regulated by urine-derived human interleukin 1 and inhibitor(s). AB - Interleukin 1 (IL-1) possesses multiple biological activities that may be blocked selectively by different inhibitors. Some known inhibitors block the lymphocyte activating factor (LAF/IL-1) but not the mononuclear cell factor (MCF/IL-1) measured by its capacity to stimulate prostaglandin E2 (PGE2) and collagenase production. The presence of IL-1 in vivo may be difficult to detect due to the presence of inhibitor(s) and the level of the inhibitor(s) may vary depending upon pathological conditions. We have found that urine from three patients with monocytic leukemia (M5) contained high levels of inhibitor(s) of MCF/IL-1, whereas urine of normal subjects did not contain significant amounts. Urine from two patients with other blood neoplasic diseases also contained little inhibitory activity. The MCF/IL-1 inhibitor(s), which also acts on human recombinant IL-1 beta, is approximately 25-35 kD, is not retained on concanavalin A-Sepharose column and can be partially destroyed with urea and boiling. At this stage of the purification the fraction containing the MCF/IL-1 inhibitor(s) also inhibits the LAF/IL-1 assay. However, this inhibitor(s) is probably distinct from other inhibitors already described. PMID- 3020093 TI - Parvovirus infection associated with aplastic crisis in a patient with HEMPAS. AB - An aplastic crisis associated with parvovirus infection occurred in a patient suffering from hereditary erythrocytic multinuclearity associated with a positive acidified (Hams) test (HEMPAS). This case emphasises that any patient who has a shortened red cell survival is susceptible to an aplastic crisis induced by parvovirus. PMID- 3020092 TI - T lymphocyte cloning from rejected human kidney allografts. Growth frequency and functional/phenotypic analysis. AB - Mechanically harvested lymphocytes invading an irreversibly rejected human kidney allograft were seeded at limiting dilution to calculate the frequency of growing precursors. Optimal growth frequency (1/13) was obtained when Epstein-Barr virus (EBV)-transformed donor B lymphocytes were used as stimulators (D-BLCL) in the presence of interleukin 2 (IL-2). The 55 clones analyzed were all T11+ and T3+, and all expressed DR antigens (45% were T8+ and 55% T4+). Only one clone had a double-labeled (T4+ T8+) surface. All cells proliferated significantly against D BLCL, although T4+ clones had a significantly shorter average doubling time than T8+ clones. Nearly all T8+ clones were specifically cytotoxic for D-BLCL, while both T4 and T8 did not react against K562, autologous EBV-BLCL, and third-party EBV-BLCL. Detectable IL-2 was found in the culture supernatants of only a minority of clones (all T4+). PMID- 3020091 TI - Properties and physiologic roles of the plasma membrane sodium-hydrogen exchanger. PMID- 3020094 TI - Human lung tumours may coexpress different classes of intermediate filaments. AB - Ninety four pulmonary neoplasms were examined immunocytochemically with two or three different monoclonal antibodies against the intermediate filament proteins cytokeratin, neurofilament, vimentin, and desmin. In normal tissues these have a different and non-overlapping distribution, and it is generally believed that tumours maintain the same pattern of expression as the tissues from which they arise. In this report, however, the coexpression of at least two (and less commonly three or four) different intermediate filaments was seen in 40% (37 of 94) of the cases of lung cancer. These results, especially if confirmed in other common types of human malignancy, have considerable implications for the use of anti-intermediate filament antibodies in diagnostic pathology. PMID- 3020096 TI - Growth fractions in breast cancers determined in situ with monoclonal antibody Ki 67. AB - The growth fractions of 160 mammary carcinomas and 30 benign mammary lesions were determined in situ by immunostaining with the monoclonal antibody Ki-67. Benign lesions had a mean value of 3% Ki-67 positive cells, whereas the mean value of mammary carcinomas was 16.6%. A comparison of the mean values of Ki-67 positive cells with the histological grade of the tumours showed a correlation between these two variables--that is, histological grade 1 showed 9%, grade 2 16%, and grade 3 26% proliferating cells. Considering the individual Ki-67 values in the different histological grades, it was evident that there was considerable scatter in the number of proliferating cells, so that the proliferation rates of grades 1, 2, and 3 overlapped each other. This indicates a dissociation between histological grade of malignancy and size of the growth fraction in most breast cancers. Follow up studies are needed to establish which of the two variables- that is, morphological degree of malignancy, or the proportion of Ki-67 positive cells--correlates better with response to treatment and survival in individual cases. PMID- 3020095 TI - Immunocytochemical study of 18 tumours causing ectopic Cushing's syndrome. AB - Eighteen cases of Cushing's syndrome caused by ectopic production of peptide hormones were investigated by histological and immunocytochemical methods and the findings correlated with clinical and biochemical observations. Immunocytochemistry showed immunoreactive adrenocorticotrophic hormone (ACTH) or peptides derived from the ACTH precursor (pro-opiomelanocortin (POMC], or both, in a total of 10 cases: five of these also contained immunoreactive-alpha melanocyte stimulating hormone, indicating more extensive translational processing of POMC than normally occurs in healthy corticotrophs of the anterior pituitary; in two further cases peptides capable of stimulating ACTH release from the anterior pituitary were present. In the remaining six cases immunocytochemistry failed to show the presence of ACTH, other POMC derived peptides, or peptides with ACTH releasing properties. These findings correlate well with the histological and clinical observations, in that the six tumours had been clinically overt, caused rapid death, and histologically seemed to be highly malignant. In contrast, the 12 other tumours were occult to radiological examination, patients had a much improved survival rate, and histologically the tumours seemed to be less aggressive. All but one of the tumours in this series showed a degree of neuroendocrine differentiation, indicated by the presence of neuron specific enolase. These results suggest that one feature of highly malignant tumours, which cause an ectopic endocrine syndrome, is a high secretion of peptide hormones, leaving amounts that are too small to be shown by immunocytochemistry. PMID- 3020098 TI - On the role of protein kinase subunits in the control of eukaryotic gene expression. AB - Transcriptional regulation by cAMP has been demonstrated for several eukaryotic genes; however, the identity of the protein kinase subunit involved has been a source of debate. Based on homologies with the procaryotic cAMP-binding catabolite activator protein, a recent hypothesis has invoked the regulatory protein RII as the mediator. The evidence currently available on the effects of microinjected kinase subunits suggests, however, that the catalytic subunit is the active factor. Moreover, the proposed homologies between the catabolite activator protein and RII are difficult to reconcile with its proposed mediatory role. We suggest as an alternative hypothesis that a phosphoprotein other than RII may mediate the effects of cAMP on eukaryotic gene expression. PMID- 3020097 TI - High proliferative activity of Reed Sternberg associated antigen Ki-1 positive cells in normal lymphoid tissue. AB - An improved immunoenzymatic double labelling method was developed, which simultaneously shows the Ki-1 membrane antigen and the nuclear proliferation associated antigen, defined by monoclonal antibody Ki-67. This new approach permits in situ discrimination of cells that are proliferating or not with a particular membrane antigen. Most Ki-1 positive cells in normal lymphoid tissue also express the Ki-67 nuclear antigen and thus appear to be proliferating. As proliferating cells are more susceptible to malignant transformation than quiescent cells, it is possible that the small number of normal Ki-1 positive cells might, none the less, cause a large proportion of all lymphomas in man. PMID- 3020099 TI - cGMP-dependent protein kinase is present in high concentrations in contractile cells of the kidney vasculature. AB - There is increasing evidence that the recently discovered Atrial Natriuretic Factor, a potent vasorelaxing, natriuretic and diuretic hormone, may achieve some of its diverse physiological effects by regulating the cellular level of cGMP. Since activation of a cGMP-dependent protein kinase is an established effect of cGMP, we have studied the cellular localization of cGMP-dependent protein kinase within the rat kidney cortex using both immunocytochemical and immunochemical procedures. cGMP-dependent protein kinase is selectively concentrated in contractile cells of the kidney vasculature, including both intra- and extraglomerular mesangial cells, vascular smooth muscle cells and microvascular pericytes, as well as in interstitial myofibroblasts. cGMP-dependent protein kinase is not detectable in significant amounts in tubular epithelial cells, podocytes, or endothelial cells. These results support the hypothesis that Atrial Natriuretic Factor may achieve some, and maybe all, of its renal effects by regulating hemodynamic parameters via activation of cGMP-dependent protein kinase within vascular contractile cells. PMID- 3020101 TI - Influence of dietary protein degradability and energy concentration on growth of heifers and steers and intraruminal protein metabolism. AB - Two studies with 4-mo old Holstein steers and heifers and one abomasal digesta collection study were used to determine the influence of altering the dietary energy content and crude protein escape on growth, intraruminal protein metabolism, and site of digestion of dry matter, starch, cellulose, hemicellulose, and protein. Dietary energy concentration was increased by tallow, and rumen protein escape was increased by dehydrated alfalfa and distillers dried grains with solubles substituted for soybean meal. Tallow appeared to depress intake and gain, but gain was increased by protein escape from the rumen. Total and particulate digesta crude protein reaching the abomasum was increased by protein escape; however, percentage crude protein digested was decreased in these treatments due to increased acid detergent insoluble nitrogen. Decreasing crude protein solubility decreased ruminoreticular digestion of dry matter, cellulose, hemicellulose, and starch; however, lower intestinal starch digestion compensated for decreased ruminoreticular digestion. In a third study, with growing steers and heifers, corn gluten meal provided the escape protein and soybean meal the degradable protein. Total and average daily gain were greater for the corn gluten meal diet, but intake was not influenced by energy concentration or protein escape. PMID- 3020100 TI - Phorbol ester-induced augmentation and inhibition of epinephrine-stimulated adenylate cyclase in S49 lymphoma cells. AB - The effects of 4-beta phorbol 12-myristate 13-acetate (PMA) on hormone and forskolin-stimulated adenylate cyclase were evaluated in S49 lymphoma cells. Treatment of wild type (WT) S49 cells with PMA caused stimulation, inhibition or had no effect on epinephrine stimulation of cAMP accumulation. The effect observed was dependent on the length of PMA treatment, the concentration of PMA and the concentration of hormone (or forskolin) used to stimulate cAMP accumulation. Longer treatment times with PMA and higher PMA concentrations favored the inhibitory effects. Pretreating WT with 0.5 microM PMA for 18 min caused an increase in the EC50 and maximal levels for epinephrine stimulation of cAMP accumulation. Thus inhibition was seen at relatively low epinephrine concentrations and augmentation with high concentrations. The inhibitory effects of PMA on epinephrine-stimulated adenylate cyclase activity were observed only at low free Mg++ concentrations (0.75 mM). The effects of PMA on PGE1-stimulated cAMP accumulation were similar to those observed for epinephrine. In S49 WT cells 100 nM PMA augmented 5 microM forskolin-stimulated cAMP accumulation; however with 100 microM forskolin, PMA effects were minimal. PMA also attenuated Gi mediated Gpp(NH)p inhibition of forskolin-stimulated adenylate cyclase in both WT and cyc- membranes, resembling the effects of pertussis toxin. The effects of various phorbol analogues on epinephrine-stimulated cAMP accumulation were as follows: 4 beta-phorbol 12,13-didecanoate had similar effects to PMA, 4 alpha phorbol 12,13-didecanoate had no effects and 1-oleoyl, 2-acetylglycerol augmented epinephrine-stimulated cAMP accumulation at concentrations greater than or equal to 5 microM. Our results are consistent with a dual mechanism of PMA action on adenylate cyclase involving protein kinase C-mediated phosphorylation of Gi and of the beta-adrenergic receptor, the former leading to augmentation and the latter to inhibition of hormone-stimulated adenylate cyclase. PMID- 3020103 TI - Adrenal response during periparturient period to adrenocorticotropin in dairy cattle fed corn silage and grass legume silage. AB - Glucocorticoid response to exogenous adrenocorticotropin was used as an indicator of stress in cows fed different diets ad libitum during the dry period and throughout lactation. Twenty-three Holstein cows were administered adrenocorticotropin at 2 d after initiation of the dry period and at 2 d and 35 d postpartum, and plasma glucocorticoid response was evaluated. Cows were fed diets using only corn silage as forage or mixed forage with or without supplemental concentrate. Mean basal plasma glucocorticoid concentrations were 6.1 ng/ml on d 2 after dry off, 4.7 ng/ml on d 2 postpartum, and 6.7 ng/ml on d 35. Cows on the 32.5:32.5:35 corn silage:grass-legume silage:concentrate diet had the lowest basal glucocorticoid concentrations on d 2 postpartum but highest concentrations on d 35 of lactation. Mean glucocorticoid response was 33.2 ng/ml on d 2 of lactation compared with 50.6 ng/ml on d 35 and 48.5 ng/ml plasma on d 2 following drying off. Diet interactions suggest that feed components may be involved with ability to tolerate stresses that occur during lactation and the dry period and thus may lead to altered adrenal function. PMID- 3020102 TI - Effects of feed intake and protein degradability on ruminal characteristics and site of digestion in steers. AB - Four multiple-fistulated Hereford steers were used in a 4 X 4 Latin square design with a 2 X 2 factorial arrangement of treatments [two intakes (9.1 and 6.1 kg dry matter/d) and two protein sources differing in ruminal degradability (dry distillers grains and dry corn gluten feed)]. Steers fed at the high intake had faster fluid dilution rates (7.63 versus 6.52%/h), higher ruminal fluid outflows (120.2 versus 91.7 L/d), lower apparent ruminal digestibilities of organic matter (41.3 versus 44.3%) and neutral detergent fiber (56.0 versus 60.2%), and lower total tract digestibilities of neutral detergent fiber (64.3 versus 68.7%) than when they were fed at the low intake. Steers fed dry corn gluten feed had higher apparent ruminal digestibilities of organic matter (45.5 versus 40.1%) and neutral detergent fiber (60.2 versus 56.0%) and lower duodenal flows of nonammonia-nonbacterial N (40.1 versus 52.2% of N intake) than when they were fed dry distillers grains. Efficiency of ruminal bacterial growth was higher when steers were fed at the high versus low intakes. Efficiency of ruminal bacterial growth and site and extent of fiber digestion, especially hemicellulose, but not ruminal escape of protein, can be readily altered by manipulation of feed intake of moderately high forage diets. PMID- 3020104 TI - Kinetic analysis of Streptococcus sanguis adhesion to artificial pellicle. AB - Studies of equilibria between Streptococcus sanguis and artificial pellicle have suggested that there are multiple binding sites for the organism. In the present study, adhesion of S. sanguis to saliva-coated hydroxylapatite was examined by means of kinetic methods. Cell-pellicle complex formation was measured from initiation of binding to equilibrium. Rate constants were calculated for forward reactions (adsorption) and reverse reactions (desorption). Initial binding obeyed reversible, first-order kinetics, whereas desorption of bound cells followed biphasic kinetics. Initial desorption proceeded approximately ten times faster than the slower second rate. The results are consistent with the mechanism C + P reversible CP* in equilibrium with CP in which CP* represents the reversible equilibrium that shifts at a discrete rate to the high-affinity CP state. Thus, the biphasic binding behavior that has been previously deduced from equilibrium studies may be attributed to a time-dependent shift from close apposition to pellicle, stabilized by low-specificity forces, to a higher-affinity binding. PMID- 3020105 TI - Survival of herpes simplex virus during cryosurgery with liquid nitrogen. AB - Because many viruses can survive extremely cold temperatures for extended periods of time, transmission of viral diseases from person to person using improper cryosurgical techniques might conceivably occur. To investigate the survivability of herpes simplex virus during cryotherapy, virus was first inoculated onto cotton-tipped applicators from either tissue cultures or from active lesions on patients, and then frozen for variable times in liquid nitrogen (-196 degrees C). After thawing, the applicators were cultured for the virus. The virus survived 12 hours of freezing (the maximum time of freezing in the study), which suggests that herpes simplex could be transmitted between cryosurgical patients if care is not taken to use separate cotton-tipped applicators and liquid nitrogen containers for each case. PMID- 3020106 TI - UVA induced darkening of lower epidermal cells as an in vitro system of immediate pigment darkening (IPD) and mechanisms of IPD. PMID- 3020107 TI - The use of sodium bicarbonate in the therapy of organic acidosis. PMID- 3020108 TI - Opiate receptor sensitivity in depressed patients before and after clomipramine treatment. AB - To measure opiate receptor sensitivity, the effects of naloxone on beta-endorphin and cortisol serum levels were determined before and after 3 weeks of clomipramine treatment (75 mg/day i.v.) in 12 patients with major depressive disorder. The opiate antagonist significantly increased both hormonal levels. The only significant endocrine differences between pre- and post-treatment were basal and maximal cortisol levels, which were elevated before antidepressant therapy. The rise in the cortisol or beta-endorphin serum level after naloxone injection was not affected by clomipramine treatment. There was no significant relationship between depression score and basal or stimulated endocrine variables. These data do not indicate a distinct effect of antidepressants on opiate receptor sensitivity. PMID- 3020109 TI - Antiarrhythmic efficacy of propranolol: comparison of low and high serum concentrations. AB - Propranolol has been effective in suppressing ventricular arrhythmias in up to 70% of patients in some series; however, a wide range of concentrations was required to produce this degree of efficacy. In one series, 40% of responders required high serum concentrations (greater than 500 ng/ml) in excess of those required for physiologic beta-receptor blockade (25 to 150 ng/ml). To assess the relative contribution of high concentration electrophysiologic effects to antiarrhythmic efficacy the results of programmed electrical stimulation were compared at high and low (beta-blocking) concentrations in 28 patients with inducible sustained ventricular tachycardia. Propranolol was given as a series of loading and maintenance infusions producing first a mean concentration of 130 +/- 72 ng/ml (beta-blocking) and then a mean concentration of 743 +/- 523 ng/ml (high). Beta-blockade was assessed by the percent reduction in exercise-induced tachycardia. Near maximal beta-blockade was achieved by a concentration of 150 ng/ml. At a low concentration, 6 of 28 patients had a response to propranolol (complete in 5 and partial in 1). At a high concentration, one additional patient had a complete response while three had a partial antiarrhythmic response. At high concentrations of propranolol there was a significant shortening of the QTc interval relative to that seen during the low dose infusion. No other significant electrophysiologic changes occurred at high versus low concentration. In summary, an antiarrhythmic response to propranolol occurs most frequently at a beta blocking concentration. High concentration electrophysiologic effects occur and these appear to contribute to antiarrhythmic efficacy in some patients. PMID- 3020110 TI - Aminophylline exposure alters mouse bone marrow-derived mast cell adenosine responsiveness. AB - Aminophylline-treated mouse bone marrow-derived mast cells exhibit a hyperresponsiveness to adenosine addition (10(-6) to 10(-4) mol/L) at the time of secretagogue challenge coincident with an up regulation of adenosine receptor numbers. This effect on beta-hexosaminidase release is maximal at 100 mumol/L of aminophylline, evident after 5 days of aminophylline exposure, and reversed by 6 days after washing. Neither radiolabeled arachidonic acid nor leukotriene C4 release in the absence or presence of adenosine was altered by aminophylline treatment. Although cAMP levels were dynamically changed by adenosine or secretagogue, these changes were not different in the two cell populations; resting and challenged mast cell adenosine levels were similarly unaffected by aminophylline. The long-term action of aminophylline on mouse bone marrow-derived mast cells appears to be primarily on adenosine receptors and thereby on preformed mediators, and this action may have some importance in the pathophysiology and treatment of asthma. PMID- 3020111 TI - Nutrient intakes of women in NHANES II, emphasizing trace minerals, fiber, and phytate. AB - Nutrient intakes of 1,066 young women 18 to 24 years of age were estimated using a shortened nutrient data base (the University of California, Berkeley Minilist) containing 235 food items. The dietary data consisted of 24-hour recalls collected during the Second National Health and Nutrition Examination Survey (NHANES II). A cross-reference index, recipe file, and concentration factors were developed to substitute foods on the data base for the 1,267 foods reported by the young women. Estimates of energy, protein, fat, carbohydrate, and iron intakes using the NHANES II nutrient data base and the UCB Minilist were within 3% of one another, and had correlation coefficients above 0.97. Mean +/- S.E. daily intakes of copper, zinc, dietary fiber, and phytate, which are not included in the NHANES II data base, were estimated to be 1.16 +/- 0.02 mg, 8.11 +/- 0.14 mg, 13.2 +/- 0.3 gm, 395 +/- 14 mg, respectively. The values agree closely with other published values for young women's intakes. A shortened nutrient data base can be a valid tool for estimating intakes of populations. PMID- 3020112 TI - Neuroendocrine control of reproductive function in the aging female rodent. PMID- 3020113 TI - Treatment of amiodarone associated thyrotoxicosis by simultaneous administration of potassium perchlorate and methimazole. AB - Amiodarone iodine induced thyrotoxicosis occurs frequently in patients residing in areas of mild iodine deficiency and in patients with preexisting goiter. Drug therapy of the hyperthyroidism is often unsuccessful. Twenty-three patients with amiodarone induced thyrotoxicosis were either not treated, treated with 40 mg methimazole daily or with methimazole and 1 gm potassium perchlorate daily for up to 40 days and then with methimazole alone. Thyrotoxicosis was more likely to spontaneously remit in patients without goiter. Therapy with methimazole alone was unsuccessful in inducing euthyroidism in 5 patients with goiter. However, combined therapy with methimazole and potassium perchlorate rapidly alleviated hyperthyroidism in almost all patients with goiter. This drug combination is successful because perchlorate inhibits the active transport of iodine into the thyroid and methimazole blocks the intrathyroidal synthesis of thyroid hormones. PMID- 3020114 TI - Effects of beta non-selective and beta 1 selective adrenergic blocking agents on glucagon secretion from isolated perfused rat pancreas. AB - To characterize beta-receptors which affect pancreatic A-cell activity, the effects of propranolol (beta non-selective blockade) and metoprolol (beta 1 selective blockade) were evaluated on epinephrine modulated insulin (IRI) and glucagon (IRG) release both in basal state and during metabolic stimulus (arginine 20 mM). The isolated perfused rat pancreas model with the exclusion of stomach and duodenum was used. Epinephrine infusion (at 10(-7) M) caused a prompt and sustained increase in basal IRG secretion and significantly potentiated glucagon release in response to metabolic stimulus. Insulin secretion was markedly suppressed by epinephrine both in basal conditions and during metabolic stimulus. Propranolol (at 10(-7) M) and metoprolol (at 10(-7) M) infusion clearly and similarly counteracted epinephrine stimulatory effects on IRG secretion but failed to elicit any significant effect on the epinephrine inhibited IRI release either in basal state or during the metabolic stimulus. These results suggest that, at least in the rat, the adrenergic stimulation of IRG release is mediated through a beta 1 receptor. PMID- 3020115 TI - Urinary catecholamine excretion in patients with secondary adrenocortical insufficiency. AB - To evaluate the relationship between the secretion of cortisol and the activity of adrenal medulla in the secondary adrenocortical insufficiency, the excretion of epinephrine and norepinephrine was documented in 8 patients suffering from panhypopituitarism. Plasma levels and urinary excretion of cortisol were very low in baseline conditions, and the increase in these parameters of cortisol secretion occurring upon ACTH infusion was significantly reduced with respect to the response to ACTH documented in normal subjects. The mean value of urinary epinephrine excretion was at the lower limit of normal values, and a highly significant positive correlation was found between cortisolemia or cortisoluria and urinary epinephrine excretion in these patients. Despite a significant increase in cortisolemia and cortisoluria upon ACTH administration, this acute increase in adrenocortical activity was without any stimulatory effect on epinephrine or norepinephrine excretion. But, as in baseline conditions, a significant correlation was documented for the degree of adrenocortical activity and epinephrine excretion on the day of ACTH administration. It appears, therefore, that in severe secondary adrenocortical insufficiency the excretion of epinephrine is reduced proportionally to the decrease in adrenocortical activity. PMID- 3020116 TI - Sodium loading raises urinary cortisol in man. AB - It has been suggested that cortisol secretion is modified by sodium intake. We therefore studied the pituitary-adrenal axis by measuring diurnal rhythms of ACTH and cortisol levels in serum of 10 normal control subjects after 4 days of low sodium diet (intake 40 mEq/day) and after 6 days of high sodium diet (intake 320 mEq/day). Urinary excretion of aldosterone-18-glucuronide and free cortisol were determined at the end of each diet. Urinary aldosterone excretion declined from 17.9 +/- 2.6 to 2.8 +/- 1.1 microgram/day and urinary cortisol increased from 26.2 +/- 6.2 to 36.8 +/- 13.8 micrograms/day during low and high sodium intake. In contrast, plasma ACTH and serum cortisol measured every two hours for a 24-h period were similar both during low and high sodium intake. The results suggest an altered handling of cortisol by the kidney during high salt intake. PMID- 3020117 TI - Infantile digital fibromatosis after web construction in syndactyly. AB - A case of infantile digital fibromatosis with an unusual onset is reported. A Japanese girl, with a simple syndactyly of the right ring and little fingers, had an operation at the age of 2 1/2 years. Three months after the operation, multiple nodules appeared at the skin graft edge. The nodules were excised but soon recurred. When the patient was 8 years old, a large-scale excision of the tumors was performed. Intracytoplasmic inclusion bodies were shown by phosphotungstic acid-hematoxylin stain, and the case was diagnosed as infantile digital fibromatosis. PMID- 3020118 TI - Malignant fibrous histiocytoma in a child's hand. AB - A 3-year-old girl had a 4-month history of a tumor in her right hand. The tumor was located in the subcutaneous and soft tissues of the palm and the long, ring, and small fingers. Histologic studies showed a malignant fibrous histiocytoma that was confirmed by the ultrastructural study as having a fibroblastic and histiocytic origin. The long, ring, and small fingers were amputated. The postoperative course was normal, and 18 months later no recurrence or metastases were observed. PMID- 3020119 TI - Spontaneous rupture of the normal stomach after sodium bicarbonate ingestion. AB - Spontaneous rupture of the normal stomach is an unusual and highly lethal event. We present a case of spontaneous rupture of a normal stomach after ingestion of sodium bicarbonate, and review the seven similar cases previously reported. PMID- 3020120 TI - Hepatocellular adenoma and nodular regenerative hyperplasia of the liver in a young man. AB - A 26-year-old man had a massive intraabdominal hemorrhage from a hepatocellular adenoma (HCA). The tumor arose within a liver that demonstrated generalized nodular regenerative hyperplasia. The patient had no factors predisposing to either HCA or nodular regenerative hyperplasia (NRH) of the liver. Although rare, HCA should be included in the differential diagnosis of spontaneous intraperitoneal hemorrhage even in young men. The coexistence of HCA and NRH of the liver in this patient may indicate a common pathogenesis. PMID- 3020121 TI - On the virtues and pitfalls of the molecular evolutionary clock. AB - "Informational" macromolecules--i.e., proteins and nucleic acids--have in their sequences a register of evolutionary history. Zuckerkandl and Pauling suggested in 1965 that these molecules might provide a "molecular clock" of evolution. The molecular clock would time evolutionary events and make it possible to reconstruct phylogenetic history--the branching relationships among lineages leading to modern species. Kimura's neutrality theory postulates that rates of molecular evolution are stochastically constant and, hence, that there is a molecular clock. A variety of tests have shown that molecular evolution does not behave like a stochastic clock. The variance in evolutionary rates is much too large and thus inconsistent with the neutrality theory. This, however, does not invalidate the clock, but rather leaves it without a theoretical foundation to anticipate its properties. Sequence comparisons show that molecular evolution is sufficiently regular to serve in many situations as a clock, but uncertainty concerning the properties of the clock (for example, about the circumstances that may yield large oscillations in substitution rates from time to time or from lineage to lineage) demands that it be used with caution. Few DNA or protein sequences are known from organisms that range from closely related, e.g., different mammals, to very remote, e.g., mammals and fungi. One example is cytochrome c, which has an acceptable clockwise behavior over the whole span, in spite of some irregularities. Another example is the copper-zinc superoxide dismutase (SOD), which behaves like a very erratic clock. The SOD average rate of amino acid substitution per 100 residues per 100 million years (MY) is 5.5 when fungi and animals are compared, 9.1 when comparisons are made between insects and mammals, and 27.8 when mammals are compared with each other. The question is which mode is more common over broad evolutionary spans: the regularity of cytochrome c or the capriciousness of SOD? Additional data sets will be required in order to obtain the answer and to develop expectations about the accuracy of the clock in particular instances. Until such data exist, conclusions solely based on the molecular clock are potentially fraught with error. PMID- 3020122 TI - Membrane IgM, IgD, and IgG act as signal transmission molecules in a series of B lymphomas. AB - Increases in intracellular free calcium concentration ((Ca2+)i) were observed in response to anti-immunoglobulin (Ig) antibodies in each of six B cell tumors or B cell hybridomas bearing mu or delta chains on their cell surface. The BAL17 cell line, bearing mu and delta chains on its surface, behaved similarly to mature B cells in the following respects. Anti-IgM and anti-IgD antibodies caused increases in (Ca2+)i and inositol phospholipid metabolism; the initial increases in (Ca2+)i were derived partly from an intracellular Ca2+ pool; lipopolysaccharide, phorbol myristate acetate (PMA), B cell stimulatory factor-1, and antibodies to class I and class II major histocompatibility molecules and to the Fc gamma receptor failed to cause increases in (Ca2+)i or in inositol phospholipid metabolism; and increases in (Ca2+)i and inositol phospholipid metabolism in response to anti-Ig were inhibited by pretreatment with PMA. Furthermore A20, an IgG2a bearing lymphoma, showed increases in (Ca2+)i in response to anti-IgG2a, and a lymphoma cell line (6G8-2E10) expressing membrane IgG2b as a result of DNA-mediated transfer of the gamma 2b H chain gene, showed increases in (Ca2+)i in response to anti-IgG2b. These results indicate that Ig bearing lymphomas display early events in B cell activation after receptor cross linkage and can be used for detailed studies of the activation process. PMID- 3020123 TI - Differential effects of type I IFN and IFN-gamma on the binding of tumor necrosis factor to receptors in two human cell lines. AB - The effect of IFN-alpha and IFN-beta on the expression of cell surface receptors for tumor necrosis factor (TNF) was examined in two human cell lines. In HeLa cells, IFN-alpha and IFN-beta increased 125I-TNF binding, whereas in HT-29 cells these two IFN either slightly decreased or had no effect on 125I-TNF binding. In contrast, IFN-gamma increased 125I-TNF binding in both cell lines. Both IFN-alpha and IFN-beta exerted an antagonistic effect on IFN-gamma-induced stimulation of TNF receptor expression in HT-29 cells, but did not inhibit TNF receptor induction by IFN-gamma in HeLa cells. IFN-gamma and, to a lesser extent, IFN-beta were synergistic with TNF in producing cytotoxic/cytostatic activity in HT-29 cells. Despite the inhibitory effect of IFN-beta on the IFN-gamma-induced stimulation of TNF receptor expression, IFN-beta did not inhibit the synergistic enhancement of TNF cytotoxicity by IFN-gamma in HT-29 cells. The dissociation between the effects of IFN-beta on TNF receptor expression and on the cytotoxic activity of TNF in HT-29 cells suggests that TNF receptor modulation is not a major mechanism of synergism between IFN and TNF. PMID- 3020124 TI - Susceptibility/resistance to mouse hepatitis virus strain 3 and macrophage procoagulant activity are genetically linked and controlled by two non-H-2-linked genes. AB - The mode of inheritance of susceptibility/resistance to mouse hepatitis strain 3 (MHV-3) was determined by typing the set of AXB/BXA recombinant inbred (RI) strains derived from resistant A/J (A) and susceptible C57BL/6J (B) progenitors for susceptibility to infection as determined by the severity of liver pathology. The strain distribution pattern for susceptibility showed a discontinuous variation: one strain was fully resistant (A-like), four strains were fully susceptible (B-like), and 16 strains showed an intermediate degree of susceptibility. The fully susceptible strains developed fulminant hepatitis and died; the fully resistant strain developed no liver disease, whereas a range of disease ranging from mild focal hepatitis to widespread hepatocellular necrosis was seen in the semisusceptible strains. This SDP best fits the two-recessive gene model of inheritance, and neither of these two loci is linked to the H-2 complex. Macrophage procoagulant activity (PCA) segregated among the RI strains in a strain distribution pattern identical to that of susceptibility/resistance. PCA levels were greater than sevenfold elevated in fully susceptible RI mice and fourfold elevated in semisusceptible mice with no increase in resistant mice. These observations suggest genetic linkage of susceptibility/resistance to MHV-3 infection and macrophage PCA. PMID- 3020125 TI - Ecto-5'-nucleotidase expression during human B cell development. An explanation for the heterogeneity in B lymphocyte ecto-5'-nucleotidase activity in patients with hypogammaglobulinemia. AB - Ecto-5'-nucleotidase (ecto-5'-NT) activity was measured in human B cells at different stages of development. Ecto-5'-NT activity of B cell preparations from fetal spleen and cord blood was 5.08 and 5.59 +/- 2.8 nmol/hr/10(6) cells, respectively; that of B cell preparations from adult peripheral blood, spleen, or lymph node was fivefold to sixfold higher (27.9 +/- 12, 29.2 and 33.8 nmol/hr/10(6) cells, respectively). The increased enzyme activity in B cell preparations from adult peripheral blood as compared with cord blood paralleled increased percentages of 5'-NT+ cells (69 +/- 12% vs 32 +/- 17%) and an average of twice as much enzyme activity per positive cell. Small, resting B cells that cannot synthesize Ig in vitro in response to pokeweed mitogen (PWM) were isolated from adult peripheral blood by mouse erythrocyte rosetting. Total ecto-5'-NT activity and the percentage of 5'-NT+ cells were equivalent in total B cells and the mouse erythrocyte rosette-positive subpopulation. Thus, ecto-5'-NT activity is acquired before B cells gain the ability to differentiate into Ig-secreting plasma cells in response to PWM. Ecto-5'-NT activity was also measured in B cell preparations from eight patients with common variable immunodeficiency. Six had reduced ecto-5'-NT activity (2.83 to 15.4 nmol/hr/10(6) cells), and two had normal activity (34.7 and 58.2 nmol/hr/10(6) cells). B cells from all six patients with low ecto-5'-NT activity failed to synthesize Ig when cultured with PWM and normal irradiated T cells. Of the two patients with normal B cell ecto-5' NT activity, one also had B cells unresponsive to PWM, but B cells from the other patient appeared to more normal, in that they synthesized IgM and IgG when cultured with PWM plus irradiated allogeneic T cells. Thus, measurement of B cell ecto-5'-NT activity allows the subclassification of patients who have a common inability to synthesize immunoglobulin in vitro response to PWM. B cells with low ecto-5'-NT activity are presumably blocked at an earlier stage in development than B cells with normal ecto-5'-NT activity. Evaluation of ecto-5'-NT activity along with the expression of other B cell surface antigens should aid in the definition of discrete stages of B cell development. PMID- 3020126 TI - Characterization of two distinct DR beta chain alleles at the beta III locus of the DR5 haplotype: beta III alleles are highly conserved. AB - The HLA-DR beta region of at least two members of the DRw52 (MT2) supertypic group (DR3, DR5, DRw6, and DRw8) has recently been shown to contain three beta chain genes--beta I, beta II, and beta III, ordered in the direction of transcription. beta III is apparently a duplication of beta I. Both beta I and beta III are expressed, whereas beta II is a pseudogene. We previously reported the sequence of the DR5 beta I cDNA from the homozygous DR5 cell line Swei. We report here the sequence of two different DR5 beta III cDNA from the same cell line. The assignment of the genes of this and other members of the DRw52 supertypic group to specific loci allow comparisons between products of known alleles and between products of distinct loci. beta I allelic products showed approximately 11% divergence in the first domain, whereas beta I and beta III products showed 17% difference. These differences were clustered and found predominantly in the previously described variable regions (amino acid residues 9 through 13, 26 through 38, and 67 through 74). These data, coupled with the finding of shared variable regions among different DR beta chains, suggest gene conversion as a means of generating polymorphism in the beta I alleles. In contrast, the beta III allelic products showed less than 1% amino acid and nucleotide divergence in the first domain. These differences were outside the variable regions, and attributable to single nucleotide changes. The polymorphism at the beta I locus and the conservation at the beta III locus suggest selective pressure conserving the beta III locus and/or generating polymorphism at the beta I locus, and further suggest that gene conversion may be acting unidirectionally from beta III to beta I. PMID- 3020127 TI - Analysis of DR beta and DQ beta chain cDNA clones from a DR7 haplotype. AB - A cDNA library was constructed from a DR7, DRw53, DQw2 homozygous cell line, cDNA clones corresponding to DR beta and DQ beta chains were isolated, and the nucleotide sequences of the polymorphic first domains of these chains were determined. A novel screening strategy allowed rapid and simple identification of cDNA clones corresponding to both DR beta chains (DR7 beta1 and DR7 beta2): DR7 beta2 clones have a recognition site for the enzyme BssHII, whereas DR7 beta1 clones do not. The DR7 beta 1 sequence differs significantly from all previously described DR beta chains. As predicted by the presence of the BssHII site in DR7 beta 2 clones, the DR7 beta 2 sequence differs from the DR7 beta 1 sequence. The sequence of the DRw53-associated DR7 beta 2 chain is identical to the reported sequence of the DRw53-associated DR4 beta 2 chain. In addition, the sequence of the DQ beta chain from the DR7, DQw2 cell line is identical to the reported sequence of a DQ beta chain from a DR3, DQw2 cell. These findings raise interesting questions about the evolution of the DR3, DR4, and DR7 haplotypes. PMID- 3020128 TI - Identification of distinct activation pathways of the human neutrophil NADPH oxidase. AB - The stimulation of the human neutrophil NADPH-oxidase is initiated by a variety of agonists, which appear to utilize more than one activation pathway. We have discerned that opsonized zymosan (OZ) stimulates O2- release by a mechanism distinct from that of phorbol myristate acetate (PMA). PMA differs from OZ stimulation in its susceptibility to H-7 (a protein kinase inhibitor) inhibition of O2- release and the lack of PMA-initiated release of radiolabeled arachidonic acid ([3H]AA) from prelabeled cells. That AA release was linked to O2- generation in OZ-stimulated cells was suggested by the finding that mepacrine, a phospholipase inhibitor, exhibits parallel dose response inhibition for both O2- generation and [3H]AA release, whereas mepacrine did not significantly inhibit the O2- generation induced by PMA. The specific involvement of phospholipase A2 (PLA2) in the release of AA was indicated by the lack of release of [3H]oleate, which is not released by PLA2 in intact cells; [3H]AA released from phosphatidylinositol and phosphatidylcholine and not accompanied by the formation of [3H]-arachidonyl phosphatidic acid, thus eliminating the involvement of phospholipase C; and the inhibition of [3H]AA release by p-bromophenacyl bromide, a specific PLA2 inhibitor. The reduction of O2- formation by inhibitors of AA metabolism (BW755C, acetylsalicylic acid, and indomethacin) further supports a linkage between AA release and O2- generation. That [3H]AA release, like O2- generation, in OZ-stimulated cells was calcium dependent further differentiates OZ from calcium-independent PMA activation. These studies in toto suggest that OZ stimulation of the NADPH-oxidase differs from PMA, in that the particulate stimulus is PLA2 mediated and independent of protein kinase C. PMID- 3020130 TI - Some effects of flavonoids on lymphocyte proliferative responses. AB - A number of representative flavonoids reversibly inhibit human lymphocyte proliferative responses in a concentration-dependent manner. The flavonoids quercetin and tangeretin are most effective when added during the early phase of exposure of lymphocytes to the mitogenic stimuli but become progressively less effective when added after increasing lengths of time following stimulation, suggesting an early flavonoid-sensitive step(s) in cell activation. In the proliferative response to phytomitogens, they do not act by inhibiting the early increase in calcium influx. They do not augment cellular cyclic-AMP concentration in basal or phytomitogen-stimulated lymphocytes nor reduce its increment in the presence of inhibitors of phosphodiesterase. At concentrations inhibitory to the proliferative response, quercetin (but not tangeretin) inhibits the calcium activated, phospholipid-dependent protein kinase (C kinase). Certain flavonoids powerfully inhibit the uptake of thymidine into phytomitogen-stimulated lymphocytes but do not directly affect incorporation of already transported thymidine into newly synthesized DNA. PMID- 3020129 TI - Syngeneic monoclonal internal image anti-idiotopes as prophylactic vaccines. AB - A syngeneic monoclonal anti-idiotope that behaves as an internal image of the mammalian reovirus type 3 cellular attachment protein (viral hemagglutinin) was used in the syngeneic host for the induction of a prophylactic anti-viral antibody response. These studies were performed without the aid of co-stimulation by viral antigens. The high stringency of this system enables us to define the maximum constraints on the use of anti-idiotopes as anti-viral vaccines. We have used the murine BALB/c monoclonal IgM anti-idiotope 87.92.6 to study the idiotope and antigen specificity, kinetics, dose dependence, adjuvant, carrier, and valency requirements of anti-idiotope-induced anti-viral antibody responses. These studies show that the production of high titer neutralizing antibody requires a lengthy (60 day) immunization protocol, which includes the use of adjuvant and multivalent anti-idiotope, and is dependent on anti-idiotope concentrations of greater than 50 micrograms. When administered in this manner anti-idiotope can stimulate serotype-specific antibody responses across species barriers at levels comparable with those obtained after inoculation with virus. The practical efficacy of these reagents and procedures is documented by the ability of maternal immunization with anti-idiotope to confer complete protection in neonates from a potentially lethal reovirus type 3 viral infection. PMID- 3020131 TI - In vitro generation of chemotactic leukotrienes from unfractionated murine epidermal cells. AB - Single cell suspensions of murine epidermal cells were studied for the generation of leukotrienes (LTs), using in vitro bioassays for chemotaxis, reverse-phase high-pressure liquid chromatography (HPLC), and radioimmunoassays (RIAs). A combination of arachidonic acid (AA) at 10(-3)-10(-4) M with the calcium ionophore A 23187 at 5 X 10(-6) M was the most potent stimulus, causing release of LTs within 10-30 min. Other stimuli, like the N-formyl-methionyl-leucyl phenylalanine, at 10(-7) M and bradykinin at 10(-3) M, were less effective, and the tumor promotor phorbol-myristate-acetate (10(-5)-10(-8) M) caused no release at all. AA induced release at cytotoxic concentrations, but the other stimuli did not, and keratinocytes from different body regions were equally good sources of the LTs. In vivo or in vitro pretreatment of keratinocytes with UV radiation did not alter spontaneous or stimulated secretion of LTs, while pretreatment of cells with Ia, but not with Thy-1, monoclonal antibodies caused a moderate decrease of release. Analyses by HPLC indicated the release of 20-OH-LTB4 in addition to LTB4 in cell supernatants. Murine keratinocytes and epidermal dendritic cells serve therefore as a source of chemotactic leukotrienes after appropriate in vitro stimulation with agents that are known to play a role in cutaneous inflammation. PMID- 3020132 TI - Isolation and characterization of type V collagen from human post-burn granulation tissues. AB - Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of pepsin solubilized collagens from post-burn granulation tissues revealed that type V collagen consisted of 3 alpha chains: alpha 1(V), alpha 2(V), and alpha 3(V). The mean value (0.12 +/- 0.01 SD) of the type V/type I ratio in the granulation tissues was significantly higher (p less than 0.001) than that (0.03 +/- 0.01 SD) of the ratio in normal skin. The average ratio of alpha 1(V):alpha 2(V):alpha 3(V) of type V collagen purified from the granulation tissues was determined to be about 5:3:1. SDS-polyacrylamide gel electrophoresis patterns of 3 alpha chains were not affected in the presence or absence of 2-mercaptoethanol. Purified type V collagen was degraded by bacterial collagenase, but remained intact after tadpole collagenase digestion, in contrast to type I and type III collagens. Amino acid analyses of each alpha chain separated on SDS-gel electrophoresis of type V collagen revealed that all 3 alpha chains of type V collagen were poor in alanine, rich in hydroxylysine, and had high ratios of hydroxylysine/lysine, which are typical features of type V collagen. The purified type V collagen was further fractionated by ammonium sulfate into 2 molecular species, [alpha 1(V)]2 alpha 2(V) and alpha 1(V)alpha 2(V)alpha 3(V). Our data demonstrate that type V collagen in preparations from human post-burn granulation tissues consists of 3 alpha chains and can be resolved into 2 distinct heterotrimers. PMID- 3020134 TI - [Detection of immunoglobulin M antibodies against cytomegalovirus by enzyme linked immunosorbent assay]. PMID- 3020133 TI - [Immunological background of patients with dilated cardiomyopathy and myocarditis: clinical and experimental studies]. AB - Immunogenetic mechanism may be involved in the pathogenesis of idiopathic cardiomyopathy. Viral myocarditis is considered a cause of dilated cardiomyopathy. In this study, we examined the major histocompatibility complexes (human leukocyte antigens: HLA) by microdroplet cytotoxicity test, lymphocyte subsets by laser flow cytometry, and the activity of lymphocyte blastoformation induced by phytohemagglutinin (PHA) and concanavalin A (Con A) in patients with cardiomyopathies (DCM) and myocarditis (MC). We also examined the incidence and histopathology of encephalomyocarditis (EMC) virus myocarditis in inbred strains of mice, and serial changes of T- and B-lymphocytes in the peripheral blood of DBA/2 mice inoculated with EMC virus by immunofluorescence techniques. The results were as follows: Major histocompatibility complex (HLA and H-2); HLA-B12 in patients with DCM and HLA-DR8 in patients with MC were more frequent than in controls. In EMC virus infection, differences were found in the frequency of occurrence of MC in inbred strains of A/J (H-2a), C57BL/6 (H-2b), BALB/c (H-2d), DBA/2 (H-2d) and C3H/He (H-2k) mice. Genetic mechanism may play a role heart similar to lesions in patients with DCM were seen in DBA/2 mice in the chronic stage of MC. in susceptibility to virus infection. Lymphocyte population study; OKT 8 (suppressor T-cell) was significantly lower in patients with DCM and hypertrophic cardiomyopathy (HCM) than in controls. There were no significant changes of lymphocyte subsets in patients with MC as compared with the controls.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020135 TI - [Malignant fibrous histiocytoma arising in the pulmonary artery]. PMID- 3020138 TI - [Effect of arterial injection of lipiodol-adriamycin emulsion on hepatocellular carcinoma]. PMID- 3020136 TI - Phagocytosis of unopsonized zymosan particles by trypsin-sensitive and beta glucan-inhibitable receptors on bone marrow-derived murine macrophages. AB - Murine bone marrow cells, plated at 4 X 10(4) cells/well and cultured in 50% fibroblast CM, yielded pure populations of large, individual, adherent cells that were phagocytic and morphologically indistinguishable from macrophages. Adherent macrophages appeared in small numbers with 24 h of culture, increased to maximal cell numbers within 10 days of culture, and remained at these cell densities for at least 11 weeks in culture. The capacities of adherent macrophages to ingest unopsonized zymosan particles and EsIgG, at inputs of 1.25 X 10(7) targets, were expressed by 7 and 40% of the cells derived from 24-hour cultures, respectively, were increased at nearly identical rates to comparable maximal levels within 10 14 days of culture and were exhibited by essentially all adherent cells derived from 2-11-week cultures. The percentage of adherent macrophages from twelve 3-6 week cultures ingesting greater than or equal to 1, greater than or equal to 6 and greater than or equal to 10 zymosan particles was 89 +/- 5, 47 +/- 11 and 14 +/- 9% (mean +/- SD, n = 12), respectively, and the percentage ingesting greater than or equal to 1, greater than or equal 6 and greater than or equal to 10 EsIgG was 86 +/- 5, 49 +/- 10 and 14 +/- 8%, respectively. Incubation of adherent macrophages with mannan-free ss-glucan particles at inputs of 5 X 10(5)-5 X 10(7)/ml initiated a phagocytic response comparable to that obtained with the same doses of zymosan particles which contained mannan and beta-glucan. Preincubation of adherent macrophages with 100 micrograms/ml of a fully soluble beta-glucan, laminarin, and solubilized barley beta-glucan reduced subsequent macrophage phagocytosis of greater than or equal to 6 zymosan particles by 53 and 40%, respectively. In contrast, yeast alpha-mannan was less than 1% as active, and 10 mg/ml reduced the number of adherent macrophages ingesting greater than or equal to 1 zymosan particles by 64%. At concentrations as high as 2 mg/ml, laminarin and barley beta-glucan had no effect on Fc receptor-mediated ingestion of EsIgG, and mannan at 20 mg/ml also failed to inhibit EsIgG ingestion. Pretreatment of adherent macrophages with 20 micrograms/ml of trypsin reduced the number of cells ingesting greater than or equal to 1 zymosan particles from 89 to 10% and those ingesting greater than or equal to 6 zymosan from 43 to 0%, whereas pretreatment with as much as 100 micrograms/ml of trypsin failed to decrease macrophage ingestion of EsIgG.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3020137 TI - [Effect of radiotherapy on two cases with walk disturbance by paralysis of vertebral metastases]. PMID- 3020139 TI - Chemodectoma of the larynx. AB - Two cases of chemodectoma of the larynx are presented. Both of them arose from the superior laryngeal glomus in men late in their lives. Pain was the landmark of the disease. Both cases showed very aggressive behaviour. In conclusion, these are malignant tumours which should be treated by total laryngectomy. There is no place for radiotherapy in their treatment. PMID- 3020140 TI - Intraoral angiofibroma: a case report. PMID- 3020141 TI - Dopamine does not attenuate phosphoinositide hydrolysis in rat anterior pituitary cells. AB - The hydrolysis of membrane phosphatidylinositol to yield [3H]labelled inositol phosphates by anterior pituitary cells was stimulated significantly by angiotensin II, TRH and neurotensin over a broad range of concentrations. These secretagogues also stimulated release of prolactin. Although the coincident incubation of dopamine with these agents resulted in a marked diminution of prolactin release, no concomitant reduction in inositol phosphate production was observed. In addition, bromocriptine, a potent agonist of dopamine, also proved ineffective in blunting stimulated phosphatidylinositol catabolism. Although it slightly inhibited basal rates of inositol tris-, bis- and monophosphate production, these results show that the secretagogue-mediated enhancement of phosphatidylinositol catabolism may be correlated with an increased release of prolactin and that the inhibition of hormone release produced by dopamine is not achieved by reducing basal or secretagogue-mediated inositol phosphate production. PMID- 3020143 TI - Adrenocortical functions in a macropodid marsupial Thylogale billardierii. AB - In a study of adrenocortical functions in macropodid marsupials, measurements were made of the effects of ACTH infusion, ether stress and adrenaline infusion on plasma corticosteroid and glucose concentrations in wallabies (Thylogale billardierii) provided with indwelling venous catheters. The mean plasma total glucocorticoid concentration in undisturbed males and females was 80 +/- 5 (S.E.M.) micrograms/l, of which more than 90% was cortisol. This fraction declined to 68% of the total at the highest ACTH-stimulated concentration of 225 micrograms/l, due to an increase in the contribution by 11-deoxycortisol. Although maximal ACTH stimulation (4.5 i.u./kg per h) caused a five- to sixfold increase in cortisol secretion rate, as measured by isotope dilution during constant-rate tracer infusion, plasma cortisol concentration rose only two- to threefold, due to a marked increase in metabolic clearance. Plasma glucose concentration did not change significantly during either short-term (1 h) i.v. infusion or long-term (8 days) i.m. injection of ACTH, even though plasma cortisol concentration was significantly increased. Ether anaesthesia caused a marked hyperglycaemia that preceded an increase in plasma cortisol concentration and was not sustained while plasma cortisol concentration continued to increase. Infusion of adrenaline i.v. at rates sufficient to cause a similar hyperglycaemia had no significant effect on plasma cortisol concentration. A marked hyperglycaemia during xylazine anaesthesia was not associated with an increase in plasma cortisol concentration and was attributable to suppression of insulin secretion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020142 TI - Effects of alpha-melanocyte-stimulating hormone on the cyclic AMP and phospholipid metabolism of rat adrenocortical cells. AB - Results on the effects of peptides on the phospholipid metabolism and steroid and cyclic AMP (cAMP) outputs of rat adrenal capsular cells (96% zona glomerulosa, 4% zona fasciculata) were obtained in a series of three batch experiments. Their significance was examined by analysis of variance. Incorporation of [32P] into phosphatidylcholine, phosphatidic acid and phosphatidylinositol was measured. Production of [3H]inositol-1 monophosphate, inositol-1,4 bisphosphate and inositol-1,4,5 tris-phosphate was estimated after prelabelling with [3H]inositol followed by 1 min incubation with a steroidogenic stimulus. Angiotensin II (0.25 nmol/l to 0.25 mumol/l) highly significantly (P less than 0.01) stimulated aldosterone and corticosterone outputs, [32P] incorporation into phosphatidic acid and phosphatidylinositol (but not into phosphatidylcholine) and the production of the three [3H]inositol phosphates. Aldosterone and corticosterone outputs were stimulated by alpha-MSH (above 0.1 nmol/l). However, incorporation of [32P] was not significantly increased until 10 mumol alpha-MSH/l but, unlike with angiotensin II, incorporation into phosphatidylcholine was also then stimulated. Also, the production of the inositol phosphates was not increased significantly (P greater than 0.05) by any dose of alpha-MSH (10 nmol/l, 1 mumol/l and 0.1 mmol/l) used. Therefore, it can be concluded that alpha-MSH does not stimulate phospholipase C in rat zona glomerulosa cells. In further experiments, it was also found that there were significant increases in cAMP as well as in steroid outputs above 1 nmol alpha MSH/l (highly significant above 10 nmol alpha-MSH/l). There were plateaux of the outputs of both steroids and cAMP from 0.1 to 1 mumol alpha-MSH/l. However, there were further increases in steroid and cAMP outputs of the capsular cells at higher doses. Concomitant results on the stimulation of corticosterone output by zona fasciculata-reticularis cells indicate that this additional increase was mostly due to the stimulation of the contaminating zona fasciculata cells. It was also confirmed that alpha-MSH preferentially stimulates steroidogenesis by the zona glomerulosa. However, under our conditions, alpha-MSH highly significantly increased the output of cAMP by both zona fasciculata and glomerulosa cells. PMID- 3020144 TI - Are catechol oestrogens obligatory mediators of oestrogen action in the central nervous system? I. Characterization of pharmacological probes with different receptor binding affinities and catechol oestrogen formation rates. AB - In an attempt to define pharmacological probes with which to test the role of catechol oestrogen formation in the central nervous system, five oestrogens (oestradiol-17 beta, oestradiol-17 alpha, 4-fluoro-oestradiol, 2-fluoro oestradiol and moxestrol (11 beta-methoxy-17 alpha-ethynyloestradiol) were studied for binding to oestrogen receptors and conversion to catechol metabolites. Binding to cytosol oestrogen receptors was measured in the hypothalamus-preoptic area-amygdala (HPA), pituitary gland and uterus of ovariectomized rats. Conversion to catechol oestrogens was tested in microsomes from the HPA, pituitary gland and liver, using a catechol-O-methyltransferase coupled radioenzymatic assay. Oestradiol-17 alpha was the only weak oestrogen receptor ligand. Binding affinities of the other compounds tested were much higher and comparable to those of oestradiol-17 beta. In contrast, oestradiol-17 alpha was rapidly converted to catechol metabolites, while moxestrol was a relatively poor substrate for catechol oestrogen formation. 4-Fluoro-oestradiol could be 2-hydroxylated but not 4-hydroxylated. 2-Fluoro-oestradiol exhibited impaired 2-hydroxylation but normal 4-hydroxylation. PMID- 3020145 TI - Are catechol oestrogens obligatory mediators of oestrogen action in the central nervous system? II. Potencies of natural and synthetic oestrogens for induction of gonadotrophin release and female sexual behaviour in the rat. AB - The role of catechol oestrogen formation in the mechanism by which circulating oestrogens facilitate gonadotrophin release and female sexual behaviour was explored in adult female rats. The effects of oestradiol-17 beta were compared with those of a group of oestrogens with either a reduced affinity for oestrogen receptors (oestradiol-17 alpha) or a reduced ability to act as substrates for catechol oestrogen formation (2-fluoro-oestradiol, 4-fluoro-oestradiol and moxestrol (11 beta-methoxy-17 alpha-ethynyloestradiol]. Rats were ovariectomized on the evening of dioestrus day 1 of the 4-day oestrous cycle and implanted s.c. 12 h later with infusion pumps containing either one of the test oestrogens or vehicle alone. Infusion rates for oestradiol-17 beta, moxestrol, 2-fluoro oestradiol and 4-fluoro-oestradiol were adjusted to give concentrations of nuclear oestrogen receptors in the brain and pituitary gland within the range of those found in intact female rats during pro-oestrus. Oestradiol-17 alpha was infused at the same and at a tenfold higher rate than that of oestradiol-17 beta; neither of these treatments with oestradiol-17 alpha significantly increased brain or pituitary gland nuclear oestrogen receptor levels. On the day after the pump was implanted, samples of tail vein blood were withdrawn at 12.00, 14.00, 16.00 and 18.00 h for LH assay. All animals were then injected s.c. with 1 mg progesterone in propylene glycol, and tested for feminine sexual behaviour 5 h later. Oestradiol-17 beta, moxestrol, 2-fluoro-oestradiol and 4-fluoro-oestradiol all elicited pronounced LH surges and facilitated progesterone-triggered proceptive and lordosis behaviours.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020146 TI - Amino acid receptor-mediated transmission at primary afferent synapses in rat spinal cord. AB - Intracellular recording techniques have been used to provide information on the identity of excitatory transmitters released at synapses formed between dorsal root ganglion (DRG) and spinal cord neurones in two in vitro preparations. Explants of embryonic rat DRG were added to dissociated cultures of embryonic dorsal horn neurones and synaptic potentials recorded intracellularly from dorsal horn neurones after DRG explant stimulation. More than 80% of dorsal horn neurones received at least one fast, DRG-evoked, monosynaptic input. In the presence of high divalent cation concentrations (5 mmol l-1 Ca2+, 3 mmol l-1 Mg2+) the acidic amino acid receptor agonists, L-glutamate, kainate (KA) and quisqualate (QUIS) excited all dorsal horn neurones which received a monosynaptic DRG neurone input, whereas L-aspartate and N-methyl-D-aspartate (NMDA) had little or no action. 2-Amino-5-phosphonovalerate (APV), a selective NMDA receptor antagonist, was relatively ineffective at antagonizing DRG-evoked synaptic potentials and L-glutamate-evoked responses. In contrast, kynurenate was found to be a potent antagonist of amino acid-evoked responses and of synaptic transmission at all DRG-dorsal horn synapses examined. The blockade of synaptic transmission by kynurenate appeared to result from a postsynaptic action on dorsal horn neurones. Intracellular recordings from motoneurones in new-born rat spinal cord were used to study the sensitivity of the Ia excitatory postsynaptic potential (EPSP) to antagonists of excitatory amino acids. Superfusion of the spinal cord with APV did not inhibit the Ia EPSP but did suppress later, polysynaptic components of the afferent-evoked response. Kynurenate was a potent and selective inhibitor of the Ia EPSP, acting via a postsynaptic mechanism. These findings indicate that L-glutamate, or a glutamate-like compound, but not L aspartate, is likely to be the predominant excitatory transmitter that mediates fast excitatory postsynaptic potentials at primary afferent synapses with both dorsal horn neurones and motoneurones. PMID- 3020148 TI - Inositol lipid metabolism and signal transduction in clonal pituitary cells. AB - A number of clonal cell lines derived from a rat pituitary tumour, collectively termed GH cells, have retained a range of differentiated cell functions, including their ability to secrete the hormones prolactin and growth hormone in response to stimuli such as thyrotropin-releasing hormone (TRH). The mechanisms underlying this release process involve, at least in part, an increase in cytosolic free calcium levels, and the cells have proved useful as a model system in studies of receptor-controlled calcium mobilization. The initial response of the cells to the addition of TRH now appears to be the interaction of the occupied TRH receptor with a GTP-binding protein. A sophisticated signalling system is then activated which initially involves the phosphodiesteratic hydrolysis of phosphatidylinositol 4,5-bisphosphate to 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. Both of these products are important intracellular messengers, and their formation leads to a plethora of biochemical and electrical changes which culminate in the biphasic release of hormone from the cell. The changes in cytosolic free calcium that occur following TRH addition follow a complex temporal pattern. Within 1 s, the concentration starts to increase from a resting level, in the range 100-150 nmol l-1, to a peak value of around 1 mumol l 1 which is attained within 6-8 s. This 'spike' of calcium is almost exclusively derived from intracellular stores, probably the endoplasmic reticulum, in response to the formation of inositol 1,4,5-trisphosphate. With high concentrations of the peptide, the cytosolic free calcium concentration declines promptly, due to the activation of a protein kinase C-mediated extrusion and/or sequestration process. This inhibitory phase is less marked at low agonist concentrations but, in all cases, is superseded by a second increase in free calcium, which is due to the stimulated influx of the cation through dihydropyridine-sensitive calcium channels. These biphasic changes in calcium, in concert with the activation of protein kinase C, appear sufficient to regulate prolactin secretion. PMID- 3020147 TI - Slow synaptic transmission in frog sympathetic ganglia. AB - Bullfrog ganglia contain two classes of neurone, B and C cells, which receive different inputs and exhibit different slow synaptic potentials. B cells, to which most effort has been directed, possess slow and late slow EPSPs. The sEPSP reflects a muscarinic action of acetylcholine released from boutons on B cells, whereas the late sEPSP is caused by a peptide (similar to teleost LHRH) released from boutons on C cells. During either sEPSP there is a selective reduction in two slow potassium conductances, designated 'M' and 'AHP'. The M conductance is voltage dependent and the AHP conductance is calcium dependent. Normally they act synergistically to prevent repetitive firing of action potentials during maintained stimuli. Computer stimulation of the interactions of these conductances with the other five voltage-dependent conductances present in the membrane allows a complete reconstruction of the effects of slow synaptic transmission on electrical behaviour. PMID- 3020149 TI - Perfusion-secretion relationships in the isolated elasmobranch rectal gland. AB - Perfusion and sodium secretion parameters were measured in the isolated rectal gland of Scyliorhinus canicula L. perfused at in vivo pressures, and the effect of stimulation of secretory activity by cyclic AMP and theophylline on these parameters was determined. Stimulation resulted in large increases in secretion flow rate, percentage extraction of sodium from the perfusing fluid, and arteriovenous sodium concentration difference, but did not affect perfusion flow rate or the sodium concentration of the secreted fluid. Reduction of perfusion flow rate to values below 65% of the control level, achieved by reducing perfusion pressure, produced a marked decline in sodium secretion--a process accompanied by increases in the percentage extraction of sodium and arteriovenous concentration difference of sodium, but again without any change in the sodium concentration of the secreted fluid. The in vivo consequences of these findings are discussed with reference to related findings for the avian nasal salt gland. The normal rate of secretion, its sodium concentration, and the nature of the dependence of secretion rate on perfusion flow below certain levels, were essentially unaffected by a reduction in the availability of oxygen to the gland by approximately 80%. It is concluded that the observed relationship between perfusion flow and sodium secretion rate in the stimulated gland is not related to oxygen availability, and hence that the primary underlying function of the synchronized secretion-related vasodilation seen in the gland is not to increase the supply of oxygen to the stimulated secretory tissue. We discuss possible reasons why this erroneous conclusion has been reached by other workers. PMID- 3020150 TI - Thymic B lymphocyte clones from patients with myasthenia gravis secrete monoclonal striational autoantibodies reacting with myosin, alpha actinin, or actin. AB - Striational autoantibodies (StrAb), which react with elements of skeletal muscle cross-striations, occur frequently in patients with thymoma associated with myasthenia gravis (MG). Dissociated thymic lymphocytes from 22 of 72 MG patients secreted StrAb when cultured with PWM. A high yield of EBV-transformed B cell lines was established from thymus, thymoma, and peripheral blood of seven patients with MG, but clones secreting StrAb arose only from the three patients who had StrAb in their sera. The monoclonal StrAb bound to A bands or I bands in skeletal muscle of human, rat, and frog. One bound to mitochondria in addition to myofibrillar I bands. None bound to nuclei, smooth muscle, or gastric mucosal cells. In immunoblot analyses and ELISAs the monoclonal StrAb bound to muscle and nonmuscle isotypes of myosin, alpha actinin, and/or actin. All bound to contractile proteins common to thymus and muscle, and one selectively immunostained epithelial cells of the thymic medulla. From these antigenic specificities we suggest that StrAb might arise as an immune response directed against the cytoskeletal anchoring proteins associated with nicotinic acetylcholine receptors in thymic epithelial cells undergoing neoplastic transformation to thymoma. PMID- 3020152 TI - Nonhematopoietic cells selected for resistance to tumor necrosis factor produce tumor necrosis factor. AB - TNF-resistant lines of L cells can be derived from TNF-sensitive populations by repeated exposure to TNF, and these resistant L cells, in contrast to sensitive L cells and other types of cells, lack demonstrable cell surface receptors for TNF. We have now found that TNF-resistant L cells produce a factor that is cytotoxic for L cells and has the following distinguishing characteristics of mouse TNF: it is a protein of 43 kD, composed of 16 kD subunits, that competes with TNF for receptor binding, induces hemorrhagic necrosis of the TNF-sensitive mouse sarcoma Meth A, has synergistic cytotoxic action with interferon, and its activity is neutralized by antibody to TNF. The two conclusions of this study are that cells selected for TNF resistance spontaneously produce a molecule resembling macrophage TNF, and that cells of nonhematopoietic origin are capable of producing TNF. PMID- 3020153 TI - Influence of neuraminidase treatment on the electrophoretic behaviour of angiotensin converting enzyme from human tissues. AB - Angiotensin converting enzyme (dipeptidyl carboxypeptidase, kininase II; EC 3.4.15.1), is a membrane bound glycoprotein, playing an important role in the renin-aldosterone system. The enzyme contains a carbohydrate moiety, consisting of fucose, mannose, galactose, N-acetylglucosamine and N-acetylneuraminic acid. Treatment with neuraminidase (EC 3.2.1.18) removes sialic acid from the molecule. The influence of this treatment on the electrophoretic mobility of the enzyme was studied in 29 human tissues and body fluids. Results obtained showed differences in the sialic acid content of the enzyme in the tissues examined. PMID- 3020151 TI - Macrophage deactivation. Altered kinetic properties of superoxide-producing enzyme after exposure to tumor cell-conditioned medium. AB - Incubation of activated mouse peritoneal macrophages with tumor cell-conditioned medium (TCM) results in their deactivation, as measured by ability to release reactive oxygen intermediates and kill protozoal pathogens. The mechanism of suppression by macrophage deactivation factor (MDF) was studied. Inhibition of H2O2 release could not be overcome by increasing the concentration of phorbol diesters used to trigger the respiratory burst. Deactivated macrophages consumed H2O2 at the same rate as activated cells (t1/2, 35-40 min for 25 nmol H2O2 per 10(6) peritoneal cells). They transported glucose with the same kinetics (Km, 1 mM; Vmax, approximately 100 nmol per 6 min per milligram cell protein), and maintained similar intracellular concentrations of NADPH and NADP (approximately 0.62 mM and approximately 0.11 mM, respectively), as measured by enzymatic cycling methods and determinations of the volume of cell water (3.6 microliter/mg cell protein). To study the kinetics of the PMA-triggered NADPH oxidase in cell lysates, mixed detergents were used (deoxycholate and Tween 20). These stabilized the oxidase for approximately 3.3-fold longer than deoxycholate alone, which was used in previous studies. Incubation of activated macrophages in MDF resulted in a marked increase in the Km of the oxidase for NADPH, from 0.06 mM to 0.67 mM. The Vmax fell approximately 1.7-fold. These kinetic changes, together with the measured intracellular concentration of NADPH, account quantitatively for the suppression of H2O2 release by deactivated macrophages, and are nearly the mirror image of the kinetic changes observed during macrophage activation. PMID- 3020154 TI - Location of the site-specific recombination system of R46: a function necessary for plasmid maintenance. AB - The R46 site-specific recombination system comprises a per (plasmid-encoded recombinase) gene and a site at which the gene product acts, the per site. The two functions have been cloned into pACYC184. They are encoded by sequences within a region of approximately 2 kb on the R46 genome. These R46 sequences are closely related to the site-specific recombination systems of the ampicillin resistance transposons collectively designated TnA. The R46 per function is interchangeable with the tnpR gene product of TnA. Both enzymes can mediate recombination between the related res and per sites in R46::TnA recombinant plasmids to generate site-specific deletions and inversions. Similar DNA rearrangements occur when TnA inserts into pACYC184 derivatives carrying the cloned R46 per functions. Carriage of this site-specific recombination system contributes to the stable maintenance of R46. By converting plasmid dimers to monomers the R46 per functions help to ensure equal partitioning at cell division. PMID- 3020156 TI - The nucleotide sequence of a type 3 poliovirus isolated during a recent outbreak of poliomyelitis in Finland. AB - We have cloned and sequenced the complete genome of a strain of poliovirus type 3 (23127) isolated during an outbreak of poliomyelitis in Finland. The genome is 7435 nucleotides long excluding the 3' poly(A) stretch and is 95.5% homologous at the amino acid level to the previously sequenced type 3 strain, P3/Leon/37. The most striking feature of the presented sequence is the extent of amino acid substitutions relative to P3/Leon/37 and other type 3 strains in areas of known antigenic importance. The major antigenic determinant for virus neutralization (site 1), located at residues 89 to 100 of VP1, has three amino acid substitutions and there are six substitutions in site 3, a composite site made up of sequences from VP1 and VP3. The variation in these regions probably accounts for the observed unusual antigenicity and may explain why the virus was able to spread in a well-vaccinated community. Sequence comparisons imply that the virus is not derived from the currently used live attenuated vaccine. PMID- 3020155 TI - Isolation of a cDNA encoding a portion of the cyclic nucleotide phosphodiesterase of Dictyostelium discoideum. AB - The cyclic nucleotide phosphodiesterase (phosphodiesterase) of Dictyostelium discoideum is one of a group of developmentally regulated proteins which enable cells to aggregate by chemotaxis during the early stages of development. We report the identification and DNA sequence of a cDNA clone encoding the amino terminal region of the phosphodiesterase. The clone, pPD-3, was selected from a cDNA library created by priming first strand synthesis using a set of oligonucleotides with sequences predicted from the amino-terminal amino acid sequence of purified phosphodiesterase. The DNA sequence of pPD-3 encodes perfectly the available phosphodiesterase amino acid sequence, and pPD-3 selects an mRNA which can be translated into material recognized by phosphodiesterase antisera. The nucleotide sequence of pPD-3 indicates there are 49 amino acids, which contain a segment possessing the characteristics of a signal peptide, that separate the amino-terminal residue identified in the purified protein from the methionine codon at which translation originates. DNA blot analysis demonstrates that the phosphodiesterase gene exists as a single copy in the nuclear genome. Analysis of RNA indicates that the phosphodiesterase transcript is 2.1 kb long, which is approximately 0.8 kb more than the minimum required to encode this protein. PMID- 3020157 TI - Polymorphonuclear leukocyte dysfunction associated with feline leukaemia virus infection. AB - The chemiluminescent characteristics of enriched (greater than 95%) peripheral blood polymorphonuclear leukocyte populations (PMN) from normal and feline leukaemia virus (FeLV)-infected cats were investigated. FeLV-infected cats demonstrated a significantly lower (P less than 0.001) PMN chemiluminescent response when compared to the response of normal age-matched controls. Normal PMN treated with FeLV-infected cat serum exhibited a depressed response in comparison to control cells. A titration of serum from infected cats supplemented with normal serum revealed a titratable suppression of chemiluminescence with increasing concentration of serum from the infected cats. However, PMN from FeLV infected cats treated with normal serum displayed a slight increase in chemiluminescence over the same cells in autologous serum. The addition of inactivated FeLV to normal PMN caused a titratable decrease in chemiluminescence. PMID- 3020158 TI - Intranuclear localization of herpes simplex virus immediate-early and delayed early proteins: evidence that ICP 4 is associated with progeny virus DNA. AB - The localization of ICP 4, ICP 8, DNA polymerase and alkaline exonuclease within herpes simplex virus type 1 (HSV)-infected cells has been examined by immunofluorescence using specific antibodies to these proteins. Cells were simultaneously counterstained with the DNA-binding fluorochrome 4,6-diamidino-2 phenylindole (DAPI) to reveal the intranuclear distribution of DNA. These studies showed that in the absence of virus DNA replication ICP 4, ICP 8 and DNA polymerase were diffusely distributed throughout the nucleus but during virus DNA replication these proteins accumulated at specific foci within the nucleus. Initially these foci were near the nuclear membrane but with continuing virus DNA replication they increased in size until the whole of the nucleus became affected. The increase in size of these foci was coincident with a redistribution of nuclear DNA and margination of chromatin at the nuclear membrane, as revealed by DAPI staining. The number of foci initially present in an infected cell was dependent on the multiplicity of infection. The distribution of ICP 4, ICP 8 and DNA polymerase within the nucleus was altered by treating the cells with DNase. The majority of alkaline exonuclease was diffusely distributed throughout the nucleus during virus DNA replication and did not localize at specific foci within the nucleus. Autoradiographic examination of the incorporation of [3H]thymidine in cells infected with HSV showed that viral DNA replication occurred in restricted areas within the nucleus that were similar, in terms of number, location and size, to the foci where ICP 4, ICP 8 and DNA polymerase accumulated. Furthermore, in cells blocked in mitosis following infection with HSV, ICP 4, ICP 8 and DNA polymerase, but not alkaline exonuclease, localized in areas outside the condensed chromatin structures. DAPI staining revealed the presence of DNA in these areas and, as such structures were never seen when uninfected cells had entered mitosis, it is suggested that this extrachromosomal DNA is of viral origin. These studies therefore suggest that ICP 4 is associated with progeny virus DNA and that while its intranuclear localization is initially at non-viral sites, as DNA replication proceeds so ICP 4 is recruited into areas of virus DNA transcription and replication. PMID- 3020159 TI - Properties of natural and hybrid murine alpha interferons. AB - Four natural murine interferon-alpha genes (MuIFN-alpha 1, -alpha 2, -alpha 4 and -alpha 6) and four hybrid genes (alpha 1 alpha 4, alpha 2 alpha 4, alpha 4 alpha 1 and alpha 4 alpha 2) were transiently expressed in monkey COS cells under the transcriptional control of the simian virus 40 early promoter. The proteins were labelled with [35S]methionine during a 16 h incubation and proteins secreted by the cells during this period were separated by polyacrylamide gel electrophoresis and subsequently visualized by fluorography. Under the conditions used, the IFNs represented 5 to 10% of the total amount of secreted proteins. All genes were found to encode biologically active IFN subspecies, including alpha 4 which has a deletion of five amino acids. When the specific activities of the proteins were compared, it appeared that the specific antiviral activity of alpha 4 on mouse cells was three- to sixfold higher than the activities of the other natural IFN subspecies. The specific activities of the hybrid proteins were similar to those of the natural proteins, except for the alpha 2 alpha 4 hybrid which had a higher specific activity than the original proteins. The ability of the natural and hybrid subspecies to protect hamster cells against viral infection was determined using MuIFN-alpha 1 as a standard. Large differences in activity were found, with alpha 6 as the most and alpha 4 as the least active subspecies. PMID- 3020160 TI - Independent regulation of the antiviral states induced by MuIFN-alpha/beta and by MuIFN-gamma. AB - The stability profiles of the antiviral states induced in L-929 cells against mengovirus by murine interferons MuIFN-alpha/beta and by MuIFN-gamma were shown to be different. Treatment of cells with combinations of IFN-alpha/beta and IFN gamma have previously been shown to result in a potentiated expression of the antiviral state. Here we report studies of the stabilities of the antiviral states induced by various combinations of IFN-alpha/beta and IFN-gamma and that the regulation of the antiviral states induced by IFN-alpha/beta and IFN-gamma were independent of each other. PMID- 3020161 TI - Reactions of sera from patients with rheumatoid arthritis, systemic lupus erythematosus and infectious mononucleosis to Epstein-Barr virus-induced polypeptides. AB - P3HR-1 and Ramos cells induced with sodium butyrate and 12-O-tetradecanoylphorbol 13-acetate were used in the protein immunoblot technique to identify Epstein-Barr virus (EBV)-specific antibodies present in sera from clinically normal individuals and patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and infectious mononucleosis (IM). Sixteen EBV-specific polypeptides were detected ranging in mol. wt. from 22,000 (22K) to 140K. Many of the sera contained antibodies to different subsets of these antigens, and a high proportion expressed autoantibodies which reacted with cellular components from an EBV genome-negative cell line. About 50% of the sera from each category reacted with the 44K to 48K and 36K and 38K early antigen (EA) components. A high proportion of the SLE sera (64%) were found to contain anti-EA antibodies, suggesting an association between EBV and SLE. Almost all of the EBV-seropositive sera examined contained antibodies against a 22K late antigen, but none of the sera from IM patients reacted with this polypeptide. PMID- 3020162 TI - Acyclovir efficiently inhibits oropharyngeal excretion of Epstein-Barr virus in patients with acute infectious mononucleosis. AB - Shedding of Epstein-Barr virus (EBV) into saliva was studied in 31 patients with verified acute infectious mononucleosis. The patients had been randomized for intravenous treatment with acyclovir (ACV) at 10 mg/kg body weight at 8 h intervals for 7 days, or placebo, in a double-blind trial. EBV in centrifuged throat washings was detected by transformation of umbilical cord lymphocytes and by immunofluorescence staining for EBV-associated nuclear antigen in fixed cell smears. Saliva samples were obtained before and during treatment, and after 4 weeks and 6 months, respectively. ACV effectively but transiently interrupted EBV production (P less than 0.001), but virus shedding resumed at the initial level within 3 weeks of cessation of the treatment. Initially, 93.5% of the patients had detectable EBV in the saliva compared with 83% in the 4th week and 58% after 6 months. PMID- 3020163 TI - Chromosome damage induced by herpes simplex virus type 1 in early infection. AB - The chromosome damage induced by herpes simplex virus type 1 (HSV-1) in vitro was examined up to 6 h after infection using HT-1080 cells. Initial damage occurring within 3 h was specific, involving uncoiling of chromosome 1q12-21 and to a lesser extent the pericentric regions of chromosomes 9, 16 and satellited chromosomes. For the initial unwinding, synthesis of the immediate early class of HSV proteins needed to occur as was demonstrated using HSV-1 temperature sensitive mutants tsK and tsB7 and two viral inhibitors, beta-propiolactone and psoralen plus long wavelength u.v. light. Later damage included chromatid breaks, acentric fragments and pulverization which did not take place until synthesis of delayed early proteins had begun. PMID- 3020164 TI - DNA sequence of the major capsid protein gene of herpes simplex virus type 1. AB - The DNA sequence of the region of the herpes simplex virus type 1 genome encoding the major capsid protein was determined. The predicted protein contains 1374 amino acid residues and has a molecular weight of 149,075. Comparisons of the amino acid sequence of this protein with those predicted from the published DNA sequences of two other herpesviruses, varicella-zoster virus and Epstein-Barr virus, resulted in the identification of the major capsid protein gene in each genome. PMID- 3020165 TI - Potential treatment of herpes simplex virus encephalitis by brain-specific delivery of trifluorothymidine using a dihydropyridine in equilibrium pyridinium salt type redox delivery system. AB - A newly described, drug-carrier delivery system in which a lipophilic derivative is enzymatically converted to a hydrophilic compound was used to treat experimental herpes simplex virus (HSV) encephalitis. Because trifluorothymidine (TFT) does not cross the blood brain barrier, the lipophilic dihydropyridine derivative 3'-(N-methyl-1, 4-dihydronicotinoyl)-5-'pivaloyltrifluorothymidine (DHTFT) was synthesized and characterized by HPLC. After intravenous administration of 20 mg/kg of DHTFT to rats, the quaternary, intermediate compound 3'-N-methyl-1,4-nicotinoyltrifluorothymidine was measured at levels of 7 8 micrograms/g brain at 1 hour and 13.5 +/- 0.8 micrograms/g brain at 4 hours. This compound had antiviral activity equivalent to that of TFT against HSV-1 in a plaque reduction assay (ID 50 = 0.5-1.0 microgram/ml), either directly or by conversion to TFT. Although survival was not prolonged in a rat model of HSV encephalitis, a statistically significant reduction in titer of HSV/g brain was achieved with daily intravenous treatment with DHTFT. TFT was not detected in brains of rats at 1 and 4 hours after intravenous DHTFT, but a low level was observed at 18 hours, 0.3 +/- 0.05 microgram/g brain. These data suggest that the lipophilic compound DHTFT or a lipophilic metabolite crossed the blood brain barrier and was converted to a quaternary compound, which accumulated in the brain and which was either active directly or was converted to TFT. The drug carrier delivery system described here can potentially be used in the treatment of HSV or other viral encephalitides. PMID- 3020167 TI - Lack of complement-dependent cytolytic antibodies in hepatitis A virus infection. AB - Sera collected from patients with acute hepatitis A virus (HAV) infection and convalescent sera were examined for cytolytic activity against HAV-infected human embryo lung fibroblasts (HAV carrier fibroblasts). Using the 51chromium release assay, no complement dependent antibody mediated cytolytic activity against HAV carrier cells could be detected. In control experiments with identical cell strains, anti-herpes simplex virus (HSV) positive sera and complement caused specific lysis of HSV type 1 infected target cells. The data presented here do not support the hypothesis that in the possible immunopathogenesis of HAV infection, complement-dependent cytolytic antibodies play an essential role. PMID- 3020166 TI - Excretion of cytomegalovirus in semen associated with HTLV-III seropositivity in asymptomatic homosexual men. AB - We studied 56 asymptomatic homosexual male volunteers in Pittsburgh for 1 1/2 yr for relationships between cytomegalovirus (CMV) and human T-lymphotropic virus type III (HTLV-III) infections. CMV was most frequently isolated from semen (8%) as compared with throat washings (5.9%) and urine (0%) on initial testing of CMV seropositive subjects. Other viruses commonly isolated from immunosuppressed patients (herpes simplex virus, adenovirus) were rarely detected in this cohort. Seropositivity to HTLV-III was significantly associated with isolation of CMV from semen in our asymptomatic cohort (odds ratio = 9.5, p = .008). These results suggest that HTLV-III infection is associated with selective, temporal activation of CMV in the genital tract of asymptomatic homosexual men. PMID- 3020168 TI - Chronic neurologic disease in Junin virus-infected rats. AB - The purpose of this study was to determine whether Junin virus persistence in CNS of rats was capable of inducing late neurologic disease. Following intracerebral inoculation of newborn animals with XJ strain, three distinct stages could be discerned: an early phase of acute disease, up to 30 days pi, with 5% mortality; an intermediate one, extending to 280 days pi, without clinical signs but with evident viral persistence; and a final period of chronic illness, featuring clinical neurologic syndrome, severe perivascular inflammatory reaction, PAP labeled viral antigen in a few cerebral and cerebellar neurons, and virus recovery only by coculture. Late neurologic disease seems associated to the lack of effective clearance of brain virus, leading to viral persistence and long lasting immunologic stimulation. The importance of animal models for pathogenic studies on CNS persistent viral infections leading to late neurologic disease is stressed. PMID- 3020169 TI - Herpes simplex virus detection by ELISA: effect of enzyme amplification, nature of lesion sampled and specimen treatment. AB - The relative sensitivity of two enzyme detection procedures was investigated in a simultaneous "monoclonal" ELISA for herpes simplex virus (HSV). A cyclical enzyme amplified detection system with alkaline phosphatase, rather than horse-radish peroxidase and a conventional chromogenic substrate, gave an increase in absolute sensitivity and a 20 to 30% increase in the detection of HSV in routine isolation positive genital specimens collected in transport medium. The HSV detection rate, with both procedures, was shown to vary with the site and clinical stage of lesion sampled; it was highest with penile vesicular lesions. Direct extraction of the swab specimen in a small volume of diluent further increased the sensitivity of antigen detection giving positive and negative predictive values of 100 and 96% respectively. The overall sensitivity of HSV detection was equivalent to that obtained by isolation in cell culture. The amplified ELISA offers an alternative, rapid, simple, non-culture technique for routine HSV diagnosis that does not rely upon retention of virus viability. PMID- 3020171 TI - Regulation of glycerol phosphate dehydrogenase and lactate dehydrogenase activity by forskolin and dibutyryl cyclic AMP in the C6 glial cells. AB - We have compared the effects of norepinephrine, forskolin, and dibutyryl cyclic AMP (Bt2cAMP) on the regulation of the cytosolic enzyme glycerol phosphate dehydrogenase (GPDH) in the C6 rat glioma cell line. Forskolin and Bt2cAMP elicit a dose-dependent increase in the levels of the enzyme that was, however, unaffected by norepinephrine. The half-maximal effect of forskolin was obtained at 7-8 microM, and the effect was maximal at 30 microM. Dexamethasone at a 50 nM concentration produced a two- to sixfold induction of GPDH after 48 h. The combination of dexamethasone with forskolin or Bt2cAMP leads to an elevation in GPDH levels that is higher than that produced by one of the compounds alone. This potentiation is found when both agents are added together with or after the glucocorticoid. The increase in uninduced and dexamethasone-induced GPDH activity was blocked by cycloheximide and actinomycin D, indicating that de novo protein and RNA synthesis are required. The activity of cytosolic lactate dehydrogenase activity did not change after incubation with dexamethasone, but increased with forskolin or Bt2cAMP. PMID- 3020170 TI - The production of hydroxyl radical from the reaction between hydrogen peroxide and NADH. PMID- 3020172 TI - Changes in levels of purine and pyrimidine nucleotides during acute hypoxia and recovery in neonatal rat brain. AB - Neonatal rat brains were examined for changes in levels of ATP, ADP, AMP, cyclic AMP, GTP, GDP, UTP, UDP, UMP, and CTP during exposure to 100% nitrogen for 20 min and subsequent recovery in air. During hypoxia, ATP, GTP, UTP, and CTP levels and the GTP/GDP ratio decreased to 38, 50, 26, 21, and 21%, respectively, of control levels. No significant change in cyclic AMP level was observed. The decrease in the total uridine nucleotide pool during hypoxia was markedly greater (to 53% of control levels) than that in the total adenine nucleotide pool (to 92% of control levels). During recovery, ATP and GTP levels were rapidly and almost completely restored. On the other hand, CTP levels returned slowly to control values after a 2-h recovery period. Restoration of the UTP level was slow and incomplete (87% of the control value even after a 3-h recovery period). The GTP/GDP ratio also did not return to normal. These data suggest that hypoxic insult to the neonate may have an effect on the synthesis of nucleotidyl sugars, phospholipids, and proteins in the brain, resulting in significant problems with developmental processes of the brain. The present study also showed that the delayed restorations of the UTP level and the GTP/GDP ratio were not seen in the brains of adult rats subjected to acute severe hypoxic insult. PMID- 3020173 TI - Differential sensitivity of basal and opioid-stimulated low Km GTPase to guanine nucleotide analogs. AB - In membranes derived from NG108-15 cells, the opioid peptide [D-Ala2,D Leu5]enkephalin (DADLE) stimulates a low Km GTPase. The nucleotide analogs guanosine 5'-O-(2-thio)diphosphate (GDP beta S), guanosine 5'-(beta,gamma imido)triphosphate [Gpp(NH)p] and guanosine 5'-O-(3-thio)-triphosphate (GTP gamma S) inhibit the basal enzymatic activity with the order of potency GTP gamma S greater than Gpp (NH)p greater than GDP beta S. In the presence of DADLE, the inhibition isotherms of GDP beta S and Gpp(NH)p are shifted to the right five- and fourfold, respectively, compared to the inhibition observed in the absence of DADLE. In contrast, the IC50 of GTP gamma S for inhibiting the enzyme is reduced by 55% in the presence of the opioid. Both Gpp(NH)p and GTP gamma S produce a concentration-dependent increase in the Km(app) of GTPase, without affecting its Vmax, indicating a competitive inhibition. However, the replots of Km(app) versus inhibitor concentration are hyperbolic, suggesting a partial type of inhibition. Both Gpp(NH)p and GTP gamma S, but not GTP, induce an increase in the EC50 of DADLE for stimulating GTPase. These findings indicate that the basal and the opioid-stimulated low Km GTPase differ in their respective sensitivities to inhibition by guanine nucleotide analogs. PMID- 3020174 TI - Vesicular storage of 3,4-dihydroxyphenylethylamine and noradrenaline in terminal sympathetic nerves of dog spleen and kidney. AB - The subcellular distribution of 3,4-dihydroxyphenylethylamine (DA, dopamine) and noradrenaline was examined in preparations of dog spleen and renal cortex following ultracentrifugation on a discontinuous sucrose gradient. In both tissues, only half the total tissue DA was localized to the soluble phase, and 30 50% was found in association with noradrenaline in the large vesicular fraction, suggesting that both catecholamines may be stored together and released by nerve stimulation. The vesicular fraction from renal cortex contained more DA than could be attributed to its presence in noradrenergic axons alone, supporting other evidence for the existence of dopaminergic renal nerves. PMID- 3020175 TI - Molecular characteristics and peptide specificity of vasoactive intestinal peptide receptors from rat cerebral cortex. AB - Vasoactive intestinal peptide (VIP) receptors have been identified in CNS by their chemical specificity and molecular size. Using synaptosomes isolated from rat cerebral cortex, it was shown that central VIP receptors discriminated among natural and synthetic VIP-related peptides, because half-maximal inhibition of [125I]VIP binding to synaptosomes was obtained for 0.6 nM VIP, 9 nM peptide histidine isoleucineamide (PHI), 50 nM VIP 2-28, 70 nM secretin, 100 nM rat growth hormone-releasing factor (GRF), and 350 nM human GRF. Other peptides of the VIP family, such as glucagon and gastric inhibitory polypeptide, did not interact with cortical VIP receptors. The molecular components of VIP receptors in rat cerebral cortex were identified after [125I]VIP cross-linking to synaptosomes using the cross-linker dithiobis(succinimidyl propionate). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of synaptosomal proteins revealed two major [125I]VIP-protein complexes of Mr 49,000 and 18,000. The labeling of the Mr 49,000 component was specific, because it was abolished by native VIP, whereas the labeling of the Mr 18,000 component was not. Natural VIP agonists reduced the labeling of the Mr 49,000 component with the following order of potency: VIP greater than PHI greater than secretin approximately equal to rat GRF. In contrast, glucagon and octapeptide of cholecystokinin were without effect, a result indicating its peptide specificity. Densitometric scanning of autoradiographs showed that the labeling of the Mr 49,000 component was inhibited by low VIP concentrations between 10(-10) and 10(-6) M (IC50 = 0.8 nM), a result indicating the component's high affinity for VIP.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020176 TI - Enhancement of histamine H1-receptor agonist activity by 1,4-dithiothreitol in guinea-pig cerebellum and cerebral cortex. AB - The disulphide bond-reducing agent 1,4-dithiothreitol (1 mM) produced a marked potentiation of histamine-stimulated accumulation of [3H]inositol phosphates in lithium-treated slices of guinea-pig cerebellum and cerebral cortex. This was seen as a parallel shift of the concentration-response curve for histamine to lower agonist concentrations, with no significant effect on the maximal response or Hill coefficient. Dithiothreitol similarly potentiated the augmentation of adenosine-stimulated cyclic AMP accumulation elicited by histamine in guinea-pig cerebral cortex. Studies with partial agonists suggested that this potentiating effect was associated with an increase in agonist efficacy rather than a change in agonist binding affinity. Thus, dithiothreitol increased the maximal accumulation of [3H]inositol phosphates produced by both 2-pyridylethylamine and 2-methylhistamine, which appeared to act as partial agonists in guinea-pig cerebral cortex. Dithiothreitol similarly increased the maximal extent of the augmentation of adenosine-stimulated accumulation of cyclic AMP produced by 2 methylhistamine. The site of action of dithiothreitol is not known; however, a comparison of the effect of dithiothreitol on muscarinic and histamine H1 receptor-mediated phosphoinositide responses in guinea-pig cerebral cortex suggests that it is before the stage at which the receptor-effector pathways are shared by these two receptor systems. PMID- 3020178 TI - Nerve and muscle microvasculitis in peripheral neuropathy: a remote effect of cancer? AB - In a series of 50 cases in which nerve and/or muscle microvasculitis was seen on biopsy, seven were associated with malignancy. In two cases, the cancer was found after the discovery of microvasculitis. All patients exhibited sensory-motor neuropathy, which was often painful and asymmetrical, with a progressive course. ESR and CSF protein levels were always elevated. Motor conduction velocity was slightly reduced in three cases, unmeasurable in one case, and normal in three. Cancers involved were adenocarcinoma in five cases (three prostate and two lung), Hodgkin's disease in one and immunoblastic lymphadenopathy in one. A thorough search for cancer should be performed when microvasculitis is seen in nerve or muscle biopsy specimens, especially when ESR and CSF protein levels are elevated. PMID- 3020177 TI - Cyclic AMP-dependent protein phosphorylation in chemosensory neurons: identification of cyclic nucleotide-regulated phosphoproteins in olfactory cilia. AB - Chemosensory dendritic membranes (olfactory cilia) contain protein kinase activity that is stimulated by cyclic AMP and more efficiently by the nonhydrolyzable GTP analog guanosine-5'-O-(3-thio)triphosphate (GTP gamma S). In control nonsensory (respiratory) cilia, the cyclic AMP-dependent protein kinase is practically GTP gamma S-insensitive. GTP gamma S activation of the olfactory enzyme appears to be mediated by a stimulatory GTP-binding protein (G-protein) and adenylate cyclase previously shown to be enriched in the sensory membranes. Protein kinase C activity cannot be detected in the chemosensory cilia preparation under the conditions tested. Incubation of olfactory cilia with [gamma-32P]ATP leads to the incorporation of [32P]phosphate into many polypeptides, four of which undergo covalent modification in a cyclic nucleotide dependent manner. The phosphorylation of one polypeptide, pp24, is strongly and specifically enhanced by cyclic AMP at concentrations lower than 1 microM. This phosphoprotein is not present in respiratory cilia, but is seen also in membranes prepared from olfactory neuroepithelium after cilia removal. Cyclic AMP-dependent protein kinase and phosphoprotein pp24 may be candidate components of the molecular machinery that transduces odor signals. PMID- 3020179 TI - Recent progress on an endogenous digitalislike factor in hypertension. AB - Evidence exists that demonstrates the relationship between a natriuretic factor, or Na+, K+-ATPase inhibitor, and volume expansion in man. Patients having extracellular volume expansion have been studied for the effect of their plasma on erythrocyte [3H]ouabain binding. High levels of ouabainlike activity were found in plasma from acromegalic patients and patients with chronic renal failure. High levels were also observed in some hypertensive patients. A partial purification of such a compound was performed from the urine of hypertensive patients. The various steps of purification achieved a 400,000-fold purified compound of apparent homogeneity. The inhibitor was extracted from 140 liters of urine of 21 donors (hypertensive patients and normotensive offspring of hypertensive patients). The purification steps included flash chromatography, anionic exchange, and reversed-phase HPLC on RP 18, diphenyl and phenyl packings. Nuclear magnetic resonance and mass spectrometry indicated a nonpeptidic compound, which was possibly a steroid with a low molecular mass (less than 500 daltons). PMID- 3020180 TI - Small-cell lung cancer--whither late intensification? PMID- 3020181 TI - Late intensive combined modality therapy followed by autologous bone marrow infusion in extensive-stage small-cell lung cancer. AB - To attempt to improve the poor prognosis of extensive-stage small-cell lung cancer (SCLC) patients, we tried to administer late intensive combined modality therapy (LICMRX) to patients with good tumor regression after 12 weeks of conventional chemotherapy. Twenty-nine consecutive extensive-stage SCLC patients received 6 weeks of cyclophosphamide, methotrexate, and lomustine (CMC) induction therapy, followed by 6 weeks of vincristine, doxorubicin, and procarbazine (VAP). After restaging for assessment of tumor response, autologous bone marrow (ABM) was collected in patients in good medical condition with complete response (CR) or partial response (PR) and no tumor on marrow examination. LICMRX consisted of irradiation with 2,000 rad in five fractions for five days to sites of initial tumor involvement, followed by cyclophosphamide, 60 mg/kg for 2 days, and etoposide, 200 mg/m2 for 3 days and then by ABM infusion. Prophylactic cranial irradiation (PCI) was administered thereafter, but no further chemotherapy was used. Due to lack of tumor regression or poor medical condition, only ten of the original 29 patients were eligible for LICMRX; two refused, so only eight (28%) received therapy. Three patients who began LICMRX in CR developed recurrence of SCLC after an additional 4, 8, and 15 months. Of five patients with PR, one attained CR but relapsed at 3 months, two remained in PR and progressed at 2 and 4 months, and two died of infection without recovery from LICMRX. Mean time from ABM infusion to recovery of granulocyte count to 500/microL was 15.8 days in the six surviving patients (range, 12-22). The major non-hematologic toxicity of LICMRX was severe esophagitis. Among all 29 patients, there were six CRs (21%) and no 2-year survivors, compared with a CR rate of 36% and 10% 2-year survivors in 78 extensive-stage patients previously treated with CMC plus VAP without LICMRX. We conclude that the LICMRX given in this study can be administered to only a minority of extensive-stage SCLC patients and is very unlikely to yield substantial improvement in the fraction of 2-year survivors (95% confidence limits for 2-year survival 0% to 10%). PMID- 3020182 TI - An EBV genome carrying pre-B cell leukemia in a homosexual man with characteristic karyotype and impaired EBV-specific immunity. AB - A Burkitt-like lymphoma/leukemia confined to bone marrow was detected in a human T cell leukemia virus (HTLV)-III/LAV- and Epstein-Barr virus (EBV)-seropositive homosexual man. The tumor cells were EBNA-positive and contained at least 22 EBV genomes per cell. They were totally immunoglobin negative, but showed other markers for B cells detected with monoclonal antibodies. The patient had an impaired cellular immunity to EBV antigens and EBV-infected cells at diagnosis, but these reactions normalized during treatment. Cell clones derived from the bone marrow tumor in vitro also carried EBV and had six different marker chromosomes, including the typical 14q+ chromosome and a t(8 - ;8), which resulted in trisomy for the largest part of 8q. Partial trisomy for 12q was also observed. The patient completed six courses of combination chemotherapy and remains in excellent health after 34 months of follow-up. PMID- 3020183 TI - Gonadal dysfunction in patients treated for metastatic germ-cell tumors. AB - The effects of chemotherapy on endocrine function were assessed in 22 previously treated patients with germ-cell tumors and compared with the endocrine function of six previously untreated patients. Baseline and stimulated serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, thyroid-stimulating hormone (TSH), prolactin, and thyroxine (T4) were obtained. Baseline LH levels were elevated in both groups of patients, whereas basal FSH levels were significantly elevated only in treated patients (P less than .001). Following gonadotropin-releasing hormone (GnRH), levels of LH (P = .051) and FSH (P = .003) were greater in treated patients than in untreated control patients. No abnormalities of thyroid function or prolactin responsiveness were observed. Patients younger than 25 years of age at the time of treatment had lower serum levels of LH and FSH following chemotherapy than patients older than 25. Evidence for partial recovery of gonadal function was present with patients treated more than 18 months before study having lower levels of LH and FSH than those patients studied less than 18 months after treatment. These data demonstrate that frequent gonadal dysfunction exists in untreated patients with germ-cell tumors and that chemotherapy induces additional injury to both Leydig cells and the germinal epithelium. Further studies with long-term follow-up are necessary to define the pattern of gonadal recovery and to assess the potential sequelae of endogenous gonadotropin hypersecretion. PMID- 3020184 TI - In vivo electrophysiological evidence for the regulatory role of autoreceptors on serotonergic terminals. AB - The present in vivo studies were undertaken to evaluate electrophysiologically the modulatory role of the terminal 5-HT autoreceptor on 5-HT neurotransmission. In a first series of experiments, the effect of the electrical activation of the ascending 5-HT pathway on the firing activity of CA3 hippocampal pyramidal neurons was measured before and after the intravenous administration of methiothepin, a terminal 5-HT autoreceptor antagonist. Methiothepin significantly increased the duration of the suppression of firing activity of these neurons by the electrical stimulation of the 5-HT pathway, without modifying their responsiveness to microiontophoretically applied 5-HT. This suggests that endogenously released 5-HT activates the 5-HT terminal autoreceptor and that methiothepin enhances the efficacy of 5-HT synaptic transmission by blocking this activation. In a second series of experiments, further evidence for the activation of terminal 5-HT autoreceptors by 5-HT released by the electrical stimulation was sought by assessing the effectiveness of 2 series of stimulations of the ascending 5-HT pathway delivered at different frequencies while recording the same postsynaptic neuron. Increasing the frequency of stimulation (from 0.8 to 5 Hz) significantly reduced the duration of suppression of firing activity of the postsynaptic neurons. This difference between the 0.8 and 5 Hz stimulations was decreased by intravenous methiothepin, suggesting that the reduced effectiveness of the stimulations delivered at the higher frequency is attributable to a greater activation of the terminal 5-HT autoreceptor. These results provide direct electrophysiological evidence for the modulatory role of the 5-HT terminal autoreceptor on 5-HT neurotransmission. PMID- 3020185 TI - Transmembrane topology and subcellular distribution of the benzodiazepine receptor. AB - The distribution and transmembrane topology of benzodiazepine receptors were investigated in situ using intact and saponin-treated neurons of brain cell cultures. Reversible ligand binding and photoaffinity labeling, using 3H flunitrazepam as a probe, were employed in conjunction with trypsin-induced exhaustive proteolysis and competition binding using a novel benzodiazepine (Ro7 0213) that contains a quaternary ammonium moiety bearing a full positive charge. About 80% of the benzodiazepine receptors, with apparent subunit molecular weights of 48,000 and 51,000, are located in the surface membrane and contain one or more trypsin-sensitive sites exposed at the extracellular surface. The ligand recognition sites of the surface receptors are orientated toward the extracellular space and are sensitive to both competition by Ro7-0213 and trypsin attack. Approximately 20% of the receptors are intracellular or sequestered and are insensitive to both trypsin and competition by Ro7-0213 in intact cells. Strikingly, all of the 3H-flunitrazepam photolabeled sites inaccessible to competition by Ro7-0213 are also resistant to extracellular trypsin, but are sensitive to trypsin and Ro7-0213 in saponin-treated cells. This result provides strong evidence for protected receptors. Surface receptors are differentially sensitive to trypsin-induced inactivation such that 57% (of total) are inactivated, while 24% (of total) are cleaved but, surprisingly, retain the capacity to bind 3H-flunitrazepam. The observation that the ability of the receptor fragment to bind ligand is unaltered demonstrates that the integrity of the benzodiazepine binding site is not contingent upon the presence of about half of the intact polypeptide chain.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020186 TI - Interspecies selective motoneuron projection patterns in chick-quail chimeras. AB - During normal development chick motoneurons have been shown to project selectively to appropriate muscles by responding to a series of cues, both specific and nonspecific, within the limb. We tested the ability of motoneurons from another avian species, the Japanese quail, to respond to these cues by transplanting chick limb buds onto quail embryos and quail limb buds onto chick embryos between stages 17 1/2 and 19. Feulgen staining, which distinguishes chick from quail cells on the basis of nuclear chromatin, revealed that all limb tissue, including muscle, was of donor origin, indicating that the migration of somite-derived muscle precursor cells had been completed by the time of transplantation. Normal quail motoneuron pools for most muscles were located in the same relative positions as homologous chick pools. In chick-quail chimeras we found that the motoneuron pools of one species selectively innervated the homologous muscles in the limb of opposite species with considerable precision. This was determined by defining the segmental innervation pattern of the muscles electrophysiologically and by retrogradely labeling motoneuron pools with HRP. Selective innervation was confirmed by using the functional activation patterns of the motoneuron pools as an additional means of identifying motoneurons. We conclude that any limb-derived cues required by motoneurons to project to their appropriate muscles must be similar in chick and quail and that the growth cones of both species must have similar detector systems for responding to these cues. Only 7 spinal segments were found to innervate the quail limb (versus 8 for the chick), resulting in an anterior shift in the spinal segments innervating several posterior quail muscles.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020187 TI - Alterations in corticotropin-releasing factor-like immunoreactivity in discrete rat brain regions after acute and chronic stress. AB - Corticotropin releasing factor (CRF) may regulate endocrine, autonomic, and behavioral responses to stress. Evidence indicates that CRF-like immunoreactivity (CRF-LI) is widely distributed throughout the CNS. In this study, the distribution of CRF-LI was determined in 36 rat brain regions by combined radioimmunoassay-micropunch dissection techniques and the effect of stress on CRF LI was investigated, using a chronic stress model that induces endocrine changes in rats similar to those seen in depressed humans. A control group of rats was handled daily. An acute stress group was subjected to 3 hr of immobilization at 4 degrees C, while a chronic stress group was exposed to unpredictable stressors. Thirty-six brain regions were microdissected by the technique of Palkovits and assayed for CRF-LI, using a specific antiserum to ovine CRF. CRF-LI was detected in most regions. In controls, the highest concentrations were found in the arcuate nucleus/median eminence, the hypothalamic paraventricular (PVN) nucleus, and the periventricular nucleus. The next highest levels were found in the raphe nuclei and dorsal vagal complex. CRF-LI was well represented in the locus coeruleus (LC); in the central, cortical, and medial amygdaloid nuclei; and in the bed nucleus of the stria terminalis. Low concentrations occurred in the hippocampus and cerebrocortical regions. Appreciable concentrations were detected in midbrain and brain stem regions. Acute stress reduced CRF-LI in the arcuate nucleus/median eminence (ME) (by 52%) and in the median preoptic (MPO) nucleus (by 32%) and doubled its concentration in the locus coeruleus.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020189 TI - Differentiation and central projections of peripheral sensory cells with action potential block in Drosophila mosaics. AB - The ultrastructural differentiation and central projection of identified bristle mechanosensory neurons were examined in Drosophila mutants lacking action potentials. Two mutations, parats1 and napts, are known to block axonal conduction in centrally located neurons at high temperatures. Their effects on epithelial sensory cells, which are derived from imaginal disks during pupation, have not been determined. Furthermore, the parats1 napts double-mutant flies are lethal at all temperatures; thus the synergistic effect of these mutations on neurons has not yet been studied. It is possible to examine the above questions in genetic mosaics. By monitoring a reflex response involving identified bristle sensory cells, we found that the 2 mutations exert similar effects on these epithelial sensory cells as seen in central neurons. This also indicates that the action potential mechanisms in both epithelial sensory cells and central neurons are under similar genetic control. The parats1 napts double-mutant sensory cells in mosaics are nonfunctional at all temperatures, providing an opportunity to examine, at the single cell level, the development of neurons with activity block. Ultrastructural specializations typical of epithelial sensory cells were found in the double-mutant cells. Cobalt backfilling experiments showed that central projections of these nonfunctional sensory cells were not altered, as compared with the active contralateral sensory cells. Therefore, blockage of the action potential mechanism in individual sensory cells has no effect on their pathfinding and arborization. PMID- 3020188 TI - Connectional basis for frequency representation in the nuclei of the lateral lemniscus of the bat Eptesicus fuscus. AB - To study the role of the lateral lemniscus as a link in the ascending auditory pathway, injections of neuronal tracers were placed in the anteroventral cochlear nucleus (AVCN) and in the inferior colliculus of the bat Eptesicus fuscus. To correlate the anatomical results with tonotopic organization, the characteristic frequency of cells at each injection site was determined electrophysiologically. Pathways from AVCN diverge to 3 major targets in the lateral lemniscus, the intermediate nucleus and 2 divisions of the ventral nucleus (VNLL). Projections from these 3 nuclei then converge at the inferior colliculus. One cell group is particularly notable for its cytoarchitectural appearance. It is referred to here as the columnar area of VNLL because its cells are organized as a tightly packed matrix of columns and rows. The connections of the columnar area are organized in sheets that are precisely related to the tonotopic organization of both AVCN and the inferior colliculus. Sheets of cells in the dorsal part of the columnar area receive projections from low-frequency parts of AVCN and project to low-frequency parts of the inferior colliculus. These sheets of connections occupy successively more ventral locations as the tonotopic focus of the injection site increases in frequency. The entire range of frequencies audible to the bat is systematically represented along the dorsal-ventral dimension of the columnar area. Because each column is only 20-30 cells in height, frequency representation must be compressed in this dimension. Within the columnar area there is an overrepresentation of frequencies between 25 and 50 kHz, which corresponds roughly to the range of the FM echo-location call in Eptesicus. The connections of the other nuclei of the lateral lemniscus are not as precisely related to the tonotopic organization of the system as are those of the columnar area. PMID- 3020190 TI - Anatomical evidence for direct projections from the entorhinal area to the entire cortical mantle in the rat. AB - The entorhinal area is the most highly differentiated cortical field of the hippocampal formation from an anatomical point of view, and is best known as the origin of the perforant pathway, a massive association projection to the molecular layer of the dentate gyrus and Ammon's horn. This pathway is important as the first link in the so-called "trisynaptic circuit," which is thought to form the basic unit of information processing in the hippocampal formation and has been implicated in the elaboration of short-term memory and the more permanent storage of selected events in other parts of the cortical mantle. We have reexamined the efferent projections of the lateral entorhinal area with a sensitive new method that utilizes the anterograde axonal transport of a lectin, Phaseolus vulgaris leukoagglutinin (PHA-L), that is not internalized by fibers of passage, and displays labeled axons with the clarity of Golgi impregnations. The results of 5 experiments with injections confined entirely to the lateral entorhinal area suggest that this area sends fibers to innervate the entire cortical mantle, as well as to a longitudinal zone extending the length of the striatum (nucleus accumbens and medial caudoputamen) and the basolateral complex of the amygdala. In an additional series of experiments, injections of the fluorescent retrograde tracer fast blue that were centered in medial prefrontal, somatosensory, auditory, and motor areas of the cortex invariably labeled many neurons in layer IV of the lateral entorhinal area, as well as in other layers, depending on the site of injection. Finally, the results of double retrograde tracer experiments indicated that the 2 densest projections from the lateral entorhinal area--to the medial prefrontal region and to the dentate gyrus and Ammon's horn--arise from essentially separate populations of neurons. These findings serve to clarify the neural mechanisms underlying the role of the hippocampal formation in learning and memory, as well as in locomotor activity associated with goal-oriented behavior. PMID- 3020191 TI - Radiation dosimetry from breast milk excretion of radioiodine and pertechnetate. AB - Measurements were made of the activity in samples of breast milk obtained from a patient with postpartum thyroiditis following administration of [123I]sodium iodide and subsequently [99mTc]pertechnetate 24 hr later. Both 123I and 99mTc were found to be excreted exponentially with an effective half-life of 5.8 hr and 2.8 hr, respectively. Less than 10% of the activity was incorporated into breast milk protein. After administration of [123I]sodium iodide breast feeding should be discontinued for 24-36 hr to reduce the absorbed dose to the child's thyroid. PMID- 3020192 TI - Visualization of specific binding sites of benzodiazepine in human brain. AB - Using 11C-labeled Ro15-1788 and positron emission tomography, studies of benzodiazepine binding sites in the human brain were performed on four normal volunteers. Rapid and high accumulation of 11C activity was observed in the brain after i.v. injection of [11C]Ro15-1788, the maximum of which was within 12 min. Initial distribution of 11C activity in the brain was similar to the distribution of the normal cerebral blood flow. Ten minutes after injection, however, a high uptake of 11C activity was observed in the cerebral cortex and moderate uptake was seen in the cerebellar cortex, the basal ganglia, and the thalamus. The accumulation of 11C activity was low in the brain stem. This distribution of 11C activity was approximately parallel to the known distribution of benzodiazepine receptors. Saturation experiments were performed on four volunteers with oral administration of 0.3-1.8 mg/kg of cold Ro15-1788 prior to injection. Initial distribution of 11C activity following injection peaked within 2 min and then the accumulation of 11C activity decreased rapidly and remarkably throughout the brain. The results indicated that [11C] Ro15-1788 associates and dissociates to specific and nonspecific binding sites rapidly and has a high ratio of specific receptor binding to nonspecific binding in vivo. Carbon-11 Ro15-1788 is a suitable radioligand for the study of benzodiazepine receptors in vivo in humans. PMID- 3020194 TI - Does the baccalaureate make a difference?: Differentiating nurse performance by education and experience. AB - The nursing profession continues to debate the question of whether level of education matters in performance once the nurse is oriented to a particular setting, or whether the assumed advantages of baccalaureate education can be substituted by on-the-job experience. In this study, nurses with a baccalaureate degree demonstrated more nursing competencies when compared with their associate degree or diploma colleagues. Results also suggest that education promotes a broader range of abilities than does experience. PMID- 3020193 TI - A clinical evaluation tool for nursing students based on the nursing process. AB - An integrated curriculum was implemented in an upper division baccalaureate nursing program which required letter grades for clinical courses. The second semester clinical course involved student evaluation in four different settings. The original evaluation tool lacked both the discrimination needed for letter grades and the competencies common to all the settings. A computerized evaluation tool was developed to identify behaviors which could be evaluated and assigned a letter grade in all four clinical settings, and to provide formative and summative evaluation of performance for students throughout the semester. The tool focuses on the four areas of the nursing process, each of which is weighted according to the conceptual framework of the curriculum and the ability of a student at this level. In each of these four areas, entry level behaviors were identified and then built upon by progression from fundamental skills to more complex and independent behaviors. Students are rated on a criterion-referenced rating scale. A statistician verified that the tool was mathematically sound. Once developed, the tool was placed on computer where both students and faculty evaluate student clinical performance every two to four weeks. Because the tool was placed on computer, students receive immediate feedback, which facilitates formative evaluations. Trends in student performance can be identified easily, and faculty paperwork is decreased. The tool also promotes objectivity in evaluation and student awareness of expected behaviors. Finally, faculty and students have become more familiar and more comfortable with computers. PMID- 3020195 TI - Criterion-related validity of a clinical simulation. AB - This study explored the criterion-related validity of a clinical simulation by examining nurse practitioner's (NP) performance as measured by a chart audit and direct observation of care with performance on a clinical simulation. The clinical problem for all three instruments was the assessment and management of hypertension. Seventeen NPs completed all three instruments. The findings suggest that the chart audit and observation data were significantly correlated, but neither was significantly correlated with the clinical simulation. No evidence of criterion-related validity was noted, although several limitations of the study prohibited a definitive conclusion. Nurse educators are cautioned about using clinical simulations for evaluating student's performance until there is more support for criterion-related validity. PMID- 3020196 TI - Oral communication instruction for a career in nursing. AB - The reason for this study was to discover the specific oral communication skills necessary for success in nursing, and those skills most in need of improvement, according to Directors of Nursing. The findings supported claims in nursing literature that many specific oral communication are critical to the success of nurses. Other findings showed that most undesirable communication behaviors are only used either "rarely" or "sometimes" by nurses and that, of seven communication variables studied, communication skills associated with listening were most important and in greatest need of improvement by nurses. PMID- 3020197 TI - A model for constructing a conceptual framework for education in the clinical specialty. AB - A "Process Model" is described as an approach to developing and refining conceptual frameworks for education in the clinical specialties. A major characteristic of this approach is the borrowing of concepts from selected nursing models to construct the framework. Conceptual and empirical phases of the Model are discussed as they relate to clarifying the conceptual base of the clinical specialty. Applications of the Process Model to undergraduate and graduate education are presented. PMID- 3020198 TI - Teaching students to write for publication. PMID- 3020199 TI - Copyright law and nursing educators. PMID- 3020200 TI - Bridging the gap between humanism and behaviorism in nursing education. PMID- 3020201 TI - Faculty practice in a prison setting: implications for teaching. PMID- 3020203 TI - Purified reference diets for weanling rats: effects of biotin and cellulose. AB - Standardized purified diets limited to required nutrients are needed for nutritional and toxicological studies. In the present study, we formulated a biotin- and cellulose-free diet of reproducible mineral composition (diet A), based on diet AIN-76, and fed it to weanling Long-Evans rats for 3 wk. Inductively coupled argon plasma atomic emission spectrometry was used to determine Ca, Cu, Fe, K, Mg, Mn, Na, P and Zn in liver, duodenum, kidney, spleen and femur. Results were compared with those obtained with rats fed biotin- and/or cellulose-supplemented variations of diet A, diet AIN-76 and diet NIH-31 (an open formula stock diet). Weanling rats grew slowly and steadily on purified diet A. Growth rates increased when diet A was supplemented with biotin and cellulose. In general, differences among tissue mineral levels in rats fed diet NIH-31 and those fed diet AIN-76 were more pronounced than those among groups fed our purified diets. Values for hemoglobin and hematocrit were significantly lower in rats fed all purified diets than in those fed diet NIH-31. Diets A + biotin, A + cellulose and A + cellulose + biotin appear satisfactory as reference diets for measuring mineral interactions at near-requirement levels as well as effects of fiber on mineral utilization or for studies on vitamins whose endogenous synthesis may be influenced by dietary fiber. PMID- 3020202 TI - Copper deficiency-induced hypercholesterolemia: effects on HDL subfractions and hepatic lipoprotein receptor activity in the rat. AB - Male Sprague-Dawley rats (10 per group) were fed diets adequate (control) or deficient (CuDef) in copper for 6 wk. In the CuDef group, plasma total cholesterol, high density lipoprotein (HDL) cholesterol and apoA-I levels were significantly higher than in controls. Apolipoprotein analysis of the HDL fractions revealed a relative enrichment of apoE in the CuDef group. Size analysis of 1.21 density lipoproteins by nondenaturing gradient gel electrophoresis demonstrated the presence of more material migrating in the size range of HDL1 in the CuDef group. Separation of HDL into apoE-rich and apoA-I rich fractions by heparin-affinity chromatography confirmed the presence of increased apoE-rich HDL in the CuDef group. To determine the mechanism responsible for higher apoE-rich HDL in the CuDef group, the lipoprotein receptor binding activity in hepatic membranes from the control and CuDef group was assayed. Kinetic analysis of the binding data revealed that the lipoprotein binding assay was primarily measuring the activity of the HDL receptor, which is not apoE mediated. When corrected for differential enrichment of plasma membrane, hepatic membranes from the CuDef group bound significantly fewer lipoproteins than did controls. Furthermore, the hepatic receptor binding activity was negatively correlated with the proportion of HDL enriched with apoE. PMID- 3020204 TI - Effect of dietary protein level on the first steps of glucagon action in rat liver plasma membranes. AB - Binding of glucagon and glucagon-stimulated cyclic AMP production were studied in highly purified liver plasma membranes from growing rats fed a 12% protein diet (group 1) or 20% protein diet; this latter was given either in normal (group 2) or restricted (group 3) amounts. Groups 2 and 3 exhibited significantly higher peripheral glucagonemia than group 1 (amounting to 286 and 160% of group 1, respectively). Specific [125I]iodoglucagon binding to plasma membranes was similar in all groups. Scatchard analysis revealed no further differences between affinity constants and binding capacities in the three groups. Hormone degradation was constant. As membrane recovery and membrane purity were unaltered, these results suggest that hyperglucagonemia caused by increasing dietary protein level is not associated with any significant modification of glucagon binding sites in rat liver. In the presence of a potent phosphodiesterase inhibitor (3-isobutyl-1-methyl xanthine 0.2 mM) the glucagon stimulated cyclic AMP production was higher in rats fed the 20% protein diet, which was given in normal amounts, than in rats fed the 12% protein diet. In contrast when the 20% protein diet was given in restricted amounts the glucagon stimulated cyclic AMP production was similar to that in the 12% protein-fed rats. PMID- 3020205 TI - Regulatory effect of calcium-regulating hormones on the synthesis and/or secretion of bone gamma-carboxyglutamic acid-containing protein in chick embryonic calvaria in vitro. AB - Regulatory effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3), 24,25 dihydroxyvitamin D3 (24,25-(OH)2-D3), parathyroid hormone (PTH) and calcitonin (CT) on BGP synthesis and/or secretion were studied using chick embryonic calvaria in vitro. BGP contents in calvaria and culture medium were determined by radioimmunoassay using antiserum to purified chick BGP. After 72 h culture, 1,25 (OH)2-D3 (10(-10)-10(-7) M) increased the BGP content in culture medium significantly, and the effect was maximum at 10(-8)M, while the BGP content in calvaria was not changed by 1,25-(OH)2-D3. 24,25-(OH)2-D3 increased the BGP content both in medium at 10(-6) M-10(-5) M after 24 h culture and in calvaria at 5 X 10(-7)-10(-5) M after 48 h culture. PTH increased BGP contents both in calvaria after 72 h culture and medium at 1 U/ml after 24 h culture, while it decreased them at 5-10 U/ml after 24 h culture. CT (0.5-10 U/ml) had no effect on BGP contents in calvaria and medium. 1,25-(OH)2-D3-induced increase of BGP in culture medium was observed from 24 to 72 h of culture and then reached a plateau at 120 h of culture. 1,25-(OH)2-D3-induced increase in the BGP level in medium after 72 h culture was 3-4 times the control value. On the other hand, the BGP content in calvaria was not affected by 1,25-(OH)2-D3 until 72 h of culture and then significantly increased at 120 h. The addition of 1,25-(OH)2-D3 (10(-8) M), 24,25-(OH)2-D3 (10(-6) M), and 1 U PTH (and 1 U CT added to those hormones) into culture medium of chick embryonic calvaria significantly increased BGP contents in calvaria and medium at 72 h after culture. The effect of three hormones was not synergistic, but had a tendency to be greater than that of a single addition of 1,25-(OH)2-D3, 24,25-(OH)2-D3 or PTH. Further addition of 1 U CT into culture medium did not affect the combined effects of 1,25-(OH)2-D3, 24,25-(OH)2-D3 and PTH except for the calcium content in calvaria. The addition of individual calcium-regulating hormones into culture medium of chick embryonic calvaria did not affect the calcium and phosphorus contents in calvaria, but the calcium content in calvaria was significantly increased by the addition of 1,25-(OH)2-D3, 24,25-(OH)2-D3 and PTH to the culture medium.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3020206 TI - Effect of dietary vitamin D3 and cadmium on the lipid composition of rat intestinal brush border membranes. AB - Vitamin D deficiency in cadmium-exposed rats was observed along with enhanced tissue cadmium accumulation. In relation to the barrier function, the changes in the lipid composition have been studied in the intestinal brush border membranes prepared from rats raised on diets differing in vitamin D status in the absence or presence of cadmium. In an analysis of lipid composition, vitamin D3 treatment resulted in an increase of phospholipid content, and cadmium ingestion resulted in a decrease of cholesterol and glycolipid contents in the duodenal brush border membranes. On the other hand, vitamin D3 and cadmium showed no significant effect on the lipid composition of the jejunal brush border membranes. Further analysis of the fatty acid composition in duodenal brush border membrane lipids showed that vitamin D3 treatment led to an increase of the proportion of fatty acids (18:1 and 18:2 in the total and phospholipid fraction) and also shorter chain fatty acids in neutral lipid fractions in the absence of cadmium. However, vitamin D3 treatment in the presence of cadmium led to a decrease of the proportion of fatty acid (18:2 in the total and phospholipid fraction) and also shorter chain fatty acids in neutral lipid fractions. Vitamin D-dependent alterations of the membranes might act as a barrier in cadmium-exposed rats. PMID- 3020208 TI - Effect of fiber on protein, fat and calcium digestibilities and fecal cholesterol excretion. AB - Five female subjects were given four types of test diets containing various levels of protein for four consecutive 5-day periods and their dietary fiber and feces were collected throughout the experimental period. Diet A was a high-fiber, low-protein diet containing brown rice. Diet B was a semi-purified, low-protein diet containing agar agar as the sole source of dietary fiber. Diet C was a low fiber, normal-protein diet containing polished rice. Diet D was a high-fiber, normal-protein diet containing brown rice. A fecal marker was given at breakfast on the first day of each five-day test period and on the day after the end of the experiment. Fecal weight increased during the period on high-fiber diets (diets A and D). The apparent digestibilities of protein and fat were significantly depressed by high-fiber diet. Fecal excretion of calcium did not increase on the high-fiber diets. A decrease in the apparent digestibility on a high-fiber, low protein diet was partly due to the low intake of calcium during this period. Fecal excretion of cholesterol increased markedly during the periods on high fiber diets. The difference between the intake and fecal excretion of dietary fiber suggested that the fiber was partially digested in the colon. PMID- 3020209 TI - The cyclic AMP, the cyclic GMP and the protein kinase system in the rat parotid. PMID- 3020207 TI - Chemiluminescence in vitamin E-deficient erythrocytes initiated by xanthine oxidase reaction, in relation to the accumulation of thiobarbituric acid reactive substances. PMID- 3020210 TI - Late posttraumatic enophthalmos corrected by dense hydroxylapatite blocks. PMID- 3020212 TI - Intra-oral salivary gland neoplasms: a retrospective study of 98 cases. AB - The findings of a retrospective study of 98 minor salivary neoplasms are reported. The patient's ages ranged from 13-79 years and there was an equal sex distribution. Sixty-one of the lesions were benign, 53 being pleomorphic adenomas and 8 monomorphic adenomas. Of the malignant tumors, 19 were muco-epidermoid tumors, 12 adenoid cystic carcinomas, 4 adenocarcinomas, 1 carcinoma ex pleomorphic adenoma and 1 epidermoid carcinoma. One striking finding was the difference in age at the time of presentation for patients with muco-epidermoid tumors compared with those with adenoid cystic carcinomas. Seventy-four percent of the patients with muco-epidermoid tumors were under 50 years of age, but 75% of those with adenoid cystic carcinomas were over 50 years. PMID- 3020211 TI - The occurrence of blood group substances (A, B, H, Le-a, Le-b) in salivary glands and salivary gland tumors. An immunohistochemical investigation. AB - The distribution of blood group substances A, B, H, Le-a and Le-b in normal and neoplastic salivary gland tissue was evaluated by means of immunohistochemistry. The serological ABH blood group status of one third of the patients was known. Lewis blood group and secretory status were not known. In normal tissue, expression of blood group antigens corresponded to the serological blood group. Blood group substance H was present in almost every gland, regardless of the serological blood group. In submandibular glands, Le-b was rather selective for mucous acini. In tumors, a relationship of blood group expression to a glandular pattern and a high differentiation could be observed. Blood group substances were expressed at a high level in benign and highly differentiated malignant tumors. In poorly differentiated malignant tumors, they were mostly absent. Blood group expression evaluation could be of value in establishing the level of functional differentiation in salivary gland tumors. PMID- 3020213 TI - Particle and pore size distributions of investments. AB - The particle-size distributions of investment powders and the pore-size distributions of set-fired investments were investigated by electrozone size analysis, and by mercury intrusion scanning porosimetry, respectively, for a number of different investments, representing all three types available, namely: the gypsum-, phosphate- and silicate-bonded. The pore volume (V), the pore surface area (S), and their first derivatives with respect to radius, r, dV/dr and dS/dr, were obtained with pressures up to 414 MPa corresponding to spherical pores of 0.0018 micron radius if the Washburn equation is to apply. Results distinguished pores with a range of different sizes. Besides the porosity due to the matrix binder, porosity was also attributable to the refractory components. This latter type of porosity occurred with pore radii below about 0.03 micron, whereas matrix porosity occurred above this pore size. Matrix porosity for the phosphate materials was smallest at about 0.03 to 0.5 micron in radius, followed by the gypsum materials at about 0.3 to 0.6 micron, and by the silicate materials at about 1 to 10 micron. The particle-size distribution of the investment powders may affect both types of porosities, whereas the setting chemistries of the particular investment types may affect matrix porosity, by the generation of gaseous by-products. No major distinctions in pore size and distributions occurred between air-set and hygroscopic-set materials. This study emphasizes that at present, knowledge of the total pore volume only is inadequate for characterizing porosity distribution and hence investment permeability. PMID- 3020214 TI - Renal autoregulation and prostaglandin E2 release. Hemodynamic conditions for PGE2 and renin release. PMID- 3020215 TI - [Pleomorphic adenoma of the neck. Report of two cases]. PMID- 3020217 TI - Survival in breast cancer related to tumour oestrogen receptor status and immunohistochemical staining for NCRC 11. AB - NCRC 11 is a monoclonal antibody raised against dissociated breast cancer cells. Using this antibody, it has been shown in an earlier study that staining of a high proportion of tumour cells is strongly associated with superior survival. Many investigators have found that positive tumour oestrogen receptor (OER) status is associated with better survival in breast cancer. In the present study of 136 breast cancer patients followed up for a minimum of 30 months, tumour oestrogen receptor assays were carried out, and using the indirect immunoperoxidase technique, histological sections of paraffin embedded material were stained for NCRC 11; for the purpose of this study, tumours showing 25 per cent or less cells staining were regarded as 'negative'. Patients whose tumours were OER positive had a significantly better prognosis (logrank test P less than 0.05). Patients whose tumours were graded as negative for NCRC 11 had a poorer prognosis compared with the positive group but this did not attain significance. Our failure to demonstrate convincingly a better prognosis for the NCRC 11 positive patients may relate to the shorter duration of our study, or to different tissue fixation techniques. In this study staining for NCRC 11 was positively correlated with oestrogen receptor status (P less than 0.02). PMID- 3020216 TI - Effect fixation on T and B lymphocyte surface membrane antigen demonstration in paraffin processed tissue. AB - The identification of lymphoid surface membrane antigens in tissue sections using immunohistochemical techniques is becoming increasingly important for the diagnosis and classification of lymphoproliferative disorders. Many of the lymphocyte specific monoclonal antibodies used, however, can only be applied to frozen tissue sections. In this paper we report the successful application of a number of these antibodies to paraffin processed tissue utilizing alternative fixatives and the highly sensitive immunogold-silver staining method. The best fixatives for this purpose were formol dichromate, periodate-lysine paraformaldehyde (PLP) and a novel fixative formed from the addition of a dichromate solution to PLP. PMID- 3020218 TI - Noma in children with severe combined immunodeficiency. AB - Three Native American children with severe combined immunodeficiency developed noma, a necrotizing gingivostomatitis not previously reported in this country. The similarity between the clinical findings and those observed in monkeys with simian AIDS prompted us to evaluate our patients and their families for human retroviral infection. Antibodies to HTLV-I or HTLV-III/LAV proteins were not identified in patients nor in their family members. Standard bacterial and viral cultures similarly failed to identify a suspect pathogen. PMID- 3020219 TI - Calcium absorption during development: experimental studies of the rat small intestine. AB - During the period of natural weaning in the rat (beginning during the third week) the mechanisms of calcium absorption from the duodenum change from an efficient nonsaturable process, which is insensitive to vitamin D, to a combination of a less efficient nonsaturable process and a saturable vitamin D-dependent component. The stimulatory effect of vitamin D on active calcium transport requires the development of mucosal receptors for 1,25-(OH)2D3 and increasing circulating levels of 1,25-(OH)2D3. Concurrent increases in the concentration of the cytosolic calcium-binding protein (CaBP) support the suggestion that the CaBP mediates, in part, the effect of 1,25-(OH)2D3 on active calcium transport. The decline in the efficiency of nonsaturable calcium absorption during the third week coincides with, but is probably not due to, the loss of pinocytotic capacity of the small intestine. The time of appearance of the receptors for 1,25-(OH)2D3 and the decline in nonsaturable calcium absorption are under glucocorticoid control as are several other maturational events in the intestine at this time. PMID- 3020221 TI - Endosseous dental implantology from the periodontist's viewpoint. AB - Oral implantology has been a controversial dental therapeutic procedure for many years. Periodontics, as a specialty, did not really get involved until the word, osseointegration, was placed in the implant nomenclature by Dr. Per-Ingvar Branemark and the attendant success rate for osseointegrated prostheses was presented in a fully documented format. In March 1985, the definition of the scope of periodontics was provisionally changed by the Executive Council of the American Academy of Periodontology to include the discipline of oral implantology. Requirements for Advanced Specialty Education Programs in Periodontics with an effective date of January 1, 1986, include a statement as to the desirability of including implantology in some form in the postdoctoral curriculum. Many implant materials and designs are presently being used in endosseous dental implantology; studies are in progress to evaluate the short- and long-term response of hard and soft tissues to these materials. Continuing, nonbiased research is needed to fully understand and use this promising modality. PMID- 3020220 TI - Effects of neonatal undernutrition and subsequent nutritional rehabilitation or administration of thyroxine and hydrocortisone on the inositol phosphatase activities in rat intestine. AB - Undernutrition during the suckling period imposed by maternal protein deficiency during lactation resulted in elevated inositol triphosphatase activity (units per gram of wet tissue) in the ileum and lower phytase activity in the duodenum and jejunum. Activities of inositol triphosphatase in the duodenum and jejunum and phytase in ileum were unaffected. Postweaning nutritional rehabilitation resulted in elevated specific activities of both enzymes in all segments; however, activities of whole segments were similar to the corresponding control values. Elevation of inositol triphosphatase (ileum) and decline of phytase (duodenum and jejunum) activities due to undernutrition were reversed by the administration of hydrocortisone or thyroxine during undernutrition. These results suggest that maturation of activities of inositol triphosphatase in ileum (by hydrocortisone) and phytase in all segments (by both hydrocortisone and thyroxine) is under hormonal regulation, and the effects of neonatal undernutrition may be partly due to hormonal imbalances. PMID- 3020222 TI - Enhancement of mitomycin C uptake by isoproterenol in rat ascites hepatoma. AB - The amounts of mitomycin C (MMC) taken up into rat ascites hepatoma cells were determined by measuring the decrease in the absorbance of the incubation medium at 363 nm. The extracellular concentration of MMC decreased progressively in AH130 cell suspension and no peaks other than MMC were detected by analytical high-performance liquid chromatography (HPLC). Isoproterenol (IPN) enhanced the uptake of MMC into AH44 and AH130 cells but not AH13 cells in which the uptake increased by N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP). The increase of uptake of MMC by IPN was inhibited by propranolol and the uptake of MMC increased in a dose-dependent manner by theophylline in AH130 cells. The maximum combined cytotoxicity was observed when AH130 cells were treated with IPN at 10(-7) M for 30 min before the exposure to MMC and, in this pretreatment condition, the uptake of MMC was enhanced in parallel with the increase of cyclic adenosine 3':5'-monophosphate (cyclic AMP) level in the cells. On the other hand, MMC, which had no stimulating effect on the intracellular cyclic AMP level, nevertheless maintained a high level of intracellular cyclic AMP elevated by IPN for a longer period than in the treatment with IPN alone and prolonged the period of acceleration of the uptake of MMC. In the in vivo combined treatment with IPN and MMC, the life span of AH44-bearing rats was prolonged and 2 out of 6 rats were cured, while the mean survival time of the rats treated with MMC alone was 11.3 d.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020223 TI - Involvement of opioid receptors in hypo- and hyperthermic effects induced by phencyclidine in mice. AB - Experiments were conducted in order to examine the mechanism of changes in body temperature induced by phencyclidine (PCP) in mice. It is well known that morphine changes body temperature in a biphasic manner. PCP also produced hyperthermia at low doses (5 and 10 mg/kg) and hypothermia at high dose (40 mg/kg). The changes in body temperature induced by PCP were blocked by naloxone, a mu antagonist. Pretreatment with morphine (2.5 mg/kg), a mu agonist, or ethylketocyclazocine (EKC: 2.5 mg/kg), a kappa agonist, potentiated hypothermia induced by high dose of PCP. Effects of morphine and EKC on PCP-induced hypothermia were antagonized by naloxone. N-Allylnormetazocine (SKF 10 047: 20 mg/kg), a kappa and mu antagonist, antagonized PCP- and EKC + PCP-induced hypothermia but not morphine + PCP-induced hypothermia. Furthermore, Mr 2266, a kappa antagonist, antagonized PCP (10mg/kg)-induced hyperthermia and EKC + PCP induced hypothermia. It is suggested that PCP may affect thermoregulation through mu and/or kappa opioid receptor mechanisms. PMID- 3020224 TI - Inhibition by cyclic GMP of p-aminohippurate uptake by basolateral membrane vesicles isolated from rat kidney cortex. AB - We studied the effect of cyclic guanosine 3',5'-monophosphate (cyclic GMP) on p aminohippurate (PAH) uptake by basolateral membrane vesicles isolated from rat kidney cortex. Cyclic GMP inhibited PAH uptake dose-dependently. Dibutyryl cyclic GMP inhibited the uptake of PAH to the same extent. However, cyclic adenosine 3',5'-monophosphate, cyclic cytidine 3',5'-monophosphate and guanosine monophosphate had no effect on PAH uptake by membrane vesicles. Therefore, the inhibition of PAH uptake was specific to cyclic GMP and not common to nucleotides. In the presence of probenecid, an inhibitor of PAH transport, cyclic GMP did not affect PAH uptake. Thus, cyclic GMP had an inhibitory effect on probenecid-sensitive PAH transport. Inhibition by cyclic GMP of PAH uptake by basolateral membrane vesicles as described this study may contribute to the decrease in PAH accumulation in kidney cortical slices caused by the cyclic nucleotide which we previously reported. PMID- 3020226 TI - [The periodontist confronting the viruses: herpes, hepatitis B, AIDS]. PMID- 3020225 TI - Molecular properties of cetiedil and its interactions with erythrocyte membranes. AB - Cetiedil, an antisickling agent and a vascular smooth muscle relaxant, is an amphiphilic molecule. The critical micelle concentration in 5 mM phosphate buffer with 150 mM NaCl is approximately 8.8 mM. The molecule, as the citrate salt, is highly acidic at millimolar concentrations. The UV absorption extinction coefficient at 233 nm, E233, is 2796 M-1 cm-1. The studies of free cetiedil concentrations in the presence of membrane ghosts show that large amounts of cetiedil associate with membrane samples. Spin label electron paramagnetic resonance experiments showed that the lipids and the proteins of erythrocyte membrane samples were both affected by the addition of cetiedil. However, the cetiedil effects on membrane components are reversible. The protein spin label results demonstrate the binding of cetiedil to the membrane with an apparent equilibrium dissociation constant of approximately 2 mM. The binding is saturable. The apparent half-saturation concentrations for the binding at physiological ionic strength and temperature are in the range of 1-3 mmoles of cetiedil per gram of membrane proteins. Our studies also indicate that binding is not affected by the removal of the spectrin and actin network from the membranes. Interaction of cetiedil with Band 3 molecules in the erythrocyte membrane is suggested. The regions near the lipid head groups in the membrane samples are also affected by cetiedil. PMID- 3020227 TI - Effects of pindolol and propranolol on beta adrenergic receptors on human lymphocytes. AB - The cardiovascular supersensitivity observed after discontinuation of administration of beta adrenergic receptor antagonists may be explained by an increase in the density of beta adrenergic receptors. Thus, a change in the density of receptors has been observed in human lymphocytes after administration of propranolol (Aarons et al., 1980). The effects of pindolol, a beta adrenergic receptor antagonist with intrinsic sympathomimetic activity, were compared with those of propranolol or placebo. Pindolol (10 mg q.i.d.), propranolol (40 mg q.i.d.) or placebo were administered to 12 subjects for 8 days. The density of beta adrenergic receptors was determined by Scatchard analysis of the specific binding of [125I]iodopindolol on membranes prepared from human lymphocytes. Administration of pindolol resulted in a 30 to 50% decrease in the density of beta adrenergic receptors. This decrease was apparent within 1 day of beginning pindolol administration and it persisted for at least 8 days after discontinuation of drug administration. The reversibility of the decrease in receptors observed after pindolol administration was studied in 27 subjects given propranolol, pindolol or placebo for 4 days in a double-blind cross-over trial. Propranolol consistently induced a small increase in the density of beta adrenergic receptors. The density of receptors returned to predrug values within 2 days after discontinuation of propranolol administration. Pindolol induced a 30 to 50% decrease in the density of receptors which, as observed previously, persisted for at least 10 days after discontinuation of treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020228 TI - Pharmacodynamic supersensitivity and opioid receptor upregulation in the mouse. AB - The analgesic potency and toxicity (lethality) of morphine were increased 2.5 times after implantation of 7.5-mg s.c. naltrexone pellets in the mouse for 8 days. Implantation for 8 days also resulted in a 41% [3H][D-Ala2-D Leu5]enkephalin and 55% [3H] [D-Ala2-MePhe4-Gly(ol)5]enkephalin increase in radiolabeled opioid binding in mouse brain relative to placebo-implanted controls. Treatment for 1 day did not produce any significant increases in binding or morphine's analgesic potency. Brain morphine concentrations did not differ after a dose of morphine (8 mg/kg) that produced analgesia in 86% of 8-day naltrexone-treated mice vs. 39% of placebo-treated mice. The increase in the analgesic potency of morphine by chronic naltrexone treatment in the mouse is particularly striking as it is approximately 3 times greater than that observed for the rat. The decrease in the LD50 of morphine after 8 days of naltrexone treatment raises the possibility that the toxicity of opiates may be increased in patients who discontinue naltrexone maintenance treatment and resume opiate abuse. PMID- 3020229 TI - Stereoselective effect of beta-endorphin on the production of analgesia and the spinal release of met-enkephalin in rats. AB - The author has previously reported that i.c.v. beta-endorphin increases the release of immunoreactive Met-enkephalin from the spinal cord. The present studies were conducted to study the effects of beta-endorphin and its position 5 amino acid-substituted analogs on the production of analgesia and the spinal release of Met-enkephalin in rats. Analgesia was measured by tail-flick test in conscious rats, and the release of Met-enkephalin from the spinal cord was studied in urethane-anesthetized rats using a technique of spinal superfusion. beta-Endorphin (0.8-40 micrograms i.c.v.) inhibited the tail-flick response in a dose-related manner, whereas D-Met5-beta-endorphin, even at the highest dose, 40 micrograms, was unable to inhibit the tail-flick response. L- and D-Leu5-beta endorphin inhibited the tail-flick response only at high doses, with the D-isomer being slightly more potent than the L-isomer. Human beta-endorphin (0.8-16 micrograms i.c.v.) caused a dose-dependent increase of Met-enkephalin release from the spinal cord, whereas D-Met5-beta-endorphin, even at a high dose (16 micrograms), showed no release of Met-enkephalin. Leu5-beta-endorphin and D-Leu5 beta-endorphin showed little or no release of Met-enkephalin. The results indicate that beta-endorphin shows stereoselectivity on the production of analgesia and the release of Met-enkephalin. PMID- 3020230 TI - Inhibition of human placental Na+-H+ exchanger by cimetidine. AB - The Na+-H+ exchanger of human placental brush-border membrane was strongly inhibited by cimetidine, a histamine type II receptor antagonist. The inhibition was freely reversible and the apparent inhibition constant for the process was 38 microM. The nature of inhibition was found to be competitive with respect to Na+. Cimetidine inhibition was specific as other related compounds had no inhibitory effect. Treatment of the membrane vesicles with diethylpyrocarbonate inactivated the exchanger, but the presence of cimetidine during the treatment provided complete protection from this inhibition. These results show that histidine residues are located at the external Na+-binding site of the exchanger and cimetidine interacts with this site. The Na+-H+ exchanger of rabbit renal brush border membrane was also susceptible to inhibition by the drug. However, transport of proline and glucose in placental brush-border membrane vesicles was not affected by cimetidine. The placental Na+-H+ exchanger was also inhibited by amiloride and the inhibition was freely reversible and competitive with respect to Na+. These results show that the characteristics of the inhibition of Na+-H+ exchanger by cimetidine and amiloride are very similar. PMID- 3020231 TI - Digitalis-induced mechanical toxicity: protection by slow Ca++ channel blockers. AB - In the in vitro perfusion of the isolated heart, toxic doses of cardiac glycosides produce an inotropic response which is followed by a decline in contractile force and an increase in the resting tension. Several reports in the literature indicate that the subsequent decline in contractile force may be related to cardiac cellular Ca++ overload. The purpose of the present study was to determine if the slow Ca++ channel blockers such as verapamil and nifedipine, which block Ca++ influx through voltage-dependent gated channels, can reduce or prevent the digitalis-induced decline in contractile force (mechanical toxicity). Langendorff preparations of isolated perfused guinea pig heart were used for the present study. The data obtained demonstrate that 1 to 2 microM ouabain in the perfusion medium produced mechanical toxicity in the hearts after an initial inotropic response. Verapamil or nifedipine, when combined with ouabain in the perfusion medium, increased the magnitude of the inotropic response and delayed or abolished the mechanical toxicity in a dose-dependent manner. No changes in the sarcolemmal Na+,K+-adenosine triphosphatase or ouabain binding were observed in the presence of verapamil or nifedipine. The data suggest that simultaneous use of verapamil or nifedipine may protect against digitalis-induced mechanical toxicity. PMID- 3020232 TI - A re-evaluated role for cyclic AMP in uterine relaxation. Differential effect of isoproterenol and forskolin. AB - Our previous observations suggested that beta adrenergic-mediated relaxation of the rat myometrium could not be ascribed solely to cyclic AMP. The present study examines the relationships between relaxation and cyclic AMP accumulation in the myometrium in response to isoproterenol, forskolin and the combination of both. The diterpene enhanced cyclic AMP generation and potentiated the rises in cyclic AMP due to isoproterenol and prostaglandin (PG) E2. Isoproterenol-induced relaxation of a carbachol-contracted myometrium was associated with modest increments in cyclic AMP (6-12 pmol/mg of protein) in contrast to forskolin whose relaxing effect could be expressed only when associated with large increases in cyclic AMP (80-180 pmol/mg of protein). PGE2, although elevating cyclic AMP to the same extent as isoproterenol, caused contractions which were antagonized by isoproterenol and forskolin, respectively, associated with low and high cyclic AMP concentrations. Both PGE2 and forskolin, by virtue of their stimulatory effect on cyclic AMP generation, enhanced the efficiency of isoproterenol to cause relaxation. Likewise, the greater efficacy of forskolin to relax a PGE2- as opposed to a carbachol-contracted myometrium, was ascribed to its potentiated cyclic AMP response when combined with PGE2. It is proposed that the beta adrenoceptor-linked relaxation results from the concerted effects of both a cyclic AMP-dependent (sensitive to low cyclic AMP) and a cyclic AMP-independent process; the latter is postulated to operate at the membrane level with an ultimate reduction in cytosolic Ca++. On the other hand, cyclic AMP, provided it reached a critical concentration essential to mediate intracellular Ca++ sequestration, would be the sole determinant for forskolin-elicited relaxation. PMID- 3020233 TI - Visualization of vasoactive intestinal peptide receptors in human and guinea pig lung. AB - Binding of [125I]vasoactive intestinal peptide (VIP) to human and guinea pig lung sections was characterized and VIP receptors localized by light-microscopic autoradiography. Inhibition of [125I] VIP binding by unlabeled VIP, peptide histidine isoleucine, peptide histidine methionine, secretin and VIP fragment VIP10-28 indicated that [125I]VIP bound to specific receptors and binding was shown to be reversible. Scatchard analysis showed two binding sites; human lung and a dissociation constant (Kd) of 0.27 +/- 0.04 and 23.3 +/- 3.5 nM, with maximum binding capacities (Bmax) of 11.2 +/- 3.1 and 589 +/- 98 fmol mg-1 of protein for the high and low affinity sites, respectively. Guinea pig lung similarly had a high affinity Kd of 0.3 +/- 0.05 nM and Bmax of 226 +/- 61.1 fmol mg-1 of protein and a low affinity Kd value of 18.8 +/- 2.4 nM and Bmax of 1730 +/- 260 fmol mg-1 of protein. In both human and guinea pig lung there was labeling of cellular structures and no specific labeling pattern was found in sections incubated in the presence of an excess of unlabeled VIP. A high density of labeling was found over airway epithelium of all airways, submucosal glands and vascular smooth muscle. There was also labeling of airway smooth muscle of large, but not of small, airways. This localization corresponds to the pattern of VIPergic innervation. In addition, there was labeling of alveolar walls. This indicates that VIP has a physiological role in the lung. PMID- 3020234 TI - Carbachol- and norepinephrine-stimulated phosphoinositide metabolism in rat brain: effect of chronic cholinesterase inhibition. AB - Activation of cholinergic muscarinic receptors leads to several biochemical events including an increased turnover of phosphoinositides. In this study we have investigated whether repeated administration of the organophosphorus insecticide disulfoton, known to cause the development of tolerance to this compound, would affect phosphoinositide metabolism in rat brain. Basal and carbachol-stimulated phosphoinositide metabolism were measured in cerebral cortex slices, by measuring the accumulation of inositol phosphates (InsPs) in the presence of lithium. In control animals carbachol caused a 600% increase in InsPs accumulation with an EC50 of 100 microM. Maximal effect occurred with a LiCl concentration of 7.5 mM and required the presence of calcium. Administration of disulfoton for 10 days (2 mg/kg/day by gavage), decreased the number of muscarinic receptors in cortex from 1.1 to 0.7 pmol/mg of protein without changing the affinity of the receptors (both measured by binding of [3H]quinuclidinyl benzilate). Acetylcholinesterase was inhibited by 85%. Basal InsPs accumulation was unchanged in disulfoton-treated rats, whereas carbachol stimulated InsPs accumulation decreased by 18%. No changes of norepinephrine stimulated InsPs formation and of alpha-1 adrenoceptors were present in cortices from disulfoton-treated rats. Recovery of muscarinic receptor binding and carbachol-stimulated InsPs accumulation occurred at a similar rate and was completed 2 to 3 weeks after the end of the treatment, whereas acetylcholinesterase activity was still 38% inhibited 3 weeks later. These results support the hypothesis that a functional adaptation of muscarinic receptors is involved in the development of tolerance to organophosphates. PMID- 3020236 TI - Serum calcium level of freshwater snake, Natrix piscator, in response to vitamin D3 administration. AB - The effect of i.m. injection of vitamin D3 (25 IU/100 g b.wt) on serum calcium level was investigated in Natrix piscator. This treatment evokes hypercalcemia at day 3 which progresses up to day 5. Thereafter, a decline was observed in the serum calcium level at day 10 and day 15. PMID- 3020235 TI - Leukotriene D4 receptor-mediated synthesis and release of arachidonic acid metabolites in guinea pig lung: induction of thromboxane and prostacyclin biosynthesis by leukotriene D4. AB - The effects of the peptidoleukotrienes C4 (LTC4), D4 (LTD4), E4 (LTE4) and a series agonists and antagonists on arachidonic acid metabolism were characterized in minced guinea pig lung. In response to LTD4, guinea pig lung utilized and converted the endogenous arachidonic acid into a variety of cyclooxygenase metabolites [prostaglandins (PGs) E2, F2 alpha, 6-keto-F1 alpha and thromboxane (Tx) B2] and lipoxygenase metabolites (5,12-dihydroxy-6,8,11,14-eicosatetraneoic acid and 5,15-dihydroxy-5,9,11,13-eicosatetraneoic acid). Using radioimmunoassays, the stable, pharmacologically important metabolites of the cyclooxygenase pathway, 6-keto-PGF1 alpha and TxB2, were quantitated. LTD4, LTE4 and several synthetic agonists induced dose-dependent synthesis and release of TxB2 and 6-keto-PGF1 alpha. Agonist-induced synthesis and release of 6-keto-PGF1 alpha and TxB2 was time-dependent and was maximal after 10 to 12 min of incubation. The slow-reacting substance of anaphylaxis antagonist, FPL 55712, and the newly reported dithioacetal LTD4 receptor antagonists (SKF 102922 and SKF 102081) did not induce synthesis and release of TxB2 and 6-keto-PGF1 alpha in concentrations that blocked the agonist-induced smooth muscle contraction. Preincubation with these antagonists inhibited the synthesis and release of prostanoids induced by LTD4 in the guinea pig lung. Islet activating protein, which inactivates the inhibitory guanine nucleotide binding protein (Gi protein), partially inhibited the agonist-induced synthesis and release of prostanoids. Furthermore, the receptor binding affinities and/or the myotonic activities of the LTD4 agonists correlated linearly with the agonist-induced prostanoid synthesis and release effect.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020237 TI - In vitro effect of acidulated phosphate fluoride gel on the surface of composites with different filler particles. PMID- 3020238 TI - Role of rota-virus in acute gastroenteritis in young children--a preliminary report. PMID- 3020239 TI - The irritable bowel syndrome. AB - The clinical characteristics and putative pathogenetic mechanisms of the irritable bowel syndrome are described. The complex inter-relationships between stressful life situations, psychoneurotic and somatic factors involved in the production, maintenance and management of alimentary symptoms are discussed using a behavioural model and catastrophe theory. PMID- 3020240 TI - [Blue tympanic membrane. Apropos of 3 cases]. AB - A CT scan allowed simple diagnosis of the affection in three patients with the blue tympanic membrane syndrome. Lesions included prolapse of the internal jugular vein sinus into the tympanic cavity, an aberrant trajectory of the internal carotid artery in the middle ear and a tympano-jugular glomus tumor. Angiographic investigation of the latter lesion was performed more as a pre treatment procedure rather than for diagnostic purposes, since it served as a guide to surgery, consisting usually of embolization. PMID- 3020242 TI - Arthralgia from parvovirus infection. PMID- 3020241 TI - [Dissolution of obstructive uric acid calculi by percutaneous nephrostomy and in situ alkalinization]. AB - Following the introduction of one or two percutaneous nephrostomy drains, 16 obstructive uric acid calculi were treated by perfusion of excretory cavities with 14% sodium bicarbonate solution. Twelve calculi had dissolved within an average of 5 days, 1 required percutaneous lithotripsy because of incomplete dissolution and 3 were unaltered. Success of this treatment appears to be dependent on the total radiotransparency of calculi, a feature difficult to demonstrate. There were no reports of major complications. The only weakly invasive nature of alkalinization in situ makes it a method of choice to be reserved for obstructive calculi, and a therapeutic strategy is proposed. PMID- 3020243 TI - Adrenal and gonadal contribution to circulating androgens in spotted hyaenas (Crocuta crocuta) as revealed by LHRH, hCG and ACTH stimulation. AB - Single venous blood samples were collected from 52 hyaenas and serial samples via indwelling jugular catheters from 8 hyaenas. Social status of the hyaenas was unknown. Radioimmunoassay estimations were specific for testosterone, androstenedione, cortisol and LH. No significant differences could be found between the sexes for concentrations of testosterone (adult males 2.9 +/- 1.3 nmol/l; adult females (non-pregnant) 1.6 +/- 0.3 nmol/l). Androstenedione concentrations in sub-adult males were significantly lower than those in females (2.3 +/- 0.05 and 5.5 +/- 1.2 nmol/l). Testosterone and androstenedione concentrations were significantly higher in parous pregnant than in parous non pregnant females (4.3 +/- 1.4 and 1.6 +/- 0.3; and 23.7 +/- 11.6 and 6.7 +/- 0.9 nmol/l respectively). Both LHRH and hCG elicited elevated androgen concentrations in females in different reproductive categories. Androgens are produced by the ovary in the absence of follicular or luteal structures, indicating androgen production by the interstitial tissue of the ovarian stroma. PMID- 3020244 TI - Opioid agonist and antagonist bivalent ligands. The relationship between spacer length and selectivity at multiple opioid receptors. AB - Bivalent ligands containing the oxymorphamine or naltrexamine pharmacophores connected to spacers of varying length were synthesized and evaluated for their selectivity at mu, kappa, and delta opioid receptors. The oxymorphamine bivalent ligands (1-8) behaved as mu agonists on the electrically stimulated guinea pig ileum longitudinal muscle preparation (GPI). The spacer that conferred peak agonist activity in these series contains a total of four glycyl units (n = 2). Binding studies with guinea pig brain membranes showed a qualitatively similar profile at mu receptors as a function of spacer length. Also, delta receptor selectivity increased as the spacer was lengthened. The naltrexamine bivalent ligands (9-13) effectively antagonized the mu receptor agonist morphine in the GPI at the same optimal spacer length (n = 2) as in the agonist series. However, the peak antagonism of ethylketazocine, a kappa receptor agonist, occurred with the bivalent ligand 9 containing the shortest spacer (n = 0), and it was found that 9 is the most selective kappa antagonist in the series. While receptor binding roughly parallels that of kappa antagonist activity in the GPI, no correlation between binding and antagonist activity was observed at mu opioid receptors. The possible significance of these results is discussed. PMID- 3020245 TI - Synthesis and biological activity of analogues of beta-chlornaltrexamine and beta funaltrexamine at opioid receptors. AB - beta-Chlornaltrexamine and beta-funaltrexamine analogues 4-7 with different length "arms" to which an electrophilic moiety is attached were synthesized in an effort to obtain affinity labels that would selectively and irreversibly block specific opioid receptor types and subtypes. One of the compounds, 4, was a potent, irreversible blocker of opioid receptors in the guinea pig ileum and mouse vas deferens preparations. The results of this study suggest that nucleophiles that are remote from the recognition locus are capable of alkylation by reactive electrophiles. PMID- 3020246 TI - Irreversible blockage of opioid receptor types by ester homologues of beta funaltrexamine. AB - A series of ester homologues 2-5 of the mu receptor nonequilibrium antagonist beta-funaltrexamine (1, beta-FNA) was synthesized. These ligands were of interest in our investigation of the relationship between the structure of the ester function and the ability to irreversibly block mu opioid receptors. While all of the ligands were potent reversible agonists in the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations, most appeared to behave as irreversible antagonists of morphine. The benzyl 5 and phenethyl 6 esters possessed irreversible mu antagonist potency that was of similar magnitude to that of beta FNA in the GPI. In the MVD, all esters appeared to irreversibly block the agonist effect of morphine, but none of the compounds irreversibly antagonized [D-Ala2,D Leu5]enkephalin to a significant degree. [3H]Dihydromorphine displacement studies revealed no relationship between the affinity of the esters 1-6 and the irreversible blockage of mu receptors in the GPI or MVD. Possible reasons for the observed structure-activity relationship are discussed. PMID- 3020247 TI - Conformation and activity of tetrahydrofuran lignans and analogues as specific platelet activating factor antagonists. AB - The six (racemic or meso) isomers of 3,4-dimethyl-2,5-bis(3,4 dimethoxyphenyl)tetrahydrofuran and four corresponding desmethyl analogues were prepared and assayed as inhibitors of platelet activating factor (PAF) receptor binding to rabbit platelet plasma membranes. The inhibition by these isomers is stereodependent and varies with the gross shape of the molecules as determined by the molecular mechanics program MM2. The most potent PAF antagonist in this group of compounds is trans-2,5-bis(3,4,5-trimethoxyphenyl)tetrahydrofuran (L-652,731, 14) with an IC50 of 0.02 microM. PMID- 3020248 TI - Cardiac glycosides. 7. Sugar stereochemistry and cardiac glycoside activity. AB - Digitoxigenin alpha-L-, beta-L-, alpha-D-, and beta-D-glucosides; alpha-L-, beta L-, alpha-D-, and beta-D-mannosides; and alpha-L- and beta-L-rhamnosides were stereoselectively synthesized from the corresponding sugar tetrabenzyl trichloroacetimidates. The Na+,K+-ATPase receptor inhibitory activities of these glycosides (as a measure of receptor binding) were compared with those of digitoxigenin, digitoxigenin 6'-hydroxy-beta-D-digitoxoside, digitoxigenin beta-D galactoside, and digitoxigenin beta-D-digitoxoside. The observed activities reveal that a given sugar substituent may have a role in binding of some glycoside stereoisomers, but not others. With alpha-L- and possibly beta-L rhamnosides, the 5'-CH3 and 4'-OH appear to have a predominant role in binding to the Na+,K+-ATPase receptor. Addition of a 6'-OH to form the corresponding mannosides dramatically disrupts the effect of both the 5'-CH3 and 4'-OH in prompting receptor binding of the alpha-L isomer. However, with the beta-L isomer, some influence of 4'-OH, 3'-OH, and 2'-OH binding remains. With beta-D glycosides, binding via the "5'-CH3 site" appears to be of little importance and addition of a 6'-OH diminishes activity only slightly. With these beta-D glycosides, an equatorial 4'-OH, axial 3'-OH, and equatorial 2'-OH groups appear to contribute to binding. PMID- 3020249 TI - Synthesis of novel angiotensin converting enzyme inhibitor quinapril and related compounds. A divergence of structure-activity relationships for non-sulfhydryl and sulfhydryl types. AB - The synthesis and angiotensin converting enzyme (ACE) inhibiting activities of quinapril (CI-906, 22), its active diacid (CI-928, 33), and its dimethoxy analogue (CI-925, 25) are reported. These tetrahydro-3-isoquinolinecarboxylic acid derivatives possess equivalent in vitro potency and in vivo efficacy to enalapril. Sulfhydryl analogues with the same structural variation are also highly potent. In contrast, tetrahydro-1-isoquinolinecarboxylic acid and homologous isoindoline-1-carboxylic acid analogues show a striking divergence in potency between the two types, sulfhydryl analogues being essentially inactive, while non-sulfhydryl analogues are equipotent with the proline prototype. This is the first evidence suggesting that alternate binding modes may exist for the two major structural classes of small molecule ACE inhibitors. PMID- 3020250 TI - Synthesis and adrenergic activity of ring-fluorinated phenylephrines. AB - 2-Fluoro-, 4-fluoro-, and 6-fluorophenylephrine (6-FPE) were synthesized from the corresponding fluorinated 3-hydroxybenzaldehydes. New routes to 2-fluoro- and 6 fluoro-3-hydroxybenzaldehydes were developed based on regioselective lithiation of 2- and 4-[(dimethyl-tert-butylsilyl)oxy]fluorobenzene ortho to fluorine. As with norepinephrine and isoproterenol analogues, the adrenergic properties of phenylephrine were markedly altered by ring fluorination. The order of potency of the fluoro analogues as alpha 1-adrenergic agonists in the stimulation of contraction of aortic strips and of phosphatidylinositol turnover and potentiation of cyclic AMP accumulation in guinea pig synaptoneurosomes was 6-FPE greater than PE greater than 4-FPE greater than 2-FPE. The same pattern was observed for the displacement of radioligands specific for alpha 1- and alpha 2 adrenergic receptors on brain membranes. The order of potency for the displacement of [3H]dihydroalprenolol, a beta-specific adrenergic ligand from brain membranes, was 2-FPE greater than 4-FPE = PE much greater than 6-FPE. 6-FPE was much more selective for alpha-adrenergic receptors compared to beta-receptors than was phenylephrine. A rationale for the observed fluorine-induced alterations in potency and selectivity of the FPEs for alpha- and beta-adrenergic systems is presented based on fluorine-induced conformations due to electrostatic repulsion of fluorine and the benzyl hydroxyl group. PMID- 3020251 TI - Cyclic analogues of 2-amino-4-phosphonobutanoic acid (APB) and their inhibition of hippocampal excitatory transmission and displacement of [3H]APB binding. AB - Conformationally restricted analogues of 2-amino-4-phosphonobutanoic acid (APB,2) were prepared where the structure of APB was incorporated into cyclopentane (3) or cyclohexane (4) rings. Hydrophosphinylation of the appropriate cycloalkenones followed by Strecker amino acid syntheses provided the desired analogues. Assignments of the relative configurations for 3a (trans), 3b (cis), 4a (cis), and 4b (trans) were determined through 13C NMR studies. Compounds 3b, 4a, and 4b possessed low activity as inhibitors of excitatory synaptic field potentials in the rat hippocampal perforant path. Analogues 4a and 4b also showed little activity in displacing [3H]APB from synaptic plasma membranes. The cyclopentyl APB analogue 36, on the other hand, was extremely potent in inhibiting the binding of [3H]APB, possessing an IC50 = 4.7 microM, thus giving further credence to the idea that the APB binding site in the rat brain synaptosomal membrane preparation is not the same as the receptor mediating APB-induced inhibition of the lateral perforant path. Of the four cyclic APB analogues, 3a most resembled APB in its spectrum of biological activity. It showed significant potency (IC50 = 130 microM) in inhibiting lateral entorhinal projections to hippocampal granule cells. Analogous to APB, 3a also showed selectivity for the lateral perforant path over the medial perforant path. Its activity in the radioligand binding assay paralleled its activity in inhibiting the lateral perforant path. It thus appears that 3a comes closest to mimicking the active conformation of APB and suggests that a folded conformation wherein the amino and phosphonate moieties are in a cis relationship to one another may approximate the active conformation of APB. PMID- 3020252 TI - Investigation of 4-(3-hydroxyphenyl)-4-methylpipecolic acid as a conformationally restricted mimic of the tyrosyl residue of leucine-enkephalinamide. AB - Analogues of leucine-enkephalinamide containing N-terminal cis- or trans-4-(3 hydroxyphenyl)-4-methylpipecolic acid were prepared to examine the conformational requirements of the N-terminal tyrosyl residue in opioid activity. The diastereomeric amino acids were prepared and purified by semipreparative HPLC before incorporation into the peptide. Spectroscopic analysis based on proton nuclear Overhauser enhanced differential spectroscopy (NOEDS) allowed assignment of the cis and trans stereochemistry. Despite spatial analogy between the trans isomer 5 and leucine-enkephalinamide, it possessed neither opioid agonist nor antagonist activity in the guinea pig ileal longitudinal muscle (GPI) or mouse vas deferens (MVD) preparations. Possible explanations for this inactivity are discussed. PMID- 3020253 TI - Chiral DNA gyrase inhibitors. 1. Synthesis and antimicrobial activity of the enantiomers of 6-fluoro-7-(1-piperazinyl)-1-(2'-trans-phenyl-1'-cyclopropyl)-1, 4 dihydro-4-oxoquinoline-3-carboxylic acid. AB - New quinolone antimicrobial agents (racemic, (1'S,2'R)- and (1'R,2'S)-6-fluoro-7 (1-piperazinyl)-1-(2'-trans-phenyl-1'-cyclopropyl)- 1, 4-dihydro-4-oxoquinoline-3 carboxylic acids) were synthesized, and their in vitro antimicrobial potencies and spectra were determined. As compared to their conceptual parents, these agents retained a considerable amount of the antimicrobial potency and spectra of ciprofloxacin and of 6-fluoro-1-phenyl-7-(1-piperazinyl)-1,4-dihydro-4 oxoquinoline-3-carboxy lic acid against Gram-positives. Gram-negatives were considerably less sensitive. The (-)-(1'S,2'R) analogue was the more potent of the enantiomers, but the degree of chiral discrimination by most bacteria was only 4-fold. The 4-fold chiral discrimination was observed also using purified DNA gyrase obtained from Micrococcus luteus, whereas the two enantiomers were essentially equiactive against the enzyme derived from Escherichia coli. These results confirm that there is a substantial degree of bulk tolerance available at N-1 of quinolone antimicrobial agents and suggest that electronic factors controlled by substitution at that site are of considerable importance. On the other hand, chiral recognition brought about by attachment of optically active groups to the N-1 position in these derivatives is relatively small. PMID- 3020254 TI - The effect of clindamycin on macrophage and monocyte Fc receptors. AB - The effect of clindamycin on Fc receptor expression has been investigated with mouse peritoneal macrophages and human peripheral blood monocytes. When monolayers of these cells were treated with therapeutic concentrations of clindamycin for periods up to 1 hr at 37 degrees C binding of IgG2b coated sheep erythrocytes (EAG) was inhibited. No effect was observed at 4 degrees C or 22 degrees C. An independent effect of the antibiotic on the sheep erythrocytes was noted when they were pretreated with clindamycin that resulted in enhanced binding to the mouse but not the human phagocytes. These findings are discussed in relation to possible in vivo actions of the antibiotic. PMID- 3020255 TI - Indomethacin induces the suppression of plasma neutral proteinase activity in mice: possible relationship to efficacy as an anti-inflammatory drug and induction of alterations in the immune system. AB - Treatment of BALB/c, C57Bl/6 or C3H/HeJ mice with non-toxic concentrations of indomethacin (75-100 micrograms/day) led to a depression of plasma neutral proteinase activity as determined with an (125I)-caseinolytic assay. Lower concentrations of indomethacin (50 micrograms/day), aspirin (1 mg/day), LiCl (3 meq/kg/day), Sulindac (100 micrograms/day), indomethacin analogs (MK-410, MK-555) or lipopolysaccharide (100 micrograms) did not induce depression in proteinase activity. Indomethacin did not directly inhibit the proteinase activity of normal plasma in vitro. The in vivo effects of indomethacin were reversible and plasma proteinase activity returned to normal values within 8 days of cessation of treatment. These results indicate that indomethacin can uniquely alter plasma proteinase homeostasis in normal mice. While effective in depressing the plasma proteinase activity of normal mice, treatment of mice bearing either the BCL1 leukemia or the B16-F10 melanoma with indomethacin did not depress the elevated plasma proteinase levels detected in tumor-bearing animals. Thus the elevation in proteinases detected in tumor-bearing animals may not be the result of increased prostaglandin synthesis and plasma proteinase activity in such disease states may be regulated differently than in normal mice. However, the ability of this potent anti-inflammatory agent to alter proteinase metabolism may contribute to its therapeutic efficacy in the management of inflammatory disease. PMID- 3020258 TI - Chronic antidepressant treatment and mouse brain 3H-imipramine binding. AB - Chronic pretreatment of mice with the monoamine oxidase type B inhibitor ( )deprenyl resulted in an increase in the density of cerebral cortical 3H imipramine binding sites and a decrease in the density of cerebral cortical beta adrenergic receptors. In contrast, pretreatment of mice with the tricyclic antidepressants imipramine and desipramine did not alter the density of cerebral cortical 3H-imipramine binding sites. Imipramine and desipramine treatment decreased the density of beta-adrenergic receptors. Haloperidol pretreatment resulted in an increase in the density of striatal D-2 dopamine receptors, but did not alter the density of cerebral cortical 3H-imipramine binding sites or beta-adrenergic receptors. These data suggest that brain 3H-imipramine binding sites can be regulated by pharmacological pretreatment, but that this regulation may not occur for all antidepressants. PMID- 3020259 TI - Germ cell tumors of the testicle among aircraft repairmen. AB - A cluster of testicular germ cell tumors occurred among 3 of 153 white men who worked in a shop engaged in repair of exterior surfaces and electrical components of the airframes of F4 Phantom Jet aircraft. Evaluation of an occupationally identical shop at a second F4 rework facility at which there had been no previous reports of excess neoplasms revealed 4 additional men with a history of testicular germ cell tumors (p less than 0.01, Poisson, compared to the expected number of cases based on national incidence rates). Our investigation raises but does not prove a hypothesis of association between subsequent development of testicular germ cell cancer and history of extensive exposure to a mixture containing dimethylformamide, which had been used in F4 repair work at these facilities in the 1960s and 1970s. This represents the first report of 2 corresponding mini-epidemics of testicular tumors among workers in occupationally identical industrial settings. PMID- 3020256 TI - Serum stimulation of sodium transport in human fibroblasts containing low and high levels of intracellular sodium. AB - The relationships between intracellular sodium content, sodium transport and serum effects were investigated in human fibroblasts. In the cells with low intracellular sodium (Na+iL; 0.04 mumol sodium/mg protein), serum stimulated the sodium-potassium pump as measured by ouabain-sensitive sodium efflux and rubidium influx and also exerted a transstimulation of ouabain-insensitive sodium transport resulting in net influx. In cells with high intracellular sodium (Na+iH; 0.42 mumol sodium/mg protein) all aspects of sodium transport were increased compared to Na+iL cells. In these cells serum caused no change in sodium-potassium pump activity but significantly increased the ouabain insensitive sodium fluxes resulting in net efflux. In Na+iL cells, serum promoted net sodium influx through an amiloride-sensitive pathway that was undetectable in the basal state. In Na+iH cells the serum-stimulated net efflux was amiloride sensitive but this pathway also contributed to a major portion of sodium transport in the basal state. This study demonstrated that sodium-potassium pump activity is directed by the supply of internal sodium and that serum can increase this supply by promoting net influx, and that serum-induced sodium transport can be modified by intracellular sodium content. PMID- 3020257 TI - Effects of the mobilization of aged tissue cadmium by chelating agents. AB - An examination of the kidney and liver subsequent to the mobilization of aged cadmium deposits from them by the use of both dithiocarbamates and 1,2-dimercapto 1-propranolol (BAL) was carried out. No striking permanent effects due to cadmium mobilization were noted in the kidneys or livers for most of the chelating agents used. After the mobilization of part of the cadmium burden, the remaining cadmium gives evidence of undergoing a redistribution, leading to a renewed increase in both the kidney and liver levels of this element. PMID- 3020260 TI - Chemolysis of cystine calculi. AB - Historically, cystine stone chemolysis has been approached with 2 different categories of compounds--alkalizing agents (sodium bicarbonate and tromethamine) and, more recently, protonated thiols and disulfide compounds (alpha mercaptopropionylglycine, N-acetylcysteine and penicillamine). To establish the relative efficacy of these agents an in vitro model was devised that simulates the clinical setting. The optimal molar concentrations for sodium bicarbonate, N acetylcysteine and tromethamine were determined initially and then compared at these strengths. Lastly, a variety of solution combinations were made to determine if a synergistic effect could be demonstrated. Results of this study demonstrate that the combination of acetylcysteine, a protonated thiol, with the strong alkalizing agent sodium hydroxide yields the most effective solution for chemolysis of cystine stones. The mechanism of action is believed to occur by a synergistic combination of the pH dependent increase in cystine solubility, with a simultaneously occurring thiol disulfide interchange. PMID- 3020262 TI - Leads from the MMWR. Human T-lymphotropic virus type III/lymphadenopathy associated virus: agent summary statement. PMID- 3020261 TI - Intermediate filament proteins as tissue specific markers in normal and malignant urological tissues. AB - Immunocytochemical techniques have become valuable tools in many fields of clinical pathology and medical research. Especially the development of highly specific (monoclonal) antibodies to a large variety of tissue antigens has in recent years led to the establishment of sensitive tissue markers. One of the most promising types of tissue specific markers so far is represented by the intermediate filament proteins. Since the findings of this rapidly expanding field are also being applied in urology, we have reviewed the current data in order to describe the new insights in tumor biology and histogenesis, as well as their application in diagnostic pathology. PMID- 3020263 TI - The inspection of international cruise ships. PMID- 3020264 TI - Primary cytomegalovirus infection in pregnancy. Incidence, transmission to fetus, and clinical outcome. AB - We studied 16 218 pregnant women from two income groups to determine the incidence of primary cytomegalovirus (CMV) infection and its consequences for the offspring. In the high-income group, 64.5% of the women were seronegative for CMV and 1.6% had primary CMV infection. In the low-income group, only 23.4% of the women were seronegative for CMV, but 3.7% experienced a primary infection. The rate of transmission in utero was similar in the two groups (39% and 31%). Congenital infections were more frequent in the low-income group; however, primary CMV accounted for 25% of the congenital infections in this group, in contrast to 63% of the high-income cases. Infections acquired early and late in gestation had similar rates of transmission in utero, but three infants (8%) with symptomatic congenital infection and five infants (13.5%) who have developed significant handicaps were exposed in the first half of pregnancy. Primary CMV infection during pregnancy poses a 30% to 40% risk of intrauterine transmission and adverse outcome is more likely when infection occurs within the first half of gestation. PMID- 3020265 TI - Cytomegalovirus is not an occupational risk for nurses in renal transplant and neonatal units. Results of a prospective surveillance study. AB - The risk of inpatient-to-nurse transmission of cytomegalovirus (CMV) was assessed in a five-year prospective seroepidemiologic study. We enrolled 263 renal transplant/hemodialysis nurses, 204 neonatal intensive care nurses, 225 student nurses, and 251 blood donor controls. Prevalence of CMV antibody in these 943 subjects was 33.7% and did not differ significantly among the four study groups. Sixteen subjects experienced primary CMV infections. The yearly seroconversion rate, calculated from age-related seroprevalence data, was 1.9%. The rate of seroconversion during the study, established by observing 519 seronegative subjects for 10 420 person-months, was 1.84% per year and did not differ significantly among the study groups. The proportions of patients with CMV infection (1.1% to 11.9%) and disease (0.5% to 3.4%) on study wards were not related to seroconversion in the nurses. The slow rate of acquisition of CMV in susceptible adults suggests that transmission requires prolonged, intimate contact. Nurses and nursing students who practice good personal hygiene are no more likely to acquire CMV than their peers in the community. PMID- 3020266 TI - Rectal bleeding in a 4-month-old boy. PMID- 3020267 TI - Effects of alternate and simultaneous administrations of calcium and phosphorus on calcium metabolism in children receiving total parenteral nutrition. AB - The effects of alternate and simultaneous administrations of calcium (Ca) and phosphorus (P) on Ca metabolism in children receiving total parenteral nutrition (TPN) were examined. Eight children, aged 2 to 36 months, were studied. The following three solutions were administered: solution 1 contains Ca (533 mg/liter); solution 2 contains P (413 mg/liter); and solution 3 contains Ca (267 mg/liter) and P (207 mg/liter). Solutions 1 and 2 were administered alternately for 24-hr periods. (Results) I. During administration of solution 1, significant hypophosphatemia (4.39 +/- 0.26 mg/dl) and hypercalcemia (9.96 +/- 0.15 mg/dl) were observed and, conversely, during administration of solution 2, significant hypocalcemia (8.36 +/- 0.18 mg/dl) and hyperphosphatemia (6.16 +/- 0.27 mg/dl) were observed. During administration of solution 3, the serum levels of both minerals were maintained within the normal ranges (Ca 9.46 +/- 0.12 mg/dl, P 5.65 +/- 0.21 mg/dl). II. The urinary excretion of cyclic AMP was significantly lower during administration of solution 1 (6.67 +/- 0.45 nmol/mg creatinine (Cr] as compared with solution 3 (7.50 +/- 0.61 nmol/mg of Cr). On the other hand, the excretion was significantly higher during administration of solution 2 (11.55 +/- 1.58 nmol/mg of Cr) as compared with solution 3, indicating the existence of secondary hyperparathyroidism. III. The Ca and P retention rates were significantly higher with solution 3 (Ca 79.0 +/- 5.5%, P 73.2 +/- 7.2% of the intake) than with solutions 1 and 2 alternately (Ca 62.7 +/- 4.5%, P 49.2 +/- 9.3%). (Conclusions) Simultaneous administrations of Ca and P are preferable to their alternate administrations for Ca metabolism in children receiving TPN. PMID- 3020268 TI - Viral encephalitis. PMID- 3020269 TI - [Gastrointestinal decontamination procedure in bone marrow transplantation]. PMID- 3020270 TI - [Progress in chemotherapeutic agents--antiviral agents]. PMID- 3020271 TI - [Drug resistance of bacteria and countermeasures--with special reference to the mechanism of drug resistance]. PMID- 3020272 TI - [Progress in emergency medicine and cardiovascular imaging methods: RI imaging- single photon emission CT and positron emission tomography]. PMID- 3020273 TI - [Cell physiology of the exocrine glands]. PMID- 3020274 TI - [Regulation of salivary secretion and receptors]. PMID- 3020275 TI - [Dysfunction of digestive system secretion: intestinal secretion]. PMID- 3020276 TI - [Fabry's disease]. PMID- 3020277 TI - [A case of early uptake of hepatobiliary scan agent in hepatocellular carcinoma]. PMID- 3020278 TI - [Transcatheter embolization of hepatoma through a collateral artery by the balloon occlusion technique in cases of occluded hepatic artery]. PMID- 3020279 TI - [Ultrasonography of the liver: hepatocellular carcinoma]. PMID- 3020281 TI - [Epidemiological study of mucocutaneous herpes simplex virus infection--research on the possible source of infection other than the main foci]. PMID- 3020280 TI - [Bone metastases detected by radionuclide angiography]. PMID- 3020282 TI - Protective efficacy of intranasal vaccination with subunit Sendai virus vaccine in mice. AB - Mice and rats were vaccinated with tween 80-ethyl ether disrupted subunit vaccine of Sendai virus by the subcutaneous or intranasal route. The intranasal vaccination rendered mice resistant to intranasal challenge with active Sendai virus as the subcutaneous vaccination did. The former vaccination induced low titered hemagglutination-inhibiting (HI) antibody to the virus in 3 of 4 vaccinated mice, whereas the latter vaccination readily produced high-titered HI antibody. However, intranasally vaccinated rats produced no HI antibody and showed no resistance to the intranasal challenge, whereas subcutaneously vaccinated rats produced high-titered HI antibody and resisted to the intranasal challenge. PMID- 3020283 TI - Bone marrow cell-dependency of delayed-type hypersensitivity response in mice infected with mouse hepatitis virus. AB - The mechanisms underlying delayed-type hypersensitivity (DTH) response in mouse hepatitis virus (MHV) infection was studied using high responder B10.D2 and low responder DBA/2 mice. Bone-marrow chimeras expressed the DTH responses at the same degree as the donors. The involvement of suppressor cells in low DTH response in DBA/2 mice seems unlikely since transfer of DBA/2 spleen cells to B10.D2 mice did not suppress the response. Transfer of B10.D2 spleen cells to DBA/2 mice caused no changes in DTH response of the recipients. MHV-infected mice, which were irradiated with 500 rad of gamma-ray and then given B10.D2 bone marrow cells 8 days p.i., showed higher DTH responses than those given DBA/2 bone marrow cells. These results suggest that the difference in DTH responsiveness to MHV between the two mouse strains is dependent on the activity of bone-marrow cells. PMID- 3020284 TI - [Study of long-surviving hepatocellular carcinoma patients after transhepatic arterial embolization (TAE)]. PMID- 3020285 TI - [A case of small cell carcinoma of the esophagus with SIADH]. PMID- 3020286 TI - [Serum vitamin K content in patients with hepatocellular carcinoma with special reference to plasma PIVKA-II levels]. PMID- 3020287 TI - [The effects of biological response modifier on murine fulminant hepatitis caused by inoculation of mouse hepatitis virus-type II (MHV-II)]. PMID- 3020288 TI - [An organic peroxide, methyl ethyl ketone peroxide, damage to cytochrome P-450 peroxidase and microsomal enzymes activity]. PMID- 3020289 TI - [Health hazards in workers of small scale polyurethane production factory]. PMID- 3020290 TI - Viral conjunctivitis with special reference to adenovirus type 37 and enterovirus 70 infection. AB - The clinical and etiological findings in 727 patients with viral conjunctivitis treated from January 1982 through December 1984 at Aoki Eye Clinic in Sapporo, Japan, were presented. The age of the patients ranged from 11 days to 88 years, and the monthly incidences of the disease from 18 to 131 cases, with a clustering in the summer season. The etiological diagnosis was established in 399 (54.9%) of the 727 patients: adenovirus (Ad) 3 in 45 cases; Ad4 in 98 cases; Ad8 in 57 cases; Ad19 in 23 cases; Ad37 in 51 cases; untyped Ad in 33 cases; enterovirus (EV) 70 in 60 cases; herpes simplex virus type I in 24 cases and varicella-zoster virus in 4 cases. The clinical pictures of adenovirus 37 conjunctivitis were generally similar to those of adenovirus 8 conjunctivitis, and subconjunctival hemorrhage was more frequently seen in cases of EV70 conjunctivitis. EV70 cases showed nosocomial infection. In addition to Ad8, Ad37 seems to play an important role as a causative agent of epidemic keratoconjunctivitis in Japan. PMID- 3020292 TI - [Evaluation of gamma-enolase as a tumor marker for lung cancer]. PMID- 3020291 TI - Afferent and efferent connections of the cat abducens nucleus: a study by injection of wheat germ agglutinin conjugated with horseradish peroxidase. AB - The ascending projection of the abducens internuclear neurons and the afferent pathway to the abducens nucleus were examined after injection of wheat germ agglutinin conjugated with horseradish peroxidase (HRP) into the abducens nucleus of the cat. Orthograde neural terminations of the internuclear neurons of the abducens nucleus were found in the contralateral medial rectus subdivision of the oculomotor complex and the medial longitudinal fasciculus (MLF). Furthermore, HRP positive cells were also found in the ipsilateral pontine reticular formation. These findings indicate that the axons of the neurons within the pontine reticular formation project to the ipsilateral abducens nucleus, and that the internuclear neurons of the abducens nucleus project to the contralateral medial rectus subdivision of the oculomotor complex and the MLF. Clinically, the pontine reticular formation is thought to be a center for the horizontal eye movements which generates impulses to the ipsilateral abducens motor neurons and excites the internuclear neurons of the abducens nucleus projecting to the contralateral MLF as well as to the oculomotor complex. These pathways are essential for conjugate horizontal eye movements. PMID- 3020293 TI - [A case of acquired immunodeficiency syndrome (AIDS) in a homosexual man]. PMID- 3020294 TI - Histopathological characteristics as diagnostic indicators in canine parvoviral enteritis. PMID- 3020295 TI - Ouabain-sensitive potassium-dependent p-nitrophenylphosphatase activity on choroid plexus in N-methyl-nitrosourea-induced dysgenetic hydromicrocephalic rat offsprings. PMID- 3020296 TI - Growth of feline panleukopenia virus and canine parvovirus in vitro. PMID- 3020297 TI - Natural killer cell activity and head and neck cancer: a clinical assessment. AB - In 127 patients with squamous cell carcinoma of the upper aerodigestive system, an assessment of natural killer (NK) cell function was performed. The mean lytic unit (LU) value of this cancer population was noted to be less than the mean value of 67 age-matched controls assessed concurrently. The major determinant of cytolytic function was related to the growth pattern of the tumor. Increased NK cell function was observed in patients with lesions that were more locally or regionally aggressive, i.e., that infiltrated surrounding anatomic structures. The magnitude of NK cell response also correlated with increased amounts of circulating IgG immunoglobulin to herpes simplex virus-type 1-associated antigens; elevated IgG levels were also associated with locally aggressive lesions. The clinical significance of NK cell activity in these patients is shown by its relationship to disease-free prognosis. Patients with elevated NK activity followed for a mean of 12 months had an improved disease-free survival as compared to the survival of the remaining population. Furthermore, NK LU values were not reflected in standard staging methods, which suggests that the measurement of NK cell function represents an independent prognostic parameter in the patient with head and neck cancer. PMID- 3020298 TI - Silica dust and lung cancer: results from the Nordic occupational mortality and cancer incidence registers. AB - Autopsy studies of the relationship between silicosis and lung cancer have been mainly negative; but recent epidemiologic studies have indicated a positive association, and an excess lung cancer risk has been observed in some occupational groups with exposure to silica dust. For the further shedding of light on the possible association between silica dust and lung cancer, analysis was made on mortality and cancer incidence data available in census-based record linkage studies from the Nordic countries for males in occupational groups with potential exposure to silica dust. The study showed an excess lung cancer risk for foundry workers in all the Nordic countries and for miners in Sweden. These results were consistent with findings from previous in-depth epidemiologic studies. The lung cancer risk did not differ significantly from that of the respective national populations for males working in excavation; stone quarries; sand and gravel pits; and glass, porcelain, ceramic, and tile manufacture. Stonecutters, who are probably not exposed to known lung carcinogens at the workplace but in some places to high concentrations of silica dust, showed a significant excess lung cancer risk in both Finland and Denmark. Excess lung cancer risks furthermore were seen for Finish miners, for Finnish males in excavation work, and for Danish glassworkers. PMID- 3020299 TI - Influence of age on induction of mammary tumors by diethylstilbestrol in C3H/HeN mice with low murine mammary tumor virus titer. AB - C3H/HeN female mice with low murine mammary tumor virus titer (MTV-) were fed diets containing a targeted concentration of 640 ppb diethylstilbestrol [(DES) CAS: 56-53-1; 4,4'-(1,2-diethyl-1,2-ethenediyl)bis-phenol]. Mice were started on DES at 3, 5, 7, or 9 weeks of age. Some continued on the diet throughout the rest of their life-spans, whereas others were killed as soon as they had been fed DES for 2, 4, 6, 8, 10, or 12 weeks. Controls were also examined throughout the study. Among mice killed early, the only observation significantly influenced by age at the start of DES treatment was the presence or absence of corpora lutea (CL). DES did not prevent CL from appearing in mice started on DES at 7 or 9 weeks of age, but it did prevent their appearance in about 25% of the mice started at 5 weeks and in up to 75% of the mice started at 3 weeks of age. In the life-span-exposure groups, CL either disappeared or were never formed in 88% or more of the mice, regardless of age at the start of treatment. Neoplastic or presumptive preneoplastic lesions apparently influenced by DES in the life-span treatment groups included ovarian tubular adenomas; granulosa cell tumors and luteomas; pituitary cystoid degeneration, hyperplasia, and adenomas; uterine adenocarcinomas and cervical adenosis; mesotheliomas; and mammary hyperplastic alveolar nodules (HANs) and adenocarcinomas. Luteoma and granulosa cell tumor incidences were reduced by DES, regardless of age at the start of treatment. Influence of age at the start of treatment was minimal or not apparent for mesotheliomas, uterine adenocarcinomas, or pituitary adenomas; however, pituitary cystoid degeneration and hyperplasia and cervical adenosis occurred in higher frequency and/or with shorter duration of DES exposure the earlier that treatment was started. A delay in the start of DES treatment was associated with a remarkable delay in HAN and mammary adenocarcinoma development. This was especially apparent in young mice (3-7 wk old) in which a 2-week delay in treatment resulted in a 20-week delay in HAN or tumor onset. Age at the start of treatment was a major factor in susceptibility of C3H/HeN-MTV- female mice to DES induced mammary tumorigenesis. PMID- 3020300 TI - Relationship of success in classical immunotherapy to the relative immunorejective strength of the tumor. AB - Six tumors of varying immunorejective strengths were used to compare the response of their isogenic hosts to standardized regimens of immunoprophylaxis and immunotherapy. The tumors were generated spontaneously or induced chemically [with 9,10-dimethyl-1,2-benzanthracene (CAS: 57-97-6)], virally (with murine leukemia virus), or radiogenically (with strontium-90). The hosts were C57BL/6J or BALB/cByJ mice. Immunoprophylaxis and immunotherapy were performed with isogenic irradiated tumor cells, with Corynebacterium parvum, or with both. The results of challenge experiments were quantified as the doses of viable tumor cells that produced 50% tumor deaths for immunized and for control mice. The results for these quantitative "classical" immunoprophylaxis and immunotherapy experiments were consistent with two theses: that only immunorejective tumors give positive results with classical immunotherapy and that classical immunoprophylaxis is more effective than classical immunotherapy when identical materials are used for immunizations. These results have important consequences for the clinical use of classical immunotherapy. PMID- 3020302 TI - Immunodeficiency in rhesus monkeys associated with the original Mason-Pfizer monkey virus. AB - The Mason-Pfizer monkey virus (MPMV) was reisolated from a cryopreserved sample of the original MPMV-containing rhesus breast carcinoma, and complete integrated MPMV provirus was detected in chromosomal DNA of this tumor. Reanalysis of the in vivo pathogenicity and molecular character of MPMV reisolated from the rhesus breast tumor and analysis of the original MPMV after long-term in vitro propagation in human and rhesus cells show that the original MPMV produces an acquired immunodeficiency similar to that caused by the recently described simian acquired immune deficiency syndrome type D retroviruses, and the MPMV genome and its immunosuppressive effect in vivo have remained stable despite prolonged in vitro passage in human and rhesus cells. PMID- 3020301 TI - Inhibition of DNA synthesis by a small-cell lung carcinoma-derived protein. AB - A tumor-derived factor that inhibits cellular DNA synthesis was identified. The factor was extractable from a small-cell lung carcinoma cell line grown in either chemically defined medium or nu/nu mice and inhibited tritiated thymidine ([3H]dThd) incorporation by tumor cell lines of autologous, allogeneic, and xenogeneic origins. The viability of nonproliferating cells from normal tissue was not affected. Tumor extract inhibitory activity was trypsin labile but was resistant to other proteases, neuraminidase, lipase, DNase, RNase, glucosidase, extremes of pH-temperature, and reducing conditions. Inhibitory activity was reversibly bound to helix pomatia lectin but not to lentil, wheat germ, or concanavalin A lectins. Purification by size-exclusion high-performance liquid chromatography yielded a bioactive unimodal 12-kilodalton (kd) peak. The bioactive 12-kd moiety could be eluted from sodium dodecyl sulfate-polyacrylamide gels. Redosing of populations of the T-lymphoblastoid cell line CEM achieved an early (24 hr) sustained depression of pulse [3H]dThd incorporation and ultimately led to decreased population density of factor-treated populations. DNA histogram analysis demonstrated no change in cell cycle phase distribution after factor treatment. 5-Bromo-2'-deoxyuridine (BrdUrd) vs. propidium iodide with the two parameter Fluorescence-Activated Cell Sorter analysis showed relative inhibition of non-S-phase BrdUrd uptake at 24 hours. A cell-free DNA polymerase assay demonstrated significant inhibition of non-alpha-polymerase-associated DNA synthesis in factor-treated cells. These studies suggest that this tumor-derived inhibitor of DNA synthesis represents a class of cellular products involved in the autoregulation of growth by regulation of DNA synthetic activity. PMID- 3020303 TI - [Vasopressin content of the blood in ischemic heart disease patients and its interrelation with other hormones]. AB - Blood vasopressin, tri-iodothyronine, total thyroxine, thyrotrophic hormone, cortisol, ACTH and insulin levels were measured in 60 chronic coronary patients aged 35 to 70. Coronary patients showed elevated blood vasopressin, particularly in the presence of frequently recurring anginal attacks. There was no significant difference in vasopressin levels of patients with and without attendant essential hypertension, those with atherosclerotic and postinfarction cardiosclerosis, or in relation to body weight. Insulin, cortisol, adrenocorticotrophic and thyrotrophic hormones were significantly increased, and tri-iodothyronine and total thyroxine, significantly decreased, in coronary patients as compared to the controls. PMID- 3020304 TI - Renal clearance and isolated kidney perfusion techniques. PMID- 3020305 TI - Isolation and use of specific nephron segments and their cells in biochemical studies. PMID- 3020306 TI - [Diagnosis and treatment of angiodysplasia of the head and neck]. PMID- 3020307 TI - [Pathogenic action of free radicals and the potentials of anti-inflammatory agents]. PMID- 3020308 TI - [Naloxone treatment of shock]. PMID- 3020309 TI - [Bilateral cytomegalovirus retinitis in an infant with fatal congenital AIDS]. AB - The authors report on a baby born on August 25, 1984 with a birthweight of 1100 g. Her parents used drugs intravenously and her mother had anti-HTLV III antibody. In the clinical course an immune deficiency syndrome with pathologic reduction of T4:T8 (T-helper: T-suppressor cells) was apparent. The child had no anti-HTLV III antibody, but exposition, typical clinical course and immunological data led to the diagnosis Acquired Immune Deficiency Syndrome (AIDS). In the 9th month bilateral progressive retinitis developed; it was thought to have been induced by cytomegalovirus. Complement-fixation antibody titer for cytomegalovirus was negative, but the virus was found in the urine. In the 12th month the baby died of cardiac arrhythmia caused by cytomegalic myocarditis. Macroscopic and light-microscopic illustrations of the inflammatory changes in both eyes are presented; in the retina there were large multinucleated cells which were visible macroscopically. PMID- 3020310 TI - [Progress in molecular endocrinology]. PMID- 3020311 TI - [Results of a 5-year study with captopril in patients with severe therapy resistant hypertension]. AB - The angiotensin converting enzyme (ACE) inhibitor captopril proved to be an effective antihypertensive drug during a 5-year follow-up study of patients with severe hypertension who had been resistant to a triple-drug regimen. Of the 42 patients, 41 had to be treated additionally with diuretics. Because of hypokalemia, potassium supplements were necessary in 26 patients, despite the use of "potassium-saving" diuretics in 12 patients. Blood pressure was controlled sufficiently in 3/4 of the patients during the 5 years. Patients with a large elevation in plasma renin activity showed the best response to the treatment. Six patients died during the 5 years. Therapy had to be stopped in 11 patients because of complications. The following complications and adverse effects were observed: cerebral ischemia (n = 10), vertigo and orthostasis (10), exanthema (9), hypogeusia (7), circulatory failure (7), myocardial infarction (6), and scintigraphically demonstrable decrease of renal perfusion (5). One patient with bilateral renal artery stenosis suffered from acute renal failure, which was reversible after withdrawal of captopril. Significant changes of red and white blood cell counts, transaminases, lipids, urine protein excretion, and heart rate were not observed. PMID- 3020312 TI - Converting enzyme inhibitor ramipril stimulates prostacyclin synthesis by isolated rat aorta: evidence for a kinin-dependent mechanism. AB - The present study was performed to investigate the effect of the angiotensin I converting enzyme inhibitor ramipril on vascular synthesis of prostacyclin (PGI2). Administration of ramipril (Hoe 498) to rats significantly stimulated prostacyclin (PGI2) synthesis, quantified by radioimmunoassay of its stable hydrolysis product 6-keto-PGF1 alpha, by portions of the animals' isolated aorta. This effect was maximal at a dose range of 10(-7) mol/kg ramipril. The addition of the active ramipril metabolite ramipril diacid directly into the incubation buffer at final concentrations of 10(-9), 10(-6), and 10(-4) M resulted in a dose dependent stimulation of 6-keto-PGF1 alpha released by isolated aortic tissue. Pretreatment of rats with aprotinin (40,000 U s.c. 60 min before the incubations) attenuated the ramipril-induced effect on aortic 6-keto-PGF1 alpha synthesis. Our results show that the angiotensin I-converting enzyme inhibitor ramipril stimulates PGI2 synthesis in vascular tissue and that this effect may be secondary to changes in the activity of the kinin system. PMID- 3020313 TI - Effect of glucagon on adenylate cyclase activity and acid production of isolated human parietal cells. AB - The direct effect of glucagon on human parietal cell function in vitro was tested by measuring adenylate cyclase (AC) activity and H+ production in homogenates of human gastric mucosa obtained during surgery or at biopsy. Cells isolated from mucosa obtained during surgery showed an increase in AC with histamine and glucagon. In parietal cell enriched fractions (75%) glucagon and histamine stimulated AC much more effectively than in parietal cell depleted fractions (15% and 7%). In contrast, glucagon did not affect basal or histamine stimulated 14C amino pyrine uptake. In homogenates of mucosal biopsy specimens 2 X 10(-7) mol/l glucagon enhanced AC activity by 76% (corpus) and 20% (antrum). In the same homogenates 10(-4) mol/l histamine caused a stimulation by 161% (corpus) and 38% (antrum). In fundic biopsy specimens glucagon displayed a biphasic concentration response curve with an increase at 10(-10) mol/l (46% above basal AC activity) and a maximum at 2 X 10(-7) mol/l (97%). Histamine elicited the maximal response (192%) at 10(-3) mol/l. Increasing histamine and glucagon concentrations caused additive stimulation of AC. Ranitidine did not change AC in response to glucagon but abolished the effect of histamine. Data suggest that the glucagon action is mediated by separate (glucagon?) receptors. As H+ production was not affected by glucagon, the coexistence of two AC systems in the human parietal cell is postulated: One that is activated via histamine H2-receptors and which stimulated H+ production; another that is activated by glucagon and is directed towards other, possibly metabolic effects. PMID- 3020316 TI - [After care and after treatment of patients receiving loco-regional chemotherapy from a nurse's viewpoint]. PMID- 3020314 TI - Studies on ouabain-like endogenous natriuretic factors in human urine. Inhibition of Na-K-ATPase and 3H-ouabain binding. AB - An endogenous inhibitor of sodium transport and of the Na-K-ATPase enzyme was previously detected in the small molecular weight postsalt fraction SIV of serum from saline-loaded rats after gel filtration on Sephadex G-25. In addition, a natriuretic factor present in this fraction of urine from salt-loaded subjects was found to bind to a specific digoxin antibody. Therefore, in the present study the small molecular weight natriuretic and digoxin antibody-binding activities present in the urine of salt-loaded healthy volunteers were purified by reverse phase chromatography and by immunoprecipitation with the digoxin antibody and were studied for their in vitro effects on Na-K-ATPase derived from hog cerebral cortex. Na-K-ATPase inhibitory activities were found to roughly parallel the natriuretic activities at the various stages of purification. After reverse-phase chromatography, the material of fraction SIV which was bound to the digoxin antibody revealed the highest specific natriuretic activity of 3.95 +/- 0.29 mumol/min X mg injected material and showed strongest inhibition of the enzyme with I50 at a concentration of 0.08 microgram/ml, as compared with 2.4 micrograms/ml of the original postsalt urine fraction SIV. Fraction SIV revealed a noncompetitive inhibition of the enzyme with respect to potassium, but also significantly inhibited 3H-ouabain binding to the enzyme. Thus, the natriuretic substance(s) present in the urine of salt-loaded healthy subjects exhibit a potent inhibitory effect on the Na-K-ATPase enzyme which is similar, but not identical to that of ouabain. PMID- 3020315 TI - [Terminal renal failure in aniridia-Wilms syndrome]. AB - Missing iris combined with debility and incidence of Wilms' tumor seem to be a complex syndrome which appears in 1:100,000 people. It is caused by an interstitial deletion on the short arm of chromosome no. 11. We refer to a patient who developed end-stage renal failure caused by a focal-segmental nephrosclerosis. He underwent renal transplantation because chronic hemodialysis was impossible due to his lack of compliance. The deletion of chromosome 11 could be recognized by chromosomal analysis after transplantation. An aniridia-Wilms' tumor association (AWTA) with following focal segmental nephrosclerosis could be diagnosed. PMID- 3020317 TI - Effects of alcohol and other psychotropic drugs on eye movements: relevance to traffic safety. AB - The effects of alcohol and other psychotropic drugs on eye movements are reviewed with particular attention to the possible relevance of these effects for traffic safety. Alcohol has been shown to have diverse effects, including reduction of the velocity of both saccadic and smooth pursuit eye movements, increased saccadic latency, impairment of convergence and induction of nystagmus. These effects probably contribute to impaired visual information processing, which reduces driving ability. Barbiturates have been reported to produce effects similar to alcohol, and the effects of benzodiazepines and opioids seem to be more limited but still substantial. Marihuana has relatively little effect on eye movements. PMID- 3020318 TI - Increased marihuana-induced fetotoxicity by a low dose of concomitant alcohol administration. AB - Many pregnant women use both alcohol and marihuana. To evaluate the effects of this combination on fetotoxicity, pregnant mice in the experimental group were injected with a relatively low dose of alcohol (1 g/kg) and with one of two doses of marihuana extract (equivalent of 50 or 100 g/kg delta 9-THC). Control mice received marihuana extract or alcohol alone. The combination of alcohol and the high dose of marihuana produced a greater effect on fetotoxicity (83%) than either marihuana or alcohol alone or that due to the additive effects of either of these substances (63%). The combination of alcohol and the lower dose of marihuana extract did not increase fetotoxicity significantly. Doses of alcohol that are otherwise without effect on pregnancy may thus have the potential for greatly increasing the effects of drugs on pregnancy outcome. PMID- 3020320 TI - Immortalization of human T lymphocytes by HTLV-I: phenotypic characteristics of target cells and kinetics of virus integration and expression. AB - In-vitro infection of normal human lymphocytes with HTLV-I (human T-cell lymphotropic retrovirus type I) has been carried out to study the target cell specificity and the kinetics of infection. Cord blood (CBL) and adult peripheral blood lymphocytes (PBL) have been co-cultivated with irradiated HTLV-I donor cells (MT2 and C91PL lines). Established ('immortalized') HTLV-I positive cell lines were obtained only from CBL: in comparison with PBL, a less mature phenotype of T-cell subsets and a lower interferon-gamma production was evidenced in CBL. A progressive variation of differentiation antigen representation and of exogenous T-cell growth factor (TCGF, interleukin-2, IL-2) medium concentration was observed with increasing time from infection. The four established lines obtained showed a predominant T3+, T4+, T8-, Tac+ phenotype and a reduced TCGF requirement. Studies on kinetics of HTLV-I infection showed that p19 and p24 viral antigens became expressed after a lag phase of 5 weeks. DNA Southern blot analysis indicated that a shift from polyclonal to monoclonal pattern of proviral integration occurred with time of culture, both complete and defective copies being transmitted from donor to recipient cells. PMID- 3020319 TI - Resection of thoracic and abdominal teratoma in patients after cisplatin-based chemotherapy for germ cell tumor. Late results. AB - Fifty-one patients with primary testicular (N = 46) or mediastinal germ cell cancer (N = 5) were treated from April, 1975, through May, 1981, and had teratoma resected from residual disease after cisplatin-based combination chemotherapy. All patients had normal serum markers before resection of pulmonary (N = 12), mediastinal (N = 5), thoracoabdominal (N = 8), supraclavicular (N = 1) or abdominal disease (N = 25). Teratoma was classified as mature teratoma (N = 29), immature teratoma (N = 15), or immature teratoma with non-germ cell elements (N = 7). Thirty of 51 (60%) patients remain free of recurrent disease, whereas 20 patients have either recurrent carcinoma (N = 10) or teratoma (N = 10). One patient has a presumed second malignancy. After additional chemotherapy, four patients with recurrent carcinoma are alive and disease free and six have died. After an additional operation, eight of 10 patients with recurrent teratoma are long-term survivors. In four patients the initial relapse of carcinoma developed more than 2 years after therapy; in an additional patient carcinoma recurred after a 32 month disease-free survival period. Univariate factors predicting for relapse include tumor burden, immature teratoma with non-germ cell elements, and site (mediastinum), whereas only immature teratoma with non-germ cell elements and site predicted for survival. Immature teratoma and mature teratoma had similar relapse-free intervals and overall survival intervals. According to a multivariate analysis, primary tumor site at the mediastinum is the most significant adverse factor predictive for both relapse and survival (two of five patients survived). This study appears to support the various preclinical models that demonstrate multipotential capabilities of teratoma. Complete surgical excision of teratoma remains the most effective treatment with continued close follow-up recommended for high-risk patients (immature teratoma with non-germ cell elements, large tumor burden, or primary mediastinal tumors. PMID- 3020321 TI - Differences in oncogenicity among murine leukemia virus isolates are not correlated with the magnitude of H-2 regulated anti-viral envelope antibody responses. AB - The antibody response against the envelope proteins of a variety of cloned highly and poorly oncogenic dualtropic mink cell focus-inducing (MCF) murine leukemia viruses (MuLV) was studied and compared with the antibody response against ecotropic isolates. MCF viruses evoke stronger antibody responses than ecotropic MuLV isolates. Although these MCF viruses are highly polymorphic with respect to their gp70 and p15(E) envelope proteins, generally a similar H-2-linked immune response gene control of anti-viral antibody responses is observed. Neonatally infected BALB/c (H-2d) and C57BL/10 (B10,H-2b) mice are high responders and B10.A (H-2a) mice, congenic at the major histocompatibility complex (H-2) with B10, low responders. No correlation was found between the expression of any single gp70 or p15(E) epitope of the infecting MCF virus and the magnitude of the antibody response. This indicates that the level of the H-2 regulated anti-MuLV envelope antibody response is most likely determined by a MuLV antigen shared by all MuLV isolates examined. The magnitude of the antibody response against highly oncogenic and against poorly oncogenic MCF virus does not differ significantly. The combined data indicate that the intrinsic oncogenic potency encoded by the viral genome is a more important feature of oncogenesis than the level of antiviral envelope antibody response evoked by the MCF virus. However, the oncogenic properties of a single murine leukemia virus may vary among H-2 congenic mice, correlated with their H-2-dependent capacity to produce antiviral antibodies. PMID- 3020322 TI - Mental health cost models. Refinements and applications. AB - This paper describes a 2-year study whose goal was to refine Burton Weisbrod's cost model for public programs for the chronically mentally ill. The authors made comprehensive cost assessments of all the resources, including treatment programs, used by a small sample of severely disturbed chronically ill patients. Refinement of the model included a method to assess capital costs of public facilities. The use of disaggregated patient information permitted analysis of cost differences between patients when adjusted for case mix. Patient costs over the study period ranged from $24,000 to $99,000. Patient characteristics and change in clinical status account for 30% of the variance. PMID- 3020323 TI - [Preliminary restriction analysis of the DNA of selected Staphylococcus aureus phages]. PMID- 3020326 TI - Phosphoinositide metabolism and cGMP levels are not coupled to the muscarinic cholinergic receptor in human erythrocyte. AB - Recently, Tang et al. [BBA 772, 235 (1984)] reported that cholinergic agonists stimulate calcium uptake and cGMP formation in the human erythrocyte. We undertook this investigation since polyphosphoinositide breakdown precedes calcium mobilization and cGMP formation in several tissues. In [32P]-prelabeled erythrocyte ghosts, calcium (0.5 mM) but not carbachol (0.1 mM) caused a 2- and 20-fold increase in the accumulation of IP2 and IP3, respectively. This was accompanied by a 50% decrease in PIP2 and PIP. In intact erythrocytes prelabeled with [32P], 1 microM A23187 but not carbachol (0.1 mM) produced a 300% increase in radioactivity in PA after a 30-min incubation. cGMP levels after a 2-min incubation with saline, A23187 (1 microM), or carbachol (0.1 mM) were 0.27 +/- .03, 0.27 +/- .04, and 0.34 +/- .04 fmol/10(6) cells. Our studies indicate that the muscarinic receptor in the erythrocytes is "non-functional" insofar as its stimulation is not accompanied by phosphoinositide breakdown or cGMP formation. PMID- 3020324 TI - [Preoperative staging of rectal cancer with computer tomography]. AB - The correlation of the preoperative staging by CT with the postoperative staging was prospectively investigated in 112 patients with carcinoma of the rectum. The influence of CT on the choice of surgical treatment was also proven. The evaluation of the infiltration of perirectal tissue and especially of other organs is possible with CT. According to TNM classification the pre- and postoperative staging showed identical results by conventional diagnostic methods in T1 in 7/16, in T2 in 22/38, in T3 in 37/49 and in T4 in 5/9 patients. With the additional CT identical results were found in T1 in 7/14, in T2 in 25/31, in T3 in 49/53 and in T4 in 13/14 cases. Thus, the preoperative staging turned out to be correct with CT in 94/112 cases (83.9%). By conventional diagnostic methods identical results were found in 71/112 patients (63.4%). The infiltration of other organs was suspected preoperatively in 24 cases with CT and was found intraoperatively in 30/112 (accuracy 94.6%, sensitivity 88%, specificity 96%). Metastases of lymph nodes were suspected in the tomograms in 32/49 patients (65.3%). By the differentiated interpretation of the tumor growth with special regard to the "Grenzlamellen" of the rectum the CT gives important information for planning therapy. PMID- 3020325 TI - Analgesic (sub anesthetic) nitrous oxide interacts with the endogenous opioid system: a review of the evidence. AB - The concept that anesthesia and analgesia are distinct states and therefore are possibly mediated by different mechanisms is stressed. Analgesic nitrous oxide is shown to act at specific rather than non specific central nervous system sites, as well as having a large number of actions similar to morphine the classical opioid. This includes the fact that specific opioid antagonists attenuate the effects of both morphine and analgesic nitrous oxide. Evidence is also provided showing that nitrous oxide may be a partial agonist and that it may interact with the endogenous opioid system by the release of endogenous opioids, and/or by direct action at the mu, delta, sigma and kappa receptors. PMID- 3020328 TI - Interaction of aflatoxin and hepatitis B virus in the pathogenesis of hepatocellular carcinoma. AB - Aflatoxin-B1 (AFB) and chronic hepatitis B virus (HBV) infection epidemiologically correlate with the geographic distribution of hepatocellular carcinoma (HCC). Integration of HBV DNA into the cellular genome of HCCs and the in vivo formation of adducts between AFB and nucleic acids lead us to suggest that hepatocytes with integrated HBV DNA preferentially accumulate AFB; the AFB adducts formed may then initiate cell transformation by modifying the expression of critical host genes. The altered molecular biology of liver cells in HCC is evidenced by the fact that HBV does not replicate in HCC tissues or cell lines. The effect of AFB on the expression of cellular genes such as endogenous retrovirus(es) and possibly cellular oncogene(s) can be analyzed in HCC cell lines with and without integrated HBV DNA. In addition, human HCC tissues can be probed for HBV sequences and AFB-DNA adducts at the single-cell level. The presence of HBV and AFB can be correlated with the expression of putative transforming genes, providing a new insight into the interaction between liver cells, HBV and AFB in the pathogenesis of HCC. PMID- 3020327 TI - Adrenocorticotropin reversal of experimental hemorrhagic shock is antagonized by morphine. AB - ACTH-(1-24) dose-dependently improved cardiovascular function in rats and dogs subjected to experimental hemorrhagic shock, and intravenous dose of 160 and 100/microgram/kg, respectively, completely restoring arterial blood pressure and pulse amplitude. All saline-treated animals died within 30 min of bleeding, while all ACTH-treated animals were still alive at the end of the observation period (2 hr). The injection of ACTH-(1-24) also dramatically improved the respiratory function. Morphine, i.v. injected into rats at the dose of 2.5 mg/kg, antagonised the effect of ACTH-(1-24) to a greater or lesser degree, depending on the dose of peptide employed: at 160/microgram/kg, antagonism was complete, at 320/microgram/kg antagonism was only partial, while at 480/microgram/kg antagonism was almost completely overcome. These data further support the idea that melanocortins are physiological antagonists of opioids, and suggest that melanocortin peptides may prove to be rational and effective drugs in the treatment of hypovolemic shock. PMID- 3020329 TI - Aluminum increases cyclic AMP in rat cerebral cortex in vivo. AB - In vivo concentrations of cyclic AMP were elevated in the cerebral cortex of rats that were administered aluminum citrate by either of two routes; in the diet for one month or via a single intracerebroventricular injection two weeks prior to the experiment. These treatments did not alter the in vivo concentrations of cyclic GMP or acetylcholine. Aluminum treatment also altered the response of cortical cyclic AMP to the administration of pilocarpine and of apomorphine. It is proposed that the neurotoxicity of aluminum is not due to effects on acetylcholine but may be due to altered protein phosphorylation as a consequence of the chronic elevation of cyclic AMP. PMID- 3020330 TI - Binding of 3',4'-dideoxykanamycin B to ATP and its related compounds. AB - The interactions of aminoglycoside, 3',4'-dideoxykanamycin B(DKB) with ATP and its related compounds were investigated. ATP, ADP, cyclic AMP and FAD bound to the DKB-conjugated Sepharose 4B column. The binding of DKB to ATP was also confirmed by equilibrium gel filtration. In the acidic pH region, the fluorescence of nucleotides was quenched by DKB. The Stern-Volmer plots showed that the molar ratios of the complexes were 1:1. The apparent stability constant was dependent on the number of the phosphate groups of nucleotides and was in the order of ATP greater than ADP greater than AMP. PMID- 3020331 TI - Cyclic AMP, the hyperresponsiveness factor from hog kidney. AB - The isolation and identification of a material present in the plasma of hypertensive dogs and hypertensive human patients has been under study since 1972. The earliest experiments in relation to this work, noted that plasma from hypertensive dogs cause a hyperresponse to norepinephrine when both were administered by way of the vein. Employing a rat assay system that consisted of an anesthetized rat with polyethylene catheters in the vein for giving norepinephrine and the test fractions and a catheter in the artery for blood pressure monitoring, fractions from hog kidney were tested for hyperresponsiveness activity. The active material is very comparable to cyclic AMP in molecular weight, ultraviolet spectrum, paper chromatography, Enzyme hydrolysis and activity in the anesthetized rat system. This evidence indicates that the hyperresponsiveness factor of renal origin is cyclic AMP. PMID- 3020332 TI - Central, stereoselective receptors mediate the acute effects of opiate antagonists on luteinizing hormone secretion. AB - Although a central site of acute opiate action in regulating luteinizing hormone (LH) secretion has been suggested by the ability of centrally implanted opiate antagonists to increase LH levels, opiate antagonists are lipophilic and could influence the pituitary in situ. Also, the physiological significance of opiate receptor blockade with antagonists rests on the assumed, but untested, stereoselectivity of these receptors. Therefore, a lipophobic quaternized derivative of naltrexone (MRZ 2663-Naltrexone methobromide) and dextro- (+) and levo- (-) stereoisomers of naloxone were used to study the site- and stereoselectivity of gonadotropin responses to opiate antagonists in vivo. Male rats were injected intracerebroventricularly (icv) or intravenously (iv) with the quaternary or tertiary congeners of naltrexone and subcutaneously (sc) with (-) or (+)-naloxone. Rats injected icv with 20 ug of quaternary naltrexone displayed significant increases in serum luteinizing hormone (LH). The onset of the response was rapid with serum LH levels being significantly elevated 15 minutes after the injection and returning to basal levels 30 minutes later. Rats injected iv with 10 mg/kg of quaternary naltrexone failed to show significant LH responses. Rats injected either centrally or periphally with equivalent doses of tertiary naltrexone showed LH responses that were similar to those found in animals injected icv with quaternary naltrexone. As little as 0.5 mg/kg of (-) naloxone resulted in significant elevations in serum LH that were higher than those elicited by up to 10 mg/kg of (+)-naloxone, indicating that this effect of naloxone is stereoselective. These data support the argument that opioids can acutely modulate LH secretion through actions at stereoselective opioid receptors in the central nervous system. PMID- 3020333 TI - Down-regulation of rat liver beta-adrenergic receptors by cysteine. AB - Down-regulation of hepatic beta-adrenergic receptors was indicated by a 56% decrease in the specific activity of 125I-iodocyanopindolol bound to rat liver membrane preparations from rats fed diets containing 15% of casein supplemented with cysteine, instead of methionine or unsupplemented. Down-regulation of hepatic beta-adrenergic receptors by cysteine appears to be mediated through an effect of cysteine on the tissue concentration of S-adenosyl methionine (SAM). The liver tissue concentration of SAM in rats fed cysteine-supplemented diets decreased 53% compared to those fed diets supplemented with methionine. The decrease in liver SAM in rats fed the diet supplemented with cysteine appears to reflect a non-competitive inhibition of methionine adenosyl-transferase by cysteine. Lineweaver-Burk plots demonstrated a dose-related Vmax response to cysteine but did not change the apparent Km at any concentration tested. PMID- 3020334 TI - Reversibility of cholinephosphotransferase in lung microsomes. AB - The effect of cytidine 5'-monophosphate (CMP) on the incorporation of cytidine 5' diphosphate (CDP) [methyl-14C]choline or [1-14C]dipalmitoylglycerol into phosphatidylcholine (PC) catalyzed by rabbit lung microsomal CDPcholine:1,2 diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied. In the presence of 0.85 mM CMP and nonsaturating diacylglycerol concentration, the incorporation of CDP[14C]choline into PC was markedly stimulated, but the incorporation of [14C]dipalmitoylglycerol into PC was inhibited. This was due to the increase of endogenous diacylglycerol generated from microsomal PC by the cholinephosphotransferase reverse reaction. However, the newly synthesized PC was not readily hydrolyzed in the presence of CMP. The results of this study suggest that the endogenous membranous diacylglycerol is utilized more preferentially for PC synthesis than the exogenous diacylglycerol and that the newly synthesized PC could rapidly equilibrate with the endogenous membrane PC pool. PMID- 3020335 TI - Cyclic AMP increases incorporation of exogenous fatty acids into triacylglycerols in hamster fibroblasts. AB - Incorporation of various exogenous saturated or unsaturated [14C]labeled fatty acids (palmitic, stearic, oleic, linoleic and arachidonic) into triacylglycerols by hamster fibroblasts was markedly enhanced (two- to fourfold) in the presence of theophylline or dibutyryl cyclic adenosine monophosphate (dbcAMP). This effect was observed for short-term (1-6 hr) as well as long-term (15-24 hr) preincubation with dbcAMP. In the presence of sodium fluoride, a phosphoprotein phosphatase inhibitor, measurement of diacylglycerol acyltransferase (DGAT) activity in cells pretreated with dbcAMP pointed out a marked increase (3 X) in specific activity. The results suggest that DGAT activity in fibroblasts could be activated by a cAMP-dependent phosphorylation process. PMID- 3020337 TI - [Radionuclide diagnosis of kidney calculi]. AB - Radionuclide investigations were conducted in 322 patients with nephrolithiasis. Unilateral calculosis was established in 46.3% of the patients, bilateral calculosis in 50.6%. The nature of changes on renograms, scintigrams and in clearance values was shown to depend on the localization of concrements, their size and the presence of concomitant infection. A conclusion has been made as to the usefulness of the methods with relation to operative treatment, especially in a bilateral localization of a pathological renal process. PMID- 3020336 TI - Inhibition of the antidiuretic hormone hydroosmotic response by phospholipids and phospholipid metabolites. AB - Phospholipid metabolities and phospholipids containing arachidonic acid (AA) inhibited the antidiuretic hormone (ADH)-induced increase in transepithelial water flow in the toad urinary bladder, but had no effect on basal water flow when added to the serosal bathing solution. Other fatty acid-substituted phospholipid metabolites had no effect on osmotic water movement in the presence or absence of ADH. Indomethacin attenuated the inhibitory effects of the AA containing phospholipid metabolities (PMAA), suggesting that the PMAA response required AA release and prostaglandin (PG) formation. PMAA increased PGE formation as measured by radioimmunoassay. PG have been reported to inhibit ADH stimulated water flow by inhibiting adenylcyclase. PGE2 (10(-8) M) had no effect on cyclic AMP-stimulated water flow, whereas exogenous AA and PMAA attenuated the hydroosmotic response to added cyclic AMP. Indomethacin only partially reversed the inhibition by AA of the cyclic AMP-associated water movement, suggesting that the inhibition by AA and PMAA may involve other metabolites of AA than PG. PG and the AA cascade have been implicated as cellular modulators of the ADH hydroosmotic response. The present results offer additional support to the theory that this system may regulate the intracellular events that are transduced following receptor activation by ADH. PMID- 3020338 TI - Infection of Cebus monkeys with Junin virus. PMID- 3020339 TI - [Inactivated Junin virus antigens]. PMID- 3020340 TI - Mucinous carcinoma of the colon and rectum and its relation with adenomas. PMID- 3020341 TI - [Junin virus activity in humans and rodents of non-endemic areas of the province of Buenos Aires]. PMID- 3020342 TI - Angiotensin-converting enzyme: characteristics in human skin fibroblasts. AB - Angiotensin-converting enzyme, although most prominent in vascular endothelium, has been identified in numerous tissues. Recent studies have indicated that several hormones, including glucocorticoids and thyroid hormone, may affect the activity of this enzyme. In the present study, angiotensin-converting enzyme was examined in homogenates of cultured human skin fibroblasts. Angiotensin converting enzyme activity was measured by a radiometric assay using [Glycine-1 14C] Hippuryl-L-histidyl-L-leucine (1.1 mmol/L) as substrate, and was expressed as nmol hippuric acid formed per minute/mg protein. Angiotensin-converting enzyme was identified in all five cell strains tested, and the activity observed was 0.97 +/- 0.18 nmol/min/mg protein (mean +/- SE). The optimum pH was between 6.9 and 7.6, and optimum temperature was 37 degrees C, with loss of activity of 55 degrees C and higher. Buffer strength was optimized at Tris 0.025 mol/L, and 1.0 mol/L NaCl. Activity increased linearly with protein concentration and with time, and the Km = 1.14 mmol/L. The most potent inhibitor of fibroblast ACE was captopril (SQ 14,225) with an IC50 = 10(-10) mol/L; other inhibitors included SQ 20,881, EDTA, and phenanthroline. Competitive substrates included angiotensin-I, substance P, and bradykinin. Four hormones, T3 (10(-9)-10(-7) mol/L), 1,25 (OH)2D3 (10(-8)-10(-7) mol/L), dexamethasone (10(-7)-10(-6) mol/L), and a synthetic androgen, R1881 (10(-8)-10(-7) mol/L) were incubated with cells for 72 hours. In all incubations, there was no significant effect on cellular ACE activity induced by any agent. Angiotensin-converting enzyme activity in serum free media was less than 1% of cell activity and was unaltered by hormone treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020343 TI - Insulin-stimulated conversion of D-[5-3H] glucose to 3HOH in the perifused isolated rat adipocyte. AB - Characteristics of basal and insulin-stimulated glucose utilization by perifused adipocytes have been investigated by measuring the formation of 3HOH from D-(5 3H) glucose. At a glucose concentration of 0.55 mmol/L, basal glucose utilization ranged from 0.5 to 1.0 nmol/min/10(6) cells. Perifused adipocytes showed a maximal response to insulin of a threefold to fourfold increase in the conversion of (5-3H) glucose to 3HOH with a half-maximal response at an insulin concentration of 20 microU/mL. The response to insulin was blocked by phlorizin and cytochalasin B, competitive inhibitors of glucose transport, consistent with an effect of insulin on glucose transport. Insulin increased the Vmax for glucose metabolism but had no effect on the apparent affinity for glucose utilization. The characteristics of glucose utilization and the stimulation of glucose metabolism by insulin in the perifused adipocyte are therefore similar to characteristics previously observed with incubated adipocytes. Because insulin can readily be removed from the system, perifused adipocytes are especially suited for studying the termination of insulin action. The termination of insulin stimulated glucose metabolism occurred at the same rate in the presence of tracer (1 nmol/L) (5-3H)-glucose alone as when 0.55 mmol/L glucose or 2 mmol/L pyruvate were added to the perifusion buffer. The halftime for this process in both cases was approximately 40 minutes. These data suggest that the presence of metabolizable substrate is not required for the termination of the insulin response, but the time course suggests that termination requires more than simply insulin-receptor dissociation. PMID- 3020344 TI - Effects of different proportions of carbohydrates, polysaccharides/monosaccharides, and different fibers on the metabolic control in diabetic rats. AB - Recent findings have suggested that diets with a high level of carbohydrates may impair the metabolic control of diabetes mellitus in humans. Moreover, other investigations have indicated that if the simple sugar content is increased in order to attain a proportion of polysaccharides/monosaccharides equal to 1, then neither the blood glucose nor the lipidic response show any change. We have studied the effect of increasing carbohydrates in the diet (59% v 82%), while maintaining cereal fiber levels constant (30%) and replacing cereal fiber in high carbohydrate diets by guar gum (30%) and lentil-derived leguminous fiber (30%) on the metabolic control of streptozotocin-induced diabetic rats. A study with different diets was performed for 3 weeks. An increase of carbohydrates in the diet produces an increase in the HbA1 concentration (1.9% v 3.9%, P less than 0.01) and in serum triglyceride levels (98.75 +/- 22.09 mg/dL v 144.50 +/- 3.52 mg/dL, P less than 0.05). Total cholesterol and HDL-cholesterol levels remained unchanged. The increase does not occur if the cereal fiber is replaced by lentil derived leguminous fiber. In a second experiment, we substituted 50% of the complex carbohydrates in diets with 80% carbohydrates by glucose. Blood glucose, triglycerides, and HbA1 levels rose significantly in the four groups of rats that received diets containing 50% carbohydrates in glucose form. In addition, a test meal was carried out on day 19, consisting of 2.5 g of food/kg of wt. The maximum increase in blood glucose and the area below the glucose curve response was also significantly higher in the four groups of rats who received glucose in their diet.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020345 TI - A primary defect in insulin receptor in a young male patient with insulin resistance. AB - We studied insulin binding to cells from an insulin-resistant patient, a 5-year old boy with clinical Rabson-Mendenhall syndrome. Decreased insulin binding was observed in three different cells: erythrocytes (37.4% of normal), cultured fibroblasts (53.3% of normal), and transformed lymphocytes (9.8% of normal). Decreased insulin binding in the cultured cells suggested that the patient had a primary defect in insulin receptors. In addition, insulin binding to the transformed lymphocytes from the patient was relatively high at lower pH compared with those in normal subjects. The cultured fibroblasts from the patient showed decreased glucose incorporation at the low insulin concentration with normal maximal stimulation, and the insulin dose response curve was shifted to the right. These results suggested that the defect resided in the receptor binding but not in the postreceptor steps. This was one of the rare cases showing decreased insulin binding clearly demonstrated in three different cells from a young male patient with extreme insulin resistance. PMID- 3020346 TI - Impaired thermoregulation in cold-exposed rats with hypothalamic obesity. AB - Rats with obesity-producing, hypothalamic knife cuts were fed a high fat diet and placed in the cold (2 degrees C) for six days starting 3, 11, or 24 days after surgery. Between surgery and cold exposure, knife-cut rats consumed 90% to 122% more energy and gained more weight (32 +/- 4, 112 +/- 5, and 241 +/- 9 g) than sham-operated rats (15 +/- 2, 34 +/- 2, and 58 +/- 3 g). When exposed to cold, sham-operated rats increased (22% to 30%) energy intake whereas knife-cut rats decreased (5% to 51%) intake. After 24 hours at 2 degrees C body temperatures of knife-cut rats were 1.2, 0.7, and 0.7 degrees less than those of control rats; body temperatures continued to decrease to 2.9, 3.0 and 2.5 degrees less than control rats after six days at 2 degrees C. Fasting for 12 hours at 2 degrees C caused a further reduction in body temperature to 4.9, 4.8, and 5.9 degrees less than in control rats. Cold exposure increased urinary excretion of norepinephrine and epinephrine (indicators of sympathoadrenal activity) in all rats. Guanosine diphosphate (GDP) binding to brown adipose tissue (BAT) mitochondria (an indicator of the thermogenic capacity of the tissue) was similar in cold-exposed, knife-cut, and sham-operated rats. Cold acclimation before hypothalamic knife-cut surgery prevented the cold-induced decrease in body temperatures of knife-cut rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020347 TI - Production and purification of bovine interferon beta. PMID- 3020348 TI - Radiolabeling of human interferon alphas with 125I-labeled Bolton-Hunter reagent. PMID- 3020349 TI - Large-scale production of human interferon from lymphoblastoid cells. PMID- 3020351 TI - Large-scale production and recovery of human leukocyte interferon from peripheral blood leukocytes. PMID- 3020350 TI - Use of Rous sarcoma viral genome to express human fibroblast interferon. PMID- 3020352 TI - Construction of expression vectors for secretion of human interferons by yeast. PMID- 3020353 TI - Procedures for isolation of murine alpha interferon genes and expression of a murine leukocyte interferon in E. coli. PMID- 3020354 TI - Cloning, expression, and purification of rat IFN-alpha 1. PMID- 3020356 TI - A convenient microassay for cytolysis and cytostasis. PMID- 3020355 TI - Isolation of bovine IFN-alpha genes and their expression in bacteria. PMID- 3020357 TI - Selection and screening of transformed NIH3T3 cells for enhanced sensitivity to human interferons alpha and beta. PMID- 3020358 TI - Production of human immune interferon from leukocytes cocultured with exogenous cells. PMID- 3020359 TI - Animal models for investigating antitumor effects of interferon. PMID- 3020360 TI - Assay of effect of interferon on virus-induced cell fusion. PMID- 3020361 TI - Asymmetric replication of an oriC plasmid in Escherichia coli. AB - Plasmid pTSO118 containing the Escherichia coli origin of replication, oriC, initiated replication simultaneously with the chromosome when temperature sensitive host cells were synchronized by temperature shifts. Replicating intermediates of the plasmid as well as of the chromosome were isolated from the outer membrane fraction of the cell. Plasmid DNA with eye structures was enriched when cytosine-1-beta-arabinofuranoside was introduced into the culture during replication. Electron microscopy of the replicating molecules, after digestion with restriction endonucleases, showed that the replication fork proceeds exclusively counter-clockwise towards the unc operon. We conclude that the replication of the oriC plasmid is unidirectional or, if bidirectional, is highly asymmetric. PMID- 3020362 TI - Separation and analysis of the RNA polymerase binding sites of a complex Bacillus subtilis promoter. AB - The Bacillus subtilis veg promotor site was shown to be composed of two neighboring RNA polymerase binding sites, only one of which ("Site I") produces a transcript. The relationship between these sites has been investigated following insertion of ten base pairs of DNA between the sites and subsequent cloning of each site independent of the other, and deletion of DNA sequences upstream from the productive site. The effect of DNA insertion was to permit transcription initiation from the previously nonproductive Site II, in either the presence or absence of Site I. This could be attributed to insertion of a purine residue seven base pairs downstream from the Pribnow box of Site II. Transcription from Site I was slightly increased in the presence of Site II in cis, but was still efficient in the absence of Site II, indicating that there is no absolute dependence on Site II for in vitro transcription from Site I. PMID- 3020363 TI - Isolation, characterization and physiological properties of an autolytic deficient mutant of Streptococcus pneumoniae. AB - A spontaneous mutation in the gene lyt encoding the pneumococcal autolysin has been characterized. This mutation, named lyt-32, which behaves as a high efficiency marker in pneumococcal transformation, is a single base pair GC deletion causing the appearance of two consecutive termination codons in the amino terminal part of the sequence of the autolysin gene. The mutant lyt gene did not code for a polypeptide of relative molecular mass corresponding to the pneumococcal E form amidase in Escherichia coli maxicells. Pneumococcal cells containing the lyt-32 mutation (M32) were fully transformable, multiplied at a normal growth rate forming small chains and showed a tolerant response when treated with beta-lactam antibiotics. Strain M32 represents the first example of a mutant of Streptococcus pneumoniae completely lacking amidase as a consequence of an alteration in the structural gene coding for the pneumococcal autolysin. PMID- 3020364 TI - Purified Saccharomyces cerevisiae RNA polymerase II interacts homologously with two different promoters as revealed by P1 endonuclease analysis. AB - The intergenic region of the Saccharomyces cerevisiae GAL1-GAL10 divergent promoters has been circularized in vitro in different topological states. In defined conditions, purified homologous RNA polymerase II forms two stable complexes (half-life approximately equal to 5 h) with this DNA in the presence of the four ribonucleotides, as determined by measurement (Gamper and Hearst 1983) of the amount and stability of the resulting unwinding. Each stable complex induces in the closed DNA domain a region of hypersensitivity to P1 endonuclease. The two induced hypersensitive regions are very similar: each maps on one promoter, spans over the 100 bp DNA sequence that encompasses the RNA Initiation Sites (RIS) and the TATA box, is composed by three subregions (one on the RIS, one proximal or overlapping the TATA sequence, one intermediate). We show that this promoter-localized interaction is supercoil-dependent. PMID- 3020365 TI - Deletion polymorphism in a Drosophila melanogaster heat shock gene. AB - We have continued the transcriptional analysis of the region of cytological locus 67B that contains the four small heat shock genes and other genes. Transcription from one of the heat shock genes in the region, hsp 26, takes place during high temperature treatment and at certain developmental stages, without heat shock, in several tissues, such as imaginal discs and adult ovaries. Observations of unexpected products after nuclease protection experiments provided the first indication of what genomic blot experiments showed to be small deletions. The alleles containing the deletion are expressed at the same level as the wild type allele. The deletion shortens the protein product, implying that it is in the coding region. Furthermore, flies homozygous for one of the deletion alleles are viable. PMID- 3020366 TI - Replicon fusions promoted by insertion sequences on Pseudomonas cepacia plasmid pTGL6. AB - Plasmid pMR5 (pRP1ts) failed to replicate in Pseudomonas cepacia at 47 degrees C. Selection at this temperature for maintenance of tetracycline resistance associated with this plasmid allowed isolation of cointegrate plasmids formed by fusion of pMR5 with pTGL6, a 170 kb plasmid harbored by P. cepacia 249. In the cointegrate plasmids pTGL100, pTGL101, and pTGL102, different regions of pTGL6 were involved in fusion with the same tra-2-containing region of pMR5. Formation of all three plasmids was promoted by insertion sequences on pTGL6, which were also represented in the chromosome. Two different copies of a 1.3 kb element, IS401, were involved in formation of pTGL100 and pTGL101. Another insertion sequence, IS402 (1 kb), promoted the fusion which formed pTGL102. Southern hybridization experiments indicated that each of the cointegrate plasmids contained an additional copy of the fusion mediating element. Plasmid pTGL100 was observed to resolve into two independent replicons: pTGL6 and pTGL105 (pMR5::IS401), a novel derivative of pMR5 containing a copy of IS401. The third cointegrate plasmid, pTGL102, evolved in two steps: fusion of pTGL6 and pMR5 mediated by IS402, and transposition of IS411 (1.9 kb) to a region of pMR5 distinct from that involved in the fusion. Plasmid pTGL6 contained one copy of IS402 and IS411 while pTGL102 contained two copies of each of these elements. PMID- 3020367 TI - Cloning of the lkyB (tolB) gene of Escherichia coli K12 and characterization of its product. AB - The lkyB gene of Escherichia coli K12 has been cloned from the Clarke and Carbon colony bank by selecting a ColE1 plasmid conferring cholic acid resistance to lkyB mutants. The lkyB gene was localized on hybrid plasmid pJC778 by analysis of mutated plasmids generated by Tn5 insertions. Restriction analysis and complementation studies indicated that plasmid pJC778 carried genes nadA, lkyB and sucA which mapped at min 16.5; the lkyB+ allele was dominant over the lkyB207 mutant allele. Analysis of cell envelope proteins from strains carrying plasmids pJC778 (lkyB+), pJC2578 or pJC2579 (lkyB::Tn5), as well as plasmid-coded proteins in a maxicell system, made it likely that the ikyB gene product was a membrane protein of molecular weight 42,000. PMID- 3020368 TI - Molecular cloning and expression in Escherichia coli of the structural gene for the hemolytic toxin aerolysin from Aeromonas hydrophila. AB - The structural gene for the hemolytic toxin aerolysin has been cloned into the plasmid vectors pBR322 and pEMBL8+. The gene was localized on the hybrid plasmids by analysis of plasmids generated by transposon mutagenesis. The sequence of the first 683 bases of an insert in pEMBL8+ was determined and shown to encode the amino terminus of the protein as well as a typical signal sequence of 23 amino acids. Aerolysin is produced by E. coli cells containing the cloned aerolysin gene and it is processed normally by removal of the signal sequence, however it is not released from the cell. The protein appears to be translocated across the inner membrane of E. coli as its signal sequence is removed and the processed protein can be released by osmotic shock. PMID- 3020369 TI - Deletion of the phosphoglucose isomerase structural gene makes growth and sporulation glucose dependent in Saccharomyces cerevisiae. AB - The structural gene PGI1 coding for phosphoglucose isomerase was replaced by the LEU2 gene in the genome of Saccharomyces cerevisiae. Plasmids carrying the LEU2 gene between genomic regions flanking the PGI1 gene were constructed and used to transform a PGI1/pgi1 diploid strain. Stable transformants lacking the PGI1 allele were isolated. Southern analysis of their meiotic products showed that haploid strains with a deletion of 1.6 kb within the 2.2 kb PGI1 coding region were viable. Thus, the PGI1 gene is not essential in yeasts. However, unlike pgi1 mutants with residual phosphoglucose isomerase activity, no growth was detected in the pgi1 delta haploid strains when fructose was supplied as sole carbon source. The wild-type growth rate could be restored by adding 0.1% glucose to the medium. Furthermore, pgi1 mutants with residual enzymatic activity grew very slowly on fructose-supplemented media containing up to 2% glucose. Strains carrying the deletion allele, however, failed to grow at glucose concentrations higher than 0.5%. Also the pgi1 delta strains did not grow in glucose as sole carbon source. On the other hand pgi1 delta/pgi1 delta diploid strains did not sporulate on the usual acetate medium. This defect could be alleviated by the addition of 0.05% glucose to the sporulation medium. Under these conditions the pgi1 delta mutants sporulated with an efficiency of 25% compared with the wild type. These results suggest that the phosphoglucose isomerase reaction is the only step catalysing the interconversion of glucose-6-P and fructose-6-P, glucose 6-P is essential in yeasts, and the oxidation of glucose-6-P through the glucose 6-P dehydrogenase reaction is not sufficient to support growth in yeasts. PMID- 3020371 TI - Incompatibility between plasmids with independent copy control. AB - Incompatibility between autonomous plasmids has been attributed, for the most part, to interaction between plasmids' negative control systems and/or partitioning systems. In this report it is shown that indirectly regulated plasmids with non-interactive negative control systems are incompatible on the basis of their shared initiator protein. This principle was demonstrated for a family of Staphylococcus aureus plasmids whose copy number is regulated by inhibitory RNAs that control the production of a rate-limiting, trans-active, initiator protein. We have constructed a pair of plasmids that have the same regulation systems and different initiator proteins and another pair with different regulation systems and the same initiators. Both of these pairs of plasmids were shown to be incompatible. PMID- 3020370 TI - Rescue of a tk-plasmid from transgenic mice reveals its episomal transmission. AB - This communication demonstrates the usefulness of the plasmid rescue procedure for recovery of plasmids from transgenic mice. We have microinjected the plasmid pSK1 harbouring the Herpes simplex virus thymidine kinase gene into fertilized mouse oocytes and succeeded in recovering plasmids from newborns by transformation of E. coli either with HindIII cut cellular DNA or with uncut DNA. The majority of the rescued plasmids were indistinguishable from pSK1 by restriction analysis. The rescued plasmids proved to be functionally active in a transient expression assay in mouse Ltk- cells. The pSK1 DNA sequences were inherited by up to 90% of the second generation progeny mice, which is not in agreement with a Mendelian transmission of heterozygous markers integrated into a single site of the chromosome. These data support the assumption that germ line transmission of non-integrated episomal plasmids can occur. PMID- 3020372 TI - Molecular analysis of the argB gene of Aspergillus nidulans. AB - The transcriptional organization and sequence of the Aspergillus nidulans argB gene, encoding ornithine carbamoyl transferase (OCTase; E.C. 2.1.3.3.), was determined. Transcription of the gene begins within a methionine-initiated open translation reading frame, indicating that a second methionine codon of the open reading frame is used for translation initiation. The predicted length of the OCTase precursor peptide is 359 amino acids, and it contains a highly basic amino terminus that is probably involved in mitochondrial targeting. There is extensive homology between Aspergillus OCTase and mammalian and bacterial OCTases and weaker homology between the Aspergillus polypeptide and bacterial arginine carbamoyl transferase. PMID- 3020373 TI - Single amino acid replacements affecting the thermostability of kanamycin nucleotidyltransferase. AB - Amino acid residues of the carboxyl-terminal region of kanamycin nucleotidyltransferase were modified using segment-directed mutagenesis. Six different mutant enzymes with single amino acid replacements were selected out of 59 clones by DNA sequence analyses. The mutant enzymes were purified and it was found that the thermostability of one mutant enzyme was identical to the wild type, whereas the other five were less thermostable at varying degrees. The data suggested that changes in the enzyme thermostability depend not only on the position but also on the species of amino acid residue replaced. PMID- 3020375 TI - Transcription of genes encoding enzymes involved in DNA synthesis during the cell cycle of Saccharomyces cerevisiae. AB - We have examined the pattern of transcription exhibited by four genes in the dTTP biosynthetic pathway of Saccharomyces cerevisiae. Consistent with the results reported previously by Storms et al. (1984), the TMP1 (or CDC21) gene encoding thymidylate synthase was found to be transcribed in a periodic manner during the cell cycle with maximal mRNA levels occurring just prior to the onset of DNA replication. Three other genes in this pathway DCD1, DUT1 and DFR1 encoding dCMP deaminase, dUTP pyrophosphatase and dihydrofolate reductase, respectively, exhibited relatively constant levels of transcription throughout the cell cycle. These results, particularly for DFR1, are in marked contrast with those obtained in other eukaryotic systems which have suggested that, in general, genes encoding enzymes involved in DNA precursor synthesis are subject to cell cycle regulation. Thus, periodic transcription is not a property common to all genes involved in DNA replication in this eukaryote. PMID- 3020374 TI - sub, a host mutation that specifically allows growth of replication-deficient gene B mutants of coliphage P2. AB - Bacteriophage P2 normally requires the products of its early genes A and B for lytic growth in its host, Escherichia coli C. A host mutation, sub-1, which allows P2 to grow without a functional B gene product is described. The sub-1 mutation is recessive and maps at approximately 10 min on the E. coli genetic map. PMID- 3020376 TI - Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli. AB - DNA fragments of 3.4 kb containing the gyrB gene were cloned from Escherichia coli KL-16 and from spontaneous nalidixic acid-resistant mutants. The mutations (nal-24 and nal-31) had been determined to be in the gyrB gene by transduction analysis. Nucleotide sequence analysis of the cloned DNA fragments revealed that nal-24 was a G to A transition at the first base of the 426th codon of the gyrB gene, resulting in an amino acid change from aspartic acid to asparagine, and nal 31 was an A to G transition at the first base of the 447th codon, resulting in an amino acid change from lysine to glutamic acid. This indicates tha mutations in the gyrB gene are responsible for nalidixic acid resistance. PMID- 3020377 TI - Yeast LEU5 is a PET-like gene that is not essential for leucine biosynthesis. AB - Alpha-IPM synthase catalyzes the first committed step in leucine biosynthesis in the yeast S. cerevisiae. LEU4 is known to encode this enzyme activity. A second gene, LEU5, has been proposed to encode a second enzyme with this activity. We cloned LEU5 and genetically defined the locus. LEU5 maps to chromosome VIII and is tightly linked to CEN8. Five different mutations in LEU5 were analyzed: a site directed deletion and a disruption, as well as three distinct mutations produced by chemical mutagenesis. In a leu4 background, each leu5 mutation causes a Leu- phenotype; in a LEU4 background, none of the mutations alters the Leu+ phenotype. This shows that LEU5 is not essential for leucine biosynthesis. In either a leu4 or LEU4 background, each leu5 mutation causes a glycerol--phenotype. This operationally defines LEU5 as a PET gene. Two distinct suppressors of the Pet- phenotype of leu5 strains have been isolated. These suppressors revert the Pet- phenotype of each of four mutant leu5 alleles that were tested. Suppression occurs regardless of the allele at LEU4. Moreover, the suppressors co-revert the Leu--phenotype for each of the four leu5 mutations that is combined with a leu4 allele. This establishes the presence of a gene other than LEU5 that encodes a second alpha-IPM synthase. Further analysis provided no evidence for synthase activity that is encoded by LEU5. PMID- 3020378 TI - Expression of Tn5-encoded streptomycin resistance in E. coli. AB - Four Tn5 mutations able to express streptomycin resistance in E. coli were obtained independently. These mutations (called Tn5) were localized and sequenced. All of them consist of a 6 bp deletion in the str gene near the 3' end. The mutation affects a region peculiar for its repetition of an identical 6 bp sequence. The mutation does not affect the level of transcription of the kan, ble, str operon of Tn5, neither does it increase the level of translation of str. The mutation seems to interfere with a post-translational event. PMID- 3020379 TI - Reduced superhelicity of plasmid DNA produced by the rho-15 mutation in Escherichia coli. AB - The plasmid pJSF6, a derivative of pBR327, could be maintained at 30 degrees C in strains of Escherichia coli containing the strong rho mutation, rho-15. Plasmids extracted from rho-15 cells were always less negatively supercoiled than plasmids from rho+ cells. Transduction experiments designed to separate the rho gene from possible extragenic suppressors showed that the rho allele consistently determined the degree of plasmid superhelicity. Comparison of the superhelicity of plasmids extracted from the rho-15 and from a gyrB mutant showed that at 30 degrees C the negative supercoiling was reduced by the amounts delta Wrho = 4.0 +/- 0.3 and delta Wgyr = 6.0 +/- 0.3 turns; the effect of the rho-15 mutation on supercoiling was thus comparable to that of the gyrB mutation. A similar effect of the rho-15 mutation on the superhelicity of pBR329 was observed. The observation that the Rho protein has a role in determining DNA superhelicity (though not necessarily a direct role) provides a new point of view for studying the pleiotropic properties of rho mutants. PMID- 3020380 TI - Iron hydroxamate transport of Escherichia coli: nucleotide sequence of the fhuB gene and identification of the protein. AB - The genes fhuA, fhuC and fhuD encode products involved in the transport of ferrichrome into cells of Escherichia coli. The nucleotide sequence of an additional locus contiguous to fhuD and formerly designated fhuB was determined. It contains an open reading frame for a polypeptide that consists of 659 amino acids. Expression of plasmids containing the fhuB region in an in vitro transcription/translation system and in E. coli minicells revealed a polypeptide with an electrophoretic mobility on SDS-polyacrylamide gels extremely dependent on the experimental conditions employed. This property could be explained by the very hydrophobic nature of the protein and it was concluded that the fhuB locus consists of a structural gene that encodes a membrane protein with a molecular weight of 82,181. The fhuB gene is required for all iron transport systems which operate via hydroxamate compounds. The order of the genes in the fhu operon is fhuA fhuC fhuD fhuB, and transcription proceeds from fhuA to fhuB. PMID- 3020381 TI - Molecular cloning of the phosphate (inorganic) transport (pit) gene of Escherichia coli K12. Identification of the pit+ gene product and physical mapping of the pit-gor region of the chromosome. AB - The pit+ gene, encoding the phosphate (inorganic) transport system of Escherichia coli, was isolated from a library of E. coli genes inserted in the cosmid vector pHC79. A 25.5-kb chromosomal DNA fragment was shown also to carry the gor locus encoding glutathione oxidoreductase. Physical mapping placed the two genes about 10 kb apart, confirming bacteriophage P1 mapping of the 77-min region. Subcloning and deletion analysis indicated that the entire pit+ gene was located within a 2.2-kb Sal1-Ava1 fragment. The pit+ gene product was identified by SDS polyacrylamide gel electrophoresis as a 39-kdal inner membrane protein by two methods: 35S-methionine-labelling of minicells carrying pit+ plasmids or plasmids from which all or part of the pit+ gene was deleted. Overproduction of the Pit protein using a thermoinducible "runaway" replication plasmid. Complementation of the pit-1 mutant allele using a unit-copy-number pit+ plasmid indicated that the pit-1 mutation is recessive. Strains carrying a multicopy pit+ plasmid show a 10 fold increase in the initial rate of phosphate uptake; however there is no change in the steady-state level of 32Pi accumulation. PMID- 3020382 TI - Genetic manipulations in Rhizobium meliloti utilizing two new transposon Tn5 derivatives. AB - Two derivatives of the prokaryotic transposon Tn5 were constructed in vitro. In Tn5-233, the central area of Tn5, which carries resistance to kanamycin/neomycin, bleomycin and streptomycin, is replaced by a fragment carrying resistance to the aminocyclitol antibiotics gentamycin/kanamycin and streptomycin/spectinomycin. In Tn5-235, the Escherichia coli beta-galactosidase gene is inserted within the streptomycin resistance gene of Tn5, and constitutively expressed from a Tn5 promoter. Both constructs transpose with about the same frequency as Tn5 in Escherichia coli and Rhizobium meliloti. When a Tn5-derivative is introduced into an R. meliloti strain which already contains a different Tn5-derivative, in situ transposon replacement is obtained at high frequency, presumably by a pair of crossovers between the IS50 sequences at the ends of the incoming and resident transposons. In this way we converted a previously isolated recA::Tn5 mutant into the corresponding recA::Tn5-233 strain, which can now be used as a genetic background in the study of complementation of other Tn5-induced mutations. We also replaced the drug markers of several Tn5-induced exo mutants, which we were then able to map relative to each other by transduction with phage phi M12. In a strain carrying Tn5-235 located near Tn5-233, we were able to isolate deletions of the intervening markers, presumably resulting from general recombination between the two transposons, by screening for loss of the Lac+ phenotype. Unlike Tn5 itself, resident Tn5-233 does not appear to suppress transposition of another incoming Tn5-derivative. PMID- 3020384 TI - Structure and function of the repressor of bacteriophage lambda. III. Molecular cloning of the high-affinity mutant cI gene of lambda and studies of the properties of the clones. AB - The high-affinity mutant cI gene of lambda cIha (Nag et al. 1984) was cloned in the multicopy plasmid pBR322. In the resulting plasmid, pMD 102, a lacUV5 promoter was inserted giving the lacUV5-cIha fusion plasmid pMD 205. Bacteria carrying pMD 102 and pMD 205 contain 2.5 and 15 times, respectively, the level of repressor in a monolysogen of lambda cIha. Results of the study of certain properties of the bacteria carrying these plasmids suggest that the ha repressor also has a higher affinity for the virulent mutant operators as well as the prm promoter of lambda. PMID- 3020383 TI - Cloning and expression of a tylosin resistance gene from a tylosin-producing strain of Streptomyces fradiae. AB - A gene conferring high-level resistance to tylosin in Streptomyces lividans and Streptomyces griseofuscus was cloned from a tylosin-producing strain of Streptomyces fradiae. The tylosin-resistance (Tylr) gene (tlrA) was isolated on five overlapping DNA fragments which contained a common 2.6 Kb KpnI fragment. The KpnI fragment contained all of the information required for the expression of the Tylr phenotype in S. lividans and S. griseofuscus. Southern hybridization indicated that the sequence conferring tylosin resistance was present on the same 5 kb SalI fragment in genomic DNA from S. fradiae and several tylosin-sensitive (Tyls) mutants. The cloned tlrA gene failed to restore tylosin resistance in two Tyls mutants derived by protoplast formation and regeneration, and it restored partial resistance in a Tyls mutant obtained by N-methyl-N'-nitro-N nitrosoguanidine (MNNG) mutagenesis. The tlrA gene conferred resistance to tylosin, carbomycin, niddamycin, vernamycin-B and, to some degree, lincomycin in S. griseofuscus, but it had no effect on sensitivity to streptomycin or spectinomycin, suggesting that the cloned gene is an MLS (macrolide, lincosamide, streptogramin-B)-resistance gene. Twenty-eight kb of S. fradiae DNA surrounding the tlrA gene was isolated from a genomic library in bacteriophage lambda Charon 4. Introduction of these DNA sequence into S. fradiae mutants blocked at different steps in tylosin biosynthesis failed to restore tylosin production, suggesting that the cloned Tylr gene is not closely linked to tylosin biosynthetic genes. PMID- 3020385 TI - Magnetic resonance imaging--first human images in Australia. AB - The use of magnetic resonance imaging, in the demonstration of internal human anatomy and in the diagnosis of disease, has the major advantages that the technique is noninvasive, does not require the use of ionizing radiation and that it can demonstrate neurological and cardiovascular lesions that cannot be diagnosed easily by other imaging methods. Magnetic resonance imaging is derived from the principle that certain atomic nuclei in a strong magnetic field will absorb pulses of radiofrequency energy; when the pulse is finished the nuclei will emit radiowaves at the same frequency. These radiowaves are received by specially designed aerials or coils and the information is collected by a computer which reconstructs an image of internal anatomy in a similar way to that of x-ray computed tomography (CT). By changing the strength of the magnetic fields and the frequency of the radiowave pulses, it is possible to examine different sections within the body. The first magnetic resonance images of humans were obtained in Australia in October 1985 on the research instrument of the Queensland Medical Magnetic Resonance Research Centre, which is based at the Mater Hospital in Brisbane, and is part of the University of Queensland's Department of Radiology. PMID- 3020387 TI - Lactic acidosis B associated with solid tumors. PMID- 3020388 TI - Orthopoxvirus infections. PMID- 3020386 TI - Pathogenesis of genital herpes simplex virus infection in mice. IV. Quantitative aspects of viral latency. AB - Experiments in the mouse model of herpes simplex virus (HSV) infection involving the intact genital mucous membranes as inoculation site yielded the following results. In untreated mice the extent of latency was correlated with the degree of peripheral virus replication. This correlation could not be observed when the course of infection was interrupted by chemotherapy, interferon, or passive immunization. Acyclovir had little effect on peripheral virus multiplication, but markedly reduced latent ganglionic infection. As acute ganglionic infection and virus concentration in the spinal nerves were already reduced, acyclovir is assumed to inhibit either virus penetration into the nerve endings or virus replication in the ganglia. Interferon apparently has an active role in the elimination of virus infected cells from the ganglia, as its effect was restricted to a reduced rate of latency and of lethality. Passive immunization with antiserum led to similar results as ACV-treatment. While lacking a pronounced effect on virus replication in the mucous membranes, specific antibody was found to influence both virus elimination from the ganglia, and conversion from productive to latent ganglionic infection. Immune lymphocytes proved to be the only agent capable of suppressing peripheral infection, thereby inhibiting the neural spread of the virus. These results suggest that the decrease in latency may result from modulations occurring at different stages in the course of infection. PMID- 3020390 TI - Agonist photoaffinity labeling of A1 adenosine receptors: persistent activation reveals spare receptors. AB - This study describes experiments investigating the mechanism of activation of A1 adenosine receptors. Isolated rat fat cells were used as a cellular model. The A1 receptors of these cells were covalently labeled with the agonist photoaffinity label R-2-azido-N6-p-hydroxyphenylisopropyladenosine. The covalent incorporation of the label into the binding subunit of the receptor was verified by demonstration of specific labeling of a peptide with Mr = 35,000 by the radioiodinated label. Such covalent labeling followed by removal of label not covalently bound led to a concentration-dependent reduction of cellular cAMP levels. This persistent effect of covalent labeling occurred with an IC50 value of 9 nM compared to an IC50 value of 0.9 nM for the direct reduction of cAMP levels by the label. The affinity of the label was determined in binding experiments. The Ki value of 19 nM was about 20 times higher than the corresponding IC50 value of cAMP reduction. Finally, the comparison between covalent binding and its effects suggests that covalently labeled receptors were fully activated. The data are interpreted as evidence for a receptor activation according to the occupancy theory. The analysis of the various concentration response curves reveals the presence of spare receptors, which can be demonstrated by the method of agonist photoaffinity labeling. PMID- 3020389 TI - Further studies on the interactions between the calcium mobilization and cyclic AMP pathways in guinea pig hepatocytes. AB - Isoproterenol (50 nM) potentiated the effects of angiotensin (1-50 nM) on 86Rb efflux and 45Ca efflux from guinea pig hepatocytes. This effect occurred in the presence or absence of extracellular Ca2+ and required the simultaneous presence of both isoproterenol and angiotensin. Neither the divalent cationophore, A23187, nor 4 beta-phorbol dibutyrate could substitute for angiotensin. The effects of isoproterenol were greatest with submaximal concentrations of angiotensin, whereas maximal concentrations of angiotensin were affected little. Isoproterenol did not substantially increase the formation of [3H]inositol triphosphate or the ratio of isomers [3H]inositol 1,4,5-trisphosphate and [3H]inositol 1,3,4 trisphosphate formed in response to angiotensin. Isoproterenol also enhanced the phase of Ca2+ mobilization involving Ca2+ entry which is consistent with the previously proposed functional linkage between receptor-regulated Ca2+ release and Ca2+ entry. These findings suggest that isoproterenol may act by increasing the sensitivity of the endoplasmic reticulum to the Ca2+-releasing action of inositol 1,4,5-trisphosphate. PMID- 3020392 TI - A rapid and efficient method to remove labelled molecular probes from nucleic acids immobilized on solid supports. AB - A rapid and efficient method is described for the removal of radio-active molecular probes from nucleic acids immobilized on nylon membranes. This method involves boiling in distilled water in a microwave oven. This procedure can be completed within ten minutes, does not require the use of any buffers or reagents, and produces results comparable with conventional buffer-wash procedures recommended by the suppliers of the transfer membranes. PMID- 3020391 TI - Sequence analysis of the larval beta II-globin gene of Xenopus laevis. AB - The 1822 bp sequence of the larval Xenopus laevis beta II-globin gene is reported together with 240 bp upstream of the gene and 190 bp beyond the site of polyadenylation. The mRNA start point was determined by primer extension as well as nuclease S1 mapping and the polyadenylation site by comparison of the gene sequence to the mRNA sequence derived from a corresponding cDNA clone. Like other vertebrate globin genes, this gene comprises three exons interrupted by two intervening sequences (IVS). IVS I spans over 582 nucleotides and interrupts the exon sequences within codon 30. IVS II is located between the codons 104/105 and spans over 617 nucleotides. The 5' region of the gene contains the canonical TATAA homology at position -31. Comparison of the upstream sequence to that of Xenopus laevis larval beta I-globin gene revealed a conserved sequence, located between nucleotide positions -60 and -87, which might function as regulatory element of transcription. Whereas the upstream region of the larval beta II globin gene does not contain a CAAT box, we notice a reiterated AAATGA motif and discuss its possible significance. PMID- 3020393 TI - Correlations of repetitive and AT-rich DNA segments within the chicken globin gene domains. AB - The repetitive DNA segments were mapped within a 30 Kbp genomic domain including (in 5' to 3' order) the chicken embryonic pi and adult alpha D (minor) and alpha A (major) globin genes. Two repeats map 5 and 8 Kbp upstream from the embryonic pi gene and another 3 Kbp downstream of the adult alpha A gene. These repetitive DNA sequences are placed within, or immediately adjacent to the AT-rich DNA segments framing this domain. Similar correlations exist also within the chicken beta globin gene domain. The positions of these AT-rich and repetitive DNA segments framing the alpha globin gene domain also correlate with other already explored features of long range DNA organisation, as clusters of sites of DNAse I hypersensitivity and differential methylation, sites of Matrix-DNA attachment, and with the beginning and end of the transcribed domain. PMID- 3020395 TI - Papillomaviruses and neoplasia in man. PMID- 3020394 TI - [Expression of aminoglycoside phosphotransferase gene is under the control of recA promoter]. AB - A novel expression vector using the 300 bp promotor-operator fragment of the recA gene of Escherichia coli has been constructed. The strength of the recA promotor was examined by assaying aminoglycoside phosphotransferase (APT) activity expressed from APT gene placed downstream of the promotor. We have observed, that some plasmids, containing N-portion of recA gene caused a large increase in radiosensitivity of host bacteria cells. PMID- 3020396 TI - Carcinoma of the breast and of the ovary as a model for the application of monoclonal antibodies to diagnostic pathology. PMID- 3020397 TI - Clinic and pathologic study of a case of inflammatory fibrous histiocytoma. PMID- 3020398 TI - The characterization of chromosome breaks in Drosophila melanogaster. II. Molecular analysis of gamma-ray-induced deficiencies in the 14F-15A region. AB - In the previous paper of this series, a total of 446 mutations of the para gene were isolated following gamma- or X-irradiation. Of these, 180 were shown to be chromosome deficiencies. In this analysis we examine the molecular distribution of breakpoints in a subset of strains [38] which have an endpoint in the 14F-15A5 region. We find that although the breakpoints are distributed throughout the entire region, there is some regional specificity to the distribution of those endpoints. PMID- 3020400 TI - Establishment of cell lines derived from ataxia telangiectasia and xeroderma pigmentosum patients with high radiation sensitivity. AB - Four human fibroblast cell lines, three of which were derived from a patient with ataxia telangiectasia and the other from a patient with xeroderma pigmentosum, were established after transfection with cloned SV40 DNA. These 4 cell lines showed some phenotypes characteristic of neoplastically transformed cells, and had a human karyotype with heteromorphisms identical to those of the parental fibroblasts. Their sensitivity to the cytotoxic effects of gamma-rays or ultraviolet irradiation was as high as those of their parental fibroblasts. PMID- 3020399 TI - Characterization of a simian virus 40-transformed Fanconi anemia fibroblast cell line. AB - We have characterized a SV40-transformed human fibroblast cell line (GM6914) derived from a patient with Fanconi anemia (FA) in order to establish its usefulness for biochemical and genetic experiments, including DNA-mediated gene transfer. GM6914 cells have a growth rate similar to that of SV40-transformed normal human fibroblasts and an indefinite lifespan in culture. As has been established for other FA cell types, GM6914 cells have an increased sensitivity to DNA-crosslinking agents such as mitomycin C (MMC). The D10 for GM6914 cells is 8 times lower than for equivalent controls. GM6914 cells also have an elevated frequency of spontaneous chromosome aberrations and this frequency can be increased by MMC concentrations which show no effect on control cells. Genetic complementation studies with lymphoblasts derived from two affected sibs of the donor of GM6914 cells show that GM6914 belongs to FA complementation group A. In DNA-transfection studies using plasmid pRSVneo, colonies of GM6914 cells resistant to the drug G-418 were observed at frequencies ranging from 1.7 to 16 X 10(-4), values similar to those observed with several other SV40-transformed human cell lines. GM6914 should be a useful recipient cell line in experiments using DNA-mediated gene transfer to clone the normal allele of the gene which is defective in FA complementation group A. GM6914 would also be an excellent cell line for studies on mutagenesis, recombination and repair using plasmid vectors. PMID- 3020401 TI - Restriction endonuclease Bam H I induces chromosomal aberrations in Chinese hamster ovary (CHO) cells. AB - Treatment of Chinese hamster ovary (CHO) cells with the restriction endonuclease Bam H I (recognition site: G/GATCC) leads to high frequencies of chromosomal aberrations. Experiments with bromodeoxyuridine-labelled chromosomes show that the aberrations occur nearly exclusively in first post-treatment metaphases. The results are interpreted to mean that only some of the cells take up the enzyme and that these cells are the ones showing the aberrations. Cells which do not take up the enzyme show up as differentially stained metaphases and have no aberrations. Why some cells take up the restriction enzyme and others not is not known, possibly this is dependent on the physiological condition of the cells. PMID- 3020403 TI - Peptide hormone antagonists that are effective in vivo. Lessons from parathyroid hormone. PMID- 3020402 TI - Neuropathy associated with cryoglobulinemia. AB - A patient with severe subacute sensory ataxia was found to have an IgM (kappa) cryoglobulin. Clinical, electrophysiologic, and sural nerve biopsy studies indicated that axonal degeneration and segmental demyelination both played a role in the pathogenesis of this neuropathy. Corticosteroid therapy was associated with notable clinical improvement and a 50% decrease in cryoglobulin concentration. PMID- 3020405 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 42-1986. A 28-year-old man with a renal transplant and recent disorientation. PMID- 3020404 TI - Human papillomavirus in clinically and histologically normal tissue of patients with genital cancer. AB - To study the association of human papillomavirus (HPV) and herpes simplex virus (HSV) with genital cancer, we collected specimens of cervical, vulvar, endometrial, and vaginal tumors at the time of operation in patients with cancer. In some patients, matched internal-control (histologically normal) tissue was also collected. DNA extracted from the tissue was probed with radiolabeled HPV type 16 DNA, HPV type 18 DNA, and cloned fragments of HSV type 2 DNA. Hybridization to the HindIIIa clone of HSV-2 was detected in only 1 cervical tumor and 1 vulvar tumor (9 percent) among the 22 tumors tested. However, DNA sequences hybridizing to HPV-16 were detected in 21 of 25 tumors (84 percent) and in 8 of 11 (73 percent) of the DNA samples from clinically and histologically normal, paired, internal-control tissues from the patients with cancer. HPV-16 DNA was found in one of nine normal cervixes (11 percent) of women without genital neoplastic disease or abnormal cytology. HPV-18 DNA was detected in only 2 of 24 tumors (8 percent), 1 cervical and 1 vulvar. Our results show a strong association between the presence of HPV-16 genomes and genital tumors and between HPV-16 genomes and histologically normal tissue within 2 to 5 cm of the tumors. The implications of these findings remain to be explored. PMID- 3020406 TI - On human papillomaviruses. PMID- 3020407 TI - Chemotherapy versus hormonal therapy in advanced breast carcinoma. PMID- 3020408 TI - Cytomegalovirus infection in parents of children at day-care centers. PMID- 3020409 TI - Is diet the cornerstone in management of diabetes? PMID- 3020411 TI - Structure-activity relationship and pharmacology of the highly selective mu opioid agonist, morphiceptin. PMID- 3020410 TI - Donor strains of cytomegalovirus in renal-transplant recipients. PMID- 3020413 TI - Affinity labels as probes for opioid receptor types and subtypes. PMID- 3020412 TI - Design of conformationally constrained cyclic peptides with high delta and mu opioid receptor specificities. PMID- 3020414 TI - Opioid peptides: analysis of specificity and multiple binding modes through computer-aided drug design and structure-activity studies. PMID- 3020416 TI - Frontiers of research in the medicinal chemistry and molecular pharmacology of opioid peptides. PMID- 3020415 TI - Mechanistic structure-activity studies of peptide and nonpeptide flexible opioids: an interdisciplinary approach. PMID- 3020417 TI - Structure-activity relationships of opioid peptides. PMID- 3020418 TI - Design principles: enkephalins with predictable mu/delta receptor specificity. PMID- 3020419 TI - Beta-endorphin: naturally occurring or synthetic agonists and antagonists. PMID- 3020421 TI - Progress in the characterization of the opioid receptor subtypes: peptides as probes. Future directions. PMID- 3020420 TI - Enkephalin degrading enzyme inhibitors: a physiological way to new analgesics and psychoactive agents. PMID- 3020422 TI - Regulation of agonist binding to opioid receptor types of sodium and GTP: relevance to receptor function. PMID- 3020423 TI - Recent developments in bioassay using selective ligands and selective in vitro preparations. PMID- 3020424 TI - The analysis of endogenous opioid peptides with HPLC, radioreceptor assay, radioimmunoassay, and mass spectrometry. PMID- 3020425 TI - Progress in the potential use of enkephalin analogs. PMID- 3020426 TI - Structure of the receptor for platelet-derived growth factor helps define a family of closely related growth factor receptors. AB - The primary structure of the receptor for platelet-derived growth factor (PDGF), determined by means of cloning a cDNA that encodes the murine pre-PDGF receptor, is closely related to that of the v-kit oncogene product and the receptor for macrophage colony stimulating factor (CSF-1). Common structural features include the presence of long sequences that interrupt the tyrosine-specific protein kinase domains of each molecule. The PDGF and CSF-1 receptors also share a characteristic distribution of extracellular cysteine residues. Ubiquitin is covalently bound to the purified PDGF receptor, the human gene for which is on chromosome 5. PMID- 3020427 TI - Substrate phosphoprotein availability regulates eclosion hormone sensitivity in an insect CNS. AB - The final step in the moulting of all insects is ecdysis, the shedding of the cuticle of the previous instar, which is triggered in Lepidoptera by the neurosecretory peptide eclosion hormone. This hormone acts directly on the nervous system to release the appropriate motor patterns for larval, pupal and adult ecdysis, but there are only brief periods near the end of each moult when the nervous system is competent to respond to the hormone. Previous experiments have shown that the action of eclosion hormone on the nervous system at pupal ecdysis in the tobacco hornworm, Manduca sexta, is mediated by the second messenger cyclic GMP. Here we report that the hormone-stimulated increase in cGMP results in the phosphorylation of two proteins, each with an apparent relative molecular mass (Mr) of 54,000. Moreover, the brief periods during which the central nervous system (CNS) is responsive to eclosion hormone seem to result from the transient presence of these substrate proteins within the nervous system. This provides a novel mechanism by which hormonal responsiveness can be regulated. PMID- 3020428 TI - A cyclic AMP- and phorbol ester-inducible DNA element. AB - Many cellular processes are regulated by hormones and neurotransmitters which interact with cell-surface receptors to produce intracellular second messengers that activate protein kinases. Cyclic (c) AMP is a second messenger whose intracellular level is determined by receptor-mediated activation or inhibition of adenylate cyclase. Phorbol esters directly activate protein kinase C, a Ca2+ and phospholipid-dependent protein kinase and a component of a different second messenger system, the phosphatidylinositol pathway. Proenkephalin messenger RNA levels are regulated in response to cAMP analogues, activators of adenylate cyclase, nicotinic agonists and depolarization, suggesting that expression of the gene encoding proenkephalin is regulated by trans-synaptic events involving cell surface-receptor activation. Here we report that cAMP analogues and activators of adenylate cyclase regulate a proenkephalin-chloramphenicol acetyl transferase fusion gene when transiently expressed in tissue culture cells. Phorbol ester regulates the fusion gene in a similar fashion, but requires the presence of phosphodiesterase inhibitors for large effects. The DNA sequences required for regulation by both cAMP and phorbol ester map to the same 37-base pair (bp) region located 107-71 bp 5' to the mRNA cap site of the proenkephalin gene. This highly conserved region is composed of three closely related 12-bp sequences and has properties similar to those of previously characterized transcriptional enhancers. PMID- 3020429 TI - Cumulative effect of intragenic amino-acid replacements on the thermostability of a protein. AB - The marginal net stability of a folded protein is thought to depend on a small difference between large, compensating individual forces. Therefore, the net free energy of stabilization of proteins is unexpectedly small (approximately 10 kcal mol-1). The contribution of individual forces such as hydrogen bonds and salt bridges to the stabilization is evaluated as 1-3 kcal mol-1, and several additional forces are thought to be sufficient to account for the extra thermostability of thermophilic proteins. The native conformation of a protein is determined by the total number of interatomic interactions and hence by the amino acid sequence. If the few amino-acid residues that individually contribute to the stabilization could be implemented concurrently into the sequence, the multiple replacement would enhance the overall stability of the protein molecule. Here we report evidence to support this argument. Thermal inactivation kinetics and proteolytic resistance for mutants of a kanamycin nucleotidyltransferase reveal that a few intragenic amino-acid replacements stabilize the protein cumulatively. Our experiments not only demonstrate the feasibility of elevating the thermostability of a protein but also lead to better understanding of the forces that are responsible for protein stability. PMID- 3020430 TI - Mapping of the class II region of the human major histocompatibility complex by pulsed-field gel electrophoresis. AB - The class II region of the human major histocompatibility complex (MHC) encodes a polymorphic set of cell surface glycoproteins involved in the regulation of the immune response. Each glycoprotein is a heterodimer composed of a alpha-chain of relative molecular mass (Mr) 34,000 (34 K) and a beta-chain of Mr = 28K. The products of the class II region have been characterized by the mixed lymphocyte reaction, serology, primed lymphocyte typing and DNA cloning. DR, DQ and DP, three subregions containing both alpha- and beta-chains, and two additional loci, DZ alpha and DO beta, locate this gene cluster on the short arm of chromosome 6. The precise genomic organization of these loci have been difficult to determine. Here we describe the use of pulsed-field gel electrophoresis together with restriction endonucleases having few genomic restriction sites and Southern blotting, to determine the order of the subregions and to derive a map for the human class II region. The order of these loci is similar to that of the homologous loci in the murine class II region. Our study establishes the use of pulsed-field gel electrophoresis in mapping large regions of the genome in higher eukaryotes. PMID- 3020431 TI - Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin-binding domain. AB - Gelsolin is representative of a class of actin-modulating proteins found in lower eukaryotes to mammals, which sever actin filaments. Gelsolin found in the cytoplasm of cells is functionally similar to a mammalian plasma protein of similar size, originally called ADF or brevin. Human plasma and rabbit macrophage gelsolins differ by the presence of a 25-amino-acid residue extension on plasma gelsolin which appears to account for the difference in relative molecular mass (Mr) between the proteins as assessed by SDS-polyacrylamide gel electrophoresis (PAGE), 93,000 (93K) and 90K, respectively. Here we report the isolation of full length human plasma gelsolin complementary DNA clones from a HepG2 library. The inferred amino-acid sequence reveals the presence of a signal peptide, a long tandem repeat that matches the actin-binding domains of gelsolin, a tetrapeptide present in actin and extended regions of identical sequence with rabbit macrophage gelsolin. Southern blot analysis indicates that a single gene in the haploid genome encodes both protein forms. PMID- 3020432 TI - Bending of promoter DNA on binding of heat shock transcription factor. AB - The Drosophila heat-shock transcription factor (HSTF) has been shown to bind to three domains of the heat shock protein 70 gene (hsp 70) control region. The most critical of these for transcriptional activation appears to be the one closest to the TATA-homology region. This domain, spanning sequences from -40 to -95, consists of two contiguous HSTF binding sites (sites 1 and 2) that are occupied in a cooperative manner (see Fig. 1). Recent alkylation interference and protection studies suggest a conformational change occurs in the protein-DNA complex at site 1 upon sequential HSTF binding at site 2 (ref. 5). We report here that HSTF binding to a single site or to both contiguous sites results in the introduction of a specific DNA bend within this domain of the hsp 70 promoter. PMID- 3020433 TI - Cloning and sequence analysis of cDNA for bovine carboxypeptidase E. AB - Carboxypeptidase E (enkephalin convertase) was first identified as the carboxypeptidase B-like enzyme involved in the biosynthesis of enkephalin in bovine adrenal chromaffin granules. A similar enzyme is present in many brain regions and in purified secretory granules from rat pituitary and rat insulinoma. Within the secretory granules, carboxypeptidase E (CPE) activity is found in both a soluble and a membrane-bound form, which differ slightly in relative molecular mass (Mr). Here, to investigate whether the CPE activities in the various tissues are produced from a single gene, purified CPE was partially sequenced and oligonucleotide probes were used to isolate a clone encoding CPE from a bovine pituitary complementary DNA library. This cDNA hybridizes to bovine pituitary poly(A)+ RNAs of approximately 3.3, 2.6 and 2.1 kilobases (kb), with the 3.3-kb messenger RNA the predominant species. The predicted amino-acid sequence of the cDNA clone contains the partially determined sequences of CPE, several pairs of basic amino acids and displays some homology with both carboxypeptidases A and B. Restriction analysis of bovine genomic DNA suggests only one gene for CPE. This is consistent with a broad role for CPE in the biosynthesis of many neuropeptides. PMID- 3020434 TI - Cell-type specific protein binding to the enhancer of simian virus 40 in nuclear extracts. AB - Enhancers are cis-acting activators of transcription from homologous or heterologous promoter elements of viral and cellular genes (see refs 1-6 for reviews). The activity of the simian virus 40 (SV40) (refs 7-9) and immunoglobulin heavy-chain gene (IgH) (refs 10, 11) enhancers has been reproduced to some extent in vitro and appears to be mediated by trans-acting factors both in vitro and in vivo. The SV40 enhancer consists of multiple sequence motifs in two domains, A and B (Fig. 1, see ref. 14): domain B contains GT-I and -II and two TC motifs, of which only TC-II is important for enhancer activity in HeLa cells; and domain A contains the P and the two Sph motifs, the repetition of which generates the sequence 5'-ATGCAAAG-3', similar to the 'octameric' sequence of the IgH enhancer, (Fig. 4i; refs 14, 16), where it is important for enhancing activity. Each SV40 enhancer motif is a binding site for a protein or proteins present in HeLa cell nuclear extracts. Unlike the SV40 enhancer, which is active in HeLa and lymphoid B cells, the IgH enhancer is preferentially active in B cells, suggesting that not all the trans-acting factors necessary for its activity are present in HeLa cells. However, the IgH enhancer can compete with the SV40 enhancer in vitro in HeLa or lymphoid cell extracts and in vivo in B cells. Here we show that both human HeLa and BJA-B lymphoid B-cell nuclear extracts contain proteins that bind to specific, sometimes overlapping, motifs of the SV40 enhancer. Some binding is cell-specific, suggesting that it is not the same set of sequence motifs and proteins that is responsible for the enhancer activity in the two cell types. This is confirmed by our results obtained in vivo with mutated SV40 enhancers. PMID- 3020436 TI - Distribution of Meynert cells in primate striate cortex. Spatial relationships with cytochrome oxidase blobs. PMID- 3020435 TI - Phorbol ester induces the transcriptional stimulatory activity of the SV40 enhancer. AB - Phorbol ester tumour promoters can induce the transcription of a number of genes, including c-myc and c-fos. These genes are part of a group referred to as 'competence' genes because they are expressed very early after quiescent cells are stimulated to enter the cell cycle. The 'competence' genes are coordinately induced by serum and by factors such as platelet-derived growth factor and fibroblast growth factor. These factors, as well as the tumour promoter, 12-O tetradecanoyl phorbol-13-acetate (TPA), are thought to exert their action by a mechanism involving the activation of protein kinase C. It is likely that these factors induce the transcription of the 'competence' genes either by activating specific transcription factors or by increasing their intracellular concentration; either mechanism may be mediated by protein kinase C. One approach to identifying such a putative transcription factor is to characterize the cis acting transcriptional control elements that serve as a target site for the factor. Here we report that, in a human hepatoma cell line, TPA can specifically induce the activity of the simian virus 40 (SV40) transcriptional enhancer element. Since the SV40 enhancer is a thoroughly characterized cis-acting element, this system may facilitate the eventual identification of the trans acting factor(s) whose activity is modified by TPA treatment. PMID- 3020437 TI - Inhibition of noradrenergic neurotransmission by apomorphine and pergolide in the in situ autoperfused rat renal and superior mesenteric vascular beds. AB - In vitro studies have provided evidence that presynaptic dopamine receptors are present in the rat renal and superior mesenteric vascular beds. To confirm this in vivo, the effects of locally administered apomorphine and pergolide were studied in the in situ autoperfused renal and superior mesenteric vascular beds. Local infusion of apomorphine (1 microgram X kg-1 X min-1 for 5 min) or pergolide (1 microgram X kg-1 X min-1 for 5 min) into either the renal or the superior mesenteric artery had no effect on perfusion pressure per se. In the renal vascular bed, the pressure response to electrical stimulation (4 Hz, 1 ms, supramaximal voltage) was reduced to 49.8 +/- 4.8% by apomorphine and to 54.8 +/- 2.7% by pergolide; in the mesenteric vascular bed, apomorphine reduced the pressure response to electrical stimulation (4 Hz, 1 ms, supramaximal voltage) to 53.8 +/- 2.9, pergolide to 52.0 +/- 1.8%. Increases of perfusion pressure in the renal and in the mesenteric vascular bed induced by locally administered noradrenaline were not modified by apomorphine or pergolide. In both vascular beds, the inhibition of the stimulation-evoked pressure responses by apomorphine or pergolide was completely antagonized by local administration of the dopamine receptor antagonist haloperidol in a dose (1 microgram X kg-1) which did not influence the inhibitory effect of the alpha 2-adrenoceptor agonist UK-14,304; the alpha 2-adrenoceptor antagonist rauwolscine, in a dose (100 micrograms X kg 1) which completely antagonized the inhibitory effect of UK-14,304, did not antagonize the inhibitory effects of apomorphine and pergolide.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020439 TI - Interactions of MEN 935 (adimolol), a long acting beta- and alpha-adrenolytic antihypertensive agent, with postsynaptic alpha-adrenoceptors in different isolated blood vessels--influence of angiotensin II. AB - MEN 935 [1-(3-[3-(1-naphthoxy)-2-hydroxypropyl) amino)-3,3-dimethylpropyl)-2 benzimidazolinone-hydrochloride monohydrate, adimolol] is a long acting antihypertensive agent with beta- and alpha-adrenolytic properties. Preliminary experiments in pithed rats had led to the suggestion that the alpha-adrenolytic activity was of the alpha 2-subtype. The alpha-adrenolytic properties of MEN 935 were now tested in isolated vascular preparations of rat aorta, rabbit vena ischiadica and rabbit vena cava inferior against the selective alpha 1-adrenergic agonist phenylephrine (PE) and the selective alpha 2-adrenergic agonist B-HT 920 [2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-(4,5-d)azepine]. The experiments were performed in absence and in presence of 5 X 10(-9) mol/l angiotensin II (A II). MEN 935 antagonized contractions to phenylephrine as well as those to B-HT 920 in each vessel. A twofold shift to the right of the concentration-response curves to both agonists was obtained with concentrations between 1.9 X 10(-8) and 1.4 X 10(-5) mol/l, depending on the vessel under investigation. A II modulated the adrenolytic properties of MEN 935 in each vessel. However, irrespective of the presence or absence of A II, no pharmacologically relevant difference between antagonism against PE or B-HT 920 could be seen. In isolated vessels, MEN 935 exerts a nonselective alpha-adrenergic antagonism. In receptor binding studies in rat cerebellar cortex, MEN 935 showed a Ki of 5.2 X 10(-7) mol/l at alpha 1 adrenoceptors and a Ki of 1.3 X 10(-5) mol/l at alpha 2-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020438 TI - Receptor protection experiments confirm the identity of presynaptic alpha 2 autoreceptors. AB - Receptor protection experiments were carried out in cerebrocortical slices from rabbits in order to study the sites at which drugs with alpha-adrenoceptor affinity modulate the release of noradrenaline. The slices were preincubated with 3H-noradrenaline. They were then superfused with 3H-noradrenaline-free medium and stimulated electrically (3 Hz) twice for 2 min each, after 60 and 250 min of superfusion (S1, S2). Phenoxybenzamine was added from 85 to 95 min of superfusion. Potential protecting drugs were present for 5 min before and during the exposure to phenoxybenzamine and then washed out together with the latter. Phenoxybenzamine 0.1 and 1 mumol/l increased the evoked overflow of tritium by 77 and 287%, respectively, as indicated by the S2/S1 overflow ratio. When cocaine was present throughout superfusion, phenoxybenzamine 0.1 and 1 mumol/l increased the evoked overflow by 97 and 353%, respectively. Clonidine 0.1-100 mumol/l, when added before and during the contact with phenoxybenzamine, reduced or even abolished the increase caused by the latter. This interaction was not changed when cocaine was included in the superfusion fluid. The increase caused by phenoxybenzamine was also reduced or abolished by noradrenaline 1-100 mumol/l (tested in the presence of cocaine), yohimbine 0.01-1 mumol/l and phentolamine 0.1-10 mumol/l. Only high concentrations of clonidine, noradrenaline, yohimbine and phentolamine changed the evoked overflow when given alone (and subsequently washed out). The effect of phenoxybenzamine was not modified by prazosin 1 mumol/l, morphine 1 mumol/l and naloxone 10 mumol/l.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020441 TI - Variation in behavioral response to baclofen: correlation with benzodiazepine binding sites in mouse forebrain. AB - Mice treated with (+/-)baclofen (2 mg/kg i.p.) displayed profound differences to the motor depressant action of this drug. Mice were divided into three groups termed as responders (20%), moderate responders (65%) and nonresponders (15%). No differences were detected in the spontaneous motor activity between the selected groups. When the animal population in the home cages was kept unchanged the different responses to baclofen were reproducible three weeks after selection. A borderline dose of diazepam (1.5 mg/kg) decreased highly significantly motor activity in baclofen responders but failed to do this in nonresponders group. Ex vivo studies with 3H-flunitrazepam (labelling in vivo and binding carried out in vitro) demonstrated that 3H-flunitrazepam binding in mouse forebrain was significantly smaller in baclofen responders than in nonresponders. Further, the apparent number of 3H-flunitrazepam binding sites in vitro was significantly decreased in the forebrain of baclofen responders. It is concluded that some actions of baclofen depend on the functional state of benzodiazepine recognition sites. PMID- 3020440 TI - Increased sensitivity to alpha-adrenoceptor stimulation but intact purinergic and muscarinergic effects in prehypertensive cardiac hypertrophy of spontaneously hypertensive rats. AB - The effects of phenylephrine, isoprenaline and adenosine, (-)-N6 phenylisopropyladenosine (PIA) or carbachol alone and in the presence of isoprenaline on force of contraction were studied in isolated electrically driven papillary muscles of spontaneously hypertensive rats (SHR) and age-matched Wistar control rats. In SHR an increased heart to body weight ratio was observed when blood pressure was not yet elevated. During this stage of the syndrome (i.e. between the 27th and 35th day of life) phenylephrine was about 3.4 times more potent to increase force of contraction in SHR (mean EC50: 2.8 mumol l-1) than in control rats (mean EC50: 9.4 mumol l-1). The positive inotropic effect of isoprenaline was similar in SHR and control rats. Also no difference could be detected in the isoprenaline-antagonistic effect of adenosine, the adenosine receptor agonist PIA or carbachol. We conclude that an increased sensitivity to cardiac alpha-adrenoceptor stimulation might be related to prehypertensive cardiac hypertrophy in SHR. PMID- 3020442 TI - [Calcium channel blockers and angiotensin converting enzyme inhibitors in the treatment of high blood pressure]. PMID- 3020443 TI - [Nuclear magnetic resonance spectroscopy in medicine: biochemistry of intact tissues]. PMID- 3020444 TI - [It is the little things which count ... parvoviruses]. PMID- 3020445 TI - [Human parvovirus B19]. PMID- 3020446 TI - [Acute joint symptoms in a parvovirus infection]. PMID- 3020448 TI - [A patient with an aplastic crisis in spherocytosis caused by a parvovirus infection]. PMID- 3020447 TI - [A young woman with a liver tumor and hypercalcemia]. PMID- 3020449 TI - Experience with continence-preserving procedures at University of Nebraska Hospital. PMID- 3020450 TI - Consensus conference on cytomegalovirus and herpes simplex in children: report of the Medical/Scientific Subcommittee. PMID- 3020451 TI - [Dendritic action potentials of pyramidal neurons of the sensomotor cortex of the cerebral cortex of the cat]. AB - The intracellular activity of pyramidal tract neurons during electrical stimulation of ventro-lateral and ventro-postero-lateral nuclei of thalamus was studied in acute experiments on cats immobilized by myorelaxants. Both somatic and presumably dendritic spikes (d-spikes) were observed. The latter were characterized by relatively low and variable (5-60 mV) amplitude; d-spikes occurred both spontaneously and in response to single shock and tetanic (8-14/s) stimulation of the thalamus. They were also induced by intracellular depolarizing current pulses and thalamic stimulation following iontophoretic application of strychnine. Simultaneously generated somatic and d-spikes revealed no collision between each other. Intracellular hyperpolarizing current pulses abolished only somatic spikes, while d-spikes were not affected. Dendritic origin with multiple generation zones of these variable spikes is suggested. Possible functional role of d-spike is discussed. PMID- 3020452 TI - [Effect of intracellular injections of chloride ion on the inhibitory postsynaptic potential and post-burst hyperpolarization of sensomotor cortex neurons in the cat]. AB - Inversion of the early component of IPSPs in pyramidal neurons of the sensorimotor cortex by intracellular injection of chloride ions was demonstrated in cats immobilized by myorelaxants in acute experiments under moderate composed anesthesia (40 mg/kg nembutal and 20 mg/kg chloralose intraperitoneally). The late component of IPSPs as well as the post-burst hyperpolarization in pyramidal neurons were not inverted. It is concluded that during the early component of IPSPs of both pyramidal and nonpyramidal neurons the membrane permeability is increased for chloride ions, while both the late component of IPSPs and the post burst hyperpolarization in pyramidal neurons are less dependent on the chloride permeability. PMID- 3020453 TI - [Effect of changes in membrane potential and temperature on the post-synaptic potential on the neuromuscular junction of the frog]. AB - The decay time-constants of the 2nd and 1st nerve-evoked paired end-plate currents (epc) were recorded in transversely cut muscle preparations of frog. After the inhibition of synaptic acetylcholinesterase by prostigmine (3 X 10(-6) mol/l) the decay of the 2nd epc was 39 +/- 8% slower than the decay of the 1st epc (the interstimulus interval being 100 ms) due to postsynaptic potentiation (PSP). It was found that PSP does not depend on the membrane potential level in the range of-30-120 mV. A drop in temperature from 22 degrees to 12 degrees resulted in several effects: an increase in the decay time constant of epc and meps; a slight decrease in mepc amplitude; a fall of epc quantal content. The comparison of paired epc of equal quantal content showed that PSP was more pronounced at lower temperature. The temperature coefficient (Q10) for the ratio of decay time constants of the 2nd and the 1st epc was 2.0 +/- 0.2. Evidently, the trace of preceding activity of the transmitter does not depend on the membrane potential level but becomes stronger with a fall of the temperature. PMID- 3020454 TI - [Reversal potential of the EPSP of frog motoneurons]. AB - The current-chop technique has shown that I-V characteristic of the membrane are nonlinear in lumbar motoneurons of isolated perfused frog spinal cord. Input resistance of the membrane decreased with depolarization when constant current was applied during 0.1-1.0 s. However, injection of current 40-60 nA during 1-2 min led to an increase of the membrane resistance to the initial value. As a result the membrane potential could be shifted to the positive level up to +50 mV and more. Monosynaptic excitatory postsynaptic potentials evoked by stimulation of the brainstem or by microstimulation of ventrolateral tract fibres were found to reverse completely at a positive level of the membrane potential. In most cases the reversal potentials ranged between 0 mV and -10 mV. PMID- 3020455 TI - Electron microscopy in assessment of the biological behavior of human papillomavirus infections in the uterine cervix. AB - To asses the natural history of human papillomavirus (HPV) infections in uterine cervix, currently implicated in etiology of cervical cancer, a prospective follow up study has been conducted for 418 women at our clinic since 1981. The present communication summarized the current follow-up data of these patients, with special emphasis on detection of the virus in cervical punch biopsies, as correlated with other characteristics pertinent to the clinical behavior of cervical HPV infections. On each attendance, the patients are subjected to colposcopy accompanied either by Papanicolaou (PAP) smears or punch biopsies. The latter are analyzed for the cytopathic changes of HPV, for concomitant cervical intraepithelial neoplasia (CIN), for HPV structural proteins with IP-PAP technique as well as on transmission electron microscopy (TEM) for the presence of HPV particles. The local immunocompetent cell (ICC) infiltrates are analyzed using ANAE technique to define B cells, MPS cells and T cells and monoclonal antibodies (McAb) for T cell subsets, NK (natural killer) cells and Langerhans cells. HPV particles were disclosed with equal frequency (approx. 65%) in all three types of HPV lesions. Surprisingly, HPV particles were present in 70% of the biopsies derived from the regressed lesions (e. g. in those without histological evidence of HPV lesions), suggesting a possibility of a latent HPV infection. Presence of viral particles did not bear any direct correlations with the expression of HPV antigens, intensity or cellular composition of the ICC infiltrate, defined by ANAE or using McAb. Presence of HPV particles was not a major prognostic determinant, whereas the clinical course was most significantly influenced by the grade of HPV-associated CIN, to which regression was inversely and progression directly related. The results clearly confirm that cervical HPV infections are capable of progressing into carcinoma in situ and thus present with a natural history equivalent to that of classical CIN. PMID- 3020456 TI - Immunological reactivity in children with Wilms' tumor. AB - Cellular immune reactivity was investigated in 49 newly diagnosed children with Wilms' tumor and compared to age-matched control. The level of total T (T4 degrees) and B lymphocytes was normal while the relative number of T lymphocytes with high affinity receptors for sheep erythrocytes (T29 degrees) was significantly decreased in the patients studied. The lymphocyte response to PHA in vitro was diminished but PHA-induced lymphokine production was not altered. The depression of T29 degrees level and lymphocyte reactivity to PHA was associated with high grade tumor rather than with the clinical stage. Lymphocytes of 42-47% patients reacted with autochthonous and allogeneic KCl tumor extracts in the migration inhibition test and the degree of reactivity was related to the histological differentiation of the tumor. PMID- 3020457 TI - [Comparison of the lipid composition and physicochemical characteristics of membrane preparations of transport ATPases]. PMID- 3020458 TI - [Mechanisms of energy transfer in biological structures: the role of hydrogen bonds]. PMID- 3020459 TI - [Gluconokinase characteristics of the brain tissue of white rats]. PMID- 3020460 TI - [Effect of glucocorticoid hormones on NADH and succinate oxidation by rat brain and liver mitochondria]. PMID- 3020461 TI - Effect of perinatal exposure to methadone on brain opioid and alpha 2-adrenergic receptors. AB - Prenatal exposure to psychotropic drugs has recently been shown to induce behavioral and brain neurochemical changes in the newborn. The long-term effects of pre- and postnatal exposure to methadone on the opioid (delta and mu) and adrenergic (alpha 2) receptors were investigated in two areas of the rat brain. Rat pups received prenatal treatment with methadone through a minipump implanted in the pregnant dams and were cross-fostered to methadone- or non-methadone implanted dams until weaned. The rats were sacrificed when fully grown and brains removed. Receptor binding assays using 3H-D-ala-leu-enkephalin and 3H-naloxone for delta and mu opioid receptors respectively and 3H-rauwolscine for alpha 2 adrenergic receptors were performed in two areas of the brain. In the hypothalamus, prenatal methadone treatment induced sustained decreases in both delta and mu opioid receptors but did not cause significant changes in the alpha 2-adrenergic receptors. In the cerebral cortex, prenatal methadone treatment induced significant decreases not only in delta and mu opioid receptors but also in alpha 2-adrenergic receptors. Postnatal methadone treatment alone did not cause significant changes in opioid receptors in the two brain areas, but did induce reduction in the cerebral cortical alpha 2-adrenergic receptors. The significance of these results is discussed. PMID- 3020462 TI - Effects of postweaning administration of delta-9-tetrahydrocannabinol (THC) on adult behavioral and neuroendocrine function in Sprague-Dawley and Fischer-344 rats. AB - Delta-9-tetrahydrocannabinol (THC) was administered to young male Fischer-344 (CDF) and Sprague-Dawley (CD) rats on days 30-50 of age. Doses of THC consisted of 20, 10 or 5 doses of 10 mg/kg spaced over the 20-day period. On day 140 animals were exposed to a 15 sec 1.5 mA scrambled foot-shock. Latency for withdrawal from 55 degrees C water was used as a measure for analgesia. Both CDF and CD rats showed a foot-shock induced analgesia (FSIA). Animals which had received 5 or 10 doses of THC in youth showed an enhanced response to foot-shock in the CDF rat. The foot-shock was then paired with an unconditioned stimulus (shock environment) and a conditioned analgesia developed over 4 days. At weekly intervals thereafter animals were tested in the shock environment only for extinction of the analgesic response. Over 4 weeks, analgesia did not show extinction in the CDF rat. Extinction of the response was observed in the veh and 20 dose groups in the CD rat; whereas a resistance to extinction was observed in the other groups. The CDF rats were then sacrificed following the last extinction trial and serum corticosterone and prolactin measured. Five and 10 doses of THC decreased prolactin levels; stress, however, increased these levels above the levels in VEH treated animals exposed to stress. Extinction of a fixed ratio 10 as well as exposure to fixed ratio strain in the CD rat were not affected by THC. These data suggest that THC administered during postweaning development alters endogenous systems which mediate the animals response to stress. PMID- 3020463 TI - [Clinicopathological study on malignant glioma--with special reference to GFAP]. AB - In surgical specimens of 91 cases of malignant glial tumor, the correlation between the clinical malignancy of the tumors and the presence of GFA protein in the surgical specimen was examined. In anaplastic astrocytomas, 20 of 27 cases revealed positive staining of GFAP. In the cases of glioblastoma multiforme 27 of 35 cases showed GFAP positive staining. On the other hand, in medulloblastomas, all 29 cases were negative. In anaplastic astrocytomas and glioblastoma multiforms, the difference between the survival curves of GFAP positive groups and negative groups were examined. Between two groups, there was no statistical differences. In this study, the presence of GFAP was not proportional to the degree of tumor differentiation, and the GFAP staining of the tumor tissue was not a valuable examination showing the prognosis of the patient. PMID- 3020464 TI - Use of 125I-Tyr27 beta-endorphin for the study of beta-endorphin binding sites in rat cortex. AB - An iodine-labelled derivative of beta-endorphin, 125I-Tyr27 beta h-endorphin, was used in carrier-free form to study the binding of beta-endorphin to brain opioid receptors. The experiments were carried out with rat cortex membranes in vitro under conditions that gave a high degree of naloxone reversible binding. Beta h Endorphin and nonradioactive iodo-Tyr27 beta h-endorphin were found to be identical in their ability to inhibit the binding of 125I-Tyr27 beta h-endorphin. Competition experiments demonstrated the existence of binding sites with higher affinity for beta-endorphin than for a variety of other opioids, including naturally occurring fragments of beta-endorphin. The experiments show that 125I Tyr27 beta-endorphin possesses similar binding properties to the unmodified peptide and can be used with the advantages of iodine-125 as an isotope for the investigation of beta-endorphin receptors in brain. In experiments employing 125I Tyr27 beta-endorphin 1-27 as the radioiodinated ligand, binding curves were obtained which showed that beta-endorphin 1-31 was more potent than beta endorphin 1-27 in inhibiting the binding of the labelled 27 residue peptide. With both the 27 and 31 residue radioligands, magnesium ion enhanced the specific binding whereas sodium ion and guanylyl-imidodiphosphate had a strong inhibitory effect. The data indicate that beta-endorphin 1-27 bindings with reduced affinity to the same receptors as beta-endorphin 1-31 and like the 31 residue peptide exhibits properties characteristic of an agonist. PMID- 3020465 TI - Naloxone does not improve cardiovascular or blunt vasopressin responses in spontaneously hypertensive rats following graded hemorrhage. AB - The effects of continuous intravenous infusion of naloxone or vehicle on the blood pressure and vasopressin responses to step-wise hemorrhage were examined in conscious, age-matched spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rats (WKY). Step-wise hemorrhage progressively lowered blood pressure and increased plasma vasopressin levels in both SHR and WKY. The WKY were relatively resistant to the hypotensive effect of hemorrhage. No significant differences were noted in blood pressure responses between naloxone-treated and vehicle treated SHR while naloxone treatment attenuated hypotension only slightly in WKY. Plasma vasopressin levels were also elevated by naloxone treatment in SHR following a nonhypotensive hemorrhage equivalent to 0.5% of body weight. However, no differences were observed between plasma vasopressin levels in naloxone treated and vehicle-treated SHR at greater degrees of hemorrhage. In addition, plasma vasopressin levels were similar at all times in hemorrhaged WKY, regardless of treatment. Plasma vasopressin levels were increased by naloxone in both time-control SHR and WKY. The data demonstrate that naloxone-sensitive systems exert only minimal effects on the immediate cardiovascular responses to hypovolemia in normotensive rats and no measurable effects in SHR. It does appear that naloxone-sensitive mechanisms contribute a small, but significant, tonic inhibitory influence over vasopressin secretion in both normotensive and hypertensive rats under basal conditions and in SHR in response to a small reduction in blood volume. PMID- 3020466 TI - Melanotic neuroectodermal neurocranial tumor of infancy of extra-intra-and subdural right temporal location: CT examination, surgical treatment, literature review. AB - A melanotic neuroectodermal neurocranial tumor of infancy of extra-intra-subdural right temporal location, examined by CT and successfully operated in a 33-month old boy is reported in context with the literature. Out of 30 cases (including our case) of melanotic neuroectodermal neurocranial and intracranial tumors (MNNIT) 18 were located in the neurocranium (MNNT) and 12 intracranially, i.e. in the brain and cerebellum (MNIT). 23 tumors were located medially (11 at the anterior fontanel, one at the posterior fontanel, one in the 3rd ventricle, one in the sella turcica, one in the pineal region, 8 in the cerebellar vermis) and 7 laterally (five involved the temporal bone, one of which with intracranial propagation (our case), one in the temporal lobe of the brain, one in the skull posterior to the mastoid process). Twenty-eight cases were children (16 boys, 8 girls, four not stated), two adults. The location is important in respect to biology and surgery: MNNIT can be grouped as to its relation to the arachnoid barrier layer into malignant and benign. MNIT localised medially subarachnoidally are malignant. MNNT of small dimensions localised medially and laterally extraarachnoidally are benign. MNNT of extreme dimensions localised medially extra-intradurally are also benign, but difficult to treat. In the MNNT when diagnosed early and operated radically, the mortality can be lowered. Our case, presented on CT (1978) as first case of combined extra-intra-subdural location, represents the 173rd MNT and the 18th related to neurocranium. PMID- 3020468 TI - Microtubule disassembly increases the number of opioid receptor binding sites in rat cerebrum membranes. AB - The role of microtubules in opioid receptor binding was studied by using microtubule assembly inhibitors. Preincubation of rat cerebrum membranes with podophyllotoxin or colchicine provoked a marked increase in the number of binding sites as judged by [3H]-naloxone, [3H]-morphine and [3H]-D-Ala2-Leu5-enkephalin binding experiments. These results indicate microtubule involvement in regulation of opioid receptor expression. PMID- 3020467 TI - Influence of vagotomy and of atropine on the anti-shock effect of adrenocorticotropin. AB - ACTH-(1-24), intravenously injected at the dose of 160 micrograms/kg to rats bled to the point of otherwise irreversible hypovolemic shock, causes a prompt and sustained increase in blood pressure and pulse amplitude, all treated rats surviving at the end of the experiment (2 hr). Bilateral vagotomy, as well as atropine sulphate (2 mg/kg i.p. immediately before bleeding), almost completely abolishes the anti-shock activity of ACTH. These data indicate that a central cholinergic pathway and vagal afferent (but not efferent) fibers play an important role in the anti-shock effect of ACTH. PMID- 3020469 TI - Low affinity inhibition of opioid receptor binding by FMRFamide. AB - The ability of the molluscan neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) to inhibit the binding of opioid-receptor radioligands to mammalian neural tissue was examined. Rabbit brain membrane preparations were exposed to tritiated dihydromorphine and ethylketocyclazocine in the presence of various concentrations of FMRFamide. FMRFamide inhibited the specific binding of both ligands in a dose-related manner, suggesting that the neuropeptide can inhibit binding to at least two subtypes of opioid receptors (mu and kappa). These data are consistent with the recent proposal that FMRFamide, or the immunoreactive FMRFamide-like material in mammalian brain, spinal cord, and gastrointestinal tract, can act as an endogenous opioid antagonist. However, the low binding affinity of FMRFamide might suggest an alternative mechanism for FMRFamide antagonism of opioid action in vivo. PMID- 3020470 TI - Is oxytocin-induced grooming mediated by uterine-like receptors? AB - In this study, we examined whether the mechanisms mediating the induction of grooming behavior by oxytocin (OT) is similar to mechanisms mediating the effects of OT on uterine contractility. Sprague-Dawley strain female rats were injected intracerebroventricularly (ICV) with OT or OT analogues and then were observed for grooming behaviors 25 minutes later for 30 minutes. The uterotonic analogue deamino-OT injected ICV at equimolar doses to 1 microgram OT significantly elevated grooming scores although less than did OT. Other agonist analogues were not effective in inducing an increase in grooming behavior. The simultaneous ICV injection of the analogue [Pen1, Phe2, Thr4, delta 3, 4Pro7, Orn8]-OT, which blocked the effects of OT on uterine contractility, also blocked the effect of OT on grooming behavior. Injection of the same dose of this antagonist analogue did not effect the increased grooming behavior after AVP injection. Pretreatment with 5 mg/kg of the prostaglandin synthetase inhibitor indomethacin significantly inhibited OT-induced grooming. We have concluded from these data that the mechanism underlying the effect of OT on grooming is similar to its effects on uterine contractility in some respects. However, observations that the OT antagonist analogue blocked OT- but not AVP-induced grooming may suggest that more than one receptor or mechanism exists by which nonapeptides initiate excessive grooming. PMID- 3020472 TI - Comparison of two penicillamine-containing enkephalins: mu, not delta, activity produces analgesia. AB - Two penicillamine-containing enkephalin analogs were compared for inhibition of mouse writhing after intracerebroventricular administration. The delta receptor selective analog was less potent in inhibiting writhing than the non-selective analog. Inhibition of writhing produced by the delta-selective analog was antagonized by very low doses of naloxone, and produced additive analgesic effects with the delta receptor antagonist, ICI 174864. These results suggest that inhibition of writhing was produced by mu receptor agonist activity, and not delta receptor agonist activity. PMID- 3020471 TI - Enkephalinase and angiotensin converting enzyme activities in human venous and arterial plasma. AB - Circulating opioids, particularly enkephalins, can act on specific receptors located on the neurovascular sympathetic junction. These peptides are quickly metabolized by enkephalinase and angiotensin converting enzyme (ACE). According to the parallel distribution of enkephalinase with opioid receptors in the rat brain, and its location in the vascular bed, putative differences of enkephalinase and ACE activity between arterial and venous plasma of the same subjects was researched. Venous enkephalinase activity was found to be greater than arterial activity. No arterovenous differences were present in ACE activity. The activities of both enzymes presented a positive correlation between venous and arterial plasma in the same subjects. The arterovenous difference in enkephalinase activity supports a release of the enzyme from microvessels. PMID- 3020473 TI - Irreversible labelling of rat brain opioid receptors by enkephalin chloromethyl ketones. AB - Chloromethyl ketone derivatives of leucine enkephalin (LE), D-Ala2-Leu5 enkephalin (DALE) and D-Ala2-D-Leu5-enkephalin (DADLE) were synthesized. They all show high affinity for rat brain opioid binding sites. Preincubation of the membrane fraction with enkephalin chloromethyl ketones causes a significant inhibition of /3H/-naloxone binding which cannot be reversed by extensive washing. It was found that the irreversible inhibition is selective for the high affinity (KD less than 1 nM) /3H/-naloxone binding site (putative mu-1 site). The irreversible blockade of opioid binding was partially protected by opiate alkaloids and opioid peptides, suggesting that non-specific labelling also occurs. Affinity of enkephalin chloromethyl ketones toward the mu sites is greater than that of the parent compounds. It was also found that the covalent inhibition of mu sites (/3H/-dihydromorphine and /3H/-DAGO binding) is more effective than that of delta sites (/3H/-DALE binding). We conclude that these chloromethyl ketone derivatives can be used as affinity labels for the opioid receptors, allowing us to study the structure of the mu receptor subtype. PMID- 3020474 TI - Perineuritis and ulcerative colitis. AB - We describe the association of chronic polyneuropathy with ulcerative colitis. Electrophysiologic studies disclosed a severe neuropathy with both axonal and demyelinating features. The CSF protein content was 875 mg/dl. Sural nerve biopsy revealed perineuritis. Peripheral neuropathy with perineuritis may be an immunologically mediated extraintestinal manifestation of ulcerative colitis. PMID- 3020475 TI - Benzodiazepine receptor binding is not altered in human epileptogenic cortical foci. AB - We measured benzodiazepine binding in cortical tissues from 13 patients with partial seizures. No differences in receptor density (Bmax) or affinity (Kd) were observed when epileptic foci were compared with nonspiking cortex. PMID- 3020476 TI - Acute pure sensory paraneoplastic neuropathy with perivascular endoneurial inflammation: ultrastructural study of capillary walls. AB - We studied a patient with epidermoid carcinoma of the lung (treated surgically 1 year earlier) and an acute symmetric pure sensory neuropathy that regressed almost completely within 1 month. Superficial peroneal nerve biopsy 15 days after onset showed evidence of demyelination with perivascular endoneurial inflammation. On ultrastructural examination, lymphocytes were seen passing through endothelial cells of endoneurial capillaries. PMID- 3020477 TI - Serial neuropsychological studies of a child with acute lymphoblastic leukemia and subsequent glioblastoma multiforme. AB - A 10-year-old boy had a right posterior parietal glioblastoma 5 years after completing treatment for acute lymphoblastic leukemia. Interim findings included seizures, leukoencephalopathy, diffuse mineralizing microangiopathy, and abnormal changes in neuropsychological test performance, which, in retrospect, provided information about the location of the tumor. This case highlights unusual sequelae of childhood leukemia and its treatment, as well as the value of neuropsychological procedures in assessing functional status and integrity of the brain. PMID- 3020478 TI - [Scintigraphy in the study of varicocele]. AB - Varicocele which is relatively common after puberty may cause even severe alterations to fertility. Sequential Isotopic Scrotal Angiography (SISA) was used for the diagnosis of this condition in both the recognition and confirmation phase. This dynamic and static survey based on the use of 99mTc-pyrophosphate proved able to discriminate between the various degrees of varicocele. No statement can as yet be made on the specificity and sensitivity of the examination since so few cases have so far been checked with other techniques (Doppler-Telethermography-Phlebography) and an insufficient number of postsurgical controls came to our observation. PMID- 3020479 TI - Induction of taurine responsiveness in Xenopus oocytes by messenger RNA from mouse brain. AB - A taurine response was induced in the surface membrane of the oocytes of Xenopus laevis by injection of the mRNA from the neonatal mouse brain, and the response was studied electrophysiologically. The permeability of mRNA-injected oocytes to chloride ions was increased by the application of taurine in a dose-dependent manner. The same oocyte also responded to GABA, but bicuculline suppressed only the GABA response. These results suggest a possibility that taurine could be a neurotransmitter of certain neurons as yet unknown in the central nervous system. PMID- 3020481 TI - [The dental management of patients with hypophosphatemic resistant rickets]. PMID- 3020480 TI - Natural history of partial molar pregnancy. PMID- 3020482 TI - [Pathogenesis and etiologic treatment of herpetic stomatitis]. PMID- 3020483 TI - [Granular cell tumor of the tongue. Review of the literature and report of 2 cases]. PMID- 3020484 TI - [Carcinoma in pleomorphic adenoma. Review of the literature and report of a case]. PMID- 3020486 TI - [Bronchial papillomas of various origin]. PMID- 3020485 TI - The question of confidentiality of a patient's HIV antibody test in a psychiatric treatment unit. PMID- 3020487 TI - The modification by systemic morphine of the responses of serotonergic and non serotonergic neurons in nucleus raphe magnus to heating the tail. AB - In anaesthetised rats, 63 raphe-spinal units in nucleus raphe magnus (NRM) have been identified by means of electrophysiological methods and further classified into serotonergic (5-HT) and non-5-HT units according to their conduction velocity and spontaneous discharge rate. All units, except one, showed either excitatory (n = 39) or inhibitory (n = 23) responses to noxious heating of the tail with hot water at 52 degrees C. The threshold temperature was around 44 degrees C and both types of responses increased with stepwise increases in temperature. The excitatory or inhibitory responses of raphe-spinal units induced by noxious heating of the tail correlated well with those elicited by noxious pinching, but did not correlate with the different transmitter populations (5-HT or non-5-HT) of the NRM unit. The effects of morphine or Fentanyl administration on the heat-induced responses of NRM units was observed. The possible involvement of NRM raphe-spinal units in 'diffuse noxious inhibitory controls' (DNICs) was discussed, particularly in relation to the 'lifting of DNICs' produced by morphine. PMID- 3020489 TI - Alpha thalassemia British type (alpha alpha/--Brit) in an Australian family. AB - Alpha thalassemia is rarely diagnosed in Australian families of British or Northern European ancestry. In 1972, a third generation Australian was shown to have alpha thalassemia. In the absence of known Mediterranean or South East Asian ancestry it was reported as being the first example of alpha thalassemia in an Australian family. Further study of the proposita in 1985 using DNA mapping of the alpha globin gene complex, shows a distinctive molecular defect identical to the British type of alpha thalassemia. The latter is clearly different from the commonly encountered Mediterranean and South East Asian alpha zero haplotypes. Recognition that alpha zero thalassemia occurs in Australians is important since it may produce a microcytic hypochromic anemia. Its inheritance together with other forms of alpha thalassemia may lead to severe Hb H disease or Hb Bart's hydrops fetalis. PMID- 3020488 TI - [HLA typing in patients with chronic adenopathies in a population at risk for AIDS]. AB - HLA-A B and DR typing were performed in 77 patients with AIDS related complex (ARC)--69 lymphadenopathy associated syndrome and 8 thrombocytopenic purpura LAV/HTLV III related--and 21 symptom free homosexual males. A significant increase in the frequency of HLA DR5 antigen was observed in patients with ARC mainly in purpura thrombocytopenic patients. We suggest that increase of HLA DR5 antigen support the view that DR5 antigen could be one of the factors necessary at the spreading out clinical symptoms. PMID- 3020490 TI - Clinching the diagnosis: an approach to the investigation of hypercalcemia. AB - Hypercalcemia is a common biochemical abnormality. It is usually possible to make a presumptive diagnosis of its cause by relatively simple means. In a large majority of cases this involves differentiating between primary hyperparathyroidism and malignancy-associated hypercalcemia. Clinical evaluation and parathyroid hormone assay are particularly useful. Since the management of hypercalcemia in the short term generally involves non-specific measures such as rehydration, the inability to make an immediate accurate diagnosis presents less of a problem than might otherwise be the case. The definitive diagnosis of hyperparathyroidism, malignancy and sarcoidosis requires histological confirmation. PMID- 3020491 TI - ["So-called" sclerosing hemangioma of the lung]. PMID- 3020492 TI - Human immunodeficiency virus seroprevalence in pediatric patients 2 to 14 years of age at Mama Yemo Hospital, Kinshasa, Zaire. AB - Seroprevalence to human immunodeficiency virus (HIV) was determined among 368 children 2 to 14 years of age who were admitted to the pediatric service at Mama Yemo Hospital in Kinshasa, Zaire. Forty (11%) of these patients and only one (1%) of 92 healthy siblings of these patients were HIV seropositive (chi 2 = 8.68, P less than .01). Seropositivity was associated with previous hospitalization, receipt of a blood transfusion prior to the current hospitalization (odds ratio 3.1; 95% confidence interval, 1.5 to 6.4), receipt of medical injections during the past year, and smaller household size. Clinically, HIV seropositivity was associated with the diagnoses of malnutrition and pneumonia. A higher proportion of seropositive children died during the current hospitalization (4/40 v 10/328); when patients with malaria were excluded, the in-hospital mortality of seropositive children was more than eight times higher than that of seronegative children (Fisher exact test, P = .006). Clarification of clinical, immunologic, and epidemiologic features of childhood HIV infection is urgently required because HIV appears to account for or complicate a substantial proportion of pediatric hospitalizations in Kinshasa. PMID- 3020493 TI - Clinical overview of varicella vaccine: development and early studies. AB - A live varicella vaccine was developed in 1973 and has been used effectively and safely in normal as well as high-risk children. Some precautions are necessary for use in the high-risk children. PMID- 3020494 TI - Varicella vaccine studies in healthy children and adults. AB - Immunization of normal children and adults with Oka strain live varicella vaccine from several manufacturers has been studied in our laboratory and elsewhere. This paper summarizes clinical trials designed to obtain information on minimum dose immunogenicity pre- and postexposure prophylaxis, immunization of various age groups, and booster immunizations for seropositive individuals. These studies documented a 94% to 100% seroconversion rate with 95% to 100% persistence of antibodies at 3 to 4 years. Protective efficacy was more than 90%, and the vaccine was successful in preventing varicella when a high dose was given postexposure. Clinical reactions were limited to temperature elevations and minor papulovesicular rashes that occurred in 5% to 10% of vaccinees. Herpes zoster has been absent in vaccinated healthy individuals. PMID- 3020495 TI - Live attenuated varicella vaccine use in immunocompromised children and adults. AB - Live attenuated varicella vaccine has been administered to 307 children with leukemia in remission and to 86 healthy adults. The vaccine was well tolerated and immunogenic. The major side effect in leukemic children receiving maintenance chemotherapy was development of a vaccine-associated rash. Vaccinees in whom a rash developed were potentially somewhat infectious to others about 1 month after immunization. Vaccination was not associated with an increase in the incidence of herpes zoster or in relapse of leukemia. Vaccination provided excellent protection against severe varicella. It was associated with a significant decrease in the attack rate of chickenpox following an intimate exposure to varicella-zoster virus, conferring about 80% protection in leukemic children. The cases of breakthrough varicella that occurred were mild. Thus, the vaccine may either prevent or modify varicella in high-risk individuals. It may also have use for prevention of nosocomial varicella. PMID- 3020496 TI - A study of alternating leadership for group psychotherapy in an aftercare clinic. PMID- 3020497 TI - The beta-adrenoceptor-adenylate cyclase complex. From model to biochemical reality. AB - Developments in the receptor concept have greatly influenced our current knowledge of the beta-adrenoceptor. The triad of pharmacology, organic chemistry and studies in structure-activity relationships is discussed along historical lines, as it has been and still is an impetus for progress in the biochemistry of ligand-receptor interactions. With respect to the beta-adrenoceptor complex these advances which have led to a model in which three protein structures are functionally interacting within the frame of the cell wall: the beta adrenoceptor, the regulatory guanine nucleotide binding protein, and the enzyme adenylate cyclase, are reviewed. PMID- 3020498 TI - [Studies on relative biological effectiveness and therapeutic gain factor of high energy protons modulated for radiotherapy]. PMID- 3020499 TI - [Experimental evaluation of pulmonary artery embolization for the treatment of lung cancer]. PMID- 3020500 TI - Analysis of the yeast SPT3 gene and identification of its product, a positive regulator of Ty transcription. AB - Previous work has demonstrated that the yeast SPT3 gene is required for transcription from delta sequences, the long terminal repeats that flank yeast Ty elements. In spt3 null mutants, transcription fails to initiate in delta sequences and instead initiates farther downstream. Null mutations in SPT3 cause other mutant phenotypes, including defects in sporulation and diploid formation. In this paper we report further genetic and physical characterization of the SPT3 gene and protein. By extensive linker insertion mutagenesis, we have delimited the region necessary for SPT3 function. From DNA sequence analysis, SPT3 encodes a protein of 337 amino acids. We have identified this protein with an anti-SPT3 antibody. Finally, we show that overproduction of the SPT3 gene product does not alter the level of Ty transcription. PMID- 3020501 TI - ISH51: a large, degenerate family of insertion sequence-like elements in the genome of the archaebacterium, Halobacterium volcanii. AB - We describe a new family of repetitive elements in the genome of the archaebacterium Halobacterium volcanii. There are some 20-30 copies of this element, which we designate ISH51. Sequenced copies show typical insertion sequence characteristics (terminal inverted repeats, direct flanking repeats of "target site" DNA). However, members of the ISH51 family are highly heterogeneous, showing on average only 85% primary sequence homology; and some genomic copies appear to be severely truncated. Some ISH51 elements are clustered together in regions of relatively AT-rich DNA. There are at least five such AT rich "islands" in the H. volcanii genome. Repetitive sequences homologous to ISH51 are found in the genomes of most Halobacterium and Halococcus species. PMID- 3020502 TI - Expression strategies of the yeast retrotransposon Ty: a short sequence directs ribosomal frameshifting. AB - The Ty element of yeast is a member of a class of eukaryotic transposons which bear a striking resemblance to retroviral proviruses in their structure and expression strategies (1,2). A direct comparison can be drawn between the production of a fusion protein encoded by Ty, resulting from a frameshift event which fuses two out-of-phase open reading frames TYA and TYB, and the production of Pr180gag-pol in a retrovirus such as Rous Sarcoma Virus (RSV) (3,4). We present data which shows, definitively, that RNA splicing is not responsible for the frameshift in Ty. By in vitro mutation of a class I element, Ty1-15, we demonstrate that 31 nucleotides contained within the region where the TYA and TYB open reading frames overlap direct the frameshift. Within this short sequence there is a region of homology with a class II element which we show is also able to frameshift. PMID- 3020503 TI - Restriction endonucleases from Herpetosiphon giganteus: an example of the evolution of DNA recognition specificity? AB - We describe the partial purification and characterisation of five Type II restriction endonucleases from two strains of Herpetosiphon giganteus. One of the activities, HgiJII, was the first enzyme found that cleaves DNA at the family of related sequences 5'-G-R-G-C-Y/C-3'. This enzyme may be related to the enzyme HgiAI from a different strain of the same species, and which cleaves at the sites 5'-G-W-G-C-W/C-3'. We have shown that DNAs from the strains producing HgiAI and HgiJII are resistant to both of these restriction endonucleases. The remaining four enzymes described here share recognition and cleavage specificities with other restriction endonucleases. The evolution of Type II restriction modification systems and their role in vivo are discussed. PMID- 3020505 TI - Guanine and 7-methylguanine amino proton exchange rates as a function of buffer pK: implications for the exchange mechanism. AB - Using the stopped-flow kinetic method we have measured the deuteration rate of the amino protons in 2'deoxyguanosine 5'monophosphate and 7-methylguanosine 5'monophosphate. For both compounds the exchange rates are accelerated with increasing concentration of a large number of buffers with widely differing pKs. The results obtained, in conjunction with a theoretical model study, give rise to serious doubts concerning the normally accepted mechanism of amino proton exchange involving a pre-protonation at N7. PMID- 3020504 TI - Nucleotide sequence of the tetM tetracycline resistance determinant of the streptococcal conjugative shuttle transposon Tn1545. AB - The nucleotide sequence of the tetracycline resistance gene tetM encoded by streptococcal conjugative shuttle transposon Tn1545 has been determined. The resistance gene was identified as a coding sequence of 1917 base pairs corresponding to a protein with a Mr of 72,500 daltons. This value is in good agreement with that, 68,000 daltons, estimated by SDS-polyacrylamide gel electrophoresis of Escherichia coli minicell extracts. The tetM gene product does not exhibit any sequence homology with either the Gram-negative (tetA, tetB and tetC), or the Bacillus and Staphylococcus tetracycline resistance proteins. The average hydropathy value of the tetM gene product (-0.21) contrasts with those calculated for the other TET proteins which are markedly hydrophobic (0.76 to 0.93). Hybridization experiments performed with an intragenic tetM probe do not support the claim [Taylor, D. (1986), J. Bact. 165, 1037-1039)] that tetracycline resistance in Campylobacter is due to acquisition of tetM. PMID- 3020507 TI - EMBL 12, a new lambda replacement vector with sites for SalI, XbaI, BamHI, SstI and EcoRI. PMID- 3020506 TI - Epstein-Barr virus mRNAs produced by alternative splicing. AB - The structure of Epstein-Barr virus mRNAs transcribed in B95-8 cells has been studied by cDNA cloning and sequencing. We present here the analysis of four cDNAs. The corresponding mRNAs are probably transcribed from a single promoter located in the US region. They are produced by alternative splicing of exons transcribed from the US, IR and UL regions. The exons are spread over 100 kbp. The exons from the IR region constitute a unit which is repeated several times. The cDNAs share the exons from the US and IR regions. Some of the cDNAs also share some of the exons from the UL region. Each cDNA contains a long open reading frame or the 5' end of a long open reading frame which ends several hundred nucleotides downstream on the viral genome. The 5' untranslated regions are unusually long. Three mRNA species differing in their 5' untranslated regions may encode for the nuclear antigen EBNA-1. The other mRNAs encode for polypeptides which may not have any common region. PMID- 3020508 TI - Butyrate induced accumulation of a 2.3 kb polyadenylated H1(0) histone mRNA in HeLa cells. AB - Sodium butyrate was used to induce the accumulation of human H1(0) mRNA in HeLa cells. The length of this mRNA (2,300 nucleotides) was determined by Northern blot hybridization and S1 nuclease analysis using a human H1(0) gene probe. The mRNA shows long 5' and 3' non coding segments and it is polyadenylated. The signal for this step of mRNA maturation (cleavage and polyadenylation) appears to be the hexanucleotide AAUAAA in analogy to most (other than histone) mRNA species. Thus, the mode of maturation of H1(0) mRNA differs, on one hand, from that of the cell cycle dependent mRNA species, where it is based on a specific stem-and-loop structure. On the other hand, the 3' end of H1(0) mRNA varies from H5 mRNA, which is characterized by two unique dyad symmetry structures at its 3' end. PMID- 3020509 TI - Site-specific recombination and topoisomerization by Tn21 resolvase: role of metal ions. AB - The resolvase from the transposon Tn21 catalyses site-specific recombination between the two res sites on its DNA substrate both in the absence and presence of Mg2+ ions. This contrasts with reports on the resolvase from gamma-delta (Tn1000) and on other recombinational proteins that are homologous to Tn21 resolvase but which need Mg2+ for their activity. Magnesium ions could enhance the activity of Tn21 resolvase, as did a number of other cations but some metal ions such as Ni2+ inhibit recombination. The metal ions are not directly involved in the catalytic process but probably affect recombination by altering the conformation of the DNA. Tn21 resolvase relaxes its DNA substrate in the presence and in the absence of Mg2+, and also in ionic conditions that inhibit recombination. Hence, the topoisomerization reflects an activity of resolvase that is distinct from recombination. However, the two activities are functions of the same DNA-protein complex. The complex contains about 6 molecules of the resolvase dimer per molecule of DNA. PMID- 3020511 TI - Genomic analysis II: isolation of high molecular weight heteroduplex DNA following differential methylase protection and Formamide-PERT hybridization. AB - Understanding the nature of DNA sequence differences among individuals is important to the understanding of fundamental questions in biology. To analyze such differences in complex genomes new approaches must be developed. We report two new techniques which aid in this effort. First, we have developed a modification of the Phenol Emulsion Reassociation Technique (PERT) that allows hybridization of long (20 kb and longer) single copy heteroduplex DNA fragments from human genomic DNAs. Secondly, by using a differential methylase protection technique we have shown that double methylase resistant heteroduplex DNA molecules can be size fractionated away from reannealed single methylase resistant homoduplex DNA molecules. These methods will be useful in obtaining DNA from chromosomal subregions linked to the inheritance of a specific trait or condition as described in the preceding paper and could also be used to create a map of the chromosomal subregion which includes the gene for the trait. PMID- 3020510 TI - Methylation and restriction endonuclease cleavage of linear Z-DNA in the presence of hexamminecobalt (III) ions. AB - These studies employed the synthetic linear DNA, poly dGdC, in the B and cobalt hexammine chloride (Co)-induced Z form to determine the effect of conformation on protein-DNA interactions. The rate of the reaction of the restriction endonucleases, Hha I and Cfo I, are reduced with Z DNA as compared to B DNA. The ability of both restriction endonucleases to react with an aggregate form of Z DNA (Z* DNA) is found to depend upon how the Z* DNA is formed. When Z* DNA is induced by low concentrations of Co (50 microM), the endonucleases remain active. In the presence of 100 microM Co, which causes increased aggregation, the endonucleases are inactive. The Hha I DNA methyltransferase reacts at equal rates with the B, Z and low cobalt Z* forms and at a greatly reduced rate with the high cobalt Z* form. These results are significantly different than those observed with Z form dGdC tracts inserted into circular DNA molecules. PMID- 3020512 TI - RecA independent recombination of poly[d(GT)-d(CA)] in pBR322. AB - Short sequence tracts composed of alternating guanosine and thymidine nucleotide residues poly[d(GT)-d(CA)] carried in a derivative of pBR322 were recombinogenic in a recA host. Recombination brought about by poly[d(GT)-d(CA)] tracts displayed two interesting properties: (i) the reaction was quasi-sequence-specific in that while recombination usually occurred between two poly[d(GT)-d(CA)] tracts, recombination also occurred between sequences bordering the dinucleotide repeats. (ii) recombination was enhanced when two poly[d(GT)-d(CA)] tracts were clustered within 250 base pairs of each other, but not when the repeats were separated by 3 kilobase pairs. The mechanism by which poly[d(GT)-d(CA)] stimulated recombination remains to be determined, but the behavior of these sequences is consistent with the idea that general recombination in E. coli may involve formation of Z-DNA. PMID- 3020513 TI - The 52-protein subunit of T4 DNA topoisomerase is homologous to the gyrA-protein of gyrase. AB - T4 gene 52 encodes one of the three subunits of T4 DNA topoisomerase. The T4 enzyme is required for normal phage DNA replication. I have cloned the entire gene, and it is expressed in uninfected E. coli cells. The sequence of 1966 nucleotides of T4 deletion delta sa9 surrounding gene 52 has been determined. The reading frame of the gene was established by identifying the first ten amino acids in the large open reading frame derived from the DNA sequence as those at the amino-terminus of the purified 52-protein. Based on the DNA sequence, 52 protein has 441 amino acids and a calculated peptide molecular weight of 50,583 daltons. This T4 topoisomerase subunit shares significant amino acid sequence homology with the gyrA subunit of bacterial gyrases and the carboxyl-half of yeast topoisomerase II in spite of the large differences in their sizes, confirming their functional equivalence in type II enzyme directed DNA topoisomerization. Amino acid sequence homology is highest in the amino-terminal portions of the equivalent peptides. The homology alignment suggests a consensus sequence organization surrounding the reactive tyrosine which is used to form the transient protein-DNA intermediate in the double-stranded DNA passing reaction. The delta sa9 deletion in T4 brings gene 52 to a location 30 nucleotides 3' from the rIIB gene whose C-terminal 167 codons are also reported here. PMID- 3020514 TI - Alkyl phosphotriester modified oligodeoxyribonucleotides. V. Synthesis and absolute configuration of Rp and Sp diastereomers of an ethyl phosphotriester (Et) modified EcoRI recognition sequence, d[GGAA(Et)TTCC]. A synthetic approach to regio- and stereospecific ethylation-interference studies. AB - Protected deoxynucleoside 3'-O-ethyl-N,N-diisopropylphosphoramidite reagents were prepared for use in the automated synthesis of ethyl phosphotriester (Et) modified oligonucleotides. The title diastereomers were separated by reversed phase HPLC, and chirality at phosphorus was assigned by an improved configurational correlation scheme that was verified by NMR spectroscopic studies (accompanying paper, Part VI). This generally applicable correlation scheme involved enzymatic digestions of each diastereomer to give the corresponding diastereomer of d[A(Et)T]; phosphite triester sulfurization to obtain diastereomeric O-ethyl phosphorothioates, d[AS(Et)T], which were separated by HPLC for stereoretentive oxidation with H2O2 to give d[A(Et)T], and stereoretentive de-ethylation with PhSH-Et3N to give diastereomeric phosphorothioates, d[AST], whose configurations at phosphorus had been assigned previously. Neither the Rp-Rp nor Sp-Sp duplex, (d[GGAA(Et)TTCC])2, was cleaved by EcoRI endonuclease under conditions that led to cleavage of both the unmodified duplex, [d(GGAATTCC)]2, and the mixture of diastereomeric phosphorothioate-modified duplexes, [d(GGAASTTCC)]2. Cleavage of the latter substrates was Sp-selective. PMID- 3020515 TI - Nucleotide sequence of Rhizobium meliloti RCR2011 genes involved in host specificity of nodulation. AB - A 6 kb DNA segment of the R. meliloti 2011 pSym megaplasmid, which contains genes controlling host specificity of root hair infection and of nodulation, was cloned and sequenced. The DNA sequence analysis, in conjunction with previous genetic data, allowed identification of four nod genes designated as E, F, G and H. nodH is divergently transcribed with respect to nodFE and nodG. A conserved nucleotide sequence was found around 200 bp upstream of the translation start of nodF, nodH and nodA. This sequence is also present upstream of common nodA and species specific nodF genes of other Rhizobium species. The predicted protein products of nodF and nodG show homology with acyl carrier protein and ribitol dehydrogenase, respectively. The nodH product contains a rare sequence of four contiguous proline residues. Comparison with the nod gene products of R. leguminosarum shows that species specific nodFE products are as well conserved as those of common nodABC and nodD genes. PMID- 3020517 TI - Nucleotide sequence of a protein coding region in Chlamydomonas reinhardtii mitochondrial DNA. PMID- 3020518 TI - Difference in the nucleotide sequence of human angiotensinogen cDNA. PMID- 3020516 TI - Secretion of heterologous gene products to the culture medium of Escherichia coli. AB - Different constructs containing fragments of the Staphylococcal protein A gene have been introduced in Escherichia coli and the effect on expression and translocation of the various heterologous gene products have been studied. By reversing the orientation of the different protein A gene constructions in the plasmid vector, a dramatic 20-fold difference in expression was obtained, accompanied with secretion of the gene product to the culture medium. Similar results were obtained by "heat-shock" treatment of the E.coli host cells. These results suggest the presence in the protein A gene of a stress induced promoter, functional in E.coli. The system was used to efficiently secrete a fusion protein consisting of a protein A fragment and human insulin-like growth factor I (IGF-I) to the culture medium of E.coli HB101. The fusion protein was purified from the culture medium by IgG affinity chromatography in a one-step procedure giving more than 95% yield. PMID- 3020519 TI - Chronic Epstein-Barr virus infection in children. PMID- 3020520 TI - Recurrent herpes simplex in a neonate. PMID- 3020521 TI - Preliminary evaluation of the efficacy of rhesus rotavirus vaccine strain MMU 18006 in young children. PMID- 3020522 TI - Fatal varicella-zoster infection in a newborn treated with varicella-zoster immunoglobulin. PMID- 3020523 TI - Cytomegalovirus and neonatal resuscitation. PMID- 3020524 TI - The ACTH-(4-9) analog ORG 2766 and desglycinamide9-(Arg8)-vasopressin reverse the retrograde amnesia induced by disrupting circadian rhythms in rats. AB - Disrupting circadian organization by exposing rats to a shifted illumination schedule after training for passive avoidance and shuttle box avoidance behavior resulted in retrograde amnesia as evidenced by impaired performance during retention and extinction testing respectively. A single treatment with either the ACTH-(4-9) analog ORG 2766 or desglycinamide9-(Arg8)-vasopressin (DGAVP) 1 hour prior to the retention of passive avoidance or extinction of shuttle box avoidance behavior restored the behavioral impairment. It is suggested that these peptides may be useful to relieve memory deficits induced by disturbances in circadian organization. PMID- 3020526 TI - Pressor, tachycardic and behavioral excitatory responses in conscious rats following ICV administration of ACTH and CRF are blocked by naloxone pretreatment. AB - Experiments were conducted to compare the blood pressure and heart rate responses of conscious rats given intracerebroventricular (ICV) injections of adrenocorticotropin (ACTH 1-24) and corticotropin releasing factor (CRF). Under sodium pentobarbital anaesthesia, rats were implanted with a stainless-steel cannula into the lateral cerebral ventricle and had their right femoral artery and vein cannulated. Upon recovery (24-48 hr later) conscious, unrestrained rats were given ICV injections (total volume 5 microliter by gravity flow) of sterile saline, ACTH (1-24) (0.85 and 1.7 nmoles) or CRF (0.55 and 1.1 nmoles) and blood pressure and heart rate were monitored over the next 2 hr (from the abdominal aorta via the femoral arterial catheter). Both ACTH and CRF caused mean arterial pressure (MAP) to increase, which was paralleled with increases in mean heart rate (MHR). Moreover, these elevations in MAP and MHR were temporally associated with excessive grooming (for ACTH) and locomotor activity (for CRF), which occurred before and lasted as long as MAP and MHR were enhanced. Intravenous (IV) pretreatment whereby naloxone was given 10 min before ICV administration of ACTH (1.7 nmoles) or CRF (1.1 nmoles), showed that naloxone blocked the behavioral, pressor and tachycardic effects of both ACTH and CRF. The results demonstrate that the pressor, tachycardic and locomotor effects evoked in conscious rats by ICV administration of ACTH or CRF are antagonized by naloxone and that their hemodynamic changes may, in part, be mediated by prior behavioral activation. PMID- 3020527 TI - ACTH acts via an anterior ventral third ventricular site to elicit grooming behavior. AB - Intracerebroventricular but not parenteral application of ACTH has been shown to elicit excessive grooming behavior in rats and mice. This behavior is elicited by administration of ACTH into the lateral, third, or fourth ventricles. Plugging of the cerebral aqueduct with cold cream fails to prevent grooming in response to lateral ventricle injection of ACTH. However, cold cream plugs in the third ventricle can prevent the subsequent induction of grooming behavior by lateral ventricle injection of ACTH, but only when the plugs are located in the anterior ventral third ventricle in the region of the organum vasculosum laminae terminalis (OVLT) and median eminence. These data suggest the anterior ventral third ventricle as the periventricular site of action of ACTH in eliciting excessive grooming, although it is possible that peptides taken up in this area are transported to other regions to elicit the behavioral response. PMID- 3020525 TI - Existence of different forms of adrenocorticotropin in rat hypothalamus. AB - Adrenocorticotropin (ACTH)-like immunoreactivity and bioactivity were extracted from rat hypothalamus and fractionated by high pressure liquid chromatography. Analysis of the fractions either by radioimmunoassay or bioassay (corticosteroid production from rat adrenal cells) revealed several peaks of immunoreactivity and bioactivity. Only 20-25% of total ACTH-like immunoreactivity and bioactivity eluted with the same retention time as authentic ACTH 1-39. The results suggest that different forms of ACTH exist in rat hypothalamus. PMID- 3020528 TI - [Kinin aminopeptidase in lung tissue and activity of kininase II in the serum of patients with silicosis]. PMID- 3020529 TI - [Treatment of patients with small-cell lung cancer by the method of chemotherapy combined with Co-60 irradiation]. PMID- 3020530 TI - A mucinous carcinoma of the thyroid. AB - A primary mucinous carcinoma of the thyroid is reported. The tumour cells, arranged in small nests and trabecules, were surrounded by abundant intercellular substance which was positively stained for mucicarmine, alcian blue and PAS before and after diastase treatment. Diastase-resistant, PAS-positive material was also present in the tumour cells. Electron microscopy revealed neoplastic cells linked by desmosomes and not surrounded by basal lamina; the nuclei had irregular contours, prominent nucleoli and numerous pseudoinclusions; mucin granules and rough endoplasmic reticulum were the prominent organelles in the cytoplasm. Follicles and follicle-like structures were not found, either by light or by electron microscopy. A definite diagnosis could only be established after the disclosure of intense immunostaining for thyroglobulin in several groups of tumour cells. Criteria for the diagnosis are discussed and the question of histogenesis raised. PMID- 3020531 TI - The effect of repeated doses of (-) deprenyl on the dynamics of monoaminergic transmission. Comparison with clorgyline. AB - The action of clorgyline and (-)deprenyl on the dynamics of dopaminergic and serotonergic transmission in the rat brain was compared. It was found, that daily administration of 0.25 mg/kg sc clorgyline, a specific MAO A inhibitor, reduced the turnover rate of both dopamine and serotonin after two weeks of injections. The treatment for two or four weeks with 0.25 mg/kg sc (-)deprenyl, a specific MAO B inhibitor, enhanced the turnover rate of dopamine and the fractional rate constant of dopamine efflux, reflecting an increased utilization rate of this amine in the striatum. Beside the augmentation of the dopamine turnover rate, the dopaminergic tone was also elevated by the reduction of the dopamine uptake in the striatum. Two week injections with the same dose of (-)deprenyl did not change the dynamics of serotonergic transmission. PMID- 3020532 TI - Antagonistic effects of some metabolites and analogues of mianserin on serotonin and alpha 1- and alpha 2-adrenergic receptors in the flexor reflex model in vivo. AB - Mianserin, its two metabolites desmethylmianserin (Org OH-46) and 8 hydroxymianserin (Org GF-45), and its two analogues R(-)-6-aza-mianserin (Org 44 19) and S(+)-6-aza-mianserin (Org 44-20) were tested for their antagonistic action on the central serotonin (5-HT) receptor and alpha-adrenoceptors in the flexor reflex model in vivo in the spinal rat. The ability to reduce the increase in the flexor reflex activity induced by quipazine (a 5-HT receptor agonist), St 587 (an alpha 1-adrenoceptor agonist), or clonidine (an alpha 2-adrenoceptor agonist) was a measure of their potencies as antagonists of the respective receptors. All the compounds occurred to be potent antagonists of the central 5 HT receptor (S(+)-6-aza-mianserin was most active) and, to a lesser degree, of alpha-adrenoceptors with preferential antagonistic action on alpha 2-subtype. PMID- 3020533 TI - Frostbite and bone scanning: the use of 99m-labeled phosphates in demarcating the line of viability in frostbite victims. AB - Early diagnosis of the extent of bone and soft tissue damage is a very important step in treating a frostbite victim. The diagnostic use of Tc-99m-phosphates in assessing the viability of soft tissue and bone in frostbite was evaluated in the early post-thaw period. Four patients were treated with a combination of warm baths, rehydration, vasodilators, epidural block, fasciotomy, and debridement. Six scans were done to stage involvement. In three of the four cases, final involvement could be determined as early as the third day. When a specific level of soft tissue or bone uptake was determined, future scanning showed either improvement, or no change in three of the four patients. In our experience, Tc 99m-phosphate scans represent an improvement over other diagnostic tests for viability of tissues. PMID- 3020534 TI - Epithelial polyps of the large intestine. A classification based on biologic behavior. AB - Classifications of colonic polypoid lesions are often based on their pathogenesis. Little attention is paid to the biologic behavior (or predicted behavior, based on experience) of these lesions. Classification based on biologic behavior separates these lesions, regardless of histogenesis, according to possible malignant potential. This helps the clinician to explain the implications of the histopathologic diagnosis to the patient and to understand when aggressive or prophylactic therapy should be pursued. PMID- 3020536 TI - Nuclease activity of 1,10-phenanthroline-copper: sequence-specific targeting. AB - The nuclease activity of 1,10-phenanthroline-copper ion can be targeted to specific DNA sequences by attachment of the ligand to the 5' end of complementary deoxyoligonucleotides via a phosphoramidate linkage. To synthesize the adduct, the phosphorimidazolide of the deoxyoligonucleotide is prepared using a water soluble carbodiimide and is then coupled to 5-glycylamido-1,10-phenanthroline. After hybridization to the target DNA, sequence-specific cleavage is observed upon the addition of cupric ion and 3-mercaptopropionic acid. Two methods of assaying the cutting of the operator sequence of the lac operon have been employed using the oligonucleotide 5'-AATTGTTATCCGCTCACAATT-3' representing sequence positions 21-1 of the template strand. In the first, the single-stranded DNA of the phage M13mp8 was the target, and cuts were detected using a primer extension assay. In the second, the substrate was an EcoRI fragment 3' labeled in the nontemplate strand. After denaturation and reannealing to the oligonucleotide 1,10-phenanthroline adduct, cupric ion and 3-mercaptopropionic acid were added, and the products were analyzed directly on a sequencing gel. With the phenanthroline moiety attached to position 21 of the oligonucleotide carrier, cutting was observed at positions 20-25 using both assays. PMID- 3020537 TI - ATP-independent type II topoisomerase from trypanosomes. AB - We have characterized in Trypanosoma cruzi a DNA topoisomerase capable of decatenating complex trypanosomal kinetoplast DNA networks in the absence of ATP. The enzymatic activity requires Mg2+ and K+. Using a defined DNA topoisomer we showed that the linking number changes by steps of 2, which characterizes the enzyme as a type II topoisomerase. The enzyme can catenate supercoiled DNA molecules, unknot DNA, and cleave double-stranded DNA. The enzyme has no ATPase activity. The native enzyme has an Mr of about 200,000. Crude extracts and partially purified fractions contain an aggregating factor that can substitute spermidine in catenating reactions. Because of the presence of this factor, the kinetoplast DNA can only be decatenated by purified fractions. The enzyme is inhibited by certain drugs and provides a potential target for chemotherapy. Such an enzyme was also characterized in Trypanosoma equiperdum. PMID- 3020535 TI - [Effect of hydrocortisone on the activity of angiotensin-converting and renin like enzymes and kininase I in the brain and hypophysis of rats]. AB - The paper is concerned with a study of the effect of an excess of corticosteroids in the body, resulting from single and multiple hydrocortisone administration to rats, on the activity of enzymes involved in the pathway of the renin-angiotensin and kinin systems in different parts of the brain and hypophysis. Hydrocortisone administration to rats resulted in an increase in the activity of the angiotensin converting enzyme in the hypothalamus, hippocamp, striate body and hypophysis, an increase in the renin-like activity in the hypothalamus and hippocamp and its decrease in the striate body. The nature of changes in kininase I activity after the hormone administration was different in the examined parts of the brain. The results obtained indicated that one of the links in the mechanism of corticosteroid action on the renin-angiotensin and kinin system of the brain was the effect of these hormones on the activity of the renin-like, angiotensin converting enzymes and kininase I. PMID- 3020538 TI - Expression of the complete human T-cell leukemia virus type I pX coding sequence as a functional protein in Escherichia coli. AB - Human T-cell leukemia virus type I (HTLV-I), a virus associated with adult T-cell leukemia, contains a long open reading frame (LOR) in the 3' end of its genome between the env region and the 3' long terminal repeat (LTR). This open reading frame encodes a 40-kDa protein (designated p40x) that has been implicated as a positive control element for transcription from the HTLV-I LTR in a phenomenon known as trans-activation. We now report the expression of the complete p40x coding sequence as a 40-kDa protein in Escherichia coli. The p40x protein produced in bacteria is shown, using the protoplast fusion technique, to possess biological activity by its ability to trans-activate a HTLV-I LTR-chloramphenicol acetyltransferase plasmid that is stably integrated into the genome of mouse L cells. This stimulatory activity could be detected within 2 hr after fusion, suggesting the possibility of a direct role for p40x in trans-activation of the HTLV-I LTR. The production of p40x in large quantities in E. coli, together with the rapid protoplast fusion assay for its biological activity, should facilitate the analysis of p40x mutants and the elucidation of the molecular mechanism of trans-activation. PMID- 3020539 TI - Human melanoma cell lines of primary and metastatic origin express the genes encoding the chains of platelet-derived growth factor (PDGF) and produce a PDGF like growth factor. AB - Normal human melanocytes and five human melanoma cell lines were analyzed for production of platelet-derived growth factor (PDGF)-like activity. Three of the melanoma cell lines released an activity that inhibited binding of 125I-labeled PDGF to human foreskin fibroblasts and stimulated [3H]thymidine incorporation in such cells. These activities were inhibited by the addition of anti-PDGF antibodies. All three factor-producing cell lines were derived from the same patient--one originated from the primary tumor (WM 115), and two were from individual lymph-node metastases (WM 239A and WM 266-4). The factor produced by WM 266-4 cells was characterized biochemically in detail. Immunoprecipitated, the metabolically labeled factor migrated in NaDod-SO4/gel electrophoresis as a homogeneous Mr 31,000 species, which under reducing conditions was resolved into two species of Mr 16,500 and Mr 17,000, implying a dimeric structure of the molecule. The factor was purified to homogeneity. Analysis by reverse-phase high pressure liquid chromatography of reduced and alkylated factor revealed an elution pattern identical to that of PDGF A chains. Thus, the native molecule appears to be a homodimer of PDGF A chains. Blot-hybridization analysis of poly(A)+ RNA from the cell lines with 32P-labeled PDGF A chain and B chain (SIS product) cDNA probes revealed a relative abundance of B chain transcripts in the cell line originating from the primary tumor tissue only but expression of A chain in all three cell lines. We conclude that the two structural genes encoding each of the subunit chains of PDGF can be expressed in human melanoma cells and that the two genes can be independently expressed in such cells. PMID- 3020541 TI - Cloning, characterization, expression, and chromosomal localization of a human ferritin heavy-chain gene. AB - A genomic phage clone containing a full-length copy of a functional human gene for ferritin heavy chain has been isolated. The gene consists of four exons spanning approximately 3 kilobases and has been localized to chromosome 11. The functionality of the gene was demonstrated by the fact that both transient transfectants and stable transformants of murine fibroblasts actively transcribe human ferritin heavy-chain mRNA. PMID- 3020540 TI - Phospholipase A2 and phospholipase C are activated by distinct GTP-binding proteins in response to alpha 1-adrenergic stimulation in FRTL5 thyroid cells. AB - In FRTL5 rat thyroid cells, norepinephrine, by interacting with alpha 1 adrenergic receptors, stimulates inositol phosphate formation, through activation of phospholipase C, and arachidonic acid release. Recent studies have shown that GTP-binding proteins couple several types of receptors to phospholipase C activation. The present study was undertaken to determine whether GTP-binding proteins couple alpha 1-adrenergic receptors to stimulation of phospholipase C activity and arachidonic acid release. When introduced into permeabilized FRTL5 cells, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]), which activates many GTP-binding proteins, stimulated inositol phosphate formation and arachidonic acid release. Neomycin inhibited GTP[gamma-S]-stimulated inositol phosphate formation but was without effect on GTP[gamma-S]-stimulated arachidonic acid release, suggesting that separate GTP-binding proteins mediate each process. In addition, pertussis toxin inhibited norepinephrine-stimulated arachidonic acid release but not norepinephrine-stimulated inositol phosphate formation. Norepinephrine-stimulated arachidonic acid release but not inositol phosphate formation was also inhibited by decreased extracellular calcium and by TMB-8, suggesting a role for a phospholipase A2. To confirm that arachidonic acid was released by a phospholipase A2, FRTL5 membranes were incubated with 1-acyl-2 [3H]arachidonoyl-sn-glycero-3-phosphocholine. GTP[gamma-S] slightly stimulated arachidonic acid release, whereas norepinephrine acted synergistically with GTP[gamma-S] to stimulate arachidonic acid release. The results show that phospholipase C and phospholipase A2 are activated by alpha 1-adrenergic agonists. Both phospholipases are coupled to the receptor by GTP-binding proteins. That coupled to phospholipase A2 is pertussis toxin-sensitive, whereas that coupled to phospholipase C is pertussis toxin-insensitive. PMID- 3020542 TI - Identification and purification of a bacterial ice-nucleation protein. AB - The protein product of a gene (inaZ) responsible for ice nucleation by Pseudomonas syringae S203 has been identified and purified after overexpression in Escherichia coli. The amino acid composition and the N-terminal sequence of the purified, denatured protein corresponded well with that predicted from the sequence of the inaZ gene. The product of inaZ was also found to be the major component in preparations of ice-nucleating, proteinaceous particles, obtained after extraction with and gel filtration in a mixture of urea and the nondenaturing detergent octyl beta-D-thioglucopyranoside. The activity of these preparations in the absence of added lipid implies that the protein participates directly in the nucleation process. PMID- 3020543 TI - Production of platelet-derived growth factor-like molecules by cultured arterial smooth muscle cells accompanies proliferation after arterial injury. AB - The migration and proliferation of smooth muscle cells (SMCs) within the intima of arteries following mechanical injury is thought to be initiated by vessel wall injury and release of growth factors, in particular the platelet-derived growth factor (PDGF). However, the mechanism by which SMC proliferation is regulated after platelet interaction with the vessel wall has ceased is unknown. Here we show that SMCs derived from the intima of injured rat arteries (intimal SMCs) are phenotypically distinct from SMCs from unmanipulated vessels (medial SMCs). Intimal SMCs secrete 5-fold greater amounts of PDGF-like activity into conditioned medium in culture, have fewer receptors for 125I-labeled PDGF, and are not mitogenically stimulated by exogenous purified PDGF. This study demonstrates that two SMC phenotypes can develop in the adult rat artery and suggests that SMC proliferation in vivo may be controlled, in part, by SMCs that produce PDGF-like molecules. PMID- 3020544 TI - Multiple chromosomal rearrangements in a spontaneously arising t(6;7) rat immunocytoma juxtapose c-myc and immunoglobulin heavy chain sequences. AB - Spontaneously arising immunocytomas in Lou/Wsl rats contain a consistent translocation between chromosomes 6 and 7. The c-myc gene has been localized to chromosome 7 and has been shown to be rearranged in the majority of the rat immunocytomas. We now report the cloning of the rearranged 11-kilobase EcoRI c myc fragment from the IgE-secreting IR75 tumor. Sequence analysis revealed that the cytogenetically visible t(6;7) translocation must have involved several events in this tumor. One event has led to the juxtaposition of c-myc and the switch mu region, in a head-to-head orientation. The breakpoint is approximately 850 base pairs upstream from the proximal c-myc promoter on chromosome 7. This area is distinct from the more common mouse plasmacytoma- and Burkitt lymphoma associated translocation breakpoints and also differs from the known murine retroviral insertion sites. A second rearrangement has led to the transposition of sequences upstream from the switch gamma 1 region to the c-myc-distant end of the switch mu region, tail-to-tail. This requires at least two events, including one inversion. In addition to showing that identical loci (c-myc, immunoglobulin) are juxtaposed via chromosomal translocations in three different tumors (Burkitt lymphoma, mouse plasmacytoma, and rat immunocytoma) in different species (human, mouse, and rat), the multiple rearrangements in IR75 and some other tumors emphasize the selective value of c-myc activation by an immunoglobulin locus in the tumorigenic process. PMID- 3020546 TI - Vasoactive intestinal peptide induces the synthesis of the cholesterol side-chain cleavage enzyme complex in cultured rat ovarian granulosa cells. AB - Vasoactive intestinal peptide (VIP) has been identified in ovarian nerves and stimulates steroid secretion from immature ovaries. To gain insight into its mechanism of action, the effect of VIP on the synthesis of the cholesterol side chain cleavage enzyme complex was studied in ovarian granulosa cells from immature estrogen-primed rats. The cells were cultured for 48 hr in serum-free medium; the proteins were labeled with [35S]methionine; and the synthesis of cytochrome P-450, iron-sulfur protein, and NADPH:iron-sulfur protein reductase was evaluated by electrophoretic analysis after immunoisolation with polyclonal antibodies directed against the bovine adrenal enzymes. VIP at concentrations ranging from 0.001 to 1 microM stimulated 3- to 5-fold the synthesis of cytochrome P-450 and iron-sulfur protein. Peptide NH2-terminal histidine, COOH terminal isoleucine, which has greater than 50% sequence homology of VIP, stimulated the synthesis of both proteins at approximately 50% of VIP effectiveness. Secretin, another member of the glucagon-secretin family of peptides, which has only 30% sequence homology to VIP, was without effect. Similar results were observed with the NADPH:iron-sulfur protein reductase. VIP induced synthesis of the cholesterol side-chain cleavage enzyme complex was accompanied by a dose-related increase in cAMP accumulation and progestin formation. It is concluded that VIP regulates the synthesis of the ovarian cholesterol side-chain cleavage enzyme complex, which catalyzes the rate-limiting reaction in progesterone biosynthesis, and that the VIP effect is at least partially mediated through cAMP. It is suggested that a stimulatory action of VIP on the synthesis of ovarian progesterone may contribute to regulating the functional development of the ovary. PMID- 3020545 TI - Expression of T-cell-activating protein in peripheral lymphocyte subsets. AB - T-cell-activating protein (TAP) is an allelic 12-kDa membrane protein that participates in T-cell activation. Soluble anti-TAP monoclonal antibodies can trigger antigen-specific, major histocompatibility complex-restricted T-cell hybridomas to produce interleukin 2 and are mitogenic for normal T cells and thymocytes. TAP is expressed on 10% of thymocytes, which are mainly cortisone resistant and mature. In the periphery, TAP is expressed on 70% of resting T cells but not on resting B cells. In this report, we analyze in detail the nature of TAP expression on peripheral lymphocyte subsets by immunofluorescence techniques. We show that all inducer (L3T4+) T cells are TAP+. In contrast, only 50% of Lyt-2+ T cells express detectable TAP. Functional studies demonstrated that at least part of the heterogeneity of TAP expression is present in the Lyt 2+ cytolytic T-cell (CTL) subset. Unstimulated CTL precursors are TAP- but are induced to express TAP in the effector state. Furthermore, this reflects actual synthesis of TAP, as TAP is detectable on activated Lyt-2+ CTLs passaged in vitro under conditions where passive acquisition can be ruled out. To extend this observation, we have studied the expression of TAP on activated T and B cells. Upon activation, all T and B cells became TAP+. Furthermore, the TAP molecules on B and T cells are indistinguishable by NaDodSO4/polyacrylamide gel electrophoresis. This suggests that TAP expression defines further heterogeneity of lymphocytes, with activation being one parameter influencing its expression. PMID- 3020547 TI - Identification and properties of N-methyl-D-aspartate receptors in rat brain synaptic plasma membranes. AB - The excitatory amino acid receptors selectively activated by N-methyl-D-aspartate (N-Me-D-Asp) (also known as NMDA) are a major determinant of central nervous system neuronal excitability. We report here that rat brain synaptic plasma membranes contain a distinct population of L-[3H]glutamate binding sites with pharmacological properties indicative of the N-Me-D-Asp receptor. The N-Me-D-Asp sites are readily distinguished from other L-[3H]glutamate binding and uptake sites by their sharp pH optimum, more rapid association rate, preferential localization in synaptic structures, and lack of dependence on temperature and inorganic ions. As with other receptor systems, ligand binding at the N-Me-D-Asp site is reduced by guanine nucleotides but not by adenosine nucleotides. Binding is insensitive to ketamine and cyclazocine, indicating that sigma opiates inhibit N-Me-D-Asp excitation at a site different from that of the N-Me-D-Asp binding site. The quantitative pharmacological properties of N-Me-D-Asp-sensitive L [3H]glutamate binding sites determined in a well-defined dendritic field (stratum radiatum of CA1) by quantitative autoradiography closely correlate to those of both the electrophysiologically identified N-Me-D-Asp receptors in the same dendritic field and the N-Me-D-Asp sites studied in membrane preparations. Under conditions that selectively reveal N-Me-D-Asp receptors, these sites are found to exhibit considerable anatomical specificity as evidenced by variations within cortical, striatal, and thalamic regions. Autoradiography also showed that regions in rodent and primate brain that are especially sensitive to anoxic and excitotoxic neuronal damage (e.g., Sommer's sector or CA1) have a high level of N Me-D-Asp sites. Since N-Me-D-Asp receptors are known to contribute to these causes of neuronal loss, their selective distribution partially accounts for the pattern of selective damage seen in these pathological conditions. PMID- 3020548 TI - Cloning and expression of cDNA for human diazepam binding inhibitor, a natural ligand of an allosteric regulatory site of the gamma-aminobutyric acid type A receptor. AB - Diazepam binding inhibitor (DBI) is a protein that displaces ligands bound to the beta-carboline/benzodiazepine recognition site, an allosteric modulatory site of the type A gamma-aminobutyric acid receptor complex. An incomplete rat cDNA clone coding for DBI was isolated. This rat sequence was utilized to identify a cDNA clone that encoded the entire 104 residues of human DBI. This sequence was engineered for expression in E. coli, and recombinant DBI exhibits identical biochemical and antigenic characteristics of natural human DBI. DBI is encoded by a multigene family of at least five members, but a single gene appears to account for the majority of DBI expression. DBI is expressed in a tissue-specific manner. Expression is found in central nervous system tissues and appears to extend to peripheral tissues rich in the peripheral type of high-affinity benzodiazepine recognition sites. The role of these sites and DBI in adrenal gland, testis, and kidney remains to be determined. PMID- 3020549 TI - Endogenous inhibitor of nonlysosomal high molecular weight protease and calcium dependent protease. AB - An endogenous inhibitor of high molecular weight protease was purified from human erythrocytes and partially characterized. The inhibitor was isolated by DEAE Sephacel ion-exchange chromatography followed by separation on a Bio-Gel A-0.5m column. The inhibitor displayed a native Mr of 240,000 and contained a single subunit of Mr 40,000 after NaDodSO4/polyacrylamide gel electrophoresis. The Mr 240,000 hexamer inhibited high molecular weight protease noncompetitively (Ki = 8.3 X 10(-8) M) and showed marked susceptibility to proteolytic digestion and heat treatment. The purified factor was also a potent inhibitor of calcium dependent protease (Ki = 2.8 X 10(-8) M), whereas it had no effect on trypsin, chymotrypsin, or papain. Heat treatment (50-70 degrees C X 10 min) caused loss of inhibition against high molecular weight protease; however, inhibition of calcium dependent protease was stable under the same conditions. This result is consistent with different domains on the inhibitor that interact with high molecular weight protease and calcium-dependent protease. Together with earlier studies in which repression of inhibitor by an ATP-ubiquitin-dependent process was proposed, the present results suggest a general mechanism for regulation of multiple nonlysosomal proteases that are complexed with endogenous inhibitors. PMID- 3020550 TI - On the spin trapping and ESR detection of oxygen-derived radicals generated inside cells. AB - Recently several attempts to identify oxygen-derived radicals in whole cells by spin trapping and electron spin resonance have been reported by using 5,5 dimethyl-1-pyrroline-N-oxide as the spin trap. In the present study, the feasibility of this method is examined. Chinese hamster V79 cells and human erythrocytes served as the test systems, while OH radicals were generated by gamma radiolysis. Several spin traps were used to scavange the radicals and a distinction between exo- and endocellular ESR observable species was achieved using tri(oxalato) chromiate(III) as a line broadening agent. To distinguish between exo- and endocellular sites of radical formation, we studied the effects of high molecular weight scavengers (polyethylene glycols), which do not enter the cell. Various possible obstacles associated with trapping and detecting the radicals inside the cells were examined. The results indicate that the primary radicals react with the spin traps. However, these spin adducts decayed within the cells. Cellularly induced decay of 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl radical presented the major difficulty in detecting the endogenous radicals, and potential experimental approaches to overcome this difficulty are discussed. PMID- 3020551 TI - Evidence that a human soluble beta-galactoside-binding lectin is encoded by a family of genes. AB - Two cDNA clones were isolated by immunoscreening a human hepatoma cDNA library with an antiserum that bound specifically to a human soluble beta-galactoside binding lectin with Mr of approximately 14,000. The deduced amino acid sequences of the inserts of these two clones show considerable homology with each other, the sequence of chicken skin beta-galactoside-binding lectin, and eight peptides derived from purified human lung lectin of Mr approximately 14,000. However, the sequence differences between the two hepatoma clones as well as among each clone and the lung peptides suggest that at least three variants of the gene encoding this lectin are expressed in human tissue. PMID- 3020553 TI - Availability of chemosensory receptors is down-regulated by habituation of larvae to a morphogenetic signal. AB - Larvae of the gastropod mollusc Haliotis rufescens are induced to settle from the plankton and metamorphose in response to exogenous gamma-aminobutyric acid (GABA) and a number of GABA-mimetic compounds, including a GABA-mimetic inducer uniquely associated with the surfaces of the naturally recruiting algae. Previous evidence has shown that recognition of these inducers is mediated by specialized chemosensory receptors on the larval epithelium and that transduction of the morphogenetic signal then is mediated by cAMP and excitatory depolarization. We demonstrate here the specific and saturable labeling of a population of larval receptors with the GABA analog beta-(p-chlorophenyl)-[3H]GABA ([3H]baclofen); identification of these labeled receptors with those controlling metamorphosis is suggested by four independent criteria: the effectiveness of GABA and its close structural analogs to induce metamorphosis is closely correlated with the effectiveness of these compounds to compete for binding to this receptor; the natural inducer purified from the recruiting algae competes for binding to this receptor; (-)-[3H]baclofen specifically bound to the receptors is shed from the larvae after approximately 20 hr, at the time corresponding to the metamorphic abscission and shedding of sensory cilia and other structures from the larvae; and the availability of the receptors for labeling and the ability of the larvae to respond to GABA and GABA analogs can be down-regulated in parallel by habituation of the larvae early in their development. These down-regulated larvae are fully capable of settlement and metamorphosis in response to agents that elevate intracellular cAMP or depolarize the chemosensory membrane, confirming that down-regulation is confined to the receptors, with no effect on the postreceptor pathway. The results reported here thus suggest that the sensitivity of marine invertebrate larvae to morphogenetic stimuli from the environment can be down-regulated by reduction in the number of chemosensory receptors available for interaction with the molecules that induce settlement and metamorphosis. In this respect, chemosensory receptors for environmental and morphogenetic signals are demonstrated biochemically to respond to habituation in a similar manner to neuronal and hormonal receptors. PMID- 3020552 TI - Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: localized unwinding of duplex DNA by a six-protein reaction. AB - The O protein of bacteriophage lambda localizes the initiation of DNA replication to a unique site on the lambda genome, ori lambda. By means of electron microscopy, we infer that the binding of O to ori lambda initiates a series of protein addition and transfer reactions that culminate in localized unwinding of the origin DNA, generating a prepriming structure for the initiation of DNA replication. We can define three stages of this prepriming reaction, the first two of which we have characterized previously. First, dimeric O protein binds to multiple DNA binding sites and self-associates to form a nucleoprotein structure, the O-some. Second, lambda P and host DnaB proteins interact with the O-some to generate a larger complex that includes additional DNA from an A + T-rich region adjacent to the O binding sites. Third, the addition of the DnaJ, DnaK, and Ssb proteins and ATP results in an origin-specific unwinding reaction, probably catalyzed by the helicase activity of DnaB. The unwinding reaction is unidirectional, proceeding "rightward" from the origin. The minimal DNA sequence competent for unwinding consists of two O binding sites and the adjacent A + T rich region to the right of the binding sites. We conclude that the lambda O protein localizes and initiates a six-protein sequential reaction responsible for but preceding the precise initiation of DNA replication. Specialized nucleoprotein structures similar to the O-some may be a general feature of DNA transactions requiring extraordinary precision in localization and control. PMID- 3020554 TI - all-trans-Retinal stimulates superoxide release and phospholipase C activity in neutrophils without significantly blocking protein kinase C. AB - all-trans-Retinal was previously shown to stimulate high levels of superoxide release by guinea pig neutrophils. When the cells, previously labeled with [3H]inositol, are treated with all-trans-retinal, they exhibit a decrease in the levels of [3H]inositol phospholipids and an increase in the accumulation of [3H]inositol phosphates. The maximal accumulation of inositol phosphates and the optimal rate of superoxide release occurred together at approximately 7 min after stimulation. The levels of [3H]inositol phosphates accumulated were comparable to those observed when the cells were stimulated with a chemotactic peptide. In direct measurements, using concentrations that stimulate intact cells maximally, all-trans-retinal was found not to inhibit protein kinase C from the cytosol of neutrophils significantly. This contrasts with the situation with this kinase obtained from other sources. These observations represent additional effects of vitamin A on cells. PMID- 3020555 TI - Development of the smooth muscle foam cell: uptake of macrophage lipid inclusions. AB - A possible mechanism for the formation of smooth muscle foam cells in the atherosclerotic lesion was explored. Cultured macrophages (J774 cell line) were induced to form cytoplasmic cholesteryl ester inclusions by exposure to acetylated low density lipoprotein in the presence of cholesterol-rich phospholipid dispersions. The macrophages were disrupted by brief sonication, and the inclusions were isolated by flotation. When these inclusions were placed in direct contact with cultured smooth muscle cells, cellular uptake of the inclusions in a time- and dose-dependent manner was observed. Light and electron microscopy indicated the presence of lipid inclusions throughout the cytoplasm of the cells. Uptake of inclusion lipid by the smooth muscle cells was inhibited by several metabolic inhibitors, indicating that the process is dependent on metabolic activity. A modest but significant hydrolysis of the cholesteryl ester was observed, showing that the stored cholesteryl esters are metabolically available. PMID- 3020556 TI - Enhancement of galactose/N-acetylgalactosamine receptor activity on the surface of freshly isolated rat hepatocytes: evidence for masking of receptor sites by inhibitors derived from collagenase preparations. AB - Rat hepatocytes prepared by collagenase perfusion of the liver are known to exhibit increased binding of asialoorosomucoid (ASOR) after prior treatment with EDTA or after warming at 37 degrees C. The cause of the apparent increase in the surface binding activity of the galactose/N-acetylgalactosamine (Gal/GalNAc) receptor on freshly isolated rat hepatocytes was investigated. Binding experiments using three different galactose-terminated ligands revealed up to a 2 to 6-fold increase in the level of surface receptor sites on rat hepatocytes upon prior incubation at 4 degrees C with 10 mM GalNAc or 10 mM EDTA or at 37 degrees C compared to untreated cells. With digitonin-permeabilized cells, it was shown that the newly exposed receptor sites most likely originated from masked surface receptor sites, as no alteration in the size of the internal pool of receptor was observed. Collagenase preparations were found to inhibit the binding of 125I-labeled ASOR to the Gal/GalNAc receptor. Exposure of hepatocytes to collagenase resulted in a significant decrease in 125I-labeled ASOR binding, which was reversible upon treatment with GalNAc or EDTA at 4 degrees C or upon warming at 37 degrees C. Perfusion of EDTA through the isolated whole liver at 0 2 degrees C to remove any possible bound endogenous ligands did not result in a significant increase in the level of 125I-labeled ASOR binding, while perfusion of collagenase caused a marked decrease in the binding activity of the liver. We conclude that the enhancement of Gal/GalNAc receptor activity on the surface of freshly isolated hepatocytes by temperature and EDTA is potentially an artifact of the collagenase perfusion method. PMID- 3020558 TI - Codon insertion mutants of the adenovirus terminal protein. AB - A series of codon insertion mutants was isolated following restriction site directed linker insertion mutagenesis of the open reading frame for the type 5 adenovirus terminal protein precursor. The conditionally lethal mutant H5sub100 bears an insertion mutation upstream of the first AUG in the reading frame, fails to replicate its DNA under nonpermissive conditions, and was assigned to the terminal protein complementation group. These data establish that terminal protein is an essential polypeptide required for DNA replication in vivo and indicate that the NH2-terminal region of the precursor is encoded in an upstream mRNA leader. The extended eclipse period of the viral replication cycle in H5in179-infected cells is probably a consequence of delayed onset of DNA replication. Analysis of DNA replication in coinfections with wild-type virus shows that the in179 mutation has cis and trans effects. The trans-dominant, negative-complementing in179 terminal protein precursor inhibits wild-type DNA replication in a dose-dependent manner. Replication of parental in179 templates is not stimulated by an excess of coinfecting wild-type virus, indicating that the mutant terminal protein covalently bound to the in179 template in some way interferes with the replication of that template. The implications of these results for the structure and function of the terminal protein are discussed. PMID- 3020557 TI - Differential effects of transforming growth factor type beta on the growth and function of adrenocortical cells in vitro. AB - Transforming growth factor type beta (TGF-beta) suppresses basal as well as corticotropin (ACTH)-stimulated steroid formation by bovine adrenocortical cells in culture. The effect is dose dependent and is not accompanied by any change in adrenocortical cell growth. The minimum effective dose of TGF-beta is 4 X 10(-13) M (10 pg/ml), and maximal inhibition is observed at a concentration of 4 X 10( 11) M (1 ng/ml). A 16- to 20-hr incubation with TGF-beta is required to decrease steroidogenesis, and 12-18 hr are required before cells treated with TGF-beta recover complete responsiveness to corticotropin. Increases in cAMP mediated by corticotropin, forskolin, and isobutylmethylxanthine are not modified by the addition of TGF-beta; thus adenylate cyclase activity is unaffected by TGF-beta. Although TGF-beta inhibits the formation of all of the delta 4-steroids measured (including cortisol, corticosterone, aldosterone, and androstenedione), its effect can be completely reversed by the addition of 25-hydroxycholesterol, pregnenolone, or progesterone to the cells. In contrast, the addition of low density lipoprotein has no effect suggesting that TGF-beta targets the conversion of cholesterol precursors to cholesterol. The results demonstrate a highly potent effect of TGF-beta on the differentiated function of the adrenocortical cell. The inhibition of steroidogenesis can be dissociated from any effect on cell proliferation, and it occurs distal to the formation of cAMP but proximal to the formation of cholesterol. The results suggest that in the adrenal, TGF-beta or TGF-beta-like proteins may be playing an important role in modifying the differentiated state of the adrenocortical cell. PMID- 3020559 TI - A 24-base-pair DNA sequence from the MAT locus stimulates intergenic recombination in yeast. AB - HO nuclease is a site-specific double-strand endonuclease present in haploid Saccharomyces cerevisiae undergoing mating type interconversion. HO nuclease initiates mating type interconversion by making a double-strand break within the MAT locus. To define the recognition site for the enzyme in vitro, we have constructed a number of point mutations and deletions within or adjacent to the HO recognition site. Digestion of these substrates with HO in vitro reveals that the minimal recognition site is 18 base pairs long, although several shorter substrates and substrates containing point mutations are cleaved at low levels in vitro. A 24-base-pair HO recognition site stimulates homologous recombination when present in a region unrelated to MAT. Recombinants arise from both gene conversion and crossover events. The identification of the HO recognition site provides a way of introducing a defined initiation site for recombination. PMID- 3020560 TI - Transformation of a human poliovirus receptor gene into mouse cells. AB - The first step in poliovirus replication is binding of virus to a cellular receptor. Mouse L cells, which are resistant to poliovirus infection because they do not bear a poliovirus receptor, were transformed with HeLa cell (human) DNA to poliovirus sensitivity at a frequency of approximately 1 in 50,000 transformants. Monoclonal antibody directed against the HeLa cell poliovirus receptor site was used in rosette assays to identify poliovirus-sensitive L-cell transformants in a background of L-cell tk+ transformants. A cloned cell line, CM-1, was isolated that displayed a surface component recognized by the anti-poliovirus receptor antibody. CM-1 cells were susceptible to infection with all three poliovirus serotypes, and infection could be blocked by the antireceptor antibody. Poliovirus formed plaques in CM-1 and HeLa cells with equal efficiency. CM-1 and HeLa cells produced infectious poliovirus at a similar rate, although yield of virus in CM-1 cells was about 33% less than the yield in HeLa cells. These results suggest that DNA encoding the HeLa cell poliovirus receptor has been introduced into mouse cells, resulting in the expression of the receptor and susceptibility to poliovirus infection. PMID- 3020561 TI - Two-component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC. AB - We report that the ntrB and ntrC proteins of Bradyrhizobium sp. [Parasponia] strain RP501 share homology with other regulatory proteins. There is extensive conservation of C-terminal regions between products of RP501 ntrB; Klebsiella pneumoniae ntrB; Escherichia coli envZ, cpxA, and phoR; Agrobacterium tumefaciens virA; and, to a lesser extent, E. coli cheA. There is also extensive conservation of N-terminal regions between products of RP501 ntrC; K. pneumoniae ntrC; E. coli ompR, sfrA, phoB, cheY and cheB; Salmonella typhimurium cheB and cheY; Bacillus subtilis spoOA and spoOF; and A. tumefaciens virG. We propose that these regulatory genes comprise two-component regulatory systems that evolved from a common ancestral system that involved transduction of information about the status of the environment by one protein domain (the C-terminal regions conserved among ntrB, envZ, etc.) to a second one (the N-terminal region conserved among ntrC, ompR, etc.). The ntrC-set protein then acts upon a specific responding mechanism, typically as a transcriptional activator but also as an effector of the maturation of outer membrane proteins or as a modulator of the direction of flagella rotation. PMID- 3020562 TI - Localization and comparative nucleotide sequence analysis of the transforming domain in herpes simplex virus DNA containing repetitive genetic elements. AB - The 7.5-kilobase BamHI E fragment (BamHI-E) of herpes simplex virus type 2 (HSV 2) DNA (map position 0.533-0.583) encodes the 144-kDa subunit of ribonucleotide reductase and induces the neoplastic transformation of immortalized cell lines. To define the minimal transforming region of BamHI-E, a series of subclones were constructed that spanned the entire fragment. These subclones were assayed for focus formation in Rat-2 cells. Removal of the promoter region from the viral 144 kDa-protein gene left the transforming activity of DNA clones intact. A 481-bp Pst I-Sal I subclone of BamHI-E was capable of inducing focus formation and tumorigenic conversion. The nucleotide sequence of this fragment and the colinear nontransforming region of HSV-1 DNA was determined and compared. Striking differences were detected in the structure and organization of repeated sequence elements. Specifically, transforming HSV-2 DNA contains multiple regions of alternating purines and pyrimidines, G + C-rich sequences that are potential binding sites for transcription factor Sp1, and insertion-like sequence elements that are interrupted by base substitutions in nontransforming HSV-1 DNA. These results define a distinct transforming domain in HSV-2 DNA composed of repetitive elements implicated in gene rearrangement and activation. PMID- 3020564 TI - Salicylate effects on proton gradient dissipation by isolated gastric mucosal surface cells. AB - The effects of salicylate were examined on Na+/H+ exchange by isolated gastric mucosal surface cells loaded with H+ and resuspended in a buffered medium. Choline salicylate (pH 7.4) increases the dissipation of an intracellular proton gradient which was measured using acridine orange. The exchange of extracellular Na+ with intracellular H+ by surface cells not only remains intact but also is enhanced upon exposure to salicylate. This was confirmed by cellular uptake of 22Na and titration of cellular H+ efflux. Salicylate increases Na+/H+ exchange via a pathway predominantly sensitive to amiloride. However, the data also suggest that salicylate dissipates an intracellular proton gradient by an additional mechanism. The latter is independent of extracellular Na+ and not due to a generalized increase in cellular permeability. PMID- 3020563 TI - In vivo evidence that cGMP is the second messenger for atrial natriuretic factor. AB - cGMP generation has been associated with many of the vascular and endocrine actions of atrial natriuretic factor (ANF) in vitro. To examine the role of cGMP as a second messenger for the renal hemodynamic action of ANF in vivo, we measured glomerular filtration rate (GFR) and cGMP concentration in systemic artery, renal vein, and urine as well as in Bowman's space and end-proximal tubule (by free-flow micropuncture) after administration of ANF. ANF increased GFR by 45% and simultaneously induced a greater than 5-fold increase of cGMP concentration in glomerular ultrafiltrate (Bowman's space) when compared to controls. There was no significant increase in either systemic artery or renal vein cGMP concentration. Thus, the source of increased Bowman's space cGMP is not from the blood via filtration but rather from either glomerular mesangial or epithelial cells, which are not in direct contact with the circulation. Although a small amount of tubular handling of cGMP occurred along the length of the nephron, the augmented cGMP production from the glomerulus accounted for most of the 10- to 12-fold higher urinary cGMP excretion observed after ANF administration. Intrarenal arterial infusion of dibutyryl cGMP, but not dibutyryl cAMP, increased GFR in a dose-dependent fashion (from 10 to 1000 microM) by a mechanism similar to that of ANF--an increase in glomerular hydraulic pressure. Thus, ANF markedly stimulated glomerular production of cGMP, which coincided with a marked increase in GFR. Since dibutyryl cGMP itself was capable of increasing GFR, cGMP is the likely second messenger for ANF in vivo. PMID- 3020566 TI - Proteoglycan- and collagen-degrading enzymes from human interleukin 1-stimulated chondrocytes from several species: proteoglycanase and collagenase inhibitors as potentially new disease-modifying antiarthritic agents. AB - Human IL-1-stimulated chondrocytes derived from rabbit, bovine, and human articular cartilage produce proteoglycan- and collagen-degrading enzymes. These studies demonstrate that the biological activity of IL-1 is not species specific. Several thiol, carboxyalkyl, and hydroxamic acid peptide inhibitors showed differential effects. The thiols were equipotent inhibitors of both the collagen- and proteoglycan-degrading enzymes whereas the carboxyalkyls appear to inhibit solely the proteoglycan-degrading enzyme(s). The hydroxamic acid peptides, the most potent inhibitors, appear to be more active against the proteoglycan degrading enzymes. These synthetic inhibitors of proteoglycan- and/or collagen degrading enzymes may represent a new class of disease-modifying antiarthritic agents. PMID- 3020565 TI - Oxygen consumption and oxidative capacity of hepatocytes from young male obese and nonobese Zucker rats. AB - The contribution of the liver to the increased metabolic efficiency of the obese rat (fa/fa) was examined. Oxygen consumption of isolated hepatocytes and isolated mitochondria, and hepatic activities of mitochondrial enzymes were measured. Hepatocyte oxygen consumption was similar in the obese and nonobese rats for all substrates tested. Mitochondrial respiration also was similar in both phenotypes for all substrates tested. Activities of citrate synthase, succinate dehydrogenase, and cytochrome oxidase were similar for obese and nonobese rats. Taken together, these data show that in vitro hepatic oxygen consumption and oxidative capacity are similar in obese and nonobese rats. Rates of mitochondrial respiration with palmitoylcarnitine further show that the capacity for hepatic lipid oxidation is similar in obese and nonobese rats. Therefore, the increased metabolic efficiency of the obese rat probably cannot be attributed to an intrinsic decreased hepatic oxidative capacity. Further, there is no defect in hepatic lipid oxidative capacity in the young obese rat. PMID- 3020568 TI - The effect of cold storage on the response of rat aorta to biogenic amines. PMID- 3020567 TI - High vanadate interferes with the Fiske-Subbarow determination of inorganic phosphate. AB - We evaluated the possibility that oxyions of vanadium might react with molybdate and, in that manner, interfere with the Fiske-Subbarow colorimetric determination of inorganic phosphate. Phosphate (Pi) standard curves were prepared (0.03-0.30 mumole/ml) in the presence and absence of oxyvanadium solutions (2 X 10(-4) M) prepared from ortho- and metavanadate. Molybdate prepared in 5 N sulfuric acid was added to each standard. Upon addition of a reducing agent to develop color of the phosphomolybdate complex, a less intense color was observed at any given Pi concentration in the presence of oxyvanadium species. The slope of the regression line for the Pi standard curve in the presence of 2 X 10(-4) M oxyvanadium species was markedly depressed. The effect of oxyvanadium was similar when solutions were prepared from ortho- and metavanadate, despite differences in pH of these solutions. In addition, in the final reaction the pH was similar in the presence and absence of oxyvanadium, independent of the source of vanadate used to prepare solutions. Thus, interference by oxyvanadium did not appear to be related to changes in pH of samples containing vanadium oxyions. Interference was concentration dependent and the minimal concentration of vanadium oxyions that interfered was 5 X 10(-5) M. The effects of oxyvanadium (2 X 10(-4) M) on Mg+2 dependent and Na+-K+-ATPase activities in a renal microsomal preparation were then evaluated through the measurement of inorganic phosphate generation. Enzyme activities were determined with and without correction for interference by oxyvanadium with the method of Fiske and Subbarow. A significant artifactual depression of Mg+2 ATPase activity, but not Na+-K+-ATPase activity, was consistently observed when enzyme activities were not corrected for interference by oxyvanadium with the measurement of inorganic phosphate. These data indicate that when effects of high vanadate concentrations (5 X 10(-5) M) on ATP hydrolyzing enzymes are evaluated through changes in Pi generation, artifactual depression of enzyme activity may occur. PMID- 3020569 TI - Verapamil reduces muscle twitch in chronically treated preparations. PMID- 3020570 TI - Experimentally induced effects of diabetes on the rat myocardial beta adrenoceptor system. PMID- 3020571 TI - Genetics of ACTH action in Y1 mouse adrenocortical tumor cells. PMID- 3020572 TI - Receptor regulation of phosphoinositide and calcium metabolism. PMID- 3020573 TI - Role of Ca2+, diacylglycerol and cyclic AMP stimulated protein kinases in hormone action. PMID- 3020574 TI - Cholera toxin induced negative chronotropic and positive cyclic AMP response in cardiac myocytes. PMID- 3020575 TI - Opioid receptor effects of two aminotetralin derivatives using the electrically driven mouse vas deferens. PMID- 3020577 TI - Structure and function of transforming growth factor-beta. PMID- 3020576 TI - Effects of prolonged phorbol ester exposure on ACTH secretion from mouse pituitary tumor cells. PMID- 3020578 TI - Regulation of cAMP metabolism in adult ventricular myocytes: effects of histamine. PMID- 3020579 TI - Potentiation of 5-lipoxygenase metabolism by human neutrophils using a combination of activators. AB - Platelet-derived 12-hydroperoxy-5,8,10,14-icosatetraenoic acid (12-HPETE) cannot increase the synthesis of leukotriene B4 metabolites by human neutrophils activated by 1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (Paf Acether) at 10(-9) and 10(-8) M in contrast to 1 microM arachidonic acid. The combination of the 3 inducers at various concentrations resulted in a potentiation of the formation of leukotriene B4 metabolites suggesting that multiregulatory steps play a key role to govern the 5-lipoxygenase activity. These results suggest that the use of agents that can act synergistically to produce leukotrienes may be worthwhile investigating to understand some aspects of the leukotriene biosynthesis. PMID- 3020580 TI - Stimulus-related difference in the formation of leukotrienes and PGD2 after immunological and non-immunological challenge of human lung parenchyma "in vitro". AB - The stimulation of human lung parenchymal fragments with A-23187 induces formation of leukotrienes as well as of PGD2: LTE4 is the compound found in larger amount and independently of the intensity of the stimulus the % of metabolized precursor which is converted to leukotrienes or PGD2 is remarkably similar. However, under conditions of IgE-Anti IgE challenge the predominant conversion of arachidonic acid occurs to PGD2, LTB4 is almost negligible and LTD4 and E4 together represent less than 30% of the oxidative products of arachidonic acid. Whether PGD2 and leukotrienes derive from the same or different subset of cells is unresolved. PMID- 3020581 TI - Essential fatty acid deficiency: a new look at an old problem. AB - Essential fatty acid (EFA) deficiency is a useful tool to study the role of arachidonate and its metabolites in various physiologic and pathologic states. Recent studies have clarified the effects of EFA deficiency on membrane arachidonate and its metabolites, and have demonstrated that 20:3(n-9) (which accumulates in EFA deficiency) can be metabolized to a variety of eicosanoids. EFA deficiency has been shown to exert an anti-inflammatory effect. The mechanism of this effect may in part be mediated through a decrease in leukocyte leukotriene formation. In contrast, studies using the novel fatty acid, columbinic acid, have shown that the epidermal dysfunction seen in EFA deficiency may be a function of linoleate and its lipoxygenase metabolites rather than of arachidonate and the prostaglandins. Finally, it has recently been shown that EFA deficiency potentiates the effects of volatile anesthetics. EFA deficiency may thus provide a useful tool to investigate the molecular mechanism of these drugs. PMID- 3020582 TI - The effect of endogenous eicosapentaenoic acid on PMN leukotriene and PAF biosynthesis. AB - Caseinate elicited peritoneal PMNs were obtained from rats fed Menhaden fish oil or control rat chow. Total polyeneoic fatty acids were measured and compared with PAF and leukotriene biosynthesis after stimulation with the calcium ionophore A23187. PAF was measured by acetate incorporation and its molecular species were measured by GCNICIMS after conversion to the PFB-diglyceride. Eicosapentaenoic acid enrichment lowered the total amount of leukotriene B4 but increased the amount of LTB5 proportionally to the increase in the fatty acid. Although concentrations of 20:5 exceeded those of 20:4, PAF biosynthesis was not inhibited nor were the relative amounts of its molecular species dramatically altered. PMID- 3020583 TI - Neutrophil LTA4 hydrolases and leukotriene B4 receptors: effects of leukotriene epoxides and their enzymatic products. AB - A selection of inhibitors of rat and human neutrophil LTA4 hydrolases have been studied in vitro using partially purified enzymes. 5(S)trans 5,6 oxido-7,9-trans 11-cis-eicosatrienoic acid (LTA3) and 5(S)trans 5,6 oxido, 7,9-trans, 11,14,17 cis-eicosapentaenoic acid (LTA5) have been shown to inhibit neutrophil LTA4 hydrolases in a time-dependent manner. The products of hydrolysis of LTA3, LTA4 and LTA5 by human and rat neutrophil LTA4 hydrolase have been shown to displace [3H] LTB4 binding to human and rat neutrophil membranes. The order of displacement of [3H] LTB4 is LTB4 = LTB3 greater than LTB5 and this correlated well with their biological potencies for enhancement of neutrophil aggregation and chemokinesis. PMID- 3020584 TI - Contrasting effects of fatty acids on oocyte maturation in several starfish species. AB - Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine. In some species (group 2) oocyte maturation can be induced by micromolar concentrations of a few fatty acids such as arachidonic and eicosapentaenoic acids or by nanomolar concentrations of hydroxyeicosatetraenoic acid. Complete maturation is triggered: increased protein phosphorylation, appearance of the cytoplasmic "maturation-promoting factor", germinal vesicle breakdown, emission of the two polar bodies and formation of the female pronucleus. In other species (group 1), however, no maturation can be induced by the fatty acids active in the species of group 2, despite a large variety of experimental conditions. PMID- 3020585 TI - Role of icosanoids in alveolar macrophage phagocytosis and aggregation. AB - Rat alveolar macrophages (AM) collected by bronchoalveolar lavage were incubated in the presence or absence of various icosanoids or inhibitors of the arachidonic acid cascade with either chrysotile asbestos (50 micrograms/ml) to determine cell aggregation, or human red blood cells (Rh+; preincubated with anti-D globulin) to measure phagocytosis. Phorbol myristate acetate (PMA) and ionophore A23187, two agents which stimulate arachidonic acid metabolism, reduced red blood cell phagocytosis by AM whereas arachidonic acid had no effect on this cellular event. Neither arachidonic acid nor its metabolites (prostaglandins E2, I2, F2 alpha, the thromboxane mimick U44069 and leukotrienes A4, B4, C4, D4) had a significant effect on phagocytosis. Inhibition of the synthesis of cyclooxygenase products with various concentrations of indomethacin, aspirin and OKY-1581 or of lipoxygenase products with eicosatetraynoic acid, BW755c, diethylcarbamazine and phenidone did not affect phagocytosis either. On the other hand, asbestos-induced aggregation was significantly reduced by nordihydroguaiaretic acid (NDGA), BW755C, benoxaprofen, and high concentrations of indomethacin but not by aspirin. These results suggested that metabolites of arachidonic acid (especially lipoxygenase products) play a modulatory role in non specific alveolar macrophage aggregation but not in specific, Fc receptor-mediated phagocytosis. PMID- 3020587 TI - Stimulation of interleukin 2 and interferon gamma production by leukotriene B4 in human lymphocyte cultures. AB - Interleukin 2 (IL-2) and interferon-gamma (IFN) production by human lymphocytes was significantly enhanced in the presence of leukotriene B4 (LTB4) and indomethacin. Depletion of the T8+ suppressor cell subset of human T cells resulted in enhanced lymphokine production and further augmentation by LTB4. These findings suggest that LTB4 can regulate immune responses through its modulation of lymphokine production. PMID- 3020586 TI - Comparison of 5- and 15-lipoxygenase activities in blood and alveolar leukocyte preparations from normal subjects and patients with eosinophilia. AB - The arachidonic acid lipoxygenase metabolites of polymorphonuclear leukocyte (PMNL) preparations obtained from 31 subjects with eosinophilia (eosinophil content of PMNL preparations: 27.0%, 4-73, median with range) and 29 normal donors (4.5%, 0-15) were analyzed by reversed phase high performance liquid chromatography. More leukotriene (LT) C4 and 15-hydroxyeicosatetraenoic acid (HETE) (p less than 0.001) and less LTB4 (p less than 0.005) were found in eosinophil-rich preparations in comparison to controls on incubations with ionophore and arachidonic acid. In incubations with arachidonic acid alone, only small amounts of 5-lipoxygenase metabolites were produced by both groups, but the PMNL preparations from patients with eosinophilia showed higher capacity to release LTC4 and 15-HETE (p less than 0.005). In the eosinophil-rich preparations, the percentage of eosinophils correlated positively with the production of LTC4 and negatively with LTB4 (p less than 0.05, incubations with ionophore +/- arachidonic acid), and positively with 15-HETE (p less than 0.01, incubations with arachidonic acid +/- ionophore). Moreover the eosinophil-rich preparations produced more LTC4 per eosinophil in incubations with arachidonic acid alone (p less than 0.001) than normal PMNL preparations. The predominant release of LTC4 and 15-HETE by eosinophils was further confirmed by the analysis of bronchoalveolar lavage cells. Lavage cells containing eosinophils released LTC4 and 15-HETE while normal bronchoalveolar lavages, which were poor in eosinophils, did not produce detectable amount of LTC4 and 15-HETE. These results show that eosinophils release preferentially LTC4 and 15-HETE in contrast to LTB4 for the neutrophils. PMID- 3020588 TI - Meclofenamate sodium is an inhibitor of both the 5-lipoxygenase and cyclooxygenase pathways of the arachidonic acid cascade in vitro. AB - Meclofenamate sodium was compared to other nonsteroidal antiinflammatory drugs in terms of its potency to inhibit the formation of 5-HETE and LTB4 in human leukocytes and the formation of prostaglandin E2 in bovine seminal vesicles as measures of its ability to inhibit the 5-lipoxygenase and cyclooxygenase pathways of the arachidonic acid cascade. Meclofenamate sodium was about 2-4 times less potent than BW-755C in inhibiting 5-lipoxygenase enzyme activity and three times more potent than benoxaprofen, while naproxen, ibuprofen, and indomethacin showed IC50 greater than 100 microM. Meclofenamate sodium and indomethacin were the most potent inhibitors of the formation of PGE2 in bovine seminal vesicles followed by ibuprofen, naproxen, and benoxaprofen in this order. Meclofenamate sodium, like BW-755C, can be considered a dual inhibitor of 5-lipoxygenase and cyclooxygenase pathways of arachidonic acid cascade. This finding may explain in part the antiinflammatory activity of meclofenamate sodium. PMID- 3020589 TI - Inhibitory effect of prostaglandin D2 on DNA synthesis in nuclei. AB - Prostaglandin (PG) D2 treatment inhibited DNA synthesis in isolated nuclei of mastocytoma P-815, 2-E-6 cells. On treatment with PGD2 (10 micrograms/ml), the inhibition was distinct by 8 hrs, and complete after 18 hrs. This effect of PGD2 on DNA synthesis in nuclei was not direct or mediated by cyclic AMP, but was a cell-mediated reaction. The cytoplasmic fractions of PGD2-treated and untreated cells both had stimulatory effects and their potencies were the same except for that of the cytoplasmic fraction of 8 hr-treated cells, which was less than that of the cytoplasmic fraction of untreated cells. On treatment with PGD2, inhibition of DNA synthesis in the nuclei began after 8 hrs, and this inhibition could not be reversed, even by adding the cytoplasmic fraction from untreated cells to the assay system. A nuclear salt extract prepared by adding 0.3 M NaCl to nuclei of cells that had been treated with PGD2 for 18 hrs had a much smaller stimulatory effect on DNA synthesis of salt-treated nuclei than an extract of nuclei from untreated cells. It is suggested that inhibition of cell growth by PGD2 is not mediated by intracellular cyclic AMP, but that PGD2 induces a factor(s) that inhibits nuclear DNA synthesis. PMID- 3020590 TI - Antitumor effect of gamma-linolenic acid on cultured human neuroblastoma cells. AB - gamma-linolenic acid (GLA) was found to suppress the cell growth of 4 human neuroblastoma cell lines. GOTO was the most sensitive, followed by SK-N-DZ and NKP, while NCG was much less sensitive to GLA. In terms of GLA cytotoxicity, neither cyclo-oxygenase inhibitors nor a lipoxygenase inhibitor showed any effect. On the other hand, 4 antioxidants (Coenzyme Q, (D) alpha-tocopherol, (DL) alpha-tocopherol, butylated hydroxytoluene) reduced the growth inhibitory effect of GLA, but not in proportion to the decrease of GLA-stimulated lipid peroxidation. Accordingly, prostaglandins and leukotrienes probably do not play a role, and lipid peroxide may only be partly involved in the GLA effect. PMID- 3020591 TI - The binding of 6-oxo-prostaglandin E1 to platelet prostanoid receptors. AB - 6-oxo-PGE1 has been shown to cause a dose dependent stimulation of platelet adenylate cyclase (Km 1.6 X 10(-7)M). Since this compound also displaced specifically bound 9-3H-PGI2 from intact washed platelets (Ki 1.6 X 10(-6)M), the coupling to platelet PGI2 receptors has been suggested to account for the observed platelet cyclase stimulation. However, the Ki/Km-ratio of 6-oxo-PGE1 appears to be high (10.3) as compared to PGI2, ZK 36 374, and PGE1 (1.9, 2.4 and 3.1, resp.). Thus, one might expect that 6-oxo-PGE1 interacts with a heterogeneous population of platelet receptors mediating the same biological response, eg cyclase activation. In this study, we present evidence supporting this concept and report that 6-oxo-PGE1 also displaces specifically bound 3H-PGD2 from its platelet receptor (Ki 1.6 X 10(-5)M). PMID- 3020593 TI - Dopaminergic mediation of a behavioral effect of l-cathinone. AB - Ten male rats were trained to discriminate between the stimulus properties of 0.6 mg/kg l-cathinone and saline in a two-lever food-motivated operant task. Once trained, rats showed a dose-dependent increase in discrimination over a dosage range of 0.15-1.2 mg/kg l-cathinone. Analysis of this dose-response relationship indicated an ED50 of 0.27 mg/kg. Pretreatment with 0.2 mg/kg of the specific dopamine blocking drug haloperidol increased this ED50 to 0.47 mg/kg and significantly decreased discriminative performance when co-administered with either 0.15, 0.3, or 0.6 mg/kg l-cathinone. Since the dose-effect curves for cathinone with and without haloperidol pre-treatment were parallel, it is suggested that l-cathinone, the active constituent in khat, produces its discriminative properties, in part, by mediation of dopaminergic neuronal systems. PMID- 3020592 TI - Effects of REM sleep deprivation on central alpha 1- and beta-adrenoceptors in rat brain. AB - In the present experiment the effects of 'rapid-eye-movement' sleep deprivation (REMd) on cortical alpha 1- and beta-adrenoceptor binding sites in the rat brain were investigated. REMd was induced for 72 hr in two different ways: by the platform and the pendulum technique. In addition, three control groups were run. Determination of alpha 1- and beta-adrenoceptor sites in the cortex was done by 3H-prazosin and 3H-dihydroalprenolol binding studies, respectively. Both REM sleep deprived groups showed a small but significant decrease in the number of beta-adrenoceptor sites along with a small increase in affinity. On the other hand, alpha 1-adrenoceptor binding and affinity were not changed. These results agree with the effects of tricyclic antidepressant drug treatment. Common effects of REMd and tricyclic drugs are discussed in terms of modulation of tonic arousal processes. PMID- 3020594 TI - Cannabimimetic activity (delta 1-THC cue) of cannabidiol monomethyl ether and two stereoisomeric hexahydrocannabinols in rats and pigeons. AB - Animals (rats and pigeons) trained to discriminate between the presence and absence of the effects of delta 1-tetrahydrocannabinol (delta 1-THC; 3 and 0.56 mg/kg, respectively) were tested for generalization with graded doses of delta 1 THC as well as with two 7-hydroxyhexahydrocannabinol epimers which differ in the stereochemistry at the C-1 position only, and a cannabidiol (CBD)-like compound, cannabidiol monomethyl ether (CBDM). delta 1-THC produced dose/time related effects in both rats and pigeons. Both 7-hydroxyhexahydrocannabinols generalized with delta 1-THC in both species. Greater cannabimimetic activity was observed when the substituent at the C-1 position was equatorial (as in compound NL-105) than when the substituent was axial (compound NL-106) (for chemical structures see Fig. 1, below). Thus in the absence of other substituents the planarity at the C-1 position determines cannabimimetic activity. CBDM induced only vehicle appropriate responding at the doses of 3 and 10 mg/kg in both species; 30% delta 1-THC appropriate responding occurred with 17.5 mg/kg (only tested in pigeons), a dose which also appeared to excert rate depressant effects. Thus, like CBD, CBDM has a low degree of cannabimimetic activity. PMID- 3020595 TI - Effects of benzodiazepines on taste aversions in a two-bottle choice paradigm. AB - Diazepam (DZ) and chlordiazepoxide (CDP) were tested for their ability to antagonize LiCl-established conditioned taste aversions (CTAs) to saccharin in a two-bottle free-choice paradigm. CTAs to saccharin were established in male Sprague-Dawley rats on a chronic fluid-deprivation schedule by the administration of LiCl (3 mEq/kg, IP) following a forced-choice exposure to a novel saccharin solution (0.1%, w/v). Three days later, rats were provided with a two-bottle choice presentation of saccharin and distilled water. Conditioned rats drank distilled water almost exclusively while unconditioned animals preferred saccharin. Pretreatment with DZ (6, 9, 12 mg/kg, IP) and CDP (12 mg/kg, IP) significantly increased the saccharin intake of conditioned rats indicating an attenuation of the manifestation of the CTA. While these results are consistent with the known disinhibitory effects of benzodiazepines, alternative mechanisms involving polydipsia or interactions with the taste characteristics of saccharin could not be excluded. Both hypertonic saline (16%, w/v NaCl) and Barbital Sodium (100 mg/kg) produced polydipsia without attenuating CTAs suggesting that the two bottle procedure is capable of distinguishing between polydipsic effects and anti aversion effects for these drugs. PMID- 3020597 TI - An EEG investigation on nabilone, a synthetic cannabinoid, in rabbits. AB - In this paper, the electroencephalographic (EEG) and behavioural effects of nabilone, a derivative compound from delta 9-tetrahydrocannabinol (delta 9-THC), are studied in comparison with those of delta 8-tetrahydrocannabinol (delta 8 THC) in rabbits. The results show that nabilone requires lower doses than delta 8 THC to elicit cortical spike-and-wave complexes, but higher doses to disrupt hippocampal theta rhythm. These data are discussed in connection with the clinical actions of nabilone in man. PMID- 3020596 TI - Possible involvement of prostaglandins in cataleptic behavior in rats. AB - Involvement of prostaglandin (PG) in cataleptic behavior was investigated by a high bar test method in rats. PG F2 alpha (F2a) and E2 administered intracerebroventricularly (ICV) elicited cataleptic behavior in a dose-dependent manner. The cataleptic behaviors produced by PGs were markedly inhibited by ICV pretreatment with propranolol. The cataleptic behaviors induced by haloperidol were also inhibited by propranolol. The PG F2a- and haloperidol-induced cataleptic behaviors were almost abolished by the thermal coagulation of bilateral striatum where the dopaminergic and cholinergic link is found. The pilocarpine-induced cataleptic behavior was potentiated by ICV treatment with PG F2a. On the other hand, the cataleptic behavior elicited by haloperidol was reduced after oral treatment with aspirin, a PG synthesis inhibitor. These results suggest that PGs seem to be participated in incidence of cataleptic behavior, which might involve alteration of brain beta-adrenoceptor activity. PMID- 3020598 TI - Characterization and control of cytosolic binding proteins for 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) in the rat lung. AB - The presence of a high-affinity, low-capacity 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) binding complex was demonstrated in cytosol from rat lung. When analyzed by sucrose density gradient centrifugation, the 3H-TCDD binding complex sedimented at 8-9 S and 6.5 S in low and high ionic media, respectively. The apparent dissociation constant (Kd) of the 3H-TCDD binding complex was determined to be 2.9 nM and the number of binding sites (Bmax) was approximately equal to 69 fmol/mg of cytosolic protein. The entity was sensitive to heat and to proteases but not to DNAse or RNAse, indicating that it was a protein. An excess of unlabeled TCDD, benzo[a]pyrene, 3-methylcholanthrene, 7,12 dimethylbenzo[a]anthracene and 9-hydroxy ellipticine all competed with 3H-TCDD for binding. A single injection of either benzo[a]pyrene (20 mg/kg body weight) or 9-hydroxy ellipticine (20 mg/kg body weight) had different effects on the concentrations of 3H-TCDD binding proteins in the lung but none of the chemicals had a significant effect on the synthesis of surfactant as assayed by total phospholipid content, 72 h after the injection. PMID- 3020599 TI - Metabolic effects of chronic ACTH administration, interaction with response to stress. AB - The present experiment was undertaken to study the metabolic response to stress of single or chronic ACTH-treated male rats. It was found that chronic ACTH treated rats showed a slight reduction in food intake and a decrease in body weight gain. This treatment increased basal serum triglyceride and insulin levels. In addition, some differences in response to stress was found in chronic ACTH-treated rats. Thus, these latter animals, unlike the other two groups, showed a decrease in circulating triglyceride and insulin levels in response to short-term stress. Moreover, 24 h after onset of stress a more marked fall in liver weight and glucose levels were found in chronic ACTH-treated rats. It suggests that chronic ACTH treatment might alter the metabolic response to prolonged acute stress what could result in lower resistance to severe stresses. PMID- 3020601 TI - A simple and rapid orientation procedure for the estimation of affinities (1/Kd) and maximum binding capacities (Bmax) of ligand binding to two receptor subpopulations. AB - This paper describes a simple and rapid procedure for the estimation of specific parameters (dissociation constants, Kd and maximum binding capacities, Bmax) of ligand binding to two receptor subpopulations. This procedure provides, in a few minutes, the investigator, performing the actual binding studies, the necessary information about receptor heterogeneity, enabling the investigator to plan further experiments. The procedure is based on the graphical comparison of experimental binding data (ligand binding to one or two receptor subpopulations) with the theoretical values of ligand binding to one receptor population at four levels). The values of Kd and Bmax for high- and low-affinity receptors are derived from 4 horizontal deviations of experimental data from a theoretical data plot at these levels by their comparison with tabulated deviations. The correctness of the estimated parameters can be confirmed by the comparison of experimental data with those simulated on the basis of applying the values of Kd and Bmax found in the formula for ligand binding to two receptor subpopulations. The practical applicability of this procedure was demonstrated both on simulated and experimental data, and confirmed by the well known computer programs for evaluating receptor heterogeneity, namely "LIGAND" and "Affinity spectra". PMID- 3020600 TI - Comparison of the effect of gamma-aminobutyric acid and its derivative baclofen on the activity of Purkinje cells of frog cerebellum in vitro. AB - The inhibitory effect of gamma-aminobutyric acid (GABA) and its synthetic derivative baclofen were compared in frog cerebellum in vitro. Baclofen inhibited synaptic transmission from parallel fibres to the Purkinje cells in EC50 concentrations approximately 200-fold lower than for GABA. In addition to its inhibitory effect, GABA induced temporary facilitation of responses in the dendrite zone by a mechanism dependent on the presence of a normal Cl- concentration; the inhibitory phase was only partly sensitive to reduction of the Cl- concentration in the medium and to the administration of picrotoxin. The action of baclofen, which was unaffected by these treatments, requires an intact catecholamine and serotonin pool, since it is ineffective in reserpine-treated animals. Both substances also influence the excitability of parallel fibres. In solutions with a high Mg2+ and a low Ca2+ concentrations GABA inhibits the spontaneous activity of Purkinje cells by acting on the postsynaptic membrane of the soma and the primary dendrites. The effect of baclofen is evidently the outcome of inhibition of transmitter release from presynaptic endings. PMID- 3020602 TI - The importance of sodium and chloride ions for the development of DOCA-NaCl hypertension: a haemodynamic study. AB - The role of sodium and its accompanying anion for the development of DOCA-salt hypertension was studied in uninephrectomized DOCA-treated weanling Wistar rats which were fed a diet containing either sodium chloride or sodium bicarbonate (170 mmol/kg). The blood pressure was increased in both groups of rats with sodium overload as compared to rats fed a low-salt diet only. A decreased cardiac output and substantially elevated systemic resistance were demonstrated in both groups of rats with high sodium intake in comparison with rats kept on a low-salt diet. However, these haemodynamic changes were more pronounced in rats with sodium chloride overload than in animals with a high sodium bicarbonate intake. On the other hand, the rigidity of major arteries which was estimated as the pulse pressure/stroke volume ratio, was increased only in rats fed a diet with sodium chloride but not in rats with sodium bicarbonate overload. Thus high sodium intake was responsible for the changes of systemic resistance in DOCA treated animals and its action was only slightly augmented by a high chloride intake. In contrast to this, the chloride overload seemed to be essential for the induction of increased arterial rigidity. PMID- 3020603 TI - Ergot alkaloids inhibit 3H-naloxone binding to opiate receptors in the rat striatum and hippocampus. AB - The authors tested direct effect of selected ergot alkaloids (lisuride, terguride, DH-ergotoxine, DH-ergotamine and DH-ergocristine) on specific 3H naloxone binding in the rat striatum and hippocampus. In the striatum they found that DH-ergotoxine (a substance with high affinity for noradrenergic receptors) inhibited specific 3H-naloxone binding much more strongly than lisuride and terguride (substances with a greater affinity for dopaminergic and serotoninergic receptors). DH-ergotoxine, which inhibited binding significantly more in the striatum than in the hippocampus, displayed the greatest activity. The results show differences in the degree of inhibition by the various groups of ergot alkaloids in the striatum. In the case of DH-ergotoxine there was also a difference in the degree of inhibition in the striatum and the hippocampus. PMID- 3020604 TI - Functional significance of the serotoninergic influence on neuronal activity of the somatosensory cortex in 12-day-old kittens. AB - Monoamines, which participate in synaptic transmission as transmitters, also accomplish the modulation of neuronal activity. The role of serotonin (5 hydroxytryptamine--5-HT) was investigated in 8 to 14-day-old kittens by means of iontophoretic application onto neurones of the somatosensory cerebral cortex. The most typical response was the inhibition of neuronal activity. Another type of reaction was generated by inhibitory interneurones. After the microiontophoretic application of 5-HT, the tonic response of cortical neurones to the stimulation of the sciatic nerve changed into a phasic response. It is being suggested that the application of 5-HT to cortical neurones increases the excitability of inhibitory neurones. Hence, the serotoninergic regulation of neuronal activity in the somatosensory cortex may be operational from the 11th to the 12th day of postnatal life in cats. PMID- 3020605 TI - Conditioned slowing of stomach emptying produced by Pavlovian pairings of a drug CS or a place with lithium chloride. AB - Lithium chloride, in common with other drugs with emetic effects, prolongs stomach emptying. In different experiments, a drug state induced by a low dose of pentobarbital in Experiment 1 and morphine in Experiment 2 or a distinctive place (Experiments 3, 4) was the conditioned stimulus paired with lithium chloride as the unconditioned stimulus. In each case, Pavlovian conditioning occurred and the conditioned response mimicked lithium's unconditioned effect on stomach emptying. PMID- 3020606 TI - Calcified lymph node metastases in bronchioloalveolar carcinoma. AB - A 45-year-old man was found by cytopathologic examination of bronchial washings to have bronchioloalveolar carcinoma. A computed tomography (CT) scan of the chest showed diffuse calcifications in the consolidated left upper lobe. Similar calcifications were seen in several mediastinal lymph nodes; these were shown by biopsy to be metastases with calcified psammoma bodies. When CT demonstrates diffuse calcifications in a bronchioloalveolar carcinoma, the finding of identical calcifications in the mediastinal lymph nodes should raise a strong suspicion of metastatic involvement. PMID- 3020607 TI - Gestational trophoblastic neoplasm of the uterus: MR assessment. AB - Magnetic resonance (MR) imaging characteristics of uterine gestational trophoblastic neoplasia were prospectively studied in nine women (aged 21-58 years). MR imaging was done at the time of initial clinical diagnosis, after each of the first two cycles of chemotherapy, and 6-9 months after initiation of chemotherapy. Sagittal and transverse MR images of the pelvis were generated with a 0.35-T superconducting magnet and the double spin-echo technique with short and long repetition times (TRs). The neoplasm distorted the MR appearance of uterine zonal structures (myometrium, endometrium, and junctional zone) and demonstrated hypervascular masses of heterogeneous signal intensity. Favorable response to chemotherapy was determined by a decrease in serum beta-subunit human chorionic gonadotropin (HCG) concentrations, and was accompanied by MR findings of regression of vascular abnormalities, development of intralesional hemorrhage, and return of normal appearance of uterine zones. The return of uterine zonal anatomy on MR images antedated definitive decrease in uterine volume. All eight patients imaged 6-9 months after initial imaging showed normal uterine volume and zonal anatomy. PMID- 3020609 TI - Breast tumors: evaluation with P-31 MR spectroscopy. AB - Differences in the energetics of breast carcinomas and benign breast tumors were monitored by recording their high-energy phosphate concentrations using phosphorus-31 magnetic resonance (MR) spectroscopy. Analysis of the P-31 spectra of biopsy samples from five benign breast tumors and nine breast carcinomas showed that the concentrations of nucleoside triphosphates and phosphomonoesters are consistently higher in the carcinomas than in the benign tumors by an average factor of about three. In addition, the chemical shift differences between the alpha-adenosine triphosphate (alpha-ATP) and beta-ATP signals and between the gamma-ATP and beta-ATP signals were larger by about 0.2 ppm in the carcinomas. These differences result from the lower fraction of magnesium-bound ATP found in the carcinomas under our experimental conditions and reflect a decrease of about two in the concentration of free magnesium ions in the carcinomas relative to that in the benign tumors. Despite the limited number of tumors studied, the results suggest that in vivo P-31 MR spectroscopy may become a reliable method for the diagnosis of breast tumors. PMID- 3020608 TI - Frostbite: experimental assessment of tissue damage using Tc-99m pyrophosphate. Work in progress. AB - We designed an experimental model using a new method of freezing to study the pathogenesis and treatment of frostbite. Frostbite was simulated in a manner that closely resembles that which occurs in a natural environment. We used a radionuclide imaging technique to monitor the evolution and extent of tissue damage relative to temperature, rate of freezing, and controlled rewarming. Characteristic sequential changes were demonstrated on sequential nuclear scans. Nonperfusion, followed by perfusion, and finally again by nonperfusion occurred in all areas in which necrosis developed. The reappearance of nonperfusion corresponded to vascular injury and thrombosis evidenced at pathologic examination. We determined that lack of tissue perfusion corresponded to tissue injury. We believe that our experimental model provides an effective means of evaluating potential therapeutic regimens. PMID- 3020610 TI - Parathyroid imaging: comparison of double-tracer (T1-201, Tc-99m) scintigraphy and high-resolution US. AB - Parathyroid scintigraphy using a double-tracer (T1-201, Tc-99m) subtraction technique depicted 17 of 23 (74%) parathyroid adenomas in patients with and without previous neck operations. High-resolution (10-MHz) ultrasound (US) depicted 18 (78%) of these adenomas. Average tumor size depicted by US was 17 X 10 X 8 mm (excluding a giant adenoma) and 19 X 10 X 9 mm by scintigraphy. Alone, neither modality was particularly sensitive in the depiction of primary hyperplasia of the parathyroid glands, but combined techniques were more effective than the use of a single modality. With both US and T1-201 scintigraphy, only two of 23 cases of parathyroid adenoma in the neck were missed, and none of the eight cases of secondary hyperplasia were missed. In 11 patients who had previously undergone neck surgery, parathyroid tumors were identified in eight by either US or double-tracer scintigraphy. Preoperative parathyroid imaging with double-tracer scintigraphy and high-resolution US is suggested for patients with hyperparathyroidism, particularly in those patients who have had previous parathyroid surgery. PMID- 3020611 TI - Hepatocellular carcinoma: combined hepatic, arterial, and portal venous embolization. AB - Transcatheter arterial embolization (TAE) is an effective means of treating primary hepatocellular carcinoma (HCC). However, in many cases of HCC the tumor recurs after treatment. In an attempt to obtain complete tumor necrosis, the authors studied the clinical and histologic effect of simultaneous embolization of both the hepatic artery and portal vein in ten patients with HCC. In those cases in which combined embolization caused infarction, tumor cells in the main tumor, tumor cells that had invaded the tumor capsule, and small intrahepatic metastases had become totally necrotic following treatment. No viable tumor cells were detected in four patients who subsequently underwent operations; nor were viable tumor cells present in one other patient who later died as a result of a perforated duodenal ulcer. Five patients who did not subsequently undergo operations were still free of the disease 2-17 months after combined arterial and portal embolization. The impact of combined embolization on liver function was nearly the same as that produced when TAE was performed alone. Combined embolization may be a viable alternative to hepatectomy for the treatment of HCC. PMID- 3020612 TI - US-guided percutaneous alcohol injection of small hepatic and abdominal tumors. AB - Fourteen lesions (nine hepatocellular carcinomas, four hepatic metastases from gastric carcinoma, and one peritoneal metastasis from transitional cell carcinoma) in 12 patients were treated with percutaneous injection of 95% ethyl alcohol under guidance with ultrasound. Requirements for this procedure included inadequate response to conventional treatment or refusal of surgery, easy identification of the tumor with sonography, and a tumor diameter less than 4 cm. Three to nine administrations (75 in all) were performed for each lesion according to its size and to the results of the fine needle biopsy. All lesions had posttreatment sonographic structural changes of fibronecrotic degeneration. All lesions less than 3.2 cm in diameter (11 cases) that underwent the final fine needle biopsy were negative for malignant cells and had volume reductions up to 100%. No biochemical changes or untoward clinical sequelae were detected. Percutaneous intratumoral alcohol injection is inexpensive, easy to perform, and potentially valuable in the treatment of selected small neoplastic lesions of the liver and abdomen. PMID- 3020613 TI - [Composite restorations]. PMID- 3020614 TI - Ebselen reduces the formation of LTB4 in human and porcine leukocytes by isomerisation to its 5S, 12R-6-trans-isomer. AB - Ebselen, a new organoselenium compound with pronounced anti-inflammatory properties, selectively inhibits the formation of leukotriene B4 in human and porcine leukocytes with half-maximal inhibition at 4.0 and 2.7 mumol/1, respectively. The underlying mechanism was found to be a cis-trans-isomerisation of leukotriene B4 to the 5S, 12R-6-trans-isomer. 5-Hydroxy-eicosatetraenoic acid was also isomerised to the 8-trans-isomer. At higher concentrations, ebselen induces a dose-dependent decrease in the sum of total 5-lipoxygenase products with half-maximal inhibition at 30 mumol/1. Additionally, the effects of ebselen on human platelet 12-lipoxygenase and cyclooxygenase were investigated. Human platelet 12-lipoxygenase and cyclooxygenase were inhibited in a dose dependent manner with half-maximal inhibition at 20 mumol/1 and 5 mumol/1, respectively. We suggest that suppression of leukotriene B4-formation by isomerisation to a biologically inactive compound represents a promising approach to the therapy of inflammation. PMID- 3020615 TI - Roles of leukotrienes in two rat allergic inflammatory models; IgE-mediated and IgG-antigen complex-induced pleurisies. AB - Rat IgE pleurisy was induced by the injection of di-nitrophenol-conjugated bovine serum albumin (DNP-BSA) 48 hours after the intrapleural injection of rat anti-DNP IgE serum. IgG-BSA complex pleurisy was also induced by the intrapleural injection of IgG-BSA complexes produced at the optimum ratio in vitro. Plasma exudation was markedly increased in the first 20 minutes, but not observed thereafter, in IgE pleurisy, whereas marked plasma exudation in the first 20 minutes was followed by weak exudation at three and five hours in IgG-BSA complex pleurisy. Leukotrienes (LTs) E4 (100 ng/rat), D4 (32) and B4 (16) were detected on HPLC in the pleural exudate in the first 20 minutes of IgG-BSA complex pleurisy, but less (9 ng/rat) LTE4 alone was detected in the five-hour exudate. The first 20-minute pleural exudate contained 13 ng/rat of LTE4 in IgE pleurisy. The plasma was completely inhibited by simultaneous treatment of rats with pyrilamine (2.5 mg/kg, i.p.) and methysergide (3 mg/kg, i.p.), as it was in compound 48/80-induced pleurisy. In IgG-BSA complex pleurisy, 90% of the pleural exudate for the first 20 minutes was inhibited by the same treatment, and the rest was completely suppressed by simultaneous treatment with an intrapleural injection of AA-1777, a selective 5-lipoxygenase inhibitor. AA-1777 alone did not reduce the plasma exudation significantly. The 5-lipoxygenase inhibitor was also very effective in reducing the migrating numbers of polymorphonuclear and mononuclear leukocytes to half, without affecting the eosinophils of mast cells. PMID- 3020617 TI - Eicosapentaenoic acid metabolism in human and rabbit anterior uvea. AB - Eicosapentaenoic acid (EPA) metabolism into 3 series cyclooxygenase and 5 series lipoxygenase products was assessed in human and rabbit anterior uvea. Both tissues synthesized 3 series cyclooxygenase products such as delta17 6-keto-PGF1 (PGI3 metabolite), PGE3 alpha, PGE3, PGD3 and TxB3 (a stable product of TxA3) and lipoxygenase products 12-hydroxyeicosapentaenoic acid (HEPE), 5-HEPE and 5,12 diHEPE from 14C-EPA. EPA-derived cyclooxygenase product synthesis was considerably greater than the formation of lipoxygenase products from EPA in both tissues. PMID- 3020616 TI - Further studies on the mechanism of action of leukotrienes and histamine on guinea pig lung parenchyma. Role of calcium, phospholipase and methyltransferase. AB - The contractile activity of leukotriene B4 (LTB4), leukotriene D4 (LTD4) and histamine on strips of guinea pig lung parenchyma was shown to be dependent on the calcium concentrations of the Krebs solution. The calcium channel blocker verapamil (2.0 to 15 microM) had an additive effect on the inhibitory activity of low calcium (0.1 mM) on contractions of guinea pig parenchyma to leukotrienes and histamine. Cobalt chloride, a divalent cation, also produced dose-dependent reductions of the myotropic activities of LTB4, LTD4 and histamine. An antagonist of calmodulin, trifluoperazine (1-200 microM), dose-dependently inhibited the contractile activity of the three agonists on the parenchyma strip. The IC50 of this compound for inhibition of histamine was much lower (2-3 microM) than the IC50 for inhibition of leukotrienes (75 microM). Valinomycin, a potassium ionophore, also interfere with the contractile activities of leukotrienes and histamine whereas a blocker of sodium channel, tetrodotoxin, had no effect on the activity of these agonists. Furthermore, an inhibitor of methyltransferase, 3 deazaadenosine, significantly diminished the responses of the parenchyma to leukotrienes and histamine. These results confirmed the important role of extracellular and intracellular calcium in the myotropic activity of leukotrienes and histamine in guinea pig lungs and showed that compounds which interfere either directly or indirectly with calcium mobilization into the lung smooth muscles, decreased the tissue responsiveness. PMID- 3020618 TI - Arachidonic acid metabolites in a nephroblastoma associated with paraneoplastic hypercalcemia. AB - Blood concentration of PGE2, F2a, 6 keto PGF1a (6kF1a), TxB2 and 13, 14 dehydro 15 keto PGE2 (13, 14 OH 15 k E2) were measured in renal artery and vein of a patient with a PGs producing nephroblastoma. The tumor tissue produced PGs in the following order: PGF2a greater than PGE2 greater than TxB2 greater than 6kF1a greater than 13, 14 OH 15 k E2. However, renal artery concentration of the substances were as follows: 13, 14 OH 15 k E2 greater than TxB2 greater than 6kF1a greater than PGF2a greater than PGE2. Since arterial concentration is critical to postulating a calcium mobilizing effect on bone tissue, PGE2 arterial level seems to be too low to exert a pathogenetic role on hypercalcemia, at least in the patient reported here. PMID- 3020619 TI - Stereoselective hydrogen abstraction in leukotriene A4 synthesis by purified 5 lipoxygenase of porcine leukocytes. AB - Arachidonate 5-lipoxygenase purified from porcine leukocytes transformed arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid. By the leukotriene A synthase activity of the same enzyme the product was further metabolized to leukotriene A4 (actually detected as 6-trans-leukotriene B4, 12 epi-6-trans-leukotriene B4, and 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids). The enzyme was incubated with [10-DR-3H]- or [10-LS-3H]-labeled arachidonic acid, and 6-trans-LTB4 and its 12-epimer were analyzed. More than 90% of 10-DR-hydrogen was lost while about 100% of 10-LS-hydrogen was retained, indicating a stereospecific hydrogen elimination from C-10 during the formation of leukotriene A4. PMID- 3020621 TI - [Multicentric chemodectoma]. PMID- 3020620 TI - Tissue differences in vascular permeability induced by leukotriene B4 and prostaglandin E2 in the rat. AB - The activity of synthetic LTB4 and PGE2, in increasing vascular permeability was tested simultaneously in seventeen different organs in the rat. Rats were injected in the aortic arch through a cannula in the carotid artery with 125I albumin, 51Cr-erythrocytes, and 57Co-EDTA. The rats were then injected through the carotid artery cannula with LTB4, PGE2 or a combination of LTB4 and PGE2. Eight minutes later the rats were killed and the activity of 125I, 51Cr, and 57Co measured in different organs. Changes in vascular permeability were inferred from changes in the ratios of the isotope activities. LTB4 (15 micrograms/kg) induced enhanced permeability in caecum, small bowel, skin, fat pad, stomach, pancreas, and aorta, but not in the heart, brain, colon, testes, diaphragm, forelimb, cremaster muscle, lung, kidney or eye. A lower dose of LTB4, 3 micrograms/kg, enhanced vascular permeability in caecum, small bowel, skin, stomach, and aorta. PGE2 (1 microgram/kg) enhanced vascular permeability only in the caecum. A combination of LTB4 (3 micrograms/kg) and PGE2 (1 microgram/kg) was more potent than either alone. Rats depleted of neutrophils with anti-neutrophil serum were less sensitive to LTB4 than intact rats. These findings suggest that the vasculatures of different tissues in the rat vary markedly in their susceptibility to LTB4 induced increases in permeability. PMID- 3020622 TI - [Antibodies against rubella and herpes virus in women attending hospitalized newborn infants]. PMID- 3020623 TI - [Acute focal bacterial nephritis]. AB - Acute focal bacterial nephritis is a very rare type of infective nephritis. It is characterised by groups of abscesses of 1 to 5 mm. situated in the renal cortex with pus tracking to the papillae. Urography is normal or suggests a non-specific enlargement. On sonography, non-homogeneous foci with reduced echogenicity are observed. Unenhanced CT shows indefinite lesions of reduced density, which do not enhance as much as the surrounding parenchyma after contrast injection. On angiography these areas appear as hypovascular lesions. The disease must be differentiated from a malignant renal tumour and from an acute renal abscess. The clinical findings and the results of sonographic and radiological observations on five patients with acute focal bacterial nephritis are described. PMID- 3020624 TI - [Magnetic resonance tomography of the kidneys: comparison with anatomic correlates]. AB - Specimens of human kidneys (n = 24) were investigated using magnetic resonance imaging (MRI), and compared with the corresponding cross-sections that had been prepared later from the same kidneys. The tomographs reveal detailed informations of renal fine structure. In particular, parenchyma and sinus can be easily differentiated from vasculature. It is possible to visualize vessel arborization, including the arcuate vessels of the renal cortex. The clinical significance of renal MR tomographs is discussed. PMID- 3020625 TI - [Computed tomography of an aortoiliac aneurysm with ureteral stenosis]. AB - 10 patients with uni- or bilateral ureteral stenosis due to an aortoiliac aneurysm were evaluated by computed tomography. In 6 patients the complicating ureteral stenosis was caused by an inflammatory aneurysm, in 3 patients by an arteriosclerotic aneurysm, and in 1 patient by a secondary expanding aneurysm of the internal iliac artery following aortofemoral reconstruction. CT allowed non invasively to document the association between ureteral stenosis and aneurysm. Post-operative follow-up CT studies of the 6 patients with an inflammatory aneurysm revealed a time dependent regression or complete disappearance of the perivascular fibrosis. PMID- 3020626 TI - [Intra-arterial dynamic computed tomography in characterizing liver metastases of colorectal cancer]. AB - Locoregional chemotherapy of liver metastases from colorectal tumours is a promising new approach. The results of CT in fifteen patients after intravenous and intra-arterial contrast injection are compared. The liver metastases are supplied mainly by the arterial route and this permits a means of assessing therapy. Contrast injections through a hepatic catheter may result in flow phenomena which limits the value of this investigation. It is unknown whether such flow phenomena may influence the result of intra-arterial chemotherapy. Although the margins of the metastases were more clearly defined, diagnostic accuracy was not increased, as compared with intravenous CT enhancement. PMID- 3020627 TI - [Solitary black pigment stones]. AB - Solitary pigment stones of the gallbladder are rare (1.7%). 82.5% are radiopaque, 17.5% radiolucent. 64.8% of radiopaque solitary pigment stones have the structure of a cockade. Such cockades take years to develop. Solitary cholesterol stones with a nucleus of a radiopaque pigment stone should not be treated for litholysis. 8% of solitary cholesterol stones with a cross diameter below 15 mm. possess a radiolucent pigment stone nucleus. X-ray diagnosis for selecting litholytic treatment remains the safest method, especially if the radiologist compares his films regularly with the specimens after surgery. PMID- 3020628 TI - [Detection of narcotic-containing packages in "body-packers" using imaging procedures. Studies in vitro and in vivo]. AB - The increase in narcotic smuggling through swallowing small packages ("body packing") has created problems in detection and in avoiding acute intoxication. Five such cases are reported. Experiments have been carried out in vitro in order to investigate how such narcotic packages could best be shown by radiological examination, by CT or by MRT. PMID- 3020629 TI - [Real-time sonography in the diagnosis of neck cysts]. AB - The diagnostic possibility of high resolution real time sonography has been investigated in 31 patients with cysts in the neck. The method is of special value preoperatively by demonstrating neighbouring structures, such as the parotid and thyroid glands and the major vessels in the neck; it contributes to differential diagnosis and surgical planning. Lateral cysts, because of their contents, may be highly echogenic and may be difficult to distinguish from lymphomas. The differential diagnosis of various cysts in the neck is discussed. An important aspect of sonography of the neck is the experience of the operator and his detailed knowledge of the local anatomy. PMID- 3020631 TI - [Sonography as a new diagnostic procedure for resolving shoulder problems]. AB - Eighty-one sonographic examinations of patients with complaints relating to the shoulder joint have shown that this is the method next in value to radiological examination. So far, lesions of the rotator cuff and of the long head of the biceps could only be demonstrated by invasive procedures such as arthrography or arthroscopy. In these situations, sonography attains a similar accuracy. Diffuse lesions can also be diagnosed correctly, making arthrography and arthroscopy unnecessary. In addition, sonography can demonstrate inflammatory and degenerative changes and incomplete sub-acromial and intermediary tears of the rotator cuff, unlike conventional diagnostic methods. In future, arthrography and arthroscopy will only be necessary as additional diagnostic methods if sonography remains inconclusive. PMID- 3020630 TI - [Gray-scale histogram analysis in thyroid sonography. Critique of visual evaluation of echogenicity]. AB - Three hundred and eighteen grey scale histograms of thyroid glands in 121 patients have been analysed. The arithmetic average grey scale value (GWMi) and the most commonly appearing grey value (GWMa) were determined electronically. The normal range of GWMi was 24 +/- 4.6 (x +/- s) grey scale units (GWE), for GWMa it was 26 +/- 5.6 GWe (x +/- s). Macrofollicular thyroid adenomas (8 cases) and thyroids with diffuse autonomy (12 cases) differed with a GWMi of 21 +/- 3.8 and 23 +/- GEW (x +/- s) respectively and a GWMa of 23 +/- 3.9 and 25 +/- 4.8 GWE and were within the normal range (p greater than 0.05). Microfollicular thyroid adenomas (12 cases), immunological thyroid lesions (25 cases) and parathyroid adenomas (6 cases) had reduced GWMi (6 +/- 3.1 and 8 +/- 1.8 GWE respectively) and GWMa (5 +/- 3 and 7 +/- 2 GWE, p less than 0.005). Thyroid carcinomas (9 cases) and lymph node metastases (8 cases) were also significantly below normal, with a GWMi of 11 +/- 2.3 and 11 +/- 3.4 respectively (p less than 0.05). Analysis of grey scale histograms makes it possible to evaluate changes in the thyroid without reference to surrounding tissues. PMID- 3020632 TI - [Arthrography of the temporomandibular joint. Indications, technic and interpretation]. AB - Articular dysfunction of the TMJ with anterior displacement of the disc ("internal derangement") is an entity which has been separated from other types of the "myofascial pain syndromes" and which can be treated conservatively or by surgery. Arthrography of the TMJ has contributed greatly to an understanding of normal and abnormal function and, in many cases, it can provide a diagnosis. On the basis of our experience with 80 investigations we discuss technical problems and the clinical indications. The indications for arthrography are in the pre operative diagnosis, when clinical findings are uncertain, in order to demonstrate perforation, in order to confirm a suspected diagnosis and to assist in prosthetic treatment. PMID- 3020633 TI - [The "unreadable" knee arthrogram. The problem and proposal of a new method: air- and magnification-technic]. AB - The problem of the difficulty experienced by non-radiologists in reading a convention knee arthrogram is discussed. A new method is proposed, consisting of air as contrast medium in combination with direct radiological magnification. Operative control of the first 100 patients showed correct arthrographic diagnosis in 91%. The advantage of the new method is a better presentation of meniscus pathology to the operating surgeon and the possibility of video documentation. PMID- 3020634 TI - [Importance of rapid gradient echo sequences for nuclear magnetic tomographic diagnosis of joint diseases]. AB - The possibilities and limitations of rapid echo acquisition methods in various posttraumatic, degenerative and inflammatory diseases of the joints are demonstrated and discussed on the basis of a pilot study. It is evident that specific information important for clarifying effusions and meniscopathies is supplied especially by "water"-like images. Limitations of spatial resolution are at present still a limiting factor. PMID- 3020635 TI - MR imaging of 22 Charnley-Muller total hip prostheses. AB - To find out whether MR imaging is contraindicated in patients with metallic implants or can be a routine diagnostic procedure, MR investigations in 18 patients with 22 Charnley-Muller total hip prostheses were performed on a 0.5 T Gyroscan S 5, Philips. No adverse reactions during or post MR investigation were encountered. The imaging of the soft tissue was superior to CT and showed less distortion. The diagnosis of loosening, by detection of demarcation lines at the interfaces was at its best in the distal part of the femoral stem prosthesis and was poor in the acetabular component and in the upper part of the stem prosthesis due to artifacts. PMID- 3020636 TI - [Pathology of the craniocervical junction as shown by magnetic resonance tomography. 68 cases]. AB - In cases of a suspected lesion at the level of the cranio-cervical junction, MR should be considered as the imaging technique of choice. Tumours, inflammation and degenerative processes can be reliably demonstrated and diagnosed. Administration of contrast media, for example, gadolinium-DTPA, is necessary in selected cases only. Comparing "examination time" of x-ray myelography and high resolution x-ray CT with MR, one can well tolerate the long "acquisition" of MR images. PMID- 3020637 TI - [Diagnosis of spondylitis by MR tomography]. AB - The results of MR tomography in 13 patients with specific and nonspecific spondylitis are reported. There were characteristic changes in signal intensity and in the configuration of the vertebral bodies and the intervertebral discs. The extent of intraspinal stenosis can be demonstrated accurately by MR. Paravertebral abscesses can be shown accurately and their position can be clarified by means of multiplanar sections. The early diagnosis of spondylitis by MR tomography is discussed. PMID- 3020638 TI - [Magnetic resonance tomography of spinal masses]. AB - We examined 22 intramedullary, 17 intradural extramedullary and 20 extradural lesions, with direct involvement of the spinal cord by magnetic resonance tomography, using surface coils and 5 mm., or narrower continuous sections. Spinecho pulse sequences are suitable for multi-slice examinations. T.1-weighted examinations are suitable for demonstrating the syrinx in syringomyelia, in all other circumstances T1 and T2 sequences are essential. Gd-DTPA is unnecessary. The results of these recommendations are illustrated. PMID- 3020639 TI - [Rare anomalies of craniocervical vessels accompanied by subarachnoid hemorrhage]. AB - Seven patients presenting rare vascular anomalies of the craniocerebral vessels are reported, six of whom suffered from subarachnoid haemorrhage (SAH). Angiography demonstrated typically located saccular aneurysms as the cause of bleeding in three of these cases. An increased coincidence of rare vascular anomalies and aneurysms is known from the literature. Our findings suggest that in cases of SAH and rare cerebrovascular anomalies additional aneurysms can be demonstrated almost as often as in unselected cases of SAH (70%). Despite controversial aspects regarding the pathogenesis of cerebral aneurysms, abnormalities of the vessel walls of genetic or embryologic origin seem to be the most likely reason for this high coincidence. PMID- 3020640 TI - [111Indium-thrombocyte scintigraphy and percutaneous transluminal dilatation (angioplasty) of supra-aortic vascular stenoses. A new method for deciding therapy and for follow-up control]. AB - Scintigraphy with 111indium-marked autologous platelets has been used during percutaneous transluminal dilatation of supra-aortic arterial stenoses; there were 14 successful dilatations and one attempted dilatation. The material consisted of nine stenoses of the internal carotid artery, two stenoses at the origin of the vertebral artery and four stenoses of the first part of the left subclavian artery. Deposition of thrombi could be demonstrated by scintigraphy, both before and after catheter dilatation. The value of this method for deciding on the type of treatment and for observing the course of percutaneous dilatation of cerebral vessels is discussed. PMID- 3020641 TI - [DSA of the pulmonary vessels during endobronchial laser- and afterloading therapy of malignant bronchial stenoses]. AB - The findings are reported of the DSA examination of the pulmonary vessels in 16 patients with malignant bronchial stenoses, for whom laser or after-loading therapy was being considered. In these patients an improvement of dyspnoea can be expected only of there is adequate perfusion of the involved lung segment. DSA demonstrated stenoses or occlusions of main pulmonary or lobar arteries in six patients. Ten patients had a normal pulmonary circulation. In most cases the result of the DSA examination was important in determining treatment. PMID- 3020643 TI - Pan-sialadenitis due to Staphylococcus aureus septicemia. PMID- 3020644 TI - Primary chyluria. Lymphographic and CT findings. PMID- 3020642 TI - [Radiologic diagnosis of benign fibrous pleural mesotheliomas]. PMID- 3020645 TI - The "empty" right iliac fossa. Radiological features of a large retroperitoneal liposarcoma. PMID- 3020646 TI - Improved MR imaging of the bladder by using a new surface coil. PMID- 3020648 TI - Histopathology of infectious bursal disease in non-lymphoid organs of chickens. PMID- 3020647 TI - [Bilateral symmetric obliteration syndrome of the subclavian-brachial artery system in elderly women]. PMID- 3020649 TI - [Involvement of motor fibers in the ulnar nerve syndrome at the elbow]. AB - In order to improve the diagnosis of the ulnar nerve neuropathy at the elbow, we have retrospectively analysed the data obtained in the course of the electrophysiological examinations of the ulnar nerve motor component in three groups of adult subjects of both sexes (210 control subjects; 111 patients with more or less marked clinical symptoms suggestive of ulnar nerve neuropathy; 21 patients who after our investigation have undergone an operation for an ulnar nerve neuropathy at the elbow). The comparison of the data obtained in the three groups permits us to propose the following parameters as signs of ulnar neuropathy at the elbow: the proximal conduction time (stimulation in the axilla) greater than or equal to 12 msec for males and greater than or equal to 11 msec for females; motor conduction velocity of ulnar motor nerve fibers at the arm comprise in the range of normal values and similar to that measured on the contralateral ulnar nerve; conduction velocity of ulnar nerve motor fibers when it is measured on all the length of the upper limb less than 45 m/sec-1 (males) or less than 50 m/sec-1 (females). PMID- 3020650 TI - Relations between basal ganglia and hippocampus: action of substantia nigra and pallidum. AB - Several interrelationships exist between basal ganglia and hippocampus. The ventral striatum appears to be involved in the control of the dopaminergic nigro striatal pathway. The caudate, in turn, seems to influence the hippocampal theta rhythm and to inhibit hippocampal spikes. In the present work the role played by globus pallidus pars interna and substantia nigra pars compacta on hippocampal bioelectrical activity is studied. Injection of sodium penicillin i.v. produces steady interictal spikes in the hippocampus. Substantia nigra stimulation induces regular theta rhythm and inhibits the spikes. Pallidal stimulation, on the contrary, appears to strongly enhance epileptiform activity, proceeding to generalised seizure activity. The results are discussed in the light of a putative feedback loop from basal ganglia to hippocampus, probably underlying co participation of the two subcortical structures in the control of motor behaviour. PMID- 3020652 TI - Myocardial adaptations to dietary sucrose in spontaneously hypertensive rats. AB - The effects of dietary sucrose upon specific myocardial and hemodynamic parameters were examined in spontaneously hypertensive rats (SHR). Following a 6 wk program, SHR consuming a supplement of 10% sucrose in the drinking water exhibited increased heart weight, heart mass, left ventricular free wall thickness and increased resting heart rate when compared to the hypertensive control group. Total caloric intake was similar between groups. Cardiac alpha 1 adrenoceptor density and affinity were also altered following sucrose feeding. These data suggest that modest intake of dietary sucrose is associated with cardiovascular adaptations that may further burden a heart already compromised by the presence of systemic hypertension. PMID- 3020651 TI - [Symptomatic therapy of rheumatoid arthritis]. PMID- 3020654 TI - Dynamic (18F)-2-fluoro-2-deoxy-D-glucose (FDG) scintigraphy of normal and tumor bearing rats. AB - Five normal rats, 14 rats bearing the Rous sarcoma intrarenally, and four rats with DMBA-induced mammary carcinomas were imaged by dynamic (18F)-2-fluoro-2 deoxy-D-glucose ((18F)FDG) scintigraphy and (99mTc)DTPA renography. The brain, heart, liver, kidneys, and tumor were selected as regions of interest (ROI) for time activity curves; exponential functions were fitted to the decay-corrected curves, which yielded biologic half-lives of (18F)FDG in the ROI. It was found that all organs ROI's exhibited average elimination of activity, whereas activity accumulated in the tumor ROI for the duration of the study (5 h). The (99mTc)DTPA renographies showed that the excretory function in kidneys bearing the Rous sarcoma is severely impaired. This study shows that small laboratory animals can be successfully scintigraphied with a conventional gamma camera using (18F)FDG and that the pharmacokinetics of this radiopharmaceutical may be evaluated. PMID- 3020653 TI - Effect of halothane on brain 2',3'-cyclic nucleotide 3'-phosphodiesterase during neurodevelopment in the rat. AB - Chronic exposure to anesthetic concentrations of halothane during the prenatal and early postnatal periods inhibits the incorporation of the leucine into myelin subcellular fractions in the rat. The enzyme 2',3' - cyclic nucleotide 3' - phosphodiesterase (CNPase) has been widely used as a myelin marker. To determine the effect of halothane on the developmental profile of CNPase, two groups of pregnant Sprague Dawley rats were exposed to 500 p.p.m. or 250 p.p.m. halothane, eight hours per day, five days per week from the third day after conception through postnatal day ten. Control animals were exposed to air alone. CNPase activity was significantly decreased by 500 p.p.m. halothane (34%) and by 250 p.p.m. halothane (29%) at postnatal day 17. Brain and body weights in both halothane treated groups were also less than control animals throughout the measurement period. The data indicates that chronic pre- and postnatal halothane exposure at low levels delays myelination in the rat. PMID- 3020656 TI - Passage of duck hepatitis virus in cell cultures derived from avian embryos of different species. AB - An egg-attenuated strain of duck hepatitis virus was successfully passaged through cell cultures of avian embryos derived from goose, turkey, guinea fowl, Japanese quail, pheasant and chicken. Two field strains of the virus were passaged in a more limited range of species. PMID- 3020655 TI - Further studies on the effects of prolonged prolactin administration on the zona glomerulosa of the rat adrenal cortex. AB - The effects of a prolonged treatment with prolactin on the morphology and hormone secretion of adrenal zona glomerulosa of gonadectomized testosterone-replaced rats were investigated by coupled morphometric and radioimmunologic techniques. The dexamethasone/captopril-induced atrophy of zona glomerulosa cells (-42%) and fall in the blood level of aldosterone (-37%) were partially counteracted by chronic prolactin administration: the values had increased by about 15% and 17%, respectively, but remained lower than in the control animals. The prolonged treatment with prolactin of dexamethasone/captopril-administered ACTH/angiotensin II-replaced rats provoked a striking increase in the zona glomerulosa cell volume (24%) and in the blood level of aldosterone (33%) above the control values. The possibility is discussed whether prolactin may be directly involved in the stimulation of the growth and steroidogenic capacity of rat zona glomerulosa, without interacting with the gonads and interfering with the hypothalamo hypophyseal axis and the renin-angiotensin system. PMID- 3020657 TI - Response of young pigs to foot-and-mouth disease oil emulsion vaccination in the presence and absence of maternally derived neutralising antibodies. AB - Serum samples and bodyweights were taken at regular intervals for six to seven months from piglets, born to foot-and-mouth disease (FMD) vaccinated or unvaccinated sows, which had been vaccinated at one, two, four or eight weeks old. Young pigs, devoid of maternally derived antibodies (MDA), were capable of responding to FMD vaccination at one week old, with no deleterious effects on their growth rate. However, their immunity to experimental infection at six to seven months old was poor (33.3 per cent). Vaccination of four or eight-week-old piglets, born to unvaccinated sows, protected 87.5 per cent from a similar challenge. In piglets, born to FMD vaccinated sows, the MDA had a suppressive effect on the early vaccination response. This suppression, which was affected by the titre of MDA present in the piglets at the time of vaccination, was complete in one-, two- and four-week-old piglets and partial in eight-week-old piglets. Furthermore, none of these piglets were immune to experimental infection at six to seven months old. PMID- 3020658 TI - Exposure of sheep to natural aerosols of foot-and-mouth disease virus. AB - Sheep taken individually and allowed to inhale air being drawn along a duct from a cabinet containing pigs acutely infected with foot-and-mouth disease virus for 10 or 15 minute periods were infected by doses as low as 10 TCID50 of virus. The most consistent and reliable indicators of infection were viraemia and seroconversion. The mean times from exposure to onset of viraemia, pyrexia and the appearance of vesicular lesions were 2.5, 3.8 and 4.7 days, respectively. Neither the time from exposure to first detectable viraemia nor vesication correlated with dose. Around 27 per cent of sheep which were known to have been infected did not develop vesicles. PMID- 3020659 TI - Magnesium metabolism in open-heart surgery. AB - The levels of magnesium in serum, urine and erythrocytes were studied in 22 patients undergoing cardiac surgery for valvular prosthesis. Magnesium values were correlated with serum albumin and non-esterified fatty acids (NEFA). Data were collected before anesthesia, 10 min after sternotomy, heparinization and declamping of the aorta and in the 1st postoperative day. A slight decrease in magnesemia was observed before extracorporeal circulation (ECC) and was mainly due to haemodilution. The correlation of magnesium with NEFA was significant only after heparinization. The use of the St Thomas solution as cardioplegia fully corrected the hypomagnesemia previously reported during ECC as well as in the 1st postoperative day. A moderate hypermagnesemia was observed at the end of ECC, but no patient reached dangerous levels of serum magnesium. Urinary losses increased during and after ECC. Red blood cell magnesium showed a slight increase before ECC, followed by a significant reduction at the end of ECC. PMID- 3020661 TI - A new model for total cerebral ischemia in dogs. AB - We have developed a new method producing total cerebral ischemia (TCI) in dogs; clamping ascending aorta with aorto-atrial bypass formation. Clamping ascending aorta provides TCI, the duration of which can be controlled up to the periods of 10 min. Beyond this interval, it is difficult to maintain TCI because of heart failure from high afterload. Blood outflow from left ventricle is completely obstructed except for coronary circulation which is small relative to the blood volume expelled from left ventricle, even if venous return to the heart is reduced. Aorto-atrial bypass formation during aortic clamping provides two distinctive advantages. First, adjusting aortic pressure in an appropriate level low enough not to overload myocardium but still high enough to maintain sufficient coronary blood flow is possible by regulating the blood flow through the bypass tubing, and secondly drug administration and blood volume control is possible through the tubing. These result in better preservation of myocardium, enabling longer TCI and longer survivals after TCI. We were successful in having up to 18 min of TCI with this method. Seventy-five percent of dogs of 12 min TCI and 40% of 15 min TCI survived 7 days, limit of experiment, after TCI, but no dogs of 18 min TCI survived for more than 3 days. PMID- 3020660 TI - Hemodynamic effects of naloxone in anaphylactic shock. AB - Recent reports suggest that endorphins may contribute to hemodynamic depression in septic and hemorrhagic shock. There is also evidence that reversal of endorphin effects with high dose naloxone may improve hemodynamic function and improve survival in shock states. The purpose of this study was to examine the effects of naloxone on hemodynamic parameters in anaphylactic shock. Anaphylactic shock was induced in sensitized rabbits with horse serum. Three minutes after serum challenge, rabbits were treated with a 3 mg/kg bolus of naloxone followed by a 3 mg/kg per h infusion (group I, n = 8), or by injection with an equal volume of saline (group II, n = 8). Cardiac output, blood pressure, heart rate and body temperature were monitored continuously for 60 min and the experiment was terminated. There was a significant increase in cardiac index in group I animals at 10 min (P less than 0.01) and 15 min (P less than 0.01). Stroke volume index was also higher in naloxone treated animals at 10 min and 15 min (P less than 0.05). Although mean blood pressure was higher in group I animals at all time intervals after naloxone was begun, the difference was statistically significant only at 60 min (P less than 0.05). Peripheral vascular resistance index was not significantly different for the two groups. PMID- 3020662 TI - Continuous intravenous infusion of sodium thiopental for managing drug withdrawal syndromes. AB - This report describes the successful use of sodium thiopental by continuous infusion in three patients with drug withdrawal syndromes and the concomitant need for mechanical support of ventilation. This approach provided the sedation necessary so the patients could be effectively ventilated while simultaneously controlling the manifestations of drug withdrawal. PMID- 3020663 TI - The comparative pathology of open chest vs. mechanical closed chest cardiopulmonary resuscitation in dogs. AB - We compared the pathologic changes following open-chest cardiopulmonary resuscitation (OCCPR) vs. closed chest cardiopulmonary resuscitation (CCCPR) in 28 healthy mongrel dogs subjected to experimentally induced ventricular fibrillation (VF). VF was induced in 29 dogs. No treatment was given for 3 min, then mechanical CCCPR was given for the next 12 min. External defibrillation (80 joules) was then attempted twice. One dog was resuscitated. The remaining 28 dogs were divided into 2 groups of 14 each. Group A received continued CCCPR and group B received OCCPR. All dogs received advanced cardiac life support and were followed until resuscitated or dead. All dogs were autopsied and gross pathology scores and histopathology scores were determined for each animal, and for each of 19 separate tissues within each animal. The mean gross pathology scores for the following tissues were significantly greater for dogs that received OCCPR vs. those that received CCCPR: skin (3.4 vs. 1.2; P less than 0.001), subcutaneous tissue (3.7 vs. 0.6; P less than 0.001), chest wall muscle (3.7 vs. 0.5; P less than 0.001), and pleura (1.9 vs. 0.1; P less than 0.001). The mean total gross pathology score was also greater in dogs that received OCCPR vs. those that received CCCPR (17.2 vs. 7.7; P less than 0.001). The mean histopathology scores for the following tissues were significantly greater for dogs that received OCCPR vs. those that received CCCPR: skin (2.5 vs. 0.0; P less than 0.001), subcutaneous tissue (2.2 vs. 0.1; P less than 0.001), muscle (2.3 vs. 0.1; P less than 0.001), pleura (1.6 vs. 0.0; P less than 0.001), pericardium (1.4 vs. 0.2; P less than 0.01), epicardium (2.5 vs. 0.2; P less than 0.001), myocardium (2.5 vs. 0.3; P less than 0.001), and endocardium (1.9 vs. 0.5; P less than 0.01). The mean total histopathology score was also greater in dogs that received OCCPR vs. those that received CCCPR (20.1 vs. 7.4; P less than 0.001). The histopathology score for brain tissue was greater for the CCCPR group than for the OCCPR group (1.9 vs. 0.4; P less than 0.05). This study showed that OCCPR in dogs following VF caused more severe pathologic changes than CCCPR. These changes were attributed to thoracotomy-induced chest wall injury and to internal defibrillation induced myocardial injury. However, OCCPR caused less severe microscopic brain lesions than CCCPR. PMID- 3020664 TI - Ischemia modifies protein distribution in gerbil brain subcellular fractions. AB - This work deals with changes in sedimentation of proteins in subcellular fractions isolated from the ischemic brain of Mongolian gerbils. We analysed protein content and the activity of marker enzymes in P2, S2, M and S3 subfractions isolated after 10 min ischemia, ischemia combined with apnea or 1 h later, after recirculation. On the basis of protein balance, the distribution of marker enzymes and electrophoretic analysis of proteins in brain subfractions it was shown that in ischemia some fraction of proteins which are normally cytosolic and microsomal, cosediment with the crude mitochondrial pellet. This effect, aggravated in ischemia complicated by apnea, is reversible and after 1 h recirculation the protein distribution in the brain normalizes completely. PMID- 3020665 TI - [Endorphin and respiratory regulation]. PMID- 3020666 TI - [Follow-up of genital herpes infection in volunteer couples]. PMID- 3020667 TI - [Current treatment of small-cell anaplastic bronchial cancer (II)]. PMID- 3020668 TI - [Frequency of the rotavirus antigen, detected by the ELISA method, in the feces of patients with infantile enteritis]. PMID- 3020669 TI - [Pectins--important components of dietary fiber with complex implications in nutrition, medicine and pharmacy]. PMID- 3020670 TI - Detection of group rotavirus antigen with Romanian ELISA kits in the acute gastroenteritis of the infant and young child. PMID- 3020671 TI - Experimental data on the antiulcerous action of a new pharmacological combination. PMID- 3020672 TI - [Germ cell tumors of the mediastinum]. PMID- 3020673 TI - [Hypoglycemia in the adult caused by a tumor or endocrine hyperplasia of the pancreas]. PMID- 3020674 TI - REM sleep deprivation antagonizes morphine-induced akinesia and catalepsy. AB - An examination was made of the effect of REM sleep deprivation (REMSD) on some forms of altered motor activity, such as akinesia and catalepsy, induced by intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) administration of morphine in adult, male Wistar rats. Administration of morphine (25 mg/kg i.p.) induced an akinetic-cataleptic syndrome and decreased spontaneous vertical motor activity (SVMA) in animals allowed undisturbed sleep. REMSD decreased the morphine-induced akinesia and catalepsy that are known to be mediated by an inhibitory mu-opiate system. The locomotor depressant action of morphine was converted to excitation (manifested as increased SVMA and hopping behavior) by REMSD. Similarly, decreased motor activity following i.c.v. administration of morphine (25 micrograms) was replaced by excitation in the form of jumping behavior after REMSD. Naltrexone (1 mg/kg i.p.) blocked the akinetic and cataleptic effects, but not the excitatory effects, of morphine. It is suggested that REMSD is associated with a functional insufficiency of an inhibitory mu opiate system, thus unmasking the excitatory morphine effects. The proposed insufficiency of an endogenous opioid system might explain an increase in neuronal excitation during REMSD and the therapeutic effect of REM deficiency in some types of depression. PMID- 3020675 TI - [Present status of rotavirus infection in nursing infants and children]. PMID- 3020676 TI - [Acute, recurrent medio-cubital nerve compression of the wrist caused by deposits of pyrophosphate crystals. A case]. PMID- 3020677 TI - Inhibition of erythropoiesis by human parvovirus-containing serum from a patient with hereditary spherocytosis in aplastic crisis. AB - Aplastic phase serum from a patient with aplastic crisis of hereditary spherocytosis, which was demonstrated to contain human parvovirus, inhibited in vitro erythroid colony formation almost completely. Human parvovirus was resistant to heating for 30 min at 56 degrees C. The suppressive effect of the serum was completely abrogated by adding convalescent phase serum from another patient with aplastic crisis of hereditary spherocytosis. Some normal sera had similar neutralizing ability. The results suggested that aplastic crisis of a patient with hereditary spherocytosis is caused by human parvovirus and that the neutralizing test could offer a tool for predicting the future occurrence of aplastic crisis in the patients with chronic hemolytic anemia. PMID- 3020679 TI - Why do some individuals produce autoreactive antibodies against receptors and/or their ligands? A possible answer to the question. A review with implications. PMID- 3020678 TI - Bone-marrow regeneration after therapy-induced hypoplasia monitored by serum measurements of lactoferrin, lysozyme and myeloperoxidase. AB - Serum levels of lactoferrin, lysozyme and myeloperoxidase were measured sequentially after induction treatment in 30 patients with AML in order to test the hypothesis that these proteins may be used to monitor activity and different stages of myelopoiesis in the bone-marrow. The results showed that myeloperoxidase and lysozyme started to rise again 6-8 days after initiation of treatment as compared to 12 d for lactoferrin and 16 d for blood polymorphonuclear leukocytes. The rate of marrow regeneration was exponential and estimated to be 9-20% per d. The comparison between two different chemotherapy regimes showed that the initial reduction in lysozyme, in contrast to other measured variables, was significantly larger with the therapy that contained vincristine and glucocorticosteroids. This might reflect a reduction in lysozyme secretion in addition to the effects on the leukemic cell mass. We conclude that the measurements of lactoferrin, lysozyme and myeloperoxidase in serum under certain circumstances may be used to monitor the myelopoietic activity in the bone-marrow. PMID- 3020680 TI - The role of superoxide anion and lysosomal enzymes in anti-listerial activity of elicited peritoneal macrophages. AB - The in vitro effect of superoxide anion and lysosomal enzyme activity on the killing of Listeria monocytogenes EGD (listeria) by peritoneal macrophages (PM) was investigated. Generation of superoxide anion by PM stimulated with phorbol myristate acetate (PMA) was significantly increased by intraperitoneal injection of Lactobacillus casei YIT 9018 (LC9018) or Corynebacterium parvum (CP), but not by injection of peptone. However, superoxide anion generation by LC9018-elicited PM stimulated with listeria was not increased any more than that by peptone elicited PM, and generation of superoxide anion by the PM was affected by the difference in stimuli. The killing of listeria by LC9018- or CP-elicited PM in vitro was significantly less than that by peptone-elicited PM or resident PM. Significant correlation was observed between the anti-listerial activity of PM and the intracellular killing of listeria by PM. On the other hand, beta glucuronidase and beta-N-acetyl-D-glucosaminidase activities of LC9018-elicited, CP-elicited, or resident PM were significantly weaker than those of peptone elicited PM, and no significant correlation was observed between the increase in beta-glucuronidase and beta-N-acetyl-D-glucosaminidase activities and the increase in anti-listerial activity. These results suggest that increase in the activity of lysosomal enzymes such as beta-glucuronidase and beta-N-acetyl-D glucosaminidase is not correlated with the anti-listerial activity of PM, and that superoxide anion has very little effect on the anti-listerial activity of PM in vitro. PMID- 3020681 TI - [Clinical value of gastrointestinal bleeding scintigraphy in vivo using 99mTc labeled erythrocytes]. AB - 99mTc labeled red blood cell imaging was performed in 13 patients with clinical evidence of gastrointestinal bleeding from an unknown source. In all these patients the bleeding sites had remained unclear after the standard diagnostic evaluation, including upper gastrointestinal endoscopy (13 patients), colonoscopy (12 patients) and angiography (4 patients). Nine of 13 patients (70%) had a scan indicating active bleeding. In the 10 patients in whom the bleeding site was definitely identified by endoscopy, arteriography, or surgery, scintigraphy correctly localized the bleeding site in 6 (60%). One false positive localization was noted. Bleeding was detected in 7 of 9 patients with melena and in 2 of 4 patients with occult bleeding and chronic anemia. In all but 1 patient, additional diagnostic investigations were needed to finally establish the bleeding site. In patients in whom a potential bleeding site has been identified by standard diagnostic tests, 99mTc red blood cell imaging may provide a reliable noninvasive test by which to document active bleeding from the suspected source. PMID- 3020683 TI - [Vasoactive intestinal polypeptide (VIP)--new radioimmunoassay and its possible clinical applications]. AB - Vasoactive intestinal polypeptide (VIP), a basic polypeptide mainly found in neural tissue, has been associated with specific endocrine pancreatic tumors. In the present study, a sensitive and specific radioimmunoassay for VIP is described and its clinical application discussed. PMID- 3020682 TI - [Estrogen and progesterone receptors in human liver: does their study contribute to a better understanding of benign contraceptive-associated liver tumors?]. AB - The estrogen and progesterone receptor content of liver cytosol was measured in female patients with focal nodular hyperplasia associated with oral contraceptive use and compared with the receptor content of non-tumorous liver and of primary hepatocellular carcinomas. Receptors were found in very low concentrations or were not measurable at all. In one case of focal nodular hyperplasia the estrogen receptor content of the tumor was higher than that in the adjacent normal liver. Malignant liver tumors and the male liver were characterized by a low or non measurable receptor content. The study of nuclear receptors combined with the use of monoclonal antibodies may be more helpful in elucidating the complex relationship between oral contraceptive use, benign liver tumors and hepatic steroid receptors. PMID- 3020685 TI - [Fibrolamellar hepatoma]. AB - Unlike hepatocellular hepatoma (HCC), the so-called fibrolamellar hepatoma (FLH) is found almost exclusively in the non-cirrhotic, non-infected liver. Patient characteristics and the course of the disease in FLH differ markedly from HCC. FLH represents only a small portion of hepatomas in general (approx. 2%), but accounts for over 40% of hepatomas in young adults. We present the case of a 38 year-old woman showing the typical histological findings of polygonal eosinophilic tumor cells and characteristic lamellar bundles of fibrous stroma, which led to the diagnosis of FLH. The approx. 140 cases of FLH published in the world literature are also presented and discussed. The usefulness of additional examinations (neurotensin, vitamin B12 binding capacity and copper stains) is mentioned. The difficulty in diagnosing FLH lies in its histological differentiation from focal nodular hyperplasia. When diagnosed early, however, FLH is characterized by good resectability with a chance of cure or at least a markedly better survival rate than HCC. PMID- 3020684 TI - [Hepatocellular carcinoma, hepatitis B virus and the immune system]. AB - Epidemiologically, two main geographical areas for hepatocellular carcinoma (HCC) can be distinguished: China, other parts of Asia and parts of Africa are high incidence zones for HCC. The HBsAg-associated form accounts for more than 90% of all cases. HBV-infections are frequent and most often occur early in life. Also, the prevalence of HBsAg carriers is high. HCC most often appears in midlife and in patients with formerly quiescent chronic hepatitis and cirrhosis. The USA, Western and Northern Europe represent low incidence zones for HCC, with the HBsAg associated form accounting for less than 40% of all cases. HBV infections occur less frequently and most often after adolescence. The HBsAg carrier rate is low. HCC most often develops in elderly men with formerly active chronic hepatitis and cirrhosis. For the development of HBsAg-associated HCC the integration of viral DNA (HBV-DNA) into the host genome seems crucial; it may occur after a longterm productive HBV infection which is then associated with a semi- or non-productive infection. Initially the HBV-DNA is integrated at random and later becomes monoclonal. HBsAg negative HCC may be due to oncogenic stimuli resulting from recurrent bouts of liver destruction and regeneration induced by alcohol, other hepatotoxic substances or by the virus(es) responsible for chronic non-A/non-B hepatitis. Defective immune elimination of HBV-infected cells also appears crucial for the development of HCC, in three stages: initially not all HBV infected cells are destroyed and chronic infection results. Then cells with latent HBV infection are insufficiently eliminated and finally HCC-cells escape from immune elimination altogether. Hepatitis B vaccination is the most promising measure for prevention of HBsAg-associated HCC. Other strategies such as immune modulation and/or virostatic principles are considered. PMID- 3020686 TI - Vitamins, fiber, and cancer. PMID- 3020687 TI - Human monoclonals from antigen-specific selection of B lymphocytes and transformation by EBV. AB - Hybridoma technology has made it possible to prepare monoclonal antibodies with the use of murine lymphocytes. Attempts to extend this technology to the human level, however, have met with difficulties. A method has been developed for making human monoclonal antibodies of predetermined specificity. Biotinylated antigens (human thyroglobulin or tetanus toxoid) were incubated with human B lymphocytes from peripheral blood. The lymphocytes to which the antigens bound were selected by fluorescence-activated cell sorting. Positively selected (high fluorescence) and negatively selected (low fluorescence) cells were then transformed with Epstein-Barr virus (EBV) and grown in microculture wells. All wells from the positively selected fraction produced antigen-specific antibody (95 to 1800 nanogram-equivalents per milliliter), whereas fewer than 6% of the wells from negatively selected fraction made any detectable antibody (less than 10 nanogram-equivalents per milliliter). When the positively selected EBV transformed cells were cultured in limiting dilution, clones were obtained that made antigen-specific monoclonal antibodies. By this method, monoclonal antibodies to both foreign antigens and autoantigens can be prepared from the normal human B-cell repertoire. PMID- 3020688 TI - Synchronized rearrangement of T-cell gamma and beta chain genes in fetal thymocyte development. AB - Kinetics of mouse T-cell gamma gene rearrangements in ontogeny were determined as an approach to understanding the possible role of these genes in the development of fetal thymocytes. Two of these genes (C gamma 1 and C gamma 2) rearranged rapidly during days 14 to 17 of the gestational period in BALB/c mice. Moreover, these rearrangements seemed to be tightly synchronized with rearrangements of T cell receptor beta chain genes in the same cells. It is suggested that the early transcriptional activity of gamma genes, which precedes that of beta chain genes, may not reflect the functional activation of these genes. Nevertheless, productive and therefore potentially functional gamma gene rearrangements precede surface expression of T-cell receptors in the thymus by 2 to 3 days, which is compatible with a role for gamma gene products in thymocyte development prior to antigen-specific stages. PMID- 3020689 TI - Mystery disease at Lake Tahoe challenges virologists and clinicians. PMID- 3020690 TI - Neuroleukin: a lymphokine product of lectin-stimulated T cells. AB - Neuroleukin is a lymphokine product of lectin-stimulated T cells that induces immunoglobulin secretion by cultured human peripheral blood mononuclear cells. Neuroleukin acts early in the in vitro response that leads to formation of antibody-secreting cells, but continued production of immunoglobulin by differentiated antibody-secreting cells is neuroleukin-independent. Although the factor is not directly mitogenic, cellular proliferation is a late component of the response to neuroleukin. Neuroleukin does not have B-cell growth factor (BCGF) or B-cell differentiation factor (BCDF) activity in defined assays. Neuroleukin-evoked induction of immunoglobulin secretion is both monocyte- and T cell-dependent. PMID- 3020691 TI - Genomic analysis of the human B-lymphotropic virus (HBLV). AB - The human B-lymphotropic virus (HBLV) has a double-stranded DNA genome of greater than 110 kilobase pairs, which is consistent with its morphological classification as a herpesvirus. A 9000-base pair cloned probe of HBLV detected specific sequences in DNA and RNA of infected cells but did not hybridize to the genomic DNA of other human herpesviruses including the Epstein-Barr virus, human cytomegalovirus, herpes simplex type I, and varicella-zoster virus. Conversely, while probes obtained from each of the known human herpesvirus readily detected the homologous viral DNA, they did not hybridize to genomic HBLV DNA. This evidence, in addition to serological and morphological distinctions and the biological effects of this virus demonstrate that HBLV is a novel human herpesvirus. PMID- 3020693 TI - Therapy with cisplatin and etoposide for non-small-cell lung cancer. PMID- 3020692 TI - The use of VP-16 plus cisplatin during induction chemotherapy for small-cell lung cancer. AB - In an attempt to circumvent innate or acquired tumor-cell resistance to chemotherapy, patients with small-cell lung cancer (SCLC) were treated with induction therapy that incorporated two active and potentially non-cross resistant chemotherapy regimens on two National Cancer Institute of Canada (NCI C) trials. Patients with limited disease (LD) SCLC were treated with cyclophosphamide, doxorubicin (Adriamycin [Adria Laboratories, Columbus, Ohio]) and vincristine (CAV) and VP-16 plus cisplatin in two different sequences. One arm was randomized to receive CAV alternating with VP-16 plus cisplatin for a total of six treatment cycles, and the other arm received three courses of CAV followed by three courses of VP-16 plus cisplatin. Both treatment strategies produced similar response rates and survival curves, and each treatment group has a projected 2-year survival of 20%. Patients with extensive disease (ED) were treated with either six cycles of CAV (standard regimen) or CAV alternating with VP-16 plus cisplatin for a total of six treatment cycles. In this study, the alternating regimen produced a higher complete response (CR) rate (40% v 27%) and overall response rate (61% v 39%; P less than .01). The progression-free survival was also superior for the alternating arm (P = .001), as was overall survival (P less than .05). The frequency of thrombocytopenia and severe gastrointestinal toxicity was slightly greater in the alternating arm, but the frequency of neutropenia and infection was less. The alternation of CAV and VP-16 plus cisplatin during induction therapy is an effective treatment strategy in the management of SCLC and superior to CAV alone in extensive SCLC. PMID- 3020694 TI - First-line therapy with VP-16 and cisplatin for small-cell lung cancer. AB - VP-16 and cisplatin were used as first-line therapy in 31 patients with small cell lung cancer (SCLC) in whom chemotherapy regimens that contained doxorubicin (Adriamycin [Adria Laboratories, Columbus, Ohio]) were contraindicated because of severe cardiac or hepatic disease. Eight patients who had cerebral metastases at presentation were also included in the study. There were 11 patients with limited disease (LD) and 20 with extensive disease (ED). Of the 28 evaluable patients, 12 (43%) had a complete response (CR) and 12 (43%) had a partial response (PR). Four patients (14%) failed to respond. The median duration of response (MDR) for LD patients was 39 weeks and for ED patients was 26 weeks. Patients with LD who responded (CR and PR) had a median survival time (MST) of 70 weeks (range, 28 to 232+ weeks), whereas ED patients who responded had an MST of 43 weeks (range, 17 to 68 weeks). Gastrointestinal toxicity was mild, but leukopenia and thrombocytopenia were common. Mild degrees of reversible nephrotoxicity occurred in 15 patients, but required discontinuation of cisplatin in only two. The results of this study are compared with several other recently published reports of VP-16 and cisplatin used as first-line therapy in SCLC. PMID- 3020695 TI - Alternating chemotherapy and thoracic radiotherapy with concurrent cisplatin etoposide for limited-stage small-cell carcinoma of the lung. AB - Seventy patients with limited-stage small-cell lung cancer (SCLC) were given six courses of chemotherapy alternating two drug combinations: a combination of cyclophosphamide, doxorubicin (Adriamycin [Adria Laboratories, Columbus OH]) and vincristine (CAV) was alternated with cisplatin and etoposide at 3-week intervals. Thoracic radiotherapy was administered concurrently with the first cisplatin-etoposide chemotherapy. Prophylactic cranial irradiation (PCI) was administered after the completion of all chemotherapy. No maintenance treatment was used. Seventy-six percent of patients achieved a complete clinical response. The median survival was 78 weeks and the 2-year survival rate was 32% with an average follow-up of 3 1/2 years. Seventeen percent are currently alive and disease free. Cisplatin and etoposide can be administered concurrently with thoracic irradiation with acceptable toxicity. Our results justify further clinical research using alternating chemotherapy and concurrent thoracic irradiation and cisplatin-etoposide chemotherapy. PMID- 3020697 TI - Concurrent chemotherapy and radiotherapy for limited small-cell carcinoma of the lung: a Southwest Oncology Group Study. AB - This pilot study in limited-stage small-cell carcinoma of the lung using concurrent cisplatin (Platinol) and etoposide (VePesid) chemotherapy with radiotherapy has yielded a high complete response rate in 23 of 40 patients evaluable for response. Five of these responders have survived greater than 2 years off all therapy with a stable, high performance status. Median survival of all patients is 18 months. Toxicity has been acceptable, the most common being neutropenia. Radiation toxicities include 17 of 40 patients experiencing mild to moderate esophagitis, with one severe toxicity; and three of 40 patients developing mild to moderate radiation pneumonitis. The high complete remission observed with this program and the long tumor-free interval seen off maintenance therapy deserve further exploration. Toxicities appear only moderately greater than with other programs currently utilized. PMID- 3020696 TI - Cisplatin plus VP-16 in small-cell lung cancer. PMID- 3020698 TI - High-dose pilot studies in extensive-stage small-cell lung cancer. AB - Small-cell lung cancer is rarely cured with standard chemotherapy, despite initial good responses. The concept of high-dose induction therapy for extensive stage patients has been explained in a series of pilot studies. The rationale of this approach and the most recent preliminary results will be presented. PMID- 3020700 TI - Cyclophosphamide, doxorubicin, and etoposide as first-line therapy in the treatment of small-cell lung cancer. AB - The discovery that etoposide is one of the most active drugs in small-cell lung cancer (SCLC) led to its incorporation into a number of first-line combination chemotherapy regimens. The cyclophosphamide/doxorubicin/etoposide (CAE) regimen was shown to be as active or more active than other standard regimens in nonrandomized studies. In a randomized study presented in this report, the CAE regimen was significantly superior to the cyclophosphamide/doxorubicin/vincristine (CAV) regimen for response duration and survival in extensive-stage patients. In limited-stage patients the results were slightly better with CAE. In addition, CAE lacked the neurotoxicity of CAV. PMID- 3020699 TI - Clinical trials of cyclophosphamide, etoposide, and vincristine in the treatment of small-cell lung cancer. AB - Etoposide is one of the most active drugs used in the treatment of small-cell lung cancer (SCLC). Recently, studies were completed that evaluated the substitution of etoposide for doxorubicin (Adriamycin) used in combination with cyclophosphamide and vincristine. This study has shown that equivalent antitumor activity, as measured by objective response, can be obtained with the combination of cyclophosphamide, etoposide, and vincristine (CEV) as compared with the CAV combination. A longer response duration and median survival are seen in extensive disease patients treated with the CEV combination. As expected, no cardiotoxicity is associated with CEV therapy, and interestingly, there is no potentiating neurotoxicity with CEV. A study recently completed has shown that CEV can be effectively combined with intensive radiation therapy to the chest administered simultaneously. CEV appears to be an effective alternative to CAV, and it can be readily combined with aggressive radiation therapy. PMID- 3020701 TI - Initial therapy with cisplatin plus VP-16 in small-cell lung cancer. AB - Cisplatin plus VP-16 has become a widely used salvage regimen for CAV (cyclophosphamide, doxorubicin [Adriamycin], and vincristine) failures. However, the major value of this two-drug combination will probably be as an integral part of the initial therapeutic strategy. Cisplatin plus VP-16 has been used as induction therapy in four separate published studies involving 238 patients. The response rates ranged from 71% to 94% and complete remission (CR) rates varied from 30% to 53%. Cisplatin plus VP-16 has also been alternated with CAV in several phase II studies with encouraging results. A Southeastern Cancer Study Group (SECSG) protocol in extensive disease is currently testing this hypothesis in a random prospective study. A recently completed SECSG protocol in limited small-cell lung cancer tested the concept of late intensification with two courses of cisplatin plus VP-16 following six courses of CAV, v six courses of CAV alone. Presently, there is a statistically significant survival advantage for cisplatin plus VP-16. PMID- 3020702 TI - Doxorubicin, cyclophosphamide, etoposide and platinum, doxorubicin, cyclophosphamide and etoposide for small-cell carcinoma of the lung. AB - Small-cell lung cancer (SCLC) is a disseminated disease regardless of our ability to document all sites. Chemotherapy is thus the cornerstone of treatment. There are multiple active single agents, resulting in many combination chemotherapy regimens. Optimal combinations are probably derived from the use of synergistic drug interactions. A three drug combination of doxorubicin (Adriamycin), cyclophosphamide, and etoposide (ACE) has been used at the University of Maryland Cancer Center (UMCC) for more than a decade in sequential studies. Two hundred four patients, 143 men and 61 women, were treated on these studies. Eighty-five had limited disease (LD) and 119 had extensive disease (ED). The complete response (CR) frequencies were 65% and 43% for LD and ED, respectively, and the median survivals were 15 and 9.5 months, respectively. Twenty-two percent of the LD patients were alive at 2 years. To improve upon response or survival, and because of synergy, cisplatin was added to ACE (PACE). PACE chemotherapy was administered in two studies--study 1 at UMCC for both LD and ED, and study 2 by Cancer and Acute Leukemia Group B (CALGB) for ED only. Preliminary review suggests that CR frequencies for LD (53%, study 1) and ED (44%, study 1; 37%, study 2) were similar to prior studies, but median survivals (LD, 18+ months, study 1; ED, 15 months, study 1, 10.5 months, study 2) appears superior to previous studies. However, PACE is more toxic than ACE. Further studies of PACE are needed to assess if the additional toxicities are warranted. PMID- 3020703 TI - Small-cell lung cancer: a 10-year perspective. AB - Over the past 10 years, four clinical trials have been conducted with a total of 528 patients with previously untreated small-cell lung cancer. Trials I to III were randomized phase III studies testing the value of prophylactic cranial irradiation, immunotherapy, and etoposide, respectively. They were done in the setting of combined modality treatment with the same local chest radiotherapy (3,000 rad in 10 fractions) and combined agent chemotherapy using a nucleus of CAV (cyclophosphamide, doxorubicin, vincristine). Trial IV was a pilot study evaluating sequential hemibody radiotherapy in a combined modality treatment program with CAV. Overall, the best treatment results to date have been observed following the combination of etoposide with CAV in trial III, particularly for patients with extensive disease. This combination with or without other agents also appears to be a common component of many trials that have reported particularly good survival results in this disease. PMID- 3020704 TI - Oral etoposide in small-cell lung cancer. AB - Etoposide can be administered orally. On the average, the bioavailability of etoposide administered in a soft gelatin capsule is approximately 50%. For both the oral and intravenous (IV) routes, there is a significant amount of inter- and intrapatient variability. In spite of the variability, clinical studies show that oral etoposide is clearly active in small-cell lung carcinoma (SCLC). In addition, combination chemotherapy studies using etoposide have yielded toxicity equivalent to IV administered programs such as CAV (cyclophosphamide, doxorubicin, vincristine). Oral etoposide has been used in several large combination chemotherapy studies safely and with clear efficacy. PMID- 3020705 TI - Outpatient administration of VP-16 and cisplatin. PMID- 3020706 TI - Brief overview of combination chemotherapy in non-small-cell lung cancer. PMID- 3020707 TI - A multicenter phase II trial of cisplatin and oral etoposide (VP-16) in inoperable non-small-cell lung cancer. AB - Sixty patients with inoperable non-small-cell lung cancer (NSCLC) were entered into a phase II study that tested the combination of cisplatin (80 mg/m2, day, etoposide intravenously (IV) (100 mg, days 1 and etoposide orally (200 mg/m2, days 3 and 5). The regimen was repeated every 28 days for six courses, after which patients were allowed to receive additional treatment at the discretion of their physician. Overall objective response rate in 51 evaluable patients was 69% (95% confidence interval: range, 56% to 81%), with 16% sustaining complete remission (CR), 53% partial remission (PR), 17% stable disease (SD), and 14% progressive disease (PD). CR was pathologically confirmed by bronchoscopy and biopsy. One patient with a clinical PR underwent surgery and was shown to have a pathologic CR. Median survival of all evaluable patients was 52 weeks, greater than 75 weeks for CR patients, 52 weeks for PR patients, 42 weeks for SD patients, and 13 weeks for PD patients. Eleven patients (21.5%) developed CNS metastases, which resulted in the deaths of ten. Survival was significantly correlated with extent of disease, performance status, and albumin level, but not with histology or weight loss. Tumor response was significantly correlated only with histology (squamous-cell and large-cell undifferentiated carcinoma greater than adenocarcinoma). Side effects were nausea, vomiting, anorexia, alopecia, bone marrow suppression, and nephrotoxicity. One patient died from leukopenia and sepsis. Pharmacokinetic studies in ten patients showed the continuous presence of etoposide in plasma for six days at a level of at least 220 to 480 ng/mL. In order to investigate whether this very effective combination of cisplatin and etoposide can prolong survival in NSCLC, it will be tested as preoperative chemotherapy in a randomized trial in operable patients with T1N1 and T2N0-1 disease. PMID- 3020708 TI - Elastic tissue in salivary gland tumours. PMID- 3020709 TI - Cost effectiveness of potential immunization interventions against diarrhoeal disease. AB - Estimates are made of the costs per death averted and the costs per case prevented by three possible immunization interventions against diarrhoeal disease in children. These estimates are based on cost information collected from a number of on-going national immunization programmes and from effectiveness estimates reported in previously published reviews. The first part of the paper reviews the state of current knowledge regarding immunization costs and converts data from 9 different studies into a common set of price equivalents. The second section assesses the composition of typical immunization programme costs and estimates the likely effect on existing costs of introducing new vaccines. Compatibility between existing EPI activity and the administration schedule of the new vaccine is likely to be a major determinant of increments in cost per fully immunized child. The third section brings together the cost information with estimates of the likely impact of measles, rotavirus and new cholera vaccines on mortality and morbidity from diarrhoea. PMID- 3020710 TI - Dietary fiber: its role in preventing gastrointestinal disease. AB - Only in relatively recent years has the role of dietary fiber, once thought to be an unnecessary and even undesirable by-product, begun to be appreciated in the maintenance of health. Research now indicates that a deficiency of fiber in the modern western diet may contribute to a host of diseases. Inadequate dietary fiber produces low fecal bulk, which in turn causes a high intraluminal pressure in the colon and may contribute to diverticular disease, appendicitis, and even carcinoma. This paper examines the evidence for these conclusions and the mechanisms for production of chronic gastrointestinal disease. PMID- 3020711 TI - Assignment of human nerve growth factor receptor gene to chromosome 17 and regulation of receptor expression in somatic cell hybrids. AB - Nerve growth factor (NGF) is a polypeptide hormone which plays a central role in the development and growth of sympathetic and sensory neurons. The effects of NGF on target cells are mediated by a specific cell surface structure, nerve growth factor receptor (NGFr), which has been identified in human cells as a 75,000-mol wt glycoprotein. We have used a monoclonal antibody to human NGFr to study cell surface expression of the receptor on a panel of mouse-human neuroblastoma hybrids, and the serological typing results permit assignment of the gene coding for NGFr (NGFR) to chromosome 17q21-qter. In addition to mouse-human neuroblastoma hybrids, human NGFr was also detected on hybrids derived from fusions between mouse L-cell fibroblasts and human neuroblastoma and melanoma cells. Furthermore, induction of human NGFr expression was observed in hybrids derived from NGFr- human kidney epithelial cells and mouse L cells, but not in hybrids derived from human kidney epithelial cells and mouse RAG kidney carcinoma cells. These results suggest that cell-surface expression of human NGFr is controlled by trans-acting regulatory signals. PMID- 3020712 TI - Analysis of hybridoma mutants defective in synthesis of immunoglobulin M. AB - Hybridoma mutants defective in the expression of IgM have been analyzed by molecular and somatic cell hybridization techniques. The frequency of kappa light chain mutants in the hybridoma PC7 was much higher than for other cell lines. In contrast to the mutations which we observed previously, the kappa mutants examined here resulted from complete or partial deletion of the kappa gene. Mutants defective in mu chain synthesis were of more diverse types including deletions, gross rearrangements, and more subtle changes. One mutant containing a cis-acting mutation resulting in reduced expression of the mu chain had an associated partial duplication of the mu gene, while others making low or undetectable levels of mu had no gross alterations in genetic structure. The usefulness of this approach to the study of gene structure and expression is discussed. PMID- 3020713 TI - Chinese hamster cells with a minichromosome containing the centromere region of human chromosome 1. AB - We describe a series of primary and secondary hamster-human hybrids which have selectively retained a small amount of human DNA. The hybrid XJM12.1.3 contains an estimated 4000-8000 kb of human DNA, and for a secondary hybrid derived from it, XEW8.2.3, our estimate is 1000-2000 kb. The hybridization of Southern blots of DNA from these hybrids with a variety of human satellite DNA probes reveals that these lines include centromere sequences of human chromosome 1. The identifiable human DNA is in the form of a minichromosome, as detected by in situ hybridization in the light microscope and in the electron microscope. At mitosis, the minichromosome can be observed to have kinetochores and to be associated with microtubules. Therefore, it can segregate in a stable fashion. It may be significant that in the selection of the hybrids we had selected for a human gene which has been mapped on human chromosome 1. PMID- 3020714 TI - Expression of human argininosuccinate synthetase after retroviral-mediated gene transfer. AB - The cDNA sequence for human argininosuccinate synthetase (AS) was introduced into plasmid expression vectors with an SV40 promoter or Rous sarcoma virus promoter to construct pSV2-AS and pRSV-AS, respectively, and human enzyme was synthesized after gene transfer into Chinese hamster cells. The functional cDNA was inserted into the retroviral vectors pZIP-NeoSV(X) and pZIP-NeoSV(B). Ecotropic AS retrovirus was produced after calcium-phosphate-mediated gene transfer of these constructions into the packaging cell line psi-2, and viral titers up to 10(5) CFU/ml were obtained. Recombinant AS retrovirus was evaluated by detecting G-418 resistant colonies after infection of the rodent cells, XC, NRK, and 3T3. Colonies were also obtained when infected XC cells were selected in citrulline medium for expression of AS activity. Southern blot analysis of infected cells demonstrated that the recombinant retroviral genome was not altered grossly after infecting some rodent cells, while other cells showed evidence of rearrangement. A rapid assay for detecting AS retrovirus was developed based on the incorporation of [14C]citrulline into protein by intact 3T3 cells or XC cells. PMID- 3020716 TI - [Results and value of mammography in male breast cancer]. PMID- 3020715 TI - Chromosomal assignment of gene encoding the largest subunit of RNA polymerase II in the mouse. AB - The gene encoding the largest subunit of RNA polymerase II was mapped to mouse chromosome 11 by Southern blotting analysis of mouse-Chinese hamster somatic cell hybrids and by in situ hybridization. This assignment extends the previously defined homology between mouse chromosome 11 and human chromosome 17. PMID- 3020717 TI - [Differential diagnosis of disseminated periventricular calcification]. PMID- 3020718 TI - Paediatric meningitis in the western Cape. A 3-year hospital-based prospective survey. AB - Between July 1981 and June 1984 1223 cases of meningitis were seen in the Department of Paediatrics, Tygerberg Hospital. The commonest form in each population group was aseptic meningitis. Positive viral cultures were obtained from the CSF in 108 cases. The median age of white children with aseptic meningitis, 64 months, was significantly greater than that of coloured children, 45 months (P greater than 0.0001), and black children, 26 months (P greater than 0.014). The commonest cause of confirmed bacterial meningitis was Neisseria meningitidis (140 cases; 11.5%), which continues to affect mainly young coloured children (median age 16.9 months). Resistance to sulphonamides was found among 21% of 114 N. meningitidis isolates. Among white children Haemophilus influenzae was responsible for 9 of the 18 cases of confirmed bacterial meningitis. Tuberculosis was responsible for 62 cases of meningitis (5%) and was a commoner cause of meningitis than either H. influenzae (47 cases) or Streptococcus pneumoniae (34 cases). Thirty-four confirmed cases of bacterial meningitis were seen in children less than 1 month old. Klebsiella species were responsible for 8 cases (24%), Escherichia coli for 6 cases (12%), group B beta-haemolytic Streptococcus for 5 cases (15%) while 4 cases each were due to N. meningitidis and Strept. pneumoniae. PMID- 3020719 TI - The spermicide nonoxynol-9 does not inhibit Chlamydia trachomatis in vitro. AB - The antimicrobial effects of the active ingredient, nonoxynol-9, and the base component of a commercially available spermicide were tested in vitro against Chlamydia trachomatis. The infectivity of cell-free elementary bodies was not affected by nonoxynol-9 or the base after incubation for 30, 60, or 180 min in direct contact with serial twofold dilutions of each agent. The spermicide's effect on the in-vitro growth of C. trachomatis was evaluated after exposure of C. trachomatis-infected McCoy cells to serial dilutions of each agent for 2 hr and for 72 hr. A significant cytopathic effect of nonoxynol-9 on the host cell membrane was observed at concentrations of spermicide between 100% and 0.0014%. However, neither agent was effective in inhibiting the intracellular growth of C. trachomatis at concentrations (0.0014-0.0004%) of nonoxynol-9 that produced no evident cytopathic effect on McCoy cells. Thus, nonoxynol-9 was found to be ineffective in inhibiting infectivity or subsequent growth of C. trachomatis. PMID- 3020720 TI - Lack of evidence for intertypic recombinants in the pathogenesis of recurrent genital infections with herpes simplex virus type 1. AB - Clinical observations indicate that herpes simplex virus type 1 (HSV-1) is significantly less likely than herpes simplex virus type 2 (HSV-2) to establish latency in (or reactivate from) sacral ganglionic tissue. In an effort to identify viral functions associated with latency, we analyzed HSV-1 isolates from three patients with established recurrent genital herpes and sought evidence of DNA sequences and proteins similar to those found in HSV-2. By restriction endonuclease cleavage patterns and by DNA hybridization analysis using either whole HSV-2 DNA or several cloned segments of HSV-2 DNA as probes, we found that the three HSV-1 isolates from patients with recurrent genital herpes showed no unusual homology to HSV-2 as compared with other HSV-1 isolates. Similarly, the proteins of these isolates could not be distinguished from those of other HSV-1 isolates and were distinct from those of HSV-2. At this level of resolution, there was no evidence to suggest that these recurrent genital HSV-1 isolates were intertypic recombinants, nor did they show any other unusual similarity to HSV-2. PMID- 3020721 TI - Transfer of plasmid-mediated ampicillin resistance from Haemophilus to Neisseria gonorrhoeae requires an intervening organism. AB - Haemophilus species have been implicated as the source of plasmid-mediated ampicillin resistance in Neisseria gonorrhoeae. Previous attempts to transfer conjugally the resistance plasmids from Haemophilus species to N. gonorrhoeae have met with limited success. Using both biparental and triparental mating systems, it was found that transfer will occur if the commensal Neisseria species, Neisseria cinerea, is used as a transfer intermediate. This organism stably maintains resistance plasmids of Haemophilus and facilitates transfer of these plasmids to N. gonorrhoeae, in a triparental mating system, at a transfer frequency of 10(-8). Both Haemophilus ducreyi and N. gonorrhoeae carry mobilizing plasmids capable of mediating conjugal transfer of the same resistance plasmids. However, restriction endonuclease mapping and DNA hybridization studies indicate that the mobilizing plasmids are distinctly different molecules. Limited homology is present within the transfer region of these plasmids. PMID- 3020723 TI - A review of the tumors of the salivary gland. AB - Neoplasms of the salivary gland are an interesting, diverse group of tumors with many associated factors, which make them a therapeutic challenge. Their long natural history, the wide spectrum of biologic activity which they exhibit, the difficulty in diagnosis and their high rate of recurrence contribute to the complexity of their management. Operation is the principle method of treatment for these tumors, but radiation therapy plays a significant role in the control and palliation of malignant tumors of the salivary gland. It is the hope for the future that effective chemotherapy agents will contribute significantly to the treatment of malignant tumors of the salivary gland. PMID- 3020722 TI - Effect of an experimental dentifrice on plaque accumulation and gingival inflammation. PMID- 3020724 TI - Brain metastasis: a rare manifestation of adenoid cystic carcinoma of the breast. AB - Adenoid cystic carcinoma of the breast is a rare neoplasm that usually has a benign biological behavior. A patient who was operated upon for removal of this tumor developed metastases to the lungs and brain 12 years after mastectomy. This is probably the first report in the literature of brain metastasis from adenoid cystic carcinoma of the breast. Radiological and histological evidence of both primary breast tumor and the metastasis to the brain are presented. PMID- 3020725 TI - Modified stage I (T1N0M0, T2N0M0), nonsmall cell lung cancer: treatment results, recurrence patterns, and adjuvant immunotherapy. AB - We analyzed 96 patients who had surgery with T1N0M0 or T2N0M0 nonsmall cell lung cancer (NSCLC) to identify survival rates and recurrence patterns in well-staged patients and to evaluate adjuvant therapy. Preoperative staging included chest x ray, gallium 67 scanning, and bronchoscopy in all patients. At thoracotomy, multiple mediastinal lymph node sites were routinely sampled. The results included an operative mortality rate of 5.2%, and the actuarial 5-year survival rate of all patients was 70.0%. Survival of T1N0 (n = 44) and T2N0 (n = 47) patients was 72.1% and 68.3%, respectively (p = NS). Survival was not affected by type of surgery, cell type, sex, age, or race. Late death was due to recurrence in 12 patients, a new airway malignancy in three, and a noncancer problem in six. Disease recurred in 15 patients: four (9.1%) T1N0 patients versus 11 (23.4%) T2N0 patients, p less than 0.05. Recurrence was local in four patients and distant in 11. Second lung cancers developed in six patients at a mean interval of 65.7 months after resection. A prospective, randomized trial of systemic immunotherapy with bacillus Calmette-Guerin (BCG) skin scarification was carried out in 29 patients. Survival in those patients receiving BCG was 85.9% compared with 63.9% for control subjects (p = 0.075) and 69.6% for patients not in the study (p = 0.077). The following conclusions can be made: Resection for well-staged, modified stage I NSCLC results in a 5-year survival rate of 70%. Nearly half the deaths are unrelated to recurrence of the original cancer. Recurrences are more frequent in T2N0 patients, but there is no survival difference compared with T1N0 patients. Systemic recurrences are more frequent than local recurrences, and there is an appreciable incidence of second lung cancers. Adjuvant chemotherapy or radiation therapy does not seem justified, but systemic immunotherapy holds sufficient promise to warrant further investigation. PMID- 3020726 TI - [Changes in the cardiovascular system in adenovirus infection in adults]. AB - The effect of adenovirus infection on the cardiovascular system of 37 adult patients with CHD and without signs of CHD was studied. It was shown that adenovirus infection caused lesion of the cardiovascular system with few clinical signs but with distinct ECG changes. They occurred in the 1st and 2nd weeks and were rather unstable. The most stable and noticeable ECG changes were recorded in CHD patients with a previous history of myocardial infarction and when adenovirus infection was complicated by pneumonia. PMID- 3020727 TI - [Various urgent problems of clinical enterology]. PMID- 3020728 TI - [Cyclic AMP level in T- and B lymphocytes in patients with rheumatoid arthritis and systemic lupus erythematosus]. AB - The content of cAMP in blood T and B lymphocytes was measured in 30 patients with rheumatoid arthritis (RA), 10 patients with systemic lupus erythematosus (SLE) and 28 donors. A statistically significant increase in the cAMP content in T lymphocytes of RA and SLE patients was established. On the contrary, in B lymphocytes of blood of RA patients and of SLE patients, in particular, the cAMP content was below normal. In female patients with RA, the cAMP content in T lymphocytes was appreciably higher than in male patients. PMID- 3020729 TI - [Mechanisms of hypertension in patients with terminal kidney failure on programmed hemodialysis]. AB - The state of some mechanisms involved in AP regulation was studied in 18 patients with terminal renal failure (TRF) on programmed hemodialysis for 7-9 mos. The imbalance of extra- and intracellular sodium, potassium and water causing hypertension, was noted in TRF patients. TRF patients revealed (against a background of the normal activity of plasma renin) a high activity of carboxycathepsin resulting in the creation of conditions for enhanced kinin degradation playing a depressor role and for intense angiotensin II formation, being an important mechanism of persistent hypertension. An increase in the activity of carboxycathepsin can be one of the reasons of hyperaldosteronemia detected in the patients. A single session of hemodialysis does not significantly influence the activity of carboxycathepsin. PMID- 3020731 TI - Oto-rhino-laryngological interventions performed in arduan relaxation. PMID- 3020730 TI - Caffeine effects on cyclic AMP levels in the mouse embryonic limb and palate in vitro. AB - Caffeine is a teratogen that causes limb and palate malformations in rodents. Since the ability to raise cyclic nucleotide levels is a known biological action of caffeine, cyclic AMP levels were measured in CD-1 mouse embryonic forelimb from whole embryo culture and embryonic limb and palate cells grown in primary culture following treatment with various concentrations of caffeine (0, 1, 3, or 10 mM). In forelimb buds from whole embryo culture, a dose-dependent response was observed. Caffeine at 1 mM concentration stimulated cyclic AMP levels to 151% of control value at 60 min. Even greater stimulation of cyclic AMP occurred at higher caffeine concentrations. A dose-dependent response was seen in both limb and palate cell culture. In limb cell culture, all caffeine concentrations significantly stimulated cyclic AMP after 10 min compared to control. In palate cell culture, there was a twofold increase in cyclic AMP at the 1-mM caffeine concentration. At higher caffeine concentrations, cyclic AMP was significantly increased after 60 min. In addition, stimulation of cyclic AMP in cultured limb and palate cells by isoproterenol, a beta-adrenergic agonist, was used as a positive control. Isoproterenol stimulated a 2.5-fold greater response in the palate cells than in the limb bud cells at isoproterenol levels of 10(-5) or 10( 4) M. The increase of cyclic AMP may be influential in the process of abnormal limb or palate development. PMID- 3020733 TI - [Sarcoidosis of the nervous system]. PMID- 3020734 TI - [Canine parvovirus infection in dogs: a consideration]. AB - The current knowledge of canine parvovirus (CPV) and the clinical symptoms associated with CPV infection within seven years after the first outbreaks of the disease are reviewed in the present paper. The most important symptoms result from the occurrence of acute enteritis and/or acute myocarditis. Besides characteristics of the virus, symptoms of disease and (histo)pathological findings, particular attention is focussed on recent developments in diagnosis and prevention. A protocol for the prevention of CPV infections in situations in kennels, consisting of combined vaccination and hygienic procedures is presented. PMID- 3020732 TI - Effects of a fish oil supplement on platelet function, haemostatic variables and albuminuria in insulin-dependent diabetics. AB - A randomised trial of the effects of 15 gm per day of a fish oil supplement (MaxEPA) on blood lipids, haemostatic variables (including platelet function) and albuminuria was undertaken in 41 insulin dependent diabetics. Compared with the control group there was a significant reduction in thromboxane production by platelets stimulated by collagen in vitro in the group who took the fish oil supplement. The extent of platelet aggregation was not altered but the lag phase before aggregation was prolonged. There were also statistically significant increases in plasma LDL cholesterol, fibrinogen and clotting factor X in the group who took the fish oil supplement. No other significant differences were noted. PMID- 3020735 TI - [Cattle calcinosis in the Lower Austria Alpine foothills area]. AB - It is reported on cases of calcinosis during winter 1979/80 occurring on 42 farms and leading to 34 forced slaughters and on the control of this disease in a localized region of the Alpine foot-hills. Instruction of the farmers, ploughing up of the meadows with following new sowing of grass without golden oat (Trisetum flavescens) allowed to push back calcinosis in high situated especially endangered farms. The different calcinogenic activity of golden oat in the different stages of growing, pushing back the golden oat by repeated utilisation of the pastures and keeping the cattle in the stable were successful methods. Additionally it was possible to influence the quantity of golden oat in pastures and meadows by methods influencing the growing such as climate, altitude, quality and quantity of fertilizer, age of the meadows. The importance of the Alpine pastures for prevention of calcinosis in cattle is pointed out. PMID- 3020736 TI - [Infectious diarrheal diseases in swine: morphologic and microbiologic findings]. AB - Infectious diarrhea in piglets can be caused by TGE, EVD and rota virus. The present report shows another viral agent (para-rota virus) to be present. In addition the report describes light and electronmicroscopic findings of the enteric mucosa caused by the agents mentioned. The degree and distribution of villous atrophy, one of the main histological findings, of infections with TGE, EVD, rota and para-rota virus is compared. A correlation of the results obtained with fluorescent antibody staining and ultrastructural diagnosis of agents was done. An application of the latter seems advisable in case of negative FA staining results. PMID- 3020737 TI - Ovarian membrane receptors for LH, FSH and prolactin during the menstrual cycle and in polycystic ovary syndrome. AB - To investigate the role of ovarian membrane receptors for LH, FSH and prolactin of patients with developing polycystic ovary (PCO) syndrome, seven patients were selected and the receptor function was studied in relation to changes of several serum hormone levels. The results were compared with those from individuals with regular menstrual cycles in the late follicular (LF; n = 6) and midluteal (ML; n = 6) phases. LH receptor binding in the normal cycle remained low (1.73 +/- 0.14 fmole/mg homogenate protein) in the LF phase and elevated 3 fold in the ML phase. LH receptors in the PCO patients maintained a higher binding level than that in the LF phase, being close to the ML level. FSH receptors were at a high level in the LF phase (4.09 +/- 0.40 fmole/mg homogenate protein), but decreased by 23% in the ML phase. In the PCO group the ovarian FSH receptor showed a high level, near to that in the LF phase. Prolactin receptors showed no significant changes among the two controls and PCO group. PCO patients showed increased levels of serum LH and testosterone and a raised ratio of estrone to estradiol, although there was no change in the serum FSH level. These data suggested that the LH receptor binding in PCO was not so low as to the LF level. A lack of the down-regulation mechanism of LH receptors, in spite of the high level of serum LH in PCO, might be one of the clues to elucidate the pathophysiological mechanism in developing PCO syndrome. Elevated levels of the gonadotropin receptors, especially FSH receptors, seem to be involved in the high incidence of ovarian hyperstimulation during hormone treatment. PMID- 3020738 TI - Immunohistochemical localization of carcinoembryonic antigen in carcinoma in pleomorphic adenoma of salivary gland: use in the diagnosis of benign and malignant lesions. AB - The localization of carcinoembryonic antigen (CEA) and lysozyme (LZM) was immunohistochemically studied in 34 carcinomas arising in benign pleomorphic adenomas and 25 normal salivary glands in order to assess its potential diagnostic value. CEA in the normal salivary gland was located in luminal cell membranes of intercalated duct cells and serous acinar cells. Strongly positive cell surface and intraluminal staining of CEA appeared in the areas of gland forming pattern in pleomorphic adenoma. CEA activity was detected in 7/9 cases (78%) of adenocarcinoma, 10/11 cases (91%) of epidermoid carcinoma, 3/8 cases (38%) of anaplastic carcinoma, 5/5 cases (100%) of mucoepidermoid carcinoma, and 1/1 case (100%) of adenoid cystic carcinoma. CEA was always present in the cytoplasm of epithelial cells and luminal contents of neoplastic glands. CEA in epidermoid carcinoma may occasionally react strongly in the cytoplasm. Lysozyme immunoreactivity was detected in the cytoplasm of intercalated duct cells and serous acinar cells of the normal salivary gland but little or no LZM was observed in any of the tumors. These results suggest that the presence of CEA could be a useful marker that provides valuable information for the differential diagnosis between benign and malignant areas of carcinoma in pleomorphic adenoma of the salivary gland. Moreover, LZM could be of valuable use for discriminating neoplastic from non-neoplastic tissue of salivary glands. PMID- 3020739 TI - The effects of perfluoro-n-decanoic acid in the rat heart. AB - Perfluoro-n-decanoic acid (PFDA) is a synthetic chemical resembling a 10-carbon fatty acid. Several studies have suggested that the toxic mechanism of PFDA may involve impaired lipid metabolism and/or altered cell membrane function. We examined the possibility that altered cell membrane structure in the heart might lead to changes in the functional activity of the organ. Functional characteristics were determined in the isolated perfused rat heart by measuring the ability of the heart to respond to either sympathetic nerve stimulation or infused norepinephrine. PFDA reduced the intrinsic resting heart rate and the inotropic response to a stimulus with maximal effects occurring 8 days after dosing. In addition, resting heart rate measured in vivo was found to be reduced in PFDA-treated rats 6 to 8 days after dosing. beta-Receptor binding studies conducted 8 days after a single dose of PFDA showed that the maximum binding capacity was reduced by PFDA treatment without significant changes in receptor affinity. It is concluded that the reduction in the inotropic response to catecholamines following PFDA treatment may be explained in part by lower beta receptor density in the myocardial cell membrane. These effects may be related to the early fall in serum thyroid hormone levels as previously reported. PMID- 3020740 TI - Macrocytic-megaloblastic anemia in male NIH Swiss mice following repeated exposure to 1,3-butadiene. AB - Thymic lymphoma/leukemia is the major cause of death in B6C3F1 mice chronically exposed to 1,3-butadiene (BD). Similar to radiation-induced murine thymic lymphoma, the bone marrow is also a major target organ. Because of the association of murine thymic lymphoma with endogenous type-C murine leukemia retroviruses (MuLV) present in the germ line of most strains of laboratory mice, including B6C3F1 and its parent strains, we examined the effects of BD exposure on NIH Swiss mice which do not possess intact endogenous ecotropic MuLV. Male NIH Swiss mice exhibited a macrocytic-megaloblastic anemia following inhalation of 1250 ppm BD for 6 weeks. Treatment-related changes included decreases in circulating erythrocytes, total hemoglobin, and hematocrit and an increase in mean corpuscular volume. An eightfold increase in circulating micronuclei was also observed. The anemia was not accompanied by a significant alteration in mean corpuscular hemoglobin concentration, an increase in circulating reticulocytes, or an increase in circulating nucleated erythrocytes. These findings are consistent with a treatment-related macrocytic-megaloblastic anemia and indicate that the bone marrow is an important target for BD toxicity in mice independent of MuLV background and expression. PMID- 3020741 TI - Polychlorinated terphenyls: alterations in liver morphology and induction of cytochrome P-450. AB - Exposure of male rats to the polychlorinated terphenyl (PCT) mixtures Aroclor 5460 and Aroclor 5432 containing 60% and 32% (w/w) of chlorine, respectively, showed that the PCT mixture with a low degree of chlorination, Aroclor 5432, was a potent inducer of liver microsomal cytochrome P-450, aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase whereas Aroclor 5460 and the unchlorinated isomers o-, m- and p-terphenyl were weak inducers. Ultrastructurally, proliferation of SER but not RER, frequent occurrence of lysosomes containing partially degraded lipid material as well as an increased number and size of cytoplasmic lipid droplets were observed. These changes were most pronounced in Aroclor 5432 treated rats. Competition experiments with [3H]2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD) for binding to the cytosolic receptor protein indicated the presence in Aroclor 5432, but not in Aroclor 5460, of components with receptor affinity. These components represent only a minor fraction of the mixture as judged by the 1900-fold excess necessary to displace 50% of the specific [3H]TCDD binding. Based on the biochemical results and the ultrastructural findings it is concluded that the PCT mixture Aroclor 5432 is a mixed type inducer of hepatic cytochrome P-450 in the rat. The presence in Aroclor 5432 of compounds capable of inducing AHH in vivo and of binding to the TCDD-receptor might be highly relevant with regard to the potential toxicity in view of the apparent correlation between affinity for the TCDD-receptor, induction of AHH activity and toxic properties for chlorinated aromatic hydrocarbons. PMID- 3020743 TI - Time-related asymmetric changes of brain microvessel beta-adrenergic receptors in the two hemispheres after carotid occlusion. AB - The effect of short term and long term ischemia induced by right carotid occlusion was studied on beta-adrenergic receptor function in rat cerebral microvessels. The results show a different time-dependent responsiveness of the two hemispheres to ischemia, with a pronounced and more persistent decrease in the number of capillary beta-receptors in the left side of the brain. The data suggest the existence of asymmetries in the control of brain microvasculature which may mediate the different time-course of beta-receptor changes in response to ischemia. PMID- 3020742 TI - Subarachnoid hemorrhage and granulomatous angiitis of the basilar artery: demonstration of the varicella-zoster-virus in the basilar artery lesions. AB - A 70-year-old man, with regional herpes zoster (C2) of 10 weeks duration, died following subarachnoid hemorrhage caused by the rupture of an aneurysm in the basilar artery. Granulomatous angiitis, with multinucleated giant cells, was found at autopsy in the wall of the aneurysm. Electron microscopy of the basilar artery disclosed intracytoplasmic viral particles with an envelope which measured 150-220 nm in diameter. Immunohistochemistry studies revealed varicella-zoster virus-related antigen in the cytoplasm and/or in the nucleus of histiocytes in the vessel wall. These findings suggest that varicella-zoster virus may be linked to the development of granulomatous angiitis. PMID- 3020744 TI - [Electron paramagnetic resonance method in the diagnosis of intravital formation in mechanical injuries to the large blood vessels]. PMID- 3020745 TI - Chemodectoma of the neck presenting with hemoptysis and a thyroid mass. PMID- 3020746 TI - Conversion from seropositive to seronegative for cytomegalovirus antibody in plateletapheresis donors. PMID- 3020747 TI - CMV infection, class II antigen expression, and human kidney allograft rejection. AB - After successful transplantation, the major histocompatibility complex (MHC) antigens of kidney parenchymal cells are lost and no longer detectable in the graft, presumably due to administration of glucocorticosteroids. During rejection, the MHC antigens reappear in the graft parenchymal cells. The upregulation is possibly due to gamma-interferon released in situ by the allograft activated T (blast) cells. In this communication we demonstrate that cytomegalovirus (CMV) infection is invariably associated with an upregulation of the antigen display in the graft. In 12 of 14 (86%) cases with proved CMV disease, the display of class II antigens was associated with a cytological and/or clinical episode of rejection. In 223 transplant recipients without proved CMV disease transplanted during the same period, the frequency of late rejections was 17% (P less than 0.001). The results suggest that the display of class II antigens on the graft, mediated presumably by gamma-interferon as a consequence of CMV infection, is the reason for graft rejection in context of CMV disease. PMID- 3020749 TI - [Characteristics of human-mouse hybrid cell lines and the changes induced in them by the poliomyelitis virus]. AB - Cells of the four hybrid lines between continuous mouse cells Rag and human diploid embryonal fibroblasts were polymorphic and had mitotic activity in fully formed monolayers. Most of the these mitoses were pathological. Hybrid cells examined 8 months after hybridization were susceptible to the poliomyelitis virus infection with partial cytopathologic effect, they produced virus antigens and the infectious virus. Small hybrid cells displayed a more pronounced cytopathologic effect than did big, polynuclear and mitotic cells. Hybrid cells that were passaged 1.5 months after infection did not excrete any infectious poliovirus but contained poliovirus antigens. PMID- 3020748 TI - Increased serum beta 2 microglobulin during rejection, cyclosporine-induced nephrotoxicity, and cytomegalovirus infection in renal transplant recipients. AB - In 83 renal transplant recipients, serum beta 2 microglobulin (beta 2m) levels were significantly elevated during pretransplant uremia, rejection, cyclosporine induced nephrotoxicity, and infections. In patients with normal serum creatinine, 74% had elevated serum beta 2m levels. None of the cyclosporine-treated patients had normal levels of beta 2m. Patients with stable renal allograft function receiving cyclosporine showed significantly higher serum beta 2m (P less than 0.001) and serum creatinine (P less than 0.01) levels than azathioprine treated patients. Patients with an irreversible rejection showed significantly higher serum concentrations of beta 2m than patients experiencing a reversible rejection (P less than 0.001). During cytomegalovirus (CMV) infection the serum beta 2m levels were elevated compared with other infections (P less than 0.001), while the serum creatinine was not. However, infected patients had higher serum levels of beta 2m and creatinine than patients with stable renal allograft function (P less than 0.001). Serum beta 2m may therefore be useful in the early diagnosis of CMV infection. To conclude, serum beta 2m levels cannot distinguish between rejection, cyclosporine nephrotoxicity, or infection. PMID- 3020750 TI - Concurrent outbreak of pseudo-lumpy skin disease and acute Trypanosoma vivax infection in cattle. AB - Pseudo-lumpy skin disease and acute Trypanosoma vivax infections occurred simultaneously in a dairy herd which had no previous history of trypanosomiasis. The onset of the outbreak was sudden and 29 out of the 40 adult Friesian and exotic x zebu cattle were found to have skin lesions. Five out of the nine animals sampled had fulminating T. vivax parasitaemias with packed red cell volumes ranging from 0.09 to 0.28 litres/litre. Whereas carried tsetse may have introduced T. vivax, the failure to trap tsetse and the presence of large numbers of biting flies strongly suggested that in this outbreak both aetiological agents were transmitted mechanically. PMID- 3020751 TI - Epidemic of goat dermatitis in India. PMID- 3020753 TI - Long-term survival and complete response to adriamycin and etoposide in a case of hepatocellular carcinoma. AB - Hepatocellular carcinoma has a poor prognosis and is poorly responsive to systemic chemotherapy. Here we describe a case with unresectable hepatocellular carcinoma who achieved a complete response and has survived for more than 2 years, following treatment with a combination of adriamycin and etoposide (VP 16 213). PMID- 3020752 TI - Growth characteristics of human colorectal and non-small cell lung tumors xenografted into nude mice: possible correlation with prognosis. AB - Specimens from human colorectal tumors and from non-small-cell lung tumors obtained at surgery were subcutaneously implanted as xenografts in athymic Swiss mice of both sexes to investigate to what extent the properties of the original tumors were maintained. A successful take was obtained in 5 of 9 colorectal tumor and 6 of 11 non-small-cell lung tumor xenografts. Moreover, 44% and 45% of the respective tumors could be established as tumor lines. Neither metastases nor local tumor invasion was observed in tumor-bearing mice. Seven of 9 serially transplantable tumors had a short latency period (14-30.5 days) when first xenografted. No significant changes in tumor histopathology were noted after growth into nude mice. Tumor take was partially related to clinical stage and prognosis of patients. In fact, 8 of 12 specimens from N0 patients failed to grow, whereas 7 of 8 tumors from patients with nodal invasion and/or metastasis grew in nude mice. Moreover, for the group of patients whose tumor was "take - ", the one-year survival was 85%, compared to 40% for the "take + " group (p less than 0.05). PMID- 3020754 TI - Cisplatin plus vindesine versus cisplatin plus VP16 versus doxorubicin plus cytoxan in non-small-cell carcinoma of the lung. A randomized study. AB - From March 1981 to January 1984, 116 patients with advanced non-small-cell carcinoma of the lung (NSCCL) were randomly assigned to 3 combinations as follows: CDDP + DVA, CDDP + VP16 and DXR + CTX. 94 patients were evaluable for response, 106 for toxicity and survival. Of 31 patients, 15 (48%; 3 CRs and 12 PRs) responded to CDDP + DVA; of 33 patients, 12 (36%, 2 CRs and 10 PRs) responded to CDDP + VP16; of 30 patients, 3 (10%) obtained a PR with DXR + CTX (CDDP + DVA vs DXR + CTX, P less than 0.005; CDDP + VP16 vs DXR + CTX, P less than 0.05; CDDP + DVA vs CDDP + VP16, P = NS). The median duration of response was 22 weeks in the CDDP-DVA group, 17 weeks in the CDDP-VP16 group, and 16 weeks in the DXR + CTX group. No significant difference in survival was observed among the 3 groups (median: 43, 47, 41 weeks, respectively). Hematologic and neurologic toxicities were significantly higher in the DVA-containing regimen. Despite the lack of improvement of overall survival with the CDDP-containing combinations over the DXR + CTX control group, the good response rate makes them suitable to be used in combined therapeutic strategies. PMID- 3020755 TI - Calcium pyrophosphate dihydrate microcrystal-associated arthropathy. PMID- 3020756 TI - A small cell tumor. PMID- 3020757 TI - The effect of DMSA loading on the renal handling of technetium-99m in rats. AB - The renal handling of technetium-99m dimercaptosuccinic acid (99mTc DMSA) was studied in rats treated with high doses of nonradioactive DMSA to inhibit the renal uptake mechanism(s). A static scan was obtained 1 hour after the intravenous (iv) injection of 99mTc DMSA and the radioactivity in kidneys and bladder was calculated as a percentage of the injected amount. Total glomerular filtration rate (GFR) and effective renal plasma flow were also determined. Preloading with DMSA caused a fall in the renal accumulation of 99mTc DMSA together with a small increase in the amount excreted into the urinary bladder. Despite a stable GFR, the total amount of 99mTc DMSA handled by the kidneys (i.e., renal plus bladder activity) was reduced. These findings are compatible with the hypothesis that peritubular uptake and subsequent intracellular fixation are of importance in the renal accumulation of 99mTc DMSA. On the other hand, the radioactivity excreted into the urine probably stems from non-reabsorbed 99mTc DMSA initially filtered by the glomeruli. PMID- 3020759 TI - [Detection of bovine leukemia virus antibodies using the cytotoxicity test in comparison with other serologic methods]. AB - In ninety-five serum samples taken in a herd of five-year to seven-year cattle that was heavily infected by bovine leukosis virus, the four serological assays were used for demonstration of the antibodies to bovine leukosis virus; cytotoxic test, immunodiffusion test in agar-agar, immunoenzymatic test and serum neutralizing test. The serum neutralizing test was found to be the most sensitive: further seven positive reagents were diagnosed in comparison with immunoenzymatic test; cytotoxic and immunodiffusion tests in agar-agar have the lowest sensitivity and the results of these tests are almost identical. It was found out in forty titrated samples that serum neutralizing test was by as much as 20 times more sensitive than immunoenzymatic test, the latter being about 50 times more sensitive than cytotoxic and immunodiffusion tests. PMID- 3020760 TI - [Radioimmunologic detection of antibodies to bovine leukemia virus]. AB - The radioimmunologic assay (RIA) was elaborated for a demonstration of serum antibodies to bovine leukosis virus. The procedure makes use of the viral antigen bond to the fixed phase of a polystyrene carrier. The method was compared with the ELISA method and pseudoneutralizing and immunodiffusion tests. High congruence of the results of the RIA and ELISA methods was achieved, making 95%. The RIA method is more sensitive than the immunodiffusion test. PMID- 3020758 TI - The use of micropuncture, isolated tubule, and vesicle technique in the study of the action of thyroid hormones on the proximal tubule function. AB - In hypothyroid rats (TX), the isotonic fluid reabsorption (Jv), that is closely linked to the transepithelial sodium transport (JNa), is impaired. The administration of physiological doses (10 micrograms/kg body weight per day) of tri-iodothyronine (T3) doubles Jv in three days (TX+T3). This phenomenon could be explained by several mechanisms: a direct stimulation of Na-K-ATPase, an increase in the Na+ entry step, changes in the permeability properties of the luminal and/or basal lateral membranes. Using a kinetic microassay, Na-K-ATPase activity was measured in early (S1) and late (S2) proximal tubules segments isolated from control, TX, and TX+3T3 animals. In TX rats the enzyme activity was lower (70%) in both segments versus control rats, it remained unchanged after 3 days, and it increased after 7 days of T3 substitution. The Na+ permeability of brush border membrane (BBM) vesicles isolated from TX and TX+T3 rats was identical. However the valuation of the K+ membrane permeability by in vivo perfusion of the lumen and peritubular space of proximal tubules of TX rats, with perfusate containing the K+ ionophore valinomycin (1 microgram/ml), induced a significant increase in Jv that accounted for 40% of that elicited by T3. Taken together, the in vivo and in vitro experiments suggest that the early effect on Jv of physiological doses of T3 cannot be explained by a direct action of T3 either on the Na+ entry step across the BBM or on the Na+ exit step (i.e., the Na-K-ATPase), but rather by an increase in K+ permeability of proximal tubular cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020761 TI - [Electron microscopy of Aujeszky's disease virus in explants of Gasser's ganglion from pigs with a latent infection]. AB - Different developmental stages of the Aujeszky's disease virus were demonstrated by electron microscopy in the ultra-thin slices by the cultivated fragments of the Gasserian ganglion (G. g.) of two pigs latently infected with the Aujeszky's disease virus (ADV). In a pig vaccinated with the inactivated vaccine against the disease, the virus was detected in the G. g. cells 186 days after virus challenge, the reactivation of latency being obtained after immunosuppression with dexamethasone. In the non-vaccinated pig the virus was detected in G. g. cells after three months from experimental infection. In the ultra-thin slices the largest amount of virus was located in the nuclei and cytoplasm of satellite and Schwann's cells, in the connective-tissue cells and in the extracellular space. In the ganglion cells the virus was present in the cytoplasm and sporadically in the myelinized axons. PMID- 3020762 TI - [Differential diagnosis of viral diarrheas in calves]. AB - There is a description of the enzymoimmunologic method (ELISA), which was used for demonstration of rotaviruses and coronaviruses in the samples of excrements of the calves suffering from diarrheas. It is shown by a comparison with the results obtained by electron microscopy that the ELISA method provides by up to 50% higher capture rate and the reaction is highly specific. The method can also be applied to a detection of human rotavirus in the children's stool. PMID- 3020763 TI - [The effect of short-term isolation of vaccinated chicks on the incidence of Marek's disease]. AB - In chickens immunized by normal and tenfold doses of MARVAK vaccine (100 and 1,000 PFU) we investigated protective effects against the natural contact infection of chickens that were exposed to the infection immediately after vaccination, or at the intervals of 48 and 72 hours, and 7 and 14 days. Studying the elimination of Marek's disease virus by feather follicles, the health condition of chickens, tumor frequency and mortality rate we demonstrated that neither of these doses could protect the chickens from the disease if they were exposed to contact infection just after vaccination. An expressive protective effect was observed in chickens isolated for 7-14 days and there was not recorded any difference in the protection against the disease after the doses of 100 and 1,000 PFU. The main and feasible measure against MD is to prevent an early infection of newborn chickens by improving the zoohygienic conditions. PMID- 3020764 TI - How drugs act in the body. PMID- 3020765 TI - Efficacy of porcine parvovirus vaccines. AB - Three inactivated porcine parvovirus vaccines were tested for efficacy in 66 susceptible gilts. The gilts were challenged with virulent virus on the 40th day of gestation. All the vaccines provided excellent protection against fetal mortality despite insignificant serological responses to one of them. Good protection was obtained with two of the vaccines even when the dose was substantially reduced. Unvaccinated controls had very few viable fetuses. PMID- 3020766 TI - First simultaneous isolation of influenza A virus and duck enteritis virus from commercial ducks in France. PMID- 3020767 TI - A togavirus-like agent in the pancreatic duct of chickens with infectious stunting syndrome. PMID- 3020768 TI - [Pneumonia-like form of bronchioalveolar lung cancer]. PMID- 3020769 TI - [Morphological x-ray signs of lung cancer depending on the rate of growth of the tumor]. PMID- 3020770 TI - Effect of exogenous corticosteroids on circulating virus and neutralizing antibodies in striped bass (Morone saxatilis) infected with infectious pancreatic necrosis virus. AB - Corticosteroids have been reported to induce immunosuppression in fish exposed to many types of bacterial antigens. We document a similar phenomenon in fish exposed to infectious pancreatic necrosis virus (IPNV). Fingerling striped bass that were injected with the steroid triamcinolone acetonide (100 mg/kg body weight) 24 hours before receiving intraperitoneal inoculation with IPNV became viremic 3 days post inoculation (dpi) and virus was still detected in the buffy coat cells 14 dpi. In contrast, viremia could not be detected after 7 dpi in fish that received virus but not steroids. Circulating virus neutralizing antibodies were first detected in steroid treated fish at 10 dpi compared to 7 dpi for the virus injected fish and titers were consistently lower in the steroid group. Steroid treatment of chronic IPNV-carriers did not induce detectable viremia nor alter circulating antibody levels in chronic IPNV-carriers. None of the striped bass demonstrated clinical signs of viral disease. PMID- 3020771 TI - Detection of impaired T cell-mediated immune responses to herpesvirus (BHV-1) in cattle. AB - Interleukin 2 (IL-2) functions in the regulation of cell-mediated immune responses both in vivo and in vitro. Therefore, IL-2 production has been studied in patients with immunological disorders to determine the level of the immune defect. However, the cause(s) of low responses to selected antigens by cells from clinically normal individuals has not been examined. The ability of cells from clinically normal individual cattle to produce and respond to IL-2 was investigated as a measure of specific cell-mediated immune response. Cells from the majority of animals (6/7 in a representative experiment) could produce IL-2 in response to mitogen or a specific antigen, Bovine Herpesvirus 1 (BHV-1). Proliferation of lymphocytes from the majority of low responding animals (2/3 and 4/4 in separate experiments) could be restored to a level similar to high responders by the addition of exogenous IL-2 after endogenous IL-2 depletion. IL 2 receptor expression was indirectly assessed by the ability of activated cells to absorb IL-2. One individual's cells were incapable of absorbing exogenous IL-2 and failed to proliferate indicating a lack of activation and expression of receptors. In addition, exogenous IL-2 was able to enhance proliferation of both high and low responders in the presence of endogenous IL-2. These results suggest that proliferation of bovine peripheral blood mononuclear cells is dependent on the presence of IL-2. In addition, IL-2 production can be used to measure specific cell-mediated immune responses. PMID- 3020772 TI - Phagocytosis and intracellular killing of Pasteurella multocida by porcine alveolar macrophages after infection with pseudorabies virus. AB - The purpose of this study was to determine if pseudorabies virus (PrV) interfered with normal alveolar macrophage phagocytic functions. Porcine alveolar macrophages (PAM) obtained by pulmonary lavage were exposed to PrV. At 1 hour postinfection, cells were challenged with Pasteurella multocida labeled with 3[H] thymidine. The phagocytosis assay was performed by measuring total radioactivity 1 hour after Pm challenge in a soft-beta spectrophotometer. Intracellular killing was measured by counting viable bacteria 3 hours after P. multocida challenge. Phagocytic values of PrV-infected and control PAM ranged from 11% to 20%, a non significant difference. Values for intracellular killing for PrV-infected PAM ranged from 7.1 X 10(5) to 1 X 10(6) in contrast to 5.1 X 10(1) to 1.8 X 10(2) for the control PAM. This difference in killing function was significantly lower in PrV-infected PAM than in control cells (P less than 0.01). This alteration of macrophage function may be a factor in the pathogenesis of PrV-Pm mediated pneumonia in pigs. PMID- 3020773 TI - Cultured bovine monocytes exhibit decreased release of superoxide anion and increased levels of lysosomal enzymes but do not secrete detectable lysozyme activity. AB - In this study we determined the effects of in vitro maturation on the phagocytic activity, lysosomal enzyme content and oxidative response of bovine monocytes. Intracellular levels of the lysosomal enzymes acid phosphatase, beta glucuronidase, and beta glucosaminidase increased as bovine monocytes matured in vitro. However, in marked contrast to the mononuclear phagocytes of other mammalian species, lysozyme activity was undetectable in the culture supernatants and cell lysates of adherent bovine blood monocytes cultured for one to fifteen days. In vitro maturation of bovine monocytes also increased their phagocytic activity as determined by the ingestion of opsonized yeast. A greater percentage of monocyte-derived macrophages that were stimulated with opsonized yeast and phorbol myristic acetate (PMA) reduced nitroblue tetrazolium than did similarly treated monocytes. Monocyte-derived macrophages stimulated with PMA released significantly less superoxide anion than did PMA-stimulated monocytes. Bovine monocytes and macrophages also failed to bind the monoclonal antibody Mac-1, which binds to human and mouse macrophages. Bovine monocytes demonstrated both similarities and differences with other mammalian mononuclear phagocytes, thus making them a useful model for further study of the comparative and developmental biology of mononuclear phagocytes. PMID- 3020776 TI - [Use of a microvirus-neutralizing reaction in diagnosing transmissible gastroenteritis (TGE)]. AB - A micro virus-neutralization reaction was designed and tested to detect antibodies to the virus of transmissive gastroenteritis. Use was made of a stable cell line, SPEV, and a laboratory strain of the virus that had been adapted to it. The optimal concentration values of the cell suspension and the normal calf serum contained in it were determined. A total of 90 blood serum samples from pigs were comparatively investigated for the presence of virus-neutralising TGE antibodies, the tube test being performed trough the inoculation of the cells in suspension. On the other hand, the micro virus-neutralization test was carried out in two variants: the sera were diluted via Mikrotiter micropipettes and micropipettes of the Takachi apparatus. It was found that the micro virus neutralization test in its two variants was not inferior in terms of sensitivity to the tube test, was more readily applicable, and was less material consuming. PMID- 3020775 TI - [Immunofluorescence demonstration of bovine pestivirus]. AB - A high-titer hiperimmune calf serum was obtained via the manifold hyperimmunization with local strains of the bovine pestivirus, cultivated in homologous cell cultures. Its globulin fractions were treated with fluorescein isothiocyanate to produce a specific conjugate. The specificity of the immunofluorescent serum was demonstrated through the comparative study of cell cultures infected with various viruses. The method worked out was employed to investigate tissue cross sections from diseased calves, whereupon the opportunity for the rapid and specific diagnosis of mucous disease was demonstrated. The replication dynamic of a bovine pestivirus strain in cell cultures was followed up. PMID- 3020774 TI - [Rotavirus infections in pigs]. AB - Rotaviruses were found in the study of 123 pigs and 132 fecal samples from animals with diarrhea. Employed were the method of immunofluorescence and the high concentration of virions (10(4) up to 10(9)/cm3). As high as 57.3 per cent of the fecal extracts proved positive for rotaviruses. In 14.3 per cent of the extracts a group-specific rotavirus antigen was established via the agar gel immunodiffusion, and 30.9 per cent of the intestinal cross sections were positive as demonstrated through immunofluorescence. Conditionally, the cases of rotavirus gastroenteritis are divided into two types--early and late. The first ones have been seen in sucking piglest at the age of 10-15 days, and with the growing of the epizootic wave--in 3-day-old ones, too. The second type of rotavirus gastroenteritis have been described in young pigs fifteen days after weaning, The death rate with sucking pigs was found to vary from 7 to 17 per cent, and it was 5 per cent with weaned ones. High mortality rate (82 per cent) was observed on a farm where a mixed infection with transmissive gastroenteritis was noted. The diagnosed cases of rotavirus enteritis prevailed in the cold months of the year (autumn-winter). On two farms the disease was established in the summer. The serologic examination of the blood sera of sows revealed group-specific antibodies to rotaviruses in 71 per cent of the cases. The percent of seroagents with the young sows ranged from 50 to 63.3 per cent, while with the adult ones it was 87 to 100 per cent. Discussed is the link between the low titer of natural immunity and the aspects of the epizootic process with rotavirus diarrhea. PMID- 3020777 TI - In situ hybridization for the detection of cytomegalovirus (CMV) infection. Application of biotinylated CMV-DNA probes on paraffin-embedded specimens. AB - Five autopsy cases of cytomegalovirus (CMV) infections were studied. Conventional light microscopy disclosed characteristic cytopathic effects in lungs, kidneys, and brain. In one case, electron microscopy was carried out and revealed typical herpesvirus particles. In situ hybridization was done with biotin-labeled CMV-DNA probes and an avidin-alkaline phosphatase detection system. 4/5 cases were observed to contain hybridizing cells in different organs. Intensity of hybridization was related to the severity of CMV infection, roughly estimated by counting cytomegalic cells. In addition to cytomegalic cells, a high number of normal-looking epithelial and mesenchymal cell types were positive. These latter cells showed nuclear hybridizations in contrast to cytomegalic cells which hybridized both within the nuclei and the cell bodies. This modified in situ hybridization procedure is a rapid and valuable tool for the detection and final demonstration of virus infection, and will be of particular help for the examination of paraffin-embedded specimens. PMID- 3020779 TI - Comparative studies on experimental latent herpesvirus infections. AB - Two different models of virus persistence in laboratory animals, that of herpes simplex virus (HSV-1) and that of murine herpesvirus (MHV), are described. While HSV-1 is harboured in a nonproductive form in the regional sensory ganglion of rabbits and mice, the persistence of MHV in mouse lungs and spleen is of a productive (dynamic) type. Since cultivation of kidney and trigeminal ganglion fragments enhanced the MHV recovery rate, one may assume that MHV persisted in a static form too, at least in some of the latter tissues. In contrast to HSV, MHV is predominantly disseminated by hematogenous route. So far no evidence of neural transmission of MHV has been found. PMID- 3020778 TI - The action of mouse liver and lung homogenates on the ESR signal of a spin labelled antiviral compound. AB - A spin-labelled derivative of amantadine was prepared and added to mouse liver and lung homogenates in vitro in the presence and in the absence of inhibitors of oxidative enzymes; the kinetics of the quenching of ESR signals was followed up in different experimental variants. PMID- 3020780 TI - Phosphoprotein NS of vesicular stomatitis virus: phosphorylated states and transcriptional activities of intracellular and virion forms. AB - The phosphorylation and transcriptional competence of the free cytoplasmic form and the virion form of NS protein of vesicular stomatitis virus (VSV-Indiana/Mudd Summers) were compared. NS protein is known to exist in two distinct phosphorylated states, NS1 and NS2, that are resolvable by gel electrophoresis. In vitro phosphorylation of virion NS protein by the viral L protein-associated protein kinase resulted in the phosphorylation of both NS1 and NS2. However, in the presence of the N-RNA complex, the NS2 form was preferentially phosphorylated. A cellular protein kinase activity, found in cytoplasmic extracts from VSV-infected or uninfected cells, preferentially phosphorylated NS1, which did not undergo dephosphorylation by cellular phosphatase and also did not convert to NS2. In contrast, the virion or cellular NS2 which had been phosphorylated in vivo or in vitro could be rapidly dephosphorylated by a cellular phosphatase. Cytoplasmic NS protein was found to be fully capable of binding to the virion N-RNA template, and in conjunction with L protein, it participated in synthesis of the leader RNA and five mRNA species of VSV. Moreover, under these conditions, neither cellular phosphatase nor cellular ribonuclease was able to bind to reconstituted nucleocapsids. Binding of cytoplasmic NS to the virion N-RNA template in the presence of L protein resulted in a large and preferential enhancement of NS2 phosphorylation. A protein kinase activity, which phosphorylated NS protein in vitro, was found to be associated with the N-RNA template. This activity appeared to be very tightly bound to N-RNA and exhibited absolute specificity for NS protein of the homologous serotype. PMID- 3020781 TI - Activation of int-1 and int-2 loci in GRf mammary tumors. AB - The Mtv-2 locus is known to be associated with a high mammary tumor incidence (97%) and early development of mammary tumors (3-13 months) in GR mice. However, it was not previously known whether the provirus which resides at the Mtv-2 locus is tumorigenic in and of itself or whether reintegration of proviruses generated from Mtv-2 is required for tumorigenesis. Foster-nursing GR mice on C57/BL mice eliminates the milk-borne source of GR virus, and allows the study of Mtv-2 derived proviruses alone. Using this approach, we have tested predictions which follow from the "positional" versus "reintegrational" models of tumorigenesis. Specifically, we have examined tumors from primary foster-nursed (GRf) mice to determine if MMTV proviruses derived from Mtv-2 were scattered randomly throughout the genome or were clustered in the vicinity of the int-1 and int-2 loci, which are thought to be associated with mammary tumorigenesis. It was found that the majority of spontaneous GRf mammary tumors that were tested have MMTV proviral integrations in either or both of the int-1 and int-2 loci and have transcription of either or both of the int loci. Tumors induced by Mtv-2, therefore, appear to have arisen via a mechanism similar to the activation of the int loci by exogenous (milk-borne) MMTV proviruses. PMID- 3020782 TI - Gag-derived but not abl-derived determinants are exposed on the surface of Abelson virus-transformed cells. AB - The organization of the transforming protein encoded by Abelson murine leukemia virus (A-MuLV) in transformed lymphoid and fibroblast cells was examined using immunofluorescent analysis. Antibodies specific for v-abl were capable of detecting cytoplasmic Abelson protein molecules in fixed cells, but none were able to stain the surface of live A-MuLV transformed cells. However, a series of monoclonal antibodies selected for the ability to bind to the surface of A-MuLV transformed cells did stain live cells. These antibodies were shown to react with a determinant within the helper virus-derived p15 sequences that are present at the amino terminus of the Abelson protein, indicating that gag-derived determinants are exposed on the surface of transformed cells. The inability of a p12-specific monoclonal antibody to stain live cells indicates that only a small portion of the amino terminal sequences are exposed. Examination of the ability of these antibodies to react with Abelson protein encoded by a series of gag deletion mutants suggests that the determinant recognized by these antibodies lies between amino acids 38 and 114 of p15. PMID- 3020783 TI - The hamster papovavirus: evolutionary relationships with other polyomaviruses. AB - The hamster papovavirus (HaPV) is a polyoma virus with a restricted tumor spectrum. It is actively replicated in hair follicle tumors arising spontaneously in young Syrian hamsters. It can also induce lymphomas and leukemias in newborn hamsters. The complete nucleotide sequence of a cloned HaPV has been established recently. This report presents a comparison of this sequence with other polyomavirus genomes (polyoma, SV40, BKV, LPV) by matrix dot analysis and electron microscopy heteroduplex mapping. The results demonstrate a close relationship between the HaPV and the murine polyoma virus and designate the LPV as the closest relative among the primate polyomaviruses. PMID- 3020784 TI - Complementation between SV40 and RFV defectives and acquisition of SV40 origins by late RFV genomes. AB - EL SV40 and RFV are variants of SV40 and BKV which contain bipartite or dual genomes. One molecule contains all the early viral sequences (E-SV40, E-RFV) and the other all the late viral sequences (L-SV40, L-RFV). Early and late genomes complement one another during productive infection. Experiments were designed to determine if E-genomes of one virus could complement L-genomes of another virus. If complementation did occur, intermolecular recombination events which lead to a more efficient infection or an altered host range might occur, and the sequences involved could than be identified. Two combinations were generated by direct transfection of BSC-1 green monkey cells. E-RFV and L-SV40 DNA complementation resulted in hybrid virus growth and cell killing. The hybrid demonstrated a narrow host range. Following serial passage, some E-RFV genomes contained SV40 origin region sequences but these recombinants did not overgrow prototype E-RFV genomes, even after many virus passages. In addition, no significant alterations in host range could be detected. Complementation between E-SV40 and L-RFV yielded a virus with a relatively wider host range. Virus growth and cell killing appeared very slowly at first. However, with each passage of E-SV40/L-RFV, cell killing occurred progressively more rapidly, until passage 7 when it became extensive in 7 days rather than 6-8 weeks. Infected cells contained 10-20 times more E-SV40 than L-RFV DNA during the first passage. However, by passage 7, both genomes were equally represented. During serial passage, L-RFV DNA acquired SV40 sequences from around the origin and the terminus of replication, such that recombinant (r) L-RFV genomes contained three SV40 origins [corrected] (including the 72-bp repeat) and 2 termini, and prototype L-RFV DNA was lost. E-SV40/rL-RFV demonstrated an altered host range propagating in some cell lines which did not support E-SV40/L-RFV growth. Both the host range change and the increased growth of rL-RFV genomes were shown to be at least partly caused by the acquisition of the SV40 sequences. PMID- 3020785 TI - A simple repetitive sequence common to herpes simplex virus type 1 and human ribosomal DNAs. AB - The simple sequence GGC was tandemly repeated in herpes simplex virus type 1 DNA and human 28S rDNA and its mature 28 S rRNA transcript. The sequence homology was responsible for the observed hybridization between the two DNAs under high stringency blot hybridization conditions. PMID- 3020786 TI - Characterization of a new type of human papillomavirus (HPV) related to HPV5 from a case of actinic keratosis. AB - Human papillomavirus (HPV) DNA sequences, related to the genomes of HPVs associated with epidermodysplasia verruciformis (EV), were detected in DNA samples extracted from biopsied lesions in 2 of 24 cases of actinic keratosis found in the general population. An HPV DNA was molecularly cloned from one of these samples. Blot hybridization experiments, performed under stringent conditions, revealed a significant cross-hybridization only between this HPV DNA and the DNAs of HPV5 and of the HPV5-related types. The extent of homology between them ranged from 7 to 30%, as evaluated by hybridization in liquid phase at saturation followed by nuclease S1 analysis. This showed that the cloned HPV represented a new type, tentatively named HPV36. HPV36 was not found in the other 22 cases of actinic keratosis, but was detected in scrapings of benign lesions of 7 of 18 (39%) EV patients. PMID- 3020787 TI - Analysis of six distinct integrated hepatitis B virus sequences cloned from the cellular DNA of a human hepatocellular carcinoma. AB - Six distinct hepatitis B virus (HBV) integrations and the flanking cellular sequences were cloned from a hepatoma DNA preparation. None of the cloned fragments retains the entire HBV sequences but the surface antigen (HBsAg) gene and the HBV enhancer are retained in three of the six clones. The other three clones carry only short and possibly highly rearranged HBV genomic sequences and seem to contain some GC-rich clusters. Members of the repetitive Alu family are also found in the vicinity of five of the six integration regions which may have contributed to genome instability. In these six clones, the preferred integration sites are shown to lie within the single-strand region of the HBV genome. None of the clones carries in the flanking cellular sequences any of the 17 oncogenes tested, although the possibility still exists that an oncogene may be found on the side of the genome which has not been cloned. This work thus paves the way for detailed sequence analysis of virus-host junctions, for transfection studies of the HBV integration events, and for a search of genes in the flanking cellular sequences which may have been activated by the retained HBV enhancer using the clones described. PMID- 3020788 TI - Transfection and recombination with molecularly cloned derivatives of avian sarcoma virus UR2. AB - A cloned version of avian sarcoma virus UR2, plasmid pKD6, which includes the full, nonpermuted proviral sequence between two LTR regions, has been prepared. The plasmid is biologically active in transfection experiments, even when intact. Two transformation-defective mutants with nonoverlapping deletions within the transforming gene ros were constructed from pKD6. These mutants recombine to produce transforming virus when mixed DNA from both is used to transfect chick embryo fibroblasts along with helper virus DNA. However, recombination was not readily detected when cells were coinfected with fluids harvested from cultures separately transfected with DNA from each mutant. This, and marker rescue experiments with a temperature-sensitive mutant of UR2 defective in transformation but able to replicate, suggest that deletion mutants of UR2 do not propagate efficiently. PMID- 3020789 TI - Specific sequences of the env gene determine the host range of two XC-negative viruses of the Rauscher virus complex. AB - Two viruses which do not give rise to XC plaques in the standard XC assay (XC negative) have been isolated from the Rauscher virus (RV) complex. These viruses differ in their host range. One, R-MCF-1, is dualtropic and will therefore infect both murine and non-murine cells. However, unlike other mink cell focus-inducing (MCF) viruses, it cannot infect NIH 3T3 cells. The other, R-XC-, is ecotropic. It will infect murine cells, including NIH 3T3 cells, but does not infect mink lung cells. Analysis of hybrid viruses, in which homologous regions of the genomes of R-MCF-1 and R-XC- virus were exchanged, indicated that the NH2-terminal portion of the gp70 is responsible for the particular host ranges of these viruses. The nucleotide sequence of the env gene of R-XC- virus was therefore determined and compared with the known env sequences of ecotropic MLVs and dualtropic MCF viruses of the Rauscher and Friend virus complexes. R-XC- virus was found to be a recombinant virus. Its env gene contained sequences derived from an endogenous env gene which were closely related to those of the MCF viruses but differed from any previously described sequences. The particular properties of R-MCF-1 and R-XC virus suggest that the two viruses arose by recombination between R-MLV and two endogenous env sequences which differ from those of the known MCF viruses. If so, this suggests that the mouse genome contains at least five env sequences which can give rise to MCF-like viruses. In addition, since the host range and interference properties of R-XC- virus are very similar to those of the previously described ecotropic recombinant viruses, it may be that the ecotropic recombinant viruses arose by recombination with the same endogenous env sequences as did R-XC- virus. PMID- 3020790 TI - [Radioprotective action of cystamine and gammaphos in gamma ray exposure (Czechoslovak Socialist Republic)]. PMID- 3020792 TI - [Calcium metabolism and the level of active metabolites of vitamin D3 in the rat serum during bone regeneration]. AB - Fracture of rat femur bone was accompanied by an increase in content of calcium and 25(OH)D3, by a decrease in content of 1,25(OH)2D3 while the 24,25(OH)2D3 level was unaltered in blood serum as well as an augmented absorption of calcium was found in small intestine during formation and mineralization of callus. The elevated consumption of the vitamin D3 active metabolites in small intestine mucose and in bone tissue and/or stimulation of their biosynthesis in these tissues appear to occur in response to transformation of the calcium metabolism and to remodelation of rat bone tissue in fractures. PMID- 3020791 TI - [Biochemical and functional characteristics of thymus and spleen lymphocytes in C3HA mice during the growth of hepatoma 22 and after immunization with sheep erythrocytes]. AB - Activities of key enzymes of purine metabolism [adenosine deaminase (AD); purine nucleoside phosphorylase (PNP); 5'-nucleotidase] were studied; changes in DNA content, nucleus ploidity in thymocytes, T- and B-lymphocytes in the C3HA mouse spleen during solid 22 hepatoma growth and after the immunization were monitored. Immunological properties of lymphocytes were also investigated measuring antibody formation and the reaction of blasttransformation in response to phytohemagglutinin, concanavalin A and lipopolysaccharide. Within the first 48 hrs after the tumor implantation and immunization certain nonspecific biochemical mechanisms of lymphocytes activation (elevated AD activity, decreased activity of 5'-nucleotidase, augmented intracellular DNA levels, polyploidity) were revealed. As the solid 22 hepatoma reached the maximum growth rate specific alterations in the activities of the purine metabolism key enzymes were observed reflecting the response of thymus and spleen lymphocytes to the presence of the malignant tumor. PMID- 3020794 TI - [Malignant glomus tumor of the stomach (case report)]. PMID- 3020793 TI - [Genetic heterogeneity and approaches to the prenatal diagnosis of phenylketonuria (review)]. AB - Analysis of the data on structure polymorphism of phenylalanine hydroxylase in mammals including man is of importance in elucidation of the enzyme structural alterations in the patients with phenylketonuria. Molecular-genetic approaches are developed for prenatal diagnosis of hereditary diseases; approaches for studies on the phenylketonuria genetic heterogeneity have to be developed. The most promising methods for the prenatal diagnosis appear to be based on the oligonucleotide probes or on use of specific restrictases, recognizing mutant sites in the gene. This suggests that analysis of the intralocus genetic heterogeneity of phenylketonuria is one of important problems in studies of the genetic heterogeneity. PMID- 3020795 TI - [Determination of cyclic AMP and protein kinases in the diagnosis of colonic tumors]. AB - The cAMP level and the activity of cAMP-dependent and independent protein kinases were measured in adenomatous polyps, villous polyps (premalignant condition) and adenocarcinomas. The cAMP concentration and ratio of cAMP level versus cAMP independent casein kinase activity were significantly lower in adenocarcinomas compared to adenomatous polyps, thus permitting differentiation between benign and malignant lesions. The cAMP versus cAMP-dependent histone kinase level ratio was used as a test for differentiation between malignancies and premalignant condition. It was markedly lower in adenocarcinomas than in villous polyps. PMID- 3020796 TI - [Erroneous diagnosis of nephroblastoma (Wilm's tumor) based on SIOP data]. AB - Available films of 21 cases of erroneously diagnosed Wilms' tumor in the European Wilms' material have been studied. The diagnosis has been reassessed and the reasons for agreement in 6 cases and disagreement in the remaining 15 are discussed. General diagnostic recommendations are given to help secure optimal diagnostic information. PMID- 3020797 TI - [Nitrate and nitrite content of food products of plant origin grown with the use of mineral fertilizers]. AB - Introduction of mineral fertilizers into soil results in the nitrate accumulation in the vegetables grown in this soil. However, under conditions of the utilization of nitrogen fertilizers in a dose of 300 kg/hectar in the soil of Byelorussia, the nitrate content in vegetables does not exceed the permissible value. No direct relationship has been established between the accumulation of nitrates and nitrites in the vegetables and cereals and the dose of mineral fertilizers and nitrite accumulation in the soil. PMID- 3020798 TI - Diminishing morbidity and mortality of bone marrow transplantation. AB - The reasons for the operational failure of bone marrow transplantation (BMT) are familiar and varied. They are the following: nonmarrow toxicity of the preparative regimen, marrow toxicity of the preparative regimen, acute graft versus-host disease (GVHD), opportunistic infections, especially viral infections and recurrence of hematologic malignancy in the case of transplants for leukemia. This review examines operational failure of BMT and presents measures for its prevention. PMID- 3020799 TI - [Use of a conjugate of staphylococcal protein A with peroxidase in an immunoenzyme test system for determining antibodies to the herpes simplex virus]. AB - An enzyme-immunoassay system for the detection of specific antibody to herpes simplex virus was developed in which the antigen layered on the solid phase consisted of a crude extract of infected cells, and the detecting agent was a conjugate of protein A with horseradish peroxidase. This assay system was used for serological examination of 22 serum specimens from patients suffering from either meningoencephalitis or recurrent herpes of the skin and mucous membranes of the genitalia, as well as 6 specimens of the cerebrospinal fluid from patients with meningoencephalitis. The enzymeimmunoassay was found to be more sensitive than CFT. The advantages of using protein A/peroxidase conjugate for rapid estimation of the total amount of specific antibodies at any stage of herpes infection are discussed. PMID- 3020800 TI - [Cytomegalovirus infection and immunodeficiency]. AB - The results of detailed clinical, hematological, immunological, and virological investigations of a female patient who within 16 months after hemotransfusion acutely developed a long-term disease with a undulant course accompanied by high fever, lymphadenopathy, hepatolienal syndrome, stomatitis, leukopenia, and thrombocytopenia, are presented. Significant disorders in cell-mediated and humoral immunity were detected. Cytomegalovirus was isolated from the urine and saliva. The pathogenetic role of cytomegalovirus in the development and course of the disease is evaluated, and its etiological importance is discussed on a broader scale with regard to a possible association with retroviruses. PMID- 3020802 TI - [Virus-specific antibodies in persons inoculated with a bivaccine against eastern and western equine encephalomyelitis]. PMID- 3020801 TI - [Structure and functions of cloned adenovirus DNA]. PMID- 3020803 TI - [New data on the acquired immunodeficiency syndrome (AIDS) and its causative agent]. PMID- 3020804 TI - [Experimental models of insulin-dependent diabetes]. PMID- 3020805 TI - [Use of proteolysis inhibitors for treating parainfluenza infections in an experiment and in the clinic]. PMID- 3020806 TI - [Association of the hepatitis A virus with the membranes of infected cells]. AB - "Light" viral antigen (HAAg) with buoyant density 1.20 g/cm2 and sedimentation coefficient 92S are accumulated together with mature viral particles in Hepatitis A virus (HAV) infected FRhK-4 cells. This HAAg is localized predominantly in endoplasmic reticulum fraction of infected cells, while nature virions are localized in cytosol. In contrast to mature virus, "light" HAAg is sensitive to trypsin digestion and is not able to hybridize with synthetic oligodeoxinucleotide which is complementary to structural part of HAV RNA. Antigenic properties of mature virus and "light" antigen are compared. PMID- 3020807 TI - [Inhibition of the infectivity of enveloped viruses while retaining the specific biological activity of the viral proteins]. AB - A method of inactivation of enveloped viruses by treatment of virus-containing material with a nonionic detergent MESK was studied. Treatment with the detergent of allantoic or culture fluids containing influenza, parainfluenza, herpes simplex, and Venezuelan equine encephalomyelitis viruses was shown to result in significant (to 10 lg ID50) decrease of the infectivity of the viruses. At that, the specific biological activity of viral proteins: the hemagglutinating and neuraminidase activities in the case of influenza and parainfluenza viruses, and the hemagglutinating activity in the case of Venezuelan equine encephalomyelitis virus, remained unchanged. Treatment of the viruses with the detergent in the presence of 2% BSA, 5% gamma-globulin, or 50% blood serum also inactivated the infectivity of virions. The inactivating effect of the MESK detergent is associated with solubilization of external glycoproteins of the virus envelope. The method of mild nondenaturating inactivation of enveloped viruses with the MESK detergent may prove to be useful in laboratory practice. PMID- 3020809 TI - [Hepatocellular carcinoma--its echographic characteristics and diagnostic potentials]. AB - Echography is an approved method for establishing focal processes in liver. Forty five patients, from Bulgaria and Kuwait, with confirmed hepatocellular cancer (HCC) were studied by echographies aiming at the elucidation of the possibilities of ultrasound examination in making the diagnosis of HCC in patients with and without cirrhosis of the liver. Five types of images have been established- hyperechogenic (29%), hypoechogenic (27%), mosaic (24%) mixed (11%) and type "bovine eye" (9%). The echographic characteristics that could suggest the character of the lesion are low-echogenic zone in the periphery and non homogenous (mosaic) tissue structure. HCC, in 55.6 per cent of the patients, proved to be with multiple localization in liver. The comparison of the patients from Kuwait and Bulgaria revealed no differences in the mean age, carriership of HBsAg, but in the latter--the HCC was significantly more often combined with cirrhosis of the liver. Serum alpha-fetoprotein was increased in 15 out of 20 patients with HCC. The establishment of the tumour in part of the patients by ultrasound initially, suggests that echography is a useful method for the early diagnosis of HCC and for a systematic follow up of the patients with cirrhosis of the liver. Guided fine-needle biopsy was performed in 15 patients, and in 86.7 per cent of the cases--accurate cytologic diagnosis was obtained. Echography, combined with guided biopsy and determination of alpha-fetoprotein, guarantee in a considerable part of the cases, also the correct diagnosis of HCC in patients with and without cirrhosis of the liver. PMID- 3020808 TI - [Production of infectious virus and the accumulation in Vero cells of Pichinde virus antigens detectable by an indirect fluorescent antibody method]. AB - The dynamics of accumulation of infectious Pichinde virus in the culture medium and virus-specific antigens in cells was studied in relation to multiplicity of infection in a multicycle experiment. Differences in the fluorescence pattern of Pichinde virus antigens in IFAT were found to depend on the use of acetone or formaldehyde for fixation of the infected cells. PMID- 3020810 TI - [A 62-year-old patient with recurring venous thromboses of the leg and lung infarcts despite therapy with oral anticoagulants]. PMID- 3020811 TI - [Diagnostic value of the combined determination of carcinoembryonic antigen (CEA) in pleural effusion and serum with an enzyme immunoassay (EIA). Sensitivity, specificity and relation to tumor type]. AB - Measurement of carcinoembryonal (CEA) levels in pleural fluid are suggested to improve the unsatisfactory sensitivity of pleural cytology in the differential diagnosis of malignant pleural effusions. We evaluated simultaneously determined pleural and serum CEA levels in 117 patients with pleural effusions of different aetiology (74 malignant, 30 inflammatory exudates and 13 transudates) by use of an enzyme immunoassay (EIA). Despite considerable scatter, pleural levels of CEA in malignant effusions were significantly higher (p less than 0.001) than the values in benign effusions. Using a cut off level of 5 ng/ml, 41% (= sensitivity) of malignant pleural effusions showed elevated concentrations of CEA. Only one out of 43 benign effusions showed a level of 5 ng/ml, which is equivalent to a specificity of 98%. However, malignant effusions due to small cell lung cancer, pleural mesothelioma and metastasising ovarian carcinoma never showed elevated levels of CEA. Highest pleural values of CEA were observed in cases of alveolar cell or adenocarcinoma of the lung or metastasising breast cancer. Although pleural and serum CEA levels correlated significantly (rs = 0.77), the evaluation of serum CEA levels alone yielded a lower sensitivity (36%) and specificity (93%) than pleural levels. 77% of cases with malignant pleural effusions showing elevated pleural CEA levels were characterized by an increased ratio Pleura/Serum greater than 1, particularly in effusions due to lung cancer. The CEA ratio was significantly higher (p less than 0.05) in patients with malignant than with benign effusions. EIA appears to be more specific by avoiding false positive results in benign effusions as compared with determination by conventional RIA. In conclusion, evaluation of pleural CEA levels in patients with malignant effusions by using an EIA because of its high specificity is a valuable adjunct to pleural cytology in improving the diagnosis of malignant effusions. However, a normal CEA level in either pleural effusion or in serum is of no clinical significance. Simultaneous measurement in pleural effusion and serum is of greater value. PMID- 3020812 TI - Menadione (2-methyl-1,4-naphthoquinone)-induced Ca2+ release from rat-liver mitochondria is caused by NAD(P)H oxidation. AB - Incubation of rat-liver mitochondria with menadione in the presence of succinate and rotenone resulted in rapid glutathione and NAD(P)H oxidation followed by Ca2+ release and mitochondrial swelling. Ca2+ release, NAD(P)H oxidation and mitochondrial swelling, were also observed in mitochondria from selenium deficient rats. Glutathione was only slowly oxidized, suggesting that glutathione oxidation, and subsequent NAD(P)H oxidation via the glutathione peroxidase glutathione reductase system were not required for Ca2+ release by menadione. Isocitrate prevented and reversed Ca2+ release dose-dependently but dicoumarol had no effect indicating that NADH-ubiquinone oxidoreductase and not DT diaphorase was responsible for NAD(P)H oxidation. Superoxide anion radical was formed by cyanide-resistant respiration, suggesting that menadione undergoes a one-electron reduction to an autoxidizable semiquinone radical by NADH-ubiquinone oxidoreductase. The inability of menadione to oxidize glutathione in selenium deficient mitochondria indicates that the metabolism of the superoxide dismutation product, H2O2, by glutathione peroxidase was probably responsible for the glutathione oxidation in selenium-replete mitochondria. PMID- 3020813 TI - [Acute adrenal cortex insufficiency]. PMID- 3020814 TI - [The endocrine system and stress]. PMID- 3020815 TI - [Value of bronchoalveolar lavage in the diagnosis and follow-up of lung diseases]. AB - In a group of 100 patients with different lung diseases the diagnostic value of BAL was estimated comparing with conventional broncho-bioptical methods. Final diagnosis was obtained by BAL only in 3% of all cases in contrast to conventional broncho-bioptical methods with 49% positive findings. In a further group of 77 patients with acute and chronic forms of sarcoidosis in stage I and II a significant difference in BAL-lymphocytosis between acute and chronic forms was observed. Our findings demonstrate the limited value of BAL for diagnostic purpose but its valuability in estimation of the activity and the course of different lung diseases especially of granulomatous and fibrotic origin. PMID- 3020816 TI - [Blockade of postsynaptic adrenergic alpha receptors--a new principle for inhibiting uterine contraction]. AB - In analogy to beta 2 mimetic agents, also alpha 1 adrenoblockers should show an uterus-relaxing effect. Therefore, the tocolytic effect of prazosin, which is supposed to be the most potent alpha 1 blocking agent, was investigated in myometrial strips. Uterine activity was highly significantly (p less than 0.001) decreased by reduction of frequency and amplitude, but not by duration and area of spontaneous uterine contractions. PMID- 3020818 TI - A computer-aided analysis of the effect of peripheral nerve transection on TMPase activity of substantia gelatinosa Rolandi. PMID- 3020817 TI - [Electron microscopy and histochemical studies of the origin and function of myoepithelioid cells in glomangioma and peripheral barrier arteries]. AB - Epithelioid cells (myoepithelioid cells, glomus cells) are a structural element characteristic of Hoyer-Grosser shunts (glomus organs, arterio-venous epithelioid cell glomerula) and Bailey's glomangioma (Masson's angio-neuromyome-arteriel, glomus tumors). Their functional importance cannot be estimated without knowledge of their histogenetic origin. The myoepithelioid cells are derived from smooth muscle cells, which can be shown ultrastructurally, histochemically, or by light microscopy. PMID- 3020819 TI - The quest for a herpes simplex virus vaccine: background and recent developments. AB - Herpes simplex virus infections in humans range from localized skin infections of the oral, ocular and genital regions, to severe and often fatal disseminated infections of immunocompromised hosts. Following primary infection, the virus often becomes established in a latent form in the neurons of sensory ganglia and can reactivate to excrete virus asymptomatically or produce recrudescent lesions. This review describes some of the mechanisms involved in the immune response against HSV infections and examines the different strategies adopted to develop a vaccine against this seemingly intractable disease. PMID- 3020820 TI - Development of an attenuated strain of chikungunya virus for use in vaccine production. AB - An attenuated chikungunya (CHIK) virus clone was developed for production of a live vaccine for human use. CHIK strain 15561 was subjected to 18 plaque-to plaque passages in MRC-5 cultures before CHIK 181/clone 25 was selected as vaccine seed based on homogeneous small plaque size, suckling mouse avirulence, reduced monkey viraemia and genetic stability. Oligonucleotide mapping demonstrated differences between parent and clone. Vaccine (pilot-lot production) elicited neutralizing antibody and protected mice and rhesus monkeys against challenge. After challenge, viraemias were absent in vaccinated monkeys. Vaccine was then produced and tested in accordance with governmental regulatory requirements of human use. PMID- 3020822 TI - [Hormone interactions in varicose veins--receptor analysis and hormone level]. PMID- 3020821 TI - Characterization and immunogenicity of HSV-1 antigens obtained following zwitterionic detergent treatment. AB - A preparation was obtained from herpesvirus hominis type 1 (HSV-1) infected cells using the zwitterionic detergent, Empigen BB. The preparation was partially purified by ultracentrifugation over a cushion of 20% sucrose. Serological characterization by ELISA and immuno double diffusion, using both polyclonal and monoclonal antibodies shows this preparation to contain HSV glycoproteins, including gC, gD and gE. Immunization of Balb/c mice elicited serum antibody responses against both HSV-1 and HSV-2 viruses, complete protection against HSV-1 (strain WAL) and partial protection against HSV-2 (strain 333). PMID- 3020823 TI - [Methodological aspects of the determination of the capillary filtrate in the extremities. Simultaneous measurement of leg and regional blood volume by plethysmography and Tc-99m-labeled erythrocytes]. PMID- 3020824 TI - [Interaction of the cyclase and transmethylase systems]. PMID- 3020825 TI - [Self-regulation of brain opiate receptors in rats chronically administered morphine]. PMID- 3020826 TI - [Alternative pathways of substrate transformation in reactions catalyzed by thiamine enzymes]. PMID- 3020827 TI - [Ca2+-activated neutral proteinases and their regulatory importance]. PMID- 3020828 TI - [Role of the ATP-Mg generated by plasma membranes in mediating signal transmission from the insulin receptor and other regulators to membrane kinase]. PMID- 3020829 TI - Replacement of the deletion in the genome (0.762-0.789 mu) of avirulent HSV-1 HFEM using cloned MluI DNA fragment (0.7615-0.796 mu) of virulent HSV-1 F leads to generation of virulent intratypic recombinant. AB - The HFEM strain of HSV-1 is apathogenic for the tree shrew by the intraperitoneal (i.p.) route because of a deletion in the genome coordinates 0.762-0.789. Insertion of the MluI DNA fragment (coordinates 0.7615-0.789) cloned from HSV-1 strain F, which is pathogenic for the tree shrew, restored the i.p. pathogenicity to strain HFEM. The recombinant designated R-M1-C1 was highly pathogenic for the tree shrew, but slightly virulent for inbred mouse strain A. It thus appears that the viral DNA sequence involved in the i.p. pathogenicity of HSV-1 is located within the genome coordinates 0.761-0.796. This sequence is recognized differently by the cellular elements involved in HSV-1 infection in the tree shrew and the mouse. PMID- 3020830 TI - Passage of herpes simplex virus type 1 on chick embryo fibroblasts confers virulence for chick embryos. AB - The pathogenesis of chorioallantoic membrane (CAM) infection with herpes simplex virus 1 and 2 (HSV-1 and HSV-2) as well as chick embryo fibroblast (CEF) passaged HSV-1 was studied. It was found that HSV-2 is at least a million-fold more virulent than HSV-1 as measured by pfu/LD50 ratios for the embryo. Serial passage of HSV-1 in vitro on CEF cells selected for a virus (CEFP10) which, unlike its parental HSV-1 strain, is able to kill the embryo. The restriction endonuclease maps of CEFP10 and its parental strain are indistinguishable and the mechanism of the increased virulence of CEFP10 was demonstrated to be its enhanced replication in chick embryo cells both in vivo and in vitro. In contrast, despite its inferior replicative ability, HSV-2 was found to have a biologically important specific invasiveness function that is not simply related to overall viral replication. Finally, the ability to isolate HSV-1 (CEFP10) virulent for the chick embryo after passage in vitro illustrates that tissue culture passage of HSV in appropriate cells may actually increase virulence for the animal host. PMID- 3020831 TI - Genomic relationship between capripoxviruses. AB - Capripoxvirus DNAs from field isolates and vaccine samples were analysed by digestion with the restriction enzyme Hind III. The patterns of fragments generated by digestion with Hind III are sufficiently similar to show that all capripoxviruses are closely related, although patterns of different isolates can be grouped in a way which correlates with the animal of origin. The close relatedness was also demonstrated by the high level of sequence homology detected using the Southern Cross hybridization system. Despite the sequence homology, the molecular weights of the genomes of different isolates varied from 73 to 91 MDa. The presence of two rapidly reannealing restriction fragments in the Hind III digests of capripoxvirus DNA indicated the presence of terminal cross-links. PMID- 3020832 TI - [Strengthening interhemispheric interferential inhibition in man with diazepam]. AB - The influence of oral diazepam administration (10 mg) on interhemispheric proactive and retroactive interferential inhibition during detection of test verbal signals was tachistoscopally studied in eight healthy right-handed subjects, in conditions of forward and backward contralateral masking according to the scheme of double blind investigations. Strengthening of effects of forward and backward contralateral masking was revealed which is considered as a testimony to participation of GABA-ergic inhibitory systems in the mechanisms of interhemispheric proactive and retroactive interferential inhibition. PMID- 3020833 TI - [Comparative characteristics of septal "burst" neurons after elimination of afferent effects of the reticular formation in the rabbit]. AB - Comparative analysis of characteristics of rhythmic theta-activity in the neurones of the medial septal nucleus and nucleus of diagonal band was performed in intact rabbits after. i. v. injection of pentobarbital, and in rabbits with chronic lesion of the ascending brain-stem afferent fibers. In both conditions theta-bursts disappeared in some cells with unstable periodic rhythmic modulation; substantial population of the septal units preserved regular burst activity. Main characteristics of theta-bursts were almost identical in both states, their mean frequency decreased to 3.5 Hz. The theta-rhythm in hippocampal EEG was usually absent; but low-frequency rhythmic activity could be evoked by electrical or sensory stimulation as well as by injection of bemegrid or physostigmine. The data show that the ascending brain-stem afferents control: the frequency of the bursts in a population of septal units regarded as bursting pace maker cells; the total number of the septal cells secondarily (synaptically) involved into rhythmic activity. The effect of pentobarbital upon theta-rhythm results from elimination of these influences upon the septal cells. PMID- 3020834 TI - [Contour of auto- and cross-correlation histograms of the spike flows of monosynaptically connected neurons]. AB - By methods of neuronal interaction modelling--biomathematical (computer controlled experiment on molluscs neurones) and mathematical--in wide physiological ranges of parameters values, characterizing properties and conditions of neurones and synapses functioning, the forms were studied of auto- and cross-correlation histograms of impulse flows of neurones at forward and backward monosynaptic connections between them. Specific form is established of cross-correlation histogram of impulse flows of interconnected neurones in conditions typical of CNS of mammals, when the neurones are subjected to intensive random afferent synaptic bombardment and do not reveal any pace-maker properties. It is also shown that random afferent synaptic bombardment prevents the appearance of excitation reverberation in closed neuronal circuits. PMID- 3020835 TI - [Comparative cytophotometric and cytomorphometric studies of concave cells and atypical squamous epithelial cells of the cervix uteri]. AB - Cytologic slides from 11 women with clinical, cytological and histological signs of a papillomavirus infection of the cervix uteri are investigated by cytophotometry and cytomorphometry. The mean values of DNA content, chromatin density, nuclear area and nuclear circumference of koilocytes were notably higher than for atypical or normal squamous cells. In those patients with concurrent cervical intraepithelial neoplasia the DNA content of koilocytes was inversely related to the degree of atypia. Squamous cells with HPV-induced lesions can be characterized by cytophotometry and cytomorphometry as a distinct cell population forming a continuum with the preneoplastic epithelium. PMID- 3020836 TI - [Ultrastructural studies of metastases of anaplastic tumors and non-Hodgkin's lymphoma as a contribution to differential diagnosis]. AB - Difficulties in morphologic classification frequently arise in distinguishing between non-Hodgkin lymphoma and lymph node metastases from anaplastic tumors, mainly germ cell tumors and small cell carcinomas. The present study explores the role of ultrastructural features as a supplement to light microscopy in 14 cases of metastatic disease and 6 non-Hodgkin lymphomas. It appeared that the ultrastructure of the nucleoli, the arrangement of the nuclear bodies and the shape of nuclear invaginations are ultrastructural cell characteristics, which may contribute to the definitive diagnosis. PMID- 3020837 TI - [Pathogenetic correlation between chronic pancreatitis and pancreatic carcinoma]. AB - The autopsy material from the Institute of Pathology of the Faculty of Medicine of the Humboldt University (Charite) in Berlin for the years 1970 to 1984 was analyzed with respect to the presence of pancreatic carcinoma and a long history of chronic, non-obstructive pancreatitis. A total of 20,515 adult sections were reviewed. 331 (1.6%) of these had carcinoma of the exocrine pancreas. 75 pancreata were dissected in tail-to-head direction into 10 blocks. In 12 (16%) of them a chronic non-obstructive pancreatitis or pancreatic scarring as a result of pancreatitis could be demonstrated histologically. The possibility of a relationship between pancreatitis and pancreatic carcinoma is considered, in particular with respect to pre-malignant changes and latency of the latter. Comprehensive analysis and evaluation of the present study material, together with a critical evaluation of the literature, support the viewpoint that long term, chronic pancreatitis and pancreatic carcinoma have a common pathogenic basis and that chronic pancreatitis may be regarded as an antecedent event for the neoplasm. PMID- 3020838 TI - [A hitherto unknown reaction pattern in vertebrate cells (RiV). 2. The protective effect of RiV particle preparations against foot-and-mouth disease in guinea pigs]. AB - Further observations concerning the previously described RiV-particles are reported. They were isolated from a diploid cell line of bovine origin, embryonal duck fibroblasts and BHK-21 cells. A protective effect against foot-and-mouth disease virus in guinea pigs could be observed following inoculation with the RiV preparation of bovine origin. All 3 preparations isolated from the 3 cell lines showed immunologic cross reactions. PMID- 3020839 TI - [Value of computerized tomography in the evaluation of the operability of bronchial cancers]. AB - 65 computer tomographs were taken of 79 patients with bronchial carcinoma for the purpose of determining operability. Included were 49 computer tomographs of the thoracic region, 7 of the epigastrium, and 9 of the skull. CT, a noninvasive radiological diagnostic method, was used in the above cases not only for diagnosis but even more for high-accuracy staging and thus therapy planning. CT accuracy regarding assessment of local operability amounted to 97.9 per cent (n = 49). There was only one case in which tumour infiltration into the aorta had been overlooked. Diagnosis restricted to conventional tomography gave rise to four thoracotomies which proved to be useless for local inoperability due to tumour infiltration of the mediastinum. The advantages of computed tomography over conventional radiological approaches were found to be primarily based on possible detection of mediastinal lymph node metastases as well as tumour infiltration of organs adjacent to the mediastinum and of the thoracic wall. The diagnostic potential implied in computed tomography could not be optimally utilised until CT was simultaneously applied to epigastric organs. Therefore, basic examinations for preoperative staging of bronchial carcinoma should by all means include computed tomography in addition to conventional radiological methods and bronchoscopy. PMID- 3020840 TI - [Bifurcated resection in the surgical treatment of central non-small cell T3 carcinoma]. AB - Reported in this paper is experience obtained since 1978 from surgical removal of the tracheal bifurcation in combination with pneumonectomy in 38 cases. Squamous cell carcinoma centrally growing from the upper lobe was recorded from 24 patients to whom 36 interventions had been applied for bronchial carcinoma. 50 per cent of all operations in conjunction with pneumonectomy were performed on patients in the pT3NOMO stage. The cumulated 3-year survival rate amounted to 37 per cent. Mortality in hospital accounted for 18.4 per cent and was thus clearly higher than what had been recordable from standard techniques in carcinoma surgery. Causes of postoperative deaths included insufficient sutures in three cases, pulmonary embolism in 2, and pneumonia in the contralateral residual lung in another 2. Standardised surgical and anaesthesiological techniques were used. Modifications of resections are discussed. PMID- 3020841 TI - [Estrogen receptor status of breast cancer patients]. AB - Reported in this paper are studies conducted into oestrogen receptor levels of 103 patients with mammary carcinoma, with the dextran-carbon method being used. The findings thus recorded are discussed for their clinical relevance. Recorded were correlations between quantitative oestrogen receptor levels, on the one hand, and menopausal status, grading, histological typing, and tumour size, on the other. PMID- 3020842 TI - [Malignant transformation of cystosarcoma phylloides]. PMID- 3020843 TI - [Poland syndrome. Possibilities for treatment]. PMID- 3020844 TI - Occurrence and properties of hly-plasmids in E. coli from patients of the Berne area. AB - 1818 E. coli wild-type strains from patients of the region of Berne were screened for their haemolytic property and for carriage of hly-plasmids. Among 885 strains of fecal and 993 strains of urinary tract isolates 136 (15%) and 259 (28%) were haemolytic respectively. In five fecal, but none of the urinary strains the haemolytic activity was encoded by transmissible plasmids. In spite of independent origin, the five isolated hly-plasmids proved to be very similar. They all showed a high transfer-frequency according to their derepressed state and coded for F-sexpili. Four belonged to the rare F VI- and one to the F IV incompatibility group. Restriction patterns after Eco R1- and Hind III-digestion were identical in three and similar in the other two plasmids. Comparing the plasmids from Berne with hly-plasmids isolated in Essex, similarities as well as specific differences were detected. PMID- 3020845 TI - Mycobacterium marinum infection in gnotobiotic nu/nu mice pretreated with cyclophosphamide and silica. AB - Germfree nude mice (NMRJ-nu/nu) and germfree nu/+ mice were infected with Mycobacterium marinum in the footpad test. After pretreatment with 400 mg/kg cyclophosphamide, significantly higher amounts of M. marinum were reached in the footpad and in the liver. On the other hand, the yield of M. marinum could not be elevated by in vivo inhibition of macrophages with silica. PMID- 3020846 TI - [Possible participation of lipids in information gathering and the amplification of biological signals]. PMID- 3020847 TI - [Phylogenetically ancient regulatory peptides in the most recent systems of the higher vertebrates]. PMID- 3020848 TI - Do non-steroidal anti-inflammatory drugs influence the effects of antihypertensive drugs? PMID- 3020849 TI - Long-term treatment of idiopathic hyperaldosteronism using trilostane. AB - Three patients with idiopathic hyperaldosteronism were continuously treated with trilostane, a competitive inhibitor of adrenal 3 beta-hydroxysteroid dehydrogenase (3 beta-HSDH) (3 to 4 2/3 years). Trilostane, in conjunction with antihypertensive drugs, effectively decreased plasma aldosterone levels and improved hyperaldosteronism symptoms without undesirable side effects. Trilostane continued to be effective even when treatment was continuous. Rapid ACTH testing (iv bolus of 0.25 mg alpha 1-24 ACTH) was done on the day without trilostane after long-term treatment, and plasma levels of aldosterone and cortisol were compared to those obtained during a pre-treatment period. Results suggest that the inhibitory effect of trilostane on steroid biosynthesis rapidly disappears following discontinuance of trilostane administration even after long-term treatment, and that continuous treatment causes no significant or irreversible change in steroid biosynthesis. These results suggest that trilostane is a safe, feasible therapeutic agent for long-term treatment of idiopathic hyperaldosteronism. PMID- 3020850 TI - Potentiation of cholinergic tone by pyridostigmine bromide re-instates and potentiates the growth hormone responsiveness to intermittent administration of growth hormone-releasing factor in man. AB - It is known that in normal subjects repeated administration of the growth hormone releasing factor (GRF) induces a state of partial refractoriness of the somatotropes to GRF. Studies were conducted to verify whether the cholinergic system plays a role in the mechanism(s) underlying the reduced GH responsiveness to the neuropeptide. In five healthy men, the GH response to three consecutive injections of GRF (50 micrograms iv), administered at 2 h intervals, was considerably blunted after the second and third GRF bolus. Administration of the inhibitor of cholinesterase, pyridostigmine bromide (120 mg orally) 30 min before the second GRF bolus, not only restored but greatly potentiated the GH responsiveness to the second GRF bolus. The GH response to the third GRF bolus was not apparently influenced by pre-treatment with pyridostigmine. These data reinforce the view that cholinergic neurotransmission plays an important role in the control of GH secretion in human. PMID- 3020852 TI - Secretion of adrenocorticotropin, beta-endorphin and calcitonin by cultured medullary thyroid carcinoma cells. Effects of synthetic corticotropin-releasing factor and lysine vasopressin. AB - The present study was performed to investigate the potential of human medullary thyroid carcinoma (MTC) cells to secrete ACTH, beta-LPH/beta-EP. In addition, these studies might shed further light on the possible synthesis of a common precursor molecule for calcitonin (CT), ACTH and beta-LPH/beta-EP. MTC tissue was obtained from 10 patients (6 familial, 4 sporadic) without clinical and biochemical signs of Cushing's syndrome. Single cell suspensions were cultured for 1 to 2 weeks. Mean basal release of beta-LPH/beta-EP was 0.76 +/- 0.29 (SE) ng/10(6) cells/4 h (n = 10). Dibutyryl cyclic AMP (3 mM) stimulated beta-EP release significantly in 3 out of 7 cultures, while Ca2+ (2 mM) had no effect at all. The effects of the physiological regulators of pituitary ACTH and beta LPH/beta-EP secretion, synthetic corticotropin-releasing factor (CRF-41) and vasopressin (LVP) were also studied in these MTC cell cultures. LVP (100 nM) had no effect on beta-EP release from MTC cells of all 8 cultures investigated. CRF 41 (10 nM) stimulated beta-EP release from 5 cultures and was without effect on 4. Maximal stimulation was noticed with 10 nM, while the effect of 100 nM CRF-41 was lower or absent. Stimulation was most outspoken in 3 cultures of familial MTC, whereas in 2 cultures of sporadic MTC CRF-41 stimulated beta-EP release marginally only after a 24 h incubation. LVP and/or CRF-41 stimulated CT release significantly in 3 cultures from sporadic MTC, while in these cultures the effect of CRF-41 on beta-EP release was either very small or absent.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020853 TI - Hormonal modification of the growth and metabolic effects of a transplantable rat insulinoma. AB - The growth and metabolic effects of a transplantable radiation-induced rat insulinoma were examined in intact male and female New England Deaconess Hospital (NEDH) rats, and in parathyroidectomised or adrenalectomised male NEDH rats. Subscapular transplantation of insulinoma fragments in intact male rats consistently produced a highly vascularised encapsulated tumour associated with hyperphagia, hyperinsulinaemia and hypoglycaemia which progressed to fatal neuroglycopaenic coma by 30 +/- 0.8 days (mean +/- SEM) and 19 +/- 0.5 days for slow-growing and fast-growing tumour sublines respectively (P less than 0.001). In intact female rats transplanted with the slow-growing subline, the onset of hyperphagia was advanced by 4 days and the severity of hyperinsulinaemia and hypoglycaemia increased (21% and 36%; P less than 0.01 and P less than 0.001, respectively), resulting in a 10% decrease of survival time (P less than 0.05) and a 65% reduction of tumour weight (P less than 0.01). Transplantation of the fast-growing subline into parathyroidectomised male rats, which exhibited a 15 24% (P less than 0.05 - less than 0.01) decrease of plasma calcium, did not modify either the growth or metabolic effects of the tumour. In contrast, transplantation of this subline into adrenalectomised male rats decreased survival time by 32% (P less than 0.001) and reduced final tumour weight by 88% (P less than 0.02) without markedly affecting the onset or magnitude of the hyperinsulinaemia. These results indicate that the growth and metabolic effects of the transplantable NEDH rat insulinoma are modified by the presence of ovarian hormones and by adrenal hormones.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020854 TI - Formation of lattice-like inclusions in circulating erythroblasts of the laboratory mouse (hea/hea) with hereditary congenital erythroblastic anemia. PMID- 3020851 TI - The effects of conjugated equine oestrogens with and without a cyclical progestogen on lipoproteins, and HDL subfractions in postmenopausal women. AB - Serum lipoprotein levels were followed for 24 weeks in 21 oophorectomised women treated with conjugated equine oestrogens (0.625 mg/day) and 21 women who had had a natural menopause and were treated with a combined preparation consisting of conjugated equine oestrogens (0.625 mg/day) with the addition of dl-norgestrel (0.15 mg/day) for the last 12 days of each treatment cycle. Conjugated equine oestrogens caused a significant (P less than 0.05) increase in triglyceride, HDL cholesterol, especially HDL2 cholesterol, and a significant decrease (P less than 0.05) in LDL cholesterol. Those subjects treated with conjugated equine oestrogens plus cyclical norgestrel showed a significant (P less than 0.05) decrease in LDL cholesterol levels only. PMID- 3020855 TI - [Prognosis of malignant tumors of the exocrine pancreas: effect of clinical and pathologico-anatomic variables on patient survival]. AB - Survival patterns of 112 patients with histologically ascertained pancreatic cancer were analysed retrospectively in an attempt to determine the relationship to various clinical and pathologic-anatomic prognostic factors. Stage of disease, localisation of the primary tumor, as well as histologic grade were found to influence the patient's survival significantly. A limited anatomic involvement with tumor, localisation within the head of the pancreas, and high-grade differentiation were associated with an increased median survival. An evaluation of non-anatomic prognostic variables suggested a relative survival advantage for females, younger age groups, patients with favourable initial performance status and short symptom duration. There was no obvious relationship between survival and qualitative or quantitative indication of weight loss. In order to permit a critical evaluation of the value of any treatment program in pancreatic cancer, our observations reemphasize the need to define characteristics of patients under study. PMID- 3020856 TI - Peripheral neuropathy in IgD myeloma. Cerebrospinal fluid paraprotein analysis in three cases. AB - High resolution polyacrylamide gel isoelectric focusing (IEF), followed by direct immunofixation with anti-delta chains monospecific antibodies, were used to detect and identify IgD paraprotein in the cerebrospinal fluid (CSF) of 3 patients affected by IgD myeloma. Two of these patients presented a paraproteinemic neuropathy. Blood-brain barrier damage was investigated by means of CSF/serum albumin ratio. IgG index and CSF and serum IgD/albumin ratio were also evaluated. An intrathecal origin of the IgD paraprotein was excluded. The correlation between the presence of the paraprotein in the CSF and the possible neurological involvement was also examined. PMID- 3020857 TI - Visual and auditory evoked potentials in the early diagnosis and follow-up of neurosarcoidosis. AB - Visual and brainstem auditory evoked potentials (VEPs, BAEPs) were recorded in 23 patients with neurosarcoidosis. Eight patients (35%) had abnormal BAEPs, and 10 (43%) had abnormal VEPs. Four of the 8 patients with abnormal BAEPs had facial paresis, one had impaired memory and only 3 had symptoms and signs compatible with brainstem lesion. Seven of the patients with abnormal VEPs had no visual symptoms. These findings suggest that BAEP and VEP can reveal subclinical nervous system involvement in sarcoidosis and can also help in the early diagnosis of neurosarcoidosis. Successive recordings of 5 patients showed that BAEP and VEP were useful in the follow-up of these patients. PMID- 3020858 TI - Immunohistochemical demonstration of vimentin in human cerebral tumors. AB - The distribution of vimentin (VIM) has been histochemically investigated in 53 cerebral tumors and compared in gliomas to that of glial fibrillary acidic protein (GFAP). In gliomas VIM is less positive than GFAP, but shows the same distribution. It cannot be considered as indicating immaturity of glial tumor cells. VIM is also positive in glial processes of cerebellar pilocytic astrocytomas, in Schwann cells of neurinomas and in endothelial cells of all oncotypes. In medulloblastomas, VIM decorates reactive glia cells. A diffuse positive reaction has been observed in meningiomas. In hemangioblastomas, besides intervascular and endothelial cells, groups of polygonal cells are intensely positive for both VIM and GFAP. The interpretation of VIM in cerebral tumors is largely based on the distribution patterns of this intermediate filament in the developing CNS of rodents. PMID- 3020859 TI - Role of VSV G antigen in the development of experimental spongiform encephalopathy in mice. AB - The ts G31 VSV mutant induced spongiform encephalopathy without any inflammatory response when injected i.c. into the mouse. In electron microscopy, no virions could be detected in spongiform lesions. In contrast, with the wild VSV strain inflammatory lesions were seen, which contained viral particles in great abundance. As previously shown in vitro, when using the ts G 31 mutant at the nonpermissive temperature, the G antigen can spread from membrane to membrane to distant sites, fusing a great number of cells even in the absence of virus multiplication. Therefore, we postulate that a comparable mechanism is responsible for extensive brain lesions originating probably from a relatively small number of G antigen-producing cells. Indeed, the spongious regions seen mainly in the grey matter contained vacuoles, whose walls were clearly stained by peroxidase-labelled immune serum to G antigen, without detectable virions or inflammatory lesions. The vacuoles probably represent altered and swollen dendritic cell membranes. The relationship between spongiosis development and antigen diffusion in the absence of significant virus replication is discussed. PMID- 3020860 TI - Formation of psammoma bodies in meningocytic whorls. Ultrastructural study and analysis of calcified material. AB - Psammoma bodies in meningocytic whorls were investigated by electron microscopy. In some whorls, connective tissue fibers were seen and membrane-bound vesicles were contiguous to degenerated cells. Some small vesicles, 0.1 to 0.5 micron in diameter, were outlined by plasma membrane (matrix vesicles), other larger ones, about 1 to several micron in diameter, were invested by a thick wall (matrix giant bodies). Mineralized deposits were frequent in these vesicles and occasionally large masses of mineralized connective tissue fibers (psammoma bodies) were seen. Analysis of the material in the mineralized vesicles and fibers, using an energy dispersive X-ray microanalyzer, showed that both calcium and phosphorous were evident and hydroxyapatite was substantiated using an X-ray diffractometer. Psammoma body formation in the meningocytic whorls may represent degeneration in some whorls of the central cells which contain connective tissue fibers, producing cell debris such as membrane invested vesicles. Subsequently, calcification occurs in these vesicles, and the mineralization process extends to neighboring connective tissue fibers. The calcified mass forms a psammoma body. PMID- 3020861 TI - Pyruvate kinase inhibition in the diagnosis of gliomas with an intermediate degree of malignancy. AB - The aim of the investigation was to see if the histological diagnosis of brain tumors showing an intermediate degree of malignancy can be improved by the measurement of L-alpha-alanine inhibition of pyruvate kinase isoenzymes. The inhibition of pyruvate kinase activity was measured in 51 gliomas with different grades of malignancy. It was confirmed that benign tumors have a low level of inhibition (less than 50%) and that the more malignant the tumor, the higher the level of inhibition became, reaching more than 75%. However, when grade II and III astrocytomas and grade II and III oligodendrogliomas were analyzed, their level of inhibition was found to be variable. Grade II showed low and moderate levels of inhibition and grade III moderate and high levels. In turn, inhibition levels ranging from 50 to 75% were not only found in brain tumors with an intermediate grade of malignancy, but also in a number of benign and malignant tumors. When the survival times of patients with brain tumors were compared with both the histological diagnosis and pyruvate kinase inhibition, the prediction of the survival time on the basis of low and high levels of inhibition correlated well with the histological diagnosis. In contrast, when moderate levels of inhibition were measured, the prediction of the patients' survival remained uncertain and no improvement was found in the prediction for tumors showing an intermediate degree of malignancy on the basis of histology. PMID- 3020862 TI - An immunohistochemical study of glial and neuronal markers in primary neoplasms of the central nervous system. AB - Paraffin-embedded tissues from 56 primary neoplasms of the central nervous system and seven cases of non-neoplastic reactive astrocytosis were examined by immunoperoxidase techniques on serial sections using monoclonal antibodies to glial fibrillary acidic protein (GFAP) and the 68 kDa neurofilament subunit and monospecific polyclonal antibodies to alpha- and gamma-enolase. gamma-Enolase was present in all neoplasms of neuronal origin, but was also present in anaplastic gliomas (particularly in giant cells), in some well-differentiated astrocytomas and reactive astrocytes. The cells containing gamma-enolase in these cases appeared morphologically identical to those containing alpha-enolase and GFAP in adjacent serial sections. No relationship was found between gamma-enolase immunoreactivity and cellular anaplasia in the gliomas studied. Subependymal neoplasms from patients with tuberose sclerosis exhibited evidence of both astrocytic and neuronal differentiation, sometimes in morphologically distinct cell populations, consistent with their suggested origin from a primitive cell line. PMID- 3020863 TI - Neuronal intranuclear hyaline inclusion disease in a nine year old. AB - Thirteen previous cases have been reported as neuronal intranuclear hyaline inclusion disease. The majority of patients have presented with movement disorders at less than 12 years of age followed by a progressive worsening of symptoms and, frequently, loss of cognitive function. Death has usually occurred by the second or third decade. Three have presented in the fifth through seventh decade with either movement disorders or dementia. These cases have been linked by the presence of eosinophilic neuronal intranuclear inclusions diffusely within the CNS and in peripheral ganglion cells. The patient in this case report also presented with a rapidly progressive movement disorder and at autopsy showed the characteristic intranuclear inclusions. Investigation of these inclusions did not reveal shared epitopes with neurofilaments or other intermediate filaments. PMID- 3020864 TI - Co-expression of glial fibrillary acidic protein- and vimentin-type intermediate filaments in human astrocytomas. AB - The expression of intermediate filament (IF) proteins was studied in 71 cases of malignant human astrocytoma and in 17 cases of reactive gliosis, using immunocytochemical techniques with polyclonal and monoclonal antibodies to glial fibrillary acidic protein (GFAP) and vimentin. In all cases of astrocytoma, varying in degree of malignancy from grade I to grade IV, co-expression of GFAP and vimentin was found. No change in vimentin- or GFAP-IF expression with increasing anaplasia was seen. In addition astrocytic cells in reactive gliosis showed simultaneous expression of GFAP and vimentin. The intracellular distribution of these IF proteins differed. Vimentin was found to be located in a more juxta-nuclear position, whereas GFAP immunoreactivity showed a more intense staining of the cellular processes. Astrocytes in reactive gliosis behaved more or less like neoplastic cells. However, thin cell processes of reactive astrocytes in the cortex and superficial white matter only contained GFAP immunoreactivity. Simultaneous expression of GFAP and vimentin and their proportion in malignant and reactive glial cells are discussed in the light of earlier reports on the IF content of glial cells during development and maturation, in which vimentin precedes GFAP-expression. The existence of two separate (functional) IF systems in astroglia is suggested. PMID- 3020865 TI - Induction of undifferentiated tumors by JC virus in the cerebrum of rats. AB - Newborn Sprague-Dawley rats were inoculated intracranially with JC virus (Tokyo 1), a human polyomavirus, which had been isolated by Nagashima et al. from the autopsied brain of a patient with progressive multifocal leukoencephalopathy in Japan. Twenty-one to 70 weeks later, 21 of 27 rats developed brain tumors in the cerebrum, but not in the cerebellum. Most of the tumor cells were of an undifferentiated neuroectodermal nature and showed nuclear palisades and pseudorosettes. In some tumor cells glial fibrillary acidic protein was positive immunohistochemically, and many glial filaments were demonstrated ultrastructurally. Neuronal differentiation was not proved. Two continuous lines of cultured tumor cells were established, and T antigen of JCV (Tokyo-1) was present in both cell lines. Glial differentiation was confirmed also in the tumors produced by subcutaneous transplantation of cultured tumor cells. PMID- 3020866 TI - Primary small cell carcinoma of skin. Histogenetical study. AB - Four cases of primary small cell carcinoma of the skin (PSCCS) are presented (ages ranging from 60 to 68 years). Ultrastructurally, two cell types were identified, with both presenting electron-dense secretory granules and paranuclear intermediate filaments. Argyrophylia was positive in one case. Intense solar elastosis in two cases and actinic keratosis in one case suggest a possible role from solar damage in the pathogenesis of this tumor. According to comparative ultrastructural features, different histogenetic possibilities in Merkel cells (MC), peripheral neuroblastic tissue, and totipotential cells are discussed. Some neurosecretory-like granules were observed in basal cell carcinoma (BCC). We consider that PSCCS reproduces cells similar to MC and probably originates in stem cells with totipotential capacity. PMID- 3020868 TI - [Effects of 5 derivatives of 3-azabicyclo [3,3,1]nonanes on isolated guinea pig ileum myenteric plexus-longitudinal muscle]. PMID- 3020867 TI - Comparison of characteristics of human small cell carcinoma of the lung in patients, in vitro and transplanted into nude mice. AB - Specimens from 24 patients with metastatic small cell carcinoma of the lung were explanted in vitro as well as transplanted directly into nude mice. A method to obtain fibroblast-free cultures is described. This method resulted in cell lines which could be grown for more than one year in 79% of the cases. Fifty-four % of the tumours could be established as serially transplantable tumours in nude mice. The tumours were characterized by histology, electron microscopy, DNA index, and cell cycle distribution. The in vitro cell lines were furthermore characterized by the plating efficiency and by doubling time. The macroscopic growth of the heterotransplanted tumours was ascribed to a transformed Gompertz function. The tumour cells preserved their light microscopic constitution of small cell carcinoma of the lung in the model systems. The heterogeneity of the original tumours was reflected in vitro and in nude mice and the model systems thus allows an expression of the inherent heterogeneity and instability. The panel of transplantable tumours and the in vitro cell lines offer the study of biology inclusive of tumour progression of SCCL. PMID- 3020870 TI - [Effects of aconitine on neuromuscular transmission and the superior cervical ganglion]. PMID- 3020869 TI - [Analgesic effect of metorphamide on skin pain and visceral pain]. PMID- 3020871 TI - [Inhibitory action of ribostamycin sulfate on neuromuscular transmission]. PMID- 3020872 TI - [Relation between the bronchospasmolytic action of sophocarpine and cAMP content of the mesencephalon]. PMID- 3020873 TI - Interactions of angiotensin II and GABA-ergic agents at the threshold of convulsive reactions. AB - The effects of angiotensin II (AT II), GABA, muscimol (administered i.c.v.) and of amino-oxiacetic acid (AOAA) and baclofen (administered i.p.), as well as of the combinations of AT II with these substances and bicuculline, were studied with respect to the threshold of the convulsive seizures induced by timed intravenous infusion of pentylenetetrazol (PTZ) in mice. It was found in the experiments that the threshold-increasing effect of GABA, muscimol and AOAA was potentiated by AT II (applied in a dose which did not change essentially the convulsive threshold). The potentiated effect of GABA, muscimol and AOAA on the convulsive threshold, when applied in combination with AT II, was antagonized by bicuculline. The threshold-increasing effect of baclofen was not affected substantially by AT II; in this case bicuculline had no effect. On the basis of the results obtained it may be assumed that AT II performs the functions of an antocoid predominantly for the GABA-A receptors in the central nervous system. PMID- 3020874 TI - Effects of picrotoxin on the turtle's electroretinogram under different background illumination. AB - The effect of 0.4 mM solution of picrotoxin (PT) on the electroretinogram (ERG) of isolated eyecup preparations of turtle (Emys orbicularis) was investigated under two different backgrounds (Ib)--scotopic (0.009 lx) and mesopic (9 lx). The test stimulus intensity was 46 lx. An increase of the amplitudes of all ERG waves was observed after PT application. The relative increase of the d-wave was the most pronounced one and was similar under the two Ib. The relative increase of the b-wave, however, was greater in mesopic than in scotopic conditions. The two subcomponents of the a-wave were differentially influenced by PT. The development of a new steady level of b-wave sensitivity was delayed after PT. PMID- 3020875 TI - Cyclic AMP, adenylate cyclase and cyclic AMP-phosphodiesterase activities in diabetic rat adipocytes. AB - The effect of streptozotocin-induced diabetes on cyclic AMP content, adenylate cyclase and cyclic-AMP phosphodiesterase activities in rat adipocytes was investigated. The results show that diabetes induced an increase in intracellular cyclic AMP. Basal adenylate cyclase activity and in response to norepinephrine were higher in fat cell membranes from diabetic rats. Adenylate cyclase activity stimulated by fluoride was the same in both normal and diabetic preparations. Low and high Km phosphodiesterase activities in adipocytes from diabetic rats were higher than the controls. The results suggest that the deficiency of insulin present in the diabetic state induces an increase in adenylate cyclase activity which increases the cyclic AMP production. This increase in cyclic AMP promotes a higher cyclic AMP-phosphodiesterase activity which is not sufficient to hydrolyze all the newly formed cyclic AMP. PMID- 3020877 TI - Anterior pituitary response to thyrotrophin releasing hormone in senile dementia (Alzheimer type) and elderly normals. AB - Sixteen patients with senile dementia of the Alzheimer type (SDAT) were compared with 11 age-matched normal subjects with respect to their responses of thyrotrophin (TSH) and prolactin (PRL) to thyrotrophin releasing hormone (TRH) challenge. Both groups showed a continuing rise in TSH response at 60 min, and no difference was found between the groups. A significantly exaggerated PRL response at 20 min was found in the SDAT group. There was no difference between sexes with respect to either the TSH or PRL response. These findings, albeit preliminary, may have implications for dopaminergic impairment in SDAT. PMID- 3020876 TI - Projection of Ia and Ib afferents from forelimb muscles to the C3-C4 segments of the cat spinal cord. AB - Group Ia muscle spindle afferents were activated separately by small stretches applied to the tendons of antibrachial muscles in the forelimb in the cat. Group Ib tendon organ afferents were stimulated electrically after a selective increase of the threshold of the Ia afferents. Recordings of focal synaptic potentials were made in the C3-C4 segments in the medial part of the base of the dorsal horn. It has been found that both Ia and Ib afferents have monosynaptic connections with neurones in this medial region. Quantitatively, these two groups of afferents produced focal synaptic potentials of approximately the same size. The connections may be to inhibitory interneurones projecting to the C3-C4 propriospinal neurones, which are known to receive disynaptic IPSPs from group I muscle afferents. PMID- 3020879 TI - Thyroid function after postoperative radiation therapy in patients with breast cancer. AB - Thyroid stimulating hormone (S-TSH) and thyroxine (S-T4) were measured in sera from 80 patients with breast cancer using radioimmunoassay. The patients had been irradiated after mastectomy 7.2 +/- 1.0 years earlier with 45 Gy in 3 to 4 weeks to parasternal, ipsilateral supraclavicular and axillary areas. Five patients (6%) had hypothyroidism with low S-T4 and elevated S-TSH levels. In addition, 12 patients (15%) had elevated (greater than 5 mU/l) S-TSH levels and normal S-T4 values. Shielding of the thyroid during irradiation and long-term follow-up of thyroid function with repeated hormone assays are recommended. PMID- 3020878 TI - Postoperative radiation therapy and adjuvant chemoimmunotherapy in breast cancer. Aspects of timing and immune competence. AB - The effects of radiation therapy and adjuvant chemoimmunotherapy on the immune competence of patients with breast cancer were investigated. The tests performed included intradermal tuberculin tests, T- and B-lymphocyte counts, and lymphocyte blast transformation tests; phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM) were used as mitogens. Enhancement in lymphocyte proliferative response to mitogenic stimulation by PHA and PWM was seen in patients after 3 courses of chemotherapy + levamisole, whereas irradiation given after chemotherapy caused long-lasting depression in response to PHA (p = 0.039) and PWM (not significant). T-lymphocyte counts were also lower after irradiation than after chemoimmunotherapy (p = 0.007). Clinically, the 16 patients treated with radiation therapy after chemotherapy exhibited a higher recurrence rate than the 24 patients treated first by irradiation. Enhanced reactivity to tuberculin tests occurred generally in patients receiving a planned treatment including irradiation, chemotherapy (5-fluorouracil, doxorubicin, cyclophosphamide) and levamisole. Enhancement of reactivity was seen more often in patients who had not relapsed. PMID- 3020880 TI - Enzyme immuno assay of the estrogen receptor in breast cancer biopsy samples. A comparison with isoelectric focusing. AB - The estrogen receptor (ER) was measured by isoelectric focusing (IF) and enzyme immuno assay (EIA) in 127 breast cancer samples. When comparing the two techniques quantitatively, a very high correlation coefficient was obtained (rs = 0.98; p less than 0.001). ER recovery increased, however, by a mean factor of 2.5 when EIA was used instead of IF. This increase seemed to be influenced by the ER concentration level, being lower at higher levels. The difference between IF and EIA did not seem to be due to menopausal status. A possible field of application for EIA might be measurement of ER in samples from patients on tamoxifen therapy. In five out of nine such samples, a considerably higher measurable amount of ER was found with EIA than with IF. EIA was also found to be a simpler and less time consuming method than IF. These advantages should, however, be weighed against the present higher costs. PMID- 3020882 TI - Different fractionation schedules in radiation treatment of cerebral metastases. AB - Multiple daily fractionation (MDF) was compared, in a cooperative study, with conventional fractionation (CF) in the radiation treatment of brain metastases. The 103 patients treated by MDF and the 44 given CF showed a similar response, in terms of neurologic improvement, survival, quality of residual life, and treatment-related morbidity. The hypothesis that MDF might give a better therapeutic ratio than CF was not confirmed in the present series. The short time period required gives MDF advantages in clinical practice. PMID- 3020881 TI - Combination chemotherapy with dacarbazine and lomustine in disseminated malignant melanoma. AB - Thirty-eight patients with disseminated malignant melanoma (stage IV) who had not received previous chemotherapy were given lomustine 50 to 80 mg/m2 orally on day 1 and dacarbazine 400 mg intravenously on days 1 to 3 with intervals of 6 weeks. Three of the 36 evaluable patients showed complete response (8%), 4 partial response (11%), and 5 had stable disease for at least 3 months (13%). The responding patients had metastases confined to cutaneous, nodal or pulmonary sites. None of the patients with liver, osseous or cerebral metastases, or patients with Karnofsky's status of less than 80, responded. Patients with more than two years from the diagnosis to the start of the chemotherapy were more likely to achieve objective response (p less than 0.05). Eighty-four per cent of the patients had nausea or vomiting, but otherwise toxicity was minimal. PMID- 3020884 TI - Irradiation of the thoracic esophagus. Prone versus supine treatment positions. AB - A vast majority of patients with esophageal cancer receive radiation therapy for cure or palliation. Because of the close anatomic proximity of the esophagus to the spinal cord, and unusually long fields used in the irradiation of esophageal cancer, staying within the spinal cord tolerance is crucial. The present investigation shows how this can be achieved by delivering the radiation in prone position. PMID- 3020883 TI - Daily multiple session radiation therapy in advanced oral carcinoma. A preliminary investigation. AB - Radiation therapy using conventional fractionation schedules has not achieved adequate disease control in patients with advanced oral carcinoma. Hence a trial was conducted to investigate the efficacy of daily multiple session irradiation in this disease. Of the 32 patients entered in the study, 25 were eligible for evaluation. Sixteen of the 25 patients had no evidence of disease during a follow up period of 24 months. Acute mucosal reactions were observed in all the patients, but they subsided in 2 to 4 weeks after completion of treatment. The results obtained in this pilot study justify evaluation of this method in a larger number of patients with advanced oral carcinoma. PMID- 3020885 TI - Primary extraosseous osteogenic sarcoma in the retroperitoneal space. A case report and review of the literature. AB - One case of primary, extraosseous osteogenic sarcoma in the retroperitoneal region is presented, which is the tenth case hitherto reported in the literature. The 3 females and 7 males reported had a median age at diagnosis of 59 1/2 years. The tumours were all large at diagnosis and the symptoms short and uncharacteristic. There was no characteristic set of laboratory or roentgenologic findings; 2 of the 10 cases had elevation of alkaline phosphatase, and in 4 cases roentgen examination revealed tumour calcifications. The prognosis was poor and 8 of the 10 patients died, with a median survival of 9 1/2 months. In 3 cases, surgery was supplemented by chemotherapy without obvious improvement in survival. PMID- 3020886 TI - Enhancement of hemopoietic recovery by indomethacin after sublethal whole-body gamma irradiation. AB - The effect of the non-steroid anti-inflammatory drug, indomethacin, a potent prostaglandin synthesis inhibitor, on the recovery of hemopoiesis was investigated in sublethally gamma irradiated mice. Treatment with indomethacin after irradiation was found to increase the granulocyte and lymphocyte counts in peripheral blood. Furthermore, an increased rate of the restitution of bone marrow cellularity and of the spleen weight was observed. Using the method of 125iodo-deoxyuridine uptake in the spleen, the ability of indomethacin to potentiate cell proliferation was demonstrated. PMID- 3020887 TI - Evidence of C-cell destruction in the thyroid gland of mice exposed to high 131I doses. AB - The parafollicular cells of the thyroid gland were visualized by means of the Sevier-Munger silver technique in normal mice and in mice receiving 131I in amounts sufficient to completely destroy the thyroid tissue. The destruction of the C-cells was observed in all 131I injected mice, and no histologic signs of recovery were seen during a period of 40 days following the treatment. PMID- 3020888 TI - Late radiation enteropathy following split-dose irradiation of rat small intestine. AB - In female Wistar rats roentgen irradiation of a 10 cm long exteriorized mid small intestinal segment was performed. The radiation dose was 23 Gy, given as a single exposure or divided in two equal fractions separated by intervals of 4 to 96 hours. Radiation injury was assessed 2, 8 and 26 weeks following irradiation using a semiquantitative histopathologic scoring system. An increased fractionation interval led to a reduced degree of radiation injury at all 3 observation times. The greatest difference was found between the single dose and 4 hour groups, indicating a relatively large capacity for repair of sublethal damage. The degree of radiation injury also decreased significantly when the interval between fractions was increased from 4 hours to 96 hours. This suggests that the phenomenon of slow repair may occur in cells involved in the development of late radiation enteropathy. However, an indirectly protective effect due to mucosal repopulation between fractions may also explain some of the differences. PMID- 3020889 TI - Effect of irradiation on early enzymatic changes in healing mandibular periosteum and bone. A histochemical study on rats. AB - The influence of irradiation was studied histochemically in healing mandibular periosteum and bone. After a cut line had been made on both sides of the mandible the rats were exposed to roentgen ray irradiation. The single doses were 15, 20, 30, 35 or 40 Gy. The animals were killed 1, 2, 4, 8, 10, 12, 16 and 24 hours after irradiation, for histochemical analysis. All enzymes, acid phosphatase, cytochrome oxidase, lactate, isocitrate, glucose-6-phosphatase and succinate dehydrogenase, showed a greater increase in enzyme staining in the irradiated cut lines than in the non-irradiated control lines. The intensity of the staining increased with time and dose over 24 hours. The observation time included an inflammatory phase with vascular, enzymatic and cellular responses to periosteal and bone injury. The increase in staining was dependent on the time after surgical trauma and radiation dose. The increase in enzyme staining probably represents the initial cell damage after irradiation. PMID- 3020890 TI - Dosimetry and quality specification of high energy photon beams. AB - A number of quality descriptors are defined characterizing the photon attenuation and lepton contamination properties of high energy photon beams for radiation therapy. The dependence of the quality parameters on the design of the clinical beams such as the incident electron energy, target and filter thicknesses, field size and depth in the phantom are analyzed in some detail using analytical and Monte Carlo techniques. It is shown that the mean attenuation coefficient of the beam for a standard field size of 10 cm X 10 cm is related very accurately to the mean stopping power ratio for ionizing chamber dosimetry but also approximately to the equilibrium absorbed dose in the beam for a given photon energy fluence. This means that accurate photon dosimetry can be performed without knowing the acceleration potential, target design or filter thickness for the beam in use. Furthermore, the mechanism behind beam hardening and softening in the phantom are quantitized and suitable quality parameters for the lepton contamination are identified. The latter allow a determination of the lepton contamination for correction of the stopping power ratio near the surface if the contamination is large. PMID- 3020891 TI - Primary cultures of human livers and their albumin-producing capacity. AB - Primary cultures of surgically obtained noncancerous portions of human liver tissues were made. Liver tissues were poorly dissociated with collagenase, but well dissociated with dispase. The yield and viability of cells were improved somewhat when dissociated with collagenase followed by dispase. The mean cell yield was 1.1 X 10(6) cells/g liver. The epithelial-like morphology of the dissociated liver cells was maintained for about one week, but thereafter degenerative alteration of cells was observed. In liver explant culture, an active outgrowth of cells was observed for more than one month. Albumin production in culture fluids from dissociated livers was detectable for about 2 weeks, but later became undetectable, while that from explant culture was detectable for at least one month. These data demonstrate that adult human hepatocytes can be isolated from noncancerous portions of livers with relatively high yield, and that albumin production of the dissociated cells is detectable for several days. PMID- 3020893 TI - Participation of GABA B binding sites on brain control of pituitary secretion: effect of baclofen. PMID- 3020892 TI - The hypothalamo-pituitary GABAergic regulation of prolactin release in the rat. PMID- 3020894 TI - Functional links between benzodiazepine and GABA receptors and pineal activity. PMID- 3020895 TI - Participation of GABA/benzodiazepine receptor system in the adrenal chromaffin cell function. AB - Histochemical studies have shown that GABA-containing nerve terminals impinge upon the chromaffin cells and that approximately 40% of the chromaffin cells store and release GABA. These observations are compatible with the possibility that GABA receptors located on specific populations of chromaffin cells can be activated either by GABA released from nerve terminals or by GABA released from adjacent chromaffin cells. Indeed, experiments with bicuculline indicate that in bovine chromaffin cells in culture and in the adrenal medulla of dog in vivo, the secretion of CA and opioid peptides mediated by activation of nicotinic receptors is under tonic control of GABA. In a series of pharmacological experiments in dog, we have shown that appropriate doses of GABA or other GABA-mimetic drugs release CA into the circulation. This release, comparable in its magnitude to that obtained by injecting a full pharmacological dose of carbamylcholine or by maximally efficient electrical stimulation of the splanchnic nerve, was not blocked by hexamethonium, naloxone or splanchnicotomy, but instead, was prevented by treatment with bicuculline methiodide. These data suggest that GABA-induced CA release is not the consequences of activation of transynaptic mechanisms involving either acetylcholine, enkephalin or GABA acting at the GABAB receptors, but rather the result of stimulation of GABAA receptors linked to C1- channels located on the membranes of the adrenal chromaffin cells. Because the administration of GABA was found to induce depolarization in autonomic mammalian ganglia in electrophysiological studies (DeGroat, 1970), we propose that the GABA mediated release of CA from dog adrenal medulla is the consequence of chromaffin cell depolarization.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020898 TI - GABA receptors in the developing chick visual system: influence of light and darkness. PMID- 3020896 TI - Gamma amino butyric acid (GABA) controls anterior pituitary hormone secretion. AB - Intraventricular injection (3V) of GABA can lead to a dose-related increase in plasma LH in ovariectomized (OVX) and OVX, estrogen-primed animals. Since the effects were blocked by the GABA receptor antagonist, bicucculine (B), they appear to be specific. These alterations in plasma LH were accompanied by alterations in hypothalamic LHRH, dopamine (DA) and norepinephrine (NE) content which suggests roles for all three compounds in the genesis of the increase in plasma LH. Since the DA receptor blocker pimozide (P) failed to block the elevation in LH induced by GABA it appears that the effect of GABA on LH release is mediated by NE. Others have found that the GABA agonist, muscimol, can lower plasma LH under certain conditions. Consequently, it appears likely that there may be opposite actions of GABA on LHRH release depending on the site of action within the hypothalamus. Intravenous (iv) injection of B in OVX rats produced an initial fall in plasma LH followed by a prolonged rise which again suggests several sites of action of GABA on LH release. GABA had no effect on FSH release consistent with separate control of this hormone. 3V injection of various doses of GABA produced a dose-related lowering of plasma TSH in OVX rats which appeared to be mediated by the dopaminergic system since P abolished the TSH-lowering of GABA. Following iv injection of B in normal male rats, there was a dramatic decline in TSH suggesting that under these conditions GABA would have a stimulatory action. Similar results were seen in OVX rats. The results indicate an important stimulatory action of GABA on TSH release in both males and OVX females. Perhaps the discrepancy between the results with B and GABA can be explained again by multiple sites of action of GABA of opposite sign. 3V injection of GABA induced a dose-related stimulation of growth hormone (GH) secretion; however, more recent evidence from other laboratories suggests that under certain conditions GABA has an inhibitory role in GH secretion. Again, we speculate that GABA had dual sites of action of opposite sign to affect GH. In contrast to the effects of GABA on these pituitary hormones, it appears to have a direct inhibitory effect on prolactin (Prl) secretion via the lactotrophs. Both stimulatory and inhibitory actions of GABA have been found following its injection into the brain. Studies with iv B also support dual actions on Prl release. PMID- 3020899 TI - Occurrence of GABA in rat testis and its effect on androgen production. PMID- 3020897 TI - GABAergic control of anterior pituitary function in humans. PMID- 3020900 TI - GABAergic responses to lithium chloride: dependence on dose, treatment length and experimental condition. PMID- 3020901 TI - Adenosine receptors on human T lymphocytes and human thymocytes. PMID- 3020902 TI - Regulation of deoxyadenosine and nucleoside analog phosphorylation by human placental adenosine kinase. AB - The enzymes responsible for phosphorylation of adenosine and nucleoside analogs are important in the pathogenesis of adenosine deaminase deficiency and for the activation of specific anticancer and antiviral drugs. We examined the role of adenosine kinase in catalyzing these reactions using an enzyme purified 4000-fold (2.1 umol/min/mg) from human placenta. The Km values of adenosine and ATP are 135 uM and 4 uM, respectively. Adenosine kinase phosphorylates adenine arabinoside with an apparent Km value of 1 mM using adenosine kinase assay conditions. The Km values for 6-methylmercaptopurine riboside and 5-iodotubercidin, substrates for adenosine kinase, are estimated to be 4.5 uM and 2.6 nM, respectively. These data indicate that dadenosine phosphorylation by adenosine kinase is primarily regulated by its Km, and the concentrations of Mg2+, ADP and AMP. The high Km values for phosphorylation of dadenosine and adenine arabinoside suggest that adenosine kinase may be less likely to phosphorylate these nucleosides in vivo than other enzymes with lower Km values. Adenosine kinase appears to be important for adenosine analog phosphorylation where the Michaelis constant is in the low micromolar range. PMID- 3020903 TI - The biosynthesis of deoxyguanosine triphosphate in herpes simplex type-1 infected Vero cells treated with acyclovir and hydroxyurea. PMID- 3020904 TI - 5-Fluoro-5'-deoxyuridine is an inhibitor of uridylate nucleotidase in L1210 leukaemia. PMID- 3020905 TI - Genetic analysis of deoxyadenosine toxicity in dividing human lymphoblasts. PMID- 3020906 TI - Regulation of enzymes by ligand induced change in polymerization. PMID- 3020907 TI - Identification of a purine 5'-nucleotidase in human erythrocytes. PMID- 3020909 TI - Regulation of the cytosol 5'-nucleotidase of the heart by adenylate energy charge. PMID- 3020908 TI - Human placental cytoplasmic 5'-nucleotidase: kinetics and molecular properties. PMID- 3020910 TI - Characterization of hydroxyurea (HYU) resistant S49 T lymphoma cells. PMID- 3020911 TI - Purification and properties of human deoxycytidine kinase. AB - The purification of deoxycytidine kinase from human leucemic spleen reported here result in a pure protein of molecular weight 28K. The enzyme eluates during gel filtration as a dimer and the same enzyme phosphorylates both deoxycytidine, deoxyguanosine and deoxyadenosine, but with different Km and Vmax values. Our results are in agreement with earlier studies with partially purified calf thymus deoxycytidine, but clearly different from some studies on human deoxycytidine kinase. PMID- 3020912 TI - Production and properties of monoclonal antibodies against human ecto-5' nucleotidase. PMID- 3020913 TI - AMP-deaminase and cytosolic 5'nucleotidase in human and murine lymphocyte subpopulations. PMID- 3020914 TI - An explanation for the heterogeneity in B lymphocyte ecto-5'-nucleotidase activity in patients with hypogammaglobulinemia. PMID- 3020915 TI - Purine enzyme activities as markers of lymphocytic differentiation: studies of lymphocytes from horses with severe combined immunodeficiency (SCID). PMID- 3020916 TI - The effects of PNP inhibition on rat lymphoid cell populations. PMID- 3020917 TI - Ectoenzymes of nucleotide metabolism on human lymphoid cells. PMID- 3020918 TI - Separate mechanisms for cellular uptake of purine nucleotides by B- and T lymphoblasts. PMID- 3020919 TI - Ecto-5'-nucleotidase can use IMP to provide the total purine requirements of mitogen-stimulated human T cells and human B lymphoblasts. PMID- 3020920 TI - Morphine: sites of action in guanosine nucleoside pathway. PMID- 3020922 TI - Production and degradation of AMP in cultured rat skeletal and heart muscle: a comparative study. PMID- 3020921 TI - Changes of purine metabolism during differentiation of rat heart myoblasts. PMID- 3020924 TI - Cell culture models for the study of purine metabolism in human placental tissue. PMID- 3020923 TI - Enzyme activities of purine catabolism and salvage in human muscle tissue. PMID- 3020925 TI - Evidence for a membrane adenosine receptor in Leishmania mexicana mexicana (WR 227). PMID- 3020926 TI - Purine salvage enzymes in Trichomonas vaginalis. PMID- 3020927 TI - Genetic and biochemical characteristics of three different types of mutants of mammalian cells affected in adenosine kinase. PMID- 3020928 TI - Adenosine receptors on human lymphocytes. PMID- 3020929 TI - Hypoxanthine transport through human erythrocyte membranes. PMID- 3020930 TI - Mechanism of thymineless death. PMID- 3020931 TI - Enhancement of methotrexate cytotoxicity by uracil analogues that inhibit deoxyuridine triphosphate nucleotidohydrolase (dUTPase) activity. PMID- 3020932 TI - Metabolism of diethylstilbestrol by horseradish peroxidase and prostaglandin synthase--evidence for a free radical intermediate. PMID- 3020933 TI - Thiyl radicals--their generation and further reactions. PMID- 3020934 TI - Glutathione peroxidase and reactive oxygen species in TCDD-induced lipid peroxidation. AB - Previous studies have shown that high doses of TCDD induce hepatic lipid peroxidation and inhibit selenium dependent glutathione peroxidase (GSH-Px) activity. The dose dependent effects of TCDD on hepatic lipid peroxidation (malondialdehyde content) and GSH-Px activity were determined. A dose as low as 1 microgram/kg induced hepatic lipid peroxidation and inhibited GSH-Px. Based on the use of scavengers of reactive oxygen species, lipid peroxidation (malondialdehyde formation) by hepatic microsomes from both control and TCDD treated rats appears to be due primarily to H2O2. The results indicate that superoxide, hydroxyl radical and singlet oxygen are also involved. The differences in the reactive oxygen species involved in microsomal lipid peroxidation between control and TCDD treated animals appear to be quantitative rather than qualitative. A 5.9-fold greater rate of malondialdehyde (MDA) formation by microsomes from TCDD treated animals occurred as compared to controls, while livers of TCDD rats had an MDA content that was 5.0-fold greater than the controls. These differences may be due in part to an enhanced production of H2O2 as well as a decrease in the activity of selenium dependent glutathione peroxidase which metabolizes H2O2. PMID- 3020935 TI - One- and two-electron oxidation of reduced glutathione by peroxidases. AB - The oxidation of glutathione by horseradish peroxidase or lactoperoxidase forms a thiyl free radical, as demonstrated with the spin-trapping ESR technique. Reactions of this thiyl free radical result in oxygen consumption, which is inhibited by the radical trap 5,5-dimethyl-1-pyrroline-N-oxide. In contrast to L cysteine oxidation, glutathione oxidation is highly hydrogen peroxide-dependent. The oxidation of glutathione by glutathione peroxidase forms GSSG without forming a thiyl radical intermediate except in the presence of the thiyl radical generating horseradish peroxidase. PMID- 3020936 TI - Active oxygen and toxicity. PMID- 3020937 TI - Formation of a ring-opened product from benzene in a hydroxyl radical generating system. PMID- 3020938 TI - Conversion of phenytoin to reactive metabolites in rat liver microsomes. PMID- 3020939 TI - Bacterial oxidation of methane and methanol. PMID- 3020940 TI - Characterization of macrophages elicited by intraperitoneal injection of hyaluronate. AB - Hyaluronate of 120,000 molecular weight has been injected in the peritoneal cavity of mice to study its effect on migration of inflammatory cells in vivo. After one day a dose-dependent granulocyte migration is observed. Three days later the number of granulocytes is greatly reduced and macrophages form about half of the total cell population. Hyaluronate-elicited macrophages show a decreased 5'-nucleotidase and an increased acid phosphatase activity as compared to resident macrophages. The production of superoxide anion in response to the phorbol ester tetradecanoyl-phorbolacetate, and the phagocytic activity are also enhanced. Macrophages elicited by hyaluronate secrete growth factor(s) for non lymphoid mesenchymal cells. It is concluded that hyaluronate in vivo stimulates the migration of inflammatory cells, thus causing the recruitment of a population of stimulating macrophages. These effects may explain previous reports on the acceleration of wound healing by hyaluronate. PMID- 3020941 TI - In vitro interactions between endogenous polyamines and superoxide anion. AB - Endogenous polyamines with anti-inflammatory activity scavenge superoxide and possibly other oxy-radicals produced by xanthine oxidase or from stimulated polymorphonuclear leucocytes. Polyamines incubated with stimulated cells are in part metabolised. Putrescine is converted to metabolites tentatively identified as gamma-aminobutyraldehyde, delta'-pyrolline and gamma-aminobutyric acid. The metabolism of spermidine, spermine and cadaverine was not as extensively studied but metabolites were formed that gave positive reaction to Schiffs reagent on tlc plates. The possible relevance of the results to the anti-inflammatory action of polyamines is discussed. PMID- 3020944 TI - Subtypes and functions of alpha-adrenoceptors. PMID- 3020942 TI - Potentiation by adrenaline of human platelet activation and the inhibition by the alpha-adrenergic antagonist nicergoline of platelet adhesion, secretion and aggregation. AB - Adrenaline (1 to 10 microM) can induce the aggregation of human platelets suspended in citrated plasma but does not induce the aggregation of washed human platelets at doses as high as 1 mM, although these platelets respond normally to ADP, PAF-acether, collagen, arachidonic acid, thrombin, the endoperoxide analog U 46619 and the Ca2+ ionophore A23187. Adrenaline (0.5 microM) potentiates the aggregation and secretion induced by all the previous agonists in citrated platelet-rich plasma (cPRP) or in washed platelets. The activation by adrenaline of human platelets is mediated by alpha 2-adrenergic receptors, as demonstrated by inhibition with a series of adrenergic antagonists. The alpha-adrenergic antagonist nicergoline inhibits the activation of human platelets by adrenaline in the following situations: nicergoline inhibits the aggregation and secretion caused by adrenaline in cPRP (IC50 0.22 microM and 0.28 microM respectively); nicergoline inhibits the aggregation and secretion induced by the combination of adrenaline and each aggregating agent listed above in cPRP (IC50 ranging from 0.1 to 2.5 microM) or in washed platelets (IC50 ranging from 0.1 to 0.8 microM); nicergoline inhibits the binding of 3H-yohimbine to washed human platelets (IC50 0.26 microM); the intravenous administration of nicergoline (0.5 mg/kg per day) to patients inhibits significantly the ex vivo response of their platelets to adrenaline in cPRP. High concentrations of nicergoline also inhibit the aggregation and secretion induced by the aggregating agents listed above in cPRP (IC50 range 108 to 670 microM) and in washed platelets (IC50 range 27 to 140 microM) and the adhesion of platelets to collagen-coated surfaces. This latter effect is not mediated through blockade of alpha-adrenoceptors. A possible role of adrenaline in platelet activation in vivo could justify the use of nicergoline (Sermion), an alpha-adrenergic antagonist in combination therapy to prevent arterial thrombosis. PMID- 3020943 TI - [Malignant fibrous histiocytoma of the perirenal tissue: report of a case--a statistical study of 58 cases of urological malignant fibrous histiocytoma in Japanese literature]. AB - A 78-year-old man was admitted in June 18, 1982 with a two-year history of general fatigue and loss of appetite. Physical examination revealed a child's head sized, firm, not tender, right upper quadrant mass which had an almost smooth surface and had respiratory displacement. Preoperative diagnosis was a hypovascular renal tumor presenting at the lower part of the right kidney. Right nephrectomy was performed on July 6, which displayed a specimen 1,300 g and 17 X 12 X 10 cm. The light yellow tumor appeared between the renal parenchyma and large fatty masses. The tumor was histologically diagnosed as storiform pleomorphic malignant fibrous histiocytoma (MFH) and disclosed infiltration of both the fibrous renal capsule and adjacent perirenal fatty tissue. There was no invasion of the tumor into the renal parenchyma and the case was considered to arise from the fibrous renal capsule or the perirenal tissue. Although he had been treated with ifosfamide and adriamycin three times after operation and with immunotherapy of 3 g of PSK per day for about five months, he died three years and one month after operation. We reviewed 58 cases of MFH arising from the retroperitoneum and genitourinary tract (urological MFH) in the Japanese literature. PMID- 3020945 TI - The role of alpha-adrenoceptors in the regulation of vigilance and pain. PMID- 3020946 TI - Detection of bacterial spoilage in some fruit flavoured ultra-high temperature milks in Nigeria. AB - Statistical analyses of pH measurements and microbiological methods have been employed to monitor microbial activity (spoilage) in a brand of non-refrigerated mango and vanilla flavoured ultra-high temperature (UHT) milk drinks in Nigeria. The mango flavoured milks were more sensitive to changes in storage conditions than was the vanilla flavoured milks. The aerobic bacterial flora in the spoiled flavoured milks was dominated by Gram-positive cocci, both catalase-positive and catalase-negative types. Coliforms and pseudomonads were also detected. The importance of this study to the methods for detecting spoilage in quality control and to the storage conditions of such products in the tropics is discussed. PMID- 3020947 TI - Fertility after salpingostomy following mechanically produced hydrosalpinx in rabbits. AB - Microsurgical salpingostomy results in 90% of tubes remaining patent following surgery. Although pregnancy rates have improved to a slight extent, the percentage of patients having live babies after salpingostomy is still disappointing. The discrepancy between patency rates and pregnancy rates seems to be due mainly to long standing tubal damage after the original infection. Possibly, raised intraluminal pressure from accumulated fluid may be a factor. Mechanically produced hydrosalpinges in rabbits show similar morphological characteristics to human hydrosalpinges (Vasquez et al., 1981, Otubu et al., 1984). This rabbit model was used in this study to examine the relationship between the severity of these changes and fertility after salpingostomy. Mechanical hydrosalpinges were produced in thirty mature New Zealand white rabbits by double clip application. After a period varying from 8 to 52 weeks of clip application, cuff salpingostomy and ampullary-isthmic anastomosis was done. One week after surgery, the animals were mated with bucks of proven fertility. There were pregnancies in all the uterine horns on the unoperated side (control) in all animals. There were pregnancies on the operated side in only two animals giving a pregnancy rate of 6.7%. A total of sixteen tubes were patent out of thirty operated, giving a patency rate of 53.3%. Adhesion was a major problem. We conclude that fertility is reduced after salpingostomy in mechanically produced hydrosalpinges in rabbits. The significance of this finding is discussed. PMID- 3020948 TI - Ovarian function after salpingostomy following mechanically produced hydrosalpinx in rabbits. AB - The discrepancy between pregnancy and patency rates after salpingostomy is explained largely by the residual functional damage to both the mucosa and smooth muscles of the fallopian tube. The function of the ovary after salpingostomy has not been extensively studied. Hydrosalpinx was mechanically induced by double clip application in thirty adult female New Zealand white rabbits. After varying periods of hydrosalpinx formation, ampullary-isthmic anastomosis and cuff salpingostomy were carried out using full microsurgical techniques. The animals were mated 1 week after surgery. Ten days after mating, laparotomy was performed. The contralateral oviducts and oviducts from unoperated animals served as controls 1 and 2 respectively. Corpora lutea counts and ovarian venous estradiol 17B (E2) and progesterone (P) levels were measured to assess ovarian function. The mean values (+/- s.e.) for corpora lutea count on Control 1, Control 2 and operated sides were 6.46 +/- 0.43, 5.69 +/- 0.44 and 4.03 +/- 0.32 respectively. The mean values for E2 and P were 159.0 +/- 17.43 and 281.769 +/- 13.58; 133.75 +/- 18.45 and 265.89 +/- 18.12; 128.57 +/- 14.66 and 206.103 +/- 13.296 respectively. Only the differences between the mean number of corpora lutea and mean progesterone were statistically significant (P less than 0.001) (0.02 greater than P greater than 0.01) respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020950 TI - Heat-labile prolactin immunoreactivity in normal human tissues: artifacts of peptide hormone radioimmunoassay in human tissues. AB - We recently demonstrated that a substantial proportion of the previously reported widespread distribution of human chorionic gonadotropin-like material in nonplacental normal human tissues is due to non-specific activity of tissue proteases (Adejuwon & Segal, 1984). The study was extended to another polypetide hormone, measured by the same general method of radioimmunoassay. Normal tissue samples of colon (n = 1) and testes (n = 5) obtained at surgery were analysed by a highly specific and sensitive radioimmunoassay (RIA) for human prolactin (hPRL). All tissues investigated appeared to contain immunoreactive hPRL-like material (6.7 +/- 5.3 ng/g weight, mean +/- s.d.) Heat at 55 degrees C for 15-20 min was found to have no effect on the immunoreactivity of purified hPRL. Exposure of normal tissues to these conditions was, however, found to completely eliminate the hPRL-like immunoreactivity. The factor present in these normal tissues giving positive radioimmunoassay results for hPRL is not identical to native hPRL. It may be due to tissue proteases interfering with the radioligand assay. True polypeptide hormone immunoreactivity must be distinguished from proteolytic activity by treatment with heat or protease inhibitor. PMID- 3020949 TI - Endocrine function and haemoglobinopathies: biochemical assessment of thyroid function in children with sickle-cell disease. AB - Thyroid function was assessed in ninety children with homozygous sickle-cell disease (haemoglobin genotype SS) in forty-five children with heterozygous sickle cell trait (AS) and in 162 control children with haemoglobin genotype AA. Serum levels of thyroxine, the in vitro triiodothyronine resin uptake and the calculated index of 'free thyroxine' were not significantly different in the three groups. The distribution of individual thyrotropin (TSH) values showed that only 11% of the HbSS subjects had values below the 95% confidence limits for the HbAA controls. However, the mean level of TSH was significantly lower in the HbSS than the other two groups of children. PMID- 3020951 TI - Cefoxitin: single agent treatment of septic abortion. AB - Using strict diagnostic criteria, a clinical trial of cefoxitin as the only antimicrobial agent in the treatment of twenty-five cases of septic abortion was carried out. The success rate of 77% (seventeen out of twenty-two) was significant in this group of seriously ill patients of low social class. Presence of pelvic abscess does not seem to preclude the use of cefoxitin. While the infections were of mixed bacterial flora, anaerobes were the predominant organisms isolated. Most organisms isolated were sensitive to cefoxitin but Pseudomonas aeruginosa was markedly resistant. No untoward reactions were observed. It appears that cefoxitin was successful as a single agent in the treatment of septic abortion. PMID- 3020952 TI - Haemoglobin A1 levels in Nigerian diabetic patients using microcolumn affinity chromatography. AB - Haemoglobin A1 (HbA1) levels were measured in forty-five diabetic Nigerians and thirteen non-diabetic controls using disposable microcolumn kits based on affinity chromatography. The effect on HbA1 results of storage of whole blood samples for 1 week at room temperature and in a refrigerator was also evaluated. The mean +/- s.d. of HbA1 levels in diabetic patients and controls were 12.1 +/- 5.6% and 5.2 +/- 0.90% respectively. The difference was highly significant, P less than 0.001. Newly diagnosed diabetic patients who had not been on any treatment had significantly higher mean value of HbA1, 14.4 +/- 5.0%, than old patients already on treatment, 9.5 +/- 2.9%, P less than 0.01. In a group of nine patients, the mean +/- s.d. of baseline HbA1 value was 10.3 +/- 4.2% and after 1 week storage of different portions of the samples at room temperature and in a refrigerator, the mean +/- s.d. respectively were 9.8 +/- 3.6% and 11.0 +/- 4.2%. There was thus a slight decrease on storage at room temperature and a slight increase during refrigeration but the observed differences were not statistically significant. It is concluded that the glyc-affinity micro-column chromatography is a satisfactory and potentially useful method for measuring HbA1 levels in tropical developing countries. The method was easy to use and the assays could be run under average ambient room temperature without recourse to use of an air conditioned room or a special chromatography chamber. PMID- 3020953 TI - Levels of immunoglobulins (G, A and M) and circulatory immunocomplexes in Nigerians with joint pains. AB - Serum immunoglobulin (G, A and M) and circulating immune complex concentrations were measured in eighty-three Nigerians having joint pains and forty apparently healthy Nigerians by the single radial immune-diffusion and polyethylene glycol precipitation methods respectively. The IgA level was significantly lower and IgM statistically higher in patients with joint pains than the controls. There was however no significant difference between the mean IgG concentrations in patients with joint pains and controls. The observed low circulating IgA levels could be as a result of depressed thymic activity, development of autoimmunity or utilization in immune complex formation. The mean concentration of soluble immune complexes was significantly higher in patients with joint pains than in the apparently healthy subjects. These immune complexes some of which may remain localized in the surface of cartilages, ligaments and menisci would cause activation of complement resulting in persistent inflammation and joint pains in these patients. PMID- 3020954 TI - Pediatric hypertrophic gastropathy. AB - Four previously healthy children presented in a 6-week period with marked hypoproteinemia without liver disease, malnutrition, or significant proteinuria. They all had strikingly similar radiographic findings consisting of enlarged folds confined to the fundus and body of the stomach. Three of the children had prodromal symptoms suggesting a viral illness. Cytomegalovirus was cultured from the urine in all cases and from the gastric biopsy specimens in three patients. Two of these patients also showed intranuclear inclusions in their biopsy specimens compatible with cytomegalovirus. It is not certain if cytomegalovirus was the cause of the illness. PMID- 3020955 TI - Benign pancreatic insulinoma: preoperative and intraoperative sonographic localization. AB - Twenty-nine patients with surgically proved benign pancreatic insulinoma were studied by preoperative or intraoperative sonography. Twenty-five patients had solitary pancreatic tumors; four had multiple tumors. Six of the patients with solitary insulinomas and one of the patients with multiple insulinomas had undergone previous unsuccessful exploration. Preoperative sonography was performed in 24 patients with solitary insulinomas, and 15 (63%) were localized. Intraoperative sonography was performed in 22 patients with solitary insulinomas, and 19 (86%) were visualized without having been previously located by palpation. Four of these visible solitary tumors (18%) were not detected by palpation at surgery. All the solitary insulinomas were detected with the combination of palpation and intraoperative sonography. In each of the six patients with solitary insulinoma who had undergone previous surgery, the tumor was visible with intraoperative sonography, which also demonstrated nonpalpable insulinomas in two of the four patients with multiple tumors. Preoperative real-time sonography is a sensitive, noninvasive, inexpensive method for localization of insulinoma. Intraoperative high-frequency sonography is a highly sensitive method for the detection of insulinoma. Intraoperative sonography is also valuable to determine the relationship of the insulinoma to pancreatic and bile ducts and thereby facilitate safe enucleation. PMID- 3020956 TI - Amiodarone-induced ultrastructural changes in canine myocardial fibers. AB - Chronic clinical toxicity with amiodarone, a unique antiarrhythmic drug, is associated with intracellular myelinoid inclusion bodies in skin, cornea, lung, liver and lymph nodes. The present study was designed to develop a canine animal model to study amiodarone concentration and onset of formation of myelinoid inclusion bodies in the cardiac tissue. Amiodarone was given intravenously in a single dose (40 mg/kg body weight) or multiple doses (40 mg/kg IV + 10 mg/kg IV X 7 days). Amiodarone concentration in the heart was high after a single dose but electron microscopic examination showed a normal ultrastructure. Numerous myelinoid inclusion bodies were found in the myofibrils of the left atria and right and left ventricles after only 7 days of amiodarone treatment. Myelinoid inclusion bodies were identified in several subcellular locations including intercalated disc but most were in close proximity of the mitochondria, sometimes touching and indenting the mitochondrial membrane. The antiarrhythmic effect of the schedule of intravenous amiodarone for 7 days used in this study was minimal, and this correlated with unexpectedly low myocardial levels of the drug and its metabolite. The results are consistent with our previous data of an antiarrhythmic effect with a single intravenous dose of 40 mg/kg body weight associated with high myocardial levels of amiodarone. We conclude that a single large dose of amiodarone with high tissue level may not cause myelinoid inclusion bodies, but they can be readily identified in all heart chambers after only 1 week of amiodarone treatment. This model would be useful to study amiodarone induced ultrastructural changes in the heart. PMID- 3020958 TI - Respiratory hazard from removal of ceramic fiber insulation from high temperature industrial furnaces. AB - Ceramic fiber insulation is being used increasingly as a refractory lining for heat treating and preheating furnaces in the iron and steel industry. This is largely due to its superior thermal resistance per unit thickness when compared to insulating fire brick, which was the previous mainstay of refractory linings. Although toxicity data to date have found these ceramic fibers to be innocuous, recent studies have shown the fibers to devitrify and undergo partial conversion to cristobalite when exposed to elevated temperatures. This paper presents the exposure hazards to cristobalite found during the removal of various brands of ceramic fiber insulation from heat treat furnaces and the extent of fiber transformation to cristobalite. PMID- 3020957 TI - Asbestos and disease: an industrial hygienist's perspective. AB - I have traced a personal thirty year journey of involvement with asbestos in the workplace and the nonoccupational environment, one still in progress, in an attempt to extract selected lessons for the industrial hygienist. Lessons for other professions, namely medicine, law and management have been studiously avoided in the interests of the scope of this presentation and permissible time. There are certainly numerous other lessons to contemplate. The movement of asbestos from the occupational to the nonoccupational environment is a case study that will undoubtedly be followed in the future by other potentially toxic materials. We must determine our position as a profession with regard to the tools we utilize, the procedures we follow, and our understanding of our moral and legal obligations to prepare for the often scientifically and technically unsupportable positions assumed by others, including governmental agencies, in their zeal to bring about change. We must discourage the opportunistic distortion of professional practices in these matters, and must issue well considered statements as a profession, statements relevant to the way these matters are addressed by society. We have a responsibility to lend perspective to these issues, to not permit understandably emotional responses to documented past severe health effects in other areas, such as the case of asbestos in the workplace, to carry over into conditions of very low exposures in the public domain. We must remind people of the relevance of dose-response and toxicological principles to assessment of risk.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3020959 TI - Activities of the hepatic enzymes of vitamin B6 metabolism for patients with cirrhosis. AB - Patients with cirrhosis and other hepatic diseases frequently exhibit lower concentrations of plasma pyridoxal 5'-phosphate (PLP), which is derived primarily from liver. To determine the biochemical basis for this abnormality, the enzymes of vitamin B6 metabolism--pyridoxal kinase, pyridoxine (pyridoxamine) 5' phosphate oxidase, PLP phosphatase(s), and pyridoxal oxidase(s)--were analyzed in liver. The activities of the two biosynthetic enzymes, pyridoxal kinase and pyridoxine (pyridoxamine) 5'-phosphate oxidase were similar for both. The phosphatase activities were significantly higher (mean +/- SD of 9.55 +/- 8.03 versus 3.97 +/- 2.36 nmol X min X mg protein, p less than 0.05) for cirrhotics. Pyridoxal oxidase activities appeared slightly lower for cirrhotics. There was considerable variation in many indices of liver function, which suggests that the defects contributing to altered vitamin B6 metabolism may be complex and individualistic. These analyses have shown that cirrhotics are capable of apparently normal PLP synthesis and that increased hepatic dephosphorylation may be responsible for low levels of plasma PLP. PMID- 3020960 TI - Short-term effects of calcium carbonate, lactate, and gluconate on the calcium parathyroid axis in normal elderly men and women. AB - We evaluated the impact of three calcium salts on the calcium-parathyroid axis in healthy elderly volunteers. Fasting subjects were administered a standardized 1-g calcium load on three occasions, using calcium carbonate, lactate, and gluconate in random sequence with 8 oz milk or orange juice as carrier. Blood and urine were collected at baseline and for 6 h following the calcium load. Each salt rapidly increased the serum-calcium concentration and urinary-calcium excretion. Response duration was shorter with gluconate than with the other salts, but peak responses were similar for all preparations. Urinary-calcium excretion was slightly lower when orange juice replaced milk, but calcemic responses were not different. Changes in immunoreactive-parathyroid hormone and renal-phosphorus handling did not differ among calcium salts. Except for a shorter duration of effect with gluconate, all three calcium salts provided similar short-term suppression of the parathyroid axis. PMID- 3020961 TI - Neuroendocrine differentiation in mullerian neoplasms. An immunohistochemical study of a "pure" endometrial small-cell carcinoma and a mixed mullerian tumor containing small-cell carcinoma. AB - Two neoplasms of the endometrium exhibiting histologic features of small cell carcinoma (SCC) were studied immunohistochemically for the presence of antigens indicative of neuroendocrine differentiation. The first case, a pure SCC, was positive for neuron-specific enolase (NSE), Leu-7, and chromogranin. The second case, a mixed mullerian tumor, had an extensive SCC component; the latter element was reactive for NSE and Leu-7. The first patient has had an unexpectedly long survival, while the second patient died with multiple distant metastases, containing only SCC. PMID- 3020962 TI - The histopathology of fundic gland polyps of the stomach. AB - To assess the histopathologic features of fundic gland polyps (FGP) of the stomach, 83 specimens from 25 patients were examined. In nine patients, FGP were associated with an inherited adenomatous polyposis syndrome; the other 16 patients had nonsyndromic FGP. FGP were microscopically characterized by the presence of fundic glands with variably disordered architecture, a feature not previously emphasized. Disordered architecture, found in 80 of 83 specimens (96%), comprised a spectrum of abnormalities, ranging from prominent glandular and cellular budding, found in 51 biopsies (61%), to irregular tortuous glands, noted in 61 biopsies (75%), to microcysts lined by fundic epithelium, seen in 72 biopsies (87%). The continuous range of these changes suggested that FGP arise through progressive formation and unfolding of secondary glandular buds, to result in cystic dilatation. No differences were found between syndromic and nonsyndromic FGP with respect to histologic features or mucin histochemistry, including presence of O-acylated sialomucins. PMID- 3020963 TI - Evaluation of cytomegalovirus (CMV) antibody screening tests for blood donors. AB - Four technics were compared to find the most suitable screening test for the cytomegalovirus (CMV) antibody status in blood donors. One hundred thirty-five donor samples were tested by two enzyme immunoassays, EIA(Litton) and EIA(Abbott), and by latex agglutination (LA) and complement fixation (CF). The seroreactivity of the tests were judged by concordance of three or more methods. The authors found that the test performance of the EIA(Litton) could be improved with adjustment of sample color variation, which increased the test sensitivity and specificity from 48.8% and 87.2% to 83.8% and 96.4%, respectively. Both the EIA(Abbott) and LA technics proved to be ideal screening tests with 100% sensitivity and negative predictive values. However, the rapid turnaround time and the simplicity in technics and equipment used with the LA test make it the test of choice for blood donor screening. PMID- 3020965 TI - Fever in respiratory virus infections. AB - The case records of 258 children with adenovirus; influenza A or B virus; parainfluenza 1, 2, or 3 virus; or respiratory syncytial virus infections were studied retrospectively with special attention to the degree and duration of fever. A temperature of 39.0 degrees C or higher was most frequently recorded in adenovirus, influenza A, and influenza B virus infections (in 68%, 84%, and 65%, respectively). The mean highest degree of fever in respiratory virus infections (39.2 degrees C +/- 0.6 degrees C) during hospitalization did not differ from that in defined serious bacterial infections, ie, meningitis, epiglottitis, sepsis, and urinary tract infections (39.3 degrees C +/- 0.7 degrees C). The mean duration of fever varied from 2.5 days (parainfluenza 2) to 5.2 days (influenza B). Of all children with respiratory virus infections, 37% had fever lasting five days or longer. The data show that high and prolonged fever is frequently associated with respiratory virus infections in hospitalized children and that it does not differ significantly from fever in severe bacterial infections. PMID- 3020964 TI - Childhood near-death experiences. AB - We nonselectively interviewed 11 patients aged 3 through 16 years who had survived critical illnesses, including cardiac arrests and profound comas. Any memory of a time they were unconscious was considered to be a near-death experience (NDE) and was recorded. Seven of these children had memories that included being out of the physical body (six patients), entering darkness (five patients), being in a tunnel (four patients), and deciding to return to the body (three patients). We also interviewed 29 age-matched survivors of illnesses that required intubation, narcotics, benzodiazepines, and admission to an intensive care unit. None of them had any memories of the time they were unconscious. In our study population, NDEs were clearly associated with surviving a critical illness. The elements of NDEs reported are similar to those previously described in adults. No children described elements of depersonalization as part of their NDEs. A core NDE, triggered by the process of dying or resuscitation efforts, may be a natural developmental experience. We present a neurophysiologic hypothesis as to the cause of NDEs. PMID- 3020967 TI - Fiber and health: an overview. PMID- 3020966 TI - Modulation of rotavirus enteritis during breast-feeding. Implications on alterations in the intestinal bacterial flora. AB - A cohort of 197 infants was followed up prospectively for a single rotavirus (RV) season, 1983 to 1984, to examine the effect of long-term feeding method on RV infection. The feeding classification distinguished breast vs formula milk intake over the long term, for at least 18 weeks from birth (approximately four months). During the follow-up period, relative numbers of RV particles in feces were compared by electron microscopy, and positive specimens were confirmed by an enzyme-linked immunosorbent assay. There was no apparent difference in the infection rates of rotavirus enteritis in breast-fed (20%) as compared with bottle-fed (17%) infants. However, clinical manifestation of illness was milder in breast-fed infants. Among the breast-fed subjects, fecal flora identified by bacterial cultures, biochemical reaction, and gas-liquid chromatography revealed a significant growth of bifidobacteria lasting as long as the period of lactation. Colonization by this organism above the detection level of log 10(5)/mL was not observed in the feces of bottle-fed infants. These data suggest that alterations in enteric flora induced by breast-feeding may be correlates of intraluminal events, mediated by human milk, that modulate the clinical course of RV gastroenteritis. PMID- 3020968 TI - Dietary fiber: diabetes and obesity. PMID- 3020969 TI - Dietary fiber: hyperlipidemia, hypertension, and coronary heart disease. PMID- 3020970 TI - Fiber and starchy foods: gut function and implications in disease. AB - Increased intake of fiber and starchy foods has been recommended in the treatment or prevention of a range of diseases including dumping syndrome, hyperlipidemia, gallstones, diabetes, Crohn's disease, constipation, irritable bowel, diverticular disease, and colonic cancer. The nature and physiological effects of fiber are diverse. However in general, insoluble fibers increase fecal bulk and decrease transit time. On the other hand, soluble fibers have metabolic effects secondary to reducing the rate of small intestinal absorption. In the colon, along with undigested starch, they are largely fermented yielding short-chain fatty acids which may have further metabolic effects. At present although much further work is required, the clinical management of hyperlipidemia, diabetes, constipation, and diverticular disease have already been significantly influenced as a result of the ideas and experimental evidence generated by the fiber hypothesis. PMID- 3020971 TI - Cancer risk: possible protective role of high carbohydrate high fiber diets. AB - Epidemiological data from different populations have suggested positive relationships between the incidence of colon cancer and meat and fat intake and a negative relationship with dietary fiber consumption. Within population comparisons have been less clearcut. Current theories on colonic carcinogenesis in man involve increased concentrations of bile acids and their metabolites, alterations in colonic pH, low Ca++, raised NH3 and long chain fatty acid levels, and alterations in bacterial numbers, type, and metabolic capabilities. The many laboratory studies in rats have been difficult to interpret since powerful initiators of carcinogenesis are always required and this rather than the promotion of spontaneous neoplastic change is the sine qua non for tumor growth in this situation. The current dilemma highlights the lack of knowledge of most aspects of human colonic physiology. Until these issues are more clearly resolved the epidemiological leads would point to low fat diets rich in less processed starchy foods with increased fiber as possible protection. Such advice is in common with the pronouncements of heart foundations, diabetes associations, and recommendations of official bodies to the general public. PMID- 3020972 TI - Practical aspects of implementing increased dietary fiber intake. AB - Two hundred twenty dietitians participated in a workshop conference on Health Implications of Dietary Fiber. They were given lectures to increase their knowledge base, and then in group sessions answered questions and wrote concensus opinions. The results are the content of this paper. The topics covered and responses are reported in four categories, diabetes and obesity, hyperlipidemia, hypertension and coronary heart disease, gut function and gastrointestinal disease, and cancer. Specific recommendation for implementing high fiber diets are made in each category. However, the dietitians expressed caution on accepting all of the conclusions expressed in the literature on the value of fiber and believed much education and instruction is needed in order to increase dietary fiber intake. PMID- 3020973 TI - Cytomegalovirus infection of the alimentary tract: a clinicopathological correlation. AB - Alimentary tract cytomegalovirus (CMV) infections of 24 patients were reviewed, including 19 with the acquired immune deficiency syndrome. CMV inclusion bodies (CMV-IB) were calibrated per mm2 of tissue. CMV-IB counts were correlated with biopsy site, inflammatory response, and clinical parameters. Colonic biopsies showed the highest counts. Biopsies of the right colon had about three times as many CMV-IBs as those of the left. Upper alimentary tract biopsies had low counts. Mesenchymal cells were most affected (97%); 35% were identified as endothelial and 6% as smooth muscle. Only 3% of CMV-IBs were in epithelial cells. Grades of inflammation, 1-5, correlated directly with CMV-IB counts up to grade 4. In grade 5 inflammation tissue destruction was so severe that CMV-IBs were difficult to recognize. Ulcers were demonstrated in more than half of all patients, either histologically or endoscopically. The inflammatory response was nonspecific, except for patchy infiltrates and the absence of lymphoid follicles, crypt abscesses, or granulomas. Gastrointestinal infections, such as shigellosis, candidiasis, mycobacteriosis, and cryptosporidiosis coexisted in 17 patients. No correlation was found between CMV-IB counts and severity of symptoms or length of survival. Alimentary tract CMV infections was the first manifestation of the acquired immune deficiency syndrome in 11 patients. Survival ranged from 2 wk to 19 months. PMID- 3020974 TI - Necrosis of hepatocellular carcinoma as a result of subintimal injury incurred by hepatic angiography: report of two cases. AB - This report described two patients with hepatocellular carcinoma in whom angiographic procedure caused an inadvertent subintimal injury of the hepatic artery, resulting in tumor necrosis. In the first case of a 38-year-old male, complete obstruction of the common hepatic artery occurred on the initial angiography. It was followed by marked reduction of the tumor vessels on repeat angiography, and necrosis of about half of the tumor as confirmed by computed tomography. In the other 58-year-old female, severe subintimal injury occurred in the proper hepatic artery followed by obstruction of the feeding arteries. Subsequent computed tomography scan disclosed necrosis of the tumor. Both patients presented the postembolization syndrome that consisted of a transient fever and elevation of blood enzymes. When spontaneous regression or reduction of hepatocellular carcinoma is observed, special attention should be paid regarding whether or not hepatic angiography was performed and clinical symptoms followed it. PMID- 3020975 TI - Solitary hamartomatous polyp of the duodenum in the absence of familial polyposis. AB - A large hamartomatous polyp of the Peutz-Jegher type was discovered in the distal duodenum by endoscopy in a patient with occult gastrointestinal bleeding. There were no other polyps in the gastrointestinal tract and the patient lacked any stigmata associated with the familial polyposis syndromes. This is the second well-documented case of an isolated hamartomatous polyp of the Peutz-Jegher type in the small intestine occurring in the absence of familial polyposis. PMID- 3020976 TI - Conjugated estrogens and fibrocystic breast disease. AB - The association between replacement estrogen use and subsequent development of fibrocystic breast disease was studied at Group Health Cooperative of Puget Sound. Included were 142 women aged 50-64 years diagnosed by biopsy as having fibrocystic breast disease during the years 1978 through 1983. The relative incidence of hospitalization for estrogen users compared with non-users was 1.9 (95% confidence interval 1.3-2.7). The association became stronger with increasing duration of estrogen use. PMID- 3020978 TI - Human parvovirus and aplastic crisis in chronic hemolytic anemias: a study of 24 observations. AB - From March 1984 through November 1985, 24 children and adults with aplastic crises were admitted in several Parisian hospitals. Twelve patients had known hemolytic anemia. Aplastic crisis revealed hemolytic anemia in the remaining patients. The detection of human parvovirus antigen was performed by counter immunoelectrophoresis, and specific IgM antibodies were detected by IgM-antibody capture-radioimmunoassay, in order to establish the incidence of human parvovirus infection in the genesis of the aplastic crisis. Twenty-one patients had acute infection with human parvovirus. In the three remaining patients, no marker of human parvovirus infection was found. The features of the human parvovirus linked aplastic crisis are described. We consider that human parvovirus infection, and unknown hemolytic anemia, must be systematically researched in any case of unexplained acute aplastic anemia. PMID- 3020977 TI - Spectrum of HTLV-III infection in a hemophilic cohort treated with blood products from a single manufacturer. AB - One hundred fifty-eight hemophilia A, B, and von Willebrand disease (VWD) patients treated with clotting factor concentrates from a single manufacturer were tested for antibody to the human T-lymphotropic virus type III (HTLV-III). Antibody was detected in 63% and 40% of those with severe hemophilia A and B, respectively, 12% and 0% of those with mild hemophilia A and B, and two patients with recessive VWD. Forty-two antibody-positive and 20 antibody-negative patients were studied for clinical and laboratory features of infection. Eleven seropositive patients had clinical signs of infection including Pneumocystis carinii pneumonia, lymphadenopathy, splenomegaly or diarrhea; however, only one patient had developed acquired immune deficiency syndrome (AIDS), and only two had significant impairment of their performance status. Thirty-one patients remained totally asymptomatic. Eight patients had a history suggestive of acute HTLV-III infection. Thrombocytopenia was observed in 18% of seropositive patients, lymphopenia in 60%, depressed T-helper cells in 43%, reduced T-helper:T suppressor ratios (TH:TS) in 33%, and elevated platelet-bound immunoglobulin in 53%. The antibody-negative group had normal T-helper cell levels (except one patient) and TH:TS ratios, and normal platelet immunoglobulin levels. Both groups demonstrated a significant elevation of immunoglobulin levels and a high prevalence of antinuclear factor and antismooth muscle antibodies. The mean level of IgG was significantly higher in the antibody-positive group. This study confirms the correlation between HTLV-III infection and reduced T-helper cells in hemophiliacs but demonstrates a low incidence of clinical symptomatology. There was evidence of polyclonal B-cell hyperactivity in the antibody-negative group as well as the seropositive group. PMID- 3020979 TI - Procedures for the inactivation of viruses in clotting factor concentrates. AB - A considerable body of data has accumulated on the viral inactivated clotting factor concentrates. This information strongly indicates considerable safety and expected efficacy of these products. Probably most procedures are capable of inactivating large amounts of the LAV/HTLV-III virus, but there is considerable variability between products in the ability to inactivate the hepatitis viruses and perhaps also the LAV/HTLV-III. This variability is probably related to the mechanism of viral inactivation, the time of exposure to heat or chemical additives, and also the presence and type of stabilizer. So-called "second generation" products will probably show improved elimination of more resistant viruses including the non-A, non-B forms. PMID- 3020980 TI - Chromosomal localization of a human band 3-like gene to region 7q35----7q36. AB - Band 3, the major transmembrane protein of erythrocytes, mediates the exchange of anions across the membrane and anchors the erythroid membrane skeleton. Proteins immunologically related to Band 3 have been detected in a variety of nonerythroid cells. We have isolated a human cDNA clone that encodes a protein related to but distinct from the erythroid form of Band 3, based on the comparison of the amino acid sequence for the two proteins. The presence of the gene for the Band 3-like protein in a panel of mouse-human somatic cell hybrids containing subsets of human chromosomes correlated with the presence of human chromosome 7. In situ hybridization analysis using the c-DNA for this nonerythroid Band 3 gene further localized the gene to region 7q35----7q36 of human metaphase chromosomes. PMID- 3020981 TI - Human placental opioid peptides: correlation to the route of delivery. AB - The levels of opioid peptides in placental extracts were determined by means of a radioreceptor assay with two prototypical opioid ligands, 3H-ethylketocyclazocine and 3H-D-ala2-enkephalinamide. Opioid peptide levels were significantly higher in the placentas obtained by the vaginal route than in those by the abdominal route. The different peptides in the extract were detected by reverse phase high performance liquid chromatography and a radioreceptor assay for all fractions. The presence of dynorphin-like peptides in several fractions suggests that one or more could be the "natural" ligand of the opioid receptors in the human placenta. A role for these peptides in parturition is a viable possibility. PMID- 3020982 TI - Human genital papilloma infections: an evaluation of immunologic competence in the genital neoplasia-papilloma syndrome. AB - Immunologic evaluations of women with genital neoplasia-papilloma syndrome demonstrated the presence of subclinical immunodeficiency when compared with results in 20 control women. All patients with genital neoplasia-papilloma syndrome were previously found to have human papillomavirus deoxyribonucleic acid in genital neoplasias or papillomas occurring either synchronously (in at least two genital organs at the same time) or metachronously (at different times during a period of months to years). Immunologic tests included blastogenic responses of lymphocytes to mitogens (phytohemagglutinin, concanavalin A, pokeweed mitogen, and tetanus antigen) and lymphocyte phenotyping with the use of monoclonal antibodies (OKT3, OKT4, OKT8, and OKT11). As compared with those of control subjects, the responses of the lymphocytes of patients with genital neoplasia papilloma syndrome to mitogens were significantly decreased. The group with genital neoplasia-papilloma syndrome had a significantly higher percentage of suppressor-cytotoxic T cells (OKT8-positive cells) when compared with that of control subjects (mean 33% versus 18%) and a lower proportion of helper T cells (OKT4-positive cells) when compared with that of control subjects (35% versus 50%). The mean helper-to-suppressor/cytotoxic T-cell ratio (mean OKT4/OKT8 ratio) in the human papillomavirus-infected women was 1.72 +/- 0.29 (SE) as compared with 3.21 +/- 0.33 (SE) in the control group, demonstrating a significant reduction of the ratio in the patients with genital neoplasia-papilloma syndrome. These findings suggest that patients with genital neoplasia-papilloma syndrome have a reduced suppressor/cytotoxic T-cell ratio (mean OKT4/OKT8 ratio; that in the human papillomavirus-infected women was 1.72 +/- 0.29 (SE) as compared with 3.21 +/- 0.33 (SE) in the control group, demonstrating a significant reduction of the ratio in patients with genital neoplasia-papilloma syndrome. These findings suggest that patients with genital neoplasia-papilloma syndrome have reduced immunocompetence of unknown etiology. PMID- 3020984 TI - Treatment of cytomegalovirus optic neuritis with dihydroxy propoxymethyl guanine. PMID- 3020983 TI - Parity and use-effectiveness with the contraceptive sponge. AB - The results of a randomized United States study indicated that the Today contraceptive sponge was less effective than the diaphragm (1-year cumulative life-table rate of 17.4 versus 12.9 pregnancies per 100 women, p = 0.01). However, this overall comparison is misleading. Using univariate and multivariate analyses to account for the effects of user characteristics we found parity to be the most important single determinant of effectiveness for users of the sponge, but parity was unimportant as a risk factor for pregnancy among diaphragm users. For nulliparous women the sponge was as effective as a physician-prescribed barrier method (13.9 for sponge, 12.8 for diaphragm, p = 0.45); however, parous women using the sponge were twice as likely to become pregnant (28.3 for sponge, 13.4 for diaphragm, p = 0.001). The effect of parity among sponge users is consistent with the results of international studies of the contraceptive sponge. PMID- 3020985 TI - Therapeutic modality comparisons in day treatment. AB - The deinstitutionalization of patients with chronic mental illness and shorter hospitalizations of individuals recently diagnosed as mentally ill has resulted in the establishment of an enlarged network of community mental health services. Diminished federal financial support calls for greater efficiency and accountability in the delivery of community-based mental health services. The purpose of this study was to determine whether differences in treatment approach relate to differences in outcome measures of symptom reduction, community tenure, and relapse. In a study of two day treatment centers, one offering twice as much activity therapy as verbal therapy, and the other offering twice as much verbal therapy as activity therapy, it was found that clients receiving primarily activity therapy achieved four times more symptom reduction, equivalent community tenure, and a three and a half times greater relapse rate than clients receiving primarily verbal therapy. PMID- 3020986 TI - Structure-activity relationships in the induction of mammary gland neoplasia in male rats with substituted aminopyrazoles. AB - In toxicity studies with a potential antipsychotic agent, N-(4-[2-fluorobenzoyl] 1,3-dimethyl-1H-pyrazol-5-yl)-2-([3- (2-methyl-1-piperidinyl)propyl]amino) acetamide(Z)-2-butenedioate (1:2) (FP-1), mammary gland neoplasia in male rats was induced within 13 weeks. Tumor induction by the parent compound (FP-1) and structural analogs was also explored. Rats were given 50 mg/kg/day of FP-1 or the diethyl glycine analog, FP-2. Other experimental groups received the FP nucleus, a benzoylpyrazolylacetamide, or the FP side chains alone or administered concurrently with the nucleus. Most animals survived the 13 weeks without significant clinical effects. Clinically detectable, gross subcutaneous mammary nodules developed only in rats given FP-1 or the FP nucleus coadministered with the FP-1 side chain. Additional mammary gland neoplasms were found at necropsy or on histopathologic examination of mammary glands from rats receiving FP-1, FP-2, and the FP nucleus. The neoplastic effect was not influenced by the structure of the side chains. Since these substituted aminopyrazoles are novel chemicals, the mechanism for this neoplastic effect is not yet clearly established; however, the proliferating effect resides in the nucleus of this series of compounds and is likely related to alteration of DNA in target mammary tissue. PMID- 3020987 TI - Uptake of human eosinophil peroxidase by human neutrophils. AB - A cytochemical analysis was carried out for study of the interaction between human eosinophil peroxidase (EPO) and human neutrophils. To this end, neutrophils with a genetic deficiency of myeloperoxidase (MPO) were used to avoid the otherwise inevitable interference of the high endogenous MPO activity of normal neutrophils. The data show that human neutrophils incubated with EPO (1 GU/ml) rapidly bind the enzyme all over the cell surface and internalize it in small vesicles. Part of bound EPO concentrates in a limited area on the cell surface and is then internalized by means of coarse tubular channels. Fusion of the small vesicles to each other or possibly with the tubular channels gives rise ultimately to EPO-containing multivesicular bodies, which, after 30 minutes of incubation, are the only peroxidase-positive structures in the cytoplasm. Under identical experimental conditions, no binding of human MPO to the neutrophils was detected. At concentrations 10 times as high as those used for EPO, a minority of neutrophils bound MPO, but the binding pattern remained diffuse on the plasma membrane and the internalization was negligible. It seems, therefore, that the EPO trapping system of human neutrophils exhibits specificity at least among leukocyte peroxidases. Furthermore, it operates at much lower concentrations of EPO than those reported for EPO uptake by mast cells and basophils. The uptake of EPO by neutrophils may serve to sequester a potentially toxic agent, thus limiting damage to the tissue in eosinophil-rich inflammatory lesions. PMID- 3020988 TI - Effects of calcitonin on cytosolic Ca in a suspension of rabbit medullary thick ascending limb tubules. AB - A basal level of cytosolic free Ca (Caf) of 112 +/- 7 nM (n = 15) was measured in rabbit medullary thick ascending limb tubules that were loaded with the fluorescent Ca indicator quin 2. Calcitonin addition (10(-6) M) caused a greater than twofold increase in Caf. The KD for calcitonin stimulation of Caf was 8 X 10(-11) M. The rise in Caf had a half time of approximately 1.5 min. Neither parathyroid hormone (10(-6) M), dibutyryladenosine 3',5'-cyclic monophosphate (10(-4) M), nor forskolin (2 X 10(-5) M) had any significant effect on Caf levels, consistent with the rise in Caf being independent of an elevation in adenosine 3',5'-cyclic monophosphate. The effects of ouabain and the mitochondrial uncoupler, 1799, were also tested to obtain further information regarding the regulation of Caf. Ouabain (10(-4) M) caused no significant elevation in Caf; in contrast, 15 min of incubation with 1799 (10(-5) M) produced a twofold increase in Caf. PMID- 3020989 TI - Target molecular weights for red cell band 3 stilbene and mercurial binding sites. AB - Radiation inactivation was used to measure the target sizes for binding of disulfonic stilbene anion transport inhibitor 4,4'-dibenzamido-2,2'-disulfonic stilbene (DBDS) and mercurial water transport inhibitor p-chloromercuribenzene sulfonate (pCMBS) to human erythrocytes. The measured target size for erythrocyte ghost acetylcholinesterase was 78 +/- 3 kDa. DBDS binding to ghost membranes was measured by a fluorescence enhancement technique. Radiation (0-26 Mrad) had no effect on total membrane protein and DBDS binding affinity, whereas DBDS binding stoichiometry decreased exponentially with radiation dose, giving a target size of 59 +/- 4 kDa. H2-4,4'-diisothiocyano-2,2'-disulfonic stilbene (H2-DIDS, 5 microM) blocked greater than 95% of DBDS binding at all radiation doses. pCMBS binding was measured from the time course of tryptophan fluorescence quenching in ghosts treated with the sulfhydryl reagent N-ethylmaleimide (NEM). Radiation did not affect the kinetics of tryptophan quenching, whereas the total amplitude of the fluorescence signal inactivated with radiation with a target size of 31 +/- 6 kDa. These results support the notion that DBDS and pCMBS bind to the transmembrane domain of erythrocyte band 3 in NEM-treated ghosts and demonstrate that radiation inactivation may probe a target significantly smaller than a covalently linked protein subunit. The small target size for the band 3 stilbene binding site may correspond to the intramembrane domain of the band 3 monomer (52 kDa), which is physically distinct from the cytoplasmic domain (42 kDa). PMID- 3020990 TI - Kinetics of the Na+-H+ antiporter as assessed by the change in intracellular pH in MDCK cells. AB - The Na+-H+ antiporter regulates both H+ secretion and cell pH in renal epithelia. The present study was designed to further define the Na+ dependency and kinetics of proton efflux in MDCK cells. Intracellular pH (pHi) was determined with a pH sensitive fluorescent probe, 2,7-bis-carboxyethyl-5,6-carboxyfluorescein (BCECF). Data were obtained from confluent monolayers grown on plastic cover slips and studied in media free of added CO2 and HCO-3, pH = 7.2 (pHo). Monolayers in NaCl maintain a pHi of 7.48 +/- 0.03 (n = 13). When cells were acidified with NH4Cl, pHi decreased to 6.73 +/- 0.07 (n = 10) and remained stable in Na+-free choline chloride. When monolayers were subsequently exposed to NaCl, pHi increased 0.28 +/- 0.04 pH units/min. Amiloride (2.5 mM) inhibited this Na+-dependent rise in pHi by more than 75%. The rate of pHi recovery after exposure to Na+ exhibited saturation kinetics; the apparent Km(Na) was 30.4 +/- 4.2 mM and maximum velocity was 107.1 +/- 17.1 delta [H+]i nmol X l-1 X min-1. We conclude that pHi regulation in the MDCK cell is in part mediated by a Na+-H+ antiporter, and the kinetics of this process can be reliably assessed by the pH-sensitive fluorometric probe, BCECF. PMID- 3020991 TI - Chronic stimulation of mammalian muscle: enzyme changes in individual fibers. AB - Single fibers of rabbit fast-twitch tibialis anterior (TA) muscles were analyzed after continuous low-frequency stimulation for up to 8 wk. After 2-5 wk, every fiber showed higher levels of citrate synthase, hexokinase, and 3-oxoacid CoA transferase than any control fiber; in some cases these levels were 2-10 times higher (well above any found even in the control soleus, a slow-twitch muscle). Average levels of malate dehydrogenase and alanine transaminase also rose dramatically, but peak single fiber levels were not much above the highest in controls. These differential effects confirm at the single fiber level that chronic stimulation can alter mitochondrial composition. Lactate dehydrogenase, fructose-bisphosphatase, and adenylate kinase declined to levels far below those of any control TA fiber, and, in the case of fructose-bisphosphatase, to within the activity range of control soleus fibers. According to their staining reaction for myofibrillar ATPase, TA fibers were initially 23% type IIA, and 74% type IIB, but by 5 wk these had been converted to a mixture of type I, IIA, and IIC fibers. At 5 wk, levels of lactate dehydrogenase, adenylate kinase, and malate dehydrogenase were characteristic of their (new) ATPase type, but 3-oxoacid CoA transferase had increased to levels 6-15 times higher than in control fibers of the same type. PMID- 3020992 TI - Stimulation of pancreatic acinar cell growth by CCK, epidermal growth factor, and insulin in vitro. AB - Effects of regulatory molecules on growth of mouse pancreatic acinar cells in culture were examined. The cholecystokinin (CCK) analogue caerulein and cholecystokinin octapeptide (CCK-8) each led to threefold increases in incorporation of [3H]thymidine into DNA. Gastrin, which interacts weakly with the CCK receptor, stimulated DNA synthesis, but only at much higher concentrations. In contrast, other secretagogues that utilize Ca2+ as an intracellular messenger, including carbachol, bombesin, substance P, and the ionophore A23187, did not induce trophic responses. Factors that affect intracellular cAMP concentration, such as secretin, somatostatin, VIP, DBcAMP, and forskolin, did not increase DNA synthesis in cultured pancreatic cells. Insulin and epidermal growth factor induced two- and threefold increases in [3H] thymidine incorporation into DNA, respectively. The effects of insulin were mediated via insulin-like growth factor I receptors. Steroid hormones had little effect on pancreatic acinar cell DNA synthesis. The stimulatory effects of CCK, insulin, and EGF were additive. The combination of caerulein, EGF, and insulin in a hormonally defined medium led to a tenfold increase in the incorporation of [3H]thymidine into DNA. These data indicate that CCK, EGF, and insulin directly increase DNA synthesis in pancreatic acinar cells. PMID- 3020993 TI - Response of isolated rat jejunum to angiotensin peptides. AB - Following intravenous infusion, angiotensin I (ANG I), angiotensin II (ANG II), and angiotensin III (ANG III) enhance Na and water absorption across the jejunum by increasing sympathetic nerve activity. Increased absorption following intravenous infusion of angiotensin peptides may be secondary to an increase in ion transport. To test this hypothesis, the effect of angiotensin peptides on ion and water absorption from the isolated jejunum was studied. At a dose of 100 pM, serosal addition of ANG II stimulated jejunal Na and water absorption. In contrast, mucosal additions of the peptide were ineffective at doses up to 1 microM. ANG II enhanced jejunal absorption in the presence of prazosin. Thus ANG II does not appear to stimulate absorption from the isolated intestine through mediation of sympathetic fibers. ANG III and the substituted analogue [Sar1,Leu8]ANG II stimulated jejunal absorption at a dose of 100 pM. At a dose of 1 nM ANG I also stimulated jejunal absorption. The effect of ANG I on absorption could be prevented by prior treatment of the animals with the converting enzyme inhibitor MK 422. Thus ANG I must first be converted to ANG II to stimulate jejunal absorption. PMID- 3020995 TI - Proximal tubular reabsorption and Na-K-ATPase activity in remnant kidney of young rats. AB - In rats unilaterally nephrectomized (NX) in infancy, the compensatory growth of the remnant kidney is due first to hypertrophy and hyperplasia, but after 2 wk only to hyperplasia. We studied proximal tubular adaptation (reabsorption, Na-K ATPase activity, length, and basolateral membrane area) 2 and 8 wk after NX. The rats were NX at 5 days of age. The size of remnant kidney obtained from uninephrectomized rats was 125% at 2 wk and 179% at 8 wk relative to the appropriate time-related controls. Single-nephron glomerular filtration rate in the uninephrectomized group was doubled relative to controls, whereas fractional reabsorption by the proximal tubule was unchanged. Na-K-ATPase activity per millimeter of proximal convoluted tubule (PCT) was significantly increased in the uninephrectomized group relative to controls at 2 but not 8 wk. The area of the basolateral cell membrane per millimeter PCT was unchanged at both 2 and 8 wk, which suggests that the density of enzyme units inserted in the membrane was increased at 2 but not at 8 wk. PCT length was increased at 8 but not at 2 wk. There was a close correlation between total PCT Na-K-ATPase activity and filtered sodium (r = 0.999) and between total PCT Na-K-ATPase activity and PCT sodium reabsorption (r = 0.998). We conclude that the proximal tubule can adapt to an increased filtered load by increasing the density of transporting units or by increasing the tubular length. The latter stage may be attained only in young growing animals. PMID- 3020994 TI - Xanthine oxidase and neutrophil infiltration in intestinal ischemia. AB - A growing body of experimental data indicates that reactive oxygen metabolites such as superoxide, hydrogen peroxide, and hydroxyl radical may mediate the mucosal injury produced by reperfusion of ischemic intestine. Xanthine oxidase has been proposed as the primary source of these reduced O2 species because pretreatment with xanthine oxidase inhibitors such as allopurinol or pterin aldehyde prevent postischemic mucosal injury. Another potential source of oxygen radicals is the inflammatory neutrophil. To ascertain whether neutrophils could play a role in the pathogenesis of ischemia-reperfusion injury in the small bowel we examined the effect of ischemia and reperfusion on neutrophil infiltration and tissue levels of reduced glutathione, superoxide dismutase, and catalase. Our studies demonstrate that reperfusion of ischemic intestines results in a dramatic increase (1,800%) in neutrophil infiltration and a concurrent loss of reduced glutathione and superoxide dismutase of 60 and 30%, respectively. Catalase activity was unaffected by ischemia-reperfusion. Pretreatment with allopurinol or administration of superoxide dismutase prevented the influx of neutrophils and retarded the drop in reduced glutathione levels. These results suggest a relationship among xanthine oxidase-generated oxy radicals, neutrophil extravasation, and mucosal damage. We propose that ischemia and reperfusion results in xanthine oxidase-generated, superoxide-dependent accumulation of inflammatory neutrophils in the mucosa where neutrophil-derived reactive oxygen metabolites mediate and/or exacerbate intestinal injury. PMID- 3020996 TI - Control of transepithelial Na+ transport and Na-K-ATPase by oxytocin and aldosterone. AB - Short- and long-term effect of oxytocin on Na+ transport and Na-K-ATPase biosynthesis in the toad bladder, and the potential interaction of this hormone with aldosterone have been studied, leading to the following observations. An early Na+ transport response (oxytocin, 50 mU/ml) peaked at 10-15 min of hormone addition. At maximal stimulation a three- to fourfold increase in Na+ transport was observed, a sustained Na+ transport response (about two-fold control base line) was observed as long as the hormone was present in the medium and for up to 20 h of incubation. Pretreatment for 30 min with actinomycin D (2 micrograms/ml) did not inhibit the early response, but significantly impaired the sustained response, suggesting that de novo protein synthesis was required. The simultaneous addition of the two hormones led within 60 min to a marked potentiation of the action on Na+ transport. This synergism could be mimicked by exogenous cyclic adenosine monophosphate (cAMP). Oxytocin alone (18 h exposure, 50 mU/ml) increased the relative rate of synthesis of both alpha and beta subunits of Na-K-ATPase (1.9- and 1.6-fold, respectively; P less than 0.05), whereas aldosterone (80 nM) increased the relative rate of synthesis of the same subunits (2.6- and 2.2-fold, respectively; P less than 0.02). Finally, in contrast to what was observed at the physiological level, the interaction of oxytocin and aldosterone did not lead to a similar potentiation at the biochemical level, i.e., induction of Na-K-ATPase biosynthesis (2.7- and 2.9 fold, for alpha and beta subunits, respectively; P less than 0.025). PMID- 3020997 TI - Effect of cAMP on prostaglandin E2 production in cultured rat inner medullary collecting tubule cells. AB - Studies were performed to determine whether cAMP impairs prostaglandin (PG) E2 production in a homogeneous population of cultured rat inner medullary collecting duct cells. Three structurally different cAMP analogues were shown to decrease PGE2 synthesis by 48.4% in the basal state and by 49.3% in response to the divalent cation ionophore A23187 (5 microM). Thromboxane B2 production was similarly suppressed. An increase in endogenous cAMP by forskolin also decreased PGE2 synthesis. To determine the locus of the cAMP effect we examined the response to exogenously added arachidonic acid. At a concentration of arachidonic acid (5 micrograms/ml) sufficient to render the phospholipase-dependent fraction negligible (as evidenced by the lack of a mepacrine effect), cAMP had no effect on PGE2 production, suggesting phospholipase as the site of cAMP action. Further evidence for a phospholipase-mediated mechanism derives from studies employing [5,6,8,9,11,12,14,15-3H(N)]arachidonic acid in which cAMP analogues had no effect on the rate of cellular arachidonic acid incorporation, but did impair the release of tritiated arachidonic acid in response to ionophore. These results suggest the existence of a negative feedback system that, by impairing phospholipase activity and PGE2 synthesis, could enhance the action of cAMP in the antidiuretic state. PMID- 3020998 TI - Electrically neutral Na+-H+ exchange in endosomes obtained from rabbit renal cortex. AB - Endocytotic vesicles (i.e., endosomes) were prepared from rabbit renal cortex following the intravenous injection of horseradish peroxidase. The endosomal population was derived from a 100,000-g pellet and was found at equilibrium in the lightest area of a sucrose density gradient. This population was separate from other organelles as determined by enzyme markers and had a 2.5-fold enrichment of horseradish peroxidase specific activity compared with the homogenate specific activity corrected for soluble horseradish peroxidase activity. The endosomes contain an oligomycin-insensitive, electrogenic H+ translocating ATPase. They also contain an electroneutral Na+-H+ exchanger. This exchanger is not inhibited by amiloride, and lithium is not a substrate for the exchanger. Lithium does inhibit the Na+-H+ exchanger when added prior to the addition of sodium. The Michaelis constant for sodium of the endosomal Na+-H+ exchanger was found to be 10.0 mM. These data indicate that a population of endosomes from rabbit renal cortex contain an electrogenic H+-ATPase and an electroneutral Na+-H+ exchanger and that the exchanger is distinct from the brush border membrane Na+-H+ antiporter. PMID- 3020999 TI - Zona glomerulosa cell responses to atrial natriuretic factor in genetically hypertensive rats. AB - We examined whether abnormalities in target cell responsiveness to atrial natriuretic factor (ANF), similar to those previously found in the kidney, could also be present in the zona glomerulosa cells of spontaneously hypertensive rats (SHR) and salt-sensitive Dahl rats (S rats). We found an attenuated aldosterone (Aldo) response to angiotensin II (ANG II) in zona glomerulosa cell suspensions isolated from hypertensive SHR compared with those from Wistar-Kyoto (WKY) rats, whereas cells derived from hypertensive S rats showed a significantly higher Aldo response to the maximum stimulatory dose of ANG II than those from salt-resistant Dahl rats (R rats). The maximum observed Aldo responses to ACTH stimulation were not different in SHR or S rats compared with their respective controls. ANF exerted a potent inhibitory action on both ANG II- and ACTH-stimulated secretions of glomerulosa cell suspensions, without any difference in its potency between hypertensive and control rats. The equipotent inhibitory action of ANF on the ANG II- and ACTH-stimulated secretion of Aldo in those cell suspensions suggests that the previously observed alterations in the target cell responsiveness to ANF do not exist in the adrenal zona glomerulosa cells of SHR and Dahl S rats. PMID- 3021000 TI - Systemic and regional hemodynamic effects of leukotrienes D4 and E4 in the conscious rat. AB - The effect of leukotrienes (LTs) D4 and E4 on systemic and regional hemodynamic variables were studied in the conscious rat (n = 5-9). Renal (R), mesenteric (M), and hindquarter (HQ) blood flow (BF) were monitored by directional pulsed Doppler velocimetry, and mean arterial blood pressure (MAP) and heart rate were recorded through a catheter in the femoral artery. In a separate series of experiments, cardiac index (CI) was measured by the thermodilution method. Systemic injection of LTD4 or LTE4 (0.1-10 micrograms/kg) produced dose-dependent pressor responses; BF in the M, HQ, and R vessels declined, due to increased vascular resistance (VR) at the following order: M much greater than HQ greater than R. Low doses of LTD4 or LTE4 produced vasodilation in the HQ area. Infusion of LTD4 (3 micrograms X kg-1 X min-1) for 10 min produced progressive and pronounced vascular constriction in the M and HQ regions along with reduction in BF. The LTD4 infusion also markedly decreased CI with a concomitant rise in total peripheral resistance index (TPRI). Indomethacin (5 mg/kg iv) pretreatment did not modify any of the hemodynamic effects of LTD4 or LTE4. FPL 55712 (10 mg/kg iv) and LY 171883 (30 mg/kg iv), two different LT-receptor antagonists, partially blocked the constriction effects of these LTs. LY 171883, but not FPL 55172, blocked the HQ vasodilation produced by LTE4. LY 171883 alone increased HQ-BF and reduced HQ VR. These data indicate that LTD4 and LTE4 are potent constrictors of the M vascular bed, but at low doses they also produce dilation of the HQ blood vessels. Furthermore, no escape from the effects of prolonged infusion of the LTs was demonstrated in this species. Finally, the hemodynamic responses to LTD4 and LTE4 in the conscious rat are independent of cyclooxygenase products of LTs and are only partially blocked by FPL 55712 or LY 171883. These studies taken together suggest a differential distribution of multiple LT receptors in the rat vasculature. PMID- 3021001 TI - Selective vagal innervation of sinoatrial and atrioventricular nodes in canine heart. AB - Parasympathetic pathways mediating chronotropic and dromotropic responses to cervical vagal stimulation were determined from sequential, restricted, intrapericardial dissection around major cardiac vessels. Although right cervical vagal input evoked significantly greater bradycardia, supramaximal electrical stimulation of either vagus produced similar ventricular rates, both with and without simultaneous atrial pacing. Dissection of the triangular fat pad at the junction of the inferior vena cava-inferior left atrium (IVC-ILA) invariably eliminated all vagal input to the atrioventricular (AV) nodal region. Yet IVC-ILA dissection had minimal influence on evoked-chronotropic responses to either cervical vagal or stellate ganglia stimulation. Respective intrapericardial projection pathways, from either right or left vagi, are sufficiently distinct to allow unilateral parasympathetic denervation of the sinoatrial (SA) and atrioventricular (AV) nodal regions. Left vagal projections to the SA and AV nodal regions course primarily along and between the right pulmonary artery and left superior pulmonary vein. Right vagal projections to the SA and AV nodal regions are somewhat more diffuse but concentrate around the right pulmonary vein complex and adjacent segments of the right pulmonary artery. We conclude there are parallel, yet functionally distinct, inputs from right and left vagi to the SA and AV nodal regions. PMID- 3021002 TI - Cystic fibrosis and beta-adrenergic response of airway epithelial cell cultures. AB - Confluent cell sheets were cultured from the tracheal epithelium of normal humans or from tracheal and nasal epithelia of patients with cystic fibrosis (CF). Changes in short-circuit current (Isc) or cyclic AMP (cAMP) levels in response to 10(-5) M isoproterenol were measured. In CF tracheal cells the response to isoproterenol was transient, and the maximal increase in Isc was one-tenth normal. In CF nasal cells, isoproterenol or epinephrine caused only small transient increases in Isc. However, in both CF nasal and tracheal cells, the Ca ionophore, A23187, caused relatively large increases in Isc that were inhibited by the Cl transport blocker, bumetanide, suggesting that Cl secretion can be induced by raising intracellular levels of Ca. In normal tracheal cell sheets, cAMP levels increased within 15 s of isoproterenol addition and continued to increase for up to 20 min. Resting levels of cAMP in CF tracheal cells were not statistically different from those of normal cells and showed linear increases for up to 4 min after addition of isoproterenol. Changes in cAMP in CF nasal cells were similar to the changes in CF tracheal cells. After 2 min, all three cell types showed cAMP levels elevated approximately equal to 10-fold. These results suggest that receptor-activated stimulation of adenylate cyclase is normal in CF. However, though raised cAMP levels stimulate Cl secretion in normal, they are unable to do so in CF airway epithelial cells. PMID- 3021003 TI - Posthospital mandatory outpatient treatment. AB - Mandatory outpatient treatment invoked after the patient has improved in the hospital is a relatively new development. Tennessee instituted this policy by statute in 1981. While people placed under the constraints of that law showed a reduction in rate of readmission, comparison with control groups failed to support the conclusion that these results are due to the forced outpatient constraints. The author discusses some procedure and policy considerations that stem from these findings. PMID- 3021004 TI - Rights, wrongs, and the dilemma of coerced community treatment. AB - An outpatient treatment approach directed to patients with histories of psychotically based dangerousness, poor compliance, and recidivism is described. Cases are presented that suggest favorable outcomes of this approach, but the coercive nature of the treatment raises questions about the psychiatrist's violation of patients' rights and transgression of ethical standards. If psychiatrists are to successfully treat the most difficult chronic patients, can we do it without legally sanctioned, benevolent, coercive treatments? One model of such treatment is outpatient commitment. There is concern that without sound outpatient commitment statutes, we may witness the reemergence of asylums. PMID- 3021005 TI - Outpatient commitment: the problems and the promise. PMID- 3021007 TI - Varicella-zoster dilemma: common sense in medical education. PMID- 3021006 TI - A randomized controlled trial of low carbohydrate and low fat/high fiber diets for weight loss. AB - Among 135 overweight subjects, we conducted a three-month randomized controlled trial of two sets of dietary advice, each providing approximately 1,000 calories per day but differing in fiber, carbohydrate, and fat content. Information on weight and eating habits, as well as measures of lipoprotein and glucose metabolism were obtained at entry and one and three months later. We found that dieters given low carbohydrate/low fiber dietary advice tended to lose more weight than those given a higher carbohydrate/higher fiber regimen (5.0 vs 3.7 kg on average at three months). This pattern was particularly marked among women, and among participants who were under age 40 or of lower social class. There were no differences between the diet groups in the proportion complaining of hunger but, in general, members of the low carbohydrate group complained of more problems in dieting. There were only minor differences in the serum lipoprotein patterns during the diet period. In view of these results, we believe previous claims of the benefits of fiber for weight loss may have been overstated. PMID- 3021008 TI - The occurrence of sarcomatous components in primary mediastinal germ cell tumors. AB - The occurrence of a sarcomatous component in germ cell tumors is an uncommon phenomenon; seven cases with such an association are presented. The sarcomatous elements were rhabdomyosarcomatous in four cases, angiosarcomatous in two, and a combination of these two types in one case. Immunohistochemical studies supported the endothelial and skeletal muscle differentiation of the sarcomatous components. All patients were treated surgically, and some received various chemotherapeutic agents and radiation. On follow-up, four patients had died of their disease, one developed recurrence and pulmonary metastases, one was free of disease after 4 years, and one is a recent case. Chemotherapy protocols may need to be altered to include sarcoma-oriented drugs for this particular group of patients. PMID- 3021009 TI - Isolation of Japanese encephalitis virus from clinical specimens using a continuous mosquito cell line. AB - During the 1983 Japanese encephalitis (JE) epidemic in northern Thailand, we systematically attempted to isolate JE virus (JEV) from clinical specimens collected from 49 consecutive JE patients at 1 provincial hospital. Fresh acute plasma and cerebrospinal fluid (CSF) samples and postmortem brain samples were immediately inoculated onto cultured monolayers of Aedes pseudoscutellaris (LSTM AP-61) cells which had been shipped to the epidemic site. None of 49 plasma samples yielded virus. None of 30 fresh CSF samples from nonfatal cases yielded virus, but 5 of 15 (33%) CSF samples from fatal cases did. Inoculation of fresh brain specimens obtained at autopsy yielded virus in every case attempted (7 of 7), whereas postmortem needle biopsy specimens of brain yielded virus in only 1 of 4 cases. Isolates were most frequently successful using thalamic tissue (6 of 7 cases), but isolates were also commonly obtained from frontal cortex (4/7), occipital cortex (4/7), cerebellum (4/7), medulla (4/7) and pons (2/7). PMID- 3021010 TI - Facial nerve sacrifice and tumor recurrence in primary and recurrent benign parotid tumors. AB - Three hundred eight patients underwent parotidectomy for a benign parotid tumor between 1948 and 1979. Two hundred seventy-four had operation for primary tumor, and 34, for recurrent tumor. Ninety-eight percent of those with primary tumors had superficial or total parotidectomy, and 2 percent had local excision with a wide margin of normal tissue. In those with recurrent tumor, 91 percent had superficial or total parotidectomy and 9 percent had local excision with a wide margin of normal tissue. There were nine recurrences in the primary group (3.2 percent) and 10 in the recurrent group (29 percent), at an average follow-up of 10 and 13 years, respectively. The time to recurrence in the primary group was between 5 and 20 years, whereas, second recurrences in the recurrent group generally took place within 5 years. Seven patients in the primary group (2.5 percent) and 9 in the recurrent group (26 percent) had sacrifice of the facial nerve. Most facial nerve sacrifices in the primary group were minor, involving a branch of the nerve only. Facial nerve sacrifice in the recurrent group, however, usually involved division of the nerve or the nerve trunk. These findings demonstrate that the major morbidity associated with managing benign parotid tumors occurs in dealing with recurrent tumors. Recurrence is uncommon if superficial or total parotidectomy is performed for a primary tumor. PMID- 3021012 TI - Mitochondrial cytopathy with lactic acidosis, carnitine deficiency and DeToni Fanconi-Debre syndrome. AB - We reported a 6-year-old girl with mitochondrial cytopathy with lactic acidosis. The patient developed hypotonia, hearing loss, mental retardation, short stature, cataracta, hypoparathyroidism, DeToni-Fanconi-Debre syndrome and carnitine deficiency. Histological examination disclosed ragged red fibers and moderate lipid storage in skeletal muscle tissue and several structural abnormalities of mitochondria both in muscle tissue and proximal renal tubules. Biochemical examination of muscle tissue revealed a partial deficiency of pyruvate dehydrogenase complex and normal activities of cytochrome c oxidase, succinate cytochrome c reductase and NADH cytochrome c reductase. This is the first report of mitochondrial cytopathy representing DeToni-Fanconi-Debre syndrome associated with partial deficiency of pyruvate dehydrogenase complex and normal cytochrome c oxidase activity. PMID- 3021011 TI - [Serum ferritin in ovarian tumors]. PMID- 3021014 TI - Nephroblastomatosis and deletion of 11p. The potential etiologic relationship to subsequent Wilms' tumor. AB - Both nephroblastomatosis and deletions of the short arm of chromosome 11 (11p-) have been associated independently with Wilms' tumor. The finding of 11p- in a specimen of nodular renal blastema in the currently described patient represents a previously unknown association with this chromosomal lesion. The possibility that 11p- produced an abnormal renal substrate (nephroblastomatosis), upon which the action of a second postzygotic genetic alteration led to Wilms' tumor, is considered. It is suggested that, in the present case, tumorigenesis may have been the result of two postzygotic events, one of which may have been postnatal. Recent cytogenetic observations in both Wilms' tumor and retinoblastoma support such an hypothesis. PMID- 3021013 TI - A case of hereditary motor and sensory neuropathy type III with a decrease in unmyelinated fibers. AB - We report a 3-year-old girl with hereditary motor and sensory neuropathy type III with a decrease in unmyelinated fibers. There have been few reports of such cases. The present findings suggest the possibility that the primary lesion in this disease is in the axons. We consider that more attention should be paid to the changes in unmyelinated fibers and axons in further studies on this disease. PMID- 3021015 TI - Human parvovirus-associated red cell aplasia in the absence of underlying hemolytic anemia. AB - Human parvovirus (HPV) infection has recently been implicated as the cause of aplastic crisis in patients with hemolytic anemias such as congenital spherocytosis and sickle cell anemia. The virus causes a transient red cell aplasia which, in patients with a shortened red cell life span, is manifested as a rapid worsening of the anemia and an absence of peripheral reticulocytosis. Recovery is associated with the presence of giant pronormoblasts in the bone marrow, and several days later, a brisk peripheral reticulocytosis. In normal subjects, HPV causes erythema infectiosum (fifth disease) but is not associated with symptomatic anemia, probably because of the duration of the normal red blood cell life span. A case of HPV infection producing severe anemia in an immunocompromised patient without an underlying hemolytic anemia is presented here. Infection in this patient, a 3-year-old boy with acute lymphoblastic leukemia in remission, may have been prolonged by immunosuppression, leading over a 4-week period to a severe anemia. The immunosuppressed appear to be another group of patients at risk of developing symptomatic anemia when infected by HPV. PMID- 3021016 TI - Fulminant metastasizing chondroid syringoma of the skin. AB - A case of malignant chondroid syringoma of skin with widespread metastases is recorded. Several features of mixed tumors are discussed, and the pertinent literature is reviewed. PMID- 3021017 TI - Chemotactic activity of LTB4 in man. AB - An improved skin window chamber technique has been developed and used for a quantitative study of the chemotactic effect of leukotriene B4 (LTB4). LTB4 (0.5 microM) was exposed to a skin window on the forearm of eight healthy volunteers, while phosphate buffered saline served as control in a skin window on the other forearm. Skin window exudates and samples of blood draining the skin window areas were collected after 1, 2, 4, 8, and 24 h. The samples were quantitated for the different types of leukocytes as well as the intra- and extracellular concentration of the eosinophilic cationic protein and lactoferrin as markers of eosinophil and neutrophil granulocytes. A significantly increased migration of neutrophil granulocytes into the skin window chamber containing LTB4 was found from the 2nd to the 8th hour after the initial LTB4 exposure. The eosinophils reached a significant peak at the 4th hour. The rise in the actual number of eosinophil cells did not reach significance, whereas measurements of the eosinophilic cationic protein in the cellular fraction of the exudate exhibited a significant increase as a reflection of the number of eosinophils. This highlights the potential clinical value of eosinophilic cationic protein measurements to reveal eosinophilia instead of the traditional eosinophil counts. Extracellular eosinophilic cationic protein and lactoferrin did not change significantly in the LTB4-exposed skin window, implying that LTB4 does not activate the eosinophils and neutrophils to exocytosis of their enzymes. The present in vivo results support the concept of LTB4 being a potent chemoattractant to neutrophil and less so to eosinophil granulocytes in humans, a chemoattractant that recruits the leukocytes but does not seem to activate them. PMID- 3021018 TI - A quantitative decatenation assay for type II topoisomerases. AB - Type II topoisomerases catalyze decatenation of the catenated network of kinetoplast DNA [J. C. Marini, K. G. Miller, and P. T. Englund (1980) J. Biol. Chem. 255, 4976-4979]. The individual DNA circles and small catenanes produced during the decatenation reaction can be separated from the large network of substrate DNA by 5 min centrifugation at 13,000g and quantitated. The appearance of these decatenated DNA molecules which appear in the supernatant first showed a lag, whose duration depended on the enzyme concentration, and then increased linearly with time until it reached a plateau. The slope of the linear part of the kinetic curve was directly proportional to the enzyme concentration, whether a purified or crude preparation of type II topoisomerase from mammalian cells was used. These findings led us to a rapid quantitative assay of type II topoisomerases not involving electrophoresis. The method was developed with purified enzyme but was also useful for assay of the activity in crude extracts. Surprisingly, the type I topoisomerase, even when present in large excess, failed to decatenate the nicked DNA circles often present in the kinetoplast DNA. This renders the assay virtually free from interference by type I enzyme. The method is sensitive and allowed quantitative estimation of the enzyme activity present in the crude extracts corresponding to that derived from 500 to 700 cultured mammalian cells. Since various type II topoisomerases from procaryotic, eucaryotic, and viral sources decatenate kinetoplast DNA and generate similar DNA products, the assay method is likely to be generally applicable. PMID- 3021020 TI - Comparison of glycoproteins by two-dimensional mapping of glycosylated peptides. AB - We describe here a two-dimensional mapping procedure which is capable of resolving glycopeptides isolated by lectin affinity chromatography from radioiodinated tryptic digests of glycoproteins. Glycopeptide maps were successfully produced for the model proteins alpha 1-acid glycoprotein and fetuin, as well as for the two surface glycoproteins gp90 and gp45 from equine infectious anemia virus (EIAV). Differences were detected in the glycopeptide maps obtained for the gp90 and gp45 components from two antigenically distinct strains of EIAV, demonstrating the ability of this procedure to detect variations in glycosylation in closely related glycoproteins. Thus this glycopeptide mapping technique provides a simple, rapid method to study changes in glycopeptides requiring only micrograms of glycoprotein. PMID- 3021019 TI - Analysis of whole blood manganese by flameless atomic absorption spectrophotometry and its use as an indicator of manganese status in animals. AB - To investigate the use of whole blood manganese (Mn) as an indicator of total body Mn, we measured Mn in whole blood and liver of rats fed purified diets containing adequate (45 micrograms Mn/g diet) or deficient (1 microgram Mn/g diet) Mn. The mean hepatic Mn concentration was significantly lower (P less than 0.001) in the Mn-deficient group compared to the control group, 0.36, microgram Mn/g and 1.73 micrograms Mn/g, respectively. Furthermore, whole blood Mn was significantly reduced (P less than 0.001) in the deficient group when compared to the control group, 4.0 ng Mn/ml and 8.6 ng Mn/ml, respectively. Hepatic Mn linearly regressed against whole blood Mn yielded a statistically significant (P less than 0.001) correlation coefficient of 0.775. These data suggest that whole blood Mn is a valid indicator of body Mn status and thus may be useful, in addition to the measurements of serum copper and zinc, for the diagnosis and prognosis of diseases in which the metabolism of trace elements is affected. In addition, this paper describes and delineates operational parameters for the measurement of whole blood Mn using the IL 551 atomic absorption spectrophotometer and the IL 555 B flameless atomizer. PMID- 3021021 TI - Ultracytochemistry of ouabain-sensitive K+-dependent p-nitrophenyl phosphatase in rat incisor enamel organ. AB - Sprague-Dawley strain rats of 4-5 weeks old were perfusion-fixed with either a mixture containing 0.1 or 0.25% glutaraldehyde and 2% formaldehyde, or a 2% formaldehyde in 0.1 M sodium cacodylate buffer for 10 minutes. Non-decalcified 30 50-micron sections of the enamel organ taken from lower incisors were then processed for ultracytochemical demonstration of ouabain-sensitive, K+-dependent, p-nitrophenyl phosphatase, by use of the one-step lead method, representing the second dephosphorylative step of Na+-K+-ATPase. Throughout the secretory, transition, and maturation stages of amelogenesis, the enzymatic activity was demonstrated along the cytoplasmic side of the plasma membranes of the stratum intermedium and the papillary layer cells, especially along their numerous microvilli. The plasma membranes forming gap junctions and desmosomes were free of reaction or showed slight focal precipitates of reaction products. The stellate reticulum and the outer enamel epithelium exhibited either a weak reaction or were reaction negative. Secretory ameloblasts showed a weak trace like reaction along the basal and lateral cell surfaces; however, the latter surfaces were sometimes completely free of reaction. Tomes' processes were usually reaction negative. Ameloblasts in the transition and maturation stages were devoid of enzymatic activity, except for a slight reaction along the plasma membranes of the basal cell surfaces of transition ameloblasts facing the papillary layer. The enzymatic activity described above was completely dependent on the presence of potassium and substrate in the incubation media and was almost completely inhibited by an addition of 10 mM ouabain to the incubation media. PMID- 3021023 TI - Pharmacokinetic differences could explain the lack of reversal of nitrous oxide analgesia by low-dose naloxone. PMID- 3021022 TI - The effect of beta-adrenergic blockade on the cardiovascular response to diltiazem or verapamil in dogs. AB - Diltiazem or verapamil were each given at two different infusion rates to pentobarbital-anesthetized dogs with or without a concurrent infusion of propranolol. Changes in cardiovascular function, in reflex activation as reflected by circulating catecholamine levels, and in the chronotropic response to an exogenous beta-adrenergic agonist, isoproterenol, were measured. When administered alone, diltiazem or verapamil, at plasma concentrations of 160 and 370 ng/ml, or 230 and 500 ng/ml, respectively, prolonged atrioventricular conduction and caused systemic vasodilation with a decrease in mean arterial pressure. Cardiac index increased, associated with an increase in arterial norepinephrine level. Heart rate increased with the lower level of verapamil; left ventricular dP/dt increased with both levels of verapamil and at the higher level of diltiazem. Plasma propranolol levels of approximately 35 ng/ml were well tolerated in the absence of diltiazem or verapamil. When added to diltiazem or verapamil, propranolol resulted in an increase in systemic vascular resistance to near control values; a decrease in cardiac index, left ventricular dP/dt, and heart rate; and worsened atrioventricular conduction. Three of nine animals in the high verapamil-propranolol group were unable to maintain a mean arterial pressure greater than 50 mm Hg, and developed a low cardiac index with an elevated systemic vascular resistance, despite very high levels of circulating catecholamines. Compared to the anesthetized state, greater amounts of isoproterenol were needed to effect the same increase in heart rate with the addition of diltiazem, verapamil, or propranolol alone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021024 TI - Transmission of bovine leukosis virus by blood inoculation. AB - The experimental transmission of bovine leukosis virus (BLV)-infected whole blood was studied in 2 groups of Holstein calves. The 1st group of 4 BLV-seronegative calves was given 10 microliters of whole blood by either the IM, IV, subcutaneous, or intradermal routes. All 4 calves seroconverted to BLV within 8 weeks after they were inoculated. The 2nd group, also comprising 4 calves, was given the equivalent of 1 microliter of whole blood by the described routes. These calves seroconverted to BLV by 14 weeks after they were inoculated. The results indicated that small volumes of whole blood administered by 4 different routes were effective in the spread of BLV. PMID- 3021026 TI - Identification of a bovine enteric syncytial virus as a nongroup A rotavirus. AB - An atypical or nongroup A rotavirus was identified in feces obtained from gnotobiotic calves in which fecal preparations originally derived during an epizootic of neonatal calf diarrhea had been serially passaged. The epizootic was previously reported to be caused by a noncharacterized viral agent that induced the formation of epithelial syncytia on small intestinal villi of experimentally infected calves. This bovine, nongroup A rotavirus was found to be antigenically related to a described atypical rotavirus of rats by immunofluorescence and by enzyme immunoassay. Complementary DNA derived from the atypical rat rotavirus cross hybridized with RNA obtained from the bovine virus, but not with RNA extracted from group A rotaviruses. Complementary DNA derived from SA-11 group A rotavirus cross hybridized with other group A rotavirus RNA, but not with RNA obtained from either the rat or bovine nongroup A isolates. Additionally, another similar, if not identical, bovine atypical rotavirus was identified in a second epizootic of neonatal calf diarrhea that occurred several hundred kilometers and 8 months apart from the original epizootic. PMID- 3021025 TI - Antiviral effectiveness of butylated hydroxytoluene against pseudorabies (Aujeszky's disease) virus in cell culture, mice, and swine. AB - Butylated hydroxytoluene (BHT) was evaluated for antiviral effectiveness on pseudorabies virus (PRV) in cell culture, mice, and swine. When relatively small amounts of BHT were mixed with PRV and incubated at 37 C for 30 or 60 minutes before inoculation into cell cultures, the cell cultures did not become infected with virus. The PRV was not infectious when the virus was treated with BHT and then inoculated intraperitoneally into mice, but was infectious when BHT and PRV were inoculated simultaneously or when BHT was inoculated either 30 or 60 minutes before PRV. Swine fed BHT-medicated feed for 10 days before they were intranasally exposed with virulent PRV did not have overt signs of pseudorabies, had a lower concentration of PRV in nasal mucus than did control swine, and had acceptable blood enzyme and cholesterol concentrations during the experiment. The BHT was detected in tissues of 2 swine after they were fed BHT-medicated feed for 10 days, and higher concentrations of BHT were detected in tissues of 3 swine given BHT feed for 29 days. PMID- 3021027 TI - Responses of horses vaccinated with avirulent modified-live equine arteritis virus propagated in the E. Derm (NBL-6) cell line to nasal inoculation with virulent virus. AB - Nineteen horses with no prior experience with equine arteritis virus (EAV) were inoculated IM with an avirulent live-virus vaccine against equine viral arteritis; the vaccinal virus had been passaged serially 131 times in primary cell cultures of equine kidney, 111 times in primary cell cultures of rabbit kidney, and 16 times in an equine dermis cell line (EAV HK-131/RK-111/ED-16). Three or 4 of the vaccinated horses each, along with appropriate nonvaccinated controls, were inoculated nasally with virulent EAV at each of months 3, 6, 9, 12, 18, and 24 after they were vaccinated. The following was concluded: Vaccination did not induce clinical signs of disease in any horse and, thus, seemed safe for use in the field. All vaccinated horses (n = 19) developed serum neutralizing antibodies to EAV. Fourteen of the vaccinated horses were completely protected from clinical arteritis when exposed to large doses of virulent EAV. Four were partially protected, and one had little or no protection. Six of 13 nonvaccinated horses died of acute arteritis, and the remaining 7 horses experienced severe signs of disease, but survived the infection. All horses (n = 32), whether vaccinated or not, became infected when inoculated nasally with virulent EAV. Virus was recovered from 17 of the 19 vaccinated horses, and all 19 had a secondary humoral immune response. The duration and severity of thermal reaction and persistence of virus were more transitory in vaccinated horses than in the nonvaccinated controls. Protection afforded by this vaccine can persist for at least 24 months, the maximal time after horses were vaccinated that immunity was challenged in the present study.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021028 TI - Immunologic profiles of cats with persistent, naturally acquired feline leukemia virus infection. AB - Several immunologic responses were measured in 13 healthy cats with naturally acquired, persistent feline leukemia virus (FeLV) viremia from 4 multiple-cat households and were compared with responses from 28 of their healthy, non-FeLV viremic housemates. Significant differences (P = less than 0.05) were not observed between results of FeLV-viremic and nonviremic cats for peripheral blood leukocyte or lymphocyte count, percentage of peripheral blood mononuclear cells able to form rosettes with guinea pig RBC or with antibody- and complement-coated sheep RBC, lymphocyte proliferative response to concanavalin A or pokeweed mitogen, or serum immunoglobulin G concentration. Seemingly, persistent FeLV viremia, when naturally acquired, may exist for some time without lymphopenia or a marked loss of mitogen-induced lymphocyte proliferation. PMID- 3021029 TI - Regulation of phytohemagglutinin-induced bovine lymphocyte blastogenesis by leukotrienes. AB - Leukotriene (LT) B4, a 5-lipoxygenase metabolite of arachidonic acid, is a potent inducer of suppressor cells in phytohemagglutinin-stimulated cultures of bovine peripheral blood mononuclear cells. In contrast, LTC4 and LTD4 have little activity. Incubation of T lymphocytes with LTB4 at concentrations as low as 1 X 10(-12)M rendered these lymphocytes suppressive of [3H]thymidine incorporation in subsequent phytohemagglutinin-stimulated cultures of fresh autologous lymphocytes. This LTB4-induced cell was radiosensitive to irradiation at 2,000 rads. Leukotriene B4 may have an important part in immunoregulation during hypersensitivity reactions. PMID- 3021030 TI - Progesterone secretion by the bovine fetoplacental unit and responsiveness of corpora lutea to steroidogenic stimuli at two stages of gestation. AB - To evaluate the response of luteal cells to in vitro stimulation with luteinizing hormone (LH) or dibutyryl cyclic adenosine monophosphate (dbcAMP) and determine the secretion of progesterone by the fetoplacental unit, the corpora lutea were removed surgically in 10 cows (luteectomy) at 250 days (5 cows) or 270 days (5 cows) of gestation. During surgery, but before luteectomy, catheters were placed in the middle uterine artery and vein, carotid artery, and jugular vein. Blood samples were collected from all catheters just before luteectomy and at 8-hour intervals after luteectomy for 72 hours or until calving, whichever occurred first. Luteal tissue was prepared as a dispersed cell preparation and incubated with 0, 0.1, 1.0, 10, or 100 ng of LH/ml of medium or was incubated with 0, 0.5, or 2 mM dbcAMP. Synthesis of progesterone in response to LH by dispersed cells prepared from corpora lutea at 270 days was less (P less than 0.01; analysis of variance) than that by similar preparations at 250 days because a dose-response relationship was not observed for incubations of luteal tissue with LH at 270 days of gestation. Progesterone synthesis in response to the addition of dbcAMP also was less (P less than 0.01) at 270 than at 250 days of gestation. This difference in responsiveness to LH and dbcAMP between the 2 stages of gestation was not reflected by a significant difference between stages of gestation in systemic concentrations of progesterone before luteectomy.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021031 TI - Effects of estrone infusion on secretion of F series prostaglandins and on function of the corpus luteum during late gestation in cattle. AB - Catheters were surgically implanted in the carotid artery, jugular vein, and middle uterine vein of 8 cows at 245 days of gestation. Four cows were given 4 mg of estrone/hour via continuous jugular infusion from 0800 hours on day 246 of gestation through 0800 hours on day 250 of gestation; the remaining 4 cows (controls) were given the vehicle for estrone at 10 ml/hour for the same period. Blood samples were collected from the carotid artery every 3 hours during the infusion. Samples were collected hourly from the middle uterine vein from 0 through 8, 54 through 66, and 112 through 120 hours of the infusion periods. After completion of the infusion, corpora lutea were enucleated and blood samples were collected from the carotid artery and uterine vein at hourly intervals for an additional 8 hours. Dispersed cell preparations of the corpora lutea were incubated with and without luteinizing hormone (LH) or dibutyryl cyclic adenosine monophosphate (dbcAMP). Circulating concentrations of unconjugated and conjugated estrone and estradiol-17 beta were higher (P less than 0.05) in the group infused with estrone than in the vehicle-infused group. Mean and base-line concentrations of F series prostaglandins (PGF) for each blood collection period tended to increase (P less than 0.10) during infusion with estrone, but not during infusion with vehicle. After luteectomy, mean and base-line concentrations of PGF also tended (P less than 0.10) to be greater in the estrone-infused cows than in the control cows, but a surge in PGF concentrations due to removal of the ovarian source of progesterone did not develop.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021032 TI - Channel catfish virus: use of nucleic acids in studying viral relationships. AB - Restriction digestion patterns were used to determine differences in the DNA of various isolates of channel catfish virus (CCV). All viruses were different from each other and from the type strain of CCV. The differences in the digestion patterns were used to relate the viruses quantitatively as to sequence divergence between all pairs of viruses. A range of values from 104 nucleotide changes to 1,690 nucleotide changes/total DNA of CCV was found for the various pairs. A cladistic analysis produced a phylogenetic network relating the viruses by possible ancestory. This network indicated that some viruses were relatively more separated from other viruses that were clustered in the network. A phenetic analysis indicated that the viruses that were clustered in the network were also reasonably similar to one another. PMID- 3021033 TI - Detection of mink enteritis virus in mink feces, using enzyme-linked immunosorbent assay, hemagglutination, and electron microscopy. AB - Twenty-five mink were inoculated with mink enteritis virus (MEV). Fecal specimens were collected daily and were simultaneously evaluated for MEV antigen by use of a direct enzyme-linked immunosorbent assay (ELISA), hemagglutination (HA), and electron microscopy. Results of the evaluations indicated that MEV was shed in the feces on postinoculation days 5 and 6. The virus was not detectable by ELISA or HA after postinoculation day 6, although viruses were found in reduced numbers by use of electron microscopy. The ELISA was specific for MEV, and the sensitivity of the ELISA for MEV was comparable with that of HA. PMID- 3021034 TI - Analysis of plasmids in Mycobacterium avium-intracellulare isolates from persons with acquired immunodeficiency syndrome. AB - Twenty-six strains of the Mycobacterium avium complex isolated from persons with AIDS were examined for plasmid content. The strains were of serotypes 4, 8, 4/6, and 4/8. Plasmid content was determined by agarose gel electrophoresis. All 26 strains carried small plasmids (9 to 15 Mdal), with 11 strains carrying 1 small plasmid and 15 carrying 2. Ten strains also carried large plasmids (greater than 100 Mdal), and 1 strain carried a 60 Mdal plasmid. We have reported molecular cloning of plasmid pLR7, a small plasmid derived from a serotype 4 strain. Cloned segments of pLR7 were used as hybridization probes to detect homologous plasmids in the AIDS-associated strains. Each of the 26 strains carried a plasmid closely related to pLR7. Considerable heterogeneity of size and restriction sites was observed. The finding of plasmids in all strains raises the possibility that they may play a role in virulence. The plasmids will serve as a useful marker for epidemiology and might be useful as probes for detecting M. avium-intracellulare in clinical material. PMID- 3021035 TI - Treatment of murine histoplasmosis with UK 49,858 (fluconazole). AB - Fluconazole was compared with ketoconazole and with amphotericin B in treatment of pulmonary histoplasmosis in immunologically intact mice and in congenitally athymic nude mice. Both fluconazole and amphotericin B increased survival and reduced fungal burden in normal mice. All 3 drugs equally prolonged survival of nude mice challenged with Histoplasma capsulatum, and all effectively reduced the fungal burden. Fluconazole may be a useful antifungal drug in treatment of murine histoplasmosis. PMID- 3021037 TI - Elevated serum chromogranin A concentrations in small-cell lung carcinoma. AB - Serum chromogranin A concentrations measured by radioimmunoassay in patients with small-cell lung carcinoma were compared with values from healthy adults and patients with non-small-cell lung carcinoma or chronic obstructive pulmonary disease. The mean (+/- SE) level was significantly higher (p less than or equal to 0.02) in patients with small-cell lung carcinoma (815 +/- 290 ng/mL, n = 46) than in normal controls (123 +/- 6 ng/mL, n = 20) or patients with chronic obstructive pulmonary disease (169 +/- 18 ng/mL, n = 39), lung adenocarcinoma (180 +/- 22, ng/mL, n = 62), large-cell lung carcinoma (183 +/- 23 ng/mL, n = 18), or lung epidermoid carcinoma (203 +/- 37 ng/mL, n = 78). The mean concentration in extensive-stage small-cell lung carcinoma (1155 +/- 449 ng/mL, n = 29) was significantly greater (p = 0.026) than in limited disease (234 +/- 56 ng/mL, n = 17). Elevated serum chromogranin A values were seen in 53% of patients with limited and 72% with extensive disease. Four patients originally classified as having non-small-cell lung carcinomas with raised chromogranin A levels were subsequently found to have mixed small-cell and non-small-cell tumors. Serum chromogranin A concentrations may be a useful marker of small-cell lung carcinoma disease activity. PMID- 3021036 TI - Non-Hodgkin's lymphoma after treatment of Hodgkin's disease: association with Epstein-Barr virus. AB - Non-Hodgkin's lymphoma occurs infrequently as a late complication of obscure cause after treatment of Hodgkin's disease. We investigated the possible role of Epstein-Barr virus in the pathogenesis of such secondary malignancies of B-cell lineage. Two patients, aged 25 and 43 years, developed high-grade non-Hodgkin's lymphomas 12 and 8 years after radiation therapy for Hodgkin's disease. Serologic profiles in these patients showed evidence of acute and past Epstein-Barr virus infections, respectively. Molecular hybridization analysis showed the presence of multiple cellular equivalents of virus genome in tumor specimens from each patient. Our findings suggest that Epstein-Barr virus may play an integral role in the pathogenesis of non-Hodgkin's lymphoma of B-cell lineage that develops after treatment of Hodgkin's disease. PMID- 3021038 TI - Arthritis, vasculitis, and cryoglobulinemia associated with relapsing hepatitis A virus infection. AB - Hepatitis A virus, unlike hepatitis B virus, has rarely been associated with extrahepatic features. Two patients developed relapsing hepatitis A complicated by arthritis in both cases and cutaneous vasculitis in one. Both patients had cryoglobulinemia, with cryocrit values of 4.3% and 8.6%. Serologic studies showed that the cryoglobulin consisted of polyclonal IgM and IgG. The washed cryoglobulin was analyzed by sucrose density gradient ultracentrifugation under neutral (pH 7.4) and acidic (pH 2.8) conditions. Enzyme-linked immunosorbent assay techniques were used to characterize the native and dissociated cryoglobulin. The cryoglobulin contained acid-dissociable IgG complexes greater than 19S, and high molecular weight rheumatoid factors of both IgG and IgM isotypes that could be dissociated to 7S and 19S forms, respectively. Dissociation of the cryoglobulin augmented 7S anti-hepatitis A virus IgG 2.27 fold, but augmented total 7S IgG only 1.12-fold, suggesting enrichment of antiviral antibody in the cryoglobulin. PMID- 3021039 TI - Nosocomial transmission of a strain of Staphylococcus aureus causing toxic shock syndrome. AB - A strain of Staphylococcus aureus producing toxic shock syndrome toxin-1 was repeatedly isolated from the nares of a neurosurgeon. This strain was identical to strains cultured from two of his patients who developed toxic shock syndrome after laminectomy. The relatedness of the isolates was shown by Southern blot hybridization analyses using chromosomal transposons as probes. This approach should be considered, in addition to standard bacteriologic techniques, as an effective method to analyze the relatedness of nosocomial isolates. PMID- 3021040 TI - Monovalent influenza A(H1N1) vaccine, 1986-1987. Recommendations of the Immunization Practices Advisory Committee. Centers for Disease Control, Department of Health and Human Services. AB - These supplemental recommendations provide guidelines for a monovalent influenza A(H1N1) vaccine for protection against a newly emerged variant of influenza that has recently caused outbreaks among children and young adults in Asia. Guidance is provided for the use of this monovalent vaccine, which contains 15 micrograms of A/Taiwan/1/86(H1N1) antigen, as a supplement to the standard trivalent influenza vaccine. Recommendations for the use of the standard trivalent influenza vaccine for the 1986-1987 season and the use of antivirals for the prevention and treatment of influenza remain in effect and should be referred to in conjunction with this supplemental recommendation. The trivalent vaccine is intended to protect against currently circulating strains of influenza A(H3N2) and influenza B viruses and may provide partial protection against the new influenza A(H1N1) variant. PMID- 3021042 TI - The assay of 4-hydroxy-3-methoxymandelic acid in urine by HPLC with electrochemical detection using bonded-phase silica sorbents for rapid, simple and selective extraction. AB - A method is reported for the determination of 4-hydroxy-3-methoxymandelic acid (HMMA) by HPLC with electrochemical detection. The preparation of sample prior to HPLC has been studied and an efficient and selective extraction procedure described. Bonded-phase silica extraction columns and a vacuum manifold were used for the simple and rapid processing of batches of urine samples. Combining a reverse-phase C18 and an anion exchange column ensures selective isolation of HMMA. This simplified greatly the subsequent chromatography. The method was combined into a simple scheme for the routine analysis of urine HMMA, catecholamines and 5-hydroxyindole-3-acetic acid. The HPLC was standardised such that a single mobile phase was used with minor modification for each of the assays. PMID- 3021041 TI - Cochlear and retrocochlear immune-mediated inner ear disorders. Pathogenetic mechanisms and diagnostic tools. AB - Three different forms of immune-mediated sensorineural hearing loss are described. The pathogeneses of these three cases with severe audiovestibular deficits are completely different. To make an appropriate diagnosis remains a dilemma. Autoimmunity plays a certain role, but is not always present. Vascular and neural tissue can become involved. Immune-mediated forms of sensorineural hearing loss can be of cochlear or retrocochlear origin. PMID- 3021043 TI - [Regulation of enkephalin biosynthesis in chromaffin cells]. AB - Enkephalin peptides (ENK) are co-released with catecholamines from bovine chromaffin cells in culture. Drugs mimicking the effects of c-AMP increase ENK biosynthesis by increasing ENK mRNA, but are uneffective on ENK secretion. Nicotine, which causes a rapid release of ENK from these cells, also induces an increase in ENK biosynthesis and ENK mRNA. Reserpine enhance ENK precursor processing. The actions of these different pharmacological agents show that ENK biosynthesis is regulated at different levels in chromaffin cells. PMID- 3021044 TI - [Electrophysiological and biochemical studies of GABA receptors in a primary cell culture of the pars intermedia of the porcine pituitary]. AB - Using a primary culture of intermediate lobe (IL) we are investigating how GABA modulates stimulus-secretion coupling in the hypophysis. Two classes of GABA receptors have been described: GABA-A and GABA-B. We have characterised the GABA A receptor using the patch clamp technique (Whole Cell Recording). GABA (10-100 microM) and isoguvacine (50 microM) a specific GABA-A agonist elicit an inward current with an equilibrium potential that coincides with Ecl-, this could explain the depolarising action of GABA-A agonists. When applying baclofen, the specific GABA-B agonist, no modifications of membrane potential were seen. Perfusion studies on alpha MSH release showed GABA (50 microM) and isoguvacine (50 microM) to potentiate Ba++-evoked secretion. The effects were antagonised by bicuculline and SR 95103, confirming GABA-A receptor action. Baclofen stereospecifically inhibited both basal and Ba++-evoked release. Together the results suggest the co-existence of the two classes of GABA receptors on the endocrine cells of the IL. PMID- 3021045 TI - [Characterization and modulation of anterior pituitary binding sites for rat corticotropin releasing factor (r-CRF)]. AB - Specific binding sites for rat CRF (r-CRF) have been characterized on rat anterior pituitary membranes. The binding of the radioiodinated analog of r-CRF (125I Tyr-r-CRF) was time, temperature, pH and protein dependent. No interaction was found with other neurohormones except with Arginine Vasopressin, but at supra physiological levels. Two classes of specific binding sites (high affinity and low affinity) for r-CRF were identified. Bilateral adrenalectomy provoked, since the 24th hour and up to 7 days, in addition of an increase of ACTH plasmatic levels, an abolition of the high affinity binding site; corticosterone treatment reversed these changes. This finding suggests that circulating glucocorticoids may control the anterior pituitary binding sites for CRF, either by a direct action on the anterior pituitary, or by a modulatory effect on hypothalamic CRF secretion. PMID- 3021046 TI - [Effect of kerogen oxidation products on the biosynthesis of beta-lactamases by Escherichia coli]. AB - Shale acid concentrate (SAC) and ozonid, the products of water-alkaline oxidation of kerogen stimulated production of beta-lactamases by E. coli. The effect was most pronounced when the SAC was added to the fermentation nutrient medium. Kerogen ozonid was inferior by its activity. Cultivation of the seed material on media containing both the products of kerogen did not influence the enzyme production. Stimulating effect on production of either penicillinases or cephalosporinases was observed. SAC and ozonid had no influence on the culture growth, multiplication and biomass accumulation. PMID- 3021047 TI - [Cytotoxic action of doxorubicin and daunomycin on human lung cancer cells]. AB - The cytotoxic activity of two anthracycline antibiotics, i. e. doxorubicin and daunomycin against the cells of human lung cancer was estimated. Tumors resected from 19 patients, including 8 with adenocarcinoma, 10 with squamous cell cancer and 1 with small cell cancer were used. The tumor fragments were placed into diffusion chambers and implanted into the abdominal cavity of mice CBA. The effect was estimated autoradiographically on the 6th-7th day of cultivation after administration of doxorubicin or daunomycin to the animals in the maximum tolerance doses. The number of the tumors sensitive to doxorubicin and daunomycin amounted to 79 and 89 per cent, respectively. As compared to doxorubicin, daunomycin lowered the proportion of the DNA-synthesizing cells in the drug sensitive xenografts of lung cancer more significantly. With the use of daunomycin the synthesis of DNA in the tumor cells was inhibited more intensively than with the use of doxorubicin. The cytotoxic effect of daunomycin and doxorubicin did not depend on the tumor histological structure. The experimental data suggested that daunomycin would be highly efficient in chemotherapy of patients with lung cancer. PMID- 3021049 TI - Genetic studies of kanamycin resistance in Campylobacter jejuni. AB - Campylobacter jejuni 3H40 and 4B20 harbored 59-kilobase (kb) self-transmissible plasmids encoding resistance to kanamycin and tetracycline. Although the two antibiotic resistances were more frequently inherited together, some transconjugants and ethidium bromide segregants which were resistant to only one of these antibiotics were recovered. The kanamycin-susceptible, tetracycline resistant segregants carried plasmids 4 kb smaller than the 59-kb plasmids of their parents, whereas the kanamycin-resistant, tetracycline-susceptible segregants contained no detectable plasmid DNA. Restriction endonuclease maps of deleted forms of the 59-kb plasmids revealed that deletions and rearrangements of 4-kb lengths of DNA were associated with loss of kanamycin resistance. Translocation of the kanamycin resistance determinant between plasmid and chromosomal DNA was demonstrated. Such phenomena have not been previously described in C. jejuni spp. and are consistent with the interpretation that the kanamycin resistance determinant is encoded by a translocatable element. PMID- 3021048 TI - In vitro activity of ketoconazole against herpes simplex virus. AB - The effects of ketoconazole alone and in combination with acyclovir and adenine arabinoside upon the replication of herpes simplex virus types 1 and 2 (HSV-1 and -2) were investigated by using a yield reduction assay. Ketoconazole demonstrated antiviral activity against HSV-1 and -2 and synergistic antiviral activity when it was combined with acyclovir. Combinations of ketoconazole with adenine arabinoside resulted in either interference or indifference. The effects of ketoconazole upon the protein synthesis of HSV-2-infected cells were also determined in an effort to define the mechanism of action for the antiviral activity of ketoconazole. There was no reduction of HSV proteins when compared with acyclovir. These findings suggest that further investigations of the use of ketoconazole for the treatment of HSV infections are warranted. PMID- 3021050 TI - High-pressure liquid chromatographic assay of sulbactam in plasma, urine, and tissue. AB - A reverse-phase high-pressure liquid chromatography method for the quantitation of sulbactam in plasma, urine, and tissue is described. The assay used the formation of an imidazole derivative followed by extraction with acetonitrile and dichloromethane and used UV absorbance for detection. The mobile phase consisted of acetonitrile, tetrabutylammonium hydroxide, and phosphate buffer. The assay was linear from 100 micrograms/ml (g of tissue) to 1 microgram/ml (g). Within- and between-batch recovery was greater than 90%. The coefficient of variation was generally less than 15%. There were no interfering peaks in the quantitation of sulbactam. PMID- 3021051 TI - Diversity of determinants encoding carbenicillin, gentamicin, and tobramycin resistance in nosocomial Pseudomonas aeruginosa. AB - Plasmid pFMH1010, an 89-megadalton R plasmid, is endemic among members of the family Enterobacteriaceae at Hines Veterans Administration Hospital, Hines, Ill. It encodes resistance to nine antibiotics, including resistance to carbenicillin (Cb), gentamicin (Gm), and tobramycin (Tm). Pseudomonas aeruginosa strains resistant to carbenicillin, gentamicin, and tobramycin were isolated from five patients at Hines Veterans Administration Hospital from whom Serratia marcescens strains harboring pFMH1010 were also obtained. The P. aeruginosa strains were investigated to determine whether their Cb, Gm, and Tm characteristics derived from pFMH1010. One of the isolates, Ps559, was shown by Southern hybridization to contain approximately 76% of pFMH1010. Several lines of evidence suggested that the pFMH1010 sequences in Ps559 are integrated in the chromosome. Southern hybridization also demonstrated that the beta-lactam resistance of pFMH1010 is most probably due to the presence of sequences homologous with Tn3 and that these sequences are retained in Ps559. In two other Pseudomonas isolates, resistance to carbenicillin, gentamicin, tobramycin, and kanamycin was encoded by R plasmids unrelated to pFMH1010. In the last two isolates, resistance to gentamicin and tobramycin and several other antibiotics appeared to be chromosomally encoded, and it was rescuable from one of these strains by RP4-mediated mobilization. PMID- 3021054 TI - EMC virus infection in baboons as a model for studies on antiviral substances. AB - EMC virus causes a lethal infection in baboon monkeys within 4-8 days following subcutaneous injection with 10(4)-10(8) pfu of virus. The infection is accompanied by viremia, invasion of heart muscle and of brain. Monkeys infected with 10(6) pfu of EMC virus were treated with human leukocyte interferon. The interferon was injected intramuscularly first 0, 0.5, 6 and 24 h post-infection, then twice daily with a dose of 3 X 10(6) units for 5 consecutive days. All the monkeys treated with interferon remained alive and healthy. Animals infected with EMC virus, but not treated with interferon died within 6 days with evidence of myocarditis. The EMC virus-interferon interaction in baboon monkeys seems to provide a useful primate model system for testing the prophylactic and therapeutic antiviral activity of interferons or other antiviral substances. PMID- 3021052 TI - Molecular cloning of amikacin resistance determinants from a Klebsiella pneumoniae plasmid. AB - A multiresistant Klebsiella pneumoniae strain, JHCK1, harbored several plasmids. One of these, plasmid pJHCMW1, carried determinants for resistance to amikacin in addition to kanamycin, tobramycin, and ampicillin. The amikacin resistance determinant(s) was cloned and studied by restriction mapping, insertion, and deletion analysis. The amikacin resistance gene(s) was localized in a 1.5 kilobase DNA fragment. This pJHCMW1 DNA region was responsible for not only the resistance to amikacin but also the resistance to kanamycin and tobramycin. The cloned DNA fragment specified both an acetyltransferase activity and a low level of phosphotransferase activity. The two activities were absent from mutants that did not confer resistance to amikacin, kanamycin, and tobramycin. PMID- 3021055 TI - In vitro and in vivo activities of phosphate derivatives of 9-(1,3-dihydroxy-2 propoxymethyl)-guanine against cytomegaloviruses. AB - The anti-cytomegalovirus activities of four phosphate derivatives of 9-(1,3 dihydroxy-2-propoxymethyl)guanine (DHPG) were evaluated against human, monkey and murine viruses. The 5'-mono-, 3'5'-bis(mono-), and 3',5'-cyclic monophosphate and 5'-homophosphonate forms of DHPG inhibited virus plaque formation at 1-15 microM. The cyclic phosphate and homophosphonate were more active than the other compounds against murine cytomegalovirus (MCMV) in vitro. In an in vivo MCMV infection model, DHPG homophosphonate and DHPG were equally effective at reducing mortality at greater than or equal to 10 mg/kg. The cyclic phosphate was active at 10-20 mg/kg but toxic at greater than or equal to 40 mg/kg. The phosphorylation of DHPG phosphate and DHPG phosphonate, as well as the inhibition of human cytomegalovirus DNA polymerase by their respective triphosphates, were also examined. PMID- 3021053 TI - In vitro cytotoxicity and antibiotic activity of polymyxin B nonapeptide. AB - Polymyxin B nonapeptide, prepared by enzymic removal of the fatty acyl diaminobutyric acid side chain from polymyxin B, was about 100-fold less toxic to K562 cells than polymyxin B. MICs of polymyxin B nonapeptide against a test panel of bacteria were 2- to 64-fold lower than those of polymyxin B. PMID- 3021056 TI - Mechanism of inactivation of enteric viruses in fresh water. AB - Fresh water obtained from nine sources was shown to cause inactivation of poliovirus. Further testing with four of these water samples showed that enteric viruses from different genera were consistently inactivated in these freshwater samples. Studies on the cause of inactivation were conducted with echovirus type 12 as the model virus. The results revealed that the virucidal agents in the waters tested could not be separated from microorganisms. Any treatment that removed or inactivated microorganisms caused loss of virucidal activity. Microbial growth in a sterilized creek water seeded with a small amount of stream water resulted in concomitant production of virucidal activity. When individual bacterial isolates obtained from a stream were grown in this sterilized creek water, most (22 of 27) produced a large amount of virucidal activity, although the amount varied from one isolate to the next. Active and inactive isolates were represented by both gram-positive and gram-negative organisms. Examination of echoviruses inactivated in stream water revealed that loss of infectivity first correlated with a slight decrease in the sedimentation coefficient of virus particles. The cause appeared to be cleavage of viral proteins, most notably, VP 4 and, to a lesser extent, VP-1. Viral RNA associated with particles was also cleaved but the rate was slower than loss of infectivity. These results suggest that proteolytic bacterial enzymes inactivate echovirus particles in fresh water by cleavage of viral proteins, thus exposing the viral RNA to nuclease digestion. PMID- 3021057 TI - Development of a method for concentration of rotavirus and its application to recovery of rotaviruses from estuarine waters. AB - As part of our studies on the ecology of human enteric viruses, an improved method for detection of rotaviruses in water was developed, and their presence in Galveston Bay was monitored. Samples (378 liters) of estuarine water adjusted to pH 3.5 and a final AlCl3 molarity of 0.001 were filtered through 25-cm pleated cartridge-type filters (Filterite Corp., Timonium, Md.) of 3.0- and 0.45-micron porosity. Adsorbed virus was eluted with 1 liter of 10% tryptose phosphate broth, pH 9.5. Primary eluates were reconcentrated to a final volume of 10 to 20 ml by a simple and rapid magnetic iron oxide adsorption and elution procedure. Two percent casein at pH 8.5 effectively eluted rotavirus from iron oxide. A total of 21 of 72 samples of water, suspended solids, fluffy sediments, and compact sediments collected in different seasons in Galveston Bay yielded rotaviruses. Recovery of rotaviruses varied from 119 to 1,000 PFU/378 liters of water, 1,200 PFU/1,000 g of compact sediment, 800 to 3,800 PFU/378 liters of fluffy sediment, and 1,800 to 4,980 PFU from suspended solids derived from 378 liters of water based on immunofluorescent foci counts on cover slip cultures of fetal monkey kidney cells. PMID- 3021059 TI - Activation of human erythrocyte 2,3-bisphosphoglycerate phosphatase at physiological concentrations of substrate. AB - In human erythrocytes the reactions of the 2,3-bisphosphoglycerate shunt are catalyzed primarily by one protein, 2,3-bisphosphoglycerate synthase-phosphatase. At low concentrations of 2,3-bisphosphoglycerate the phosphatase is activated by several anions including inorganic phosphate and sulfite, and the phosphate activation is inhibited by low concentrations of 3-phosphoglycerate [Z. B. Rose and J. Liebowitz (1970) J. Biol. Chem. 245, 3232-3241]. Phosphate and sulfite also activate at high but physiological concentrations of 2,3-bisphosphoglycerate (5 mM), but the inhibition by 3-phosphoglycerate is much weaker. The basal activity (without added phosphate or sulfite) was also found to be higher and to be 3-phosphoglycerate sensitive; this is attributed to activation either by 2,3 bisphosphoglycerate itself or by a contaminant in it. These results allow previous observations of 2,3-bisphosphoglycerate hydrolysis in intact erythrocytes to be reconciled with the properties of the purified enzyme under near-physiological conditions. PMID- 3021058 TI - Humic acid interference with virus recovery by electropositive microporous filters. AB - The effects of humic acid on poliovirus type 1 recovery from water by Zeta Plus 60S filters were investigated. The humic acid interfered by preventing virus adsorption to the filters, and the interference increased as a function of the amount of humic acid filtered. Humic acid decreased virus adsorption when filtered before the virus, but did not elute virus which had adsorbed to the filters. The effects on virus recovery were not due to alterations in virus titer or neutralizability. The addition of AlCl3, which improved virus recovery by electronegative filters in the presence of humic acid, did not aid in overall virus recovery by the Zeta Plus filters in the presence or absence of humic acid. However, the salt and humic acid in combination improved virus adsorption and concurrently reduced virus elution efficiency. The addition of NaH2PO4 had no direct effect on virus recovery and did not alter the effect of humic acid. In an attempt to identify the components of humic acid responsible for the interference, humic materials were fractionated by size by using Sephadex gel chromatography and dialysis, and the fractions were tested for interfering activity. Interference was not associated with specific size fractions of the humic materials. PMID- 3021060 TI - The vanadate-stimulated oxidation of NAD(P)H by biomembranes is a superoxide initiated free radical chain reaction. AB - Rat liver microsomes catalyze a vanadate-stimulated oxidation of NAD(P)H, which is augmented by paraquat and suppressed by superoxide dismutase, but not by catalase. NADPH oxidation was a linear function of the concentration of microsomes in the absence of vanadate, but was a saturating function in the presence of vanadate. Microsomes did not catalyze a vanadate-stimulated oxidation of reduced nicotinamide mononucleotide (NMNH), but gained this ability when NADPH was also present. When the concentration of NMNH was much greater than that of NADPH a minimal average chain length could be calculated from 1/2 the ratio of NMNH oxidized per NADPH added. The term chain length, as used here, signifies the number of molecules of NMNH oxidized per initiating event. Chain length could be increased by increasing [vanadate] and [NMNH] and by decreasing pH. Chain lengths in excess of 30 could easily be achieved. The Km for NADPH, arrived at from saturation of its ability to trigger NMNH oxidation by microsomes in the presence of vanadate, was 1.5 microM. Microsomes or the outer mitochondrial membrane was able to catalyze the vanadate-stimulated oxidation of NADH or NADPH but only the oxidation of NADPH was accelerated by paraquat. The inner mitochondrial membrane was able to cause the vanadate-stimulated oxidation of NAD(P)H and in this case paraquat stimulated the oxidation of both pyridine coenzymes. Our results indicate that vanadate stimulation of NAD(P)H oxidation by biomembranes is a consequence of vanadate stimulation of NAD(P)H or NMNH oxidation by O-2, rather than being due to the existence of vanadate-stimulated NAD(P)H oxidases or dehydrogenases. PMID- 3021061 TI - Stimulation by dolichol phosphate-mannose of N-acetylglucosaminyl-lipid biosynthesis by membranes from class E Thy-1-negative mutant mouse lymphoma cells which are defective in dolichol phosphate-mannose biosynthesis. AB - Dolichol phosphate-mannose has been shown previously to stimulate the biosynthesis of N-acetylglucosaminyl-diphosphate-dolichol (E. L. Kean (1985) J. Biol. Chem. 260, 12561-12571). Although the class E Thy-1-negative mutant mouse lymphoma cells are unable to synthesize dolichol phosphate-mannose, the addition of this compound exogenously to membranes from the mutant cells brought about a stimulation of N-acetylglucosaminyl-lipid synthesis similar to that obtained with membranes from wild type cells. The retention of this activity by the mutant cells supports the suggestion of a regulatory role for dolichol phosphate-mannose as an intrinsic property of the glucosaminyltransferase which catalyzes the initial reaction of the dolichol pathway. PMID- 3021062 TI - Cleavage of the rat intestinal 1,25-dihydroxyvitamin D3 receptor by an endogenous protease to a form with defective DNA binding. AB - In this report we describe a form of the 1,25(OH)2D3 receptor which no longer binds to DNA. The defective form of the receptor was produced by the action of an endogenous protease. Rat intestinal receptors, obtained by a two-step procedure of a low salt homogenization followed by extraction of the chromatin pellet with high salt, fail to bind to DNA-cellulose. Inclusion of various serine protease inhibitors during the preparation protects against the loss of DNA binding. Sedimentation analysis in sucrose gradients indicates that the defective receptor is measurably smaller than the native receptor and is unable to aggregate normally under low salt conditions. The size difference, as determined by gel chromatography, is approximately 9,000 Da (56,000 for the protected receptor, 47,000 for the cleaved form). The elution from DEAE-cellulose indicates that the overall charge of both intact and cleaved receptor forms is very similar. Cell fractionation and mixing experiments suggest the enzyme may be located in the lysosomal compartment, organelles which are susceptible to breakage during the extraction procedure. The results demonstrate that an endogenous enzyme preferentially cleaves the 1,25(OH)2D3 DNA binding site resulting in a receptor with altered characteristics. Such an enzymatic activity has not been previously described for the 1,25(OH)2D3 receptor from other tissues or species. Since rat intestine is a classically studied target organ, these findings have additional relevance in receptor purification or other studies to characterize the receptor. PMID- 3021063 TI - Rapid purification of adenylate kinase from human erythrocytes and skeletal muscle. AB - Adenylate kinase from human erythrocytes and skeletal muscle can be purified to homogeneity by a new procedure based on DEAE-Sepharose and Blue Sepharose affinity chromatography and Sephadex G-75 fractionation. For the enzyme purified from erythrocytes the specific activity is 3,000 U/mg of protein, and the overall yield is 70%. For the enzyme purified from skeletal muscle the specific activity is 2,075 U/mg of protein, and the overall yield is 44%. The sequence of steps takes advantage of the high isoelectric point, the high affinity for Blue Sepharose, and the low molecular weight of the isoenzyme from these two human tissues. PMID- 3021064 TI - Isolation and identification of a 23,000-dalton heparin binding fragment from the amino terminus of bovine thrombospondin. AB - Bovine freeze-thaw lysed platelets were fractionated by dextran sulfate affinity chromatography and a purified protein of 23,000 Da was subsequently obtained by G 75 gel filtration of the 0.5 M NaCl fraction. This protein had an amino terminal sequence of Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-Asp-Ile-Phe-Glu Leu- Thr-Gly-Ala-Ala-Trp-Lys-, a sequence identical to that reported for human thrombospondin. Thrombin-released platelets, fractionated in an identical manner, yielded a protein of 30,000 Da. Immunoblotting of purified bovine platelet thrombospondin and the 150,000- and 30,000-Da plasmin-generated thrombospondin fragments indicated that polyclonal antisera raised against the 23,000-Da protein cross-reacted with intact thrombospondin and the 30,000-Da fragment but not the 150,000-Da fragment. The 23,000-Da protein possessed weak heparin neutralization activity. PMID- 3021066 TI - [Treatment of epithelial ovarian carcinoma with cisplatin and adriamycin: analysis of factors influencing prognosis in advanced cases]. AB - Thirty previously untreated patients with epithelial ovarian carcinoma were treated with cisplatin and adriamycin (PA). Of eight evaluable patients, six were responders (two CRs and four PRs). The three-year survival rates were 83% for stage I, 67% for stage II, 50% for stage III, and none for stage IV. Toxicities included moderate myelo-suppression, mild nephrotoxicity, alopecia, and severe vomiting in almost all patients. Patients with residual lesions smaller than 2 cm had excellent prognosis when PA was given for more than five courses. Patients with residual lesions larger than 2 cm, however, had poor prognosis irrespective of the number of courses. No specific relation between histological grade and prognosis could be found. From the present data, aggressive cytoreductive surgery followed by PA therapy repeated more than five times is recommended for achieving a good outcome in the treatment of epithelial ovarian carcinoma. PMID- 3021065 TI - Purification and properties of beta-mannosyltransferase that synthesizes Man-beta GlcNAc-GlcNAc-pyrophosphoryl-dolichol. AB - The beta-mannosyltransferase that adds mannose, from GDP-mannose, to GlcNAc GlcNAc-pyrophosphoryl-dolichol, to form Man-beta-GlcNAc-GlcNAc-pyrophosphoryl dolichol was solubilized from pig aorta microsomal preparations, using 0.5% NP 40, and was purified about 116-fold using conventional methods. The purified enzyme was mostly free of alpha 1,3- or alpha 1,6-mannosyltransferase activities, since Man beta-GlcNAc-GlcNAc-PP-dolichol (PP = pyrophosphoryl) accounted for more than 95% of the product when enzyme was incubated with GDP-[14C]mannose and GlcNAc-GlcNAc-PP-dolichol. Very little Man-beta-GlcNAc-GlcNAc-PP-dolichol was formed when GDP-[14C]mannose was replaced by dolichol-phosphoryl-[14C]mannose, indicating that GDP-mannose was the mannosyl donor. The oligosaccharide portion of this lipid was released by mild acid hydrolysis and was characterized by gel filtration as well as by susceptibility to beta-mannosidase and resistance to alpha-mannosidase. The partially purified enzyme could be stabilized by the addition of 20% glycerol and 0.5 mM dithiothreitol to the buffer, and could be kept in this solution for 5 or 6 days in ice. The enzyme was greatly stimulated by the addition of detergent (NP-40) with optimum activity being observed at 0.1%. However, no stimulation was seen with any phospholipid. The partially purified enzyme had a pH optimum of about 7.0, and showed an almost absolute requirement for Mg2+ with optimal activity occurring at about 5 mM Mg2+. Mn2+ and Ca2+ were only slightly active. The Km for GDP-mannose was about 5 X 10(-7) M and that for GlcNAc-GlcNAc-PP-dolichol about 1 X 10(-6) M. Beta-Mannosyltransferase activity was inhibited competitively by a variety of guanosine nucleotides with GDP and GDP-glucose being most active, but GTP, GMP, guanosine, and periodate oxidized guanosine were also effective. The enzyme was strongly inhibited by p chloromercuribenzenesulfonic acid and this inhibition was partially prevented by the addition of dithiothreitol. PMID- 3021067 TI - [A combination chemotherapy of cisplatin and etoposide in small cell lung cancer]. AB - Fourteen patients with small cell lung cancer were treated with cisplatin (80 mg/m2 i.v.) and etoposide (300, 400, 500 mg/m2 i.v.). This combination chemotherapy was administered over a three- or four-week period. Eleven of 13 evaluable patients showed a greater than 50% tumor reduction, but there were 3 complete responses and 5 partial responses, giving a response rate of 61%. Four patients who were initially treated achieved major responses. In 9 patients who had received prior chemotherapy, 4 achieved a major response. Of 3 complete responders, 2 patients had previously received etoposide treatment alone. The renal toxicity of this regimen was minimal and no patients developed any clinical symptoms. Nausea and vomiting were well controlled by high-dose metoclopramide and methylprednisolone. All patients, however, experienced appetite loss after treatment. The dose-limiting toxic effect of this regimen was hematologic toxicity. We therefore concluded that the combination of etoposide (300 mg/m2 i.v.) and cisplatin (80 mg/m2 i.v.) is repeatable at 3 or 4 week intervals and effective in patients with small cell lung cancer. PMID- 3021068 TI - [A phase II study of UFT in non-small cell lung cancer]. AB - A phase II evaluation of UFT, a mixture of tegafur and uracil, was performed in 13 patients with non-small cell lung cancer (eight patients with adenocarcinoma and five patients with squamous cell carcinoma). UFT at a dose of 600 mg was given per os every day for more than four weeks. Among 12 evaluable patients, one patient with adenocarcinoma of the lung showed partial response. The response rate for UFT was 8.3%. Toxic effects included anorexia (31%), nausea (15%), liver disorder (15%), and pigmentation (8%). PMID- 3021070 TI - Neuropathy in a petrol sniffer. AB - A 4 year old boy developed a profound motor neuropathy after repeated deliberate inhalation of petroleum vapour. The condition was characterised by extreme slowing of the nerve conduction velocity. He made a gradual recovery over six months. The neuropathy was attributed to the N-hexane component of petroleum. PMID- 3021069 TI - Genital tract papillomavirus infection in children. AB - Genital tract papillomas in five children were examined for the presence of human papillomavirus (HPV) DNA by molecular hybridization. Papillomavirus DNA was detected in each sample and was identified as HPV-6 (three cases), HPV-6 or HPV 11 (one case), or HPV-16 (one case). These viruses are the same as are responsible for genital papillomas (condylomata) of adults. The transmission of adult genital tract viruses to children occurs primarily by a venereal route but may occur by a nonvenereal route. PMID- 3021071 TI - The extent and distribution of cancer in breasts with palpable primary tumors. AB - The term multicentricity has been employed to describe cancer cells beyond the borders of the primary tumor. However, it is not clear if there are multiple independent sites of origin or if the process simply represents spread of the cancer. The present study was designed to examine the distribution and extent of cancer in the breast and identify factors that bear on these events. All mastectomy specimens between 1980 and 1983 were systematically examined by means of multiple sections. One hundred seventy-nine of 657 patients (27%) were found to have separate foci. The most common histologic type (invasive ductal) was least likely to have multifocal disease (19%), while it was extremely common in the small group of patients with intraductal lesions (81%). Size was a factor in ductal but not in lobular lesions. Ninety per cent of the secondary foci were found in close proximity to the primary, suggesting spread rather than multicentricity. This implies a more limited and predictable distribution of cancer cells and opens the way to more rational selection and surgical preparation of patients for breast preservation. PMID- 3021072 TI - Utility of operative ultrasound in the surgical management of liver tumors. AB - In this study the utility of operative ultrasound in the surgical management of 98 consecutive patients with liver and gastrointestinal tumors was assessed. All patients had preoperative work-up including ultrasound study of the liver as well as selective hepatic arteriography (50 patients) and computerized tomography of the liver (45 patients). At surgery, inspection and palpation of the liver as well as operative ultrasound examination were performed in all cases. Fifty-six patients were known to have liver tumors before operation, while 42 patients had their liver examined as part of the treatment of a primary gastrointestinal malignancy. A total of 126 liver tumors were found in 58 patients, all of whom were confirmed histologically. Eighteen nodules unsuspected before operation were found at surgery--nine by inspection and palpation of the liver, and nine others that were nonpalpable were found by operative ultrasound only. Eighteen lesions that were missed by all diagnostic modalities were found as secondary lesions on pathologic examination of the resected specimens. In addition to diagnostic applications, operative ultrasound was useful in localizing nodules and permitting guided biopsies deep in the hepatic parenchyma. In eight cases, segmental resections were performed with operative ultrasound to localize the plane of section and to catheterize the intrahepatic portal vein branch afferent to the tumor in order to perform balloon catheter occlusion of the vessel for control of bleeding. Operative ultrasound was found to be important in the surgical management of 19 of 98 patients (19%). PMID- 3021074 TI - Anticatatonic effect of clonidine: its interaction with dopaminergic, anticholinergic and GABA-ergic drugs. AB - Anticatatonic effects of systemic (50 micrograms/kg, i.p.) and intracerebroventricularly (5 micrograms/10 microliters/rat) administered clonidine was studied in rats against perphenazine-induced catatonia. Clonidine was found to possess anticatatonic actions in rats as it significantly blocked perphenazine-induced catatonia. When clonidine was administered simultaneously with dopaminergic (dopamine, bromocriptine), anticholinergic (scopolamine, diphenhydramine) and GABA-ergic (GABA, muscimol) agents there was a potentiation of the anticatatonic effect. However, clonidine-anticholinergic combination showed a better protective response as compared to clonidine-dopaminergic combination. The protective effect of clonidine and its modification by anticholinergic agents has been explained on the basis of direct central adrenergic as well as central anticholinergic actions of clonidine. Although higher dose of GABA-ergic agents was not studied due to toxicity, the combined effect of clonidine and GABA-ergic agents has been explained on neurotransmitter interactions. PMID- 3021073 TI - Selective affinity of one enantiomer of suriclone demonstrated by a binding assay with benzodiazepine receptors. AB - The binding of racemic 3H-suriclone on the BZD receptor was performed in special conditions in order to discriminate between the affinity of the two enantiomers: a rebinding method was used. In the first incubation 40% of 3H-suriclone was specifically bound to the BZD receptor. In the second incubation performed with the supernatant coming from the first incubation, less than 1% of the radiolabelled suriclone was specifically bound to the BZD receptor. These results suggest that most probably only one of the two enantiomers of suriclone may bind the BZD receptor. It appears that this enantiomer has the greatest affinity constant ever found for a BZD receptor agonist (Kd = 20 pM at 37 degrees C in presence of GABA and 70 pM in absence of GABA). PMID- 3021075 TI - Are there any presynaptic 5-HT receptors in frog atria? AB - The effect of 5-hydroxytryptamine (5-HT) on the stimulation-induced inotropic responses was investigated in the isolated frog atria and ventricles. 5-HT (1 X 10(-5)-2 X 10(-4) M) inhibited the contractile responses elicited by intramural nerve stimulation (INS) in atria. The inhibitory action of 5-HT was antagonized by raising the external Ca++ concentration. Pizotifen and methysergide (1 X 10( 6) M) antagonized the inhibitory action of 5-HT on high frequency INS responses. Higher concentrations of methysergide by itself depressed the responses to low frequency INS. Ketanserin, metoclopramide and phentolamine (1 X 10(-6) M) failed to antagonize the inhibitory action of 5-HT. The results suggest that the inhibitory effect of 5-HT in frog atria is mediated through a subtype of 5-HT receptors which is neither of 5-HT2 nor of "M" type but may correspond to the 5 HT1 subtype of serotoninergic receptors. In contrast with atria, 5-HT-induced inhibition of INS responses was not observed in frog ventricles. PMID- 3021076 TI - Selectivity of raubasine stereoisomers for alpha 1- and alpha 2-adrenoceptors in the rat. AB - The selectivity of raubasine and its two isomers tetrahydroalstonine (THA) and akuammigine (AKU) for alpha 1- and alpha 2-adrenoceptors has been investigated in pithed normotensive rats. alpha 1-Adrenoceptor blockade was measured by inhibition of the pressor response to (-)-phenylephrine. alpha 2-Adrenoceptor blockade was measured by antagonism of the inhibitory effect of clonidine both on the tachycardia produced by electrical stimulation of the cardiac accelerator nerves and on the pressor response to B-HT 933. Pressor responses to (-) phenylephrine were reduced in a dose-dependent manner by raubasine but not by THA and AKU. The inhibitory effect of clonidine and the pressor response to B-HT 933 were antagonized in a dose-dependent manner by THA but not by raubasine and AKU. These results indicate that, in pithed rats, AKU is a very weak antagonist at alpha 1- and alpha 2-adrenoceptors, raubasine is a preferential alpha 1 adrenoceptor antagonist, and THA is a selective alpha 2-adrenoceptor antagonist. PMID- 3021078 TI - Angiotensin I level and sporadic hypokalemic periodic paralysis. AB - A patient with secondary (sporadic type) hypokalemic periodic paralysis with relative hypertension had reninism with a high concentration of plasma angiotensin I (ANG-I) but no hyperaldosteronism or high angiotensin II value. Angiotensin-converting enzyme (ACE) activity was usually normal. Results of other hormonal analyses were also normal. However, the glomerular filtration rate and filtration fraction of the kidneys were greatly elevated. Despite severe hypokalemia, the patient's potassium clearance was high. No evidence of distinct hyperplasia of the juxtaglomerular cells was obtained. These results suggest that decreased affinity of ACE to the substrate ANG-I (so-called ACE dysfunction syndrome) produced the reninism and high concentration of plasma ANG-I, and that the latter induced an increase in the glomerular filtration rate of the kidneys with sequential occurrence of secondary hypokalemic periodic paralysis. PMID- 3021077 TI - Serum creatine kinase release and alpha- and beta-adrenergic receptors. AB - Changes in the serum creatine kinase (CK) activity and the mechanism of its release after administration of two kinds of beta-blockers to rats were studied in order to determine the mechanism of the increase in serum CK accompanying administration of beta-blockers. When pindolol or propranolol was administered at a dose of more than 10 mg/kg of body weight, the serum CK activity was increased in proportion to the dose. Also when an alpha-stimulator or an alpha, beta stimulator such as noradrenaline and adrenaline was administered, the serum CK activity was increased. When the beta-stimulator isoproterenol or the alpha 1 blocker prazosin was administered, there was no increase in the serum CK activity. In terms of consecutive administration of a combination of beta blockers and adrenaline, the serum CK activity was not increased by the second and succeeding administrations. As for the use of adrenaline alone, on the other hand, the serum CK activity after the second administration was equal to that after the first administration. The increase in CK activity after administration of adrenaline as the second treatment following administration of a beta-blocker as the first treatment was approximately the same as that after single administration of adrenaline. From the fact that the serum CK activity in rats was increased by administration of an alpha-stimulator and of a beta-blocker, it is possible that CK release in the blood is due to the action of alpha-receptors rather than beta-receptors. In the case of consecutive administration of a beta blocker, the serum CK activity was not increased after the second administration. However, the serum CK activity was increased by the administration of an alpha stimulator. This finding suggests the possibility that CK release is due to the disturbance of the balance between alpha-receptor and beta-receptor. Most of the CK isoenzymes released in the blood after administration of a beta-blocker were of the MM type. PMID- 3021079 TI - [Radioimmunoassay for the determination of serum corticosterone in ducks (Anas platyrhynchos)]. PMID- 3021080 TI - [Rotavirus infections in calves. 1. Adaptation of calf-pathogenic strains of rotavirus to cell culture]. PMID- 3021081 TI - [Radiological case of the month. Nephroblastoma with right auricular and caval malignant vascular thrombus]. PMID- 3021082 TI - Alpha 2-adrenergic and opiate receptor blockade. Synergistic effects on anxiety in healthy subjects. AB - To evaluate interactions between the opiate and adrenergic systems in healthy humans, concomitant administration of the opiate antagonist, naloxone hydrochloride, and the alpha 2-adrenergic receptor antagonist, yohimbine hydrochloride, was compared with the administration of placebo and of each drug separately. A synergistic effect of the combination (larger than the sum of the effects of the two drugs separately) was observed on subject ratings of nervousness, anxiety, tremors, palpitations, nausea, hot and cold flashes, and increased plasma cortisol concentrations. In addition, following the combination, each of the male subjects studied reported a full penile erection lasting at least 60 minutes, an effect not reported when each drug was given separately. These results demonstrate that interactions between the opiate the adrenergic systems have important implications for our understanding of the cause and treatment of anxiety disorders and male impotence. PMID- 3021084 TI - DNA distributions in human normal, precancerous and cancerous breast tissue. I. Ploidy and cell cycle distribution. AB - DNA distributions were recorded flow cytometrically in 19 normal, 23 precancerous tissues with epithelial proliferation and cell atypia (P1), 7 carcinomata lobularia in situ and intraductale carcinomas (P2) and in 44 invasive carcinomas of the human breast. The results were compared to histological findings, tumor staging and grading, lymph node involvement and age of the patients. DNA diploidy was found in all normal, 21 P1 tissues, 6 P2 tissues and 12 carcinomas; DNA aneuploidy in 2 P1 tissues, 1 P2 tissue and 32 carcinomas. Lobular carcinomas were preferentially involved in the DNA diploid group, duct carcinomas in the DNA aneuploid group. Cell cycle distribution analysis resulted in significantly higher percentages of cells in S- and G2M phase in DNA aneuploid carcinomas compared to normal and precancerous (P1, P2) tissues. The percentage of cells in G2M phase was significantly higher in DNA aneuploid carcinomas originating from patients younger than 50 years compared to patients older than 50 years. Correlations between the DNA-index, cell cycle distribution, lymph node involvement, tumour staging or histological type were not evident. PMID- 3021083 TI - Abnormal regulation of noradrenergic function in panic disorders. Effects of clonidine in healthy subjects and patients with agoraphobia and panic disorder. AB - Clonidine hydrochloride, an alpha 2-adrenergic receptor agonist that decreases noradrenergic function, was administered to 21 healthy subjects and 26 drug-free patients with agoraphobia and panic attacks. Clonidine produced significantly greater decreases in plasma MHPG levels and sitting and standing diastolic blood pressure and significantly smaller increases in growth hormone levels and self rated drowsiness in the patients. These findings indicate that the regulation of noradrenergic activity is aberrant in some patients with panic disorder, since a previous study demonstrated that patients with panic disorder exhibit increased plasma MHPG levels, blood pressure, and behavioral responses to the alpha 2 adrenergic receptor antagonist yohimbine. The increased dynamic range of noradrenergic activity observed as an increased sensitivity to both clonidine and yohimbine may reflect abnormalities in the regulatory inputs to noradrenergic neurons, or dysfunction in the alpha 2-adrenergic receptor effector coupling mechanism or the intracellular effector system. PMID- 3021086 TI - Hepatic disease associated with ground-glass inclusions in hepatocytes after cyanamide therapy. AB - In a retrospective review of 2400 consecutive liver biopsy specimens, 60 cases with ground-glass hepatocytes were identified, 41 specimens gave a positive reaction to orcein stain and 19 a negative staining. These 19 specimens were obtained from chronic alcoholics who had been admitted to a detoxication program that used aversive drugs and who were hepatitis B surface antigen negative. The use of cyanamide (Colme), an inhibitor of aldehyde dehydrogenase could be documented in 11 instances. In addition to ground-glass hepatocytes, which were periodic acid-Schiff positive and had a periportal or paraseptal distribution, these liver specimens showed a variety of hepatic lesions: cirrhosis in five cases, portal and periportal inflammation in six, triaditis in five, portal fibrosis in two, and minimal changes in one. Patients with shorter courses of cyanamide were those who had less severe histologic lesions. In three patients who had a liver biopsy carried out before the cyanamide treatment ground-glass hepatocytes were not found. These data indicate that ground-glass hepatocytes that stain with periodic acid-Schiff may develop after cyanamide treatment. They are associated with structural hepatic damage of varied severity in patients submitted to a long-term treatment. PMID- 3021085 TI - Three types of growth hormone cells of the rat anterior pituitary as revealed by immunoelectron microscopy using a colloidal gold-antibody method. AB - Immunoelectron microscopy using a colloidal gold-antibody method with anti-rat GH serum demonstrated three morphologically different types of GH cells in the rat anterior pituitary. They were distinguished as Types I, II and III GH cells, containing only large secretory granules about 350 nm in diameter, mixed large and small granules, and only small granules about 150 nm in diameter, respectively. Double gold labeling with large gold particles for GH and small particles for PRL or ACTH indicated that neither GH and PRL nor GH and ACTH were contained in the same cell. In adult male rats, Type I cells (68%) predominated over Type II (22%), while Type III cells were rare (9.7%). On the contrary, in the adult female rats, Type II cells (47%) slightly dominated over Type I (44%) though the rate of Type III cells was the same as in the male. In neonatal infants, the frequency of occurrence of Type III cells was as high as about 20%; sex differences between Types I and II were indistinct. The Type III cells were therefore thought to represent an immature type. PMID- 3021088 TI - The clinical aspect of retained gastric antrum. AB - Thirty symptomatic patients with retained gastric antrum proved pathologically and/or by isotopic visualization were studied and treated from 1968 to 1983. The latent periods from the antral exclusion to the occurrence of anastomotic ulcers after a subtotal gastrectomy with Billroth's type II reconstruction varied from a few days to 19 years, with an average of 2.8 years. Fasting serum gastrin levels were normal in 14 of 21 patients and were intermittently high in some patients. The basal to maximal acid-output ratio was greater than 0.6 in 69% of the patients; primary cimetidine treatment was effective in three of five patients. Thirty-five operations on 27 patients were divided into six groups; all of these 27 patients eventually underwent resection of retained gastric antrum. We concluded that resection remains the best treatment for anastomotic ulcer related to retained gastric antrum. Additional truncal vagotomy did not provide additional benefit to these patients. Furthermore, cimetidine can be useful to control the symptoms for preoperative preparation or definitive treatment in high risk patients. PMID- 3021087 TI - Amyloid tumor (amyloidoma) of a peripheral nerve. AB - We report a case of amyloidoma of a peripheral cervical nerve situated in the right anterior aspect of the neck. Such a localization has not been previously described, to the best of our knowledge. PMID- 3021090 TI - BW B759U for cytomegalovirus retinitis: intraocular drug penetration. PMID- 3021089 TI - [Effects of exposure to sulfuric acid mist on the induction of experimental asthma in guinea-pigs: studies on the breathing curve pattern and pulmonary beta adrenergic receptor]. PMID- 3021091 TI - Effect of ethanol on human lymphocyte adenylate cyclase system. PMID- 3021092 TI - A review of 58 Rh-isoimmunized cases. PMID- 3021093 TI - Determinants of risk for developing invasive mole and choriocarcinoma following hydatidiform mole. PMID- 3021094 TI - A single alpha-globin gene deletion in Australian aborigines. AB - alpha- and beta-thalassaemias and other haemoglobinopathies have not so far been reported in Australian Aborigines. Using a DNA mapping technique, we tested groups of Aborigines for a deletion form of alpha-thalassaemia and found that there was a single alpha-globin gene deletion (-alpha/alpha alpha) in some populations. The alpha-globin gene deletion was detected in Aboriginal DNA samples collected from Kalumburu in the Kimberley region of Western Australia. It was found also in one sample from Mowanjum, near Derby in Western Australia, and in one from Mornington Island in the Gulf of Carpentaria. It was not observed in Aboriginal DNA samples from the central desert. Further analysis of the alpha globin gene deletion revealed that it was of the 3.7 kilobase (Kb) (-alpha 3.7) type. However, the -alpha 3.7 deletion in the Aborigines is apparently different from that found in southern Papua New Guinea as it is linked to a different zeta globin gene polymorphism. The presence of this silent alpha-thalassaemia in several populations of Aborigines may be explained in several ways. The most likely is through contact with Macassans or other voyagers from the Indonesian and Southeast Asian areas. PMID- 3021095 TI - Combination chemotherapy with CDDP and 5-FU in head and neck cancer. AB - Combination chemotherapy with CDDP and 5-FU was performed in 19 patients with evaluable head and neck cancer and 2 CR patients and 7 PR patients were obtained; thus the response rate was 47.4%. Histologically, the present therapy is considered to be especially effective against well differentiated squamous cell carcinoma (83.3%). The present therapy is considered to be useful as a neo adjuvant chemotherapy (75.0%), but it is desirable to perform at least 2 courses of treatment. The side effects observed were nausea, vomiting, anemia, leukocytopenia and alopecia, etc., and most of them were reversible. However, there were 2 patients in which the continuation of chemotherapy was impossible due to renal disorders. PMID- 3021096 TI - Role of route of exposure, age, sex, and type of chicken on the pathogenicity of avian reovirus strain 81-176. AB - Chickens were evaluated by age, sex, and type for susceptibility to reovirus strain 81-176 inoculated subcutaneously. Chicks were most susceptible to the lethal effects of reovirus infection at hatching, after which resistance increased rapidly. By 1 week of age, mortality was negligible, but chicks were still susceptible to the less lethal effects of the virus. Mortality rates of males and females were equal. Leghorn and broiler-type chicks did not differ appreciably in their response to viral inoculation. An effort was made to find a more "natural" means of exposure to reovirus than parenteral inoculation. Neither oral nor aerosol exposure was as effective as subcutaneous inoculation. Attempts to transmit reovirus to susceptible hatching chicks, starting when they were in ovo (19 days of incubation), also failed. Decreased weight gain proved to be a valid criterion for judging reovirus infection. Reovirus infection lowered the mean values of body weights, and the standard deviations were substantially greater, indicating an unevenness in size of the affected chickens. PMID- 3021097 TI - Comparison of the nephropathogenicity of four strains of infectious bronchitis virus. AB - Experiments were conducted to characterize renal lesions in chickens induced by four strains of infectious bronchitis virus (IBV); each has been described as nephropathogenic. Those strains were also compared in vaccinated and unvaccinated older chickens for nephropathogenicity. The younger birds were much more susceptible to the nephritogenic effects of the strains. All four strains produced acute renal changes consisting of tubular damage and interstitial inflammatory cell infiltration and edema. Although both cortex and medulla were involved, the latter was generally affected more severely. The Holte strain proved to be the least pathogenic, followed by the more pathogenic Gray and Italian strains and finally by the Australian strain. All four strains produced similar chronic renal changes in unvaccinated birds, with no correlation to the severity of lesions seen at the acute phase. Chronic active and inactive types of interstitial nephritis were seen at the chronic phase. Vaccinated birds challenged with the Australian strain had the highest prevalence of the chronic active type of interstitial nephritis. The implication of renal viral persistence in the development of chronic active interstitial nephritis is discussed. PMID- 3021098 TI - Observations on an enzyme-linked immunosorbent assay for the detection of antibodies against avian leukosis-sarcoma viruses. AB - In the detection of antibodies against exogenous subgroup A avian leukosis viruses (ALVs) using a representative subgroup A virus, concordance between enzyme-linked immunosorbent assays (ELISAs) and serum neutralizations ranged from 83 to 95%. In ELISAs, subgroup A- and subgroup B-specific neutralizing antisera were equally reactive against ALVs of subgroups A, B, and E. Conversely, little cross-reactivity of high-titered subgroup E antisera was observed against subgroup A viruses. Significant cross-reactivities of spontaneously induced subgroup E-neutralizing antisera were observed when tested against a representative subgroup B ALV. Because some normal chickens spontaneously mount antibodies against infectious endogenous viruses, misleading results may be obtained if subgroup B or E ALVs are the source of target antigens in ELISAs. PMID- 3021099 TI - Primary isolation and identification of avian rotaviruses from turkeys exhibiting signs of clinical enteritis in a continuous MA 104 cell line. AB - Avian rotaviruses were isolated from turkeys with enteritis using MA 104 cell line. MA 104 cells were suitable for primary isolation and propagation of avian rotaviruses. Trypsin appeared essential for the enhancement of infectivity and the occurrence of cytopathic effect (CPE). Serum neutralization (SN), electron microscopy (EM), and analysis of genomic RNA were done to identify and confirm the identity of rotaviruses. Electrophoretic migration patterns of genomic RNA from avian rotaviruses were examined, and they were compared with those from mammalian rotaviruses. The migration patterns differed between these groups. PMID- 3021101 TI - Experimental infection of specific-pathogen-free chickens with avian rotaviruses. AB - One-to-70-day-old specific-pathogen-free chickens were infected with rotaviruses isolated from chickens, turkeys, and pheasants. Chicks infected at 1 or 7 days of age remained largely free of infection: virus could be isolated from a few chicks of these ages, but virus antigen could not be detected in the epithelial cells of the intestinal villi. Treating the virus with trypsin before inoculation did not markedly enhance virus infectivity. The same batches of virus successfully infected chickens at 56 and 70 days of age: virus could be isolated between 1 and 5 days postinfection, and virus antigen was detected in the epithelial cells of the intestinal villi. Rotaviruses isolated from pheasants were infectious for 49 day-old chickens. All infections in the older chickens remained subclinical. PMID- 3021100 TI - Infectious bursal disease virus infection in the quail-chicken hybrid. AB - Hybrids produced from crossing Cornell K-strain white leghorn chickens and Line II Japanese quails were studied for susceptibility to infection with infectious bursal disease virus (IBDV). Quail-chicken hybrids were infected successfully following inoculation with IBDV at 14, 21, or 52 days of age. In most cases, precipitating antibodies were detected in serum by 10 days postinoculation (PI). Although no clinical signs or gross lesions were evident in the bursa of Fabricius of hybrids, histologic changes in the bursa were detected upon microscopic examination using hematoxylin and eosin staining. Chickens were successfully infected also; they had gross and microscopic lesions in the bursa and produced precipitating antibodies. In addition, staining of bursal sections with low concentrations of peroxidase-conjugated concanavalin A revealed a rearrangement of a leukocyte cell type (probably macrophages) in infected chickens and hybrids. Japanese quails were refractory to infection; they showed no bursal changes and did not form precipitating antibodies. PMID- 3021102 TI - Vaccination of day-old broiler chicks against Newcastle disease and infectious bursal disease using commercial live and/or inactivated vaccines. AB - Day-old broilers were administered live and/or inactivated vaccines to assess vaccine efficacy against challenge with Newcastle disease (ND) and infectious bursal disease (IBD). Chicks were from commercial breeder pullets vaccinated against ND and IBD using several live vaccine primers followed by an inactivated ND-IBD vaccine at 18 weeks. The most efficacious initial ND-IBD vaccination program was live ND virus by eye drop and live IBD vaccine injected subcutaneously (SQ) followed 2 hours later with inactivated ND-IBD vaccine SQ. The next two most efficacious programs were live vaccine alone and the inactivated vaccine only. Inactivated vaccine given SQ had no adverse effect on live IBD vaccine given 2 hours earlier in a similar site. Administration of inactivated vaccine by vent was not as efficacious as administration SQ. A booster of a second live ND-IBD vaccine drinking water at 18 days significantly increased levels of circulating antibody, regardless of the initial vaccination program. PMID- 3021105 TI - Viral arthritis in fryers related to reovirus infection in breeders. AB - In 1983, twenty-two outbreaks of viral arthritis/tenosynovitis were diagnosed in a 6-month period on 18 fryer farms of one commercial operation located in western Washington. The main source of the reovirus infection was traced to a breeder flock that supplied progeny chicks to all of the affected farms. PMID- 3021103 TI - Studies on orthoreoviruses isolated from young turkeys. III. Pathogenic effects in chicken embryos, chicks, poults, and suckling mice. AB - The pathogenicity of four clone-purified reoviruses (81-51, 81-68, 81-311, and 82 88) was studied in experimentally infected specific-pathogen-free (SPF) chicken embryos and chicks. SPF and specific-antigen-and-antibody-negative (SAAN) turkey poults, and suckling mice. In SPF embryos, all four viruses caused death or lesions characteristic for avian reoviruses. SPF chicks inoculated orally with isolate 81-68 showed no signs of overt disease but did develop antibodies to reovirus. In some experiments, poults inoculated orally with isolate 81-68 exhibited increased mortality, abnormal feather development, lower body weight gain, and pasted vents. Body tremors, uncoordinated motor movement, and oily hair coats were seen in suckling mice inoculated intracerebrally with isolates 81-51, 81-68, and 82-88. Mice inoculated intracerebrally with isolates 81-68 and 82-88 exhibited retarded growth. PMID- 3021104 TI - Intestinal cryptosporidiosis and reovirus isolation from bobwhite quail (Colinus virginianus) with enteritis. AB - An acute enteric disease of young pen-raised bobwhite quails was studied. Affected quails had white, watery diarrhea accompanied by dehydration and subsequent death. Mortality from hatch to 17 days of age ranged from 30 to 45% in the three flocks examined. Small intestines were thin-walled and distended with fluid and gas. Microscopic lesions in the intestinal tract consisted of villus atrophy, villus fusion, and sloughing of cells at the tip of the villi in duodenum, jejunum, and ileum. Cryptosporidium sp. and reovirus were identified in affected quails. PMID- 3021107 TI - Atypical condylomas of the cervix uteri in Singapore women: a histopathological and immunohistochemical study. AB - Subclinical flat cervical warts are commonly recognized by means of colposcopy. These warty lesions frequently contain cellular atypia of a varying degree showing all the features of cervical intraepithelial neoplasia (CIN) together with the features of wart virus infection e.g. koilocytosis. Human papillomavirus (HPV) antigens and DNA have been found in these lesions which can be called atypical condylomas. The presence of such markers in lesions regarded as having neoplastic potential implicates HPV in cervical carcinogenesis. We investigated a group of Chinese women in Singapore to see whether this hitherto unstudied group showed HPV markers, in particular, HPV antigens by means of the peroxidase antiperoxidase (PAP) staining technique. We found that histological warty change and HPV antigens were commoner in early compared with advanced CIN. These results, which are similar to previous studies, suggest that the aetiology of cervical carcinoma is probably no different in Singapore than in the West. They also suggest that HPV infection of the cervix is endemic in non-Western countries as well as in the West and may play a role in cervical carcinogenesis in diverse racial groups. PMID- 3021106 TI - Subcutaneous clostridial infection in broilers. AB - A flock of 12,500 broilers 36 days of age experienced a sudden increase in mortality. Post-mortem lesions were emphysema, severe enteritis, and a serosanguineous fluid in the subcutaneous tissue of the breast and thighs; there was no evidence of a loss in the integrity of the skin. Clostridium perfringens and C. septicum were isolated from the affected subcutaneous tissue. Histopathological and serological examination indicated previous infection with infectious bursal disease virus. The subsequent immunosuppression and severe enteritis may have permitted the clostridia access to the circulatory system, with localization in the subcutaneous areas of the breast and thighs. Mortality returned to normal 48 hours after potassium penicillin G was administered via the drinking water. PMID- 3021108 TI - Spontaneous rupture of an hepatic adenoma in pregnancy with survival of mother and fetus. AB - A case of spontaneous rupture of an hepatic adenoma during pregnancy is reported with survival of both mother and baby. The mother had previously taken the contraceptive pill for 9 years. Attention is drawn to the relationship between the contraceptive pill and the previously rare condition of benign liver adenoma, the need for prompt recognition, and for an intensive team effort in the management of rupture of this neoplasm. PMID- 3021109 TI - Schizophrenia as a disorder of cerebral state transition. AB - This paper offers a speculative consideration of the schizophrenic process in the light of recent findings concerning the wave nature of electrocortical activity. These findings indicate that changes of brain state can be described in the terminology of finite-state machines, and both the instantaneous states and the state transitions can be specified. It is suggested that the mental phenomena of schizophrenia may be reducible to events (some specific type of instability) which could be observed by appropriate analytic techniques applied to EEG. Present empirical EEG findings in schizophrenics are reviewed in this light, and the role of dopamine blockade in treatment is also considered. PMID- 3021110 TI - The influence of neurotensin, naloxone, and haloperidol on elements of excessive grooming behavior induced by ACTH. AB - Naloxone, haloperidol, and neurotensin suppress ACTH-induced grooming. The suppressive effects of naloxone and of haloperidol on ACTH-induced grooming are observed following subcutaneous as well as intracerebroventricular administration. The suppression of ACTH-induced grooming by these drugs is not accompanied by a change in the relative distribution of grooming elements. From previous data and from the results of the present study it is suggested that the underlying substrate involved in ACTH-induced excessive grooming may differ from that of bombesin-induced grooming. PMID- 3021112 TI - Genetic analyses of the roles of umuDC and mucAB in mutagenesis. PMID- 3021111 TI - Opioid peptidergic systems modulate the activity of beta-adrenergic mechanisms during memory consolidation processes. AB - Post-training administration of the opioid receptor antagonist naloxone (0.1 mg/kg) facilitated 48-hr retention, in mice, of a one-trial step-through inhibitory avoidance response. The naloxone-induced memory facilitation was blocked in animals given the selective brain-noradrenergic neurotoxin DSP4 (N-(2 chloroethyl)-N-ethyl-2-bromobenzylamine) (50.0 mg/kg, ip) 7 days before training. Pretreatment with the norepinephrine-uptake inhibitor desmethylimipramine (10.0 mg/kg, ip, 30 min), but not with the serotonin-uptake inhibitor fluoxetine (5.0 mg/kg, ip, 30 min), prevented this antagonism. The simultaneous administration of the central beta-adrenoceptor blocker l-propranolol (2.0 mg/kg, ip), also blocked the effects of naloxone on memory. The effects of naloxone were not blocked by d propranolol (2.0 mg/kg, ip), the peripheral beta-adrenoceptor blocker sotalol (2.0 mg/kg, ip), the alpha-adrenoceptor blocker phenoxybenzamine (10.0 mg/kg, ip), or the predominantly peripheral alpha-adrenoceptor blocker phentolamine (10.0 mg/kg, ip). These findings suggest that central beta-adrenergic mechanisms are involved in the effects of naloxone on memory. Naloxone (0.1 mg/kg, ip) potentiated the effects of the central beta-adrenoceptor agonist clenbuterol (0.001-1.00 mg/kg, ip), which, when administered alone, facilitates or impairs retention as a function of the dose injected. The simultaneous administration of beta-endorphin (0.1 micrograms/kg, ip) exerted effects opposite to those elicited by naloxone, that is, shifted the dose-response curve of clenbuterol to the right. Considered together, these findings are consistent with the view that the facilitatory action of naloxone on memory results from the release of central beta-adrenergic mechanisms from an inhibition induced by opioid peptides released during or immediately after training. PMID- 3021113 TI - Mechanisms of spontaneous mutagenesis: clues from altered mutational specificity in DNA repair-defective strains. PMID- 3021114 TI - Dietary promoters and antipromoters. PMID- 3021115 TI - [Detection of combined hydrocyanic acid-carbon monoxide poisoning in indoor fires]. PMID- 3021116 TI - [Thin layer chromatographic analysis of the lipoid content of smokers' cells]. PMID- 3021117 TI - [Occupational therapy after-care in distal radius fractures]. PMID- 3021118 TI - [Effect of maternal antibodies on the vaccination of young swine against porcine parvovirus (PPV)]. PMID- 3021120 TI - Induction of vascular relaxation by hydroperoxides. AB - Hydrogen peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, and 3 chloroperoxybenzoic acid (CPB) and 15-HPETE relaxed, in a concentration dependent manner rat aortic rings contracted with PGF2 alpha (1 X 10(-5)). Relaxation is not inhibited by either indomethacin (2 X 10(-5) M), a cyclo-oxygenase inhibitor or eicosatetraynoic acid (1 X 10(-5) M), a dual cyclo-oxygenase and lipoxygenase inhibitor. Rings with intact endothelium relaxed to a greater degree on exposure to CPB and 15-HPETE. Methylene blue, a soluble guanylate cyclase inhibitor (1 X 10(-5) M) blocked the relaxation elicited by the five peroxides, whereas both superoxide dismutase (scavenger of superoxide anion) and mannitol (scavenger of hydroxyl radical) have no effect. We conclude that relaxation of vascular smooth muscle is a general property of peroxides and that the endothelium may in some instances facilitate this effect. PMID- 3021119 TI - Glucocorticoids and cyclic AMP synergistically regulate the abundance of preproenkephalin messenger RNA in neuroblastoma-glioma hybrid cells. AB - Regulation of preproenkephalin gene expression was studied in NG108-15 neuroblastoma-glioma hybrid cells. Untreated cells contain 20-120 fg preproenkephalin mRNA per microgram cellular RNA. Treatment of cells with a glucocorticoid (e.g. dexamethasone) for 24 hr or 8 days elevated the abundance of this mRNA to 3 or 9 times the control, respectively. Treatment with 8-bromo cyclic AMP or an adenylate cyclase activator such as prostaglandin E1 or forskolin elevated preproenkephalin mRNA to twice the control or less. Treatment with both glucocorticoid and forskolin for 24 hr or 8 days markedly increased preproenkephalin mRNA to 5-8 and 30 times the control, respectively. Intracellular Met-enkephalin immunoreactivity was increased in parallel with the mRNA abundance. The results demonstrate that preproenkephalin gene expression is synergistically regulated by glucocorticoids and cAMP. PMID- 3021121 TI - Identification of a protease gene of human T-cell leukemia virus type I (HTLV-I) and its structural comparison. AB - We determined the nucleotide sequence of a region between the gag and pol genes of a replication-competent proviral clone of a human T-cell leukemia virus type I (HTLV-I) from MT-2 cells. This region overlapping the gag and pol genes contains an open reading frame with a different phase from others. The deduced amino acid sequences show significant homology with the known protease gene of other retroviruses, and harbors highly conserved amino acid sequences that are well conserved in other retroviral protease domains. These results indicate that this open reading frame encodes a HTLV-I protease. PMID- 3021123 TI - Presence of an insulin-stimulated serine kinase in cell extracts from IM-9 cells. AB - Insulin responsive protein kinase activities of wheat germ purified glycoproteins were examined. Glycoproteins were first incubated without or with insulin, and then exposed to a serum containing antibodies to insulin receptor. Thereafter, both immunoprecipitates and supernatants were studied for their kinase activity toward histone. Incubation with anti receptor antibodies promoted insulin receptor beta subunit and histone phosphorylation. More important insulin receptor depleted extract contained a kinase activity toward histone, that was increased by preincubation with insulin. This stimulation was observed only when insulin was added before the immunoprecipitation of insulin receptors. Alkali treatment and phosphoamino acids analysis revealed that the kinase activity remaining in the supernatant is serine specific. These findings suggest, that a serine kinase activity is associated with the insulin receptor, that it can be separated from the insulin receptor with anti receptor antibodies, that the serine kinase is activated by the hormone-receptor complex. PMID- 3021122 TI - 1,25-Dihydroxyvitamin D3 inhibits the clonogenic growth of transformed cells via its receptor. AB - Anchorage-independent growth in soft agar is a unique property of transformed cells which is known to be correlated with tumorigenicity. We report here that 1,25-dihydroxyvitamin D3 suppresses colony formation by a number of cultured cancer cell lines in soft agar in a dose dependent manner with an ID50 of 5-7 X 10(-10) M. This effect is also achieved with analogues of 1,25-dihydroxyvitamin D3 in accordance with their binding affinity for the hormone's receptor. Only cells with 1,25-dihydroxyvitamin D3 receptor protein are inhibited in their colony formation by vitamin D analogs indicating that the hormone receptor complex may be integrally involved in the in vitro suppression of the anchorage independent phenotype. PMID- 3021124 TI - Reversible activation and inactivation of phosphofructokinase from Ascaris suum by the action of tissue-homologous protein phosphorylating and dephosphorylating enzymes. AB - In the presence of ATP-Mg2+, purified phosphofructokinase from Ascaris suum muscle was effectively phosphorylated and activated in vitro by a protein kinase purified from the same tissue. Both effects were reversed by the action of a purified protein phosphatase from the same tissue. The findings suggest the presence of a highly potent interconversion mechanism for phosphofructokinase in the muscle of the parasitic nematode. PMID- 3021125 TI - Phorbol esters down-regulate protein kinase C in rat brain cerebral cortical slices. AB - The effect of phorbol esters on cyclic AMP production in rat cerebral cortical slices was studied using a prelabelling technique to measure cyclic nucleotide accumulation. Cholera toxin-stimulated cyclic AMP accumulation was enhanced approximately 2-fold by phorbol 12-myristate, 13-acetate (PMA) which alone had no effect on cyclic AMP production. The augmentation by PMA was maximal within the first hour of incubation, decreasing progressively thereafter. Protein kinase C activity was decreased 80-90% during a 3 hr exposure to PMA, as was 3H-phorbol 12,13-dibutyrate binding. Both phosphatidyl serine and arachidonic acid were found to enhance protein kinase C activity in a concentration-dependent manner, an effect that was attenuated by prolonged incubation of the brain tissue with PMA. The results indicate that exposure of brain slices to phorbol esters causes a down-regulation of rat brain protein kinase C, and that this modification corresponds with a decrease in the ability of PMA to augment cyclic AMP production, suggesting a functional relationship between the two systems in rat brain. PMID- 3021126 TI - Computer-based search for steroid and DNA binding sites on estrogen and glucocorticoid receptors. AB - The primary amino acid sequences of proteins that are receptors for estrogen, glucocorticoids, and ouabain were compared with each other using computer programs designed to detect and quantify similarities between proteins. Three regions of similarity between the estrogen receptor (ER) and the glucocorticoid receptor (GR) were identified. On the ER, residues 173-250, 323-395, and 426-458 are similar to residues 409-486, 540-612, and 644-676, respectively, on the GR. The ALIGN computer analysis of these segments on the ER and the GR gave comparison scores that were 16.8, 13.7, and 6.8 standard deviations higher, respectively, than that obtained with a comparison of randomized sequences of these proteins. The probability of getting these scores by chance is less than 10(-60), 10(-40), and 10(-11), respectively. Others have proposed that the segment on the ER and GR that is nearest their amino terminus (e.g. residues 173 250 of the ER) is part of their DNA binding domain and that the other two similar segments on each receptor, which are closer to their carboxy terminus, are part of their steroid binding domain. Here, we present evidence to support both of these hypotheses. First, an Align computer analysis indicates that residues 323 395 of the ER and residues 570-612 of the GR contain a region that is similar to a part of the alpha-subunit of the (Na+ + K+)ATPase that is hypothesized to bind the steroid ouabain. This similarity provides additional support for the proposed location of the steroid binding site on the ER, GR, and (Na+ + K+)ATPase. Second, a computer search of the protein sequence database revealed that protamine, a DNA binding protein, has some similarity to residues 255-281 of the ER, which are thought to be part of the DNA binding domain in the ER. Further, we find that residues 276-281 of the ER contain a structure that has been found at the nucleotide binding domain of some protein kinases. If this region on the ER binds ATP, then it may be involved in phosphorylation/dephosphorylation of the ER, which is thought to be important in its mechanism of action. PMID- 3021127 TI - The role of superoxide radicals in lactoperoxidase-catalysed H2O2-metabolism and in irreversible enzyme inactivation. AB - Irreversible inactivation of lactoperoxidase in the presence of excess H2O2 has been investigated. Serial overlay absorption spectra of the Soret region show that the rate and total amount of enzyme inactivation depend on the proton concentration. Perhydroxyl or superoxide radicals (HO.2 or O-2) cannot be established as the inactivating species in this mechanism, but they influence the rate of reconversion of the intermediate lactoperoxidase-compound III back to the resting ferric form of the enzyme. PMID- 3021128 TI - Characterization of the defective calpain-endogenous calpain inhibitor system in erythrocytes from Milan hypertensive rats. AB - In mature red cells of rats from Milan normal (MNS) and hypertensive strains (MHS), the soluble Ca2+ dependent neutral proteinase (calpain) is present in similar amounts with identical Mr of 110 kDa and a dimeric structure composed of two unequal subunits of Mr of 84 and 26 kDa. Conversely, the amount of the endogenous inhibitor is now confirmed by analysis of the specific activity to be approximately 10 times less in red cells of MHS rats. The inhibitor is present in red cells of both strains in three different oligomeric forms of Mr of 240, 120 and 64 kDa. This last molecular species corresponds to the single basic constituent subunit which is the reacting inhibitor form. The apparent equilibrium between the three oligomeric structures is Ca2+-dependent. The high (0.1 mM) Ca2+ requirement for the activity of calpain from erythrocytes of both strains is reduced to 1-5 microM in the presence of plasma membrane phospholipids. Activation of the enzyme in these conditions is prevented by the natural inhibitor. These results strongly support and further emphasize the hypothesis that the structural and functional abnormalities in MHS rats red cells result from an impairment in the modulation of intracellular calpain activity by interaction with its endogenous inhibitor. PMID- 3021129 TI - Fucosterol decreases angiotensin converting enzyme levels with reduction of glucocorticoid receptors in endothelial cells. AB - The modulation of angiotensin converting enzyme (ACE) levels was studied using fucosterol, one of phytosterols, in cultured bovine carotid endothelial cells. Addition of fucosterol to the culture medium resulted in the decrease of ACE activity of endothelial cells; however, fucosterol did not directly inhibit ACE activity. Dexamethasone elevated the levels of ACE in normal cells, but this effect was not seen in the fucosterol-treated cells. Receptor assays showed that the amount of glucocorticoid receptors in fucosterol-treated cells decreased to an undetectable level. These results indicate that fucosterol lowers the ACE levels on the endothelial cells by inhibiting the synthesis of glucocorticoid receptors involved in the regulation of ACE levels. PMID- 3021130 TI - Interleukin 1 alpha mRNA in virus-transformed T and B cells. AB - IL-1 alpha cDNA clone was isolated from a T cell line infected by the human T lymphotropic retrovirus type-I (HTLV-I/ATLV). We found significant amounts of mRNA hybridizing to IL-1 alpha cDNA not only in HTLV-I-transformed T cells but also in Epstein-Barr Virus-transformed B cells. A part of IL-2 receptor inducing activity in Adult T cell leukemia (ATL) cell line seems to be due to IL-1 alpha. PMID- 3021131 TI - Isolation of angiotensin-converting enzyme inhibitor from porcine plasma. AB - An inhibitory peptide of angiotensin-converting enzyme was purified from porcine plasma. The final preparation showed IC50 values of 4.2, 0.6, and 0.9 microgram/ml for angiotensin-converting enzymes from guinea pig serum, rabbit lung, and monkey brain, respectively. The amino acid sequence of the inhibitor was determined as leucyl-valyl-leucine by Edman procedure. This structural observation was supported by mass spectrometric analysis. PMID- 3021132 TI - Specific binding of estrogen receptor to sites upstream and within the transcribed region of the chicken ovalbumin gene. AB - By means of the DNA-cellulose competitive binding assay, the interaction of estrogen receptor complexed to 17 beta-estradiol with fragments of a cloned DNA region of the estrogen responsive chicken ovalbumin gene spanning from 1343 bps upstream to 373 bps within the transcribed region of the gene (p0V 1.7) was investigated. Only DNA fragments including either the 5'-flanking region from -21 to -140 bps or the region within the gene from +41 to +143 bps showed binding affinity for the estrogen receptor higher than calf thymus DNA. DNA fragments from human alpha 1-globin gene and glucocorticoid responsive murine mammary tumor provirus corresponding to the same DNA region investigated for ovalbumin showed affinity for the estrogen receptor no higher than that of calf thymus DNA. These results suggest that two specific binding sites for estrogen receptor are located upstream and within the ovalbumin gene, near the start-site of transcription. These receptor binding sites overlap with the 'estrogen response element' identified by Dean et al. (1) and the DNase I Hypersensitive region I found by Kaye et al. (2). PMID- 3021133 TI - Modification of brain fructose-1,6-bisphosphatase activity by chelators: "induction" of 5'-AMP sensitivity. AB - Brain fructose-1,6-bisphosphatase (EC 3.1.3.11) from various sources are ordinarily insensitive to 5'-AMP. In addition to stimulation and conferring a "neutral" behaviour, prior treatment with histidine, EDTA or imidazole renders the brain enzyme sensitive to 5'-AMP. The histidine treated enzyme(s) bind to Blue-Sepharose CL-6B column and are specifically eluted by 5'-AMP in contrast to the untreated enzyme(s) which do not bind to the affinity column at all. The histidine effect in inducing 5'-AMP sensitivity was abolished by treatment of the native enzyme by subtilisin or by a number of divalent cations including Zn++. PMID- 3021134 TI - Calcium uptake by intracellular compartments in permeabilised enterocytes. Effect of inositol 1,4,5 trisphosphate. AB - Treatment of rat small intestine with EDTA produced isolated enterocytes with plasma membranes which were permeable to small ions. When resuspended in a medium designed to resemble the intracellular medium, Ca2+ was accumulated into the cells. Both mitochondrial and a non-mitochondrial (presumably endoplasmic reticulum) compartments were responsible for sequestering the cation, as indicated by the effects of the mitochondrial inhibitors oligomycin and antimycin and of the Ca-ATPase inhibitor sodium orthovanadate assayed at low (0.9 microM) and high (12 microM) free Ca2+ concentrations. Addition of inositol (1,4,5) trisphosphate induced a rapid release of Ca2+ from the non mitochondrial compartment. The effect of inositol trisphosphate was concentration dependent and showed 50% of maximal release at 2 M. Neither cyclic AMP nor dibutyryl cyclic AMP caused release of Ca2+. These findings lend novel support to the possibility that Ca-mediated control of ionic transport in the small intestine is exerted through the phosphatidylinositol-protein kinase C transduction mechanism. PMID- 3021136 TI - Characterization of the soluble hydrogenase from Desulfovibrio africanus. AB - The soluble hydrogenase from Desulfovibrio africanus has been isolated and characterized. The enzyme consists of two subunits of 65 kDa and 27 kDa. Its absorption spectrum is typical of an iron-sulfur protein. The protein contains 12 iron atoms, 10 labile sulfur atoms and 0.9 nickel atom per molecule. D. africanus hydrogenase is rapidly activated under reducing conditions and exhibits a specific activity of 570 mumoles H2 evolved/min/mg. The EPR spectrum of the oxidized enzyme shows no Ni(III) signals. Upon reduction under hydrogen, the protein sample exhibits signals due to nickel with g values at 2.21, 2.17 and 2.01 correlating with the active state of the enzyme. PMID- 3021135 TI - Regulation of ferritin synthesis in malignant and non-malignant lymphoid cells. AB - The different amounts of H-rich and L-rich isoferritins found in malignant and non malignant lymphoid cells are accompanied by proportional variations in the relative quantity of messenger RNAs for the H and L subunits of ferritin. The correlation between levels of messenger RNAs and proteins suggests that the amount of messenger RNA plays an important role in ferritin biosynthesis in these cells. The enhanced expression of ferritin messenger RNAs in some neoplastic cells is not caused by gross alterations in the structure of ferritin genes. PMID- 3021137 TI - Effect of tolbutamide on fructose-6-phosphate,2-kinase and fructose-2,6 bisphosphatase in rat liver. AB - The effects of tolbutamide on the activities of fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase were examined using rat hepatocytes. Tolbutamide stimulated fructose-6-phosphate,2-kinase activity and inhibited fructose-2,6 bisphosphatase activity, resulting in an increase of fructose-2,6-bisphosphate level. Changes in the activities of the enzyme by tolbutamide were due to variation in the Km value, but not dependent on alteration of Vmax. Glucagon inhibition of fructose-2,6-bisphosphate formation resulting from an inactivation of fructose-6-phosphate,2-kinase and an activation of fructose-2,6-bisphosphatase was released by tolbutamide. Tolbutamide stimulation of fructose-2,6-bisphosphate formation through regulation of fructose-6-phosphate,2-kinase/fructose-2,6 bisphosphatase may produce enhancement of glycolysis and inhibition of gluconeogenesis in the liver. PMID- 3021138 TI - Type beta transforming growth factor is a potent modulator of differentiated adrenocortical cell functions. AB - Whereas TGF-beta exhibited no detectable effect on DNA synthesis, it was found to exert a striking inhibitory effect on the steroidogenic activities of bovine adrenocortical cells in culture. Basal, as well as ACTH- and angiotensin II- activated adrenocortical cortisol productions were inhibited in a time and dose dependent manner following TGF-beta treatment. Half-maximum inhibition of ACTH- and AII-activated steroidogenesis was observed with TGF-beta concentrations of 0.40 and 0.12 ng/ml, respectively. This effect was half maximal after 6 hours of cell exposure to optimally effective TGF-beta concentrations (1 ng/ml) and reached a plateau after 12-15 hours, resulting in an average 60% inhibition in the steroidogenic response to ACTH and 90% in the case of AII. Supply of different exogenous steroid substrates to support steroidogenesis in adrenocortical cells pointed to a marked loss in steroid-17 alpha hydroxylase activity as a major alteration following TGF-beta treatment. TGF-beta thus appears as a potent modulator of differentiated adrenocortical cell functions in vitro; in this regard it may play a significant role in the development and the regulation of adrenal cortex in vivo. PMID- 3021139 TI - Beta thalassemia due to a novel mutation in IVS 1 sequence donor site consensus sequence creating a restriction site. AB - During a systematic screening of Algerian thalassemics by determining the DNA polymorphism haplotypes in the beta globin gene cluster, a novel haplotype was identified. The DNA of a homozygous individual was cloned and sequenced. The mutation, a G----A change, at position 5 of the small intervening sequence, probably interferes with normal splicing events, and, moreover, creates a new Eco RV restriction site that provides a useful diagnostic tool for detecting this condition. PMID- 3021140 TI - Solubilization of the iron molybdenum cofactor of Azotobacter vinelandii nitrogenase in dimethylformamide and acetonitrile. AB - The iron molybdenum cofactor of Azotobacter vinelandii nitrogenase has been solubilized for the first time in dimethylformamide and acetonitrile. These solutions have the ability to reconstitute the inactive nitrogenase of the UW 45 mutant of A. vinelandii and exhibit an S = 3/2 EPR signal similar to that for the cofactor in N-methylformamide. Our ability to obtain solutions of FeMoco in these solvents seemingly refutes a previous hypothesis concerning the necessity of solvents with a dissociable proton for iron molybdenum cofactor solubility and should facilitate the spectroscopic characterization of this important species. PMID- 3021141 TI - 1-amino-3,7,8-trichlorodibenzo-p-dioxin: a specific antagonist for TCDD-induced myelotoxicity. AB - It was recently reported that suppression of murine bone marrow hematopoiesis is a very sensitive indicator for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity (1). We report here that a structural analog of TCDD, 1-NH2-3,7,8 trichlorodibenzo-p-dioxin (NH2-TriCDD), is a specific and effective antagonist for TCDD-induced myelotoxicity and enzyme induction. When administered to mice or added directly into culture at a 100-fold excess, relative to TCDD, NH2-TriCDD completely abrogated the ability of TCDD to inhibit granulocyte-macrophage progenitor cells (CFU-C) formation, an indicator of hematopoiesis. Further, NH2 TriCDD inhibited TCDD-induced activation of cytochrome P1-450 monooxygenase activity. Studies designed to measure specific binding of TCDD to the cytosolic Ah receptor indicated that NH2-TriCDD effectively inhibited binding of TCDD to the receptor by acting as a competitive antagonist (Ki = 0.72 nM). PMID- 3021142 TI - Generation of transgenic mice producing a human transthyretin variant: a possible mouse model for familial amyloidotic polyneuropathy. AB - Type I familial amyloidotic polyneuropathy (FAP) results from the systemic deposition of a plasma transthyretin (TTR) variant with a Val----Met change at position 30. In an attempt to establish a model of this disease, we generated transgenic mice producing the variant TTR. A DNA fragment containing the mouse metallothionein-I promoter fused to the structural gene coding for the human TTR variant was microinjected into fertilized mouse eggs. Among 72 mice that developed from these eggs, ten carried the fusion gene and three of these showed significant concentrations of the variant TTR in their serum. These mice may be useful in elucidating the pathogenesis of FAP and in establishing a therapy for this intractable disorder. PMID- 3021143 TI - Transforming growth factor-beta regulates the expression of luteinizing hormone receptors in ovarian granulosa cells. AB - In cultured granulosa cells, addition of 1 to 50 ng follicle-stimulating hormone induced a 350-fold rise in luteinizing hormone receptors, while larger amounts of gonadotropin up to 200 ng reduced these receptors to approximately 50% of peak levels. Transforming growth factor-beta (16 pM) enhanced the stimulatory actions of low levels of gonadotropin (2.5-10 ng) by 2 to 3-fold, and inhibited the induction of luteinizing hormone receptors by higher levels of follicle stimulating hormone (greater than or equal to 50 ng) by 30-50%. The actions of the growth factor were concentration-dependent over the range from 0.8 to 16 pM and included a similar biphasic effect upon gonadotropin-induced cAMP production. Modulation of cAMP formation and luteinizing hormone receptor expression by transforming growth factor-beta could influence the ability of the granulosa cell to respond to luteinizing hormone during ovarian follicular maturation and ovulation. PMID- 3021144 TI - Electron transfer reaction of cytochrome c peroxidase at tin oxide electrodes. AB - The unmediated one-electron reduction and reoxidation of ferric cytochrome c peroxidase at fluoride-doped tin oxide electrodes is reported. A long range interfacial electron transfer distance of 17 A is postulated by analogy to the Poulos/Kraut model for the cytochrome c peroxidase/cytochrome c electron transfer complex. The utility of the interfacial electrochemical approach for investigating cytochrome c peroxidase electron transfer behavior is discussed. PMID- 3021145 TI - Effect of phosphorylation by different protein kinases on the behaviour of glycogen synthase as a substrate for hepatic synthase phosphatases. AB - Glycogen synthase a from skeletal muscle was phosphorylated in vitro and then used as substrate for the two major synthase phosphatases from liver. Synthase phosphorylated by cAMP-dependent protein kinase (1.4-1.7 P/subunit) was preferentially activated by the cytosolic S-component; in contrast, progressive phosphorylation by casein kinase-1 (0.9-6.5 P/subunit) yielded substrates that were always better dephosphorylated and activated by the glycogen-bound G component. We have previously isolated from dog liver several types of synthase b that differ by their need for the S- and/or G-component for prompt activation. After additional phosphorylation by a mixture of synthase kinases the activation of these enzyme preparations required the presence of both components. PMID- 3021146 TI - Periportal and perivenous hepatocytes retain their zonal characteristics in primary culture. AB - Periportal and perivenous hepatocytes from rat liver were isolated by combined digitonin-collagenase perfusion, and gluconeogenesis, urea synthesis and fatty acid synthesis was measured both in freshly isolated cells and in primary culture. A periportal zonation of gluconeogenesis and urea synthesis of about 3 and 1.5 fold, respectively, was observed. This zonation persisted unchanged for 23 hours in culture under identical conditions of incubation for periportal and perivenous cells. Fatty acid synthesis was not zonated. PMID- 3021148 TI - Superoxide photogeneration by chlorophyll a in water/acetone. Electron spin resonance studies of radical intermediates in chlorophyll a photoreaction in vitro. AB - In this paper we report the use of DMPO (5,5-dimethyl-1-pyrroline-1-oxide) as spin trap in the ESR observation of O2-. photogeneration by in vitro Ch1 a in oxygen-saturated 50:50% (v/v) water/acetone. The observed hyperfine parameters for the spin adducts of DMPO are identical to those obtained from H2O2 decomposition, and to those reported by earlier workers for the formation of 02-. in oxygen-saturated preparations of spinach chloroplasts. PMID- 3021147 TI - mRNA from human colon tumor and mucosa related to the pol gene of an endogenous A type retrovirus. AB - The expression of endogenous retroviral genes in mammals may be etiologically related to genetic diseases including cancer. Recently, A-type human endogenous retroviral genomes have been cloned using the reverse transcriptase (pol) genes of rodent intracisternal A-particles (IAP). In this report, RNA from human colon adenocarcinoma and surrounding mucosa was hybridized to mouse IAP pol and gag genes to examine the expression of human endogenous A-type retroviruses. Abundant, heterogeneous size, polyadenylated transcripts homologous to the mouse IAP pol gene were detected in both tissues. Transcripts homologous to the mouse IAP gag region were not found. PMID- 3021149 TI - Induction of glucose 6-phosphatase in cultured hepatoma cells by dexamethasone. AB - The synthetic glucocorticoid, dexamethasone, induced high levels of glucose 6 phosphatase in a line of cultured hepatoma cells (2S FAZA). This conflicts with current theory that glucocorticoids induce a microsomal translocase, specific for glucose 6-phosphate, but not the hydrolase itself. The earlier conclusion was based on experiments with rat livers in vivo in which cortisone was the inducing agent. In the present study, 5 X 10(-6) M dexamethasone induced a tenfold increase in glucose 6-phosphatase activity in "intact" as well as disrupted microsomes using either glucose 6-phosphate or mannose 6-phosphate as substrate. This induction was blocked by cycloheximide (50 micrograms/ml). The broad pH maxima between 5.5 and 7.0 were similar for both "intact" and disrupted microsomes. PMID- 3021150 TI - Cleavage site specificity of vertebrate collagenases. AB - A series of synthetic peptide substrates for vertebrate collagenase having the structure Ac-Pro-Leu-Gly-X-Leu-Gly-OC2H5, where X is Leu, Ile, Val, Phe and Ala, have been prepared. Collagenolytic enzymes from various sources cleave these substrates with differing relative rate patterns. This series of peptides should be valuable for characterization of collagenases. PMID- 3021151 TI - Parathyroid hormone secretion does not respond to changes of free calcium in electropermeabilized bovine parathyroid cells, but is stimulated with phorbol ester and cyclic AMP. AB - The secretion of parathyroid hormone (PTH) is suppressed in bovine parathyroid cells by raised extracellular [Ca2+], and 12-0-tetradecanoyl-phorbol-13-acetate (TPA) stimulates the release of PTH from cells suppressed by high extracellular [Ca2+]. Extracellular and cytosolic free [Ca2+] are proportionally related in intact cells. To assess the role of cytosolic free [Ca2+] on PTH secretion, bovine parathyroid cells were rendered permeable by brief exposure to an intense electric field. PTH secretion was comparable at 40 nM, 500 nM, 5 microM, 28 microM, 0.5 mM and 2 mM [Ca2+] (release of total cellular PTH 3.7 +/- 0.5%, 3.9 +/- 0.4%, 3.4% +/- 0.3%, 3.9 +/- 0.4%, 3.1 +/- 0.3%, 3.5 +/- 0.7%, respectively), but the secretion was stimulated twofold (P less than 0.05 vs. control) in a dose and ATP dependent manner with TPA (100 nM) and cyclic AMP (1 mM). As a result, free [Ca2+] in the range of those observed in intact cells during regulation of PTH secretion by changes of extracellular [Ca2+] did not affect the release of PTH in permeabilized cells. The [Ca2+] independent stimulation of PTH release by TPA and cyclic AMP indicates that changes of cytosolic free [Ca2+] may represent a secondary event not related to the regulation of PTH secretion. PMID- 3021152 TI - Inhibition of inositol phospholipids metabolism and calcium mobilization by cyclic AMP-increasing agents and phorbol ester in neutrophils. AB - Cyclic AMP-increasing agents such as PGE2 and dibutyryl cAMP inhibited the fMLP induced inositol phospholipids metabolism mainly through the suppression of the conversion of phosphatidylinositol(PI) to phosphatidylinositol 4,5 bisphosphate(PIP2). A part of this inhibition was found to be caused by the inhibitory effect of cAMP on PI kinase using isolated plasma membranes. On the other hand, 12-O-tetradecanoyl phorbol acetate(TPA) mainly inhibited the conversion of phosphatidylinositol 4-phosphate(PIP) to PIP2 without a significant effect on the fMLP-induced breakdown of PIP2, though direct effect of TPA on PI and PIP kinases was not demonstrated in isolated plasma membranes. Concerning Ca2+ mobilization, both cAMP-increasing agents and TPA inhibited the fMLP-induced second phase of Ca2+ elevation, while they did not affect the first phase of Ca2+ rapid increase. However, Ca2+ ionophore ionomycin-induced Ca2+ elevation was only inhibitable by TPA but not PGE2. These results suggest that cAMP inhibits the fMLP-induced Ca2+ influx, while TPA stimulates Ca2+ removal from cytosol. PMID- 3021153 TI - 1,25-Dihydroxyvitamin D3 enhances the growth of tumors in athymic mice inoculated with receptor rich osteosarcoma cells. AB - We tested the influence of daily subcutaneous injections of 12.5 and 25 pmol of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the growth of tumors arising from intracutaneous inoculations of athymic nude mice with rat osteogenic sarcoma cells (ROS) and human melanoma cells. Both doses of 1,25(OH)2D3 increased plasma calcium levels after 3 weeks and produced a striking enhancement in tumor weight when the mice received 1,25(OH)2D3 receptor-rich ROS17/2.8 cells. In contrast, 1,25(OH)2D3 caused no consistent effect on tumor weight in mice given G-361 melanoma cells with low receptor copy number or receptor deficient ROS 24/1 cells. Thus, 1,25(OH)2D3 stimulated tumor growth in a receptor dependent fashion, in vivo, instead of inhibiting it as predicted from the reduction of proliferation of cultured cells in the presence of 1,25(OH)2D3. PMID- 3021154 TI - Methylation of the c-myc gene changes during aging process of mice. AB - The degree of methylation at c-myc proto-oncogene was found to change during aging process of mice by the use of methylation-sensitive restriction enzymes. The spleen DNA showed hypomethylation as mice aged, while hypermethylation was observed in the liver DNA. The brain DNA on the contrary revealed no appreciable difference between young and old mice. When the DNAs were examined at actin and dihydrofolate reductase (DHFR), no significant change was observed. It suggests that an age-related change of oncogene structure may be one of the factors which are related to an age-associated increase of cancer incidence rate. PMID- 3021155 TI - (Na+ +K+)-ATPase activity in kidney basolateral membranes of non insulin dependent diabetic rats. AB - Insulin resistant, Type II diabetes mellitus (NIDD) in a rat animal model results in profound changes in basal and insulin-stimulated membrane (Ca2+ +Mg2+)-ATPase activity in kidney basolateral membrane (BLM) preparations. We find that NIDD in these animals does not result in similar changes in membrane (Na+ +K+)-ATPase activity. Basal enzyme activity was the same in diabetic and control animals. Insulin treatment of diabetic animals in vivo resulted in hyperinsulinemia and increased BLM (Na+ +K+)-ATPase, while food restriction for 18 hr resulted in lowered enzyme activity. There was no direct effect of insulin on (Na+ +K+) ATPase activity in isolated membranes from any of the animal groups. Thus, physiologic perturbations which alter insulin sensitivity and glucose homeostasis are accompanied by altered levels of (Na+ +K+)-ATPase activity. Lower levels of this membrane enzyme activity appear to be associated with optimal insulin action. PMID- 3021156 TI - Reconstitution of liver monooxygenase system in solution from cytochrome P-450 and NADPH-specific flavoprotein monomers. AB - Microsomal monooxygenase system was reconstituted in the presence of non-ionic detergent Emulgen 913 from cytochrome P-450 and NADPH-specific flavoprotein isolated from phenobarbital-induced rabbit liver microsomes. At Emulgen 913 concentration of 0.05 g/l mixed complex between flavoprotein and cytochrome was formed with 5: 5 protein molar ratio and molecular weight of 700 kD. The 2-hour incubation of the enzymes with 0.25 g/l Emulgen 913 at 4 degrees C was accompanied by dissociation of protein oligomers to monomers. The reconstituted systems containing flavoprotein and cytochrome as mixed complexes or monomers were able to catalyze NADPH-dependent cytochrome P-450 reduction and benzphetamine N-demethylation. Taking into consideration the effective concentrations of the enzymes the apparent second order rate constants of these reactions with monomers were 100 times those with complexes. PMID- 3021157 TI - Enhanced instability of IncFII basic replicon by the polA mutation. AB - IncFII plasmids consisting of a basic replicon and of an additional fragment(s) unrelated to plasmid maintenance were all less stable in polA1 than in its wild type. Reversion to UV-resistance recovered stability and vice versa. UV irradiation promoted instability in polA1 cells. Larger plasmids showed a greater instability and a fewer number of copies in a same host. Surprisingly, polA1 cells with Tn3 on the plasmid showed a higher ampicillin resistance than the wild type, apparently suggesting that the polA1 mutation increases the copy number. The size-dependency of instability was less marked in polA1 than in its parent. Comparison of the generation times has suggested a detrimental effect exerted by a basic replicon in polA1 hosts. PMID- 3021158 TI - The association of ribulose-1,5-bisphosphate carboxylase with phosphoriboisomerase and phosphoribulokinase. AB - RuBPCase from peas showed Ribose-5-phosphate and Ribulose-5-phosphate dependent CO2 fixation when purified on sucrose gradients or by conventional methods. If purification was done in the presence of 20 mM MgCl2 and 20-25 mM NaHCO3 RuBPCase showed higher Ribose-5-phosphate and Ribulose-5-phosphate dependent CO2 fixation rates. Partially purified phosphoriboisomerase, phosphoribulokinase and RuBPCase from spinach could be reassociated in vitro. PMID- 3021159 TI - Evidence that divalent cations bind to diphtheria toxin: an ESR approach. AB - We have used electron spin resonance (ESR) techniques to study the binding of divalent cations to diphtheria toxin (DT). Addition of DT to Mn(II) solution at a stoichiometry of 2:1 (DT:Mn) induces a 79% loss in the intensity of the ESR spectrum of Mn(II) suggesting a strong binding of Mn(II) to DT. Inclusion of Ca(II) at a ratio of 1:2:1 (Ca:DT:Mn) in the reaction mixture restores the intensity of the Mn(II) signal to 64%. This indicates that Ca(II) and Mn(II) share same binding site(s) in DT. The results presented in this communication suggest that DT is a Ca(II) binding protein. PMID- 3021161 TI - Cellular resistance to vinblastine is associated with altered respiratory function. AB - Human leukaemic T lymphoblasts made resistant to low levels (20-40 ng/ml) of vinblastine have altered respiratory capacity. Cellular oxygen uptake was greater in resistant cells compared with sensitive cells, and vinblastine (40 ng/ml) caused immediate inhibition of oxygen uptake in sensitive cells, but not in resistant cells. Isolated mitochondria reflected the changes observed in the intact cells. Rates of oxidation of cytochrome c, succinate and glutamine were higher in mitochondria from resistant cells and were little affected by challenge with vinblastine, whereas vinblastine at 40 ng/ml was completely inhibitory for sensitive cell mitochondria. Azide inhibited vinblastine efflux from sensitive and resistant cells in both the presence and absence of glucose. Levels of protein, total lipid, free cholesterol and cardiolipin were elevated in vinblastine-resistant lymphoblasts. PMID- 3021160 TI - Opiate receptor binding characteristics of dimeric analogues of mu-selective DAGO enkephalin. AB - DAGO-enkephalin ([ D-Ala2, MePhe4, Gly-ol5]enkephalin), a highly selective ligand for mu opiate receptors, was dimerized with a series of alpha,omega-alkanedioic acids (n = 2-12) at the OH-terminus. In the radioligand receptor binding assays with rat brain, most of the DAGO-enkephalin dimers with cross-linking methylene chain n (DEDn) were more potent than DAGO monomer. For delta receptors, affinity of DEDn was maximized with n = 8, which might be related to an optimal distance between two binding sites. For mu receptors, an increase in chain length resulted in a progressive loss of potency. Although all of DEDn are considerably mu selective, with a mu/delta ratio of 15-50, DEDn exhibited fairly flat binding curves with 15-50% smaller sloped than that of DAGO, suggesting that the dimers interact more strongly with one of the possible two mu binding sites. PMID- 3021162 TI - Receptor-mediated heme uptake from hemopexin by human erythroleukemia K562 cells. AB - Using human erythroleukemia K562 cells, existence of receptors for hemopexin has been investigated. Hemopexin was bound to the cells in saturable, time- and temperature-dependent manner. The cells exhibited approximately 8,400 binding sites/cell for hemopexin and apohemopexin. The dissociation constants (Kd) for hemopexin and apohemopexin were 4.79 nM and 10.8 nM, respectively. Specific binding of labeled hemopexin was inhibited with increasing concentrations of unlabeled hemopexin and apohemopexin, but unaffected by transferrin and serum albumin. Heme bound to hemopexin was incorporated into the cells at 37 degrees C, but not at 4 degrees C. These results indicate that heme in hemopexin was taken up by K562 cells via the receptors for hemopexin. PMID- 3021164 TI - Collagenolytic metalloenzymes of the human neutrophil. Characteristics, regulation and potential function in vivo. PMID- 3021163 TI - A simple assay of the superoxide generation rate with Tiron as an EPR-visible radical scavenger. AB - Tiron (1,2-dihydroxybenzene-3,5-disulfonate) is oxidized to an EPR-visible semiquinone by superoxide radicals produced by xanthine oxidase. The steady-state level of the Tiron radicals increases with an increased xanthine oxidase concentration. A calibration plot has been obtained relating the steady-state concentration of the Tiron semiquinone determined by EPR-spectroscopy to the rate of 0.2 production as measured by the superoxide dismutase-sensitive cytochrome c reduction. This approach allows for a simple and sensitive assay of 0.2 generation rate in biological systems in the range of ca.0.1-4.0 microM/min using Tiron as a spin trap. The rate of 0.2 generation by antimycin-inhibited ischemic rat heart mitochondria has been measured by this method. PMID- 3021165 TI - Methylcholanthrene: a possible pseudosubstrate for adrenocortical 17 alpha hydroxylase and aryl hydrocarbon hydroxylase. AB - In cultured bovine adrenocortical cells, loss of 17 alpha-hydroxylase activity was observed after incubation with 3-methylcholanthrene (3-MC). The suppression of 17 alpha-hydroxylase by 3-MC was rapid (50% loss of activity in 10 hr at 1 microM 3-MC), did not exhibit a lag period, and was not affected by cycloheximide. Direct effects of 3-MC on 17 alpha-hydroxylase were observed only at high concentrations, but the concentration for 50% loss of activity was 0.3 microM when 3-MC was added for 24 hr prior to assay of 17 alpha-hydroxylase. High concentrations (to 40 microM) of substrate (progesterone), did not affect the loss of activity due to 3-MC. Loss of 17 alpha-hydroxylase activity was specific; 11 beta-hydroxylase was unaffected and cell growth was unaltered. However, 22 amino-23,24-bisnorchol-5-en-3 beta-ol, an inhibitor of 17 alpha-hydroxylase, partially prevented the loss of 17 alpha-hydroxylase at 1-30 nM. 3-MC is thought to induce cytochrome P-450s via a receptor with high affinity for 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). TCDD was without effect on 17 alpha hydroxylase over the range of 10 nM to 10 microM. Benz[a]anthracene, 7,12 dimethylbenz[a]anthracene, benzo[a]pyrene, chrysene, and methylphenanthrenes suppressed 17 alpha-hydroxylase at high concentrations (10-50 microM for 50% loss of activity). Some steroids that lack a substituent at position 17 also caused loss of 17 alpha-hydroxylase. Like 17 alpha-hydroxylase, bovine adreno-cortical cell AHH was found to be suppressed by exposure to 3-MC. Compounds that caused loss of 17 alpha-hydroxylase caused loss of AHH, with a similar order of potency and at similar concentrations. Suppression of AHH by 3-MC did not require protein synthesis and was prevented by an inhibitor of enzymatic activity, alpha naphthoflavone. This implies a degree of similarity of the cytochrome P-450s for 17 alpha-hydroxylase and adrenal AHH, but the activities were shown to be likely due to different enzymes. The suppression of 17 alpha-hydroxylase and AHH by 3-MC appears not to occur by a receptor-mediated mechanism but to be similar to the suppression of 11 beta-hydroxylase and 21-hydroxylase by steroid pseudosubstrates previously observed. PMID- 3021166 TI - Origin of differences of inhibitory potency of cardiac glycosides in Na+/K+ transporting ATPase from human cardiac muscle, human brain cortex and guinea-pig cardiac muscle. AB - The inhibitory potency of altogether 95 steroidal compounds (including cardenolides, bufadienolides and their glycosides) on the Na/K-ATPases (Na+/K+ transporting ATPases, EC 3.6.1.37) from human cardiac muscle, human brain cortex and guinea-pig cardiac muscle was compared to probe the complementary chemotopology of the inhibitor binding site areas on the three enzyme variants. The changes of potency, resulting from systematic variations of the geometry of steroid skeleton and the character as well as the structure of side chains at C3 or/and C17 of steroid backbone, allowed the following major conclusions. With the human cardiac and cerebral enzyme forms, the paired K0.5 (K'D) values for 77 steroid derivatives, covering seven orders of ten, were highly correlated. On an average, the total of compounds showed a 1.5-fold higher affinity to the cardiac enzyme. This tiny differentiation did not appear to be connected with an important difference in the chemotopology of the complementary subsites for steroid nucleus binding on the two enzyme forms. With the human and guinea-pig cardiac enzyme variants, the K0.5 values for 69 steroid derivatives, covering six orders of ten, were determined. For 41 5 beta, 14 beta-androstane derivatives only, the paired K0.5 values showed a close correlation. Here, the human enzyme variant exhibited 27-fold higher affinity. However, the paired K0.5 values determined on both enzymes for 28 steroid derivatives of differing structural features were but poorly correlated. Essentially, the geometries of the steroid nucleus determined the differential contributions of the side chains at C3 and C17 to the integral inhibitory potency on the two enzyme variants. Thus, the species differences in the potency of cardiac glycosides were traced to species differences in the complementarity of the steroid binding subsites. Hence, estimates of the potency of new steroidal compounds obtained on the guinea-pig cardiac enzyme can be neither quantitatively nor qualitatively easily extrapolated to the human cardiac enzyme. The extrathermodynamic analysis of the data opened major new insights in the structure-activity relationships concerning the role of C14 beta-OH, the character of the lead structure in cardioactive steroid lactones, and the significance of the configuration of A/B ring junction. PMID- 3021167 TI - Serum albumin and the metabolism of disulfiram. AB - The effectiveness of tetraethylthiuram disulfide (DSF) as a drug used in the treatment of alcohol abuse has been limited by the fact that it is degraded rapidly in the tissues and in the serum. Hence, a useful dose-response curve for this drug cannot be determined easily. The degradation in the tissues has been well characterized; however, its fate in the serum is less well understood. Here we kinetically describe the first steps in the degradation of DSF in the serum which results from a covalent interaction of this drug with the free sulfhydryl of serum albumin. DSF and its cleavage product diethyldithiocarbamate (DDC) both absorb significantly in the ultraviolet region. The reduction of DSF with mercaptoethanol to two molecules of DDC resulted in a large change in absorption in this region. The reaction of serum albumin with DSF produced a similar but much slower change in the ultraviolet absorption. As a result of the existence of this slow spectral change, we have been able to directly and continuously monitor the interaction of serum albumin and DSF and have determined that it is an overall first-order process. A model is proposed wherein DSF and serum albumin rapidly form a noncovalent adduct and, subsequently, in a slow unimolecular process, DSF is reduced to one mole of free DDC and one mole of the serum albumin DDC mixed disulfide. At pH 9 the half-time for this process was 30 to 40 sec, and at pH 7.4 the half-time for this process was 1 to 1.5 min. These results suggest that degradation of DSF by serum albumin is physiologically and clinically important since the drug is maximally active only many hours after administration. PMID- 3021168 TI - Effects of ibuprofen on chemotactic peptide-receptor binding and granulocyte response. AB - Inhibition of complement-mediated granulocyte aggregation has been proposed recently as a mechanism of action of high-dose corticosteroids and ibuprofen in shock states. Such inhibition by corticosteroids may be effected through alteration of receptor function, and we have therefore examined the effect of ibuprofen on the extent and kinetics of binding of the synthetic chemotactic peptide formylmethionine-leucine-phenylalanine (FMLP) to its specific receptor on the granulocyte surface. Dose-dependent inhibition of binding was observed at ibuprofen concentrations paralleling plasma levels achieved with 30 mg/kg intravenous bolus therapy, and also at concentrations achieved with oral therapy. Ibuprofen did not affect the receptor number, but did decrease the association rate constant for the FMLP-receptor interaction (30% of normal for 0.125 mg/ml ibuprofen), leading to a decrease in receptor affinity for ligand. Dissociation kinetics, as determined by cold chase experiments, were unaltered by ibuprofen. We conclude that ibuprofen, like corticosteroids, can slow the rate of association of FMLP with its receptor on the granulocyte surface while allowing dissociation to proceed; altered kinetics of receptor-FMLP interaction may explain the inhibition of granulocyte aggregation. Blockade of granulocyte surface receptors for inflammatory stimuli may be important in the clinical effects of very high-dose corticosteroids and ibuprofen such as are administered in shock; such effects are seen at blood levels of ibuprofen that occur with oral therapy. Similar observations may hold for other physiologic stimuli. PMID- 3021170 TI - Effects of serum-fractions from patients with Eaton-Lambert syndrome on rat cortical synaptosomal [3H]acetylcholine release. PMID- 3021169 TI - Demonstration of cellular retinoic acid binding protein (CRABP) in chick embryo tendon cells and effects of retinoids on collagen synthesis in tendon and sterna. AB - The binding of all-trans-retinoic acid (all-trans RA) to specific cytosol proteins and the effects of retinoids on procollagen synthesis were studied in chick-embryo tendon cells. For the receptor assay, tendon cytosols were incubated with [3H]all-trans-RA in the presence or absence of 100-fold excess of nonlabeled all-trans-RA up to 20 hr at +4 degrees and unbound retinoid was removed by charcoal-dextran treatment or by gel filtration chromatography. The results indicated that chick-embryo tendon cells contained cellular retinoic acid binding protein (CRABP). The binding of [3H]all-trans-RA could be displaced by 13-cis retinoic acid, but not by retinol or etretinate. In contrast no CRABP could be found in cartilage cells isolated from sterna or in whole sterna. The treatment of tendon cytosol with proteases (pronase, trypsin, chymotrypsin) abolished the specific binding of [3H]all-trans-RA. Gel filtration studies on Sephadex G-100 indicated an apparent molecular weight of 14,500-15,000 daltons for the all-trans retinoic acid binding protein. All-trans-RA markedly decreased procollagen synthesis in isolated chick-embryo tendon cells, the inhibition being concentration dependent; the decrease was about 58% of the control in the presence of 10(-5) M all-trans-RA. Similar decrease was noted with 13-cis-RA and etretinate, while retinol was less effective. In isolated cartilage cells the dose of 10(-5) M of all-trans-retinoic acid decreased drastically total protein and collagen synthesis. The mannosylation of procollagen, the conversion of procollagen to collagen and the size of procollagen chains were not significantly affected. The results of the present study indicate that CRABP is not expressed in sterna of chick-embryos, and in contrast high levels of CRABP could be found in tendons. However, retinoids modulated collagen synthesis in both tissues. Thus it is possible that retinoids can affect the metabolism of different collagen types also in clinical use. PMID- 3021171 TI - Respective involvements of high- and low-affinity digitalis receptors in the inotropic response of isolated rat heart to ouabain. AB - High- and low-affinity digitalis receptors coexist in rat cardiac sarcolemma. In this study, their relative involvement in the inotropic effect of ouabain was evaluated on an isolated Langendorff rat heart preparation working under isovolumic conditions at a low external calcium concentration (0.25 mM). This involvement was estimated according to both the development of the inotropic response to ouabain (10(-8)-10(-4)M) and the time course of the washing out of the biological effect. In each phenomenon considered, and whatever the index of inotropy chosen, the high-affinity digitalis receptor (EC50: 1-2 X 10(-8) M) contributed to 25-40% of the maximal inotropy (evoked by 10(-4) M ouabain). Low affinity receptors (EC50: 1-2 X 10(-5) M) accounted for 60-75%. These apparent affinities were identical to those previously determined in sarcolemma isolated from rat heart perfused with 0.25 mM Ca2+. The biphasic effect of ouabain was related to both the inhibition of high- and low-sensitivity Na+, K+-ATPase forms and the corresponding number of ouabain-binding sites occupied. These results support the concept that the Na+, K+-ATPase highly sensitive to ouabain as revealed by lowering calcium is the in vivo manifestation of the high-sensitivity inotropic component. PMID- 3021173 TI - A comparative study on the effects of inhibitors of the lipoxygenase pathway on neutrophil function. Inhibitory effects on neutrophil function may not be attributed to inhibition of the lipoxygenase pathway. AB - The effects of five inhibitors of the lipoxygenase pathway were evaluated on oxygen radical production, degranulation, chemotaxis, leukotriene B4 (LTB4) production by neutrophils. The lipoxygenase inhibitors tested were nordihydroguaiaretic acid (NDGA), esculetin, eicosatetraynoic acid (ETYA), 2-(12 hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoqu inone (AA-861), and 6,9 deepoxy-6, 9-(phenylimino)-delta 6.8-prostaglandin I1 (U-60,257). Neutrophils were activated by n-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), A23187, or platelet activating factor (PAF). The effects of these inhibitors on NADPH oxidase activity and phospholipase A2 activity of isolated particulate fraction of neutrophils were also evaluated. ETYA inhibited neutrophil function induced by all the stimulators except PMA. AA-681 was unique in that it did not inhibit PAF-induced neutrophil activation. U-60,257 had virtually no effect on oxygen radical production and degranulation, but chemotaxis was moderately suppressed. NDGA effectively inhibited neutrophil function, except for chemotaxis. Esculetin inhibited only oxygen radical production, but this was due to inhibition on NADPH oxidase activity of neutrophil membrane. The inhibitory effect on neutrophil function and that of LTB4 production were not closely correlated. It is suggested that lipoxygenase inhibitors may modify neutrophil function by the mechanism not involving the lipoxygenase pathway. It is also suggested that LTB4 may not be a mediator in neutrophil oxygen radical production and degranulation induced by the stimulators used in the present study. PMID- 3021172 TI - Calcium channel antagonist induced inhibition of superoxide production in human neutrophils. Mechanisms independent of antagonizing calcium influx. AB - Three calcium channel antagonists, verapamil, diltiazem and nisoldipine, inhibited superoxide production in human neutrophils that were stimulated by phorbol 12-myristate 13-acetate (PMA) in a buffered saline lacking calcium. Concentrations of these drugs giving 50% control activity (IC50) were 0.3, 0.45 and 0.01 mM respectively. This inhibition was also observed in the presence of ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) and was not reversed by the addition of calcium. This suggests that calcium channel antagonists inhibited superoxide production independently of extracellular calcium. These calcium channel antagonists inhibited the mobilization of membrane associated calcium, and protein phosphorylation probably catalyzed by C-kinase, both of which are thought to be involved in the signal transmission for the induction of superoxide production. Calcium channel antagonists also inhibited NADPH oxidase, responsible for superoxide production, with IC50 = 0.5, 3 and more than 0.08 mM, respectively, for verapamil, diltiazem and nisoldipine. The results indicate that calcium channel antagonists inhibit superoxide production by affecting not only the catalytic activity by also the activation of NADPH oxidase. Inhibition of superoxide production by calcium channel antagonists suggests that these antagonists do not affect cell functions merely by affecting calcium influx. PMID- 3021174 TI - [Structural-functional relations of short analogs of enkephalin]. AB - The similarity of action of narcotic analgesics and opioid peptides is due to activation of a common opiate receptor as the primary step in initiating biochemical chains responsible for diverse morphine-like effects. The most widely used assays for opioid and analgesic activities are presented and evaluated. Approximately 180 short enkephalin analogues (di-, tri- and tetrapeptides), described in the literature, are systematized and their opioid and systemic analgesic activities compared with methionine-enkephalin and morphine as the reference compounds, respectively. The analysis of structure-opioid activity relationships among these enkephalin analogues substantiates the hypothesis that only a limited N-terminal region of the peptide molecule is essential for the binding of opioid peptides to the subclass of opiate receptors interacting with narcotic alkaloids (mu-receptors). An attempt has been made to identify minimal structural elements responsible for the mu-receptor activation. Shortening of the molecule and modification of its elements are examined with regard to the mu- and delta-receptor selectivity. It is emphasized that the aromatic structure of the C terminal region of the peptide is not obligatory for the mu-receptor binding. Modifications of short enkephalin analogues which might confer them antagonistic properties are reviewed. The correlation between the ability of short enkephalin analogues to interact with mu-receptors and their antinociceptive properties is discussed along with some structural features pertinent to the analgesic effect after systemic administration of peptides. On the basis of this analysis, peptides containing no more than four amino acids are considered as the most probable morphine-like analgesics. PMID- 3021175 TI - [Monomeric form of E. coli pyrophosphatase. Regulatory properties, stability and interaction with affinity inhibitors]. AB - A comparative study on the E. coli inorganic pyrophosphatase subunit and native oligomer disclosed that the quaternary structure is not an essential prerequisite for exhibiting by the enzyme its regulatory properties. However, association of the subunits enhances the stability of the protein molecule and is obligatory for irreversible affinity inhibition. PMID- 3021176 TI - Heterogeneity of proteoglycans extracted before and after collagenase treatment of human articular cartilage. I. Physical properties related to age. AB - Proteoglycans were isolated from young and mature human articular cartilage 4 different ways: by direct extraction with 4M guanidine hydrochloride (GuHCl); after digestion of the residue from this first extraction with collagenase, by extraction with 4M GuHCl; associatively with 0.5M GuHCl after digestion of the cartilage with collagenase; and dissociatively with 4M GuHCl after digestion of the cartilage with collagenase. The structural properties of these proteoglycans were compared. Proteoglycan aggregates and monomers isolated from second extractions and from young cartilage were of larger hydrodynamic size than proteoglycans isolated from first extractions and mature cartilage, respectively. The same applied to the chondroitin sulfate chain lengths of these proteoglycans. The proteoglycan fraction from second extractions of cartilage contained a larger proportion of monomers than the fraction from first extractions. Associative extraction of mature collagenase-digested cartilage yielded mainly proteoglycan monomers, whereas an appreciable amount of proteoglycan aggregate was also liberated from young collagenase-digested cartilage. Our results indicate that, because of their larger size, proteoglycans from second extractions of cartilage are more entrapped in the collagen network. These large proteoglycans can only be liberated from the matrix after extraction of the smaller proteoglycans, followed by digestion of the residue with collagenase. This indicates that proteoglycans overlap and entangle with the collagen and protect it from degradation by collagenase. PMID- 3021178 TI - Rat cytomegalovirus infection enhances type II collagen arthritis in rats. AB - The effect of rat cytomegalovirus (RCMV) infection on type II collagen-induced arthritis was studied in DA rats. Rats were infected with RCMV 5 days before, simultaneously with, or 5 days after immunization with calf type II collagen. Control rats were either given type II collagen alone or were injected with normal rat salivary gland (NRSG) simultaneously with collagen immunization. Severity of arthritis in each limb was graded on a scale of 1-4 (maximum score 16). In 5 experiments, peak arthritis scores in the RCMV groups were twice those of the control groups which received NRSG or collagen only (8-9 versus 4-6). Radiographs of involved joints showed greater destruction of cartilage and articular bone in the RCMV rats than in the NRSG control group. Repeated attempts to culture RCMV from joint tissues were unsuccessful. Our results indicate that RCMV infection enhances the arthritic process in this experimental model of an autoimmune arthritis. PMID- 3021179 TI - Thalidomide neurotoxicity and rheumatoid arthritis. PMID- 3021180 TI - Binding of [3H]-nitrendipine and [3H]-diltiazem to rat myocardial sarcolemma. AB - The binding properties of two major and chemically distinct calcium antagonists, [3H]-nitrendipine and [3H]-diltiazem, were investigated in highly purified rat cardiac sarcolemma. In the case of [3H]-nitrendipine, there appeared a single set of high affinity binding sites with a B max of approximately 0.9 pmol X mg-1 protein and a KD of approximately 0.15 nmol/l. Because of the extremely high value obtained for KD (29 mumol/l), the specificity of [3H]-diltiazem binding seemed questionable. The specific binding of 0.1 nmol/l [3H]-nitrendipine to cardiac sarcolemma was inhibited by nitrendipine and nifedipine (1 mumol/l), only partly inhibited by verapamil (1 mumol/l), and was enhanced by diltiazem (0.1-10 mumol/l). The stimulation of [3H]-nitrendipine binding by diltiazem was associated with an increase in the number of binding sites, Bmax, but with no change in the KD or the Hill coefficient. An enantiomer of diltiazem (1-cis) neither stimulated nor inhibited the [3H]-nitrendipine binding. These results strongly suggest that major prototype calcium antagonists have distinct and variously interacting sites of action in the rat cardiac sarcolemma. PMID- 3021177 TI - Heterogeneity of proteoglycans extracted before and after collagenase treatment of human articular cartilage. II. Variations in composition with age and tissue source. AB - Proteoglycans (A1 fractions) were extracted with 4M guanidine hydrochloride (GuHCl) from human articular cartilage samples of a wide age range. Distinctions were made between hip and knee, and upper and lower layers. The residues of these extractions were digested with purified collagenase, and a second extraction with 4M GuHCl was performed, which yielded appreciable amounts of proteoglycans. When proteoglycans from second extractions were compared with those from first extractions, the following changes were observed: an increase in chondroitin sulfate; a relative decrease in keratan sulfate; a decrease in protein content; and a decrease in the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate. The same changes were found when nonaggregating proteoglycans were compared with proteoglycan aggregates, when proteoglycans from young cartilage were compared with those from mature cartilage, when proteoglycans from knee cartilage were compared with those from hip cartilage, and when proteoglycans from upper layers of cartilage were compared with those from deeper layers. It is suggested that the differences found between first and second extractions of cartilage, between upper and lower layers of cartilage, and between knee and hip cartilage are caused by variations in the relative amount of nonaggregating proteoglycans and/or variations in proteoglycan size. PMID- 3021181 TI - Early handling increases hippocampal long-term potentiation in young rats. AB - The hippocampal formation undergoes major anatomical and physiological changes postnatally, and thus might be expected to be particularly sensitive to early handling effects. Long-term potentiation (LTP) in the hippocampal formation, a form of brain plasticity thought to be important in learning and memory, was examined in young rats following early handling or control treatments. The amplitude of LTP was reliably greater in rats receiving the early handling regime. Possible mechanisms and consequences of enhanced LTP were discussed. PMID- 3021182 TI - 31P-NMR studies on ATP X Mg2+, p21 X nucleotide, and adenylate kinase X nucleotide complexes. Chemical shifts, rate and equilibrium constants. PMID- 3021183 TI - Platelet-activating factor binding to human platelet membranes. AB - Previously reported methods for quantifying platelet-activating factor (PAF) binding to rabbit platelet membranes were modified for studies of PAF binding to human platelet membranes. The membranes were prepared by the "glycerol lysis" method and PAF binding was quantified by using polyethylene glycol precipitation to recover membrane-bound PAF. Optimal PAF binding required buffers containing 3 to 10 mm KCl and either 5 to 10 mM MgCl2 or 5 to 10 mM CaCl2. NaCl was not as effective as KCl and concentrations of NaCl greater than 3 mM strongly inhibited PAF binding. Maximal binding occurred after incubation for 60 min at 0 degree C and was reversed by the addition of excess unlabeled PAF. PAF binding was saturable. Scatchard analysis of PAF binding to 50 micrograms of membrane protein revealed 10.3 +/- 1.7 x 10(11) receptors per milligram of membrane protein and the receptors had a Kd of 7.6 +/- 1.9 nM. The calculated receptor number, binding affinity, and specificity of binding are similar to those previously calculated for PAF binding to intact human platelets, suggesting that the membrane binding site for PAF is the PAF receptor. PMID- 3021184 TI - The RPO31 gene of Saccharomyces cerevisiae encodes the largest subunit of RNA polymerase III. AB - The 5' end of the RNA transcript of the Saccharomyces cerevisiae RNA polymerase related gene, RPO31, was localized on the RPO31 locus using Northern analyses and primer extension experiments. The amino terminal protein sequence was determined for the largest (relative mass approximately 160,000) subunit polypeptide of S. cerevisiae RNA polymerase III. The N-terminal amino acid sequence of this polypeptide was shown to be identical to that predicted for a putative RPO31 polypeptide initiated at the first ATG codon located 208-216 base pairs 3' to the major RPO31 mRNA start sites. The RPO31 locus thus encodes the largest subunit of S. cerevisiae RNA polymerase III. PMID- 3021186 TI - Glycosaminoglycans in human breast cancer: morphological and biochemical study. AB - The authors evaluate the distribution and the content of glycosaminoglycans (GAG) in neoplastic tissue from mastectomy specimens of 15 patients treated for an infiltrating ductal carcinoma NOS, in comparison to normal tissue of the opposite quadrant. Neoplastic tissues contain a marked amount (5 mg/g dry defatted tissue) of GAG, in comparison to normal ones (2.2 mg/g dry defatted weight). In stellate carcinomas GAG are localized near the interface with the host tissue and are constituted mainly of chondroitin sulfates and hyaluronate. These data are confirmed by electrophoretic analysis. PMID- 3021187 TI - Collagen in human breast cancer: morphological and biochemical study. AB - The authors studied the distribution and the composition of collagen in neoplastic tissue from mastectomy specimens of 15 patients treated for an infiltrating ductal carcinoma NOS, in comparison to normal tissue of the opposite quadrant. The study was performed by light microscopy, microspectrophotometry, and electrophoretic methods. The results demonstrate a characteristic stromal organization of breast cancer in relation to collagen type I content. PMID- 3021188 TI - Glycosaminoglycan-enriched extracellular matrix surrounds intraductal carcinoma of human breast: histochemical study. AB - The presence of glycosaminoglycans and hyaluronate was histochemically detected in connective tissue surrounding the intraductal carcinoma of human breast. The implications of the observation in the interaction between neoplasia and host tissues are discussed. PMID- 3021189 TI - Dose-dependent inhibition of phosphoinositide metabolism in human platelets by aspirin in vitro and in vivo. AB - The effects of aspirin on the metabolism of phosphoinositides in human platelets were studied in vitro and in vivo. Eight volunteers received, at two-weekly intervals, a single dose of 10, 30, 100 or 600 mg aspirin. Before the first dose platelets were taken and incubated in vitro with a range of concentrations (10 nM 100 microM) of aspirin. Formation of inositol phosphates (IP) was measured in [3H]-inositol labelled platelets after incubation with collagen and thrombin for 30 min, a time at which a maximal increase in [3H]-IP was observed. The in vitro IC50 for inhibition of the response to collagen by aspirin was approximately 1 microM; the in vivo ID50 was 40-50 mg. Aspirin did not fully inhibit the collagen stimulated IP formation either in vitro or in vivo, and the response to thrombin was unaffected. The ID50 and IC50 of aspirin is thus in accord with the doses of aspirin associated with inhibition of platelet aggregation and thromboxane production in other studies. The possible relevance of these data to the clinical uses of aspirin is discussed. PMID- 3021185 TI - A steryl glycoside fraction from Momordica charantia seeds with an inhibitory action on lipid metabolism in vitro. AB - A saponin fraction was isolated from Momordica charantia seeds by delipidation, saline extraction, ammonium sulfate precipitation, and extraction of the resulting supernatant with n-butanol. Thin-layer chromatography, in the upper phase of the n-butanol--ethyl acetate--water (4:1:5, by volume) system on plastic sheets coated with silica gel 60 F254, revealed the presence of a single spot after spraying with 10% sulfuric acid. The lack of contamination of the saponin preparation with proteins was judged by the absence of protein bands in sodium dodecyl sulfate--polyacrylamide gel electrophoresis, agarose electrophoresis and agarose diffusion, and by the absence of an absorption maximum around 278 nm. The saponin acted as a noncompetitive inhibitor of corticotropin, glucagon, and epinephrine in lipolysis in isolated rat adipocytes, and it also antagonized dibutyryl cAMP induced lipolysis. The antilipolytic activity was resistant to heat, trypsin, chymotrypsin, pronase, and glutathione, in keeping with the chemical nature of saponin. Incorporation of [3-3H]glucose into lipid was inhibited. Adipocyte viability and ATP content were not affected by the saponin, suggesting that its inhibitory effects on lipolysis and lipogenesis were not due to an adverse effect on cell viability. PMID- 3021190 TI - Expression of cytochrome P-450 and albumin genes in rat liver: effect of xenobiotics. AB - Thioacetamide, a hepatocarcinogen and an inhibitor of heme synthesis, blocks the phenobarbitone-mediated increase in the transcription of cytochrome P-450b+e messenger RNA in rat liver. This property is also shared by CoCl2 and 3-amino 1,2,4-triazole, two other inhibitors of heme synthesis. Thus, it appears feasible that heme may serve as a positive regulator of cytochrome P-450b+e gene transcription. Thioacetamide enhances albumin messenger RNA concentration, whereas phenobarbitone decreases the same. However, these changes in albumin messenger RNA concentration are not accompanied by corresponding changes in the transcription rates. Therefore, drug-mediated changes in albumin messenger RNA concentration are due to posttranscriptional regulation. The property of thioacetamide to enhance the albumin messenger RNA concentration is not shared by CoCl2 and 3-amino-1,2,4-triazole. Therefore, heme does not appear to be a regulatory molecule mediating the reciprocal changes brought about in the concentrations of cytochrome P-450b+e and albumin messenger RNAs. PMID- 3021191 TI - Protein complement of rod outer segments of frog retina. AB - Rod outer segments (ROS) from frog retina have been purified by Percoll density gradient centrifugation, a procedure that preserves their form and intactness. One- and two-dimensional electrophoretic analysis reveals a smaller number of proteins than is observed in many cell organelles and permits quantitation of the 20 most abundant polypeptides. Rhodopsin accounts for 70% of the total protein (3 X 10(9) copies/outer segment), and approximately 70 other polypeptides are present at more than 6 X 10(4) copies/outer segment. Another 17% of the total protein is accounted for by the G-protein (3 X 10(8) copies/outer segment) that links rhodopsin bleaching and the activation of cyclic GMP phosphodiesterase (PDE). The phosphodiesterase accounts for 1.5% of the protein (1.5 X 10(7) copies/outer segment), and a 48,000-dalton component that binds to the membrane in the light accounts for a further 2.6%. The function of approximately 90% of the total protein in the outer segment is known, and two-thirds of the non rhodopsin protein is accounted for by enzyme activities associated with cyclic GMP metabolism. The relative molar abundance of rhodopsin, G-protein, and PDE is 100:10:1. Apart from these major membrane-associated proteins, most of the other proteins are cytosolic. Thirteen other polypeptides are found at an abundance of one or more copies per 1000 rhodopsins, nine soluble and four membrane-bound, and their abundance relative to rhodopsin has been quantitated. ROS have been separated into subcellular fractions which resolve three classes of soluble, extrinsic membrane, and integral membrane proteins. A listing of the proteins that are phosphorylated and their subcellular localization is given. Approximately 25 phosphopeptides are detected, and most are in the soluble fraction. Fewer phosphorylated proteins are associated with the purified outer segments than with crude ROS. Distinct patterns of phosphorylation are associated with intact rods incubated with [32P]Pi and broken rods incubated with [gamma 32P]ATP. PMID- 3021192 TI - Fluorescence photobleaching recovery as a method to quantitate viral envelope cell fusion: application to study fusion of Sendai virus envelopes with cells. AB - A method to quantitate viral envelope-cell fusion at the single-cell level is presented. The method is based on the incorporation of nonquenching concentrations of a fluorescent lipid probe into the viral envelope; fluorescence photobleaching recovery (FPR) is then applied to measure the lateral mobilization of the probe in the cell membrane following fusion. In adsorbed (unfused) viral envelopes, the probe is constricted to the envelope and is laterally immobile on the micrometer scale of FPR. After fusion, the envelope lipids intermix with the plasma membrane, the probe becomes laterally mobile, and the fraction of fused viral envelopes can be extracted from the fraction of mobile probe molecules. The method has several advantages: (i) It clearly distinguishes fused from internalized envelopes, as probes in the latter are immobile in FPR studies; (ii) focusing the laser beam on specific regions of the cell enables region-specific measurements of the fusion level; (iii) one cell is measured at a time, enabling studies on the distribution of the fusion level within the cell population. The new method was employed to study fusion of reconstituted Sendai virus envelopes (RSVE) containing N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine with several cell types. Experiments with human erythrocytes demonstrated that the lateral mobilization measured is due to fusion and not the result of exchange processes. The extent of RSVE-erythrocyte fusion determined by FPR was similar to that measured by two other independent methods (fluorescence dequenching and removal of adsorbed RSVE by dithiothreitol).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021193 TI - Effects of vanadate on the rotational dynamics of spin-labeled calcium adenosinetriphosphatase in sarcoplasmic reticulum membranes. AB - We have studied the effects of vanadate on the rotational motion of the calcium adenosine-triphosphatase (Ca-ATPase) from sarcoplasmic reticulum (SR), using saturation-transfer electron paramagnetic resonance (ST-EPR). Vanadate has been proposed to act as a phosphate analogue and produce a stable intermediate state similar to the phosphoenzyme. This study provides evidence about the physical state of this intermediate. In particular, since ST-EPR provides a sensitive measure of microsecond protein rotational mobility, and hence of protein-protein association, these studies allowed us to ask (a) whether the vanadate-induced protein association observed in electron micrographs of SR vesicles also occurs under physiological (as opposed to fixed, stained, or frozen) conditions and (b) whether vanadate-induced changes in protein association also occur under conditions sufficient for enzyme inhibition but not for the production of large arrays detectable by electron microscopy (EM). At 5 mM decavanadate, a concentration sufficient to crystallize the ATPase on greater than 90% of the membrane surface area in EM, ST-EPR showed substantial immobilization of the spin labeled protein, indicating protein-protein association in the unstained vesicles. Conventional EPR spectra of lipid probes showed that lipid hydrocarbon chain mobility is unaffected by decavanadate-induced protein crystallization in SR, suggesting that changes in protein-protein contacts do not involve the lipid hydrocarbon region. At 5 mM monovanadate, a concentration sufficient to inhibit the ATPase but not to form crystals detectable by EM, no changes were observed in ST-EPR or conventional EPR spectra of either protein or lipid.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021194 TI - Amino acid sequence of the b subunit of human factor XIII, a protein composed of ten repetitive segments. AB - Factor XIII is a plasma protein that participates in the final stages of blood coagulation. The complete amino acid sequence of the b subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gt11 cDNA library prepared from human liver mRNA was screened with an affinity-purified antibody against the b subunit of human factor XIII. Nine positive clones were isolated from 2 X 10(6) phage and plaque-purified. The largest cDNA insert was sequenced and shown to contain 2180 base pairs coding for a portion of the leader sequence (19 amino acids), the mature protein (641 amino acids), a stop codon (TGA), a 3' noncoding region (187 nucleotides), and a poly(A) tail. When the b subunit of human factor XIII was digested with cyanogen bromide, nine peptides were isolated by gel filtration and reverse-phase high performance liquid chromatography. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 299 amino acid residues were identified. These amino acid sequences were in complete agreement with the amino acid sequence predicted from the cDNA. The b subunit of factor XIII contained 10 repetitive homologous segments, each composed of about 60 amino acids and 4 half cystine residues. Each of these repeated segments is a member of a family of repeats present in human beta 2-glycoprotein I, complement factor B, and haptoglobin alpha 1 chain. Three potential Asn-linked carbohydrate attachment sites were also identified in the b subunit of factor XIII. PMID- 3021195 TI - 57Fe and 1H electron-nuclear double resonance of three doubly reduced states Escherichia coli sulfite reductase. AB - We have employed electron-nuclear double resonance (ENDOR) spectroscopy to study the 57Fe hyperfine interactions in the bridged-siroheme [4Fe-4S] cluster that forms the catalytically active center of the two-electron-reduced hemoprotein subunit of Escherichia coli NADPH-sulfite reductase (SiR2-). Previous electron paramagnetic resonance (EPR) and Mossbauer studies have shown that this enzyme oxidation state can exist in three distinct spectroscopic forms: (1) a "g = 2.29" EPR species that predominates in unligated SiR2-, in which the siroheme Fe2+ is believed to be in an S = 1 state; (2) a "g = 4.88" type of EPR species that predominates in SiR2- in the presence of small amounts of guanidinium sulfate, in which the siroheme Fe2+ is in an S = 2 state; and (3) a classical "g = 1.94" type of EPR species that is seen in SiR2- ligated with CO, in which the siroheme Fe2+ is in an S = 0 state. In all three species, the cluster is in the [4Fe-4S]1+ state, and two distinct types of Fe site are seen in Mossbauer spectroscopy. ENDOR studies confirm the Mossbauer assignments for the cluster 57Fe in the g = 1.94 state, with A values of 37, 37, and 32 MHz for site I and ca. 19 MHz for site II. The hyperfine interactions are not too different on the g = 2.29 state, with site I Fe showing more anisotropic A values of 32, 24, and 20 MHz (site II was not detected).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021196 TI - Interaction of spin-labeled nicotinamide adenine dinucleotide phosphate with chicken liver fatty acid synthase. AB - The spatial relationships between the four reduced nicotinamide adenine dinucleotide phosphate (NADPH) binding sites on chicken liver fatty acid synthase were explored with electron paramagnetic resonance (EPR) and spin-labeled analogues of NADP+. The analogues were prepared by reaction of NADP+ with 2,2,5,5 tetramethyl-1-oxy-3-pyrroline-3-carboxylic acid, with 1,1'-carbonyldiimidazole as the coupling reagent. Several esterification products were characterized, and the interaction of the N3' ester of NADP+ with the enzyme was examined in detail. Both 1H13, 14N and 2H13, 15N spin-labels were used: the EPR spectrum was simpler, and the sensitivity greater, for the latter. The spin-labeled NADP+ is a competitive inhibitor of NADPH in fatty acid synthesis, and an EPR titration of the enzyme with the modified NADP+ indicates four identical binding sites per enzyme molecule with a dissociation constant of 124 microM in 0.1 M potassium phosphate and 1 mM ethylenediaminetetraacetic acid (pH 7.0) at 25 degrees C. The EPR spectra indicate the bound spin-label is immobilized relative to the unbound probe. No evidence for electron-electron interactions between bound spin-labels was found with the native enzyme, the enzyme dissociated into monomers, or the enzyme with the enoyl reductase sites blocked by labeling the enzyme with pyridoxal 5'-phosphate. Furthermore, the EPR spectrum of bound ligand was the same in all cases. This indicates that the bound spin-labels are at least 15 A apart, that the environment of the spin-label at all sites is similar, and that the environment is not altered by major structural changes in the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021197 TI - Hydrolysis of peptides by carboxypeptidase A: equilibrium trapping of the ES2 intermediate. AB - The cobalt absorption and electron paramagnetic resonance (EPR) spectra of cobalt carboxypeptidase undergo unique variations on formation of catalytic peptide and ester intermediates as previously recorded in cryoenzymologic experiments employing rapid-scanning spectroscopy and cryotrapping [Geoghegan, K. F., Galdes, A., Martinelli, R. A., Holmquist, B., Auld, D.S., & Vallee, B. L. (1983) Biochemistry 22, 2255-2262]. We here describe a means of stabilizing these intermediates, which we have termed "equilibrium trapping". It allows peptide intermediates to be observed for longer periods (much greater than 1 min) at ambient as well as subzero temperatures. The reaction intermediate with the rapidly turned over peptide substrate Dns-Ala-Ala-Phe is trapped when the cobalt enzyme (greater than 10 microM) has catalyzed the attainment of chemical equilibrium between high concentrations of the hydrolysis products Dns-Ala-Ala, 10 mM, and L-phenylalanine, 50 mM, and the product of their coupling Dns-Ala-Ala Phe. Under these conditions, Dns-Ala-Ala-Phe is present in the equilibrated substrate-product reaction mixture at a level that exceeds the one predicted on the basis of K'eq for hydrolysis of this substrate and is close to the enzyme concentration. Other pairs of peptide hydrolysis products yield similar results. Visible absorption and EPR spectra of the cobalt enzyme show that the synthesized peptide binds to the active site in the mode previously recognized as the ES2 catalytic intermediate in peptide hydrolysis. Equilibrium trapping of the ES2 intermediate allows analysis of its physicochemical properties by methods that could not be employed readily under cryoenzymological conditions, e.g., circular dichroic and magnetic circular dichroic spectra.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021198 TI - Carbohydrate substrate specificity of bacterial and plant pyrophosphate-dependent phosphofructokinases. AB - Pyrophosphate-dependent phosphofructokinase from the facultative anaerobic bacterium Propionibacterium freudenreichii and from the mung bean Phaseolus aureus has been purified to homogeneity. Potential utilization of carbohydrate substrate analogues for each enzyme was initially screened by using Fourier transform 31P NMR at pH 8 and 25 degrees C and monitoring the appearance of the phosphate resonance in the direction of D-fructose 6-phosphate phosphorylation (forward reaction direction) and, with the bisphosphate analogues, the appearance of the pyrophosphate resonance in the direction of phosphate phosphorylation (reverse reaction direction). Both enzymes are strict in their requirements for the sugar phosphate substrate, with only D-fructose 6-phosphate, D-sedoheptulose 7-phosphate, and 2,5-anhydro-D-mannitol 6-phosphate, or their respective bisphosphates in the reverse reaction direction, utilized as substrates at detectable levels. The dissociation constants for D-psicose 6-phosphate, D tagatose 6-phosphate, and L-sorbose 6-phosphate are an order of magnitude larger than that for D-fructose 6-phosphate, indicating a stringent steric requirement for the D-threo (trans) configuration at the two nonanomeric furan ring hydroxyl groups. These results strongly suggest that the anomeric, epimeric, and tautomeric form of the sugar phosphate substrates favored by both enzymes is the beta-D-fructofuranose form. Dissociation constants for nonsubstrate analogues were used to provide information on the nature of the active site. Competitive inhibition patterns vs. fructose 1,6-bisphosphate were obtained for a series of 1,n-alkanediol bisphosphates (where n = 2-9).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021199 TI - Kinetic studies on the activation of pyrophosphate-dependent phosphofructokinase from mung bean by fructose 2,6-bisphosphate and related compounds. AB - Pyrophosphate-dependent phosphofructokinase (PPi-PFK) was purified from the mung bean Phaseolus aureus. The enzyme is activated by fructose 2,6-bisphosphate at nanomolar concentrations. The enzyme exhibits Michaelis-Menten kinetics, and the reaction mechanism, deduced from initial velocity studies in the absence of inhibitors as well as product and dead-end inhibition studies, is rapid equilibrium random in the presence and absence of fructose 2,6-bisphosphate. In the direction of fructose 6-phosphate phosphorylation, saturating fructose 2,6 bisphosphate (1 microM) increases V congruent to 9-fold and increases V/KMgPPi and V/KF6P about 30-fold. In the reverse direction (phosphate phosphorylation), the same concentration of activator has little if any effect on V or the Km for inorganic phosphate (Pi) and Mg2+ but does increase V/KFBP about 42-fold. No changes were observed in any of the other rate constants. The binding affinity of fructose 2,6-bisphosphate to all enzyme forms is identical. The activator site of the mung bean PPi-PFK binds fructose 2,6-bisphosphate with a Kact of 30 nM with the 2,5-anhydro-D-glucitol 1,6-bisphosphate (the most effective analogue) 33-fold less tightly. Of the alkanediol bisphosphate series, 1,4-butanediol bisphosphate exhibited the tightest binding (Kact congruent to 3 microM). These and a series of other activating analogues are discussed in relation to the activator site. PMID- 3021201 TI - Inactivation of Escherichia coli glycerol kinase by 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide: evidence for nucleotide regulatory binding sites. AB - Glycerol kinase (EC 2.7.1.30, ATP:glycerol 3-phosphotransferase) from Escherichia coli is inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and by N ethylmaleimide (NEM) in 0.1 M triethanolamine at pH 7 and 25 degrees C. The inactivation by DTNB is reversed by dithiothreitol. In the cases of both reagents, the kinetics of activity loss are pseudo first order. The dependencies of the rate constants on reagent concentration show that while the inactivation by NEM obeys second-order kinetics (k2app = 0.3 M-1 s-1), DTNB binds to the enzyme prior to the inactivation reaction; i.e., the pseudo-first-order rate constant shows a hyperbolic dependence on DTNB concentration. Complete inactivation by each reagent apparently involves the modification of two sulfhydryl groups per enzyme subunit. However, analysis of the kinetics of DTNB modification, as measured by the release of 2-nitro-5-thiobenzoate, shows that the inactivation is due to the modification of one sulfhydryl group per subunit, while two other groups are modified 6 and 15 times more slowly. The enzyme is protected from inactivation by the ligands glycerol, propane-1,2-diol, ATP, ADP, AMP, and cAMP but not by Mg2+, fructose 1,6-bisphosphate, or propane-1,3-diol. The protection afforded by ATP or AMP is not dependent on Mg2+. The kinetics of DTNB modification are different in the presence of glycerol or ATP, despite the observation that the degree of protection afforded by both of these ligands is the same.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021200 TI - Investigation of the regiospecificity and stereospecificity of proton transfer in the yeast inorganic pyrophosphatase catalyzed reaction. AB - The regiospecificity and stereospecificity of proton transfer in the yeast inorganic pyrophosphatase (PPase) catalyzed hydrolysis of P1,P2-bidentate Mg(H2O)4(PPi)2- were probed with exchange-inert metal complexes of imidodiphosphate (PNP) and thiopyrophosphate (PPS). PPase was unable to catalyze the hydrolysis of Mg(H2O)4PNP and P1,P2-bidentate Co(NH3)4PNP under conditions that resulted in rapid hydrolysis of the corresponding metal-PPi complexes. PPase was found to catalyze the hydrolysis of Mg(H2O)4PPS at 17% the rate of Mg(H2O)4PPi hydrolysis. The Km of Mg(H2O)4PPS was determined to be 300 microM, which is a value 10-fold greater than that observed for Mg(H2O)4PPi. P1,P2 Bidentate Cr(H2O)4PPS and Co(NH3)4PPS (prepared from PPS) were both found to be substrates for PPase. The enzyme specifically catalyzed the hydrolysis of the Rp enantiomers of these complexes and not the Sp enantiomers. These results are accommodated by a reaction mechanism involving enzyme-mediated proton transfer to the pro-R oxygen atom of the incipient phosphoryl leaving group of the bound P1,P2-bidentate Mg(H2O)4PPi2- complex. PMID- 3021202 TI - Induction of collagenase production in U937 cells by phorbol ester and partial purification of the induced enzyme. AB - The U937 cell line is a monoblast-like cell line that can be induced to differentiate when treated with phorbol ester or a variety of other agents. Collagenase was detected in the media of U937 cell cultures after treatment with phorbol myristate acetate (PMA) at concentrations of 5 ng/mL or greater. In general, no collagenase was detected in the media of untreated cells. The induced collagenase cleaved native type I collagen into the 3/4 and 1/4-length fragments and showed the inhibition by ethylenediaminetetraacetic acid characteristic of the action of mammalian collagenases. Collagenase activity could be detected in the media of treated cells 12-18 h after the addition of PMA. Secretion of collagenase continued for 2-3 days after PMA addition. The production of collagenase by PMA-treated U937 cells was inhibited by actinomycin D and cycloheximide, suggesting that the induction of the enzyme is the result of de novo synthesis. The collagenase secreted by U937 cells induced with PMA has been purified 12-fold by using DEAE-Sephacel followed by wheat germ agglutinin-agarose chromatography. The apparent molecular mass of this U937 collagenase, determined by gel filtration chromatography on the partially purified enzyme, was 29-36 kilodaltons. PMID- 3021203 TI - Use of lectin affinity chromatography for the purification of collagenase from human polymorphonuclear leukocytes. AB - Polymorphonuclear leukocytes (PMNLs) store collagenase in an inactive form in secretory granules. The enzyme can be activated in vitro by limited proteolysis or by sulfhydryl-modifying agents such as N-ethylmaleimide (NEM). We have enriched NEM-activated collagenase 820-fold using granule isolation, gel filtration, and wheat germ agglutinin (WGA)-agarose chromatography. The use of WGA-agarose resulted in a 55-fold enrichment of collagenase in a single step with very little loss of activity. The chromatographic behavior of collagenase on other lectin matrices was explored and gave information about the type of complex asparagine-linked oligosaccharide found on collagenase isolated from PMNLs. PMID- 3021204 TI - Membrane vesicles containing the Sendai virus binding glycoprotein, but not the viral fusion protein, fuse with phosphatidylserine liposomes at low pH. AB - Membrane vesicles containing the Sendai virus hemagglutinin/neuraminidase (HN) glycoprotein were able to induce carboxyfluorescein (CF) release from loaded phosphatidylserine (PS) but not loaded phosphatidylcholine (PC) liposomes. Similarly, fluorescence dequenching was observed only when HN vesicles, bearing self-quenched N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (N-NBD PE), were incubated with PS but not PC liposomes. Thus, fusion between Sendai virus HN glycoprotein vesicles and the negatively charged PS liposomes is suggested. Induction of CF release and fluorescence dequenching were not observed when Pronase-treated HN vesicles were incubated with the PS liposomes. On the other hand, the fusogenic activity of the HN vesicles was not inhibited by treatment with dithiothreitol (DTT) or phenylmethanesulfonyl fluoride (PMSF), both of which are known to inhibit the Sendai virus fusogenic activity. Fusion was highly dependent on the pH of the medium, being maximal after an incubation of 60-90 s at pH 4.0. Electron microscopy studies showed that incubation at pH 4.0 of the HN vesicles with PS liposomes, both of which are of an average diameter of 150 nm, resulted in the formation of large unilamellar vesicles, the average diameter of which reached 450 nm. The relevance of these observations to the mechanism of liposome-membrane and virus-membrane fusion is discussed. PMID- 3021205 TI - Nucleotide sequence of the cDNA coding for human complement C1r. AB - C1r is a zymogen of a serine protease that is involved in the activation of the first component of the classical pathway of the complement system. cDNAs coding for human C1r have been isolated from libraries prepared from poly(A) RNA from human liver and Hep G2 cells. From DNA sequence analysis, the overlapping cDNA inserts were shown to span 2493 nucleotides of the C1r mRNA, not including the poly(A) tail. The cDNA sequence coding for C1r contained a 5' noncoding region, 2115 nucleotides coding for a polypeptide precursor of 705 amino acids, and a 3' noncoding region. Some variability in the length of the 3' noncoding sequence was observed with the cDNA inserts, although most contained a polyadenylation signal followed by a poly(A) tail. The A or noncatalytic chain of C-1r, which originates from the amino-terminal end of the precursor molecule, contains a potential growth factor domain and two different pairs of internal repeats. One pair of these internal repeats is closely related to the amino-terminal sequence of C1s, while the other pair of repeats is homologous to the tandem repeats present in beta 2-glycoprotein I, complement factor B, the b subunit of factor XIII, and a single region present in the alpha 1 chain of haptoglobin. The B chain of C-1r contains the catalytic portion of the enzyme and is homologous to the trypsin family of serine proteases. PMID- 3021207 TI - Covalent attachment of sulfhydryl-specific, electron spin resonance spin-labels to Fab' fragments of murine monoclonal antibodies that recognize human platelet membrane glycoproteins. Development of membrane protein specific spin probes. AB - A general method for the production of high-affinity, nitroxide-labeled, protein specific spin probes is described in this paper. Fab' fragments are generated from protein-specific, murine monoclonal antibodies by pepsin digestion and mild reduction with cysteine. The free sulfhydryl group located in the carboxy terminal region of these molecules and produced de novo by this manipulation is then alkylated by reaction with 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO-maleimide), thereby generating spin-labeled Fab' fragments of these monoclonal antibodies. Two prototypic monoclonal antibodies were tested, each specific for a different integral membrane glycoprotein of human blood platelets. The results indicate that Fab' spin probes generated by this method retain the ability to bind to these glycoproteins within the membrane of intact platelets. These reagents thus represent probes that can be generally used to monitor integral membrane protein mobility on the surface of the intact cell. PMID- 3021206 TI - Human liver fibronectin complementary DNAs: identification of two different messenger RNAs possibly encoding the alpha and beta subunits of plasma fibronectin. AB - Human fibronectin polymorphism arises from variation in the C-terminal region [e.g., Sekiguchi, K., Siri, A., Zardi, L., & Hakomori, S. (1985) J. Biol. Chem. 260, 5105-5114]. In order to verify the chemical basis of the fibronectin polymorphism, cDNAs encoding the C-terminal region of human liver fibronectin have been isolated, sequenced, and compared with cDNAs encoding so-called "cellular fibronectin" (i.e., fibronectin produced by cultured cells in vitro). Among the five independent cDNAs thus isolated, two cDNAs, named pLF2 and pLF4, differed in the nucleotide sequence at the "type III connecting segment" (IIIcs) region. pLF4 contained 192 bases in this region whereas pLF2 completely lacked these bases. S1 mapping analysis indicated that both cDNAs with and without the 192 bases are faithful copies of two fibronectin mRNA species abundantly present in human liver. Comparison of the liver cDNAs with those coding for cellular fibronectin indicates that the latter cDNAs contain the 75-base and/or 93-base extra segments at the 5' and 3' boundaries of the 192-base IIIcs region. These extra segments have the consensus sequences for the 3' splice sites at their 3' ends, suggesting that fibronectin mRNAs with partial or complete deletion of the IIIcs sequence result from alternative splicing of a primary RNA transcript. Liver fibronectin cDNAs also lacked the 270-based "extra domain" (ED) segment present in some, but not all, cDNAs encoding cellular fibronectin. Thus, cellular fibronectin appears to have three extra peptide segments, encoded by the 75-base and 93-base segments in the IIIcs region and by the 270-base ED region, that are mostly absent in the liver fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021209 TI - Structure and activities of a variant ubiquitin sequence from bakers' yeast. AB - Ubiquitin is an extremely conserved protein, with an identical sequence throughout the animal kingdom. However, the gene sequence of the yeast protein [Ozkaynak, E., Finley, D., & Varshavsky, A. (1984) Nature (London) 312, 663-666] predicts three amino acid differences. This implies that some functions or binding interactions of ubiquitin are different in yeast and animal cells. In an effort to define these differences, ubiquitin has been purified to homogeneity from bakers' yeast and characterized. Amino acid analysis of the protein and the isolated tryptic peptides confirms the primary structure of this protein as predicted from the gene sequence. This result indicates that the gene sequenced is the transcriptionally active gene from yeast. The conformation of yeast ubiquitin is similar to human ubiquitin as judged by circular dichroism, sensitivity to trypsin, and Stokes radius. Yeast and animal ubiquitins show identical activities in supporting ubiquitin-dependent protein degradation and in the ATP-pyrophosphate exchange reaction catalyzed by the purified ubiquitin adenylating enzyme. Thus, the three conservative amino acid differences between yeast and animal ubiquitins have very little effect on the structure of ubiquitin or its activity in the ubiquitin-dependent proteolytic system. These results suggest that at least some of the evolutionary pressure preventing sequence variation among animal ubiquitins stems from one or more of its nonproteolytic functions. PMID- 3021208 TI - Amplification of phosphodiesterase activation is greatly reduced by rhodopsin phosphorylation. AB - In the vertebrate rod outer segment (ROS), the light-dependent activation of a GTP-binding protein (G-protein) and phosphodiesterase (PDE) is quenched by a process that requires ATP [Liebman, P.A., & Pugh, E.N. (1979) Vision Res. 19, 375 380]. The ATP-dependent quenching mechanism apparently requires the phosphorylation of photoactivated rhodopsin (Rho*); however, a 48-kilodalton protein (48K protein) has also been proposed to participate in the inactivation process. Purified species of phosphorylated rhodopsin containing 0, 2, or greater than or equal to 4 (high) phosphates per rhodopsin (PO4/Rho) were reconstituted into phosphatidylcholine (PC) vesicles and reassociated with a hypotonic extract from isotonically washed disk membranes that were depleted of 48K protein; PDE activation, in response to bleaching from 0.01% to 15% of the rhodopsin present, was measured. PDE activity was reduced by at least 30% at high fractional rhodopsin bleaches and by greater than 80% at low fractional rhodopsin bleaches in high PO4/Rho samples when compared to the activity measured in O PO4/Rho controls. A phosphorylation level of 2 PO4/Rho produced PDE activities that were intermediate between O PO4/Rho and high PO4/Rho samples at low bleaches, but were identical with the O PO4/Rho samples at high rhodopsin bleaches. Rhodopsin phosphorylation is thus capable of producing a graded inhibition of light stimulated PDE activation over a limited range of (near physiological) bleach levels. This effect become less pronounced as the bleach levels approach those that saturate PDE activation. These results are consistent with increasing levels of phosphorylation, producing a reduction of the binding affinity of G-protein for Rho*. PMID- 3021210 TI - Molecular characterization of the catalytic domains of human complement serine protease C1r. AB - Limited cleavages of human C1r by extrinsic proteases of various specificity (plasmin, elastase, chymotrypsin, thermolysin) yield dimeric associations of two globular domains, each comprised of the intact B chain disulfide linked to gamma, the C-terminal fragment of the A chain. These (gamma-B)2 domains, which are homologous to those obtained from C1r by autolytic cleavage [Villiers, C. L., Arlaud, G. J., & Colomb, M. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4477 4481], represent the core of the C1r molecule and are associated with the catalytic properties of the serine active site. V8 protease also yields (gamma B)2 associations, although additional cleavages occur in the B chain. Sequence analysis shows that all cleavages generating the gamma fragments occur within a 13-residue sequence extending from positions 274 to 286 of the C1r A chain. Chemical cross-linking with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide of the (gamma-B)2 catalytic domains obtained from C1r autolytic cleavage indicates that each gamma-B domain interacts with its neighbor in a "head to tail" configuration, the gamma region of one domain interacting with the B chain of the other domain, and conversely. No evidence is found of gamma-gamma or B-B interactions. Such a head to tail configuration, placed in the context of the model proposed for the C1s-C1r-C1r-C1s catalytic subunit of C1 [Colomb, M. G., Arlaud, G. J., & Villiers, C. L. (1984) Philos. Trans. R. Soc. London, B 306, 283 292], is compatible with autolytic activation of C1r through an intramolecular cross-mechanism and with subsequent activation of C1s by activated C1r. PMID- 3021211 TI - Purification of human collagenases with a hydroxamic acid affinity column. AB - Human collagenase has been isolated from skin fibroblasts and rheumatoid synovium by using an affinity matrix, prepared by coupling Pro-Leu-Gly-NHOH to agarose. Following the methodology described herein, the skin enzyme was isolated in two steps in 76% yield and the synovial enzyme was purified in three steps in 71% yield. Importantly, each enzyme hydrolyzed collagen into 3/4-1/4 cleavage fragments, indicating that a true collagenase had been isolated. The column was specific for the human enzyme since the collagenase from Clostridium histolyticum did not bind. The affinity ligand was designed according to the formalism proposed by Holmquist and Vallee [Holmquist, B., & Vallee, B. L. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6216] that effective metalloenzyme inhibitors can be synthesized by coupling a suitable metal-coordinating group to a substrate analogue. In this case, the hydroxamic acid probably coordinates to the active site metal and the Pro-Leu-Gly moiety is similar to the carboxyl side of the cleavage site of collagen, the enzyme's substrate. The IC50 for N (benzyloxycarbonyl)-Pro-Leu-Gly-NHOH is 4 X 10(-5) M for both enzymes. The affinity chromatographic procedures described here should aid in future studies on vertebrate collagenases. PMID- 3021212 TI - Bacillus subtilis mutant succinate dehydrogenase lacking covalently bound flavin: identification of the primary defect and studies on the iron-sulfur clusters in mutated and wild-type enzyme. AB - Succinate dehydrogenase consists of two protein subunits and contains one FAD and three iron-sulfur clusters. The flavin is covalently bound to a histidine in the larger, Fp, subunit. The reduction oxidation midpoint potentials of the clusters designated S-1, S-2, and S-3 in Bacillus subtilis wild-type membrane-bound enzyme were determined as +80, -240, and -25 mV, respectively. Magnetic spin interactions between clusters S-1 and S-2 and between S-1 and S-3 were detected by using EPR spectroscopy. The point mutations of four B. subtilis mutants with defective Fp subunits were mapped. The gene of the mutant specifically lacking covalently bound flavin in the enzyme was cloned. The mutation was determined from the DNA sequence as a glycine to aspartate substitution at a conserved site seven residues downstream from the histidine that binds the flavin in wild-type enzyme. The redox midpoint potential of the iron-sulfur clusters and the magnetic spin interactions in mutated succinate dehydrogenases were indistinguishable from the those of the wild type. This shows that flavin has no role in the measured magnetic spin interactions or in the structure and stability of the iron-sulfur clusters. It is concluded from sequence and mutant studies that conserved amino acid residues around the histidyl-FAD are important for FAD binding; however, amino acids located more than 100 residues downstream from the histidyl in the Fp subunit can also effect flavinylation. PMID- 3021213 TI - The role of phospholipids in the binding of antimycin to yeast Complex III. AB - Treatment of yeast Complex III with hexane or repeated fractionation with ammonium sulfate-cholate abolishes the ability of antimycin to bind to the complex. Antimycin binding is partially restored by addition of phospholipids to the depleted complex; this action of phospholipids can be enhanced by including Q6 in the reconstitution mixture. PMID- 3021214 TI - Kinetic investigations of the reactions of cytochrome c oxidase with hydrogen peroxide. AB - The reaction of H2O2 with reduced cytochrome c oxidase was investigated with rapid-scan/stopped-flow techniques. The results show that the oxidation rate of cytochrome a3 was dependent upon the peroxide concentration (k = 2 X 10(4) M-1 X s-1). Cytochrome a and CuA were oxidised with a maximal rate of approx. 20 s-1, indicating that the rate of internal electron transfer was much slower with H2O2 as the electron acceptor than with O2 (k greater than or equal to 700 s-1). Although other explanations are possible, this result strongly suggests that in the catalytic cycle with oxygen as a substrate the internal electron-transfer rate is enhanced by the formation of a peroxo-intermediate at the cytochrome a3 CuB site. It is shown that H2O2 took up two electrons per molecule. The reaction of H2O2 with oxidised cytochrome c oxidase was also studied. It is shown that pulsed oxidase readily reacted with H2O2 (k approximately 700 M-1 X s-1). Peroxide binding is followed by an H2O2-independent conformational change (k = 0.9 s-1). Resting oxidase partially bound H2O2 with a rate similar to that of pulsed oxidase; after H2O2 binding the resting enzyme was converted into the pulsed conformation in a peroxide-independent step (k = 0.2 s-1). Within 5 min, 55% of the resting enzyme reacted in a slower process. We conclude from the results that oxygenated cytochrome c oxidase probably is an enzyme-peroxide complex. PMID- 3021216 TI - Effect of saturated and unsaturated fatty acids on the oxidative metabolism of human neutrophils. The role of calcium ion in the extracellular medium. AB - The ability of fatty acids to stimulate the generation of superoxide anion (O2-) by human neutrophils was investigated with respect to their Krafft points. Saturated (myristic acid) and unsaturated (elaidic and oleic acid) induced a marked O2- generation and release from human neutrophils at pH 7.4 in the absence of Ca2+, while 0.3 mM Ca2+ inhibited both myristic acid and elaidic acid-induced O2- release. [14C]Myristic acid association with neutrophils was reduced by addition of Ca2+, whereas oleic acid association was not affected. When the pH of the reaction mixture was lowered to 6.4, 0.6 mM Ca2+ did not inhibit the O2- generation by human neutrophils. These results indicate that the inhibitory effect of Ca2+ on the fatty acid-induced O2- generation might be due to the ionic interaction between the carboxyl group of the fatty acid and Ca2+. Furthermore, 11-methyltridecanoic acid, a branched isomer of myristic acid, which showed the low Krafft point even in the presence of Ca2+, stimulated O2- generation by human neutrophils not only in the absence but also in the presence of 0.6 mM Ca2+. The effect of Ca2+ on the fatty acid-induced O2- generation by neutrophils was discussed with reference to its possible relationship to the Krafft point. PMID- 3021215 TI - The O2- -forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function. PMID- 3021217 TI - Modification of phospholipids in erythrocyte membranes by phospholipase D. A fluorescence and ESR spectroscopic study. AB - The conversion of more than 65% of the phospholipids in human erythrocyte membranes to phosphatidyl-methanol and phosphatidic acid by incubation with phospholipase D and methanol increased the dissociation constant of the fluorescence probe ANS compared to untreated membranes, but did not affect the number of binding sites and the limiting fluorescence enhancement at maximal binding (Imax). On the contrary, the cationic fluorescence probe dansylcadaverin showed additional binding sites without a change in Kd and an increase of Imax upon incubation with phospholipase D treated erythrocyte membranes compared to incubations of membranes with the original phospholipid pattern. The characteristic temperature-dependence of the quenching of the membrane protein fluorescence by a membrane-bound nitroxide-labeled stearic acid was not influenced by the modification of the phospholipids. A slight reduction of the order parameter, S, determined by ESR-spectroscopy with the same nitroxide spin labeled fatty acid incorporated into modified membranes compared to controls was found at 40 degrees C, but not at 25 degrees C. The results were interpreted as an indication of membrane domains that retained their physical properties and lipid composition during the incubation with phospholipase D. PMID- 3021218 TI - Uptake of aminoglycoside antibiotics into brush-border membrane vesicles and inhibition of (Na+ + K+)-ATPase activity of basolateral membrane. AB - The effects of aminoglycoside antibiotics on plasma membranes were studied using rat renal basolateral and brush-border membrane vesicles. 3',4'-Dideoxykanamycin was bound to the basolateral membrane and brush-border membrane vesicles. They had a single class of binding sites with nearly the same constant, and the basolateral membrane vesicles had more binding sites than those of the brush border membrane. Dideoxykanamycin B was transported into the intravesicular space of brush-border membrane vesicles, but not into that of basolateral membrane vesicles. The (Na+ + K+)-ATPase activity of the plasma membrane fraction prepared from the kidney of rat administered with dideoxykanamycin B intravenously decreased significantly. Aminoglycoside antibiotics entrapped in the basolateral membrane vesicles inhibited (Na+ + K+)-ATPase activity, but those added to the basolateral membrane vesicles externally failed to do so. The activity of (Na+ + K+)-ATPase was non-competitively inhibited by gentamicin. It is thus concluded that aminoglycoside antibiotics are taken up into the renal proximal tubular cells across the brush-border membrane and inhibit the (Na+ + K+)-ATPase activity of basolateral membrane. This inhibition may possibly disrupt the balance of cellular electrolytes, leading to a cellular dysfunction, and consequently to the development of aminoglycoside antibiotics' nephrotoxicity. PMID- 3021219 TI - Amphotericin B enhanced anomalous potential difference response to changes in aqueous K+ in frog cornea. AB - An increase in aqueous K+ from 0 to 4 mM increased the potential difference (anomalous response of electrogenic (Na+ + K+)-ATPase antiport) by 1.1 mV in Cl( )-free solutions compared to 6.8 mV in Cl- solutions. With amphotericin B added to the tear solution in Cl(-)-free solutions, the anomalous PD response for the addition of 4 mM K+ to the aqueous solution was about 20 mV, significantly greater than in Cl- solutions. This anomalous response was inhibited by ouabain. These data support the electrogenicity of the (Na+ + K+)-ATPase pump. It is also evident that, for the pump to respond, Na+ should readily enter the cell. This may be accomplished experimentally, either across the basolateral membrane in Cl- solutions or across the apical membrane in Cl(-)-free solutions with amphotericin B present in the tear solution. PMID- 3021220 TI - Membrane effects of aminoglycoside antibiotics measured in liposomes containing the fluorescent probe, 1-anilino-8-naphthalene sulfonate. AB - The mechanism of membrane disturbance by aminoglycoside antibiotics was investigated in liposomes containing the fluorescent probe, 1-anilino-8 naphthalene sulfonate (ANS). Liposomes of PC and different anionic phospholipids (1:1 to 15:1 molar ratios) were challenged with aminoglycosides in the presence of low (1 microM) and high (3 mM) concentrations of calcium. Liposomes containing PIP2 showed the greatest drug-induced changes in ANS fluorescence in the presence of high and low concentrations of calcium and at all PC:PIP2 molar ratios tested. Liposomes containing other anionic phospholipids (PS, PI and PIP) were not reactive toward aminoglycosides in the presence of 3 mM calcium or when the ratio of PC to anionic lipid was increased to 10:1. The aminoglycoside-induced changes of ANS fluorescence were not due to any changes in the emission spectrum of ANS, nor to changes in quantum yield, nor to a change in the binding affinity of ANS. It is concluded that a specific aminoglycoside-PIP2 interaction results in phase separation of PC and PIP2 and thus increases the number of available ANS binding sites in PC:PIP2 liposomes. PMID- 3021222 TI - The plasma-membrane-associated form of SV40 large tumor antigen: biochemical and biological properties. PMID- 3021224 TI - A cloned fragment of HeLa DNA containing consensus sequences of satellite II and III DNA hybridizes with the Drosophila P-element and with the 1.8 kb family of human KpnI fragments. AB - We have cloned a repetitive EcoRI fragment from the human genome which displays weak homologies with the Drosophila melanogaster transposable P-element. This cloned DNA appeared not to be a mobile element but, instead, a divergent member of human satellite II or III DNAs. We present here the first complete nucleotide sequence of a 1.797 kilobase pair (kb) satellite-like DNA. Moreover, this EcoRI satellite monomer contains a unique sequence of 49 basepairs (bp) that is devoid of the satellite consensus repeat 5'TTCCA3'. Southern hybridization analysis revealed that the cloned insert is closely related to a highly repetitive 1.8 kb KpnI family of tandemly organized satellite DNAs. Thus, the relationships among these satellite DNA families appear to be complex and may be a factor in their copy number, position and spatial organization. PMID- 3021221 TI - Inhibition of erythrocyte Ca2+-ATPase by activated oxygen through thiol- and lipid-dependent mechanisms. AB - We have studied erythrocyte Ca2+-ATPase as a model target for elucidating effects of activated oxygen on the erythrocyte membrane. Either intracellular or extracellular generation of activated oxygen causes parallel decrements in Ca2+ ATPase activity and cytoplasmic GSH, oxidation of membrane protein thiols, and lipid peroxidation. Subsequent incubation with either dithiothreitol or glucose allows only partial recovery of Ca2+-ATPase, indicating both reversible and irreversible components which are modeled herein using diamide and t-butyl hydroperoxide. The reversible component reflects thiol oxidation, and its recovery depends upon GSH restoration. The irreversible component is largely due to lipid peroxidation, which appears to act through mechanisms involving neither malondialdehyde nor secondary thiol oxidation. However, some portion of the irreversible component could also reflect oxidation of thiols which are inaccessible for reduction by GSH, since we demonstrate existence of different classes of thiols relevant to Ca2+-ATPase activity. Activated oxygen has an exaggerated effect on Ca2+-ATPase of GSH-depleted cells. Sickle erythrocytes treated with dithiothreitol show a heterogeneous response of Ca2+-ATPase activity. These findings are potentially relevant to oxidant-induced hemolysis. They also may be pertinent to oxidative alteration of functional or structural membrane components in general, since many components share with Ca2+-ATPase both free thiols and close proximity to unsaturated lipid. PMID- 3021223 TI - Virology, genetics and immunology of murine lymphomagenesis. PMID- 3021225 TI - Nucleoside 5'-triphosphates with modified sugars as substrates for DNA polymerases. AB - A number of nucleoside 5'-triphosphate analogs were tested with Escherichia coli DNA polymerase I and Klenow fragment of the enzyme, bacteriophage T4 DNA polymerase and calf thymus DNA polymerase alpha. It was shown that 3'-amino-2',3' dideoxynucleoside 5'-triphosphates as well as a number of 3'-derivatives of dTTP(3'NH2) are able to terminate DNA synthesis catalyzed by each enzyme if the reaction is performed in the absence of natural substrates. ddNTP and dNTP(3'F) were found to be inactive with DNA polymerase alpha only, but araNTP(3'NH2) was inactive with E. coli DNA polymerase I. dTTP(3'N3), dGTP(3'N'3), dCTP(3'N3), araNTP(3'N3) and (alpha-thio)dTTP(3'F) were unable to inhibit any of the above mentioned DNA polymerases, in contrast to reverse transcriptase, accessible to the most nucleotide analogs tested. PMID- 3021226 TI - Eicosapentaenoic acid inhibits synthesis and secretion of triacylglycerols by cultured rat hepatocytes. AB - Primary cultures of rat hepatocytes were used to study the effects of eicosapentaenoic and oleic acid on synthesis and secretion of triacylglycerols associated with very low density lipoproteins. From the experiments the following was observed. Oleic acid markedly stimulates secretion as well as synthesis of triacylglycerols, whereas eicosapentaenoic acid causes very little or no increase in secretion or synthesis as compared to a fatty-acid-free medium. The effects could already be observed after 15 min incubation. The inhibitory effect of eicosapentaenoic acid is reversible within 1-2 h. Eicosapentaenoic acid inhibits much of the stimulatory effect of oleic acid on synthesis and secretion of triacylglycerols. The cellular uptake of eicosapentaenoic acid is somewhat higher than that of oleic acid and the metabolism of these fatty acids to acid-soluble materials is similar. Eicosapentaenoic acid does not affect the secretory pathway of triacylglycerols per se. From these results it may be concluded that the mechanism for the inhibitory effect of eicosapentaenoic acid on triacylglycerol secretion is probably via reduced triacylglycerol synthesis. PMID- 3021227 TI - High-affinity binding site for leukotriene C4 in human erythrocytes. AB - A specific, high-affinity binding site for leukotriene C4 was identified in human erythrocyte particulate fraction and in vesicle preparation. The binding was saturable, reversible and specific. Vesicle preparations showed that binding sites were localized on the outside of the plasma membrane. The dissociation constant and site density were found to be Kd = 15.9 +/- 3.2 nmol X 1(-1) and N = 152 +/- 35 sites per cell, respectively, as calculated from Scatchard analysis. The effect of leukotriene C4 did not modify the calcium influx and did not inhibit the ATPase-dependent calcium efflux. In this paper, the physiological significance of these sites is discussed. PMID- 3021228 TI - Spin-labelled analogues of GDP and GTP as site-specific reporter groups for guanosine nucleotide-binding proteins. AB - New derivatives of GDP and GTP have been synthesized for the spectroscopic investigation of the interaction between guanosine nucleotides and guanosine nucleotide-binding proteins. The 3'-hydroxyl group in these nucleotides was replaced by a 3'-amino group, which was further derivatized by the introduction of a spin-label reporter group. The biological activity of 3'SL-GDP and 3'SL-GTP could be demonstrated by measuring the interaction of these spin-labelled derivatives with bacterial elongation factor Tu. The amino modification and spin labelling only slightly influenced the affinity of the guanosine nucleotides for EF-Tu from Escherichia coli or Thermus thermophilus. Electron paramagnetic resonance (EPR) measurements revealed a strong immobilization of the labelled nucleotides upon binding to T. thermophilus EF-Tu. Significant differences between the spectra of EF-Tu X 3'SL-GDP, EF-Tu X 3'SL-GTP and aminoacyl-tRNA X EF Tu X 3'SL-GTP ternary complexes were observed. Our data demonstrate that spin labelled guanosine nucleotides can be used as sensitive spectroscopic probes for the investigation of the local environment of the nucleotide-binding site during distinct functional states of a guanosine nucleotide-binding protein. PMID- 3021229 TI - Purification of S1 nuclease to homogeneity and its chemical, physical and catalytic properties. AB - An S1 nuclease preparation was used to purify the enzyme to homogeneity. The enzyme had an isoelectric point of 4.2, and a high content of hydrophobic amino acids, especially tyrosine. It exhibited low 3'-ribonucleotidase activity. Circular dichroism analysis suggested that the contents of alpha-helix, beta structure and random coil are 25%, 31% and 44%, respectively. The enzyme contained about 3 g atoms Zn/mol and the removal of Zn from the enzyme by addition of EDTA resulted in disruption of its secondary structure with resultant inactivation. From Con A-Sepharose chromatography, we suggest that the enzyme is a high-mannose glycoprotein. After treatment with endo-beta-N acetylglucosaminidase H under moderate conditions, a small part of the enzyme was converted to a form lacking the sugar side chain. This form of the enzyme was as thermostable as the parent enzyme, suggesting that the sugar side chain may not be involved in thermostability of the enzyme. PMID- 3021230 TI - Mechanism of adenosine production by isolated rat heart mitochondria. AB - Adenosine production by isolated rat heart mitochondria was examined and was observed to be dependent on an active adenine nucleotide transporter and a functional 5'-nucleotidase. It was found that mitochondria do not transport adenosine. These results suggest that mitochondria provide AMP for an extramitochondrial 5'-nucleotidase and this was verified by direct measurement of extramitochondrial levels of AMP and adenosine. A possible role for mitochondria in myocardial adenosine production is discussed. PMID- 3021231 TI - Human placental ferritin receptor. AB - Brush-border membranes from human placenta were prepared and their purity was clarified by biochemical and morphological methods. Ferritin binding to these prepared membranes was examined using horse spleen 125I-apoferritin, and was found to be completed within 10 min at 37 degrees C and pH 7.5. The amount of ferritin bound to the membranes was found to be proportional to the amount of membrane added and saturable for a given amount of the membrane in the presence of excess ligand. The membranes exhibited specific ferritin binding with a Ka of 2.3 X 10(7) M-1 at pH 7.5. A competitive binding assay indicated that horse spleen 125I-apoferritin binding was inhibited by a 10-fold molar excess of horse spleen ferric ferritin and a 500-fold molar excess of human transferrin. These results suggest that human placental brush-border membranes have specific receptors for horse spleen apoferritin molecules. PMID- 3021232 TI - Interaction between enzyme-generated triplet carbonyls and molecules intercalated into DNA. AB - The phosphorescence from enzyme-generated and -protected triplet acetone is very efficiently quenched by dyes intercalated into DNA. The process is unlikely to be due to energy transfer and is tentatively ascribed to electron transfer occurring within the DNA helix complex with the acting enzyme. This quenching markedly protects DNA from breaks induced by triplet acetone. In the case of some barely emissive enzyme-generated triplet carbonyl species, it is possible to detect a weak emission resulting from the interaction with dye X DNA; this emission may be associated with back electron transfer. PMID- 3021234 TI - Vanadium complexes of transferrin and ferritin in the rat. AB - Vanadium associates with serum transferrin of rats administered vanadyl(IV) sulfate or ammonium metavanadate(V) by gastric intubation. Low molecular weight species account for only 3% of the vanadium present in plasma. The element distributes between the two major isotransferrins in proportion to their concentrations. Rat apotransferrin binds both vanadium(IV) and vanadium(V), forming 2:1 metal-protein complexes in both instances. Although the two isotransferrins apparently differ in their physiological properties, they exhibit identical vanadyl(IV) (VO2+) EPR spectra, indicating identical or very similar metal binding sites for both proteins. In contrast to other transferrins, the two sites of the rat protein are spectroscopically indistinguishable and exhibit a VO2+ EPR spectrum similar to that of the C-terminal metal binding site of human serum transferrin. VO2+ EPR signals are observed with liver, spleen, and kidney tissue samples from animals maintained on a vanadium-supplemented diet. These signals arise from a specific intracellular VO2+ complex with the iron storage protein ferritin. PMID- 3021233 TI - Membrane-bound 5'-nucleotidase inhibitor in Ehrlich ascites tumour cells and newborn mouse liver. AB - 5'-Nucleotidase activity in Ehrlich ascites tumour cells was undetectable. The cell homogenate, when mixed with adult mouse liver homogenate, inhibited the 5' nucleotidase activity of the latter, without affecting its p-nitrophenyl phosphate-hydrolysing activity. The inhibitor activity was enriched (6.8-fold) in a membrane fraction which was enriched in (Na+ + K+)-ATPase (14-fold) and alkaline phosphatase (8-fold). 5'-Nucleotidase activity in this membrane fraction could be detected only after separating the inhibitor activity from the enzyme on Sephadex G-50. The inhibitor activity was decreased by 27% when heat-treated, 33% when treated with 6 M urea and was almost completely lost when treated with trypsin. It was dialysable from a tubing with a molecular exclusion limit of 10,000, but was retained in a tubing with an exclusion limit of 3000. From these results we conclude that a small molecular weight protein inhibitor(s) of 5' nucleotidase is present in the plasma membrane of Ehrlich ascites tumour cells. Also, the presence of such an inhibitor in the newborn mouse liver but not in the adult liver suggests that it may have some role in cellular ageing and cancer. PMID- 3021235 TI - Effect of pertussis toxin on the phosphodiesteratic cleavage of the polyphosphoinositides by guanosine 5'-O-thiotriphosphate and thrombin in permeabilized human platelets. AB - It has recently been shown in this laboratory that permeabilization of human platelets with 15-25 micrograms/ml saponin allows ADP-ribosylation by pertussis toxin of the alpha i-subunit of Gi (Ni), a guanine nucleotide-binding regulatory protein. The same assay conditions have been used to determine phospholipase C in permeabilized platelets. Guanosine 5'-O-thiotriphosphate- (GTP[gamma S]-) activated phospholipase C in permeabilized platelets whose inositol phospholipids were prelabeled with [3H]inositol. Phospholipase C activity was measured by [3H]polyphosphoinositide decreases and formation of [3H]inositol bisphosphate and [3H]inositol trisphosphate. Prostacyclin, cyclic AMP or pretreatment of permeabilized platelets with pertussis toxin did not alter this effect under conditions in which the alpha i-subunit was effectively ADP-ribosylated by pertussis toxin. This information indicated that ADP-ribosylation of Gi-protein was not directly related to activation or inhibition of platelet phospholipase C by GTP [gamma S]. Thrombin also activated phospholipase C in permeabilized platelets and, surprisingly, this action was enhanced by pertussis toxin pretreatment. This indicates that ADP-ribosylation of Gi-protein facilitates the action of thrombin on phospholipase C. PMID- 3021236 TI - Stimulation of oxygen consumption at the cytochrome A3 level inhibits aldosterone biosynthesis from 18-hydroxycorticosterone. AB - A mitochondrial preparation from duck adrenal gland was used, under aerobic conditions, to show that the oxygen requirement for the last step of aldosterone biosynthesis (transformation of 18-hydroxycorticosterone into aldosterone) is at the cytochrome P-450 level only. Vitamin C and tetramethyl-p-phenylene-diamine (TMPD) were used to increase oxygen consumption at the cytochrome a3 level, thereby decreasing its availability to cytochrome P-450. The vitamin C plus TMPD system acts as an 'oxygen trap'. Results show that despite reducing equivalents provided by L-malate, vitamin C plus TMPD strongly inhibits aldosterone biosynthesis from 18-hydroxycorticosterone (89%). Moreover, we used KCN in order to block oxygen consumption, even in the presence of vitamin C plus TMPD. Under these conditions, the inhibition of aldosterone biosynthesis from 18 hydroxycorticosterone is reduced by 51%. The reversal of this inhibition by KCN was evident but only partial. According to polarographic and electron microscopy studies, the reversal of inhibition can only be explained by an increased availability of oxygen at the cytochrome P-450 level. Experiments performed under aerobic conditions, without a nitrogen atmosphere, show that oxygen is required in the transformation of 18-hydroxycorticosterone into aldosterone, at the cytochrome P-450 level. This suggests that a classical hydroxylating mechanism is involved. PMID- 3021237 TI - Modulation of prostacyclin generation in cultured human vascular endothelial cells and the effects of human alpha-natriuretic polypeptide. AB - Vascular endothelial cells have been known to possess not only a barrier function but also other biologically important functions maintaining vascular homeostasis. Among these, the generation of prostacyclin is one of the most conspicuous functions, and the modulation of its synthesis and liberation has been of interest with reference to the interaction with several vasoactive substances, including human atrial alpha-natriuretic polypeptide. This paper investigates the regulatory mechanism of prostacyclin generation using cultured human vascular endothelial cells as far as Ca2+ flux, (Na+ + K+)-ATPase activity, and Na+-Ca2+ exchange systems are concerned. Through these experimental studies the following results were obtained. Prostacyclin generation was triggered by an increase of Ca2+ influx, and an increase in intracellular Na+ also enhanced it, and this was accompanied by a decreased Ca2+ efflux arising from suppression of Na+-Ca2+ exchange systems. (Na+ + K+)-ATPase activity as well as prostacyclin generation was also enhanced by the increase of intracellular Na+. These results indicate a possible link between the mechanism which generates prostacyclin in the human vascular endothelial cells and the mobilization of electrolytes; however, in this aspect human atrial alpha-natriuretic polypeptide had no effect on the endothelial cells. PMID- 3021238 TI - Effect of cellular fatty acid composition on the phospholipase A2 activity of bone marrow-derived macrophages, and their ability to induce lucigenin-dependent chemiluminescence. AB - Mouse bone marrow macrophages were obtained by cultivation in serum-free medium. Addition of specific fatty acids to the medium leads to macrophage populations which differ in their fatty acid composition. The fatty acid composition of the cellular membranes directly modulates functional abilities of the macrophages such as the generation of superoxide anion and phospholipase A2 activity in response to phorbol ester and zymosan. Both capacities were lowest in macrophages cultured serum-free without lipids. Incorporation of unsaturated fatty acids into macrophage phospholipids leads to an increase of O2- production as measured by lucigenin-dependent chemiluminescence and to an increased phospholipase A2 activity after challenge with phorbol ester or zymosan. PMID- 3021239 TI - Altered physiological responsiveness and decreased cyclic AMP levels in rat atria after dietary cod liver oil supplementation and its possible association with an increased membrane phospholipid n-3/n-6 fatty acid ratio. AB - Rats were given a cod liver oil supplemented diet and a standard diet for 4 months. The cod liver oil supplementation resulted in a marked increase in the 20:5(n-3) and 22:6(n-3) fatty acids and a marked decrease in the 20:4(n-6) fatty acid in phosphatidylcholine and ethanolamine of the atrial membrane. Atria from the cod liver oil treated rats showed a marked decrease in contractile force, heart rate and cyclic AMP (cAMP) levels under basal conditions. Stimulation with noradrenaline (1 X 10(-6) M) during high oxygen saturation and reoxygenation resulted in an equal increase in the mechanical responses of the two groups in spite of the significantly different levels of cAMP, whereas in hypoxia, both the cAMP level and the contractile force were significantly lower in the cod liver oil treated group. These results indicate that changes in the fatty acid composition of heart membrane phospholipids is associated with changes in adenylate cyclase activity and physiological function of the rat heart and that an increase in the n-3/n-6 fatty acid ratio in membrane phospholipids of the heart results, when oxygen is abundant in enhanced cAMP-independent contractile activity. PMID- 3021240 TI - [Changes in the properties of brain opiate receptors during the development of morphine tolerance in rats]. AB - The effects of prolonged administration of morphine on the properties of opiate receptors of rat brain were studied. For this purpose the isotherms of binding of labeled mu-, delta-, and chi-ligands--morphine, D-Ala2, D-Leu5-enkephalin and ethylketocyclazocine--with brain membrane preparations of morphine-tolerant rats as well as those of control animals were analyzed. For quantitative determination of dissociation constants of the ligand-receptor complexes (K) and receptor concentrations ([Q]), the difference and simulation methods were used. It was shown that the values of K and [Q] vary within broad ranges in individual animals, whereas the individual variations of the [Q]/[K] ratios in controls or in morphine-tolerant rats are not so significant. This suggests [Q]/K to be one of the basic criteria for a comparison of properties of opiate receptors in different groups of animals. The use of this criterion and of the simulation method demonstrated that the development of tolerance causes changes in the properties of delta-receptors (the [Q]/K ratio decreases by greater than 50%). Unlike delta-receptors, the tolerance has no appreciable effect on the properties of mu- or chi-receptors or on the superhigh affinity binding sites of the ligands tested. PMID- 3021242 TI - [Active transport of Na+ by modified Na,K-ATPase]. AB - The ability of ATP, CTP, ITP, GTP and UTP to induce ouabain-sensitive accumulation of Na+ by proteoliposomes with a reconstituted Na/K-pump was studied. At low Na+/K+ ratio (20 mM/50 mM), a correlation was observed between the proton-accepting capacity of the nucleotide and its efficiency as an active transport substrate. In order to test the hypothesis on the role of the negative charge in position 1 of the purine (3-pyrimidine) base of the nucleotide in the reversible transitions from the Na- to the K-conformations of Na,K-ATPase, two ATP analogs (N1-hydroxy-ATP possessing a proton-accepting ability and N1-methoxy ATP whose molecule carries a negative charge quenched by a methyl group) were used. The first substrate provides for active accumulation of Na+ by proteoliposomes at a rate similar to that of ATP, whereas the second substrate is fairly ineffective. PMID- 3021241 TI - [Peroxidase activity of catalase modified by progesterone]. AB - Catalase conjugates with 3, 7, 9 and 42 progesterone molecules were obtained by the reaction between the enzyme and N-oxy-succinimide ether of 3-0 carboxymethyloxime of progesterone. The enzyme modified by 42 progesterone molecules is effective in o-dianisidine oxidation by hydrogen peroxide and has a kcat/KM value of 512 M-1 s-1. The catalase conjugates with 3, 7 and 9 progesterone molecules exhibit a high activity during o-dianisidine oxidation by cumene hydroperoxide. The activity of conjugates is higher than that of the native non-modified enzyme in the same reaction. The maximum effectiveness was observed for catalase modified by 7 progesterone molecules. This conjugate is characterized by kcat/KM of 99,000 M-1 s-1 at 30 degrees C. The effect of the degree of enzyme modification on the kinetic parameters of o-dianisidine oxidation by H2O2 and cumene hydroperoxide is discussed. PMID- 3021243 TI - [Affinity modification of creatine kinase and ATP-ADP translocase in heart mitochondria: determination of their stoichiometric ratio]. AB - The interaction of mitochondrial creatine kinase and ATP-ADP translocase with 2.3 dialdehyde derivatives of ADP and ATP (oADP and oATP) has been studied. It was shown that these compounds are irreversible and specific inhibitors of creatine kinase (KioADP = 0.6mM, KioATP = 1.12 mM) and ATP-ADP translocase (KioADP = 0.065mM, KioATP = 0.14 mM). The substrates protect both enzymes from inactivation by these compounds. The maximal pseudo-first order rate constants for the 2,3 dialdehyde nucleotide derivative interaction with creatine kinase are 0.2 min-1 for oADP (pH 6.5) and 0.11 min-1 for oATP (pH 7.0). A decrease in the creatine kinase activity correlates with the incorporation of the reagent into the protein. The completely inactivated, isolated and purified enzyme contains 1 mol of oADP per mole of active sites. A procedure for simultaneous determination of the creatine kinase and translocase content in mitochondria and mitoplasts has been developed, which is based on the application of [3H]oADP in combination with specific treatment of mitochondria (or mitoplasts) with carboxyatractyloside 2,4 dinitrofluorobenzene and a mixture of creatine kinase substrates (MgADP + phosphocreatine). It has been found that for heart mitochondria from different animals the content of creatine kinase and translocase is 2.1-2.6 and 2.4-2.9 mol per mol of cytochrome c oxidase, respectively. Thus, the stoiochiometric ratio of creatine kinase and ATP-ADP translocase is close to 1.0 for all mitochondrial preparations under study (i.e. rat, dog, rabbit and chicken). PMID- 3021244 TI - [Cleavage of DNA adsorbed on the surface of phospholipid membranes by restrictases type II]. AB - Cleavage of phage lambda DNA by restriction endonucleases in the presence of model phosphatidylcholine membranes was studied. Bsp1, Pst1 and Bam H1 were found to cleave DNA under these conditions to a considerably decreased extent. This effect does not result from irreversible inactivation of the enzymes or their direct interaction with the membranes. The most probable explanation of the membrane inhibitory effect is the change of DNA substrate properties resulting from its Mg2+-mediated binding to the membranes. PMID- 3021245 TI - [Fatty acid composition of total lipids and phospholipids of membrane preparations of transport ATPases]. AB - The fatty acid composition of total lipids and phospholipids of duck salt gland Na,K-ATPase (outer plasma membrane) and of rabbit skeletal muscle Ca-ATPase (intracellular membrane) was investigated. The bulk of Na,K-ATPase fatty acids is represented by palmitic (16:0), oleic (18:1), stearic (18:0) and arachidonic (20:4) acids. The duck salt gland is characterized by rather a high content of unsaturated fatty acids, especially of arachidonic acid. The unsaturation index of total-lipid fatty acids increases during purification of these preparations in the following order: homogenate greater than microsomal fraction greater than purified enzyme. The fatty acid composition of Na,K-ATPase total lipids and phospholipids reveals certain differences. Phospholipids contain more stearic and liholeic (18:2) acids than total lipids, but the level of arachidonic acid in them is twice as low. Besides, phospholipids were found to contain polyunsaturated docosohexaenic (22:6) acid. The bulk of fatty acids of rabbit skeletal muscle Ca-ATPase total lipids and phospholipids is represented by 16:0, 18:0, 18:1 and 18:2 acids. The content of polyunsaturated fatty acids in this preparation is much lower than in duck salt gland Na,K-ATPase. The fatty acid composition of total lipids and phospholipids in rabbit skeletal muscle Ca-ATPase differ insignificantly. The differences in the fatty acid composition of membrane preparations under study is conditioned mainly by the fractional composition of their lipids. PMID- 3021246 TI - Inositol phosphatase in developing rat duodenum, jejunum and ileum. AB - Inositol phosphatase and phytase activities in different segments of the rat small intestine were measured during postnatal development. In the duodenum and jejunum, inositol phosphatase activity (units/g tissue) was low during the suckling period and increased at weaning, reaching a peak of activity at 4 weeks of age. In the ileum, peak activity was observed during the suckling period with a sharp decline at weaning. Phytase activity was very low during the suckling period in all segments, and increased to exhibit a peak at 4 weeks of age in the duodenum, and to a lesser extent in the jejunum (low activity was maintained in the ileum). The content of inositol phosphatase activity in the duodenum increased rapidly during the suckling period to reach its maximum at 4 weeks of age. This suggests a relationship to cell proliferation rate in the small intestinal mucosa. PMID- 3021247 TI - Effect of endogenous opioid blockade on fetal cardiovascular and sympathoadrenal responses to hypoxemia induced by umbilical cord constriction. AB - The cardiovascular and plasma catecholamine responses to hypoxemia following endogenous opioid blockade were evaluated in 18 late gestation fetal lambs (0.75 0.83 gestation) in utero. Hypoxemia was produced by progressive constriction of the umbilical cord with an inflatable silicone rubber cuff while fetal heart rate, blood pressure, and arterial blood gases were monitored. Fetal arterial blood samples were obtained for plasma catecholamine analysis using a radioenzymatic method. The animals were divided into 4 experimental groups. Control animals during normoxemia and following hypoxemia are represented by groups I and III, respectively. During umbilical constriction, arterial oxygen tension declined from 21.4 +/- 0.3 to 13.4 +/- 0.3 torr (p less than 0.001, mean +/- SE) and was accompanied by an increase in blood pressure (44 +/- 1 to 53 +/- 1 torr), decrease in heart rate (176 +/- 4 to 114 +/- 4 beats/min) and rise in plasma norepinephrine (437 +/- 90 to 3,410 +/- 617 pg/ml) and epinephrine (53 +/- 10 to 1,074 +/- 325 pg/ml). Naloxone infusion had no significant effect on the fetus during either normoxemic (group II) or hypoxemic conditions (group IV). Plasma catecholamines rose less in group IV than in hypoxemic control fetuses (norepinephrine: 2,407 +/- 406 vs. 3,410 +/- 617 pg/ml; epinephrine: 966 +/- 641 vs. 1,074 +/- 305 pg/ml), but the differences were not significant (p greater than 0.2). It is concluded that endogenous opioids do not play a major role in the modulation of cardiovascular and sympathoadrenal adaptations to moderate hypoxemia in the fetus. PMID- 3021250 TI - Sequence-dependence of the CD of synthetic double-stranded RNAs containing inosinate instead of guanylate subunits. PMID- 3021249 TI - The effects of ethylene dimethane sulphonate (EDS) on rat Leydig cells: evidence to support a connective tissue origin of Leydig cells. AB - Ethylene dimethane sulphonate (DS) administered to adult male rats in a single dose of 75 mg/kg body weight results in a rapid destruction of Leydig cells which, in turn, is associated with a marked decline in levels of serum testosterone. For 24-72 h after treatment with EDS (post-EDS) the Leydig cells undergo degenerative changes consisting of chromatin condensation and cytoplasmic vacuolation, and testicular macrophages progressively remove Leydig cells from the intertubular tissue by phagocytosis. This results in the total absence of Leydig cells on Days 7-14 and the absence of any detectable specific 125I-hCG binding to testis homogenates. Associated with the low levels of serum testosterone, levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in serum rise, LH to levels found in castrate rats. Morphometric and 125I hCG binding studies indicate that a new generation of Leydig cells develop from Day 21 and reach control levels by Day 49. Morphologic observations suggest that the Leydig cells arise by differentiation from a pool of connective tissue cells that includes fibroblasts, lymphatic endothelial cells and pericytes. The new Leydig cells, which appear around Day 21 post-EDS, have the features of fetal Leydig cells. The latter appear to transform into Leydig cells typical of normal adult rats between 35-49 days post-EDS. The differentiation of new Leydig cells is associated with a reestablishment of normal levels of testosterone 21 days post-EDS. Serum LH and FSH return to normal at 28 days and 49 days respectively. PMID- 3021248 TI - Testosterone production by collagenase-dispersed cells from baboon fetal testis. AB - We determined whether dehydroepiandrosterone (DHA) and androstenedione (A) were converted to testosterone (T) by the midgestation primate fetal testis in the absence of gonadotropins. Testes from six baboon (Papio anubis) fetuses, obtained by cesarean section at Day 100-107 of gestation (term = Day 184) were dispersed with 0.2% collagenase. Cells (1.1 X 10(6)) were suspended in 4 ml Eagle's Minimum Essential Medium containing penicillin/streptomycin (MEM) and incubated for 20 h (37 degrees C) with or without DHA, A, pregnenolone (P5), 17 alpha hydroxypregnenolone (17OH-P5), progesterone (P4) or 17 alpha-hydroxyprogesterone (17OH-P4). Concentrations of T, A, P4, and 17OH-P4 in the medium and cells were measured by radioimmunoassay. Mean secretions of T and A, in the absence of exogenous substrates, were 0.5 +/- 0.2 and 0.8 +/- 0.3 ng/mg testis, respectively, and were not elevated by human chorionic gonadotropin (hCG). Addition of DHA at 100, 500, or 1000 ng/4 ml increased (p less than 0.05) the production of T to 6 +/- 0.6, 33 +/- 10, and 64 +/- 26 ng/mg testis and the production of A to 13 +/- 5.5, 54 +/- 10, and 67 +/- 22 ng/mg testis, respectively. Similarly, addition of A at 100, 500, or 1000 ng/4 ml increased (p less than 0.05) production of T to 27 +/- 5.3, 155 +/- 29, and 254 +/- 79 ng/mg testis, respectively. In contrast, production of T and A remained near baseline concentrations when cells were incubated with 1000 ng/4 ml of P5, P4, 17OH-P5, or 17OH-P4.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021251 TI - Pharmacokinetics of oral 500-mg penicillamine: effect of antacids on absorption. PMID- 3021252 TI - Effect of alkylation with streptozotocin on the secondary structure of DNA. AB - S1 nuclease hydrolysis and hydroxyapatite chromatography were used to study the effect of the alkylating antibiotic, streptozotocin, on the secondary structure of DNA. Native calf thymus DNA was alkylated in vitro with increasing concentrations of streptozotocin and subjected to S1 nuclease hydrolysis. An increasing degree of DNA degradation was seen, suggesting a destabilization of the secondary structure. Indirect evidence, deduced from alkaline hydrolysis, effect of NaCl on S1 nuclease hydrolysis, and hydroxyapatite chromatographic analysis of alkylated DNA, suggested a significant alkylation of DNA phosphates in addition to DNA bases. Nicotinamide has been reported to alter the cytotoxic and carcinogenic effects of streptozotocin. Our experiments indicate that in the presence of nicotinamide, streptozotocin causes the formation of a greater proportion of alkylated bases in relation to alkyl phosphotriesters. This may have significance in relation to the differential cytotoxicity of streptozotocin in the absence and presence of nicotinamide. PMID- 3021254 TI - Isolation of glycoproteins of parainfluenza I (Sendai) by Helix pomatia Lectin column. AB - Glycoproteins of Sendai virus were successfully isolated on a column of Helix pomatia Lectin-Sepharose 6MB following solubilization with Nonidet P-40. This technique can be used as a preparative step for the study of viral glycoproteins. PMID- 3021253 TI - Interaction of a bovine thymic peptide extract with vasoactive intestinal peptide (VIP) receptors. AB - Bovine thymic peptide extract (1-100 micrograms/ml) is shown to completely inhibit the binding of [125I]VIP to rat blood mononuclear cells, lymphoid cells of spleen, and liver plasma membranes. In the three models, the bovine thymic peptide extract inhibits [125I]VIP binding with a potency that is 4000-7000 times lower than that of the native VIP, on a weight basis. In rat liver plasma membranes, the bovine thymic peptide extract stimulates adenylate cyclase with a maximal efficiency that is similar to that of VIP. At maximal doses, VIP and thymic peptide extract do not exert an additive effect on adenylate cyclase, suggesting that the activation of the enzyme by the bovine thymic peptide extract occurs through VIP receptors. Finally, no VIP-like immunoreactivity was detected in the thymic peptide extract using an antiserum raised against mammalian VIP. All these data suggest the presence in the bovine thymic peptide extract of a new substance which behaves as a VIP agonist in rat. PMID- 3021255 TI - [Effect of preliminary adaptation on 99mTc pyrophosphate accumulation and the development of structural changes in the heart muscle under stress]. AB - It is established that preliminary adaptation of animals to physical exertion and short-term stress prevents the contracture and necrotic changes, as well as 99mTc pyrophosphate accumulation in the heart muscle caused by durable emotional stress. PMID- 3021257 TI - [In vitro effect of synthetic water-soluble antioxidants of the class of screened phenols on the beta-adrenergic system in the cardiocyte plasma membranes of rats]. AB - Influence of synthetic water-soluble antioxidants gamma-(4-oxi-3,5-ditret butylphenol) propionate (phenozan) and the potassium salt of phenozan on the signal processing in beta-receptor-adenylate-cyclase complex of rat cardiocyte membranes has been studied. It was demonstrated that these compounds act at the level of signal transduction from receptor to adenylate cyclase catalytic subunit rather than at the level of ligand-receptor complex formation. At concentrations exceeding-10 microM the antioxidants inhibit both isoproterenol-stimulated synthesis of cyclic 3',5'-AMP and accumulation of the products of lipid peroxidation in membranes. It is proposed that in vitro addition of antioxidants on cardiocyte beta-receptor-adenylate-cyclase complex is a result of alteration of physico-chemical properties of membrane lipids caused by these inhibitors of free radical reactions. PMID- 3021256 TI - [Metabolic level and the function of the peripheral catecholaminergic systems in immobilization hypothermia in rats]. AB - Metabolic peculiarities were studied on the model of prolonged immobilization hypothermia in rats (body temperature +20 degrees C for 24 h). Stress reactions and the state of peripheral catecholaminergic systems were compared in hypo- and normothermia. A direct correlation was established between the intensity of metabolism and the mediator activity in adrenergic nerve structures. PMID- 3021258 TI - [Cyclic GMP- and AMP-modified calcium binding by the myocardial sarcolemma in circulatory hypoxia]. AB - 10(-6) M cAMP were shown to induce a 61% and 21% increase in 45Ca binding to sarcolemma proteins in intact and injured (circulatory hypoxia) hearts, respectively. The addition of exogenous protein kinase equalized 45Ca-binding levels in normal and impaired sarcolemma. The decrease in 45Ca-binding capacity by 16 and 36% was detected in the presence of 10(-7) and 10(-6) M of cGMP, respectively. In impaired hearts, cAMP and Ca-ATPase activity levels remain constant, while cGMP content increases, as compared to normal myocardial level. PMID- 3021259 TI - [Changes in the phospholipid-phospholipid ratios and the enzyme activity of antioxidant protection in the rat lung during dehydration]. AB - It was established that water deprivation during 3, 6, 9 days caused a distinct decrease in phospholipid level and disturbances of phospholipid composition in the rat lung tissue. It was accompanied by alterations in the activity of antioxidant defense system enzymes (superoxide dismutase, glutathione peroxidase, glutathione reductase, catalase, glucose-6-phosphate dehydrogenase). These data are indicative of lipid peroxidation intensification in the rat lungs during water deprivation. PMID- 3021260 TI - [Effect of mineralocorticoids on the Na, K-ATPase activity of the rat brain]. AB - The effect of desoxycorticosterone (DOC) on Na, K-ATPase activity was studied in vivo and in vitro on microsomal rat brain fractions. An hour after intramuscular administration of DOC a noticeable increase in the enzyme activity was observed. Preincubation of microsomal brain fractions with 5 and 15 mkg/ml of DOC caused a decrease in Na, K-ATPase activity, with the results evident 3-5 minutes after the addition of the hormone into the incubation medium. The idea of a two-phase hormonal effect is suggested. It is likely that desoxycorticosterone effect is realized both by the direct influence, on Na, K-ATPase of the brain plasma membrane and by the influence on the biosynthesis. PMID- 3021261 TI - [Contrasting effect of piracetam and proline on the release of 3H-D-aspartic acid from the cerebral cortical synaptosomes of rats]. AB - Piracetam (at concentrations of 10(-6) and 10(-5), but not 10(-4) and 5 X 10(-4) M) decreased K+-stimulated 3H-D-aspartate release. Proline enhanced K+-stimulated D-aspartate release, and its effect was antagonized by piracetam at a concentration that had no effect on K+-stimulated release. Quisqualic acid attenuated K+-stimulated D-aspartate release, with the effect antagonized by GDEE. GDEE also blocked the effect of piracetam, but not proline. The data are discussed in terms of the role of excitatory amino acid neurotransmission in the mechanisms of amnestic and antihypoxic piracetam action. PMID- 3021262 TI - [Effect of transposons on the system regulating derepressed plasmid tra-genes]. AB - The ability of standard plasmids of six Fin-groups to inhibit the functions of genes transferring derepressed F-like plasmids has been studied. It is shown that transposons incorporation into the structure of individual plasmids alters the regulatory system of plasmid tra-genes. PMID- 3021263 TI - Decrease in subunit aggregation of phosphoribosylpyrophosphate synthetase: a mechanism for decreased nucleotide concentrations in pyruvate kinase-deficient human erythrocytes. AB - Pyruvate kinase (PK)-deficient RBCs have several unexplained metabolic abnormalities, such as decreased concentrations of total adenine nucleotides (AMP, ADP, and ATP) and total (oxidized and reduced) nicotinamide adenine dinucleotide (NAD). Because 5-phosphoribosyl-1-pyrophosphate (PRPP) is an intermediate in the synthesis of adenine nucleotides and NAD, we investigated PRPP synthetase (PRPPS), the enzyme responsible for PRPP synthesis. This enzyme is regulated, in part, by changes in its state of subunit aggregation. The proportion of aggregated PRPPS can be altered in vitro by ATP and 2,3 diphosphoglycerate (DPG). Because PK-deficient RBCs have decreased ATP and increased DPG concentrations, we examined the state of subunit aggregation of PRPPS in RBCs from normal and PK-deficient subjects, using gel permeation chromatography. Young normal RBCs have more aggregated PRPPS than do older RBCs. In contrast, due to their decreased ATP and increased DPG concentrations, PK deficient RBCs contain less aggregated PRPPS than do RBCs of comparable age without PK deficiency. These data suggest that PRPPS should be less active in vivo in PK-deficient RBCs. This may play a key role in mediating the decreases in total adenine nucleotide and total NAD concentrations in these RBCs. PMID- 3021265 TI - Neuromuscular transmission defect in inherited polyneuropathy. AB - A 51-year-old woman with an inherited polyneuropathy had fatiguability and a dramatic historical response to prostigmine. Repetitive motor nerve stimulation produced a prominent decrement of the compound muscle action potential in distal muscles with marked facilitation after brief exercise. Defective neuromuscular transmission paralleled the polyneuropathy in distribution and severity. We hypothesize that deficient release of acetylcholine by regenerating or degenerating nerve terminals likely caused the defect of neuromuscular transmission in this patient. PMID- 3021264 TI - Frequent and extensive deletion during the 9,22 translocation in CML. AB - Chromosomal translocation is one mechanism by which cellular oncogenes may be activated during tumorigenesis. The translocation of the abl oncogene to the Philadelphia chromosome in chronic myelogenous leukemia (CML) results in a new RNA transcript that fuses sequence from chromosome 22 to sequence from the abl oncogene. This RNA presumably codes for a new abl-related protein product found in CML, the activity of which is different from the normal abl protein. The molecular structure of the translocation varies from patient to patient, and the individual variation in RNA transcript and protein product remains to be defined. This report describes the frequent occurrence of chromosomal deletion within the 9q+ chromosome during these translocations. The location of the deletions suggests that some mechanism maintains the chromosomal breakpoint on the Philadelphia chromosome within a limited region. These deletions complicate the interpretation of Southern blots as a means of detecting the translocation. PMID- 3021266 TI - Cytological and ultrastructural changes in the pituitary pars distalis of the goldfish kept in calcium-free environments. AB - The cytology and ultrastructure of the pars distalis, mainly that of prolactin (PRL) cells, were investigated in goldfish adapted to fresh water (FW) or deionized water (DW) for 20 and 40 days, or gradually adapted to 1/3 artificial sea water (ASW) or 1/3 Ca-free sea water. When compared to PRL cells of goldfish kept in FW, those of goldfish adapted to DW did not show signs of increased activity. The lack of exocytotic activity and the low development of various organelles suggested that cell activity was slightly reduced. In 1/3 ASW, PRL cells were smaller and less active. In 1/3 Ca-free ASW, PRL cells appeared slightly stimulated compared with those of fish in 1/3 ASW. The Golgi area was more developed and a few lamellae of endoplasmic reticulum were observed in some cell islets. However, there was no significant difference between PRL cells of goldfish kept in 1/3 Ca-free ASW and in FW. In 1/3 ASW, which is isosmotic to the blood, thyrotrophs (TSH cells) corticotrophs (ACTH cells) and somatotrophs (STH cells) were not clearly affected. In DW, these cells and their nuclei were significantly enlarged. Their stimulation was also evident in 1/3 Ca-free ASW; values for cellular and nuclear areas were maximal in this environment and significantly higher than those of fish in FW and 1/3 ASW. These data suggest that in addition to the PAS-positive cells of the pars intermedia, highly stimulated in Ca-free environments, other cell types of the pars distalis may be involved in osmoregulation, and that the role of PRL cells is not primordial in the goldfish. PMID- 3021267 TI - Serologic screening for cytomegalovirus, rubella virus, herpes simplex virus, hepatitis B virus, and Toxoplasma gondii in two urban populations of pregnant women in Chile. PMID- 3021269 TI - Serum concentrations of sex hormone binding globulin in lung cancer. PMID- 3021268 TI - Assessment of need for coordinated approach in families with victims of head injury. AB - Forty two men with severe head injury, and 41 with minor head injury, together with their families, were assessed at home after the injury. Despite significant impairment with respect to physical symptoms, personality difficulties, and occupational status in severely injured patients after one year, there was a very poor uptake of hospital rehabilitation facilities. In addition, patients' relatives showed significant psychosocial impairment throughout this period. There is a need for a specialist to coordinate rehabilitation services for patients with head injury and their relatives and, in particular, to integrate physical and psychological aspects of management with a multidisciplinary team approach. Although this task will require specialist hospital teams for future development, at present general practitioners have some specialised knowledge that would enable them to coordinate rehabilitation. PMID- 3021270 TI - Hyoplastic [correction of Hyperplastic] anaemia and parvovirus infection. PMID- 3021271 TI - Insulinoma producing progressive neurological deterioration over 30 years. PMID- 3021272 TI - Viral warts: a new look at an old problem. PMID- 3021273 TI - Prevalence of antibody to poliovirus in England and Wales 1984-6. AB - Serum from 995 subjects aged 6 months to 99 years was screened at a dilution of 1/8 for neutralising antibodies to poliovirus. Of these samples, 975 (98%) contained antibody to at least one serotype, and 763 (77%) contained antibody to all three, an improvement since previous studies. Children aged 8 to 15, however, had a low prevalence of antibody to poliovirus type 3, with only four (40%) of those aged 12 protected. This finding is possibly due to the waning of antibodies induced by the type 3 component of oral poliovirus vaccine and emphasises the continued importance of a booster dose of vaccine for those leaving school. PMID- 3021274 TI - Separation of microtubule populations in rat brain homogenates by differential centrifugation. AB - Subcellular fractions were separated from rat homogenates at 600 g for 5 min (P1), 15,000 g for 10 min (P2), 48,000 g for 60 min (P3), 100,000 g for 150 min (P4) and a final supernatant (S). They contained 5.0, 14.3, 14.7, 24.7 and 41.3% of the total tubulin in the homogenate measured by colchicine binding. The microtubules recovered in a pool of fractions P1 + P2 + P3 underwent a faster depolymerization than those of P4 when the sediments were suspended in buffered solutions, poor in the stabilizing agent glycerol. As was observed with the electron microscope, P4 was richer than the other fractions in microtubules associated with membranous structures (MTA) by filamentous lateral connections. Subcellular distribution and lability properties may be the result of the connection of microtubules with membranous structures; the attached membranes would decrease the sedimentation velocity of microtubules, and lateral bridges could increase microtubular stability. Differences in length were not a cause of microtubule separation since this parameter did not vary among the microtubule of the different fractions or between MTA and non-associated microtubules of the same fraction. PMID- 3021275 TI - Studies on the effects of intrathalamically injected DADL and morphine on nociceptive thresholds and electroencephalographic activity: a thalamic delta receptor syndrome. AB - Bilateral microinjections of DADL (D-Ala2-D-Leu5-enkephalin) and morphine were carried out in rats in a systematic fashion at histologically identified medial and lateral thalamic sites. DADL produced a dose-dependent (1.5-15.0 nmol), naloxone-reversible (1 mg/kg, i.p.) increase in the hot-plate (HP), tail-flick (TF) and catalepsy (CAT) response latencies with a predominance of activity occurring at lateral as opposed to medial thalamic sites. These effects were seen within 5 min of microinjection. At a significant number of sites, DADL precipitated convulsive seizure activity. Equimolar doses of morphine had a negligible effect on nociceptive indices and were not productive of seizures even at sites where DADL was found to be active. To further examine seizure activity, rats were prepared with bilateral frontal cortical electrodes and microinjected also at medial and lateral thalamic sites with equimolar doses of DADL and morphine (15 nmol). DADL was found to produce electrographically defined seizures unaccompanied by convulsive motor behavior (cataleptic seizures), as well as convulsive seizures. All animals in this group exhibiting analgesia and catalepsy had electrographic evidence of a seizure with markedly abnormal EEG tracings showing postictal spiking and changes in baseline frequency and amplitude. These seizures appeared to be naloxone-reversible. Morphine on the other hand was not productive of seizures, but did produce changes in electroencephalographic activity including spindle bursting, high-voltage slow-frequency activity as well as spiking. As noted, these changes were not associated with any effects on nociceptive measures. PMID- 3021276 TI - Anti-S-100 serum blocks long-term potentiation in the hippocampal slice. AB - S-100 is a calcium-binding, glial protein which has been shown to be involved in behavioral learning and memory tasks. Long-term potentiation (LTP) in the hippocampus is a long-lasting enhancement of synaptic efficacy evoked by repetitive afferent stimulation. When anti-S-100 serum is applied by pressure ejection onto the stratum radiatum of area CA1 of the hippocampal slice, the amplitude of the extracellularly recorded population spike is not affected. However, repetitive stimulation of the afferents during S-100 application failed to produce LTP. At a distant site in the same slice, LTP occurs normally. Preimmune normal rabbit serum had no effect on the development of LTP. It appears that S-100 protein is involved in the establishment of LTP. PMID- 3021277 TI - The parafasciculus thalami as a site for mediating the antinociceptive response to GABAergic drugs. AB - Electrophysiological (single cell) experiments were undertaken to examine whether neurons in the rat parafasciculus thalami (PF) are involved in mediating the antinociceptive response to GABAergic drugs. The results indicated that: noxious stimuli excite most PF neurons; microiontophoretic application of morphine, GABA or the GABA agonist, THIP, attenuated the spontaneous firing rate of PF neurons; morphine, THIP and GABA reduced the neuronal excitation induced by noxious stimuli; application of the GABA receptor antagonist, bicuculline, prevented the effects of THIP and GABA on PF activity; while naloxone blocked the response to morphine on PF neurons, it failed to influence the actions of GABA and THIP; and the injection of THIP or GABA into the PF produced an antinociceptive response as assessed by the rat tail-immersion assay, whereas pentobarbital was inactive. The findings suggest that GABA receptors located in the PF may mediate the antinociceptive response to GABAergic drugs, and that the action of these agents is unrelated to opiate receptors. PMID- 3021278 TI - An octadecaneuropeptide (ODN) derived from diazepam binding inhibitor increases aggressive interactions in mice. AB - The effects of intracerebroventricular administrations of an octadecaneuropeptide (ODN) derived from the polypeptide, diazepam binding inhibitor (DBI), on offensive and defensive aggression were examined in male mice. During the initial period after administration (1-5 min) ODN inhibited social and agonistic behavior. At 30 min after treatment, ODN increased, in a dose-dependent manner, the incidence of and intensity of offensive aggression in dominant resident mice. ODN also increased the number of bites required to obtain defeat in subordinate mice during aggressive interactions, as well as reducing subsequent defeat induced analgesia. These changes in offensive and defensive aggressive behavior that were induced by ODN were reduced by the benzodiazepine receptor antagonist Ro 15-1788. These results suggest that ODN has significant modulatory effects on aggression. PMID- 3021279 TI - Aminophylline shortens thiopental sleep-time and enhances noradrenergic neurotransmission in rats. AB - We investigated the effect of aminophylline on thiopental sleep-times and monoamine neurotransmitter turnover rates in discrete brain areas. Aminophylline treated rats had shorter thiopental sleep-times than saline-treated controls. Noradrenergic neurotransmission was greater following aminophylline treatment in thiopental-anesthetized rats in all brain areas while turnover in other monoaminergic pathways was unchanged. These data suggest that acute aminophylline treatment increases central noradrenergic neurotransmission which pharmacodynamically diminishes the hypnotic response to thiopental. PMID- 3021280 TI - Saccadic eye movement deficits in the MPTP monkey model of Parkinson's disease. AB - Saccadic eye tracking was studied in a monkey given i.v. injections of N-methyl-4 phenyl-1,2,3,6-tetrahydropyridine (MPTP). The Parkinson-like symptoms which appeared in the animal's general motor behavior (akinesia, bradykinesia, hypokinesia) were also observed in its eye tracking. Similar oculomotor deficits are seen in patients with idiopathic Parkinsonism. The MPTP model offers excellent possibilities for studying the mechanisms underlying the motor disabilities of Parkinson's disease. PMID- 3021281 TI - Corticosterone reduces the excitability of hippocampal pyramidal cells in vitro. AB - In the rat brain, the hippocampus (HC) is a major target area for corticosterone (CT). In this study, we investigated the effects of CT in the in vitro slice preparation of the rat HC. Population spikes (PS) evoked by stratum radiatum stimulation were recorded in CA1. Bath-applied CT (10(-7)-10(-5) M) induced a decrease in the PS amplitude. This effect occurred within 10-15 min after the onset of CT perfusion, reached a plateau after 20-40 min and was reversible after 20 min washout. Neither dexamethasone (10(-5) M) nor vehicle produced any significant change in PS amplitude. Paired-pulse stimulation showed that the degree of inhibition of the second PS produced by the conditioning stimulus was either unchanged or decreased by CT. In the latter case, CT also reduced the inhibitory effect of gamma-aminobutyric acid on PS and enhanced the excitatory action of the opioid peptide D-Ala2-D-Leu5-enkephalin. These results show that CT reduces the excitability of HC pyramidal cells with a time course which may be compatible with a genomic action of CT. The fact that paired-pulse inhibition was either not changed or reduced, suggests that the decrease in PS size by CT is not due to an indirect excitatory effect of CT on inhibitory interneurons. Instead, CT may hyperpolarize pyramidal cells thus lowering their excitability and depressing the interneurons involved in recurrent inhibition. PMID- 3021283 TI - Is synaptic transmission modulated by progesterone? AB - The influence of the gonadosteroid hormone progesterone on synaptic transmission was studied using the frog neuromuscular preparation. Intracellular recording of synaptic potentials revealed enhanced release of acetylcholine from motor nerve terminals exposed to progesterone (3 nM-3 mM). The following effects were observed. An augmented quantal content of evoked release of transmitter; an elevation in synaptic facilitation; and a substantial increase in the rate of spontaneously occurring miniature endplate potentials. It is suggested that synaptic transmission at the neuromuscular junction may be naturally modulated by the physiologically oscillating level of progesterone. PMID- 3021282 TI - Presynaptic glutamate receptor--possible involvement of a K+ channel. AB - Intra-axonal recording was made from the excitatory axon of the lobster walking leg near the nerve terminal and the effects of L-glutamate were studied. Topical application of glutamate to the synapse produced hyperpolarization in the presynaptic membrane with increase in conductance. The glutamate-induced hyperpolarization was reversed to a depolarization at about -100 mV. The reversal potential was not significantly changed by altering the external Cl- but was shifted to more positive values by increasing the external K+. A spider toxin (JSTX), which specifically blocks the postsynaptic glutamate receptor, failed to block the presynaptic glutamate potential. The results suggest that the presynaptic membrane of the lobster neuromuscular synapse has a different type of glutamate receptor from those in the postsynaptic membrane. PMID- 3021284 TI - Amino acid sequence of toxin F, a snake venom toxin that blocks neuronal nicotinic receptors. AB - The amino acid sequence was determined for toxin F, a component of Bungarus multicinctus venom that blocks nicotinic transmission in the chick ciliary ganglion and the rat superior cervical ganglion. Toxin F was purified by a procedure that includes preparative isoelectric focusing and ion exchange chromatography. Seventy nanomolar toxin F blocks nicotinic transmission in the chick ciliary ganglion; however, the toxin only weakly blocks the binding of 125I alpha-bungarotoxin to membranes derived from Torpedo californica electroplax (IC50 = 1 microM). These data raise the possibility that toxin F may preferentially recognize neuronal nicotinic receptors. Toxin F focused identically on an isoelectric focusing gel with samples of two similar toxins, bungarotoxin 3.1 and kappa-bungarotoxin. The sequence of toxin F is identical with that recently reported for kappa-bungarotoxin. When the N-terminal portion of bungarotoxin 3.1 was sequenced, it was found to be identical to the other two toxins. These and other data suggest that the 3 toxins are, in fact, the same. PMID- 3021285 TI - Kappa opioid receptor-mediated analgesia in the developing rat. AB - The prototypic kappa opiate ketocyclazocine produced robust analgesia in 10-day old rats in the tail-flick nociceptive test. The kappa-opiate behavioral response coincided with the onset of a rapid rise to adult levels in brain kappa receptor site density. In contrast, morphine (prototypic mu opiate) was without marked effect until 14 days of age. The period of rapid mu receptor increase did not take place until days 14-16, which was after kappa receptor levels had already plateaued. Further, there was no or incomplete cross-tolerance between ketocyclazocine and morphine at 14 days of age. The present study, therefore, establishes a role for the kappa binding site in thermal analgesia in the tail flick test and differentiates its ontogenetic pattern from that of the mu receptor. PMID- 3021286 TI - Differential ontogeny of rat brain peptidases: prenatal expression of enkephalin convertase and postnatal development of angiotensin-converting enzyme. AB - We quantitated the levels of two peptidases in the developing rat brain as a means to determine their function. Enkephalin convertase (EC 3.4.17.10), a carboxypeptidase B-like enzyme detected by [3H]guanidinoethylmercaptosuccinic acid (GEMSA) autoradiography, is present in high concentration throughout the brains of rat fetuses 3 days prior to birth. During the first 3 postnatal weeks, the density of [3H]GEMSA-labeled enkephalin convertase drops to adult levels. The expression of enkephalin convertase prior to that of most neuropeptides supports a role for this enzyme in propeptide processing. The regional distribution of [3H]GEMSA binding is similar in fetal and adult rats except that the thalamus exhibits the highest levels of [3H]GEMSA binding prenatally, and among the lowest levels in adult rats. Thus, peptide(s) formed in high concentration in the prenatal thalamus may be substrates for enkephalin convertase. Angiotensin converting enzyme (ACE, EC 3.14.15.1) was visualized in the perinatal period by [3H]captopril autoradiography. Striatonigral ACE is undetectable at birth and increases to adult levels by two weeks of age. The expression of ACE after the initial presence of known peptides in the basal ganglia implies that the enzyme is not essential for peptide synthesis, suggesting instead a degradative role. In contrast to the striatonigral system, the choroid plexus contains high concentrations of ACE prior to birth, consistent with previous proposals of different substrates for ACE in the choroid plexus and the basal ganglia. PMID- 3021287 TI - Prenatal protein malnutrition affects synaptic potentiation in the dentate gyrus of rats in adulthood. AB - Long-term potentiation (LTP) was studied in the dentate gyrus of anesthetized normal and prenatally protein malnourished rats in adulthood. LTP was initiated by high-frequency stimulation of the perforant path. Potentiation of both population excitatory postsynaptic potential (EPSP) slope and population spike was studied at various times after conditioning out to 5 h. The results indicate that prenatal protein malnutrition has a differential effect on LTP. Although potentiation of the population spike was relatively unaffected, prenatal protein malnutrition did lead to a significant reduction in potentiation of the population EPSP. Several possibilities are proposed as to the cause of the differential effect. PMID- 3021288 TI - Response properties of the non-pyramidal tract neuron in the kitten motor cortex during early postnatal development: an intracellular HRP study. AB - Response properties of the non-pyramidal tract neurons (non-PTNs) of the kitten motor cortex were examined with intracellular horseradish peroxidase (HRP) staining under Nembutal anesthesia. Thirty-one neurons were identified as non PTNs and their responses were analyzed with stimulation of the cerebellar nuclei (CN) and/or the medullary pyramid (Py). Excitatory postsynaptic potentials (EPSPs) were induced both by CN and Py stimulation even at birth, whereas inhibitory postsynaptic potentials (IPSPs) were first detected during the third postnatal week in layers II-III pyramidal neurons, which is much later than in PTNs. This indicates that the intracortical circuits responsible for IPSPs in the superficial cortical layers develop later than in layer V. PMID- 3021289 TI - Effects of hypothalamic lesions on active sodium-potassium transport in the extensor digitorum longus muscles of hypokalemic rat. AB - The CNS-induced suppression on muscle Na+-K+ pump was studied in "twitch" muscle, extensor digitorum longus (EDL), of hypokalemic rats which were fed a K+ deficient diet for several weeks. Peripheral nerve section or bilateral lesion of the ventromedial hypothalamic nucleus had no effect on the Na+ and K+ contents in EDL of hypokalemic rats. However, lesions of the paraventricular nucleus caused the net Na+ loss and the net K+ uptake in the muscles. Lesions in either the dorsomedial nucleus or anterior hypothalamus also caused significant net K+ uptake but the net Na+ loss was not significant. The results were compared with those of "tonic" muscle, soleus, reported previously. PMID- 3021290 TI - In vivo down-regulation of rat striatal opioid receptors by chronic enkephalin. AB - Administration of methionine enkephalin (ICV) to rats for 5 days resulted in the development of physical dependence as exemplified by a hypothermic response which peaked 2-8 hours after initiation of withdrawal. Twenty-four hours post withdrawal, opioid receptor binding was determined in the striatum using a selective delta receptor ligand. These studies revealed a decreased in the number of receptors. Bmax decreased from 193 +/- 20.4 fmoles/mg protein in controls to 136 +/- 9.7 fmoles/mg protein in enkephalin treated rats. This difference is significant at p less than 0.001. Existing evidence suggests that this decrease in binding is predominantly due to a decrease in delta receptors. Hence, the present findings indicate that delta receptor down-regulation in vivo may be an important mechanism in the adaptive response to chronic exposure to an endogenous opioid peptide. PMID- 3021291 TI - Calcium-induced long-term potentiation in the hippocampal slice: characterization of the time course and conditions. AB - A transient increase in extracellular calcium concentration causes a long-lasting enhancement of radiatum fibers evoked excitatory postsynaptic potential and population spike responses of CA1 pyramidal neurons which resembles long-term potentiation (LTP). The duration of this potentiation is much longer than described previously and is probably limited by the survival of the preparation itself (greater than 8 hr). Therefore, Ca-induced LTP can be used for the investigation of a postulated late phase of LTP. Ca effects were activity independent, since the subsequently evoked responses were facilitated even when the presynaptic fibers were not concurrently stimulated during or immediately after superfusion with the high Ca medium. In contrast, if too frequent testing of the synaptic input was done during the high Ca pulse, a short lasting depression instead of potentiation was observed. A lower extracellular magnesium concentration in the standard medium (1.3 instead of 2.0 mM MgSO4) prevents the potentiation of the EPSP at least for the first few hours. Presumably, both tetanus- and Ca-induced LTP share some common mechanisms, since an additional tetanization after Ca induction was not followed by an additional LTP. Compared to the potentiation following tetanization, the Ca-induced LTP was, however, not accompanied by a potentiation of the EPSP/spike ratio within the range of the population spike threshold intensity. PMID- 3021292 TI - Demonstration of depressed polymorphonuclear leukocyte function in nonviremic FeLV-infected cats. AB - Polymorphonuclear leukocytes (PMN) from healthy, normal control cats and FeLV infected cats were analyzed for differences in phagocytic capabilities. One week after experimental infection, PMNs from FeLV-infected cats exhibited a marked decrease in phagocytic function as determined by the luminol-dependent chemiluminescent response. Depressed PMN function was observed in these cats during the viremic stage of infection and subsequently remained depressed after the cessation of viremia. The data presented here suggest that while nonviremic cats are reported as clinically normal, they may in fact be exhibiting a suppressed PMN function. PMID- 3021293 TI - Growth factor-mediated proliferative pathways and the neoplastic process. PMID- 3021294 TI - Chemotherapy in the treatment of non-small cell lung cancer. PMID- 3021295 TI - [Ultrastructural study on cytoplasmic inclusions in human hepatocellular carcinoma]. PMID- 3021296 TI - [Atrial natriuretic polypeptide-like material in the rat lung]. PMID- 3021297 TI - [Pathomorphologic study of viral pneumonia]. PMID- 3021298 TI - [Diet and carcinoma: the status quo of research]. PMID- 3021299 TI - Effect of pH-adjustment of bupivacaine on onset and duration of epidural analgesia in parturients. AB - Previous studies have reported that elevation of the pH of local anaesthetics is associated with enhanced quality and duration of block. This study investigated the effect, on time to onset and duration of analgesia, of pH adjustment of 0.25 per cent bupivacaine immediately prior to injection into the epidural space in parturients. Addition of 0.1 ml of 8.4 per cent sodium bicarbonate to 20 ml of 0.25 per cent bupivacaine consistently raised the pH of the local anaesthetic from 5.65 to 7.26 (mean values). Thirty parturients received an epidural injection of 8 ml of pH-adjusted 0.25 per cent bupivacaine and a control group of 30 parturients received 8 ml of the standard commercial preparation of 0.25 per cent bupivacaine. Elevation of the pH of the local anaesthetic significantly increased the speed of onset of analgesia from 6.0 minutes to 3.2 minutes and the duration of analgesia was significantly lengthened from 79.4 minutes to 96.5 minutes. There was no significant influence on time to peak effect, nor on mean maternal plasma levels of bupivacaine. PMID- 3021301 TI - Adriamycin cardiotoxicity and calcium entry blockers: the need for caution in the combination. PMID- 3021300 TI - Onset of pancuronium and d-tubocurarine blockade with priming. AB - The synergistic effect of pancuronium bromide (PCB) and d-tubocurarine (DTC) on the onset time of neuromuscular blockade was tested in 108 ASA physical status I and II adults anaesthetized with thiopentone, nitrous oxide and halothane. Either saline or a small (priming) dose (DTC, 0.04 mg X kg-1, or PCB, 0.007 mg X kg-1) was administered 3 min before a paralyzing dose of either DTC or PCB. The total dose of relaxant was equivalent to DTC, 0.4 mg X kg-1, or PCB, 0.07 mg X kg-1. Neuromuscular activity was measured using train-of-four stimulation applied every 12 s. Time to 50 per cent first twitch blockade was 63 +/- 4.6 s (mean +/- SEM) with DTC and 88 +/- 5.2 s with PCB (p less than 0.002). Times to 90 per cent blockade were not different between the two drugs (161 +/- 20 s and 141 +/- 21 s respectively). Priming a DTC blockade with either DTC or PCB or priming a PCB blockade with PCB produced an acceleration of less than 10 s at all levels of blockade. Compared with PCB alone, priming PCB blockade with DTC reduced the time to 50 per cent blockade to 71 +/- 4.5 s (p less than 0.02) and to 90 per cent blockade to 111 +/- 8 s (p less than 0.05). Priming did not affect the duration of action significantly, except in the case of PCB priming of DTC, where duration was increased from 39 +/- 4.4 to 57 +/- 4 min (p less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021302 TI - Beta-adrenergic stimulation of sarcolemmal phosphorylation in the perfused rabbit heart. AB - To study the mechanism of catcholamine-induced positive inotropy, sarcolemmal membrane proteins were isolated from freeze-clamped rabbit hearts perfused with [32P] and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The major phosphorylated proteins were of apparent molecular mass 27,000 and 14,000 Da. Bolus injections of epinephrine increased phosphorylation of the 27,000 Da protein 2- and to 3-fold coincidentally with increases in developed tension. With epinephrine washout contractility returned to control levels in 2 minutes but dephosphorylation was not complete by 10 minutes. Phosphorylation of the 27,000 Da protein induced by epinephrine was dose dependent and could also be stimulated by isoproterenol and blocked by propranolol. Phenylephrine and ouabain did not stimulate phosphorylation. Forskolin, the beta-adrenergic acting drug, stimulated contractility and also increased phosphorylation. The results suggest that phosphorylation of the 27,000 Da protein may be involved in mediating increases in tension, though the lack of coincidence between dephosphorylation and decline in tension with time suggests a complicated relationship possibly involving other factors. PMID- 3021303 TI - Forskolin and myocardial function in the normal, ischemic and reperfused rat heart. AB - Forskolin increases intracellular cyclic AMP content by direct stimulation of adenylate-cyclase independently of the beta receptor. Studies have been carried out in two different types of isolated rat heart preparation to compare the cardiovascular effects of forskolin and isoprenaline. The inotropic and chronotropic properties of these drugs, together with their ability to increase myocardial cyclic AMP content, have been studied under conditions of aerobic perfusion and also post-ischemic reperfusion. In studies with isolated rat hearts perfused in the Langendorff mode, the dose-response characteristics of forskolin have been characterized. At a concentration of 2 X 10(-7) moles/L, forskolin increased heart rate, coronary flow and left ventricular pressure by 24 +/- 4%, 33 +/- 6% and 33 +/- 3% respectively, and cyclic AMP content was increased seventeen fold (4.0 +/- 0.3 to 68.0 +/- 8.0 nanomoles/g dry wt). Investigation of the effects of three concentrations of forskolin in the isolated working heart revealed that some increases in contractile state could again be achieved. Thus, at a concentration of 5 X 10(-7) moles/L, forskolin increased heart rate by up to 27%, coronary flow by up to 18%, aortic flow by up to 10%, cardiac output by up to 8%, peak aortic pressure by up to 18%, pressure rate product by up to 45% and minute work by up to 25%. These increases, although relatively small, were greater and longer sustained than those seen with isoprenaline (10(-6) moles/L). In additional studies, forskolin (10(-7) moles/L) was included in the reperfusion solution of hearts recovering from a 30 minute period of ischemic cardioplegic arrest. In comparison with controls, which were reperfused without forskolin, small increases in contractile recovery (mostly relating to an increased chronotropic state) were observed. However, upon withdrawal of the drug the functional indices declined such that they were indistinguishable from control hearts which had not received the drug during reperfusion. It is speculated that the relatively poor response to forskolin in the working as opposed to the Langendorff perfused heart may be a reflection of the greater metabolic rate of the working heart and that it may be the rate of turnover of cyclic AMP and other metabolites rather than their steady state cellular level which might determine the responsiveness of tissue to forskolin. PMID- 3021305 TI - The ontogeny of placental Na+-K+ ATPase in the mouse and its impairment by ethanol. AB - We have investigated the normal ontogeny of Na+-K+ ATPase in the mouse placenta and the possibility that impairment in placental transport capacity, as reflected in reductions in NA+-K+ ATPase activity, is associated with alcohol-related embryonic growth restriction. We have demonstrated that over the normal course of pregnancy there is a dramatic increase in placental NA+-K+ ATPase activity which occurs in concert with the embryofetal body growth spurt. Maternal ethanol administration during the early period of placental enzymogenesis (days 7-9) resulted in a significant reduction (up to 40%) of placental Na+-K+ ATPase activity on day 15. Both the severity and the frequency of the reduction were dose dependent. The effect was associated with significant reductions in embryonic body and brain weight but no change in body length or prenatal mortality. Incubation of term placental fragments for 2 h in increasing concentrations of ethanol resulted in a comparable reduction in enzyme activity. Our studies demonstrate that direct ethanol exposure produces a reduction of placental Na+-K+ ATPase activity, that exposure during the early stages of enzymogenesis results in persistent reductions in Na+-K+ ATPase activity in the mature placenta, and that this effect is associated with deficits of embryonic body and brain growth. A direct causal relationship has not been proven; however, it is conceivable that the correlation between reduced placental Na+-K+ ATPase activity and impaired embryofetal growth reflects a common causal pathway. PMID- 3021304 TI - Isoproterenol-induced myocardial alterations in alloxan-diabetic rabbits. AB - This study was undertaken to characterize catecholamine-induced myocardial necrosis in 10-week alloxan-diabetic rabbits. Myocardial injury was induced by administering graded doses of isoproterenol (ISO) for 15 days. Injection of ISO to control and diabetic rabbits resulted in atrial tachycardias and ventricular fibrillation. The severity of the arrhythmias and the overall mortality was the same in both groups of animals. Analyses of serum biochemical parameters revealed significant increases in blood glucose, free fatty acids and total cholesterol in the ISO-treated diabetic animals relative to ISO-treated controls. ISO-treatment of both control and diabetic animals showed similar increases in heart weight, left ventricular weight and myocardial total water content. Analyses of various subcellular organelle marker enzyme activities indicated a significant decrease in the K+, Ca2+-stimulated sarcoplasmic reticulum, mitochondrial (azide sensitive) and sarcolemmal Na+, K+-stimulated ATPase activities, decreases in ATP and glycogen and increases in myocardial sodium content in both the ISO-treated control and diabetic animal hearts. In addition, significant accumulation of Ca2+ and hydroxyproline were evident in the ISO-treated diabetic animal hearts. PMID- 3021306 TI - Neuronal experience modifies synaptic long-term facilitation. AB - In a crayfish phasic neuromuscular junction, we have demonstrated low-frequency depression (LFD), high-frequency depression (HFD), and long-term facilitation (LTF) in response to different regimens of stimulation. Chronic stimulation of the phasic axon supplying the closer muscle of the claw in Procambarus clarkii resulted in diminished expression of HFD and LTF. Conversely, when impulse production in the phasic motoneuron was reduced by claw immobilization, both HFD and LTF were enhanced. LFD was insensitive to these manipulations. These results provide further evidence for long-term adaptation of the phasic neuromuscular junction to ongoing levels of impulse activity and illustrate the importance of a neuron's past history for synaptic plasticity. The ability of the neuron to adjust its short-term plasticity in response to altered experience constitutes an adaptive response that could be of general significance. PMID- 3021307 TI - The effect of oxidized isoprenaline on the chick embryonic heart. AB - Intra-amnial administration of isoprenaline (IPRO) to chick embryos induces a number of myocardial lesions. The purpose of the present study was to investigate whether similar changes may also be induced after injection of spontaneously oxidized isoprenaline and commercially obtained adrenochrome. Cardiotoxicity of these substances has been demonstrated in adult animals. IPRO, oxidized IPRO, or adrenochrome were administered intra-amnially to 10-day-old chick embryos at doses of 0.1, 1.0, 10.0, and 100.0 mg X kg-1. Parallel experimental groups received propranolol at a dose of 1 mg X kg-1, 15 s before injection of IPRO or oxidized IPRO. The cAMP level in the heart was determined by radioimmunoassay 2 and 30 min after administration of IPRO, oxidized IPRO, or adrenochrome at a single dose of 10.0 mg X kg-1. It has been found that in embryos the effect of IPRO and oxidized IPRO is dose dependent. The rise in mortality and development of cardiomegaly together with increased hydration and disturbances of the development of coronary vascularization were highly significant starting from the dose of 10 mg X kg-1. Furthermore, both drugs significantly increased cAMP levels in the embryonic heart. On the other hand, the administration of adrenochrome was without any effect. The changes induced by IPRO were prevented by the administration of the beta-blocking agent propranolol; the lesions induced by spontaneously oxidized IPRO were, however, prevented only partially. PMID- 3021308 TI - The effects of morphine on sympathetic transmission in the stellate ganglion of the cat. AB - The aim of this study was to investigate which of the processes involved in synaptic transmission are affected by morphine in concentrations comparable to those used during surgical procedures. The effects of morphine sulfate on ganglionic transmission were studied in the stellate ganglion of the cat using intracellular and extracellular recordings in vitro. The neurons of the stellate ganglion were depolarized using preganglionic nerve stimulation, postganglionic nerve stimulation, and intracellular stimulation before and after introduction of morphine sulfate (up to 20 micrograms/mL). Tissue concentrations of morphine were estimated using radiolabeled morphine. Axonal transmission and the excitability of the postganglionic neurons to direct intracellular stimulation was not affected at the concentrations of morphine studied. In addition, morphine had a dose-dependent depolarizing effect on the resting membrane potential of most of the neurons in the stellate ganglion. Such neuronal depolarizations alone could initially produce excitation in some cell populations, followed by inhibition, secondary to the membrane depolarization, leading to depression of sympathetic nerve activity. The overall ganglionic transmission as recorded using an evoked potential was biphasic. At low doses morphine facilitated transmission, while at larger doses morphine attenuated evoked potentials. These effects do not appear to be mediated through classical opiate receptors since they are not blocked by naloxone. PMID- 3021310 TI - Characterization of a plasmid in isolates of Corynebacterium sepedonicum. AB - Lysis of mid-log phase cells of the Gram-positive bacterium, Corynebacterium sepedonicum, by a combination of lysozyme treatment and incubation with alkaline sodium dodecyl sulfate at 56 degrees C led to the recovery of a single plasmid. The plasmid was purified in CsCl density gradients, and its molecular weight estimated to be 31 megadaltons (46 kilobases), as determined from its relative mobility in agarose gels, from its contour dimensions in electron micrographs, and from the size of the fragments generated when it was cleaved with various restriction endonucleases. Thirteen widely divergent isolates of C. sepedonicum were screened for the presence of plasmid, and of these, 11 were shown to harbour a single plasmid at a level of about 30 copies per cell. Cleavage of the plasmid with PstI gave an identical banding pattern in agarose gels for the fragments from all of the plasmid-carrying isolates. The relevance of plasmid incidence and distribution in C. sepedonicum is discussed in relation to the possibility of developing a test for the detection of bacterial ring rot by using plasmid DNA as a hybridization probe. PMID- 3021311 TI - Survival of indigenous enteric viruses during storage of waste water sludge samples. AB - The stability of indigenous enteric viruses in samples of settled primary and mixed-liquor activated sludges was studied at 2, 23, and -70 degrees C. Changes of virus titer which occurred in these samples were followed during an 84-day observation period, with rates of change then calculated by least-squares regression. Virus survival was found to be statistically dependent (p less than or equal to 0.05) upon storage temperature but not sludge solids content. Based upon the observed rates of inactivation, the average times which would be required for a 90% decrease in virus titer are 26 days at 23 degrees C, 180 days at 2 degrees C, and 163 days at -70 degrees C. As a group, the rates of virus inactivation observed at 2 degrees C were statistically different (p less than or equal to 0.05) from those observed at 23 degrees C, but not different from those observed at -70 degrees C. The three study temperatures were selected to approximate holding of samples in an air-conditioned room, on wet ice (H2O), and on dry ice (CO2). PMID- 3021312 TI - Stability of viruses in waste water sludge eluates. AB - The survival of indigenous enteric viruses in samples of unconcentrated and concentrated waste water sludge eluates, which had been prepared using a combination beef extract elution - organic flocculation concentration procedure, was studied at 2, 23, and -70 degrees C. Changes of virus titer occurring in the samples were followed during an 84-day observation period, with rates of change then calculated by least-squares regression. Virus survival in both types of eluates was statistically dependent (p less than or equal to 0.05) upon storage temperature. Based upon the observed rates of inactivation the average times which would be required for a 90% decrease (one log10 unit) in virus titer for unconcentrated eluates are 27 days at 23 degrees C, 198 days at 2 degrees C, and 375 days at -70 degrees C. The calculated average times required for a 90% decrease in virus titer for concentrated eluates are 22 days at 23 degrees C, 132 days at 2 degrees C, and 246 days at -70 degrees C. In both types of eluates the rates of virus inactivation at 2 degrees C were statistically different from those observed at 23 degrees C, but not different from those observed at -70 degrees C. The three study temperatures were selected to approximate holding of samples in an air-conditioned room, fluid on wet ice (H2O), and frozen on dry ice (CO2). PMID- 3021309 TI - The modulation of sensory transmission through the medullary dorsal horn during cortically driven mastication. AB - During mastication, reflexes are modulated and sensory transmission is altered in interneurons and ascending pathways of the rostral trigeminal sensory complex. The current experiment examines the modulation of sensory transmission through the most caudal part of the trigeminal sensory system, the medullary dorsal horn, during fictive mastication produced by cortical stimulation. Extracellular single unit activity was recorded from the medullary dorsal horn, and multiple unit activity was recorded from the trigeminal motor nucleus in anesthetized, paralyzed rabbits. The masticatory area of sensorimotor cortex was stimulated to produce rhythmic activity in the trigeminal motor nucleus (fictive mastication). Activity in the dorsal horn was compared in the presence and absence of cortical stimulation. Fifty-two percent of neurons classified as low threshold and 83% of neurons receiving noxious inputs were influenced by cortical stimulation. The cortical effects were mainly inhibitory, but 21% of wide dynamic range and 6% of low threshold cells were excited by cortical stimulation. The modulation produced by cortical stimulation, whether inhibitory or excitatory, was not phasically related to the masticatory cycle. It is likely that, when masticatory movements are commanded by the sensorimotor cortex, the program includes tonic changes in sensory transmission through the medullary dorsal horn. PMID- 3021313 TI - Severe coughing during captopril and enalapril therapy. PMID- 3021314 TI - Regional intra-arterial infusion of cisplatin in primary hepatocellular carcinoma. A phase II study. AB - Ten consecutive adults with localized nonresectable hepatocellular carcinomas were selected as a nonrandom sample for an investigation into the effectiveness of cisplatin (DDP) as a single agent when administered regionally via the hepatic artery from 1981 to 1984. The dose of DDP was 50 mg/m2 (normally, 80 mg). Complete remission (CR) was observed in one patient, partial remission (PR) in three patients, and in five patients, there were no significant changes in tumor size; the disease progressed in one patient. The mean period of survival of the group was 19.7 months. All patients suffered from severe nausea and vomiting, ordinarily until the afternoon of the day after treatment. One patient died of uremia, which related to the cytostate. The authors consider cisplatin useful in the intra-arterial treatment of hepatocellular carcinoma with favorable prognostic factors in patients for whom surgical treatment is not suitable. PMID- 3021315 TI - Sclerosing hemangioma of the lung. Immunohistochemical demonstration of mesenchymal origin using antibodies to tissue-specific intermediate filaments. AB - A case of pulmonary sclerosing hemangioma of the lung was studied by light microscopy and indirect immunofluorescence using tissue-specific antibodies against intermediate filament subunits. All the tumor cells stained positively and exclusively with antivimentin antibodies thus indicating their mesenchymal origin. In addition, positive staining with cytokeratin antibodies was observed in cells lining cystic spaces and elongated slit-like spaces were occasionally encountered throughout the tumor, disclosing residual epithelial elements. Using brightfield microscopy, the keratin-positive areas were identified as distorted alveolar spaces lined by hyperplastic respiratory epithelium entrapped within the tumor. It is proposed that these entrapped epithelial elements may account for the conflicting results obtained by different investigators in previous attempts to determine the histogenesis of this tumor. PMID- 3021316 TI - Abnormal muscle fructose bisphosphatase activity in malnourished cancer patients. AB - Malnourished patients without cancer have abnormal glucose metabolism, low activities of the key enzymes of glycolysis in muscle, and abnormal muscle fiber type distribution. Malnutrition in cancer is also associated with altered glucose metabolism, and therefore muscle enzyme activities and fiber types were measured in 17 malnourished patients with gastrointestinal cancer (weight loss, 18.1% +/- 7.9 SD). These patients were matched with 17 depleted noncancer patients (weight loss, 22.8% +/- 10.25 SD) and 17 normal controls. Results of in vitro measurement of the maximal activity of phosphofructokinase (PFK), hexokinase (HK), and oxogluterate dehydrogenase (OGD) were similar in both undernourished groups and lower than that of normal controls. Both groups also had reduced Type II fiber size and number. The activity of fructose bisphosphatase (FBP) was significantly higher in cancer patients (0.62 mu ml min-1 g +/- 0.26 SD) than in noncancer patients (0.39 +/- 0.15), but similar to that in controls (0.65 +/- 0.29). As FBP is involved in substrate cycling, inappropriately high activity reflects an inability to adapt to malnutrition that could lead to high rates of cycling and wasteful energy expenditure at times of maximal activation of the cycle. PMID- 3021317 TI - Diagnostic accuracy of fibronectin in the differential diagnosis of ascites. AB - To evaluate the diagnostic accuracy of fibronectin levels in ascites to differentiate malignant from non-malignant ascites, the authors studied 30 patients with sterile uncomplicated ascites in chronic liver disease, 18 patients with malignant ascites and four patients with spontaneous bacterial peritonitis. Fibronectin concentration was significantly higher in malignant ascites than in sterile ascites (P less than 0.001). High values (greater than 85 mg/l) were found in four of six cases of hepatocellular carcinoma in liver cirrhosis with negative cytologic examination, and in six of seven peritoneal carcinomatoses. Low values (less than 85 mg/l) were found in four patients with liver metastases and in one patient with intrahepatic biliary duct carcinoma in cirrhosis. In four patients with infected ascites, the fibronectin level was low. Among all other parameters (total protein concentration, lactate dehydrogenase, gamma-glutamyl transpeptidase, pH, amylase, triglycerides, leukocyte count, and cytologic examination), fibronectin yielded the best degree of discrimination (diagnostic accuracy, 79%). PMID- 3021318 TI - Flow cytometric analysis of nephroblastomas and related neoplasms. AB - A total of 59 cases of nephroblastoma and related neoplasms were studied by flow cytometry of paraffin-embedded tissue. According to clinical prognosis, cases were subdivided into three groups: Group 1 (low risk) consisted of congenital mesoblastic nephroma (n = 13) and cystic, partially differentiated nephroblastoma (n = 2). Group 2 (intermediate risk) comprised the various subtypes of "typical" nephroblastoma (n = 24) including cases of fetal rhabdomyomatous nephroblastoma (n = 4). In group 3 (high risk) there were cases of anaplastic nephroblastoma (n = 3), clear cell sarcoma of the kidney or "bone metastasizing renal tumor of childhood" (n = 7), and malignant rhabdoid tumor of the kidney (n = 6). The three clinically different groups of tumors also varied in the proportion of cases with aneuploid tumor DNA stemlines, in S-phase fractions, and in proliferation indices (PI = S + G2 + M). Group 1 was generally characterized by a small number of cases with aneuploid tumor DNA stemlines and low values for S-phase fractions and PI, whereas Group 3 showed the largest number of cases with aneuploid tumor DNA stemlines and high values for S-phase fractions and PI. Group 2 was in between. It is concluded that flow cytometry on paraffin-embedded tissue from pediatric tumors may be a useful adjunct in determining prognosis, and that the subdivision of nephroblastomas and related neoplasms into three prognostically different groups is warranted. PMID- 3021319 TI - Clinicopathologic features and prognosis for Wilms' tumor patients with metastases at diagnosis. AB - Comparisons were made between 236 Wilms' tumor patients with metastasis to the lungs and/or liver at initial diagnosis who were registered on the National Wilms' Tumor Study (NWTS) during 1969 to 1983, and 1755 patients who did not have overt metastases at diagnosis. Patients with evidence of regional spread of disease beyond the kidney, especially if to the renal vein or lymph nodes, were much more likely to have overt metastases present at diagnosis than those with apparently localized disease. The presence of metastases was also correlated with age at diagnosis, ranging from 1% among infants younger than 1 year of age to 24% for those aged 6 years or older. The percentage of tumor deaths for patients with metastases at diagnosis (Stage IV) and a primary tumor of favorable histology (FH) declined from 29% at 2 years postdiagnosis on the first therapeutic trial (NWTS-1) to 9% for the most recent one (NWTS-3), and is now comparable to that for patients without metastases but with nonresectable local invasion at diagnosis (Stage III). The local extent of disease also influenced the survival outcome for Stage IV/FH patients. Survival was poor for those with anaplastic or sarcomatous (unfavorable) histology, regardless of local staging or trial. There was no difference in survival according to metastatic site (liver +/- lung vs. lung only) if present prior to treatment. By contrast, patients who developed liver metastases during or after treatment had an especially poor chance for survival as compared with those who developed lung deposits at those times. PMID- 3021320 TI - Detection of bone marrow relapse in patients with small cell carcinoma of the lung. AB - From a total of 874 patients with small cell lung cancer (SCC), a series of 104 patients were reviewed to determine if bone marrow relapses are detectable by routine clinical investigations. Autopsy, including microscopical bone marrow examination, was performed in all the 104 patients and bone metastases were disclosed in 36 patients (35%). After retrospective evaluation it was concluded that dose modification, occurrence of leukopenia plus fever, need of blood transfusion, leukopenia, and thrombopenia during treatment all were without predictive value in the detection of bone marrow relapse. Increased concentrations of lactate dehydrogenase and alkaline phosphatase were, however, positively correlated to the finding of bone marrow relapse. PMID- 3021322 TI - Use of the human liver cell line Hep G2 in a modified Salmonella reversion assay. AB - The human hepatoma cell line Hep G2 was used to activate promutagenic chemicals to mutagens in a modified Salmonella typhimurium reversion assay. Hep G2 cells mediated positive mutagenic responses in tester strain TA98 with 5 and 25 micrograms/plate of 2-aminofluorene, but these responses were consistently lower than those seen using primary rat hepatocytes. In addition, 3 and 6 X 10(6) Hep G2 cells per assay produced positive mutagenic responses with 2-aminoanthracene, benzidine, acetylbenzidine and aflatoxin B1, while benzo[a]pyrene, 7,12 dimethylbenz[a]anthracene, 3-methylcholanthrene, 4-aminobiphenyl and 4- and 11 aminobenzo[a]pyrene were nonmutagenic with Hep G2-cell activation. These results indicate that Hep G2 cells may be a useful intact cellular metabolizing system of human origin for predicting the genotoxicity of promutagenic agents, but that the use of Salmonella as a target cell may limit the classes of mutagens detected. PMID- 3021321 TI - Hu-ets-2 is translocated to chromosome 8 in the t(8;21) in acute myelogenous leukemia. AB - The human genome contains two distinct loci with homology to the viral ets gene, the transforming sequence of the E26 avian erythroblastosis virus; these loci, Hu ets-1, and Hu-ets-2, have been mapped to 11q23 and 21q22, respectively. Using in situ chromosomal hybridization, we have demonstrated that Hu-ets-2 is translocated to chromosome #8, the chromosome containing the critical or conserved junction, as a result of the t(8;21) (q22;q22) in acute myelogenous leukemia. Another protooncogene, c-mos, is also retained at the conserved junction, suggesting that one or both of these genes may play a role in the pathogenesis of acute myelogenous leukemia. PMID- 3021323 TI - Restless legs syndrome and periodic movements in sleep: physiopathology and treatment with L-dopa. AB - Seven patients suffering from restless legs syndrome (RLS) and periodic movements in sleep (PMS) were investigated before and after treatment with L-Dopa. The effect of treatment was evaluated by polysomnography, structured interviews, and daily questionnaires. Sleep organization and subjective complaints improved during treatment with 100 to 200 mg of L-Dopa. Polysomnographic recordings also revealed a significant decrease of periodic leg movements during the first third of the night and a rebound during the last third. These results and previous biochemical findings raise the hypothesis that RLS and PMS may both result from reduced dopaminergic activity in the CNS, perhaps resulting from decreased sensibility of postsynaptic receptors. PMID- 3021324 TI - Effect of sulpiride on chronic abstinence syndrome in addicted patients. AB - A total of 410 patients (342 men and 68 women) addicted to heroin for at least 38 months, with or without previous methadone treatment experience, were treated with sulpiride before the appearance of withdrawal syndrome. The drug was injected intramuscularly in a single administration at a dosage of 600 mg. Successively, sulpiride was injected intramuscularly at a dosage of 200 mg 3 times per day from days 2-21 of hospitalization, and then at a dosage of 100 and 50 mg 3 times per day (days 22-25 and 26-29, respectively). All patients showed a suppression of withdrawal signs and symptoms within 6 days of treatment, as assessed by subjective and objective scores. The effect of sulpiride was compared with that of placebo administered to a control group of 10 heroin addicts. Because prolactin has been shown to reduce the dependence of rats to heroin and the naloxone-precipitated withdrawal syndrome in morphine-addicted animals, the effects of sulpiride on heroin addiction may be related to the hyperprolactinemic action of this drug. PMID- 3021325 TI - Phase II study of combined carmustine, 5-fluorouracil, hydroxyurea, and 6 mercaptopurine (BFHM) for the treatment of malignant gliomas. AB - The Neuro-oncology Service of the University of California Brain Tumor Research Center conducted a nonrandomized phase II study to evaluate, in patients with recurrent malignant glioma, the benefit of a four-drug combination (BFHM) consisting of carmustine (1,3-bis (2-chloroethyl)-1-nitrosourea), 5-fluorouracil, hydroxyurea, and 6-mercaptopurine. There were 29 evaluable glioblastoma multiforme patients and 45 nonglioblastoma anaplastic glioma patients available for analysis. Tumor progression was analyzed as the primary study endpoint. Of the glioblastoma patients, 16 of 29 (55%) responded or stabilized on therapy; of the other anaplastic gliomas, 32 of 45 (71%) responded or stabilized. For patients who stabilized or responded to treatment, BFHM achieved a median time to tumor progression of 46 weeks with a 25th percentile time to tumor progression of 68 weeks for anaplastic gliomas and a median time to tumor progression of 23 weeks with a 25th percentile time to tumor progression of 36 weeks for glioblastoma multiforme patients. A Cox multivariate analysis demonstrated that age and Karnofsky score were important prognostic variables for these patients. PMID- 3021327 TI - Clinical trial of doxifluridine in the treatment of primary hepatocellular carcinoma. PMID- 3021326 TI - Phase II study of acivicin in non-small cell lung cancer: a National Cancer Institute of Canada Study. AB - Thirty-six previously untreated patients with metastatic non-small cell lung cancer received acivicin at a starting dose of 15 mg/m2, given over 5 days and repeated every 21 days. Hematological toxicity was dose-related; one patient died of neutropenic sepsis at 18 mg/m2. Nonhematological toxicity was mild, with gastrointestinal symptoms being the most prominent. Neurological toxicity was seen in 48% of the patients and consisted of confusion, hallucinations, and sleeping difficulty. A minority of patients required dose reduction because of these symptoms. In 33 evaluable patients, two partial remissions were documented, with seven additional patients showing evidence of minor responses. Although modest, these responses warrant further study of acivicin in non-small cell lung cancer in combination with other agents. PMID- 3021328 TI - The use of an immobilised cyclic multi-enzyme system to synthesise branched penta and hexa-saccharides associated with blood-group I epitopes. AB - The branched hexasaccharide beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)- [beta-D Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Galp-(1----4)-beta -D- GlcOMe (1) was obtained on a 0.1-mmol scale by enzymic bigalactosylation of the tetrasaccharide beta-D-GlcpNAc-(1----3)-[beta-D-GlcpNAc-(1----6)]-beta-D-Galp-(1- --4)-b eta- D-GlcOMe by the use of an immobilised multi-enzyme system which regenerated UDP-alpha-D-galactose in situ. The formation of 1 proceeded in two steps. An intermediate compound was identified on the basis of 1H- and 13C-n.m.r. data as the pentasaccharide beta-D-GlcpNAc-(1----3)-[beta-D-Galp-(1----4)-beta-D GlcpNAc-(1----6)]-b eta- D-Galp-(1----4)-beta-D-GlcOMe. PMID- 3021329 TI - Lysosomal-enzyme targeting: the phosphorylation of synthetic D-mannosyl saccharides by UDP-N-acetyl-glucosamine:lysosomal-enzyme N-acetylglucosamine phosphotransferase from rat-liver microsomes and fibroblasts. AB - Phosphorylation of the D-mannose residues of lysosomal enzymes is essential for the uptake and intracellular transport of these enzymes to lysosomes. The GlcNAc P-transferase which is involved in the phosphorylation reaction seems to recognize a signal, probably a protein conformation, common to many lysosomal enzymes. To evaluate the role of the carbohydrate portion of the enzyme in these phosphorylation reactions, the acceptor specificity of GlcNAc-P-transferase from rat-liver microsomes and fibroblasts was examined with the aid of synthetic D mannosyl disaccharides and derivatives that are closely related to the high mannose type of oligosaccharides. Four methyl D-mannobiosides were synthesized, and their structures were established by 13C-n.m.r. spectroscopy. Of all the D mannosyl saccharides tested, alpha-D-Man-(1----2)-alpha-D-Man-(1----OMe) was found to be the best acceptor, thereby suggesting that oligosaccharide structure may also have a role to play in recognition by this enzyme. PMID- 3021330 TI - Biomedical nuclear magnetic resonance: principles and progress. PMID- 3021331 TI - Clinical applications of magnetic resonance imaging (MRI) of the heart. AB - The rapid progress of MRI has been remarkable, and it is clear that it will become an important method for cardiac imaging. Its major advantages are the lack of ionizing radiation and the ability to obtain excellent global images of the cardiac walls and chambers without the need for contrast injection or cardiac catheterization. High resolution surface coil imaging, tissue spectroscopy, and other improvements and applications should be rapidly forthcoming. PMID- 3021332 TI - Abnormal membrane composition and membrane-dependent transduction mechanisms in cluster headache. AB - Previous studies have indicated that membrane structure and function may be abnormal in cluster headache. This has been further investigated by analysis of membrane phosphatidylcholine, total phospholipids, and cholesterol in erythrocytes and by assay of receptor-mediated transduction. The stimulation of lymphocyte adenylate cyclase with isoprenaline and prostacyclin was used as the test system. A significant increase in the ratio of membrane phosphatidylcholine to cholesterol without change in cholesterol was found in cluster headache patients as compared with control subjects. This indicated a reduced turnover of phosphatidylcholine, since erythrocyte choline is significantly reduced in this condition. Abnormal membrane function was also indicated from the significant depression of high-affinity prostaglandin receptor stimulation of lymphocyte adenylate cyclase and the similar trend in the beta-adrenoceptor response. Since no change in agonist affinity and beta-adrenoceptor density occurred, this depression indicates a generalized defect in coupling of receptors to adenylate cyclase. It is hypothesized that the impaired function that would result might contribute to the aetiology of cluster headache. PMID- 3021333 TI - One binding site determines sequence specificity of Tetrahymena pre-rRNA self splicing, trans-splicing, and RNA enzyme activity. AB - The specificity of reactions catalyzed by the Tetrahymena pre-rRNA intervening sequence (IVS) was studied using site-specific mutagenesis. Two sequences required for 5' splice-site selection during self-splicing were defined. Single base changes in either a 5' exon sequence or a 5' exon-binding site within the IVS disrupt their ability to pair and result in inefficient or inaccurate splicing. Combinations that restore complementarity suppress the effect of the single-base changes. Sequence alterations in the 5' exon-binding site also change the specificity of two other reactions: intermolecular exon ligation (trans splicing) and the enzymatic nucleotidyltransferase activity of the IVS RNA. Thus the substrate specificity of an RNA enzyme can be changed in a manner predictable by the rules of Watson-Crick base-pairing. PMID- 3021334 TI - DNA replication in vitro erases a Xenopus 5S RNA gene transcription complex. AB - Replication of the Xenopus laevis 5S RNA gene in vitro is unimpeded by the presence of a complete transcription complex assembled on the internal control region of the gene. The transcription complex is disrupted by passage of the replication fork, specific transcription factors are displaced, and the daughter 5S RNA genes are inactivated. We have been unable to demonstrate that any "memory" of the preexisting transcription complex is transmitted to the daughter DNA duplexes following replication. PMID- 3021335 TI - SV40 enhancer-binding factors are required at the establishment but not the maintenance step of enhancer-dependent transcriptional activation. AB - We have used temperature-sensitive COS cells to design delayed competition experiments in which competition for simian virus 40 (SV40) enhancer factors occurs after enhancer-dependent transcription has been established. The results demonstrate that competition for SV40 enhancer-binding factors has no effect on enhancer-dependent transcription after transcription has been established at the SV40 early promoter. These data show that the enhancer and factors that bind to it are involved in the establishment of stable transcription complexes, although they do not show whether enhancer factors are an integral part of the transcription complexes. Furthermore, the results of delayed competition experiments with a replicating test plasmid are consistent with the possibility that enhancer-dependent stable transcription complexes could be maintained after DNA replication. PMID- 3021336 TI - 3' end formation of U1 snRNA precursors is coupled to transcription from snRNA promoters. AB - Promoters of small nuclear RNA (snRNA) genes are partly responsible for 3' end formation of snRNA precursors. In injected X. laevis oocytes, substitution of an mRNA promoter (HSV tk) for the snRNA promoter significantly reduces the utilization of a conserved snRNA 3' end signal and permits recognition of a downstream polyadenylation site. Neither the U1 enhancer nor the U1 coding region is essential for recognition of the snRNA 3' end signal. Deletion of the U1 3' end signal from genes with a U1 promoter results in utilization of "cryptic" signals resembling the consensus sequence. However, these snRNA gene-promoted transcripts are not polyadenylated, in spite of the functional polyadenylation signal they contain. Thus, the ability to recognize 3' end signals is determined during initiation, presumably by interaction of transcription complexes with specific processing or termination factors. PMID- 3021337 TI - Alternative splicing of RNAs transcribed from the human abl gene and from the bcr abl fused gene. AB - The primary structure of normal abl protein was determined by sequencing the coding region of its cDNA. abl contains two alternative 5' exons spliced to a common set of 3' exons to yield the two major abl RNA transcripts. These transcripts initiate in different promoter regions and give rise to proteins that vary in their N-termini. In the human cell line K562, abl is translocated from chromosome 9 to within the bcr gene on chromosome 22. Within the fused bcr-abl gene, abl exon II alternatively splices to two adjacent bcr exons. This phenomenon is seen in many patients with chronic myeloid leukemia. PMID- 3021338 TI - Transposable elements generate novel spatial patterns of gene expression in Antirrhinum majus. AB - The pallida gene of A. majus encodes a product required for the synthesis of red flower pigment. We have shown that the unstable pallida(recurrens) mutation is due to the insertion of the Tam3 transposable element near the promoter of the gene. Imprecise excision of Tam3 alters pallida gene expression and generates new spatial patterns or different intensities of flower pigmentation. Distinct spatial patterns may also result from rearrangements induced by Tam3 that alter the relative position of the pallida gene. Changes in Tam3 structure or position result in new unstable phenotypes. These findings suggest that genes may be rendered genetically hypervariable as a consequence of transposable element insertion and excision. PMID- 3021339 TI - Replicative and conservative transposition in bacteria. PMID- 3021340 TI - The mechanism of RNA recombination in poliovirus. AB - We have investigated RNA recombination among poliovirus genomes by analyzing both intratypic and intertypic recombinant crosses involving the same defined genetic markers. Sequence analysis of the recombinant junctions of 13 nonsibling intertypic recombinants showed that intertypic RNA recombination is not site specific, nor does it require extensive homology between the recombining parents at the crossover site. To discriminate between breaking-rejoining and copy choice mechanisms of RNA recombination, we have inhibited the replication of the recombining parents independently and found opposite effects on the frequency of genetic recombination in intratypic crosses. The results strongly support a copy choice mechanism for RNA recombination, in which the viral RNA polymerase switches templates during negative strand synthesis. PMID- 3021342 TI - Augmentation of the edentulous ridge prior to the fabrication of a fixed partial denture. PMID- 3021341 TI - The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease. AB - Tumor formation by Agrobacterium tumefaciens involves the transfer and integration of a defined segment (T-DNA) of tumor-inducing (Ti) plasmid DNA into the plant nuclear genome. A set of plasmid genes outside the T-DNA, the vir genes, are thought to mediate the transfer process. We report here that the virD operon encodes a site-specific endonuclease that cleaves at a unique site within each of the 24 bp direct repeats that flank the T-DNA. The endonuclease function was further localized to the 5' end of this operon by demonstrating that cleavage does not occur in virD mutant strains of Agrobacterium and that the 5' end of the virD operon is sufficient to direct cleavage in E. coli. Analysis of nucleotide sequence and protein data indicate that two proteins of 16.2 and 47.4 kd are involved. PMID- 3021343 TI - Rapid proteolysis of brain MAP-1 related cytoskeleton-associated 350kd protein by purified calpain. AB - Microtubule associated protein-1 of brain and its intracellular 350kd analogues were highly sensitive to purified Ca2+-dependent cysteine proteinase (calpain). After 15 second digestion, we detected intermediate degradation products of MAP-1 by immunoblotting using anti-MAP-1 antibody as 290, 260, 220, 170, 140, 112, 80, 68, and 32kd polypeptides. These values corresponded to the molecular weights of the immunoreactive polypeptides of microtubule-enriched cytoskeletons isolated from HeLa and SV-3Y1 cells, suggesting the action of endogenous calpain on intracellular MAP-1 analogues in vivo or during the course of preparation. PMID- 3021344 TI - The (Na+, K+)ATPase of rat kidney: purification, biosynthesis, and processing. AB - (Na+, K+)ATPase was purified from rat renal outer medulla by concanavalin A- and wheat germ agglutinin-lectin Sepharose affinity chromatographies. The antibody, which was raised in rabbits, markedly inhibited ATPase activity. The monospecificity of this antibody was assayed by the Ouchterlony double immunodiffusion and Western blotting tests. The endoplasmic reticulum (ER)-rich, and Golgi-rich subfractions were prepared from the rat kidney microsomal fraction by sucrose density gradient centrifugation. On the immunoblot, the molecular weight of the alpha subunit in both fractions was 95 kilodalton (Kd); whereas, that of the beta subunit was 50 Kd in the ER-rich fraction and 54 Kd in the Golgi rich fraction. When treated with endoglucosidase H, the 50 Kd component was converted to 38 Kd, but the 54 Kd component was endoglucosidase H resistant. These results suggest that the beta subunit (38 Kd) is glycosylated cotranslationally in the ER (50 Kd) then is converted to the mature type subunit (54 Kd) in the Golgi apparatus. PMID- 3021345 TI - Establishment of mutant murine mammary carcinoma FM3A cell strains transformed with the herpes simplex virus type 2 thymidine kinase gene. AB - To establish cell systems appropriate for investigating the mode of action of antiherpetic nucleoside analogues, mutant cell strains were constructed from murine mammary carcinoma FM3A cells, which were deficient in TK, but were transformed with a recombinant plasmid DNA containing the HSV-2 TK gene. The transformed cells incorporated the viral DNA, expressed viral TK activity and showed unusually high sensitivity to the cytostatic action of the antiherpetic nucleoside analogues ACV and IVDU, both of which were only weakly inhibitory to the growth of the parent cells. Curiously, the FM3A cell strains transformed with HSV-2 TK gene showed a higher sensitivity to ACV and IVDU than the previously established cell line transformed with HSV-1 TK gene. This contrasts with the inhibitory effects of ACV and IVDU on acute HSV infection, since HSV-2 infection is slightly or considerably less susceptible than HSV-1 infection to inhibition by ACV or IVDU, respectively. PMID- 3021346 TI - Phase II studies on weekly cisplatinum plus epirubicin or etoposide in the treatment of advanced non-small cell bronchogenic carcinoma. AB - Thirty-six patients with advanced non-small cell bronchogenic carcinoma were divided in 2 groups for treatment with two different platinum-based combination therapy regimens. All 16 patients who received the weekly administered combination of 10 mg/m2 cisplatin (CDDP) plus 10 mg/m2 epirubicin (4EPIDX) experienced no clinical response. Among the 20 patients who received the combination CDDP (10 mg/m2) plus etoposide (VP16, 60 mg/m2) weekly, 4 of them (20%) showed partial remission (PR). The side effects of both combinations were of mild grade. Further study is needed to verify the effectiveness of the weekly combination of CDDP and VP16 by comparing the above regimen with the "standard" intermittent doses. PMID- 3021347 TI - Hepatic microsomal mixed-function oxidase activity in ethanol-treated hamsters and its consequences on the bioactivation of aromatic amines to mutagens. AB - Male golden Syrian hamsters were maintained on ethanol-containing liquid diets for 4 weeks, corresponding to an average daily intake of 17 g/kg body wt. The p hydroxylation of aniline was markedly enhanced by this treatment while minimal effects were seen in benzphetamine N-demethylase and ethoxyresorufin O-deethylase activities; there was no change in the microsomal levels of cytochromes P-450. Hepatic microsomal preparations from the ethanol-treated hamsters were more efficient than controls fed isocaloric diets in converting 2-aminofluorene, 4 aminobiphenyl, benzidine and 2-acetylaminofluorene into mutagens in the Salmonella mutagenicity test. The same treatment had no effect on the metabolic activation of 2-naphthylamine and even inhibited the mutagenicity of 2 aminoanthracene. No increase was seen in the activation of the two polycyclic aromatic hydrocarbons, benzo[a]pyrene and 3-methylcholanthrene to mutagens and an inhibitory effect was seen with the former. The ethanol-induced increase in the mutagenicity of 2-aminofluorene was inhibited by 2-butanol but not by the hydroxyl radical scavenger dimethylsulphoxide. It is concluded that chronic ethanol ingestion modulates the bioactivation of aromatic amines and amides to mutagens, the effect being substrate dependent. This effect of ethanol may be catalysed by unique form(s) of cytochrome P-450 whose synthesis is induced by such treatment. PMID- 3021348 TI - The effects of 4-hydroxy-2,3-trans-nonenal on beta-adrenoceptors of rat lung membranes. AB - Lung membranes are susceptible to oxygen radicals, formed during inflammation, redox cycling of toxic agents, exposition to ozon etc. Oxygen radicals may modify the beta-adrenergic response. However, at the same time beta-adrenoceptors of the lung are frequently addressed in therapy. We embarked upon this problem by studying the effects of the aldehyde 4-hydroxy-2,3-transnonenal (HNE), one of the major products of lipid peroxidation, on the density of beta-adrenoceptors of rat lung membranes. It is shown, that the physiological important sulfhydryl blocking agent HNE inactivates the beta-adrenoceptors in a time- and concentration dependent (0.5-2.5 mM) way, indicated by a decrease in (-)-[3H]dihydroalprenolol (DHA) binding to lung membranes. Moreover, it is shown that combined treatment of HNE with (-)-isoproterenol (0.5 microM) or 1-alprenolol (0.5-10 nM) does not influence the extent of inactivation of beta-adrenoceptors by HNE. This is in contrast with previous studies, conducted with other, synthetic, sulfhydryl blocking agents, such as N-ethylmaleimide (NEM), suggesting that an other mechanism of inactivation is involved upon HNE treatment. PMID- 3021349 TI - Aerobic and anaerobic reduction of nitrated pyrenes in vitro. AB - Nitrated pyrenes are mutagenic and tumorigenic environmental pollutants that are activated to DNA-binding derivatives via nitroreduction. We have investigated the enzymatic nitroreduction of 1-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene to determine if differences in the extent of nitroreduction may help explain differences in their biological potencies. Each nitrated pyrene was incubated aerobically and anaerobically with 105,000 X g supernatant (S105) from Salmonella typhimurium TA98 and the nitroreductase-deficient strain, TA98NR, and with cytosol and microsomes from rat and human liver. Under anaerobic conditions, 1 nitropyrene and 1,3-dinitropyrene were reduced by TA98 S105 to a lesser extent than 1,6- and 1,8-dinitropyrene. The extent of 1,6- and 1,8-dinitropyrene metabolism was not altered relative to TA98 when using TA98NR S105, but the nitroreduction of 1-nitropyrene and 1,3-dinitropyrene was decreased. Both rat and human liver cytosol and microsomes reduced 1,6- and 1,8-dinitropyrene to greater extents than 1-nitropyrene and 1,3-dinitropyrene. Under aerobic conditions rat and human liver cytosols were similar to TA98 S105 in that aminopyrene decreased while nitrosopyrene formation increased. By comparison, oxygen decreased the microsomal formation of both nitrosopyrenes and aminopyrenes. The reduction of succinoylated cytochrome c was measured during the hepatic metabolism of nitro- and nitrosopyrenes under aerobic conditions. The data indicated that reduced nitro- and nitrosopyrene intermediates were directly reducing succinoylated cytochrome c and that the assay could be used as a measure of aerobic nitroreduction. These studies demonstrate that 1,6- and 1,8-dinitropyrene are reduced to a greater extent than 1-nitropyrene and 1,3-dinitropyrene, which corresponds to their relative biological potencies as mutagens and carcinogens. Furthermore, although more extensive nitroreduction is detected under anaerobic conditions, the nitroreduction that occurs aerobically may be important for the mutagenic and tumorigenic properties of these compounds. PMID- 3021351 TI - Angiotensin converting enzyme-inhibitory triterpenes from Ganoderma lucidum. PMID- 3021350 TI - Design and synthesis of N-[N-[(S)-1-ethoxycarbonyl-3-phenylpropyl]-L -alanyl]-N (indan-2-yl)glycine (CV-3317), a new, potent angiotensin converting enzyme inhibitor. PMID- 3021352 TI - [Endemic emergence of yellow fever in the Ivory Coast: the place of anti-yellow fever IgM detection in the strategy of surveillance]. PMID- 3021353 TI - Apparent synergy in lung carcinogenesis: interactions between N nitrosoheptamethyleneimine, particulate cadmium and crocidolite asbestos fibres in rats. AB - Environmental carcinogenesis in man is widely accepted to be a multifactorial process, and in the causation of lung cancers it is suspected that low levels of systemic carcinogens may act synergistically with inhaled particulates so that some exposed individuals are at increased risk. In the present study the carcinogenic effects of low levels of industrially and environmentally significant particulate materials (crocidolite asbestos and metallic cadmium) and a putative systemic carcinogen, N-nitrosoheptamethyleneimine (NHMI), were investigated in the laboratory rat, using this as a model of potential human exposure. The overall lung tumour incidence rate in the group of animals receiving crocidolite, cadmium and NHMI (14/45) was significantly higher than in the groups of animals receiving either crocidolite and cadmium together (2/51) or crocidolite and NHMI together (7/42). The results demonstrated an apparent synergy between cadmium and NHMI in the presence of crocidolite in the causation of lung cancer in rats, a finding which was confirmed statistically. This study helps to further evaluate and define the roles of asbestos and particulate cadmium in the causation of lung cancer. It is suggested that people who are exposed through occupation and/or environment to cadmium and asbestos and to low levels of systemic carcinogens may show a significantly elevated risk of lung cancer. PMID- 3021354 TI - Novobiocin does not inhibit DNA repair in an active gene. AB - Novobiocin, an inhibitor of type II topoisomerase, has been reported to inhibit u.v.-induced DNA repair in a number of established mammalian cell lines; we have confirmed this general observation in primary cultures of human epidermal keratinocytes. Using a recently developed technique for measuring pyrimidine dimer frequencies in genomic restriction fragments, we have determined the extent of DNA repair in the active, essential dihydrofolate reductase (DHFR) gene. Novobiocin did not affect repair of the DHFR gene in keratinocytes or in a Chinese hamster ovary (CHO) cell line over a 24-h period following irradiation with 20 J/m2 u.v. These findings suggest that qualitative differences exist in the repair pathways in different genomic regions; topoisomerase II may not have an essential role in repair of active genes but may be required for repair of other regions in the genome. PMID- 3021355 TI - Local renal ischemia during burn shock in rat effected by thromboxane A2 and serotonin. AB - Intermittent patchy ischemia in the renal cortex during traumatic shock has previously been observed in dogs and rats. In recent experiments on rats a high rate of abrupt changes in local blood flow was observed after scalding. To try to reveal endogenous factors causing such ischemic episodes, we scalded six series of anesthetized rats (50% of body surface for 30 s in 80 degrees C water) and measured arterial pressure (AP), hematocrit (Hct), and local renal cortical blood flow (RCF). RCF was recorded by the local H2 washout technique. After scalding, RCF decreased markedly in all series whereas AP was relatively well preserved. In accordance with previous experiments, 11% of the washout curves recorded 0-75 min after scalding showed abrupt changes in local blood flow, rising to 27% during the next 60 min in drug-untreated rats. In contrast, blocking of serotonin S2 receptors with Ketanserin abolished the phenomenon. However, in rats treated with the prostaglandin synthesis blockers, indomethacin (general) or 3-ethyl pyridin (thromboxane A2 blocker), the phenomenon was observed in only 2-3% of the washout curves. Furthermore, after blocking the AngII receptors by saralasin or alpha receptors by phentolamine in separate series, the frequency of abrupt flow shifts was reduced in comparison to the frequency in untreated rats. The results indicate that intermittent, patchy vasoconstriction is mediated by serotonin and thromboxane A2 (TxA2), probably released from platelets. The occurrence of ischemic episodes also depends on the local AngII and alpha-adrenergic tonus present after scalding. PMID- 3021356 TI - Sympathetic reflex control of skeletal muscle blood flow in patients with congestive heart failure: evidence for beta-adrenergic circulatory control. AB - Mechanisms controlling forearm muscle vascular resistance (FMVR) during postural changes were investigated in seven patients with severe congestive heart failure (CHF) and in seven control subjects with unimpaired left ventricular function. Relative brachioradial muscle blood flow was determined by the local 133Xe washout technique. Unloading of baroreceptors with use of 45 degree upright tilt was comparably obtained in the patients with CHF and control subjects. Control subjects had substantially increased FMVR and heart rate to maintain arterial pressure whereas patients with CHF had decreased FMVR by 51 +/- 11% (mean +/- SEM, p less than .02) and had no increase in heart rate despite a fall in arterial pressure during upright tilt. The autoregulatory and local vasoconstrictor reflex responsiveness during postural changes in forearm vascular pressures were intact in both groups. Further investigations were carried out in the patients with CHF. The left axillary nerve plexus was blocked by local anesthesia in the seven patients. No alterations in forearm vascular pressures were observed. This blockade preserved the local regulation of FMVR but reversed the vasodilator response to upright tilt as FMVR increased by 30 +/- 7% (p less than .02). Blockade of central neural impulses to this limb combined with brachial arterial infusions of phentolamine completely abolished the humoral vasoconstriction in the tilted position. Infusions of propranolol to the contralateral brachial artery that did not affect baseline values of heart rate, arterial pressure, or the local reflex regulation of FMVR reversed the abnormal vasodilator response to upright tilt as FMVR increased by 42 +/- 12% (p less than .02). Despite augmented baseline values, forearm venous but not arterial plasma levels of epinephrine increased in the tilted position, as did arterial rather than venous plasma concentrations of norepinephrine in these patients. The results suggest a beta-adrenergic reflex mechanism elicited by spinal or supraspinal neural impulses and probably modulating a cotransmitter release in the patients with CHF. PMID- 3021357 TI - Enhancement of cardioplegic protection with the free-radical scavenger peroxidase. AB - This study assesses the ability of the free-radical scavenger peroxidase to enhance cardioplegic protection when given during or before myocardial ischemia. Forty-four isolated isovolumetric buffer-perfused rat hearts were studied. In a first series of experiments that consisted of three groups, hearts were subjected to 90 min of normothermic global ischemia followed by 45 min of reperfusion. One group received a crystalloid cardioplegic solution given as a single dose at the onset of arrest. A second group received cardioplegic solution supplemented with superoxide dismutase (200,000 U/liter), and a third group received cardioplegic solution supplemented with peroxidase (6000 U/liter). Based on comparisons of postreperfusion coronary flow, left ventricular developed pressure, maximum dP/dt, and diastolic pressure, we found that the best protection was provided by peroxidase-enriched cardioplegia. A second series of experiments was then undertaken to assess the effects of the latter enzyme given as a pretreatment. Hearts were subjected to 3 hr of global ischemia, during which myocardial protection was provided by hypothermia (15 degrees C) along with multidose cardioplegia. The treatment group was given peroxidase (10,000 U/liter) added to the perfusate fluid for 15 min before the onset of cardioplegic arrest without further enzyme supplementation during ischemia or reperfusion. Hearts perfused with standard buffer for an equal period of time served as controls. While the two groups demonstrated the same degree of postischemic increase in myocardial stiffness, peroxidase-pretreated hearts had a significantly better recovery of contractile indexes at 30 and 45 min of reflow.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021358 TI - Errors in measurements of CO2 with the use of drying agents. AB - To assess the errors in measurements of CO2 concentration that arise from the widespread use of drying agents in clinical and physiological studies, continuous measurements of CO2 concentration were made with an infra-red analyser before and after standard gas mixtures were passed through a tube containing one of several drying agents: calcium chloride; calcium sulphate (Drierite); alumina; silica gel; and magnesium perchlorate. The response time of the infra-red analyser was independent of the concentration of CO2 used (0-1%), but it was related to flow rate, dead space and the amount and drying agent used. At a low rate of 1 litre/min, alumina (145 g anhydrous weight) trapped virtually all the CO2 (0.8% in air) passing over it; silica gel (145 g anhydrous weight, mesh 6-20) adsorbed approximately 2 mmol CO2 (38% of total) over a period of 15 min; calcium sulphate (Drierite) absorbed a variable amount, depending on the bath; and calcium chloride adsorbed virtually no CO2. Increasing hydration of the drying agents reduced their capacity to adsorb CO2. Nitrogen was found to elute CO2 from the drying agents at about the same rate as it had been adsorbed. It is concluded that awareness of these adsorption/elution phenomena by drying agents should prevent errors from being made in the calibration of CO2 analysers, in the analysis of CO2, and in the measurement of specific activity or isotopic enrichment of CO2. PMID- 3021360 TI - Mitochondrial myopathy: tissue-specific expression of a defect in ubiquinol cytochrome c reductase. AB - We have expanded our studies on a patient with a mitochondrial myopathy caused by a defect at the level of complex III of the respiratory chain. Using activity measurements, electron microscopy, protein synthesis in the presence of emetine, and antibody binding, we have demonstrated that the defect is not expressed in cultured skin fibroblasts from this patient. Electron microscopy of peripheral blood leukocytes and activity measurements in transformed lymphoid cells indicated that the defect was not expressed in these cells either. These results imply that there are either isoforms of complex III components which show differential tissue expression or that independent segregation and assortment of defective mitochondria has occurred during development. PMID- 3021359 TI - Sarcoplasmic reticulum Ca2+-ATPase and acylphosphatase activities in muscle biopsies from patients with Duchenne muscular dystrophy. AB - Sarcoplasmic reticulum Ca2+-ATPase, acylphosphatase and other soluble enzymes (creatine kinase, lactate dehydrogenase, aldolase and pyruvate kinase) were assayed in muscle biopsies from patients affected by Duchenne muscular dystrophy (DMD) and from normal controls. Specific activities of all the soluble enzymes were decreased in dystrophic muscle, acylphosphatase exhibiting the most marked and significant decrease comparable to that of creatine kinase, in spite of a moderate increase in serum levels. Also, Ca2+-ATPase, particularly the calcium dependent activity, was decreased in dystrophic muscle. A positive correlation, higher than with the other soluble enzymes, was obtained between acylphosphatase specific activity and the percentage of Ca2+-activation of Ca2+-ATPase. These findings: suggest an impairment of microsomal calcium uptake which could be, at least in part, responsible for sarcoplasmic calcium accumulation observed in DMD; do not disagree with an hypothesized role of acylphosphatase in intracellular calcium homeostasis, consistent with the enzyme's demonstrated hydrolytic activity on the phosphorylated intermediate of Ca2+-ATPase. PMID- 3021361 TI - Spin-echo and 2-dimensional 1H nuclear magnetic resonance studies on urinary metabolites from patients with 2-methylacetoacetyl CoA thiolase deficiency. AB - Spin-echo 1H nuclear magnetic resonance (nmr) spectra of urine from two unrelated patients with 3-oxoacylthiolase deficiency are presented. The metabolite profile revealed by these spectra is probably of diagnostic value. Two of the major abnormal metabolites known to accumulate in this disorder (tiglylglycine and 2 methyl-3-hydroxybutyrate) can both be detected in the spectra. The technique offers the advantage over combined gas chromatography/mass spectrometry that no pre-treatment of the sample is required and that the data can be obtained in less than 2 min. In addition 2-dimensional nmr techniques (J-resolved and chemical shift correlated) were used to further characterise the spectra. This allowed assignment of resonances which could not be detected in the spin-echo spectra, in particular 2-methylacetoacetate and butanone, which are known to accumulate in this disease. PMID- 3021362 TI - Direct demonstration of an inhibitor of sodium, potassium dependent adenosine triphosphatase (Na+, K+-ATPase) in plasma from normotensive and hypertensive subjects. AB - An endogenous inhibitor of Na+, K+-ATPase was extracted from human plasma and sera and concentrated by a novel reverse-phase octadecylsilane chromatography method. The active extracts (eluates) were dried and reconstituted in the minimum volume of the non-adsorbed fraction of the plasma from which they had been derived. Reconstituted eluates, non-adsorbed plasma fractions and native plasma samples were then tested for their ability to inhibit phosphate production in standard Na+, K+-ATPase incubation mixtures. In a pilot study 31 samples of pooled normal human sera were assayed. The eluates gave a significantly lower production of phosphate than the non-adsorbed fractions or the native sera (n = 31, p less than 0.0025). Further concentration of the eluates by repeated chromatography increased the inhibitory power of the eluate proportional to the concentration achieved, as quantified by ouabain dose-equivalents. In clinical studies, samples from 12 normotensive subjects and from 12 untreated patients with essential hypertension were tested. Significant inhibition of the ATPase by the eluates, as compared to the corresponding non-adsorbed fractions was seen for samples from both normotensive (p less than 0.05) and hypertensive (p less than 0.05) subjects. There was no significant difference in incidence or degree of inhibition between the normotensive and hypertensive groups. This study provides direct evidence for the presence of an endogenous inhibitor of Na+, K+-ATPase in human plasma. PMID- 3021363 TI - A sandwich immunoassay for human prolyl 4-hydroxylase using monoclonal antibody. AB - Monoclonal antibody was used in a sandwich enzyme immunoassay and in a radioimmunoassay for human serum immunoreactive prolyl 4-hydroxylase. The enzyme immunoassay utilized a monoclonal antibody as a solid phase and horseradish peroxidase-labeled rabbit antibody (Fab') to human prolyl 4-hydroxylase as a conjugate. Sensitivity was 0.1 ng (0.4 fmol) of enzyme per tube. With a conjugate purified by an enzyme-bound affinity column, sensitivity was increased to 0.01 ng (0.04 fmol) per tube, and linearity was obtained between 0.01 to 30 ng (0.04-125 fmol) per tube. The radioimmunoassay used a 125I-labeled rabbit antibody (IgG) as the conjugate. Sensitivity of this technique was 0.4 ng of enzyme per tube. The enzyme immunoassay gave reproducible quantitation and evidenced a higher enzyme concentration in the serum of patients with liver disorders. Protein immunoblotting showed that the serum immunoreactive prolyl 4-hydroxylase trapped in the sandwich immunoassay was mainly the beta-subunit. PMID- 3021364 TI - Changes in immunoreactive beta-endorphin, methionine-enkephalin and ACTH in bone marrow cells and fluid from leukemic children. AB - The present study demonstrates the presence of the endogenous opioid peptides, beta-endorphin (beta-EP) and methionine-enkephalin (MET-ENK), and of ACTH in cell homogenates and interstitial fluid from sternal biopsy of leukemic children. The peptides were identified by chromatography and radioimmunoassay. In leukemic children with lymphoblastic cells present in the sternal sample, concentrations of immunoreactive (ir) beta-EP in the cell homogenate, but not in the fluid, were significantly higher than in leukemic children with normal bone marrow. In contrast, ir MET-ENK and ir ACTH did not differ between the two study groups either in the cell homogenate or in the fluid. These data suggest the presence of a complex system of opioid peptides in the cells and interstitial fluid of bone marrow of leukemic children with the highest concentrations of ir beta-EP appearing in samples collected during the active phase of the disease, and may suggest a possible role of opioid peptides as immunomodulatory substances. PMID- 3021366 TI - [A case of adrenoleukomyeloneuropathy(ALMN) with pseudobulbar symptom- predominancy case of brainstem and cerebellum atrophies]. PMID- 3021365 TI - The effect of N-(2-carboxyphenyl)-4-chloroantranilic acid disodium salt (CCA) on concanavalin A-induced superoxide anion release in human mononuclear phagocytes. AB - Superoxide anion (O2-), generated and released by mononuclear phagocytes (M phi), plays significant roles in tissue damage accompanying inflammatory and immunological reactions. N-(2-carboxyphenyl)-4-chloroantranilic acid disodium salt (CCA), is a newly synthesized immunomodulator for which therapeutic efficacy in the treatment of rheumatoid arthritis (RA) has been reported. In this investigation, we have examined the effect of CCA on concanavalin A (Con A) induced O2- release in human M phi. Exposure of M phi to Con A induced a large increase of O2- release. Preincubation of M phi with CCA suppressed O2- release induced by Con A almost to a control level. On the contrary, CCA itself did not have a significant effect on basal O2- release from M phi. These results indicate that the inhibition of O2- release from activated M phi may be an important factor in the anti-inflammatory effects of this drug. PMID- 3021367 TI - [A case of vasculitic neuropathy with pulmonary tuberculosis, anti-endothelial cells activity and increased serum level of immune complexes]. PMID- 3021368 TI - Splenic "disappearance" during gated exercise nuclear angiocardiography. AB - Decreasing splenic activity occurred during the maximum exercise phase of gated stress testing in 24 patients who underwent 26 such studies. This event was related to increasing exercise, and correlated with the relative increase in heart rate, pulse-pressure product, and cardiac output, but not ejection fraction. A multiple linear regression analysis showed that the combination of increases in pulse-pressure product and cardiac output had the best correlation with decreasing splenic activity at the conclusion of exercise. Redistribution of cardiac output toward muscle and away from splanchnic viscera is a consequence of exercise, and is graphically demonstrated by decreasing splenic visualization, which in some patients was so marked as to result in disappearance of the organ from the video monitor. PMID- 3021369 TI - A patient with primary hyperparathyroidism and an abnormal bone scan. AB - A patient with primary hyperparathyroidism whose bone scan showed signs of extensive pulmonary and gastric calcifications is described. The patient also had renal insufficiency. A review of the literature and of data of 13 patients with primary hyperparathyroidism who were seen in the Department of Internal Medicine at the University Hospital of Maastricht, led to the conclusion that only in patients with renal insufficiency could ectopic calcifications be expected to occur. Phosphate retention, rather than the hyperphosphaturia that occurs in that particular situation, is cited as the cause. PMID- 3021371 TI - Diffuse uptake of technetium-99m pertechnetate in a patient with metastases from thyroid carcinoma. PMID- 3021372 TI - Budd-Chiari syndrome complicating hepatocellular carcinoma. Demonstration by multiple radiotracer scintigraphy. AB - A case of hepatocellular carcinoma (HCC) complicated by the Budd-Chiari syndrome is described. The antemortem diagnosis of both conditions was made with the unique findings of multitracer scintigraphy. The difficulty of diagnosing these two conditions by the conventional approach is reviewed. The advantages of using multitracer scintigraphy for evaluation of hepatic lesions are also discussed. PMID- 3021370 TI - Detection of hemangiomas using whole-body imaging with technetium-99m labeled RBCs. AB - Hemangiomas are easily, quickly and relatively noninvasively detected using whole body imaging with Tc-99m labeled RBCs. A case in which this method was used to confirm known lesions and detect additional unsuspected lesions is described. This method can also be useful in the detection and evaluation of other types of vascular malformations, such as arteriovenous communications. The lesions must be of sufficient size and flow to be seen. Applications include the examination of patients known to have one vascular malformation in a search for additional lesions and the screening of relatives of patients with familial conditions that include hemangiomas. PMID- 3021373 TI - Septal infarction demonstrated on technetium-99m PYP SPECT. AB - The case of a 62-year-old man with an acute myocardial infarction detected by planar Tc-99m PYP imaging is presented. The use of SPECT imaging provided more information with regard to infarct localization by demonstrating uptake by the septum, a finding not apparent on the conventional planar images. PMID- 3021374 TI - Intraperitoneal hemorrhage diagnosed by technetium-99m labeled RBC imaging. AB - During examination for occult gastrointestinal bleeding, Tc-99m labeled RBC imaging demonstrated intraperitoneal bleeding into Morrison's pouch from a hepatocellular carcinoma. PMID- 3021376 TI - Dynamic or early imaging in dual-tracer parathyroid scintigraphy. AB - The dual tracer radionuclide method using Tl-201 and Tc-99m subtraction imaging has proven to be effective for evaluation of parathyroid lesions. Ninety-two percent sensitivity for detection of parathyroid adenomas has been reported. The importance of dynamic or early imaging, however, has not been emphasized. A case of surgically proven parathyroid adenoma was detected using dynamic scanning after Tl-201 injection and was less apparent on delayed image due to rapid washout. PMID- 3021375 TI - Dual isotope parathyroid imaging. AB - Eleven patients who had clinical and biochemical evidence of primary hyperparathyroidism were studied using dual isotope technetium-thallium parathyroid subtraction imaging. The parathyroid scans correctly identified all surgically proven parathyroid adenomas. Three abnormal foci also were identified that were not parathyroid adenomas. These were caused by adenocarcinoma metastatic to a lymph node, primary papillary carcinoma of the thyroid, and parathyroid hyperplasia. This report also demonstrates the importance of surgically examining all abnormal sites of uptake. PMID- 3021377 TI - Colon carcinoma metastatic to the thyroid gland. AB - Metastatic carcinoma to the thyroid gland rarely is encountered in clinical practice; however, autopsy series have shown that it is not a rare occurrence. A case of adenocarcinoma of the colon with metastases to the thyroid is reported. A review of the literature reveals that melanoma, breast, renal, and lung carcinomas are the most frequent tumors to metastasize to the thyroid. Metastatic disease must be considered in the differential diagnosis of cold nodules on radionuclide thyroid scans, particularly in patients with a known primary. PMID- 3021378 TI - Myocardial imaging. Coxsackie myocarditis. AB - A 3-week-old male neonate with heart failure associated with Coxsackie virus infection was imaged with Tc-99m PYP and TI-201. The abnormal imaging pattern suggested myocardial infarction. Autopsy findings indicated that the cause was myocardial necrosis secondary to an acute inflammatory process. Causes of abnormal myocardial uptake of Tc-99m PYP in pediatrics include infarction, myocarditis, cardiomyopathy, bacterial endocarditis, and trauma. Myocardial imaging cannot provide a specific cause diagnosis. Causes of myocardial infarction in pediatrics are listed in Table 1. PMID- 3021380 TI - The (Na+-K+-Cl-) co-transport system. PMID- 3021379 TI - [Phantom substances for the quantitative evaluation of MR T images. I. Paramagnetic agar gels for the optimal simulation of tissue specific MR T parameters]. AB - Reference material for quantitative magnetic resonance imaging (MRI) has been developed by combining water, gelling agent agar, and paramagnetic ions (Gd3+). By in-vitro measurement of the relaxation times T1 and T2 it can be shown that tissue-relevant values can be attained. The substances described are physically and chemically stable and can be handled without problems. PMID- 3021381 TI - Respiratory pharmacology. Antibiotics. II. Aminoglycosides, polymyxins, vancomycin, trimethoprim-sulfamethoxazole, and pentamidine. AB - This article provides the pulmonary specialist with a summary description of these commonly used antimicrobial agents, including mode of action, antimicrobial spectrum, pharmacology, toxicity, indications for use, and recommended dosages. PMID- 3021382 TI - The pharmacology of agents used in the treatment of pulmonary mycoses. AB - The different antifungals currently used in pulmonary mycoses consist of polyene antibiotics, the antimetabolite 5-fluorocytosine, and azole derivatives. The pharmacologic profiles, mechanisms of action, pharmacokinetics, clinical indications for use, dosage recommendations, side effects, and drug interactions for these agents are presented. PMID- 3021383 TI - Hydroxyapatite formation in collagen, gelatin, and agarose gels. AB - Collagen has long been suspected to be a promoter of hydroxyapatite (HA) deposition in bone. This theory was tested by comparison of HA formation in gels composed of collagen, gelatin or agarose. Collagen gels supported substantially more HA precipitation than gelatin gels, but slightly less than agarose gels. Analysis of the relative diffusion rates for calcium in these matrices indicated that, in this system, amount of HA formation is dependent upon the rate of diffusion. Under conditions in which the diffusion rates for collagen and agarose gels were comparable, similar amounts of HA were formed. This suggests that fibrillar collagen is not per se a promoter of HA deposition. Extracellular matrix macromolecules may influence calcification by restricting ionic diffusion through connective tissues. PMID- 3021384 TI - Homology between exon-containing portions of rabbit genomic clones for synovial cell collagenase and human foreskin and synovial cell mRNAs. AB - Portions of rabbit genomic clones containing exons cross-hybridize with human synovial cell and human foreskin fibroblast mRNAs. These cross-hybridizing genomic fragments have been subcloned into pBR328 and may be useful probes in isolating cDNA and genomic clones from human tissue, and perhaps from other species as well. In addition, DNA sequence data on a 530 bp cDNA clone for rabbit synovial-cell collagenase indicate that this clone is composed primarily of 3' untranslated region. As such, it is probably not useful in cross-species hybridizations. PMID- 3021385 TI - A study of a herpesvirus isolated from dairy cattle with a history of reproductive disorders. AB - Three strains of herpesvirus were recovered from cows with vulvovaginitis. The three isolates (85/BH 16TV, 85/BH 17TV, 85/BH 18TV), when compared by cross serum neutralization (SN) tests, were found to be antigenically identical. They were serologically distinct from infectious bovine rhinotracheitis (IBR) virus and Bovid herpesvirus 2 (BHV2), while they cross reacted with bovine herpesvirus DN 599. Besides the serologic aspects, the three isolates appeared to share common biological, physical and morphological properties with the newly recognized bovine herpesviruses, of which DN-599 is a representative strain. PMID- 3021386 TI - Neutralization kinetics of bovine viral diarrhea virus by hyperimmune serum: one or multi-hit mechanism. AB - The kinetics of bovine viral diarrhea (BVD) virus neutralization (VN) by hyperimmune serum followed first order kinetics at low dilutions (1:8 and 1:16) of hyperimmune bovine serum. A lag phase in the VN curve occurred when the serum was further diluted. Addition of rabbit anti-bovine IgG, but not anti-IgM, significantly increased the degree of VN after BVD virus was reacted with further diluted (1:256 dilution) anti-BVD bovine hyperimmune serum. Incubation of virus hyperimmune serum (1:64 dilution) at 4 degrees C for 0 to 60 min, followed by incubation at 37 degrees C indicated VN occurred as a two-stage process: a binding (temperature-independent) phase that was followed by a triggering (temperature-dependent) phase. The data favor the thesis that neutralization of BVD virus occurs by a multi-hit mechanism and requires combination of at least two molecules of antibody with each virus. PMID- 3021388 TI - Superoxide generation by pig alveolar macrophages. AB - Pig alveolar macrophages generate superoxide anions at a rate of 1.8 nanomoles/1 X 10(6) cells/min. The intracellular value of ATP in resting cells was 4.0 +/- 0.1 X 10(-16) mole/cell; in contrast the value in cells generating superoxide anions was 2.0 +/- 0.6. Superoxide generation was increasingly inhibited by exposing cells to adenosine from 0.1 to 1.0 mM. Unlike human macrophages, pig cell production of superoxide anions was not inhibited by exposure to the adenosine analog, 2-Cl-adenosine. PMID- 3021387 TI - Quantitation of natural antibodies to infectious bursal disease in Nigerian indigenous chickens. AB - Serum samples collected from 687 indigenous chickens located in small scattered groups in four states of Nigeria were examined for antibodies to infectious bursal disease (IBD) virus by the agar-gel precipitation (AGPT) and counterimmunoelectrophoresis (CIEOP) tests. 51 of the positive samples were further titrated by each of the two techniques. CIEOP detected more positive reactors (74.59%) than AGPT (58.95%). CIEOP also detected higher antibody levels among the reactors [geometric mean titre (GMT) of 51 samples was 23.02] when compared to AGPT. GMT of the same 51 samples was 21.8. The prevalence of antibodies to IBD virus in the indigenous chickens ranged from 64.7 to 77.7% CIEOP reactors between states. Since reports of IBD outbreaks among these chickens are rare unlike the situation among commercial poultry flocks, it was concluded that local chickens probably act as carriers of IBD virus. PMID- 3021389 TI - Vasoactive role of endogenous digoxin-like factor in rats with experimental hypertension. AB - Acute blockade of circulating digoxin-like factor endoxin lowered blood pressure (BP) by a decrease in systemic resistance which was partially compensated by an increased cardiac output. The important participation of circulating endoxin in the maintenance of elevated BP was proved in young animals with DOCA-salt and one kidney, one-clip (1K1C) Goldblatt hypertension but not in young spontaneously hypertensive rats (SHR). In rats with DOCA-salt as well as with 1K1C hypertension, the role of endoxin differed according to the age at which the hypertensive stimulus was applied. The significant role of the "digoxin-like" factor in BP regulation was found only in young animals. On the other hand, in SHR the participation of endoxin in the maintenance of elevated BP rises with the age. High salt intake prior to sexual maturation seems to be critical for later participation of the "digoxin-like" factor in long-term BP regulation. PMID- 3021390 TI - Myocardial changes in influenza virus and staphylococcus infection. AB - Experiments carried out on 134 newborn mice showed that influenza virus and staphylococcus infection and their combination induces local necrosis in the myocardium. Morphometric measurement has shown that the extent of necrotic changes amounted with the action of the influenza virus to 1.37%, with the action of staphylococcus to 1.4% and with their combined action to 1.8%. Electron microscopy revealed dystrophic changes in cardiomyocytes with subsequent intracellular regeneration. PMID- 3021391 TI - Effect of dichloroacetate in the treatment of anoxic lactic acidosis in dogs. AB - Lactic acidosis is seen frequently after severe anoxia and circulatory failure. Because dichloroacetate (DCA) has been shown to be effective in the treatment of lactic acidosis, we studied its effect on lactate levels and pH in arterial and sagittal sinus blood specimens in a pediatric canine model of anoxic cardiac arrest followed by CPR. Lactate levels rose steadily in all puppies receiving DCA alone (group 1), DCA plus bicarbonate (group 2), bicarbonate alone (group 3), or neither drug (group 4). Arterial and sagittal-sinus lactate levels were in the range of 2 mmol/L during the baseline period, 6 mmol/L after anoxic arrest, and 10 mmol/L after 20 min of CPR. Bicarbonate, but not DCA, significantly raised arterial pH. Neither drug reversed the progression of acidosis in the sagittal sinus; mean pH ranged from 6.85 to 6.92 among the four groups after 20 min of CPR. We speculate that DCA did not decrease lactate levels or raise the pH in either the peripheral circulation or the CNS (sagittal sinus) because of poor perfusion achieved during closed-chest cardiac compression. PMID- 3021392 TI - A quantitative evaluation of the extent of inner mitochondrial membrane destruction after freezing-thawing based on functional studies. AB - An assay based on comparative investigation of ATPase and ATP synthetase changes in the activity in rat liver mitochondria after low temperature and uncoupler 2,4 dinitrophenol treatment is considered. By varying the activity of the respiratory chain, three extents of membrane cryoinjury could be distinguished by monitoring the changes in ATPase activity under different types of cryoinfluence. The first extent corresponds to a minimum membrane destruction, where the action of the respiratory chain compensates the changes in proton permeability and the cryotreatment does not change the ATPase activity. The second extent corresponds to changes in ion permeability which is partially compensated by the respiratory chain action. The third extent corresponds to a maximum membrane destruction and, therefore, maximum increase in permeability which is related to the irreversible stimulation of the ATPase activity and complete inhibition of the phosphorylation. In this study the extent of cryoinjury in mitochondrial preparations frozen and thawed at different rates was evaluated using this method. PMID- 3021393 TI - Lens proteins are substrates for the reticulocyte ubiquitin conjugation system. AB - In the aged lens postsynthetically altered molecules comprise the majority of lens proteins. Many proteolytic activities have been observed in lens supernatants. Since damaged or altered proteins are usually selectively and rapidly degraded in other cells and tissues, the accumulation of these species in the lens seemed enigmatic. Initiation of proteolysis has been studied most extensively in reticulocytes and ts 85 cells. In these systems proteolysis is absolutely ATP dependent, occurs effectively on high molecular weight substrates and, at least for a majority of proteolytic reactions, requires conjugation of ubiquitin to putative substrates. It seemed plausible that the accumulation of high molecular weight protein aggregates in older lenses might be due to the attenuated function of these ubiquitin- and ATP-dependent components in the initial committing processes of proteolysis. This research shows that: ubiquitin is present in the lens; lens proteins are conjugated to 125I-ubiquitin using reticulocyte conjugating systems; the reaction is ATP dependent; proteins from lens epithelium/outer cortex and core form different ubiquitin conjugates; lens proteins compete with lysozyme and reticulocyte proteins for the ubiquitin conjugating apparatus; most of the conjugates are of very high molecular weight; there is a temporal nature to the pattern of conjugates observed; and the ubiquitin conjugation system shows extreme selectivity. PMID- 3021395 TI - Relationship between lacrimal gland, isolated cells (lacrimocytes) and tears: biochemical and histological studies in the rabbit eye. AB - The rabbit defense system has a number of specific features: no lactoferrin and lysozyme are detectable and peroxidase activity is only demonstrated in the cubic epithelial cells of the ducts. Experiments carried out with radioactive amino acid, demonstrate the absence of secreted proteins with molecular weights corresponding to those of albumin and transferrin, indicating that these proteins are not synthesized by the lacrimal gland tissue. Rabbit tear pattern presents a set of acidic proteins secreted by the lacrimal gland tissue, with small molecular weight and acidic pI's. PMID- 3021394 TI - Herpesvirus simplex in chronic human stromal keratitis. AB - A method is described for the isolation of Herpesvirus simplex (HSV) from corneal discs of patients suffering from chronic stromal keratitis. The discs were removed during penetrating keratoplasty. Virus was successfully isolated from 2 out of 8 discs maintained in vitro. PMID- 3021396 TI - Pheochromocytoma: a clinical and experimental overview. PMID- 3021398 TI - Cyclic nucleotide levels in plasma and cerebrospinal fluid during transient ischemic attacks of brain. PMID- 3021397 TI - Hepatic resection for primary liver cancer. Report of 400 cases. PMID- 3021399 TI - [Retrorectal and presacral tumors]. PMID- 3021400 TI - [Spinal cord metastasis in malignant trophoblastic diseases: report of 15 cases]. PMID- 3021401 TI - [Detection of herpes simplex virus antigen in human cervical carcinoma tissue]. PMID- 3021403 TI - [Infantile spasm]. PMID- 3021402 TI - Characterization of SV40 chromatin by mass determination on STEM. AB - Direct mass determination of purified SV40 minichromosomes was obtained by scanning transmission electron microscopy. Twenty to thirty percent of the minichromosomes were found with an Mr of 6.9 +/- 0.4 X 10(6). The rest of the molecules formed a spread Mr distribution ranging from 7.3 X 10(6) to 9.5 X 10(6) due possibly to different contents of the virus-coded proteins, mainly VP1. The apparent mass histogram of individual SV40 nucleosomes presents three maxima at Mr 2.1 X 10(5), 2.6 X 10(5) and 3.1 X 10(5) that could correspond to partially unravelled nucleosomes, complete nucleosomes and complete nucleosomes with the addition of VP1. Beaded structures with a higher mass were also measured; some were found at either side of the open nucleosome-free region. PMID- 3021404 TI - [An investigation of phosphatic manure worker pneumoconiosis]. PMID- 3021405 TI - State-of-the-art barium examination in opportunistic esophagitis. AB - This report presents a comparison of state-of-the-art esophagography and endoscopy in the diagnosis of pathologically proven esophagitis. The modern multiphasic esophagogram is shown to have a sensitivity of 92% for the detection of opportunistic esophagitis in the immunocompromised patient. State-of-the-art esophagography provides a sensitive and inexpensive method for investigating patients in whom opportunistic esophagitis is suspected and for monitoring their response to therapy. PMID- 3021406 TI - Role of adrenergic alpha-receptors in regulation of acetylcholine release evoked by distension of guinea pig ileum. AB - The significance of adrenergic nerves in the regulation of acetylcholine (ACh) release evoked by distension of the guinea pig ileum was evaluated by pharmacological manipulations. ACh release was measured by bioassay. Release in response to distension was completely abolished by epinephrine and norepinephrine. Tyramine also suppressed the distension-evoked ACh release, while dopamine was ineffective. The release of ACh in the anal segment, adjacent to the distended part, was abolished by epinephrine and norepinephrine. Dibenamine and phentolamine abolished ACh release anal to the distension, but augmented release orally, while dichloroisoproterenol and propranolol were ineffective. The present results give direct evidence that adrenergic nerves modulate cholinergic transmission in the myenteric plexus through alpha-receptors. PMID- 3021407 TI - Nutritional risk of high-carbohydrate, guar gum dietary supplementation in non insulin-dependent diabetes mellitus. AB - Dietary supplementation with high-carbohydrate, guar gum fiber (HCF) is effective in acutely blunting postprandial blood glucose levels. We report the effect of such supplementation on the diet and nutritional status of a group of 16 subjects with non-insulin-dependent diabetes mellitus (NIDDM) who incorporated either HCF bars (35.7 g carbohydrate and 6.6 g guar gum/bar) or placebo bars (identical except for the absence of guar gum) into the diet for 6 mo as part of a double blind, randomized clinical trial. The HCF subjects achieved mean daily intake of 4.8 +/- 0.4 bars, constituting 51.2 +/- 3.1% of total calories and providing 29.7 +/- 2.6 g guar gum daily. Energy intakes and body weight did not change significantly in either group. Food consumption patterns and nutrient intakes did change, although not enough to impair the nutritional integrity of the diet because the bars themselves served as a source of nutrients. The bars were rich in thiamin, B6, folacin, phosphorus, iron, zinc, and copper, adequately replacing any decrease in nutrient intake as a result of foods being dropped from the diet. In fact, daily intakes of B6, folacin, and copper actually increased due to contributions from the bars. Nutrients in which the bars were poor (vitamins A, C and B12) resulted in suboptimal intakes (less than 66% RDA). Although no significant change in nutritional status of the HCF group occurred as determined by arm muscle area, arm fat area, hemoglobin, hematocrit, or serum albumin, transferrin, iron, ferritin, calcium, phosphate, B12, and magnesium levels, these indicators of nutritional status are rather insensitive.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021408 TI - Pulmonary cytology of the acquired immune deficiency syndrome: an analysis of 36 cases. AB - The infectious pathogens and associated cellular reactions in 75 pulmonary cytological specimens obtained largely by fiberoptic bronchoscopy from 36 patients with the acquired immune deficiency syndrome are described and correlated with the biopsy specimens. An opportunistic pathogen was diagnosed in 33% of cytological preparations. Pneumocystis carinii was encountered most frequently; Cryptococcus neoformans and cytomegalovirus were also seen. The polymorphonuclear neutrophil was the predominant inflammatory cell. Cells of secondary import were bronchial cells and type II pneumocytes with atypia and hyperplasia. Bronchoalveolar lavage had the highest yield, positive predictive value, and sensitivity for all pathogens and for P. carinii. An opportunistic pathogen was diagnosed in 69% of patients utilizing histological material and in 33% using cytological samples; the rate of diagnosis increased to 72% when the samples were combined. PMID- 3021410 TI - Hyperplastic and neoplastic endocrine cells of the pancreas in aspiration biopsy. AB - Aspiration biopsy of four primary endocrine tumors of the pancreas showed single and large sheets of tumor cells. Tumor cells were polygonal in shape with ill defined, filmy or granular cytoplasm and regular round nuclei containing a finely granular chromatin pattern and inconspicuous or prominent nucleoli. Focal gland like arrangement was seen in several sheets of tumor cells. Aspirate from liver metastases of an endocrine tumor of the pancreas showed single and clustered oval cells with granular cytoplasm and eccentrically located round and hyperchromatic nuclei. Smears prepared from aspirates of three fibrotic pancreata containing nodules of hyperplastic endocrine cells showed smaller fragments of endocrine cell epithelium with focal gland-like arrangement. Individual hyperplastic endocrine cells displayed granular, filmy, and ill-defined cytoplasm and round to oval hyperchromatic nuclei showing a finely granular chromatin pattern. Nuclear pleomorphism was noted in some cell groups. PMID- 3021409 TI - Pulmonary cytology in patients with the acquired immunodeficiency syndrome (AIDS). AB - Over a 2-yr period, 1982-1984, 181 pulmonary cytology specimens were obtained from 45 patients clinically suspected of having AIDS in an attempt to identify the various organisms and the cytologic atypias present in pulmonary cells. Cytologic correlations with autopsy findings were available for 28 of these cases (62%). Specimens were collected from sputum, bronchial washes and brushes, bronchoalveolar lavages, and pleural fluid. Of the 181 specimens, 121 (67%) were of diagnostic value. Depending on the cytologic technique, Pneumocystis carinii, cytomegalovirus, herpesvirus, and Candida were detected with varying frequency. The bronchial epithelial and glandular cell atypias, ranging from mild inflammatory reactive changes to marked cellular atypia, were noted most often in specimens from patients with pulmonary infections who were concurrently receiving oxygen therapy and/or chemotherapy for Kaposi's sarcoma or central nervous system lymphoma. An awareness of the wide range of AIDS-associated pulmonary cell atypias is required to rule out a diagnosis of malignancy. PMID- 3021411 TI - Aspiration biopsy cytology of metastatic endometrial stromal sarcoma and extragenital mixed mesodermal tumor. AB - Aspiration biopsy from metastatic tumors in two cases of endometrial stromal sarcoma and one case of endometrial adenosarcoma revealed malignant endometrial stromal cells with ill-defined cytoplasm and round or oval hyperchromatic nuclei showing irregular chromatin clumping and conspicuous nucleoli. They were seen mainly in clusters. Aspirate from a metastatic tumor of a mixed mesodermal tumor arising from the omentum showed similar malignant endometrial stroma cells, irregular tight clusters of malignant glandular cells having scanty but well defined cytoplasm and vesicular nuclei with conspicuous nucleoli, and fragments of atypical smooth muscle tissue. The diagnostic malignant endometrial stromal cells in those reported cases did not display any distinctive cellular features permitting their cytologic identification. They were difficult to differentiate from those of other types of sarcoma. In a clinical setting, with a known primary endometrial stromal sarcoma or mixed mesodermal tumor, however, a cytodiagnosis of its metastases may be suggested when malignant endometrial stromal-cell-like cells are seen in aspirated material, oviating an open-tissue biopsy. PMID- 3021412 TI - [Labelling of DNA by iodination and its application on the detection of EBV-DNA]. AB - The authors developed a simple and efficient method--the iodination of the single stranded DNA catalyzed by thallium chloride in vitro. The 125I-labelled DNA has a specific activity higher than 10(8) cpm/micrograms DNA. This reaction does not involve any enzyme. The optimal conditions of the iodinating reaction, including the high temperature, low pH and the concentrations of DNA and iodine, are described. 125I-labelled DNA inserted with the fragment of EBV-DNA are used to detect EBV-DNA by blot and spot and in situ hybridization. PMID- 3021413 TI - [Gastroscopy plus cytology for the diagnosis of gastric cancer or precancerous diseases--report of 127 cases]. AB - One hundred and twenty-seven patients with gastric tumor or precancerous diseases were examined by gastroscopy plus cytology, and the results were compared with that of histopathology after operation. Out of 127 cases, 70 were diagnosed as gastric cancer (15 cases as early cancer, 55 as advanced cancer). The positive rate for early cancer was 21.4%, it comprised 11.2% of all gastric cancers diagnosed by gastroscopy during the same period. The preoperative positive rate of early gastric cancer was 100% (13/13) as examined by the combination of touch, brush cytology and mucosal biopsy. The combined method may increase the positive rate and the accuracy, not only for advanced but also for early gastric cancers. It is suggested that the combination of these methods be routinely used in the diagnosis of suspicious cancer in the gastric mucosa. PMID- 3021414 TI - [Preoperative radiotherapy and ultrastructural pathology of lung cancer]. AB - The results of 26 patients with lung cancer treated by preoperative radiotherapy from March 1978 to October 1983 are reported. The pathological and ultrastructural findings as well as the reduction rate on the roentgenograph before and after radiation are observed in 8 patients. These 26 patients comprised 14 squamous cell carcinomas, 7 adenocarcinomas and 5 small cell carcinomas. The total tumor dose was 3,500-4,008 rad. The treatment results of 26 patients show that surgical resection rate was 92.3% and there were no operative mortality or death due to complications. The improvement in 1 year survival rate was 3.9% and 3 year survival rate was 17.5% as compared with resection alone in the same period. In pathology and ultrastructural studies, the preoperative radiation injuries directly the cancer cells in the tumor mass and also its surrounding tissues. Preoperative radiation is of benefit in the radical resection and the prevention of spread and metastasis of lung cancer. PMID- 3021415 TI - [Prognostic significance of pathology in 3, 147 cases of colorectal cancer. National Cooperative Group on Pathology and Prognosis of Colorectal Cancer]. AB - This paper analyses the pathological features and prognosis in 3,147 cases of colorectal carcinoma collected from thirteen colleges, institutes and hospitals. The results reveal: The mean 5 year survival rate of 3,147 cases of colorectal carcinoma is 50.21%. The age of patient, tumor location, size, gross typing, histologic classification, growth pattern, the depth of infiltration, the metastasis into lymph nodes, the lymphocyte infiltration and fibrous proliferation surrounding the cancer are all prognostic. The depth of infiltration and metastasis into lymph nodes are the chief determinant of prognosis. The prognosis is much better in cases without penetrating the musculature or prognosis. The prognosis is much better in cases without penetrating the musculature or metastasis into lymph nodes than otherwise. It is better in those with metastases into 1-2 2 lymph nodes than with 3 or more. The histological types reflect the biologic nature of the cancer, exercising the most decisive influence upon the prognosis. The various pathologic changes are all closely associated with it. There is much difference in prognosis among well, moderately and poorly differentiated adenocarcinomas (70.3%, 49.6% and 26.6%) whereas almost no difference exists among the different histologic types with the same degree of differentiation. This indicates that it is the cancer differentiation which plays the important role in the prognosis. 4. The immune response in the regional lymph nodes, the lymphocyte infiltration and fibrous proliferation surrounding the cancer reflect the immune status of the organism against the cancer and favorably affect the prognosis. PMID- 3021416 TI - [Prognostic significance of immunoreactivity of regional lymph nodes in colorectal cancer--an analysis of 2,502 cases. National Cooperative Group on Pathology and Prognosis of Colorectal Cancer]. AB - This paper presents an analysis of the relation between immunoreactivity of the regional lymph node and survival rate. 2,502 cases of colorectal cancer collected from 13 areas in China were studied. The immunomorphology of the regional lymph node in this study was shown by sinus histiocytosis (SH), paracortical hyperplasia (PH) and germinal hyperplasia (GH). Each of these reactions was divided into 4 grades (-, +, ++, +++). The results show: A significant correlation exists between the extent of immunoreaction and prognosis. The 5 year survival rates of patients with SH (++, +++), PH (++, +++), GH (++, +++) are 59.0%, 58.1% and 62.1% respectively, much higher than those in the negative groups (P less than 0.001). The effect of these three immunoreactive features on prognosis is diverse. The 5 year survival rate of cases with simple GH (++, +++) is 62.9%, statistically higher than those of cases with simple PH (++, +++) or SH (++, +++). It seems that the B-cell may play a very important role in the host defence mechanism. It was found that SH (++, +++), PH (++, +++) and GH (++, +++) occurred more frequently in Dukes' A stage than those in Dukes' C stage. The same phenomenon is also identified in comparing the well-differentiated with the poorly-differentiated cases. PMID- 3021418 TI - [Pathology and prognosis of colorectal cancer in Chinese young patients--an analysis of 319 cases. National Cooperative Group on Pathology and Prognosis of Colorectal Cancer]. AB - The clinicopathologic factors that may influence the survival rate of colonic cancer were analyzed in 319 cases under 30 years old. These patients comprised 10.1% of 3147 cases collected from 13 institutions in the cooperative group. The 5 year survival rate was 40.1% which is apparently higher than that reported in the western literature. Although similar in the primary site of the tumor between the young and elderly patients, their histopathologic features and the number of metastasis in the lymph nodes are different. In the young patients, the main pathologic patterns are moderately or poorly differentiated adenocarcinoma but they are well or moderately differentiated in the elderly. The number of lymph nodes involved in the young patients is more than that in the elderly. These result in poorer prognosis. PMID- 3021417 TI - [Study of Dukes' staging of colorectal carcinoma by the prognosis. National Cooperative Group on Pathology and Prognosis of Colorectal Cancer]. AB - The authors make a recommendation of modified Dukes' staging basing on the prognosis of 3122 patients with colorectal cancer. The fact that there are no significant differences in the 5 year survival rates is observed in the lesions limited to the mucosa and those invading into the submucosa (P greater than 0.2), the two groups should be classed as stage A1. The fact that differences present in the 5 year survival rates of cancers invading into the superficial and deep layers of muscularis is statistically significant (P less than 0.001), the lesion should be subdivided into stages A2 and A3. The cancer penetrating through the muscular layer is classed as stage B. Because no significant differences in the 5 year survival rates are found between the metastasis in the local and mesenteric nodes (P greater than 0.1), stages C1 and C2 should be merged into stage C. Distant metastasis is classed as stage D. The Dukes' staging of colorectal cancer and other staging systems are briefly discussed in this paper. PMID- 3021420 TI - [Malignant transformation of mouse embryo cells by estrogenic hormones in vitro]. AB - Mouse embryo cells (MEC) taken from a LACA pregnant female mouse, were cultured in RPMI 1640 medium with 20% horse serum. In the experimental group (10 bottles), the first 3 generations of MEC were treated with estradiol valerate (E) and hydroxyprogesterone caproate (P) at doses of 5.2 micrograms/ml and 261 micrograms/ml medium in the original and the 2nd generations, and at double doses in the 1st generation. In the control group (10 bottles), the MEC were cultured without E and P. The MEC in both groups was subdivided into 3 bottles from 1 every seven days. In the experimental group (44 bottles), the morphological transformation emerged in 2 bottles of MEC (the 5th and 7th generations) after 79 day's treatment by E and P. Meanwhile, the nonmorphological transformed cells (the 3rd, 5th and 7th generations) had the ability to grow in ouabain-contained medium (Anti-Oua+). It implies that the mutation had already occurred before transformation. The transformed cells had the ability to form colonies in soft agar medium, could be agglutinated by concanavalin A and form fibrosarcoma in the subcutaneous region after inoculation. Basing on the above results, we suggest that E and P directly act as a carcinogen on the MEC and cause the latter to undergo malignant transformation. PMID- 3021422 TI - [Double recombinants of the vaccinia virus expressing hepatitis B virus surface antigen and herpes simplex virus thymidine kinase]. PMID- 3021419 TI - [Schistosomiasis and its prognostic significance in patients with colorectal cancer. National Cooperative Group on Pathology and Prognosis of Colorectal Cancer]. AB - This paper analyses 430 cases of colorectal cancer complicated with schistosomiasis. The 5 year survival rate was 45.6%, lower than that without schistosomiasis. The lowered 5 year survival rate may have been associated with higher percentage of negative immune response in the regional lymph nodes and higher percentage of infiltrating growth pattern of the tumor. The infection of schistosome should be considered as one of the important factors in prognosis. PMID- 3021421 TI - [MDG4 transposition in cases of invariable localization of other mobile elements in the mutator strain of Drosophila melanogaster characterized by genetic instability]. PMID- 3021423 TI - [Inactivation of active forms of oxygen generated by myeloperoxidase and serum proteins]. PMID- 3021424 TI - [Cloning of the human natriuretic factor gene and its use in evaluating the effectiveness of gene expression]. PMID- 3021425 TI - [Scintigraphy and sonography in the diagnosis of thyroid autonomy. A retrospective study of 526 patients]. AB - The place of scintigraphy and ultrasonography in diagnosis was analysed retrospectively in a series of 526 patients with confirmed thyroid autonomy. Male:female ratio was 1:4.6; peak age incidence was in the sixth decade. 5.6% of patients were younger than 30 years and in each case had a sonographic focus. A normal thyroid size was found in 6.5% on palpation and in 10% by ultrasound. Normal thyroid function was found in 287 patients (56%), of whom 48% had a positive TRH test, the remainder being hyperthyroid. Unilocular autonomy was present in 57%, multilocular in the remainder. With increasing degree of autonomy there was an increase in echo-density. In 351 patients (74%) the adenoma was echo poor, in 16% echo-normal and in 10% echo-dense. Focal findings on sonography were noted in 94% of patients. The incidence of thyroid carcinoma was 1.5% (2 of 136 patients with goitre resection), the carcinoma not being located in the autonomous tissue. It is concluded that a thyroid of normal size does not exclude autonomy. Patients younger than 30 years require scintigraphic examination only if there are focal signs. Echogenicity of a thyroid focus does not correlate with function. The incidence of cancer in the presence of autonomy is low. There was no case of thyroid carcinoma in the autonomous region. PMID- 3021426 TI - [Status of immunity against poliomyelitis in Germany]. AB - Neutralizing antibodies against poliovirus types 1, 2 and 3 were determined in serum of 4,039 persons of different ages and of 879 persons at the beginning of their training (male recruits at their initial examination; female teaching trainees and student nurses). The sera were supplied by eleven institutions. Compared with similar studies in 1969, 1972 and 1978, the immunity against individual poliovirus types as well as that against the three types in the different age groups has improved. About three quarters of those older than seven years have antibodies against types 1, 2 and 3. The present study, which for the first time used a more sensitive neutralization test, revealed that about 85% of probands had antibodies against all three types. The trivalent immunity of those aged from 0 to six years has improved by 11 percent points since 1978. There was no decrease in immunity among those older than 20 years. PMID- 3021427 TI - [Cytomegalic virus infection]. PMID- 3021428 TI - Schwann cell differentiation in vitro: extracellular matrix deposition and interaction. AB - Neonatal rat Schwann cells isolated in culture proliferate slowly and do not form a basement membrane although they express laminin continuously. We demonstrate here that isolated Schwann cells express other basement membrane components, including entactin and heparan sulfate proteoglycan. Treatment with ascorbate, and to a lesser extent with cyclic adenosine 3',5'-monophosphate, modulates the synthesis of extracellular matrix components by cultured Schwann cells. After this treatment, fibronectin and collagen type IV are detected on the Schwann cell surface, which form, with the other components, a membrane-bound extracellular matrix. Electron microscopy shows that these elements are organized in a filamentous matrix which resembles a basement membrane and may be a precursor form of a basement membrane. We also show the effect of complete basement membrane matrices on Schwann cell behavior in culture. These matrices support Schwann cell proliferation in both serum-containing and serum-free media. The extracellular matrix from endothelial cells mimics fibronectin and induces a flat phenotype whereas the reconstituted basement membrane gel from the EHS tumor mimics laminin and allows an elongated phenotype. Thus, the basement membrane matrices interact with Schwann cells in vitro and may play an important role in Schwann cell proliferation in vivo. PMID- 3021430 TI - [Sensorimotor and autonomous polyneuropathies in diabetic children and adolescents]. AB - In 28 juvenile and adolescent diabetics (14 males, 14 females) ranging in age between 9 and 20 years (mean: 14.9) we investigated several electrophysiological parameters, the vibration threshold and the cardiovascular reflexes in order to determine the incidence of sensomotor and autonomic polyneuropathy. 6 patients showed evidence of sensomotor polyneuropathy, 2 more had signs of autonomic polyneuropathy. Both groups with polyneuropathy did not differ significantly from the whole group of diabetics concerning the mean onset of illness and several metabolic parameters. The mean duration of illness and the average age was clearly higher in the patients with sensomotor polyneuropathy than in the whole group. Out of 5 patients with diabetic retinopathy 3 had signs of sensomotor polyneuropathy. No patient was clinically affected by polyneuropathy. In 1 patient the distal latency and the amplitude of the compound muscle action potential of the peroneal nerve showed pathologic values, in a second patient the maximal conduction velocity of the median nerve was slowed. 4 patients had abnormally high vibration threshold. The heart rate variation during rest was reduced in 2 patients. Significant differences between the diabetics and the controls could only be found concerning the maximum conduction velocities of the peroneal and the median nerve. Our results will be compared and discussed. PMID- 3021429 TI - [Cerebral single-photon emission tomography]. PMID- 3021431 TI - Hormonal regulation of collagen synthesis in a clonal rat osteosarcoma cell line. AB - Collagen synthesis in rat osteosarcoma cell line 17/2 (ROS 17/2) was assessed by measuring the incorporation of [3H]proline into collagenase-digestible protein and the formation of [3H]hydroxyproline. PTH and 1,25-dihydroxyvitamin D3 [1,25 (OH)2D3] inhibited collagen synthesis in ROS 17/2 cells in a time- and dose dependent manner. PTH reduced collagen synthesis after a 3-h incubation, whereas the effect of 1,25-(OH)2D3 was somewhat slower. Maximal and half-maximal inhibition of collagen synthesis occurred at approximately 1 and 0.1 nM of each hormone, respectively. At confluency, ROS 17/2 cells synthesized 96% type I and 4% type III collagen. PTH reduced the synthesis of type I, but not type III, collagen. PTH and 1,25-(OH)2D3 also reduced procollagen mRNA levels, as determined by a dot blot hybridization assay. Thus, ROS 17/2 cells are a convenient model system for studying the hormonal regulation of collagen metabolism and gene expression in a cloned cell line with the osteoblastic phenotype. PMID- 3021432 TI - Establishment of a parathyroid hormone-responsive phosphate transport system in vitro using cultured renal cells. AB - A new system was established using primary cultured mouse kidney epithelial cells to study the effect of PTH on renal tubular phosphate transport. The cells used in our study had alkaline phosphatase activity. They showed increased cAMP content in response to PTH, calcitonin, and vasopressin. Thus, these cells were thought to be a mixture of cells originating from the proximal and distal renal tubules. To explore the mechanism of phosphate handling in these cells, the accumulation of radioactive phosphate from the medium into the cells and the spaces between the cell layer and culture plate (submonolayer spaces) was measured. The accumulation of phosphate by the cells was a sodium-dependent, energy-dependent process, which was demonstrated by inhibition both in the absence of sodium and in the presence of ouabain or 2,4-dinitrophenol. Furthermore, phosphate accumulation was decreased significantly by PTH presumably acting through cAMP. PTH inhibited phosphate accumulation not by affecting the efflux process, but by affecting the uptake process through the apical membrane of the cultured cells without altering the compartmental mental distribution of the accumulated phosphate. These characteristics of phosphate accumulation resemble those of renal tubular phosphate transport. PMID- 3021433 TI - Effects of phorbol esters on metabolic variables in the thyroid. AB - Since 12-O-tetradecanoyl-phorbol-13-acetate (TPA) reproduced some of the effects of TSH on phosphorylation of polypeptides in the thyroid, its effects on several thyroid metabolic variables were investigated. Like TSH, TPA stimulated glucose oxidation, iodide organification, and 32P incorporation into phospholipids in thyroid slices. However, in contrast to TSH, it did not augment cAMP accumulation. An inactive phorbol ester, 4 alpha-phorbol, did not reproduce any of the effects of TPA. An initial incubation of thyroid slices with TPA decreased the stimulation of cAMP, glucose oxidation, and colloid droplet formation induced by TSH. However, an initial incubation with TPA did not modify the subsequent stimulation of glucose oxidation induced by (Bu)2 cAMP. TPA potentiated the ability of TSH to desensitize the adenylate cyclase system. Although both TPA and TSH increased 32P incorporation into phospholipids, the patterns were different when individual phospholipids were examined. These results indicate another regulatory mechanism for thyroid cell functions independent of cAMP. PMID- 3021434 TI - Functional substructure of the rat somatotroph immediate release pool: definition by responses to N6,2'-O-dibutyryl cyclic adenosine 3',5'-monophosphate, potassium ion, and/or prostaglandin E1. AB - Previous results from our laboratory suggest that stored rat GH (rGH) in the pituitary is divisible into at least two functional compartments. An immediate release pool (IRP) responds quickly and can be exhausted. A larger and less labile pool responds continuously to long term stimulation. We previously demonstrated that the sum of IRP rGH discharged by (Bu)2cAMP and potassium ion (K+) in separate experiments exceeds by one third the amount released by the two agents administered simultaneously. This overlap suggested an IRP substructure. We used prelabeled rat pituitary fragments in an in vitro perifusion immunoprecipitation system to define intracellular hormone storage and to track release of stored rGH and rat PRL (rPRL). We tested three secretagogues: K+ to induce release without altering pituitary cAMP levels, (Bu)2cAMP to introduce cAMP into cells without activating adenylate cyclase, and prostaglandin E1 (PGE1) to produce a temporary, localized cAMP increase through adenylate cyclase activation. Prelabeled tissue in basal perifusion was first exposed to one secretagogue for 90 min. Then, while the first secretagogue was continued, a second secretagogue was added for a second 90-min period. Demonstrable alterations in tissue responses to secretagogues included: K+ diminished PGE1 induced rGH release from the IRP by 69% but had a mixed effect on the response to (Bu)2cAMP; (Bu)2cAMP enhanced K+-induced rGH release from the IRP by 71% but reduced PGE1-induced rGH release by 72%; PGE1 diminished K+-induced rGH release by 13% and (Bu)2cAMP-induced rGH release by 23%; combined K+ and (Bu)2cAMP reduced the rGH response to PGE1 stimulation by 81% whereas prior PGE1 enhanced the response to subsequent combined K+ and (Bu)2cAMP by 16%. We conclude that the somatotroph IRP consists of a K+-sensitive portion which overlaps with, but is not identical to, a (Bu)2cAMP-sensitive portion. The PGE1-sensitive portion of the IRP appears to be roughly equivalent to the shared fraction of the K+- and (Bu)2cAMP-sensitive portions of the IRP. These agents define a similar rPRL compartmentalization. However, the K+-sensitive portions of the somatotroph and lactotroph IRP differ in that the former is larger and expandable, whereas the latter is smaller and appears to be of limited capacity. PMID- 3021435 TI - Norepinephrine and thyroid-stimulating hormone induce inositol phosphate accumulation in FRTL-5 cells. AB - 3H-Labeled inositol phosphate accumulation is observed when prelabeled FRTL-5 cells (a rat thyroid cell line) are exposed to norepinephrine (NE) or TSH. The presence of inositol trisphosphate among the products implicates a phosphodiesterase-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate. The response to NE is much greater than that to TSH. This may be explained by the ability of cAMP to inhibit inositol phosphate accumulation in these cells. The stimulation by NE is inhibited by alpha 1-adrenergic receptor antagonists and is markedly potentiated in medium of reduced Ca2+ concentration. After chronic withdrawal of TSH from the growth medium, the magnitude of the response to NE is considerably reduced; however, there is no substantial shift in the dose-response curve. This reflects the dependency of alpha 1-adrenergic receptor expression on TSH in the FRTL-5 cell. In contrast, the characteristics of inositol phosphate accumulation induced by acute treatment with TSH are similar in cells maintained in the presence or absence of a low concentration of this hormone, and correlate well with the iodide efflux and iodination of thyroglobulin observed in response to TSH. These results support the hypothesis that TSH may mediate certain of its physiological effects through cAMP-independent mechanisms, such as phospholipid/Ca2+ and C-kinase pathways. PMID- 3021436 TI - Differential effects of aging on adrenocorticotropin receptors, adenosine 3'5' monophosphate response, and corticosterone secretion in adrenocortical cells from Sprague-Dawley rats. AB - This study examined the effect of aging on adrenal cell function in Sprague Dawley rats, as judged by the binding of iodinated Phe2, Nle4-ACTH-(1-38) to the adrenal cells, and the ability of the cells to respond to ACTH stimulation by the production of cAMP and corticosterone in vitro. Collagenase-dispersed adrenal cells obtained from 2-, 12-, and 18-month-old-rats were used. The maximum corticosterone concentration after incubation with ACTH was significantly lower (P less than 0.001) in the 12-month-old (40 +/- 7 ng/micrograms DNA) and 18-month old rats (28 +/- 3 ng/micrograms DNA) compared to that in 2-month-old controls (102 +/- 9 ng/micrograms DNA). The ED50 values of ACTH-induced corticosterone production measured in the cell suspension were similar in 2- and 12-month-old groups (30 pg/ml), and the diminished production of corticosterone in the 12-and 18-month-old rats persisted after incubation with N6,O2-dibutyryl cAMP. Neither the number nor the binding affinity of adrenal receptors for [125I]I Tyr23,Phe2,Nle4-ACTH-(1-38) changed from 2-12 months of age. Furthermore, increases in concentrations of intra- and extracellular cAMP after ACTH stimulation were not significantly different in the 2-, 12-, and 18-month-old groups. Similarly, adrenal hydrolysis of cAMP by low and high Km phosphodiesterases did not change significantly with advancing age. These results provide strong evidence that there is a diminished capacity for corticosterone production with aging in the rat, and that the site of the defect lies distal to binding of trophic hormone to its receptor and to the production of its secondary messenger. Finally, an age-related decline in adrenal steroidogenic capacity could be viewed as a counterregulatory mechanism invoked in old rats to compensate, at least partially, for elevated plasma ACTH and corticosterone concentrations. PMID- 3021437 TI - 1,25-dihydroxyvitamin D3 receptors and hormonal responses in cloned human skeletal muscle cells. AB - Although skeletal muscle is a major calcium-regulated organ, there remains uncertainty about whether muscle is a target organ for the action of 1,25 dihydroxyvitamin D3 [1,25-(OH)2D3]. In this study we examine pure populations of clonally derived human muscle cells for the presence of 1,25-(OH)2D3 receptors and direct responses to the hormone. All of the clones tested exhibited specific [3H]1,25-(OH)2D3 binding, with values ranging from 5-70 fmol/mg protein. Scatchard analysis of binding data revealed a dissociation constant (approximately 100 pM) comparable to that of classical receptors in other target organs. The 1,25-(OH)2D3 receptors sedimented at 3.3S on hypertonic sucrose gradients. Specificity for [3H]1,25-(OH)2D3 was demonstrated on gradients by substantially better competition by 1,25-(OH)2D3 than 25-hydroxyvitamin D3 for the 3.3S receptor binding peak. The 1,25-(OH)2D3 receptor complex bound to DNA cellulose and eluted as a single peak at 0.2 M KCl. Myoblasts and myotubes did not show significant differences in either the amount or characteristics of the 1,25-(OH)2D3 receptor. In addition to the presence of receptors, cells were tested for functional responsiveness to 1,25-(OH)2D3. Both cell types exhibited a dose-dependent induction of 25-hydroxyvitamin D3-24-hydroxylase enzyme activity after treatment of monolayers with 1,25-(OH)2D3. Incorporation of both leucine and thymidine into growing myoblasts and fused myotubes was inhibited in a dose dependent fashion after treatment with 1,25-(OH)2D3. In summary, cloned human skeletal muscle cells contain a binding protein compatible with classical 1,25 (OH)2D3 receptors as well as functional responsiveness to 1,25-(OH)2D3 at physiological concentrations of hormone. PMID- 3021438 TI - Effect of calmodulin inhibitors on thyroid hormone secretion. AB - The effect of calmodulin inhibitors, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) and trifluoperazine, on TSH-induced thyroid hormone secretion from rat thyroid was examined in vivo and in vitro. The ip administration of 5 mg W-7 to the rat inhibited T4 and T3 secretion from rat thyroids at 2, 3, and 4 h after the ip injection of 2 IU TSH, and so did the ip injection of trifluoperazine at 3 and 4 h. However, the ip injection of N-(6-aminohexyl)-1 naphthalene sulfonamide as a control substance did not show any significant inhibition of T4 and T3 release. To identify the site of action of calmodulin, the effect of W-7 on (Bu)2cAMP-induced thyroid hormone secretion was tested in vitro. One hundred micromolar W-7 completely inhibited T4 release from the rat thyroid when it was enhanced by TSH or (Bu)2cAMP, suggesting that the inhibitory effect of W-7 is subsequent to cAMP formation. These results suggest that calmodulin may play a role in thyroid hormone secretion from the thyroid, acting beyond cAMP formation. PMID- 3021439 TI - Reset of feedback in the adrenocortical system: an apparent shift in sensitivity of adrenocorticotropin to inhibition by corticosterone between morning and evening. AB - There is evidence in man and rats that higher circulating levels of glucocorticoids are required to normalize basal unstimulated ACTH levels at the peak of the circadian rhythm than at the trough. To explore this phenomenon, we tested the inhibitory effect of constant levels of corticosterone on plasma ACTH in the morning (AM) and evening (PM) in young male rats implanted with fused pellets of corticosterone-cholesterol at the time of adrenalectomy (ADX+B) and studied 5 days later. There was a marked shift of the plasma corticosterone-ACTH inhibition curve to the right between AM and PM, demonstrating that the efficacy of corticosterone feedback inhibition of ACTH is less in the PM. Comparison of plasma ACTH and corticosterone levels during 24 h in sham-adrenalectomized rats (SHAM-ADX), adrenalectomized rats (ADX), and ADX+B revealed constantly low ACTH in SHAM-ADX, constantly high ACTH in ADX, and biphasic ACTH levels in ADX+B. Corticosterone levels were biphasic in SHAM-ADX and were constant in the other two groups. These results again showed a shift in corticosterone feedback efficacy as a function of the time of day and also suggested that basal ACTH secretion is maintained in the low normal range in intact rats because of the marked diurnal rhythm in corticosterone. The sensitivity of the pituitary ACTH response to exogenous CRF did not change between AM and PM in either intact or ADX+B showing that the shift in feedback sensitivity to corticosterone does not reside in the pituitary. The response of the entire adrenocortical system to histamine stress was shown to be equivalent in both the AM and PM, suggesting that feedback sensitivity of the entire system to corticosterone does not change as a function of the time of day. We conclude from these results that there is an apparent diurnal change in ACTH sensitivity to corticosterone feedback that can be defined operationally as reset. We believe that the site of feedback being tested shifts solely from the pituitary in the AM (at the nadir of the rhythm) to the brain and the pituitary in the PM (at the peak of the rhythm). The lack of the normally high transients of corticosterone that occur in SHAM-ADX rats results in increased brain drive of the pituitary in ADX+B. PMID- 3021440 TI - Ontogeny of the in vitro growth hormone stimulatory effect of thyrotropin releasing hormone in the rat. AB - The ontogenic changes in the somatotroph's responsiveness to TRH were examined in enzymatically dissociated rat pituitary cells in primary monolayer culture. Exposure to TRH (10(-8) M) caused a significant increase in GH release in cultured pituitary cells from rats ranging in age from -1 day (20 days of gestational age) to 90 days. The magnitude of the response, expressed as a percent increment above control rat GH (rGH) release, rose progressively until it reached a maximum of 209 +/- 5% (mean +/- SE) on postnatal day 12. Thereafter, the response declined to values ranging from 10-30% above control rGH release. In cultured adenohypophyseal cells of rats on postnatal day 12, the effect of TRH was dose related; the effective concentration range was 10(-10)-10(-7) M, with an EC50 of 2.5 +/- 0.6 X 10(-9) M. TRH (10(-8) M) potentiated the GH stimulatory effect of a submaximally effective concentration of human GH-releasing factor-40 (hGRF-40; 10(-9) M) in cultured pituitary cells of developing rats, aged -1 to 30 days, and that of (Bu)2cAMP (5 X 10(-4) M) in cultured pituitary cells of rats aged -1 to 45 days. The rGH response to the combined addition of TRH with either hGRF-40 or (Bu)2cAMP was up to 2 times greater (P less than 0.05) than the sum of the individual responses. When the interaction of TRH (10(-8) M) with multiple concentrations of hGRF-40 (10(-10), 10(-9), and 10(-8) M) was tested in cultured pituitary cells of 4- to 36-day-old rats at 4-day intervals, synergism was least at the lowest and greatest at the highest concentration of hGRF-40; synergistic interaction decreased progressively after 20 days of age to undetectable levels by 36 days. In cultured anterior pituitary cells of 12-day-old rats, maximally stimulatory TRH (10(-7) M) potentiated the GH stimulatory effects of both hGRF-40 and (Bu)2cAMP at concentrations at the EC50 value or greater, with synergism being most pronounced at maximally effective concentrations. Whereas the GH response to the combined addition of maximal hGRF-40 (10(-7) M) and (Bu)2cAMP (1.5 X 10(-3) M) was not greater than that to maximal hGRF alone, TRH potentiated the responses to both secretagogues whether added separately or combined.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3021441 TI - Identification of corticotropin-releasing factor (CRF) target cells and effects of dexamethasone on binding in anterior pituitary using a fluorescent analog of CRF. AB - A fluorescein-conjugated bioactive analog of corticotropin-releasing factor (CRF) was synthesized and used to label cells that have high affinity CRF-binding sites. Of cultured bovine anterior pituitary cells, 6.1 +/- 0.6% were visible by fluorescence microscopy after incubation with the analog. Fluorescence was eliminated by coincubation with a 200-fold excess of unlabeled CRF. Treatment with dexamethasone (10(-9)-10(-7) M) decreased visible fluorescence in a dose dependent manner. These results demonstrate the utility of a fluorescent CRF analog for identification of cells with specific CRF-binding sites and suggest that binding of CRF to anterior pituitary cells is altered by glucocorticoids. PMID- 3021442 TI - Oxytocin receptors in rat adenohypophysis: evidence from radioligand binding studies. AB - The nature of purported receptors for oxytocin (OT) in the rat adenohypophysis was investigated by means of radioligand binding. Tritium-labeled oxytocin (3H OT) was used as the radioligand. Particulate fractions prepared from the adenohypophysis of estrogen-treated female rats bound 3H-OT reversibly and with high affinity (Kd = 1.5 nM). Scatchard plots indicated the presence of a single, low-capacity binding-site (Bmax = 33 fmol/mg protein). However, ligand displacement experiments suggested that 3H-OT labels OT- as well as vasopressin preferring sites in adenohypophysial membrane-suspensions. When the number of pituitary vasopressin receptors was reduced by adrenalectomy, it was revealed that the ligand-specificity of the high-affinity OT binding site is similar to that of classical OT receptors. The results show the presence of separate receptors for OT and vasopressin in the rat adenohypophysis, and hence reinforce the notion that OT may function as a hypophysiotrophic hormone. PMID- 3021443 TI - A comparison of two types of anaesthesia on the endocrine and metabolic responses to anaesthesia and surgery. AB - The influence of halothane-nitrous oxide anaesthesia and normotensive epidural analgesia (level of sensory block T8-T10), respectively, on the plasma concentrations of adrenocorticotrophic hormone (ACTH), beta-lipotropin (beta LPH), cortisol, dehydroepiandrosterone (DHA) and aldosterone as well as on the metabolites glucose, lactate and free fatty acids (FFA) was studied in 20 healthy patients who underwent elective orthopaedic procedures on the lower limbs. ACTH and beta-LPH concentrations in plasma rose significantly (P less than 0.001) during halothane-nitrous oxide anaesthesia. DHA secretion closely followed the secretory profile of cortisol. Increased renin levels and increased ACTH release seemed to be responsible for increased aldosterone secretion, intra-operatively. The hormonally induced rise of glycogenolysis and lipolysis did not produce important intra-operative increases in metabolite concentrations in the blood. In normotensive epidural analgesia no significant increase in stress response could be demonstrated, even with low levels of sensory block. PMID- 3021444 TI - In vitro effects of halothane on lymphocytes. AB - Many reports indicate that anaesthesia affects several immunological functions that decrease the immune response, but the mechanisms involved are still unknown. We investigated the in vitro effect of halothane on human lymphocyte metabolism and plasma membrane function by evaluating the intracellular concentration of 3',5'-cyclic adenosine-monophosphate (cAMP), phosphodiesterase enzyme activity, NAD+/NADH intralymphocytic ratios and the degree of antibody and lectine-induced 'capping' of surface markers. Our results demonstrated an impaired lymphocyte capping of surface immunoglobulins and concanavalin A receptors 60 min after exposure to halothane at the concentration of 1% in oxygen. This phenomenon was reversible after 24 h and it was unrelated to the presence of adherent cells during the culture. Furthermore, halothane was able to induce a persistent increase in cAMP intracellular concentrations, which was reversible within 48 h. This effect was not dependent on adherent cells or on phosphodiesterase enzyme inhibition. Finally, no alteration in NAD+/NADH ratios after halothane exposure was observed. PMID- 3021446 TI - The B and Z forms of the d(m5C-G)3 and d(br5C-G)3 hexamers in solution. A 300-MHz and 500-MHz two-dimensional NMR study. AB - The non-exchangeable proton resonances of the hexadeoxynucleoside pentakisphosphates d(m5C-G)3 and d(br5C-G)3 in the B form as well as in the Z form were assigned by means of two-dimensional correlated spectroscopy and two dimensional nuclear Overhauser enhancement spectroscopy. The complete proton NMR spectrum of the B form of the methylated compound was assigned in a pure 2H2O solution as well as in a 2H2O/C2H3O2H mixed solvent, containing 5 mM MgCl2. In the latter solvent the B form occurs in slow equilibrium (on the NMR time scale) with the Z form, the resonances of which also were fully assigned. The proton resonances of the B and Z forms of the brominated fragment were assigned in a 2H2O/C2H3O2H solution containing 5 mM MgCl2. A new and general method is described for the sequential assignment of the non-exchangeable proton resonances of oligonucleotides in the Z form. PMID- 3021445 TI - The effect of sodium bicarbonate and sodium citrate ingestion on anaerobic power during intermittent exercise. AB - The effect of sodium bicarbonate and sodium citrate ingestion on cycling performance in three 30 s Wingate Anaerobic Tests separated by 6 min recovery periods has been studied using 6 male subjects. Subjects ingested either sodium bicarbonate (B), sodium bicarbonate plus sodium citrate (BC), sodium citrate (C) or sodium chloride (P) 2.5 h prior to exercise in a dose of 0.3 g kg-1 body weight. Pre-exercise blood pH was 7.44 +/- 0.06, 7.42 +/- 0.05, 7.41 +/- 0.05 and 7.38 +/- 0.04 in the C, BC, B and P conditions respectively. Mean and peak power output were significantly reduced by successive Wingate tests but not significantly affected by the treatments. Performance in the second and third tests was highest following C, BC and B ingestion. The total work done in the 3 tests was 103%, 102% and 101% of that achieved in the P condition after C, BC and B ingestion respectively. The increased alkali reserve recorded subsequent to bicarbonate and citrate treatment reduced mean post-exercise acidosis, although pH was significantly higher only in the C condition (p less than 0.05) compared to P after each exercise bout. No significant differences in plasma lactate concentration were recorded at any time. Citrate ingestion appears to be most effective in elevating blood pH and [HCO3-], and in enhancing performance in short-term intermittent exercise. This study demonstrates that alkali ingestion results in significant shifts in the acid-base balance of the blood and has a small, but non-significant, effect on anaerobic power and capacity as measured in a series of 3 Wingate Anaerobic Tests. PMID- 3021447 TI - Conformational analysis of the B and Z forms of the d(m5C-G)3 and d(br5C-G)3 hexamers in solution. A 300-MHz and 500-MHz NMR study. AB - The B and the Z forms of the DNA hexamers d(m5C-G)3 and d(br5C-G)3 were investigated by means of NMR spectroscopy. It is demonstrated that the low-salt form of d(m5C-G)3 is a B DNA structure. The form, which becomes increasingly predominant when increasing amounts of MgCl2 and/or methanol are added to the solution, has Z DNA characteristics. It is shown that the major geometrical features of the Z form of d(m5C-G)3 in the crystal structure are maintained in solution, with the dC residues S sugar conformation, gamma + and the base in the anti orientation and the dG residues N (except the 3'-terminal residue), gamma t and syn. Neither the Z form of the methylated nor that of the brominated compound resembles the Z' form, in which the deoxy guanosine sugar rings adopt a C1'-exo conformation. Substitution of m5C by br5C causes no perceptible conformational changes in either the B or in the Z forms. PMID- 3021448 TI - Pseudomonas aeruginosa cytochrome oxidase. Product inhibition by low thermodynamic driving force. AB - The steady-state kinetics of Pseudomonas aeruginosa cytochrome oxidase were studied. Reduced cytochrome c551 and azurin from the same bacteria were used as the electron-donating substrates, while dioxygen served as the electron acceptor. Oxidized cytochrome c551 and azurin exhibited product inhibition of the reaction. However, apo-azurin and azurin derivatives in which the copper was substituted by the redox-inert ions Ni2+, Co2+, Cd2+ and Zn2+, did not show any effect on the kinetics. These observations implied that complex formation between the substrates or the products and the enzyme is not a rate-limiting step and is not the cause for product inhibition. The integrated rate law for a reaction scheme in which we assumed that complex formation was not rate limiting was fitted to the complete reaction traces. The results suggested that it is the low thermodynamic driving force, expressed in the small differences in redox potential between the substrates and heme c of the enzyme, which cause the observed product inhibition. PMID- 3021449 TI - Simultaneous synthesis and hydrolysis of ATP regulated by the inhibitor protein in submitochondrial particles. AB - Coupled submitochondrial particles from bovine heart with ATP synthases devoid of control by the inhibitor protein of Pullman and Monroy [J. Biol. Chem. 238, 3762 3769 (1963)] can be prepared by incubation of Mg-ATP particles in 50 mM phosphate, 250 mM sucrose, and greater than 95% D2O (pD 7.8) at 38 degrees C. As monitored with oxonol, the respiring particles build up and maintain a delta psi about 5-10% lower than that of the starting preparation. With oligomycin delta psi of the two preparations is the same. In the presence of an ATP trap (hexokinase and glucose), the two types of particles carry out oxidative phosphorylation at comparable rates. Low concentrations of oligomycin induce a small enhancement of the rate of ATP synthesis in non-controlled particles. In the absence of an ATP trap, net accumulation of ATP, as driven by electron transport in particles without control by the inhibitor protein, is low. Apparently this is due to lack of control by the inhibitor protein of ATP hydrolysis that occurs during oxidative phosphorylation. PMID- 3021450 TI - Effects of guanine nucleotides on the properties of 5-hydroxytryptamine secretion from electropermeabilised human platelets. AB - Guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta,gamma imido]triphosphate enhance Ca2+-dependent 5-hydroxytryptamine secretion from electropermeabilised human platelets. GTP has little such effect except when the platelets are permeabilised, and incubated with this nucleotide, at 2 degrees C and pH 7.4. The lag phase observed in the time course of 5-hydroxytryptamine secretion induced by addition of guanosine 5'-[gamma-thio]triphosphate is markedly longer than that characterising secretion induced by Ca2+ alone, by thrombin +/- GTP or by guanosine 5'-[gamma-thio]triphosphate in the presence of thrombin. GTP causes competitive inhibition of the enhancement of the Ca2+ dependent secretory response induced by guanosine 5'-[gamma-thio]triphosphate when both nucleotides are added simultaneously. The extent of inhibition is decreased if guanosine 5'-[gamma-thio]triphosphate is added prior to GTP. GTP markedly enhances the effect of thrombin on Ca2+-dependent 5-hydroxytryptamine secretion by increasing the maximal extent of the response and decreasing the thrombin concentration required to give half-maximal response. A similar effect is observed on addition of guanosine 5'-[gamma-thio]triphosphate in the presence of thrombin at short incubation times. On more prolonged incubation the effects of thrombin and guanosine 5'-[gamma-thio]triphosphate are additive. Guanosine 5' [beta-thio]diphosphate completely inhibits the response induced by guanosine 5' [gamma-thio]triphosphate or guanosine 5'-[beta,gamma-imido]triphosphate but has little effect on the response induced by Ca2+ when added alone or in the presence of thrombin. Partial inhibition is observed for the response induced by thrombin + GTP. Cyclic-AMP effectively inhibits the response induced by thrombin + GTP but has little effect on that induced by guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta,gamma]imidotriphosphate. The results provide further support for the proposal [Haslam, R.J. & Davidson, M.M.L. (1984) FEBS Lett. 170, 90-95], that receptor--phospholipase-C coupling in platelets is mediated in part by a guanine-nucleotide-binding (Np) protein but that a coupling mechanism may also exist which is independent of such a protein. The properties of guanine nucleotide-dependent coupling resemble those previously described for receptor- adenylate-cyclase coupling. PMID- 3021451 TI - Transport and metabolism of 5'-nucleotidase in a rat hepatoma cell line. AB - The biosynthesis of the ectoenzyme 5'-nucleotidase in the rat hepatoma cell line H4S has been studied by pulse-labeling with [35S]methionine and subsequent immunoprecipitation of the cell lysate. 5'-Nucleotidase is a membrane glycoprotein with an apparent molecular mass on SDS-gels of 72 kDa. The enzyme is initially synthesized as a 68-kDa precursor which is converted to the mature 72 kDa form in 15-60 min (t1/2 = 25 min). The molecular mass of the unglycosylated enzyme is approximately 58 kDa. Culturing the cells in the presence of varying concentrations of tunicamycin, an inhibitor of N-glycosylation, revealed six species of 5'-nucleotidase after sodium dodecyl sulfate/polyacrylamide electrophoresis. This indicates the presence of five N-linked oligosaccharide chains accounting for the difference between the 58-kDa polypeptide backbone and the 68-kDa species. The 68-kDa precursor is susceptible to cleavage by endo-beta N-acetylglycosaminidase H; the 72-kDa mature protein is converted to several bands upon this treatment. This result indicates that part of 5'-nucleotidase keeps one or two high-mannose or hybrid chains in the mature form, even after prolonged pulse-chase labeling. The newly synthesized mature enzyme reaches the cell surface after 20-30 min. The half-life of 5'-nucleotidase is about 30 h in H4S cells. No immunoprecipitable 5'-nucleosidase is released into the culture medium. PMID- 3021452 TI - The Na+/H+ exchange system in glial cell lines. Properties and activation by an hyperosmotic shock. AB - The properties of the Na+/H+ exchange system in the glial cell lines C6 and NN were studied from 22Na+ uptake experiments and measurements of the internal pH (pHi) using intracellularly trapped biscarboxyethyl-carboxyfluorescein. In both cell types, the Na+/H+ exchanger is the major mechanism by which cells recover their pHi after an intracellular acidification. The exchanger is inhibited by amiloride and its derivatives. The pharmacological profile (ethylisopropylamiloride greater than amiloride greater than benzamil) is identical for the two cell lines. Both Na+ and Li+ can be exchanged for H+. Increasing the external pH increases the activity of the exchanger in the two cell lines. In NN cells the external pH dependence of the exchanger is independent of the pHi. In contrast, in C6 cells, changing the pHi value from 7.0 to 6.5 produces a pH shift of 0.6 pH units in the external pH dependence of the exchanger in the acidic range. Decreasing pHi activates the Na+/H+ exchanger in both cell lines. Increasing the osmolarity of the external medium with mannitol produces an activation of the exchanger in C6 cells, which leads to a cell alkalinization. Mannitol action on 22Na+ uptake and the pHi were not observed in the presence of amiloride derivatives. Mannitol produces a modification of the properties of interaction of the antiport with both internal and external H+. It shifts the pHi dependence of the system to the alkaline range and the external pH (pHo) dependence to the acidic range. It also suppresses the interdependence of pHi and pHo controls of the exchanger's activity. NN cells that possess an Na+/H+ exchange system with different properties do not respond to mannitol by an increased activity of the Na+/H+ exchanger. The action of mannitol on C6 cells is unlikely to be mediated by an activation of protein kinase C. PMID- 3021453 TI - Effects of serum vitamin-D-binding protein on actin in the presence of plasma gelsolin. AB - The interaction of serum vitamin-D-binding protein (DBP) and plasma gelsolin with actin was studied using fluorescent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole-actin or N-pyrenylcarboxyamidomethyl-actin. DBP and gelsolin formed very tight complexes with one or two monomeric actin subunits respectively. Kd values of about 10 nm have been found for both complexes. When DBP and gelsolin were added simultaneously to G-actin no ternary complex was observed and the DBP-actin complex was formed preferentially. In the presence of CaCl2 no transfer of actin occurred from the 1:2 gelsolin:actin molar complex to free DBP while in the presence of EGTA one actin monomer could be transferred. DBP did not affect the severing activity of gelsolin. The effects of a mixture of gelsolin and DBP on F actin suggest that only individual interactions of these two plasma proteins with actin occurred in solution. PMID- 3021454 TI - Pyrophosphate-caused inhibition of the aminoacylation of tRNA by the leucyl-tRNA synthetase from Neurospora crassa. AB - Inorganic pyrophosphate inhibits the aminoacylation of tRNALeu by the leucyl-tRNA synthetase from Neurospora crassa giving very low Kapp.i, PPi values of 3-20 microM. The inhibition by pyrophosphate, together with earlier kinetic data, suggest a reaction mechanism where leucine, ATP and tRNA are bound to the enzyme in almost random order, and pyrophosphate is dissociated before the rate-limiting step. A kinetic analysis of this mechanism shows that the measured Kapp.i values do not give the real dissociation constant but it is about 0.4 mM. Other dissociation constants are 90 microM for leucine, 2.2 mM for ATP and 1 microM for tRNALeu. At the approximate conditions of the living cell (2 mM ATP, 100 microM leucine and 150 microM PPi) the leucyl-tRNA synthetase is about 85% inhibited by pyrophosphate. PMID- 3021455 TI - Purification, characterization and origin of rat gastric peroxidase. AB - A membrane-bound peroxidase (EC 1.11.1.7) from rat stomach has been solubilized by 0.2% cetyltrimethylammonium bromide in the presence of 1.2 M NH4Cl. The enzyme was purified 3355-fold to apparent homogeneity as judged by acid polyacrylamide gel electrophoresis and appears to be a cationic protein. In sodium dodecyl sulfate gel electrophoresis, the enzyme shows single polypeptide band of Mr 45,000. In gel permeation, the Mr has been estimated as 47,000. Spectral properties indicate the presence of Soret band at 412 nm which shifts to 425 nm on complexation with CN- and to 430 nm on reduction with dithionite. The velocity constant, k1 for the reaction of the peroxidase with H2O2 is 1.38 X 10(7) M-1 s-1 and Km for H2O2 is 0.1 mM. The enzyme contains active sulphydryl groups and is inhibited by sulphydryl reagents of which p-hydroxymercuribenzoate is more reactive than mersalyl or N-ethylmaleimide. The enzyme is very resistant to thermal denaturation up to 65 degrees C and also to chaotropic reagents at least up to 2 M above which it is inactivated. The enzyme shows similarity with the intestinal eosinophil peroxidase as regards the molecular mass, spectral, kinetic and some of the catalytic properties. However, they differ significantly in terms of their interaction with fluoride ion, sulphydryl reagents, chaotropic reagent and also with the antiserum against the gastric peroxidase. Histochemically, the gastric peroxidase is shown to be localised in the gastric gland proper of the fundic stomach, rich in parietal and chief cells. PMID- 3021456 TI - Rate constants and equilibrium constants for binding of the gelsolin-actin complex to the barbed ends of actin filaments in the presence and absence of calcium. AB - The equilibrium constant for binding of the gelsolin-actin complex to the barbed ends of actin filaments was measured by the depolymerizing effect of the gelsolin actin complex on actin filaments. When the gelsolin-actin complex blocks monomer consumption at the lengthening barbed ends of treadmilling actin filaments, monomers continue to be produced at the shortening pointed ends until a new steady state is reached in which monomer production at the pointed ends is balanced by monomer consumption at the uncapped barbed ends. By using this effect the equilibrium constant for binding was determined to be about 1.5 X 10(10) M-1 in excess EGTA over total calcium (experimental conditions: 1 mM MgCl2, 100 mM KCl, pH 7.5, 37 degrees C). In the presence of Ca2+ the equilibrium constant was found to be in the range of or above 10(11) M-1. The rate constant of binding of the gelsolin-actin complex to the barbed ends was measured by inhibition of elongation of actin filaments. Nucleation of new filaments by the gelsolin-actin complex towards the pointed ends was prevented by keeping the monomer concentration below the critical monomer concentration of the pointed ends where the barbed ends of treadmilling actin filaments elongate and the pointed ends shorten. The gelsolin-actin complex was found to bind fourfold faster to the barbed ends in the presence of Ca2+ (10 X 10(6) M-1 s-1) than in excess EGTA (2.5 X 10(6) M-1 s-1). Dissociation of the gelsolin-actin complex from the barbed ends can be calculated to be rather slow. In excess EGTA the rate constant of dissociation is about 1.7 X 10(-4) s-1. In the presence of Ca2+ this dissociation rate constant is in the range of or below 10(-4) s-1. PMID- 3021457 TI - The effect of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide on the rat liver microsomal glucose-6-phosphatase system. AB - The effect of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC) on the reactions catalyzed by the glucose-6-phosphatase system of rat liver microsomes was studied. Modification of the intact microsomes by CMC leads to the inhibition of the glucose-6-phosphatase, pyrophosphate:glucose and carbamoyl-phosphate : glucose phosphotransferase activities of the system. The activities are restored by the disruption of the microsomal permeability barrier. The mannose-6 phosphate, pyrophosphate, and carbamoyl-phosphate phosphohydrolase activities of the intact as well as the disrupted microsomes were not affected by CMC. It follows from the results obtained that CMC inactivates the microsomal glucose-6 phosphate translocase, the inactivation is a result of the modification of a single sulfhydryl or amino group of the translocase; pyrophosphate, carbamoyl phosphate and inorganic phosphate are transported across the microsomal membrane without participation of the glucose-6-phosphate translocase; pyrophosphate and carbamoyl phosphate may act as the phosphate donors in the glucose phosphorylation reactions in vivo. PMID- 3021458 TI - Isolation of a mutant LLC-PK1 cell line defective in hormonal responsiveness. A pleiotropic lesion in receptor function. AB - A mutant LLC-PK1 cell line, M18, was isolated after a single treatment of the parent culture with N-methyl-N'-nitro-N-nitroso-guanidine. In contrast to LLC-PK1 cells, the mutant did not exhibit production of urokinase-type plasminogen activator (uPA) in response to the hormones calcitonin and vasopressin, but produced the expected levels of uPA upon stimulation by the receptor-independent adenylate cyclase activators forskolin and cholera toxin, as well as by the phosphodiesterase inhibitor isobutylmethylxanthine and the 8-bromo analogue of adenosine cyclic monophosphate, Br8cAMP. The patterns of activation of cAMP dependent protein kinase were identical to those of uPA induction: calcitonin and vasopressin were without effect, but the response to all other agents was normal. In similar fashion, mutant cell homogenates displayed normal activation of adenylate cyclase upon treatment with sodium fluoride, forskolin, or the non hydrolyzable GTP analogue guanosine 5'-[beta, gamma-imino]triphosphate, but were unresponsive to calcitonin or vasopressin. The ability of M18 cells to bind radioactively labelled calcitonin and vasopressin was measured. The mutant possessed less than 4% of the normal levels of the receptor binding activity for both hormones. Somatic cell hybrids formed between M18 and LLC-PK1 cells were found to retain normal hormone binding activity and responsiveness to hormones, indicating that the defect in M18 cells was recessive. M18 was concluded most probably to contain a single mutation impairing the function of two distinct polypeptide hormone receptors. PMID- 3021459 TI - New thiol inhibitor of Achromobacter iophagus collagenase. Specificity of the enzyme's S3' subsite. AB - New synthetic mercaptotripeptides (HS-CH2-CH2-CO-Pro-Yaa) which inhibit Achromobacter iophagus collagenase were produced in order to obtain more powerful bacterial collagenase inhibitors than currently available, and to investigate the specificity of the S3' subsite of the enzyme. Since similar binding constants were found for inhibitors carrying uncharged residues of various sizes in the P3' position (Yaa = Ala, Leu, Phe, Pro, Hyp) steric hindrance at the collagenase S3' appears relatively limited. The compound (HS-CH2-CH2-CO-Pro-Arg), which carries an arginine residue in the position P3' and had the highest inhibition constant of the series tested (Ki = 0.5 microM), proved to be the strongest inhibitor so far reported in the literature. The weakest in the present series was the compound (HS-CH2-CH2-CO-Pro-Asp) which carries an aspartic residue in position P3' and had a Ki = 70 microM. The present work revealed that the charged groups in the P3' position play a key role in the interaction of the inhibitors with the enzyme. PMID- 3021461 TI - Oat cell carcinoma of the esophagus: unusual radiological appearances. AB - Primary oat cell carcinoma of the esophagus is a very rare tumour. The radiographic appearance of the three cases described in this paper are unusual because they resemble benign lesions such as leiomyoma, fibrous polyp and candidiasis. It would be interesting to investigate whether such an unusual appearance is common for this neoplasm. PMID- 3021460 TI - Partial characterization of the human beta-myosin heavy-chain gene which is expressed in heart and skeletal muscle. AB - A human myosin heavy-chain gene, cloned in gamma Charon 4A phage (and as a clone designated lambda gMHC-1), was shown to code for a cardiac myosin heavy chain of the beta-type. The 5' end of the 14,200-base-pair genomic DNA clone is located in the head region of the myosin chain. The 3' end was shown to extent to the COOH terminus and includes the 3'-nontranslated sequence of the corresponding mRNA. The identification of lambda gMHC-1 as coding for a cardiac beta-myosin heavy chain was achieved by heteroduplex mapping using genomic cardiac myosin heavy chain DNA of rabbit as a probe and, furthermore, by DNA sequence analysis of three selected subregions of the clones DNA including the 3'-nontranslated sequence. It was demonstrated by the S1 nuclease protection technique that the beta-myosin heavy-chain gene is transcribed in human heart muscle. In addition, we have found by the same technique that it is also expressed in human skeletal muscle. PMID- 3021462 TI - Treatment of childhood idiopathic thrombocytopenic purpura with Rhesus antibodies (anti-D). AB - We have recently reported that a rise of platelet numbers in ITP can be induced by blockade of the RES with antibody-coated red blood cells. We now present a collaborative study in which 15 Rhesus-positive children with ITP (nine boys and six girls aged 1-15 years) were treated with low-dose anti-D. Ten patients had chronic ITP (duration 6-47 months), five had acute ITP. Doses of 28-50 micrograms anti-D/kg bodyweight per course were given intravenously. In all patients clinical signs of bleeding ceased and platelet counts were elevated. An excellent, good or fair response with platelet increments of greater than 100, 50 100, or 20-50 X 10(9)/l, respectively, was observed in 19, 7, and 12 out of 45 courses in chronic ITP, and in 4, 1, and 2 out of 8 courses in acute ITP. The platelet increase (greater than 40 X 10(9)/l) persisted for 10 to over 360 days in chronic ITP. There were no untoward side reactions. Haemoglobin values remained stable in all patients but laboratory signs of mild, compensated haemolysis ensued. The direct antiglobulin test became positive in all cases due to anti-D IgG. Previous therapy of patients with chronic ITP included high-dose immunoglobulins and prednisone. These regimens were both effective but remissions were short. We conclude that anti-D therapy is an effective and safe form of treatment in childhood ITP. PMID- 3021463 TI - C-reactive protein in the differentiation of adenoviral, Epstein-Barr viral and streptococcal tonsillitis in children. AB - Sixty-two children with febrile exudative tonsillitis were studied to explore whether quantitative measurements of serum C-reactive protein (CRP) are useful in differentiating viral from streptococcal tonsillitis. There were 23 cases of adenoviral tonsillitis, 21 of EB viral tonsillitis and 18 of streptococcal tonsillitis. Measurements of CRP, WBC counts and erythrocyte sedimentation rates (ESR) were not useful in distinguishing viral from streptococcal tonsillitis. Seventy-four percent of patients with adenoviral tonsillitis were under the age of 3 years and 71% of the patients with Epstein-Barr (EB) viral tonsillitis were under the age of 6 years whereas 72% of the patients with streptococcal tonsillitis were over the age of 6 years. Age was clearly the most important factor in distinguishing between viral and bacterial tonsillitis in children. PMID- 3021464 TI - IgG subclass of anti-HSV antibodies following neonatal HSV infections. AB - Ten survivors of neonatal HSV encephalitis had IgG1 and IgG3 antibodies to HSV 6 or more months after their infection. Six had IgG4 antibodies but only one had IgG2 antibodies. Titers of anti-HSV antibodies were similar following neonatal and later HSV infections. Antibody deficiency is thus not part of the selective deficiency of HSV-specific immunity following neonatal infection. PMID- 3021466 TI - Influence of surgical resection prior to chemotherapy on the long-term results in small cell lung cancer. A study of 150 operable patients. AB - The effect of surgical resection, prior to chemotherapy, on the long-term results obtained in treatment of operable patients with small cell lung cancer (SCC) was evaluated in a consecutive series of 874 patients treated with intensive combination chemotherapy with or without irradiation between 1973 and 1981. Evaluation of disease stage and operability was based on broncho-mediastinoscopy, chest X-ray, bone marrow examination, peritoneoscopy with liver biopsy and lung function tests. The same staging procedures were applied for restaging performed after 18 months of chemotherapy. The series comprised 440 patients with extensive disease and 437 with limited disease of whom 150 were regarded operable. Fifty four operable patients received no thoracotomy because the treatment policy of SCC did not include surgery at the hospitals from which they were referred. These patients served as a reference with which data on operated patients were compared. Resections were performed in 52 patients while 44 were regarded to be irresectable at the thoracotomy. Thirty-six resections were regarded histologically complete while 16 patients proved to have microscopic (9 pts) or macroscopic (7 pts) residual tumor. The number and per cent of 30 months disease free survivors in the various categories of the 874 patients were as follows: Completely resected, 12/36 patients (33%); Resected with residual tumor, 2/16 (12.5%); Operable but non-operated, 7/54 (13%); Irresectable, 3/44 (6.8%); Non operable patients with limited disease, 15/284 (5.3%) and with extensive disease, 11/440 (2.5%). The similarity between rates of long-term survival observed in resected patients with residual tumor and operable, non-operated patients suggests that resection, per se, has no significant influence on long-term results in SCC. The relatively high rate of long-term survival in completely resected patients may therefore primarily be a result of early stage disease at the initiation of chemotherapy. PMID- 3021465 TI - Growth kinetics and in vivo radiosensitivity in nude mice of two subpopulations derived from a single human small cell carcinoma of the lung. AB - The growth kinetics and the in vivo radiosensitivity of two human small cell carcinomas of the lung (SCCL) grown in nude mice were investigated. The tumors, CPH SCCL 54A and 54B, were derived by in vitro cloning of a single SCCL and were subsequently serially grown in nude mice. The growth curves were described according to a transformed Gompertz function, and the cell kinetics were examined by flow cytometric DNA analysis (FCM) and by the technique of labelled mitoses. The effect of single-dose irradiation was estimated by the specific growth delay calculated from the growth curves, and by the cell cycle distribution changes monitored by FCM. The results showed that the tumors differed in the in vivo radiosensitivity despite similarities in the growth kinetics. The results support the concept that difference in sensitivity among tumor subpopulations is an important reason for therapeutic failures. PMID- 3021468 TI - Cell lines as an investigational tool for the study of biology of small cell lung cancer. AB - Tumors, whether they be of clonal or polyclonal origin, are dynamic processes, constantly undergoing alterations, both in vivo and in vitro. However, in many if not most tumors, certain properties are relatively stable. There must be selective advantages for tumor populations to maintain these properties. A careful comparison of the properties of tumors and their cell lines, and correlating these data with the clinical history of the tumor is essential. From such studies we conclude that cell lines are suitable models to study the biology of SCLC and many important contributions would have been impossible without a large comprehensive panel of cell lines. These lines may be suitable for the selection of the best in vitro regimen to treat individual patients from whom the lines were derived, a hypothesis currently being tested in our Branch. Finally, in vitro studies already (and will continue to) suggest newer, more rational approaches to tumor control. PMID- 3021467 TI - Serum ferritin levels in small cell lung cancer. AB - Serum ferritin levels were measured before treatment, using an immunoradiometric method, in 39 patients with small cell lung cancer. In 11 patients serial estimations were also made. The median serum ferritin level for male patients was 660 micrograms/l (range 13-1329) and for females 306 (range 134-5300), the normal range being 32-501. This increase is significant (P less than 0.001). Serum ferritin levels were not related to metastatic, haematological or iron status. Serial ferritin levels did not reflect the clinical course of the disease. Patients with a pre-treatment serum ferritin of less than 600 micrograms/l had a significant prolongation of median survival compared to those with an initial serum ferritin of greater than 600 micrograms/l (P less than 0.02). Serum ferritin levels are not of value in staging small cell lung cancer nor in monitoring its progress. However, the initial serum ferritin is of prognostic significance. PMID- 3021469 TI - The relationship between oestradiol metabolism and adrenal steroids in the endometrium of postmenopausal women with and without endometrial cancer. AB - The aim of the present study was to investigate the hypothesis that adrenal androgens are contributory to the development of endometrial cancer either by the oestrogenic action of 5-androstene-3 beta, 17 beta-diol (androstenediol) or through the inhibition of oestradiol metabolism. Concentrations of androstenediol, dehydroepiandrosterone (DHA), DHA sulphate (DHAS), oestrone and oestradiol were measured in plasma and endometrium from postmenopausal women with and without endometrial cancer. There was no difference between normal postmenopausal women and endometrial cancer patients with respect to either tissue or plasma adrenal androgens although there was a tendency for plasma DHAS levels to be increased in cancer patients (normal women: 640 +/- 156 ng/ml; cancer patients: 808 +/- 159 ng/ml). There was a positive correlation between endometrial tissue concentrations of androstenediol and DHA in both normal women (P less than 0.05) and cancer patients (P less than 0.01) but for DHAS the relationship was only significant for non-malignant tissue (androstenediol: DHAS, P less than 0.05; DHA: DHAS, P less than 0.02). A significant positive correlation was found between all three plasma adrenal androgens for both groups. In cancer patients there was a trend towards an inverse correlation between endometrial tissue concentrations of DHAS and the enzyme 17 beta-hydroxysteroid dehydrogenase (17OHSD) although the relationship was not significant (r = 0.49). In endometrium, oestradiol was present in significantly higher concentrations than oestrone whereas in plasma the reverse was the case. There was also a tendency for plasma oestradiol levels to be elevated in the cancer subjects. These data do not support a substantial role for adrenal androgens in endometrial cancer but suggest that a relationship may exist between DHAS and 17OHSD and that an imbalance between sulphatase and sulphotransferase activities may be involved. PMID- 3021470 TI - T90/44 (9.3 antigen). A cell surface molecule with a function in human T cell activation. AB - T90/44 is a cell surface antigen which is present on human T cells of the helper and cytotoxic subsets and which binds the 9.3 monoclonal antibody (9.3 mAb). It is expressed in the form of 90-kDa disulfide-bonded dimers of a 44-kDa polypeptide and of free 44-kDa subunits. The function of T90/44 was investigated in a series of T cell function assays. 9.3 mAb was found to inhibit the activation of class II-restricted cloned T helper cells derived from leprosy patients and reactive with M. leprae antigens. The inhibition was first found at 1-10 ng/ml 9.3 mAb and regularly increased with the antibody concentration. The extent of the inhibition varied among different T cell clones in proportion to the respective different levels of T90/44 expression at their cell surface. The proliferative responses of peripheral blood lymphocytes (PBL) to purified protein derivative of M. tuberculosis (PPD) and tetanus toxoid were enhanced by the 9.3 mAb resulting in up to 20-30-fold increase of [3H]-thymidine incorporation. After phytohemagglutinin-induced activation of PBL, the number of T90/44 molecules per cell expressed at the cell surface rose from day 0 to day 7 by a factor of about 10. High concentrations of 9.3 mAb (5-10 micrograms/ml) at low cell densities and in the presence of monocytes in culture media supplemented by fetal calf serum were directly mitogenic for resting lymphocytes. The cytolytic effector functions of class I-restricted cytotoxic T lymphocytes (CTL) were not modulated by 9.3 mAb. The mixed lymphocyte reactions of three class I-restricted CTL to their specific target cells were found not to be significantly influenced by 9.3 mAb. In conclusion it is proposed that an antigen-independent T cell activation pathway can be entered at T90/44. PMID- 3021471 TI - Binding and effects of catecholestrogens on adenylate cyclase activity, and adrenoceptors, benzodiazepine and GABA receptors in guinea-pig hypothalamic membranes. AB - Catecholestrogen (CE) binding to guinea-pig hypothalamic membranes was assessed by using [3H]2-hydroxyestrone (2-OHE1) as a ligand. Binding was maximal at pH 7.4 and 37 degrees C, and after 10 min incubations. A high affinity binding site with dissociation constant (KD) = 0.20 +/- 0.02 nM and site concentration (Bmax) = 38 +/- 2 fmol/mg protein, and a low affinity binding site with KD = 235 +/- 10 nM and Bmax = 4.2 +/- 1.0 pmol/mg protein (n = 7) were detected. The order of affinity (Ki, microM) for displacement of 10 nM [3H]2-OHE1 from hypothalamic binding sites was 2-OHE1 (0.8), 2-hydroxyestradiol (2-OHE2) (1.0), epinephrine (5.9), norepinephrine (NE) (7.7), dopamine (270), estradiol, estrone, propranolol, phentolamine, domperidone (greater than 10 000). NE inhibition of 2 OHE1 binding was non-competitive, Only 0.5 mM 2-OHE2 depressed in a non competitive way the hypothalamic beta-adrenoceptor binding (measured by using [3H]dihydroalprenolol) without affecting alpha-adrenoceptor binding (measured by using [3H]dihydroergocryptine). Both 2-OHE2 and 2-OHE1 impaired NE-stimulated adenylate cyclase activity in hypothalamic membranes with EC50 of about 5 and 10 microM, respectively. CE decreased [3H]gamma-aminobutyric acid binding by hypothalamic membranes with Ki = 8 microM (2-OHE2) and 50 microM (2-OHE1). The binding of [3H]flunitrazepam to the same membrane preparation was not affected by CE. These results support the existence of significant CE binding and effects in guinea-pig hypothalamic membranes. PMID- 3021472 TI - The elevation of cyclic GMP as a response to acute hypervolemia is blocked by a monoclonal antibody directed against atrial natriuretic peptides. AB - Substantial volume expansion in conscious rats induces a strong diuresis and natriuresis that is caused by the increase in plasma levels of atrial natriuretic peptides (ANP) as measured by a radioimmunoassay. This renal response could be blocked by monoclonal antibodies directed against ANP. Parallel to the change in ANP, the cyclic GMP levels in plasma, urine and kidney tissue were increased after volume loading and reduced after additionally given antibodies. From this study it seems to be clear that the cyclic GMP rise is not a direct effect of volume expansion but is specifically mediated by the released ANP. PMID- 3021473 TI - Alpha 2-receptor control over release of noradrenaline in rat thalamus. AB - Rat brain thalamic tissue was examined for the presence of alpha 2-receptor control over noradrenaline (NA) utilization. Systemically administered clonidine (0.1 mg/kg i.p.) decreased NA turnover, yohimbine (10 mg/kg i.p.) increased NA turnover, and prazosin (0.5 mg/kg i.p.) had no effect. In vitro, yohimbine increased K+-stimulated overflow of endogenous NA in a dose-dependent fashion. It appears that locus coeruleus neurons which project to thalamus exhibit the same type of alpha 2-receptor control as those terminating in other forebrain regions. PMID- 3021474 TI - Effects of chronic treatment with benzodiazepine receptor ligands on cortical adrenoceptors. AB - We report the effects of kindling with the benzodiazepine partial inverse agonist, FG 7142 on adrenoceptor binding in mouse cerebral cortex. Twelve once daily injections of FG 7142 caused a statistically significant increase in the density of both alpha 2- and beta-adrenoceptor binding sites, seven days after the last injection. Despite the marked effects of FG 7142 on adrenoceptors, there were no changes in either alpha 2- or beta-adrenoceptor binding after prolonged treatment with the benzodiazepine, flurazepam. PMID- 3021476 TI - Irreversible selective blockade of kappa-opioid receptors in the guinea-pig ileum. AB - The irreversible non-selective opioid antagonist beta-chlornaltrexamine (beta CNA) was used in combination with selective mu receptor protection by [D-Ala2, MePhe4, Gly(ol)5]enkephalin (DAGO) to produce an effective kappa receptor antagonism in the guinea-pig field-stimulated ileum preparation. Using a standard pre-treatment of 10(-7) M beta-CNA incubated for 15 min, DAG (10(-6)-10(-4) M) protected the response to the mu agonist normorphine while reducing the antagonism of the kappa agonist U50488 to a lesser extent. The concentration of DAGO which produced the most selective protection was 10(-5) M. This method was used to find the kappa selectivity of a series of opioid agonists. Of the compounds tested, butorphanol, dynorphin-(1-17), U50488, tifluadom, bremazocine and Mr 2034 were the most kappa-selective. The correlation with kappa agonist selectivity in vitro and effects in vitro on urine output in the rat is demonstrated. PMID- 3021477 TI - D-2 receptor stimulation inhibits cyclic AMP formation brought about by D-1 receptor stimulation in rat neostriatum but not nucleus accumbens. PMID- 3021475 TI - The effects of prenatal chlordiazepoxide administration on avoidance behavior and benzodiazepine receptor density in adult albino rats. AB - Adult male and female offspring of rats injected daily with 10 mg/kg chlordiazepoxide during their pregnancy exhibited a facilitated acquisition of a two-way active avoidance response. Benzodiazepine receptor assays carried out on cortex and cerebral samples taken from experimental and control female rats showed a significant decrease in the density of benzodiazepine receptors, without changes in receptor affinity. PMID- 3021479 TI - Effects of beta-carboline-3-carboxylic acid ethyl ester on suppressed and non suppressed responding in the rhesus monkey. AB - The effects of the putative 'anxiogenic' compound beta-carboline-3-carboxylic acid ethyl ester (beta-CCE) were studied on non-suppressed and suppressed responding of rhesus monkeys. Responding was maintained under a fixed-interval 2 min schedule of food presentation (non-suppressed responding) and a fixed interval 2 min schedule of food presentation where each 10th response produced a 3-5 mA footshock (suppressed responding). Rates of suppressed responding were 60 90% lower than those for non-suppressed responding. beta-CCE reliably decreased non-suppressed responding over the range of 0.03-0.3 mg/kg (ED50 approximately 0.04 mg/kg) while consistent decreases in suppressed responding were not obtained in all animals until doses of 1-3 mg/kg (ED50 approximately 2 mg/kg) were given. Doses of beta-CCE greater than 0.01 mg/kg slightly increased blood pressure, moderately increased heart rate and greatly increased plasma ACTH levels for both types of response. In contrast, diazepam increased suppressed responding without affecting non-suppressed responding at low doses (0.3-1 mg/kg), while higher doses were required to decrease suppressed responding. Diazepam had little effect on blood pressure or mean heart rate. Ro 15-1788 (1 mg/kg) blocked the rate decreasing effects of beta-CCE on non-suppressed responding, suggesting the decrease is mediated via a benzodiazepine recognition site. These results show that under conditions where relatively low doses of diazepam have an anxiolytic effect (i.e. selectively increase rates of suppressed responding), relatively low doses of beta-CCE selectively decrease non-suppressed responding, questioning current notions of how to define an anxiogenic drug effect. PMID- 3021478 TI - Naloxonazine actions in vivo. AB - Naloxonazine is a relatively selective mu 1 affinity label in binding studies. Like naloxazone, naloxonazine has proven valuable in vivo in establishing a role for mu 1 sites in specific opiate actions. We now report a detailed characterization of naloxonazine's actions in mice and rats. Naloxonazine antagonized morphine analgesia for greater than 24 h without altering lethality. This prolonged action could not be easily explained by a slow rate of elimination since the terminal elimination half-life was estimated at less than 3 h. Furthermore, these actions were associated with a wash-resistant inhibition of binding lasting 24 h which was relatively selective for mu 1 sites. At a fixed morphine dose, increasing naloxonazine doses lowered peak tailflick latencies in a biphasic manner, suggesting that morphine analgesia consisted of both mu 1 (naloxonazine-sensitive) and a non-mu 1 (naloxonazine-insensitive) components. The ratio of mu 1 to non-mu 1 analgesia was greater at low morphine doses, implying that morphine activated mu 1 analgesic mechanisms with higher affinity. Our studies also emphasized that naloxonazine has reversible actions similar to those of naloxone; only its irreversible actions are relatively mu 1-selective. In addition, the selectivity of naloxonazine's irreversible actions is dose dependent. High naloxonazine doses will irreversibly antagonize receptors other than mu 1. PMID- 3021480 TI - Genetic marker patterns and endogenous mammary tumor virus genes in inbred mouse strains of Japan. AB - In order to establish the genetic relatedness of the inbred mouse strains kept in Nara, genetic marker patterns were determined in conjunction with a study on endogenous mammary tumor viral genes in these strains. Isoenzyme patterns combined with patterns of other genetic markers, show that the unrelatedness between various inbred strains of the dd stock is as high or even higher as between strains of known different origin and geneology. Based on endogenous viral gene patterns the dd stock derived mice can be subdivided into three group, DDD, DDN, DDO, KF and DD/Tbr. The DD/Tbr and its foster-nursed substrain (DD/Tbrf) have the lowest number of endogenous viral genes, i.e. two, while the other strains carry 4-6 such genes. The SLN and SHN strains, derived from a Swiss stock, have a similar pattern of viral genes different that of all other strains studied, also strains of Swiss origin from other sources, such as the NFS and the GR. PMID- 3021481 TI - [Serodiagnosis of microbiosis in mice with a quantitative assay of immunoglobulins by a microcomputer-introduced ELISA system. 1. Anti-Sendai virus antibodies in naturally infected mouse sera]. AB - An enzyme-linked immunosorbent assay system combined with microcomputer data analysis was established as a quantitative assay method of immunoglobulins. The assay system was applied to measure IgG and IgM levels of anti-microbe antibodies in animals, especially mouse and rat. And now the measurement of IgG and IgM levels (ng/ml) of anti-Sendai virus (HVJ) antibodies in naturally infected mice is available. The assay system could improve serodiagnosis in the specificity and sensitivity and in the rapid treatment of many serum samples. The operation of this system was performed by a microcomputer, FM 8 connected Titertek Multiskan MC. The limited sensitivity of this assay for IgG and IgM was 10 ng/ml and 30 ng/ml, respectively. Ninety-one of serum samples were positive for IgG and/or IgM (45 samples for IgG and IgM, 44 samples for IgG, 2 samples for IgM) to Sendai virus in the tested 279 mouse sera, and serum titers were ranged from 1: 10 to 1: 12,800 in the IgG, and from 1: 20 to 1: 160 in the IgM. In these titers, serum IgG and IgM amounts were estimated to be 0.1 to 154 micrograms/ml and 0.5 to 4.8 micrograms/ml, respectively. Relationships of serum titers and antibody amounts were almost consisted, being judged like that approximately 10 micrograms/ml is 1: 400, 30 micrograms/ml is 1: 1,600 in IgG, and 2.4 micrograms/ml is 1: 80, 4 micrograms/ml is 1: 160 in IgM. PMID- 3021482 TI - Transformation of human cultured fibroblasts with plasmids carrying dominant selection markers and immortalizing potential. AB - The disadvantages of using human cultured cells for biochemical and genetic studies are their limited lifespan in vitro and their lack of chemical selection markers. These problems are now overcome by transfecting human cultured fibroblasts with the pSV3-gpt and pSV3-neo plasmid DNA which carry genes coding for the immortalizing SV40 large T-antigen and dominant selection markers. Transformed human fibroblasts were obtained at a frequency of about 10(-5) with both selection systems. These transformed cells showed a twofold increase in growth rate and three to tenfold increase in cell number at confluence. The improved growth characteristics were associated with the expression of the SV40 T antigen detected with immunoprecipitation. These cell lines also changed from their usual spindle shapes to an epithelioid morphology characteristic of transformed cells. From 60 to 100% of the cells transfected with pSV3 plasmid DNA demonstrated numerical and structural abnormalities in their karyotypes. Cells transfected with DNA from a similar plasmid, pSV2-neo, which differed from the pSV3-neo plasmid only by missing the sequence encoding the complete early region of SV40, neither expressed T-antigen nor showed any change in morphology, improvement in growth characteristics or abnormalities in karyotype. However, they were still selectable with the aminoglycoside G-418. Therefore, by appropriate choice of vector plasmids, dominant selection markers and improved growth characteristics can be imparted separately or simultaneously to human fibroblasts. The morphological, biochemical and chromosomal changes resulting from such transformations must be recognized in using this approach for biochemical and genetic studies. PMID- 3021483 TI - UV-induced DNA excision repair in rat fibroblasts during immortalization and terminal differentiation in vitro. AB - UV-induced DNA excision repair was studied as DNA repair synthesis and dimer removal in rat fibroblast cultures, initiated from either dense or sparse inocula of primary cells grown from skin biopsies. During passaging in vitro an initial increase in DNA repair synthesis, determined both autoradiographically as unscheduled DNA synthesis (UDS) and by means of the BrdU photolysis assay as the number and average size of repair patches, was found to be associated with a morphological shift from small spindle-shaped to large pleiomorphic cells observed over the first twenty generations. In cell populations in growth crisis, a situation exclusively associated with thin-inoculum cultures in which the population predominantly consisted of large pleiomorphic cells, UDS was found to occur at a low level. After development of secondary cultures into immortal cell lines, both repair synthesis and morphology appeared to be the same as in the original primary spindle-shaped cells. At all passages the capacity to remove UV induced pyrimidine dimers was found to be low, as indicated by the persistence of Micrococcus luteus UV endonuclease-sensitive sites. These results are discussed in the context of terminal differentiation and immortalization of rat fibroblasts upon establishment in vitro. PMID- 3021484 TI - The major nucleoside triphosphatase of nuclear scaffold is distinct from actin. AB - The major nucleoside triphosphatase of rat liver nuclear scaffold, a 46 kD protein thought to participate in nucleocytoplasmic RNA translocation, is distinct from immunologically-identified scaffold actin on Western blots, has a substantially different amino acid composition, and its enzymatic activity is not affected by anti-actin antibodies. Thus, although the contractile protein actin is found in nuclear scaffold and appears to interact with RNA, it is not associated with the nucleoside triphosphatase activity in such preparations. PMID- 3021485 TI - Inflammation: its clinical relevance in airway diseases. Proceedings of an international symposium. Amsterdam, 16-17 March 1986. PMID- 3021486 TI - The clinical development of a new agent for the treatment of airway inflammation, nedocromil sodium (Tilade). AB - Nedocromil sodium is the disodium salt of a novel pyranoquinoline dicarboxylic acid which has physicochemical and pharmacokinetic properties which make it suitable for delivery to the respiratory tract by inhalation. Its biological properties suggest that it will display anti-allergic effects in man and that it will also have anti-inflammatory activity in chronic reversible obstructive airways disease. The clinical development of nedocromil sodium has been influenced by its anticipated effects in man and also by the knowledge that preclinical and early clinical tests are poor predictors of efficacy for agents of this type. Thus, although nedocromil sodium prevents immediate and delayed reactions to bronchial antigen challenge, and also prevents exercise-induced bronchoconstriction, the clinical development programme of this drug was designed to provide therapeutic double-blind trials of 1 month's treatment with the drug at the earliest opportunity, because this was thought to be the definitive test for activity. Studies on a larger scale and of longer duration followed the positive outcome of the first therapeutic trials. This series of studies, conducted to different designs, demonstrated the safety and efficacy of nedocromil sodium. They also permitted a view to be gained of the place of nedocromil sodium in the management of patients with airways inflammation. PMID- 3021487 TI - An open assessment study of the acceptability, tolerability and safety of nedocromil sodium in long-term clinical use in patients with perennial asthma. AB - Fifty-one patients (22 male, 29 female) aged 22-60 years (mean age 41.2 years), predominantly extrinsic asthmatics, took part in this study, a follow-up to a 28 day, double-blind trial (Lal et al., Thorax 1984: 39: 809). Forty-four patients completed 12 months of treatment after a 4-week baseline; seven withdrew. A number of symptoms (e.g. coughing, wheezing, sore throat) were reported but none appeared particularly frequently; most were attributable to the technique of inhalation. After 4 weeks of treatment with nedocromil sodium (Tilade 4 mg q.i.d.), patients were encouraged to reduce use of inhaled corticosteroids (48 patients) and sodium cromoglycate (16). Inhaled bronchodilators were to be used as required and other medication was to continue as before. At the end of the study, 28 patients had stopped using inhaled steroids and 10 had significantly reduced the dosage (p less than 0.001, week 5 to end). Ten patients had stopped using sodium cromoglycate. Inhaled bronchodilator use was significantly reduced (p less than 0.001, weeks 1-8; p less than 0.05, weeks 9-12) early in the study but returned to baseline as inhaled steroid usage was reduced. Diary card assessments of wheezing and shortness of breath showed significant improvement, particularly in the early part of the study. Diary card PEFRs showed no marked changes but significant decreases, though small, were found in FEV1, FVC and PEFR on clinic visits. Clinical assessment showed improvement in the first half of the study; the differences were less marked as inhaled steroid usage declined. Final opinions of treatment effectiveness significantly favoured nedocromil sodium. This study demonstrates the acceptability, tolerability and safety of nedocromil sodium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021488 TI - A group comparative study of the safety and efficacy of nedocromil sodium (Tilade) in reversible airways disease: a preliminary report. AB - Nedocromil sodium in a dose of 4 mg b.i.d. or q.i.d. by inhalation was used to treat asthmatic patients in a double-blind, placebo-controlled, randomized, parallel study. 80 patients (39 active drug and 41 placebo) received q.i.d. dosage and 87 patients (45 active drug and 42 placebo) received b.i.d. dosage. The patients selected were not on oral or inhaled steroids but required oral sustained-release theophylline with or without an inhaled beta-agonist aerosol. There was a significant reduction in daytime asthma symptoms (p = 0.04) and asthma severity (p less than 0.01) 6 weeks after the onset of therapy in the nedocromil sodium q.i.d. treated group as compared with placebo. In the nedocromil sodium b.i.d. treated group, the following variables showed statistically significant improvements compared with placebo: daytime and night time asthma (p = 0.002 in both instances), wheezing assessed by auscultation (p = 0.03) and overall asthma severity (p less than 0.01). All these variables showed improvements within 2 weeks of the onset of therapy and became statistically significant at 6 weeks. Side-effects were minor and occurred only in a small number of patients. It can be concluded from this study that nedocromil sodium is a safe and effective drug in the management of asthma. PMID- 3021490 TI - Preliminary report on the effect of nedocromil sodium on antigen-induced early and late reactions in allergic sheep. AB - Allergic sheep may be considered in many respects to be an animal model of human bronchial asthma. This study investigated the effects of pretreatment with nedocromil sodium and sodium cromoglycate on antigen-induced smooth muscle contraction in vitro, and antigen-induced early and late bronchial responses and inflammatory cell influx in allergic sheep in vivo. Nedocromil sodium was found to be more potent than sodium cromoglycate against the antigen-induced smooth muscle contraction in vitro. Both drugs appeared equally effective against acute antigen-induced bronchoconstriction in sheep; both gave protection against the late response, and both agents had a similar effect on the antigen-induced response in cells obtained by bronchoalveolar lavage. PMID- 3021489 TI - Clinical evaluation of nedocromil sodium in asthma. AB - The recognition that inflammatory events in the airways play a key role in the pathogenesis of asthma has led to a relentless search for pharmacological agents which modify these processes. Nedocromil sodium (Tilade) represents one such agent. Nedocromil sodium, when inhaled by patients with asthma (0.05-0.50% nebulized, 0.5-4.0 mg m.d.i.), has been shown to inhibit immediate bronchoconstriction provoked by challenges with allergen (10 studies), exercise (five studies), isocapnic hyperventilation, fog and sulphur dioxide (one study each) and adenosine (two studies). With these challenges, inhibition of bronchoconstriction exhibited dose-dependency up to 4 mg, with nedocromil sodium being up to four times more potent than sodium cromoglycate. When inhaled prior to allergen provocation, nedocromil sodium inhibited the late asthmatic reaction; when taken regularly during the pollen season, it attenuated the allergen-induced increase in non-specific bronchial responsiveness. The efficacy of nedocromil sodium (4 mg q.i.d.) in the treatment of clinical asthma was initially shown in four open studies and subsequently confirmed in nine double-blind, placebo controlled 4-12 week studies on patients with seasonal and perennial asthma. Further clinical trials (eight studies) identified some difficulty in replacing inhaled corticosteroids with nedocromil sodium, especially if the corticosteroids were reduced rapidly (four studies). However, two studies have shown that nedocromil sodium produced further improvement in asthma symptoms when used in addition to bronchodilators and inhaled corticosteroids. Treatment with nedocromil sodium (4 mg q.i.d.) for up to 52 weeks demonstrated a progressive reduction in bronchodilator usage throughout the whole treatment period. During clinical assessment, nedocromil sodium was well tolerated, side-effects being unpleasant taste, nausea and headache. In most cases the adverse reactions were mild and transient, although in approximately 3% of patients they resulted in withdrawal from clinical trials. Thus, nedocromil sodium is a novel drug of proven efficacy in the treatment of asthma. Its position in the therapeutic armamentarium is likely to be as an adjunct to bronchodilators and inhaled steroids, to produce improvement in symptoms beyond that achieved with the already established drugs. PMID- 3021492 TI - Effect of nedocromil sodium on TXB2, LTB4 and LTD4 synthesis by alveolar macrophages from asthmatic patients. AB - Arachidonic acid metabolites may play a role in the pathophysiology of bronchial asthma, influencing bronchial tone, airways inflammation and bronchial hyperreactivity. This study was designed to investigate the effect of nedocromil sodium, at a range of concentrations, on the metabolism of arachidonic acid released from alveolar macrophages (AMs) obtained by bronchoalveolar lavage in healthy and asthmatic subjects. The only effect of nedocromil sodium observed in this study was a slight decrease in LTD4 synthesis by AMs from asthmatic patients. This possible effect of nedocromil sodium on LTD4 metabolism deserves further investigation. PMID- 3021491 TI - Monkeys infected with Ascaris suum (a new in vivo model of airway disease): protective effect of nedocromil sodium and sodium cromoglycate against bronchial antigen challenge. AB - The experimental infection of Macaca arctoides monkeys with the nematode parasite Ascaris suum induces an IgE-mediated immediate hypersensitivity reaction similar to that seen in naturally sensitive animals. In the present study, the effects of inhalation of Ascaris suum antigen on lung function in infected monkeys were investigated, together with the effects of aerosolized nedocromil sodium and sodium cromoglycate (each 4 mg) given 5 min prior to antigen challenge. Ascaris suum-infected Macaca arctoides monkeys bronchoconstricted when they inhaled Ascaris antigen, maximal effects being seen 2-5 min after antigen inhalation. This bronchoconstriction was inhibited by nedocromil sodium pretreatment but not by sodium cromoglycate. This animal model therefore provides a new experimental model of airways disease. PMID- 3021493 TI - Nedocromil sodium inhibits IgE-dependent activation of rat macrophages and platelets as measured by schistosome killing, chemiluminescence and enzyme release. AB - The IgE-dependent activation of peritoneal macrophages and blood platelets can be measured by anti-parasite cytotoxicity, chemiluminescence and, in macrophages, lysosomal enzyme activity. Using these parameters, the present study demonstrates an inhibition by nedocromil sodium of the IgE-mediated stimulation of these non mast cell inflammatory populations in the rat. These observations suggest that nedocromil sodium may be of value in the treatment of diseases of the lung where inflammatory mediator release is implicated. PMID- 3021494 TI - The differential effects of nedocromil sodium and sodium cromoglycate on the secretory response of rabbit peritoneal neutrophils. AB - Activation of neutrophils, through the formation of inositol triphosphate (IP3) and diacylglycerol, results in lysosomal enzyme secretion, a release process which involves calcium and protein kinase C. Rabbit peritoneal neutrophils were used in this study to compare the effects of nedocromil sodium cromoglycate on enzyme release and IP3 accumulation induced by the activators fMLP (a chemotactic peptide which generates diacylglycerol and produces a rise in cytosolic Ca2+) and phorbol dibutyrate (PDBu), which activates only protein kinase C. Both drugs produced a small but significant (p less than 0.05) inhibition of fMLP-induced lysozyme secretion. With PDBu-induced enzyme secretion, however, nedocromil sodium caused a 25% decrease in the secretory response whilst sodium cromoglycate had no effect, suggesting that the two compounds have different mechanisms of action or that nedocromil sodium has an additional mode of action not shown by sodium cromoglycate. In addition, nedocromil sodium did not block fMLP-induced IP3 accumulation. These results indicate that nedocromil sodium inhibits the secretory response of neutrophils via an effect on protein kinase C rather than on calcium homeostasis. PMID- 3021496 TI - Adenosine-induced bronchoconstriction: premedication with chlorpheniramine and nedocromil sodium. AB - The mechanism for adenosine-induced bronchospasm was investigated in asthmatic patients. Nedocromil sodium 4 mg given by aerosol 10 min before challenge effectively inhibited adenosine-induced bronchospasm. The H1-antagonist chlorpheniramine 4 mg given orally 1 h before challenge did not inhibit adenosine induced bronchospasm, suggesting either that histaminergic pathways are not involved or that the dose was not sufficient to elicit an effect in the lung. Nedocromil sodium did not reduce the bronchial response to methacholine, supporting the view that adenosine-induced bronchospasm is not mediated through cholinergic pathways. The mechanism(s) by which nedocromil sodium inhibits the bronchial response requires further investigation. PMID- 3021495 TI - Nedocromil sodium preclinical safety evaluation studies: a preliminary report. AB - The preclinical safety evaluation programme for nedocromil sodium was conducted to examine the toxicity potential of the compound in animals and to support its chronic use in man by the inhalation route. Tests, from single-dose to 'lifetime' studies, were conducted in mouse, rat, rabbit and dog at high doses and by various routes of administration. This programme evaluated nedocromil sodium in terms of its general and reproductive toxicology, mutagenic and carcinogenic potential and other areas of interest. Nedocromil sodium proved to be safe, with a low order of toxicity and high safety margins. The results of the preclinical safety programme support the chronic use of nedocromil sodium in humans. PMID- 3021497 TI - Adenosine-induced bronchoconstriction: comparison between nedocromil sodium and sodium cromoglycate. AB - The purpose of this study was to compare the protective effect of nedocromil sodium with that of sodium cromoglycate against adenosine-induced bronchoconstriction in 15 asymptomatic asthmatic patients. Airways response was evaluated as FEV1 and adenosine was administered as an aerosol diluted in 0.9% saline to produce a concentration range of 0.03-4.00 mg/ml. When FEV1 was decreased by 20% of the post-saline value, inhalation was discontinued and the PD20 adenosine calculated. Patients were studied on four separate days. On the first day the adenosine challenge was performed and on subsequent days patients were pretreated with either placebo or test drug in a randomized double-blind manner. In this study both sodium cromoglycate and nedocromil sodium showed a significant protective effect against adenosine in comparison with placebo. Their activity was not related to a bronchodilator action. Nedocromil sodium showed a greater protective effect against adenosine challenge which may be indicative of a broader spectrum of activity against nonantigenic stimuli. PMID- 3021498 TI - A double-blind comparative trial of nedocromil sodium and placebo in the management of bronchial asthma in patients routinely using oral bronchodilators. AB - The efficacy and tolerability of nedocromil sodium, 4 mg four times daily, were investigated in a 4-week, randomized, placebo-controlled, double-blind trial in 54 asthmatic patients. Significant improvements were seen in the nedocromil sodium treated patients with respect to asthma severity (recorded by patients on diary cards); lung function (assessed by PEFR); daytime and night-time asthma (patients' diary cards), and morning and evening peak flow rates (measured by patients at home). Unusual symptoms were few and of a minor nature, differing little between active and placebo treatment. Nedocromil sodium, at the dosage used in this study, was well tolerated and provided a useful addition to asthma therapy. PMID- 3021500 TI - Withdrawal of inhaled corticosteroid under cover of nedocromil sodium. AB - Patients satisfactorily controlled on both an inhaled corticosteroid and a regularly inhaled bronchodilator were randomly allocated to 12 weeks of treatment with either nedocromil sodium (4 mg q.i.d.) or matching placebo. After 3 weeks of treatment, inhaled steroids were abruptly withdrawn. Severity of asthma deteriorated in both groups but more rapidly in the placebo group (p less than 0.05). Lung function tests showed little between-group difference. Nedocromil sodium had greater efficacy than placebo in the control of asthma in patients requiring an inhaled corticosteroid but abrupt corticosteroid withdrawal is not recommended. However, there are some clinical suggestions of the benefits of the combined use of nedocromil sodium and an inhaled corticosteroid. PMID- 3021501 TI - A 4-week Australian multicentre study of nedocromil sodium in asthmatic patients. AB - The use of nedocromil sodium 4 mg q.i.d. in asthmatics with mildly unstable asthma induced by a reduction of their inhaled beclomethasone dipropionate dose was investigated in a double-blind, parallel group, placebo-controlled study. Compared with placebo, nedocromil sodium improved night-time and daytime asthma score, night-time and daytime bronchodilator use, and evening PEFR at some stage during the 4-week treatment. It is concluded that nedocromil sodium exerts a beneficial effect in chronic asthma. PMID- 3021499 TI - A multicentre double-blind group comparative trial of two dose levels of nedocromil sodium and placebo in the management of perennial extrinsic asthma. AB - The efficacy of nedocromil sodium 4 mg or 2 mg q.i.d. in the management of perennial extrinsic asthma was evaluated in a double-blind, placebo-controlled, multicentre trial. Nedocromil sodium, especially at the dose of 4 mg q.i.d., was consistently rated higher than placebo on clinical assessment and diary card measurement. Compared with placebo, there were significant improvements for nedocromil sodium (4 X 4 mg) in asthma severity scores (p less than 0.05 after 1 and 2 weeks of treatment) and clinician's opinion of treatment (p less than 0.001). The results suggest that nedocromil sodium is effective in the maintenance treatment of patients with perennial extrinsic asthma and further studies with longer-term treatment periods are suggested. PMID- 3021502 TI - The addition of nedocromil sodium to maintenance therapy in the management of patients with bronchial asthma. AB - The addition of nedocromil sodium to maintenance asthma therapy was investigated in a 12-week double-blind group comparative trial. Maintenance therapy consisted of inhaled corticosteroid and inhaled bronchodilator; oral bronchodilator usage at constant dose was allowed but sodium cromoglycate was forbidden. Compared with placebo, the nedocromil sodium group showed greater improvement in severity of asthma and changes in lung function assessed at clinic visits, and in night-time asthma, morning tightness and PEFR recorded on diary cards. The nedocromil sodium group also showed a reduction in the diurnal variation of PEFR. PMID- 3021503 TI - A pharmacological study of group I muscle afferent terminals and synaptic excitation in the intermediate nucleus and Clarke's column of the cat spinal cord. AB - When administered microelectrophoretically GABA and piperidine-4-sulphonic acid depolarized the central terminations of muscle group Ia and Ib afferent fibres in the lumbar intermediate nucleus and Clarke's column of cats anaesthetised with pentobarbitone sodium. Both this depolarization, and primary afferent depolarization, generated by impulses in other primary afferent fibres which produce prolonged bicuculline-sensitive inhibition of the firing of group I afferent fibre-excited interneurones in the intermediate nucleus and cells in Clarke's column, are reduced by microelectrophoretic bicuculline methochloride. Systemically administered (+/-)-baclofen hydrochloride (maximum dose 8 mg kg-1) depressed the monosynaptic excitation of Clarke's column neurones by impulses in muscle and cutaneous afferent fibres. Microelectrophoretically administered (-) baclofen reduced the bicuculline-sensitive primary afferent depolarization of group I terminations without, however, reducing the depolarizing action of GABA or piperidine-4-sulphonic acid. The depression by (-)-baclofen of the group I monosynaptic excitation of intermediate nucleus neurones is not reduced by concentrations of bicuculline methochloride adequate to suppress prolonged inhibition of these neurones. PMID- 3021504 TI - On the probable absence of GABA receptors on the terminations of motor axon collaterals in the cat spinal cord. AB - When administered microelectrophoretically, GABA and the GABA-mimetic piperidine 4-sulphonic acid (P4S) appear to have no direct hyperpolarizing or depolarizing effect on the terminations of motor axon collaterals excited electrically in the ventral horn of the lumbar spinal cord of the cat. This lack of effect on axon terminals of motoneurones, which contrasts with the bicuculline-sensitive depolarization by P4S of the spinal terminals of primary afferent fibres, is consistent with previous reports of the probable absence of pharmacologically detectable GABA receptors on the spinal terminals of other central excitatory neurones, namely those of the red and lateral vestibular nuclei. PMID- 3021505 TI - Development of postural control in children: short-, medium-, and long latency EMG responses of leg muscles after perturbation of stance. AB - Short (SL), medium (ML), and long (LL) latency EMG responses of leg muscles were recorded after perturbation of stance by means of a sudden toe-up tilt of a movable platform. 56 healthy children varying in age between 14 months and 15 years were investigated. All three responses were present when children were able to stand on the recording platform. The SL-response in the triceps surae muscle, which corresponds to the mono- and oligo-synaptic spinal stretch reflex, showed a decreasing latency up to the age of 5 years. This reflects the increasing peripheral nerve conduction velocity. The ML-response in the triceps surae muscle, which as the SL-response has no stabilizing effect in this experiment, showed somewhat delayed maturational changes. The LL-response in the relaxed anterior tibial muscle helps to restore upright posture even in the youngest children. Its maturational changes in terms of latency by far exceed the range that can be explained by the increase of peripheral and spinal conduction velocities. Its mechanisms of maturation, besides the biophysical optimalization of a polysynaptic network, may include learning in terms of selecting the shortest pathways by way of synaptic potentiation within structures involved in the supposedly transcortical pathway of the LL-response. Qualitative observations made during the trials showed that the pattern of postural adaptation changed with age, suggesting the development of additional intersegmental mechanisms. PMID- 3021506 TI - Evidence of rhythmic inhibitory synaptic influences in hindlimb motoneurons during fictive locomotion in the thalamic cat. AB - Intracellular recordings of various motoneurons of proximal hindlimb muscles were performed on thalamic paralyzed cats, during fictive locomotion that was either spontaneous or evoked by stimulation of the subthalamic region. In motoneurons innervating sartorius (medialis and lateralis), vasti (intermedius, medialis and lateralis) and anterior biceps-semimembranous, one depolarization occurred in each locomotor cycle, alternating with a phase of repolarization that was synchronous with the activation of the antagonistic muscle nerve. This latter phase could be decreased or reversed by intracellular injection of chloride ions or current, revealing the presence of inhibitory inputs onto motoneurons. The pattern of membrane potential variations was more complex in motoneurons of rectus femoris and posterior biceps-semitendinosus muscles, but phases of chloride dependent inhibition were nevertheless identified, mainly during the sartorius nerve activation in the case of rectus femoris, and during the vasti and anterior biceps-semimembranosus nerve activations in the case of posterior biceps-semitendinosus. These inhibitory influences were shown to be controlled by the level of activity in exteroceptive afferents. The characteristics of the excitatory and inhibitory inputs to the hindlimb motoneurons identified here are discussed in relation with the organization of the central pattern generator for locomotion. PMID- 3021507 TI - Projection of tooth pulp afferents to the thalamus of the cat. II. Distribution and characteristics of single units. AB - Single unit activity was recorded extra- and intracellularly in the thalamus of the cat following electrical stimulation of tooth pulp afferents. Cells activated by noxious stimuli were found in the basal ventromedial nucleus (VMB), the marginal zones of the arcuate and external nuclei of the ventrobasal complex (VBA and VBX) and the intralaminar complex. Cells found in any of these locations showed a variety of properties; they were activated at different latencies and had different patterns of input. However, cells with responses of short latency and low convergence were with few exceptions found in or close to VMB. Stimulation of tooth pulp evoked both EPSP's and IPSP's in these cells. A subpopulation of cells were found that responded to stimulation of either right or left tooth pulp afferents. 22 units, mainly located in VMB, were found that responded exclusively to stimulation of tooth pulp afferents. In the laterodorsal part of VMB cells influenced by tooth pulp stimulation were found to be antidromically activated by stimulation of the cortical tooth pulp projection area. No such cells were seen at other locations. The results of this study is in agreement with the conclusions in the companion paper (Rydenhag et al. 1986a) that VMB and the border zone between VMB and VBA are the most likely relay nuclei between tooth pulp nociceptive afferents and the cortex. The intralaminar complex is suggested to be important in the cortical arousal reactions and direction of attention following a painful stimulus. PMID- 3021508 TI - Actions of pentylenetetrazol (PTZ) on CA3 neurons in hippocampal slices of guinea pigs. AB - The actions of the convulsant drug pentylenetetrazol (PTZ) were studied on CA3 neurons of hippocampal slices (300-400 microns thick). In 9 out of 109 neurons, epileptic reactions were elicited during a single application of PTZ. After repeated applications of PTZ, 53 neurons showed periodic paroxysmal activity. They developed according to the following sequence: (i) Paroxysmal hyperpolarizations, (ii) burst activity, and (iii) typical paroxysmal depolarization shifts (PDS). The rate of occurrence was about 8/min. Paroxysmal hyper- and depolarizations appeared synchronously in pairs of neurons. The developmental sequence occurred in reverse during washing. After the onset of paroxysmal activity, bursts and PDS persisted if PTZ concentration in tissue ranged between about 2 and 10 mmol/l. When this range was exceeded in either direction, epileptic activity was abolished at a calcium concentration of 2.75 mmol/l. Decreasing and increasing the calcium concentration shifted the epileptogenic concentration range to lower and higher levels, respectively. It is concluded that repetition of PTZ application alters membrane properties of neurons and thus leads to paroxysmal events triggered by synaptic processes. PMID- 3021510 TI - Cholesterol feeding to rats does not modulate the expression of binding sites for HDL on liver membranes. AB - The binding of HDL, Apo-E-free, was studied in rats fed a cholesterol rich diet for 2, 4 and 7 days. Plasma cholesterol increased up to 16-fold (from 55 to 900 mg/dl); liver cholesterol was also raised, from 0.5 to 16 mg/g of tissue. The HDL binding to membrane preparations was not affected while the binding of beta VLDL was reduced to about 50% of the controls. These data show, therefore, that liver binding sites for HDL are refractory to regulation by dietary cholesterol. PMID- 3021509 TI - Developmental genetics. AB - Of particular concern to the human geneticist are the effects of genetic abnormalities on development. To gain an understanding of these effects it is necessary to engage in a reciprocal process of using knowledge of normal developmental events to elucidate the mechanisms operative in abnormal situations and then of using what is learned about these abnormal situations to expand our understanding of the normal. True developmental genes have not been described in man, although it is likely that they exist, but many developmental abnormalities are ascribable to mutations in genes coding for enzymes and structural proteins. Some of these even produce multiple malformation syndromes with dysmorphic features. These situations provide a precedent for asserting that not only monogenic developmental abnormalities, but also abnormalities resulting from chromosome imbalance must ultimately be explicable in molecular terms. However, the major problem confronted by the investigator interested in the pathogenesis of any of the chromosome anomaly syndromes is to understand how the presence of an extra set of normal genes or the loss of one of two sets of genes has an adverse effect on development. Several molecular mechanisms for which limited precedents exist may be considered on theoretical grounds. Because of the difficulties in studying developmental disorders in man, a variety of experimental systems have been employed. Particularly useful has been the mouse, which provides models for both monogenic and aneuploidy produced abnormalities of development. An example of the former is the mutation oligosyndactylism which in the heterozygous state causes oligosyndactyly and in the homozygous state causes early embryonic mitotic arrest. All whole arm trisomies and monosomies of the mouse can be produced experimentally, and of special interest is mouse trisomy 16 which has been developed as an animal model of human trisomy 21 (Down syndrome). In the long run, the most direct approach to elucidating the genetic problems of human development will involve not only the study of man himself but also of the appropriate experimental models in other species. PMID- 3021511 TI - Genetics of B-cell neoplasia. AB - Somatic cell hybridization experiments between tumorigenic and nontumorigenic cells indicate that the differentiated state of the cells carrying the activated oncogene is important in the regulation of their expression. PMID- 3021513 TI - Epidemiology of human rotaviruses in a maternity unit as studied by electrophoresis of genomic RNA. AB - An epidemiological survey of Rotavirus infections in a maternity unit was carried out between May 1983 and August 1984. Rotaviruses have been detected throughout the survey in 184 of 528 fecal samples obtained from 3 and 6 day-old newborns. Rotavirus excretion could be detected neither from the mothers nor from the medical attendants. Rotavirus RNA was analyzed by polyacrylamide gel electrophoresis. All the strains isolated exhibited the same electropherotype. This result contrasts with the genomic variability amongst Rotavirus strains usually observed in pediatric wards, and is in agreement with some other studies conducted in maternity wards; in these locations Rotavirus infection seems to have a special epidemic status as compared with other communities. PMID- 3021514 TI - Plasmid-specified aminoglycoside-modifying enzymes in clinical isolates of Klebsiella pneumoniae. AB - In 106 clinical isolates of multiresistant Klebsiella pneumoniae strains, we found that aminoglycoside-resistance was due mostly to two adenylating enzymes: AAD (2") (56.6%), that modifies gentamicin, kanamycin, tobramycin and sissomicin, and AAD (3") 9 (56.6% + 19.8%) that modifies streptomycin and spectinomycin. The identification of these enzymes was possible by MICs determination against a set of aminoglycosides antibiotics. AAD (2") + AAD (3")9 were coded by conjugative plasmid of about 120 Md. PMID- 3021512 TI - Molecular epidemiology of human rotavirus infections. AB - Recognition of rotaviruses as a major aetiological agent of acute gastroenteritis in infants and young children has prompted the investigation of their epidemiology by molecular techniques. Genome analysis by electrophoretic separation of the RNA segments has been widely used to distinguish virus isolates and to monitor patterns of virus transmission. Examination of virus isolates from different epidemics has clearly demonstrated the existance of extensive genomic variation in viruses circulating in large communities; with the co-circulation of a number of viruses of differing electrophoretype. Preliminary studies using the more advanced techniques of oligonucleotide mapping and hybridization analysis have suggested that variation among the viruses may occur by processes involving both "drift" and "shift". Because of their ease and specificity the new hybridization analysis techniques should greatly facilitate both the rapid diagnosis of rotavirus infections, and the solution of many epidemiological and evolutionary questions. Continued and expanded use of molecular techniques for the study of the epidemiology of rotavirus infections will be required to manage future outbreaks and to effect long term control measures. PMID- 3021515 TI - Antibody response against early antigens in Herpesviridae infections. AB - For the serological diagnosis of Herpesvirus infection, increasing use is made of the determination of the antibodies against virus-specific early antigens. The presence of serum antibody to early antigens is a widely accepted marker of acute Epstein-Barr virus infection or Varicella-Zoster virus infection. Controversial data are present in the literature about the significance of Cytomegalovirus early antigens antibody response. This is discussed in view of the possibility that controversial results could be due to different methods used to produce early antigens by different authors. PMID- 3021516 TI - Role of antecedent mumps and reovirus infections on the development of type 1 (insulin-dependent) diabetes. AB - Type 1 diabetes mellitus is thought to derive from organ-specific autoimmune reactions, probably triggered by environmental factors. In view of the possible involvement of mumps virus and reoviruses in the pathogenesis of autoimmune endocrine disease, serum antibody levels to these viruses were measured in newly diagnosed diabetic patients aged 5 to 25 years and in matched control subjects. Diabetic patients showed a significantly lower prevalence and reduced titers of antibodies to mumps and reoviruses. By contrast, the antibody response to measles virus (a non-diabetogenic agent) was remarkably similar in the two groups. It is suggested that individuals with an impaired humoral response to some viral agents are at increased risk of developing diabetes when exposed to pancreotropic viruses. PMID- 3021517 TI - Human papillomavirus (HPV) type as an important determinant of the natural history of HPV infections in uterine cervix. AB - The present report summarizes our current observations on the natural history of cervical HPV (Human papillomavirus) infections, based on data from 418 women prospectively followed-up in our clinic for a mean of 20 +/- 15 (M +/- SD) months. On each attendance at the clinic (at 6-month intervals), the patients are subjected to colposcopy accompanied by PAP smears and/or punch biopsy, both being analysed for the cytopathic changes of HPV, and for concomitant CIN (cervical intraepithelial neoplasia). In the biopsies, the expression of HPV structural proteins was assessed using an indirect immunoperoxidase (IP-PAP) technique. HPV typing was accomplished by spot hybridization with the DNA probes for HPV 6, 11, 16 and 18. During the follow-up, 24% of the HPV lesions regressed, 55% remained persistent, and 21% progressed, 10.6% having been coned due to progression into CIS. The clinical progression was significantly associated with the grade of HPV associated CIN. On DNA hybridization, HPV 6 was found in 8%, HPV 11 in 36%, HPV 16 in 11% and HPV 18 in 8% of the 103 lesions typed for HPV DNA so far. HPV-CIN lesions were more frequently than HPV-NCIN associated with HPV 16 and HPV 18, as was the expression of HPV structural proteins. The progression rate was highest (45.5%) in HPV 16 lesions, followed by that (27.3%) in HPV 18 lesions, as contrasted to 0% and 13.3% for HPV 6 and 11, respectively. The natural history of cervical HPV lesions seems to be identical with that of classical CIN lesions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021518 TI - A comparison of rotavirus strains of bovine, simian and porcine origin. AB - Three rotavirus strains of bovine, simian and porcine origin, respectively, were compared. The 3 viruses induced a classic rotaviral infection in newborn, conventionally reared calves. The cross neutralization tests revealed an antigenic identity of simian and porcine rotaviruses and a slight serologic correlation of these two viruses with the bovine rotavirus strain. However, in reciprocal cross protection tests carried out in calves, the simian rotavirus antiserum afforded weak protection to challenge infection with either the porcine or the bovine viruses. By contrast, the protective level of the bovine and the porcine rotavirus antisera was relatively high. It was speculated that the 81/36F bovine rotavirus could be considered, tentatively, as an antigenic reassortant rotavirus strain. PMID- 3021520 TI - Antibody to hepatitis A virus after overt and asymptomatic hepatitis A infection. AB - The titres of antibody to hepatitis A virus (anti-HAV) were found to fall rapidly during 3-4 years after hepatitis A and more slowly thereafter, though they never reached undetectable levels in at least 23 years. The levels of anti-HAV in convalescents after overt hepatitis A were found to be significantly much higher than in those who had anti-HAV as a result of asymptomatic infection. PMID- 3021521 TI - Plasmid profiles of Legionella pneumophila strains isolated in Spain. AB - An assay for plasmid DNA content was carried out in 100 strains of Legionella pneumophila of distinct serogroups and isolated from various sources (clinical, environment). The strains were isolated from different geographic regions in our country. The presence of plasmids was proved in one of the 11 clinical isolates and in 68 of the 89 isolated of environmental origin studied. In the strains belonging to serogroup 1 and isolated in our region (Cantabria), three plasmid profiles were observed, whereas in strains of the same serogroup from other geographic regions, two profiles were shown which exhibited differences compared to the former ones. Analysis by means of restriction endonucleases suggested that plasmids of similar size in serogroup 1 strains of different source, were related. The results obtained do not appear to reveal any correlation between plasmid profile and source of isolation or serogroup. PMID- 3021522 TI - Electropherotyping of human rotaviruses: an epidemiological survey of rotavirus infections in Sicily. AB - An electrophoretic analysis of rotavirus RNA segments was carried out on 522 faecal specimens, obtained from children hospitalized in Sicily in the period 1981/85. One hundred and one viral isolates could be characterized with respect to the electrophoretic pattern of their genomic RNAs. This analysis revealed that in 1981/82 different electropherotypes cocirculated in the infant population. In 1983 one of the patterns became prevalent; in 1984/85 only one electropherotype was detected, both in Palermo and Catania specimens. The serotyping showed that all viral strains with the prevalent electropherotype were subgroup II and serotype 1. These results contrast with the extensive genome variability of rotavirus strains observed in urban areas. PMID- 3021519 TI - Ribavirin: a clinical overview. AB - Ribavirin, a broad spectrum, non-interferon-inducing virustatic chemotherapeutic agent, demonstrates activity against a wide range of RNA and DNA viruses, including the retrovirus known to cause the acquired immune deficiency syndrome. The drug's proposed mechanism of action, as well as pharmacokinetics are discussed, and preclinical toxicity, safety and clinical efficacy studies are presented. To date, the best success has occurred in the use of ribavirin to treat respiratory syncytial virus infection in infants and young children and to treat influenza A and B virus infections in young adults. Viral infections, particularly viral pneumonia, are often life-threatening in infants with severe combined immunodeficiency disease (SCID), and ribavirin aerosol has been used successfully to treat respiratory syncytial virus and parainfluenza virus infection of immunodeficient children. Special note is taken of ribavirin's clinical benefit in treating severe and life-threatening infections caused by the Lassa fever virus and the significant improvement over either the use of immune plasma or supportive therapy alone. Indeed, ribavirin thus emerges as the first antiviral drug that is able to reduce mortality in a highly lethal systemic disease by more than 90%. Additional studies demonstrate the drug's efficacy in acute viral hepatitis, herpesvirus infections, and measles. Controlled clinical trials are underway to test the drug in patients infected with the AIDS virus. PMID- 3021523 TI - Epidemiologic and genomic study of rotavirus strains infecting young children and calves in the same rural environment. AB - The epidemiological profile of rotavirus infection in young calves and young children form the same rural environment in France was studied during autumn winter 1983-1984. Viruses were isolated from calves on each farm and were identified by their electropherotype for 8 out of the 9 farms under study. Among 13 children regularly checked on 9 farms for 25 weeks, 7 were found to be rotavirus positive. Nevertheless, no transfer of virus between child and bovine was documented by the electropherotype migration. In the case of the calves, the predominance of the Ib IIa IIIe IVa electropherotype at 5 of the 9 farms is similar to the epidemiological situation observed among newborn in some maternity wards. PMID- 3021524 TI - Prevalence of antibody to human coronaviruses 229E, OC43 and neonatal calf diarrhea coronavirus (NCDCV) in patients of Northern Italy. AB - A seroepidemiological study for detection of antibody to human coronaviruses OC43, 229E, and neonatal calf diarrhea coronavirus (NCDCV), has been carried out using sera collected from hospitalized patients or healthy persons through routine laboratory tests in Northern Italy. Patients tested were children and adults with different pathological diseases. Antibody detection was performed by using an indirect immunoperoxidase staining technique (for all viruses) and, in the case of OC43 and NCDCV, antibody detection was obtained even with a hemagglutination inhibition test and a plaque reduction neutralization assay. Results obtained show a significant difference in the prevalence of antibody to 229E between children and adult group. Furthermore, a different titer was observed, within the two groups, between patients affected by hematological diseases (leukemia) and patients with other diseases. Finally, our data seem to confirm previous studies reporting a very high prevalence of antibody to coronavirus OC43 but a less detectable seropositivity to coronavirus 229E. PMID- 3021525 TI - Sequence homologies and structural similarities between the polypeptides of yeast and beef heart cytochrome c oxidase. AB - The homologous polypeptides in yeast and beef heart cytochrome c oxidase have been identified by sequence comparisons and structural similarities. The properties of individual polypeptides have been used to specify which components are extrinsic, and which intrinsic and bilayer spanning, in the cytochrome c oxidase complex. PMID- 3021526 TI - A novel member of the serpin superfamily is encoded on a circular plasmid-like DNA species isolated from rabbit cells. AB - A novel member of the serpin family of serine protease inhibitors is presented. A plasmid-like DNA was isolated from rabbit cells by its homology to the genome of Shope fibroma virus (SFV), a tumorigenic poxvirus of rabbits, and was shown elsewhere to encode a serpin-like protein [(1986) Mol. Cell. Biol. 6, 265-276]. Although significant DNA homology exists between the rabbit plasmid serpin open reading frame and the SFV terminal inverted repeat DNA there is no intact serpin counterpart encoded by this region of the SFV genome. The alignment of the novel plasmid-borne polypeptide with the serpin family of proteins confirms its status within this group. PMID- 3021527 TI - Phosphorylation of the modulator protein of the ATP, Mg-dependent protein phosphatase by casein kinase TS. Reversal by PCS phosphatases and control by distinct phosphorylation site(s). AB - The phosphorylation by casein kinase TS (II) of the modulator protein of the ATP, Mg-dependent phosphatase increases after preincubation with the PCSH1 phosphatase or with the catalytic subunit of the ATP, Mg-dependent phosphatase. Dephosphorylation by the two phosphatases combined leads to the incorporation of 2 mol phosphate per mol modulator (at Ser residues). Occupancy of the ATP, Mg dependent phosphatase phosphorylation site(s) is a negative determinant in the phosphorylation of the modulator by kinase TS. Among the PCS phosphatases PCSH1 shows the highest activity toward the 32P-Ser residues labeled by kinase TS in untreated or previously dephosphorylated modulator, while the ATP, Mg-dependent phosphatase is totally ineffective. Protamine stimulates all phosphatase activities, so that the catalytic subunit of the ATP, Mg-dependent phosphatase becomes almost as effective as the PCSC phosphatase in dephosphorylating the kinase TS sites. PMID- 3021528 TI - A 48 kDa protein arrests cGMP phosphodiesterase activation in retinal rod disk membranes. AB - Photolyzed rhodopsin (R) catalyzes GTP-binding to alpha-transducins (T alpha); T alpha X GTPs then activate cGMP phosphodiesterase (PDE). PDE activation is arrested by ATP in two ways: (i) initial velocity is suppressed, and (ii) PDE velocity rapidly returns to preactivation levels (turnoff). Arrestin (a 48 kDa protein) markedly enhances turnoff while not affecting initial velocity. Arrestin in the presence of ATP achieves rapid turnoff by directly inhibiting activated PDE, as indicated by its ability to inhibit the direct activation of PDE by T alpha X GMP--PNP (guanylyl-imidodiphosphate). Double reciprocal plots reveal a competition between arrestins and activated transducins for sites on PDE. Blocking R phosphorylation blocks initial velocity suppression but does not disturb rapid turnoff. Our data suggest a 2-fold mechanism for PDE deactivation: (i) formation of T alpha X GTPs is suppressed by R phosphorylation, while (ii) activation of PDE by T alpha X GTPs is competitively inhibited by arrestins when ATP is present. PMID- 3021529 TI - Spermine antagonises the effects of dexamethasone, glucagon and cyclic AMP in increasing the activity of phosphatidate phosphohydrolase in isolated rat hepatocytes. AB - Rat hepatocytes were incubated in monolayer culture, under serum free conditions, for 8 h. Glucagon (10 nM), 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (100 microM) and dexamethasone (100 nM) increased the activity of phosphatidate phosphohydrolase by approx. 2-, 3.6- and 3.3-fold, respectively. Spermine alone had no significant effect. Spermine (2.5 mM) almost completely inhibited the glucagon induced increase in phosphohydrolase activity. It only partially inhibited the dexamethasone and cyclic AMP mediated inductions. Spermidine had no significant effect in this respect. The results are discussed in relation to the known effects of polyamines on glycerolipid synthesis, in particular, and on intermediary metabolism. PMID- 3021530 TI - Specific effects of ATP on the kinetics of reconstituted bovine heart cytochrome c oxidase. AB - Bovine heart cytochrome-c oxidase was reconstituted in liposomes and the kinetics of cytochrome c oxidation were measured by the polarographic and photometric method under uncoupled conditions in the presence of various polyvalent anions. In order to distinguish between specific and unspecific ionic effects of ATP, the photolabelling reagent 8-azido-ATP was applied. Covalently bound ATP at the enzyme complex caused the same increase of Km for cytochrome c as free ATP, if measured by the photometric assay. The increase of Km by photolabelling with 8 azido-ATP was completely prevented by ATP, but not by ADP. The data indicate the occurrence of a specific binding site for ATP at the cytosolic side of cytochrome c oxidase, which, after binding of ATP, changes the kinetics of cytochrome c oxidation. PMID- 3021531 TI - A peculiar repetitive sequence in the rat genome. AB - We report here a new type of peculiar repetitive sequence, A15T(TC)9T12, which was detected at 750 base pairs (bp) upstream of a rat calmodulin processed pseudogene by DNA sequencing of cloned DNA fragments. This sequence element could possibly form a cruciform structure with a 12-AT-pair stem, exposing (CT)9 sequences as a loop. S1 nuclease protection experiments failed to identify this element as a cruciform structure but instead detected an alternating purine pyrimidine tract at 50 bp downstream of this element. Total genomic Southern blotting showed that the rat genome contains only a few of these elements. PMID- 3021532 TI - Measurement of the oxidation-reduction potentials of amicyanin and c-type cytochromes from Paracoccus denitrificans. AB - The oxidation-reduction potentials of four periplasmic electron carrier proteins from Paracoccus denitrificans have been determined. Their midpoint potentials are: amicyanin, 294 +/- 6 mV; cytochrome c-550, 253 +/- 5 mV; cytochrome c-551i, 190 +/- 4 mV; and cytochrome c-553i, 148 +/- 5 mV. Although rapid amicyanin mediated transfer of electrons from methylamine dehydrogenase to cytochrome c 551i was observed, reduced amicyanin did not reduce oxidized cytochrome c-551i in the absence of methylamine dehydrogenase. PMID- 3021533 TI - Stimulation of phosphatidylinositol 4-phosphate phosphorylation in human placenta membranes by GTP gamma S. AB - In human placenta membranes the rate limiting enzyme for PIP2 formation from PI is PIP kinase. GTP gamma S is shown to activate PIP kinase by increasing Vmax of the enzyme. It is suggested that a guanine nucleotide regulatory protein is involved in the activation of PIP kinase although coupling with a specific receptor is not yet known. Since PIP2 is the preferred substrate of phospholipase C, the possibility exists that an increase of PIP2 due to activation of PIP kinase leads to an enhancement of phospholipase C activity and hence to an increased production of IP3 and DAG. PMID- 3021534 TI - Regulation of protein synthesis in rabbit reticulocyte lysates. Thiophosphorylation of initiation factor eIF-2 by heme-regulated protein kinase. AB - The heme-regulated protein kinase, which specifically phosphorylates the 38-kDa subunit of initiation factor eIF-2, can utilize adenosine 5'-O-(3 thiotriphosphate) (ATP[gamma S]) as a substrate. The rate of thiophosphorylation is 5-6-times slower than that observed with ATP. It is of special interest that thiophosphorylated derivatives of eIF-2 are resistant to dephosphorylation catalyzed by eIF-2 phosphoprotein phosphatase. The thiophosphorylated eIF-2 is less effective in promoting protein synthesis in hemin-deficient lysates under physiological conditions. In addition, ATP[gamma S] could also be utilized by the self-phosphorylation activity intrinsically associated with HRI. PMID- 3021535 TI - Gold sodium thiomalate activates latent human leukocyte collagenase. AB - Gold sodium thiomalate, a drug used widely in the therapy of rheumatoid arthritis, was found to be an activator of latent human polymorphonuclear leukocyte collagenase. The activation was demonstrated by two distinct and independent collagenase assays: by recording with a spectrophotometer at 227 nm the enzyme-induced increase in ultraviolet difference absorbance of native type I collagen connected to the cleavage of collagen at 37 degrees C [(1986) Eur. J. Biochem. 156, 1-4] and by SDS-polyacrylamide gel electrophoresis analysis of formation of specific products of collagen resulting from collagenase cleavage at 25 degrees C. Activation of latent collagenase by gold sodium thiomalate appeared to be of the same magnitude as by the known activator phenylmercuric chloride. PMID- 3021536 TI - Radioimmunoassay for leukotriene E4. Use for determination of total sulfidopeptide-leukotriene release from rat gastric mucosa. AB - A conjugate of leukotriene (LT) E4 and bovine serum albumin (BSA) was prepared by covalently linking the free amino group of the hapten to the protein using dimethyl pimelindiimidate (DMP) as coupling reagent. Anti-LTE4 antibodies were raised in rabbits immunized with the conjugate. Binding of [3H]LTE4 to the antibodies is inhibited by 50% with 0.63 ng LTE4, while the relative cross reaction of LTC4 and LTD4 is 46.3% and 12.6%, respectively. Using the radioimmunoassay release of sulfidopeptide-LT (SP-LT) from rat gastric mucosa incubated in vitro was determined after quantitative enzymatic conversion of SP LT to LTE4. It could be demonstrated that this method is suitable for determination of SP-LT in biological material. PMID- 3021537 TI - Sliding-end-labelling. A method to avoid artifacts in nucleosome positioning. AB - A method, termed 'sliding-end-labelling', has been devised to avoid a frequent artifact in nucleosome positioning by indirect end labelling, namely the appearing of DNA fragments originated by two nuclease cuts, one of them lying within the region covered by the probe. The method is applied to the nucleosome positioning in the yeast SUC2 gene for invertase. PMID- 3021539 TI - The phage Mu repressor c and IS30 transposase proteins are significantly related. AB - The IS30 transposase exhibits significant amino acid sequence homology to the phage Mu repressor c in the amino- and carboxy-terminal regions of the proteins. The conserved sequences include the proposed Mu repressor DNA binding site, which is also related to the proposed Mu and D108 transposase DNA binding sites. The carboxy-terminal homologies are characterised by two almost complete, and one partial, somewhat diverged amino acid sequence repeats. Only weak homologies to this domain are present in the Mu transposase (Mu A). Nevertheless, a clear link between an insertion sequence and a bacteriophage has been established. PMID- 3021538 TI - Involvement of three intracellular messenger systems, protein kinase C, calcium ion and cyclic AMP, in the regulation of c-fos gene expression in Swiss 3T3 cells. AB - In quiescent cultures of Swiss 3T3 cells, platelet-derived growth factor or fibroblast growth factor known to induce both protein kinase C activation and Ca2+ mobilization raised c-fos mRNA. This action of the growth factors was mimicked by the specific activators for protein kinase C, such as phorbol esters and a membrane-permeable synthetic diacylglycerol, and also by the Ca2+ ionophores, such as A23187 and ionomycin. Prostaglandin E1 known to elevate cyclic AMP also raised c-fos mRNA, and this action was mimicked by 8-bromo-cyclic AMP, dibutyryl cyclic AMP and forskolin. These results suggest that expression of the c-fos gene is regulated by three different intracellular messenger systems, protein kinase C, Ca2+ and cyclic AMP, in Swiss 3T3 cells. PMID- 3021540 TI - [Effect of estradiol on the adrenocortical function of the silver fox]. PMID- 3021541 TI - Matrix degrading proteinases from human granulocytes: type I, II, III collagenase, gelatinase and type IV, V-collagenase. A survey of recent findings and inhibition by gamma-anticollagenase. AB - Three human matrix degrading leukocyte proteinases, type I collagenase, gelatinase and a new type IV collagenase were isolated in latent and active form. Activation of all three latent enzymes could be achieved by treatment with either organomercurials or with trypsin. In addition the 90 kDa latent type I collagenase could be activated by disulfides, while a newly discovered 70 kDa latent form could be activated with organomercurials or with trypsin. The active type I collagenase was inhibited by gamma-anticollagenase from human serum (and the leukocyte type I collagenase inhibitor, while the newly found type IV collagenase was inhibited only partially. The complexes formed from gamma anticollagenase with type I collagenase, i. e. latent enzyme, are not reactive site associated complexes. The binding is not of a substrate-like and competitive manner. After inhibition of the enzyme though inactive against its natural substrates it is still hydrolyzing the synthetic low molecular weight octapeptide DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH. PMID- 3021542 TI - Activation of latent human granulocyte gelatinase by rat mast cell protease. AB - Rat peritoneal mast cell extract contains an activator of latent human granulocyte gelatinase. The activator has been partially purified and characterized. It shows a similarity to rat mast cell chymase in several properties including its molecular weight, substrate specificity and sensitivity to inhibitors. The activation of latent gelatinase with rat mast cell protease is dependent on protease concentration, incubation time and is mediated through the catalytic site of the activator. The significance of mast cell protease in the regulation of collagenolytic enzymes is discussed. PMID- 3021543 TI - The biosynthesis of neutrophil and eosinophil granule proteins. AB - Composition of azurophil and specific granules from human polymorphonuclear neutrophils and granules from eosinophils is presented. Biosynthesis of the granule proteins is discussed in detail with particular emphasis on neutrophil myeloperoxidase (MPO) and eosinophil cationic protein (ECP). PMID- 3021544 TI - Subacute lupus erythematosus-like gyrate erythema. Report of a case associated with a breast cancer. AB - We present a case of an annular skin lesion in association with a breast cancer. For differential diagnosis we had to consider subacute cutaneous lupus erythematosus and the group of gyrate erythemas. Lupus band test and immune serology were negative. The parallel course of the erythema and the internal malignancy were striking. PMID- 3021545 TI - Extrapituitary neuroendocrine melanoderma. Unique association of extensive melanoderma with macromelanosomes and extrapituitary secretion of a high molecular weight neuropeptide related to pro-opiomelanocortin. AB - A patient developed an Addison-like melanoderma over the past decade. An abnormal neuropeptide related to, but bigger than pro-opiomelanocortin was found in large amount in the serum. It has most likely a stimulative action upon melanogenesis with formation of macromelanosomes. PMID- 3021547 TI - Follow-up of patients with epidermodysplasia verruciformis treated with etretinate. PMID- 3021546 TI - Triple extramammary Paget's disease. Immunohistochemical studies. AB - A 75-year-old man developed three lesions of Paget's disease which involved the pubic area and both axillae. Histologically, there was no invasion into the dermis in any of the three lesions. Various kinds of histochemical and immunohistochemical studies including those for carcinoembryonic antigen showed that both genital and axillary lesions were identical in nature. Cases with triple involvement of Paget's disease are thought to be extremely rare in the literature. PMID- 3021548 TI - Effect of diabetes and adrenocortical state on intestinal transport capacity and (Na+ + K+)-activated adenosine triphosphatase activity. AB - (Na+ + K+)-activated adenosine triphosphatase activity in the mucosal homogenate of rat small intestine estimated under conditions of experimental diabetes, application of corticosteroids, adrenalectomy or inhibition of adrenocortical function suggests a general parallelism between the capacity for monosaccharide absorption and enzyme activity. Studying the kinetic parameters it has been found that the maximal velocities of monosaccharide uptake as well as of (Na+ + K+) activated adenosine triphosphatase reaction are significantly different, whereas sugar concentrations for halfmaximal transport velocities and enzyme-substrate affinity constants remain unaltered. From studies with purified brush borders and basolateral plasma membranes it has been shown that the activity changes were caused exclusively by that part of (Na+ + K+)-activated adenosine triphosphatase which is localized in the basolateral plasma membranes. The enzyme activity in the brush border region remains unchanged. These findings support a concept of intestinal transport mechanism which suggests that the basolateral part of (Na+ + K+)-activated adenosine triphosphatase is responsible for metabolic energy supply. PMID- 3021550 TI - Isolation and characterization of proteolytic fragments of the sea urchin sperm receptor that retain species specificity. AB - The sea urchin sperm receptor isolated from the eggs of Strongylocentrotus purpuratus is a high molecular weight proteoglycan-like molecule. Previous studies in our laboratory suggested that the sperm receptor has two functional components, glycosaminoglycan chains that are responsible for sperm binding and polypeptide chains that control species specificity in the binding process. We have investigated this idea further by generating fragments of the receptor by limited proteolytic digestion of the egg cell surface. The results of experiments with these receptor preparations support the hypothesis that the species specificity of inhibition of fertilization observed in a competitive bioassay is conferred by the polypeptide portion of the receptor molecule. Studies with various receptor preparations reveal that the presence of at least 30% of the polypeptide by weight is required to inhibit fertilization species specifically. Receptor preparations containing less than 10% protein lack species specificity and inhibit fertilization in both S. purpuratus and Arbacia punctulata. PMID- 3021549 TI - Cartilage proteoglycan core protein gene expression during limb cartilage differentiation. AB - Changes in the steady-state cytoplasmic levels of mRNA for the core protein of the major sulfated proteoglycan of cartilage were examined during the course of limb chondrogenesis in vitro using cloned cDNA probes. Cytoplasmic core protein mRNA begins to accumulate at the onset of overt chondrogenesis in micromass culture coincident with the crucial condensation phase of the process, in which prechondrogenic mesenchymal cells become closely juxtaposed prior to depositing a cartilage matrix. The initiation of core protein mRNA accumulation coincides with a dramatic increase in the accumulation of mRNA for type II collagen, the other major constituent of hyaline cartilage matrix. Following condensation, there is a concomitant progressive increase in cytoplasmic core protein and type II collagen mRNA accumulation which parallels the progressive accumulation of cartilage matrix by the cells. The relative rate of accumulation of cytoplasmic type II collagen mRNA is greater than twice that of core protein mRNA during chondrogenesis in micromass culture. Cyclic AMP, an agent implicated in the regulation of chondrogenesis elicits a concomitant two- to fourfold increase in both cartilage core protein and type II collagen mRNA levels by limb mesenchymal cells. Core protein gene expression is more sensitive to cAMP than type II collagen gene expression. These results suggest that the cartilage proteoglycan core protein and type II collagen genes are coordinately regulated during the course of limb cartilage differentiation, although there are quantitative differences in the extent of expression of the two genes. PMID- 3021551 TI - Glucose transport by radiation-induced insulinoma and clonal pancreatic beta cells. AB - Sugar uptake was measured in dispersed cells prepared from radiation-induced insulinomas transplantable in NEDH rats and in three clonal beta-cell lines maintained in continuous culture (RIN m5F, RIN 1046, HIT). Uptake of D-glucose and 3-O-methyl-D-glucose by insulinoma cells was rapid so that the intracellular concentration of D-hexoses approximated the concentration in the incubation medium by 15-30 s. L-Glucose was taken up only slowly. 3-O-methyl-D-glucose uptake by RIN m5F, RIN 1046, and HIT cells was slow; with 1 mM 3-O-methylglucose in the medium, equilibrium was attained at 20 min, but with 10 mM 3-O methylglucose, equilibrium was not attained even at 20 min. In HIT cells incubated with D-glucose for 30 min, the intracellular concentration of glucose was less than the medium glucose concentration, indicating glucose transport is a nonequilibrium reaction in this cell line. These data indicate that radiation induced insulinoma cells retain the capacity of normal beta-cells to transport sugar at high rates. RIN m5F, RIN 1046, and HIT cells transport sugar slowly, however, and thus differ from normal beta-cells. In RIN m5F, RIN 1046, and HIT cells, unlike in normal beta-cells, glucose transport may be the site regulating glucose metabolism. PMID- 3021552 TI - Copper and hepatocellular carcinoma. AB - The presence of copper and copper-binding protein (CBP) within tumor cells was searched by histochemical methods (rhodanine, rubeanic acid and Shikata's orcein) in a group of 39 autopsies, all consecutive cases of hepatocellular carcinoma (HCC) associated with cirrhosis (HCC + C). In 2 cases, fibrolamellar carcinoma (FLC) not associated with cirrhosis was observed at surgery. Considerable amounts of copper and CBP were found within tumor cells only in the 2 FLC cases and in 1 HCC + C case, in a portion of the tumor showing FLC-like features. Copper positive tumor cells had an oncocytic appearance, which was confirmed by ultrastructural examination. In 64% of the HCC + C cases (25 out of 39), copper and CBP deposits were found in nonneoplastic hepatocytes mainly distributed along the periphery of cirrhotic nodules. The present study indicates that the storage of copper inside tumor cells is a peculiarity of the FLC type of HCC with a close relationship to the oncocytic nature of neoplastic hepatocytes. The significance of copper deposits in nonneoplastic cirrhotic hepatocytes remains a matter for further investigations. PMID- 3021553 TI - Prevalence of sensorineural hearing loss in premature and sick term infants with perinatally acquired cytomegalovirus infection. AB - Audiologic follow-up was obtained on 40 premature or sick term infants with perinatally acquired cytomegalovirus (CMV) infection and on 40 prospectively matched control subjects. Final evaluation was postponed until 3 years of age to assess any long-term hearing sequelae of perinatal CMV infection in this population, and to obtain reasonably complete audiometric results. One experimental subject had a bilateral sensorineural hearing loss above 4000 Hz. Four control subjects had sensorineural hearing losses, three requiring binaural hearing aids. The prevalence of confirmed hearing loss requiring amplification (3.75%) in this study group was consistent with that observed in all graduates of the Intensive Care Nursery who were considered at risk for hearing loss in the same time period (4.2%). These data suggest that perinatally acquired CMV infection is not associated with significant sensorineural hearing loss in premature or full term infants through age 3. PMID- 3021554 TI - Differentiation of F9 embryonal carcinoma cells. Differences in the effects of retinoic acid, 5-bromodeoxyuridine, and N'-N'-dimethylacetamide. AB - We found that monolayer cultures of F9 cells induced to differentiate with trans retinoic acid (RA) contain two major subpopulations of cells. These two cell types can be distinguished by their cellular morphology, their pattern of laminin accumulation, and their ability to undergo further differentiation in response to N6-O2-dibutyryl adenosine 3':5' cyclic monophosphoric acid (dBcAMP). Furthermore, the developmental pathway induced by RA appears to lead to two alternative pathways, and differentiation at the branch point is either directly or indirectly controlled by cAMP. Differentiation along one branch of this pathway can be induced by 5-bromodeoxyuridine, whereas differentiation along an unrelated pathway is induced by N'-N'-dimethylacetamide. In all cases, differentiation is closely paralleled by suppression of the tumorigenic phenotype, indicating that these two processes are tightly linked and probably share a common step. PMID- 3021555 TI - Primary adenocarcinoma of the terminal ileum simulating Crohn's disease. AB - An unusual case of histologically proven ileal adenocarcinoma mimicking regional enteritis in a 22-year-old man is reported. Although in 2 previous reports the patient's age was stressed as an important factor for the differential diagnosis, this was not of diagnostic help in our case. This report emphasizes the need to include other diagnostic possibilities when regional enteritis has an anomalous evolution and responds poorly to treatment. PMID- 3021557 TI - Central alpha 2-adrenergic control of the pattern of small intestinal motility in rats. AB - The effects of central and peripheral administration of alpha 2-adrenoceptor agonists and antagonists on small intestinal motility were examined in conscious rats chronically fitted with electrodes implanted in the duodenojejunal wall and a cannula placed in a cerebral lateral ventricle. In fasted rats, intracerebroventricular or intraperitoneal administration of clonidine (5 micrograms) immediately disrupted the migrating myoelectric complex pattern with a total inhibition of spiking activity during the first hour, followed by a period of irregular spiking activity for 2 h. The inhibition was abolished by previous intramuscular administration of yohimbine (600 micrograms), and the period of irregular activity was suppressed by intracerebroventricular yohimbine (30 micrograms). Naphazoline, an alpha 2-agonist that poorly crosses the blood brain barrier, only inhibited spiking activity when administered intraperitoneally (1 microgram) and induced only a period of irregular spiking activity when administered intracerebroventricularly at the same dose. In fed rats, intracerebroventricular administration of yohimbine or phentolamine (30 micrograms), and to a lesser extent prazosin, restores a migrating myoelectric complex pattern typical of the fasted state. Peripheral administration of these three antagonists at a dose 20 times higher was ineffective. Finally, both feeding and central administration of alpha 2-agonists disrupt the migrating myoelectric complex pattern. Such pharmacologic data suggest a possible role of central alpha 2-adrenoceptors in the regulation of intestinal motility in rats. PMID- 3021556 TI - Rat pancreatic zymogen granules. An actively acidified compartment. AB - In this study, we looked for acidification in pancreatic zymogen granules as recently reported for other secretory vesicles. In intact dispersed acinar cells, acidic intracellular compartments identified by fluorescence microscopy using acridine orange corresponded exactly to the distribution of zymogen granules visualized by light microscopy. Acridine orange fluorescence in zymogen granules was reversibly dissipated by protonophores (carbonyl cyanide m chlorophenylhydrazone, monensin) and NH4Cl; and the percentages of cytoplasmic area occupied by the acidic compartments and by zymogen granules were identical under fasting conditions and decreased in parallel after in vivo cholinergic stimulation. Zymogen granules released acutely from hypotonically disrupted cells without homogenization also accumulated acridine orange. Red-orange fluorescence in released granules was also abolished by protonophores and NH4Cl; and it reappeared after washout of protonophores in the presence, but not absence of adenosine triphosphate. Dicyclohexylcarbodiimide, which inhibits all proton pumps, and N-ethylmaleimide, which inhibits the proton pump of endocytic vesicles and lysosomes, but not mitochondria, prevented this adenosine triphosphate dependent reappearance of acridine orange fluorescence, whereas vanadate did not. In contrast to these observations with zymogen granules in situ or acutely released from disrupted cells, granules isolated by conventional multistep homogenization/centrifugation procedures did not exhibit adenosine triphosphate dependent acidification or development of a positive membrane potential as measured by quenching of acridine orange or Oxonol V, respectively. The latter findings may indicate release of inhibitors or granule damage during isolation. Collectively, the present results provide direct evidence that zymogen granules contain an active acidification mechanism which appears similar to that of other secretory vesicles and endosomes. This acidification process may have important implications for the storage, stabilization, and secretion of intragranular proteins including proenzymes. PMID- 3021558 TI - Comparison of the adrenal sensitivity to ACTH of laying hens with immobilization and plasma baseline levels of corticosterone. AB - The effects of mammalian ACTH (0.01-10.0 IU/kg) given intraarterially on plasma corticosterone levels in laying hens were determined. A rectilinear dose-response relationship was found, y = 24.545 + 8.691x. Daily ACTH injections of 0.1 IU/kg on 3 successive days showed a good reproducibility in response in plasma corticosterone levels. Immobilization by hand also increases plasma corticosterone to a plateau level of about 4 ng/ml. The effects of ACTH and immobilization in individual birds showed a good correlation (r = 0.87). A rather low correlation (r = 0.49) existed between individual baseline levels of plasma corticosterone and adrenal sensitivity for ACTH. The adrenal sensitivity for ACTH was nearly two times higher in the morning than in the evening. A diurnal pattern, however, could not be shown in the corticosterone response to immobilization. PMID- 3021559 TI - ACTH-related material in the pituitary gland of the chinook salmon (Oncorhynchus tschawytscha). AB - The distribution and nature of ACTH-related material in the pituitary gland of chinook salmon (Oncorhynchus tschawytscha) was studied. Pars distalis (PD) and neurointermediate lobe (NIL) extracts were analyzed by gel filtration on BioGel P6 and the resultant fractions assayed for ACTH immunoreactivity (ACTH-IR) using six different antibodies, all of which were specific for ACTH when applied to mammalian samples, and for alpha-MSH. In the PD a number of distinct peaks of ACTH-IR were observed, which were detected to different degrees by the different ACTH antibodies. Three of the ACTH antibodies produced essentially similar results. The other ACTH antibodies produced results quite distinct from these, and distinct from one another. Thus ACTH-IR is present in the salmon PD in a number of distinct forms, all of which appear to have molecular weights between about 2000 and 5000. No alpha-MSH was detected in the PD. All of the ACTH antibodies also detected material in the NIL. In general the different antibodies produced similar results. There were fewer peaks of ACTH-IR in the NIL than in the PD. The major one of these, which eluted between the void volume and 1-39 ACTH, was detected by all the antibodies. One of the antibodies also detected a peak of ACTH-IR which eluted close to where alpha-MSH eluted. All of the ACTH antibodies detected more material in the PD than in the NIL, the ratio being about 3:1 in every case. alpha-MSH-IR eluted as a minor peak at the void volume, and a major peak in the position of authentic 1-13 alpha-MSH. PMID- 3021560 TI - The development and validation of a radioimmunoassay to measure plasma ACTH levels in salmonid fishes. AB - A radioimmunoassay (RIA) capable of determining blood ACTH levels in salmonid fishes was developed and validated. The RIA used an antibody raised against mammalian ACTH, iodinated human ACTH as tracer, and human 1-39 ACTH as standard. Incubation of the standard or unknown with antibody for 3 days before addition of as little high-specific activity tracer as practicable (1500 cpm; equivalent to 5 pg ACTH) produced a very sensitive RIA; the operating range was 5 to 200 pg ACTH/ml. Extracts of both pars distalis and neurointermediate lobe of the pituitary glands from a range of salmonid species diluted parallel to the ACTH standard in the RIA. There was always considerably more ACTH-immunoreactivity (ACTH-IR) in the pars distalis extracts than in the neurointermediate lobe. Generally plasmas also diluted parallel to the ACTH standard, with the exception only of the plasma from sexually mature female salmonids, which diluted very non parallel to the standard, leading to unrealistically low estimates of the ACTH-IR level. The use of heparin as an anticoagulant during collection of samples caused problems when these plasmas were immunoassayed; instead EDTA was found to be a suitable anticoagulant. When the ACTH-IR was extracted from a pool of plasma obtained from acutely stressed salmon and chromatographed on a column of BioGel P6, followed by subsequent ACTH RIA of the fractions, only a single sharp peak of ACTH-IR was detected, which eluted in the position of authentic 1-39 ACTH. The plasma ACTH-IR level in unstressed fish was low, and near the detection limit of the RIA. An acute stress, produced by crowding and confinement for 30 min, increased ACTH-IR approximately 10-fold, and plasma cortisol levels 50-fold, but the plasma alpha-MSH level was not affected. Dexamethasone-treated fish did not respond to this stressor with any increase in either ACTH or cortisol levels. PMID- 3021561 TI - The effects of stress on plasma ACTH, alpha-MSH, and cortisol levels in salmonid fishes. AB - Handling and confinement caused a steady increase in the plasma ACTH level in both coho salmon and rainbow trout. Within 2 min plasma ACTH levels had increased significantly, and by 30 min they were 5- to 8-fold higher than the basal ACTH level in unstressed fish. This type of stress also caused a pronounced elevation in plasma cortisol, which lagged behind the ACTH increase, although the degree of change was greater, the level rising between 20- and 50-fold. The plasma alpha MSH level was unaffected by handling and confinement stress. A second series of experiments assessed the effects of a more severe stress, which consisted of 5 min out of water, during which the fish were restrained, followed by 25-min confinement in a small volume of water. This caused a very rapid, pronounced increase in the plasma ACTH level of sterile rainbow trout, the level reaching a peak at 5 min, and remaining elevated for the next 25 min. Plasma cortisol levels, which were low at the beginning of the experiment, remained so for the first 5 min, and rose thereafter. This type of stress also caused a rapid and pronounced elevation of the plasma alpha-MSH level. It rose in a very similar way, and at the same time, as the plasma ACTH level, but instead of remaining elevated it fell during the 25 min of confinement which followed the 5 min of restraint, to finish one-third of the peak value reached after 5 min. PMID- 3021562 TI - Immunocytochemical demonstration of pituitary cell types in the teleost Poecilia latipinna, by light and electron microscopy. AB - Using the unlabelled antibody method at the light microscope level, and the immunogold method at the electron microscope level, the distribution of the different adenohypophysial cells was demonstrated in the teleost Poecilia latipinna, by means of antisera to both teleostean and mammalian pituitary hormones and their subunits. Anti-salmon prolactin, but not anti-rat or -ovine prolactin, gave a specific staining of the acidophils of the rostral pars distalis (RPD), while anti-trout growth hormone (GH), but not anti-rat GH, stained similar but always separate cells in the proximal pars distalis (PPD). Antisera to the whole molecules of mammalian glycoprotein hormones stained the entire population of basophils in the PPD, but separate populations of gonadotrophs and thyrotrophs could be discriminated using anti-salmon gonadotrophin and anti-human thyrotrophin beta subunit. Antisera to ACTH (1-24) and (11-24) sequences, as well as beta-endorphin and met-enkephalin, stained the lead haematoxylin-positive cells of the RPD and pars intermedia (PI), whereas anti-alpha-MSH stained only the PI cells. Ultrastructural examination showed that these immunoreactivities were present in the same secretory granules, and were always greater in pale granules rather than electron dense granules. In the RPD, blebs of ACTH-immunoreactive cytoplasm were found to protrude through the gaps in the basement membrane into the neurohypophysis. The second "PAS-positive" cell type of the PI showed a strong cross-reaction with anti-salmon gonadotrophin, suggesting that it may produce a glycoprotein chemically related to the gonadotrophin(s). PMID- 3021564 TI - Cu(II)-BSA complexes and their identification by the ESR method. PMID- 3021563 TI - Effects of chronic administration of melanin-concentrating hormone on corticotrophin, melanotrophin, and pigmentation in the trout. AB - This paper reports the effects of salmonid melanin-concentrating hormone (MCH), administered via an Alzet minipump, on pigmentation and secretion of pituitary melanotrophin (MSH) and corticotrophin (ACTH) by black-adapted, adult rainbow trout. The drug induced melanin concentration in the skin melanophores and prevented melanogenesis. Both the cytological appearance of the pituitary pars intermedia and determinations of plasma alpha-MSH suggest that MCH prevented the increase in secretory activity of the melanotrophic cells seen normally in black adapted trout. Resting plasma cortisol titres were similar in all groups of fish but anterior pituitary glands taken from stressed, MCH-treated fish released less ACTH in vitro than those from corresponding saline-treated fish. PMID- 3021566 TI - Transmembrane outward hydrogen current in intracellularly perfused neurones of the snail Helix pomatia. AB - The ionic nature and pharmacological properties of the outward current activated by membrane depolarization were studied on isolated neurones of the snail Helix pomatia, placed in Na+- and Ca2+-free extracellular solutions and intracellularly perfused with K+-free solution ("nonspecific outward current"). It was shown that the amplitude and reversal potential of this current (estimated from instantaneous current-voltage characteristics) are determined mainly by the transmembrane gradient for H+ ions. Lowering of pHi induced an increase in the current amplitude and a shift of the reversal potential to more negative values; the shift magnitude was comparable with that predicted for the hydrogen electrode. Raising pHi, as well as lowering pHo, induced a decrease in the current amplitude and a displacement of the current activation curve to more positive potentials. Addition of EGTA (8 mmol/l) to the intracellular perfusate did not affect the current amplitude. Extracellular 4-aminopyridine (10 mmol/l), verapamil (0.25 mmol/l) or Cd2+ (0.5 mmol/l) blocked the current. It is concluded that the current studied is carried mainly by H+ ions. In the same neurones the nature of the fast decay of the calcium inward current was also studied (in the presence of extracellular Ca2+ ions). This decay considerably slowed when pHi was raised or pHo was lowered, and it became less pronounced upon extracellular application of 4-aminopyridine or upon intracellular introduction of phenobarbital (4 mmol/l) and tolbutamide (3 mmol/l). It is suggested that the fast decay of the calcium inward current is due to activation of a Ca-sensitive component of the hydrogen current which depends on accumulation of Ca2+ ions. The possible physiological role of the transmembrane hydrogen currents is discussed. PMID- 3021565 TI - Interaction of the antivirus agents remantadine and amantadine with lipid membranes and the influence on the curvature of human red cells. AB - Surface potential difference, conductance, and elasticity changes of bilayer lipid membranes induced by the antivirus drugs amantadine and remantadine were measured. An influence on the human erythrocyte shape was shown. Both drugs are stomatocytogenic. The adsorption at the cytoplasmatic membrane was electrophoretically proved. The heat-induced vesiculation is partly inhibited. No microvesicles were observed. Instead, large tails which did not detach from the cell body were seen. The general conclusion is that these amphiphilic adamantane derivatives are membrane agents which modify membrane interaction processes, possibly by influencing the bending properties. PMID- 3021567 TI - Hybrid dysgenesis-induced revertants of insertions at the 5' end of the rudimentary gene in Drosophila melanogaster: transposon-induced control mutations. AB - Mutations in the 5' control region of the rudimentary (r) gene of Drosophila melanogaster were generated by using hybrid dysgenesis to mobilize P elements that were already inserted within this region. Eighteen new mutations out of 7793 chromosomes were isolated. Among the mutations were small insertions, deletions and inversions. All five of the deletions deleted into the 5' coding region of the r gene resulting in a severe mutant phenotype. The inversions and small insertions left the coding region intact, but altered the 5' control region. Northern analyses of the message levels in adult females indicated that these mutations alter the amount of the wild-type rudimentary message. These data also indicated that there is a region about 800 bp upstream of the start of transcription which is necessary for normal r expression. This region is not involved with the temporal and spatial regulation of r gene expression, but with the quantitative expression. An analysis of the DNA changes associated with each of the mutations demonstrates that P elements usually mobilize in an imprecise manner. Of the 22 mutations that have been isolated to date, only three are precise excisions. This means that P elements are potent mutators of the genome. PMID- 3021568 TI - Excess polymorphism at the Adh locus in Drosophila melanogaster. AB - The evolutionary history of a region of DNA encompassing the Adh locus is studied by comparing patterns of variation in Drosophila melanogaster and its sibling species, D. simulans. An unexpectedly high level of silent polymorphism in the Adh coding region relative to the 5' and 3' flanking regions in D. melanogaster is revealed by a populational survey of restriction polymorphism using a four cutter filter hybridization technique as well as by direct sequence comparisons. In both of these studies, a region of the Adh gene encompassing the three coding exons exhibits a frequency of polymorphism equal to that of a 4-kb 5' flanking region. In contrast, an interspecific sequence comparison shows a two-fold higher level of divergence in the 5' flanking sequence compared to the structural locus. Analysis of the patterns of variation suggest an excess of polymorphism within the D. melanogaster Adh locus, rather than lack of polymorphism in the 5' flanking region. An approach is outlined for testing neutral theory predictions about patterns of variation within and between species. This approach indicates that the observed patterns of variation are incompatible with an infinite site neutral model. PMID- 3021570 TI - Two alternative transcripts coding for alcohol dehydrogenase accumulate with different developmental specificities in different species of picture-winged Drosophila. AB - Two alternate transcripts of the single copy Alcohol dehydrogenase (Adh) gene accumulate with developmental specificity in all of 12 species of Hawaiian picture-winged Drosophila which have been examined. Relative to the paradigm species D. affinidisjuncta, the Adh transcript normally restricted to larvae is found to accumulate in both larval and adult tissues in D. formella. The other Adh transcript, which normally accumulates only in adults, accumulates in third instar D. prostopalpis larvae as well. In species hybrids, the D. formella phenotype shows additive inheritance. These observations document the existence of a novel type of genetic variability. Furthermore, such variants suggest specific properties for the biological systems that regulate ADH expression in Drosophila, and they should facilitate further experimental investigations. PMID- 3021569 TI - Frequency and directionality of gene conversion events involving the CYC7-H3 mutation in Saccharomyces cerevisiae. AB - The CYC7-H3 mutation is a 5-kb deletion that causes overproduction of iso-2 cytochrome c. Unlike most mutations in yeast, the CYC7-H3 mutation is preferentially lost when it is involved in a gene conversion event. We have shown that cloned copies of CYC7-H3 DNA that are inserted into the yeast genome are associated with a high frequency of recombination and aberrant segregation events. Since parity in conversion frequency was observed when the extensive insertion/deletion heterozygosity at this locus was eliminated, we conclude that the CYC7-H3 sequences are inherently capable of acting as donors or recipients in gene conversion events, although they are unlikely to act as donors when they are located opposite a large heterology. DNA sequence comparisons revealed similarities between the CYC7-H3 junction region and the 2-micron circle DNA region that is involved in site-specific recombination. PMID- 3021572 TI - Evidence for evolutionary duplication of genes in the dopa decarboxylase region of Drosophila. AB - The region surrounding the dopa decarboxylase gene (Ddc) of Drosophila contains a cluster of genes, many of which appear to be functionally related by virtue of their effects on cuticle development and/or catecholamine metabolism. In this report we describe evidence that the Ddc gene and the closely linked alpha methyldopa hypersensitive (amd) gene share extensive sequence homology and are the products of a gene duplication event. The two genes are transcribed convergently and are separated by 2.4 kb. A gene located between Ddc and amd expresses a 2.0-kb mRNA and appears to partially overlap the Ddc gene. The organization of these transcripts implies a complex series of events giving rise to the present pattern. The patterns of expression of these genes do not support a model of coordinate regulation, but are more consistent with a pattern of duplication and divergence to various related metabolic subspecialties. These data provide the first evidence for structural relationships among genes in the 37C cluster. PMID- 3021571 TI - Molecular localization, developmental expression and nucleotide sequence of the alpha-methyldopa hypersensitive gene of Drosophila. AB - The region surrounding the dopa decarboxylase gene of Drosophila contains a cluster of functionally related genes, many of which affect cuticle development and/or catecholamine metabolism. In this report we describe the molecular mapping and sequencing of a full-length cDNA copy of a transcript that maps to the alpha methyldopa hypersensitive region. Developmental RNA blots show stage-specific patterns of transcription and multiple RNA transcripts. A 2-kb transcript is most abundant at about 12 hr of embryogenesis, and lower levels are detected throughout most of embryogenesis. Lower levels of this transcript are also detected in adults. Smaller stage-specific transcripts are detected in late third instar larvae. This pattern of transcription is consistent with the known lethal phases and phenotypes of the alpha-methyldopa hypersensitive gene (amd). Based on map position, pattern of transcription, homology with dopa decarboxylase and association with altered DNA in mutants, we conclude that this transcript represents the amd gene. PMID- 3021573 TI - Meiotic segregation and male recombination in interspecific hybrids of Drosophila. AB - Male hybrids between three pairs of Drosophila species show no substantial distortion of Mendelian segregation and no appreciable male recombination. These results do not support the theories that meiotic drive alleles of large effect are often fixed within species and that transposable genetic elements cause speciation. PMID- 3021574 TI - A quantitative model for nonrandom generalized transduction, applied to the phage P22-Salmonella typhimurium system. AB - A mathematical model for nonrandom generalized transduction is proposed and analyzed. The model takes into account the finite number of transducing particle classes for any given marker. The equations for estimation of the distance between markers from contransduction frequency data are derived and standard errors of the estimates are given. The obtained relationships depend significantly on the number of classes of transducing fragments. The model was applied to estimate the number of transducing fragment classes for a given marker in transduction with phage P22 of Salmonella typhimurium. It was found that the literature data on frequencies of contransduction in crosses with mutual substitution of selective and nonselective markers can be rationalized most accurately by assuming that the mean number of classes is equal to 2. An improved method for analysis of cotransduction data is proposed on the basis of our model and the results of calculation. The method relies on solving a set of algebraic equations for cotransduction frequencies of markers located within one phage length. The method allows a relatively precise determination of distances between markers, positions of transducing particle ends and deletion or insertion lengths. The approach is applied to the trp-cysB-pyrF and aroC-hisT-purF-dhuA regions of the Salmonella typhimurium chromosome. PMID- 3021575 TI - [Molecular cloning and functional analysis of DNA regions of plasmid RP4 determining the incompatibility properties]. AB - Sau3A-generated DNA fragments determining incompatibility functions of the plasmid RP4 were cloned on the vectors pTK16 and pBR322. Inc+ recombinant plasmids were divided into two types: 1) expressing incompatibility only towards the homologous RP4 replicon, 2) expressing incompatibility - both towards the homologous RP4 replicon and towards the heterologous replicons of plasmids R906 and R751. For one member of the first type plasmids it was shown that the cloned Inc+-specific insertion derived from the region of location of the EcoRI restriction site. The majority of the Inc+ recombinant plasmids showed asymmetric expression of incompatibility, predominantly eliminating the resident IncP plasmid. PMID- 3021577 TI - [Phasmids. Properties and the use in genetic engineering. II. Packing in vitro. Lysogeny of Escherichia coli K-12]. AB - Circular monomeric lambda DNA molecules were used as a substrate for packaging reaction in vitro. For obtaining lambda DNA in circular monomeric form only, Escherichia coli recA plasmid bearing cells were used. This hybrid DNA molecule which we designated phasmid lambda pMYF11, is the pBR322 plasmid in which lambda 47.1 DNA was introduced in vitro. The phasmid can exist in the plasmid form or as a non-defective phage. The efficiency of packaging reaction in vitro proved to be similar for monomeric circular and linear form of phasmid DNA molecules. The cI- variant of the phasmid is not able to exist as a plasmid even in the cells containing homoimmune prophage. Still, cI+ phasmid variants capable of lysogenizing arise with low frequency, as a result of recombination between the resident cI+ prophage and infecting cI- phasmid. PMID- 3021576 TI - [Phasmids. Properties and the use in genetic engineering. I. Construction of the phasmid vector]. AB - A phasmid vector molecule designated pMYF11 has been constructed. The vector combines some useful features of plasmid and phage vector molecules. lambda pMYF11 is a hybrid of lambda 47.1 vector and pBR322 plasmid. CI- marker of pMYF11 is replaced with cI+ marker by recombination between the plasmid and prophage 434. The phasmid molecule can be used as a replacement vector for BamHI, HindIII, SalGI endonucleases. The maximum size of fragments to be cloned is 21 kilobase pairs. Positive selection for hybrid molecules is possible because of the Spi phenotype expression after replacement of the central HindIII or BamHI DNA fragment with foreign DNA. A library of Escherichia coli genes is constructed with the help of lambda pMYF11 as a vector molecule. A hybrid phage harboring genes of the proline operon is detected by means of complementation. PMID- 3021578 TI - [Effective, plasmid RP4-dependent replication-transposition of DNA of the transposable phage D3112 of Pseudomonas aeruginosa in a heterologous Escherichia coli system]. AB - The processes of replication and transposition of Pseudomonas aeruginosa transposable phage D3112 in cells of Escherichia coli (D3112) and E. coli (RP4::D3112) were studied. D3112 genome is a "silent cassette" ("conex-phage"- conditionally expressible) in E. coli cells incubated at 42 degrees C. Two compulsory conditions for D3112 genome expression are incubation at 30 degrees C and the presence in cells of RP4 plasmid. Processes of replication and transposition in E. coli are coupled. RP4 plasmid stimulates D3112 DNA synthesis in E. coli at least by two order of magnitude. In correspondence with this observation is the fact that when Mg2+ is present in high concentration (0.1 M) in a cultural medium, the production of mature phage is enhanced by two order of magnitude in E. coli (RP4::D3112) or in E. coli (D3112, RP4) cells, and is approx. 10(-1)-10(-2) phage per cell. No influence of Mg on phage production is observed in E. coli (D3112) cells. PMID- 3021579 TI - [Integration of SV40 DNA into the cell genome and viral mutagenesis]. AB - Integration of DNA of a temperature-sensitive SV40 mutant (tsA239) into the cell genome was studied. The viral A gene (the oncogene) encodes the tumour T antigen which is ts in the mutant and is devoid of mutagenic and transforming activity under non-permissive conditions (40 degrees C). Clones of Chinese hamster cells infected by tsA239 mutant were analysed. Those infected by wild-type SV40 served as controls. As shown by dot-hybridization, SV40 DNA was detected in cells of 14 out of 18 clones infected by tsA mutant and incubated at 40.5 degrees C, and in all 20 clones infected by tsA mutant and incubated under permissive conditions (33 degrees C), the difference between the two groups being insignificant (p greater than 0.05). By means of blot-hybridization it was established that viral DNA was integrated into the cell genome of all 12 clones analysed, belonging to the three experimental series: infection by tsA mutant, incubation at 40.5 and 33 degrees C, infection by wt SV40, incubation at 40.5 degrees C. The number of integration sites ranged from one to four in different clones. Integration of SV40 DNA in tandems was observed. The data presented allow to conclude that integration per se does not play a crucial role in determining the mutagenic and transforming effect of the virus. Obviously, what matters is the activity of viral oncogene product - the T antigen. PMID- 3021580 TI - [Regulator sequences in the kappa-chain gene expressed in the hybridoma PTF.02]. AB - Two kappa genes, one (5.7 kb) from parental myeloma cells that were used for fusion, and the other (7.5 kb) from lymphocytes have been detected in the genome of PTF.02 hybridoma. Functionally important regions of the second gene were sequenced. In the 5'-region, positions and nucleotide sequences of the L fragment, TAATA- and CAAT-boxes were established. Deca (dc)- and pentadecanucleotide (pd) sequences, obligatory for effective transcription of Kappa genes, were localized at the distance of 91 and 118 bp from the ATG initiating codon of the leader sequence. Together with a true pd, a shadow pd sequence was localized. This sequence was overlapped with the first sequence and shifted by one helical twist. The structure of the region of the enhancer localization was established. Comparison of this sequence with known consensus of the papova virus enhancer allows us to suggest the following structure of the enhancer core for kappa chains: TGTGGCTAA... 10 bp... TGTGGTTA. In the kappa gene under study, the variable fragment is linked to the J5 segment. In the latter, a point mutation C----G was found, to which a conservative substitution Ala - Gly in a hypervariable region of the chi-chain should correspond. Somatic mutations were also observed within the intron region adjacent to the J5 segment. PMID- 3021581 TI - [Genetic analysis of Escherichia coli min81 mutation blocking the development of bacteriophage Mu]. AB - Data characterizing mim81 mutation obtained by the method for direct selection of transposition mutations are presented. The development of Mu is shown to be dramatically suppressed in the mutant strain both upon infection and after induction from the lysogenic state. Frequencies of lysogenization and mini-Mu dependent formation of cointegrates in the mutant strain are comparable with those in the wild-type strain. Mu development prohibition is removed if expression of early Mu gene is provided from the modified Pe promoter. The results obtained make us believe that the mechanism of mim81 mutation action involves reduction of early gene expression to the level that is sufficient for Mu DNA integration into the chromosome during infection and for single replicative events, but insufficient for vegetative development of bacteriophage Mu. PMID- 3021582 TI - Promoter region of the human pro-alpha 1(II)-collagen gene. AB - We have isolated a genomic clone containing the 5'-terminal portion of the human pro-alpha 1(II)-collagen gene. This clone, HC2C, contains 10 kb of the gene and 6 kb of 5'-flanking sequences. It was selected by cross hybridization using a [32P]DNA probe containing the promoter region of the rat alpha 1(II)-collagen gene. We have determined the nucleotide sequence of the first exon and of 400 bp upstream. There is considerable homology between this human sequence and the corresponding rat sequence. As in the rat, the first exon contains a 155-bp untranslated segment and a 85-bp sequence coding for the signal peptide and a part of the N-propeptide of type-II procollagen. The segment preceding the transcription initiation site contains a conserved 'ATATAA' element, and several similar G + C-rich stretches. As in the rat gene, no 'CAT' element is evident between -70 and -120. We also find the sequence 5'-GTGGTCAGA-3' reported as an enhancer element in both viral and cellular genes, located around -290 bp. A high degree of homology exists between the atypical rat and human promoter structures; however, such homology is absent among the alpha 1(I), alpha 2(I) and alpha 1(III) promoters, which suggests that the unique alpha 1(II) sequences may be related to the specific expression of the alpha 1(II)-collagen gene. PMID- 3021583 TI - Cloning and expression of a chloramphenicol acetyltransferase gene in cytosine substituted T4 bacteriophage. AB - We have developed an efficient method for transferring foreign genes into the T4 phage genome. Any foreign genes inserted into the T4 uvsY gene cloned on plasmids can be transferred into a cytosine-substituted T4dC(delta NB5060) phage genome by a replacement type of recombination. To achieve this, we constructed chimeric plasmids which had a chloramphenicol acetyltransferase gene (cat) derived from transposon Tn9 inserted into the Bg/II site within the T4 uvsY gene on pBR322. The cat gene was then transferred by in vivo recombination into the T4dC(delta NB5060) phage genome. Moreover, it was demonstrated that the cat gene in the hybrid T4dC phage was expressed upon phage infection and development. PMID- 3021584 TI - Identification of ten additional nucleotides in the primary structure of yeast 18S rRNA. AB - The ten-nucleotide-long sequence have been omitted while sequencing the 18S rRNA gene from yeast Saccharomyces cerevisiae [Rubtsov et al., Nucl. Acids Res. 8 (1980) 5779-5794]. This GAAGAUGAUC sequence and some other minor corrections are reintroduced into the yeast 18S rRNA primary structure. PMID- 3021585 TI - Neurospora crassa ribosomal DNA: sequence of internal transcribed spacer and comparison with N. intermedia and N. sitophila. AB - Using [32P]DNA probes from a clone containing 17S, 5.8S and 26S rRNA of Neurospora crassa, the remainder of the repeat unit (RU) for ribosomal DNA (rDNA) has been cloned. Combining restriction analysis of the cloned DNA and restriction digests of genomic DNA, the RU was found to be 8.7 kb. The nucleotide sequence was determined for the internal transcribed spacer (ITS) regions one and two, for 5.8S rRNA and for portions of 17S and 26S rRNAs immediately flanking the ITS regions, and compared to the corresponding region of Saccharomyces carlsbergensis. In addition, a comparative restriction analysis of two other Neurospora species was performed using twelve restriction endonucleases. Genomic DNA blots of rDNA from N. intermedia and N. sitophila revealed rDNA RUs of 8.4 kb. The majority of differences in restriction patterns were confined to sequences outside the mature rRNA regions. However, one SmaI recognition site was found in 26S rRNA of N. crassa and N. sitophila but not in N. intermedia. PMID- 3021586 TI - Production of restriction endonucleases using multicopy Hsd plasmids occurring naturally in pathogenic Escherichia coli and Shigella boydii. AB - A convenient procedure has been devised for detection of restriction endonucleases in the Escherichia coli-Shigella group. With this procedure, two restriction endonucleases, designated Sbo 13 and Eco T22, were found and later were identified as isoschizomers of NruI and AvaIII, respectively. These endonucleases were shown to be produced from small multicopy plasmids. They were isolated from nonpathogenic E. coli into which the plasmids had been introduced by transformation, and purified from contaminating nuclease activity. The yield was high, 1,000 units/g of wet cells for Sbo 13 and 500 units/g for Eco T22. Sbo 13 and Eco T22 should be preferable to NruI and AvaIII because of the high yield and ease in handling the producer cells. PMID- 3021587 TI - Isolation of Trypanosoma cruzi DNA fragments which function as ARS elements in Saccharomyces cerevisiae. AB - Genomic banks from Trypanosoma cruzi total cell DNA and kinetoplast DNA were constructed in a bacterial plasmid carrying the yeast URA3 gene marker. This vector is by itself unable to be maintained in Saccharomyces cerevisiae since it can neither replicate as an extrachromosomal element, nor readily integrate into the chromosome of ura3-52 yeast hosts. Using this system, we isolated sequences from nuclear and kinetoplast DNA of T. cruzi which enabled the vector to replicate autonomously in S. cerevisiae. The cloned DNA fragments fit the description of ARS sequences previously isolated from other organisms. PMID- 3021588 TI - Transcription of the speC (ornithine decarboxylase) gene of Escherichia coli is repressed by cyclic AMP and its receptor protein. AB - The speC gene encoding ornithine decarboxylase (ODC) in Escherichia coli is negatively regulated by cAMP and the cAMP receptor protein (CRP). In minicells transformed with the plasmid pODC bearing speC, cAMP supplementation repressed ODC synthesis. In a cell-free protein synthesizing system directed by pODC, cAMP at 10(-5) M repressed ODC synthesis by 90%. This repression required a functional CRP as cAMP failed to repress ODC synthesis in vitro in an extract prepared from a crp- strain; the addition of purified CRP to the crp- extract restored the ability of cAMP to repress ODC synthesis. In a prototroph transformed with the plasmid pCOD bearing a speC::tet chimeric gene, cAMP supplementation decreased tetracycline (Tc) resistance. In contrast, in crp- strains transformed with pCOD or pTET (TcR), cAMP supplementation did not change their Tc resistance. When a cya- strain was supplemented with 2 mM cAMP, steady state levels of ODC mRNA were repressed by 80%. However, when a isogenic crp- strain was supplemented with 2 mM cAMP, no repression of ODC mRNA was observed. These results indicate that the cAMP-CRP complex exerts negative transcriptional control of ODC synthesis as a function of the speC promoter. PMID- 3021589 TI - Molecular cloning of a recA-like gene of Methylophilus methylotrophus and identification of its product. AB - A recombinant plasmid, pPF5, has been constructed which contains a 5.5-kb fragment of Methylophilus methylotrophus DNA inserted into pAT153, and which confers resistance to UV and mitomycin C (MC) upon Escherichia coli recA mutants. Hybridisation analysis indicates that sequence homology exists between the cloned DNA and a fragment of E. coli chromosomal DNA that contains the recA gene. Tn1000 insertions have been isolated within pPF5 that inactivate its ability to complement recA mutations, and the site of each insertion has been determined by restriction mapping. A 36-kDa protein is synthesised by pPF5 but not by any of the Tn1000 derivatives, indicating that this protein is the product of the gene responsible for the complementation. Comparison of the size of truncated polypeptides produced by selected pPF5::Tn1000 derivatives with the position of the insertion sites of the transposon, gave the direction of transcription of the M. methylotrophus recA+ gene. SOS proteins are induced in E. coli recA mutants harbouring pPF5 following MC treatment. PMID- 3021590 TI - Molecular cloning and characterization of the purE operon of Escherichia coli. AB - The purE operon of Escherichia coli has been cloned and localized to a 1.7-kb HpaI fragment. The operon has been further characterized by subcloning into the lac fusion vector, pMC1403, and by the construction of BAL 31-generated deletions. The purE regulation region has been identified by assay of beta galactosidase produced by pur-lac fusion plasmids and by RNA polymerase binding to end-labelled restriction fragments. Two purE promoters have been identified; one strong that is regulated by purines, the other weaker which is not regulated. The latter may be internal to the purE1 structural gene. PMID- 3021591 TI - Molecular cloning and expression in Escherichia coli of the lethal factor gene of Bacillus anthracis. AB - We have cloned and expressed in Escherichia coli the lethal factor (LF) gene of Bacillus anthracis. At least two of the six LF recombinant plasmids produce full length LF protein. Transcription of the LF gene in E. coli appears to be under the control of its own B. anthracis promoter. Recombinant LF protein produced in E. coli remains intracellular and is not secreted. However, this LF protein is biochemically active and displays the same lethal effects as LF secreted by B. anthracis in the mouse macrophage assay. The LF gene, like that of the protective antigen gene, is present on the large B. anthracis toxin plasmid pXO1. PMID- 3021592 TI - Cloning of a gene encoding a DNA polymerase-exonuclease of Streptococcus pneumoniae. AB - A procedure was developed for cloning and characterizing genes that encode proteins with nuclease activity in the Streptococcus pneumoniae [pLS1] host/vector system. Clones are screened for nuclease activity by a DNase colony assay and the nucleases that they produce are characterized by detection of enzyme activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The method was used to clone the gene encoding a DNA polymerase (Pol)-exonuclease of S. pneumoniae. The activity of this enzyme, the predominant DNA Pol of S. pneumoniae, is tenfold greater in cells carrying the multicopy recombinant plasmid than in cells without the plasmid. The enzyme corresponds to a 100-kDa polypeptide, and its properties are similar to PolI of Escherichia coli. A restriction map of the pSM22 plasmid containing the pneumococcal polA gene was obtained. The gene was transferred into Bacillus subtilis and E. coli, and it was expressed in both species. Its direction of transcription was determined by placement of the gene in both orientations in an E. coli hyperexpression plasmid. In one of the orientations the pneumococcal PolI enzyme was produced at a level 50-fold greater than normally found in S. pneumoniae, and it comprised 5% of the total protein. PMID- 3021593 TI - Organization of the adenyl cyclase (cya) locus of Rhizobium meliloti. AB - A cya-like gene encoding adenyl cyclase from Rhizobium meliloti was localized to a 0.8-kb PstI-EcoRI fragment by subcloning experiments. Experiments in Escherichia coli 'maxicells' identified a R. meliloti cya gene product of 28 kDa, which is significantly smaller than the corresponding protein from enteric bacteria. A control region for the expression of the cya gene in E. coli was found on an adjacent 2.6-kb BglII-BamHI sequence by insertional mutagenesis with Tn5 and phage MudI (ApR lac). The direction of transcription of the cya gene was also determined using a cya::MudIlac fusion. Promoter activity of this cya::lac fusion was not decreased when glucose was added to the culture. The R. meliloti cya gene is conserved among R. meliloti strains but no homology could be detected to other Rhizobium species or to E. coli in DNA hybridization experiments. PMID- 3021594 TI - [Relation between the cytotoxic effect of silicosogenic dusts and intensified lipid peroxidation in macrophages]. PMID- 3021595 TI - [Fibrogenic activity of type Y zeolite and mordenite dusts]. PMID- 3021596 TI - [Molecular mechanism of the biological activity of asbestos]. PMID- 3021597 TI - [Incidence of pleural mesothelioma after exposure to chrysotile and asbestos dust]. PMID- 3021598 TI - Immunohistochemical demonstration of papillomavirus antigen in cervical dysplasia and vulvar condyloma. AB - Biopsies from 30 cases of vulvar condyloma, 460 cases of cervical dysplasia, 30 cases of carcinoma in situ and 75 cases of invasive carcinoma of the cervix were screened for the presence of human papillomavirus (HPV) antigen by means of the peroxidase-antiperoxidase method. Positive reaction for HPV was detected in 14 cases of condyloma and 80 cases of dysplasia as a brown intranuclear precipitate in the superficial layer of the epithelium. None of the cases of carcinoma in situ and invasive cancer were positive for HPV. The mean age of the women with HPV-positive dysplasia was significantly lower than that of the women with HPV negative dysplasia. Condylomatous dysplasia showed a significantly higher positive rate than did noncondylomatous dysplasia. Cases of condylomatous dysplasia with severe stromal inflammation were negative for HPV more frequently than those with mild stromal inflammation. PMID- 3021600 TI - Effect of dithiocarbamates on citric acid production by Aspergillus niger. AB - Dithiocarbamates were found to enhance the production of citric acid under solid state fermentation conditions by Aspergillus niger. Maximum increase was observed with tetramethylthiuram disulfide (TMTD). Percent increases observed were 74.2% with 2.5 micrograms/mL of TMTD, 19.6% with 2.5 micrograms/mL of sodium dimethyldithiocarbamate and 33.1% with 0.6 micrograms/mL of zinc dimethyldithiocarbamate. PMID- 3021599 TI - An in vivo cointegrate of two plasmids from incompatibility group X. AB - Formation of a recombinant plasmid designated pNH603 was observed when two plasmids from incompatibility group X, the multicopy plasmid pNH602 (a higher copy-number deletion derivative of R6K) and the oligocopy plasmid R485, coexisted in a single Escherichia coli cell. According to its size and its restriction endonuclease cleavage pattern, plasmid pNH603 is a true cointegrate of pNH602 and R485. An insertion-sequence-like element coming from plasmid R485 is supposed to mediate the fusion of both replicons. The pNH603 copy number (1-2 per chromosome) indicates that the mechanism of replication of the low-copy-number plasmid is dominant in this cointegrate. No dissociation of pNH603 to parental plasmids was observed even in E. coli K-12 recA+ cells. On the other hand, deletion derivatives of four size classes originate from pNH603 in both recA+ and recA hosts. A miniplasmid designated pNH604, a representative of the most frequent 7 Mg/mol size class, was found, in a low number of copies per host chromosome. PMID- 3021601 TI - Interference with sporulation can stimulate the rate of a proteinase synthesis in Bacillus megaterium. AB - Bacillus megaterium, in which sporulation was blocked either by mutation or with netropsin, synthesizes during the stationary phase more exocellular proteinase than the sporulating culture. The asporogenic mutant synthesizes the enzyme at a higher rate and for a longer time than does the sporulating population. The culture, whose sporulation was inhibited by netropsin, produces the proteinase at a higher rate, although for only a limited time interval. PMID- 3021602 TI - Evaluation and therapy after injury to peripheral nerves. AB - Peripheral nerve injuries from whatever cause should be classified according to the degree of axon, fascicle, and main nerve trunk damage. This approach is useful in assessing the chances for spontaneous recovery and planning for surgery directed at improving neural recovery. Evaluation over time may be required to better classify the degree of injury. Once recovery slows or ceases, the surgical approaches available include neurolysis, nerve repair, nerve transposition, autograft reconstruction, and various procedures directed at painful neuromata. With proper mobilization and protection of neural and vascular structures, even extensive orthopaedic reconstructive procedures may be performed. Ischemic neuritis and myopathies may be improved by such combined approaches. PMID- 3021603 TI - [Combined hyperthermia and proton irradiation: a new method in the treatment of malignant melanoma of the choroid?]. PMID- 3021604 TI - [Side effects of gyrase inhibitors]. PMID- 3021605 TI - [Tumor markers in urologic neoplasms]. PMID- 3021606 TI - Isolation of transcribed DNA sequences from chromosome 21 using mouse fetal cDNA. AB - A technique for isolating DNA sequences that are likely to be transcriptionally active in both humans and mice has been developed. This method is based on the screening of a human genomic DNA library with single stranded cDNA prepared from late gestation mouse fetal RNA. Using a human chromosome 21 DNA library, we have isolated and characterized two clones which are entirely composed of single copy human DNA sequences and are 2.7 and 6.7 kilobases in length. The 2.7 kilobase clone is homologous to a transcript found predominantly in nonpolyadenylated human fibroblast RNA. It also shows homology to mouse genomic DNA and recognises several polyadenylated RNAs in mouse testis. This cloned sequence has been mapped by in situ hybridization to the distal third of chromosome 21, the region involved in the causation of Down syndrome. PMID- 3021607 TI - The same "TATA" box beta-thalassemia mutation in Chinese and US blacks: another example of independent origins of mutation. AB - A Chinese beta +-thalassemia gene in a new haplotype was chosen for cloning and sequencing. The mutation identified was an A-G transition at position -29 in the TATA box of the beta-globin gene. This mutation has not been seen previously in Chinese but has been documented in American blacks on a different chromosomal background. This observation provides further evidence for independent origins of the same mutation in distinct ethnic groups. PMID- 3021609 TI - Cytomegalovirus infection in provincial homosexual men. PMID- 3021608 TI - Coexistent chlamydial infections related to natural history of human papillomavirus lesions in uterine cervix. AB - To assess the role of Chlamydia trachomatis in the development of cervical intraepithelial neoplasia (CIN) and to evaluate possible synergism between chlamydiae and human papillomavirus (HPV) in this process, 418 women who had been prospectively followed up for cervical HPV infections at our clinic since 1981 were tested for chlamydiae. At each visit the patients were examined by colposcopy, and other investigations, such as Papanicolaou (Pap) smears, punch biopsies, urethral, and cervical swabs, were undertaken as indicated. In biopsy specimens the cytopathic changes of HPV, concomitant CIN, and the local immunocompetent cell infiltrates were analysed. The latter were measured and further identified using an alpha naphthyl acetate esterase (ANAE) technique to define B cells, macrophages, and T cells and using monoclonal antibodies to define T cell subsets, NK (natural killer cells), and Langerhans cells. Chlamydial isolation (4.1% in the cervix, and 3.6% in the urethra) did not positively correlate with the degree of cytological atypia in PAP smears or with the degree of CIN lesions associated with HPV. Chlamydial cervicitis did not affect the ANAE definable cell composition of the immunocompetent cell infiltrates in HPV lesions, or that of the immunocompetent cell subsets, including the ratios of T helper to T suppressor cells and the numbers of NK cells. Chlamydial infection did not alter the natural history of HPV lesions, of which 30% regressed, 53% persisted, and 17% progressed during follow up. The present results do not provide evidence to substantiate the hypothesis that chlamydiae and HPV might act synergistically in cervical carcinogenesis, or the view that C trachomatis may be a major aetiological agent of CIN lesions. Chlamydiae and HPV are covariables of sexual behaviour, and their concomitant appearance in sexually promiscuous women is best explained by this fact. As we do not have more direct evidence for the oncogenic potential of C trachomatis (as we have of HPV), it seems reasonable to consider that this agent is not a major cause of CIN, but rather a sexually transmitted agent commonly found in women with CIN because of their promiscuous sexual behaviour. PMID- 3021610 TI - Endogenous protein inhibitor of mitochondrial c-AMP phosphodiesterase in tumour bearing rats. PMID- 3021611 TI - Purification of choline-ethanolamine kinase from chicken liver and its regulation by cAMP. PMID- 3021612 TI - Modulation of glycolysis by dibutyryl cyclic AMP in rat lung. PMID- 3021613 TI - Cystosarcoma phyllodes--a histological profile of 38 cases. PMID- 3021614 TI - Strong adjuvant properties of cholera toxin on gut mucosal immune responses to orally presented antigens. AB - There is a great need for substances that can act as adjuvants on local mucosal immune responses to perorally (p.o.) administered immunogens and which could be included in future oral vaccines. In this study we show that in mice cholera toxin (CT) is a potent adjuvant on enteric mucosal immune responses to related (cholera B subunit) as well as unrelated (KLH) antigens presented by the p.o. route. The adjuvant action of CT was dose-dependent and was achieved only when CT was given p.o. and together with the antigen. Both priming (memory induction) and boosting of the gut mucosal immune system by the oral route were greatly potentiated by CT. High numbers of specific antibody-producing cells as well as substantial mucosal memory in the lamina propria were stimulated by p.o. priming immunizations if CT adjuvant was included. Anamnestic responses could be elicited by a single p.o. booster immunization for at least 10 weeks and probably much longer. The adjuvant action of CT is suggested to involve activation of adenylate cyclase and cyclic AMP-mediated signals with differential effects on B and regulatory T intestinal lymphocytes. The adjuvant-active dose of CT, 100-500 ng, was lower than the immunogenic dose (2 micrograms) and much below the p.o. dose needed for detectable net fluid secretion in mouse intestine (5-10 micrograms). Cholera B subunit (10 micrograms) administered p.o. together with 500 ng of CT was 50 times more effective in stimulating gut mucosal anti-toxin responses compared with B subunit vaccine alone. Our results suggest that CT or substances that use similar adjuvant mechanisms may substantially increase the mucosal immunogenicity and efficacy of non-replicating oral vaccines. PMID- 3021615 TI - The effects of cyclosporin A on the early responses of B and T cells to Epstein Barr virus infection. AB - The immunosuppressive properties of the fungal metabolite Cyclosporin A (CsA) on the human lymphocyte response to the polyclonal B cell activator Epstein-Barr virus (EBV) in vitro were assessed. CsA showed both stimulatory and inhibitory effects on EBV-induced IgM secretion, the net effect being dependent on the dose of virus used. T cells were required for both these effects to occur. A model is proposed to account for the complex interactions of CsA in this system. PMID- 3021616 TI - Generation of phenotypically different T cell populations by cell-free or cell bound preparations of varicella zoster virus. AB - We used three different preparations of varicella zoster virus (VZV) to sensitise mononuclear cells obtained from VZV immune donors. These were autologous infected fibroblasts, live cell free virus and heat inactivated cell free virus. After 14 days of in vitro sensitisation and expansion with interleukin-2, the mononuclear cells which had been exposed to autologous infected fibroblasts had generated mainly cells of the cytotoxic/suppressor phenotype (CD8) while those stimulated with cell free virus (live or heat inactivated) had generated cells of the helper/inducer phenotype (CD4). Functional assays showed that the effector cells generated after exposure to autologous infected fibroblasts lysed autologous virus infected target cells but not uninfected cells. Effector cells generated in the same way but lacking HLA identity with the virus infected target cells failed to demonstrate cytotoxicity. None of these effector cells showed any significant natural killer cell activity. No specific cytotoxicity was obtained by effector cells generated after exposure to cell-free virus. We conclude that the way in which VZV antigen is presented to the mononuclear cells influences the cell type responding in tissue culture. These findings would be useful in the generation of T cell clones of different cell surface phenotype and function. PMID- 3021617 TI - Volume regulation of natural killer cells under hypotonic stress: comparison with T and B cell subpopulations. AB - In response to hypotonic stress, human T and B cells vary in the ability to adjust their volume. Whereas T cells exhibit a regulatory volume decrease (RVD) under these conditions, B cells do not. We studied the ability of peripheral blood-derived natural killer (NK) cells to regulate their volume in this way. Percoll density gradient purified NK subpopulation showed variable RVD which suggested the presence of mixtures of regulatory and non-regulatory cells within these subgroups. Further purification by flow cytometry into Leu 7+ and Leu 11+ cells showed that these NK cells displayed RVD similar to thymocytes but distinct from B cells and more mature T cells. These data support the hypothesis that NK cells may be derived from T cell precursors which, upon differentiation to NK cells, retain the RVD characteristic of pre-T cells. This finding may also be useful in further characterizing neoplastic clones which display NK-like activity but phenotypic heterogeneity. PMID- 3021618 TI - Functional reconstitution of class II major histocompatibility complex molecules, I-A alpha k and I-A beta k. AB - Genomic DNA fragments carrying the genes, I-A alpha k and I-A beta k, encoding the class II major histocompatibility complex (MHC) molecule I-Ak were inserted into a transducing vector pRSVneo. This vector was introduced into a B lymphoma line expressing the class II molecules (H-2d). The resultant transformants expressed I-Ak molecules on the membrane surface. These transformants were capable of presenting antigen to I-Ak restricted helper T (Th) cells and were susceptible to lysis by class II specific killer T cells. The vector pR-A alpha kA beta k which contains both I-A alpha k and I-A beta k genes will provide a useful tool to analyze function of Ia molecules in cellular interaction, recognition, regulations and stimulation. PMID- 3021619 TI - Lysis by natural killer cells requires viral replication and glycoprotein expression. AB - Several lines of evidence suggest that natural killer cells (NK) have an important role in antiviral defense. To be thus effective, NK cells have to recognize and cause lysis of virus-infected cells. The mechanism underlying this interaction was investigated in a murine system using vesicular stomatitis virus (VSV). A large number of cell lines was screened to identify a permissive target for VSV infection and the B16 murine melanoma cells were chosen since VSV replicates in these cells without producing cytopathic effects 24 h after infection. Spontaneous lysis of B16 melanoma cells by spleen cells occurred only if the target cells were previously infected with VSV. Treatment of spleen cells with anti NK 1.1 or anti Thy 1.2 plus complement decreased the specific lysis by 50%, therefore, the phenotype of the effector cells in this system corresponds to the NK cell subset. Immunofluorescent staining with polyclonal and monoclonal anti-VSV antibodies demonstrated that the viral glycoprotein G is abundantly expressed on the target cell surface. Treatment of the VSV-infected targets with tunicamycin prevented glycosylation of newly synthesized VSV glycoprotein G on the cell membrane. This treatment abrogated completely NK-mediated killing of the infected B16 melanoma cells. These results indicate that virus replication and membrane expression of glycosylated protein G are essential for recognition and lysis of VSV-infected targets by NK cells. PMID- 3021621 TI - Evolving of live modified sheep pox virus strains: evaluation of immunological responses. PMID- 3021620 TI - Evolving of live modified sheep pox viral strains: modification of indigenous viral strains. PMID- 3021622 TI - Viral & Mycoplasma pneumoniae antibodies in chronic pancreatitis of tropics. PMID- 3021623 TI - Metabolic rate at rest & after meals with varying fibre content in obese & nonobese women. PMID- 3021624 TI - Vasodepressor role of endogenous bradykinin assessed by a bradykinin antagonist. AB - This study was designed to examine the contribution of bradykinin to the depressor effect of different antihypertensive drugs in two-kidney renovascular hypertensive rats, using a new specific antagonist of bradykinin. First, the inhibitory capacity of this peptide for exogenously injected bradykinin (75-200 ng) was tested. An inhibition of the vasodepressor action of bradykinin by over 50% was found when the bradykinin inhibitor was infused at a rate of 40 micrograms/min, with little difference at higher rates of infusion. This inhibitor then was infused in three groups of renovascular hypertensive rats after their blood pressure had been decreased by pretreatment with the converting enzyme inhibitor enalapril (MK 421), saralasin, or sodium nitroprusside, respectively. Infusion of the inhibitor produced an immediate 30% increase in blood pressure only in the enalapril-treated group. These results indicate that bradykinin is involved in the decrease of blood pressure produced by converting enzyme inhibition in experimental renovascular hypertension. PMID- 3021625 TI - Mechanisms by which nephrectomy stimulates adrenal renin. AB - Renin has been identified in the adrenal gland by several investigators. Nephrectomy is the most potent stimulator of adrenal renin, and in the present study we investigated the mechanism by which nephrectomy stimulates adrenal renin. The pituitary plays a permissive role since hypophysectomy abolished the response of adrenal renin to nephrectomy (from 117.3 +/- 14.55 to 10.37 +/- 1.63 ng angiotensin I/mg protein/hr) and adrenocorticotropic hormone (ACTH) treatment restored the response to nephrectomy in hypophysectomized rats to 120 +/- 20.62 ng angiotensin I/mg protein/hr. However, large doses of ACTH given to intact rats did not increase adrenal renin to the high level observed after nephrectomy. Potassium also plays an important role, since prevention of hyperkalemia after nephrectomy by treatment with a cation exchange resin, sodium polystyrene sulfonate (Kayexalate), significantly reduced the adrenal renin response to nephrectomy. A third factor involved is the lack of negative feedback by plasma angiotensin II. Infusion of angiotensin II intraperitoneally prevented the rise in adrenal renin after nephrectomy (from 65.25 +/- 7.60 to 9.27 +/- 0.99 ng angiotensin I/mg protein/hr) despite an increase in plasma potassium and corticosterone. In conclusion, three factors influence the response of adrenal renin to nephrectomy: 1) the pituitary through the release of ACTH, 2) a direct stimulation by high plasma potassium levels, 3) the lack of angiotensin II feedback inhibition. Whether the high adrenal renin contributes to the high aldosterone observed in rats after nephrectomy remains to be established. PMID- 3021626 TI - Transformation of Streptococcus mutans with chromosomal and shuttle plasmid (pYA629) DNAs. AB - Transformation (i.e., DNase-sensitive genetic transfer) of strains of Streptococcus mutans representing serotypes c and e was accomplished by using chromosomal DNA from a Rifr Strr Spcr isolate of strain GS5 (UAB525) and a chimeric plasmid, pYA629. Shuttle plasmid pYA629 comprises the S. mutans plasmid pVA318, an inducible erythromycin resistance determinant originally isolated from a group A streptococcal strain, the tetracycline resistance gene and replication region of the Escherichia coli plasmid pBR322, and the promoter region of the S. mutans gene for aspartate beta-semialdehyde dehydrogenase. The strains examined for recipient ability included those known to lack a cryptic plasmid (GS5, UA130, UA159, and MT8148) and those known to contain a widely disseminated 5.8-kilobase cryptic plasmid (LM7, V318, UA101, UA174, and 3098791). The transformation frequencies in GS5 for GS5 chromosomal antibiotic resistance markers were comparable to those reported by others, but UA101, UA130, UA159 and UA174 were transformed with both chromosomal and plasmid markers at much higher efficiencies. In a larger strain survey, strains containing the 5.8-kilobase cryptic plasmid were more frequently transformable with both chromosomal and pYA629 DNAs than were strains lacking this cryptic plasmid. All plasmid containing strains except LM7 lost their resident cryptic plasmids when transformed with pYA629. LM7 transformed with pYA629 retained pLM7. There are therefore at least two incompatibility groups among S. mutans cryptic plasmids. yPA629 DNA isolated from either E. coli or S. mutans transformed S. mutans with equal efficiency. pYA629 DNA isolated from S. mutans transformed both restriction deficient and restriction-proficient E. coli recipients. Therefore, the strains of S. mutans used lack a restriction-modification system for pYA629 DNA sequences. S. mutans strains that are readily transformable, display maximal cariogenicity in gnotobiotic rats, and give high scores for in vitro measures of important virulence attributes have been identified to facilitate studies on the genetic basis and control of virulence. PMID- 3021627 TI - DNA sequence and product analysis of the virF locus responsible for congo red binding and cell invasion in Shigella flexneri 2a. AB - The DNA sequence of virF, a locus associated with virulence and the ability to bind Congo red in Shigella flexneri 2a that is located on a 140-megadalton (230 kilobase) plasmid, was determined and analyzed. It was rich in A and T. The direction of transcription of virF was determined by using a chloramphenicol resistance cartridge. An open reading frame readable in this direction was found. Three proteins, 30, 27, and 21 kilodaltons, all corresponding to those predicted from the above sequence, were produced in minicells containing the virF locus. The three proteins were expressed only weakly in minicells with the 230-kilobase plasmid. PMID- 3021628 TI - Hemolysin production and cloning of two hemolysin determinants from classical Vibrio cholerae. AB - The hemolytic activity of 20 classical and 3 El Tor strains of V. cholerae O1 was examined phenotypically and genetically. The El Tor strains lysed bovine, chicken, human, rabbit, and sheep erythrocytes (RBCs), while the classical strains lysed only chicken and rabbit RBCs. The assay was done with RBCs in Tris NaCl buffer, since phosphate-buffered saline was found to inhibit hemolytic activity. Hemolytic activity in culture supernatants from El Tor strains was more sensitive to heat inactivation than that in supernatants from the classical strain 395. A gene library of strain 395 was examined for hemolytic activity, and two distinct hemolytic clones were identified. One clone appeared identical to the previously cloned hemolysin structural gene from El Tor V. cholerae, while the other did not hybridize to the El Tor hemolysin probe, had a unique restriction enzyme digestion pattern, and encoded a hemolysin whose activity differed from that of the El Tor hemolysin clones. We suggest that the hemolysin specified by the determinant originally cloned from an El Tor vibrio be designated hemolysin I and the second hemolysin, cloned from the classical vibrio, be designated hemolysin II. PMID- 3021630 TI - Characterization of Shigella flexneri sequences encoding congo red binding (crb): conservation of multiple crb sequences and role of IS1 in loss of the Crb+ phenotype. AB - The ability to bind Congo red (Crb+) is associated with virulence of Shigella flexneri and is encoded by a large, 220-kilobase plasmid. We cloned fragments of this plasmid to isolate the sequences encoding Congo red binding, to determine the degree of conservation of these sequences among S. flexneri strains, and to study the molecular basis for loss of the Crb+ phenotype. At least two separate BamHI fragments cloned into plasmid vectors encode Congo red binding in E. coli or S. flexneri. One Crb+ clone, pTKS2, contains a copy of IS1 adjacent to the crb sequences. IS1 appears to be responsible for deletions leading to loss of Congo red binding in this clone. In addition, this clone was found to integrate into the chromosome at relatively high frequency. Integration resulted in loss of the Crb+ phenotype. A second clone, pTKS15, which has only limited homology to pTKS2, also encodes Congo red binding. The Crb+ phenotype of transformants carrying pTKS15 was detected at 37 degrees C but not at 30 degrees C, and thus it resembles Congo red binding in wild-type S. flexneri. HindIII digests of plasmid DNA from 10 different S. flexneri strains were hybridized to both of these Crb+ clones and to an IS1 probe. More than one fragment hybridized to pTKS2 or pTKS15. In general, the sizes of these fragments were the same in S. flexneri strains of different serotypes, indicating conservation of these sequences. Three of five copies of IS1 were also found on the large S. flexneri plasmids. Two of the copies were on fragments of the same size in each strain. Analysis of Crb- derivatives of the 10 strains indicated that, although IS1 may be closely linked to crb sequences on the 220-kilobase plasmid, it is not responsible for the majority of deletions of this plasmid associated with loss of Congo red binding. PMID- 3021629 TI - A low-Ca2+ response operon encodes the V antigen of Yersinia pestis. AB - Yersinia pestis has a virulence regulon called the low-Ca2+ response that is mediated by the plasmid pCD and manifested as regulation of growth and of expression of several virulence-associated properties by Ca2+ and temperature. We used Mu dI(Ap lac) to obtain a mutation in pCD1 of Y. pestis KIM that rendered the bacteria unable to express one of these properties, the V antigen. This mutant also had lost the Ca2+ requirement for growth at 37 degrees C and was avirulent in mice. Two-dimensional protein gel electrophoresis showed that the Mu dI(Ap lac) insertion had eliminated 13,000- and 18,000-molecular-weight proteins in addition to the V antigen. We mapped the Mu dI(Ap lac) insertion within pCD1, cloned the HindIII fragment spanning the insertion location, prepared two subclones of this fragment, and identified the proteins these clones expressed in Escherichia coli minicells. The data indicated that the V gene lies within an operon containing three genes; lcrG (encoding the 13,000-molecular-weight protein), lcrV (encoding the 38,000-molecular-weight V antigen), and lcrH (encoding the 18,000-molecular-weight protein). Therefore, the V operon contains the structural gene for V antigen, at least one virulence gene, and at least one Ca2+-dependence gene. PMID- 3021631 TI - Cloning and expression of the major 47-kilodalton surface immunogen of Treponema pallidum in Escherichia coli. AB - Monoclonal antibodies directed against the 47-kilodalton (kDa) major outer membrane surface immunogen of virulent Treponema pallidum subsp. pallidum were used to select Escherichia coli recombinant clones expressing the 47-kDa immunogen. The phenotype of the clones was dependent on the presence of recombinant plasmid in the host cell. Southern hybridization revealed that the cloned T. pallidum subsp. pallidum DNA sequence was an accurate representation of the T. pallidum subsp. pallidum genomic DNA arrangement. Purified immunoglobulin G from rabbits experimentally infected with T. pallidum subsp. pallidum and human secondary syphilitic sera specifically reacted with the clones, while normal human serum or immunoglobulin G from normal rabbit serum did not. Results of Southern hybridization indicated that a homologous 47-kDa immunogen gene was absent in at least four species of nonpathogenic treponemes tested, as well as from total rabbit genomic DNA. Rabbit anti-T. phagedenis biotype Reiter (treponemal nonpathogen) antiserum and a monoclonal antibody directed against a common treponemal determinant were unreactive with the clones. Western blotting and radioimmunoprecipitation experiments with specific monoclonal antibodies revealed that the recombinant (E. coli) and native (T. pallidum subsp. pallidum) forms of the antigen had identical electrophoretic mobilities. The availability of recombinant 47-kDa immunogen provides a new opportunity for biochemical analysis of the protein, structure-function studies, examination of its role in microbial pathogenesis, and assessment of its diagnostic and vaccinogenic potentials. PMID- 3021632 TI - Cloning and expression of the Bacillus anthracis protective antigen gene in Bacillus subtilis. AB - The gene encoding the protective antigen (PA) moiety of the tripartite exotoxin of Bacillus anthracis was cloned from the recombinant plasmid pSE36 into Bacillus subtilis 1S53 by using the plasmid vector pUB110. Two clones, designated PA1 and PA2, were identified which produced PA in liquid cultures at levels of 20.5 to 41.9 micrograms/ml. This PA was identical to B. anthracis Sterne PA with respect to migration on sodium dodecyl sulfate-polyacrylamide gels and to Western blot antigenic reactivity. Addition of lethal factor or edema factor to PA1 and PA2 supernatants generated biologically active anthrax lethal toxin or edema producing toxin, respectively. The recombinant plasmid in PA1 (pPA101) was 7.8 kilobases, whereas the PA2 strain plasmid (pPA102) was 6.1 kilobases. Both plasmids had deletions extending into the insert sequence but not into the DNA encoding the PA protein. Immunization with the live recombinant strains protected guinea pigs from lethal challenge with virulent B. anthracis spores, and the immunization partially or completely protected rats from intravenous challenge with anthrax lethal toxin. PMID- 3021634 TI - Rearranged HPV 16 molecules in an anal and in a laryngeal carcinoma. AB - By hybridization under stringent conditions, one out of two anal carcinomas and one out of 36 laryngeal carcinomas were shown to harbor HPV 16 DNA in high copy number. Further analysis of both tumor DNAs indicated a rearrangement of the viral DNA in the tumor cells. HPV 16 DNA in the anal carcinoma could chiefly be found episomally in two different forms: a minority as 7.9-kb oligomeric episomes with no apparent modifications; as 10.7-kb rear-ranged oligomeric episomes with a duplication of the part of the viral genome encoding the open reading frames (ORF) E7, E1 and parts of E6 and E2. In the laryngeal carcinoma, integrated and episomal HPV 16 DNA molecules of 7.9 kb were present, together with rearranged molecules of approximately 18 kb with multiple duplications of the ORF E4 and parts of the ORFs E2, E5, L1 and L2. Possible consequences for transcription of the modified viral genomes are discussed. PMID- 3021633 TI - Specific immunological modulation of lymphocyte adenylate cyclase in asthmatic patients after allergenic bronchial provocation. AB - Adenylate cyclase responses to isoproterenol, histamine, 5' guanylylimidodiphosphate (GppNHp) and NaF were measured in lymphocyte membranes of allergic asthmatic patients and healthy control subjects, just before and 24 h after inhalation challenge with house dust mite allergen or histamine. In the nonacute phase before the challenges, the adenylate cyclase responses in the cell membranes of the patients were not significantly different from those in membranes of the control subjects. House dust mite challenge caused a significant decrease of all adenylate cyclase responses in the cell membranes of the patients by about 40-50%. By contrast, histamine provocation of the patients had no effect on these parameters, nor was there any effect of both challenges on the adenylate cyclase activity of the control membranes. The results indicate that allergen induced asthmatic reactions may cause nonspecific refractoriness of lymphocyte adenylate cyclase. Since histamine-induced bronchial obstruction had no effect in the patients, it appears that the adenylate cyclase response is specifically modulated by the allergic process. PMID- 3021635 TI - Lymphoblastoid cell lines and Burkitt-lymphoma-derived cell lines differ in the expression of a second Epstein-Barr virus encoded nuclear antigen. AB - Twenty-seven Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and 27 EBV-carrying Burkitt-lymphoma-derived lines were analyzed for expression of the second EBV-encoded nuclear antigen (EBNA-2) by immunoblotting and anticomplement immunofluorescence with EBNA-2-specific sera. While all lymphoblastoid cell lines expressed EBNA-2, only 10 of the 27 BL lines were EBNA 2-positive. Comparison of the EBNA-2 coding BamHI W-, Y- and H-fragments of EBV DNA in the different cell lines by restriction enzyme analysis suggests that EBNA 2 negativity is due either to sequence diversity or to a deletion in the BamHI WYH region. PMID- 3021636 TI - Rearrangement of c-myc, pim-1 and Mlvi-1 and trisomy of chromosome 15 in MCF- and Moloney-MuLV-induced murine T-cell leukemias. AB - Provirus insertion near the c-myc, pim-1 or Mlvi-1 genes occurred in 7 out of 59 virally induced T-cell leukemias. C-myc was exclusively rearranged in approximately 10% of MCF247-induced tumors while Mlvi-1 was rearranged to a similar frequency in Moloney-virus-induced lymphomas. Out of 25 karyotyped tumors, 9 (36%) showed trisomy of chromosome 15. Provirus insertion near c-myc, pim-1 or Mlvi-1 occurred both in diploid lymphomas and in tumors with trisomy 15. These results suggest that the molecular and cytogenetic changes observed in murine T-cell leukemias are independent tumor-associated events and that trisomy of chromosome 15 is a common tumor-progression-related event. PMID- 3021638 TI - Stress inoculation training in the control of THC toxicities. AB - Fifty-four marijuana-naive cancer patients undergoing chemotherapy and about to use THC (delta-9-tetrahydrocannabinol) as an antiemetic for the first time were randomly assigned to one of two conditions: stress inoculation or placebo/information training. It was hypothesized that subjects exposed to stress inoculation training would experience fewer adverse subjective effects of THC, experience less stressful nausea and vomiting, and be less likely to voluntarily withdraw from subsequent THC trials. None of the hypotheses was confirmed. PMID- 3021637 TI - Interview with Prof. Raphael Mechoulam, codiscoverer of THC.. Interview by Stanley Einstein. PMID- 3021639 TI - Neutron-induced free radicals in oriented DNA. AB - Samples of oriented DNA containing 30 per cent water were irradiated with neutrons at 77 K. The electron spin resonance (e.s.r.) spectra obtained from these irradiated DNA samples show that the formation of radicals is different when the incident neutrons are parallel or perpendicular to the DNA helix. When the incident neutrons are perpendicular to the DNA helix the e.s.r. spectra of thymine and guanine ionic radicals (T-., G+.) are observed. An additional e.s.r. spectrum corresponding to the hydrogen addition radical on thymine (TH.) is observed when the incident neutrons are parallel to DNA helix. The TH. radical appears to be formed by protonation of T-. . PMID- 3021640 TI - EPR study of hydrated testosterone monoclinic and orthorhombic single crystals gamma-irradiated at 295 K. AB - The molecular structure of free radicals formed in gamma-irradiated monoclinic and orthorhombic single crystals of hydrated testosterone were investigated by EPR spectroscopy. Two different types of radical were observed. In the monoclinic form, the radical arises by the loss of a hydrogen atom from the carbon atom C(2), whereas, in the orthorhombic form, it arises by addition of a hydrogen atom to the oxygen atom O(3). The hyperfine spectrum of the radical formed in the monoclinic single crystal originates from the interaction of the unpaired electron with one alpha-proton in position 2 and two non equivalent beta-protons in position 1. In the orthorhombic single crystal, we observed interaction of the unpaired electron, which is delocalized on the carbons C(3), C(4) and C(5) with one alpha-proton in position 4 and with four nonequivalent beta-protons connected with the carbon atoms C(2) and C(6). The hyperfine tensors of the coupling and the g-tensor of the radicals are given. PMID- 3021641 TI - Radiation protection of E. coli strains by cysteamine in the presence of oxygen. AB - The survival of various E. coli K12 strains with defects in the rec system have been measured after gamma-irradiation in air in the presence (0.1 mol dm-3) or in the absence of cysteamine. The results confirm those of Bresler et al. (1978) indicating that the protection by cysteamine in the presence of oxygen is due to an influence on enzymatic repair. The low protection by cysteamine of wild-type cells pretreated with chloramphenicol which prevents protein synthesis, supports the above conclusion. The reason for the absence of a protective effect by OH radical scavenging and H-atom donation is discussed. It is proposed that DNA peroxyl radicals are formed during irradiation in the presence of oxygen and that they are transformed into hydroperoxides by H-atom donation from the intracellular glutathione and the added cysteamine. These hydroperoxides are still dangerous for the cell as indicated by the protective action of glutathione peroxidase observed by Marklund et al. (1984). PMID- 3021642 TI - Generation of radical anions from metronidazole, misonidazole and azathioprine by photoreduction in the presence of EDTA. AB - Ultraviolet irradiation of the nitroimidazole derivatives metronidazole, misonidazole, azathioprine and 1-methyl-4-nitroimidazole in aqueous solution with various reductants produced the respective nitro radical anions, as detected by electron spin resonance spectroscopy. The most effective reductant, yielding high concentrations of the radical anions, was EDTA at pH 10. NADH, NADPH, formaldehyde glutathione and methanol were also tested but were less efficient as reductants. PMID- 3021643 TI - Surgical treatment of malignant tumors of the Oddian region. AB - From 1974 through 1984, 24 patients with malignancies of the Oddian region underwent surgery. Four ampullectomies were carried out with one more than 10 year survivor. Nineteen duodenopancreatectomies were performed with no operative mortality, a 21% complication rate and a 4% reoperation rate; mean survival was 42 months; 1-, 3- and 5-year survival was 95%, 53%, and 37% respectively. Nodal metastasis was the major determinant of long-term survival. Duodenopancreatectomy should be considered the operation of choice for malignancies of the Oddian region. PMID- 3021644 TI - Stress as a principal cause of calcium oxalate urolithiasis. AB - Epidemiological information on calcium oxalate urolithiasis is reviewed and interpreted according to a proposed stress-related mechanism. This mechanism involves hypothalamo-hypophyseal secretion firstly of vasopressin which acts directly to produce hypertonic urine and secondly of adrenocorticotropin which acts via a secondary hyperparathyroid mechanism to raise serum calcium levels. PMID- 3021645 TI - The role of renal prostaglandin E as a possible modulator of cyclic AMP production in nephrotic syndrome. AB - Urinary prostaglandin E (PGE) and cyclic AMP (cAMP) excretions were studied by radioimmunoassay in children with nephrotic syndrome and in a control population. In cases with nephrotic syndrome there was a significant elevation in urinary PGE excretion and cAMP excretion was decreased. A positive correlation was found between urinary cAMP excretion and urinary osmolality (Uosm) and the ratio urine to plasma osmolality (Uosm/Posm); and a negative correlation between urinary cAMP excretion and urine volume. A negative correlation was observed between the values of PGE excretion and urinary cAMP. These data confirmed the role of PGE as a modulator of cAMP production, which was inhibited in the nephrotic syndrome. PMID- 3021647 TI - Deficiency in light-dependent opsin phosphorylation in Irish setters with rod cone dysplasia. AB - A deficiency in light-dependent opsin phosphorylation and a slight reduction in opsin synthesis were observed during photoreceptor cell development (22-26 days) preceding photoreceptor cell loss in Irish setters with rod-cone dysplasia. In addition to opsin, two other phosphoprotein bands were found associated with the photoreceptor cell layer; synthesis and phosphorylation of one of these (band 3; 44-48 Kd) appeared reduced, while synthesis and phosphorylation of the other (band 1; 29-31 Kd) was within the normal range in 25-day-old affected setters. The deficiency in light-dependent opsin phosphorylation in affected setters was not due to a deficiency in opsin kinase, since soluble proteins from affected or normal outer segments catalyzed equally well opsin phosphorylation in partially kinase-depleted outer segment membranes from normal, while both kinase preparations failed to promote light-dependent opsin phosphorylation in those from affected setters. A deficiency in light-dependent opsin phosphorylation was also observed in rd/rd mice at all ages studied. In contrast, in Royal College of Surgeons (RCS) rats, light-dependent opsin phosphorylation was within the normal range prior to photoreceptor loss, and became nondetectable only after 50% or more of the photoreceptors had degenerated. PMID- 3021646 TI - Transfer of secretory proteins through the membrane of the endoplasmic reticulum. PMID- 3021648 TI - Pigment granule migration in isolated cells of the teleost retinal pigment epithelium. AB - In the teleost eye, the melanin granules of the retinal pigment epithelium (RPE) move in response to changes in light conditions. In the dark, pigment granules aggregate toward the cell base, and in the light, they disperse into long apical projections. Isolated RPE cells from the green sunfish (Lepomis cyanellus) were used to investigate the mechanism and regulation of pigment movement. Changing light conditions did not elicit pigment migration in isolated cells. However, pigment aggregation was induced by 3',5' cyclic-adenosine monophosphate (cAMP), dibutyryl cAMP (dbcAMP), and forskolin (an adenylate cyclase activator). The effectiveness of forskolin suggests that an endogenous adenylate cyclase participates in regulating aggregation. Pigment dispersal was induced by the catecholamines epinephrine, phenylephrine, clonidine, dopamine, and apomorphine. Together the authors' studies suggest: that RPE cells contain the necessary motile machinery to support pigment granule transport in the absence of retina, but not the ability to respond to light; that elevating cAMP induces pigment aggregation; and that catecholamines induce dispersion by binding to receptors on the RPE cell. The authors' observations are consistent with previous suggestions that light regulation of RPE pigment migration is mediated by the retina. PMID- 3021649 TI - Location of parathyroid adenomas by thallium-201 and technetium-99m subtraction scanning. PMID- 3021650 TI - Modulation of pulmonary macrophage superoxide release and tumoricidal activity following activation by biological response modifiers. AB - Following immunologic activation, pulmonary macrophages may prevent or cause regression of lung metastases by mechanisms which remain largely unknown. The studies described here were designed to determine if enhanced oxygen metabolite release was related to postactivation tumoricidal activity. We have shown that in vitro activation of Fischer 344 rat pulmonary macrophages by either free or liposome-encapsulated muramyl dipeptide leads to both enhanced release of superoxide anions and marked tumoricidal activity against syngenic (Fischer 13762), allogeneic (Schmidt-Ruppin RR 1022) and xenogeneic (Fibrosarcoma MCA-F) 125I-deoxyuridine-labeled target cells. This immune modulator did not, however, metabolically activate pulmonary macrophages as effectively as liposome encapsulated lipopolysaccharide. A 24-h in vitro incubation with either 150 U or 300 U of interferon-gamma (3 X 10(6) U/mg) or 30 U, 150 U or 300 U of interferon alpha (6 X 10(5) U/mg) caused a significant elevation in superoxide release above controls, whereas short-term exposure (2 or 4 h) had little or no effect. Free or encapsulated 6-O-stearoyl muramyl dipeptide, on the other hand, did increase superoxide levels at all 3 time periods. When either interferon-gamma or free or encapsulated muramyl dipeptide derivative were administered to intact rats by either i.v. injection, intratracheal instillation or osmotic minipump infusion, pulmonary macrophage tumoricidal activity was observed 96 h after cell harvesting. Zymosan-stimulated superoxide release, however, was not consistently elevated above control or empty liposome treatment following this course of in vivo activation. The data collectively suggest that in vivo pulmonary macrophage activation to a tumoricidal state and metabolic activation resulting in enhanced superoxide may be separable events. PMID- 3021652 TI - Relative effectiveness of alpha emitters for bone sarcoma induction. PMID- 3021651 TI - Characterization of platelet activating factor (PAF)-acether-induced contractions of guinea-pig lung strips by selected inhibitors of arachidonic acid metabolism and by PAF-acether antagonists. AB - The myotropic activities of PAF-acether, leukotriene B4, leukotriene D4 and histamine were compared on superfused guinea-pig lung parenchymal strip and were shown to have the following order of potency: PAF-acether greater than LTD4 greater than LTB4 greater than histamine. The contractile response of the lung parenchyma to PAF-acether was inhibited by aspirin, imidazole and OKY-046, which suggested that thromboxane A2 might play a mediator role in PAF-induced contractions. Neither an antagonist of leukotriene D4, FPL-55712, nor an antihistamine, mepyramine, had any effect on PAF contractions. The activity of a novel antagonist of PAF-acether, BN 52021, was also studied on superfused lung parenchyma contracted by histamine, leukotriene B4, leukotriene D4 and PAF acether. This compound was without effect on the histamine response but it slightly reduced the contractions elicited by leukotriene D4 and potentiated those by leukotriene B4. BN 52021 (7.1 X 10(-6) M) inhibited by 63% the contraction induced by 5.7 X 10(-13) M PAF-acether and by 52% that induced by 5.7 X 10(-10) M PAF-acether and kadsurenone (8.4 X 10(-6) M), another PAF-acether antagonist, inhibited the same PAF-induced contractions by 75% and 20% respectively. PMID- 3021653 TI - Test of a versatile on-line distortion corrector for a gamma-camera. AB - A gamma-camera on-line distortion corrector, based on a fast microprocessor, has been tested on two cameras. The original electronic design and the software algorithm are described. Performance measurements on phantoms and clinical results show an improvement of the pictures. PMID- 3021655 TI - Syntheses of selenium labelled compounds--II. The exchange reaction with elemental selenium. AB - 73Se labelled 2-selenouracil was prepared by the isotope exchange reaction method. It has been well known that in order to prepare labelled compounds in high specific activity by exchange reaction, the substrate compounds have to be used in small quantities. For 2-selenouracil, however, the reaction in low concentration is not always preferable for the labelling of high specific activity within a limited time. The optimum conditions for the preparation of [2 73Se]-2-selenouracil of maximum specific activity were determined. PMID- 3021654 TI - Preparation of no carrier added fluorine-18 labeled 16-fluorohexadecanoic and fluoroacetic acids from highly reactive tetraethylammonium [18F]fluoride. AB - No carrier added tetraethylammonium [18F]fluoride prepared via synthesis and hydrolysis of fluorotrimethylsilane was reacted with methyl 16-iodohexadecanoate in acetonitrile. At 80 degrees C, yields of up to 80% were found with reaction times of 20-30 min. As would be expected for fluoride-for-iodide exchange, reactions were slower than previously-reported cyclic sulfate and triflate displacements. TLC and reverse-phase HPLC indicated that a single pure radioactive product was formed, which was hydrolyzed with alkali to the free acid. NMR spectroscopy of a carrier added preparation was consistent with assignment as 16-fluorohexadecanoic acid and no rearrangement to the 15-fluoro compound. [18F]Fluoroacetic acid was prepared analogously in high yield from ethyl iodoacetate. The results are discussed in terms of guidelines for syntheses of other 18F compounds and of the properties of near anhydrous tetraethylammonium fluoride in acetonitrile. PMID- 3021656 TI - HPLC separated fractions of 99mTc(NaBH4)-HEDP as myocardial infarct imaging agents. AB - Component fractions of 99mTc(NaBH4)-HEDP mixtures, isolated by anion exchange high performance liquid chromatography (HPLC), have been evaluated as myocardial infarct imaging agents in two animal models. Results from both the isoproterenol induced myocardial infarction model, and the heat-induced myocardial necrosis model, show that the several HPLC isolated components exhibit significantly different abnormal/normal heart uptake ratios. In addition, the HPLC isolated component of shortest chromatographic retention time exhibits a higher abnormal/normal heart uptake ratio than does 99mTc(Sn)-PyP, the current agent of choice for clinical myocardial infarct imaging. PMID- 3021657 TI - The effect of formulation conditions on the distribution of 99mTc(NaBH4)-HEDP components separated by anion exchange HPLC. AB - Reduction of 99mTcO4- by NaBH4 in the presence of HEDP leads to a mixture of 99mTc-containing complexes which can be separated by anion exchange high performance liquid chromatography (HPLC). The distribution of complexes within this mixture can be varied by controlling pH, the concentration of HEDP, the concentration of technetium, and the presence of air. Suitable control of these formulation conditions can yield mixtures which consist of essentially (greater than 85%) one component. Arguments are presented to support the view that the components of 99mTc(NaBH4)-HEDP and 99mTc(NaBH4)-MDP mixtures are in fact oligomeric or polymeric complexes that can contain technetium centers in at least two different oxidation states. PMID- 3021659 TI - Stable labelling of serum albumin microspheres with gallium-68. AB - A highly efficient and rapid technique for labelling serum albumin microspheres with 68Ga is described. Measurements of the in vivo stability of the radiopharmaceutical in the rabbit and the baboon show that less than 0.2% of the injected activity is eluted from the microspheres in 2 h. PMID- 3021658 TI - Simple quantitative radioiodination of (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) by exchange labelling. AB - The synthesis of [125I]-(E)-5-(2-iodovinyl)-2'-deoxyuridine [( 125I]IVDU) by a halogen isotope exchange reaction has been monitored in various reaction conditions. Quantitative incorporation of radioiodine into IVDU was obtained within 20 min by a simple and safe method that is suitable for application in any nuclear medicine laboratory. The radiolabelled antiviral agent can be used without further purification. PMID- 3021660 TI - In the procurement of stable 99mTc labeled protein using bifunctional chelating agent. AB - Recently, technetium-99m (99mTc) labeling of proteins through the use of a bifunctional chelating agent (BCA) has been reported. In previous work, a BCA containing a di(N-methylthiosemicarbazone) (DTS) moiety for 99mTc has offered good potential; however, conjugation with bulky proteins has induced some steric interference in the 99mTc labeling step using the stannous chloride method. In order to minimize this effect, changes in the hydrocarbon chain length (spacer) between the DTS and the protein binding site (carboxyl group), as well as the chemical state of the reducing agent, stannous chloride, were considered of interest. DTS molecules with variable spacer length (n = 0, 2, 4) were synthesized, coupled with human serum albumin and labeled with 99mTc. Also, stannous chloride prepared in different media was evaluated. Validity of the present approach is tested and discussed. PMID- 3021661 TI - Reversed-phase HPLC of 99mTc methylene diphosphonate bone imaging kits with quantification of pertechnetate. AB - Ion-pairing reversed-phase HPLC is used to separate a mixture of 99mTc methylene diphosphonate (MDP) complexes prepared by tin reduction of pertechnetate in the presence of ligand. Chromatographic conditions allow for the quantification of total pertechnetate concentration, as well as the determination of chemical purity of generator eluents from which 99mTc is derived. Both determinations can be made prior to actual labeling. Commercially available MDP kits from three manufacturers are evaluated. All three MDP kits, when labeled with 99mTc, produce multiple, chromatographically separable components. The formation of these Tc-MDP complexes is time dependent. The labeling procedure produces several products within the first 20 min, with up to fifteen complexes observed after 4 h. The results of this work demonstrate the presence of substantial chemical contamination in generator eluents. PMID- 3021662 TI - Synthesis and biodistribution of 18F-labeled fluoronitroimidazoles: potential in vivo markers of hypoxic tissue. AB - Three 18F labeled fluoronitroimidazoles have been prepared as potential in vivo markers of hypoxic cells in tumors, and ischemic areas of the heart and brain. 1 (2-Nitroimidazolyl)-3-[18F]fluoro-2-hydroxypropanol (18F]fluoro normethoxymisonidazole) 4, 1-(2-[18F]fluoroethyl)-2-nitroimidazole 7, and 1-(2 [18F]-fluoroethyl)-2-methyl-5-nitroimidazole ([18F]fluoro norhydroxymetronidazole) 10 were prepared in average radiochemical yields of less than 1%, 23% and 15-43% (8% at the no carrier-added level) respectively at end-of synthesis. The in vivo biodistribution in rats was determined for each of the 18F labeled fluoronitroimidazoles. At 1 and 3 h after administration, the tissue distribution of each of the 18F labeled nitroimidzaoles was quite uniform and consistent with that of nitroimidazoles previously studied. These results suggest the need for a suitable animal model to evaluate their potential as in vivo markers of hypoxic tissue in the brain. PMID- 3021664 TI - Essential elements in fresh and irradiated strawberries and strawberry marmalade. PMID- 3021665 TI - Radiopharmaceuticals labelled with halogen isotopes. PMID- 3021663 TI - 2-[18F]fluoroputrescine: preparation, biodistribution, and mechanism of defluorination. AB - A new radiolabeled analog of putrescine, 18F labeled 2-fluoroputrescine, has been prepared as a potential in vivo imaging agent for prostate and prostate derived tumors. The radiosynthesis yielded 1.5-4.5 mCi amounts of the 18F labeled putrescine analog (1-3% yields at end-of-synthesis), with specific activities ranging from 0.85-4.3 Ci/mmol, in a synthesis time of 2 h. The in vivo biodistribution in mature male rats showed considerable uptake by the bone, indicating defluorination of the compound. In animals pretreated with aminoguanidine, an inhibitor of diamine oxidase, the enzyme important in the metabolism of putrescine, defluorination was markedly reduced, but uptake by the prostate did not improve. These results suggest the need for development of a 18F labeled analog of putrescine which does not defluorinate in vivo, and which has biological properties similar to putrescine. PMID- 3021666 TI - Production of reactive fluorine-18. AB - Production conditions are reviewed from the perspective of maximizing activity- both total and specific--of 18F as electrophilic or nucleophilic precursors, with minimal levels of interfering species. Careful target design can avoid many pitfalls, and telemetry of a few key variables can guide the way to reproducible yields, consistent with prediction. Whether this 18F is chemically reactive or refractory is determined by the care taken at each step, and dictates the final success of the labeling effort. PMID- 3021667 TI - 2-Deoxy-2-[18F]fluoro-D-glucose for metabolic studies: current status. PMID- 3021669 TI - Chemistry of fluorine-18 radiopharmaceuticals. PMID- 3021668 TI - [18F]fluoro-L-dopa for the in vivo study of intracerebral dopamine. AB - 6-[18F]Fluoro-L-dopa was designed to trace the dopamine metabolism in the brain with positron tomography. 6-[18F]Fluoro-L-dopa resembles natural L-dopa biochemically, it crosses the blood-brain barrier with the similar kinetics, it is decarboxylated by dopa decarboxylase and is stored intraneuronally in vesicles. In addition the rate of O-methylation of 6-[18F]fluoro-L-dopa by catechol-O-methyl transferase is only 1/4 of that of natural L-dopa. The low rate of O-methylation, especially in the periphery, is particularly beneficial for PT investigations of the brain. The radiotracer has been synthesized using a variety of electrophilic fluorinating agents. PMID- 3021671 TI - Radiopharmaceuticals labelled with bromine isotopes. PMID- 3021670 TI - Synthesis and purification of 2-deoxy-2-[18F]fluoro-D-glucose and 2-deoxy-2 [18F]fluoro-D-mannose: characterization of products by 1H- and 19F-NMR spectroscopy. AB - A procedure has been developed that allows the separation of 2-deoxy-2 [18F]fluoro-D-glucose from 2-deoxy-2-[18F]fluoro-D-mannose employing selectively optimized ion-moderated partition chromatography. Both compounds can be obtained with a greater than 98% chemical and radiochemical purity in about one half-life of 18F. Both the alpha- and beta-anomers of both sugars were completely characterized by high-resolution 1H- and 19F-NMR spectroscopy. Various convenient preparation methods for 2-deoxy-2-[18F]fluoro-D-glucose were compared. PMID- 3021672 TI - The preparation and use of [77Br]p-bromospiperone for clinical dopamine D2 receptor measurements. PMID- 3021673 TI - Radiobrominated spiroperidol for the study of dopamine D2 receptors. AB - Some neuropsychiatric disorders have been suggested to be related to the CNS dopaminergic system. In order to probe this neurotransmitter system, radiobrominated spiroperidol, a potent dopamine (DA) D2-receptor antagonist, was developed. This review deals with the routine synthesis of [75, 76, or 77Br]-p bromospiroperidol (BrSP), its validation as a radiopharmaceutical directed to DA D2 receptors, and the imaging studies which have been done using this radioligand with either single photon tomography (SPECT) or positron tomography (PET). [*Br]BrSP will be compared to other D2 ligands currently available. The present state and future direction of DA receptor research, in particular, the quantitative aspects, will be discussed. PMID- 3021674 TI - Radioiodinated monoclonal antibodies: a critical review. PMID- 3021675 TI - Iodine-labelled monoclonal antibodies for cell labelling: principles and prospects. PMID- 3021676 TI - Iodine-131-labeled diphosphonates for the palliative treatment of bone metastases -III. Considerations of interaction, binding and absorbed dose. AB - General considerations about the possible mechanisms of action of rather low dose ionizing radiation with bone metastases and stimulated nerve fibers reveal that only minute amounts of chemicals are produced by direct interaction of energetic electrons. Thus changes of the chemical milieu due to direct interaction must be ruled out in favour of a radiation-induced trigger reaction which may then initiate a cascade of cellular responses. Organ distribution studies of a series of radioiodinated benzylidenediphosphonates with H-, HO- and H2N- in the alpha- and p-position revealed best results for pHO-NH2 (BDP3). The microscopic distribution of 131I-DBP3 in bone tissue was monitored by autoradiography. Elevated uptake in normal (tibia) and neoplastic bone (experimental osteosarcoma) corresponded with the degree of vascularization and formation of new hydroxylapatite. Unlike the uptake in human osteoblastic bone metastases the experimental osteosarcoma of SD-rats accumulated 131I-BDP3 less than normal bone. This was due to the short volume doubling time, the delay of hydroxyl-apatite deposition and the formation of necroses. Theoretical replacement of 131I in iodinated BDP3 with radioisotopes emitting higher energy electrons yielded best bone metastasis/organ ratios for 32P labeled BDP3. The bone metastasis/bone marrow dose ratio by comparison with 131I labeled BDP3 is, however, almost equal. The isotopes 130I and 133I are not suited to the achievement of higher tumor/background doses although they are higher energy beta- -emitters than 131I. Because of their short physical half life and absence of different kinetics in normal and neoplastic bone no dose enhancement in bone metastases can be attained. PMID- 3021677 TI - [123I]iodoamphetamine SPECT imaging. AB - SPECT imaging of [123I]IMP is reviewed. Methods for radiopharmaceutical production are discussed with an emphasis on labeling small quantities of IMP. Limited angle tomography and full angle SPECT with standard cameras and special imaging systems are reviewed. Selection of collimator and methods of reconstruction are discussed. Clinical studies are described with emphasis on stroke, epilepsy and dementia. The efforts to perform quantitative imaging of rCBF with [123I]IMP are reviewed. PMID- 3021678 TI - Radioiodinated iodobenzylguanidines for diagnosis and therapy. AB - Radiolabelled meta-iodobenzylguanidine has been one of the most successful of recent radiopharmaceuticals. In the short period of five years it has been shown to have high diagnostic sensitivity and specificity for many tumours of neuroectodermal origin. Furthermore its excellent concentration in many malignant tumours of this type offers a novel alternative treatment to those available at present. PMID- 3021680 TI - Astatine-211: its possible applications in cancer therapy. AB - The cyclotron-produced radiohalogen, 211At, is eminently suitable as a possible therapeutic radionuclide. It decays by the emission of 6.8 MeV mean energy alpha particles, which from a radiobiological viewpoint are of near optimal therapeutic LET. This paper reviews developments in the possible application of [211At]astato labelled molecules as potential anti-tumour agents. Additionally, radio dosimetric evidence is presented, and its implications for human cancer therapy are discussed. PMID- 3021679 TI - Radioiodinated fatty acids for cardiological diagnosis. AB - The development of fatty acids labelled with iodine-123 is reviewed. The variety of methods for producing 123I and introducing radioiodine into the molecule is discussed and the important points of the biochemical background are recalled with the aim of finding a broad application for 123I-labelled fatty acids. The results of the pharmacokinetic studies and biochemical analyses are presented as they prove that both 17-123I-heptadecanoic acid (IHA) and 15-(p-123I phenyl)pentadecanoic acid (IPPA) exhibit analogous behaviour to that of the naturally occurring fatty acids. Clinical applications demonstrated two fields of importance: applications solely for imaging the heart and assessment of myocardial turnover rates of fatty acids for functional diagnosis. Moreover, very recent studies show that the provision of information about prognosis of myocardial diseases and the applied cardiological therapy appear to be possible. PMID- 3021681 TI - Organoastatine chemistry. Astatination via electrophilic destannylation. AB - Substantial interest is currently focused on the alpha-emitting radiohalogen 211At, principally because of its potential use in the radiation therapy of cancer. Knowledge of organoastatine chemistry is incomplete and existing methods for its incorporation restrict the range of compounds which may be labeled. Aryl and vinyl Sn(IV) compounds are notably susceptible to substitution of tin by a variety of electrophiles. We have investigated the reaction of aryltrialkylstannanes with astatine and report here the first examples of astatodestannylation. Formation of aryl astatides proceeds rapidly, cleanly and under mild conditions. The data further elucidate aspects of astatine reactivity and suggest a general route to synthesize astatinated compounds. PMID- 3021682 TI - Proceedings of the International Symposium on Radiohalogens. Banff, Alberta, Canada, 10-11 September 1985. PMID- 3021683 TI - 18F-labelled N,N-dimethylamphetamine analogues for brain imaging studies. AB - The radiochemical yields of nine N,N-dimethyl-2-(substituted phenyl) isopropylamines (amphetamine analogues) were determined following reaction with [18F]acetyl hypofluorite in a 0.1 M HCl solution at room temperature. The meta dimethoxy substituted amphetamines gave the highest radiofluorination yields (24 32% at EOB). Purification of the 18F-labelled amphetamines was achieved in 10-20 min. 5-18F-2,4-Dimethoxy-N,N-dimethylamphetamine (5-18F-2,4-DNNA) was utilized to determine brain and lung uptake in rates. Positron emission tomography studies were conducted in a dog to determine the dynamic brain uptake and retention of this agent. The 5-18F-2,4-DNNA exhibited decreased initial uptake and more rapid loss of radioactivity in cerebral tissue compared to the iodinated homologue. PMID- 3021684 TI - Synthesis of carbon-11 labelled glycine and the dipeptides L-phenylalanylglycine and L-leucylglycine. AB - Carbon-11 labelled glycine has been prepared in 30 min by carboxylation of alpha lithiomethylisocyanide with a radiochemical yield of 10-15%. After coupling with L-phenylalanine-N-carboxyanhydride and L-leucine-N-carboxyanhydride followed by HPLC purification, the corresponding dipeptides were obtained in 20 min with a radiochemical yield of 30-40%. Consequently, starting with 11CO2, non carrier added L-phenylalanyl[1-(11)C]glycine and L-leucyl-[1-(11)C]glycine in 0.1 N NaH2PO4 were obtained in 50 min with a radiochemical yield of 3-6%. The radiochemical yield figures are not corrected for decay. PMID- 3021685 TI - A carbon particulate, packed bed electrolysis flow cell for the production of technetium complexes. AB - A carbon particulate packed bulk electrolysis flow cell is described and detailed construction plans are provided. The rapid generation of technetium formate complexes from the electrolysis of a pertechnetate/formate buffer solution demonstrates the electrolytic efficiency of the flow cell. PMID- 3021687 TI - In vivo study of receptors for neuromediators with PET. PMID- 3021686 TI - Radionuclide determination of absolute LV volumes: interstudy, interobserver and intraobserver variances. AB - The results of 22 absolute left ventricular volume (LVV) determinations by a radionuclide (RN) method are compared to the results obtained by contrast ventriculography (CV). Another 10 patients were analysed in order to evaluate the interstudy, interobserver and intraobserver variances. Good correlation was shown between the RN and CV measurements of the end diastolic volume (EDV), end systolic volume (ESV), stroke volume (SV) and ejection fraction (EF), but the RN method overestimates the EDV and ESV. The EF was underestimated, but no difference could be shown for the SV. On the inter- and intraobserver levels, regression analysis yielded excellent correlation (r greater than 0.99 in all cases) with no statistically significant difference (P less than 0.05). The interstudy variance was minimal as indicated by regression analysis (r greater than 0.87) and no statistically significant difference (P less than 0.05) could be shown between studies. The results indicate that the RN method of LVV determination can be used in intervention studies over a limited period. PMID- 3021688 TI - Receptors labeled with gamma-emitting radiotracers. PMID- 3021689 TI - Properties of multilamellar liposomes containing 99mTcO4-: effect of distearoylphosphatidylcholine to sphingomyelin ratio. AB - Multilamellar liposomes composed of eight different mixtures of distearoylphosphatidylcholine (DSPC) and sphingomyelin (SM) were prepared containing [99mTc]pertechnetate. The in vitro permeability of the liposome preparations in buffer and serum were measured by both dialysis and direct exposure. Liposomes composed of 25-33% SM were least permeable exhibiting leakage half-times of approximately 70 h in buffer and 52 h in serum. Pertechnetate containing liposomes of three different lipid compositions were injected into mice and the biodistribution of 99mTc activity was determined. All preparations were taken up primarily by the lungs and liver. The half-time for clearance of 99mTc activity ranged from 24.8 to 30.6 h for the liver and 25.3 to 33.0 h for the lungs. PMID- 3021690 TI - Quantitative determination of arachidonate 5-lipoxygenase metabolites in human polymorphonuclear leukocytes by reverse-phase high-performance liquid chromatography. PMID- 3021691 TI - Dietary sources of energy, protein, fat and fibre in 375 English adolescents. AB - The aim of this report of the dietary sources of energy, protein, fat and fibre in 375 English adolescents aged 11-14 years is to contribute to the development of health education. Each child recorded their dietary intake five times over a 2 year period using a 3-day diary with interview. Food tables (Paul & Southgate, 1978) were used to calculate nutrient intake. The results are the mean of all 15 days intake. Potatoes (chips and crisps) were the largest single source of energy but 'meat' was the main source of protein and fat, other important sources of fat were the spreading fats, milk, chips and crisps. Chips were the main source of fibre but white bread, crisps and baked beans also contributed similar proportions of fibre. Some differences between the sexes and social classes were observed; the lower social class children appeared to have more undesirable eating habits. PMID- 3021692 TI - Sequential hemibody irradiation integrated into a chemotherapy-local radiotherapy program for limited disease small cell lung cancer. AB - Twenty-four patients with limited disease small cell lung cancer (SCLC) were treated with sequential hemibody irradiation (SHB) integrated into a conventional chemotherapy-local radiotherapy (LRT) program. Among 23 evaluable patients, 12 (52%) attained a complete response (CR) and 8 (35%) attained a partial response for an overall major response rate of 87%. The median time since study entry is 29 months. Durations of response are 9.9 months for all patients and 16.5 months for patients who achieved a CR. The primary site was the predominant area of recurrence. The median survival is 13.2 months for all patients and 23.2 months for the 12 patients who attained a CR. Myelosuppression, especially thrombocytopenia, was the major toxicity. Acute radiation toxicities and subacute pneumonitis previously associated with hemibody radiotherapy were well controlled or prevented using the current dose, premedication, and shielding techniques. This integrated program of systemic therapies with SHB and combination chemotherapy plus LRT is feasible for limited disease SCLC; it may prolong survival in patients who attain a CR but compared to similar programs without hemibody irradiation, there was no improvement in overall response rate, response duration, or survival. PMID- 3021693 TI - Influence of WR 2721 on radiation response of canine soft tissue sarcomas. AB - Seventy-three dogs with soft tissue sarcomas were randomized to 2 dose response assays to receive irradiation alone or with the radioprotector WR-2721. Nausea and vomiting were the major side effects of WR-2721 administration although one death occurred because of cardiac and respiratory failure which may have been caused by the WR-2721. There was no change in blood counts or serum chemistry values. The TCD50/lyr was 52 Gy delivered in 10 fractions for both groups. However, there was an indication of tumor protection at lower doses because at all comparable dose levels the percentage tumor control was lower in dogs given WR-2721. There was no decrease in acute skin reactions of dogs treated with WR 2721 and little, if any, protection against fibrosis, soft tissue necrosis or bone necrosis. The lack of protection of normal tissues may have been caused by fractionation. The agent may be more useful combined with large single doses such as given in intraoperative radiotherapy. PMID- 3021694 TI - Computed tomographic assessment of radiation induced damage in the lung of normal and WR 2721 protected LAF1 mice. AB - LAF1 mice were irradiated with single, graded doses of X rays to the thorax in the range of 0 to 14 Gy unprotected, or 0 to 18 Gy after injecting the radioprotective aminothiol compound, WR 2721. Computed tomographic (CT) scanning of the thorax was performed at intervals for a period of 42 weeks after irradiation. The gravimetric density was determined for both left and right lungs by averaging the CT numerical data within lung slices traced on a magnified video image of the thorax. Significant elevations in CT density occurred at post irradiation times corresponding to pneumonitis and late phase, as evidenced by the pneumopathic decline in survival. The threshold dose yielding a significant increase in CT density in the pneumonitis phase was 11 Gy, a dose at which only 3% of the animals died. A single peak of increased CT density was observed for the pneumonitis phase for unprotected animals, whereas a transient return of CT density toward control values at 21-22 weeks produced two peaks from the WR 2721 treated group. The CT density of lung increased in a stepwise manner in the dose range of 11-14 Gy. For the isoeffect dose that produced equal animal survival (14 Gy and 18 Gy + WR 2721), the lung density increased by approximately 27% over control values for both treatments, suggesting that CT density is related to survival. Periodic computed tomographic analysis of the lungs of patients sustaining radiotherapy to large pulmonary fields may be of value in assessing the degree and progression of pulmonary complications. PMID- 3021695 TI - CNS relapse despite prophylactic cranial irradiation (PCI) in small cell lung cancer (SCLC). AB - CNS relapse after PCI may reflect either suboptimal radiation dose schedules or reseeding from other sites of active disease. A retrospective study has been undertaken to investigate these alternative mechanisms of treatment failure. Between August 1981 and December 1983, 30 patients with SCLC treated by induction chemotherapy, followed by high-dose cyclophosphamide (7 Gm/m2), were selected for PCI on the basis of clinical, radiological, and bronchoscopic CR. Pretreatment CT brain scans were normal in all patients, and 20 Gy mid-plane dose in 5 daily fractions were delivered by lateral fields to whole brain using megavoltage X rays and localizing check films. Progressive focal neurological signs of cranial metastases developed in 7/30 (23%) patients 3-11 months after PCI, confirmed on CT scanning in 4 patients. Relapse at other sites, predominantly the thorax, occurred in all seven patients at intervals of 1-6 months prior to CNS relapse. Published clinical data of tumor volume doubling times in SCLC predict longer CNS relapse-free intervals after PCI than those observed in our patients if reseeding was responsible for relapse. This suggests that incomplete eradication of intracranial disease is the main cause of CNS relapse after PCI. Higher equivalent radiation doses may improve the results of PCI. PMID- 3021697 TI - Insulinoma in a ferret. AB - Insulinoma was diagnosed in a 7-year-old female ferret examined because of generalized seizures, intermittent paraplegia, and abnormal behavior. Low serum glucose, high serum insulin, and infinite amended insulin/glucose ratio values in this ferret supported the clinical diagnosis of insulinoma. Histologic examination of the pancreas confirmed the diagnosis of insulinoma. The clinical signs and laboratory evaluations in this case and in a previously reported case of insulinoma in a ferret were consistent with variations reported in dogs with insulinoma. PMID- 3021696 TI - Transmissibility and abortogenic effect of equine viral arteritis in mares. AB - A group of 14 pregnant mares was exposed via contact to 4 mares bred to stallions infected with equine viral arteritis virus. There was a demonstrable febrile response in each donor mare and in 12 of the pregnant mares. All 18 mares became seropositive after exposure. Equine viral arteritis virus was isolated from the nasopharynx of 5 pregnant mares, but not from the donor mares. Ten of the pregnant mares aborted, and virus was isolated from fetal specimens or placenta of 8. PMID- 3021698 TI - The frequency of papillomavirus infection in cervical precancerous lesions in Japan: an immunoperoxidase study. AB - In order to establish the frequency of human papillomavirus (HPV) infection in cervical precancerous lesions of Japanese patients, cervical materials routinely biopsied in the past year were examined immunohistochemically for the papillomavirus genus-specific antigen. Of a total of 832 cervical biopsy specimens, 46 (5.5%) were immunohistochemically positive for HPV. In this study, 206 patients were diagnosed as having dysplasia or carcinoma in situ (CIS), and HPV antigen was found in 15% of these patients. It was found in 13% of patients with mild dysplasia, 28% of those with moderate dysplasia, 17% of those with severe dysplasia and 4.5% of those with CIS. However, HPV antigen was detected only in the epithelium with dysplastic change, not in cancerous areas. PMID- 3021699 TI - Colon carcinoma K-ras 2 oncogene of a familial polyposis coli patient. AB - The DNA of a colon carcinoma-derived cell line (KMS-4) and that of skin fibroblasts from a familial polyposis coli patient were transfected into NIH3T3 cells in order to detect oncogenes associated with the disease. No transformation was observed with the normal skin fibroblast DNA, while the KMS-4 cell DNA was able to transform NIH3T3 cells. Through hybridization with known oncogene probes, the KMS-4 transforming gene was found to be a human activated c-K-ras 2 oncogene. Sequence analysis of the molecularly cloned KMS-4 c-K-ras 2 oncogene showed a single nucleotide transition from G to T at the 12th codon. This results in substitution of cysteine for glycine at this position. On using labeled synthetic oligonucleotides to detect the mutation in codon 12, we found the G to T transition in colon carcinoma cells. This suggests that activation of the c-K-ras 2 oncogene could be associated with colon carcinoma induction. PMID- 3021700 TI - Quantitative distributions of aspartate aminotransferase and glutaminase activities in the rat cochlea. AB - The intra-cochlear distributions of aspartate aminotransferase and glutaminase, prominent enzymes of aspartate and glutamate metabolism, have been studied by quantitative microchemical techniques. Also measured was choline acetyltransferase, the enzyme synthesizing acetylcholine, and a marker for the olivocochlear bundle. Aspartate aminotransferase activity was highest in the stria vascularis, about half this high in the organ of Corti synaptic (hair cell) zones, somewhat lower in the organ of Corti non-synaptic (Hensen's cell) zones, lower yet in Reissner's and lowest in the tectorial membrane. Glutaminase, on the other hand, had its highest activity in synaptic zones, about a third of that activity in the organ of Corti non-synaptic zones, and a barely detectable activity in Reissner's and tectorial membranes, and stria vascularis. Seven days after transection of the olivocochlear bundle, no significant difference was found between lesion- and control-side aspartate aminotransferase or glutaminase activities, even though no choline acetyltransferase activity remained in the lesion-side of the organ of Corti. Both the distribution of aspartate aminotransferase activity and the lesion results would seem to implicate it in energy more so than neurotransmitter metabolism. The distribution of glutaminase activity could be consistent with a role in neurotransmission; however, the lesion data were unable to demonstrate a specific association with the olivocochlear bundle. PMID- 3021702 TI - Mefluidide effects on smooth brome composition and grazing cow-calf performance. AB - Mefluidide, a plant growth regulator, was evaluated in two cow-calf grazing trials and one herbage trial on smooth brome (Bromus inermis) pastures stocked at recommended densities in eastern Nebraska. Mefluidide-treated pasture increased cow and calf production during August of 1982 (P = .03) and calf production was greater during July of 1983 (P = .09). Mefluidide-treated smooth brome pastures increased calf production over the 1982 grazing season (P = .11) and cow gain over the 1982 (P = .12) and 1983 grazing seasons (P = .13). Mefluidide decreased neutral detergent fiber (NDF) content and increased crude protein content of smooth brome during both years (P less than .05), and increased in vitro dry matter disappearance (IVDMD) in 1982 (P less than .05). In ungrazed smooth brome, mefluidide treatment appeared to shift dry matter production to green leaves from green stem and brown leaf and stem fractions. Cell wall components [NDF, acid detergent fiber (ADF) and lignin] of green leaves were not affected significantly by mefluidide treatment, although green stems treated with mefluidide were lower in ADF and lignin (P less than .05). PMID- 3021703 TI - The voluntary forage intake and nutrition of goats and sheep in the semi-arid tropics of northeastern Brazil. AB - The nutritive value of diets selected by free-ranging goats and sheep, and estimates of forage intake, were obtained during the wet (January to May, 1982) and the dry season (June to December, 1981) in the Brazilian state of Ceara. Esophageally fistulated animals were used to collect dietary samples for nutritive evaluation. Organic matter intake (OMI) was estimated by total fecal collections. Goats selected diets higher in crude protein (CP) than did sheep (16.3 and 15.5%, respectively; P = .03). Contrary to current hypotheses, goats did not generally select diets of higher nutritional quality than did sheep. Sheep diets had lower (P less than .05) levels of lignin and equal (P greater than .1) levels of cell wall fiber compared with goats' diets. No difference in in vitro organic matter digestibility (IVOMD) was found between sheep and goats (P greater than .1), averaging 54%. However, major differences in IVOMD occurred during the wet season, as sheep diets were 4 to 10 digestibility units higher than were goats' diets. This may have been due to problems in using the in vitro procedure for dietary samples high in browse material. The OMI averaged 2.2 and 2.1% of body weight for sheep and goats, respectively (P = .08). Lowest levels of OMI (1.2% for sheep and goats) were noted during the latter portion of the wet season when forage biomass was high but nutrient quality was declining due to maturation. Daily digestible energy (DE) intake (kcal) differed (P = .04) between sheep (1,665.5 +/- 23.7) and goats (1,329.7 +/- 27.5).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021701 TI - Purines, prostaglandins and peptides--nature and cellular mechanisms of action of local assist and assassin agents in the ovary. AB - The evidence for a paracrine, progonadotropic role of adenosine in ovarian cells is summarized along with a capsule review of the origin and mechanisms of release and action of adenosine in other tissues. Briefly, adenosine markedly amplified rat and human luteal cell cyclic AMP and progesterone accumulation in the presence, but not the absence, of LH. The site of action of adenosine was found to be intracellular, linked to its phosphorylation, which resulted in increased levels of ATP. In rat luteal cells, adenosine blocked the acute antigonadotropic (luteolytic) action of PGF2 alpha. In the follicle, adenosine release from granulosal cells appeared to be stimulated by FSH. Adenosine and a nonmetabolized adenosine analog, augmented FSH-dependent inhibition of oocyte maturation in the presence or absence of an adenosine transport inhibitor. Inhibition of oocyte maturation by adenosine thus appears to be mediated by extracellular purinergic receptors. Paracrine, antigonadotropic agents also appear to regulate ovarian function. For example, GnRH elicits antigonadotropic activity in rat granulosal and luteal cells. We describe a novel, GnRH-like, ovarian hormone (GLOH) which may be the physiological ligand whose action GnRH mimics in rat ovarian cells. This protein was shown to be distinctly different from GnRH and a variety of other cyclic and noncyclic peptides. PGF2 alpha is a well known leutolytic agent and a summary of the antigonadotropic mechanism of PGF2 alpha action in rat luteal cells is presented. In these cells, the action of GnRH (or possibly the GnRH-like protein) and PGF2 alpha are mediated by separate membrane receptors but they appeared to share the same intracellular second messenger. Evidence for a role of products of phosphoinositol as a mediator of these antigonadotropic agents is summarized. We suggest that the ultimate mediator of antigonadotropic agents is Ca2+ which is released in the luteal cell in response to the intracellular mediator of antigonadotropic agents. For example, pharmacological agents which increase intracellular levels of Ca2+, mimicked the antigonadotropic action of GnRH and PGF2 alpha in rat luteal cells. Also, Ca2+ directly inhibited LH-sensitive adenylate cyclase activity in isolated luteal membranes, a paradigm in which GnRH and PGF2 alpha were inactive. The mechanism of Ca2+ action appeared to be linked to interference with GTP activation of adenylate cyclase. However, removal of extracellular Ca2+ did not abrogate the action of either GnRH or PGF2 alpha.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3021704 TI - Markers for estimating digesta flow in pigs and the effects of dietary fiber. AB - Effects of diluting the energy content of a corn-soybean meal diet with either alfalfa meal or corn cobs on nutrient digestibility and rate of passage of feed residues and particle markers were measured in crossbred (Yorkshire X Landrace X Chester White X Large White) barrows with a mean body weight of about 80 kg. The excretion pattern in the feces of Cr-mordanted diet and of rare earths initially bound individually to the mixed diet or to the corn or soybean meal suggested a model having a single age-dependent compartment with time delay. The compartmental turnover rate parameter (lambda 1) estimated by this model did not differ for the rare earths individually used to mark the corn-soybean meal diet (Yb), the corn (La) or the soybean meal (Sm). In contrast, lambda 1 for Cr was smaller (P less than .001) than that of the mean for the three rare earths. The residence time due to displacement flow (tau) did not differ among markers. These results were interpreted to indicate that the high specific gravity of Cr mordanted feed slowed flow due to mixing but not due to displacement. Correlations between lambda 1 and tau were less than .71. These results suggested that the flow of rare earths initially bound to feed ingredients provides a reasonable estimate of the flow of their undigested residues through the gastrointestinal tracts of nonruminant animals. Inclusion of the fibrous feeds reduced digestibility of dry matter, cell contents, crude protein and acid detergent lignin and increased digestibility of cell walls, cellulose and acid detergent fiber. Digestibilities of cellulose and acid detergent fiber were greater with alfalfa than with corn cobs as the fiber source. Differences in digestibility of crude protein and acid detergent fiber existed due to litter in one replicate of the experiment. Variation in digestibility was not significantly related to variation in lambda 1 or tau within or among treatments and litters. This suggests that variations in lambda 1 and tau were not important causes of the observed variation in digestibility. PMID- 3021706 TI - Effects of whole cottonseed, cottonseed oil or animal fat on digestibility of wheat straw diets by steers. AB - Two completely random digestion trials were conducted, each with 12 beef steers (325 kg initial weight), to measure changes in digestibilities of fat and of forage components when fat was added to diets containing 62 to 76% wheat straw. Trial 1 diets contained either no added fat or 6.3% added fat from whole cottonseed (30% of the diet), cottonseed oil or animal fat; diets were formulated to contain equal levels of cottonseed hulls and cottonseed meal. Trial 2 diets contained 0, 2, 4 or 8% added animal fat. In all forms and at all levels, added fat increased apparent digestibility of dietary lipid (P less than .05). However, estimated true digestibility of lipid decreased (from 94 to 71%) as added fat was increased from 0 to 8% (P less than .05). Up to 6.3% added fat increased digestible energy (DE) content of the diet. Fat additions of 2 and 4% increased daily DE intake (P less than .05) and did not depress digestibility of diet components (P greater than .05). Fat additions of 6.3% or greater, either as free fats or as whole cottonseed, reduced (P less than .05) mean acid detergent fiber digestibility from 40 to 28%. In addition to depressing fiber digestibility, 8% added fat reduced (P less than .05) digestibilities of dry matter (from 54 to 47%), organic matter (60 to 52%) and gross energy (60 to 51%). Oil fed as whole cottonseed caused digestibility depressions similar to free fat addition at the same level.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021705 TI - Effect of sulfur fertilization on chemical composition, ensiling characteristics and utilization of corn silage by lambs. AB - Effects of S fertilization, 0 or 67 kg S/ha, as (NH4)2SO4, as a single or split application on chemical composition, ensiling characteristics and utilization of corn (Zea mays L.) silage by wether lambs was investigated. Corn forages were ensiled at hard-dent stage at 35% dry matter (DM). Sulfur fertilization increased (P less than .01) total S and SO4 but had no effect on N, Mg, Ca or fiber components in silages. Sulfur-fertilized corn silages (N:S = 42 and 43) and non-S fertilized silage (N:S = 62) were fed alone and supplemented with Na2SO4 at two rates to achieve N:S ratios of 12 (high) and 45 (low, to approximate S-fertilized silage), to 30 wether lambs (Suffolk and Dorset crosses) in metabolism and palatability trials. Both trials were randomized complete-block designs. Lambs weighed 36 and 41 kg, respectively. All silages were supplemented with 6.7 g urea/d. Digestibility of DM and cell wall components, and apparent absorption of S and N were increased (P less than .05) by S fertilization and supplementation. Nitrogen retention was increased (P less than .01) by S fertilization and supplementation. Retention was higher (P less than .001) for lambs that were fed S-fertilized silage, compared with supplementation to achieve a similar N:S ratio. It appears that S fertilization was more effective than S supplementation in improving efficiency of N utilization. PMID- 3021707 TI - Diarrhea: the nemesis of the artificially reared, early weaned piglet and a strategy for defense. AB - Rearing early weaned piglets artificially for the purpose of increasing the efficiency of the sow is an attractive management concept. However, high death losses resulting from diarrhea in artificially reared piglets have dampered enthusiasm for early weaning. Enterotoxigenic Escherichia coli, transmissible gastroenteritis virus and rotavirus are the three main enteropathogens responsible for causing the diarrhea. The enteropathogens infect the small intestine, which produces a secretory or malabsorptive diarrhea. In nature, the nursing piglet is protected from the enteropathogens by antibody bathing his gut. The source of the antibody is the dam's colostrum and milk. It should be possible to protect artificially reared, early weaned piglets from enteropathogens by feeding them diets that contain antibodies to putative enteropathogens. PMID- 3021708 TI - Uptake of gentamicin by Staphylococcus aureus possessing gentamicin-modifying enzymes: enhancement of uptake by puromycin and N,N'-dicyclohexylcarbodiimide. AB - Uptake of gentamicin by a gentamicin-resistant strain of Staphylococcus aureus possessing the aminoglycoside-modifying phosphotransferase enzyme APH(2") was enhanced by the protein synthesis inhibitor, puromycin or by the proton translocating ATPase inhibitor, N,N'-dicylohexylcarbodiimide. Such enhanced uptake was inhibited by carbonyl cyanide p trifluoromethoxyphenylhydrazone or by valinomycin in the presence of potassium ions, suggesting a role for the transmembrane proton motive force in the process. The accumulated gentamicin did not cause loss of cell viability and exhibited altered chromatographic mobility compared with a control (unmodified) preparation of gentamicin. PMID- 3021709 TI - In-vitro activity of cefoperazone-sulbactam against Bacteroides species. AB - The in-vitro activity of two combinations of sulbactam and cefoperazone against 187 strains of Bacteroides fragilis were evaluated; their activity was compared with that of the drugs alone, and correlated with the production of beta lactamase by the bacterial strains. The results indicated that both combinations had a synergistic effect: the addition of 8 mg/l of sulbactam to the MIC of cefoperazone reduced the percentage of resistant strains from 58% to 0.5%; while the combination of two parts of cefoperazone to one pact of sulbactam lowered the resistance rate to 1%. Synergy was observed most frequently with beta-lactamase positive strains, but it also occurred among beta-lactamase negative strains. PMID- 3021710 TI - Antifungal effects of fluconazole (UK 49858), a new triazole antifungal, in vitro. AB - Fluconazole is a novel triazole antifungal intended for oral treatment of superficial and systemic mycoses. In tests done in standard mycological media, the compound had minimal inhibitory concentrations against pathogenic Candida species that were usually in excess of 100 mg/l. By contrast, its 'relative inhibition factors' against Candida species (calculated from areas under the antifungal dose-response curves) were of the same order as those of other imidazole and triazole antifungal agents. Against pathogenic Aspergillus species and dermatophytes, the mean relative inhibition factors were the highest so far recorded for an azole antifungal, indicating a relatively weak inhibitory activity against these fungi. Fluconazole inhibited branching and hyphal development in C. albicans at concentrations as low as 10(-6) M (0.3 mg/l), but miconazole and ketoconazole were still active in these tests at concentrations 100 times lower than this. The new antifungal did not suppress ATP concentrations in C. albicans spheroplasts, in common with other weakly lipophilic azole antifungals. This overall poor activity of fluconazole in vitro corresponds badly with its high activity in animal models of mycoses in vivo, and provides more evidence for the unreliability of tests with azole antifungals in vitro as predictors of potential efficacy in vivo. PMID- 3021711 TI - Determination of trace elements in foods by HCl-HNO3 leaching and flame atomic absorption spectroscopy. AB - Aluminum, iron, tin, zinc, calcium, magnesium, nickel, copper, chromium, cadmium, and potassium in foods can be extracted by HCl-HNO3 leaching and determined quantitatively using flame atomic absorption spectroscopy (AAS), with recoveries ranging from 90 to 110%. Thirty to 40 samples of almost any type of food sample can be analyzed routinely for 2 elements in 4-5 h. In contrast, one or 2 days are required when a wet-ash or dry-ash technique is used. Extraction consists of weighing 2-10 g samples into 125 mL Erlenmeyer flasks, adding 20 mL concentrated HCl-HNO3 (9 + 1), then heating in a 82- 93 degrees C water bath for 30 min. After cooling, samples are diluted to volume in 50 mL Nessler tubes and then filtered through No. 541 or 540 Whatman paper. The filtrate is analyzed directly by AAS. PMID- 3021712 TI - Intraoperative ultrasonography in endocrine pancreatic surgery: preliminary results in 6 cases of insulinoma. PMID- 3021713 TI - Treatment of chronic Epstein-Barr virus disease with H2 blockers. PMID- 3021714 TI - The random collision model and a critical assessment of diffusion and collision in mitochondrial electron transport. AB - This review focuses on our studies over the past ten years which reveal that the mitochondrial inner membrane is a fluid-state rather than a solid-state membrane and that all membrane proteins and redox components which catalyze electron transport and ATP synthesis are in constant and independent diffusional motion. The studies reviewed represent the experimental basis for the random collision model of electron transport. We present five fundamental postulates upon which the random collision model of mitochondrial electron transport is founded: All redox components are independent lateral diffusants; Cytochrome c diffuses primarily in three dimensions; Electron transport is a diffusion-coupled kinetic process; Electron transport is a multicollisional, obstructed, long-range diffusional process; The rates of diffusion of the redox components have a direct influence on the overall kinetic process of electron transport and can be rate limiting, as in diffusion control. The experimental rationales and the results obtained in testing each of the five postulates of the random collision model are presented. In addition, we offer the basic concepts, criteria and experimental strategies that we believe are essential in considering the significance of the relationship between diffusion and electron transport. Finally, we critically explore and assess other contemporary studies on the diffusion of inner membrane components related to electron transport including studies on: rotational diffusion, immobile fractions, complex formation, dynamic aggregates, and rates of diffusion. Review of all available data confirms the random collision model and no data appear to exist that contravene it. It is concluded that mitochondrial electron transport is a diffusion-based random collision process and that diffusion has an integral and controlling affect on electron transport. PMID- 3021716 TI - Regulation of electron transfer by the quinone pool. AB - Strong evidence for a random collisional mechanism for ubiquinone-mediated electron transfer is provided by the characteristic kinetic properties of respiratory chains originally explored by Kroger, A., and Klingenberg, M. (1973), Eur. J. Biochem. 34, 313-323. A kinetic model which leads to this so-called "simple Q-pool behavior" has been described and we use this in reviewing evidence that electron transfer is diffusion-controlled as well as diffusion-coupled. We also consider mechanisms by which the kinetics of electron transfer might deviate from simple Q-pool behavior and how these might be implicated in the regulation of electron transport. PMID- 3021715 TI - Is ubiquinone diffusion rate-limiting for electron transfer? AB - The different possible dispositions of the electron transfer components in electron transfer chains are discussed: random distribution of complexes and ubiquinone with diffusion-controlled collisions of ubiquinone with the complexes, random distribution as above, but with ubiquinone diffusion not rate-limiting, diffusion and collision of protein complexes carrying bound ubiquinone, and solid state assembly. Discrimination among these possibilities requires knowledge of the mobility of the electron transfer chain components. The collisional frequency of ubiquinone-10 with the fluorescent probe 12-(9-anthroyl)stearate, investigated by fluorescence quenching, is 2.3 X 10(9) M-1 sec-1 corresponding to a diffusion coefficient in the range of 10(-6) cm2/sec (Fato, R., Battino, M., Degli Esposti, M., Parenti Castelli, G., and Lenaz, G., Biochemistry, 25, 3378-3390, 1986); the long-range diffusion of a short-chain polar Q derivative measured by fluorescence photobleaching recovery (FRAP) (Gupte, S., Wu, E. S., Hochli, L., Hochli, M., Jacobson, K., Sowers, A. E., and Hackenbrock, C. R., Proc. Natl. Acad. Sci. USA 81, 2606-2610, 1984) is 3 X 10(-9) cm2/sec. The discrepancy between these results is carefully scrutinized, and is mainly ascribed to the differences in diffusion ranges measured by the two techniques; it is proposed that short-range diffusion, measured by fluorescence quenching, is more meaningful for electron transfer than long-range diffusion measured by FRAP, or microcollisions, which are not sensed by either method. Calculation of the distances traveled by random walk of ubiquinone in the membrane allows a large excess of collisions per turnover of the respiratory chain. Moreover, the second-order rate constants of NADH ubiquinone reductase and ubiquinol-cytochrome c reductase are at least three orders of magnitude lower than the second-order collisional constant calculated from the diffusion of ubiquinone. The activation energies of either the above activities or integrated electron transfer (NADH-cytochrome c reductase) are well above that for diffusion (found to be ca. 1 kcal/mol). Cholesterol incorporation in liposomes, increasing bilayer viscosity, lowers the diffusion coefficients of ubiquinone but not ubiquinol-cytochrome c reductase or succinate-cytochrome c reductase activities. The decrease of activity by ubiquinone dilution in the membrane is explained by its concentration falling below the Km of the partner enzymes. It is calculated that ubiquinone diffusion is not rate-limiting, favoring a random model of the respiratory chain organization.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3021717 TI - Reaction center and UQH2:cyt c2 oxidoreductase act as independent enzymes in Rps. sphaeroides. AB - Turnover of the ubiquinol oxidizing site of the UQH2:cyt c2 oxidoreductase (b/c1 complex) of Rps. sphaeroides can be assayed by measuring the rate of reduction of cyt b561 in the presence of antimycin (AA). Oxidation of ubiquinol is a second order process, with a value of k2 of about 3 X 10(5)M-1. The reaction shows saturation at high quinol concentrations, with an apparent Km of about 6-8mM (with respect to the concentration of quinol in the membrane). When the quinone pool is oxidized before illumination, reduction of the complex shows a substantial lag (about 1 ms) after a flash, indicating that the quinol produced as a result of the photochemical reactions is not immediately available to the complex. We have suggested that the lag may be due to several factors, including the leaving time of the quinol from the reaction center, the diffusion time to the complex, and the time for the head group to cross the membrane. We have suggested a minimal value for the diffusion coefficient of ubiquinone in the membrane (assuming that the lag is due entirely to diffusion) of about 10(-9) cm 2 sec-1. The lag is reduced to about 100 microseconds when the pool is significantly reduced, showing that quinol from the pool is more rapidly available to the complex than that from the reaction center. With the pool oxidized, similar kinetics are seen when the reduction of cyt b561 occurs through the AA-sensitive site (with reactions at the quinol oxidizing site blocked by myxothiazol).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021718 TI - On the role of physical parameters in the regulation of electron transport: diffusion, collision, and complex formation. PMID- 3021719 TI - Inorganic pyrophosphatase activity in a plaque calcifying microorganism: Bacterionema matruchotii. AB - The presence and activity of an alcaline pyrophosphatase of Bacterionema matruchotii, a Gram positive filamentous organism present in the bacterial plaque and calculus was studied. A strong enzymatic activity was demonstrated. The optimal conditions for this activity (alcaline pH, magnesium cofactor) have to be related to the pyrophosphatase conditions found in other calcifiable structures. The activity was shown in intact bacteria as well as in the culture medium devoid of micro-organisms. The strongest activity having been demonstrated in broken cells, it can be postulated that the enzyme is active inside the cell. The hypothesis of a contribution of Bacterionema matruchotii to the mineral phase formation is proposed; it is possible that the pyrophosphatase produced by this microorganism contributes to the mineralization process involved in dental calculus formation. PMID- 3021721 TI - Characterization of one- and two-electron oxidations of glutathione coupled with lactoperoxidase and thyroid peroxidase reactions. AB - Glutathione (GSH) was oxidized to GSSG in the presence of H2O2, tyrosine, and peroxidase. During the GSH oxidation catalyzed by lactoperoxidase, O2 was consumed and the formation of glutathione free radical was confirmed by ESR of its 5,5'-dimethyl-1-pyrroline-N-oxide adduct. When lactoperoxidase was replaced by thyroid peroxidase in the reaction system, the consumption of O2 and the formation of the free radical became negligibly small. These results led us to conclude that, in the presence of H2O2 and tyrosine, lactoperoxidase and thyroid peroxidase caused the one-electron and two-electron oxidations of GSH, respectively. It was assumed that GSH is oxidized by primary oxidation products of tyrosine, which are phenoxyl free radicals in lactoperoxidase reactions and phenoxyl cations in thyroid peroxidase reactions. When tyrosine was replaced by diiodotyrosine or 2,6-dichlorophenol, the difference in the mechanism between lactoperoxidase and thyroid peroxidase disappeared and both caused the one electron oxidation of GSH. Iodides also served as an effective mediator of GSH oxidation coupled with the peroxidase reactions. In this case the two peroxidases both caused the two-electron oxidation of GSH. PMID- 3021720 TI - Expression of uncoupling protein mRNA in thermogenic or weakly thermogenic brown adipose tissue. Evidence for a rapid beta-adrenoreceptor-mediated and transcriptionally regulated step during activation of thermogenesis. AB - A cloned cDNA sequence for the unique mitochondrial uncoupling protein of rat brown adipose tissue has been used to assay the corresponding mRNA in several situations. When thermogenesis in brown adipose tissue is stimulated (exposure of adult rats to the cold, birth) a rapid and prolonged increase in the level of uncoupling protein mRNA is observed. Such an increase can be mimicked by injection of animals with a new beta-adrenoreceptor agonist BRL 26830A. Conversely it is known that mice and rats with genetic or surgical obesity have a weakly thermogenic brown adipose tissue with a reduced norepinephrine turnover. A reduced level of uncoupling protein mRNA was measured in obese fa/fa rats 10 days or 10 weeks old and in obese rats with a lesion of the ventromedial hypothalamic area but not in obese ob/ob mice. Moreover, exposure of obese animals to cold or dosing with BRL 26830A strikingly increased the level of uncoupling protein mRNA. Measurement of the relative concentration of nascent Ucp transcripts in nuclei isolated from brown adipose tissue indicates that Ucp gene is acutely (within 15 min) regulated at the level of transcription and is controlled via activation of beta-adrenoreceptors of plasma membrane. Ucp gene transcription is decreased in obese fa/fa rats but can be fully and rapidly turned on after injection of BRL 26830A. PMID- 3021722 TI - Hydrophobic association of calpains with subcellular organelles. Compartmentalization of calpains and the endogenous inhibitor calpastatin in tissues. AB - Calpains I and II isolated from diverse tissues possess both Ca2+-independent, and Ca2+-dependent accessible hydrophobic regions. Possible subcellular organelle association of calpains involving these hydrophobic regions was studied. By homogenizing rat tissues directly in Ca2+ (50 microM), about 30-60% of the cytosolic calpain I and II activity reversibly associated with isolated subcellular fractions (microsomal greater than plasma membrane greater than nuclear). After binding to the particulate fraction, calpain II converted to a calpain I-like form exhibiting stronger Ca2+-independent binding to phenyl Sepharose and a lower Ca2+ requirement for optimal activity. However, it retained its DEAE-cellulose chromatographic pattern, and precipitated with monospecific anti-calpain II antibodies. Although purified calpastatin (endogenous inhibitor) is known to form a Ca2+-dependent complex with calpains, it was not able to reverse the binding of calpains to the particulate fraction upon short incubation. It was, however, effective in blocking calpain binding when the isolated cytosolic fraction or a mixture of purified calpain and calpastatin was preincubated in the presence of Ca2+, and then added to the particulate fraction. Extraction of tissues under controlled conditions revealed that in fact calpains are already loosely associated with subcellular organelles even in the absence of Ca2+. This is the reason why in the crude homogenates with the addition of Ca2+, calpains strongly bind to the particulate fraction without interference by cytosolic calpastatin. Although calpastatin by complexing initially to calpain can prevent the association of this protease with subcellular organelles, it cannot dissociate calpains already bound to these subcellular fractions. By prior Ca2+-independent association with the hydrophobic proteins present in the subcellular fractions, calpains overcome the 3- to 30-fold inhibitory excess of calpastatin in tissues. PMID- 3021724 TI - Identification of a vanadate-sensitive potassium-dependent proton pump from rabbit colon. AB - Membrane vesicles prepared from rabbit distal colon were used to study colonic transport mechanisms. Differential and sucrose-Ficoll density gradient centrifugation of the mucosal homogenate yielded fractions which supported ATP dependent proton transport, as measured with the fluorescent weak base acridine orange. Quenching of acridine orange absorbance in light microsomes and microsome derived density gradient fractions was MgATP-dependent and was reversed with nigericin; these characteristics suggest the presence of one or more ATP-driven proton pumps. Proton transport in the microsomal fraction was inhibited by N ethylmaleimide more than by orthovanadate, and was dependent on extravesicular chloride. Vesicles in a microsome-derived gradient fraction were inhibited by orthovanadate more than by N-ethylmaleimide. N-ethylmaleimide pretreatment of this gradient fraction uncovered a vesicle population with characteristics similar to the gastric H+,K+ATPase: proton transport was abolished by orthovanadate and the experimental anti-ulcer drug SCH 28080, was enhanced by potassium, and was not affected by chloride. ATP-generated proton gradients in this fraction were not dissipated by the proton ionophore 3,3',4',5 tetrachlorosalicylanilide. We conclude that two ATP-driven proton pumps are present in mucosa from distal rabbit colon; one with characteristics of N ethylmaleimide-sensitive organelle associated proton pumps, and one similar to the gastric proton-potassium exchanger. PMID- 3021723 TI - Pressure effects on sarcoplasmic reticulum. AB - The irreversible effects of pressure (1-2000 atm) upon the enzymatic activity and structure of the Ca2+-ATPase of sarcoplasmic reticulum were investigated. Sarcoplasmic reticulum vesicles suspended in a medium of 0.1 M KCl, 10 mM imidazole, pH 7.0, 5 mM MgCl2, and 0.5 mM EGTA irreversibly lose their Ca2+ transport and Ca2+-stimulated ATPase activities on exposure to pressures of 800 2000 atmospheres. The pressure-induced inactivation of Ca2+-ATPase is accompanied by inhibition of the formation of phosphorylated enzyme intermediate, an increase in the passive Ca2+ permeability of the membrane, and structural changes in the Ca2+-ATPase as shown by disruption of Ca2+-ATPase membrane crystals, increased susceptibility to tryptic digestion, unmasking of SH groups, and loss of the conformational responses to Ca2+ and vanadate. The sensitivity to pressure is influenced by enzyme conformation. Ca2+ or vanadate + EGTA protect the Ca2+ ATPase against pressure-induced inactivation, implying a greater stability of the enzyme in the E1 and E2 states than in the conformational equilibrium that prevails at low [Ca2+] in the absence of vanadate. Protection against pressure inactivation was also observed in the presence of sucrose, glycerol, ethylene glycol and 1 M KCl, suggesting that water density modifying groups significantly affect the stability of Ca2+-ATPase under pressure. PMID- 3021725 TI - Cytochrome P-450-catalyzed rearrangement of a peroxyquinol derived from butylated hydroxytoluene. Involvement of radical and cationic intermediates. AB - The p-peroxyquinol derived from butylated hydroxytoluene, 2,6-di-t-butyl-4 hydroperoxy-4-methyl-2,5-cyclohexadienone, was degraded by the ferric form of rat liver cytochrome P-450, and the resulting products and their mechanisms of formation were investigated. Quinoxy radical BO. from homolysis of the O-O bond reacted by competing pathways; beta-scission yielded 2,6-di-t-butyl-p benzoquinone, and rearrangement with ring-expansion produced an oxacycloheptadienone free radical (X(.)). This rearranged radical was stabilized by the captodative effect that facilitated competitive interactions with the P 450 iron-oxo complexes formed during O-O bond scission. Approximately 15% of X(.) was captured by oxygen rebound with a hydroxyl radical from the P-450 complex (FeOH)3+ to form a hemiketal, that led to the ring-contracted product 2,5-di-t butyl-5-(2'-oxopropyl)-4-oxa-2-cyclopentenone by spontaneous rearrangement. The major fraction of X(.), however, underwent electron transfer oxidation to form the corresponding cation. Hydration of this cation produced the ring-contracted product, and proton elimination (or, alternatively, direct H(.) removal from X(.) led to the product 2,7-di-t-butyl-4-methylene-5-oxacyclohepta-2,6-dienone. The findings indicate that cytochrome P-450 intermediate complexes are mainly responsible for oxidation of X(.). The results complement our previous study with 2,6-di-t-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (Thompson, J. A., and Wand, M. D. (1985) J. Biol. Chem. 260, 10637-10644), demonstrating competitive heterolytic and homolytic mechanisms of O-O bond cleavage, and competitive rebound and oxidation processes when a substrate-derived radical interacts with P 450 complexes. PMID- 3021726 TI - Adrenic acid content in rat adrenal mitochondrial phosphatidylethanolamine and its relation to ACTH-mediated stimulation of cholesterol side chain cleavage reaction. AB - We have isolated various phospholipids from adrenal mitochondria of adrenocorticotropic hormone (ACTH)-treated (stimulated) and cycloheximide/ACTH treated (unstimulated) rats. When the effects of these phospholipids were examined on the formation of pregnenolone from endogenous cholesterol by adrenal mitochondria of unstimulated rats, phosphatidylethanolamine and phosphatidylserine from stimulated mitochondria were effective in enhancing the cleavage reaction in unstimulated mitochondria, whereas these phospholipids from unstimulated mitochondria were all ineffective. Cardiolipins from both stimulated and unstimulated mitochondria were effective. When the compositional changes in fatty acid moiety of phospholipids were examined, a significant increase in C22:4 (adrenic) acid was observed only for phosphatidylethanolamine under the influence of ACTH. A linear relationship between the contents of C22:4 acid in various phospholipids and respective steroidogenic activities was obtained (r = 0.880), suggesting an important role of this fatty acid moiety. The separation of active phosphatidylethanolamine by high performance liquid chromatography revealed that a fraction containing 25% C22:4 acid was most effective in the activation. Based on these results, it is most likely that 1-stearoyl-2-adrenoyl phosphatidylethanolamine is an active species. C22:4 acid was liberated together with C20:4 acid from adrenal triglycerides by the action of ACTH but the liberation was insensitive to cycloheximide inhibition. Finally, cardiolipin which enhances the transfer of cholesterol to cytochrome P-450scc may not be a physiological mediator of ACTH action. PMID- 3021727 TI - H+ coupled uphill transport of aminocephalosporins via the dipeptide transport system in rabbit intestinal brush-border membranes. AB - The transport characteristics of aminocephalosporin antibiotics, possessing an alpha-amino group and a carboxyl group, in brush-border membranes isolated from rabbit small intestine have been studied by a rapid filtration technique. The uptake of cephradine by brush-border membrane vesicles was stimulated by the countertransport effect of dipeptides, which indicates the existence of a common carrier transport system. An inward H+ gradient ([pH]i = 7.5 to 8.4, [pH]o = 6.0) stimulated cephradine uptake against a concentration gradient (overshoot phenomenon), and this stimulation was reduced when the H+ gradient was subjected to rapid dissipation by the presence of carbonyl cyanide p trifluoromethoxyphenylhydrazone, a protonophore. A valinomycin-induced K+ diffusion potential (interior-negative) stimulated H+ gradient-dependent cephradine uptake without altering the equilibrium value. The uptake of other aminocephalosporins (cefadroxil, cefaclor, cephalexin) was also stimulated in the presence of an inward H+ gradient, while the uptake of cephalosporins without the alpha-amino group (cefazolin, cefotiam) was not changed in the presence or absence of the H+ gradient. These results suggest that the transport of aminocephalosporins can be driven actively by an inward H+ gradient via the dipeptide transport system in the intestinal brush-border membranes, and that the process results in the transfer of a positive charge. PMID- 3021728 TI - Construction of DNA substrates modified with psoralen at a unique site and study of the action mechanism of ABC excinuclease on these uniformly modified substrates. AB - Psoralens bind to DNA noncovalently and upon exposure to near UV (320-400 nm) light produce covalent adducts. Thymidine residues in DNA, especially those at 5' TpA-3' sequences, are most susceptible to the photochemical reaction. This property of the reaction and the recent advances in oligonucleotide synthesis and separation has enabled us to construct DNA fragments containing psoralen adducts at a specific site. The octanucleotide 5'-TCGTAGCT-3' was photoreacted (in the presence of the complementary strand) with the synthetic psoralen 4' hydroxymethyl-4,5',8-trimethylpsoralen to obtain oligonucleotides adducted via the furan or pyrone ring at the internal thymine. These modified octanucleotides were ligated to nonmodified oligonucleotides to obtain a 40-base pair DNA fragment containing a psoralen adduct at a central location. The modified fragment having the thymine-furan side 4'-hydroxymethyl-4,5',8-trimethylpsoralen adduct was irradiated with 360 nm of light to produce an interstrand cross-link, and this cross-linked DNA was purified to homogeneity. These uniquely modified DNAs were used as substrates for Escherichia coli ABC excinuclease to determine its incision mechanism unambiguously and to determine the contact sites of the enzyme. ABC excinuclease mediates the cleavage of the 8th and 5th phosphodiester bonds 5' and 3', respectively, to psoralen monoadducts, and the 9th (5') and 3rd (3') phosphodiester bonds to the furan-side thymine of the cross-link. Preliminary DNaseI footprinting studies show that ABC excinuclease protects the whole 40-base pair fragment from DNaseI, and binding of the A and B subunits to the furan side-monoadducted substrate produces two hypersensitive phosphodiester bonds in the vicinity of the 5' incision site of ABC excinuclease. PMID- 3021729 TI - Rapid declines in cyclic GMP of rod outer segments of intact frog photoreceptors after illumination. AB - Considerable disagreement has resulted from experiments designed to test whether light-induced falls in cGMP in outer segment (OS) of photoreceptors precede their light-induced electrical responses. Different studies have reported initial declines at 50 ms, at s, or not at all for physiological stimuli. Such studies have employed whole retinas, isolated rod OS, or isolated rod OS with attached inner segments and involved a variety of techniques. We developed an apparatus that illuminates intact pieces of dark-adapted frog retinas at 22 degrees C for known brief durations and then rapidly (47 ms) presses their OS surface against a copper mirror cooled by liquid helium. Freezing occurs in less than 2 ms. Cyclic GMP was then assayed in cryostat sections of the OS layer. Six illumination intensities that bleached from 90 to 9 X 10(8) rhodopsin molecules per s were delivered for durations of 0.1-2 s. Compared to dark-adapted values, progressive losses of cGMP were seen with all illumination intensities. Because a significant loss in cGMP was seen after a 100 ms exposure to our dimmest stimulus, it appears that a loss of cGMP could play a role in rod visual transduction. PMID- 3021730 TI - Purification and characterization of bovine lung calmodulin-dependent cyclic nucleotide phosphodiesterase. An enzyme containing calmodulin as a subunit. AB - A rabbit lung cyclic nucleotide phosphodiesterase (PDE) prepared by successive chromatography on DEAE-cellulose and G-200 Sephadex columns in the presence of EGTA was activated by Ca2+ and contained calmodulin (CaM), suggesting that the enzyme exists as a stable CaM X PDE complex (Sharma, R. K., and Wirch, E. (1979) Biochem. Biophys. Res. Commun. 91, 338-344). An enzyme with similar properties was demonstrated to exist in bovine lung extract. C1, a monoclonal antibody previously shown to react with the 60-kDa subunit of bovine brain PDE isozymes (Sharma, R. K., Adachi, A.-M., Adachi, K., and Wang, J. H.) (1984) J. Biol. Chem. 259, 9248-9254), cross-reacted with the lung enzyme. Purification of the lung enzyme by C1 antibody immunoaffinity chromatography rendered the enzyme dependent on exogenous CaM for Ca2+ stimulation. Further purification was achieved by CaM affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed a predominant polypeptide of Mr 58,000 and a minor band of about 50,000. The purified enzyme could be reconstituted into a PDE X CaM complex upon incubation with CaM in the presence of either Ca2+ or EGTA. The reconstituted protein complex did not dissociate in buffers containing 0.1 mM EGTA. Analysis of the purified and reconstituted lung phosphodiesterase by Sephacryl S-300 gel filtration indicated that the lung enzyme is a dimeric protein and that the reconstituted enzyme contained two molecules of calmodulin. Analysis of the reconstituted phosphodiesterase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis also showed it to contain equimolar calmodulin and the enzyme subunit. The CaM antagonists, fluphenazine, compound 48/80, and calcineurin at concentrations abolishing CaM stimulation of bovine brain PDE had little effect on the activity of reconstituted bovine lung phosphodiesterase. PMID- 3021731 TI - Rabbit alveolar macrophages contain a Ca2+-sensitive, 41,000-dalton protein which reversibly blocks the "barbed" ends of actin filaments but does not sever them. AB - A 41,000-dalton Ca2+-sensitive actin-modulating protein has been purified from rabbit alveolar macrophages using ion exchange and gel filtration chromatography. On sodium dodecyl-polyacrylamide gel electrophoresis, this macrophage protein migrates more rapidly than actin and fails to cross-react with polyclonal anti actin antibody. It has a Stokes radius of 3.0 nm and an isoelectric point of 6.6. In the presence of micromolar Ca2+ this 41,000-Da protein: reduces the viscosity of polymerized actin, nucleates actin filament assembly, causes a nearly instantaneous increase in fluorescence intensity of subcritical concentrations of pyrenyl-actin (estimated KD of the pyrene actin-macrophage protein complex, 5 X 10(-8) M), increases the critical concentration of actin by 0.65 microM (molar ratios of protein/actin, 1/100-1/10), blocks actin monomer depolymerization from the "barbed" filament ends, and does not sever preformed actin filaments. The ability of this protein to block filament ends is rapidly and completely inhibited by lowering free calcium ion concentration below the micromolar range. PMID- 3021732 TI - On the interaction of the finger and the kringle-2 domain of tissue-type plasminogen activator with fibrin. Inhibition of kringle-2 binding to fibrin by epsilon-amino caproic acid. AB - The binding of tissue-type plasminogen activator (t-PA) to fibrin is mediated both by its finger domain and by its kringle-2 domain. In this report, we investigate the relative affinities of these domains for lysine. Human recombinant t-PA deletion-mutant proteins were prepared and their ability to bind to lysine-Sepharose was investigated. Mutants containing the kringle-2 domain bound to lysine-Sepharose, whereas mutants lacking this domain but containing the finger domain, the epidermal growth factor domain or the kringle-1 domain did not bind to lysine-Sepharose. Mutant proteins containing the kringle-2 domain could be specifically eluted from lysine-Sepharose with epsilon-amino caproic acid. This lysine derivative also abolished fibrin binding by the kringle-2 domain but had no effect on the fibrin-binding property of the finger domain. Thus, a lysine binding site is involved in the interaction of the kringle-2 domain with fibrin but not in the interaction of the finger domain with fibrin. The implications of the nature of these two distinct interactions of t-PA with fibrin on plasminogen activation by t-PA will be discussed. PMID- 3021733 TI - Localization of cytochrome c-binding domain on NADPH-cytochrome P-450 reductase. AB - A covalent complex between purified rat liver microsomal NADPH-cytochrome P-450 reductase and horse cytochrome c was formed through cross-linking studies with 1 ethyl-3-(3-dimethylaminopropyl)carbodiimide at low ionic strength. The purified cross-linked derivative shows that this product is a 1:1 complex containing one molecule each of the flavoprotein and cytochrome. The covalent complex had almost completely blocked the electron transfer from NADPH to exogenous cytochrome c or the rabbit liver microsomal cytochrome P-450 induced by phenobarbital, indicating that the cross-linked cytochrome c covers the electron-accepting site of the reductase. These results suggest that the covalently cross-linked derivative is a valid model of the noncovalent electron transfer complex. Although the exact number and site of the cross-linked location were not determinable, in cytochrome c the amide bond originates from Lys-13 and in reductase it might be at any one of six different side chain carboxyl groups in the two neighboring cluster acidic residues, Asp-207, -208, and -209, and Glu-213, Glu-214, and Asp-215. It is therefore proposed that the six clustered carboxyl groups on reductase are in an exposed location near the area where one heme edge comes close to the molecular surface. PMID- 3021734 TI - Phospholipid binding and biophysical activity of pulmonary surfactant-associated protein (SAP)-35 and its non-collagenous COOH-terminal domains. AB - Surfactant-associated protein of Mr = 35,000, SAP-35, is the major glycoprotein present in mammalian pulmonary surfactants. In this study, canine SAP-35 and several of its COOH-terminal peptides were purified and characterized by amino acid composition and NH2-terminal sequencing analysis. These proteins were then studied in terms of their specific lipid-binding characteristics and surface activity when combined with a synthetic phospholipid mixture, SM, chosen as an approximation of lung surfactant phospholipids. Purified, delipidated SAP-35 bound SM strongly. In contrast, SAP-21 (a non-collagenous fragment generated by collagenase digestion) bound phospholipid weakly; SAP-18 (an acidic COOH-terminal fragment comprising residues Gly-118 to Phe-231) did not bind phospholipid, demonstrating the importance of hydrophobic amino acid residues Gly-81 to Val-117 and the NH2-terminal collagenous domain in interaction of the SAP-35 with phospholipids. In surface activity experiments, purified SAP-35 enhanced the adsorption of SM phospholipids in terms of both rate and overall surface tension lowering. However, the adsorption facility of the SM-SAP-35 mixture did not approach that of either whole surfactant or the surfactant extract preparations, calf lung surfactant extract or surfactant-TA, used in exogenous surfactant replacement therapy for the neonatal respiratory distress syndrome. In addition, the dynamic surface activity of the SM-SAP-35 mixture was well below that of natural surfactant or surfactant extracts. This was also true of mixtures of SM phospholipids combined with the SAP-18 and SAP-21 fragments of SAP-35. PMID- 3021735 TI - Synthesis of growth hormone-prolactin chimeric proteins and processing mutants by the exchange and deletion of genomic exons. AB - To test the feasibility of synthesizing recombinant peptide hormones by exon deletion and exchange, we have constructed and expressed hybrid human growth hormone (hGH)-rat prolactin (rPrl) genes in which the third and fourth exons of the hGH gene are deleted and separately replaced by the corresponding exons of the rPrl gene. These exon deletion and exon exchange genes were inserted into an SV40 viral vector, packaged, and expressed following acute viral infection of monkey kidney cells. Expression of the hGH gene lacking the third exon (hGHd3) was not detectable at the level of protein production. However, replacement of the deleted third exon in the hGHd3 gene with exon 3 of the rPrl gene (hGHP3) resulted in the efficient synthesis of a secreted chimeric protein. When the fourth exon of the hGH gene was deleted (hGHd4), the encoded protein was found only in the cytosol despite signal sequence processing. Replacement of the deleted fourth exon in this hGHd4 gene with exon 4 of rPrl resulted in the synthesis and secretion of both a chimeric protein (hGHP4) as well as a larger protein corresponding in size to prehGHP4. These results suggest that exon exchange among distantly related genes in the GH family may be used to produce high levels of chimeric GH-related proteins, and regions internal to the hGH protein may be critical in establishing normal protein processing and secretion. PMID- 3021737 TI - Inhibition of simian virus 40 DNA replication by specific modification of T antigen with oxidized ATP. AB - We covalently bound periodate-oxidized ATP (oATP) to purified simian virus 40 (SV40) large T-antigen and determined the effect of this modification on viral DNA replication and three other biochemical activities of T-antigen. The oATP bound specifically to T-antigen, inhibiting the ATPase activity and preventing T antigen from activating SV40 DNA replication in vitro. In contrast, binding of oATP had no effect on the DNA-binding activity of T-antigen nor on its ability to form a complex with DNA polymerase alpha. These results provide direct biochemical evidence suggesting that the T-antigen ATPase activity is necessary for viral DNA replication. PMID- 3021736 TI - Antibodies directed against ubiquitin inhibit high affinity [3H]choline uptake in rat cerebral cortical synaptosomes. AB - Sodium-dependent [3H]choline uptake and coupled [3H]acetylcholine synthesis were inhibited in rat cerebral cortical synaptosomes in a dose- (1-10 micrograms/ml) and time-dependent manner by affinity-purified antibodies directed against ubiquitin (anti-Ub). Neither sodium-independent [3H]choline uptake nor [3H]acetylcholine release was affected by up to 10 micrograms/ml anti-Ub, indicating that the cholinergic terminals were not depolarized by the anti-Ub. Binding of anti-Ub to synaptosomes, as measured with 125I-protein A, was saturable and occurred over the same concentration range (1-10 micrograms/ml) at which uptake inhibition was observed. Although preimmune IgG bound to the synaptosome preparation to a greater extent and was apparently not readily saturable, this fortuitous binding was without effect on high affinity choline uptake and conversion to acetylcholine. The results suggest the presence of a ubiquitin-protein conjugate on the synaptosomal surface and a functional relationship between this protein conjugate and the sodium-dependent choline transport system. PMID- 3021738 TI - Norepinephrine-induced Na+ influx in brown adipocytes is cyclic AMP-mediated. AB - To examine the involvement of Na+ ions in adrenergic responses in brown adipose tissue, a method is described for measuring Na+ influx into isolated brown adipocytes, using short (30 s) incubations with 22Na+, followed by a two-step centrifugation recovery procedure. Using this method, a clear norepinephrine stimulated accumulation of intracellular 22Na+ was observed, which was enhanced by the addition of ouabain, was insensitive to amiloride (a Na+/H+ exchange blocker), and could not be mimicked by the total removal of oxygen from the incubation medium. The norepinephrine-stimulated Na+ influx was dose-dependent for the hormone with an EC50 of 250 nM, was blocked by the beta-antagonist propranolol but not by the alpha 1-antagonist prazosin, and could be induced by adrenergic agonists with the order of potency: isoproterenol greater than norepinephrine greater than phenylephrine, indicating a beta-receptor-mediated process. The Na+ influx was found to be cAMP-dependent since it could be induced by both theophylline (a phosphodiesterase inhibitor) and forskolin (an adenylate cyclase activator), but it was independent of other known cellular cAMP-dependent responses since neither addition of fatty acid substrates (octanoate or palmitate), nor of the mitochondrial uncoupler carbonyl cyanide p trifluoromethoxyphenyl-hydrazone could induce the phenomenon, despite having significant stimulatory effects on cellular respiration. Furthermore, total respiratory inhibition with rotenone, or total oxygen depletion of the medium with dithionite, did not prevent the normal norepinephrine-induced Na+ influx. The possibility that this beta-mediated norepinephrine-stimulated Na+ influx plays an important physiological role in brown adipose tissue activity is discussed, perhaps as one of the, as yet undefined, signals initiating tissue growth in the chronically beta-stimulated tissue of animals facing long-term increases in thermogenic demands. PMID- 3021739 TI - Purification and characterization of a paired basic residue-specific prohormone converting enzyme from bovine pituitary neural lobe secretory vesicles. AB - The neuropeptides arginine vasopressin and oxytocin are generated from their prohormones in the hypothalamoneurohypophysial system by enzymatic cleavages at paired basic residues (i.e. Lys-Arg). This study describes the purification of an enzyme from bovine neural lobe secretory vesicles, the putative site of this processing, which is capable of cleaving several prohormones at paired basic residues. The enzyme is a glycoprotein of Mr approximately 70,000 and has an acidic pH maximum. It processes the heterologous precursors pro-opiomelanocortin and insulin at paired basic residues in a manner similar to a pro opiomelanocortin-converting enzyme derived from bovine intermediate lobe secretory vesicles which has been described previously. In addition, the neural lobe-derived converting enzyme cleaves the human vasopressin prohormone in vitro to yield arginine vasopressin-Gly10-Lys11-Arg12 as the major vasopressin cleavage product. This indicates that the enzymatic cleavage in the vasopressin precursor occurred primarily on the carboxyl side of the arginine in the pair of Lys-Arg basic residues separating the vasopressin peptide from the neurophysin moiety in the precursor. The properties of the neural and intermediate lobe-derived enzymes are virtually identical, raising the possibility that a family of similar enzymes may be responsible for cleaving a number of prohormones at paired basic residues in different tissues. PMID- 3021740 TI - The mechanism by which oxygen and cytochrome c increase the rate of electron transfer from cytochrome a to cytochrome a3 of cytochrome c oxidase. AB - When cytochrome c oxidase is isolated from mitochondria, the purified enzyme requires both cytochrome c and O2 to achieve its maximum rate of internal electron transfer from cytochrome a to cytochrome a3. When reductants other than cytochrome c are used, the rate of internal electron transfer is very slow. In this paper we offer an explanation for the slow reduction of cytochrome a3 when reductants other than cytochrome c are used and for the apparent allosteric effects of cytochrome c and O2. Our model is based on the conventional understanding of cytochrome oxidase mechanism (i.e. electron transfer from cytochrome a/CuA to cytochrome a3/CuB), but assumes a relatively rapid two electron transfer between cytochrome a/CuA and cytochrome a3/CuB and a thermodynamic equilibrium in the "resting" enzyme (the enzyme as isolated) which favors reduced cytochrome a and oxidized cytochrome a3. Using the kinetic constants that are known for this reaction, we find that the activating effects of O2 and cytochrome c on the rate of electron transfer from cytochrome a to cytochrome a3 conform to the predictions of the model and so provide no evidence of any allosteric effects or control of cytochrome c oxidase by O2 or cytochrome c. PMID- 3021741 TI - Solubilization of the liver vasoactive intestinal peptide receptor. Hydrodynamic characterization and evidence for an association with a functional GTP regulatory protein. AB - Vasoactive intestinal peptide (VIP) receptors were solubilized using the nondenaturing detergent Triton X-100 after occupancy of rat liver membrane-bound receptors with 125I-VIP. Gel filtration and ultracentrifugation on sucrose density gradients revealed the existence in the soluble macromolecular fraction of two labeled components: a major (80%) heavy component and a minor (20%) light one. The two components exhibit the following hydrodynamic parameters: Stokes radius, 5.8 nm: s20,w, 5.98 s; Mr, 150,000; frictional ratio, 1.52 for the major; and Stokes radius, 3.0 nm: s20,w, 3.98 s; Mr = 52,000; frictional ratio, 1.12 for the minor component. The labeling of these components was specific in that it dramatically decreased when unlabeled VIP was added together with 125I-VIP. The pharmacological specificity was also assessed by using 10 nM histidylisoleucineamide (a VIP agonist). Many lines of evidence indicate that the light component (Mr = 52,000) is the VIP-receptor complex while the heavy component (Mr = 150,000) is a ternary complex consisting of VIP, the receptor, and a guanine nucleotide regulatory protein, probably Ns. GTP is required to dissociate 125I-VIP from the heavy component whereas it is ineffective on the light component. This effect is nucleotide specific. After cholera toxin-induced [32P]ADP ribosylation of liver membranes, a high peak of 32P radioactivity containing the alpha subunit (Mr = 42,000) of the Ns protein is coeluted with the heavy component on Sephacryl S-300. By mild urea (2 M) treatment, the heavy component is converted into the light without significant dissociation of 125I VIP. When a Triton extract of membranes prelabeled with 125I-VIP is treated with dithiobis(succinimidyl propionate) subsequent sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis reveals a major band corresponding to Mr = 150,000. Alternatively, when prelabeled membranes are directly treated with the cross-linker, a major complex of Mr = 51,000 is observed. This may be related to different accessibility of the cross-linker to the site at which the receptor and the Ns protein interact in the two conditions. In conclusion, these data represent initial reports on the successful solubilization of functional VIP receptor complexes and provide evidence for an interaction between liver VIP receptor complexes and a GTP-binding protein. PMID- 3021742 TI - Calcium-dependent isolation of the 36-kilodalton substrate of pp60src-kinase. Fractionation of the phosphorylated and unphosphorylated species. AB - In this paper, we present a new and simple purification of the 36-kDa protein, a major substrate of both viral and growth factor-receptor associated tyrosine protein kinases, and its complex from normal and Schmidt-Ruppin strain Rous sarcoma virus-transformed chicken embryo fibroblasts that employs a DEAE-Sephacel column and introduces the calcium-dependent adsorption of 36-kDa protein. The use of EGTA step gradients differentially elutes the 36-kDa molecules from the DEAE Sephacel column. An average total yield of the 36-kDa protein in all fractions approached 80% and represented 0.78% of the [35S] methionine-labeled cellular protein. A purity of 95-99% was obtained with a yield of 60% in the central elution fractions from normal or Rous sarcoma virus-transformed chicken embryo fibroblasts. Furthermore, 2 mM EGTA elutes poorly phosphorylated molecules while heavily phosphorylated 36-kDa protein requires 4 or 6 M EGTA; a small residual fraction is released at 8-10 mM EGTA. If the EGTA step gradients were neutralized with Ca2+ ion, elution of the 36-kDa protein is inhibited. The complex of the 36 kDa protein and the 6-10-kDa protein may not be dependent on the phosphorylation as the associated 6-10-kDa peptide is seen in all fractions containing the 36-kDa protein. Tyrosine phosphorylation of the 36-kDa protein is increased 2-3-fold following a short term incubation of whole cells with micromolar vanadate. The elution pattern (but not intensity) of the 36-kDa protein obtained from lysates of vanadate-treated cells was identical to untreated cell lysates. The additional phosphorylation appears to result from a recruitment of unphosphorylated 36-kDa protein as the position (but not intensity) of the phosphorylated tryptic peptides is unchanged. We conclude that the function of the 36-kDa protein may be calcium ion-dependent and may be influenced by the phosphorylation state of the protein. PMID- 3021743 TI - The hormone-sensitive hepatic Na+-pump. Evidence for regulation by diacylglycerol and tumor promoters. AB - Ouabain-sensitive 86Rb+ uptake by isolated rat hepatocytes was studied to elucidate how Ca2+-mobilizing hormones stimulate the Na+-pump. Stimulation of this uptake was observed with concentrations of vasopressin ([8 arginine]vasopressin, AVP), angiotensin II, and norepinephrine which elicited Ca2+ mobilization and phosphorylase activation. These results suggested that changes in cytosolic Ca2+, mediated by inositol trisphosphate, might trigger sodium pump stimulation by AVP. However, in hepatocytes incubated in Ca2+-free Krebs-Henseleit buffer, Na+-pump activity was not altered over 15 min by either 1.5 mM EGTA or 1.5 mM Ca2+. Furthermore, incubation of cells in 5 mM EGTA for 15 30 min drastically impaired the ability of AVP to increase cytosolic Ca2+, but only modestly attenuated AVP-stimulated Na+-pump activity. Two tumor promoters, phorbol myristate acetate (PMA) and mezerein, stimulated Na+/K+-ATPase-mediated transport activity. Similarly, addition of synthetic diacylglycerols or of exogenous phospholipase C from Clostridium perfringens to increase endogenous diacylglycerol levels also resulted in a stimulation of the Na+-pump in the absence of changes in cytosolic or total cellular Ca2+ levels. Stimulation of the Na+-pump by the combination of maximal concentrations of PMA and AVP did not produce an additive response, and both agents displayed a transient time course, suggesting that the two agents share a common mechanism. Stimulation of the Na+ pump by AVP and PMA was not blocked by amiloride analogs which inhibit Na+/H+ exchange, but these compounds blocked the action of insulin. These data suggest that the elevated Na+/K+-ATPase-mediated transport activity observed in hepatocytes following exposure to Ca2+-mobilizing hormones is a consequence of stimulated diacylglycerol formation and may involve protein kinase C. PMID- 3021744 TI - Mammalian beta 1- and beta 2-adrenergic receptors. Immunological and structural comparisons. AB - Beta 1- and beta 2-adrenergic receptors, pharmacologically distinct proteins, have been reported to be structurally dissimilar. In the present study three techniques were employed to compare the nature of mammalian beta 1- and beta 2 adrenergic receptors. Antibodies against each of the receptor subtypes were raised separately. Polyclonal antisera against beta 1-receptors of rat fat cells were raised in mice, and antisera against beta 2-receptors of guinea pig lung were raised in rabbits. Receptors purified from rat fat cells (beta 1-), S49 mouse lymphoma cells (beta 2-), and rat liver (beta 2-) were probed with these antisera. Each anti-receptor antisera demonstrated the ability to immunoprecipitate purified receptors of both beta 1- and beta 2- subtypes. The mobility of beta-receptors subjected to polyacrylamide gel electrophoresis was probed using antireceptor antibodies and nitrocellulose blots of the gels. Fat cell beta 1-adrenergic receptors display Mr = 67,000 under reducing conditions and Mr = 54,000 under nonreducing conditions, as previously reported (Moxham, C. P., and Malbon, C. C. (1985) Biochemistry 24, 6072-6077). Both beta 1- and beta 2 receptors displayed this same shift in electrophoretic mobility observed in the presence as compared to the absence of disulfide bridge-reducing agents, as detected both by autoradiography of the radiolabeled receptors and by immunoblotting of native receptors. Finally, isoelectric focusing of purified radioiodinated beta 1- and beta 2-adrenergic receptors revealed identical isoelectric points. These data are the first to provide analyses of immunological, structural, and biochemical features of beta 1- and beta 2 subtypes in tandem and underscore the structural similarities that exist between these pharmacologically distinct receptors. PMID- 3021745 TI - Proton and phosphorus-31 NMR study of the dependence of diadenosine tetraphosphate conformation on metal ions. AB - Adenosine 5'-tetraphospho-5'-adenosine (Ap4A) plays a role in cellular metabolism in a wide variety of organisms. Because the divalent cations Mg2+ and Zn2+ are involved in the synthesis and function of Ap4A, the effect of divalent cations on the dinucleotide's conformation is of interest. 1H and 31P chemical shift experiments were carried out as a function of Mg2+ concentration and pH. We propose that Mg2+ stabilizes the unusual ring-stacked conformation of Ap4A at pH greater than 2 by interacting with the beta-phosphates. To further probe conformational effects, stable complexes of Ap4A with Co3+ were studied using 1H and 31P NMR. Co3+ forms two different bidentate complexes with Ap4A, independent of whether the other four octahedral coordination sites are occupied by ammonia or trimethylenediamine. NMR results suggest that in one complex the Co3+ is coordinated to two beta-phosphates and ring stacking is stabilized. In the other complex, Co3+ is coordinated to an alpha-phosphate and its neighboring beta phosphate and ring stacking is destabilized. These results further support the hypothesis that Mg2+ stabilizes the ring-stacked conformation by interacting symmetrically with the two beta-phosphate groups. PMID- 3021746 TI - Immunological comparison of the b and c1 cytochromes from bovine heart mitochondria and the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26. AB - Antibodies against cytochromes b and c1 of bovine heart mitochondria and the photosynthetic bacterium, Rhodopseudomonas sphaeroides R-26, were raised in rabbits. The purified antibodies showed high titers against their respective antigens in enzyme-linked immunosorbent assays. Less than 15% cross-reactivity between the mitochondrial and bacterial cytochromes was detected. Although antibodies against mitochondrial cytochrome b did not inhibit the mitochondrial cytochrome b-c1 complex, a 70% inhibition was obtained when these antibodies were incubated with delipidated mitochondrial cytochrome b-c1 complex prior to reconstitution with phospholipids indicating that the catalytic site(s) of mitochondrial cytochrome b are masked by phospholipids. On the other hand, antibodies against bacterial cytochrome b showed significant inhibition of the intact bacterial cytochrome b-c1 complex, indicating that some of the catalytic site epitopes of bacterial cytochrome b are exposed to the hydrophilic environment. Similar to antibodies against mitochondrial cytochrome b, antibodies against bacterial cytochrome b inhibited 50% activity of the mitochondrial cytochrome b-c1 complex only when they were incubated with the delipidated mitochondrial cytochrome b-c1 complex prior to reconstitution with phospholipids, indicating that the common epitopes between the cytochromes b are masked by phospholipids. Antibodies against mitochondrial and bacterial cytochromes c1 completely inhibited their respective cytochrome b-c1 complexes but no cross immunoinhibition was observed. However, when antibodies against bacterial cytochrome c1 were incubated with the delipidated mitochondrial cytochrome b-c1 complex before reconstitution with phospholipids, a 65% inhibition was observed, indicating that the common epitopes between the cytochromes c1 were also somewhat masked by phospholipids. Antibodies against mitochondrial cytochrome c1 inhibited 70% of the succinate oxidase activity in the intact mitochondria preparation, but no inhibition was observed in submitochondrial particles, indicating that some mitochondrial cytochrome c1 epitopes are exposed to the cytoplasmic side. PMID- 3021747 TI - Isolation and properties of fibroblast mutants overexpressing an altered Na+/H+ antiporter. AB - A new method based on the toxicity of low intracellular pH (pHi) was developed to isolate fibroblast variants overexpressing Na+/H+ antiport activity. Chinese hamster lung fibroblasts (CCL39) were incubated for 60 min in medium containing 50 mM NH4Cl. Removal of external NH+4 induced a rapid and lethal intracellular acidification when the Na+/H+ antiporter was inhibited during the 60 min of the pHi recovery phase. The inhibition was provoked either by adding 5-(N-methyl,N propyl)amiloride (MPA, LD50 = 0.3 microM) or by reducing external [Na+] (LD50 = 25 mM). Progressively increasing the MPA concentration during the acid-load selection led to the isolation of two stable variants: AR40 and AR300, resistant, respectively, to 40 and 300 microM MPA. In response to an acid-load, these variants display a much higher rate of pHi recovery due to an overexpression of Na+/H+ antiport activity. In addition, AR40 and AR300 have an altered Na+/H+ antiporter: in AR300 cells K0.5 of MPA for inhibiting Na+/H+ exchange is shifted from 5 X 10(-8) to 1.5 X 10(-6) M, Km (Na+) is decreased 2-fold, and Vmax is increased 4.5-fold. Alternatively reducing Na+ concentration of the pHi recovery saline medium in a stepwise manner led to the selection of another class of variants (DD8 and DD12) also characterized by an altered Na+/H+ antiporter and an increased expression level. The 10-fold increased rate of amiloride-sensitive Na+ influx of DD12 is accounted for by a 4-fold increase in Vmax and a 2.5-fold increase in affinity for Na+ or Li+ at the external site. Interestingly, the affinity for the amiloride analog MPA and for external H+ is unchanged in DD12. In conclusion, the genetic approach presented here: provides a general and specific method for selecting variants of the Na+/H+ antiporter with increased expression levels and/or with structural alterations and demonstrates that the external Na+- and amiloride-binding sites are not identical, since they can be genetically altered independently of each other. PMID- 3021748 TI - Differential recognition of calmodulin-enzyme complexes by a conformation specific anti-calmodulin monoclonal antibody. AB - An anti-calmodulin monoclonal antibody having an absolute requirement for Ca2+ has been produced from mice immunized with a mixture of calmodulin and calmodulin binding proteins. Radioimmune assays were developed for the determination of its specificity. the epitope for this antibody resides on the COOH-terminal half of the mammalian protein. Plant calmodulin or troponin C had little reactivity. The apparent affinity of the antibody for calmodulin was increased approximately 60 fold in the presence of heart calmodulin-dependent phosphodiesterase. The presence of heart phosphodiesterase in the radioimmune assay greatly enhanced the sensitivity for calmodulin. The intrinsic calmodulin subunit of phosphorylase kinase and calmodulin which was bound to brain phosphodiesterases was also recognized with high affinity by the antibody. The antibody reacted poorly with calmodulin which was bound to heart or brain calcineurin, skeletal muscle myosin light chain kinase, or other calmodulin-binding proteins. In direct binding experiments, most of the calmodulin-binding proteins studied were unreactive with the antibody. This selectivity allowed purification of heart and two brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes on immobilized antibody affinity columns. Phosphodiesterase activity was adsorbed directly from crude samples and specifically eluted with EGTA. Isozyme separation was accomplished using a previously described anti-heart phosphodiesterase monoclonal antibody affinity support. The brain isozymes differed not only in reactivity with the anti-phosphodiesterase antibody, but also in apparent subunit molecular weight, and relative specificity for cAMP and cGMP as substrates. The calmodulin activation constants for the brain enzymes were 10-20-fold greater than for the heart enzyme. The data suggest that the binding of ligands to Ca2+/calmodulin induce conformation changes in calmodulin which alter reactivity with the anti calmodulin monoclonal antibody. The differential antibody reactivity toward calmodulin-enzyme complexes indicates that target proteins either induce very different conformations in calmodulin and/or interact with different geometries relative to the antibody binding site. The anti-calmodulin monoclonal antibody should be useful for the purification of other calmodulin-dependent phosphodiesterases as well as isozymes of phosphorylase kinase. PMID- 3021749 TI - Carbachol induces a rapid and sustained hydrolysis of polyphosphoinositide in bovine tracheal smooth muscle measurements of the mass of polyphosphoinositides, 1,2-diacylglycerol, and phosphatidic acid. AB - The effects of carbachol on polyphosphoinositides and 1,2-diacylglycerol metabolism were investigated in bovine tracheal smooth muscle by measuring both lipid mass and the turnover of [3H]inositol-labeled phosphoinositides. Carbachol induces a rapid reduction in the mass of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-monophosphate and a rapid increase in the mass of 1,2 diacylglycerol and phosphatidic acid. These changes in lipid mass are sustained for at least 60 min. The level of phosphatidylinositol shows a delayed and progressive decrease during a 60-min period of carbachol stimulation. The addition of atropine reverses these responses completely. Carbachol stimulates a rapid loss in [3H]inositol radioactivity from phosphatidylinositol 4,5 bisphosphate and phosphatidylinositol 4-monophosphate associated with production of [3H]inositol trisphosphate. The carbachol-induced change in the mass of phosphoinositides and phosphatidic acid is not affected by removal of extracellular Ca2+ and does not appear to be secondary to an increase in intracellular Ca2+. These results indicate that carbachol causes phospholipase C mediated polyphosphoinositide breakdown, resulting in the production of inositol trisphosphate and a sustained increase in the actual content of 1,2 diacylglycerol. These results strongly suggest that carbachol-induced contraction is mediated by the hydrolysis of polyphosphoinositides with the resulting generation of two messengers: inositol 1,4,5-trisphosphate and 1,2 diacylglycerol. PMID- 3021750 TI - ATP-coupled transport of vesicular stomatitis virus G protein between the endoplasmic reticulum and the Golgi. AB - The temperature and ATP dependence of transport of the vesicular stomatitis virus strain ts045 G protein from the endoplasmic reticulum (ER) to an early Golgi compartment containing mannosidase I was studied in the mutant Chinese hamster ovary cell clone 15B. Appearance of G protein containing the Man5GlcNAc2 oligosaccharide species occurred after a shift to the permissive temperature with a lag period of 5 min and without detectable formation of the intermediate Man7GlcNAc2 and Man6GlcNAc2 species. Two biochemically distinct transport steps were detected during transport from the ER to the Golgi. An initial step is temperature sensitive, thermoreversible, and requires a high threshold of cellular ATP for maximal rate of transport (80% of the normal cellular ATP pool). Export from the ER is inhibited at 65% of the normal cellular ATP pool. Prolonged incubation at reduced levels of cellular ATP or at the restrictive temperature resulted in the accumulation of G protein in either the Man8GlcNAc2 species or the Man7GlcNAc2 and Man6GlcNAc2 species, respectively. Reversal of the temperature-sensitive block is ATP coupled. A second step is insensitive to incubation at the restrictive temperature and proceeds efficiently when the cellular ATP pool is reduced to 20% of the control. G protein accumulates at this intermediate step during prolonged incubation at 15 degrees C. The data suggest a functional division of processes required for transport of protein between the ER and Golgi compartments. The two steps may reflect the export (budding) and delivery (fusion) of proteins through vesicular trafficking between the ER and Golgi. PMID- 3021751 TI - ATP-coupled transport of vesicular stomatitis virus G protein. Functional boundaries of secretory compartments. AB - The oligosaccharide processing intermediates of the vesicular stomatitis virus strain ts045 G protein were used to identify ATP- and temperature-sensitive steps in the constitutive pathway of protein transfer to the cell surface. In addition to the initial ATP-sensitive step required for export from the endoplasmic reticulum (Balch, W. E., Elliott, M. M., and Keller, D. S. (1986) J. Biol. Chem. 261, 14681-14689), two distinct ATP-sensitive steps functionally dissect the Golgi into at least 3 compartments: a cis compartment containing the trimming enzyme mannosidase I, a medial compartment conferring resistance to endoglycosidase H, and a trans compartment containing terminal glycosyl transferases. A fourth ATP-sensitive step is required for export of G protein from the trans Golgi to the cell surface. A high threshold of cellular ATP (70% of the control) was required for maximal rates of transport between Golgi compartments. Transport between compartments is inhibited at 40% of the normal cellular ATP pool. Only a single temperature-sensitive step localized to the endoplasmic reticulum inhibited transport of ts045 G protein to the cell surface. The data suggest that ATP-sensitive steps punctuate transport of protein between compartmental boundaries of the secretory pathway. PMID- 3021752 TI - Sequence-dependent S1 nuclease hypersensitivity of a heteronomous DNA duplex. AB - Using cloned (dG-dA)n X (dC-dT)n DNA duplexes [GA)n) as models of homopurine homopyrimidine S1-hypersensitive sites, we show that cleavage of the alternate (non-B, non-Z) DNA structure by S1 nuclease is length-dependent, in both supercoiled and linear forms, which are similar because of the identity of their nicking profiles. However, the length of flanking sequences, the presence of borders, and the DNA topology affect the equilibrium between the alternate structure and B-DNA. The B form of (GA)38 has a 10.4-base pair helical repeat, but the two phosphodiester backbones have different conformations (heteronomous DNA with a dinucleotide repeat unit). Extension experiments reveal that the alternate structure is also heteronomous, in agreement with the nicking patterns generated by S1 and mung bean nucleases and by venom phosphodiesterase. Sensitivity to the latter enzyme at pH 9.0 indicates that the alternate DNA does not appear only in the low pH of the S1 nuclease reaction. Moreover, Hoogsteen G CH+ base-pairing does not seem to be a prerequisite for the appearance of sensitivity because S1 still recognizes the structure even when all Gs are methylated at N-7. This is consistent with the results of chemical probing of the structure using dimethyl sulfate and diethyl pyrocarbonate at various pH values, which show absence of protection at guanine N-7. However, diethyl pyrocarbonate treatment at low pH results in hyper-reactivity of A residues. PMID- 3021753 TI - Anti-immunoglobulin and phorbol ester induce phosphorylation of proteins associated with the plasma membrane and cytoskeleton in murine B lymphocytes. AB - Protein phosphorylations are rapidly induced in intact B cells by antibodies to surface immunoglobulin (anti-IgM) and by phorbol 12-myristate 13-acetate (PMA). A comparison of the molecular weight, isoelectric points, phosphopeptides, and phosphoamino acids of the phosphoproteins induced by anti-IgM and by PMA suggests that anti-IgM acts through the activation of protein kinase C. This conclusion is strengthened by the observation that prolonged treatment with PMA ablates the ability of anti-IgM to induce phosphorylation, presumably by depleting cellular protein kinase C. Furthermore, the effects of dibutyryl cyclic AMP on protein phosphorylation are quite distinct from the effects of anti-IgM. The six most prominent phosphoproteins induced by PMA, with approximate Mr values of 47, 55, 62, 68, 68, and 65-70 X 10(3), are associated with the plasma membrane. Of these, four are apparently associated with the cytoskeleton, suggesting that the phosphorylation of cytoskeletal proteins may be important events early in B cell activation. Examination of protein phosphorylation in cell lines derived from different tissues has identified one major B cell phosphoprotein (Mr 65-70 X 10(3), which is absent in T cells, and two phosphoproteins (Mr 55 and 68 X 10(3), which are observed in cells of hematopoietic origin but which are absent or uncommon in other cell types. PMID- 3021755 TI - Protein phosphatase type-1 and type-2 catalytic subunits both bind inhibitor-2 and monoclonal immunoglobulins. AB - Protein phosphatases involved in cellular regulation have been categorized functionally into two major types by substrate specificity and sensitivity to protein inhibitors. In this classification type-1 phosphatases are inhibited by the heat-stable protein inhibitor-2 (I-2), whereas type-2 phosphatases are considered insensitive to inhibition by this protein. This study demonstrates that the phosphorylase phosphatase activity of both purified type-1 and type-2 catalytic subunits can be blocked by micromolar concentrations of I-2. Heparin also was more effective at inhibiting the type-1 compared to type-2 phosphatase but required thousandfold higher concentrations than I-2. The specificity of the interaction with I-2 indicates that the tertiary structures of the two phosphatase catalytic subunits closely resemble one another. However, only the type-1, not the type-2, protein phosphatase activity was neutralized by immunoglobulins affinity-purified against the Mr = 33,000 catalytic fragment of the type-1 phosphatase. Preparations of rabbit skeletal muscle type-1 phosphatase catalytic fragment and of bovine cardiac type-2 phosphatase catalytic subunit were compared by "Western" immunoblotting with sheep polyclonal and mouse monoclonal immunoglobulins raised against the respective proteins. Monoclonal anti-type-2 immunoglobulins preferentially stained the type-2 phosphatase catalytic subunit used as antigen, but displayed cross-reaction with 10-50 times more of the type-1 phosphatase. In contrast, as found with their effects on activity, sheep anti-type-1 immunoglobulins were specific; immunoblotting detected the type-1, not the type-2, catalytic protein. We conclude that the two catalytic proteins have at least one common primary structural epitope recognized by the monoclonal immunoglobulins. These data, taken together with other recent immunochemical results, support a hypothesis that this family of enzymes was derived from a common ancestral protein phosphatase catalytic subunit. PMID- 3021754 TI - Species specificity of human and murine tumor necrosis factor. A comparative study of tumor necrosis factor receptors. AB - Recombinant murine and human tumor necrosis factor (mTNF and hTNF, respectively) were radioiodinated to high specific activity using a solid-phase lactoperoxidase method. A single class of high affinity receptors for 125I-TNF was identified on TNF-sensitive murine L cells and human HeLa S2 cells. Competitive radioligand binding assays were used to study the species specificity of TNF preparations. Unlabeled hTNF competed 30-fold less effectively than mTNF for binding to L cell receptors, whereas mTNF competed to approximately the same extent as hTNF for binding to HeLa cell receptors. A similar species specificity was observed in cytotoxicity assays; hTNF was more cytotoxic for HeLa cells than mTNF. Conversely, mTNF was more growth inhibitory and cytotoxic for L cells than hTNF. mTNF. and hTNF.receptor complexes were compared by gel filtration chromatography and polyacrylamide gel electrophoresis before and after cross-linking with bis[2 (succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES). These complexes eluted in gel filtration at a position corresponding to a globular protein of 350,000 Mr. Gel autoradiographs of the fractions containing cross-linked complexes showed bands of 95,000 and 75,000 Mr as well as small amounts of higher Mr bands. mTNF and hTNF treated with BSOCOES formed cross-linked dimers and trimers. Therefore, we were unable to determine whether the 95,000 and 75,000 Mr bands represented two distinct subunits of receptors or one subunit to which either a dimer or a monomer of TNF was cross-linked. These results demonstrate species specificity in the TNF-receptor interaction. In addition, the affinity labeling studies in two species give an identical pattern for the TNF X receptor complexes, suggesting that the receptors have similar subunit composition. PMID- 3021756 TI - Escherichia coli exonuclease VII. Cloning and sequencing of the gene encoding the large subunit (xseA). AB - We have determined the sequence of the gene encoding the large subunit of Escherichia coli exonuclease VII (xseA) and the amino acid sequence of the protein it encodes. The coding region of the xseA gene is 1368 base pairs. The protein encoded by the gene contains 456 amino acids and has a calculated molecular weight of 51,823. The promoter for xseA is close to that for guaB, and these two genes are transcribed in opposite directions: xseA clockwise and guaB counterclockwise on the standard E. coli genetic map. The cloned xseA gene can complement an xseA deletion mutant strain. In an xseA+ genetic background production of large quantities of the xseA gene product appeared to decrease the amount of exonuclease VII activity in cell extracts. In fact, no exonuclease VII activity at all could be detected following induction of strains in which the xseA gene was under lambda pL regulation. These observations suggest that the proper ratio of the large and small exonuclease VII subunits must be maintained in order to produce active enzyme. PMID- 3021757 TI - Absence of iron transfer from uteroferrin to transferrin. AB - Transfer of iron from native porcine uteroferrin to apotransferrin was investigated using EPR spectroscopy. Purple (oxidized) or pink (reduced) forms of uteroferrin were incubated with porcine or human apotransferrin under conditions of temperature (37 degrees C) and pH (6.8) approximating those found in the allantoic fluid of the pregnant sow. Studies were also performed in the presence of mediators such as ascorbate, citrate, and ATP in concentrations previously claimed to be effective in promoting large-scale transfer of iron (Buhi, W. C., Ducsay, C. A., Bazer, F. W., and Roberts, R. M. (1982) J. Biol. Chem. 257, 1712 1723). Our experiments indicate that even in the presence of mediators, less than 20% of the iron in uteroferrin is transferred to apotransferrin at the end of 24 h and such transfer may be accompanied by denaturation of uteroferrin. We therefore conclude that the direct transfer of iron to apotransferrin is unlikely to be a physiological role of uteroferrin. PMID- 3021758 TI - Intrinsic differences of insulin receptor kinase activity in red and white muscle. AB - The sensitivity and responsiveness of glucose uptake and glycogen synthesis to insulin are 3-4-fold greater in red than in white skeletal muscle (James, D. E., Jenkins, A. B., and Kraegen, E. W. (1985) Am. J. Physiol. 248, E567-E574). In the present study, the insulin receptor tyrosine kinase activity has been examined in red and white muscle of rats. Partially purified insulin receptors were obtained from muscle following solubilization in detergent, ultracentrifugation, and lectin affinity chromatography. Total insulin receptor number per gram of tissue was slightly higher in red (30%) than in white muscle. In contrast, basal and insulin-stimulated autophosphorylation, normalized for receptor number, were 2.3 fold higher in red muscle. A similar difference was observed in the ability of partially purified receptors to phosphorylate the exogenous substrate polyglutamate/tyrosine. The integrity of the insulin receptor preparation in the two fiber types was identical as determined by affinity cross-linking of [125I TyrB26]insulin to the receptor. Mixing partially purified receptors from red and white muscle resulted in an additive response for exogenous substrate phosphorylation, suggesting that the difference in tyrosine kinase activity was not due to the presence of an inhibitor or activator. The results suggest that there are differences in the insulin receptors of red and white muscles that lead to discordance in their basal and insulin-stimulated intrinsic tyrosine kinase activity. The correlation between these differences and insulin action in red and white muscle supports the concept that the insulin receptor tyrosine kinase activity is involved in the initiation of insulin action. PMID- 3021759 TI - Effect of depletion of phosphate and bicarbonate ions on insulin action in rat adipocytes. AB - The effect of insulin on rat adipocytes was studied in isotonic buffers (pH 7.4) containing NaCl, CaCl2, MgSO4, KCl, and bovine serum albumin but no phosphate or bicarbonate anions. In phosphate- and bicarbonate-free buffers the dose-response curve to insulin is shifted to the right, the effects of the hormone on hexose uptake, glucose metabolism, and inhibition of lipolysis being observed at much higher (nearly 2 orders of magnitude) concentrations of insulin. The insulin binding capacity of the cells is only slightly changed. The dose-response curve for isoproterenol which stimulates lipolysis in the same cell type is almost the same in both Krebs-Ringer bicarbonate buffer and phosphate- and bicarbonate-free buffers. The dose-response curves for agents that mimic the action of insulin such as wheat germ agglutinin or vanadate ions are also shifted to the right. The dose-response curve to insulin can be returned to "normal" by readdition of either bicarbonate or phosphate. Almost complete recovery is obtained at either 10 mM bicarbonate or 24 mM phosphate, respectively. External Ca2+ ions which are not required for the proper action of insulin in fat cells maintained in Krebs Ringer bicarbonate buffer, become essential for insulin action in bicarbonate free buffer. The study indicates that depletion of bicarbonate and, to a lesser extent, phosphate anions, interferes with an essential insulin-dependent post binding event. Also, in bicarbonate-free medium, external Ca2+ ions are essential for insulin-mediated processes. The implications of this study to the mode of action of insulin, and to physiological and clinical states of insulin desensitization are discussed. PMID- 3021760 TI - Effect of depletion of bicarbonate or phosphate ions on insulin action in rat adipocytes. Further characterization of the receptor-effector system. AB - In the preceding paper (Shechter, Y., and Ron, A. (1986) J. Biol. Chem. 261, 14945-14950) we have shown that in fat cells, prepared and maintained in an isotonic buffer (pH 7.4) containing neither phosphate nor bicarbonate anions (Buffer A), the dose-response curve to insulin shifted to the right by about 2 logarithms and insulin binding affinity or capacity was only slightly decreased. In the current paper we demonstrate that progressive loss of insulin binding, either by treatment with trypsin or preincubating the cells with isoproterenol, correlates well with the reduced ability of the cells to elicit maximal lipogenesis in response to insulin. We further demonstrate in the "new" system that: the dissociation of labeled insulin from fat cells is not accelerated by the inclusion of unlabeled insulin in the medium; termination of lipogenesis in Buffer A occurs immediately; ligand-induced receptor internalization is grossly defective; and insulin is unable to stimulate lipogenesis at 15 degrees C. The data support the hypothesis that in the new experimental system all measurable binding sites are linked to a coupling mechanism. Each site behaves as an independent, separate entity and there are no site to site interactions. This leads to a linear relationship between binding and bioactivation, lack of negative or positive cooperatively, accelerated rate of termination, defective internalization, a shift to the right in the dose-response curve to insulin, and a lack of insulin response at a lower temperature. In more general terms, the study indicates that all measurable insulin receptors are chemically homogeneous in their potential capability to be coupled to an insulin effector (biologically relevant) system, and they do so under particular experimental conditions. PMID- 3021761 TI - Separate subunits for agonist and benzodiazepine binding in the gamma aminobutyric acidA receptor oligomer. AB - The gamma-aminobutyric acidA (GABAA) agonist muscimol can be photoactivated by 254 nm illumination to affinity label its binding site in the GABAA receptor. We have conducted this reaction on the pure receptor from bovine cerebral cortex in detergent solution, showing that [3H]muscimol can produce then a specific saturable labeling. In the detergent solution, the receptor alone is sensitive to 254 nm irradiation; this reduces the efficiency of incorporation to below that in the membranes, but the competing photoreaction with [3H]muscimol is sufficient and occurs at a representative set of the muscimol-binding sites, such that it can be employed for the photolabeling of those sites. The affinity of [3H]muscimol displayed in this irreversible reaction is indistinguishable from that of its reversible binding. gamma-Aminobutyric acid and bicuculline compete in the photolabeling reaction according to their known affinities at the gamma aminobutyric acid-binding site. The labeling is shown to occur at the beta subunit (apparent Mr 57,000) in the pure receptor. The binding sites for gamma aminobutyric acid agonists, on the beta-subunits, and the benzodiazepine binding sites, on the alpha-subunits, are linked allosterically so that a strongly cooperative hetero-oligomeric structure of this receptor is deduced. PMID- 3021762 TI - The primary structure and the functional domains of an elongation factor-1 alpha from Mucor racemosus. AB - We have determined the complete nucleotide sequence for TEF-1, one of three genes coding for elongation factor (EF)-1 alpha in Mucor racemosus. The deduced EF-1 alpha protein contains 458 amino acids encoded by two exons. The presence of an intervening sequence located near the 3' end of the gene was predicted by the nucleotide sequence data and confirmed by alkaline S1 nuclease mapping. The amino acid sequence of EF-1 alpha was compared to the published amino acid sequences of EF-1 alpha proteins from Saccharomyces cerevisiae and Artemia salina. These proteins shared nearly 85% homology. A similar comparison to the functionally analogous EF-Tu from Escherichia coli revealed several regions of amino acid homology suggesting that the functional domains are conserved in elongation factors from these diverse organisms. Secondary structure predictions indicated that alpha helix and beta sheet conformations associated with the functional domains in EF-Tu are present in the same relative location in EF-1 alpha from M. racemosus. Through this comparative structural analysis we have predicted the general location of functional domains in EF-1 alpha which interact with GTP and tRNA. PMID- 3021763 TI - Nucleotide sequence of the structural genes for an anion pump. The plasmid encoded arsenical resistance operon. AB - The structural genes for the arsenical pump of the conjugative R-factor R773 contained on a HindIII fragment of 4.3 kilobase pairs were cloned into bacteriophage M13. A series of ordered deletions was created using Bal31 digestion, and the nucleotide sequence of the operon determined. Three open reading frames for genes arsA, arsB, and arsC were found. The arsA gene encodes a hydrophilic protein of 63,169 Da with two potential adenylate-binding sites. The arsB gene encodes a potentially membrane protein of 45,577 Da. The arsC gene encodes a 15,811-Da hydrophilic protein. The arsA and arsC gene products correspond to cytosolic proteins previously identified from minicell experiments. Isolated ArsA protein was shown to bind to dye-agarose columns which act as affinity resins for nucleotide-binding proteins. A model is proposed in which these gene products form an anion translocating ATPase for extrusion of arsenite and arsenate from resistant cells. PMID- 3021764 TI - sn-1,2-Diacylglycerol kinase of Escherichia coli. Structural and kinetic analysis of the lipid cofactor dependence. AB - The lipid cofactor requirement of Escherichia coli sn-1,2-diacylglycerol kinase was studied using a beta-octylglucoside mixed micellar assay (Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 6239-6247). The enzyme was shown to have an absolute requirement for a lipid activator. sn-1,2-Dioleoylglycerol was both an activator and a substrate for the enzyme, 1,3-dioleoylglycerol was an activator but not a substrate, and sn-1,2-dioctanoylglycerol was a substrate but not an activator. Activation was observed with a large number of phospholipids, sulfolipids, neutral lipids, and detergents. Lipids with longer alkyl/acyl chains stimulated activity to a greater extent and at lower concentrations than their shorter chain homologs. Anionic lipids were the best activators, and neutral lipids were somewhat less effective. Cationic lipids were poor activators. Lipid activation was cooperative in all cases, with Hill coefficients ranging from 2.9 to 4.7. Lipid activators stabilized the enzyme against inactivation induced by diacylglycerols. The effectiveness of several lipids in stabilizing the enzyme correlated with their effectiveness as kinetic activators, suggesting a common mechanism. Kinetic analyses also suggested that a lipid cofactor-induced conformational change occurs as a part of the activation process. beta Octylglucoside was shown not to function as a lipid cofactor for diacylglycerol kinase. The requirement for detergent in the assay was related, instead, to the need to disperse and deliver water-insoluble substrates and cofactors to the enzyme. beta-Octylglucoside also provided an inert matrix to which lipid substrates and cofactors could be added, enabling study of their concentration dependencies. PMID- 3021765 TI - Regulation by phorbol esters of asialoglycoprotein and transferrin receptor distribution and ligand affinity in a hepatoma cell line. AB - We have investigated the simultaneous regulation of cell surface distribution and ligand binding of the asialoglycoprotein (ASGP) receptor and the transferrin receptor in a hepatoma cell line by phorbol esters. One hour exposure to phorbol esters causes a redistribution of both receptors to the cell interior as shown by radioligand binding at 4 degrees C and selective immunoprecipitation from the plasma membrane. This effect is temperature- and dose-dependent and is not seen with 4-alpha-phorbol, an inactive tumor promoter. The mechanism and kinetics of the ASGP receptor response to phorbol esters appears to differ from that of the transferrin receptor in this cell line. Within the first 10 min there is a decrease in binding of iodinated ligands for both receptors to the HepG2 cell surface. For the transferrin receptor this results from a net internalization of receptor molecules from the plasma membrane pool, while for the ASGP receptor this decrease is accounted for by a 3.5-fold reduction in ligand binding affinity (6.6 X 10(-8) M to 24.0 X 10(-8) M), with essentially no change in the number of ASGP receptors recoverable from the plasma membrane pool by immunoprecipitation. The altered affinity of the ASGP-R is transient; the Kd returns to control levels by 20 min of continued exposure to the agent. The transferrin receptor shows no change in binding affinity during the course of exposure to phorbol esters. ASGP receptors in cells exposed to phorbol esters for 1 h maintain their competence to deliver exogenous ligand to intracellular sites of degradation and to participate in the recycling pathway of receptor-mediated endocytosis, although at a lower rate than in control cells. We conclude that under identical conditions phorbol esters modulate the binding capacity of two receptors at the cell surface by separate mechanisms. Furthermore, the transient nature of the altered ASGP-R binding affinity suggests that at least two mechanisms, receptor redistribution as well as decreased binding affinity, are operative in the modulation of ASGP-R cell surface binding during the first hour of exposure to the phorbol esters. PMID- 3021766 TI - Cloning and nucleotide sequence of wild type and a mutant histidine decarboxylase from Lactobacillus 30a. AB - Prohistidine decarboxylase from Lactobacillus 30a is a protein that autoactivates to histidine decarboxylase by cleaving its peptide chain between serines 81 and 82 and converting Ser-82 to a pyruvoyl moiety. The pyruvoyl group serves as the prosthetic group for the decarboxylation reaction. We have cloned and determined the nucleotide sequence of the gene for this enzyme from a wild type strain and from a mutant with altered autoactivation properties. The nucleotide sequence modifies the previously determined amino acid sequence of the protein. A tripeptide missed in the chemical sequence is inserted, and three other amino acids show conservative changes. The activation mutant shows a single change of Gly-58 to an Asp. Sequence analysis up- and downstream from the gene suggests that histidine decarboxylase is part of a polycistronic message, and that the transcriptional promotor region is strongly homologous to those of other Gram positive organisms. PMID- 3021767 TI - Antibody-induced receptor loss. Different fates for asialoglycoproteins and the asialoglycoprotein receptor in HepG2 cells. AB - The human asialoglycoprotein receptor (ASGP-R) is a membrane glycoprotein which participates in receptor-mediated endocytosis and delivery of its ligands to lysosomes for degradation. In order to examine the pathways and mechanisms responsible for the turnover and degradation of the ASGP-R we have followed the fate of the ASGP-R in HepG2 cells during exposure to anti-receptor antibody as well as inhibitors of lysosomal processing and receptor recycling. Incubation of cells at 37 degrees C with anti-ASGP-R antibody results in the rapid (t 1/2 30 min) loss of mature 46,000-Da ASGP-R (control, t 1/2 20 h). This process requires whole IgG, since Fab fragments do not induce loss of receptor. Furthermore, this antibody-induced loss is specific, since incubation with antibody to the transferrin receptor does not alter cellular ASGP-R content. Of note, weak bases (e.g. primaquine) abrogate this antibody-induced loss of ASGP-R. Inhibitors of lysosomal proteases (EC64 and leupeptin) do not alter this antibody-mediated loss. Furthermore, this effect occurs at 18 degrees C, a temperature at which delivery of ligand to the lysosome is blocked. Thus, the present observations suggest a unique pathway for antibody-induced ASGP-R loss which is distinct from the pathway of lysosomal delivery of ligand. PMID- 3021768 TI - Developmental changes in cardiac sarcoplasmic reticulum in sheep. AB - Physiologic studies suggest that the myocardium from fetal and newborn sheep functions at a higher contractile state with decreased contractile reserve when compared to the myocardium of adult sheep. To investigate the role of Ca2+ transport by the sarcoplasmic reticulum (SR) in this phenomenon, we studied functional properties and protein composition of cardiac SR vesicles isolated from fetal and maternal sheep. Active accumulation of Ca2+ and the density of the Ca2+ pump protein were decreased 60% (p less than 0.01) in fetal SR vesicles; however Ca2+-dependent ATPase activity was decreased only 30% (p less than 0.01). This decreased difference in Ca2+-dependent ATPase activities was accounted for by the higher turnover number measured for the Ca2+ pump of fetal SR vesicles (1.6-fold increased, p less than 0.01). Ryanodine, an alkaloid which blocks Ca2+ efflux from cardiac SR vesicles, stimulated Ca2+ uptake more effectively in fetal SR vesicles, suggesting that these vesicles had a higher passive Ca2+ permeability during conditions of active Ca2+ transport. Protein compositional studies showed that the content of phospholamban was decreased in fetal SR vesicles and was correlated with the decrease in the density of Ca2+ pumps. In contrast, the content of calsequestrin and the density of [3H]nitrendipine binding sites were increased approximately 2-fold in fetal SR vesicles. These functional and compositional differences between SR vesicles isolated from fetal and maternal sheep may indicate that there is relatively more junctional SR in fetal hearts. Since the SR regulates muscle contraction by modulating intracellular Ca2+ concentration, it is possible that developmental alterations in cardiac SR may contribute to the decreased myocardial contractile reserve noted in fetal sheep. PMID- 3021770 TI - Isolation and characterization of a second nitrogenase Fe-protein from Azotobacter vinelandii. AB - Wild-type Azotobacter vinelandii strain UW was transformed with plasmid pDB12 to produce a species (LS10) unable to synthesize the structural proteins of component 1 and component 2 of native nitrogenase. A spontaneous mutant of this strain was isolated (LS15) which can grow by nitrogen fixation in the presence or absence of either Mo or W. It is proposed that LS15 fixes nitrogen solely by an alternative nitrogen-fixing system which previously has been hypothesized to exist in A. vinelandii. Under nitrogen-fixing conditions, LS15 synthesizes a protein similar to component 2 (Av2) of native nitrogenase in that it can complement native component 1 (Av1) for enzymatic activity. Isolation and characterization of this second component 2 shows it to be a 4Fe-4S protein of molecular mass about 62 kDa and is antigenically similar to Av2. This protein is also similar to Av2 in that in the reduced state it possesses a rhombic ESR spectrum in the g = 2 region, which changes to an axial spectrum upon addition of MgATP. It is suggested that this second Fe-protein is associated with the alternative nitrogen-fixing system in A. vinelandii. PMID- 3021769 TI - Structural requirements for the transmembrane activation of the insulin receptor kinase. AB - Tetrameric insulin holoreceptor (alpha 2 beta 2) was reduced with dithiothreitol into alpha beta dimers such that they maintain up to 50% of insulin binding at tracer ligand concentrations. Scatchard analysis of insulin binding to dimers revealed that they had a reduced affinity for ligand by a factor of 3-6 compared to holoreceptor, whereas the maximum number of high affinity binding sites was not affected. The alpha beta dimers can be separated from holoreceptor by sucrose density gradient centrifugation, and hence, they are not associated by noncovalent interactions. Insulin-dependent autophosphorylation of alpha beta dimers isolated from low ionic strength sucrose density gradients was minimal and was always accompanied by reoxidation of dimers to the tetrameric holoreceptor. The reformed tetramer exhibited a strong insulin-dependent autophosphorylation reaction. Reoxidation was prevented by isolating alpha beta dimers in sucrose density gradients containing 0.15 M NaCl. Under these conditions, no insulin dependent autophosphorylation was observed. When insulin receptor was first autophosphorylated and then reduced, receptor kinase activity, as assayed by histone phosphorylation, was not affected. Also, the insulin-independent, basal autophosphorylation was maintained after reduction into alpha beta dimers. We conclude that alpha beta-alpha beta interaction is not necessary for the maintenance of basal kinase activity or for insulin-activated kinase activity once autophosphorylation occurs. However, dimer-dimer interaction appears critical for the insulin-dependent activation of the receptor's intrinsic kinase activity. PMID- 3021771 TI - A single Mr approximately 103,000 125I-beta-nerve growth factor-affinity-labeled species represents both the low and high affinity forms of the nerve growth factor receptor. AB - Both high and low affinity receptors for nerve growth factor (NGF) have been described, but only the former appear to mediate NGF actions and uptake. To specifically characterize the molecular identity of the high affinity site and to compare it with the low affinity site, the water-soluble carbodiimide EDC was used to cross-link 125I-NGF to NGF receptors on: rat PC12 cells, PC12nnr5 cells (PC12 mutants that have only low affinity NGF binding), SH-SY5Y human neuroblastoma cells (which have only high affinity binding sites), and cultured rat sympathetic ganglion cells. A variety of criteria were used to distinguish the two classes of affinity-labeled receptors: competition with unlabeled NGF, dissociation rate, and selective solubilization by 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that cross-linking generated only a single Mr approximately 103,000 125I-NGF affinity-labeled species which represents both the low and high affinity forms of the receptor. The 125I-NGF X receptor complexes formed with both affinity classes of the receptor were quantitatively immunoprecipitated by the monoclonal anti-NGF receptor antibody 192-IgG and both showed identical shifts in mobility when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. These findings indicate that both high and low affinity NGF receptors possess apparently identical NGF-binding moieties. The differences between the kinetic and functional properties of the two receptor types may therefore result from their interactions with other membrane components or with cytoplasmic proteins. PMID- 3021772 TI - GABA-related activities of amino phosphonic acids on guinea-pig ileum longitudinal muscle. AB - The effects of phosphonic analogues of GABA, beta-alanine and glycine on guinea pig ileum longitudinal muscle were measured. Aminomethylphosphonic acid (AMPh) and 2-aminoethylphosphonic acid (2-AEPh) were devoid of any effect both in non stimulated preparations and in electrically-stimulated preparations. The phosphonic analogue of GABA, 3-aminopropylphosphonic acid (3-APPh) possessed a GABAB agonistic effect (relaxation and inhibition of twitch response) at doses of 10(-3)M. No agonistic effect on GABAA receptors was observed. 3-APPh at doses tested (2 X 10(-4)M and 10(-3)M) also displayed antagonistic action on the effects of GABAB agonists producing a parallel shift of the log dose-effect curves of GABA- and (-)-baclofen-inhibition of twitch responses. In contrast 3 APPh did not antagonize the inhibitory effect of morphine and noradrenaline. The contractile effect of GABA, mediated via GABAA receptors, was unaffected by 3 APPh(10(-3)M). It is concluded that 3-APPh is a partial agonist at the GABAB site in guinea-pig ileum. PMID- 3021773 TI - Beta 2-adrenoreceptor binding sites in circular and longitudinal myometrial layers of the virgin guinea-pig: the influence of ovarian steroids. AB - Membranes prepared from both circular and longitudinal muscle layers of the uteri of two groups of virgin adult guinea-pigs were used to study the influence of ovarian steroids upon beta-adrenoreceptor binding sites. Animals were (i) treated for 14 days with oestradiol cypionate, beginning on day 9-10 of the oestrous cycle; or (ii) treated as in (i) and in addition, with progesterone for the last four days of oestradiol administration. (-)-[125I]-iodocyanopindolol ([125I]-CYP) was used to determine the numbers and characteristics of beta-adrenoreceptor binding sites in the four membrane preparations. In all cases, binding displayed characteristics of a saturable bimolecular reaction; the estimates of receptor site density (Bmax; 0.26-0.33 pmol g-1 wet weight) were similar in all four preparations, as were those of binding affinity KD; 18-31 pmol l-1). The mean negative logarithms of apparent dissociation constants (pKD) for the inhibition of specific [125I]-CYP binding by ICI 118, 551 (beta 2-adrenoreceptor selective antagonist) ranged from 8.4 to 8.6; and those for L 643, 717-01J10 (beta 1 adrenoreceptor selective antagonist) were 5.7-6.2. Thus the [125I]-CYP binding sites in all four membrane preparations displayed the characteristics expected of homogeneous populations of adrenoreceptors of the beta 2-subtype. The pKD values for isoprenaline were also similar in each type of membrane preparation (5.8 6.0). It is concluded that the clearcut differences in the contractile responsiveness, to adrenoreceptor agonists, of the circular and longitudinal myometrial preparations from oestrogen-treated guinea-pigs are not due to differences in the numbers, subtype or binding affinities of beta-adrenoreceptor binding sites.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021775 TI - Dedifferentiated chondrosarcoma. A report of the clinicopathological features and treatment of seventy-eight cases. AB - Dedifferentiated chondrosarcoma is a highly malignant variant of chondrosarcoma. Approximately 11 per cent of chondrosarcomas can be expected to dedifferentiate into more anaplastic lesions. In this report, we analyze the clinicopathological features and treatment of seventy-eight lesions of this type. The ages of the patients ranged from nineteen to eighty-two years (average, 54.6 years). The cartilaginous precursor was central in most patients. Eleven of the lesions developed in the site of a previously resected low-grade chondrosarcoma. Dedifferentiation was from low-grade chondrosarcoma to osteosarcoma in forty-two patients, to fibrosarcoma in thirty-three, and to malignant (fibrous) histiocytoma in three. Perforation of the cortex and a soft-tissue mass were found in most of the patients. Widespread metastatic disease within two years after resection was a frequent finding. The over-all five-year-survival rate was 10.5 per cent. Any potential for a "cure" is related to early diagnosis and adequate surgical treatment by amputation or resection. PMID- 3021774 TI - Picric acid functions as a releaser of [14C]acetylcholine in isolated ileal synaptosomal preparations of guinea-pig. AB - The effect of picric acid on the release of [14C]acetylcholine has been investigated in isolated ileal synaptosomes of guinea-pig. Nicotine, high K depolarization (50 mM KCl) and electrical field stimulation were employed to characterize the specificity of picric acid. Picric acid induced the release of labelled acetylcholine in a dose-dependent manner and this action was negated by the removal of calcium ions from the bathing medium. Tetrodotoxin (0.1 microM) abolished the actions of picric acid, nicotine or electrical field stimulation (0.1 Hz). It reduced but did not totally suppress the effect of high K depolarization. Agents capable of affecting the content of cyclic AMP, such as forskolin and alloxan, modified the effects of picric acid or nicotine but did not influence the effects of high K-depolarization or electrical field stimulation. Indomethacin, at a concentration (1 microM) effective in inhibiting the synthesis of prostaglandins, reduced the release of acetylcholine evoked by picric acid or nicotine, but did not affect the responses to high K depolarization or electrical field stimulation. [3H]5-hydroxytryptamine was also released by high K-depolarization at a concentration sufficient to induce the release of acetylcholine. Similar results were obtained when the frequency of electrical field stimulation was raised to 10 Hz. However, picric acid did not initiate the release of 5-hydroxytryptamine. These results suggest that picric acid functions as a releaser of acetylcholine through a mechanism different from that of other stimulants. PMID- 3021777 TI - Benign fibrous histiocytoma of bone. AB - The cases of seven patients who had a lytic lesion that was histologically similar to a metaphyseal fibrous defect (non-ossifying fibroma) of bone were studied. The patients all were adults and had pain without a fracture. These features were considered distinctive for the lesion, which has the same histological appearance as benign fibrous histiocytoma of soft tissue. The lesion is a benign tumor with fibroblastic and histiocytic differentiation. This picture may be seen in foci in other lesions of bone (aneurysmal bone cyst, fibrous dysplasia, and giant-cell tumor). Ten cases of giant-cell tumor of bone that had a large component of the same foci were also reviewed. It should be emphasized that these areas are secondary reactive tissue rather than the true neoplastic tissue of benign fibrous histiocytoma. PMID- 3021776 TI - Hyperbaric oxygen reduces edema and necrosis of skeletal muscle in compartment syndromes associated with hemorrhagic hypotension. AB - This study examined the effect of exposures to hyperbaric oxygen on the development of the edema and necrosis of muscle that are associated with compartment syndromes that are complicated by hemorrhagic hypotension. A compartment syndrome (twenty millimeters of mercury for six hours) was induced by infusion of autologous plasma in the anterolateral compartment of the left hind limb of seven anesthetized dogs while the mean arterial blood pressure was maintained at sixty-five millimeters of mercury after 30 per cent loss of blood volume. These dogs were treated with hyperbaric oxygen (two atmospheres of pure oxygen) and were compared with six dogs that had an identical compartment syndrome and hypotensive condition but were not exposed to hyperbaric oxygen. Forty-eight hours later, edema was quantified by measuring the weights of the muscles (the pressurized muscle compared with the contralateral muscle), and necrosis of muscle was evaluated by measuring the uptake of technetium-99m stannous pyrophosphate. The ratio for edema was significantly (p = 0.01) greater in dogs that had not been exposed to hyperbaric oxygen (1.15 +/- 0.01) than in the dogs that had been treated with hyperbaric oxygen (1.01 +/- 0.03), and the ratio for necrosis of muscle was also significantly (p = 0.04) greater in dogs that had not had hyperbaric oxygen (1.96 +/- 0.41) than in those that had been treated with hyperbaric oxygen (1.05 +/- 0.11). Comparisons were also made with the muscles of four normal control dogs and separately with the muscles of six normotensive dogs that had an identical compartment syndrome and normal blood pressure and were not treated with hyperbaric oxygen.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021778 TI - Morphological predictors of survival in advanced gastric carcinoma univariate and multivariate analysis. AB - In 76 curative resected advanced gastric carcinomas, the relationships between macro- and microscopy of the carcinoma and the survival rate were studied by univariate and multivariate survival analyses. In the Kaplan-Meier survival analysis (product limit estimator), significant influence on survival rate was found for tumor size, Lauren type, number of lymphocytes, tumor fibrosis, and N stage. In the multivariate survival analysis of covariates according to the Cox regression model, in macroscopically evaluable variables the tumor size was effective, in bioptically evaluable variables the number of plasma cells and the histological type. Concerning the primary tumor, the same set of variables presents significant correlations to survival time. Adding the involvement of lymph nodes, Lauren type, and N stage express all significant correlations of the tumor to survival rate. In parametric multivariate stepwise regression analyses of survival time, all variables working in the Cox proportional hazard model were very ineffective. But adaption of the Kaplan-Meier test to the effective variables of multivariate survival analysis elucidates the tight relationships between survival rate and these variables. No distinct relationships are present between variables of primary tumor and presence of lymph nodes metastases or N stage respectively. PMID- 3021779 TI - The signal recognition particle receptor is a complex that contains two distinct polypeptide chains. AB - Signal recognition particle (SRP) and SRP receptor are known to be essential components of the cellular machinery that targets nascent secretory proteins to the endoplasmic reticulum (ER) membrane. Here we report that the SRP receptor contains, in addition to the previously identified and sequenced 69-kD polypeptide (alpha-subunit, SR alpha), a 30-kD beta-subunit (SR beta). When SRP receptor was purified by SRP-Sepharose affinity chromatography, we observed the co-purification of two other ER membrane proteins. Both proteins are approximately 30 kD in size and are immunologically distinct from each other, as well as from SR alpha and SRP proteins. One of the 30-kD proteins (SR beta) forms a tight complex with SR alpha in detergent solution that is stable to high salt and can be immunoprecipitated with antibodies to either SR alpha or SR beta. Both subunits are present in the ER membrane in equimolar amounts and co-fractionate in constant stoichiometry when rough and smooth liver microsomes are separated on sucrose gradients. We therefore conclude that SR beta is an integral component of SRP receptor. The presence of SR beta was previously masked by proteolytic breakdown products of SR alpha observed by others and by the presence of another 30-kD ER membrane protein (mp30) which co-purifies with SR alpha. Mp30 binds to SRP-Sepharose directly and is present in the ER membrane in several-fold molar excess of SR alpha and SR beta. The affinity of mp30 for SRP suggests that it may serve a yet unknown function in protein translocation. PMID- 3021780 TI - Isolated secretion granules from parotid glands of chronically stimulated rats possess an alkaline internal pH and inward-directed H+ pump activity. AB - Secretion granules have been isolated from the parotid glands of rats that have been chronically stimulated with the beta-adrenergic agonist, isoproterenol. These granules are of interest because they package a quantitatively different set of secretory proteins in comparison with granules from the normal gland. Polypeptides enriched in proline, glycine, and glutamine, which are known to have pI's greater than 10, replace alpha-amylase (pI's = 6.8) as the principal content species. The internal pH of granules from the treated rats ranges from 7.8 in a potassium sulfate medium to 6.9 in a choline chloride medium. The increased pH over that of normal parotid granules (approximately 6.8) appears to reflect the change in composition of the secretory content. Whereas normal mature parotid granules have practically negligible levels of H+ pumping ATPase activity (Arvan, P., G. Rudnick, and J. D. Castle, 1985, J. Biol. Chem., 260, 14945-14952) the isolated granules from isoproterenol-treated rats undergo a time-dependent internal acidification (approximately 0.2 pH unit) that requires the presence of ATP and is abolished by an H+ ionophore. Additionally, an inside-positive granule transmembrane potential develops after ATP addition that depends upon ATP hydrolysis. Two independent methods have been used that exclude the possibility that contaminating organelles are the source of the H+-ATPase activity. Together these data provide clear evidence for the presence of an H+ pump in the membranes of parotid granules from chronically stimulated rats. However, despite the presence of H+-pump activity, fluorescence microscopy with the weak base, acridine orange, reveals that the intragranular pH in live cells is greater than that of the cytoplasm. PMID- 3021781 TI - Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor. AB - Treatment of cells with tumor-promoting phorbol diesters, which causes activation of protein kinase C, leads to phosphorylation of the epidermal growth factor (EGF) receptor at threonine-654. Addition of phorbol diesters to intact cells causes inhibition of the EGF-induced tyrosine-protein kinase activity of the EGF receptor and it has been suggested that this effect of phorbol diesters is mediated by the phosphorylation of the receptor by protein kinase C. We measured the activity of protein kinase C in A431 cells by determining the incorporation of [32P]phosphate into peptides containing threonine-654 obtained by trypsin digestion of EGF receptors. After 3 h of exposure to serum-free medium, A431 cells had no detectable protein kinase C activity. Addition of EGF to these cells resulted in [32P] incorporation into threonine-654 as well as into tyrosine residues. This indicates that EGF promotes the activation of protein kinase C in A431 cells. The phosphorylation of threonine-654 induced by EGF was maximal after only 5 min of EGF addition and the [32P] incorporation into threonine-654 reached 50% of the [32P] in a tyrosine-containing peptide. This indicates that a significant percentage of the total EGF receptors are phosphorylated by protein kinase C. A variety of external stimuli activate Na+/H+ exchange, including EGF, phorbol diesters, and hypertonicity. To ascertain whether activation of protein kinase C is an intracellular common effector of all of these systems, we measured the activity of protein kinase C after exposure of A431 cells to hyperosmotic conditions and observed no effect on phosphorylation of threonine-654, therefore, activation of Na+/H+ exchange by hypertonic medium is independent of protein kinase C activity. Since stimulation of protein kinase C by phorbol diesters results in a decrease in EGF receptor activity, the stimulation of protein kinase C activity by addition of EGF to A431 cells contributes to a feedback mechanism which results in the attenuation of EGF receptor function. PMID- 3021784 TI - Stimulation of bovine endothelial cell angiotensin-I-converting enzyme activity by cyclic AMP-related agents. AB - Bovine pulmonary artery endothelial cells in culture were used to assess the influence of cyclic nucleotides, isoproterenol (beta adrenergic agonist), and theophylline (phosphodiesterase inhibitor) on angiotensin-I-converting enzyme (ACE) activity of the cells and culture medium. Dibutyryl cAMP (10(-3) M) but not cAMP or dibutyryl cGMP stimulated angiotensin-I-converting enzyme (ACE) activity of cells in culture approximately 50-100% but had little influence on ACE activity of the medium. Theophylline at 10(-3) M concentration produced a three- to fourfold stimulation of both cellular and medium ACE activity. Isoproterenol by itself had no effect on cellular ACE activity but produced a stimulatory effect at 10(-7)-10(-5) M concentration after pretreatment of cells for 24 hr with 10(-4) M theophylline. The results support the concept that ACE activity of endothelial cells is influenced by the cyclic AMP system. ACE activity in cells and that released into medium may be under different regulatory controls. PMID- 3021782 TI - The actin filament-severing domain of plasma gelsolin. AB - Gelsolin, a multifunctional actin-modulating protein, has two actin-binding sites which may interact cooperatively. Native gelsolin requires micromolar Ca2+ for optimal binding of actin to both sites, and for expression of its actin filament severing function. Recent work has shown that an NH2-terminal chymotryptic 17-kD fragment of human plasma gelsolin contains one of the actin-binding sites, and that this fragment binds to and severs actin filaments weakly irrespective of whether Ca2+ is present. The other binding site is Ca2+ sensitive, and is found in a chymotryptic peptide derived from the COOH-terminal two-thirds of plasma gelsolin; this fragment does not sever F-actin or accelerate the polymerization of actin. This paper documents that larger thermolysin-derived fragments encompassing the NH2-terminal half of gelsolin sever actin filaments as effectively as native plasma gelsolin, although in a Ca2+-insensitive manner. This result indicates that the NH2-terminal half of gelsolin is the actin severing domain. The stringent Ca2+ requirement for actin severing found in intact gelsolin is not due to a direct effect of Ca2+ on the severing domain, but indirectly through an effect on domains in the COOH-terminal half of the molecule to allow exposure of both actin-binding sites. PMID- 3021783 TI - Expression and function of a putative cell surface receptor for fibronectin in hamster and human cell lines. AB - We have previously reported the use of monoclonal antibodies to identify a 140-kD cell surface glycoprotein in mammalian cells that is specifically involved in fibronectin-mediated cell adhesion. We now report the purification of this molecule using immunoaffinity chromatography and the subsequent generation of polyclonal antibodies that selectively immunoprecipitate 140-kD putative fibronectin receptor glycoprotein (gp140) extracted from rodent or human cells; these antibodies also specifically block fibronectin-mediated cell adhesion but not adhesion mediated by other factors in serum. Expression of gp140-like molecules was detected on the surfaces of several adherent human cell lines (HDF, WISH, and EFC) but not on erythrocytes; however, gp140 was also detected on a nonadherent human lymphoid line (DAUDI). Analysis of gp140 on nonreducing SDS gels revealed two closely migrating bands. Protease digestion and peptide mapping suggests that the two bands are closely related polypeptides. PMID- 3021786 TI - The role of cyclic GMP in cells with the properties of smooth muscle cultured from the rat myometrium. AB - Cells growing in culture with previously described properties of rat uterine smooth muscle accumulated 45Ca2+ from the medium. Ca2+ uptake by these cells was stimulated by the addition to the medium of 8-bromo-cGMP but not by 8-bromo-cAMP. Ca2+ uptake was also stimulated by carbachol and by the nitro-vasodilator nitroprusside. Although cholinergic agonists have been shown previously to stimulate contraction but not cGMP synthesis in the rat myometrium, both carbachol and nitroprusside stimulated cGMP production by the cultured cells. These results suggested the cells had cholinergic receptor-mediated functions that reflected some neurotransmitter-sensitive properties of uterine smooth muscle in situ. When determined by a specific radioligand binding assay, subcellular fractions of the cultured cells bound muscarinic cholinergic agonists and antagonists with affinities expected of the muscarinic receptor. The cells were also sensitive to the beta-adrenergic catecholamine agonist isoproterenol, which stimulated cAMP production but not Ca2+ uptake. Carbachol failed to inhibit isoproterenol-dependent cAMP production, which is an important property of the cholinergic receptor in uterine smooth muscle in situ. These results suggest some but not all acetylcholine-sensitive properties of uterine smooth muscle may be retained in cell culture. PMID- 3021785 TI - Alpha-thrombin-induced inositol phosphate formation in G0-arrested and cycling hamster lung fibroblasts: evidence for a protein kinase C-mediated desensitization response. AB - In resting Chinese hamster fibroblasts (CCL39) alpha-thrombin rapidly induces the breakdown of phosphoinositides. Accumulation of inositol phosphates (IP), measured in the presence of Li+, is detectable within 5s (seconds) of thrombin stimulation. Formation of inositol tris- and bisphosphates slightly precedes that of inositol monophosphate, indicating that thrombin activates primarily the phospholipase C-mediated generation of inositol trisphosphate from phosphatidylinositol 4,5-bisphosphate. Initial rates of IP production increase with thrombin concentration, with no apparent saturability over the range 10(-4) 10 U/ml. Thrombin-induced phosphoinositide hydrolysis rapidly desensitizes (t1/2 less than 5 min), but a residual activity, corresponding to about 10% of the initial stimulation is sustained for at least 9 h, in contrast with the undetectable activity of G0-arrested cells. This apparent desensitization may be due to a feedback regulation by protein kinase C, since pretreatment with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) markedly inhibits (by up to 70%) subsequent thrombin-induced inositol phosphate formation. Conversely, growth factor deprivation of CCL39 cells results in a progressive increase of thrombin-induced phosphoinositide hydrolysis, from the very low level of exponentially growing cells to the maximal level of G0-arrested cells. This "up regulation" was found maximal in A51, a very well growth-arrested CCL39 derivative, and reduced or virtually abolished in two tumoral and growth factor relaxed derivatives of CCL39. Although preliminary, this observation suggests that a persistent activation of phosphatidyl inositol breakdown might operate in variants selected for autonomous growth. PMID- 3021787 TI - Both normal and tumor cells produce basic fibroblast growth factor. AB - We have previously purified from human placenta a basic fibroblast growth factor (FGF)-like molecule which stimulates the production of plasminogen activator (PA) and collagenase, induces DNA synthesis, produces an increase in motility in cultured bovine capillary endothelial (BCE) cells, and induces angiogenesis in vivo. The ability of basic FGF to stimulate PA production in BCE cells was used as an assay for the presence of basic FGF-like molecules in extracts of both normal and tumor-derived cultured cells. The identity of the PA-stimulatory activity with basic FGF was confirmed by its high affinity for heparin and by its cross-reactivity with antibodies to human placental basic FGF. Basic FGF-like molecules were identified in eight of ten cell lines tested, and the amount of FGF-like activity present in these cells bore no relation to their origin from normal or tumor tissue. The test cells, BCE cells, had one of the highest levels of FGF-like activity, suggesting that it may have an autocrine role in these cells. PMID- 3021788 TI - Occupational neurology and the hand. Differential diagnosis. AB - Occupationally related dysfunction of the peripheral nervous system is a common problem. The signs and symptoms of neurologic problems of the upper extremity and neck that can alter hand function and sensation are described in detail. PMID- 3021789 TI - Role of guanine nucleotide regulatory protein in polyphosphoinositide degradation and activation of phagocytic leukocytes by chemoattractants. AB - Leukocyte activation by chemoattractants provides an important model to study the biochemical mechanisms of stimulus-response coupling in these cells. Well-defined chemotactic factors induce readily quantifiable responses in phagocytic leukocytes. These include directed migration and the production and release of toxic substances including oxygen radicals and lysosomal enzymes. The development of radiolabeled synthetic oligopeptides with potent chemotactic activity allowed the demonstration of chemoattractant receptors on polymorphonuclear leukocytes (PMNs) as well as macrophages. In membrane preparations from these cells, these receptors exist in high- and low-affinity states which are regulated by guanosine di- and triphosphates. This suggested that chemoattractant receptors interact with guanine nucleotide regulatory proteins (N or G proteins). Although chemoattractants elicit a rapid but transient increase in intracellular cAMP levels, they neither stimulate nor inhibit membrane-bound adenylate cyclase, suggesting a novel role for N proteins in certain receptor-transduction mechanisms. Stimulation of phagocytes by chemoattractants is also associated with a rapid increase in cytosolic Ca2+ concentrations ([ Ca2+]i) which appears to result from the production of inositol 1,4,5-triphosphate (IP3) as a consequence of the diesteric cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2). Treatment of phagocytes with pertussis toxin (PT), which ADP-ribosylates and thereby inactivates certain N proteins, abolishes the cells' responsiveness to chemoattractants. More direct evidence for a role of a PT-sensitive N protein in leukocyte activation was provided by the demonstration that chemoattractants stimulate the hydrolysis of PIP2 in PMN membranes only in the presence of GTP. This receptor-mediated hydrolysis of PIP2 is not observed in plasma membranes prepared from PT-treated PMNs. Therefore, these studies suggest that occupancy of chemoattractant receptors activates a PT-sensitive N protein. The activated N protein shifts the Ca2+ requirement for phospholipase C activity from supraphysiological levels to ambient cytosolic Ca2+ concentrations. Cleavage of PIP2 results in the formation of the second messenger molecules, IP3 and 1,2 diacylglycerol, which can initiate cellular activation. These messengers also seem to activate responses which feed back to attenuate receptor stimulation of phospholipase. PMID- 3021790 TI - Inhibition of mammalian collagenases by thiol-containing peptides. AB - The following thiol-containing peptide analogues of the carboxyl side of the collagenase-sensitive bond of collagen were synthesized and tested as inhibitors of collagenases partially purified from homogenates of rabbit V-2 tumor and culture medium of pig synovium: HSCH2CH(CH3)CO-Ala-OEt (I), HSCH2CH(CH2Ph)CO-Ala OEt (II), HSCH2CH[CH2CH(CH3)2]CO-Ala-OEt (III); HSCH2 CH-[CH2CH(CH3)2]CO-Ala-Gly OEt (IV); HSCH2CH[CH2CH(CH3)2]CO-Ala-Gly-Gln (V). The compounds are listed in order of their inhibitory potency when assayed with nonfibrillar-acid-soluble calf skin collagen at pH 7.6, 35 degrees C. The best inhibitor (III) gave 50% inhibition between 1 and 4 microM. II was a competitive inhibitor with a Ki value of 75 microM. The enzymes preferred an isobutyl side chain at the 2-carbon position, and, where tested (III, IV), did not discriminate strongly between stereoisomers at the chiral 2-carbon. Increasing the length of the inhibitor did not markedly increase potency. PMID- 3021791 TI - Pain, increasing girth in healthy Japanese man. PMID- 3021792 TI - High-pressure affinity chromatography of calmodulin on a phenothiazine-silica. PMID- 3021793 TI - Ligand-exchange chromatography of nucleases. AB - The synthesis of chelating sorbents for ligand-exchange chromatography of enzymes is described. An inorganic support "Silochrom" and organic "Spheron", TSK-Gel HW 55 and cellulose were used as initial supports. The chelating sorbents contained iminodiacetic acid and iminodimethylphosphonic acid as stationary ligands. In order to obtain monofunctional sorbents, iminodiacetic acid was added in the form of its dimethyl ester. The concentration of stationary ligands on the sorbents varied from 10 to 100 mumol per ml sorbent. A chelating sorbent (in nickel form) was shown to be effective in the purification of exonuclease A5 from actinomyces. Electrophoretically homogeneous exonuclease A5 was obtained with a 25% yield. A chelating sorbent with iminodiacetic groups (in copper form) was applied to the isolation of endonuclease from Serratia marcescens directly from the culture medium. The capacity of the chelating sorbents for the endonuclease was studied as a function of the stationary ligand concentration. After one stage of purification, more than 70% pure enzyme was obtained with a yield exceeding 80%. PMID- 3021794 TI - Chromatographic separation of alpha 1-acid glycoprotein from alpha 1-antitrypsin by high-performance liquid chromatography using a hydroxyapatite column. PMID- 3021795 TI - A rapid microneutralization assay for cytomegalovirus. AB - A rapid, simple and reproducible microneutralization test for human cytomegalovirus is described. The results can be read in 1-2 days and the neutralization titer detected in human and guinea pig sera and in monoclonal antibody-containing supernatants is consistent with that derived by the plaque reduction neutralization test. PMID- 3021797 TI - Early detection of cytomegalovirus in cell culture by a monoclonal antibody. AB - A commercially available monoclonal antibody directed against early cytomegalovirus (CMV) antigen was used for the demonstration of CMV by immunofluorescence (IF) in cell culture within 2 days. The results were compared with the appearance of CMV-specific cytopathogenic effect (CPE). Urine specimens from 31 healthy children in day-care centers were inoculated on human embryonic fibroblasts. In addition, 45 CMV strains that had been stored at -70 degrees C were reinoculated. CMV was detected in 8/31 urine specimens by IF and 7 of these gave a specific CPE at an average of 16 days post-inoculation. One specimen was negative by IF but specific CPE was found at day 13. After reinoculation, CMV was detected in 76% by IF while 44 specimens developed CPE within a 6-week period. Demonstration of early CMV antigen in cell culture was found to be a rapid method for early diagnosis of CMV. Since the conventional cell culture with detection of CPE was more sensitive it may be useful to combine the two methods. PMID- 3021796 TI - ELISA for detection of IgG and IgM antibodies to HSV-1 and HSV-2 in human sera. AB - A rapid, enzyme-linked immunoassay (ELISA) was applied to identify and measure specific IgG and IgM antibodies to herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2). Detergent solubilized infected cells and mock-infected cells were used as antigens in the assay. Identification of type-specific antibodies was achieved by a competition assay in which clinical sera mixed with HSV-1 or HSV-2 antigens were assayed for reactivity to identical antigens coating wells of polystyrene microtiter plates. Reactivity and the specificity of the reactive immunoglobulin class was quantitated using biotinylated goat anti-IgG and biotinylated goat anti-IgM. Five paired sera from patients with diagnosed herpes simplex genital infections and one human anti-HSV-1 reference serum were tested with this assay and results were compared to results previously obtained using a complement fixation test and micro-SPRIA. The results indicate that the ELISA is a specific, sensitive and simple test which confirms the herpes simplex virus infection history of patients. PMID- 3021798 TI - Enumeration of enterovirus particles by scanning electron microscopy. AB - Enumeration of virus particles requires relatively concentrated and uniformly dispersed virus preparations, which is difficult to achieve by the usual methods of negative staining and transmission electron microscopy. We have developed an electrophoretic method that concentrates enteroviruses onto a polycarbonate membrane for examination by high-resolution scanning electron microscopy. The electrophoretic apparatus comprises three chambers in electrical series, each containing 3.5 ml of dilute buffer. The center chamber is inoculated with virus. A 15-nm porosity membrane, which does not pass virus, separates the center from the side chambers. A constant current is applied, and chilled buffer is pumped past the electrodes for 2 h. The virus suspension is recovered, and changes in titer (or radioactivity if labeled virus is used) due to electrophoresis are measured. Buffer pH, relative to the viral isoelectric points, determines the direction of virus migration. Particle counts are calculated from the mean of 25 randomly chosen fields photographed at 35-60,000 X magnification and related to titers measured by plaque assay. PMID- 3021799 TI - An electroblotting technique for assessing the integrity of the major immunogenic protein in foot-and-mouth disease virus vaccines. AB - A method has been developed whereby VP1, the major immunogenic protein of foot and-mouth disease virus can be detected after electroblotting on nitrocellulose paper. Proteins can be examined in unfractionated virus harvests and after formulation as aluminium hydroxide-adjuvanted vaccines. The limit of detection is approximately 10 ng of VP1 and up to 20 samples can be analysed simultaneously. The technique allows the integrity of VP1 to be examined in fully formulated vaccines. PMID- 3021800 TI - The selection and characterization of human monoclonal antibodies to human cytomegalovirus. AB - This communication describes the application of Epstein-Barr virus lymphocyte transformation technology to the production of human monoclonal antibodies specific for human cytomegalovirus. A group of such IgG antibodies have been characterized in terms of subclass, light-chain composition, specificity for particular viral proteins and neutralizing capacity. These results have shown that the production of antibodies by transformed lymphocytes is representative of the in vivo human immune response; the antibodies produced may therefore be of therapeutic value. This approach should prove to be useful for the identification of specific virion proteins which are antigenic in humans and for the in vitro evaluation of the immune responses to synthetic peptide vaccines. PMID- 3021801 TI - A novel glycoprotein for detection of herpes simplex virus type 1-specific antibodies. AB - A novel herpes simplex virus type 1 (HSV-1)-specific glycoprotein reactive with monoclonal antibody H1379 was purified by affinity chromatography. This glycoprotein, provisionally designated as gG-1, forms two sets of bands with molecular weights of 40-44,000 and 60-88,000. When used in an immunodot enzymatic assay, gG-1 reacted strongly with rabbit antisera to HSV-1, but not with sera hyperimmune to HSV-2. Specificity of the assay was further established by the lack of reactivity of convalescent sera collected from 20 patients with primary genital HSV-2 infections, and from 100 sero-negative individuals. In contrast, antibodies to gG-1 were detected in 9 of 10 patients with primary HSV-1 infection, and in 63/67 patients with culture-positive, recurrent oral or genital HSV-1 infection. Reproducibility of the gG-1 immunodot assay for HSV-1 antibody detection was 96%. Serological assay with purified gG-1, done in parallel with the assay using purified gG-2 described in an earlier report, provides simple and reliable methods to detect type-specific HSV-1 and HSV-2 antibodies for seroepidemiological studies. PMID- 3021802 TI - Antibody against adult diarrhoea rotavirus among healthy adult population in China. AB - This paper deals with a method of using unknown serum as the first antibody to coat wells of the microtiter plate directly in application of the sandwich ELISA method with double antibodies for the assay of the antibody against adult diarrhoea rotavirus. This method was used for assay of the antibody against adult rotavirus in 1,380 sera of healthy adults obtained from some provinces and cities of China, of which 141 showed a positive result with a total positive rate of 10.2%. The infection rate varied in different regions. In order to confirm the accuracy of these results a blocking test was carried out on 25 positive specimens, and the results revealed that the P/N ratios after blocking were all lower than those before blocking. Seventeen of these 25 positive serum specimens were obtained from rural areas of Qian'an County, Hebei Province where epidemic adult diarrhoea had occurred two years ago. The antibody against adult diarrhoea rotavirus was not detected in 50 adult sera from Xizang Autonomous Region. Only one out of 50 adult sera from Hainan Island was positive for the antibody. PMID- 3021803 TI - Aggregation of encephalomyocarditis virus induced by radio-iodination. AB - Radio-iodination causes encephalomyocarditis virus to behave aberrantly when examined by affinity chromatography and to sediment rapidly during analysis on sucrose density gradients suggesting that aggregation had taken place. The change in physical properties of the virus occurred whether iodination was carried out with 125I or 131I, with radio-iodine from two different sources, or using two different iodination procedures. The changes were not observed in virus subjected to an iodination procedure in the absence of radio-iodine suggesting that modification of tyrosine residues was involved rather than a side reaction such as amino acid oxidation. It is recommended that caution be exercised when following the fate of radio-iodinated virus in any particular study because its behaviour may not reflect that of normal, non-iodinated virus present. PMID- 3021804 TI - Purification of individual varicella-zoster virus (VZV) glycoproteins gpI, gpII, and gpIII and their use in ELISA for detection of VZV glycoprotein-specific antibodies. AB - We have utilized monoclonal antibodies in immune affinity chromatography to purify each of the 3 major glycoproteins of varicella-zoster virus (VZV), gpI, gpII, and gpIII, in immunologically active form. Upon injection into guinea pigs, each preparation elicited the production of specific antibodies capable of immunoprecipitating the homologous glycoprotein and of neutralizing VZV infectivity in vitro. Also, total glycoproteins from VZV-infected cells have been purified by lectin affinity chromatography. Each of the individual purified glycoproteins, as well as total VZV glycoproteins and appropriate uninfected cell protein controls, have been employed as solid-phase reagents in enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies directed against specific VZV glycoproteins. The specificity of the purified glycoproteins as ELISA reagents was verified by the ability of individual monoclonal antibodies to bind specifically to individual glycoprotein preparations. We have demonstrated the utility of the glycoprotein-specific ELISA by detecting antibodies in sera from post-zoster and post-varicella patients. The assay detects antibodies directed against each of the 3 major glycoproteins and is sensitive enough to detect antibodies in a 1:320 000 dilution of some sera. This assay, as well as the purified individual glycoproteins per se, should prove to be very useful reagents in understanding the role of each of gpI, gpII, and gpIII in immunity to VZV. PMID- 3021805 TI - ELISA for detection of group-common and virus-specific antibodies in human and simian sera induced by herpes simplex and related simian viruses. AB - A rapid (3.5 h) enzyme-linked immunosorbent assay (ELISA) was developed for the detection of serum antibodies to herpes simplex virus type 1 (HSV-1) and to two antigenically related monkey viruses, simian agent 8 (SA8) and Herpesvirus simiae (B virus). Crude preparations of detergent solubilized infected cells and similarly treated control mock-infected cells served as antigens for coating wells in microplates. Biotinylated protein A and avidin-conjugated alkaline phosphatase were used to detect antibodies in sera from different species (humans, monkeys and rabbits). Three prototype assays are described with three degrees of specificity. Common or specific determinants on the viral antigens could be assayed in simple competition tests using similar antigen preparations to those coating the wells. The specific assays permitted rapid differential serodiagnosis of antibodies to human and simian herpesviruses. PMID- 3021806 TI - Alterations in human leukocyte function induced by ingestion of eicosapentaenoic acid. AB - Two groups of six adults with persistent asthma, who were identical clinically, received 0.1 or 4 g of purified eicosapentaenoic acid ethyl ester (EPA) daily for 8 weeks. Both doses increased significantly the generation of leukotriene B5 (LTB5) from EPA by polymorphonuclear (PMN) and mononuclear leukocytes, while only the high dose decreased leukocyte arachidonic acid (AA) and the generation of LTB4 and prostaglandin E2 from AA. Only the high dose led to inhibition of PMN leukocyte chemotaxis to multiple stimuli by a mean of 57-70% (P less than 0.01), without altering monocyte chemotaxis, the production of platelet-activating factor by mononuclear leukocytes, or the IgE-dependent release of histamine from basophils. Both doses of EPA increased the responses of T lymphocytes to phytohemagglutinin by a mean of 73% or more (P less than 0.01) without modifying the numbers of helper and suppressor T lymphocytes. EPA affects the functions of several types of leukocytes critical to inflammation and immunity. PMID- 3021808 TI - In vitro effect of ascorbic acid on infectivity of herpesviruses and paramyxoviruses. AB - Suspensions of herpes simplex virus types 1 and 2, cytomegalovirus, and parainfluenzavirus type 2 were inactivated within 24 h when treated at 37 degrees C with 1 mg (5.05 mM) of copper-catalyzed sodium ascorbate per ml. The infectivity titer of respiratory syncytial virus was reduced substantially after 24 h but required 48 h for inactivation. Under these conditions, inactivation of these viruses was also successfully achieved with 5.68 mM catalyzed ascorbic acid. Copper (Cu2+), when added with the ascorbate solution at 5 micrograms/ml (0.022 mM), exhibited a catalytic effect on the inactivation of these viruses. The rate of inactivation was affected by the incubation temperature, time of exposure, and the virus concentration. Ascorbate concentrations as high as 10 mg/ml (50.5 mM) demonstrated only a minimum increase in effect on viral inactivation. The loss of infectivity did not alter either the hemagglutination or complement fixation qualities of the antigens. PMID- 3021807 TI - Detection of antibodies and antigens of human parvovirus B19 by enzyme-linked immunosorbent assay. AB - Acute-phase serum from a patient with aplastic crisis provided sufficient human parvovirus B19 to make a monoclonal antibody against B19 and to develop antigen and immunoglobulin M (IgM) and IgG antibody detection enzyme-linked immunosorbent assays (ELISAs). The indirect capture antibody method was used for all three assays. Antigen was detected in 8 of 29 sera drawn within 2 days of onset of illness from patients with aplastic crisis. These sera had high titers of virus by electron microscopy and DNA hybridization and had no detectable B19 antibody. Antigen was not detected in serum specimens that had low titers of B19 DNA and had B19 antibody. With the IgM ELISA, we detected B19 IgM in over 85% of clinical cases of aplastic crisis and fifth disease and less than 2% of controls. The prevalence of B19 IgG antibodies increased with age. Approximately 2% of children less than 5 years of age and 49% of adults greater than 20 years of age had B19 IgG antibodies. The B19 antibody ELISAs are sensitive and specific tests to detect B19 infections. PMID- 3021809 TI - Elimination of toxicity and enhanced cytomegalovirus detection in cell cultures inoculated with semen from patients with acquired immunodeficiency syndrome. AB - Although semen is a particularly rich source of cytomegalovirus (CMV) and is useful for monitoring CMV shedding, its culturing is associated with extensive monolayer toxicity, isolation failures, and lengthy detection times. Inoculation of fractionated semen with immunoperoxidase staining of monolayers eliminated virtually all toxicity, increased isolation rates and monolayer infectivity, and greatly reduced detection times. Semen specimens (n = 73) were processed conventionally (C) or separated into supernatant (SF) and cellular pellet (PF) fractions, and 35% of C and SF inocula produced extensive toxicity. In contrast, virtually no toxicity was observed in monolayers inoculated with PF. C and SF isolation rates were 41 of 73 and 38 of 73, respectively, whereas that for PF was 51 of 73. Although monolayer infectivity at initial CMV detection was often less than 10% for C and SF, it was as much as 25% for PF. Average detection times were reduced from 13 days for C and SF to 6 days with PF and were further reduced to 3 days when PF inoculation was combined with immunoperoxidase staining. Thirty percent of specimens negative by C were positive by PF. PMID- 3021810 TI - Elimination of Fc receptor binding of human immunoglobulin G in immunofluorescence assays for herpes simplex virus antibodies. AB - The binding of human immunoglobulin G by Fc receptors in herpes simplex virus (HSV)-infected cells can cause false-positive interpretations in the immunofluorescence test for HSV antibody. When the infected cell smears were treated with 10% glacial acetic acid for 5 min and rinsed in phosphate-buffered saline before the immunofluorescence test was performed, the Fc receptors were completely inactivated, resulting in a reliable method for HSV antibody detection. PMID- 3021811 TI - Suitability of new chlamydia transport medium for transport of herpes simplex virus. AB - A new chlamydia transport medium (ChlamydiaPort; Scott Laboratories, Inc., Fiskeville, R.I.) was evaluated for its suitability as a transport medium for herpes simplex virus (HSV). Two laboratory HSV strains (McIntyre and 333) and two clinical isolates (AO218 and AO301) were suspended in ChlamydiaPort, ViraPort (Scott Laboratories), and cell culture medium and maintained at 2 and 22 degrees C. Samples were tested at various time intervals to determine surviving virus. The range of half-lives of the HSV strains held at 2 degrees C in ChlamydiaPort medium was from 3.5 to 10 days, while virus stability was greater in ViraPort and less in cell culture medium. These HSV strains held at 22 degrees C in ChlamydiaPort had half-lives from 1.5 to 6 days, which were significantly greater than the half-lives of the viruses held in either tissue culture medium or ViraPort. Clinical specimens were tested for virus by using the Selecticult-HSV (Scott Laboratories) system to determine the performance of the transport medium under field conditions. Clinical specimens maintained up to 5 days at ambient temperatures in ChlamydiaPort medium appeared suitable for diagnostic testing without detectable loss of positive specimens. In addition, there was a significant decrease in the average time required for diagnosis when compared with a standard transport system, Virocult (Microdiagnostics, Cleveland, Ohio). These results show that HSV infections can be successfully diagnosed in distant virology laboratories by shipping specimens in ChlamydiaPort transport medium at ambient temperatures. PMID- 3021812 TI - Rapid detection of cytomegalovirus in clinical specimens by using biotinylated DNA probes and analysis of cross-reactivity with herpes simplex virus. AB - A method for rapid identification of human cytomegalovirus (HCMV) was developed with biotinylated DNA probes. BamHI restriction fragments from HCMV strain AD169 were selected and tested for their ability to detect virus in patient urine samples. All probes detected 30 pg of HCMV AD169 DNA. The BamHI B fragment detected 15 of 29 cell-culture-positive samples (sensitivity, 52%). There were four samples which were probe positive and cell culture negative (specificity, 87%). The D and H fragments used as combined probes detected 17 of 21 cell culture-positive samples (sensitivity, 81%). There were five probe-positive and cell-culture-negative samples (specificity, 68.8%). The H fragment, when used alone, detected 11 of 14 culture-positive samples, and 5 samples were culture negative and probe positive. Sensitivity (78.6%) and specificity (76.2%) for the H fragment were similar to those for the combined probes, but the color intensity of the positive reactions detected by the H fragment alone was lower. There was unexpected cross-reactivity with herpes simplex type 1 and 2 controls when the combined D and H probes were used. Specific hybridization was demonstrated between subfragments of the HCMV BamHI D fragment and the herpes simplex virus type 1 EcoRI M fragment. PMID- 3021813 TI - Relevance of detection of immunoglobulin M antibody response in birds used for arbovirus surveillance. AB - Young chickens were inoculated with 5,000 PFU of eastern equine encephalitis (EEE) virus and bled at intervals thereafter for determinations of hemagglutination-inhibiting (HI), neutralizing (N), immunoglobulin M (IgM), and IgG antibodies. HI, N, and IgM antibodies were first detected 4 days after infection, and IgG was detected 7 days after infection. All four antibodies persisted through day 90 after infection. HI, N, and IgM antibody titers remained elevated and were not cross-reactive with the related alphavirus western equine encephalitis (WEE) virus. IgG antibody titers also remained high, but heterologous reactivity to WEE virus increased with time after infection. Serum samples from sentinel chickens and wild birds infected in nature with EEE, WEE, or St. Louis encephalitis virus and submitted to this laboratory from state and local health departments were tested for IgM antibody by using anti-chicken IgM for capture and for IgG antibodies to the EEE and WEE viruses. There was essentially complete correlation between HI, N, and either IgM (indicating recent infections) or IgG (indicating more remote infections) antibody. We conclude that the IgM antibody capture enzyme immunoassay can be used as a specific and sensitive assay to replace the routinely used HI test for detecting antibody in sentinel chickens and in young, wild birds used for arbovirus surveillance. The test is rapid and relatively inexpensive and can be performed in essentially all adequately supplied laboratories. PMID- 3021814 TI - Single gene substitution rotavirus reassortants containing the major neutralization protein (VP7) of human rotavirus serotype 4. AB - A series of reassortants was isolated from coinfection of cell cultures with wild type bovine rotavirus (UK strain [serotype 6]) or rhesus rotavirus (strain MMU18006 [serotype 3]) and a tissue culture-adapted human rotavirus strain, ST3 (serotype 4). Monospecific antiserum or a set of monoclonal antibodies to the major outer capsid neutralization glycoprotein, VP7, of the animal rotavirus parent was used to select for reassortants with human rotavirus serotype 4 neutralization specificity. The majority of reassortants contained only gene 9 of the human rotavirus parent, ST3, whereas the remaining genes were derived from the animal rotavirus parent. These single human rotavirus gene substitution reassortants were neutralized to high titer by hyperimmune serum directed at ST3, thus demonstrating that gene 9 of ST3 codes for the major neutralization protein of this strain. Moreover, these single gene substitution, reassortants were also neutralized to low titer by antiserum directed at their animal rotavirus parent, probably because they derived gene 4, which codes for another outer capsid protein, VP3, from their animal rotavirus parent. None of the reassortants derived gene 4, which had previously been shown to be responsible for host range restriction of human rotaviruses in tissue culture, from ST3, despite the fact that the ST3 strain used for gene reassortment had been tissue culture adapted. PMID- 3021815 TI - Detection of cytomegalovirus antibody by enzyme immunoassay and lack of evidence for an effect resulting from strain heterogeneity. AB - A total of 259 serum samples from random blood donors were assayed by an enzyme immunoassay for antibody to cytomegalovirus (CMV) with antigens prepared from eight genetically unrelated clinical isolates of CMV and the laboratory-adapted strains AD169 and Towne. A total of 144 serum samples were reactive against all 10 strains, and 115 serum samples lacked immunoglobulin against all 10 strains. The results support the continued use of the AD169 strain of human CMV as the reference antigen for CMV serologic testing. PMID- 3021816 TI - Salivary antibodies as a means of detecting human T cell lymphotropic virus type III/lymphadenopathy-associated virus infection. AB - Of 45 individuals seropositive for human T cell lymphotropic virus type III/lymphadenopathy-associated virus, 45 were found to have detectable salivary antibodies to viral antigens by a radioimmunoprecipitation assay. The results also showed that a Western blot assay for salivary antibodies may be possible. The feasibility of a diagnostic test for human T cell lymphotropic virus type III/lymphadenopathy-associated virus not requiring venipuncture is discussed. PMID- 3021818 TI - Induction of prostacyclin biosynthesis is closely associated with increased guanosine 3',5'-cyclic monophosphate accumulation in cultured human endothelium. AB - Stimuli of prostacyclin (PGI2) biosynthesis such as thrombin, bradykinin, histamine, and A23187 increase guanosine 3',5'-cyclic monophosphate (cyclic GMP) levels in primary monolayer cultures of human umbilical vein endothelium by about twofold. This effect is dependent on the presence of extracellular Ca2+. Increases of about tenfold are observed when cyclic GMP phosphodiesterase activity is inhibited, which suggests that the observed increases in cyclic GMP involve the activation of guanylate cyclase. Activation of guanylate cyclase appears to involve an early event in the induction of PGI2 biosynthesis, as neither arachidonic acid nor its metabolites stimulate cyclic GMP accumulation. Although activators of guanylate cyclase such as atriopeptin III, sodium nitroprusside, and tert-butylhydroperoxide increase cyclic GMP levels by approximately 2-3-fold, they do not stimulate or modulate PGI2 production. We conclude that cyclic GMP does not play a primary role in mediating the induction or regulation of PGI2 biosynthesis in vascular endothelium. PMID- 3021817 TI - Recombinant human granulocyte-macrophage colony-stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression, and survival. AB - A purified recombinant human granulocyte-macrophage colony stimulating factor (rH GM-CSF) was a powerful stimulator of mature human eosinophils and neutrophils. The purified rH GM-CSF enhanced the cytotoxic activity of neutrophils and eosinophils against antibody-coated targets, stimulated phagocytosis of serum opsonized yeast by both cell types in a dose-dependent manner, and stimulated neutrophil-mediated iodination in the presence of zymosan. In addition, rH GM-CSF enhanced N-formylmethionylleucylphenylalanine(FMLP)-stimulated degranulation of Cytochalasin B pretreated neutrophils and FMLP-stimulated superoxide production. In contrast, rH GM-CSF did not promote adherence of granulocytes to endothelial cells or plastic surfaces. rH GM-CSF selectively enhanced the surface expression of granulocyte functional antigens 1 and 2, and the Mo1 antigen. rH GM-CSF induced morphological changes and enhanced the survival of both neutrophils and eosinophils by 6 and 9 h, respectively. These experiments show that granulocyte macrophage colony stimulating factor can selectively stimulate mature granulocyte function. PMID- 3021820 TI - Breakpoints on chromosomes 9 and 22 in Philadelphia chromosome-positive chronic myelogenous leukemia (CML). Amplification of rearranged c-abl oncogenes in CML blast crisis. AB - We surveyed 20 Philadelphia chromosome (Ph1) positive chronic myelogenous leukemia (CML) samples by Southern blot hybridization to determine the location of the breakpoints that occur on chromosomes 9 and 22 in the Ph1 translocation. Only 3 of 20 samples exhibited breakpoints on chromosome 9 within 18 kilobases (kb) of the v-abl homologous sequences. Mapping of these three chromosome 9 breakpoints indicates that each is at a separate location within this 18-kb region, indicating that there are no breakpoint "hot spots" in this area. In contrast, all 20 CML samples exhibited breaks on chromosome 22 within a 5.0-kb Bgl II fragment that lies within the previously described breakpoint cluster region (bcr). Several patients with CML blast crisis exhibiting multiple Ph1 chromosomes/metaphase exhibited amplified and rearranged c-abl-related fragments. These additional Ph1 chromosomes in blast crisis cells do not arise from a second, independent 9:22 translocation but rather result from a duplication of the preexisting Ph1 chromosome. PMID- 3021821 TI - Rapidly expanding mass in a neonate. PMID- 3021822 TI - A review and proposal for the etiology of acute necrotizing gingivitis. AB - There are numerous correlations between features of acute necrotizing gingivitis (ANG) and virus infections, notably cytomegalovirus (CMV) infections. The age range of occurrence of seroconversions to CMV positive and occurrence of ANG correlate both in industrialized countries (young adults) and in underdeveloped countries (young children). A depression in cell-mediated immunity, as evidenced by a decrease in T-lymphocyte helper/suppressor ratio and decreased responsiveness to the mitogen ConA, has been shown to occur in both CMV infection and ANG. There is a higher incidence of both CMV infection and ANG in young male homosexuals. These and other correlations, taken together, argue for a fundamental role for virus in ANG etiology. PMID- 3021819 TI - Hormonal regulation of proton secretion in rabbit medullary collecting duct. AB - With the exception of aldosterone, little is known about the hormonal regulation of distal nephron acidification. These experiments investigated the effects of prostaglandin E2, indomethacin, lysyl-bradykinin, 8-bromo-cyclic AMP, and forskolin on proton secretion in the major acidifying segment of the distal nephron, the medullary collecting duct from inner stripe of outer medulla. Using in vitro microperfusion and microcalorimetry, net bicarbonate reabsorption (proton secretion) was measured in rabbit medullary collecting ducts before, during, and after exposure to each test substance. PGE2 reduced proton secretion 12.2%, while the following substances stimulated proton secretion: indomethacin 14.2%; 8-bromo-cyclic AMP 34.5%; forskolin 39%. Lysyl-bradykinin was without effect. These studies demonstrate that distal nephron acidification, in addition to being stimulated by aldosterone, is significantly inhibited by the hormone PGE2. The stimulation of proton secretion by cAMP suggests that other hormones known to activate adenylate cyclase may also influence distal nephron acidification. PMID- 3021824 TI - Collateralization of descending pathways from the brainstem to the spinal cord in a lizard, Varanus exanthematicus. AB - With the multiple fluorescent retrograde tracer technique, the collateralization in the spinal cord of descending supraspinal pathways was studied in a lizard, Varanus exanthematicus. Fast Blue (FB) gels were implanted unilaterally in the spinal gray matter of the cervical enlargement and Nuclear Yellow (NY) gels were implanted ipsilaterally in two series of experiments in all spinal funiculi of the lumbar enlargement or in midthoracic spinal segments, respectively. All brainstem nuclei known to project to the spinal cord in reptiles were found to give rise to branching axons that may influence widely separate levels of the spinal cord. The number of double-labeled FB-NY cells varied in these brainstem nuclei from none to half the number of neurons projecting to the cervical enlargement. Highly collateralizing projections (expressed as the percentage of double-labeled neurons, DL) were found to arise from the nucleus raphes inferior, the contralateral nucleus reticularis superior pars lateralis, the contralateral nuclei vestibulares ventromedialis and descendens, and the ipsilateral nucleus reticularis inferior pars ventralis. A lower percentage of DL neurons was noted for the contralateral nucleus ruber and bilaterally for the nucleus reticularis medius and nucleus reticularis inferior. Extensive brainstem projections directed to cervical and high thoracic spinal levels originate from the area lateralis hypothalami, the nucleus of the fasciculus longitudinalis medialis, the contralateral nucleus cerebellaris medialis, and from the nucleus tractus solitarii. Projections preferentially directed to midthoracic or lower levels of the spinal cord were found to arise from the ipsilateral locus coeruleus, the contralateral nucleus reticularis superior pars lateralis, the nucleus reticularis inferior pars ventralis, the nucleus reticularis inferior, and the nucleus raphes inferior. In contrast to findings in mammals, in Varanus exanthematicus the red nucleus, the nucleus vestibularis ventrolateralis, and certain parts of the reticular formation did not display a clear-cut somatotopic organization. In general two different patterns of collateralization can grossly be discerned: a gradual decrease of spinal collaterals caudalward, which can be interpreted as a certain specificity of such projections; and a constant number of collateral nerve fibers throughout the spinal cord that can be interpreted as either a nonspecific or, in contrast, a highly specific system, focussed exclusively on the cervical and lumbar enlargements. PMID- 3021823 TI - Afferent and efferent projections of the inferior area 6 in the macaque monkey. AB - The rostral part of the agranular frontal cortex (area 6) can be subdivided on the basis of its cytoarchitecture, enzymatic properties, and connections into two large sectors: a superior region, lying medial to the spur of the arcuate sulcus, and an inferior region, lying lateral to it. In this study we traced the afferent and efferent connections of the inferior region of area 6 by injecting small amounts of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) and fluorescent tracers (fast blue and diamidino yellow) into restricted parts of inferior area 6 and in physiologically determined fields of area 4. There is an ordered topographic pattern of connections between inferior area 6 and area 4. The region near the spur of the arcuate sulcus (hand field) projects to the area 4 hand field while the lateral part of inferior area 6 (mouth field) is connected with the corresponding field in area 4. The organization of the connections between the two fields is, however, different. The hand fields in area 6 and 4 have direct reciprocal projections, whereas the mouth field in the postarcuate cortex relays information to area 4 via a zone intermediate between the arcuate and the central sulcus. This zone corresponds to the cytochrome oxidase area F4 (Matelli, Luppino, and Rizzolatti: Behav. Brain Res. 18: 125-137, '85). The inferior area 6 also has topographically organized connections with the supplementary motor area. The inferior area 6 receives and sends fibers to a series of discrete cortical areas located in the lower cortical moiety (Sanides: The Structure and Function of the Nervous Tissue, Vol. 5. New York: Academic Press, pp 329-453, '72). These areas that form a broad ring around the central sulcus are the ventral bank of the principal sulcus and the adjacent area 46, the precentral operculum (PrOC), area SII (Jones and Burton: J. Comp. Neurol. 168:197 248, '76), the parietal operculum, and the rostral part of the inferior parietal lobule including the lower bank of the intraparietal sulcus. Finally, the inferior area 6 has sparse but consistent connections with insular and cingulate cortices. The functional significance of this complex pattern of connections is discussed. PMID- 3021825 TI - Course of retinogeniculate projection fibers in the cat optic nerve. AB - The fiber courses of the cat optic nerve were studied by using wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), which was iontophoretically applied to electrophysiologically defined positions in the lateral geniculate nucleus (LGN). Grossly, there were two distinct bends along the length of the optic nerve. The ventrally flexing anterior bend was located approximately 2 mm from the eyeball, while the dorsally flexing posterior bend was found at some 6 mm distant. The optic nerve fibers showed a tendency to scatter toward the chiasm. At the optic nerve head, the fibers from the different retinal areas maintained the retinal topography in a simplified form according to the trajectory of optic fibers surrounding the optic disc. Between the anterior and posterior bends, the fibers from the pericentral, middle-temporal, and most upper areas of the dorsal retina migrated ventrally and were arranged in the middle of the lateral, middle, and medial parts of the optic nerve, respectively, while fibers from the middle-temporal area of the ventral retina migrated dorsally and scattered into the lateral half. The fibers from the temporal and nasal horizontal meridian areas tended to hold their respective positions in the lateral and medial halves of the optic nerve. As a result, in this level they displayed a complex retinotopy in that the fibers from each part of the retina were mixed. Passing the posterior bend, as the optic nerve proceeded toward the chiasm, the characteristic pattern became less defined. Near the chiasm, the retinotopy became very scattered, showing a partial dorsoventral inversion of the retinal topography with substantial overlapping. It was noted from the present findings that the fibers from ventral retina scattered more quickly than the fibers from the dorsal retina, which tended to hold their grouping until the anterior bend, but the central or pericentral retinal fibers proceeded without significant scatter as far as the posterior bend. The analysis of labelled ganglion cells suggested that even if fibers that arise from one certain "mode" of ganglion cells are selected, it is unlikely that they maintain their initial fiber topography along the entire length of the optic nerve. PMID- 3021826 TI - Treatment of cutaneous leishmaniasis with an intralesional antimonial drug (Pentostam). AB - In major American textbooks of dermatology and medicine the use of intralesional sodium stibogluconate (Pentostam) in the treatment of cutaneous leishmaniasis is not mentioned. In this brief informal communication I have summarized personal experience with this effective treatment modality. PMID- 3021827 TI - Retinoic acid: biochemistry and metabolism. AB - Retinoic acid, unlike the naturally occurring vitamin A (retinol), is a minor component of the human diet. It is formed in vivo from retinol and has many metabolites. The biological activity of the metabolites is not higher than that of retinoic acid itself, indicating that the metabolites must be products of retinoic acid catabolism. Little is known about the enzymatic systems responsible for forming retinoic acid or about how it enters the cell. Discovering the molecular mechanism(s) of retinoic acid activity in cellular metabolism is important to understanding its physiologic role. The pharmacologic effects of high doses of retinoic acid may be caused by its action on cellular membranes. Conversely, low concentrations appear to produce physiologic effects. Results of experiments with animals and with cell cultures indicate that the primary physiologic role of retinoic acid is in cellular differentiation. Retinoic acid influences genomic expression, inducing the appearance of some proteins while suppressing the expression of others. The existence of an intracellular retinoic acid-binding protein suggests that it may mediate the physiologic effects of retinoic acid on cellular differentiation. PMID- 3021828 TI - Pharmacology of topical retinoids. AB - The goal of research into retinoic acid is to maintain or improve the pharmacologic activities of topically applied retinoic acid while decreasing both local and systemic toxicity and improving chemical stability. Data are presented from a wide range of in vitro and in vivo test systems for retinoic acid, its congeners such as etretinate, and recently synthesized derivatives. The in vitro tests involved cell culture and biochemical assays: rat testes cytosol (receptor binding), mouse embryo teratocarcinoma cells (morphologic evaluation of differentiation), transformed human keratinocytes (cross-linked envelope formation), and polymorphonuclear leukocytes (lipoxygenase enzyme). The in vivo tests were carried out in the hairless rat (ornithine decarboxylase inhibition), the rabbit (irritation), and the guinea pig (ultraviolet erythema). Results from these experiments were used to determine biologic indices for the test substances. Subsequently, these test systems were used to evaluate a series of new compounds designed to control the chemical nature of a key double bond in the retinoic acid structure. Results are presented to show that different actions on the proliferative, differentiating, and inflammatory processes can be obtained by progressive aromatization of the retinoic acid structure. PMID- 3021829 TI - Mechanism of action of retinoids. AB - In several recent reviews, we have suggested that the mechanism of action of retinoids in controlling cell differentiation is related to their effects on the expression of oncogenes and peptide growth factors. It is currently believed that oncogenes control metabolic pathways that involve peptide growth factors and their receptors, as well as postreceptor signaling mechanisms. Retinoids, therefore, have been valuable probes to study the function of oncogenes and peptide growth factors. In several tumor cells, including human promyelocytic leukemia, human and murine neuroblastoma, and murine teratocarcinoma, retinoic acid induces terminal differentiation, accompanied by suppression of the expression of either the c-myc or the N-myc gene. Many studies have indicated that retinoic acid can markedly increase the number of cellular receptors for epidermal growth factor, which is partially encoded by another oncogene, erb-B. We have shown that retinoic acid greatly inhibits the anchorage-independent growth of a rat fibroblast cell line that has been transfected with the c-myc gene, particularly when these cells are stimulated by the combination of platelet derived growth factor and transforming growth factor-beta. At present, the mechanisms by which retinoids control oncogene and growth factor expression are unknown. A wide range of new compounds, including the retinoidal benzoic acid derivatives, are now available to study these mechanisms, and will necessitate the identification of a high-affinity receptor for retinoids and the elucidation of the interaction of this receptor with the genome of the cell. The recent synthesis of new terephthalic acid anilides and chalcone carboxylic acid derivatives, which have retinoid-like activity, offers a particularly useful approach to this problem. PMID- 3021830 TI - Local and systemic effects of orally applied sodium salts. AB - This study determines whether the oral application of a baking soda-3% hydrogen peroxide dentifrice and a nearly saturated sodium chloride mouthwash, as a home care method for treating periodontal disease, creates a sodium burden for human subjects. The dietary intake and urinary excretion of sodium and potassium were monitored in participating subjects. Urinary sodium did not increase in subjects using the method. Desquamative gingival lesions, however, were seen in all treated subjects. Further study is needed to determine safe salt concentrations for this home care regimen. PMID- 3021832 TI - T cell reactivity to penicillin: phenotypic analysis of in vitro activated cell subsets. AB - Patients with penicillin allergy demonstrate a T cell proliferative response after in vitro stimulation with penicillin G (Pen G) and other beta-lactam antibiotics. To understand better penicillin-allergic reactions, T cell subset stimulation with Pen G was studied and compared with other soluble (tetanus toxoid and purified protein derivative [PPD]) and membrane-bound viral (influenza A and Epstein-Barr viruses) antigens. A double fluorescence method for flow cytometry was used to evaluate the activated cells simultaneously by pyronin Y staining of RNA and by indirect immunofluorescence of cell surface T4, T8, or Leu 8 antigens. The antigens used stimulated mainly the T4+ subset (greater than 90%), whereas the number of activated T8 cells was slightly increased only in Pen G- and influenza A-triggered cultures (5% to 15%). Leu 8 antigen was used to analyze more precisely the activated T4+ cells. Pen G and influenza A and Epstein Barr viruses stimulated both T4+, Leu 8+ (greater than 50% of activated cells, inducers for suppressor cells), and T4+, Leu 8- (helpers for B cells) subsets, whereas PPD activated mainly T4+, Leu 8- subpopulations. These results indicate that penicillin-allergic patients with skin symptoms demonstrate a T cell subset stimulation that resembles more the reaction versus viral antigens (membrane incorporated) than to soluble antigens like PPD. These results suggest that Pen G is presented to T cells like viral proteins and might thus cause allergic reactions resembling skin symptoms observed in viral diseases. PMID- 3021831 TI - Slowly enlarging, nontender mass in the right submandibular region. AB - Slow-growing, soft tissue masses in the submandibular region of the neck frequently are caused by tumors of the submandibular salivary gland. These neoplasms are most commonly benign and characteristically develop during a prolonged period--often, during 15 or more years. The recommended treatment is excision of the gland, and, if adequately performed, the probability of the tumor's recurring is insignificant. PMID- 3021834 TI - An assay for proteolytic activity using spin-labeled substrates. AB - A method is proposed for the assay of proteolytic activity based on the measurement of changes in the electron spin resonance spectra (increase in the ratio of weakly to strongly immobilized spin label residues) of substrate proteins labeled with a maleimide nitroxide derivative. PMID- 3021833 TI - Relationships between adenosine, cyclic nucleotides, and xanthines in asthma. AB - Methylxanthines have been used for the treatment of asthma for more than 60 years, but their mechanism of action is poorly understood. Their ability to inhibit cyclic adenosine monophosphate phosphodiesterase has attracted much attention. However, this is clearly demonstrable only in high doses and is more likely to be related to toxicity. An alternative mechanism is antagonism of adenosine receptors in the lung. Adenosine has been shown to be released in asthma and cause bronchoconstriction in patients with asthma. Its effects are selectively inhibited by concentrations of theophylline that do not block histamine-induced bronchoconstriction. Neither phosphodiesterase inhibition nor adenosine receptor antagonism explains the action of enprofylline in asthma. Consequently, additional actions of methylxanthines are likely to contribute to their beneficial effects. They may include adrenaline release from the adrenal medulla, an effect on cell calcium distribution, inhibition of the generation of contractile prostaglandins, and an improvement of diaphragmatic contractility. PMID- 3021835 TI - The ontogeny of renal alpha 1- and alpha 2-adrenoceptors in the Dahl rat model of experimental hypertension. AB - [3H]prazosin (PRAZ) and [3H]rauwolscine (RAUW) were used to examine the ontogeny of renal alpha 1- and alpha 2-adrenoceptors in the inbred Dahl hypertension sensitive (S/JR) and -resistant (R/JR) rat. PRAZ and RAUW each bound to a single population of non-interacting sites. The binding of each ligand was saturable and reversible. The greatest proliferation of each receptor subtype occurred between 5 and 25 days of age. During this period, a 4 to 5-fold increase in the density of each was observed. Adult levels of each were reached by 50 days of age. The alpha 2-adrenoceptor was the predominant subtype present in renal tissue. However, its ratio to the alpha 1 subtype was influenced by strain and age: the ratio was greatest in the S/JR strain and decreased with age in both strains. The profile of alpha 1-adrenoceptor development was similar in S/JR and R/JR rats. In contrast, the density of alpha 2-adrenoceptors was similar in S/JR and R/JR rats at 5 and 15 days of age but significantly greater in the S/JR rat between 25 and 150 days of age. The elevated density of alpha 2-adrenoceptors could not be explained by strain-related differences in blood pressure or alterations in the affinity of the receptor. The results suggest that a relationship may exist between elevated renal alpha 2-adrenoceptor density and the genetic predisposition to hypertension in the S/JR rat. However, because this relationship is not apparent during the neonatal period of development, the possibility that the elevated density of sites may be secondary to some other event should also be considered. PMID- 3021836 TI - Regulation of the right coronary circulation during a controlled behavioral stress in the conscious dog. AB - Right coronary blood flow (CBF) was measured in 11 mongrel dogs during classical aversive conditioning (a 30 s tone followed by a 1 s 2-6 mA electrical shock). The cardiovascular response consisted of significant (P less than 0.01) increases in: mean arterial pressure (12.9%), systolic right ventricular pressure (RVP, 31.8%), d(RVP)/dt (49.9%) and heart rate (56.2%). The coronary vascular response to behavioral stress consisted of an immediate and significant increase in mean CBF (56.9%) coupled with a significant decrease in mean coronary vascular resistance (CVR, -28.5%). The mean CVR decrease was reduced by cardioselective beta-blockade (CBB) or cardiac pacing and eliminated by right stellectomy (RSGx). The combination of CBB with cardiac pacing resulted in a significant increase in mean CVR. alpha-adrenergic blockade with either phentolamine or prazosin, RSGx, and cardiac pacing significantly reduced the control mean CVR values. Thus, these data suggest that the right coronary response to stress is primarily mediated by the release of metabolic factors secondarily to an increased inotropic or chronotropic state. However, when these metabolic effects were controlled, mean coronary resistance no longer decreased but, rather, increased in response to the aversive stress. This increase could be eliminated by the addition of an alpha adrenergic antagonist. These data further suggest that the coronary response to behavioral stress activates an alpha-adrenergic vasoconstriction. PMID- 3021837 TI - Use of salicylate with high pressure liquid chromatography and electrochemical detection (LCED) as a sensitive measure of hydroxyl free radicals in adriamycin treated rats. AB - Hydroxyl free radicals react with salicylate to form 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA). Utilizing the technique of high pressure liquid chromatography with electrochemical detection (LCED), it is possible to detect DHBAs at the level of femtomoles. Since salicylate is relatively non-toxic, we have administered it as a trapping agent in a first attempt to examine the use of the LCED method as a sensitive measure of in vivo OH production. Utilizing adriamycin administration as a model to induce oxygen free radical tissue damage, we found that the level of DHBAs present in drug treated rats versus controls was increased 100-fold in heart and muscle, 30-fold in lung, and 3- and 4-fold in brain and blood, respectively. These first observations support the theory that adriamycin induces OH in tissue and indicates that the LCED method may prove to be useful to measure oxygen free radical production in vivo. PMID- 3021838 TI - Superoxide production and chemiluminescence induced in differentiated HL-60 cells by the chemoattractant formyl-methionyl-leucyl-phenylalanine. AB - Superoxide production and chemiluminescence induced in differentiated HL-60 cells by the chemoattractant formylmethionyl-leucyl-phenylalanine: In order to study the generation of oxidative metabolites in relation to cell differentiation, dimethyl sulfoxide (DMSO) and retinoic acid (RA) differentiated HL-60 cells were stimulated with the chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP). The oxidative response was measured as luminol-dependent chemiluminescence, lucigenin-dependent chemiluminescence, and cytochrome c reduction. Cells grown in the presence of DMSO or RA progressively expressed morphological changes, and when the mature cells were exposed to FMLP the cells produced oxidative metabolites. Quantitatively the HL-60 cells grown in the presence of DMSO gave rise to the most pronounced response. No correlation was obtained between superoxide production, luminol-chemiluminescence and lucigenin dependent chemiluminescence, indicating that different aspects of the oxidative response are elucidated by the three different methods. Furthermore, the experiments show that DMSO and RA-induced differentiation of HL-60 cells leads to granulocyte-like cells with different abilities to produce oxidative metabolites, possibly due to differences in receptor function. PMID- 3021839 TI - Horseradish peroxidase-catalyzed oxidation of mitoxantrone: spectrophotometric and electron paramagnetic resonance studies. AB - The clinical anticancer agent mitoxantrone is subject to irreversible oxidation by hydrogen peroxide catalyzed by horseradish peroxidase (HRP). The characteristic absorption changes that result provide evidence for an initial metabolite which is further oxidized enzymatically. The formation of the metabolite is accompanied by the concomitant generation of a free radical species detected by EPR spectroscopy. The intensity of the latter is dependent on the ratio of mitoxantrone to oxidant as well as on the pH of the medium. The metabolite in its oxidized form is a strong electrophile and can be reduced by biologically and physiologically relevant electron donors including ascorbic acid, L-cysteine and reduced glutathione. The results establish a new facile metabolic conversion of this clinically useful anticancer agent that may be relevant to its mode of action. PMID- 3021840 TI - The role and mechanism of metal ions and their complexes in enhancing damage in biological systems or in protecting these systems from the toxicity of O2-. AB - Copper complexes of 1,10-phenanthroline and some substituted 1,10-phenanthroline cleave DNA in the presence of a reducing agent and molecular oxygen. Generally, the damage is attributed to hydroxyl radicals which are formed through the Haber Weiss reaction. It is assumed that this reaction occurs with the ternary metal complexes with the biological target and the mechanism is defined as the "site specific mechanism." In these systems, O2- drives the cycle through the reduction of copper(II). On the other hand, these same copper complexes catalyze the dismutation of O2- and thus should protect the systems from O2- toxicity. In this article, the toxicity of these complexes is explained on kinetic grounds. A general discussion on the various factors which could cause the metal ions or their complexes to act either as protectors from O2- toxicity or as sensitizers of toxic effects of O2- is given. PMID- 3021842 TI - Studies of the reactivity of trolox with Mn3+/Fe3+ complexes by pulse radiolysis. AB - The rates and mechanisms for the reactions between Trolox (6-hydroxy-2,5,7,8 tetramethylchroman-2-carboxylic acid), a synthetic analogue of alpha-tocopherol, Br2- and Mn3+-phosphate were determined under aerobic and anaerobic conditions by fast kinetic methods. In addition, the reaction between Fe(OH)2+ and Trolox at pH 6.0 was shown not to occur under the conditions of the experiment, leading to a limiting rate for that reaction of k less than 10(3) M-1 s-1. These results are compared to studies of the reactivity of Trolox with HO2/O2- and are discussed in light of the known antioxidant properties of vitamin E. PMID- 3021841 TI - Hemolysis and membrane lipid changes induced by xanthine oxidase in vitamin E deficient red cells. AB - A procedure to induce hemolysis by the hypoxanthine-xanthine oxidase reaction was developed and applied to vitamin E deficient red blood cells (RBCs) in rats. The reaction system was as follows: 0.16 mM hypoxanthine, 0.05 U/ml xanthine oxidase in 2.5% RBC suspensions with an isotonic buffer (pH 7.4) containing 10 mM phosphate buffer and 125 mM saline (277 mOsm). Hemolysis was observed to depend on the vitamin E concentrations in the RBCs. Hemolysis was inhibited by catalase but not by SOD. After the reaction with vitamin E deficient RBCs, an increase in TBARS in the aqueous phase of the reaction mixture was observed. This accompanied the increase in fluorescent substances in the lipid extracts, in association with a significant decrease in the PE and PS of the RBCs, and a decrease in arachidonic acid in membrane lipids. The above changes were almost completely inhibited by tocopherol incorporated into vitamin E deficient RBCs. PMID- 3021843 TI - Influence of aging and protein deficiency on neutrophil function. AB - This study examined the effect of age and protein deficiency on the function of mouse neutrophils. Compared with appropriate controls aging and protein deficiency caused significant reductions in respiratory burst activity, exocytosis, and enzyme release from neutrophils. Individually, neither aging nor protein deficiency caused decreases in the ability of the neutrophil to phagocytose or kill bacteria. When aged animals were fed a protein deficient diet, however, further reductions in neutrophil function occurred that resulted in significant decreases in phagocytosis and bacterial cell kill. These findings indicate that both aging and protein deficiency compromise neutrophil function. When both variables are present the abnormalities become sufficient to affect the neutrophils' most critically important functions. The results emphasize the importance of protein deficiency in the aged and help explain the high prevalence of bacterial infection in malnourished older individuals. PMID- 3021844 TI - Mobilization of intracellular calcium from the sarcoplasmic reticulum of B lymphocytes using cyclic guanosine 3'5'-monophosphate (cGMP). AB - Cyclic guanosine-3'5' monophosphate (cGMP) can mobilize intracellular calcium from the microsomal fraction of B-lymphocytes of the mouse spleen as a result of activation of cGMP-dependent microsomal proteinkinases. The existence of such a mechanism makes B-lymphocytes independent of extracellular calcium in response to agents whose effect on B-lymphocytes is mediated by calcium mechanisms. PMID- 3021845 TI - A clinical approach for the identification of peripheral pre- and post-synaptic alpha-adrenergic receptors. AB - The present study examines the possibility that pre- and post-synaptic alpha adrenergic receptors can be differentiated by a clinical pharmacological approach. We compared the ability of the alpha 1-selective antagonists, prazosin and urapidil, with the ability of the alpha 2-selective antagonist, yohimbine, to inhibit cardiovascular responses to the non-selective alpha-adrenergic agonist norepinephrine in 26 healthy volunteers. Urapidil (50 mg i.v.) and prazosin (5 mg orally) induced significant shifts to the right in the blood pressure dose response curve of norepinephrine. Yohimbine (10 mg orally), on the other hand, increased norepinephrine sensitivity, as indicated by a significant shift to the left of the norepinephrine dose-response curve. In addition, urapidil and prazosin decreased norepinephrine potency on heart rate, whereas no effect on norepinephrine-induced heart rate elevation was observed with yohimbine. From the studies in which yohimbine was used it can be assumed that the post-synaptic response to norepinephrine is not counteracted by the drug-induced inhibition of sympathetic neurotransmitter release. Hence, our findings confirm the presence of pre- and post-synaptic alpha-adrenergic receptors in man. PMID- 3021846 TI - Effect of interferon-alpha on immunoglobulin synthesis by human B cells. AB - We have investigated the effect of human recombinant interferon-alpha (IFN-alpha) on mitogen-induced immunoglobulin (Ig) production by peripheral blood mononuclear cells from normal individuals. Low concentrations (1 to 100 IU/ml) of IFN-alpha enhanced pokeweed mitogen-stimulated Ig production. In contrast, high concentrations of IFN-alpha (10(5) IU/ml) suppressed pokeweed mitogen-induced Ig production. Irradiation of T cells did not ablate the high dose suppression, indicating that suppression was not due to a radiation-sensitive T cell. Kinetic experiments revealed that IFN-alpha needed to be added to 10 day cultures within the first 72 hr for either enhancement or suppression to be noted. Preincubation of purified B cells with IFN-alpha suppressed Ig production as completely as when unfractionated mononuclear cells were incubated with IFN-alpha. On the other hand, preincubation of T cells or monocytes with IFN-alpha had no effect on subsequent Ig production in reconstituted mononuclear cell cultures. Mitogen induced proliferation of purified B cells was not affected by IFN-alpha at any concentration, but Ig production by purified B cells stimulated with Staphylococcus aureus Cowan I or anti-mu and B cell differentiation factors responded to IFN-alpha with low concentration enhancement and high concentration suppression. Studies of Ebstein-Barr virus-transformed B cell lines showed that IFN-alpha caused a similar effect on the CESS line as on peripheral blood B cells, with low dose enhancement and high dose suppression of Ig production. Thus one IFN-alpha effect is to modulate Ig production, and this appears to be a direct effect on B cells. Combined with the data in the accompanying paper, the effects of IFN-alpha on B cell function are similar in vivo and in vitro. PMID- 3021847 TI - Detection of Epstein-Barr virus-associated antigens and DNA in salivary gland biopsies from patients with Sjogren's syndrome. AB - Sjogren's syndrome (SS) is an autoimmune disorder characterized by lymphocytic infiltration of salivary and lacrimal glands. To determine whether Epstein-Barr virus (EBV) might play a role in the pathogenesis of this disorder, we used monoclonal antibodies and DNA probes to detect evidence of viral gene products and genomes in these patients' tissue biopsies and saliva. Cytoplasmic staining of epithelial cells (i.e., ductal and/or acinar cells) with monoclonal antibody against the EBV-encoded early antigen (EA-D) was noted in 8/14 salivary gland biopsies from SS patients. This antibody did not react with normal salivary glands or salivary gland tumors, nor with other tissues from SS patients. The reactive antigen in SS biopsies had a m.w. of 52,000 on the basis of immunoblotting experiments, similar to the EA-D antigen found in lymphoblastoid cells lytically infected with EBV. EBV DNA was detected in parotid biopsies from two SS patients in amounts ranging from 0.1 to 3 pg per 20 micrograms of cellular DNA. Southern blotting was used to demonstrate the reactivity with Bam V, Eco D, and Bam M probes. Parotid saliva samples from 8/20 SS patients contained EBV DNA detectable by slot blot hybridization. EBV DNA was not detected in saliva of age matched controls, rheumatoid arthritis patients lacking sicca symptoms, or patients with benign parotid tumors. The presence of EBV in salivary gland and saliva samples was associated with clinically more severe SS, as manifested by extraglandular symptoms such as vasculitis, occurrence of "pseudolymphoma", and marked abnormalities in immunoglobulin levels. These results demonstrate an elevated content of EBV in salivary glands of SS patients, and suggest that this virus may play a role in pathogenesis. PMID- 3021848 TI - Prostaglandins posttranscriptionally inhibit monocyte expression of interleukin 1 activity by increasing intracellular cyclic adenosine monophosphate. AB - The effect of prostaglandins and cyclic 3',5'-adenosine monophosphate (cAMP) on expression of human interleukin 1 (IL 1) activity was investigated in the promonocytic tumor cell line U937 and peripheral blood monocytes. After in vitro stimulation by bacteriotoxins, monocytes express IL 1 activity, as measured by the thymocyte costimulation assay. Although high doses of bacteriotoxins impaired expression of IL 1, this effect was reversed by indomethacin. When stimulated monocytes were cultured with exogenous prostaglandins, including PGE2 and PGI2, expression of IL 1 was reversibly inhibited. Interaction of U937 cells with PGE2 resulted in a transient increase in cellular cAMP concentration during the first hour of exposure. Other agents that cause an increase in levels of cellular cAMP, including theophylline, isobutylmethylxanthine, dibutyryl cAMP, or cholera toxin, also reversibly reduced expression of IL 1 by stimulated monocytes. The effect of these agents on levels of IL 1 mRNA was analyzed. TSS-stimulated increase in levels of IL 1-encoding mRNA was studied both by DNA-RNA hybridization analysis performed with an IL 1-beta cDNA probe and by injecting U937 polyadenylated mRNA into frog oocytes and then measuring expression of IL 1 activity in the oocyte supernatant. Agents that increased levels of cellular cAMP did not alter levels of IL 1 mRNA accumulation or global protein synthesis in TSS-stimulated U937 cells. IL 1 stimulates synthesis of prostaglandins that reach high levels during immune and inflammatory reactions. Our data suggest that prostaglandins participate in an autoregulatory pathway that posttranscriptionally reduces expression of IL 1 activity. PMID- 3021849 TI - Isolation of soluble yeast beta-glucans that inhibit human monocyte phagocytosis mediated by beta-glucan receptors. AB - The trypsin-sensitive receptor that mediates phagocytosis of unopsonized zymosan particles by human monocytes has been designated as a beta-glucan receptor because of its functional inhibition by specific algal and plant beta-glucans. Soluble ligands that are chemically and structurally identical to beta-glucan constituents of zymosan were isolated from a carbohydrate-enriched fraction of yeast extract by sequential chromatography on DE-cellulose, SP-Sephadex, and Con A-Sepharose. Preincubation of adherent human monocytes with 278, 210, and 2.5 micrograms/ml hexose equivalents in pooled chromatographic fractions from DE cellulose, SP-Sephadex, and Con A-Sepharose, respectively, effected 50% reductions in subsequent phagocytosis of zymosan particles without affecting Fc mediated ingestion of IgG-coated sheep erythrocytes (ESIgG). The purified yeast extract-derived beta-glucans, which contained 92% glucose and 8% mannose by gas chromatographic analysis and eluted from a Sephacryl S-200 column as a broad peak with a Kav of 0.39 and estimated molecular sizes of from 20,000 to 70,000 m.w., required only 3.5 +/- 0.9 micrograms/ml (mean +/- SD, n = 6), as compared with 31.5 micrograms/ml of the algal beta-glucan laminarin to achieve 50% decreases in zymosan ingestion. Alternatively, soluble yeast beta-glucans with estimated molecular sizes of from 2 X 10(5) to 2 X 10(6) were prepared from yeast glucan particles, which contained 98% glucose and 0% mannose, by sonication and sequential centrifugation at 15,000 and 100,000 X G for 30 and 60 min, respectively. Monocyte ingestion of zymosan was reduced by 50% by pretreatment with 60 ng/ml of the soluble beta-glucans in 15,000 X G supernatants, whereas ingestion of ESIgG was unaffected by as much as 50 micrograms/ml of this material. Partial acid hydrolysis of soluble glucan-derived beta-glucans in 15,000 X G supernatants followed by gel filtration on Bio-Gel P-4 revealed two well-defined peaks within the inclusion volume of the column with phagocytosis inhibiting activity. Oligoglucosides that eluted at a Kav of 0.46 had an estimated molecular size of 2,000 m.w. and effected a 48% reduction in zymosan ingestion at inputs of 2 to 5 micrograms/ml, and smaller oligoglucosides with a Kav of 0.82 and an estimated molecular size of 1,000 m.w. effected a 50% reduction at inputs of 25 micrograms/ml. Preincubation of monocytes for 2 min with 25 micrograms/ml of the oligoglucosides with estimated molecular size of 1,000 m.w. and with 50 ng/ml of soluble glucan-derived beta-glucans in 100,000 X G supernatants reduced zymosan ingestion by 41% +/- 4 and 44% +/- 3 (mean +/- SD, n = 3), respectively.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3021850 TI - The effects of adenosine agonists on human neutrophil function. AB - Adenosine is a potent physiologic substance with a variety of biologic activities. Many of the effects of adenosine appear to be mediated by two populations of cell-surface adenosine receptors (A1 and A2). We have examined the effects of several adenosine receptor agonists on human neutrophils stimulated with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). The results indicate that both superoxide anion generation and degranulation (as assessed by lysozyme release) are inhibited. Inhibition correlated most strongly with A2 receptor affinity for both parameters and was reversible by the adenosine receptor antagonist 8-phenyltheophylline. Because toxic oxygen metabolites and degradative enzymes are implicated in a variety of inflammatory disorders, adenosine agonists may be useful probes to help expand our knowledge of the role of these mediators in human disease. PMID- 3021851 TI - Increased superoxide anion release from human endothelial cells in response to cytokines. AB - To study the effects of macrophage and lymphocyte-derived factors on superoxide anion (O2-) generation and release from human umbilical vein endothelial cells (EC), cultured EC were stimulated by ultrapure interleukin 1 (IL 1) and recombinant interferon-gamma (IFN-gamma), and the O2- released into the supernatant was measured. Both of these cytokines enhanced O2- release in a dose and time-dependent manner. Addition of a combination of IL 1 and IFN-gamma, each in submaximal concentration, produced an additive effect on O2- release. It would appear from these findings that cytokines released by macrophages and lymphocytes during inflammatory reactions can promote O2- generation and release from human EC. O2- released from EC may alter the basement membrane of blood vessels and the surrounding connective tissue, and in this way promote the vascular injury and angiogenesis associated with local inflammation. PMID- 3021853 TI - Regulation of guinea-pig immune functions by interleukin 2: critical role of natural killer activity in acute HSV-2 genital infection. AB - We have previously demonstrated that recombinant interleukin 2 (rIL 2) has a protective effect against acute HSV-2 infection in guinea pigs with a biphasic dose response which peaked between 4 and 20 X 10(4) U/kg, whereas 8 X 10(5) U/kg showed no effect on disease. Animals that escaped infection appeared lack immunologic memory to HSV-2, suggesting a nonspecific immune mechanism. In this study we have found that NK activity of fresh splenocytes measured against HSV-2 infected human foreskin fibroblast (HFF) is stimulated in vitro and in vivo by rIL 2 in a biphasic dose range similar to that determined for protection against disease. In contrast, lymphokine-activated killer (LAK)-mediated lysis of P815 showed a linear response to increasing concentrations of rIL 2 both in vitro and in vivo. Transfer of LAK cells did not alter the rate of infection after HSV-2 challenge. Anti-asialo GM-1 eliminated rIL 2 protection against HSV-2 infection. It also blocked HSV-2/HFF lysis and partially decreased P815 lysis in vitro; however, in vivo it inhibited both natural killer (NK) activity and LAK generation, failing to distinguish which of the lytic cells was responsible for the effect against infection. Early IgG production (7 days post-infection) was enhanced by rIL 2 administration before viral inoculation, but it did not influence the rate of infection as compared with controls. Polyclonal IgM secretion was not found to play a role in acute protection. Circulating serum interferon levels were enhanced with increasing concentrations of rIL 2 but did not correlate with the biphasic dose curve for protection. Therefore of these mechanisms the one that is most closely related to the protective effect of rIL 2 against primary HSV-2 infection appears to be NK-mediated lysis, although the other mechanisms may add to this effect. PMID- 3021852 TI - Antibody binding to CD5 (Tp67) and Tp44 T cell surface molecules: effects on cyclic nucleotides, cytoplasmic free calcium, and cAMP-mediated suppression. AB - T cells can be activated to proliferate by antibodies to the T cell antigen receptor or the molecularly associated CD3 complex if monocytes are present. We have shown previously that monoclonal antibodies to the human T cell differentiation antigens CD5 (Tp67) and Tp44 each augment and prolong proliferative responses of anti-CD3-activated T cells, even in the absence of monocytes. Here we show that the functional and biochemical mechanisms of CD5 and Tp44 signal transmission are distinct. T cell proliferation is suppressed by agents that increase the concentration of intracellular cAMP. We found that antibody binding to the Tp44 surface molecule overcomes this suppression, whereas antibody binding to CD5 does not, indicating that ligand-Tp44 interaction changes T cell sensitivity to cAMP-mediated growth inhibition. The ability of anti-CD3, anti-Tp44, and anti-CD5 monoclonal antibodies to directly alter cyclic nucleotide levels in the Jurkat T cell line was examined. Anti-CD3 alone caused a rapid four to sixfold increase in cAMP levels, but did not affect cGMP levels. However, anti-Tp44 and anti-CD5 each caused a rapid three- to fourfold increase in cGMP levels without affecting cAMP levels. In other experiments, cytoplasmic free calcium levels were measured in resting T cells after CD5 or Tp44 stimulation by using the dye indo-1 and flow cytometry. This sensitive method showed that anti CD5 alone caused an increase in cytoplasmic calcium free levels within 3 min of antibody addition, whereas anti-Tp44 had no effect. Finally, anti-Tp44 and IL 1 each augmented proliferation of phorbol ester-stimulated lymphocytes, whereas anti-CD5 did not. The effects of IL 1 and Tp44 could be further distinguished in that the effect of anti-Tp44 was resistant to inhibition by dBcAMP whereas IL 1 was not. These data suggest that the receptor function of both Tp44 and CD5 involves changes in cyclic nucleotides levels, and that the mechanism by which anti-Tp44 and anti-CD5 antibodies affect T cell proliferative responses may be related to their selective effects on cGMP levels and cytoplasmic calcium concentrations. PMID- 3021854 TI - A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. I. Development and method of ELISA. AB - A liquid-phase blocking sandwich ELISA has been developed for the quantification of antibodies against foot-and-mouth disease virus which may replace the virus neutralisation (VN) test. This test employs the incubation of a constant amount of antigen with a range of test serum dilutions in the liquid-phase before being assayed using a trapping ELISA. Thus it does not rely on the availability or growth of tissue culture cells. The assay is rapid and relatively simple to perform, reagents are used economically and results may be recorded within 24 h. The ELISA is sensitive and results are more specific and more reproducible than those obtained by VN. Results are expressed as reciprocal antibody titres which are analogous and of a similar order to those recorded by VN. Individual titres, therefore, may be easily assessed by workers in the field who are already familiar with VN. PMID- 3021855 TI - A new enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. II. Application. AB - The liquid-phase blocking sandwich ELISA has been evaluated for the serological study of antibodies against foot-and-mouth disease virus (FMDV). The titres recorded for sera from a population of more than 300 British uninfected, unvaccinated cattle which were examined against each of the seven immunologically distinct FMDV types were less than 1 in 40. A positive correlation between ELISA and VN titres was recorded for sera either vaccinated or involved in outbreaks of FMDV. The overall regression between the ELISA/VN data showed that 1 in 16 by VN was equivalent to 1 in 40 by ELISA. Thus ELISA is sensitive, specific and reproducible and results may be directly correlated to those recorded by VN. Serum titres may be interpreted as positive or negative and the number of sera which require retesting would be considerably less than by VN. It is suggested that this ELISA may be used as an alternative to the existing VN test for the quantification of antibodies against FMD virus. PMID- 3021856 TI - Detection of receptors for polymerised human albumin by immunoperoxidase and immunoadherence in liver tissue of HBsAg chronic carriers. AB - Receptors for polymerised human albumin (pHSA-Rs) were detected in unfixed cryostat sections from HBsAg chronic carriers using direct immunoperoxidase and immunoadherence methods. Although pHSA-Rs were detected by both methods, the receptors detected by immunoperoxidase were associated with HBV and showed properties different from the receptors detected by immunoadherence. The double immunocytochemical staining which detected contemporaneously pHSA-Rs and HBsAg in the same cell showed that there are two types of infected hepatocytes: one capable of synthesizing pHSA-Rs and HBsAg and the other capable of synthesizing only HBsAg. The intrahepatocyte synthesis of pHSA-Rs does not correlate with the severity of chronic liver disease or with the presence of tissue HB core antigen. PMID- 3021857 TI - Reductive amination for solid-phase coupling of protein. A practical alternative to cyanogen bromide. AB - For coupling proteins to Sephacryl gels periodate oxidation of these gels was investigated as an alternative method to cyanogen bromide activation. Optimum conditions were studied with respect to periodate concentration, time of oxidation, pH and type of coupling buffer, concentration of protein, temperature and time of protein uptake, and protein leakage after coupling. The effects of sodium cyanoborohydride and ascorbic acid as reducing agents, and of manganese ions as a potential catalyst were investigated. Using the derived optimum conditions, stable solid-phased antibodies were produced in high yield and used to adsorb factor VIII from plasma. These gels were stable for many weeks, as was the intermediate oxidised gel. Reductive amination for coupling proteins to oxidised Sephacryl gels results in increased binding and lower leakage than is obtained with cyanogen bromide activated agarose. PMID- 3021858 TI - An unusual case of paraparesis with multiple congenital defects. PMID- 3021859 TI - Glucocorticoid-induced modulation of the beta-adrenergic adenylate cyclase response of epidermis: its relation to epidermal phospholipase A2 activity. AB - It has been suggested that glucocorticoids produce their biologic effects through the synthesis of phospholipase A2 inhibitor protein (lipocortin) in various cell systems. Recent studies from our laboratory revealed that glucocorticoids augment the beta-adrenergic adenylate cyclase response of epidermis and that this effect depends on a protein synthesis mechanism. In order to elucidate the possible mechanism of this glucocorticoid effect in terms of phospholipase A2 activity, an in vitro pig skin incubation system was employed. Mepacrine, a phospholipase A2 inhibitor, augmented the beta-adrenergic adenylate cyclase response of epidermis as glucocorticoids. The effect of mepacrine was stronger and was observed earlier than that of glucocorticoid (hydrocortisone). The addition of both mepacrine and hydrocortisone at their optimal concentrations in the incubation medium, resulted in neither an additive nor a synergistic effect on the beta-adrenergic augmentation. On the other hand, melittin, a phospholipase A2 stimulator, depressed the beta-adrenergic adenylate cyclase response. The addition of both melittin and hydrocortisone in the incubation medium resulted in the inhibition of the hydrocortisone-induced beta-adrenergic augmentation effect. Following long term incubation with hydrocortisone, the epidermal phospholipase A2 activity was significantly decreased. These results indicate that glucocorticoids might affect the beta-adrenergic adenylate cyclase response of epidermis through the synthesis of phospholipase A2 inhibitor protein (lipocortin) as in other cell systems. PMID- 3021860 TI - Elevated levels of human collagenase inhibitor in blister fluids of diverse etiology. AB - Blister fluids from a variety of bullous disorders were examined for the presence of human collagenase inhibitor. A protein immunologically identical to the collagenase inhibitor produced by human skin fibroblasts was found in high concentrations within bullae of diverse etiologies. Levels of collagenase inhibitor in blister fluids ranged from 0.9-12.5 micrograms/ml, averaging 4.9 micrograms/ml. The mean values were 3- to 4-fold greater than those present in the sera of corresponding patients and exceeded plasma levels by 6- to 8-fold. The time course of collagenase inhibitor accumulation in blister fluid was studied using heat- and suction-induced bullae. The concentration in newly formed blisters was approximately 0.5 micrograms/ml, virtually identical to plasma inhibitor levels, and remained constant for approximately 4 h. Inhibitor concentrations then rose rapidly, reaching peak values of approximately 6 micrograms/ml after 48 h. We speculate that the role of this inhibitor in blister fluid involves the inhibitions of active proteinases within the bulla cavity and may occur to limit the extent of blister formation or to assist in wound repair. PMID- 3021862 TI - Epidermis and lymphocyte interactions during a tuberculin skin reaction. II. Epidermis contains specific lymphocyte chemotactic factors. AB - Lymphocyte chemotaxis was studied in a blind-well chamber assay by measuring the passage of 51Cr-labeled cells through a polycarbonate filter with a pore size of 5 micron. Monocyte-depleted lymphocytes were divided into T cells (E receptor positive lymphocytes) and non-T cells. T lymphocytes showed pronounced migration after exposure to leukotriene B4 (LTB4) and casein, and weak migration after exposure to N-formyl-methionyl-leucyl-phenylalanine (FMLP). Non-T cells showed strong migration after exposure to FMLP, but weak migration after exposure to casein and LTB4. Supernatants of homogenized suction blisters from normal skin did not induce active migration. However, if the epidermis came from an area overlying a positive tuberculin skin reaction, there was a significant migration mostly of T, but also of non-T cells. Supernatants from phytohemagglutinin (PHA) stimulated lymphocyte cultures also contained lymphocyte chemotactic factor(s), which, however, had an effect only on T lymphocytes. Purified protein derivative of tuberculin (PPD)-stimulated lymphocytes did not produce chemoattractants either for T or for non-T cells. These studies show that lymphocytes can show active, directed migration following exposure to well-known chemotaxins for granulocytes and monocytes although their migrational capability differs for different subpopulations. Epidermis overlying a cell-mediated immune reaction (tuberculin) contains epidermal lymphocyte chemotactic factor(s). This factor(s) may be of importance for the type of cell infiltrate occurring in certain dermatologic disorders. PMID- 3021861 TI - Antenatal diagnosis of recessive dystrophic epidermolysis bullosa: collagenase expression in cultured fibroblasts as a biochemical marker. AB - We performed fetoscopy and skin biopsy on a 19-week fetus at risk for recessive dystrophic epidermolysis bullosa (RDEB). Ultrastructural analysis of the tissue revealed dermolytic blister formation in the skin characteristic of the disease. To develop a biochemical test for use in antenatal diagnosis of RDEB, we established skin fibroblast cultures from the 20-week aborted fetus. The collagenase production by fetal RDEB fibroblast cultures was greater than seen in normal fetal fibroblast cultures. The concentration in culture medium from fetal RDEB cultures was 5.42 +/- 0.74 micrograms/ml (mean +/- SE) compared with 2.24 +/ 1.11 micrograms/ml in normal adult control cultures and 2.05 +/- 0.61 micrograms/ml in cultures from patients with other genetic forms of epidermolysis bullosa (p less than 0.025). In contrast, the concentration of collagenase in the fetal RDEB culture medium was not different from that seen in cell cultures from known patients with RDEB (5.34 +/- 1.12 micrograms/ml). Collagenase activity of the fetal RDEB medium was also increased approximately 3.5-fold. These data indicate that enhanced expression of collagenase by fetal RDEB skin fibroblasts can serve as a biochemical adjunct, and possibly an alternative, to morphologic examination of tissue for antenatal diagnosis. PMID- 3021863 TI - Anthralin (1,8-dihydroxyanthrone) is a potent inhibitor of leukotriene production and LTB4-omega oxidation by human neutrophils. AB - The effect of anthralin and its oxidation products danthrone and anthralin-dimer on the production of 5-lipoxygenase products (5-HETE, leukotriene B4, omega oxidized LTB4) by Ca-ionophore A 23187-stimulated human neutrophils has been studied in vitro. Anthralin exhibited dose-dependent inhibitory activity showing 50% inhibition at 7 microM with 10(7) neutrophils. Inhibitory effects strongly depended upon cell densities and maximal inhibition occurred at low cell concentrations, whereas inhibitory rates of anthralin were low at high cell densities. Inhibition of leukotriene production persisted after washing of anthralin-treated neutrophils. Also, with increasing amounts of arachidonic acid as substrate only slight changes of inhibitory activity were detected, indicating a noncompetitive way of action. In addition to the inhibition of leukotriene production, the formation of omega-OH-LTB4 from LTB4 as well as omega-COOH-LTB4 from omega-OH-LTB4 was inhibited with IC50 (half maximum inhibition concentration) near 4.4 microM and 2.2 microM, respectively. In contrast to anthralin, both metabolites--danthrone as well as anthralin-dimer--did not show any effect on leukotriene production and omega-oxidation even at high concentrations (up to 70 microM and 44 microM, respectively). PMID- 3021864 TI - Ultraviolet-irradiated urocanic acid suppresses delayed-type hypersensitivity to herpes simplex virus in mice. AB - Ultraviolet radiation is known to induce a transient defect in epidermal antigen presentation which leads to the generation of antigen-specific suppression of the delayed-type hypersensitivity (DTH) response. The putative receptor in skin for the primary event in UV-suppression is urocanic acid (UCA) which may then interact locally, or systemically, with antigen presenting cells or initiate a cascade of events resulting in suppression. We present the first direct evidence that UCA, when irradiated with a dose (96 mJ/cm2) of UVB radiation known to suppress the DTH response to herpes simplex virus, type 1 (HSV-1) in mice, can induce suppression following epidermal application or s.c. injection of the irradiated substance. This suppression is transferable with nylon wool-passed spleen cells. PMID- 3021865 TI - Relation of cytosolic calcium to the microbicidal activation of blood monocytes by recombinant gamma interferon. AB - Oxygen metabolism and calcium ion (Ca++) handling in blood monocytes were greatly affected by treatment with recombinant gamma interferon (rIFN-gamma). Incubating the monocytes with rIFN-gamma resulted in increased basal production of superoxide anion, as well as a significantly enhanced respiratory burst in response to concanavalin A (Con A). A concomitant increase in microbicidal activity against Listeria monocytogenes was observed, and cell membrane permeability to Ca++ was enhanced by treatment with rIFN-gamma. Con A stimulation was also associated with acute rises in cytosolic-free calcium, and these calcium transients were greatly augmented in cells pretreated with rIFN-gamma. The enhanced respiratory burst and the increased calcium transients were completely neutralized by a monoclonal antibody to rIFN-gamma. Furthermore, enhancement of the respiratory burst and calcium transients occurred in a parallel dose-response range for rIFN-gamma. Blocking the intracellular release of calcium into cytosol by TMB-8 abrogated the capacity of rIFN-gamma to enhance the monocyte respiratory burst. Thus, not only was rIFN-gamma treatment of monocytes associated with altered calcium metabolism, but calcium from intracellular pools was essential to the expression of this activated state during Con A stimulation. PMID- 3021866 TI - Changes in the structural and functional properties of human eosinophils during experimental hookworm infection. AB - Normal volunteers were infected with hookworm larvae Necator americanus. Peripheral blood counts showed a mean of 524 +/- 29 eosinophils/mm3 of blood before infection and a mean of 3,008 +/- 456 eosinophils/mm3 of blood during infection (P less than .01). Absolute numbers of neutrophils did not change. Eosinophils and neutrophils from the infected period were compared with the noninfected state in each subject. The percentage of hypodense eosinophils increased from a mean of 34% +/- 13% to 80% +/- 7% during infection (P less than .05). Superoxide production of eosinophils increased from a mean of 56 +/- 9 to 97 +/- 12 nmol of O2-./10(6) cells per 60 min (P less than .05) during infection. Chemotaxis of eosinophils to Escherichia coli endotoxin-activated serum increased from a mean average distance migrated of 19 +/- 2 micron (P less than .05), whereas neutrophil responsiveness did not change. This is the first report of changes in eosinophil density and stimulation of eosinophil function in normal hosts experimentally infected with hookworm. The data indicate that hookworm infection preferentially increases eosinophil production and activity with little effect on neutrophils. PMID- 3021867 TI - Epstein-Barr virus infections in families: the role of children with infectious mononucleosis. AB - We performed a prospective evaluation of Epstein-Barr virus (EBV) infections in 78 families with a childhood index case of EBV-infectious mononucleosis (EBV-IM). During the acute index episode, adult family (household) contacts, compared with control adults, had a greater rate of oropharyngeal EBV excretion and high serum antibody responses, which suggested a recent antecedent reactivation of an old EBV infection. The increased prevalence of oropharyngeal EBV and acute serological responses found in sibling family contacts during the early surveillance period suggested that these contacts had experienced an increased rate of primary-type EBV infections shortly before or concurrent with the index case. Although nonimmune sibling contacts seroconverted at a slow rate, five of nine manifested IM with their eventual documented primary EBV infection. This study noted significant intrafamilial EBV activity surrounding an episode of childhood EBV-IM. Reactivation of an old EBV infection in adults may be an important source of virus for susceptible children within these families. Siblings of a childhood case of EBV-IM appear to be at increased risk for manifesting IM with their primary EBV infection. PMID- 3021868 TI - Enhanced serological and virological findings of Epstein-Barr virus in patients with AIDS and AIDS-related complex. AB - The potential involvement of Epstein-Barr virus (EBV) in AIDS was examined by determining the type of EBV-specific antibody responses and the EBV content or lymphoproliferative ability present in selected body fluids of patients with AIDS or AIDS-related complex. The results were compared with two control groups. An enhanced antibody response to a broad spectrum of EBV antigens was found in patients with AIDS or AIDS-related complex. The pattern of virus-specific antibody responses resembled that associated with a persistent or reactivated infection. The content of EBV in oropharyngeal secretions and the lymphoproliferative ability in peripheral blood from patients with AIDS or AIDS related complex was significantly greater than that from healthy controls and approached levels detected in the control group with infectious mononucleosis. These findings, together with recent reports of cellular-level interaction between EBV and human T lymphotropic virus type III, suggest that EBV may have a contributory role in these disorders. PMID- 3021869 TI - Human rotavirus studies in volunteers: determination of infectious dose and serological response to infection. AB - An unpassaged, safety-tested strain (CJN) of human rotavirus from a stool specimen of a hospitalized child was administered orally to 62 adult volunteers for determination of the dose required to produce infection with or without illness. Subjects ingested doses ranging from 9 X 10(-3) to 9 X 10(4) focus forming units in buffered salt solution after consumption of 50 ml of 4% NaHCO3. The amount of virus in the inoculum required to cause infection (shedding of virus, seroconversion, or both) in study subjects was comparable to the minimum detectable in cultures of primary monkey kidney cells. Seventeen of 30 infected subjects became ill with doses equivalent to that required for infection. Although the preinoculation titers of serum neutralizing antibody to the challenge virus in study subjects ranged from less than 1:2 to 1:1,600, the concentration of serum antibody could not be correlated with protection from infection or illness in subjects given an infectious dose of virus. PMID- 3021870 TI - Role of the monocyte in cytomegalovirus-mediated immunosuppression in vitro. PMID- 3021871 TI - A synthetic peptide for detecting antibodies to Epstein-Barr virus nuclear antigen in sera from patients with infectious mononucleosis. PMID- 3021872 TI - Vaginal transmission of human immunodeficiency virus (HIV) to a chimpanzee. PMID- 3021873 TI - The effect of hydrocortisone on the production of gamma-interferon and other lymphokines by human peripheral blood mononuclear cells. AB - Concentrations of hydrocortisone as low as 0.08 microgram/ml significantly reduced the yields of gamma-interferon (IFN-gamma) when phytohemagglutinin (PHA) or concanavalin A (ConA) were used as inducers; however, when staphylococcal enterotoxin A was utilized, higher concentrations (5.0 micrograms/ml) were required to achieve the same effect. Yields of interleukin-2 (IL-2) and lymphotoxin were also found to be sensitive to the effects of the steroids, but expressions of TAC antigen was not generally affected by these agents. In contrast to the effects of steroids on cell proliferation, lymphokine production remained suppressed after steroid withdrawal. Hydrocortisone appeared to influence the concentrations of cyclic nucleotides following lectin stimulation, but attempts to correct these alterations or to add exogenous IL-2 failed to restore lymphokine production to normal levels. Addition of the calcium ionophore A23187 partially restored IFN-gamma production. We conclude that the effects of corticosteroids on the yields of lymphokines, including IFN-gamma, are profound. The depression of lymphokine production appears to be associated with a number of alterations in the cell, including depression of protein synthesis, alterations in cyclic nucleotides, and diminution of the production of cofactors necessary for IFN-gamma production. Enhancement of the flux of calcium into the cell may restore some of the ability to produce IFN-gamma. PMID- 3021875 TI - Presynaptic inhibition of primary afferents. PMID- 3021874 TI - Antiviral and antitumor effects of a human interferon analog, IFN-alpha Con 1, assessed in hamsters. AB - An analog of human alpha-and beta-interferons (IFN-alpha and -beta) (generally consisting of the most frequently observed amino acid residue at each position in the molecule) has pronounced antiviral and antiproliferative activity in human and hamster cells. Intraperitoneal administration of this analog (designated IFN alpha Con 1) to hamsters at 10(6) to 10(8) U/kg resulted in proportional increases in plasma concentrations through 6 h of monitoring. IFN-alpha Con 1 at these doses effectively limited encephalomyocarditis virus (EMCV) infections of hamsters. A natural human IFN-alpha preparation was also active against virus infections in hamsters. The antitumor activity of IFN-alpha Con 1 and natural human IFN-alpha was assessed in hamsters inoculated with lethal TBD932 lymphosarcoma. Various IFN treatment schedules resulted in prolonged survival following tumor challenge. IFN-alpha Con 1 administered at 10(5) to 10(6) U/hamster daily for 9-12 days following tumor challenge was effective in delaying tumor development, as was a natural human IFN-alpha preparation. The efficacies of combined IFN and cyclophosphamide therapies were determined. Unlike the natural human subtype IFN-alpha A, IFN-alpha Con 1 did not diminish the efficacy of cyclophosphamide (2.5 mg/hamster for 3 days) against the lymphosarcoma. However, an ineffective dose of cyclophosphamide (0.05 mg/hamster for 3 days) when combined with IFN-alpha Con 1 treatment showed enhanced antitumor activity. Combinations of cimetidine (16 mg/hamster for 4 days) and IFN-alpha Con 1 treatment did not prolong survival in this model system. PMID- 3021876 TI - Immunocytochemical investigation of native matrix granules of the rat heart mitochondrion. AB - We have examined the ultrastructure and the protein content of native matrix granules (NMG) in rat heart mitochondria, by postembedding immunocytochemistry. Cytochrome c oxidase was found to be present in these granules. It is believed that these granules contain incomplete inner mitochondrial membrane fractions, which can be incorporated in the membrane after stimulation of the metabolism. PMID- 3021877 TI - [Experimental study on porous hydroxyapatite ceramics for onlay implantation- experiment on onlay implantation in the mandible]. PMID- 3021879 TI - Role of the Vi antigen of Salmonella typhi in resistance to host defense in vitro. AB - The virulence of Salmonella typhi is associated with the presence of the Vi antigen. Mechanisms of Vi antigen virulence were examined in vitro. The Vi antigen-containing strain Quailes was significantly (P less than 0.025) more resistant to lysis by nonimmune serum than S. typhi 0901, which does not have the Vi antigen, and resulted in less activation of complement by the alternative pathway (P less than 0.05). Human polymorphonuclear leukocytes (PMNs) ingested strain Quailes significantly (P less than 0.01) more slowly and less completely than strain 0901 as assessed by three measures of phagocytic rate. In contrast to prior reports, the Vi antigen did not prevent an oxidative burst, measured by O2- production, chemiluminescence, and O2 consumption. The extent of the oxidative burst correlated directly and closely with the rate of phagocytosis. When the rate of PMN phagocytosis for both strains was equalized by opsonizing strain 0901 with 1% and strain Quailes with a 3% concentration of serum, the PMN oxidative burst was equal. C3 binding to strain Quailes was significantly (P less than 0.005) less than to strain 0901. Hence the Vi antigen inhibited phagocytosis by preventing C3b binding and solely as a consequence of this induced a lesser PMN oxidative burst. Furthermore, strain Quailes was significantly (P less than 0.025) less susceptible to killing by H2O2 than strain 0901. To ensure that these observations were a consequence of the Vi antigen and not other strain differences, another pair of S. typhi with and without the Vi antigen were similarly compared, and the results were the same as with strains Quailes and 0901. Strains 0901 and Quailes were killed by PMNs from a patient with chronic granulomatous disease but more slowly than by normal PMNs, indicating that S. typhi is susceptible to nonoxidative killing. PMID- 3021878 TI - Neutrophil defect associated with malignant infantile osteopetrosis. AB - We studied the phagocytes from three infants with malignant osteopetrosis and from their families in an attempt to define further the phagocyte abnormalities associated with this disorder. The rapid membrane potential depolarization in response to the soluble stimuli formylmethionylleucylphenylalanine (FMLP) and phorbol myristate acetate (PMA) served as a measure of neutrophil activation, with 3,3-dipentyloxacarbocyanine (diOC5 [3]) used as the probe. A fluorescence activated cell sorter (FACS) allowed us to use small volumes of blood in a new quantitative evaluation of neutrophil response. The neutrophils from the infants with malignant osteopetrosis were very minimally activated with either stimulus, as demonstrated by incomplete membrane potential depolarization (10% to 15% of normal controls). This finding indicates that malignant osteopetrosis is accompanied by a significantly reduced neutrophil response to stimulation. The abnormal activation process was also reflected in the respiratory burst response of the patients' neutrophils and monocytes. Fifty percent to 60% of the infants' neutrophils totally failed to reduce nitro blue tetrazolium dye (NBT), 30% to 40% of the cells showed only slight reduction after PMA or FMLP stimulation, and only 5% to 10% demonstrated normal reduction. Peripheral blood monocytes failed to reduce NBT in 35% to 70% of the cells tested. Similar testing of granulocyte macrophage colonies grown in vitro from circulating progenitor cells also showed an abnormal distribution of response to PMA, with a majority of colonies showing a decrease or absence of NBT reduction. Thus control of expression of the osteopetrotic defect occurs at or before the progenitor cell level. PMID- 3021881 TI - Glomus vagale: an unusual presentation. AB - An unusual case of glomus vagale tumour is presented where severe pain was a predominant feature. The pathological and clinical features of this rare tumour are reviewed and the investigations and treatment are discussed. PMID- 3021880 TI - Adenoid cystic carcinoma presenting as a mass in the neck. AB - Three of a personal series of 82 patients with adenoid cystic carcinoma presented with a node in the neck. Two of these patients had no discoverable primary tumour. One of the latter died rapidly, and might have had an undisclosed primary tumour in the lung. The other remains well 3 years after radical neck dissection, and it is possible that he had a tumour arising in an aberrant cervical salivary rest. PMID- 3021882 TI - Biochemical events associated with the stimulation of rabbit neutrophils by platelet-activating factor. AB - The functional and biochemical responses evoked by the addition of platelet activating factor (PAF) to a suspension of rabbit neutrophils have been characterized in an effort to define the mode of action of this lipid mediator. PAF was found to elicit a secretory response and to stimulate a rapid breakdown of the polyphosphoinositides, an increase in the cytoplasmic level of free calcium (as monitored by quin2), a decrease in the fluorescence of cell associated chlortetracycline, an enhanced activity of the sodium/hydrogen antiport, a transient depolarization, and an increase in the level of cytoskeletal actin. The quin2 response to PAF was found to be detectable at concentrations as low as 0.01 nM, to be very dependent on the presence of extracellular calcium, and to be sensitive to inhibition by phorbol esters. On the other hand, the increase in free calcium induced by PAF in the presence of extracellular calcium was essentially unaffected by pertussis toxin. PAF-induced neutrophil degranulation was similarly extracellular calcium dependent and phorbol ester sensitive. The secretory activity of PAF was evident only at concentrations in excess of 1 nM. All of the other effects of PAF were found to be independent of the presence of external calcium and to be demonstrable only at concentrations larger than 1 nM. In addition, all neutrophil responses to PAF (with the above noted exception of quin2) were potently inhibited by pertussis toxin. These results are interpreted in terms of the possible existence of two functionally distinct populations of receptors. The occupation of one set (of apparent high affinity) induces an increase in permeability to calcium in a phorbol-ester-, but not pertussis-toxin-, sensitive manner. The activation of the other set of receptors at higher concentrations of PAF stimulates the polyphosphoinositide-specific phospholipase C and induces the attendant biochemical responses. These latter responses appear to be mediated by a guanine nucleotide-binding regulatory protein. PMID- 3021883 TI - Transient catecholamine modulation of neutrophil activation: kinetic and intracellular aspects of isoproterenol action. AB - Modulation of neutrophil activation by catecholamines may reflect regulatory mechanisms that couple beta-adrenergic and N-formyl peptide receptors to antagonistic biochemical pathways. We examined kinetic and mechanistic aspects of the inhibition by catecholamines of neutrophil activation by formyl peptides. Inhibition of oxidant production by isoproterenol (ISO) was detected as low as 3 nM, had an ID50 of 10(-7) M, and could be blocked and reversed by propranolol. Recovery of cell function occurred over a period of minutes when the concentration of ISO was less than 10(-6) M. These observations are discussed in terms of the interaction of ISO with the adrenergic receptors. The site of catecholamine action is addressed. ISO neither influences formyl peptide-receptor interaction nor does it inhibit oxidant production by phorbol ester. These results suggest an impairment of intracellular signalling processes that couple the formyl peptide-receptor binding to cell activation. We observed inhibition of intracellular Ca++ elevation by ISO only at low formyl peptide concentrations. This inhibition is consistent with a partial inhibition of phosphoinositide metabolism, which was observed. Several other cell responses, including actin polymerization and right angle light scatter, are minimally inhibited by 10(-6) M ISO indicating that the cell activation process is not entirely obliterated. The presence of catecholamine and formyl peptide results in a synergistic elevation of cAMP. The intracellular targets of ISO action may be regulated by cAMP dependent kinases and could follow a branchpoint in the activation sequence that leads distinctly to oxidase activation and cytoskeletal activation. PMID- 3021884 TI - Very low and low density lipoprotein synthesis and secretion by the human hepatoma cell line Hep-G2: effects of free fatty acid. AB - The liver is a major source of the plasma lipoproteins; however, direct studies of the regulation of lipoprotein synthesis and secretion by human liver are lacking. Dense monolayers of Hep-G2 cells incorporated radiolabeled precursors into protein ([35S]methionine), cholesterol ([3H]mevalonate and [14C]acetate), triacylglycerol, and phospholipid ([3H]glycerol), and secreted them as lipoproteins. In the absence of free fatty acid in the media, the principal lipoprotein secretory product that accumulated had a density maximum of 1.039 g/ml, similar to serum low density lipoprotein (LDL). ApoB-100 represented greater than 95% of the radiolabeled apoprotein of these particles, with only traces of apoproteins A and E present. Inclusion of 0.8 mM oleic acid in the media resulted in a 54% reduction in radiolabeled triacylglycerol in the LDL fraction and a 324% increase in triacylglycerol in the very low density lipoprotein (VLDL) fraction. Similar changes occurred in the secretion of newly synthesized apoB-100. The VLDL contained apoB-100 as well as apoE. In the absence of exogenous free fatty acid, the radiolabeled cholesterol was recovered in both the LDL and the high density lipoprotein (HDL) regions. Oleic acid caused a 50% decrease in HDL radiolabeled cholesterol and increases of radiolabeled cholesterol in VLDL and LDL. In general, less than 15% of the radiolabeled cholesterol was esterified, despite the presence of cholesteryl ester in the cell. Incubation with oleic acid did not cause an increase in the total amount of radiolabeled lipid or protein secreted. We conclude that human liver-derived cells can secrete distinct VLDL and LDL-like particles, and the relative amounts of these lipoproteins are determined, at least in part, by the availability of free fatty acid. PMID- 3021885 TI - Parameters of cholesterol metabolism in the human hepatoma cell line, Hep-G2. AB - The human hepatoma cell line Hep-G2 has been shown to express the major enzymes of intra- and extracellular cholesterol metabolism. These include lecithin:cholesterol acyltransferase, acyl coenzyme A:cholesterol acyltransferase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and cholesterol 7 alpha-hydroxylase. Regulatory mechanisms that have been described in other hepatic systems also appear to be active in Hep-G2 cells: perturbations of cholesterol and triglyceride metabolism affected the enzyme activities and the accumulation of specific apolipoproteins in the culture media. The results indicate that studies of Hep-G2 cells may provide useful information for the elucidation of mechanisms of regulation of human hepatocyte cholesterol, lipoprotein, and biliary metabolism. PMID- 3021886 TI - Stimulated platelets release equivalent amounts of arachidonate from phosphatidylcholine, phosphatidylethanolamine, and inositides. AB - Thrombin-induced changes in arachidonate content of platelet phospholipids were quantitated to establish the ultimate origins of this eicosanoid precursor. Fifteen seconds following thrombin addition (15 U/5 X 10(9) platelets), phosphatidylcholine lost 11.8 nmol of arachidonate and phosphatidylethanolamine lost 10.5 nmol. Arachidonate in phosphatidate, phosphatidylinositol, and phosphatidylinositol-4,5-bisphosphate combined decreased by 11.0 nmol. Increases in free and oxygenated arachidonate (41 nmol) exceeded decreases in inositides. Thus phospholipase A2 released at least twice as much arachidonate as phospholipase C-diglyceride lipase. Phosphatidylinositol-4-phosphate levels remained unchanged upon stimulation. Therefore, increases in phosphatidylinositol 4,5-bisphosphate indicated the minimum rate of phosphorylation of phosphatidylinositol to resynthesize phosphatidylinositol-4,5-bisphosphate, following stimulus-induced breakdown by phospholipase C. Phosphatidylinositol-4, 5-bisphosphate increased 1.4 nmol between 10 and 15 sec following thrombin, markedly less than phosphatidylinositol decreased (2.1 nmol). This could be due to phospholipase A2, in addition to phospholipase C, acting directly on phosphatidylinositol to a greater extent than estimated by accumulation of lysophosphatidylinositol, degraded rapidly by lysophospholipase. Thus, upon high dose thrombin stimulation of human platelets inositide metabolism via phospholipase C directs initial formation of intracellular second messengers, and sequentially, or in parallel, arachidonate release by phospholipase A2 supplies the larger proportion of arachidonate for syntheses of eicosanoids involved in intercellular communication. PMID- 3021887 TI - Recognition of vesicular lipoproteins by the apolipoprotein B,E receptor of cultured fibroblasts. AB - Vesicular lipoproteins (e.g., lipoprotein-X) are found in plasma in cholestasis or following infusion of Intralipid or phospholipid. To investigate the metabolism of vesicular lipoproteins, we isolated them from the plasma of subjects with cholestasis or following chronic or single Intralipid infusion. Cholestasis and chronic Intralipid therapy were found to be associated with elevated plasma concentrations of apoE, as determined by radioimmunoassay. Vesicular lipoproteins purified from each of the three types of plasma contained apoE, as well as other proteins. In cholestasis, in which levels of apoE were up to five times normal, a major portion of the plasma apoE was on vesicular lipoproteins. Normalized for apoE content, all preparations of vesicular lipoproteins displaced 125I-labeled LDL from apoB,E receptors of cultured fibroblasts identically. This displacement was inhibited by monoclonal antibodies that block receptor binding of apoE. Vesicular lipoproteins containing 125I labeled apoE were internalized and degraded by fibroblasts. Different preparations caused small losses or gains of cellular cholesterol, with appropriate stimulation or suppression of apoB,E receptors. Thus, vesicular lipoproteins contain apoE, and apoE mediates their interaction with the apoB,E receptor. Our results suggest that the catabolism of cholesterol-rich vesicular lipoproteins, formed during cholestasis or following infusions of Intralipid or phospholipid, may be receptor-mediated. PMID- 3021888 TI - Congenital cytomegalovirus infection: predisposing maternal factors. AB - In a prospective study of cytomegalovirus (CMV) infection in pregnancy 69 congenitally infected infants were identified. The age, race, marital status, social class, and parity of the mothers of congenital CMV infants were compared with those of the screened population of women with non-infected infants. These factors were all individually strongly associated with the prevalence of congenital CMV. However, once age, marital status, and race were accounted for, neither social class nor parity had any additional effect. The overall congenital CMV rate was 3 per 1000 livebirths, ranging from 25/1000 for single black women under 20 to 1.6/1000 in married or cohabitating white women over 25. PMID- 3021889 TI - Month of birth of men with malignant germ cell tumours of the testis. AB - A recent paper has suggested that there is a temporal cycle in birth rates of men with testis cancer with a peak in April/May. We have investigated the suggestion using data from the South Thames and Los Angeles cancer registries. There is some slight evidence of a peak in August, which is confined to teratomas. The effects demonstrated are, however, too small and inconsistent to provide any real clue to the aetiology of testis cancer. PMID- 3021890 TI - Epidemiology of rotavirus gastroenteritis. PMID- 3021891 TI - Anthrax toxin blocks priming of neutrophils by lipopolysaccharide and by muramyl dipeptide. AB - We studied the pretreatment of human polymorphonuclear neutrophils (PMN) with purified preparations of the anthrax toxin components--protective antigen (PA), edema factor (EF), and lethal factor (LF)--and their effects on release of superoxide anion (O-2) after stimulation with the chemotactic peptide N-formyl methionyl-leucyl-phenylalanine (FMLP). PMN isolated in the absence of lipopolysaccharide (LPS) (less than 0.1 ng/ml) released only small amounts of O-2 after FMLP stimulation; pretreatment with anthrax toxin had little effect. The release of O-2 was increased fivefold by prior treatment with 3 ng/ml LPS for 1 h at 37 degrees C, an effect referred to as priming. PMN were primed to an equivalent extent by treatment with 100 ng/ml N-acetyl-muramyl-L-alanyl-D isoglutamine (muramyl dipeptide [MDP]). Pretreatment of PMN with anthrax toxin components PA plus EF or PA plus LF inhibited priming by LPS or MDP, as shown by the reduction in the release of O-2 up to 90% relative to controls not treated with toxin; single toxin components were inactive. The inhibition was markedly reduced when priming with LPS or MDP was carried out before exposure to toxin. O 2 release after stimulation by phorbol myristate acetate was not increased by priming, and pretreatment with toxin did not inhibit O-2 release after this stimulus. Evidently, anthrax toxin inhibits the priming that is normally induced in PMN by bacterial products and is necessary for the full expression of antimicrobial effects. PMID- 3021893 TI - Isolation and partial characterization of the plasma membrane from human spermatozoa. AB - Ejaculated human spermatozoa were subjected to nitrogen cavitation (600 psi for ten min) to remove the plasma membrane (PM). Electron microscopic examination of the cavitated cells revealed that 33% of the PM was removed from the sperm which includes both the head and tail regions. The released membrane was separated from the cavitated cells by centrifugation followed by a discontinuous sucrose density gradient centrifugation. A single membrane population was resolved at the 1.0 M sucrose interface. Examination of the isolated membranes by electron microscopy revealed vesicles of various sizes displaying unit membrane structures. Biochemical analysis of the isolated membranes showed a threefold enrichment in the surface membrane marker 5' nucleotidase and also suggested little contamination by enzymes from the cytosol (lactate dehydrogenase) or mitochondria (cytochrome oxidase). Analytical lipid analysis of the isolated membranes revealed a 26-fold enrichment in the distribution of cholesterol, an 11-fold enrichment of phospholipids, and a cholesterol:phospholipid molar ratio of 0.83. Also found was a twofold increase in glycosphingolipids which are ubiquitous components of PM in eukaryotic cells. These data indicate that the membrane vesicles isolated after nitrogen cavitation are primarily PM. PMID- 3021892 TI - Retroviral activation of interleukin 2 gene in a gibbon ape T cell lymphoma line. AB - The gibbon ape leukemia virus (GaLVSF)-infected T cell line, MLA 144, was established from the lymphoma of a gibbon ape. The cell line constitutively expresses IL-2 and its receptor, implying that an autocrine mechanism could be responsible for or contribute toward its growth. To explore the mechanism of constitutive IL-2 expression in MLA 144, we have isolated and characterized cosmid clones representing a normal and a doubly inserted IL-2 allele in this cell line. The map of the normal MLA 144 IL-2 allele closely resembles that of the normal human IL-2 gene. The abnormal allele contains a 3' insertion that is a GaLVSF provirus with two long terminal repeats (LTR) and an internal 3.25 kb deletion. At the 5' end of the abnormal allele is a second insertion that DNA sequencing showed to be an isolated GaLVSF LTR with a transcriptional orientation opposing that of the IL-2 gene. We demonstrate by Northern blotting analysis that the vast majority of transcripts are from the abnormal allele, implying that one or both retroviral insertions are responsible for constitutive expression of the allele. PMID- 3021894 TI - Control of the cAMP pathway by the cell cycle start function, CDC25, in Saccharomyces cerevisiae. AB - We investigated the relationship in Saccharomyces cerevisiae between the cell cycle start function, CDC25, and two mutants defining components of the cAMP pathway. The thermolabile adenylate cyclase mutant cyr1-2(ts) is phenotypically similar to the temperature-sensitive mutant cdc25(ts) in that both mutants, when shifted to the restrictive temperature, arrest in G1 of the cell cycle and permit the initiation of meiosis and sporulation. The mutant bcy1 [a lesion resulting in a low level of regulatory (R) subunit and a high level of active, catalytic (C) subunit of the cAMP-dependent protein kinase] suppresses the temperature sensitive phenotype of cyr1-2(ts) and confers an asporogenous phenotype. We found that cdc25(ts) complemented cyr1-2(ts), and, unlike cyr1-2(ts), was not suppressible by bcy1, demonstrating that CYR1 and CDC25 must encode different functions. Also our results indicate that CDC25 does not encode the R subunit of the cAMP-dependent protein kinase. In addition, although the cdc25(ts)bcy1 double mutant was temperature sensitive like cdc25(ts), we found that the cdc25(ts)bcy1 homozygous diploid was asporogenous like bcy1/bcy1. The inability of the cdc25(ts)bcy1 double mutant to sporulate demonstrated that CDC25 does not encode the C subunit of the cAMP kinase, and indicated that the CDC25 function modulates the cAMP pathway to control meiosis and sporulation. Further, the temperature sensitive phenotype of the double mutant, and hence the inability of bcy1 to suppress cdc25(ts), suggested that a second CDC25 cell cycle function exists which is independent of the cAMP pathway.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021896 TI - Cloning and expression of Clostridium acetobutylicum endoglucanase, cellobiase and amino acid biosynthesis genes in Escherichia coli. AB - Clostridium acetobutylicum P262 endoglucanase and cellobiase genes, cloned on a 4.9 kb DNA fragment in the recombinant plasmid pHZ100, were expressed from their own promoter in Escherichia coli. Active carboxymethylcellulase and cellobiase enzymes were produced, but there was no degradation of Avicel. The endoglucanase activities observed in cell extracts of E. coli HB101(pHZ100) differed in their pH and temperature optima from those previously reported for C. acetobutylicum P270. Complementation of E. coli arg and his mutations by cloned C. acetobutylicum DNA was also observed. PMID- 3021895 TI - The oxidation of glucose by Acinetobacter calcoaceticus: interaction of the quinoprotein glucose dehydrogenase with the electron transport chain. AB - The coupling of the quinoprotein glucose dehydrogenase to the electron transport chain has been investigated in Acinetobacter calcoaceticus. No evidence was obtained to support a previous suggestion that the soluble form of the dehydrogenase and the soluble cytochrome b associated with it are involved in the oxidation of glucose. Analysis of cytochrome content, and of reduction of cytochromes in membranes by substrates, and of sensitivity to cyanide indicated that glucose, succinate and NADH are all oxidized by way of the same b-type cytochrome(s) and cytochrome oxidases (cytochrome o and cytochrome d). Mixed inhibition studies [with KCN and hydroxyquinoline N-oxide (HQNO)] showed that the b-type cytochrome(s) formed a binary complex with the o-type oxidase and that there was thus no communication between the electron transport chains at the cytochrome level. Measurements of the reduction of ubiquinone-9 by glucose and NADH, and inhibitor studies using HQNO, indicated that the ubiquinone mediates electron transport from both the glucose and NADH dehydrogenases. In some conditions the quinone pool facilitated communication between the 'glucose oxidase' and 'NADH oxidase' electron transport chains, but in normal conditions these chains were kinetically distinct. PMID- 3021897 TI - Coxsackievirus group B replication in cultured fetal baboon aortic smooth muscle cells. AB - All six coxsackievirus B (CVB) serotypes replicated to various extents in fetal baboon aortic smooth muscle cells (SMC) in culture. CVB3 and CVB4 replicated to the highest titers and induced no cytopathology at the level of light microscopy. Maximum yields of CVB3 were produced between 12 and 24 hr postinoculation. Up to 15% of SMC cells became infected, as determined by immunofluorescence assays with anti-CVB3 antiserum, yet overall cell division in infected cultures did not differ from infected SMC cultures. Electron microscopy of CVB3-inoculated SMC cultures revealed changes in some cells: viruslike particles, secondary lysosomes containing dense bodies, and peripheral nuclear chromatin condensation. CVB3 replicated well in SMC passages up to the eighth, but did not replicate in eleventh-passage cells. Because of the cardiotropic and myotropic potential of this virus and its ability to replicate in aortic SMC with associated ultrastructural alterations, CVB3 (and other CVB) should be further examined as an etiologic agent(s) that could induce atherosclerosis. PMID- 3021898 TI - Binding and internalization of herpes simplex virus-antibody complexes by polymorphonuclear leukocytes. AB - We studied the interactions between rabbit polymorphonuclear leukocytes (PMN) and the RE strain of herpes simplex virus type 1 (HSV-1) to determine better the role of inflammatory cells in herpetic stromal keratitis. PMN were found to be nonpermissive for HSV replication and were unable to bind virus in the absence of antibody. However, PMN did bind and internalize HSV-antibody complexes in vitro as was demonstrated visually by electron microscopic studies and quantitatively by measurement of activity associated with radiolabeled HSV-antibody complexes. Virus used for immune complex formation was labeled with either 125Iodine or 35S methionine. In some experiments, anti-HSV IgG used for immune complex formation was labeled with 125Iodine before incubation with virus. Use of all three radiolabeling approaches resulted in the same general pattern of binding, indicating a requirement for both antibody and virus for interaction with PMN. The activity associated with PMN was increased by preincubation with complement. The results suggest an active role for PMN in controlling HSV infection through their ability to bind and ingest virus-antibody complexes. PMID- 3021899 TI - New findings in live, attenuated hepatitis A vaccine development. AB - Strain CR326F of hepatitis A virus, derived from a fecal specimen of Costa Rican patient 033-03, was passed 15 times in fetal rhesus monkey kidney (FRhK6) cell cultures plus eight times in human diploid lung (MRC5) cell cultures to yield variant F and 16 times in MRC5 cell cultures to yield variant F'. Both variants were purified by limit dilution passages. Virulence for marmosets was assessed at six different passage levels, including variants F and F'. There was a gradual loss of virulence with in vitro passage. Variant F retained slight virulence for marmosets; variant F' showed no evidence of virulence. Both variants induced hepatitis A antibody in most marmosets that received them, and the animals were immune to infection when challenged. Variants F and F' were also assessed in chimpanzees. As in marmosets, F retained slight virulence but F' did not. Experimental vaccines made from variants F and F' were then inoculated parenterally into adult human volunteers. A portion of recipients of variant F showed brief, low-order enzyme elevations; none was seen in recipients of F', although their occurrence could not be totally ruled out. As in the animal models, F' appeared more attenuated than F. Most persons developed hepatitis A antibody, indicating the feasibility of developing a live, attenuated hepatitis A vaccine for human beings. PMID- 3021900 TI - Prevalence of antibodies to enteroviruses and varicella-zoster virus among residents and overseas volunteers at agricultural settlements in Israel. AB - Within the framework of a comprehensive study of the correlation between enteric diseases and wastewater utilization in agricultural settlements (kibbutzim) the prevalence of several viral antibodies was examined among kibbutz residents and overseas volunteers. The latter were assumed to be a group highly susceptible to local pathogens. For the purpose of this study the presence of antibodies against eight enteroviruses [Coxsackieviruses (COX) types A9, B1, B3, and B4, echoviruses (ECHO) types 4, 7, and 9, and hepatitis A virus (HAV)] and a nonenterovirus, varicella-zoster virus (VZV), was tested in their sera. The prevalence of these viral antibodies among 342 volunteers (aged 18 to 34 years) upon their arrival at the kibbutzim was compared with that of 176 kibbutz residents of the same age. Seroconversion (ie, acquisition of viral antibodies) was tested in 115 of the volunteers two months after their arrival at the kibbutz. The prevalence of antibodies against each of the eight enteroviruses studied was found to be significantly higher among the kibbutz residents, but the prevalence of antibodies to VZV was similarly high in both groups (52% for volunteers and 59% for kibbutz residents). The mean antibody prevalence for the seven COX and ECHO viruses was 2.1 antibodies/person in the volunteer group v 4.7 antibodies/person among the kibbutz residents. Fifty-eight percent (58%) of the residents had antibodies to HAV as compared with 14% of the volunteers. No correlation was found between seropositivity (ie, previous exposure) to various enteroviruses and the immune status to HAV or VZV in both kibbutz residents and volunteers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021901 TI - Transient depletion of serotonin in the nervous system of Helisoma. AB - The present study shows that the drug 5,7-dihydroxytryptamine (5,7-diHT) can be used reliably to deplete the neurotransmitter serotonin (5-HT) from the nervous system of the snail Helisoma. The depletion is more effective in axonal and synaptic regions (85-90%) than in the somata (55%), is reasonably specific for serotonin (dopamine is affected to a much lesser extent), and is transient, with normal levels of neurotransmitter being restored by 2 months. A physiological correlate of 5-HT depletion has been shown in that an EPSP elicited by a cerebral serotonergic neuron (C1) onto a buccal motoneuron (B19) is much smaller during depletion and also recovers with time as 5-HT regains normal concentration. Despite the severe 5-HT depletion and physiological impairment, the gross morphology of neuron C1 remains indistinguishable from controls. Serotonergic depletion is not accompanied by development of receptor supersensitivity nor by the production of serotonin in extraneuronal sources. PMID- 3021902 TI - High-affinity receptor sites and rapid proteolytic inactivation of neurotensin in primary cultured neurons. AB - The present article describes the interaction of neurotensin with specific receptors in pure primary cultured neurons and the mechanisms by which this peptide is inactivated by these cells. Neurotensin binding sites are not detectable in nondifferentiated neurons and appear during maturation. The binding at 37 degrees C of [monoiodo-Tyr3]neurotensin to monolayers of neurons 96 h after plating is saturable and characterized by a dissociation constant of 300 pM and a maximal binding capacity of 178 fmol/mg of protein. The binding parameters as well as the specificity of these receptors toward neurotensin analogues reveal close similarities between the binding sites present in primary cultured neurons and those described in other membrane preparations or cells. Neurotensin is rapidly degraded by primary cultured neurons. The sites of primary inactivating cleavages are the Pro7-Arg8, Arg8-Arg9, and Pro10-Tyr11 bonds. Proline endopeptidase is totally responsible for the cleavage at the Pro7-Arg8 bond and contributes to the hydrolysis mainly at the Pro10-Tyr11 site. However, the latter breakdown is also generated by a neurotensin-degrading neutral metallopeptidase. The cleavage at the Arg8-Arg9 bond is due to a peptidase that can be specifically inhibited by N-[1(R,S)-carboxy-2-phenylethyl]-alanyl-alanyl-phenylalanyl-p- aminobenzoate. The secondary processing occurring on neurotensin degradation products are: a bestatin-sensitive aminopeptidasic conversion of neurotensin11-13 to free Tyr11, and a rapid cleavage of neurotensin8-13 by proline endopeptidase. A model for the inactivation of neurotensin in primary cultured neurons is proposed and compared to that previously described for purified rat brain synaptic membranes. PMID- 3021903 TI - Agonist-induced phosphoinositide hydrolysis in choroid plexus. AB - 5-Hydroxytryptamine (5-HT, serotonin) stimulates phosphoinositide hydrolysis in choroid plexus by interacting with the 5-HTlc site. In the present study, the effects of 5-HT were compared with those of other agonists. 5-HT stimulates a rapid release of all three inositol sugars in a mianserin-sensitive manner. Inositol bisphosphate and inositol trisphosphate levels increase about twofold within 2.5 min, whereas inositol monophosphate levels are not appreciably elevated until 5 min. In contrast, glutamate, carbachol, histamine, substance P, and vasopressin, agents that increase phosphoinositide hydrolysis in other tissues, do not stimulate this response in choroid plexus. High concentrations of norepinephrine increase inositol phosphate release in choroid plexus, but this effect is apparently mediated by activation of the 5-HTlc site. The depolarizing agents KCl and veratrine also fail to stimulate phosphoinositide hydrolysis in choroid plexus. These results, combined with the finding that the phosphoinositide response to 5-HT is insensitive to tetrodotoxin, suggest that the effects of 5-HT are not secondary to neurotransmitter release. Furthermore, an indirect effect mediated via arachidonic acid metabolism is unlikely, since inhibitors of cyclooxygenase and lipoxygenase do not reduce the 5-HT response. We conclude, therefore, that phosphoinositide hydrolysis is the transducing mechanism of the 5-HT 5-HTlc receptor and that the choroid plexus will serve as a useful model system for studies of this receptor. PMID- 3021905 TI - Sexual dimorphism in the response of the GABAergic system to estrogen administration. AB - Administration of estradiol benzoate to gonadectomized female rats results in up regulation of CNS gamma-aminobutyric acid (GABA) receptors. The increase of [3H]muscimol binding activity is observed in six of the seven brain areas examined. The same treatment, performed in castrated male or androgenized female rats, induced an increase of [3H]muscimol binding only in the striatum. Evidence is provided suggesting that the dimorphic sensitivity of GABA receptor is not correlated with the difference in spontaneous motor activity reported between male and female rats. PMID- 3021904 TI - Estrogen-induced up-regulation of gamma-aminobutyric acid receptors in the CNS of rodents. AB - Previous studies have identified an effect of estrogen administration on the number of central GABAergic binding sites of rat. We have further characterized this effect by performing a series of experiments in vitro where we analyzed the changes of gamma-aminobutyric acid (GABA) binding in slices of nervous tissue incubated in a physiological medium in presence of estradiol. The tissues were dissected from ovariectomized rats. In such a system, estrogen augmented the amount of [3H]muscimol binding within 3 h of incubation. The effect was dose dependent and could be blocked by the addition of the anti-estrogen tamoxifen. The increase in [3H]muscimol binding could not be observed by addition of estradiol to broken membranes or by incubation of the slices with steroids deprived of estrogenic activity. Furthermore, the estrogen-induced increase of GABA binding sites could be prevented by addition of cycloheximide and alpha amanitin in the incubation medium. Our data indicate that the estrogen may increase the number of GABA binding sites by direct interaction with the GABA receptor gene or genes involved in the metabolism of GABA receptor. PMID- 3021906 TI - Superoxide radical-mediated alteration of synaptosome membrane structure and high affinity gamma-[14C]aminobutyric acid uptake. AB - Mouse cortical synaptosomal structure and function are altered when exposed to hypoxanthine/xanthine oxidase (HPX/XOD)-generated active oxygen/free radical species. The structure of both the synaptic vesicle and plasma membrane systems are altered by HPX/XOD treatment. The alteration of synaptic vesicle structure is exhibited by a significant increase in the cumulative length of nonsynaptic vesicle membrane per nerve terminal. With respect to the nerve terminal plasma membrane, the length of the perimeter of the synaptosome is increased as the membrane pulls away from portions of the terminal in blebs. The functional lesion generated by HPX/XOD treatment results in a reduction in selective high-affinity gamma-[14C]aminobutyric acid (GABA) uptake. Kinetic analysis of the reduction in high-affinity uptake reveals that the Vmax is significantly altered whereas the Km is not. Preincubation with specific active oxygen/free radical scavengers indicates that the super-oxide radical is directly involved. This radical, most probably in the protonated perhydroxyl form, initiates lipid peroxidative damage of the synaptosomal membrane systems. Low-affinity [14C]GABA transport is unaltered by the HPX/XOD treatment. The apparent ineffectiveness of free radical exposure on low-affinity [14C]GABA transport coupled with its effectiveness in reducing high-affinity transport supports the idea that two separate and different amino acid uptake systems exist in CNS tissue, with the high-affinity being more sensitive (lipid-dependent) and/or more energy-dependent (Na+,K+ ATPase) than the low-affinity system. PMID- 3021907 TI - Subcellular localization of angiotensin-converting enzyme in cultured neuroblastoma cells. AB - Angiotensin-converting enzyme (ACE) activity of Neuro-2A mouse neuroblastoma cells was found predominantly in particulate fractions. Density gradient centrifugation of the particulate fractions showed ACE activity in light fractions of the gradient, a result suggesting a plasma membrane localization. This was confirmed using the aqueous two-phase polymer system of plasma membrane isolation. The rapid and energy-independent hydrolysis of exogenous substrate by ACE of intact cells and the sensitivity of the enzyme of intact cells to proteases indicate further that the active site of ACE is oriented extracellularly. PMID- 3021908 TI - Changes in the expression of beta and gamma actins during differentiation of PC12 cells. AB - Cells of the rat pheochromocytoma line PC12 cease proliferation and develop neurites in response to nerve growth factor (NGF). Quantification of beta and gamma isoforms of nonmuscle actin in extracts of these differentiating cells showed that the beta:gamma ratio decreased from 1.30 +/- 0.05 to 0.99 +/- 0.05 after 6 days of NGF treatment. Cells treated with N6,O2-dibutyryl cyclic AMP (dbcAMP) also showed a shift in the ratio of beta:gamma isoforms, although few of these cells extended neurites. Administration of dbcAMP or both NGF and dbcAMP to cells accelerated the decrease in the beta:gamma actin isoform ratio relative to treatment with NGF alone. Those cells treated with both NGF and dbcAMP also showed an accelerated rate of neurite outgrowth. Suspension-grown PC12 cells treated with NGF showed neither an isoform ratio decrease nor neurite development. Our results suggest that either cyclic AMP may be a "second messenger" for NGF or it may effect the isoform ratio change by an independent mechanism. In addition, our data demonstrate an alteration in actin isoform expression, which accompanies the morphological differentiation of PC12 cells. PMID- 3021909 TI - Insulin reverses enhanced incorporation of 32P into polyphosphoinositides in peripheral nerve of the streptozotocin diabetic rat. AB - The ability of insulin treatment to reverse altered phosphoinositide metabolism in sciatic nerve from streptozotocin diabetic rats was studied. Diabetes was induced in rats by means of a single injection of streptozotocin. Enhanced incorporation of 32P into phosphatidylinositol-4,5-bisphosphate (PIP2) was detectable as early as 8 days following intravenous injection of streptozotocin and was maximal after 4 weeks. Hormone treatment was initiated at this time by daily injections of protamine zinc insulin followed by the implantation of long acting insulin osmotic minipumps, and 4 weeks later sciatic nerves were removed and incubated in the presence of [32P]orthophosphate. The increased labeling of PIP2 was completely reversed by hormone administration. In contrast, insulin (0.1 and 1.0 mU/ml) added to the incubation medium failed to reverse the altered pattern of 32P incorporation into PIP2. The uptake of 32P into PIP2 was greater than 80% higher into the proximal than into the distal portion of normal sciatic nerve when these were incubated separately. This metabolic difference was abolished in diabetic rats, although the incorporation into both segments was still significantly higher than in controls. These results strengthen the association of altered nerve PIP2 metabolism with the diabetic state and are consistent with the concept that experimental diabetic neuropathy is a distal axonopathy. PMID- 3021910 TI - gamma-Aminobutyric acid receptor complex of insect CNS: characterization of a benzodiazepine binding site. AB - The specific binding of [N-methyl-3H]flunitrazepam ([3H]FNZP) to a membrane fraction from the supraoesophageal ganglion of the locust (Schistocerca gregaria) has been measured. The ligand binds reversibly with a KD of 47 nM. The binding is Ca2+-dependent, a property not found for the equivalent binding site in vertebrate brain. The pharmacological characteristics of the locust binding site show similarities to both central and peripheral benzodiazepine receptors in mammals. Thus binding is enhanced by gamma-aminobutyric acid (GABA), a feature of mammalian central receptors, whereas the ligand Ro 5-4864 was more effective in displacing [3H]FNZP than was clonazepam, which is the pattern seen in mammalian peripheral receptors. The locust benzodiazepine binding site was photoaffinity labelled by [3H]FNZP, and two major proteins of Mr 45K and 59K were specifically labelled. In parallel experiments with rat brain membranes a single major protein of Mr 49K was labelled, a finding in keeping with many reports in the literature. We suggest that the FNZP binding site described here is part of the GABA receptor complex of locust ganglia. The insect receptor appears to have the same general organization as its mammalian counterpart but differs significantly in its detailed properties. PMID- 3021911 TI - 2',3'-cyclic nucleotide 3'-phosphodiesterase in CNS and PNS myelin: two molecular forms. PMID- 3021912 TI - A new class of synthetic biological response modifiers: the methylfurylbutyrolactones (Nafocare B). AB - A new class of synthetic biological response modifiers (BRMs) has been produced by combining a highly electrophilic reactant, 2-methyl-2, 5-dihydrofuran (a cyclic acetal of cis-3-acetyl acrolein), with L-ascorbic acid. The parent class of compounds can be referred to as methylfurylbutyrolactones (MFBL), previously termed Nafocare B. This parent molecule is amorphous, has a molecular weight of 252.7, and the chemical name [3,6] cyclohemiketal of 2-(5-methyl-2-furyl)-3-keto L-butyrolactone. Two crystalline forms were produced by a reaction of the MFBL parent molecule with either succinic anhydride or succinimide, to create MFBL-SA (Nafocare B2) and MFBL-S (Nafocare B3) dimers, respectively. The structure of these compounds has been confirmed by modern methods of analytical chemistry, including x-ray crystallography. All three forms of the MFBLs showed negligible toxicity in single-dose acute LD-50s in mice. Also, the MFBLs did not demonstrate genotoxic activity at 800 mg/kg in the mouse micronucleus assay. The MFBLs are immunostimulatory in assays involving T- and B-lymphocytes, but not in immunoassays on macrophages derived from resident- or thioglycollate-elicited peritoneal exudate cells (PEC). Spleen cells from mice treated 4 days via the intraperitoneal, intravenous, or the oral routes responded significantly over controls to suboptimal stimulatory concentrations of polyclonal mitogens in the lymphocyte stimulation assay. The MFBLs were not mitogenic, since they did not increase DNA synthesis in resting spleen cells from MFBL-treated mice. Antibody production is also amplified by the MFBLs. Mice immunized with sheep erythrocytes, a T-cell-dependent antigen, and treated with MFBLs had a 200-800% increase in the numbers of antibody-producing splenic lymphocytes detected by the Jerne hemolytic plaque assay. Also, mice immunized with soluble bovine serum albumin (BSA), and treated with a MFBL, demonstrated at least a fourfold increase in IgG-specific antibodies to BSA, when compared with controls. To demonstrate effects of MFBLs on macrophages, we used the Fc receptor (FcR) surface marker and superoxide anion assays. Only at the highest in vitro dose of MFBL (16 micrograms/ml) was a significant increase in erythrocyte antibody rosette formation detected, using resident macrophages isolated from PEC. In the superoxide anion release assay neither resident- nor thioglycollate-elicited PECs, obtained from in vivo-treated mice or macrophages treated in vitro, showed increased production of superoxide anion.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3021913 TI - Ultrastructural immunohistochemical localization of poliovirus during virulent infection of mice. AB - Ultrastructural immunohistochemistry was used to localize type 2 human poliovirus (HPV 2) during virulent infection of mice caused by the Lansing strain. In the spinal cord, immune-reaction product was exclusively localized within neurons and their processes. The absence of viral antigen in glial, endothelial and inflammatory cells further supports the strict neuronotropicity of HPV. In addition, viral antigen and virus-like particles were localized in synaptic complexes and axons, including preterminal axons. This clear demonstration of HPV in neuronal cell bodies, their axons, and synaptic elements strongly supports the hypothesis of HPV dissemination in the central nervous system via axonal transport. PMID- 3021914 TI - Neuropathology of acquired immunodeficiency syndrome (AIDS): an autopsy review. AB - In the brains and spinal cords of 153 adult patients dying with acquired immunodeficiency syndrome (AIDS) at New York and Memorial Hospitals a subacute encephalitis with multinucleated cells was present in 28% of all patients. This encephalitis was characterized by multinucleated cells primarily located in the white matter and associated with myelin pallor and sparse infiltrates of rod cells, macrophages, gemistocytic astrocytes and lymphocytes. The incidence per 12 month period ranged from 0 to 43% and significantly increased between 1983-84 (14%) and 1984-85 (43%). Recent virologic and pathologic studies suggest that this encephalitis may be caused by direct LAV/HTLV-III infection of the central nervous system (CNS). Cytomegalovirus encephalomyelitis and toxoplasmosis were the most common opportunistic infections (26% and 10%, respectively). Progressive multifocal leukoencephalopathy, herpes simplex ventriculitis, varicella-zoster leukoencephalitis and fungal infections were infrequent (less than 3% each). A nonspecific encephalitis with microglial nodules and with mild white matter changes occurred in 17%, vacuolar myelopathy in 29% and CNS lymphoma in 6%. Less than 20% of patients had either normal brains or terminal metabolic encephalopathies. This survey shows that neuropathologic complications of AIDS are frequent. Infections are the most common complication and are caused by probable LAV/HTLV-III infection, or by opportunistic organisms. PMID- 3021915 TI - A glial fibrillary acidic protein-expressing and tumorigenic cell line derived from an avian sarcoma virus-induced rat astrocytoma. AB - A permanent cell line, S635c15, was derived from an anaplastic astrocytoma induced by the Schmidt-Ruppin strain of avian sarcoma virus (ASV) in a female F 344 rat. Persistent expression of the astrocytic differentiation protein, glial fibrillary acidic protein (GFAP), was detected both in cultured cells after 100 passages in vitro and in transplanted tumors. Subcutaneous and intracerebral transplantation of S635c15 cells in syngeneic rats resulted in a 100% tumor incidence and a reproducible mortality distribution. S635c15 cells formed discrete masses after subcutaneous injection but grew intracranially as infiltrative lesions. Tumor blood flow and blood-to-tissue transport studies yield comparable values to other rat glioma models; S635c15 intracranial tumors proved to be a homogeneous model with little variation within and between tumors with respect to morphology, GFAP expression, blood flow, and permeability. This cell line provides a GFAP-expressing brain tumor model that extends the use of autochthonous ASV-induced astrocytomas by allowing in vitro and in vivo studies. It may be useful for further studies in neurobiology and brain tumor biology, diagnosis, and therapy. PMID- 3021916 TI - Absence of both internal carotid arteries. AB - A 63-year-old man developed a slight left hemiparesis. CT scan showed an intracerebral tumour, which was later identified as glioblastoma multiforme. Angiographic examination revealed the absence of both internal carotid arteries. Blood supply of anterior and middle cerebral arteries was provided by communication between a tortuous megadolichobasilar artery and the circle of Willis through enlarged posterior communicating arteries. The case is reported with reference to clinical symptoms as well as angiographic and anatomical findings of 17 comparable cases mentioned in literature. PMID- 3021919 TI - Genetic linkage studies in hereditary motor and sensory neuropathies. PMID- 3021917 TI - Anti-herpes simplex type 1 activity in IgG subclasses produced systemically and intrathecally in patients with herpes encephalitis. AB - The role of the humoral immune response in herpes simplex encephalitis (HSE) is largely unknown. The finding that herpes simplex virus type 1 (HSV 1) induced IgG Fc receptor binds to all IgG subclasses except IgG 3 prompted an investigation of anti-HSV activity in IgG subclasses from serum and cerebrospinal fluid (CSF) in ten patients with proven or highly probable HSE by means of a monoclonal antibody IgG subclass-specific solid-phase radioimmunoassay (SPRIA). In contrast to serum, CSF contained no or low anti-HSV IgG titres during the first 2 weeks of disease in five of seven patients tested. The IgG titres rose thereafter for at least 4 weeks after the start of illness and remained high in both serum and CSF up to 393 days. The anti-HSV IgG subclass distribution in serum was IgG 1 (ten of ten), IgG 2 (two of ten), IgG 3 (six of ten), and IgG 4 (six of ten). Two patients had a simultaneous anti-HSV IgG 3 and IgG 4 response. With the exception of one patient lacking anti-HSV IgG 4 and two patients lacking anti-HSV IgG 2, the subclass distribution in CSF was the same as in serum. The anti-HSV subclass distribution in sera from ten seropositive patients without evidence of recent herpes infection did not differ from that of the HSE patients, except that five of ten patients had simultaneous anti-HSV IgG 3 and IgG 4 responses. Thus we could not correlate the anti-HSV subclass response in patients with HSE with the subclass preference of the HSV-induced Fc receptor. PMID- 3021918 TI - CSF cyclic AMP and CSF adenylate kinase in cerebral ischaemic infarction. AB - The severity of neurological deficits, size of hypodense zone on CT, concentration of cAMP and activity of adenylate kinase in cerebrospinal fluid (CSF) were evaluated at predefined intervals in the acute stage of supratentorial cerebral ischaemic infarction in 52 patients. Patients with cerebral infarction had raised activities of adenylate kinase CSF as compared with normal persons. Patients with marked neurological deficits, only slight improvement of neurological signs and large infarction zones on CT had higher average activities of adenylate kinase and lower concentration of cAMP in CSF. Alterations of CSF adenylate kinase and CSF cAMP values were most distinct on the 3rd day after the stroke. Reasons for the changes may be metabolic disorders following brain ischaemia. PMID- 3021920 TI - Frequency and prognostic importance of pretreatment clinical characteristics in patients with advanced non-small-cell lung cancer treated with combination chemotherapy. AB - To determine the frequency and prognostic importance of pretreatment clinical characteristics in patients currently undergoing treatment for stage III non small-cell lung cancer (NSCLC), data were collected on 378 patients receiving high-dose (120 mg/m2) cisplatin plus vinca alkaloid combination chemotherapy regimens since 1978. Variables analyzed included age, sex, weight loss, performance status, histologic subtype, presence of extrathoracic metastases, number of metastatic organ sites, presence of liver, bone, or brain involvement, prior radiation or surgery, and serum lactate dehydrogenase (LDH). The effect of a major response to chemotherapy on survival was also investigated. Using multivariable analyses, the following were found to be associated with outcome: initial performance status, with patients having a performance status of 80% to 100% having an increased major objective response rate and survival; bone metastases, which were adversely predictive of response rate and survival; elevated serum LDH and male sex, both of which were associated with shortened survival and remission duration; and the presence of two or more extrathoracic metastatic organ sites, which was associated with shorter survival. When major objective response with chemotherapy was included in a conditional multivariable analysis, it was strongly associated with longer median survival. Information from this analysis may be useful when comparing the response data of completed studies in similar patients, in designing future trials, and in the selection of cisplatin plus vinca alkaloid therapy for individual patients with advanced NSCLC. PMID- 3021921 TI - Effects of brain irradiation and chemotherapy on myelosuppression in small-cell lung cancer. AB - The effect of brain irradiation on myelosuppression was studied in patients with untreated small-cell lung cancer (SCLC) by comparing 24 patients who received brain irradiation for brain metastasis at presentation (irradiated patients) with 24 control patients who were selected by matching ages and non-CNS metastatic sites with those of irradiated patients. All patients were evaluated during the first three courses of chemotherapy. More irradiated patients than control patients had chemotherapy dose reductions from the starting dose level for the second (nine of 22 v two of 24; P = .03) and the third (nine of 18 v three of 20; P = .05) courses of chemotherapy. Overall, more irradiated patients had chemotherapy dose reductions than did control patients (11 of 22 v three of 24; P = .01). The difference was highly significant even after other variables were considered in a multivariate analysis (P less than .001). Myelosuppression was more severe in irradiated patients for WBCs (P = .01) and for platelets (P = .05). When the second course of chemotherapy was administered at the same dose levels as in the first course, irradiated patients had greater decreases in nadir counts after the second course compared with the first course than did control patients. Irradiated patients had a higher incidence of infectious complications than did control patients (14 of 24 v six of 24; P = .02), particularly after the second course of chemotherapy (seven of 22 v one of 24; P = .04). There were four treatment-related deaths due to sepsis in irradiated patients. Following brain irradiation given concurrently with intensive chemotherapy, close monitoring of myelosuppression and adjustments of chemotherapy doses are advised. PMID- 3021922 TI - Cerebrospinal fluid bombesin and calcitonin in patients with central nervous system metastases from small-cell lung cancer. AB - To determine whether levels of mammalian bombesin (MB) or calcitonin would be useful in detecting CNS metastases in patients with small-cell lung cancer (SCLC), we measured their concentrations in the CSF of 94 patients who underwent lumbar puncture for suspected CNS involvement. The MB concentration was significantly elevated in the 51 patients with definite CNS metastases as compared with the 30 patients without apparent CNS involvement (P less than .01). This significance was due to increased levels of MB in 18 patients with meningeal carcinomatosis. Whereas CSF MB was detectable (greater than 10 fmol/mL) in only 7% of patients without apparent CNS involvement, CSF MB was detectable in 21% with parenchymal CNS metastases and in 78% of those with meningeal carcinomatosis. Interestingly, 93% of patients having MB concentrations above 20 fmol/mL had meningeal metastases. The calcitonin concentration was significantly elevated in 42 patients with CNS metastases as compared with 27 patients without CNS involvement (P less than .01). Both the 15 patients with meningeal carcinomatosis and the 27 patients with only parenchymal metastases had significantly elevated levels of CSF calcitonin as compared with those without CNS metastases. Fifty-three percent of patients with meningeal carcinomatosis and 48% with parenchymal metastases had a CSF calcitonin level above 18 fmol/mL, whereas only 7% without apparent CNS metastases exceeded this level. Sixty-seven percent of all patients with CNS metastases had increased CSF levels of one of the two hormonal markers. Thus, in SCLC patients, an elevated CSF calcitonin strongly suggested CNS metastases and an elevated MB was very suggestive of the presence of meningeal carcinomatosis. However, only the latter observation seems of clinical importance due to the difficulties in establishing this diagnosis with current diagnostic measures. PMID- 3021923 TI - Increased plasma renin and aldosterone in patients treated with cisplatin-based chemotherapy for metastatic germ-cell tumors. AB - Twenty-four normotensive males in complete remission (CR) for 9+ to 54+ months after cisplatin-based chemotherapy for metastatic germ-cell tumors were evaluated for evidence of alterations in the renin-aldosterone axis and renal function. Abnormally high ambulatory plasma renin activity was seen in 14 of 19 patients with 24-hour urine sodium excretion greater than 50 mEq. This was correlated with elevated ambulatory plasma aldosterone (P = .009) and 24-hour urinary aldosterone excretion (P = .01). The mean serum magnesium value (1.34 +/- .05 mEq/L) was subnormal. Therapy resulted in an increase in serum creatinine during treatment (P less than .0001), an increase in BUN (P less than .01), and decrease in serum phosphorus (P less than .001). The relationship between the alterations in the renin-aldosterone axis and abnormal renal tubular function remains to be determined. In view of reports of cardiovascular toxicity after treatment for germ-cell tumors, and evidence individually linking both magnesium deficiency and increased plasma renin activity (PRA) to cardiovascular consequences, these abnormalities in renin and magnesium metabolism suggest that patients treated with cisplatin-based chemotherapy should be carefully observed for the development of delayed cardiovascular toxicities. PMID- 3021924 TI - Distribution of calpains I and II in rat brain. AB - Calpains I and II are calcium-dependent proteases that have been implicated in several aspects of brain function, including neurofilament turnover, Wallerian degeneration, and excitatory synaptic transmission. In this study, specific affinity-purified antibodies against each of the enzymes were used to determine their cellular distribution in rat brain. Differences between the two were found throughout the brain, with calpain I being located primarily in neurons, whereas calpain II was more prominent in glial cells. In myelinated axons, calpain II was present at low levels but calpain I was not detectable. In all brain areas, both enzymes were concentrated in cell bodies, with lesser amounts in neuronal and glial processes. Calpain I was only detectable proximally in dendrites and was not found in spiny branchlets of either pyramidal or Purkinje cells. These results suggest that calpain II is the likely form of the enzyme involved in calcium-activated proteolytic phenomena in axons. They do not support the existence of a role for calpain at excitatory axospinous synapses. PMID- 3021925 TI - Electrophysiological interactions of isomers of cyclazocine with the phencyclidine antagonist metaphit in rat cerebellar Purkinje neurons. AB - Metaphit, 1-(1-(3-isothiocyanatophenyl) cyclohexyl) piperidine, an analog of phencyclidine (PCP) has been shown previously to selectively block PCP receptors and to irreversibly antagonize the depressant effect of PCP in cerebellum. In this study, we examined the electrophysiological interactions of metaphit and naloxone with stereoisomers of cyclazocine, an agent known to have analgesic and psychotomimetic activity in behavioral studies, effects that have been ascribed to opiate and PCP receptor activity. A dose-dependent and reversible slowing of Purkinje neuron discharge was seen with local application of (+)- or (-) cyclazocine. We found that the blockade of (-)-cyclazocine effects required both high doses of naloxone and the presence of metaphit, whereas the responses to (+) cyclazocine were blocked by metaphit alone on most cerebellar Purkinje neurons. These findings suggest that the depressant reaction of (+)-cyclazocine in cerebellar Purkinje neurons is primarily mediated through PCP receptors. (-) Cyclazocine responses, on the other hand, appear to be due to activity at both PCP and kappa opioid receptors. PMID- 3021927 TI - Excitatory synaptic transmission between interneurons and motoneurons in chick spinal cord cell cultures. AB - We have examined the development of synaptic transmission between interneurons and motoneurons in spinal cord cell cultures. Unitary excitatory synaptic currents and complex bursts of excitatory currents develop rapidly: EPSCs (excitatory postsynaptic currents) were detected in 100% of the motoneurons by the 4th day after plating. Inhibitory synaptic currents develop more slowly: IPSCs (inhibitory postsynaptic currents) were detected in only 10% of the motoneurons on day 5 and 40% on day 8. During the 1st and 2nd days in vitro, 24% of the motoneurons tested were dye (Lucifer Yellow) coupled to nearby interneurons. The incidence of dye coupling declined during the first week in culture. No coupling was observed between motoneurons. Our data imply that both G1 and G2 receptors are activated at each synapse. The amplitude of spontaneous excitatory synaptic currents did not change when the motoneuron was hyperpolarized from -50 to -80 mV. This behavior is similar to that of currents induced by glutamate, an agonist that activates 2 types of receptors (G1 and G2) on motoneurons. In addition, a concentration of 2-amino-5-phosphonovaleric acid sufficient to inhibit all G1 receptors only partially inhibited the excitatory synaptic currents. Given the conductance of G1 and G2 channels and the ratio of channels activated during unitary EPSCs, we estimate that as few as 25 G1 channels and 5 G2 channels may mediate excitatory interaction between interneurons and motoneurons during the first week in culture. PMID- 3021926 TI - The role of spinal cord cyclic AMP in the acoustic startle response in rats. AB - Drugs thought to increase intracellular levels of cAMP were infused intrathecally into the subarachnoid space of the lumbar spinal cord, and the effects on the acoustic startle response in rats were measured. Intrathecal infusions of the cAMP analogs dibutyryl cAMP or 8-bromo cAMP (12.5-100 micrograms) produced marked, dose-dependent increases in startle amplitude compared to the infusion of artificial cerebrospinal fluid (CSF). Local infusions of dibutyryl cAMP at more rostral levels of the spinal cord or brain failed to mimic the excitatory effect seen following lumbar intrathecal infusion. No excitation of startle was seen following intrathecal infusion of cAMP itself, ATP, 5'-AMP, or dibutyryl cGMP. A weak excitation of startle was seen following intrathecal, but not intraventricular, infusion of the water-soluble adenylate cyclase activator forskolin 7-deacetyl-7-O-hemisuccinic acid (forskolin-DHA; 5.0-100 micrograms, in artificial CSF), whereas forskolin itself [0.01-200 micrograms, in dimethyl sulfoxide (DMSO)] was without consistent effect. Finally, intrathecal infusion of the selective phosphodiesterase inhibitor Rolipram (12.5-200 micrograms) produced a marked excitation of startle similar in magnitude to the effects produced by cAMP analogs. The excitatory effects of intrathecally infused dibutyryl cAMP, 8 bromo cAMP, forskolin-DHA, or Rolipram support a functional link between spinal cord cAMP and the acoustic startle reflex. Possible sites of cAMP action on startle are discussed. PMID- 3021928 TI - Modulation of embryonic chick motoneuron glutamate sensitivity by interneurons and agonists. AB - Embryonic chick motoneurons grown in culture together with other spinal cord cells are more sensitive to L-glutamate than are sorted motoneurons grown in isolation. After 6 d in vitro, the difference in peak sensitivity reached 6-fold. Comparable increases in aspartate and kainate currents were observed, indicating that both G1 and G2 amino acid receptors were affected. Elimination of proliferating non-neuronal cells from mixed spinal cord cell cultures by addition of cytosine arabinoside (ara C) did not prevent the increase in motoneuron chemosensitivity, so the induction is probably due to the presence of interneurons. In contrast to their effect on glutamate response, interneurons did not affect the sensitivity of motoneurons to the inhibitory neurotransmitters GABA and glycine. Glutamate receptors expressed by sorted and unsorted motoneurons are identical in terms of their ED50, reversal potential, mean channel open time, and conductance, implying that the increased sensitivity of motoneurons in mixed cultures is due to an increase in the number of open channels. In addition to an increase in the number of channels, the distribution of glutamate sensitivity over the surface of individual motoneurons was altered in interneuron-containing cultures. The sensitivity of isolated motoneurons was greatest at the soma and decreased with distance along major processes, but the sites of highest sensitivity on motoneurons in mixed cultures occurred along their processes. Sharp peaks identified by focal iontophoresis of glutamate were separated by areas of lower sensitivity. The inductive effect of interneurons cannot be due to glutamate, the most likely excitatory interneuron-motoneuron transmitter in 6 d chick cultures.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021929 TI - Cell surface changes accompanying the neural differentiation of an embryonal carcinoma cell line. AB - The murine embryonal carcinoma cell line P19S18O1A1 develops into neuronlike cells after treatment with retinoic acid (Edwards and McBurney, 1983). We have analyzed the expression of cell surface carbohydrate antigens and intracellular cytoskeletal antigens in differentiating O1A1 cells in order to identify the cell types present in the cultures and to characterize the differentiation process. Undifferentiated O1A1 cells express the SSEA-1 antigen, GD3 ganglioside, and the D1.1 ganglioside antigen, carbohydrate markers that are found on early embryonic cells and neuroepithelial germinal cells in vivo. The cells also bind tetanus toxin, cholera toxin, and monoclonal antibody A2B5, probes that bind to gangliosides found on the surfaces of neurons and immature astrocytes in vivo and in vitro. They contain vimentin-type intermediate filament antigens but have no detectable neurofilament or glial filament protein antigens. After aggregation of the cells in medium containing retinoic acid followed by growth in a serum-free chemically defined medium, over 80% of the cells differentiate into neurons as determined by immunofluorescent labeling with antibodies against neurofilament protein antigens. The differentiated cells no longer express either the embryonic or neuroepithelial carbohydrate antigens, but they continue to express the cell surface markers characteristic of neurons. These changes in the expression of cell surface antigens are accompanied by changes in ganglioside metabolism, including a shift towards the synthesis of more complex gangliosides. Thus, the retinoic acid-induced changes in O1A1 cells in vitro resemble the in vivo development of neurons. This establishes the O1A1 cell line as a relevant model system for studies of the molecular basis of neuronal differentiation and development. PMID- 3021930 TI - High concentrations of N-acetylaspartylglutamate (NAAG) selectively activate NMDA receptors on mouse spinal cord neurons in cell culture. AB - We examined the membrane action of the endogenous dipeptide and putative neurotransmitter N-acetylaspartylglutamate (NAAG) on the excitatory amino acid receptors of cultured mouse spinal cord neurons using electrophysiological methods. Responses to NAAG (1 microM-5 mM) were compared to those elicited by N methyl-D-aspartate (1 microM-1 mM) and L-glutamate (0.5-500 microM). Under voltage clamp, concentration-response curves of agonist-evoked currents demonstrated that NAAG was much less potent than either L-glutamate or N-methyl-D aspartate (NMDA), so that inward currents could be evoked only at NAAG concentrations above 300 microM. Analysis of the dipeptide by high-pressure liquid chromatography showed no evidence of contamination by excitatory amino acids, suggesting that NAAG has an intrinsic, although weak, neuroexcitatory action on spinal neurons. Previous studies have shown that activation of NMDA receptors produces a voltage-dependent response. The current-voltage relationship of responses evoked by NAAG was also voltage-dependent. The peptide-activated conductance decreased with hyperpolarization in the presence of extracellular Mg2+, such that little inward current could be evoked at a membrane potential of 80 mV. In addition, responses to NAAG were completely antagonized by 250 microM DL-2-amino-5-phosphonovaleric acid, a specific NMDA-receptor antagonist. Application of NAAG in Mg2+-free medium resulted in an inward current with a large increase in membrane current noise. The spectral density function of this current noise could be fitted with a single Lorentzian with a decay time constant near 5 msec and a calculated single-channel conductance of 50-60 pS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021931 TI - Development of multiple lesions during radiation therapy and chemotherapy in patients with gliomas. AB - To determine the percentage of patients who developed multiple central nervous system (CNS) gliomas during postoperative radiation therapy and chemotherapy, the authors reviewed the records of 1047 patients treated between December 2, 1976, and August 16, 1985, who had an original diagnosis of supratentorial glioblastoma multiforme or other anaplastic glioma. The occurrence of multiple lesions was verified by neurodiagnostic studies (computerized tomography or myelography) or by findings at operation or autopsy. Twelve patients (1.1%) who presented with multiple lesions were excluded from this analysis. There were 405 patients with glioblastoma multiforme; their median age was 46.5 years (range 22 to 70 years). Eighteen (5%) of these patients had multiple CNS lesions, five of which were in the spinal cord. The median time from diagnosis to detection of the second lesion in this group was 59.5 weeks (range 10 to 182 weeks). There were 630 patients with anaplastic glioma (which included mixed malignant glioma and highly anaplastic, gemistocytic, moderately anaplastic, and anaplastic astrocytomas); their median age was 30 years (range 2 to 62 years). Fifty-four (8.6%) of these patients had multiple lesions, 10 of which were in the spinal cord; only one case of extraneural metastasis was found. The median time from diagnosis to detection of the second lesion in this group was 101 weeks (range 14 to 459 weeks). These results show that more than 90% of CNS gliomas recur at the site of the original tumor. Considering the high frequency of intellectual dysfunction after whole brain radiation therapy, the use of focal radiation fields appears to be the most judicious approach to the treatment of patients with gliomas. PMID- 3021933 TI - Cylindromas of the base of the skull. Report of four cases. AB - Cylindromas are rarely encountered in the neurosurgical field. Four cases of this rare tumor are presented. Conventional and computerized tomography scanning were most useful in establishing the diagnosis; angiography did not provide any further information. Because these tumors are well demarcated from surrounding structures, even in cases of recurrence, surgery is the treatment of choice. Radiation therapy is useful in the postoperative management. Long survival times with multiple recurrences are the rule. PMID- 3021932 TI - Computerized tomography in the prognosis of malignant cerebral gliomas. AB - Ninety-seven patients with supratentorial malignant gliomas who received postoperative radiation therapy and chemotherapy at the University of California, San Francisco, from 1977 through 1984 showed improvement in their follow-up computerized tomography (CT) scans. Twenty-one of these 97 "CT responders" were designated "complete responders" because on serial CT scans they had complete disappearance of the tumor mass and contrast enhancement, which had been present postoperatively. In the remaining 76 patients, CT scans showed reduction in the size, but not disappearance, of the lesions, and these were designated "partial responders." Fifty-eight partial responders had glioblastoma multiforme (GM); their median survival time was 72 weeks. The median survival time for the 11 complete responders with GM has not yet been achieved, but survival at the 53rd percentile is 172 weeks. Among patients with highly anaplastic astrocytoma, the median survival time was 211 weeks for the 10 complete responders and 125 weeks for the 18 partial responders. Eleven of the 21 complete responders are alive at a median postoperative follow-up time of 163 weeks (range 114 to 470 weeks). Eighteen of these patients had subtotal resection of tumor; three patients had gross total tumor resections, but postoperative CT scans showed evidence of residual or possibly recurrent tumor within 1.5 to 4.5 months. Resolution of the tumor mass and contrast enhancement took 9 to 151 weeks; the time to resolution did not depend upon the configuration of the remaining tumor mass and contrast enhancement after surgery. In this study, patients with malignant gliomas whose CT scans eventually showed sustained complete disappearance of the tumor mass and contrast enhancement had a more favorable prognosis than did patients whose CT scans showed improvement, but not complete disappearance, of the tumor. These CT findings may prove useful in determining the prognosis of patients with malignant gliomas. PMID- 3021934 TI - Localization of technetium-99m pertechnetate in peripheral nerve tumors. AB - Technetium-99m pertechnetate scintigraphy in three patients with pathologically proven peripheral nerve tumors (six in total) demonstrated its ability to assess accurately the size, location, and the extent of all lesions. Pertechnetate localized only in areas of abnormal nerve involvement and all lesions were better seen in delayed images than the earlier ones. Pertechnetate imaging appears to be a promising method of noninvasive evaluation of clinically evident and occult tumors of peripheral nerve origin. PMID- 3021935 TI - Collimator selection for SPECT brain imaging: the advantage of high resolution. AB - We compared a prototype long-bore (LB) high-resolution collimator with a low energy, general-purpose collimator (LEGP) using 99mTc and 123I. The LB collimator provided a 56% improvement in tomographic resolution (autocorrelation width) over the LEGP for 99mTc; for 123I, the gain was 79%, providing substantially improved contrast for small structures. The sensitivity of the LB collimator, however, is only 32% of that of the LEGP. The imaging tasks to be performed on [123I]IMP brain scans involve localization and discrimination of small, high-contrast brain structures and detection of abnormalities in shape, size, or uptake, rather than simple detection of lesions. Observer performance in such higher-order imaging tasks is known to depend on high spatial resolution, even at the cost of sensitivity. Patient studies confirmed that, for resolution-limited tasks, the increase in resolution outweighs the increased noise due to a loss in sensitivity. When the tomographic resolution of the LB collimator was degraded by smoothing to that of the LEGP, the noise in the LB images was lower than that of the LEGP by a factor of 2.9 for the same imaging time, demonstrating the advantage of high-resolution detectors and a smooth reconstruction filter over low-resolution detectors without smoothing. Therefore, collimators designed for high resolution, even at substantial cost in sensitivity, are expected to yield significant improvements for brain SPECT. Geometric calculations show that commercially available low-energy, high-resolution cast collimators promise to meet these requirements. PMID- 3021936 TI - Iodine-131 MIBG uptake in a small cell carcinoma of the lung. PMID- 3021938 TI - Screening occupational populations for asymptomatic or early peripheral neuropathy. AB - Traditional quantitative methods for evaluating neuropathy may be problematic or insufficiently sensitive as screening tools; standard electrophysiological techniques (nerve conduction velocities and electromyography) are often no more sensitive or reproducible, and in some cases are less sensitive, than the clinical neurological examination. Several promising methods for the evaluation of subclinical neurotoxic effects, including somatosensory and visual evoked potentials, quantitative sensory testing of temperature and vibration, and specialized nerve conduction testing, will be discussed. The crucial issues of sensitivity, standardized test procedures, reproducibility, and practical application in the field setting are currently being examined for these and other techniques. It should be recognized, however, that a brief, directed clinical examination may currently serve as the best screening battery for certain neurotoxins. The most appropriate screening tests can be devised when the neurotoxin is identified and its effects known. PMID- 3021937 TI - The prevalence of screening in industry: report from the National Institute for Occupational Safety and Health National Occupational Hazard Survey. AB - Data from 4,500 workplaces surveyed by the National Institute for Occupational Safety and Health (NIOSH) in the National Occupational Hazard Survey (1972 to 1974) and National Occupational Exposure Survey (1981 to 1983) show an increase in both preplacement and periodic medical screening in US industries during the past decade. The distribution of screening is primarily related to plant size, but also varies considerably by industry type; further, plants with industrial hygiene and safety programs and/or unions are more likely to provide screening examinations than those without, irrespective of plant size. As for workers potentially exposed to selected chemical hazards, the first survey provides no consistent evidence that such workers were more likely to receive exposure specific tests than other workers. The significance of these findings is discussed in the context of the proposed framework for medical screening practices developed by NIOSH researchers. PMID- 3021939 TI - In pursuit of the abnormal serum alkaline phosphatase: a clinical dilemma. AB - The serum alkaline phosphatase (ALP) is often included among the tests used for case-finding among ambulatory patients. To determine the positive predictive value of the ALP, test results for all adults screened by a health maintenance organization between March and December 1969 were obtained by computer. The authors reviewed the charts of all 661 patients with abnormal tests whose primary source of medical care was at this facility. Complete two-year follow-up data were available for 91% of these patients. There were 56 patients (9%) with a diagnosis that could have explained an abnormal ALP. Of those cases in which ALP would have been clinically useful all but one could have been diagnosed by a simple, noninvasive workup, and in that one case, no management change would have occurred. The authors conclude that in the absence of a small number of specific indications, extensive testing need not be performed to evaluate an isolated abnormal ALP obtained from a screening examination. PMID- 3021940 TI - Intracellular action of renin, angiotensin production and release. AB - The enzyme renin has been purified and characterized by structural analysis. Pure renin protein was used to produce a specific antibody to renin, which was useful in demonstrating the presence of a specific renin in many tissues other than kidney. In these cells angiotensins I and II and angiotensin converting enzyme were found to coexist with renin by immunohistochemical studies and by the direct determination with cultured cells. Studies with these cells indicated the local production of renin, angiotensinogen and angiotensins in these cells. Angiotensin II produced in the cultured cells was secreted to the outside of the cells while more than 95% renin remained within the cells. Secretion of angiotensin II from the angiotensin producing cells was demonstrated with perfused mesenteric artery. The secretion was stimulated by the adrenergic beta-agonist isoproterenol in a dose-dependent manner and specifically inhibited by a beta 2-antagonist. Angiotensin II secreted from the vascular bed by the beta 2-adrenoceptor stimulation acts locally to facilitate norepinephrine release. These studies demonstrate local production and secretion of angiotensin II and define its physiological role. PMID- 3021941 TI - Cytochrome P450-dependent arachidonate metabolism in renomedullary cells: formation of Na+K+-ATPase inhibitor. AB - The medullary portion of the thick ascending limb of the loop of Henle (mTALH) has one of the highest concentrations of Na+K+-ATPase found in mammalian tissues, reflecting the importance of this nephron segment in the regulation of extracellular fluid volume, as active sodium transport is driven by Na+K+-ATPase. We have isolated cells derived primarily from the mTALH of the outer medulla of rabbit kidney and have identified a cytochrome P450-dependent mono-oxygenase system which metabolizes arachidonic acid to two biologically active oxygenated peaks, each containing two or more products. One of the peaks potently inhibits cardiac Na+K+-ATPase and the other relaxes blood vessels. We report that formation of these oxygenated arachidonate metabolites is stimulated by arginine vasopressin (AVP) and salmon calcitonin (SCT). In mTALH cells obtained from rabbits made hypertensive by aortic constriction there was a selective increase in P1 and P2 formation compared to other renomedullary cells. PMID- 3021943 TI - Breast carcinoma metastatic to the gingiva. PMID- 3021942 TI - Mechanisms of sodium balance in hypertension: role of pressure natriuresis. AB - This paper summarizes the role of the renal pressure natriuresis and diuresis mechanisms in maintaining sodium and water balance in hypertension. In all forms of chronic hypertension studied to date, the renal pressure natriuresis and diuresis mechanisms are abnormal, since increased arterial pressure is required to maintain normal excretion of sodium and water, and therefore fluid balance. When renal perfusion pressure is prevented from increasing in various forms of experimental hypertension, caused by infusion of mineralocorticoids, angiotensin II, vasopressin, or norepinephrine and adrenocorticotrophic hormone (ACTH), sodium and water retention continues until ascites, pulmonary oedema and circulatory collapse occur within a few days. Thus, chronic hypertension appears to be an essential homeostatic response that permits sodium and water balance to be maintained despite various abnormalities which tend to decrease renal excretory capability. The intrarenal mechanisms by which increased renal perfusion pressure maintains sodium and water balance in hypertension have not been fully elucidated, but appear to involve small changes in glomerular filtration rate (GFR) and reductions in fractional sodium reabsorption, due either to the direct hydraulic effects of pressure or to various indirect effects, such as changes in angiotensin II formation. PMID- 3021944 TI - Virilization of two siblings by maternal androgen-secreting adrenal adenoma. PMID- 3021946 TI - Interaction of microspheres with blood constituents II: Uptake of biodegradable particles by macrophages. PMID- 3021945 TI - Lithium for treatment of neutropenia in glycogen storage disease type Ib. PMID- 3021947 TI - Treatment of a retentive encopretic child using contingency management and diet modification with stimulus control. PMID- 3021948 TI - Angiotensin I converting enzyme activity in the kidney of bullfrog (Rana catesbeiana). AB - In the present investigation, the occurrence of angiotensin I converting enzyme (EC 3.4.15.1; ACE) was first demonstrated in the kidney of bullfrog (Rana catesbeiana). Namely, a large amount of hydrolyzing activity toward N alpha hippuryl-L-His-L-Leu-OH (HHL), a synthetic substrate of ACE, was detected in a 100,000 X g pellet fraction of the bullfrog kidney. The renal HHL hydrolyzing enzyme was solubilized from the 100,000 X g pellet with sodium deoxycholate. The solubilized bullfrog renal enzyme liberated H-His-Leu-OH from HHL. The enzyme activity was strongly activated by NaCl and slightly activated by cobalt sulfate in the presence of NaCl. These properties of the bullfrog renal enzyme agreed well with those of mammalian ACE previously reported. The bullfrog renal HHL hydrolyzing activity was completely inhibited by typical ACE inhibitors, ethylenediamine tetraacetic acid (10(-3) M), captopril (10(-5) M), SA-466 (10(-5) M) and MK-422 (10(-6) M). Furthermore, [Ile5]-angiotensin I converting activity was also detected in this enzyme fraction of bullfrog kidney and this converting activity was completely inhibited by MK-422 (10(-6) M). Thus, enzyme activities having characteristics of ACE were detected in the 100000 X g pellet of bullfrog kidney. We also detected [Val5, Asn9]-angiotensin I (bullfrog angiotensin I) converting activity in the bullfrog renal extract. This converting activity was also completely inhibited by MK-422 (10(-6) M). PMID- 3021949 TI - Effect of melatonin on the force of spontaneous contractions of in vitro rat small and large intestine. AB - Segments from various locations of the small and large intestine of the rat were removed, bathed in Tyrode's solution and attached to a force displacement transducer. Melatonin, while not influencing the frequency of contraction, did reduce the force of spontaneous contractions of duodenal and colon segments of rat intestine by 92 and 52%, respectively compared to only 25 and 22% for the ileum and jejunum, respectively. Areas with greatest responsiveness to melatonin were those that previous studies have shown to contain the largest concentrations of endogenous melatonin. Cyclic guanosine monophosphate, when tested in similar preparations, did not produce an inhibitory response characteristic of melatonin. It is hypothesized, therefore, that this hormone has physiological action within the gut, including motility; however, its action may not be directly on smooth muscle contraction but may be through an indirect action inhibiting the contractile response of serotonin, as suggested by other investigators. PMID- 3021950 TI - Directed selection of differentiation mutants of Streptomyces noursei using chemostat cultivation. AB - A nourseothricin-producing Streptomyces noursei strain was continuously cultivated in a chemostat equipped with a stirrer for mechanical fractionation of the mycelium. Different cultivation conditions allowed the selection of six types of differentiation mutants after the culture had reached a population genetically stationary state. The mutants showed an altered control pattern of sporulation as well as altered antibiotic biosynthesis and antibiotic resistance. In addition, the stability of the recombinant plasmid pIJ385 in several differentiation type mutants as host strains was tested. The results suggest that there exists a strong correlation between the cultivation conditions employed and the type of differentiation mutants selected. PMID- 3021951 TI - Molecular orbital calculations of proton transfer involving amines as models for the clastic binding of opiates with their receptor. AB - Semi-empirical (CNDO) molecular orbital calculations, based on a previously reported ammonia-amine model system, were performed on an extended series of methyl-, ethyl-, and propylamines as models for the analgesic receptor. Methyl-, dimethyl-, and trimethylamines were chosen to represent the opiate molecules. Interatomic distances were varied within normally expected biological values. The results for the larger systems are similar to more elaborate calculations previously reported using smaller molecules. At internuclear distances of greater than 0.275 nm, the potential energy curves had two minima. At 0.2731 nm, the optimized N-N distance, the "depth" of the minima in the potential energy curve were not as great. Energy differences as well as population differences suggest deviation from the currently stated clastic binding theories mechanism for the analgesic response of the tertiary amines. The dimethylamine energy profile and population data indicate that the hypothesis of N-demethylated opiate as the active molecule needs further consideration and investigation. Investigation of larger systems is also indicated to develop increasingly realistic models for the analgesic response. PMID- 3021952 TI - Dependence on tetrahydrocannabinol in rhesus monkeys. AB - The present studies examined whether dependence could be induced by continuous infusion of delta-9-tetrahydrocannabinol (THC), as evidenced by behavioral disruptions during withdrawal of THC administration. Four rhesus monkeys lever pressed under fixed-ratio schedules for their daily food rations during four, daily, 0.5 -hr sessions. Initially, the monkeys were tested before, during and after continuous i.v. infusions of THC. A dosage regimen of 0.05 mg/kg/hr for 10 days for three of the monkeys and a somewhat greater regimen in the fourth had little direct effect on rates of respondings for food during THC infusion, but marked decreases in response rates occurred in each monkey during withdrawal. These effects generally did not occur until the second day of withdrawal, and often lasted for over a week. These initial demonstrations were replicated subsequently with lower solvent concentrations. In the final study, THC was administered for a greater number of days and at higher dosages than during any of the previous studies. Response rate reductions again occurred within 2 to 3 days of withdrawal. When THC was readministered, reversal of the withdrawal effects occurred in that withdrawal-induced rate decreases were halted and rates increased subsequently. Administration of THC was discontinued after 2 to 3 days of its readministration. Response rates again decreased within the first 3 days after this withdrawal and then recovered slowly with time. Withdrawal of THC administration can disrupt operant behavior, and these disruptions can be reversed by resumption of THC administration, indicating that dependence upon this drug can be produced in rhesus monkeys. PMID- 3021954 TI - Reversible and irreversible binding of beta-funaltrexamine to mu, delta and kappa opioid receptors in guinea pig brain membranes. AB - The effect of beta-funaltrexamine (beta-FNA), an irreversible mu receptor blocker in isolated tissue bioassays, on mu, kappa and delta opioid receptor binding and the binding of beta-[3H]FNA were determined in guinea pig brain membranes. beta FNA inhibited the binding of mu, kappa and delta opioid ligands to their receptors with Ki values of 2.2, 14 and 78 nM, respectively. Pretreatment of brain membranes with beta-FNA (less than 2 microM) followed by extensive washing inhibited mu binding and to a lesser degree delta binding, without changing kappa binding. The extent of the irreversible inhibition was dependent on the concentration of beta-FNA, and this inhibition on mu binding could be observed with as little as 1 nM beta-FNA. The irreversible inhibition of mu binding by beta-FNA pretreatment was due to a decrease in the number of binding sites with little change in Kd, and was more pronounced in the presence of increasing concentrations of NaCl. Specific binding of beta-[3H]FNA to opioid receptors was demonstrated. The rate of specific binding with 2 nM beta-[3H]FNA was rapid in the initial 10 min and did not reach maximum in 90 min. The dissociation of bound beta-[3H]FNA (5 nM added) by the addition of excess unlabeled naloxone reached maximum at 30 min with approximately 35% of specifically bound beta-[3H]FNA remaining. Mu opioids were most effective in preventing specific binding of beta [3H]FNA when added before beta-[3H]FNA. Opioids added 1 hr after 2 nM beta [3H]FNA could displace maximally only 70 to 75% of specific binding.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021953 TI - Increase of cyclic AMP concentrations in cultured vascular smooth muscle cells by vasoactive peptide hormones. Role of endogenous prostaglandins. AB - We have evaluated the hypothesis that vasoactive hormones increase cellular cyclic AMP (cAMP) levels in cultured vascular smooth muscle cells from rat mesenteric arteries by stimulating endogenous prostaglandin (PG) synthesis. Vasopressin and angiotensin II, which were shown previously to provoke the synthesis of PGs in cultured vascular smooth muscle cells, increased cellular cAMP concentrations by about 2-fold, whereas a peptide analog of vasopressin, 1 desamino-8-D-arginine vasopressin, mostly lacking vasopressin's ability to elicit PG synthesis, was ineffective. Two other chemically dissimilar effectors that provoked the synthesis of PGs in cultured vascular smooth muscle cells, namely arachidonate and ionophore A23187, also increased cellular cAMP levels. The increase of cAMP by vasopressin and angiotensin II was transient, reaching a maximum at 1 to 2 min of incubation, followed by a decline to basal levels. Acetylsalicylic acid, a specific inhibitor of PG synthesis, completely prevented vasopressin- and arachidonate-evoked increases of cAMP but did not affect basal cAMP concentrations. Exogenous prostacyclin and prostaglandin E2 dose-dependently increased cAMP concentrations although prostacyclin was more effective than prostaglandin E2. The ability of exogenous prostacyclin to evoke cAMP increases was not inhibited by acetylsalicylic acid. The results support the hypothesis that the stimulation of endogenous PG synthesis by vasoactive hormones in turn modulates cellular cAMP levels in cultured vascular smooth muscle cells from rat mesenteric arteries. PMID- 3021955 TI - Molecular basis for the in vitro and in vivo cardiotonic activities of AR-L100. AB - Imidazo[4,5-b]pyridines, such as AR-L57, AR-L100 and AR-L115 (Vardax), have been of interest as inotropic agents for the management of congestive heart failure. Although it has been presumed that their activities derive from inhibition of phosphodiesterase, it is now apparent that similar structural analogs possess surprisingly diverse pharmacologies and mechanisms of action. AR-L100 increased the contractile state of cat papillary muscles in a concentration-dependent manner; these effects were not blocked by either alpha, beta or H2-receptor antagonists. To determine whether the contractile responses resulted from intracellular cyclic AMP accumulation, the cardiotonic actions of AR-L100 were assessed in the presence of carbachol. Muscarinic receptor stimulation did not alter inotropic responses to AR-L100; in addition, AR-L100 did not potentiate the inotropic actions of isoproterenol. These results imply that cyclic AMP is not involved in the cardiac responses to this agent. AR-L100 inhibited Na+,K+ adenosine triphosphatase activity of either canine kidney or cardiac sarcolemmal vesicles. Inhibition of this enzyme paralleled inotropic responses in vitro; that is, in papillary muscle, the EC50 for contractility was 11.5 microM compared with an IC50 for inhibition of Na+,K+-adenosine triphosphatase of 8 microM. By contrast, the IC50 for inhibition of phosphodiesterase (isozyme III) was 280 microM. AR-L100 also inhibited sodium pump activity in intact cat papillary muscles. Concentrations of 30 and 100 microM AR-L100 resulted in 13 and 45% decreases in ouabain-sensitive 86Rb+ uptake determined at 3 Hz. In anesthetized dogs, AR-L100 increased contractility but did not alter either heart rate or mean arterial blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3021956 TI - Sympathetic and parasympathetic nerves regulate postsynaptic alpha-2 adrenoceptor in salivary glands. AB - The effects of sympathetic denervation or parasympathetic decentralization on the inhibitory effects of postsynaptic alpha-2 adrenoceptors were studied in the submaxillary and the sublingual gland of the rat. Chronic sympathetic denervation enhanced by a factor of 10 the potency of clonidine to inhibit the secretory responses of the submaxillary gland to either norepinephrine or methacholine. In denervated glands, clonidine (1 microgram/kg), reduced markedly the response to norepinephrine, but potentiated this response in control glands. Blockade of postsynaptic alpha-2 adrenoceptors with idazoxan (3 micrograms/kg) enhanced the secretory responses of denervated glands to norepinephrine. Parasympathetic decentralization also potentiated the inhibitory effects of the alpha-2 agonists. In the submaxillary gland the potency of guanabenz to decrease the secretory response to methacholine was increased by a factor of 30. Supersensitivity to the inhibitory effects of clonidine was also observed in parasympathetically decentralized sublingual glands. Parasympathetic decentralization increased the maximum binding site of [3H]clonidine binding by about 50% in both the submaxillary and sublingual glands. No changes in KD were detected. This surgical procedure also increased the maximum binding site of [3H]prazosin binding in submaxillary glands. The present findings show clearly that interruption of either branch of the autonomic nervous system induces supersensitivity of the inhibitory response mediated through postsynaptic alpha-2 adrenoceptors. The enhanced inhibitory activity could mask alpha-1 adrenoceptor supersensitivity after postganglionic sympathetic denervation. PMID- 3021958 TI - Hydroxyapatite for alveolar ridge augmentation: a clinical study. PMID- 3021957 TI - Correlative studies on the effect of carbachol on myo-inositol trisphosphate accumulation, myosin light chain phosphorylation and contraction in sphincter smooth muscle of rabbit iris. AB - Previously, we have reported that activation of muscarinic cholinergic receptors in the iris smooth muscle results in a rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,2-diacylglycerol and myo-inositol trisphosphate (IP3) and that the stimulated hydrolysis of this phospholipid correlates well with contraction. To determine whether or not there is a causal relationship between PIP2 breakdown and contraction, we have conducted correlative studies on the effects of carbachol (CCh) on PIP2 breakdown, measured as IP3 accumulation, myosin light chain (MLC) phosphorylation and contraction in the rabbit iris sphincter. We have also investigated the effects of time, temperature, atropine antagonism, Ca++ and C-kinase activators on the three measured responses. The data obtained can be summarized as follows: dose-response studies for IP3 accumulation, MLC phosphorylation and contraction revealed a close correlation between these responses; kinetic data on atropine antagonism showed that the three measured responses are competitively inhibited by the muscarinic antagonist; time course studies conducted at low temperature showed that the CCh induced IP3 accumulation and MLC phosphorylation may precede contraction; time course studies on the effect of Ca++ on the three measured responses showed that IP3 release may account for the rapid phase of CCh-induced contraction and that extracellular Ca++ is essential for sustained MLC phosphorylation and the slow phase of contraction; the activity of phospholipase C, the enzyme involved in PIP2 hydrolysis, in membrane fragments from 32P-labeled sphincter muscle was found to be highly sensitive to Ca++, with half-maximal stimulation at about 1.1 microM Ca++; and phorbol 12,13-dibutyrate, but not phorbol 12-myristate 13 acetate, induced MLC phosphorylation and muscle contraction in a dose- and time dependent manner. Phorbol 12,13-dibutyrate and ionomycin acted in a synergistic manner to elicit contraction. In conclusion, contractions by CCh in the iris sphincter may be explained on the basis of enhanced PIP2 turnover and its derived second messenger molecule(s); that there are consistent correlations, using different concentrations of CCh, atropine antagonism, time, temperature and Ca++, between the stimulated hydrolysis of PIP2, MLC phosphorylation and contraction. Finally, whereas the data presented favor the involvement of IP3 in the phasic component of the contractile response, the studies with phorbol 12,13-dibutyrate suggest that contractile regulation by 1,2-diacylglycerol, through activation of C-kinase, may be important during the tonic component of smooth muscle contraction. PMID- 3021959 TI - The isolation of fungi from laboratory dental pumice. AB - Samples of used dental laboratory pumice from the two dental laboratories were cultured for the isolation of fungi. The resulting supernatant fluid from sedimentation of each pumice sample after suspension in sterile saline was serially diluted and plated onto Sabouraud agar. After incubation, fungal colonies observed were enumerated, isolated, and identified. The mean number of fungal colonies recovered from 10 pumice samples in laboratories I and II was 51.0 X 10(2) and 22.6 X 10(2), respectively. In both laboratories the predominant fungi recovered were Aspergillus niger and Fusarium sp. Other fungi recovered included Cephalosporium and Penicillium species and A. flavus. Many of these organisms have been involved in human disease. It is suggested that the presence of fungi in used dental laboratory pumice presents an unhygienic condition in the dental laboratory and may place dental laboratory technicians and denture patients at increased risk of fungal sensitization and disease. PMID- 3021960 TI - Congenital mesoblastic nephroma of infancy. Study of four cases and review of the literature. PMID- 3021961 TI - Ultrastructure of preneoplastic lesions of the vulva. AB - Ultrastructural analysis using electron microscopy was performed on 16 cases of preneoplastic lesions of the vulva. They included specimens from patients with condylomatous dysplasia, carcinoma in situ, melanoma in situ and extramammary Paget's disease. Each showed significant differences from normal in the ultrastructural details. PMID- 3021962 TI - Membranolytic effects of monosodium urate monohydrate: influence of grinding. AB - The effect of grinding on the membranolytic interaction between monosodium urate monohydrate (MSUM) crystals and intact erythrocyte membranes was studied. Crystals were ground for between 1-72 h, and percent hemolysis and zeta potentials determined. A cationic amphiphilic probe (CAT12) was incorporated into the erythrocyte membrane and incubated with MSUM. Increasing grinding times caused a decrease in both the crystallinity and zeta potential of the samples, a decrease in percent hemolysis values and a change in the distribution of free and bound spin label populations. The probe redistribution is thought to be due to an electrostatic interaction between negatively charged MSUM and the CAT12 probe. PMID- 3021964 TI - Effects of polychlorinated biphenyls and lipemia on serum analytes. AB - Twelve serum analytes [triglycerides, cholesterol, total and conjugated bilirubin, high-density-lipoprotein cholesterol (HDL-C), alkaline phosphatase (AP), gammaglutamyl transferase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), beta-glucuronidase (beta-glu), alanine aminopeptidase (AAP), and 5'-nucleotidase (5'nuc)] were measured to investigate their correlation with exposure to polychlorinated biphenyls (PCBs) and 1,1,1-trichloro 2,2-bis(p-chlorophenyl)ethane (DDT). The relationship between serum lipids, lipophilic toxicants, and the analytes was also evaluated. The beta-glu, 5'nuc, triglycerides, cholesterol, and total bilirubin correlated positively and significantly with log concentrations of serum total PCBs and 1,1-dichloro-2,2 bis(p-chlorophenyl)ethylene (DDE), a metabolite of DDT. The more highly chlorinated PCBs (Aroclor 1260) had significant, positive correlations with several serum analytes, but the less chlorinated PCBs (Aroclor 1242) correlated significantly and negatively only with HDL-cholesterol. Triglyceride- and cholesterol-rich lipoproteins were added to serum to determine the effects of lipids on these assays. Several were spuriously elevated. AP and beta-glu were not affected by lipoprotein addition with the methods used in this study. AAP was increased significantly only at triglyceride concentrations exceeding 400 mg/dl. Lipoproteins may be elevated because of deranged lipid metabolism in response to PCBs, or PCBs may be elevated because elevated lipoproteins are present, as in familial triglyceridemia, a relatively common dyslipoproteinemia. Because this relationship is not well understood with respect to cause and effect, we propose the further use in epidemiological investigations of assay methods that are little affected by blood lipids yet are correlated with PCB concentrations. Congener-specific quantification of PCBs would help elucidate the effects of PCBs on assays used to monitor health effects. PMID- 3021963 TI - Kaposi's sarcoma and HTLV-III: a study in Nigerian adult males. AB - Sera from 37 adult Nigerian men with Kaposi's sarcoma (KS), 30 contemporaneous controls bearing primary cell carcinoma of the liver (PCL), and 150 healthy non tumour-bearing negative controls were tested for antibody to human T-cell lymphotropic virus type III/lymphadenopathy associated virus (HTLV-III/LAV) by enzyme-linked immunosorbent assays (ELISA). Certain immunocellular functions were also measured: the chemotactic locomotion of peripheral blood monocytes towards casein, delayed-type cutaneous hypersensitivity reaction to tuberculoprotein and opportunistic infection with the fungus Candida albicans. Sera from all these groups were also tested for markers of previous infections with the viruses cytomegalovirus (CMV), Epstein-Barr virus (EBV), hepatitis B (HBV) and hepatitis A (HAV). All serum samples tested were reproducibly and consistently negative for anti-HTLV-III/LAV. Peripheral blood monocytes from both KS and PCL patients showed profound depression of chemotaxis; similarly all tumour patients gave markedly depressed cutaneous reactivity to tuberculoprotein and uniformly exhibited seropositivity to CMV, EBV, HBV and HAV. A great majority showed evidence of infection with Candida albicans. It is concluded that tropical African KS is not associated with HTLV-III/LAV infection. PMID- 3021966 TI - The morphological spectrum of peripheral intrahepatic cholangiocarcinoma--an attempt at histological classification of twenty-eight autopsy cases. PMID- 3021965 TI - Direct noninvasive assessment of brain metabolism during increased intracranial pressure: potential therapeutic vistas. AB - Intracranial pressure was increased in cats by infusing 'mock' CSF intracranially, thus decreasing cerebral perfusion and oxygenation. The cats then randomly received either 50% O2 or 50% O2-5% CO2 by inhalation. As monitored by in vivo near-infrared spectroscopy (NIR), no improvement was noted after 50% O2 whereas 50% O2-5% CO2 resulted in increased perfusion, an oxidation of cytochrome a,a3, an increase in oxyhemoglobin, and reduced quantities of de-oxyhemoglobin (p less than 0.01) despite a further increase in intracranial pressure. The authors conclude that: NIR is a useful means of noninvasively and directly assessing brain metabolism and has advantages over simple ICP monitoring; and continued investigations of CO2 as a possible therapeutic modality after head injury appear warranted. PMID- 3021967 TI - Seroepidemiological study on kala-azar in Baringo District, Kenya. AB - This paper reports on 164 cases of kala-azar observed in the Baringo District of Kenya between February 1981 and February 1983. All were confirmed serologically by enzyme-linked immunosorbent assay (ELISA) and all but 20 by parasitological examination as well. Following the standard treatment with a 30 day course of sodium stibogluconate (Pentostam) two non-responders and four relapses were observed. Children between 2 and 15 years old were found to be the most affected age group; male patients predominated slightly at 57%. All cases occurred in the semi-arid and arid parts of the district below 1500 m, where pastoralism predominates. Besides scattered cases, certain kala-azar foci could be identified. Two of these--Endao with 49 households, 228 inhabitants and 13 cases of kala-azar, and Koriema with 22 households, 93 inhabitants and 11 cases--were subject to a house to house survey. People were examined physically, their weight and height recorded and fingerprick blood collected on blotting paper for later serological testing. Each household was mapped and the relevant environmental factors recorded. A positive correlation could be demonstrated between kala-azar cases and the vicinity of their homesteads to seasonal rivers and also between kala-azar cases and people living in timber houses, rather than mud and wattle houses. Eroded termite hills were not found to be of epidemiological importance. No satisfying explanation could be found for the striking temporal and local clustering of cases. The homestead was identified as an important site of transmission with optimum conditions for transmission occurring during supper in the evening. Based on spleen rates, Endao was classified as hyperendemic for malaria and Koriema as mesoendemic. Diagnostic ELISA values above 0.2 were observed in all cases of active kala-azar. However, ELISA values above 0.04, taken as the borderline non-specific reaction, could be found in about half of the study areas population. Therefore we conclude that asymptomatic infection must be common. Observations demonstrated that spontaneous recovery may follow clinical illness and visceralization of the parasite. Comparison of parasitological and serological data suggest that this may be expected in more than 15% of cases. PMID- 3021968 TI - Seroepidemiological study of para-influenza viruses in preschool children in Ghana. PMID- 3021969 TI - Characterization of monoclonal antibodies reactive to several biochemically distinct human cytomegalovirus glycoprotein complexes. AB - Three monoclonal antibodies were characterized by examining their reactivity to human cytomegalovirus (HCMV) glycoproteins under reducing and nonreducing conditions and their reactivity to glycoproteins and disulfide-linked glycoprotein complexes isolated by ion-exchange high-performance liquid chromatography. One monoclonal antibody, 9E10, reacted with glycoprotein complexes which had molecular weights of 93,000 and 450,000 and eluted from the ion-exchange column at 0.3 and 0.9 M NaCl, respectively. All glycoproteins associated in these complexes could be immunoprecipitated under reducing conditions by 9E10, suggesting that they were related to one another. The most abundant glycoproteins immunoprecipitated by 9E10 had molecular weights of 50,000 to 52,000. In contrast to this antibody, two other monoclonal antibodies, 9B7 and 41C2, reacted with glycoprotein complexes which had molecular weights of 130,000 and greater than 200,000 and eluted from the ion-exchange column at 0.6 M NaCl. All glycoproteins associated in these complexes could be immunoprecipitated by 9B7 or 41C2 under reducing conditions, suggesting that they were also related to one another. The most abundant glycoprotein immunoprecipitated by 41C2 or 9B7 had a molecular weight of 93,000. In addition, it was also determined that a 93,000 molecular-weight glycoprotein which was not associated with other glycoproteins by disulfide bonds could not be precipitated by any of the three antibodies, suggesting that it was different from the other glycoproteins. The monoclonal antibodies were also examined for specificity and neutralizing activity. Monoclonal antibodies 41C2 and 9B7 were specific to HCMV as determined by immunofluorescent staining of skin fibroblast cells infected with several different viruses. However, 41C2 did not neutralize Towne strain HCMV, while 9B7 did. The neutralizing activity of 9B7 did require complement. These results suggested that 41C2 and 9B7 reacted with different antigenic sites on the same glycoproteins. Unlike 41C2 and 9B7, monoclonal antibody 9E10 was found to cross react with adenovirus and herpes simplex virus as determined by immunofluorescent staining of infected skin fibroblast cells. Furthermore, 9E10 neutralized the Towne and Toledo strains of HCMV in the absence of complement. PMID- 3021970 TI - Suppression of the translation defect phenotype specific for a virus-associated RNA-deficient adenovirus mutant in monkey cells by simian virus 40. AB - Human cells infected with adenovirus type 2 (Ad2) or Ad5 require VAI RNA for efficient translation of viral mRNAs at late times after infection. The Ad5 mutant dl-sub720 synthesized neither virus-associated I (VAI) nor VAII RNAs, and infection of human cells with this mutant resulted in reduced virion polypeptide synthesis. Infection of monkey cells with this mutant also resulted in drastic reduction of polypeptide synthesis compared with wild-type (WT) adenovirus infections. Steady-state levels of hexon-specific mRNA were found to be comparable in WT- and mutant-infected monkey cells. The in vitro translation experiments showed that double-mutant- and WT-infected cells contained comparable levels of translatable hexon mRNA (and other adenovirus late mRNAs), suggesting that the severe inhibition of hexon protein synthesis in the VA mutant involves a translation block. Preinfection of monkey cells with simian virus 40 fully restored the efficient translation of this mRNA in the VA mutant infections to the level observed in WT-infected cultures. These results raise the possibility that simian virus 40 may encode or induce factors that suppress the translation block that occurs during adenovirus infections in the absence of the VA RNAs. PMID- 3021971 TI - Detection by monoclonal antibodies of an early membrane protein induced by Epstein-Barr virus. AB - Two monoclonal antibodies, E8B3 and E8D2, were raised against Epstein-Barr virus (EBV)-producing cells and were shown to immunoprecipitate a protein with an approximate molecular weight of 105,000 (p105). The protein was detectable only in EBV-containing cells which were supporting the virus lytic cycle, and its synthesis increased after cells were induced with phorbol esters. The molecule was radiolabeled and immunoprecipitated from virus-producing cells that had been extrinsically labeled with 125I, and the antibodies E8B3 and E8D2 reacted in immunofluorescence assays with infected cells; the molecule was also associated with virion particles. Synthesis of p105 was not blocked by phosphonoacetic acid and could be induced in Raji cells by superinfection with virus derived from P3HR1 cells. These data support the conclusion that p105 is an EBV-specific early membrane protein. PMID- 3021972 TI - Encephalomyocarditis virus 3C protease: efficient cell-free expression from clones which link viral 5' noncoding sequences to the P3 region. AB - All picornaviral peptides are derived by progressive posttranslational cleavage of a giant precursor polyprotein. Translation of encephalomyocarditis virus (EMC) RNA in rabbit reticulocyte extracts produces active viral peptides, including protease 3C, which is responsible for many cleavage reactions within the processing cascade. DNA plasmids containing 5' noncoding sequences of EMC linked to other portions of the viral genome were constructed and transcribed into RNA. Like virion RNA, the clone-derived transcripts directed efficient protein translation in vitro. The 5'-linked constructions may represent examples of a general method for cell-free expression of any cloned gene segment. One construction produced a self-cleaving P3 region precursor, which contained active 3C protease. A genetically engineered insertion within the 3C sequences eliminated endogenous self-cleavage activity without altering the ability of the P3 peptide to serve as substrate in bimolecular reactions with added 3C. Another plasmid encoding the L-VP0 portion of the capsid region was used to demonstrate that scission between the leader peptide (L) and capsid protein VP0 can be catalyzed by 3C. The enzyme responsible for this step was previously unidentified. A rapid purification scheme for isolation of 3C from EMC-infected HeLa cells is also presented. PMID- 3021974 TI - A single species of pX mRNA of human T-cell leukemia virus type I encodes trans activator p40x and two other phosphoproteins. AB - Human T-cell leukemia virus type I (HTLV-I) contains the pX sequence which codes for the trans-activator of the long terminal repeat (LTR) and is thus postulated to be associated with leukemogenesis in adult T-cell leukemia. Overlapping open reading frames (ORF) in the pX sequence were recently found to code for p27x-III and p21x-III by ORF III, in addition to p40x coded for by ORF IV. The mechanism of expression of these newly identified proteins and their possible association with trans-activation were studied. On transfection of an expression plasmid that contains a cDNA sequence of the pX mRNA, products from both ORFs III and IV were detected in the cells. The RNA was synthesized in vitro from the cDNA clone by SP6 RNA polymerase and translated in a rabbit reticulocyte lysate. As translation products, two proteins, p27x-III and p21x-III, were detected in addition to p40x. Elimination of the first and second ATG codons in ORF III resulted in loss of the ability to code for p27x-III and p21x-III, respectively, which indicated that the translations from these two ATG codons were independent. A mutant that lacked both ATG codons was fully active in trans-activation of chloramphenicol acetyltransferase gene expression directed by the LTR. These results indicate that a 2.1-kilobase pX mRNA of HTLV-I independently encodes three proteins, p40x, p27x-III, and p21x-III, by different ORFs and that the last two proteins are not involved in trans-activation of the unintegrated LTR. PMID- 3021973 TI - Nucleotide sequence and transcriptional activity of the caprine arthritis encephalitis virus long terminal repeat. AB - Caprine arthritis-encephalitis virus (CAEV) and visna virus are pathogenic lentiviruses of goats and sheep which share morphologic features and sequence homology with human T-cell lymphotropic virus type III (HTLV-III), the etiologic agent of the acquired immune deficiency syndrome. The nucleotide sequence of the CAEV long terminal repeat (LTR) was determined, and it was found to be 450 base pairs long, with U3, R, and U5 regions of 287, 85, and 78 base pairs, respectively. Portions of the CAEV LTR are closely homologous to analogous regions of visna virus. The CAEV LTR is not significantly homologous with the HTLV-III LTR; however, like HTLV-III, visna virus, and equine infectious anemia virus, CAEV uses tRNA lysine as a primer for reverse transcription. The transcriptional activity of the CAEV and visna virus LTRs was measured by a chloramphenicol acetyltransferase assay, and the activity of the visna virus LTR was generally higher in a variety of uninfected cell types. Infection of cells with visna virus markedly increased gene expression directed by either the CAEV or visna virus LTR, but in contrast, infection of cells with CAEV had little effect on the activity of either LTR. The lack of trans-activation by CAEV, a virus which causes debilitating arthritis and encephalitis in goats, suggests that trans-activation may not be a general property of pathogenic lentiviruses. PMID- 3021975 TI - Activity of simian virus 40 late promoter elements in the absence of large T antigen: evidence for repression of late gene expression. AB - We used chloramphenicol acetyltransferase transient expression to examine the activity of the promoter elements of the simian virus 40 late promoter in the absence of large T antigen. Since the experiments were done in permissive CV-1 cells, these conditions mimic the state which exists early in the viral lytic cycle before the onset of replication and T-antigen-mediated trans activation. Our data, using deletion analysis, indicate that removal of the 21-base-pair (bp) repeat region causes as much as a 10-fold increase in activity of the late promoter elements. This result suggests that the 21-bp repeat sequences may be involved in repression of the late promoter elements during the early phase of the lytic infection. This is supported by competition analysis which indicates that increasing amounts of competitor containing only the 21-bp repeat region results in increased activity of the intact promoter. A model for the activity of the late promoter through the course of lytic infection is presented. PMID- 3021976 TI - Map location of the gene for a 130,000-dalton glycoprotein of bovine herpesvirus 1. AB - A bovine herpesvirus 1 variant (mar6) containing a mutation in a viral glycoprotein with a molecular weight of 130,000 (g130) was isolated by selecting for resistance to a neutralizing monoclonal antibody (130-6) directed against g130. Mar6 was completely resistant to neutralization by monoclonal antibody 130 6 in the presence and absence of complement, but was neutralized by polyvalent immune sera. The mar6 mutant synthesized and processed g130, but produced plaques which failed to react with monoclonal antibody 130-6 in an in situ immunoassay (black plaque). However, monoclonal antibody 130-6 was capable of binding and immunoprecipitating g130 from infected-cell extracts produced by lysis of mar6 infected cells with nonionic detergents. The mutation in mar6 was mapped by marker rescue with cloned bovine herpesvirus 1 restriction enzyme fragments to a 3.8-kilobase fragment at approximate map units 0.405 to 0.432. In addition, it was found that a DNA probe containing the glycoprotein B gene of herpes simplex type 1 hybridized uniquely to the same 3.8-kilobase fragment which was shown by marker rescue to contain the mutation site in the gene for bovine herpesvirus 1 g130. PMID- 3021977 TI - Construction and characterization of CV-1P cell lines which constitutively express the simian virus 40 agnoprotein: alteration of plaquing phenotype of viral agnogene mutants. AB - The simian virus 40 (SV40) agnoprotein is a 61-amino-acid polypeptide encoded in the leader region of some late viral mRNAs. Its function is unclear, although previous investigations suggest that the agnoprotein may function in late transcriptional regulation and virus assembly. To define the specific role(s) of agnoprotein in the SV40 lytic cycle, CV-1P cell lines were constructed in which the agnogene was stably integrated and constitutively expressed under the control of a retroviral long terminal repeat. Two types of cell lines were isolated. One group, typified by the cell line Ag18, produced low levels of agnogene-specific mRNA and agnoprotein. The other type, represented by a single isolate named Ag8, produced high levels of agnogene-specific mRNA and correspondingly high levels of agnoprotein. By indirect immunofluorescence, the agnoprotein was located predominantly in the cytoplasmic and perinuclear region of both cell lines; this is its site of localization in wild-type (WT)-infected CV-1P cells. Viruses that were agnoprotein-minus formed small plaques on normal CV-1P cells, but produced nearly WT-sized plaques on Ag18 cells. Conversely, the plaques formed on Ag8 cells infected with agnoprotein-minus mutants of WT SV40 were markedly smaller than the plaques formed by these viruses when they were grown on control cells. Overall, our results suggest that the agnoprotein is a trans-effector of virus production. The opposite effects on plaquing that were observed with Ag8 and Ag18 cells correlated with the very different levels of agnogene expression in the two cell lines. This suggests that the nature of the effect of the agnoprotein on virus production may vary depending on its intracellular concentration. PMID- 3021978 TI - Prevention of simian acquired immune deficiency syndrome with a formalin inactivated type D retrovirus vaccine. AB - Experimental induction of simian acquired immune deficiency syndrome (SAIDS) by inoculation of juvenile rhesus monkeys with a type D retrovirus was prevented by immunization with Formalin-killed whole SAIDS retrovirus serotype 1 containing the adjuvant threonyl muramyl-dipeptide. All six immunized animals developed neutralizing antibody after three injections, while six age-matched cagemates receiving adjuvant alone were antibody free. All 12 monkeys were challenged intravenously with a potentially lethal dose of SAIDS retrovirus serotype 1. The six immunized animals failed to develop persistent viremia and remained clinically normal 8 months postchallenge. In contrast, five of six nonvaccinates developed persistent viremia, four of six developed clinical SAIDS, and two of six died with SAIDS at 10 weeks and 8 months postchallenge, respectively. These results show that prevention of a common spontaneous retrovirus-induced immunosuppressive disease in macaques is now possible by vaccination. PMID- 3021979 TI - Conserved TAAATG sequence at the transcriptional and translational initiation sites of vaccinia virus late genes deduced by structural and functional analysis of the HindIII H genome fragment. AB - The sequence of the 8,600-base-pair HindIII H fragment, located at the center of the vaccinia virus genome, was determined to analyze several late genes. Seven major complete open reading frames (ORFs) and two that started from or continued into adjacent DNA segments were identified. ORFs were closely spaced and present on both DNA strands. Some adjacent ORFs had oppositely oriented overlapping termination codons or contiguous stop and start codons. Nucleotide compositional analysis indicated that the A-T frequency was consistently lowest in the first codon position. The sizes of the polypeptides predicted from the DNA sequence were compared with those determined by polyacrylamide gel electrophoresis of cell free translation products of mRNAs selected by hybridization to cloned single stranded DNA segments or synthesized in vitro by bacteriophage T7 RNA polymerase. Six transcripts that initiated within the HindIII H DNA fragment were detected, and of these, four were synthesized only at late times, one was synthesized only early, and one was synthesized early and late. The sites on the genome corresponding to the 5' ends of the transcripts were located by high-resolution nuclease S1 analysis. For late genes, the transcriptional and translational initiation sites mapped within a few nucleotides of each other, and in each case the sequence TAAATGG occurred at the start of the ORF. The extremely short leader and the absence of A or G in the -3 position, relative to the first nucleotide of the initiation codon, distinguishes the majority of vaccinia virus late genes from eucaryotic and vaccinia virus early genes. PMID- 3021980 TI - A single regulatory region modulates both cis activation and trans activation of the herpes simplex virus VP5 promoter in transient-expression assays in vivo. AB - A detailed analysis of the expression of the bacterial chloramphenicol acetyltransferase gene controlled by the herpes simplex virus major capsid protein (VP5) promoter showed that this promoter can be functionally separated into an 80-base core region, which has the minimal information required to serve as a pol II promoter but which is not fully activated by viral superinfection or by cotransfections with plasmids bearing functional alpha (immediate-early) genes, and an approximately 100-base regulatory region upstream of the core, which allowed full induction of VP5 promoter-driven chloramphenicol acetyltransferase activity but which repressed the ability of the VP5 core promoter to be cis activated by the simian virus 40 enhancer. This was in distinct contrast to the situation with the alkaline exonuclease promoter (a model early promoter) and defined the regions of this promoter which can be used to study the interaction between viral promoters and putative regulatory proteins induced by viral infection. PMID- 3021981 TI - Binding of complement component C3b to glycoprotein gC of herpes simplex virus type 1: mapping of gC-binding sites and demonstration of conserved C3b binding in low-passage clinical isolates. AB - The sites on glycoprotein gC of herpes simplex virus type 1 (HSV-1) which bind complement component C3b were evaluated by using anti-gC monoclonal antibodies and mutants which have alterations at defined regions of the glycoprotein. Monoclonal antibodies were incubated with HSV-1-infected cells in a competitive assay to block C3b binding. Each of 12 different monoclonals, which recognize the four major antigenic sites of gC, completely inhibited C3b binding. With this approach, no one antigenic group on gC could be assigned as the C3b-binding region. Next, 21 gC mutants were evaluated for C3b binding, including 1 which failed to synthesize gC, 4 which synthesized truncated forms of the glycoprotein such that gC did not insert into the cell's membrane, and 16 which expressed gC on the cell's surface but which had mutations in various antigenic groups. Eleven strains did not bind C3b. This included the 1 strain which did not synthesize gC, the 4 strains which secreted gC without inserting the glycoprotein into the cell membrane, and 6 of 16 strains which expressed gC on the cell surface. In these six strains, the mutations were at three different antigenic sites. One hypothesis to explain these findings is that C3b binding is modified by changes in the conformation of gC which develop either after antibodies bind to gC or as a result of mutations in the gC gene. Attachment of C3b to gC was also evaluated in 31 low-passage clinical isolates of HSV-1. Binding was detected with each HSV 1 isolate, but not with nine HSV-2 isolates. Therefore, although mutants that lack C3b binding are readily selected in vitro, the C3b-binding function of gC is maintained in vivo. These results indicate that the sites on gC that bind C3b are different from those that bind monoclonal antibodies, that antibodies directed against all sites on gC block C3b binding, and that C3b binding is a conserved function of gC in vivo. PMID- 3021982 TI - Isolation of a lentivirus from a macaque with lymphoma: comparison with HTLV III/LAV and other lentiviruses. AB - A retrovirus has been isolated on the human T-cell line HuT 78 after cocultivation of a lymph node from a pig-tailed macaque (Macaca nemestrina) that had died with malignant lymphoma in 1982 at the University of Washington primate center. This isolate, designated MnIV (WPRC-1) (M. nemestrina immunodeficiency virus, Washington Primate Research Center) shows the characteristic morphology of a lentivirus and replicates to high titers in various lymphocyte lines of human and primate origin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified MnIV revealed multiple bands of structural proteins, including a major viral gag protein of 28 kilodaltons, that did not comigrate with the viral proteins of a human immunodeficiency virus (HIV [FRE-1]) that was also isolated on HuT 78 cells. The relatedness of MnIV to other lentiviruses (HTLV-III/LAV, EIAV, and visna) was examined in radioimmunoassays, by immunoblot techniques, and by N-terminal amino acid sequence analysis of the viral p28 gag protein. The immunoassays revealed cross-reactivity only between MnIV p28 and HTLV-III/LAV p24, and sequence analysis showed that 14 of the 24 N-terminal residues of MnIV p28 and HTLV-III/LAV p24 are identical. These results indicate that MnIV belongs to the same lentivirus family as HTLV-III/LAV but is only partially related to these human acquired immune deficiency syndrome retroviruses. PMID- 3021983 TI - Reassortant rotaviruses containing structural proteins vp3 and vp7 from different parents induce antibodies protective against each parental serotype. AB - Genetic studies of reassortant rotaviruses have demonstrated that gene segments 4 and 9 each segregate with the serotype-specific neutralization phenotype in vitro. Reassortant rotaviruses derived by coinfection of MA-104 cells with the simian strain SA11 and the antigenically distinct bovine strain NCDV were used to determine which viral genes coded for proteins which induced a protective immune response in vivo. In addition, reassortant rotaviruses containing only the gene segment 4 or 9 protein products (vp3 and vp7, respectively) from SA11 or NCDV were used to determine the serotypic specificities of both vp3 and vp7 in several mammalian rotavirus strains. vp3 and vp7 from the murine strain Eb were shown to be indistinguishable from the corresponding proteins from strain SA11. Adult mice orally inoculated with strain Eb developed neutralizing antibodies to both vp3 and vp7. The two naturally occurring bovine rotavirus strains NCDV and UK were shown to contain antigenically similar vp7 but distinct vp3 proteins. Mouse dams orally immunized with a reassortant virus containing only gene 9 from NCDV passively protected their progeny against UK challenge, whereas mouse dams orally immunized with a reassortant virus containing only gene 4 from NCDV did not. Finally, we constructed reassortant viruses that immunized against rotaviruses of two distinct serotypes. SA11 X NCDV reassortants that contained vp3 and vp7 from different parents induced a protective immune response against both parental serotypes. vp3 and vp7 were independently capable of inducing a protective immune response after oral immunization. An understanding of the serotypic specificities of both vp3 and vp7 of human rotavirus isolates will be necessary for the development of successful strategies to protect infants against severe rotavirus infections. PMID- 3021984 TI - Differential transcription from the long terminal repeats of integrated avian leukosis virus DNA. AB - In avian leukosis virus-induced lymphoma and erythroblastosis, the expression of the proto-oncogenes c-myc and c-erbB is activated by downstream or readthrough transcripts initiated within integrated proviral DNA. To determine the relative abundance of viral RNAs extending into the downstream cellular sequences independently of the effects that may be exerted by specific sites of proviral integration, we examined the RNA of infected avian fibroblasts. Using a nuclease protection strategy to detect downstream, readthrough, and normal viral RNAs and to distinguish them from each other, we found that transcripts initiated within the 3' long terminal repeat, i.e., downstream transcripts, were undetectable in infected fibroblasts and could not have amounted to more than 1 to 2% of the total viral RNA. However, readthrough RNAs, which are transcripts initiated within the 5' long terminal repeat and extended beyond the viral polyadenylation site into the downstream cellular DNA, were present at relatively high levels, making up approximately 15% of the total viral RNA. PMID- 3021985 TI - Latent infection of KB cells with adeno-associated virus type 2. AB - Adeno-associated virus (AAV) is a prevalent human virus whose replication requires factors provided by a coinfecting helper virus. AAV can establish latent infections in vitro by integration of the AAV genome into cellular DNA. To study the process of integration as well as the rescue of AAV replication in latently infected cells after superinfection with a helper virus, we established a panel of independently derived latently infected cell clones. KB cells were infected with a high multiplicity of AAV in the absence of helper virus, cloned, and passaged to dilute out input AAV genomes. AAV DNA replication and protein synthesis were rescued from more than 10% of the KB cell clones after superinfection with adenovirus type 5 (Ad5) or herpes simplex virus types 1 or 2. In the absence of helper virus, there was no detectable expression of AAV specific RNA or proteins in the latently infected cell clones. Ad5 superinfection also resulted in the production of infectious AAV in most cases. All mutant adenoviruses tested that were able to help AAV DNA replication in a coinfection were also able to rescue AAV from the latently infected cells, although one mutant, Ad5hr6, was less efficient at AAV rescue. Analysis of high-molecular weight cellular DNA indicated that AAV sequences were integrated into the cell genome. The restriction enzyme digestion patterns of the cellular DNA were consistent with colinear integration of the AAV genome, with the viral termini present at the cell-virus junction. In addition, many of the cell lines appeared to contain head-to-tail concatemers of the AAV genome. The understanding of the integration of AAV DNA is increasingly important since AAV-based vectors have many advantages for gene transduction in vitro and in vivo. PMID- 3021986 TI - Molecular cloning and analysis of three cDNA clones homologous to human cytomegalovirus RNAs present during late infection. AB - Three virus-specific clones were isolated from a cDNA library synthesized from human cytomegalovirus (AD169)-infected cell RNA and cloned into the expression vector lambda gt11. These clones, designated C3, D10, and H10 were each found to express a human cytomegalovirus/beta-galactosidase fusion protein that was reactive with antibody prepared against purified virions. By using the cloned cDNA, we were able to identify the transcripts that code for each gene product and study the kinetics of expression during permissive infection. Our results suggest that at least two of the RNAs undergo posttranscriptional processing and appear in infected cells at immediate-early times. The authentic C3 gene product was identified by probing Western blots with antibody prepared against fusion protein fpC3. PMID- 3021987 TI - Persistence of vesicular stomatitis virus in cloned interleukin-2-dependent natural killer cell lines. AB - We have investigated virus-lymphocyte interactions by using cloned subpopulations of interleukin-2-dependent effector lymphocytes maintained in vitro. Cloned lines of H-2-restricted hapten- or virus-specific cytotoxic T lymphocytes (CTL) and alloantigen-specific CTL were resistant to productive infection by vesicular stomatitis virus (VSV). In contrast, cloned lines of natural killer (NK) cells were readily and persistently infected by VSV, a virus which is normally highly cytolytic. VSV-infected NK cells continued to proliferate, express viral surface antigen, and produce infectious virus. Furthermore, persistently infected NK cells showed no marked alteration of normal cellular morphology and continued to lyse NK-sensitive target cells albeit at a slightly but significantly reduced level. The persistence of VSV in NK cells did not appear to be caused by the generation of temperature-sensitive viral mutants, defective interfering particles, or interferon. Consequently, studies comparing the intracellular synthesis and maturation of VSV proteins in infected NK and mouse L cells were conducted. In contrast to L cells, in which host cell protein synthesis was essentially totally inhibited by infection, the infection of NK cells caused no marked diminution in the synthesis of host cell proteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of immunoprecipitates of viral proteins from infected cells showed that the maturation rate and size of VSV surface G glycoprotein were comparable in L cells and NK cells. Nucleocapsid (N) protein synthesis also appeared to be unaffected in NK cells. In contrast, the viral proteins NS and M appeared to be selectively degraded in NK cell extracts. Mixing experiments suggested that a protease in NK cells was responsible for the selective breakdown of VSV NS protein. Finally, VSV-infected NK cells were resistant to lysis by virus-specific CTL, suggesting that persistently infected NK cells may harbor virus and avoid cell-mediated immune destruction in an immunocompetent host. PMID- 3021988 TI - Identification of the major structural and nonstructural proteins encoded by human parvovirus B19 and mapping of their genes by procaryotic expression of isolated genomic fragments. AB - Plasma from a child with homozygous sickle-cell disease, sampled during the early phase of an aplastic crisis, contained human parvovirus B19 virions. Plasma taken 10 days later (during the convalescent phase) contained both immunoglobulin M and immunoglobulin G antibodies directed against two viral polypeptides with apparent molecular weights of 83,000 and 58,000 which were present exclusively in the particulate fraction of the plasma taken during the acute phase. These two protein species comigrated at 110S on neutral sucrose velocity gradients with the B19 viral DNA and thus appear to constitute the viral capsid polypeptides. The B19 genome was molecularly cloned into a bacterial plasmid vector. Restriction endonuclease fragments of this cloned B19 genome were treated with BAL 31 and shotgun cloned into the open reading frame expression vector pJS413. Two expression constructs containing B19 sequences from different halves of the viral genome were obtained, which directed the synthesis, in bacteria, of segments of virally encoded protein. These polypeptide fragments were then purified and used to immunize rabbits. Antibodies against a protein sequence specified between nucleotides 2897 and 3749 recognized both the 83- and 58-kilodalton capsid polypeptides in aplastic plasma taken during the acute phase and detected similar proteins in the tissues of a stillborn fetus which had been infected transplacentally with B19. Antibodies against a protein sequence encoded in the other half of the B19 genome (nucleotides 1072 through 2044) did not react specifically with any protein in plasma taken during the acute phase but recognized three nonstructural polypeptides of 71, 63, and 52 kilodaltons present in the liver and, at lower levels, in some other tissues of the transplacentally infected fetus. PMID- 3021989 TI - Quantitation, with a new assay, of Theiler's virus capsid protein in the central nervous system of mice. AB - We developed a quantitative assay for antigens at the single-cell level. Tissue sections were reacted with a primary antibody, a biotinylated secondary antibody, or 35S-streptavidin. Binding of streptavidin to cells was quantitated by microscopic autoradiography. We showed that the number of autoradiographic grains was proportional to the amount of antigen per cell. With this assay, we studied the synthesis of Theiler's virus capsid proteins VP1, VP2, and VP3 in permissive BHK cells grown in vitro and in mouse central nervous system (CNS) cells during a persistent infection. We found that synthesis of the three capsid proteins was restricted in mouse CNS cells. Restricted virus replication could play a major role in the persistence of Theiler's virus in mouse CNS cells. PMID- 3021990 TI - Metabolic activation of 9([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine in human lymphoblastoid cell lines infected with Epstein-Barr virus. AB - 9-([2-Hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U) is more potent and has a more prolonged inhibitory effect against Epstein-Barr virus (EBV) in vitro than does acyclovir (ACV). To assess the mechanism of this difference, we first compared the extent of phosphorylation of the two drugs in superinfected Raji cells. BW B759U is phosphorylated to levels 100-fold higher than is ACV. In addition, lower levels of phosphorylation of BW B759U and ACV were observed in uninfected Raji cells. Studies on the kinetics of formation of BW B759U triphosphate in superinfected Raji cells indicated that drug-phosphorylating activity was detected as early as 3 h after superinfection; this activity was steadily maintained for the first 7 h, followed by a burst of activity between 7 and 10 h and a doubling of phosphorylation between 10 and 25 h. During the superinfection cycle, the pool sizes of deoxyribonucleoside and ribonucleoside triphosphates were increased and reached their maxima at 10 h after infection. The maximal amount of triphosphorylated drug in a virus producer cell, P3HR-1 (LS), was obtained at 21 h after drug treatment. During long-term drug treatment, approximately 44 and 77% reduction in EBV genome copies per cell was observed on days 3 and 7, respectively. In a separate experiment, after treatment of P3HR-1 (LS) cells with BW B759U for 36 h, 4.2 pmol of BW B759U triphosphate per 10(6) cells was achieved. After the cells were released into drug-free medium, drug triphosphate was rapidly decreased to 11% of the original level in 1 day. Thereafter, the decrease was slow but steady, down to 0.22 pmol/10(6) P3HR-1 cells by 5 days. We calculated that 0.22 pmol of BW B759U triphosphate per 10(6) cells represents a cellular concentration of 0.22 microM, which is theoretically enough to inhibit EBV replication. This is based upon a comparison with the 50% effective dose of BW B759U (0.05 microM) for inhibition of genome replication and a Ki of 0.08 microM for BW B759U triphosphate inhibition of EBV DNA polymerase. PMID- 3021992 TI - Human cytomegalovirus: demonstration of permissive epithelial cells and nonpermissive fibroblastic cells in a survey of human cell lines. AB - To more clearly define the characteristics which render a cell permissive for human cytomegalovirus (HCMV), we screened a panel of human cell lines differing in morphology, ploidy, and extent of differentiation for the ability to sustain productive HCMV replication. Cells were exposed to HCMV at 5 to 20 PFU per cell and examined at 4 to 14 days postinfection to detect the production of infectious virus by a plaque assay and the assembly of progeny virions by electron microscopy. By these criteria, high-titer HCMV replication (10(6) to 10(7) PFU/ml) occurred in a well-differentiated, diploid, epithelial cell line, HCMC, which had been derived from normal human colonic mucosa. In contrast, all aneuploid human cell types proved to be nonpermissive, including a fibroblastic cell line designated HT-144. These results indicate that HCMV replication in cultures is not strictly limited to fibroblasts and conversely that not all human fibroblastic cells are permissive for HCMV. Nonpermissive cell types were further investigated by attempts to chemically induce HCMV replication. Treatment of nonpermissive cell types with 25 to 500 micrograms of 5-iodo-2'-deoxyuridine per ml prior to infection did not convert them to the permissive state. The implications of these findings for the possible mechanisms maintaining the nonpermissive state are discussed. PMID- 3021991 TI - Selective tropism of a neurotropic coronavirus for ependymal cells, neurons, and meningeal cells. AB - The ability of a neurotropic virus, mouse hepatitis virus type 3 (MHV3), to invade the central nervous system (CNS) and to recognize cells selectively within the brain was investigated in vivo and in vitro. In vivo, MHV3 induced in C3H mice a genetically controlled infection of meningeal cells, ependymal cells, and neurons. In vitro, purified MHV3 bound to the surface of isolated ependymal cells and cultured cortical neurons but not to oligodendrocytes or cultured astrocytes. MHV3 replicated within cultured cortical neurons and neuroblastoma cells (NIE 115); infected cultured neurons nonetheless survived and matured normally for a 7 day period postinfection. On the other hand, MHV3 had a low affinity for cortical glial cells or glioma cells (C6 line), both of which appear to be morphologically unaltered by viral infection. Finally, MHV3 infected and disrupted cultured meningeal cells. This suggests that differences in the affinity of cells for MHV3 are determinants of the selective vulnerability of cellular subpopulations within the CNS. In vivo, a higher titer of virus was needed for CNS penetration in the genetically resistant (A/Jx) mice than in the susceptible (C57/BL6) mouse strain. However, in spite of viral invasion, no neuropathological lesions developed. In vitro viral binding to adult ependymal cells of susceptible and resistant strains of mice was identical. Genetic resistance to MHV3-CNS infection appeared to be mediated both by a peripheral mechanism limiting viral penetration into the CNS and by intra-CNS mechanisms, presumably at a stage after viral attachment to target cells. PMID- 3021994 TI - Abelson murine leukemia virus variants with increased oncogenic potential. AB - A number of strains of Abelson murine leukemia virus (A-MuLV) with various abilities to transform cells have been identified. Among these is the A-MuLV-P90 strain, a mutant derived from A-MuLV-P120 that encodes an A-MuLV protein missing sequences that are normally present at the extreme carboxy terminus of P120 (N. Rosenberg and O. N. Witte, J. Virol. 33:340-348, 1980). This virus transforms NIH 3T3 cells efficiently but does not transform a high frequency of lymphoid cells in vitro or in vivo. In this communication, we show that of the relatively few tumors induced by A-MuLV-P90 nearly all contained new variant viruses that stably expressed either larger or smaller A-MuLV proteins. Strains that expressed larger A-MuLV proteins behaved like A-MuLV-P120 in transformation assays, whereas those expressing smaller A-MuLV proteins induced a high frequency of tumors after a short latent period in vivo but failed to transform large numbers of lymphoid cells in vitro. Thus, these latter viruses separated the requirements for in vitro transformation of lymphoid cells from those for tumor induction. All of the variants differed from A-MuLV-P90 in the carboxy-terminal region of the A-MuLV protein, suggesting that sequences in this region play a key role in the ability of the virus to interact with hematopoietic cells in vivo and in vitro. PMID- 3021993 TI - Nucleotide sequence of human endogenous retrovirus genome related to the mouse mammary tumor virus genome. AB - We determined the complete nucleotide sequence of the human endogenous retrovirus genome HERV-K10 isolated as the sequence homologous to the Syrian hamster intracisternal A-particle (type A retrovirus) genome. HERV-K10 is 9,179 base pairs long with long terminal repeats of 968 base pairs at both ends; a sequence 290 base pairs long, however, was found to be deleted. It was concluded that a composite genome having the 290-base-pair fragment is the prototype HERV-K provirus gag (666 codons), protease (334 codons), pol (937 codons), and env (618 codons) genes. The size of the protease gene product of HERV-K is essentially the same as that of A- and D-type oncoviruses but nearly twice that of other retroviruses. A comparison of the deduced amino acid sequences encoded by the pol region showed HERV-K to be closely related to types A and D retroviruses and even more so to type B retrovirus. It was noted that the env gene product of HERV-K structurally resembles the mouse mammary tumor virus (type B retrovirus) env protein, and the possible expression of the HERV-K env gene in human breast cancer cells is discussed. PMID- 3021995 TI - The promoter for the late gene encoding Vp5 of herpes simplex virus type 1 is recognized by cell extracts derived from uninfected cells. AB - The ability of whole-cell extracts from uninfected HeLa cells to recognize the promoter for the herpes simplex virus type 1 late gene encoding the major capsid protein Vp5 was investigated by using both in vitro transcriptional and S1 nuclease protection analysis. This gene promoter was recognized by the cell extracts and produced abundant amounts of transcript in the absence of any other virus-encoded factors. This transcript was shown to arise, in vitro, from specific initiation at or very near the physiological mRNA start site. Thus, it appears that cell extracts from uninfected HeLa cells can efficiently recognize both early- and late-gene promoters. PMID- 3021996 TI - Bovine papillomavirus type 1 3' early region transformation and plasmid maintenance functions. AB - We examined bovine papillomavirus type 1 (BPV-1) DNAs mutated in the E2 open reading frame (ORF) to determine their ability (i) to transform C127 cells and (ii) to remain extrachromosomal in transfected cells. Results obtained with deletion mutants and insertion mutants containing a linker with translational termination codons in all possible reading frames indicated that an E2 ORF gene product(s) is necessary for efficient transformation, as well as viral plasmid replication and maintenance in the context of the full BPV-1 genome. Complementation assays in which mutant BPV-1 DNAs were transfected into cell lines expressing some viral functions from integrated BPV-1 cDNAs demonstrated that the E2 ORF product, when provided in trans, could allow BPV-1 E2 mutants to remain extrachromosomal. The E2 function could also augment transformation of some, but not all, BPV-1 E2 mutants, allowing identification of another region of BPV-1 involved in cellular transformation. It is likely that the role of the BPV 1 E2 product(s) in transformation and plasmid maintenance is indirect. A BPV-1 mutant altered in the E5 ORF is transformation defective and unable to replicate as a stable plasmid in C127 cells. PMID- 3021997 TI - Multiple transforming regions of human cytomegalovirus DNA. AB - The transforming (focus forming) activity of defined cloned DNA fragments from human cytomegalovirus Towne and AD169 was carried out in immortalized rodent cells. The frequency of focus formation in NIH 3T3 cells by Towne XbaI fragment E was 80- to 100-fold higher than that observed with Towne XbaI fragments AO, O, C, or carrier DNA alone but was similar to that observed with pCM4127, a transforming fragment from HCMV AD169 (J. A. Nelson, B. Fleckenstein, D. A. Galloway, and J. K. McDougall, J. Virol. 43:83-91, 1982; J. A. Nelson, B. Fleckenstein, G. Jahn, D. A. Galloway, and J. K. McDougall, J. Virol. 49:109-115, 1984). Foci were first detected in Towne XbaI fragment E-transfected NIH 3T3 cells at 5 to 6 weeks posttransfection, whereas foci were detected at 2 to 3 weeks after transfection with AD169 pCM4127. Digestion of Towne XbaI fragment E with BamHI did not significantly reduce its focus-forming activity. When BamHI subclones of Towne XbaI fragment E were assayed individually for focus formation in NIH 3T3 and Rat-2 cells, transforming activity was localized within each terminal fragment (EJ and EM). Foci induced by EJ or EM DNA alone were smaller compared with those induced by Towne XbaI fragment E. Isolated focal lines exhibited growth in soft agar and were tumorigenic in immunocompetent syngeneic animals. High-molecular-weight DNAs from transformed and tumor-derived lines were analyzed by Southern blot hybridization with intact EM and a 1.5-kilobase subfragment lacking cell-related sequences. Virus-specific EM sequences were detected at less than one copy per cell in Towne XbaI fragment E-transformed NIH 3T3 cells and at multiple copies in rat tumor-derived cell lines. In contrast, virus-specific EJ sequences were barely detected in EJ-transformed and tumor derived lines with intact EJ as probe. PMID- 3021998 TI - Functional analysis of the adenovirus type 5 DNA-binding protein: site-directed mutants which are defective for adeno-associated virus helper activity. AB - We generated four point mutations in the DNA-binding protein (DBP) gene of adenovirus type 5 by oligonucleotide-directed site-specific mutagenesis. The sites mutated were in the three conserved regions (CR; amino acids 178-186 [CR1], 322-330 [CR2], and 464-475 [CR3]) identified previously by comparative sequence analysis (G. R. Kitchingman, Virology 146:90-101, 1985). The mutations resulted in changes in amino acids 181 (Trp to Leu), 323 (Arg to Leu), 324 (Trp to Leu), and 469 (Phe to Ile). The mutated DBP genes were put under the control of the simian virus 40 early promoter and analyzed by transfection for their ability to help adeno-associated virus replicate its DNA in COS-1 monkey cells. Mutations in the aromatic amino acids 324 and 469 reduced the amount of AAV DNA replication approximately 10-fold, while the mutation in Arg 323 produced a reduction of approximately fourfold. The Trp-to-Leu mutation in amino acid 181 had no effect on AAV DNA replication. The decreased helper activity of the 323, 324, and 469 mutations was not caused by any effect of the mutation on the stability of the DBP. These results suggest that CR2 and CR3 are involved in AAV helper activity, specifically in AAV DNA replication. The relevance of these findings to the identification of residues important for the functions of DBP in adenovirus infection is discussed. PMID- 3022000 TI - Humoral immune response of asymptomatic cats naturally infected with feline leukemia virus. AB - The humoral immune response of cats that were naturally infected with the feline leukemia virus (FeLV) was examined after antigenic stimulation with the synthetic antigen poly(L-Tyr, L-Glu)-poly(DL-Ala)-poly(L-Lys). The primary humoral antibody response in FeLV-infected cats was both delayed and greatly reduced, compared with that seen in uninfected control cats. A similar discordance was observed after secondary stimulation with the antigen, in the FeLV-infected cats had both a delayed response and a reduced response, compared with uninfected cats. The levels of total immunoglobulins of the immunoglobulin G and immunoglobulin M classes in the sera of FeLV-infected cats were significantly higher (two- and threefold, respectively) than were those of the uninfected control animals. The presence of an impaired humoral immune response to newly presented antigens in the presence of elevated immunoglobulin levels has been thoroughly documented in the case of people with the acquired immunodeficiency syndrome. This further emphasizes the potential value of FeLV-infected cats as a model for human acquired immunodeficiency syndrome. PMID- 3021999 TI - Pseudotyped retroviral vectors reveal restrictions to reticuloendotheliosis virus replication in rat cells. AB - Reticuloendotheliosis viruses (Rev) replicate in chicken and dog cells, but not in rat cells. Amphotropic murine leukemia viruses (Am-MLV) replicate in chicken, dog, and rat cells. Transcription from the Rev long terminal repeat, determined by the chloramphenicol acetyltransferase assay, was not significantly different from transcription from the MLV long terminal repeat in rat cells. To determine further the step(s) in the retroviral life cycle that is blocked for Rev replication in rat cells, we took advantage of the wide host range of Am-MLV (S. Rasheed, M. B. Gardner, and E. Chan, J. Virol. 19:13-18, 1976) and the ability to form Rev-Am-MLV pseudotypes. Data from these pseudotypes indicate that the block to Rev replication in rat cells is posttranscriptional. PMID- 3022001 TI - Construction and properties of a cell line constitutively expressing the herpes simplex virus glycoprotein B dependent on functional alpha 4 protein synthesis. AB - We report the construction of a cell line constitutively expressing the glycoprotein B (gB) of herpes simplex virus (HSV) 1. The cell line was constructed in two steps. In the first, a baby hamster kidney cell line was transfected with the DNA of a plasmid containing the neomycin phosphotransferase gene that confers resistance to the antibiotic G418 and the gene specifying a temperature-sensitive (ts-) alpha 4 protein of HSV-1, the major viral regulatory protein. A clonal cell line, alpha 4/c113, selected for resistance to the antibiotic G418, expressed high levels of alpha 4 protein constitutively. Superinfection of these cells with HSV-2 resulted in twofold induction of the resident HSV-1 alpha 4 gene. In the second step, alpha 4/c113 cells were transfected with the DNA of a plasmid carrying the gB gene and the mouse methotrexate resistance dihydrofolate reductase gene. A clonal cell line, alpha 4/c113/gB, selected for methotrexate resistance expressed gB constitutively. Expression of both gB and alpha 4 continued unabated for at least 32 serial passages. Cells passaged serially in medium containing both methotrexate and G418 after passage 10 contained a higher copy number of the alpha 4 gene and produced larger amounts of both gB and alpha 4 proteins than did cells maintained in medium containing methotrexate alone. Expression of gB was dependent on the presence of functional alpha 4 protein inasmuch as expression of gB ceased on shift up to nonpermissive temperatures, when shifted to permissive temperatures, the cell line reinitiated expression of gB after a delay commensurate with the length of incubation at the nonpermissive temperature, and the cell-resident HSV 1 gB gene was expressed at the nonpermissive temperature in cells infected with a recombinant expressing a ts+ alpha 4 protein and an HSV-2 gB. The properties of the alpha 4/c113 cell line suggest that it may express other viral genes induced by alpha 4 protein constitutively, provided that the product is not toxic to the cells. PMID- 3022002 TI - Genomic organization of the related Bacillus subtilis bacteriophages SPP1, 41c, rho 15, and SF6. AB - The genomes of the related virulent Bacillus subtilis bacteriophages SPP1, 41c, rho 15, and SF6 are partially circularly permuted and terminally redundant. Heteroduplex molecules were produced with various combinations of these DNAs. Their electron-microscopic analyses showed a consistent pattern of homologous and heterologous regions of DNA. Restriction maps of the phage DNAs were established. A comparison of these maps showed a pattern of conserved and variable DNAs compatible with the electron-microscopic analyses. In all phage genomes, regions specifying early and late functions were conserved. In each phage genome, such regions were separated by short segments of heterologous DNA characteristic for each phage. PMID- 3022004 TI - Reovirus type 1 is secreted into the bile. AB - Reovirus type 1, known to be a cause of systemic and intestinal disease in mice, is secreted into the bile of adult A/J mice after viremia. Virus found in the bile in concentrations higher than those in blood may indicate that reovirus type 1 is actively transported into the bile. The transport of virus was independent of levels of virus-specific immunoglobulin A antibody. Modifications of the virus that occurred during transport did not discernibly affect the infectivity of the virus. Entry of virus into the bile may be an important mechanism by which an enteric virus that produces systemic disease reenters the intestine for transmission. PMID- 3022003 TI - N protein is the predominant antigen recognized by vesicular stomatitis virus specific cytotoxic T cells. AB - The specificity of anti-vesicular stomatitis virus (VSV)-specific cytotoxic T cells was explored with cell lines expressing VSV genes introduced by electroporation. Low levels of nucleocapsid (N) protein were detected on the surface of VSV-infected cells, but N protein could not be detected on the plasma membrane of transfected EL4 cells. Intracellular N protein was detectable by enzyme-linked immunosorbent assay or immunoprecipitation in some of the transfected cell lines but not in others, unless the transfected genes were induced by sodium butyrate. However, all of the stably transfected EL4 cell lines expressing the VSV-Indiana N protein were efficiently lysed by serotype-specific and cross-reactive anti-VSV cytotoxic T cells (CTLs). Primary cross-reactive anti VSV CTLs appeared to be specific solely for N protein, based on cold-target competition assays using infected and transfected target cells. Cell lines expressing 100- to 1,000-fold less N protein than did VSV-infected cells were efficiently lysed by both primary and secondary anti-VSV CTLs. Cell lines expressing 100-fold less G protein than did VSV-infected cells were not lysed by either population of effectors. Significantly, cold-target competition studies with secondary CTLs demonstrated that N protein-expressing cell lines were more efficient competitors than were VSV-infected cells even though the latter expressed 100- to 1,000-fold more N protein. This was not an artifact of viral infection since infection of the transfected cell lines did not affect their ability to compete. The possibility that cell lines constitutively expressing internal virus proteins present antigen more effectively than infected cells do is discussed. PMID- 3022005 TI - A bovine papillomavirus type 1-encoded modulator function is dispensable for transient viral replication but is required for establishment of the stable plasmid state. AB - A bovine papillomavirus (BPV) type 1-encoded function (M) which is a negative regulator of viral plasmid replication has been described elsewhere (Berg et al. Cell, in press; Roberts and Weintraub, Cell, in press). We report here that expression of M, which is a repressor of transient BPV replication and is not required as a positive factor in these assays, is required for the establishment of the viral genome as a stable nuclear plasmid. This function is encoded in part by the 5' portion of the BPV E1 open reading frame, whereas the 3' part of this open reading frame encodes a positive replication function (R). The R function is required for early replication events. We used transient replication assays to define the phenotypes of mutants in both the R and M genes and complementation tests to show that R and M define two separate genes. We showed that R- and M- mutants could also complement each other in stable assays. In cotransfection experiments, M- mutants had a lethal effect on the growth of G418-resistant colonies, and in addition their morphological transformation efficiencies were reduced. The rare colonies which did appear contained the mutant DNA integrated into the cellular genome. R- mutants transformed with wild-type efficiency, and the mutant DNA was also found integrated. When cotransfected, R- and M- mutants could each be established as unrearranged plasmids. PMID- 3022006 TI - Structure of a human retroviral sequence related to mouse mammary tumor virus. AB - Human sequences that are related to the mouse mammary tumor virus (MuMTV) genome were cloned from breast tumor cell DNA. Of 100 recombinants, only 1 hybridized with two different probes from separate regions of the MuMTV genome (gag-pol and long terminal repeat [LTR]). This sequence, NMWV 4, was shown to have a proviruslike structure. Hybridization to digests of normal and tumor cell DNA indicated that NMWV 4 and a few closely related sequences are endogenous to the human genome. The regions that contain homology to the MuMTV LTR were sequenced. Long repeated sequences with the hallmarks of retroviral LTRs were identified. The NMWV 4 LTR contains transcription initiation and termination signals and is flanked by a polypurine tract (5' LTR) and a primer-binding site (3' LTR). The primer-binding site is complementary to tRNA lysine, the primer used by MuMTV and HTLV-III. The polypurine tract is also similar to those of these two retroviruses. PMID- 3022007 TI - Transduction of host cellular sequences by a retroviral shuttle vector. AB - We studied the frequencies and types of excision events which can occur with a retroviral shuttle vector containing the simian virus 40 origin of DNA replication. Analysis of the recloned vector plasmids by size and restriction enzyme mapping indicated that most contain one long terminal repeat. By hybridizing the plasmids to a mouse genomic repetitive DNA probe, we also determined that approximately 1 to 3% contain transduced cellular DNA sequences. PMID- 3022009 TI - Polyomavirus small T antigen enhances replication of viral genomes in 3T6 mouse fibroblasts. AB - Transfection of 3T6 cells with a cloned polyomavirus genome encoding only large T antigen resulted in DNA replication with only about 1/10 the efficiency of wild type viral DNA coding for all three T antigens. This replication defect was at least in part overcome by the simultaneous transfections of polyomavirus genomes which allowed the expression of small T antigen. We conclude that polyomavirus small T antigen has a (probably indirect) role in replication. PMID- 3022008 TI - Complex formation of simian virus 40 large T antigen with cellular protein p53. AB - We investigated the formation of native complexes between simian virus 40 large T antigen and the cellular protein p53 (T-p53) by using simian virus 40 tsA58 transformed mouse fibroblasts (tsA58 F2b). We observed that newly synthesized p53 bound to all structural subclasses of large T antigen detectable on sucrose density gradients. This led to various intermediates of T-p53 complexes which converted within 2 h into typical mature aggregates. The final levels of stable T p53 complexes seemed to be determined by p53 rather than by large T antigen. PMID- 3022010 TI - Molecular cloning of the temperature-sensitive 371 Kirsten murine sarcoma virus and expression in Escherichia coli of the mutant and wild-type viral Kirsten ras p21 proteins. AB - Rodent fibroblasts infected with the ts371 Kirsten murine sarcoma virus (KiMuSV) are temperature sensitive for the maintenance of transformation because of the production of an abnormal p21 protein. We cloned the ts371 KiMuSV provirus from the genome of a conditionally transformed nonproducer cell line, ts371 KiMuSV NRK clone 5 (T. Y. Shih, M. O. Weeks, H. A. Young, and E. M. Scolnick, J. Virol. 31:546-556, 1979). The molecularly cloned virus had 1,000-fold lower transformed focus-forming activity at 39 degrees C than at 34 degrees C. The ts371-v-Ki-ras gene differed from the wild type (wt) by a single point mutation, resulting in the substitution of arginine for glutamine at amino acid residue 43 of the encoded p21. A second difference from the published sequence for wt v-Ki-ras (N. Tsuchida, T. Ryder, and E. Ohtsubo, Science 217:937-939, 1982) at amino acid residue 37 was found. However, on sequencing the wt v-Ki-ras in this region, we found that it also contained a glutamate at residue 37. Preliminary characterization of bacterially expressed wt and ts371-v-Ki-ras p21 proteins is discussed. PMID- 3022011 TI - Cloning the complete rIIB gene of bacteriophage T4 and some observations concerning its middle promoters. AB - We cloned the intact T4 rIIB gene by joining plasmids carrying gene fragments. rIIB was expressed at a low level under control of the lac promoter, and the clone complemented rIIB mutants. We suspect that earlier attempts to clone the intact gene were unsuccessful because of transcription from T4 middle-mode promoters. These promoters are silent early in infection but are recognized when resident on a plasmid in an uninfected cell. PMID- 3022013 TI - Role of the inner protein capsid on in vitro human rotavirus transcription. AB - The inner protein shell of human rotavirus consists of a single polypeptide called VP6 which was removed from the single-shelled virus by treatment with CaCl2, leaving the viral core. The core thus obtained was unable to transcribe. However, the addition of a supernatant containing VP6 in the absence of Ca2+ restored the transcriptional activity. VP6 obtained from different electropherotypes and serotypes was able to restore transcriptional activity to homologous and heterologous cores. Viral cores obtained after incubation with purified VP6 had electron microscopic characteristics, polypeptide compositions, and transcription products similar to those of the single-shelled virus. The results suggested the successful in vitro reconstitution of the single-shelled virus. PMID- 3022012 TI - Production of guanidine-resistant and -dependent poliovirus mutants from cloned cDNA: mutations in polypeptide 2C are directly responsible for altered guanidine sensitivity. AB - cDNA fragments representing the region in polypeptide 2C containing mutations in a guanidine-resistant or -dependent mutant were cloned into the wild-type background of an infectious clone. Transfection of COS-1 cells with these plasmids yielded viruses that were either completely resistant to 2.0 mM guanidine hydrochloride or dependent on this concentration of drug for growth. PMID- 3022014 TI - Oligomerization of herpes simplex virus glycoprotein B. AB - Glycoprotein B (gB) specified by herpes simplex virus can be extracted from virions or infected cells in the form of detergent-stable, heat-dissociable oligomers. The composition of the oligomers and requirements for their formation were investigated. Evidence is presented that the faster-migrating forms of the oligomers are homodimers of gB. Dimerization was shown to occur within minutes of polypeptide synthesis and did not depend on glycosylation, the expression of other viral proteins, or virion morphogenesis. The multiple, electrophoretically distinct forms of gB dimers differ in extent or rate of N-linked oligosaccharide processing and also have other differences that influence electrophoretic mobility. PMID- 3022017 TI - Cytomegaloviral epididymitis in a patient with the acquired immune deficiency syndrome. AB - We report on a patient with cytomegaloviral epididymitis and the acquired immune deficiency syndrome. The diagnosis was suggested by epididymitis that was refractory to antibiotics, and by isolation of cytomegalovirus from the urine and semen. The definitive diagnosis was made with immunohistochemical staining for cytomegaloviral antigens in the epididymal ductal cells. Because of the ineffectiveness of antimicrobial agents in this disorder, epididymectomy is the current treatment of choice. PMID- 3022015 TI - Construction and properties of a viable herpes simplex virus 1 recombinant lacking coding sequences of the alpha 47 gene. AB - The five alpha genes, alpha 0, alpha 4, alpha 22, alpha 27, and alpha 47, are the first set of herpes simplex virus 1 genes to be transcribed and expressed in productively infected cells. We report here the construction of a viral recombinant from which all of the coding sequences of the alpha 47 gene were deleted. In addition to the alpha 47 protein, infected cell lysates did not contain detectable amounts of two polypeptide bands with apparent molecular weights of 18,000 and 21,000 which could be specified by a gene whose regulatory domain and 5' transcribed noncoding sequences overlap with the coding sequences of the alpha 47 gene. The alpha 47- virus grew as well as the wild-type parent virus in Vero, baby hamster kidney, and Rat-1 cell lines. PMID- 3022018 TI - Orchiectomy in advanced germ cell cancer following intensive chemotherapy: a comparison of systemic to testicular response. AB - A total of 16 patients with advanced germ cell cancer underwent initial chemotherapy that was followed by a delayed orchiectomy for an unrecognized primary in 3 and for life-threatening distant metastatic cancer in 13. Of these patients 13 had a complete and 3 had a partial remission at the time of the delayed orchiectomy. Of the former 13 patients 3 (23 per cent) had persistent viable tumor in the testis. To date all 3 patients have remained free of disease for more than 12, 20 and 30 months, respectively, without further therapy. One early relapse (1 month) was found in the remaining 10 patients with a complete remission and without viable disease in the testis. Of the 3 patients with a partial remission 1 had residual tumor in the testis and disease progressed despite further therapy. There was no evidence of tumor in the testis in the other 2 patients. These data document the presence of a differential response of germ cell tumors in the primary and metastatic sites. Post-chemotherapy orchiectomy for a suspicious primary tumor of the testis is necessary because of the risk of persistent primary disease. The post-chemotherapy pathological findings in the resected primary tumor do not reflect the systemic response. PMID- 3022016 TI - Fluctuation of simian virus 40 (SV40) super T-antigen expression in tumors induced by SV40-transformed mouse mammary epithelial cells. AB - Higher-molecular-weight forms of the simian virus 40 (SV40) large tumor antigen (T-Ag), designated super T-Ag, are commonly found in SV40-transformed rodent cells. We examined the potential role of super T-Ag in neoplastic progression by using a series of clonal SV40-transformed mouse mammary epithelial cell lines. We confirmed an association between the presence of super T-Ag and cellular anchorage-independent growth in methylcellulose. However, tumorigenicity in nude mice did not correlate with the expression of super T-Ag. In the tumors that developed in nude mice, super T-Ag expression fluctuated almost randomly. Cell surface iodination showed that super T-Ag molecules were transported to the epithelial cell surface. The biological functions of super T-Ag remain obscure, but it is clear that it is not important for tumorigenicity by SV40-transformed mouse mammary epithelial cells. Super T-Ag may be most important as a marker of genomic rearrangements by the resident viral genes in transformed cells. PMID- 3022019 TI - Synchronous presentation of nonseminomatous germ cell tumor and renal cell carcinoma: a case report. PMID- 3022020 TI - Renal failure obfuscates the diagnosis of Cushing's disease. PMID- 3022021 TI - [The mechanism of aldosterone secretion during partial gastrectomy]. PMID- 3022022 TI - [Phosphoenolpyruvate:sugar phosphotransferase systems in a strain of Lactobacillus casei subsp. casei]. PMID- 3022023 TI - [Monoclonal antibody against human HLA class II antigens. I. Analysis of sero reactive patterns]. PMID- 3022025 TI - [Four cases of male breast cancer including one synchronously combined with gastric cancer]. AB - Four men with primary breast cancer were seen between 1972 and 1985 at the Sasebo Municipal Hospital. They were admitted complaining of breast mass and/or bloody nipple discharge. There was no delay between the onset of symptoms and seeking medical advice. They had relatively early stages of disease (three patients had stage I and one had stage II). All patients were treated by modified radical or radical mastectomy. Histopathological study revealed ductal carcinomas and no lymph node metastasis in all patients. Multiple bone metastasis and death occurred in one case. One patient (61 years old) had two separate synchronous primary cancers of the breast and stomach, which is very uncommon. PMID- 3022024 TI - [Morphometric studies of blood vessels in human livers with hepatocellular neoplasms, with special reference to "tumor-related arteriopathies" of pre existing host arteries]. AB - Autopsy or surgical liver materials from 36 patients with hepatocellular carcinomas or adenomas were submitted to histometric treatment of the hepatic arteries supplying these tumors. Not only the medial smooth muscles were extremely hypoplastic in the arterioles contained in the tumors, but also the media of tumor-supplying extraneoplastic host arteries were significantly thinner than in the control, even in branches distant from the tumor, suggesting more or less lowered activities of blood flow regulation. Under these circumstances, dissecting injuries of host arterioles were frequently found to develop. Computer aided 3-D reconstruction visualized constant spatial transition from such "tumor related arteriopathies" to media-free intraneoplastic arterioles, showing their forerunning character preceding the development of typical "tumor vessels". PMID- 3022026 TI - [Splenic metastasis of hepatocellular carcinoma]. AB - It is known that hepatocellular carcinoma (HCC) frequently metastasises to various organs, but metastasis to the spleen is quite rare. We experienced two cases of splenic metastasis of HCC revealed by ultrasonography (US) or computed tomography (CT) and confirmed by autopsy. We report the morphological characteristics of the US or CT findings in these two cases. The first patient was a 68-year-old woman who was admitted with the chief complaint of abdominal fullness. US revealed a hypoechoic lesion in the liver and spleen. She died of hepatic failure about a month later, and splenic metastasis of HCC was confirmed by autopsy. The second patient was a 61-year-old man who was admitted with the chief complaint of swelling a cervical lymph nodes. US showed a mosaic pattern of a mass lesion in the spleen. The patient died of hepatic failure about two weeks later, and metastasis of HCC to the spleen was confirmed by autopsy. PMID- 3022027 TI - [Two adult cases of malignant hepatic mixed tumor]. AB - Two adult males, aged 20 and 47 years, suffered from malignant hepatic mixed tumor. The common initial symptom was low grade fever and a large palpable tumor in the right quadrant of the abdomen. Macroscopically, these tumors were huge and elastic firm in consistency. Microscopic findings confirmed that the tumor cells possessed totipotential cell characteristics. Therefore, it might be conceivable that these two cases were malignant hepatic mixed tumor originating from the hepatic premordial cell. PMID- 3022028 TI - Workshop on small cell lung cancer: biology, pathology and therapy. Tokyo, June 28, 1986. PMID- 3022029 TI - Oncogenes and a specific chromosomal abnormality associated with small-cell lung cancers. AB - Studies on oncogenes and a specific chromosomal abnormality involved in small cell lung cancers are reviewed. Amplification and/or expression of one of the three myc family oncogenes (c-myc, N-myc, L-myc) were found in 21 of 31 small cell lung cancers. However, oncogenes were not detected in DNAs of small-cell lung cancers by the NIH3T3 transfection assay. In addition, a specific deletion on the short arm of chromosome number 3 was often associated with small-cell lung cancers. These characteristics are quite different from those of non-small-cell lung cancers. PMID- 3022030 TI - Changes in cell characteristics due to culture conditions in cell lines from human small cell lung cancer. AB - Eight cultured cell lines were established from human small cell lung cancers. Every cell line showed the morphological and biochemical characteristics of small cell cancer. Changes in cell characteristics were observed in many of these cell lines when culture conditions were changed: "oat cell type" changed to "intermediate cell type" and vice versa when serum-free medium was changed to serum-supplemented medium; a deficiency of vitamin A in the medium caused a change to squamous cells and vice versa; and a tumor promoter (teleocidin B) enhanced the adherence of these cells to the surface of plastic culture dishes. These findings provide evidence that many small cell lung cancer cell lines can change their morphology with changes in the environment of the cells. PMID- 3022031 TI - Biological and clinical implication of neuron-specific enolase and creatine kinase BB in small cell lung cancer. AB - The specificity of neuron-specific enolase (NSE) and creatine kinase BB (CK-BB) for small cell lung cancer (SCLC) was determined by biological and immunohistochemical procedures in lung cancer tissues and cultured cell lines. Average values of extractable NSE and CK-BB of SCLC tissues were significantly higher than those of non-SCLC and normal lung tissues. A large amount of NSE and CK-BB was demonstrated in SCLC cell lines. Immunohistochemical examination showed positive staining for NSE and CK-BB in most cases of SCLC and in a few cases of non-SCLC. From these data NSE and CK-BB should be considered to be highly specific for SCLC. In a clinical study serum values exceeding 10 ng/ml for NSE and 1.5 ng/ml for CK-BB were set as positive for the enzymes. Positive rates in SCLC were 71.4% for NSE and 65.3% for CK-BB, which were significantly higher than those in non-SCLC. All positive cases were in an advanced stage. Consecutive daily NSE determinations during induction chemotherapy showed transient elevation immediately after the initiation of drug administration (tumor lysis syndrome), followed by a decline to normal range in responders. This phenomenon seems to indicate tumor sensitivity to cytotoxic drugs. NSE positive non-SCLC was as sensitive to cytotoxic drugs as SCLC. These findings indicate that lung cancer with elevated serum NSE and CK-BB levels at diagnosis should be strongly suspected of being SCLC in the advanced stage. PMID- 3022032 TI - Peptide hormone production in small cell lung carcinomas with particular reference to gastrin-releasing peptide. AB - Tissues of 50 small cell lung carcinomas were examined for production of 17 peptide hormones. Only when the concentration of a peptide detected in the tumor was 10 pmol or more per g wet weight, was the peptide considered to be produced by the tumor. The frequency of production of at least one of these peptide hormones was 84%, and that of two or more hormones was 50%. These results indicate that peptide hormone production is a very common phenomenon in small cell lung carcinoma. Of the peptide hormones examined, gastrin-releasing peptide is produced with the highest frequency, suggesting that this peptide could play an important role in small cell lung carcinoma. PMID- 3022034 TI - Chemosensitivity test for human small cell lung cancer cell lines in vitro. AB - The in vitro response to seven chemotherapeutic drugs of three established human small cell lung cancer (SCLC) cell lines (NCI H69, H128, N231) was tested by a double soft agar clonogenic assay. Colony formation by the three cell lines was universally reduced more than 50% by continuous exposure to peak plasma concentrations of all the drugs. However by exposure to one-tenth of the peak plasma concentrations, the colony growth of H69 was reduced to 25.6% and 37.7% by etoposide and teniposide, respectively, and that of N231 was reduced to 46.7%, 39.0%, 27.5% by carboplatin, etoposide and teniposide, respectively. On the other hand colony formation by the three cell lines was not suppressed more than 50% by one-hour exposure to any of the drugs tested at one-tenth of the peak plasma concentrations. By one-hour exposure to drugs at the peak plasma concentrations, colony formation by H69, H128 and N231 was reduced more than 50% by cisplatin, etoposide, teniposide and nimustin, by adriamycin, teniposide and ACNU, and by adriamycin, etoposide, teniposide and nimustin, respectively. It was concluded that these three cell lines have similar sensitivity to seven drugs commonly used against small cell lung cancer. PMID- 3022033 TI - Diagnostic and therapeutic applications of monoclonal antibodies to small cell carcinoma of the lung. AB - For diagnosis and treatment of small cell lung cancer (SCLC), we have developed two different types of monoclonal antibodies, one of which is useful for specific diagnosis of SCLC. The other has been used for purging tumor cell infiltrates from bone marrow in high-dose chemotherapy followed by autologous bone marrow rescue. The monoclonal antibody, TFS-4, is highly specific for SCLC. It reacts with SCLC but is unreactive with squamous cell carcinoma or adenocarcinoma of the lung. It can distinguish SCLC from non-SCLC in histologic diagnosis at transbronchial biopsy, and in cytological identification of tumor cells in malignant effusions and in infiltrated bone marrow. Radiolabeled TFS-4 specifically accumulated into SCLC tumors and depicted a clear-demarcated tumor image with a gamma scintillation camera. The TFS-4 labeled with 131I inhibited the growth of SCLC tumors transplanted into nude mice. TFS-2 antibody demonstrated "pancarcinoma" reactivity, showing binding to SCLC, non-SCLC, and carcinomas derived from other organs such as the colon, pancreas, and stomach. This antibody failed to react with a variety of normal tissues including lung, bone marrow, spleen, stomach, colon, and pancreas. TFS-2 showed complement mediated cytolysis of target cells. TFS-2 did not inhibit the growth of hemopoietic progenitors committed to granulocyte-macrophage, or erythroid lineage. In mixed cell populations of tumor cells and bone marrow cells, the antibody effectively killed the tumor cells. PMID- 3022035 TI - Alternating non-cross resistant chemotherapy for small cell lung cancer. AB - After stratification for the extent of disease, previously untreated patients with small cell lung cancer randomized to receive therapy with the four-drug combination of cyclophosphamide, oncovin, nimustine hydrochloride (ACNU), and procarbazine (CONP) every four weeks (continuous regimen) or to receive CONP alternating with the three-drug combination of etoposide (VP-16), adriamycin and cisplatin (VAD) at four-week intervals (alternating regimen). Sixty-nine patients were entered in the study. Of 34 evaluable patients receiving the continuous regimen, six (17.6%) achieved complete response (CR) and 16 (47.1%) achieved partial response (PR). Of 31 evaluable patients receiving the alternating regimen, 10 (32.3%) achieved CR, and 16 (51.6%) achieved PR. There was a tendency in favor of the alternating regimen in CR and over-all response rates (0.05 less than p less than 0.1). There were no significant differences between the regimens in response duration or survival. The projected median survival times were 9.2 months and 9.4 months for the continuous and alternating regimens, respectively. One patient receiving the continuous regimen and three receiving the alternating regimen have been living for more than two years. The major toxicity was myelosuppression in both regimens. One patient died of hemorrhage due to thrombocytopenia during induction with CONP, and one patient died of cisplatin induced renal failure. We conclude that alternating non-cross resistant chemotherapy leads to improved CR and response rates, but does not improve survival. PMID- 3022036 TI - Randomized trial comparing chemotherapy alone and chemotherapy plus chest irradiation in limited stage small cell lung cancer: a preliminary report. AB - In order to assess the effectiveness of chest irradiation in addition to intensive chemotherapy in limited stage small cell lung cancer, 50 patients were randomized to receive either chemotherapy alone or chemotherapy plus chest irradiation, between April 1981 and October 1985. The chemotherapy regimen consisted of a four-drug combination of cyclophosphamide, vincristine, methotrexate, and procarbazine, and a three-drug combination of etoposide, adriamycin, and nimustine, given alternately every 8 weeks. One group of 26 patients received the chemotherapy alone, and another group of 24 patients received chest irradiation with 40 Gy between cycles 1 and 2 of the chemotherapy. Complete response rates were quite similar in the two groups; 50% for those receiving chemotherapy alone, and 59% for those receiving chemotherapy plus chest irradiation. There were no significant differences in median survival (15 months versus 12 months) and in long-term survival rates between the two groups with a median follow-up period of 26 months. The combined modality treatment was more toxic than chemotherapy alone; two patients receiving such treatment died of radiation pneumonitis. It is concluded that chest irradiation combined with chemotherapy does not affect the response rate, survival, or pattern of recurrence in patients with limited stage small cell lung cancer. PMID- 3022037 TI - The role of surgical resection in the management of small cell carcinoma of the lung. AB - To assess the role of surgical resection in the management of small cell carcinoma of the lung, experience with 118 patients who were treated between 1973 and 1985 was reviewed. Twenty-five patients underwent surgical resection followed by combination chemotherapy in all except one. The remaining 93 patients were treated by combined chemotherapy and radiation therapy. The 5-year survival rate for patients with stage I disease undergoing surgical resection was 50.8%. For all 25 patients operated on, the 5-year survival rate was 30.7%. In the patients not operated on, only those with complete response had long-term survival, for whom the 5-year survival rate was 11.9%. We consider that surgical resection is definitely indicated in patients with stage I disease. If the response to initial chemotherapy is very good, patients with stage II or T3N0M0 disease also probably should receive resection. Patients with N2 disease are not candidates for resection, unless distant metastases are controlled completely by intensive chemotherapy. PMID- 3022038 TI - A clinico-pathological study of surgical treatment for small cell carcinoma of the lung. AB - Thirty-seven patients with histologically confirmed small cell carcinoma (SCLC), who underwent surgical resection at the National Cancer Center Hospital between 1963 and 1983, were reviewed. They were divided into two groups, 25 patients who were operated on between 1963 to 1979 and 12 who were operated on between 1980 and 1983. When these two groups were compared, a significant difference in 5-year survival was found (8% vs 50%). An accumulation of various factors including adjuvant chemotherapy was considered to contribute to the improvement in survival. After carefully analyzing these factors, we have come to the conclusion that adjuvant chemotherapy was the most important factor among them. An additional six patients with SCLC, who were operated on in 1984 and 1985, were also studied. They were either those who were given an adequate dose of combination chemotherapy before surgical resection or those whose local carcinoma which recurred after complete response was achieved by chemotherapy and/or chest radiation was surgically removed. In two cases, a tumor-like mass which was clearly visible on X-ray film and in the surgeon's hand at the time of thoracotomy revealed a histopathological "cure." In another two cases, tissue diagnosis of SCLC which was obtained without thoracotomy before chemotherapy and/or radiation was started was reported as NSCLC after the resected specimen was histo-pathologically examined. In both of them, the cancer tissue was made up of NSCLC of small cell type. A discrepancy between clinical TNM after treatment and pathological TNM was noted in two cases. Microinvasion and micrometastases, which were the reasons for the discrepancy, are considered to be a core of eventual recurrence following induction of complete response. PMID- 3022039 TI - Long-term survival in patients with small cell lung cancer. AB - Of the 66 patients with small cell lung cancer (SCLC) who were treated by combination chemotherapy with or without radiation therapy from July 1978 to December 1983 at the National Cancer Center Hospital, Tokyo, 12 (18%) survived over two years and nine (14%) have remained disease-free over three years. The two-year survival rates were compared according to the patient characteristics of sex, performance status (PS), extent of disease, histologic subtype, regimen of the initial chemotherapy and response to treatment. Sex, extent of disease and response to the initial chemotherapy were the most important prognostic factors. The prognosis for patients with liver or bone metastasis was poor. All disease free survivors, except for two patients who were treated by surgical resection after chemotherapy, achieved complete response (CR) with chemotherapy with or without radiation therapy. Eleven of the 12 two-year survivors achieved CR. Because of the small number of patients in our study, it will be necessary to evaluate further the influence of prognostic factors in patients with SCLC in future studies. PMID- 3022040 TI - [Liver function tests: analysis of HAV antigen and antibody]. PMID- 3022041 TI - [Magnetic resonance imaging of hepatoma]. PMID- 3022042 TI - [67Ga-scintigraphy in patients with primary and secondary hepatic malignant neoplasms]. PMID- 3022043 TI - [Measurement of serum angiotensin-converting enzyme in pneumoconiosis--comparison of Kasahara's method with Cushman's modified method]. PMID- 3022044 TI - Antitumor, antiviral and natural killer-inducing activities of human recombinant interferon-alpha in the guinea pig. AB - It was found that human recombinant interferon-alpha (Hu rIFN-alpha) was biologically active in the guinea pig. Namely, intratumor injection of either Hu rIFN-alpha A or A/D regressed tumors and prevented metastasis of guinea-pig line 10 tumor. These rIFNs augmented splenic natural killer activity of the guinea pig and reduced the cytopathic effect of virus on guinea-pig cells. Hu IFN-gamma showed no such activity. PMID- 3022045 TI - [Relationship between serum myo-inositol and peripheral neuropathy in patients with chronic renal failure]. PMID- 3022047 TI - Increase in membrane resistance during noradrenaline-induced depolarization in arterial smooth muscle. AB - The effects of noradrenaline on membrane resistance of smooth muscle cells of the guinea-pig main pulmonary artery were assessed from the change in amplitude of electronic potentials produced by extracellularly applied current pulses. Noradrenaline was applied exogenously by bath application or endogenously by stimulating perivascular nerves. Exogenous noradrenaline depolarized the smooth muscle membrane with an associated increase in amplitude of electrotonic potentials and produced spike responses. Perivascular nerve stimulation evoked an excitatory junction potential with slow time course. The amplitude of electrotonic potential was slightly decreased during generation of the excitatory junction potential, but the reduction was less than that observed during current induced depolarization. These observations suggest that the depolarizations produced by either endogenous or exogenous noradrenaline are accompanied by an increase in membrane resistance of arterial smooth muscle. PMID- 3022046 TI - Clinical significance of blood cell differentiation. PMID- 3022049 TI - Head-twitches induced by p-hydroxyamphetamine in mice. AB - Head-twitches have been regarded as an experimental model of hallucination, and we have recently observed that p-hydroxyamphetamine (p-OHA) markedly induced head twitches in mice. The present work was undertaken to study possible participation of a serotonergic system in the mechanism of head-twitches induced by p-OHA. Head twitches induced by p-OHA continued for 20-80 min, and the peak time of this effect was approximately 30-40 min after the administration. The i.c.v. administration of p-OHA (20, 40, 80 and 160 micrograms/mouse) produced characteristic head-twitches in a dose-dependent manner. Simultaneous injection of serotonin (10 micrograms/mouse, i.c.v.) and p-OHA caused a 2-2.5-fold increase in the number of head-twitches compared with non-serotonin controls. Pretreatment with p-chlorophenylalanine (200 mg/kg, i.p. and 500 micrograms/mouse, i.c.v.), in contrast, reduced head-twitches as did the pretreatment with cyproheptadine or dimethothiazine. These results suggest that p-OHA-induced head-twitches may involve the central serotonergic system which may exert an excitatory effect on head-twitches. PMID- 3022048 TI - Effects of prostaglandin D2 on the activity of hypothalamic neurons in the rat. AB - The effects of electrophoretically applied prostaglandin D2 (PGD2) on neuronal activity in the rat lateral preoptic area (LPOA) and posterior hypothalamic area (PHA) were examined. In the LPOA, 20% of the tested neurons were excited, 26% inhibited, and 6% showed bidirectional response. The direct effects often showed desensitization after repeated applications. Neurons excited by PGD2 were significantly sensitive (excitation) to acetylcholine (ACh). The ACh excitatory effect was sometimes (38%) attenuated, blocked, or reversed by concurrent PGD2 application. Excitatory or inhibitory effect of noradrenaline (NA) was not related to the effects of PGD2; however, modulation of the NA responses by PGD2 was common (58%). Inhibition, the predominant NA response, was changed to no effect or to excitation during simultaneous PGD2 application. Changes of the NA responses from inhibition to excitation, or from excitation to inhibition excitation sequences were observed after PGD2 infusion into the third cerebral ventricle at low concentrations. In 43% of the cells, neurotransmission in the LPOA following ventral noradrenergic bundle stimulation was modified by PGD2 application. PGD2 application tended to reduce the duration of inhibition and to extend that of excitation. The direct effects of PGD2 in the PHA were similar to those in the LPOA. Desensitization was also observed in the PHA, but no interrelations were observed among the effects of PGD2, ACh, and NA. Modulation of ACh and NA responses by PGD2 was rarely seen in the PHA. Possible contributions of PGD2 to sleep and thermoregulation are discussed. PMID- 3022050 TI - A difference in alpha-adrenoceptor mechanisms in vasa deferentia isolated from young and old rats. AB - A difference in alpha-adrenoceptor mechanisms in vasa deferentia isolated from 3 weeks old and 40 weeks old rats were studied by analysis of the concentration response curve of norepinephrine and Scatchard plot of specific [3H]-prazosin binding to microsomal fractions. The efficacy of norepinephrine and capacity of the maximum binding sites of [3H]-prazosin estimated in 40 weeks old rats were larger than those in the 3 weeks old rats, while there was no difference in the affinity of norepinephrine estimated in the 3 weeks old rats and in the 40 weeks old rats. The increase of efficacy for norepinephrine in the vas deferens from the 40 weeks old rats is due to the increase in the total concentration of alpha adrenoceptors. PMID- 3022051 TI - Effects of 4-aminopyridine on the fade response to vagal nerve stimulation in the isolated dog atrium. AB - Intramural parasympathetic nerve stimulation (5 Hz, 2 min) after propranolol treatment evoked negative chronotropic and inotropic responses in the isolated dog atrium. The cardiac responses were not maintained during stimulation; they reached maximum values and then faded back toward their control values ("fade" response). 4-Aminopyridine increased the maximum values of both responses, and it increased the fade of the chronotropic response but not that of the inotropic response. These results suggest that the fade response to parasympathetic nerve stimulation is modulated at the parasympathetic nerve terminals of the isolated dog atrium. PMID- 3022052 TI - Peroxidase-antiperoxidase staining for estrogen and progesterone in scirrhous type of gastric cancer: possible existence of the estrogen receptor. AB - Tissue specimens from patients with the scirrhous type of gastric carcinoma were stained using the peroxidase-antiperoxidase (PAP) method. Nine out of thirty seven specimens (24 per cent) showed positive estrogen staining, and here tissues from male or older patients were usually stained. Cumulative survival rate in patients whose tissue showed a positive estrogen staining was higher than that in case of a negative estrogen staining. Four out of thirty-one specimens (13 per cent) stained positively for progesterone, all four patients being male. These results suggest that estrogen and progesterone may relate to the growth of the scirrhous type of gastric cancer. PMID- 3022053 TI - [A case of mixed dust fibrosis with lesions akin to idiopathic interstitial pneumonia]. PMID- 3022054 TI - Proliferative rates of cloned malignant mammary epithelial cells as a measure of clonal heterogeneity in human breast carcinomas. AB - Malignant mammary epithelial cells (MMECs) were isolated from 8 human breast carcinomas and 1 adenoma as single cells or organoids and established in vitro. Depending on the cellularity of the tumor, between 9 X 10(4) and 4 X 10(6) cells were released per gram of tumor tissue. With the use of conditioned media and growth-promoting agents, a high proportion of cells (ranging from 0.5 to 11.4%) could be established in culture. A high degree of tumor cell heterogeneity in breast carcinomas was suggested by the observation that significantly different proliferative rates were found for 50 mammary epithelial cells cloned from 2 different tumors during the first subpassage in vitro prior to significant expansion of the cell colonies. The computed doubling times of these clones varied between 16 hours and more than 48 hours. The mammary epithelial nature of the cells was confirmed by their surface reactivity with monoclonal antibodies specific for MMECs. Studies on clones from 2 tumors revealed a positive correlation between proliferative rates of MMECs, their lactate production, and specific proteins synthesized as analyzed by two-dimensional macromolecular protein maps. PMID- 3022055 TI - Discrimination of human small cell and non-small cell lung tumors by a panel of monoclonal antibodies. AB - The immunohistochemical reactivity of biopsy specimens from different human lung tumor cell types was examined with 4 human small cell lung carcinoma (SCLC) reactive monoclonal antibodies (MoAbs) (SCLC 1096, SCLC 2051, SCLC 3121, and SCLC 5023) generated in this laboratory, with the use of Formalin-fixed, paraffin embedded tissue sections. Each MoAb reacted preferentially with SCLC tumors (62 97% of 29 cases tested), with low-to-moderate cross-reactivity with non-SCLC tumors (SCLC 1096, 13.7%; SCLC 2051 and SCLC 3121, 9.8% each; SCLC 5023, 41%, of 51 cases tested). None of the 4 MoAbs reacted with nonmalignant lung biopsy specimens. SCLC tumors were characterized by their collective immunoperoxidase reactivity with the MoAb panel of SCLC 2051, SCLC 3121, and SCLC 5023. Based on unequivocal positive reactions with at least 2 of the 3 MoAbs, this MoAb panel correctly identified 97% (28/29) of SCLC cases of 80 lung tumor cases tested. By contrast, only 4 of 51 (7.8%) non-SCLC cases were positive by the same criterion. These observations suggest that immunohistochemical analysis with these SCLC reactive MoAbs could help in discriminating small cell from other human lung carcinoma cell types. PMID- 3022056 TI - Effects of cyclosporin A and alpha-difluoromethylornithine on the growth of hamster pancreatic cancer in vitro. AB - The growth and survival of hamster H2T pancreatic ductal adenocarcinoma in vitro are known to be significantly reduced by inhibitors of polyamine biosynthesis. alpha-Difluoromethylornithine (alpha-DFMO) is a specific and irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis. alpha-DFMO treatment inhibits the growth of H2T pancreatic cancer cells and decreases H2T cell survival in vitro and in vivo. In the present study, the effects of cyclosporin A (CsA) were examined on growth, survival, and polyamine levels in H2T pancreatic ductal adenocarcinoma in vitro. CsA had inhibitory effects on H2T pancreatic cancer growth similar to those of alpha DFMO; these effects were blocked by the addition of the polyamine putrescine. Polyamine levels were found to be significantly altered in cells treated with CsA and/or alpha-DFMO. The combination of CsA (8.3 X 10(-4) mM) and alpha-DFMO (0.5 mM or 1.0 mM) inhibited H2T cell survival to a greater extent than either agent alone. These results suggest that CsA in combination with other agents that inhibit polyamine synthesis may be useful for the treatment of pancreatic cancer. PMID- 3022058 TI - Reversible suppression of mouse mammary tumor virus replication by prolonged glucocorticoid exposure. AB - Expression of mouse mammary tumor virus (MuMTV) in MJY-alpha mammary tumor cells was only transiently stimulated by exposure to 14 microM hydrocortisone (HC). Short-term HC treatment for 24-48 hours resulted in twofold-to-fivefold increases in levels of MuMTV polypeptide synthesis, MuMTV surface antigen expression, and MuMTV production. HC treatment also induced quantitative alterations in glycosylation of MuMTV precursors Pr79env and Pr76env. In contrast, prolonged exposure to 14 microM HC for 14-21 days decreased MuMTV polypeptide synthesis, surface antigen expression, and virion production to levels similar to untreated MJY-alpha cells. The pattern of MuMTV precursor glycosylation following prolonged HC treatment was identical to that detected in unexposed control cultures. Attenuation of glucocorticoid-mediated MuMTV stimulation was reversed either by exposure of treated cells to elevated (28 or 60 microM) concentrations of HC or by intermediate passage of treated cells in HC-free medium, suggesting additional levels of control of MuMTV replication. PMID- 3022057 TI - Biological activity of Pityrosporum. II. Antitumor and immune stimulating effect of Pityrosporum in mice. AB - The antitumor activity of Pityrosporum (P. orbiculare, P. ovale, P. pachydermatis, and Pityrosporum sp.) on Ehrlich ascites carcinomas (EACs) implanted into outbred ICR mice was studied. Pityrosporum significantly prolonged the survival of mice, regardless of the administration mode. In the case of P. orbiculare, the maximum survival time was 32.3 days on the mean and was obtained by injection ip of 1 mg (dry weight) P. orbiculare for 7 consecutive days following inoculation of the tumor cells. In contrast, the mean survival time of the nontreated mice was 14.9 days. For the investigation of the mechanisms of this antitumor activity, an examination was done on the ability of intracellular killing of Salmonella typhimurium and oxygen intermediate release by Pityrosporum, as elicited by mouse peritoneal exudate cells (PEC) or mouse peritoneal macrophages (PM). With about 40-minute incubation, 60-80% of S. typhimurium phagocytized by Pityrosporum elicited PEC or PM or were killed. The amounts of superoxide released from Pityrosporum-elicited cells were 1.5 times higher than those of P. acnes-elicited ones. Furthermore, three serum proteins (LA, LB, and LC), which closely related to the anti-tumor activity of immunomodulators, increased in the mice given Pityrosporum. These results indicated that the better survival rate seen in the case of Pityrosporum administration to mice with an implanted EAC may relate to the potent activation of phagocytes and to the increase in serum proteins LA, LB, and LC. PMID- 3022059 TI - Plaque assay and neutralization test for porcine transmissible gastroenteritis virus in cultures of swine cell lines. PMID- 3022061 TI - Intracellular regulatory cascades: examples from parathyroid hormone regulation of renal phosphate transport. AB - The knowledge about intracellular regulatory cascades in hormone action has increased considerably over the last few years. Receptor occupation at the plasma membrane level results in a production of intracellular messengers, such as cyclic nucleotides (cAMP, cGMP), inositoltrisphosphate (IP3), diacylglycerol (DAG) and a rise in cytosolic calcium concentration. These messengers control the activity of different regulatory mechanisms which operate either in sequence or in parallel to generate the final biological response. In PTH-dependent regulation of renal phosphate transport, cAMP-dependent and calcium-dependent mechanisms are involved: Recent experiments with cultured renal epithelial cells have confirmed that activation of adenylate cyclase is the initial event. However, the cAMP signal can be bypassed and direct activation of protein kinase C seems to mimic PTH induced inhibition of phosphate transport. The final event in the regulatory cascade is most likely a removal of the phosphate transport system followed by a degradation. PMID- 3022063 TI - Comparison of serological tests for detection of antibodies to Sendai virus in rats. AB - The sensitivities of the enzyme-linked immunosorbent assay, complement fixation and hemagglutination inhibition tests for detection of serum antibodies to Sendai virus were compared. Virus antibody-free, 4-week-old, female Fischer 344 rats were inoculated intranasally with either egg-propagated Sendai virus or a noninfectious egg control preparation. None of the rats became ill clinically or developed pulmonary lesions characteristic of Sendai virus infection. The enzyme linked immunosorbent assay was the most sensitive of the three tests, particularly in detection of early antibodies to Sendai virus and detection of small amounts of antibody. PMID- 3022060 TI - [The significance of eicosanoids in glomerular diseases]. AB - Prostanoids are local cyclooxygenase products, synthesized by mesangial and epithelial cells of the glomerulus as well as by a variety of inflammatory cells and platelets. Prostaglandins and thromboxane have direct vasodilatory and vasoconstrictory effects and can modulate glomerular function. Arachidonic acid, the main substrate for cyclooxygenase, can also be metabolized by the lipoxygenase pathway to leukotrienes, substances which are primarily synthesized in inflammatory cells. In several models induction of immunologic glomerular injury is associated with an increased glomerular formation of cyclooxygenase and lipoxygenase products. The changes in cyclooxygenase products have been shown to account for some hemodynamic changes found in some of these models. Increased renal prostanoid formation is also present in patients with glomerular disease. There is some evidence that increased renal PG-formation in patients with moderate glomerular disease regulates GFR and mediates proteinurie in some of these patients. Leukotrienes are chemotactive substances which modulate the function of inflammatory cells, stimulate the growth of mesangial cells, and constrict mesangial cells in culture. Thus, these compounds might be mediators in the induction of immune mediated glomerular disease. PMID- 3022064 TI - Characterization of membrane vesicles circulating in the serum of patients with common acute lymphoblastic leukemia. AB - Common acute lymphoblastic leukemia antigen detected by radioimmunoassay in the serum of patients with common acute lymphoblastic leukemia was found to be exclusively associated with the pellet of the serum samples obtained by ultracentrifugation at 100,000 X g. The pellets were shown to contain membrane vesicles or fragments which were characterized by electron microscopy and determination of enzymatic activity. The pelleted fragments had an apparent diameter ranging between 60 and 260 nm and showed a trilaminar membrane structure. On freeze-fracture preparations, the fragments with concave profile, corresponding to the external fracture face of plasma membrane, displayed an intramembrane particle density (ranging from 0 to 750 particles per micron2) which is similar to that recorded on the corresponding fracture face of intact cells from the common lymphoblastic leukemia antigen positive leukemic cell line (Nalm-1) or of vesicles shed in the culture medium by Nalm-1 cells. Furthermore, analysis of the membrane enzyme marker 5'-nucleotidase in the pellet of patient's sera, showed that the presence of this enzyme correlated with that of common lymphoblastic leukemia antigen, but the quantitative relationship between the two surface constituents was not linear. The results suggest that the two markers are located on the same membrane fragments, but that their individual distribution on the shed fragments is heterogeneous. PMID- 3022065 TI - Use of brachytherapy in lung cancer. PMID- 3022066 TI - A method for the determination of 11-nor-delta-9-tetrahydrocannabinol-9 carboxylic acid in urine using high performance liquid chromatography with electrochemical detection. AB - 11-Nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) was isolated using solid phase extraction and identified with high performance liquid chromatography (HPLC) with electrochemical detection (EC). HPLC/EC was compared to an established HPLC procedure using ultraviolet (UV) detection. Statistical analysis revealed good precision, linearity, and correlation between the HPLC/EC and HPLC/UV methods. Within-run and between-run precision for the HPLC/EC vs. HPLC/UV analyses yielded coefficients of variation of 4.1 vs. 6.7% and 8.3 vs. 10.9%, respectively. Standard curves over a range of 20 to 160 ng/mL of THC-COOH in fortified urine on four separate analytical runs yielded correlation coefficients of greater than 0.99 for each method. Quantitative results from 15 positive urine specimens also compared favorably between the two methods (r = 0.9923). Using an immunoassay screening technique with confirmation by HPLC/UV and high efficiency thin layer chromatography (HETLC), further evaluation of the HPLC/EC method was achieved by examining 50 cannabinoid positive and 50 cannabinoid negative clinical urine specimens and 500 sequentially collected postmortem urine specimens. PMID- 3022062 TI - Intracellular control of renin release--an overview. PMID- 3022067 TI - Evaluation of the Ion Trap Detector for the detection of 11-nor-delta 9-THC-9 carboxylic acid in urine after extraction by bonded-phase adsorption. AB - The Ion Trap Detector (ITD), a relatively new gas chromatographic detector from Finnigan MAT, was evaluated for its suitability in confirming urine specimens found positive for cannabinoids by immunoassay techniques. Also presented is a procedure for the isolation of 11-nor-delta 9-THC-9-carboxylic acid (THC-COOH) from human urine by bonded-phase absorption chromatography. PMID- 3022069 TI - A long-term field experience with breath ethanol collection employing silica gel. AB - Breath specimens (N = 143) from human subjects initially analyzed for ethanol content by infrared spectroscopy were collected onto silica gel. The specimens were retained 1.25 to 2.75 years following adsorption and subsequently analyzed by headspace gas chromatography. The results revealed good overall correlation between the direct and delayed ethanol determinations (r = 0.900). Specimens were collected by police officers following a defined protocol and the results are presented to illustrate practical use and limitations of such a technique. PMID- 3022068 TI - Evaluation of EMIT and RIA high volume test procedures for THC metabolites in urine utilizing GC/MS confirmation. AB - Results of EMIT, Abuscreen RIA, and GC/MS tests for THC metabolites in a high volume random urinalysis program are compared. Samples were field tested by non laboratory personnel with an EMIT system using a 100 ng/mL cutoff. Samples were then sent to the Army Forensic Toxicology Drug Testing Laboratory (WRAMC) at Fort Meade, Maryland, where they were tested by RIA (Abuscreen) using a statistical 100 ng/mL cutoff. Confirmations of all RIA positives were accomplished using a GC/MS procedure. EMIT and RIA results agreed for 91% of samples. Data indicated a 4% false positive rate and a 10% false negative rate for EMIT field testing. In a related study, results for samples which tested positive by RIA for THC metabolites using a statistical 100 ng/mL cutoff were compared with results by GC/MS utilizing a 20 ng/mL cutoff for the THCA metabolite. Presence of THCA metabolite was detected in 99.7% of RIA positive samples. No relationship between quantitations determined by the two tests was found. PMID- 3022070 TI - Growth of intestinal neomucosa on prosthetic materials. AB - The short bowel syndrome is a potential complication of the surgical management of diseases of the large and small intestine. Recently methods have been examined for expanding the small bowel absorptive area using prosthetic materials. We investigated the feasibility of growing intestinal mucosa on prosthetic patches and tubes. Ileal defects were patched with a 2 X 5-cm patch of either Dacron (n = 15), polyglycolic acid mesh (PGA) (n = 9), or polytetrafluroethylene (PTFE) (n = 5) prosthesis in New Zealand white male rabbits. Gross and microscopic analysis at 2, 4, and 8 weeks revealed that the serosal surface was covered with neomucosa by 4 weeks. Dacron and PTFE grafts were either minimally attached or extruded and PGA grafts had dissolved. At 8 weeks, none of the patches were present but with all three materials the resultant area of neomucosa was only 15% of the original defect. The neomucosa was functional as determined by glucose uptake and disaccharidase activity. Three centimeter Dacron tubes were interposed in the distal ileum of 10 rabbits and in a bypassed ileal segment in 11 rabbits. There was an 80% mortality within 2 weeks, and no evidence of neomucosal growth. Although prosthetic patches support the growth of functional neomucosa, there is a minimal increase in the final surface area. The type of prosthesis did not influence the outcome. The use of Dacron tubes is associated with high mortality and no neomucosal growth. The use of prosthetic materials is not likely to be useful in the clinical management of the short bowel syndrome. PMID- 3022071 TI - 1,25-Dihydroxyvitamin D3 receptor replenishment in the chicken intestine. AB - 1,25-Dihydroxyvitamin D3 intestinal receptor replenishment was examined in rachitic chickens after hormone administration. A single injection of 1,25 dihydroxyvitamin D3 caused an increase in the level of occupied receptors with a concomitant decrease in the amount of unoccupied receptors. Maximum occupancy occurred 1 h after hormone injection. The metabolic inhibitor of protein synthesis, cycloheximide, was employed to obtain additional information concerning the fate of 1,25-dihydroxyvitamin D3 receptor complexes. Cycloheximide, at a dose that effectively blocked protein synthesis, had no effect on the time-course or the magnitude of replenishment of nuclear receptors. Additionally, repletion with vitamin D3 or administration of several injections of 1,25-dihydroxyvitamin D3 did not lead to a lag in replenishment time or a significant decrease in total receptor levels. These findings demonstrate that recycling of receptors plays an important functional role for the replenishment of unoccupied 1,25-dihydroxyvitamin D3 intestinal receptors. PMID- 3022072 TI - Solubilization of 1,25-dihydroxyvitamin D3 receptor with pyridoxal 5'-phosphate from hen intestinal mucosa. AB - 1,25-Dihydroxyvitamin D3(1,25-(OH)2D3) receptor was solubilized in cytosol fractions upon homogenization of hen intestinal mucosa with pyridoxal 5' phosphate contained in a low ionic strength buffer. Pyridoxal 5'-phosphate did not inhibit the binding of 1,25-(OH)2D3 to its receptor. The receptor solubilized with pyridoxal 5'-phosphate was similar to the KCl-solubilized receptor in its binding affinity to the hormone and sedimentation coefficient. A majority (greater than 90%) of the mucosal 1,25-(OH)2D3 receptors were obtained as associating with crude chromatin which was prepared with a low ionic strength buffer, and this fraction of the receptor was solubilized with pyridoxal 5' phosphate. Ten millimolar pyridoxal 5'-phosphate was as effective as approx 0.2 M KCl in solubilizing the receptor from the crude chromatin. Pyridoxal 5'-phosphate also showed a potency to dissociate the 1,25-(OH)2D3-receptor complex previously bound to DNA-cellulose. Pyridoxal 5'-phosphate-related compounds such as pyridoxamine 5'-phosphate and pyridoxal did not show this potency. These results suggest that pyridoxal 5'-phosphate reduced the interaction of 1,25-(OH)2D3 receptor with its nuclear binding components without inhibiting the binding of the receptor to the hormone. PMID- 3022073 TI - Androgens inhibit the formation of catechol estrogens by estrogen 2-hydroxylase present in rat liver microsomal preparations. AB - The inhibition of estrogen 2-hydroxylase by androgens was demonstrated in screening assays and has been further investigated under initial velocity conditions. The ability of testosterone, 5 alpha-dihydrotestosterone, and dehydroepiandrosterone to block the conversion of estradiol to 2-hydroxyestradiol by male rat liver microsomal preparations was determined using two radiotracer methods--the conversion of [4-14C]estradiol to [4-14C]2-hydroxyestradiol and the release of 3H2O from [2-3H]estradiol. The apparent Ki's for the androgens ranged from 12.0 to 14.0 microM, with the apparent Km for the substrate estradiol in these assays of 2.08 microM. Multiple inhibition studies with the androgens and 2 bromoestradiol, an effective estrogen inhibitor, in male rat liver microsomes resulted in Dixon plots consisting of a series of nonparallel, intersecting lines. Thus, the androgens and 2-bromoestradiol are non-exclusive inhibitors, i.e. the binding of one compound to the enzyme does not interfere with the binding of the other. These interactions of androgens suggest that the steroid hormonal environment be considered in the examination of the physiological role(s) of the estrogen 2-hydroxylase and the catechol estrogen products. PMID- 3022074 TI - Direct effects of heparin on basal and stimulated aldosterone production in rat adrenal glomerulosa cells. AB - Direct effects of heparin (0.1-10 IU/ml) on basal and stimulated aldosterone production have been studied using intact rat adrenal glomerulosa cells. Heparin at any dose did not affect basal aldosterone production when added to the incubation medium. Heparin at a 0.01 IU/ml dose had no effect on aldosterone production maximally stimulated by angiotensin II (AII, 4.8 X 10(-8) M), ACTH (4.3 X 10(-9) M) or potassium (8.0 mM). However, heparin at 0.1 and 0.3 IU/ml doses selectively blocked aldosterone production maximally stimulated by AII but not by ACTH or potassium, while the compound at 1 and 10 IU/ml doses inhibited aldosterone production maximally stimulated by these three stimuli. In addition, the inhibitory effect of 0.3 IU/ml heparin occurred as early as 30 min after incubation with heparin. These data suggest that heparin at 0.1 and 0.3 IU/ml doses acts directly on adrenal zona glomerulosa to selectively block the stimulatory action of AII, while the compound at 1 and 10 IU/ml doses inhibits all the stimulatory actions of AII, ACTH and potassium. PMID- 3022076 TI - Affinity of corticosteroids for mineralocorticoid and glucocorticoid receptors of the rabbit kidney: effect of steroid substitution. AB - Corticosteroid derivatives coupled in the C3, C7 or C17 position with a long aliphatic chain were synthesized in order to select a suitable ligand for the preparation of a biospecific affinity adsorbent for mineralocorticoid receptor purification. The affinity of these derivatives for mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) was explored in rabbit kidney cytosol. In this model, aldosterone bound to a single class of receptors with high affinity (Kd 1 nM) and mineralocorticoid specificity. RU26988, a highly specific ligand for GR, did not compete for these sites. The C7 and C17 positions were found to be of crucial importance in the steroid's interaction with the mineralocorticoid receptors, since the linkage of a long side chain in these positions induced complete loss of affinity. Hence, deoxycorticosterone no longer bound to MR after 17 beta substitution with a 9-carbon aliphatic chain. This loss of affinity was not observed for glucocorticoids. The 17 beta nonylamide derivative of dexamethasone still competed for GR. Increasing the length of the C7 side of the spirolactone SC26304 suppressed its affinity for MR. Finally, C3 was an appropriate position for steroid substitution. The 3-nonylamide of carboxymethyloxime deoxycorticosterone bound to MR but not to GR, and therefore constitutes a suitable ligand for the preparation of a mineralocorticoid adsorbent. PMID- 3022075 TI - The difference of biological activity among four diastereoisomers of 1 alpha,25 dihydroxycholecalciferol-26,23-lactone. AB - All four possible diastereoisomers of 1 alpha,25-dihydroxycholecalciferol-26,23 lactone (1 alpha,25-(OH)2D3-26,23-lactone) were chemically synthesized and were compared to 1 alpha,25-dihydroxycholecalciferol (1 alpha,25(OH)2D3) in terms of their stimulation, in vivo, of intestinal calcium transport and mobilization of calcium from bone in vitamin D-deficient rats (the two classic vitamin D-mediated responses), and their relative binding to the chick intestinal cytosol receptor for 1 alpha,25-(OH)2D3. The receptor binding affinity results are expressed as relative competitive index (RCI), where the RCI is defined as 100 for 1 alpha,25(OH)2D3. The RCI obtained for 23(S)25(S)-1 alpha,25(OH)2D3-26,23-lactone was 7.90, for 23(R)25(R)-1 alpha,25(OH)2D3-26,23-lactone was 2.27, 23(S)25(R)-1 alpha,25(OH)2D3-26,23-lactone was 0.17, for 23(R)25(S)-1 alpha,25(OH)2D3-26,23 lactone 0.22 and for the in vivo produced 1 alpha,25(OH)2D3-26,23-lactone the RCI was only 0.17. Also the four diastereoisomers of 1 alpha,25(OH)2D3-26,23-lactone all stimulated intestinal calcium transport, reaching a maximum 8 h after administration. Compared with the stimulation of intestinal calcium transport by 1 alpha,25(OH)2D3, 23(S)25(S)-1 alpha,25(OH)2D3-26,23-lactone was 1/4 as effective, 23(R)25(R)-1 alpha,25(OH)2D3-26,23-lactone was 1/20 as effective, 23(S)25(R)-1 alpha,25(OH)2D3-26,23-lactone was 1/74 as effective and 23(R)25(S)-1 alpha,25(OH)2D3-26,23-lactone was 1/53 as effective. Similarly, 23(S)25(S)-1 alpha,25(OH)2D3-26,23-lactone and 23(R)25(R)-1 alpha,25(OH)2D3-26,23-lactone were estimated to be 3 and 20 times less active than 1 alpha,25-(OH)2D3 in elevation of serum calcium. However, 23(S)25(R)-1 alpha,25(OH)2D3-26,23-lactone and 23(R)25(S)-1 alpha,25(OH)2D3-26,23-lactone decreased the serum calcium levels 24 h after administration. 23(S)25(R)-1 alpha,25(OH)2D3-26,23-lactone reduced serum calcium concentrations to a greater extent than 23(R)25(S)-1 alpha,25(OH)2D3 26,23-lactone. These results indicate that the biological activities of the diastereoisomers of 1 alpha,25(OH)2D3-26,23-lactone were quite different among four stereochemical configurations. PMID- 3022077 TI - Investigations on the effects of long-term administration of a methionine enkephalin analogue on the adrenal zona fasciculata of rats treated with dexamethasone or dexamethasone and ACTH. AB - Prolonged (7 days) methionine-enkephalin (DALA) treatment provoked a dose dependent increase in the volume of zona fasciculata cells of dexamethasone administered rats, along with a notable rise in the plasma concentration of corticosterone and the activity of 11 beta-hydroxylase. Comparable dose-dependent effects were observed after chronic administration of ACTH to dexamethasone suppressed rats. The chronic administration of the maximum dose of DALA (500 micrograms/kg/day) was found to significantly further the trophic action of ACTH on the zona fasciculata of dexamethasone-treated animals. It is suggested that enkephalins act independently of and synergistically with ACTH in stimulating the growth and steroidogenic capacity of the rat adrenal zona fasciculata. PMID- 3022078 TI - Fatal metastatic cystosarcoma phyllodes in an adolescent female: case report and review of treatment approaches. AB - The third reported case of fatal malignant cystosarcoma phyllodes in an adolescent female is described. The patterns of local recurrence and distant spread in this case, including the response to treatment, were similar to those reported in the first reported case in this age group. A review of the treatment recommendations for cystosarcoma phyllodes revealed that the surgical procedure of choice for the malignant variant has remained controversial, and the conclusions regarding the ineffectiveness of radiation and chemotherapy have been based on insufficient data handed down through the years. Our observations in this case and the information we have obtained from the literature have prompted us to recommend a multidisciplinary approach for malignant cystosarcoma phyllodes, particularly in young women, and we are calling for a multi institutional study group to further investigate this disease. PMID- 3022079 TI - Serologic studies in hairy cell leukemia: high prevalence of Epstein-Barr and cytomegalovirus antibodies and absence of human T-cell lymphotrophic viruses antibodies. AB - Serum from 60 patients with hairy cell leukemia (HCL) were studied for the presence and the titers of antibodies to Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human T-cell lymphotrophic viruses (HTLV). Eighty-three percent of the patients were seropositive for EBV, with a (reciprocal) geometric mean titer (GMT) of 960. Seventy-eight percent of the patients had antibodies to CMV with a GMT of 435. All 21 patients tested for HTLV I and HTLV III were seronegative; only one patient showed detectable antibodies to HTLV II. The potential role of these infections in the physiopathology of HCL is discussed. PMID- 3022080 TI - Infectious feline leukaemia virus is erythrosuppressive in vitro. AB - The direct effect of the feline leukaemia virus (FeLV) on erythroid colony formation in vitro was investigated. Bone marrow mononuclear cells (BMMC) from FeLV-naive, specific-pathogen-free (SPF), adult cats were inoculated with FeLVs of characterized strains and biologically cloned subgroups and the subsequent development of colony forming units-erythroid (CFUE) and burst forming units erythroid (BFUE) and colony forming units-granulocyte-macrophage (CFUGM) was monitored. Exposure to the anaemia-causing Kawakami-Theilen strain of FeLV (FeLV KT), a phenotypic mixture of subgroups A, B, and C, caused constant depression of day 2 CFUE (to 47% of sham-inoculated controls), day 4 CFUE (41% of controls), and day 10 BFUE (38% of controls). CFUGM were unaffected. The lymphoma-causing Rickard strain of FeLV (FeLV-R-TL) caused sporadic depression of CFUE and BFUE. In contrast, neither FeLV-R passaged through feline embryonic kidney fibroblasts (FeLV-R-CRFK) nor biologically cloned, subgroup-specific, FeLVs of fibroblast origin, caused decrements in CFUE or BFUE, suggesting that fibroblast passage attenuated the direct erythrosuppressive effect of FeLV. Suppression of CFUE and BFUE by lymphoma cell-origin FeLV was dependent on infectious virus and was associated with FeLV replication by the cultured myelomonocytic precursor cells. Attenuation of infectivity by heat or u.v. restored CFUE and BFUE development. Examination of the relationship between viral infectivity (VI), viral protein concentration, and CFUE suppression showed that the infectious FeLV was 20-fold more effective than u.v.-inactivated FeLV as an inhibitor of erythrogenesis in vitro. PMID- 3022081 TI - Role of marrow stromal cells in the establishment of a transformed lymphoblastic B-cell line from a normal human subject. AB - A monoclonal human B-lymphoblastoid cell line (UTMB-460) arose spontaneously from the bone marrow of a normal healthy woman who was seropositive for an EB-virus infection. Chromosomally, the UTMB-460 cells are near tetraploid, with a specific translocation (8;9) (p11.2; p24), and have surface IgMk. The UTMB-460 cells are resistant to killing in vitro by spontaneous and rIFN alpha 2 and rIL-2 stimulated NK cells from the patient and other normal subjects, but are killed by lymphokine activated killer cells. The index patient has not developed leukemia/lymphoma during the follow-up interval of 22 months. The growth of UTMB 460 cells is supported by undefined growth factors in FCS and by BCGF in the absence of FCS. rIL-2 stimulates DNA synthesis by UTMB-460 cells. The UTMB-460 cells were adherent to the normal MSC in the primary culture and show specific heterotypic adherence to normal MSC when compared to skin fibroblasts. In addition, 6/6 normal marrow stromal cells and 4/6 normal skin fibroblasts induced growth of colonies from UTMB-460 cells. These data suggest that MSC interacted with the transformed cells (UTMB-460) in vitro and played a critical role in the establishment of the UTMB-460 cell line. PMID- 3022082 TI - A patient follow-up system for stomach cancer. AB - The concept of PASK (Patient follow-up System of Kyushu University Hospital) has been innovated in order to manage clinical data of patients in the hospital-wide scope over long periods of time by using computers. PASK represents a collection of subsystems, each tuned to a specific disease. For the initial implementation, the subsystem for stomach cancer (STOMAC) was constructed according to the Cancer Center of Kyushu University Hospital project. STOMAC began to work in October 1984 and has proved effective. Using STOMAC, doctors in a clinical section can easily access patient data gained in another section. Besides providing a data reference function, STOMAC automatically gives alarms for abnormalities in clinical examination results, anti-tumor drug dosage, visitation cycles of patients, etc. The database and STOMAC programs are strongly standardized and easily applicable to another specific disease. PMID- 3022084 TI - Apparent pA2 estimation of benzodiazepine receptor antagonists. AB - The in vivo equivalent pA2 values for benzodiazepine (BZ) antagonists Ro15-1788 and CGS 8216 were estimated using diazepam as agonist. ED50 value of diazepam was established against pentylenetetrazole-induced seizures in rats. This value was raised by pretreatment with BZ antagonists Ro15-1788 and CGS 8216, in a dose dependent manner. The apparent pA2 values for Ro15-1788-diazepam pair and CGS 8216-diazepam pair were found to be 5.28 and 5.48, respectively, which indicates almost equal antagonistic potency at the BZ receptor in reversing the protective effect of diazepam against pentylenetetrazole seizures in rats. PMID- 3022083 TI - Effects of two structurally different angiotensin-converting enzyme inhibitors, captopril and quinapril (CI-906), in rats with one-kidney deoxycorticosterone salt hypertension. AB - Possible non-renin antihypertensive actions of two angiotensin-converting enzyme inhibitors, the sulfhydryl compound captopril and the new nonsulfhydryl inhibitor quinapril (CI-906), were compared in rats with one-kidney deoxycorticosterone salt hypertension. Plasma renin activity remained low during the 12-day treatments and showed very strong suppression of the renin-angiotensin system. Quinapril did not influence the rapidly increasing blood pressure. Although captopril tended to reduce the development of hypertension (p = 0.08), it did not have any significant effect, either. The results indicate that these ACE inhibitors with different chemical structures lack any significant blood pressure lowering mechanism in severe deoxycorticosterone-salt hypertension, a model in which they cannot act through their established antihypertensive mechanism, the inhibition of the renin-angiotensin system. PMID- 3022085 TI - Small cell carcinoma of the larynx: results of therapy. AB - Primary small cell carcinoma of the larynx is a rare malignancy with a dismal prognosis. A survey of the long-term follow-up from reported cases of small cell carcinoma of the larynx and a review of the recent experience with this tumor at the University of Michigan Hospitals was undertaken to determine if newer treatment approaches incorporating adjuvant chemotherapy were associated with prolonged survival. Median survival for those patients receiving adjuvant chemotherapy was 19 months compared to 11 months for patients treated with surgery and/or radiation therapy alone. Among patients treated initially with primary radiation therapy and adjuvant chemotherapy median survival was 55 months, which was significantly longer than any other treatment regimen (P = 0.02). Systemic chemotherapy and therapeutic irradiation appears to offer the least disabling and most efficacious form of current therapy. PMID- 3022086 TI - 13-Propylberberine reduces response of guinea-pig myocardium to inotropic interventions including changes in extracellular Ca2+. AB - Berberine has been shown to increase developed tension in cardiac muscle but its derivatives have been reported to inhibit the catalytic subunit of adenylate cyclase. In the present study, the cardiac actions of the most potent derivative, 13-propylberberine, were examined. It produced a transient increase followed by a sustained decrease in developed tension in paced left atrial muscle preparations isolated from guinea-pig heart. In the presence of 13-propylberberine, isoproterenol caused only a transient increase in developed tension; marked desensitization to the positive inotropic effect of isoproterenol occurred within 20 min. After washout of isoproterenol and an additional 15-min incubation in the presence of 13-propylberberine, the muscle lost its sensitivity to isoproterenol. Moreover, the positive inotropic effect of ouabain or effects of decrease or increase in extracellular Ca2+ concentration on the force of muscle contraction were markedly attenuated. Isoproterenol-induced elevation of tissue cyclic AMP concentration was inhibited by 13-propylberberine; however, 13-propylberberine did not alter the basal cyclic AMP concentration and its effects on inotropic actions of ouabain or extracellular Ca2+ appear unrelated to tissue cyclic AMP concentration. PMID- 3022087 TI - 1-Thioglycerol: inhibitor of glycerol kinase activity in vitro and in situ. AB - The infantile form of glycerol kinase (GK) deficiency (McKusick No. 30703) (1) is characterized by adrenal cortical insufficiency, adrenal hypoplasia and developmental delay. The underlying biochemical mechanism(s) responsible for the observed clinical presentations are undetermined. Pursuant to our examination of the molecular pathogenesis of this enzyme deficiency, we have endeavored to develop a model for this disorder. 1-thioglycerol (1-TG) was investigated as a potential GK inhibitor in adrenal gland, an organ consistently affected, and in cultured fibroblasts, available from affected individuals. In 105,000 g bovine adrenal supernatant the Ki for 1-TG was 1.9 mM. In human fibroblast 105,000 g supernatant, the Ki for 1-TG was 3.4 mM. In both tissues the inhibition was purely competitive with respect to glycerol. Using incorporation of [14C(U)] glycerol into protein as an index of GK activity in situ in human skin fibroblasts, GK deficient fibroblasts incorporate less than 10% of that observed in normal fibroblasts. Addition of 1-TG to normal fibroblasts resulted in inhibited incorporation rates. The specificity of these effects in situ was examined. Our findings indicate that 1-TG may be a suitable inhibitor of GK activity for the development of a model for glycerol kinase deficiency. PMID- 3022088 TI - Effects of treadmill activity on cerebellar cyclic GMP. AB - Ovariectomized, female Sprague-Dawley rats were trained to run on a treadmill for up to several minutes. On the day of the experiment the treadmill speed was adjusted to either 680, 1360 or 2720 cm/minute and separate groups of 5 rats were required to run at each of these speeds for either 15, 30, 45 or 60 seconds. Immediately after running on the treadmill, each rat was killed by microwave irradiation, and the cerebellum was collected for subsequent determination of cyclic guanosine monophosphate (cGMP). The time to the onset of an elevation of cGMP and the maximal elevation obtained were functions of the speed of running. The first significant increase in cerebellar cGMP over that observed in inactive control rats occurred within 15 seconds after the onset of running. Regardless of the speed of running, the cGMP content reached a plateau after 45 seconds. PMID- 3022089 TI - Kinin release from kininogens by calpains. AB - During the investigation of inhibitory activity of kininogens toward calpains [EC 3.4.22.17], we found that lysyl-bradykinin was liberated from both high molecular weight (HMW) and low molecular weight (LMW) kininogens by the action of the calpains. The kinin liberation occurred in a limited range of calpain to kininogen molar ratios of 0.5:1 to 8:1, and in that condition calpains were simultaneously inhibited 20 to 80% by kininogens. The maximum level of kinin release from HMW and LMW kininogens by calpain I was about 25% and that by calpain II was 20%. These results suggest that in case of inflammation the kininogens play two physiologically distinct roles by interaction with calpains: to release lysyl-bradykinin and to inhibit proteinase activity of calpains derived from the damaged tissues. PMID- 3022091 TI - Nociceptive responses to altered GABAergic activity at the spinal cord. AB - GABA agonists and antagonists were injected intrathecally at the spinal cord, to determine their effect on nociceptive thresholds. Tactile stimulation, applied against the flank by a medium diameter von Frey fiber (5.5 g force), elicited distress vocalizations after, but not before injection of the GABA antagonists, bicuculline MI or picrotoxin (0.25 and 1 microgram dosages). Vocalization threshold to tail shock was significantly reduced by bicuculline MI or picrotoxin. Tail flick withdrawal latency from radiant heat was not altered by GABA antagonists. The GABA agonist, muscimol, significantly elevated vocalization threshold to tail shock at a 5 micrograms dose. At a lower dose level (1 microgram), muscimol significantly reduced vocalization threshold to tail shock. Tail flick latency was significantly prolonged by the 5 micrograms dose of muscimol; however, flaccid paralysis of the hind limbs was also evident. Nociceptive thresholds were not altered by GABA or saline injection. These findings indicate that GABAergic activity contributes to the tonic modulation of nociception at the spinal cord. PMID- 3022090 TI - Action of pro-opiomelanocortin products on the rat vas deferens. AB - Behavioral study of pro-opiomelanocortin products indicates that beta-endorphin and corticotrophin-like peptides have antagonistic effects. However, these peptides have similar actions on the rat vas deferens. beta-endorphin, alpha-MSH and ACTH each inhibit electrically evoked contraction of the duct, but the corticotrophin derived peptides are tenfold more potent on a molar basis (ED50 = 9 nM). Pharmacological analysis shows that the action of corticotrophin-derived peptides does not involve an opiate receptor mechanism. The results are discussed in terms of the central action of the peptides. PMID- 3022092 TI - Effect of ethanol on receptor-stimulated phosphatidic acid and polyphosphoinositide metabolism in mouse brain. AB - The effects of chronic ethanol consumption as well as the effects of ethanol added in vitro on phosphoinositide metabolism were determined in mouse forebrain. [32P] incorporation into synaptosomal phosphatidic acid (PhA) was stimulated through both M1 muscarinic cholinergic and alpha 1 adrenergic receptor activation. Similarly, [3H]inositol 1-PO4 accumulation in brain slices was stimulated through these same receptors, but could also be stimulated by histamine1 receptor activation. In mice made physically dependent to ethanol, the magnitude of receptor-mediated [32P] incorporation in PhA did not differ from that of control animals. However, ethanol (100mM) added in vitro to synaptosomes from control mice significantly inhibited the carbamylcholine stimulated PhA response, but had no effect on the response to norepinephrine. Carbamylcholine stimulated [32P] incorporation into PhA, however, was no longer significantly inhibited by the addition of 100mM ethanol to synaptosomes from physically dependent-tolerant animals indicating that a cellular tolerance had developed. In contrast, receptor mediated [3H]inositol 1-PO4 accumulation in brain slices was not significantly affected by either chronic ethanol treatment or the in vitro addition of ethanol as high as 200mM. It is concluded that the muscarinic cholinergic stimulation of [32P] incorporation into PhA, but not [3H]inositol 1 PO4 accumulation is relatively more sensitive to the direct effects of ethanol than are the other receptor mediated phospholipid responses examined in the present investigation and that this sensitivity is lost in animals made behaviorally tolerant and physically dependent to ethanol. PMID- 3022094 TI - Vasopressin and oxytocin reduce plasma prolactin levels of conscious rats in basal and stress conditions. Study of the characteristics of the receptor involved. AB - Subcutaneous administration of arginine vasopressin (AVP) to conscious rats induced a dose-dependent increase of plasma ACTH and beta-endorphin levels and decrease of plasma prolactin (PRL) levels 30 min later. AVP similarly reduced PRL increase induced by exposure to a novel environment stress. Oxytocin (OT) was also active but 5-fold less potent than AVP. The study of several analogs with specific agonistic and antagonistic activity on the oxytocic, vasopressor and antidiuretic receptors of OT and AVP suggests that the receptor involved in this effect does not fit into this classification. PMID- 3022093 TI - Evidence for coupling of the kappa opioid receptor to brain GTPase. AB - In membranes from guinea pig cerebellum, a tissue which predominantly contains kappa opioid receptors, exogenous and endogenous kappa-selective opioid agonists stimulated low-km GTPase activity by 11-20% with concentrations for half-maximal stimulation of 3-23 microM. Opioid ligands of the mu and delta type had no effect on GTPase in these membranes. Similar stimulation of GTPase by kappa opiates was obtained in rat and monkey brain membranes pretreated with beta-funaltrexamine (beta-FNA) and cis-(+/-)-3-methylfentanyl isothiocyanate (superfit) to alkylate the mu and delta receptors, respectively. The stimulation of brain GTPase by kappa opiates in both types of membranes was inhibited by naloxone with IC50's of 0.35 microM and 0.40 microM. The results demonstrate the coupling of the kappa opioid receptor to high affinity GTPase, the Ni regulatory protein of the adenylate cyclase complex. PMID- 3022095 TI - Estimation of the affinity of naloxone at supraspinal and spinal opioid receptors in vivo: studies with receptor selective agonists. AB - The apparent affinity of naloxone at cerebral and spinal sites was estimated using selective mu [D-Ala2, Gly-o15]-enkephalin (DAGO) and delta [D-Pen2, D Pen5]enkephalin] (DPDPE) opioid agonists in the mouse warm water tail-withdrawal test in vivo; the mu agonist morphine was employed as a reference compound. The approach was to determine the naloxone pA2 using a time-dependent method with both agonist and antagonist given intracerebroventricularly (i.c.v.) or intrathecally (i.th.); naloxone was always given 5 min before the agonist. Complete time-response curves were determined for each agonist at each site in the absence, and in the presence, of a single, fixed i.c.v. or i.th. dose of naloxone. From these i.c.v. or i.th. pairs of time-response curves, pairs of dose response lines were constructed at various times; these lines showed decreasing displacement with time, indicative of the disappearance of naloxone. The graph of log (dose ratio-1) vs. time was linear with negative slope, in agreement with the time-dependent form of the equation for competitive antagonism. From this plot, the apparent pA2 and naloxone half-life was calculated at each site and against each agonist. The affinity of naloxone was not significantly different when compared between agonists after i.c.v. administration. A small difference was seen between the affinity of i.th. naloxone against DPDPE and DAGO; the i.th. naloxone pA2 against morphine, however, was not different than that for DPDPE and DAGO. The naloxone half-life varied between 6.6 and 16.9 min, values close to those previously reported for this compound. These results suggest that the agonists studied may produce their i.c.v. analgesic effects at the same receptor type or that alternatively, the naloxone pA2 may be fortuitously similar for mu and delta receptors in vivo. Additionally, while the affinity of naloxone appears different for the receptors activated by i.th. DAGO and DPDPE, further work may be necessary before firm conclusions regarding the nature of the spinal analgesic receptor(s) can be drawn. PMID- 3022098 TI - State of hepatitis B viral DNA in the liver of patients with hepatocellular carcinoma and chronic liver disease. AB - In order to clarify the relationship between the integration of hepatitis B virus (HBV) DNA and human hepatocellular carcinoma (HCC), the states of HBV DNA in liver tissues were examined by the Southern blot hybridization procedure. Integration of HBV DNA was found in 12 of 25 HCC cases and 5 of 12 cirrhotic cases. Of these 17 cases, 11 were positive for serum hepatitis B virus surface antigen (HBsAg) and the remaining six were positive for more than one of the serum HBV-related antibodies. In all three cases of chronic hepatitis and 18 controls, integration of HBV DNA could not be detected. Free viral DNA was found in 12 of 15 cases with serum HBsAg. One patient without serum HBsAg also had free viral DNA in the liver. Integration of HBV DNA could be observed in HCC cases positive for serum HBsAg at the highest frequency. However, there was one HCC case from an HBsAg carrier in whom integration of HBV DNA might not have a causal effect on malignant transformation, because integrated HBV DNA could be detected only in a non-tumor region. Since integrated HBV DNA could not be detected in 13 of 25 HCC cases, other etiologic factors than the integration of HBV DNA must also be taken into consideration for HCC. PMID- 3022096 TI - Prostaglandins and cannabis XV. Comparison of enantiomeric cannabinoids in stimulating prostaglandin synthesis in fibroblasts. AB - Stereospecificity has been reported for a number of actions of the cannabinoids in a variety of systems. In the present report, we have shown that this effect can also be demonstrated when human lung fibroblasts in monolayer culture are stimulated by cannabinoids to produce prostaglandin E2 (PGE2). Three enantiomeric pairs of cannabinoids, (+) and (-)-delta 1-tetrahydrocannabinol (THC), (+) and ( )-delta 6-THC and (+) and (-)-delta 6-dimethylheptyl (DMH) THC were tested. In each case the (-) isomer was significantly more potent in agreement with the findings of others using different systems. Interestingly, very little stereospecificity was found in fibroblasts when the release of arachidonic acid, the precursor of PGE2, was monitored. This suggests that cannabinoids may act at several sites within the cell some of which show comparatively greater stereoselectivity for these agonists. PMID- 3022097 TI - Research topics in the medicinal chemistry and molecular pharmacology of opioid peptides--present and future. PMID- 3022099 TI - Natural killer (NK) cell activity and its in vitro response to interferon alpha(Le) in chronic liver diseases and hepatocellular carcinoma. AB - The natural killer (NK) cell activity of peripheral blood lymphocytes (PBL) in patients with various chronic liver diseases, and its in vitro response to human interferon-alpha(Le) were investigated using a 16-h 51-Cr releasing cytotoxicity assay against YAC-1 or RSa target cells. The NK cell activity was found to be higher in chronic active hepatitis (CAH) and liver cirrhosis (LC) patients without HCC, whereas it was slightly lower in LC patients with hepatocellular carcinoma (HCC), than in normal controls. By the addition of IFN-alpha(Le) in vitro, the NK cell activity was clearly and dose-dependently augmented, even in chronic liver diseases, as well as in normal controls. The magnitude of this augmentation by 10,000 IU/ml of IFN-alpha(Le) in the various chronic liver diseases was not significantly different from that in normal controls. The results suggested that the response of NK cells to IFN-alpha(Le) is not impaired even in chronic liver disease conditions, while the level of NK cell activity may vary according to the type of chronic liver disease and may decrease in patients with HCC. PMID- 3022100 TI - [Therapeutic use of 198Au-comisol]. PMID- 3022101 TI - [Ultrasonic and scintigraphic diagnosis of thyroid diseases]. AB - The paper is concerned with the results of ultrasonic scanning and scintigraphy in various thyroid diseases and neck tumors on the basis of an analysis of the results of the examination of 133 patients and 18 controls. The efficacy of ultrasonic and scintigraphic investigations in some thyroid diseases as well as their comparative informative value in the assessment of anatomotopographic, structural and functional peculiarities of the thyroid were demonstrated. A combined approach to the use of the above methods was substantiated and their priority was indicated. PMID- 3022102 TI - [Pre-radiation care in proton irradiation of tumors of the cavernous sinus]. AB - The authors described methods of the individual precision anatomodosimetric planning of proton-beam irradiation of tumors of the cavernous sinus. The results of computerized tomography in 2 planes (axial and frontal) were used for planning. Manual tumor reconstruction with subsequent dose planning was done on direct and lateral telecraniograms. The procedure was tested in 25 patients. Complications and noticeable radiation reactions in adjacent tissues were undetectable during 3.5 yrs. PMID- 3022103 TI - Rabbit oviduct mucosa healing after accidental transmucosal suture passage. AB - The purpose of this study is to show by SEM the characteristics of mucosal healing related to the presence of intraluminal sutures of nylon (nonabsorbable) or dexon (absorbable) in the rabbit oviduct. Altogether, 30 animals with anastomosis in the isthmus were killed 2, 4, 8, or 12 weeks after the operation. Tubular structures or pieces of thread partially or completely covered by the epithelium were found n 36.8% of these cases. Independently of the suture material used, already 2 weeks after surgery, the thread was covered by an epithelium composed mostly of squamous cells and some rare ciliated cells. At 4 weeks, the proportion of ciliated cells was increased. Regions with the usual cellular morphology and repartition of the different cellular types were also observed. However, at 8 and 12 weeks, islets of atypical squamous cells persisted in areas of transmucosal passage of the suture material. At 12 weeks, the dexon suture was not yet completely absorbed. PMID- 3022104 TI - [Meningoencephalitis caused by the St. Louis encephalitis virus]. PMID- 3022105 TI - [Effect of cyclophosphamide on the development of infection in Calomys musculinus with Junin virus]. PMID- 3022106 TI - [Biological effect of fibrous mineral dust. II. The neurotoxic effect of products of the thermal degradation of chrysotile]. AB - In experiments on animals the effect of heating chrysotile at 600 degrees C upon their biological action has been tested. Three chrysotile samples used in the Polish industry (MX-22 from deposits in Rhodesia, N-239 from Canada and PRZ-1-71 from deposits in the USSR (have been investigated). An X-ray diffraction analysis of heated chrysotile samples exhibited the total lack of lines characteristic for crystal substances in MX-22 and N-239 samples, whereas in the PRZ-1-71 sample the chrysotile content was about 20% of that in a non-heated sample. Intraperitoneal administration of a suspension of 100 mg of the dust brought about nervous system disturbances (shambling, atrophy of the response to ache, drop of internal body temperature and other (and finally-death of all experimental animals. Male rats survived longer from dust administration till the disturbances followed by death, i.e. from 8 hrs to 3 days, whereas female rats survived from 45 minutes to 24 hours only. PMID- 3022107 TI - Structure of the mutant prealbumin gene responsible for familial amyloidotic polyneuropathy. AB - Familial amyloidotic polyneuropathy (FAP) is a genetic disorder showing autosomal dominant inheritance. Amyloid fibrils of FAP patients from various origins have been shown to contain a prealbumin variant with Val30----Met30 substitution as a major component. However, the structure of the prealbumin gene responsible for the variation has not been characterized. We determined the complete nucleotide sequence of the prealbumin gene from a patient with the Japanese type of FAP. In comparison with a normal prealbumin gene sequence, the patient's gene was found to be carrying seven base substitutions. The substitution responsible for the Val ---Met change was found in exon 2, as expected, and the others were in introns. Hybridization analyses of normal and FAP patient DNAs showed that the base substitution in exon 2 was specific for FAP but the others were polymorphic changes. It was concluded that the mutation responsible for the Val----Met change is the only base change specific for FAP in the prealbumin gene. PMID- 3022108 TI - Structure and expression of the mutant prealbumin gene associated with familial amyloidotic polyneuropathy. AB - We have cloned a chromosomal DNA segment that covers the entire sequence for the mutant prealbumin gene associated with familial amyloidotic polyneuropathy, and determined the sequence of the gene including 581 base-pairs of the 5'-flanking region and 95 base-pairs of the 3'-flanking region, except for the internal portions of the second and third introns. This sequence was aligned with that of the previously determined normal prealbumin gene and both sequences matched perfectly except for a single-base substitution present in the second exon. This substitution is responsible for the transition from valine (GTG) to methionine (ATG) in the mutant prealbumin and creates NsiI and BalI restriction sites in the mutant prealbumin gene. We also analysed prealbumin mRNAs in the livers of a disease-free individual and one with familial amyloidotic polyneuropathy and confirmed that there is no difference in the levels and sizes of the prealbumin mRNAs between the two. As we have demonstrated already that individuals with familial amyloidotic polyneuropathy are heterozygous for the prealbumin gene, carrying one normal and one mutant gene, levels of the two different mRNAs within the liver of an individual with familial amyloidotic polyneuropathy were separately estimated and were demonstrated to be approximately equal. PMID- 3022110 TI - In vitro interferon production: induction in human leukocytes after exposure to cytomegalovirus antigens. AB - In vitro lymphocytes from healthy donors with crude (HCMV), heat inactivated (HCMVi) and infected human fetal fibroblasts (HCMVFF) were stimulated. In all the experiments peak titers of alfa Interferon (IFN) were present 24 h after exposure to antigens. On the other hand, at least after seven days, only lymphocytes of seropositive donors stimulated by HCMVFF and HCMV showed gamma IFN production. Our HCMVFF gave much higher IFN yields than free virus. Our data suggest that HCMVFF and HCMV are recognised by lymphocytes of HCMV specific memory and HCMVFF give additional and more reproducible information on HCMV specific cellular immunity. PMID- 3022109 TI - Protein conformational dynamics measured by hydrogen isotope exchange techniques. PMID- 3022111 TI - Fluorescence polarization immunoassay to determine aminoglycoside modifying enzymes activity. AB - The possible use of polarized-immunofluorescence to evaluate aminoglycoside modifying enzyme activities has been investigated. The TDX instrument was used to determine the residual quantity of aminoglycoside following inactivation during the assay procedure. Through the spectrum of the modified aminoglycoside antibiotics the enzymes encoded on eleven R-factors from clinical isolates of Enterobacteriaceae have been identified. PMID- 3022112 TI - Comparative study of some biological activities of porins extracted from various microorganisms. AB - The outer membrane of Gram-negative bacteria contains some major proteins, called porins, with particular chemical and physical characteristics which reflect some specialized functions peculiar to the outer membrane. In recent years the biological properties of Salmonella typhimurium SH5014 porins have been the focus of our experimental studies. The aim of the present research was to make a comparative investigation of the biological activities of porins extracted from other microorganisms classified in the Enterobacteriaceae, together with a taxonomically different one, Eikenella corrodens frequently isolated from gum pockets of patients with periodontal disease. Porins from E. coli K12, Proteus mirabilis and Eikenella corrodens were extracted, purified and separated from LPS by means of phenol extraction. The purity of preparation was checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis in slabs. We studied the interactions of these proteins with some cellular and humoral systems within the host. Mouse peritoneal macrophages in the presence of these proteins show morphological and functional modifications, depending upon the type of porins and the concentrations used. The ability of porins to interact with the complement system was investigated in vitro. A pool of sera from 20 blood donors served as a source of human complement. The results obtained with porins of various sources were shown comparatively. PMID- 3022113 TI - Viral childhood gastroenteritis. AB - Viruses were detected in 150 hospitalized subjects with gastroenteritis during the years 1983-1984. The samples were collected on admission in the day care unit and after three and seven days. The research was carried out using electron microscopy (E.M.) and isolation in cell cultures in vitro. Faecal samples were also collected from 70 control subjects. Observed and/or isolated viruses were identified by structural features at E.M. and by typical cytopathic effects in cultures. Rotavirus and not cultivable adenovirus proved the most widespread agents in the aetiology of acute viral gastroenteritis in infants. PMID- 3022114 TI - Serum antibodies to individual cytomegalovirus structural polypeptides in renal transplant recipients during viral infection. AB - In a longitudinal study we examined by immunoblotting (IB) the development and the evolution of the humoral immune response against individual cytomegalovirus (CMV) structural polypeptides in a total of 80 serum samples from 13 renal transplant recipients showing serological evidence of CMV infection and five renal transplant recipients with an anti-CMV antibody level unchanged over the observation period. The results showed that the IB reactivity at the time of transplantation may be a good index of the host's humoral immune status against CMV; by using this procedure it is possible to identify a seroconversion by the detection of antibodies reacting with some intermediate molecular weight proteins in sera examined at high dilution. Furthermore, IB is a very sensitive procedure also for IgM detection as it anticipates the positivity of the enzyme immune assay for IgM. PMID- 3022115 TI - No involvement of bovine leukemia virus in sporadic bovine lymphosarcoma. AB - Using various portions of a molecularly cloned bovine leukemia virus (BLV) DNA as probes, the possible integration of a BLV genome or a BLV-related sequence into the chromosomal DNA of sporadic bovine leukosis (SBL) tumor cells was investigated by Southern blotting analysis. Under stringent as well as nonstringent conditions of hybridization, neither BLV nor BLV-related sequence specific to SBL DNAs was detected in any SBL tumor examined. These results provide conclusive evidence for lack of the relation of BLV or a BLV-related agent to SBL. PMID- 3022116 TI - [A method for detecting new strains producing DNA modification and restriction enzymes--isoschizomers and isomethylomers of known restrictases and methylases]. AB - The paper describes a technique for the detection of new strains producing enzymes which mediate DNA modification and restriction, and isoschizomers and isomethylomers of the known restriction endonucleases and methylases. Three Bacillus subtilis strains whose DNA carries a BamH1 modification have been found. Two of these strains exert the restrictase activity with an R BamH1 specificity. PMID- 3022117 TI - [Comparison of complement fixation and Tzanck smear tests for the diagnosis of herpes labialis]. AB - In this study, the serum antibody titer of Herpes simplex virus type 1 (HSV-1) in 82 patients with herpes labialis and 100 controls was determined using the complement fixation test. In own experience, the Tzanck smear test (reported to be an aid in the diagnosis of HSV infection) was of no value. In comparison to patients in an early stage of infection, convalescent patients had an antibody titer that was 4 or more times higher (1/64-1/256). Fifty-eight percent of the control group was negative. In own opinion, the variation in titer between the acute and convalescent stages in Herpes simplex cases would be of particular value in diagnosing cases of urethritis, cystitis encephalitis, meningitis, conjunctivitis, primary and recurrent eczema herpeticum and erythema multiforme when HSV is suspected to play a role in the etiology. PMID- 3022118 TI - The change of plasma glucose after the embolization of hepatic arteries. PMID- 3022119 TI - H1(0) histones of normal and cancer human cells. Amino acid composition of H1 purified by polyacrylamide gel electrophoresis. AB - H1 histones were purified by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis from human lung carcinoma (line DMS79), human hepatoblastoma (HepG2), human adult lung and human adult and fetal liver. The purified human H1 histones were analyzed for their amino acid composition and terminal residues. The comparative analysis of the amino acid compositions of the different human H1 histones showed that: all the H1 preparations have the characteristically high lysine content associated with a low arginine content, which distinguishes outer histones from core histones; H1 is distinguishable from other H1 histones by the presence of methionine and histidine; H1 histones from human adult, fetal and cancer cells are very similar in amino acid composition, and in cancer cells the level of the H1 histone is not inversely related with cell growth rate nor with the expression of the alpha-fetoprotein gene. PMID- 3022121 TI - [Structural polymorphism of the provirus integrated in the genome of mammalian cells transformed by Rous sarcoma virus]. AB - The structural organization of integrated Rous sarcoma virus (RSV) genome in 8 different mammalian tumour cell lines has been studied. The different types of provirus rearrangements were found, e.g.: breakpoint mutations, deletions of RSV structural genes, elimination of the entire viral genome. The structural changes of proviruses appeared during long-term cultivation of tumour cell lines in vitro. The ability of transformed cells to produce complete infectious RSV correlate with the structural peculiarities of proviruses, although the tumorigenic properties of these cells are retained in all cases. PMID- 3022125 TI - [Isolation of recipient yeast strains for the stable reproduction of 2-micron DNA vectors]. AB - Most Leu- clones of yeast transformants (cir0, pJDB219) can stabilize the replication of 2 micron-vectors with REP3, the stability obtained being comparable to the one for the standard cir0 strain. One of the Leu- clones was used to isolate a plasmid with Rep 1.2 functions ("Rep-helper plasmid"). The plasmid was shown to carry a partially active LEU2 gene by transforming both E. coli and S. cerevisiae to Leu+ phenotype. A restriction analysis performed demonstrated that the Rep-helper plasmid has lost approximately 1.9 kb compared to the parent pJDB219, deletion and rearrangement having taken place at the bacterial and 2 mem components boundary. The Rep-helper plasmid carrying host strains allows to quantify the REP3 function on different 2 microns vectors. Some but not all cir+ stabilized vectors show greater stability in Rep-helper strains compared to the standard cir0 ones. Manipulating the Rep-helper plasmid level, by selecting for Leu+ phenotype, stabilized REP3 +/- plasmid p3030, but mostly destabilizes REP3+ plasmid YEp13HIS3. PMID- 3022120 TI - Clearance of thrombin in vivo: significance of alternative pathways. AB - The clearance of thrombin seems to occur at more than one site and by different mechanisms. This contributes to maintaining thrombin at the right concentrations to act optimally on its various substrates, and thus, to produce the proper amount of proteolytic conversions so that coagulation is precisely controlled. The vascular endothelium plays a major role in thrombin regulation and clearance. It contains heparin-like binding sites and thrombomodulin which serve as cofactors for the thrombin-antithrombin III reaction and the activation of protein C, respectively. In addition, thrombomodulin also serves as a receptor for endothelial cell mediated thrombin endocytosis. Thrombin clearance, which occurs following reaction with antithrombin III or thrombomodulin, probably takes place at different stages in hemostasis. PMID- 3022122 TI - [Transfer of active oncogenes and promoters into the mouse cell genome]. AB - Different cell DNA's (normal NIH 3T3 DNA; human osteosarcoma cell DNA; human malignant glioma cell DNA with amplified c-Ha-ras) were cotransfected onto NIH 3T3 cells with cloned long terminal repeat (LTR) sequences of Rous sarcoma virus. LTR RSV and normal NIH 3T3 DNA c-fos oncogen expression was detected in tumors induced in nude mice. In the same system human tumour cell DNA with amplified c Ha-ras gene was used, that to the integration and amplification of LTP sequences with simultaneous maintenance of c-Ha-ras amplification. Nude mouse tumour DNA with integrated LTR sequences was active in successive rounds of transfection. PMID- 3022124 TI - [Study of the interaction of proflavine and isomeric diribonucleoside monophosphates CpG and GpC by proton magnetic resonance]. AB - A comparative study of the interaction of proflavine with isomeric diribonucleoside monophosphates CpG and GpC has been made by the method of 1H NMR (270 MHz). A method of calculation of the parameters of complex formation from the concentration dependences of proton chemical shifts of the dye has been proposed. The equilibrium constants of 1:1 and 1:2 complexes association of these molecules and the most probable structures of the complexes have been determined. PMID- 3022123 TI - [Activation of ras and myc proto-oncogenes in human breast carcinoma and neuroblastoma]. AB - DNA from human breast carcinoma (SK-BR-3) and neuroblastoma (LA-N-1) cell lines are capable of inducing foci of transformed NIH 3T3 cells after DNA-mediated gene transfer. The blot hybridization analysis of DNA from primary and secondary NIH 3T3 transformants identified additional sequences homologous to the c-Ha-ras 1 oncogene, and revealed amplification of nucleotide sequences homologous to the v myc oncogene. Restriction fragments of the amplified myc-related sequences correspond to c-myc (SK-BR-3) and N-myc (LA-N-1) loci of the human genome. The results show that active Ha-ras oncogenes can coexist with altered myc oncogenes in breast carcinomas and neuroblastomas. This suggests that a multi-step mechanism involves both ras and myc genes and their cooperation in the development of these tumors. PMID- 3022126 TI - [Interaction of restriction and modification enzyme EcoRII with synthetic DNA fragments. VII. The study of complex-formation of endonuclease EcoRII with substrates containing natural and modified recognition sites]. AB - As shown by a nitrocellulose filter binding assay, in the absence of Mg2+ EcoRII restriction endonuclease binds specifically to a set of synthetic concatemer DNA duplexes of varying chain length, containing natural and modified recognition sites of this enzyme. The binding of the substrates with the central AT, TT or AA pair in the recognition site decreases at AT greater than TT much greater than AA. Substitution of the pyrophosphate bond at the cleavage site for the phosphodiester or phosphoramide bond produces little influence on the stability of the complexes. The affinity of the enzyme for nonspecific sites is two orders of magnitude less than that for the specific EcoRII sequences. Equilibrium association constant for a substrate with one recognition site is 3.9 X 10(8) M 1. Addition of Mg2+ leads to the destabilization of the EcoRII endonuclease complex with DNA duplex, containing pyrophosphate bonds. The dissociation rate constants and the lifetime of the EcoRII endonuclease--synthetic substrates complexes have been determined. PMID- 3022127 TI - Absence of a structural basis for intracellular recognition and differential localization of nuclear and plasma membrane-associated forms of simian virus 40 large tumor antigen. AB - The simian virus 40 large tumor antigen (T-ag) is found in both the nuclei (nT ag) and plasma membranes (mT-ag) of simian virus 40-infected or -transformed cells. It is not known how newly synthesized T-ag molecules are recognized, sorted, and transported to their ultimate subcellular destinations. One possibility is that these events depend upon structural differences between nT-ag and mT-ag. To test this possibility, we compared the structures of nT-ag and mT ag from simian virus 40-infected cells. No differences between the two forms of T ag were detected by migration in polyacrylamide gels, by Staphylococcus aureus V8 partial proteolytic mapping of methionine- or proline-containing peptides, or by two-dimensional tryptic peptide mapping of methionine-containing peptides. The carboxy-terminal, methionine-containing tryptic peptide was identified in the two dimensional maps and was shown to be identical in nT-ag and mT-ag. Thus, a structural basis for the recognition and differential localization of T-ags could not be demonstrated. The carboxy terminus of the T-ag encoded by mutant dlA2413 is derived from the alternate open reading frame of the simian virus 40 early region, in analogy with the theoretical early gene product, T*-ag. We used this mutant to identify peptides unique to T*-ag. None of these peptides were detected in maps of mT-ag; only wild-type T-ag-specific peptides were found. These findings suggest that T*-ag does not represent the membrane-associated form of T ag, but that mT-ag is encoded within the same reading frame used for nT-ag. PMID- 3022128 TI - Structural characterization of a heterogeneous family of rat brain mRNAs. AB - A large heterogeneous family of RNAs derived from a single rat gene contains members that differ from each other at one or more of three positions. Their 5' ends are nested and transcription can begin at 22 or more sites covering 265 nucleotides. Many of the 5' ends are detectable only in brain RNAs, and even 5' ends common with other tissues appear with different absolute and relative abundances in brain RNA. The central portions of the RNAs are of two forms, differing only by the presence or absence of 17 nucleotides; these forms are probably produced by alternative splicing. Polyadenylation occurs at either of two sites. This complicated family of 88 RNAs encodes two novel putative proteins that differ at their C termini. PMID- 3022130 TI - Quantitative analysis of gene suppression in integrated retrovirus vectors. AB - Previously, we described a retrovirus vector that contained two genes: a 5' gene under transcriptional control of the viral long terminal repeat and a 3' gene under transcriptional control of the herpes simplex virus thymidine kinase promoter. By using a biological assay, we found that expression of the 5' gene is suppressed when there was selection for the 3' gene and expression of the 3' gene is suppressed when there is selection for the 5' gene (M. Emerman and H. M. Temin, Cell 39:459-467, 1984). In the present study, we replaced the thymidine kinase promoter with stronger promoters, and we measured expression of the genes in the retrovirus vectors by enzyme activity and RNA analysis. We found that all of the vectors displayed gene suppression when analyzed biochemically, although not when analyzed biologically. The suppressed genes produced about 10 to 50% as much product as when they were selected. The amount of suppression depended on whether the suppressed gene was 5' or 3' to the selected gene and from which promoter the suppressed gene was transcribed. The amount of suppression correlated with a decrease in the amount of steady-state RNA transcribed from the suppressed gene's promoter. PMID- 3022129 TI - Nucleosomal instability and induction of new upstream protein-DNA associations accompany activation of four small heat shock protein genes in Drosophila melanogaster. AB - We investigated in detail the structural changes that occur in nuclear chromatin upon activation of the four small heat shock protein genes in D. melanogaster. Both the chemical cleavage reagent methidiumpropyl-EDTA X iron(II) [MPE X Fe(II)] and the nuclease DNase I revealed a complex pattern of four or five hypersensitive sites upstream of each gene before activation. In addition, MPE X Fe(II) detected a short positioned array of nucleosomes located on each coding region. Upon heat shock activation a number of changes in the patterns occurred. For each gene, at least one of the upstream hypersensitive regions was eliminated or substantially shifted in position. Regions were established which became highly refractile to digestion by either MPE X Fe(II) or DNase I and, as such, appeared as small "footprints" in the pattern. The location of these refractile regions relative to the cap site varied for each gene examined. The coding regions themselves became highly accessible to DNase I. The nucleosomal arrays detected by MPE X Fe(II) were characterized by a considerable loss of detail and significantly enhanced accessibility, the extent of which probably reflected the relative transcription rate of each gene. Careful mapping of the location and extent of each upstream footprint and comparison with the DNA sequence revealed the presence at each location of two (or more) contiguous or overlapping segments that bear high homology to the heat shock consensus sequence C-T-N-G-A-A-N-N-T-T C-N-A-G. A specific protein factor (or factors) is most likely bound at or near these sequence in heat-shocked Drosophila cells. PMID- 3022131 TI - Spatial and temporal regulation of a foreign gene by a prestalk-specific promoter in transformed Dictyostelium discoideum. AB - We analyzed a developmentally regulated prestalk-specific gene from Dictyostelium discoideum encoding a cathepsin-like protease. A hybrid gene was constructed by fusing 2.5 kilobases of 5' flanking sequences and part of the coding region of the gene in-frame to the Escherichia coli beta-glucuronidase gene and was transformed into D. discoideum cells. In cells transformed with this vector, the gene fusion showed the same temporal regulation as the endogenous gene during multicellular development and, like endogenous prestalk genes, was highly inducible by cyclic AMP in in vitro cell cultures. Moreover, immunofluorescence studies showed that the fusion protein had the same spatial distribution within the migrating pseudoplasmodium as the endogenous gene. The results indicate that the regions of the D. discoideum prestalk-specific cathepsin gene contain all the necessary information for proper temporal, spatial, and cyclic AMP regulation of a prestalk cell-type gene in D. discoideum transformants and leads the way for experiments to identify the cell-type-specific regulatory elements. PMID- 3022132 TI - Molecular organization and expression of the genetic locus glued in Drosophila melanogaster. AB - The Glued locus of Drosophila melanogaster is genetically defined as the functional unit which is affected by the dominant Glued mutation Gl. Genomic DNA was cloned from the region of the Glued locus, at 70C2 on chromosome 3, by using a P element insertion in the region as a molecular marker. Three genes encoding polyadenylated transcripts were detected within a 30-kilobase span of the cloned DNA. The gene nearest the P element insertion site was identified as a Glued gene on the basis of alterations in its DNA and encoded transcript associated with the Gl mutation and with reversions of Gl which eliminate the dominant effect by inactivation of the mutant allele. Expression of the wild-type Gl+ gene is temporally regulated during development; the amount of the encoded transcript is highest in the embryonic stage, decreasing in the first and second larval instars, and then increasing in the third instar and pupal stages. There is a maternal contribution of the Gl+ transcript to the embryo, which probably accounts for the maternal lethal effect of Glued mutations on early development. In situ hybridizations of Gl+ DNA to RNA in tissue sections showed that the Gl+ transcript is present in virtually all tissues of the embryo, late larva, and pupa. The general distribution of this transcript is consistent with genetic evidence indicating that the Glued locus controls a generally essential cell function (P. J. Harte and D. R. Kankel, Genetics 101:477-501, 1982). Different Glued mutations produce distinct phenotypic effects, including adults with severe visual defects, larvae lacking imaginal discs, and early lethality. These diverse mutant phenotypes are discussed in terms of quantitative changes in the Glued function. Closely adjacent to Gl+ is another gene which is transcribed in a divergent direction and expressed coordinately with Gl+ throughout Drosophila development. It remains to be determined whether this gene is also involved with the Glued function. PMID- 3022133 TI - Cloning, sequencing, and evolutionary analysis of the mouse erythropoietin gene. AB - The gene for mouse erythropoietin was cloned and sequenced. We present here a preliminary analysis of the overall genomic organization of the coding portions and the two flanking regions of the gene. This is the third mammalian erythropoietin for which the sequence is available, but it represents the first from a nonprimate species. We investigated the evolutionary divergence of sequence and structure of the three erythropoietins and identified specific regions of the molecules that are apparently under various degrees, and perhaps different types, of functional constraint. PMID- 3022134 TI - Complementation of a bovine papilloma virus low-copy-number mutant: evidence for a temporal requirement of the complementing gene. AB - We identified a bovine papilloma virus function encoded by the E6/E7 gene, which is required for both BPV high-copy-number replication and maintenance of transformation of cultured cells. A cDNA copy of this gene was isolated and expressed from a retrovirus vector. We found that complete complementation of a BPV low-copy-number mutant (dl576) by the cDNA encoding the E6/E7 gene was temporally dependent. When both the E6/E7 cDNA and dl576 were introduced together into cells, wild-type replication and stable transformation resulted. In contrast, introduction of the complementing cDNA into cells already carrying dl576 led to only partial amplification of the resident mutant DNA accompanied by a restoration of the transformed phenotype. These results, along with other findings, suggest that the establishment of BPV plasmid replication occurs in two stages: an initial amplification of the incoming DNA followed by stable homeostatic replication which maintains the existing copy number. PMID- 3022135 TI - Differential responsiveness of myc- and ras-transfected cells to growth factors: selective stimulation of myc-transfected cells by epidermal growth factor. AB - To identify functional relationships between oncogenes and growth factors, we compared the effects of transfected myc and ras oncogenes on the responsiveness of Fischer rat 3T3 cells to three growth factors: epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta). Control cells did not grow in soft agar under any conditions. ras Transfected cells grew in soft agar under all conditions tested and were insensitive to the stimulatory effects of exogenous growth factors. These cells secreted elevated levels of both EGF-like factors and TGF-beta, suggesting that the lack of responsiveness of these cells to exogenous growth factors arose from autocrine stimulation. myc-Transfected cells displayed conditional anchorage independent growth: they formed numerous colonies in soft agar in the presence of EGF but relatively few colonies in the presence of PDGF or TGF-beta. Secretion of EGF-like factors and TGF-beta by these cells was not elevated above that of control cells. These results suggest a model for the mechanism of cooperation between myc and ras oncogenes in which ras-like genes induce growth factor production, while myc-like genes increase the responsiveness of cells to these factors. PMID- 3022136 TI - 5' Nucleotide sequences influence serum-modulated expression of a human dihydrofolate reductase minigene. AB - Human dihydrofolate reductase (DHFR) gene sequences were isolated from DHFR gene amplified breast cancer cell line MCF-7. These genomic sequences plus human DHFR cDNA sequences were used to construct a DHFR minigene. Calcium phosphate-mediated transfer of minigene DNA into DHFR gene-deleted Chinese hamster ovary cells converted these cells to a DHFR+ phenotype at a frequency of 0.12%. Minigene transfected cells contained 20 to 30 minigene copies per cell and had DHFR enzyme levels similar to those of wild-type MCF-7 human cells (1.4 pmol/mg of protein). In contrast to gene-amplified MCF-7 cells, which contained multiple DHFR mRNA species (1.1, 1.6, 3.8, and 5.3 kilobases), only a single 3.8-kilobase DHFR mRNA was found in minigene-transfected cells. Previous studies on normal cells demonstrated modulation of DHFR levels by a variety of conditions which altered cell growth. When cell growth was induced in minigene-transfected cells by release from serum deprivation and DHFR levels were assayed at the time of maximum DNA synthesis, these levels were increased 2.4 to 3.7-fold. In contrast, the DHFR levels in cells transfected with a construct made from DHFR cDNA and viral promoter, intron, and termination sequences were unchanged. Minigene deletions were made and analyzed to determine the DHFR gene sequences responsible for regulation. Deletion of sequences upstream from 322 base pairs 5' to the start of transcription or 90 base pairs downstream from the termination of translation (which removed most of the 3' nontranslated region of the gene) did not alter the responsiveness of minigene-transfected cells to serum deprivation. However, when sequences between 322 and 113 base pairs 5' to the start of transcription were deleted, serum-dependent expression in minigene-transfected cells was affected. PMID- 3022138 TI - Methylation of the mouse hprt gene differs on the active and inactive X chromosomes. AB - It has been proposed that DNA methylation is involved in the mechanism of X inactivation, the process by which equivalence of levels of X-linked gene products is achieved in female (XX) and male (XY) mammals. In this study, Southern blots of female and male DNA digested with methylation-sensitive restriction endonucleases and hybridized to various portions of the cloned mouse hprt gene were compared, and sites within the mouse hprt gene were identified that are differentially methylated in female and male cells. The extent to which these sites are methylated when carried on the active and inactive X chromosomes was directly determined in a similar analysis of DNA from clonal cell lines established from a female embryo derived from a mating of two species of mouse, Mus musculus and Mus caroli. The results revealed two regions of differential methylation in the mouse hprt gene. One region, in the first intron of the gene, includes four sites that are completely unmethylated when carried on the active X and extensively methylated when carried on the inactive X. These same sites are extensively demethylated in hprt genes reactivated either spontaneously or after 5-azacytidine treatment. The second region includes several sites in the 3' 20kilobases of the gene extending from exon 3 to exon 9 that show the converse pattern; i.e., they are completely methylated when carried on the active X and completely unmethylated when carried on the inactive X. At least one of these sites does not become methylated after reactivation of the gene. The results of this study, together with the results of previous studies by others of the human hprt gene, indicate that these regions of differential methylation on the active and inactive X are conserved between mammalian species. Furthermore, the data described here are consistent with the idea that at least the sites in the 5' region of the gene play a role in the X inactivation phenomenon and regulation of expression of the mouse hprt gene. PMID- 3022137 TI - Adenovirus E1A coding sequences that enable ras and pmt oncogenes to transform cultured primary cells. AB - Plasmids expressing partial adenovirus early region 1A (E1A) coding sequences were tested for activities which facilitate in vitro establishment (immortalization) of primary baby rat kidney cells and which enable the T24 Harvey ras-related oncogene and the polyomavirus middle T antigen (pmt) gene to transform primary baby rat kidney cells. E1A cDNAs expressing the 289- and 243 amino acid proteins expressed both E1A transforming functions. Mutant hrA, which encodes a 140-amino acid protein derived from the amino-terminal domain shared by the 289- and 243-amino acid proteins, enabled ras (but not pmt) to transform and facilitated in vitro establishment to a low, but detectable, extent. These studies suggest that E1A functions which collaborate with ras oncogenes and those which facilitate establishment are linked. Furthermore, E1A transforming functions are not associated with activities of the 289-amino acid E1A proteins required for efficient transcriptional activation of viral early region promoters. PMID- 3022139 TI - Size threshold for Saccharomyces cerevisiae chromosomes: generation of telocentric chromosomes from an unstable minichromosome. AB - A 9-kilobase pair CEN4 linear minichromosome constructed in vitro transformed Saccharomyces cerevisiae with high frequency but duplicated or segregated inefficiently in most cells. Stable transformants were only produced by events which fundamentally altered the structure of the minichromosome: elimination of telomeres, alteration of the centromere, or an increase of fivefold or greater in its size. Half of the stable transformants arose via homologous recombination between an intact chromosome IV and the CEN4 minichromosome. This event generated a new chromosome from each arm of chromosome IV. The other "arm" of each new chromosome was identical to one "arm" of the unstable minichromosome. Unlike natural yeast chromosomes, these new chromosomes were telocentric: their centromeres were either 3.9 or 5.4 kilobases from one end of the chromosome. The mitotic stability of the telocentric chromosome derived from the right arm of chromosome IV was determined by a visual assay and found to be comparable to that of natural yeast chromosomes. Both new chromosomes duplicated, paired, and segregated properly in meiosis. Moreover, their structure, as deduced from mobilities in orthogonal field gels, did not change with continued mitotic growth or after passage through meiosis, indicating that they did not give rise to isochromosomes or suffer large deletions or additions. Thus, in S. cerevisiae the close spacing of centromeres and telomeres on a DNA molecule of chromosomal size does not markedly alter the efficiency with which it is maintained. Taken together these data suggest that there is a size threshold below which stable propagation of linear chromosomes is no longer possible. PMID- 3022141 TI - Tn10 transposition does not respond to environmental stress. PMID- 3022142 TI - Purification and properties of cytochrome c555 from Leishmania donovani. AB - A hemoprotein present in minute quantities in Leishmania donovani promastigotes was isolated, purified and spectrally characterized as cytochrome c555. Physicochemical characterization revealed electrophoretic mobility of 1.6 X 10( 5) cm2 V-1 s-1, an isoelectric point (pI) of 9.9, a relative molecular weight (Mr) of 11.9 kDa and half-wave potential (Ep) of -1.058 V. Values obtained were compared with those of horse heart cytochrome c subjected to the same analyses. Heme c555 was also prepared and spectrally characterized. The results indicated that leishmanial cytochrome c555 is similar but not identical to its crithidial counterpart. PMID- 3022144 TI - Follow-up of a patient with somatostatinoma. PMID- 3022140 TI - Tx1: a transposable element from Xenopus laevis with some unusual properties. AB - A family of transposable genetic elements in the genome of the frog, Xenopus laevis, is described. They are designated Tx1. Transposability of the elements was deduced by characterization of a chromosomal locus which is polymorphic for the presence or absence of a Tx1 element. Nucleotide sequence analysis suggested that Tx1 elements show target site specificity, as they are inserted at the pentanucleotide TTTAA in all four cases that were examined. The elements appear to have 19-base-pair (bp) inverted terminal repeats, and they are flanked by 4-bp target duplications (TTAA), although the possibility that they do not create target site duplications is discussed. Tx1 elements have several unusual characteristics: the central portion of each element is comprised of a variable number of two types of 393-bp repeating units; the rightmost 1,000 bp of the element contains separate regions potentially capable of forming bends, left handed Z-form DNA, and alternative stem-loop structures. Comparisons among single frogs suggest that germ line transposition is relatively infrequent and that variations in numbers of internal repeats accumulate quite slowly at any locus. PMID- 3022143 TI - Sequence diversity in the kinetoplast DNA minicircles of Trypanosoma cruzi. AB - Minicircles are the most abundant component of the mitochondrially located kinetoplast DNA in the members of the order Kinetoplastida. Minicircle sequences differ among most trypanosomatid species. To learn about the molecular mechanisms that give rise to this diversity, we sequenced a complete minicircle (pTckAWP-2) and two homologous but polymorphic minicircle fragments isolated from different Trypanosoma cruzi clones. Comparison of these sequences revealed 23 point mutations, 19 of which were transitions. A single base pair insertion was also detected in one of the two minicircle fragments sequenced. Analysis of pTckAWP-2 sequence showed the following features: the presence of four internal 118 base pairs conserved regions with 80% or higher homology; the fact that these four conserved regions also differed mainly by point mutations, although in this case a bias in favor of transversions was observed; the existence in each of these four regions of the highly conserved 13 bp sequence 5'GGGGTTGGTGTAA3', detected in all trypanosomatid minicircles, which is thought to be the origin of replication; and the presence of several direct and inverted repeat sequences of 8 base pairs or longer, scattered throughout the minicircle molecule. Comparison of the T. cruzi conserved minicircle region with that of other trypanosomatids showed a higher homology of T. cruzi with T. lewisi, another stercorarian trypanosome, than with African trypanosomes or Leishmania. PMID- 3022145 TI - Isolated failure of autonomic noradrenergic neurotransmission. PMID- 3022146 TI - Detection of malignant tumors. Water-suppressed proton nuclear magnetic resonance spectroscopy of plasma. AB - A sensitive and specific blood test for cancer has long been sought. The water suppressed proton nuclear magnetic resonance (NMR) spectrum of plasma is dominated by the resonances of plasma lipoprotein lipids. We measured the mean line widths of the methyl and methylene resonances, which were found to be correlated with the presence or absence of malignant tumors. Values for the average line width were lower in patients with cancer. We analyzed plasma from 331 people (normal controls, patients with malignant and benign tumors, patients without tumors, and pregnant patients); NMR analysis and measurement of line widths were blinded to diagnosis or patient group. The mean line width for 44 normal controls (+/- SD) was 39.5 +/- 1.6 Hz. For 81 patients with untreated cancer, demonstrated by biopsy, the line width was 29.9 +/- 2.5 Hz. Patients with malignant tumors were reliably distinguished from normal controls by this method (P less than 0.0001), and differed from patients with diseases that did not involve tumors (line width, 36.1 +/- 2.6 Hz; P less than 0.0001). Patients with benign tumors (e.g., those of the breast, ovary, uterus, and colon) had line widths of 36.7 +/- 2.0 Hz and were different from those with malignant tumors (P less than 0.0001). However, pregnant patients and those with benign prostatic hyperplasia had line widths consistent with the presence of malignant tumors. The narrowing of lipoprotein-lipid resonances with cancer is consistent with the response of a host to tumor growth. We conclude that these preliminary results demonstrate that water-suppressed proton NMR spectroscopy is a potentially valuable approach to the detection of cancer and the monitoring of therapy. PMID- 3022147 TI - Lung cancer: scalpels, beams, drugs, and probes. PMID- 3022148 TI - Glucagon receptor number and the MHC. PMID- 3022149 TI - Calbindin immunoreactivity alternates with cytochrome c-oxidase-rich zones in some layers of the primate visual cortex. AB - Calcium ions have a pivotal role in many neuronal activities, but little is known about their involvement in the cortical processing of visual information. Using immunohistochemical methods, we have now detected a calcium-binding protein, calbindin-D-28K, which may confer on certain compartments of cortical area 17 the ability to modulate Ca2+ metabolism. Thus, calbindin occurs in the primate striate cortex in a pattern almost complementary to that displaying strong cytochrome c-oxidase activity. From this and other observations, we deduce that the distribution of calbindin-immunoreactive sites corresponds mainly to extra geniculocortical connections of the primary visual cortex. This implies that the geniculocortical and extra-geniculocortical compartments of area 17 differ in an intracellular system for Ca2+ homeostasis. PMID- 3022150 TI - Isolation and expression of a complementary DNA that confers multidrug resistance. AB - The emergence and outgrowth of a population of tumour cells resistant to multiple drugs is a major problem in the chemotherapeutic treatment of cancer. We have used highly drug-resistant cell lines developed in vitro to study the molecular basis of multidrug resistance. In these cell lines high levels of resistance are frequently associated with amplification and overexpression of a small group of genes termed mdr or gp170. Direct evaluation of the role of these genes in multidrug resistance has awaited the isolation of a member of this gene family in a biologically active form. Here we report the isolation of DNA clones complementary to the cellular messenger RNA transcripts of mdr genes and show that high-level expression of a full-length complementary DNA clone in an otherwise drug-sensitive cell confers a complete multidrug-resistant phenotype. Our results demonstrate that overexpression of a single member of the mdr group is sufficient to confer drug resistance. Furthermore, because the cDNA was isolated from a drug-sensitive cell, mutations in the primary sequence of mdr are not required to produce a multidrug-resistance phenotype. PMID- 3022151 TI - A 3' enhancer is required for temporal and tissue-specific transcriptional activation of the chicken adult beta-globin gene. AB - The chicken adult beta-globin gene is one of the more intensively investigated developmentally regulated loci in higher eukaryotes. Detailed molecular analysis of the locus allows precise examination of the chromosomal changes that occur on activation of the gene during erythroid maturation. The best studied of these changes are the acquisition of DNase I hypersensitivity, developmentally correlated alteration of CpG-specific cytosine methylation patterns and in vitro assembly of erythroid-specific protein complexes 5' to the gene that mimics in vivo creation of the 5' DNase I hypersensitive 'region' lying 60 to 260 nucleotides 5' to the beta-globin cap site in red blood cell chromatin. Here we demonstrate that proximal beta-globin DNA sequences lying greater than 112 base pairs (bp) 5' to the cap site are not involved in determining the erythroid specific induction characteristics of this gene in transient expression assays, whereas an enhancer sequence within a 300-bp PvuII fragment lying approximately 400 nucleotides 3' to the polyadenylation signal is intimately involved in determining the erythroid cell specificity and correct time of induction of beta globin transcription during red cell maturation. PMID- 3022152 TI - Origin of HTLV-I virus in Japan. PMID- 3022153 TI - Compensatory mutations suggest that base-pairing with a small nuclear RNA is required to form the 3' end of H3 messenger RNA. AB - Processing of the 3' end of sea urchin H3 histone pre-mRNA requires conserved sequence elements and the presence of U7 snRNA. A mutation in the conserved CAAGAAGA sequence of the H3 pre-mRNA that renders 3' processing of this precursor defective is shown to be suppressed by a compensatory change in the U7 snRNA, restoring the base-pairing potential of the two RNAs. RNA-RNA contacts between these two molecules appear to be an essential feature of the 3' processing reaction. PMID- 3022155 TI - Antiviral effects of recombinant tumour necrosis factor in vitro. AB - Tumour necrosis factor (TNF) was first described as a factor in the serum of mice injected with tubercle bacilli (BCG) and several days later with lipopolysaccharide (LPS). The gene encoding TNF has recently been cloned and pure recombinant human TNF is now available. TNF is known for its in vivo antitumour effect and in vitro cytotoxicity on certain transformed cell lines. Similarities in amino acid sequence and biological activity to lymphotoxin and cachectin have been reported, and very recently a growth-factor-like activity on diploid fibroblasts was observed. There is no similarity between these proteins and interferons (IFNs), which are also induced during in vivo induction of TNF. Here we describe the antiviral activity of pure recombinant human TNF in a typical in vitro antiviral assay which we discovered while investigating the possible role of TNF as an inducer of IFN. PMID- 3022154 TI - cGMP-dependent protein kinase enhances Ca2+ current and potentiates the serotonin induced Ca2+ current increase in snail neurones. AB - Protein phosphorylation catalysed by cyclic AMP-dependent, Ca2+/calmodulin dependent and Ca2+/diacylglycerol-dependent protein kinases is important both in the modulation of synaptic transmission and in the regulation of neuronal membrane permeability (for reviews see refs 5-7). However, there has previously been no evidence for the involvement of cyclic GMP-dependent protein kinase (cGMP PK) in the regulation of neuronal function. Serotonin induces an increase of Ca2+ current in a group of identified ventral neurones of the snail Helix aspersa. This effect is probably mediated by cGMP because it is mimicked by the intracellular injection of cGMP or the application of zaprinast, an inhibitor of cGMP-dependent phosphodiesterase. We have now found that the effect of either serotonin or zaprinast on the Ca2+ current is potentiated by the intracellular injection of cGMP-PK. Moreover, the intracellular injection of activated cGMP-PK (cGMP-PK + 1 microM cGMP) greatly enhances the Ca2+ current of the identified ventral neurones seen in the absence of serotonin. These results indicate that cGMP-PK has a physiological role in the control of the membrane permeability of these neurones. PMID- 3022156 TI - Treatment of locally advanced breast cancer using primary induction chemotherapy with hormonal synchronization followed by radiation therapy with or without debulking surgery. AB - Fifty-one patients with stage IIIA or IIIB locally advanced breast cancer received primary induction chemotherapy (hormonal synchronization, 46 patients) to a maximum objective clinical response before proceeding to local therapy. Patients achieving a pathological complete response (CR) received radiation therapy, while patients with residual disease, partial response (PR), or stable disease (NC) received debulking surgery prior to radiation therapy; in all patients, 6 additional months of chemotherapy were administered. Chemotherapy consisted of cyclophosphamide (500 mg/m2 iv) and doxorubicin (30 mg/m2 iv) on day 1; tamoxifen (40 mg/m2 orally) on days 2-6; Premarin (0.625 mg/m2 orally) every 12 hours for three doses, beginning on day 7; and methotrexate (300 mg/m2 iv) followed in 1 hour by 5-fluorouracil (500 mg/m2 iv) on day 8 and leucovorin rescue (10 mg/m2 orally) every 6 hours for six doses, beginning 24 hours after methotrexate. Fifty patients are evaluable with respect to response, time to disease progression, and survival. The rate of objective response to chemotherapy was 90%; 52% of the patients had CR, 38% had PR, and 10% had NC. The median numbers of chemotherapy cycles to achieve CR, PR, and NC were five, four, and four, respectively. Twenty-four patients with CR to chemotherapy were pathologically assessed, 22 by multiple biopsies and 2 by mastectomy. Seventy-one percent of the patients were proven to have pathological CR. All patients completing combined therapy thus far are disease free. Nine of 29 patients with stage IIIB disease have relapsed; 7 of 23 with inflammatory histology have relapsed; and 2 of 20 with stage IIIA disease have relapsed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022158 TI - Evidence for prejunctionally located beta 2-adrenoceptors in the cat spleen. AB - The number of beta-adrenoceptors in myocardium and spleen from 6-hydroxydopamine (6-OH-DA) treated cats was determined by radioligand binding with [125I] iodohydroxybenzylpindolol (IHYP). The effectiveness of 6-OH-DA pretreatment was assessed by analyses of the tissue content of catecholamines and the contractile response of isolated splenic strips to electrical stimulation. Since no effect on the splenic strip was produced by the beta-agonist isoprenaline, whereas noradrenaline caused contraction, it is concluded that the smooth muscle of the splenic capsule is controlled by postjunctional alpha-adrenoceptors. The number of specific IHYP binding sites were reduced by 70% in whole spleen tissue and totally abolished in the splenic capsule by pretreatment with 6-OH-DA. Subclass analysis revealed that the reduction in total splenic beta-adrenoceptor number was due to a loss of beta 2-adrenoceptors. However, the 6-OH-DA induced chemical sympathectomy did not produce any alteration either in beta-adrenoceptor density or the relative distribution of the beta-adrenoceptor subtype in the myocardium. It is suggested that a loss of prejunctional beta-adrenoceptors, due to chemical sympathectomy, might be compensated for by an increased number of postjunctional beta-adrenoceptors in the myocardium due to the development of denervation supersensitivity in this tissue. In conclusion, the findings provide direct biochemical evidence for existence of prejunctional beta 2-adrenoceptors on the sympathetic nerve terminals of the cat spleen. PMID- 3022159 TI - Activation of presynaptic alpha 2-adrenoceptors attenuates the inhibitory effect of mu-opioid receptor agonists on noradrenaline release from brain slices. AB - 3H-noradrenaline release from rat neocortical slices induced by 15 mM K+ was concentration-dependently inhibited by morphine, [D-Ala2-D-Leu5] enkephalin (DADLE) and the calcium entry blocker Cd2+. Blockade of presynaptic alpha 2 adrenoceptors with phentolamine, almost doubling K+-induced 3H-noradrenaline release, slightly enhanced the relative inhibitory effects of morphine and DADLE, whereas that of Cd2+ remained unaffected. In contrast, activation of presynaptic alpha 2-adrenoceptors with clonidine (1 microM) or TL-99 (1 microM), inhibiting release by about 50%, completely abolished the inhibitory effects of morphine and DADLE without affecting that of Cd2+. When in the presence of 1 microM clonidine adenylate cyclase was activated with forskolin (10 microM), which restored release to the drug-free control level, the opioids still did not display their inhibitory effects. Therefore, mu-opioid receptor efficacy appears to be dependent on the degree of activation of alpha 2-adrenoceptors in central noradrenergic nerve terminals, probably through a local receptor interaction within the nerve terminal membrane. PMID- 3022157 TI - Effects of a selective 5-HT reuptake blocker, citalopram, on the sensitivity of 5 HT autoreceptors: electrophysiological studies in the rat brain. AB - Citalopram (CIT), is a selective serotonin (5-HT) reuptake blocker and a clinically effective antidepressant. The present electrophysiological studies were undertaken to investigate in vivo the acute and long-term effects of CIT administration on 5-HT neurotransmission. In a first series of experiments, a single dose of CIT (0.05-0.5 mg/kg) was administered intravenously to naive rats while recording the activity of a 5-HT-containing neuron in the nucleus raphe dorsalis. A dose-response relationship of the inhibitory effect of CIT on the firing activity of 5-HT neurons was obtained with an ED50 of 0.23 +/- 0.03 mg/kg. In a second series of experiments, rats were treated with CIT (20 mg/kg/day, i.p.) for 2, 7 and 14 days. In rats treated for 2 days, there was a marked reduction in the firing activity of 5-HT neurons in the nucleus raphe dorsalis; there was a partial recovery after 7 days and a complete recovery after 14 days of treatment. The response of 5-HT neurons to intravenously administered LSD was decreased in rats treated for 14 days with CIT, indicating a desensitization of the somatodendritic 5-HT autoreceptor. In a third series of experiments, carried out in rats treated with CIT (20 mg/kg/day, i.p.) for 14 days, the suppression of firing activity of CA3 hippocampal pyramidal neurons produced by microiontophoretically-applied 5-HT and by the electrical activation of the ascending 5-HT pathway was measured. Long-term treatment with CIT did not modify the responsiveness of these neurons to microiontophoretically-applied 5 HT.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022160 TI - Evidence for a displaceable non-specific [3H]neurotensin binding site in rat brain. AB - Levocabastine is a potent antihistamine drug, structurally unrelated to neurotensin. In rat and mouse brain but not in other animal species, it inhibited 60% of the [3H]neurotensin binding displaced by unlabelled neurotensin or neurotensin(8-13). The levocabastine-sensitive site or "site 1" displayed high affinity properties for levocabastine (IC50 = 25 nM) and was highly sterospecific (IC50-value higher than 10 microM for one of the isomers). Binding to the "site 1" in rat brain corresponded to the [3H]neurotensin binding displaceable by 1 microM levocabastine, whereas binding to the "site 2" corresponded to the binding displaced by 1 microM neurotensin when the "site 1" was occluded by 1 microM levocabastine. Both "site 1" and "site 2" appeared to be saturable. Scatchard plots obtained in rat bulbus olfactorius allowed to calculate a KD-values of 7.1 nM and a Bmax-values of 37.2 fmol/mg original tissue for "site 1", while "site 2" displayed a KD-value of 0.7 nM and a Bmax-value of 16.3 fmol/mg original tissue. The regional distributions of both sites showed marked differences. The "site 1" was homogeneously distributed throughout all rat brain areas, whereas the amount of "site 2" binding was markedly different in separate brain areas: bulbus olfactorius and substantia nigra had the highest amounts (8.9 and 7.8 fmol/mg tissue) while cerebellum had the lowest (0.4 fmol/mg tissue). In spite of its high affinity and stereospecificity, "site 1" has to be considered as an acceptor or recognition site for [3H]neurotensin because of its species-link, low saturability and homogeneous distribution in all rat brain areas.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022163 TI - [Cisplatin in the treatment of testicular carcinoma]. PMID- 3022162 TI - [Calcium entry blockers and angiotensin converting enzyme antagonists in the treatment of high blood pressure]. PMID- 3022161 TI - Effects of sulfhydryl agents on neuromuscular transmission in the presence or absence of cadmium. AB - To test whether thiol groups are involved in neuromuscular transmission and its blockade by cadmium, the influence of sulfhydryl reagents were investigated on neuromuscular transmission, in the presence or absence of cadmium, using isolated mouse phrenic nerve-diaphragm muscles. Cadmium attenuated twitches evoked by indirect shocks but had almost no effect on twitches by direct shocks in the presence of d-tubocurarine (2.5 microM). The inhibitory effect was reversed by cysteine or by an increase in external calcium. The sulfhydryl oxidizing agent DTNB 5,5'-dithiobis(2-nitrobenzoic acid) potentiated the inhibitory effect of cadmium. Conversely, the reducing agent dithiothreitol (DTT) inhibited this effect. The potentiation by DTNB could be reversed by subsequent application of DTT. The sulfhydryl reagents had no influence on neuromuscular transmission in the absence of cadmium. Neuromuscular transmission was inhibited by the removal of calcium. This inhibition could be reversed by readmission of calcium. This restoration was neither potentiated nor reduced in the presence of these sulfhydryl reagents: It is suggested that an interaction of cadmium with thiol groups is independent of its calcium antagonistic action on neuromuscular transmission. Thiol groups may protect the transmission from the effect of cadmium. PMID- 3022164 TI - [The extra-mammary form of Paget's disease]. PMID- 3022165 TI - [Inhibitory action of dopamine on excitatory transmission in the isolated spinal cord of the rat]. AB - The effect of dopamine on the ventral root potential evoked by single supramaximal dorsal root stimulation was studied in the experiment in the isolated superfused spinal cord of 10-16 days old rats. It was discovered that application of dopamine caused the reversible dose-dependent inhibition of the mono- and polysynaptic ventral root reflex responses. The minimal effective concentration was 1 X 10(-8) mol/l. Dopamine applied in concentrations of 1 X 10( 4) mol/l and 1 X 10(-3) mol/l decreased the amplitude of the monosynaptic ventral root potential by 20% and 87% as against the control, respectively. Under the same conditions the amplitude of the polysynaptic ventral root potential decreased by 18% and 87% as against the control, respectively. The obtained results demonstrate that the dopaminergic brainstem-spinal pathways take part in the control of the transmission in the segmentary reflex arc. PMID- 3022166 TI - [Effect of thiamine on various types of synaptic junctions]. AB - Thiamine (1.10(-14) -- 1.10(-4) mol/l) reversibly increased the frequency of miniature excitatory postsynaptic potentials, amplitude and quantal content of excitatory postsynaptic potentials in crayfish glutaminergic synapse. Thiamine also increased spontaneous electrical activity and amplitude of synaptic potentials in guinea-pig taenia coli. In synaptosomes from the rat brain thiamine produced depolarization of nerve endings. The role of thiamine in the regulation of synaptic transmission and mechanism of its action are discussed. PMID- 3022167 TI - [Postsynaptic potentiation and desensitization at the myoneural synapse of the frog induced by rhythmic stimulation of a motor nerve]. AB - The contribution of postsynaptic potentiation (PSP) and desensitization (DS) to the changes in the amplitude and time course of miniature end-plate currents (mepc) recorded after 10 Hz repetitive stimulation of the motor nerve during 5 or 60 s was studied in the experiments on "cut" nerve-muscle preparation with inhibited acetylcholinesterase. After the short (5 s) train the mepc amplitude did not differ from the initial one while the decay time constant (tau mepc) increased by 32% (indication of PSP). The decrease of mepc amplitude by 23% was observed after the end of the long (60 s) train, while tau mepc did not differ from the original one (indication of DS). Similar but more marked two-phase changes occurred in the time course of end-plate currents. These effects were not observed when acetylcholinesterase was active. The PSP and DS manifestations were reproduced with acetylcholine addition into the bath. It was possible to change the ratio of PSP and DS under the action of aprodifen. PMID- 3022169 TI - [Changes in the excitability of motor neurons and synaptic effects on them during the formation of a locomotor generator in the cat]. AB - It is shown on decorticated (high-decerebrated) and spinal cats and on spinal cats injected with DOPA that the activation of the spinal locomotor generator is accompanied by an increase of the extensor and a decrease of the flexor motoneurons' excitability, an increase of the recurrent and reciprocal Ia inhibition, a decrease of Ib and flexor reflex afferent influences on motoneurons. The possible physiological role of changes in tuning of the spinal segmental apparatus in the generation of the locomotor rhythm is discussed. PMID- 3022168 TI - [Effect of thiamine on auditory evoked potentials of the guinea pig]. AB - Application of thiamine (1 X 10(-11)-1 X 10(-3) mol/l) on the membrane of cochlear round window increased the amplitude and decreased the latency of the auditory nerve action potential, waves I and II of brainstem auditory-evoked electrical potentials in response to acoustic clicks of different intensity. The mechanism of thiamine action on auditory-evoked electric potentials is discussed. PMID- 3022170 TI - [Effect 4-aminopyridine on the recovery of segmental reflexes after sectioning of the sciatic nerve in the rat]. AB - L5 ventral root reflex discharges evoked by dorsal root stimulation were studied 3-4 weeks after transection of sciatic nerve in rats; only polysynaptic transmission was found on the side of operation. Injection of 4-aminopyridine (500-600 mg/kg, intraperitoneally) resulted in facilitation of reflex discharges on the intact side and in reappearance of intensive monosynaptic component in such discharges on the affected side. It is concluded that 4-aminopyridine recovers monosynaptic transmission between primary afferents and chromatolysed motoneurons because of increased transmitter liberation. PMID- 3022171 TI - Linitis plastica of the stomach and entire intestine. PMID- 3022172 TI - Small bowel carcinoma and Crohn's disease. PMID- 3022173 TI - [Use of the F wave for detection of preclinical changes in the peripheral nerves in alcoholics]. AB - Orthodromic conduction in motor fibres and F wave were analysed in the median, ulnar, peroneal and tibial nerves in groups of 30 subjects: one control and one comprising patients with chronic alcoholism without detectable clinically damage to the peripheral nervous system. Abnormalities were demonstrated in both these analysed parameters, but they were more pronounced and were present in a greater proportion of cases in the assessment of the F wave. In the light of these results the determination of this response may be a useful supplementary method in the investigation of peripheral nerves, especially in the stage of preclinical lesions. PMID- 3022174 TI - Peripheral neuropathy and refractory periods of motor nerves. PMID- 3022175 TI - A potent factor in extracts of the skin of the Australian frog, Pseudophryne coriacea--III. Potentiation of contractions elicited in avian and mammalian isolated skeletal muscle preparations by direct and indirect electrical stimulation. AB - Extracts of the skin of the Australian frog Pseudophryne coriacea displayed a striking potentiating effect on contractions evoked in isolated skeletal muscle preparations of mammals (phrenic nerve diaphragm) and birds (chick biventer cervicis and semispinalis muscles) by indirect and direct electrical stimulation. There was both a conspicuous increase in the amplitude of the twitch, up to 10 fold, and a remarkable prolongation of the duration of the twitch. The effect was dose- and frequency-dependent. In the presence of the extract, fusion of twitches after tetanic stimulation occurred earlier. No tachyphylaxis upon repeated stimulation by the extract was observed and the response to large doses persisted, declining slowly, for hours. These effects must be ascribed to an alkaloid related in structure to pumiliotoxin B. Response to the extract of Pseudophryne coriacea by indirectly-stimulated preparations was potentiated by physostigmine and blocked by tubocurarine and alpha-bungarotoxin, demonstrating that in these preparations the extract acted pre-synaptically to facilitate the release of acetylcholine from motor nerve endings. However, the extract of Pseudophryne coriacea displayed equally potent effects in directly stimulated preparations, insensitive to physostigmine and to blockers of nicotinic acetylcholine receptors, indicating a direct action on the skeletal muscle. It is suggested that, like pumiliotoxin B, the Pseudophryne coriacea alkaloid may interfere in the regulation of calcium channels in both nerve and muscle fibres. PMID- 3022176 TI - The enkephalinase inhibitor, GB 52, does not affect nociceptive flexion reflexes nor pain sensation in humans. AB - The effects of intravenous administration of 2.5 mg/kg of GB 52, a highly potent derivative of the enkephalinase inhibitor, thiorphan, were studied on the threshold of both the nociceptive reflex (Tr) and sensation of pain (Tp) as well as on the thresholds of both recruitment of the maximal nociceptive reflex response (Tmr) and tolerable pain (Tip), elicited by electrical stimulation of the sural nerve in normal and relaxed volunteers. It was found that neither the nociceptive motor responses (Tr and Tmr) nor the subjective reports of pain (Tp and Tip), were significantly affected by GB 52. It is concluded that, in the experimental conditions used, the transmission of nociceptive messages at the spinal level is not tonically modulated by any enkephalinergic system. PMID- 3022177 TI - The adenylate cyclase rebound response to naloxone in the NG108-15 cells. Effects of etorphine and other opiates. AB - The adenylate cyclase (AC) of the neuroblastoma-glioma hybrid cells (NG108-15), is generally considered to be a model for the study of the biochemical correlates of opiate tolerance and dependence. However, the naloxone-induced rebound response of adenylate cyclase, described in some recent reports, is much smaller than that originally described by Sharma, Klee and Nirenberg (1975). Possible explanations for these discrepancies are: (1) a marked down-regulation of opioid receptors and tolerance produced by the use of delta agonists or (2) the use of etorphine, a relatively hydrophobic drug which has slower dissociation rates than morphine. To test these possibilities, neuroblastoma-glioma hybrid cells were treated cells with morphine, etorphine, [D-Ala2,D-Leu5]enkephalin (DADLE), [D Ala2]Leu-enkephalinamide (DALAMID) or vehicle. In addition, some of the cells treated with etorphine were washed with DADLE to replace the etorphine without producing the rebound response of adenylate cyclase prior to the addition of naloxone. The cells treated with morphine, DADLE and DALAMID, and incubated with prostaglandin E1 (PGE1) and naloxone showed a significant rebound of adenylate cyclase when compared with control groups and opiate-treated cells, incubated only with PGE1. In contrast, naloxone did not induce any significant rebound response in cells treated with etorphine unless they were previously washed with DADLE. These results demonstrate that the lack of a rebound response in cells treated with etorphine was due to the slow dissociation rates of the opiate and not to tolerance or to down-regulation of opioid receptors produced by agonists of high intrinsic activity. PMID- 3022178 TI - Tifluadom-induced diuresis in rats. Evidence for an opioid receptor-mediated central action. AB - Tifluadom dose-dependently induced diuresis in rats after subcutaneous injection and oral application. (+)Tifluadom was at least 100-fold more potent than the ( )enantiomer in inducing diuresis. The diuretic action of tifluadom was dose relatedly reduced by the opioid receptor antagonists naloxone and MR 2266. Naloxone methobromide did not antagonize the diuretic effect of tifluadom nor did the benzodiazepine receptor blocker Ro 15-1788. These data demonstrate that the diuretic effect of tifluadom is mediated centrally via an agonistic interaction between the drug and opioid receptors. PMID- 3022179 TI - Evidence for a role of 5-hydroxytryptamine in the responses of rat raphe magnus neurones to peripheral noxious stimuli. AB - The role of serotoninergic mechanisms in the responses of neurones in the nucleus raphe magnus to peripheral noxious stimuli was investigated in rats anaesthetized with halothane. In normal animals a large proportion of neurones responded to peripheral noxious stimuli with excitation or inhibition, the direction of the response being dependent on the spontaneous activity of the neurone. Pretreatment with p-chlorophenylalanine (pCPA; 300 mg/kg X 3 days) led to a marked reduction in the number of cells responding to peripheral stimuli. Similarly, the injection of metergoline (5 mg/kg, i.v.) in normal animals caused a reduction in the magnitude of the neuronal responses to noxious stimuli. The results are discussed with regard to the role of serotoninergic mechanisms in the control of nociceptive transmission by this nucleus. PMID- 3022180 TI - Pharmacological analysis of the behavioural and thermoregulatory effects of the putative 5-HT1 receptor agonist, RU 24969, in the rat. AB - The roles of recognition sites for central neurotransmitters in the mediation of the behavioural effects of the putative 5-hydroxytryptamine (5-HT1) receptor agonist, RU 24969 [5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)1H indole] in the rat have been examined. The drug RU 24969 was found to have high affinity for 5 HT1A and 5-HT1B recognition sites. Hyperlocomotion, induced by RU 24969, was enhanced in animals depleted of 5-HT with 5,7-dihydroxytryptamine, suggesting an involvement of 5-HT receptors in the mediation of this behaviour. However, results of experiments with 5-HT receptor antagonists argued against the receptors being of either the 5-HT1 or 5-HT2 type. Despite the negligible affinity of RU 24969 for catecholamine receptors, hyperlocomotion induced by RU 24969 was clearly dependent on intact catecholamine systems. When hyperlocomotion was blocked by treatment with reserpine, reciprocal forepaw-treading and a flat body posture, behavioural responses which are consistent with activation of the putative 5-HT1A receptor, became evident. When animals were restrained from moving, RU 24969 dose-dependently reduced body temperature, an effect that may also be associated with activation of the 5-HT1A recognition site. Thus, although the mechanism by which RU 24969 induces hyperlocomotion is not yet established, the agonist nevertheless can induce functional responses consistent with its high affinity for the 5-HT1A recognition site. PMID- 3022181 TI - Folate induced-hypermotility response after bilateral injection into the nucleus accumbens of the rat. Possible mediation through dopaminergic mechanisms. AB - Folic acid (FA) and certain of its reduced congeners produce excitatory effects when applied to neuronal tissue. Recent evidence has suggested that folates have other biological properties in common with the excitatory amino acids. The purpose of this study was to determine the activity of folate compounds in a system sensitive to excitatory amino acids. Bilateral injection of folic acid into the nucleus accumbens resulted in a marked increase in locomotor activity at doses of 2.5 and 5 micrograms. Larger doses resulted in behavioral responses, such as body tremor and labored breathing, which interfered with the locomotor response. Similarly, 5-formyltetrahydrofolic acid (FTHF) produced a marked hypermotility response after bilateral injection into the nucleus accumbens (2.5 25 micrograms), while dihydrofolic acid, tetrahydrofolic acid, and 5 methyltetrahydrofolic acid were ineffective. Pretreatment with reserpine (10 mg/kg, i.p.) markedly reduced the hypermotility response elicited by folic acid and FTHF as did pretreatment with haloperidol in both peripheral (0.8 mg/kg) and direct (5 micrograms) injection into the nucleus accumbens. In addition, injection of muscimol (30 ng), which depresses hypermotility induced by dopamine and amphetamine, produced a significant decrease in the hypermotility response produced by folic acid. In contrast, pretreatment with phentolamine (5 mg/kg, i.p.) or propranolol (4 mg/kg, i.p.) did not decrease folic acid or FTHF-induced responses. These results suggest that folic acid and FTHF produce an increase in locomotor activity by facilitating dopaminergic neurotransmission in the nucleus accumbens, possibly by inducing the release of dopamine from the nerve terminals. Thus, these folates have effects similar to those of the excitatory amino acids when injected into the nucleus accumbens. PMID- 3022182 TI - The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on striatal and limbic catecholamine neurones in white and black mice. Antagonism by monoamine oxidase inhibitors. AB - Albino mice and pigmented mice were treated for 6 days with 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) at the maximum tolerated doses (2 days at 30 mg/kg i.p., 2 days at 40 mg/kg i.p. and 2 days at 50 mg/kg i.p. in white mice, 6 days at 30 mg/kg i.p. in pigmented mice) and the effects of simultaneous treatment with the monoamine oxidase inhibitors, deprenyl (1 mg/kg, i.p.), MDL 72145 (0.5 mg/kg, i.p.) and clorgyline (5 mg/kg, i.p.), determined behaviourally (daily for 6 days and for 4 days after withdrawal) and biochemically (92 hr after withdrawal of drug). In albino mice MPTP caused depletions of dopamine (90%), dihydroxyphenylacetic acid (DOPAC; 82%) and homovanillic acid (HVA; 65%) in the striatum and in dopamine (54%), DOPAC (51%) and HVA (53%) in the nigra. However, MPTP was not selective in its action since the levels of dopamine and its metabolites were also reduced in limbic tissue. Further, MPTP affected the function of noradrenaline, with reduced levels in tissues of the striatum (74%) and nigra (46%). Pigmented mice were as susceptible as albino mice to the actions of MPTP to reduce the levels of dopamine and metabolites in the striatum. However, the limbic areas and substantia nigra of the pigmented mouse were more resistant to the actions of MPTP. Treatment with deprenyl and MDL 72145 (but not clorgyline) could be shown to reduce the biochemical and behavioural consequences of the action of MPTP (although behavioural changes, development of severe motor incapacitation and prostrate appearance, appeared to be non-specific).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022184 TI - Is histamine an anticonvulsive inhibitory transmitter? AB - This study was done to investigate the possible role of histaminergic systems in electroshock seizures. Brain histamine concentrations in rats were elevated by metoprine (i.p.), an inhibitor of histamine-N-methyltransferase. Animals were tested for their response to maximal electroshock (MES) at different times after the injection. Metoprine raised brain histamine concentrations and inhibited maximal hindleg extension after MES in a dose-dependent manner. Sensitivity to seizures correlated inversely with histamine concentrations. These results suggest that histaminergic neurones are involved in mechanisms which inhibit generalizations of epileptic discharges in the brain. PMID- 3022183 TI - The use of cyclic AMP efflux studies in attempts to determine the effects of morphine on cyclic AMP formation in striatal slices. AB - The effect of morphine and naloxone on basal and forskolin-stimulated efflux of cyclic AMP from rat striatal slices was examined. Neither morphine nor naloxone had any consistent effect on the basal efflux of cyclic AMP. Forskolin produced a time and dose-dependent enhancement of cyclic AMP efflux. Neither morphine nor naloxone affected this forskolin-enhanced release. These results suggest that measurements of cyclic AMP released from brain slices do not accurately reflect effects on adenylate cyclase inhibition by opiates. PMID- 3022185 TI - The effects of intracerebroventricular injection of clonidine on conditioned pressor and adrenergic responses in rats. AB - Studies from this laboratory have shown that the first filial offspring of female spontaneously-hypertensive rats and male Wistar-Kyoto (WKY) normotensive rats develop stress-induced hypertension. The present study sought to examine the effects of intracerebroventricular administration of clonidine (8 micrograms) on cardiovascular and sympathoadrenal responses to aversive classical conditioning in these borderline hypertensive rats (BHR) and in normotensive WKY control rats. Clonidine caused significant reductions in resting arterial pressure, vascular resistance, heart rate and concentrations of epinephrine (E) in plasma for both hypertensive and normotensive rats. Central administration of normal saline to control rats of each strain did not alter basal cardiovascular or sympathoadrenal function. The presentation of a conditioned stimulus (CS) elicited a significant increase in arterial pressure and total peripheral resistance in hypertensive rats treated with saline and clonidine and in normotensive rats treated with saline. In contrast, normotensive rats treated with clonidine showed no increases in arterial pressure or vascular resistance following the onset of the conditioned stimulus. The aversive conditioning session instigated significant increases in the concentrations of norepinephrine (NE) and E in plasma in saline treated rats. Hypertensive and normotensive rats treated with clonidine-showed a blunted increase in plasma concentrations of NE and E during this period; however, concentrations of E in hypertensive rats increased significantly from the baseline period after injection. These data suggest that an abnormality in central alpha 2-adrenoceptor-mediated inhibition of sympathoadrenal discharge and sympathetic vasomotor tone may predispose the hypertensive rat to develop stress induced hypertension. PMID- 3022186 TI - Studies on the mesolimbic loop of antinociception--II. A serotonin-enkephalin interaction in the nucleus accumbens. AB - In a previous report we have shown that the antinociceptive effect elicited by microinjection of morphine into the periaqueductal gray is due, at least in part, to the activation of an ascending serotonergic pathway which releases 5 hydroxytryptamine in the nucleus accumbens. We now report that antinociception induced by intra-periaqueductal gray injection of morphine can be attenuated also by the narcotic antagonist naloxone or the enkephalin antibodies administered into the nucleus accumbens, and potentiated by D-phenylalanine, a putative inhibitor of the degradation of enkephalins. Moreover, the antinociceptive effect induced by 5-hydroxytryptamine administered into nucleus accumbens could be blocked by naloxone injected into the same site, whereas the antinociception elicited by intra-accumbens injection of [D-Ala2,D-Leu5]enkephalin was not affected by cinanserin, a 5-hydroxytryptamine blocking agent. It is concluded that morphine administered to the periaqueductal gray is capable of activating an ascending serotonergic pathway to release 5-hydroxytryptamine in the nucleus accumbens, which in turn activates an enkephalinergic mechanism within the same nucleus, resulting in an antinociceptive effect. PMID- 3022188 TI - Alzheimer's disease and hypothalamic-mediated endorphinergic mechanisms. PMID- 3022187 TI - Prenatal ontogeny of the GABAergic system in the rat brain: an immunocytochemical study. AB - Prenatal development of the GABAergic system in the rat brain has been studied using an antiserum to GABA-glutaraldehyde-hemocyanin conjugates, specific for GABAergic neurons. The gamma-aminobutyric acid (GABA) system has been found to differentiate very early relative to other transmitter-identified neurons, such that by embryonic day 13 a well developed fiber network exists in the brainstem, mesencephalon and diencephalon, including a large projection in the posterior commissure and adjacent areas on the surface of the mesencephalon and tectum. Although no cell bodies are visible at this time, it appears that these fibers originate from the caudal brainstem and spinal cord. GABAergic cell bodies begin to appear on embryonic day 14 in the lateral cortical anlage. By embryonic day 16, they are also visible in the basal forebrain and in all regions of cortex where they are located in three zones: in layer I, below the cortical plate, and in the intermediate zone. Also contained in the outer part of layer I is a dense fiber plexus which stains intensely for GABA. These fibers may be part of the first contingent of cortical afferents to invade the telencephalic vesicle, an event which is thought to be a stimulus for the beginning of neuronal differentiation in this region. By E18, two bands of immunoreactivity are visible in layer I, which probably contain both cell bodies and fibers. The trajectories taken by growing GABAergic fibers in the brainstem, mesencephalon and diencephalon at embryonic day 13 and at subsequent stages of development are coincident with regions of both monoaminergic and peptidergic differentiation and appear to correspond to recently reported patterns of benzodiazepine receptors which appear slightly later. The early differentiation of the GABAergic system could indicate a trophic role for GABA in early brain development, possibly involving receptors for this neurotransmitter or related substances. PMID- 3022189 TI - Perianal mucinous adenocarcinoma and fistula-in-ano. PMID- 3022190 TI - 4-Aminopyridine-mediated increase in long-term potentiation in CA1 of the rat hippocampus. AB - The effect of 4-aminopyridine (4-AP) on long-term potentiation (LTP) was studied in the hippocampal slice preparation of the rat. Field excitatory postsynaptic potentials (EPSPs) were recorded and evoked in the stratum radiatum of the CA1. Both the low frequency EPSP and LTP of the EPSP were significantly increased by treatment with 4-AP. These effects were inhibited by increasing the magnesium concentration from 1 to 4 mM. Pretreatment with 20 microM DL-2-amino-5 phosphonovalerate antagonized only the increase in LTP produced by 4-AP. It is suggested that 4-AP enhances Ca influx either pre- or postsynaptically and thereby increases LTP. PMID- 3022191 TI - The enhancement of diazepam and muscimol binding by pentobarbital and (+) etomidate: size of the molecular arrangement estimated by electron irradiation inactivation of rat cortex. AB - Synaptosomal membranes were prepared from frozen rat cortices irradiated by 10 MeV electrons and the enhancement of [3H]diazepam and [3H]muscimol binding by pentobarbital (PB) and (+)-etomidate was studied. The target sizes of the corresponding parts of the receptor complex were estimated from the decrease in the enhancement as a function of irradiation dose. Different radiation inactivation constants suggest different regulatory units for the enhancement by PB and (+)-etomidate. Target sizes for PB and (+)-etomidate enhancement of [3H]diazepam binding were 127 +/- 14 and 360 + 124 kDa, respectively. PMID- 3022192 TI - Long-term potentiation of guinea pig mossy fiber responses is not blocked by N methyl D-aspartate antagonists. AB - Receptors preferentially activated by the excitatory amino acid N-methyl-D aspartate (NMDA) do not mediate synaptic transmission in the hippocampus but are involved in initiating long-term potentiation (LTP) in hippocampal region CA1. We have examined the role of NMDA receptors in LTP of the commissural/associational and mossy fiber pathways to region CA3 pyramidal neurons. In the commissural/associational pathway, NMDA receptor blockers did not reduce synaptic responses but reversibly blocked the induction of LTP. In contrast, NMDA receptor blockers had no effect on mossy fiber LTP. These results suggest that induction of commissural/associational LTP differs from mossy fiber LTP, although the mechanisms underlying expression of LTP along these pathways could be similar. Kynurenate and L-2-amino-4-phosphonobutyrate, which potently reduce mossy fiber responses, also did not block induction of mossy fiber LTP. PMID- 3022193 TI - Augmentation of succinylcholine-induced neuromuscular blockade by calcium channel antagonists. AB - The block of endplate currents (EPCs) by succinylcholine, in frog cutaneous pectoris muscles, is significantly potentiated by certain organic calcium channel antagonists, at concentrations at which the calcium channel antagonists have no effect on EPCs themselves. Succinylcholine alone blocked the current in a dose dependent manner with an IC50 = 2.7 microM in voltage-clamped muscle fibers. The ability of succinylcholine to block endplate currents was potentiated by nicardipine (3.5- and 31-fold by 1 and 10 microM, respectively), by bepridil (4.3 fold by 1 microM), and by verapamil (7.7-fold by 10 microM). The dihydropyridine nifedipine (10 microM), did not potentiate the succinylcholine effect. Since the calcium channel antagonists do not affect endplate currents directly at these concentrations, a channel blocking or receptor antagonistic effect of these drugs is not indicated. We therefore suggest that the enhancement of block by succinylcholine may be due to the increased desensitization of the receptor. Furthermore, this enhancement of desensitization may explain the effects of these calcium channel antagonists seen in whole animal studies, where the contractures caused by acetylcholine and succinylcholine, applied systemically, are blocked. PMID- 3022194 TI - Glutamate-containing dipeptides do not modulate ligand binding at excitatory amino acid receptors. AB - Dipeptides of the structure X-Glu (e.g. X = Phe, Leu) have been proposed as allosteric modulators of excitatory amino acid receptors in rat brain membranes. Here we report that these dipeptides reduce the binding of L-[3H]Glu (predominantly N-methyl-D-aspartate-sensitive sites) and of [3H]kainate to postsynaptic density preparations isolated from rat brain. However, several observations indicate that the effects of these dipeptides are mediated not by allosteric modulation, but by free L-Glu liberated by the actions of a membrane associated aminopeptidase. The absolute and relative potencies of the dipeptides are similar at all acidic amino acid binding sites examined to date, suggesting the involvement of a factor with similar activity at each site (e.g. L-Glu). N Acetyl-Met-Glu is a weak inhibitor of L-Glu and kainate binding, and N-blocked peptides are known to be poor substrates of aminopeptidases. Bestatin, an inhibitor of aminopeptidases, decreases or abolishes the effects of substrate dipeptides on L-Glu and kainate receptor binding, while having no effect itself. PMID- 3022195 TI - Resistance of afterhyperpolarizations in hippocampal pyramidal cells to prostaglandins and vasoactive intestinal polypeptide (VIP). AB - The afterhyperpolarization (AHP) that follows repetitive stimulation was recorded intracellularly from CA1 pyramidal neurons in the guinea pig hippocampal slice preparation. Although the late AHP could be blocked by histamine (1-10 microM), forskolin (10 microM) and 8-bromo-cyclic AMP (100 and 500 microM), neither prostaglandins D2, E1 and F2 alpha (0.5 microM) nor vasoactive intestinal polypeptide (0.5 microM) had any effect on the AHP, membrane potential, membrane resistance or action potential properties. PMID- 3022196 TI - Lattice of high oxidative metabolism in the intermediate grey layer of the rat and hamster superior colliculus. AB - Patches of high oxidative metabolic activity were observed in sagittal sections through the rat and hamster colliculus. These patches were demonstrated with the histochemical markers succinate dehydrogenase and cytochrome oxidase. In surface parallel sections the patches were found to form a continuous lattice in both species. The lattice was composed of strips of high enzyme activity, about 60-200 micron wide, that surrounded islands of low activity. This lattice occurs at the same depth as the lattice of acetylcholinesterase activity, which also occurs in the intermediate grey layer, but the two lattices do not correspond. PMID- 3022197 TI - GABA enhances acetylcholine release from hippocampal nerve endings through a mechanism blocked by a GABA uptake inhibitor. AB - The effect of gamma-aminobutyric acid (GABA) on the release of [3H]acetylcholine [( 3H]ACh) was investigated using superfused rat hippocampal synaptosomes. GABA enhanced the basal efflux of [3H]ACh. The effect of GABA was bicuculline insensitive. Muscimol, (+/-)-baclofen or (-)-baclofen did not increase [3H]ACh release. The effect of GABA was counteracted by SK&F 89976 A (N-(4,4-diphenyl-3 butenyl)-nipecotic acid), a GABA uptake inhibitor. One possible interpretation of the results is that a GABA transport system is present on cholinergic terminals, suggesting that GABA and ACh may coexist in some rat hippocampus nerve endings. Another possibility is that the effect of GABA is mediated by a novel subtype of GABA receptor sensitive to SK&F 89976 A. PMID- 3022198 TI - gamma-Aminobutyric acid release in the globus pallidus in vivo after a 6 hydroxydopamine lesion in the substantia nigra of the rat. AB - This experiment was designed to determine whether the release of gamma aminobutyric acid (GABA) in the globus pallidus (GP) is affected by a lesion of the nigrostriatal pathway. Rats were lesioned with 6-hydroxydopamine 4 weeks prior to study of the in vivo release of GABA. A microdialysis probe was stereotaxically implanted in halothane-anesthetized animals in a vertical position on both GPs. The perfusates were analyzed for their GABA content with a high performance liquid chromatography technique. Compared to unlesioned controls, a marked decrease in the overflow of GABA was observed in the GP contralateral to the lesion, whereas the ipsilateral GP showed a slight increase. The differences between the sides were exaggerated after KCl (100 mmol) administration. The results are discussed in terms of a possible bilateral influence of dopamine terminals in the striatum on GABA transmission. PMID- 3022199 TI - Reduced responsiveness of locus coeruleus neurons to cutaneous thermal stimuli in capsaicin-treated rats. AB - Recent electrophysiological experiments have shown that brain norepinephrine (NE) neurons in the locus coeruleus (LC) are activated by cutaneous thermal stimuli of both non-noxious and noxious character. In the present study the LC neuronal response to thermal stimuli was used to evaluate cutaneous thermal sensitivity in capsaicin-treated rats, a treatment that is described to cause impaired thermoregulation. Capsaicin treatment, of neonates as well as of adult rats, caused a reduced responsiveness of brain LC neurons to thermal stimuli. The results suggest that a reduction in peripheral thermal afferent transmission may be one mechanism underlying the capsaicin-induced thermoregulatory dysfunction. PMID- 3022200 TI - Bipolar-amacrine synaptic transmission: effect of polarization of bipolar cells on amacrine cells in the carp retina. PMID- 3022201 TI - Synaptic transmission without calcium. PMID- 3022202 TI - Morphology and physiology of catfish cone horizontal cells. PMID- 3022203 TI - White-noise analysis in retinal physiology. PMID- 3022204 TI - Does the random distribution of discrete photoreceptor events limit the sensitivity of the retina? PMID- 3022205 TI - The role of cGMP control in visual receptor transduction. PMID- 3022207 TI - The investigations of N. Tsukahara on relocation of synapses on red nucleus neurones. PMID- 3022206 TI - Properties and functions of GABA-induced responses in turtle photoreceptors. PMID- 3022208 TI - Factors influencing the efficiency of dendritic synapses on hippocampal pyramidal cells. PMID- 3022210 TI - The magnitude of long-term potentiation of field potentials induced monosynaptically in region CA3 of guinea pig hippocampus. AB - The magnitude of long-term potentiation (LTP) was re-examined for monosynaptic transmission between mossy fibers and CA3 neurons in thin sections of the hippocampus of the guinea pig. Although an initial negative deflection in the field potential showed apparent short-term potentiation, this deflection was concluded to reflect non-synaptic activation of CA3 neurons. Accordingly, the magnitude of LTP of monosynaptic transmission was re-calculated at 3.4 +/- 0.9. PMID- 3022209 TI - Synaptic currents at interpositorubral and corticorubral excitatory synapses measured by a new iterative single-electrode voltage-clamp method. AB - A new iterative single-electrode voltage clamp method was applied to the measurement of synaptic currents in the red nucleus (RN) neuron of the cat. Voltage clamp was attained within 10 repetitions with great stability and the new algorithm was demonstrated to be superior to the original algorithm of iterative voltage clamp. With a conventional microelectrode, it was possible to measure the synaptic current with the time resolution of 50 microseconds. The synaptic currents evoked by stimulation of the contralateral interpositus nucleus (IP) had time-to-peak ranging from 200 to 540 microseconds and fitted well to alpha functions. Corticorubral (CR) synaptic current was also measured by making use of synaptic plasticity. The stimulation of the ipsilateral cerebral peduncle in cats with chronic lesion of the contralateral IP evoked fast rising EPSPs, as reported previously. The CR-EPSPs with times-to-peak less than 1 ms were subjected to voltage clamp. The CR synaptic currents had times-to-peak ranging from 350 to 880 microseconds. Since most of the interpositorubral (IR) synapses and a part of the CR synapses in IP-lesioned cats are situated on the somatic membrane of RN neurons and some of the CR synaptic currents were as rapid as the IR synaptic currents, the observed synaptic currents evoked by stimulation of the IP and those of the fast-rising CR-EPSPs were taken to originate from the synaptic membrane under space-clamp, i.e. soma. The present study provided additional evidence for the sprouting of the CR fibers as well as the time course of the synaptic current at the dendritic synapses remote from the soma, for the first time. PMID- 3022211 TI - The effect of nitrogen fertilization on forest blueberries. AB - In a randomized prospective trial with hens it is shown that artificially fertilized forest blueberries are toxic. In a high dose (1.7 1/hens) the birds died within 72 hours (p less than 0.01). In a lower dose (0.33 1/hens) all survived but a lifelong change in the egg production occurred implying defects of the shells (p less than 0.0000001). It seems that fertilizers change the microflora in the ground, which releases soluble aluminiumnitrate which is absorbed in the berries. Aluminium poisoning has been found to have clinical significance in dialysis patients and probably also in senile dementia. In smaller doses it can be difficult to establish this relation as the symptoms can have a latency period of more than 10 years. PMID- 3022212 TI - Idiopathic giant cell myocarditis: case report. AB - Idiopathic giant cell myocarditis has not been previously reported from Australasia; a typical case is presented, and the clinical and pathological findings discussed. Possible aetiologies are discussed in the light of positive serology to coxsackie B viruses. PMID- 3022214 TI - A simple objective method of recognizing goitre during parathyroid scintigraphy. AB - The presence of diffuse or multinodular goitre can lead to a false negative study of 10 to 20% of parathyroid investigations when the thallium-pertechnetate subtraction technique is used. A simple quantitative index is described that aids recognition of scintiscans whose diagnostic value may be limited by goitre. The index, referred to as the thallium thyroid index (TTI), is obtained from the ratio of thyroidal thallium counts above background to the mean background count density (expressed as counts cm-2) measured in regions just above and below the thyroid image. It correlates linearly with thyroid mass over the range 7 to 50 g, and goitre is likely to adversely affect the diagnostic quality of parathyroid scintiscans for values of TTI greater than 30 cm2 (corresponding to thyroid masses exceeding 35 g). TTI is insensitive to the time of commencement of image acquisition for times between 2 to 30 min following injection of the patient, and its correlation with thyroid mass has been confirmed by independent series of scans in two centres. PMID- 3022213 TI - A Keyes comment. PMID- 3022215 TI - Formation of labelled colloid in 99Tcm-DMSA due to the presence of bactericidal fluid. AB - The possibility that traces of bactericidal fluid in 99Tcm-DMSA could lead to the formation of labelled colloid, was explored. In vitro investigations were undertaken using ultracentrifugation techniques and photon correlation spectroscopy. The latter showed that both contaminated and uncontaminated DMSA contained colloidal (or particulate) material. However the presence of 10 microliters bactericidal fluid as contaminant was shown by ultracentrifugation to result in labelling of this colloidal material when 99Tcm was added to DMSA. Studies in a normal volunteer confirmed the results of the in vitro studies, in that significant liver and spleen uptake was observed after the administration of contaminated 99Tcm-DMSA. PMID- 3022216 TI - Representation of the path of a non-disintegrating capsule in the gastrointestinal tract using external scintigraphy and computer graphics. AB - The intestinal transit rate of a single non-disintegrating object has been measured using gamma scintigraphy. Using computer graphics, a three-dimensional visualization of the data has been developed. The display enables viewing from any angle and should be of value in assessing the effect of variables on the transit of pharmaceuticals and other materials. PMID- 3022219 TI - [10 years' therapy of bronchial carcinoma]. PMID- 3022218 TI - The objective evaluation of the phase image: a comparison of different automated methods. AB - One hundred and twenty eight patients with suspected or proven CAD were investigated using both X-ray ventriculography and equilibrium gated radionuclide angiography. In order to diagnose regional wall motion abnormalities, the parametric images obtained by Fourier analysis of the radionuclide images were analysed by different automated methods based on the measurement of the homogeneity of the phase values within the LV ROI. The effect of a diastolic frames exclusion, smoothing the original data, weighting the phase histogram, using Bacharach's error corrected phase distribution functions, using different descriptors of the spread of the phase histograms or distribution functions were tested. Using the results of the X-ray examination as the gold standard, ROC curves were plotted for each method. The ROC curves were modelled by a binormal model using the maximum likelihood method. Statistical tests were applied on the area under the ROC curves. The results show that the diagnostic value of the automated methods depends mainly on the way the histograms or distribution functions are described and to a lesser extent on the type of histograms or distribution functions used. The best result is obtained after smoothing, diastolic frames exclusion, weighting the phase histogram by the amplitude and describing it by its standard deviation. Nevertheless, this result is not significantly different from the result obtained by visual analysis of the phase and amplitude images. PMID- 3022220 TI - Ocular findings in juvenile nasopharyngeal angiofibroma. AB - Juvenile nasopharyngeal angiofibroma (JNA) is the most common benign neoplasm of the nasopharynx. While histologically benign, it has the propensity for aggressive local growth. This highly vascular tumor predominantly occurs in adolescent males. The literature fails to provide a thorough description of ocular complications and their incidence in JNA. This report summarizes the data from those clinical series detailing ocular findings in a total of 218 JNA cases. Exophthalmos was found in 14% of all cases. Decreased visual acuity and partial ophthalmoplegia occurred in 5% and 2% respectively. Recognition of ocular involvement in JNA is of the utmost importance, for it is often a manifestation of orbital or intracranial extension or both. We describe the diagnosis and management of a case of JNA in a five-year-old white male. The patient developed ocular findings of marked exophthalmos and optic atrophy. Early multidisciplinary diagnostic evaluation (otolaryngological, neurosurgical, and ophthalmological) followed by a team surgical approach to excision is most likely to yield efficacious results. PMID- 3022217 TI - Biodistribution in rats of 99Tcm-labelled human serum albumin. AB - Three different 99Tcm-human serum albumin (HSA) preparations were simultaneously studied by high pressure liquid chromatography (HPLC) and gamma camera techniques in rats. The products contained variable amounts of 99Tcm-human albumin, 99Tcm tin colloid and a 99Tcm-low molecular weight complex. The biological half life varied from 55 to 132 min and the bladder excretion for the first 60 min from 2 to 31%. Separated HPLC fractions of 99Tcm-albumin and 99Tcm-tin colloid both showed considerably longer biological half life when injected separately than when injected in the unfractionated preparation. A final evaluation of the quality of such products seem to require evaluation in humans. PMID- 3022221 TI - Synovial chondromatosis of the temporomandibular joint presenting as a parotid mass: possibility of confusion with benign mixed tumor. AB - Synovial chondromatosis is a rare metaplastic disorder of synovium in which cartilaginous nodules are produced within joint spaces. An unusual case involving the temporomandibular joint, with extension of the lesion beyond the joint capsule into the parotid gland, is described. The patient had a history of previous superficial parotidectomy for a "benign mixed tumor." Review of the histologic features revealed both lesions to be identical. The reason for confusion between the two diagnoses is discussed. PMID- 3022222 TI - Benign mixed tumor of the mandible 17 years after the occurrence of a similar lesion in the parotid gland. AB - Presented is a salivary tumor occurring in the posterior mandible. This tumor was preceded by a mixed tumor of the ipsilateral parotid gland, which was removed surgically 17 years earlier. The actual connection between the two lesions is discussed, but the true relationship can only be speculative. The mandibular lesion may represent a recurrence of the parotid tumor, a second primary tumor, or may possibly be metastatic in origin. PMID- 3022223 TI - Chemotherapy (CAV) and surgical resection as combined modality in the therapy of small-cell lung cancer. PMID- 3022224 TI - Clinical observations on mechanical ventilation for respiratory failure in bronchiolitis. AB - An unusually large number of infants (82) were admitted to Stanford University Hospital from November 1, 1983, through May 31, 1985, with a diagnosis of bronchiolitis requiring oxygen therapy. A larger percentage of these infants (17/82 = 21%) than generally expected required mechanical ventilation for respiratory failure. Fourteen infants had respiratory syncytial virus (RSV) infections, and three had parainfluenza virus infections. Ten patients had respiratory difficulties as neonates. The mechanical ventilation of the children requiring respiratory assistance was characterized by high minute ventilation with high tidal volumes (15 to 20 ml/kg) and slow respiratory rates (16 to 22 breaths/min). Peak inspiratory pressure averaged (mean +/- SD) 35 +/- 6 cm H2O in the RSV group and 34 +/- 6 cm H2O in the parainfluenza group. The mean number of days on the ventilator was 9.7 +/- 3.1 for the RSV group and 8.3 +/- 2.9 for the parainfluenza group. All were extubated within 17 days of presentation and discharged within 28 days. The complications encountered included pneumothorax and acute pulmonary hypertension. PMID- 3022225 TI - Hypercalcemia in association with mesoblastic nephroma: report of a case and review of the literature. AB - Hypercalcemia, often associated with certain types of adult tumors, has also been described in pediatric neoplasms. In childhood, the more common associations include lymphoma, leukemia, rhabdomyosarcoma and rarely neuroblastoma. However, recently, several infants with hypercalcemia were described having renal tumors without bone metastases. The following is a case report of a 2-month-old infant who presented with severe hypercalcemia and a large right-sided abdominal mass, which at surgery was diagnosed as a cellular mesoblastic nephroma. PMID- 3022226 TI - Effects of leukotrienes C4, D4, and E4 on cerebral arteries of newborn pigs. AB - We examined effects of topical application of leukotrienes (LT) C4, D4, and E4 on cerebral arteries of newborn pigs in vivo. Diameters of pial arteries were measured using a cranial window method during application of artificial cerebrospinal fluid without drug, and cerebrospinal fluid containing LT C4, D4, and E4 (1, 10, 100, 1000, and 5000 ng/ml). Control diameters ranged from 51-345 micron. All three LT constricted pial arteries in a dose-dependent manner, with a threshold for detectable response at 10 ng/ml (7 +/- 3% for LTD4). The magnitude of constrictor response at the highest dose was 23 +/- 3% for LTC4, 17 +/- 2% for LTD4, and 17 +/- 3% for LTE4. The specific receptor antagonist FPL 55712 blocked the constrictor response to LT. We conclude that LT are potent constrictors of cerebral arteries in newborn pigs. PMID- 3022227 TI - Tubeless pancreatic function tests used in combination for screening of pancreatic disease. AB - The diagnostic efficacy of some tubeless screening tests was examined in chronic pancreatitis patients with different degrees of pancreatic insufficiency. Results of the Pancreolauryl and Lipiodol tests were equal in the same patients. Sensitivity of the Lipiodol test, starch tolerance test and PABA test was 80%, 90% and 57%, respectively. Specificities were 48%, 57% and 100%. Sensitivity increased considerably when two tubeless tests were used in combination: 99% and 90% of patients had at last one abnormal result with starch tolerance + Lipiodol and PABA + Lipiodol tests, respectively. The specificity did not change significantly. Combination of tubeless tests seems to be useful for screening of mild and moderate pancreatic diseases because false negative results of single tests caused possibly by non-parallel enzyme secretion can be avoided. PMID- 3022228 TI - Lack of stimulation of kidney Na-K-ATPase by thyroid hormones in long-term thyroidectomized rabbits. AB - The effects of long-term thyroidectomy and of subsequent triiodothyronine administration on kidney Na-K-ATPase were studied at the level of single nephron segments and were compared to the short-term effects previously reported. After 8 11 weeks, thyroidectomy resulted in a marked decrease in Na-K-ATPase activity in all the segments of the rabbit nephron, the proximal tubule, the thick ascending limb of Henle's loop, the distal convoluted tubule and the collecting tubule. Within this delay, thyroidectomy also decreased the ouabain-insensitive Mg-ATPase activity, the basal and hormone-stimulated adenylate-cyclase activity, and the volume of tubular epithelium in all the segments where these parameters were measured. Administration of 50 micrograms/kg body weight triiodothyronine to 8-11 weeks thyroidectomized rabbits did not restore Na-K-ATPase activity in any nephron segment within 48 h. These observations are different from those reported in animals thyroidectomized only 1 week before study since, within this latter delay, thyroidectomy altered specifically Na-K-ATPase activity, this action was observed on the proximal and collecting tubules exclusively and, triiodothyronine administration corrected Na-K-ATPase alterations after 48 h. Results of the present study indicate that in the long term, thyroidectomy has a wide spectrum of renal effects which involves the whole nephron and most cellular functions. The tubular involution induced by long-term thyroidectomy is probably responsible for the inability of kidney cells to quickly increase their Na-K-ATPase activity in response to hormonal stimulation. PMID- 3022230 TI - Na,K-ATPase activity and 25(OH)vitamin D3 hydroxylation in rat proximal tubules. AB - The proximal tubule is the target site for parathyroid hormone (PTH), and conversion of 25(OH)vitamin D3 hormones which impinge on calcium (Ca) homeostasis, as well as a major site for sodium (Na) reabsorption. The effect of changes in PTH and vitamin D status on Na,K-ATPase activity, as a measure of Na transport, were studied in the proximal tubules of adult rat kidneys where Na and Ca reabsorption rates are in parallel. Na,K-ATPase activity and 25(OH)D3 metabolism were determined in cortical and juxtamedullary proximal tubule segments from normal, parathyroidectomized (PTX), and vitamin D-deficient (-D) rats. Na,K-ATPase activity was highest in cortical segments. PTX led to a decrease in activity in convoluted segments but increased activity in straight segments. In -D rats, Na,K-ATPase activity decreased in cortical segments but increased in juxtamedullary segments. 25(OH)D3 was metabolized more to 24,25(OH)2D3 than to 1,25(OH)2D3 in all normal segments. Juxtamedullary segments were more sensitive to PTX and -D conditions. These findings suggest that cortical and juxtamedullary nephrons are inherently different in basal Na,K ATPase activity, in conversion of 25(OH)D3 to active metabolites, and in response to altered PTH and vitamin D3 status. PMID- 3022229 TI - Effects of isoproterenol on rubidium transport in slow- and fast-twitch muscles from euthyroid and hyperthyroid rats. AB - The influence of experimental hyperthyroidism on the catecholamine induced stimulation of rubidium ion transport in the soleus (SOL), a slow-twitch muscle and the extensor digitorum longus (EDL), a fast-twitch skeletal muscle of rats was studied. Thyroxine administration (800 micrograms/kg/day), for ten days induced a rise of ouabain-sensitive 86Rb uptake in SOL muscle, without affecting the ouabain-insensitive uptake, whereas both fractions of 86Rb uptake were increased in EDL muscle from hyperthyroid rats. Isoproterenol (5 mumol/l) caused a two-fold rise in ouabain-sensitive Rb uptake of euthyroid SOL muscle, while in hyperthyroid SOL it could stimulate only the ouabain-insensitive fraction of 86Rb influx. On the other hand, the stimulating action of isoproterenol on euthyroid EDL muscle was due to an enhancement of ouabain-insensitive Rb uptake, but in hyperthyroid EDL it failed to stimulate the ouabain-insensitive transport and caused a marked rise in ouabain-sensitive Rb uptake. The changes in catecholamine mediated transport properties in SOL muscle may be related to fibre type transformation induced by thyroid hormone, although in EDL the changes of catecholamine stimulation are unlikely due to fibre type conversion. Basal and isoproterenol stimulated cAMP levels were significantly reduced in both EDL and SOL muscles from hyperthyroid rats, in contrast with an insignificant decrease in net rubidium uptake caused by isoproterenol at the same concentration. PMID- 3022231 TI - Regulation of inter- and intramolecular ligation with T4 DNA ligase in the presence of polyethylene glycol. AB - Polyethylene glycol (PEG) stimulates ligation with T4 DNA ligase. In 10% (w/v) PEG 6,000 solutions, only intermolecular ligation is enhanced by monovalent cations, while both inter- and intramolecular ligation occur without their presence. Similar stimulation was also caused by divalent cations or polyamines in the PEG 6,000 solutions. Such properties of the ligase could be applied to control the extent of inter- and intramolecular ligation. Ligation with cations or polyamines in 10% PEG 6,000 solutions was effective for intermolecular ligation. Ligation without cations or polyamines in 6.0% to 10% PEG 6,000 solutions was effective for intramolecular ligation. PMID- 3022232 TI - Nucleotide sequences of the gal E gene and the gal T gene of E. coli. AB - The nucleotide sequences of the gal E gene coding for UDP-galactose-4-epimerase and the gal T gene coding for galactose-1-P uridyltransferase of Escherichia coli have been determined. UDP-galactose-4-epimerase and galactose-1-P uridyltransferase are predicted to consist of 338 and 347 residues, respectively, NH2-terminal methionines included. PMID- 3022233 TI - Nucleotide sequence of a type II DNA topoisomerase gene. Bacteriophage T4 gene 39. AB - T4 DNA topoisomerase is a type II enzyme and is thought to be required for normal T4 DNA replication T4 gene 39 codes for the largest of the three subunits of T4 DNA topoisomerase. I have determined the nucleotide sequence of a region of 2568 nucleotides of T4 DNA which includes gene 39. The location of the gene was established by the identification of the first fifteen amino acids in the large open reading frame in the DNA sequence as those found at the amino-terminus of the purified 39-protein. The coding region of gene 39 has 1560 bases, and it is followed by two in-frame stop codons. The gene is preceded by a typical Shine Dalgarno sequence as well as possible promoter sequences for E. coli RNA polymerase. T4 39-protein consists of 520 amino acids, and it has a calculated molecular weight of 58,478. By comparing the amino acid sequences, T4 39-protein is found to share homology with the gyrB subunit of DNA gyrase. This suggests that these topoisomerase subunits may be equivalent functionally. Some of the characteristics of the 39-protein and its structural features predicted from the DNA sequence data are discussed. PMID- 3022234 TI - Analysis of the Kluyveromyces lactis positive regulatory gene LAC9 reveals functional homology to, but sequence divergence from, the Saccharomyces cerevisiae GAL4 gene. AB - The galactose metabolism positive regulatory gene from Kluyveromyces lactis, LAC9, has been isolated through its ability to activate expression of galactose metabolism enzyme genes in Saccharomyces cerevisiae. The LAC9 gene also activates expression of the S. cerevisiae alpha-galactosidase (MEL1) and K. lactis beta galactosidase (LAC4) genes in S. cerevisiae. Although LAC9-activated gene expression in K. lactis is not glucose repressed, activation of MEL1 gene expression by LAC9 in S. cerevisiae is. The LAC9 gene is expressed at an extremely low level as a approximately 2.9-kb mRNA, and encodes a protein of 865 amino acids. Although the LAC9 gene is functionally analogous to the S. cerevisiae GAL4 gene, the bulk of its protein sequence shows little homology to that of GAL4. Two of the three regions of homology that do exist, however, are restricted to areas of GAL4 protein already implicated in nuclear localization, DNA binding, and transcriptional activation. PMID- 3022235 TI - Comparative structural analysis of 1-methyladenosine, 7-methylguanosine, ethenoadenosine and their protonated salts IV: 1H, 13C, and 15N NMR studies at natural isotope abundance. AB - The 1H, 13C, and 15N NMR spectra of neutral and protonated forms of the nucleosides 1-methyladenosine (m1A), 7-methylguanosine (m7G) and ethenoadenosine (EA), as a model compound, have been analyzed in order to assign the site of protonation in m1A and m7G. Protonation of these nucleosides occurs in the pyrimidine ring of m1A and EA and in the imidazole ring of m7G, with the charge being distributed rather than localized. Structural differences for both m1A and m7G were observed in solution and compared with those existing in the crystal state of monomers as well as in tRNA where these nucleosides occur quite often. The protonated nucleoside structures in solution compared favorably in sugar pucker and glycosidic bond conformations with x-ray crystallographic data. Methyl group carbon chemical shifts of the protonated mononucleosides corresponded to those of the methyls of the respective nucleosides in native tRNA structures. Therefore, the tRNA methyl group carbon chemical shifts are indicative of fully protonated nucleosides in the native, three dimensional structure of the nucleic acid. PMID- 3022236 TI - The nucleotide sequence of the extreme 5' end of the avian coronavirus genome; implications for the discontinuous mRNA synthesis. PMID- 3022237 TI - Transposition of a bacterial IS3 element into a Xenopus Vi-element. PMID- 3022238 TI - Nucleotide sequence of the Saccharomyces cerevisiae MET25 gene. AB - To elucidate further the molecular basis of the specific regulatory mechanism modulating the expression of the genes implicated in methionine metabolism, we have cloned and characterized two genes, MET3 and MET25, and shown that the regulation of their expression is transcriptional. The sequence of the cloned yeast MET25 gene which encodes the O-acetyl homoserine - O-acetyl serine (OAH OAS) sulfhydrylase is reported here along with its 5' and 3' flanking regions. The amino acid composition predicted from the DNA sequence is in good agreement with that determined by hydrolysis of the purified enzyme. In the 5' flanking region the signal for general amino acid control was not found, corroborating our previous finding that the synthesis of OAH-OAS sulfhydrylase is not submitted to general control. The transcription start points have been determined. The 5' and 3' flanking regions of the MET25 gene suggest initiation and termination signals similar to those associated with other yeast genes. PMID- 3022239 TI - Tn7 transposition: a multigene process. Identification of a regulatory gene product. AB - Tn7 transposition is abolished by the deletion of a 2.2kb HindIII fragment from the central region of the transposon. Transposition is restored when the fragment is present in trans. When this fragment is present in trans with wild-type Tn7, transposition frequencies are stimulated 10-100-fold. The DNA sequence of this fragment has been determined and found to contain one long open reading frame coding for a protein of molecular weight 61 187. We have visualised this protein using a DNA-directed prokaryotic transcription-translation system. This gene may fill a regulatory role in the mechanism of Tn7 transposition. When present in trans the 2.2kb HindIII fragment alleviates a transcriptional block of promoter activity detected in Tn7. PMID- 3022240 TI - A variable tandem repeat locus mapped to chromosome band 10q26 is amplified and rearranged in leukocyte DNAs of two cancer patients. AB - A highly polymorphic locus associated with the variable tandem repetition of a 35 bp consensus sequence was mapped to chromosome 10, band q26. Examination of leukocyte DNA from a cancer patient revealed the twenty-fold amplification of one allelic fragment of this locus, while the other allelic fragment demonstrated a normal copy number. In another patient, Southern blotting of leukocyte DNA detected the deletion of the 3'-flanking region from one tandem repeat allele. These results indicate that variable tandem repeats may mark highly unstable regions of DNA in the human genome which can be altered by changes more extensive than simple tandem repeat variation. PMID- 3022241 TI - Cloning the DdeI restriction-modification system using a two-step method. AB - DdeI, a Type II restriction-modification system from the gram-negative anaerobic bacterium Desulfovibrio desulfuricans, recognizes the sequence CTNAG. The system has been cloned into E. coli in two steps. First the methylase gene was cloned into pBR322 and a derivative expressing higher levels was constructed. Then the endonuclease gene was located by Southern blot analyses; BamHI fragments large enough to contain the gene were cloned into pACYC184, introduced into a host containing the methylase gene, and screened for endonuclease activity. Both genes are stably maintained in E. coli on separate but compatible plasmids. The DdeI methylase is shown to be a cytosine methylase. DdeI methylase clones decrease in viability as methylation activity increases in E. coli RR1 (our original cloning strain). Therefore the DdeI system has been cloned and maintained in ER1467, a new E. coli cloning strain engineered to accept cytosine methylases. Finally, it has been demonstrated that a very high level of methylation was necessary in the DdeI system for successful introduction of the active endonuclease gene into E. coli. PMID- 3022243 TI - Molecular characterisation of subgenomic single-stranded and double-stranded DNA forms isolated from plants infected with tomato golden mosaic virus. AB - A subgenomic single-stranded DNA present in particles of the geminivirus, tomato golden mosaic virus, has been shown by electron microscope heteroduplex mapping and Southern hybridisation analysis to consist of circular molecules, ca. 1.2 kb in size, derived from the smaller of the two genomic DNA components, DNA B, by deletion of open reading frame (ORF) BR1 and the C-terminal portion of ORF BL1. A covalently closed circular, supercoiled, double-stranded form of the subgenomic DNA has been isolated from virus-infected plants and cloned into pEMBL9. Analysis of the sequence of 22 clones across the deletion boundaries revealed only four different deletion boundaries, derived from four different left hand borders and three different right hand borders. Each border was within a region of 11 nucleotides and gave rise to a narrow size range (1248-1261 nucleotides) for the population of 22 subgenomic DNAs. However apparently smaller subgenomic DNAs were sometimes formed when plants were inoculated with cloned subgenomic DNA, or a construct derived from a subgenomic DNA in which a neomycin phosphotransferase gene had been inserted, together with the genomic DNA components. Mechanisms to account for the size, specificity and formation of the subgenomic DNA are discussed. PMID- 3022244 TI - Chromatin 3'-phosphatase/5'-OH kinase cannot transfer phosphate from 3' to 5' across a strand nick in DNA. AB - Rat liver chromatin contains a 3'-phosphatase/5'-OH kinase which may be involved in the repair of DNA strand breaks limited by 3'-phosphate/5'-OH ends. In order to determine whether the phosphate group can be transferred directly from the 3' to the 5' position, a polynucleotide duplex was synthesized between poly (dA) and oligo (dT) segments which had 3'-[32P]phosphate and 5'-OH ends. The oligo (dT) segments were separated by simple nicks as shown by the ability of T4 DNA ligase to seal the nick after the 3'-phosphate was removed by a phosphatase and the 5' end was phosphorylated with a kinase. The chromatin 3'-phosphatase/5'-OH kinase was unable to transfer phosphate directly from the 3' to the 5' end of the oligo (dT) segments in the original duplex; ATP was needed to phosphorylate the 5'-OH end. It is concluded that the chromatin 3'-phosphatase/5'-OH kinase is unable to convert a 3'-phosphate/5'-OH nick which cannot be repaired by DNA ligase directly into a 3'-OH/5'-phosphate nick which can be repaired by DNA ligase; the chromatin enzyme rather acts in two steps: hydrolysis of the 3'-phosphate followed by ATP mediated phosphorylation of the 5'-OH end. PMID- 3022242 TI - kDNA minicircles of the major sequence class of C. fasciculata contain a single region of bent helix widely separated from the two origins of replication. AB - The major sequence class of Crithidia fasciculata minicircles is shown to have a single region of bent helical DNA widely separated from the two replication origins located 180 degrees apart on the minicircle map. The position of the bend in the DNA has been mapped both by gel electrophoretic methods and by direct electron microscopic observation of the DNA. This sequence directed bending is apparently the result of homopolymeric dA X dT tracts 4-6 base pairs long repeated in phase with the helix screw. The region of the bend contains nineteen such homopolymeric tracts in a region of about 200 base pairs with sixteen of the tracts oriented in the same direction. PMID- 3022246 TI - Nucleotide sequence of the DNA polymerase gene of herpes simplex virus type 1 strain Angelotti. PMID- 3022245 TI - Comparison of the late H1 histone genes of the sea urchins Lytechinus pictus and Strongelocentrotus purpuratus. AB - We have isolated and sequenced a gene encoding a late H1 histone subtype from the sea urchin species L. pictus. The primary structure of the late H1 subtype encoded by this gene is 209 amino acids in length, and has a net positive charge of 67. This gene is present in a single copy per haploid genome and encodes an mRNA of 752 nucleotides. Late H1 transcripts are detected in the unfertilized egg and are most prevalent in gastrulating embryos. Comparison of 375 bp of 5' flanking sequences of the L. pictus late H1 gene and the H1-gamma gene of a distantly related sea urchin species, S. purpuratus, reveals large blocks of sequences that are identical between the two genes. To determine if these conserved 5' sequences are present in other members of the sea urchin H1 gene family, the analogous region of S. purpuratus H1-alpha, an early H1 gene, was sequenced. The homology between the flanking sequences of the early and late families was limited to consensus sequences which are found upstream of all H1 genes. The possible regulatory implications of these findings are discussed. PMID- 3022249 TI - Addisonian crisis in an adolescent female: a case report. AB - Acute adrenal insufficiency is rarely diagnosed in the pediatric age group. In this case report, an 11-year-old female with three previous admissions for dehydration was seen for dehydration and shock. Adrenal insufficiency was considered as a working diagnosis in the emergency department. Rapid intervention with intravenous hydrocortisone and fluid resuscitation resulted in a good outcome. PMID- 3022247 TI - Anorexia, serum zinc, and immunologic response in small cell lung cancer patients receiving chemotherapy and prophylactic cranial radiotherapy. AB - Anorexia is a major clinical problem for patients with certain types of cancer. The specific mechanisms that result in this spontaneous decline in food intake remain unknown. In noncancer populations, zinc has been shown to play a role in maintaining normal appetite, taste acuity, and immunocompetence. One purpose of this prospective, longitudinal study of cachexia in ten males with small cell lung carcinoma was to determine if anorexia (caloric intake), perceived taste changes, zinc intake, and impaired cellular immunity were associated with serum zinc concentrations. The average daily caloric intake declined 490 kcal from time of diagnosis to seven months after diagnosis (mean caloric intake = 72% of RDA). Daily zinc intake ranged from 6.5 to 25.4 mg over the seven months. During this period, the mean serum zinc concentrations, although low (71 micrograms/dl), remained within the normal range. The average weight declined from 81.7 to 74.1 kg. There was no identifiable pattern of perceived taste changes; most of the perceived changes were recorded during the period coinciding with prophylactic cranial radiation. At the initial testing, four of nine subjects were anergic to a battery of skin test antigens (mumps, candida, tuberculin purified protein derivative). The only subject who remained responsive to two antigens throughout the study remained alive at 12 months. Caloric intake was inadequate to maintain weight. While zinc intake was low, low normal serum zinc concentrations were maintained; thus in this sample, serum zinc does not appear to be the anorexigenic factor. PMID- 3022248 TI - The effect of diets high in fat and/or fiber on colonic absorption of DMH in the rat. AB - The possibility that long-term feeding of diets high in fat or fiber could alter the colonic mucosa and subsequent colonic absorption of 1,2-dimethylhydrazine (DMH) in situ was examined in the rat model. Male Sprague-Dawley rats were fed one of four experimental diets for six weeks prior to studies of DMH absorption and bile acid excretion; dietary treatments consisted of two levels of fat (12 and 47% of calories from corn oil) fed at each of two levels of fiber (plus or minus 15% wheat bran). Two sets of DMH absorption studies (Studies 1 and 2) were performed; the first used a 10- and the second a 20-minute test period. In Study 1, DMH absorption was greater in those animals that had been fed the high level of corn oil when additional fiber was not present in the diet. When a longer absorption period was used (Study 2), this effect of diet on DMH absorption was not apparent. The level of fiber, not the fat intake, altered bile acid excretion. Bile acid concentration (mg/g dry wt) decreased with added fiber, whereas total bile acid excretion (mg/day) increased. These results indicate that high levels of dietary fat may result in small increases in DMH absorption which are unrelated to changes in bile acid concentration. PMID- 3022250 TI - In vitro interaction of ACTH with rat brain muscarinic receptors. AB - ACTH-(1-24) inhibits the in vitro binding of the muscarinic antagonist [3H]QNB to membranes from rat brain. The magnitude of inhibition is dependent on the concentration of ACTH-(1-24). Kinetic analysis indicates a pure competitive inhibition which is suggestive of a reversible interaction of ACTH with muscarinic receptors. A mechanism involving an interaction of ACTH-(1-24) with the phospholipid core of the receptors is suggested. Structure activity studies point to a relation with reported effects of intracerebroventricularly administered ACTH on the turnover rate of acetylcholine and the ACTH-induced stretching and yawning syndrome. PMID- 3022251 TI - The effects of pro-opiomelanocortin peptides on cyclic AMP and tyrosinase in melanoma cells. AB - Des-, mono-, and diacetylated melanotropin (des-, mono-, and di-Ac MSH, respectively) were compared for their dose-related effects on content of adenosine 3':5'-monophosphate (cAMP) and tyrosinase activity in the Cloudman S91 mouse melanoma tumor. Des-Ac MSH was more potent than the acetylated forms of MSH at increasing cellular levels of cAMP; mono- and di-Ac MSHs, however, were more potent than des-Ac MSH at elevating the activity of the enzyme, tyrosinase. Lysine-gamma1 MSH, a melanotropin from the amino terminus of pro opiomelanocortin, exhibited slight stimulatory effects on tyrosinase and these actions were less than additive to those of mono-Ac MSH. Unlike their actions on amphibian skin-darkening or in mammalian behavior, neither beta-endorphin1-31 nor its derivatives, N-Ac-beta-endorphin1-27 or beta-endorphin30-31 (glycylglutamine), exhibited any influence on tyrosinase activity evoked by mono Ac MSH in the tumor cells. PMID- 3022252 TI - The role of calcium in the mechanism of corticotropin releasing factor mediated ACTH release. AB - To investigate the role of calcium (Ca+2) in CRF stimulated ACTH release, we studied the effect of the following conditions on CRF (10 nM) mediated ACTH release in primary pituitary monolayer culture: different concentrations of Ca+2; EGTA; lanthanum (La+3) and nifedipine, blockers of calcium cell influx and penfluridol, trifluoperazine, and pimozide, inhibitors of calmodulin activation. Higher concentrations of Ca+2 in the culture medium led to greater amounts of CRF induced ACTH release. EGTA at 3 mM decreased the amount of CRF stimulated ACTH release by 60% but did not alter the spontaneous release of ACTH. At 0.5 mM and 1.0 mM La+3, ACTH release induced by CRF was inhibited by 23% and 35% respectively (p less than 0.01). Nifedipine (both 10(-5) and 10(-4) M) inhibited CRF stimulated ACTH release but only to a maximum of 30%. This inhibition was completely overcome by the addition of 12 mM calcium. Penfluridol, pimozide, and trifluoperazine blocked the release of ACTH induced by CRF by 63%, 26%, and 0% respectively. In conclusion, extracellular Ca+2, Ca+2 influx, and calmodulin play a role in the mechanism of CRF stimulated ACTH in vitro. PMID- 3022253 TI - Magnetic fields differentially inhibit mu, delta, kappa and sigma opiate-induced analgesia in mice. AB - An exposure for 60 min to a 0.5 Hz rotating magnetic field (1.5-90 G) significantly attenuated the daytime analgesic effects of the mu and kappa opiate agonists, morphine and U50,488H, respectively, and significantly inhibited the analgesic actions of the delta agonist, D-Ala2-D-Leu5-enkephalin, in mice. The magnetic stimuli had no significant effects on the analgesic effects of the prototypic sigma opiate agonist (+/-) SKF-10,047. These results show that exposure to relatively weak magnetic stimuli has significant and differential inhibitory influences on various opioid systems. PMID- 3022255 TI - High performance liquid chromatographic properties of peptides and proteins on a dihydroxyalkyl bonded silica stationary phase. AB - The chromatographic behavior of biologically relevant peptides and proteins in the molecular weight range between 200 and 200,000 dalton units were studied on a size exclusion matrix column consisting of an aqueous compatible dihydroxyalkyl bonded silica support. The mechanism of separation appears to be dependent on hydrodynamic radius, hydrophobic and ionic interactions. Support for this contention is based on the chromatographic properties of these peptides and proteins at different mobile phase ionic strengths and pH, oxidation state of amino acid residues and total hydrophobicity of the peptide or protein. This column is also capable of separating native angiotensin-I from its iodinated congener. Recoveries of proteins and peptides from this column ranged between 70 100%. Unlike typical reverse phase separations, this modified silica chromatographic media allows for an alternative technique employing aqueous eluents for rapid separation/isolation and purification of peptides and proteins from natural or synthetic sources. PMID- 3022256 TI - Derivatives of speract are associated with the eggs of Lytechinus pictus sea urchins. AB - Two peptides associated with the eggs of the sea urchin, Lytechinus pictus, which stimulate L. pictus but not Arbacia punctulata sperm respiration rates, were purified and their amino acid sequences determined. The peptides (Gly-Phe-Asp-Leu Thr-Gly-Gly-Gly-Val-Gln and Phe-Asp-Leu-Thr-Gly-Gly-Gly-Val-Gln) were found to be structurally similar to the peptide, speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val Gly). Chemical synthesis of the two peptides confirmed their ability to activate sperm respiration. The peptides had equivalent biological activity with half maximal stimulation of respiration rates and of cyclic nucleotide concentrations occurring at 60 pM and 700 pM, respectively. Addition of the peptides to intact spermatozoa resulted in the rapid appearance of a newly-stained protein on Na X dodecyl X SOl polyacrylamide gels (Mr = 140,000); one-half maximal formation of the Mr 140,000 protein occurred at about 20-100 nM peptide. PMID- 3022259 TI - Lack of effect of chronic imipramine administration on beta-adrenoceptor density on rat lymphocytes. AB - Chronic administration of imipramine with drinking water to Wistar rats for two months did not affect the [3H]dihydroalprenolol binding to lymphocytic membranes, though the treatment depressed the radioligand binding to cerebral cortical membranes. The result suggests that the beta-adrenoceptor downregulation caused by the antidepressant in the central nervous system is not reflected in the lymphocytes. PMID- 3022257 TI - Patterns of use and psychopathology in chronic marijuana users. AB - After an epidemic increase in the use of marijuana from the mid 1960s to the late 1970s, there appears to be a recent decrease in the prevalence of use. Patterns of use of marijuana vary considerably depending upon the socio-cultural and psychobiologic characteristics of the user. There may also be a natural history of marijuana use in individuals that is influenced importantly by the age and perspectives of the user. Marijuana use has been shown to be an important cause of psychopathology in some people and an attempt at self-medication in others. PMID- 3022254 TI - Receptor-mediated uptake of GnRH agonist and antagonists by cultured gonadotropes: evidence for differential intracellular routing. AB - The binding and uptake of the GnRH agonist D-Lys6-GnRH and of the antagonists [N Ac-D-(pyro)-Cl-Phe1,2-D-Trp3-Lys6-D-Ala10]-GnRH and D-p-Glu1-D-Phe2-D-Trp3-D-Lys6 GnRH by dispersed pituitary gonadotropes was studied with electron microscopy. The peptides were coupled to colloidal gold markers with a diameter of 6 or 20 nm which were incubated separately or together for time periods between 15 and 180 min. Both antagonists could be found after 45 and 180 min at 37 degrees C in lysosomes as well as at the plasma membrane of gonadotropes. Co-incubation of both antagonists or of agonist and either antagonist resulted in uptake of the conjugates into separate lysosomes as well as mixed together into the same lysosome. Localization of the antagonists in structures associated with the Golgi apparatus was not observed at the time points studied. The results show that both GnRH agonist- and antagonist-conjugates are biologically active and that they are internalized by the gonadotropes via receptor mediated endocytosis. The failure to detect antagonist conjugates in the Golgi apparatus may indicate that passage through this organelle requires activation of the receptors by agonists and that the uptake of antagonist into lysosomes due to normal membrane protein turnover. PMID- 3022258 TI - On the role of noradrenergic neurotransmission in the action of desipramine and amitriptyline in animal models of depression. AB - Three weeks of treatment with desipramine (DMI) and amitriptyline (AMI) reduced the hypothermic action of clonidine in rats. Both electrolytic and 6 hydroxydopamine lesions of the locus coeruleus (LC) and administration of DSP-4 counteracted the reduction of clonidine hypothermia produced by antidepressants. Lesions of the LC and DSP-4 administration also antagonized the anti-immobility action of single doses of DMI but failed to modulate the action of AMI in the forced swim test. Chronic DMI action on the rat immobility was reduced by 6 hydroxydopamine lesions of the LC: other lesions (electrolytic, DSP-4) were ineffective. Electrical stimulation of the LC increased the rat activity in the forced swim paradigm, producing an effect similar to that of antidepressants. The anti-immobility effect of DMI as well as LC stimulation were antagonized by drugs blocking alpha-adrenoceptors (phenoxybenzamine, prazosin) but not by propranolol, a non-selective antagonist of beta-adrenoceptors. On the other hand, the anti immobility action of AMI was unchanged by all adrenolytics used in that study. The results indicate that the LC system and alpha 1-adrenoceptors play an important role in the antidepressive action of DMI, but not AMI, in the forced swim test. PMID- 3022260 TI - Characteristics of the beta-adrenergic stimulation of adenylate cyclase activity in rat ventral prostate and its modulation by androgens. AB - Beta-adrenergic agents cause a 2.5-3-fold stimulation of adenylate cyclase activity in rat ventral prostate membrane preparations with an order of potency (KD values) typical of a beta 2-subtype receptor: (-)isoproterenol (20 nM) greater than (-)epinephrine (70 nM) much greater than (-)norepinephrine (1 microM) much greater than dopamine (70 microM). The stimulatory effect exerted by high concentrations of dopamine (greater than 0.1 mM) is completely reversed by propranolol but not by haloperidol or sulpiride, thus indicating an action of dopamine mediated by the beta-adrenergic receptor. One week after castration, basal adenylate cyclase activity in prostatic membranes is 50% reduced. In the same group, the stimulation by isoproterenol is completely abolished in the absence of GTP, while the effect of GTP alone is reduced by 75%. The inhibitory effect of castration on basal as well as isoproterenol- and GTP-stimulated adenylate cyclase activity can be completely reversed by treatment of castrated animals with dihydrotestosterone, thus demonstrating the marked androgen dependency of adenylate cyclase activity in prostatic tissue. Since the response to direct stimulation of adenylate cyclase activity (assessed by NaF and forskolin) is only reduced by 33%-60% while the response to isoproterenol is 100% abolished, the present data indicates that the complete loss of beta-adrenergic responsiveness of prostatic adenylate cyclase following castration includes many steps, including those preceding adenylate cyclase activity, namely the beta adrenergic receptor itself and/or its coupling via the GTP-binding protein. The large amplitude of the effects observed should facilitate study of the mechanisms involved in the marked regulation of the beta-adrenergic receptor-adenylate cyclase system by androgens in prostatic tissue. PMID- 3022262 TI - Blue rubber bleb nevus syndrome. AB - Blue rubber bleb nevus syndrome (BRBN) is a rare disease entity of which at least 22 cases of pediatric origin have been described since 1958. Only a few known cases have developed in adulthood. This syndrome is characterized by cutaneous, usually multiple, cavernous hemangiomas associated with similar lesions of the gastrointestinal tract. The cutaneous hemangiomas are a variant of the cavernous type, which are generally soft, rubbery, and compressible. Most cases are sporadic, although autosomal dominant inheritance has been described. Complications of this syndrome may include gastrointestinal bleeding leading to anemia, amputations of extremities, and hematologic disturbances such as chronic consumption coagulopathy. Children suffering from BRBN syndrome should be surveyed extensively for comprehensive care. This syndrome is important to consider in cases of unexplained intestinal bleeding. Therapy is mainly symptomatic and directed to complications. PMID- 3022261 TI - [Dietary fiber: Cinderella becomes a queen]. PMID- 3022264 TI - [Neuropeptides and the pathogenesis and treatment of endogenous mental disorders]. PMID- 3022263 TI - [Endogenous opioid systems]. PMID- 3022265 TI - [Expression of genes introduced into mammalian cells by transfection]. PMID- 3022266 TI - [Enzymes participating in the metabolism of 2'-deoxynucleotides in mitochondria]. PMID- 3022267 TI - Treatment of disseminated non-small cell lung cancer. Guidelines for the practicing physician. PMID- 3022268 TI - Clarification of lisinopril cough. PMID- 3022269 TI - Human serum parvovirus associated vasculitis. AB - A 48 year old man had a severe symmetrical arthritis of knee and ankle joints accompanied by an extensive purpuric rash of both legs and several areas of skin necrosis. No other system was clinically involved. Human serum parvovirus specific IgM was present in a blood sample taken 2 weeks after the onset of the clinical illness indicating recent infection with this virus. The patient was treated with complete bed rest, and the application of saline soaks to both legs. He had a recurrence of the rash 5 weeks after onset, but otherwise made a complete recovery. Purpura with skin necrosis has not previously been reported in association with this virus. PMID- 3022271 TI - Effect of nifedipine in hypertension not controlled by converting enzyme inhibitor and diuretic. AB - Nifedipine, in a slow release preparation, was given at a mean daily dosage of 47 +/- 4 mg to 12 patients with severe hypertension in whom arterial pressure was not satisfactorily controlled (mean blood pressure, 172 +/- 6/111 +/- 4 mmHg) by the association of a converting enzyme inhibitor and a diuretic. Nifedipine administration induced a marked decrease in blood pressure (to 133 +/- 3/85 +/- 3 mmHg), serum potassium and plasma aldosterone. Following adequate control of hypertension and because of severe hypokalaemia in some patients, the diuretic was discontinued in 10 subjects. After 1.7 +/- 0.5 months of treatment by the converting enzyme inhibitor and nifedipine, no change in arterial pressure occurred whilst serum potassium returned to normal in most patients. These results demonstrate that nifedipine may be useful in patients with residual elevation of arterial pressure when treated by converting enzyme inhibitor and diuretic. However, in such patients serum potassium level should be carefully monitored. In addition, our observations suggest that calcium blockers may be an effective alternative to diuretics in patients receiving a converting enzyme inhibitor. PMID- 3022272 TI - Adverse effects of converting-enzyme inhibition in patients with severe congestive heart failure: pathophysiology and management. AB - Although converting-enzyme inhibition is of established value in the management of patients with severe chronic congestive heart failure, troublesome adverse reactions occur frequently during the course of treatment and may cause physicians to interrupt effective therapy. The three most common adverse reactions that are seen in patients with heart failure following treatment with captopril and enalapril (symptomatic hypotension, functional renal insufficiency, hyperkalaemia) are predictable consequences of interfering with the homeostatic functions of the renin-angiotensin system, which evolved millions of years ago to preserve life in sodium-depleted states. It is not surprising, therefore, that these untoward effects can be prevented or reversed by increasing the dietary intake of salt or reducing the dose of concomitantly administered diuretics; their occurrence rarely requires discontinuation of drug therapy. Recognition of this link between sodium balance and the adverse effects of converting-enzyme inhibition is important, because most patients with severe heart failure who experience such untoward reactions can nevertheless be expected to improve clinically during long-term therapy, if effective treatment is not interrupted. PMID- 3022270 TI - Successful control of Cushing's disease in the elderly with long term metyrapone. AB - A 77-year old woman with pituitary-driven Cushing's disease is described. The condition was completely controlled on long-term treatment with metyrapone. PMID- 3022273 TI - Influence of ACE inhibition on pulmonary haemodynamics and function in patients in whom beta-blockers are contraindicated. AB - The effects of captopril have been studied in 19 patients suffering from chronic obstructive pulmonary disease (COPD). In 9 of these patients with pulmonary hypertension (mean pulmonary artery pressure greater than 20 mm Hg), we studied the effects of the drug on pulmonary haemodynamics, blood gases and systemic circulation. Haemodynamic data were recorded before oral captopril 50 mg and for the next 60 minutes. Following captopril administration, significant reductions in mean pulmonary artery pressure (PAP) (P less than 0.05), in mean pulmonary wedge pressure (PWP) (P less than 0.05) and in total pulmonary resistance (TPR) were noted; significant reductions in mean brachial artery pressure (BAP) and systemic vascular resistance (SVR) were also recorded, while cardiac output, heart rate and blood gas tensions showed no significant changes. Furthermore, the higher the hypoxaemia was, the greater the reduction in BAP (P less than 0.05). In the other 10 COPD patients with moderate essential hypertension, captopril was given as monotherapy (75-10 mg/day) for 60 days. We found no significant modification of the various respiratory function tests except for an increase in the vital capacity (P less than 0.05). Systolic blood pressure and diastolic blood pressure were reduced both in the supine and in the standing position and there were no side effects, in particular no bronchospasm even in the patients responsive to bronchodilator drugs. Our data suggest that captopril is an effective and safe drug when administered to COPD patients with essential hypertension. Moreover, in these patients, it may protect the pulmonary circulation from hypoxic pulmonary vasoconstriction. PMID- 3022274 TI - Angiotensin converting enzyme inhibitors in diabetes: experimental and human experience. AB - Peripheral glucose metabolism was studied during the influence of elevated systemic kinin levels by either application of exogenous bradykinin (BK) or inhibition of endogenous kinin degradation by angiotensin converting enzyme inhibitors (ACEI). In infusion experiments in streptozotocin-diabetic rats, both BK (0.3 micrograms/min) and ACEI (300 micrograms captopril/100 g) showed a significant reduction of elevated blood glucose levels. In man, non-insulin dependent diabetics (NIDD) and healthy subjects in postoperative (POP) stress were examined. Peripheral insulin-sensitivity and muscular glucose balance were determined using the glucose clamp technique and the forearm technique. In NIDD as well as in POP subjects, impaired peripheral insulin responsiveness, as evaluated by whole body glucose consumption, was improved up to 50% by application of BK (80 micrograms/h i.v.) or ACEI (25 mg captopril p.o.), respectively. This was most probably due to accelerated muscular glucose uptake which was assessed in the same experiment and found to be increased 2- to 3-fold under these conditions. As a molecular basis for the observed effects on muscular glucose uptake, phosphofructokinase was found to be markedly stimulated by BK (8 X 10(-10)mol/l) in the isolated perfused rat heart, suggesting accelerated glycolytic flux. It may be concluded that ACEI increase muscular insulin responsiveness, thus being beneficial in insulin-resistant states as well as in hypertension. This metabolic effect is most probably due to elevated systemic kinin levels. Further investigations are required to evaluate the clinical relevance of these findings. PMID- 3022275 TI - Metastasis of Rous sarcoma tumors in chickens is influenced by the major histocompatibility (B) complex and sex. AB - Six-week-old second generation progeny from the cross of inbred Lines 6(1) and 15(1), segregating into three major histocompatibility (B) complex groups (B2/B2, B2/B5, and B5/B5), were inoculated subcutaneously in the wingweb with one of three pseudotypes of Rous sarcoma virus. Chickens that died during a 10-week period after inoculation were necropsied and scrutinized for gross metastasis and histological sections of at least one lesion per affected organ examined for Rous sarcoma-transformed cells. By definition, a metastatic tumor was one located in an organ or tissue other than the primary inoculation site and having the histological appearance of a Rous sarcoma. Sarcomas developed in 1144 chickens, 390 of which died with tumor. For B2/B2, compared to B5/B5 hosts, mortality was 8 vs. 93%, median days to death were 45 vs. 31, and metastatic frequency was significantly lower, 32 vs. 58%. Disseminated lesions were significantly less frequent in females than males and grew preferentially in the heart and pericardial sac. Because the frequency of metastasis was significantly lower in B2/B2 than in B5/B5 chickens, a gene(s) within, or closely linked to, the B complex sharply retards the spread of Rous sarcoma virus-induced tumors. PMID- 3022276 TI - Use of ammonium chloride and sodium bicarbonate in acute heat exposure of broilers. AB - Ammonium chloride (NH4Cl) and sodium bicarbonate (NaHCO3) were added separately to the drinking water of 42- to 52-day-old broilers. Birds were given access to the water ad libitum for a total of 42.5 hr consisting of 18.5 hr prior to an 8 hr interval of severe heat exposure and a further 16 hr-post exposure. Water and feed intake during the treatment period were unaffected by either NH4Cl at 6.25 g/liter (.63%) of distilled water (DW) or NaHCO3 at 3.15 g/liter (.32%) DW. Water intake was increased by approximately 20% in birds given water containing 6.25 g of NaHCO3/liter (.63%) DW, while both feed and water intake were severely limited by NH4Cl at 31 g/liter (3.1%) DW. Blood pH of birds was substantially lowered by consumption of NH4Cl, while consumption of NaHCO3 did not significantly affect blood pH. Blood pH of all treatments increased during the heat exposure period and declined afterward; however, blood pH change appeared to be more pronounced for birds receiving the NH4CL. A correlation coefficient (r = -.31) existed between blood pH and mortality, while a correlation (r = -.72) was demonstrated between water consumption and mortality. PMID- 3022277 TI - Chemical carcinogenesis: the liver as a model. PMID- 3022278 TI - Cultured amniotic fluid cells for prenatal diagnosis of lysosomal storage disorders: a methodological study. AB - The influence of culture conditions on the ultrastructure and enzyme activities of amniotic fluid cells are reported. Morphological changes were determined as a function of the number of lysosomal-like inclusion bodies per cell, and these results correlated to the activity of beta-hexosaminidase, alpha-mannosidase, beta-glucuronidase, arylsulphatase C and 5' nucleotidase. The parameters examined were pH of the culture media, type of media, increasing cell passage and day of harvest. Our results indicate that enzyme activities are less sensitive to changes in culture conditions as compared to ultrastructural changes. We therefore recommend that in order to obtain reliable ultrastructural results for the diagnosis of storage disorders, cultures should be grown in MEM as the culture medium, the pH of the medium carefully monitored to remain below pH 7.4, examining the cultures no later than the eighth cell passage and no later than the 10th day after subculture. PMID- 3022279 TI - [Fine needle aspiration biopsy of breast sarcomas]. PMID- 3022281 TI - Expression of the RNA genome of an animal virus in Saccharomyces cerevisiae. AB - The nucleocapsid of vesicular stomatitis virus (VSV) was introduced into the cytoplasm of Saccharomyces cerevisiae by low pH-dependent fusion of the viral envelope with the spheroplast plasma membrane. This led to de novo synthesis of the three major structural proteins of the virus--the G, N, and M proteins--as shown by immunoprecipitation of [35S]methionine-labeled spheroplast lysates. In NaDodSO4/polyacrylamide gel electrophoresis, M and N proteins comigrated with those of the virion, whereas the yeast-made G protein migrated as two bands with apparent molecular sizes of 60 and 70 kDa. Both polypeptides appeared to be N glycosylated, since only one polypeptide with the apparent molecular mass of approximately equal to 55 kDa was produced in the presence of tunicamycin. Phase separation into Triton X-114 suggested that the unglycosylated G protein was membrane bound. According to immunofluorescent surface staining of live spheroplasts, at least part of the G protein was transported to the plasma membrane. Spheroplasts expressing the VSV genes could be fused together by low pH to form polykaryons, indicating that G protein synthetized by yeast was fusogenic -i.e., biologically active. PMID- 3022280 TI - Requirement of multiple copies of a 21-nucleotide sequence in the U3 regions of human T-cell leukemia virus type I and type II long terminal repeats for trans acting activation of transcription. AB - The cis-acting regulatory sequence of transcription from long terminal repeats (LTRs) of human T-cell leukemia virus type I and type II (HTLV-I and HTLV-II), which is essential for action of the virally encoded trans-acting transcriptional factor(s) designated pX(s), in HTLV-I and -II was identified. Deletion of most of the U3 region of the HTLV-I LTR resulted in loss of trans-acting transcriptional activation. However, when a tandem repeat of a 21-nucleotide sequence (GAAGGCTCTGACGTCTCCCCC) that is present in the U3 region of HTLV-I and -II LTRs was inserted into the deleted U3 region of the HTLV-I LTRs, chloramphenicol acetyltransferase activity was restored. The extent of restoration of activity was proportional to the number of copies of the sequence inserted. To test the possibility that the 21-nucleotide sequence alone is necessary for trans activation, a sequence (AGGAACTGAAA) homologous to a type-specific viral enhancer sequence and present in the U3 region of HTLV-II LTR, but not in the same region of the HTLV-I LTR, was inserted together with the 21-nucleotide sequence into the deleted U3 region of the HTLV-I LTR. However, no significant differences of the levels of activities of those LTRs compared to the LTRs with only the 21 nucleotide sequence repeats were observed. PMID- 3022283 TI - Possible involvement of RAS-encoded proteins in glucose-induced inositolphospholipid turnover in Saccharomyces cerevisiae. AB - Incubation of yeast Saccharomyces cerevisiae at very low (0.02%) glucose levels led to arrest of the cell cycle at the G0/G1 phase. Readdition of glucose to these "starved" yeast resulted in cell proliferation. In glucose-starved yeast, glucose stimulated 32P incorporation into phosphatidic acid, phosphatidylinositol, phosphatidylinositol monophosphate, and phosphatidylinositol bisphosphate but not into phosphatidylethanolamine and phosphatidylcholine. Preincubation of yeast with [3H]inositol and subsequent exposure to glucose resulted in rapid formation of [3H]inositol monophosphate and [3H]inositol trisphosphate, presumably derived from phosphatidylinositol and phosphatidylinositol bisphosphate. Under similar conditions, glucose elicited both efflux and influx of Ca2+ in yeast. Glucose-induced 32P incorporation into inositolphospholipids and formation of [3H]inositol phosphates were more pronounced in RAS-related mutants such as ras1, ras1 ras2 bcy1, and RAS2Val19 than in the wild-type strain. These results strongly suggest that glucose stimulates inositolphospholipid turnover, Ca2+ mobilization, and subsequent cell proliferation in a manner similar to that of growth factors with mammalian cells, and that RAS-encoded proteins are involved in regulation of this glucose-induced inositolphospholipid turnover in yeast. PMID- 3022282 TI - Linking functional domains of the human insulin receptor with the bacterial aspartate receptor. AB - A hybrid receptor has been constructed that is composed of the extracellular domain of the human insulin receptor fused to the transmembrane and cytoplasmic domains of the bacterial aspartate chemoreceptor. This hybrid protein can be expressed in rodent (CHO) cells and displays several functional features comparable to wild-type insulin receptor. It is localized to the cell surface, binds insulin with high affinity, forms oligomers, and is recognized by conformation-specific monoclonal antibodies. Although most of the expressed protein accumulates as a 180-kDa proreceptor, some processed 135-kDa receptor can be detected on the cell surface by covalent cross-linking. Expression of the hybrid receptor inhibits the insulin-activated uptake of 2-deoxyglucose by CHO cells. Thus, this hybrid is partially functional and can be processed; however, it is incapable of native transmembrane signaling. The results indicate that the intact domains of different types of receptors can retain some of the native features in a hybrid molecule but specific requirements will need to be satisfied for transmembrane signaling. PMID- 3022284 TI - Transformation-dependent increases in endogenous cytochalasin-like activity in chicken embryo fibroblasts infected by Rous sarcoma virus. AB - Transformation of chicken embryo fibroblasts by infection with Rous sarcoma virus has been shown to cause disruption of actin filament organization as seen with fluorescence staining techniques. This study is an attempt to use quantitative biochemical techniques to compare actin-related parameters in normal and transformed cells. Normal cells and cells infected with a temperature-sensitive mutant virus (NY68) and grown at the restrictive temperature of 41.5 degrees C have normal bundles of actin filaments, or F-actin; these cells also have about the same number of high-affinity cytochalasin binding sites at the ends of F actin (approximately 5 pmol of sites per mg of cellular protein; Kd, 20 nM). In contrast, infected cells grown at the permissive temperature of 37 degrees C have a more diffuse pattern of actin filaments, and the number of cytochalasin binding sites in these transformed cells was below the level of detection. DNase I inhibition assays showed that the percent of unpolymerized actin, or G-actin, in cell extracts was not significantly different between normal and transformed cells (approximately 50%). In assays of cell extracts for endogenous cytochalasin like activity on actin filaments (i.e., retardation of filament assembly at the fast-growing end, inhibition of cytochalasin binding to actin "nuclei," and decrease of low-shear viscosity of solutions of actin filaments), infected cells at 37 degrees C showed a higher level of activity per mg of protein than did uninfected cells or infected cells at 41.5 degrees C. These results suggest that the increase in endogenous cytochalasin-like activity in transformed cells may relate to the decrease in measurable cytochalasin binding sites and the abnormal distribution of actin filaments previously seen by fluorescence staining techniques. PMID- 3022285 TI - Type beta transforming growth factor is an inhibitor of myogenic differentiation. AB - We have investigated the effect of type beta transforming growth factor (TGF beta) on the differentiation of skeletal muscle myoblasts. TGF-beta potently (ID50 approximately 10 pM) prevents established cell lines and primary cultures of rat and chicken embryo myoblasts from fusing into multinucleated myotubes. Inhibition of morphological differentiation by TGF-beta correlates with inhibition of the expression of muscle-specific mRNAs and proteins, strong induction of extracellular matrix type I collagen and fibronectin, and a marked tendency of the treated myoblasts to aggregate into densely multilayered arrays or clusters. Myogenic differentiation can resume after removal of TGF-beta from the medium. Examination of the time of action of TGF-beta shows that myoblasts stochastically reach a point beyond which they become insensitive to the inhibitory action of TGF-beta. This resistance of committed myoblasts to the inhibitory action of TGF-beta is not associated with any measurable change in the number or affinity of TGF-beta receptors in those cells. The results indicate that TGF-beta is a potent inhibitor of myogenesis and may regulate muscle development in vivo. PMID- 3022286 TI - Trophic molecules and evolution of the nervous system. AB - Although recent work has reemphasized the general importance of ontogeny in evolution, underlying developmental molecular mechanisms are largely undefined. What heritable ontogenetic mechanisms result in the evolution of new morphologies and functions? Such questions are particularly difficult in the nervous system, in which each of 10(11) neurons forms approximately equal to 10(4) specific interconnections. I propose that specific heritable, trophic interactions during development, which determine cell survival and pathway size, form a substrate for neural evolution. This model is based on the observation that neurons are vastly overproduced during ontogeny; neurons, their pathways and connections are dependent on target-derived trophic factors for developmental survival; and co innervating, functionally and anatomically distinct neural populations compete for common trophic factors for survival. Focusing on sympathetic and sensory neurons, which require the target-derived, trophic protein nerve growth factor at different times for developmental survival, and which innervate common targets, different classes of ontogenetic evolutionary mechanisms may be characterized. Evolution may occur from heritable changes in the structure of trophic gene products or altered timing of expression. Molecular mechanisms underlying heterochrony are thereby described. The model is directly applicable to evolution of the brain and is testable in a variety of situations. PMID- 3022287 TI - Oxygen-dependent mutagenesis in Escherichia coli lacking superoxide dismutase. AB - Escherichia coli double mutants (sodA sodB) completely lacking superoxide dismutase (SOD) have greatly enhanced mutation rates during aerobic growth. Single mutants lacking manganese SOD (MnSOD) but possessing iron SOD (FeSOD) have a smaller increase, and single mutants lacking FeSOD but possessing MnSOD do not show such an increase. The enhancement of mutagenesis is completely dependent on the presence of oxygen, and treatments that increase the flux of superoxide radicals produce even higher levels of mutagenesis. The presence of a plasmid overproducing either form of SOD reduces the level of mutagenesis to that of wild type, showing that the O2-dependent enhancement results from a lack of SOD. The enhancement of mutagenesis is RecA-independent, and a complete lack of SOD does not induce the SOS response during aerobic growth. However, the enhanced mutagenesis in aerobically grown sodA sodB mutants is largely dependent on functional exonuclease III, suggesting that the increased flux of superoxide radicals results in DNA lesions that can be acted on by this enzyme, leading to mutations. PMID- 3022288 TI - A gene essential for Agrobacterium virulence is homologous to a family of positive regulatory loci. AB - The vir region of Agrobacterium tumefaciens spans at least six transcriptional loci required for crown gall tumorigenesis. The transcriptional induction of two of these vir loci in response to cocultivation with tobacco suspension cells was measured by using bacteria containing mutations in each of the six vir loci located on the Ti plasmid. Induction of these vir genes occurred only in bacteria that had functional copies of virA and virG. The nucleic acid sequence of a 1.25 kilobase clone encompassing virG contains one open reading frame capable of coding for a protein of about 30,000 daltons. The amino acid sequence of the predicted virG product is homologous to that of eight bacterial proteins, including that of the ompR gene of Escherichia coli. Most, although not all, of these proteins, like VirG, are positive regulatory elements. PMID- 3022289 TI - Structure-function analysis of murine interleukin 1: biologically active polypeptides are at least 127 amino acids long and are derived from the carboxyl terminus of a 270-amino acid precursor. AB - Murine interleukin 1 (IL-1) is initially synthesized as a 270-amino acid precursor protein. Guided by amino-terminal end sequence analyses of mouse macrophage-derived IL-1, it was shown that expression of the carboxyl-terminal 156 amino acids (i.e., amino acids 115-270) of this precursor in Escherichia coli yields biologically active recombinant IL-1 (rIL-1) protein. To answer questions about precursor processing and the size of the smallest biologically active IL-1 fragment, we have engineered deletions of the rIL-1 (115-270) gene to encode two amino-terminal deletion analogs, rIL-1 (131-270) and rIL-1 (144-270), and a carboxyl-terminal deletion analog, rIL-1 (131-257, 270). The analogs were produced in E. coli, purified to homogeneity, and assayed for biological activity on murine thymocytes, human rheumatoid synovial cells, and human dermal fibroblasts and for their ability to bind to IL-1 receptors on murine EL-4 thymoma cells. The amino-terminal deletion analog rIL-1 (131-270) possessed a specific activity in the murine thymocyte proliferation assay equivalent to that of the 115-270 parent protein and exhibited significant biological activity in stimulating the production of collagenase and prostaglandin E2 by synovial cells and fibroblasts. The more extensive amino-terminal deletion analog rIL-1 (144 270) was inactive in all biological assays and failed to compete in the receptor binding assay. The carboxyl-terminal deletion analog rIL-1 (131-257, 270) competed less efficiently (by a factor of 100) in the receptor binding assay, retained weak biological activity on synovial cells and fibroblasts, and only demonstrated full intrinsic activity in the thymocyte proliferation assay when 100-200 times more protein was assayed. These results suggest that biologically active murine IL-1 polypeptides are at least 127 amino acids long and are derived from the carboxyl terminus of the 270-amino acid precursor. Furthermore, it appears that the integrity of the carboxyl terminus of the 270-amino acid precursor is important for activity but that different amino termini can be utilized to generate molecules with equivalent specific activities. This amino terminal end flexibility supports a processing model for IL-1 maturation that partially explains IL-1 polypeptide heterogeneity. PMID- 3022290 TI - Hepatitis B virus integration site in hepatocellular carcinoma at chromosome 17;18 translocation. AB - Integrated hepatitis B virus (HBV) DNA is almost invariably found in hepatocellular carcinomas (HCC) which develop in HBV carriers. Integrated HBV DNAs from two single-integration HCCs (C3 and C4) have been cloned, and the cellular integration sites have been analyzed. Integrated HBV DNA of C3 is present in chromosome 6 and contains a nearly complete linear HBV genome. The HBV DNA integration in tumor C3 was not associated with major rearrangements of cellular DNA. In contrast, the integrated HBV DNA in C4 contains a large inverted repeat of HBV DNA, in which each repeat consists of a linear HBV DNA segment similar to that present in C3. The C4 integration was also accompanied by a cellular DNA translocation at the HBV integration site. The translocation occurred between chromosomes 17 and 18, along with a deletion of at least 1.3 kilobases of chromosome 18 DNA at the translocation site. Our data support a model in which postintegration rearrangement of integrated HBV and cellular DNA results in the generation of chromosomal aberrations. These chromosomal aberrations may function in a multistage mechanism leading to fully malignant HCC. PMID- 3022291 TI - Cardiac myocyte hypertrophy is associated with c-myc protooncogene expression. AB - The mechanism of hormonally induced cell hypertrophy is unknown. Stimulation of cardiac myocytes by alpha 1-adrenergic agents, phorbol esters, and serum induces an increase in the cell size of nondividing cardiac myocytes in primary culture. Expression of the c-myc gene, known to be increased in growth factor-induced cell division, was studied in this model of cell hypertrophy. The alpha-adrenergic agonist norepinephrine (0.002-20 microM) increased levels of c-myc-encoded mRNA to 10-fold over control levels. This increase was detectable at 30 min, peaked at 2 hr, and returned to baseline by 6 hr after stimulation. The norepinephrine response was abolished by the alpha 1-antagonist terazosin (2 microM) but was not affected by the beta-adrenergic antagonist propranolol (2 microM) and was only slightly (25%) attenuated by the alpha 2-adrenergic antagonist yohimbine (2 microM). Serum and the phorbol ester tumor promoter phorbol 12-myristate 13 acetate also enhanced c-myc expression in cardiac myocyte cultures. These findings show that the induction of cardiac myocyte hypertrophy is associated with enhanced expression of the c-myc gene and suggest that hormonally induced cell hypertrophy and cell division share common mechanistic pathways. PMID- 3022292 TI - Neural map of interaural phase difference in the owl's brainstem. AB - Neurons of the barn owl's (Tyto alba) nucleus laminaris, the first site of binaural convergence, respond in a phase-locked fashion to a tone delivered to either ear. It may take longer to elicit phase-locked spikes from one ear than from the other. This disparity in delay differs from neuron to neuron and is independent of tonal frequency. In binaural stimulation, neurons respond best when sound in one ear leads that in the other by an amount equal to their delay disparities but opposite in sign. This condition causes simultaneous arrival of phase-locked spikes from the two sides. Laminaris neurons can thus be described as coincidence detectors. The phase of a tone-induced evoked potential, termed "neurophonic," varies systematically with position in nucleus laminaris. From dorsal to ventral within the nucleus, the phase delay of a contralaterally elicited potential decreases and that of its ipsilateral counterpart increases. Therefore, if the neurophonic delay is due to the delay of phase-locked spikes, an orderly representation of delay disparities is shown. Because they act as coincidence detectors, laminaris neurons should show selectivity for interaural phase difference based on their place in the nucleus. Thus, nucleus laminaris presumably measures and maps interaural phase differences by using the principles of delay lines and coincidence detection. PMID- 3022293 TI - Altered effector specificities in regulators of gene expression: TOL plasmid xylS mutants and their use to engineer expansion of the range of aromatics degraded by bacteria. AB - Stimulation of transcription from positively regulated promoters involves regulatory proteins that have been activated, generally as a consequence of binding low molecular weight effector molecules. To define essential structural features of effectors for one positively acting gene regulator, the xylS-encoded protein, which activates the TOL plasmid meta-cleavage pathway operon promoters, effector activities of a wide range of benzoate derivatives have been systematically analyzed, and mutant xylS-encoded proteins exhibiting altered effector specificities have been generated and characterized. Cloned mutant xylS genes were trans dominant in partial diploids containing the wild-type xylS allele and could therefore be used to effect expansion of the range of aromatic compounds completely or partially degraded by Pseudomonas bacteria. The method developed to isolate mutant xylS-encoded proteins has general applicability and could in principle be used to isolate gene regulator specificity mutants of any inducible regulatory system. PMID- 3022294 TI - Mouse thymidylate synthase messenger RNA lacks a 3' untranslated region. AB - Analysis of the sequence of cDNA corresponding to mouse thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) mRNA revealed that the termination codon TAA was followed immediately by a poly(A) sequence. This raised the possibility that mouse thymidylate synthase mRNA lacks a 3' untranslated region. In the present study, we have further investigated this possibility. DNA corresponding to the 3' end of the thymidylate synthase gene was isolated from a genomic library. The sequence of the genomic DNA was identical to that of the cDNA in the coding region. However, the termination codon was TAG in the genomic sequence rather than TAA, and poly(A) was not present in the genomic DNA. Sequences flanking the site of poly(A) addition were in good agreement with polyadenylylation consensus sequences. S1 nuclease analysis revealed that approximately 80% of the thymidylate synthase mRNA molecules were polyadenylylated at the termination codon. A secondary polyadenylylation site was detected 190-200 nucleotides downstream of the primary site. We conclude that the major species of mouse thymidylate synthase mRNA lacks a 3' untranslated region and that the final A of the termination codon is added by poly(A) polymerase. It appears that a 3' untranslated region is not essential for the accumulation or translation of this mRNA. PMID- 3022295 TI - Phosphatidylinositol 4,5-bisphosphate turnover is transient while phosphatidylinositol turnover is persistent in thyrotropin-releasing hormone stimulated rat pituitary cells. AB - Stimulated inositolphospholipid turnover has been proposed to be initiated and sustained by hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], which may be replenished by an enhanced flux of phosphatidylinositol (PtdIns) to PtdIns 4-phosphate (PtdIns4P) to PtdIns(4,5)P2. To determine whether there is continued hydrolysis and resynthesis of PtdIns(4,5)P2 in rat pituitary cells (GH3 cells) during stimulation by thyrotropin-releasing hormone (TRH), we investigated the turnover kinetics of the inositolphospholipids and of phosphatidic acid (PtdOH). In cells incubated with 32Pi for 1 min, TRH rapidly and persistently (for at least 30 min) enhanced the rate of 32P-labeling of PtdOH. After a lag time of 1 min, TRH markedly and persistently increased 32P-labeling of PtdIns also. In contrast, TRH caused only a transient increase in 32P-labeling of PtdIns(4,5)P2 that lasted less than 2 min. There was no rapid (before 10 min) effect of TRH on 32P-labeling of PtdIns4P. By 2 min of TRH stimulation, specific 32P radioactivity in PtdOH increased from 3.6% (control) of that in the gamma phosphate of ATP to 15%; in PtdIns, from 0.07% to 1.3%; and in PtdIns(4,5)P2, from 3.8% to 5.4% (specific 32P radioactivity in PtdIns4P was 1.7% of that in ATP in control and TRH-stimulated cells). In cells exposed to TRH for 4 min and then to 32Pi, 32P-labeling of PtdOH and PtdIns increased, but that of PtdIns(4,5)P2 was not affected. Last, persistent turnover of PtdOH and PtdIns was not caused by initial hydrolysis of PtdIns(4,5)P2 because the turnover of PtdOH and PtdIns could be terminated by displacement of TRH from its receptor by chlordiazepoxide and restarted by reoccupying the receptors with TRH. These data demonstrate that turnover of PtdIns(4,5)P2 is stimulated only transiently, whereas turnover of PtdIns and PtdOH is stimulated persistently by TRH in GH3 cells. Hence, inositolphospholipid turnover in GH3 cells does not occur via continued hydrolysis of PtdIns(4,5)P2 accompanied by enhanced flux of PtdIns to PtdIns4P to PtdIns(4,5)P2, but there is direct and persistent hydrolysis of PtdIns. The dissociation of these actions suggests that there are separate mechanisms involved in coupling TRH-receptor complexes to stimulation of PtdIns(4,5)P2 and PtdIns hydrolysis in GH3 cells. PMID- 3022296 TI - Interaction of a nuclear factor with the polyomavirus enhancer region. AB - We have identified a factor present in nuclear extracts of undifferentiated F9 murine embryonal carcinoma cells that specifically interacts with the polyomavirus enhancer region. Nuclease "footprint" analysis was used to define the binding site that corresponds precisely to the boundaries of polyoma enhancer element C defined by Veldman et al. [Veldman, G. M., Lupton, S. & Kamen, R. (1985) Mol. Cell. Biol. 5, 649-658] that is required as an enhancer for efficient viral DNA replication and early and late region transcription. The region of nuclease protection contains a 6-base-pair inverted repeat, separated by 3 base pairs, and symmetrical flanking DNase I hypersensitive cleavage sites, suggesting that this factor may bind as a dimer. A cloned 29-base-pair polyoma DNA fragment contains an intact binding domain. Similar levels of binding activity were found in nuclear extracts prepared from differentiated murine F9 cells, as well as murine L cells and human HeLa cells. The factor has been termed "EF-C" for enhancer binding factor to polyoma element C. PMID- 3022297 TI - Differential sensitivity of two functions of the insulin receptor to the associated proteolysis: kinase action and hormone binding. AB - Since we observed that after purification the receptor kinase activity is rapidly lost under conditions where insulin binding function seems to be preserved, we have studied the cause(s) of receptor kinase inactivation. Highly purified placental insulin receptor preparations were analyzed by NaDodSO4/PAGE followed by silver staining or immunostaining using domain-specific antibodies raised against synthetic peptides corresponding to the amino acid sequences of the beta subunit. These studies revealed the intact 90-kDa beta subunit is degraded first to an 88-kDa form and then to a 50-kDa beta 1-subunit form by proteolysis even after purification when stored at 4 degrees C. The 88-kDa beta subunit, which lacks the carboxyl-terminal approximately equal to 2-kDa portion exhibits almost no autophosphorylation activity, nor does insulin stimulate autophosphorylation. The loss of kinase activity as measured by phosphorylation of the src-related peptide is correlated with the loss of the intact 90-kDa beta subunit. Degradation of the beta subunit to the 50-kDa form seems to be facilitated by the removal of the approximately equal to 2-kDa peptide. Present studies thus suggest that only the intact form of the beta subunit has full kinase activity in an insulin-dependent manner and that other forms, such as the 88-kDa beta subunit show little kinase activity. The inactivation appears to arise from a conformational change of the 90-kDa form, which makes it susceptible to proteolysis at the carboxyl-terminal end. These results imply that the carboxyl terminal of the beta subunit is important for the manifestation of the tyrosine kinase activity of the insulin receptor. PMID- 3022298 TI - Structural homology of reaction centers from Rhodopseudomonas sphaeroides and Rhodopseudomonas viridis as determined by x-ray diffraction. AB - Crystals of the reaction center (RC) from Rhodopseudomonas sphaeroides with the space group P2(1)2(1)2(1), have been studied by x-ray diffraction. The Patterson search (molecular replacement) technique was used to analyze the data, with the structure of the reaction center from Rhodopseudomonas viridis as a model system. A preliminary electron density map of the reaction center from R. sphaeroides has been obtained. Comparison of the structure of the RC from R. sphaeroides with that from R. viridis showed the following conserved features: five membrane spanning helices in each of the L and M subunits, a single membrane-spanning helix in the H subunit, a 2-fold symmetry axis, and similar positions and orientations of the cofactors. Unlike the RCs from R. viridis, both quinones are retained in the RCs from R. sphaeroides. The secondary quinone is located near the position related by the 2-fold symmetry axis to the primary quinone. PMID- 3022299 TI - cAMP induction of prespore and prestalk gene expression in Dictyostelium is mediated by the cell-surface cAMP receptor. AB - Extracellular adenosine 3',5'-cyclic monophosphate (cAMP) is required for cell type-specific gene expression in developing Dictyostelium discoideum. We have developed a microassay for the expression of these genes, using antibodies directed against their protein products. To characterize the transduction mechanism, we have used in this assay cAMP analogues that preferentially activate either the cell-surface cAMP receptor or the internal cAMP-dependent protein kinase. N6-(aminohexyl) cAMP activates the Dictyostelium cAMP-dependent protein kinase but does not bind to the cell-surface cAMP receptor and does not cause cell-type-specific gene expression. 2'-Deoxy-cAMP does not activate the cAMP dependent protein kinase but binds to the receptor and causes cell-type-specific gene expression. Cyclic AMP-induced accumulation of prestalk mRNA in shaking cultures still occurs in the presence of caffeine, which blocks the receptor coupled activation of adenyl cyclase. This suggests that the extracellular cAMP induction of cell-type-specific gene expression in developing Dictyostelium cells is mediated by the cell-surface cAMP receptor and that activating adenyl cyclase by this receptor is not essential. Using the N6-(aminohexyl) cAMP to competitively inhibit phosphodiesterase, we show that 30 nM cAMP is sufficient to induce prestalk or prespore gene expression. PMID- 3022301 TI - Charomids: cosmid vectors for efficient cloning and mapping of large or small restriction fragments. AB - Charomids are cosmid vectors up to 52 kilobases (kb) long, bearing 1-23 copies of a 2-kb spacer fragment linked in head-to-tail tandem arrays. Like cosmids and lambda phage, charomids can be packaged in vitro for efficient introduction into bacteria. Charomids contain a polylinker with nine unique restriction sites for cloning and can be used without preparing vector arms. Using a charomid of appropriate size, one can clone inserts of any size up to 45 kb. For example, charomid 9-36 (9 cloning sites, 36 kb long) is too small to be packaged efficiently without an insert and can be used to clone fragments of 2-16 kb. The structure of charomids facilitates restriction mapping of the insert DNA and, after cloning, all the spacer fragments can be removed easily. After enrichment by size fractionation in an agarose gel, a specific single-copy genomic sequence can be cloned rapidly from approximately 3 micrograms of DNA. Using charomid 9 36, we have cloned and mapped an amplified novel DNA fragment from a cell line resistant to N-(phosphonoacetyl)-L-aspartate and carrying about 100 copies of the CAD (carbamoyl-phosphate synthetase/aspartate carbamoyltransferase/dihydroorotase) gene. The fragment lies at the center of an inverted duplication of this gene. PMID- 3022300 TI - Suppression of tumor growth by senescence in virally transformed human fibroblasts. AB - Normal human cells whether embryonic, neonatal, or adult are resistant to experimentally induced tumorigenesis in contrast to rodent or chicken cells. We showed previously that neither transformation with simian virus 40 DNA nor transfection with human mutant HRAS DNA immortalized FS-2 cells (diploid, neonatal human fibroblasts). Further, tumorigenicity was not induced, despite expression of the respective transforming gene products tumor (T) antigen or p21. Here we describe treatment of FS-2 and FSSV cells with baboon endogenous virus pseudotyped Kirsten murine sarcoma virus. FSSV cells were derived from individual foci of simian virus 40-transformed FS-2 cells. The retrovirus-treated FS-2 cells (called FSK) appeared heavily granulated and expressed viral p21 but senesced during passage in culture and were not tumorigenic. The retrovirus-treated FSSV 27 cells (called FSVK-27) expressed simian virus 40 tumor antigen, had elevated levels of viral p21 protein, and formed transient tumors in nude mice. Whether grown in culture or explanted from small tumors, the FSVK-27 cells senesced. The FSVK-46 cells senesced before tumor growth occurred. On the contrary, Kirsten murine sarcoma virus (baboon endogenous virus) treatment of immortalized nontumorigenic human fibroblasts expressing simian virus 40 tumor antigen (Va2 cells) led to consistent tumor formation. The results illustrate the importance of senescence in restricting the tumor-forming ability of human cells. PMID- 3022302 TI - Molecular structure of a somatically unstable transposable element in Drosophila. AB - A transposable element has been isolated from an unstable white mutation in Drosophila mauritiana, a sibling species of Drosophila melanogaster. The unstable white-peach (wpch) allele exhibits a spectrum of germ-line and somatic mutability more similar to insertion mutations in maize and in the nematode Caenorhabditis elegans than has been reported for insertion mutations in Drosophila. The inserted element mariner is 1286 nucleotides long and has terminal inverted repeats. The element contains a single open reading frame encoding 346 amino acids. A duplication of 2 base pairs of white sequence is present at the insertion site. Mariner is present in approximately 20 copies in the D. mauritiana genome, is present from 0 to 7 copies in other members of the sibling species group, and is apparently absent from the genome of D. melanogaster. PMID- 3022303 TI - Transformation of human ciliary epithelial cells by simian virus 40: induction of cell proliferation and retention of beta 2-adrenergic receptors. AB - Ciliary epithelial cells derived from human eye were successfully propagated through many generations after transformation with simian virus 40. The cell clone 8-SVHCE was isolated and characterized by immunoprecipitation and pharmacological studies that demonstrated the presence of several functional properties observed in the parent cells of this tissue. Immunoprecipitation revealed the presence of large tumor (T) antigen, and Southern blot analysis showed the incorporation of viral DNA into high molecular weight ciliary epithelial cell DNA. The presence of beta-adrenergic receptors was demonstrated by direct binding of a radiolabeled antagonist, [125I]iodopindolol, to membrane preparations of 8-SVHCE cells (Kd = 41.8 pM and Bmax = 67.1 fmol/mg of protein). Competition experiments with [125I]iodopindolol and selective drugs suggested that the receptors are of the beta 2-adrenergic subtype. Studies of catecholamine stimulated cellular cAMP production and of isoproterenol-dependent protein phosphorylation of vimentin in 8-SVHCE indicated the functional conservation of beta-adrenergic receptor-mediated processes that are thought to be important in the regulation of aqueous humor production by the ciliary epithelium in vivo. PMID- 3022304 TI - A human cytomegalovirus mutant resistant to the nucleoside analog 9-([2-hydroxy-1 (hydroxymethyl)ethoxy]methyl)guanine (BW B759U) induces reduced levels of BW B759U triphosphate. AB - We have isolated a human cytomegalovirus mutant that is resistant to the antiviral drug 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U), yet exhibits wild-type sensitivity to inhibitors of herpesvirus DNA polymerases such as phosphonoformic acid and aphidicolin. Cells infected with the mutant accumulate approximately equal to 1/10th the amount of drug triphosphate as do those infected with the wild-type parent. This reduction in drug triphosphate could not be attributed to altered drug uptake or to reduced stability of the triphosphate, once formed. The induction of normal nucleoside and deoxynucleoside triphosphates and certain cellular nucleoside kinases was comparable in mutant and wild-type virus infections. These results provide strong evidence for the importance of phosphorylation in the selectivity of this antiviral compound and raise the possibility that human cytomegalovirus encodes a nucleoside kinase. The mutant may identify the existence of a cytomegalovirus function whose properties could facilitate genetic analysis of this important pathogen. PMID- 3022305 TI - Brain opioid receptors in the hibernating bat, Myotis lucifugus: modification by low temperature and comparison with rat, mouse and hamster. AB - Several studies have provided evidence that brain opioid peptides may be involved in the control of the hibernation cycle. We have now examined the influence of hibernation on central opioid receptors. We have characterized the receptor binding properties of [3H]-naloxone in slices of the cerebral cortex and hypothalamus of the bat Myotis lucifugus. These receptors possess those characteristics expected of an opioid site namely high affinity, stereospecificity and saturability. Under normal incubation conditions (30 degrees C) we observed no effect of hibernation on [3H]-naloxone binding. However, when binding assays were performed at temperatures corresponding to the appropriate body temperature (e.g., 4 degrees C for hibernation) we detected a significant low temperature-induced increase in hypothalamic binding. Similar experiments in rat, mouse and hamster revealed that [3H]-naloxone binding was also increased at 4 degrees C when compared to 30 degrees C. This was true in hypothalamus and cortex. Additional studies in the rat demonstrated that the opioid receptor is of higher affinity at low temperatures. The behavioural and neurochemical consequences of this change in the opioid receptor, and whether this might be involved in the regulation of hibernation, remain to be studied. PMID- 3022306 TI - Food hoarding and ingestion in the deer mouse, Peromyscus maniculatus: selective responses to mu and kappa opiate agonists. AB - The feeding behavior of the deer mouse, Peromyscus maniculatus, includes food hoarding as well as ingestion. Administration of the prototypical mu opiate agonist, morphine sulfate, 1-20 mg/kg, produced over three hours a significant dose-dependent stimulation of hoarding by free feeding deer mice. The specific kappa opiate agonist, U-50,488H, 0.10-10 mg/kg, markedly increased ingestion without having any augmentatory effects on hoarding. The mixed mu and kappa opiate agonist, ketocyclazocine hydrochloride, 1-10 mg/kg, as well as various combinations of morphine sulfate and U-50,488H, augmented both hoarding and ingestion. Food restriction for 24 hr caused a significant, naloxone (1.0 mg/kg) reversible, increase in food intake. Food deprivation also modified the hoarding and ingestion responses of the deer mice to the mu and kappa opiate agonists, reducing the relative amounts of food that were hoarded. These results indicate that mu and kappa opioid systems are differentially involved in the mediation of various aspects of feeding. This also suggests that environmental factors, such as food restriction, can modify the relative roles of mu and kappa opioid systems in the expression of feeding behavior. PMID- 3022307 TI - Behavior and receptor changes after kainate lesioning of nodular cerebellum. AB - A study was undertaken on the effects of kainic acid lesioning on the nodulus of the rat cerebellum on behavior and various brain receptors in conscious, freely moving rats. The basis for the study was the observation that barrel rotation and other motor effects induced by intraventricular administration of vasopressin and nicotine could be elicited by their administration into the nodular area of the cerebellum. Histology revealed a marked destruction of Purkinje, stellate, and Golgi cells in the area surrounding the site of kainate administration, with little effect on the granular cells. Immediately after administering 4-12 ng of kainic acid into the nodular cerebellum, rats exhibited circling movements, barrel rotation, and clonic convulsions accompanied by stereotypic head movements, aggressiveness, and gnawing-biting; effects gradually diminishing over 3 days. Receptor binding studies 4-14 days after kainate lesioning revealed a marked increase in 3H-nicotine and 3H-QNB binding in the surrounding cerebellar region, caudate nucleus, and hypothalamus, with no change in 3H-dihydromorphine binding. The findings are consistent with the hypothesis that nicotinic and muscarinic pathways in the vestibular cerebellum, along with its connection to nigrostriatal dopaminergic systems, are involved in the mediation of barrel rotation, ataxia, and other motor disturbances resulting from administration of vasopressin on nicotine intraventricularly. PMID- 3022308 TI - Potentiation of the propunishment, but not the convulsant action of the beta carboline DMCM by naltrexone. AB - The ability of naltrexone (NTX) to potentiate the propunishment and convulsant properties of DMCM, a benzodiazepine receptor inverse agonist, was studied in mice. Doses (0.39 and 1.56 mg/kg) of DMCM which were below the threshold for propunishment effects showed a marked ability to enhance the suppressive effects of punishment on locomotor activity in the presence of naltrexone (0.5 or 2.5 mg/kg IP), higher doses of DMCM and NTX (3.13 mg/kg and 10 mg/kg, respectively) had a depressant effect of their own on both punished and unpunished locomotor activity. DMCM given alone induced clonic convulsions (ED50: 5.7 mg/kg IP) but this activity was not changed in the presence of naltrexone. These results suggest an interaction of BZ receptors and opioid systems in the control of anxiety. PMID- 3022309 TI - Scopolamine impairs learning performance of rats in a 14-unit T-maze. AB - To assess involvement of muscarinic cholinergic systems in performance of a shock motivated 14-unit T-maze task, 3-month old Fischer-344 rats were given an IP injection of scopolamine (0.1, 0.3, 1.0 or 3.0 mg/kg), methylscopolamine (1.0 mg/kg), or saline 30 min prior to maze training on 2 consecutive days. Scopolamine, but not methylscopolamine, impaired all components of acquisition performance. Measures of error performance, run time, shock duration, and number of shocks received were significantly increased but only at the 1.0 and 3.0 mg/kg scopolamine doses. The cognitive component of the task, measured by error performance, appeared most affected. Cognitive performance deficits observed following scopolamine administration in the present study resembled age-related impairments in rats and mice previously observed in this task. The cholinergic hypothesis of geriatric memory dysfunction appears to be implicated by these findings; however, the degree to which memory systems are involved remains unclear. Other performance variables such as discriminative control of stimuli or mechanisms of attention are implicated and discussed. PMID- 3022311 TI - Stimulus amplification by PAF and LTB4 in human neutrophils. PMID- 3022310 TI - Comparative effects of leukotrienes and PAF-acether electrophysiological activity of the rat retina. PMID- 3022312 TI - Comparative effects of drugs on leukotrienes-, PAF-acether- and histamine- induced contractions of guinea-pig lung parenchymal strips. AB - Dose-response curves to leukotrienes B4 (LTB4), D4 (LTD4), PAF-acether and histamine applied as single or cumulative doses are studied on guinea-pig lung parenchymal strips (GPLP). A mole to mole comparison shows that PAF-acether is the most potent agonist, when GPLP contracts maximally to histamine. GPLP possesses specific receptors to histamine, LTD4 and PAF-acether which can be blocked by selective receptors antagonists: mepyramine as antihistaminic, FPL 55712 as anti-SRS-A substance, BN 52021 and kadsurenone as anti-PAF-receptor antagonists; meanwhile, a part of their action is mediated through the release of arachidonate metabolites. Histamine, LTB4, LTD4 and PAF-acether-induced contractions are all dependent on cyclooxygenase. Thromboxane A2 (TXA2) could be one of the cyclooxygenase products involved. A part of contractile effects of LTB4 and LTD4, but not of histamine, is liked to the generation of lipoxygenase metabolites as their contractions are partially inhibited by NDGA and ETYA. Contraction-induced by PAF-acether is reduced in presence of NDGA but not by FPL 55712, which indicates that lipoxygenase products other than peptidoleukotrienes are involved in the PAF-response. PMID- 3022314 TI - Induction of cross-links in viral DNA by naturally occurring photosensitizers. PMID- 3022313 TI - Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers. PMID- 3022315 TI - Effect of manganese on human placental spin-lattice (T1) and spin-spin (T2) relaxation times. AB - Human placentas were obtained immediately following delivery and incubated with manganese chloride (MnCl2) in concentrations ranging from 0.002 to 2.0 mM. Proton density, T1 and T2 were measured at times ranging from 5-200 minutes. There was rapid uptake of manganese by the placenta producing a dose-dependent decrease in placental T1 and T2. The major effect of manganese uptake was shortening of T1 suggesting that the contrast between placenta and myometrium will be enhanced predominantly for T1-dependent imaging pulse sequences. PMID- 3022316 TI - Effect of magnetic field on the process of cell respiration in mitochondria of rats. AB - Exposure of rats to static magnetic field 1 hour daily for a period of 7 weeks (7 days a week) leading to disturbances of the respiration processes in the mitochondria of liver cells. The rate of respiration through NADH dehydrogenase, succinic dehydrogenase and cytochrome oxidase was dependent on both the duration and the intensity value of the field applied. The animals showed greater sensitivity to the action of a 0.008 T magnetic induction field than to that of 0.15 T. The observed changes were reversible after 3 months since the everyday exposure had been stopped. PMID- 3022317 TI - Conduction velocity in slower motor fibres. AB - Measuring conduction velocity in slower fibres (CVSF), through a partial antidromic block (Hopf-Seppalainen method), allows estimating the dispersal of conduction velocity in motor fibres by the difference between the maximal motor conduction velocity (MCV) and CVSF. Investigations performed in workers exposed to medium concentrations of carbon disulphide show difference between MCV and CVSF as providing a more sensitive indicator for early diagnosis of peripheral neuropathies than MCV. PMID- 3022319 TI - Effects on long-term sensitivity to pain and morphine of stress induced in the newborn rat by pain or manipulation. AB - Four times daily from postnatal day 1 to 15, rats were stressed either by being removed from the maternity cage (manipulation stress, MS) or by being placed on a hotplate at 55 degrees C (pain stress, PS). When 70 days old, they were examined for sensitivity to pain and to the analgesic effect of morphine, and for brain opiate receptors. Pain sensitivity of MS and PS rats was not significantly different from that of controls. The analgesic activity of morphine, assessed by the hotplate test at 49 degrees C, was significantly reduced in MS rats, while in PS rats it was similar to that in controls. 3H-dihydromorphine binding studies performed on whole brain synaptic membranes showed a reduction in the maximum number of binding sites in both MS and PS rats; on the other hand, the affinity constant was higher in PS rats, while in MS rats it was similar to that of controls. These data show that the repeated stress of removal from the mother during the first 15 days of life induce a reduction in the number of brain opiate receptors with reduced activity of morphine, while in rats exposed to repeated removal stress associated with painful stimuli the reduction in the number of brain opiate receptors seems to be counterbalanced by their higher affinity. PMID- 3022318 TI - Food ingestion is more important to plasma corticosterone dynamics than water intake in rats under restricted daily feeding. AB - The roles of food and/or water ingestion in the regulation of plasma corticosterone level were examined in rats under restricted daily feeding. When the time of food-pellets and water supply was restricted to 2 hours in the early light period (meal feeding) for 2 weeks, the corticosterone level increased prior to meal (prefeeding peak). A similar prefeeding hormone peak was observed when supply of food-pellets was restricted to 2 hours with free-access to water (food restriction). In contrast, when water supply was restricted to 2 hours with free access to food-pellets (water restriction), the hormone level before water supply did not increase as much as that under meal feeding or food restriction. Shortening of an available time for water under water restriction or prolongation of the restriction schedule failed to elevate the hormone level furthermore. On the other hand, the high prefeeding corticosterone level before meal decreased subsequently to meal feeding (prandial fall), which was not observed when rats were kept fasting during the meal time. This prandial fall of the hormone level was not observed by water intake alone, and closely related to food-pellets ingestion. It is concluded that food ingestion is more important than water intake to the formation of the prefeeding corticosterone peak and to the prandial fall of the hormone level under restricted daily feeding. PMID- 3022321 TI - Determination of the sizes of the opioid receptors in their membrane environment by radiation inactivation. AB - Opioids, like other drugs, are thought to initiate their effects by association with their specific receptors. However, very little is known about the opioid receptor as a molecular entity. The binding components have been solubilized in detergent and purified by different approaches, but the molecular size of soluble opioid receptor complexes reported by different groups varied from 23,000 to 750,000. In this study, the technique of radiation inactivation by gamma rays was used to investigate the apparent size of the opioid receptor in rat brain membranes under different conditions. The molecular sizes of opioid receptor complexes were estimated as 313,000 +/- 13,500 in the presence of [D-Ala2, D Leu5] enkephalin, NaCl and Gpp (NH)p; as 165,000 +/- 8,500 in the presence of NaCl only, or of both NaCl and Gpp (NH)p; as 217,000 +/- 6,600 in the presence of Gpp (NH)p only; and as 286,000 +/- 60,900 in the presence of MgCl2 only. A simple model has been proposed to explain these different apparent target sizes of opioid receptors obtained under different conditions. PMID- 3022320 TI - Effect of estrogen (Premarin) replacement therapy on serum level of total estrogen and urinary excretion of calcium and hydroxyproline in postmenopausal Chinese women. AB - The study subjects included a total of 30 postmenopausal Chinese women, including 14 natural menopausal women, 13 castrated menopausal women and 3 post-irradiated menopausal women. Premarin 1.25 mg/day was given orally for 3 weeks and off one week, repeated for 6 cycles. Fasting morning urine and blood samples were collected before hormone treatment and at the end of 3 weeks, 11 weeks, and 23 weeks of Premarin therapy. Serum total estrogen level was measured by radioimmunoassay. Urinary calcium and creatinine were measured by atomic absorption and Jeffe's reaction, respectively. The concentration of urinary hydroxyproline was determined by Kivirikko's method. After Premarin therapy, the mean concentration of serum total estrogen increased 3 to 4 times from the pretreatment level of 71.7 pg/ml. On the other hand, the mean value of calcium/creatinine (Ca/Cr) molar ratio dropped down from 0.249 to 0.098. The mean value of hydroxyproline/creatinine (HOPr/Cr) molar ratio was reduced from 0.028 to 0.012. In view of the hormonal and biochemical changes after Premarin therapy, it is concluded that estrogen (Premarin) replacement should be effective in the treatment of enhanced bone loss or osteoporosis in postmenopausal women. The relationship of estrogen and calcitonin in the regulation of bone metabolism is also discussed. PMID- 3022322 TI - The role of the locus coeruleus-limbic noradrenergic transmission in the action of antidepressant drugs. PMID- 3022323 TI - Psychosis and water intoxication as presenting symptoms of pulmonary carcinoma. PMID- 3022324 TI - Hepatocellular carcinoma in immigrants to the United Kingdom. AB - Analysis of the origin of patients with hepatocellular carcinoma admitted to the Liver Unit between 1970 and July 1985 showed an increase in frequency of this tumour in immigrants to the United Kingdom from none between 1970 and 1973, to 15 per cent between 1981 and 1985. The 19 immigrant cases had resided in this country for between four and 28 years and when compared with a group of 30 patients born in the United Kingdom three striking differences emerged. Firstly, the immigrants were significantly younger. Secondly, although the frequency of underlying cirrhosis (approximately 80 per cent) was similar the aetiology differed with HBV-related disease being the largest group (93 per cent) in the immigrants and alcohol or cryptogenic cirrhosis in the other patients amongst whom HBV markers were detected in only 20 per cent. Thirdly, whilst signs and symptoms attributable to hepatocellular carcinoma represented the first evidence of liver disease in 89 per cent of the immigrants, symptoms of cirrhosis in patients born in the United Kingdom had been evident for several years before tumour development. PMID- 3022326 TI - Amplification of oncogenes and integrated SV40 sequences in mammalian cells by the decay of incorporated iodine-125. AB - Iodine-125, in the form of 5-[125I]iododeoxyuridine (I-UdR), was incorporated into the DNA of SV40 transformed Chinese hamster embryo cells. Disintegration of the 125I led to increased cell killing with increasing dose as measured by the colony-forming ability of single cells. The D37 (the dose at which 37% of the cells survive) amounts to 95 decays per cell, corresponding to 0.66 Gy. Variations in the copy number of specific DNA sequences was measured by using dispersed cell blotting with sensitive DNA hybridizations. A 13-fold amplification of the viral DNA sequences (SV40) and a twofold amplification of two cellular oncogenes of the ras-family (Ki-ras and Ha-ras) were found. Other cellular genes, like the alpha-actin gene, were not amplified, and no variation in gene copy number was detected after incubation of cells with cold I-UdR. We suggest the observed gene amplifications are induced by the densely ionizing radiation emitted by the decay of the incorporated 125I atoms. PMID- 3022327 TI - [Localization of metronidazole in the organs of tumor-bearing animals using electron paramagnetic resonance]. AB - The low-temperature ESR-spectroscopy was used to study the irradiated organs and blood of tumor-bearing animals before and after the administration of metronidazole. The ESR signals of metronidazole anion-radicals were registered in tissues of the animals injected with metronidazole: the intensity of these signals correspondent with the tissues preparation content. PMID- 3022325 TI - OH-induced free radicals in uridine studied by a method combining ESR, spin trapping, and liquid chromatography. AB - Free radicals produced by the reactions of OH radicals with uridine were investigated by a method combining ESR, spin-trapping, and liquid chromatography. A N2O-saturated aqueous solution of uridine, containing 2-methyl-2-nitrosopropane as a spin-trap, was X-irradiated and the resulting spin-adducts were separated by gel permeation chromatography and reverse-phase HPLC. ESR and uv-absorbance spectra obtained from the separated spin-adducts show that 5-yl and 6-yl radicals are produced by OH addition to the 5,6 double bond of the base moiety. It is also shown that radicals due to H abstraction from the sugar moiety at the C-4' and C 5' positions are produced. PMID- 3022328 TI - Effect of S-2(3-aminopropylamino) ethyl phosphorothioic acid on ATPase activity in the testis of irradiated mouse. PMID- 3022330 TI - [Giant ovarian cystadenoma and bilateral ovarian theco-fibroma. Presentation of a case]. PMID- 3022329 TI - [Infection with human Cytomegalovirus]. PMID- 3022331 TI - [Comparative x-ray and nuclear medical studies of osteosarcomas to evaluate the effectiveness of preoperative chemotherapy]. AB - In 16 patients with a long bone osteosarcoma treated after the therapy regimen of COSS 80/82 (Cooperative Osteosarcoma Study) the tumour response to preoperative chemotherapy was assessed by conventional roentgenograms (plain films, tomograms, angiograms). The histological grade of regression determined in the operative specimen was used as a reference for the extent of tumour devitalisation. By their different typical roentgenological course the osteosarcomas with a very good and those with a rather poor response could be correctly identified (100%). On following the clinically used differentiation between "good responders" (histological grades I to III) and "poor responders" (grades IV to VI) the corresponding classification of these tumours by roentgenograms and functional scintigraphic imaging was found to be correct in 94% of the cases. The coincidence of the correct as well as the false results in both radiological methods was as high as 100% if those osteosarcomas showing only a "medium response" in their roentgenograms were added to the clinical group of good responders. There were no fundamentally discrepant results obtained by each of the two radiological techniques. With the combined workup of the roentgenological and the scintigraphic material any incorrect preoperative estimation of tumour regression with a harmful influence on therapy (date and type of surgery, mode of postoperative chemotherapy) could be avoided. PMID- 3022332 TI - [Real-time sonography. An important imaging procedure in the diagnosis and treatment planning of bone metastases]. AB - The value of sonography for diagnosis and therapy planning of bone metastases is shown in 110 affected skeletal regions (60 patients with malignant diseases). Whereas sonography is inferior to conventional x-ray in respect of defects of the spongiosa, it is equal with regard to defects of the cortical layer but superior in respect of changes of the periosteum and the surrounding soft tissue. Moreover, there is an excellent correlation (97%) between pain and the reaction of periosteum and soft-tissue layer. Because of the three dimensional representation of the tumourous processes sonography has proved valuable in radiotherapy planning (field size, radiation method, risk organs etc.). PMID- 3022333 TI - [Selective arterial DSA of the femur head vessels]. AB - Selective arterial DSA of the arteries of the femoral head was carried out in 39 patients. The appearances were studied in nine patients without any hip abnormality, in 13 idiopathic and eight post-traumatic cases of femoral head necrosis and in nine patients with recent fractures. In every patient the vessels were well demonstrated. In the presence of idiopathic aseptic necrosis there was always hypervascularity around the necrotic area. As in the normal controls, pathological findings were seen in the deep branch of the medial circumflex femoral artery and in the nutrient artery in one-third of patients. Post traumatic aseptic necrosis, recent fractures and dislocations always showed abnormalities of the vessels to the femoral head. The changes in the femoral head vessels demonstrated by DSA are probably not of great significance for the development of idiopathic aseptic necrosis, however they are found regularly following trauma. PMID- 3022334 TI - [Magnetic resonance tomography and safety. Electroencephalographic and neuropsychologic findings before and after MR studies of the brain]. AB - The influence of MR on the EEG and neuropsychological functions was investigated. An EEG was done in 8 of 20 patients before and after 0.5 Tesla MRI. Computerized EEG analysis showed no significant differences in respect of the dominant frequency and the power spectrum. The memory functions of 12 patients were investigated by clinically proven neuropsychological tests before and after MRI (0.5 and 1.5 tesla). The results demonstrated no measurable influence of MRI on cognitive functions. PMID- 3022335 TI - [Magnetic resonance tomography of the orbit using surface coils. Study technic, anatomy and initial clinical experience]. AB - MRI of the orbit is strongly improved by the use of surface coils due to a higher signal-to-noise ratio. Oblique views without moving the patient present the optic nerve in full length on one slice. First experience with a small number of cases demonstrates normal anatomy and lesions in detail only at T1-weighted pulse sequences. Losses in contrast variation and detail accuracy are caused by movements of the eyeballs. Edge artifacts due to chemical shifting impair the image quality. So far there are no pointers towards tissue-specific signal intensity behaviour. Procedure and most favourable parameters at 1 tesla are given. PMID- 3022336 TI - [Lumbosacral computed tomography and lumbosacral functional myelography. Dosimetric comparison and comparison of indications]. AB - The integral dose and organ dose produced by spinal lumbosacral CT and lumbosacral functional myelography were compared, using a phantom. The value of these methods of investigation is discussed. PMID- 3022337 TI - [Computed tomographic stereotaxy: development of a new system]. AB - We present a newly developed stereotactic apparatus for use with conventional roentgenograms and computed tomography. The geometrical dimensions and proportions of the apparatus render a calculation of target coordinates redundant. It incorporates all the required safety and precision. Two independent targeting devices can be mounted to the headholder. Thus the apparatus encompasses, to a large extent, present and future developments towards efficient and less invasive brain operations. PMID- 3022338 TI - [Computed tomographic findings in extrinsic allergic alveolitis. Comparison with conventional x-ray findings]. AB - Seventeen patients with extrinsic allergic alveolitis or bird-fancier's lung were examined by standard radiological techniques and classified after Hapke's classification. In addition, the patients were examined by CT. The CT patterns have been analysed and compared with standard radiological findings. The methodological advantages of CT are discussed. Radiological investigation is of limited value in the diagnosis of extrinsic allergic alveolitis. Conventional radiography remains the standard of initial x-ray examination. In early cases, however, CT may be a valuable addition within the diagnostic strategy of a diagnostic imaging department. PMID- 3022339 TI - [Differential analysis of ventricular contraction using digital subtraction angiography]. AB - One hundred and thirty-six patients with various cardiac abnormalities that had been diagnosed by standard methods, were examined by I-V DSA; images of the left and right ventricles during a representative cardiac cycle were submitted to amplitude and phase analysis, using a Fourier transformation. Temporal differentiation of ventricular function was derived from the best available right anterior oblique projection; the amplitude of cardiac movement of abnormal areas in the myocardium were obtained from a grey scale or colour coding at a fixed point. Comparison with a control group of 35 individuals showed the following pathological findings: hypokinetic segments (33 cases) showed delay, dyskinesias (20 cases) showed complete separation of maximal phases, frequently with a double peak in the phase histogram; disturbances of cardiac rhythm (14 cases) showed atypical localisation of initiation of contraction: in Wolfe-Parkinson-White syndrome this is basal-anterior, in left bundle branch block and VVI stimulation it is apical inferior; in hypertrophic obstructive cardiomyopathy (eight cases) it is postero-basal inferior with high, narrow peaks on the phase histogram. Differential phase analysis on I-V DSA enables one to define cardiac contraction in a simple manner. PMID- 3022340 TI - Ultrasound guided percutaneous drainage of pericardial fluid with an indwelling catheter. AB - The technique of ultrasonographic guided percutaneous drainage of pericardial fluid, applied in three patients, is reported. The primary disease was synovial sarcoma, rheumatoid arthritis and prostatic carcinoma, respectively. Although three slightly different techniques and catheters were used all patients were sufficiently drained and the clinical symptoms promptly relieved. The catheters were left for drainage 3 months, 5 days and 14 days respectively. There were no major complications. One patient complained of transient palpitations. Percutaneous ultrasound-guided catheter drainage seems to be a safe method in patients with pericardial fluid where an indwelling catheter is considered. PMID- 3022342 TI - [Digital subtraction angiography of the aorta]. AB - The results of 300 digital subtraction angiographies of the thoracic and abdominal aorta are analysed. Following intraarterial injection of contrast, all examinations were of good quality. Following I-V DSA, two thoracic (1.8%) and two abdominal (1.2%) aortograms were non-diagnostic. Anomalies in the position of the aorta, dilatation (aneurysm) or stenoses (coarctations, arteriosclerosis, occlusions) could be diagnosed by I-V DSA. One I-A DSA was performed for aneurysm of the ascending aorta in order to evaluate function of the aortic valve and for demonstrating the coronary artery origins, and pre-operatively for an aortic dissection. PMID- 3022341 TI - [Atresia of the aortic isthmus from the radiological point of view]. AB - The most important angiographic features of atresia of the aortic isthmus are illustrated by three patients. The first patient to have reached an advanced age despite atresia of the aortic isthmus is reported and the diagnosis was confirmed at autopsy. The haemodynamic effects, the age at operation and the incidence of other associated cardiovascular malformations are discussed with reference to the literature. PMID- 3022343 TI - [Are thoracic radiographs in the recumbent position useful in the diagnosis of chronic left heart insufficiency? Comparison of x-ray findings with left ventricular hemodynamics]. AB - 108 patients (88 male, mean age 50 years, 20 female, mean age 49) having coronary heart disease (40), cardiomyopathy (39), hypertension (9), valvular disease (9), pulmonary diseases (5) and without cardiac diseases (6) underwent chest plain radiographs in standing and recumbent position immediately before being investigated invasively by left and right heart catheterisation, angiocardiography and coronary angiography. Two experienced radiologists evaluated the plain films with special reference to the findings of the heart and lung vessels, particularly the pulmonary veins. Dilatated veins of the upper lobes were observed more often if the radiograph was obtained from the recumbent patient. A significant close correlation with increased LVEDP was, however, revealed in both positions. A possible simulation of pathological dilatation of the venous trunci of the upper lobes merely due to the redistribution of the blood flow in the horizontal position was excluded. The results show that reliable conclusions can be drawn concerning chronic left ventricular failure if the characteristic pulmonary vein findings are visualised in patients both in the standing and in the recumbent position. PMID- 3022344 TI - Arterial embolism of the upper extremities. AB - The angiographic signs, the frequency and the site of distribution of acute emboli of the arteries of the upper extremity are described. The conclusions are based on the author's own experience gained from selective studies of acute arterial embolism of the upper limb, during a period of 15 years. A comparison is made with the results of two of the largest series reported in the literature. In addition, a brief review of the aetiology, pathogenesis, the clinical and roentgenological signs of the condition is given. PMID- 3022345 TI - Gastrooesophageal reflux during liquid and solid meals. A reevaluation of the de Carvalho test. AB - Gastrooesophageal reflux during liquid and solid meals was investigated in 43 patients with upper abdominal dyspepsia. Gastrooesophageal reflux occurred in 45% of the patients after a solid meal and in 90% after a liquid meal. Differences in oesophageal motility during liquid and solid meals are discussed as a possible explanation of the phenomenon. PMID- 3022346 TI - [Magnetic resonance tomographic findings in esophageal cancer]. AB - The value of MRT and CT for staging and for determining operability of carcinoma of the oesophagus was assessed in 20 patients with histologically confirmed diagnoses. Both methods are limited in this respect. In some cases magnetic resonance provides more accurate concerning the extent of the tumour; this, however, is true only for lesions in the upper and middle thirds of the oesophagus. Where tumours of the distal third of the oesophagus are concerned, and particularly in those extending into the fundus of the stomach, CT is clearly superior. Both methods are of limited value in assessing the depth of tumour penetration, infiltration of neighbouring organs and involvement of regional lymph nodes. PMID- 3022347 TI - Graded compression sonography in acute appendicitis. AB - Acute appendicitis never was a radiologically established diagnosis. A study on sonography in acute appendicitis was published recently in which in 89% of cases the inflamed appendix could be demonstrated by ultrasound, utilising 5 and 7.5 MHz intraoperative linear array transducers. Our study was started to evaluate the possibilities of a standard 7.5 MHz sector transducer in detecting acute appendicitis. In our study sonography was correctly diagnosed in 72% of cases, but it became apparent that with the larger format transducer used in our study the retrocaecal appendix could not be demonstrated in the majority of cases. The reliability of sonography in detecting acute appendicitis is not high enough to be used on a large scale but sonography can play a role in doubtful cases of suspected acute appendicitis. PMID- 3022348 TI - [Angiographic and computed tomographic findings in intra-arterial cytostasis of colorectal liver metastases]. AB - Between October 1983 and January 1986, sixteen patients with inoperable liver metastases from colorectal carcinomas were treated by single or cyclical intraarterial chemotherapy introduced either through an angiographic catheter or by means of a subcutaneously implanted access point (Port-A-Cath). Before each treatment cycle, the position of the catheter and the state of the vessels was examined by subtraction angiography in order to avoid complications and to confirm that the cytostatic agents were actually perfusing metastases, as shown by angio-CT. The complementary nature of the information obtained by these methods and their superiority over scintigraphy with 99mTc macroaggregated albumin particles is documented and the most frequent complications of regional tumour therapy are described. PMID- 3022349 TI - [CT diagnosis of focal nodular hyperplasia]. AB - The paper analyses the findings of a CT study of 21 lesions of focal nodular hyperplasia in 17 patients. In addition to subjective valuatin of the dynamic studies, densitometric measurements on a time basis were carried out (gamma-fit curves) and a density profile was obtained. These tumour-like lesions show typical density changes with rapid enhancement in the arterial phase and rapid fall in density after one or two minutes. Various patterns of the hyperdense lesions (non-homogeneous, those resembling vessels or homogeneous lesions) may be of significance in differential diagnosis. Atypical density patterns were found particularly in foci projecting from the liver. PMID- 3022350 TI - [CT of tongue and mouth floor amyloidosis. A case contribution to the differential diagnosis of macroglossia]. PMID- 3022351 TI - [Pulmonary blastoma: evolution into an adenocarcinoma]. PMID- 3022352 TI - [Large adenoma of the gallbladder]. PMID- 3022353 TI - [Unusual metastasis of a prostatic carcinoma]. PMID- 3022354 TI - Carotid aneurysm formation due to focal exogenous arteritis documented by serial angiography. PMID- 3022355 TI - [Aneurysm of the splenic artery]. PMID- 3022356 TI - [Poxvirus infections in Mauritanian sheep. Atypical sheep pox or lumpy skin disease?]. PMID- 3022357 TI - Studies on the possible roles of naturally infected Nigerian local chickens and vaccine virus in the epidemiology of infectious bursal disease. PMID- 3022358 TI - Effect of temperature on beta receptor responsiveness in guinea pig pulmonary tissues. AB - Cumulative concentration-effect relationships of isoproterenol (isoprenaline) on guinea pig tracheal spirals and lung parenchymal strips were determined in organ baths kept at 37 degrees C or at 18 degrees C. The IC50's for isoproterenol at 18 degrees C were significantly (p less than .01) higher than those for isoproterenol at 37 degrees C in both the tissues; although the parenchymal response was affected more than that of the trachea by cooling. pA2 values for propranolol were determined on both tracheal spirals and parenchymal strips at 37 degrees C and 18 degrees C. The pA2 values were decreased in both the tissues by cooling. The decrease was more prominent in the parenchyma than in the trachea, a finding consistent with the concentration-effect relationships studies mentioned above. PMID- 3022359 TI - Fine structural and cytochemical studies of eosinophils from fowls and ducks with eosinophilia. AB - The ultrastructure and cytochemistry of eosinophils from adult fowl and ducks with either spontaneous or experimentally induced eosinophilia were examined. The results showed that a high proportion of the eosinophils in the peripheral blood of eosinophilic birds had ultrastructural features different from those of normal eosinophils. In both species, there was a reduction in cell size. Fowl eosinophil granules showed similar morphological changes to those seen in the quail with many crescentic and vacuolated forms being present. In eosinophilic ducks, the crystalline interna of the specific granules were often fragmented or were either partially or completely lysed. Cytochemically, peroxidase activity in both species was generally unaltered in abnormal eosinophils compared with those from normal birds. This correlates with the findings in man. However, amounts of acid phosphatase and trimetaphosphatase were reduced in many cells, with a large proportion of granules being non-reactive. The latter observation corresponds with that in quail but differs from man, in which stimulated eosinophils have increased enzymatic activity. PMID- 3022360 TI - Ultrastructural and cytochemical studies in normal Japanese quail (Coturnix coturnix japonica) eosinophils and in those from birds with experimentally induced eosinophilia. AB - Normal eosinophil development in the Japanese quail (Coturnix coturnix japonica) was similar to that described in the fowl and the duck, with granulogenesis occurring in the Golgi apparatus. The characteristic lipid droplets were small in the immature eosinophils, and after staining specifically for lipid, small moieties were also traced to the Golgi apparatus. In mature eosinophils the lipid droplets measured between 1.0 and 1.5 micron in diameter and they were surrounded by profiles of smooth endoplasmic reticulum. Eosinophilia was difficult to induce in quails; injections of either horse serum or bovine serum albumin (BSA)/aluminium hydroxide produced a poor response. In some quails in which eosinophilia was produced, however, eosinophil granules showed many crescentic and vacuolated forms. The lipid droplets in the activated eosinophils were fused in many cells to form large intracellular aggregates of lipid. Quail eosinophils, which hitherto have been regarded as peroxidase-negative, had strong activity in the lipid droplets of cells from stimulated birds. It is postulated that this peroxidase-positive reaction may represent a form of ceroid or lipofuscin pigment resulting from lipid peroxidation. Acid phosphatase and trimetaphosphatase reactions were reduced in many activated cells, with a large proportion of granules being non-reactive. The results of dietary manipulations in quails appear to suggest that in stressful situations the eosinophil metabolism is altered and there is a reduction in the number of lipid droplets in the cell. PMID- 3022361 TI - Passive protection of sheep against capripoxvirus. AB - The close antigenic relationship between strains of capripox was shown by passively immunising sheep with serum against capripoxviruses isolated from a sheep and from a goat. Sheep immunised with immune serum to Oman sheep pox or Yemen goat pox resisted challenge with Yemen goat pox or Nigeria sheep pox respectively. Lambs born to sheep previously infected with isolates of capripox from the Sudan, India and Nigeria were also protected against challenge with Yemen goat pox. PMID- 3022362 TI - Incidence of group A and atypical rotaviruses in Brazilian pig herds. AB - The incidence of rotaviruses as a gastroenteritis causal agent in piglets was studied in 19 pig herds of Sao Paulo State, Brazil, during 1985. From 302 diarrhoea samples collected during January (summer), 65 were positive for rotavirus when analysed by enzyme-linked immunosorbent assay (ELISA) and polyacrylamide gel electrophoresis (PAGE). Sixty-two of these samples belonged to the classical group A rotavirus, three to atypical rotaviruses (ELISA negative and probably group B) and one elicited a mixed electropherotype of group A and atypical rotavirus and was ELISA positive. Atypical viruses appear to be very fragile and were rapidly degraded upon storage of samples at -20 degrees C. Three herds where atypical rotaviruses were present in January were sampled again in August (winter). Nine atypical isolates out of a total 21 positive samples (assayed by electron microscopy and PAGE) were detected again in two of them. PMID- 3022363 TI - Demonstration of the carrier state in naturally acquired equine arteritis virus infection in the stallion. AB - The chronic carrier state was virologically confirmed in 15 thoroughbred stallions naturally infected with equine arteritis virus based on the results of test matings and, or, isolations of the virus from semen. Carrier stallions were shown to shed equine arteritis virus in the semen for at least one to two years. Existence of a short-term or convalescent carrier state was also demonstrated in five additional stallions. The frequency of the long-term carrier state in stallions naturally infected with equine arteritis virus was 35 per cent; it varied considerably between groups of stallions on different farms. The carrier stallion would appear to play an important epidemiological role in the dissemination and perpetuation of equine arteritis virus. PMID- 3022364 TI - Protection from radiation-associated small bowel injury with the aid of an absorbable mesh. AB - Radiation associated small bowel injury results from aggressive treatment of pelvic malignancies with radiation therapy. The incidence increases when radiation therapy follows pelvic surgery due to adhesions that form between the small bowel and the operated site. Application of an absorbable polyglycolic acid mesh to keep the small bowel from descending into the pelvis and around the operated site prevents this complication. Use to date in humans and non-human primates has not been associated with any complications. PMID- 3022365 TI - [Dietary fiber]. PMID- 3022366 TI - [Dopaminergic receptors]. AB - The functions of multiple brain dopamine receptors are discussed in relation to the physiological role of dopamine in CNS. PMID- 3022367 TI - [The acquired immunodeficiency syndrome, a new disease of uncertain future]. PMID- 3022368 TI - [Epidemiology and immunological study of LAV virus infection]. PMID- 3022369 TI - [Viral etiology of AIDS]. PMID- 3022370 TI - Immunologic aspects of peripheral nerve disease. PMID- 3022371 TI - Endoscopic ultrasonography of bile duct malignancy and the preoperative assessment of local resectability. AB - Endoscopic ultrasonography (EUS) was performed before surgery in 12 patients with Klatskin tumour and in 8 patients with distal common bile duct carcinoma. The usual EUS findings consisted of an intraductular mass lesion together with adjacent lymph node involvement. The depth of malignant infiltration into the surrounding tissues could clearly be seen, which was of utmost importance in assessing local resectability. A clearly demarcated malignant growth with or without evidence of loco-regional lymph node involvement was considered compatible with local resectability. Involvement of distant lymph nodes along major blood vessels was thought to indicate palliative resection. Evidence of deep infiltration of the malignancy into the surrounding tissues (mesenteric artery, coeliac trunk, aorta) and/or organs (liver metastasis) was considered to indicate non-resectability. From this study EUS appears to be a sensitive modality for detecting, staging, and preoperative assessment of local resectability in bifurcation tumours and in distal bile duct malignancy. Technical improvements enabling guided cytological puncture and reducing the length of the rigid tip may further enhance its diagnostic value. PMID- 3022372 TI - [Severe electrolyte disorders during the therapy of heart failure with the therapy of heart failure with the ACE-inhibitor enalapril]. AB - Angiotensin I converting enzyme inhibition by captopril and enalapril may influence sodium and potassium homeostasis. In patients without cardiac failure and with normal renal function significant electrolyte disturbances rarely occur. We report on four patients who developed life-threatening electrolyte disturbances following treatment with enalapril for severe cardiac failure (NYHA class II-IV). There were important concomitant factors in all four cases: in one case under additional medication with a thiazide diuretic and a nonsteroidal antiinflammatory, hyponatremia of 107 mmol/l occurred. In two further cases severe hyperkalemia of 7.4 and 7.3 mmol/l was observed in the presence of acute renal failure due to enalapril-induced hypotension and concomitant therapy with a nonsteroidal antiinflammatory drug respectively. In a fourth case the combination of enalapril with a potassium-sparing diuretic provoked severe hyperkalemia of 7.9 mmol/l. PMID- 3022373 TI - [Outbreak of infectious laryngotracheitis (ILT) in a flock of young hens]. PMID- 3022374 TI - [Prevalence of bovine herpesvirus 4 in the Swiss cattle population and possible serologic cross reaction with bovine herpesvirus 1 (IBR/IPV virus)]. PMID- 3022375 TI - Researcher reprimanded for pseudorabies test. PMID- 3022376 TI - Two avirulent herpes simplex viruses generate lethal recombinants in vivo. AB - While it is widely appreciated that infection with a virulent virus can produce disease in an animal, the ability of a mixture of avirulent viruses to produce disease by means of complementation or recombination in vivo has not been established. In this study, two weakly neuroinvasive herpes simplex virus type 1 (HSV-1) strains were simultaneously inoculated onto the footpads of mice. Many (62%) of the animals that received a 1:1 mixture of the viruses died, whereas the animals that received a similar or 100-fold higher dose of each agent alone survived. Of fourteen viruses isolated from the brains of ten mice that died after receiving the mixture of the two weakly neuroinvasive viruses, eleven were recombinants; three of these recombinants were lethal when reapplied to the footpads of mice. These results show that two avirulent HSV-1 variants may interact in vivo to produce virulent recombinants and a lethal infection. They also suggest that different genetic lesions account for the weakly neuroinvasive character of the HSV-1 strains ANG and KOS after footpad inoculation. PMID- 3022378 TI - Debate about epilepsy: what initiates seizures? PMID- 3022377 TI - Chromosomal localization of muscle nicotinic acetylcholine receptor genes in the mouse. AB - The chromosomal localization of the genes encoding the four subunits of muscle nicotinic receptor was determined by analyzing restriction fragment length polymorphisms between two mouse species Mus musculus domesticus (DBA/2) and Mus spretus (SPE). Analysis of the progeny of the interspecies mouse backcross (DBA/2 X SPE) X DBA/2 showed that the alpha-subunit gene cosegregates with the alpha cardiac actin gene on chromosome 17, that the beta-subunit gene is located on chromosome 11, and that the gamma- and delta-subunit genes cosegregate and are located on chromosome 1. PMID- 3022380 TI - Glucose-6-phosphatase activity in brain. PMID- 3022379 TI - AIDS in Africa: an epidemiologic paradigm. AB - Cases of the acquired immune deficiency syndrome (AIDS) have been reported in countries throughout the world. Initial surveillance studies in Central Africa suggest an annual incidence of AIDS of 550 to 1000 cases per million adults. The male to female ratio of cases is 1:1, with age- and sex-specific rates greater in females less than 30 years of age and greater in males over age 40. Clinically, AIDS in Africans is often characterized by a diarrhea-wasting syndrome, opportunistic infections, such as tuberculosis, cryptococcosis, and cryptosporidiosis, or disseminated Kaposi's sarcoma. From 1 to 18% of healthy blood donors and pregnant women and as many as 27 to 88% of female prostitutes have antibodies to human immunodeficiency virus (HIV). The present annual incidence of infection is approximately 0.75% among the general population of Central and East Africa. The disease is transmitted predominantly by heterosexual activity, parenteral exposure to blood transfusions and unsterilized needles, and perinatally from infected mothers to their newborns, and will continue to spread rapidly where economic and cultural factors favor these modes of transmission. Prevention and control of HIV infection through educational programs and blood bank screening should be an immediate public health priority for all African countries. PMID- 3022381 TI - Estrogen memory effect in human hepatocytes during repeated cell division without hormone. AB - Transient stimulation of target tissues by sex steroids can cause long-lasting changes that may facilitate or alter responses to subsequent hormonal treatment. How these altered characteristics are propagated during cell division in the absence of the stimulating hormone is unknown. The human hepatocarcinoma cell line HepG2 was used as a model to examine the effects of estrogen on the synthesis of serum apolipoproteins in vitro. Treatment with low concentrations of estrogen for 24 to 48 hours resulted in long-lasting alterations in the kinetics with which the cells responded to subsequent stimulation with estrogen. Manifestation of this memory effect was correlated quantitatively with the induction and propagation of a moderate-affinity, nuclear, estrogen-binding protein with the characteristics of a type II estrogen receptor. The data indicate that transient exposure of these cells to estrogen can induce changes in their response characteristics and composition of nuclear proteins that are inherited by daughter cells grown in the absence of hormone for more than ten generations. PMID- 3022382 TI - Diminished response of Werner's syndrome fibroblasts to growth factors PDGF and FGF. AB - Patients with Werner's syndrome, an autosomal recessive disorder, undergo an accelerated aging process that leads to premature death. Fibroblasts from such patients typically grow poorly in culture. Here it is shown that fibroblasts from a patient with Werner's syndrome have a markedly attenuated mitogenic response to platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). In contrast, they have a full mitogenic response to fetal bovine serum. Both PDGF binding and receptor numbers per cell are unaltered. The Werner's syndrome cells express high constitutive levels of collagenase in vitro. Although PDGF enhances collagenase expression through increased levels of hybridizable collagenase messenger RNA in normal skin fibroblasts, no induction of collagenase occurs in the Werner's syndrome fibroblasts. Moreover, the failure to respond to this agonist effect of PDGF is not restored by fetal bovine serum. The data suggest that failure of one or more PDGF-mediated pathways in Werner's syndrome cells may contribute to the phenotypic expression of the disorder. PMID- 3022383 TI - A selective imidazobenzodiazepine antagonist of ethanol in the rat. AB - Ethanol, at pharmacologically relevant concentrations of 20 to 100 mM, stimulates gamma-aminobutyric (GABA) receptor-mediated uptake of 36Cl-labeled chlorine into isolated brain vesicles. One drug that acts at GABA-benzodiazepine receptors, the imidazobenzodiazepine Ro15-4513, has been found to be a potent antagonist of ethanol-stimulated 36Cl- uptake into brain vesicles, but it fails to antagonize either pentobarbital- or muscimol-stimulated 36Cl- uptake. Pretreatment of rats with Ro15-4513 blocks the anticonflict activity of low doses of ethanol (but not pentobarbital) as well as the behavioral intoxication observed with higher doses of ethanol. The effects of Ro15-4513 in antagonizing ethanol-stimulated 36Cl- uptake and behavior are completely blocked by benzodiazepine receptor antagonists. However, other benzodiazepine receptor inverse agonists fail to antagonize the actions of ethanol in vitro or in vivo, suggesting a novel interaction of Ro15-4513 with the GABA receptor-coupled chloride ion channel complex. The identification of a selective benzodiazepine antagonist of ethanol stimulated 36Cl- uptake in vitro that blocks the anxiolytic and intoxicating actions of ethanol suggests that many of the neuropharmacologic actions of ethanol may be mediated via central GABA receptors. PMID- 3022385 TI - Thrombin binding and stimulation of cyclic guanosine monophosphate formation in neuroblastoma cells. PMID- 3022384 TI - Double-signal hypothesis for thrombin initiation of cell proliferation. PMID- 3022386 TI - Histologic spectrum of alcoholic liver disease. PMID- 3022387 TI - Pathologic characteristics of hepatocellular carcinoma. PMID- 3022388 TI - [Alpha-adrenoceptors in the rat spinal cord: its role in baroreflex and hemorrhagic shock]. PMID- 3022389 TI - [Studies on the content and effects of opiate-like substances in rabbit ear artery strips]. PMID- 3022391 TI - [The mechanism of inhibition of anisodamine on platelet aggregation induced by E. coli endotoxin]. PMID- 3022390 TI - [Alpha-adrenergic control of segmental coronary arterial resistance in the dog]. PMID- 3022392 TI - Case report 394: Malignant fibrous histiocytoma (MFH) arising in an infarct of bone. PMID- 3022393 TI - Preoperative localization of parathyroid adenomas using thallium-technetium subtraction scintigraphy. AB - Primary hyperparathyroidism can be diagnosed by laboratory values, yet localization of the adenomas preoperatively has always presented difficulties. Dual isotope scintigraphy with technetium Tc 99m sodium pertechnetate and thallium Tl-201 chloride has recently been used for the localization of parathyroid adenomas. In this paper, we review our experience with scintigraphy in 53 patients suspected of having hyperparathyroidism. Based on favorable results, we recommend this technique for preoperative detection of parathyroid adenomas. PMID- 3022394 TI - Natural history of Kluver-Bucy syndrome after treated herpes encephalitis. AB - We present the natural history of three patients with features of Kluver-Bucy syndrome after treated herpes encephalitis. Although cognitive and behavioral disturbances following herpes encephalitis are often severe, improvement can occur over an extended period, and chronic residual sequelae may be relatively mild. The pattern of memory impairment is consistent with the current hypothesis that medial temporal lobe structures mediate memory consolidation. PMID- 3022395 TI - [Criteria of the reliability of neurohormonal mechanisms of adaptation during intensive therapy of cerebral infarct]. PMID- 3022397 TI - [Hypothalamic puberty syndrome]. PMID- 3022396 TI - [Neutrophilic leukocyte function in pneumococcal meningitis]. PMID- 3022398 TI - [Medullary breast cancer]. PMID- 3022399 TI - [Hormonally active tumors of the pancreas]. PMID- 3022400 TI - [Acquired immune deficiency syndrome in Mexico: basis for its prevention and control]. PMID- 3022402 TI - [Detection of liver hemangioma using 2-phase scintigraphy with radiocolloid and 99mTc labeled erythrocytes]. PMID- 3022401 TI - [Class M immunoglobulins (IgM) against rubella and cytomegaloviruses in the blood of newborn infants with a low weight]. PMID- 3022404 TI - [Herpes simplex virus 1 and herpes labialis]. PMID- 3022403 TI - [Pleomorphic adenoma of the palate]. PMID- 3022406 TI - Mosquito-borne virus diseases of man in southern Africa. PMID- 3022405 TI - Virus-associated tumours in man. PMID- 3022407 TI - Radionuclide evaluation of left ventricular function in a distantly located intensive coronary care unit. AB - Regular evaluation of left ventricular (LV) function is important in assessing patients in an intensive coronary care unit (ICCU). An adapted first-transit (FT) technique was applied to 21 patients for calculation of LV ejection fraction (LVEF). In this method the radionuclide is administered directly into the right pulmonary artery through a Swan-Ganz catheter. High-quality LV images are obtained because of minimal activity in the right ventricle and left lung. Data were acquired on a distantly located on-line computer system to restrict equipment in the ICCU. LVEF from FT compared well with LVEF from gated blood pool studies (r = 0.91) but gave consistently lower values. The adapted FT method ensures improved counting statistics during LVEF determination and facilitates evaluation of LV wall motion in the ICCU patient. PMID- 3022408 TI - Occupational history-taking in the RSA. AB - Medical practitioners need to elicit a comprehensive occupational history from their patients, since this may play an integral part in establishing the diagnosis, may indicate the most appropriate form of management, and will ensure that claims for Workmen's Compensation are initiated when appropriate. This history can also assist in identifying undetected workplace hazards and in formulating and testing hypotheses concerning the relationship of work to health. The particular problems encountered in taking an occupational history effectively in the RSA are discussed. PMID- 3022409 TI - Managing the discharge crisis following catastrophic illness or injury. AB - Discharge from the acute care setting frequently represents a serious crisis for the catastrophically ill. Family members and the patient are expected to resume responsibility quickly for ongoing care while simultaneously coping with significant alterations in established role and behavior patterns. The dynamics of this transition are discussed and suggestions for helping the family cope with this crisis are offered. PMID- 3022410 TI - Intracranial seeding from an intramedullary malignant astrocytoma. AB - The authors report a case of intracranial seeding from a spinal intramedullary astrocytoma. The possible mechanisms of seeding and the literature are reviewed. PMID- 3022411 TI - Imaging sacral nerve root cysts. AB - Sacral nerve root cysts usually are an incidental finding but may be found in patients with a history of pain. On computed tomography they are demonstrated as a soft tissue mass, possibly with associated bony erosion. Demonstrating the fluid-filled nature of these masses is important in establishing the diagnosis. The imaging of these cysts in two patients, including magnetic resonance, is described. PMID- 3022412 TI - [Value of neuraminic acid as a clinical parameter in 42 patients with small-cell bronchial cancer]. AB - The serum concentrations of neuraminic acid, CEA, gamma-Gt, 5-nucleotidase, haptoglobin, and alkaline phosphatase were determined before therapy and three and six months after the initiation of therapy in 42 patients with small cell bronchial carcinomas. Before therapy, a significantly increased serum concentration as compared to normal values was found for neuraminic acid in 97% of patients (43/44), for CEA in 54.7% (23/42), for gamma-Gt in 19% (8/42), for 5 nucleotidase in 11.9% (5/42), for haptoglobin in 36.3% (14/44), and for alkaline phosphatase in 9.5% of all patients (4/42). Three and six months later, these laboratory investigations did not give any valuable hint with respect to therapy results, with the exception of neuraminic acid (p less than 0.05). Prior to therapy, the concentrations of neuraminic acid were considerably increased in patients (mean = 3.16 +/- 0.47 mumol/ml) with regard to normal values (mean = 1.90 +/- 0.14 mumol/ml). Within the total group of 42 patients suffering from small cell bronchial carcinomas, there was no significant difference between the serum concentrations of neuraminic acid of eleven patients with localized tumors (mean = 3.12 +/- 0.53) and those of 31 advanced tumor patients (mean = 3.16 +/- 0.45) (p less than 0.05). During the six months' treatment period, the concentration of neuraminic acid was valuable as clinical parameter in 36 patients, i.e. 85% of all cases (0.001 less than p less than 0.01). It was shown that the serum concentration of neuraminic acid indicated a regression of the disease in 23 patients (p less than 0.001), a progression in 8 patients (0.02 less than p less than 0.05), and a regression with subsequent progression of the tumor in 5 patients (0.001 less than p less than 0.01). PMID- 3022413 TI - Selective portal branch occlusion by balloon catheter during liver resection. AB - An intraoperative ultrasonographically guided introduction of a balloon catheter into labor or smaller branches of the portal vein within the liver parenchyma made it possible to temporarily occlude them and perform regional staining during resection for tumors. The technique minimized blood loss without hilar dissection for vascular control, and the presence of the catheter facilitated intraparenchymal dissection of the portal stalk to the part of the liver to be resected. PMID- 3022414 TI - In vitro incorporation and metabolism of icosapentaenoic and docosahexaenoic acids in human platelets--effect on aggregation. AB - Washed human platelets were pre-loaded with icosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or EPA + DHA and tested for their aggregation response in comparison with control platelets. In fatty acid-rich platelets, an inhibition of the aggregation could be observed when induced by thrombin, collagen or U 46619. The strongest inhibition was observed with DHA-rich platelets and it was reduced when DHA was incorporated in the presence of EPA. Study of fatty acid distribution in cell lipids after loading showed that around 90% of EPA or DHA taken up was acylated into phospholipids and a very small amount (less than 2%) remained in their free and hydroxylated forms. DHA was more efficiently acylated into phosphatidylethanolamine (PE) than into phosphatidylinositol (PI) in contrast to what observed with EPA, and both acids were preferentially incorporated into phosphatidylcholine (PC). EPA inhibited total incorporation of DHA and increased its relative acylation into PE at the expense of PC. In contrast, DHA did not affect the acylation of EPA. Upon stimulation with thrombin, EPA was liberated from phospholipids and oxygenated (as judged by the formation of its monohydroxy derivative) whereas DHA was much less metabolized, although consistently transferred into PE. It is concluded that EPA and DHA might affect platelet aggregation via different mechanisms when pre-loaded in phospholipids. Whereas EPA is known to alter thromboxane A2 metabolism from endogenous arachidonic acid, by competing with it, DHA might act directly at the membrane level for inhibiting aggregation. PMID- 3022415 TI - The platelet reactivity of collagen type I: evidence for multiple platelet reactive sites in the type I collagen molecule. AB - In this study, the ability of peptides, obtained by fragmentation of the collagen type I molecule, to induce platelet aggregation has been examined. In order to satisfy requirements for tertiary and quaternary structure, peptides were first renatured (where necessary) to restore triple-helical configuration and then polymerised. Fragmentation with mammalian collagenase indicated the presence of platelet-reactive sites in both the N-terminal three-quarter and C-terminal one quarter fragment of the collagen molecule. Cleavage with cyanogen bromide indicated the presence in the constituent alpha 1(I)-chain of at least four platelet-reactive sites. Our results suggest a relatively wide distribution of platelet-binding sites situated throughout the length of the collagen (type I) molecule, each probably of relatively low affinity and low structural specificity, at least in terms of amino acid sequence, and probably of a similar nature to those that might be expected to exist in any collagen-like species. PMID- 3022416 TI - [The cellular immune system. Possible adverse effects of drugs]. PMID- 3022417 TI - [Late sequelae after Coxsackie virus B myocarditis]. PMID- 3022418 TI - [A simple method for detection of rotaviruses in feces]. PMID- 3022419 TI - [Trends in the control of Aujeszky's disease in the Netherlands]. PMID- 3022420 TI - Brain macrophages in cerebellar cell cultures. AB - Brain macrophages were studied in dispersed monolayer cultures of post-natal mouse cerebella. The phagocytotic activity, binding of immunoglobulins, presence and characteristics of nucleoside diphosphatases as well as argentophilia and lipid distribution were tested, with results similar to those for brain macrophages in vivo and for other macrophages in vitro. The macrophages in the cultures showed four different forms with continuous transition from one form to the following and subsequent cell death. No signs of proliferation of this cell type was found. Dynamic investigations showed repelling of neuronal growth cones by certain macrophages, resulting in the formation of areas devoid of neuronal and glial processes around the macrophages. An in vitro model for these cells, described by other authors as being important in post-natal reshaping of the brain, is presented and investigated in this study. PMID- 3022421 TI - Characterization of functional beta-adrenoceptor subtypes in rabbit urinary bladder smooth muscle. AB - Spontaneous contractile force of muscle strips isolated from male rabbit urinary bladder dome (detrusor) and base (trigonal muscle) was significantly inhibited by isoproterenol (10(-7)-10(-5) M), a non-specific beta-adrenoceptor agonist or by terbutaline (10(-8)-10(-5) M), a selective beta 2-adrenoceptor agonist. The EC50 values for isoproterenol and terbutaline in detrusor were the same as those in trigonal muscle but the maximum relaxant response to isoproterenol or terbutaline was significantly greater in detrusor than in trigonal muscle. Dobutamine (10(-5) 10(-4) M), a relatively specific beta 1-adrenoceptor agonist caused a small but significant relaxant response in trigonal muscle but no change in detrusor. In trigonal muscle the relaxant response to dobutamine was less than that to terbutaline. The relaxant response to 10(-6) M isoproterenol in detrusor was completely blocked by butoxamine (10(-4) M), a selective beta 2-antagonist or by propranolol (10(-6) M), a non-specific beta-antagonist but not by metoprolol (10( 6)-10(-4) M), a selective beta 1-antagonist. Relaxation of trigonal muscle induced by 10(-6) M isoproterenol was inhibited by 10(-5) M metoprolol by 30%, by 10(-4) M butoxamine by 70%, or completely by 10(-6) M propranolol. These findings are consistent with the view that the density of beta-adrenoceptors is higher in the detrusor than in trigonal muscle, and that the relaxant response to beta adrenoceptor stimulation is mediated by beta 2-subtype in the detrusor and by both of beta 1- and beta 2-subtypes in trigonal muscle of the male rabbit. PMID- 3022423 TI - Stimulation of melanogenesis by cholecalciferol in cultured human melanocytes: a possible mechanism underlying pigmentation after ultraviolet irradiation. AB - An increase in the amount of tyrosinase was demonstrated in the cultured human melanocyte after 6-day culturing with cholecalciferol (vitamin D3) by increased intensity of the immunofluorescent staining using monoclonal antibody against tyrosinase. Furthermore, the melanocytes became more dendritic as noted in those in the skin after the irradiation of ultraviolet. However, 7-dehydrocholesterol (pro-vitamin D3) or 1 alpha, 25-dihydroxy-vitamin D3 (activated vitamin D3) did not induce any such effect on the cultured human melanocytes. Since cholecalciferol is known to be photo-chemically converted by the ultraviolet irradiation from pro-vitamin D3 produced in the skin, the so-far-unknown mechanism of human skin pigmentation after the ultraviolet irradiation may be partly explained by this stimulating effect of vitamin D3 on the melanocytes. PMID- 3022422 TI - The effect of trypsin on the growth and infectivity of human rotavirus. AB - The effect of trypsin on the infectivity of human rotavirus (HRV) was examined using HRV strains that are cultivable only when treated with trypsin. Their infectivity in cell culture systems was enhanced in relation to enzyme concentration. The primary effect of trypsin appeared to be on the virus. Trypsin untreated HRV virions were capable of being adsorbed on the cell (MA104) but not of passing into it without trypsin. For multiple cycles of replication the presence of trypsin in the maintenance medium was required. PMID- 3022424 TI - Biochemical improvement after treatment by bone marrow transplantation in I-cell disease. AB - The first case of successful bone marrow transplantation (BMT) in a patient with I-cell disease is reported. A 8-month-old girl with I-cell disease (N acetylglucosaminylphosphotransferase deficiency) has had successful reconstitution with bone marrow from her HLA-MLC-matched brother who has heterozygous level of the transferase activity. The following biochemical and clinical improvements have occurred: the transferase in peripheral lymphocytes increased to donor's level, and lymphocytic alpha-neuraminidase, beta galactosidase and alpha-mannosidase increased to normal levels. Plasma acid hydrolase activities, which had been 10 to 60 times higher in the patient than normal control levels, have slowly but steadily decreased from one month after the graft. Such decreases were observed in the activities of alpha-mannosidase, N acetyl-beta-glucosaminidase, alpha-fucosidase, arylsulfatase A and acidic beta galactosidase. There was also a marked decrease of vacuolated peripheral lymphocyte after the BMT. Three-months after the engraftment, hepatomegaly gradually decreased in size, corneal clouding has not progressed, and tight skin seems to have improved. PMID- 3022425 TI - Response of pulmonary energy metabolism to phosgene. AB - Rats were exposed to phosgene at a concentration of 1.0 ppm for 4 hours in a Rochester-type chamber. At intervals thereafter over a 4 day period, lungs were obtained for histological and biochemical assessments. Edema was estimated by histological examination and by measurement of lung wet and dry weights. In parallel studies, pulmonary mitochondrial respiratory activity was measured using Clark oxygen electrodes. The significant reduction in respiratory control index (State 3 respiration/State 4 respiration) found immediately following phosgene exposure coincided with the highest level of % lung water. There was a concomitant decrease of ATP concentration that persisted on the third day after exposure. Na-K-ATPase activity was reduced 1 day after exposure, thus a lowered ATP level preceded a reduction in Na-K-ATPase or sodium pump activity. The reduction in ATP level and Na-K-ATPase activity may play a major role in damage to lung tissue following exposure to phosgene. PMID- 3022426 TI - Acute responses to phosgene inhalation and selected corrective measures (including surfactant). AB - We exposed 22 mongrel dogs to 94 ppm phosgene for 20 min from a non-rebreathing system. We expressed exposure to phosgene as ppm X VI X min X kg 1, i.e., the amount of gas containing a known phosgene concentration that was actually inhaled per min standardized to body weight, the "exposure index" (EI). In contrast, the conventional expression of exposure, i.e., ppm X min, fails to take volume inhaled (VI) and body weight into account. Five dogs received no intervention and served as controls. Fourteen dogs received basic therapy of oral cortisone (40 mg/kg) and NaHCO3 (3 mEq/kg) plus 100% O2 (FiO2 = 1.0) for 30 min after the exposure period. These animals were further divided according to the following selected additional therapies, which were started 30 min after exposure: Theophylline, 5 mg/kg iv for 20 min followed by 1 mg/kg/hr for 70 min (n = 5). Three dogs of this group were given a trial of 5 cm H2O expiratory resistance during the period of basic therapy. Because of the untoward response, expiratory resistance was discontinued and not used in other experiments. PGE2-hi, [1 microgram/kg/min] iv for 90 min (n = 3). PGE1-lo, [0.04 microgram/kg/min] iv for 90 min (n = 3). Atropine, 0.5 mg/kg iv at 30 and 50 min after exposure (n = 3). Three dogs [group 5] received oral cortisone and NaHCO3 plus inhaled supplementary surfactant, 2.7 mg/min ultrasonically nebulized (FiO2 = 0.5; phosphate buffer), for 30 min after exposure. All treated dogs, groups [1] through [4] and the surfactant group [5], received cortisone (40 mg/kg/hr iv), NaHCO3 to correct base deficit, and O2 to correct hypoxemia from 30 min to 120 min after exposure. Because of its clearly beneficial effect in group [1], theophylline was also given to all other treatment groups during this period. At the end of the study, all lungs were excised, examined and prepared for light microscopy. We found that EI, which varied among subjects because of spontaneous variations of VI during exposure, correlated significantly with the changes in base deficit induced by phosgene inhalation. We also found that the change in minute ventilation, delta VI X kg-1, correlated significantly with changes in lung compliance, peak flow and base deficit. Evaluation of the various therapeutic modalities revealed the following: Immediate therapy with O2 is vital and and FiO2 of 0.4 to 0.5 is sufficient.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3022427 TI - Phosgene: a practitioner's viewpoint. AB - Clinical experience with five patients exposed to phosgene is described. The treatment of phosgene poisoning was focused upon the presenting problem, pulmonary edema. Arterial hypoxemia was treated with a face mask with 10 cm CPAP with the FiO2 adjusted as needed or with a volume ventilator with controlled ventilation. Ventilation was controlled to reduce the work of breathing. Metabolic acidosis was treated with NaCHO3 to produce a normal pH. A vigorous program of diuresis was used to treat the pulmonary edema. Lasix was administered to produce a negative fluid balance while maintaining a good urinary output. The negative fluid balance correlated well with reduced oxygen requirements. PMID- 3022428 TI - Increment of prostaglandin E2 in association with elevation of urinary sodium excretion following lead administration. AB - Lead administered to semi-fasted rats caused an elevation of urinary sodium excretion but no increase of urinary potassium excretion as previously observed in fasted rats. Moreover, in this experiment it was found that urinary and serum prostaglandin E2 (PG E2) contents were increased in association with the elevation of urinary sodium excretion by lead and that natriuresis was suppressed by indomethacine treatment. These findings suggest a stimulation of prostaglandin synthetase rather than to a suppression of the renin-angiotensin-aldosterone system as a primary reaction caused by the lead administration. PMID- 3022429 TI - Chronic lead exposure alters dopaminergic mechanisms in rat pituitary. AB - The effect of chronic lead treatment on pituitary dopamine (DA) D2 receptors was studied by measuring (-)sulpiride-displaceable [3H]spiroperidol-binding and DA inhibited adenylate cyclase. Receptor number was reduced in lead-exposed animals and bromocriptine was less able to inhibit cyclase activity in pituitary homogenates. In addition, the capacity of DA to inhibit the VIP-stimulated cAMP formation was decreased. PMID- 3022430 TI - Rat liver plasma membrane damage in hexachlorobenzene intoxication and its potentiation by ethanol. AB - Chronic feeding of male Wistar rats with food containing hexachlorobenzene (HCB) at 17.5 mmol/kg induced elevation of serum amino-transferases and bilirubin content, increase of microsomal cytochrome P-450 concentration, and decrease of 5'-nucleotidase, K+,Na+- and Mg2+-adenosine triphosphatase activities in liver plasma membrane preparations. These changes were potentiated by ethanol consumption suggesting a possible role of liver plasma membrane damage in the pathogenesis of HCB intoxication. PMID- 3022431 TI - The WHO-UNDP epidemiological study on the health effects of exposure to organophosphorus pesticides. AB - A multinational epidemiological study on the neurotoxic effects of long-term, low level exposure to organophosphorus pesticides (OPs) is being supported in ten European countries by the United Nations Development Programme (UNDP) jointly with the World Health Organization (WHO) Regional Office for Europe. The protocol developed for the study is directed at the assessment of OP exposure and absorption, abnormal neurological findings, and behavioral changes in both agricultural and industrial workers. The biological monitoring tests adopted in the study have been standardized and submitted to quality assurance programmes. PMID- 3022433 TI - Treatment of visceral leishmaniasis in Bihar. PMID- 3022432 TI - Field studies on health effects from the application of two organophosphorus insecticide formulations by hand-held ULV to cotton. AB - Two field studies to assess the health implications for farmers applying two different formulations containing organophosphorus (OP) pesticides to cotton by hand-held ULV are described. The first study, carried out in the Ivory Coast, involved the application of an endrin/DDT/methylparathion (MEP) formulation in an aromatic hydrocarbon solvent. The second study took place in Indonesia with a 20% monocrotophos formulation in a mixture of a glycol and a glycol ether. Both studies were carried out under actual field conditions. The purpose of the studies was to get a good assessment of the health hazards of the particular formulation, used under the specific circumstances and agronomic requirements of the area of application and taking into account all local, climatic and cultural conditions that could be of possible influence. The results showed that in both studies skin exposures took place during application and especially during handling, filling and cleaning, and that inhalation of spray mist was negligible. Absorption was confirmed by the presence in urine of metabolites of endrin and methylparathion in the Ivory Coast study, and of dimethyl phosphate in the Indonesia study. No clinical signs or symptoms of intoxication were discovered in either study, nor were inhibitions of cholinesterase (ChE) activity of health significance established under the conditions of the studies. In addition, various practical aspects such as choice of apparatus, of formulation, the application procedures etc. are discussed. PMID- 3022435 TI - Odd men out: a quantitative objective procedure for identifying anomalous members of a set of noisy images of ostensibly identical specimens. AB - Achievement of an optimal improvement in signal-to-noise ratio from image averaging techniques depends crucially on the assumption that all members of the set of images to be averaged are fundamentally alike. In HREM of biological macromolecules, this assumption may be invalid for such reasons as variations in viewing geometry, non-uniformity of staining, or structural perturbations caused by specimen preparation procedures of radiation damage. Inclusion of data that are compromized these or other factors will degrade the information content of the averaged image. Here we present an algorithm which provides an objective quantitative method for the identification and elimination of anomalous members of a set of pre-aligned images. Based on a statistical criterion of mutual consistency, the algorithm forms an ordered list in which the individual images are ranked from most to least reliable. On specification of the noise statistics- in the formulation given here, of stationary white noise--an acceptability threshold in this ordered list is imposed. The derivation and implementation of this algorithm are presented, its properties discussed, and its application illustrated using both real and model electron micrograph data. PMID- 3022434 TI - [Cytochemical detection of actin in the structure of the synaptic apparatus of hippocampal field CA3]. AB - Ultrastructural distribution of actin in dendrites, dendritic spines and presynaptic boutons of the hippocampal area CA3 of the guinea pig was investigated using decoration and immunocytochemical methods. The distribution of actin was non-homogeneous in all the parts of neurons. The highest concentration of this contractile protein was revealed in the spine cytoplasm. Here actin forms a dense cytoskeleton meshwork and is present also in postsynaptic densities. An intimate interaction between the spine actin cytoskeleton and the postsynaptic actin densities has been revealed. This feature may indicate the involvement of actin cytoskeleton in the organization and maintenance of dimensions, location and geometry of active zones. PMID- 3022436 TI - Effects of platelet antagonists on the reduction in platelet density caused by microbubbles in vitro. AB - Platelet-rich plasma (PRP) was stirred and incubated at 37 degrees C with N2 microbubbles in vitro in the presence and absence of platelet antagonists. The N2 microbubbles acted as a platelet agonist, like "classical" agonists such as ADP, collagen, and thrombin, causing an agonist-induced aggregation requiring extracellular Ca2+. This aggregation is abolished by 2-ethylamineglycolether (N',N',N',N') tetraacetate (chelator of extracellular Ca2+), and 2-deoxy-D glucose plus antimycin A (inhibitors of glycolysis and electron transport, and thereby of ATP production). Furthermore, this aggregation could be depressed pharmacologically by several antagonists of the aggregation induced by "classical" platelet agonists. The greatest inhibition of microbubble-induced aggregation was obtained by substances that increase the intracellular levels of cyclic AMP. Medications that are in common clinical use, such as theophylline, seem promising in this respect. The prostaglandin-thromboxane pathway did not seem to be involved in the N2 microbubble-induced platelet aggregation in vitro, since acetylsalicylic acid and indomethacin were without effect, nor did the 3',5'-cyclic guanosine monophosphate pathway seem to be linked to this aggregation mechanism. PMID- 3022438 TI - [Round cell undifferentiated neoplasms. Ultrastructural and immunohistochemical study]. PMID- 3022437 TI - Effect of pressure on oxidative metabolism of drugs by mouse hepatic microsomes. AB - The metabolism of antipyrene and delta-9-tetrahydrocannabinol (delta-9-THC) was studied in vitro with mouse liver supernatant and 105,000 g microsomes at 450 atm pressure. No significant change in rate of antipyrene metabolism was detected, whereas a slight (5.6%) but nonsignificant reduction in the rate of metabolism of delta-9-THC was found. Pressure did, however, produce a significant (23%) reduction in the formation of the 11-hydroxy metabolite from delta-9-THC, but at the same time had no effect on the production of the other major metabolite, 8 alpha-hydroxy-delta-9-THC. This is possibly due to a differential effect on cytochrome P-450 isozymes. Mechanisms for the possible cause of the inhibition of metabolism are discussed. PMID- 3022439 TI - Impaired adaptation to salt-induced urinary calcium losses in postmenopausal osteoporosis. PMID- 3022440 TI - Coupling of receptors to signal transducing components: use of anti-idiotypic antibodies to map the specific sites of interaction of receptors and guanyl nucleotide-binding proteins. PMID- 3022441 TI - The first seconds of neutrophil activation: phosphoinositides, protein kinase C, and calcium movements. AB - Activation of the neutrophil by interaction of a ligand such as f-Met-Leu-Phe with its specific receptor elicits a prompt breakdown of PIP2 and the generation of PA via diacylglycerol. There is a rapid elevation of cytosolic calcium and activation of protein kinase C. Calcium and protein kinase C act synergistically to elicit the physiological responses. Although PIP2 breakdown and PA generation are prompt responses of neutrophils to receptor occupancy by chemoattractants, these steps can be bypassed by stimuli which directly activate protein kinase C or increase cytosolic calcium. Elevation of cytosolic Ca and activation of protein kinase C did not elicit breakdown of PIP2, indicating that phosphoinositide remodeling is not caused by activation of protein kinase C or by elevation of cytosolic calcium, nor is such a breakdown or the generation of phosphatidic acid required for the subsequent responses. The evidence indicates that PIP2 breakdown is an early event in stimulus-response coupling and is correlated with receptor-initiated generation of the signal. PMID- 3022442 TI - Specific atrial natriuretic factor receptors mediate increased cyclic GMP accumulation in cultured bovine aortic endothelial and smooth muscle cells. PMID- 3022445 TI - A monocyte-derived inhibitor of interleukin 1 induced by human cytomegalovirus. PMID- 3022444 TI - Monoclonal antibody to the Epstein-Barr virus receptor induces human B lymphocyte activation and differentiation. PMID- 3022443 TI - Diacylglycerol activates protein kinase C and modulates oxidative metabolism in human neutrophils. PMID- 3022446 TI - Liver endothelium binds, transports, and desialates ceruloplasmin which is then recognized by galactosyl receptors of hepatocytes. AB - Incubation of 125I-labeled ceruloplasmin with various fractions of liver cell suspensions at 4 degrees C led to its exclusive binding to the endothelium. At 37 degrees C the uptake was followed by internalization and subsequent release of the labeled molecule which, then, had acquired the capacity to bind to the hepatocytes. Unlabeled CP did not inhibit this hepatocyte binding, but two galactose-containing molecules, asialofetuin and asialoceruloplasmin, did. Incubation of double-labeled CP (sialic acid with 3H, protein with 125I) with endothelial cells led to the dissociation of two labels, indicating desialation of CP. The findings indicate that liver endothelium takes up and desialates CP which is subsequently released and recognized by hepatocytes through their membrane galactosyl receptors. This work offers a physiological function for the galactosyl receptors on hepatocyte membrane. PMID- 3022447 TI - Renal sites of action for atrial natriuretic factor. PMID- 3022448 TI - Physiologically regulated atrial natriuretic peptide receptors in renal glomeruli. PMID- 3022449 TI - [Experience in the clinico-genetic examination of patients with juvenile angiofibroma of the base of the skull]. PMID- 3022450 TI - [Detection of enzootic bovine leukemia virus using the syncytium test]. AB - BLV detection by the syncytial test was performed in 27 heifers experimentally and naturally infected by the enzootic bovine leukosis virus (BLV). The presence of BLV was demonstrated in 94.7% of the animals. The bovine foetal spleen cells (FBS) were found to be suitable for the syncytial test. Positive animals not reacting to infection by the production of anti-BLV antibodies were identified during the syncytial-test investigation. The importance of this finding for the programme of controlling enzootic bovine leukosis on farms is discussed. As suggested by the results, temporary occurrence of anti-BLV antibodies followed by their disappearance can be observed together with a negative result of the syncytial test in some circumstances. The discussion deals with the problems of the determination of anti-BLV antibodies in milk, and/or milk secretion, by the ELISA method. PMID- 3022451 TI - [Use of immunoenzyme (ELISA) diagnosis of Aujeszky's disease in pigs during the recovery period]. AB - Samples of blood and blood serums of pigs were examined for the presence of antibodies to the Aujeszky's disease virus. The virus-neutralizing (VN) test and the enzymoimmunologic (ELISA) method were used for this examination. As indicated by comparison of the average titres of antiviral antibodies determined by both methods, the ELISA method is 60 to 600 times more sensitive than the VN test. The high sensitivity of the ELISA method enabled to detect antiviral antibodies even in samples considered as negative after VN-testing. The method has been used with success for the sanitation of three swine stocks where the Aujeszky's disease was eradicated without interruption of operation. PMID- 3022453 TI - Acute interstitial pneumonia in mink kits: experimental reproduction of the disease. AB - Organ homogenates from kits that died of interstitial pneumonia were inoculated into adult Aleutian disease virus (ADV)-negative mink and shown to contain infectious ADV. Acute interstitial pneumonia was experimentally reproduced with the organ homogenate but only by inoculation of newborn kits born from ADV negative dams. Older kits and kits from ADV-positive dams did not develop interstitial pneumonia, but later developed the classic form of Aleutian disease. Electron microscopic examination was done on purified suspensions of defined ADV isolates and on purified organ homogenates from kits with spontaneous or experimental interstitial pneumonia. In kits from both groups a virus, morphologically resembling the defined ADV isolates, was demonstrated. Findings of intranuclear inclusion bodies and intranuclear ADV antigen in alveolar type-II cells in affected lungs and the lack of immunologically mediated lesions suggest that lung lesions result from primary viral injury to alveolar type-II cells. Experiments also showed that infection of dams with ADV before pregnancy decreased the number of kits per mated dam and infection with ADV in mid pregnancy caused fetal death, fetal resorption, or abortion. PMID- 3022452 TI - [The effectiveness of vaccinating gilts against parvoviruses in a herd with endemic parvovirus infection]. AB - An inactivated vaccine against swine parvovirosis with a lipoid adjuvant was tested in a herd infested with parvoviruses. The titre of the haemagglutination inhibition antibodies was studied at the time of the first pregnancy in two groups of gilts included in the herd at the age of 7.5 months - one group vaccinated, the other left untreated. In the vaccinated group the geometrical means of the titres were significantly higher than in the non-vaccinated group throughout the time of study. The difference in the average number of piglets per litter between the two groups was evaluated after parturition. On an average, the gilts of the vaccinated group had 1.5 more live pigs per litter (P less than 0.05). As also found, when the antibody titre increases by log 10, the number of piglets per litter increases by 1.55. On the basis of the results the vaccination of gilts against swine parvovirosis in endemically infested herds is considered an efficient preventive measure having a high economic effect. PMID- 3022454 TI - Pulmonary lesions in lambs experimentally infected with ovine adenovirus 5 strain RTS-42. AB - Twelve lambs were inoculated transtracheally and intranasally with Mastadenovirus ovi 5 strain RTS-42 and killed sequentially. Pulmonary lesions were studied by light and electron microscopy. Four lambs served as sham inoculated controls. Pulmonary lesions consisted of multifocal areas of bronchiolitis and alveolitis associated with necrosis and sloughing of isolated type I and type II alveolar epithelial cells and nonciliated bronchiolar epithelial cells. This was followed rapidly by hyperplasia of the remaining epithelium and repair of the damage. A cellular infiltrate of neutrophils and macrophages began at 2 days after inoculation, peaked at 4 days after inoculation, gradually diminished until minimal at 12 days after inoculation, and was resolved at 21 days after inoculation. Surfactant was abundant and, along with debris, was removed from the alveoli by macrophages. Clinical disease was not seen, but lesions were believed to be sufficient to allow bacteria to colonize the lungs and cause severe disease. PMID- 3022455 TI - Poxvirus infection in a donkey. PMID- 3022456 TI - Peripheral neuropathy in cats with inherited primary hyperchylomicronaemia. AB - Primary hyperlipoproteinaemia (hyperchylomicronaemia) with a slight increase in very low density lipoprotein) is described in 20 cats. Fasting hyperlipaemia, lipaemia retinalis and peripheral neuropathies were the most frequently detected clinical signs. The disease is thought to be inherited as an autosomal recessive trait but the exact mode of inheritance has not been determined. Affected cats showed reduced lipoprotein lipase activity measured after heparin activation compared with the response in normal cats. Plasma triglyceride and cholesterol were increased in all the cats with the major proportion of triglyceride and cholesterol being present in chylomicrons. The peripheral nerve lesions were caused by compression of nerves by lipid granulomata. It is probable that the lipid granulomata result from trauma because the nerves most often affected were at sites like the spinal foraminae where they were susceptible to trauma. PMID- 3022457 TI - New porcine coronavirus? PMID- 3022458 TI - Treatment of canine colitis. PMID- 3022459 TI - Kangaroo gait in ewes. PMID- 3022460 TI - Photodynamic action of erythrosin B as a toxic mechanism for infective larvae of bovine gastrointestinal nematodes. AB - The effect of erythrosin B on adults and 3rd stage larvae of gastrointestinal nematodes of cattle was studied by treating calves per os with 20 mg kg-1, 40 mg kg-1, and 60 mg kg-1 of the dye daily for 21 days and monitoring its effect. Erythrosin B had no detectable effect on adult nematode fecundity or viability. Data collected did indicate, however, that erythrosin B produced a consistent toxic effect on 3rd stage larvae after exposure to fluorescent light. This toxic effect was dependent upon dosage of erythrosin B administered, time of light exposure and, to a much lesser extent, the length of time the larvae were left in culture in the presence of the dye. PMID- 3022461 TI - Inhibition of glucose-6-phosphatase and adenosine-triphosphatase activity in rats treated with toxic Spanish rapeseed oil or synthetic anilides. AB - After having demonstrated the impairment of the microsomal oxidation process in rats treated with toxic Spanish cooking oil or fatty acid anilides, we studied the possibility that the function of the cytoplasmic membrane had been affected. For this, the activities of glucose-6-phosphatase and adenosintriphosphatase were determined, these being enzymes of utmost importance in transport at the liver cell membrane level. Wistar rats received disease-related toxic rapeseed oil (1 mg/kg/day) or linoleilanilide (50 mg/kg/day) for 6 days, and the animals were sacrificed immediately after the administration period and after a 4-, 8- and 12 week latency period. Enzyme activities were measured in the membrane fraction of liver and lung. The results showed considerable reduction (up to 80%) in the activity of these enzymes, confirming that transport mechanisms through cell membranes must have been impaired. PMID- 3022462 TI - Preliminary serological characterization of bovine viral diarrhoea virus strains using monoclonal antibodies. AB - Five monoclonal antibodies against the bovine viral diarrhoea (BVD) viral strain NADL were isolated and characterized by an indirect immunofluorescence assay. Extensive cross-reactions were detected when the antibodies were tested with 12 heterologous BVD and four hog cholera (HC) viral strains. One antibody reacted with all strains tested. Two antibodies were specific for cytopathogenic BVD viruses, but failed to react with HC virus. The other antibodies reacted to varying degrees with BVD and HC viral strains. PMID- 3022463 TI - Expression of the malignant phenotype in rat fibroblasts transfected with the polyomavirus transforming genes. AB - As a step toward understanding the molecular mechanism of cooperation between viral and cellular genes in oncogenic transformation, we examined various properties of rat cells transfected with the polyomavirus transforming genes without selecting for a neoplastic phenotype. The cell lines displayed a phenotype ranging from nontumorigenic (flat) to fully transformed (tumorigenic). In the established FR3T3 cell line, acquisition of the fully transformed phenotype correlated with effective expression of the polyomavirus middle T (pmt) antigen. Flat cells carrying silent copies of pmt mutated spontaneously to the fully transformed state with a frequency of 2 to 6 X 10(-5) per cell per generation. In unestablished rat fibroblasts, simultaneous transfer of either pmt and small T or pmt and large T in the presence of the neo marker conferred only a partially transformed phenotype to most of the cell lines. The same results were obtained when wild-type genomic DNA was cotransfected with pSV2-neo. The flat transformants progressively acquired properties characteristic of fully transformed cells with passage in culture. However, in contrast to FR3T3 cells, the generation of fully transformed variants from the flat, unestablished fibroblasts was not caused by activation of pmt expression. This indicates that the functions conferred by the large and small T antigens, alone or in combination with each other, cannot substitute for all the functions expressed by the FR3T3 cell line as a result of in vitro establishment. Thus, polyomavirus mediated transformation may require additional cellular alterations beyond the acquisition of the three viral oncogenes. PMID- 3022464 TI - Human papillomavirus types 16 and 18 sequences in early cervical neoplasia. AB - A total of 100 colposcopic biopsies from patients with abnormal Papanicolau's tests were surveyed for the presence of human papillomavirus (HPV) types 16 and 18 sequences by spot-blot hybridization. HPV 16 and 18 DNA sequences were detected in 58% of the biopsies. None of the cervical intraepithelial neoplasia grade I (CIN I) contained HPV 16 while 50% of the CIN III lesions (carcinoma in situ, CIS) contained HPV 16. HPV 18-related sequences were equally represented in CIN I, II, and III. Southern-blot hybridization of total undigested cellular DNA revealed the presence of HPV DNA sequences only in an episomal form. While the restriction enzyme patterns in HPV 16-positive samples were mostly identical to the originally cloned sequence, the restriction enzyme pattern for HPV 18 positive samples were different from that of HPV 18 but identical to each other. Furthermore, this DNA hybridized more strongly to HPV 18 under nonstringent conditions, suggesting a new type. PMID- 3022465 TI - The transformation-related protein p53 is not bound to the SV40 T antigen in BALB 3T12 cells expressing T antigen. AB - In most murine cells transformed by the SV40 virus, virtually all of the cellular phosphoprotein p53 is in a complex with the SV40 T antigen. Here, we report that, in SV40-infected T-antigen-positive Balb 3T12 mouse cells, most (approximately 80%) of the p53 is not in complex. Complex formation was determined by measuring the amounts of [35S]methionine-labeled p53 which coprecipitated with T antigen when using monoclonal antibody to T antigen. The amount of complex formation was expressed as a percentage of total p53 present, measured by the amount of p53 precipitated with the monoclonal antibody to the p53. The values were confirmed by Western blotting procedure, in which the steady-state levels of the proteins were measured. In these measurements after complete precipitation with antibody to T antigen, the residual p53 in the supernatant was precipitated by antibody to p53, and this amount was denoted as free p53. There was no significant difference seen between the [35S]methionine-labeled tryptic peptides of complexed and the free p53 (or between complexed and free T antigens) as determined by two dimensional gel electrophoresis and chromatography. Virus rescue experiments and retransformation by the rescued virus showed that there was no mutation in the SV40 DNA coding for the T antigen which could account for the lack of complex formation. Both p53 and T antigen were underphosphorylated in cells which exhibited reduced complex formation. Tumorigenicity in syngeneic mice and anchorage-independent cell growth in culture of various cloned mouse cells with or without T antigen expression was compared. The changes in the biologic properties were explainable solely on the basis of known or expected effects of expression of the T antigen and were independent of complex formation or of absence of complex formation between p53 and T antigen. PMID- 3022466 TI - An E1A mutant of adenovirus type 3: Ad3hr15 has reiterated DNA sequences 5' to its E1A gene. AB - Defective variants of adenovirus type 3 (Ad3) have been isolated from a heterogeneous, high multiplicity passage stock of the virus. A strikingly defective variant, Ad3hr15, fails to propagate on normally permissive A549 cells, yet has greater infectivity than wild type Ad3 in the adenovirus type 5 (Ad5) DNA transformed 293 cell line. Investigation of its genomic alterations revealed that Ad3hr15 bears two short tandem duplications of viral DNA sequences near its left end, 5' to the E1A gene. The variant also bears a large tandem triplication at its right end. Marker rescue experiments with plasmid-cloned left end DNA sequences of Ad3 implicate that the duplications 5' to E1A are responsible for the Ad3hr15 defect and the E1A structural gene of the variant is functional. Northern analysis revealed no detectable E1A transcripts early after Ad3hr15 infection of A549 cells. The 293 cell line, however, supports high levels of transcription of the Ad3 E1A gene by the mutant Ad3hr15 E1A promoter. PMID- 3022467 TI - Organization and expression of the major genes from the long inverted repeat of the human cytomegalovirus genome. AB - The long inverted repeat (TRL:IRL) of the human cytomegalovirus (CMV) genome is a major transcription unit in productively infected human fibroblasts. Cloned DNA fragments of the CMV IRL and complementary DNA (cDNA) copies of RNAs transcribed from the TRL:IRL were used as probes in RNA filter hybridization experiments to characterize and map the RNAs transcribed from this region of the virus genome. In human fibroblasts, three poly A+ RNAs of 2.7, 2.0, and 1.2 kb were detected during the early (E) and late (L) phases of virus gene expression. Analysis of cloned cDNAs and RNA mapping studies indicate that the TRL:IRL can be divided into three transcriptionally active regions. The most highly transcribed region lies between 0.805 and 0.816 map units and encodes a major abundant poly A+ RNA of 2.7 kb that is expressed at E and L times postinfection (p.i.). The second region spans map coordinates 0.792-0.797 and encodes a 1.2-kb poly A+ RNA that is relatively low in abundance at E times p.i. but achieves nearly the same abundance as the 2.7-kb transcript at L times p.i. The third region encompasses map units 0.796-0.804 and encodes a less abundant poly A+ RNA of 2.0 kb that attains maximum expression at E times. The 1.2- and 2.7-kb RNAs are transcribed in the same direction, while the 2.0-kb RNA is transcribed in the opposite direction. The 2.7-, 2.0-, and 1.2-kb RNAs, as well as 5.7- and 1.8-kb transcripts were detected at immediate early times p.i. when human fibroblasts were treated with cycloheximide, but not in cells treated with anisiomycin. PMID- 3022468 TI - Characterization of a major early gene from the human cytomegalovirus long inverted repeat; predicted amino acid sequence of a 30-kDa protein encoded by the 1.2-kb mRNA. AB - The human cytomegalovirus (CMV) long inverted repeat (TRL:IRL) encodes three major early mRNAs. One of these RNAs is a 1.2-kb transcript that maps between 0.792 and 0.797 map units on the human CMV genome. Primer extension experiments, in addition to nucleotide sequence analyses of cloned cDNA transcripts and human CMV IRL DNA fragments, demonstrated that the 1.2-kb mRNA was not spliced. A single major open reading frame of 254 amino acids was identified, encoding a basic polypeptide of approximately 30 kDa. This polypeptide contains 19% Arg, Lys, and His residues, and would have a net positive charge of 31 at neutral pH. Examination of the deduced amino acid sequence revealed several potential phosphorylation sites and a hydrophobic carboxy terminus which resembles a membrane anchor sequence. In vitro translation of human CMV infected cell RNA, hybrid selected with either cloned cDNA or human CMV IRL DNA fragments specific for the 1.2-kb mRNA, resulted in a unique translation product that migrated on SDS-polyacrylamide gels with an apparent molecular mass of 37 kDa. Potential transcriptional regulatory sequences were also identified upstream of the gene encoding the 30-kDa protein. PMID- 3022469 TI - The effect of viral transformation on prostaglandin production depends on cell type. AB - The role of prostaglandins in cellular differentiation and transformation has been widely studied. We have found previously that prostaglandin E2 production was greatly diminished in dog kidney cells (MDCK) after transformation by Harvey murine sarcoma virus. In the present study, we have shown that viral transformation can have differing effects in the ability to modify the production of prostaglandin in cultured cells. For example, the prostaglandin E2 production in rat kidney cells (NRK) is decreased after transformation by Rous sarcoma virus, while production in 3T3 cells is increased markedly after transformation by the same virus. Similarly, SV40 transformation increases prostaglandin E2 production of 3T3 cells and decreases the production in rat thyroid cells (FRTL). These results indicate that the biosynthetic pathway for prostaglandin production has varying susceptibility following viral transformation and the effect of transformation depends more on the type of cell than virus. Taking advantage of the well-defined transforming proteins encoded by polyomavirus, we have further studied the relationship between prostaglandin production in cells and the expression of T antigens in transformed cells. We showed that the expression of middle T antigen, which is associated with a protein kinase and is responsible for phenotype of transformed cells, is required for the change in prostaglandin production in cells. How these changes of prostaglandin production relate to the progression of viral transformation remains to be explored. PMID- 3022471 TI - The primary structure of crossover regions of intertypic poliovirus recombinants: a model of recombination between RNA genomes. AB - The nucleotide sequence of crossover sites in the genome of four intertypic (type 3/type 1) poliovirus recombinants has been determined. The approximate boundaries of the crossover regions were first estimated by RNase T1 oligonucleotide mapping of the recombinant genomes; then appropriate regions were sequenced by the chain termination method using oligonucleotide-primed reverse transcription of the recombinant RNAs. The crossover sites (defined as the contiguous sequences shared by the recombinant and both parental genomes flanked, in the recombinant genome, by heterotypic RNA segments) are 5, 5, 7, and 11 nucleotides long, respectively. The recombination was precise and was not accompanied by any other genetic alterations. The recombination sites were found to be located within genome segments having a potential to form secondary structure elements. Based on this observation, a model of recombination between picornaviral RNA genomes has been proposed. The essence of this model consists in bringing together homologous regions of two recombining RNA genomes via formation of intermolecular duplexes, detachment of the nascent 3' end of the newly synthesized complementary RNA from a "parting" site on the first template and its subsequent "jumping" to the identical (or closely related) "anchoring" site on the other template. Features of this model are discussed in some detail. PMID- 3022470 TI - Sequences homologous to two separate transforming regions of herpes simplex virus DNA are linked in two human genital tumors. AB - Ten human genital invasive squamous cell carcinomas and five human premalignant tissues were analyzed for the presence of selected sets of herpes simplex virus 2 (HSV-2) DNA sequences. Two vulvar tumors and one vulvar dysplastic tissue were found to contain DNA sequences homologous to the BglII O fragment (coordinates 0.38-0.42) and the BglII N fragment (coordinates 0.58-0.63) of HSV-2 DNA. These two fragments overlap the subsets of HSV-1 and HSV-2 DNA sequences (respectively) shown previously to transform cells in culture. Sequences homologous to an additional HSV-2 DNA probe (BglII G) were not detected in the same tumors. Surprisingly, in each of the two positive vulvar tumors, the BglII N and BglII O sequences appeared to be linked, whereas in the standard HSV-2 genome the two fragments are separated by approximately 26 kb. This finding suggested that the two sets of sequences may have rearranged prior to or following the association of the HSV DNA sequences with the tumor cells. The same set of 10 tumors were analyzed for the presence of sequences complementary to human papillomavirus 16 (HPV16) DNA. The HPV16 DNA probe hybridized to three of six cervical tumors, whereas no hybridization was detected with the two vulvar tumors which contained the HSV DNA sequences. PMID- 3022472 TI - Dominance of temperature-sensitive phenotypes. I. Studies of the mechanism of inhibition of the growth of wild-type vesicular stomatitis virus. AB - It has been reported previously that temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV) with an RNA- phenotype interfere with the growth of wild-type (wt)-VSV at both the permissive (34 degrees) and nonpermissive (39.5 degrees) temperatures (J. S. Youngner and D. O. Quagliana (1976) J. Virol. 19, 102-107). Investigation of the mechanism of this interference has revealed the following information. In double infection with RNA- ts mutants and wt-VSV, the cumulative synthesis of viral RNA is inhibited. By varying the relative multiplicities of the two viruses, it was observed that the level of RNA synthesis reflects the level of interference with wt-VSV growth. Although viral RNA synthesis was severely compromised in double infections, this inhibition was not at the level of primary transcription or the translation of primary transcripts. Rather, secondary transcription and genome RNA replication were drastically reduced. Sequential infection with wt-VSV and the ts mutants revealed that there is an early point in the replication cycle of wt-VSV (1 to 2 hr) after which the ts mutants can no longer interfere with the growth of wt-VSV. Ultraviolet irradiation of ts G 41, a mutant belonging to complementation group IV, was used to determine the target size of the interference function. The calculated value for the target size was very close to the target size of the N gene. Additional experiments showed that RNA+ ts mutants representing complementation groups III and V also were able to interfere with the growth of wt-VSV. PMID- 3022473 TI - Mouse mammary tumor virus mediated transfer and expression of neomycin resistance to infected cultured cells. AB - The mammary gland specific, glucocorticoid controlled expression of MMTV makes it an ideal candidate for the basis of a nonpromiscuous regulated retroviral vector system. We have previously constructed an MMTV proviral variant that gives rise to virus particles upon introduction into cultured cells. This was used to construct a defective MMTV provirus in which the MMTV env gene was replaced by the neomycin resistance gene under the control of the herpes simplex thymidine kinase promotor. The defective provirus was packaged after transfection into two distinct MMTV producing cell lines. Conditioned medium from these cells contains virus particles which are able to infect cells of fibroblast and epithelial origin and to confer neomycin resistance upon them. This indicates that the defective MMTV provirus contains the sequences required for packaging of the genomic viral RNA. Transfection of the same MMTV-neo recombinant provirus into the MoMLV packaging cell line, psi 2, did not result in any infectious virus particles. Thus the packaging signals for MMTV and MoMLV appear to be distinct. Analysis of the MMTV infected cells reveals the presence of the MMTV-neo recombinant provirus. Expression of both MMTV and neomycin is detectable and augmented when the infected cells are grown in the presence of glucocorticoid hormone. PMID- 3022474 TI - Thymidine kinase (TK) activity in herpes simplex virus type 1 recombinants that carry insertions affecting regulation of the TK gene. AB - Determinations of the possible importance of herpes simplex virus type 1 (HSV) thymidine kinase (TK) expression in the pathogenesis of viral latency depend in part on the use of defined mutants. In a recent study by A. E. Sears, B. Meignier, and B. Roizman (J. Virol. 55, 410-416 (1985], in which they utilized genetically engineered viral recombinants considered to be TK-, the role of HSV TK expression in latency was reported to be minimal. To further investigate this conclusion we intensively studied the TK phenotypes of their M316-2 and M316-10 HSV-1 mutants. TK activity was investigated by phosphorylation of thymidine, by arabinosylthymine (ara-T) inhibition and by virus plaque autoradiography. TK activity of the M316-2 and M316-10 HSV mutants was not detected in 5-min assays (as performed by Sears et al.), but in longer assays substantial activity was apparent. In contrast, in assays of control TK- viruses, activity was minimal or absent at all time points. In ara-T inhibition assays the M316-2 and M316-10 viruses were inhibited more than 10-fold, consistent with viruses of intermediate TK activity. By plaque autoradiography both of these viruses produced plaques which incorporated significant amounts of thymidine. Based on these results we conclude that the M316-2 and M316-10 viruses should likely be considered to express intermediate levels of TK activity. HSV latency results using these mutants may need to be interpreted with this in mind. PMID- 3022476 TI - A herpes simplex virus mutant is temperature sensitive for reactivation from the latent state: evidence for selective restriction in neuronal cells. AB - When the replicative defect in the HSV-1 temperature sensitive mutant tsI was repaired, the agent derived (RI-1) was found to possess an additional temperature sensitive lesion limiting its reactivation from the latent state. Thus, when spinal ganglia from latently infected mice were scored for reactivation by cocultivating them with indicator cells in vitro, significantly more were found to be positive at 31 degrees than at 38.5 degrees. To assess a possible relationship between reactivation and replication in neurons, the replication of RI-1 in murine C1300 neuroblastoma cells was studied. In these cells, RI-1 was severely restricted, and viral replication was delayed at 38.5 degrees. Serial passage of RI-1 in neuroblastoma cells at the restrictive temperature resulted in selection of an agent which gained both the capacity to replicate efficiently in neuroblastoma cells and reactivate from the latent state at 38.5 degrees. However, the replication pattern of this neuron adapted virus in mouse embryo fibroblasts remained unchanged from the parental RI-1. Taken together, these results indicate that RI-1 possesses a neuron specific temperature sensitive replicative lesion which is also manifest during reactivation from the latent state. PMID- 3022475 TI - Genomic organization of the simian virus 40-adenovirus 7 hybrid virus, PARA(cT), that encodes a nuclear transport defective simian virus 40 T antigen. AB - The genomic organization of the simian virus 40 (SV40)-adenovirus (Ad)7 hybrid virus, PARA(cT), was examined. A deletion of approximately 5529 bp of Ad7 DNA extends from 78.8 map units to 94.0 map units and is replaced by an SV40 DNA insert of 3809 bp. The left-hand end of the insertion begins at SV40 nucleotide 5168, 5 bp upstream of the ATG initiation codon for T-ag synthesis. The sequence extends counterclockwise through the T-ag encoding sequences and into SV40 late region DNA. Most of the late region DNA has been removed in a deletion between nucleotides 2464 and 301. One of the 72-bp repeats has also been deleted. The right-hand end of the SV40 DNA insert is at nucleotide 4366. Thus, a portion of the SV40 DNA early region is repeated at both ends of the insert (nucleotides 5168-4366). PMID- 3022477 TI - Molecular cloning and sequence analysis of the human parainfluenza 3 virus mRNA encoding the P and C proteins. AB - The sequence of the mRNA encoding the phosphoprotein (P protein) of the human parainfluenza virus 3 (PF3) was determined by molecular cloning. In other Parmyxoviridae the P protein mRNA is functionally bicistronic and encodes an additional smaller nonstructural protein termed C. In this report three open reading frames (ORF) are described. These consist of a single long ORF encoding the P protein, and two shorter ORFs encoding the structural Vp18 protein (analogous to the Sendai C protein) and a putative polypeptide termed D protein. The encoded phosphoprotein consists of 603 amino acids and has a predicted molecular weight of 67,683. The C protein consists of 199 amino acids and has a predicted molecular weight of 23,288. The D protein consists of 140 amino acids and has a predicted molecular weight of 16,270. Although the D protein has not yet been demonstrated in vivo its synthesis could be demonstrated in vitro using a rabbit reticulocyte lysate system. Thus it appears that unlike the other paramyxoviruses, the PF3 P protein mRNA may be functionally tricistronic. PMID- 3022478 TI - Defective interfering particles of VSVNJ (Ogden), generated by heat treatment, contain multiple internal genomic deletions. AB - Defective interfering (DI) particles have been isolated from a heat-resistant strain of the New Jersey (Ogden) serotype of vesicular stomatitis virus (VSV). Most of these DI particles contain various portions of all five cistrons of VSV. The two largest DI particles, NJ-121 and NJ-PG2, represent approximately 60% of the standard virus genome and contain both the positive and negative strand leader RNA templates. These two DI particles are transcriptionally active and synthesize both the positive and negative strand leader RNAs in vitro. Virion RNA probe-mRNA hybridizations and cDNA probe-virion RNA hybridizations have shown that NJ-121 contains mainly sequences from the L and G genes. In contrast, NJ-PG2 has portions of the sequences from all five genes of VSV. Smaller DI particles, NJ-121a, NJ-121b, NJ-PG1, and NJ-JM2 representing approximately 50, 38, 28, and 25% of the standard virus genome respectively, were also generated. These DI particles did not have sequences complementary to the positive strand leader RNA template. The mRNA hybridization patterns and results of the genomic RNAs hybridizing to cDNAs of N, NS, M, and G genes of these DI particles showed that they contain parts of information from all five cistrons. Most of the DI particles appear to be generated by multiple deletions throughout the standard virus genome. None of these DI particles interfered heterotypically with VSVIND HR in BHK21, R(B77), or L2 cells. However, they interfered well with infection by VSVNJ (Hazelhurst). PMID- 3022479 TI - Cloning full-length dengue type 4 viral DNA sequences: analysis of genes coding for structural proteins. AB - DNA sequences (approximately 11,000 nucleotides) representing the full-length genome of the dengue virus type 4 were cloned. The sequence of the first 2,429 nucleotides at the 5' terminus which includes the coding region for the structural proteins is presented. The virion structural proteins are encoded in one long open reading frame specifying a polyprotein precursor which is apparently proteolytically cleaved by a mechanism resembling that proposed for expression of structural proteins of other flaviviruses such as yellow fever (YF) and West Nile (WN) viruses. The N terminus for each of the dengue virus structural proteins was tentatively assigned by homology alignment to the corresponding sequence of YF or WN virus. Comparison of sequence homology of structural proteins suggests that dengue virus is more closely related to WN virus than to YF virus or Murray Valley encephalitis virus. Finally, analysis of the extreme 5'- and 3'-terminal nucleotides of the dengue virus genome revealed sequences that may be involved in transcription, replication, and packaging of viral RNA. PMID- 3022480 TI - [Regulatory region of the polyomavirus genome and regulation of gene expression in the developing mouse embryo]. PMID- 3022481 TI - [Property of polymorphonuclear leukocytes in generating superoxide in osteomyelitis]. AB - Rate of superoxide anion production was decreased in blood polymorphonuclear leukocytes of patients with acute and chronic osteomyelitis both in absence of an activator and after stimulation of the cells by latex particles and menadion in vitro. PMID- 3022482 TI - [Changes in collagen protein biosynthesis in oncotransformed human fibroblasts]. PMID- 3022483 TI - [Incorporation and release of cholesterol from plasma membranes of hepatocytes and Zajdela hepatoma cells]. AB - Molar ratio of cholesterol/phospholipids was decreased in Zajdela hepatoma cell membranes as compared with rat normal hepatocytes. Microviscosity of lipid bilayer was increased in membranes of normal and malignant cells, while the activity of Na+, K+-ATPase and 5'-nucleotidase was decreased during introduction of cholesterol into the cell membranes. As compared with control groups the duration of life was increased in the group of animals implanted with Zajdela hepatoma cells modified by cholesterol. The data obtained suggest that an increase in microviscosity of membranes in tumoral cells, enriched with cholesterol, might inhibit their mitotic divisions. PMID- 3022485 TI - [Stimulation of mitochondrial oxidative enzymes in acute cooling and its catecholamine mechanisms]. AB - Acute cooling of rats led to stimulation of NAD+-dependent isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH) and NAD(P)+-transhydrogenase (TH) but did not affect the NADP+-ICDH activity in liver, heart and skeletal muscle mitochondria. After pretreatment of the animals with propranolol the stimulating effect was decreased, thus suggesting that endogenous catecholamines and beta-adrenoreceptors are of importance in activation of NAD+-ICDH, SDH and TH. The effects of cooling, noradrenaline and cAMP did not summarize. Role of catecholamines in stimulation of mitochondrial oxidative enzymes under conditions of cooling is discussed. PMID- 3022486 TI - [A case of coexistence of pregnancy and ovarian cyst initially diagnosed as trophoblastic disease of pregnancy]. PMID- 3022484 TI - [Analysis of thermodenaturation of Na,K-ATPase in the rat myocardial sarcolemma and possible role of damage of this enzyme in the pathogenesis of arrhythmias]. AB - Activity of Na+, K+-ATPase and kinetics of the enzyme thermodenaturation were estimated in preparations of heavy sarcolemma isolated from rat myocardium. Emotional-painful stress (EPS) decreased the enzymatic activity by 20%, whereas the rate of the enzyme thermodenaturation was increased 2-3-fold. A thermodynamic analysis enabled to find a decrease in the energy of activation of the process after the stressory effects as well as alterations in entalpy and entropy under conditions of practically constant changes in free energy; this suggests the possible conformational transformations in the Na+, K+-ATPase molecules in EPS, resembling the state of denaturation. Lipid peroxidation was essential for the decrease in the enzyme thermostability. Activation of lipid peroxidation in the sarcolemma native membranes was accompanied by impairments typical for EPS. Preadministration of an antioxidant ionol before stress protected both the activity and thermostability of Na+, K+-ATPase. Adaptation to EPS by means of multiple short-term stressory actions prevented also the enzyme impairment. Mechanisms of the stress arrhythmogenic effect are discussed. PMID- 3022488 TI - Preoperative portal vein embolization for hepatocellular carcinoma. PMID- 3022487 TI - Effect of ethyl alcohol on the TSH-receptor-cyclase system in thyroid and nonthyroid tissues. PMID- 3022489 TI - Papillary cystic neoplasm of the pancreas: a curable pancreatic tumor. PMID- 3022491 TI - [Changed intraluminal conditions in hemochromatosis]. AB - The influence of the pancreatic secretion on the absorption of iron supposed by several authors could be ascertained by the investigation of pancreatic juice which was obtained by means of cannulation of the pancreatic duct and subsequent stimulation of the organ with secretin pancreozymin in patients with haemochromatosis. The absorption of iron increased in this disease can on the one hand be caused by a protracted secretion of hydrogen carbonate and the slower decrease of the pH-value in the upper duodenum, on the other hand by an increased secretion of pancreatic protein. The quantity of hydrogen carbonate decides on the speed of neutralisation of the formerly acid chyme, however, together with the proteins being at the disposal on the formation of absorption-promoting iron chelates or of not bioavailable, partly polymeric iron hydroxides. PMID- 3022490 TI - Comparison of sonography, computed tomography, angiography, and percutaneous transhepatic portography in detection of portal tumor thrombus in hepatoma. PMID- 3022492 TI - [cAMP concentration, cAMP metabolism and fluid secretion in the jejunum, ileum and colon of the rat caused by cholera toxin]. PMID- 3022493 TI - Chemically induced microencapsulated rat insuloma as a bioartificial endocrine pancreas. PMID- 3022494 TI - [Malignant fibrous histiocytomas]. AB - Malignant fibrous histiocytomas of the storiform pleomorphic type were found in the lower legs of two patients, 79 and 42 years of age. In the first, dermatofibrosarcoma protuberans, in the second, squamous cell carcinoma, had to be discussed. In the first patient, the tumor arose in the area of a preexisting paresis of the superficial peroneal nerve; the second patient developed the tumor in fistulous osteomyelitis existing since childhood. PMID- 3022495 TI - [Use of protein A for immune electron microscopic demonstration of hepatitis A viruses]. PMID- 3022496 TI - Interaction between spin labels and DPPC vesicles. AB - 1H NMR studies on DPPC vesicles labeled with the spin labels (1,14) or (12,3) have shown that both of the spin labels influence the fatty acid side chains as well as the choline head groups. The spin label (12,3) affects, naturally, the head groups stronger than the spin label (1,14), while the reversed effect can be observed at the side chains. This effect progresses with time after vesicle preparation and is fully developed after about 24 h. Addition of Na-ascorbate, at this time, seems to reverse the effect indicating a stabilizing effect of the vitamin on the membrane. These results could be confirmed by ESR investigations according to which the high-field signal seems to be indicative for changes occurring at the side chains, while the low-field signal seems to reflect modifications of the head groups. Since the spin label (1,14) affects considerably the head groups at temperatures less than 6 degrees C, in which case the spectrum is very similar to that obtained with spin label (12,3) at 24 degrees C, one might conclude that there might be a phase transition in regard to the head groups. PMID- 3022498 TI - Inhibition of Epstein-Barr virus infection in vitro by recombinant human interferons alpha and gamma. AB - The inhibitory effects of pure recombinant human interferons alpha A and gamma (reIFN-alpha A and -gamma) on Epstein-Barr virus (EBV) infection of a human EBV negative B cell line, BJAB, and of normal adult B lymphocytes were studied. With pretreatment for 24 h, both types of reIFNs were effective in suppressing the production of EBV specific nuclear antigen (EBNA-1) in BJAB cells 24 h after EBV infection, as determined by the immunoblotting technique. ReIFN-alpha A was, however, a much more potent inhibitor than reIFN-gamma. With treatment starting 1 h after EBV infection, both types of reIFNs were less effective in the suppression of EBNA production. Neither of the reIFNs showed any inhibitory effect on EBNA production in the latently EBV-infected cell lines, Raji and Daudi. These results suggest that reIFNs act in the early phase of EBV infection. Both types of reIFNs were also effective in inhibiting EBV infection of normal adult B lymphocytes as demonstrated by a reduction both in [3H]thymidine incorporation 6 days after EBV infection and in the total number of proliferating cells 21 days after EBV infection. Again, reIFN-alpha A showed a greater inhibitory effect than reIFN-gamma. We also showed that in BJAB cells, reIFN alpha A strongly induced (2'-5')oligoadenylate synthetase activity, whereas reIFN gamma increased the surface expression of HLA class I antigens. PMID- 3022497 TI - Antibody response of mice to lactate dehydrogenase-elevating virus during infection and immunization with inactivated virus. AB - BALB/c and Swiss mice were infected with lactate dehydrogenase-elevating virus (LDV) or immunized with glutaraldehyde-inactivated or ether-extracted virus and their plasma was monitored for anti-LDV IgG and IgM levels by ELISA and indirect fluorescent antibody staining, for neutralizing antibodies, for sensitized antibody-virus complexes, for immune complexes, and for total plasma IgG and IgM. In infected mice, anti-LDV IgM was transiently formed during the first 2 weeks post infection (p.i.) but only at a low level. Anti-LDV IgG was produced in a biphasic manner with an initial peak at about 10 days p.i. and a secondary rise reaching a maximum level 30-80 days p.i. which was retained throughout the persistent phase of infection. The concomitant appearance of comparable levels of low molecular weight immune complexes suggests that most anti-LDV IgG was complexed with LDV proteins. Also, as early as 10 days p.i., infectious antibody LDV complexes developed, which were neutralizable by rabbit anti-mouse IgG, whereas antibodies that neutralize the infectivity of exogenously added LDV appeared only 1-2 months p.i. Throughout infection, most of the anti-LDV IgG was directed to VP-3, the envelope glycoprotein of LDV, which was found to exist in at least 10 distinct forms ranging in molecular weight from 24 to 42 kDa. Anti LDV IgG levels as high as those observed in infected mice developed in mice immunized with inactivated LDV. Antibodies to glutaraldehyde-inactivated LDV were also mainly directed to VP-3, but exhibited no neutralizing activity. The polyclonal B cell activation associated with a persistent LDV infection and the formation of immune complexes were not observed in mice immunized with inactivated virus. PMID- 3022499 TI - Effects of intermittent and continuous haloperidol administration on the dopaminergic system in the rat brain. AB - The effects of intermittent and continuous treatment with haloperidol (HPD) on dopamine turnover in the rat brain were studied. Each rat was treated with HPD for 14 days by either once daily intraperitoneal injections (2 mg/kg/day) (intermittent HPD group) or continuous infusion by a subcutaneously implanted pump which was adjusted to release 28 mg/kg of HPD over 14 days (continuous HPD group). Seven days after cessation of injections or removal of the pump, the regional changes in DA turnover following an acute dose of HPD (1 mg/kg, intraperitoneally) were examined. The continuous HPD group showed more prominent tolerance to HPD effect of augmenting DA turnover than the intermittent HPD group. It is presumed that continuously administered HPD produces greater tolerance to the acute effect of HPD than intermittently administered HPD. PMID- 3022500 TI - [Quartz-dust determination in the dental technician's workplace]. PMID- 3022501 TI - [Effect of silver compounds on viruses in water]. AB - Two commercial substances, Certisil and Micropur, containing microbicidal silver compounds and destinated for decontamination as well as preservation of water were examined for virus inactivating activity against ECBO-, influenza A, Newcastle Disease, pseudorabies and vaccinia viruses in drinking water. In the recommended concentration as well as higher concentrated the lability of the viruses was increased by the silver compounds. This activity which cannot be designated as a true virucidal effect was clearly evident in the case of ECBO and vaccinia viruses, moderate on influenza and pseudorabies viruses but insignificant on Newcastle disease virus. Two combined silver compounds, Certisil Combina and Sanosil, each containing an immediate microbicidal part besides silver differed in their antiviral activity. The chlorine separating part of Certisil-Combina didn't cause an improvement or acceleration of the destabilizing effect on viruses compared to the pure silver compound, while the hydrogen peroxide part of Sanosil led to a better and continuing inactivating influence on the viruses which were merely reduced in infectivity by 99,9% within one day. Only in the case of evident or suspected contamination of water reservoirs by viruses the addition of a combined silver drug with oxygen separating part seems to be useful. PMID- 3022502 TI - A blocking enzyme-linked immunosorbent assay (ELISA) for bovine virus diarrhoea virus serology. PMID- 3022503 TI - Usefulness of the direct fluorescent antibody technique for diagnosis of influenza in swine. PMID- 3022504 TI - [Pathogenesis and treatment of migraine (neuroendocrinologic aspect)]. AB - In 24 women with simple and ophthalamic migraine the authors employed neuropeptide 8-arginine-vasopressin under control of plasma levels of ACTH and cortisol and rheoencephalographic parameters. The authors observed a marked clinical effect, normalization of hypothalamoadrenal function and improvement in the cerebral hemodynamics. PMID- 3022505 TI - [Meralgia paresthetica in patients with osteochondrosis of the spine]. AB - Clinical and electrophysiologic examinations of 1220 patients with spinal osteochondrosis syndromes have made it possible to distinguish two variants of meralgia paresthetica, permanent and transitory ones. Different methods for the management of the syndrome were developed, with due consideration for the aforementioned variants. PMID- 3022506 TI - [Neurologic manifestations in aseptic necrosis of the head of the femur and structural features of its innervation]. AB - Examination of 54 patients with aseptic necrosis of the femoral head showed that the most characteristic alterations related to this pathology include vegetative trophic disturbances in zones of autonomic innervation of neurometamers LIII = LIV. Characteristics of the innervation of the femoral head were studied in man and experimental animals. PMID- 3022508 TI - Symposium: Converting enzyme inhibition. New trends in the treatment of hypertension and congestive heart failure. Brussels, Belgium, June 21, 1985. PMID- 3022507 TI - [Increased efficacy of teturam combined with L-DOPA in experimental alcoholism in the rat]. AB - In an experiment on Wistar male rats the authors studied the effects of teturam, diethyldithiocarbamate and Esperale in combination with L-DOPA on the consumption of alcohol and metabolism of biogenic amines in the brain. The use of L-DOPA prolonged the inhibitary effect of teturam on alcohol consumption. The administration of L-DOPA following Esperale implantation prevented the development of the abstinence syndrome. An increase in the efficacy of teturam and Esperale under the effect of L-DOPA was attended by elevated synthesis of cerebral noradrenaline. PMID- 3022509 TI - Converting enzyme inhibition: search for additional mechanisms of action. AB - Although the therapeutic usefulness of angiotensin converting enzyme (ACE) inhibitors in patients with hypertension and congestive heart failure has been clearly demonstrated, important unanswered questions remain about these drugs, including patient selection criteria, side effects, long-term effects, and especially their precise mechanism of action. The principal explanation of the effect of ACE inhibitors remains the inhibition of the renin-angiotensin system (RAS). However, in chronic treatment with ACE-inhibitory drugs, this relationship may not held true. Additional mechanisms of action postulated to explain the effect of ACE-inhibitors include inhibition of angiotensin II formation in the central RAS, neutralization of renin activity in the vascular wall, blockade of vasoconstrictor response to sympathetic nerve stimulation, and possible involvement of prostaglandins linked, for instance, to bradykinin accumulation. The search for additional mechanisms of action should lead to clinically important findings, provide a better understanding of the pathophysiology of cardiovascular disease, and improve patient treatment with ACE inhibitory drugs. PMID- 3022510 TI - Clinical pharmacology of the angiotensin converting enzyme inhibitor, enalapril. AB - Enalapril is a new angiotensin converting enzyme inhibitor which, when absorbed by the digestive tract, is converted to enalaprilat. This metabolite is, in fact, the active form of enalapril. Its duration of action is sufficiently long so that enalapril can be administered in a single daily dose. Enalapril has been proved to be a highly effective therapeutic agent which is well tolerated by patients with arterial hypertension or severe congestive heart failure. PMID- 3022511 TI - Effect of enalapril on ambulatory blood pressure, renal hemodynamics and cardiac function in essential hypertension. PMID- 3022513 TI - Treatment of dialysis resistant hypertension with enalapril. PMID- 3022512 TI - Vasodilator effects of enalapril in patients with arterial hypertension. AB - Although it has been recognized that enalapril lowers blood pressure by reducing the total peripheral vascular resistance, its direct effect on blood vessels is largely unknown. Little information is available about the influence of enalapril on the different vascular regions. Ten patients with moderate essential hypertension were treated with enalapril 20 mg daily in a double-blind, placebo controlled cross-over study for six weeks during each period. Blood pressure and heart rate were measured in supine, sitting and standing position. Venous capacity was derived from pressure volume curves plotted simultaneously at forearm and calf. Arterial blood flow at rest and during reactive hyperemia was measured at calf and finger by plethysmography. Enalapril increases venous capacity in upper and lower limbs in patients with moderate essential hypertension. Also, there is vasodilation of calf and finger arteries both at rest and during reactive hyperemia. Finger and calf arteries contribute to the decrease of the total peripheral vascular resistance during treatment with enalapril; thus, ACE inhibition is capable of correcting the increased peripheral resistance which often is the main cause of arterial hypertension. PMID- 3022514 TI - Why are the angiotensin converting enzyme inhibitors rational in the treatment of congestive heart failure? AB - In chronic congestive heart failure, there is stimulation of the renin angiotensin-aldosterone system, in addition to other homeostatic mechanisms. In addition to increased tone in the arteriolar and venous beds, there is salt and water retention by the kidney and excitation of the central nervous system by angiotensin. Interference with the generation of angiotensin reduces preload and afterload without a reflex tachycardia as the effects of angiotensin on the central nervous system are reduced. ACE inhibition is, therefore, a logical approach to the management of chronic congestive heart failure. PMID- 3022515 TI - Acute and chronic hemodynamic effects of enalapril in patients with congestive heart failure. PMID- 3022516 TI - Fine needle aspiration cytology of "minimal" breast cancer. AB - The value of fine needle aspiration (FNA) cytology in the diagnosis of "minimal" breast cancer was studied. Sixteen (76.2%) of 21 cases of invasive breast cancer less than 1.0 cm in diameter and 14 (73.3%) of 19 cases of noninvasive breast carcinoma were given a positive diagnosis by FNA cytology. One "suspicious" and the five false-negative diagnoses occurred in cases of invasive carcinoma; the reasons were considered to be either a faulty technique of needling the tumor or the presence of prominent fibrosis in the tumor. In noninvasive carcinoma, atypical cells were misdiagnosed in two of the five smears that had been originally reported as negative. The results of the retrospective analysis showed that FNA cytology had a higher accuracy in the diagnosis of small lesions than did radiologic and echographic criteria, and FNA cytology was thus used as the main criterion for deciding on the necessity for preoperative surgical biopsies. PMID- 3022517 TI - Characterization of alpha-MSH-like immunoreactivity in human plasma. AB - We report new information on the presence of alpha-melanotropin(alpha-MSH)-like immunoreactivity in the peripheral circulation of the adult human. Pooled blood from 20 patients with non-endocrine diseases was subjected to a Sep-pak pre purification followed by a high pressure liquid chromatographic (HPLC) fractionation. The eluate from the HPLC column was analyzed by a radioimmunoassay (RIA) specific for the C-terminal part of the alpha-MSH molecule. From this it appeared that alpha-MSH was the major alpha-MSH-like immunoreactive peptide present in human blood with a level of 2-5 pg/ml. This level is similar to the one determined by direct measurements in Sep-pak pre-purified human plasma (median 2.0 pg/ml, n = 11). Des-acetyl alpha-MSH was present in human blood in only a minor quantity. We discuss this finding in view of earlier reports on alpha-MSH-like immunoreactivity in human pituitary tissue. PMID- 3022518 TI - Compensated 131I-therapy of solitary autonomous thyroid nodules: effect on thyroid size and early hypothyroidism. AB - Thyroid function and thyroid gland volume, ultrasonically determined, were investigated in 27 hyperthyroid patients with solitary autonomous thyroid nodules before and during one year after 131I-treatment. Total thyroid volume decreased gradually from 40.9 +/- 3.5 ml (mean +/- SEM) before treatment to 23.9 +/- 1.8 ml (P less than 0.001) at 3 months after 131I-treatment. No further change was observed. All but two patients received only one dose of 131I, and in spite of a significant decrease also of the non-adenoma side of the gland, none became hypothyroid. We conclude that 131I-therapy has an important place in the treatment of solitary autonomous thyroid nodules since all our patients became euthyroid within 3 months, only 2 of 27 patients needed more than one dose of 131I, no cases of hypothyroidism occurred, and thyroid volume was substantially decreased. PMID- 3022519 TI - Steroidogenesis and cAMP production in isolated avian granulosa cells during follicular maturation: lack of positive correlation. AB - Luteinizing hormone- and forskolin-induced acute steroidogenesis and cAMP production were studied in dispersed chicken granulosa cells in relation to follicular maturation. Cells isolated from the 6 largest preovulatory follicles (F1-F6) were used in this study. Basal steroidogenic activity increased with progressive maturation of the follicle without a corresponding rise in basal cAMP production. Both LH- and forskolin-promoted progesterone production was directly correlated with the maturational stage of the follicle and the dose of the agonists. On the other hand, LH caused the greatest increase in cAMP production in the third and fourth largest preovulatory follicles (F3-F4), whereas forskolin stimulated cAMP accumulation was maximal in the least mature granulosa cells (F5 F6). Thereafter it decreased in proportion with follicular development. It is concluded that LH-induced acute steroidogenesis in chicken granulosa cells is not positively correlated with cAMP production during the last few days of follicular maturation. Therefore the steroidogenic responsiveness of granulosa cells of the largest follicle to LH cannot be ascribed solely to receptor-coupled adenylate cyclase activity. As shown by the forskolin experiment, the intrinsic activity of the adenylate cyclase-cAMP system is greatest in the small F5, F6 follicles which respond only minimally in terms of steroidogenesis to either a receptor dependent agonist (LH) or a nonreceptor agonist (forskolin). PMID- 3022520 TI - Effect on growth of patients with Turner's syndrome treated with low estrogen doses. AB - In 33 patients with Turner's syndrome growth during a one year period of treatment with low doses of estrogens was evaluated (group A: (N = 12) Presomen 5 9 micrograms/kg d; group B: (N = 9) Presomen 10-21 micrograms/kg d; group C: (N = 12) ethinylestradiol 45-155 ng/kg d) and compared to a group (N = 37) of untreated patients. The auxological evaluation was made using SDS derivations based on control data derived from 150 untreated patients. Based on chronological age (CA) SDS levels for height velocity and the increments in height at the end of treatment increased marginally. Compared to untreated patients no effect was seen when calculations were based on bone age (BA) due to an advancement in bone maturity. It is concluded that low doses of estrogens are not suitable to improve the height development in Turner's syndrome. PMID- 3022521 TI - Plasma ACTH in diagnosis and control of adrenal disorders. AB - Unstimulated plasma ACTH concentrations remain at or below the detection limit of conventional immunoassays. Grossly elevated ACTH concentrations are diagnostic in suspected adrenal insufficiency, remain elevated well above 200 ng/l during substitution therapy and obviate the need of further tests. For the diagnosis of secondary adrenal failure, plasma ACTH, cortisol and 11-desoxycortisol response to a single midnight dose of metyrapone (1.2 g/m2 = 30 mg/kg) discriminates between a normal (morning ACTH above 100 ng/l), diminished (morning ACTH detectable, but below 100 ng/l), and an absent (ACTH below 20 - 40 ng/l) ACTH reserve. In congenital adrenal hyperplasia, plasma ACTH concentrations mirror, together with 17-alpha-hydroxyprogesterone, the extent of ACTH suppression. Elevated ACTH concentrations were suppressed by prednisolone (25%), dexamethasone (2% of the hydrocortisone dose) or by addition of cyproterone acetate (100 mg/m2/day). Using selective venous catheterisation in clinically and biochemically proven Cushing's syndromes, a pituitary adenoma could be identified and localized in 6 of 8 patients by measuring ACTH concentrations in the left and right petrosal sinus before and after stimulation with corticotrophin releasing hormone. PMID- 3022522 TI - Usefulness of plasma pregnenolone sulfate in testing pituitary-adrenal function in children. AB - In normal subjects, plasma pregnenolone sulfate (PS) levels high at birth, decreased during the first year of life in relation to the pattern of involution of the fetal adrenal zone. Thereafter, PS levels, in contrast with those of DHAS, did not show the abrupt rise characteristic of the adrenarche, but increased very progressively till adulthood. The response of PS to various provocative tests of adrenal and pituitary function (ACTH and Metyrapone stimulation, dexamethasone suppression), has been established in normal subjects. The measurement of plasma PS levels in basal conditions as well as in response to dynamic tests was very useful in the diagnosis of various adrenal and pituitary diseases in children. PMID- 3022523 TI - Is heterozygosity for the steroid 21-hydroxylase deficiency responsible for hirsutism, premature pubarche, early puberty, and precocious puberty in children? AB - We applied the ACTH-stimulation test developed in our laboratory for the detection of heterozygous carriers of the 21-hydroxylase deficiency gene to patients suffering from hirsutism (n = 89), premature pubarche (n = 75), early puberty (n = 37), and precocious puberty (n = 22). While, in the general population, this test is positive in less than 2%, we found in 33% of hirsute patients, in 41% of patients with premature pubarche, and in 33% of patients with early puberty a hormonal response similar to the one seen in heterozygous carriers for the 21-hydroxylase defect. In contrast, only 18% of patients with precocious puberty responded abnormally. Thus we speculate that, at least in some patients with hirsutism, premature pubarche, and early puberty, heterozygosity for the 21-hydroxylase defect plays a major role in the pathogenesis of these disorders. PMID- 3022524 TI - Congenital adrenal hyperplasia: molecular mechanisms resulting in 21-hydroxylase deficiency. AB - 21-Hydroxylase deficiency resulting in congenital adrenal hyperplasia (CAH) is a HLA-linked autosomal recessive disorder that has a wide range of phenotypic expression. Two homologous 21-hydroxylase genes (21-OHA and 21-OHB) occur within the Class III region of the major histocompatibility complex, but only one (21 OHB) appears to function in adrenal steroidogenesis. Our restriction maps, and initial sequence data from White et al. (Pediatr Res 20:274A (1986)) for the two human 21-OH genes reveal a high degree of homology between these genes and a reading frame shift mutation in the 21-OHA gene respectively. Among fourteen control subjects, the intragenic restriction patterns of the 21-OHA and 21-OHB genes are invariant. The few restriction fragment length polymorphisms (RFLPs) found in some controls result from polymorphic restriction sites outside the 21 OH genes. In patients with CAH, several different mechanisms for mutation of the 21-OHB gene have been described: deletion of the unique sequences of the 21-OHB gene, conversion of the unique sequences of the 21-OHB gene to those of 21-OHA, and mutations of 21-OHB which do not result in a detectable alteration of restriction pattern (e.g., point mutations). Duplication of the 21-OHA gene has been found in some patients with attenuated CAH; however, the significance of this finding remains unclear. PMID- 3022525 TI - The functional response to furosemide in a case of de Toni-Debre-Fanconi disease. AB - The case of a 13-year-old boy with the advanced clinical picture of the idiopathic DeToni-Debre-Fanconi syndrome is described, on whom acute studies of proximal tubular functions and of the effect of furosemide thereon were performed. Sodium bicarbonate loading corrected the hyperchloremic acidosis, but induced an increase of urinary bicarbonate loss of over 20% of the filtered amount. Furosemide corrected bicarbonate reabsorption in spite of the presence of metabolic alkalosis. The urinary excretion of alpha-amino nitrogen, glucose, and phosphates decreased and tubular reabsorption of the two latter increased under furosemide. On a chronic treatment with furosemide and dietary sodium chloride restriction, correction of hyperchloremic acidosis, hypophosphatemia and rickets was achieved. PMID- 3022526 TI - Adult respiratory distress syndrome after renal transplantation. A case report. AB - A potentially lethal case of "air-borne" adult respiratory distress syndrome, most likely consequent to cytomegalovirus (CMV) pneumonitis, is described in a kidney transplant patient. It was characterized by confluent densities on both lung fields with peripheral zones of normal radiographic pattern and with one of the highest values of extravascular lung water reported in the literature, in the presence of a normal pulmonary capillary wedge pressure. When specific conservative therapy for curing a potentially lethal CMV pneumonitis after kidney transplantation fails, we suggest that transplantectomy should be considered. PMID- 3022527 TI - Heterogeneity of hereditary motor and sensory neuropathy type I (HMSN I): electroneurographical findings, visual evoked potentials and blood group markers in a family with Charcot-Marie-Tooth disease (CMT). AB - 21 members of a large kinship with autosomal dominant CMT showing typical clinical findings were studied electroneurographically, with visual evoked potentials (VEP) and with blood group markers. In clinically affected members, the mean motor nerve conduction velocity (NCV) of the median nerve was found to be 17.5 m/s (SD 2.4). Contrary to previous genetic linkage studies in CMT families with comparable slow motor NCV, blood group typing in this family excluded close linkage of HMSN I to Duffy locus, which may indicate the existence of another subgroup in CMT neuropathy. Mean latencies of VEP, in both clinically affected and unaffected members, showed no pathological alterations when compared to normals. There was no correlation between NCV and P 100 latencies, but significant variation of P 100 latencies in families of twin brothers could be demonstrated. As already suggested by other authors, our findings may also indicate heterogeneity in this neuropathy. PMID- 3022528 TI - Search for HTLV-I and LAV/HTLV-III antibodies in serum and CSF of multiple sclerosis patients. AB - In order to confirm the findings on the presence of antibodies against human T lymphotropic retroviruses in subjects affected by multiple sclerosis we studied paired serum and CSF samples from 32 MS patients. Both ELISA and Western blot procedures were employed to detect antibodies against HTLV-I and LAV/HTLV-III antigens. Negative results were obtained in all samples examined, except one which was reactive to HTLV-I in ELISA but not in Western blot. PMID- 3022529 TI - A comparison of the effects of epilepsy and ageing on learning and hippocampal physiology. PMID- 3022530 TI - A new type of inclusion body in human spinal cord. AB - New pericapillary inclusion bodies were found in 17 cases of sporadic amyotrophic lateral sclerosis (ALS). The inclusion bodies consisted of paracrystalline arrays with 5-7 nm electron-dense subunits, were discernible with the light microscope and had the staining properties of protein. They were surrounded by capillary wall basement membrane and were often associated with peripheral fibrils. Astrocytic fibrillary bodies, without paracrystalline material, were also found. The ultrastructure and staining of the fibrils suggests that the paracrystalline material is within pericapillary astrocytes. The nature and significance of the inclusion bodies are unknown, but their presence suggests that there may be pericapillary abnormalities in the spinal cord in ALS and possibly other disorders. PMID- 3022531 TI - Long-term culture of human corticotropin-secreting adenomas on extracellular matrix and evaluation of serum-free conditions. Secretory aspects. AB - Tissues from 12 human corticotropin-secreting adenomas, obtained during surgery for Cushing's disease (CD, ten cases) or Nelson's Syndrome (NS, two cases), were exclusively mechanically dispersed. Single cells and cell aggregates were plated on extracellular matrix derived from bovine corneal endothelia. Functional responses to physiological stimuli were analyzed by measuring human beta endorphin (beta h-EP) immunoreactivity (IR) by radioimmunoassay in the culture medium. All adenomas responded with stimulated secretory activity to arginine vasopressin (AVP), corticotropin-releasing factor (CRF), or both. Cortisol higher than 10(-8) M suppressed basal secretion and CRF- or AVP-stimulated beta h-EP-IR secretion. There was no consistent difference in response of the cells when cultured in medium containing 10% fetal calf serum (FCS) or in serum-free conditions. A change of cells from serum to serum-free conditions usually resulted in 10%-50% reduction in the basal secretion level that remained stable for at least 2 weeks and, in one case (NS), 10 weeks. In cells maintained in medium supplemented with 5% serum obtained from the respective patients 40 min after adenoma removal, basal secretion was suppressed to 60% of the baseline level in a 10% FCS control. Long-term incubation with CRF (10(-9) M) showed sustained stimulation of hormone secretion. No remarkable cell proliferation was observed under basal conditions or during long-term, low-dose incubation with cholera toxin (10(-12) M) in two cases (CD), or CRF (10(-9) M) in two cases (NS, CD). Parallel beta-EP-IR and adrenocorticotropin secretion was verified in selected cases. PMID- 3022532 TI - Long-term culture of human corticotropin-secreting adenomas on extracellular matrix and evaluation of serum-free conditions. Morphological aspects. AB - Tissues from 12 human corticotropin-secreting adenomas, obtained during transsphenoidal surgery for Cushing's disease (CD, ten cases) or Nelson's syndrome (NS, two cases), were mechanically dispersed. The resulting single cells and cell aggregates were plated on extracellular matrix derived from bovine corneal endothelia. CD and NS cells showed distinct morphological differences initially, CD cells being much more spherical than the flattened NS cells. By 10 days at the latest after plating, however, CD and NS cells were indistinguishable morphologically. Cultured cells from both entities responded with rounding to cortisol (hydrocortisone, 10(-6) M) within 4-6 h. Synthetic ovine corticotropin releasing factor (10(-8) M) produced flattening and extension of cytoplasmic processes after as early as 2 h. PMID- 3022533 TI - Immunoenzymatic labelling of JC papovavirus T antigen in brains of patients with progressive multifocal leukoencephalopathy. AB - Formalin-fixed, paraffin-embedded autopsy sections of brains from two patients with progressive multifocal leukoencephalopathy (PML) were stained by peroxidase antiperoxidase methods for human papovavirus T antigen, a nonstructural protein expressed in cells lytically infected or transformed by JC, BK, and SV40 viruses. Adjacent sections were stained for papovavirus common structural antigen, a component of JC, BK, and SV40 virions which is synthesized in productively infected but not transformed cells. Intense immunoperoxidase labelling specific for T antigen was detected in large numbers of oligodendrocytes at the edges of demyelinated areas and in occasional oligodendrocytes within otherwise normal brain. Occasional morphologically normal astrocytic cells exhibited similar specific staining, but only rate atypical astrocytic cells contained detectable amounts of T antigen. Examination of adjacent sections stained with antisera to common structural antigen revealed an identical pattern of immunoenzymatic labelling, indicating that most of the cells expressing T antigen were also expressing viral structural proteins. The present study demonstrates that T antigen can be identified by immunoperoxidase methods in routinely processed autopsy material from cases of PML, but that detectable amounts of antigen are found almost exclusively in cells undergoing lytic infection. PMID- 3022534 TI - Hirano body in an inflammatory cell of leptomeningeal vessel infected by fungus Paecilomyces. AB - An intracytoplasmic microfilamentous lattice, ultrastructurally identical to Hirano body, was found in an inflammatory cell within a leptomeningeal vessel wall infected by fungus Paecilomyces javanicus. The structure was well preserved and not associated with phagosomes. This is the first report of Hirano body found in an inflammatory cell at the site of fungal infection. The present finding suggests that the formation of Hirano body is non-specific and not restricted to the cell of the neuro-muscular system. PMID- 3022535 TI - The structure analysis of Hirano bodies by digital processing on electron micrographs. AB - To clarify the structure of Hirano bodies, electron micrographs of Hirano bodies taken at various tilting angles have been studied by digital image analysis. On the electron micrographs, the beaded filaments of Hirano bodies were turned into a pattern of lattice-like arrays by changing the tilting angles. Based on computer-processed diffraction patterns and filtered images, it is proposed that the filaments of Hirano bodies are helical strands with a pitch of 185 A. A model for the helical strand drawn by microcomputer at various angles of rotation is in accordance with the filtered images of the tilted filaments. Computer simulation also reveals that the helical strands appear to be lattice-like when they are arranged in parallel. PMID- 3022536 TI - Pick body-like inclusions in the dentate fascia of the hippocampus in Alzheimer's disease. AB - Pick body-like inclusions are described in the granular neurons of the dentate fascia in Alzheimer's disease. The inclusions are round, argyrophilic and stained by thioflavine-S. Immunocytochemically they contain antigenic determinants of neurofilaments and of Alzheimer neurofibrillary tangles. Ultrastructurally they are composed primarily of 15-18 nm straight filaments similar to the neurofibrillary pathology of progressive supranuclear palsy and Pick's disease. The dentate fascia inclusions, as well as cerebellar plaques but not amyloid angiopathy, are found most frequently in association with severe neurofibrillary degeneration. PMID- 3022538 TI - Cytochemical demonstration of steroid binding in human ovarian carcinoma and thecofibroma. A preliminary report. PMID- 3022537 TI - The axon reaction in spinal ganglion neurons of acrylamide-treated rats. AB - Rats were given acrylamide in doses of either 30 or 50 mg/kg (5 days each week) for up to 3 weeks and killed at weekly intervals. The right sciatic nerve was tied tightly at the level of the major trochanter 4 days before killing the animals by perfusion fixation when ipsilateral and contralateral sensory ganglia (L5 and L6) were removed. The effects on neuronal perikarya of axotomy alone, of acrylamide alone and of these combined were studied by light and electron microscopy. The responses to axotomy and to acrylamide intoxication shared certain features, namely peripheral Nissl substance and to a lesser degree nuclear eccentricity, nucleolemmal crenation and mitochondrial enlargement. Neurofilament loss was present only with acrylamide. In combined axotomy and acrylamide all these five features were prominent. These findings indicate firstly that the individual responses to axotomy and to acrylamide, while sharing several features, are subtly different and secondly that acrylamide appears to impede the vital neuronal responses directed towards repair of the axon. PMID- 3022539 TI - Colposcopy in assessment of the natural history of prospectively followed-up human papillomavirus (HPV) lesions in the uterine cervix. AB - As a part of the long-term prospective follow-up study conducted in our clinic since 1981 for women with cervical HPV (Human papillomavirus) infections (either with or without cervical intra-epithelial neoplasia, HPV-CIN and HPV-NCIN, respectively) the colposcopic data on 292 consecutive patients with a mean follow up of 16 months are reported. The results are based on 786 colposcopies performed at 6-month intervals according to conventional methods. Colposcopic findings are categorized into one of the following patterns: normal, warty, punctate, mosaic, leukoplakia, and combination. Colposcopic pattern bore an excellent correlation to the natural history (clinical course) of the HPV lesions, in that colposcopy was normal in 75% of the regressing lesions, in 38% of the persistors, and in only 16% of the progressors. The percentage of regression increased parallel with the frequency of normal pattern during the follow-up. Leukoplakia and combination were the two most frequent (55.7%) patterns in progressor lesions, as compared with only 12.9% in regressors and in 33.2% of the persistors. The same was true of the 15 lesions progressing into carcinoma in situ (CIS), and treated by cone. In the progressor lesions, an abnormal colposcopy pattern was disclosed in 65 100%, as compared with 14-35% only in the regressors. The present study provides evidence that colposcopy is an applicable and relatively accurate diagnostic method for assessing cervical HPV lesions. Colposcopy also seems to have some prognostic value in the follow-up of these lesions, as shown by the fact that normal colposcopic pattern is frequently associated with regression, whereas leukoplakia and combination are encountered more frequently than others in HPV lesions with CIN, shown to be more prone to progress during the follow-up. PMID- 3022540 TI - Polyhydramnios caused by giant alveolar granular cell myoblastoma. AB - Congenital granular cell myoblastoma of the newborn is a rare tumor. The combination of polyhydramnios caused by obstruction of the infant's mouth by this tumor is extremely rare, and this is the first published report of such a condition. PMID- 3022541 TI - Detection of mucus glycoconjugates in human conjunctiva by using the lectin colloidal gold technique in TEM. I. A quantitative study in normal subjects. AB - We applied a specific cytochemical reaction to characterize the glycoconjugates produced by goblet and non-goblet epithelial cells of normal human conjunctiva. For this purpose we utilized the lectins, proteins of vegetal origin, which are extremely sensitive in binding glycosidic residues. In particular, we used WGA, PNA, SBA and ConA conjugated with colloidal gold as ultrastructural marker for Transmission Electron Microscopy. This technique allowed us also to perform a quantitative analysis, by counting colloidal gold particles present on mucus granules. In this way we analyzed the content both of goblet and non-goblet epithelial cells. In the former, WGA, PNA, SBA and ConA receptors, here reported in decreasing density, were present. In the latter WGA was always positive, SBA and PNA sometimes were negative, ConA was always negative. We speculate the different contribution to mucus production by these two sources may be important in evaluating tear film stability alterations occurring in those diseases in which non-goblet epithelial cell vesicles increase. PMID- 3022542 TI - Detection of mucus glycoconjugates in human conjunctiva by using the lectin colloidal gold technique in TEM. II. A quantitative study in dry-eye patients. AB - The mucus glycoconjugates produced by conjunctival goblet cells in dry-eye patients were studied by a specific cytochemical reaction in Transmission Electron Microscopy (TEM). Four lectins, proteins of vegetal origin which specifically bind glycosidic residues, (WGA, PNA, SBA and ConA) were used conjugated with colloidal gold as ultrastructural marker. We performed a quantitative analysis by counting the colloidal gold particles present on mucus granules. The results were compared with normal conditions. We found a decrease in sialic acid, N-acetyl-glucosamine, N-acetyl-galactosamine and galactose-N acetyl-galactosamine and an increase in mannose. The different content of glycoconjugates in goblet cells may reflect in the change of physical and functional properties of mucus. We think these data may be useful in the search for a therapeutic mucomimetic drug. PMID- 3022543 TI - Pathologic features of small hepatocellular carcinoma. AB - Twenty-one nodules of small hepatocellular carcinoma (HCC) were examined. Histologically, the nodules often presented formation of plump trabeculae, marked nuclear atypism, or aggressive growth comprising capsular invasion, vascular invasion, and replacement of adjacent pseudolobules. Aside from these characteristic findings of HCC, it was important to reveal the following features for the diagnosis of well differentiated type of small HCC: variable thickening or distortion of trabecular structure in association with nuclear crowding, acinar formation, selective cytoplasmic accumulation of Mallory bodies, nuclear abnormalities consisting of thickening of nucleolus, hepatic cords in close contact with bile ducts or blood vessels, and hepatocytes growing in a fibrous environment. During the invasive growth, the tumor cells may well be subtly blended with benign hepatocytes, giving rise to a pattern of "mixed cellularity". It is also emphasized that connective tissue septa of pseudolobules could be a route of rapid tumor spreading. PMID- 3022544 TI - Hypothalamic gangliocytoma. Selective appearance of neurofibrillary changes, granulovacuolar degeneration, and argentophilic bodies. AB - A hamartomatous gangliocytoma was observed in the hypothalamus of a 54-year-old woman. The ganglion cells were atypical, highly pleomorphic and often multinucleated, and they possessed neurofibrillary changes, granulovacuolar degeneration, and argentophilic bodies. The neuronal changes were highly selective and were not found in other parts of the brain. The tumor was also characterized by the presence of angiomatous blood vessels having such degenerative changes as fibrosis and thrombosis. The angiomatous blood vessels were found only in the lesion. The ultrastructural features of the neurofibrillary changes were similar to those observed in Alzheimer's disease. Vascular alterations have been suggested to be a possible contributor to the morphogenesis of neurofibrillary changes. In this case the exact etiology of these neuronal changes remains unclear, however, the possibility of a regional vascular role is considered with respect to their morphogenesis. PMID- 3022545 TI - Cytoplasmic inclusions and virus-like particles in blast cells in acute lymphoblastic leukemia. AB - Cytoplasmic inclusions and virus-like particles are described in blast cells of peripheral blood from a 16-year-old female with acute lymphoblastic leukemia. Three kinds of inclusions were identified on electron microscopy. The first type of inclusion was single membrane-bounded vacuoles, some of which contained virus like particles, the second was lysosome-like structures, and the third appeared to be of mitochondrial origin. Virus-like particles were round in shape and had a diameter of 26 to 58 nm. They consisted of an electron-dense outer membrane and an electron-lucent core. At the present time the exact nature and significance of these virus-like particles still remain unclear. PMID- 3022546 TI - Long term treatment with nomifensine or lithium does not change 3H-naloxone binding to opioid receptors in rat brain. PMID- 3022547 TI - Selenium deficiency in Finnish foods and nutrition: research strategy and measures. PMID- 3022548 TI - Comparison of four enzymes in zinc-deficient rats as possible indicators of marginal zinc status. PMID- 3022549 TI - Mechanism of chromium carcinogenesis. PMID- 3022550 TI - The antioxidative ability of erythrocyte of children with different selenium status. PMID- 3022551 TI - Trace elements and rheumatoid arthritis (RA)--pathogenetic and therapeutic aspects. AB - Rheumatoid arthritis is characterized by increased activity of macrophages which produce toxic forms of oxygen. Such oxygen has been suggested as mediator also of rheumatoid inflammation. Gold accumulates in lysosomes of the macrophages and stabilizes lysosomal and other cell membranes leading to reduced liberation of toxic oxygen. Intracellular production of metallothionein can be induced. Zinc in high doses parenterally can immobilize macrophages and also induce metallothionein-like proteins. Copper and zinc are components of SOD which detoxifies oxygen, and copper-thiolate complexes are reported to be anti inflammatory. The therapeutic effect of penicillamine and other thiols like aurothiomalate may also be related to an anti-oxidative action. Therapeutic induction of increased intracellular levels of glutathione or administration of selenium in such a form that it incorporates into glutathione-peroxidase and increases the efficacy of the enzyme may lead to accelerated metabolism of toxic oxygen. PMID- 3022553 TI - Effects of dietary factors on g.i. Cd absorption in mice. PMID- 3022552 TI - Effect of DDC on acute oral Cd toxicity. PMID- 3022554 TI - Quantitative autoradiography of central neurotransmitter receptors: methodological and statistical aspects with special reference to computer assisted image analysis. AB - In the last few years, quantitative receptor autoradiography has been extensively employed to study the distribution and the functional role of area-specific transmitter receptors in the central nervous system. In the present paper we have developed quantitative methodologies for the analysis of autoradiograms using computerized image analysers coupled with standard TV camera input for the microdensitometrical evaluations. These procedures include the assessment of the film response to radioactivity using appropriate standards calibrated according to brain tissue quenching and non-linear conversion of density measurements in radioactivity values adopting the best mathematical model fitting to give as little variability as possible in the transformations. The reliability of the proposed approach has also been evaluated by means of a computer-assisted Monte Carlo simulation and parallel biochemical determinations. PMID- 3022555 TI - Receptor autoradiographical evidence for high densities of 125I-neuropeptide Y binding sites in the nucleus tractus solitarius of the normal male rat. AB - By means of quantitative receptor autoradiography using 125I-neuropeptide Y (125I NPY) as a radioligand, the distribution of 125I-NPY binding sites has been evaluated in coronal sections at various rostrocaudal levels of the medulla oblongata of the male rat. High densities of neuropeptide Y (NPY) binding sites were demonstrated in the nucleus tractus solitarius (nTS), in the nucleus paratrigeminalis, in the area postrema, in the medial nuclei of the inferior olive and in the substantia gelatinosa of the caudal part of the spinal trigeminal nucleus. Low densities were present in the dorsal motor nucleus of the vagus (dmnX) and in the hypoglossal nucleus. Other regions of the medulla oblongata showed only a very low density or no specific binding of 125I-NPY. These results indicate that the central cardiovascular actions of NPY at least in part may be mediated via an action in the nTS, in this way controlling the baroreceptor reflex activity. Neuropeptide Y mechanisms may also play a role in the regulation of other visceral afferents such as those involved in gastrointestinal control (dmnX) and of cerebellar function (inferior olive). Finally, the results indicate that a high density of NPY immunoreactive terminals in some regions of the medulla oblongata is associated with a low density of high affinity 125I-neuropeptide Y binding sites and vice versa. PMID- 3022556 TI - A correlation analysis of the regional distribution of central enkephalin and beta-endorphin immunoreactive terminals and of opiate receptors in adult and old male rats. Evidence for the existence of two main types of communication in the central nervous system: the volume transmission and the wiring transmission. AB - By means of semiquantitative immunocytochemistry and quantitative receptor autoradiography a correlation analysis has been performed on the pre- and post synaptic features of enkephalin and beta-endorphin immunoreactive neuron systems of the 3- and 24-month-old male rat. A parallel disappearance of enkephalin- and beta-endorphin-like immunoreactivity and of the density of mu and delta opiate receptors is shown during ageing. Furthermore, the lack of an overall correlation between the amount of pre- and post-synaptic components of the enkephalin and beta-endorphin synapses give evidence for the existence of a volume type of transmission in such systems in the telencephalic, diencephalic and mesencephalic areas analysed. PMID- 3022558 TI - Tumor vessels and contrast enhancement of hepatocellular carcinoma demonstrated by percutaneous transhepatic portography. Report of a case. AB - A patient with an Edmondson type I-II hepatocellular carcinoma had, at celiac angiography, a poor arterial supply but a rich portal supply as observed at percutaneous transhepatic portography, an observation not previously reported in this disease. The importance of demonstrating the vascular supply of the tumor previous to planned intravascular treatment is obvious. PMID- 3022557 TI - Capsaicin-sensitive nerves and ureteric motility: opposing effects of tachykinins and calcitonin gene-related peptide. PMID- 3022559 TI - Angiography of histopathologic variants of synovial sarcoma. AB - Synovial sarcomas are rare soft tissue tumors which histopathologically can be divided into monophasic, biphasic and mixed variants. As part of a protocol for intra-arterial chemotherapy 12 patients with biopsy proven synovial sarcoma underwent angiography. The angiograms on these patients were reviewed to determine whether synovial sarcomas and their variants demonstrated a characteristic angiographic appearance. Synovial sarcomas appeared angiographically as soft tissue masses which showed a fine network of tumor vessels with an inhomogeneous capillary blush. Their degree of vascularity varied according to their histopathology. Monophasic synovial sarcomas demonstrated in general a higher degree of neovascularity than the biphasic form. This finding was also suggested by histopathologic analysis of the vessels in the tumor. Although angiography did not show a distinctive vascular pattern it may be useful to evaluate tumor size and vascularity. PMID- 3022560 TI - Structure enhancement by subtraction in magnetic resonance imaging. AB - Two patients with tumours in the pituitary gland and the liver, respectively, were studied with MRI. Regions of different transverse relaxation times were found in the tumours. Subtraction of images recorded with different echo times demonstrated the tumour regions better than the original images although these were recorded with 2 repetition times and double and multiple echo sequences. Subtraction techniques may thus aid diagnosis in MRI. PMID- 3022561 TI - Autoradiographic localization of GABAergic and muscarinic cholinergic receptor sites in the visual system of the kitten. AB - The distribution of binding sites for [3H] muscimol (which binds to GABA, receptor) and [3H] QNB (which binds to muscarinic cholinergic receptor) was studied in visual areas of 5 week-old kittens using contact film autoradiography after incubation in vitro of brain sections. A distinct laminar localization of [3H] muscimol receptor sites was observed in young animals with the highest density in layers II/ III and IV of all cortical areas that were investigated. For [3H] QNB binding a laminar pattern of distribution was also found with labeling in layer V exceeding that in adjacent layers. A small intracortical regional variability was noted, for both of the investigated neuroreceptors. [3H] muscimol labeling of the lateral geniculate nucleus and superior colliculus was weaker in comparison to that found in the cortex. The superficial layers of the superior colliculus revealed a high density of [3H] muscimol and [3H] QNB binding sites. The laminar and regional variations in the distribution of [3H] muscimol and [3H] QNB binding sites observed in young kittens, may be related to the possible role of GABAergic and muscarinic receptors in regulation of plastic changes that occur after visual deprivation. PMID- 3022562 TI - [The F-wave in uremic neuropathy]. PMID- 3022563 TI - Therapy of bronchogenic carcinoma with the drug platidiam lachema. PMID- 3022564 TI - Oncogene amplification in tumor cells. PMID- 3022565 TI - Transcription activation by viral and cellular oncogenes. PMID- 3022567 TI - Worldwide method effectiveness of the Today vaginal contraceptive sponge. AB - The Today vaginal contraceptive sponge is made of polyurethane and contains 1 gram of the spermicide nonoxynol-9. Following preclinical and phase I and II clinical trials, extensive worldwide phase III trials were conducted. These multiclinic trials were conducted according to a common protocol with regularly scheduled follow-up visits and examinations. The cumulative first year method effectiveness rate (life table) was 90 per 100 women. The second year rate was 97 per 100 women. No statistically significant difference was found in method failures between nulliparous and parous women. No serious complications occurred in over 1000 women-years of sponge use. PMID- 3022568 TI - Oxygen-derived free radicals in the pathogenesis of parasitic disease. PMID- 3022566 TI - Oncogenes in retroviruses and cells: biochemistry and molecular genetics. PMID- 3022569 TI - Characteristics of an established cell line (KU-2) derived from human renal cell carcinoma: I. Cloning of cells and morphological study of clones; II. Cell kinetics of KU-2 cells; III. Detection of type C virus in the culture. AB - A KU-2 cell line derived from human renal cell carcinoma was established by an indirect culture system using the nude mouse in November, 1976. These cells have been examined from different points of view including light and electron microscopic observation, and chromosomal analysis. Histopathological characteristics of the KU-2 cell line, even after being transplanted back to nude mouse, remain similar. However, the characterization of this established cell line has not been fully elucidated. In the present experiments, attempts have been made to study the cloning of KU-2 cells and morphological feature of clones, cell growth and kinetics, and detection of the type C virus in the culture. These results suggested that the KU-2 cell line was not homogeneous but composed of a heterogeneous population of cells based on morphological difference of 6 clones and discrepancy between population doubling time and generation time when calculated from the growth curve and synchronous culture of the KU-2 cells, which may be explained by the cytotoxic effect of excess thymidine. Also the type C virus was negative in the medium. PMID- 3022570 TI - Effects of alcohol on bone, muscle and nerve. AB - The acute and chronic effects of alcohol on bone, muscle and peripheral nerves are not well appreciated. Bone complications include traumatic fractures, osteoporosis and osteonecrosis. In muscle, a sustained bout of heavy drinking may cause rhabdomyolysis, while chronic alcohol abuse may produce proximal myopathy. In peripheral nerves, acute alcohol intoxication may lead to pressure neuropathy and chronic abuse may cause peripheral neuropathy. PMID- 3022571 TI - Chronic hiccups. AB - Patients with chronic hiccups should be carefully examined for an underlying disorder while receiving symptomatic treatment. Treatment includes physical maneuvers, drugs such as chlorpromazine, metoclopramide, anticonvulsants or quinidine, and other, less tested modalities such as hypnosis. Only those patients with disabling hiccups that do not respond to conservative treatment should be considered for phrenic nerve surgery. PMID- 3022572 TI - Salute to the CDC. PMID- 3022573 TI - Nuclear DNA content in mammary carcinomas in women aged 35 or younger. AB - Nuclear DNA content was studied in cytologic preparations obtained from 50 patients aged 35 or less with primary mammary carcinoma. As many as 90% of the tumors were aneuploid, i.e., exhibited DNA profile types III and IV. The cytologic diagnosis was confirmed by histologic examination in 46 patients. The majority of these tumors, 83%, were invasive ductal carcinomas, while medullary carcinomas constituted 13% of the surgical material. As judged from their DNA profiles, most mammary carcinomas in this age group would be tumors of high malignancy potential. This does not seem, however, to influence the prognosis of young women with breast cancer as much as would be expected, possibly because of a better-functioning immune surveillance system in this age group. PMID- 3022574 TI - Fast neutron radiotherapy for soft tissue sarcomas. University of Washington experience and review of the world's literature. AB - Sixteen patients with inoperable soft tissue sarcomas were treated definitively with fast neutrons at the University of Washington between August, 1970 and May, 1982. Eleven of these 16 patients were treated with curative intent and form the basis of this report. Actuarial plots are shown for local tumor control and survival. This work is placed in the context of worldwide experience in using fast neutrons to treat unresectable soft tissue sarcomas. PMID- 3022575 TI - Large granular lymphocytes in bronchoalveolar lavage fluids from immunocompromised patients with cytomegalovirus pneumonitis. AB - Natural killer (NK) cells activities had been demonstrated to be depressed in patients with fatal cytomegalovirus (CMV) pneumonitis. NK cells can be identified by morphologic features characteristic of large granular lymphocytes (LGLs). Bronchoalveolar lavage (BAL) cells from 16 immunocompromised patients with CMV pneumonitis were analyzed. Two different groups of patients could be distinguished depending on the course of the CMV pneumonitis: nine patients who recovered (Group A), seven patients with a fatal outcome (Group B). Except for the increase in polymorphonuclear cells (PMN) in Group B (12.4 +/- 11.6%), no significant difference in the macrophage or the total lymphocyte population was observed. A differential count excluding alveolar macrophages specified the percentage of LGLs from the total lymphocyte population. The LGLs in Group A (7.1 +/- 9.9%) were similar to those previously reported in normal lung. A significant increase in LGLs was observed in the BAL cells from patients of Group B (28.1 +/- 22%). The discrepancy between the high percentage of LGLs in patients with a fatal outcome and their expected protective effects is discussed. PMID- 3022576 TI - EMA and small cell anaplastic carcinomas: further evidence for an epithelial origin needed. PMID- 3022577 TI - Presence of vaccine-strain poliovirus in cerebrospinal fluid of patient with near miss sudden infant death syndrome. PMID- 3022579 TI - Update on the clinical utility of converting enzyme inhibitors in cardiovascular disease. November 10, 1985, Washington, D.C. PMID- 3022578 TI - Cytomegalovirus cholangitis in a homosexual man with acquired immune deficiency syndrome. AB - A 29-year old white homosexual man with acquired immune deficiency syndrome presented initially with right upper quadrant pain and progressive cholestasis. Diffuse mucosal irregularities were seen at endoscopic retrograde cholangiography. Histopathological examination of the gallbladder and wedge liver biopsy showed evidence of cytomegalovirus infection. A repeat endoscopic retrograde cholangiography for persistent symptoms of right upper quadrant pain and cholestasis showed progressive mucosal irregularities of the intra- and extrahepatic bile ducts consistent with progressive cholangitis. Subsequently the patient developed evidence of disseminated infection and died. Postmortem examination revealed histologic features of cytomegalovirus infection in lungs, pancreas, small bowel, adrenal glands, and liver. Immunohistochemical staining of liver confirmed the presence of cytomegalovirus infection of the biliary duct system. PMID- 3022580 TI - Pharmacology of angiotensin converting enzyme inhibitors. A review. AB - Two angiotensin converting enzyme inhibitors are currently available for clinical use. Captopril, a sulfhydryl-containing compound, is a direct acting enzyme inhibitor. Enalapril is a non-sulfhydryl pro-drug that requires enzymatic conversion to enalaprilic acid, a potent angiotensin converting enzyme inhibitor. Both drugs lower blood pressure, suppress blood pressure response to angiotensin I infusion, and elevate plasma renin activity. Enalapril bioavailability is unaffected by food, whereas captopril availability is suppressed by food. Dose adjustments are necessary for patients with congestive heart failure and renal failure. PMID- 3022581 TI - Renal effects of angiotensin converting enzyme inhibitors in hypertension. AB - This review focuses on the renal effects of the angiotensin converting enzyme inhibitors, captopril and enalapril. Emphasis is placed on the renal response to these drugs in patients with primary essential hypertension, and in hypertension accompanying renal parenchymal disease. Specifically reviewed are the renal function and hemodynamic, salt and water, body fluid composition, and urinary protein excretion responses. The interruption of the renin-angiotensin aldosterone axis has the potential to produce a variety of favorable renal responses, including reduction of renal vascular resistance, enhancement of renal blood flow, enhancement of glomerular filtration rate, acute natriuresis, sustained diuresis, and a decrease in urinary protein excretion. Data in support of these potential renal perturbations are presented and discussed. The results suggest that the angiotensin converting enzyme inhibitors are important therapeutic agents in the treatment of hypertensive disease, in that they may modify pathophysiologic renal abnormalities encountered in this disease state. PMID- 3022582 TI - Clinical utility of angiotensin converting enzyme inhibitors in hypertension. AB - Oral angiotensin converting enzyme inhibition was introduced eight years ago and is becoming increasingly popular for the treatment of hypertension and congestive heart failure. This treatment causes blood pressure lowering associated with suppression of angiotensin and aldosterone, lack of orthostatic hypotension or metabolic disturbances, redistribution of regional blood flows in favor of vital organs and, in the long term, decreased sympathetic drive and regression of left ventricular hypertrophy. It is effective as monotherapy in more than 50 percent of unselected patients; addition of a diuretic increases the percentage of responders to more than 80 percent. It is the treatment of choice for patients with concurrent diabetes, asthma, gout, depression, or very active life-style. Side effects, observed originally in patients with severe hypertension and renal failure treated with very high doses of captopril, are rare in otherwise healthy hypertensive patients receiving smaller doses of this drug and virtually absent with second-generation angiotensin converting enzyme inhibitors like enalapril. PMID- 3022583 TI - Angiotensin converting enzyme inhibitors in congestive heart failure. Overview in comparison of captopril and enalapril. AB - In this study, the use of enalapril and captopril is compared in the treatment of congestive heart failure. Although both drugs act on the renin-angiotensin system via converting enzyme inhibition, their different chemical structures may dispose them to different pharmacologic and physiologic activity. Both drugs exert a vasodilator effect, with reduction of left and right ventricular filling pressures and aortic impedance. In short-term hemodynamic studies, the onset of action and peak effect are earlier with captopril. Enalapril has a much more gradual onset and longer duration of action. Both drugs have a shallow dose response curve and both produce comparable hormonal changes: an increase in plasma renin activity and a decrease in aldosterone levels. Captopril also increases prostaglandin production. Long-term efficacy trials have demonstrated symptomatic improvement in patients given captopril and those receiving enalapril who were also receiving digitalis and diuretics. Baseline hemodynamics may not predict long-term improvement. There are few adverse effects for the two drugs, but their incidences differ, suggesting a relationship to chemical structure. Recent studies in congestive heart failure suggest a reduction in mortality with various drug regimens. PMID- 3022584 TI - Safety profiles of the angiotensin converting enzyme inhibitors captopril and enalapril. AB - The safety profiles of the angiotensin converting enzyme inhibitors, captopril and enalapril, are the focus of this review. Adverse effects are reviewed as those associated with sulfhydryl compounds and as those considered class-specific adverse effects of angiotensin converting enzyme inhibitors. Specifically discussed are the incidences of the adverse effects of rash, taste disturbance, neutropenia, and proteinuria, which are characteristic of compounds containing sulfhydryl moieties, such as captopril. It is concluded from the review of these safety data that enalapril is well tolerated, has few class-specific adverse effects, and may offer a potential advantage over captopril by having fewer sulfhydryl-related adverse effects. PMID- 3022585 TI - The renin-angiotensin-aldosterone complex. AB - Both the thiazide and loop diuretics have long been known to induce both potassium and magnesium wastage with resultant negative balances of these important intracellular cations. The negative potassium and magnesium balances resulting from diuretic therapy are due primarily to stimulation of the renin angiotensin-aldosterone complex or secondary hyperaldosteronism. A long experience with an attempt to modify or reduce diuretic-induced potassium depletion by dietary sodium restriction and by the use of antikaluretics has been recorded. PMID- 3022586 TI - Herpes zoster and zosteriform herpes simplex virus infections in immunocompetent adults. AB - Among 111 immunocompetent patients referred to a general hospital setting with the clinical diagnosis of herpes zoster, viral cultures were obtained from 47 patients. Six of these patients (13 percent) had herpes simplex virus isolated, with four of the six infections involving the facial distribution, and the other two involving the T4 (breast) distribution. Excluding those in whom herpes simplex virus was isolated, the mean age (+/- SD) of the remaining 105 patients was 50 +/- 19 years. Thirty-two percent of the patients were at least 65 years old; however, 39 percent were younger than 40 years of age. Thus, herpes zoster frequently occurs in young, immunocompetent adults. Also, since zosteriform rashes may be caused by herpes simplex virus, viral cultures of lesions are useful to differentiate infections caused by herpes simplex virus from those due to varicella-zoster virus. The need to distinguish between these two viruses may be important with the advent of antiviral drugs and for use of the proper epidemiologic isolation procedures. PMID- 3022588 TI - Diffuse ecchymoses, hemoptysis, and atrial fibrillation in a patient with a lung mass. PMID- 3022587 TI - Serum angiotensin converting enzyme levels in patients with alpha 1-antitrypsin variants. AB - Serum angiotensin converting enzyme levels were measured in 184 subjects having either MM, MZ, ZZ, or MS Pi-types of alpha 1-antitrypsin. Elevated angiotensin converting enzyme levels were detected in 31 percent of the patients with the MZ Pi-type, 20 percent of the patients with the ZZ Pi-type, and 20 percent of the patients with the MS Pi-type compared with 1.33 percent of those with normal MM Pi-type. The mean serum angiotensin converting enzyme levels were also significantly higher in those with the MZ, ZZ, and MS Pi-types. Multiple family members of two families were found to have both the Z variant and angiotensin converting enzyme elevations, suggesting the possibility of a genetic linkage. Alpha 1-antitrypsin deficiency must be added to the list of disease states potentially associated with elevated serum angiotensin converting enzyme levels. PMID- 3022589 TI - Effects of endogenous redox-active compounds on coupling of human beta 2 adrenergic receptors. AB - Altered redox states such as metabolic acidosis may impair beta-adrenergic receptor responsiveness. Beta-adrenergic receptor function requires formation of a high affinity, "coupled" state of the receptor. The degree of coupling is reflected in the ratio of dissociation constants, KL/KH, for the low and high affinity states of the receptor. It has previously been demonstrated that 16 mM lactate and pH 7.1 induce independent defects in beta-adrenergic receptor function. The purpose of this study was to examine further how endogenous redox agents might alter high affinity state formation. Normal neutrophil membrane preparations containing beta-adrenergic receptors were exposed to several concentrations of three redox couplets native to plasma: lactate (L)-pyruvate (P), beta-hydroxybutyrate (BOHB)-acetoacetate (AcAc), and glutathione (GSH-GSSG). BOHB, AcAc, and P had no isolated effect on high affinity state formation while 10 mM lactate diminished KL/KH by 30% (p less than 0.001). Dropping the pH from 7.4 to 7.1 resulted in a 50% to 70% reduction in KL/KH (p less than 0.001), independent of metabolite present. GSH or GSSG exposure resulted in a concentration-dependent fall in KL/KH value. Thus, high affinity state formation is regulated by redox couplets and pH independently. The reduced responsiveness of beta-adrenergic receptors observed in such states as metabolic acidosis could result from direct effects of redox couplets in addition to those of low pH. PMID- 3022590 TI - Intracellular neurofibrillary tangle-like aggregations. A constantly present amyloid alteration in the aging choroid plexus. AB - Intracellular neurofibrillary tangles is one of the most characteristic findings in Alzheimer's disease and senile dementia of Alzheimer type. In the present paper the authors show that intracellular accumulation of paired helical filaments is also a constant finding in the epithelial cells of the choroid plexus of aging persons. Like the neurofibrillary tangles, the fibrils of the choroid plexus show staining properties typical of amyloid. The nature of the fibrils could not be clarified by electron microscopy or by immunohistochemistry with the use of antisera to gamma-trace or to amyloid fibril proteins of AA and prealbumin type. Amyloid protein AP, found in all amyloid substances except for neurofibrillary tangles and amyloid of senile plaques in the brain, was not demonstrated in the inclusions of the choroid plexus. PMID- 3022592 TI - Resolution of apical from basolateral membrane of shark rectal gland. AB - Membrane fractions were isolated from the rectal gland of Squalus acanthias using differential centrifugation and a sucrose gradient run in the presence of 1 M KBr. Using the basolateral membrane marker Na+-K+-ATPase, we obtained a sixfold purification with the most highly purified fraction from the gradient (sp act = 336 +/- 37 mumol X mg protein-1 X h-1). Electrogenic Br- transport was used as a marker activity of the apical membrane, which enabled the identification and purification of a membrane fraction that is highly resolved from the basolateral membrane. The most active fraction was purified approximately 50-fold compared with the crude homogenate. In this fraction, the specific activity of electrogenic anion transport was 296 +/- 87 nmol X mg protein-1 X min-1, whereas the ATPase was only 17.6 +/- 5.7 mumol X mg protein-1 X h-1, representing about a 4-5% contamination of the apical fraction with the basolateral membrane. PMID- 3022591 TI - Ovine lentivirus lymphoid interstitial pneumonia. Rapid induction in neonatal lambs. AB - For examination of the characteristics of lentivirus-induced pulmonary disease in an animal model, neonatal lambs were given intratracheal injections of high-and low-passage ovine lentivirus (OvLV) isolates. In 6 of 6 lambs inoculated with low passage OvLV or OvLV from lung lavage fluid, lesions of lymphoid interstitial pneumonia (LIP) developed. In none of 7 lambs inoculated with a high-passage OvLV or 4 control lambs inoculated with medium alone or ultrafiltered lung fluid did lung lesions develop. Systemic distribution of lentivirus was greater and development of lentivirus antibody was more rapid in lambs inoculated with low passage OvLV, compared with lambs inoculated with high passage OvLV. The number of lymphocytes in bronchoalveolar lavage samples was increased in lambs with lymphoid interstitial pneumonia. The development of lymphoid interstitial pneumonia was markedly accelerated, in comparison with previous reports of experimentally induced lentivirus pneumonia in sheep. In lentivirus-inoculated lambs pulmonary lesions developed comparable to lymphoid interstitial pneumonia associated with the acquired immunodeficiency syndrome and other human benign lymphoid disorders of the lung. Similarities between the disease manifestations and virologic properties of OvLV and human T-cell lymphotropic virus III argue for the relevance of OvLV-induced disease as a model for human retrovirus diseases. The ability of OvLV to cause accelerated pulmonary disease in neonates may be due to age-related susceptibility factors that enhance the pathogenicity of lentiviruses. PMID- 3022593 TI - Forskolin, a tool for rat parotid secretion studies: 45Ca efflux is not related to cAMP. AB - In the present work, we investigated, by use of forskolin, whether adenosine 3',5'-cyclic monophosphate (cAMP) level and Ca movements were modulated sequentially or in parallel by the activation of the beta-adrenergic receptor in the rat parotid gland. Forskolin-induced [3H]protein secretion was dependent on external Ca, whereas isoproterenol-induced secretion was not. This effect was not due to a requirement of adenylate cyclase for Ca, since the cAMP level increase induced by forskolin was not Ca dependent. Furthermore isoproterenol induced 45Ca efflux, whereas forskolin did not. 45Ca efflux was correlated neither to cAMP nor to secretion, since when there was a massive augmentation of cAMP there was no change in 45Ca efflux, and forskolin, which induced much secretion, was unable to induce Ca efflux. Carbachol potentiated the secretion induced by forskolin in the absence of Ca, whereas it did not potentiate the isoproterenol-induced response. From these results we suggest that beta-adrenergic receptor activation would lead to two parallel events, cAMP accumulation and Ca movements, which together would lead to maximal secretion. PMID- 3022594 TI - Use of monoclonal antibodies to culture rat proximal tubule cells. AB - Current renal cell culture techniques are limited by either a low yield of cells or by heterogeneity of cell types. We have used monoclonal antibodies to microvillus membrane proteins to isolate and culture a pure population of proximal tubule cells. The cells were characterized as proximal tubule cells by phase microscopy, enzyme histochemistry for alkaline phosphatase, butyrate esterase, and gamma-glutamyltransferase, electron microscopy, and specific reactivity with a variety of monoclonal antibodies for proximal tubule cells. Growth over 2-7 days yielded cell numbers up to 1,000-fold greater than obtained by single tubule microdissection. Dome formation was observed, suggesting intact fluid transport. In addition, Na+-H+ exchange and Na+-dependent D-hexose transport, known transport processes of the proximal tubule, were demonstrated by microfluorimetry of single cells and methyl-alpha-D-glucopyranoside uptake, respectively. Our results indicate that large numbers of homogeneous, cultured rat proximal tubule cells that maintain characteristics of in vivo proximal tubule cells can be obtained using a monoclonal antibody technique of isolation. PMID- 3022595 TI - Adenosine 3',5'-cyclic monophosphate stimulates chloride secretion in A6 epithelia. AB - Basal and aldosterone-stimulated short-circuit current (Isc) of A6 epithelia are known to be equivalent to net apical to basal Na flux and are completely inhibited by 0.05 mM amiloride added to the solution bathing the apical surface of the epithelium. In the absence of amiloride, the Isc stimulated by adenosine 3',5'-cyclic monophosphate (cAMP) is also equivalent to net apical to basal Na flux. However, amiloride does not completely inhibit the cAMP-stimulated Isc. In this study, the cAMP-stimulated, amiloride-insensitive Isc was characterized, using vasopressin or forskolin to raise cell cAMP. After basal Isc is inhibited by amiloride, forskolin stimulates Isc, conductance, and bidirectional 36Cl flux. Stimulation of Isc depends on the presence of both Na and Cl; stimulation of conductance depends on the presence of Cl. 36Cl flux studies showed that the cAMP stimulated, amiloride-insensitive Isc is equivalent to net Cl flux. It is inhibited by ouabain and by furosemide or bumetanide added to the solution bathing the basal surface of the epithelium. In view of the effect of cAMP in some other epithelia, we suggest that cAMP activates apical membrane Cl channels that are in series with a Na-K-Cl cotransporter in the basolateral plasma membrane. PMID- 3022596 TI - Isolation and characterization of a Na-H antiporter-deficient mutant of LLC-PK1 cells. AB - LLC-PK1 is an established cell line derived from pig kidney epithelium; it differentiates in vitro to form a polarized epithelial sheet, capable of the vectorial transport of solutes and water. We have used a modification of the "proton-suicide" method of Pouyssegur et al. (Proc. Natl. Acad. Sci. USA. 81:4833 4837, 1984) to isolate a mutant of LLC-PK1 cells that is deficient in Na-H antiporter activity. The mutant grows normally at pH 7.0 and above in the presence and absence of bicarbonate; at pH 6.5, however, it requires bicarbonate for growth. Alkalinization of the cytoplasm by the Na-H antiporter thus appears to be essential for growth at acidic pH in the absence of bicarbonate. The antiporter is also essential for the formation of domes (a consequence of vectorial water transport) in the absence of bicarbonate. PMID- 3022597 TI - Evidence for reflex adrenergic inhibition of acid secretion in the conscious rat. AB - In conscious gastric fistula rats, gastric distension with saline to a pressure of 7 cm caused a threefold reduction of basal gastric acid secretion. Distension with 6.25% peptone solution to the same pressure doubled basal acid secretion. The saline distension-induced inhibition was abolished by guanethidine and markedly reduced by propranolol; phentolamine was ineffective. The response to peptone was unaffected by guanethidine. The results suggest that in the rat, gastric distension at physiological pressures inhibits acid secretion by a beta adrenergic reflex. The inhibition can be masked by concurrent chemical stimulation of the gastric mucosa by the digestion products of food. PMID- 3022599 TI - Influence of phenobarbital on the hepatic handling of [3H]vitamin D3 in the dog. AB - The effect of phenobarbital (PB) on the hepatic handling of vitamin D3 (D3) and its metabolism to 25-hydroxyvitamin D3 [25(OH)D3] was studied in eight mongrel dogs. The hepatic uptake and clearance of [3H]D3 were evaluated by the multiple indicator-dilution curve technique, and the formation of [3H]25(OH)D3 was evaluated by sampling the hepatic effluent. The hepatic enzyme induction was assessed in six dogs by the 14CO2 breath excretion test. The results show that the hepatic uptake of [3H]D3 was not significantly affected but that its hepatic clearance was significantly increased during PB treatment. The [3H]25(OH)D3 production was increased during PB administration by a factor of 3-5 times over the pre- or post-PB period. Evaluation of the enzyme induction produced by PB revealed that two of the dogs studied had a blunted response to PB; furthermore, these dogs were the only animals that showed no increase in [3H]25(OH)D3 production during PB treatment and that in the presence of similar serum PB, endogenous 25(OH)D3 and 1,25-dihydroxyvitamin D3 pools, and hepatic uptake and clearance of [3H]D3. Strong positive correlation coefficients were observed between the breath excretion of 14CO2 and the [3H]25(OH)D3 production during PB treatment, whereas no correlation was present in the absence of PB. These observations show that, in most animals, PB is accompanied by an increased hepatic clearance of [3H]D3 and by an increased production of [3H]25(OH)D3. The data obtained during the present study also show that the response to PB is heterogeneous and that some animals escaped PB-mediated enzyme induction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022598 TI - Dietary potassium modulates active potassium absorption and secretion in rat distal colon. AB - To determine the effect of variations in body stores of potassium on the rate of active potassium transport in the large intestine, unidirectional 42K fluxes were performed under short-circuit conditions across isolated distal colonic mucosa of control, dietary potassium-depleted and dietary potassium-loaded rats. Potassium depletion stimulated net potassium absorption (JK net) (0.87 +/- 0.19 vs. 0.49 +/ 0.04 mu eq X h-1 X cm-2, P less than 0.025) due to a 40% increase in mucosal-to serosal potassium transport (JK m----s). In sodium-free Ringer solution JK net in the potassium-depleted group was also significantly greater than in controls (1.93 +/- 0.26 vs. 1.01 +/- 0.11 mu eq X h-1 X cm-2, P less than 0.005). In contrast, in chloride-free Ringer solution JK net was identical in the control and potassium-depleted groups (0.39 +/- 0.05 vs. 0.46 +/- 0.07 mu eq X h-1 X cm 2, P = NS). Potassium loading reversed net potassium absorption to net potassium secretion (-0.76 +/- 0.08 mu eq X h-1 X cm-2, P less than 0.001) as the result of a decrease in JK m----s and an increase in serosal-to-mucosal potassium transport (JK s----m). Net potassium secretion was abolished in the absence of either sodium or chloride from the bathing solution but not by mucosal amiloride. In sodium-free Ringer solution JK net was similar in control and potassium-loaded groups, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022600 TI - Taurocholate transport and Na+-K+-ATPase activity in fetal and neonatal rat liver plasma membrane vesicles. AB - The ontogenesis of Na+-K+-ATPase activity and Na+-taurocholate cotransport was studied in basolateral plasma membrane vesicles from fetal and neonatal rat liver. Membrane vesicles from each age group were 30-fold enriched in the basolateral marker enzyme Na+-K+-ATPase, 4- to 7-fold enriched in the bile canalicular membrane marker enzymes alkaline phosphatase and Mg2+ ATPase, and not significantly enriched in activities of marker enzymes for intracellular organelles. Na+-K+-ATPase activity was significantly lower in basolateral membranes from late fetal (day 21-22) and neonatal (day 1) rat liver. Kinetic analysis of Na+-K+-ATPase activity at various concentrations of ATP revealed that the maximum velocity of enzyme reaction (Vmax) for Na+-K+-ATPase was 70 and 90% of adult activity in the fetus and the neonate, respectively. The ATP Km was significantly lower in the neonate than the adult, suggesting a higher affinity of the neonatal enzyme for ATP. In contrast to the early maturation of Na+-K+ ATPase, transport of taurocholate was markedly lower in both fetal and neonatal vesicles compared with the adult. Taurocholate uptake on day 19 of gestation did not differ in the presence of a Na+ or K+ gradient, and uphill transport, as indicated by an overshoot, did not occur. On day 20 taurocholate uptake was stimulated by a Na+ compared with a K+ gradient, and accumulation of isotope above equilibrium was demonstrated. Na+-dependent transport of taurocholate by late fetal (day 22) and neonatal vesicles was saturable but the Vmax at each age was significantly lower and the apparent Km higher in developing compared with adult membrane vesicles.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022601 TI - Suggestion of a role for calmodulin and phosphorylation in regulation of rabbit ileal electrolyte transport: effects of promethazine. AB - Suggestion of a role for protein phosphorylation in the regulation of intestinal active NaCl transport was found by studying the effects of low concentrations of promethazine on Ca2+-calmodulin (CaM)-dependent protein phosphorylation of ileal microvillus membranes and on active ileal electrolyte transport. Ca2+-CaM increased the phosphorylation of six microvillus peptides (Mr 137,000, 116,000, 77,000, 58,000, 53,000, and 50,000) in a concentration-dependent manner. Promethazine inhibited the Ca2+-CaM-induced increases in each of these phosphorylations. The effect of promethazine was concentration dependent, with concentrations of 5-12 microM (mean 8 microM) causing 50% inhibition. Promethazine also caused a concentration-dependent increase in net Cl absorption and decrease in the ileal short-circuit current, with 9 microM promethazine causing a change in short-circuit current 50% of maximum. The promethazine effect on microvillus membrane phosphorylation was specific, since neither cAMP- and cGMP-induced phosphorylation in the microvillus membrane nor the stimulation by Ca2+-CaM of myosin light chain kinase phosphorylation of myosin light chain were affected by promethazine. The similar, and unusual sensitivity to low concentrations of promethazine on ileal microvillus membrane phosphorylation increased by Ca2+-CaM and on ileal electrolyte transport is consistent with Ca2+ CaM-dependent microvillus membrane phosphorylation being involved in the regulation of active electrolyte transport in ileal absorptive cells. PMID- 3022602 TI - Reactive oxygen species: production and role in the kidney. AB - Reactive oxygen species (ROS) are formed by incomplete reduction of molecular oxygen. They include superoxide anion (O2-.), hydrogen peroxide (H2O2), hydroxyl radical (OH.), and singlet oxygen (1O2). ROS may induce different types of cell injury, particularly lipid peroxidation and membrane damage. ROS have been shown to play an essential role in the mechanisms of experimental models of several renal diseases: ischemic acute renal failure, renal graft rejection, acute glomerulonephritis, and toxic renal diseases. They are produced by the renal cells and also by the inflammatory bone marrow-derived cells invading the renal tissue. ROS, regardless of their origin, may degrade the glomerular basement membrane and alter the glomerular and tubular cell functions. Particularly, they produce an increase in cyclic AMP synthesis and prostaglandin production in the glomeruli. Recent studies have shown that the glomerular mesangial cells themselves generated ROS on stimulation by phagocytosis of foreign particles or exposure to the complement membrane attack complex or platelet-activating factor. Production of ROS is in narrow relationship with the metabolism of arachidonic acid. Conversion of this fatty acid via the lipoxygenase pathway is associated with an increase of ROS, whereas its transformation into prostaglandins via the cyclooxygenase pathway results in the opposite effect. Production of ROS in activated mesangial cells can be inhibited by glucocorticoids via a receptor mediated mechanism. The fact that some of these characteristics are different in leukocytes suggests the possibility in the future of the more specific pharmacological control of the inflammatory process in the glomerular mesangium. PMID- 3022603 TI - Effect of serotonin on prostaglandin synthesis in rat cultured mesangial cells. AB - Serotonin (5-hydroxytryptamine) (5-HT) is a potent vasoactive amine that reduces renal blood flow and glomerular filtration rate. Vasodilator prostaglandins (PGs) modulate the effects of several vasoconstrictors on the renal circulation. Since mesangial cells are smooth muscle-like cells that may regulate glomerular hemodynamics, we studied the effect of 5-HT on PGs synthesis in rat cultured mesangial cells. 5-HT (10(-6)-10(-3) M) resulted in progressive stimulation of prostaglandin E2 (PGE2) synthesis. Significant stimulation in response to 10(-4) M 5-HT started after 2 min of incubation and progressively increased for at least 30 min. This effect was structurally specific for the 5-HT receptor since indole containing precursors and metabolites of 5-HT as well as the aminergic compounds, adenosine, and dopamine were without effect. Moreover, 5-HT receptor antagonists, but not histaminergic or beta-adrenergic antagonists, abolished 5-HT-stimulated PGE2 synthesis. 5-HT also stimulated prostacyclin (measured as 6 ketoprostaglandin F1 alpha) but not thromboxane synthesis in the same cell cultures. 5-HT-stimulated PGE2 synthesis was not affected by extracellular calcium depletion but was abolished by preincubating the cells with the intracellular calcium antagonist 8-(N,N-diethylamine)-octyl-3,4-5 trimethoxybenzoate (10(-5) M). These studies show that 5-HT stimulates PGE2 and prostacyclin (PGI2) synthesis in mesangial cells via a mechanism dependent on intracellular calcium. These vasodilator PGs may modulate the effect of 5-HT on renal and specifically glomerular hemodynamics. PMID- 3022604 TI - In vitro stimulation of Na-K-ATPase in rat thick ascending limb by dexamethasone. AB - Dexamethasone has been reported to stimulate Na-K-ATPase activity in the medullary thick ascending limb of adrenalectomized animals within a few hours. The present study was aimed at characterizing the mechanism of this action by investigating the stimulatory effect of the hormone in vitro. Dexamethasone (10( 8) M) added in vitro to segments of the medullary thick ascending limb of Henle's loop, which were microdissected from adrenalectomized rats, restored in a dose dependent manner the depressed Na-K-ATPase activity within one h of incubation. This stimulation of Na-K-ATPase was inhibited by cycloheximide and actinomycin D. Dexamethasone also stimulated the component of oxidative metabolism coupled to sodium transport. These results, which confirm previous in vivo observations, demonstrate that dexamethasone-induced stimulation of Na-K-ATPase is a direct tubular action of the hormone mediated by protein synthesis. They suggest that this short-term effect of dexamethasone corresponds to the stimulation of sodium reabsorption by the dilution segment. PMID- 3022605 TI - Fluctuations in intracellular pH associated with vasopressin stimulation. AB - Intracellular pH (pHi) was estimated in paired hemibladders isolated from Dominican toads (Bufo marinus) by the tissue distribution of [14C]5,5'-dimethyl 2,4-oxazolidinedione and [3H]inulin. Tissues were incubated with isotopes for 30 min to correlate changes in pHi with the approximate time of peak vasopressin (VP)-induced water flow. At serosal pH 7.1 in the presence of an osmotic gradient, the intracellular hydrogen ion concentration [( H+]i) after 30 min of VP (20 mU/ml) stimulation was 8.29 +/- 0.23 X 10(-8) M (pHi 7.08) compared with 5.19 +/- 0.46 X 10(-8) M (pHi 7.28) in unstimulated paired controls (n = 5, P less than 0.001). The cyclic AMP (cAMP) analogue 8-(p-chlorophenylthio)-cAMP (10( 5) M) mimicked the VP effects. A similar change was observed at serosal bath pH 8.2, where [H+]i was 1.67 +/- 0.06 X 10(-8) M (pHi 7.78) with VP vs. 1.11 +/- 0.04 X 10(-8) M (pHi 7.95) in matched controls (n = 8, P less than 0.001). In all cases, the hydroosmotic response was associated with a significant decrease in inulin space. When the osmotic gradient was eliminated with Ringer solution or isotonic sorbitol in the mucosal bath, VP produced a smaller decrease in pHi (approximately 0.08 pH units) at both serosal pH. 31P-nuclear magnetic resonance spectra showed a similar downward trend in pHi with cell swelling. When vasopressin was removed from the bath, pHi and inulin space in stimulated hemibladders returned to pretreatment values within 30 min, and the tissues were again capable of a maximum hydroosmotic response if rechallenged with the hormone.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022606 TI - Pulmonary vasoactive effects of exogenous and endogenous AVP in conscious dogs. AB - Our objectives were to investigate the extent to which both exogenously administered and endogenously released arginine vasopressin (AVP) exert a direct, vasoactive influence on the pulmonary circulation of conscious dogs. Multipoint pulmonary vascular pressure-cardiac index (P/Q) plots were constructed during normoxia in conscious dogs by stepwise constriction of the thoracic inferior vena cava (IVC) to reduce Q. In intact dogs, AVP infusion (7.6 ng X kg-1 X min-1 iv) increased (P less than 0.01) plasma AVP from 2.3 +/- 0.4 to 280 +/- 23 pg/ml, and increased (P less than 0.01) the pulmonary vascular pressure gradient (pulmonary arterial pressure minus pulmonary capillary wedge pressure, PAP-PCWP) over the entire range of Q studied. Following administration of autonomic nervous system antagonists and a converting-enzyme inhibitor, exogenous AVP again increased (P less than 0.01) PAP-PCWP over the entire range of Q. Generation of P/Q plots via IVC constriction resulted in systemic hypotension (58 +/- 4 mmHg) and a concomitant increase (P less than 0.01) in endogenous AVP release from 2.1 +/- 0.2 to 109 +/- 22 pg/ml. Following administration of the specific AVP receptor antagonist [d(CH2)5]AVP (10 micrograms/kg iv), systemic arterial pressure, but not PAP - PCWP, was decreased to significantly lower levels as Q was reduced during IVC constriction. A similar response was observed in dogs pretreated with the neurohumoral blockers. Thus exogenous administration of AVP results in active, non-flow-dependent pulmonary vasoconstriction. This effect is not dependent on reflex activation of the autonomic nervous system or on the increased production of angiotensin II.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022607 TI - Alpha 1-adrenergic stimulation of rat myocardial cells increases protein synthesis. AB - The effects of adrenergic stimulation on the rates of protein synthesis, degradation, and accumulation were examined in primary cultures of neonatal rat heart cells. Treatment of myocardial cells with norepinephrine increased total cellular protein content and the rate of incorporation of radiolabeled tyrosine into trichloroacetic acid insoluble protein. alpha 1-Adrenergic, but not alpha 2- or beta-adrenergic blockade, inhibited these norepinephrine induced increases. The rate of protein synthesis estimated from the kinetics of equilibrium labeling and from combined equilibrium and pulse labeling was increased by norepinephrine stimulation, whereas protein degradation estimated by release of previously incorporated radiolabeled tyrosine or in pulse-chase experiments was unaffected. To determine whether alpha 1-adrenergic stimulation produced similar effects on the turnover of myofibrillar proteins, rates of synthesis and degradation were estimated for a myofibrillar-enriched protein fraction and for myosin heavy chain and actin. Norepinephrine treatment produced increases in the synthesis of myofibrillar protein without significantly altering degradation rates. These experiments suggest that alpha 1-adrenergic stimulation increases myocardial cell protein content by accelerating protein synthesis. PMID- 3022608 TI - Renal vasodilatation after inhibition of renin or converting enzyme in marmoset. AB - The relative importance of angiotensin II for the renal vasodilatory response after converting-enzyme inhibition was evaluated by a comparison of the effects of converting-enzyme and renin inhibition on renal vascular resistance. Renal, mesenteric, and hindquarter blood flows were measured with chronically implanted ultrasonic-pulsed Doppler flow probes in conscious, mildly volume-depleted marmosets after administration of a converting-enzyme inhibitor (enalaprilat, 2 mg/kg iv), a synthetic renin inhibitor (CGP 29,287, 1 mg/kg iv), or a renin inhibitory monoclonal antibody (R-3-36-16, 0.1 mg/kg iv). Enalaprilat reduced blood pressure (-16 +/- 4 mmHg, n = 6) and induced a selective increase in renal blood flow (27 +/- 8%, n = 6). CGP 29,287 and R-3-36-16 induced comparable reductions in blood pressure (-16 +/- 4 mmHg, n = 6 and -20 +/- 4 mmHg, n = 5, respectively) and selective increases in renal blood flow (36 +/- 12%, n = 6 and 34 +/- 16%, n = 4, respectively). The decrease in renal vascular resistance was of similar magnitude for all of the inhibitors (enalaprilat -28 +/- 3%, CGP 29,287 -32 +/- 6%; and R-3-36-16 -33 +/- 7%). These results indicate that the renal vasodilatation induced after converting-enzyme or renin inhibition is mainly due to decreased formation of angiotensin II. PMID- 3022609 TI - Total peripheral resistance during cardiac tamponade: adrenergic and angiotensin roles. AB - During progressive cardiac tamponade in conscious dogs, cardiac output falls continuously while arterial blood pressure is maintained until cardiovascular decompensation by increases in total peripheral resistance (TPR). Plasma renin activity (PRA) is known to increase at decompensation. We hypothesized that the increase in TPR during cardiac tamponade was mediated by alpha-adrenergic and renin-angiotensin mechanisms. Twelve adult dogs were instrumented to measure cardiac output (electromagnetic flow probe), aortic and right atrial blood pressures, and intrapericardial pressure (IPP). TPR was calculated as the conscious euvolemic animals underwent cardiac tamponade induced by intrapericardial saline infusion at 20 ml/min. Six dogs underwent cardiac tamponade in the control condition (no medications) and during independent alpha- and beta-adrenergic and angiotensin-converting enzyme (ACE) inhibition. PRA and angiotensin II (ANG II) were measured during control tamponade. We found that TPR increased continuously to levels of greater than 200% of base line as IPP rose during cardiac tamponade (P less than 0.01). This increase in TPR was unaffected by beta-adrenergic or ACE blockade but was blunted by alpha-adrenergic blockade. PRA and ANG II increased only at decompensated tamponade (P less than 0.05) when arterial blood pressure had fallen by 30%. These changes in PRA and ANG II during tamponade were not altered by beta-blockade in six separate animals. We conclude that cardiac tamponade stimulates renin release and ANG II generation by a non beta-receptor-mediated mechanism. The increase in TPR during cardiac tamponade is primarily dependent on alpha-adrenergic mechanisms, with a limited late contribution from the renin-angiotensin system. PMID- 3022610 TI - Caudolateral areas of medulla-mediating release of ACTH in cats. AB - The effect of electrical stimulation of the caudolateral brain stem on plasma adrenocorticotropin (ACTH) was assessed in cats anesthetized with alpha chloralose-urethan. To examine the influence of stimulus pattern on ACTH release, an equal number of pulses was presented in a continuous pattern and in a burst pattern at each electrode site. Stimulation of the magnocellular portion (layers 4-6) of trigeminal nucleus caudalis evoked a significant (P less than 0.01) and equal peak change in plasma ACTH after continuous pattern (+121 +/- 32 pg/ml) and after burst pattern stimuli (+126 +/- 30 pg/ml, n = 21). In contrast, stimulation of more ventromedial portions (layers 7-8) of nucleus caudalis had no significant effect on plasma ACTH. Stimulation of the trigeminal lateral cervical region the caudal extent of the A1 noradrenergic cell group, or the lateral reticular nucleus evoked significant peak increases in plasma ACTH regardless of stimulus pattern. Transient changes in arterial pressure accompanied brain stem stimulation and were not correlated with the changes in ACTH. The results indicate that stimulation of trigeminal subnucleus caudalis, a brain stem region that processes nociceptor afferent information, evokes a prompt increase in plasma ACTH. Stimulation of brain stem regions that process autonomic and cardiovascular afferent information (A1 region, lateral reticular nucleus) also facilitate ACTH release. No significant influence of stimulus pattern on brain stem-evoked ACTH release was seen. The results support the hypothesis that the influence of the central nervous system on ACTH release may be processed by parallel pathways at the caudal brain stem level. PMID- 3022611 TI - Sodium absorption coupled to ammonia excretion in osmoconforming marine invertebrates. AB - Evidence was sought for the presence of amiloride-sensitive Na-NH4 and NaH exchange systems in four species of marine osmoconformers. When the crabs Cancer antennarius and Petrolisthes cinctipes were in seawater (SW), amiloride (10(-4) M) reduced NH3 efflux by approximately 33 and 60%. Inhibition was reversible on removing the amiloride. In dilute (60%) SW, inhibition of NH3 output by C. antennarius was even greater than in SW. Na+ uptake by P. cinctipes was reduced by approximately 20% in the presence of amiloride (the measurement was not made on C. antennarius). Amiloride had no effect on proton efflux in either crab. The data suggest that Na-NH4 exchange occurs in these animals but that Na-H exchange does not occur. The inhibitor had no effect on NH+4 excretion by the polychaete worm Nephtys caecoides and the mussel Mytilus californianus; it was also without effect on proton output by the worm. The data suggest that the exchange systems are absent from these animals. Implications of these observations for the evolution of such cation exchange systems are discussed. PMID- 3022612 TI - The corticotropin-releasing hormone stimulation test in chronic schizophrenia. AB - During a drug-free period a group of schizophrenic subjects (N = 9) showed normal mean basal plasma ACTH and cortisol levels in association with normal plasma ACTH and cortisol responses to an infusion of corticotropin-releasing hormone (CRH). Administration of fluphenazine had no effect on basal ACTH and cortisol levels or their responses to CRH (N = 8). These data differ from those previously reported in depressed patients, who showed elevated basal cortisol values in association with a blunted ACTH response to CRH, and add to a growing body of literature which suggests that hypothalamic-pituitary-adrenal regulation is less disturbed in schizophrenia than in depression. PMID- 3022614 TI - Mammary fibroadenoma with multinucleated stromal giant cells. AB - We have studied an otherwise typical mammary fibroadenoma in a 42-year-old female in which numerous, bizarre, mitotically inactive, multinucleated giant cells were present throughout the stroma. Immunohistochemical and ultrastructural studies confirmed the mesenchymal, specifically fibroblastic, nature of the giant cells. Cells of this type, which are more commonly an incidental finding within the interlobular stroma of the breast, are benign, and should not be mistaken for malignant cells on microscopic examination. PMID- 3022613 TI - Virilizing Leydig cell adenoma of adrenal gland. AB - We describe the third report testosterone-producing, virilizing, adrenal Leydig cell adenoma, which was identified in a oophorectomized postmenopausal female patient. Light- and electron-microscopy demonstrated typical Leydig cell differentiation, including numerous intracytoplasmic and intranuclear Reinke crystals and rice-like bodies (elementary tubular inclusions). Testosterone production by the adenoma was demonstrated by the immunoperoxidase technique. We propose that adrenal Leydig cell adenomas arise as a result of ovarian gonadal stromal metaplasia associated with elevated follicle-stimulating hormone and luteinizing hormone in the postmenopausal female patient. Adrenal Leydig cell lesions must be considered in the virilized woman with elevated testosterone levels and normal levels of the adrenal androgens and their 17-ketosteroid metabolites. PMID- 3022616 TI - ATP sulfurylase-dependent assays for inorganic pyrophosphate: applications to determining the equilibrium constant and reverse direction kinetics of the pyrophosphatase reaction, magnesium binding to orthophosphate, and unknown concentrations of pyrophosphate. AB - A continuous, coupled, spectrophotometric assay is described in which the enzyme ATP sulfurylase is employed to measure the concentration of inorganic pyrophosphate (PPi) at equilibrium with known concentrations of inorganic orthophosphate (Pi) in the presence of excess inorganic pyrophosphatase (PPitase). In agreement with previous reports, the apparent equilibrium constant (Keq,app) of the PPi hydrolysis reaction was shown to decrease as the concentration of Mg2+ is increased. At pH 7.3, 30 degrees C, in the presence of 150 mM NaCl and 1 mM free Mg2+, Keq,app (calculated as [Pi]t2/[PPi]t) was 1950. Measurements of Keq,app at different total concentrations of Mg2+ and Pi permitted the determination of K0, the dissociation constant of the Mg-Pi complex. In 0.05 M Tris-Cl, pH 8.0, at 30 degrees C, K0 was 3.6 mM. In the presence of excess ATP sulfurylase, yeast PPitase catalyzed PPi formation from Pi with a specific activity (Vmax) of 9 units X mg protein-1 at pH 8.0, 30 degrees C, and 1 mM free Mg2+. Half-maximum reverse reaction velocity was observed at a total Pi concentration of 18 mM. (Under the same conditions, Vmax of the PPi hydrolysis reaction was 530 units X mg protein-1.) A radiochemical end point ("reaction-to-completion") assay for measuring unknown concentrations of PPi was devised. In the presence of excess 35S-adenosine-5'-phosphosulfate ([35S]APS) as the cosubstrate, 35SO2-4 formation was stoichiometric with added PPi. (The 35SO2 4 and [35S]APS are separated by adsorption of the latter onto charcoal.) The sensitivity of the assay can be adjusted by varying the specific radioactivity of the [35S]APS. In the absence of interfering substances, as little as 2 pmol of PPi per 1.0 ml assay volume can be measured. The sensitivity of the assay is reduced in the presence of ATP plus perchlorate (which synergistically inhibit the enzyme). However, if the bulk of the ATP is removed from perchloric acid extracts of tissues with glucose and hexokinase, initial intracellular levels as low as 1 microM can be measured. The possibility that most of the cellular PPi extracted with perchloric acid was originally enzyme bound is discussed. PMID- 3022615 TI - Acute poisoning with gold cyanide. AB - A case of deliberate ingestion of an electroplating solution containing gold cyanide is described. Despite the use of an antidote, and supportive treatment for cyanide poisoning, the patient died after 13 hours. Sublethal cyanide and high red blood cell gold levels suggest acute gold toxicity as the most likely cause of death. Evidence for this is discussed and recommendations are made for the treatment of cyanide poisoning. PMID- 3022617 TI - Determination in urine of diisocyanate-derived amines from occupational exposure by gas chromatography-mass fragmentography. PMID- 3022618 TI - Significance of the increase in glucose 6-phosphatase activity in skeletal muscle cells of the mouse by starvation. AB - The effects of starvation on glucose 6-phosphatase (G6Pase; EC 3.1.3.9., D glucose 6-phosphate phosphohydrolase) and glycogen phosphorylase (EC 2.4.1.1.) activities, and on glycogen content, were studied in skeletal muscles (m. rectus femoris) of mice. In the muscle cells from fed animals, the cytochemical reaction product for G6Pase activity was observed in moderate amounts in the terminal cisternae of sarcoplasmic reticulum and in small amounts in the nuclear envelope, and was rare or absent in the intermyofibrillar sarcoplasmic reticulum. After 4 days of starvation, however, the reaction product became abundant in all of the terminal cisternae, intermyofibrillar sarcoplasmic reticulum, and nuclear envelope. Biochemical G6Pase and glycogen phosphorylase a (active form) activities were higher in the muscles of starved mice than in those of fed animals. The glycogen content decreased markedly in the muscles of starved mice. The results suggest that the role of the increased G6Pase in skeletal muscle cells of starved mice is to release glucose into the blood by hydrolyzing glucose 6-phosphate produced through the increased phosphorylase activity. PMID- 3022620 TI - Opiate antagonist in traumatic shock. AB - In experimental animal studies, opiate receptor antagonists (such as naloxone) and physiological opiate antagonists (thyrotropin-releasing hormone [TRH] have been used with some success to improve outcome and physiological variables following traumatic shock associated with hypovolemia, spinal cord trauma, and head injury. Naloxone at high doses (in the mg/kg range) improves blood pressure and survival following hypovolemic shock in some species subjected to fixed pressure shock. Similarly, naloxone treatment in the same dose range improves blood pressure and outcome following traumatic spinal shock as well as shock associated with traumatic head injury in selected animal models. The high doses of naloxone required in these studies suggest that the beneficial effects may be due to actions at relatively naloxone-insensitive opiate receptors, such as the kappa-receptor. Changes in the putative kappa-receptor ligand dynorphin are found after hypovolemic shock and traumatic injury to the brain or spinal cord. Opiate receptor antagonists with increased selectivity for the kappa-receptor may be superior to naloxone in the treatment of these conditions. TRH or TRH analogs similarly improve blood pressure and outcome following hypovolemic or spinal shock. Clinical trials of naloxone (at high doses) in human spinal cord injury have begun, and there are plans for clinical trials of naloxone in human head trauma and of TRH in human spinal cord injury. PMID- 3022619 TI - The benzodiazepine receptor. AB - The benzodiazepines are among the most widely used drugs in the world. When first introduced, little was known about their mechanism of action. However, in the last 20 years, our understanding of the chemistry and function of the central nervous system (CNS) has increased substantially. This knowledge has shed some light on the mechanism of action of the benzodiazepines and other centrally acting drugs. It is well established that the benzodiazepines act by combining with specific receptors in the central nervous system. These receptors are anatomically in close association with gamma amino butyric acid (GABA) receptors and appear to reside on the neuronal membrane in the same supramolecular protein complex. GABA is the major inhibitory neurotransmitter of the CNS. The benzodiazepines act by increasing the affinity of the GABA receptor for its ligand, thereby augmenting the inhibitory effect of a given concentration of GABA. Two hypotheses of benzodiazepine ligand-receptor interactions in this supramolecular protein complex have been proposed: (1) multiple receptor subtypes analogous to the opioid receptors; (2) single receptor with multiple conformations. The multiple receptor hypothesis suggests that each pharmacologic effect of the benzodiazepines (i.e., anxiolysis) is mediated by interaction with a specific receptor subtype. On the other hand, the alternative hypothesis suggests that only one receptor exists which has a dynamic conformation. Experimental evidence in support of each hypothesis is presented and critically evaluated. PMID- 3022621 TI - Detection of transmissible gastroenteritis virus in feces from pigs by reversed passive hemagglutination. AB - A reversed passive hemagglutination (RPHA) method was developed for the detection of transmissible gastroenteritis (TGE) virus in the fecal specimens from pigs. Ovine erythrocytes fixed with glutaraldehyde and treated with tannic acid were coated with anti-TGE virus swine antibodies, which were purified by affinity chromatographic technique linked with purified TGE virus. The RPHA test was done by the Microtiter method. Erythrocytes coated with purified specific antibodies were agglutinated by TGE virus, but not by porcine rotavirus or porcine enterovirus. The reaction was specifically inhibited by antiserum against TGE virus, confirming the specificity of the reaction. A litter of seven 3-day-old pigs was orally inoculated with TGE virus, and fecal specimens were obtained once a day and serum was obtained every 4th day. With the RPHA test, TGE virus was detected in the diarrheal feces; all of the inoculated pigs developed virus neutralization antibody for the TGE virus. The RPHA test detected TGE virus in feces from pigs with naturally occurring diarrhea. The RPHA test detected TGE virus in 5 of 6 fecal specimens (80%), whereas the positive rate was only 50% (3/6) for the immunofluorescent staining of primary cultures of porcine kidney cells inoculated with the specimens. The advantages of the RPHA method are simplicity, high sensitivity, and rapid to do. PMID- 3022622 TI - Restriction endonuclease analysis of various strains of Mycobacterium paratuberculosis isolated from cattle. AB - Deoxyribonucleic acid (DNA) preparations from 3 reference strains of Mycobacterium paratuberculosis and from 23 isolates of M paratuberculosis obtained from cattle in New Zealand were characterized by restriction endonuclease analysis, using the enzymes BstE II, Pvu II, and Bcl I. Patterns of DNA fragments for strain 18 (one of the reference strains) differed markedly from patterns of other strains, indicating genetic differences between strain 18 and the other strains of M paratuberculosis evaluated. The other 2 reference strains (TMC 1613 and Weybridge strain 316) and all but 1 of the isolates from cattle had identical patterns with the 3 enzymes. These 2 reference strains differed from each other in their dependence on exogenous mycobactin, but this was not reflected in their restriction patterns. The single variant isolate from cattle had patterns identical to those of the other isolates, using Pvu II and Bcl I, and had only 1 fragment line difference with BstE II. Although close genetic homogeneity of cattle strains of M paratuberculosis prevented development of a typing system on the basis of restriction endonuclease analysis, the results provided a basis for genomic comparison with other closely related organisms. PMID- 3022624 TI - Genetic relatedness of disease-associated field isolates of bovid herpesvirus type 4. AB - Eight field isolates of bovid herpesvirus type 4 (BHV-4) were examined by restriction analysis and Southern blot hybridization with respect to their relatedness to one another and to the BHV-4 prototype strain DN-599. Isolates were obtained from cattle exhibiting a range of disease states including abortion, pneumonia, enteritis, metritis, and vaginal blisters. Initial growth studies of all 9 viruses were performed and revealed that the overall rate of virus growth was slow when compared with that of other herpesviruses. Infection with each virus also resulted in the formation of large fused cells, which in addition to the slow growth rate, indicated that the isolates were of the cytomegalovirus type. Further studies to characterize and compare the various BHV 4 isolates were undertaken by obtaining cell-free virus from infected cell populations. Viral isolates were purified and used as a source of BHV-4 DNA. Purified DNA, representing each of the 8 field isolates and the prototype strain DN-599, were each cleaved with 3 restriction enzymes and were separated by agarose-gel electrophoresis, and the resultant fragment patterns were compared. In general, genomic fragments of the field isolates corresponded to those generated by cleavage of DN-599 DNA, with the exception of the abortion associated isolate 83-3572. Additional minor differences were also seen between DN-599 DNA and DNA from the other field isolates, but the overall restriction patterns were similar. To confirm that all isolates were members of the BHV-4 type, hybridization studies were performed using DN-599.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022623 TI - Effect of suramin on serum viral replication in feline leukemia virus-infected pet cats. AB - Suramin treatments were administered (IV) to 2 healthy adult cats infected with naturally acquired feline leukemia virus. Serum viral infectivity--as assessed by focus induction by the method of Fischinger, using serial serum samples titrated on clone 81 cells--ceased transiently in both cats during treatment with suramin, but returned to significantly high levels approximately 14 days after treatment was stopped. Both cats tested positive for FeLV internal antigens in peripheral blood cells and serum before, during, and after treatment. Both cats tested negative for antibody to feline oncornavirus membrane antigen before, during, and after treatment. The major adverse effects of suramin in the 2 cats were transient vomiting and anorexia. PMID- 3022625 TI - Quantitative studies of erythropoiesis in the clinically normal, phlebotomized, and feline leukemia virus-infected cat. AB - Erythropoiesis was evaluated in 5 cats at base line with normal PCV and then in the same cats with anemia induced by phlebotomy and in 5 other cats with nonregenerative anemia from community-acquired feline leukemia virus (FeLV) infection. The hematologic evaluation included complete blood cell and reticulocyte counts, marrow morphologic features, determination of serum erythropoietin concentrations by radioimmunoassay, ferrokinetic studies, and in vitro marrow culture of early erythroid progenitors (erythroid burst-forming units; BFU-E) and late erythroid progenitors (erythroid colony-forming units; CFU E). Phlebotomized cats developed marrow erythroid hyperplasia and an increased reticulocyte count. Ferrokinetic studies revealed an increase in plasma iron turnover from 1.4 to 3.8 mg of Fe/dl of blood/day and RBC use from 50.4% to 78.5%. The mean CFU-E number and CFU-E/BFU-E ratio increased after phlebotomy, but the increase was not significant (P greater than 0.05). Serum erythropoietin values did increase significantly. In FeLV-infected cats, a nonregenerative anemia was demonstrated by marrow erythroid hypoplasia and a low total reticulocyte count. An increased percentage of rubriblasts and prorubricytes was observed in 4 of the 5 cats. Although serum erythropoietin values were high (321 +/- 123 mU/ml vs normal 14 +/- 1 mU/ml), ferrokinetic data revealed decreased erythropoiesis. Marrow culture studies in the FeLV-infected cats also revealed low numbers of BFU-E and CFU-E, but normal numbers of granulocyte-macrophage progenitors remained. Seemingly, the FeLV infection impaired the ability of feline marrow to respond physiologically to anemia. PMID- 3022626 TI - Digestion in horses after resection or ischemic insult of the large colon. AB - The effect of 60% resection of the large colon vs ischemic insult without resection on the ability of horses to digest grass hay was investigated. Digestion trials were performed on 9 horses before surgery (base line) and 3 weeks, 6 weeks, and 6 months after surgery. The percentage of apparent digestion of crude protein, crude fiber, nitrogen-free extract, calcium, phosphorus, magnesium, manganese, copper, and zinc was calculated. Horses that had resection (n = 5) had decreased apparent digestion of crude protein, crude fiber, and phosphorus 3 weeks after surgery, compared with those in horses with ischemic insults (n = 4) and with base-line values. Horses with ischemic insults also had a decrease in crude protein digestion 3 weeks after surgery, compared with base line values. All horses returned to base-line values of digestion at the 6-month trials, although horses that had resection had higher fecal concentrations of phosphorus and nitrogen-free extract than did horses with ischemic insult. During the study, all horses had maintained good body condition. PMID- 3022627 TI - Angiotensin-converting enzyme. Investigation of diurnal variation, the effect of a large dose of prednisolone, and prednisolone pharmacokinetics in patients with sarcoidosis. AB - Serial assay of serum angiotensin-converting enzyme concentrations (SACE) is advocated for monitoring disease progress in sarcoidosis. Because little is known of nondisease factors affecting SACE, 10 patients with histologically proved sarcoidosis were assessed for diurnal fluctuation in SACE and as to whether a large dose of corticosteroid had an immediate effect on SACE independent of disease. The pharmacokinetics of prednisolone in 8 of these patients was also evaluated. On Day 1, serum samples were obtained for 24 h after placebo, and the next day at the same times after 75 mg of orally administered prednisolone. There was no obvious diurnal pattern on either day, and there was no significant difference in SACE after prednisolone. The mean maximal difference obtained within or between days was 8.8%. Prednisolone pharmacokinetics were comparable to normal volunteers. SACE concentrations can be confidently determined at any time of day, and changes of greater than 9% are probably significant. The use of prednisolone in patients with sarcoidosis can be safely based upon pharmacokinetic data obtained from normal volunteers. PMID- 3022628 TI - Improvement in ventilation-perfusion relationships by almitrine in patients with chronic obstructive pulmonary disease during mechanical ventilation. AB - Although the respiratory stimulant effects of almitrine bismesylate (AB) via an action on the peripheral chemoreceptors have been demonstrated, the mechanism of its intrapulmonary action has not yet been elucidated. In order to abolish the stimulation of ventilation, observed in studies on spontaneously breathing patients, an investigation of patients suffering from severe COPD under constant mechanical ventilation, with FIO2 = 0.21, during the weaning period was carried out. Eighteen patients were randomly divided into 2 groups (9 receiving 1.5 mg/kg AB and 9 receiving placebo). The ventilatory and hemodynamic variables, blood and alveolar gases, and the VA/Q ratio distributions using the multiple inert gas technique were collected before treatment with drug or placebo, as well as 90 and 180 min afterwards. The PaO2 was found to be raised 90 min after AB administration (+57 +/- 3.9 mm Hg, p less than 0.01) and remained above the baseline value at 180 min (+5.4 +/- 4.6 mm Hg, p less than 0.01). Compared with those in the placebo group, these increases were significant (p less than 0.01). A slight decrease in PaCO2 but similar in the 2 groups was observed despite constant ventilation. The hemodynamic data were the same for the 2 groups. The changes in overall criteria of the distributions (mean VA/Q and SD) were small. The main finding was a decrease in the percentage of the perfusion flowing through the true shunt and the underventilated areas after AB treatment. In the control group, the blood flow percentage in the true shunt and low VA/Q units was either stable or increased.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022630 TI - Classification of infections with human immunodeficiency virus. PMID- 3022629 TI - The ovine corticotropin-releasing hormone stimulation test and the dexamethasone suppression test in the differential diagnosis of Cushing's syndrome. AB - We gave a standard dexamethasone suppression test and an ovine corticotropin releasing hormone (CRH) stimulation test to 41 patients with adrenocorticotrophic hormone (ACTH)-dependent hypercortisolism to determine the efficacy of each test in the differential diagnosis of Cushing's syndrome. Twenty-nine of thirty-three patients with Cushing's disease and 0 of 8 patients with ectopic secretion of ACTH responded to the ovine CRH test with increased levels of cortisol. When a cortisol response was judged as positive for Cushing's disease, the CRH test had a diagnostic sensitivity, specificity, and accuracy of 88%, 100%, and 90%, respectively. Twenty-nine patients with Cushing's disease and 1 patient with ectopic secretion of ACTH responded to the dexamethasone suppression test. A combined-test strategy requiring negative results from both tests to exclude a diagnosis of Cushing's disease yielded superior sensitivity (100%) and diagnostic accuracy (98%). Thus, the ovine CRH test works as well as the standard dexamethasone suppression test in discriminating between Cushing's disease and ectopic ACTH secretion. The diagnostic power of each test is enhanced when the two tests are combined. PMID- 3022631 TI - [Value of evoked sacral potentials in studying bladder sphincter disorders in peripheral neuropathies and central nervous system diseases. A study of 110 cases]. AB - The sacral spinal cord is one of the main sites of integration of bladder and anal sphincter and sexual function. Clinical tests (bulbocavernous reflex, perineal reflexes) are often inadequate and urodynamic investigations (cystomanometry, urecholine test, Susset's test) are sometimes unhelpful. The integrity of this structure is essential for normal sphincter function and disease at this level (or of its afferent or efferent limbs) produces a peripheral type of disorder. Sacral evoked potentials are an electrophysiological means of testing the bulbocavernous reflex by external stimulation of the dorsal nerve of the penis or clitoris and recording by a needle bipolar electrode in the striated muscle of the sphincter or the bulbocavernous muscle. An objective study of the sacral reflex are (S2, S3, S4) can thus be performed. One hundred and ten patients have undergone this investigation. Normal latency established in a series of 26 healthy volunteers was less than 42.1 ms. Prolongation of this interval is a sign of disease of the reflex are and correlated with urinary or sexual problems in 46 cases of clinical or electrical peripheral neuropathy. The reflex time was normal in all patients with CNS disease (32 cases) and an impotence of psychological origin (6 cases). PMID- 3022632 TI - Fine biogenic silica fibres in sugar cane: a possible hazard. PMID- 3022633 TI - [Effects of a diet with low fiber and thiamine contents on erythrocyte transketolase activity in goats]. AB - To test the effects of an insufficient fiber content, a high percentage of readily fermentescible carbohydrate and non protein nitrogen uptake on thiamin nutrition, three goats were submitted during periods of 40 or 90 days to three diets with low fiber contents (1: 16.53%; 2: 12.37%; 3: 12.84%) and low thiamin contents (1: 0.07; 2: 0.08; 3: 0.05' mg/kg). The diets were basically composed of beet pulp and equilibrated in protein either with copra meal or with urea. Diets 2 and 3 were enriched with 20% lactose. Animals had no access to coarse forage or litter. Variations of blood transketolase activity, pyruvicemia and lactacidemia were followed. Preliminary standard values of transketolase activity were determined with a hay diet (38 +/- 10 Ul/l total blood) or a grass diet (47 +/- 13 Ul/l). In the same way, no decrease of food intake or clinical signs of thiamin deficiency could be observed. In none of the cases were noted impairment of blood transketolase activity or increase in lactacidemia or pyruvicemia. It was concluded that a diet deficient in fiber and rich in fermentescible carbohydrate cannot by itself induce a decrease in thiamin synthesis in so far as transitions between diets are not done abruptly. PMID- 3022634 TI - [Diagnosis of enzootic bovine leukosis by the ELISA test of mixed and individual milk]. AB - The ELISA test was applied to mixed milk from 325 cowsherds of the Landes region of France. From the 88 cowsheds giving a positive or inconclusive response, individual samples of milk were studied by the ELISA test, and individual samples of serum by the agar immunodiffusion test (1734 cows). The same procedure was carried out on 1250 animals in a sample of 49 cowsheds chosen at random from amongst the 237 cowsheds whose mixed milk gave a negative response to the ELISA test. The results confirmed the importance of the ELISA test applied to mixed milks, provided that the samples are studied several times per year, so as to minimize default errors. The study of individual milk allowed the identification of 92 to 94% of the infected animals, with a specificity of 98 to 99%. The systematic and repeated testing of mixed milks by the ELISA test should allow initial detection of infection and supervision of a satisfactory cost-efficiency report in the decontaminated cowsheds. PMID- 3022635 TI - [Experimental inoculation of bovine herpesvirus 4 (strain LVR 140) in pregnant and nonpregnant cows]. AB - Ten cows (5 pregnant and 5 non pregnant) were inoculated with a BHV 4 (Bovine Herpes Virus 4) strain LVR 140. The infection caused metritis symptoms, but only after parturition and even if the calving occurred several weeks after the inoculation. The metritis was accompanied by leucopenia. The virus was reisolated from the lochia and the lymph nodes, in some cases several weeks after parturition. A number of unexplained mortalities was observed during the experiment. The evolution of antibodies detected by the indirect immunofluorescence test (IIF) showed two levels: the first after inoculation and the second after parturition. PMID- 3022636 TI - [2 methods of studying in vitro the virucidal action of disinfectants]. AB - The assessment of the virucidal activity of disinfectants and antiseptics is a permanent care. That is why, in recent years, studies have been developed in order to standardize these techniques. The present work describes two methods, the method of instantaneous dilution and that of gel filtration. The kinetics of inactivation of two viruses (the Talfan and the Canine Contagious Hepatitis viruses) which were obtained after contact with 10 disinfectants commonly used in agriculture and the food industry are compared. Usually these products exhibit a weak virucidal activity at the dosages recommended by the manufacturer. The present study emphasizes the necessity to have available viruses with a high titer and the inconvenience of using only the gel filtration technique when a precipitation takes place between the disinfectant and the viral suspension. PMID- 3022637 TI - Results of surgical treatment in bronchioloalveolar carcinoma. AB - The study relates to patients with bronchioloalveolar carcinoma who had undergone operation. On reassessment of histological specimens, 92 patients were considered to have been suffering from bronchioloalveolar carcinoma. Bronchioloalveolar carcinoma was further classified according to histological findings as typical or of mixed type. The latter included cases on which there was differentiation towards pulmonary adenocarcinoma. A third group consisted of 32 cases of peripheral pulmonary adenocarcinoma originally diagnosed as bronchioloalveolar carcinoma. Pulmonary tuberculosis was found to have occurred oftener in bronchioloalveolar carcinoma cases than in mixed bronchioloalveolar cases (p less than 0.005). A history of pneumonia was commoner in mixed bronchioloalveolar and adenocarcinoma patients than in bronchioloalveolar patients (p less than 0.05). Lobectomy or more conservative resection had been possible in the majority of cases. There had been no surgical or hospital mortality. No differences existed between the groups as regards surgical treatment, postoperative radiotherapy or chemotherapy. Local recurrence was commoner in bronchioloalveolar patients than in mixed bronchioloalveolar patients (p less than 0.001) or adenocarcinoma patients (p less than 0.025). Mixed bronchioloalveolar and adenocarcinoma patients had distant metastases oftener than bronchioloalveolar patients (p less than 0.025 and p less than 0.001). Adenocarcinoma patients also had more metastases than mixed bronchioloalveolar patients, but the difference was not statistically significant. Most metastases (82%) were discovered within three years of operation. The incidence of local recurrences increased from three years after operation. The five-year survival rate was 57% in the bronchioloalveolar group, 45% in the mixed bronchioloalveolar group and 17% in the adenocarcinoma group.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022638 TI - [What have we learned about inhibitors of the renin-angiotensin system?]. AB - With orally active angiotensin converting inhibitors it is now possible to block the renin-angiotensin system chronically. These agents given alone normalize blood pressure of many hypertensive patients. In the remaining, an additional salt subtraction, induced for example by diuretics, is necessary to further reduce blood pressure. In patients with congestive heart failure, angiotensin converting enzyme inhibitors increase cardiac output and exercise capacity, both acutely and chronically. Adverse reactions resulting from blockade of the renin angiotensin system can be predicted to a large extent and therefore are most often easily avoided. Angiotensin converting enzyme inhibitors like captopril and enalapril, because of their efficacy and good acceptability are likely to become important drugs for the treatment of hypertension and congestive heart failure. PMID- 3022639 TI - [Hepatocellular carcinoma in cirrhosis: prolonged survival (4 years 9 months) after tumor excision and portacaval shunting]. PMID- 3022640 TI - Conjugative transposons and the dissemination of antibiotic resistance in streptococci. PMID- 3022641 TI - Activity of fluconazole (UK 49,858) and ketoconazole against Candida albicans in vitro and in vivo. AB - Fluconazole (UK 49,858), a new orally administered bis-triazole, was compared with ketoconazole for activity in synthetic broth dilution susceptibility tests against Candida albicans and also in treatment of experimental systemic candidal infections in rats. In vitro studies indicated that fluconazole activity is less sensitive to acidic medium than is that of ketoconazole. At physiologic pH, fluconazole was approximately 16-fold less active than ketoconazole against 35 representative isolates of C. albicans. Two additional isolates (K-1 and K-3) recovered from patients who had failed ketoconazole therapy were 32- to 64-fold more resistant than the median of each drug for other isolates. In animal studies, fluconazole was very effective in prolonging survival of rats infected with a representative candidal strain. With an inoculum sufficient to kill 29 of 38 sham-treated animals, only 1 of 18 animals treated with 0.5 mg of fluconazole per kg per day died compared with 13 of 20 animals treated with 10.0 mg of ketoconazole per kg per day. However, when similar fluconazole treatment was administered to rats infected with the more resistant strain, K-1, no prolongation of survival was found. Thus, in vivo and in vitro results between strains correlated well for fluconazole. However, in comparing results between drugs, ketoconazole was 16-fold more active in vitro and fluconazole was 20-fold more active in vivo. This discrepancy may be due to drug distribution, modes of drug metabolism, or other pharmacologic differences between the two agents. PMID- 3022642 TI - Mutation within the herpes simplex virus DNA polymerase gene conferring resistance to (R)-9-(3,4-dihydroxybutyl)guanine. AB - Five herpes simplex virus mutants known or presumed to contain mutations in their DNA polymerase genes conferring resistance to acyclovir and arabinosyladenine also proved to exhibit some degree of resistance to (R)-9-(3,4 dihydroxybutyl)guanine (buciclovir). For one mutant, a buciclovir resistance mutation was mapped to a region of the viral DNA polymerase gene proposed to encode the deoxynucleoside 5'-triphosphate binding domain. These data implicate the viral polymerase as a target of buciclovir action that contributes to its antiviral selectivity. PMID- 3022643 TI - Efficacy of 2'-nor-cyclicGMP in treatment of experimental herpes virus infections. AB - 9-[(2-Hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]guanine P-oxide (2'-nor cGMP), the cyclic phosphate of 2'-nor-deoxyguanosine (2'-NDG) was synthesized by phosphorylation of 2'-NDG and evaluated for antiherpetic activity in cell cultures and in animal protection studies. 2'-nor-cGMP was effective in cell culture against both thymidine kinase deficient and wild-type herpes simplex virus type 1 strains and also against herpes simplex virus type 2. The anti herpes activity of 2'-nor-cGMP against thymidine kinase deficient HSV-1 was confirmed by animal protection studies. Also, in comparative cell culture protection studies, the ED50 (microM) of 2'-nor-cGMP was approximately 10-fold lower than that of 2'-NDG against three strains of varicella zoster virus. In addition, 2'-nor-cGMP was effective orally in preventing HSV-1 orofacial infection and HSV-2 genital infection of mice. Topical therapeutic applications of 2'-nor-cGMP prevented orofacial HSV-1 lesion development in mice and development of HSV-2 genital lesions in guinea pigs. Subcutaneous application of 2'-nor-cGMP to intracerebral HSV-1 challenged weanling mice significantly prolonged survival. These studies indicate that 2'-nor-cGMP is not dependent on viral thymidine kinase for its antiviral activity and is highly effective in preventing experimental HSV infections. PMID- 3022644 TI - Effect of selenazofurin on influenza A and B virus infections of mice. AB - The inhibitory effects of selenazofurin and ribavirin on influenza A and B virus infections in mice were compared. Both compounds, when administered intraperitoneally (i.p.), reduced lung consolidation and prolonged mean day of death, but ribavirin more effectively increased survivor number and lowered lung viral hemagglutinin (HA) titers. Lung HA titers often increased in selenazofurin treated animals. To determine the most appropriate i.p. treatment schedule, influenza A virus-infected mice were treated once, twice or thrice daily for 7-9 days, or once only. Treatment once daily for 9 days beginning 4 h pre-virus exposure, for 3 days beginning 24 h post-virus exposure, or once only 48 h post virus exposure was most effective. Body temperature, which usually declined during infection, increased to near-normal levels in animals treated with selenazofurin, especially in animals treated a single time or for 3 days with high dose levels. Selenazofurin was well tolerated at a dose of 50 mg/kg administered twice daily, and at 400 mg/kg administered once only. Rectal temperatures temporarily declined following every other day treatment with 400 mg/kg. PMID- 3022645 TI - Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes. AB - The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10(4) physical particles of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hybridization stringency, 32P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments. PMID- 3022646 TI - Heparin-activated protein kinase from rabbit muscle: relationship to enzymes of the glycogen synthase kinase-3 category. AB - A heparin-activated protein kinase has been previously identified in rabbit skeletal muscle extracts (Z. Ahmad et al. (1985) FEBS Lett. 179, 96-100). Further study has indicated that this enzyme phosphorylates rabbit muscle glycogen synthase in the same tryptic peptide(s) as the protein kinase FA/GSK-3 (glycogen synthase kinase-3) and is able to activate the ATP-Mg2+-dependent protein phosphatase. These results indicate similarities in properties between the two protein kinases. Exposure of the heparin-activated enzyme to trypsin resulted in loss of heparin activation, from 3-fold to 1.3-fold. One hypothesis suggested by this result is that the enzyme FA/GSK-3 could be a derivative of the heparin activated enzyme that has lost heparin sensitivity. The conceptual importance of this hypothesis is that it may provide a clue to the mode of regulation of this important class of protein kinases. PMID- 3022647 TI - Potassium activation and its relationship to a highly reactive cysteine residue in fructose 1,6-bisphosphatase. AB - The specific chemical modification by sodium cyanate of highly reactive cysteine residues at pH 7.5 in pig kidney fructose 1,6-bisphosphatase results in the reversible loss of activation of the enzyme by monovalent cations. No loss of activation by potassium ions occurs when modification is carried out in the presence of fructose 2,6-bisphosphate. The effect of Mg2+ on native and cyanate modified enzyme activities implicates the above cysteine residue as being directly linked to the inhibition by both the divalent cation and fructose 2,6 bisphosphate. Incorporation of [14C]cyanate to the enzyme shows that the blockage of two reactive residues per tetramer is sufficient to eliminate the activation of the enzyme by K+. PMID- 3022649 TI - Alcohol-induced conformational changes of ubiquitin. AB - Ubiquitin has been found to be soluble in ethylene glycol and alcohols as the perchlorate or hydrochloride salt. When the effect of alcohol on the structure of ubiquitin is examined, two reversible conformational transitions are observed. Upon lowering the dielectric constant of aqueous alcohol solutions of ubiquitin from 80 to 45, the native structure of ubiquitin is converted to a form consistent with 50% helical structure. This conformational change results in a change in exposure to solvent of the single methionine and the single tyrosine residues of ubiquitin. In agreement with crystallographic results, these residues are buried in the native conformation but become fully exposed to solvent upon undergoing this transition. Further lowering of the dielectric constant to 20 results in the accumulation of a conformation with almost complete helical structure. Thus, hydrophobic interactions cause facile conformational changes in the ubiquitin structure. These results are discussed in terms of a preferential solvation model. It is shown that the results obtained with different alcohols can be normalized by the use of a dielectric constant scale. This normalization corrects for the different molar volumes of different alcohols, allows comparison of results obtained with different alcohols, and should be useful in studying this phenomenon with different proteins. PMID- 3022648 TI - Phospholipid base exchange in human leukocyte membranes: quantitation and correlation with other phospholipid biosynthetic pathways. AB - The biosynthesis of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) by base-exchange reactions, and of PC and PE by the CDP pathways, was assessed in the membrane phospholipids of human leukocytes (neutrophils, lymphocytes, T lymphocytes, non-T lymphocytes, and monocytes). Of the three base-exchange activities, ethanolamine exchange was the highest and choline exchange the lowest in each leukocyte membrane. In the CDP pathways, ethanolaminephosphotransferase (EPT) and cholinephosphotransferase (CPT) had comparable activities. Among subpopulations of leukocytes, T lymphocytes showed the highest levels of each enzyme activity, and neutrophils showed the least. In contrast to the enzymes of the CDP pathways, each base-exchange activity was directly proportional to the Ca2+ concentration, but markedly inhibited by Mg2+. Despite this Ca2+ dependence, the base-exchange activities were increased in a dose-dependent manner by calmodulin antagonists and, except for ethanolamine exchange, inhibited by the addition of calmodulin; EPT and CPT activities were only slightly inhibited by calmodulin antagonists and were unaffected by calmodulin. PE formation in both neutrophil and lymphocyte base-exchange reactions was enhanced in a dose-dependent manner by the presence of low concentrations of bioactive stimulants (zymosan, 0.05-0.2 mg/ml; Con A, 0.5-2 micrograms/ml), while EPT and CPT activities were not increased by these cell stimulants. Taken together, our data suggest that base-exchange activity, the biological significance of which has been hitherto unclear, may be related to cell activation; in contrast, the CDP pathways appear primarily to involve the constitutive biosynthesis of phospholipids. Our data further suggest that ethanolamine required for base-exchange reactions is a precursor of PE, N transmethylation of which can serve as a source of cell activation, leading to production of arachidonic through PC by mediation of phospholipase A2 activity. PMID- 3022650 TI - Role of ubiquitin conformations in the specificity of protein degradation: iodinated derivatives with altered conformations and activities. AB - Three iodinated derivatives of ubiquitin have been synthesized and these derivatives have been characterized in the ubiquitin-dependent protein degradation system. With chloramine-T as the oxidant, a derivative containing monoiodotyrosine is formed in the presence of 1 M KI and a derivative containing diiodotyrosine is produced in the presence of 1 mM KI. These derivatives exhibit phenolate ionizations at pH 9.2 and 7.9 with absorbance maxima at 305 and 314 nm, respectively. In addition to modification of the tyrosine residue, these conditions lead to the oxidation of the single methionine residue and iodination of the single histidine residue [M.J. Cox, R. Shapira, and K.D. Wilkinson (1986) Anal. Biochem. 154, 345-352]. Iodination of ubiquitin under these conditions renders the protein sensitive to hydrolysis by trypsin and results in an enhanced susceptibility to alcohol-induced helix formation. When the derivatives are tested in the ATP: pyrophosphate exchange reaction catalyzed by the ubiquitin adenylating enzyme, they are found to exhibit activity comparable to the native protein. When these derivatives are tested for the ability to act as a cofactor in the ubiquitin-dependent protein degradation system, they are both found to support a rate of protein degradation that is twice that of native ubiquitin. At high concentrations of derivatives, the rate of protein degradation is inhibited, while the steady state level of conjugates increases. Thus, the free derivatives inhibit the protease portion of the reaction, but are fully active in the activation and conjugation portions of the reaction. With iodine as the modification reagent, monoiodination of tyrosine is the predominant reaction. This derivative exhibits activity similar to native ubiquitin. Thus, it appears that modification of the histidine residue is responsible for the increased activity of the more highly iodinated derivatives. The enzymes of the system must recognize different portions of the ubiquitin structure, or different conformations of ubiquitin that are affected by the iodination of the histidine residue. These results suggest a conformational change of the ubiquitin molecule may be important in determining the rate and specificity of proteolysis. PMID- 3022651 TI - The enzymic synthesis of ribose-1,5-bisphosphate: studies of its role in metabolism. AB - Ribose-1,5-bisphosphate is synthesized in a reaction that uses ribose-1(or 5)-P as the phosphoryl acceptor and the acyl-P of 3-phosphoglyceryl phosphate as the donor. Glucose-1,6-bisphosphate is synthesized in a similar reaction. The relative activity with the two substrates remains unchanged over almost 300-fold purification of the enzyme, indicating that glucose-1,6-bisphosphate synthase catalyzes both reactions. The relative V/Km values for alternative phosphoryl acceptors are ribose-1-P (1); glucose-1-P (0.30); mannose-1-P and ribose-5-P (0.11); glucose-6-P (0.10); 2-deoxyglucose-6-P (0.03); and 2-deoxyribose-5-P (0.02). Fructose-1- and 6-phosphates are not substrates. The synthesis of both ribose-1,5-bisphosphate and glucose-1,6-bisphosphate is inhibited by physiologically significant levels of fructose-1,6-bisphosphate, glycerate-2,3 bisphosphate, glycerate-3-phosphate, citrate, and inorganic phosphate. Ribose-1,5 bisphosphate is a strong activator of brain phosphofructokinase. PMID- 3022653 TI - [Stability of adriamycin-in-oil emulsion in hepatic artery chemoembolization]. PMID- 3022652 TI - [An unresectable case of hepatoma completely disappearing after oral administration of an anticancer drug (UFT)]. AB - A 78-year-old man was admitted to our hospital complaining of hypochondralgia. Since he was diagnosed as having advanced and unresectable hepatoma. UFT was administered as an oral chemotherapy, resulting in reduction of the liver tumor, and a decrease in AFP (ng/ml) from 1,200 to 18. Twenty-six months have already passed since the tumor was detected and 21 months since the start of UFT administration. The patient is still enjoying a good general condition and is presently under follow-up at our out-patient clinic. By the standard for the efficacy of cancer chemotherapy proposed by Koyama and Saito, this case corresponds to the state of complete response. This case suggested the clinical usefulness of UFT as an oral chemotherapy for advanced hepatoma. PMID- 3022654 TI - [Control release of adriamycin from water-in-lipiodol emulsion and a comment on the measurement of drug release]. PMID- 3022656 TI - Immunohistochemical detection of carcinoembryonic antigen in infiltrating duct carcinomas of the breast: correlation with morphofunctional parameters and prognostic significance. PMID- 3022655 TI - Effects of dietary supplementation of fish oil on neutrophil and epidermal fatty acids. Modulation of clinical course of psoriatic subjects. AB - Findings from an eight-week fish oil-supplemented diet given to 13 psoriatic patients demonstrated that eicosapentaenoic acid (20:5,n3 [EPA]) and docosahexaenoic acid (22:6,n3 [DCHA]) are rapidly incorporated into the sera, neutrophils, and epidermis of participating patients, and that the incorporation of EPA and DCHA into epidermal lipids increased with weeks of supplementation with minimal alteration of arachidonic acid (AA) in the epidermal lipids. Global clinical evaluation showed that eight patients demonstrated mild to moderate improvement in their psoriatic lesions. Improved clinical response correlated with high EPA/DCHA ratios attained in epidermal tissue specimens. These findings underscore the need for further investigations into the role of dietary n3 fatty acids, particularly the possibility of pentaenoic acid as a potential protective agent and/or therapeutic adjunct for the clinical management of psoriasis. PMID- 3022657 TI - Primary aortic malignant fibrous histiocytoma: a successfully treated case by surgical excision. AB - A man with a short history of retrosternal discomfort and weight loss was found to have a large malignant fibrous histiocytoma of the ascending aorta. The tumor was resected on partial cardiopulmonary bypass. We believe this is the only malignant fibrous histiocytoma of the ascending aorta reported and successfully treated in the literature. PMID- 3022658 TI - Role of mediastinoscopy in lung cancer. PMID- 3022659 TI - Vaginal fluid adenosine 3',5'-cyclic monophosphate (cAMP) in the rat: interaction with sperm cAMP-dependent protein kinase regulatory subunits. AB - Cyclic AMP (adenosine 3',5'-cyclic monophosphate) concentrations were determined in rat vaginal fluids throughout the estrous cycle. Radioimmunoassay results demonstrated that estrus and early metestrus vaginal fluids had significantly (p less than 0.01) elevated cAMP concentrations compared to proestrus, late metestrus, and early and late diestrus. Ovariectomy reduced RIA-detectable cAMP in vaginal fluid. When cauda sperm were preincubated for 5 min with vaginal fluids from each stage of the estrous cycle, results demonstrated that only estrus- and early metestrus-stage vaginal fluids caused a decrease in [32P] 8N3cAMP (8-azido photoaffinity analogue of cAMP) photolabeling of sperm cAMP dependent protein kinase regulatory subunits RI and RII. To examine if this reduction in [32P]-8N3cAMP photoincorporation by sperm RI and RII could be due to endogenous cAMP, vaginal fluids were boiled, trypsinized, and/or incubated with EGTA or phosphodiesterase. Only phosphodiesterase-treated vaginal fluids restored sperm regulatory subunit photoincorporation of [32P]-8N3cAMP. It is suggested that cAMP is present in rat vaginal fluids during the estrous cycle in a concentration sufficient to bind the regulatory subunits of rat sperm cAMP dependent protein kinase. PMID- 3022660 TI - Effect of enalapril in subjects with hypertension associated with moderate to severe renal dysfunction. AB - The purpose of this study was to define the effect of the angiotensin-converting enzyme inhibitor, enalapril maleate, on blood pressure, renal function, protein excretion, and potassium homeostasis in patients with hypertension associated with moderate to severe renal dysfunction. Nine patients, having an initial inulin clearance between 9 and 48 mL/min/1.73 m2, were treated with enalapril monotherapy (n = 6) or enalapril/furosemide (n = 3) combination therapy. Systolic and diastolic blood pressures were well controlled. Supine plasma renin activity was stimulated; the supine plasma aldosterone level was suppressed, with a resultant increase in the serum potassium level. Clinical hyperkalemia was not observed. Glomerular filtration rate, assessed by inulin and creatinine clearances, was not significantly changed. Effective renal plasma flow, assessed by paraaminohippurate clearance was significantly increased, with a resultant decrease in filtration fraction. Importantly, urinary protein excretion was markedly reduced. These results suggest that enalapril therapy produces efferent arteriolar dilitation with preservation of the glomerular filtration rate. Enalapril therapy may also blunt the effects of angiotensin II on transglomerular passage of protein, as demonstrated by reduced proteinuria. These findings suggest that interruption of the renin-angiotensin system in patients with preexisting renal disease may have renal protective effects. PMID- 3022661 TI - [West's syndrome. A clinical, therapeutic and prognostic study. Apropos of 66 cases]. AB - The charts of 66 children suffering from West syndrome followed by the paediatric department of the University Central Hospital, Lille, between 1957 and 1984 were reviewed: 24 cases were followed for more than 10 years; 20 cases from 5 to 10 years; 22 cases from 1 to 5 years. Age at onset of the disease ranged from 3 to 5 months (37.8%) and 6 to 8 months (30.3%) with a slight male predominance. Prenatal causes were the most important (42.4%) with anomalies and cerebral malformations in 15.2% and neurocutaneous syndromes in 9.1% of cases. Etiology remained unknown in 27.3% of cases. Some clinical aspects were characteristic (flexion or extension spasms, psychomotor retardation) in 84.8% of cases. Hyparrhythmia occurred in 62.1% of cases. There were no significant differences in the results of the varying therapies used (ACTH, Synacten, hydrocortisone) or its associations. The best results were obtained with a treatment started as early as possible in the cryptogenic forms and in older infants (6 to 8 months). PMID- 3022662 TI - [Auto-immune hemolytic anemia revealed by erythroblastopenia linked to a parvovirus infection]. AB - In a 12 year-old boy presenting with auto-immune hemolytic anemia of the IgG type, human parvovirus infection was responsible for acute erythroblastopenia. Aplastic crisis quickly and spontaneously recovered but auto-hemolysis was durable. Human parvovirus induced erythroblastopenia has only been reported in patients with constitutional hemolytic anemia. On the other hand, in this case, human parvovirus infection revealed the auto-immune hemolytic anemia. PMID- 3022663 TI - Diazepam-binding inhibitor. A brain neuropeptide present in human spinal fluid: studies in depression, schizophrenia, and Alzheimer's disease. AB - Diazepam-binding inhibitor is a novel peptide purified to homogeneity from rat and human brain. Diazepam-binding inhibitor is present, though not exclusively, in gamma-aminobutyric acid (GABA)-containing neurons where it is believed to inhibit GABAergic neurotransmission mediated by GABA by binding to the benzodiazepine-GABA receptor complex. Since an impairment of central GABAergic tone has been postulated to be associated with a number of neuropsychiatric disorders, we measured human diazepam-binding inhibitor immunoreactivity in the cerebrospinal fluid (CSF) of patients suffering from endogenous depression, schizophrenia, and dementia of the Alzheimer's type. Patients with major depression had significantly higher concentrations of human diazepam-binding inhibitor immunoreactivity in CSF when compared with age- and sex-matched normal volunteers, while no difference in CSF diazepam-binding inhibitor immunoreactivity was found in schizophrenics or patients with dementia of the Alzheimer's type when compared with controls. The possibility is discussed that the increased CSF human diazepam-binding inhibitor immunoreactivity observed in depressed patients may represent a functional disinhibition of GABAergic neurotransmission associated with depression. PMID- 3022665 TI - Mast cells in regional lymph nodes responding to syngeneic and xenogeneic tumour MSVC and HeLa cell lines in BALB/c mice. AB - In response to primary and secondary stimulation by xenogeneic HeLa carcinoma cells or by syngeneic sarcoma MSVC cells the regional lymph nodes of BALB/c mice are enlarged, however, their absolute mast cells content does not increase and in relative terms a decrease of mast cells concentration is observed. These results are in accordance with our earlier observations that during antigeneic and T-cell mitogen stimulation the lymphatic mast cells population does not increase but is rather reduced. This may indicate the engagement of these cells in delayed-type hypersensitivity. PMID- 3022664 TI - Desipramine-yohimbine combination treatment of refractory depression. Implications for the beta-adrenergic receptor hypothesis of antidepressant action. AB - Preclinical investigations have shown that combined administration of the alpha 2 adrenergic receptor antagonist yohimbine hydrochloride and the tricyclic antidepressant desipramine hydrochloride produces a reduction in brain beta adrenergic receptor function within four days. Since the ability of antidepressant treatments to reduce beta-adrenergic receptor function has been hypothesized to mediate antidepressant efficacy, it was predicted that combined desipramine-yohimbine treatment would be a more rapid-acting and potent antidepressant regimen than desipramine alone. In the present investigation, the effects of desipramine (N = 11) and desipramine-yohimbine (N = 10) treatment on depressive symptoms, norepinephrine turnover, and blood pressure were determined in patients with major depression who had a history of nonresponse to standard antidepressant treatments. Neither desipramine nor desipramine-yohimbine proved to be an effective treatment, although concomitant yohimbine administration did attenuate the ability of desipramine to decrease plasma free and 24-hour urinary 3-methoxy-4-hydroxyphenyl-ethyleneglycol levels and blood pressure. Fifteen of the 21 patients eventually had a good response to pharmacologic treatments, particularly a desipramine-lithium carbonate or lithium carbonate-tranylcypromine sulfate combination treatment (11 of 14 responded). This study provides evidence against the beta-adrenergic receptor hypothesis of antidepressant action. PMID- 3022666 TI - Histogenesis of the development of Langhans' giant cells. AB - Microscopic analysis of the development of Langhans' giant cells in lymph nodes of hyperimmunized animals was performed. It seems that they originate from the primitive mesenchymal cells, arranged along the blood vessels especially along the capillaries. PMID- 3022668 TI - Pure small-cell carcinoma of the prostate with fatal liver metastasis. AB - A case of small-cell carcinoma (SCC) of the prostate is described with extensive liver metastases in a 60-year-old man. The case is unique because of the rare histologic pattern coupled with death secondary to hepatic failure. A spectrum of neuroendocrine tumors are found in the prostate similar to those found in the lung. Ten cases of prostatic SCC or carcinoid, identified in the English-language literature over a period of 14 years, are summarized. In the absence of an apparent pulmonary lesion, surgical pathologists should also consider the prostate as a primary site for extrapulmonary SCC when confronted with biopsy material of metastatic SCC. PMID- 3022667 TI - Correlation of polyalbumin receptors with hepatitis B surface antigen polypeptides in human sera. AB - The molecular nature of the association between hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), polymerized human serum albumin receptors (pHSA-R), and HBsAg polypeptides is controversial. Therefore, we studied HBsAg, HBeAg, and pHSA-R by radioimmunoassay in sera of 27 patients with acute or chronic hepatitis B virus infection. In addition, the HBsAg polypeptides were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining and immunoblot analysis. As a group, HBeAg-positive sera contained significantly more HBsAg and higher pHSA-R activity than HBeAg negative sera. At a given amount of HBsAg, pHSA-R activity was significantly higher in HBeAg-positive sera than in HBeAg-negative sera. However, there was no correlation between the presence or absence of a particular HBsAg polypeptide and pHSA-R activity, suggesting that posttranscriptional events such as glycosylation may modulate pHSA-R activity associated with native HBsAg. PMID- 3022669 TI - Mammary adenoid cystic carcinoma with sebaceous differentiation. A morphologic study of the cell types. AB - Nine examples of mammary adenoid cystic carcinoma with sebaceous differentiation were studied. Each one of the nine tumors contained basaloid, eosinophilic, and sebaceous cells. Two of these tumors were examined by electron microscopy and compared with four mammary adenoid cystic carcinomas without sebaceous differentiation. The sebaceous cells were similar to normal adnexal sebaceous cells. The eosinophilic cells reflected adenosquamous differentiation and were identical to superficial cells of eccrine sweat ducts. The basaloid cells showed no distinctive cytoplasmic differentiation. Cells with features between the basaloid, sebaceous, and adenosquamous cells were present, indicating that basaloid cells have the potential to differentiate toward skin adnexal structures giving rise to both sebaceous and adenosquamous cells. Myoepithelial cells were not identified in any of the six tumors evaluated by electron microscopy. PMID- 3022670 TI - Endocrine and tumor differentiation markers in poorly differentiated small-cell carcinoids of the cervix and vagina. AB - Immunoreactivity for endocrine peptides (serotonin, gastrin, somatostatin, insulin, corticotropin, calcitonin, neurotensin, vasoactive intestinal peptide, and bombesin), cytoskeletal proteins (high and low molecular weight keratins), and tumor differentiation markers (chromogranin, neuron-specific enolase, carcinoembryonic antigen, S100 protein, and Grimelius stain) was sought on nine cervical and one vaginal poorly differentiated small-cell carcinoids. Dense-core secretory granules were ultrastructurally identified in all cases (seven of ten) in which tissue was available for electron microscopy. Immunoreactivity for endocrine secretory products was rarely noted, and only in a minority cell population (serotonin in two of ten). The majority of the tumors exhibited immunoreactivity for low molecular weight keratin (AE1/AE3 in eight of ten; CAM 5.2 in seven of nine), and three of ten tumors focally expressed high molecular weight keratin. Among the markers of neuroendocrine differentiation, neurospecific enolase was more frequently expressed (ten of ten) than chromogranin (five of ten) or argyrophilia (three of ten). Carcinoembryonic antigen was present in eight of ten tumors. S100 protein was absent in all cases. In summary, poorly differentiated small-cell carcinoids of the lower female genital tract, similarly to other small-cell endocrine tumors, occasionally exhibit focal glandular and squamoid differentiation, and only relatively infrequently or focally express immunohistochemically detectable endocrine secretory products, chromogranin, and argyrophilia. PMID- 3022671 TI - Liquefactive necrosis in Coxsackie B encephalitis. AB - Coxsackieviruses may cause serious illness in infants and children, specifically myocarditis and meningoencephalitis. Central nervous system lesions have been characterized as inflammatory in nature with mononuclear cell infiltration, neuronophagia, and glial nodule formation largely confined to the brain stem and spinal cord. We present two infants with documented Coxsackie B virus infection who also had widespread multifocal areas of liquefaction necrosis unassociated with inflammation. Such areas of bland necrosis, especially in the cerebrum, are unusual in Coxsackie B virus infection, and may provide the morphological substratum of permanent neurologic impairment in children who survive. PMID- 3022672 TI - Lewy bodies of Parkinson's disease. Immune electron microscopic demonstration of neurofilament antigens in constituent filaments. AB - Recently it has been shown by light microscopic immunochemistry that Lewy bodies (LBs) react with antibodies raised against neurofilament proteins (NFPs). Because of the ubiquity of the NFPs within neurons, the heterogeneous makeup of the inclusions, and the varying patterns of immunolabeling, we undertook to determine whether the labeled elements are indeed constituent filaments. Employing a preembedding technique, we investigated sections of the same LBs by light and electron immunochemistry. Decoration of the filaments was obtained with a monoclonal anti-NFP antibody. Whereas cores of mature LBs were unreactive by light microscopy, these same cores yielded a positive reaction at the ultrastructural level. Early LBs were intensely labeled in both the core and periphery. These results demonstrate that the filamentous profiles that form the LBs are antigenically identical to neurofilaments and suggest a posttranslational modification of the filaments as they "age" within the inclusion. PMID- 3022673 TI - Tumor cell embolism to pulmonary alveolar capillaries. Cause of sudden cor pulmonale. AB - A 51-year-old woman, with a 13-month history of widely metastatic breast carcinoma treated with radical mastectomy and chemotherapy, developed sudden shortness of breath and chest pain. Rapidly progressive pulmonary hypertension was documented that failed to respond to supportive measures, and the patient died. The lungs at autopsy demonstrated tumor microemboli in the form of noncohesive, individual cells within the capillaries of approximately 40% of the pulmonary alveolar septae. This case is remarkable for widespread involvement of the alveolar septal capillaries as the cause of acute cor pulmonale. PMID- 3022675 TI - Polyneuropathy due to glue exposure: case report and 16-year follow-up. AB - Polyneuropathy resulting from industrial exposure to solvent n-hexane is widely recognized and reported in the medical literature. We report a case followed for 16 years with a series of electromyographic and nerve conduction studies. A 63 year-old man was exposed to glue fumes in a cabinet-making factory. This exposure resulted in polyneuropathy affecting the upper and lower extremities with more severe involvement of the legs. Electrodiagnostic examination revealed mixed type, predominantly motor polyneuropathy. Intensive medical treatment and physical rehabilitation failed to retard the progression of the disease and the patient never fully recovered. He was fitted with short leg orthoses with Klenzak joints, adjustable springs, and stirrups attached to orthopedic shoes. Only with the help of the braces could he ambulate and stay functionally independent. There was good correlation between electromyographic and nerve conduction findings and the clinical picture. PMID- 3022674 TI - A pilot study of the value of ceramics for bone replacement. AB - We report on eight cases in which ceramic implants were used to replace bone grafts. The results of this pilot study show definite usefulness of tricalcium phosphates, whereas hydroxyapatite did not prove suitable. PMID- 3022676 TI - Intraductal carcinoma. Analysis of presentation, pathologic findings, and outcome of disease. AB - A retrospective analysis of 52 patients with intraductal carcinoma or ductal carcinoma in situ (DCIS) and 30 patients with microinvasive DCIS was performed. All patients but one were treated by mastectomy. The average follow-up was 5 1/2 years. The clinical presentation of the patients having DCIS only included the presence of a mass in 33% (17/52), nipple discharge in 34% (18/52), or suspicious mammographic finding in 33% (17/52), whereas in those patients having DCIS with microinvasion, the initial presenting symptom was a mass in 63% (19/30) of the patients, nipple discharge in 13% (4/30), and mammographic finding in 23% (7/30). The presence of axillary lymph node metastasis was identified in one of the 52 patients with DCIS and six (20%) of the 30 patients with DCIS and microinvasion. Associated carcinomas in the mastectomy specimens of patients with DCIS were as follows: DCIS, 18% (9/51); lobular carcinoma in situ, 13% (7/51); Paget's disease, 8% (4/51); and invasive carcinoma, 2% (1/51). In the 30 patients with microinvasion, DCIS was found in other quadrants in 23% (7/51) of the patients; lobular carcinoma in situ, 7% (2/51); Paget's disease, 13% (4/51); and invasive carcinoma, 7% (2/51). There was one death due to cancer in the patients with DCIS only. Of the patients diagnosed as having DCIS with microinvasion, seven patients have developed metastasis and four have died of the disease. PMID- 3022677 TI - Neutralization of poliovirus by polyclonal antibodies requires binding of a single IgG molecule per virion. AB - Neutralization of poliovirus type 1 was studied using radioactively labelled polyclonal IgG. With nonsaturating antibody concentrations various virus-antibody complexes were produced which were isolated by sucrose gradient centrifugation and identified by electron microscopy as virus monomers, dimers, trimers, tetramers and pentamers. The neutralization rate (n.r.) of each of the virus antibody complexes relative to non-neutralized virus and the stoichiometry have been estimated. The monomer fraction showed that about every fifth virion was associated with one IgG molecule and neutralized. The antibody was bivalently attached. The majority of virus particles formed aggregates of different sizes, which were cross-linked by antibodies. The following neutralization rates and ratios of IgG to virus (IgG/V) were determined for the oligomers: dimers, 59.2 per cent n.r. and 0.55 IgG/V; trimers, 67.3 per cent n.r. and 0.66 IgG/V; tetramers, 79.0 per cent n.r. and 0.75 IgG/V; pentamers, 86.3 per cent n.r. and 0.98 IgG/V. Two different mechanisms of neutralization are proposed: i) an antibody-mediated mechanism specifically inhibits infectivity of the monomer virus-antibody complexes and ii) reduction of infectivity of oligomer virus antibody complexes is caused simply by reduction of the actual number of infectious units. Immunoprecipitation of the denatured capsid proteins showed that only VP 1 was recognized by the polyclonal IgGs. PMID- 3022678 TI - Comparative biological characterization of mouse adenovirus strains FL and K 87 and seroprevalence in laboratory rodents. AB - The growth, stability and seroprevalence in laboratory rodents of the two known strains of mouse adenovirus were compared. The FL strain of mouse adenovirus grew in both L 929 murine fibroblasts and in CMT-93 murine rectal carcinoma cells, whereas the K 87 strain grew only in CMT-93 cells. The bulk of the FL progeny virus was released from the host cells. K 87 virus was largely cell-associated. Both virus strains were stable at 37 degrees C in liquid medium. The K 87 strain was completely inactivated after 5-15 minutes at 56 degrees C, whereas FL infectivity was still detected after two hours at this temperature. Both virus strains were stable in the dessicated state for 14 days, although FL viability was more dependent on the presence of protein in the virus diluent. Seroepidemiologic data suggest that viruses antigenically related to mouse adenovirus are more prevalent among laboratory rats than among laboratory mice and that the virus(es) infecting rats differ from those infecting mice. Results of retrospective serologic testing suggest an association between mouse adenovirus and an outbreak of disease in a mouse breeding colony. PMID- 3022679 TI - Mouse hepatitis virus nasoencephalopathy is dependent upon virus strain and host genotype. AB - Mouse hepatitis virus (MHV) S induced typical MHV spongiform lesions in brainstem 28 days following intranasal inoculation of adult A/J, BALB/cByJ, CBA/J, C 3 H/HeJ and C 3 H/RV, but not SJL mice. In all but SJL mice, brain lesions occurred at or near the infectious dose level, based on seroconversion by the indirect immunofluorescence assay. During the acute phase of infection (day 5), lesions were limited to the nose and brain in most genotypes. Exceptions were BALB mice, which had mild hepatitis and SJL mice, which had lesions restricted to the nose. No mortality occurred in any genotype. Following intranasal inoculation of adult mice, MHV-1, -3, -A 59, -JHM and -S all caused brain lesions at 28 days after inoculation. MHV-1 and -3 caused lesions that were usually restricted to the anterior olfactory tracts, while MHV-A 59, -S and -JHM also caused more generalized and pronounced lesions involving the midbrain and pons. These studies suggest that avirulent MHV-S given intranasally to most mouse genotypes is a good model for induction of brain infection in the absence of mortality. They also confirm observations made by others in which MHV-JHM, -S and -A 59 are relatively more neurotropic than other MHV strains, such as MHV-1 and -3. PMID- 3022680 TI - Involvement of actin-containing microfilaments in HSV-induced cytopathology and the influence of inhibitors of glycosylation. AB - Two and a half hours after infection with a high dose of different strains of HSV 1 which induce rounding of cells, breakdown of actin containing microfilaments can be observed. At the periphery of the cell, actin containing knob-like protuberances were visible. Later on, actin seems to be located exclusively on the surface of cells. Observations were done by immunofluorescence microscopy, scanning electron-microscopy and immunoperoxidase staining of ultrathin sections. The envelope of HSV appears to be stained by anti-actin. Strain IES produces rounding of cells at a high dose of infection before fusion proceeds at 37 degrees C. Similar alterations were not observed with the fusing strains MP and HFEM. Incubation of infected cells at 39 degrees C revealed strain dependent differences of the fusion activity. At 41 degrees C no "fusion from within" of cells but only rounding was detectable. Application of tunicamycin resulted in complete inhibition of fusion by all strains. The fusion activity of some strains of HSV-1 (ANG, HFEM, and MP) was not inhibited by addition of 2-deoxy-D-glucose and 2-fluoro-deoxy-D-glucose. A variant from strain MP could be isolated, which is sensitive to the effects of 2-deoxy-D-glucose. Inhibitors of processing of glycoproteins did not affect fusion of cells. PMID- 3022681 TI - DNA restriction analysis of adenovirus prototypes 1 to 41. AB - DNA restriction patterns of all known human adenovirus prototypes (1 to 41), ordered according to subgenera A to F, are presented for restriction endonucleases BamHI, BglII, BstEII, HindIII, and SmaI. This catalogue is a prerequisite for typing of adenoviruses by DNA restriction analysis in diagnostic laboratories and for strain identification in reference laboratories. To determine the genetic relationship of human adenoviruses within a subgenus and between subgenera, a pairwise analysis of comigrating fragments was performed. Adenoviruses of subgenus C were closely related. Adenoviruses of subgenus B showed two related clusters of four types each, whereas the numerous serotypes of subgenus D did not show any corresponding clustering. Little comigration was observed between DNA restriction fragments from members of subgenus A or F respectively. PMID- 3022682 TI - Characterization of a herpes simplex virus regulatory protein: aggregation and phosphorylation of a temperature-sensitive variant of ICP 4. AB - The viral polypeptide ICP 4 (or Vmw 175) is synthesized during the immediate early phase of infection by herpes simplex virus (HSV) and is required during the viral reproductive cycle for efficient transcription of delayed early viral genes. Replication of mutant strains of HSV-1 such as tsLB 2 that encode a temperature-sensitive variant of ICP 4 does not proceed beyond the immediate early phase in cells that are infected and maintained at the nonpermissive temperature (NPT). Under these conditions, the immediate early viral polypeptides accumulate to levels that are 10 to 100 fold greater than normal. We have investigated the use of tsLB 2-infected cells maintained at the NPT as a source for substantial amounts of ICP 4 for further characterization. Extraction of ICP 4 from tsLB 2-infected cells requires 0.5 M NaCl and yields aggregates that contain ICP 4, ICP 6, ICP 27, and lesser amounts of other proteins. These large aggregates cannot be disrupted under nondenaturing conditions and thus are not a suitable source for native ICP 4. We have used this overproduced ICP 4 as an antigen to generate ICP 4-specific antibody and for characterization of the primary structure of ICP 4. Analysis of acid-hydrolysed 32P-labeled ICP 4 revealed that the major phosphorylated residues in ICP 4 are phosphoserine and phosphothreonine. PMID- 3022683 TI - Autonomic nervous system involvement in experimental genital infection by herpes simplex virus type 2. AB - Peroxidase-antiperoxidase technique and histology were employed to elucidate the peripheral routes involved in HSV-2 progression from vagina towards the central nervous system in mice. 12 week-old female Balb/c mice were intravaginally infected with 5 X 10(5)LD50 of HSV-2. Sixty per cent of animals developed vulvovaginitis, perigenital alopecia and hind-limb paresia. Death occurred at 9 11 days post-infection. Colon dilatation and urinary bladder distention were observed in all cases. Complete transversal sections from vulva to kidneys were obtained of each animal, including the spinal cord in situ. Herpes antigen were regularly detected in vulvovaginal epithelium, intramural, perigenital and perivesical small nerves. Besides, their invariable presence in Auerbach's plexus and sympathetic ganglia, strongly suggests preferential autonomic nervous system involvement in the progression of HSV-2 intravaginal infection towards the spinal cord. PMID- 3022685 TI - Cytomegalovirus: the relationship of nucleocapsid assembly to the organization of cellulae. AB - Ultrastructural analysis of cytomegalovirus (CMV)-induced nuclear inclusions (NI) revealed a consistent organizational pattern that may have a relationship to the assembly of virus particles. The NI consisted of cellulae that were divided into three zones based on the extent of development of virus particles. PMID- 3022684 TI - Skin Langerhans cells play an essential role in the defense against HSV-1 infection. AB - The role of the Langerhans cells (LC) during HSV-1 infection in murine skin was examined. LC function as very potent antigen-presenting cells and represent the most peripheral immune cellular elements in the body. The footpad route was used to study the response of LC to infection with a pathogenic and non-pathogenic strain of HSV-1. Following infection with a pathogenic HSV-1 strain, there was an increase in the number of LC in the footpad skin. Depletion of LC from the skin by treatment with 10 per cent aqueous saline and abrasion led to the enhancement of HSV-1 virulence and the nonpathogenic virus became highly pathogenic. Progressive recovery of LC in the skin during the healing process was accompanied by a gradual increase in resistance to HSV-1 infection. PMID- 3022686 TI - A correlation between the weather and the incidence of ocular adenovirus infections. AB - Cyclical changes in the frequency of adenovirus isolation from patients with non epidemic conjunctivitis were shown to correlate with monthly total hours of sunshine. Sunlight may predispose to clinical infection eyes already contaminated with adenovirus. PMID- 3022687 TI - Equine herpesvirus genomes: heterogeneity of naturally occurring type 4 isolates and of a type 1 isolate after heterologous cell passage. AB - The restriction endonuclease DNA fingerprints of 20 low passage, epidemiologically unrelated isolates of equine herpesvirus 4 (equine rhinopneumonitis virus) showed considerable heterogeneity in certain fragments, the positions of which were assigned to quite restricted positions on the 141 kilobase (kb) genome. We note that the heterogeneity observed in the restriction endonuclease DNA fingerprints of EHV 1 (equine abortion virus) and of pseudorabies virus also tend to map to these same restricted regions. The restriction endonuclease DNA fingerprints of an EHV 1 strain was invariant using low (less than 1) multiplicity of infection during 20 passages in equine cells but when adapted to hamster cells developed an approximately 0.8 kb deletion in the unique short region of the genome between 8 and 11 passages. PMID- 3022689 TI - [The effect of noise on the secretion of ACTH, cortisol and catecholamines]. PMID- 3022688 TI - [Characterization of B cell lines spontaneously established from the peripheral blood of Sjogren's syndrome]. PMID- 3022690 TI - Cyclic AMP and cyclic GMP content in a short-term organ culture of normal and atherosclerotic human aorta. AB - Cyclic AMP and cyclic GMP content was measured in intima and media of unaffected and atherosclerotic areas of human aorta in a short-term organ culture. It was demonstrated that during short-term cultivation the content of both cyclic nucleotides in tissues is constant. The cyclic AMP content in fatty streaks and atherosclerotic plaques is significantly (2 to 7-fold) lower than in unaffected intima. The cyclic GMP level in atherosclerotic lesions is 1.5 to 3-fold higher than in normal. The content of both cyclic nucleotides in the media underlying fatty streaks is the same as in the normal tissue. In the media underlying atherosclerotic plaques, the cyclic AMP level is decreased compared to the normal. The obtained data indicates serious disorders in the system of cyclic nucleotides during atherosclerosis. Possible consequences of this disfunction are discussed. PMID- 3022692 TI - [Adenoid cystic cancer of the breast]. AB - A case of a mammary adenoid cystic carcinoma in a female of 45, is described. The tumour was histologically identical to the salivary gland tumours of the same name. PMID- 3022691 TI - [Ultrastructure and electron-histochemical properties of the glandular endometrial epithelium in women]. AB - Ultrastructural and ultracytochemical characteristics of glandular epithelium of endometrium in 10 women with two-phase cycle were studied. Secretory transformation of glandular epithelium at ultrastructural level is manifested by deposits of glycogen in cytoplasm, giant mitochondria, hypertrophic Golgi complex and nuclear channel system. In organ endometrial cultures formation of the nuclear channel system at the stage of proliferation is induced by progesterone. Localization of ribonucleoproteins and activity of nucleoside phosphatases (ATPase and 5'-nucleotidase) in the nuclei and the channel system is described. Ultrastructural and ultracytochemical data permit to exclude nucleolar origin of the nucleolar channel system. PMID- 3022693 TI - [Nodular fibrohistiocytosis of the gallbladder]. AB - A case of nodular fibrohistiocytosis with tissue eosinophilia located in the submucous layer of the gallbladder in a woman of 42 is presented. Similar cases have been described so far only in the stomach. Clinically there existed conditions for the immune system disturbances (bronchial asthma). PMID- 3022694 TI - Infantile spasms. Comparative trial of nitrazepam and corticotropin. AB - Fifty-two patients were enrolled in a four-week randomized multicenter study comparing nitrazepam and corticotropin in the treatment of infantile spasms. The drugs' efficacy was evaluated in 48 patients, all less than 2 years of age. Both treatments resulted in a statistically significant reduction in spasm frequency from that at baseline, but the difference between treatments was not significant. The number of patients who experienced side effects was similar in the two treatment groups, but the adverse effects encountered among the patients treated with corticotropin were qualitatively more severe and required the discontinuation of treatment in six patients. PMID- 3022695 TI - Myopathy and fatal cardiopathy due to cytochrome c oxidase deficiency. AB - A 3-day-old girl had a syndrome of lethargy and lactic acidosis. Pregnancy and delivery had been normal; there was no consanguinity or family history of neuromuscular disease. At age 4 1/2 months, she had generalized weakness, hypotonia, areflexia, and macroglossia. She developed cyanosis and respiratory failure, and marked cardiomegaly was noted. She died at age 8 1/2 months of cardiac arrest. Results from a muscle biopsy specimen obtained at age 4 1/2 months showed ragged-red fibers and increased glycogen and lipid droplets. With the cytochrome c oxidase reaction, only 5% of the fibers stained positively in the biopsy specimen. Cytochrome c oxidase activity was 7.3% of normal in muscle mitochondria and 12.2% of normal in heart mitochondria. Reduced-minus-oxidized cytochrome spectra showed lack of the cytochrome aa3 peak. Immunotitration using antibodies against purified human heart cytochrome c oxidase showed normal amount of cross-reacting material in both heart and muscle. The genetic error could have involved a cytochrome c oxidase isozyme common to heart and muscle. PMID- 3022696 TI - Brachial plexus lesions. PMID- 3022697 TI - Nonfamilial prealbumin-type amyloid polyneuropathy. AB - A 53-year-old man with nonfamilial prealbumin-type amyloid polyneuropathy had severe motor, sensory, and autonomic polyneuropathy, beginning at age 48 years. These clinical features closely resembled familial amyloid polyneuropathy (FAP), but abnormal serum prealbumin levels, specific to FAP (Japanese type), were not detected by radioimmunoassay; DNA sequence for prealbumin was normal. Thus, the diagnosis of FAP was excluded. A possible diagnosis of systemic senile amyloidosis was also considered. PMID- 3022698 TI - Cyclic 3',5'-adenosine monophosphate modulates retinal pigment epithelial cell migration in vitro. AB - Retinal pigment epithelial (RPE) cell migration has been implicated in the pathogenesis of proliferative vitreoretinopathy (PVR). Using a modified Boyden chamber assay, we have examined the effect of cyclic nucleotides on human RPE cell migration in vitro. Dibutyryl cyclic 3',5'-adenosine monophosphate (cAMP) (10(-3) mmol/L) inhibits RPE cell random migration by 83%, fibronectin-induced chemotaxis by 61%, and platelet-derived growth factor-induced chemotaxis by 68%. Random and directed migration of RPE cells is not significantly affected by 8 bromo cyclic 3',5'-guanosine monophosphate. Agents that significantly increase intracellular levels of cAMP are also inhibitors of RPE cell migration. Though there is a fairly good correlation for most drugs for their ability to stimulate cAMP production and their ability to inhibit cell migration, it is not perfect, suggesting that some drugs may modulate migration by more than one mechanism. Timolol blocked both the isoproterenol-induced stimulation of RPE adenylate cyclase and attenuated the ability of isoproterenol to inhibit RPE migration. These data suggest that cAMP may modulate RPE cell migration in an inhibitory fashion. Elucidation of the biochemical events involved in RPE cell migration could provide information that might be useful in planning a strategy to attempt pharmacologic control of proliferative vitreoretinopathy. PMID- 3022700 TI - The effectiveness of acyclovir. PMID- 3022699 TI - Time course of release into the medium of newly synthesized proteins by cultured aortic endothelial cells. Role of serum in preventing proteolytic degradation. AB - Endothelial cells from pig aortas were labeled with 35S-methionine, and the soluble proteins that were released into the culture medium were examined by SDS PAGE. Proteins were collected during labeling, and during the intervals 0 to 3, 3 to 6, 6 to 12, and 12 to 24 hours of chase after a 1-hour labeling period, and during the intervals 0 to 12, 12 to 24, and 24 to 48 hours of chase after a 24 hour labeling period. Release of radiolabeled soluble protein from cells into the medium continued over the longest time periods examined. If the cells were labeled for 1 hour, characteristic patterns of proteins appeared in the medium during the labeling period, early (0- to 3-hour postlabeling) and late (6- to 24 hour postlabeling). The time course of release of fibronectin (Fn) and of angiotensin-converting enzyme (ACE) was determined by using immunoprecipitation. ACE appeared only after 6 hours of chase. Fn appeared throughout the chase period. The effects of serum and of the protease inhibitors alpha-2 macroglobulin, pepstatin, and leupeptin on protein release were examined. In the absence of serum, endothelial cell culture medium contained substantial protease activity capable of completely degrading most of the released proteins; a major effect of serum was to protect newly released protein from degradation. PMID- 3022701 TI - A preliminary report of a comparison of epizootic haemorrhagic disease viruses from Australia with others from North America, Japan and Nigeria. PMID- 3022702 TI - Radiographically visible calcification in colloid carcinoma of the stomach. PMID- 3022703 TI - A photometric analysis of free alveolar macrophages (FAMs) in smoking and nonsmoking firefighters. AB - The effects of cigarette smoking and chronic smoke inhalation were evaluated in free alveolar macrophages (FAMs) in firefighters and police officers from the city of Denver, CO. Evaluation was accomplished by comparing statistical morphometric and photometric data taken from digital images of FAMs generated by the microscope photometer. Although our results failed to show significant differences between occupations and smoking status in FAM size, degree of size variability, or nuclear/cytoplasmic area ratios, they did demonstrate a significant difference in the degree of nuclear and cytoplasmic optical density (O.D.) between both occupation and smoking status. Firefighters consistently showed significantly greater O.D. values than police officers while smokers demonstrated a significantly greater O.D. than nonsmokers. While the meaning of these findings remains illusive, they do, however, present quantitative data supporting the biological response of the FAM to occupational and cigarette smoke inhalation. PMID- 3022704 TI - Factors that influence test session performance measured 0, 3, or 6 h after inhibitory avoidance training. AB - Rats were trained in a step-down inhibitory avoidance task using a 0.3-mA, 60-Hz footshock, and were tested at 0, 3, and 6 h from training. Retrieval scores (test session minus training session step-down latencies) were higher in control groups at 0 than at 3 or 6 h. Test session performance at 0 h was unaffected by the pretraining ip injection of ACTH1-24 (0.2 microgram/kg), epinephrine-HCl (5.0 micrograms/kg), human beta-endorphin (1.0 microgram/kg), or naloxone-HCl (0.4 mg/kg); or by a pretreatment with dexamethasone phosphate (2.0 mg/kg in divided doses 24 and 12 h before training); or by anterior or posterior hypothalamic deafferentation. Test session performance at 0 h was depressed by prior bilateral transection of the fornix, which suggests it depends on hippocampal function. The effect of the fornix lesion on test session performance at 0 h was not counteracted by ACTH, epinephrine, or beta-endorphin administration. When animals were tested 3 h after training, the post-training administration of ACTH and epinephrine caused an enhancement of test session performance; neither post training beta-endorphin or naloxone, nor pretest ACTH, epinephrine, or beta endorphin administration, had any effect in these animals. At 6 h from training, the post-training facilitatory action of ACTH and epinephrine was still present and the post-training depressant effect of beta-endorphin and the post-training facilitatory effect of naloxone became manifest, and so did the naloxone reversible pretest facilitation induced by ACTH, epinephrine, or beta-endorphin. The influence of post-training naloxone or pretest beta-endorphin on retrieval scores at 6 h was not observed in the fornix-lesioned animals. IN CONCLUSION: Test session performance of this task at 0 h from training is regulated by different mechanisms than those which regulate test session performance at 3 or 6 h; in particular, it is less susceptible to modulation by the drugs used in the present study and it depends on the fornix; At least two major classes of modulatory factors influence retrieval scores at later times: consolidation enhancing effects of ACTH and epinephrine, which become manifest at 3 h, and mechanisms related to beta-endorphin, which involve a form of state dependency and only become manifest at 6 h from training. PMID- 3022705 TI - Aversive and attractive marking of toxic and safe foods by Norway rats. AB - The present series of studies was undertaken to investigate the hypothesis (von F. Steiniger, 1950, Zeitschrift fur Tierpsychologie, 7, 356-379; K. A. Stierhoff, & M. Lavin, 1982, Behavioral and Neural Biology, 34, 180-189) that rats poisoned after eating a novel food will mark that novel food in such a way as to dissuade naive conspecifics from ingesting it. Our results provided no evidence of aversive marking of a novel food by rats poisoned after ingesting it. We did, however, find evidence of attractive marking of feeding sites by rats exploiting those sites. This attractive marking rendered exploited feeding sites more attractive to naive conspecifics than other portions of an enclosure that rats had visited. The present findings are consistent with the results of a number of experiments conducted in our laboratory over the last decade indicating that rats directly communicate learned food preferences, but not learned food aversions. PMID- 3022707 TI - [Artificial long-term nutrition of neurologic patients]. PMID- 3022706 TI - Myocardial cAMP and calcium levels in the calcium paradox. AB - In a graded model (minimal, subtotal, total) of the calcium paradox phenomenon induced by a progressive increase in the flow rate and volume (5 ml, 10 ml, 45 ml) of calcium-free perfusion (5 min) the severity of tissue injury on calcium repletion was related to a potential elevation of cAMP during calcium depletion. In the subtotal and total models of the calcium paradox a 50% increase was found for tissue cAMP after calcium-free perfusion, but no such rise could be associated with the minimal calcium paradox. In the study tissue injury, as assessed by whole tissue and mitochondrial accumulation of calcium and by myocardial leakage of creatine kinase, could only partly be related to cAMP changes. It is concluded that calcium-free coronary perfusion induces a complex series of events favouring excessive calcium entry, only one of which may be related to a change in cAMP. PMID- 3022708 TI - Susceptibility of varicella-zoster virus to thymidine analogues. AB - Ten strains of varicella-zoster virus (VZV) were tested for susceptibility to 17 nucleoside analogues by a plaque reduction assay using human embryonic lung fibroblast cells. The compounds employed were 5-substituted arabinosyluracils and 2'-deoxyuridines, 2'-fluoro-arabinosylpyrimidines (F-araPyr) and acyclovir. In terms of the 50% plaque reduction dose (PD50), 4- to 40-fold difference were found between the 10 strains of VZV in susceptibilities to each compound. VZV was highly susceptible to 5-halogenovinyl-arabinosyluracils (XV-araUs); the PD50 values of these compounds were less than 0.001 micrograms/ml. VZV was much more susceptible than herpes simplex virus (HSV) type 1 to XV-araUs, but less susceptible than either HSV type 1 or type 2 to 5-ethyl-2'-deoxyuridine, 5-ethyl arabinosyluracil and acyclovir. PMID- 3022709 TI - Susceptibilities of phosphonoacetic acid and acyclovir resistant varicella-zoster virus mutants to 9-beta-arabinofuranosyladenine and 1-beta arabinofuranosylcytosine. AB - The DNA polymerase activity, and susceptibilities to 9-beta-D arabinofuranosyladenine(ara-A) and 1-beta-arabinofuranosylcytosine(ara-C) of a phosphonoacetic acid resistant mutant (PAA-R) of varicella-zoster virus (VZV) selected in the presence of PAA were examined. The DNA polymerase activity of PAA R was inhibited less than that of the parent strain by PAA in vitro. PAA-R was resistant to acyclovir and also to both ara-A and ara-C. The susceptibilities to ara-A and ara-C of four acyclovir resistant mutants selected in the presence of acyclovir, and also resistant to PAA, were examined. Two variants were resistant, one was slightly resistant, and one was sensitive to both drugs. These cross resistances and susceptibilities of VZV variants to PAA, ACV, ara-A and ara-C should be considered in chemotherapy of VZV infections. PMID- 3022710 TI - Development of a micro-neutralization test for chikungunya virus. AB - A rapid, micro-scale focus reduction neutralization test for chikungunya virus was developed. In the test, cell monolayers are prepared in a 96-well tissue culture plate and the PAP (peroxidase-antiperoxidase) staining technique is used for detection of foci of chikungunya virus infected cells. This test is suitable for rapid diagnosis and epidemiological studies of the virus. PMID- 3022711 TI - Thymidine kinase with altered substrate specificity of acyclovir resistant varicella-zoster virus. AB - The acyclovir resistant mutant of varicella-zoster virus ACV-R (A 8) induced the same level of thymidine kinase activity in infected cells as the parent Kawaguchi strain. However, it induced less deoxycytidine kinase activity and did not induce phosphorylating activity for the nucleotide analogue, 9-(2 hydroxy-ethoxymethyl) guanine-(acyclovir). Another acyclovir resistant mutant, ACV-R (A 4), which is cross-resistant to phosphonoacetate and is thought to be a viral DNA polymerase mutant, induced the same level of phosphorylating activities for thymidine, deoxycytidine and acyclovir as the parent strain. The altered substrate specificity of thymidine kinase induced by ACV-R (A 8) is concluded to confer resistance to acyclovir on ACV-R (A 8). PMID- 3022712 TI - The reactions of SacI, PvuII and EcoRI endonucleases. AB - A new approach to the mechanism study of DNA restriction is suggested. It is based on enzymatic hydrolysis of DNA in the presence of organic solvent. We found that in the presence of DMSO (10% v/v), the superhelical cleavage of pBR322 DNA by restriction endonucleases SacI and EcoRI proceeds with extensive accumulation of an intermediate, the single-nicked circular DNA, while with PvuII endonuclease the superhelical form is converted directly to the linear form. PMID- 3022713 TI - Double stimulation with FMLP and Con A restores the activation of the respiratory burst but not of the phosphoinositide turnover in Ca2+-depleted human neutrophils. A further example of dissociation between stimulation of the NADPH oxidase and phosphoinositide turnover. AB - The results reported here show that the activation of the NADPH oxidase in neutrophils by formyl-methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A) may occur with a stimulus response coupling sequence that bypasses the activation of phosphoinositide hydrolysis, monitored as accumulation of inositol phosphates and glycerophosphoinositol, and the increase in [Ca2+]i. In fact: in Ca2+-depleted neutrophils FMLP and Con A do not induce the respiratory burst and the activation of phosphoinositide hydrolysis. The addition of Ca2+ restores both the respiratory and the phosphoinositide responses; the double treatment of Ca2+ depleted neutrophils with FMLP and Con A in sequence, before FMLP and then Con A and vice versa, or simultaneously, restores the capacity to respond to the second stimulus with the respiratory burst but not with the activation of phosphoinositide hydrolysis. These findings suggest that, for the activation of the NADPH oxidase by FMLP and by Con A: the transduction pathway including the stimulation of phosphoinositide turnover, the Ca2+ changes and the activity of the protein kinase C is not required, or is not the unique, and one stimulus may trigger more than one transduction pathway. Possible transduction pathways are discussed. PMID- 3022715 TI - Identification of NGF receptor in chromatin of melanoma cells using monoclonal antibody to cell surface NGF receptor. AB - A 230 KDa species of Nerve Growth Factor (NGF) receptor was immunoprecipitated from EcoRI-digested chromatin of melanoma cells using a monoclonal antibody to the 75 KDa cell surface NGF receptor. The chromatin NGF receptor was shown to exist tightly bound to DNase II-sensitive sequences which, upon growth factor binding, became resistant to DNase II digestion. PMID- 3022714 TI - Phorbol esters alter muscarinic receptor binding and inhibit polyphosphoinositide breakdown in human neuroblastoma (SH-SY5Y) cells. AB - Many recent reports have indicated that the effect of the phorbol ester tumor promoters is mediated through the Ca2+/phospholipid dependent protein kinase C. We have investigated the effect of two biologically active phorbol esters, 4 beta phorbol 12 beta-myristate 13 alpha-acetate (PMA) and 4 beta-phorbol 12 beta,13 alpha-didecanoate (beta PDD) on muscarinic agonist binding and receptor stimulated phosphoinositide breakdown in cultured human neuroblastoma (SH-SY5Y) cells. Preincubation of these cells with phorbol esters significantly reduced the carbachol-stimulated breakdown of inositol phospholipids and caused a decrease of agonist affinity for [3H](-)methyl quinuclidinyl benzilate ([3H](-)MQNB) binding without affecting the affinity of antagonist to the muscarinic receptor. The nontumor promoting 4 alpha-phorbol 12 beta,12 alpha-didecanoate (alpha PDD) was ineffective in our studies. These results suggest that the activation of protein kinase C may play an important role in regulating the muscarinic receptor system. PMID- 3022716 TI - Identification and characterization of Mg2+-dependent phosphotyrosyl protein phosphatase from rat liver cytosol. AB - Although highly purified preparations of Mg2+-dependent phosphoseryl protein phosphatase (also designated phosphatase IA or phosphatase 2C) dephosphorylated phosphotyrosyl histone, the activity has been resolved from phosphatase IA by polyacrylamide gel electrophoresis at pH 9.5. This novel phosphotyrosyl-specific protein phosphatase absolutely requires Mg2+ or Mn2+ for activity, is inhibited by Zn2+, vanadate and fluoride, and has an optimal pH of 9.0 and Mr = 50,000. Certain properties of this phosphatase so closely resemble those of phosphatase IA that the two enzymes tend to be copurified through various separation procedures. PMID- 3022717 TI - Specific receptors for atriopeptin III in rabbit lung. AB - Binding studies revealed the presence of a single class of high affinity binding sites for atriopeptin III on rabbit lung membranes. An apparent dissociation constant (Kd) of 0.32 nM and a binding capacity of 166 fmol/mg protein was determined. Binding was time-dependent and saturable. The relative binding affinities of atrial peptide analogs correlated well with their potencies in eliciting relaxation of norepinephrine-contracted rabbit aorta strips. Unrelated peptide hormones did not compete for the atriopeptin binding site on rabbit lung membranes. The atrial peptide binding data are similar to those obtained for other tissues and indicate the presence of a physiologically relevant atrial peptide receptor in lung. PMID- 3022718 TI - Modulation of calcineurin phosphotyrosyl protein phosphatase activity by calmodulin and protease treatment. AB - Calcineurin (CN) dephosphorylated [32P] phosphotyrosyl glutamine synthetase, a model phosphoprotein substrate containing approximately 1 mol of phosphotyrosine per mol subunit. Phosphatase activity with and without calmodulin (CaM) was greatly stimulated by Mn2+; with Ca2+, even in the presence of CaM, activity was very low. CaM-stimulated phosphatase activity exhibited deactivation with time; initial rates declined markedly after 2-3 min. The Michaelis constant for substrate (3 microM) was identical whether 2 or 12 min assays (with CaM) were used suggesting that the decreased rate of hydrolysis did not result from a decrease in affinity for the phosphoprotein substrate. Limited proteolysis of CN by chymotrypsin increased phosphatase activity 2-3 times that of CaM-supported activity; however, addition of CaM to assays with protease-activated CN reduced activity to that observed for non-proteolyzed enzyme. These data suggest that, in addition to stimulation, CaM can inhibit certain activated conformations of the phosphatase. PMID- 3022719 TI - Separable function of platelet release reaction and clot retraction. AB - Amiloride, a known Na+/H+ exchange inhibitor, inhibited platelet serotonin release in a dose-dependent manner (100 microM for 50% inhibition, and 1mM for the nearly complete inhibition), although amiloride (1mM) accelerated clot retraction when it was measured at decreased platelet concentration. On the contrary, cytochalasin B (10 micrograms/ml) accelerated platelet serotonin release, but it inhibited clot retraction. These results demonstrate that release reaction and clot retraction, both of which are important processes involved in platelet activation, can be functionally separated. PMID- 3022720 TI - Antibody to human lymphocyte actin regulates immunoglobulin secretion by an EBV transformed human B-cell line. AB - Antibody against actin isolated from a human EBV-transformed lymphoblastoid B cell line exerted an inhibitory effect on in vitro IgM secretion by a different lymphoblastoid B-cell line, LA350. This effect was dose dependent showing from 24 40% inhibition at a dilution of 1:100 and 68-80% inhibition at a dilution of 1:50. This effect was noted in the absence of changes in either total cell count or [3H]-thymidine incorporation and was reversed by co-incubation with purified rabbit thymus actin (100 micrograms/ml) but not bovine serum albumin at the same concentration. These data demonstrate regulation of immunoglobulin synthesis by antibodies against human lymphocyte derived actin in a lymphoblastoid B-cell line. PMID- 3022721 TI - The hepatic galactosyl receptor system: two different ligand dissociation pathways are mediated by distinct receptor populations. AB - After internalization of 125I-asialo-orosomucoid (ASOR) by isolated rat hepatocytes, ligand dissociates by two kinetically distinct pathways (Oka and Weigel, J. Biol. Chem. 257, 10,253, 1983). These slow and fast dissociation pathways correspond to two functionally different subpopulations of cell surface galactosyl receptors designated, respectively, State 1 and State 2 receptors. Freshly isolated cells or cells equilibrated below 24 degrees C express only State 1 receptors. Cells equilibrated at 37 degrees C express both State 1 and State 2 receptors. Ligand dissociation after internalization of surface-bound 125I-ASOR was measured using the permeabilizing detergent, digitonin. The slow dissociation pathway was mediated by State 1 receptors and was the only pathway expressed by cells which were freshly isolated or had been equilibrated at 24 degrees C. State 2 receptors are expressed at temperatures above about 20 degrees C, and both the fast and slow dissociation pathways occurred in cells equilibrated at 37 degrees C. State 2 receptors therefore mediate the rapid dissociation pathway. Dissociation and subsequent degradation of specifically bound ligand routed in either pathway were complete, respectively, within 3 and 6 hrs. PMID- 3022722 TI - A derivative of bisbenzylisoquinoline alkaloid is a new and potential calmodulin antagonist. AB - A new derivative of bisbenzylisoquinoline (berbamine type): 0-(4-ethoxylbutyl) berbamine (EBB) was found to possess powerful and specific calmodulin (CaM) inhibitory properties. It inhibited CaM-stimulated Ca2+-Mg2+-ATPase in human erythrocyte membrane with IC50 value of 0.35 microM compared to that of 60 microM of berbamine. CaM-independent basal Ca2+-Mg2+-ATPase, Na+-K+-ATPase and Mg2+ ATPase were not effect at 1.0 microM of EBB at which CaM-dependent Ca2+-Mg2+ ATPase was already potently inhibited. The inhibition of CaM-dependent Ca2+-Mg2+ ATPase was competitive with respect to CaM. Higher amount of CaM reversed the inhibition caused by higher concentration of EBB. Using dansyl-CaM (D-CaM), it was shown that EBB binds directly to CaM and caused a conformational change of CaM polypeptide chain. From fluorescence titration curve we obtained evidence that in the presence of Ca2+, CaM has two specific binding sites for EBB and additional unspecific binding sites. The Ca2+-dependent binding sites of EBB on CaM were novel region different from the binding sites for TFP. PMID- 3022723 TI - Detection of active oxygen in rat hepatocyte suspensions with the chemiluminigenic probe lucigenin. AB - The chemiluminigenic probe lucigenin has been employed to detect the production of active oxygen species in suspensions of intact rat hepatocytes. Light emission from lucigenin arises from oxygenation by superoxide anion; hydrogen peroxide or a species derived from it may contribute to the reaction. The inhibitory action of antioxidants on the availability of active oxygen species produced by hepatocytes was tested. Propyl gallate was the most potent inhibitor, butylated hydroxyanisole and butylated hydroxytoluene were less active. The latter compounds cause an alteration of the cell membrane at high concentrations. PMID- 3022724 TI - Deficiency of subunits in heart mitochondrial NADH-ubiquinone oxidoreductase of a patient with mitochondrial encephalomyopathy and cardiomyopathy. AB - The heart mitochondria isolated from a patient with hypertrophic cardiomyopathy associated with mitochondrial encephalomyopathy were analyzed by immunoblotting using specific antibody against each of the purified mitochondrial energy transducing complexes from beef heart. Subunits of NADH-ubiquinone oxidoreductase (Complex I) were markedly decreased and those of cytochrome c oxidase (Complex IV) were decreased to some extent, but the deficiency of any of these subunits was only partial. On the other hand, the contents of subunits of ubiquinol cytochrome c oxidoreductase (Complex III) were normal. These results suggest that the decreased levels of some of the Complex I subunits might be the primary cause of disorder in this patient. PMID- 3022725 TI - Evidence for regulatory control of canine platelet phosphodiesterase. AB - Forskolin, epinephrine, and prostaglandin I2 were used to examine the adenylate cyclase-phosphodiesterase system of intact thrombopathic and normal canine platelets. The results provide indirect support for the hypothesis that the elevation of intraplatelet c-AMP in this unique hereditary defect is due to impaired phosphodiesterase activity. The inhibitory (Nj) and stimulatory (Ns) components of adenylate cyclase appeared functionally intact. Cytosolic fractions of normal and thrombopathic platelets had similar cAMP hydrolytic activities. The failure of intact forskolin-stimulated thrombopathic platelets to return elevated cAMP to non-stimulated levels after 15 min, despite significant phosphodiesterase activity in cytosolic fractions, implies that the platelet isoenzymes are under regulatory control. PMID- 3022726 TI - Mu-opioid receptor is associated with phosphatase activity. AB - A preparation of purified mu opioid receptor from bovine brain hydrolyzes p nitrophenylphosphate. This phosphatase activity has a pH optimum of 9.0, a Km of 9.0 microM, and is stimulated by Mn++ and Mg++ ions. Evidence that the observed activity is not due to a contaminant in the opioid receptor preparation includes 1) the activity is associated primarily with 60,000 molecular weight material which is much smaller than bovine brain alkaline phosphatase; and 2) the activity could not be absorbed by antibodies specific for bovine alkaline phosphatase. Thus this appears to be the first demonstration of enzymatic activity associated with an opioid receptor. PMID- 3022727 TI - Taurine protection against experimental arterial calcinosis in mice. AB - Combined administration of vitamin D3 and nicotine produced severe myocardial and aortic calcinosis in mice within 4 days. Treatment of these mice with taurine increased the survival rate of the mice and reduced the elevation of calcium content in both aorta and myocardium as did diltiazem treatment. The present study suggests that taurine which is capable of regulating calcium flux may also prevent the progression of arteriosclerosis. PMID- 3022728 TI - Altered cellular functions in a PC-12 cell clone chronically infected with retrovirus. AB - We characterized retrovirus-induced changes in PC-12 cell function and neuronal differentiation. PC-12 cells were infected with a neurotropic retrovirus (temperature-sensitive Moloney murine leukemia virus, mutant BA-1). We isolated a cell clone from this infected culture that displayed altered response to nerve growth factor; increased choline acetyltransferase activity; and decreased basal and nerve growth factor-stimulated acetylcholinesterase activity. In addition, Kirsten murine sarcoma virus infection of and subsequent expression of the v-ras oncogene in PC-12 cells induced neurite extension, enhanced choline acetyltransferase activity, and limited the growth potential of the infected cells. PMID- 3022730 TI - Wheat germ agglutinin behaves as a GnRH antagonist but induces gonadotrope desensitization. AB - Preincubation of cultured rat pituitary cells with 10 micrograms/ml of either wheat germ agglutinin (WGA) or concanavalin A inhibited LH release stimulated with GnRH (0.5 nM) by 55% and 40%, respectively. WGA-inhibition of LH release stimulated by GnRH was dose-dependent, reaching a plateau of 75% inhibition at 50 micrograms/ml. Concomitantly, WGA induced a dose-dependent inhibition of 125I Buserelin specific binding to pituitary cells, with a maximal inhibition of 45%. The inhibition of 125I-Buserelin binding by WGA is due to GnRH receptor internalization and not to persistent occupancy of the receptors. In addition to the effect of WGA on receptor internalization, WGA also induced partial desensitization of pituitary cells but was ineffective in modulating GnRH-induced desensitization. These findings indicate that WGA has all the characteristics of a GnRH antagonist, nevertheless, it does induce desensitization of cultured rat pituitary cells to further stimulation with GnRH. PMID- 3022729 TI - Potent and selective anti-HTLV-III/LAV activity of 2',3'-dideoxycytidinene, the 2',3'-unsaturated derivative of 2',3'-dideoxycytidine. AB - 2',3'-Dideoxycytidinene (ddeCyd), the 2',3'-unsaturated derivative of 2',3' dideoxycytidine (ddCyd) is, like ddCyd itself, a potent and selective inhibitor of HTLV-III/LAV in vitro. This conclusion is based on the relatively high ratio of effective antiviral dose (0.3 microM) versus cell growth inhibitory concentration (20-35 microM) and the lack of any appreciable inhibitory activity against a series of non-oncogenic RNA and DNA viruses. Both compounds were considerably more inhibitory to human lymphoid cell lines than human nonlymphoid or murine cell lines. They were highly dependent on prior activation by deoxycytidine kinase to exert their anti-HTLV-III/LAV and cytostatic effects. In contrast with ddCyd, ddeCyd lost part of its anti-retrovirus effect upon prolonged incubation (10 days) with the virus-infected cells in culture. PMID- 3022731 TI - Analysis of the Cu, Fe, and Zn contents in cytochrome C oxidases from different species and tissues by proton-induced X-ray emission (PIXE). AB - The Cu, Fe and Zn contents in isolated cytochrome c oxidase preparations from heart, liver, diaphragm or kidney of bovine, pig and rat was measured by proton induced X-ray emission (PIXE). The average Cu/2Fe ratio was 2.73 and Zn/2Fe ratio 0.98. Correspondingly a Cu/Zn ratio of 2.76 was found. Dialysis of the bovine heart enzyme against increasing EDTA concentrations up to 30 mM did not change this result. It is concluded that all isozymes of mammalian cytochrome c oxidase contain 3 Cu, 2 Fe and 1 Zn per monomeric catalytic unit. PMID- 3022732 TI - Inositol phospholipid and intracellular calcium metabolism in B lymphocytes stimulated with antigen. AB - Stimulation of B lymphocytes by anti mu antibody can activate the phosphatidylinositol pathway, but B cell activation by LPS does not involve this pathway. This study was done to determine if stimulation of B lymphocytes by their specific antigen involves this important activation pathway. We showed that levels of IP2 and IP3 increase while PIP and PIP2 decline when dinitrophenyl specific B lymphocytes are stimulated with the antigen DNP-Ficoll. Intracellular calcium concentration also increases with this stimulus. Thus, antigen stimulation of B lymphocytes is associated with activation of phosphatidylinositol pathway. PMID- 3022734 TI - Reversible self-phosphorylation of asparaginase complex in Leptosphaeria michotii: characterization of associated protein kinase and protein phosphatase activities. AB - Regulation of the asparaginase activity rhythm in L. michotii has previously been shown to be dependent on a reversible phosphorylation process. Asparaginase was isolated as a purified protein complex having self-phosphorylating capacities, which were analyzed. In vivo phosphorylation of asparaginase complex was performed synchronously with the rhythm of asparaginase activity. In vitro self phosphorylation of asparaginase complex resulted from the activity of an ATP-Mg2+ dependent protein kinase, which phosphorylated protein at threonine residues and was not dependent on cyclic AMP, Ca2+ or calmodulin. Dephosphorylation of this complex was due to a Mg2+-Zn2+-dependent protein phosphatase, molybdate inhibited, the specificity of which, for low-molecular-weight nonprotein phosphoesters, was broad. PMID- 3022733 TI - Activation of NADPH oxidase and phosphorylation of membrane proteins in human neutrophils: coordinate inhibition by a surface antigen-directed monoclonal antibody. AB - Exposure of human neutrophils to low concentrations of phorbol myristate acetate (PMA) results, after a brief lag, in the production of superoxide anion and the phosphorylation of membrane proteins. Evidence that these responses are linked has now been obtained using a monoclonal antibody directed against an undefined macrophage surface antigen. The addition of this antibody, which recognizes a 90 kDa neutrophil membrane protein, caused dose-dependent delays in the onset of both phosphorylation of neutrophil membrane proteins and in the appearance of superoxide anion, following addition of PMA to the cell suspensions. For each response the lag period increased with increasing concentrations of antibody, but the onset of phosphorylation always preceded by a few minutes the initial appearance of superoxide anion. PMID- 3022735 TI - A use for principal coordinate analysis in the comparison of protein sequences. AB - Principal Coordinate analysis (PCO) was applied to the comparison of protein sequences. A similarity matrix was derived from a dataset containing 21 c-type cytochrome sequences and this was analysed using PCO to produce a plot of the first three principal axes. The relationships indicated from this plot are considered in conjunction with those derived by cluster analysis using the UPGMA method, and the advantages offered by a nonhierarchical method of sequence comparison discussed. PMID- 3022736 TI - Insulin receptor autophosphorylation and kinase activity in streptozotocin diabetic rats. Effect of a short fast. AB - Insulin receptor associated kinase activity and its relationships with the insulin resistance of streptozotocin-induced diabetes were investigated in rats, using solubilized, partially purified insulin receptors from liver membranes. Insulin receptor kinase activity was measured by means of both autophosphorylation and phosphorylation of the exogenous substrate Glu4:Tyr1. Diabetes was associated with a 45% reduction in kinase activity, in the same number of insulin receptors, with no change in insulin binding affinity. To investigate the independent roles of hyperglycemia and hypoinsulinemia on the observed impairment of receptor kinase activity, diabetic rats were fasted for 24 h in order to normalize blood glucose levels only. After this short fast, no change in kinase activity, from the values measured in fed diabetic animals, was observed. Our findings suggest that streptozotocin diabetes is associated with a reduction of insulin receptor kinase activity, which a short fast is not able to reverse. PMID- 3022737 TI - Human genetic defect in leukotriene C4 synthesis. AB - Normal human platelets metabolise [3H]-LTA4 into [3H]-LTC4. Platelets from patients with glutathione synthetase deficiency possessing 10-30% of normal levels of cellular glutathione showed marked reduction in capacity to form [3H] LTC4 (8-10% of normal) even though exogenous reduced glutathione was added to the incubation medium. To our knowledge this is the first demonstration of a genetic defect in LTC4 synthetase coupled to a defect in cellular glutathione levels. PMID- 3022738 TI - Determination of the rate of rapid pH equilibration across isolated sarcoplasmic reticulum membranes. AB - The rate of the transmembrane pH equilibration in the isolated vesicles of sarcoplasmic reticulum of skeletal muscle after extravesicular pH jump was determined for the first time. A highly water-soluble pH sensitive fluorescent dye, 8-hydroxy-1,3,6-pyrenetrisulphonic acid (pyranine), was used as intravesicular pH indicator in the stopped-flow fluorophotometry. The pH of the medium was controlled with 20 mM HEPES-K or PIPES-K. The fluorescence intensity changed monophasically as much as expected from its pH dependency for imposed delta pH. The half time for initial pH of 7.53 or 6.83 was about 6 msec. The H+/OH- permeability was 11 cm/sec. The results suggested that each vesicle contained large numbers of H+/OH- channels. PMID- 3022739 TI - Specific binding and proteolytic inactivation of bradykinin by membrane vesicles from pig intestinal smooth muscle. AB - A preparation of closed membrane vesicles derived from the longitudinal and circular smooth muscle of pig small intestine was enriched eight-fold in the activity of 5'-nucleotidase and six-fold in the activity of peptidyl dipeptidase A relative to the tissue homogenate. The membrane vesicles specifically bound [3H]bradykinin and the concentration of bradykinin required to inhibit 50% binding was 0.76 +/- 0.05 nM. This concentration was not significantly different from the corresponding concentration of lysyl-bradykinin (0.45 +/- 0.13 nM) but was less (P less than 0.05) than the concentration of methionyl-lysyl-bradykinin (1.25 +/- 0.10 nM). The concentration of des-Arg9 bradykinin (7.5 microM) required for 50% inhibition was greater than 10(3) times less than bradykinin indicating the presence of a B2-type receptor. The membrane vesicles also degraded bradykinin and the principal metabolite was identified as bradykinin. Des-Arg1 bradykinin, des-Arg9 bradykinin and bradykinin were also formed in low yield. Cleavage of the Pro7-Phe8 bond was inhibited by phosphoramidon but not by enalapril or captopril indicating that proteolytic inactivation of bradykinin in the muscle layer of the intestine is mediated through endopeptidase 24.11 ("enkephalinase") but not through peptidyl dipeptidase A ("angiotensin-converting enzyme"). PMID- 3022740 TI - Induction of functional beta-adrenergic receptors in rat aortic smooth muscle cells by sodium butyrate. AB - Rat aortic smooth muscle cells in culture (A-10; ATCC CRL 1476) exhibited low levels of beta-adrenergic receptors as determined by specific binding of [125I]cyanopindolol ([125I]CYP) and marginal stimulation of adenylate cyclase in plasma membranes by (-)isoproterenol. When these cells were exposed to 5 mM sodium butyrate, the number of beta-adrenergic receptors and the beta-agonist stimulated adenylate cyclase activity increased markedly. However, basal, GTP, Gpp(NH)p, and fluoride-stimulated activities did not change. The induction of beta-adrenergic receptors and beta-agonist stimulated adenylate cyclase activity was time- and dose-dependent, and was relatively specific for sodium butyrate. Propionate and valerate were less effective than butyrate, while isobutyrate, succinate, and malonate were ineffective. The induction involved RNA and protein synthesis because induction was prevented by treatment with cycloheximide, puromycin, and actinomycin D. Butyrate did not cause a general increase in cell surface receptors, because the number of vasopressin receptors did not change. The sustained presence of butyrate appeared to be necessary for the maintenance of the induced beta-receptors. When butyrate was removed, receptor number and beta-agonist-stimulated adenylate cyclase activity were decreased by 90% over 24 hr. We conclude that the poor response of rat aortic smooth muscle cell plasma membranes to beta-adrenergic agonists is due to the presence of a low number of beta-adrenergic receptors. Butyrate markedly increased the number of beta receptors which resulted in a proportional increase in beta-agonist-stimulated adenylate cyclase activity. The increase in receptor number was dependent on RNA and protein synthesis. Butyrate treatment did not affect the activity of the cyclase unit and the efficiency of coupling between the receptors and the guanine nucleotide regulatory protein, Ns. PMID- 3022741 TI - Modification of sarcolemmal enzymes by chagasic IgG and its effect on cardiac contractility. AB - It has been shown that sera from chagasic patients contain an antibody which binds to beta-adrenoceptors of myocardium and modulates their activity. Chagasic IgG triggered a marked stimulation of myocardial contractility with an increase in intramyocardial cyclic AMP and inhibition of (Na+ + K+)-ATPase activity. Both the mechanical and enzymatic effects of the IgG could be prevented by beta adrenoceptor blockade or after the absorption of chagasic IgG with turkey red blood cells. In contrast, guinea pig red blood cells were unable to remove the beta-reactivity of chagasic IgG. These findings suggest that the IgG from chagasic patients increases myocardial contractility by behaving as a beta agonist. This effect is likely related to stimulation of the adenylate cyclase coupled to the cardiac beta-adrenoceptor. PMID- 3022742 TI - Multiple muscarinic receptors in the CNS. Significance and prospects for future research. PMID- 3022743 TI - Effects of acute and chronic ethanol administration on regional thiamin pyrophosphokinase activity of the rat brain. AB - Thiamin pyrophosphokinase (TPKase) activity was determined in supernatants of cerebral cortex, cerebellum, pons, medulla, hypothalamus and corpus callosum homogenates obtained from normal rats and from rats given ethanol acutely (a single dose of 4.7 g X kg-1 body wt) or chronically (4.7 g X kg-1 body wt daily for 35 days) by gastric gavage. Regional cell densities (derived from DNA content) and protein contents were also determined. TPKase was detected in all brain regions investigated, the highest activity being found in the cerebellum or in the pons depending on whether it was expressed per mg of protein or per number of cells, respectively. In samples taken following acute ethanol administration protein content was unaffected, while TPKase activity was significantly reduced in the cerebellum, cerebral cortex and hypothalamus at 90 min and in the cerebellum and cerebral cortex at 300 min. Chronic ethanol intake was associated with a significant decrease in regional cell densities, protein contents and TPKase activity. The addition of ethanol to the incubation medium of normal tissue supernatant caused a dose-dependent inhibition of TPKase activity. These results suggest that ethanol markedly impairs thiamin cellular utilization, which may result in depression of brain metabolism. PMID- 3022744 TI - Interaction of clonidine with human placental Na+ -H+ exchanger. AB - The effect of clonidine, an alpha 2-adrenergic receptor agonist, on the Na+ -H+ exchanger in human placental brush-border membrane vesicles was examined. The exchanger was inhibited by clonidine. The inhibition was freely reversible, and the apparent inhibition constant for the process was 250 microM. The nature of inhibition was found to be competitive with respect to Na+. The Dixon plot (1/v versus clonidine concentration) was linear (r2 = 0.998), indicating the interaction of the drug with a single site on the exchanger protein. Similar kinetic analyses with amiloride, a potassium-sparing diuretic, and cimetidine, a histamine type II receptor antagonist, revealed that these drugs also inhibited the Na+ -H+ exchanger by interacting with a single site on the protein. The presence of clonidine increased the intercepts without affecting the slopes of the l/v versus amiloride concentration and the l/v versus cimetidine concentration plots. These results demonstrate that all three drugs, amiloride, cimetidine and clonidine, interact with the human placental Na+-H+ exchanger at a single site in a mutually exclusive manner, and the site of interaction is identical with the Na+-binding site on the external surface of the exchanger protein. PMID- 3022745 TI - Effects of the affinity ligands 14-beta-chloroacetylnaltrexone and 14-beta bromoacetamidomorphine on [3H]-dihydromorphine binding sites in rat brain. AB - The aim of the present study was to examine the inhibitory effects in vitro of the affinity ligands 14-beta-chloroacetylnaltrexone (CAN) and 14-beta bromoacetamidomorphine (BAM) to characterize the pharmacological specificity of the ligands for high and low affinity opioid binding sites. Rat brain membranes were incubated with 2.0 microM BAM or CAN, or their parent compounds (morphine and naltrexone, respectively) at 37 degrees for 45 min, and the membranes were washed extensively to remove the unbound ligand. The specific binding of 0.3 nM [3H]dihydromorphine ([3H]DHM) was reduced 32 +/- 7% in membranes treated with CAN and BAM, whereas specific binding in preparations treated with morphine and naltrexone was not significantly different from controls. An increased affinity of BAM and CAN relative to morphine and naltrexone could not account for the observed irreversible inhibition, since the relative affinity of CAN was similar to that of naltrexone and that of BAM was 10-fold less active than morphine. Saturation binding assays revealed that the affinity ligands selectively abolished a high affinity binding site (Kd = 0.3 nM, Bmax 95 fmoles/mg protein), which comprised approximately one-third of the total number of sites. The affinity of the remaining site (Kd = 4.0 nM) was not altered significantly. The results indicate that the inhibition caused by the affinity ligands is irreversible and represents inactivation of high affinity opioid binding sites in a relatively selective manner. PMID- 3022747 TI - Comparison of the action of cholecystokinin, carbachol and vasoactive intestinal peptide on receptor-activated formation of cyclic GMP and cyclic AMP in the striatum and the pancreas. AB - Sulphated cholecystokinin octapeptide (CCK-8S) and sodium nitroprusside (SNP) increased the formation of cyclic GMP in rat striatal slices with no effect on cyclic AMP. CCK-8S, SNP and carbachol increased the formation of cyclic GMP in guinea-pig pancreatic lobules, but had no effect on levels of cyclic AMP. Vasoactive intestinal peptide (VIP) significantly stimulated the formation of cyclic AMP in both striatal and pancreatic tissue without effect on levels of cyclic GMP in these tissues. In rat striatal slices carbachol significantly inhibited the VIP-stimulated increase in cyclic AMP. PMID- 3022746 TI - The effect of ketoconazole on adrenal and testicular steroidogenesis in vitro. AB - The effect of the anti-fungal agent, ketoconazole, on cortisol secretion from adrenal cells stimulated with ACTH (1-24, 50 ng/l) and on testosterone secretion from Leydig cells stimulated with LH (5 i.u./l), has been tested. The concentration of drug which inhibited cortisol and testosterone secretion by 50% (ED50) was 3.6 +/- 0.7 mumol/l and 0.61 +/- 0.03 mumol/l, respectively. The effect of ketoconazole on adrenal and testicular steroidogenesis was completely reversible. Thus, adrenal and testicular cells which had been washed after exposure to greater than 95% inhibitory dose of ketoconazole responded in a similar manner to hormone stimulation as cells similarly washed and which had not been exposed to drug. The sites of the anti-steroidogenic effect of ketoconazole have been established using a method based upon the sequential stimulation by the exogenous precursor steroids of the various steps leading to the biosynthesis of cortisol by adrenal cells and testosterone by Leydig cells. We conclude that ketoconazole reversibly inhibits the sequence between ACTH/LH binding and pregnenolone production and also inhibits testicular C-17-C-20, lyase activity. PMID- 3022749 TI - Modulation of the number of muscarinic receptors in mouse neuroblastoma cells by soman. AB - Long-term preincubation at 37 degrees of mouse neuroblastoma cells (clones NS-20 and N1E-115) with soman, a potent and irreversible cholinesterase inhibitor, resulted in a significant decrease in the number of [3H]N-methylscopolamine binding sites and in the inhibition of carbamylcholine-induced cyclic GMP formation. The disappearance of surface muscarinic receptors and the desensitization of the receptor-mediated response seem to occur via accumulation of acetylcholine in the culture medium. The significance of these findings is discussed. PMID- 3022748 TI - Differential effects of the cytochrome P-450/reductase ratio on the oxidation of ethanol and the hydroxyl radical scavenging agent 2-keto-4-thiomethylbutyric acid (KMBA). AB - NADPH-cytochrome P-450 reductase catalyzes a low rate of oxidation of hydroxyl radical scavenging agents such as ethanol and 2-keto-4-thiomethylbutyric acid (KMBA), in a reaction markedly stimulated by the addition of ferric-EDTA. The effect of various ratios of cytochrome P-450 (phenobarbital-inducible isozyme)/reductase on the oxidation of ethanol and KMBA was determined: There was essentially no increase in KMBA oxidation over the range of ratios from 0.5 to 5 as compared to the reductase-catalyzed rate. High ratios actually caused some decrease in KMBA oxidation, which was especially notable under conditions of increased rates of hydroxyl radical generation (presence of increasing amounts of ferric-EDTA). This decrease at high P-450/reductase ratios could reflect tight coupling of reductase to P-450-PB, therefore decreasing electron transfer from reductase to ferric-EDTA, or could involve non-specific scavenging of .OH by P 450-PB. Indeed, native, but not boiled, P-450 inhibited KMBA oxidation by the xanthine oxidase system. By contrast, the oxidation of ethanol was stimulated at all concentrations of P-450-PB, and this increase was not sensitive to desferrioxamine. However, under conditions of high rates of .OH production, the ethanol oxidation profile tended to resemble the KMBA oxidation profile with respect to the effect of P-450-PB, whereas the two profiles were different under conditions of low rates of .OH production. These results suggest that P-450-PB does not catalyze the oxidation of .OH scavengers or promote the production of .OH, even at ratios of P-450/reductase approaching those found with intact microsomes and even in the presence of excess iron-EDTA, whereas ethanol is directly oxidized by P-450-PB, as are typical drug substrates. However, the P-450 PB-dependent oxidation of ethanol can be masked under conditions in which .OH production is increased. PMID- 3022750 TI - Chemical modification of alpha 2-adrenoceptors. Possible role for tyrosine in the ligand binding site. AB - Tetranitromethane (TNM) is a reagent which reacts with the tyrosine and cysteine residues of proteins. Chemical modification of partially purified human platelet alpha 2-adrenoceptors with TNM resulted in an irreversible loss of binding activity. Typically, an 80-90% decrease in binding activity occurred with a 60 min exposure to 320 microM TNM. The loss of alpha 2-adrenoceptor activity caused by TNM could be prevented if alpha 2-adrenergic ligands were present during exposure of the receptor to TNM. The protection afforded by alpha 2-adrenergic ligands was dose-dependent and showed a positive correlation with the affinity of the ligand for the alpha 2-adrenoceptor. Prazosin, an alpha 1-specific antagonist, and propranolol, a beta-adrenergic antagonist, did not protect alpha 2-adrenoceptors against the inactivation caused by TNM. Saturation curve analysis revealed that the decrease in alpha 2-adrenoceptor activity caused by TNM was due to a decrease in Bmax with no change in Kd. alpha 2-Adrenoceptors were also inactivated with the sulfhydryl-specific reagent phenylmercuric chloride (PMC). The receptor inactivation caused by PMC could be reversed completely by subsequent treatment with dithiothreitol. Treatment of alpha 2-adrenoceptors with combinations of TNM and PMC showed that the receptor inactivation caused by TNM was most likely due to an interaction with tyrosine residues. These results indicate that tyrosine residues have a function in the conformational stability of alpha 2-adrenoceptors and may be directly involved with ligand binding to the receptor. PMID- 3022751 TI - Alpha-adrenoceptor-mediated actions of optical isomers and desoxy analogs of catecholimidazoline and norepinephrine in human platelets: in vitro. AB - Adrenoceptor-mediated effects of the enantiomers of optically active imidazoline, 2-(3,4,alpha-trihydroxybenzyl imidazoline (catecholimidazoline; CI), and norepinephrine (NE), and the corresponding desoxy derivatives, 2-(3,4 dihydroxybenzyl)imidazoline (desoxy-CI) and dopamine, have been investigated in human platelets. Differences between responsiveness of platelets from donor to donor were observed in the presence of the isomers and the desoxy analogs of NE and CI. In certain platelet preparations, all compounds gave concentration dependent stimulatory responses, whereas in other preparations, only R(-)-NE and R(-)-CI were inducers of platelet aggregation and serotonin release. The rank order of stimulatory potencies (EC50; microM) for CI and NE was R(-)-NE (1.3) greater than R(-)-CI (7.5) greater than S(+)-NE (19) = S(+)-CI (20) = dopamine (22) greater than desoxy-CI (greater than 35). Unlike R(-)-CI, both S(+)-CI and desoxy-CI were either agonists or antagonists of human platelet function. In preparations unresponsive to the S(+)-isomers or desoxy analogs, the potencies (EC50) for R(-)-NE and R(-)-CI were 1.7 and 7.7 microM respectively. The corresponding inactive CIs [S(+)-CI and desoxy-CI] were inhibitors of both primary and secondary phases of aggregation and serotonin release responses to R( )-CI and R(-)-NE, respectively. In contrast, the aggregation responses to ADP, arachidonic acid or U46619 were not blocked by S(+)-CI or desoxy CI. The rank order of inhibitory potencies for selected alpha-adrenoceptor agents against R(-) NE was phentolamine greater than clonidine greater than desoxy-CI greater than S(+)-CI. Moreover, the relative inhibitory potencies of phentolamine and desoxy CI against aggregation responses to R(-)-NE and R(-)-CI, respectively, were the same. These results suggest that the enantiomers and desoxy derivatives of CI and NE mediate their effects in human platelets by an interaction with alpha adrenoceptors; catecholamines and imidazolines interact with the same alpha adrenoceptors in human platelets; the stereochemical requirements of both chemical classes for stimulatory activity in human platelets adhere to the Easson Stedman hypothesis in this alpha 2-adrenoceptor system; and desoxy-CI possessed the highest potency as an antagonist of alpha-adrenoceptors which suggests that the hydroxy group at the benzylic carbon atom of these imidazolines may not be required for maximal binding to adrenoceptors in platelets. PMID- 3022752 TI - Evidence that the carbocyclic analog of adenosine has different mechanisms of cytotoxicity to cells with adenosine kinase activity and to cells lacking this enzyme. PMID- 3022754 TI - [A new specific endodeoxyribonuclease from Escherichia coli RFL 72]. AB - A restriction endonuclease Eco72I with a novel substrate specificity has been isolated from Escherichia coli strain RFL 72. The enzyme recognizes (Formula: see text) hexanucleotide palindromic sequence and cleaves it, as indicated by the arrows, to produce blunt-ended fragments. PMID- 3022753 TI - [Nucleotide sequences in DNA showing preferential binding with distamycin A and acridine derivatives]. AB - The specificity of DNA X dye binding was studied. Antibiotic distamycin A was bound most strongly to the DNA sequences composed of three or more neighboring A X T pairs. Acrichin and 7-aminoacrichin proved to be weak specific inhibitors binding predominantly within the A X T regions. PMID- 3022755 TI - [Plasmid vector pSSC1 for cloning synthetic polynucleotides and their regeneration with a unique nucleotide sequence]. AB - For subcloning separate synthetic gene fragments, a plasmid vector pSSC1 was constructed by inserting a synthetic polylinker into plasmid pBR 327 at the EcoRI PstI sites. There are two FokI and HgaI sites at the ends of this polylinker in the opposite orientation, with the EcoRI and PstI sites between them. DNA fragments cloned at the EcoRI and PstI sites can be regenerated by either FokI or HgaI, the EcoRI and PstI sites being deleted from the cloned sequences. Such fragments have unique cohesive ends that allows their directed ligation into longer DNA (genes). PMID- 3022756 TI - [Insertion of the cos-site into DNA of phage M13 and its packing in proteins of phage lambda]. AB - The cos-site of lambda phage from pHC79 cosmide is transferred to DNA from M13 mp18 phage. The recombinant DNA thus obtained (MC18) is efficiently packaged into lambda proteins in vitro. The BamHI-HindIII fragment of pGP588 (a pBR322 derivatives containing fragment of human DNA) is subcloned into MC18. Although this pGP588 fragment contains numerous Alu repeats, no essential rearrangements of the insert were revealed. The efficiency infection by recombinant DNA packaged with lambda proteins is about 1 X 10(5) pfu/microgram DNA, whereas in the similar conditions the efficiency of lambda EMBL3A was 1 X 10(6) pfu/microgram. It is assumed that the MC vectors might be suitable for cloning and sequencing large fragments either with cohesive or blunt ends. It opens also the way to construct genomic libraries in single-stranded phages. PMID- 3022757 TI - [Nucleotide sequence of the genome region of the tick-borne encephalitis virus coding for structural virion proteins]. AB - RNA of a flavivirus-tick-borne encephalitis virus (strain Sofjin) was subjected to reverse transcription and the DNA copy was transformed into double-stranded DNA by action of E. coli DNA-polymerase I (Klenow's fragment). This DNA was annealed with pBR322 plasmid. The recombinant plasmids were cloned in E. coli K802. The nucleotide sequence of the inserts of the clones coding for region of structural proteins C, pre-M, E and nonstructural protein ns1 was determined by the Maxam-Gilbert method. The nucleotide sequence of these regions is translatable into an amino acid sequence of proteins without interruption. The amino acid sequences of proteins and nucleotide sequence of genome of the tick borne encephalitis virus are extensively homologous to that found for the flaviviruses Yellow Fever and West Nile. PMID- 3022758 TI - Characterization of rabbit genes for synovial cell collagenase. AB - To provide tools for understanding collagenase gene expression in rheumatoid arthritis, we have isolated and characterized genomic clones for rabbit synovial cell collagenase. These clones represent 2 types of collagenase gene, at least 1 of which is transcribed in synovial fibroblasts. By examining the rabbit genome in situ, we provide evidence that there are only 2 different synovial cell collagenase genes found in a haploid genome. Amplification of these genes is not a mechanism for collagenase messenger RNA induction by phorbol esters. PMID- 3022759 TI - Prevalence of Coxsackie B virus antibodies in patients with juvenile dermatomyositis. AB - A number of viruses have been implicated as being the cause of various forms of myositis, including acute transient myositis, chronic polymyositis, and dermatomyositis. However, the cause of juvenile dermatomyositis (JDM) has remained elusive. Our study of serum samples taken within 4 months of the onset of disease in 12 children with JDM showed that 83% had detectable titers of complement-fixing (CF) antibody to 1 or more coxsackie B viral antigens. Detectable titers were found in only 25% of age-, sex-, and date-matched control sera taken from 24 patients with juvenile rheumatoid arthritis (JRA), and in 25% of serum samples taken from 2,192 "normal" children who had been hospitalized because of viral syndromes. Titers of CF antibody to coxsackie B1, B2, and B4 were positive in 58%, 50%, and 58%, respectively, of the JDM patients. In matched JRA controls, the respective values were 8%, 13%, and 8%. There were no significant antiviral titers and no significant differences in the results of tests for 13 other viral CF antigens, hepatitis B surface antigen, and Mycoplasma pneumoniae CF antigen in the JDM patient sera compared with the JRA patient sera. When titers of neutralizing antibody were determined, 58%, 58%, and 67% of the JDM patients were positive for coxsackie B2, B4, and B5, respectively, whereas 16%, 26%, and 21%, respectively, of the JRA controls were positive for the 3 antigens. These data suggest that the host response to coxsackie B virus might be related to the pathophysiology of JDM. PMID- 3022760 TI - Congenital cytomegalovirus infection and maternal systemic lupus erythematosus: a case report. AB - We describe an infant with symptomatic congenital cytomegalovirus infection, who was born to a mother with active systemic lupus erythematosus. Infection in the child resulted from reactivation of maternal cytomegalovirus infection. The mother's use of prednisone may have contributed to the reactivation. The role of maternal immunosuppression in the acquisition of congenital viral infection by the neonate is discussed. PMID- 3022762 TI - Free radicals in skin exposed to dithranol and its derivatives. AB - The generation of persistent free radical species derived from antipsoriatic drugs in their target tissue under use conditions is reported. Dithranol (anthralin, 1,8-dihydroxy-9-anthrone) and chemical analogues were applied topically to the ear of the pig and the time course of the production of the resulting free radicals was followed by electron spin resonance spectroscopy in biopsy samples. The spectral characteristics observed correspond to data previously reported in vitro for the anthralin-10-yl radical. The role of these radical species in the mode of action of the drug is discussed. PMID- 3022761 TI - Biochemical and physiological evidences for antiserotonergic properties of naftidrofuryl. AB - In experimental and clinical investigations, 2-(diethylamino)ethyl-tetrahydro alpha-(1-naphthyl-methyl)-2-furanpro pionate (naftidrofuryl, Praxilene) appears to improve blood flow and microcirculation in ischemic areas. In order to define the mechanism by which the drug may exert its vascular effects, the binding affinity of naftidrofuryl toward various receptors was studied. From this biochemical study, it appeared that, in therapeutic doses, naftidrofuryl selectively inhibited the binding of spiperone or ketanserin to serotonin S2 receptors. This finding was corroborated in physiological models such as isolated rat caudal arteries or preparations of aortic myocytes. Naftidrofuryl effectively blocked the constrictive effect of serotonin on the artery in a dose-dependent, competitive manner. It also strongly inhibited the formation of serotonin stimulated inositol triphosphate in the myocytes. From these good correlations between biochemical and physiological data it is concluded that the beneficial effects of naftidrofuryl on ischemic tissue perfusion may be partly explained on the basis of selective antiserotonergic S2 properties. PMID- 3022763 TI - The effects of marijuana smoke on gas exchange in ovine pregnancy. AB - The effects of marijuana smoke on maternal respiratory rate and gas exchange were examined in nine chronically instrumented, late gestation ewes carrying singletons. The magnitude of exposure was randomly varied producing peak plasma levels of delta-9-tetrahydrocannabinol (delta-9-THC) ranging from 0 to 161 ng/ml. delta-9-THC levels, respiratory rate and arterial blood gas tensions were monitored before and for two hours after inhalational exposure. When compared to placebo, marijuana smoke produced a dose dependent and sustained decrease in maternal respiratory rate and arterial oxygen tension without evidence of either systemic acidosis or carbon dioxide retention. A logarithmic relationship was observed between the blood level of delta-9-THC and the change in respiratory rate. The change plateaued at 30% of control at levels above 80 ng/ml. However, the relationship between the blood level of delta-9-THC and the change in arterial oxygen tension had a linear fit with a maximum decrease of 45% at a blood level of 160 ng/ml. No change was detected in minute ventilation. Fetal oxygen tension fell significantly and remained depressed after maternal values had returned to control levels. We conclude that, in this species, inhalational exposure to marijuana smoke induces a prolonged maternal ventilation/perfusion imbalance and limits fetal oxygen availability by one or more mechanisms. PMID- 3022764 TI - Application of toxicological concepts to the occupational history. PMID- 3022765 TI - Neurotoxicity of organic solvents. PMID- 3022766 TI - Dopamine in the lateral caudate-putamen of the rat is essential for somatosensory orientation. AB - The present study examined the possible localization of somatosensory orientation in the caudate-putamen (CP) of the rat. In the first experiment, 6 hydroxydopamine (6-OHDA) was injected into either the anterodorsal (AD), anteroventral (AV), posterodorsal (PD), or posteroventral (PV) CP. Only rats with PV-CP 6-OHDA injections showed impaired orientation scores. However, these PV injections often caused widespread CP dopamine (DA) depletions, and no specific CP region appeared to be particularly associated with somatosensory orientation. In the second experiment, multiple injections of 6-OHDA were directed toward the medial or lateral halves of the CP to assess their relative contributions directly. DA depletions confined to the lateral (but not medial) CP resulted in orientation deficits; these deficits were greater than would be predicted from the volume of CP/DA loss. Furthermore, the magnitude of the DA fluorescence loss in the lateral CP was more highly correlated with the orientation impairment than was the medial CP fluorescence loss. Thus the lateral CP contributes to sensorimotor functions to a greater extent than does the medial CP, but the volume of CP/DA depletion also appears important. PMID- 3022767 TI - Stressor controllability and the pituitary-adrenal system. AB - Stressor controllability can alter both behavior and pituitary-adrenal activity. Potential mediation of these behavioral effects by differential pituitary-adrenal output requires that the precise conditions that lead to differential behavioral consequences also produce differential pituitary-adrenal activity. Both plasma ACTH and corticosterone levels were measured at various times following escapable and yoked inescapable electric shock conditions known to produce differential behavioral outcomes. The escapable and inescapable shock procedures did not produce a detectable differential effect. Both shock conditions produced equivalent elevation of ACTH and corticosterone. Neither decay rates nor the ACTH and corticosterone response to shock reexposure differed among shocked groups. PMID- 3022768 TI - Pairing pentobarbital with one toxin causes it to attenuate taste aversions produced by a different toxin: implications for conditioned antisickness theory. AB - Normally, if pentobarbital and then a toxin are injected after a rat drinks saccharin solution, a taste aversion produced by the pentobarbital summates with that produced by the toxin. An opposite effect is obtained after a preconditioning series in which pentobarbital is injected prior to a toxic dose of lithium or amphetamine in the absence of saccharin drinking: The pentobarbital attenuates the saccharin aversion normally produced by the toxin. Lett (1983) theorized that a conditioned antisickness response (CAR) to pentobarbital is responsible for this conditioned attenuation of saccharin aversion. It is reported here that this attenuation of taste aversion occurs even if the toxin paired with pentobarbital is different from the toxin used during saccharin aversion conditioning. Preconditioning pentobarbital with a high dose of amphetamine allows it to attenuate saccharin aversions produced by lithium and by gamma radiation (as well as by amphetamine itself). Preconditioning pentobarbital with a high dose of lithium allows it to attenuate aversions produced by amphetamine, gamma radiation, cisplatin, mechlorethamine, dactinomycin, and doxorubicin (as well as by lithium itself). This means that the CAR cannot be due to conditioned amelioration of specific effects of specific toxins (which would not be effective if the toxin were changed) and suggests a central alleviation of nausea, perhaps like the alleviation of pain by endogenous opiates. However, aversions produced by intraperitoneal copper sulfate were not attenuated by lithium-conditioned pentobarbital. PMID- 3022770 TI - Enzymatic dephosphorylation of retinyl monophosphate by rat liver. AB - Rat liver has been shown to contain an enzyme that catalyzes the dephosphorylation of retinyl monophosphate. This activity was extracted with 0.1 M Tris buffer (pH 7.5). Maximum reaction rate was observed at a pH range of 7.0 7.5. It did not require metal ions for activity and was sensitive to fluoride ion. The retinyl monophosphate phosphatase activity was proportional to time and protein and substrate concentration. Triton X-100 (range of 0.05-0.10%) increased the activity 100%, whereas other detergents (Tween 80, cholate, and deoxycholate) did not activate the enzyme. A number of phosphorylated compounds tested as inhibitors of retinyl monophosphatase activity, such as glucose 6-phosphate (20 mM), glycerophosphate (20 mM), phosphatidic acid (8 mM), and dolichyl phosphate (3 mM), did not compete with retinyl monophosphate as substrate. However, at 20 mM concentration, ATP, ADP, 5'-AMP, and pyrophosphate were inhibitors of the enzyme. It is not possible at present to give further details about the specificity of the phosphatase activity. The enzyme described could play a regulatory role in retinol-mediated glycosylations, by altering the endogenous level of retinyl monophosphate. PMID- 3022769 TI - Noradrenaline and sensory preconditioning in the rat. AB - Two experiments were performed to investigate the effect of noradrenaline (NA) depletion following systemic administration of the neurotoxin N-(2-chloroethyl)-N ethyl-2-bromobenzylamine (DSP4; 50 mg/kg, ip) on sensory preconditioning in the rat. For sensory preconditioning, a taste (saccharin, CS2) and a special type of drinking bottle (noisy bottle) were paired during Phase 1. During Phase 2, the noisy bottle (CS1) was paired with lithium chloride, and, finally, during Phase 3 the aversion to saccharin (CS2) was tested for in saccharin preference tests. The DSP4 treatment disrupted rats' ability to form sensory preconditioning, and this effect could not be explained on the basis of enhanced neophobia, stimulus generalization, or a deficit in first-order conditioning in DSP4-treated rats. These findings are closely related to these and other issues of associative learning such as contextual control of latent inhibition and extinction. The evidence from the present data suggests that NA-depleted rats fail to form associations between the CS1 and CS2 during sensory preconditioning and, as such, are consistent with other data from various compound conditioning experiments on the functional role of NA in learning and memory. PMID- 3022771 TI - Purification and polyamine activation of a high-affinity 3',5'-cAMP phosphodiesterase from rabbit muscle. AB - A high-affinity phosphodiesterase, termed PDE II, has been purified about 1400 fold from rabbit skeletal muscle. This enzyme is activated by treatment with proteases. It is also activated specifically by polyarginine and arginine-rich histones, but not by other polyanions. The activation is counteracted nonspecifically by polycations, such as heparin and chondroitin sulphate. When the enzyme is fully activated by polyarginine it is no longer susceptible to activation by proteases. A conformational or structural change must thus occur in the enzyme by the binding of the polyanions. PMID- 3022772 TI - Active immunization of children exposed to varicella infection in a hospital ward using live attenuated varicella vaccine given subcutaneously or intracutaneously. AB - Active immunization using Takahashi OKA live attenuated varicella vaccine was carried out fire 5 times to prevent the spread of "imported" varicella in a hospital ward. Susceptibility was previously tested by serological examinations: 14 children were vaccinated subcutaneously, the other 19 received the vaccine intracutaneously. Vaccination within a few days following exposure provided complete immunity in the great majority of cases. Intracutaneous administration was nearly as protective as the subcutaneous one. PMID- 3022773 TI - Hepatic effects of phenobarbitone in female mice fed sublethal levels of dietary cyanide. AB - Female albino mice were fed sublethal doses of KCN (approx. 10 micrograms/mouse/day) for 7 days, injected intraperitoneally with phenobarbitone (50 mg/kg body wt/day) in the subsequent 3 days, and sacrificed 24 hr after the last injection. Phenobarbitone sleeping time was increasingly shortened (16-27%) daily in cyanide-fed mice in comparison with cyanide-free controls. Both compounds administered singly or simultaneously increased the liver weight/body weight ratios by not more than 10%. Aniline hydroxylase, glucose-6-phosphatase, NADPH- and NADH-cytochrome c reductase activities were similarly increased. Aniline hydroxylase activity was most markedly increased (by a factor of 4). The toxicological implications of these results are discussed. PMID- 3022774 TI - Polymyxin B sulfate-induced pH-dependent increase in calcium influx in cultured fibroblasts. AB - Polymyxin B sulfate treatment induced an increase in calcium influx in mouse fibroblasts (3T6) and normal human skin fibroblasts. This increase in calcium influx occurred in a dose- and time-dependent fashion and was dependent on pH but independent of the electrochemical gradient of calcium across the plasma membrane. This effect was prevented when cycloheximide (20 micrograms/ml) was added with polymyxin B sulfate. Addition of actinomycin D (2 micrograms/ml) also remarkably reduced this effect. In view of these findings, it is possible that polymyxin B sulfate therapy-induced side effects, such as neuromuscular blockade and kidney dysfunction, are conditional and may be due to an increase in calcium influx. PMID- 3022775 TI - Characteristics of cultured human renal cortical epithelia. AB - Nine human kidney epithelial cell lines, isolated from small biopsied material and from whole kidney, were propagated in both a hormonally defined medium and a medium supplemented with serum. At confluency, hemicysts or domes, typical of cultured epithelial cells, were formed by these cells. Monolayers had junctional complexes between cells and the presence of numerous microvilli on the cell surface. Parathyroid hormone markedly stimulated these cells to produce cyclic AMP. They also contained high levels of gamma-glutamyltranspeptidase, leucine aminopeptidase, and maltase, enzymes that are associated with the brush-border membrane of the proximal tubule. The cultured cells demonstrated the ability to transport amino acids and alpha-methylglucoside, a substrate actively transported only by the proximal tubule in the kidney. Based on these findings, the cultured cells reflected a number of characteristics associated with the proximal tubule. These renal epithelial cell lines may provide a useful model for studying various aspects of human renal physiology and biochemistry. PMID- 3022776 TI - Effect of erythropoietin on the different ATPases and acetylcholinesterase of rat RBC membrane. AB - The effect of Ep on different ATPases and acetylcholinesterase of rat RBC membrane was studied. Starvation caused a slight decrease in Mg2+-, Ca2+-, and Na+ + K+-ATPases. However, these enzyme activities were markedly increased on Ep treatment of starved rats. Specific activities of all three ATPases increased linearly with increasing concentration of Ep. Under identical conditions the hormone failed to stimulate the ATPase activity of liver plasma membrane. Desensitization by fluoride of allosteric inhibition of erythrocyte membrane bound Na+ + K+-ATPase was observed under starvation which showed a return to normal n values on Ep administration. The enzyme from normal animals was inhibited almost completely at 0.1 mM fluoride whereas enzyme from starved and Ep treated animals showed only about 50% inhibition at that fluoride concentration. Ep increased the acetylcholinesterase activity of normal RBC membrane to a small extent whereas the stimulation was much higher under starvation. The fluoride inhibition curve of this enzyme changed from sigmoidal to hyperbolic under starvation which again changed to allosteric on administration of Ep. These changes were closely correlated to n values. Red blood cells of Ep-treated animals became more susceptible to osmotic shock under the experimental conditions. PMID- 3022777 TI - A bioartificial pancreas prevents chronic complications of diabetes in rats. PMID- 3022778 TI - Ventilation, ventilatory carbon dioxide and hormonal response during halothane anaesthesia and surgery in children after midazolam premedication. AB - In 14 intubated, spontaneously breathing children with body weight (bw) ranging from 8.3 to 25.6 kg, the influence of midazolam 0.1 mg kg-1 i.m. (group M0.1, n = 7) and 0.2 mg kg-1 i.m. (group M0.2, n = 7) as premedication, on sedation, ventilation, ventilatory response to carbon dioxide and hormonal stress response was studied in connection with minor surgical procedures during halothane anaesthesia. The concentrations of catecholamines, ACTH and cortisol were measured immediately after induction, during undisturbed anaesthesia, during surgery and 15 min after the end of the surgical procedure. Sedation was better and plasma catecholamine concentrations during undisturbed anaesthesia were less in children receiving the larger dose of midazolam. During surgery and in recovery there were no differences in hormone concentrations. In recovery, the concentrations of all hormones were significantly greater compared with during undisturbed anaesthesia. During surgery, VE and respiratory rate were somewhat lower in group M0.2 while E' CO2 was similar. A dose dependent depression of the response to carbon dioxide was found. However, clinically, the ventilatory response to carbon dioxide after surgery was considered to be adequate in both groups. PMID- 3022779 TI - Drug sensitivity of non small cell carcinoma of lung by clonogenic assay in several media. AB - Lung tumours of non small cell pathology were cultured by clonogenic assay in several media. Culture was successful in spleen conditioned medium, but only 57% grew and low plating efficiencies (PE) meant that only 23% of the original number produced significant drug results. Comparison of rat erythrocyte lysate (REL) medium with serum free defined medium (HITES) and HITES + 10% FBS demonstrated clear enhancement of PE in REL although growth was 100% successful in all these media. Ninety-three percent of samples tested against drugs in REL produced significant results. A later comparison of REL with McCoy's 5A + rbc +/- hydrocortisone produced relatively poor culture success for these 3 media and equivocal growth patterns. Low PE was attributed to age of rats used for rbc. Vindesine and cis-platinum cytotoxicity in spleen conditioned medium were 61% and 15% sensitivity respectively. These do not concur with clinical experience but the figures for overt resistance, at 39% and 69%, correspond with expected non responders to these regimes. Drug testing in REL produced figures correlating more closely with clinical performance at 45% sensitivity to platinum and 36% of patients sensitive to both drugs, but the vindesine sensitivity at 55% is again discrepant with performance of this drug as a single agent. PMID- 3022780 TI - The use of expert surrogates to evaluate clinical trials in non-small cell lung cancer. AB - One hundred and eighteen doctors who treat pulmonary neoplasms in Ontario were asked how they would wish to be treated if they had non-small cell lung cancer. Four different scenarios were given. The physicians were then asked if they would consent to take part as subjects in one or more clinical trials for which they would be eligible in those situations. The proportion of respondents who would consent to each study ranged from 11% to 64%. Reasons given for refusing to participate as subjects in each trial were varied, but many felt that the trials offered unacceptable options for treatment. Medical oncologists consented to each study more frequently than radiation oncologists, respirologists or thoracic surgeons but all disciplines ranked the 6 studies in the same order of acceptability. It is concluded that some patients with non-oat cell lung cancer currently receive experimental therapies with high risk/benefits ratios which experts in the field would not accept for themselves. It is suggested that the expert surrogate system may be useful as an adjunct to the institutional review board in evaluating new trials before they are activated. PMID- 3022781 TI - Time trends in prevalence of cervical cytological abnormality in women attending a sexually transmitted diseases clinic and their relationship to trends in sexual activity and specific infections. AB - Trends in prevalence of cytological evidence of cervical intraepithelial neoplasia (CIN) and cervical infection with human papilloma virus (HPV), as indicated by HPV infection and dyskeratosis, were studied in 2,992 new attenders at a sexually transmitted diseases (STD) clinic between 1978 and 1982. Crude prevalence of CIN increased from 1.3% to 4.3% (P less than 0.001) and crude prevalence of HPV infection increased from 2.8% to 9.3% (P less than 0.001). Age adjustment had little effect on these trends. Review, in 1984-85, of samples of smears taken in 1978 and 1982 showed that recognition of koilocytosis by the laboratory had increased substantially over time while a tendency had developed to downgrade nuclear changes in the presence of koilocytosis. Correction of the 1978 and 1982 smear results to the 1984-85 classifications suggested that prevalence of koilocytosis had increased little (from 13.4% to 16.1%, P = 0.20) while there had been a substantial real increase in CIN (0.8% to 2.4%, P less than 0.001). To try to explain the trend in CIN, other characteristics of a sample of attenders at the STD clinic were studied. There were no appreciable trends in prevalence of past STD, number of sexual partners in the last 3 months, method of contraception, genital warts and culture of N. gonorrhoea, T. vaginalis, C. albicans and Chlamydia sp. from the vagina. There was an increase in the proportions in socioeconomic group I, as classified by postcode of residence (17.0% to 26.9%, P = 0.04), referred as contacts rather than with symptoms (24.0% to 41.6%, P less than 0.001), with a clinical diagnosis of genital herpes (5.0% to 8.6%, P = 0.08) and with herpes virus cultured from the cervix (2.1% to 6.3%, P = 0.03). The trend in prevalence of herpes virus infection was not explained by the other trends. It may explain the trend in prevalence of CIN. PMID- 3022782 TI - Degradation of porcine dermal connective tissue by collagenase and hyaluronidase. AB - Selective destruction of connective tissue may be a useful therapeutic tool in conditions associated with abnormal deposition of scar tissue. We have investigated intradermal injections of clostridial collagenase and bovine testicular hyaluronidase alone and in combination in Yucatan miniature hairless pigs. Collagenase in combination with hyaluronidase was quite efficient at destroying the connective tissue matrix, although elastic tissue appeared to be completely spared. Collagenase alone at higher doses degraded collagen, but hyaluronidase had little effect on connective tissue architecture. PMID- 3022784 TI - An open study of vitamin D3 treatment in psoriasis vulgaris. AB - Active forms of vitamin D3, 1 alpha-hydroxyvitamin D3 and 1 alpha,25 dihydroxyvitamin D3, were administered in an open-design study to 40 patients with psoriasis vulgaris in three ways: to 17 patients 1 alpha-hydroxyvitamin D3 was given orally at a dose of 1.0 micrograms/day for 6 months, to four patients 1 alpha,25-dihydroxyvitamin D3 was given orally at a dose of 0.5 microgram/day for 6 months, and 19 patients were given 1 alpha,25-dihydroxyvitamin D3 applied topically at concentration of 0.5 microgram/g of base for 8 weeks. Improvement was observed at the end of the individual study periods in 13 (76%) patients in Group 1 with a mean period of treatment (+/- SD) of 2.7 +/- 0.6 months, in one patient in Group 2 at 3 months after the start of treatment, and in 16 (84%) patients in Group 3 when the chemical was applied for 3.3 +/- 1.2 weeks. No side effects were observed in any of these trials. These data suggest that psoriasis may respond to active metabolites of vitamin D3 and that abnormalities in vitamin D metabolism or in responsiveness of the skin cells to active metabolites of vitamin D may be involved in the pathogenesis of this skin disease. PMID- 3022783 TI - Immunolocalization of collagenase and tissue inhibitor of metalloproteinases (TIMP) in hypertrophic scar tissue. AB - Normal, mature and hypertrophic dermal scars were examined by indirect immunofluorescence for the presence of collagenase, tissue inhibitor of metalloproteinases (TIMP) and cathepsin D. Significant extracellular immunoprecipitation of both collagenase and TIMP were found in areas of all scars judged to be actively remodelling, whereas inactive areas were predominantly negative. TIMP was also present in endothelial cells of patent blood vessels, but found not to be present in the enlarged endothelial cells of occluded vessels. Negligible amounts of extracellular cathepsin D were found. PMID- 3022785 TI - The effect of interferon on human papillomaviruses associated with cervical intraepithelial neoplasia. AB - A double-blind, placebo-controlled, trial of leucocyte interferon showed that, contrary to previous reports, interferon had no significant effect on cervical intraepithelial neoplasia (CIN) when applied topically in a gel. DNA hybridization of cervical scrapes was used to monitor the effect of interferon on the human papillomaviruses (HPV) associated with CIN. There was, however, no significant difference in the expression of HPV 6 or 16 in the cervical epithelium of patients treated with interferon compared with those given a placebo. By using superficial cells scraped from the surface of the cervical epithelium as a source of DNA for viral studies, we were able to investigate the relation between HPV and CIN without interfering with the natural history of the disease. HPV 16 was detected in lesions which persisted while HPV 6 only was detected in one lesion that regressed. Regression was clearly associated with reduction in the number of copies of viral DNA per cell in this case. Dual infection with HPV types 6 and 16 were recorded in two patients with persistent lesions. In one patient, hybridization studies indicated that infection with HPV 16 could have occurred after infection with type 6 was established, and it is postulated that this may have changed the nature of the cervical lesion. PMID- 3022786 TI - Carcinoma of the gall bladder metastatic to the cervix. Case report. PMID- 3022787 TI - Past, present, and future surgical management of malignant epithelial neoplasms of the lacrimal gland. PMID- 3022788 TI - Complementary DNA derived structure of the amino-terminal domain of human apolipoprotein B and size of its messenger RNA transcript. AB - In this paper the sequence of a 5.2-kilobase (kb) cDNA covering the amino terminal domain of human apolipoprotein B-100 (apoB-100) is reported. The cDNA derived protein sequence provides the primary structure of 1748 amino acids. This segment of apoB-100 is more hydrophilic than hydrophobic and contains short stretches of predicted helical and beta structures that are interrupted by beta turns. Blotting analysis of RNA isolated from fetal human and adult monkey tissues and various human cell lines showed synthesis of apoB mRNA by liver and intestine and by cells of hepatic (HepG2) and intestinal (Caco-2) origin. The isolation and characterization of overlapping cDNA clones, which provide a nearly full-length copy of human apoB-100, are also reported. From the length of these clones the size of the cytoplasmic apoB mRNA is estimated to be 14.0 kb and codes for a protein of approximately 512,000 daltons. PMID- 3022789 TI - Expression of bovine intestinal calcium binding protein from a synthetic gene in Escherichia coli and characterization of the product. AB - Intestinal calcium binding proteins (ICaBP's) constitute a group of small vitamin D inducible proteins considered to play an important role in the absorption of dietary calcium. The mammalian ICaBP's are representatives of the "EF-hand" family of calcium binding proteins. As a first step in the application of protein engineering techniques to the study of structure-function relationships in mammalian ICaBP's, we have synthesized a gene encoding the minor A form (the native form lacking the two N-terminal amino acids) of bovine ICaBP employing a rapid, microscale gene synthesis technique based on "shotgun ligation" of sets of oligonucleotides. Expression of the synthetic gene from a plasmid containing the tac promoter in a lon protease deficient strain of Escherichia coli yielded the desired product at a level of about 1-2 wt % of total protein. During the purification of the ICaBP expressed in E. coli, a contaminant was strongly adhering to it but was efficiently removed by gel filtration after denaturation with urea. The minor A form of ICaBP produced in E. coli was characterized by its mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by its total amino acid composition, partial amino acid sequence, UV absorption spectrum, and 360-MHz 1H NMR spectrum, showing beyond reasonable doubt its identity with the minor A form of ICaBP obtained from bovine intestines. PMID- 3022790 TI - Mechanism of inactivation of human leukocyte elastase by a chloromethyl ketone: kinetic and solvent isotope effect studies. AB - The mechanism of inactivation of human leukocyte elastase (HLE) by the chloromethyl ketone MeOSuc-Ala-Ala-Pro-Val-CH2Cl was investigated. The dependence of the first-order rate constant for inactivation on concentration of chloromethyl ketone is hyperbolic and suggests formation of a reversible "Michaelis complex" prior to covalent interaction between the enzyme and inhibitor. However, the observed Ki value is 10 microM, at least 10-fold lower than dissociation constants for complexes formed from interaction of HLE with structurally related substrates or reversible inhibitors, and suggests that Ki is a complex kinetic constant, reflecting the formation and accumulation of both the Michaelis complex and a second complex. It is proposed that this second complex is a hemiketal formed from attack of the active site serine on the carbonyl carbon of the inhibitor. The accumulation of this intermediate may be a general feature of reactions of serine proteases and chloromethyl ketones derived from specific peptides and accounts for the very low Ki values observed for these reactions. The solvent deuterium isotope effect (SIE) on the inactivation step (ki) is 1.58 +/- 0.07 and is consistent with rate-limiting, general-catalyzed attack of the active site His on the methylene carbon of the inhibitor with displacement of chloride anion. The general catalyst is thought to be the active site Asp. In contrast, the SIE on the second-order rate constant for HLE inactivation, ki/Ki, is inverse and equals 0.64 +/- 0.05.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022791 TI - Rhodopsin kinase prepared from bovine rod disk membranes quenches light activation of cGMP phosphodiesterase in a reconstituted system. AB - Rhodopsin kinase was extracted into a buffer containing 200 mM KCl and no MgCl2. The activity of the enzyme was stabilized with the use of a mixture of protease inhibitors, aprotinin, benzamidine, leupeptin, and pepstatin. The extract consisted of three major proteins of molecular weight (Mr) 65,000, 56,000, and 37,000, of which the Mr 65,000 protein was identified with the kinase activity since preparations containing the other proteins had no kinase activity and the Mr 65,000 protein was phosphorylated when the extract was incubated with ATP. A reconstituted cGMP phosphodiesterase (PDE) system consisting of peripheral protein-depleted rod disk membranes (RDM), GTP binding protein (G-protein), and PDE was used to test the effectiveness of the rhodopsin kinase preparation in mediating the ATP-dependent quench of light activation of PDE. In the absence of kinase, light-activated PDE activity lasted several minutes. In its presence, ATP and to a lesser extent GTP quenched the activation about as rapidly as in rod disk membranes. The influence of kinase was unaffected by increasing G-protein or PDE content of the reconstituted system but was slowed down by brighter flashes, showing that quench was caused by the inactivation of bleached rhodopsin and not of PDE or G-protein. PMID- 3022792 TI - Target-sensitive immunoliposomes: preparation and characterization. AB - A novel target-sensitive immunoliposome was prepared and characterized. In this design, target-specific binding of antibody-coated liposomes was sufficient to induce bilayer destabilization, resulting in a site-specific release of liposome contents. Unilamellar liposomes were prepared by using a small quantity of palmitoyl-immunoglobulin G (pIgG) to stabilize the bilayer phase of the unsaturated dioleoylphosphatidylethanolamine (PE) which by itself does not form stable liposomes. A mouse monoclonal IgG antibody to the glycoprotein D of Herpes simplex virus (HSV) and PE were used in this study. A minimal coupling stoichiometry of 2.2 palmitic acids per IgG was essential for the stabilization activity of pIgG. In addition, the minimal pIgG to PE molar ratio for stable liposomes was 2.5 X 10(-4). PE immunoliposomes bound with HSV-infected mouse L929 cells with an apparent Kd of 1.00 X 10(-8) M which was approximately the same as that of the native antibody. When 50 mM calcein was encapsulated in the PE immunoliposomes as an aqueous marker, binding of the liposomes to HSV-infected cells resulted in a cell concentration dependent lysis of the liposomes as detected by the release of the encapsulated calcein. Neither uninfected nor Sendai virus infected cells caused a significant amount of calcein release. Therefore, the release of calcein from PE immunoliposomes was target specific. Dioleoylphosphatidylcholine immunoliposomes were not lysed upon contact with infected cells under the same conditions, indicating that PE was essential for the target-specific liposome destabilization.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022793 TI - Unique rearrangement of ergocalciferol side chain in vitro: production of a biologically highly active homologue of 1,25-dihydroxyvitamin D3. AB - In vitro incubation of 24-epi-25-hydroxyvitamin D2 with chicken kidney homogenate produced several compounds, one of which had an affinity equal to that of 1,25 dihydroxyvitamin D2 for the chick intestinal receptor. The affinity of 24-epi 1,25-dihydroxyvitamin D2 for the same receptor was found to be half that of 1,25 dihydroxyvitamin D2. The unknown compound was produced only when homogenate was prepared from pooled kidneys taken from both vitamin D deficient and replete chickens. The compound has been tentatively identified as 1,25-dihydroxy-22 dehydro-26-homovitamin D3 by ultraviolet absorption spectrophotometry and mass spectrometry. Chemical synthesis of 1,25-dihydroxy-22-dehydro-26-homovitamin D3 provided additional evidence for the structure. Administration of this 26 homologue of 1,25-dihydroxyvitamin D3 at the dose level of 650 pmol/rat stimulated bone calcium mobilization in the hypocalcemic rat equal to that of 1,25-dihydroxyvitamin D3. Thus, this paper demonstrates unique methyl migration on the side chain of 24-epi-1,25-dihydroxyvitamin D3 to form a more biologically potent analogue. PMID- 3022794 TI - Magnetic resonance studies of fredericamycin A: evidence for O2-dependent free radical formation. AB - Fredericamycin A, a newly described potent antitumor antibiotic, exhibits unusual spectroscopic and physical properties. The drug shows a striking color change from red to blue on exposure to O2, with the appearance of an optical absorption band at 675 nm; on addition of acid these changes are readily reversed. 1H and 13C NMR spectra of fredericamycin A show that the resonances from the quininoid half of the molecule disappear after exposure to O2 but reappear on acidification in parallel with the observed optical spectral shift. These unusual NMR data are explained by electron spin resonance studies which demonstrate that fredericamycin A spontaneously forms an oxidized free radical with electron transfer to O2. The observed hyperfine structure of this radical is consistent with one-electron oxidation of the quininoid group. After fredericamycin A is exposed to O2, an EPR signal is observed with axial symmetry with temperature and power saturation behavior suggestive of .O2-. Spin-trapping EPR studies demonstrate that the drug reduces O2 to .O2- and H2O2 to .OH. This spontaneous mechanism of O2 reduction with the generation of oxidized drug free radicals and reduced oxygen free radicals is unprecedented among anticancer drugs, suggesting that fredericamycin A could be the forerunner of a new class of anticancer drug. PMID- 3022795 TI - Role of a second catabolite activator protein molecule in controlling initiation of transcription at the galactose operon of Escherichia coli. AB - The molecular mechanisms whereby RNA polymerase, catabolite activator protein (CAP), and cyclic AMP (cAMP) participate in transcriptional regulation at the galactose operon have been probed by a variety of in vitro techniques. Interactions between purified proteins and promoter-containing DNA fragments were assayed by gel electrophoresis, by resistance to restriction endonuclease digestion, and by monitoring runoff transcripts. The data bear on the multiple functions that CAP performs in gal control. A CAP-cAMP complex can exclude RNA polymerase from one of the two overlapping promoter regions (P2), thereby targeting the enzyme to the other (P1); this process is markedly influenced by the cAMP level. In addition, a second CAP molecule is involved in a cooperative process, which, at low cAMP, is required for efficient formation of transcriptionally competent complexes at P1. This second CAP may serve to stabilize the 1:1:1 CAP-polymerase-gal DNA intermediate under physiological conditions, thus enhancing initiation from P1 relative to P2. Kinetic analysis reveals that the modest effect of CAP on the rate of P1 open complex formation can be resolved into about a 4-fold increase in the binding of RNA polymerase to the P1 region, plus a 1.5-fold elevation in the rate of isomerization of enzyme promoter complexes to the open state. PMID- 3022797 TI - Galactose-1-phosphate uridylyltransferase. Purification of the enzyme and stereochemical course of each step of the double-displacement mechanism. AB - A convenient new procedure for purifying galactose-1-phosphate uridylyltransferase from Escherichia coli is described. It departs from earlier methods by introducing the use of a Cibacron Blue-agarose (Bio-Rad Affi-Gel Blue) at an early stage. Purification is completed by ion-exchange chromatography using DEAE-Sephadex A-50. The procedure is substantially shorter than earlier methods and reproducibly yields enzyme of high specific activity suitable for use in structural work such as characterization of the intermediate uridylyl-enzyme. The first step of the galactose-1-P uridylyltransferase reaction is the transfer of the uridylyl group from UDP-glucose to N3 of a histidine residue in the enzyme to form the covalent uridylyl-enzyme and glucose-1-P. The uridylyl-enzyme intermediate then reacts in a second step with galactose-1-P to form UDP galactose. The enzyme accepts (RP)-UDP alpha S-glucose as a good substrate, converting it to (RP)-UDP alpha S-galactose, i.e., with overall retention of configuration. In this paper we show that reaction of the enzyme with (RP)-[2 14C]UDP alpha S-glucose produces a [2-14C]uridylyl alpha S-enzyme that can be converted by base-catalyzed cyclization to (RP)-[2-14C]cUMPS. Inasmuch as cyclization must have proceeded with inversion of configuration at phosphorus, the corresponding configuration in the intermediate must have been the inverse of that in the substrate. Therefore, formation of uridylyl alpha S-enzyme from (RP) UDP alpha S-glucose proceeds with inversion of configuration, and overall retention arises from inversion in each of the two steps. The results support the authenticity of the isolated uridylyl-enzyme as the true reaction intermediate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022796 TI - Structural aspects of the copper sites in cytochrome c oxidase. An X-ray absorption spectroscopic investigation of the resting-state enzyme. AB - Copper K-edge X-ray absorption spectroscopy (XAS) has been used to investigate the structural details of the coordination environment of the copper sites in eight resting-state samples of beef heart cytochrome c oxidase prepared by different methods. The unusual position and structure of the resting-state copper edge spectrum can be adequately explained by the presence of sulfur-containing ligands, with a significant amount of S----Cu(II) charge transfer (i.e., a covalent site). Quantitative curve-fitting analysis of the copper extended X-ray absorption fine structure (EXAFS) data indicates similar average first coordination spheres for all resting-state samples, regardless of preparation method. The average coordination sphere (per 2 coppers) mainly consists of 6 +/- 1 nitrogens or oxygens at an average Cu-(N,O) distance of 1.99 +/- 0.03 A and 2 +/- 1 sulfurs at an average Cu-S distance of 2.28 +/- 0.02 A. Quantitative curve fitting analysis of the outer shell of the copper EXAFS indicates the presence of a Cu...Fe interaction at a distance of 3.00 +/- 0.03 A. Proposed structures of the two copper sites based on these and other spectroscopic results are presented, and differences between our results and those of other published copper XAS studies [Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465-498] are discussed. PMID- 3022798 TI - Mechanism of action of isopentenyl pyrophosphate isomerase: evidence for a carbonium ion intermediate. AB - Isopentenyl pyrophosphate isomerase catalyzes the interconversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate. The isomerase from yeast has been purified to near homogeneity (purity greater than 90%). The substrate analogue (Z)-3-(trifluoromethyl)-2-butenyl pyrophosphate reacts at less than 1.8 X 10(-6) times the rate of dimethylallyl pyrophosphate. The enzyme is irreversibly inactivated by 2-(dimethyl-amino)ethyl pyrophosphate (I). These observations are consistent with a carbonium ion mechanism for the isomerization. Compound I is an analogue of the intermediate carbonium ion and probably acts as a transition state analogue. For I, kon' = 2.1 X 10(6) M-1 min-1. No off-rate was detected and, therefore, Ki less than 1.4 X 10(-11) M. Upon denaturation of the inactivated enzyme, I is released unchanged. 2-(Trimethylammonio)ethyl pyrophosphate also inhibits with Ki' = 7 X 10(-7) M, kon' = 4.4 X 10(4) M-1 min 1, and koff = 0.03 min-1. Substrate analogues without a positively charged nitrogen were relatively poor inhibitors. The best inhibitor of these is ethyl pyrophosphate, Ki = 10(-4) M. The enzyme is inactivated by sulfhydryl-selective reagents. These reagents also prevent binding of I to the enzyme. The inactivation by iodoacetamide is dependent upon one ionizable group (pK = 9.3). The pH dependence of V and V/K for the isomerase-catalyzed reaction also depends upon a group with pK = 9.3. PMID- 3022799 TI - Interaction of a spin-labeled phenylalanine analogue with normal and sickle hemoglobins: detection of site-specific interactions through spin-label-induced 1H NMR relaxation. AB - In a preliminary report, we have previously shown that N-[(2,2,5,5-tetramethyl-1 oxypyrrolidin-3-yl)carbonyl]-L-phenyl ala nine tert-butyl ester (SL-Phe) exhibits specific binding to hemoglobin and an antiaggregation activity more than 2 orders of magnitude greater than that of phenylalanine [Lu, H.-Z., Currie, B. L., & Johnson, M. E. (1984) FEBS Lett. 173, 259-263]. Transverse 1H NMR relaxation measurements have been used to investigate the interaction of SL-Phe with hemoglobin molecules by use of the resonances assigned to the C2 protons of the beta 2 His, the beta 143 His, and the beta 146 or beta 97 His residues as intrinsic probes. Distance calculations using the paramagnetically induced relaxation data suggest that the SL-Phe binding site is approximately 12-16 A away from the C2 protons of the beta 2 His and the beta 146 or beta 97 His residues in the (carbonmonoxy)hemoglobin tetramer; for deoxyhemoglobin, the distances are approximately 14-17 A between the SL-Phe binding site and the C2 protons of the beta 2 His, the beta 143 His, and the beta 146 His residues. Calculations using the (carbonmonoxy)hemoglobin crystal atomic coordinates only restrict the probable SL-Phe binding region to the full F and H helices of the beta-chain and a small section of the alpha-chain. For deoxyhemoglobin, the distance calculations provide greater restrictions on the probable binding region, limiting it to small sections of the beta-chain F, G, and H helices near the EF bend and to a few residues on the alpha-chain.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022801 TI - cAMP-dependent protein kinases I and II: divergent turnover of subunits. AB - cAMP-dependent protein kinase subunits were isolated from livers of rats that had been subjected to biosynthetic labeling with radioactive leucine. By application of ligand and antibody affinity techniques pure regulatory (R I; R II) and catalytic (C) subunits could be obtained in high yields, which allowed measurement of the apparent degradation rate constants and half-lives following a double isotope labeling protocol. In this way marked differences of apparent half lives of regulatory subunits R I (t1/2 = 31 h) and R II (t1/2 = 125 h) were observed. To avoid the negative influence of reutilization inherent in the decay experiments, specific radioactivities were determined after a short isotope pulse. This parameter, which under steady-state conditions reflects the fractional turnover rate of the subunits, was found to be different for all three protein kinase subunits. Relative to total liver protein, the ratios R I:R II:C corresponded to 3.9:0.6:2. Our data indicate that in each type of protein kinase isoenzymes regulatory and catalytic subunits turn over with similar rates. The type I isoenzyme, however, is renewed much faster than protein kinase II. Furthermore, our findings are consistent with the thesis that free subunits as generated by activation are more susceptible to degradation than the holoenzymes, leading under steady-state conditions to compensatory resynthesis. Since renewal of R I exceeded that of R II also in two other tissues, the elevated turnover of protein kinase I as an indicator of preferential activation appears to be a general phenomenon. The different turnover of the two isoenzymes, then, may relate to different cellular functions like modulation of enzyme activity vs. modulation of gene activity. PMID- 3022800 TI - Observation of manganese(II)-ligand superhyperfine couplings in complexes with proteins by electron spin-echo spectroscopy. AB - Pulsed electron paramagnetic resonance spectroscopy has been used to detect Mn(II)-ligand superhyperfine couplings in complexes with creatine kinase and in the Mn(II) metalloprotein concanavalin A. Electron spin-echo envelopes from Mn(II), bound in these complexes, are modulated by superhyperfine interactions between Mn(II) and nearby, weakly coupled nuclear spins. The characteristic frequencies of the modulations were obtained by Fourier transformation of the three-pulse, spin-echo envelopes. In transition-state analogue complexes of creatine kinase (enzyme-MnIIADP-anion-creatine), superhyperfine interactions from the directly coordinated nitrogen of the thiocyanate ligand give envelope modulations. The source of the modulations was confirmed by measurements with the 14N and 15N forms of thiocyanate. On the other hand, the nitrogen of coordinated nitrate, which is two bonds removed from the paramagnetic center, does not produce detectable modulations. In spectra for Mn(II) concanavalin A, envelope modulations are detected due to the nitrogen of the coordinated histidine residue. Complexes prepared in 2H2O give strong signals due to weakly coupled 2H. For Mn(II)-doped single crystals of sodium pyrophosphate, signals are observed in the frequency domain spectra that are due to coupling from 31P. Phosphorus signals from the ADP ligand in complexes with creatine kinase show approximately the same coupling constant but have a much broader line width. PMID- 3022802 TI - N-ethylmaleimide uncouples the glucagon receptor from the regulatory component of adenylyl cyclase. AB - 125I-Glucagon binding to rat liver plasma membranes was composed of high- and low affinity components. N-Ethylmaleimide (NEM) and several other alkylating agents induced a dose-dependent loss of high-affinity sites. This diminished the apparent affinity of glucagon receptors for hormone without decreasing the binding capacity of membranes. Solubilized hormone-receptor complexes were fractionated as high molecular weight (Kav = 0.16) and low molecular weight (Kav = 0.46) species by gel filtration chromatography; NEM or guanosine 5' triphosphate (GTP) diminished the fraction of high molecular weight complexes, suggesting that NEM uncouples glucagon receptor-N-protein complexes. Exposure of intact hepatocytes to the impermeable alkylating reagent p (chloromercuri)benzenesulfonic acid failed to diminish the affinity of glucagon receptors on subsequently isolated plasma membranes, indicating that the thiol that affects receptor affinity is on the cytoplasmic side of the membrane. Hormone binding to plasma membranes was altered by NEM even after receptors were uncoupled from N proteins by GTP. These data suggest that a sensitive thiol group that affects hormone binding resides in the glucagon receptor, which may be a transmembrane protein. Alkylated membranes were fused with wild-type or cyc- S49 lymphoma cells to determine how alkylation affects the various components of the glucagon-adenylyl cyclase system. Stimulation of adenylyl cyclase with fluoride, guanylyl 5'-imidodiphosphate, glucagon, or isoproterenol was observed after fusion of cyc- S49 cells [which lack the stimulatory, guanine nucleotide binding, regulatory protein of adenylyl cyclase (Ns)] with liver membranes alkylated with 1.5 mM NEM.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022804 TI - Depolymerization of microtubules alters membrane potential and affects the motional freedom of membrane proteins. AB - Two independent lines of evidence were obtained indicating that microtubule depolymerization affects the functions and the physical state of membranes in intact Chinese hamster ovary cells. The first type of evidence was obtained by using the dye dihexyloxacarbocyanine iodide to measure membrane potential before and after treatment with several microtubule active agents. Microtubule depolymerization resulted in a decrease in cell fluorescence, whereas stabilization of microtubules with taxol resulted in an increase in cell fluorescence. These effects of the drugs were due to their interactions with microtubules and not to direct effects of the drugs on the plasma membranes for the following reasons: effects were time dependent and required entry into the cells as indicated by the lack of fluorescence change in a multi-drug-resistant mutant that does not accumulate antimicrotubule drugs and a colcemid-resistant tubulin mutant did not show these effects on cell fluorescence. Evidence for altered motional freedom of membrane proteins in the plasma membrane was obtained by using electron spin resonance analysis of maleimide spin probe labeled cells. This study showed that depolymerization of microtubules results in increased motional freedom of maleimide-labeled sulfhydryl group containing proteins. Taken together, these data argue that microtubules function in mammalian cells to regulate the physical state of membranes and modulate membrane potential generated across cell membranes. PMID- 3022803 TI - Gelsolin inhibits nucleotide exchange from actin. AB - The effects of platelet gelsolin on the state and exchangeability of the nucleotide bound to skeletal muscle actin monomer have been investigated. In the presence of Ca2+, a stable ternary complex consisting of two actins and one gelsolin is formed. Removal of Ca2+ from this species results in the formation of a highly stable binary gelsolin-actin complex. The interaction of gelsolin with actin monomer has no effect on the virtually negligible [less than 0.01 mol of Pi X h-1 X (mol of actin)-1] intrinsic ATPase activity of actin monomer (in the absence of Mg2+). A single molecule of ATP is bound to the binary complex while two molecules of ATP are bound to the actins within the ternary complex. The ATP within the binary complex is nonexchangeable, and only one of the two ATP molecules in the ternary complex is exchangeable. In the latter case the rate constant for this nucleotide exchange is decreased compared to that for free actin monomer. These results demonstrate the nonequivalence of actin monomers within the ternary complex. The involvement of these oligomeric complexes of gelsolin and actin in the expression of the activity(ies) of gelsolin is discussed. PMID- 3022805 TI - Binding of local anesthetics to reconstituted acetylcholine receptors: effect of protein surface potential. AB - Nicotinic acetylcholine receptor isolated from Torpedo californica electric organ is reconstituted into lipid bilayers of zwitterionic dioleoylphosphatidylcholine. These membranes are labeled with a spin-labeled quaternary amine local anesthetic (C6SLMeI), which has been shown previously to be a noncompetitive blocker of acetylcholine receptor-ion channel function in the micromolar concentration range. The electron spin resonance spectral component corresponding to protein immobilized anesthetic spin-label can be resolved from the composite data spectrum by using spectral subtraction of lipid components. This protein immobilized component is shown to represent C6SLMeI bound to a finite number of sites on the receptor. We demonstrate that C6SLMeI binds to the receptor as a function of the surface potential on the protein and suggest that the acetylcholine receptor reconstituted into zwitterionic phospholipid, which has no surface potential of its own, provides an excellent model system with which to study effects of protein surface charge. We hypothesize that the primary pathway of interaction of C6SLMeI with the acetylcholine receptor is via the aqueous medium. PMID- 3022806 TI - Characterization of purified cytochrome c1 from Rhodobacter sphaeroides R-26. AB - Cytochrome c1 from a photosynthetic bacterium Rhodobacter sphaeroides R-26 has been purified to homogeneity. The purified protein contains 30 nmol heme per mg protein, has an isoelectric point of 5.7, and is soluble in aqueous solution in the absence of detergents. The apparent molecular weight of this protein is about 150,000, determined by Bio Gel A-0.5 m column chromatography; a minimum molecular weight of 30,000 is obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The absorption spectrum of this cytochrome is similar to that of mammalian cytochrome c1, but the amino acid composition and circular dichroism spectral characteristics are different. The heme moiety of cytochrome c1 is more exposed than is that of mammalian cytochrome c1, but less exposed than that of cytochrome c2. Ferricytochrome c1 undergoes photoreduction upon illumination with light under anaerobic conditions. Such photoreduction is completely abolished when p-chloromercuriphenylsulfonate is added to ferricytochrome c1, suggesting that the sulfhydryl groups of cytochrome c1 are the electron donors for photoreduction. Purified cytochrome c1 contains 3 +/- 0.1 mol of the p chloromercuriphenylsulfonate titratable sulfhydryl groups per mol of protein. In contrast to mammalian cytochrome c1, the bacterial protein does not form a stable complex with cytochrome c2 or with mammalian cytochrome c at low ionic strength. Electron transfer between bacterial ferrocytochrome c1 and bacterial ferricytochrome c2, and between bacterial ferrocytochrome c1 and mammalian ferricytochrome c proceeds rapidly with equilibrium constants of 49 and 3.5, respectively. The midpoint potential of purified cytochrome c1 is calculated to be 228 mV, which is identical to that of mammalian cytochrome c1. PMID- 3022807 TI - Inhibition of oxidative phosphorylation by Ca2+ or Sr2+: a competition with Mg2+ for the formation of adenine nucleotide complexes. AB - Intramitochondrial Sr2+, similar to Ca2+, inhibits oxidative phosphorylation in intact rat-liver mitochondria. Both Ca2+ and Sr2+ also inhibit the hydrolytic activity of the ATPase in submitochondrial particles. Half-maximal inhibition of ATPase activity was attained at a concentration of 2.5 mM Ca2+ or 5.0 mM Sr2+ when the concentration of Mg2+ in the medium was 1.0 mM. The inhibition of ATPase activity by both cations was strongly decreased by increasing the Mg2+ concentration in the reaction medium. In addition, kinetical data and the determination of the concentration of MgATP, the substrate of the ATPase, in the presence of different concentrations of Ca2+ or Sr2+ strongly indicate that these cations inhibit ATP hydrolysis by competing with Mg2+ for the formation of MgATP. On the basis of a good agreement between these results with submitochondrial particles and the results of titrations of oxidative phosphorylation with carboxyatractyloside or oligomycin in mitochondria loaded with Sr2+ it can be concluded that intramitochondrial Ca2+ or Sr2+ inhibits oxidative phosphorylation in intact mitochondria by decreasing the availability of adenine nucleotides to both the ADP/ATP carrier and the ATP synthase. PMID- 3022809 TI - (Na+ + K+)-ATPase in artificial lipid vesicles: influence of the concentration of mono- and divalent cations on the pumping rate. AB - (Na+ + K+)-ATPase from kidney outer medulla was incorporated into artificial dioleoylphosphatidylcholine vesicles. Transport activity was induced by adding ATP to the external medium. A voltage-sensitive dye was used to detect the ATP driven potassium extrusion in the presence of valinomycin. The observed substrate protein interactions of the reconstituted (Na+ + K+)-ATPase largely agree with that from native tissues. An agreement between ATP hydrolysis and transport activity is given for concentration dependences of sodium, potassium, magnesium and calcium ions. The only significant deviations were observed in the influence of pH. Protons were found to have different influence on transport, enzymatic activity and phosphorylation of the enzyme. The transport studies showed a twofold interaction of protons with the protein: competition with sodium at the cytoplasmic ion binding sites, a non competitive inhibition of transport which is not correlated with protein phosphorylation. PMID- 3022808 TI - Conserved structural domains among species and tissues-specific differences in the mitochondrial phosphate-transport protein and the ADP/ATP carrier. AB - Peptide maps were generated of the CNBr-digested mitochondrial phosphate transport protein and ADP/ATP carrier from bovine and rat heart, rat liver and blowfly flight muscle. Total mitochondrial proteins from the same sources plus pig heart were separated by SDS-polyacrylamide gel electrophoresis. The peptide maps and the total mitochondrial proteins were electroblotted onto nitrocellulose membranes and reacted with rabbit antisera raised against the purified bovine heart phosphate-transport protein and the ADP/ATP carrier. On the basis of antibody specificity, mobility in SDS-polyacrylamide gel electrophoresis, and peptide maps the following was concluded. Phosphate-transport protein alpha and phosphate-transport protein beta (pig and bovine heart) react equally with the first and also with the second of two independent phosphate-transport protein antisera. Tissue-specific structural domains exist for both the phosphate transport protein and the ADP/ATP carrier, i.e., one phosphate-transport protein antiserum reacts with the phosphate-transport protein from all assayed sources, the other only with the cardiac phosphate-transport protein. These differences may reflect tissue-specific regulation of phosphate and adenine nucleotide transport. Homologies among the different species are found for the phosphate transport protein and the ADP/ATP carrier, except for the flight muscle ADP/ATP carrier. These conserved structural domains of the phosphate-transport protein may relate directly to catalytic activity. Alkylation of the purified phosphate transport proteins and the ADP/ATP carriers by the transport inhibitor N ethylmaleimide affects electrophoretic mobilities but not the antibody binding. Neither of the two phosphate-transport protein-antisera nor the ADP/ATP-carrier antiserum react with both phosphate transport protein and ADP/ATP carrier, even though these two proteins possess similarities in primary structure and function. Possible mechanisms for generating tissue-specific structural differences in the proteins are discussed. PMID- 3022810 TI - Sulfhydryl groups are essential for organic anion exchange in canine renal brush border membranes. AB - The effect of several sulfhydryl-modifying reagents (HgCl2, p chloromercuribenzenesulfonic acid (PCMBS), N-ethylmaleimide) on the renal organic anion exchanger was studied. The transport of p-amino[3H]hippurate, a prototypic organic anion, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. HgCl2, PCMBS and N-ethylmaleimide inactivated p-aminohippurate transport with IC50 values of 38, 78 and 190 microM. The rate of p-aminohippurate inactivation by N-ethylmaleimide followed apparent pseudo-first-order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the N-ethylmaleimide concentration with a slope of 0.8. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of N-ethylmaleimide inactivates one essential sulfhydryl group per active transport unit. Substrate (1 mM p-aminohippurate) affected the rate of the N-ethylmaleimide (1.3 mM) inactivation: the t1/2 values for inactivation in the presence and absence of p aminohippurate were 7.4 and 3.7 min, respectively. The results demonstrate that there are essential sulfhydryl groups for organic anion transport in the brush border membrane. Moreover, the ability of substrate to alter sulfhydryl reactivity suggests that the latter may play a dynamic role in the transport process. PMID- 3022812 TI - Association of ferri- and ferro-cytochrome c with lipid multilayers: a 31P solid state NMR study. AB - The 31P nuclear magnetic resonance anisotropies of dispersions of diacylphosphatidic acid and diacylphosphatidylserine were slightly increased in the presence of cytochrome c: no increase was observed with cardiolipin. However, the 31P spin-lattice relaxation times (T1) for all of these lipids were reduced markedly by the protein. As similar effects were observed with ferri-cytochrome c and with the reduced protein, which is diamagnetic, we suggest that the changes in T1 reflect a reduction in the spectral density of fast motions for the lipid headgroups attendant on binding of protein, rather than paramagnetic relaxation of the phosphorus nuclear spin. PMID- 3022811 TI - Inhibition of the intestinal transport of uracil by hexoses and amino acids. AB - Various hexoses and amino acids were tested as potential inhibitors of the active mucosal to serosal transport of uracil across the everted rat jejunum. Uracil transport displayed Michaelis-Menten type kinetics with a Vmax of 10.4 +/- 0.2 mumol X g-1 X h-1 and an apparent Km of 0.047 +/- 0.002 mM (means +/- S.D.). Scilliroside, an inhibitor of the basolateral (Na+ + K+)-ATPase, dose-dependently inhibited the transport of uracil consistent with the Na+ dependency of uracil transport. Thymine was a full competitive inhibitor (Ki = 0.021 +/- 0.002 mM) of uracil transport. All actively transported substances tested including L phenylalanine, L-leucine, D-galactose, D-glucose, and 3-O-methylglucose inhibited the transport of uracil. In contrast, L-glucose and fructose, substances which are not actively transported, were without effect on uracil transport. Further studies with D-galactose indicated that it acts as a partial noncompetitive inhibitor (Ki = 6.0 +/- 1.4 mM) of uracil transport. This Ki is in good agreement with the apparent Kt (5.8 +/- 1.1 mM) for D-galactose transport. Phlorizin (0.1 mM), an inhibitor of galactose transport, blocked the inhibitory effect of galactose on uracil transport. In the ileum D-galactose had no effect on uracil transport but thymine caused the same degree of inhibition as in the jejunum. The results demonstrate that heterologous inhibition is a more general phenomenon than had previously been realized. PMID- 3022813 TI - Thiophosphorylation of hog gastric (H+ + K+)-ATPase membranes by endogenous protein kinases. AB - (H+ + K+)-ATPase-enriched membranes from hog stomachs were tested for their capacity to autophosphorylate using [gamma-32P]ATP or [gamma-35S]ATP[S] as phosphate donors. The radioactive polypeptides were characterized by SDS-PAGE. In the presence of Mg2+ and 5 microM [gamma-32P]ATP, rapid and transient incorporation of 32P occurred at 0 degrees C. Radioactivity was essentially found in the major polypeptide of the material, the 95 kDa subunit of (H+ + K+)-ATPase. Under the same experimental conditions, thiophosphorylation was slower and reached a plateau within 1 h. Incorporation levels were higher with manganese than with magnesium. After one hour at 0 degrees C, and in the presence of 10 mM manganese and 5 microM ATP[S], 0.58 +/- 0.06 nmoles of thiophosphate were incorporated per mg of protein. Twenty seven percent of the thiophosphorylated amino acids were acylphosphates i.e. likely to be the ATPase thiophosphointermediate. The remaining thiophosphorylated amino acids (73%) were thought to be produced by protein kinases. This was supported by the autoradiographies of membrane SDS-PAGE which indicated that, in addition to the 95 kDa ATPase subunit, other polypeptides were thiophosphorylated especially at 108, 58, 47, 45 and 36-40 kDa. A previous study had provided strong evidence that chloride transport in gastric microsomes, is modulated by a protein kinase dependent phosphorylation (Soumarmon, A., Abastado, M., Bonfils, S. and Lewin M.J.M. (1980) J. Biol. Chem. 255, 11682-11687). In the present work, we demonstrate that the peptidic inhibitor of cAMP-dependent protein kinases decreased thiophosphorylation of a 45 kDa polypeptide. We suggest that this polypeptide could be regarded as a candidate for the role of chloride transporter or chloride transport regulator. PMID- 3022815 TI - The formation of ES of cytochrome-c peroxidase: a comparison with lactoperoxidase and horseradish peroxidase. AB - The activation energy for the formation of the first red compound, ES, for cytochrome-c peroxidase (ferrocytochrome-c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) by i-propyl hydroperoxide and the rate constants for the formation of ES with various hydroperoxides have been determined. Multivariate data analysis by the partial least-squares model in latent variables has been used to compare the rate constants with the corresponding rate constants for the formation of compound I from lactoperoxidase and two isoenzymes of horseradish peroxidase. The results show that the rate of formation of ES from cytochrome-c peroxidase is highly correlated with the pKa of the hydroperoxides. The activation energy for the formation of ES with i-propyl hydroperoxide is close to the corresponding value for hydrogen peroxide. PMID- 3022814 TI - Resistance of horse alpha 1-proteinase inhibitor to perchloric acid denaturation and a simplified purification procedure resulting therefrom. AB - Addition of perchloric acid (6.4% w/v final concentration) to horse alpha 1 proteinase inhibitor or to horse plasma neither precipitated nor inactivated alpha 1-proteinase inhibitor. None of the isoinhibitors of alpha 1-proteinase inhibitor was altered by dilute perchloric acid. This unexpected behavior led to a simplified procedure for the purification of horse alpha 1-proteinase inhibitor, consisting of removal of the bulk of plasma proteins, by perchloric acid precipitation and by gel filtration on Sephadex G-75 and G-200. The resulting preparations of alpha 1-proteinase inhibitor were immunogenically pure. The simplified purification procedure permitted the immunochemical comparison of the isoinhibitors of alpha 1-proteinase inhibitor, which proved to be immunologically identical. PMID- 3022817 TI - ESR studies on the oxidation of N,N-dimethyl-p-anisidine and its analogues catalyzed by myeloperoxidase. AB - N,N-Dimethyl-p-anisidine (DMA) was used as a substrate to differentiate between the direct, or chloride-independent, and the indirect, or chloride-dependent, pathways characteristic of myeloperoxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7). The chemical oxidation by sodium hypochlorite and the horseradish peroxidase-catalyzed oxidation by H2O2 were also investigated for a comparison. The chemical oxidation of DMA by NaOCl (DMA/NaOCl = 1) gave the p N,N-dimethylaminophenoxy radical at pH 5 and 7. p-Benzoquinone and formaldehyde were determined as stable end-products. On the other hand, the cation radical of DMA was detected and p-benzoquinone was not obtained in the horseradish peroxidase-H2O2-Cl- system. In the presence of Cl- the myeloperoxidase-catalyzed oxidation at pH 5 gave nearly the same result as did the oxidation by NaOCl, whereas in the absence of Cl- the result of the oxidation was similar to that of the horseradish peroxidase-catalyzed oxidation, except for a low yield of formaldehyde formation, which was ascribed to the decomposition of H2O2 by the catalase activity of myeloperoxidase. Although the myeloperoxidase-catalyzed oxidation of DMA at pH 7 in the presence of Cl- gave only the cation radical of DMA, a fairly large amount of p-benzoquinone was obtained as a product. This result indicates that the indirect chloride-dependent oxidation is also operating at pH 7. The reaction mechanism for the myeloperoxidase-catalyzed oxidation of DMA is proposed. PMID- 3022816 TI - Sheep brain pyridoxal kinase: fluorescence spectroscopy of the dimeric enzyme. AB - Pyridoxal kinase (ATP:pyridoxal 5-phosphotransferase, EC 2.7.1.35) has been purified 9000-fold from sheep brain by affinity chromatography. The enzyme of 80,000 molecular weight is made up of two identical-size subunits. The interaction of the inhibitor N-dansyl-1,8-diaminooctane with the nucleotide site of the kinase was examined by means of steady and nanosecond fluorescence spectroscopy. N-Dansyl-1,8-diaminooctane is a competitive inhibitor with respect to ATP at saturating concentrations of pyridoxal. It binds to the nucleotide site of the enzyme with Kd = 2.2 microM. Bound N-dansyl-1,8-diaminooctane is shielded from collisional encounters with the external quencher acrylamide. The collisional rate constant for bound N-dansyl-1,8-diaminooctane (Kq = 1.4 X 10(8) M-1 X s-1) is 10-times lower than the value obtained for the free chromophore. Nanosecond emission anisotropy measurements yield a rotational correlation time of 42 ns for the inhibitor complexes to the kinase. Both steady and nanosecond fluorescence results are consistent with a model in which the inhibitor bound to the nucleotide site is immobilized by amino acids located at the catalytic site. PMID- 3022818 TI - Dibutyryl cyclic AMP decreases the rate of lipoprotein lipase synthesis in cultured adipocytes. AB - The regulation of avian lipoprotein lipase by dibutyryl cyclic AMP in cultured adipocytes was studied with quantitative and specific methods for the measurements of enzyme catalytic activity, enzyme protein mass, and immunoadsorption of labeled enzyme. Incubation of adipocytes in 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline results in a time-dependent decrease in cell lipoprotein lipase catalytic activity. The activity is decreased by 70% in 4 h and over 90% by 12 h. The decrease in cellular catalytic activity is due to a decrease in both enzyme content and enzyme catalytic efficiency. 4 h after exposure of adipocytes to cAMP, enzyme protein was decreased from 3.58 +/- 0.5 to 1.92 +/- 0.1 ng/dish and specific activity from 15.1 +/- 2.1 to 8.4 +/- 1.1 nmol/ng. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP mediated decrease in lipoprotein lipase activity was half-maximal at less than 25 microM dibutyryl cyclic AMP. The rate of lipoprotein lipase synthesis was estimated by measuring the incorporation of L-[35S]methionine into enzyme protein during 30 min. A method for the quantitative immunoadsorption of lipoprotein lipase from cell lysates was developed. Utilizing this immunoadsorption technique, the rate of incorporation of L-[35S]methionine into lipoprotein lipase was 0.0026 +/- 0.002%, when expressed as a percentage of that incorporated into total trichloroacetic acid-precipitable counts. By 2 h after exposure of adipocytes to 0.5 mM dibutyryl cAMP, the relative synthesis rate had already decreased to 64 +/- 4% of the control rate. After 16 h the synthesis rate was 43.2 +/- 13.8% of the control rate. The observed decreased synthesis rate could account for most of the decreased cellular enzyme content and diminished enzyme secretion rate. PMID- 3022819 TI - Regulation of lipoprotein receptors on rat hepatomas in vivo. AB - It has been shown previously that the rat hepatoma no. 7288C grown in vivo or in vitro expresses fewer receptors which recognize chylomicron remnants than does normal rat liver, and it was suggested that this may contribute to the deletion of dietary cholesterol-induced regulation of cholesterol synthesis in hepatomas (Barnard, G., Erickson, S. and Cooper, A. (1984) J. Clin. Invest. 74, 173-184). To investigate this further, Buffalo rats bearing hepatomas (HTC no. 7288C) were made hypercholesterolemic by feeding an atherogenic diet and hypocholesterolemic by ethinyl estradiol injections. Under all circumstances, tumor membranes had fewer receptors than liver membranes as measured by specific binding of [125I]chylomicron remnants. Ethinyl estradiol treatment increased the number of lipoprotein receptors 1.7-fold in liver membranes and 1.2-1.6-fold in tumor membranes, but hypercholesterolemia did not produce any significant changes in remnant binding to either liver or hepatoma membranes. Feeding an atherogenic diet induced a 2.4-fold increase in total cholesterol content in the liver, primarily as cholesterol ester; however, there was no change in total, free or ester cholesterol in the hepatomas. Acyl coenzyme A:cholesterol acyltransferase activity was low in this hepatoma line and neither treatment significantly affected its activity. One explanation for the lack of effect of the atherogenic diet on hepatoma cholesterol metabolism in addition to the decreased number of lipoprotein receptors might be the failure of access of lipoproteins to the tumor cell. To assess this, radioiodinated apo E-rich lipoproteins of various sizes were injected intravenously into rats with hepatomas. Their disappearance from the circulation was followed, and the uptake of each lipoprotein into a variety of tissues was determined. Chylomicron remnants were the most avidly removed particles. VLDLH, IDLH and HDLC were removed more slowly and less completely. None of the lipoproteins accumulated substantially in the tumors suggesting a limited access to the hepatoma tissue. Thus, in addition to the observed reduction in lipoprotein receptor number, limited lipoprotein access to the hepatoma tissue may be a significant factor in contributing to the apparent lack of feedback regulation of cholesterol synthesis by hepatoma tissue in vivo. PMID- 3022820 TI - Mitochondrial and peroxisomal oxidation of arachidonic and eicosapentaenoic acid studied in isolated liver cells. AB - The partitioning between peroxisomal and mitochondrial beta-oxidation of [1 14C]eicosapentaenoic acid (20:5(n-3] and [1-14C]arachidonic acid (20:4(n-6)) was studied. In hepatocytes from fasted rats approximately 70% of the fatty acid substrate was oxidized with oleic, linoleic, eicosapentaenoic and docosahexaenoic (22:6(n-3)) acid, even more with adrenic (22:4(n-6)) and less with arachidonic acid. When the mitochondrial oxidation was suppressed by fructose refeeding and by (+)-decanoylcarnitine, the fatty acid oxidation in per cent of that in cells from fasted rats was with 18:1(n-9) 7%, 18:2(n-6) 8%, 20:4(n-6) 12%, 20:5(n-3) 20%, 22:4(n-6) 57% and for 22:6(n-3) 29%. The fraction of 14C recovered in palmitate and other newly synthesized fatty acids after fructose refeeding decreased in the order 22:4(n-6) greater than 22:6(n-3) greater than 20:5(n-3) greater than 20:4(n-6) and was very small with 18:1(n-9) and 18:2(n-6). In cells from both fed and fructose-refed animals 20:5(n-3) was efficiently elongated to 22:5(n-3) and 22:6(n-3). 20:5(n-3) and 20:4(n-6) were not elongated after fasting. The phospholipid incorporation with [1-14C]20:5(n-3) decreased during prolonged incubations while it remained stable with [1-14C]arachidonic acid. The results suggest that peroxisomes contribute more to the oxidation of 20:5(n-3) than with 20:4(n-6) although both substrates are probably oxidized mainly in the mitochondria. PMID- 3022821 TI - Increase in the formation of leukotriene B4 and other lipoxygenase products in peritoneal macrophages of adrenalectomized rats. AB - The effect of adrenalectomy on the formation of cyclooxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined. After isolation, the cells were incubated with [1-14C]arachidonic acid and the calcium ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the controls are 6-keto-prostaglandin F1 alpha, thromboxane B2 and 12 HETE. One peak represents 5,12-di-HETE. Smaller amounts of prostaglandin F2 alpha, prostaglandin E2, prostaglandin D2, leukotriene B4 and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of leukotriene B4, 15-HETE and 12-HETE. The increase in the prostaglandins is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, 6-keto-prostaglandin F1 alpha and thromboxane B2 are produced in higher amounts than leukotriene B4. After adrenalectomy, the formation of leukotriene B4 is much more increased than that of 6-keto-prostaglandin F1 alpha. These effects are most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid-induced peptide with phospholipase A2 inhibitory activity in adrenalectomized animals. PMID- 3022822 TI - Analysis of the ESR spectrum of synthetic dopa melanin. AB - The 35 GHz ESR spectrum of frozen aqueous suspensions of synthetic melanin from autoxidation of dopa is asymmetric in the pH range 3-12. This asymmetry increases with increasing pH. A detailed computer analysis of second-derivative spectra suggests that the asymmetry is a result of two factors: approximately axial anisotropy of the g-tensor of the radical species; superposition of ESR spectra arising from a total of four radical species. The relative amounts of these individual spectra vary in a pH-dependent manner. Anisotropy of spectra varies with pH, probably due to pH-induced changes in the delocalization of the unpaired electrons. Thus the species present at high pH are suggested to be relatively localized radical anions, while the species detected at low pH are suggested to be protonated forms of the high pH species in which the unpaired electron is more extensively delocalized. PMID- 3022824 TI - Electrical stimulation of hybridoma cells producing monoclonal antibody to cAMP. AB - Electrical stimulation was applied to hybridoma cells in order to activate metabolic activities and increase the monoclonal antibody production. Hybridoma cells that produce monoclonal antibody to adenosine 3':5'-cyclic monophosphate were placed on a transparent glass electrode immersed in medium and subjected to electric pulses (pulse shape, alternating rectangular; field strength, 4 X 10(3) V X m-1; frequency, 5 kHz; pulse mode, 0.5 min application and 4.5 min pause). After 48 h of incubation, the concentration of lactic acid in the medium reached 8.4 mM, approx. 30% higher than that obtained without electric stimulation. Similarly, cell growth rate was promoted by the electric stimulation, reaching a maximum stimulation after 40 h. When the hybridoma was cultured for 48 h with electrical stimulation, the antibody concentration in the medium reached 22.3 microgram X ml-1, approx. 10% higher than the control, with a concomitant 16% increase in cell concentration. Longer periods of electric pulse application, however, caused an inhibitory effect on the hybridoma growth. The most probable cause of the inhibition are reactive oxygen species such as superoxide and hydrogen peroxide, which are inevitably generated by electrolysis. The presence of superoxide dismutase (EC 1.15.1.1) reduced the inhibitory effects. In conclusion, metabolic activities including monoclonal antibody production were activated by the electrical stimulation. PMID- 3022823 TI - Protein glycosylation regulates the externalization of two distinct classes of glucocorticoid-induced glycoproteins in rat hepatoma cells. AB - The relationship of protein glycosylation to the externalization of glucocorticoid inducible alpha1-acid glycoprotein and mouse mammary tumor virus glycoproteins was examined in M1.54, a clonal population of mouse mammary tumor virus-infected rat hepatoma cells. Multiple freeze-thaw of isolated microsomes revealed that while alpha 1-acid glycoprotein is carried through the cell as a soluble component of vesicles, extracellular viral glycoproteins are initially membrane-associated. At concentrations of tunicamycin that specifically inhibited N-linked protein glycosylation, alpha 1-acid glycoprotein fractionated between the cellular and extracellular compartments. Thus, approximately one half of the newly synthesized, nonglycosylated (22,000 Mr) alpha 1-acid glycoprotein was rapidly secreted with kinetics similar to its glycosylated counterpart (release half-time of 60 min), while the remaining species first localized in an undefined intracellular compartment prior to its slow secretion (release half-time of 24 h). The same distribution of nonglycosylated alpha 1-acid glycoprotein was observed at various absolute levels of polypeptide, suggesting that this was not due simply to the saturation of an efficient secretory pathway at high polypeptide levels. In contrast to alpha 1-acid glycoprotein, no labeled viral antigens were released by tunicamycin-treated M1.54, while a nonglycosylated viral precursor glycopolyprotein was expressed intracellularly. Taken together, these results suggest that carbohydrate attachment strongly regulates the externalization of both alpha 1-acid glycoprotein and mouse mammary tumor virus species, which represent two distinct classes of extracellular glycoproteins. PMID- 3022825 TI - Phosphate turnover of phosphatidylinositol in resting and thrombin-stimulated platelets. AB - Human platelets were pulse-labelled with [32P]Pi and extracts were analyzed for masses and radioactivities of ATP and phosphoinositides. Immediately after pulse labelling, the specific 32P radioactivity of phosphatidylinositol (PI) was only 3.4% of that of the gamma-phosphoryl of ATP. Upon incubation of the platelets at 37 degrees C, the specific 32P radioactivity of ATP (beta- and gamma-phosphoryls) remained constant. However, specific 32P radioactivity in PI increased continuously to 17% of specific [gamma-32P]ATP at 90 min of incubation. Stimulation with 0.5 U/ml of thrombin induced a 35% decrease in mass of PI which was unaffected by the time after the pulse-labelling. In contrast, the thrombin induced changes in [32P]PI differed markedly at the various times after the [32P]Pi-pulse. Immediately after pulse-labelling, [32P]PI initially decreased but increased thereafter to 260% of control values after 180 s. With increasing specific 32P-radioactivity in PI before stimulation, the thrombin-induced increase in [32P]PI gradually disappeared. After 90 min of incubation, thrombin induced a continuous decrease in [32P]PI that almost parallelled mass. The data are explained by an initial breakdown of PI to diacylglycerol through the PI cycle or the polyphosphoinositide cycle, followed by resynthesis of PI through phosphatidic acid. In contrast to pre-existing PI, the resynthesized PI is in full isotopic equilibrium with ATP. This allowed us to estimate that 14% of the PI that is consumed between 30 and 180 s of stimulation, is recycles. From our data we calculate that the rate of PI resynthesis increased from 2.4 to 20 nmol/min per 10(11) cells upon thrombin stimulation of platelets. PMID- 3022826 TI - Catalytic subunit of the polycation-stimulated protein phosphatase. Effect of proteolysis on polycation stimulation. AB - The phosphorylase phosphatase activity of the holoenzyme form of phosphatase 2A isolated from extracts of porcine renal cortex or bovine heart was stimulated 600% and 500%, respectively, by the addition of histone H1. After conversion of the phosphatase to the catalytic subunit form by treatment with ethanol at room temperature, histone H1 stimulated activity by about 150% only. Purification of the catalytic subunit from porcine renal cortex yielded two forms of the enzyme which were separated by heparin-Sepharose chromatography. These forms were designated peak 1 and peak 2 according to their order of elution from the column. Peak 1 catalytic subunit was stimulated by more than 400% by histone H1, whereas peak 2 was stimulated by about 50% only. Based on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, peak 2 had a slightly higher Mr value than peak 1 (35,500 vs. 35,000). Incubation of the peak 2 phosphatase with trypsin converted it to a form that was similar to peak 1 with respect to Mr and stimulation by histone H1. When the catalytic subunit of phosphatase 2A was purified from bovine heart only one form was obtained. Bovine heart enzyme was similar to renal peak 2 in that it had an apparent Mr of 35,500 and was only slightly stimulated by histone H1. Treatment of the bovine heart catalytic subunit with trypsin, chymotrypsin or type 2 Ca2+-dependent proteinase decreased the apparent Mr by about 500 and increased histone H1 stimulation to about 500%. Thus, when a small peptide was removed by proteolysis, histone H1 stimulation of the 'nicked' catalytic subunit was similar to that obtained with the holoenzyme. PMID- 3022828 TI - Insulin-like effect of epidermal growth factor in isolated rat hepatocytes. Modulation of the alpha-1-adrenergic stimulation of ureagenesis. AB - Insulin and epidermal growth factor (EGF) inhibit the stimulation of ureagenesis induced by adrenaline (alpha 1-adrenergic effect) in hepatocytes from control rats incubated in medium without calcium and in cells from hypothyroid rats. In hepatocytes from euthyroid rats incubated in normal buffer neither insulin or EGF diminished the alpha 1-adrenergic stimulation of ureagenesis. No effect of EGF or insulin on the alpha 1-adrenergic stimulation of phosphatidylinositol labeling was observed under any conditions. It is suggested that EGF mimics the action of insulin on one of the pathways of the alpha 1-adrenergic action: the calcium independent, insulin-sensitive pathway which predominates in hepatocytes from hypothyroid rats. PMID- 3022827 TI - The substrate specificity of the protein kinase induced in cells infected with herpesviruses: studies with synthetic substrates [corrected] indicate structural requirements distinct from other protein kinases. AB - Synthetic peptides have been used to investigate the site specificity of highly purified virus induced protein kinase, a recently discovered protein kinase isolated from cells infected with alpha-herpesviruses. The enzyme from cells infected with pseudorabies virus can catalyse the phosphorylation of both seryl and threonyl residues in peptides that contain several arginyl residues on the amino-terminal side of the target residue. At least two arginyl residues are required, and the best substrates examined contain four to six such residues. Virus induced protein kinase differs in site specificity from protein kinase C in being unable to phosphorylate peptides in which multiple arginyl residues are on the carboxyl-terminal side of the target residue, or to phosphorylate peptides in which the arginyl residues are replaced by ornithyl residues. Virus induced protein kinase from cells infected with herpes simplex virus type I had similar substrate preferences to virus induced protein kinase from cells infected with pseudorabies virus. Although virus induced protein kinase and the cyclic AMP dependent protein kinase have several peptide substrates in common, their relative preferences for these (as indicated by Km values) were found to be very different. PMID- 3022829 TI - [Binding of reversible spin-labeled inhibitors with an butyrylcholinesterase active center]. AB - The interaction of spin-labeled metacyn, procaine, carbolin and bivalent cations (Ca2+, Co2+, Ni2+) with butyrylcholinesterase (BChE) was studied by ESR and enzyme kinetic methods. The effect of pH, ionic strength and organic solvent was analysed. Spin-labeled metacyn binds at the anionic site of BChE active centre. This complex is stabilized both with coulombic and hydrophobic interactions, ionizing group of active centre with pK 6-7 also affects the binding. Spin labeled procaine appeared to be enzyme competitive inhibitor (Ki = 4 X 10(-5) M) and is located, most probably, at the same site. Activating effect of Ca2+ ions on BChE was confirmed. Simultaneous application of spin labels and paramagnetic ions demonstrates that cations Co2+ and Ni2+ bind with BChE in the close vicinity of spin-labeled inhibitor site. Paramagnetic cations are located more closely to the cationic part of the inhibitor molecule than to the hydrophobic one, and can be displaced by surplus of Ca2+ ions. The experimental data testify the model of anionic centre which consists of bivalent metal ions and aminoalcyl cationic group subsites and is located in a hydrophobic pocket of the enzyme surface. PMID- 3022830 TI - [Mechanisms of induced processes in aqueous hemoglobin solutions at 77 K]. AB - ESR spectra of gamma-irradiated and frozen at 77 K human oxyhemoglobin and partially denaturated methemoglobin solutions were analysed. The quartet signal ascribed to the anion-radical of proximal histidine was shown to dominate in the spectra of both solutions. The spectra of methemoglobin solution irradiated with relatively small doses have an intensive singlet ascribed to the stabilized electron. The formation mechanism of free radicals is discussed. PMID- 3022831 TI - [Effect of physico-chemical factors and species specificity on the structure of dinitrosyl iron complexes]. AB - Formation in mouse, rat and man's blood of iron nitrosyl complexes with pair thiol groups of proteins (complexes 2.03) was shown by ESR method. This formation was initiated by the introduction in blood in vitro or in vivo of low molecular dinitrosyl complexes of iron with phosphate, thiosulphate, cysteine or reduced gluthatione. Three forms of these complexes were found. They were characterized by ESR signals with rhombic or axial symmetry of g-factor tensor. These forms pass into one another under the effect of a number of thiol-containing compounds or at blood freezing. The life time of the complexes 2.03 in the blood in vivo is several hours. PMID- 3022832 TI - [Variability of the response types induced by cyclic 3',5'-adenosine monophosphate into identified molluscan neurons]. AB - In all investigated neurons of snail Helix pomatia an injection of cAMP evoked depolarization and spike generation. The possible experimental mistakes leading to another types of response are considered. PMID- 3022833 TI - [cDNA of cattle alpha S1-casein: cloning and nucleotide sequence]. AB - The nucleotide sequence of recombinant plasmids representing a full-size cDNA of cow alpha s1-casein was investigated. The corresponding mRNA consists of 1133 nucleotides except for poly(A) and includes 642 nucleotides of the coding region, 63 nucleotides of 5'- and 428 nucleotides of the 3'-noncoding regions. A comparative analysis of nucleotide sequences of cow alpha s1-casein and guinea pig B-casein showed that the homology in the 5'-nontranslatable region is 90.5%, that of a precasein single peptide is 82.22%, while that of the major polypeptide in the coding region is 64% without taking into account the blank spaces. The homology is higher in the 3'-noncoding region than in the coding region and makes up to 72%. The data obtained testify to the high degree of conservatism of sequences in casein mRNA noncoding regions as well as to functional and regulatory role of these sequences in gene expression of caseins. PMID- 3022834 TI - Resonance Raman spectral isolation of the a and a3 chromophores in cytochrome oxidase. AB - Resonance Raman spectra of reduced CO-bound cytochrome oxidase obtained at two different excitation frequencies (441.6 and 413.1 nm) are compared with the spectra of the fully reduced enzyme. In the spectra of the CO-bound complex only the cytochrome a modes are strongly enhanced with 441.6 nm excitation and only the modes of the CO-bound cytochrome a3 heme are strongly enhanced with 413.1-nm excitation. In the fully reduced complex with both excitation frequencies, modes of both cytochrome a and a3 are enhanced. By subtraction we are able to uncover the complete spectrum of the fully reduced ligand-free cytochrome a3 heme. Thus, we report the discrete resonance Raman spectra of cytochromes a2+, a2+3, and a2+3 (CO). The spectra of fully reduced cytochrome a and ligand-free cytochrome a3 are very different especially in the low frequency region. Binding CO to ferrous cytochrome a3 results in electronic structure changes in the heme analogous to those in hemoglobin and myoglobin, from which we conclude that there is nothing electronically unique in the ferrous cytochrome a3 heme to account for its catalytic properties. PMID- 3022835 TI - Stiffness of skinned rabbit psoas fibers in MgATP and MgPPi solution. AB - The stiffness of single skinned rabbit psoas fibers was measured during rapid length changes applied to one end of the fibers. Apparent fiber stiffness was taken as the initial slope when force was plotted vs. change in sarcomere length. In the presence of MgATP, apparent fiber stiffness increased with increasing speed of stretch. With the fastest possible stretches, the stiffness of relaxed fibers at an ionic strength of 20 mM reached more than 50% of the stiffness measured in rigor. However, it was not clear whether apparent fiber stiffness had reached a maximum, speed independent value. The same behavior was seen at several ionic strengths, with increasing ionic strength leading to a decrease in the apparent fiber stiffness measured at any speed of stretch. A speed dependence of apparent fiber stiffness was demonstrated even more clearly when stiffness was measured in the presence of 4 mM MgPPi. In this case, stiffness varied with speed of stretch over about four decades. This speed dependence of apparent fiber stiffness is likely due to cross-bridges detaching and reattaching during the stiffness measurement (Schoenberg, 1985. Biophys. J. 48:467). This means that obtaining an estimate of the maximum number of cross-bridges attached to actin in relaxed fibers at various ionic strengths is not straightforward. However, the data we have obtained are consistent with other estimates of cross-bridge affinity for actin in fibers (Brenner et al., 1986. Biophys. J. In press.) which suggest that ~60-90% of the cross-bridges attached in rigor are attached in relaxed fibers at an ionic strength of 20 mM and ~2-10% of this number of cross bridges are attached in a relaxed fiber at an ionic strength of 170 mM. PMID- 3022837 TI - Conformational considerations in the design of glucagon agonists and antagonists: examination using synthetic analogs. PMID- 3022836 TI - Redox-linked proton translocation in cytochrome oxidase: the importance of gating electron flow. The effects of slip in a model transducer. AB - In at least one component of the mitochondrial respiratory chain, cytochrome c oxidase, exothermic electron transfer reactions are used to drive vectorial proton transport against an electrochemical hydrogen ion gradient across the mitochondrial inner membrane. The role of the gating of electrons (the regulation of the rates of electron transfer into and out of the proton transport site) in this coupling between electron transfer and proton pumping has been explored. The approach involves the solution of the steady-state rate equations pertinent to proton pump models which include, to various degrees, the uncoupled (i.e., not linked to proton pumping) electron transfer processes which are likely to occur in any real electron transfer-driven proton pump. This analysis furnishes a quantitative framework for examining the effects of variations in proton binding site pKas and metal center reduction potentials, the relationship between energy conservation efficiency and turnover rate, the conditions for maximum power output or minimum heat production, and required efficiency of the gating of electrons. Some novel conclusions emerge from the analysis, including: An efficient electron transfer-driven proton pump need not exhibit a pH-dependent reduction potential; Very efficient gating of electrons is required for efficient electron transfer driven proton pumping, especially when a reasonable correlation of electron transfer rate and electron transfer exoergonicity is assumed; and A consideration of the importance and possible mechanisms of the gating of electrons suggests that efficient proton pumping by CuA in cytochrome oxidase could, in principle, take place with structural changes confined to the immediate vicinity of the copper ion, while proton pumping by Fea would probably require conformational coupling between the iron and more remote structures in the enzyme. The conclusions are discussed with reference to proton pumping by cytochrome c oxidase, and some possible implications for oxidative phosphorylation are noted. PMID- 3022839 TI - Isolation of repetitive clones from human muscle cDNA library. AB - Human fetal muscle cDNA library was screened with a beta-myosin heavy chain gene fragment containing Alu sequences. Two cDNA clones AI and BII with 1.8 and 3 kb inserts respectively were chosen for further characterization by means of RNA and DNA hybridization procedures and sequencing. The clones appeared to contain repetitive sequences as well as single copy regions. They are actively transcribed in different stages of myogenic development but not in the liver. DNA sequence analysis of short stretches from both clones revealed no sequence homology to any other published DNA sequences. PMID- 3022838 TI - alpha- and beta-adrenergic control of thermogenin mRNA expression in brown adipose tissue. AB - By the use of an earlier characterised cDNA clone, CIN-1, corresponding to a sequence of the mRNA coding for the brown-fat specific "uncoupling" protein, thermogenin, the amount of thermogenin mRNA found in the brown adipose tissue of mice was quantitatively investigated under different physiological and pharmacological conditions. It was found that a 4 hr cold stress led to a 7-fold increase in the amount of thermogenin mRNA; injection of norepinephrine had a significant but smaller effect. Most notably, isoprenaline (beta-agonist) and phenylephrine (alpha-agonist) had in themselves no effect, but when injected together were able to increase the mRNA level synergistically. In 4 hr cold stressed mice, norepinephrine, isoprenaline and cholera toxin could all further potentiate the effect of the cold stress itself on the mRNA level. Insulin and the glucocorticoid dexamethasone both had weak stimulatory effects on the mRNA level. It is concluded that an increase in intracellular cAMP levels is a necessary and perhaps sufficient stimulus for the increase in thermogenin gene expression. However, at least under in vivo conditions, this increase requires stimulation of both alpha- and beta-adrenergic pathways. PMID- 3022840 TI - Brown adipose tissue activity in hypocaloric-diet fed lactating rats. AB - Hypocaloric diet feeding reduced the mitochondrial protein content and whole tissue GDP-binding in interscapular brown adipose tissue from both virgin and lactating rats. A reduction in brown fat lipoprotein lipase activity was also detected in underfed virgin and lactating animals. These results indicate that lactation in the rat, even though it produces a reduction in brown fat activity, does not impair the capacity of the tissue to respond to a diminished caloric intake by lowering its activity further. PMID- 3022841 TI - DNA cloning and hybridization in deer species supporting the chromosome field theory. AB - The Cervidae show the largest variation in chromosome number found within any mammalian family. The eight species of deer which are the subject of this study vary in chromosome number from 2n = 70 to 2n = 6. Three species of Bovidae are also included since they belong to a closely related family. Digestion of nuclear DNAs with the restriction endonucleases Hae III, Hpa II, Msp I, Eco RI, Xba I, Pst I and Bam HI reveals that there is a series of highly repetitive sequences forming similar band patterns in the different species. There are two bands (1100 and 550 base pairs) which are common to all species although the two families separated more than 40 million years ago. To obtain information on the degree of homology among these conserved sequences we isolated a Bam HI restriction fragment of approximately 770 base pairs from red deer DNA. This sequence was 32P labeled and hybridized by the Southern blot technique with DNAs cleaved with Bam HI, Eco RI, Hpa II and Msp I. Moreover, the same sequence was cloned in the plasmid vector pBR322 nick translated with 32P and hybridized with the DNAs of 8 species of Cervidae and 3 of Bovidae. The same cloned probe was labeled with 3H and hybridized in situ with the metaphase chromosomes of red deer (2n = 68) and Muntiacus muntjak (2n = 7 male). Homologies are still present between the highly repetitive sequences of the 8 species of Cervidae despite the drastic reorganization that led to extreme chromosome numbers. Moreover, the cloned DNA sequence was found to occupy the same position, in the proximal regions of the arms, in both red deer (2n = 68) and M. muntjak (2n = 7 male) chromosomes. The ribosomal RNA genes and the centromeres in these species have also maintained their main territory despite the drastic chromosome reorganization. These results are experimental confirmation of the chromosome field theory which predicted that each DNA sequence has an optimal territory within the centromere-telomere field and tends to occupy this same territory following chromosome reorganization. PMID- 3022842 TI - Turing structures in an enzyme-induction system with gap junction-mediated non linear diffusion. AB - Two cells, each containing a reaction system modeling genetic induction, are coupled by diffusion. The substrate is moving through gap junctions, the number of which is regulated by the adjacent cells. This leads to a non-linear substrate diffusion term in the rate equations. Stability analysis reveals the conditions for the emergence of stable asymmetric solutions (dissipative structures). Due to non-linear diffusion rigid restrictions on the ratio of the two diffusion constants no longer exist. We demonstrate that substances operating as regulators of intercellular communication and participating in cellular metabolism may exhibit morphogenetic functions. PMID- 3022843 TI - [Disordered functioning of the superoxide radical-superoxide dismutase system in rat liver ischemia]. AB - The rate of O2 radical generation in microsomal membranes (VO2), the activity of cytosol superoxide dismutase (Cu, ZnSOD) and mitochondrial superoxide dismutase (MnSOD), and the activity of xanthine oxidizing system (XO) after a two-hour ischemia following a 24-hour reoxygenation of the rat liver were investigated. The high value of VO2, as compared to Cu, ZnSOD activity, may result in regulation disorders in O2-SOD system during ischemia. During reoxygenation, xanthine oxidizing system in combination with lowered Cu, ZnSOD activity may substantially contribute to the disturbance. PMID- 3022844 TI - [Effect of fusaric acid on phosphoinositide metabolism in the erythrocyte membranes of rats with spontaneous hypertension]. AB - 2- and 4-month-old male spontaneously hypertensive rats (SHR) were injected fusaric acid at a dose of 50 mg/kg body weight. Fusaric acid increased diphosphoinositide (DPI) and triphosphoinositide (TPI) levels in erythrocyte membranes of 4-month-old SHR by 41% and 20%, respectively. 32P incorporation into TPI decreased by 24% in 2- and by 20% in 4-month-old SHR. Phosphatidylinositol metabolism remained unchanged. The results also suggest that fusaric acid normalized DPI and TPI metabolism in erythrocyte membranes of SHR. PMID- 3022846 TI - Biosynthesis of factor XIII B subunit by human hepatoma cell lines. AB - The plasma transglutaminase, factor XIIIa (FXIIIa), circulates as a zymogen containing two proteins, A and B, arranged in a noncovalent tetrameric complex, A2B2. Biosynthesis of plasma FXIII has not previously been demonstrated. In the present study, direct evidence has been obtained that two human hepatoma cell lines, Hep G2 and PLC/PRF/5, synthesize and secrete FXIII B protein. Secretion of the B subunit of FXIII by Hep G2 was demonstrated by immunoblotting. De novo synthesis by Hep G2 was confirmed in 35S-methionine-labeled cultures. Radiolabeled conditioned medium was concentrated, mixed (1:1) with purified B protein, and examined by crossed immunoelectrophoresis with antiserum to the B subunit. The single protein precipitin arc of purified B protein comigrated with the radiolabeled FXIII from Hep G2 visualized by autoradiography, indicating both electrophoretic and antigenic identity. The data presented here represent the first demonstrations of biosynthesis of FXIII B protein by any cell type and suggest that the liver is the site of synthesis of FXIII B protein. Further analysis of concentrated Hep G2 serum-free conditioned medium (SFCM) and cell lysate by immunoblotting following nondenaturing agarose gel electrophoresis demonstrated the FXIII A protein as well as the B protein and also revealed synthesis and secretion of the A and B proteins by PLC/PRF/5. Crossed immunoelectrophoresis studies of Hep G2 SFCM and cell lysate suggest that Hep G2 cells also synthesize and secrete the plasma FXIII zymogen. With a specific radioimmunoassay for B protein, FXIII was found in Hep G2 SFCM at approximately 4 ng/mL; with an amplified rocket immunoelectrophoresis technique the level was approximately 5 ng/mL. PMID- 3022845 TI - [Catecholaminergic component of the action of a heptapeptide analog of ACTH4-10 on the open-field behavior of rats]. AB - Heptapeptide Met-Glu-His-Phe-Pro-Gly-Pro (ACTH4-10 analog) at a dose of 0.015 mg/kg failed to alter open field behaviour of rats in the first test series. The peptide abolished amphetamine-induced stimulation of the exploratory and grooming behaviour. Extinction of the rats' exploratory behaviour during second test series in the open field (7 days later) was disturbed when haloperidol or apomorphine were injected before the first test series. When the peptide was administered with haloperidol or apomorphine, the extinction tended to become normal. Heptapeptide failed to change noradrenaline, dopamine or 5 hydroxytryptamine content in the rat forebrain. However, this peptide at a concentration of 10(-4) M moderately diminished tyrosine hydroxylation velocity in the rat striatal or hypothalamic synaptosomes, the effect depending on tyrosine concentration. These data suggest the involvement of catecholaminergic component into the heptapeptide action on the behaviour of rats. PMID- 3022847 TI - Human myeloperoxidase gene: molecular cloning and expression in leukemic cells. AB - We have molecularly cloned the human myeloperoxidase (MPO) gene from the lambda gt11 expression library by screening with an affinity-purified MPO antibody. The cDNA clone of the MPO gene was used to study MPO gene expression in leukemic cells. The amino acid sequence predicted from the nucleotide sequence of the cDNA clone pMP401 matched exactly the 23 amino acid sequence of the NH2-terminal of the 60,000 MPO subunit. We found that MPO cDNA hybridized to a single EcoRI genomic band of 19 kb, indicating that the MPO gene represents a single gene in the human genome. Northern blot analysis of RNA isolated from leukemic cell lines and acute myelogenous leukemia (AML) patients' samples shows that MPO gene expression correlated with myeloid lineage. The intensity of MPO mRNA expression on Northern blot correlated with the level of MPO expression by cytochemical staining. Multiple species of MPO mRNA were found. This indicates that a single MPO gene may encode different RNA species through a mechanism of posttranscriptional processing or that multiple transcriptional start/termination sites exist in the MPO gene. PMID- 3022848 TI - Regional variation in beta-adrenoceptor-mediated relaxation of canine veins. AB - Three types of vasorelaxants were used to test the responses of canine veins isolated from 13 different sites: isoproterenol, papaverine and nitroglycerin. Strips were preconstricted with methoxamine (5 X 10(-6)-10(-5) M), KCl (50 mM) and PGF2 alpha (1 microgram/ml), and were relaxed by cumulative addition of relaxants. Isoproterenol caused more than 80% relaxation after preconstriction with methoxamine in cephalic, external jugular, azygos, renal, femoral, lateral saphenous veins and the supradiaphragmatic and infrarenal portions of the inferior vena cava, all of which are veins of the body wall. Pulmonary and splenic veins also showed marked relaxation with isoproterenol. However, maximal relaxation responses of portal, mesenteric veins and segment C of the inferior vena cava (a portion between liver and renal veins), which are embryologically related to the digestive tube, were less than 30%. Similar regional differences in the relaxation responses to isoproterenol were obtained after preconstriction with KCl or PGF2 alpha. Papaverine and nitroglycerin caused nearly uniform relaxation in all veins, although relaxations of segment C of the inferior vena cava were slightly less than those of other veins. These results indicated that there is a regional difference in the relaxation responses of the canine venous system to isoproterenol, and such a difference may be related to its embryogenesis. PMID- 3022850 TI - [Demonstration of fetal-type thymidine kinase in cancer of the breast]. AB - Seventy five human breast cancers were examined in order to search for the presence of thymidine kinase of the fetal-type (TK-F). The presence of TK-F was evidenced in all tumors. Its activity varied from one to another tumor, but it was evident that the increased TK activity observed in mammary cancers could exclusively be related to high TK-F activity. Some relations between TK-F activity and the presence of estradiol and progesterone receptors (ER, PR) were obvious. The highest activities were observed in cancers with high level of ER and PR. Thymidylate kinase activity (d-TTP synthesis) varied in parallel with TK F activity. In a general way, it was higher in ER+ PR+ than in ER+ PR- cancers. PMID- 3022849 TI - Cell kinetics of histologic variants of in situ breast carcinoma. AB - A thymidine labeling study of cell kinetics of 61 in situ breast carcinomas showed relationships between histological characteristics and kinetics. The thymidine labeling index (TLI) was significantly lower in cribriform-papillary intraductal carcinoma (median 1.30%, geometric mean 1.18%, mean 1.83 +/- 0.45%) and lobular carcinoma in situ (median 1.43%, geometric mean 1.12%, mean 1.63 +/- 0.46%) than in comedo intraductal carcinoma (median 4.40%, geometric mean 3.74%, mean 5.15 +/- 0.86%). The results for solid intraductal carcinoma, which is a less well defined and more heterogeneous entity, were intermediate (median 2.45%, geometric mean 2.40%, mean 3.32 +/- 0.80%). When invasive carcinoma was also available for kinetic study, the TLI of in situ and invasive components were usually similar (r = 0.66). The data indicate that the TLI usually does not change during the transition from in situ to invasive carcinoma. Cribriform papillary intraductal carcinoma is a slowly proliferating entity that gives rise to slowly proliferating invasive carcinomas with relatively high levels of estrogen and progesterone receptors. Lobular carcinoma in situ similarly has low proliferative rates and gives rise to slowly proliferating invasive carcinomas. Intraductal comedocarcinoma has relatively high proliferative rates and gives rise to invasive carcinomas with high proliferative rates that often are receptor negative. Nine of the 11 in situ carcinomas that were associated with invasive tumor and subsequent local recurrence or metastasis had TLIs above the median, and seven were comedo type with high TLIs. Our observations from thymidine labeling are consistent with a viewpoint regarding cribriform-papillary intraductal carcinoma as relatively bland, and comedo intraductal carcinoma as a distinctly more dangerous entity. Solid intraductal carcinoma seems to resemble cribriform-papillary more closely than comedo intraductal carcinoma. PMID- 3022851 TI - The MEDICYC programme. AB - The major goal of MEDICYC, the medical cyclotron programme of the Antoine Lacassagne Cancer Center (Nice, France) is radiotherapy based on protons and fast neutrons. This cyclotron has also been designed to meet the medical requirements for the Center's Nuclear Medicine Department. Technical specifications and anticipated applications are discussed. The construction of the machine is nearly finished and a first axially injected beam is scheduled for early 1987. PMID- 3022853 TI - Persistence of the herbicides [14C]chlorsulfuron and [14C]metsulfuron methyl in prairie soils under laboratory conditions. PMID- 3022852 TI - [Assessment of the quality of life of cancer patients. The preliminary applications to advanced bronchial cancers]. AB - So far there is no good evidence that chemotherapy improves survival in non small cell lung cancer. Reports of randomized trials testing chemotherapy versus supportive treatment alone are very few. In recent years, more and more emphasis has been put on the quality of life for cancer patients. Subjective tests such as Anamnestic comparative self Assessment (A.C.S.A. test, Dr. Bernheim) were designed as well as objective ones, using different types of self evaluation. However very few studies have been focused on this aspect in patients with non small cell lung cancer. Therefore the G.E.T.C.B. (Groupe d'Etude et de Traitement des Cancers Bronchiques) decided to initiate a cooperative randomized trial (02 CB 84) in metastatic epidermoid and large cell bronchus carcinoma, in order to test the potential benefit from chemotherapy in these tumors and how it affects the quality of live. In this trial, C.O.P.A.C. combination (Cyclophosphamide, Vincristine, Cis-Platinum, Adriamycin and CCNU) is randomized versus no chemotherapy. The quality of life is assessed monthly on the one hand by a modified A.C.S.A. test performed by a physician and on the other hand by a question list given to the patient. This question list attempts to evaluate the impact of disease as a reference point. Finally protocol 02 CB 84 (activated in 05/84) may give information about the effect of chemotherapy on both survival and quality of life. PMID- 3022854 TI - Primary Epstein-Barr virus infection and cervical lymphadenopathy in childhood. PMID- 3022855 TI - Pleomorphic adenoma occurring in a 9-year-old girl. PMID- 3022856 TI - [AIDS and mucocutaneous lesions]. PMID- 3022857 TI - Conservation surgery in the management of adenoidcystic carcinoma of the head and neck. PMID- 3022858 TI - The distinction between central and peripheral nervous system disease. PMID- 3022860 TI - A study of the effects of catabolin on cyclic adenosine monophosphate biosynthesis and prostaglandin E2 secretion in pig articular chondrocytes. AB - The effects of porcine leucocyte catabolin on prostaglandin E2 (PGE2) secretion and cyclic adenosine monophosphate (cAMP) production were examined in pig articular chondrocytes. Cells were cultured in monolayers on plastic substrates for either 1 or 6 days. Catabolin was found to stimulate PGE2 secretion markedly in all chondrocyte cultures, but had no effect on cAMP biosynthesis, irrespective of the duration of culture, time of exposure or concentration. It is concluded that in pig articular chondrocytes catabolin does not modulate cell function by a cAMP-dependent mechanism. PMID- 3022861 TI - Effect of corticosteroid therapy on blood monocyte superoxide generation in rheumatoid arthritis: studies in vitro and ex vivo. AB - Rates of superoxide (SA) generation by blood monocytes stimulated ex vivo were studied before and during corticosteroid treatment of rheumatoid arthritis (RA) patients, in control patients and in healthy controls. The direct effect on stimulated SA production of pre-incubating cells with prednisolone in vitro was also studied. Significant inhibition of monocyte SA output stimulated with IgG treated zymosan (ITZ) and fluoride ion (F), but not serum-treated zymosan (STZ) was demonstrated following steroid therapy in RA. No inhibitory effect of prednisolone could be demonstrated in vitro, using ITZ, STZ and F as stimuli. Our data on blood monocyte yields, size and cytochemistry suggest that the in vivo effect is due to a shift in blood monocyte traffic. PMID- 3022859 TI - Alpha 2-adrenoceptor-mediated contractile response to catecholamines in smooth muscle strips isolated from rainbow trout stomach (Salmo gairdneri). AB - The type of adrenoceptor involved in the contractile response to catecholamines in smooth muscle strips isolated from rainbow trout stomach was determined. Noradrenaline (10 nM-10 microM) and adrenaline (10 nM-3 microM) caused non sustained contractions which were markedly decreased by phentolamine (5.4 microM) but not by carteolol (5 microM). Phenylephrine (1 microM-1 mM) was less effective in causing muscle contraction and methoxamine produced no contraction. Clonidine (100 nM-300 microM) caused no mechanical response but inhibited the contraction to noradrenaline or adrenaline but not acetylcholine or 5-hydroxytryptamine. Yohimbine (10 nM-1 microM) decreased the contraction induced by noradrenaline or adrenaline but prazosin (1 microM) did not. Tetrodotoxin (780 nM) partially reduced the contraction induced by noradrenaline or adrenaline but atropine (500 nM) did not. In the presence of atropine (1 microM), electrical transmural stimulation caused frequency-dependent, tetrodotoxin-sensitive contractions. These results suggest that the contractile response induced by noradrenaline or adrenaline is mediated by alpha 2-adrenoceptors. It is also suggested that noradrenaline and adrenaline contract the smooth muscle by direct action and by indirect action through the non-cholinergic excitatory nerve. PMID- 3022862 TI - MR imaging of the intervertebral disc: a quantitative study. AB - The T1 and T2 relaxation times and the proton density of the nucleus pulposus have been measured in 107 normal and 18 surgically proven degenerate intervertebral discs. Data from total saturation recovery and spin echo sequences have been utilised in a robust multi-point method and relaxation times and proton density calculated. The results show that both the T1 and T2 values of the normal nucleus pulposus decrease with age. There was no significant correlation between proton density and age in normal discs. At all ages there was a highly significant difference between the T1 values of normal and degenerate discs. With T2 a highly significant difference in the younger age groups reduced to no distinction in the seventh decade. The observed change in the T1 and T2 values of the nucleus is in agreement with the reduction of water content known to occur with age. Our results indicate that quantitative MR imaging may assist in the diagnosis of intervertebral disc degeneration. PMID- 3022863 TI - CT sialography: utilising acinar filling. AB - The technique of computed tomographic sialography (CTS) has been demonstrated to be valuable in the diagnosis of masses of the major salivary glands. Forty-one CT sialograms were performed in 35 patients using acinar glandular filling with oily contrast material. Twenty-two mass lesions and seven cases of inflammatory disease were identified. There were two instances of mild parotitis following the procedure. CTS performed with this technique was found to be a safe, accurate method for evaluating salivary gland masses. PMID- 3022865 TI - Hereditary motor and sensory neuropathy type II. Clinicopathological study of a family. AB - A family with hereditary motor and sensory neuropathy (HMSN) type II is described in which 10 affected and 17 unaffected members in three generations were examined. The peak age of onset was in the second decade. In the youngest generation, the proportion of affected to unaffected individuals at risk significantly differed from the expected 50%. There was slight slowing of conduction velocities in 36% of nerves; however, only 3 out of 10 affected members had entirely normal conduction studies. The amplitude of the sensory potentials of median and peroneal nerves was almost uniformly reduced. In all affected patients electromyography of anterior tibial muscles showed signs of neurogenic involvement. Histological study of two sural nerves and a sciatic nerve and its branches revealed loss of myelinated fibres with a proximal-to distal gradient in this fibre loss, clusters of small regenerating fibres, and atrophic axons. Postmortem study of the proband showed loss of anterior horn and dorsal root ganglion neurons in the lumbar and sacral segments and degeneration of the fasciculus gracilis. Morphometric evaluation of L5 ventral and dorsal roots revealed a normal number of myelinated fibres, diameter histograms being shifted to the left because of a significant loss of large myelinated fibres and regeneration. These anatomical findings are consistent with the hypothesis that HMSN type II represents a primary neuronopathy affecting motor and sensory neurons. PMID- 3022866 TI - Biochemical characterization of the interaction of three pyridazinyl-GABA derivatives with the GABAA receptor site. AB - An arylaminopyridazine derivative of gamma-aminobutyric acid (GABA), SR 95103, has been shown to be a selective antagonist of GABA at the GABAA receptor site. Subsequent structure-activity studies showed that suppressing the methyl in the 4 position of the pyridazine ring, and substituting the phenyl ring at the para position with a chlorine (SR 42641) or a methoxy group (SR 95531) led to compounds which exhibited the highest affinities for the GABA receptor site in this series. In the present study we examined the biochemical interaction of these compounds with the GABA receptor as well as their biochemical selectivity for this receptor. SR 95531 and SR 42641 displaced [3H]GABA from rat brain membranes with apparent Ki values of 0.15 microM and 0.28 microM respectively and Hill numbers near 1.0. The two compounds antagonized the GABA-elicited enhancement of [3H]diazepam-binding in a concentration-dependent manner without affecting [3H]diazepam-binding per se. Scatchard and Lineweaver-Burk analysis of the interaction of the two compounds with the GABAA receptor sites, revealed that the compounds were competitive at the high affinity site, but non-competitive at the low affinity site. Neither compound interacted with other GABAergic processes or with a variety of central receptor sites. When administered intravenously, SR 95531 and SR 42641 elicited tonic-clonic seizures in mice. Based on these results, it is postulated that SR 95531 and SR 42641 are specific, potent and competitive GABAA antagonists. PMID- 3022864 TI - Human papillomavirus infection of the uterine cervix of women without cytological signs of neoplasia. AB - One hundred and six patients were studied whose cervical smears showed only non specific inflammatory changes. Screening for genital pathogens yielded only a few positive cases. Histological examination of biopsy specimens taken by colposcopically directed tissue sampling showed cervical intraepithelial neoplasia in 13 of the women (12.3%). Deoxyribonucleic acid (DNA) hybridisation techniques were used to detect human papillomavirus, which was found in 24 patients (22.6%). In a second group of 104 patients with normal cervical cytology tissue biopsy samples were obtained and examined histologically but in no case was cervical intraepithelial neoplasia found. On DNA hybridisation, however, 12 patients (11.5%) were found to be positive for human papillomavirus. In this group finding human papillomavirus DNA was usually associated with a columnar ectopy. An association between human papillomavirus type 16 DNA and both cervical intraepithelial neoplasia and cervical cancer is well established. In this study it was type 16 which occurred most frequently in both groups. PMID- 3022867 TI - Effect of protein malnutrition on hippocampal kindling: electrographic and behavioral measures. AB - Rats born to dams fed either a 6% (malnourished) or a 25% (control) casein diet during gestation and lactation and maintained on the diet of the dam after weaning were tested for electrographic and behavioral responses to electrically induced kindling of the CA1 field of the hippocampus beginning at 44 days of age. Animals in the 6% diet group had a significantly lower threshold to afterdischarge (AD), a significantly faster spread of AD activity to distal recording sites, significantly longer average duration of AD activity at all recording sites and a markedly altered behavioral progression toward seizure activity compared to control animals. These findings indicate that prenatal protein malnutrition results in hippocampal dysfunction as evidenced by both the electrographic and behavioral correlates of the kindling process. The data presented suggest that prenatal proteins malnutrition alters the response of hippocampal CA1 pyramids to electrical stimulation and that this alteration results in marked changes to both the electrographic and behavioral correlates of kindling. PMID- 3022869 TI - Alpha-adrenergic modulation of cyclic AMP formation in rat CNS: highest level in olfactory bulb. AB - The cyclic adenosine monophosphate response to catecholamines in the rat brain is mediated by beta-adrenergic receptors which activate adenylate cyclase and by alpha-adrenergic receptors which potentiate the response to beta-stimulation. We have found that the alpha-potentiation effect in the olfactory bulb is 2-3X greater than in other forebrain areas. This correlates with the extremely high density of alpha-receptors in this brain region and makes it a useful model for the study of alpha-receptor function. PMID- 3022868 TI - A procedure for measuring alpha 2-adrenergic receptor-mediated inhibition of cyclic AMP accumulation in rat brain slices. AB - The alpha 2-adrenergic receptor regulation of cyclic adenosine monophosphate (cAMP) accumulation in rat brain slices was examined. using a prelabeling technique for measuring second messenger production. The mixed alpha-adrenergic agonist 6-fluoronorepinephrine, as well as the more selective alpha 2-agonists clonidine and UK-14,304, caused a concentration-dependent inhibition of forskolin stimulated cAMP accumulation in cerebral cortical slices, whereas phenylephrine, a selective alpha 1-adrenergic agonist, had no inhibitory effect in this system. Moreover, alpha 2-adrenergic receptor antagonists were more potent than alpha 1 adrenergic antagonists in blocking the inhibitory response to UK-14,304. Neither alpha 1- nor alpha 2-adrenergic agonists displayed any inhibitory effect when cAMP accumulation was stimulated by isoproterenol, vasoactive intestinal peptide or 2-chloroadenosine. The results provide further evidence that some alpha 2 adrenergic receptors are negatively coupled to adenylate cyclase in brain, and yield a procedure for studying this phenomenon in intact central nervous system tissue. PMID- 3022870 TI - Relationships between medial hypothalamic alpha 2-receptor binding, norepinephrine, and circulating glucose. AB - These experiments examine the dependence of medial hypothalamic alpha 2-receptor binding on the availability of circulating glucose. Food deprivation and tolbutamide-induced hypoglycemia both diminished alpha 2-binding. These binding results were highly correlated with serum glucose levels. Prevention of tolbutamide hypoglycemia by concomitant injections of dextrose blocked the effects on alpha 2-binding. Injection of norepinephrine into the medial hypothalamic sites that normally elicit feeding behavior elevated serum glucose. These results suggest a homeostasis for control of glucose in a hypothalamic feeding system. PMID- 3022872 TI - Chlorpromazine protects brain tissue in hypoxia by delaying spreading depression mediated calcium influx. AB - We have investigated the possible protective effect of chlorpromazine in hypoxia of brain tissue, using rat hippocampal slices maintained at 35-36 degrees C. The recovery of synaptic transmission along the Schaffer collaterals to the CA1 pathway after 9 min hypoxia was compared in chlorpromazine-treated and in control slices. Recovery upon reoxygenation was the exception in control slices, while it was observed in approximately 50 and 100% of slices treated with 7 and 70 microM chlorpromazine, respectively. Chlorpromazine also significantly delayed the occurrence of the hypoxia-induced spreading depression (SD). Recovery took place when SD occurred late during hypoxia, not when it occurred early. In those slices in which 7 microM chlorpromazine afforded no protection, SD occurred as early as it did in control slices. In further experiments, we deliberately induced SD during hypoxia in 70 microM-treated slices by topically applying a drop of high K+ artificial cerebrospinal fluid (ACSF). Recovery was not observed when SD was induced early, but it was observed when it was induced near the end of the hypoxic period. Slices exposed to the same period of hypoxia in Ca2+-free ACSF recovered synaptic transmission (even without chlorpromazine treatment) despite early induction of SD. We conclude that: chlorpromazine protects brain tissue from hypoxia-induced irreversible loss of synaptic transmission; it does so by delaying the occurrence of SD, and hence shortening the time spent in the SD induced depolarized state; and the harm done by SD in hypoxia is related to the influx of Ca2+ into neurons. PMID- 3022871 TI - The action of natural and synthetic isomers of quisqualic acid at a well-defined glutamatergic synapse. AB - L-, D- and DL-quisqualic acid have been synthesized and their activities at the glutamatergic locust nerve-muscle junction have been compared with those of natural quisqualic acid and with glutamic acid. Two well-characterised locust nerve-muscle preparations were used in these studies, the retractor unguis nerve muscle system and the extensor tibiae nerve-muscle system. The amino acids were tested on the whole nerve-muscle system in the former, when reduction in neurally evoked twitch contraction amplitude was the measured parameter, and by ionophoretic application to single excitatory junctional sites in the latter, when amplitude of junctional depolarization was the measured parameter. Synthetic L-quisqualic acid exhibited identical potency to its natural counterpart. However, D-quisqualic acid and DL-quisqualic acid were more active than expected from the known stereospecificity of this glutamatergic system towards D- and L glutamic acid. The hydantoin analogue of quisqualic acid was inactive. X-ray crystallographic analysis of L-quisqualic acid and the hydantoin analogue showed that the ring junction in the former is pyramidal whereas in the latter it is planar. This may account for the high potency of L-quisqualic acid on a receptor system which identifies a partially folded conformation of L-glutamic acid. A pyramidal configuration of D-quisqualic acid would allow either rapid interconversion between active and inactive configurations at its ring junction or adoption of a trigonal configuration in solution. Either interpretation could explain the unexpected potency of D-quisqualic acid. PMID- 3022873 TI - Stimulation by dopamine of protein phosphorylation in rat striatal slices. AB - Incubation of rat striatal slices with dopamine enhanced the phosphorylation of two proteins with mol. wts. of 64,000 and 43,000. Although dopamine did increase cAMP levels in striatal slices, the addition of cAMP to striatal slices did not mimic the effects of dopamine on protein phosphorylation. The present results suggest that cAMP-independent protein kinases may mediate some of the effects of dopamine within the corpus striatum. PMID- 3022875 TI - Diminished interaction of norepinephrine with climbing fiber inputs to cerebellar Purkinje neurons in aged Fischer 344 rats. AB - The ability of norepinephrine (NE) to modulate climbing fiber activation of complex spike discharge in cerebellar Purkinje neurons was compared in young (3-6 months) and aged (18-20 months) Fischer 344 rats. In young rats, NE selectively inhibits spontaneous activity while climbing fiber evoked activity remains intact or increased. NE also increases the probability of observing 4 bursts of full sized action potentials rather than partially inactivated action potentials in the complex spike. In older rats, both of these modulatory actions of NE on climbing fiber complex spike activation are markedly diminished. These data support the concept that age-related reductions in catecholamine modulation of synaptic inputs may contribute to CNS dysfunction found in senescence. PMID- 3022874 TI - Serotonin 5-HT1C receptors are expressed at high density on choroid plexus tumors from transgenic mice. AB - Choroid plexus tumors develop spontaneously in adult transgenic mice carrying integrated copies of SV40 early region genes. In this communication, we report that these tumors exhibit the highest density of serotonin receptors (6600 fmol/mg protein) found in any tissue. 125I-LSD binding to choroid plexus tumors displays a pharmacological profile that matches the properties of 5-HT1C receptors in normal choroid plexus tissue. Autoradiographic localization of 125I LSD binding in brain sections from transgenic mice shows high levels of labelling in the tumors, in correlation with immunohistochemical staining for SV40 large T antigen expression. Choroid plexus tumors from these transgenic mice provide an excellent model system for the study of serotonin 5-HT1C receptors. PMID- 3022876 TI - Electrophysiological evidence for locus coeruleus norepinephrine autoreceptor subsensitivity following subchronic administration of D-amphetamine. AB - The effect produced by subchronic administration of D-amphetamine (D-AMP) on the sensitivity of norepinephrine (NE) autoreceptors in the rat locus coeruleus (LC) was studied by means of single unit recording and microiontophoretic techniques. Twice daily i.p. administration of 5 mg/kg D-AMP for one week markedly reduced the ability of i.v. D-AMP and microiontophoretic application of clonidine to suppress the firing of LC NE neurons, suggesting strongly that NE autoreceptors became subsensitive. In addition, the firing pattern of NE neurons became 'disorganized' following subchronic AMP treatment. PMID- 3022877 TI - Calcium-activated, phospholipid-dependent protein kinase and protein substrates in primary cultures of astrocytes. AB - Phosphoinositide-linked transmembrane signaling in the brain involves calcium activated, phospholipid-dependent protein kinase (protein kinase C), but little is known about the glial contribution to this system. We observed that phosphorylation of several proteins in a cytosol fraction of rat astrocytes in primary culture was increased by the addition of calcium and phosphatidylserine. These agents also stimulated phosphate incorporation into lysine-rich histone, a substrate for protein kinase C. Addition of diacylglycerol, an activator of protein kinase C, further increased histone phosphorylation, whereas polymyxin B, an inhibitor of protein kinase C, blocked the stimulatory effect of calcium and phosphatidylserine. Based on enzyme units per mg protein, the activity of protein kinase C in astrocytes appears similar to that in whole brain cytosol. These results indicate that astrocytes display protein kinase C activity and suggest that the glial enzyme may be an important component of the receptor-linked phosphoinositide response system in the brain. PMID- 3022878 TI - Hippocampal and cortical opioid receptor binding: changes related to the hibernation state. AB - In vitro opioid receptor binding in the dorsal hippocampal formation and parietal cortex was surveyed in ground squirrels (Citellus lateralis) in the contrasting physiological states of hibernation and euthermia (i.e. not hibernating). Computer-assisted autoradiographic analysis of coronal sections incubated with [3H]dihydromorphine (DMH; 4 nM) revealed statistically significant reductions in specific opioid binding associated with hibernation. In the dorsal hippocampal formation of hibernating animals, binding in the stratum radiatum of CA3, hilus of the dentate gyrus and molecular layer of the dentate gyrus exhibited decreases up to 34% compared to euthermic animals. The stratum radiatum of CA3 exhibited the smallest decrease overall. DHM binding in parietal cortex displayed significant hibernation-related reductions, although they were not uniformly observed across all laminae at the 3 different brain levels examined. These experiments present evidence of changes in brain opioid binding related to the mammalian state of hibernation. The results suggest that changes in opioid receptor binding during hibernation may contribute to the earlier reported apparent failure of morphine physical dependence to develop during hibernation. PMID- 3022879 TI - Effects of serotonin, cyproheptadine and reserpine on corticotropin-releasing factor release from the rat hypothalamus in vitro. AB - We investigated the effects of serotonin, cyproheptadine and reserpine on corticotropin-releasing factor (CRF) release from the rat hypothalamus, and the effect of cyproheptadine on CRF-induced adrenocorticotropic hormone (ACTH) secretion from the anterior pituitary (AP) in vitro using a perifusion system for rat hypothalami and AP, and a rat CRF radioimmunoassay. Cyproheptadine, 10(-8) M, had a direct inhibitory effect on both basal and 10(-9) M CRF-induced ACTH secretion from the rat AP in vitro. In addition, 10(-9)-10(-7) M cyproheptadine inhibited basal CRF release in a dose-dependent fashion, and also suppressed serotonin- and KCl-induced CRF release. Conversely, 10(-9)-10(-7) M reserpine failed to influence CRF release from the rat hypothalamus. These results indicate that a serotonergic mechanism may be involved in the CRF-releasing mechanism, and inhibition of depolarization-dependent calcium entry into cells and/or nerve endings. In addition an anti-serotonergic mechanism is involved in the inhibitory action of cyproheptadine. PMID- 3022880 TI - Effects of prostaglandin E2, forskolin and cholera toxin on cAMP production and in vitro LH-RH release from the rat hypothalamus. AB - The present study attempts to elucidate the possible role of adenosine 3',5' monophosphate (cAMP) and prostaglandin E2 (PGE2) in the function of the neural luteinizing hormone-releasing hormone (LH-RH) apparatus. To this end, in vitro LH RH release from superfused hypothalamic fragments and cAMP production by hypothalamic P2 membrane fractions were measured. Immature female rats (day 28) were ovariectomized and implanted with Silastic capsules containing estradiol (235 micrograms/ml). Two days later, animals were sacrificed and the mediobasal hypothalamic preoptic area (hypothalamic units or fragments) were removed. To examine in vitro LH-RH release from superfused hypothalamic fragments, effluents were collected into tubes on ice at 10-min intervals and LH-RH concentration was determined by radioimmunoassay (RIA). Following a 50-min control period, a step wise increment in several doses of PGE2 (each dose for a 50-min interval) evoked a dose-related increase in LH-RH release. PGE2 induced significant (P less than 0.01) increments in LH-RH release at doses of 5.68 X 10(-7), 5.68 X 10(-6), and 5.68 X 10(-5) M, respectively. When adenylate cyclase activators, such as forskolin and cholera toxin were infused in a step-wise manner (each dose for a 50-min interval) following a 50-min control period, a dose-related increase in LH RH release was also obtained; forskolin and cholera toxin significantly (P less than 0.01) stimulated LH-RH release at doses of 1 X 10(-4) and 5.4 X 10(-10) M, respectively. These two substances were ineffective in stimulating LH-RH release when hypothalamic fragments were superfused in calcium-free plus EGTA (10 mM) containing medium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022881 TI - Phencyclidine (PCP) receptors: autoradiographic localization in brain with the selective ligand, [3H]TCP. AB - Receptor binding sites for the phencyclidine (PCP) analogue, [3H]TCP, have been localized in the rat and guinea pig central nervous systems by in vitro autoradiography. Quantitation of [3H]TCP binding site densities in rat brain reveals highest levels in the forebrain, in particular the strata oriens and radiatum of the hippocampus, the molecular layer of the dentate gyrus and superficial layers of the cerebral cortex. Moderate levels of binding occur in the amygdala, thalamus, anterior olfactory nucleus, external plexiform layer of the olfactory bulb, olfactory tubercle, geniculate nuclei and deep layers of the cortex. Low levels of binding occur throughout most of the septum, diagonal band, hypothalamus, pons-medulla and cerebellum. Spinal cord grey matter also has low levels of binding. Excitotoxin lesions of the hippocampal formation, which destroy the pyramidal and granule cells, reduce the binding of [3H]TCP to strata radiatum and oriens and the molecular layer of the dentate gyrus by 60% suggesting that [3H]TCP labels intrinsic neurons in these regions. Residual binding is probably on afferent terminals. Ibotenic acid lesions of the caudate putamen reduce [3H]TCP binding by 70%, indicating that binding sites are localized on intrinsic striatal neurons. 6-Hydroxydopamine lesions do not alter [3H]TCP binding levels in the caudate, suggesting the absence of binding sites on dopaminergic terminals in the caudate. PMID- 3022882 TI - 'Catatonia' produced by alfentanil is reversed by methylnaloxonium microinjections into the brain. AB - Alfentanil, a short-acting and powerful analgesic, when injected peripherally to rats (0.5 mg/kg) produced a catatonic state characterized by a rigid akinesia. The present study was designed to explore the neuroanatomical location of the opiate receptors mediating the alfentanil induced catatonia. The catatonic effect of alfentanil was measured using a bar test and depression of locomotor activity in rats tested in photocell cages during an active nocturnal phase of their cycle. Methylnaloxonium HCl (MN), a quaternary derivative of naloxone which does not readily cross the blood-brain barrier, injected into the lateral ventricle significantly reduced the catatonia at doses of 0.125-2.0 micrograms as measured in both the locomotor and bar test. MN perfusion of similar doses directly into the nucleus raphe pontis, but not in the caudate nucleus significantly antagonized the catatonia. These data complement results on alfentanil-induced muscular rigidity (Blasco et al., see companion paper) where EMG indices of rigidity in rats were reversed by microinjections of low doses of MN (0.125 and 0.5 microgram) in the nucleus raphe pontis, but not the caudate nucleus even at a high dose (4.0 micrograms). Together these results suggest that the region of the nucleus raphe pontis is an important neural substrate for opiate-induced muscular rigidity, and that the catatonic state produced by opiates depends on more diffuse opiate receptor activation of which one important component may be the nucleus raphe pontis. PMID- 3022883 TI - Fluidity of normal and epileptogenic freeze-lesioned synaptic membranes in the cat. AB - Electron paramagnetic resonance spectra obtained with a 5-Nitroxylstearate spin label indicate that inhomogeneous lipid domains exist naturally in the bulk lipid matrix of cat synaptic plasma membranes at physiological temperatures, associated with non-cooperative phase transitions spanning broad temperature intervals between about 10 degrees C and 40 degrees C. These properties are qualitatively similar in normal membranes and in membranes isolated from epileptogenic freeze lesioned brain cortex. The fluidity of matrix lipids isolated from cortex adjacent to such lesions appears to be abnormally increased at physiological temperatures, but membrane polarity, partially reflecting water penetration of the surface of the bilayer, remains normal in membranes from lesioned areas. Both fluidity and polarity of membranes isolated from 'mirror' sites contralateral to lesions appear to remain normal, and thus unaffected by seizure activity per se in these preparations, in which independent 'mirror' foci did not develop. These findings demonstrate that lipid phase inhomogeneities are present and thus may play a role in normal and abnormal functions of synaptic membranes, and that abnormalities of membrane fluidity are in fact closely associated with epileptogenesis in the freeze-lesion model. PMID- 3022884 TI - Non-linear summation at an identified synapse in Aplysia. AB - The degree of non-linear summation of postsynaptic potentials at the RC1-R15 synapse in Aplysia californica was determined. Two independent methods were used to reduce the size of the postsynaptic potential: voltage clamp of the postsynaptic potential and pharmacologic blockade of the postsynaptic receptors. Results from both methods demonstrate that non-linear summation is significant at this synapse. The non-linear correction formula of Stevens produces a reasonable compensation of measured EPSP amplitudes for the contribution of non-linear summation. The experiments also provide additional evidence that all of the variations in EPSP amplitude seen at this synapse are due to changes in the number of postsynaptic channels opened and not due to changes in the non-synaptic membrane properties of R15. PMID- 3022885 TI - Storage and release of acetylcholine in rat cortical synaptosomes: effects of D,L 2-(4-phenylpiperidino)cyclohexanol (AH5183). AB - A post-stimulation synthesis of acetylcholine (ACh), its incorporation into a 'stable-bound' (vesicular) compartment and subsequent release, were compared in K+-stimulated synaptosomes, in the absence and presence of 10 microM AH5183. The drug depressed by 16% the net intrasynaptosomal formation of ACh from 1 microM [3H]choline (Ch) in the medium, by competitively inhibiting (Ki approximately equal to 20 microM) the high-affinity Ch transport, but it had no direct effect on the intraterminal synthesis of ACh per se. The drug reduced incorporation of newly synthesized [3H]ACh into synaptic vesicles by 55% and subsequent K+ depolarization-induced release of [3H]ACh by 83%, although it had no effect on Ca2+ influx into synaptosomes. These results are consistent with the hypothesis that AH5183 blocks cholinergic neurotransmission presynaptically by interfering with recharging of synaptic vesicles with ACh. Since the reduction of ACh release in the presence of AH5183 had no direct effect on ACh synthesis, these results also suggest that the transmitter release is not prerequisite for enhancement of Ch uptake and ACh synthesis in stimulated nerve terminals. PMID- 3022886 TI - Critical period plasticity of kitten visual cortex is not associated with enhanced susceptibility to electrical kindling. AB - The goal of this study was to determine whether the use-dependent malleability of visual cortex functions which is particularly pronounced in 4-week-old kittens correlates with enhanced susceptibility to kindling. For that purpose the effects of high-frequency electrical stimulation were compared in the visual cortex of 4 week-old kittens and of adult cats. The striate cortex of one hemisphere was stimulated with a single train of pulses whose intensity was set just above the threshold for the elicitation of afterdischarges (ADs). In kittens the AD thresholds were consistently higher than in adults and with repeated stimulation, the ADs tended to disorganize, to decrease in amplitude and duration and to become more restricted to the site of stimulation after about 6 stimulations. In the adult, by contrast, the ADs remained well organized and constant in duration throughout 30 stimulations. They showed an increase in amplitude and spike frequency and spread with increasing consistency to the other hemisphere. No electrographic or behavioural signs of epileptic activity developed in kittens, while in adults ADs were on occasion followed by irregular spike activity associated with behavioural states resembling absences. We conclude that the visual cortex possesses powerful mechanisms to prevent the development of supracritical excitatory states, these mechanisms being more effective in the kitten than in the adult. PMID- 3022887 TI - Postnatal development of [3H]yohimbine binding in the olfactory bulb of male and female rats. AB - A postnatal increase in [3H]yohimbine binding sites was observed in the rat olfactory bulb. The maximal increase in binding site density was between postnatal days 1 and 14 and corresponded to that of peak interneuron (granule and periglomerular) production and maturation, suggesting a possible interneuron location for the alpha 2-adrenoceptors in the rat olfactory bulb. No differences in [3H]yohimbine binding in the olfactory bulb attributable to sex or stage of the estrous cycle were observed. PMID- 3022889 TI - Increased vascularity and alterations in cytochrome oxidase distribution associated with abnormal hippocampal cytoarchitecture in micrencephalic rats. AB - Prenatal exposure of the developing rat brain to methylazoxymethanol acetate results in the formation of ectopic groups of pyramidal neurons in the subfields CA1 and CA2 of the mature hippocampal formation. These ectopic neurons are situated in regions of increased vascularization and increased activity of cytochrome oxidase, as demonstrated histochemically. Both these findings suggest that there may be an abnormality of distribution or intensity of oxidative metabolic activity in the ectopic pyramidal neurons found in these animals. PMID- 3022888 TI - Interaction of beta-casomorphins with multiple opioid receptors: in vitro and in vivo studies in the newborn rat brain. AB - beta-Casomorphins (beta-CMs) are peptidic fragments of bovine beta-casein with potent opioid activity. In view of a possible physiological meaning of these milk derived compounds, we have studied the affinity of some natural beta-CMs and some semisynthetic analogues for mu-, delta- and kappa-brain opioid receptors in newborn and adult rat. Moreover we have investigated whether a chronic treatment with the potent analogue D-Ala2-beta-CM-4-NH2 during the suckling period could affect mu and delta opioid receptor function. Our findings demonstrate that beta CMs are mu-oriented compounds both in adult and in newborn rat brain. They display the same mu-affinity in newborn as well as in adult animals, however delta and kappa-affinities appear different, probably due to the lower degree of maturation of these two receptors in the first days of life. A prolonged treatment with D-Ala2-beta-CM-4-NH2 during the preweanling period is able to induce a delay in the ontogenetic increase of delta-receptor affinity, whereas it affects neither the affinity nor the density of mu-receptors. This effect could be related to a general action of opioids on cerebral maturative processes; moreover we point out that a beta-CMs analogue, given orally to newborn animals, can induce modifications at central level, suggesting thus the hypothesis that beta-CMs could be a biologically active peptide in the first stages of life. PMID- 3022890 TI - Asynchronism in the neurogenesis of GABAergic and non-GABAergic neurons in the mouse hippocampus. AB - The time course of the neurogenesis of cells showing glutamic acid decarboxylase (GAD) immunoreactivity has been analyzed in the dorsal hippocampus and area dentata of the mouse. The quantitative data indicate that gamma-aminobutyric acid (GABA)ergic cells are generated prenatally, and that their peaks of neurogenesis occur before the peaks of neurogenesis of non-GABAergic cells. Additionally, GABAergic cells in the hippocampus proper follow a 'sandwich' sequence of positioning. PMID- 3022891 TI - Immunocytochemical localisation of gelsolin in oligodendroglia of the developing rabbit central nervous system. AB - Antibodies raised against gelsolin of rabbit lung macrophages were used in an immunocytochemical study during development of the rabbit central nervous system. With the exception of a transient staining of the cerebellar Purkinje cells during the first postnatal week, gelsolin was found a new marker for oligodendrocytes, especially in developing animals. In the macrophages, gelsolin is involved in the control of locomotion, secretion and endocytosis. In the oligodendrocytes, which produce the myelin sheaths in the central nervous system, gelsolin could therefore be involved in the control of the complex motile events leading to myelin wrapping. PMID- 3022892 TI - The self-regulating synapse: a functional role for the co-existence of neuroactive substances. AB - Although the co-localizations of neuroactive substances, such as transmitters and peptides, in identified neurons is now a common histochemical phenomenon, the physiological roles and functional significance of such co-existence are largely unknown. Using the vertebrate retina as a model for the central nervous system, we have examined the relationship between co-existence and co-function. We propose here that the co-localization of neuroactive substances in a synaptic terminal provides the structural configuration to ensure the co-release of two or more predetermined substances into the same synaptic cleft, resulting in the capability of the presynaptic neuron to stringently regulate its own activities and output. PMID- 3022893 TI - Centrally mediated effect of vanadate on diuresis and natriuresis in normal rats. Interaction with ouabain. AB - Prolonged infusion of vanadate (VO-3) (51 ng/microliter/hr during 15 hr), an in vitro inhibitor of Na,K-ATPase activity, into the 3rd brain ventricle (3BV) increased diuresis, sodium and potassium excretion in normal rats (p less than 0.001). Since the animals did not receive food or water during the infusion period, the effect of VO-3 was not related to hydration or volume expansion. A single injection of ouabain (a specific inhibitor of the sodium pump) 1 microliter, 50 pg/microliter, followed by a single injection of vanadate (1 microliter/51 ng/microliter) 1, 5 or 10 minutes after ouabain, prevented the diuretic and natriuretic effect of VO-3. When vanadate injections were delayed 20 or 30 minutes, diuresis reappeared. Ouabain (50 pg) decreased sodium intake induced by sodium depletion, an effect that lasted for 10 to 15 minutes after injection, a time coincident with the inhibition of the natriuretic effect of VO 3 by OUA. Then the life-span of ouabain effect on prevention of VO-3-induced diuresis as well as on inhibition of sodium intake, lasted between 10 to 15 minutes. These results provide further evidence for the powerful diuretic, natriuretic and kaliuretic effect of centrally injected vanadate. However, the finding that ouabain suppresses this effect, does not support the suggestion that the active transport system might be involved. PMID- 3022894 TI - [Morphologic and functional study of a human insulin-secreting cell line]. AB - Monolayer cell cultures were obtained from a human insulinoma (HIN) after collagenase digestion. HIN cells were initially plated on extracellular matrix (ECM) secreted by bovine corneal endothelial cells. Capsular integrity from cell clusters quickly interrupted and cell began to migrate as adhesive sheets onto ECM. After 2 months on ECM cell attachment and proliferation occurred on plastic allowing cloning of cells by limiting dilution. 9 clones were successfully cultured for 7 months with 20 subsequent passages. Immunoreactivity for insulin by indirect immunofluorescence typical secretory granules by electron microscopy and stable amounts of immunoreactive insulin in culture media suggest that HIN cells are beta cell related. One clone HIN D8 when challenged for half an hour with either 30 mM glucose, 1 mM isobutyl Methylxanthine 4 mM Tolbutamide, 10(-6) M glucagon responded respectively with a 1.5, 2, 3 and 1.5 fold increase in insulin output. Population doubling time of HIN D8 was 42 hrs. Establishment of such insulin secreting cell lines provides a valuable tool for diabetes research. PMID- 3022895 TI - [Demonstration of VIP receptors in B cells of the pancreas by quantitative ultrastructural autoradiography]. AB - Using quantitative electron microscopic autoradiography we demonstrated the existence of specific VIP receptors in B cells of the pancreatic islets. The presence of those receptors strongly suggests that the neuromediator can induce insulin release by direct stimulation of B cells. PMID- 3022896 TI - [Action of a dietary Mg2+ deficiency and overload in the male rat: measurement of certain parameters of testicular and spermatozoan activity]. AB - The specific activities of several enzymes were investigated as possible markers of the Mg2+ impact on testicular and spermatozoa glucidic metabolism: no modification was noticed. However, as a reflection of hormonal metabolism, 17 beta-OH steroid-deshydrogenase activity, AMPc and testosterone levels were measured in the testes and showed significant variations. PMID- 3022897 TI - [Viruses and human cancers: fundamental aspects and hopes of prevention]. PMID- 3022899 TI - [HDL receptor activities in patients with familial hypercholesterolemia]. PMID- 3022898 TI - [Anti-inflammatory effect of ACTH in the rat]. AB - In rats, ACTH reduced the oedemas induced by zymosan and lambda carrageenan. ACTH reduced the volume of the exudate induced by sponge implantation and its content in proteins, beta-galactosidase, beta-glucuronidase and PGE2. The inhibitory effect of ACTH was suppressed by adrenalectomy which increased the carrageenan oedema. The inhibitory effect of ACTH was also suppressed by 17 alpha methyltestosterone. Corticosterone reduced carrageenan-oedema. The inhibitory effect of corticosterone was suppressed by cycloheximide and actinomycin D. These results suggest that rat adrenal steroids, among which corticosterone, can modulate the reactivity of the animal towards irritating processes. The anti inflammatory effect of rat adrenal steroids would depend on the formation of lipocortin-like peptides. PMID- 3022900 TI - Does nalbuphine reverse opioid obtuned laryngeal reflexes? PMID- 3022901 TI - Signet-ring cell lymphoma in the orbit: a case report and review. AB - The authors report on the first case of signet-ring cell lymphoma involving the orbit. This variant of non-Hodgkin's lymphoma contains either vacuolated or eosinophilic cytoplasmic inclusions that squeeze the nucleus to one side of the cell. In this case, the inclusions were eosinophilic and stained positively for PAS, immunoglobulin M (IgM) heavy chain and kappa light chain. Electron microscopy showed granular electron-dense material trapped within distended loops of rough endoplasmic reticulum. Twenty-two cases of signet-ring cell lymphoma previously reported elsewhere in the body are reviewed, and their clinical significance is discussed. PMID- 3022902 TI - Ileal perforation due to cytomegaloviral enteritis. AB - As the AIDS epidemic unfolds, many newly described clinical syndromes resulting from unusual manifestations of opportunistic infections are being seen. The authors report the clinical and pathological features of intestinal perforation in an AIDS patient caused by extensive small-bowel ulcerations due to cytomegalovirus. The diagnosis was confirmed by ultrastructural and immunohistochemical studies. It is suggested that infection and destruction of the muscularis propria by the virus contributes to intestinal perforation. PMID- 3022904 TI - The Vancouver Lymphadenopathy-AIDS Study: 6. HIV seroconversion in a cohort of homosexual men. AB - In an ongoing prospective study of homosexual men conducted since November 1982 in Vancouver, we identified 345 men who did not have antibody to human immunodeficiency virus (HIV) at the time of enrolment and for whom results of follow-up serologic testing were available. A total of 66 cases of seroconversion were documented among the 345 men between November 1982 and October 1985. Methods of survival data analysis that take into account the varying durations of follow up were used to study the epidemiologic features of seroconversion in this group. The probability of seroconversion during the entire observation period was 23.1%. The seroconversion rates remained stable, at 10.5% and 10.0% during the last 2 years of the observation period. Cox regression analysis revealed the following variables to be independently associated with risk of seroconversion: frequent receptive anal intercourse, elevated number of male sexual partners in the year before enrolment, use of illicit drugs, a history of gonorrhea and age less than 30 years in November 1982. Multivariate analysis failed to reveal any role of oral sexual activity in the transmission of HIV. Oral ingestion of semen was not associated with seroconversion in either univariate or multivariate analysis. The observation that younger men were more likely to seroconvert suggests that young homosexual men were less likely than older men to modify their sexual behaviour. PMID- 3022905 TI - Laboratory reports of herpesvirus infections in Canada in 1985. PMID- 3022907 TI - Nongerminomatous malignant germ cell tumors in children. A review of 89 cases from the Pediatric Oncology Group, 1971-1984. AB - Clinical and morphologic features of 89 cases of childhood yolk sac tumor (YS) and embryonal carcinoma (EC) (29 associated with teratomas) submitted to the Rare Tumor Registry of the Southwest Oncology Group (1971-1979) or the Pediatric Oncology Group (1980-1984) between 1971 and 1984 were reviewed and submitted to statistical analysis. This review showed an improved survival for each 5-year period regardless of tumor site, no statistically significant difference between "pure" tumors and those mixed with other teratomatous components, no statistically significant difference between YS and EC in children, a better than reported prognosis for sacrococcygeal tumors occurring after the neonatal period, a particularly poor prognosis for neonatal "benign" sacrococcygeal teratomas resected without coccygectomy when they recur as YS, excellent survival for all testicular tumors regardless of age or the presence of EC, and the occurrence of mediastinal tumors in females. PMID- 3022906 TI - Prostaglandins, thromboxane and leukotriene in human follicular fluid. AB - The concentrations of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), thromboxane B2 (TxB2) and leukotriene B4 (LTB4) were measured in the follicular fluid of 15 hyperstimulated human follicles. All the samples contained these eicosanoids. Ova were recovered in six aspirates; the measured levels of prostanoids and LTB4 did not differ significantly from the levels of nine aspirates without ova. The presence of LTB4 in human follicular fluid suggests that the products of the lipoxygenase pathway of arachidonic acid may also have a role in the function of the follicular fluid. PMID- 3022903 TI - Chronic viral diseases. AB - Until 20 years ago the only chronic viral diseases known were those considered to be confined to the nervous system. As a result of recent advances in epidemiology, molecular biology and immunology, new viral diseases have been recognized and their clinical features and pathogenesis elucidated. Chronic disease may result from infection with the hepatitis B and D viruses and whatever agent or agents cause hepatitis non-A, non-B, the herpesviruses, Epstein-Barr virus, cytomegalovirus and human T-lymphotropic virus type III. These diseases have common features, including long-term or even lifetime asymptomatic carriage, viremia, with virus free in the plasma or attached to circulating mononuclear cells, presence of virus in body secretions, irreversible tissue injury in target organs and oncogenic potential. New information on these diseases is reviewed. Other chronic diseases for which the cause is currently unknown may eventually prove to be due to viral infection. In addition, vaccines may be developed for prophylaxis of some chronic viral diseases and associated malignant diseases. PMID- 3022908 TI - Long-term survival and morbidity in patients with metastatic malignant germ cell tumors treated with cisplatin-based combination chemotherapy. AB - Forty-three of 79 patients treated for clinically metastatic germ cell cancer survived for a median of 66 months (range, 52-83). In patients without previous chemotherapy the 5-year survival rate was 69%, whereas only 32% of patients with prior chemotherapy survived for 5 years. Limited disease, complete clinical response, histopathologically proven postchemotherapy tumor necrosis or complete resectability of a posttreatment mature teratoma indicated a favorable prognosis in patients without prior chemotherapy. Only 20% to 30% of the patients with less than a clinical complete response or with posttreatment residual malignant tumor can be salvaged by second line therapy. Posttreatment mature teratoma should be resected completely whenever possible, as this condition may lead to reactivation of the malignancy even after several years. Raynaud-like phenomena and/or gastrointestinal problems are the main long-term sequel (10%-15%) after modern multimodality treatment of advanced germ cell cancer (fertility-related problems are not considered here). In the majority of surviving patients, the lifestyle seems unaffected by the previous intensive treatment, evaluated about 5 years after discontinuation of all therapy. PMID- 3022910 TI - Chondrosarcoma of the temporal bone. Diagnosis and treatment of 13 cases and review of the literature. AB - Chondrosarcoma of the temporal bone is a rare lesion. Clinically it has been confused with multiple sclerosis, glomus jugulare tumors, meningioma, and chordomas. The cranial nerve palsies frequently observed with the tumors are related to the anatomic locations of the tumors. Thirteen patients with this entity are presented and the eleven other cases in the literature are reviewed. Histologically the tumors are low grade and exhibit myxoid features. The myxoid features must be differentiated from chordoma and chondroid chordoma. The tumor locations preclude surgical excision and conventional radiation therapy can cause unacceptable neurologic sequelae. Proton beam therapy has been effective in short term results and appears capable of avoiding serious neurologic side effects. PMID- 3022909 TI - CA-125. Use as a tumor marker with mixed mesodermal tumors of the female genital tract. AB - CA-125 levels were determined in seven patients with a mixed mesodermal tumor of the ovary or endometrium. An elevated level was detected in five of six patients with metastatic or recurrent disease. Chemotherapy with cisplatin and Adriamycin (doxorubicin) resulted in a response in four patients, and the CA-125 level correlated well with the clinical status of the disease. Cisplatin/Adriamycin is an active combination in mixed mesodermal tumors and CA-125 appears to be a useful marker for following therapy. PMID- 3022911 TI - T-cell lymphoma revealed by a peripheral neuropathy. A report of two cases with an immunohistologic study on lymph node and nerve biopsies. AB - In two patients a peripheral neuropathy was the presenting symptom of a noncutaneous peripheral T-cell lymphoma. In the first patient, the neuropathy had a relapsing and remitting course, the symptoms improved under corticosteroid therapy. The second patient suffered from a relentless neuropathy. In both cases the lymphoma infiltrated the peroneal nerve with an angiocentric and perivascular pattern resembling that observed in central nervous system lymphomas. The characterization of T-cell subsets in the lymph node showed cells with the helper/inducer and suppressor/cytotoxic phenotype in the first case and a predominance of cells with the helper/inducer phenotype in the second case. In the nerve, lymphocytes beard the helper/inducer phenotype antigen. A typical paraneoplastic vasculitis of nerve showed clearly different immunologic features. PMID- 3022912 TI - Diffuse subependymal periventricular metastases. Report of three cases. AB - Three clinicopathologic cases with a remarkable pattern of extensive diffuse subependymal periventricular spread of cerebral metastases from solid systemic cancer are reported. Two patients had a small cell carcinoma of the lung. In the third case, the histologic features of the brain metastases were consistent with a neuron-specific enolase-positive, small cell anaplastic carcinoma. Involvement of the choroid plexus and leptomeninges was moderate or absent. Intraparenchymatous nodular metastases were not found except in one case in which rare nodular superficial cortical metastases were present. The clinical data were nonspecific except for orthostatic hypotension, in one patient, which was probably due to the infiltration of the floor of the third and fourth ventricles. Results of the cerebrospinal fluid examination, available in two cases, were normal. The only diagnostic investigation was contrast-enhanced computed tomography scanning. PMID- 3022913 TI - Application of long-term collagenase disaggregation for the cytogenetic analysis of human solid tumors. AB - A method has been elaborated for obtaining chromosome preparations from different histologic types of human solid tumors and applied to 60 cases. Such tumors were mechanically dispersed, washed by settling and transferred into flasks containing collagenase (200 U/ml) in the growth medium. Tumor pieces were dissociated in enzyme for 16 hours to 6 days, depending on tumor consistency. The majority of the tumors (80%) required 16-24 hours of such treatment. After disaggregation, the suspension of cells was washed by settling and then centrifuged. Cells were cultured for 24 hours to 6 days (85% of tumors), treated with a hypotonic solution in situ and then fixed. Of the 60 tumors, 37 (60%) displayed a sufficient number of metaphases for banding analysis. The success rate depends primarily on the type of tumor: 70% for soft tissue sarcomas, 40% for bladder carcinomas, and 40% for renal adenocarcinomas. This procedure is gentle, requires no special equipment and is applicable for the study of the full spectrum of human solid tumors. PMID- 3022914 TI - DNA damage in cultured L1210 cells by 2-haloethyl esters of (methylsulfonyl)methanesulfonic acid. AB - The effects of the 2-chloroethyl, 2-bromoethyl, and 2-fluoroethyl esters of (methylsulfonyl)methanesulfonic acid upon the DNA of cultured L1210 cells have been measured and compared with each other and with the effects of chlorozotocin. Results obtained by the alkaline elution method indicated that, at equimolar and equitoxic concentrations, the esters caused more strand scission than chlorozotocin, but at compound concentrations that caused a 50% reduction in colony formation by cells following an exposure period of 2 h, they caused no detectable cross-linking, whereas chlorozotocin did cause cross-linking. Two in vitro experimental methods that are based upon the complexing of ethidium to calf thymus DNA also yielded data showing that, at equimolar concentrations, chlorozotocin caused cross-linking of calf thymus DNA, but the 2-chloroethyl ester did not. These results indicate that these esters might not kill cells by producing DNA-DNA cross-links. The three esters caused qualitatively similar effects, but the fluoro esters caused less strand scission than the chloro and bromo esters, which caused about the same extent of strand scission. PMID- 3022915 TI - Potentiation of lymphocyte responses by monoclonal antibodies to the ganglioside GD3. AB - Previous studies have shown that the ganglioside GD3 is expressed in high concentrations on melanoma cells and that monoclonal antibodies (MAbs) against GD3 may induce partial remissions in tumor growth in patients with melanoma. The present studies indicated that incubation of interleukin 2 (IL2) dependent murine natural killer cells with several MAbs against the ganglioside GD3 potentiated the proliferative response to IL2. There was no effect on cell division in the absence of added IL2. Similarly the mitogenic response of human blood lymphocytes to phytohemagglutinin (PHA) or the T3 antigen was enhanced by coculture with these MAbs. This was noted when the MAbs to GD3 were added either before or up to 48 h after addition of PHA or MAb to T3. Kinetic analysis revealed that the potentiation of the PHA induced mitogenic response followed the expected response to PHA suggesting that the MAbs amplified normal activation pathways. These effects were also seen wih MAb to GD3 and the T10 structure on T-cells but not with MAbs to the transferrin receptor or isotype MAb controls. Studies on T-cell subsets suggested that the enhanced PHA responses were confined mainly to the T8 subset and responses of the T4 subset were not enhanced. Flow cytometric analysis revealed low levels of GD3 expression on blood lymphocytes which increased during culture with PHA. IL2 receptor (Tac epitope) expression did not show close correlation with the enhanced lymphocyte responses and the mechanism of the potentiation remains uncertain. These studies raise questions concerning the role of gangliosides in T-cell activation and whether these in vitro effects of MAbs to GD3 may account in part for their antitumor effects in patients with melanoma. PMID- 3022916 TI - Calmodulin antagonism and growth-inhibiting activity of triphenylethylene antiestrogens in MCF-7 human breast cancer cells. AB - The triphenylethylene antiestrogen tamoxifen has been shown previously to inhibit both calmodulin and protein kinase C activities, which are involved in the control of cell proliferation. We have studied the effect of several derivatives of the triphenylethylene antiestrogen family on the inhibition of both calmodulin dependent cyclic adenosine 3':5'-monophosphate-phosphodiesterase activity and proliferation of breast cancer cells cultured with 0.5 microM estradiol in order to prevent interaction of these drugs with the estrogen receptor. We have observed that hydroxylation of the triphenylethylene molecule significantly decreases its ability to inhibit the calmodulin-dependent phosphodiesterase activity in vitro. Furthermore, the growth-inhibiting activity of several antiestrogens and other calmodulin antagonists [R24571, trifluoperazine, N-(6 aminohexyl)-5-chloronaphthalene-1-sulfonamide, and N-(6-aminohexyl)-1 naphthalenesulfonamide] correlated with their antagonistic effects on calmodulin activity. The level of activity was determined as follows: R24571 greater than tamoxifen = N-demethyltamoxifen = nafoxidine greater than 4-hydroxytamoxifen greater than 3,4-dihydroxytamoxifen = trifluoperazine greater than N-(6 aminohexyl)-5-chloronaphthalene-1-sulfononamide greater than metabolite A greater than N-(6-aminohexyl)-1-naphthalenesulfonamide. On the other hand both protein kinase C-activating and -inhibiting drugs (phorboltetradecanoate-13-acetate and tamoxifen, respectively) have a synergistic inhibitory effect on the growth of MCF-7 cells. Our data suggest that antiestrogen interactions with calmodulin and not protein kinase C may play a role in mediating the drug-induced estrogen independent inhibition of breast cancer cell growth. PMID- 3022917 TI - Enhanced expression of the c-myc gene in bovine leukemia virus-induced bovine tumors. AB - We have detected elevated levels of c-myc gene expression in neoplastic cells from all seven bovine leukemia virus (BLV)-induced bovine tumors examined, but not in BLV-infected, nonneoplastic lymphoid cells. No rearrangement or amplification of the c-myc gene could be demonstrated in any of the BLV-induced tumors. Furthermore, BLV proviral DNA was found to have no preferred site of integration in these tumors. The possible mechanisms of enhanced expression of the c-myc gene in BLV-induced tumors have been discussed. PMID- 3022918 TI - Presence of chromosomal abnormalities and lack of AIDS retrovirus DNA sequences in AIDS-associated Kaposi's sarcoma. AB - The frequent occurrence of Kaposi's sarcoma (KS) in association with the acquired immune deficiency syndrome (AIDS) could be due to the fact that the etiological agent of this tumor is the same retrovirus causing AIDS, to another oncogenic virus frequently found in AIDS patients, or to the unmasking of the tumorigenic potential of KS cells by immunosuppression. We have therefore investigated the presence of DNA sequences homologous to the AIDS retrovirus, cytomegalovirus (CMV), and hepatitis B virus in 13 KS necropsies and biopsies from AIDS patients. All KS DNA samples were negative for AIDS retrovirus or hepatitis B DNA sequences. Two DNAs from necropsies contained CMV DNA, but the data suggested the presence of replicating CMV DNA due to generalized infection. We have also studied cell cultures derived from KS skin biopsies of AIDS patients. These cultures had a short lifetime in vitro and expressed some markers of endothelial cells. The cells were not tumorigenic in nude mice but contained a number of chromosomal rearrangements which were often monoclonal within the same culture. However, these abnormalities were different from culture to culture and even in cultures from the same biopsy. The presence of these chromosomal abnormalities seemed to correlate with the cell positivity for endothelial markers. Taken together these results indicate that neither the AIDS retrovirus, CMV, or hepatitis B virus is directly responsible for the altered growth of KS cells, that KS may be polyclonal even within the same lesion, and that KS cells have a tendency to karyotypic rearrangements. PMID- 3022919 TI - Collagenase and elastase production by mouse mammary adenocarcinoma primary cultures and cloned cells. AB - We have shown previously that an increase in tumor invasion and metastases occurred concurrently with a decrease in collagen content of the extracellular matrix surrounding the C3H mouse mammary adenocarcinoma borne by C3H/HeJ mice. In this paper we report the production of collagenase and elastase activities by the primary tumor cultures and three types of cloned C3H mouse mammary adenocarcinoma cell cultures. The primary tumor cell cultures and tumor-associated stromal cultures produced large amounts of collagenase and elastase activities. On the other hand, the primary tumor capsule cultures produced little or no collagenase and elastase activities even though they produced type I collagen. The production of proteases by the primary tumor cultures decreased along with time and with an alteration in the morphology of cell populations and/or passage of the cultures. The three clones of tumor cell cultures produced variable amounts of collagenase in response to induction by phorbol myristate acetate, an agent that stimulates maximal collagenase production. In contrast, all three cloned cultures elaborated significant amounts of elastase that degraded insoluble ligamental elastin, and most of the elastase production was increased further in response to induction by phorbol myristate acetate. Each cloned cell population exhibited differences in their production of collagenase and elastase in parallel with their difference in growth kinetics, yet these cells still possess the distinctive properties of the tumor. However, a unit amount of collagenase produced by each of the cloned cultures, with or without induction by phorbol myristate acetate, was less than that of the primary tumor cultures. Results suggest that some cell types or combination of cell types in the heterogeneous cell population of the tumor and/or their products appear to be responsible for the increased production of collagenase and elastase activities and for the invasiveness of a malignant tumor. PMID- 3022920 TI - Phenotypic characterization of lung cancers in fine needle aspiration biopsies using monoclonal antibody B72.3. AB - Monoclonal antibody B72.3, reactive with a high-molecular-weight glycoprotein tumor-associated antigen (designated TAG-72), has been previously shown to be reactive with formalin-fixed paraffin-embedded tissue sections of adenocarcinomas of the ovary, colon, and breast and not a variety of normal adult tissues. It has demonstrated utility as an immunocytochemical adjunct to diagnose carcinoma in cell block and cytocentrifuge preparations of human serous effusions, with selective reactivity for tumor cells (particularly adenocarcinoma) over reactive mesothelium. In this study, fine needle aspiration biopsies of 127 lung cancers (93 primary and 34 metastatic tumors) as well as 18 benign lung lesions were analyzed with monoclonal antibody B72.3 using avidin-biotin-peroxidase techniques. Monoclonal antibody B72.3 showed reactivity with 100% of the 27 lung adenocarcinomas and adenosquamous carcinomas, with greater than or equal to 10% of tumor cells showing reactivity in 22 of 27. A lesser percentage of squamous cell carcinomas (24 of 31) and large cell carcinomas (7 of 13) were immunoreactive, and of these several were weakly reactive with less than or equal to 1% tumor cells reacting. In contrast, monoclonal antibody B72.3 failed to react with any of the 21 small cell carcinomas or one carcinoid tumor evaluated. In 35 patients tumor-bearing tissue was resected, and formalin-fixed tissue sections were also evaluated. The staining pattern and percentage of tumor cells positive in the aspiration biopsies were, in most cases, highly predictive of the reactivity observed in corresponding resected tumor. Metastatic adenocarcinomas to lung from various body sites were also immunoreactive with monoclonal antibody B72.3; however, a variety of other tumor types (including 13 melanomas) failed to stain. Staining by monoclonal antibody B72.3 was not noted in any of the 18 benign lesions aspirated, with the exception of occasional fine stippling in the cytoplasm of bronchial epithelial cells. Hence, monoclonal antibody B72.3 and fine needle aspiration biopsy techniques may be of potential use in the differential diagnosis and antigenic phenotyping of a spectrum of lung neoplasms prior to surgical resection. PMID- 3022921 TI - Cytotoxic T-lymphocyte recognition of simian virus 40 antigens. AB - During the transformation of mammalian cells by SV40, the cells acquire new antigenicity at the cell surface that acts as a target for cytotoxic T lymphocytes. Evidence is discussed which leads to the conclusion that the product of the SV40 tsA gene, the T antigen, is directly involved in converting a cell to malignant phenotype, is largely localized in the nucleus of transformed cells and is also present at the cell surface. Therefore, it appears that the T antigen serves a dual function, i.e. it is involved in the initiation and maintenance of transformation and provides a target for the immune effector mechanisms in the host undergoing viral carcinogenesis. PMID- 3022922 TI - Cell surface antigens of chemically induced sarcomas of murine origin. AB - Chemically induced sarcomas of murine origin are characterized by the expression of unique tumour-specific transplantation antigens. Their nature and the genetic basis of their antigenic diversity are unknown. Numerous attempts have been made to relate these antigens to products of known polymorphic gene families. A serological analysis of cell-surface antigens of sarcomas was initiated on the premise that serologically defined unique tumour-specific antigens would be related to tumour-specific transplantation antigens. This analysis led to the serological definition of two noncross-reacting tumour-specific antigens on two antigenically distinct BALB/c sarcomas, Meth A and CMS4. Assignment of the gene(s) involved in expression of the Meth A antigen to the distal region of chromosome 12, the same region encoding Igh, raised the possibility that genetic elements responsible for antibody idiotype could be involved in the antigenic diversity of tumour-specific transplantation antigens. Although several studies indicate a concordant expression of the Meth A and the tumour-specific transplantation antigen, the Meth A tumour-specific transplantation antigen identified as an 82,000-Da protein does not express the serologically defined antigen. This is in contrast to the Zajdela hepatoma of rat origin, where a serologically defined tumour-specific antigen is related to the tumour-specific transplantation antigen. Whether the serologically defined Meth A antigen has tumour-specific transplantation antigen activity is not presently known. PMID- 3022923 TI - The fms gene and the CSF-1 receptor. AB - The c-fms proto-oncogene encodes an integral transmembrane glycoprotein with tyrosine specific protein kinase activity whose properties resemble those of receptors for polypeptide growth factors. The relatively restricted expression of the feline c-fms gene in mononuclear phagocytes (peripheral blood monocytes and tissue macrophages) and their committed bone marrow progenitors suggested that c fms encoded a receptor for a macrophage specific growth factor. We found that the receptor for the mononuclear phagocyte colony stimulating factor, CSF-1 (M-CSF), is biochemically and immunologically related to the c-fms gene product, consistent with the hypothesis that c-fms is identical to the CSF-1 receptor gene (Sherr et al, 1985). Although the activity of CSF-1 has been defined through its action on haemopoietic cells, the c-fms gene is expressed in human placenta (Muller et al, 1983a) and in human choriocarcinoma cell lines derived from placental trophoblasts (Muller et al, 1983b). The latter cell lines exhibit binding sites for CSF-1, suggesting that this colony stimulating factor might also have an embryological role in placental development. The retroviral oncogene v-fms is a potent fibroblast transforming gene, whereas c-fms can be expressed at relatively high levels in certain normal tissues without causing neoplastic transformation. The glycoprotein encoded by v-fms is closely related to the c-fms gene product but differs at its extreme carboxy terminal end. Because v-fms, like c-fms, encodes a competent ligand binding domain, cells producing the v-fms gene product acquire the ability to bind CSF-1. Moreover, many fibroblast cell lines susceptible to transformation by v-fms produce the growth factor. Although this raises the possibility that v-fms transforms cells by an autocrine mechanism, antibodies to epitopes in the v-fms-coded ligand binding domain that interfere with CSF-1 binding, or antibodies to CSF-1 itself, do not affect the transformed phenotype. In membrane preparations, tyrosine-specific phosphorylation of the v fms product appears to be constitutive, whereas in vitro phosphorylation of the c fms-coded glycoprotein on tyrosine is enhanced in the presence of CSF-1. These results are most compatible with the possibility that critical alterations in the 3' coding region of the c-fms gene activate its kinase activity and unmask its latent transforming potential. An implication of these findings is that chromosomal rearrangements affecting c-fms could contribute to myeloid leukaemogenesis. PMID- 3022924 TI - The role of cAMP in regulating tumour cell growth. AB - Addition of cAMP to various cultured cell types has a dramatic effect on cell growth. Both positive and negative effects on growth have been demonstrated. Analysis of mutants with altered cAMP dependent protein kinases suggests that tumour cells do not require a functional endogenous cAMP system for normal cell cycling. Whether or not cAMP stimulates or inhibits cell growth depends on the cell type, the oncogene driving its growth, the dose of cAMP and the environment of the cell. The ras gene product does not appear to be a component of the adenylate cyclase system, and no other oncogenes have been shown to use cAMP as a second messenger. However, another class of oncogenes possesses a serine/threonine kinase activity analogous to that of cAMP dependent protein kinase. Several oncogene products and growth factor receptors are phosphorylated on serine residues, suggesting that some oncogene products, such as pp60src may be targets for the action of cAMP. The role of cAMP in tumour cell growth may be to modulate the activity of the oncogenes or their protein targets which control cell growth. PMID- 3022925 TI - SV40 large T-antigen: dual oncogene. AB - Simian virus 40 (SV40) is a small, DNA-containing tumour virus. One of its gene products, the large tumour antigen (T-ag), is essential for both viral replication and cell transformation. SV40 T-ag can be considered a dual oncogene protein; it is a composite transforming protein that provides distinct functions at different subcellular locations. In addition to its roles in virus replication, T-ag exerts numerous effects on host cells. Those cellular effects reflect viral stimulation of host cell entry into S phase. Numerous chemical modifications have been ascribed to T-ag. They might be involved in defining subpopulations of T-ag that are, in turn, responsible for mediating various T-ag biochemical functions. The T-ag polypeptide, 90,000-100,000 in molecular weight, appears to contain multiple, discrete functional domains; several biological activities have been assigned to relatively small defined regions of the molecule. The cellular progenitors of the T-ag biochemical activities are not obvious. A cellular protein, p53, thought to be involved in regulation of cell proliferation, becomes complexed with T-ag in transformed cells and is stabilized. The interaction of T-ag with this cellular substrate may play an important part in SV40 transformation. T-ag and T-ag/p53 complexes are localized in both the nucleus and plasma membrane of transformed cells. T-ag is transported to the nucleus because of a 7-residue nuclear transport signal contained within its primary sequence. Its migration to the membrane is by an unknown pathway. Only a minor fraction of the total cellular T-ag is present at the cell surface. Both amino and carboxy termini of the T-ag polypeptide are exposed on the extracellular face of the cell. Nuclear and membrane T-ag are structurally very similar, although a portion of membrane T-ag is acylated and nuclear T-ag is not. The nuclear and membrane forms of T-ag apparently provide separate and complementary functions necessary for cell transformation. Nuclear T-ag is important in immortalizing primary cells and membrane T-ag may mediate more pronounced morphological changes. A model is presented, postulating how the two forms of T-ag might cooperate to mediate phenotypic transformation. PMID- 3022926 TI - New drugs in small-cell lung cancer. PMID- 3022927 TI - Human leukocyte antigens and sister chromatid exchanges in families with multiple adenomatosis coli. AB - To study human leukocyte antigens (HLA) and sister chromatid exchanges (SCE) in multiple adenomatosis coli (MAC), eight families were evaluated. Twenty-five subjects (14 affected members and 11 healthy relatives) were studied with HLA A, B, and C typing of peripheral lymphocytes and SCE count in cells that had replicated twice. The results of the present immunogenetic study, although limited to a small number of cases, suggest a correlation between HLA, SCE, and MAC. When the results of further investigations on larger series confirmed by clinical follow-up of these subjects are available, these methods might give useful information in predicting the risk of each family member to become affected by MAC and consequently develop a colorectal carcinoma. PMID- 3022928 TI - Clinicomorphological manifestations of primary liver carcinoma (PLC) in liver cirrhosis. AB - Among 200 deaths from liver cirrhosis the clinical and autopsy records of 30 histologically confirmed cases of primary liver carcinoma (PLC) were reviewed. Male to female ratio was 5:1. Biopsy-proven liver lesions reflected chronic liver disease, mainly cirrhosis. Autopsy-PLC detected was classified as hepatocellular carcinoma (21 cases) with trabecular and pseudoglandular histological pattern, cholangiocarcinoma (two cases), and hemangiosarcoma. This retrospective analysis pointed to the relationship between PLC and liver cirrhosis, the latter being the primary risk factor for the incidence of PLC in Slovenia (Yugoslavia). PMID- 3022929 TI - Lesion formation in abraded human enamel. Influence of the gradient in solubility and the degree of saturation of buffer solutions on the lesion characteristics. PMID- 3022930 TI - [The importance of the current use of pyrophosphate scintigraphy and other noninvasive methods in the detection of cardiac damage in systemic lupus erythematosus]. PMID- 3022931 TI - [Plasma ACTH concentration in the diagnosis of various types of hypercorticism]. PMID- 3022932 TI - [The distribution of herpes simplex and cytomegalovirus antibody in differential age groups in Guangzhou]. PMID- 3022933 TI - Tumor invasion through the human amniotic membrane: requirement for a proteinase cascade. AB - To understand the role of proteinases in tumor invasion, the effects of inhibitors of metallo-, serine-, and cysteine-proteinases on this process were studied using 125I-iododeoxyuridine-labeled B16/BL6 cells grown on human amnion basement membrane. Cellular invasion was quantitated by measuring the radioactivity associated with the amniotic membrane after the B16/BL6 cells on the basement membrane were removed by lysis followed by scraping. The results obtained with proteinase inhibitors showed that inhibitors of collagenase and plasmin prevented invasion of the amnion. Tissue invasion was also blocked by antiurokinase antibodies. On the contrary, cysteine-proteinase inhibitors and anti-tissue plasminogen activator antiserum were ineffective. Mersalyl, a compound known to activate collagenase, stimulated invasion under conditions where plasmin formation or activity were inhibited. Evidence for the role of a plasminogen activator-plasmin-collagenase activation cascade in B16 invasion is provided. PMID- 3022934 TI - Mitochondrial plasmids of Neurospora: integration into mitochondrial DNA and evidence for reverse transcription in mitochondria. AB - The Mauriceville (3.6 kb) and Varkud (3.8 kb) mitochondrial plasmids of Neurospora are closely related, closed circular DNAs whose nucleotide sequences and genetic organization suggest relationships to mitochondrial introns and retrotransposons. Here we isolated mutants whose growth is impaired as a result of malevolent behavior of these plasmids. All 12 mutants contain variant plasmids that are suppressive relative to mtDNA, and ten also contain defective mtDNAs. All the suppressive plasmids contain small insertions, generally including a mitochondrial tRNA sequence, at or near the major 5' RNA start site. The structure of the suppressive plasmids suggests that they were generated via an RNA intermediate and a reverse transcription step. At least three of the mutants contain defective mtDNAs into which mitochondrial plasmid sequences have integrated. Sequences at the plasmid-mtDNA junctions are also consistent with integration via an RNA intermediate. PMID- 3022935 TI - Evidence for trans splicing in trypanosomes. AB - The 5' ends of trypanosome mRNAs consist of an identical sequence of 35 nucleotides. This "mini-exon" sequence is derived from the 5' end of a 137 nucleotide RNA (medRNA). The remainder of each mRNA is derived from a protein coding exon that is not linked to the mini-exon. We propose that medRNA is spliced in trans to de-novo-initiated transcripts of protein-coding genes. This trans splicing model predicts that the downstream portion of medRNA will be part of a branched structure and then be released as a free product (minRNA). We demonstrate that significant levels of minRNA exist in trypanosome RNA. Furthermore, minRNA can be released from high molecular weight RNA by a HeLa cell S100 "debranching" extract. We conclude that trans splicing is the physiological process by which mature mRNA molecules are synthesized in trypanosomes. PMID- 3022936 TI - Organized packaging of kinetoplast DNA networks. AB - The kinetoplast DNA (kDNA) of Trypanosoma equiperdum is organized as a complex structure of catenated circular DNA molecules. The major component of the kDNA network is the one kilobase minicircle that is present at about 10,000 copies per network. We have developed two assays to examine the structure of kDNA networks compacted in vitro with spermidine. Our results suggest that minicircles are arranged into a regular structure with an exposed domain which is DNAase I- and restriction-sensitive and a protected domain which is resistant to restriction endonucleases and DNAase I. This regularly packaged structure is dependent upon spermidine compaction and the circularity of the kDNA, but does not require supercoiled minicircles or catenated networks. PMID- 3022938 TI - Mitochondrial genome rearrangement leads to extension and relocation of the cytochrome c oxidase subunit I gene in sorghum. AB - The mitochondrial gene (COXI) encoding cytochrome c oxidase subunit I (COI) was isolated from two cytoplasmic genotypes of sorghum that synthesize different COI polypeptides. The Milo COI (Mr approximately 38,000) is encoded by a 530 codon structural gene sharing 98% homology with the corresponding maize gene. A variant COI observed in 9E cytoplasm (Mr approximately 42,000) is encoded by a 631 codon structural gene that diverges completely from the Milo COXI gene both 100 bp 5' to the presumed initiator methionine and within the 3' coding sequence. The 3' divergence results in a 101 C-terminal extension of the 9E COI that is not homologous to any known mitochondrial polypeptide. The novel 9E COXI, apparently arising from at least two rearrangements, affects transcription and gene product. PMID- 3022939 TI - DNA synthesis dependent on genetic recombination: characterization of a reaction catalyzed by purified bacteriophage T4 proteins. AB - To simulate a reaction that occurs in T4-infected cells, we have developed an in vitro DNA synthesis system that requires seven highly purified proteins encoded by this bacteriophage: the DNA polymerase "holoenzyme" (four proteins), gene 32 protein, dda DNA helicase, and uvsX protein - an enzyme that catalyzes homologous DNA pairing and is functionally homologous to the recA protein. In the reaction observed, the 3'OH end of one single-stranded DNA molecule primes DNA synthesis using a double-stranded DNA molecule of homologous sequence as the template. The uvsX protein continuously removes the new DNA chain from its template, so that DNA is synthesized by a conservative mechanism. This type of reaction, which requires the cooperation of recombination and replication enzymes, seems likely to be a general feature of DNA metabolism. PMID- 3022937 TI - Expression and structure of the human NGF receptor. AB - The nucleotide sequence for the human nerve growth factor (NGF) receptor has been determined. The 3.8 kb receptor mRNA encodes a 427 amino acid protein containing a 28 amino acid signal peptide, an extracellular domain containing four 40 amino acid repeats with six cysteine residues at conserved positions followed by a serine/threonine-rich region, a single transmembrane domain, and a 155 amino acid cytoplasmic domain. The sequence of the extracellular domain of the NGF receptor predicts a highly ordered structure containing a negatively charged region that may serve as the ligand-binding site. This domain is conserved through evolution. Transfection of a full-length cDNA in mouse fibroblasts results in stable expression of NGF receptors that are recognized by monoclonal antibodies to the human NGF receptor and that bind [125I]NGF. PMID- 3022940 TI - Nonpolarized expression of a secreted murine leukemia virus glycoprotein in polarized epithelial cells. AB - Vaccinia virus recombinants were generated which express the intact gp70/p15E of Friend mink cell focus inducing virus (F-MCFV) or truncated forms of the glycoprotein that lack the transmembrane and cytoplasmic domains. The transport of the intact and truncated envelope glycoproteins to apical or basolateral surfaces was studied in the polarized epithelial MDCK cell line. Infection of MDCK cells with the recombinant expressing the intact F-MCFV envelope glycoprotein resulted in transport exclusively to the basolateral surfaces, whereas the recombinant expressing the truncated glycoprotein was found to be secreted from both the apical and basolateral surfaces. Thus removal of the transmembrane and cytoplasmic domains of the p15E protein results in a loss of directional transport to the basolateral membrane of polarized epithelial cells. PMID- 3022941 TI - Bidirectional pigment granule movements of melanophores are regulated by protein phosphorylation and dephosphorylation. AB - Studies were conducted to investigate the molecular basis for bidirectional pigment granule transport in digitonin-lysed melanophores. Pigment granule dispersion, but not aggregation, required cAMP and resulted in the phosphorylation of a 57 kd polypeptide. cAMP-dependent protein kinase inhibitor prevented this phosphorylation as well as pigment dispersal. In contrast, both pigment aggregation and the concomitant dephosphorylation of the 57 kd polypeptide were blocked by phosphatase inhibitors. These data support a model in which pigment dispersion and aggregation require protein phosphorylation and dephosphorylation, respectively. Furthermore, studies using the ATP analog, ATP gamma S, suggest either that protein phosphorylation alone is sufficient for dispersion or that transport is mediated by a unique force-generating ATPase that can use ATP gamma S for hydrolyzable energy. PMID- 3022942 TI - The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation. AB - The linear virion form of Epstein-Barr virus (EBV) DNA has variable numbers of direct tandem 500 bp repeats at each terminus. The terminal restriction endonuclease fragments and the fused terminal fragments in the intracellular episomal form are heterogeneous in size, and vary by increments of 500 bp. The structure of the termini of EBV in carcinomas of the nasopharynx and the parotid gland was compared with the EBV termini in monoclonal and polyclonal tissues or cell lines. A single band representing the EBV joined termini was detected in each of the carcinomas and in the monoclonal lymphoid proliferations. Polyclonal cell lines contained multiple forms of the joined termini. The detection of a homogeneous episomal population suggests that EBV-associated epithelial malignancies are clonal expansions of a single EBV-infected progenitor cell. PMID- 3022943 TI - Yeast peptide pheromones, a-factor and alpha-factor, activate a common response mechanism in their target cells. AB - We show that in yeast the cell type specificity of pheromone response is determined solely by the species of receptor that a cell synthesizes. The two receptor-pheromone interactions are functionally interchangeable and involve the creation of a common intracellular signal. In particular, we find that provision of a-factor receptor or alpha-factor receptor in mat alpha 1 mutants, which normally do not express either receptor or any other a- or alpha-specific products, allows response to the appropriate pheromone. Moreover, provision of a factor receptor in a cells lacking alpha-factor receptor restores mating competence to those cells. Finally, an aspect of pheromone response that is normally unique to a-factor action on alpha cells--increased transcription from the alpha-specific STE3 gene--can also be observed following alpha-factor treatment of pseudo-a cells (mat alpha 2 ste3 ste13), special mutants that respond to alpha-factor and also have an active STE3 promoter. PMID- 3022944 TI - Transport into mitochondria and intramitochondrial sorting of the Fe/S protein of ubiquinol-cytochrome c reductase. AB - The Fe/S protein of complex III is encoded by a nuclear gene, synthesized in the cytoplasm as a precursor with a 32 residue amino-terminal extension, and transported to the outer surface of the inner mitochondrial membrane. Our data suggest the following transport pathway. First, the precursor is translocated via translocation contact sites into the matrix. There, cleavage to an intermediate containing an eight residue extension occurs. The intermediate is then redirected across the inner membrane, processed to the mature subunit, and assembled into complex III. We suggest that the folding and membrane-translocation pathway in the endosymbiotic ancestor of mitochondria has been conserved during evolution of eukaryotic cells; transfer of the gene for Fe/S protein to the nucleus has led to addition of the presequence, which routes the precursor back to its "ancestral" assembly pathway. PMID- 3022945 TI - A possible role for Mg2+ ions in the induction of meiotic maturation of Xenopus oocyte. AB - Progesterone induces in vitro the meiotic cell division of Xenopus full-grown oocytes. Microinjection into oocyte of a solution containing Mg2+ (20 mM) facilitates by one order of magnitude the dose of progesterone which induces 50% of germinal vesicle breakdown. Microinjected in the absence of hormone, Mg2+ and also Mn2+ can induce maturation with efficiencies of, respectively, 24% (SEM = 8; n = 13) and 70% (SEM = 6; n = 23). The dose-response curves of cation-induction of maturation show an optimum of 20 mM for Mg2+ and 15 mM for Mn2+ (pipet concentration); higher doses were less active. Cation-induction of maturation is inhibited when oocytes are preincubated with cholera toxin (500 ng/ml); nevertheless, it cannot be interpreted at the level of cAMP, since both Mg2+ and Mn2+ microinjections provoke an increase in the oocyte cAMP content. Mg2+ induction of maturation is more efficient when oocytes are incubated in trimethylamine at pH 8.2, which is known to increase intracellular pH suggesting an action at the level of alkali pH-sensitive enzymes. Altogether, our results indicate a positive role for Mg2+ ions in the induction of oocyte maturation and raise an attractive hypothesis about the respective roles of cAMP and Mg2+ changes involved in the mechanism of progesterone action. Our results also show that co-injection of 2-glycerophosphate and Mg2+ ions, which are both commonly used in the preparation of the MPF mitotic factors from dividing cells, induces oocyte maturation more efficiently than Mg2+ alone and drastically shortens the kinetics of germinal vesicle breakdown to 1 h 30 min to 2 h 30 min. PMID- 3022946 TI - Low degree of ubiquitination of histone 2A in the dipteran Chironomus tentans. AB - A polyclonal antibody to ubiquitin has been prepared and shown to react with both ubiquitin and ubiquitinated histone 2A (uH2A). Applying this antibody in Western blotting experiments, we have observed that the salivary glands of Chironomus tentans contain an unusually low amount of uH2A (1% of histone 2A), while the amount of free ubiquitin is as abundant as in other animal cells, e.g. HeLa cells. The same low content of uH2A was also found in diploid epidermal cells of Chironomus origin suggesting that the low amount is not a characteristic of the polytene state of chromatin in salivary gland cells but rather a property of C. tentans as a species. The significance of the low degree of ubiquitination is discussed in relation to the information available on the organization of Chironomus chromatin into unusually large chromomeric entities. PMID- 3022947 TI - Protein synthesis, ribosomal protein S6 phosphorylation in vitro and the effects of amiloride: SDS gel electrophoresis studies in the Yoshida ascites tumor (AH 130) grown in vivo. AB - Cell-free cytosolic extracts from the Yoshida (AH 130) rat ascites hepatoma cell line, grown in vivo, showed high ribosomal protein S6 kinase activity in vitro, as measured by transfer of 32P to exogenous 40S rat liver ribosomal subunits, in both exponential growing and stationary phase cells. A significant decrease of protein synthesis (3H-leucine incorporation into total cell protein) was found to occur in cells reaching the stationary phase of growth, suggesting that S6 phosphorylation was not tightly coupled to the rate of the intraperitoneal cell growth and of protein synthesis in these tumor cells. When the cell-free cytosolic extracts were prepared from cells exposed to amiloride, at concentrations that inhibit the Na+/H+ exchange, a decrease of S6 kinase activity was observed only in exponential growing cells, suggesting the possibility of coupling of the Na+/H+ exchange with phosphorylation of intracellular proteins in these tumor cells. Actually, stationary phase cells showed unchanged S6 kinase activity under the same conditions, possibly due to the extremely low Na+/H+ exchange activity, previously demonstrated (Cell Biol. Int. Rep., 1985, 9, 1017 1025). The present experiments support the hypothesis that the regulation of protein synthesis is not tightly coupled to phosphorylation-dephosphorylation cycles, at least of ribosomal protein S6, in cells characterized by a rather uncontrolled growth such as the Yoshida (AH 130) rat ascites hepatoma. In this connection, an elevated degree of protein phosphorylation, such as that of the ribosomal protein S6, could be a general phenomenon of neoplastic transformation. PMID- 3022948 TI - [Hormonal changes in the treatment of the climacteric syndrome using conjugated estrogens]. PMID- 3022949 TI - [Studies on the relationship of duck hepatoma and duck hepatitis B virus in a high incidence area of human hepatoma]. PMID- 3022951 TI - [The borderline serous tumor of the ovary]. PMID- 3022950 TI - [Effect of coal particles on the pulmonary macrophage of rabbits in vitro]. PMID- 3022952 TI - [The pathological observation of experimental infections by Coxsackie virus B4 (CVB4) in mice]. PMID- 3022953 TI - [Study on the relationship between the serum IgA antibody titres to EB VCA and the pathological changes in untreated nasopharyngeal carcinoma (NPC) patients]. PMID- 3022955 TI - [Ultrastructure of hepatocellular carcinoma and hepatoblastoma]. PMID- 3022954 TI - [Cytomegalovirus (CMV) infection in cases of infantile autopsies]. PMID- 3022956 TI - [An ultrastructural study of undifferentiated bronchogenic carcinoma]. PMID- 3022957 TI - [Relations between the depressor effect induced by stimulation of peripheral ends of the abdominal vagi and serotonin]. PMID- 3022959 TI - [Application of the computer in making physical maps of DNA by partial digestion with restriction enzymes]. PMID- 3022958 TI - [Diagnostic value of sialography for Sjogren's syndrome]. PMID- 3022960 TI - [A study of the relation of intestinal metaplasia, gastric carcinoma and the spleen deficiency syndrome using histochemical staining of the gastric mucosa and determination of cAMP and cGMP]. PMID- 3022961 TI - Visualization of chromatin events during DNA excision repair in XP cells: deficiency in localized but not generalized chromatin events. AB - Cells derived from patients with xeroderma pigmentosum (XP) are known to be compromised in excision repair after u.v. irradiation, but the exact site of the molecular defect in the repair pathway is not known. The purpose of this study was to examine the ability of XP cells to alter their chromatin in preparation for excision repair. Treatment of quiescent normal human fibroblasts with u.v. light results in two types of chromatin changes that can be visualized in G1 phase prematurely condensed chromosomes (PCC): a generalized elongation of the G1 PCC, and regions of localized elongation or gaps. Quiescent XP cells (complementation groups A, C, D and G) were treated with u.v. light, and their G1 PCC were examined for chromatin changes in response to damage. All cell lines tested showed a normal generalized chromatin elongation response to u.v. treatment. However, the XP cell lines were found to be deficient in the generation of localized regions of decondensation, even when incubated after u.v. treatment in the presence of cytosine arabinoside and hydroxyurea. Since the regions of localized decondensation in the G1 PCC have been previously shown to be the sites of unscheduled DNA synthesis, these results suggest that while XP cells do exhibit a generalized chromatin response to u.v. irradiation, they are defective in their ability to alter the chromatin in a localized fashion in preparation for excision repair after u.v. irradiation. PMID- 3022962 TI - Assessment of the beta-adrenergic receptor pathway in the intact failing human heart: progressive receptor down-regulation and subsensitivity to agonist response. AB - We developed methods for identifying beta-adrenergic receptors in human right ventricular endomyocardial biopsy tissue with the radioligand ( )[125I]iodocyanopindolol (ICYP). Specific ICYP binding in a crude, high-yield membrane preparation derived from endomyocardial biopsy tissue was high (specificity greater than 90%), of high affinity (KD around 20 pM), saturable and stereospecific for the (-) vs the (+) isomer of isoproterenol. Subjects with mild moderate and severe biventricular dysfunction had respective decreases in beta adrenergic receptor density of 38.2% and 57.7% when normalization methods were averaged, with no significant differences in ICYP dissociation constant. A subgroup of subjects was subdivided by left ventricular ejection fraction (LVEF) into those with mild cardiac dysfunction (LVEF less than 0.50 greater than 0.40) and severe heart failure (LVEF less than 0.20) and given graded sequential infusions of dobutamine and calcium gluconate. Those with severe cardiac dysfunction had marked impairment of the dobutamine dP/dt and stroke work index response, whereas these responses to calcium did not differ in the two groups. These data indicate that in the intact human heart endomyocardial biopsy may be used for direct analysis of beta-adrenergic receptors, heart failure-associated myocardial beta-adrenergic down-regulation begins with mild-moderate ventricular dysfunction, reduction in myocardial beta-receptor density is related to degree of heart failure, and beta-receptor down-regulation is associated with pharmacologically specific impairment of the beta-agonist-mediated contractile response. PMID- 3022964 TI - Cardiovascular pharmacology. I. AB - The use of bicarbonate during cardiopulmonary resuscitation remains controversial. The present standards, suggested in large part by the investigations of Bishop and Weisfeldt, and the acknowledged toxicity of treatment with bicarbonate led to aggressive use of hyperventilation, the frequent monitoring of pH, and a reduction in bicarbonate administration. However, to date no studies have indicated an improvement in outcome to support the importance of these changes. Instead, controversy continues concerning the most appropriate buffer and whether the pH gradient induced between venous and arterial beds during CPR is of importance. To date, a viable alternative regimen has not been proposed. Thus, at present there is little new data upon which to base a major change in strategy, although the logic of reducing further the use of bicarbonate seems compelling. The choice of antiarrhythmic therapy is equally difficult. Initially, experimental studies suggested a more potent antifibrillatory effect for bretylium than for lidocaine. Subsequent studies have challenged these initial experimental results and clinical data have failed to indicate the benefit of one drug over the other. There is little information to suggest that these agents are more effective than the aggressive use of defibrillation alone in patients with ventricular fibrillation. It therefore seems improbable that a definitive decision concerning the use of one or another of these agents can be made. PMID- 3022963 TI - Advanced pediatric life support: state of the art. AB - Cardiopulmonary resuscitation in children is not well studied; many of the current recommendations for advanced pediatric life support (APLS) are based on anecdotal experience rather than scientific study. The following are unique issues in APLS requiring a consensus decision: What are the best methods of vascular access and of drug delivery and dosages? What constitutes minimal paramedic training and equipment? There are also many shared controversies between APLS and ACLS, including the use of calcium, epinephrine vs isoproterenol, methoxamine, and bicarbonate. This article presents the scientific basis for these controversial issues and highlights areas where information is lacking. A discussion of these questions generated a consensus on some issues and hopefully will stimulate further study to answer the questions that were raised. PMID- 3022965 TI - Failure of sodium bicarbonate to improve resuscitation from ventricular fibrillation in dogs. AB - To determine the value of sodium bicarbonate in resuscitation from ventricular fibrillation and the prevention of spontaneous refibrillation, sodium bicarbonate (1 meq/kg) or placebo was administered on a random basis to 16 pentobarbital anesthetized dogs 18 min after the induction of ventricular fibrillation and cardiopulmonary resuscitation. Defibrillation was attempted 2 min after the administration of bicarbonate or placebo. All animals were successfully defibrillated, but three of eight bicarbonate-treated and two of eight control animals died in electromechanical dissociation (p = NS). Spontaneous refibrillation occurred in three animals in each group (p = NS). Successful resuscitation was not dependent on treatment, arterial or mixed venous Pco2, or arterial or mixed venous pH but correlated strongly with coronary perfusion pressure (p less than .003). Spontaneous refibrillation occurred without relation to any identifiable variable. The gradient between diastolic aortic and right atrial pressures was 24 +/- 2 mm Hg in controls and 23 +/- 2 mm Hg in treated animals over the entire 20 min of cardiopulmonary resuscitation (p = NS). However, among animals successfully resuscitated, mean diastolic coronary perfusion pressure averaged 27 +/- 2 mm Hg compared with 20 +/- 1 mm Hg among those dying in electromechanical dissociation (p less than .02). For the final 2 min of resuscitation, after drug administration, these gradients were 31 +/- 2 and 23 +/- 2 mm Hg, respectively (p less than .01). Microsphere determined myocardial perfusion correlated with the diastolic aortic-right atrial perfusion pressure gradient (r = .86) and was 0.43 +/- 0.03 ml/min/g in survivors and 0.22 +/- 0.01 ml/min/g in nonsurvivors (p less than .01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022967 TI - Clarification on a method for separating cells in whole blood. PMID- 3022966 TI - Assay of circulating 1,25-dihydroxyvitamin D involving a novel single-cartridge extraction and purification procedure. AB - By use of a single-cartridge system, 1,25-dihydroxyvitamin D [1,25(OH)2D] is extracted and purified from a plasma or serum sample. Proteins are removed and 1,25(OH)2D is liberated from the sample with acetonitrile. The acetonitrile extract is applied to the nonpolar octadecylsilanol silica cartridge, in which 1,25(OH)2D is retained while polar compounds are eluted. Then by "phase switching" on the same cartridge, 1,25(OH)2D is sufficiently resolved from other vitamin D metabolites and extraneous lipophilic compounds to allow its quantification by radioreceptor assay according to an established procedure. Mean (and SD) values for 1,25(OH)2D in serum of 29 normal, 27 chronic renal failure, and nine pregnant subjects were 28.2 (11.3), 10.9 (5.2), and 47.3 (12.9) ng/L, respectively. Results compared well with those of an established radioreceptor procedure. This procedure offers the advantage of a single rapid purification step not involving "high-performance" liquid chromatography or evaporation, under nitrogen, of polar solvents such as acetonitrile or methanol. PMID- 3022968 TI - Problems of measuring serum angiotensin converting enzyme in the RA-1000. PMID- 3022969 TI - Rubella and cytomegalovirus infection in pregnancy. PMID- 3022970 TI - Urinary enzyme analysis in renal allograft transplantation. AB - The urinary excretion of four enzymes (fructose-1,6-bisphosphatase, glutathione S transferase, N-acetyl-beta-D-glucosaminidase and pyruvate kinase) was assayed daily in 59 patients following renal cadaveric allografting. 51 patients were given cyclosporin A (CyA group) as an immunosuppressive, 8 patients were treated conventionally with azathioprine and prednisolone (CON-group). Urinary enzyme output was evaluated by two different mathematical models. Model A follows single enzyme excretion, whereas model B also analyzes enzyme patterns. The best results were obtained by a combined analysis of all four enzymes with model B. In the CON group the sensitivity was 1.00, the specificity 0.85, the predictive values of positive test 0.45 and all 12 graft rejections were diagnosed correctly. In the CyA group the sensitivity was 0.40, the specificity 0.99, the predictive value of positive test 0.33, and 6 out of 9 rejections were recognized. The evaluation of the single enzymes did not produce similarly good results with either model. PMID- 3022971 TI - Interference of target cell catalase with an early step of the neutrophil cytolytic pathway. AB - The hypochlorous acid (HOCL)-dependent lysis of human red blood cells (HRBC) targets by neutrophils, activated with opsonized zymosan particles (OPZ), was increased by inhibiting HRBC catalatic activity with aminotriazole (AT; HRBCAT). The inhibition of HRBC glutathione cycle activity with carmustine (BCNU; HRBCBCNU) had no effect. In addition, the recovery of hydrogen peroxide (H2O2) and HOCL from neutrophils, activated under conditions similar to those used for cytotoxicity assay, was reduced by the presence of HRBC and restored by replacing HRBC with HRBCAT, but not with HRBCBCNU. Linear relationships were found between the increments in the neutrophil-mediated lysis, observed by using HRBCAT instead of HRBC, and the increments in the H2O2 or HOCL recovery, detected by replacing HRBC with HRBCAT. Together these data, coupled with the results obtained by probing neutrophil cytolysis with chemical agents, suggest that the increased cytolytic efficiency displayed by neutrophils against HRBCAT, inhibited in their catalatic activity, is due to an enhanced availability of neutrophil-derived H2O2, with a consequent enhancement in the HOCL production (according to the following reaction: (formula; see text). Thus it appears that HRBC catalase restrains the neutrophil cytolytic activity, by interfering with an early step of the pathway through which neutrophils generate cytotoxins. PMID- 3022972 TI - Epstein-Barr virus (EBV) antigen-specific leukocyte migration inhibition (LMI) in infectious mononucleosis (IM). I. Kinetics and response to a membrane protein on EBV-transformed cells. AB - Cell-mediated immune response of mononucleosis (IM) patients to Epstein-Barr virus (EBV)-determined antigens was measured by the leukocyte migration inhibition (LMI) assay. Patients in the acute phase of the disease failed to respond to partially purified nuclear antigen, EBNA, or to cell extracts that contained EBNA as the predominant EBV antigen. They showed a strong specific response to cell extracts enriched in early antigen (EA) and virus capsid antigen (VCA). The LMI response to EBNA appeared in convalescence in parallel with EBNA specific antibodies, slightly later in children than in adults. Membrane fractions of EBV-carrying, virus nonproducer Raji cells induced an EBV-specific LMI at approximately the same time. A bacterial fusion protein containing the hydrophilic part of the virus-coded membrane antigen of latently EBV-infected cells also induced an EBV-specific response that parallelled the LMI reaction elicited by the Raji membrane fraction. This is in line with our previous finding (D. Sulitzeanu et al., J. Virol. 58, 230, 1986) that this fusion protein shares an epitope with Raji cell membranes. PMID- 3022973 TI - Pulmonary angiitis with atypical lymphoreticular infiltrates in Wiskott-Aldrich syndrome: possible relationship of lymphomatoid granulomatosis and EBV infection. AB - We describe a 12-year-old boy with Wiskott-Aldrich syndrome who developed a pulmonary vasculitis associated with lymphoreticular proliferation, consistent with the histological and clinical diagnosis of lymphomatoid granulomatosis. The lesions were responsive to cyclophosphamide and steroids. The patient has had severely depressed immune function and was shown to have abnormal Epstein-Barr virus (EBV)-specific cellular and humoral immune responses. Lymph nodes obtained at autopsy were positive for EBV genome. In this patient, reactivated EBV infection resulting from impaired immune surveillance of the virus may have been responsible for the development of this paraneoplastic disorder. PMID- 3022974 TI - Degradative enzyme systems in cartilage. AB - There appears to be a final common pathway in the pathogenesis of osteoarthritis, regardless of the initiating cause. This involves an increase in degradative enzymes that arise from the cartilage. Both proteoglycan- and collagen-degrading enzymes, active at a neutral pH, increase in proportion to the severity of the arthritis until a final end-stage state is reached. This increase in enzyme activity may be triggered by release of a synovial messenger protein similar to interleukin-1. It is suggested by studies in an animal model that inhibition of these enzymes could lead to treatment of osteoarthritis. PMID- 3022975 TI - T-lymphocyte subpopulations in peripheral blood of patients with multiple sclerosis, patients with other neurological diseases and healthy controls. AB - We report our results in profiling peripheral blood lymphocyte subpopulations with monoclonal antibodies in 17 multiple sclerosis (MS) patients, 22 patients with other neurological diseases (OND), and 11 healthy controls, using a blind experiment. Untreated patients with a chronic progressive MS have higher T-helper cell (OKT4+) counts and a higher ratio OKT4+/OKT8+ than other MS patients, OND or healthy controls. Two weeks after the onset of a relapse of MS there is a decreased T-helper and an increased T-suppressor cell percentage. Treatment with ACTH results in a significant increase of helper cells after 4 weeks of therapy. Patients with the lowest helper cell counts and the lowest helper/suppressor ratio show the best clinical improvement by ACTH. High OKT4+ cell percentages make a chronic progressive course of MS more probable. PMID- 3022976 TI - Clinical aspects of Japanese B encephalitis in North Vietnam. AB - The author reports the retrospective study of 116 children suffering from Japanese B encephalitis in recent epidemics. This disease often appeared in summer and affected 2 to 7-year-old children. In the acute stage the clinical picture included meningeal signs, motor disorders, consciousness dysfunction and neurovegetative disturbances. 90.4% of cases presented abnormal features in CSF. Serological tests with Nakayama and H.N-60 strains were positive. B.M-79 Arbo virus was isolated from 1 case. Neuropathological examination revealed typical lesions in 16 cases. Sero-, viro- and anatomical correlations with clinical course confirmed the role of J.B.E. in outbreaks of 'acute encephalitic syndrome' in North Vietnam. PMID- 3022978 TI - Growth of cavernous hemangioma with puberty. PMID- 3022977 TI - Olfactory neuroblastoma with spinal metastasis--a problem in diagnosis. AB - The olfactory neuroblastoma or esthesioneuroblastoma is a rare neuroectodermal tumor originating from the olfactory neuroepithelium, which can metastasis via cerebrospinal fluid pathways. In the present case of an extensive nasal malignancy with cervical lymph node metastases in a 75-year old woman, its difficult histology alternatively led to a diagnosis of anaplastic carcinoma and non-Hodgkin lymphoma. The patient died from complications following spread of the tumor to the spinal cord and cauda equina. Review of the literature shows that this tumor is notorious for its chameleonic character. In view of its clearly demonstrated malignancy an aggressive therapeutic approach is advocated. PMID- 3022979 TI - Two-dimensional proton magnetic resonance of human muscle extracts. AB - In continuation of our previous work on one-dimensional (1D) proton nuclear magnetic resonance (1H-NMR) of normal and diseased human muscle extracts we recorded the two-dimensional (2D) J-correlated proton magnetic resonance spectra of these extracts. Significant differences between normal and diseased muscle extracts, not observed in the 1D 1H-NMR spectra, were seen from their 2D connectivity contour patterns. Taurine was not present in cerebral palsy muscle extract while both normal and scoliosis muscles contained this metabolite. Only the normal muscle had carnitine. Carnosine was present in all muscles. alpha Ketoglutarate was found only in the diseased muscle extracts. While the amino acids lysine, cysteine and glutamine were common to normal and diseased muscles, threonine was seen only in the diseased muscles. Additional small differences were detected in the 2D patterns of human muscle extracts. PMID- 3022980 TI - Role of membranes in disease. AB - The membranes of mammalian cells are composed of an ordered array of lipids and proteins, the latter containing carbohydrate residues directed towards the exterior and important in the interaction of cells with each other and with external proteins. This external (plasma) membrane and other more simple membranes within the cell are damaged in all diseases which compromise the integrity of the cell. However, in many cases, chemical or functional changes in these membranes are central to the pathogenesis of the disease. These processes are illustrated, and a classification of membrane-related diseases is proposed. This includes: receptor-related diseases such as type II familial hypercholesterolaemia, Grave's disease, some lysosomal storage diseases and some forms of diabetes and obesity; structural instability as manifested by red cell abnormalities and multiple sclerosis; changes in lipid state as in muscular dystrophy and multiple sclerosis; altered permeability or transport as in cystic fibrosis, diseases associated with specific transport defects, and the action of many bacterial toxins, and abnormality of the cytoskeleton-membrane interface as in Chediak-Higashi disease and some diseases associated with red cell abnormalities. Different mechanisms can contribute to the membrane disorder in a single disease state and many of these are described to illustrate this diversity. PMID- 3022981 TI - Biological differences in a rat insulinoma cell line obtained from different laboratories do not affect binding of human anti-islet immunoglobulins. AB - An insulin-producing clone of rat insulinoma cells (RINm5F) has been used by several investigators as target cells for studies of both humoral and cell mediated anti-islet immunity in diabetic animals and humans. We noted that the rate of proliferation of RINm5F cells obtained from different laboratories varied considerably, and, in the present study, we have compared the proliferation rates of RINm5F cells obtained from 3 laboratories (Uppsala, Sweden [UPP], Chicago [CHI] and New York [NY]). The cells were plated at 0.5 and 2.0 X 10(4)/cm2 and changes in cell number were measured over 5 days. Basal insulin release was also determined daily. In addition, binding of IgG from sera of human diabetics by each of the cell lines was also examined by a solid-phase, quantitative assay. Plating efficiency was significantly greater in the NY and CHI cells than UPP cells at both plating densities (p less than 0.025). When plated at 2 X 10(4)/cm2, the growth rate of the NY cells was faster than the others (NY: 100.1 +/- 7.8%/day, CHI: 72.2 +/- 8.1%/day, UPP: 78.3 +/- 14.0%/day, p less than 0.025). All growth rates were lower when cells were plated at 5 X 10(4)/cm2, and the differences in growth between the NY and the other cells was greater (NY: 94.1 +/- 12.2%/day, CHI: 61.8 +/- 5.8%/day, UPP: 58.1 +/- 5.6%/day). Insulin release also differed among the cells. More insulin was released by the NY cells than by the other cells on all days, and the CHI cells released more insulin than the UPP cells (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022982 TI - Metabolism of exercising skeletal muscle during beta 1-selective adrenoceptor blockade. AB - Concentrations of glycogen, glucose, glucose-6-phosphate and lactate in the lateral vastus muscle were measured in seven subjects before and after dynamic muscle exercise at a work load of 75% of each subject's maximal working capacity, and with and without intravenous administration of the beta 1-selective beta adrenoceptor blocking agent, atenolol. Pulmonary oxygen uptake was measured during exercise. Heart rate and arterial blood pressure were measured throughout the study. Arterial concentrations of glucose, lactate and free fatty acids were measured at rest and during exercise. The muscle concentration of glycogen and the extent of glycogen depletion with exercise were not influenced by the beta 1 adrenoceptor blocker. Similarly, there was no change in the muscle concentrations of glucose, glucose-6-phosphate and lactate. Heart rate decreased at rest and during exercise. Arterial blood pressure was not influenced by beta-blockade. Pulmonary oxygen uptake decreased by 6.5%. The exercise induced rise in arterial blood concentration of free fatty acids was abolished by beta 1-selective beta blockade. It is concluded that the decrease in lactate release from exercising muscles during beta 1-adrenoceptor blockade seen in other studies cannot be explained by an impaired breakdown of muscle glycogen. It may be inferred, however, that a reduced availability of free fatty acids in the exercising muscles during beta 1-selective (and non-selective) beta-blockade may enhance the combustion of pyruvic acid and thereby decrease the production of lactate. PMID- 3022983 TI - Localization of cytochrome C oxidase and cytochrome C peroxidase in mitochondria of Hymenolepis diminuta (Cestoda). AB - The intramitochondrial localization of cytochrome c oxidase and cytochrome c peroxidase in adult Hymenolepis diminuta was investigated. Mitochondria were fractionated into inner membrane, outer membrane, intermembrane space and matrix and the efficacy of fractionation was monitored employing marker enzymes. Cytochrome c oxidase was associated with the mitochondrial inner membrane. Whereas 55% of the cytochrome c peroxidase activity was in the matrix, 32% of the activity was in the intermembrane space fraction. Based upon the distribution of marker enzymes, a dual compartmentalization of cytochrome c peroxidase is apparent in H. diminuta mitochondria. PMID- 3022984 TI - Cross-reactivity of a monoclonal antibody to the catalytic subunit of chicken gizzard desmin-specific calpain. AB - The desmin-specific calpain I from chicken gizzard smooth muscle is a dimer of 83 and 35 kDalton subunits. A monoclonal antibody to the large subunit did not cross react with chicken gizzard and hamster skeletal muscle calpain II, but it did recognize hamster skeletal muscle desmin-specific calpain I and the denatured calpain II from chicken gizzard smooth muscle. These results indicate that different desmin-specific calpains have similar large subunits which differ significantly from the large subunit of calpain II in the same tissue. PMID- 3022985 TI - Comparison of the potency of d-propranolol, chlorhexidine and nonoxynol-9 in the Sander-Cramer test. AB - The potency of two new candidate spermicides, d-propranolol and chlorhexidine, was compared with an established agent nonoxynol-9 by the Sander-Cramer test. The lowest concentrations of nonoxynol-9, d-propranolol and chlorhexidine which consistently inhibited progressive forward sperm motility within twenty seconds were found to be 0.12, 0.44, and 4.81 mg/ml, respectively. These relative potencies and other properties of d-propranolol and chlorhexidine indicate that they should be further investigated as alternatives to nonoxynol-9 in spermicidal preparations. PMID- 3022987 TI - Neurobiology of human herpesvirus infections. AB - Critical to the adaptation of herpes simplex virus type 1 (HSV-1) to its human host is a complex interaction with the peripheral nervous system. The "life cycle" of the virus depends not only on axoplasmic transport which carries virus to and from the infected ganglia, but also on variable expression of the virus within ganglionic neurons. During latency, limited viral gene expression allows the virus to persist undetected by immune defenses, while intermittent reactivation, perhaps triggered by metabolic perturbation of these neurons, leads to production of new virus with subsequent transmission. In parasitizing its host, the virus exploits a number of the specialized properties of neurons and supporting cells. A second important type of HSV-1 infection involves the central nervous system (CNS). Herpes encephalitis, an uncommon yet severe disease, represents an aberrant interaction of the virus and host that is at present poorly understood. This review examines current understanding of HSV-1 infections of both the peripheral and CNS from a neurobiological perspective. PMID- 3022986 TI - Kinetics of carbon dioxide during cardiopulmonary resuscitation. AB - CO2 kinetics during CPR was investigated in 15 anesthetized piglets. BP, blood gases, and acid-base balance were monitored through catheters in the carotid artery and a central vein, as well as in cerebrospinal fluid. Cardiac arrest was induced by a transthoracic direct current shock. CPR was begun immediately by artificial ventilation and simultaneous external chest compressions. Epinephrine was administered after 8 min of CPR. One group (n = 5) of animals received no buffer treatment while another (n = 5) received an infusion of 75 mmol sodium bicarbonate and a third group (n = 5) received an equivalent amount of tris buffer mixture. The results of these experiments, as well as previously described circulatory variables during CPR, were analyzed using a computer model describing the CO2 kinetics of the pig. Our main finding was that PaCO2 was positively correlated to cardiac output during CPR; improved cardiac output during CPR resulted in more efficient tissue CO2 elimination and was associated with increased survival rates. PaCO2 was also somewhat reduced by efficient alveolar hyperventilation. The arterial PCO2 and pH did not reflect the acid-base balance in peripheral tissues. During CPR, bicarbonate and tris-buffer mixture both quickly passed through the blood-brain barrier. When buffer treatment is indicated during CPR, a buffer which does not increase tissue PCO2 may be the drug of choice. PMID- 3022988 TI - The influence of ossein-hydroxyapatite compound ('Ossopan') on the healing of a bone defect. AB - A study was undertaken in 60 adult rabbits in order to determine the effects of ossein-hydroxyapatite compound on bone healing. Standardized bony defects were produced in the distal femoral epiphyses, after which animals were randomized into four equal groups. An untreated group served as a control. One group received ossein-hydroxyapatite compound, 830 mg per day, a second group received bone mineral (ossein-hydroxyapatite compound reduced to ash, to remove organic constituents), 510 mg per day, and a third group received calcium carbonate, 650 mg per day. A series of fluorescent vital markers was administered to the animals from the 7th to the 32nd day after production of the defect. A third of the animals in each group were sacrificed 35, 56 and 84 days, respectively, after induction of the defect. Histological sections of the region of the bone defect were examined with a fluorescence microscope and resulting photomicrographs were scored with respect to degree of fluorescence, nature and degree of defect filling and structure of the newly formed bone. All three active treatment groups resulted in significantly improved mineralization as compared with the untreated control group. Treatment with ossein-hydroxyapatite compound, but not the other two active treatments, resulted in significant improvements in the pattern and quality of bone healing, particularly when assessed at 56 or 84 days after induction of the bone defect. These results indicate that ossein-hydroxyapatite compound has a beneficial effect on the process of bone healing but that this effect is lost if the organic components of the compound are destroyed or if pure calcium carbonate treatment is substituted. This strongly suggests that organic components of ossein-hydroxyapatite compound have osteogenic effects, enhancing the utilization of the mineral intake, and is consistent with previous experimental findings. It is suggested, therefore, that ossein-hydroxyapatite compound has considerable clinical potential and should be regarded as having specific, sophisticated effects on bone metabolism, rather than as a simple dietary mineral supplement. PMID- 3022989 TI - Chronic respiratory problems in infancy. PMID- 3022990 TI - The use of peptides in studying mechanisms of immune tolerance. PMID- 3022991 TI - Immune response to synthetic herpes simplex virus peptides: the feasibility of a synthetic vaccine. PMID- 3022992 TI - Morphological correlates of follicular fluid stimulation of steroidogenesis in immature porcine granulosa cells. AB - Factors in porcine ovarian follicular fluid are known to influence steroidogenesis in cultured ovarian granulosa cells. This study examined whether ultrastructural changes characteristic of normal maturation and/or atresia accompany the steroidogenic alterations. Two and 5 day incubations of immature porcine granulosa cells were performed in media supplemented with either serum or follicular fluids (FFL) from mature follicles. Under these conditions both oestrogen and progesterone secretion were stimulated in FFL supplemented cultures as compared to serum supplemented cultures. Cells in serum exhibited increased size, number and volume of lysosomes and resembled in vivo atretic cells. In comparison, the FFL treated cells had greatly increased steroid output, numerous microvilli and increased size, number and volume of electron dense lipid droplets after 2 days of culture although the differences declined by day 5 of culture. This suggests that mature FFL contains a factor(s) stimulating granulosa maturation while inhibiting ultrastructural correlates of follicular atresia. PMID- 3022993 TI - Ultrastructure of antibody dependent cellular cytotoxicity in HSV 1 infected and uninfected Chang liver cells. AB - The binding and ultrastructural features of antibody dependent cellular cytotoxicity (ADCC) mediated by human peripheral blood lymphocytes were studied in herpes simplex virus type I (HSV-1) infected Chang liver (CL) cells plus human anti-HSV-1 serum, and in uninfected CL cells plus guinea pig anti-CL antiserum. Non-cytolytic controls included target cells treated with normal serum in place of sensitized targets and heat shocked lymphocytes instead of normal lymphocytes. By transmission electron microscopy, target cell membranes were either broadly indented by effector cells or locally invaginated by means of effector cell filopodia. In neither case did the indentation appear to break the plasma membrane of the target. Control preparations showed only non-indented areas of simple membrane contact. By scanning electron microscopy, the effector lymphocytes in both the active ADCC and normal serum control preparations had a sparse distribution of short microvilli over their surfaces. The majority of heat shock control lymphocytes appeared normal, but 12-20% demonstrated surface patches devoid of microvilli. The hypothesis that ADCC may involve a three-step process is discussed. PMID- 3022995 TI - Assignment of RAS proto-oncogenes in Chinese hamsters: implications for mammalian gene linkage conservation and neoplasia. AB - HRAS and KRAS are the cellular homologs of the oncogenic transforming genes found in the Harvey strain of murine sarcoma virus and the Kirsten murine sarcoma virus, respectively. Phyla as diverse as insects, birds, and mammals possess distinct HRAS and KRAS sequences, suggesting that these genes are essential to metazoa. In this report, we used a clone panel of Chinese hamster X mouse C11D somatic cell hybrids segregating hamster chromosomes to map those genes. Southern filter hybridization analyses of the hybrids revealed that hamster HRAS and KRAS gene sequences are on chromosomes 3 and 8, respectively. These gene assignments are consistent with the conservation of autosomal gene linkage groups observed among hamsters, humans, and mice and may provide insight into specific chromosomal alterations that have been observed during the spontaneous neoplastic transformation of Chinese hamster fibroblasts in vitro. PMID- 3022994 TI - The 724 family of DNA sequences is interspersed about the pericentromeric regions of human acrocentric chromosomes. AB - We describe the organization of the complex, interspersed 724 family of DNA sequences that is distributed in multiple copies about the pericentromeric region of human acrocentric chromosomes. 724 family members were isolated using an efficient recombination-based assay for nucleotide sequence homology to screen a human genomic library. Eight related but distinct 724 family members were isolated that hybridized to a total of 20 different human-genomic EcoRI DNA fragments spanning 100,000 base pairs. In contrast with tandemly clustered satellite and ribosomal DNA sequences also located on the short arms of human acrocentric chromosomes, 724 family members are interspersed. No evidence for local interspersion or homology between 724 family members and ribosomal or satellite DNA sequences was found. Juxtaposition of the complex 724 family to the nucleolus organizer region was a recent event in primate evolution. The unique organization of 724 family members on each of the five human acrocentric chromosomes indicates that the 724 family continues to evolve within the human karyotype. PMID- 3022996 TI - An angiotensin-converting enzyme (ACE) inhibitor in human serum. Increased sensitivity of the serum ACE assay for detecting active sarcoidosis. AB - Measurement of serum angiotensin-converting enzyme (ACE) is extremely useful as an aid in the diagnosis and longitudinal evaluation of patients with sarcoidosis. We have detected a human serum ACE-inhibitor which affects the ACE level obtained by activity measurements. The effect of the inhibitor can be eliminated by a mere eight-fold dilution of the serum sample with physiologic saline. We recommend that serum ACE be performed with 1:8 dilutions of serum to eliminate the effect of the ACE inhibitor. The inhibitor has a MW above 50,000 daltons, and reversibility of inhibition by dilution appears to be ion dependent. Dialysis of an inhibitor-containing serum sample against saline causes the inhibition to become irreversible, allowing the distinction between alinearity of the assay vs an inhibitor effect when a serum has ACE activity greater than 50 units/ml. The source of the serum ACE-inhibitor remains to be determined. PMID- 3022997 TI - Spontaneous pneumothorax. A complication of lung cancer? PMID- 3022998 TI - Centrifugal separation of cells in sputum specimens from patients with undifferentiated carcinoma. AB - Ethanol-fixed cells in sputum from patients with undifferentiated carcinoma of the lung were separated in aqueous Ficoll using a discontinuous density gradient centrifugation technique. The selective enrichment of small cell undifferentiated (e.g., oat cell) or large cell undifferentiated carcinoma cells was achieved while removing most of the leukocytes (80-90%) and macrophages (65-75%) from specimen fractions containing the greatest relative frequencies of cancer cells. The maximum purity of small cell carcinoma cells (0.04%) occurs in moderate density (rho = 1.121 g/ml) gradient fractions and results in a 2.4-fold enrichment relative to unprocessed specimens. In contrast, the maximum purity of large cell carcinoma cells (0.22%) is obtained in very high density (rho = 1.172 g/ml) gradient fractions and results in a 1.2-fold enrichment in comparison with unprocessed specimens. Microscopic examination of Papanicolaou-stained specimen fractions reveals that these enrichments were achieved while retaining diagnostically significant cytomorphologic and tinctorial features necessary for cancer screening and diagnosis. Peak purity ranges of undifferentiated cancer cells significantly overlap comparable ranges for material from bronchogenic adenocarcinoma and squamous cell carcinoma. PMID- 3023000 TI - Dioxin-inducible enhancer region upstream from the mouse P(1)450 gene and interaction with a heterologous SV40 promoter. AB - In mouse hepatoma Hepa-1 cells, polycyclic aromatic compounds such as 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) activate transcription of the mouse P(1)450 gene via trans-acting regulatory factors that include the TCDD X receptor complex. The positive control element in the P(1)450 5'-flanking region was examined in control and TCDD-treated Hepa-1 stable transformants that had been transfected with either of two expression vectors containing the chloramphenicol acetyltransferase (CAT) gene: pA10-cat, which has the simian virus 40 (SV40) early core promoter (without enhancers) immediately upstream from the CAT gene; and pSV0-cat, which has no promoter or enhancer. When the 1-kb DNA fragment from 1,647 to -611 upstream from the P(1)450 gene is inserted in either orientation- immediately upstream or almost 2 kb further upstream--from the SV40 promoter in pA10-cat, there is enhancement of CAT activity that can be further induced three- to fourfold by TCDD. When the same experiment is carried out with the -1,247 to 823 fragment or the -1,051 to -823 fragment, but not the -1,247 to -1,052 fragment, TCDD responsiveness is lost, or at least masked, because of a large increase in constitutive CAT activity. pSV0-cat mutants containing internal deletions in the upstream flanking sequences of P(1)450 were constructed. A region of 300 bases (-1,218 to -918) is shown to be required for TCDD responsiveness, and one TCDD-inducible element can be dissociated from an enhancer of constitutive gene expression, whereas one or more other TCDD inducible elements cannot.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3022999 TI - Molecular cloning and expression in Escherichia coli of equine type I interferons. AB - Using human interferon-alpha 2 (IFN-alpha 2) and IFN-beta DNA to probe an equine genomic library we isolated recombinant phages containing genes for equine interferon-alpha (EqIFN-alpha), interferon-beta (EqIFN-beta), and interferon omega (EqIFN-omega). Sequence and hybridization analyses of these genes reveal that the equine genome contains gene families of each of these three type I interferon classes. The mature proteins of EqIFN-alpha are 71-77% homologous to human IFN-alpha polypeptides, and, when expressed in E. coli, possess antiviral activity on both equine and human cells. By contrast, EqIFN-beta is only 59% homologous to its human counterpart and shows activity only on equine cells. PMID- 3023001 TI - [A new approach to creating chromosome libraries as illustrated by the X chromosome]. PMID- 3023002 TI - A comparison of the structure of two models of treatment for alcoholism. AB - A comparison is made between the services offered by a District General Hospital and a Community-based Centre, for the treatment of alcohol related problems. The two treatment models are discussed in terms of their structure and their approach to problem drinking. Differences between the Hospital and the Centre are noted, and some conclusions are drawn regarding service developments. PMID- 3023003 TI - [Significance of neuron-specific enolase (NSE) in the diagnosis of bronchial carcinomas and neuroendocrine tumors]. AB - The diagnostic significance of neuron-specific enolase in serum was examined in 54 patients with bronchial carcinoma and in 28 with neuroendocrine tumors. Control groups were 42 patients with epithelial and 39 with nonepithelial malignant neoplasms as well as 40 patients with benign pulmonary diseases. The sensitivity of neuron-specific enolase in small-cell bronchial carcinoma was 60% and increased to 87.5% in advanced stages ("extensive disease"). On the other hand, non-specific enolase showed an increase in only 13.8% of patients with other than small-cell bronchial carcinoma. The proportion of false-positive enolase values in non-malignant pulmonary diseases was 5%. Some endocrinal tumors (e.g. tumors of the APUD cell system) showed pathological serum concentrations in 7.1% of the cases only. 37.5% of epithelial malignant neoplasms had enhanced levels, but only 5.1% in nonepithelial neoplasms. Small-cell bronchial carcinoma is most probably present in patients with bronchial carcinoma and neuron-specific enolase serum concentrations above 25 micrograms/l. PMID- 3023004 TI - [Diagnosis of vipoma]. PMID- 3023005 TI - [Therapy of vipoma]. PMID- 3023006 TI - [Importance of bone scintigraphy in the after-care of breast cancer]. PMID- 3023007 TI - [Cell surface receptors in lymphoid lines studied using lectins]. AB - The nature and distribution of some carbohydrate receptors on the cell membrane of transformed human B- and T-derived lymphocyte cell lines (RPMI-1788, Daudi, Jurkat, Molt-4) are studied by Ricinus communis agglutinin, soybean agglutinin, Helix pomatia lectin, peanut agglutinin and wheat germ agglutinin conjugated with ferritin and colloidal gold. The quality of receptors and the manner of their distribution detected by different lectins are considerably different. PMID- 3023009 TI - [Effect of khingamin on the development of SV40 virus-induced and transplantable tumors in hamsters]. AB - Chloroquine was administered to 2-month hamsters inoculated at birth with an oncogenic virus SV-40 which was injected daily in a dose of 30 mg/kg for a month. Chloroquine-treated animals showed a statistically significant (6 weeks) shorter latent period of tumour development. The drug exerted no effect neither on the rate and incidence of transplantable E-1 test tumours or on the phenomenon of resistance in experiments on 4-6-month hamsters under its multiple administration in doses close to maximum tolerated ones (40 mg/kg). PMID- 3023008 TI - [Adenylate cyclase complex, lipids and the regulation of the hormonal sensitivity of tumor tissue]. AB - This review consists of the following parts: plasma membranes of cells in normal and cancer tissue; adenylate cyclase complex (ACC) and tumour development; ACC regulation and phase state of lipids in the membranes of tumour cells; interrelations of ACC with the other signal-receiving systems of normal and tumour cells; the role of ACC in providing, changing and analyzing the hormone sensitivity of tumour tissue, regulation of the hormone sensitivity of tumour tissue by means of influences on ACC. PMID- 3023010 TI - Peripheral neuropathy: comparison of sensory conduction abnormalities in upper and lower extremities. PMID- 3023011 TI - Electrodiagnostic findings of persisting polyneuropathies due to previous nutritional deficiency in former prisoners of war. PMID- 3023012 TI - Hormonal regulation of (Ca2+ + Mg2+)ATPase activity in canine renal basolateral membrane. AB - High affinity (Ca2+ + Mg2+)ATPase activity was demonstrated in proximal tubule basolateral membranes (BLM) obtained from canine kidney. The Km of the enzyme for free Ca2+ was 0.12 +/- 0.02 microM, and at 3 microM free Ca2+, the enzyme reached its maximal velocity. To evaluate hormonal regulation of this enzyme, we studied the in vitro effects of several polypeptide hormones on enzyme activity. We measured the effects of insulin and human (h) PTH-(1-34) and their inactive analogs desoctapeptide insulin, bovine (b) PTH-(3-34), and oxidized hPTH-(1-34); insulin-like growth factors (IGFs) I and II; calcitonin; and the common cellular mediator for PTH and calcitonin, cAMP. At 0.1 microM free Ca2+, insulin (25-100 microU/ml) increased (Ca2+ + Mg2+)ATPase activity in a dose-dependent manner by 35-52% (P less than 0.01) and by 8-13% (P less than 0.05 to P less than 0.01) at a 3-microM free Ca2+ concentration; hPTH-(1-34) PTH (10(-9)-10(-7) M) increased the enzyme activity at a free Ca2+ concentration of 0.1 microM by 13-25% (P less than 0.05 to P less than 0.01). IGF-I increased (Ca2+ + Mg2+)ATPase activity by 40% (P less than 0.05) at 0.1 microM free Ca2+ at high peptide concentrations (10 ng/ml). No effect was obtained at 2 ng/ml IGF-I. cAMP (10(-7)-10(-4) M) stimulated enzyme activity by 18-27% (P less than 0.05 to P less than 0.02) at 0.1 microM Ca2+ and by 8-12% (P less than 0.05 to P less than 0.01) at 3 microM Ca2+. The effects of insulin and cAMP on (Ca2+ + Mg2+)ATPase activity were additive. No effect on the enzyme activity was obtained by the inactive analogs desoctapeptide insulin, bPTH-(3-34), and oxidized hPTH-(1-34), or by calcitonin or IGF-II. The data suggest that insulin and PTH have a specific stimulatory effect on (Ca2+ + Mg2+)ATPase activity in canine kidney proximal tubular BLM. While the insulin action is independent of cAMP, a role of cAMP in the regulatory effect of PTH on this enzyme cannot be ruled out. The direct stimulatory effect of insulin and PTH on (Ca2+ + Mg2+)ATPase in canine kidney proximal tubular BLM suggests that these hormones mediate their cellular effects in part by changes in cellular calcium homeostasis. PMID- 3023013 TI - Glucose induces insulin release and a rise in cytosolic calcium concentration in a transplantable rat insulinoma. AB - An important role for calcium in the cellular events leading to insulin secretion is supported by many studies. However, simultaneous measurements of changes in intracellular free Ca2+ concentrations [( Ca2+]i) and insulin release in response to secretagogues have not been performed. Using cells isolated from a glucose responsive insulinoma, changes in [Ca2+]i were measured with the fluorescent calcium probe quin2. With the nutrient secretagogues glucose (30 mM) and D,L glyceraldehyde (GA; 20 mM), [Ca2+]i increased slowly, reaching a peak approximately 15 min after addition of the stimulus, while KCl (25 mM) and carbachol (2 mM) led to a rapid but transient increase in [Ca2+]i. Glucose increased [Ca2+]i from 104 +/- 6 (mean +/- SEM) to 248 +/- 31 mM (n = 13), and GA caused a rise in [Ca2+]i from 96 +/- 6 to 280 +/- 39 nM (n = 4). KCl and carbachol caused a rise from 107 +/- 6 to 184 +/- 5 nM and from 98 +/- 5 to 157 +/- 5 nM, respectively (n = 5 each). When insulin release was measured simultaneously with changes in [Ca2+]i and compared to unstimulated cells, the following results were obtained. During the first 5 min of stimulation, high glucose caused a 90 +/- 12% increase in insulin release and a 72 +/- 11% rise in [Ca2+]i (n = 5). GA evoked a 122 +/- 30% increase in insulin secretion, with a 82 +/- 17% rise in [Ca2+]i (n = 3). Both KCl and carbachol caused a 58 +/- 9% increase in insulin release, with 7 +/- 4% and 50 +/- 2% rises in [Ca2+]i, respectively (n = 4 each). Insulin release was also measured in a perifusion system. It was shown that glucose (30 mM), GA (20 mM), and alpha-ketoisocaproate (30 mM) caused a biphasic release of insulin, while KCl (25 mM) and carbachol (2 mM) caused a monophasic release. The results show that [Ca2+]i increases during the stimulation of insulin secretion when measured simultaneously on the same beta-cells. However, while these changes coincide, a simple direct quantitative relationship between insulin release and the rise in [Ca2+]i could not be demonstrated. PMID- 3023014 TI - Benzodiazepines inhibit thyrotropin (TSH)-releasing hormone-induced TSH and growth hormone release from perifused rat pituitaries. AB - The perifusion technique was used to investigate the action of diazepam (DZ), a benzodiazepine molecule known to compete for TRH receptor binding in rat pituitary, on TRH-induced TSH and GH release. The release kinetics for the two hormones from quartered pituitaries were measured in response to a 6-min pulse of TRH (10 nM), without or with DZ addition for a period of 30 min before and during the TRH pulse, plus an additional 15-min period. The dynamic patterns of TSH and GH release in response to TRH were characterized by a rapid increase in hormone release, declining slowly over the next 20 min. The rate of release represented 2.98 +/- 0.02 (+/- SE) and 1.75 +/- 0.06 times the corresponding basal level for TSH and GH, respectively, when evaluated over the first 15 min of the response to TRH. Addition of increasing doses of DZ suppressed the stimulatory effect of TRH in a dose-related manner, with an ID50 of 3 nM for both TSH and GH. The maximal effect of DZ was obtained with a concentration of 10 nM for both hormones. Ro 15 1788 (100 nM), a selective antagonist of the central type of benzodiazepine binding sites (added to the perifusion system 30 min before DZ and then during the whole period of DZ perifusion), completely abolished (P less than 0.01) the inhibitory effect of DZ (10 nM) on the TRH-induced TSH and GH responses. When added alone before the TRH pulse, Ro 15-1788 had no effect on the TSH response to TRH. In contrast, PK 11,195 (100 nM), a selective antagonist of the nonneuronal benzodiazepine-binding sites, was unable to abolish the inhibitory action of DZ on TRH-stimulated TSH release. In addition, the effects of four other types of benzodiazepine (flurazepam, chlordiazepoxide, midazolam, and medazepam), all tested at a 10-nM concentration, corroborated these findings. Furthermore, DZ inhibition of the TSH response was nullified by picrotoxin (1 microM), but not by bicuculline (1 microM), two gamma-aminobutyric acid antagonists that had no effect, by themselves, on this response. For comparison, the effect of DZ (10 nM) was also tested on the release of GH in response to human GH-releasing factor-(1 44)-NH2 (10 nM) and was found to be ineffective.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3023015 TI - Stimulation of active Na+ and K+ transport by thyroid hormone in a rat liver cell line: role of enhanced Na+ entry. AB - A continuous cell line derived from rat liver (ARL 15) has been identified that responds to thyroid hormone with a stimulation of active Na,K transport. Stimulation of ouabain-inhibitable K+ uptake, which is half-maximal at a T3 concentration of 1.4 X 10(-10) M, is accompanied by corresponding increases in passive Na+ influx and in passive K+ efflux. The enhancement of Na+ and K+ fluxes by T3 is shown to be accompanied by smaller and equivalent increases both in enzymatically measured Na,K-ATPase activity and in maximal ouabain-sensitive Na,K transport in the presence of the Na+ ionophore monensin. The demonstration that both passive Na+ influx and passive fractional K+ efflux are simultaneously increased by T3 supports the earlier suggestion that the stimulation of active Na,K transport by thyroid hormone is attributable, at least in part, to an enhancement of rate-limiting passive Na+ and K+ fluxes by an increase in membrane permeability. PMID- 3023017 TI - Autoregulation of iodide transport in the rabbit: absence of autoregulation in fetal tissue and comparison of maternal and fetal thyroid iodination products. AB - Maternal and fetal rabbit thyroid glands were compared as to their ability to respond to excess iodide in vitro with a reduction in subsequent iodide transport activity. Preincubation of maternal thyroid tissue slices for 2 h with excess iodide (30 microM) resulted in a 31% reduction in the subsequently measured thyroid-medium radioiodide concentration ratio. In contrast, similar iodide pretreatment had no significant effect on fetal iodide transport. In all other respects, fetal iodide transport, although it was 10 times higher, did not differ significantly from maternal transport activity. Combined radiolabeled maternal (125I) and fetal (131I) rabbit thyroid tissue was eluted on Sephadex G-25 columns. Fractions were analyzed for both 125I and 131I activity, and the maternal to fetal ratios (125I/131I) were determined for each fraction. The majority of iodoproteins eluted with the void volume, and the 125I/131I ratio was constant in these fractions. Thereafter, two peaks of elevated 125I/131I activity could be observed. Peak A eluted below lysozyme (Mr = 14,300) and above insulin (Mr = 6,000), with an apparent mol wt of 8,000 to 10,000. A second peak, peak B, eluted from the column at a site similar to that of MIT or a protein of Mr of 2,000. Ascending paper chromatography of this latter peak of 125I/131I activity and MIT was carried out in two solvent systems. In both systems, peak B comigrated with MIT. These findings suggest that the failure of fetal thyroid tissue to exhibit autoregulation may be associated with the reduced iodination of a compound with an approximate Mr of 8,000 to 10,000. The role of this substance in iodide transport remains to be identified. The reason for the apparent increase in the labeling of MIT observed in maternal vs. fetal tissue is unknown. PMID- 3023016 TI - Stimulation of luteinizing hormone (LH) release and phospholipid breakdown by guanosine triphosphate in permeabilized pituitary gonadotropes: antagonist action suggests association of a G protein and gonadotropin-releasing hormone receptor. AB - The stimulation of gonadotropin release from pituitary cell cultures by GnRH has been linked to inositol phospholipid breakdown to diacylglycerols and subsequent activation of protein kinase C as well as Ca2+ mobilization. In order to examine the means of receptor coupling to a phospholipase C-type reaction, we evaluated the role of guanine nucleotides in inositol phospholipid breakdown. In these studies ATP (50 microM) was used for cell permeabilization to allow guanine nucleotides access to the intracellular compartment. Under these conditions GTP and the GTP analog, guanylylimidodiphosphate (GMP-PNP), stimulated a time- and dose-dependent increase in LH release and inositol phosphate accumulation. These actions of GTP and GMP-PNP were not observed unless ATP was included in the treatment media. Other closely related nucleotides and nucleosides alone, or in the presence of ATP, did not elevate LH release above basal levels. We also evaluated the actions of pertussis toxin and cholera toxin on mediating the effect of GTP, GMP-PNP, and GnRH on LH release and inositol phosphate accumulation. After treatment with these agents, no changes were observed in the ability of GnRH, GTP, or GMP-PNP to stimulate either LH release or inositol phosphate accumulation. The additional observation that GnRH-, GTP-, or GMP-PNP stimulated LH release and inositol phosphate accumulation were blocked by a potent GnRH antagonist suggests that a G protein is functionally associated with the GnRH receptor recognition site. PMID- 3023018 TI - Flow cytometric analysis and sorting of live female rat anterior pituitary cell types by forward angle and perpendicular light scatter: effect of 17 beta estradiol. AB - Administration of physiological concentrations of 17 beta-estradiol (E2) to ovariectomized rats resulted in changes in the forward angle light scatter (FALS) and perpendicular light scatter (PLS) signals of anterior pituitary lobe cells. One day after steroid treatment some cells showed increases in PLS signals to an intermediate ridge position lying between the two ridges seen for cells prepared from oil-treated ovariectomized rats. Over the first 5 days of E2 treatment, the major effect was a progressive increase in the FALS signal of cells in this intermediate ridge position. Increases in PLS signals continued over the 12-day treatment period. Since the results of a cell sorting experiment showed that PRL cells predominated in the intermediate scatter ridge, it was concluded that E2 increases both size (FALS) and secretory granule content (PLS) of mammotrophs. Cells in the low and high scatter regions were unaffected by E2. Those in the low scatter region were shown, by both electron microscopy and light microscopic immunochemistry, to be enriched in agranular, unstained endothelial-follicular cells. The high-scatter region contained predominantly GH cells. Administration of E2 to pituitary cells cultured in serum-free or serum-containing media for 1 12 days had no effect on the FALS-PLS bivariate distribution. However, the light scatter ridges produced from cells in culture were compressed into a single ridge of narrow PLS and broad FALS signal intensity with increasing time in culture. PMID- 3023019 TI - Hormonal regulation of granulosa cell inhibin biosynthesis. AB - The hormonal regulation of inhibin production by cultured granulosa cells from immature hypophysectomized, estrogen-treated rats was examined using a specific RIA which detects the N-terminal portion of the inhibin alpha-chain. The RIA measured bioactive inhibin of Mr about 32,000 in granulosa cell conditioned media fractionated by fast protein liquid chromatography. In the presence of 10(-7) M androstenedione, FSH stimulated inhibin production in a dose-dependent manner during a 2-day culture. Inclusion of a phosphodiesterase inhibitor decreased the EC50 for FSH from 2.6 to 0.8 ng/ml (n = 3). The stimulatory effect of FSH could be mimicked with forskolin (an adenyl cyclase activator) and with a cAMP analog, (Bu)2cAMP, consistent with FSH action mediated through a cAMP dependent pathway. Intracellular levels of inhibin were unmeasureable, suggesting that inhibin is not stored to any great extent by the granulosa cells. This finding was consistent with in vivo studies which showed that whereas FSH treatment for 2 days doubled serum inhibin levels when compared with basal levels, there was no increase in the concentration of extractable inhibin in ovarian tissue. Granulosa cells which had been exposed to 20 ng/ml FSH for 2 days to induce LH receptors produced inhibin in response to both LH and human CG during the subsequent 2-day culture, with the levels of inhibin equalling the amount inducible by FSH. In contrast, neither PRL nor terbutaline, a beta 2-adrenergic agonist, had any effect on inhibin production even though receptors for these hormones are also induced by FSH. GnRH was found to inhibit the FSH-stimulated production of inhibin (IC50, 10(-7) M), consistent with previous observations that GnRH can act at the ovarian level to inhibit granulosa cell differentiation. This inhibition by GnRH could be reversed by inclusion of a specific GnRH antagonist. On the other hand, another regulatory peptide, vasoactive intestinal peptide, slightly stimulated inhibin production. The effect of several growth factors was also tested. Insulin-like growth factor I raised not only FSH-stimulated inhibin levels, but basal levels as well. Insulin was also effective, but only at 100 fold higher concentration. Epidermal growth factor inhibited FSH-stimulated inhibin production (IC50 = 0.1 ng/ml), whereas fibroblast growth factor had no effect. Thus, granulosa cell inhibin secretion is regulated by FSH and LH but not by PRL, presumably via a cAMP-mediated pathway.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3023020 TI - Effects of catecholamine-depleting agents and receptor blockers on basal and angiotensin II- or norepinephrine-stimulated luteinizing hormone release in female rats. AB - Depletion of hypothalamic norepinephrine (NE) and epinephrine by administration of diethyldithiocarbamate abolished the stimulatory effects of intraventricular (IVT) angiotensin II (AII) on LH release in ovariectomized rats pretreated with estradiol and progesterone. The increase in blood LH produced by IVT NE or iv LHRH was unaffected in these drug-treated animals. Selective depletion of hypothalamic epinephrine by the administration of LY134046 (8,9-dichloro-2,3,4,5 tetrahydro-1H-2-benzazepine hydrochloride) potentiated the effect of AII on LH secretion. Blockade of alpha 1-, alpha 2-, or beta-adrenergic receptors resulted in a transient increase in basal LH levels. The stimulation of LH secretion induced by IVT AII or NE was unaffected by alpha 1-receptor blockade with prazosin, but was abolished by alpha 2-receptor blockade with yohimbine. beta Receptor blockade with propranolol potentiated both NE- and AII-induced LH release. AII receptor blockade with IVT saralasin prevented the LH rise due to AII without modifying that due to NE. Taken together, these data suggest that IVT AII stimulates LH release in ovariectomized rats treated with estradiol and progesterone by releasing endogenous NE, which, in turn, acts on facilitatory alpha 2-receptors to affect LH secretion, presumably by increasing the secretion of LHRH. Exogenous NE also acts at this receptor. beta-Receptors provide inhibitory tone to this facilitatory system, and blockade of this receptor subtype results in potentiated LH responses to both AII and NE. PMID- 3023021 TI - Calcium ion and cyclic adenosine 3',5'-monophosphate regulate proopiomelanocortin messenger ribonucleic acid levels in rat intermediate and anterior pituitary lobes. AB - The role of the second messengers cAMP and Ca++ in the control of proopiomelanocortin (POMC) gene expression was investigated with the use of hybridization with cloned complementary DNA probes. The effects of cAMP-related drugs on POMC messenger RNA (mRNA) levels were assessed in primary cultures of intermediate (IL) and anterior rat pituitary cells maintained in serum-free medium. 8-Bromo-cAMP (1 mM), but not 8-bromo-cGMP (1 mM), induced a 2-fold increase in IL and anterior lobe cell after 2 days of treatment. A similar increase was obtained with the adenylate cyclase-activating drugs forskolin (1 microM) and cholera toxin (100 ng/ml) or the phosphodiesterase inhibitor RO 20 1724 (100 microM). At 48 h, all these treatments had increased beta-endorphin accumulation in the medium and transiently decreased the cellular beta-endorphin content in IL cells, suggesting a parallel effect of cAMP-related drugs on secretion and biosynthesis. Incubating the cells with the Ca++ channel antagonists D600 (50 microM), verapamil (50 microM), and the dihydropyridine nifedipine (0.1 microM) decreased basal POMC mRNA levels, whereas the dihydropyridine BAYK 8644 (0.1 microM), which activates the Ca++ channel, increased POMC mRNA levels after 2 days. In addition, nifedipine decreased the stimulatory effect of forskolin, whereas BAYK 8644 further stimulated the forskolin-increased POMC mRNA levels in IL cells. We conclude that both Ca++ and cAMP may regulate the gene expression of POMC. PMID- 3023022 TI - Constitutive steroidogenesis in the R2C Leydig tumor cell line is maintained by the adenosine 3',5'-cyclic monophosphate-independent production of a cycloheximide-sensitive factor that enhances mitochondrial pregnenolone biosynthesis. AB - These studies were designed to characterize constitutive steroidogenesis in Leydig tumor cells. Constitutive steroidogenesis was investigated by comparing constitutively active R2C Leydig tumor cells to trophic hormone-responsive MA-10 Leydig tumor cells. Unlike the MA-10 cells, R2C cells appeared to synthesize steroid hormones independently of the cAMP-protein kinase pathway. Although the adenylate cyclase of R2C cells could be stimulated in the expected manner by cholera toxin, cAMP concentrations in these cells were low, and R2C cell steroidogenesis could be dissociated from other cAMP-dependent processes. Two cAMP-dependent processes in steroidogenic cells, protein kinase activation and lactate formation, showed low basal activities in R2C cells and could be stimulated by (Bu)2cAMP with a dose dependence similar to that detected in MA-10 cells. Steroid hormone biosynthesis parallelled these other cAMP-dependent processes in MA-10 cells, but not in R2C cells. Cycloheximide, however, caused similar dose-dependent inhibition of steroidogenesis in both the R2C and MA-10 cells. Using a cell component bioassay, it was shown that R2C cells constitutively synthesize an extramitochondrial cycloheximide-sensitive factor that is functionally identical to the factor produced in response to hCG in MA-10 cells. This factor enhanced mitochondrial pregnenolone biosynthesis. Thus, constitutive steroidogenesis in R2C cells could be explained by the cAMP independent but cycloheximide-sensitive constitutive production of an extramitochondrial factor that activated mitochondrial pregnenolone biosynthesis. PMID- 3023023 TI - Effect of mestranol on cell proliferation and angiotensinogen production in HepG2 cells: relation with the cell cycle and action of tamoxifen. AB - The effects of the estrogen analog mestranol and of the antiestrogen tamoxifen on cell growth and the rate of angiotensinogen production were investigated in HepG2 cells, an hepato-carcinoma cell line of human origin. After 36 h of cell contact with high concentration of mestranol, a (10(-5) M) dose increased by 2-fold the rate of proliferation of HepG2 while reducing angiotensinogen production to below control level. Mestranol at 10(-6) M preferentially stimulated angiotensinogen production 5-fold, whereas cell growth rate was slightly increased. Comparable results were obtained for thymidine uptake in the course of the cell cycle, with a maximum increase for 10(-5) M mestranol, and an increase of angiotensinogen production for 10(-6) M mestranol. At 10(-6) M, tamoxifen acted as a pure antagonist by strongly inhibiting the stimulatory effect of mestranol and reducing angiotensinogen production to below the control level within 60 h. Tamoxifen did not affect the growth rate of HepG2 cells, either when administered alone or together with an equimolar concentration of mestranol. PMID- 3023024 TI - Stage-specific regulation of plasminogen activator secretion in the rat seminiferous epithelium. AB - The cyclic secretion of plasminogen activator (PA) by Sertoli cells in stages VII and VIII of the rat seminiferous epithelial cycle is influenced by hormones and adjacent spermatogenic cells. To understand this interaction more in detail, we have analyzed the effects of FSH, (Bu)2cAMP, testosterone, insulin, and retinoic acid (RA) on staged seminiferous tubule segments in vitro. FSH stimulated stages VIIcd to XI of the cycle; similar results were obtained with (BU)2cAMP. RA stimulated PA secretion in stages I-VIIab, but testosterone and insulin had no effect in any stage. The secreted PA was mainly of the urokinase type, although small amounts of the tissue-type PA were found after stimulation by FSH and cAMP. These results suggest that spermatogenic cells modify the responsiveness of Sertoli cells to hormonal stimulation. Stages I-VIIab are sensitive to stimulation by RA whereas stages VIIcd-XI are preferentially stimulated by FSH and (Bu)2cAMP. PMID- 3023026 TI - Synthetic atrial natriuretic factors (ANFs) stimulate guanine 3',5'-monophosphate production but not hormone release in rat pituitary cells: peptide contamination with a gonadotropin-releasing hormone agonist explains luteinizing hormone releasing activity of certain ANFs. AB - The effects of atrial natriuretic factors (ANFs) on anterior pituitary hormone secretion and cyclic nucleotide production were investigated in cultured rat pituitary cells. ANF had no effect on ACTH, GH, PRL, and TSH release or on cAMP production either on basal hormone levels or during stimulation of their secretion by the appropriate releasing factor. However, ANF markedly stimulated cGMP production in both mixed anterior pituitary cells and enriched anterior pituitary cell populations fractionated by centrifugal elutriation. Unexpectedly, certain ANF preparations, Bachem rat ANF-(5-28) and rat ANF-(5-25), markedly stimulated LH release from cultured anterior pituitary cells and gonadotroph enriched elutriated pituitary cells. The same ANFs also displaced [125I-D Lys6]GnRH ethylamide from binding to anterior pituitary membranes with potencies similar to their LH-releasing activities. Immunoprecipitation of ANF with a specific antiserum abolished the effect of ANF on cGMP production, but did not change the effect of ANF on LH release. In conclusion, ANF did not affect anterior pituitary hormone secretion or cAMP production, but stimulated cGMP formation. The effect of certain ANF preparations on LH release appears to be attributable to peptide contamination with a potent GnRH agonist. PMID- 3023025 TI - The relationship between gonadotropin-releasing hormone-stimulated luteinizing hormone release and inositol phosphate production: studies with calcium antagonists and protein kinase C activators. AB - The coupling between GnRH-stimulated phosphoinositide (PI) turnover and LH release has been investigated in rat pituitary cell cultures. Accumulation of [3H]inositol phosphates ([3H]IPs) formed by hydrolysis of PIs was measured in cells that had been preloaded with [3H]myo-inositol. GnRH stimulated both LH release and incorporation of [3H]inositol into total [3H]IPs with similar dose and time dependencies. [3H] IP production in response to GnRH could be blocked by a GnRH antagonist, but was stimulated by a compound that provokes receptor microaggregation. GnRH-stimulated IP production persisted in the presence of either the Ca2+ channel blocker D600 or the calmodulin antagonist pimozide at concentrations that reduced LH release to 60% and 20% of control, respectively. Stimulated [3H]IP production was inhibited at higher concentrations of D600. In 1 h incubations, GnRH-stimulated [3H]IP production, but not LH release, was markedly inhibited by the protein kinase C activators phorbol myristate acetate and 1,2-dioctanoylglycerol. These findings indicate that in the gonadotrope, GnRH stimulated LH release and [3H]IP production are closely coupled to receptor activation by an agonist; Ca2+ antagonists uncouple stimulated LH release from [3H]IP production; and protein kinase C activators uncouple stimulated PI turnover from LH release. Thus, GnRH-stimulated production of PI metabolites, as measured by [3H]IP accumulation, is apparently not sufficient to support LH release in the absence of Ca2+. In addition, GnRH-stimulated LH release is apparently not dependent on full expression of the PI response. PMID- 3023027 TI - Differential activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in zones of the adrenal cortex. AB - Cholesterol metabolism and steroidogenesis in the outer (zona fasciculata/glomerulosa) and inner (zona reticularis) zones of the adrenal cortex were examined in the guinea pig. It is known from previous studies that the content of cholesterol in the inner zone is considerably lower than that in the outer zone, although basal low density lipoprotein (LDL) receptor activity is similar in the two zones. To further explore cholesterol metabolism in the guinea pig adrenal cortex, the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate-limiting step in cholesterol synthesis, has been examined for which this paper forms the initial report. It was found that the basal specific activity of HMG-CoA reductase was similar in the outer and inner adrenocortical zones (approximately 230 pmol mevalonate formed/min X mg microsomal protein). The administration of ACTH caused 4- and 5-fold increases in HMG-CoA reductase activity in the outer and inner zones, respectively. In fact, the increase in HMG-CoA reductase activity with ACTH treatment was always greater for the inner zone than for the outer zone. This is in contrast to LDL receptor activity, which does not increase in the inner zone as it does in the outer zone with ACTH treatment. When dexamethasone was administered, HMG-CoA reductase activity decreased in the outer zone by about 50%, while there was no change in reductase activity in the inner zone. The latter finding is similar to what happens with LDL receptor activity during dexamethasone administration. Why suppression of endogenous ACTH had no effect on HMG-CoA reductase activity in the inner zone while exogenous ACTH administration caused a marked increase in enzyme activity is not clear, but may be related to phosphorylation/dephosphorylation mechanisms. Based on the use of sodium fluoride in solutions to block HMG-CoA reductase phosphatase, evidence is presented which indicates that a pharmacological dose of ACTH alters the phosphorylation/dephosphorylation status of HMG-CoA reductase in the inner adrenocortical zone, but not in the outer cortical zone. PMID- 3023028 TI - Agents that decrease gonadotropin-releasing hormone (GnRH) receptor internalization do not inhibit GnRH-mediated gonadotrope desensitization. AB - Exposure of pituitary cell cultures to GnRH causes gonadotropin release, receptor capping, internalization, and loss as well as altered responsiveness of the target cell. In the present study, the relationship between loss of gonadotrope secretory responsiveness to GnRH (desensitization) and internalization of the GnRH-receptor complex was examined. Pituitary cell cultures were pretreated (30 min) with vinblastine (100 microM, a concentration that prevents measurable receptor internalization) or with medium containing carrier only, incubated with 10(-7) M GnRH (a desensitizing concentration) with or without vinblastine or with medium alone for 60 min, and finally washed and rechallenged for 3 h with increasing concentrations of GnRH to assess the degree of desensitization as determined by LH release. Results indicate that vinblastine had no measurable effect on the ability of GnRH to stimulate LH release or desensitize the cells. In a second series of studies, a GnRH analog (D-Lys6-GnRH) was immobilized to a cross-linked agarose matrix. The covalent link was shown to be stable by biological, immunological, and physical criteria. This product bound to the GnRH receptor and provoked LH release, but was not internalized, as determined by GnRH receptor binding assays. Cultured cells were treated with either 10(-9) M free analog or an equivalent concentration of coupled analog (as measured by LH release) for 3 h. Cells were washed, then rechallenged with GnRH to assess desensitization. Both the free and coupled analogs provoked an equivalent degree of desensitization. While a significant degree of desensitization also occurred in the presence of 3 mM EGTA (conditions that totally inhibited GnRH-stimulated LH release), the loss of responsiveness was not as great as in the absence of EGTA, indicating that partial depletion of available LH may play a role in GnRH stimulated gonadotrope desensitization. The present findings suggest that GnRH receptor internalization and LH release can be uncoupled and that loss of the GnRH receptor by internalization is not a sufficient explanation for GnRH mediated desensitization of the gonadotrope. PMID- 3023029 TI - Evidence that stimulation of growth hormone release by epinephrine and vasoactive intestinal peptide is based on cell-to-cell communication in the pituitary. AB - Epinephrine (Epi) evoked a strong concentration-dependent (1-1000 nM) rise of GH release from perifused rat anterior pituitary cells cultured as aggregates in a serum-free defined culture medium. Dexamethasone (Dex), added to the culture medium, enhanced the secretory response to Epi. Aggregates of pituitary cells separated by gradient sedimentation at unit gravity widely differed in responsiveness to Epi, provided Dex was added to the culture medium. The poorest response was seen in aggregates composed of a population highly enriched in large somatotrophs from adult male rats even when cultured in the presence of 80 nM Dex. However, when these large somatotrophs were coaggregated with various somatotroph-poor cell populations, all of which were enriched in lactotrophs, the GH response to Epi increased by a factor of 3-4. The latter populations also enhanced GH secretion stimulated by vasoactive intestinal peptide (1-10 nM). In contrast, the GH response to rat GH-releasing factor (GRF, 0.01-0.1 nM) was not significantly potentiated in the coaggregates. The facilitation of the GH response to Epi was not seen when Dex was omitted from the culture medium. All of the lactotroph-enriched populations enhancing the response to Epi also contained corticotrophs, but none were highly enriched in the latter cell type. The magnitude of the Epi effect on GH release was not affected when the large somatotrophs were coaggregated with enriched populations of gonadotrophs, thyrotrophs, or folliculostellate cells. However, coaggregation with GH3 tumor cells provoked some stimulation. The present data suggest that GH release stimulated by Epi is modulated by facilitatory interactions of somatotrophs with other cells, the latter being most likely lactotrophs, although participation of corticotrophs in this interactions cannot be unequivocally excluded. Facilitatory interactions also modulate GH secretion in response to vasoactive intestinal peptide, but the GH response to GRF weakly, if at all. PMID- 3023030 TI - Differential effects of atrial natriuretic factor on angiotensin II- and adrenocorticotropin-stimulated aldosterone secretion. AB - The possible role of atrial natriuretic factor (ANF) in the regulation of adrenal sensitivity to angiotensin II (AII) was investigated in vivo and in vitro by analyzing the characteristics of the inhibitory effect of ANF on aldosterone production stimulated by AII and other stimuli. In isolated adrenal glomerulosa cells, ANF caused a dose-dependent inhibition of basal and stimulated aldosterone production by submaximal concentrations of ACTH, AII, and potassium, with an ED50 of about 1 nM for ANF and complete inhibition with 10 nM ANF. ANF increased the ED50 for ACTH from 14.6 +/- 3.2 to 376 +/- 104 pM with no significant decrease in the maximum aldosterone response. In contrast, ANF inhibited the aldosterone responses to all doses of AII, decreasing maximal aldosterone production by 75%, with a small increase in the ED50 for AII. In conscious rats, ANF infusion (100 ng/min) markedly decreased the plasma aldosterone response to AII infusion (5-10 ng/min). With higher AII doses (50 and 100 ng/min), which increased plasma corticosterone (and presumably ACTH secretion), the inhibitory effect of ANF was less marked. When the rise in ACTH secretion was prevented by dexamethasone treatment, ANF decreased the aldosterone response to 100 ng/min AII by 85%. Similarly, ANF had a minor although significant inhibitory effect on the primary ACTH-mediated increases in plasma aldosterone after stress by immobilization for 15 min. The data demonstrate a prominent inhibitory effect of ANF on AII stimulated aldosterone production in vivo and in vitro. Since plasma ANF levels are increased during atrial distension, these observations support a regulatory role of ANF in the control of the adrenal sensitivity to AII during alterations of extracellular volume. PMID- 3023031 TI - Transferrin messenger ribonucleic acid: molecular cloning and hormonal regulation in rat Sertoli cells. AB - Transferrin-specific cDNA clones were isolated from a rat liver cDNA library prepared from transferrin-enriched mRNA. Hybrid selection and sequence analysis confirmed that the selected clone contained the carboxy-terminal coding region of the transferrin mRNA. Northern blot analysis was used to demonstrate the presence of transferrin mRNA in liver and Sertoli cells. Transferrin mRNA levels were measured in total RNA isolated from cultured rat Sertoli cells after treatment with FSH, insulin, retinol, and testosterone. The results showed a 2- to 4-fold increase in the level of transferrin mRNA, which peaked on the fourth day of culture after initiation of treatment, with FSH, insulin, retinol, and testosterone. This induction is gene specific, since no change in the mRNA levels for either the catalytic or regulatory subunits of cAMP-dependent protein kinase was observed. The effects of hormones, vitamin A (retinol), and Bu2 cAMP on transferrin mRNA and transferrin secretion (measured by RIA) in cultured Sertoli cells were compared. In general, a direct relationship between the amount of transferrin mRNA present in the cells and the amount of transferrin secreted into the culture medium was observed. These results demonstrate the important role that vitamin A, testosterone, and peptide hormones play in modulating transferrin gene expression in Sertoli cells. PMID- 3023032 TI - The carbohydrate moiety of bovine thyrotropin is essential for full bioactivity but not for receptor recognition. AB - TSH is a glycoprotein hormone whose carbohydrate content varies among different species. Although recent studies suggest that variants of TSH deficient in carbohydrate occur naturally, the significance of the carbohydrate moiety of TSH in respect to its thyrotropic function is unclear. The present studies were undertaken, therefore, to examine this question. A highly purified preparation of bovine TSH (bTSH) was deglycosylated by treatment with anhydrous hydrogen fluoride. Amino acid and carbohydrate analyses of the original and deglycosylated preparations indicated that approximately 85% of the carbohydrate originally present had been removed and that the protein moiety was unaltered. As judged from TSH radioreceptor assays, bTSH and deglycosylated bTSH (dg-bTSH) bound to human thyroid membranes with equal affinity, since both caused a half-maximal inhibition of [125I]bTSH binding at approximately equal concentrations. Nonetheless, dg-bTSH at optimal concentration displayed only about one third the activity of intact TSH in stimulating adenylate cyclase activity in human thyroid membranes. dg-bTSH also antagonized the adenylate cyclase-stimulating activity of intact bTSH in this system, but only weakly, since abolition of the bTSH effect required an approximately 40-fold higher concentration of dg-bTSH. In cultures of FRTL5 cells, a cloned line of follicular cells derived from normal rat thyroid, both intact and dg-bTSH enhanced cell growth, as measured by [3H]thymidine incorporation and stimulated cAMP release in the medium, but the response elicited by dg-bTSH was much less than that caused by equal concentrations of the intact hormone. In accord with the findings in the in vitro assays, dg-bTSH evoked a much smaller response than bTSH did in the in vivo mouse assay. It is concluded that although not required for receptor recognition, the carbohydrate moiety of bTSH is essential for the full expression of its biological activity. PMID- 3023034 TI - The metabolism of vitamin D3 during fracture healing in chicks. AB - The metabolism of vitamin D3 was studied in chicks after experimental fractures were performed on their tibiae. The chicks were fed for 3 weeks a vitamin D deficient diet but were supplemented with radioactive labeled vitamin D3. The chicks were then divided into two groups. In the first group the right tibia was fractured, whereas the second group served as nonfractured control group. During the following days of fracture healing, the metabolites of [3H]vitamin D3 were measured in callus, epiphysis, diaphysis, plasma, duodenum, and kidney. Histological examination of calluses and bones, measurements of intestinal absorption of calcium, and renal production of dihydroxylated metabolites of vitamin D3 were performed as well. The levels of the dihydroxylated metabolites were increased in the calluses and the levels of [3H]24,25-dihydroxyvitamin D3 were found to coincide with the formation of cartilaginous tissue and with the renal production of this steroid. In the duodenum of the fractured chicks, the levels of [3H]1,25-dihydroxyvitamin D3 dropped significantly during the first week after fracture, coinciding with reduction in the intestinal absorption of calcium. In the plasma during those 3 weeks of healing process the levels of [3H]1,25-dihydroxyvitamin D3 were far below normal. These findings indicate that during the process of fracture repair, changes in the metabolism and expression of vitamin D are taking place in order to meet the new requirements of the body under stress condition of skeletal fracture. PMID- 3023033 TI - Chronic administration of corticotropin-releasing factor increases pituitary corticotroph number. AB - The effect of chronic administration of CRF on rat pituitary morphology was studied. Experimental animals received CRF (10 micrograms/day) over a period of 52 days by means of sc osmotic pumps changed at 10- to 14-day intervals. The average 0800 h plasma corticosterone levels in the treated animals were significantly greater than control values [7.52 +/- 0.99 (+/- SE) vs. 1.14 +/- 0.5 micrograms/dl; P less than 0.001]. The CRF-treated animals also had a significantly greater adrenal weight (16.44 +/- 1.38 vs. 12.24 +/- 0.85 mg; P less than 0.05) and lower thymus weight (164 +/- 12 vs. 248 +/- 27 mg; P less than 0.005). There was a marked increase in the number of ACTH-producing cells in the anterior pituitaries of the rats that received CRF (13.3 +/- 0.8% vs. 4.5 +/- 0.3% ACTH-producing cells; P less than 0.001), as determined by immunocytochemical methods. Corticotrophs of rats treated with CRF manifested a significant increase in nuclear area (24.0 +/- 0.7 vs. 21.4 +/- 0.4 micron 2; P less than 0.001) and an increased diameter of forming and storage granules (191.1 +/- 1.1 vs. 158.6 +/- 3.5 nm and 196.1 +/- 1.2 vs. 170.1 +/- 3.7 nm, respectively; P less than 0.001). There was no demonstrable increase in ACTH cell area. These data indicate that long term administration of CRF is capable of increasing the number of pituitary corticotrophs. It also supports the view that the corticotroph hyperplasia occurring after adrenalectomy, in unusual cases of ectopic CRF production, and in rare instances of Cushing's disease is a proliferative response to CRF. PMID- 3023035 TI - Stimulation of polyamine biosynthesis by follicle-stimulating hormone in serum free cultures of rat Sertoli cells. AB - Sertoli cells derived from 21-day-old rats were cultured in serum-free Ham's F-10 medium to allow a direct investigation of the effects of FSH on polyamine (PA) biosynthesis in a defined culture system. After 48 h in culture, the basal cellular content consisted predominantly of spermine (1.1 nmol/mg protein) with substantially lower amounts of spermidine (0.1 nmol/mg protein) and undetectable amounts of putrescine. Upon the addition of ovine FSH (3 X 10(-9) M), cellular spermine content became significantly elevated above the control value as early as 1 h after treatment, reaching a 2.5-fold stimulation by 4 h. Spermidine was also elevated by 4 h after FSH treatment, but remained less than 20% of the spermine concentration. At no time did the cellular content of putrescine increase to measurable levels. Extended time-course studies demonstrated that the FSH-induced cellular increase in spermine and spermidine content persisted up to 24 h during the continuous presence of FSH. Bu2cAMP (5 mM) invoked similar changes in PA levels when measured at 4, 8, and 24 h. Ornithine decarboxylase (ODC) activity, which catalyzes the production of putrescine, was increased by FSH in a temporal fashion similar to that of spermine production. Addition of alpha-difluoromethylornithine, an irreversible inhibitor of ODC, blocked increases in both ODC activity and PA in cells stimulated with FSH or Bu2cAMP. Pulse-chase experiments using [3H]ornithine demonstrate that putrescine is initially synthesized, and is subsequently converted to spermidine and spermine. These studies suggest that regulation of PA biosynthesis by FSH is largely expressed as increases in spermine, and to a lesser extent spermidine, suggesting that the more complex PAs may be involved in the regulation of Sertoli cell function. PMID- 3023036 TI - Insulin-induced hypoglycemia activates the release of adrenocorticotropin predominantly via central and propranolol insensitive mechanisms. AB - The dynamic patterns of pituitary-adrenocortical and sympatho-adrenal hormone responses to insulin hypoglycemia as well as the relative importance of central vs. peripheral control of hypoglycemia-induced ACTH secretion were evaluated. In conscious rats bearing indwelling cannulae, the changes in hormone concentrations after insulin injection were dependent on the changes in blood glucose levels with respect to both time course and magnitude. ACTH, corticosterone, epinephrine, and norepinephrine levels were found to be maximal at 60 min after 2.5 IU kg-1 insulin injected ip, whereas earlier (20 min) but smaller increases were obtained in response to 0.5 IU kg-1 insulin injected iv. In rats 6-7 days after lesions of the medial basal hypothalamus (MBH), the rise of ACTH during insulin hypoglycemia was markedly inhibited and corticosterone levels were significantly reduced. Simultaneously, the hypoglycemia-induced increase in plasma epinephrine was unchanged and that in plasma norepinephrine was significantly enhanced in rats with the MBH destroyed. The beta-adrenoreceptor blocker propranolol did not inhibit ACTH and corticosterone responses to hypoglycemia in either sham-operated or MBH-lesioned animals. We conclude that the main factors triggering ACTH release during insulin-induced hypoglycemia are of central rather than peripheral origin. The high concentrations of circulating catecholamines occurring during insulin hypoglycemia are not responsible for pituitary-adrenocortical activation by direct, beta-adrenoreceptor mediated action at the pituitary level. PMID- 3023037 TI - 1,25-Dihydroxyvitamin D3 exerts opposite effects on the regulation of human embryonic and nonembryonic alkaline phosphatase isoenzymes. AB - The regulation of alkaline phosphatase activity by steroid hormones was studied in two human breast cancer cell lines, MDA-MB-157 and BT20. MDA-MB-157 cells were shown to express the alkaline phosphatase isoenzyme produced by normal breast tissue, and the activity of this isoenzyme increased 3-fold after a 72-h treatment of these cells with 10(-7) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], 2 fold after treatment with 10(-6) M hydrocortisone (HC), and 5-fold after treatment with both hormones. BT20 cells did not express the isoenzyme phenotypic to breast, but ectopically expressed the isoenzyme phenotypic to term placenta and other embryonic tissue. Treatment of BT20 cells with 1,25-(OH)2D3 results in a 30% decrease in alkaline phosphatase activity of the embryonic isoenzyme. There was a 2-fold increase in activity after treatment with HC, and enzyme activity was similar to control values after treatment with both hormones. For both cell lines, changes in alkaline phosphatase activity correlated with changes in nanograms of isoenzyme per mg cellular protein, as measured by RIA. Increases in enzyme activity were inhibited when the cells were incubated simultaneously with the steroids and cycloheximide. Studies with receptors in each cell line showed that both cell lines bound 1,25-(OH)2D3 and that a 1,25-(OH)2D3-binding protein with the same mol wt as the D3 receptor was present in both. The BT20 cells also express a larger mol wt protein which binds 1,25-(OH)2D3 but is not as specific for the 1,25-(OH)2D3 isomer. HC receptors were similar in quantity and binding affinity in both cell lines. PMID- 3023039 TI - Ultrasound in cholangiofibromas of the liver detected by laparoscopy. AB - We investigated the question as to whether benign cholangiofibromas of the liver (biliary microharmatomas) show specific structures in ultrasound. Patients with single, sporadic, multiple or diffuse cholangiofibromas (n = 53), detected by laparoscopy and verified by histology, were examined by ultrasound. The single and sporadic type (n = 30) could not be detected by US. In patients with the multiple and diffuse type (N = 23) the descriptive US finding was: "increased content of fibrous tissue, cirrhotic transformation possible". Thus we conclude that patients with non-specific alterations of the liver in the ultrasound image must undergo further investigation by laparoscopy. Blind biopsy is not capable of verifying the presence of cholangiofibromas. PMID- 3023038 TI - Increased atrial natriuretic peptide binding sites in the rat subfornical organ after water deprivation. AB - Binding sites for rat atrial natriuretic peptide (99-126) were localized and quantified in the subfornical organ and choroid plexus of control and water deprived rats. Brain sections were analyzed after incubation with 125I-rat atrial natriuretic peptide (99-126), autoradiography, computerized microdensitometry and comparison to 125I-standards. Higher numbers of atrial natriuretic peptide binding sites were present in both the subfornical organ and the choroid plexus after 4 days of water deprivation. Our results indicate a central role for atrial natriuretic peptide in the regulation of water balance. PMID- 3023040 TI - The mechanisms by which phorbol ester inhibits LH stimulation of progesterone production in rat granulosa cells. AB - Tumor-promoting phorbol esters are believed to affect cell functions by activating a Ca+2-and lipid-dependent protein kinase (protein kinase C). 12-0 Tetradecanoyl phorbol-13-acetate (TPA) inhibits LH stimulation of progesterone (P) accumulation in rat granulosa cells. To determine the mechanisms by which TPA inhibits LH stimulation of P accumulation, TPA regulation of various ovarian steroidogenic enzymes was investigated. Cells were obtained from immature (28-29 days old) rats 48 h after injection of 20 IU PMSG and incubated for up to 5 h. TPA decreased the P responses to LH, cholera toxin, and (Bu)2cAMP by 20%, 24%, and 28%, respectively. One locus of inhibition of LH action, therefore, was after cAMP. TPA decreased LH-stimulated 3-beta-hydroxysteroid dehydrogenase activity. Furthermore, TPA stimulated 20-alpha-hydroxysteroid dehydrogenase activity. The combination of TPA and A23187 inhibited LH-stimulated P accumulation to the same extent as TPA alone. These data suggest that TPA-induced decreases in LH stimulated P production result from both the inhibition of P biosynthesis and the stimulation of P breakdown. PMID- 3023041 TI - Hypothalamic control of adrenocorticotropin secretion: advances since the discovery of 41-residue corticotropin-releasing factor. PMID- 3023042 TI - Effect of fluoride on uptake of D-glucose by isolated epithelial cells of rat intestine. AB - Inhibition on uptake of D-glucose by isolated intestinal epithelial cells (IIEC) was observed when the fluoride concentration ranged between 0.25 and 5 mM. Active transport was almost completely inhibited at 5 mM. When CaCl2 was added to fluoride, the inhibitory effect on glucose uptake was abolished. Preincubation of IIEC with different concentrations of fluoride (2.5-5.0 mM) for different intervals of time (2-20 min) at different pH levels (6.2-7.8) and temperatures (0 37 degrees C) revealed that the conditions which led to higher uptake of fluoride by IIEC produced maximum inhibition. The degree of inhibition was not appreciably altered by a change in glucose concentrations. A concentration-dependent effect of fluoride on lactic acid and carbon dioxide production by IIEC was also observed. PMID- 3023043 TI - Selective adsorption of serum proteins by chrysotile and crocidolite. AB - Two distinct and contemporaneous adsorption mechanisms occur when serum proteins are incubated with chrysotile and crocidolite. The first one appears to be reversible and nonspecific, independent of the characteristics of the protein and the fiber surface. The second process seems to depend on fiber type and to involve only some protein species that are strongly concentrated on the fiber surface. This latter adsorption mechanism appears to be electrostatic in nature. PMID- 3023044 TI - Serum angiotensin-I-converting enzyme and carboxypeptidase N in Crohn's disease and ulcerative colitis. AB - Angiotensin-I-converting enzyme (ACE) and carboxypeptidase N1 and N2 (CPN1, CPN2) inactivate kinins and might therefore play a role in the development of inflammatory reactions via an influence on the release of prostaglandins and inactivation of anaphylatoxic peptides of the complement system. In the present study, the serum activity of these enzymes was determined in 60 patients with Crohn's disease, 18 patients with ulcerative colitis and 70 healthy control subjects. ACE was significantly lowered in active Crohn's disease (CDAI greater than 150) and in ulcerative colitis (p less than 0.01), as long as the ileum or cecum was affected. Since ACE was detected in high concentrations in the human intestinal mucosa, decreased values may be explained by damage to the site of its production. CPN1 and CPN2 were raised in both diseases (p less than 0.005), irrespective of their activity and location. These alterations in the activity of the kininases investigated may play a role in the pathogenesis of inflammatory bowel diseases. PMID- 3023045 TI - Structure and expression of cDNA and genes for human interferon-beta-2, a distinct species inducible by growth-stimulatory cytokines. AB - Induced human fibroblasts produce several mRNAs encoding interferon (IFN) activity. We previously cloned cDNA for a 1.3-kb RNA designated IFN-beta 2 and distinct from the 0.9-kb IFN-beta 1 mRNA. In vitro transcription--translation mapping of the full-length IFN-beta 2 cDNA sequence, shows that it encodes a 23.7 kd protein of 212 amino acids. This cDNA, fused to the SV40 early gene promoter, was transfected and amplified in Chinese hamster ovary cells and clones were obtained which constitutively produce human interferon activity. Two IFN-beta 2 genomic clones were isolated and their expression in hamster and mouse cells also produces biologically active rIFN-beta 2. Specific immunoassays show that IFN beta 2 secreted by DNA-transformed rodent cells is a processed 21-kd protein, whose activity is cross-neutralized by antibodies to human IFN-beta 1 but not to IFN-alpha or gamma. The immunoassay also demonstrates the induction of IFN-beta 2 secretion by fibroblasts in response to growth-regulatory cytokines, such as interleukin-1 and tumor necrosis factor. The function of this IFN-beta 2 as an autoregulatory inhibitor of cell growth is discussed. PMID- 3023047 TI - A domain of SV40 capsid polypeptide VP1 that specifies migration into the cell nucleus. AB - In order to identify the determinants responsible for the nuclear migration of simian virus 40 (SV40) polypeptide VP1, the 5'-terminal portion of the SV40 VP1 gene was fused with the complete cDNA sequence of poliovirus capsid polypeptide VP1 and the hybrid gene was inserted into an SV40 vector in place of the normal SV40 VP1 gene. Deletions of various length were generated in the SV40 VP1 portion of the hybrid gene, resulting in a set of truncated genes encoding 2-40 NH2 terminal amino acids from SV40 VP1, followed by poliovirus VP1. Monkey kidney cells were infected by the deleted hybrid viruses in the presence of an early SV40 amber mutant as helper, and the subcellular localization of the fusion proteins was determined by indirect immunofluorescence using an anti-poliovirus VP1 immune serum. The presence of the first 11 NH2-terminal amino acids from SV40 VP1 was found to be sufficient to target the fusion protein to the cell nucleus. Deletions extending from the NH2- towards the COOH-terminal end of the protein were next generated. Transport of the SV40 VP1-poliovirus VP1 fusion polypeptide to the nucleus was abolished when the first eight amino acids from SV40 VP1 were deleted. Thus the sequence of the first eight NH2-terminal amino acids of SV40 VP1 appears to contain a nuclear migration signal which is sufficient to target the protein to the cell nucleus. PMID- 3023046 TI - A 5' duplication of the alpha-cardiac actin gene in BALB/c mice is associated with abnormal levels of alpha-cardiac and alpha-skeletal actin mRNAs in adult cardiac tissue. AB - We describe the structure and transcriptional activity of the 5' portion of the alpha-cardiac actin gene of BALB/c mice. Southern blotting and DNA sequencing reveal that the promoter and first three exons of the gene are present as perfect repeats in a direct duplication of 9.5 kbp situated immediately upstream of the gene. Both promoters are active in adult cardiac tissue. Transcripts from the partial gene duplication give rise to novel RNAs that are spliced correctly in the actin region and polyadenylated. The level of mature alpha-cardiac actin mRNA is only 16.5% that found in mice that do not possess the duplication. This is due, at least in part, to interference at the transcriptional level. Transcripts from the alpha-skeletal actin gene accumulate to abnormally high levels in the hearts of such mutant mice. This result suggests tight regulatory coupling for this actin gene pair. PMID- 3023048 TI - Purification of a factor specific for the upstream element of the adenovirus-2 major late promoter. AB - Stimulation of in vitro transcription by the upstream element (UE) of the adenovirus-2 major late promoter (Ad2MLP) involves a specific trans-acting factor present in a HeLa whole-cell extract. By following its transcriptional stimulatory activity and its DNase I footprint on the Ad2MLP-UE, we have purified this factor to greater than 10% purity and separated it from RNA polymerase B and the general transcription factors required for transcription from the Ad2MLP. PMID- 3023049 TI - The 3' region of bovine leukemia virus genome encodes a trans-activator protein. AB - The genome of bovine leukemia virus (BLV) contains several overlapping, long open reading frames 3' to the envelope gene. Experiments presented here show that the cDNA encompassing the X region open reading frames encodes a trans-activator function capable of increasing the level of gene expression directed by the BLV long terminal repeat sequences. This study provides further evidence of the structural and functional similarities of the bovine leukemia virus and the human T lymphotropic viruses, HTLV-I and HTLV-II. PMID- 3023050 TI - Restricted expression of EBV latent genes and T-lymphocyte-detected membrane antigen in Burkitt's lymphoma cells. AB - Certain newly established Epstein-Barr virus-containing Burkitt's lymphoma cell lines do not express the cytotoxic T-lymphocyte-detected membrane antigen (LYDMA) through which EBV infection is normally controlled by the host. When the EB virus recovered from these BL lines was used to transform peripheral blood lymphocytes from seronegative donors, the lymphoblastoid cell lines (LCLs) that arose were all LYDMA positive. This indicates that the LYDMA-negative nature of the BLs is not the result of a mutation in the resident viral genome but is rather a specific adaptation in those cells, perhaps permitting evasion of the host immune surveillance in tumour development. A comparison of the EBV gene expression in six LYDMA-negative and two LYDMA-positive BL lines and in their corresponding LCLs revealed that several of the BL lines did not express all of the viral gene products classically associated with latent transformation by EBV. Four out of eight cell lines showed restricted expression of the latent membrane protein (LMP) and/or the EB nuclear antigen, EBNA 2. A new level of EBV gene regulation therefore appears to be operating in some of the BL cell lines. The patterns of expression of EBV genes in the cell lines did not show any correlation with the known susceptibility of the lines to T cell killing. PMID- 3023052 TI - Transformation and stimulation of DNA synthesis in NIH-3T3 cells are a titratable function of normal p21N-ras expression. AB - A plasmid has been constructed which contains the normal human N-ras proto oncogene under the transcriptional control of the steroid-sensitive promoter of the mouse mammary tumor virus long terminal repeat. This plasmid has been introduced into NIH-3T3 cells producing a clone of cells, T15, which is phenotypically normal in the absence of the transcription inducer, dexamethasone, and transformed when treated with high levels of the inducer. At lower levels of dexamethasone, both morphological transformation and stimulation of DNA synthesis are titratable functions of p21N-ras levels. T15 cells have been used to demonstrate that: (i) a 20- to 50-fold over-expression of normal p21ras is required for complete cellular transformation, (ii) p21N-ras expression induces DNA synthesis and the effect can be amplified by epidermal growth factor, (iii) moderate increases in normal p21ras expression can influence cell behaviour. PMID- 3023051 TI - The v-mos and H-ras oncogene expression represses glucocorticoid hormone dependent transcription from the mouse mammary tumor virus LTR. AB - We have subjected the viral mos oncogene (v-mos), the activated human H-ras oncogene [H-ras (A)] and the normal human H-ras protooncogene [H-ras (N)] to the transcriptional regulation of glucocorticoid hormones by in vitro recombination with the promoter region of the mouse mammary tumor virus long terminal repeat (MMTV LTR) and transfection into NIH 3T3 cells. Cell clones were selected which exhibit a transformed phenotype strictly dependent on the presence of hormone in the growth medium. The expression of the chimeric genes as a function of time after hormone stimulation was studied at the level of transcriptional rate, mRNA and protein accumulation. Oncogene expression was stimulated rapidly to high levels, after hormone addition, but declined in the continuous presence of hormone. Measurements of the transcriptional rates in nuclei from LTR v-mos and LTR H-ras (A) transfected cells showed a repression of LTR v-mos and LTR H-ras (A) transcription after the initial stimulation by hormone. LTR H-ras (N) transcription was not affected. An independently transfected LTR H-2Ld construct in LTR v-mos or LTR H-ras (A) containing cells is also transcriptionally repressed. These experiments demonstrated a transcriptional repression effect of the oncogene products on the glucocorticoid hormone-dependent MMTV LTR transcription. PMID- 3023053 TI - Amplification and overexpression of the met gene in spontaneously transformed NIH3T3 mouse fibroblasts. AB - We have identified a class of transformed NIH3T3 mouse fibroblasts that arise at low frequencies in transfection experiments with DNA from both neoplastic and non neoplastic cells and that may result from a low level of spontaneous transformation of NIH3T3 cells. DNA from the transformed cells was unable to transform NIH3T3 cells in a second cycle of transfection and, where examined, the cells showed no evidence for the uptake of the transfected DNA sequences. The results of Southern analyses demonstrate that a mouse homologue of the human met oncogene is amplified 4- to 8-fold in 7 of 10 lines of these transformed NIH3T3 mouse fibroblasts. The cells containing the amplified gene also exhibit at least a 20-fold overexpression of an 8.5-kb mRNA that is homologous to met. To test the hypothesis that met encodes a growth factor receptor, we examined the binding of platelet-derived growth factor, epidermal growth factor, insulin-like growth factor I and gastrin-releasing peptide to transformed and non-transformed NIH3T3 cells. The results show that there is no significant elevation of the binding of these growth factors to cells containing amplification and overexpression of met. PMID- 3023054 TI - Sequence and structure of the dopa decarboxylase gene of Drosophila: evidence for novel RNA splicing variants. AB - In Drosophila, dopa decarboxylase (DDC) serves a dual role in neurotransmitter production and sclerotization of the cuticle. The Ddc gene is under complex hormonal and tissue-specific control and several sizes of Ddc RNA are observed at embryonic hatching, pupariation and adult eclosion. We present here the complete nucleotide sequence of the Drosophila dopa decarboxylase gene and the partial sequence of two corresponding Ddc cDNAs. The sequence allows us to account for the detailed structure of four of the five major Ddc RNA species observed. The cDNA sequence reveals the existence of previously undetected splicing events and provides evidence for two RNA splicing alternatives which appear to encode two protein isoforms. The structure, processing and developmental regulation of the Ddc transcripts and putative protein isoforms are discussed. Interestingly, the pyridoxal-binding peptide of porcine DDC matches the Drosophila sequence perfectly suggesting considerable selective pressure on at least portions of the sequence. This is the first available Ddc gene sequence from any organism and should serve as a basis of comparison for the related proteins of other species. PMID- 3023055 TI - Nuclease hypersensitive regions with adjacent positioned nucleosomes mark the gene boundaries of the PHO5/PHO3 locus in yeast. AB - The chromatin structure of two tandemly linked acid phosphatase genes (PHO5 and PHO3) from Saccharomyces cerevisiae was analyzed under conditions at which the strongly regulated PHO5 gene is repressed. Digestion experiments with DNase I, DNase II, micrococcal nuclease and restriction nucleases reveal the presence of five hypersensitive sites at the PHO5/PHO3 locus, two of them upstream of PHO5 at distances of 920 and 370 bp, one in between the two genes and two downstream of PHO3. Specifically positioned nucleosomes are located next to these hypersensitive sites as shown by indirect end-labeling experiments. The positions deduced from these experiments could be verified by monitoring the accessibility of various restriction sites to the respective nucleases. Sites within putative linker regions were about 50-60% susceptible, whereas sites located within nucleosome cores were resistant. Hybridizing micrococcal nuclease digests to a probe from in between the two upstream hypersensitive sites leads to an interruption of an otherwise regular nucleosomal DNA pattern. This shows directly that these hypersensitive sites represent gaps within ordered nucleosomal arrays. PMID- 3023056 TI - Structure of DNA formed in the first step of CAD gene amplification. AB - Thirty-three independent mutant cell lines were selected in single steps for resistance to low concentrations of N-(phosphonacetyl)-L-aspartate and the structure of their amplified DNA was probed, using a set of recombinant phage and cosmids containing a total of 380 kb of amplified DNA. In all 33 cell lines, the selected CAD gene and at least 65 kb of flanking DNA were amplified, an average of 2.6-fold. Six other regions of DNA were co-amplified in all 33 mutants, but sometimes to a different extent than CAD. Novel joints, marking recombinations which link amplified regions to each other, were found surprisingly rarely. There were only three within the 380 kb of DNA sequence examined in the total of 33 cell lines. Each novel joint was present in only one copy per cell, was found in a different cell line and was homologous to a different probe. The low frequency of novel joints is consistent either with very large amplified regions in the single-step mutants, possibly 10,000 kb of co-amplified DNA for each copy of the CAD gene, or with a strong bias against recombination in the cloned sequences used as probes. Our previous finding that CAD probes hybridize in situ to unusually large chromosome arms in several single-step mutants is most consistent with the first possibility. PMID- 3023057 TI - Molecular characterization of a meiotic recombinational hotspot enhancing homologous equal crossing-over. AB - We have cloned and sequenced a meiotic recombinational hotspot between the A beta 3 and A beta 2 genes in the major histocompatibility complex (MHC) of the mouse. This recombinational hotspot in the Mus musculus castaneus cas3 haplotype was previously localized to a region of 9.5 kb of DNA in which five independent crossing-over events occurred at the unusually high frequency of 0.6%. Aside from cas3, the hotspot appears to be absent in many other MHC haplotypes. We have now confined the five recombinational breakpoints to a stretch of 3.5 kb of DNA. From the nucleotide sequence around the recombinational breakpoints, determined in the parental cas3 and b haplotypes as well as for two recombinant haplotypes, we show that the two recombinant haplotypes were generated by homologous equal crossing over and place the breakpoints within two non-overlapping stretches of 10 and 36 bp, respectively. Comparison of the DNA sequences of the hotspot-positive cas3 and the hotspot-negative b haplotypes reveals a number of differences, in particular, a CAGA-repeat sequence which is present in CAS3 in six, but only four copies in C57BL/6 DNA. This repeat sequence is reminiscent of one in a previously characterized hotspot in the E beta gene. PMID- 3023058 TI - Growth factors induce early pre-replicative changes in senescent human fibroblasts. AB - As human fibroblasts in culture senesce their response to platelet-derived growth factor (PDGF) becomes attenuated. To clarify at which level such cells are blocked in the pre-replicative part of the cell cycle, we have analysed PDGF induced pre-replicative events in senescent (phase III) cultures. We found that phase III cells retain a normal number of PDGF receptors and that these are functional with regard to PDGF-induced receptor autophosphorylation. Phase III cells also respond to PDGF by rapid actin reorganization and increased levels of c-fos and c-myc mRNA, similar to growth-arrested phase II fibroblasts. However, the expression of the nuclear antigen K-67, which in phase II cell is induced in S-phase and continues to be expressed throughout the cell cycle, is not induced in phase III cells in response to PDGF. We conclude that phase III human fibroblasts, although blocked with regard to proliferation, still retain a functional growth factor receptor system, and display early responses when exposed to growth factors, such as changes in the cytoskeleton and the expression of proto-oncogenes. PMID- 3023059 TI - Regulation of cell surface receptors for different hematopoietic growth factors on myeloid leukemic cells. AB - There are clones of myeloid leukemic cells which are different from normal myeloid cells in that they have become independent of hematopoietic growth factor for cell viability and growth. The ability of these clones to bind three types of hematopoietic growth factors (MGI-1GM = GM-CSF, IL-3 = multi-CSF and MGI-1M = M CSF = CSF-1) was measured using the method of quantitative absorption at 1 degree C and low pH elution of cell-bound biological activity. Results of binding to normal myeloid and lymphoid cells were similar to those obtained by radioreceptor assays. The results indicate that the number of receptors on different clones of these leukemic cells varied from 0 to 1,300 per cell. The receptors have a high binding affinity. Receptors for different growth factors can be independently expressed in different clones. There was no relationship between expression of receptors for these growth factors and the phenotype of the leukemic cells regarding their ability to be induced to differentiate. The number of receptors on the leukemic cells was lower than on normal mature macrophages. Myeloid leukemic cells induced to differentiate by normal myeloid cell differentiation factor MGI-2 (= DF), or by low doses of actinomycin D or cytosine arabinoside, showed an up-regulation of the number of MGI-1GM and IL-3 receptors. Induction of differentiation of leukemic cells by MGI-2 also induced production and secretion of the growth factor MGI-1GM, and this induced MGI-1GM saturated the up-regulated MGI-1GM receptors. It is suggested that up-regulation of these receptors during differentiation is required for the functioning of differentiated cells. PMID- 3023060 TI - Abelson transformed fibroblasts lacking the EGF receptor are not tumourigenic in nude mice. AB - Cells transformed by the v-abl-oncogene produce large amounts of the tumour growth factor alpha TGF. alpha TGF is homologous to the epidermal growth factor (EGF) and stimulates cell growth via the EGF receptor pathway. To separate metabolic events in the v-abl-transformed cells mediated by alpha TGF as opposed to the v-abl-encoded protein-tyrosine kinase, we have employed the Swiss 3T3 variant cell line NR6 which lacks a functional EGF receptor. v-abl was found to transform efficiently NR6 cells in vitro. These transformed NR6 cells displayed a variety of in vitro properties which were indistinguishable from transformed wild type fibroblast lines. However, in contrast to the wild-type lines, v-abl transformed NR6 cells failed to form tumours when injected into athymic nude mice. These results imply an important function for alpha TGF and the EGF receptor in the establishment of the v-abl-induced fibrosarcomas. PMID- 3023061 TI - Evolutionary conserved close linkage of the c-fes/fps proto-oncogene and genetic sequences encoding a receptor-like protein. AB - Recently we described that genetic sequences in the immediately upstream region of the c-fes/fps proto-oncogene, designated fur, constituted a transcription unit for a 4.5-kb mRNA. Here we present characteristics of the genetic organization of fur and some features of its putative translation product which we call furin. The nucleotide sequence of a 3.1-kbp fur-specific cDNA isolated from a human cDNA library revealed an open reading frame of 1,498 bp from which the 499 carboxy terminal amino acids of the primary fur translational product could be deduced. Computer analysis indicated that furin contained a possible transmembrane domain which resembled that of class II MHC antigens. Furthermore, a cysteine-rich region was present. Significant homology, especially with respect to the topography of cysteine residues, was found between the cysteine-rich regions of the human insulin receptor, the human epidermal growth factor receptor and furin. From the data presented here we deduce that fur may encode a membrane-associated protein with a recognition function. PMID- 3023062 TI - The ral gene: a new ras related gene isolated by the use of a synthetic probe. AB - We synthesized a set of 20-mer oligonucleotides corresponding to a sequence of seven amino acids strictly conserved in all the different ras proteins, from yeast to man, as well as in rho and YPT, two proteins distantly related to p21 ras (approximately 30% amino acid homology). This oligonucleotide probe was used to search for new members of the ras family. We describe here a new ras related gene named ral, isolated from a cDNA library of immortalized simian B lymphocytes. The ral gene codes for a 206 amino acid protein of expected mol. wt 23.5 kd that shares greater than 50% homology with H-ras, K-ras or N-ras. The GTP binding regions of p21 ras and a C-terminal cysteine involved in membrane anchoring are also present in ral; this strongly suggests that ral is a GTP binding protein with membrane localization. Furthermore, several external regions of p21 ras presumably involved in the interaction with effector, receptor and/or regulatory proteins are highly homologous to the corresponding regions in ral. Therefore some of the proteins that interact with ral might be identical or closely related to those interacting with p21 ras. PMID- 3023063 TI - The hormone regulatory element of mouse mammary tumour virus mediates progesterone induction. AB - Sequences within the long terminal repeat region (LTR) of mouse mammary tumour virus (MMTV) confer progestin inducibility to either the tk-promoter or the MMTV promoter in T47D cells, a human mammary tumour cell line which possesses high constitutive levels of progesterone receptor. In a clone of MCF7 cells, another human mammary tumour cell line with a low level of progesterone receptor, as well as in rat fibroblasts, glucocorticoid but not progestin induction is observed. The effect of the progesterone analogue R5020 is much more pronounced than the effect of dexamethasone, and at the concentrations required for maximal induction, R5020 does not significantly compete with binding of dexamethasone to the glucocorticoid receptor. In conjunction with previous results on the DNA binding of the glucocorticoid and progesterone receptors, these data show that two different steroid hormones, acting through their respective receptors, can mediate the induction of gene expression by interacting with the same DNA sequences. Our results suggest that the hormone regulatory element of MMTV may primarily be a progesterone-responsive element in mammary cells. PMID- 3023064 TI - DNA topoisomerase II cleaves at specific sites in the 5' flanking region of c-fos proto-oncogenes in vitro. AB - We have analyzed 1 kb of cloned human c-fos sequence (-711 to +287) for calf thymus DNA topoisomerase II cleavage sites in vitro. Using the anti-tumor drug VP16 (demethylepipodophyllotoxin-beta-D-glucoside) with purified topoisomerase II, we identify twelve sites. Five sites are clustered around position -306 in a region that possesses enhancer-like properties. A second cluster of three sites is positioned 15 bp upstream of the TATA promoter element. With a HeLa nuclear extract as a source of topoisomerase II, a subset of cleavage sites is conserved within the two clusters. The cleavage sites in the enhancer-like element are conserved in the homologous region of the murine c-fos. These findings raise the possibility that topoisomerase II is involved in mediation of mitogen-induced c fos expression. PMID- 3023065 TI - Mammalian single-stranded DNA binding protein UP I is derived from the hnRNP core protein A1. AB - Antibodies induced against mammalian single-stranded DNA binding protein (ssDBP) UP I were shown to be cross-reactive with most of the basic hnRNP core proteins, the main constituents of 40S hnRNP particles. This suggested a structural relationship between both groups of proteins. Using the anti-ssDBP antibodies, a cDNA clone (pRP10) was isolated from a human liver cDNA library in plasmid expression vector pEX1. By DNA sequencing this clone was shown to encode in its 949 bp insert the last 72 carboxy terminal amino acids of the ssDBP UP I. Thereafter, an open reading frame continued for another 124 amino acids followed by a UAA (ochre) stop codon. Direct amino acid sequencing of a V8 protease peptide from hnRNP core protein A1 showed that this peptide contained at its amino terminus the last 11 amino acids of UP I followed by 19 amino acids which are encoded by the open reading frame of cDNA clone pRP10 immediately following the UP I sequence. This proves that ssDBP UP I arises by proteolysis from hnRNP core protein A1. This finding must lead to a re-evaluation of the possible physiological role of UP I and related ssDBPs. The formerly assumed function in DNA replication, although not completely ruled out, should be reconsidered in the light of a possible alternative or complementary function in hnRNA processing where UP I could either be a simple degradation product of core protein A1 (as a consequence of controlling the levels of active A1) or may continue to function as an RNA binding protein which has lost the ability to interact with the other core proteins.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3023067 TI - Different human cervical carcinoma cell lines show similar transcription patterns of human papillomavirus type 18 early genes. AB - Transcription of human papillomavirus type 18 (HPV18) DNA in the human cervical carcinoma cell lines HeLa, C4-1 and SW756 was studied by nucleotide sequence analysis of HPV18-positive cDNA clones isolated from a HeLa, C4-1 and SW756 cDNA library, respectively, and the cDNA sequences were used to predict the potential encoded proteins. The cDNA clones from all three cell lines were found to be derived from virus-cell fusion transcripts in which 3'-terminal host cell sequences (different for each cell line) were spliced to 5'-terminal exon sequences from the HPV18 E6-E7-E1 region. Three different types of cDNA clones can be distinguished according to the splicing patterns observed in the 5' terminal HPV18 sequences. They carry as potential protein-coding regions the HPV18 specific open reading frames E6 and E6* (generated by splicing and identical with E6 up to the E6* splice junction), E7 and E1 (only in HeLa). Translation of specific cellular genes from the chimeric viral-cellular transcripts seems to be unlikely. The mapping of the 5'-ends of the virus-cell fusion transcripts indicates that transcription is initiated at a viral promoter. The similar patterns of HPV18 transcription in the three different cervical carcinoma cell lines suggest a functional role of HPV18 early genes for the malignant phenotype of these cells. PMID- 3023066 TI - Molecular cloning and sequencing of the human erythrocyte 2,3-bisphosphoglycerate mutase cDNA: revised amino acid sequence. AB - The human erythrocyte 2,3-bisphosphoglycerate mutase (BPGM) is a multifunctional enzyme which controls the metabolism of 2,3-diphosphoglycerate, the main allosteric effector of haemoglobin. Several cDNA banks were constructed from reticulocyte mRNA, either by conventional cloning methods in pBR322 and screening with specific mixed oligonucleotide probes, or in the expression vector lambda gt 11. The largest cDNA isolated contained 1673 bases [plus the poly(A) tail], which is slightly smaller than the size of the intact mRNA as estimated by Northern blot analysis (approximately 1800 bases). This cDNA encodes for a protein of 258 residues; the protein yielded 34 tryptic peptides which were subsequently isolated by h.p.l.c. Our nucleotide sequence data were entirely confirmed by the amino acid composition of these tryptic peptides and reveal several major differences from the published sequence; the revised amino acid sequence of human BPGM is presented. These findings represent the first step in the study of the expression and regulation of this enzyme as a specific marker of the erythroid cell line. PMID- 3023068 TI - The anterobithorax and bithorax mutations of the bithorax complex. AB - The anterobithorax (abx) and bithorax (bx) genes together direct the development of the posterior second and anterior third thoracic segments of the fruit fly. We have characterized the phenotypes and DNA lesions of 19 abx and bx alleles. abx and bx mutations differ both in the nature and location of their DNA lesions, forming two clusters within a relatively small region of the Ultrabithorax transcription unit. Correlation between phenotype and DNA lesion suggests the presence of two or more genetic elements in this region distinct from the Ultrabithorax transcript. Mutant transformations do not strictly obey segmental or parasegmental boundaries. Most of the bx mutations result from insertions of the mobile element gypsy. The strength of these alleles varies in a regular way dependent on the position and orientation of the gypsy element. We propose models for gypsy element action and bithorax complex expression in the light of these results. PMID- 3023069 TI - Embryonic expression of the period clock gene in the central nervous system of Drosophila melanogaster. AB - We have examined the temporal and spatial expression of the 4.5-kb mRNA that is transcribed from the period locus of Drosophila melanogaster and is the best candidate for the per gene product. Both Northern blot analyses and hybridizations in situ to tissue sections reveal significant expression of the 4.5-kb mRNA in embryos. This expression is limited to the central nervous system of the developing embryo and is localized within the brain and ventral ganglia. The 4.5-kb mRNA is enriched in adult heads (by Northern blotting) although we were not able to detect specific localization (in situ). In addition to the physiological role the 4.5-kb mRNA might have in maintaining biological rhythms, we now suggest that it has a developmental role for establishing mechanisms that are necessary for eventual expression of clock functions. PMID- 3023070 TI - The nucleotide sequence of the fission yeast DNA topoisomerase II gene: structural and functional relationships to other DNA topoisomerases. AB - We have determined the complete nucleotide sequence of a 5.3-kb long genomic DNA fragment of the fission yeast Schizosaccharomyces pombe that encodes DNA topoisomerase II. It contains a 4293 bp long single open reading frame. The predicted polypeptide has 1431 residues (mol. wt 162,000) and shows three characteristic domains; the large C-terminal region, which consists of alternating acidic-basic stretches and might be a chromatin-binding domain, the NH2 half domain homologous to the ATP-binding gyrB subunit of bacterial gyrase and the central-to-latter part which is homologous to the NH2 domain of the catalytic gyrA subunit, suggesting a possible evolutionary consequence of the gene fusion of the bacterial gyrase subunits into the eucaryotic DNA topoisomerase II gene. We have found that the cloned fission yeast TOP2 gene can complement the budding yeast top2 mutation, although the fission yeast TOP2 protein sequence is only 50% homologous to the recently determined sequence of budding yeast (J.C. Wang, personal communication). Conversely, the budding yeast TOP2 gene can complement the fission yeast top2 mutations, indicating that their DNA topoisomerase II genes are functionally exchangeable. PMID- 3023071 TI - Nuclear location of all three influenza polymerase proteins and a nuclear signal in polymerase PB2. AB - In order to re-examine the sub-cellular location of the three influenza A/NT/60/68 polymerase proteins PB1, PB2 and PA in infected cells, specific antisera for each polymerase component have been prepared by immunizing rabbits with polymerase-beta-galactosidase fusion proteins synthesized in Escherichia coli. We show that polymerase PB1, PB2, and PA are predominantly associated with the nucleus of influenza-infected MDCK cells by immunocytochemical techniques. In the case of polymerase PB2 we investigate the possibility that nuclear accumulation is an intrinsic property of the PB2 protein. Using a vaccinia-PB2 recombinant virus, we show that PB2 accumulates intra-nuclearly in monkey CV-1 cells in the absence of any other influenza protein, suggesting it contains an intrinsic nuclear signal. PMID- 3023072 TI - Construction of cell lines that regulate by temperature the amplification and expression of influenza virus non-structural protein genes. AB - Monkey cell lines have been transformed with a mixture of plasmids pSV2neo and pSLVa232N, a derivative of plasmid pSLVa232 (Portela et al., 1985b). Plasmid pSLVa232N contained the influenza virus genes encoding non-structural proteins under the control of the SV40 late promoter in pSLts1 vector that includes the SV40 ori and the tsA209 T-antigen gene. At restrictive temperature, plasmid sequences remained stably integrated in the cell genome, but upon temperature shift-down, defined circular DNA molecules were generated and amplified up to 2000-5000 copies/cell. Restriction analysis, Southern blot hybridization and partial sequencing indicate that one such episome, pC5, was derived from the integrated plasmid sequences by a homologous recombination event that led to deletion of the pBR322 sequences included in pSLVa232N. Concomitant with gene amplification, an induction of 20-65-fold in the expression of NS1 and NS2 proteins was observed after temperature shift-down. Thus, gene cloning into vector pSLts1 and transformation at restrictive temperature of cells permissive for SV40 DNA replication, appears to be a useful strategy for the controlled amplification and expression of cloned genes. PMID- 3023074 TI - The role of ascorbic acid in etomidate toxicity. AB - Etomidate, a short-acting hypnotic used in anaesthesia, has been shown to block steroidogenesis in man. The free imidazole radical in etomidate binds to cytochrome P450. Serious side-effects of etomidate have only been observed in guinea-pigs and in man, both species relying for restoration of ascorbic acid pools upon resynthesis and upon the daily dietary intake of vitamin C. It has been shown that ascorbic acid and not ACTH can increase serum cortisol concentration during etomidate infusion. Ascorbic acid even restores the ACTH/cortisol ratio to pre-operative values. It is therefore suggested that etomidate blocks ascorbic acid metabolism. Resynthesis of ascorbic acid cannot occur because of the blockade of cytochrome P450. Depletion of the ascorbic acid pool would then cause inhibition of steroidogenesis. This is the case in man and probably also in the guinea-pig. All other species can synthesize their own ascorbic acid from alpha ketogluconic acid. PMID- 3023073 TI - Stress-induced cruciform formation in a cloned d(CATG)10 sequence. AB - The synthetic alternating purine-pyrimidine sequence, d(CATG)10.d(CATG)10, has been cloned into a 2.079-kb pBR322-derived plasmid (pLN1) and its conformation studied under torsional stress. The resultant plasmid, pLNc40, is hypersensitive to cleavage by the single strand-specific nucleases, S1 nuclease and Bal31 nuclease, and to modification by the single strand-selective reagent, osmium tetroxide. The S1-hypersensitive site of this plasmid predominates over those previously mapped in pBR322. Site-specific cleavage of pLNc40 with the resolvase T4 endonuclease VII demonstrates that this alternating purine-pyrimidine tract selectively forms a cruciform structure when stably integrated into a negatively supercoiled plasmid. Quantitative measurements of the twist change (-4.3 +/- 0.2) and free energy of formation (16.2 +/- 0.5 kcal/mol) of this cruciform have been made from two-dimensional gel electrophoresis experiments, and correspond well with the predicted values of cruciform formation for this sequence. We conclude that cruciform extrusion versus the B-Z transition is the favoured conformation of this insert under torsional stress. PMID- 3023075 TI - Comparison of four commercial assays for detection of rotavirus in childhood gastroenteritis. PMID- 3023076 TI - Relationship between Chlamydia trachomatis infection and elevated serum immunoglobulin M levels in premature infants. AB - Serum immunoglobulin M (IgM) antibodies to Chlamydia trachomatis and to human cytomegalovirus (CMV) were detected by enzyme-linked fluorescence assay and enzyme-linked immunosorbent assay, respectively in 19 premature infants with chronic lung diseases, in 43 extremely low birth weight premature infants and in 123 neonates with elevated serum IgM levels. Ten of the 19 premature infants with chronic lung diseases had elevated serum IgM levels, and five had IgM antibodies to Chlamydia trachomatis. Three of the 43 extremely low birth weight premature infants had elevated serum IgM levels, and two had IgM antibodies to Chlamydia trachomatis. Three of the 123 neonates with elevated serum IgM levels (excluding those with chronic lung diseases and extremely low birth weight) had IgM antibodies to CMV. These results suggest that chronic lung diseases in low birth weight infants might be caused by intrauterine Chlamydia trachomatis infection. PMID- 3023077 TI - Purification and characterization of DNA topoisomerase II from calf thymus associated with polypeptides of 175 and 150 kDa. AB - DNA topoisomerase II was purified from calf thymus nuclei by a simple and fast four-step procedure: selective ammonium sulfate precipitation, chromatography on blue-Sepharose and hydroxyapatite, followed by ultracentrifugation on a glycerol gradient. Starting from 300 g thymus glands, this procedure yields 0.7 mg of homogeneous topoisomerase II. The final product is free of any nucleolytic, proteolytic or topoisomerase I activity. Dodecylsulfate/polyacrylamide gel electrophoresis reveals two bands with apparent molecular masses of 175 and 150 kDa. Analytical gel filtration and sedimentation on isokinetic sucrose gradients were used to determine the Stokes' radius as 6.4 nm and the sedimentation coefficient as 9.5 S, indicating a dimeric structure for the native enzyme. The purified topoisomerase II is strictly dependent on ATP or dATP, the Km values of which were 0.14 mM and 0.5 mM, respectively. Mg2+ is an essential cofactor for the reaction at concentrations between 0.5-8 mM, with an optimum at 4 mM. Mg2+ can be substituted by Mn2+ at concentrations between 0.2-0.4 mM. Both the relaxation and the catenation reaction exhibit a salt optimum at 130 mM NaCl. At concentrations below 30 mM and above 200 mM, the enzyme is inactive. The pH is optimal between 8 and 9.5 using Tris buffers. PMID- 3023078 TI - Purification and characterization of human interleukin-1 beta expressed in recombinant Escherichia coli. AB - The high-level expression of human interleukin-1 beta in Escherichia coli is described. The protein contributes about 12% of the total cell protein and is associated with the soluble cytoplasmic fraction of the cell. A method for the purification of the protein is given which is based on anion- and cation-exchange chromatographies. The isolated protein, shown to be homogeneous by several analytical methods, has been characterized by amino acid analysis, N- and C terminal sequence analysis and analytical centrifugation. The protein is biologically active as demonstrated by two different in vitro assays, namely, the mononuclear cell factor (IL-1/MCF) assay and lymphocyte activating factor (IL 1/LAF) assay. The specific activities determined with the IL-1/MCF and IL-1/LAF assays, are 2 X 10(7) and 4 X 10(7) units mg-1, respectively. PMID- 3023079 TI - On the oxidation pathways of the mitochondrial bc1 complex from beef heart. Effects of various inhibitors. AB - We have investigated the oxidation of the reduced ubiquinol:cytochrome c reductase (bc1 complex) isolated from beef heart mitochondria. The oxidation of cytochrome c1 by both potassium ferricyanide and cytochrome c in the ascorbate reduced bc1 complex is not a first-order process. This is taken as evidence that cytochrome c1 is in rapid equilibrium with the Rieske iron-sulphur center. Among the several inhibitors tested, only 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole and stigmatellin are seen to affect this redox equilibrium between the high potential centers of the beef heart bc1 complex. The oxidation of cytochrome b by cytochrome c in both the succinate-reduced and the fully reduced bc1 complex is blocked by all the inhibitors tested. This inhibition occurs simultaneously with an acceleration in the oxidation of cytochrome c1, even after extraction of the endogenous ubiquinone which is present in the bc1 preparation. Almost complete extraction of ubiquinone from the bc1 complex has no effect upon the rapid phase of cytochrome b oxidation, nor does it alter the inhibition of cytochrome b oxidation by the various inhibitors. The oxidation of cytochrome b by exogenous ubiquinones is stimulated by myxothiazol and partially inhibited by antimycin. However, the addition of both these inhibitors together completely blocks the oxidation of cytochrome b by quinones. In contrast, the simultaneous addition of antimycin and myxothiazol has no such synergistic effect upon the oxidation of cytochrome b by cytochrome c. Our data show that intramolecular electron transfer from cytochrome(s) b to the Rieske iron-sulphur center can take place in the bc1 complex without involvement of endogenous ubiquinone-10. This electron pathway is sensitive to all the inhibitors of the enzyme. PMID- 3023080 TI - Structural relationships in the adenylate kinase family. AB - The sequences of five distantly related adenylate kinases have been aligned. The local conservation of amino acids is discussed in the light of the known three dimensional structure of one of the enzymes, the cytosolic isoenzyme 1 (AK1) from porcine muscle. The similarity profile outlines clearly the active site in the cleft of the spatial structure of AK1. The alignment reveals further that the enzyme family can be subdivided into small and large variants according to the presence or absence of a particular segment of about 30 residues in the middle of the chain. The extra segments of the large variants are strongly conserved. PMID- 3023081 TI - Substrate specificity of protein kinase C. Use of synthetic peptides corresponding to physiological sites as probes for substrate recognition requirements. AB - Although the Ca2+/phospholipid-dependent protein kinase, protein kinase C, has a broad substrate specificity in vitro, the enzyme appears considerably less promiscuous in vivo. To date only a handful of proteins have been identified as physiological substrates for this protein kinase. In order to determine the basis for this selectivity for substrates in intact cells, we have probed the substrate primary sequence requirements of protein kinase C using synthetic peptides corresponding to sites of phosphorylation from four of the known physiological substrates. We have also identified the acetylated N-terminal serine of chick muscle lactate dehydrogenase as an in vitro site of phosphorylation for this protein kinase. These comparative studies have demonstrated that, in vivo, the enzyme exhibits a preference for one basic residue C-terminal to the phosphorylatable residue, as in the sequence: Ser/Thr-Xaa-Lys/Arg, where Xaa is usually an uncharged residue. Additional basic residues, both N and C-terminal to the target amino acid, enhance the Vmax and Km parameters of phosphorylation. None of the peptides based on physiological phosphorylation sites of protein kinase C was an efficient substrate of cAMP-dependent protein kinase, emphasizing the distinct site-recognition selectivities of these two pleiotropic protein kinases. The favorable kinetic parameters of several of the synthetic peptides, coupled with their selectivity for phosphorylation by protein kinase C, will facilitate the assay of this enzyme in the presence of other protein kinases in tissue and cell extracts. PMID- 3023082 TI - Binding of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-diphosphate opens the pathway for protons through the chloroplast ATPase complex. AB - The effect of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-diphosphate (TNP-ADP) on photophosphorylation and on the proton conductivity of the thylakoid membrane has been investigated. The results show that TNP-ADP is a potent competitive inhibitor of photophosphorylation (Ki = 1-2 microM). Moreover, in the absence of ADP and Pi, TNP-ADP accelerates basal electron transport of chloroplasts. Addition of ADP, which promotes release of the analogue from CF1, completely reverses this effect of TNP-ADP; likewise Pi alone reverses stimulation of electron transport by TNP-ADP. Dicyclohexylcarbodiimide treatment, which is known to close CF0 to H+, completely abolishes the effect of TNP-ADP. The measurements of the alkalization of the medium and the acidification of the thylakoid lumen following single turnover flashes showed that binding of TNP-ADP to CF1 increased membrane permeability for H+. Further results suggest that binding of TNP-ADP to the catalytic site of CF1 opens the CF0-CF1 complex for H+. Since ADP, as well as Pi alone, reverses the effect, it is concluded that TNP-ADP induces a conformation of the CF0-CF1 complex similar to the one triggered by simultaneous binding of ADP plus Pi. This may be achieved by interaction of the TNP residue with the Pi binding site. Thus it seems that the status of the catalytic site(s) in CF1 can be transmitted to the CF0 part to control proton flux through the ATPase complex in an economically reasonable way. PMID- 3023083 TI - The phosphoenolpyruvate-dependent fructose-specific phosphotransferase system in Rhodopseudomonas sphaeroides. Distribution of EIIFru over the membranes of phototrophically grown Rps. sphaeroides. AB - The distribution of the fructose carrier over the membranes of Rhodopseudomonas sphaeroides was studied in cells grown under light saturation and light limitation. Three types of membranes were isolated after disruption of the cells in a French press. All three types were present in the cells grown either under the high or low light intensity, but they were present in different quantities. The cytoplasmic membrane could be separated from the photosynthetic membranes by Sephacryl S-1000 chromatography. The cytoplasmic membrane has the highest specific density and fructose carrier content and does not contain the light harvesting pigments. The photosynthetic membranes could be resolved into two types by sucrose density gradient centrifugation. Type A predominates when cells are grown under light saturation, whereas type B, the chromatophores, is synthesized abundantly under light limitation. The properties of type A are in between the properties of the cytoplasmic membrane and the chromatophores. It has a slightly lower specific density and contains four times less fructose carrier than the cytoplasmic membrane, but contains half of the light-harvesting bacteriochlorophyll of the chromatophore membrane. The fructose carrier content in the type B membranes, the chromatophores, is very low. PMID- 3023085 TI - Purification of the proteolytically solubilized, active catalytic subunit of adenylate cyclase from ram sperm. Inhibition by adenosine. AB - Ram spermatozoa adenylate cyclase is insensitive to all usual regulatory processes. The purification of its active catalytic subunit was accomplished after proteolytic solubilization of a particulate fraction by alpha-chymotrypsin. The purification (26,000-fold from the particulate fraction or 125,000-fold from the whole-sperm proteins) was achieved by conventional procedures (DEAE Trisacryl, Ultrogel AcA 34, DEAE-Sephacel, hydroxyapatite), in the absence of detergent, and with a yield of 5-10% and a final specific activity of 19 mumol cyclic AMP formed mg protein-1 min-1 at 30 degrees C in the presence of manganese as cosubstrate. The solubilized enzyme, stable at the beginning of the purification procedure, became unstable at the later stages. After the last step (chromatography on hydroxyapatite) half-lives of 27 min, 50 min and 160 min were obtained at 30 degrees C, 20 degrees C and 4 degrees C respectively. The enzyme was stabilized by addition of bovine serum albumin and Lubrol PX, 80% of the activity remaining after 24 h at 4 degrees C. The purified enzyme exhibited a Km value similar to that of the native enzyme (Km = 1.4 mM). Unlike the native enzyme, the purified enzyme has an absolute requirement for MnATP; no significant activity was recovered in the presence of MgATP. Adenosine inhibited the activity of both the native and purified forms of the enzyme to the same extent and in a non-competitive manner. This indicates that adenosine acts on the catalytic component itself and the inhibition site and the catalytic site are different. Data obtained with adenosine analogs indicate that adenosine interacts with the cyclase catalytic subunit with a 'P-site' specificity. The purified adenylate cyclase, which had an apparent molecular mass of 38 kDa on a high-performance liquid chromatography column [Stengel, D., Guenet, L. and Hanoune, J. (1982) J. Biol. Chem. 257, 10,818-10,826], gave a doublet of 36 kDa and 34 kDa on sodium dodecyl sulfate gel electrophoresis. This represents the smallest protein entity associated with adenylate cyclase activity so far reported. PMID- 3023084 TI - Purification of a functional receptor for calcium-channel blockers from rabbit skeletal-muscle microsomes. AB - The dihydropyridine receptor was purified from rabbit skeletal muscle microsomes in the presence of [3H]nitrendipine plus diltiazem or [3H](+)PN 200-110 to an apparent density of 1.5-2 nmol binding sites/mg protein. Sodium dodecyl sulfate gel electrophoresis in the absence of reducing agents yielded three peptide bands of 142, 56 and 30 kDa in a relative ratio of 11:1:1.3, whereas in the presence of 40 mM dithiothreitol bands of 142, 122, 56, 31, 26 and 22 kDa were obtained in a relative ratio of 5.5:2.2:1:0.9:14:0.09. This gel pattern was observed regardless of whether the receptor was purified as a complex with nitrendipine plus diltiazem or with (+)PN 200-110. cAMP-dependent protein kinase phosphorylated preferentially the 142-kDa band up to a stoichiometry of 0.82 +/- 0.07 (15) mol phosphate/mol peptide. The 56-kDa band was phosphorylated only in substoichiometric amounts. [3H]PN 200-110 bound at 4 degrees C to one site with apparent Kd and Bmax values of 9.3 +/- 1.7 nM and 2.2 +/- 0.3 (3) nmol/mg protein, respectively. The binding was stereospecific and was not observed in the presence of 1 mM EGTA. Desmethoxyverapamil interfered with the binding of [3H]PN 200-110 in an apparent allosteric manner. (-)Desmethoxyverapamil inhibited the binding of [3H]PN 200-110 at 37 degrees C and stimulated it at 18 degrees C. In agreement with these results, (-)desmethoxyverapamil increased the dissociation rate of [3H]PN 200-110 from 0.29 min-1 to 0.38 min-1 at 37 degrees C and decreased it threefold from 0.046 min-1 to 0.017 min-1 at 18 degrees C. The (+)isomer of desmethoxyverapamil inhibited PN 200-110 binding at all temperatures tested. d-cis-Diltiazem stimulated the binding of [3H]PN 200-110 at 37 degrees C with an apparent EC50 of 1.4 microM and decreased the dissociation rate from 0.29 min-1 to 0.11 min-1. The stimulatory effect of d-cis-diltiazem was temperature dependent and was seen only at temperatures above 18 degrees C. These results suggest that the purified dihydropyridine receptor retains the basic properties of the membrane-bound receptor and contains separate sites for at least dihydropyridines and phenylalkylamines. PMID- 3023088 TI - Interactions of pig plasma gelsolin with G-actin. AB - Pig plasma gelsolin forms a ternary complex with monomeric actin in 0.1 mM CaCl2 and a binary complex in EGTA (less than 0.01 microM calcium), as shown by gel filtration and fluorescence changes when actin which had been treated with N ethylmaleimide and 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole (NBD-actin) or with N-(1-pyrenyl)iodoacetamide (PI-actin) binds to gelsolin. The fluorescence enhancement per actin molecule bound is similar in the binary and ternary complexes, but the affinity of gelsolin for labelled actin is very much greater in the presence of calcium. Furthermore, the formation of ternary complex exhibits strong positive cooperativity. PMID- 3023086 TI - A 1H-NMR study of human interleukin-1 beta. Sequence-specific assignment of aromatic residues using site-directed mutant proteins. AB - Complete identification of spin systems in the aromatic region of recombinant human interleukin-1 beta has been achieved using two-dimensional homonuclear Hartmann-Hahn spectroscopy. In addition, sequence-specific assignments for the four tyrosine residues have been carried out with the help of a series of mutant proteins, obtained by site-directed mutagenesis of the cloned gene. It is shown that, for the mutant proteins investigated, either none or only local structural changes occur. The use of NMR spectroscopy to determine the structural identity of site-directed mutant proteins with respect to the wild-type protein is discussed. PMID- 3023087 TI - Preparation and characterization of pig plasma and platelet gelsolins. AB - Pig plasma gelsolin has been prepared by a revised method involving poly(ethylene glycol) precipitation, chromatography on CM-cellulose and affinity chromatography on actin-Sepharose. Pig platelet gelsolin has been prepared by chromatography on DEAE-cellulose and actin-Sepharose. Partial chemical and proteolytic cleavage shows that the two proteins are closely related in their fragmentation patterns. The amino acid sequences are identical at the N-terminus of the platelet protein, but the plasma protein has an additional nine residues on the N-terminal side of the common sequence. Calcium binding studies show that the plasma protein has similar calcium binding properties to both macrophage and platelet gelsolins. PMID- 3023089 TI - Binding of pig plasma gelsolin to F-actin and partial fractionation into calcium dependent and calcium-independent forms. AB - The interaction of pig plasma gelsolin with F-actin has been studied by a sedimentation assay using 125I-gelsolin in a Beckman Airfuge. Over 90% of the gelsolin bound to F-actin in 0.1 mM CaCl2 in experiments using 24 microM actin and 2-10 nM 125I-gelsolin, but only 40-50% bound in 1 mM EGTA. Addition of more F actin to the EGTA supernatant does not sediment this gelsolin. Demonstration of this partial calcium sensitivity depends critically on the use of F-actin that has been prepared in the absence of calcium ions. F-actin prepared from G-actin in calcium or pretreated with calcium, binds 125I-gelsolin more completely in EGTA. This suggests that gelsolin activity is influenced by transient exposure of actin to calcium. Further evidence for partial calcium sensitivity in the interactions between gelsolin and F-actin has been obtained by other methods, including viscometry and electron microscopy. The gelsolin present in the EGTA supernatant is complexed to G-actin, predominantly as binary complexes. Very low concentrations of these complexes reduce the viscosity of F-actin in calcium but not in EGTA. Whether this effect is due to severing activity, or capping with consequent depolymerization to establish the new critical concentration, is uncertain. The results suggest the presence of two types of gelsolin, one that requires micromolar concentrations of calcium for binding to F-actin and one that does not. Both bind to G-actin. Partial separation has been achieved using actin Sepharose. Pig plasma gelsolin is heterogeneous on isoelectric focussing gels in urea, but the two types of gelsolin separated on actin-Sepharose do not correspond to specific isoelectric species. PMID- 3023090 TI - Phosphatidylinositol-4-phosphate kinase from rat brain. Activation by polyamines and inhibition by phosphatidylinositol 4,5-bisphosphate. AB - Phosphatidylinositol-4-phosphate (PtdIns-P) kinase was purified approximately 30 fold from rat brain cytosol. No contaminating activity of PtdIns kinase or of phosphomonoesterase and phospholipase C using PtdIns-P or PtdIns-P2 as substrate could be detected in the enzyme preparation. The PtdIns-P kinase activity was severalfold higher when PtdIns-P/PtdEtn vesicles rather than PtdIns-P alone were used as substrate. This might be due to increased accessibility of the enzyme for the vesicular substrate, further indicated by the lower activity obtained when PtdCho or PtdIns, phospholipids with bulky head groups, was also present in the vesicles. The product PtdIns-P2 was a competitive inhibitor with respect to PtdIns-P and 50% inhibition of enzyme activity was observed at the same product concentration regardless of whether the substrate-product mixture was presented in vesicular or micellar form, or the substrate and product were added in separate vesicles. The polyamines spermine and spermidine enhanced PtdIns-P kinase activity severalfold. Spermine also caused a shift in the MgCl2 saturation curve from sigmoidal to hyperbolic, lowering the Mg2+ concentration required for optimum kinase activity to the physiological range. Myelin basic protein enhanced the enzyme activity when PtdIns-P/PtdEtn vesicles were used as substrate, whereas it was inhibitory when PtdIns-P was added alone. The possible role of polyamines and the product PtdIns-P2 in the regulation of PtdIns-P kinase activity is discussed. PMID- 3023091 TI - Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715. AB - A plasmid-encoded crystal protein gene (bt2) has been cloned from Bacillus thuringiensis berliner 1715. In Escherichia coli, it directs the synthesis of the 130-kDa protein (Bt2) which is toxic to larvae of Pieris brassicae and Manduca sexta. Comparison of the deduced amino acid sequence of this Bt2 protein with the B. thuringiensis kurstaki HD1 Dipel, B. thuringiensis kurstaki HD73 and B. thuringiensis sotto crystal protein sequences suggests that homologous recombination between the different genes has occurred during evolution. Treatment of the Bt2 protein with trypsin or chymotrypsin yields a 60-kDa protease-resistant and fully toxic polypeptide. The minimal portion of the Bt2 protein required for toxicity has been determined by analysing the polypeptides produced by deletion derivatives of the bt2 gene. It coincides with the 60-kDa protease-resistant Bt2 fragment and it starts between amino acids 29 and 35 at the N-terminus and terminates between positions 599 and 607 at the C-terminus. PMID- 3023092 TI - Purification and properties of spinach leaf phosphofructokinase 2/fructose 2,6 bisphosphatase. AB - Phosphofructokinase 2 was purified from spinach leaves by fractionation with poly(ethylene glycol) and by chromatography on blue Sepharose, anion exchanger Mono-Q and blue Trisacryl. A low-Km fructose-2,6-bisphosphatase copurified with phosphofructokinase 2 and its constitutive subunits could be easily identified by sodium dodecyl sulphate gel electrophoresis thanks to the formation of a [32P]phosphoenzyme intermediate upon short-time incubation in the presence of 1 microM fructose 2,6-[2-32P]bisphosphate. On anion-exchange chromatography, two peaks of phosphofructokinase 2/fructose-2,6-bisphosphatase were resolved. The first one, called L (light), represented about 10% of the phosphofructokinase 2 activity and was characterized by a phosphofructokinase 2/fructose-2,6 bisphosphatase activity ratio close to 1, by an Mr of 132,000 as measured by gel filtration, and by a series of subunits of Mr comprised between 44,000 and 70,000. The second and major peak of phosphofructokinase 2, called H (heavy), had a phosphofructokinase 2/fructose-2,6-bisphosphatase ratio close to 8, an Mr of 390,000 and was made of 90,000-Mr subunits. The H form of phosphofructokinase 2 had a lower Km for fructose 6-phosphate than the L form and a higher Ki for a series of physiological inhibitors. By contrast, the kinetics of fructose-2,6 bisphosphatase was the same for the two forms of the enzyme. Upon incubation in the presence of papain or of a crude spinach leaf extract, the purified H form gave rise to products made of subunits of Mr comprised between 70,000 and 44,000 but also of lower values which maintained their fructose-2,6-bisphosphatase activity. The H and L forms of phosphofructokinase 2/fructose-2,6-bisphosphatase were also detected in crude homogenates of castor bean endosperm and of Jerusalem artichoke tubers. PMID- 3023093 TI - Influence of buffer composition, membrane lipids and proteases on the kinetics of reconstituted cytochrome-c oxidase from bovine liver and heart. AB - Isolated cytochrome-c oxidases from bovine heart and liver were reconstituted in liposomes with asolectin and the kinetics of cytochrome c oxidation were measured under various uncoupled conditions. With 40 mM KCl, 10 mM Hepes, pH 7.4, the liver enzyme showed a higher Vmax in the polarographic but a lower Vmax in the photometric assay. With 125 mM phosphate buffer at pH 6.0 both enzymes revealed identical kinetics. Reconstitution with pure phosphatidylcholine leads to a low activity, which is specifically stimulated for the heart enzyme by inclusion of 10% cardiolipin. Proteoliposomes of both enzymes prepared with asolectin have a high activity, which is unaffected by cardiolipin. Exchanging the intraliposomal buffer, Hepes, for phosphate causes an opposite change of the Vmax and a similar change of the Km for both enzymes suggesting a conformational change of the extraliposomal binding domain for cytochrome c through the membrane. Proteases change the kinetics of both enzymes, but to a different degree. The data indicate a complex and tissue-specific influence of nucleus-coded subunits on the catalytic activity of cytochrome-c-oxidase. PMID- 3023094 TI - Differential response of cultured parsley cells to elicitors from two non pathogenic strains of fungi. Microsomal conversion of (+)marmesin into psoralen. AB - Microsomal fractions isolated from parsley cell suspension cultures, which had been challenged with an elicitor from either Alternaria carthami or Phytophthora megasperma f. sp. glycinea, catalyzed the formation of psoralen from synthetic [3 14C](+)marmesin. Whereas psoralen was the only product formed in incubations with Alternaria-induced microsomes, another unidentified product was isolated from incubations with Phytophthora-induced microsomes. The latter product is neither a precursor nor a product of psoralen. In contrast, microsomes isolated from non induced parsley cells lacked both of these catalytic activities. The formation of psoralen depends on NADPH as a cofactor and molecular oxygen. Blue-light reversible CO inhibition and inhibition by various synthetic chemicals known to bind to cytochromes P450 indicated that the reaction is catalyzed by an elicitor inducible cytochrome P450-dependent psoralen synthase. Fractionation of microsomal preparations by centrifugation revealed that psoralen synthase is associated with the endoplasmic reticulum. Our results suggest that the endoplasmic reticulum of cultured parsley cells is the primary target in the previously reported differential induction by elicitors from these two non pathogenic strains of fungi. PMID- 3023095 TI - Conformational analysis of hairpin oligodeoxyribonucleotides by a single-strand specific nuclease. AB - Hairpin-forming oligodeoxynucleotides d[ATCCTA(A)NTAGGAT] with n = 3-6 were subjected to nuclease digestion with the nuclease from mung bean. Cleavage occurs only in the loop region of the hairpin molecules. In detail, the number and intensity of cleavage sites were determined in relation to the length of the loop, the temperature and the salt concentration. The restricted accessibility of mung bean nuclease to the loop bases adjacent to the helical region of all hairpins is due to a reduced exposure of these bases in presence of a certain Mg2+ concentration. With increasing temperature the exposure of these bases is increased. It is deduced that the bases adjacent to the helix increase the length of the latter by stacking, the degree of which is dependent on the number of loop bases. PMID- 3023096 TI - Application of aspiration biopsy cytology in the diagnosis of breast diseases. AB - Fine-needle aspiration biopsy of the breast was performed in 198 patients; 158 benign and 8 malignant lesions were found, 24 samples were considered unsatisfactory and 8 suspicious. The cytological diagnosis was correlated with clinical follow-up and histological findings. The Author discusses the value of aspiration biopsy cytology in the clinical assessment of breast disease and describes the cytological appearances and diagnostic criteria in the major benign and malignant breast diseases. PMID- 3023097 TI - A reproducible radionuclide procedure for measurement of cerebrospinal fluid shunt flow. AB - A radionuclide technique was developed to obtain reproducible measurements of cerebrospinal fluid flow in Rickham-Holter type shunt systems. In attempting to reproduce previously described radionuclide procedures, we found that different flow rate measurements can be obtained simply by changing the radiotracer injection technique or the method of analysis of the time activity curves which are generated. The importance of these technical pitfalls cannot be over emphasized, since there is no gold standard to evaluate cerebrospinal fluid flow in vivo. An experimental model with a calibrated Harvard pump and a shunt system filled with normal saline was constructed. Aliquots of 500 microCi of pertechnetate in 0.05 ml of saline were injected serially into the center of Rickham reservoir using a size 28 needle. Time-activity curves were obtained after waiting five min to reach equilibrium, and standard curves of radiotracer clearance vs actual flow for each system used clinically in our institution were generated. In addition, a technique to quantify and thus correct for the small but significant leak of radiotracer which occurs at the site of injection in the reservoir is described. Excellent correlation was obtained between our results and the clinical and radiological findings in a preliminary clinical evaluation. PMID- 3023098 TI - Preferential accumulation of 11C in human brain tumors after intravenous injection of 11C-1-pyruvate. AB - Regional cerebral turnover of 11C following intravenous injection of 11C-1 pyruvate was investigated in 12 patients with brain tumors. The positron emission tomography (PET) image taken at 10-15 min after the injection always demonstrated a high concentration of 11C in tumor, and thus visualized precise tumor contours. This phenomenon was not influenced qualitatively by the variation of blood flow in the tumor. Brain edema associated with the tumor was not visualized. We conclude that the localization of brain tumors would be well determined by the PET scan, using 11C-1-pyruvate. PMID- 3023099 TI - Viral etiology in myasthenia gravis? Analysis of thymic tissue. AB - Thymic cells from 26 myasthenia gravis patients were investigated by viral isolation using inoculation of supernatants obtained from thymic tissue homogenates into human and monkey cells. Living cells from 14 thymuses were also cocultivated with monkey cell lines and with human embryonic lung fibroblasts. None of the thymic biopsies showed any cytopathic effect in these cell cultures sensitive to a broad spectrum of viruses. Cytomegalovirus antigen was not detected in any of 25 biopsies investigated in an imprint immunofluorescence assay. PMID- 3023100 TI - The acute haemodynamic effects of quinapril, a new non-sulfhydryl angiotensin converting enzyme inhibitor, in patients with severe congestive cardiac failure. AB - This study investigates the acute haemodynamic effects of Quinapril (CI-906) a new non-sulphydryl angiotensin converting enzyme inhibitor in 15 patients with refractory congestive cardiac failure. There were 14 males and 1 female mean age 59.5 years. After administration of Quinapril there was a significant reduction in mean arterial pressure (MAP) from 93.1 to 79 mmHg, systemic vascular resistance (SVR) from 1887 to 1349 dyn s cm-5 and PCW from 27.3 to 15.3 mmHg. This was accompanied by an increase in CO from 3.7 to 4.71/min, cardiac index (CI) from 1.97 to 2.51/min/m2 and Stroke volume index from 21.1 to 28.7 ml/m2. There was no significant change in heart rate (HR), right atrial pressure (RAP), or pulmonary vascular resistance. The peak effect on pulmonary capillary wedge pressure (PCW) and cardiac output (CO) occurred at 75-120 min after Quinapril administration. The maximum effect on mean arterial pressure (MAP) occurred slightly later at 120-150 min. SVR and CI exhibited 2 periods of peak effects, at 90 and 180 min. This time course is very similar to that observed in studies on the acute effects of Captopril. The significant improvement in haemodynamic measurements acutely, following administration of Quinapril 5 mg orally, suggests that this drug is worthy of further study in the management of patients with refractory congestive cardiac failure, in particular its long term effects. PMID- 3023101 TI - A monoclonal antibody reacting with a membrane determinant expressed on activated chicken T lymphocytes. AB - A monoclonal antibody (INN-CH-16) was prepared which reacts with a cell surface antigen termed chicken activated T lymphocyte antigen. This antigen is expressed on antigen- or mitogen-activated T lymphocytes and is not present on nonstimulated lymphocytes. It has an apparent molecular mass of 48-50 kDa under reducing conditions. The value of this antibody for the immunohistochemical characterization of infiltrating cells in the thyroid glands from Obese strain chickens with spontaneous thyroiditis is demonstrated. PMID- 3023102 TI - Human immunodeficiency virus gp120 glycoprotein detected by a monoclonal antibody to a synthetic peptide. AB - A chemically synthesized peptide corresponding to the amino acid sequence 503-532 of gp160 of human immunodeficiency virus (HIV) was used to generate monoclonal antibodies reactive with the env glycoprotein gp120. One monoclonal antibody, 120 1, was isolated that reacted with the peptide and with HIV antigen(s). Western blot analysis showed reactivity with two bands of 120 kDa and 88 kDa. 120-1 reacted in indirect immunofluorescence with 15-20% of infected human T cell line A3.01 as early as 4 days post in vitro infection, 2-3 days prior to detectable reverse transcriptase activity in supernatant fluids. PMID- 3023103 TI - ATP analogues and the guinea-pig taenia coli: a comparison of the structure activity relationships of ectonucleotidases with those of the P2-purinoceptor. AB - The dephosphorylation of adenine nucleotides and their analogues by ectonucleotidases on the guinea-pig taenia coli was studied using HPLC. The rate of dephosphorylation of each analogue was compared with its pharmacological potency relative to ATP. ATP, ADP and AMP were rapidly dephosphorylated, and substitution on the purine ring had no effect upon the rate of breakdown. The ectonucleotidases showed stereoselectivity towards the ribose, the unnatural L enantiomers of nucleotides being dephosphorylated more slowly. Analogues in which one of the oxygen atoms on the terminal phosphate had been replaced were resistant to degradation. Phosphorothioate analogues in which a sulphur was attached to the penultimate phosphorus were degraded stereoselectively. Methylene isosteres of ATP and ADP resisted degradation, except for homo-ATP which was dephosphorylated at the same rate as ATP. Overall, no correlation was found between the potency of an analogue and its rate of degradation. PMID- 3023104 TI - Chronic steroid contraceptive treatment decreases renal alpha-adrenoceptor levels in the rat. AB - Female Sprague-Dawley rats were injected s.c. with ethynyloestradiol (EE2, 0.2 microgram/day) and levonorgestrel (NG, 2.0 micrograms/day) separately and in combination (EE2/NG). Binding of [3H]rauwolscine (alpha 2-adrenoceptor specific) and [3H]prazosin (alpha 1-adrenoceptor specific) was examined in crude membrane suspensions prepared from whole rat kidney after 3, 6 and 12 weeks of steroid administration. Receptor affinity was high for both ligands (KD, equilibrium dissociation constant [3H]rauwolscine, congruent to 2.0 nM; [3H]prazosin, congruent to 0.2 nM) and was not altered in rats chronically treated with steroid contraceptives. The Bmax (maximum density of binding sites) for [3H]prazosin binding was not altered, indicating no change in the number of renal alpha 1 adrenoceptors. NG administered alone did not affect the numbers of alpha 1- or alpha 2-adrenoceptors. Catechol metabolites of endogenous oestrogens did not displace the binding of either radioligand, suggesting that these metabolites do not directly interact with renal alpha-adrenoceptors. However, after 12 weeks treatment, the number of [3H]rauwolscine binding sites was reduced in both EE2 (Bmax, 133 +/- 7 fmol/mg protein)- and combined EE2/NG (135 +/- 11 fmol/mg protein)-treated rats, compared to controls (162 +/- 9 fmol/mg protein). Since renal alpha 2-adrenoceptors inhibit renin release, this reduction in alpha 2 adrenoceptor number may contribute to increased renin levels associated with oestrogen-induced hypertension. PMID- 3023105 TI - GABA mechanisms of ventromedial thalamic nucleus in morphine-induced muscle rigidity. AB - The aim of the study was to investigate the role of the ventromedial thalamic nucleus in the rigidity induced by morphine. Muscle rigidity was assessed using an electromyographic method (EMG) in non-anaesthesized rats with electrodes implanted unilaterally in the gastrocnemius soleus muscle. Subcutaneous (s.c.) injections of morphine in doses of 5, 10 or 20 mg/kg evoked tonic EMG activity in the gastrocnemius soleus muscle; this was estimated as muscle rigidity. Picrotoxin was injected bilaterally into the ventromedial nucleus in doses of 50 400 ng/0.5 microliter 30 min after morphine administration. Picrotoxin in doses of 200 and 400 ng attenuated the tonic EMG activity induced by morphine, 10 mg/kg s.c. Picrotoxin in a dose of 400 ng reduced the tonic activity induced by morphine, 20 mg/kg s.c. The results suggest that the thalamic ventromedial nucleus mediates the morphine-induced rigidity. PMID- 3023106 TI - A comparison of the effects of doxazosin and alfuzosin with those of urapidil on preganglionic sympathetic nerve activity in anaesthetised cats. AB - Preganglionic sympathetic nerve activity, blood pressure, heart rate and femoral arterial conductance were recorded in anaesthetised, paralysed cats. Urapidil, doxazosin and alfuzosin were infused i.v. for 1 h into different animals at the rate of 2 mg kg-1 h-1. All three drugs caused a fall in thoracic preganglionic sympathetic nerve activity along with blood pressure. Although urapidil had a greater hypotensive action than doxazosin and alfuzosin its sympathoinhibitory action was delayed and weaker. Since all three drugs are alpha 1-adrenoceptor antagonists it appears that antagonism at this receptor type may cause central sympathoinhibition as well as a decrease in total peripheral resistance. However, the different effects of urapidil suggest that its action on central sympathetic tone and therefore its hypotensive action cannot be due entirely to its ability to block alpha 1-adrenoceptors. PMID- 3023108 TI - LANT-6, xenopsin and neuromedin N stimulate cyclic GMP at neurotensin receptors. AB - The naturally occurring analogs of neurotensin-(8-13), xenopsin, [Lys8,Asn9]neurotensin-(8-13) (LANT-6) and neuromedin N stimulated the production of intracellular cyclic GMP in murine neuroblastoma clone N1E-115, an adrenergic neuronal cell type. The order of potency was neurotensin-(8-13) greater than neurotensin greater than xenopsin greater than neuromedin N greater than LANT-6. Furthermore, xenopsin, LANT-6 and neuromedin N each inhibited the specific binding of [3H]neurotensin to intact N1E-115 cells in a dose-related fashion. The order of affinity of the peptides for the neurotensin receptor was neurotensin-(8 13) greater than xenopsin greater than neurotensin greater than neuromedin N greater than LANT-6. PMID- 3023107 TI - Benzodiazepine receptor GABA ratios: regional differences in rat brain and modulation by adrenalectomy. AB - GABA ratios were measured in several brain regions in sham-operated, adrenalectomized and adrenalectomized dexamethasone-injected rats. In sham operated animals, GABA ratios varied 2.5-fold among the regions. In adrenalectomized animals the GABA ratios were increased in the hypothalamus and striatum but decreased in the hippocampus. Dexamethasone injection into adrenalectomized animals reversed the increased GABA ratio in the striatum. These data indicate that glucocorticoids can modulate GABA ratios, possibly by affecting the availability of the individual receptors in the receptor complex. PMID- 3023109 TI - Ca2+-cyclic AMP interaction in chemically skinned smooth muscle. AB - Using guinea-pig taenia coli smooth muscle that is chemically skinned with Triton X-100 to functionally destroy plasmalemma as well as sarcoplasmic reticulum, we demonstrate that cAMP can enhance the extent as well as the rate of relaxation produced by lowering free [Ca2+] in a skinned fiber bundle precontracted with maximal [Ca2+]. However, these actions of cAMP are strongly dependent on the level of free [Ca2+] in the bathing medium, the effect of cAMP being greatly attenuated at higher [Ca2+]. These data support and further extend the results of our previous study indicating that modulations in [Ca2+] can have a strong influence on the ability of cAMP to produce a direct inhibitory effect on the contractile machinery in smooth muscle. PMID- 3023110 TI - Regional distribution of myocardial beta-adrenoceptors in the cat. AB - The purpose of this study was to delineate the distribution of beta-adrenoceptor density in the cat heart, with an emphasis on areas within the left ventricle. beta-Adrenoceptor densities, determined for hearts obtained from five cats, were not significantly different in the left and rights atria, i.e. 47.6 +/- 7.2 and 32.8 +/- 7.5 fmol/mg protein, respectively. beta-Adrenoceptor densities for the septum and right ventricle were 105.4 +/- 15.0 and 65.0 +/- 14.0 fmol/mg protein, respectively. The beta-adrenoceptor density for the proximal distribution of the left anterior descending artery LV1, distal distribution of the left anterior descending artery LV2 and posterior wall of the left ventricle LV3 were: 81.3 +/- 11.5, 145.1 +/- 20.8 and 165.4 +/- 35.8 fmol/mg protein, respectively. Thus, the distribution of the beta-adrenoceptor densities was greatest in the apex of the left ventricle. The data suggest that there are regional differences in the beta adrenoceptor densities among the areas of the heart and within the left ventricle. These differences may be related to functional differences. PMID- 3023111 TI - Interaction between beta 2- and alpha 2-adrenoceptor responses in the vascular system: effect of clenbuterol. AB - Subchronic treatment with the beta 2-adrenoceptor agonist, clenbuterol (0.3 mg X kg-1, twice daily for 14 days), significantly increased the median blood pressure in anaesthetized normotensive rats. The treatment produced a marked reduction in the vasodilator effect of isoproterenol. Acute clenbuterol administration (0.01 mg X kg-1 i.v.) reduced the contractile response induced by the alpha 2 adrenoceptor agonists, guanabenz or B-HT 920, and the alpha 1- and alpha 2 adrenoceptor agonist, clonidine, whereas it did not affect the vasoconstriction by the alpha 1-adrenoceptor agonist, methoxamine, in the pithed rat. Subchronic treatment with clenbuterol attenuated the effect of the beta 2-adrenoceptor agonist on the vascular alpha 2-adrenoceptor responses and enhanced the alpha 1 adrenoceptor response to phenylephrine in pithed rats. The effect of the beta 2 adrenoceptor agonist was reduced by 1- but not d-propranolol. These results suggest that subchronic treatment with clenbuterol produces a subsensitivity of the beta 2-adrenoceptors and reduced the interaction between beta 2- and alpha 2 adrenoceptors at the vascular wall. PMID- 3023112 TI - Changes in rabbit platelet alpha and beta adrenoceptor number and platelet aggregation. AB - Treatment with phenoxybenzamine caused a decrease in the number of alpha but not beta adrenoceptor ligand binding sites on platelets from male rabbits and thus a decrease in the ratio of alpha/beta adrenoceptor number. This was accompanied by decreased aggregation both in the presence and absence of propranolol. In contrast in female rabbits maturation and oestrogen treatment resulted in a decrease in both alpha and beta adrenoceptor ligand binding and no change in the alpha/beta adrenoceptor ratio or in the aggregatory response of the platelets to adrenaline in the absence of propranolol. PMID- 3023114 TI - The potential antidepressant tiflucarbine down-regulates beta-adrenoceptors in rat brain. AB - Subchronic treatment of rats with the potential antidepressant tiflucarbine down regulated the noradrenaline (NA) responses of the cAMP system in cerebral cortex. A concomitant 25% decrease in dihydroalprenolol binding sites in cerebral cortical membranes was observed. The effect was dose-dependent (ED50 = 6 mg/kg), required a 9 days' treatment period, and was reversible 5 days after discontinuation of treatment. Tiflucarbine treatment increased the specific activity of soluble calmodulin (CaM)-dependent phosphodiesterase in rat brain. Tiflucarbine bound to CaM and inhibited its interaction with the phosphodiesterase. Adrenergic denervation by 6-hydroxydopamine (6-OHDA) injection prevented both the beta-adrenoceptor down-regulation and the increase in specific activity of the phosphodiesterase. We suggest that a synergistic interaction between a presynaptic phosphodiesterase inhibition and the NA reuptake blockade was responsible for the down-regulation induced by tiflucarbine. The data are compatible with the reported antidepressant properties of this drug. PMID- 3023113 TI - Alpha-MSH and other ACTH fragments improve cardiovascular function and survival in experimental hemorrhagic shock. AB - Hypovolemic shock was produced in rats by withdrawing about 50% of the estimated total blood volume. Following mean arterial pressure stabilization in the range of 15-25 mm Hg, with a pulse pressure of 7-12 mm Hg, the rats were given intravenous bolus injections either of ACTH fragments or of saline. The following ACTH fragments or analogs were used: ACTH-(4-10), alpha-MSH, ACTH-(1-16), ACTH-(1 17), ACTH-(1-18), [Nle4,D-Phe7]alpha-MSH, [beta-Ala1,Lys17]ACTH-(1-17)-4-amino-n butilamide (alsactide). ACTH-(1-24) and human synthetic ACTH-(1-39) were used for comparison. All animals treated with saline died in 22.51 +/- 3.62 min. Treatment with ACTH fragments (160 micrograms/kg i.v.) increased blood pressure and pulse amplitude, the effect starting within a few minutes, gradually increasing, and reaching a maximum in 15-30 min. The blood and pulse pressure increases were sustained, remaining almost stable until the end of the 2 h recording. Two out of nine rats treated with alsactide, which was the least active, died within 2 h after treatment, while all rats treated with the other ACTH fragments or analogs were still surviving at that time. Both on a weight and on a molar basis, the most active was ACTH-(1-24), followed by ACTH-(1-16), by the alpha-MSH analog [Nle4,D-Phe7]ACTH-(1-13), by ACTH-(1-18) and by ACTH-(1-17). The present results show that melanocortins reverse otherwise fatal hypovolemic shock, and suggest a new therapeutic approach for shock treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3023115 TI - Pharmacology of delta-opioid receptors in the hamster vas deferens. AB - Electrically evoked contractions of the hamster isolated vas deferens are inhibited only by opioid drugs which have agonist activity at delta-opioid receptors. Opioids which are mu-, kappa- or sigma-selective were either inactive or were antagonists. The compound beta-funaltrexamine, which irreversibly blocks mu- and delta-opioid receptors, caused a flattening of the dose-response curve and a reduced maximum inhibition available to delta-opioid agonists. Analysis of the curves by the double-reciprocal null method enabled the affinity of these agonists at delta-opioid receptors to be calculated. PMID- 3023116 TI - Interaction of immunoglobulin-coupled liposomes with rat liver macrophages in vitro. AB - The interaction between liposomes coated with covalently linked rabbit immunoglobulin (RbIg-liposomes), and rat liver macrophages (Kupffer cells) in monolayer culture was studied biochemically with radioactive tracers and morphologically by electron microscopy. The attachment of immunoglobulin (Ig) to liposomes caused a five-fold increase in liposome uptake by the Kupffer cells at 37 degrees C, in comparison with uncoated liposomes. The uptake was linear with time for at least 4 h and linear with liposome concentration up to a lipid concentration of 0.2 mM. At 4 degrees C uptake, probably representing cell surface-bound liposomes, was reduced to a level of approx. 20% of the 37 degrees C values. Involvement of the Fc receptor in the uptake process was indicated by the reduction of RbIg-liposome uptake by more than 75% as a result of preincubating the cells with heat-aggregated human or rabbit Ig at concentrations (less than 2 mg/ml) at which bovine serum albumin (BSA) had virtually no effect on uptake. At high concentrations (10-35 mg/ml), however, albumin also reduced liposome uptake significantly (20-30%), which suggests an interaction of the RbIg liposomes with the Kupffer cells that is partially non-specific. RbIg-liposome uptake was dependent on the amount of RbIg coupled to the liposomes. Maximal uptake values were reached at about 200 micrograms RbIg/mumol liposomal lipid. Electron microscopic observations on cells incubated with horseradish peroxidase containing RbIg-liposomes demonstrated massive accumulation of peroxidase reaction product in intracellular vacuoles, showing that the uptake observed by label association represents true internalization. PMID- 3023117 TI - Expression of the mitochondrial uncoupling protein in brown adipocytes. Absence in brown preadipocytes and BFC-1 cells. Modulation by isoproterenol in adipocytes. AB - The expression of the uncoupling protein has been compared in cells of BFC-1 clonal line established from mouse brown adipose tissue (BAT) and in preadipocytes, as well as in adipocytes from mouse BAT, both in primary culture. The results of immunoblots show that, after one week in culture, adipocytes have a reduced level of the 32 kD protein. This level can be raised 2-3.5-fold by a 24 h exposure to isoproterenol. Thus a direct modulation by a beta-agonist drug in the expression of the uncoupling protein is observed. Under the same conditions as well as under various other conditions, preadipocytes in primary culture and BFC-1 cells do not express the uncoupling protein. At the same time these cells are able both to differentiate into adipose cells, as demonstrated by the emergence of enzyme markers and triglyceride accumulation, and to respond to isoproterenol. Thus isoproterenol is not sufficient to trigger the expression of the uncoupling protein and behaves as a mere modulator once the cells have acquired the capacity to express it. Injection of undifferentiated BFC-1 cells into athymic mice bearing catecholamine-containing mini-osmotic pumps, or co cultures of BFC-1 cells and pheochromocytoma PC-12 cells do not allow BFC-1 cells to express the uncoupling protein. Taken together, the results suggest that the formation of brown preadipocytes is critically linked during development to the release by sympathetic nerves of specific trophic factors acting locally. PMID- 3023118 TI - Control of HL-60 monocytic differentiation. Different pathways and uncoupled expression of differentiation markers. AB - Control of expression of the terminally differentiated phenotype was studied using the human promyelocytic leukemia cell line HL-60. Three known inducers of HL-60 monocytic differentiation were compared: 1,25-dihydroxy vitamin D3, tetradecanoylphorbol acetate (TPA), and sodium butyrate. At concentrations where all three inducers resulted in similar courses of G1/0-specific growth arrest, the kinetics of appearance of certain differentiation markers typically characteristic of mature monocytic cells was determined. The markers were inducible oxidative metabolism, non-specific esterase activity, and the cell surface determinants Mo1, My4, and Mo2. The results indicate that: Regulation of the expression of these markers during induced monocytic differentiation is not controlled in common. The three monocytic inducers do not induce the same metabolic cascade leading to differentiation. Similar states of differentiation could thus be reached by different pathways apparently due to the fact that control of expression of different differentiation markers was not tightly coupled. PMID- 3023119 TI - Partial transformation of mouse fibroblastic and epithelial cell lines with the v myc oncogene. AB - To investigate the role of the myc gene in mammalian cell transformation, plasmid constructs containing the v-myc oncogene and a co-selectable G418 resistance marker were introduced into both mouse fibroblasts (NIH-3T3) and bladder epithelial cells (BBN3 and BBN7). After transfection or microinjection of DNA, no transformed foci could be detected on confluent monolayers but, when the cells were cultured under conditions in which individual cells were allowed to grow and form colonies, morphological transformation was observed. Unlike ras-transformed NIH-3T3 cells, v-myc-transformed cells were unable to grow in serum-free medium and therefore still required exogenous growth factors. v-myc-transformed NIH-3T3 cells were poor at forming foci when co-cultivated with untransformed cells; however, the efficiencies could be increased by addition of EGF to the medium. Both v-myc-transformed fibroblasts and epithelial cells acquired the ability to grow in soft agar, though at efficiencies lower than the corresponding ras transformants. Subcutaneous inoculation of v-myc-transformed NIH-3T3 cells into nude mice resulted in no tumours within 6 weeks. After protracted periods (2-3 months) a few tumours were detected, but at a frequency barely above that for spontaneous tumour formation. Epithelial cells transformed by v-myc were either non-tumorigenic or gave a very low incidence of tumours. We conclude that the v myc oncogene induces morphological changes and anchorage independence in immortal mouse fibroblasts and epithelial cell lines but further events are required for the cells to become tumorigenic. PMID- 3023120 TI - Establishment and characterization of two human cell lines with amplified dihydrofolate reductase genes. AB - Two SV40-transformed human cell lines, GM637, derived from a normal human subject, and GM5849, derived from a patient with ataxia-telangiectasia (A-T), were grown in increasing concentrations of the cytotoxic agent methotrexate (MTX). The GM637 line was naturally more resistant to methotrexate than was GM5849 and, over a 5-month period, became resistant even to very high concentrations (up to 100 microM). The GM5849 line became resistant to 500 nM methotrexate during the same period. However, dot blot and Southern blot analyses showed that both cell lines had amplified their dihydrofolate reductase (dhfr) genes to about the same extent, approx. 50-fold. Using the GM5849 line with amplified dhfr, we attempted to determine if interruption of DNA synthesis by hydroxyurea would cause DNA to be replicated twice within a single cell cycle, as has been reported for Chinese hamster ovary cells. No evidence for such a phenomenon was obtained. PMID- 3023122 TI - Total iridectomy does not alter outflow facility responses to cyclic AMP in cynomolgus monkeys. AB - Total outflow facility was measured by two-level constant pressure anterior chamber perfusion in surgically untouched and totally iridectomized cynomolgus monkey eyes receiving bolus intracameral infusions of cyclic AMP, dibutyryl cyclic AMP, or 5'AMP. Cyclic AMP doses of 50 and 100 micrograms (corresponding to initial intracameral concentrations of 1.5- and 3 mM) increased facility by approximately 20 and 40% respectively in both surgically untouched and aniridic eyes. Higher doses produced no greater effect, and lower doses were ineffective. Dibutyryl cyclic AMP and 5'AMP were ineffective in doses up to 100 micrograms (2- and 3 mM respectively). These findings indicate that the iris plays no mechanical role in mediating the facility response to cyclic AMP, and that the iridectomy procedure per se does not compromise the ability of the outflow pathways to respond to cyclic AMP. Since untouched and aniridic eyes exhibit similar facility responses to epinephrine and norepinephrine, the present findings also suggest that the iris is not the source of cyclic AMP mediating facility responses to catecholamines. PMID- 3023121 TI - Persistent stimulation of lens fiber cell Na,K-ATPase by sodium thiocyanate. AB - The Na,K-ATPase partially purified from porcine lens fiber cells (Sen and Pfeiffer, 1982) is stimulated fourfold (specific activity) by treatment with sodium thiocyanate. The optimum conditions are 1.5 M NaSCN, 2 mg protein ml-1 reaction mixture, pH 7.0, with incubation continued for 30 min at 23 degrees C. Sodium docecyl sulphate-gel electrophoresis and [3H]ouabain binding studies indicate that the extent of purity is not increased significantly by the procedure. The high-activity preparation has elevated phospholipid:protein and phosphatidylethanolamine:sphingomyelin ratios compared with the deoxycholate extracted starting material. The cholesterol:phospholipid ratio and phospholipid acyl group composition are not significantly altered by SCN- treatment. Measurements of 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization show that SNC- treatment produces approximately a 5 degrees C decrease in a membrane phase transition temperature. The phase transition also affects the activation energy of the Na,K-ATPase reaction and probably reflects the onset of the gel to liquid crystalline transition rather than the midpoint location of the transition per se. p-Nitrophenylphosphatase activity and Na,K-ATPase activity in the gel state membrane are also increased by SCN- treatment. Increased specific activity may result, in part, from a membrane fluidity-dependent enzyme activation but is also due, in part, to the expression of latent enzyme activity. Using ouabain-binding data and the specific activity of the activated preparation, it can be shown that the turnover number of the fiber cell enzyme is approximately 1% of that observed in most other tissues. PMID- 3023124 TI - Severe adenovirus pneumonia in an adult civilian. PMID- 3023125 TI - Adenoid cystic carcinoma masquerading as asthma: resection by laser. PMID- 3023123 TI - Maturation-associated changes in the peripheral cytoplasm of human neutrophils: a review. AB - The process of hemopoietic stem cell differentiation and proliferation leads to a large number of precursor cells committed to the neutrophil lineage. These precursor cells undergo a further limited degree of proliferation but, most importantly, undergo a dramatic process of maturation which alters the phenotype of the cell from a myeloblast, which is incapable of normal circulation and function into a segmented neutrophil capable of chemokinesis, chemotaxis, particle ingestion, microbicidal action, and other functions required to subserve the inflammatory process. This review describes the changes in the cell surface and cell cytoplasm that occur during precursor cell maturation and, to the extent possible, correlates molecular and macromolecular changes during maturation with the development of functional capacity. PMID- 3023126 TI - Chemical mediators of anaphylaxis released by alveolar macrophages in bronchial asthma. PMID- 3023127 TI - Discrepancies in release of elastase from human peripheral polymorphonuclear leukocytes. PMID- 3023128 TI - Ten cases of peripheral neuropathy during treatment with almitrine in COPD patients. PMID- 3023129 TI - Proteolytic enzyme activities and onset of muscular dystrophy in the chick. AB - The relationship between proteolytic enzyme activities, soluble protein profiles, and progression of pathology in dystrophic chick muscle was investigated. Activities of cathepsins C and H, and calcium-activated protease were significantly higher in dystrophic patagialis and pectoralis muscles compared with normal muscles prior to the onset of extensive muscle fiber necrosis. Proteolytic enzyme activity of dystrophic muscle remained constant relative to normal muscle during development while muscle pathology progressed in both patagialis and pectoralis muscles. There were more protein bands (60-80 kDa) in the dystrophic muscle extracts compared with normal at all ages studied. Activities of calcium-activated protease in the dystrophic pectoralis and patagialis were similar although muscle pathology progressed much more rapidly in the dystrophic pectoralis. We conclude there is no causal relationship between the activity of the above proteolytic enzymes and the development of muscle fiber necrosis. The elevated activities of proteolytic enzymes in dystrophic muscle may be due to abnormally accelerated growth. PMID- 3023130 TI - Mesocestoides lineatus: trypsin induced development to adult mediated by Ca2+ and protein kinase C. AB - A mode of action of the inducible treatment with trypsin for the development of Mesocestoides lineatus tetrathyridium to adult was analyzed by administering various agents effective on Ca2+-dependent metabolic pathways in the cells: protein kinase C activators such as a synthetic diacylglycerol, 1-oleoyl-2 acetylglycerol, and a tumor promoting phorbol, 12-O-tetra-decanoyl-phorbol-13 acetate, enhanced the trypsin induced developmental processes. On the contrary, a calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide, cyclic adenosine 3',5'-monophosphate, and adenylate cyclase activators such as forskolin and cholera toxin, inhibited the triggering action of trypsin. Furthermore, a combined administration of Ca2+ ionophore (A23187) and the phorbol showed a similar effect with trypsin treatment, and sodium taurocholate acted as a potent enhancer like the activators of protein kinase C. These results strongly suggest that the initiation of development to adult in this cestode may be regulated synergistically by Ca2+ and protein kinase C, and that a bile acid may be involved in an activation mechanism of protein kinase C. PMID- 3023131 TI - Trypanosoma cruzi: differentiation after interaction of epimastigotes and Triatoma infestans intestinal homogenate. AB - Incubation of Trypanosoma cruzi epimastigotes with Triatoma infestans intestinal homogenate leads to differentiation to the metacyclic trypomastigote. Features of this interaction are presented. The morphogenetic mechanism was triggered almost at once; for the minimum interaction period assayed (15 min), the degree of differentiation achieved in Grace medium by Day 6 was 70.0 +/- 9.0%. Longer interaction periods failed to improve differentiation. The morphogenesis became irreversible at 4 hr after interaction. Epimastigotes incubated for 4 hr with T. infestans intestinal homogenate and then washed reached significant differentiation, while those washed before this time failed to do so. Treatment of epimastigotes with albumin improved the experimental conditions thereby hastening morphogenesis, the same percentage of metacyclics occurring in only 4 days. The factors capable of triggering differentiation were adsorbed by T. cruzi epimastigotes, as expected, but also by Leishmania mexicana and, to a lesser degree, by sheep red blood cells. Once the morphogenetic mechanism had been triggered following interaction of epimastigotes with intestinal homogenate for 15 min, metacyclic forms developed when parasites were transferred to Grace but not to other media. Treatment of epimastigotes with trypsin abolished their capacity to differentiate, which was completely reversed following a 5 hr incubation in LIT medium. PMID- 3023132 TI - Plasmodium falciparum: inhibition of dolichol kinase by mefloquine. AB - Dolichol kinase, a rate limiting enzyme for the supply with dolichyl monophosphate as glycosyl carrier lipid, was demonstrated in Plasmodium falciparum. The enzyme was found to be associated with the pellet fraction and to depend on cytidine triphosphate as phosphoryl donor. The apparent Km values of the plasmodial enzyme were determined to be 0.8 mM for cytidine triphosphate and 17 micrograms ml-1 for dolichol. In the presence of 0.5 mM mefloquine, the inhibition of enzyme activity was found to be about 50%. PMID- 3023133 TI - Comparative effects of ouabain, natriuretic factor and ammonium chloride in the toad urinary bladder. AB - Electrical changes and direct effects on Na-K ATPase activity induced by an endogenous digitalis-like natriuretic factor (NF), NH4Cl and ouabain were studied in toad bladders. NF inhibited the SCC and the Na-K ATPase activity in a similar manner to ouabain, but induced a greater increase in calculated direct current resistance (R) (p less than 0.05). NH4Cl was a weak inhibitor of Na-K ATPase activity, although it produced steeper SCC inhibition slopes than those observed with ouabain or NF (p less than 0.01). The data suggested the same mechanism of action of NF and ouabain on the sodium pump, with an additional effect of the former on apical sodium permeability of the cells and/or closure of paracellular routes leading to an increased tissue resistance. In contrast, the effects of NH4Cl were mostly compatible with intracellular inhibition of apical sodium entry into the cell. PMID- 3023134 TI - Epidemiological survey on bacterial, viral and parasitic agents in patients affected by acute enteritis. AB - During the period June 1983-May 1984, faecal specimens from 797 patients with acute enteritis were examined for the presence of bacterial, viral and parasitic agents; 209 (26.2%) enteritic pathogens were identified, of whom 118 (35.4%) in 333 samples from the pediatrics wards. Bacterial agents were detected in 122 (15.3%), viruses in 63 (7.9%) and parasites in 25 (3.1%) of the 797 specimens. LT producing E. coli, Salmonella and Rotavirus were the most frequent pathogens. Bacterial agents occurred most frequently in the summer and autumnal months, whereas viruses showed two peaks, the first one in summer due to cultivable agents, the second in winter to Rotavirus mainly. PMID- 3023135 TI - Sequence-imposed structural constraints in the TonB protein of E. coli. AB - The solution conformation of a 33-residue peptide segment, derived from the TonB protein which is implicated in bacterial membrane transport processes, has been investigated using high-resolution proton magnetic resonance techniques. This proline-rich peptide possesses sequence-imposed sections of elongated secondary structure that must be retained in the native protein configuration. These structural constraints provide elements of stiffness that imply a purely structural role for TonB and are relevant to the subcellular location and biological role of the protein. On the basis of these data we suggest that this protein spans the periplasmic space, linking the inner and outer membrane components of TonB-dependent transport systems. PMID- 3023136 TI - A sequence homology between the pX genes of HTLV-I/II and the murine IL-3 gene. AB - Searching the protein sequence database for amino acid sequences homologous to the x-lor sequence in the pX region of human T-cell leukemia virus types I and II (HTLV-I/II), we found that there is a region of 38 amino acids where the murine interleukin 3 (IL-3) sequence has a 40% homology with the x-lor sequence. A statistical analysis shows that this homology is highly significant with a probability of 1.57 X 10(-10). The biological implication of this homology is discussed. PMID- 3023137 TI - ATP can promote activation and deactivation of the rod cGMP-phosphodiesterase. Kinetic light scattering on intact rod outer segments. AB - The AT (amplified transient) signal is a flash-induced increase of the near infrared light scattering from isolated bovine rod outer segments and is interpreted as a monitor of cGMP-phosphodiesterase activation [(1985) FEBS Lett. 188, 15-20]. We have investigated the effects of ATP and cyclic GMP on this signal. It has been found that ATP enhances the AT signal, the relative effect being the largest for low photoexcitation (approximately 1 rhodopsin per disc membrane). At a high rhodopsin turnover, which saturates the AT amplitude, the effect of ATP is to accelerate the rise of the signal. ATP can also accelerate the falling phase of the signal. This deactivating effect depends on the simultaneous presence of cyclic GMP. The results indicate that ATP acts on the phosphodiesterase activation cycle, promoting activation as well as deactivation, dependent on cGMP as a cofactor. PMID- 3023138 TI - Affinity purification of the opioid receptor in NG 108-15 cells using an avidin biotin system with a novel elution method. AB - Affinity purification of the opioid receptor in NG 108-15 cells was carried out using an affinity resin based on the avidin-biotin interactions, but a new elution method was employed with a radioligand of sub-micromolar concentration. A synthesized biotinyl derivative of leucine-enkephalin has a high affinity for the delta-receptor, but the affinity was lowered 10-fold in the presence of avidin. The new elution method is based on this affinity decrease and resulted in a 100 fold purification over the initial crude materials in the single step. SDS polyacrylamide gel electrophoresis of the purified receptor revealed three polypeptides of 58, 65 and 71 kDa as possible components of the delta-receptor. PMID- 3023139 TI - Human chorionic gonadotropin activates the inositol 1,4,5-trisphosphate-Ca2+ intracellular signalling system in bovine luteal cells. AB - Human chorionic gonadotropin, hCG, a hormone which increases intracellular cAMP, provoked rapid (30 s) and sustained (up to 30 min) increases in the levels of inositol mono-, bis- and trisphosphates (IP, IP2 and IP3, respectively) in bovine luteal cells. LiCl (10 mM) enhanced inositol phosphate accumulation in response to hCG. Concentration-dependent increases in inositol phosphates, cAMP and progesterone accumulation were observed in hCG-treated luteal cells. hCG also induced rapid and concentration-dependent increases in cytosolic free Ca2+ as measured by quin 2 fluorescence. These findings demonstrate that hCG stimulates the phospholipase C-IP3 and diacylglycerol 'second messenger' system in the bovine corpus luteum. PMID- 3023141 TI - Interaction of stigmatellin and DNP-INT with the Rieske iron-sulfur center of the chloroplast cytochrome b6-f complex. AB - Stigmatellin and DNP-INT are effective inhibitors of the catalytic activity of the plastoquinol-plastocyanin oxidoreductase complex (cytochrome b6-f complex). Both inhibitors alter the EPR spectrum of the Rieske iron-sulfur center but do not produce band-shifts of cytochrome b-563. The midpoint redox potential of the Rieske center is unaffected by either inhibitor, although both alter the DBMIB induced g-value shifts of the Rieske center. The results are considered in terms of binding domains for inhibitors in the cytochrome b6-f complex. PMID- 3023140 TI - The glycine-rich loop of adenylate kinase forms a giant anion hole. AB - The conformation of the glycine-rich loop of adenylate kinase is described in detail. It forms a giant anion hole for a sulfate ion, which presumably mimicks a nucleotide phosphoryl group. This loop had been called flexible, because at pH values of 6 or below it is displaced in the crystal. In the region of this loop the adenylate kinases are probably homologous to the p21 proteins. Is is known that a mutation in this loop at residue 12 of p21 causes cell transformation and therefore cancer. Other potentially homologous proteins are indicated. PMID- 3023142 TI - Synovial sarcoma of the head and neck: an account of four cases and review of the literature. AB - Four cases of Malignant Synovioma of the head and neck are reported. It is a rare tumour with only 76 published cases in the world literature. Study of these 76 cases allows identification of the clinical features of this tumour and also highlights the problems of histological diagnosis. The study shows that the most effective treatment is radical primary surgery in association with post-operative radiotherapy and chemotherapy. PMID- 3023143 TI - Mucinous adenocarcinoma of the appendix presenting as an ovarian cystadenocarcinoma: case report and review of appendiceal neoplasms with ovarian metastases. AB - Primary adenocarcinoma of the vermiform appendix is a rare clinical entity that is virtually never diagnosed preoperatively. A case of mucin-producing adenocarcinoma of the appendix manifesting as a pelvic mass is presented. The ultrasonographic finding of a multilocular cystic lesion with thick septa and solid components was consistent with an ovarian cystadenocarcinoma. A review of primary appendiceal neoplasms with ovarian metastases is given. PMID- 3023144 TI - Eponyms in oncology. Sir James Paget (1814-1899). PMID- 3023146 TI - In vitro recovery of human chorionic gonadotropin-stimulated cyclic adenosine 3',5'-monophosphate production in desensitized human granulosa-luteal cells. AB - Human granulosa-luteal cell production of cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone (P) were studied in response to purified human chorionic gonadotropin (hCG) in cultured cells from hyperstimulated follicles of in vitro fertilized patients. The hCG injection given to the patients 36 hours before laparoscopy caused partial desensitization of adenylate cyclase of these cells to gonadotropins. Preincubation of the cells in hormone free medium for 2 to 3 days significantly increased their cAMP responsiveness to hCG. P production was stimulated initially by hCG and showed no desensitization. In the cells preincubated for 72 hours without hCG, a subsequent stimulus of 50 ng/ml of hCG elicited maximal cAMP response, whereas 1 ng/ml of hCG was sufficient to bring about maximal P secretion. Time-course studies indicated that maximal cAMP response to hCG was obtained in 1 to 3 hours. Both basal and hCG-stimulated P accumulation continued to rise for up to 24 hours. Preincubation of granulosa luteal cells from hyperstimulated follicles improves the cells' cAMP responsiveness to hCG, whereas P response remains unaltered. PMID- 3023145 TI - T cell-activating protein on murine lymphocytes. AB - A functional T cell surface molecule, T cell-activating protein (TAP), has been identified on murine lymphocytes. TAP is a protein with an apparent molecular mass of 10-12 kilodaltons (kDa) nonreduced, 16-17 kDa reduced. Cross-linking of TAP can result in profound activation of T lymphocytes to produce lymphokines and to enter the cell cycle. Furthermore, antibody binding to TAP can modulate antigen-driven T cell stimulation. Current data suggest that TAP is physically distinct from the T cell receptor complex. On unstimulated lymphocytes, TAP is expressed on T cells and defines heterogeneity within the major T cell subsets. Its profile of expression is rapidly altered on cell activation. Ontologically, it is first detected in the thymus, where it is found on both the most immature and the most mature cell subsets, and it is functional on both. Together, these TAP+ cells constitute a small fraction of thymocytes. TAP expression, however, defines the immunocompetent compartment of the thymus, and thus could be involved in functional maturation. Finally, the gene controlling TAP expression has been mapped, and is tightly linked to a locus of hematopoietic antigens (Ly-6). TAP is molecularly distinct from these antigens. Furthermore, all of these proteins show a markedly distinct developmental regulation in their cell surface expression. PMID- 3023148 TI - [Correlation between the in vivo dynamics of redox processes and neuronal electrical activity in response to angiotensin II and cyclic AMP]. AB - The biologically active substances induced mostly similar changes of the restorative equivalents level whereas the pattern of equivalents varied in neuronal activity of the mammal brain cortex. The effects of angiotensin II and cAMP on neuronal activity were different. The data obtained suggest that the angiotensin II effect on neurons is actualized both through the cAMP system and through other molecular mechanisms. PMID- 3023147 TI - Normal ovarian function in a mild form of late-onset 3 beta-hydroxysteroid dehydrogenase deficiency. AB - Late-onset 3 beta-hydroxysteroid dehydrogenase (HSD) deficiency was diagnosed in a 30-year-old woman with hirsutism and normal menstrual cycles. No genital abnormalities were present. Elevated basal serum levels of delta 5-3 beta hydroxysteroids were demonstrated. Serum pregnenolone (P5) was 3.0 ng/ml, and dehydroepiandrosterone sulphate (DHEA-S) 3245 ng/ml. Basal serum levels of delta 4 steroids were low or within normal limits. Serum progesterone (P) was 0.5 ng/ml, 17 alpha-hydroxyprogesterone (17-OHP) 0.2 ng/ml, androstenedione (delta 4A) 0.4 ng/ml, and testosterone (T) 0.1 ng/ml. All delta 5/delta 4 steroid ratios were elevated. Dexamethasone (DEX) administration normalized the elevated levels of delta 5-3 beta-hydroxysteroids, whereas delta 4-3-ketometabolites exhibited only minor modifications. The DHEA-S/delta 4A ratio increased more than five times over the basal ratio, and P5/P and 17 alpha-hydroxypregnenolone (17 OHP5)/17-OHP ratios did not increase after adrenocorticotrophic hormone ACTH stimulation. Studies of basal ovarian function revealed 17 beta-estradiol (E2) and gonadotropins within normal limits according to the menstrual cycle. In the follicular phase, follicle-stimulating hormone (FSH) was 101.3 ng/ml, luteinizing hormone (LH) 46.0 ng/ml, and E2 49.7 pg/ml; in the luteal phase, FSH was 180.0 ng/ml, LH 69.3 ng/ml, and E2 50.1 pg/ml. The presence of ovulatory cycles was documented on the basis of the biphasic pattern of the basal body temperature cycles and the increment in P levels. This case demonstrates the existence of normal ovulatory function in a woman with late-onset of a mild form of HSD. PMID- 3023150 TI - [Role of sympathetic vasoconstrictors in providing for the aerobic capacity of skeletal muscle]. AB - The role of sympathetic vasoconstrictors in the mechanisms controlling the redistribution of blood flow to active muscle fibers in forearm muscles during and after handgrip with 10% MVC, was studied. Functional desympathization (blockade of the stellate ganglion and alpha 1-receptors with prazosin) led to shunting of active muscle fibers, subsequent activation of anaerobic glycolysis and decrease of aerobic working capacity. Increased concentration of lactate in the interstitial space of resting muscles causes analogous disturbances of blood flow redistribution and aerobic working capacity during contraction of these muscles. PMID- 3023149 TI - [Effect of beta-adrenoreceptor blockade on the filtration-absorption function of the vascular bed of the skeletal musculature of the cat]. AB - Perfusion of the cat hindlimb with alternating stabilization of pressure and blood flow in response to electrical stimulation of sympathetic fibers prior to and after blockade of beta-adrenoreceptors decreased considerably changes of the capillary filtration coefficient and the capillary hydrostatic pressure, improving changes of the filtration-absorption balance induced with the stimulation. beta-Adrenoreceptors of vascular bed are shown to take part in neurogenic regulation of both the exchange and the capacitance functions of the vascular bed, as well as its resistance function. Activation of sympathetic fibers seems to enhance permeability of the vascular wall. PMID- 3023151 TI - [Studies on hypoaldosteronism associated with diabetes mellitus: response of plasma steroids to angiotensin II or ACTH administration]. AB - This study was designed to clarify the cause of hyporeninemic hypoaldosteronism (HH) associated with diabetes mellitus. Fourty diabetic patients (DP) were divided into 3 groups; 12 patients without neuropathy or nephropathy, 13 with neuropathy and without nephropathy, and 15 with neuropathy and nephropathy; in the third group 3 HH patients were included. Fourteen normal subjects served as controls. In all DP and the normal subjects, plasma concentration of aldosterone (PAld), 18-hydroxycorticosterone (P18-OH-B), corticosterone (PB), 11 deoxycorticosterone (PDOC) and cortisol (PF) were measured before and after intravenous administration of angiotensin II (AII, 10 ng/kg/min, for 30 min) or ACTH (1-24 ACTH, 0.25 mg). In 27 DP, plasma renin activity (PRA) increased from 0.8 +/- 0.6 SD to 2.4 +/- 2.2 ng/ml/h (normal: 1.2 +/- 0.7 to 3.2 +/- 1.7 ng/ml/h) 2 hours after intravenous administration of furosemide (1 mg/kg) and assumption of the upright posture. No significant difference in PRA was found between DP and controls, whereas the response to these stimuli decreased significantly in 9 DP with neuropathy and nephropathy (from 0.4 +/- 0.2 to 0.7 +/ 0.4 ng/ml/h). The major results were as follows: 1) The mean of PAld (5.9 +/- 2.4 ng/100 ml) in DP was significantly lower than that in controls (7.7 +/- 2.2 ng/100 ml). There was no significant difference of PAld between DP without complications and controls. PAld in DP with neuropathy alone (5.0 +/- 1.5 ng/100 ml, p less than 0.05) and that in DP with neuropathy and nephropathy (4.7 +/- 2.4 ng/100 ml, p less than 0.05) were lower than that in controls. The mean of P18-OH B (12.3 +/- 4.3 ng/100 ml) in DP was similar to that (14.2 +/- 3.2 ng/100 ml) in controls. P18-OH-B in DP without complications and that in DP with neuropathy alone were similar to that in controls. However, P18-OH-B (8.8 +/- 3.2 ng/100 ml) in DP with neuropathy and nephropathy was significantly lower than that in controls (p less than 0.001). No difference in PB, PDOC or PF was observed between DP and controls. 2) PAld increased from 6.0 +/- 2.5 ng/100 ml in DP (p less than 0.001) and from 7.6 +/- 2.2 to 15.7 +/- 5.3 ng/100 ml in controls (p less than 0.001) 30 min after A II infusion.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3023152 TI - [Pseudo-Bartter's syndrome induced by surreptitious ingestion of furosemide to lose weight: a case report and possible pathophysiology]. AB - Bartter's syndrome (B.S) is often difficult to distinguish from pseudo-Bartter's syndrome (pseudo-B.S), a condition which may be caused by "loop" diuretics abuse. Although our patient firmly denied ingestion of diuretics or laxatives, all screening of urine samples gave consistently positive results for furosemide, even during hospitalization. A 25-year-old married woman with persistent hypokalemia had many characteristics of B.S, including hypokalemic hypochloremic alkalosis, hyperactivity of the renin-angiotensin-aldosterone system, normotension, insensitivity to the pressor effect of angiotensin II (A.II), increased urinary excretion of prostaglandin E2 and kallikrein, and marked reduction of distal fractional reabsorption of chloride in Henle's loop, as estimated by CH2O/CH2O+C.Cl, under conditions of hypotonic saline diuresis. Furthermore, hypotension occurred with [1-Sar, 8-Ile] A.II and with the angiotensin converting enzyme inhibitor (Captopril). Renal biopsy revealed juxtaglomerular hyperplasia. A tentative diagnosis of B.S was made. Indomethacin (IDM), an inhibitor of prostaglandin synthesis was prescribed and the pressor response to A.II improved. Impaired fractional chloride reabsorption was also improved significantly under IDM, but the value was low compared to the normal. The value for basal plasma human atrial natriuretic polypeptide (hANP) was slightly above the normal ranges and was suppressed by IDM. We conclude that manifestations B.S in this patient may have been fostered significantly by the long-term surreptitious use of furosemide, taken to lose weight. The analysis of urine for detection of diuretics was only finding distinguishing her clinical state from "true" B.S and leading to a final diagnosis. Pathophysiologic relationships between B.S and pseudo-B.S due to furosemide are discussed. PMID- 3023153 TI - Clinical effectiveness of hydrogen peroxide-sodium bicarbonate paste on periodontitis treated with and without scaling and root planing. PMID- 3023155 TI - Itraconazole pharmacokinetics in the female genital tract: plasma and tissue levels in patients undergoing hysterectomy after a single dose of 200 mg itraconazole. AB - Twenty patients who underwent hysterectomy received a single dose of 200 mg itraconazole at different moments before surgery. At the moment of surgery, a urine sample, blood sample and tissue samples of different organs of the female genital tract were collected. Blood levels and tissue levels of itraconazole were measured by means of an HPLC method. Itraconazole blood levels were lower than the corresponding tissue levels in the various organs of the female genital tract. This finding for itraconazole is different from findings with ketoconazole, indicating that itraconazole has a higher affinity for tissue than ketoconazole. Urine levels of itraconazole were virtually undetectable in all samples. None of the patients complained of side-effects, and blood biochemical parameters all remained within the normal limits. The premedication with itraconazole had no effect whatsoever on the induction and maintenance of and recovery from the anaesthesia. No abnormal effects on ECG were observed. PMID- 3023154 TI - Blood pressure regulation in third-trimester pregnant women receiving tocolytic terbutaline infusion. AB - Terbutaline (20 micrograms/min) was infused during 30 min in 17 women in whom a manual external manipulation of a breech presentation was going to be attempted. A significant increase in systolic (P = 0.003) and a decrease in diastolic blood pressure (P = 0.04) was noted at the end of the infusion but no change in mean arterial blood pressure was obtained. At the same time aldosterone serum levels had dropped significantly (P = 0.009) and plasma angiotensin II showed a marked increase (P less than 0.001) which continued during the next 30 min. All changes were normalized after the infusion. The angiotensin-converting enzyme activity remained unchanged, as did vasopressin plasma levels. The combined results of terbutaline provocation have been interpreted to mean that blood pressure regulation in third-trimester pregnant women is similar to that in nonpregnant individuals. The increase in dehydroepiandrosterone sulphate (P less than 0.05) noted at the end of infusion was suggested to be related to the blood pressure changes and was unrelated to fluctuations in serum cortisol. The latter steroid increased between 30 and 60 min, e.g. during the manual external manipulation, and was interpreted as being due to maternal stress. PMID- 3023156 TI - Characterization of the nuclear progesterone receptor in human uterine leiomyoma. AB - Progesterone binding components were characterized and measured in the 0.4 M KCl nuclear extract of Premarin-primed human leiomyoma (LM) following progesterone injection (n = 5) using [3H]R5020 as the ligand. The receptor character of the binding was demonstrated by: high specificity for progesterone and R5020 binding, high saturable affinity (Kd approximately 2.5 nM) for R5020, sedimentation coefficient in high salt concentration (approximately 3 S), change of distribution of the receptor after progesterone administration, i.e. an increase in the nuclear and decrease in the cytosolic receptor levels. Similar results were demonstrated in the corresponding myometria and endometria. The characteristics of the nuclear progesterone receptor in all three tissues of the test group were similar to those of the cytosolic progesterone receptor in the test group and in the control group (no treatment before operation, n = 5). These results indicate that the nuclear progesterone receptor in human uterine LM shares similar characteristics to those of the myometrium and endometrium. PMID- 3023157 TI - Collagen synthesis in growing human skin fibroblasts. AB - Collagen and noncollagen protein synthesis in cultured human skin fibroblasts was studied in relation to different growth phases. In order to quantify collagen synthesis, we determined the release of incorporated radioactivity using purified bacterial collagenase. Collagen as well as noncollagen protein synthesis markedly decreased during fibroblast growth. On the other hand, we found a 3-fold increase in relative collagen synthesis (i.e. collagen synthesis compared to total protein synthesis) comparing cells in the log growth phase with cells in the stationary growth phase. PMID- 3023158 TI - Cellular growth modulation. I. Effects of extracellular matrix and tumor cell conditioned medium. AB - Earlier studies reported the enzymatic modulation of the cell surface in malignant transformation of human normal mammary epithelial cells and in conversion of mammary carcinoma. Carcinoembryonic antigen (CEA) is a neoplasm associated antigen, its production and release is used to monitor changes in cell phenotype. The present study shows that CEA production and release by human colon carcinoma (CCC), and by colon cells from patients with familial polyposia coli (FPC) and ulcerative colitis (UCC) is inhibited when the cells are cultured in contact with confluent normal colon epithelial (HNCEC) cell monolayer. Footprints left behind and/or conditioned media from HNCEC cells inhibited, whereas footprints left behind and/or conditioned media from CCC, FPC or Ucc enhanced CEA release. During sequential passages of HNCEC cells grown on footprints and/or in spent media from CCC cultures, HNCEC cells acquire the ability to produce and release CEA, and to develop tumors in athymic Nu/Nu mice. On the other hand, during sequential passages, CCC, FPC or UCC grown in spent media, or on footprints left behind HNCEC cells, showed significant decrease in CEA production and release, and in oncologic ability in athymic mice. It is concluded that both the extracellular matrix, and a growth-regulating factor(s) in the spent medium modulate cellular transformation. Quantitative data on CEA-release indicate that FPC and UCC represent an intermediary stage between normal colon epithelial cells and colon carcinoma cells, i.e. a preneoplastic stage. PMID- 3023160 TI - Retinol transport proteins. PMID- 3023159 TI - A high-affinity monoclonal antibody (GIF-1) to human gamma-interferon: neutralization of interferon mediated inhibition of retrovirus production and 2' 5' (A) synthetase induction. AB - An hybridoma clone secreting an IgG1 monoclonal antibody (GIF-1) specific for human gamma-interferon (HuIFN-gamma) has been generated using HAT medium supplemented with insulin (HIAT) at the initial stage of cell fusion. This antibody is capable of neutralizing the antiviral activity of HuIFN-gamma, the ability of HuIFN-gamma to inhibit retroviral replication in RD-114 cells, and the ability of HuIFN-gamma to induce the 2'-5' oligoadenylate (A) synthetase in RD 114 and HeLa cells. Eluate from an immunoaffinity column containing GIF-1 yielded two protein bands of molecular weight of 20 and 25 kd when subjected to SDS-PAGE. PMID- 3023161 TI - Extensive sequence homology of the goldfish ras gene to mammalian ras genes. AB - We cloned ras-related sequences from goldfish genomic libraries constructed as recombinants using the lambda phage. Restriction enzyme mapping of the clones obtained revealed three kinds of ras-related sequences among approximately 350,000 genomic clones. One of these clones was partially sequenced. Comparison with the nucleotide sequences of mammalian ras genes showed that the determined sequences covered the predicted amino acid coding regions and parts of the intervening regions. The predicted amino acid sequences of the cloned ras-related goldfish gene suggested that the coding region is localized separately in DNA, and that its exon-intron boundaries are exactly the same as those of corresponding mammalian genes. The nucleotide and amino acid sequences of the goldfish ras-related gene may have extensive homologies to mammalian p 21 protein. Among the three mammalian ras proteins, the predicted amino acid sequence of the sequenced ras-related goldfish clone is most closely homologous (96%) to the Kirsten ras protein. Differences in the predicted amino acid sequence were greatest in the sequence predicted from the fourth exon; fewer differences were found in the sequence from the third exon, and only slight or no differences were found in the sequence predicted for the first and second exons. The 12th and 61st amino acids from the N-terminal of the protein, which are thought to be critical positions for GTP binding and catalysis, are both conserved in the goldfish protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3023162 TI - Immortalization of early embryonic cell derivatives after the transfer of the early region of simian virus 40 into F9 teratocarcinoma cells. AB - F9 embryonal carcinoma (EC) cells were transformed using a recombinant plasmid carrying simian virus 40 (SV40) early genes linked to the promoter-enhancer region of the early transcription unit 1A (E1A) of adenovirus type 5. One clone of transformed cells, F9-K4B2, contained several integrated copies of the plasmid and expressed SV40 early genes (T antigens) only when it was induced to differentiate in vitro by the addition of retinoic acid and dibutyryl-cAMP. From the cell population positive for T antigen, it is possible to isolate permanent cell lines with high efficiency. Most of these cells exhibit stable traits that are characteristic of parietal endoderm. We also obtained another cell type which did not express the specific markers of either undifferentiated EC cells or endoderm cells; this type might represent a different stage of commitment in the differentiation of F9 cells. All clones are tumorigenic when injected into irradiated syngeneic mice, and they maintain their phenotype after successive passages in vivo as well as in vitro. The introduction of SV40 early genes into other EC cell lines and early mouse embryos appears to be a promising approach for the immortalization of early embryonic cells. PMID- 3023164 TI - Granular cell tumor of the esophagus. AB - A case of granular cell tumor of the esophagus in a 50-year-old man is reported. Gastrointestinal endoscopy revealed a round, sessile, non-ulcerated white-yellow elevated tumor at the lower third of the esophagus. Biopsy revealed a granular cell tumor. Immunohistochemical staining demonstrated that granules in the cytoplasm of tumor cells were positive for S-100 protein and negative for carcinoembryonic antigen. An electron microscopic study revealed that tumor cells were closely packed in clusters, surrounded by basal lamina and collagen fibers. Most cells contained dark cytoplasm filled with electron-dense granules. These granules resembled lysosomes and phagosomes. In a few cells with clear cytoplasm, some mitochondria and poorly developed endoplasmic reticulums were seen. Fibrillar internal materials, myelin-like figures and a premature angulate body were observed in the clear cytoplasm. The lesion has remained unchanged in gross appearance and in size for twenty-three months without any treatment. PMID- 3023165 TI - Polypoid synovial sarcoma of the esophagus. AB - Pure sarcomas of the esophagus are exceedingly rare. We report a case of esophageal synovial sarcoma which occurred in an adolescent. The tumor was locally resected, sparing the patient esophagectomy. After postoperative radiation therapy, the patient remains alive and well without evidence of disease 28 mo after operation. The unique nature of polypoid sarcoma of the esophagus, and the potential for cure without esophagectomy, is discussed. PMID- 3023163 TI - Cyclic AMP in gastric juice does not reflect histamine H2 receptor activity in Heidenhain pouch dog. AB - It is strongly believed that cAMP mediates histamine H2 receptor activity, but does not mediate gastrin and acetylcholine stimulation of gastric acid secretion. Therefore, cAMP production could be a marker of H2 receptor activity. Whether endogenous histamine mediates gastrin and/or acetylcholine stimulation, at least partially, remains to be elucidated. If cAMP in the gastric juice reflects H2 receptor activity, we can investigate whether endogenous histamine mediates gastrin and/or acetylcholine stimulation in vivo. In this study, we investigated whether cAMP in the gastric juice reflected histamine H2 receptor activity in the Heidenhain pouch dog in vivo using different kinds of inhibitors of gastric secretion. Our hypothesis was as follows: Upon betazole stimulation, cimetidine, an H2 receptor antagonist, should decrease cAMP output into the gastric juice, but omeprazole, an H+, K+-ATPase blocker, should not, because it blocks at a site more peripheral than the H2 receptor and the production of cAMP. Sixty minutes after betazole administration, 4.0 mumol/kg of cimetidine and 0.18 mumol/kg omeprazole were administered intravenously and they inhibited gastric juice volume to a similar degree, that is, 49.6% and 52.1%, respectively. However, omeprazole caused a greater decrease in cAMP output than cimetidine. Inhibition with 4 mumol/kg/h of cimetidine or 0.2 mumol/kg of omeprazole from the beginning of betazole stimulation also caused similar decreases in gastric juice volume, 66.6% and 60.6%, respectively. Both inhibitors decreased cAMP output into the gastric juice in a similar fashion in the first two 30 minute periods. These results do not agree with our hypothesis.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3023166 TI - Amiodarone-induced lysosomal inclusions in primary cultures of human hepatocytes. PMID- 3023167 TI - In vivo production of leukotriene B4 and leukotriene C4 in rabbit colitis. Relationship to inflammation. AB - Leukotriene B4, a proinflammatory compound, recently has been identified as the major metabolite of arachidonic acid in tissue incubations of human and animal colitis. To determine the relationship of inflammation to the in vivo production of leukotrienes, rabbit colitis was induced by formalin enema followed by intravenous infusion of immune complexes, and serial samples were collected by rectal dialysis. Leukotrienes B4 and C4 were measured by radioimmunoassay after high-pressure liquid chromatography. Prostaglandin E2 was assayed after Sephadex chromatography. Leukotrienes were not detected in control animals. Eicosanoid production progressively increased during development of inflammation and correlated with severity of inflammatory cell infiltration (p less than 0.01). Methylprednisolone decreased prostaglandin E2 but did not significantly reduce leukotrienes or inflammation. These data demonstrate that in vivo production of leukotrienes B4 and C4 correlates with indices of inflammation, consistent with the concept that these eicosanoids contribute to the inflammation of colitis. PMID- 3023168 TI - Simplified assessment of segmental colonic transit. AB - Transit times of radiopaque markers through the human gut were measured by published techniques and compared with a simplified method. Three sets of distinctive markers were ingested by 24 healthy persons on 3 successive days. In the first part of the study, daily abdominal x-rays were taken and individual stools were collected for radiography. Mouth-to-anus transits were assessed from the fecal output of markers and mean colonic and segmental colonic transits were calculated from the daily radiographs. These established methods were then compared with estimates of total colonic and segmental transits based on a single abdominal film, taken on the fourth day. The single-film technique correlated well with values obtained from the previous, but more inconvenient, methods. Using the simpler approach, colonic transit was assessed in 49 additional healthy subjects, for a total group of 73. Total colonic transit was 35.0 +/- 2.1 h (mean +/- SE); segmental transits was 11.3 +/- 1.1 h for the right colon, 11.4 +/- 1.4 h for the left colon, and 12.4 +/- 1.1 h for the rectosigmoid. Men had significantly shorter transits for the whole colon than did women (p less than 0.05), and this difference was apparent to some extent in the right (p = 0.06) and left colon (p = 0.07) but not in the rectosigmoid. Age did not influence transit significantly nor did a small dose of supplemental fiber. The technique is simple, convenient for clinical usage, and reduces the exposure to radiation to acceptable levels. There should be a role for this approach in the evaluation of colonic transit in selected patients. PMID- 3023170 TI - DIDS, an anion transport blocker, modulates ivermectin-induced enhancement of benzodiazepine receptor binding in rat brain. AB - Ivermectin enhancement of [3H]diazepam binding was abolished by pretreatment of membranes with 0.05% Triton X-100, whereas GABA-induced enhancement was not changed after this treatment. Ivermectin enhancement was neither affected by picrotoxinin nor dependent on the chloride ion. 4,4'-Diisothiocyano-2,2' disulfonic acid stilbene (DIDS) dose-dependently reduced [3H]diazepam binding enhanced by 10(-6) M ivermectin, without affecting the basal specific binding. The effects of DIDS were derived from reduction of increased binding affinity of benzodiazepine receptors by ivermectin, but were not dependent on chloride ion in the assay medium. DIDS inhibited GABA- and pentobarbital- but not chloride ion induced enhancement of [3H]diazepam binding. PMID- 3023169 TI - Adherence to and penetration of the intestinal epithelium by reovirus type 1 in neonatal mice. AB - In 10-day-old suckling and adult mice, reovirus type 1 adheres selectively to and penetrates membranous epithelial (M) cells. To determine when M cells first appear, when they first transport reovirus, and if reovirus adheres to and is endocytosed by other epithelial cells in the first postnatal week, we examined neonatal mouse intestine by transmission electron microscopy after reovirus type 1 exposure. At 2 days M cells accounted for 0.9% of dome epithelial cells. By 9 days M cells had increased to 7.4%. Reovirus type 1 adherence to the surface of villus and dome epithelial cells showed marked variation in 2-6-day-old animals, but by 7 days only a few absorptive cell profiles had adherent reovirus. Adherence to greater than 50% of M-cell profiles occurred in all but 2 animals, but adherence to the majority of Peyer's patch absorptive cell profiles was present only in some 4- and 5-day-old animals. Adherence to a majority of undifferentiated cell profiles occurred in some animals at all ages. Membranous epithelial cells endocytosed reovirus at all ages but only at 2 days did rare villus and dome absorptive cells endocytose reovirus into the apical cytoplasm. Thus, adherence of reovirus to the apical surface of mucosal epithelial cells is nonselective in newborn mice but becomes more selective within the first postnatal week with adherence by day 7 to most M-cell profiles, to a substantial but variable number of undifferentiated cell profiles, but to few absorptive cell profiles. PMID- 3023171 TI - Ethylcholine mustard aziridinium ion (AF64A) impairs cholinergic neuromuscular transmission in the guinea-pig ileum and urinary bladder, and cholinergic neuromodulation in the enteric nervous system of the guinea-pig distal colon. AB - AF64A was examined for its ability to impair cholinergic neurotransmission in the autonomic nervous system. In the guinea-pig ileum AF64A impaired cholinergic neuromuscular transmission. In the guinea-pig urinary bladder AF64A selectivity impaired the cholinergic but not the purinergic component of neuromuscular transmission. In the guinea-pig colon circular muscle AF64A impaired cholinergic neuromodulation of the inhibitory transmission but not the inhibitory transmission itself. The nature of impairment of cholinergic transmission by AF64A is discussed. PMID- 3023172 TI - Sotalol potentiates the leukotriene C4-induced contractions and thromboxane A2 release of guinea pig lung parenchymal strips. AB - The leukotriene C4 (LTC4)-induced contraction and thromboxane A2 (TxA2) release of the guinea pig lung parenchymal (GPLP) strip are both inhibited by the beta 2 adrenergic agent salbutamol. The effect of LTC4 is restored to nearly normal by the beta-adrenergic antagonist sotalol. The latter substance alone also induces a contraction in the GPLP strip and potentiates the contractions and the TxA2 release of LTC4. During the sotalol-induced contractions, no TxA2 release occurs. An antihistaminic, mepyramine had no effect on the sotalol-induced contraction. When sotalol is added repeatedly to a GPLP strip, only the first time a contraction occurs. PMID- 3023173 TI - Increase of alpha 2-presynaptic adrenoceptor response after neuronal uptake inhibition in vivo. AB - alpha 2-adrenoceptor antagonists, yohimbine or idazoxan (1 mg kg-1 i.p.), administered alone, did not change noradrenaline content in the central and peripheral tissues of the rat (hypothalamus, brain stem, frontal cortex and heart). The inhibition of neuronal uptake by desipramine (DMI) administered alone or prior to alpha 2-adrenoceptor antagonists did not affect the neurotransmitter content either. alpha-methyl-p-tyrosine (alpha-MT) 6 hr before sacrifice, induced a marked disappearance of the noradrenaline content, greater in central nervous tissues than in heart. When the catecholamine synthesis was inhibited by alpha MT, neither alpha 2-adrenoceptor antagonists nor DMI at the dose used, significantly changed the disappearance rate of noradrenaline in any of the tissues studied. Under these experimental conditions, however, the combination of DMI plus alpha 2-adrenoceptor antagonists significantly decreased the neurotransmitter content in all tissues when the values were compared with the control or DMI-treated groups. The present results might suggest evidence in favour of a functional coupling between presynaptic alpha 2-autoreceptors and noradrenaline uptake mechanism. PMID- 3023175 TI - Circulating forms of alpha MSH in the carp and trout blood: an HPLC and RIA study. AB - The three forms of MSH(desacetyl-, monoacetyl-, and diacetyl) circulate in the carp and trout blood. Their proportions there are as follows: in the carp, the desacetylated form is 2.5% of total MSH and the monoacetylated form 72% of the acetylated forms of alpha MSH. In the trout, similar proportions were found: desacetyl alpha MSH was 5% of total alpha MSH and monoacetyl-, 70% of the acetylated MSHs. PMID- 3023174 TI - Effects of antidepressants on receptor-activated and Ca2+-activated contractions of rabbit isolated aorta. AB - Helically-cut strips of rabbit aorta were used to examine the inhibitory effects of some antidepressants on the contractile responses produced by norepinephrine (NE), serotonin (5-HT) or K+. The order of potency of antidepressants in antagonizing 5-HT-induced responses was: mianserin greater than amitriptyline greater than imipramine (IMI) greater than desipramine greater than or equal to nomifensine. With NE as agonist, the order of potency was: amitriptyline greater than mianserin greater than IMI greater than desipramine greater than or equal to nomifensine. Viloxazine (10(-5) M) was ineffective on 5-HT- or NE-induced contractions. For K+-induced contraction (which is mediated by Ca2+ entry into smooth muscle cells) tricyclic antidepressants were about equipotent with mianserin; nomifensine and viloxazine were relatively ineffective. These results indicate that certain antidepressants have multiple effects on arterial smooth muscle. Such effects could underlie not only the side-effects, but also the therapeutic actions of these agents. PMID- 3023176 TI - Interrenal function in larval Ambystoma tigrinum. II. Control of aldosterone secretion and electrolyte balance by ACTH. AB - Renal clearance techniques were used to assess the role of ACTH on renal electrolyte transport in larval Ambystoma tigrinum. Radioimmunoassay was employed to evaluate changes in circulating aldosterone in these animals. Larvae were hypophysectomized and maintained for 1 week on either ACTH replacement therapy (50 ng/g) or sham injections prior to clearance measurements. Hypophysectomy significantly lowered plasma [Na+] (from 96 to 90 mM), plasma [K+] (from 6 to 4 mM), plasma aldosterone titer (from 157 to 36 pg/ml), fractional Na+ reabsorption (from 97 to 94%), and fractional K+ reabsorption (from 68 to 50%). ACTH replacement restored plasma [Na+] to 96 mM, aldosterone titer to 157 pg/ml, fractional Na+ reabsorption to 96%, and fractional K+ reabsorption to 75%. When steroid synthesis was blocked in a separate set of larvae; ACTH was unable to reverse the sodium depletion which results from adaptation to distilled water. This suggests that ACTH is not acting directly on Na+ transport but acts through a steroid like aldosterone. When larvae were injected intravenously with antialdosterone antibodies their fractional Na+ reabsorption decreased from 95 to 87%. We conclude, therefore, the ACTH works via interrenal steroids, such as aldosterone, to control renal electrolyte transport in this species. PMID- 3023177 TI - Branchial Na+ -K+ -ATPase activity in freshwater or saltwater acclimated tilapia, Oreochromis (Sarotherodon) mossambicus: effects of cortisol and thyroxine. AB - The ability of cortisol to stimulate branchial Na+ -K+ -ATPase activity in tilapia was further augmented by thyroxine. The effects of hormonal treatments were greater in the saltwater acclimated fish than in the freshwater acclimated ones. PMID- 3023178 TI - Elevation of plasma aldosterone levels of tadpoles at metamorphic climax. AB - Plasma aldosterone concentrations in Rana catesbeiana tadpoles during metamorphosis were determined by radioimmunoassay. Aldosterone levels were relatively low (0.7-1.4 ng/ml) during pre- and prometamorphosis. At the onset of climax, aldosterone concentration increased markedly and reached a peak (10.9 ng/ml) at stage XXII. Subsequently, aldosterone levels declined and at the end of metamorphosis (stage XXV), the average concentration was 3.9 ng/ml plasma. Administration of ACTH elevated plasma aldosterone levels in hypophysectomized tadpoles. When hypophysectomized animals were kept in thyroxine (T4), plasma aldosterone concentrations also increased. Combined ACTH and T4 treatment caused a marked increase in plasma aldosterone concentration. The possible involvement of thyroid hormone in the elevation of plasma aldosterone during metamorphosis is discussed. PMID- 3023179 TI - Cortisol secretion in vitro by the interrenal of coho salmon (Oncorhynchus kisutch) during smoltification: relationship with plasma thyroxine and plasma cortisol. AB - An in vitro system for the incubation of interrenal tissue (head kidney fragments) from coho salmon, Oncorhynchus kisutch, was developed in order to examine changes in interrenal sensitivity to ACTH1-24 during smoltification, using cortisol secretion as the endpoint. Time-course studies indicated that maximal cortisol accumulation in incubation media was achieved after 3 hr exposure to ACTH. There was no correlation between head kidney weight, body weight, or sex and the response of the interrenal to ACTH1-24 in vitro. Approximately monthly or bi-weekly experiments were performed during the smoltification period (February-July): tissue was preincubated in hormone-free media for 3 hr, washed twice, and then challenged with 5 X 10(-10)-5 X 10(-7) M (1.5-1500 ng/ml) ACTH1-24 for 3 hr. The pattern of cortisol secretion was similar in February, early March, and late March in the dose range of 5 X 10(-10)-5 X 10( 8) M ACTH1-24. A marked, significant increase in sensitivity to ACTH and in the steroidogenic capacity of the tissue occurred in April, but by May the response was similar to that in the pre-April period. Enhanced sensitivity and steroidogenic capacity were found in interrenal tissue taken from coho salmon in June and July. Maximal in vitro responsiveness of interrenal tissue to ACTH in April was correlated with peak plasma thyroxine titers and enhanced hypoosmoregulatory ability, but not with peak plasma cortisol titers, which occurred in May. PMID- 3023180 TI - Adenylate cyclase activators and inhibitors, cyclic nucleotide analogs, and phosphatidylinositol: effects on interrenal function of coho salmon (Oncorhynchus kisutch) in vitro. AB - The role of cyclic AMP (cAMP) as mediator of ACTH action on interrenal steroidogenesis was evaluated in juvenile coho salmon (Oncorhynchus kisutch). Head kidneys (containing the interrenal cells) were incubated in the absence or presence of putative adenylate cyclase activators (forskolin and cholera toxin), ACTH combined with putative adenylate cyclase inhibitors (hydrolysis-resistant ATP analogs), dibutyryl cyclic (dbc) AMP, dbcGMP, or phosphatidylinositol. The cortisol content of the incubation medium was subsequently determined by radioimmunoassay. Forskolin markedly stimulated cortisol secretion by interrenal cells. Adenylate cyclase inhibitors depressed the steroidogenic response to ACTH. Dibutyryl cAMP, but not dbcGMP, enhanced steroid secretion. Thus, cAMP seems to be an important "second messenger" for ACTH action on salmon interrenal cells. In contrast to findings in mammalian adrenocortical cells, exogenous phosphatidylinositol and cholera toxin failed to stimulate corticosteroid secretion in salmon interrenal cells. However, it was unclear whether these negative findings were an artifact resulting from the use of kidney tissue fragments instead of isolated interrenal cells. PMID- 3023181 TI - Cloning of random-sequence oligodeoxynucleotides. AB - Methods are described for cloning random or highly degenerate nucleotide (nt) sequences. The procedures use synthetically derived mixtures of oligodeoxynucleotides (oligos) whose heterogeneous central portions are bounded at their 5' and 3' ends by sequences recognized by restriction endonucleases. Oligo collections of defined length and nt composition are synthesized by utilizing appropriate concentrations of all four nucleotide precursors during each addition step for the central region. Single-stranded oligos with appropriate 5' and 3' ends can be ligated directly, although inefficiently, into double-stranded (ds) DNA molecules with complementary 5' and 3' extensions produced by restriction endonuclease cleavage. A more general and efficient method is to convert the oligo into a ds form by incubating it with the Klenow (large) fragment of Escherichia coli DNA polymerase I. If the 3' ends are palindromic, two oligo molecules will serve as mutual primers for polymerization. The resulting products are ds molecules containing two oligo units separated by the original 3' restriction site and bounded at each end by the original 5' restriction site. After appropriate restriction endonuclease cleavage, oligo units can be cloned by standard procedures. Analysis of 26 recombinant M13 phages indicates that the nt sequences of the cloned oligos are in good accord with what was expected on a random basis. PMID- 3023183 TI - The lac operator as a phenotypic label for DNA fragments cloned in Escherichia coli. AB - To confer a detectable phenotype on any DNA fragment cloned in Escherichia coli, one can label the fragment by ligating it to the lac operator so that host cells can be identified as blue colonies on agar plates. This screening strategy is similar to that used for the pUC and M13 series of vectors, but does not require the vector to contain the lacZ gene. Instead, the presence of the lac operator on the multicopy vector results in the induction of the host cell beta-galactosidase by titrating out the repressor. This paper describes how pUC/M13 vectors or synthetic oligodeoxynucleotides can be used to supply the operator label, and shows how this method has been used to position unique restriction sites for initiating BAL 31 deletions. This approach may be particularly helpful when a given DNA fragment is to be cloned in many different constructs or is to pass through many sequential cloning steps. PMID- 3023182 TI - Isolation and characterization of cDNA encoding rabbit reticulocyte 2,3 bisphosphoglycerate synthase. AB - We have isolated and sequenced a cDNA clone containing the entire coding region of rabbit reticulocyte 2,3-bisphosphoglycerate (DPG) synthase. The cDNA was verified by translation of the hybridization-selected RNA and by demonstrating identity of the deduced amino acid (aa) sequence to the sequences of CNBr peptides of the purified enzyme. The aa sequence of the enzyme was homologous to the reported sequence of the human enzyme [Haggarty et al., EMBO J. 2 (1983) 1213 1220], especially in the N-terminal half (aa 1-142). Northern blot analysis of rabbit reticulocyte poly(A)+ RNA revealed a single species of mRNA with about 1700 nucleotides. PMID- 3023184 TI - The metH gene from Salmonella typhimurium LT2: cloning and initial characterization. AB - A 19-kb EcoRI DNA fragment carrying the metH gene of Salmonella typhimurium LT2 was cloned into plasmid vector pACYC184 and propagated in Escherichia coli K-12. The size of the metH gene product was observed to be approx. 120 kDa, as determined by SDS-polyacrylamide gel analysis of plasmid-specific polypeptides synthesized in a minicell system. The direction of transcription of the metH gene relative to the cloned fragment was determined, and the positions of translation initiation and termination were estimated. PMID- 3023185 TI - Cloning and characterization of the glycine-cleavage enzyme system of Escherichia coli. AB - The glycine-cleavage enzyme system of Escherichia coli has been cloned in the cosmid vector pMF7. The recombinant plasmid, designated pGS64, carries two 19.4 kb EcoRI insert fragments. One of these fragments, which carries the gcv system, was subcloned from plasmid pGS64 into the plasmid vectors pACYC184 and pSC101 (creating plasmids pGS96 and pGS97, respectively). Plasmid pGS97, but not pGS96, complements a gcv mutant on glycine-supplemented plates. Enzyme assays, however, verified that both plasmids carry an inducible gcv system. The location of the gcv system in plasmid pGS97 was determined by Tn5 insertional inactivation. Subcloning experiments identified the region on the 19.4-kb fragment that inhibits growth in strains transformed with plasmid pGS96 and a region that is possibly involved in negative regulation of the gcv system. PMID- 3023186 TI - Nucleotide sequence of the regulatory gene xylS on the Pseudomonas putida TOL plasmid and identification of the protein product. AB - The xylS gene is a regulatory gene which positively controls expression of the genes on the TOL plasmid for degradation enzymes of benzoate or m-toluate in Pseudomonas putida. Cloning of the gene in Escherichia coli and determination of the nucleotide sequence revealed an open reading frame of 963 bp which corresponds to a protein with an Mr of 36,502. The xylS gene was recloned onto a tac-promoter vector, and the product was identified by the maxicell procedure as a protein with an approximate Mr of 37,000. The predicted amino acid sequence of XylS protein showed a basic character and contained a region similar to those in other DNA-binding proteins. PMID- 3023188 TI - Covalently linked sequencing primer linkers (splinkers) for sequence analysis of restriction fragments. AB - A new method for direct sequence analysis of DNA restriction fragments uses synthetic covalently linked complementary oligodeoxynucleotides, as universal sequencing primer linkers (splinkers). These splinkers were designed to contain an inverted repeat sequence which forms a double-stranded hairpin structure with a known restriction site. The splinkers were characterized by their ability to be self-ligating (dimerized) and by their restriction digest product analysis of both the monomer and dimer. They can also be ligated to dephosphorylated DNA restriction fragments which contain the appropriate end. This was evidenced by mobility shifts of the splinker-ligated restriction fragments. The splinker ligated restriction fragments, after denaturation, form a single-stranded DNA template containing an inverted repeat sequence (from splinker) at one terminus. The splinker is thus suitably oriented and serves as a primer for dideoxy nucleotide (nt) sequencing catalyzed by either Klenow fragment of Escherichia coli DNA polymerase I or avian myeloblastosis virus reverse transcriptase. As demonstrated for both pBR322 and phi X174, release of the primer extension strand by digestion at the splinker restriction site results in a ladder of labelled fragments which corresponds to a unique nt sequence. PMID- 3023187 TI - Paramecium mitochondrial DNA sequences and RNA transcripts for cytochrome oxidase subunit I, URF1, and three ORFs adjacent to the replication origin. AB - A 2-kb region adjacent to the replication origin (ori) and a 3-kb region located between the small and large ribosomal RNAs of Paramecium mitochondrial (mt) DNA have been sequenced and the locations of their transcripts determined. The ori segment contains four transcripts, some of which are overlapping, which encode a known protein and two other open reading frames. The other segment encodes, on separate transcripts, the cytochrome c oxidase subunit one gene (COI) and the URF1 gene (ND1) common to most mt genomes. All these genes have the same orientation and do not contain introns. The COI gene is the most divergent of those known and has an internal 108 amino acid 'insert' not found in COI genes from other organisms. With these data it is possible to define a probable Paramecium mt genetic code. With the exception that TGA codes for tryptophan and the use of different start codons, Paramecium mtDNA appears to follow the universal code. GTA possibly can be used as a start codon. PMID- 3023189 TI - Isolation of the Rhodopseudomonas sphaeroides form I ribulose 1,5-bisphosphate carboxylase/oxygenase large and small subunit genes and expression of the active hexadecameric enzyme in Escherichia coli. AB - A library of cloned Rhodopseudomonas sphaeroides DNA was screened by colony hybridization for form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) sequences using heterologous RuBPC/O probes. A recombinant plasmid was identified that hybridized to both the Anacystis nidulans and the R. sphaeroides form II RuBPC/O genes. Subcloning of a hybridizing 4-kb SmaI fragment allowed expression of active enzyme in Escherichia coli that was identical to form I RuBPC/O based on polyacrylamide gel electrophoresis and Western immunoblot analysis. PMID- 3023191 TI - The human tRNAVal gene family: organization, nucleotide sequences and homologous transcription of three single-copy genes. AB - At least 13 independent tRNAVal gene loci were detected in the human genome. Three of these genes were isolated and shown to occur only once in the haploid genome. No further functional tRNA genes are located on the isolated clones. Two tRNAVal genes encode the known major and minor tRNAVal isoacceptors, the third may be a pseudogene because a corresponding tRNAVal is not yet known. Comparison of extragenic sequences did not reveal significant homologies, indicating the separation of these genes early in vertebrate evolution. An Alu-type repeat was found in two of the clones within several hundred bp distance from the tDNA. All three genes are transcriptionally active in a HeLa nuclear extract. We show here for the first time that homologous in vitro transcription of mammalian tRNA genes strongly depends on extragenic control regions: interestingly, as a consequence of different flanking regions, the transcription efficiencies vary by an order of magnitude among the genes for the major and the minor tRNAVal and thus reflect the concentrations of these tRNAs in vivo. PMID- 3023190 TI - Identification of the thymidine kinase gene of infectious bovine rhinotracheitis virus and its function in Escherichia coli hosts. AB - In an attempt to understand the gene expression of the infectious bovine rhinotracheitis virus (IBRV), the viral thymidine kinase gene (tk), a well regulated viral gene has been chosen for this study. A cosmid library of IBRV has been constructed in Escherichia coli HB101 by cloning partially Sau3A-digested DNA fragments into a cosmid vector, pJB8. Recombinant cosmids were further analyzed by restriction digestions and by Southern blot hybridization. Results showed that this cosmid library comprised all of the IBRV genome with the exception of both termini. The individual recombinant cosmid clones were then used to transform E. coli tdk- mutant strains, Ky895 or C600tdk- for the selection of the IBRV tk gene. The clones able to grow on the selection plates containing 5-fluorouracil, uridine, thymidine and ampicillin were selected and further characterized. The physical location of the viral DNA inserts of one of the clones, pIBR5, was determined and the sequences complementing the tk activity have been isolated by subcloning. The plasmid, pIBRTK, was shown to grow on selection plates and therefore, retained the ability to complement the tk gene. The E. coli mutant strain C600tdk- harboring pIBRTK partially restores the tk activity by exhibiting a three and half fold increase in the level of the incorporation of [3H]thymidine into bacterial DNA over that of C600tdk- mutant. PMID- 3023192 TI - Structural analysis of ribosomal DNA homologues in nucleolus-less mutant of Xenopus laevis. AB - Sequences homologous to the ribosomal DNA (rDNA) in a Xenopus anucleolate (nucleolus-less) mutant were analyzed by Southern blot analysis. The mutant was found to possess a variety of sequences homologous to non-transcribed spacer (NTS) and/or coding region of rDNA. 65 rDNA-homologous clones were isolated from a genomic DNA library of the mutant. All the clones showed only partial homology to the normal rDNA unit and their restriction maps differed from that of the normal rDNA unit. Based on the hybridization patterns, the rDNA-homologous clones were divided into four groups (I-IV). Structure of group IV, which most strongly hybridized to normal rDNA probe, was analyzed by nucleotide sequencing. The group IV sequence was found to contain a part of the rDNA, including Bam island, enhancer element, promoter region, external transcribed spacer, and a portion of 18S rRNA gene. The blotting analysis suggested that the group IV sequence is specific for a particular strain of Xenopus. PMID- 3023193 TI - Characterization and structure of an endoglucanase gene cenA of Cellulomonas fimi. AB - Two BamHI fragments (0.8 and 5.2 kb) of Cellulomonas fimi containing an endoglucanase (Eng) gene (cenA) were individually cloned into the BamHI site of pBR322; they expressed carboxymethylcellulase activity in Escherichia coli. The nucleotide (nt) sequence of the cenA gene was determined by sequencing overlapping deletions. The cenA gene is 1350 bp long encoding a polypeptide of 449 amino acids (aa) and stop codon. The 0.8-kb BamHI component encodes the first 76 aa, whereas the 5.2-kb BamHI component encodes the rest of the Eng. The Eng lacking the N-terminal 76 aa retains its activity and antigenicity, and it forms an active fusion protein with the N-terminal portion of the TcR determinant. The C-terminal region of the Eng is crucial for activity and a deletion of as little as 12 aa from that end results in the loss of all Eng activity. The N-terminal 31 aa of the Eng constitute a leader peptide which appears to be functional in exporting the enzyme to the periplasm in E. coli. PMID- 3023194 TI - Secretion of Cellulomonas fimi exoglucanase by Escherichia coli. AB - A leader sequence of 41 amino acids (aa) has been proposed as the signal sequence for the exoglucanase (Exg) from Cellulomonas fimi. The ability of this 41-aa peptide to function as a leader sequence has been shown here by gene fusion experiments in Escherichia coli. A hybrid leader sequence containing C-terminal 37 aa of the leader peptide and N-terminal 6 aa of beta-galactosidase (beta Gal) directed export of the Exg into the periplasm of E. coli. In contrast, hybrid beta Gal-Exg proteins in which the leader sequence is not present are retained in the cytoplasm. PMID- 3023195 TI - Expression of human T-cell leukemia virus type I envelope protein in Saccharomyces cerevisiae. AB - The entire envelope gene of human T-cell leukemia virus type I (HTLV-I) was inserted into an expression vector and expressed under the control of the repressible acid phosphatase promoter in yeast (Saccharomyces cerevisiae). The product in yeast cells was glycosylated into heterodisperse proteins. PMID- 3023196 TI - Satellite DNA in the crustacean Artemia. AB - We have isolated a satellite fraction from the Artemia genome by both restriction endonuclease digestion and equilibrium density centrifugation in CsCl gradients containing ligand dye Hoechst 33258. Satellite DNA was arranged in long stretches (approx. 23 kb) of tandem repeats of a basic unit of 113 bp. The basic unit has been sequenced, showing a G + C content very close to that of total DNA. Different amounts of satellite were present in several populations of Artemia, whereas it was absent from others. PMID- 3023197 TI - An improved Escherichia coli expression vector for the construction and identification of full-length cDNA clones. AB - An Escherichia coli expression vector designed for the efficient synthesis and identification of a full-length cDNA clone is constructed. The vector allows the synthesis of double-stranded cDNAs downstream from the tandem lac control regions employing the vector-primer and linker procedure of Okayama and Berg [Mol. Cell Biol. 2 (1982) 161-170]. Full-length cDNA clones carrying the 5'-noncoding region in addition to the entire coding and 3'-noncoding regions can be expressed in E. coli cells without fusing their coding region to that of E. coli proteins; these clones are identified by colony immunoassay. The entire cDNA insert can be easily excised from the plasmid, since the multiple cloning sites in the vector are duplicated at both ends of the cDNA insert during its synthesis. PMID- 3023198 TI - Expression of the Epstein-Barr virus major membrane proteins in Chinese hamster ovary cells. AB - The gene of the major membrane antigen (gp250/350) of the Epstein-Barr virus (EBV) was isolated and inserted under the control of the SV40 early promoter into a eukaryotic expression vector, which allows selection for dhfr+ phenotype. Following transfection with this vector, Chinese hamster ovary cells express on their surfaces proteins immunologically similar to the major EBV membrane antigen. The transcript encoding gp250/350 is processed by partial splicing similarly but more efficiency than in B95-8 cells from which the DNA originates. PMID- 3023199 TI - Powerful and versatile enhancer-promoter unit for mammalian expression vectors. AB - The enhancer and promoter of the immediate early gene of human cytomegalovirus (hCMV) were tested as a transcriptional control element by fusion to the cat gene, followed by measurement of its expression from plasmid DNA in the extrachromosomal and integrated state, respectively. Comparison of the hCMV enhancer-promoter to two other viral elements was performed in six commonly used cell lines of different tissue and species origin. Irrespective of the cell line and of the state of the DNA, the hCMV enhancer-promoter was considerably stronger than both the SV40 promoter and the long terminal repeat of Rous sarcoma virus. PMID- 3023200 TI - CAT vectors for analysis of eukaryotic promoters and enhancers. AB - We have constructed two sets of plasmids for analysis of factors affecting mammalian gene expression. The pOCAT series contains a bacterial chloramphenicol resistance expression unit (cat) and no eukaryotic promoter. The pUTKAT series contains the same cat unit under the control of the thymidine-kinase promoter of Herpes simplex virus. These plasmids are designed for testing effects of inserted regulatory elements on cat expression after transient transfection of mammalian cells in culture. We demonstrate here that the pOCAT series is useful for studying activities of inserted eukaryotic promoters, and the pUTKAT series is useful for studying activities of inserted eukaryotic enhancers. PMID- 3023201 TI - Identification and nucleotide sequence of the activator gene of the externally induced phosphoglycerate transport system of Salmonella typhimurium. AB - A recent study from this laboratory (G-q. Yu, D. Goldrick, H.R. Kaback and J-s. Hong, in preparation) indicates that the externally induced phosphoglycerate transport system (pgt) of Salmonella typhimurium is positively regulated by the activator gene, pgtA, and that the pgtA is localized in the SalI-PstI restriction fragment 3.0 kb from the permease gene, pgtP. In this paper, we describe the identification of the activator gene and its gene product and the determination of the complete nucleotide (nt) sequence of the activator gene as well as of a downstream gene not required for pgtP expression. The amino acid sequence of the activator based on the nt sequence shows an N-terminal signal-like sequence which is apparently not cleaved and three potential transmembrane sequences in the C terminal half of the protein based on the hydropathy analysis. PMID- 3023202 TI - Unusual occurrence of EcoP1 and EcoP15 recognition sites and counterselection of type II methylation and restriction sequences in bacteriophage T7 DNA. AB - Selected and counterselected oligodeoxynucleotide sequences were identified in the total sequence of bacteriophage T7 DNA using a statistical criterion derived for a probability model of the Markov chain type. All extremely rare tetra- and pentadeoxynucleotides are (or contain) recognition sequences for the Escherichia coli DNA methylases dam or dcm. Most of the 37 hexadeoxynucleotides absent from T7 DNA are recognition sequences for type II modification/restriction enzymes of E. coli or related species. In contrast to most restriction sites counterselected during evolution, the EcoP1 site GGTCT occurs 126 times in the T7 genome, and phage T7 replication is severely repressed in P1-lysogenic host cells. We demonstrate that the frequency of the EcoP1 site is determined by that of the overlapping recognition sites for T7 primase, an essential phage enzyme. The recognition site of a type III enzyme, EcoP15, is also not counterselected. In T7 DNA all 36 EcoP15 sites are arranged in such a manner that the sequence CAGCAG is confined to the H strand, the complementary sequence CTGCTG to the L strand. This "strand bias" is highly significant and, therefore, very probably selected. A functional relation between this strand bias and the refractive behaviour of phage T7 to EcoP15 restriction is suspected. PMID- 3023203 TI - Dose related in vitro effects of ranitidine and cimetidine on basal and ACTH stimulated steroidogenesis. AB - Isolated bovine adrenocortical cells were incubated with and without 3 ng/ml ACTH, with various concentrations (10-1000 micrograms/ml) of either cimetidine or ranitidine. Cortisol, corticosterone, and deoxycorticosterone outputs were measured. Cimetidine and ranitidine at 320 and 1000 micrograms/ml inhibited ACTH stimulated corticosterone and cortisol synthesis and cimetidine decreased basal cortisol synthesis. The inhibitory effects of cimetidine on cortisol synthesis were approximately 10 times greater than those of ranitidine. Cimetidine (1000 micrograms/ml), but not ranitidine increased deoxycorticosterone synthesis by ACTH-stimulated cells, indicating inhibition of 11 beta-hydroxylation in the adrenal steroidogenic pathway. Although doses of cimetidine and ranitidine which produce these in vitro effects are much greater than plasma concentrations in normal clinical use, they might be important in acutely ill patients given intravenous bolus injections of cimetidine, or if either antagonist were accumulated by the adrenal to produce high intracellular concentrations. PMID- 3023204 TI - Poor prognosis metastatic gestational trophoblastic disease: experience with moderate dose methotrexate plus folinic acid rescue as initial therapy. AB - From 1971 to 1981, twenty patients with poor-prognosis metastatic gestational trophoblastic neoplasia (GTN) were treated with moderate-dose methotrexate (1 g) and folinic-acid rescue (MD-MTX-FAR) as initial therapy. Seven (35%) were cured with MD-MTX-FAR, and salvage chemotherapy was successful in an additional seven, for a total cure rate of 70%. The ultimate outcome is similar to that reported for MAC triple therapy during this era. Hematologic and mucosal toxicity were negligible and no serious complications were encountered. We now use combination chemotherapy in patients with poor-prognosis GTN as first-line treatment. However, these results suggest that there may be advantages to the incorporation of MD-MTX-FAR in combination regimens in place of low-dose methotrexate, because of reduced toxicity and potential benefits for the prophylaxis and treatment of cerebral metastases. PMID- 3023205 TI - Malignant mixed mesodermal tumors of the uterus and ovary treated with cisplatin based combination chemotherapy. AB - Twelve patients with malignant mixed mullerian tumors were treated with combination chemotherapy at Vanderbilt University Hospital from 1977 through 1981. Nine patients, all of whom received combination chemotherapy with hexamethylmelamine, cyclophosphamide, doxorubicin, and cisplatin (HCAP), were evaluable for response. Objective responses (all partial responses) were noted in 3 (33.3%) (response rate greater than 10% and less than 55% with 90% confidence limits), a minimal response was noted in one patient, and stable disease in four (50%) patients. Responders survived longer (calculated from the initiation of HCAP) than nonresponders (median 112 vs 19 weeks). These results are not at present statistically different from previous studies utilizing doxorubicin alone, cisplatin alone, the combination of doxorubicin and DTIC, or the combination of vincristine, actinomycin D, and cyclophosphamide. PMID- 3023206 TI - Paget's disease of the vulva and urogenital malignancies: a case report and review of the literature. AB - The vulva is one of the extramammary sites which has a potential for developing Paget's Disease with which an underlying sweat gland carcinoma or breast carcinoma is frequently associated. Urogenital malignancy may be a third contender. Such a case managed by the authors herein reported and a review of the literature about such lesions support this assumption. PMID- 3023207 TI - Small cell carcinoma of the endometrium associated with adenosquamous carcinoma: a light and electron microscopic study. AB - This report describes a 64-year-old woman with a primary small cell carcinoma of the endometrium associated with adenosquamous carcinoma. The light microscopic features resembled those of small cell carcinoma of the lung and those of the uterine cervix, and foci of adenosquamous carcinoma lay scattered sparsely in the small cell carcinoma. Electron microscopy revealed cytoplasmic neurosecretory type granules. The neoplasm behaved in a very aggressive manner such that at 3 months after surgery a metastatic neoplasm appeared in the vagina. This case is a rare example of an endometrial carcinoma with differentiation toward endocrine as well as adeno and squamous cell carcinoma. PMID- 3023208 TI - Signet-ring cell carcinoma of the urinary bladder primary presentation as a Krukenberg tumor. AB - A 34-year-old female presented with a right ovarian Krukenberg tumor, which initially was thought to be a granulosa cell tumor. Eight months later, necropsy revealed a primary signet-ring cell carcinoma (SRCC) of the urinary bladder. When occurring in females, this rare bladder tumor often exhibits ovarian metastases. This case appears to be the first reported example of a signet-ring cell carcinoma of the bladder presenting as a Krukenberg tumor. PMID- 3023209 TI - Micro-densitometric evaluation of human neutrophil granulocyte enzymes in normals and in different pathological states. PMID- 3023211 TI - [Urinary excretion of c-AMP, as a parathyroid function test]. PMID- 3023210 TI - Inhibition of platelet thromboxane generation by suloctidil in man. AB - Oral administration of 100 or 200 mg suloctidil to healthy volunteers resulted in serum thromboxane B2 (TxB2) inhibition. This reached a maximum level between 90 min and 4 h after drug ingestion. TxB2 levels returned to 75% of basal values 6 h after 100 mg and more than 8 h after 200 mg, reaching control values at 24 h. Different experiments were performed to define the metabolic step at which suloctidil acts to inhibit serum TxB2 generation. Suloctidil prevented TxB2 synthesis also when platelet-rich plasma was stimulated by exogenous arachidonic acid or whole blood was clotted in the presence of exogenous arachidonic acid. This rules out the possibility that it inhibits phospholipases. No rediversion of prostaglandin synthesis after suloctidil occurred concomitantly with TxB2 inhibition, suggesting that the drug is not a selective thromboxane synthase inhibitor. In contrast, a significant reduction of serum PGE2 formation was found, suggesting a mechanism of action of suloctidil involving inhibition of cyclo-oxygenase. This was supported by the finding that PGI2 production by rat smooth muscle cells stimulated with arachidonic acid was significantly prevented by suloctidil in vitro. Suloctidil, however, did not prevent aspirin-induced inhibition of serum TxB2 generation. Blockade of platelet cyclo-oxygenase activity by suloctidil is therefore exerted at a level different from that of aspirin. In conclusion, suloctidil is a relatively weak nonselective cyclo oxygenase inhibitor whose effect on platelet TxA2 production hardly accounts for its reported inhibitory effect on platelet aggregation. PMID- 3023212 TI - [Converting enzyme inhibitors and calcium antagonists in left ventricular dysfunction]. PMID- 3023213 TI - [Immunotherapy of lung cancer]. PMID- 3023214 TI - In vitro correlates of mineral dust toxicity. AB - In vitro studies of mineral fibres have shown that these materials are capable of causing the production of free radical damage (measured by the accumulation of thiobarbituric acid-reactive material) in a way that does not depend on fibre morphology but rather on a surface property, probably the presence of iron. The DNA-damaging activities of the same dusts were measured in an assay based on the production of single-stranded DNA. Activity in this assay was again not dependent on the presence of fibres. In contrast the cytotoxicity of mineral fibres towards V79-4 cells correlated better with in vivo tumorigenicity than did either of the above activities. The activity of fibres against a macrophage-like cell demonstrated that both fibre morphology and chemical properties are important determinants of biological activity. PMID- 3023215 TI - Influence of phagocyte-derived active oxygen species in tissue responses to tumour promoters and irritants. AB - In response to stimuli such as irritant chemicals, inorganic particles or micro organisms, phagocytic cells produce a variety of chemical species including superoxide and hydrogen peroxide. These reactive oxygen species are cytotoxic and genotoxic to cultured cells and may be responsible for some of the tissue damage associated with inflammation in vivo. The role of phagocyte-derived oxygen radicals in the effects of irritants, tumour promoters and carcinogenic inorganic materials has been explored using mixed cultures of phagocytes and fibroblasts. Catalase-inhibitable cytotoxicity and genetic damage were demonstrated in fibroblasts exposed to neutrophils stimulated with the irritant and tumour promoter phorbol myristate acetate or with zymosan particles. To investigate this phenomenon further, other cell systems have been developed, using epidermal keratinocytes or mesothelial cells in combination with neutrophils or macrophages. PMID- 3023216 TI - Photoaffinity labelling of the Ah receptor. AB - A series of halodibenzo-p-dioxins bearing the arylazide photolabile functional group were synthesized and tested as photoaffinity labels for the Ah receptor. 2 Azido-3-iodo-7,8-dibromodibenzo-p-dioxin (KD = 0.76 X 10(-9) M) was selected for radiosynthesis. Analysis of the 125I-photoaffinity-labelled proteins in mouse liver cytosol by denaturing gel electrophoresis revealed two peptides which had apparent molecular masses of 95,000 and 70,000 daltons respectively, were labelled in an approximately 1:1 ratio and were selectively labelled at low concentrations of the photoaffinity ligand (0.05 KD = 0.04 X 10(-9) M). In addition, their labelling was inhibited by co-incubation with an excess of unlabelled ligand. On chromatographic separation under non-denaturing conditions, these two peptides co-migrated. These studies suggest that the Ah receptor in mouse liver cytosol is a heterodimer composed of two non-covalently bound peptides (95 K and 70 K) which each have a ligand binding site. PMID- 3023217 TI - [Human cytotoxic T cell clones that recognize HLA-DQw3 antigens]. AB - Human cytotoxic T cell (CTL) clones generated against a DQw3-positive EBV transformed B cell line were analyzed in terms of immunological characteristics. The cytolytic activities of clones 1-18 and 3-34 were inhibited by anti-DQw3 monoclonal antibodies (HU-18 and HU-23), but they were not affected by anti-DR (HU-4 and HU-20) and anti-HLA-A, B, C (W6/32) monoclonal antibodies. In contrast to that the clone 1-18 cytolyzed all the human B cell lines typed as DQw3, the clone 3-34 reacted only with DR4/Dw4 and DR5-positive cells that were positive for HU-23 and typed as TA10. In addition, the clone 3-34 were cross-reactive to DQw2-typed cells. These results show that there are two epitopes that CTL recognize on the same DQw3 molecules; one is the public epitope among DQw3 antigens, and the other is the private epitope that HU-23 recognizes. This is the further evidence for CTL recognition of HLA-DQ antigens. PMID- 3023219 TI - Phosphatidylinositol kinase in rat pancreatic islets: subcellular distribution and sensitivity to calcium. AB - Plasma membrane of pancreatic islets contains a calcium sensitive phosphatidylinositol kinase. This enzyme catalyzes the first reaction in the pathway leading to the production of inositol trisphosphate, which is believed to cause a redistribution of intracellular calcium. Since the activity of this enzyme is inhibited by calcium (K0.5 = 10 microM), a loss of calcium from plasma membrane (the site of PI kinase) may be necessary for activation of the enzyme in vivo. PMID- 3023218 TI - [Quantitative and qualitative analyses of inorganic dusts in idiopathic interstitial pneumonia (IIP)]. AB - We made quantitative and qualitative analyses of dusts in lung specimens using proton-induced X-ray emission (PIXE) and analytical electron microscopy (AEM). IIP group consisted of 23 patients. Control group, that had no apparent history for dust inhalation, consisted of 21 patients. Control group was matched with IIP group with respects to sex, age, smoking index and life style. For PIXE analysis, the elements Al, Si, P, S, Cl, K, Ca, Ti, Cr, Fe, Co, Ni, Cu, W and Au were examined for this study. Tissue specimen preparation for AEM study was based upon carbon extraction method. The amounts of Al and Si were significantly larger in IIP group than in the control group (Al: IIP 24.40 +/- 19.08 ng/60.8 mm2 and control 11.90 +/- 8.66, P less than 0.01; Si: IIP 54.43 +/- 45.18 and control 28.72 +/- 15.56, P less than 0.05). The elements Co, W and Au were not detected. The amounts of Si correlated inversely with PaO2 (r = -0.454, P less than 0.05). AEM study demonstrated larger amounts of free silica and silicate in IIP group than in the control group (free silica: IIP 0.7% and control 0.2%, P less than 0.01; silicate: IIP 2.6% and control 0.8%, P less than 0.01). These results suggest that inhalation of Si, especially of free silica, may have etiologic significance in IIP. PMID- 3023220 TI - Steroid hormones in the adrenal venous effluents in idiopathic hirsutism under basal and stimulated conditions. AB - Steroid hormone concentrations in the peripheral blood and the adrenal veins were measured in the basal state and after ACTH stimulation in 5 patients with idiopathic hirsutism. The basal concentrations of the steroids in the adrenal veins of the patients with idiopathic hirsutism were not significantly different from a control group of 5 patients catheterized for investigation of pheochromocytoma. Following ACTH stimulation, the concentrations of the steroids in the adrenal veins were also not significantly different in the hirsute and the control groups except for the concentrations of DHA and DHAS which were higher in the patients with idiopathic hirsutism. 17-hydroxyprogesterone (17-OHP) concentrations after ACTH stimulation were lower in the hirsute group compared to the control population. It is concluded that patients with idiopathic hirsutism have a defect in the biosynthesis of cortisol proximal to the action of the 11 beta- and 21-hydroxylase enzymes, deficiencies of which have been previously considered to be the usual causes of hirsutism due to an adrenocortical abnormality. The lower 17-OHP concentrations in the hirsute group can be explained on the basis of deficiency of substrate for the action of the 17 hydroxylating enzyme, consequent to the postulated deficiency of 3 beta hydroxysteroid dehydrogenase. PMID- 3023221 TI - Evidence for the involvement of the opiate-receptors in the diazepam-induced human growth hormone secretion. PMID- 3023223 TI - Thyrotropin-releasing hormone and insulin in chemically induced pancreatic islet cell tumors in rats. AB - Thyrotropin-releasing hormone (TRH) and insulin were measured by radioimmunoassay in acetic-acid extracts of 19 pancreatic islet cell tumors induced by streptozotocin and nicotinamide in rats. In addition, gel filtration properties of TRH-immunoreactivity and immunoreactive insulin (IRI) were examined in 5 and 14 tumors, respectively. TRH was demonstrated in 10 of 19 tumors, with a mean of 166 +/- 47 (SEM) pg/mg wet weight, whereas the concentration was less than 3 pg/mg wet weight in the other tumors. In contrast, all tumors contained IRI, with a mean of 11.0 +/- 1.6 micrograms/mg wet weight. Ten tumors in which TRH was demonstrated contained more IRI than those in which TRH was not detected (13.1 +/ 1.8 vs 6.5 +/- 1.7 micrograms/mg wet weight, P less than 0.02). After gel filtration, all TRH immunoreactivity was eluted at the same place as synthetic TRH in the 5 tumors. In addition, gel filtration elutes showed essentially the same pattern of IRI in the 14 tumors, with 3 peaks. The predominant IRI peak comigrated with marker insulin (95.7 +/- 0.8%), another prominent peak occurred coincident with proinsulin standard (3.3 +/- 0.5%), a third peak was present in the void volume (0.28 +/- 0.04%). These distributions of IRI were similar to those in extracts of normal pancreases. The present studies demonstrate TRH immunoreactivity in pancreatic islet cell tumors induced by streptozotocin and nicotinamide in rats. Chemically induced insulinomas can serve as a model for insulin storage which is analogous to islet B cells. PMID- 3023222 TI - Effects of pro-opiomelanocortin-derived peptides on plasma levels of glucagon, insulin and glucose. AB - Pro-opiomelanocortin (POMC) is a prohormone for several peptides including corticotropin, melanocyte stimulating hormones and beta-endorphin. POMC-derived peptides have been demonstrated in many tissues, including the hypothalamus and the endocrine pancreas, which play important roles in the control of plasma levels of glucagon, insulin and glucose. This article reviews the present knowledge concerning in vitro and in vivo effects of POMC-derived peptides on glucagon, insulin and glucose levels involving several possible mechanisms: direct effects on the endocrine pancreas (including endocrine, paracrine and peptidergic regulation) and glucose production, and indirect effects involving the hypothalamus, the autonomic nervous system and the adrenal gland. PMID- 3023224 TI - Adaptive regulation of beta-adrenergic receptors in children with insulin dependent diabetes mellitus. AB - The beta-adrenergic receptors were investigated in partially purified mononucleal leukocytes (MNL) plasma membranes from 18 patients with IDDM in pediatric period, 9 healthy children and 8 normal adults. The decreased beta-adrenergic receptor number was seen in patients with IDDM (Bmax = 27.6 +/- 8.3 fM (125I) IHYP/mg protein) compared with normal children (Bmax = 40.4 +/- 10.4 fM (125I) IHYP/mg protein) and normal adults (Bmax = 36.9 +/- 6 fM (125I) IHYP/mg protein). MNL beta-receptor binding affinities (apparent Kd = 109.8 +/- 26.1 pM in IDDM, 102.8 +/- 46.6 pM in normal children, 130.0 +/- 43.1 pM in normal adults) did not differ. We divided the patients with IDDM into two groups based on their level of blood glycosylated hemoglobin (HbA1) when samples were taken. Group A IDDM (consisted of 9 diabetic patients with below 10% of HbA1) had markedly decreased beta-receptor numbers compared with group B IDDM (consisted of 9 diabetic patients with more than 10% of HbA1), whereas Kd was not significantly different. Also, there was negative correlation between Bmax and level of blood sugar or HbA1 in IDDM. This is the first report concerning the beta-adrenergic receptor in IDDM in pediatric period. We suggest that decreased Bmax in group B is a homeostatic response to restore the poorly-controlled hyperglycemic state to normoglycemia because the group B patients had high level of HbA1 and blood sugar. PMID- 3023225 TI - Prostacyclin stimulation of adenylate cyclase activity in human thyroid membranes. AB - Effect of prostacyclin (PGI2) on adenylate cyclase activity in human thyroid membranes was examined. PGI2 caused a dose- and time-dependent production of cyclic AMP (cAMP) with high potency. When GTP was added in concentrations up to 100 uM, the activation of adenylate cyclase by PGI2 was increased. In the assay medium containing 3 mM ATP, 10 uM GTP and nucleotide regenerating system, the replacement of Mg2+ by increasing concentrations of Mn2+ caused a progressive loss of PGI2 as well as TSH-stimulated adenylate cyclase activities, while high concentrations of Mg2+ (12 or 18 mM) slightly suppressed the activity stimulated by either PGI2 or TSH. Both agents had an additive effect on the stimulation of adenylate cyclase activity in the presence of either 6 mM Mg2+ or 6 mM Mn2+. Gamma-globulin fraction containing non-stimulatory TSH receptor antibody which was prepared from a patient with chronic thyroiditis, suppressed only TSH- but not PGI2-stimulation of the adenylate cyclase activity. These results suggest that PGI2 can stimulate the adenylate cyclase activity in human thyroid tissue, and that PGI2-stimulation may be mediated by the different system from TSH dependent one. PMID- 3023226 TI - Plasma renin activity, active and inactive renin concentrations, and their responses to beta 1-adrenoceptor blockade with metoprolol in hyperthyroidism. AB - Plasma renin activity (PRA), plasma renin concentration (PRC), inactive renin concentration (IRC) and total renin concentration (TRC) were measured in 31 normal controls and in 8 patients with hyperthyroidism. TRC was determined as angiotensin I generated with sheep renin substrate after an acid activation of plasma. The angiotensin I of non-acidified plasma was expressed as PRC. IRC was calculated as TRC minus PRC. The mean values for PRA, PRC, IRC and TRC were significantly (P less than 0.05 to P less than 0.01) higher in the hyperthyroid patients than in the normal or euthyroid controls. The administration of a beta 1 adrenergic blocker, metoprolol (120 mg/day for 14 days), produced a significant (P less than 0.05 to P less than 0.01) fall in levels of T4, PRA and TRC, and reduced the active renin ratio calculated from PRC/TRC significantly (P less than 0.025), as compared to the pretreatment values. Our observations support the idea that the higher PRA in hyperthyroidism is due to an increased secretion of renin. Furthermore, the results may indicate that the conversion of inactive to active renin is accelerated in hyperthyroidism, possibly by an increased sympathetic activity. PMID- 3023227 TI - The effect of glucocorticoids on the in vivo conversion of androstenedione to oestrone. AB - In vitro studies have previously shown that the activity of the aromatase enzyme system, which is responsible for the conversion of androstenedione to oestrone, can be stimulated by natural and synthetic glucocorticoids and also by ACTH. In view of the potential physiological importance of such a regulatory mechanism we have examined the effect of administration of dexamethasone, and of ACTH on the conversion of androstenedione to oestrone in vivo. The transfer constants for the conversion of androstenedione to oestrone [( rho]AEBU) measured in two women before administration of dexamethasone were 1.0% and 1.1% and after were 0.9% and 1.2%. Similarly no increase in conversion of androstenedione to oestrone [( rho]AE1BB) was detected after ACTH stimulation (pre = 0.74%, post = 0.77%). It is concluded from this study that glucocorticoids and ACTH do not have a role in regulating aromatase activity in vivo. PMID- 3023228 TI - Effect of age on basal and 3,5,3'triiodothyronine (T3) stimulated human mononuclear cell sodium-potassium adenosine-triphosphatase (Na+-K+ATP'ase) activity. PMID- 3023230 TI - Hereditary resistance to 1,25-dihydroxyvitamin D: clinical and radiological improvement during high-dose oral calcium therapy. AB - A 6-year-old boy, of consanguinous parents, presented with severe rickets and alopecia; he was found to have hypocalcaemia and elevated circulating 1,25 dihydroxyvitamin D [1,25-(OH)2D] levels. He showed no calcaemic response to 1,25 (OH)2D3 or ergocalciferol given for 3 or more months in daily doses as high as 48 micrograms and 6 X 10(6) IU, respectively. Analyses with cultured skin fibroblasts revealed a normal capacity and affinity for 1,25-(OH)2D3 in soluble extracts ('cytosol') and in nuclei of intact cells but no detectable response of 25-(OH)D3 24-hydroxylase to 1,25-(OH)2D3 in high concentration. Treatment with high doses of calcium (3-4 g elemental calcium orally per day) produced a striking clinical and radiological improvement. We conclude that high oral doses of calcium can replace many of the actions of calciferols. Therapy with high doses of calcium should be tried in similarly affected cases that appear totally or partially unresponsive to calciferols. PMID- 3023229 TI - Corticotropin-releasing factor in humans. I. CRF stimulation in normals and CRF radioimmunoassay. AB - The biological activity of ovine (o) and human (h) corticotropin-releasing factor (CRF) in normal volunteers was investigated, using bolus injections with different CRF dosages. There was a significant increase of ACTH, beta-endorphin and cortisol after the injection of all dosages. Repetitive stimulation and continuous infusion of hCRF lead to repetitive release of identical amounts of ACTH or constant elevation of ACTH levels. oCRF and hCRF serum immunoreactivity was measured with specific radioimmunoassays after bolus injection, pulsatile administration and infusion of CRF. The half-time of serum disappearance after acute injection studies was calculated as 9 min for hCRF dand 18 min for oCRF. The 'metabolic clearance' of hCRF calculated using the infusion study was 2.72 ml/min X kg. Endogenous CRF immunoreactivity was detectable in 14 patients during insulin hypoglycemia and in 86 out of 97 pregnant females. Furthermore, CRF could be extracted from human placenta. The chromatographic pattern of extracted placenta CRF, pregnancy serum CRF and CRF standard preparation was identical. Furthermore, CRF immunoreactivity was detectable in some patients with different causes of ACTH hypersecretion. PMID- 3023231 TI - Giant vascular eccrine spiradenomas: a report of two cases with histology, immunohistology and electron microscopy. AB - Two examples of a variant of benign spiradenoma are reported, both characterized by their large size and high degree of vascularity. The results of studies using light microscopy, transmission electron microscopy and immunohistology are described. The relationship of this unusual variant to other spiradenomas and their eccrine sweat gland origin is discussed. The possible misdiagnosis of this rare type of spiradenoma is emphasized. PMID- 3023232 TI - Viral aetiology for verrucous carcinoma. PMID- 3023233 TI - Toluene diisocyanate. PMID- 3023234 TI - Anti-poliovirus IgG subclass antibodies in cytomegalovirus infected mice. AB - A considerable strain difference was noted in BALB/c and C57BL/6 mice with regard to the impairment of antibody responses to poliovirus antigens in the course of infection with murine cytomegalovirus (MCMV): a long lasting reduction in antibody formation in BALB/c mice contrasted with an only moderate depression observed in C57BL/6 animals. Analysis of antibody classes and IgG subclasses revealed that anti-poliovirus VP1 antibodies in BALB/c mice were predominantly of the IgG3 subclass, a subclass most drastically affected by MCMV infection, while C57BL/6 mice produced antibodies of the IgM class and of IgG1 and IgG2 subclasses which were reduced to a lesser extent by the infection with MCMV. It is concluded that the strain difference observed may be explained on the basis of differences in the handling of poliovirus antigenic determinants by BALB/c and C57BL/6 mice. PMID- 3023235 TI - Methylation patterns of HLA-DR alpha genes in six mononuclear cell lines. AB - The relationship between DNA methylation and HLA-DR alpha gene expression was investigated in six mononuclear cell lines. RPMI-4265 (B cell) and HUT-78 (T cell) constitutively express HLA-DR. HL-60 (myelomonocyte) and U-937 (monocyte) can be induced to express HLA-DR. Jurkat and Molt-4 (T cells) do not and cannot be induced to express HLA-DR. Based on the known nucleotide sequence of the HLA DR alpha gene, methylation-sensitive restriction endonucleases Msp I, Hpa II, Hha I, Ava I, Hae II, and Sma I were used to detect the CpG methylation in three regions of the HLA-DR alpha gene: the 5'flanking region, the exon 1 region, and the coding region containing exons 2, 3, 4, and 5. This precise mapping of CpG methylation yielded no correlation between DNA hypomethylation and HLA-DR alpha gene expression. In all cell lines, exon 1 region is hypomethylated, whereas 5' and coding regions are hypermethylated. Whereas hypermethylation of the coding region does not block transcription, hypomethylation of the exon 1 region may be essential but is clearly not sufficient for HLA-DR alpha gene transcription. This exon 1 region demethylation may result in an open (deoxyribonuclease I hypersensitive) chromatin conformation around the promoter where trans-acting regulatory factors presumably bind and initiate HLA-DR alpha transcription. In the course of this study, a novel Msp I polymorphism in the intron 1 of the HLA DR alpha gene was found. PMID- 3023236 TI - Migration in vitro by blood and exudate neutrophils assessed serially during an inflammatory response. AB - Migration in vitro by blood and inflammatory neutrophils has been compared serially during an inflammatory response. Using an experimental pig model, neutrophils are isolated from the peripheral blood and from the pleural space at hourly intervals after an intrapleural challenge with zymosan activated pig serum (ZAS). Following acepromazine sedation and halothane anesthesia, blood neutrophil migration was transiently reduced. By 1 hour random and directed migration of blood neutrophils returned to normal. Directed and random migration of exudate neutrophils was markedly decreased to both a stimulus-specific (ZAS) and an unrelated (LTB4) chemoattractant. After 3 hours, migration by exudate neutrophils was similar to migration by blood neutrophils examined in parallel. These findings emphasize the importance of performing serial evaluations of cell function during an inflammatory response. PMID- 3023237 TI - Effect of follicle stimulating hormone on ovarian carbohydrate metabolism in immature bonnet monkeys. PMID- 3023238 TI - Role of mononuclear phagocytes in expression of resistance and susceptibility to Mycobacterium avium infections in mice. AB - The growth of Mycobacterium avium 702 in the spleens and livers of four inbred strains of mice varied such that the mice could be separated into naturally susceptible (BALB/c and C57BL/6) and naturally resistant (A/Tru and DBA/2) strains. This phenomenon was independent of the size of the infecting inoculum of bacteria in that both low (10(4))- and high (10(7))-dose inocula of M. avium grew progressively in susceptible strains and were eliminated from the target organs of resistant strains. Resistance and susceptibility were also demonstrated in in vitro preparations of macrophages from these strains of mice. Over a 7-day period, replication of M. avium in susceptible mouse macrophages was far greater than that in resistant macrophages. Evidence was obtained to suggest that toxic oxygen metabolites were not responsible for this difference. Though no difference was found in the rate of clearance of M. avium from the blood of susceptible or resistant mice, resident macrophages from susceptible mice ingested more M. avium in vitro than did resident macrophages from resistant animals. Growth of M. avium in spleens of susceptible mice induced a large influx of phagocytes, whereas this was not observed in resistant mice. In contrast to this it was found that, after injection of a variety of inflammatory agents, influx of leukocytes into the peritoneal cavity could not be used to distinguish susceptible and resistant strains of mice. PMID- 3023240 TI - Determination of toluene diisocyanate in air by HPLC and band-tape monitors. AB - An improved HPLC method was developed for determining the atmospheric concentration of toluene diisocyanate (TDI). 1-(2-pyridyl)-piperazine in toluene was used as reagent absorber solution together with reversed phase chromatography and a simple efficient buffer system (0.1% trifluoroacetic acid--acetonitrile, 85:15%) in an isocratic elution mode. The values for atmospheric TDI concentration obtained with two continuous band-tape monitors were checked using the values from HPLC as reference. Under identical experimental conditions the two instruments (both model 7005) gave readings varying by more than 100%. At low humidity the band-tape values were considerably lower than the HPLC values. At an absolute humidity of 11.7 g H2O/m3 (58% relative humidity) the value from instrument 1, but not instrument 2, agreed with those from HPLC. The values obtained with band-tape devices in the continuous monitoring of TDI concentration in places of work, or epidemiological studies, should be assessed with caution. HPLC offers a useful reference method for monitoring the accuracy of such devices. PMID- 3023239 TI - Potential role of lysozyme in bactericidal activity of in vitro-acquired salivary pellicle against Streptococcus faecium 9790. AB - The adherence of Streptococcus faecium 9790 to hydroxyapatite (HA) coated with whole saliva supernatant proteins (S-HA) or parotid fluid proteins was studied. The organism was labeled with [3H]thymidine, and adherence was estimated as the radioactivity remaining associated with the variously coated HA preparations after incubation and removal of unbound microbes by washing the adherence substratum. Adherence was time dependent and saturable, characteristics typical of oral streptococci in this in vitro adherence model system. However, adherence to S-HA, but not bare HA, was decreased 20-fold at 4 degrees C compared with room temperature. Furthermore, adherence at 4 degrees C to S-HA was decreased 20-fold relative to bare HA at 4 degrees C. Adherence to HA coated with parotid fluid proteins also was reduced at 4 degrees C. The magnitude of the temperature dependence and the inhibitory effect at 4 degrees C of whole saliva or parotid fluid pellicles on HA was unexpected. Of several sugars and amino sugars tested, the chitin saccharides, chitotriose, chitobiose, and N-acetylglucosamine caused greater than 90% inhibition of adherence to S-HA. These same saccharides were previously shown to inhibit lysozyme, polylysine, or autolytic lysis of the organism (N. J. Laible and G. R. Germaine, Infect. Immun. 48:720-728, 1985). Examination of unbound and adherent microbes revealed that lysis of the organism occurred during the adherence assays. A strong association (r = 0.83) between the extent of lysis and the extent of adherence was found under a variety of conditions. Depletion of lysozyme from saliva specimens used to coat HA resulted in a greater than 90% decrease in both cell lysis and adherence. Lysis of the microbe appeared dependent upon the presence of the saliva pellicle (coating) on HA, since solutions containing proteins desorbed from HA during mock-adherence incubations possessed lytic activity that was 2- to 10-fold too low to account for the extents of lysis observed with greater than or equal to 10(8) input cells. These results demonstrate the potential antibacterial activity of acquired salivary pellicle on enamel in vivo and the likely role of lysozyme in this activity. The data also serve to caution that this widely used in vitro adherence model will not distinguish whole-cell adherence from the adsorption of radiolabeled DNA released from lysing cells. Several additional controls are suggested that will indicate whether test microbes remain intact or lyse during adherence trials. PMID- 3023242 TI - Routine clinical applications of hemodialysis-hemoperfusion in chronic renal failure: case reports. AB - Five hemodialysis patients were treated with combined hemodialysis-hemoperfusion with their conventional hemodialyzer plus a 70-gram ultrathin collodion coated activated charcoal device for a total of 63 months. Indications for this therapy included pericarditis, peripheral neuropathy, clotting of conventional hemodialyzers and reduction of dialysis time and frequency. The outcome was beneficial in all cases and stable biochemical and hematological parameters were maintained. No increase in heparin requirements was noted and the therapy was thought to be cost-effective. PMID- 3023241 TI - Vibration sensitivity as a parameter for detecting peripheral neuropathy. I. Results in healthy workers. AB - Two methods of evaluating the threshold for vibration perception were compared. Surprisingly it appears that the theoretically attractive, adaptive forced choice method does not result in lower variability than the method of limits. Moreover two devices were used to evaluate the threshold: the Optacon Tactile Tester and the "multirod". Based on the characteristics of these devices and the known properties of mechanoreceptors, it is argued that the two devices test different mechanoreceptor systems. The high correlation of threshold with age (r = 0.9) found by Arezzo and Schaumburg in measurements with the Optacon could not be reproduced. PMID- 3023243 TI - Pharmacodynamics (lung function tests, tremor measurements and cAMP determinations) of a single dose of 0.5 mg terbutaline subcutaneously during sustained-release theophylline medication in patients with asthmatic bronchitis. AB - A group of 10 patients with chronic asthmatic bronchitis was titrated with slow release theophylline (Theolin Retard) to a steady state plasma theophylline level of 10 mg/l. After one week of this treatment a single s.c. dose of 0.5 mg terbutaline (Bricanyl) at 10:30 a.m was administered. Lung function at 8:15 a.m. on this day was better than on the reference day without any medication, but the differences were not statistically significant. At 11:00 p.m., 30 min after s.c. administration of terbutaline all lung function parameters (VC, FEV1, MMEF and sGaw) were significantly raised compared to the values of 8:15 p.m. cAMP levels and tremor were significantly higher after the combined medication (30, as well as 150 min after s.c. administration of terbutaline) than on the reference day. This observation implies that we have to be careful with the administration of terbutaline s.c. to patients on theophylline treatment in the daily practice of the lung function laboratory. PMID- 3023244 TI - Increased release of hydrogen peroxide (H2O2) and superoxide anion (O-2) by murine macrophages in vitro after cis-platin treatment. AB - When murine macrophage (M phi) monolayers are treated with cis-Platin (10 micrograms or 5 micrograms/ml) for 30 min, 1, 2, 4, 8 and 24 h a significant increase in the release of H2O2 and O-2 by M phi is observed. The release of H2O2 and O-2 was comparatively much more when M phi were treated with 5 micrograms/ml cis-Platin than 10 micrograms/ml. However, it is observed that 4 h cis-Platin treatment results in comparative inhibition in the release of H2O2 and O-2. Further, we compared the release of H2O2 and O-2 by M phi treated with cis Platin, LPS and PMA. PMID- 3023245 TI - Marijuana effects on immunity: suppression of human natural killer cell activity of delta-9-tetrahydrocannabinol. AB - Delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, was tested for its ability to modulate human natural killer (NK) cell function. THC was toxic for peripheral blood lymphocytes at 20 micrograms/ml but not at 10 micrograms/ml or less. This component of marijuana also was inhibitory for NK activity against K562, a human tumor cell line at concentrations down to 5 micrograms/ml when pre-incubated with the effector cells. Suppression of NK function was dependent upon the concentration of THC and the length of time of pre-incubation but was independent of the ratio of effector to target cells. Prostaglandins were not involved in suppression of NK activity. PMID- 3023246 TI - In vivo immunomodulating activity of PK 1195, a structurally unrelated ligand for "peripheral" benzodiazepine binding sites--I. Potentiation in mice of the humoral response to sheep red blood cells. AB - PK 11195, an isoquinoline carboxamide compound with the highest affinity for the peripheral type benzodiazepine binding site has been shown to enhance the humoral response of mice to sheep red blood cells, a T cell dependent antigen. The compound was active when administered i.p. 1 day following immunization. The active doses ranged from 10 ng to 1 mg/kg. Such an immunomodulating activity might be related to the presence of peripheral type benzodiazepine binding sites, previously detected on the macrophage. PMID- 3023247 TI - Assignment and conformation of neurotensin in aqueous solution by 1H NMR. AB - A complete assignment of exchangeable and unexchangeable proton resonances of neurotensin 1-13 in aqueous solution has been carried out with the help of its 1 8 and 8-13 fragments. To detect formation of a secondary structure, the effects of peptide fragmentation, temperature decrease, pH changes and addition of denaturing agents on the neurotensin 1H NMR spectrum were investigated. The small changes observed in all cases support the conclusion that neurotensin exists mainly as a flexible random coiled polypeptidic chain in aqueous solution in agreement with previous CD studies. PMID- 3023249 TI - Erythrocyte lipid peroxidation and (Na+ + K+)-ATPase activity in cholesterol fed rabbits. AB - Cholesterol, phospholipid and lipid peroxide levels in plasma and erythrocytes as well as membrane-bound (Na+ + K+)-ATPase activity were determined in rabbits fed a high-cholesterol diet for 3 months. While cholesterol feeding caused an increase in lipid peroxide levels, (Na+ + K+)-ATPase activity was found to be reduced. According to this, we can assume that, in high-cholesterol fed rabbits, elevated lipid peroxides may be one of the responsible factors for the decreased erythrocyte (Na+ + K+)-ATPase activity. PMID- 3023248 TI - Chemical composition of Italian cooked dishes. AB - 50 different food items, including "first courses", main and side dishes, cheese and pork products, have been analysed for proteins, carbohydrates, fats, fiber, thiamin, riboflavin and vitamin A. The results obtained have been compared with the values determined on food samples by means of food composition tables so as to have information about the quantitative variations occurring after food processing. It can be concluded that composition tables report sufficiently reliable data with regard to the protein, fat and carbohydrate content for most of the considered food items. On the other hand, values for vitamins are lower by analysis than by calculation, suggesting that it is necessary to continually check data. PMID- 3023250 TI - Effect of l-carnitine on human neutrophil activity. AB - The effect of l-carnitine on human neutrophil oxidative metabolism was investigated, both on superoxide production and luminol amplified chemiluminescence (CL) in phorbol-myristate-acetate (PMA) stimulated cells. L carnitine either preincubated 10 minutes with the cells, before PMA challenge, or added simultaneously to the stimulator, inhibited superoxide generation. When tested in an O2- -generating system, such as xanthine-xanthine oxidase, l carnitine did not act as an O2- scavenger. On the PMA induced CL response the drug was ineffective as an inhibitor, if preincubated with the cell suspension before activation. When added together with PMA, l-carnitine significantly inhibited the CL. Taken together the results reveal that the drug might affect the interaction of PMA with its specific receptor in human neutrophils, that is protein kinase C. PMID- 3023251 TI - The lack of effects of alkylating agents on mammalian cell membranes. AB - The importance of cell membrane components as target sites for the action of 2,3,5-tris(ethyleneimino)-benzoquinone (Trenimon), mechlorethamine hydrochloride (HN2) and tris(2-chloroethyl) amine hydrochloride (HN3) was investigated. Uptake of 2-aminoisobutyric acid (AIBA) was studied under nonsaturating conditions where the transport system was rate-limiting for the uptake. Uptake of AIBA into L5178Y leukaemic cells was either inhibited or stimulated, depending on the type of the drug, the drug concentration and the length of incubation. Treatment of human erythrocytes with 10(-3) M HN3 produced new high-molecular-weight protein bands on SDS-polyacrylamide gel electrophoresis. Conversely, HN3 had no effect on L5178Y cell membrane proteins. Neither HN2 nor Trenimon produced any detectable changes in membrane proteins of L5178Y cells or human erythrocytes. None of the three drugs at concentrations and incubation conditions which inhibited cell replication changed the stoichiometry or dissociation constant of concanavalin A (Con A) binding sites on L5178Y cells. Trenimon at highly toxic concentrations had no effect on the fluidity of phospholipid membranes or of membranes of Ehrlich ascites tumour cells as analysed by ESR spin-label methods. The results presented here do not support the hypothesis that cell membranes are the primary target sites for alkylating drugs. PMID- 3023252 TI - Age related changes of glucagon binding and activity in isolated rat hepatocytes. AB - Hepatocytes isolated from young and old rats were compared for glucagon binding and its biological effect. Liver cells from young rats showed a receptor binding of the hormone significantly higher than cells from old rats. Such a behaviour was paralleled by the activity of the hormone. In fact, the glucagon-dependent stimulation of adenylate cyclase in the hepatocytes from the former group of animals was twice as high as in the latter. PMID- 3023253 TI - Changes in the levels of coenzyme Q homologues, alpha-tocopherol and malondialdehyde in human tissue during the course of circulatory shock. AB - Following our previous findings on mitochondrial oxidative damage during the course of circulatory shock in human muscular tissue, in the present work we examined the pathogenic connections between the electron-transport-chain enzymic activity and the ubiquinone metabolism. The effects of the oxidative damage on the alpha-tocopherol content and malondialdehyde (MDA) levels were also studied. The results reveal an involvement of cytochrome oxidase and coenzyme Q10 in the oxidative damage due to shock; alpha-tocopherol seems to show a particularly increased antioxidant activity contemporary with the marked increase in MDA levels. These findings suggest that the significant fall in the mitochondrial oxidative capacity could generate an oxygen free-radical production with subsequent peroxidative damage of the mitochondrial inner-membrane bilayer. PMID- 3023255 TI - Bone mineral measurement in the human spine using computed tomography. AB - A new method to measure bone mineral mass and density in human vertebrae was developed and clinically implemented. Using single-energy CT in the lumbar spine, it is possible to measure trabecular and cortical bone mineral mass regardless of the amount of soft tissue "background" present. Although the method is sensitive to the fat that resides in the marrow space of the vertebral body, an estimate of fat content can be incorporated into the calculations. We present and demonstrate the conceptual model upon which the method rests, and show that the results derived from its clinical use are highly correlated with those obtained from the conventional method of determining bone mineral concentration. PMID- 3023254 TI - Receptor-mediated endocytosis in liver parenchymal cells. PMID- 3023256 TI - Enalapril: a new angiotensin-converting enzyme inhibitor. PMID- 3023258 TI - Leukotriene B4 generation by polymorphonuclear leukocytes: possible involvement in the pathogenesis of headache. PMID- 3023257 TI - Some basic properties of sheep liver cAMP-dependent protein kinase. AB - Sheep liver cytosol (105,000 X g supernatant) yields two major peaks of protein kinase by using DEAE-Trisacryl M as an ion-exchange resin at pH 7.0. Peak I (Type I), corresponding to 30-50% of the total activity, is not retained by the column at a starting ionic strength of ca. 0.06 M, while Peak-II (Type-II) is eluting at 0.17 M ionic strength. Both peaks are found to be dependent on cAMP and are active on histone (ATP: Protein phosphotransferase, EC 2.7.1.37). Kms apparents for histone and ATP are 1.5 +/- 0.5 mg/ml and 16 +/- 4 microM, respectively, for PrK-I while that of PrK-II are 1.8 mg/ml and 28.6 microM, respectively. Both enzymes are found to be stable for two weeks at 4 degrees C. Molecular weight determination of crude extract (105,000 X g supernatant) show three peaks corresponding to the molecular weights of 251,000; 131,800 and 43,600. PMID- 3023259 TI - Energy dependence of the dose equivalent on the primary proton energy--comparison of CASIM and Moyer model calculations. PMID- 3023260 TI - Health education about AIDS among seropositive blood donors. AB - The New York Blood Center is developing a health education and psychosocial support program for blood donors who are notified that they are HIV antibody positive. The goals of that program are: to provide accurate and intelligible information about the test results to notified donors; to encourage behavior that will reduce the likelihood of spreading the virus; to encourage notified donors to behave in ways that will reduce the probability that they will develop AIDS; and to provide support and facilitate functional coping responses. This article reviews the theoretical and empirical work which informs the intervention program, and it describes how the program is being implemented. PMID- 3023261 TI - Histochemical distribution of potassium-dependent p-nitrophenylphosphatase in the calf intestine. AB - Potassium-dependent p-nitrophenylphosphatase was demonstrated, using the lead citrate method of Mayahara et al. (1980), in frozen sections of calf intestine fixed in formalin-calcium. The calcium chloride included in the fixative was shown to improve the localization of the reaction markedly. The phosphatase activity observed in the basolateral cell borders of the surface epithelium in the small intestine and colon was reduced by 10 mM ouabain and by substitution of sodium ions for potassium ions, confirming that the reaction was representative for the second step in the Na+/K+-ATPase complex. The intensity of the basolateral enzyme reaction was in the order: colon greater than duodenum, proximal jejunum greater than ileum greater than middle and distal jejunum. The crypts reacted weakly. A reaction in the brush border of the proximal jejunum and duodenum and a granular reaction in the supranuclear cytoplasm of the epithelial cells was not influenced by ouabain. The staining pattern for the potassium dependent phosphatase differed from that of alkaline phosphatase and Mg2+ dependent ATPase, which gave a reaction that was restricted to the brush border. PMID- 3023263 TI - A peculiar phenomenon: increased polypeptide hormone binding (receptor accumulation) in the midbody region. AB - Fluorescein-labeled polypeptide hormones, hormone analogons and fragments were selectively bound by the midbody, which bound neither con-A, nor the fluorescent stain in itself. This experimental observation suggests an accumulation in the midbody region of non-glycosilated hormone receptors, apparently to present a receptor pool for the separating daughter cells. PMID- 3023264 TI - A novel type of human papilloma virus DNA from the lesion of epidermodysplasia verruciformis. AB - Two types of DNAs were clonally purified from the virion fraction which was obtained by the cesium chloride density gradient centrifugation of a homogenate of scrapings from benign lesions of typical epidermodysplasia verruciformis. Restriction-enzyme maps were made for these DNAs, and the DNA-DNA hybridization of these cloned DNAs with 25 different types of human papilloma virus DNAs indicated that one of the DNA clones isolated is a new type of human papilloma virus DNA, and the other is the DNA of human papilloma virus type 21 or its subtype. PMID- 3023262 TI - Gluconeogenic-glycolytic capacities and metabolic zonation in liver of rats with streptozotocin, non-ketotic as compared to alloxan, ketotic diabetes. AB - Activities (mumol X min-1 X g liver) and zonal distributions of key enzymes of carbohydrate metabolism were studied in livers of streptozotocin-diabetic rats and compared to the values in alloxan-diabetes. Streptozotocin led to a non ketotic diabetes with blood glucose being increased by more than fivefold but ketone bodies being in the normal range, while alloxan produced a ketotic diabetes with blood glucose, acetoacetate and beta-hydroxybutyrate being elevated by more than fivefold. Portal insulin was decreased to about 20% in streptozotocin- and more drastically to about 7% in alloxan-diabetes. Conversely, portal glucagon was increased in the two states to about 250% and 180%, respectively. The glucogenic key enzyme phosphoenolpyruvate carboxykinase (PEPCK) was enhanced in streptozotocin- and alloxan-diabetes to over 300%, while the glycolytic pyruvate kinase L (PKL) was lowered to 65% and 80%, respectively. The normal periportal to perivenous gradient of PEPCK of about 3:1, as measured in microdissected tissue samples, was maintained with elevated activities in the two zones. The normal periportal to perivenous gradient of PKL of 1:1.7 was diminished with lowered activities in the two zones. The glucogenic glucose-6 phosphatase (G6Pase) was increased in streptozotocin- and alloxan-diabetes to 130% and 140%, respectively, while the glucose utilizing glucokinase (GK) was decreased to 60% and 50%, respectively. The normal periportal to perivenous gradient of G6Pase, demonstrated histochemically, remained unaffected. Carnitine palmitoyltransferase (CPT) was increased to over 190% and acetyl-CoA carboxylase (ACC) was decreased to 60% in streptozotocin, non-ketotic diabetes, while the two enzymes were altered more drastically to 400% and 50%, respectively, in alloxan, ketotic diabetes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3023266 TI - Effects of 8-bromoadenosine 3':5'-cyclic monophosphate on proteolytic enzymes, adhesiveness and lung-colonizing ability of cloned low-metastatic Lewis lung carcinoma cells. AB - Treatment of cloned low-metastatic Lewis lung carcinoma cells (P-29) with dimethylsulfoxide or butyric acid resulted in enhancement of their lung colonizing ability. This was accompanied with increases in cathepsin B activity, the production of plasminogen activator, and adhesiveness, mainly heterotypic adhesion (adhesion to monolayers of endothelial cells) of dimethylsulfoxide treated cells and homotypic aggregation of butyric acid-treated cells. Treatment of P-29 cells with 8-bromoadenosine 3':5'-cyclic monophosphate (8-bromo-cyclic AMP) also resulted in increases in cathepsin B activity and the production of plasminogen activator. However, it did not enhance either heterotypic adhesion or homotypic aggregation of the cells. The lung-colonizing ability of 8-bromo-cyclic AMP-treated P-29 cells was examined after their intravenous injection into male C57BL/6 mice. It was found that these cells did not have enhanced lung-colonizing ability. These results suggest that high activities of proteolytic enzymes such as cathepsin B and plasminogen activator in tumor cells are not sufficient alone for completing the metastatic process, but that other properties of tumor cells such as adhesiveness are also necessary. PMID- 3023265 TI - A follow-up study of hepatitis B virus carriers at hospital. AB - For the purpose of clarifying the relation between hepatitis B virus (HBV) and hepatocellular carcinoma (HCC), a follow-up study of 1,614 HBV carriers (910 males and 704 females) was conducted at Kure National Hospital from 1970 to 1984. During the follow-up period (40.3 months on average), 247 HBV carriers died. Deaths from HCC and liver cirrhosis (LC) numbered 99 of 168 males (58.9%) and 38 of 79 females (48.2%), the chi-square test revealing no sex difference. Of 142 deaths from malignant neoplasm, HCC accounted for 52 of 96 males (54.2%) and 19 of 46 females (41.3%), the chi-square test again revealing no sex difference. The adjusted odds ratio of death from HCC among HBV carriers (1,614) with respect to HBV non-carriers (176,909) in this hospital during the study period (14-years) was 9.52 (males 7.94, females 13.39). The expected number of deaths was calculated based on the population of Hiroshima Prefecture (1978-1980) as a standard. The adjusted O/E (observed number/expected number) ratio of death from HCC was 6.66 for males and 12.13 for females, and the adjusted O/E ratio of death from LC was 5.41 for males and 11.02 for females. These findings suggest that HBV is a high risk factor of both HCC and LC, and, unlike the general population, female HBV carriers may have a rather higher risk of death from HCC and LC than male HBV carriers. PMID- 3023267 TI - Studies on antiviral agents. IV. Synthesis and in vitro antiviral activity of new N-palmitoylkanamycin A derivatives. PMID- 3023268 TI - Viriplanin A, a new anthracycline antibiotic of the nogalamycin group. I. Isolation, characterization, degradation reactions and biological properties. AB - Viriplanin A, a new anthracycline antibiotic produced by Ampullariella regularis strain SE 47, was isolated from a raw product that demonstrated activity against Herpes simplex viruses. Based on spectroscopic data, the structure of the aglycone, viriplanol, was determined, and the antibiotic was found to contain the sugar moieties 2-deoxy-L-fucose, 4-O-mesaconoyl-L-diginose and decilonitrose. In solution viriplanin A is very unstable to light. The antibiotic belongs to the nogalamycin group and is related to arugomycin and decilorubicin. PMID- 3023269 TI - Purification and characterization of a streptomycete collagenase. AB - A soil streptomycete designated as Streptomyces sp. A8 produced an extracellular collagen hydrolysing enzyme that appeared to be 'true collagenase' as it degraded native collagen under physiological conditions and cleaved the synthetic hexapeptide 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-a rginine into two tripeptides. The enzyme was purified by diethyl aminoethyl cellulose chromatography and Sephadex G-150 gel filtration. The purified enzyme had an apparent molecular weight of about 75,000 by SDS-polyacrylamide gel electrophoresis. Treatment with lithium chloride did not dissociate it into subunits. A strong inhibition was observed with chelating agents such as alpha alpha-dipyridyl and 8-hydroxyquinoline. Ethylene diamine tetraacetate completely inhibited the enzyme activity. Among the cations tested only Ca2+ and Mg2+ enhanced the collagenase activity. Heavy metal ions like Pb2+, Ag+, Cu2+ and Zn2+ strongly inhibited the enzyme. The EDTA inhibition could be reversed with Ca2+. Cysteine and reduced glutathione caused significant reduction in enzyme activity. Parachloromercuribenzoate and iodoacetamide had no effect on the collagenase. Amino acid analysis revealed the absence of cysteine and tyrosine. Many of the properties were the same as collagenases of Clostridium histolyticum and Vibrio alginolyticus. PMID- 3023270 TI - Enhanced lipolysis is not evident in adipocytes from exercise-trained SHR. AB - Adipocytes from spontaneously hypertensive rats (SHR) are not as responsive to isoproterenol or dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) stimulation compared with Sprague-Dawley or Wistar-Kyoto rats. Lipolytic activity in adipocytes from trained normotensive rats was enhanced in response to 1 microM isoproterenol and 0.5 mM dibutyryl cAMP but not in adipocytes from trained SHR. Decreases in isoproterenol-stimulated (1 microM) cAMP accumulation were evident in adipocytes from trained normotensive rats but not in adipocytes from trained SHR. Basal and agonist-induced lipolysis in fat cells isolated from both normotensive rats and SHR immediately following a 60-min run was increased in both sedentary and trained rats. Adenylate cyclase activity in fat cell membranes was blunted in sedentary and trained SHR both in the absence and presence of 100 microM 5'-guanylyl imidophosphate. No apparent differences existed in antagonist affinity of binding sites for the antagonist dihydroalprenolol in normal rats or SHR. Evidence for a change in affinity of agonist isoproterenol might be indicated based on the enhanced potency of isoproterenol to stimulate lipolysis in trained normal rats. beta-Adrenergic receptor density and antagonist affinity were not different in normotensive rats and SHR in response to training. However, displacement of [3H]dihydroalprenolol in adipocytes from SHR required greater concentrations of isoproterenol compared with adipocytes from normotensive rats, further suggestive of increased agonist affinity of binding sites in normal rats. These data suggest a postreceptor lesion of the lipolytic pathway in adipocytes from spontaneously hypertensive rats, possibly at the guanine nucleotide regulatory protein level.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3023271 TI - Plasma adrenocorticotropin and cortisol responses to brief high-intensity exercise in humans. AB - The purpose of this study is to examine plasma cortisol and adrenocorticotropin (ACTH) levels following a brief high-intensity bout of exercise. Each subject (n = 6) performed a 1-min bout of exercise on a cycle ergometer at 120% of his maximum O2 uptake. Blood samples were collected at rest, immediately following the exercise bout, and at 5, 15, and 30 min postexercise. Mean (+/- SE) plasma ACTH levels increased significantly (P less than 0.05) from 2.2 +/- 0.4 pmol/l at rest to 6.2 +/- 1.7 pmol/l immediately following exercise. Mean (+/- SE) plasma cortisol levels increased significantly from 0.40 +/- 0.04 mumol/l at rest to 0.52 +/- 0.04 mumol/l at 15 min postexercise. These data show that brief high intensity exercise results in significant increases in plasma cortisol and ACTH levels. Furthermore, the temporal sequence between the two hormones suggests that the increase in plasma cortisol levels following brief high-intensity exercise is the result of ACTH-induced steroidogenesis in the adrenal cortex. PMID- 3023272 TI - Epithelial modulation of tracheal smooth muscle response to antigenic stimulation. AB - The role of the epithelium has been studied in the contractile responses of rat trachea. The different modulations observed are discussed in respect to vagal components of the epithelial layer. Responses of rat trachea to immunologic stimulation are shown to be dependent on the presence of the epithelium, which prolongs the relaxation stage without affecting the contractions. This prolongation is abolished by neonatal capsaicin pretreatment, whereas substance P induces a significantly greater relaxation of serotonin-precontracted intact than deepithelialized trachea. Serotonin concentration-response curves are shifted to the right in intact preparations, which is partly reversed by neonatal capsaicin pretreatment, but a hyporeactivity of the tissue exists. A relaxing factor released by the epithelium is hypothesized, possibly dependent on substance P ergic innervation. Muscarinic cholinergic innervation slightly modulates the contractions but not the relaxations in antigen-induced responses, independently on the presence of the epithelial layer. 4-Aminopyridine induces epithelium dependent potentiations of contractions to antigen and to serotonin, which involves acetylcholine at one step of the reaction cascade. Epithelial-dependent contracting and relaxing factors are thus suggested in rat trachea. PMID- 3023273 TI - Distribution of cyclic AMP phosphodiesterase in adipose tissue from trained rats. AB - Fat cells were isolated from sedentary and exercise trained female Sprague-Dawley rats and cyclic AMP phosphodiesterase (cyclic AMP-PDE) activities were determined from crude homogenates of the fat cells in the whole homogenate, P5, P48, and S48 fractions. Exercise training resulted in a significant increase in the mean specific activity of cyclic AMP-PDE (pmol X min-1 X mg-1) from the whole homogenate and S48 fraction at cyclic AMP concentrations of 4, 8, and 16 microM and in the P48 fraction at 8 and 16 microM cyclic AMP. Cyclic AMP-PDE kinetic plots according to Lineweaver-Burk for the calculation of Michaelis constants (Km) and maximum enzyme velocities (Vmax) were nonlinear, indicating both a low and high enzyme form. The Michaelis constants were significantly lower in trained rats than those of its control for the high Km form of cyclic AMP-PDE in the whole and soluble fractions and for the low Km form of the P5 particulate fraction. The Vmax of the high Km form of the P48 particulate fraction from trained animals was also significantly higher than that found in its control. Phosphodiesterase inhibition by methylxanthines in the various fractions was similar in both trained and sedentary animals. These changes in specific activity, Michaelis constants, and Vmax of cyclic AMP-PDE from crude homogenates of isolated fat cells from exercise trained animals may account for the decreased intracellular levels of cyclic AMP following catecholamine stimulation of isolated fat cells from trained rats. PMID- 3023274 TI - Influence of beta-adrenergic blockade on the control of sweating in humans. AB - To evaluate the role of beta-adrenergic receptors in the control of human sweating, we studied six subjects during 40 min of cycle-ergometer exercise (60% maximal O2 consumption) at 22 degrees C 2 h after oral administration of placebo or nonselective beta-blockade (BB, 80 mg propranolol). Internal temperature (esophageal temperature, Tes), mean skin temperature (Tsk), local chest temperature (Tch), and local chest sweat rate (msw) were continuously recorded. The control of sweating was best described by the slope of the linear relationship between msw and Tes and the threshold Tes for the onset of sweating. The slope of the msw-Tes relationship decreased 27% (P less than 0.01), from 1.80 to 1.30 mg X cm-2 X min-1 X degree C-1 during BB. The Tes threshold for sweating (36.8 degrees C) was not altered as the result of BB. These data suggest that BB modified the control of sweating via some peripheral interaction. Since Tsk was significantly (P less than 0.05) reduced during BB exercise, from a control value of 32.8 to 32.2 degrees C, we evaluated the influence of the reduction in local skin temperature (Tsk) in the altered control of sweating. Reductions in Tch accounted for only 45% of the decrease in the slope of the msw-Tes relationship during BB. Since evaporative heat loss requirement during exercise with BB, as estimated from the energy balance equation, was also reduced 18%, compared with control exercise, we concluded that during BB the reduction in sweating at any Tes is the consequence of both a decrease in local Tsk and a direct effect on sweat gland. PMID- 3023275 TI - Adrenergic and local control of O2 uptake during and after severe hypoxia. AB - The distribution of whole-body O2 supply during severe hypoxia and recovery and its relation to the regional distribution of O2 deficit and repayment was studied. Mongrel dogs were anesthetized, paralyzed, and ventilated to maintain an end-tidal PCO2 between 35 and 40 Torr. In one group, the alpha- and beta adrenergic receptors were blocked to eliminate neural and humoral adrenergic influences. In a second group, alpha-adrenergic receptors were stimulated to decrease O2 delivery by excessive vasoconstriction. In a third group, beta adrenergic receptors were stimulated to increase O2 delivery. Whole-body and hindlimb muscle O2 uptake and vascular responses were measured during normoxic control, 15 or 30 min of severe hypoxia (9% O2 in N2), and 20 or 30 min of normoxic recovery, respectively. The whole-body O2 deficit and excess O2 uptake in recovery were partitioned into muscle and nonmuscle areas. The data showed that neural or humoral influences had little effect on the regional distribution of the total O2 deficit and O2 excess in recovery. The O2 deficit could be decreased somewhat by increasing delivery, but the amount of excess O2 used in recovery was unaffected. This suggested that the excess O2 use in recovery was more a function of an energy deficit during hypoxia and not an O2 deficit. PMID- 3023276 TI - Propranolol does not impair exercise oxygen uptake in normal men at high altitude. AB - Decreased maximal O2 uptake (VO2max) and stimulation of the sympathetic nervous system have been previously shown to occur at high altitude. We hypothesized that tachycardia mediated by beta-adrenergic stimulation acted to defend VO2max at high altitude. Propranolol treatment beginning before high-altitude (4,300 m) ascent reduced heart rate during maximal and submaximal exercise in six healthy men treated with propranolol (80 mg three times daily) compared with five healthy subjects receiving placebo (lactose). Compared with sea-level values, the VO2max fell on day 2 at high altitude, but the magnitude of fall was similar in the placebo and propranolol treatment groups (26 +/- 6 vs. 32 +/- 5%, P = NS) and VO2max remained similar at high altitude in both groups once treatment was discontinued. During 30 min of submaximal (80% of VO2max) exercise, propranolol treated subjects maintained O2 uptake levels that were as large as those in placebo subjects. The maintenance of maximal or submaximal levels of O2 uptake in propranolol-treated subjects at 4,300 m could not be attributed to increased minute ventilation, arterial O2 saturation, or hemoglobin concentration. Rather, it appeared that propranolol-treated subjects maintained O2 uptake by transporting a greater proportion of the O2 uptake with each heartbeat. Thus, contrary to our hypothesis, beta-adrenergic blockade did not impair maximal or submaximal O2 uptake at high altitude due perhaps to compensatory mechanisms acting to maintain stroke volume and cardiac output. PMID- 3023277 TI - Interaction of rat Sertoli cells with a collagen lattice in vitro. AB - Sertoli cells from rats aged 15, 20, and 25 d were subcultured onto collagen coated, plastic dishes. If the collagen was released from the plastic surface by rimming, the floating rafts of collagen showed uniform shrinkage. If the collagen was allowed to remain attached to the plastic, holes appeared in the collagen with cells from rats aged 25 d but not with those of 15 d. Cells from rats aged 20 d caused fewer and smaller holes to appear. The holes were associated with the formation of clumps of spherical cells from which elongated Sertoli cells extended into the surrounding collagen to end near holes. Rhodamine-phalloidin revealed diffusely distributed actin in the spherical cells in contrast to well developed microfilaments in the peripheral elongated cells. Addition of cytochalasin B (5 micrograms/ml) to the medium prevented contraction of the floating rafts and the development of holes in the attached collagen. In addition, cytochalasin B caused the peripheral cells to become spherical and to separate from the clumps. Moreover, rhodamine-phalloidin revealed that actin in the peripheral cells occurred as clumps without microfilaments when cytochalasin B was present. When Sertoli cells were subcultured onto silicone rubber films, the cells produced wrinkling of the rubber surface within 4 h of plating. These observations were interpreted to mean that Sertoli cells exert local tractional forces on various substrata. These forces require actin, presumably acting by a contractile mechanism. When the collagen is attached to plastic and the cells are organized into clumps with radiating elongated cells (cells from rats aged 25 d), the tractional forces in the elongated cells reorganize the collagen fibers to produce holes. When cells are uniformly distributed (cells from rats aged 15 d), holes are not formed. When the collagen is released from the plastic surface, tractional forces cause the floating rafts to shrink. These interactions of the cells with collagen are likely to be important in determining the shape of the Sertoli cell in vivo, the polarity of the cell, and its biochemical differentiation. PMID- 3023278 TI - Human B lymphocytes immortalization by Epstein-Barr virus in the presence of cyclosporin A. AB - A simple method was devised for the establishment of continuous lymphoblastoid cell lines (LCL). Unfractionated mononuclear cells collected from healthy donors were infected in vitro by Epstein-Barr Virus (EBV) (strain B95-8) under specific conditions: an immunosuppressive drug, Cyclosporin-A (CS-A, Sandoz), associated with the use of a feeder layer (MRC-5) led to 100% efficiency of LCL establishment. A bank of 400 LCL was set up for completion of genetic studies. Regression and kinetics of virus-induced transformation were monitored and related to donors' EBV immune status. Mean time of LCL establishment and probability of regression among seropositive donors were not linked to any given value titer of antibodies against Viral Capsid Antigen (VCA) or against Epstein Barr Nuclear Antigen (EBNA). However, when the anti-VCA:anti-EBNA ratio was considered, this parameter seemed to be linked to the kinetics of transformation but not to the probability of regression. Once LCL are established, large quantities of human cells can be produced. The complete cellular DNA is available so that any part of it can be scrutinized. Moreover, some of the phenotypic characteristics of these B cells can be used for a wide range of investigations. PMID- 3023280 TI - Gene cluster of Pseudomonas syringae pv. "phaseolicola" controls pathogenicity of bean plants and hypersensitivity of nonhost plants. AB - Loss of the ability of Pseudomonas syringae pv. "phaseolicola" NPS3121 to elicit a hypersensitive response on tobacco and other nonhost plants was associated with loss of pathogenicity on the susceptible host bean. Eight independent, prototrophic transposon Tn5 insertion mutants which had lost the ability to elicit a hypersensitive response on tobacco plants were identified. Six of these mutants no longer produced disease lesions on primary leaves of the susceptible bean cultivar Red Kidney and failed to elicit a hypersensitive response on the resistant bean cultivar Red Mexican and on the nonhost plants tomato, cowpea, and soybean. The two remaining mutants had reduced pathogenicity on Red Kidney bean and elicited variable hypersensitive responses on the other plants tested. Southern blot analysis indicated that each mutant carried a single independent Tn5 insertion in one of three EcoRI fragments of about 17, 7, and 5 kilobases. Marker exchange mutagenesis further supported the conclusion that the pleiotropic mutant phenotype was not associated with multiple Tn5 insertions. A genomic library of the wild-type strain was constructed in the cosmid vector pLAFR3. A recombinant plasmid, designated pPL6, that carried P. syringae pv. "phaseolicola" genomic sequences was identified by colony hybridization. This plasmid restored the wild-type phenotype to all but one mutant, suggesting that genes affected by the insertions were clustered. Structural analysis of pPL6 and the wild-type genome indicated that the 17- and 5-kilobase EcoRI fragments were contiguous in the strain NPS3121 genome. PMID- 3023279 TI - Translational coupling in Escherichia coli of a heterologous Bacillus subtilis Escherichia coli gene fusion. AB - The efficient expression in Escherichia coli of the Tn9-derived chloramphenicol acetyltransferase (EC 2.3.1.28) gene fused distal to the promoter and N terminus of the Bacillus subtilis aprA gene was dependent on the initiation of translation from the ribosome-binding site in the aprA gene. PMID- 3023282 TI - Cloning and regulation of Erwinia herbicola pigment genes. AB - The genes coding for yellow pigment production in Erwinia herbicola Eho10 (ATCC 39368) were cloned and localized to a 12.4-kilobase (kb) chromosomal fragment. A 2.3-kb AvaI deletion in the cloned fragment resulted in the production of a pink yellow pigment, a possible precursor of the yellow pigment. Production of yellow pigment in both E. herbicola Eho10 and pigmented Escherichia coli clones was inhibited by glucose. When the pigment genes were transformed into a cya (adenylate cyclase) E. coli mutant, no expression was observed unless exogenous cyclic AMP was provided, which suggests that cyclic AMP is involved in the regulation of pigment gene expression. In E. coli minicells, the 12.4-kb fragment specified the synthesis of at least seven polypeptides. The 2.3-kb AvaI deletion resulted in the loss of a 37K polypeptide and the appearance of a polypeptide of 40 kilodaltons (40K polypeptide). The synthesis of the 37K polypeptide, which appears to be required for yellow pigment production, was not repressed by the presence of glucose in the culture medium, as was the synthesis of other polypeptides specified by the 12.4-kb fragment, suggesting that there are at least two types of gene regulation involved in yellow pigment synthesis. DNA hybridization studies indicated that different yellow pigment genes exist among different E. herbicola strains. None of six pigmented plant pathogenic bacteria examined, Agrobacterium tumefaciens C58, Cornyebacterium flaccumfaciens 1D2, Erwinia rubrifaciens 6D364, Pseudomonas syringae ATCC 19310, Xanthomonas campestris 25D11, and "Xanthomonas oryzae" 17D54, exhibited homology with the cloned pigment genes. PMID- 3023281 TI - Complete nucleotide sequence and transcription of ermF, a macrolide-lincosamide streptogramin B resistance determinant from Bacteroides fragilis. AB - DNA sequence analysis of a portion of an EcoRI fragment of the Bacteroides fragilis R plasmid pBF4 has allowed us to identify the macrolide-lincosamide streptogramin B resistance (MLSr) gene, ermF. ermF had a relative moles percent G + C of 32, was 798 base pairs in length, and encoded a protein of approximately 30,360 daltons. Comparison between the deduced amino acid sequence of ermF and six other erm genes from gram-positive bacteria revealed striking homologies among all of these determinants, suggesting a common origin. Based on these and other data, we believe that ermF codes for an rRNA methylase. Analysis of the nucleotide sequences upstream and downstream from the ermF gene revealed the presence of directly repeated sequences, now identified as two copies of the insertion element IS4351. One of these insertion elements was only 26 base pairs from the start codon of ermF and contained the transcriptional start signal for this gene as judged by S1 nuclease mapping experiments. Additional sequence analysis of the 26 base pairs separating ermF and IS4351 disclosed strong similarities between this region and the upstream regulatory control sequences of ermC and ermA (determinants of staphylococcal origin). These results suggested that ermF was not of Bacteroides origin and are discussed in terms of the evolution of ermF and the expression of drug resistance in heterologous hosts. PMID- 3023283 TI - Evidence against direct involvement of cyclic GMP or cyclic AMP in bacterial chemotactic signaling. AB - Defects in phosphotransferase chemotaxis in cya and cpd mutants previously cited as evidence of a cyclic GMP or cyclic AMP intermediate in signal transduction were not reproduced in a study of chemotaxis in Escherichia coli and Salmonella typhimurium. In cya mutants, which lack adenylate cyclase, the addition of cyclic AMP was required for synthesis of proteins that were necessary for phosphotransferase transport and chemotaxis. However, the induced cells retained normal phosphotransferase chemotaxis after cyclic AMP was removed. Phosphotransferase chemotaxis was normal in a cpd mutant of S. typhimurium that has elevated levels of cyclic GMP and cyclic AMP. S. typhimurium crr mutants are deficient in enzyme III glucose, which is a component of the glucose transport system, and a regulator of adenylate cyclase. After preincubation with cyclic AMP, the crr mutants were deficient in enzyme II glucose-mediated transport and chemotaxis, but other chemotactic responses were normal. It is concluded that cyclic GMP does not determine the frequency of tumbling and is probably not a component of the transduction pathway. The only known role of cyclic AMP is in the synthesis of some proteins that are subject to catabolite repression. PMID- 3023285 TI - Tn5-induced mutants of Azotobacter vinelandii affected in nitrogen fixation under Mo-deficient and Mo-sufficient conditions. AB - Mutants of Azotobacter vinelandii affected in N2 fixation in the presence of 1 microM Na2MoO4 (conventional system), 50 nM V2O5, or under Mo deficiency (alternative system) have been isolated after Tn5 mutagenesis with the suicide plasmid pSUP1011. These mutants can be grouped into at least four broad phenotypic classes. Mutants in the first class are Nif- under Mo sufficiency but Nif+ under Mo deficiency or in the presence of V2O5. A nifk mutant and a mutant apparently affected in regulation of the conventional system belong to this class. Mutants in the second class are Nif- under all conditions. An FeMo cofactor-negative mutant (NifB-) belongs to this class, implying an involvement of nifB in both the conventional and the alternative N2 fixation systems. The third mutant class consists of mutants incapable of N2-dependent growth under Mo deficiency. Most of the mutants in this class are also affected in N2 fixation in the presence of 1 microM Na2MoO4, with acetylene reduction rates ranging from 28 to 51% of the rates of the wild type. Strains constructed by genetic transfer of the Kanr marker of mutants from this class into nifHDK or nifK deletion mutants showed N2-dependent growth only in the presence of V2O5, suggesting that growth in the presence of V2O5 and growth under Mo deficiency are independent phenomena. The only mutant in the fourth class shows wild-type nitrogenase activity under Mo sufficiency, but only 10% of the acetylene reduction activity of the wild type in the presence of 50 nM V2O5. The acetylene reduction rates of whole cells of this mutant are identical in Mo-deficient medium and in medium containing V2O5. The conventional nitrogenase subunits are expressed in this mutant even under Mo deficiency or in the presence of V2O5; however, the NH4+- and Mo-repressible proteins normally seen under these conditions could not be detected on two dimensional gels. The Tn5 insertion carried by this mutant makes N2 fixation dependent solely on the conventional system and consequently abolishes the vanadium effect. PMID- 3023284 TI - Regulation of phosphatidylserine synthase from Saccharomyces cerevisiae by phospholipid precursors. AB - The addition of ethanolamine or choline to inositol-containing growth medium of Saccharomyces cerevisiae wild-type cells resulted in a reduction of membrane associated phosphatidylserine synthase (CDPdiacylglycerol:L-serine O phosphatidyltransferase, EC 2.7.8.8) activity in cell extracts. The reduction of activity did not occur when inositol was absent from the growth medium. Under the growth conditions where a reduction of enzyme activity occurred, there was a corresponding qualitative reduction of enzyme subunit as determined by immunoblotting with antiserum raised against purified phosphatidylserine synthase. Water-soluble phospholipid precursors did not effect purified phosphatidylserine synthase activity. Phosphatidylserine synthase (activity and enzyme subunit) was not regulated by the availability of water-soluble phospholipid precursors in S. cerevisiae VAL2C(YEp CHO1) and the opi1 mutant. VAL2C(YEp CHO1) is a plasmid-bearing strain that over produces phosphatidylserine synthase activity, and the opi1 mutant is an inositol biosynthesis regulatory mutant. The results of this study suggest that the regulation of phosphatidylserine synthase by the availability of phospholipid precursors occurs at the level of enzyme formation and not at the enzyme activity level. Furthermore, the regulation of phosphatidylserine synthase is coupled to inositol synthesis. PMID- 3023286 TI - Cyclic AMP inhibits developmental regulation of Chlamydia trachomatis. AB - The effect of cyclic AMP (cAMP) on the chlamydial growth cycle was studied with Chlamydia trachomatis-infected HeLa cells. At concentrations of 1 mM, cAMP had a profound effect on the chlamydial developmental cycle, resulting in small, immature inclusions. Immunoblot analysis revealed the absence of elementary body (EB)-specific antigens in the cAMP-treated cells. This effect was observed only if cAMP was added within the first 12 h of incubation and continued thereafter. Its withdrawal at any time from the medium led to the reappearance of fully mature, infectious organisms. Analogs or breakdown products of cAMP exerted no inhibitory effect on chlamydial development. Intracellular inclusions from the cAMP-treated cells were unable to infect fresh HeLa monolayers, in contrast to the completely infectious nontreated inclusions. Protein profiles of the cAMP treated organisms (at any time point) resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis very closely resembled reticulate bodies (RB) and did not possess characteristic EB-binding proteins. Collectively, these observations suggest an inhibitory role for cAMP at the RB stage of intracellular development. We also identified a cAMP receptor protein which is associated with RB and not with EB, further supporting a role for this system in the developmental regulation of chlamydiae. PMID- 3023287 TI - Paraquat-mediated selection for mutations in the manganese-superoxide dismutase gene sodA. AB - We report the unexpected result that Escherichia coli isolates containing a multicopy plasmid (pDT1.5) carrying the manganese-superoxide dismutase gene sodA were more sensitive than the wild type to paraquat-mediated growth inhibition. The pDT1.5 locus responsible for the paraquat-sensitive phenotype was delimited to a 0.6-kilobase segment by transposon Tn5 mutagenesis. Moreover, superoxide dismutase activity was the same as in the wild type in strains carrying pDT1.5::Tn5 insertions mapping to the 0.6-kilobase locus. These data identify the 0.6-kilobase segment as the locus of sodA and establish an association between growth inhibition by paraquat and the function of the plasmid-borne sodA gene. PMID- 3023288 TI - Naturally occurring TOL plasmids in Pseudomonas strains carry either two homologous or two nonhomologous catechol 2,3-oxygenase genes. AB - Structural genes for catechol 2,3-oxygenase (C23O) were cloned from the TOL plasmids pWW5, pWW14, pWW74, pWW84, and pWW88 isolated from Pseudomonas strains of diverse geographical origins. Each pKT230-based C23O+ recombinant plasmid carried a 2.05-kilobase XhoI insert which showed strong homology in Southern hybridizations with the xylE gene from the archetype TOL plasmid pWW0. Fragments were mapped for restriction endonuclease sites and were classified into two closely related groups on the basis of restriction maps. C23O structural genes were located on cloned fragments by a combination of subcloning and site-specific mutagenesis. All five TOL plasmids examined yielded clones whose maps differed from that of xylE of pWW0 by only a single XbaI site, but in addition plasmids pWW5, pWW74, and pWW88 carried a second, homologous C23O gene with seven further restriction site differences. The remaining plasmids, pWW14 and pWW84, carried a second nonhomologous C23O gene related to the second C23O gene (C23OII) of TOL plasmid pWW15 described previously (H. Keil, M. R. Lebens, and P. A. Williams, J. Bacteriol. 163:248-255, 1985). Thus, each naturally occurring TOL plasmid in this study appears to carry genes for two meta cleavage dioxygenases. PMID- 3023289 TI - Lactose and melibiose metabolism in Erwinia chrysanthemi. AB - A Lac+ mutant of Erwinia chrysanthemi was isolated from the Lac- wild type on lactose agar. beta-Galactosidase was expressed independently of lactose transport in both the mutant and the wild type, and neither strain expressed thiogalactoside transacetylase. Lactose transport and alpha-galactosidase, constitutive in the Lac+ strain, were coordinately induced in the Lac- strain by melibiose and raffinose but not by isopropyl-beta-D-thiogalactopyranoside or thiomethyl-beta-D-galactopyranoside. Melibiose was a strong inhibitor of both the melibiose- and the raffinose-induced lactose permeases, whereas raffinose was a strong inhibitor of only the raffinose-induced lactose permease. PMID- 3023291 TI - recA (Srf) suppression of recF deficiency in the postreplication repair of UV irradiated Escherichia coli K-12. AB - The mechanism by which recA (Srf) mutations (recA2020 and recA801) suppress the deficiency in postreplication repair shown by recF mutants of Escherichia coli was studied in UV-irradiated uvrB and uvrA recB recC sbcB cells. The recA (Srf) mutations partially suppressed the UV radiation sensitivity of uvrB recF, uvrB recF recB, and uvrA recB recC sbcB recF cells, and they partially restored the ability of uvrB recF and uvrA recB recC sbcB recF cells to repair DNA daughter strand gaps. In addition, the recA (Srf) mutations suppressed the recF deficiency in the repair of DNA double-strand breaks in UV-irradiated uvrA recB recC sbcB recF cells. The recA2020 and recA801 mutations do not appear to affect the synthesis of UV radiation-induced proteins, nor do they appear to produce an altered RecA protein, as detected by two-dimensional gel electrophoresis. These results are consistent with the suggestion (M. R. Volkert and M. A. Hartke, J. Bacteriol. 157:498-506, 1984) that the recA (Srf) mutations do not act by affecting the induction of SOS responses; rather, they allow the RecA protein to participate in the recF-dependent postreplication repair processes without the need of the RecF protein. PMID- 3023290 TI - Escherichia coli DnaK protein possesses a 5'-nucleotidase activity that is inhibited by AppppA. AB - AppppA and the DnaK protein have both been hypothesized to function in regulating the heat shock response of Escherichia coli. The proposals are that AppppA serves as a signal (alarmone) to turn on the heat shock response, whereas the DnaK protein is necessary to turn off the heat shock response. A simple model would be that the DnaK protein turns off the response by degrading AppppA. We disproved this model by demonstrating that the DnaK protein possesses a 5'-nucleotidase activity capable of degrading many cellular nucleotides but not AppppA. Although AppppA was not a substrate, it did inhibit the 5'-nucleotidase activity of the DnaK protein. This inhibition may be specific and have biological function since the mutant DnaK756 protein, which is defective in turning off the heat shock response, is partially desensitized to AppppA inhibition. These findings led us to consider other possible mechanisms for AppppA and the DnaK protein in heat shock regulation. PMID- 3023292 TI - Cloning and expression of the Rhodobacter sphaeroides reaction center H gene. AB - The Rhodobacter sphaeroides structural gene (puhA) for the reaction center H polypeptide has been identified and cloned by using restriction fragements specific for the analogous Rhodobacter capsulatus gene as a heterologous hybridization probe. The presence of puhA on a 1.45-kilobase BamHI restriction fragment was confirmed by partial DNA sequence analysis and by the synthesis of an immunoreactive Mr-28,000 reaction center H polypeptide in an R. sphaeroides coupled transcription-translation system. Approximately 450 base pairs of DNA upstream of the puhA gene were sufficient for expression of this protein in vitro. Northern RNA-DNA blot analysis with an internal puhA-specific probe identified at least two, apparently monocistronic, transcripts present at different cellular levels under physiological conditions known to affect the cellular content of both reaction center complexes and photosynthetic membrane. Northern blot analysis with specific upstream restriction fragment probes revealed that the 1,400-nucleotide puhA-specific mRNA had a 5' terminus upstream of the 1,130-nucleotide transcript. Both puhA-specific mRNA and immunoreactive reaction center H protein were detectable in chemoheterotrophically grown cells which lacked detectable bacteriochlorophyll and photosynthetic membrane. PMID- 3023294 TI - Tn5-mediated integration of the delta-endotoxin gene from Bacillus thuringiensis into the chromosome of root-colonizing pseudomonads. AB - Gene replacement mediated by Tn5 sequences was used to integrate the Bacillus thuringiensis subsp. kurstaki HD-1 delta-endotoxin gene (tox) into the chromosome of two corn root-colonizing strains of Pseudomonas fluorescens. A Tn5 transposase deletion element containing the tox gene (delta Tn5-tox) was substituted for a Tn5 element previously present in the P. fluorescens chromosome. Two classes of delta Tn5-tox elements were made. The first class encodes kanamycin resistance in addition to the Tox protein, whereas the second class encodes only the Tox protein. Both classes of delta Tn5-tox elements can no longer transpose, owing to a 324-base-pair deletion in the transposase gene of IS50R, minimizing the potential for horizontal gene transfer of the tox gene to other bacterial species. A frameshift mutation in the transposase gene of IS50L was also constructed to eliminate the possibility of suppression or of a spontaneous reversion at the ochre termination codon that would create an active transposase. Expression of the Tox protein in P. fluorescens strains 112-12 and Ps3732-3-7 was demonstrated by an immunological assay (Western blot) and toxicity against larvae of the tobacco hornworm (Manduca sexta). PMID- 3023293 TI - Cloning, DNA sequence, and expression of the Rhodobacter sphaeroides cytochrome c2 gene. AB - The Rhodobacter sphaeroides cytochrome c2 functions as a mobile electron carrier in both aerobic and photosynthetic electron transport chains. Synthetic deoxyoligonucleotide probes, based on the known amino acid sequence of this protein (Mr 14,000), were used to identify and clone the cytochrome c2 structural gene (cycA). DNA sequence analysis of the cycA gene indicated the presence of a typical procaryotic 21-residue signal sequence, suggesting that this periplasmic protein is synthesized in vivo as a precursor. Synthesis of an immunoreactive cytochrome c2 precursor protein (Mr 15,500) was observed in vitro when plasmids containing the cycA gene were used as templates in an R. sphaeroides coupled transcription-translation system. Approximately 500 base pairs of DNA upstream of the cycA gene was sufficient to allow expression of this gene product in vitro. Northern blot analysis with an internal cycA-specific probe identified at least two possibly monocistronic transcripts present in both different cellular levels and relative stoichiometries in steady-state cells grown under different physiological conditions. The ratio of the small (740-nucleotide) and large (920 nucleotide) cycA-specific mRNA species was dependent on cultural conditions but was not affected by light intensity under photosynthetic conditions. Our results suggest that the increase in the cellular level of the cytochrome c2 protein found in photosynthetic cells was due, in part, to increased transcription of the single-copy cyc operon. PMID- 3023295 TI - Dominance relationships among mutant alleles of regulatory gene araC in the Escherichia coli B/R L-arabinose operon. AB - The araBAD operon of Escherichia coli B/r is positively and negatively regulated by the araC+ regulatory protein. Mutations in gene araC can result in a variety of different regulatory phenotypes: araC null mutants (those carrying a null allele exhibiting no repressor or activator activity) are unable to achieve operon induction; araC-constitutive (araCc) mutants are partially constitutive, inducible by D-fucose, and resistant to catabolite repression; araCh mutants are hypersensitive to catabolite repression; and araCi mutants are resistant to catabolite repression. Various mutant alleles of gene araC were cloned into a derivative of plasmid pBR322 by in vivo recombination. Various heterozygous araC allelic combinations were constructed by transformation. Analysis of isomerase (araA) specific activity levels under various growth conditions indicated the following dominance relationships with regard to sensitivity to catabolite repression: araCh greater than araC+ greater than (araCc and araCi) greater than araC. It was concluded that the araCh protein may form a repressor complex that is refractory to removal by cyclic AMP receptor protein-cyclic AMP complex. This was interpreted in terms of the known nucleoprotein interactions between ara regulatory proteins and ara regulatory DNA. PMID- 3023296 TI - Isolation of an enzyme complex with carbon monoxide dehydrogenase activity containing corrinoid and nickel from acetate-grown Methanosarcina thermophila. AB - Fast protein liquid chromatography of cell extract from methanol- or acetate grown Methanosarcina thermophila resolved two peaks of CO dehydrogenase activity. The activity of one of the CO dehydrogenases was sixfold greater in acetate-grown compared with methanol-grown cells. This CO dehydrogenase was purified to apparent homogeneity (70 mumol of methyl viologen reduced per min per mg of protein) and made up greater than 10% of the cellular protein of acetate-grown cells. The native enzyme (Mr 250,000) formed aggregates with an Mr of approximately 1,000,000. The enzyme contained five subunits (Mrs 89,000, 71,000, 60,000, 58,000, and 19,000), suggesting a multifunctional enzyme complex. Nickel, iron, cobalt, zinc, inorganic sulfide, and a corrinoid were present in the complex. The UV-visible spectrum suggested the presence of iron-sulfur centers. The electron paramagnetic resonance spectrum contained g values of 2.073, 2.049, and 2.028; these features were broadened in enzyme that was purified from cells grown in the presence of medium enriched with 61Ni, indicating the involvement of this metal in the spectrum. The pattern of potassium cyanide inhibition indicated that cyanide binds at or near the CO binding site. The properties of the enzyme imply an involvement in the dissimilation of acetate to methane, possibly by cleavage of acetate or activated acetate. PMID- 3023297 TI - Assignment of symbiotic developmental phenotypes to common and specific nodulation (nod) genetic loci of Rhizobium meliloti. AB - Rhizobium meliloti nodulation (nod) genes required for specific infection and nodulation of alfalfa have been cloned. Transposon Tn5 mutagenesis defined three nod regions spanning 16 kilobases of the pSym megaplasmid. Genetic and cytological studies of 62 nodulation-defective mutants allowed the assignment of symbiotic developmental phenotypes to common and specific nod loci. Root hair curling was determined by both common (region I) and specific (region III) nod transcription units; locus IIIb (nodH gene) positively controlled curling on the homologous host alfalfa, whereas loci IIIa (nodFE) and IIIb (nodH) negatively controlled curling on heterologous hosts. Region I (nodABC) was required for bacterial penetration and infection thread initiation in shepherd's crooks, and the nodFE transcription unit controlled infection thread development within the alfalfa root hair. In contrast, induction of nodule organogenesis, which can be triggered from a distance, seemed to be controlled by common nodABC genes and not to require specific nod genes nodFE and nodH. Region II affected the efficiency of hair curling and infection thread formation. PMID- 3023298 TI - Transport of trehalose in Salmonella typhimurium. AB - We have studied trehalose uptake in Salmonella typhimurium and the possible involvement of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in this process. Two transport systems could recognize and transport trehalose, the mannose PTS and the galactose permease. Uptake of trehalose via the latter system required that it be expressed constitutively (due to a galR or galC mutation). Introduction of a ptsM mutation, resulting in a defective IIMan/IIIMan system, in S. typhimurium strains that grew on trehalose abolished growth on trehalose. A ptsG mutation, eliminating IIGlc of the glucose PTS, had no effect. In contrast, a crr mutation that resulted in the absence of IIIGlc of the glucose PTS prevented growth on trehalose. The inability of crr and also cya mutants to grow on trehalose was due to lowered intracellular cyclic AMP synthesis, since addition of extracellular cyclic AMP restored growth. Subsequent trehalose metabolism could be via a trehalose phosphate hydrolase, if trehalose phosphate was formed via the PTS, or trehalase. Trehalose-grown cells contained trehalase activity, but we could not detect phosphoenolpyruvate-dependent phosphorylation of trehalose in toluenized cells. PMID- 3023299 TI - Oxidative phosphorylation and energy buffering in cyanobacteria. AB - The onset of respiration in the cyanobacteria Anacystis nidulans and Nostoc sp. strain Mac upon a shift from dark anaerobic to aerobic conditions was accompanied by rapid energization of the adenylate pool (owing to the combined action of ATP synthase and adenylate kinase) and also the guanylate, uridylate, and cytidylate pools (owing to nucleoside diphosphate and nucleoside monophosphate kinases). Rates of the various transphosphorylation reactions were comparable to the rate of oxidative phosphorylation, thus explaining, in part, low approximately P/O ratios which incorporate adenylates only. The increase of ATP, GTP, UTP, and CTP levels (nanomoles per minute per milligram [dry weight]) in oxygen-pulsed cells of A. nidulans and Nostoc species was calculated to be, on average, 2.3, 1.05, 0.8, and 0.57, respectively. Together with aerobic steady-state pool sizes of 1.35, 0.57, 0.5, and 0.4 nmol/mg (dry weight) for these nucleotides, a fairly uniform turnover of 1.3 to 1.5 min-1 was derived. All types of nucleotides, therefore, may be conceived of as being in equilibrium with each other, reflecting the energetic homeostasis or energy buffering of the (respiring) cyanobacterial cell. For the calculation of net efficiencies of oxidative phosphorylation in terms of approximately P/O ratios, this energy buffering was taken into account. Moreover, in A. nidulans an additional 30% of the energy initially conserved in ATP by oxidative phosphorylation was immediately used up by a plasma membrane-bound reversible H+-ATPase for H+ extrusion. Consequently, by allowing for energy buffering and ATPase-linked H+ extrusion, maximum P/O ratios of 2.6 to 3.3 were calculated. By contrast, in Nostoc sp. all the H+ extrusion, appeared to be linked to a plasma membrane-bound respiratory chain, thus bypassing any ATP formation and leading to P/O ratios of only 1.3 to 1.5 despite the correction for energy buffering. PMID- 3023300 TI - Molecular cloning and analysis of genes for production of K5, K7, K12, and K92 capsular polysaccharides in Escherichia coli. AB - With a DNA fragment from within the region encoding the transport functions for K1 production as a hybridization probe in Southern blot experiments, homologous DNA sequences were detected in the DNA from Escherichia coli strains producing K5, K7, K92, and K100 capsular polysaccharides. No homology with the laboratory strain LE392 was detected. The same DNA probe was used to prescreen cosmid libraries in LE392 by colony hybridization, as a rapid method to isolate clones encoding the genes for K5, K7, K12, and K92 antigen production. Clones carrying sequences homologous to the probe that also produced capsular material were identified by using polyclonal and monoclonal antibodies raised against the K antigen in question and K antigen-specific phages. By restriction enzyme mapping of the appropriate cosmid clones it was possible to align the genes for the production of different K antigens in terms of common restriction endonuclease cleavage sites. A DNA fragment encoding the postulated transport functions for K7 antigen production could complement deletion mutations in the transport functions for K1 antigen production. Thus the transport to the cell surface of chemically distinct polysaccharides may be by a common process. Analysis in E. coli of the proteins produced by plasmids carrying the likely transport functions for K1, K5, and K7 antigen production revealed that each region coded for a similar polypeptide. PMID- 3023303 TI - Purification and some properties of buffalo liver cathepsin B. AB - Buffalo liver cathepsin B was isolated by acid extraction, ammonium sulfate fractionation, Sephadex gel filtration, DEAE-Sephadex chromatography and Sephacryl S-300 chromatography. The enzyme preparation was found to be homogeneous by gel filtration and SDS-polyacrylamide gel electrophoresis but could be resolved into two major and four minor protein bands on polyacrylamide gel electrophoresis in the absence of SDS. The enzyme showed catheptic activity against synthetic substrates such as BANA and BAPNA as well as against denatured hemoglobin. Various physico-chemical and enzymatic properties of the enzyme, such as molecular weight, Stokes radius, frictional coefficient, pH optimum, Michaelis constant, and Vmax, were determined. The values of these parameters were 27,500, 2.41 nm, 1.2, 6.5, 2.08 mM, and 42.4 units/mg, respectively. The hydrodynamic properties suggest a compact and globular conformation for this enzyme. Various compounds were tested for their influence on the activity of cathepsin B. Of these compounds, membrane phospholipids were found to increase significantly the activity of this enzyme. This increase in activity could be of physiological importance since the concentration of phospholipids is increased after endocytosis and autophagy. PMID- 3023302 TI - Genetic mapping in Escherichia coli of tmk, the locus for dTMP kinase. AB - The genetic location of tmk, the gene for dTMP kinase, has been mapped at min 24.0 on the Escherichia coli map. PMID- 3023301 TI - T-DNA and opine synthetic loci in tumors incited by Agrobacterium tumefaciens A281 on soybean and alfalfa plants. AB - We report here the molecular characterization of transferred DNA (T-DNA) in leguminous tumors incited by Agrobacterium tumefaciens A281 harboring the tumor inducing plasmid pTiBo542. The T-DNA is composed of two regions named TL (left portion)-DNA and TR (right portion)-DNA, in accordance with the nomenclature for the octopine strains. TL-DNA is defined by several internal HindIII restriction fragments totaling 10.8 kilobase pairs (kbp) in uncloned soybean and alfalfa tumors. Alfalfa tumor DNA may contain one more HindIII fragment at the left end of TL-DNA than does soybean tumor DNA. TR-DNA has a 5.8-kbp BamHI-EcoRI internal fragment. All borders other than the left border of TL-DNA appear to be the same within the detection limits of Southern blot hybridization experiments. The two T DNA regions are separated by 16 to 19 kbp of DNA not stably maintained in tumors. The distance from the left border of TL-DNA to the right border of TR-DNA is approximately 40 kbp. Loci for the mannityl opines are situated in TR-DNA, based on genetic and biochemical criteria. PMID- 3023304 TI - Pyruvate dehydrogenase and the path of lactate degradation in Desulfovibrio vulgaris Miyazaki F. AB - Pyruvate dehydrogenase from Desulfovibrio vulgaris Miyazaki F was partially purified from the soluble fraction of the bacterial sonicate, and characterized. The enzyme catalyzes oxidative decarboxylation of pyruvate to produce acetyl-CoA, in contrast to statements in current review articles in which acetyl phosphate is indicated to be a direct decomposition product of pyruvate in sulfate-reducing bacteria. The established reaction stoichiometry is: pyruvate + CoA + FMN--- acetyl-CoA + CO2 + FMNH2. The Km values are 2.9 mM for pyruvate, 32 microM for CoA and 6.7 mumol for FMN. Participation of thiamine diphosphate in the enzymic process was not proven. 2-Oxobutyrate, but not 2-oxoglutarate, can substitute for pyruvate. The three flavin compounds, FMN, FAD, and flavodoxin, as well as clostridial ferredoxin, serve as electron carriers for the enzyme. Thus the enzyme is a kind of pyruvate synthase [EC 1.2.7.1], but acts in the direction of pyruvate degradation in the growing cells. The rate of cytochrome C3 reduction is extremely low, but in the presence of flavodoxin as an electron mediator, the reduction rate of cytochrome C3 becomes faster than the reduction rate of flavodoxin alone. It seems that the physiological electron acceptor for this enzyme is flavodoxin, which might be complexed with cytochrome C3 to produce a very efficient electron transfer system in the cell. The soluble fraction of D. vulgaris cells has been proved to contain, in addition to the pyruvate dehydrogenase, lactate dehydrogenase (Ogata, M., Arihara, K., & Yagi, T. (1981) J. Biochem. 89, 1423-1431), phosphate acetyltransferase and acetate kinase, i.e., all the enzymes necessary to convert lactate to acetate, producing ATP by substrate level phosphorylation. PMID- 3023307 TI - Regulation of myeloperoxidase gene expression by the tumor promoter 12-O tetradecanoylphorbol-13-acetate in human leukemia HL-60 cells. AB - Myeloperoxidase synthesis during induction of differentiation of human promyelocytic leukemia HL-60 cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied. Differentiation was characterized by morphological changes, arrest of cell proliferation, development of cell adherence, and increased secretion of lysozyme. The cellular myeloperoxidase activity decreased early during induction of differentiation by TPA. Pulse-labeling experiments indicated that the rate of myeloperoxidase synthesis decreased to an undetectable level in cells exposed to TPA for 22 h. The relative amounts of myeloperoxidase mRNA in TPA-treated and untreated cells were determined by measuring translatable mRNA activity in a reticulocyte lysate system. Reduction in the myeloperoxidase mRNA level was observed as early as after 3 h treatment with TPA, and no myeloperoxidase mRNA was detected after 24 h. Time course experiments indicated that the time required for 50% reduction of myeloperoxidase mRNA in TPA-treated cells was approximately 5 h. These results suggest that TPA induces decrease of myeloperoxidase activity in HL-60 cells at a pretranslational level. PMID- 3023305 TI - Induction and regulation of human interleukin 2 gene expression: significance of protein kinase C activation. AB - Phytohemagglutinin (PHA) and a tumor-promoting phorbol ester, 12-O tetradecanoylphorbol-13-acetate (TPA) act synergistically to induce interleukin 2 (IL2) mRNA in human lymphocytes in vitro. The induction was inhibited by a potent inhibitor of protein kinase C (C-kinase), 1-(5-isoquinolinylsulfonyl)-2 methylpiperazine (H-7) at less than 10 microM. H-7 inhibited C-kinase activity itself in lymphocytes at the same range of the concentration but did not interfere with the translocation of C-kinase from the cytosol to the membrane fraction of the lymphocytes induced by TPA. H-7 is also known to inhibit cAMP dependent protein kinase (A-kinase) and cGMP-dependent protein kinase (G-kinase). However, the lymphocytes cultured with dibutyryl cAMP or dibutyryl cGMP could not be activated to produce IL2 mRNA. These results show that activation of C-kinase but not A-kinase and G-kinase is necessary in signal transduction for IL2 gene expression. Prostaglandin E2, which is known to elevate intracellular cAMP level, also inhibited IL2 mRNA induction in the lymphocytes stimulated with PHA and TPA. Addition of alpha-methylornithine and methylglyoxal bis (guanyl hydrazone), which inhibit polyamine synthesis, did not affect the induction of IL2 mRNA in the lymphocytes stimulated with PHA and TPA, indicating that polyamine synthesis is not necessary for IL2 mRNA induction. PMID- 3023308 TI - NAD-glycohydrolase activity of botulinum C2 toxin: a possible role of component I in the mode of action of the toxin. AB - C2 toxin (C2T) elaborated by Clostridium botulinum types C and D is composed of two separate protein components, designated components I and II, which individually have little activity, but, when mixed and treated with trypsin, exert the potent activity. The present study provides the evidence that component I of the toxin catalyzes the hydrolysis of NAD into nicotinamide and ADP-ribose, whereas component II does not, indicating that component I of C2T has NAD glycohydrolase activity, which ability is shared with cholera and diphtheria toxins. However, C2T affected neither glycerol production of fat cells nor protein synthesis in cell-free system. Component I of C2T in the presence of [alpha-32P]NAD radiolabeled a protein of Mr 46,000 in the supernatant fractions of mouse tissue homogenates; the protein was abundant in brain, lung and intestine, whereas there was little or none of the protein in muscle. These results indicate that component I can catalyze the covalent attachment of the ADP ribose moiety of NAD to intracellular protein, which differs from those modified with cholera and diphtheria toxins. The present data, together with previous findings, suggest that the biological activity of C2T is elicited by ADP ribosylation activity of component I, which is internalized into the cells after binding to the receptor site introduced with the binding of component II to the cell surface membrane. PMID- 3023306 TI - Human mammary cancer cell mutants with altered hormone receptor activity. AB - We have recently isolated retinoic acid-resistant clones U-2 and U-3 from human breast cancer cell line MCF-7 (Ueda et al. (1985) Cancer Res. 45, 3332-3338). Growth of MCF-7 cells was found to be stimulated by estradiol but that of U-2 or U-3 was not. Cytosol from U-2 or U-3 cells contained no detectable estradiol receptor activity, whereas that from the parental MCF-7 cells showed estradiol receptor activity of 32 fmol/mg cytosol protein with a Kd of 2.6 X 10(-10) M by Scatchard analysis. Sucrose gradient centrifugation analysis of the cytosol fraction confirmed the presence of estradiol receptor activity in MCF-7 but not in U-2. Cytosol from MCF-7 and U-2 cells showed progesterone receptor activities of 106 fmol/mg protein with a Kd of 7.4 X 10(-10) M and 13 fmol/mg protein with a Kd of 9.9 X 10(-10) M, respectively. Addition of estradiol to the culture medium of the cells increased the level of progesterone receptor about 2-fold in MCF-7, but not in U-2. U-2 or U-3 cells showed about 5-fold higher resistance to an antiestrogen, tamoxifen, than MCF-7, and they were also 300- to 1,000-fold more resistant to other antiestrogens, epitiostanol and medroxyprogesterone, than MCF 7. The altered cellular sensitivity of U-2 or U-3 to the hormone antagonists is discussed in relation to the absence or presence of hormone receptors. PMID- 3023310 TI - Binding and crosslinking of 125I-labeled recombinant human tumor necrosis factor to cell surface receptors. AB - Highly purified recombinant human tumor necrosis factor (TNF) (molecular mass determined as 17 kilodaltons (kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as 36 kDa by Sephadex G-100 gel chromatography) was labeled with 125I to a specific activity of 5 microCi/micrograms without appreciable loss of activity. The binding of 125I-TNF to eighteen human and twelve animal cell lines was examined. The binding varied considerably among different cell lines. In most cell lines, the binding was inhibited up to greater than 90% by the addition of a 100-fold excess of unlabeled TNF. Some human and mouse cell lines showed no significant binding above background levels, suggesting that these cell lines had no receptors for TNF. Among the TNF receptor-positive cell lines, there was no direct correlation between the level of specific TNF binding and the level of sensitivity to the cytotoxic or cytostatic effect of TNF. Some cell lines were sensitive to TNF, whereas others were not affected at all by TNF. The TNF receptor-negative cell lines were also resistant to TNF. Therefore, although the existence of TNF receptor seems to be necessary, it does not alone determine cellular sensitivity to TNF. Scatchard analysis of the binding data revealed that human HeLa S3 and THP-1 had about 50,000 and 10,000 receptors/cell with a dissociation constant (KD) of 0.3-0.5 nM, respectively. Similarly, mouse L-929 and L-M cells had about 5,000 receptors/cell with KD of 3-5 nM. 125I-TNF bound to HeLa S3 cells was rapidly internalized at 37 degrees C, presumably by receptor mediated endocytosis, and degraded to acid-soluble products. The turnover of TNF receptors on HeLA S3 cells seemed to be rapid, since the level of specific binding quickly decreased after treatment with 100 micrograms/ml of cycloheximide at 37 degrees C with a half-life of about 1.5 h. The crosslinking of the cell bound 125I-TNF with the use of disuccinimidyl suberate yielded a complex of 105 kDa for HeLa S3 and THP-1 cells, and a complex of 100 kDa for U937 cells. The crosslinking was completely inhibited by the addition of a 100-fold excess of unlabeled TNF. Assuming that the complex was due to a one-to-one association of the dimeric form of TNF (34 kDa) with the receptor, we estimated the molecular size of the human TNF receptor to be 71 kDa for HeLa S3 and THP-1, and 66 kDa for U937. PMID- 3023309 TI - Effects of Ca2+ on ethanolaminephosphotransferase and cholinephosphotransferase in rabbit platelets. AB - The effects of Ca2+ on ethanolaminephosphotransferase [EC 2.7.8.1] and cholinephosphotransferase [EC 2.7.8.2] activities in rabbit platelet membranes were studied using endogenous diglyceride and CDP-[3H]ethanolamine or CDP [14C]choline as substrates. Both transferases required Mn2+, Co2+, or Mg2+ as a metal cofactor and the optimal concentrations of the metals for both activities were about 5, 10, and 5 mM, respectively. When 5 mM Mg2+ was used as a cofactor, both transferase activities were inhibited by a low concentration of Ca2+ (half maximal inhibition at approx. 15 microM). In the presence of 5 mM Mn2+, however, approx. 5 mM Ca2+ was required to produce half maximal inhibition. The Ca2+ induced inhibition was reversible and the rate of the inhibition was not affected either by the concentrations of the CDP-compound or by exogenously added diacylglycerol. The relationship between Ca2+ and both Mg2+ and Mn2+ on the transferase activities was competitive. 45Ca2+ binding (and/or uptake) to the platelet membranes was inhibited by Mn2+, Mg2+, and Co2+, in a concentration dependent manner. However, the inhibitory effects of the three metal ions on the total Ca2+ binding (and/or uptake) did not correlate with the activation of both transferase activities by the three metal ions in the presence of Ca2+. These results suggest that both transferase activities are regulated by low concentrations of Ca2+ in the presence of optimal concentrations of Mg2+, and that the inhibition is mediated directly by Ca2+, which interacts with a specific metal cofactor binding site(s) of the transferases. PMID- 3023311 TI - Region of cytochrome c interacting with yeast cytochrome b2: determination with singly modified carboxydinitrophenyl cytochromes c. AB - The association and reduction reactions of ten different 4-carboxy-2,6 dinitrophenyl (CDNP) horse heart cytochromes c, singly modified at lysines 8, 13, 27, 39, 60, 72, 73, 86, 87, and 99, with Saccharomyces cerevisiae cytochrome b2 were studied to determine the region of cytochrome c interacting with cytochrome b2. In the presence of higher ratios of free cytochrome c to cytochrome b2, native cytochrome c, and the CDNP-lysine 39, 60, and 99 derivatives associated with cytochrome b2 with a binding stoichiometry close to 2:1, while CDNP cytochromes c modified at lysines 8, 13, 27, 72, 73, 86, and 87 formed only 1:1 complexes. In the presence of lower ratios of free cytochrome c, modifications of lysines 8, 27, 86, and 87 had more inhibitory effects on the association of cytochrome c with cytochrome b2 than modifications of lysines 13, 39, 60, 72, 73, and 99. This tendency was similar to that on removal of free cytochrome c, except in the case of CDNP-lysine 13 and 73 derivatives. The rate of reduction of cytochrome c by cytochrome b2 was decreased by carboxydinitrophenylation of lysines 8, 13, 27, 72, 73, 86, and 87. In contrast, the rate of reduction of cytochrome c was not affected by modifications of lysines 39, 60, and 99. Since lysines 8, 13, 27, 72, 73, 86, and 87 are located on the front surface and lysines 39, 60, and 99 on the back side, and since different effects of modifying lysine residues located on the front surface may be interpreted in terms of effects on the complementary interaction of cytochrome c and cytochrome b2, these results indicate that the region of cytochrome c interacting with cytochrome b2 is located on the front surface of the cytochrome c molecule containing the exposed heme edge. PMID- 3023312 TI - Calcitonin-induced phosphorylation of rat liver cytosolic proteins. AB - Calcitonin (CT) stimulated phosphorylation of two liver cytosolic proteins whose molecular weights are 67,000 and 93,000. Stimulation of 67,000-Mr protein phosphorylation began shortly after subcutaneous injection of CT, reaching a maximum at 5 min and decreasing to below the control level at 30 min. The reaction was independent of cyclic AMP or Ca2+, and was not influenced by a calmodulin antagonist, W7. Stimulation of 93,000-Mr protein phosphorylation became evident by 30 min. This reaction was also stimulated by administration of vasopressin or epinephrine, which is known to cause increased phosphorylation of glycogen phosphorylase having the same molecular weight. The phosphorylation of 93,000-Mr protein, stimulated by CT, was dependent on Ca2+ but not on cyclic AMP, and appeared to be inhibited by W7. In addition, CT did not influence the phosphorylation of 61,000-Mr protein, a major protein phosphorylated in a cyclic AMP-dependent manner. These results suggest that CT may exert its effect on liver cells through protein phosphorylation, most probably in a cyclic AMP-independent manner. PMID- 3023313 TI - Effects of epidermal growth factor on the syntheses of DNA and polyamine in isoproterenol-stimulated murine parotid gland. AB - The effects of epidermal growth factor (EGF) on isoproterenol (IPR)-stimulated DNA synthesis and the activities of the rate limiting enzymes of polyamine synthesis (ornithine and S-adenosylmethionine decarboxylases) in parotid glands were investigated in vitro in cultured rat parotid explants and in vivo in submandibulectomized mice (mice after bilateral removal of the submandibular and sublingual glands). When the explants were cultured on siliconized lens paper floating on chemically defined synthetic medium, IPR caused the increases of both tissue cAMP level and the two decarboxylase activities in the prereplicative period and the stimulation of DNA synthesis with similar time courses to those observed in vivo. Dibutyryl cyclic AMP (DBcAMP) also increased the enzyme activities, but not DNA synthesis. EGF (1-2 ng/ml) had little effect on the IPR- and DBcAMP-dependent increases of amylase secretion and the enzyme activities, but it markedly enhanced IPR-stimulated DNA synthesis. Moreover, increase in DNA synthesis by DBcAMP was clearly observed in the presence of EGF when the explants were treated with this nucleotide analogue only during the early prereplicative period. In in vivo experiments, IPR-dependent increase in DNA synthesis was less in submandibulectomized mice than in intact animals. This decreased response to IPR of DNA synthesis was completely reversed by administration of EGF, though EGF alone did not induce either the enzymes or DNA synthesis. In submandibulectomized mice, although increases in the enzyme activities 8 h after injection of IPR were lower and they were significantly reversed by EGF, the activities at 12 h and the changes in polyamine levels at 8 and 12 h were almost the same as those in intact mice and were not affected by EGF treatment. These results obtained in vitro and in vivo suggest that EGF participates in the maximal response of IPR-dependent DNA synthesis but is not involved in the change of polyamine synthesis induced by IPR in murine parotid glands. PMID- 3023314 TI - Limited autolysis of calcium-activated neutral protease (CANP): reduction of the Ca2+-requirement is due to the NH2-terminal processing of the large subunit. AB - Calcium-activated neutral protease (rabbit mCANP), composed of large and small subunits, was converted to a lower-Ca2+-requiring form (derived microCANP) by limited autolysis in the presence of Ca2+. The NH2-terminal regions of the two subunits of mCANP were cleaved by autolysis, but the COOH-termini remained intact after autolysis. When native mCANP or derived microCANP was dissociated into subunits, the proteolytic activity of the large subunit was reduced to 2-5% of that of the native dimeric enzyme. The Ca2+-sensitivity of one hybrid CANP reconstituted from the large subunit of derived microCANP and the small subunit of native mCANP was similar to that of derived microCANP. However, the other hybrid molecule composed of the large subunit of native mCANP and the small subunit of derived microCANP required a high concentration of Ca2+ for activity, like native mCANP. These results indicate that the Ca2+-sensitivity of derived microCANP is determined by the structural change of the large subunit resulting from loss of its NH2-terminal region. The autolysis of the small subunit apparently has no effect on the reduction of the Ca2+-requirement. PMID- 3023315 TI - Cytochrome c peroxidase activity of bovine heart cytochrome oxidase incorporated in liposomes and generation of membrane potential. AB - Cytochrome oxidase vesicles catalyzed the peroxidatic oxidation of ferrocytochrome c. The maximal peroxidase activity in the absence of an uncoupling agent was 9.8 mol ferrocytochrome c oxidized/(s X mol heme a), indicating a 5-fold activation compared with the soluble enzyme system. The peroxidase activity was further enhanced 1.2 to 2.1 times upon addition of an uncoupler, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The stoichiometry of the reduction of hydrogen peroxide by ferrocytochrome c was established to be 1 : 2, indicating water formation. Potassium cyanide (0.14 mM) completely inhibited the peroxidase activity. The inhibition by 1 mM CO was 40-77% depending on the energized state of cytochrome oxidase vesicles, but in contrast, 85% inhibition was observed with the soluble enzyme. In the energized state the enzyme showed a slightly lower affinity for CO than in the deenergized state. Coupled with the peroxidase activity, a membrane potential of 72 mV was registered transiently; this may be physiologically significant in relation to the energy transduction mechanism. PMID- 3023316 TI - A mutated bovine prochymosin zymogen can be activated without proteolytic processing at low pH. AB - As a first step towards understanding how the zymogen structure of prochymosin contributes to the process by which active enzyme is produced, we altered the nucleotide sequence which encodes the amino-terminal (or propeptide) region of the protein. Of the two sites for autoproteolysis of prochymosin, one where pseudochymosin is formed at a pH of 2 and the other where chymosin is formed at pH 4-5, we changed the former by removing one codon and changing two other codons. This genetically modified prochymosin was proteolytically processed and activated normally at pH 4.5. However, at pH 2.0 we observed only partial activation of the zymogen and found no evidence of proteolytic processing. The properties of this engineered prochymosin suggest that zymogen activation does not require proteolysis and that the two different zymogen processing sites can function independently from one another. PMID- 3023317 TI - Cyanide binding to bovine heart cytochrome c oxidase depleted of subunit III by treatment with lauryl maltoside. AB - Subunit III was removed from beef heart cytochrome oxidase by incubation of the isolated enzyme at 25 degrees C for 24 h in lauryl maltoside buffer at a detergent to protein ratio of 10:1 (w:w). During the course of the incubation, the reaction of the enzyme with cyanide was followed by spectrophotometry in the Soret region. The starting material binds cyanide in a multiexponential process with 70% of the reaction occurring during the slow phase of the reaction at an observed rate of 3.85 X 10(-5) S-1 with 1 mM KCN. More of the enzyme binds cyanide during the fast phase of the reaction at an observed rate of 3.8 X 10(-3) S-1 as subunit III is removed by lauryl maltoside. After 24 h of incubation in lauryl maltoside, the enzyme reacts with cyanide completely in a rapid, single exponential process. When the protein from such an incubation is recovered by cytochrome c affinity chromatography and analyzed for its subunit content, subunit III is absent. The position of the Soret maximum of the oxidized enzyme shifts from its maximum at 418 nm in the starting material to 422 nm in the subunit III-depleted enzyme. The subunit III-depleted enzyme binds cyanide completely in a simple bimolecular reaction with a rate constant of 3.8 M-1 S-1. We discuss this result in terms of the possible structural and functional roles for subunit III in the cytochrome oxidase complex. PMID- 3023318 TI - Evidence for a second isoform of the catalytic subunit of cAMP-dependent protein kinase. AB - We have used a previously characterized mouse cDNA clone for the catalytic (C) subunit of cAMP-dependent protein kinase (Uhler, M. D., Carmichael, D. F., Lee, D. C., Chrivia, J. C., Krebs, E. G., and McKnight, G. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 1300-1304), which we designate C alpha, to isolate cDNA clones coding for a second isoform of the C subunit, C beta. C alpha cDNA clones hybridize to a 2.4-kilobase mRNA on Northern blots whereas C beta cDNA clones detect a 4.3-kilobase mRNA. Nucleotide sequence comparison between C alpha and C beta cDNA clones shows that the C beta cDNA codes for a protein which shows 91% identity with C alpha. Determination of mRNA levels for C beta in various tissues shows that it is most highly expressed in brain although it is detectable in all tissues examined. The presence of two genes coding for the C subunit of cAMP dependent protein kinase may explain past reports of heterogeneity in C subunit protein preparations. PMID- 3023319 TI - Covalent modification of the inhibitor-binding site(s) of Escherichia coli ADP glucose synthetase. Isolation and structural characterization of 8-azido-AMP incorporated peptides. AB - The photoaffinity inhibitor analog [2-3H]8-azido-AMP is specifically and covalently incorporated into Escherichia coli ADP-glucose synthetase. The reaction site(s) of [2-3H]8-azido-AMP with the enzyme was identified by reverse phase high performance liquid chromatography isolation and chemical characterization of CNBr and mouse submaxillary arginyl protease-generated peptides containing the labeled analog. Three regions of modification, represented by six labeled peptides, accounted for over 85% of the covalently bound label. The major binding region of the azido analog, composed of residues 108-128, contained approximately 55% of the recovered covalently bound radioactivity. A single residue, Tyr-113, contained between 50 and 75% of the label found in the major binding region. This site is the same as the major binding region of the substrate site-specific probe, 8-azido-ADP-[14C]glucose (Lee, Y. M., and Preiss, J. (1986) J. Biol. Chem. 261, 1058-1064). Conformational analysis of this region predicts that it is a part of a Rossmann fold, the supersecondary structure found in many adenine nucleotide-binding proteins. Two minor reaction regions of the enzyme with [2-3H]8-azido-AMP were also identified by chemical characterization. One region, containing 20% of the covalently bound label, was composed of residues 11-68. This region contains Lys-38, the previously determined pyridoxal phosphate-modified allosteric activator site (Parsons, T. F., and Preiss, J. (1978) J. Biol. Chem. 253, 7638-7645). The third minor region of modification, residues 222-254, contained approximately 15% of the covalently bound label. The three modified peptide regions may be juxtaposed in the enzyme's tertiary structure. PMID- 3023320 TI - The hydrolysis of extracellular adenine nucleotides by cultured endothelial cells from pig aorta. Feed-forward inhibition of adenosine production at the cell surface. AB - The time course of the extracellular reaction sequence ATP----ADP----AMP--- adenosine has been examined during recirculation of substrate solutions over cultured pig aortic endothelial cells attached to polystyrene beads. This permits the study of reactions at volume to cell surface ratios approaching those of small blood vessels. When endothelial cells were presented with an initial bolus of ATP, high concentrations of the intermediates ADP and AMP developed before significant conversion of AMP to adenosine occurred. Further, the higher the initial ATP concentration, the slower the conversion of AMP to adenosine. Kinetic constants for each reaction were estimated by fitting simulated reaction curves to observed time courses. Apparent Km values estimated in this way agreed well with those reported for initial velocity measurements (ATPase = 300 microM; ADPase = 240 microM; and 5'-nucleotidase = 26 microM). The ratio of maximum velocities was ATPase:ADPase:AMPase = 6:1.5:1, with absolute values varying among cell batches. The data could only be fitted if the model incorporated inhibition of 5'-nucleotidase by ATP or ADP, and satisfactory fitting was achieved with a Ki value for ADP of 5 microM. These kinetic properties maximize the time separation of the intermediate pools. In vivo, at sites of platelet degranulation, they would create a time gap proportional to the size of the initial release between release of ADP (a proaggregatory milieu) and the appearance of adenosine (an anti aggregatory milieu). PMID- 3023321 TI - Simulation of extracellular nucleotide hydrolysis and determination of kinetic constants for the ectonucleotidases. PMID- 3023322 TI - On the molecular mechanism of lactoperoxidase-catalyzed H2O2 metabolism and irreversible enzyme inactivation. AB - Lactoperoxidase-catalyzed H2O2 metabolism proceeds through one of three different pathways, depending on the nature and the concentration of the second substrate as an e- donor and/or on pH conditions. In the lactoperoxidase (LPO)-H2O2 system, at low H2O2 concentrations and/or alkaline conditions the peroxidatic cycle involves ferric LPO----compound I----compound II----ferric LPO conversion, whereas high H2O2 concentrations and/or acidic conditions favor the ferric LPO--- compound I----compound II----compound III----ferrous LPO----ferric LPO pathway. The compound III/ferroperoxidase states are associated with irreversible enzyme inactivation by cleavage of the heme moiety and liberation of iron. It is likely that either singlet oxygen or superoxide and hydroxyl radicals are involved in the attack on heme iron, because inactivation correlates with oxygen production and can be decreased to a certain degree by scavengers such as ethanol, 1 propanol, 2-propanol, or mannitol. In the LPO-H2O2-I- system, the enzyme may also be inactivated by I2 generated in the course of enzymatic I- oxidation (i.e. during ferric LPO----compound I----ferric LPO cycles). PMID- 3023323 TI - Reconstitution of Saccharomyces cerevisiae phosphatidylserine synthase into phospholipid vesicles. Modulation of activity by phospholipids. AB - Membrane-associated phosphatidylserine synthase was purified from Saccharomyces cerevisiae (Bae-Lee, M., and Carman, G. M. (1984) J. Biol. Chem. 259, 10857 10862) and reconstituted into phospholipid vesicles containing phosphatidylcholine/phosphatidylethanolamine/ phosphatidylinositol/phosphatidylserine. Reconstitution was performed by removing detergent from an octyl glucoside/phospholipid/Triton X-100/enzyme mixed micelle by Sephadex G-50 super-fine chromatography. The average diameter of the vesicles was 90 nm, and the enzyme was reconstituted asymmetrically with the active site facing outward. The enzymological properties of reconstituted phosphatidylserine synthase were determined in the absence of detergent. The enzyme was reconstituted into vesicles with phospholipid compositions approximating those of wild type and mutant strains of S. cerevisiae. Reconstituted activity was modulated by the phosphatidylinositol/phosphatidylserine ratio in the vesicles. The modulation of activity observed in the vesicles is enough to account for some of the fluctuations in the phosphatidylserine content in vivo. PMID- 3023324 TI - Metabolism and analysis of cysteinyl leukotrienes in the monkey. AB - Predominant hepatobiliary elimination from blood and subsequent enterohepatic circulation of cysteinyl leukotrienes is demonstrated in the monkey Macaca fascicularis. From intravenous [3H]leukotriene C4, about 40% were recovered as metabolites in bile and about 20% in urine within 5 h. [3H]Leukotriene E4 was a predominant metabolite of defined structure in blood plasma, bile, and urine. From intraduodenal [3H]leukotriene C4, about 5% were recovered as metabolites in bile and about 8% in urine within 8 h. Endogenous cysteinyl leukotrienes generated in vivo were measured after implantation of a subcutaneously looped biliary bypass. Tapping of the loop allowed access to bile and prevented interference by leukotrienes produced by surgical trauma (Denzlinger, C., Rapp, S., Hagmann, W., and Keppler, D. (1985) Science 230, 330-332). Endogenous cysteinyl leukotrienes were analyzed in bile, urine, and blood plasma by the sequential use of high-performance liquid chromatography and a radioimmunoassay that was optimized for leukotriene E4 as a predominant metabolite detected in the tracer studies. Biliary leukotriene E4 rose from less than 0.2 to 9 nmol/liter, when leukotriene synthesis was elicited in anesthesized monkeys by staphylococcal enterotoxin B administered intragastrically. This study provides an approach to the analysis of cysteinyl leukotrienes in primates and serves to define the role of these mediators under pathophysiological as well as physiological conditions in vivo. PMID- 3023325 TI - Purified protein kinase C phosphorylates microtubule-associated protein 2. AB - We have investigated actions of purified protein kinase C on microtubule- and microfilament-related proteins. Among the cytoskeletal proteins examined, microtubule-associated protein 2 (MAP2) was found to serve as a good substrate. Other cytoskeletal proteins, tubulin, fodrin, cofilin, tropomyosin, and 53,000-Da protein, were very poorly phosphorylated. The amino acid residues of MAP2 that were phosphorylated by the protein kinase C were almost exclusively serine. The peptide mapping analysis indicated that protein kinase C and cAMP-dependent protein kinase phosphorylate MAP2 differently. The ability of MAP2 to interact with actin was markedly reduced by this protein kinase C-mediated phosphorylation. These data raise the possibility that phosphorylation of MAP2 by activated protein kinase C may be involved in cell-surface signal transduction. PMID- 3023326 TI - Binding of single-chain prourokinase to the urokinase receptor of human U937 cells. AB - The single-chain form of human urokinase plasminogen activator (uPA) is the major form of the enzyme found in cells, tissues, and extracellular fluids. The protein, called pro-uPA, has high (Kd = 0.5 nM) affinity for the specific uPA receptor of U937 human monocyte-like cells. Its conversion to two-chain uPA by plasmin does not appreciably change the binding parameters. In addition, conversion of pro-uPA to uPA occurs with receptor-bound pro-uPA and does not lead to dissociation from the membrane. These data show that secreted pro-uPA can find its way to the specific surface receptor without previous conversion to the two chain form and that, once bound, can be activated by plasmin. PMID- 3023327 TI - Primary structure of CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) from Escherichia coli. AB - The gene coding for CTP:CMP-3-deoxy-D-mannooctulosonate cytidylyltransferase (CMP KDO synthetase), kds B, was previously cloned on a 9-kilobase Pst insert of Escherichia coli DNA into pBR 322 (Goldman, R. C., and Kohlbrenner, W. E. (1985) J. Bacteriol. 163, 256-261). Using a transposon mutagenesis approach we have now located kds B on this insert, which facilitated the isolation and sequencing of a 1.3-kilobase segment of DNA containing kds B and putative RNA polymerase and ribosome binding sites. The primary structure of CMP-KDO synthetase predicted by this nucleotide sequence was verified by amino acid composition and sequence analysis of purified CMP-KDO synthetase and cleavage fragments. Our results show that kds B consists of a 744-base open reading frame coding for a 248-amino acid peptide. The molecular weight of CMP-KDO synthetase calculated from the translated sequence is 27,486, taking into account the loss of the N-terminal methionine. These data define the transcriptional unit of kds B and its translation product in molecular terms, information prerequisite to our understanding of both the mechanism of CMP-KDO formation and the regulation of the KDO metabolic pathway in Gram-negative bacteria. PMID- 3023328 TI - Multisubstrate analogs for deoxynucleoside kinases. Triphosphate end products and synthetic bisubstrate analogs exhibit identical modes of binding and are useful probes for distinguishing kinetic mechanisms. AB - Comparative inhibition kinetics with natural dNTP end products (dNp3) and new synthetic bisubstrate-type analogs, dNp4A (deoxynucleoside 5'-adenosine 5''' P1,P4-tetraphosphate), have been studied with their target deoxynucleoside kinases from Lactobacillus acidophilus. Analysis of inhibition specificity, inhibition patterns, and Ki(app) under various conditions has revealed the following conclusions. Both dNTP and dNp4A bind to the active site of the corresponding kinase through multiple binding determinants. The deoxynucleoside moiety of dNTP fits optimally at the deoxynucleoside binding site and provides the basis for its inhibition specificity, whereas the triphosphate group interacts with the ATP binding site, reinforcing the affinity of the molecule as a potent end product inhibitor (Ki = 0.4-3 microM). The adenosine moiety of dNp4A does not contribute to the binding of this compound, whereas the tetraphosphate portion is the second binding determinant, just as in the model developed for dNTP. dNTP and dNp4A proved to be useful tools for distinguishing the kinetic mechanisms of kinases which follow sequential pathways, i.e. the rapid equilibrium Random Bi Bi for dCyd and dGuo kinases and the steady state Ordered Bi Bi mechanism for two dAdo kinases associated either with dCyd kinase or with dGuo kinase on different multifunctional proteins. PMID- 3023329 TI - Inhibition of mu and delta but not kappa opioid binding to membranes by Fab fragments from a monoclonal antibody directed against the opioid receptor. AB - Fab fragments from a monoclonal antibody, OR-689.2.4, directed against the opioid receptor, selectively inhibited opioid binding to rat and guinea pig neural membranes. In a titratable manner, the Fab fragments noncompetitively inhibited the binding of the mu selective peptide [D-Ala2,(Me)Phe4,Gly(OH)5][3H] enkephalin and the delta selective peptide [D-Pen2,D-Pen5] [3H]enkephalin (where Pen represents penicillamine) to neural membranes. In contrast, kappa opioid binding, as measured by the binding of [3H]bremazocine to rat neural membranes and guinea pig cerebellum in the presence of mu and delta blockers, was not significantly altered by the Fab fragments. In addition to blocking the binding of mu and delta ligands, the Fab fragments displaced bound opioids from the membranes. When mu sites were blocked with [D-Ala2,(Me)Phe4,Gly(OH)5]enkephalin, the Fab fragments suppressed the binding of [D-Pen2,D-Pen5][3H]enkephalin to the same degree as when the mu binding site was not blocked. The Fab fragments also inhibited binding to the mu site regardless of whether or not the delta site was blocked with [D-Pen2,D-Pen5]enkephalin. This monoclonal antibody is directed against a 35,000-dalton protein. Since the antibody is able to inhibit mu and delta binding but not kappa opioid binding, it appears that this 35,000-dalton protein is an integral component of mu and delta opioid receptors but not kappa receptors. PMID- 3023330 TI - Rapid degradation of apoplastocyanin in Cu(II)-deficient cells of Chlamydomonas reinhardtii. AB - Although plastocyanin is not detected in Cu(II)-deficient cells of Chlamydomonas reinhardtii, accumulation of messenger RNA for pre-apoplastocyanin is independent of the concentration of Cu(II) in the medium (Merchant, S., and Bogorad, L. (1986) Mol. Cell. Biol. 6, 462-469). This work shows that the synthesis, transport, and processing of pre-apoplastocyanin also appear to be unaffected in cells grown in Cu(II)-deficient medium. However, the mature protein, presumably formed after import of the precursor into the chloroplast, is rapidly degraded in Cu(II)-deficient cells. The half-life of the mature protein is estimated to be between 16 and 18 min in cells grown in Cu(II)-deficient medium. In cells grown in medium containing Cu(II), the mature protein is stable. The proteolytic activity thus appears to be specific for apoplastocyanin versus plastocyanin and thereby accounts for the absence of accumulated plastocyanin in Cu(II)-deficient cells. This process may be part of a general mechanism designed to remove chloroplast proteins which cannot be utilized. PMID- 3023331 TI - Synergistic inhibition of glucagon-induced effects on hepatic glucose metabolism in the presence of insulin and a cAMP antagonist. AB - Inhibition of hepatic glycogenolysis by an intracellular inhibitor of cAMP dependent protein kinase in glucagon-stimulated hepatocytes was potentiated by insulin. When hepatocytes isolated from fed rats were treated with 0.3 nM glucagon, which activates glycogen breakdown half-maximally, the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate [Rp-cAMPS), a cAMP antagonist, inhibited glucose production half-maximally at 3 microM. A 10-fold lower concentration of antagonist was required to half-maximally inhibit glucose production in the presence of 10 nM insulin, which alone produced only 15% inhibition. Under the same experimental conditions, the maximal effect of (Rp) cAMPS was also potentiated. In addition, the increase in the concentration of glucagon required for half-maximal activation of phosphorylase activity and inactivation of glycogen synthase activity in the presence of minimally effective concentrations of insulin and (Rp)-cAMPS were clearly synergistic. It is postulated that the synergism observed is a consequence of action at several enzymatic sites leading to, and including, alteration of the phosphorylation state of the two rate-limiting enzymes in glycogen metabolism. PMID- 3023333 TI - Activation of platelets by alpha-thrombin is a receptor-mediated event. D phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-thrombin, but not N alpha tosyl-L-lysine chloromethyl ketone-thrombin, binds to the high affinity thrombin receptor. AB - Competition binding studies have been carried out to evaluate the antagonism of TLCK-thrombin (N alpha-tosyl-L-lysine chloromethyl ketone-treated thrombin) and PPACK-thrombin (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-treated thrombin) with alpha-thrombin using computer-assisted analysis of the binding isotherms (LIGAND). alpha-Thrombin bound to high, moderate, and low affinity sites as previously described (Harmon, J. T., and Jamieson, G. A. (1985) Biochemistry 24, 58-64). PPACK-thrombin bound to all three sites accessible to alpha-thrombin (K1, 7 nM; R1, 20 sites/platelet; K2, 3 nM; R2, 1800 sites/platelet; K3, 510 nM; R3, 84,000 sites/platelet) as well as to a separate fourth site (Kx, 0.4 nM; Rx, 20 sites/platelet) for PPACK-thrombin that was not accessible to alpha-thrombin. In contrast, TLCK-thrombin did not bind to the high affinity site for alpha-thrombin but bound to the moderate and low affinity sites for alpha-thrombin with similar affinity (K2, 2 nM; R2, 890 sites/platelet; K3, 900 nM; R3, 100,000 sites/platelet) and to another site (Ky, 0.03 nM; Ry, 10 sites/platelet) which was not accessible to alpha-thrombin. As predicted from these binding studies, TLCK-thrombin did not compete with alpha-thrombin for platelet activation at concentrations as high as 1000 nM (500-fold excess). In contrast a 300-fold excess of PPACK-thrombin (670 nM) totally inhibited platelet activation by 2 nM thrombin. These results demonstrate that the high affinity binding site for thrombin on human platelets is a classical receptor, occupancy of which is necessary for platelet activation by low concentrations of thrombin; that TLCK-thrombin does not occupy this high affinity site and hence cannot inhibit platelet activation by alpha-thrombin; and that PPACK-thrombin does compete with alpha-thrombin at the high affinity site and is an antagonist of alpha-thrombin induced activation. PMID- 3023332 TI - Evidence for a free radical mechanism of styrene-glutathione conjugate formation catalyzed by prostaglandin H synthase and horseradish peroxidase. AB - We have proposed, using styrene as a model, a new mechanism for the formation of glutathione conjugates that is independent of epoxide formation but dependent on the oxidation of glutathione to a thiyl radical by peroxidases such as prostaglandin H synthase or horseradish peroxidase. The thiyl radical reacts with styrene to yield a carbon-centered radical which subsequently reacts with molecular oxygen to give the styrene-glutathione conjugate. We have used electron spin resonance spin trapping techniques to detect the proposed free radical intermediates. A styrene carbon-centered radical was trapped using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and t-nitrosobutane. The position of the carbon-centered radical was confirmed to be at carbon 7 by the use of specific 2H labeled styrenes. The addition of the spin trap DMPO inhibited both the utilization of molecular oxygen and the formation of styrene-glutathione conjugates. Under anaerobic conditions additional styrene-glutathione conjugates were formed, one of which was identified by fast atom bombardment mass spectrometry as S-(2-phenyl)ethylglutathione. The glutathione thiyl radical intermediate was observed by spin trapping with DMPO. These results support the proposed free radical-mediated formation of styrene-glutathione conjugates by peroxidase enzymes. PMID- 3023334 TI - Preliminary X-ray diffraction studies of the putative catalytic domain of gamma delta resolvase from Escherichia coli. AB - Two crystal forms of the putative catalytic domain (residues 1-140) of gamma delta resolvase from Escherichia coli have been obtained. Type I is isomorphous with crystals of the intact protein, and type II is suitable for high resolution structure analysis. Type II crystals belong to the orthorhombic space group C222(1), with a = 76.8 A, b = 191.3 A, and c = 63.4 A. They contain two molecules (15,500 daltons each)/asymmetric unit and show diffraction beyond 2.7-A resolution. Calculation of a rotation function using 7-A data shows the orientation of the noncrystallographic axes. PMID- 3023335 TI - Anion inhibition of the proton pump in rat liver multivesicular bodies. AB - Rat liver multivesicular bodies (MVB), as well as other hepatic subcellular organelles, are acidified by an electrogenic ATP-dependent proton pump that requires Cl- for maximal acidification (Van Dyke, R. W., Hornick, C. A., Belcher, J., Scharschmidt, B. F., and Havel, R.J. (1985) J. Biol. Chem. 260, 11021-11026), suggesting that Cl- serves as a permeable charge-compensating anion. However, we have observed that NO3- is unable to substitute for Cl-. This study was undertaken therefore to examine more closely the effects of Cl- on MVB acidification and to determine whether NO3- and other anions interact with the proton pump. ATP-dependent vesicle acidification and membrane potential (psi) were measured using the fluorescent dyes acridine orange and Oxonol V (bis(3 phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol), respectively. Cl- both stimulated acidification (Km = 23.2 +/- 4.2 mM) and decreased psi (IC50 = 3.4 +/- 0.6 mM) in a concentration-dependent, nonlinear fashion. In the presence of saturating Cl- (100 mM), however, NO3- (shown to be more permeable than Cl-) and the impermeant anions SO4(2-) and PO4(2-), inhibited both ATP-dependent acidification and psi in a concentration-dependent manner. Other anions, including gluconate and HCO3-, had no effect. The inhibitory effect of NO3- was reversible. Neither SO4(2-) nor PO4(2-) appeared to block Cl- movement across the vesicle membrane as assessed by the ability of Cl- to decrease an established psi. In additional experiments, the effects of anions on relaxation of a previously established pH gradient were measured. Compared to Cl- or gluconate, NO3- had no significant effect on pH gradient relaxation, even when MVB were preloaded with NO3-, indicating that rapid cycling of NO3-/HNO3 across the MVB membrane does not occur. The organic nitrate, isosorbide dinitrate, also inhibited both acidification and psi and, similar to NO3-, had no effect on pH gradient relaxation. By contrast, NO2- potently inhibited both MVB acidification and psi but also rapidly relaxed a pre established pH gradient, suggesting that NO2- increases MVB membrane proton permeability. Finally, MVB exhibited N-ethylmaleimide-sensitive ATPase activity that was inhibited 23.9% by NO3- (100 mM). In conclusion, although MVB are permeable to a variety of anions (Cl-, Br-, NO3-, NO2-), only Cl- and Br- support maximal rates of acidification by the proton pump.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3023336 TI - Synergism of glucose and fructose in net glycogen synthesis in perfused rat livers. AB - Synergism of glucose and fructose in net glycogen synthesis was studied in perfused livers from 24-h fasted rats. With either glucose or fructose alone, net glycogen deposition did not occur (p greater than 0.10 for each), whereas the addition of both together resulted in significant glycogen accumulation (net glycogen accumulation was 0.21 +/- 0.03 mumol of glucose/g of liver/min at 2 mM fructose and 30 mM glucose, p less than 0.001). To better understand this synergism, intermediary substrate levels were compared at steady state with various glucose levels in the absence and in the presence of 2 mM fructose. Independent of fructose, hepatic glucose and glucose 6-phosphate increased proportionally when glucose level in the medium was raised (r = 0.86, p less than 0.001). Unlike glucose 6-phosphate, UDP-glucose did not consistently increase with glucose (p greater than 0.10); in fact, there was a small decrease at a very high glucose level (30 mM), a result consistent with the well-established activation of glycogen synthase by glucose. With elevated glucose, the level of glucose 6-phosphate was strongly correlated with glycogen content (r = 0.71, p less than 0.01, slope = 32). Adding fructose increased the "efficiency" of glucose 6-phosphate to glycogen conversion: the effect of a given increment in glucose 6-phosphate upon glycogen accumulation was increased 2.6-fold (r = 0.73, p less than 0.01, slope = 86). A kinetic modeling approach was used to investigate the mechanisms by which fructose synergized glycogen accumulation when glucose was elevated. Based on steady-state hepatic substrate levels, net hepatic glucose output, and net glycogen synthesis rate, the model estimated the rate constants of major enzymes and individual fluxes in the glycogen metabolic pathway. Modeling analysis is consistent with the following scenario: glycogen synthase is activated by glucose, whereas glucose-6-phosphatase was inhibited. In addition, the model supports the hypothesis that fructose synergizes net glycogen accumulation due to suppression of phosphorylase. Overall, our analysis suggests that glucose enhances the metabolic flux to glycogen by inducing a build up of glucose 6-phosphate via combined effects of mass action and glucose-6-phosphatase inhibition and activating glycogen synthase and that fructose enhances glycogen accumulation by retaining glycogen via phosphorylase inhibition. PMID- 3023337 TI - Actin polymerization. The mechanism of action of cytochalasin D. AB - Fluorescence changes using actin covalently labeled with N-(1 pyrenyl)iodoacetamide have been used to determine the effect of cytochalasin D on actin polymerization. A mechanism for the effect of cytochalasin D on actin polymerization is presented, which explains the experimental observation of a cytochalasin D-induced increase in the initial rate of polymerization and a decrease in the final extent of the reaction. Central to this mechanism is the Mg2+-dependent formation of cytochalasin D-induced dimers. The dimers serve as nuclei to enhance the polymerization rate. Binding of Mg2+ to a low affinity site on the dimer induces a conformational change which can be observed as a rapid fluorescence increase. A subsequent time-dependent fluorescence decrease observed prior to polymerization appears to represent ATP hydrolysis resulting in dissociation of the dimer and release of actin monomers containing ADP. We postulate that a slow rate of exchange of ATP for bound ADP relative to hydrolysis results in the accumulation of monomers containing ADP. As these monomers have a high critical concentration, the final extent of polymerization is reduced dramatically. The Mg2+ dependence of the final extent of polymerization in the presence of cytochalasin D is also explained in the context of this mechanism. PMID- 3023338 TI - Destabilization of messenger RNA/complementary DNA duplexes by the elongating 80 S ribosome. AB - In a previous study, we demonstrated that the ability of a cDNA fragment to hybrid-arrest the translation of its complementary mRNA in rabbit reticulocyte lysate depends on the position of the mRNA/cDNA duplex within the mRNA molecule. In the present report, we further characterize the mechanisms involved in the destabilization and subsequent translation of mRNA/cDNA hybrids by mapping in detail the positional dependence of hybrid-arrested translation of the human alpha- and beta-globin mRNAs and by directly assessing the stability of mRNA/cDNA duplexes in reticulocyte lysate under a variety of translational conditions. The mapping studies in this report demonstrate that the translation of a hybridized mRNA requires exposure of the 5' nontranslated region and the AUG initiation codon, as well as those bases 3' to the AUG which are typically protected by an initiating 80 S ribosome. The translation of these mRNA/cDNA hybrids is associated with the complete removal of cDNA from the mRNA coding region; this disruption of the mRNA/cDNA duplex is blocked by inhibitors of translational initiation and elongation. cDNAs which extend into the 3' nontranslated region remain associated with the mRNA during normal translation but are completely removed from the mRNA during translation if translational termination is suppressed. Taken together, these findings demonstrate that the disruption of mRNA/cDNA duplexes in rabbit reticulocyte lysate is tightly linked to the assembly and migration of 80 S ribosomes. PMID- 3023339 TI - Nerve growth factor receptor from rabbit sympathetic ganglia membranes. Relationship between subforms. AB - The receptor for nerve growth factor (NGF) was purified from Triton X-100 extracts of sympathetic ganglia membranes by affinity chromatography on NGF Sepharose. Elution of purified receptor was accomplished at pH 5 in the presence of 1 M NaCl. Sodium dodecyl sulfate gel electrophoresis of the purified iodinated receptor showed three major bands at Mr = 126,000, Mr = 105,000, and Mr = 81,000. Affinity labeling of the purified receptor using 125I-NGF and the photoreactive agent N-hydroxysuccinimidyl-p-azidobenzoate resulted in two major cross-linked complexes corresponding to Mr = 135,000 and Mr = 110,000. This labeling pattern is similar to that observed with sympathetic ganglia membranes (Massague, J., Guillette, B. J., Czech, M. P., Morgan, C. J., and Bradshaw, R. A. (1981) J. Biol. Chem. 256, 9419-9424) and indicates that these two forms do not arise from the cross-linking procedure. Reaction of the photoaffinity labeled NGF receptors with increasing amounts of trypsin resulted in a progressive decrease in the high molecular weight complex with a concomitant increase in the low molecular weight form. When the larger complex was isolated by electroelution from a sodium dodecyl sulfate gel and treated with trypsin, a species corresponding to Mr = 100,000 was generated. These observations are best explained by a precursor product relationship for the two NGF receptor species of sympathetic neurons. PMID- 3023340 TI - Na,K-ATPase in dog red cells. Immunological identification and maturation associated degradation by the proteolytic system. AB - The Na,K-ATPase of red cells from high K+ and low K+ dogs was studied immunologically by using antibodies raised against dog kidney enzyme. Anti-alpha subunit IgGs, which also recognized alpha (+) from brain enzyme, identified the larger subunit of erythrocyte Na,K-ATPase as a homogeneous polypeptide with Mr = 96,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting. In addition, erythrocyte Na,K-ATPase, purified by immunoaffinity chromatography on a monoclonal antibody-coupled column, showed the identity of its polypeptide composition to that of the renal enzyme. Furthermore, it was shown that reticulocyte lysates from high K+ and low K+ dogs substantially degraded 125I-Bolton-Hunter reagent-labeled Na,K-ATPase. This degradation of the enzyme protein was significantly enhanced by the addition of ATP and Mg2+. These results indicate that dog reticulocytes possess some mechanism for protein breakdown involving an ATP-dependent proteolytic system, resulting in the dramatic breakdown of Na,K-ATPase activity during dog reticulocyte maturation into erythrocytes (Maede, Y., and Inaba, M. (1985) J. Biol. Chem. 260, 3337 3343). PMID- 3023341 TI - 1,25-Dihydroxyvitamin D3-mediated intestinal calcium transport. Biochemical identification of lysosomes containing calcium and calcium-binding protein (calbindin-D28K). AB - A variety of intestinal cell organelles and proteins have been proposed to mediate 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-stimulated calcium absorption. In the present study biochemical analyses were undertaken to determine the subcellular localization of 45Ca after calcium transport in vivo in ligated duodenal loops of vitamin D-deficient chicks injected with 1.3 nmol of 1,25 (OH)2D3 or vehicle 15 h prior to experimentation. Separation of Golgi, mitochondria, basal lateral membrane, and lysosome fractions in the epithelial homogenates was achieved by differential sedimentation followed by centrifugation in Percoll gradients and evaluation of appropriate marker enzyme activities. Both vitamin D-deficient and 1,25-(OH)2D3-treated chicks had the highest levels of 45Ca-specific activity in lysosomal fractions. The lysosomes were also the only organelles to exhibit a 1,25-(OH)2D3-mediated difference in calcium content, increasing to 138% of controls. Lysosomes prepared from 1,25-(OH)2D3-treated chicks also contained the greatest levels of immunoreactive calbindin-D28k (calcium-binding protein). Chloroquine, a drug known to interfere with lysosomal function, was tested and found to inhibit 1,25-(OH)2D3-stimulated intestinal calcium absorption. Neither 1,25-(OH)2D3 nor chloroquine affected [3H]2O transport. In additional experiments, microsomal membranes (105,000 X g pellets) were subjected to gradient centrifugation. The highest levels of 45Ca-specific activity and calcium-binding protein in material from 1,25-(OH)2D3-treated chicks were found in fractions denser than endoplasmic reticulum and may represent endocytic vesicles. In studies on intestinal mucosa of 1,25-(OH)2D3-treated birds fractionated after 30 min of exposure to lumenal Ca2+ or Ca2+ plus chloroquine, 45Ca was found to accumulate in lysosomes and putative endocytic vesicles, relative to controls. A mechanism involving vesicular flow is proposed for 1,25 (OH)2D3-mediated intestinal calcium transport. Endocytic internalization of Ca2+, fusion of the vesicles with lysosomes, and exocytosis at the basal lateral membrane complete the transport process. PMID- 3023342 TI - Stoichiometry and dynamic interaction of metal ion activators with calcineurin phosphatase. AB - Calcineurin, a calmodulin-regulated phosphatase, is composed of two distinct subunits (A and B) and requires certain metal ions for activity. The binding of the two most potent activators, Ni2+ and Mn2+, to calcineurin and its subunits has been studied. Incubation of the protein with 63Ni2+ (or 54Mn2+) followed by gel filtration to separate free and protein-bound ions indicated that calcineurin could maximally bind 2 mol/mol of Ni2+ or Mn2+. While isolated A subunit also bound 2 mol/mol of Ni2+, no Mn2+ binding was demonstrated for either isolated A or B subunit. When bindings were monitored by nitrocellulose filter assay, only 1 mol/mol bound Ni2+ or Mn2+ was detected, suggesting that the two Ni2+ (or Mn2+) binding sites had different relative affinities and that only metal ions bound at the higher affinity sites were detected by the filter assay. Preincubation of calcineurin with Mn2+ (or Ni2+) decreased the filter assay-measured Ni2+ (or Mn2+) binding by only 30%. Preincubation of the protein with Zn2+ decreased the filter assay-measured Ni2+ or Mn2+ binding by 90 or 17%, respectively. The results suggest that the higher affinity sites are a Ni2+-specific site and a distinct Mn2+-specific site. Preincubation of calcineurin with Mn2+ (or Ni2+) decreased the gel filtration-determined Ni2+ (or Mn2+) binding from 2 to 1 mol/mol suggesting that calcineurin also contains a site which binds either metal ion. The time course of Ni2+ (or Mn2+) binding was correlated with that of the enzyme activation, and the extent of deactivation of the Ni2+-activated calcineurin by EDTA or by incubation with Ca2+ and calmodulin (Pallen, C. J., and Wang, J. H. (1984) J. Biol. Chem. 259, 6134-6141) was correlated with the release of the bound ions, thus suggesting that the bound ion is directly responsible for enzyme activation. PMID- 3023344 TI - Purification and characterization of FAD synthetase from Brevibacterium ammoniagenes. AB - The bifunctional enzyme FAD synthetase from Brevibacterium ammoniagenes was purified by a method involving ATP-affinity chromatography. The final preparation was more than 95% pure. The apparent molecular weight of the enzyme was determined as 38,000 and the isoelectric point as 4.6. Although previous attempts to separate the enzymatic activities had failed, ATP:riboflavin 5' phosphotransferase and ATP:FMN-adenylyltransferase activities in B. ammoniagenes were believed to be located on two separate proteins with similar properties, possibly joined in a complex. The following evidence, however, suggests the presence of both activities on a single polypeptide chain. The two activities copurify in the same ratio through the purification scheme as presented. Only a single band could be detected when aliquots from the final purification step were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, nondenaturing gel electrophoresis, and isoelectric focusing. Edman degradation of the protein yielded a single N-terminal sequence. PMID- 3023343 TI - Identification of histidyl and thiol groups at the active site of rabbit renal dipeptide transporter. AB - Active transport of dipeptides in rabbit renal brush-border membrane vesicles is energized by an inward-directed H+ gradient rather than a Na+ gradient. We examined the effects of treatment of membrane vesicles with diethylpyrocarbonate (DEP), a reagent specific for histidyl groups, on this H+ gradient-dependent dipeptide uptake. DEP inhibited the uptake of all three dipeptides studied, Gly sarcosine, Gly-Gly, and Gly-Pro (Ki = 0.6-0.9 mM), and the inhibition was noncompetitive. The dipeptide transporter could be protected from DEP inhibition by the presence of dipeptide substrates during the treatment of the vesicles with the inhibitor, whereas leucine plus Na+ failed to offer the protection. Na+ dependent leucine uptake was also inhibited by DEP (Ki = 2.5 mM) and the amino acid transporter could be protected from the inhibition by leucine plus Na+, but not by dipeptides. Treatment of membrane vesicles with the thiol group-specific reagents, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole,3-bromopyruvate, p chloromercuribenzenesulfonic acid, and N-ethylmaleimide, also inhibited the H+ gradient-dependent dipeptide uptake. The potency of their inhibition was in the order: 7-chloro-4-nitrobenz-2-oxa-1,3-diazol greater than p chloromercuribenzenesulfonic acid greater than 3-bromopyruvate greater than N ethylmaleimide. The inhibition could be reversed in some cases by treatment of the membrane vesicles with reducing agents such as 2,3-dimercaptopropanol following incubation with the inhibitors. Dipeptide substrates could protect the dipeptide transporter from the inhibition. We conclude that histidyl and thiol groups are present at or near the substrate-binding site of the rabbit renal dipeptide transporter. PMID- 3023345 TI - A nicking enzyme from trypanosomatids which specifically affects the topological linking of duplex DNA circles. Purification and characterization. AB - Newly replicated duplex DNA minicircles of trypanosomal kinetoplast DNA are nicked in both their monomeric and catenated topological states, whereas mature ones are covalently sealed. The possibility that nicking may play a role during kinetoplast DNA replication by affecting the topological interconversions of monomeric DNA minicircles and catenane networks was studied here in vitro using Crithidia fasciculata DNA topoisomerase. An enzyme that catalyzes the nicking of duplex DNA circles has been purified to apparent homogeneity from C. fasciculata cell extracts. The native enzyme has a sedimentation coefficient of 6.8 S and was found to be a dimer with a protomer Mr = 60,000. Nicking of kinetoplast DNA networks by the purified enzyme inhibits their decatenation by the Crithidia DNA topoisomerase but has no effect on the catenation of monomeric DNA minicircles into networks. This differential effect on decatenation versus catenation is specific to the purified nicking enzyme. Random nicking of interlocked DNA minicircles has no detectable effect on the reversibility of the topological reaction. The potential role of Crithidia nicking enzyme in the replication of kinetoplast DNA networks in trypanosomatids is discussed. PMID- 3023347 TI - A cloned bovine cDNA encodes an alternate form of the catalytic subunit of cAMP dependent protein kinase. AB - While attempting to isolate a cDNA clone for the catalytic subunit of the bovine cAMP-dependent protein kinase, we have isolated cDNAs which code for a protein slightly different than the known amino acid sequence. The alternate cDNA was identified by screening a bovine pituitary cDNA library using synthetic oligonucleotides predicted from the known amino acid sequence of the catalytic subunit. The cDNA which we identified, encodes a protein which is 93% identical to the known amino acid sequence of the bovine catalytic subunit. It seems likely that this cDNA represents a previously undiscovered catalytic subunit of the cAMP dependent protein kinase. The mRNA for the alternate catalytic subunit is different in size from the mRNA coding for the previously known catalytic subunit and also has a different tissue distribution. These findings suggest that there are at least two different genes for the catalytic subunit. The differences in amino acid sequence and tissue distribution suggest the possibility of important functional differences in the two enzymes. PMID- 3023346 TI - Glucose 6-phosphate regulates Ca2+ steady state in endoplasmic reticulum of islets. A possible link in glucose-induced insulin secretion. AB - Glucose stimulation of islets is coupled with the rapid intracellular release of myo-inositol 1,4,5-trisphosphate (IP3) and arachidonic acid which in turn mobilize Ca2+ stored in the endoplasmic reticulum (ER). The metabolism of glucose is required for insulin secretion although the link between glucose metabolism and the cellular events resulting in insulin release is unknown. In digitonin permeabilized islets, glucose 6-phosphate (0.5-4 mM) increased significantly the ATP-dependent Ca2+ content of the ER at a free Ca2+ concentration of 1 microM. At 0.2 microM free Ca2+, glucose 6-phosphate (2-10 mM) had a smaller effect. Glucose, phosphate, mannose 6-phosphate, and fructose 1,6-diphosphate had no effect on the ATP-dependent Ca2+ content of the ER. Glucose 1-phosphate and fructose 6-phosphate also increased ATP-dependent Ca2+ content of the ER, presumably due to conversion to glucose 6-phosphate by islet phosphoglucomutase and phosphoglucoisomerase, respectively. The glucose 6-phosphate increase in the ATP-dependent Ca2+ content of the ER was shown to be mediated by glucose 6 phosphatase localized to the ER. Both arachidonic acid (10 microM) and the Ca2+ ionophore A23187 (2 microM) mobilized Ca2+ stored in the ER by glucose 6 phosphate. However, IP3-induced (10 microM) Ca2+ release from the ER was abolished in the presence of glucose 6-phosphate (0.5-10 mM). We propose that glucose 6-phosphate could provide a regulatory link between glucose metabolism and intracellular Ca2+ regulation by augmenting Ca2+ sequestered in the ER as well as attenuating IP3-induced Ca2+ release. Thus, glucose 6-phosphate would serve as an "off" signal leading to a decrease in intracellular Ca2+ when both the free Ca2+ and glucose 6-phosphate concentrations have increased following glucose stimulus. PMID- 3023349 TI - Glucose-permease of the bacterial phosphotransferase system. Gene cloning, overproduction, and amino acid sequence of enzyme IIGlc. AB - The glucose-permease (IIGlc) of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It also functions as a receptor for bacterial chemotaxis. The structural gene of the permease, ptsG, has been cloned on a multicopy plasmid, and transformants constitutively overproducing the protein 10-15 times over wild type level have been isolated. Overproduction is slightly inhibited if transformants are grown in a glucose-containing medium. The complete amino acid sequence of the glucose-permease is deduced from the nucleotide sequence. It consists of 477 residues and is moderately hydrophobic. A comparison of the glucose-permease with the mannitol-permease (Lee, C. A., and Saier, M. H., Jr. (1983) J. Biol. Chem. 258, 10761-10767) does not reveal any obvious homology at the level of amino acid sequence. PMID- 3023348 TI - Structure-function analysis of three cAMP-independent forms of the cAMP receptor protein. AB - cAMP receptor protein (CRP)-dependent operon expression in Escherichia coli requires the CRP X cAMP complex form of wild-type CRP. One class of crp mutants (crp*) activates CRP-dependent promoters in strains (cya) incapable of endogenous cAMP synthesis. Of fundamental interest is the difference in regulatory properties exhibited by crp* mutant strains, some of which exhibit glucose mediated repression of beta-galactosidase synthesis, some of which do not. To gain a better understanding of the mechanisms of cAMP-independent promoter activation and repression we have: determined through cloning and DNA sequence analysis the primary structure of three CRP* forms of CRP; purified the mutant proteins; characterized the effect of these mutations on CRP secondary structure; and studied CRP*-activated lac promoter regulation in a purified in vitro transcription system. The results of this study provide strong evidence that mutations in crp alter the conformation of CRP and result in cAMP-independent activation of CRP-dependent promoters in vitro. In addition, a CRP allele specific inhibition of CRP* activity by spermidine was observed in vitro that parallels crp* strain-specific sensitivity to glucose-mediated repression of CRP dependent enzyme synthesis in vivo. This observation provides evidence that catabolite repression in cells lacking cAMP may be mediated through a mechanism that inhibits CRP* activity. PMID- 3023350 TI - Inhibition of adenosine and thymidylate kinases by bisubstrate analogs. AB - Potential bisubstrate analogs, in which the 5'-hydroxyl group of adenosine was joined to the phosphoryl group acceptor by polyphosphoryl bridges of varying length (ApnX, where n is the number of phosphoryl groups and X is the nucleoside moiety of the acceptor), were tested as inhibitors of human liver adenosine kinase and of thymidylate kinase from peripheral blast cells of patients with acute myelocytic leukemia. Adenosine kinase was most strongly inhibited by P1,P4 (diadenosine 5')-tetraphosphate (Kd = 30 nM) and P1,P5-(diadenosine 5') pentaphosphate (Kd = 73 nM). Thymidylate kinase was most strongly inhibited by P1 (adenosine 5')-P5-(thymidine 5')-pentaphosphate (Kd = 120 nM) and by P1(adenosine 5')-P6-(thymidine 5')-hexaphosphate (Kd = 43 nM). In these enzymes, as in adenylate and thymidylate kinases, strongest inhibition was achieved in compounds containing one or two more phosphoryl groups than the substrates combined. These results support the view that nucleoside and nucleotide kinases mediate direct transfer of phosphoryl groups from ATP to acceptors, rather than acting by a double displacement mechanism. PMID- 3023352 TI - Production of platelet-activating factor by chick retina. AB - In the present study it is demonstrated that platelet-activating factor (PAF) was produced by chick retinas, upon stimulation with neurotransmitters such as acetylcholine (ACh), dopamine, or with calcium ionophore A23187, but not upon stimulation with gamma-amino-n-butyric acid, L-glycine, L-glutamate, epinephrine, or histamine. PAF produced in response to ACh, dopamine, or A23187 was not released into supernatants but was extractable from retinas. The amounts of extractable PAF increased after sonication of stimulated retinas. While no PAF activity could be recovered from unstimulated retinas, small amounts of this lipid can be detected following sonication of the tissue. The amount of extractable PAF from ACh-, dopamine-, or A23187-stimulated retinas was dependent upon the incubation time and concentration of the agonists. PAF was identified on the basis of chemical and lipase treatments, biological activity with washed rabbit platelets, behavior on thin layer chromatography, and high pressure liquid chromatography. Control cell preparations (leukocytes, erythrocytes, and embryogenic fibroblasts) did not produce PAF upon neurotransmitter stimulation. ACh and dopamine promoted PAF production by increasing dithiothreitol-insensitive cholinephosphotransferase activity, without affecting the acetyltransferase activity. In contrast, the A23187 ionophore stimulated the acetyltransferase activity but did not affect the dithiothreitol-insensitive cholinephosphotransferase. PMID- 3023351 TI - Endothelial cell leukotriene C4 synthesis results from intercellular transfer of leukotriene A4 synthesized by polymorphonuclear leukocytes. AB - Leukotriene (LT) synthesis and metabolism were studied in porcine aortic endothelial cells. Leukotrienes were identified by combinations of guinea pig lung parenchymal strip bioassay, radioimmunoassay, and UV spectrophotometry with high performance liquid chromatography. Endothelial cells stimulated with the calcium ionophore, A23187, were unable to convert arachidonic acid to detectable levels of LTA4-derived products including the biologically active metabolites, LTB4 or LTC4. However, these cells readily converted exogenous LTA4 to the potent slow-reacting substance, LTC4. Smaller quantities of 11-trans-LTC4 and LTD4 were also observed. LTB4 was not detectable in these incubations nor was LTB4 metabolism observed. The possible intercellular transfer of LTA4 between polymorphonuclear leukocytes (PMNL) and endothelial cells was tested since PMNL release LTA4 when stimulated and have significant contact with endothelium. When A23187-stimulated neutrophils were coincubated with endothelial cells, a significant increase in LTC4 levels was detected over PMNL alone. LTC4 is formed by the enzymatic conjugation of glutathione (GSH) with LTA4. Therefore in some experiments, endothelial cells were prelabeled with [35S]cysteine to allow intracellular synthesis of [35S]GSH. When unlabeled PMNL were added, as a source of LTA4 to the prelabeled endothelial cells, substantial levels of [35S] LTC4 were recovered. The data indicate that endothelial cells synthesize LTC4 from LTA4. They also demonstrate a specific PMNL-endothelial cell interaction in which endothelial cell LTC4 synthesis results from the intercellular transfer of LTA4 produced by PMNL. PMID- 3023354 TI - Guanine nucleotides stimulate soluble phosphoinositide-specific phospholipase C in the absence of membranes. AB - The effect of guanine nucleotides on platelet and calf brain cytosolic phospholipase C was examined in the absence of membranes or detergents in an assay using labeled lipid vesicles. Guanine nucleotides stimulate hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate [( 3H]PtdIns-4,5-P2) catalyzed both by enzyme from human platelets and by partially purified enzyme from calf brain. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was the most potent guanine nucleotide with a half-maximal stimulation at 1-10 microM, followed by guanosine 5'-(beta, gamma-imido)triphosphate greater than GTP greater than GDP = guanosine 5'-O-(2-thiodiphosphate). Guanosine 5'-O-(2-thiodiphosphate) was able to reverse the GTP gamma S-mediated stimulation. NaF also stimulated phospholipase C activity, further implying a role for a guanine nucleotide-binding protein. In the presence of GTP gamma S, the enzyme cleaved PtdIns-4,5-P2 at higher pH values, and the need for calcium ions was reduced 100-fold. The stimulation of PtdIns-4,5-P2 hydrolysis by GTP gamma S ranged from 2 to 25-fold under various conditions, whereas hydrolysis of [3H]phosphatidylinositol was only slightly affected by guanine nucleotides. We propose that a soluble guanine nucleotide dependent protein activates phospholipase C to hydrolyze its initial substrate in the sequence of phosphoinositide-derived messenger generation. PMID- 3023353 TI - Proton and nitrogen-15 NMR spectroscopic studies of hydrogen ion-dependent pseudo halide ion binding to chloroperoxidase. AB - The proton nuclear magnetic resonance spectra of several chloroperoxidase inhibitor complexes have been investigated. Titrations of chloroperoxidase with azide, thiocyanate, cyanate, or nitrite ions indicate that only the chloroperoxidase-thiocyanate complex exhibits slow ligand exchange on the 360-MHz NMR time scale. The temperature dependence of the proton NMR spectra of the complexes suggests that, although the complexes are predominantly low-spin ferric heme iron, a spin equilibrium is present presumably between S = 1/2 and S = 5/2 states. The pH dependence of the proton NMR spectra of the psuedo-halide chloroperoxidase complexes was examined at 360 and 90 MHz. Chloroperoxidase complexes with azide and cyanate show similar behavior; 360-MHz proton spectra are readily observed at low pH (less than 5.0) but not at high pH. At high pH, the ligand exchange rate falls in an intermediate time range. When the complexes are examined at 90 MHz, however, spectra consisting of averaged signals are observed. The chloroperoxidase-thiocyanate complex does not form at high pH values; the proton NMR spectrum observed is that of native chloroperoxidase. The pKa for the chloroperoxidase-thiocyanate heme-linked ionizable amino acid residue falls between 4.2 and 5.0. Only an averaged azide signal was observed in the nitrogen-15 NMR spectra for solutions that contained the azide complex of chloroperoxidase, horseradish peroxidase, and myoglobin. PMID- 3023355 TI - N-glycosylation in expression and function of beta-adrenergic receptors. AB - Through the use of specific staining and the analysis of the interaction of pure beta-adrenergic receptor of S49 mouse lymphoma cells with lectins immobilized to insoluble matrices, we establish that this beta-adrenergic receptor is a glycoprotein. The effects of swainsonine (0.2 microgram/ml), an inhibitor of Golgi mannosidase II, as well as those of tunicamycin (0.2 microgram/ml), an inhibitor of N-glycosylation, on the expression and function of this integral membrane glycoprotein were investigated in S49 mouse lymphoma cells grown in culture. Preexisting receptors on the cells were inactivated by alkylation with the beta-adrenergic antagonist ligand N-(2-hydroxy-3-naphthoxylpropyl)-N' bromoacetyl-ethylenediamine. Swainsonine did not alter the number of beta receptors measured in intact cells, the Bmax, or Kd of receptors measured in membranes prepared from these cells as assayed by [125I]iodocyanopindolol binding or their functional coupling to adenylate cyclase. Autoradiograms of membranes photoaffinity-labeled with [125I]iodoazidobenzylpindolol and subjected to electrophoresis on polyacrylamide gels reveal a reduction of 6,000 in the Mr of beta-receptors in membranes prepared from swainsonine-treated cells. This form of receptor was sensitive to endoglycosaminidase H, indicating its high mannose hybrid oligosaccharide nature. The number and affinity of beta-receptors in tunicamycin-treated S49 cells were normal. Whereas stimulation of cyclic AMP accumulation in cells or adenylate cyclase in membranes by prostaglandin E1 was essentially abolished by tunicamycin treatment, stimulation by isoproterenol was largely unaffected. The nonglycosylated receptor displays an Mr that is approximately 8,000-11,000 smaller than the native receptor. Thus, N glycosylation does not affect the expression (steady-state) or function of the beta-adrenergic receptor, whereas prostaglandin E1 receptor function is lost. The role of N-glycosylation in receptor function is not universal among receptors coupled to adenylate cyclase. PMID- 3023356 TI - Evidence for changes in the conformational status of rat liver microsomal glucose 6-phosphate:phosphohydrolase during detergent-dependent membrane modification. Effect of p-mercuribenzoate and organomercurial agarose gel on glucose-6 phosphatase of native and detergent-modified microsomes. AB - Comparative studies investigating influences of temperature and time of preincubation on the interactions of an organomercurial agarose gel and p mercuribenzoate with glucose-6-phosphatase of native and Triton X-114-modified rat liver microsomes were carried out. The effect of p-mercuribenzoate on glucose 6-phosphate hydrolysis is a result of two processes, a moderate membrane perturbation connected with release of some latency and temperature- and time dependent inhibition of the catalytic activity. Short-term preincubation with both organic mercurials at 37 degrees C is a necessary condition for the entire inhibition of the enzyme activity of native as well as of Triton X-114-modified microsomes. A binding site of the phosphohydrolase itself is accessible to p mercuribenzoate and the phenyl mercury residue of the affinity gel from the cytoplasmic surface even in native microsomes. Kinetic analyses reveal a formally competitive mechanism of inhibition using native microsomes, but the kinetic picture changes to a noncompetitive pattern of Lineweaver-Burk plots when the inhibitor-loaded microsomes are modified optimally by Triton X-114. This behavior can be evaluated as the first convincing evidence for drastic changes of the conformational status of the phosphohydrolase during the membrane modification process. A combined conformational flexibility-substrate transport model characterizing the microsomal glucose-6-phosphatase as an integral channel protein embedded within the hydrophobic interior of the membrane is proposed. PMID- 3023358 TI - Rubella virus cDNA. Sequence and expression of E1 envelope protein. AB - A cDNA clone encoding the entire E1 envelope protein (410 amino acid residues) and a portion of the C-terminal end of the E2 envelope protein of the rubella virus has been isolated and characterized. DNA sequence analysis has revealed a region 20 nucleotides in length at the 3' end of the cloned cDNA which may be a replicase recognition site or a recognition site for encapsidation. The proteolytic cleavage site between the E1 and E2 proteins was localized based on the known amino-terminal sequence of the isolated E1 protein (Kalkkinen, N., Oker Blom, C., and Pettersson, R. F. (1984) J. Gen. Virol. 65, 1549-1557) and the deduced amino acid sequence. The mature E1 protein is preceded by a set of 20 highly hydrophobic amino acid residues possessing characteristics of a signal peptide. This "signal peptide" is flanked on both sides by typical protease cleavage sites for trypsin-like enzyme and signal peptidase. The presence of a leader sequence in the E1 protein precursor may facilitate its translocation through the host cell membrane. The E1 protein of rubella virus shows no significant homology with alphavirus E1 envelope proteins. However, a stretch of 39 amino acids in the E1 protein of rubella virus (residues 262-300) was found to share a significant homology with the first 39 residues of bovine sperm histone. The position of 4 half-cystines and 8 arginines overlaps. The E1 protein of rubella virus has been successfully expressed in COS cells after transfecting them with rubella virus cDNA in simian virus 40-derived expression vector. This protein is antigenically similar to the one expressed by cells infected with rubella virus. PMID- 3023357 TI - In vitro mutagenesis and overexpression of the Escherichia coli trpA gene and the partial characterization of the resultant tryptophan synthase mutant alpha subunits. AB - A mutagenesis approach was initiated in order to examine further the folding behavior of the alpha-subunit of the Escherichia coli tryptophan synthase. A random single base pair saturation mutagenesis procedure (Myers, R.M., Lerman, L.S., and Maniatis, T. (1985) Science 229, 242-247) was applied in vitro to subcloned fragments of the trpA gene, which codes for this polypeptide. Mutagenesis plasmid vectors were constructed containing three fragments of the trpA gene which together code for about half of the total amino acid residues of the alpha-subunit. The vectors were constructed such that each strand of each trpA fragment could be altered. These trpA fragments were mutagenized in vitro (using nitrous acid, formic acid, hydrazine, and potassium permanganate), and several thousands of mutants have been isolated. Thirty-two mutants, contained within the first two trpA fragments (which encompass the first 206 base pairs of the trpA gene and encode the first 63 residues of the alpha-subunit) have been sequenced. Of these, 20 (63%) contained single base pair alterations, 12 (37%) contained multiple alterations, and 17 (53%) of these base pair alterations resulted in single amino acid substitutions. Selected mutant trpA fragments were subcloned into an overexpression vector in which the trpA gene is controlled by the tac promoter and is inducible by lactose. The kinetics and extent of induction show that after 22 h of induction, the wild-type alpha-subunit constituted about 30% of the total protein. A simple one-step purification procedure for the alpha-subunit is described in which 15 mg of alpha-subunit can be obtained from 200 ml of fully induced cultures. The mutant trpA genes were induced for mutant alpha-subunit expression, and an initial examination of their properties in crude extracts was performed. Of the 17 mutant proteins examined, most were overproduced to levels comparable to that for the wild-type alpha subunit. An analysis of the apparent stability, beta 2-subunit-activating activity, and intrinsic activity of this group of mutant alpha-subunits suggests that many amino acid alterations have no apparent effect; there is a variety of novel functional defects; and a sequence located near residues 28 through 54 may be particularly critical for the normal folding of the polypeptide. PMID- 3023359 TI - Carbodiimide inactivation of Na,K-ATPase, via intramolecular cross-link formation, is due to inhibition of phosphorylation. AB - We have recently shown that inactivation of renal Na,K-ATPase by 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide occurs via an intramolecular cross-link formed between an activated carboxyl group and an endogenous nucleophile (Pedemonte, C.H., and Kaplan, J.H. (1986) J. Biol. Chem. 261, 3632-3639). The modified enzyme shows the same level of Rb+ binding as untreated enzyme: 3.16 and 2.93 ATP sensitive mumol of Rb+ binding/mumol of phosphoenzyme, respectively. Thus, the Rb+ binding site and the transition accomplished by low affinity nucleotide binding which accelerates de-occlusion are not greatly affected by the carbodiimide inactivation. 1 mM K+ reduces the ADP binding to the high affinity nucleotide binding site to the same extent in normal and 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide-treated enzyme and Na+ counteracts this effect. Thus, the competition between Na+ and K+ ions for binding to the free enzyme are also largely unaltered by the modification. Phosphorylation from ATP (microM) in the presence of Na+ and Mg2+ ions and from inorganic phosphate in the presence of Mg2+ ions (in the absence or presence of ouabain) is greatly inhibited (85%) following carbodiimide treatment. The extent of inhibition of phosphorylation quantitatively correlates with the residual Na,K-ATPase activity (15%). Consequently, the rate of inactivation by carbodiimide is reduced when a greater proportion of the enzyme is in the phosphorylated form. Fluoroscein isothiocyanate, which inhibits the Na,K-ATPase by covalently modifying a lysine residue close to the high affinity binding site for ATP in the alpha-subunit does not bind to the carbodiimide-inactivated enzyme. Since high affinity nucleotide binding is only partially inhibited by the modification produced by the carbodiimide this suggests that the lysine residue to which fluoroscein isothiocyanate binds is not specifically required for competent nucleotide binding. PMID- 3023360 TI - Characterization of a DNA repair domain containing the dihydrofolate reductase gene in Chinese hamster ovary cells. AB - The formation and removal of UV-induced pyrimidine dimers were measured in restriction fragments near and within the essential dihydrofolate reductase (DHFR) gene in Chinese hamster ovary cells in order to map the genomic fine structure of DNA repair. Dimer frequencies were determined at 0, 8, and 24 h after irradiating the cells with 20 J/m2 UV light (254 nm). Within 8 h, the cells had removed more than 40% of the dimers from sequences near the 5' end of the gene, somewhat fewer from the 3' end, but only 2% from the 3' flanking region and 10% from a region upstream from the gene. The corresponding extent of repair in the genome as a whole is 5-10% in the 8-h period. Isoschizomeric restriction enzyme analysis was used to detect the level of methylation in the fragments in which repair was measured. We found that the only hypomethylated sites in and around the DHFR gene were in the fragment near its 5' end, which displayed maximal DNA repair efficiency. The size of the region of preferential DNA repair at the DHFR locus appears to be in the range of 50-80 kilobases, and this finding is discussed in relation to genomic domains and the structure of mammalian chromatin. PMID- 3023361 TI - The bacteriophage Mu N gene encodes the 64-kDa virion protein which is injected with, and circularizes, infecting Mu DNA. AB - Upon infection of Escherichia coli with bacteriophage Mu, a 64-kDa protein is injected into the host cell along with the phage DNA. This protein is involved in circularizing the infecting Mu DNA (Harshey, R. M., and Bukhari, A. I. (1983) J. Mol. Biol. 167, 427-441; Puspurs, A. H., Trun, N. J., and Reeve, J. N. (1983) EMBO J. 2, 345-352). Its possible role in the integration of infecting Mu DNA and in the infection process remains to be established. To identify the source of this protein we have prepared antiserum to the protein purified from viral particles. We have shown that the antiserum is specific for the Mu N gene product. The antiserum has been used to immunologically screen a Mu DNA library cloned into an expression vector. Four clones have been shown to produce a protein of 64 kDa that is specifically bound by the antiserum. The only Mu gene common to all four clones is the N gene, as demonstrated by physical and genetic mapping. We have also demonstrated by peptide mapping that the cloned N gene product is identical to the 64-kDa protein found complexed with the injected Mu DNA. PMID- 3023362 TI - Mechanism of interferon action. Expression of vesicular stomatitis virus G gene in transfected COS cells is inhibited by interferon at the level of protein synthesis. AB - The effect of interferon on the expression of the vesicular stomatitis virus glycoprotein G gene was examined in simian COS cells transfected with the expression vector pSVGL containing the G gene under the control of the SV40 late promoter. When COS cells were treated with interferon 24 h after transfection, the synthesis of vesicular stomatitis virus G protein was inhibited by about 80% as compared to that in untreated controls. By contrast, under the same conditions, neither the plasmid copy number nor the G gene mRNA levels were detectably affected by interferon treatment. Likewise, the synthesis of simian virus 40 large T-antigen was not inhibited by interferon treatment of transfected COS cells even though the synthesis of vesicular stomatitis virus G protein was markedly inhibited. The residual G protein synthesized in transfected, interferon treated COS cells appeared to be normally glycosylated. PMID- 3023363 TI - Cross-linking of a growth hormone releasing factor-binding protein in anterior pituitary cells. AB - Growth hormone-releasing factor (GRF) stimulates the release of growth hormone from the anterior pituitary and is related to the peptides of the glucagon/secretin family. Although the mechanism of action of this hormone has been studied in considerable detail, little is known concerning the GRF receptor itself. We have attempted to label the GRF receptor by chemically coupling the 125I-GRF analog [His1, Nle27]-hGRF(1-32)-NH2 (GRFa) (where Nle is norleucine) to plated rat anterior pituitary cells with the protein cross-linker disuccinimidyl suberate (DSS) (0.1 mM). Verification of biological activity of the 125I-GRFa was confirmed prior to the cross-linking experiments using the reverse hemolytic plaque assay. Whole cell extracts prepared from the cross-linked cells were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography of the dried gels. Four bands of 72, 50, 30, and 26 kDa were detected in autoradiograms from cells exposed to the labeled analog for 20 min (22 degrees C) followed by exposure to DSS for 2 min. The 72-kDa band was interpreted to be bovine serum albumin, which was used as a carrier in initial studies. The 50- and 30-kDa bands were very faint and probably represent nonspecific binding sites since they were unchanged in the presence of excess unlabeled GRFa. The 26-kDa band was diminished in a concentration-dependent manner by unlabeled rat GRF, GRFa, and to a lesser extent by vasoactive intestinal peptide (VIP). It is unlikely, however, that GRFa was acting at a VIP receptor since the labeled analog did not induce prolactin secretion (VIP is a prolactin secretagogue). GRFa also increased cellular cAMP to levels similar to GRF and greater than VIP. Autoradiographs from gels run under nonreducing conditions revealed the 26-kDa band as the major species, indicating that, if a polymeric form of this binding protein exists, it does not involve disulfide linkages. Thus, the best candidate for the putative GRF receptor is the 26-kDa band. We have further demonstrated that the higher concentrations of DSS used previously (5 mM) result in diffuse autoradiograms with multiple bands, suggesting that caution should be exercised when interpreting cross-linking data under these conditions. PMID- 3023365 TI - Molecular cloning and developmental expression of the cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum. AB - The cyclic nucleotide phosphodiesterase of Dictyostelium discoideum functions to maintain the responsiveness of cells to the chemoattractant cAMP during the aggregation phase of development. We have prepared a cDNA library and have isolated clones which contain a portion of the 5' untranslated region and the entire coding and 3' untranslated portions of the cyclic nucleotide phosphodiesterase gene. The primary structure of the extracellular cyclic nucleotide phosphodiesterase precursor has been deduced from the nucleotide sequence. The molecule is composed of 452 amino acids and was calculated to have a molecular mass of 51,078 daltons. Forty-nine amino-terminal residues which contain a hydrophobic leader sequence are not present in the mature extracellular enzyme. Four potential asparagine-linked glycosylation sites were found within the phosphodiesterase. An amino acid sequence homology search revealed no closely related proteins. Phosphodiesterase mRNA levels are low in growing cells and first increase soon after the onset of development. The amount of transcript then decreases before rising in abundance to maximum levels during the terminal stages of cell aggregation and apical tip formation. During formation of the fruiting body, levels of phosphodiesterase mRNA decrease. Exposure of cells to cAMP increases the amount of phosphodiesterase mRNA. Increases of mRNA abundance are correlated with increases in enzyme activity, suggesting regulation at the level of transcription. PMID- 3023364 TI - Molecular cloning of the rat stomach (H+ + K+)-ATPase. AB - We have isolated cDNA clones for the rat stomach (H+ + K+)-ATPase by employing a novel procedure involving the use of oligonucleotides corresponding to conserved amino acid sequences of related cation transport ATPase and a cross-hybridization with the sheep kidney (Na+ + K+)-ATPase alpha-subunit cDNA. The complete nucleotide sequence of the cDNA has been determined and the amino acid sequence of the protein deduced. The ATPase consists of 1,033 amino acids and has an Mr of 114,012. Amino acid homology and hydropathy plot comparisons between the gastric ATPase and the (Na+ + K+)-ATPase catalytic subunit demonstrate a striking similarity which suggests that their higher order structure and mechanism of action are virtually identical. The greatest homology occurs in the phosphorylation site region and in domains which may be involved in nucleotide binding and energy transduction. The most substantial differences occur in the N terminal region and in the transmembrane domains. In addition, we report the presence of an open reading frame 5' to the translation initiation site of the gastric ATPase, which raises the possibility that the mRNA is polycistronic. PMID- 3023366 TI - Targeted inhibition of transferrin-mediated iron uptake in Hep G2 hepatoma cells. AB - We have used a model system consisting of two human hepatoma cell lines, Hep G2, representing well differentiated normal hepatocytes, and PLC/PRF/5, representing poorly differentiated malignant hepatocytes, to demonstrate that the differential presence of asialoglycoprotein receptor activity in these cell lines can be used to influence transferrin-mediated iron uptake. We based our experiments on the following facts: Hep G2 cells possess receptors that bind, internalize, and degrade galactose-terminal (asialo-)glycoproteins; PLC/PRF/5 cells have barely detectable asialoglycoprotein receptor activity; both cell lines possess active transferrin-mediated iron uptake; transferrin releases iron during acidification of intracellular vesicular compartments; primary amines, e.g. primaquine, inhibit acidification and iron release from transferrin. When added to culture medium, [55Fe]transferrin delivered 55Fe well to both cell lines. As expected, in the presence of [55Fe]transferrin, free primaquine caused a concentration-dependent decrease in 55Fe uptake in both cell lines. To create a targetable conjugate, primaquine was covalently coupled to asialofetuin to form asialofetuin primaquine. When PLC/PRF/5 (asialoglycoprotein receptor (-)) cells were preincubated with this conjugate, transferrin-mediated 55Fe uptake was unaffected. However, transferrin-mediated 55Fe uptake by Hep G2 (asialoglycoprotein receptor (+)) cells under identical conditions was specifically decreased by 55% compared to control cells incubated without the conjugate. PMID- 3023369 TI - An electron spin resonance study of free radical intermediates in the oxidation of indole acetic acid by horseradish peroxidase. AB - The oxidation of indole-3-acetic acid by horseradish peroxidase was studied using the spin traps t-nitrosobutane and 5,5-dimethyl-1-pyrroline N-oxide to trap free radical intermediates. The major free radical metabolite of indole acetic acid was unambiguously determined by the use of indole-3-[2,2-2H2]acetic acid to be the skatole carbon-centered free radical. In the presence of oxygen, superoxide was also trapped. PMID- 3023367 TI - Regulation of the phosphoinositide hydrolysis pathway in thrombin-stimulated platelets by a pertussis toxin-sensitive guanine nucleotide-binding protein. Evaluation of its contribution to platelet activation and comparisons with the adenylate cyclase inhibitory protein, Gi. AB - In platelets activated by thrombin, the hydrolysis of phosphatidylinositol 4,5 bisphosphate by phospholipase C produces inositol 1,4,5-triphosphate (IP3) and diacylglycerol, metabolites which are known to cause Ca2+ release from the platelet dense tubular system and granule secretion. Previous studies suggest that phospholipase C activation is coupled to platelet thrombin receptors by a guanine nucleotide-binding protein or G protein. The present studies examine the contribution of this protein to thrombin-induced platelet activation and compare its properties with those of Gi, the G protein which mediates inhibition of adenylate cyclase by thrombin. In platelets permeabilized with saponin, nonhydrolyzable GTP analogs reproduced the effects of thrombin by causing diacylglycerol formation, Ca2+ release from the dense tubular system and serotonin secretion. In intact platelets, fluoride, which by-passes the thrombin receptor and directly activates G proteins, caused phosphoinositide hydrolysis and secretion. Fluoride also caused an increase in the platelet cytosolic free Ca2+ concentration that appeared to be due to a combination of Ca2+ release from the dense tubular system and increased Ca2+ influx across the platelet plasma membrane. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits G protein function, inhibited the ability of thrombin to cause IP3 and diacylglycerol formation, granule secretion, and Ca2+ release from the dense tubular system in saponin-treated platelets. Increasing the thrombin concentration overcame the effects of GDP beta S on secretion without restoring diacylglycerol formation. The effects of GDP beta S on platelet responses to thrombin which had been subjected to partial proteolysis (gamma-thrombin) were similar to those obtained with native alpha-thrombin despite the fact that gamma thrombin is a less potent inhibitor of adenylate cyclase than is alpha-thrombin. Thrombin-induced diacylglycerol formation and 45Ca release were also inhibited when the saponin-treated platelets were preincubated with pertussis toxin, an event that was associated with the ADP-ribosylation of a protein with Mr = 41.7 kDa. At each concentration tested, the inhibition of thrombin-induced diacylglycerol formation by pertussis toxin paralleled the inhibition of thrombin's ability to suppress PGI2-stimulated cAMP formation.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3023368 TI - The effect of phorbol esters and diacylglycerol on expression of the phosphoenolpyruvate carboxykinase (GTP) gene in rat hepatoma H4IIE cells. AB - The effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on expression of the P-enolpyruvate carboxykinase gene was studied in rat hepatoma H4IIE cells. Like insulin, PMA provokes a concentration and time-dependent decrease of mRNA coding for that enzyme that is due to an inhibition of P enolpyruvate carboxykinase gene transcription. This effect of PMA is rapid, reversible, specific for phorbol esters known to be active in other systems, and it does not require on-going protein synthesis. PMA overrides the stimulatory effects cAMP and glucocorticoid analogs have on the transcription of this gene. A synthetic diacylglycerol, sn-1,2-dioctanoylglycerol, also inhibits P-enolpyruvate carboxykinase gene transcription. These effects of PMA and synthetic diacylglycerol are specific, since neither affected total mRNA synthesis. We conclude that diacylglycerol and phorbol esters, specific stimulators of protein kinase C, inhibit the transcription of P-enolpyruvate carboxykinase gene in H4IIE cells. The findings support the hypothesis that diacylglycerols generated in the plasma membrane can act as an intracellular signal that regulates specific gene expression. PMID- 3023370 TI - Possible involvement of cyclic AMP and calcium ion in prostaglandin E1-induced elevation of c-myc mRNA levels in Swiss 3T3 fibroblasts. AB - Prostaglandin E1 (PGE1) caused a rapid and dose-dependent increase in cAMP levels, followed by elevation of c-myc mRNA levels and then increased DNA synthesis in quiescent cultures of Swiss 3T3 fibroblasts. The dose-response curves of PGE1 were nearly the same for each of these three processes. Both 8 bromo-cAMP and forskolin increased c-myc mRNA levels to 40-50% and DNA synthesis to 70-80% of those caused by a maximally effective dose of PGE1. Under the comparable conditions, PGE1 did not stimulate diacylglycerol formation or activate protein kinase C. However, PGE1 did elevate cytoplasmic free Ca2+ concentration as measured with the fluorescent Ca2+ indicator quin 2. 8-Bromo cAMP and forskolin were inactive in this capacity. The Ca2+ ionophore A23187 increased the level of c-myc mRNA. Diacylglycerol and Ca2+ mediate the elevation of c-myc mRNA levels which is caused by platelet-derived growth factor and fibroblast growth factor (Kaibuchi, K., Tsuda, T., Kikuchi, A., Tanimoto, T., Yamashita, T., and Takai, Y. (1986) J. Biol. Chem. 261, 1187-1192). In contrast, the present results suggest that both cAMP and Ca2+ are involved in this PGE1 induced response in Swiss 3T3 cells. PMID- 3023371 TI - The mitogenic signaling pathway of fibroblast growth factor is not mediated through polyphosphoinositide hydrolysis and protein kinase C activation in hamster fibroblasts. AB - Basic or acidic fibroblast growth factor (FGF), alone, was found to be as potent as alpha-thrombin to reinitiate DNA synthesis in G0-arrested Chinese hamster lung fibroblasts (CCL39). Basic FGF at 50 ng/ml or thrombin at 1 unit/ml rapidly initiated early events such as cytoplasmic alkalinization (0.2-0.3 pH units), rise in cytoplasmic Ca2+, phosphorylation of ribosomal protein S6 and increased c myc expression, followed by a 30-40-fold increase in labeled nuclei. Whereas thrombin is a potent activator of phospholipase C as judged by the rapid release of inositol trisphosphate, inositol bisphosphate and by the massive accumulation of total inositol phosphate (IP) in the presence of 20 mM Li+, FGF failed to induce the breakdown of polyphosphoinositides in quiescent CCL39 cells. Indeed, no inositol trisphosphate nor inositol bisphosphate could be detected in response to FGF; in presence of Li+ the total IP release never exceeded 8% of the IP released by the action of thrombin. Two additional findings indicated that FGF and thrombin activate different signaling pathways. First, we found that, in contrast to thrombin, the FGF-induced rise in the cytoplasmic free Ca2+ concentration measured by quin-2 fluorescence, is strictly dependent upon the presence of Ca2+ in the external medium. Second, we found that FGF failed to activate protein kinase C as judged by the epidermal growth factor-receptor binding assay. Treatment of the cells with either thrombin or phorbol esters, rapidly inhibited 125I-labeled epidermal growth factor binding (50-60%). Basic or acidic FGF had no effect. We conclude that: the FGF-receptor signaling pathway is not coupled to phospholipase C activation, and early mitogenic events and reinitiation of DNA synthesis can be initiated independently of inositol lipid breakdown and protein kinase C activation. PMID- 3023372 TI - Effects of lithium ions on glycogen synthase and phosphorylase in rat hepatocytes. AB - Incubation of hepatocytes from fasted rats with LiCl provoked a concentration- and time-dependent activation of glycogen synthase. This effect was observed in the absence of glucose in the incubation medium. No changes in the intracellular concentrations of ATP or glucose-6-phosphate were detected. Lithium was also able to activate glycogen synthase in the absence of extracellular calcium. If hepatocytes were incubated with lithium and insulin, an additive effect of both agents on glycogen synthase activity was observed. LiCl was also effective in activating the enzyme in hepatocytes obtained from fed rats. When hepatocytes were incubated with [33P]phosphate and then treated with LiCl, a decrease in the amount of [32P]phosphate incorporated in the enzyme was observed. This dephosphorylation affected two CNBr fragments of the enzyme (CB-2 and CB-1), suggesting that several phosphorylation sites were involved. Lithium was also able to activate glycogen phosphorylase from both fasted and fed rats. Phosphorylase activation was concentration- and time-dependent, either in the presence or absence of calcium in the incubation medium. These findings demonstrate that although lithium appears to mimic the effects of insulin on glycogen synthase activity, its mechanism of action must be different from that of the hormone. PMID- 3023373 TI - Sex differences in cytochrome P-450-dependent 25-hydroxylation of C27-steroids and vitamin D3 in rat liver microsomes. AB - Monoclonal antibodies directed against the cytochrome P-450 catalyzing 25 hydroxylation of C27-steroids and vitamin D3 (Andersson, S., Holmberg, I., and Wikvall, K. (1983) J. Biol. Chem. 258, 6777-6781) have been prepared by immunization of mice in hind footpads. Lymph node cells from the mice were fused with the Sp 2/0-Ag 14 line of mouse myeloma cells. A monoclonal antibody, designated MAb-25-6, monospecific for cytochrome P-450(25) was, after coupling to Sepharose, able to bind to cytochrome P-450(25) and to immunoprecipitate the activity for 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol and vitamin D3 as well as that for 16 alpha-hydroxylation of testosterone when assayed in a reconstituted system. Two-dimensional gel electrophoresis of adult male rat liver microsomes and immunoblotting with MAb-25-6 showed a single spot with an apparent isoelectric point of 7.4 and Mr approximately 51,000. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with MAb-25 6, cytochrome P-450(25) was shown to be male-specific and not detectable in adult female rat liver microsomes. N-terminal sequence analysis of cytochrome P-450(25) showed a structure identical with that of the male-specific steroid 16 alpha hydroxylase isolated in several laboratories. PMID- 3023374 TI - Regulation of calbindin-D28K gene expression by 1,25-dihydroxyvitamin D3 is correlated to receptor occupancy. AB - 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) induces the synthesis of calcium binding protein (calbindin-D28K) and calbindin-D28K-mRNA in the chick intestine. We have examined the relationship between nuclear uptake of 1,25-(OH)2D3, 1,25-(OH)2D3 receptor occupancy levels, and transcription of the calbindin-D28K gene and found all three parameters to be highly correlated. All three events occur rapidly (within 15-30 min) following a single dose of 6.5 nmol of 1,25-(OH)2D3 to vitamin D-deficient chicks, reaching peak values by 1-2 h; by 4 h, values of all three parameters start to decline. Calbindin-D28K-mRNA begins to accumulate by 3-5 h but does not peak until 12 h following hormone administration. The levels of calbindin-D28K start to increase by 5-8 h and do not peak until 48 h after the 1,25-(OH)2D3 dose. These observations suggest that post-transcriptional regulatory mechanisms may be involved. Measurements of basal levels of calbindin D28K gene transcription show that there is virtually no transcription in the vitamin D-deficient chick intestine, and a 12-fold induction in the intestine of vitamin D-replete chicks. This basal level of transcription in vitamin D-replete chick intestine is not inhibited by cycloheximide pretreatment. These results confirm the thesis that a major component of the mechanism of action of 1,25 (OH)2D3 is functioning as a steroid hormone, effected through the direct action of the seco-steroid-receptor complex on the initiation of transcription of specific genes. PMID- 3023375 TI - Oxidation of polycyclic aromatic hydrocarbons and dibenzo[p]-dioxins by Phanerochaete chrysosporium ligninase. AB - The lignin peroxidase (ligninase) of Phanerochaete chrysosporium catalyzes the oxidation of a variety of lignin-related compounds. Here we report that this enzyme also catalyzes the oxidation of certain aromatic pollutants and compounds related to them, including polycyclic aromatic hydrocarbons with ionization potentials less than or equal to approximately 7.55 eV. This result demonstrates that the H2O2-oxidized states of lignin peroxidase are more oxidizing than the analogous states of classical peroxidases. Experiments with pyrene as the substrate showed that pyrene-1,6-dione and pyrene-1,8-dione are the major oxidation products (84% of total as determined by high performance liquid chromatography), and gas chromatography/mass spectrometry analysis of ligninase catalyzed pyrene oxidations done in the presence of H2(18)O showed that the quinone oxygens come from water. We found that whole cultures of P. chrysosporium also transiently oxidize pyrene to these quinones. Experiments with dibenzo[p]dioxin and 2-chlorodibenzo[p]dioxin showed that they are also substrates for ligninase. The immediate product of dibenzo[p]dioxin oxidation is the dibenzo[p]dioxin cation radical, which was observed in enzymatic reactions by its electron spin resonance and visible absorption spectra. The cation radical mechanism of ligninase thus applies not only to lignin, but also to other environmentally significant aromatics. PMID- 3023377 TI - Binding of oxytocin to uterine cells in vitro. Occurrence of several binding site populations and reidentification of oxytocin receptors. AB - Myometrial and endometrial cells of sheep, rat, and calf in monolayer cell culture display at least three populations of binding sites for oxytocin, with dissociation constants (Kd) of approximately 5 X 10(-9), 4 X 10(-7), and greater than 10(-5) mol/liter, respectively. Binding of the tritium-labeled oxytocin (concentration range, 10(-11) to 5 X 10(-4) M) to the first two sites is displaceable by cold oxytocin. The ratio of binding capacities of the high to medium affinity site appears to average 1:18. Dissociation rate constants for these sites (22 degrees C) are roughly 10(-4) and 2 X 10(-3) s-1, respectively. The capacity of the low affinity site varies in individual cell preparations and is between 5 and 66 times that of the medium affinity site. The low affinity binding sites may not be fully saturable and may follow a nonasymptotic binding isotherm. Logarithms of Kd and binding capacity for individual binding sites are linearly correlated. The coexistence of the three sites was also proven by cluster analysis based on similarities between Kd, binding capacity, and Hill coefficient. Only minor systematic species and cell type differences occur in these properties. The value of Kd for the oxytocin receptor in rat myometrium, derived recently from a stepwise irreversible inhibition of uterotonic response to oxytocin, is close to 2.5 X 10(-7) mol/liter. Additional pharmacological data (pA2 values of structural analogues of oxytocin acting as competitive inhibitors) also reveal a Kd value of 3 X 10(-7). It is, therefore, concluded that the receptors for oxytocin in rat myometrium are identical with the medium affinity site. PMID- 3023376 TI - Calcium ion as a probe of the monovalent cation center of sodium, potassium ATPase. AB - Sodium and potassium ion-transport adenosine triphosphatase from dog kidney was incubated with 0.4-2 mM Ca2+ at 23 degrees C for more than 2 min in the absence of monovalent inorganic cations, cooled to 0 degrees C, and phosphorylated from 1 mM Pi with 2.4 mM MgCl2. The resultant phosphoenzyme resembled that obtained by incubating the enzyme with K+ in place of Ca2+ in six respects. It was concluded that Ca2+ can occupy the monovalent cation-binding center for K+. The rate constant for release of Ca2+ from the dephosphoenzyme at 0 degrees C was 0.17 s 1. The rate of release from the phosphoenzyme was at least 7-fold slower. Phosphorylation stabilized the binding of Ca2+ to the enzyme in contrast to its destabilization of the corresponding K X enzyme complex. K-sensitive phosphoenzyme did not respond to free Ca2+. Thus Ca2+ was not easily accepted by nor released from the phosphoenzyme and would not be an effective substrate for transport. A selective barrier against Ca2+ between the monovalent cation binding center and the extracellular solution is proposed. Release of calcium from the dephosphoenzyme yielded a conformation that was not phosphorylated from Pi. The enzyme changed the conformation of its center for phosphorylation before or at the same time that it changed the conformation of its center for ion transport. PMID- 3023378 TI - Receptor interconversion model of hormone action. I. ATP-mediated conversion of estrogen receptors from a high to lower affinity state and its relationship to antiestrogen action. AB - We have previously reported that the estrogen receptor exists in three distinct states in the oviduct of the estrogen-treated chick. Since two of these forms bind estrogen and possess a number of similar properties, the intrinsic relationship between these two receptor forms has been investigated. We now show that a quantitative conversion of the high affinity (Rx) to the lower affinity (Ry) state can be induced by mild heating (30 degrees C) in the presence of estradiol and ATP, or the synthetic analogue alpha,beta-methylene adenosine triphosphate and the divalent cation Mg2+. Other nucleotides, including ADP, GTP, CTP, cAMP, and cGMP, as well as the nonhydrolyzable analogues beta,gamma methylene adenosine triphosphate and alpha,beta-methylene adenosine diphosphate are ineffective. The conversion occurs only partially in the absence of estradiol but completely in its presence. The process is not inhibited by the protease inhibitors phenylmethylsulfonyl fluoride and alpha 2-macroglobulin, and when conversion is induced by low concentrations of ATP (1 mM), a time dependent reequilibration back to Rx occurs. These observations and the fact that the pure hormone-binding peptides Rx and Ry have similar molecular weights on sodium dodecyl sulfate-polyacrylamide gels (66,000) confirm that proteolysis is not involved in the conversion. Moreover their physical properties suggest that Rx and Ry exist in alternate conformations, with Ry being favored as a result of an ATP-mediated event involving the gamma-phosphoryl moiety. The biological relevance of the receptor conversion is suggested by studies with the antiestrogen hydroxytamoxifen. This antiestrogen binds to Rx with higher affinity than either estradiol or diethylstilbestrol but with low affinity to Ry. Hydroxytamoxifen also inhibits the conversion of Rx to Ry. Since this antiestrogen is a complete antagonist in the chick oviduct and prevents estradiol induced stimulation of ovalbumin gene transcription, it is speculated that Rx to Ry conversion is crucial for ovalbumin gene activation and that Rx may act as a transcriptional repressor. Furthermore, since Rx and Ry both bind to nuclei and DNA, it is proposed that the presence of Ry is a better predictor of ovalbumin gene activation than DNA binding alone. PMID- 3023379 TI - Receptor interconversion model of hormone action. II. Nucleotide-mediated conversion of estrogen receptors from nonsteroid binding to the lower affinity binding state. AB - Two steroid binding states of an estrogen receptor each with different equilibrium constants (Kd values) Rx (Kd = 0.06 nM) and Ry (Kd = 0.8 nM) have been identified and characterized in the hen and estrogen-stimulated chick oviduct. A third nonestrogen binding form of the receptor, designated Rnb, is now described which exists in short-term estrogen withdrawn chick oviduct cytosol. A model is presented in which the receptor can be interconverted between the three states. The interconversion is monitored by Scatchard analysis, sucrose density gradient analysis, and affinity labeling using [3H]tamoxifen aziridine followed by receptor purification with estrogen receptor monoclonal antibody affinity chromatography and sodium dodecyl sulfate-gel electrophoresis. The results are consistent with each state existing in different conformations having a common molecular weight of approximately 66,000. This paper defines the conditions and nucleotide requirements for the Rnb to Ry conversion. The conversion to the steroid binding form is induced by ATP, ADP, and GTP. Cyclic nucleotides are ineffective. There is a specific requirement for Mg2+; neither Ca2+ nor Mn2+ will substitute. Nonhydrolyzable nucleotide analogues were tested for their relative efficiency to convert Rnb to Ry. Conversion occurred with alpha,beta-methylene adenosine triphosphate, but beta,gamma-methylene adenosine triphosphate and alpha,beta-methylene adenosine diphosphate were inert. Thus, activation of Rnb to form Ry appears to be catalyzed by an event requiring the loss of the terminal phosphoryl moiety from either ATP or ADP. Receptor derived from conversion of Rnb to Ry has the same physical properties as native Ry. Activation of Rnb is to Ry specifically; no increase in the Rx form of estrogen receptor was ever observed. The accompanying paper similarly describes the Rx to Ry conversion. Since these data also explain observations made with glucocorticoid and with epidermal growth factor receptors, it is speculated that the receptor interconversion model may have general application to hormone action. PMID- 3023380 TI - Stimulated human neutrophils limit iron-catalyzed hydroxyl radical formation as detected by spin-trapping techniques. AB - Neutrophils stimulated with phorbol myristate acetate (PMA) in the presence of the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO), dimethyl sulfoxide, and diethylenetriaminepentaacetic acid (DETAPAC) fail to generate hydroxyl radical (.OH), detected as the methyl spin-trapped adduct of DMPO (2,2,5-trimethyl-1 pyrrolidinyloxyl, DMPO-CH3), unless ferric salts (Fe3+) are also added (Britigan, B. E., Rosen, G. M., Chai, Y., and Cohen, M. S. (1986) J. Biol. Chem. 261, 4426 4431). Even then, .OH formation wanes in spite of ongoing superoxide (O2-.) production. In contrast, ferric salt supplementation of a hypoxanthine/xanthine oxidase O2-. generating system containing DETAPAC produces continual .OH, suggesting that neutrophils limit the formation of this free radical. To evaluate this hypothesis, neutrophil cytoplasts (largely devoid of granules but able to generate O2-.) were stimulated with PMA in the presence of Fe3+, DETAPAC, dimethyl sulfoxide, and DMPO. This resulted in continual production of DMPO-CH3. In the presence of dimethyl sulfoxide, HL-60 (promyelocytic) cells differentiate into cells similar in morphology and O2-. generating capacity to neutrophils. However, their granules lack the iron-binding protein lactoferrin (LF). Ferric salt supplementation of HL-60 cells stimulated with PMA yielded an EPR spectrum similar to cytoplasts. Supernatant obtained following PMA-induced neutrophil degranulation (which releases LF extracellularly) suppressed DMPO-CH3 formation by the hypoxanthine/xanthine oxidase/Fe3+/DETAPAC system. Anti-LF antibody, but not anti-transferrin antibody, prevented stimulated neutrophil supernatant inhibition of hypoxanthine/xanthine oxidase/Fe3+/DETAPAC-mediated .OH formation. Similarly, neutrophils stimulated with PMA in the presence of Fe3+, DETAPAC, and anti-LF antibody (but not anti-transferrin antibody) demonstrated continual formation of .OH. Neutrophil degranulation of LF limits Fe3+-catalyzed .OH formation which in vivo could protect tissue from possible .OH-mediated injury. PMID- 3023381 TI - Cytochrome aa3 from Nitrosomonas europaea. AB - Cytochrome c oxidase has been purified from the ammonia oxidizing chemoautotroph Nitrosomonas europaea by ion-exchange chromatography in the presence of Triton X 100. The enzyme has absorption maxima at 420 and 592 nm in the resting state and at 444 and 598 nm in the dithionite-reduced form; optical extinction coefficient (598 nm minus 640 nm) = 21.9 cm-1 nM-1. The enzyme has approximately 11 nmol of heme a and approximately 11 nmol of copper per mg of protein (Lowry procedure). There appear to be three subunits (approximate molecular weights 50,800, 38,400, and 35,500), two heme groups (a and a3), and two copper atoms per minimal unit. The EPR spectra of the resting and partially reduced enzyme are remarkably similar to the corresponding spectra of the mitochondrial cytochrome aa3-type oxidase. Although the enzyme had been previously classified as "cytochrome a1" on the basis of its ferrous alpha absorption maximum (598 nm), its metal content and EPR spectral properties clearly show that it is better classified as a cytochrome aa3. Neither the data reported here nor a review of the literature supports the existence of cytochrome a1 as an entity discrete from cytochrome aa3. The purified enzyme is reduced rapidly by ferrous horse heart cytochrome c or cytochrome c-554 from N. europaea, but not with cytochrome c-552 from N. europaea. The identity of the natural electron donor is as yet unestablished. With horse heart cytochrome c as electron donor, the purified enzyme could account for a significant portion of the terminal oxidase activity in vivo. PMID- 3023383 TI - Structure and expression of the rat insulin-like growth factor II (rIGF-II) gene. rIGF-II RNAs are transcribed from two promoters. AB - Insulin-like growth factor II (IGF-II) is a mitogenic polypeptide present in rat plasma at high levels during fetal and early postnatal life and is believed to play an important, although as yet undefined, role in fetal development. Both in humans and rats, expression of the IGF-II gene results in the appearance of several mRNA species. In the present study, cDNA and synthetic oligonucleotide probes were used to isolate and characterize the rat IGF-II gene from genomic libraries. The rat IGF-II gene extends over 12 kilobase pairs and contains two 5' noncoding exons and three protein-coding exons. The two 5' exons represent alternative 5' regions of different mRNA molecules and are expressed from two distinct promoters. The two promoters are transcribed with different efficiencies but exhibit similar tissue-specific expression and regulation with developmental age. PMID- 3023382 TI - Interaction of a transcriptional activator, OmpR, with reciprocally osmoregulated genes, ompF and ompC, of Escherichia coli. AB - The ompB locus, comprised of the genes ompR and envZ, regulates the expression of the genes ompF and ompC that encode the major porin proteins in the outer membrane of Escherichia coli K-12. OmpR is believed to activate transcription of ompF and ompC in a reciprocal manner depending upon the osmolarity of the culture medium. We were able to purify OmpR to homogeneity and to characterize its interaction with the porin gene promoters. The purified OmpR was shown to bind specifically to promoter fragments of both ompF and ompC. Deoxyribonuclease I footprinting was carried out in order to determine binding sites of OmpR in the promoter regions of the ompF and ompC. OmpR was found to protect the regions -105 to -60 of ompF and -102 to -78 of ompC, respectively. Two consensus sequences were found in these protected sites and are likely to play an important role in OmpR recognition of these promoter regions. The purified OmpR was also shown to be functionally active in transcription in vitro; OmpR activates the expression of ompF and inhibits the transcription of ompC from the most distal of its three tandem promoters, P3. PMID- 3023385 TI - Characterization of COX9, the nuclear gene encoding the yeast mitochondrial protein cytochrome c oxidase subunit VIIa. Subunit VIIa lacks a leader peptide and is an essential component of the holoenzyme. AB - The gene COX9 for subunit VIIa of cytochrome c oxidase from Saccharomyces cerevisiae has been cloned with the aid of an oligonucleotide probe. From the nucleotide sequence of COX9, we deduce that subunit VIIa is derived from a precursor that is 59 amino acids in length (Mr = 6963). This precursor is longer than mature subunit VIIa by one amino acid at its NH2 terminus and four amino acids at its COOH terminus. COX9 exists as a single copy in the haploid genome of S. cerevisiae and produces one major transcript. When the genomic copy of COX9 is removed, cells lack a functional cytochrome c oxidase holoenzyme. From the predicted secondary structure of subunit VIIa, previous data concerning its relationship to the lipid bilayer of the inner membrane and the location of its hydrophobic domains (Power, S.D., Lochrie, M.A., and Poyton, R.O. (1986) J. Biol. Chem. 261, 9206-9209) and the finding that it is essential for the holoenzyme, we propose a model for subunit VIIa which suggests that this small integral protein plays a role in holoenzyme assembly or stability. PMID- 3023384 TI - Assembly of the mitochondrial membrane system. Characterization of COR1, the structural gene for the 44-kilodalton core protein of yeast coenzyme QH2 cytochrome c reductase. AB - The respiratory deficiency of yeast strains previously assigned to complementation group G7 has been ascribed to the absence in the mutants of functional cytochrome b. Since G7 mutants are capable of synthesizing the apoprotein, the primary effect of the mutations is to prevent maturation of this electron carrier. The recombinant plasmid pG7/T1 with a 6.7-kilobase pairs (kb) insert of wild type yeast nuclear DNA has been selected from a genomic library by transformation of a G7 mutant to respiratory competency. The genetically active region of the pG7/T1 insert has been subcloned on a 3-kb fragment of DNA which has been shown to contain an open reading frame encoding a protein of 50,236 Mr. In situ disruption of the reading frame causes a deficiency in cytochrome b. The strain with the disrupted gene fails to complement G7 mutants thereby confirming the correct identification of the gene henceforth referred to as COR1. The carboxyl-terminal half of the COR1 gene has been fused to the amino-terminal half of the Escherichia coli trpE gene in the high expression vector pATH2. This plasmid construct promotes a high level of expression of the trpE/COR1 hybrid protein. Antibodies against the purified hybrid protein react with a 44 kDa protein subunit of yeast coenzyme QH2-cytochrome c reductase corresponding to the largest core subunit of the complex. These data indicate that the yeast nuclear gene COR1 codes for the 44-kDa core protein and that the latter is required for the conversion of apocytochrome b to mature cytochrome b. PMID- 3023386 TI - COX8, the structural gene for yeast cytochrome c oxidase subunit VIII. DNA sequence and gene disruption indicate that subunit VIII is required for maximal levels of cellular respiration and is derived from a precursor which is extended at both its NH2 and COOH termini. AB - From the amino acid sequence of yeast cytochrome c oxidase subunit VIII published previously (Power, S. D., Lochrie, M.A., Patterson, T.E., and Poyton, R.C. (1984) J. Biol. Chem. 259, 6571-6574), we have synthesized a pair of oligonucleotide probes and used them to identify COX8, its structural gene. By genomic Southern blot analysis and disruption of the COX8 chromosomal locus, we have shown that this gene is present in one copy per haploid genome and that its product, subunit VIII, is essential for maximal levels of cellular respiration and cytochrome c oxidase activity. Alignment of the amino acid sequence predicted from the DNA sequence of COX8 with the determined amino acid sequence of subunit VIII indicates that mature subunit VIII is derived from a larger precursor that extends from both the NH2 and COOH termini of the mature polypeptide. Thus, like many other nuclear coded mitochondrial proteins, subunit VIII is derived from a precursor which carries a leader peptide. In addition, this precursor, like that for yeast cytochrome c oxidase subunit VIIa, appears to carry a four-amino acid "trailer peptide" at its COOH terminus. PMID- 3023388 TI - Epithelioid sarcoma in association with total knee replacement. A case report. AB - A case is reported in which an epithelioid sarcoma developed in an apparently benign enchondroma or bone infarct at the site of a chrome-cobalt total knee replacement. PMID- 3023387 TI - The biological performance of calcium phosphate ceramics in an infected implantation site. III: Biological performance of beta-whitlockite in the noninfected and infected rat middle ear. AB - The biological performance of macroporous beta-whitlockite implanted in the rat middle ear was evaluated. The material was studied in the non-infected middle ear and in middle ears infected by Staphylococcus aureus. beta-whitlockite was quickly covered by a normal mucosa. One week post-operatively the macropores were filled with exudate, fibrous tissue, and a small quantity of bone. Six months after the operation the greater part of the macropore area was filled with bone (74%); fibrous tissue accounted for 20%, and exudate for 5%. In histological sections, the macropore area of beta-whitlockite had increased by 68% after six months, indicating biodegradation. Macrophages and multinucleated cells were present in the vicinity of the implant and played a role in this biodegradation. Besides cytoplasmic vacuoles containing calcium phosphate, the cells showed smaller granules containing trace elements originally present in the implant material, such as silicon, titanium, aluminum, iron, and magnesium. PMID- 3023389 TI - Regulation of fibronectin receptor distribution by transformation, exogenous fibronectin, and synthetic peptides. AB - Recent studies have shown that fibronectin and its 140K membrane receptor complex are spatially associated with microfilaments to form cell surface linkage complexes which are thought to mediate adhesive interactions between fibroblasts and their substrata. We examined the regulation of the organization of these cell surface structures in transformed and fibronectin-reconstituted cells as well as in cells treated with a competitive synthetic peptide inhibitor of fibronectin binding to its receptor. Correlative localization experiments with interference reflection microscopy and double-label or triple-label immunofluorescence revealed a concomitant loss of fibronectin, 140K receptor, and alpha-actinin colocalization at cell substratum extracellular matrix contact sites after transformation of chick fibroblasts by wild-type or temperature-sensitive Rous sarcoma viruses (RSV). Western and dot immunoblot analyses established that although similar total quantities of intact 140K molecules were present in the transformed cell cultures, significantly more was released into the culture medium of transformed cells. The 140K molecules on transformed cells were available for interaction with exogenously added fibronectin, which could reconstitute fibronectin-140K linkage complexes. In such fibronectin reconstitution experiments, many cells expressed both fibronectin-140K-actin linkage complexes and RSV pp60src, indicating that the morphological reversion could occur even in the continued presence of RSV transformation. The synthetic peptide Gly-Arg-Gly-Asp-Ser derived from the sequence of the cell-binding region of fibronectin could also prevent the organization of fibronectin-140K linkage complexes. Our results suggest that fibronectin interaction with cells regulates the organization of fibronectin receptor complexes and cytoskeletal components at the cell surface. PMID- 3023390 TI - Transformed human cells release different fibronectin variants than do normal cells. AB - Fibronectin molecules are dimers composed of subunits whose primary structures may differ. This is due to alternative splicing in at least two regions (ED and IIICS) of the pre-mRNA. Using two monoclonal antibodies specific for two different epitopes of domain 5 (high affinity for heparin), we have quantitatively analyzed the expression of the IIICS sequence in human fibronectins from different sources. The results demonstrated that the percentage of fibronectin subunits containing the IIICS is higher in fibronectins from tumor derived or simian virus 40-transformed human cells than in fibronectins from human plasma or normal human fibroblasts. Furthermore, we observed that 45-65% of fibronectin subunits from transformed cells or normal embryonic fibroblasts are sialylated on the heparin-binding domain 5, whereas this occurs in only 24-28% of fibronectin subunits from normal adult fibroblasts. On the contrary, no sialylation was observed on domain 5 in fibronectin from human plasma. PMID- 3023392 TI - Selective inhibition of responses to nerve growth factor and of microtubule associated protein phosphorylation by activators of adenylate cyclase. AB - To study the influence of cAMP on cellular responses to nerve growth factor (NGF) and to use elevation of intracellular cAMP to probe the NGF mechanism, cultured PC12 pheochromocytoma cells were exposed to forskolin and cholera toxin. As in other cell types, the latter agents greatly increased PC12 cell cAMP levels. Such treatment also brought about a reversible, dose-dependent suppression of NGF promoted regeneration of neurites. In support of the role of cAMP in this effect, regeneration blockage by forskolin was potentiated by phosphodiesterase inhibitors. When tested on NGF-stimulated initiation of process outgrowth, cholera toxin and forskolin exerted a dual effect. As in previous studies, these drugs, when applied along with NGF, significantly enhanced the initial formation of short cytoplasmic extensions. However, after approximately 3 d of NGF exposure, at which time such extensions begin to acquire the morphological and ultrastructural features of neurites, these agents suppressed process outgrowth. That is, the neurites were fewer in number, significantly less branched, and much shorter than in control cultures. Such changes also occurred when these drugs were added to cultures that had been pretreated with NGF alone. Whereas forskolin and cholera toxin affect the formation and regeneration of neurites, these drugs did not interfere with the short-latency, transient changes in surface morphology that are triggered by NGF, nor did they inhibit transcription-dependent priming. In contrast, the rapidly occurring NGF-induced phosphorylation of tyrosine hydroxylase was suppressed. Moreover, forskolin and cholera toxin rapidly and selectively blocked the NGF-promoted phosphorylation of a set of microtubule associated proteins known as chartins. Previous observations have suggested a causal relationship between NGF-induced chartin microtubule-associated protein phosphorylation and the formation and outgrowth of neurites. This is supported by the present data and provides a possible mechanism whereby elevated cAMP may interfere with neurite growth and regeneration. PMID- 3023393 TI - Uptake and transepithelial transport of nerve growth factor in suckling rat ileum. AB - Nerve growth factor (NGF) is necessary for the development of sympathetic and some sensory neurons. Milk may be a source of NGF for suckling young, but sites of intestinal absorption of the protein have not been identified. To determine whether NGF is transported across the absorptive epithelium of suckling rat ileum, we assessed binding, uptake, and transport of 125I-NGF by light microscopy and EM autoradiography. Blood and tissue extracts were analyzed by biochemical and immunological methods to determine whether NGF was taken up structurally intact. NGF binding sites were identified on microvilli and apical invaginations of ileal absorptive cells in vitro. Injected into ileal loops in vivo, NGF radioactivity retained by fixation was evident after 20 min in apical regions of absorptive cells, in endocytic tubules (which mediate the uptake of membrane bound ligands), in vesicles (which mediate nonspecific endocytosis), and in the supranuclear lysosomal vacuole. At 1 and 2 h, radiolabel in these compartments increased and silver grains were evident at the basal cell surface, and in cells, matrix, and vessels of the lamina propria. In blood and liver, radiolabeled molecules that were immunologically and electrophoretically indistinguishable from NGF and that co-eluted with NGF on gel filtration columns were detected, confirming that some NGF was transported across the epithelium structurally intact. Thus, absorptive cells of suckling rat ileum can take up NGF by both receptor-mediated and nonspecific endocytosis, and direct NGF either to the lysosome for degradation, or into a transepithelial transport pathway. PMID- 3023391 TI - Dynamics of membrane-skeleton (fodrin) organization during development of polarity in Madin-Darby canine kidney epithelial cells. AB - Madin-Darby canine kidney (MDCK) epithelial cells exhibit a polarized distribution of membrane proteins between the apical and basolateral domains of the plasma membrane. We have initiated studies to investigate whether the spectrin-based membrane skeleton plays a role in the establishment and maintenance of these membrane domains. MDCK cells express an isoform of spectrin composed of two subunits, Mr 240,000 (alpha-subunit) and Mr 235,000 (gamma subunit). This isoform is immunologically and structurally related to fodrin in lens and brain cells, which is a functional and structural analog of alpha beta spectrin, the major component of the erythrocyte membrane skeleton. Analysis of fodrin in MDCK cells by immunoblotting, immunofluorescence, and metabolic labeling revealed significant changes in the biophysical properties, subcellular distribution, steady-state levels, and turnover of the protein during development of a continuous monolayer of cells. The changes in the cellular organization of fodrin did not appear to coincide with the distributions of microfilaments, microtubules, or intermediate filaments. These changes result in the formation of a highly insoluble, relatively dense and stable layer of fodrin which appears to be localized to the cell periphery and predominantly in the region of the basolateral plasma membrane of MDCK cells in continuous monolayers. The formation of this structure coincides temporally and spatially with extensive cell-cell contact, and with the development of the polarized distribution of the Na+, K+ ATPase, a marker protein of the basolateral plasma membrane. PMID- 3023394 TI - Cellular and subcellular distribution of a cAMP-regulated prestalk protein and prespore protein in Dictyostelium discoideum: a study on the ontogeny of prestalk and prespore cells. AB - We have analyzed a developmentally and spatially regulated prestalk-specific gene and a prespore-specific gene from Dictyostelium. The prestalk gene, pst cathepsin, encodes a protein highly homologous to the lysosomal cysteine proteinases cathepsin H and cathepsin B. The prespore gene encodes a protein with some homology to the anti-bacterial toxin crambin and has been designated beejin. Using the lambda gtll system, we have made polyclonal antibodies directed against a portion of the protein encoded by pst-cathepsin and other antibodies directed against the beejin protein. Both antibodies stain single bands on Western blots. By immunofluorescence and Western blots, pst-cathepsin is not present in vegetative cells or developing cells during the first approximately 10 h of development. It then appears with a punctate distribution in a subset of developing cells. Beejin is detected only after approximately 15 h of development, also in a subset of cells. Pst-cathepsin is distributed in the anterior approximately 1/10 of migrating slugs and on the peripheral posterior surfaces of slugs. Beejin is distributed in the posterior region of slugs. Expression of both pst-cathepsin and beejin can be induced in subsets of isolated cultured cells by a combination of conditioned medium and extracellular cAMP in agreement with the regulation of the mRNAs encoding these proteins. We have used the antibodies as markers for cell type to examine the ontogeny and the spatial distribution of prestalk and prespore cells throughout multicellular development. Our findings suggest that prestalk cell differentiation is independent of position within the aggregate and that the spatial localization of prestalk cells within the multicellular aggregate arises from sorting of the prestalk cells after their induction. We have also found a class of cell in developing aggregates that contains neither the prestalk nor the prespore markers. PMID- 3023395 TI - Effect of catecholamines on Na/H exchange in vascular smooth muscle cells. AB - Catecholamines were found to activate Na/H exchange in a concentration-dependent manner in primary cultures of vascular smooth muscle cells (VSMC). The potency order was found to be epinephrine greater than norepinephrine greater than isoproterenol. The major pathway for catecholamine effects appeared to be via interaction with an alpha 1 adrenergic receptor. In addition, it was found that alpha 1 receptor-mediated Na/H exchange in VSMC was increased by angiotensin II and inhibited by 12-O-tetradecanoyl phorbol-13-acetate (TPA). Adrenergic receptors have been shown to be coupled to both adenylate cyclase and to inositol phosphate release (Leeb-Lundberg, L. M. F., S. Cotecchia, J. W. Lomasney, J. F. DeBernadis, R. J. Lefkowitz, and M. G. Caron, 1985, Proc. Natl. Acad. Sci. USA, 82:5651-5655.). It was found that catecholamines increased AMP levels in the potency order isoproterenol greater than norepinephrine greater than epinephrine and the receptor involved was a beta adrenergic receptor. Since these findings did not parallel the results obtained for catecholamine stimulation of Na/H exchange, an increase in AMP levels was probably not the mechanism by which major pathway for catecholamine-stimulated Na/H exchange in VSMC (via the alpha 1 receptor) was activated. When the effects of catecholamines were measured on inositol phosphate release, the potency order for catecholamine stimulation was epinephrine greater than norepinephrine greater than isoproterenol, and the receptor involved was an alpha 1 adrenergic receptor. In addition, angiotensin II increased and TPA inhibited catecholamine-stimulated inositol phosphate release. Since these findings paralleled the results obtained for catecholamine stimulation of Na/H exchange, inositol phosphate release may be the mechanism by which the major pathway for catecholamine-stimulated Na/H exchange in VSMC (via the alpha 1 receptor) was activated. PMID- 3023396 TI - Allosteric regulation of the epidermal growth factor receptor kinase. PMID- 3023397 TI - The NH2 terminus of preproinsulin directs the translocation and glycosylation of a bacterial cytoplasmic protein by mammalian microsomal membranes. AB - To investigate putative sorting domains in precursors to polypeptide hormones, we have constructed fusion proteins between the amino terminus of preproinsulin (ppI) and the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase (CAT). Our aim is to identify sequences in ppI, other than the signal peptide, that are necessary to mediate the intracellular sorting and secretion of the bacterial enzyme. Here we describe the in vitro translation of mRNAs encoding two chimeric molecules containing 71 and 38 residues, respectively, of the ppI NH2 terminus fused to the complete CAT sequence. The ppI signal peptide and 14 residues of the B-chain were sufficient to direct the translocation and segregation of CAT into microsomal membrane vesicles. Furthermore, the CAT enzyme underwent N-linked glycosylation, presumably at a single cryptic site, with an efficiency that was comparable to that of native glycoproteins synthesized in vitro. Partial amino-terminal sequencing demonstrated that the downstream sequences in the fusion proteins did not alter the specificity of signal peptidase, hence cleavage of the ppI signal peptide occurred at precisely the same site as in the native precursor. This is in contrast to results found in prokaryotic systems. These data demonstrate that the first 38 residues of ppI encode all the information necessary for binding to the endoplasmic reticulum membrane, translocation, and proteolytic (signal sequence) processing. PMID- 3023398 TI - Glucocorticoid-regulated glycoprotein maturation in wild-type and mutant rat cell lines. AB - Glucocorticoid hormones can regulate the posttranslational maturation of mouse mammary tumor virus (MTV) precursor polyproteins in M1.54, a stably infected rat hepatoma cell line. We have used complement-mediated cytolysis to recover variants of M1.54 that fail to express MTV cell surface glycoproteins in a hormone-regulated manner (Firestone, G.L., and K.R. Yamamoto, 1983, Mol. Cell. Biol., 3:149-160). One such clonal isolate, CR4, is similar to wild-type with respect to synthesis of MTV mRNAs, production of the MTV glycoprotein precursor (gPr74env) and a glycosylated maturation product (gp51), and hormone-induced processing of two MTV phosphoproteins. In contrast, three viral cell surface glycoproteins (gp78, gp70, and gp32) and one extracellular species (gp70s), which derive from gPr74env in glucocorticoid-treated wild-type cells, fail to appear in CR4. CR4 showed no apparent alterations in proliferation rate, cell shape, or expression of total functional mRNA and bulk glycoproteins. We conclude that the genetic lesion in CR4 defines a highly selective hormone-regulated glycoprotein maturation pathway that alters the fate of a restricted subset of precursor species. PMID- 3023401 TI - Caffeine contracture in the cultured chick myotube. AB - A possible function of Ca store site in cultured chick myotubes was examined by recording contraction of the myotube with special reference to the effect of caffeine. Caffeine at low concentrations (below 1 mM), applied focally on the myotube through a micropipette with a pressure pulse, elicited focal contraction without membrane potential changes. Procaine inhibited the caffeine contracture. Deuterium oxide also inhibited the caffeine contracture at low concentrations, but enhanced the maximal contracture. These observations are similar to those in the mature frog muscle fiber in which the sarcoplasmic reticulum (SR) is a main site of caffeine action. On the basis of these similarities, it was considered that caffeine acts on SR to elicit contracture in the myotube. The ability of SR to accumulate and release Ca ion seemed to be low, because caffeine contracture decreased or disappeared in a Ca-free solution in many myotubes. PMID- 3023399 TI - Stimulus-secretion coupling in the developing exocrine pancreas: secretory responsiveness to cholecystokinin. AB - We have studied the onset of secretory responsiveness to cholecystokinin (CCK) during development of the rat exocrine pancreas. Although acinar cells of the fetal pancreas (1 d before birth) are filled with zymogen granules containing the secretory protein, alpha-amylase, the rate of amylase secretion from pancreatic lobules incubated in vitro was not increased in response to CCK. In contrast, the rate of CCK-stimulated amylase discharge from the neonatal pancreas (1 d after birth) was increased four- to eightfold above that of the fetal gland. The postnatal amplification of secretory responsiveness was not associated with an increase in the number or cell surface expression of 125I-CCK binding sites. When 125I-CCK-33 binding proteins were analyzed by affinity crosslinking, two proteins of Mr 210,000 and 100,000-160,000 were labeled specifically in both fetal and neonatal pancreas. To determine if cell surface receptors for CCK in the fetal pancreas are functional and able to generate a rise in the cytosolic [Ca++], we measured 45Ca++ efflux from tracer-loaded lobules. 45Ca++ efflux from both fetal and neonatal pancreas was comparably increased by CCK, indicating CCK-induced Ca++ mobilization and elevated cytosolic [Ca++]. The Ca++ ionophore A23187 also stimulated the rate of 45Ca++ extrusion from pancreas of both ages. Increased amylase secretion occurred concurrently with A23187-stimulated 45Ca++ efflux in neonatal pancreas, but not in the fetal gland. A23187 in combination with dibutyryl cAMP potentiated amylase release from the neonatal gland, but not from fetal pancreas. Similarly, the protein kinase C activator, phorbol dibutyrate, did not increase the rate of secretion from the fetal gland when added alone or in combination with A23187 or CCK. We suggest that CCK-receptor interaction in the fetal pancreas triggers intracellular Ca++ mobilization. However, one or more signal transduction events distal to Ca++ mobilization have not yet matured. The onset of secretory response to CCK that occurs postnatally may depend on amplification of these transduction events. PMID- 3023400 TI - The plasminogen system and cell surfaces: evidence for plasminogen and urokinase receptors on the same cell type. AB - The capacity of cells to interact with the plasminogen activator, urokinase, and the zymogen, plasminogen, was assessed using the promyeloid leukemic U937 cell line and the diploid fetal lung GM1380 fibroblast cell line. Urokinase bound to both cell lines in a time-dependent, specific, and saturable manner (Kd = 0.8-2.0 nM). An active catalytic site was not required for urokinase binding to the cells, and 55,000-mol-wt urokinase was selectively recognized. Plasminogen also bound to the two cell lines in a specific and saturable manner. This interaction occurred with a Kd of 0.8-0.9 microM and was of very high capacity (1.6-3.1 X 10(7) molecules bound/cell). The interaction of plasminogen with both cell types was partially sensitive to trypsinization of the cells and required an unoccupied high affinity lysine-binding site in the ligand. When plasminogen was added to the GM1380 cells, a line with high intrinsic plasminogen activator activity, the bound ligand was comprised of both plasminogen and plasmin. Urokinase, in catalytically active or inactive form, enhanced plasminogen binding to the two cell lines by 1.4-3.3-fold. Plasmin was the predominant form of the bound ligand when active urokinase was added, and preformed plasmin can also bind directly to the cells. Plasmin on the cell surface was also protected from its primary inhibitor, alpha 2-antiplasmin. These results indicate that the two cell lines possess specific binding sites for plasminogen and urokinase, and a family of widely distributed cellular receptors for these components may be considered. Endogenous or exogenous plasminogen activators can generate plasmin on cell surfaces, and such activation may provide a mechanism for arming cell surfaces with the broad proteolytic activity of this enzyme. PMID- 3023402 TI - System A transport activity in normal rat hepatocytes and transformed liver cells: substrate protection from inactivation by sulfhydryl-modifying reagents. AB - The transport of amino acids by normal rat hepatocytes and several hepatoma cell lines has been examined for inactivation by various protein-modifying reagents, including the sulfhydryl-preferring reagents N-ethylmaleimide (NEM) and p chloromercuribenzene sulfonate (PCMBS). Uptake of 2-aminoisobutyric acid (AIB), a specific probe for hepatic System A-mediated transport, was equally sensitive to inhibition by the organic mercurial PCMBS in each of the cell types tested. In contrast, the sensitivity of System A to inactivation by NEM was substantially different among the five cell types. Normal hepatocytes showed the greatest sensitivity, while the hepatoma cells varied in their responsiveness from moderate to no inhibition. PCMBS inactivated greater than 85% of the System A activity in rat H4 hepatoma cells within 10 min (t1/2 = 3 min). The inhibition by PCMBS was rapidly reversed by treatment of the cells with dithiothreitol. Amino acids showing a high affinity for System A protected the transport system from inactivation, whereas non-substrates produced little or no protection. Amino acid dependent protection was stereospecific and system-specific. L-norleucine competitively inhibited AIB uptake (Ki = 1.9 +/- 0.1 mM) in H4 cells and also protected System A from PCMBS-dependent inactivation (half-maximal protection occurred at an amino acid concentration of 0.6 +/- 0.1 mM). N-bromosuccinimide was completely ineffective as an inhibitor of System A activity in hepatocytes, whereas treatment of H4 rat hepatoma cells with this reagent resulted in greater than 95% inhibition. PMID- 3023403 TI - Signal transduction in human monocytes: relationship between superoxide production and the level of kinase C in the membrane. AB - Activation of monocytes and neutrophils results in an increased production of superoxide, an important cytotoxic compound. The previous finding that two agents that induce superoxide production cause opposite translocation of kinase C (Costa Casnellie et al. [1985] Biochem. Biophys. Res. Commun., 133:1139-1144). led us to study the role of kinase C in superoxide production. In monocytes induction of superoxide production by 13-tetradecanoate phorbol acetate requires translocation of kinase C from the cytosol to the membrane. Superoxide production is also induced by concanavalin A, but this induction is accompanied by a shift of kinase C from the membrane to the cytosol. Superoxide production by concanavalin A is greatly augmented by cytochalasin B. During activation by Con A and cytochalasin B the membrane kinase C is translocated to the cytosol in a manner similar to that observed in the presence of Con A alone. Under these conditions approximately 5-10% of kinase C remains associated with the membrane. Thus, induction of superoxide production by concanavalin A is independent of the levels of kinase C tightly bound to the membrane. We also show evidence that the concanavalin A-induced release of kinase C from the membrane is not due to an increase in levels of intracellular calcium or increased phosphoinositide turnover. In summary, these data indicate that concanavalin A and phorbol ester induced superoxide production by human monocytes occurs by distinct pathways and that superoxide production is not closely correlated with specific levels of membrane-associated kinase C activity. PMID- 3023404 TI - Clonal variation in response to adrenocorticotropin in cultured bovine adrenocortical cells: relationship to senescence. AB - During cellular senescence, non-clonal cultures of bovine adrenocortical cells show a continuous decline in the rate of production of cyclic AMP (cAMP) stimulated by adrenocorticotropin (ACTH), without changes in the rate of forskolin- or prostaglandin E1-stimulated cAMP production. We investigated the possible mechanisms for loss of response to ACTH by examining the properties of clones of bovine adrenocortical cells. ACTH-stimulated cAMP production rates were measured in clones immediately after isolation, during long-term growth following isolation, and after subcloning. ACTH-stimulated rates were compared with cAMP production in response to forskolin, which acts directly on the catalytic subunit of adenylate cyclase. The results show that cloning is not necessarily associated with a loss of response to ACTH, but that clones with high ACTH response can give rise to subclones with low response. Clones of adrenocortical cells, at the same approximate population doubling level (PDL), showed ACTH response levels that ranged from 12 to 135 pmol cAMP/10(6) cells/min, whereas mass cultures at this PDL showed approximately 50 pmol/10(6) cells/min. Forskolin-stimulated cAMP production rates in clones varied only over the range of 59-119 pmol/10(6) cells/min and showed no correlation with the ACTH-stimulated rates. All clones were adrenocortical cells, as shown by mitogenic response to angiotensin II and cAMP-inducible 17 alpha-hydroxylase activity. The replicative potential of clones varied widely, and there was no apparent correlation between ACTH response levels and growth potential. The level of ACTH response in each clone was stable during proliferation through at least 25 PD beyond the stage at which the clone was isolated. When clones were subcloned, a clone with a high ACTH response level produced sister subclones that had ACTH response levels ranging from 3% of that of the parent clone to a level slightly greater than that of the parent clone. The growth potential of sister subclones varied widely, as for the parent clones, and there was no obvious correlation between growth potential and ACTH response. Two subclones were cloned; in sub-subclones, levels of ACTH response were again different from each other and also from the parent subclone; in one case, the level of ACTH response was approximately eight-fold higher than that of the parent subclone. These experiments show that clonal variation in the extent of expression of a differentiated property may occur in a normal differentiated cell in culture. The loss of ACTH response and the loss of proliferative potential appear to be independent stochastic events. PMID- 3023405 TI - Enzymes of phospholipid metabolism in rat pancreatic islets: subcellular distribution and the effect of glucose and calcium. AB - The effect of glucose and calcium on the activities of the phosphatidylinositol cycle enzymes, CDP-diglyceride inositol transferase, diacylglycerokinase, and lysophosphatidylcholine 2-acyltransferase in rat pancreatic islets was studied. Calcium inhibited the activity of CDP-diglyceride inositol transferase but had no effect on lysophosphatidylcholine 2-acyltransferase and diacylglycerokinase activities. Upon preincubation of islets in a concentration of glucose known to stimulate insulin release, the activity of lysophosphatidylcholine 2 acyltransferase, but not that of diacylglycerokinase or the CDP-diglyceride inositol transferase, was stimulated. Subcellular fractionation of pancreatic islets showed that secretory granule membranes were enriched in CDP-diglyceride inositol transferase, whereas lysophosphatidylcholine 2-acyltransferase activity was highest in the microsomal membranes. The activation of 2-acyltransferase by incubating islets in insulinotropic glucose, and the calcium sensitivity of CDP diglyceride inositol transferase, suggest that these enzymes may have roles in regulation of insulin secretion. PMID- 3023407 TI - Viral therapy: prospects for protease inhibitors. AB - Antiviral activities of known protease inhibitors were assayed in virus-infected cell cultures. Some members of the cystatin superfamily, in particular chicken cystatin, were able to block virus replication. In a binding assay, using purified components, chicken and human cystatin were able to bind poliovirus protease with affinities which were reflected in their relative antiviral potencies. Prospects for application of protease inhibitors in clinical viral infections are discussed. PMID- 3023406 TI - Role of granule proteins in lymphocyte-mediated killing. PMID- 3023408 TI - The receptor for urokinase-plasminogen activator. AB - Many human cells and cell lines possess a specific receptor that binds urokinase plasminogen activator (uPA) with an affinity of about 10(-10) M. Bound enzyme is not internalized, is slowly dissociated, and retains its enzymatic activity. The amino acid sequence of uPA responsible for receptor binding is located within the first 35 aminoterminal residues, ie, in the growth factor domain. Binding, however, is not competed for by other proteins that contain the growth factor domain (including epidermal growth factor). Cells that produce uPA secrete the pro-uPA form, which subsequently binds to the receptor. A431 cells, in fact, have their receptors completely saturated with pro-uPA. It is proposed that uPA:uPA receptor interaction plays a direct role in physiological and pathological processes that require cell migration. PMID- 3023409 TI - Somatic events unmask recessive cancer genes to initiate malignancy. AB - A heritable mutation predisposes an individual to certain childhood malignancies, such as retinoblastoma and Wilms' tumor. The chromosomal locations of the genes responsible for the predisposition are known by linkage with chromosomal deletions and enzyme markers. A study of these tumors in comparison to the normal constitutional cells of the patients, using enzyme and DNA markers near the predisposing genes, has shown that these genes are recessive to normal wild-type alleles at the cellular level. Expression of the recessive phenotype (malignancy) involves the same genetic events that were observed in Chinese hamster cell hybrids carrying recessive drug resistance genes. In both the experimental and clinical situations, the wild-type allele is most commonly eliminated by chromosome loss with duplication of the mutant chromosome. Simple chromosome loss and mitotic recombination have been documented in both systems. In the remaining 30% of cases, inactivation or microdeletion of the wild-type allele are assumed to be responsible for expression of the recessive phenotype. Osteosarcoma is a common second tumor in patients who have had retinoblastoma. Studies with markers in osteosarcoma show that these tumors also result from unmasking of the recessive phenotype by loss of the normal allele at the retinoblastoma locus, whether or not the patient had retinoblastoma. Subsequent chromosomal rearrangements and amplification of oncogenes that occur in these homozygous tumors provide progressive growth advantage. In other malignancies, in which studies have so far focused on oncogene amplification and chromosomal rearrangements, unmasking of recessive mutations may also be the critical initiating events. PMID- 3023410 TI - Separation of corticosteroids by high-performance liquid chromatography on dynamically modified silica. AB - High-performance liquid chromatographic methods have been elaborated for the separation of 12 different corticosteroids by reversed-phase chromatography on bare silica, dynamically modified by cetyltrimethylammonium bromide added to the eluent. The selectivity of the systems towards a mixture of the 12 corticosteroids and towards a mixture of hydrocortisone and its related impurities were investigated using different brands of silica. The variations in selectivity were found to be substantially smaller than those of chromatographic systems based on chemically bonded ODS-silicas from the same sources. PMID- 3023411 TI - Purification of myosin by high-performance liquid chromatography on hydroxyapatite. PMID- 3023412 TI - Effect of the silica support of bonded reversed-phase columns on chromatography of some antibiotic compounds. AB - Chromatographic behavior of three types of antibiotics was investigated on bonded C18 and polymeric reversed-phase columns. With penicillins with carboxylic acid functions only, retention and separations on the two types of columns were similar. beta-Lactam antibiotics with basic functions did not give as sharp peaks on the C18 column unless a silanol blocking agent, tetramethyl ammonium chloride (TMA) was added. In 0.01 M orthophosphoric acid-acetonitrile, tetracyclines were separated on the polymeric reversed-phase columns, but not on the C18 columns. With addition of TMA, results on C18 and polymeric reversed-phase columns were nearly identical. Addition of an ion pair also improved separations on the C18 columns, but not as much as TMA. Interaction with the silica support of C18 columns was used to separate tylosin from interferences in extracts of biological materials. The results demonstrate the importance of interactions with the silica support in chromatography of basic antibiotics on C18 packings. These interactions can be either beneficial or detrimental to separations, depending on the conditions used. PMID- 3023413 TI - Effect of the mobile phase composition on the retention behaviour of diphenylsilica pre-coated plates. AB - The retention of some rifamycins and steroids on diphenyl bonded pre-coated silica gel plates, in relation to the mobile phase used, was examined by thin layer chromatography. Neat organic solvents, non-aqueous and aqueous binary mixtures were tested as eluents. By comparison of retention data for rifamycins and steroids, respectively, under non-aqueous and aqueous conditions, a dual retention mechanism on this diphenyl phase was found. Interactions with the residual silanol groups seemed to prevail when employing as mobile phase the more lipophilic solvents tested, such as chloroform or dichloromethane, whereas interactions with the aryl groups of the bonded phase prevailed when using high polarity alcohols or aqueous mixtures. As a consequence, by changing the mobile phase, a large variation in selectivity with a concomitant change in retention order of the test compounds was observed. PMID- 3023414 TI - Determination of occupational exposure to toluene diisocyanate by biological monitoring. AB - Exposure of workers to toluene diisocyanate monomer in a factory producing flexible polyurethane was measured using impinger and reagent-impregnated filter sampling. These techniques yielded highly concordant results so that the facile filter techniques can be recommended. The personal exposures were compared with the individual excretion of amine metabolites from the parent toluene diisocyanates. The excretion was linearly related to the product of sampling time and concentration. The biological half-lives of the amines proved to be short so that urine should be sampled 2 h after the exposure. The percutaneous absorption of the monomers could also be controlled. PMID- 3023415 TI - Production of "internal surface reversed-phase" supports: the hydrolysis of selected substrates from silica using chymotrypsin. AB - The degree of hydrolysis of substrates attached to silica supports with alpha chymotrypsin has been evaluated relative to the production of "internal surface reversed-phase" supports. The peptide substrates N-tert.-butoxycarbonyl-L phenylalanine (Boc-L-Phe), N-carbobenzoxy-L-valine-L-phenylalanine, N-acetyl-L phenylalanine (acetyl-L-Phe) and N-benzoyl-L-phenylalanine as well as phenylpropionic acid were attached to glycerylpropyl-bonded silica via a diamine spacer using 1,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling catalyst. The products released from the silica support on enzyme treatment were quantified by high-performance liquid chromatography. Boc-L-Phe and acetyl-L-Phe were successfully cleaved from the rigid silica matrix in high yields, whereas the remaining substrates were hydrolyzed to a lesser extent. PMID- 3023416 TI - Characterization of internal surface reversed-phase silica supports for liquid chromatography. AB - Internal surface reversed-phase (ISRP) supports synthesized from commercially available porous silica particles with a variety of nominal pore diameters and specific surface areas are characterized with regard to physical and chromatographic properties. Bonded phase coverage, pore size, capacity and efficiency measurements are made upon the various ISRP supports in order to evaluate the effect that the physical properties of silica have upon the chromatographic performance of ISRP packings. In addition, various models that describe the pore structure of silica supports are discussed. PMID- 3023417 TI - Use of perphenazine as a ligand for calmodulin affinity chromatography. PMID- 3023418 TI - Alterations in calcium, vitamin D, and parathyroid hormone physiology in normal men with aging: relationship to the development of senile osteopenia. AB - The effects of aging on calcium and bone metabolism have not been systematically examined in men. To identify age-related alterations in vitamin D and PTH physiology and to assess their impact on skeletal health, we studied 62 normal men, aged 30-92 yr. The men were in excellent health, and none had any evidence of metabolic bone disease and/or known risk factors for osteopenia. Serum 25 hydroxyvitamin D (25OHD) concentrations declined steadily with advancing age (r = -0.47; P less than 0.001), and there was a corresponding decline in serum 24,25 dihydroxyvitamin D [24,25-(OH)2D] levels (r = -0.41; P less than 0.001). Serum 1,25-(OH)2D concentrations, however, did not vary over this age range (r = -0.07; P = NS). Plasma PTH levels increased with aging (r = -0.24; P less than 0.001), and there was a concomitant increase in urinary cAMP excretion (r = 0.38; P less than 0.001). Renal function (creatinine clearance) clearly declined with increasing age (r = -0.71; P less than 0.001). In conjunction with these changes in calcium metabolism, radial and vertebral bone mineral content declined. Whereas the fall in radial bone mineral content (single photon absorptiometry) at both proximal and distal sites was slight, there was a marked decrease in vertebral bone mineral content, as measured by quantitative computed tomography (r = -0.72; P less than 0.0001). The fall in vertebral bone mineral content correlated well with the declines in serum 25OHD and 24,25-(OH)2D concentrations (r = 0.47; P less than 0.001 and r = 0.51; P less than 0.001, respectively) and with the decline in renal function (r = 0.46; P less than 0.001). Multiple regression analysis revealed that the effects of aging on bone mineral content could be accounted for in large part by concomitant changes in mineral metabolism. Both the decline in renal function and the fall in serum 24,25-(OH)2D levels were closely associated with the fall in bone mineral content. These results indicate that a decline in renal function and alterations in vitamin D metabolism occur with aging in normal men. These changes contribute to, if not cause, the associated decline in skeletal mineral content in aging men. PMID- 3023419 TI - Prolonged pulsatile administration of ovine corticotropin-releasing hormone in normal man. AB - The 24-h profiles of plasma ACTH and cortisol were determined at 15-min intervals in five normal men basally and during iv bolus injections of 25 micrograms ovine corticotropin-releasing hormone (oCRH) every 4 h for 72 h. Each oCRH injection was followed by a distinct elevation of plasma ACTH and cortisol levels, with a return to basal values before the next injection. The characteristics of the ACTH and cortisol pulses induced by 25 micrograms oCRH (i.e. 0.3-0.4 micrograms/kg) were similar to those observed in other studies with 1 microgram/kg human CRH. There was no significant blunting of oCRH-induced hormonal increments in the course of the 72-h study. On each oCRH injection day, the mean 24-h cortisol level was higher than that in the basal study, but there was no increase in the mean 24-h ACTH level. During the 72-h oCRH study, the preinjection ACTH and cortisol levels exhibited a diurnal variation, indicating persistence of the circadian periodicity of pituitary-adrenal activity. There was a diurnal variation of oCRH-induced ACTH increments, with highest responses at 0700 h. A small but not significant reverse trend was apparent for cortisol increments. Spontaneous pulses of ACTH and cortisol occurred throughout the 3 days of oCRH injections, and the total number of spontaneous and oCRH-induced pulses was similar to the number of spontaneous pulses observed in the basal study. All oCRH induced and more than 90% of spontaneous cortisol pulses occurred concomitantly with an ACTH pulse. The variability of pulse increments was greater for ACTH than for cortisol. In conclusion, prolonged pulsatile administration of oCRH did not induce pituitary desensitization and did not suppress the endogenous circadian and pulsatile ACTH and cortisol variations. PMID- 3023420 TI - Responses to glucagon infusion in pseudohypoparathyroidism. AB - Single or graded doses of glucagon (Eli Lilly) were given to patients with pseudohypoparathyroidism (PsHP) type I to examine the possible presence of hormone resistance. The doses of glucagon ranged from 0.25-15 micrograms/kg. The following individuals were studied: 13 normal subjects, 5 patients with low erythrocyte N-protein activity (PsHP type Ia), and 7 patients with normal erythrocyte N-protein activity (PsHP type Ib). Two additional patients with treated primary hypothyroidism who were relatives of a patient with PsHP type Ib were also studied. The patients with PsHP type Ia had blunted plasma cAMP responses to all glucagon doses. In contrast, the patients with PsHP type Ib had normal cAMP responses to glucagon infusion. However, the 2 relatives of the patient with PsHP type Ib had clearly decreased cAMP responses to glucagon infusion; both had normal renal responses to PTH and were clinically and biochemically euthyroid at the time of study. Glucose responses to glucagon were normal in both PsHP groups; the glucose response per unit cAMP response was slightly, but not significantly, enhanced in PsHP type Ia patients. Glucagon resistance appears to be a common finding in patients with PsHP type Ia, but not in those with PsHP type Ib. However, the observation of reduced glucagon responsivity in association with familial hypothyroidism in a kindred with PsHP type Ib suggests the possibility that this disorder may also cause disturbances in several hormone systems. PMID- 3023421 TI - Use of ketoconazole in the treatment of Cushing's syndrome. AB - The therapeutic value of ketoconazole for long term treatment of patients with Cushing's syndrome was studied. Seven patients with Cushing's disease and one with an adrenal adenoma received 600-800 mg/day ketoconazole for 3-13 months. Plasma ACTH, cortisol, and dehydroepiandrosterone sulfate levels and urinary cortisol, 17-ketosteroid, and tetrahydro-11-deoxycortisol excretion were determined periodically during the treatment period. Plasma ACTH and cortisol responses to CRH stimulation were determined before and during treatment. Rapid and subsequently persistent clinical improvement occurred in each patient; plasma dehydroepiandrosterone sulfate and urinary 17-ketosteroid and cortisol excretion decreased soon after the initiation of treatment, subsequently remaining normal or nearly so throughout the treatment period. Urinary tetrahydro-11-deoxycortisol excretion increased significantly. Plasma cortisol levels decreased. Plasma ACTH levels did not change, and individual plasma ACTH and cortisol increments in response to CRH were comparable before and during treatment. The cortisol response to insulin-induced hypoglycemia improved in one patient and was restored to normal in another. The seven patients tested recovered normal adrenal suppressibility in response to a low dose of dexamethasone during ketoconazole treatment. Ketoconazole is effective for long term control of hypercortisolism of either pituitary or adrenal origin. Its effect appears to be mediated by inhibition of adrenal 11 beta-hydroxylase and 17,20-lyase, and it, in some unknown way, prevents the expected rise in ACTH secretion in patients with Cushing's disease. PMID- 3023422 TI - Lack of suppression of insulin secretion by hyperinsulinemia in a patient with an insulinoma. AB - The regulation of insulin secretion in patients with insulinoma is known to be abnormal. For example, physiological and pharmacological stimuli often fail to stimulate insulin in such patients. Recently, insulin has been found to inhibit its own secretion in normal subjects. To determine if insulin has this effect in patients with insulinoma, we infused insulin at rates of 1 and 10 mU/kg X min in such a patient and in eight normal subjects. Euglycemia was maintained by the euglycemic glucose clamp technique, and endogenous insulin secretion was estimated by measuring plasma C-peptide levels. In the normal subjects, plasma C peptide declined from 1.60 +/- 0.22 (+/- SEM) to 1.16 +/- 0.17 and 0.82 +/- 0.11 ng/ml during the low and high dose insulin infusions, respectively, indicating 27% (P less than 0.01) and 48% (P less than 0.001) decreases in endogenous insulin secretion at moderately elevated and extremely elevated insulin levels, respectively. In the insulinoma patient, plasma C-peptide was 2.6 ng/ml basally, did not change during the low dose insulin infusion, and rose to 3.4 ng/ml during the high dose insulin infusion. We conclude that the feedback regulation of insulin secretion by insulin that occurs in normal subjects is absent in insulinoma patients. This finding could have pathophysiological and possibly diagnostic significance. PMID- 3023423 TI - FK 33-824, a met-enkephalin analog, blocks corticotropin-releasing hormone induced adrenocorticotropin secretion in normal subjects but not in patients with Cushing's disease. AB - To further elucidate the site of action of opioids on the pituitary-adrenal axis, we studied the effect of D-Ala2,MePhe4,met-(O)enkephalin-ol (Sandoz, FK 33-824) on plasma ACTH and beta-endorphin immunoreactivity and serum cortisol in 7 normal subjects and 11 patients with Cushing's syndrome (Cushing's disease, n = 7; adrenal adenoma, n = 2; ectopic Cushing's syndrome, n = 2) after administration of human corticotropin-releasing hormone (hCRH). hCRH (0.1 mg; Bachem) was injected iv after pretreatment with 0.5 mg FK 33-824, im, or 0.9% saline. In normal subjects, the hCRH-induced ACTH, beta-endorphin, and cortisol increases were almost completely abolished by pretreatment with FK 33-824. Mean peak (+/- SEM) hormone concentrations were significantly reduced (ACTH, 16.7 +/- 3.5 vs. 45.3 +/- 7.8 pg/ml; beta-endorphin, 151 +/- 25 vs. 277 +/- 51 pg/ml; cortisol, 8.1 +/- 1.2 vs. 19.5 +/- 2.6 micrograms/dl; P less than 0.02), as were secretory areas (P less than 0.02). These results indicate a direct pituitary action of the synthetic met-enkephalin. In contrast, in patients with Cushing's disease, FK 33 824 did not inhibit hCRH-induced hormone release. Instead, maximum ACTH and beta endorphin concentrations were slightly but not significantly higher after the administration of FK 33-824 (ACTH, 292 +/- 143 vs. 131 +/- 32 pg/ml; beta endorphin, 2409 +/- 763 vs. 1921 +/- 600 pg/ml). These findings indicate a defect in inhibitory opiodergic control of ACTH secretion in patients with Cushing's disease, which may contribute to the pathological ACTH hypersecretion. In patients with Cushing's syndrome due to an adrenal adenoma or ectopic ACTH secretion, neither hCRH nor FK 33-824 altered hormone concentrations. PMID- 3023424 TI - Atrial natriuretic polypeptide inhibits cortisol secretion as well as aldosterone secretion in vitro from human adrenal tissue. AB - The effect of alpha-human atrial natriuretic polypeptide (ANP) on adrenal steroidogenesis was studied in human adrenal tissues obtained surgically from four patients with Cushing's syndrome due to an adrenal adenoma and five patients with an aldosterone-producing adenoma (APA). ANP significantly inhibited basal and ACTH (3.4 X 10(-8) M)-stimulated cortisol and aldosterone secretion in both the adenomas and adjacent adrenocortical tissues from patients with Cushing's syndrome. ANP inhibited ACTH-stimulated, but not basal, secretion of cortisol and aldosterone in the adjacent tissues from patients with APA. In addition, ANP significantly inhibited both basal and ACTH-, angiotensin II (10(-6) M)-, and potassium chloride (10 mM)-stimulated secretion of aldosterone from the adenomas of patients with APA. ANP-induced changes in cortisol and aldosterone secretion were accompanied by a decrease in cAMP and an increase in cGMP secretion. These results suggest that ANP may be a possible regulator of cortisol as well as aldosterone secretion in humans, and these effects might be due to concomitant alteration in cyclic nucleotide metabolism. PMID- 3023425 TI - Inhibition of thyrotropin-induced growth of rat thyroid cells, FRTL-5, by immunoglobulin G from patients with primary myxedema. AB - We studied thyroid growth-blocking activity in immunoglobulin G (IgG) fractions of serum from 24 patients with primary myxedema, 24 patients with goitrous Hashimoto's thyroiditis, and 18 normal subjects by measuring the ability of their IgG to inhibit TSH-induced [3H]thymidine incorporation into DNA in a rat thyroid cell line, FRTL-5. Both groups of patients were receiving T4 when studied. [3H]Thymidine incorporation induced by 0.1 mU/ml bovine TSH was significantly inhibited by the addition of 2 mg/ml IgG from patients with primary myxedema (P less than 0.01), while it was not affected by IgG from the normal subjects or 23 of the 24 patients with goitrous Hashimoto's thyroiditis. IgG from patients with primary myxedema also inhibited the [3H]thymidine incorporation induced by Graves' IgG, but not that induced by forskolin, cholera toxin, (Bu)2cAMP or phorbol-12-myristate-13-acetate. The inhibition of TSH-induced [3H]thymidine incorporation by IgGs from patients with primary myxedema was significantly correlated with their inhibitory activities against both TSH-induced cAMP generation and TSH binding (P less than 0.001). These data indicate that these growth-blocking antibodies are directed against the TSH receptor and might be one of the causes of the thyroid atrophy in patients with primary myxedema. PMID- 3023426 TI - Peptide histidine-methionine immunoreactivity in plasma and tissue from patients with vasoactive intestinal peptide-secreting tumors and watery diarrhea syndrome. AB - The presence of peptide histidine-methionine (PHM)-like peptides has been determined in plasma and tumor specimens from patients with vasoactive intestinal peptide (VIP)-secreting tumors and the watery diarrhea syndrome. All patients had strikingly elevated plasma concentrations of PHM immunoreactivity (median, 1800; range, 500-6800 pmol/liter; n = 12), which were higher than those of VIP (median, 235; range, 50-580 pmol/liter). In patients with other endocrine and nonendocrine pancreatic tumors, plasma PHM concentrations were not significantly different from normal (median, 20; range, 5-60 pmol/liter; n = 28). Plasma samples from patients with diarrhea due to other illnesses also had PHM concentrations that were not significantly different from normal (median, 40; range, 10-80 pmol/liter; n = 23). The gel chromatographic profiles of plasma and tumor extracts from patients with VIP-secreting tumors revealed the presence of at least two molecular forms that reacted with an antiserum directed to the N terminus of PHM (SY1). The later peak (Kav, 0.50-0.53) corresponded in position to synthetic PHM and also reacted with the PHM-specific antiserum (SY2). The earlier peak (Kav, 0.30-0.37), not reactive with antiserum SY2, corresponded to a large molecular form of PHM-like immunoreactivity previously identified as the predominant form in normal human stomach and plasma, though not in the rest of the intestinal tract. The neuroendocrine nature of the tumors was confirmed by the demonstration of immunostaining with a battery of antisera to neuroendocrine markers. Immunocytochemistry revealed the presence of both VIP and PHM in tumor cells. The presence of high circulating concentrations of PHM-like immunoreactivity in patients with VIP-secreting tumors, as measured with a PHM N terminus-directed antiserum, SY1, suggests that use of this type of antiserum may provide valuable information in the diagnosis of such tumors. The contribution of the PHM-like peptides to the features of this syndrome is not known. PMID- 3023427 TI - Steroidogenesis in the human fetal adrenal: a role for cholesterol synthesized de novo. AB - Primary monolayer cultures of human fetal adrenal cells maintained in either lipoprotein-depleted or lipoprotein-supplemented media responded chronically to ACTH treatment with similarly increased steroid secretion. The principal steroid secreted into each medium was dehydroepiandrosterone sulfate. The presence of human low density lipoprotein (hLDL) in the medium enhanced the secretion of nonsulfoconjugated steroids, especially dehydroepiandrosterone. The secretion rate of 11 beta-hydroxyandrostenedione was similar to that of cortisol. In the absence of hLDL, ACTH increased cholesterologenesis to maintain the high rates of steroid secretion. After ACTH treatment, increased accumulation of 3-hydroxy-3 methylglutaryl coenzyme A reductase, a rate-determining enzyme of cholesterol biosynthesis, was found. Immunoblot analysis demonstrated that this enzyme was a 97K protein in human fetal adrenal cells. Interestingly, the content of this enzyme in cells treated with ACTH in lipoprotein-depleted medium was similar to that in adrenal fetal zone tissue. This finding suggests that cholesterologenesis de novo in addition to plasma LDL is important as a source of steroid precursor in vivo in the human fetal adrenal gland. PMID- 3023428 TI - Cyclical edema and hypokalemia due to occult episodic hypercorticism. AB - Yearly episodes of edema, hypokalemia, anxiety, and depression were found to be due to cortisol and deoxycorticosterone surges secondary to a pituitary adenoma in a woman without any of the usual clinical features of Cushing's syndrome. During the long clinical remissions, she had no recognizable laboratory abnormality. She had two episodes in the year following incomplete transphenoidal pituitary tumor resection, but has had none in 2 yr since receiving radiotherapy. The episodes were caused by mineralocorticoid actions of large ACTH-induced increases in cortisol and deoxycorticosterone secretion. A history of episodic edema and hypokalemia, often attributed in women to surreptitious diuretic abuse, requires a careful search for hypercorticism even in the absence of clinical Cushing's syndrome. PMID- 3023429 TI - Lack of a direct effect of gonadotropin hormone-releasing hormone agonist on human testicular steroidogenesis. AB - In an attempt to determine whether the chronic administration of GnRH agonist (GnRH-A) has a direct inhibitory effect on testicular steroidogenesis in the human, the testes of four men with disseminated prostatic cancer who were treated with GnRH-A daily for at least 1 yr were assayed for intratesticular pregnenolone (5-pregnen-3 beta-ol-20-one), progesterone, dehydroepiandrosterone, 17 alpha hydroxypregnenolone (5-pregnen-3 beta 17 alpha-diol-20-one), 17 alpha hydroxyprogesterone, androstenedione, and testosterone (T). In addition, testicular 17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase enzyme activities of the delta 4 pathway were measured. These intratesticular steroids and enzyme activities from four GnRH-A-treated patients were compared to those in five men (controls) who were orchiectomized as the primary treatment for their disseminated prostatic cancer and in three other men who were treated for 3-12 months with GnRH-A daily but received, in addition to the daily GnRH-A, 1000 IUhCG, im, every other day for 3 days immediately before their salvage orchiectomy, which was performed when their disease progressed. In the control group, the delta 5-steroids, particularly dehydroepiandrosterone and pregnenolone, represented the majority of the intratesticular steroids. Compared to control values, all intratesticular steroids except delta 4-P (for which there was no difference) were significantly lowered by treatment with GnRH-A. Intratesticular T was reduced by 98% from 328 +/- 139 (+/- SEM) ng/g testis in the control group to 8 +/- 3 in the GnRH-A-treated group (P less than 0.01). The additional treatment with hCG for 3 days in the GnRH-A-treated group reversed the inhibition of all steroids to either control or above control levels, with intratesticular T rising to 1144 +/- 273 ng/g testis. A similar trend was found for all three enzymatic activities, i.e., GnRH-A alone inhibited each of the enzymatic activities, whereas the addition of hCG reversed this inhibition by GnRH-A. These data indicate that the chronic administration of GnRH-A to elderly men results in inhibition in both the delta 4 and delta 5 pathways, with a subsequent decrease in the intratesticular T concentration. The ability of exogenous hCG to reverse both the reduction in delta 4 and delta 5 intratesticular steroid content and the intratesticular enzyme activities induced by GnRH-A treatment supports the concept that GnRH-A does not have a direct inhibitory effect on testicular T biosynthesis. PMID- 3023430 TI - High plasma concentrations of human atrial natriuretic polypeptide in aged men. AB - To examine the effect of age on the secretion and action of human atrial natriuretic polypeptide (hANP), we compared plasma atrial natriuretic polypeptide and cGMP concentrations in 19 normal young (24-28 yr old) and 31 elderly (64-91 yr old) men. The mean basal plasma hANP level was 25 +/- 5 (+/- SEM) pg/ml in the young men and 120 +/- 22 pg/ml in the elderly men (P less than 0.01). The molecular size of plasma hANP in an aged man was similar to that of alpha hANP. The responses of plasma hANP and cGMP concentrations to an iv infusion of 500 ml 0.15 M NaCl also was compared. Mean plasma hANP concentrations increased significantly in both groups, and the response in the elderly men was exaggerated compared to that in the young men. However, the increases in plasma cGMP concentrations during NaCl loading were similar in both groups. We conclude that 1) the plasma hANP concentrations in healthy elderly men are higher than those in young men, and 2) the higher plasma hANP concentrations may be a compensatory response to reduced hANP activation of its receptors. PMID- 3023431 TI - Genotype and hormonal phenotype in nonclassical 21-hydroxylase deficiency. AB - In nonclassical steroid 21-hydroxylase deficiency, the genotype may be represented as a homozygous mild (nonclassical) form of the 21-hydroxylase defect or as a compound heterozygote, with one severe (classical) and one mild (nonclassical) 21-hydroxylase deficiency allele. We examined hormone levels in patients with nonclassical 21-hydroxylase deficiency in whom pedigree analysis and/or HLA linkage disequilibrium allowed unequivocal identification of the respective haplotypes as either classical or nonclassical. The results indicated that compound heterozygotes (21-OH defsevere/21-OH defmild) have an ACTH stimulated 17-hydroxyprogesterone (17-OHP) response significantly greater than that of mild homozygotes (21-OH defmild/21-OH defmild): at 60 min, 8,131 +/- 4,205 (+/-SD) (n = 17) vs. 4,468 +/- 2,123 ng/dl (n = 31) for the respective groups (P less than or equal to 0.01); at 360 min, 11,067 +/- 5,582 (n = 17) vs. 5746 +/- 1565 (n = 8, P less than or equal to 0.01). Since serum cortisol levels were the same in both groups, the ratio of 17-OHP to cortisol was higher in the former group. Sixty minute ACTH-stimulated serum delta 4-androstenedione levels also were significantly higher in compound heterozygotes than in mild homozygotes. Serum dehydroepiandrosterone and its sulfate were not significantly different between the two groups. Notably, compound heterozygotes were no more likely to have signs of androgen excess than were homozygotes for the mild gene defect. Stimulated levels of serum 17-OHP, 17-OHP/cortisol, delta 4 androstenedione and dehydroepiandrosterone and its sulfate did not differ significantly between heterozygotes for the classical (21-OH defsevere/21 OHnormal) and nonclassical (21-OH defmild/21-OHnormal) 21-hydroxylase deficiency alleles. Thus, the presence of a single normal 21-hydroxylase allele is sufficient to obscure the difference between a severe and a mild 21-hydroxylase deficiency allele on the opposite haplotype. We conclude that the compound heterozygous patients as a group have a significantly higher response of 21 hydroxylase precursors to ACTH stimulation than do patients with the homozygous mild 21-hydroxylase deficiency state. PMID- 3023433 TI - Reduced cellular immunity to varicella zoster virus during treatment for acute lymphoblastic leukemia of childhood: in vitro studies of possible mechanisms. AB - To determine the effect of antileukemic therapy on preexisting immunity to varicella zoster virus, we studied 20 children with acute lymphoblastic leukemia maintained in complete continuous remission for greater than 1 year. Cellular immunity was tested by lymphocyte proliferation in response to varicella antigen. Antiviral antibody was measured using the fluorescent antibody to membrane antigen technique. Reduced lymphocyte proliferation was found in 9 of 16 seropositive patients when compared to an age-related control group. On the other hand, antibody titers in patients receiving chemotherapy remained positive and were essentially unchanged from pretreatment values. Shingles occurred in two of nine children with diminished and none of seven patients with normal cellular immunity, suggesting that proliferative responses to varicella antigen may have predicative value in identifying patients at risk for viral reactivation. Additional studies were done to determine if defective antigen presentation or reduced lymphocyte responder-cell frequency could account for the subnormal proliferative responses. Intact presentation of varicella antigens by patient mononuclear cells to parental, virus-specific T-cell blasts suggested that antigen processing was not defective. However, varicella-specific responder-cell frequencies measured by limiting dilution analysis were found to be depressed in most patients, including some with normal proliferative responses. Our findings indicate that therapy for acute lymphoblastic leukemia in children can be associated with depressed cell-mediated immunity to varicella zoster virus even though patients remain seropositive. Further studies suggest that while monocyte mediated antigen presentation remains intact, virus-specific lymphocyte numbers decline and probably contribute to decreased cellular immunity to varicella zoster virus in children being treated for leukemia. PMID- 3023432 TI - Establishment of Tac-negative, interleukin-2-dependent cytotoxic cell lines from large granular lymphocytes (LGL) of patients with expanded LGL populations. AB - Cell lines were established from purified large granular lymphocytes (LGL) isolated from the peripheral blood of seven patients with phenotypically homogeneous LGL expansions. LGL were stimulated with phytohemagglutinin (PHA) or recombinant interleukin-2 (rIL-2) and further expanded in vitro in IL-2 containing media. The surface phenotype of LGL, as assessed by monoclonal antibody staining, was T3+ T8+ in five patients, T3- T8- in one, and T3+ T8- in another patient. The cells also expressed Leu 7, Leu 11, and/or OKM 1 markers in various proportions and were identifiable as LGL by their morphological and cytochemical features. The original surface phenotype of the unstimulated LGL was retained in the IL-2-dependent cell lines from each individual patient, i.e., T3+ T8+ cells originated T3+ T8+ cell lines and T3- T8- cells originated T3- T8- cell lines. Other markers, such as Leu 11 and OKM 1, were generally lost in culture. LGL proliferated in response to rIL-2 but did not express detectable IL-2 receptors, even after prolonged periods of culture. All cell lines from each individual patient had the same surface phenotype, and within the single lines, all of the cells expressed the same markers. Cell lines from two patients consistently displayed chromosomal abnormalities. Although different in the two patients, the abnormalities were identical in all of the lines from the same patient and detectable in most of the cells examined, suggesting a clonal origin for the abnormally expanded LGL populations. Freshly isolated LGL did not exert NK activity. However, the IL-2-dependent LGL lines acquired the ability to kill K562 target cells and to produce gamma interferon (gamma-IFN). No direct correlation was observed between these two properties. PMID- 3023435 TI - Centers for Disease Control performance evaluation program in bacteriology: 1980 to 1985 review. AB - This report presents a summary and analysis of data from the Centers for Disease Control performance evaluation program in bacteriology for the 6-year period 1980 to 1985. During this period, the number of laboratories enrolled in the program ranged from about 750 to 1,000. Identification results reported by participating laboratories for representative species of six major groups of bacteria were placed into five response categories that were based on the level and accuracy of the identifications. Data on the performance of participants with bacterial groups and performance with selected species within each major group were analyzed. Overall, participants experienced the least difficulty in identifying species or serogroups of members of the gram-positive and gram-negative cocci. Participants encountered greater difficulties with anaerobes, gram-positive bacilli, and miscellaneous gram-negative bacteria. Identification of members of the family Enterobacteriaceae was of moderate difficulty. Problems in identifying certain bacterial species were probably related to a number of factors such as the characteristics of the species, its frequency of occurrence, the state of technology available for identification, and the state of proficiency and quality control in individual laboratories at the time of testing. Examples are given of improvements over time in the identification of certain bacterial species. Laboratories participating in an external proficiency testing program should take full advantage of the benefits of participation by instituting measures to correct testing deficiencies identified by the program. PMID- 3023434 TI - Humoral immune deficiency in multiple myeloma patients due to compromised B-cell function. AB - Patients with multiple myeloma are generally immunodeficient, with pronounced depression in primary antibody responses. We have attempted to delineate the reasons for the humoral immunodeficiency by analyzing the specificity repertoire of the surface immunoglobulin (Ig)-positive B cells in patients with multiple myeloma or monoclonal gammopathy of undetermined significance (MGUS), in comparison with normal donors. B lymphocytes from 26 patients with multiple myeloma, 12 patients with MGUS, and 8 normal donors were transformed with Epstein Barr virus (EBV) and cultured at limiting dilution for clonal analysis. The Ig secreted by each clone was analyzed for class and anti-tetanus toxoid (TT) specificity to determine the frequencies of IgM, IgG, anti-TT IgM, and anti-TT IgG antibody-secreting clones. Our objective was to establish whether the inability to mount humoral responses to common environmental pathogens was due to a lack of specific B cells or to inhibition of B-cell function. Our results indicate that the quantitative B-cell deficiency in patients was due to a nonrandom loss of selected sets of B cells. Although most patients had a reduced aggregate number of B cells, the number of TT-specific B cells was normal. There was, on average, a threefold increase in the proportion of the B-cell specificity repertoire devoted to recognition of TT. Forty-four percent of the patients with MGUS were also affected. In addition, the TT-specific B cells in multiple myeloma patients were severely compromised in their ability to secrete antibody or to differentiate to antibody-secreting cells in vivo. This arrest in differentiation appears to be extrinsic to the B cells, as they were fully able to secrete anti TT antibody after transformation and culture in vitro. We postulate the existence of an autoimmune inhibitory network mediating the arrest in B-cell differentiation and the humoral immune deficiency. PMID- 3023436 TI - Heterologous protection against rotavirus-induced disease in gnotobiotic piglets. AB - Administration per os of 2 X 10(6) fluorescent cell-forming units of a human serotype 3 rotavirus (RV-3) protected all of nine gnotobiotic piglets against severe diarrheal disease when they were challenged 10 to 14 days later with 8 X 10(3) fluorescent cell-forming units of virulent wild-type porcine rotavirus (AT/76). The porcine virus was similar antigenically to porcine prototype strain OSU, previously described as antigenically distinct from all four recognized human serotypes. Administration of RV-3 was associated with the development of serum-neutralizing antibody to both RV-3 and AT/76 in piglets that excreted RV-3. Neutralizing antibody levels to RV-3 and AT/76 increased rapidly postchallenge. Vaccinated piglets were not immune to infection with AT/76 but showed no or minimal gastrointestinal symptoms after challenge. Control nonvaccinated piglets that were fed AT/76 developed severe dehydrating diarrhea and low levels of neutralizing antibody to AT/76 alone. The apparent heterologous clinical protection observed in this study could have been predicted from results of in vitro assays. Neutralization tests with reduction of fluorescence focus indicated a one-way cross-reaction between RV-3 and AT/76 such that hyperimmune antiserum to RV-3 neutralized porcine virus to moderate titer, but not vice versa. The results emphasize the importance of neutralizing antibody in protection against disease and the need to determine reciprocal cross-neutralization titers, rather than serotype alone, in order to predict the ability of rotavirus strains to cross protect. PMID- 3023437 TI - Homotypic and heterotypic antibodies for prevention of experimental rotavirus gastroenteritis. AB - We investigated the abilities of homologous and heterologous antibody preparations to neutralize murine rotavirus in a mouse model of rotavirus infection. We found that incubation of virus with murine sera obtained from animals experimentally infected with the homologous epizootic diarrhea of infant mice strain of rotavirus resulted in inability of the virus to cause symptomatic disease in infant mice. Sera from nonimmune mice and mice infected or immunized with a heterotypic strain of rotavirus did not effectively neutralize virus in this system. On the other hand, neutralization was noted after incubation of virus with a number of immunoglobulins from other animal and human sources. The neutralizing activity of the preparations in the murine model correlated partially, but not completely, with the level of in vitro neutralizing antibody to the murine strain of rotavirus measured in a tissue culture system. In most cases, asymptomatic infection after feeding of the virus and the antibody preparation resulted in subsequent generation of an active immune response in infected animals. PMID- 3023438 TI - Molecular epidemiology of adenovirus type 21 in the Netherlands and the Federal Republic of Germany from 1960 to 1985. AB - After a period of high prevalence in the early 1960s, adenovirus serotype 21 (Ad21) was identified in The Netherlands only very sporadically for more than 20 years. From December 1984 to July 1985, Ad21 was isolated relatively often from hospitalized children living in different parts of The Netherlands. The patients in question suffered from respiratory, gastrointestinal, meningeal, or ocular disorders. An increase in the incidence of Ad21 infections was also observed in the Federal Republic of Germany during this period. The DNAs of 93 isolates of Ad21 were subjected to restriction enzyme analysis with eight endonucleases. All 50 strains isolated in The Netherlands between 1960 and 1963 proved to be DNA variant Ad21/D2/20655/Netherlands/60. This variant has already been described (T. Adrian, R. Wigand, and J. C. Hierholzer, Arch. Virol. 84:79-89, 1985) as typical for the Ad21 strains circulating since 1960. Analysis of the DNAs of the 28 Ad21 strains isolated in The Netherlands or in the Federal Republic of Germany in 1984 and 1985 showed them to belong to two new, closely related DNA variants designated Ad21/D7/1857/Netherlands/84 and Ad21/D8/5398/Netherlands/85. The BglI and KpnI restriction profiles were characteristic for these recent DNA variants. PMID- 3023439 TI - Type specificity of complement-fixing antibody against herpes simplex virus type 2 AG-4 early antigen in patients with asymptomatic infection. AB - We evaluated the type specificity of complement-fixing (CF) antibody against the AG-4 early antigen of herpes simplex virus (HSV) type 2 (HSV-2) by comparing a commercial AG-4 CF kit (Simplex-2; Gene Link Australia, Inc., Princeton, N.J.) with quantal microneutralization (MN) and absorption-Western blotting in testing sera from patients with and without a history of genital herpes. Sera characterized as HSV type 1 (HSV-1) or HSV-2 positive or negative by MN were selected and tested by CF, and those with discordant results were further analyzed for specific antibodies by absorption with HSV-1 or HSV-2 antigen and Western blotting with heterologous HSV proteins. A total of 34 of 42 (81%) sera HSV-2 positive by MN, 19 of 43 (44%) sera HSV-1 positive by MN, and 0 of 19 sera negative by MN were positive by CF. Absorption-Western blotting showed that 12 of 18 (67%) sera HSV-1 positive by MN but positive by CF had no HSV-2-specific antibody and that all 7 sera HSV-2 positive by MN but negative by CF had HSV-2 specific antibody. When MN and absorption-Western blotting data were combined to analyze patients with no history of genital herpes, 7 of 19 (37%) with no HSV-2 specific antibody were positive by CF, and 7 of 27 (26%) with HSV-2-specific antibody were negative by CF. The positive and negative predictive values for the CF test were 78 and 75%, respectively, in this group. The presence of antibody to the HSV AG-4 antigen does not discriminate sufficiently between HSV-1- and HSV-2 infected patients to be of value in predicting HSV-2 infection in the absence of symptomatic disease. PMID- 3023440 TI - Detection of poliovirus antigen by enzyme immunoassay. AB - A solid-phase enzyme immunoassay (EIA) was developed for the detection of poliovirus antigen. Rabbit and guinea pig antisera for the assay were raised against purified poliovirus type 3/Fin (strain 3/Fin/K) isolated from a fecal specimen from a meningitis patient during an outbreak of poliomyelitis in Finland in 1984. The EIA was highly specific for poliovirus type 3, and it was about 30 times more sensitive for strain 3/Fin/K than for strain 3/Saukett used in the inactivated poliovirus vaccine. The sensitivity of the EIA was 2 to 5 ng of purified strain 3/Fin/K per ml, whereas disrupted viruses and soluble viral proteins were almost undetectable by the assay. Only 5 of 51 (10%) stool specimens containing poliovirus type 3/Fin detected by virus isolation were positive by the EIA. Quantitation by the EIA, using purified poliovirus 3/Fin/K as a standard, revealed that concentrations of poliovirus type 3 in undiluted fecal specimens of patients with natural poliovirus infection were only 50 ng/ml or less. In conclusion, owing to the small amount of poliovirus in feces, the EIA is not suitable for the diagnosis of poliovirus infections directly from clinical specimens, but it can be used to detect, type, and quantitate poliovirus antigen in infected cells. PMID- 3023442 TI - Comparison of in situ hybridization and immunologic staining with cytopathology for detection and identification of herpes simplex virus infection in cultured cells. AB - Two recently developed sensitive techniques, in situ hybridization with a biotinylated cloned DNA probe and an avidin-biotin complex immunoperoxidase assay, were compared with the appearance of cytopathic changes for the early detection of herpes simplex virus infection in cell culture. By using commercially made reagents, these detection methods were evaluated in two different cell culture systems inoculated with both high- and low-input multiplicity of virus. The results revealed that both viral antigen and viral DNA detection methods could shorten the time to diagnosis of herpes simplex virus infection in cell culture; however, these methods were most useful in specimens containing low titers of virus when a less sensitive cell system was used. In this study, the avidin-biotin immunoperoxidase method was more sensitive and much cheaper than hybridization with a biotinylated probe. Significantly, when a highly sensitive cell system was used, cytopathic changes alone were comparable in rapidity and sensitivity to viral antigen or DNA detection methods applied in a less sensitive cell system. PMID- 3023441 TI - Detection of enteropathogens in fatal and potentially fatal diarrhea in Cairo, Egypt. AB - A 1-year study of the etiology of acute diarrhea complicated by severe (10%) dehydration, active bleeding, shock and cardiovascular collapse, pneumonia, acute renal failure, or seizures in infants under 18 months of age was performed in Cairo, Egypt. Of 145 infants, 19 (13%) died or left the hospital moribund; the remaining 126 patients were classified as having potentially fatal illness. A variety of enteropathogens were identified with approximately equal frequency in the fatal and nonfatal complicated cases as well as in 135 controls with severe uncomplicated diarrhea. The agents most frequently detected in infants with severe diarrhea in this population which were felt to be etiologically important were rotavirus (33%), heat-stable enterotoxin-producing Escherichia coli (20%), heat-labile enterotoxin-producing E. coli (11%), enteropathogenic E. coli (8%), and Salmonella spp. (5%). The high rate of occurrence of Giardia lamblia (35%) probably represented the high carriage rate of the protozoan in this population. Complicated (fatal and potentially fatal) cases differed from control cases in a number of ways: the onset of diarrhea was more sudden, the course was progressive and of greater initial intensity, vomiting occurred more frequently, the patients more often had visited another physician before coming to the hospital, the patients more often had respiratory symptoms and pulmonary abnormalities on auscultation, hypoactive bowel sounds and abdominal distention were more common, as was oliguria, and the patients showed lower mean body weights. PMID- 3023443 TI - Detection of immunoglobulin G antibodies to cytomegalovirus antigens by antibody capture enzyme-linked immunosorbent assay. AB - An antibody capture enzyme-linked immunosorbent assay (ELISA) that uses horseradish-peroxidase-labeled antigen for the detection of immunoglobulin G (IgG) antibodies to cytomegalovirus (CMV) is described. A microtiter plate was coated with anti-human IgG and consecutively incubated with serum specimens, enzyme-labeled CMV antigen made from CMV-infected cell nuclei, and substrate. The CMV IgG antibody content was determined spectrophotometrically and expressed as absorbance. Furthermore, to reveal any nonspecific reactions, all sera were tested against an enzyme-labeled control antigen made from uninfected cell nuclei. The problem with nonspecific reactions was small and was circumvented by the addition of unlabeled control antigen to the conjugates. For epidemiological studies the test was not as sensitive as other serological tests. On the other hand, the IgG antibody capture ELISA was highly sensitive for detecting the serological antibody response in patients with primary and recurrent CMV infections. Thus, one positive serum remained positive at a serum dilution of 1:10(7). The specificity of the test was shown by a blocking experiment and by testing 126 complement fixation-positive sera, of which 97% were positive. There was a rather good correlation between the complement fixation test and the IgG antibody capture ELISA (rs = 0.79, P less than 0.001). The test is especially useful when tests for CMV antibodies of the IgM, IgA, and IgE classes are run by similar antibody capture ELISAs, since the same procedure and conjugate are used. PMID- 3023444 TI - Class II antigen expression and T lymphocyte subsets in chronic inflammatory demyelinating polyneuropathy. AB - The inflammatory infiltrate within human sural nerve, biopsied from six patients with active chronic inflammatory demyelinating neuropathy (CIDP) was studied for T lymphocyte subsets and Class II antigen (Ia)-expressing cells. Immunohistochemical staining with mouse monoclonal antibodies, acid phosphatase staining, and electron microscopy were used to provide an alternative assessment of macrophage and other mononuclear cell numbers. In normal control nerves Class II antigen was present upon endothelial cells, very occasional mononuclear cells and sparsely within the perineurium. In CIDP nerve dense Class II antigen staining was prominent within nerve fascicles, in capillary endothelial cells and within the perineurium. T lymphocytes of suppressor and helper type were present in small numbers only. Moderate numbers of macrophage-monocytes were found in the patients within nerve fascicles but these cells accounted for only part of the dense Ia staining. Since two nerves with hypertrophic changes, Schwann cells forming 'onion bulbs', were clearly Ia positive, the dense and widespread staining in all nerves studied is best explained by Ia antigen expression upon mononuclear and some Schwann cells. PMID- 3023445 TI - Effect of cyclosporin A, silica quartz dust, and protease inhibitors on virus induced demyelination. AB - Treatment with cyclosporin A, from the time of virus infection, suppressed inflammation and demyelination in the spinal cord of SJL/J or ASW(H-2s) mice persistently infected with Theiler's murine encephalomyelitis virus. Demyelination was not decreased if treatment was given after inflammation was established. The decrease was independent of serum titers of immunoglobulin G to purified viral antigen but did correlate with decreased proliferation of T lymphocytes to virus and myelin antigens. Silica quartz dust, a direct toxin of macrophages, suppressed demyelination and inflammation if begun at time of virus infection. No therapeutic effect was seen with inhibitors of plasminogen activators or other neutral proteases found primarily in macrophages. PMID- 3023446 TI - Influence of calcium or 1,25-dihydroxyvitamin D3 supplementation on the hepatic microsomal and in vivo metabolism of vitamin D3 in vitamin D-depleted rats. AB - Hypocalcemic vitamin D (D)-depleted rats were supplemented with calcium or 1,25(OH)2D3, and the metabolism of D3 to 25(OH)D3 was studied. Infusion with 7 or 65 pmol 1,25(OH)2D3 X 24 h-1 led to normal or slight hypercalcemia associated with physiological and supraphysiological plasma concentrations of the hormone while calcium supplementation normalized plasma calcium despite 1,25(OH)2D3 concentrations as low as those observed in hypocalcemic controls. Constant administrations of [14C]D3 during the supplementation regimens uncovered a stimulation of the in vivo 25(OH)D3 production by calcium supplementation; this was further confirmed in vitro by an increase in the hepatic microsomal D3-25 hydroxylase. The group supplemented with pharmacological doses of the hormone displayed lower circulating concentrations of both D3 and 25(OH)D3 while the in vitro 25(OH)D3 production remained unaffected by 1,25(OH)2D3. Investigation of the kinetics of intravenous 25(OH)[3H]D3 revealed similar elimination constants in all groups. The data indicate that calcium supplementation of hypocalcemic D depleted rats results in an increased transformation of D3 into 25(OH)D3 while supplementation with 1,25(OH)2D3 does not affect the in vitro D3-25 hydroxylase but seems to influence the in vivo handling of the vitamin by accelerating its metabolism. PMID- 3023447 TI - Intravascular release of intact cellular fibronectin during oxidant-induced injury of the in vitro perfused rabbit lung. AB - Fibronectin (Fn) is produced by cells in blood vessels at inflammatory sites in vivo. Fn release into the circulation thus may be a marker for vascular injury. In support of this, we found that oxidant-induced vascular injury of isolated perfused rabbit lungs caused elevated circulating Fn levels. Western blot analysis indicated that Fn released from the injured blood vessels was intact, dimeric, and possessed electrophoretic mobility identical with Fn produced by fibroblasts. Unlike Fn isolated from rabbit plasma, Fn derived from lung perfusate or produced by fibroblasts reacted with antibodies raised to a synthetic peptide containing sequences from the extra type III Fn domain that is transcribed in fibroblasts but not hepatocytes. Vascular injury by protease was also associated with intravascular release of Fn, but with cleavage. Oxidant induced vascular injury causes release of tissue-derived Fn, which can be distinguished from plasma Fn by its size and content of antigenic determinants of the extra type III domain. PMID- 3023448 TI - Regulation by adrenal corticosteroids of sodium and potassium transport in loop of Henle and distal tubule of rat kidney. AB - Studies were conducted to examine the effects of adrenalectomy (ADX) and selective, physiological adrenal corticosteroid replacement on sodium and potassium transport by the superficial loop of Henle and distal tubule of rat kidney in vivo. In the loop of Henle, ADX inhibited sodium reabsorption by 33%. Whereas dexamethasone had no effect on reabsorption, aldosterone increased sodium transport to control levels. Thus, physiological levels of mineralocorticoids, but not glucocorticoids, control a fraction of sodium reabsorption in the loop of Henle. ADX also inhibited potassium reabsorption in the loop of Henle. Both dexamethasone and aldosterone reversed the inhibition, although only aldosterone increased reabsorption to control levels. In the distal tubule, ADX reduced sodium reabsorption by 44%. Both aldosterone and dexamethasone stimulated reabsorption: however, only aldosterone increased transport to control. Potassium secretion by the distal tubule was also reduced 34% by ADX. Aldosterone, but not dexamethasone, stimulated secretion. Thus, physiological levels of aldosterone regulate a fraction of sodium reabsorption and potassium secretion in the distal tubule. PMID- 3023449 TI - Cyclic adenosine monophosphate-stimulated anion transport in rabbit cortical collecting duct. Kinetics, stoichiometry, and conductive pathways. AB - Cyclic AMP stimulates HCO3 secretion and Cl self-exchange in rabbit cortical collecting tubule. We found that varying peritubular [Cl] changed the Cl self exchange rate with saturation kinetics (Km, 3-4 mM). HCO3 secretion also showed saturation kinetics as a function of mean luminal [Cl] (Km, 4-11 mM). Both Cl self-exchange and Cl-HCO3 exchange thus appear to be carrier-mediated. Addition/removal of basolateral HCO3 qualitatively changed Cl and HCO3 transport as expected for Cl-HCO3 exchange, but quantitatively changed Cl absorption more than HCO3 secretion. The diffusive Cl permeability and the transepithelial conductance in the presence of HCO3/CO2 and cAMP were higher than in their absence suggesting that HCO3/CO2 and cAMP together increase a conductive Cl pathway parallel to a 1:1 Cl-HCO3 exchanger. Thus, cAMP not only stimulates the overall process of anion exchange (probably by increasing an electroneutral exchanger and/or a series Cl conductance), but also stimulates a Cl conductance parallel to the exchange process. PMID- 3023451 TI - Platelet-activating factor. A potent chemotactic and chemokinetic factor for human eosinophils. AB - Platelet-activating factor (PAF-acether), an inflammatory mediator with a wide range of biological activities including neutrophil aggregation and chemotaxis, was studied for its effect on human eosinophil locomotion (chemotaxis and chemokinesis). Human eosinophils (25-95% purity) were obtained from donors with a variety of diseases associated with hypereosinophilia. PAF-acether elicited directional locomotion of eosinophils, in a time- and dose-dependent fashion, at concentrations from 10(-5) to 10(-8) M; lyso-PAF had minimal activity over the same dose range. Compared with PAF-acether, the eosinophil locomotory responsiveness of leukotriene B4 (LTB4), histamine, and the valyl- and alanyl eosinophil chemotactic factor of anaphylaxis (ECF-A) tetrapeptides was negligible. Conversely, neutrophil responsiveness to PAF-acether (optimum 10(-6) M) was comparable in effect to LTB4 (optimum dose 10(-8) M). It was shown that PAF-acether elicited both chemotaxis and chemokinesis of eosinophils. Comparison of normal density and light density eosinophils revealed no qualitative difference in the response to PAF-acether and the other chemoattractants, although the light density cells seemed to demonstrate a greater degree of locomotion to PAF-acether and LTB4. Thus, PAF-acether appears to be a potent eosinophilotactic agent which may play a role in inflammatory reactions characterized by eosinophil infiltration. PMID- 3023453 TI - Malignant eccrine poroma: report of three cases. AB - The characteristic clinical and histological features of three cases of malignant eccrine poroma are discussed, in addition to the metastatic disease that had occurred in two cases. These cases were compared with previously reported cases of malignant eccrine poroma that had metastasised, and it is suggested that a strict classification of malignant eccrine sweat gland tumours should be made. PMID- 3023450 TI - Effects of recombinant tumor necrosis factor on proliferation and differentiation of leukemic and normal hemopoietic cells in vitro. Relationship to cell surface receptor. AB - The clonogenic growth of myeloid leukemia cell lines was inhibited by recombinant tumor necrosis factor (rTNF) at 1-15 pM concentration. However, wild type (promyelocytic) HL-60 cells were highly resistant to growth inhibition, but responded with differentiation into monocyte-like cells at 100 pM rTNF. The clonogenic growth of fresh acute myeloid leukemia cells was inhibited by 50% at approximately 15 pM rTNF. The growth of normal granulocyte-macrophage progenitors (CFU-GM) was also inhibited (by 50 pM rTNF), as was the growth of erythroid progenitors (BFU-E) (by 150 pM rTNF). A synergistic antiproliferative effect was demonstrated between rTNF and recombinant interferon-gamma. Use of radioiodinated rTNF enabled us to detect 1,500-2,100 binding sites on myeloid cell lines at 4 degrees C with Kd of approximately 300 pM. At 37 degrees C, the transfer of bound ligand to lysosomes was followed by degradation, inhibited by NH4+. No correlation was observed between the number of binding sites or affinity at 4 degrees C and antiproliferative response to the addition of rTNF. PMID- 3023452 TI - Human and viral gene detection in routine paraffin embedded tissue by in situ hybridisation with biotinylated probes: viral localisation in herpes encephalitis. AB - A simple reproducible protocol for detecting multiple copy human genes and viral DNA in routine formalin fixed paraffin embedded tonsil and brain, by in situ hybridisation with biotinylated probes, is described. The protocol consists of digestion of formalin fixed paraffin sections, with 0.4% pepsin in 0.01 M hydrochloric acid for one hour at 37 degrees C, followed by hybridisation with biotinylated probes. The biotinylated probes used for establishing the conditions for in situ localisation of DNA were total placental DNA (TG1), pHY 2.1 (a Y chromosome probe), and herpes simplex virus I and II. In human male tonsil TG1 labelled all nuclei and pHY 2.1 reacted only with nuclear Y bodies. In herpes encephalitis the virus was detected in some glial cells and neurones. PMID- 3023455 TI - Accessory cells as primary target of human immunodeficiency virus HIV infection. PMID- 3023454 TI - Problems with serological diagnosis of Toxoplasma gondii infections in heart transplant recipients. AB - Of the first 128 patients to receive either heart or heart and lung transplants at Papworth Hospital, four developed Toxoplasma gondii infections acquired from the donor heart and two died. Six patients had passively acquired T gondii antibody as a result of blood transfusions around the time of transplantation. Eight patients developed antibodies against T gondii, which were detectable by changes in the latex agglutination test titres but not by those of the dye test. These false positive latex agglutination reactions occurred simultaneously with cytomegalovirus infection and were associated with the IgM serum fraction in the patients' sera. These reactions were not associated with cytomegalovirus specific IgM, Paul-Bunnell antibody, nor detectable rheumatoid factor. These findings emphasise the need for T gondii dye test confirmation of latex agglutination test titre rises in heart transplant recipients. PMID- 3023456 TI - Histaminergic neurons in the rat brain: correlative immunocytochemistry, Golgi impregnation, and electron microscopy. AB - Histamine-containing neurons were visualized in Vibratome--sections of rat brain with the indirect peroxidase-antiperoxidase immunocytochemical method of Sternberger (Immunocytochemistry, 2nd edition, New York: John Wiley and Sons, pp. 1-354, '79) by utilizing a primary antibody directed against L-histidine decarboxylase (HDC). Cell bodies of HDC-immunoreactive neurons are located exclusively in the posterior hypothalamus: tuberal magnocellular nucleus (TM), caudal magnocellular nucleus (CM), and post-mammillary magnocellular nucleus (PCM). With the light microscope, all the HDC-immunoreactive neurons in CM and PCM and the majority of the HDC-immunoreactive neurons in TM appear to be large neurons, with a short, thick dendrite emerging from each pole of the long axis of the oval perikaryon and one or more, thinner, nonpolar primary dendrites. In the electron microscope, it can be seen that the immunoreaction product is diffusely dispersed in the cytoplasm. The ultrastructural features of all investigated (70) HDC-immunoreactive neurons in the three nuclei, independent of their light microscopic characteristics, are remarkably similar: large, unindented, pale nucleus; a high proportion of cytoplasm to nucleus (with the exception of the medium-sized HDC-immunoreactive neurons in TM); large, perinuclear array of Golgi apparatus; numerous mitochondria; endoplasmic reticulum fragmented into numerous small cisterns; thick initial portions of the primary dendritic trunks; few axosomatic synaptic contacts. Twenty-one Golgi-Kopsch-impregnated neurons taken from CM, PCM, and TM were embedded in epoxy resin, serially sectioned, and investigated in the electron microscope. The ultrastructural characteristics typical of HDC-immunoreactive neurons were observed in all three nuclei in neurons with large cell bodies tapering into two thick, sparsely spinous primary dendrites that subsequently dichotomize into very long (up to 100 microns), nontapering, aspinous secondary dendrites. In sections taken from the posterior hypothalamic area of rats prepared in a conventional way for electron microscopy, distinct populations of large cells can be observed in TM, CM, and PCM displaying the same set of ultrastructural characteristics as the HDC-immunoreactive neurons. PMID- 3023457 TI - Treatment of recurrent genital herpes with topical alpha interferon gel combined with nonoxynol 9. AB - A double-blind, placebo-controlled study was done to evaluate the efficacy of an alpha interferon preparation in 128 patients with recurrent genital herpes. The preparation containing 10(5) or 10(7) U alpha interferon with nonoxynol 9 in a cream base (Exovir-HZ) was applied three times daily for 5 days. The treatment did not cause any adverse reactions. Patients treated with either interferon concentration became negative for viral culture at a faster rate than placebo recipients. The end of new lesion formation, scabbing, and the healing of lesions were all superior in patients treated with 10(5) U to those treated with 10(7) U interferon. End of new lesion formation and scabbing were also statistically different in patients treated with 10(7) U from those patients treated with placebo. Results suggest that topical interferon might be useful in relieving symptoms of severe cases of genital herpes. PMID- 3023458 TI - MR imaging of a hepatoma associated with Alagille syndrome. AB - Alagille syndrome is a rare cause of chronic liver failure which may be treated by liver transplantation. When underlying hepatic tumor is present, transplantation has had poor results. Magnetic resonance imaging may be a sensitive, noninvasive method to evaluate the presence of tumor before transplantation. PMID- 3023459 TI - Pancreas islet cell carcinoma with complete fatty replacement: CT characteristics. AB - We report a metastatic nonfunctioning islet cell carcinoma of the head of the pancreas associated with complete fatty replacement of the pancreatic body and tail. This mass was only appreciated because of the contrast offered by the adjacent fat-replaced pancreatic body and tail. PMID- 3023460 TI - MR imaging of hepatocellular carcinoma. AB - Forty-two patients with hepatocellular carcinoma (HCC) were examined by magnetic resonance (MR) imaging. The presence of tumor was suggested in 41 of 42 cases by a high-intensity area on T2 weighted spin-echo (SE) images with a repetition time (TR) of 1.6 s. Specific findings of HCC such as the presence of a capsule, mosaic pattern, and tumor thrombus in major veins were noted in 10, two, and seven cases, respectively. In six cases the tumor pattern changed from a well-defined mass to an irregular, ill-defined one according to pulse sequence (SE: echo time 35 and 70 ms; TR 1.6 and 0.4 s). In our early experience MR was almost equal to conventional X-ray CT in the detection of main or daughter lesions and in the determination of extent and characterization of HCC. PMID- 3023461 TI - MR imaging of hepatoma treated by embolization. AB - The magnetic resonance findings of two hepatomas treated by embolization are presented. The T2-weighted spin echo images showed an increase in signal intensity in the tumor after embolization. This phenomenon corresponded to a decrease in tumor density on CT and to necrosis observed histologically. Magnetic resonance also demonstrated gas bubbles as low signal foci within the embolized tumor. PMID- 3023462 TI - Computed tomography of ovarian masses. AB - A retrospective analysis of CT images in 138 histologically proven ovarian masses in 100 patients was undertaken to evaluate the usefulness and limitation of CT in the diagnosis of ovarian tumors. Benign masses were purely cystic in 98 (94.2%) and had solid component (including thickened walls, thickened septa, papillary projections) in five of 104 lesions (4.8%) on CT. These five masses, which are classified as benign solid tumors, could not be differentiated from malignant tumors by either the size of the solid portion or the intensity of contrast enhancement. In the malignant tumors a solid portion was detected in 32 of 34 tumors (94%). When a solid component is detected in an ovarian mass, the mass should be considered malignant although a few cases will be benign solid tumors. In Krukenberg tumors, which were all of gastric origin, the solid component was so large that it occupied more than one-half the mass. Therefore, if the solid portion of the ovarian mass is large on CT, upper gastrointestinal study should be performed to rule out Krukenberg tumor. PMID- 3023463 TI - Granular-cell tumours of the skin do not express carcino-embryonic antigen. AB - Granular-cell tumour (GCT) of the skin is an uncommon tumour of disputed histogenesis, that has been subjected to several immunohistochemical studies. The controversy existing in the literature concerning the expression of carcinoembryonic antigen (CEA) by GCT prompted us to study a series of 17 cases of cutaneous GCT by using an avidin-biotin-immunoperoxidase technique on routinely-processed tissue sections. No CEA activity was detected in any of the tumours screened. The reasons for this controversy are discussed. PMID- 3023464 TI - Sodium bicarbonate and alfalfa hay additions to wheat silage diets fed to lactating dairy cows. AB - Two experiments were conducted to examine dietary effects of .8% sodium bicarbonate and 1.4 kg/d of alfalfa hay on performance and rumen metabolism of lactating dairy cows fed 50% wheat silage and 50% concentrate (dry basis). In Experiment 1 with 12 midlactation Holsteins in a 4 X 4 Latin square design, intake, milk production, and milk composition were not affected by treatment. Dietary sodium bicarbonate and alfalfa hay did not alter blood, rumen, or fecal pH. Rumen volatile fatty acid pattern was not affected by sodium bicarbonate, but addition of hay resulted in higher molar percentage propionate and lower acetate: propionate ratios. In Experiment 2 with 32 early lactation cows (20 Holsteins and 12 Jerseys) in a complete randomized block design, supplementation of sodium bicarbonate, alfalfa hay, or both did not affect intake, milk production, or milk composition in the first 8 wk of lactation. Blood, rumen, and fecal pH were not affected by treatment. Dietary sodium bicarbonate did not alter ruminal volatile fatty acid profile, whereas addition of hay increased molar proportion acetate and decreased molar proportion butyrate. A shift in rumen fermentation was observed across treatments from wk 1 through 8 postpartum with molar proportions of acetate and butyrate increasing and molar proportion of propionate decreasing. PMID- 3023465 TI - 1985 Kreshover lecture. Molecular factors influencing neutrophil defects in periodontal disease. AB - Major advances in our understanding of the role of the neutrophil in host defense against periodontal organisms have been made through studies of localized juvenile periodontitis (LJP). Several lines of evidence suggest that LJP is an infectious process closely associated with Actinobacillus (Haemophilus) actinomycetemomitans as a causative agent, although other organisms may also participate. The immunologic profile of LJP patients suggests that a cell associated neutrophil locomotory dysfunction is a key underlying immunodeficiency resulting in increased susceptibility to periodontal infection. In addition, LJP patients often exhibit cervical lymphadenopathy and IgG-hypergammaglobulinemia, and a markedly elevated antibody response to the infecting organism, A. actinomycetemcomitans, is found in the serum and crevicular fluid of most patients. Evaluation of the locomotory properties of LJP neutrophils shows that random migration and chemokinesis are normal; however, about 70% of the LJP patients suffer from a defect in chemotaxis, with their neutrophils responding poorly to bacterial chemotactic factors, synthetic chemotactic peptides, and complement fragments (C5a). Depressed chemotaxis of LJP neutrophils is paralleled by their reduced capacity to bind the synthetic chemotactic peptide N formylmethionylleucylphenylalanine (FMLP), as well as C5a. Furthermore, there is a reduction in the amount of glycoprotein 110, a neutrophil membrane matrix component and differentiation antigen which is associated with FMLP- and possibly also C5a-mediated chemotaxis. Reduction of C5a and of FMLP ligand binding, decreased expression of GP-110, and reduced neutrophil chemotaxis are consistent with a stem cell maturation error in LJP patients. This is further supported by studies demonstrating increased expression of CR2, the C3d/EBV receptor, on peripheral blood neutrophils of LJP patients. CR2 receptors are normally present on immature human neutrophils but are lost during the maturation process. These alterations in neutrophil surface components and their reduced chemotaxis may result from a genetically determined abnormality. Studies demonstrating the familial nature of both the neutrophil chemotactic disorder and the clinical entity represented by localized juvenile periodontitis point to a strong role for genetic determinants in the disease which affect neutrophil surface receptors. PMID- 3023466 TI - Mandibular growth and histologic changes in condylar cartilage of rats intoxicated with vitamin D3 or 1,25(OH)2D3 and pair-fed (undernourished) rats. AB - The mandibular condyles of 1,25(OH)2D3 or Vitamin D3 intoxicated rats were studied and compared with those of normal as well as pair-fed controls. Experimental animals were injected with either Vitamin D3 (2 mg/kg/day) or 1,25(OH)2D3 (400 ng/kg/day) for 19 days. Controls were given the solvent only, while pair-fed animals were restricted in their food intake for the same period of time, so that they exhibited a weight-curve similar to that of the experimental rats. The length of the mandibular ramus was measured in lateral radiographs of all mandibles. Demineralized coronal sections were obtained from all mandibular condyles and were stained with Mallory's connective tissue stain. The width of each zone within the condylar cartilage was measured. Experimental animals showed significant reduction in width of all layers within the condylar cartilage, with total lack of distinction between the maturation and hypertrophic zones. They also exhibited a significant retardation in growth of the mandible. Pair-fed animals had a normal width of the chondroprogenitor layer but significantly smaller maturation + hypertrophic zones (vs. controls). They also exhibited a significant retardation in mandibular growth but not to the same degree as did the intoxicated animals. Reduction in growth attributed to 1,25(OH)2D3 or Vitamin D3 intoxication is partly caused by undernutrition, which is a by-product of this condition. A further kinetic study is indicated to elucidate the mechanism of growth retardation and the differential effect on the various cartilage layers. PMID- 3023467 TI - Genital herpes simplex at term necessitating cesarean section. PMID- 3023468 TI - [The radical enzyme ribonucleotide reductase in animal tissues with a high proliferative activity]. PMID- 3023471 TI - Cellular reactions during drug-induced cleft palate. AB - Responses of the epithelial and mesenchymal cells of the developing hamster secondary palate to hadacidin, 5-fluorouracil, and hydrocortisone were analysed. Correlation of data on cellular response with morphological and biochemical observations suggested that several aspects of differentiation of palatal cells needs to be analysed before pathogenesis of teratogen-induced cleft palate can be defined. PMID- 3023469 TI - [Hormonal status of children in the late period after combined treatment for nephro- and neuroblastomas]. PMID- 3023470 TI - The Western diet: an examination of its relationship with chronic disease. AB - Diet is a component in the etiology of the two major causes of death in the United States, namely, cardiovascular disease and cancer. During the last decade, various organizations have suggested that we alter the "typical" American diet in order to decrease the incidence of these diseases even though both diseases are indisputably of multiple etiology. An implication behind these recommendations is that individuals will increase their longevity by changing their diets. The burden of proof falls on those proposing changes to the diet that such alterations will be safe and effective. In spite of our often indicted diet, mortality from heart disease and stroke continue to fall and deaths from diet related cancers are static or dropping. Longevity in the U.S. is exceeded by only five countries, whose populations consume a diet similar to ours in four, and that in the fifth is approaching ours. While low-fat high-fiber diets probably have some beneficial effect vis-a-vis chronic diseases, it is likely that other risk factors contribute more to the total risk of disease. Therefore, it is illogical to expect dietary manipulation to offset significantly other concurrent risks such as heredity, tobacco use, hypertension, and obesity. Individuals who are at high risk for specific diseases should modify their diets to minimize this particular risk factor. Most Americans can safely reduce their intake of total calories, fat, sugar, and salt. Although this can be achieved most readily on a population basis by following a form of "prudent" diet, it is premature to promise medical benefits to individuals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3023473 TI - Is the glycogenolytic effect of clonidine mediated by alpha 2-adrenergic receptors? PMID- 3023472 TI - Iodized oil treatment for endemic goiter does not induce the surge of positive serum concentrations of anti-thyroglobulin or anti-microsomal autoantibodies. AB - Forty-three goitrous patients (grade II and III, WHO classification), living in areas of chronic iodine deficiency, were treated with an injection of iodized oil (470 mg iodine). Serial measurements of serum thyroid hormone levels after the therapy revealed increasing concentrations of both hormones, with a significantly lower serum T3/T4 ratio, and progressively significantly lower serum TSH mean values. Serum Tg mean value, initially elevated (58 +/- 9 ng/ml), decreased after 6 months and returned to the normal range at 36 months of therapy. In none of the examined patients (except for one subject with positive autoantibodies before therapy), it was observed the surge of positive anti-thyroglobulin or anti microsomal autoantibodies after the iodized oil. We conclude that iodized oil therapy does not induce an abnormal autoimmune reaction in endemic goiter patients. PMID- 3023474 TI - Alcoholic liver disease in Japan. AB - From these discussions, it is apparent that: Alcoholic liver disease is increasing at a rapid rate in conjunction with an increase of annual gross and per capita consumption of alcohol. Alcoholic hepatitis and alcoholic hyaline are much less common in Japan compared to western countries. Alcoholic hepatic fibrosis and chronic hepatitis are the common types of alcoholic liver disease in Japan. Alcoholic hepatic fibrosis may be a pathological process or entity independent of fatty liver, alcoholic hepatitis, and alcoholic cirrhosis. It is not clear at the present time whether heavy alcohol consumption per se or non-A, non-B hepatitis virus is the cause of chronic hepatitis seen in HBsAg negative alcoholics. PMID- 3023475 TI - Difficulty in diagnosing complications of Caroli's disease. AB - Congenital cystic dilatation of the biliary tree (Caroli's disease) is a rare condition that usually presents with ascending cholangitis. This report demonstrates the difficulty of recognizing other complications of Caroli's disease antemortem. A 35-year-old man developed a subphrenic abscess and malignant transformation of the biliary tree; both were clinically undetected. Episodes of pain, pyrexia, or weight loss should be assessed carefully and the complications of Caroli's disease considered before attributing such symptoms to recurrent cholangitis. PMID- 3023476 TI - Multiple immunolabeling in histology: a new method using thermo-inactivation of immunoglobulins. AB - We describe a new method for successive immunofluorescence detection of multiple peptides in single paraffin tissue sections. Immunoglobulins were inactivated at 130 degrees C after each labeling step while tissues were protected with a mixture of phosphate-buffered saline and glycerin. The feasibility of the method is demonstrated by double-staining of rat pituitary corticotrophs with anti ACTH[1-24] and anti-ACTH[17-39] antiserum. The method is applicable to brain tissue, since single neurons of ovine paraventricular hypothalamus could be triple stained with anti-vasopressin, anti-neurophysins I and II, and anti-CRF antiserum. The usual absorption controls and crossreactivity tests, as well as peptide staining, can be performed on the same tissue section. This new labeling procedure should prove useful in endocrinology, clinical pathology, and the neurosciences. PMID- 3023477 TI - A single-step silver enhancement method permitting rapid diagnosis of cytomegalovirus infection in formalin-fixed, paraffin-embedded tissue sections by in situ hybridization and immunoperoxidase detection. AB - A single-step silver enhancement method was developed which intensifies the polymerized nickel-complexed diaminobenzidine (Ni-DAB) reaction product of peroxidase. With such enhancement, an in situ hybridization procedure can be performed in less than 8 hr by using a 2-hr hybridization incubation and direct detection. Cytomegalovirus (CMV)-infected lung sections were hybridized in situ for 2 hr with a biotinylated CMV genomic-length probe. The probe was detected directly with avidin-biotinylated peroxidase using Ni-DAB as the substrate, and the reaction product was enhanced with silver. Silver was deposited only on the Ni-DAB and not on normally argyrophilic substances. Indirect detection of the probe using a sandwich technique before silver enhancement proved more sensitive, but the length of the procedure was increased without substantially changing the result (infection vs. no infection). Sensitivity was also improved by omitting the dehydration step before applying the probe, and by increasing the temperature and duration of denaturation. In a blinded study of 21 open-lung biopsies, 13 of 13 culture-negative specimens were negative by hybridization, and 7 of 8 culture positive specimens were positive by hybridization. Modified short hybridization with a biotinylated probe and silver-enhanced direct detection therefore provides a rapid but sensitive method for diagnosis of viral infection. PMID- 3023478 TI - Recognition of the cryptic plasmid, pSLT, by restriction fingerprinting and a study of its incidence in Scottish Salmonella isolates. AB - The plasmid pSLT is a cryptic plasmid of 60 megadaltons (Md) present in Salmonella typhimurium LT2. We present evidence that it has a characteristic fingerprint when digested with the restriction enzymes PstI and SmaI. Among a representative collection of S. typhimurium isolates it was present in 67% of strains and was widely distributed amongst different phage types (DT) with the exception of DT10 and U285. Furthermore, its prevalence among veterinary isolates was significantly higher than among human isolates. It was not found among any of the 96 strains representative of other salmonella serotypes currently prevalent and thus appears to be serotype-specific. PMID- 3023479 TI - Restriction enzyme fingerprinting of enterobacterial plasmids: a simple strategy with wide application. AB - Restriction enzyme fingerprints were generated from purified plasmid DNA from 324 clinical isolates that belonged to 7 enterobacterial genera and 88 single plasmids in Escherichia coli K 12 according to the following strategy. Purified plasmid DNA was digested with PstI. The number of fragments detected in a 0.8 agarose gel was used to determine which 2 of 6 restriction enzymes including PstI was most likely to provide a fingerprint comprising sufficient fragments to ensure specificity but sufficiently few to allow easy visual assessment and minimize coincidental matching. When PstI produced greater than 20 fragments, EcoRI and HindIII were used; when PstI generated less than 6 fragments Bsp 1286 and AvaII were used and SmaI was employed when between 6 and 20 fragments were obtained from PstI digests. Using a minimum of 12 fragments from a combination of 2 enzymes as the criterion for characterizing a strain/plasmid, satisfactory 2 enzyme fingerprints were obtained from 87% of the strains and plasmids studied using PstI and no more than two additional enzymes per strain. Of the remaining 54 strains, 51 harboured only small plasmids (less than 10 kb) and 3 produced satisfactory fingerprints when digested with a fourth enzyme. PMID- 3023480 TI - Plasmids in group JK coryneform bacteria isolated in a single hospital. AB - Investigation of 39 JK-type coryneform isolates from patients at a single hospital revealed that 23 possessed plasmids, which formed six groups on restriction endonuclease analysis. Four of the groups were associated with production of similar bacteriocin-like substances, and shared a minimum of 6.4 kilobase pairs of DNA. These plasmids, found in isolates from different patients, provide strong direct evidence that person-to-person transmission of JK bacteria had occurred within the hospital. PMID- 3023481 TI - Susceptibility of various animals to the vesiculoviruses Isfahan and Chandipura. AB - To determine the pathogenic potential of the vesiculoviruses Isfahan and Chandipura for domestic animals, two ponies, two steers, three sheep, three goats and three pigs were inoculated with each virus intradermally in the tongue or, in the case of the pigs, in the snout, heel and coronary band. The ponies were also inoculated intradermally in the right commissure of the mouth. Animals inoculated with each virus were housed in one room and allowed to mingle freely with an equal number of uninoculated contact animals of each species. Clinical signs of infection, consisting of ulcers at the inoculation sites, were observed in the Chandipura study in two inoculated ponies, one inoculated steer and one inoculated goat. No elevated temperature was observed. Virus was isolated from the ulcerated tongue tissue, but not from serial blood samples, oesophageal pharyngeal mucus samples, or from the tissues which were collected at necropsy. Precipitating antibody was not detected by the immunoelectroosmophoresis (IEOP) test in any of the pre- or post-serum samples except from two inoculated sheep at 29 days post-inoculation (D.P.I.). Low levels of neutralizing activity were detected in pre-inoculation serum from all steers, pigs, contact sheep, and one contact goat. By 15 D.P.I. all inoculated animals and contact ponies and steers exhibited increased neutralizing antibody titres. In studies with the Isfahan virus, lesions developed only at the inoculation sites in the two ponies, and the virus was isolated. No virus was isolated from any blood, oesophageal-pharyngeal mucus samples or tissues collected at necropsy. All pre-inoculation sera were negative for neutralizing and precipitating antibodies. By 14 D.P.I. all inoculated animals exhibited neutralizing antibody, while all the contacts remained negative. The IEOP test remained negative for all animals throughout the experiment. A sub-passage of a suspension of Isfahan-infected tongue tissue injected into ponies and steers also yielded only firm swellings of lesser extent than the original reaction at the inoculation sites. With both viruses, lethal infections were produced by intracranial or intraperitoneal inoculation of day old mice and hamsters, and by allantoic inoculation of embryonating chicken eggs. Adult mice, hamsters, guinea-pigs and rabbits produced serum antibodies but lacked clinical signs. PMID- 3023482 TI - Further characterization of 41 isolates of adenovirus types 19/37 by serum neutralization and DNA restriction enzyme analysis. AB - Forty-one strains of adenovirus type 19/37 (Ad19/37) mainly isolated from patients with keratoconjunctivitis or conjunctivitis between 1974 and 1984 were re-evaluated by serum neutralization (SN), haemagglutination inhibition (HI) and DNA restriction analysis. Of 19 isolates which were neutralized to high titre by antiserum prepared against prototype Ad19, 5 showed cross-reactivity with 32-64 units of Ad37 antiserum, while of 22 strains neutralized by high titre by Ad37 antiserum, 3 showed cross-reactivity with 32 units of Ad19 antiserum. By DNA restriction analysis, all Ad19 isolates were identical to each other and to Ad19A virus. Using endonuclease Bgl 1, three variants were observed among the Ad37 isolates. PMID- 3023483 TI - Sera from patients with multiple sclerosis react with human cell T lymphotropic virus-I gag proteins but not env proteins--Western blotting analysis. AB - To study the possible involvement of human T cell lymphotropic virus type I (HTLV I)-related agent in Japanese multiple sclerosis (MS), we performed a Western blotting analysis, using purified viral antigens, on sera from 46 patients with MS, nine patients with other neurologic diseases, and 11 healthy controls. Of 46 MS patients, 11 (24%) had antibodies reactive with antigens corresponding to the group-specific antigen (gag) proteins (p15, p19, and p24), although the prevalence was lower than that reported in a recent study using an enzyme-linked immunosorbent assay (ELISA). Despite the lower frequency of immunoreactivity, Western blotting technique had merits of identification of multiple antigens and higher specificity for detection of antibodies than ELISA. Those sero-positive patients consisted of four cases with IgG antibodies reactive mainly to the gag p24 and/or p15, four with IgM antibodies mainly to the gag p24 and/or p19, and three with both IgG and IgM antibodies. These immunostaining patterns of MS sera were clearly distinguishable from those of adult T cell leukemia patients who had antibodies to the envelope (env) proteins and its precursors in addition to the gag proteins. The antibody in MS sera was generally of low titer and reactive at a high serum concentration (1/10 dilution). None of the sera from patients with other neurologic diseases and healthy controls had the viral antibodies. These findings indicate that at least one quarter of Japanese MS patients have antibody responses to a hitherto unidentified agent related to HTLV-I, which possibly plays a part, primarily or secondarily, in the pathogenesis of those patients. PMID- 3023484 TI - Interaction of IL 1 and TPA in modulation of eosinophil function. AB - The tumor co-promotor TPA is believed to enhance a wide variety of cellular processes by interacting with protein kinase C. Interleukin (IL 1) is a family of highly active molecules which augments the host response to infection. We have explored the interactions of these activators of cell function on the modulation of selected eosinophil functions. The effects of purified monocyte-derived IL 1 on the eosinophil functions of oxidative metabolism (as measured by superoxide anion production) and degranulation (as measured by release of the granular enzymes arylsulfatase and beta-glucuronidase) have been examined. Superoxide anion production by eosinophils stimulated with standard doses of the stimulant phorbol myristic acetate (TPA) (1 microgram/ml) was augmented approximately 20% by preincubation with IL 1. However, IL 1 alone had no effect on superoxide anion production. At suboptimal doses of TPA, there was a dose-dependent inhibition of superoxide anion production in the presence of IL 1. Calcium ionophore (2 X 10( 7) M) markedly enhanced superoxide anion production elicited by 0.1 ng/ml of TPA, but had only modest effects in the absence of TPA. When IL 1 was added to eosinophils stimulated by TPA in the presence of calcium ionophore, there was a dose-dependent increase in superoxide anion production. In contrast to other cell types, degranulation as measured by the release of arylsulfatase and beta glucuronidase was not elicited by the addition of TPA (1 microgram/ml). Although calcium ionophore (2 X 10(-6) M) caused enzyme release (24.2% release of beta glucuronidase, 29.4% release of arylsulfatase), this release was inhibited by the addition of TPA. The addition of IL 1 alone caused an approximate twofold increase in enzyme release, but pretreatment with IL 1 (1 U) reduced ionophore mediated degranulation (p less than or equal to 0.05). Studies employing purified monocyte IL 1 were confirmed by recombinant IL 1-beta. These studies demonstrate for the first time that eosinophil function is modulated by IL 1. IL 1 may also modify the response of eosinophils to other stimuli such as ionophore and TPA. Because TPA is known to act by direct binding to protein kinase C, these studies also demonstrate that, in eosinophils, activation of protein kinase C by phorbol esters may augment one cellular function (oxidative metabolism) while inhibiting another cellular function (degranulation). Similarly, phorbol esters may act synergistically with calcium ionophore in regulation of one function (oxidative metabolism) and act antagonistically with another function (degranulation). The concept that IL 1 uniformly enhances cell function may need to be re-evaluated. PMID- 3023485 TI - Immunosuppression in viral oncogenesis. III. Effects of virus infection on interleukin 1 and interleukin 2 generation and responsiveness. AB - Malignant rabbit fibroma virus (MV) is an oncogenic immunosuppressive leporipoxvirus. We studied the effects of MV infection and MV-associated tumor induced suppressor factor (TISF) on the production of and responsiveness to interleukins 1 and 2. Adherent cells from MV tumor-bearing rabbits elaborate adequate amounts of IL 1 in response to E. coli endotoxin. Neither live virus nor TISF alters the production or the responsiveness to IL 1. However, when we examined spleen cells from rabbits 7 days after MV inoculation, we noted that their ability to produce and respond to IL 2 is deficient. Despite their relatively poor capacity to produce IL 2, these spleen cells express receptor for IL 2 in normal amounts, as measured by the monoclonal antibody 7D4. TISF derived from T lymphocytes from MV tumor-bearing rabbits is by itself capable of inhibiting partially normal secretion of IL 2 and also the response of the cloned murine T cell line HT-2 to added IL 2. Full expression of the immunosuppressive capacity of spleen cells from MV tumor-bearing rabbits requires cell-cell contact, however, and cannot be replaced by either live virus or spleen cell supernatants. Such spleen cells inhibit normal mitogen responsiveness, a defect not remedied by adding exogenous IL 2. Immunologic dysfunction induced by MV infection is transient, and by 11 days after virus inoculation, actively mediated recovery from immunosuppression is observed. We found that spleen cells from rabbits studied 11 days postinoculation secreted IL 2 normally. Thus, immunologic dysfunction secondary to infection with malignant rabbit fibroma virus reflects deficiencies in both elaboration of and response to IL 2, and return of immune function later in the course of the infection is associated with return of the ability of lymphocytes to secrete IL 2. PMID- 3023487 TI - The released interleukin 2 receptor binds interleukin 2 efficiently. AB - The released interleukin 2 receptor (IL 2R) molecule was characterized in order to clarify its biochemical structure and to determine its functional capacity. Enzymatic digestions demonstrated that the released IL 2R, like the cell surface IL 2R, is a complex glycoprotein, modified by the addition of both N- and O linked carbohydrates and sialic acid residues. It has a peptide backbone that is approximately 10 Kd smaller than that of its membrane-associated counterpart. Affinity chromatography demonstrated that released IL 2R from either an HTLV-I positive T cell line (HUT-102) or PHA-activated normal peripheral lymphocytes binds efficiently to purified recombinant IL 2. Furthermore, the interaction between the growth factor and the released receptor does not appear to require any accessory molecules. These observations suggest a potentially significant role for the released IL 2R in the regulation of IL 2-dependent lymphocyte functions. PMID- 3023486 TI - BSF1 induces membrane protein phosphorylation but not phosphoinositide metabolism, Ca2+ mobilization, protein kinase C translocation, or membrane depolarization in resting murine B lymphocytes. AB - The findings presented in this study provide evidence that BSF1 receptors and mIg transmit signals via dissimilar transduction mechanisms that result in a common biologic response, hyper-Ia expression. Specifically, BSF1-containing supernatant does not induce PtdInsP2 hydrolysis as determined by measurement of PtdOH and InsP3. Additionally, BSF1 does not stimulate Ca2+ mobilization, PKC translocation from cytosol to membrane, or membrane depolarization. All of these metabolic events appear to play a central role in hyper-Ia expression mediated by mIg and are initiated after treatment of resting B cells with anti-Ig antibodies. In vitro phosphorylation studies with partially purified plasma membranes from resting B cells revealed that BSF1 interaction with membrane receptors stimulates a membrane-associated protein kinase that phosphorylates an endogenous protein of 44 KDa. Anti-Ig does not stimulate phosphorylation of the 44 KDa protein, suggesting that it does not activate the membrane-associated protein kinase. This observation provides the first evidence of a signal transduction mechanism associated with BSF1-receptor ligation. It indicates that although BSF1 does not modulate events associated with PKC activation, it may function via activation of a membrane-associated protein kinase. This provides a focal point for further studies directed at elucidating signal transduction resulting from BSF1-receptor interaction. PMID- 3023488 TI - Use of the monoclonal antibody 12F1 to characterize the differentiation antigen VLA-2. AB - A monoclonal antibody, 12F1, has been produced that specifically immunoprecipitates the human cell surface structure VLA-2 from platelets and long term activated T cells, as well as from fibroblast and neuroblastoma cell lines. Cross-linking studies indicate that the VLA-2 structure exists on the cell surface as a 165,000 Mr heavy chain (alpha 2) in noncovalent 1:1 association with a 130,000 Mr light chain (beta). The monoclonal antibody A-1A5, which reacts with the beta subunit common to all VLA structures, was able to completely preclear VLA-2, indicating that all of the alpha 2 subunit was associated with VLA beta chain. The specificity of 12F1 for VLA-2 allowed independent immunoprecipitation and flow cytometry analysis of this alpha 2 beta structure separate from any other VLA structures that may have been present such as VLA-1 or free beta subunit. Subunit dissociation studies were used to demonstrate that 12F1 recognizes an epitope on the alpha 2 chain on VLA-2, which is consistent with the 12F1 specificity for VLA-2 alone among the VLA proteins. Analysis of activated T cells indicated that VLA-2, like VLA-1, is another "very late" appearing T cell activation antigen that arises concurrently with VLA-1 starting at day 7 and increasing through 2 wk. VLA-2 was found on many of the same cells as VLA-1 (inactivated T cells, T cell leukemia cells, fibroblasts, SK-N-SH neuroblastoma cells), but VLA-1 and VLA-2 can be expressed independently, because VLA-2 was also present on VLA-1-negative cells such as HSB and platelets, and VLA-1 was present on VLA-2-negative C8215 cells. PMID- 3023489 TI - Retention of T cell reactivity to mitogens and alloantigens by Peyer's patch cells of aged mice. AB - Immune function generally declines with advancing age. However, recent evidence suggests that this decline may differentially affect the lymphoid compartments comprising the immune system. The effect of age on the T cell-mediated competency of cells from two different lymphoid compartments, Peyer's patch (PP) and spleen, was studied. PP cells of aged C57BL/6 mice retained a greater capacity to proliferate in response to concanavalin A, phytohemagglutinin, and alloantigen and to generate alloimmune cytotoxic cells than did splenocytes of aged mice when compared with their young counterparts. These results could not be explained by significant shifts in the response kinetics or in the proportions of T cells in individual tissues. These results support the concept that lymphoid compartments are not equally sensitive to the deleterious effects of the aging process. PMID- 3023490 TI - Stimulation of T cells via the TAP molecule, a member in a family of activating proteins encoded in the Ly-6 locus. AB - T-cell activating protein, TAP, is a Ly-6 encoded 12,000 dalton glycoprotein involved in the activation of murine T cells. TAP is distinct from other known surface activating structures, such as the T cell receptor/T3 complex and Thy-1. This study investigates the mechanism of activation via the TAP molecule. Soluble alpha TAP monoclonal antibody (MAb) is sufficient for T cell activation. This, however, requires cross-linking because F(ab) monovalent antibody is not stimulatory unless cross-linked by a second antibody. Immediately after cross linking, there is a rapid influx of calcium, which is comparable with concanavalin A or T cell receptor triggered responses. Subsequently, interleukin 2 (IL 2) is produced, and IL 2 receptors (IL 2R) are expressed. TAP-stimulated T cell proliferation is driven by this autocrine pathway because it is inhibited by alpha IL 2R MAb. Thus TAP-mediated activation appears to share a common final pathway with other mitogenic stimuli. A nonactivating alpha TAP MAb was observed to stimulate T cells upon additional cross-linking. Given this observation, we asked whether other Ly-6 linked proteins might share similar activating potential. We show that at least three distinct Ly-6 linked molecules are capable of stimulating T hybrid clones and/or heterogeneous T lymphocytes. Thus it appears that the Ly-6 locus encodes a family of activating cell surface molecules. PMID- 3023491 TI - A new cloning method for antibody-forming lymphoblastoid cells. Increase in cloning efficiency by inclusion of human fibroblasts into semisolid agarose growth layer. AB - We report the development of a method for cloning human EBV-transformed cells which has greater efficiency than techniques used presently. In this new method lymphoblastoid cells are cultured in semisolid agarose in close physical association with human fibroblasts. The results indicate a 10-fold increase in the cloning efficiencies. The average cloning efficiency, depending on the age of cell lines, was from 1 to 14%, and colonies appeared 7-9 days sooner than in the traditional soft agarose method. The new method has allowed us to develop several stable lymphoblastoid cell lines producing antibody cytotoxic to human B lymphocytes. This method may make it more practical to obtain monoclonal human antibodies from lymphoblastoid cell lines which had previously been unstable due to heterogeneity. PMID- 3023492 TI - A human T cell line established from a patient with Sezary syndrome. Application for assay of human interleukin 2 (IL-2). AB - A human T cell line, designated Sez 627, was established from a patient with Sezary syndrome. These clonal T cells have been cultured for more than 2 years in the presence of IL-2 without any antigen stimulation. The surface phenotype of Sez 627 was OKT3+, OKT4-, OKT8+, and Tac+, and infection with human T lymphotrophic virus type I (HTLV-I) was demonstrated by Southern blot hybridization analysis. In human IL-2 assay, Sez 627 cells were found to be superior to murine CTLL-2 cells with respect to their unresponsiveness to phorbol 12-myristate 13-acetate (PMA), and species specificity for human IL-2, as well as better cryopreservation, although they were less sensitive to IL-2 than CTLL-2 cells. Using Sez 627 cells, IL-2 production by lymphocytes from patients with systemic lupus erythematosus (SLE) was examined. Decreased IL-2 production was observed in the patients with active SLE but not in most patients with inactive SLE. These findings suggest that Sez 627 is a useful human T cell line for human IL-2 assay. PMID- 3023493 TI - The efficient production of stable, human monoclonal antibody-secreting hybridomas from EBV-transformed lymphocytes using the mouse myeloma X63-Ag8.653 as a fusion partner. AB - The mouse myeloma X63-Ag8.653 was fused to peripheral blood lymphocytes (PBL) from apparently healthy individuals, autoimmune patients and volunteers immunised with Rhesus (D) positive erythrocytes. Fusions were performed with or without prior transformation of PBL with Epstein-Barr virus (EBV). Using untransformed PBL, under the best conditions a mean fusion frequency of 8.4 X 10(-6) was obtained, with 22% of the resulting hybridomas secreting human immunoglobulin. Fusions with EBV-transformed cells gave fusion frequencies of 1.0 X 10(-4), with 85-90% of hybridomas secreting human immunoglobulin. The heterohybridomas formed in both cases cloned efficiently and had doubling times of 24-30 h. The heterohybridomas secreted human IgM, IgG and IgA of both kappa and lambda isotypes and culture supernatants contained up to 50 micrograms ml-1 of human immunoglobulin. Mouse immunoglobulin was not detected in the culture supernatants. 28 hybrids were selected for vigorous growth and antibody production by repeated cloning. Immunoglobulin synthesis was stabilised in 26 of these hybridomas after two or three cloning steps. The heterohybridomas have been successfully grown in large volumes for periods up to 15 months. It is concluded that the mouse myeloma X63-Ag8.653 is a suitable fusion partner with EBV transformed B cells in the efficient production of human monoclonal antibodies. PMID- 3023494 TI - A rapid and simple method for efficient coating of microtiter plates using low amounts of antigen in the presence of detergent. AB - Bio-Beads SM-2 have previously been used for the removal of non-ionic detergents from protein solutions. Addition of Bio-Beads SM-2 to detergent solubilized antigen significantly enhanced the immobilization of antigen to microtiter wells. Depending on the incubation time used 35-45% of the applied antigen could be immobilized to the microtiter wells. Using this method and a subsequent ELISA procedure it was possible to detect monoclonal antibodies in hybridoma supernatants after coating microtiter wells with 100 microliters of a solution containing 16 ng antigen/ml in the presence of 0.01% Triton X-100. PMID- 3023495 TI - Spin labeling study on membrane fluidity of epidermal cell (cow snout epidermis). AB - Cow snout epidermal cells digested by 0.25% trypsin were separated into three regions of keratinocytes by Percoll density gradient centrifugation. The membrane fluidity of keratinocytes in each region was measured by electron spin resonance using 5-doxyl stearic acid (5-DSA) as a labeling agent. The order parameter(s) showed higher values as the depth of the epidermis decreased: lower region of epidermis, 0.632; upper region, 0.645; and horny cells, 0.680. These data indicated that membrane fluidity of epidermal cells decreased as cells approached the surface. PMID- 3023496 TI - Natural killing of herpes simplex virus-infected fibroblasts. PMID- 3023497 TI - Safety and immunogenicity of live attenuated rhesus monkey rotavirus vaccine. PMID- 3023498 TI - Genetic and antigenic relatedness of human and animal strains of antigenically distinct rotaviruses. AB - We used nucleic-acid hybridization and enzymatic and immunofluorescence assay to examine the relatedness of human and animal strains of antigenically distinct rotaviruses (ADRVs). ADRVs isolated from rats and humans in Baltimore, Maryland, were shown to be closely related to bovine and porcine strains of group B rotavirus. A human group B rotavirus associated with epidemics of gastroenteritis in China was also found to share antigenic determinants and genome sequence homology with ADRVs passaged in rats in the United States. Closely related strains of group B rotavirus thus appear to infect human and animal populations in widely separated geographic areas. PMID- 3023499 TI - Molecular hybridization study of plasma hepatitis B virus DNA from different carriers. AB - We have used molecular hybridization techniques to show that hepatitis B virus (HBV) DNA from different individuals may have substantial differences in sequence homology. Seven specimens of HBV DNA were isolated from plasma of different blood donors. Samples were applied as dots to membranes and nick-translated to form probes. Densitometry of the radioautograms showed that hybridization was most extensive with probe prepared from the same specimen. The hybridization bias was statistically significant (P less than .02) and visible to the naked eye. Hybridization to probes that were digested with nuclease S1 before nick translation did not eliminate the bias. Nor was the bias related to the d/y subdeterminants; on the average, 15 other specimens hybridized equally well to probes prepared from HBV with the same or different subdeterminant. Many specimens among 37 other serum samples showed greater or lesser degrees of homology to different probes, as demonstrated by reprobing of samples fixed to nylon membranes. PMID- 3023500 TI - Postburn serum inhibits in vitro production of colony-stimulating factor by mononuclear peripheral blood cells. AB - The effects of postburn serum (PBS) on the production of colony-stimulating factor (CSF) was evaluated in 13 burned patients by adding PBS to normal peripheral blood mononuclear cells (MNC) and assaying the MNC-conditioned media for CSF content. PBS inhibited CSF production by at least 50%. PBS from non survivors significantly inhibited CSF production more than PBS from survivors. The addition of lithium chloride restored production of CSF in the presence of day 15 PBS but could not overcome the inhibitory effects of day 1 or day 8 PBS. The nature of the inhibitor(s) is uncertain, but correction of the CSF production defect by lithium chloride later in the course of thermal injury suggests that the defect may be reversible. PMID- 3023501 TI - On the treatment of cutaneous leishmaniasis in Egypt. PMID- 3023502 TI - [ErbA and steroid receptor]. PMID- 3023503 TI - [Effect of rotenone on rabbit ovulation and histochemical activities of cytochrome oxidase and 3 beta-hydroxysteroid dehydrogenase of the follicle]. AB - Various doses of rotenone, an inhibitor of respiratory chain, were administered to mature female rabbits concomitantly with an ovulatory dose of hCG. The effect of rotenone on ovulation was studied by counting the ovulated stigma under a dissecting microscope. Histochemical studies on the activities of 3 beta hydroxysteroid dehydrogenase (3 beta-HSD) and cytochrome oxidase (CYO) in follicles at various intervals after hCG-rotenone injections were also performed to investigate the role of mitochondrial oxidation in the ovulatory process. Rotenone inhibited the hCG-induced ovulation in a dose respondent manner and reduced the sudden hCG-induced increase in the histochemical activities of 3 beta HSD and CYO of granulosa cells. It is suggested that the activation of mitochondrial oxidation in the ovulating follicle is mandatory for ovulation and the induction of steroidogenic enzymes in granulosa cells. PMID- 3023504 TI - [Phase II study of etoposide (NK 171) in trophoblastic disease. Study Group on Etoposide (NK 171) for Trophoblastic Disease]. PMID- 3023505 TI - [Experimental combination chemotherapy with carboquone and 5-fluorouracil of human germ cell tumors transplanted into nude mice]. PMID- 3023506 TI - [A case of hepatoma effectively treated by TAE and intra-arterial and intraportal infusion chemotherapy]. PMID- 3023507 TI - [Treatment of circulatory diseases: recent progress. 1. Hypertension]. PMID- 3023508 TI - Virus p30-related protein in follicular fluids and C virus-like particles on the cell membrane of the human oocyte. AB - Fluids from 208 follicles were analyzed with respect to the concentration of estradiol and progesterone and to the presence of RD 114 retrovirus p30-related protein (rp), assuming that such a protein indicates the presence of C-virus particles in the follicular fluids. The results obtained showed that 60-80% of the follicular fluids within the same range of steroid hormone levels contained virus p30 rp. Electron microscopy showed 80-nm virus-like particles, more or less well preserved, in about half of the number of oocytes examined. Negative staining of purified follicular fluid particles showed ring-like structures with a size of 100-200 mm. It is concluded that the presence of virus p30 rp in follicular fluids is correlated neither to a certain steroid hormone concentration nor to the oocyte capability to become fertilized and cleave. The observed expression of an endogenous virus genome during oocyte maturation strengthens the hypothesis that this type of genome is active at early developmental stages. PMID- 3023510 TI - Plasma 1,25(OH)2D3 response to parathyroid hormone, cyclic adenosine monophosphate, and phosphorus depletion in the spontaneously hypertensive rat. AB - Spontaneously hypertensive rats (SHR) have several abnormalities of calcium metabolism compared with normotensive control Wistar-Kyoto (WKY) rats. Previously the vitamin D metabolite 1,25-dihydroxycholecalciferol (1,25[OH]2D3) was found to be inappropriately low in SHR in view of their ionized hypocalcemia and hyperparathyroidism. We examined the responses of plasma 1,25(OH)2D3 to several known stimuli. Baseline plasma 1,25(OH)2D3 levels tended to be lower in SHR than WKY rats (51.5 +/- 4.3 vs. 82.3 +/- 14.1 pg/ml, P = 0.06). Infusion of a pharmacologic dose of parathyroid hormone (8 U/hr over a period of 17 hours) resulted in a plasma 1,25(OH)2D3 level of 504 +/- 77 pg/ml in SHR vs. 1016 +/- 211 pg/ml in WKY rats (P less than 0.03). Cyclic adenosine monophosphate infusion (1 mumol/hr/100 gm over a period of 17 hours) in thyroparathyroidectomized animals resulted in a 1,25(OH)2D3 level of 121 +/- 24 pg/ml in SHR vs. 557 +/- 26 pg/ml in WKY rats (P less than 0.01). After dietary phosphorus depletion for 3 weeks, SHR also had lower 1,25(OH)2D3 levels than WKY rats (83 +/- 13 vs. 300 +/- 42 pg/ml, P less than 0.001) even though a comparable degree of hypophosphatemia was achieved. Thus, the response of plasma 1,25(OH)2D3 levels to several known stimuli is submaximal in SHR as compared with WKY rats, suggesting defective synthesis or enhanced metabolic clearance of this hormone. PMID- 3023509 TI - Methylprednisolone accelerates the ontogeny of sodium-taurocholate cotransport in rat ileal brush border membranes. AB - The effect of methylprednisolone on the postnatal maturation of taurocholate transport was studied by using isolated ileal brush border membrane vesicles. Vesicles were prepared from 14-day-old control, 14-day-old methylprednisolone treated, and untreated 21-day-old rats. Methylprednisolone treatment resulted in a significant stimulation of taurocholate uptake by an inwardly directed Na+ gradient when compared with a choline gradient incubation. These differences occurred at 20 seconds and 1, 2, and 5 minutes of incubation (P less than 0.05). In 14-day-old controls, uptake was similar for Na+ and choline gradients. A plot of active uptake velocity vs. taurocholate concentration (0.1 to 1.0 mmol/L) in 14-day-old controls was linear and approached the abscissa, indicating the absence of active transport. Plots for methylprednisolone-treated rats showed saturability. An inwardly directed Na+ gradient stimulated initial taurocholate uptake rates by twofold at 37 degrees C (P less than 0.01), but not at 4 degrees C. Glycocholate and glycodeoxycholate inhibited Na+-stimulated taurocholate uptake by 50% (P less than 0.01) and 20% (P less than 0.05), respectively. These data indicate that pharmacologic doses of methylprednisolone accelerate the postnatal acquisition of Na+-dependent taurocholate cotransport in rat ileal brush border membranes. PMID- 3023511 TI - Cumulative effects of quinidine and aspirin on bleeding time and platelet alpha 2 adrenoceptors: potential mechanism of bleeding diathesis in patients receiving this combination. AB - We observed quinidine-induced prolongation of bleeding time without thrombocytopenia in three subjects. In addition, we noticed a cumulative prolongation of bleeding time by a combination of quinidine and aspirin. We postulated that because both quinidine and aspirin inhibit epinephrine-induced platelet aggregation, a cumulative effect of the two drugs might be responsible for the hemostatic defect. In studies using normal human platelets, we confirmed a marked reduction in epinephrine-induced platelet aggregation by the combination of these two agents. To further study the potential mechanism of this cumulative effect, platelet lysates were incubated with the alpha 2-adrenoceptor antagonist tritiated yohimbine in the presence of quinidine and aspirin. On the basis of the radioligand binding data, the dissociation constant (KD) of alpha 2-adrenoceptors was observed to increase in the presence of quinidine as well as aspirin. The combination of these two agents caused a marked increase in the KD of platelet alpha 2-adrenoceptors without alteration in the number of receptor sites. These data suggest that the cumulative effects of quinidine and aspirin on platelet alpha 2-adrenoceptor KD may relate to the significant reduction in epinephrine induced platelet aggregation. This phenomenon, coupled with other well-known effects of aspirin on the platelet release reaction and arachidonate metabolism, may lead to bleeding problems in some patients receiving this combination. PMID- 3023512 TI - 1,25-Dihydroxyvitamin D3 receptors in lymphocytes from patients with rheumatoid arthritis. AB - It has been previously found that activation of human lymphocytes in vitro causes the expression of receptors for the hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and that 1,25(OH)2D3 has immunoregulatory properties including the ability to inhibit interleukin-2, to suppress lymphocyte proliferation, and to inhibit antibody production. In the present study we found that 13 of a group of 17 (76%) seropositive patients with rheumatoid arthritis had lymphocytes that possessed 1,25(OH)2D3 receptors (without activation in vitro) compared with only three of 17 (18%) normal individuals. The biochemical characteristics of the 1,25(OH)2D3 receptor, including affinity, sedimentation coefficient, and DNA binding properties in the rheumatoid arthritis lymphocytes were indistinguishable from those established for this receptor in the classic target tissue of the hormone. This finding raises the possibility that 1,25(OH)2D3, acting through its receptor, might play a previously unsuspected role on lymphocytes of patients with rheumatoid arthritis. PMID- 3023514 TI - Effect of pentoxifylline on the phagocytic activity, cAMP levels, and superoxide anion production by monocytes and polymorphonuclear cells. AB - The effect of pentoxifylline (Trental) on the phagocytic capacity, cAMP levels, and superoxide anion production of human peripheral blood monocytes and polymorphonuclears (PMNs) was studied. The drug inhibited the phagocytosis of latex particles by both monocytes and PMNs in a dose-dependent manner. In addition, superoxide anion production during the phagocytic process was also reduced following incubation of the cells with pentoxifylline. It is suggested that this inhibitory effect is due to the increased intracellular levels of cAMP induced by the drug. PMID- 3023513 TI - Enhanced superoxide production by rat alveolar macrophages stimulated in vitro with biological response modifiers. AB - The kinetics of superoxide release and the effects of several biological response modifiers (BRM) on superoxide release from rat pulmonary alveolar macrophages (AM) have been studied. These cells produced superoxide anion both spontaneously and in response to phorbol myristate acetate (PMA) in a dose-related manner. The response to PMA peaked in approximately 2 hr and maintained plateau levels for an additional 2-3 hr before subsiding. Pretreatment of the macrophages in vitro with a number of immunostimulants enhanced the production of superoxide above that of controls. The release of superoxide in response to the immunostimulants was a slow phenomenon that took place over a 3-5 hr time period. Lymphokine-containing supernatants from concanavalin A (con A)-stimulated rat spleen cells (LK-Sup), murine recombinant gamma interferon (rMuIFN-gamma), nigeran, and muramyl dipeptide (MDP) enhanced this response in a dose-related manner. Poly I:C and Salmonella typhosa lipopolysaccharide (LPS) stimulated rat alveolar macrophages at low but not high concentrations. In contrast to the alveolar macrophages, rat peritoneal exudate cells were not activated by immunostimulants to produce increased amounts of superoxide. PMID- 3023516 TI - A performance-based management system to reduce prematurity and low birth weight. AB - A performance system is a management tool for ensuring that programs accomplish their objectives. Using Delphi and nominal group process techniques, the authors established objectives for a system to reduce prematurity and low birth weight. A task force consisting of state, regional, and local public health personnel, consultants from the Centers for Disease Control, and faculty from the School of Public Health then developed the activities, standards, and guidelines of the performance system. Finally, an assessment tool for evaluation of the system was designed. The system, which is intended to complement initiatives to improve the medical care of maternity patients, should be useful to maternity clinic managers and supervisors who wish to develop performance standards specifically targeted to the achievement of objectives for reducing prematurity and low birth weight. PMID- 3023515 TI - Model for leukocyte regulation by chemoattractant receptors: roles of a guanine nucleotide regulatory protein and polyphosphoinositide metabolism. AB - Binding of chemoattractants to their receptors on phagocytes activates a guanine nucleotide regulatory (N) protein through the substitution of GTP for GDP on N. The activated N protein in turn stimulates a membrane-associated phospholipase C by lowering the Ca2+ concentration required to activate this enzyme from supraphysiologic levels to ambient intracellular concentrations. The phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate into the Ca2+ mobilizer inositol 1,4,5-trisphosphate and the protein kinase C activator 1,2 diacylglycerol. In addition to promoting cellular activation, the products of this hydrolysis initiate processes which feed back to inhibit poly phosphoinositide breakdown. The regulatory model proposed herein may be relevant to other receptors which stimulate polyphosphoinositide metabolism. PMID- 3023517 TI - Mechanism of restoration of ACTH release in rats with long-term lesions of the paraventricular nuclei. AB - The effects of lesions in the paraventricular nucleus (PVN) on the adrenocortical response to ether stress were investigated in neurohypophysectomized and intact rats. During the first 4 days after placement of lesions in the PVN, the corticosterone response to ether stress was almost completely inhibited. It then gradually increased and, within 4-6 weeks of surgery, was restored to about 60% of that in sham-operated rats. Basal plasma concentrations of corticosterone were low in rats after placement of lesions in the PVN and/or after neurointermediate lobectomy (NILX). Corticosterone responses to ether stress were similar in groups submitted to PVN lesions and/or NILX, and lower than those in the appropriate sham-operated groups. In all lesioned groups, plasma ACTH concentrations after a combination of stressors (ether plus laparotomy) were also lower than those in the sham-operated groups. Six weeks after lesioning of the PVN, immunoreactive rat corticotrophin-releasing factor-41 (rCRF-41) concentrations in stalk-median eminence (SME) extract fell to about 5% of that in sham-operated rats, while immunoreactive arginine vasopressin (AVP) concentrations did not change. Immunohistochemistry revealed a substantial decrease in rCRF-41 immunostaining of the median eminence 6 weeks after lesioning of the PVN, though randomly located clusters of stained terminals were still seen in the whole rostro-caudal extent of the median eminence. A mixture containing synthetic rCRF-41 and AVP, in proportions similar to those in SME extracts from sham-operated rats, caused significantly less release of ACTH from anterior pituitary cell cultures than did SME extracts from sham-operated rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3023519 TI - Synthetic peptides from the circumsporozoite proteins of Plasmodium falciparum and Plasmodium knowlesi recognize the human hepatoma cell line HepG2-A16 in vitro. AB - Several lines of evidence have emphasized the importance of the malaria circumsporozoite (CS) protein as a factor in sporozoite invasion of the hepatocyte; however, the specific mechanism of cell recognition and invasion has not been explained. In this study we present evidence that a highly conserved region of the CS protein immediately adjacent to the repeat region, the N1 region, specifically recognizes receptors on the human hepatoma cell line HepG2 A16 under conditions where invasion by sporozoites can occur. Peptides consisting of sequences from the repeat region or of the more extensive N2 region showed no such specific association. Antibody against the N1 peptide could inhibit sporozoite invasion in vitro. Covalent coupling of radiolabeled N1 peptide to HepG2-A16 cells identified two hepatic cell proteins to be closely associated with the peptide. We suggest that these proteins could act as receptors or mediators, via the N1 region of the CS protein, for the P. falciparum sporozoite in the process of invasion of the hepatocyte. PMID- 3023518 TI - Assembly of the prothrombin activator complex on rabbit alveolar macrophage high affinity factor Xa receptors. A kinetic study. AB - Efficient prothrombin activation occurs after assembly of factors Va, Xa, and phospholipid surface cofactor as a multimolecular complex. These components are provided by platelets and plasma within the vascular space, but molecules and membranes for prothrombin activator assembly in extravascular spaces have not been identified. In the present study, purified alveolar macrophages were found to produce high-affinity factor Xa receptors that mediate formation of enzymatic prothrombinase complexes and rapid prothrombin to thrombin conversion in the absence of exogenous factor V/Va or platelets. Thus, in reaction mixtures with alveolar macrophages cultured for 20 h in serum-free medium, the thrombin formation rate was 152 nM/min/0.66 X 10(6) cells, after adding prothrombin (1.5 microM), Ca2+ (5 mM), and factor Xa (3.7 nM). The observed Kd of factor Xa interaction with macrophage receptors is 2.1 +/- 0.94 X 10(-10) M. Kinetic analysis and inhibition studies using isolated factor V and anti-factor V antibody show that macrophage Xa receptors are functionally and antigenically similar to plasma factor V. By contrast, freshly isolated cells lacked receptors promoting prothrombin conversion at high rates. Inhibitors of protein synthesis and glycosylation, puromycin and monensin, respectively, abrogated production of Xa receptors in culture. Additionally, subcellular fractionation and enzyme marker studies (alkaline phosphodiesterase I) indicate that internal and external membranes of alveolar macrophages have phospholipid surface cofactor activity required for prothrombinase complexes. Pulmonary surfactant is also shown to express this cofactor activity. Alveolar macrophages and surfactant comprise an efficient prothrombin activator system that is independent of plasma factor V. This system may facilitate rapid extravascular alveolar thrombin formation even at very low concentrations of factor Xa during lung defense reactions to inflammation or edema. PMID- 3023522 TI - Determination of cyclic 3'-5'-adenosine monophosphate in plasma by RIA methods in the presence of EDTA. AB - Calcium ions definitively increase the ability of cyclic 3',5'-adenosine monophosphate (cAMP) to bind to its antibody. In contrast, ethylenedinitrolotetra acetic acid as its disodium salt (EDTA) shows a dose-dependent inhibition of the binding of cAMP to its antibody. The less sensitive protein binding methods are not affected by EDTA. This is inconvenient, because the EDTA-plasmas can be stored frozen without breakdown of cAMP, but are unsuitable for sensitive radioimmunoassays. The aim of this investigation was to determine how calcium ions and EDTA affect the binding of cAMP to its antibody. Based on these results, we describe an alternative procedure for commercial RIA methods for the determination of cAMP in EDTA-plasma. The almost complete inhibition of the hapten-antibody reaction by EDTA can be abolished by adding an equivalent concentration of calcium ions to the reaction medium together with trichloroacetic acid. Thus a simple and rapid procedure was found for the storage of plasma and for the determination of plasma cAMP. PMID- 3023521 TI - A conserved human germline V kappa gene directly encodes rheumatoid factor light chains. AB - The full-length gene that encodes the light chain variable regions of an idiotypically related group of human IgM kappa rheumatoid factors (RFs) has been cloned and sequenced. The deduced amino acid sequence is identical to four separate RF proteins. These results prove that genes capable of encoding human anti-IgG autoantibody light chains without any somatic mutation are present in the kappa gene repertoire of normal people. PMID- 3023523 TI - A double origin electrophoretic method for the simultaneous separation of adenosine deaminase, adenylate kinase, and carbonic anhydrase II. AB - A rapid, reliable method for the simultaneous separation of adenosine deaminase, adenylate kinase, and carbonic anhydrase II by agarose gel electrophoresis is presented. This method uses a double origin sample application system. Unreduced sample extracts for adenylate kinase analysis are applied 13.0 cm from the anode. Reduced sample extracts for the remaining proteins of interest are applied 7.0 cm from the anode. The use of applicator foils and an increased voltage gradient result in superior resolution, linearity, and band sharpness of the allozyme patterns. Further, there is no masking of the adenylate kinase 2 band as a result of the use of a reducing agent, and carbonic anhydrase II is resolved without interference from hemoglobin as has been observed with other multisystem methods. PMID- 3023520 TI - Adult-onset cyclic neutropenia is a benign neoplasm associated with clonal proliferation of large granular lymphocytes. AB - Human cyclic neutropenia occurs in children and adults. Adult-onset cyclic neutropenia is an acquired disease characterized by increased numbers of large granular lymphocytes (LGL), in contrast to childhood-onset cyclic neutropenia in which LGL counts are normal. We investigated the clonality of lymphocytes in these two groups of patients by assessing the rearrangement status of the T cell receptor beta chain gene. Patients with adult-onset cyclic neutropenia showed clonal rearrangement of the T beta gene whereas the children did not. Since LGL are known to have multiple regulatory effects on normal hematopoiesis, the finding of a clonal proliferation of this lymphocyte population implicates these cells in the pathogenesis of cyclic neutropenia. PMID- 3023524 TI - The investigation of alleged insecticide toxicity: a case involving chlordane exposure, multiple sclerosis, and peripheral neuropathy. AB - A man with no previous medical problems had two documented exposures to an insecticide containing the organophosphorous compounds chlordane and heptachlor. Six months to one year later, he began to experience neurological symptoms which progressed until his death. At autopsy, his brain showed classic findings of multiple sclerosis, and he had a severe peripheral neuropathy. Review of the literature indicates that the findings are not compatible with chlordane toxicity. Some of the factors to be used in determining the casual relationship between toxic exposure and disease processes are discussed. PMID- 3023525 TI - Simultaneous typing of erythrocyte acid phosphatase, adenylate kinase and adenosine deaminase in human hair root sheaths. PMID- 3023526 TI - Comparative expression of hepatitis B virus antigens in several cell model systems. AB - In this paper the kinetics of hepatitis B virus (HBV) gene expression were investigated in natural and experimentally transfected cell systems. These systems included four human hepatocellular carcinoma cell lines containing HBV DNA (TONG/PHC, HEp 3B2, PLC/PRF/5 and HA22T/VGH) as well as a mouse and a rat cell line both experimentally transfected with HBV DNA. Comparative results on the kinetics of hepatitis B surface antigen in these cell systems suggested that the S gene in the integrated state is expressed at different levels. No human cell line derived from HBV-associated hepatocellular carcinoma produced hepatitis B e antigen (HBeAg) when medium was concentrated by ultrafiltration. In distinct contrast, the two experimentally transfected cell systems produced e antigen at different levels. When all HBV-containing cell lines were grown as tumours in nude mice, no HBeAg was detected in the serum of these mice inoculated with human hepatocellular carcinoma cell lines, in the tumour homogenates, or in the tumour derived lines, whereas e antigen was expressed both in vivo and in vitro in the experimentally transfected cell lines. These observations indicate that C gene expression is restricted to transfected cell cultures and this suggests a distinct difference in the mechanisms of HBV gene expression between the two types of in vitro model systems. PMID- 3023527 TI - Establishment of a mouse model for human rhinovirus infection. AB - We describe here a mouse model for rhinovirus infection using a variant of human rhinovirus type 2 (HRV2/H) which replicated 50- to 300-fold in the lungs of BALB/c mice. The variant virus differed only marginally from HRV2/H according to various biochemical parameters. Use of a photosensitive inoculum and pretreatment of the animals with actinomycin D were necessary for detection of reproducible and significant levels of virus replication. This mouse model of rhinovirus infection is the first example of human rhinovirus replication in a non-primate mammal, and provides an important link for the development of rhinovirus therapy or prophylaxis. PMID- 3023528 TI - Selection and characterization of an interferon-responsive clonal cell line of HeLa cells. AB - HeLa cells generally do not respond well to interferon (IFN). We have used is-1, an IFN-sensitive mutant of mengovirus, to select a clone of IFN-responsive HeLa cells (F-H12). At moderate levels of human alpha/beta IFN, is-1 yields were fivefold lower in these cells than in similarly protected control cells. In contrast, wild-type mengovirus, vesicular stomatitis virus and a wild-type and thymidine kinase-negative strains of herpes simplex virus type 1 grew equally well in both cell lines. By a cell survival assay, the F-H12 line was up to 100 times more responsive to IFN than the parental line when challenged by is-1. 2' 5'-Oligo(A)-dependent endonuclease activity was the same in both lines. These observations cannot be accounted for by enhanced induction of IFN following infection. PMID- 3023530 TI - Characterization of transforming viruses rescued from a hamster tumour cell line harbouring the v-src gene flanked by long terminal repeats. AB - The organization of proviruses derived from infecting transforming viruses rescued from hamster tumour cells was studied. Southern blot analysis indicated that the provirus from the F6 cell line was organized as long terminal repeat (LTR)-src-LTR, and S1 mapping experiments suggested that it was probably derived by reverse transcription of src mRNA followed by integration. In the E6 cell line, the provirus unit was arranged as LTR-delta gag-src-LTR, indicating a recombination event between the rescued transforming virus and the helper virus. These results suggest that transforming defective viruses containing only the src gene can be rescued from nonpermissive mammalian cells. PMID- 3023531 TI - The genome-linked proteins of aphthoviruses: specific immunoprecipitation of the three species detected on virus RNA and identification of possible precursors. AB - Synthetic peptides have been made corresponding to the C-terminal portion of each of the three presumptive genome-linked proteins (VPgs) of foot-and-mouth disease virus type A10. Antisera against each of these peptides efficiently precipitated only the homologous VPg, and the reactions were inhibited by prior absorption with homologous, but not heterologous synthetic peptide. The peptide antisera precipitated a number of proteins from infected cell extracts with mol. wt. of 100, 84, 56, 36, 27, 25 and 20, all X 10(3); all these reactions were inhibited by absorption with homologous peptide, indicating that they were probable precursors of VPg. The relationship between these proteins is at present unclear. PMID- 3023529 TI - Characterization of the IE110 gene of herpes simplex virus type 1. AB - We have determined the DNA sequence of the herpes simplex virus type 1 (HSV-1) gene encoding the immediate early protein IE110, which is involved in transcriptional activation of later virus genes. The locations of the 5' and 3' termini of IE110 mRNA together with the positions of two introns, were identified. Examination of the DNA sequence suggested that translation starts at the first ATG after the 5' terminus of the mRNA, and that both introns occur in protein-coding sequence. The predicted IE110 polypeptide contains 775 amino acids, and has a molecular weight of 78452. It contains a cysteine-rich region resembling regions found in several proteins which interact functionally with DNA. An antiserum was raised to the predicted C terminal amino acid sequence of the IE110 polypeptide and was shown to immunoprecipitate the native protein from HSV-1-infected cell extracts. The functional importance of regions of the protein was evaluated by construction of frameshift and deletion mutants of a plasmid borne IE110 gene. The mutants were tested for IE110 function by short-term transfection assays, and the results were correlated with the DNA sequence and RNA mapping studies. PMID- 3023532 TI - VP7 serotype-specific glycoprotein of OSU porcine rotavirus: coding assignment and gene sequence. AB - With a reassortant from a cross of human rotavirus DS-1 (serotype 2) and OSU (serotype 5) it was determined that the OSU major neutralization glycoprotein antigen (VP7) was encoded by gene segment 9. A full-sized cloned cDNA copy of the OSU gene 9 was produced and sequenced. Hybridization of such labelled cDNA with the corresponding segment of a reassortant DS-1 X OSU virus confirmed the coding assignment. Comparison of the deduced amino acid sequence of the VP7 of OSU with those previously determined for five other rotavirus strains, representing four distinct serotypes, revealed some hydrophilic regions that exhibited significant homology and other hydrophilic domains with greater amino acid divergence. In one of the latter hydrophilic domains each of the five serotypes had a distinct amino acid substitution at residue 146, suggesting that it may be involved in serotype specificity. PMID- 3023533 TI - Antigenic analysis of rotavirus isolates using monoclonal antibodies specific for human serotypes 1, 2, 3 and 4, and SA11. AB - Five neutralizing monoclonal antibodies produced against human rotavirus (HRV) serotypes 1, 2, 3 and 4 and the simian rotavirus (SA11) were used to study 59 rotavirus isolates of human, simian and feline origin previously serotyped using polyclonal antisera. In neutralization tests, 19 of 26 HRV serotype 1 isolates, both strains of HRV serotype 2, 14 of 24 HRV serotype 3 isolates and all of seven serotype 4 isolates were neutralized by the homologous serotype-specific monoclonal antibodies. Use of the panel of monoclonal antibodies revealed antigenic differences between strains within serotypes 1 and 3 and, in the case of the serotype 3 strains, each variant had a unique RNA electropherotype. An enzyme immunoassay (EIA) which utilized the monoclonal antibodies essentially confirmed the neutralization results. Preliminary results show that direct serotyping in faecal extracts by EIA using these monoclonal antibodies is specific but lacks sensitivity. PMID- 3023534 TI - Establishment of herpes simplex virus latency in vitro with cycloheximide. AB - Human embryonic lung cells were infected with herpes simplex virus (HSV), treated with 10 micrograms/ml or more of cycloheximide for 24 h, incubated at 37 degrees C, and then shifted to 40.5 degrees C for various periods of time (0 to 40 days) without cycloheximide treatment. No infectious virus was detected after freezing and thawing of the cultures; however, infectious virus was recovered after temperature shift-down to 37 degrees C or superinfection with human cytomegalovirus (HCMV). The time course for formation of infectious centres after temperature shift-down was examined with and without HCMV superinfection during incubation at 40.5 degrees C. Two patterns of latently infected cells were identified: one pattern showed spontaneous reactivation of virus after temperature shift-down, and the second showed reactivation of HSV after superinfection with HCMV. The first pattern showed a rapid decrease in the number of infectious centres with time, whereas the second maintained a steady reactivation rate up to 40 days at 40.5 degrees C. The same tendency was observed for infectious centre formation at 37 degrees C with and without HCMV superinfection in the HSV latency system established with (E)-5-(2-bromovinyl)-2' deoxyuridine and interferon treatment. PMID- 3023536 TI - The products of herpes simplex virus type 1 (HSV-1) immediate early genes 1, 2 and 3 can activate HSV-1 gene expression in trans. AB - Expression of the early and late genes of herpes simplex virus type 1 (HSV-1) during infection of tissue culture cells requires the prior expression of the immediate early (IE) genes. The requirement for the product of IE gene 3, Vmw175, for the activation of early promoters has been revealed by studies with temperature-sensitive virus mutants. Recent experiments using transfection assays have shown that both Vmw175 and the product of IE gene 1, Vmw110, are involved in the transactivation of a variety of HSV-1 early promoters. This paper describes experiments which compared the activation of two early promoters [those of the glycoprotein gD and thymidine kinase (tk) genes] with that of a member of a later class of genes (the major capsid protein, VP5). Plasmids containing these promoters linked to the chloramphenicol acetyltransferase (CAT) gene were transfected into HeLa cells with plasmids containing one or more HSV-1 IE genes. Promoter activity was estimated by measurement of CAT activity in extracts of transfected cells. The gD and tk promoters were activated by both Vmw175 and Vmw110, and the combination of these two IE gene products resulted in very high levels of activation. Addition of further IE gene products did not result in any significant increase in the activation seen with the combination of Vmw175 and Vmw110. In contrast, the activation of the VP5 promoter brought about by the combination of Vmw175 and Vmw110 was relatively slight, but was increased further when plasmids containing IE gene 2, encoding Vmw63, were included in the transfection. These data suggest that Vmw63, like Vmw175 and Vmw110, is also involved in the activation of transcription from HSV-1 promoters. The effect of Vmw63 may be limited to the activation of a subset of HSV-1 genes. PMID- 3023535 TI - Restoration of wild-type pathogenicity to an attenuated DNA polymerase mutant of herpes simplex virus type 1. AB - The drug-resistant variant, RSC-26, which was derived from the herpes simplex virus type 1 wild-type strain SC16, expresses an altered DNA polymerase and has reduced pathogenicity in animal models. To determine whether the attenuation in pathogenicity was due solely to mutation in the polymerase gene, a fragment of the wild-type gene was cloned, transferred into the genome of RSC-26 and recombinants were isolated. Three recombinants examined had similar properties to wild-type virus with respect to their sensitivity to antiviral drugs, DNA polymerase activities and their pathogenicity for mice. These results strongly suggest that expression of the altered polymerase of RSC-26 results in attenuated pathogenicity. PMID- 3023537 TI - Detection of bovine herpesvirus type 1 RNA in trigeminal ganglia of latently infected rabbits by in situ hybridization. AB - At times after conjunctival inoculation with bovine herpesvirus type 1 (BHV-1), representing the acute and latent phases of infection, rabbit trigeminal ganglia were examined for the presence of BHV-1 nucleic acids by in situ hybridization using a 3H-labelled BHV-1 DNA probe. During the acute phase of virus infection, both BHV-1 DNA and RNA were detected in ganglionic neurons and occasionally in adjacent satellite cells. However, during the latent phase of infection only viral RNA was detectable in involved neurons. Viral RNA appeared restricted to the nucleus of latently infected cells and was present in varying amounts in individual cells. These results indicate that the BHV-1 genome is transcriptionally active in ganglionic neurons during latent infection. PMID- 3023538 TI - Sequence reiteration required for the efficient growth of BK virus. AB - Compared with wild-type BK virus DNA having tandem triplication of a 68 base pair (bp) element in its transcriptional control region, a mutant viral DNA with a single copy of the 68 bp element induced remarkably delayed virus production in human embryonic kidney (HEK) cells. We molecularly cloned the DNA of progeny viruses using plasmid vector pAT153. Nucleotide sequence analysis of representative clones revealed that all of the altered viral DNAs examined duplicated various segments extending over origin-distal portions of the 68 bp element and their flanking regions. After transfection of HEK cells, most of these rearranged viral DNAs induced viral growth slightly slower than, or at the same rate as, the wild-type viral DNA. Comparison of the structures of these rearranged viral DNAs suggests that reiteration of a 13 bp sequence, which is located in an origin-distal portion of the 68 bp element, is required for the efficient replication of BK virus. PMID- 3023539 TI - Nucleotide sequence of a portion of the Autographa californica nuclear polyhedrosis virus genome containing the EcoRI site-rich region (hr5) and an open reading frame just 5' of the p10 gene. AB - The nucleotide sequence of a 1587 bp region lying within the HindIII-Q fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) DNA has been determined. It begins in the EcoRI-S-EcoRI-X region, continues to the HindIII-P/Q boundary and contains an open reading frame that codes for a polypeptide of 240 amino acids (p26). This open reading frame is also included in the 1100 and 1500 base transcripts previously mapped to this region. The sequence reveals that the 5' ends of the 1100 and 1500 base transcripts are located 20 bp downstream from the end of a putative TATA box (TAATTAAAT) and 19 bp upstream from the translation start codon (ATG) of the p26 open reading frame. The translation termination codon (TAA) falls in the immediate 5' flanking region of the major late p10 gene of AcMNPV, 3 bp downstream from the putative TATA box. The probable polyadenylation site for the 1100 base transcript lies 23 bp downstream from the cap site for the 750 and 2500 base transcripts encoding the p10 protein. The 5' flanking region of the p26 open reading frame contains the EcoRI site-rich region, hr5, whose sequence is included here. The EcoRI site-rich region, hr5, consists of six imperfect tandem repeats of a sequence that includes the EcoRI recognition site. These direct repeats also include many inverted repeats. PMID- 3023541 TI - Therapeutic effect of the antiviral agent ribavirin in Junin virus infection of primates. AB - In order to assess the effect of the antiviral Ribavirin on the course of Junin virus infection in Callithrix jacchus, seven inoculated monkeys were treated with 15 mg of the drug, twice a day, starting 6 days after infection when all animals were viremic. The three untreated controls showed typical signs of Junin virus infection at 14 days pi and their mean time of death was 18 days. In contrast, no signs of illness were detected in Ribavirin-treated animals until 24 days pi, when marmosets showed signs of neurological involvement: 5 of these animals died (mean day of death: 36) but the two remaining treated monkeys improved and survived infection without sequelae. The comparison of survival rates (0% vs 28%) and the delay of the mean day of death observed indicates that the Ribavirin treatment used has therapeutic effect on Junin virus infection in vivo. PMID- 3023540 TI - Treatment of junin virus-infected guinea pigs with immune serum: development of late neurological disease. AB - Guinea pigs infected with Argentine hemorrhagic fever virus (Junin) were treated with pooled, homologous convalescent sera. Use of 15,000 or 5,000 therapeutic units of immune sera prevented all signs of illness when administered within 24 hr of infection. We could also prevent illness and death in infected guinea pigs as late as 6 days after infection if we used more antisera (30,000 therapeutic units/kg). In some treatment groups, surviving animals developed a late neurological syndrome with prominent rear-limb paralysis. Treated animals typically expressed higher viral titers in the brain than in any other organ. There appeared to be no acute exacerbation of disease by antibody administration. Our data suggest that, after replicating peripherally, Junin virus infects the brain where circulating immunoglobulins may not eliminate viable virus. Subsequent replication of virus in the brain may generate a neurological phase of the illness. Histological examination of brains from guinea pigs in treatment groups favoring the neurological phase of illness showed encephalitis, meningitis, and swollen astrocytes, suggestive of neuronal degeneration. There is likely a delicate balance among presence of virus in the brain, the amount of antibody transported into the central nervous system, and the occurrence of this late neurological aspect of experimental Argentine hemorrhagic fever. Further study of this model may elucidate factors relevant in understanding the continuing problem of the late neurological syndrome seen in some human cases of Argentine hemorrhagic fever treated with immune plasma. PMID- 3023542 TI - Detection of human papillomavirus DNA by the nucleic acid sandwich hybridization method from cervical scraping. AB - Cervical scrapes collected from 100 consecutive patients participating in a prospective follow-up study for cervical human papillomavirus (HPV) infections were tested for the presence of HPV 11 DNA by the nucleic acid sandwich hybridization method, which allows testing the specimens in a crude form. Part of each specimen was processed through phenol extraction and DNA purification to a dot blot hybridization assay. The dot blots were serially hybridized with HPV 6, 11, 16, and 18 probes as well as with an Alu-repeat probe to estimate the number of cells in the specimen. In PAP smears, HPV-infection was suspected in 63 patients whereas in 37 patients the smear was negative. In the first group, the dot blot assay revealed three cases of HPV 11, two of HPV 16, and one of HPV 18 infection. In the second group with normal PAP smear, one additional HPV 18 infection was found. The sandwich hybridization assay detected 5 HPV 11 infections, including the three mentioned above. All HPV DNA-positive samples contained at least 1.6 X 10(6) cells. Since we considered this a prerequisite for successful diagnosis, only 25 specimens in the first group and 15 in the second were adequate specimens. Thus the HPV-DNA detection rate was 32% (8/25) in the first group and 1/15 in the second. This study demonstrates that sandwich hybridization, detecting 1-3 X 10(5) HPV 11 molecules is a reliable diagnostic method. Cervical scrape is a valuable alternative to punch biopsy, but the number of cells collected is critical for the outcome of the assay. PMID- 3023543 TI - Detection of human rhinoviruses and their molecular relationship using cDNA probes. AB - We describe here a cDNA:RNA hybridization system for the study of human rhinoviruses. We have constructed an M13 probe from the 5' end of the genome of rhinovirus 14 (HRV-14) and used this to detect directly viral RNA. Of the 56 human rhinoviruses so far investigated 54 or 96.4% gave clearly positive hybridization signals. However, the strength of this signal depended very much on the molecular relationship of these viruses. Thus, HRV-3, 4, 17, 72, and, to a slightly lesser extent, HRV-2, 6, 9, 13, 19, 31, 42, 49, 64, and 69 appear to be closely related to HRV-14 whereas HRV-5, 7, 8, 16, 32, 40, 45, 55, 56, 63, 80, 82, and 85 appear to be relatively divergent. Further, evidence is provided in this study that indicates that it would be feasible to use cDNA probes to detect human rhinoviruses in nasal washings. However, the sensitivity of detection was clearly affected by both the inclusion of inhibitors of endogenous RNase activity in the RNA extraction mixture and also in the method of extracting the viral RNA. From reconstruction experiments in nasal washings and under optimal conditions, we can detect virus at 10(2.8) TCID50/ml. PMID- 3023544 TI - Endogenous benzodiazepine receptor agonist in human and mammalian plasma. AB - Using ultra-filtration steps and HPLC-separation, a low molecular weight ligand of the benzodiazepine receptor was isolated from plasma of various mammalian species including man. The endogenous ligand acts on benzodiazepine receptors agonistically and apparently has a receptor affinity similar to Diazepam. The ligand is not identical with Diazepam as indicated by HPLC and UV-spectroscopy. PMID- 3023545 TI - Noradrenaline-stimulated inositol phospholipid breakdown as a measure of alpha 1 adrenoceptor function in rat hippocampal miniprisms after repeated antidepressant treatment. AB - Noradrenaline-stimulated inositol phospholipid (PI) breakdown in rat hippocampal miniprisms was used as a measure of alpha 1-adrenoceptor function after repeated antidepressant treatment. After 24-29 days of oral treatment with either desipramine, mianserin, maprotiline or zimeldine (all at doses of 10 mg/kg b.i.d.), there was no significant difference in the degree of stimulation of hippocampal PI breakdown by 0.4, 2, 10 or 100 microM noradrenaline. It is concluded that there is no supersensitivity of hippocampal alpha 1-adrenoceptors coupled to PI breakdown after repeated antidepressant treatment under the conditions used. PMID- 3023547 TI - Effects of capsaicin on central monoaminergic mechanisms in the rat. AB - The acute and chronic effects of capsaicin (s.c.) on the monoamines in the preoptic region + hypothalamus (RPO-H), spinal cord, substantia nigra and striatum were studied. Levels of DOPA, DA, DOPAC, HVA, 3-MT, NA, Trp, 5-HTP, 5-HT and 5-HIAA were determined by means of liquid chromatography (HPLC-EC). In response to acute capsaicin treatment, the levels of DA, DOPAC and DA synthesis rate (DOPA formation) were increased in a dose-dependent manner in the RPO-H and spinal cord. The disappearance rate of NA was accelerated in both regions. In substantia nigra, increased DOPAC levels were found whereas the levels of 3-MT were decreased in striatum after acute capsaicin treatment. Only minor changes on the levels of 5-HT and 5-HIAA in the regions studied were noted. Neonatal or adult capsaicin treatment failed to affect the levels of NA, DA and 5-HT (measured two months or five weeks after injection, respectively) in the regions studied. A capsaicin injection to rats pretreated with the drug as adults did not affect either the monoamines in the RPO-H and spinal cord or the body temperature. In contrast, in rats pretreated with capsaicin as neonates, a second injection of the drug to adult animals elicited hypothermia and changes in monoamines similar to those observed in naive animals. PMID- 3023546 TI - Adrenoceptor modulated flow through the rabbit ampulloisthmic region studied in vivo and in vitro. AB - In adult rabbit does ovulation was induced by human choriongonadotropin (hCG) 48 hours before experiments. At laparotomy the oviducts were cannulated from the ovarian and uterine ends. In vivo as well as in vitro the patency of the isthmus was studied with low viscous fluid perfusion of the ampulloisthmic region in antegrade direction. Intraluminally applied norepinephrine (NE) or phenylephrine (PhE) caused dual changes in transisthmic flow; administration of a low dose increased the flow, while high doses decreased the flow in vivo. In vitro, application of PhE only induced a dose-dependent reduction of flow. The PhE induced reduction of flow was prevented by pretreatment with phenoxybenzamine in vivo and in vitro, suggesting activation of an alpha-adrenoceptor mechanism. Intraluminal application of terbutaline (T) caused a dose-dependent increase of flow, which was most prominent in vivo. Such an increase of flow was prevented by blockade of beta-adrenoceptors with propranolol or by selective blockade of beta 2-adrenoceptors with IPS 399 both in vivo and in vitro, indicating activation of a beta 2-adrenoceptor mechanism. The biochemical and hormonal changes 48 hours after ovulation imply a role for the sympathetic transmitter NE in causing a contractile state of the ampulloisthmic region ("tube locking") for retention of ova prior to nidation in the uterine cavity. The isthmus would then hypothetically act as a sympathetically innervated smooth muscle sphincter. The present results demonstrate a constrictory response of this region to high-dose stimulation of alpha-adrenoceptors in support of such a hypothesis. However, it must be noted that this region also possesses a population of beta-adrenoceptors at this time interval, which may interfere with a constrictor mechanism via circulating epinephrine. PMID- 3023548 TI - Purification and characterization of tribulin, and endogenous inhibitor of monoamine oxidase and of benzodiazepine receptor binding. AB - A low molecular weight fraction of human urine (less than 500 daltons) which both inhibits monoamine oxidase and benzodiazepine binding to central and peripheral receptors has been purified by ethyl acetate extractions, HPLC and thin layer chromatography. This material extracted equally well at acid and basic pH and was insoluble in heptane. It competitively inhibited binding of 3H-clonazepam, a central benzodiazepine receptor agonist and, in addition, displaced 3H-Ro 5-4864, a specific peripheral benzodiazepine receptor ligand, from its binding sites. It showed no GABA shift with the benzodiazepine receptor antagonist, Ro-15 1788. MAO A and B were inhibited approximately equipotently and the material competitively inhibited tyramine oxidation by rat liver. It was stable on boiling and is unlikely to be a peptide. PMID- 3023549 TI - Forskolin enhancement of acetylcholine-evoked cyclic AMP formation and catecholamine release in perfused dog adrenals. AB - Unstimulated efflux of cyclic AMP from perfused dog adrenal glands was not altered by 0.1 microM of forskolin and was slightly increased by 0.3 and 1.0 microM of forskolin. ACh stimulated efflux of cyclic AMP which preceded CA release and the efflux was dose-dependently enhanced by forskolin. Forskolin did not affect the spontaneous CA release but enhanced ACh-evoked catecholamine (CA) release. There was a close correlation between the dose relationship of forskolin enhancement of stimulated-cyclic AMP efflux and that of evoked-CA release. ACh evoked CA release in the presence of forskolin was further potentiated by R020 1724, a phosphodiesterase inhibitor. CA release evoked by excess K+, or by caffeine in the presence or absence of external Ca2+ was also potentiated by forskolin. These results suggests that cyclic AMP generation may increase in response to stimulation of adrenal chromaffin cells and that the resulting increase of the nucleotide may function as a facilitating modulator of CA release. PMID- 3023550 TI - Hormonal modulation of central dopaminergic transmission. AB - Central dopaminergic transmission is subjected to influences of various hormones of hypophyseal and extra-hypophyseal origin. Ablation of hypophysis prevents the supersensitivity of central dopamine receptors induced by chronic treatment with the dopamine receptor antagonist, haloperidol. Furthermore, hypophysectomized rats show a potentiation of hypomotility induced by low doses of the dopamine receptor agonist, apomorphine. Among the hypophyseal hormones, prolactin (PRL) seems to exert a modulatory activity on central dopamine transmission. Exogenous administration of PRL increases the density of dopamine receptors in intact and hypophysectomized rats. Furthermore, hyperprolactinaemic rats show alterations of the concentration and turnover of dopamine in various brain areas, and a potentiation of apomorphine-induced stereotypies. Endorphins related to gamma endorphin also modulate central dopamine transmission. In particular, des enkephalin-gamma-endorphin seems to interfere specifically with apomorphine sensitive dopamine receptors. The administration of this endorphin is followed by a potentiation of hypomotility induced by low doses of apomorphine, and restores the sensitivity of hypophysectomized rats to these doses. The administration of estrogens also influences the sensitivity of central dopamine receptors. Depending on the doses, these hormones can potentiate or inhibit the dopaminergic neurotransmission in the brain. PMID- 3023552 TI - Immuno-gold silver staining in the diagnosis of herpes encephalitis. PMID- 3023551 TI - Visual evoked potentials in parkinsonism and dopamine blockade reveal a stimulus dependent dopamine function in humans. AB - VEPs were recorded with three different spatial frequencies of stimulation in patients affected by idiopathic Parkinsonism and by Parkinsonian syndromes. The detection of VEP abnormalities in Parkinson's disease was dependent on the spatial frequency of the visual stimulus (a vertical square wave grating). The VEP latency was normal in Parkinsonian syndrome patients (except in one patient affected by familial Parkinsonism). Dopamine precursor therapy differently reduced the VEP latency, depending on the spatial frequency of the visual stimulus. These findings suggest that the dopaminergic mechanism involved in the generation of VEP delays is sensitive to stimulus spatial frequency. The study of VEPs before and after the administration of haloperidol confirmed this hypothesis. VEP latency did not correlate with the major clinical symptoms of Parkinson's disease and could not predict the results of chronic dopaminergic therapy. PMID- 3023554 TI - Acute renal effects of captopril in patients with congestive heart failure. AB - The effect of acute administration of the angiotensin-converting enzyme inhibitor captopril on arterial pressure, glomerular filtration rate, and renal plasma flow was assessed in 16 patients with severe congestive heart failure. Following administration of captopril, mean arterial pressure (MAP) fell in all cases, whereas effective renal plasma flow increased from 27% to 88% in 10 patients, remained unchanged in 4, and decreased by 60% and 93% in 2 patients in whom MAP fell to 54 and 47 mmHg, respectively. Effective renal plasma flow and glomerular filtration rate values achieved after captopril were both positively correlated with postcaptopril MAP. The results of this study suggest that the renin angiotensin system plays a major role in the regulation of MAP and the renal vasoconstriction associated with severe congestive heart failure. However, angiotensin blockade may induce a deterioration in renal function in patients in whom arterial pressure falls to markedly low values, thus suggesting an influence in angiotensin in renal autoregulation in these patients. PMID- 3023553 TI - Twenty-four hour changes in active and inactive renin after various oral doses of the converting enzyme inhibitor ramipril (HOE498) in normal man. AB - Different oral doses (5, 20, 50 mg) of the new orally active nonsulfhydryl converting enzyme inhibitor, ramipril (HOE498), were given to 12 normotensive healthy males, and the pattern of changes in plasma active and inactive renin concentration was evaluated. Active and inactive renin increased after ramipril, and the magnitude of the response was clearly dose related. Active renin rose markedly by 4 hours and tended to decrease thereafter, although remaining higher than basal at 24 hours. In contrast, inactive renin rose more slowly, and the increase was sustained throughout the 24-hour period. The pattern of these changes is consistent with the hypothesis that circulating inactive renin is a biosynthetic precursor of the active form. PMID- 3023555 TI - Enalaprilat: an intravenous substitute for oral enalapril therapy. Humoral and pharmacokinetic effects. AB - Thirteen subjects with essential hypertension controlled on oral enalapril (20 mg/day) therapy were entered into a protocol to assess serially 24-hour blood pressure, the renin-angiotensin-aldosterone system, angiotensin-converting enzyme activity, and plasma and urine enalaprilat drug levels, following both chronic oral administration of enalapril and its replacement with intravenous enalaprilat. Results indicate that systolic and diastolic blood pressures remain well controlled following cessation of oral enalapril and replacement with intravenous enalaprilat. Enalaprilat drug levels, following oral enalapril and intravenous enalaprilat, remained above the therapeutic levels required for angiotensin-converting enzyme inhibition. However, therapeutic enalaprilat levels can probably be achieved with one fourth of the total cumulative dose of enalapril, administered as enalaprilat at 6-hour intervals. Intravenous enalaprilat stimulated plasma renin activity and decreased immunoreactive plasma angiotensin II and plasma aldosterone concentrations. However, immunoreactive plasma angiotensin II concentrations were not suppressed below pretreatment values, suggesting that chronic enalapril/acute enalaprilat therapy controls blood pressure through a nonangiotensin-mediated antihypertension mechanism. PMID- 3023556 TI - A randomized study comparing a high and a standard dose of cisplatin in combination with etoposide in the treatment of advanced non-small-cell lung carcinoma. AB - We conducted a randomized trial comparing a high (120 mg/m2 day 1) v a standard (60 mg/m2 day 1) dose of cisplatin in combination with etoposide (120 mg/m2 days 3, 5, and 7) in advanced non-small-cell lung carcinoma (NSCLC). Two hundred forty one patients were evaluable for survival and 207 for response. We obtained a 25% objective response rate in the standard-dose arm and 29% in the high-dose arm; this difference was not statistically significant. There was no significant improvement in the overall survival or survival of responders with the high-dose regimen. However, toxicity (mainly myelosuppression) was significantly increased in the patients receiving the higher dose of cisplatin. An analysis of prognostic factors showed that disease progression, loss of body weight, performance status, and prior therapy were predictive parameters of survival. PMID- 3023558 TI - Steroid receptor status difference in recurrent intracranial meningioma and breast cancer in the same patient. AB - The authors report a case of multiple recurrent intracranial meningioma associated with breast cancer in a menopausal woman. High affinity estrogen (ER) and progestin receptors (PR) were assayed independently in the meningioma and the tumor. ER were found to be very low in the meningioma and high in the breast tumor. On the contrary PR were found to be high in the meningioma and could be not detected in the breast tumor. This unique case suggests that meningioma and breast cancer are not under the same type of hormonal influence. PMID- 3023557 TI - High-dose cisplatin in hypertonic saline: reduced toxicity of a modified dose schedule and correlation with plasma pharmacokinetics. A Northern California Oncology Group Pilot Study in non-small-cell lung cancer. AB - Although increased efficacy has been described with a five-day schedule of high dose cisplatin (CDDP) in hypertonic saline, severe myelosuppression and cumulative neurotoxicity have limited the usefulness of this therapy. In order to evaluate a possible dose-response relationship in non-small-cell lung cancer (NSCLC), 17 patients with metastatic disease were treated with a modified dose schedule delivering the same total dose (200 mg/m2) in a divided day 1 and 8 schedule. During a pilot study, a total of 47 cycles of therapy were administered, with a median of three cycles per patient and a median total cumulative dose of 600 mg/m2. Nine of 17 patients received at least 600 mg/m2. While nephrotoxicity was similar to previous reports of the five-day schedule, the incidence and severity of myelosuppression and peripheral neuropathy were markedly reduced. Using this modified schedule, severe myelosuppression did not occur. Clinically severe peripheral neuropathy developed in only one patient (6%). The overall response rate was 47% (eight of 17 patients). Plasma platinum pharmacokinetics during five cycles of the modified day 1 and 8 schedule were compared with pharmacokinetics of the five-day schedule. Accumulation of plasma ultrafiltrate platinum occurred in the five-day schedule, but not in the day 1 and 8 schedule. This difference in pharmacokinetics is one possible explanation for the reduced toxicity of this modified schedule. Although the degree of activity seen in this pilot study is encouraging, the efficacy of high-dose CDDP in NSCLC remains to be defined. In view of reduced myelosuppression and neurotoxicity, further trials with this modified schedule are indicated. PMID- 3023560 TI - Metastatic epidural osteosarcoma initially diagnosed as cisplatin neuropathy. AB - Unexpected early epidural spinal metastasis in a case of osteosarcoma occurred in a patient receiving treatment with cis-diamminedichloroplatinum-II (cisplatin). The initial neurologic symptomatology manifested as paresthesias in the feet which developed 2 months after initiation of treatment (cumulative dose of cisplatin 450 mg/M2) at which stage the primary tumor demonstrated a marked response. Concurrently two small pulmonary metastases appeared. Epidural metastasis in osteosarcoma is generally considered a late complication and is usually associated with disseminated disease. This communication draws attention to changes in the metastatic pattern which may occur with the administration of seemingly effective treatment and the potential for confusing the symptomatology of epidural spinal metastasis with cisplatin neuropathy. PMID- 3023559 TI - Diffuse leptomeningeal seeding from a malignant spinal cord astrocytoma in a child with neurofibromatosis. AB - A 5-year-old child typical clinical features of neurofibromatosis presented with a history of suspected basilar meningitis and CT findings of enlarged optic nerves and an expanding left cavernous sinus mass. CSF cytologies and meningeal biopsy were unremarkable. At craniotomy, a mass confluent with the left trigeminal nerve was resected which had histologic characteristics of a nerve sheath tumor but was GFAP (glial fibrillary acidic protein) stain positive. Postmortem examination, 1 month following surgical resection, demonstrated a clinically unsuspected primary thoracic spinal cord astrocytoma with dissemination throughout the subarachnoid space, invasion of the trigeminal nerve and encasement of other cranio-spinal nerves. This unusual case emphasizes the occurrence of leptomeningeal spread in a clinically silent spinal cord glioma and the diagnostic value of immunohistochemistry. PMID- 3023561 TI - Quantitative analysis of synaptic potentiation during kindling of the perforant path. AB - Synaptic transmission was studied during the development of kindling in the pathway from entorhinal cortex (EC) to dentate gyrus (DG) of unrestrained unanesthetized rats using chronic neurophysiological techniques. Extracellular field potentials were recorded from the DG in response to activation of the perforant pathway with 0.1-ms constant current square-wave pulses. The evoked field potentials consisted of a population EPSP (a reflection of excitatory synaptic activation) and a population spike (a measure of synchronous postsynaptic discharge of granule cells). Synaptic efficacy was quantitated in this pathway by measurement of the population EPSP slope and population spike amplitude across a range of stimulus intensities from threshold to maximal evoked response. Input-output relationships for population EPSP and population spike were determined at regular intervals during the course of kindling, corresponding to the stages of evoked behavioral seizures. Increases in the population EPSP and population spike were observed after a single kindling stimulus that evoked afterdischarge (AD) when behavioral seizures were minimal. Evaluation of the input-output relationships for the group of kindled animals at the various stages of evoked behavioral seizure activity revealed that increases in the population EPSP continued to slowly evolve with repeated stimulations but that increases in the population spike were maximal after one or at most a few stimulations that evoked AD. The increases in both population EPSP and population spike persisted for the duration of the recording, i.e., through induction of generalized motor convulsions. To evaluate the translation of synaptic activation into cell discharge during kindling, we made use of the population spike/population EPSP ratio across a range of stimulus intensities. The spike/EPSP ratios revealed a dissociation of the population spike and population EPSP early in the course of kindling during class 1 seizures. Specifically, after induction of an AD, an extracellular population EPSP of a given size evoked a larger population spike than an EPSP of comparable size before the induction of an AD by kindling stimulation. The development of generalized motor convulsions (class 5 seizures) was associated with a reduction in the spike/EPSP ratio. The mechanism of this reduction in spike/EPSP ratio is uncertain, but since synaptic activation (as reflected by population EPSP) did not decline during class 5 seizures, the reduction in the spike/EPSP ratio could be consistent with increased inhibition after generalized motor convulsions, or could reflect a decrease in granule cell excitability.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3023562 TI - The effect of axotomy on posttetanic potentiation of group Ia synapses in the cat. AB - Posttetanic potentiation (PTP) of composite Ia excitatory postsynaptic potentials (EPSPs) has been studied in normal cat alpha-motoneurons and in motoneurons axotomized 2-3 wk earlier by ventral root section. The maximal amount of PTP of EPSP amplitude (expressed relative to unpotentiated amplitude) was considerably less in the axotomized population compared with the normal population. The decrease in PTP provoked by axotomy occurs in association with a postaxotomy increase of input resistance, the net effect being that PTP in axotomized cells was much the same as that observed by others in normal motoneurons possessing similarly high input resistance. In agreement with previous results, EPSP peak amplitudes were decreased after axotomy. This decrease seemed to be largely related to an absence of the largest EPSPs, since otherwise the EPSP distributions of normal and axotomized motoneurons showed considerable overlap. It is suggested that the observed decrease in PTP after axotomy is related to a change in synaptic release properties and not secondary to changes in the electrical properties of motoneurons. A previous analysis has suggested that axotomy causes an alteration of the distribution of passive electrical properties among motoneurons such that axotomized cells resemble normal high-resistance motoneurons. The present results suggest that axotomy may affect the distribution of Ia synaptic release properties in a similar manner, since PTP in axotomized motoneurons resembles that observed in normal high-resistance motoneurons. PMID- 3023563 TI - Radiation therapy for neoplasms of the brain. AB - The effectiveness and complications of radiation therapy for brain neoplasms are reviewed. While the available data suggest a favorable influence and outcome, randomized studies are needed to further optimize radiation therapy techniques and to integrate new therapeutic modalities. PMID- 3023565 TI - Renal handling of technetium-99m DMSA: further evidence for glomerular filtration and proximal tubular reabsorption. PMID- 3023564 TI - Images of the brain: past as prologue. AB - The invention of the Anger scintillation camera and the development of 99mTc tracers brought about a tenfold increase in nuclear brain scanning between 1963 and 1973, an increase that plateaued with the introduction of x-ray computed tomography. A second growth curve began in 1976 at which time there were four PET centers in the United States, a number that grew to 60 worldwide over the next decade. PET, SPECT, MRI, and MRS are leading us into a new era of in vivo brain chemistry, based on regional bioenergetics and neurotransmission. The immediate impact is in epilepsy, stroke, brain tumors and the dementias, with psychiatric diseases becoming a major focus of research. Receptivity has become a biochemical as well as a psychological approach to mental functions. The finding of elevated D2 dopamine receptors in schizophrenia in living patients may be the forerunner of a new biochemical approach to psychiatry. PMID- 3023566 TI - Marketing weekend college for women nursing students. PMID- 3023567 TI - Top Ranked Schools of Nursing: the role of the dean. AB - The Top Ranked School of Nursing Study (TRSN) was a nationwide investigation for the purpose of delineating a composite of elements that constitute a top ranked school. The role of the dean was identified as the most frequent, if not the most significant, element of administration. The influence and scope of the administration in a TRSN were described as were the ways in which these endeavors are pursued and supported. The findings will be of benefit to anyone interested in promoting and improving nursing scholarship. PMID- 3023568 TI - An investigation of decision theory: what are the effects of teaching cue recognition? AB - As currently formulated, decision theory assumes that care givers learn cue recognition primarily by experience. However, it seems probable that the ability to receive and recognize cues can be taught. To investigate cue recognition abilities of junior and senior baccalaureate nursing students, five computer simulations were developed. The specific question investigated was: What are the effects upon students' cue recognition and clinical decision-making abilities of teaching cue recognition? Following teaching of cue recognition and decision making, a statistically significant difference was noted in both junior and senior students in relation to accuracy of cue recognition and clinical decision making. The conclusions were that cue recognition and cue sorting can be taught. Also, linking or grouping of related cues can be taught. In this study, the teaching of cue recognition and linking of cues improved the accuracy of clinical decisions made by students who were presented computer simulations of a variety of clinical situations. PMID- 3023569 TI - Belief systems which influence research in nursing: implications for preparing future investigators. AB - In the view of the authors, students enrolled in their first research course should become aware of the different belief systems held by investigators about research. All nurse researchers, including faculty who teach undergraduate research courses, have either consciously or unconsciously adopted a belief system. That philosophy determines what the educator sees as appropriate nursing theory, research questions, research designs and methods for nursing inquiry. Students may be confused by the apparent discrepancies in the content and methods of teaching a research course between two and among faculty who possess divergent philosophical views. They should be aware of some of the important belief systems which can influence theory development and research in nursing in order to use nursing research in clinical practice. Two dichotomous belief systems influencing approaches to the study of the phenomenon of pain are discussed: Empiricism and Phenomenology. Teaching strategies which help students know and appreciate different philosophical orientations are proposed. Finally, a rationale for including this content within an undergraduate research course will be given. PMID- 3023570 TI - Using humor in evaluating student performance. AB - This article describes an investigation of the effects of deliberate teacher humor as an evaluative strategy on anxiety and performance levels of beginning associate degree nursing students in a stress-producing simulated laboratory setting. While quantitative findings did not show that deliberate teacher humor reduced anxiety, and only partially demonstrated that humor improved performance, qualitative findings indicate that humor influenced both anxiety and performance. The authors suggest that this additional evaluative strategy may enhance the nurse educator's effectiveness in carrying out stress-producing evaluations. PMID- 3023572 TI - Use of behavioral observation to augment quantitative data when studying clinical teaching. AB - Observation of behavior to study clinical teaching provides advantages over the use of questionnaires alone. The great advantage is the richness of the data. Problems encountered in the process, e.g., gaining access to subjects in multiple institutions, selecting non-volunteer subjects, obtaining informed consent in a manner which will not bias the data, becoming unobtrusive and non-threatening to subjects, coping with the unpredictability of the clinical setting, and staying in the role of researcher in spite of temptations to enact other roles within the setting, are all foreseeable and to some degree preventable. Being aware of the potential obstacles permits the investigator to plan for them and to be less emotionally aroused as each new problem arises. Other nurse researchers are encouraged to utilize these techniques; the experience can be not only valuable in terms of realistic data obtained, but also can be an extremely rewarding experience. PMID- 3023571 TI - Nursing fundamentals texts: where's the ethics? AB - A primary source of information on professional ethics for nursing students is the nursing fundamentals text used in the initial courses. This study systematically evaluated nursing fundamentals texts for their coverage of ethically relevant content. Forty-two nursing fundamentals texts published from 1965 to 1985 were evaluated for: inclusion of a professional code of ethics; interpretive statements, discussion and examples; and guidelines for ethical decision making. Forty-five percent of the texts contained no content on ethics, with the remainder varying in their depth of coverage. Analysis revealed a moderate correlation (r = 0.59) between the year of publication and the average number of pages accorded content on ethics in that year. The results reflect a gradual, although irregular, trend toward inclusion of greater amounts of content on ethics. Recommendations were proposed that would facilitate the inclusion of content on ethics into nursing curricula. These included: greater communication between faculty and text publishers regarding the importance of content on ethics, inclusion of ethics as a curricular thread, small-group workshops to increase faculty sensitivity to the need for preparation in ethical decision making, and an elective course in nursing ethics. PMID- 3023573 TI - Textbook selection: perils and pointers. PMID- 3023575 TI - Playing for proficiency: a new approach to motivation and psychomotor learning. PMID- 3023574 TI - Professional role synthesis for nursing faculty: a redefinition of faculty practice. AB - Nursing faculty role is the synthesis of theory, education, practice, and research within a school of nursing environment. This model of nursing faculty role presents a synthesis and implementation of professional nurse role that nursing faculty might use to systematically meet the goals of developing nursing theory based on clinical practice, redefining and evaluating this theory through research, and educating students. PMID- 3023576 TI - Doctoral programs in nursing: an examination of curriculum similarities and differences. AB - In this exploratory study, eight doctoral programs in nursing (four PhD and four DNS/DSN/DNSc) were surveyed to examine curricular similarities and differences between research-oriented and professional-oriented programs. Content analysis was utilized to examine manifest content of 101 units of analysis derived from questionnaire items. These items reflected environmental input, curricular design, and outcome variables. Findings suggested that more curricular similarities than differences existed between the two program types. Curricular design variables reflected the most differences in the areas of program purposes and objectives. Several areas were identified for additional research in each variable category. PMID- 3023577 TI - A comparative study of adult developmental patterns of RN and generic students in a baccalaureate nursing program. AB - The current influx of RN students into baccalaureate nursing programs has led to many curriculum changes that are based on the unverified assumption that the RN students are "different." This study examined the differences and similarities among RN and generic nursing students using a conceptual framework designed by Weathersby (1977) which incorporated into a single framework Levinson's life stage, Loevinger's ego development, and Kolb's learning styles theories in order to trace the adult development patterns of generic and RN students. Three instruments were used: The Washington University Sentence Completion Test, the Kolb Learning Style Inventory, and Tarule's Educational Experience Inventory. The sample consisted of 49 RNs and 30 generic students enrolled at a large southern state university. The results of the analyses revealed significant difference between RN and generic students on the variables life stages and ego development but not on learning styles. These results substantiated the premise that RN and generic students differ significantly in their stages of adult development, and consequently affect the students' perception and expectations of the educational process. PMID- 3023578 TI - Performance predictors for nursing courses and NCLEX-RN. PMID- 3023579 TI - The impact of expectations on faculty job satisfaction. AB - Although job satisfaction of nurses within the clinical area has been heavily studied, the satisfaction of nurse faculty members has seldom been examined. This study looks at the relationship of the role of the department chairperson to the satisfaction of faculty. The sample was composed of 163 faculty in eight state supported baccalaureate/masters programs. Age, length of service, size of the department, and the size of the program served as control variables. Two self designed instruments (CPQ-E and CPQ-P) based on Need Fulfillment Theory created an Expectation Discrepancy Score for each faculty member. Job satisfaction was measured by the JDI. The relationship between the Expectation Discrepancy Score and job satisfaction was found to be significant (Fg = 15.786, p less than 05). Several recommendations were suggested. PMID- 3023580 TI - All real living is meeting: the task of international education in a nursing curriculum. AB - Our experience suggests two significant benefits of shaping an international education focus in a nursing curriculum. First, a genuine understanding and appreciation of a foreign health care system is strengthened by identification of and confrontation with the socio-political context in which it is embedded. The second, and perhaps most fundamental benefit, is not so easy to verbalize. This involves the participant's personal confrontation with another culture. What is external, the experience of a foreign culture, translates into an encounter with the foreign territory of one's own thinking. The participant is challenged to overcome the traditional limitations of professional discipline, experience and vision and to move toward notions of world community and human solidarity. It seems to us that the development of concepts, resources and methods to move American nursing in the direction of greater solidarity with the world health care community is a legitimate task for nursing education. PMID- 3023581 TI - Pairing students with the well elderly: a unique relationship for learning. PMID- 3023582 TI - Group decision making: teaching the process--an introductory Guided Design project. AB - The Guided Design experience gives students excellent grounding in the decision making process and knowledge of a decision-making language that will serve them well in all group decision-making contexts. The detailed component analysis, constraint identifications and the who, what, when, where, why and how questions applied to information gathering and analysis are simple terms, easily understood. The steps are applicable to problems needing a cause identification, solve process, and for anticipating potential problems. The process can be taught early in the curriculum and utilized throughout with special re-emphasis in leadership and management classes. The strength of the described unit lies in the provision of an introductory, nonthreatening classroom opportunity for practice in the use of decision-making language and an experience with the process using familiar content. The students experience the complexity of group decision making and the interesting variety of solutions possible for each complex problem. Like the Guided Design process, the projects used are improved with each successive experience. Student and colleague feedback provides the challenge. PMID- 3023583 TI - Mammary tumors in feral mice lacking MuMTV DNA. AB - Two mammary tumors developed in feral mice treated with dimethylbenzanthracene. The tumors, livers, and spleens of these animals contained no MuMTV proviral DNA. Neither tumor had amplified or rearranged int-1 or int-2 loci. This demonstrates that mammary tumorigenesis can occur in mice in the absence of endogenous and exogenous MuMTV. PMID- 3023584 TI - Release of low infectivity vesicular stomatitis virus particles from tunicamycin treated cells. AB - The effect of tunicamycin (TM) treatment on the production, glycosylation, morphology and infectivity of vesicular stomatitis virus (VSV) released from mouse (LB), monkey (Cos-1 and VERO), bovine (MDBK), hamster (BHK) and human (HeLa, U and GM2504) cells was investigated. The yield of VSV particles released from TM treated cells, as measured by total viral proteins, was inhibited only 2 10 fold while the infectivity of these particles was greatly reduced (10-1000 fold). The morphology of VSV particles formed in the presence of TM appeared similar to that of VSV particles released from untreated cells as demonstrated by electronmicroscopy. Analysis of intracellular and extracellular virus specific proteins by SDS polyacrylamide gel electrophoresis revealed that TM blocks the glycosylation of VSV glycoprotein G in all cells tested. These results suggest that inhibition of glycosylation of VSV G protein could affect the biological infectivity of the VSV particle released from the treated cells. PMID- 3023585 TI - Detection of dihydroorotate dehydrogenase activity as a marker of de novo nucleic acid synthesis in germinal centers of spleens following antigen administration. AB - A histochemical technique, developed for the localization of DHO-D activity in tissue, has been utilized to study the distribution of this activity in rabbit spleen. This enzyme is involved in the de novo synthesis of pyrimidines of nucleic acids via orotic acid as an intermediate. The enzyme was found to be most active at the periphery of follicles in the resting spleen. Following antigenic challenge, enzyme activity became prominent for several days in macrophages within germinal centers, but not in the intensely pyroninophilic cells which are the dominant cell type of these structures. Antigenicity of the injected compound appeared to be necessary for the production of this effect. The pattern of localization of DHO-D is similar to the pattern of distribution of antigen within splenic tissue. PMID- 3023586 TI - Sympathetic nervous system development in hypertensive rats. AB - The functional development of cardiac and adrenal medullary responses to reflex activation of the sympathetic nervous system was studied in preweanling spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive rat pups. Pups from the two strains received injections of insulin or saline at 2, 4, 8, 12 or 16 days of age and were killed 3 h later. The induction of ornithine decarboxylase (ODC) in the heart and the depletion of epinephrine (EPI) from the adrenal medulla served as tissue markers of functional sympathetic neurotransmission. Compared with WKY pups, SHR pups showed a greater induction of ODC activity in the heart at 2, 4, and 8 days of age. There were no differences between SHR and WKY pups in the adrenal medullary response to insulin-induced hypoglycaemia. These altered patterns of sympathetic-target tissue development in early life may contribute to the higher arterial pressures maintained by SHR throughout life. PMID- 3023587 TI - Alpha 1-adrenoceptor blockade and altered renal alpha 2-adrenoceptors: a model(?) for the study of genetically altered alpha 2-adrenoceptors in hypertensive rats. AB - Renal nerve stimulation (RNS)-induced sodium retention is normally mediated by alpha 1-adrenoceptors. Clinically, chronic prazosin therapy increases rather than decreases sodium retention. In preliminary studies, chronic prazosin (3 days, 3 mg/kg per day) increased renal alpha 2-adrenoceptor density (mean +/- s.e.m., 153 +/- 14 versus 209 +/- 11 fmol/mg protein) in normal rats. We therefore postulated that following chronic prazosin treatment, alpha 2-adrenoceptors may mediate RNS induced sodium retention. The effect of chronic prazosin pretreatment on the alpha-adrenoceptor subtype mediating RNS-induced antinatriuresis was studied in the perfused rat kidney. In control rats, subpressor levels of RNS (approximately 1 Hz, 10 V, 1 ms) decreased sodium and water excretion. Prazosin 28 nmol/l (alpha 1-blockade) reversed this effect of RNS. Yohimbine 300 nmol/l (alpha 2-blockade) had no effect. In rats treated with prazosin, RNS again decreased sodium and water excretion. In these rats, yohimbine as well as prazosin was necessary to reverse the effects of RNS. Thus, following prazosin pretreatment postjunctional alpha 2- as well as alpha 1-adrenoceptors mediated RNS-induced antinatriuresis. A similar function of the elevated renal alpha 2-adrenoceptors in genetic hypertension in rats could possibly explain the mechanism by which excess sodium retention occurs and thereby the elevation of blood pressure. PMID- 3023589 TI - Dietary prevention of stroke and its mechanisms in stroke-prone spontaneously hypertensive rats--preventive effect of dietary fibre and palmitoleic acid. AB - Previously it was reported that dietary protein, some amino acids and potassium are effective in preventing stroke in stroke-prone spontaneously hypertensive rats (SHRSP). The present study revealed that other dietary factors could also prevent cerebral lesions, and the mechanism of this effect was studied. In SHRSP given 1% NaCl in their drinking water, a diet containing 10% active fibre (powdered brown seaweed converted to K+ form) significantly lowered blood pressure (BP) and markedly reduced the incidence of stroke (0 versus 100% in controls on the 30th day of experiment). Since the faecal to urinary sodium (Na) excretion ratio was increased in this group and a similar increase in faecal Na content was noted in SHRSP given a diet containing 10% alginic acid, the inhibition of intestinal Na absorption by alginic acid in the seaweed fibre was considered to be a possible preventive mechanism. Among SHRSP given various fatty acids, a diet containing 1% palmitoleic acid (POA) significantly improved the survival rate, with concomitant reduction in the incidence of stroke in spite of their excess NaCl intake through 1% NaCl water for drinking. Since neither BP nor urinary Na excretion was changed by POA which had high affinity for the vascular wall, the preventive effect was ascribed to the possible direct metabolic improvement of vascular smooth muscle cells. PMID- 3023588 TI - Atrial natriuretic peptide (6-33) binding sites: decreased number and affinity in the subfornical organ of spontaneously hypertensive rats. AB - Binding sites for rat atrial natriuretic peptide (6-33) (rANP) were identified, localized and quantified in the subfornical organ and the choroid plexus of young and adult spontaneously (genetic) hypertensive rats (SHR) and normotensive, age matched Wistar-Kyoto (WKY) controls. Our methods allowed the study of binding kinetics in discrete brain areas from single rats. We used newly developed autoradiographic techniques coupled to image analysis, microdensitometry and comparison with 125I-standards. Brain sections were first incubated with 125I rANP to quantitate rANP sites. The number of rANP binding sites was much lower in both the subfornical organ and the choroid plexus of young and adult SHR when compared with normotensive controls. Analysis of binding kinetics in adult SHR showed lower maximum binding capacity (Bmax) in both the subfornical organ and the choroid plexus, and lower binding affinity (Ka) in the subfornical organ only, when compared to WKY. The results indicate a central role of rANP in the development and maintenance of spontaneous (genetic) hypertension in the rat. PMID- 3023590 TI - Hypotensive effects of eicosapentaenoic acid (EPA) and its influence on eicosanoid metabolism in spontaneously hypertensive rats. AB - Oral administration of pure eicosapentaenoic acid (EPA) ethyl ester caused a reversible hypotensive effect in spontaneously hypertensive rats. Treatment with EPA also lowered the platelet response to ADP and inhibited the conversion of leucotrienes (LTs) A4 and LTB4 and other sulphidopeptide leucotrienes. Therefore, diet supplemented with EPA may lower blood pressure in hypertensive rats and affect cardiovascular functions which may be mediated through eicosanoid metabolism. PMID- 3023591 TI - Antihypertensive action and inhibition of tissue converting enzyme (CE) by three prodrug CE inhibitors, enalapril, ramipril and perindopril in stroke-prone spontaneously hypertensive rats. AB - The active diacids of the new converting enzyme (CE) inhibitors ramipril and perindopril proved to possess a similar inhibitory potency against rat plasma CE in vitro. Both diacids were more active than enalaprilic acid or captopril. In stroke-prone spontaneously hypertensive rats (SHRSP) chronic oral treatment for 2 weeks with enalapril (30 mg/kg per day), ramipril or perindopril (each 1 mg/kg normalized blood pressure. The CE inhibitor-induced changes in parameters of the plasma renin-angiotensin system [angiotensin I (ANG I), angiotensin II (ANG II), PRC and CE activity] followed the expected pattern, but were not quantitatively related to the antihypertensive action of the three CE inhibitors. Four weeks of oral equi-dose treatment with the three CE inhibitors (10 mg/kg per day) inhibited tissue CE activity in various organs including kidney, heart, vascular wall and brain. Ramipril and perindopril lowered blood pressure and tissue CE activity more potently than enalapril did. These results are consistent with the hypothesis that CE inhibition in tissue with subsequent local reduction of ANG II synthesis may contribute to the antihypertensive mechanisms of CE inhibitors. PMID- 3023592 TI - Systemic and regional pre- and post-junctional sympathoinhibitory effect of perindopril in spontaneously hypertensive rats. AB - The interaction between converting enzyme inhibition and sympathetic system activity at systemic and regional (renal, mesenteric, hindlimb) levels was investigated in adult pithed spontaneously hypertensive rats (SHR) using perindopril (5 mg/kg orally every day for 8 days) and the pulsed Doppler technique. Perindopril induced a decrease in systolic blood pressure and in regional vascular resistance which was abolished by binephrectomy. In addition, the drug had a sympathoinhibitory effect on the systemic vasopressor and the regional vasoconstrictor responses to cirazoline, especially UK 14304, an effect which was kidney-dependent (abolished by binephrectomy) and postjunctional, and spinal cord stimulation, an effect which was kidney-independent and most likely prejunctional in its mechanism. PMID- 3023593 TI - Effect of antihypertensive treatment on myogenic properties of brain arteries from the stroke-prone rat. AB - Using an in vitro pressurized technique, we measured the myogenic pressure range and degree of myogenic activity in posterior cerebral arteries from stroke-prone spontaneously hypertensive rats (SHRSP) and rats of the same strain maintained by antihypertensive therapy. Treatment lowered the upper myogenic pressure limit and increased the strength of the myogenic response to levels comparable with those previously found in normotensive (Wistar-Kyoto, WKY) rats, suggesting that blood pressure history is the primary determinant of myogenic properties in the cerebral circulation. PMID- 3023594 TI - Alterations of presynaptic and endothelial functions in SHR: effects of adenosine. AB - The effects of adenosine on stimulation-evoked 3H-efflux from perfused mesenteric arteries pretreated with 3H-norepinephrine and on norepinephrine-induced contractile responses of the thoracic aorta before and after mechanical removal of endothelial cells were examined in spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto rats (WKY). Adenosine inhibited the stimulation-evoked 3H-efflux; this inhibitory effect was smaller in young and adult SHR than age matched WKY. Mechanical removal of endothelial cells rendered the SHR aorta much less responsive to adenosine, while in the WKY aorta endothelial removal had no influence on the relaxation response to adenosine. These results support the hypothesis that pre-synaptic factors are contributory whereas endothelial factors are compensatory to hypertension in SHR. PMID- 3023596 TI - [Nasopharyngeal angiofibroma. The effect of superselective angiographic embolization (SSAE) at operation]. PMID- 3023595 TI - Metastatic breast cancer in the maxilla. PMID- 3023597 TI - Stromal changes in early invasive and non-invasive breast carcinoma: an ultrastructural study. AB - Six examples of histologically diagnosed, non-invasive breast carcinomas were studied by electron microscopy to elucidate the ultrastructural features for an accurate diagnosis of in situ carcinoma. The results obtained revealed two patterns of basal lamina/stromal cells relationship. One pattern showed intact basal lamina with associated periductal stromal cells consisting entirely of fibroblasts, the other pattern showed disruption of basal lamina by gaps and malignant cell protrusions with associated stromal cells consisting of both fibroblasts and myofibroblasts. As myofibroblasts are not a component of normal breast stroma but are known to be a prominent feature in the stroma of infiltrating breast carcinoma, the present observations suggest that myofibroblastic proliferation around in situ carcinoma represents an early sign of carcinomatous infiltration. Hence the definitive diagnosis of non-invasive carcinoma of the breast requires an intact basal lamina and a complete absence of a myofibroblastic reaction. PMID- 3023598 TI - Spironolactone-reversible rickets associated with 11 beta-hydroxysteroid dehydrogenase deficiency syndrome. AB - A 7-year-old girl had growth retardation, hypertension, and hypokalemic alkalosis. Baseline serum aldosterone concentration and plasma renin activity were low and unresponsive to sodium deprivation and to orthostatic changes. Baseline serum progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, and cortisol levels were normal and adequately responsive to ACTH stimulation. No steroid was found abnormally elevated. A diagnosis of 11 beta-hydroxysteroid dehydrogenase deficiency was established on the basis of elevated urinary tetrahydrocortisol plus allotetrahydrocortisol/tetrahydrocortisone ratio, determined by gas chromatography-mass spectrometry. Evaluation of bone mineral metabolism and parathyroid function, and skeletal radiographs, revealed the presence of rickets and secondary hyperparathyroidism. Treatment with spironolactone alone for 2 months corrected hypertension, hypokalemic alkalosis, and all laboratory and radiologic evidence of rickets and hyperparathyroidism, resulting in acceleration of growth rate. The response to spironolactone suggests that a hypermineralocorticoid state is responsible for the hypertensive syndrome and that rickets and hyperparathyroidism could be a consequence of excess mineralocorticoid activity. PMID- 3023599 TI - Hypophosphatemic rickets/osteomalacia in linear sebaceous nevus syndrome: a variant of tumor-induced osteomalacia. AB - A severe form of vitamin D-resistant rickets is associated with the linear sebaceous nevus syndrome. We investigated the pathophysiology underlying defective bone mineralization in two individuals and examined the effects of 1,25 dihydroxyvitamin D (1,25(OH)2D, calcitriol) therapy on the clinical and biochemical abnormalities. Both patients had fasting hypophosphatemia, markedly diminished TmP/GFR, and elevated alkaline phosphase activity in the presence of normocalcemia. Before treatment with calcitriol, serum 1,25(OH)2D concentrations were reduced but serum 25-hydroxyvitamin D (25(OH)D) concentrations were normal. Administration of calcitriol increased serum 1,25(OH)2D concentrations and led to an increase in TmP/GFR and serum phosphorus levels and to a decrease in alkaline phosphatase activity. However, the renal tubular maximum for reabsorption of inorganic phosphate, normalized according to glomerular filtration rate, and serum phosphorus levels remained abnormally low even in the patient who also received phosphate supplementation. Bone histomorphologic studies in the adult patient showed extreme osteomalacia, which partially improved with calcitriol. These data demonstrate that the putative skin lesion-derived factor results in both a renal tubular defect in phosphate reabsorption and in 1,25-(OH)2 D deficiency. The vitamin D-resistant rickets of linear sebaceous nevus syndrome is a variant of tumor-induced osteomalacia. PMID- 3023600 TI - Interactions of anticancer quinone drugs, aclacinomycin A, adriamycin, carbazilquinone, and mitomycin C, with NADPH-cytochrome P-450 reductase, xanthine oxidase and oxygen. AB - The properties of the interactions of anticancer quinone drugs, aclacinomycin A, adriamycin, carbazilquinone, and mitomycin C with nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase and xanthine oxidase under anaerobic and aerobic conditions were studied. Km values of NADPH cytochrome P-450 reductase for these drugs were in the range of 40-227 microM, and that of deflavo xanthine oxidase in the range of 39-over 200 microM. Under anaerobic conditions, when xanthine was used as an electron donor, deflavo xanthine oxidase catalyzed the reductive glycosidic cleavage reaction of anthracyclines and nicotinamide adenine dinucleotide was ineffective as an electron donor. In the electron spin resonance study, the formation of the semiquinone or free radical state of the quinone drugs in both enzyme systems were evidenced. A weak and symmetric signal was obtained from aclacinomycin A, and a symmetric signal from adriamycin was changed into an asymmetric and strong. The hyperfine structure was obtained from carbazilquinone in the oxidase system. In the studies of ultraviolet-visible spectra of the quinone drugs in the reductase system, the spectra of aclacinomycin A and adriamycin were changed to their 7-deoxylaglycones, and the formation of small amounts of the fully reduced form were observed after long incubations. The spectrum of carbazilquinone was changed to the hydroquinone form and mitomycin C was converted into mitosene analogues. Under aerobic conditions, superoxide radicals and hydrogen peroxide were effectively produced in the presence of anticancer quinone drugs in both enzyme systems. The superoxide-dependent hydroxy radical production, which was measured by ethylene production from methional, was observed in the presence of aclacinomycin A and adriamycin in the deflavo xanthine oxidase system. From these results, the possible reactions in the interactions of anticancer quinone drugs with these enzymes and oxygen are discussed. PMID- 3023601 TI - In vitro and in vivo inhibition of cysteine proteinases by EST, a new analog of E 64. AB - E-64 isolated from a culture of Aspergillus japonicus is a specific inhibitor of cysteine proteinases. E-64-c, a synthetic analog of E-64, was effective in model animals of muscular dystrophy only when it was given intraperitoneally and by means of osmotic minipump. It showed no effects due to its low absorbability from intestine when it was administered orally. EST, the ethyl ester of E-64-c, was expected to be readily absorbed through intestinal membrane, since it is more lipophilic than E-64-c. Both EST and E-64-c have a high specificity to cysteine proteinase similar to E-64 but E-64-c was 100 to 1000 times stronger than EST in in vitro cathepsin inhibition. However, EST was stronger than E-64-c in cathepsin inhibition when given orally. The cathepsin B&L activities (whole activities of cathepsins B and L) in the skeletal muscle, heart and liver of hamsters were strongly inhibited soon after oral administration of 100 mg/kg body weight of EST. The inhibition continued for at least 3 h and then disappeared gradually. E 64-c was found in plasma of hamster treated with EST, but unchanged EST was not found. These results suggested that EST was converted to E-64-c, a more active form, during the permeation through intestinal membrane. The conversion of EST to E-64-c was also indicated by the absorption experiment using in situ loop method. EST was thus shown to be useful as an oral drug and expected to be effective in therapeutic trials using model animals. PMID- 3023602 TI - Inhibitory characteristics of styrene & styrene oxide on Na+,K+ activated adenosine triphosphatase. AB - Styrene, an important ingredient of plastic, is reported to cause it neurotoxic effects through its important metabolite styrene oxide. Na+, K+ -ATPase (NKA), an important enzyme in the neurotransmission processes, is found to be inhibited by styrene (200 mg/kg) and styrene oxide (55 mg/Kg) by 15 and 45% respectively. Kinetic evaluations show that Km values, number of interaction sites and rate of reaction, with respect to K+ ions, decreased in styrene oxide treated NKA samples. However, high Km values of Na+ sites suggest for low affinity with respect to Na+ ions. Na+ and K+ ion activation, ATP hydrolysis and ouabain titration patterns indicate for a conformational change in NKA mainly due to styrene oxide. PMID- 3023603 TI - Initiation of swimming activity by trigger neurons in the leech subesophageal ganglion. II. Role of segmental swim-initiating interneurons. AB - Cell Tr1, a trigger neuron found in the subesophageal ganglion of the leech, Hirudo medicinalis, is part of a network of subesophageal ganglion neurons which control swimming activity, and makes apparently direct connections to swim initiating interneurons (SIIs; cells 204 and 205). In this study, we investigated the role of SIIs in swim initiation by cell Tr1. We also examined how brief Tr1 activity controls swim initiation at the levels of the SIIs and of the oscillator neurons. We found: In shortened nerve cord preparations consisting of the head ganglion (supra- and subesophageal ganglia) through segmental ganglia 11 or 12, the effectiveness of swim initiation by Tr1 stimulation was highly correlated with the concurrent injection of depolarizing or hyperpolarizing current into a single cell 204. Tr1 stimulation causes sustained excitation in SIIs, serotonin containing interneurons and Retzius cells, independent of whether or not swimming is initiated. A short, depolarizing current pulse injected simultaneously into as many as three 204 cells does not replicate the sustained excitation evoked in these cells by Tr1 stimulation. An oscillator neuron, cell 208, is inhibited when Tr1 stimulation fails to elicit swimming, but receives excitatory input from Tr1 otherwise. In another oscillator neuron, cell 115, stimulation of Tr1 suppressed an unidentified source of inhibitory synaptic potentials only on trials which resulted in swim initiation. We conclude that Tr1 stimulation triggers swimming by activating a long-lasting ramp depolarization in the SIIs which, in turn, provide excitatory drive to the swim oscillator. Moreover, Tr1 initiates swimming only when inhibitory inputs to the swim oscillator are suppressed.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3023605 TI - Double spontaneous rupture of the common bile duct. PMID- 3023604 TI - Initiation of swimming activity by trigger neurons in the leech subesophageal ganglion. III. Sensory inputs to Tr1 and Tr2. AB - In papers I and II of this series, we described two pairs of interneurons, Tr1 and Tr2, in the leech subesophageal ganglion which can trigger swimming activity in the isolated central nervous system (CNS). In this paper, we describe sensory inputs to these trigger neurons from previously identified mechanosensory neurons. We found that: Weak mechanical stimulation (stroking) of a body wall flap attached to a segmental ganglion in an otherwise isolated CNS excites the contralateral Tr1 slightly. Strong mechanical stimulation (pinching) of a mid body wall flap evokes a burst of impulses in the contralateral Tr1. For both means of stimulation the effects on the ipsilateral Tr1 and on the Tr2 cell pair were much weaker. Stroking a body wall flap attached to the head ganglion (supra- and subesophageal ganglia) in an otherwise isolated CNS excites both Tr1s and both Tr2s, although the effect is weaker for the Tr2s. Pinching strongly excites both trigger neurons bilaterally. Pressure and nociceptive mechanosensory neurons (P and N cells) in the subesophageal ganglion and the first segmental ganglion appear to make direct excitatory synapses with the contralateral Tr1 and Tr2. Mechanosensory interactions with the ipsilateral trigger neurons appear to be indirect. Functional inactivation of Tr1 by hyperpolarization does not prevent swim initiation either by weak mechanical stimulation of a body-wall flap or by intracellular stimulation of P cells.2+ We conclude that the trigger neurons, Tr1 and Tr2, provide an excitatory pathway by which mechanosensory stimulation can initiate leech swimming activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3023606 TI - Left-sided portal hypertension from malignant islet cell tumour of the pancrease: review with a case report. PMID- 3023608 TI - Deceptive vulvar papillomavirus infection. A possible explanation for certain cases of vulvodynia. AB - Use of the colposcope for vulvar and vaginal examination in four cases of long standing vulvodynia led to the identification of lesions with an unusual appearance. In the vulvar vestibule, epithelial projections resembling cactus, camel humps or stony colonial pavement were observed. In the vagina, the lesions looked more like cerebral folds. Biopsies of these lesions showed histopathologic changes diagnostic of human papillomavirus infection, scored according to Reid's criteria. In one case, capsid antigen was detected with the peroxidase technique in the nuclei of the superficial cells. Past history and positive findings in the sexual partners of some of the patients suggested long-standing herpesvirus activity in the lower genital tract. In some cases of recalcitrant vulvodynia, colposcopic examination of the vulva and vagina may lead to a viral explanation for symptoms previously considered psychosomatic in origin. PMID- 3023607 TI - Immunosuppressive properties of human placenta: study of supernatants from short term syncytiotrophoblast cultures. AB - Using collagenase and mechanical treatment to attempt to eliminate cellular contamination such as macrophages and decidual cells, trophoblast enriched cell suspensions were isolated from the human placenta. With a view to assessing the role of trophoblast in impairing maternal rejection of the fetus, supernatants (SPl4) were prepared from these placental cells after short-term culture (4 h). The immunosuppressive activity of these supernatants was studied following application to mitogen-stimulated human lymphocyte cultures and mixed lymphocyte cultures. In both cases a reproducible inhibition was observed. The ability of these substances to induce a non-specific inhibitory effect was ascertained by observing mouse lymphocyte responses to mitogens or alloantigens. To gain further insight into in vivo fetal protection against anti-paternal cells, we also examined the effects of SPl4 on CTL generation. It was found not only that CTL generation was markedly depressed but also that SPl4 drastically impaired cell mediated lympholysis at the effector level. To characterize the factors involved in our observations, SPl4 was subjected to dialysis and to chromatography. In the first case, it was found that these factors were not amenable to dialysis. In the second case, we obtained on an Ultrogel AcA 44 column two fractions with immunosuppressive activity. Following our previous work on human syncytiotrophoblast, we analyzed only the low molecular weight inhibitory fraction, which was chromatographed again on Ultrogel AcA 202. The molecular weight of the immunosuppressive factor(s) was estimated to be around 3.5 kDa. We postulate that human trophoblast releases soluble factors around the fetus which may act to protect it against maternal immunological rejection. PMID- 3023610 TI - Synthesis and radioprotective activity of new cysteamine and cystamine derivatives. AB - A variety of N-(aminoalkanoyl)-S-acylcysteamine and N,N' bis(aminoalkanoyl)cystamine salt derivatives were synthesized. Toxicity and radioprotective activity (as the dose reduction factor DRF) were determined in vivo on mice and compared to WR 2721 and S-acetylcysteamine hydrochloride. One of the most interesting compounds of this series was N-glycyl-S-acetylcysteamine trifluoroacetate (16, I 102). Structure-activity relationships are discussed. PMID- 3023611 TI - Comparison of (-)-eseroline with (+)-eseroline and dihydroseco analogues in antinociceptive assays: confirmation of rubreserine structure by X-ray analysis. AB - The enantiomers of eseroline bind to opiate receptors of rat brain membranes with equal affinities and show opiate agonist properties as inhibitors of adenylate cyclase in vitro. However, only (-)-eseroline shows potent narcotic agonist activity in vivo, similar to that of morphine. Neither (-)-noreseroline, (+) eseroline, nor the open dihydroseco analogue (-)-8 shows analgetic effects in vivo. The structure of rubreserine being a resonance hybrid of an o-quinone with its zwitterionic mesomer is confirmed by solid-state X-ray diffraction analysis. PMID- 3023609 TI - Probes for narcotic receptor mediated phenomena. 13. Potential irreversible narcotic antagonist-based ligands derived from 6,14-endo ethenotetrahydronororipavine with 7-(methoxyfumaroyl)amino, (bromoacetyl)amino, or isothiocyanate electrophiles: chemistry, biochemistry, and pharmacology. AB - N-Allyl-, N-(cyclopropylmethyl)-, and N-propyl-endo-ethenotetrahydronororipavines (N-substituted 6,14-endo-etheno-4,5-epoxy-3-hydroxy-6-methoxymorphinans) were synthesized with potential acylating or alkylating moieties at the C-7 position (isothiocyanato, (bromoacetyl)amino, and (methoxyfumaroyl)amino) and examined in vivo for their narcotic agonist and antagonist activities and for their ability to interact with opioid receptors in vitro. The N-(cyclopropylmethyl)-substituted compounds were found to have the highest affinity for opioid receptors among these N-substituted compounds, although all of them were found to be reasonably potent narcotic antagonists in the mouse tail flick vs. morphine assay. Their in vivo potency ranged from 1/8 to 4 times that of nalorphine on intravenous injection in mice. Rat brain membrane binding studies indicated that the compounds interacted with opioid receptors with potencies that ranged from 0.5 times that of morphine (8c, 9c, and 10c) to 0.017 that of morphine (8b). Among the compounds studied here, only the previously reported isothiocyanato compound (10c) and (methoxyfumaroyl)amino compound (8c) interacted irreversibly and selectively with mu or delta opioid receptors, respectively, in assays using NG108-15 neuroblastoma-glioma hybrid cells and/or in a rat brain membrane preparation. Both 8c and 10c were found to interact irreversibly, to a limited extent, with kappa opioid sites in rat brain membranes in which the mu and delta opioid receptors were depleted by interaction with the mu-selective irreversible ligand BIT and the delta-selective irreversible ligand FIT. Neither compound showed irreversible actions in the electrically stimulated mouse vas deferens preparation. PMID- 3023612 TI - Spin trapping of superoxide and hydroxyl radicals with substituted pyrroline 1 oxides. AB - The synthesis of three nitrones, 5-butyl-5-methyl-1-pyrroline 1-oxide (BMPO), 5,5 dipropyl-1-pyrroline 1-oxide (DPPO), and 2-aza-2-cyclopentenespirocyclopentane 2 oxide (CPPO), was conducted with use of the zinc/ammonium chloride reduction of appropriately substituted gamma-nitrocarbonyl compounds. The lipophilicity of these nitrones was estimated by determining their partition coefficients in a 1 octanol/water system. These nitrones were found to possess more lipophilic character than the most frequently used cyclic nitrone, 5,5-dimethyl-1-pyrroline 1-oxide (DMPO), which exhibits a partition coefficient of only 0.02. Hyperfine coupling constants for the spin trapping of superoxide and hydroxyl radical by the various nitrones were determined. The rate of spin trapping of superoxide with each nitrone was conducted by competitive kinetics with superoxide dismutase (SOD). In addition, the ability of DPPO and BMPO to spin trap free radicals generated during the metabolism of menadione by rat enterocyte cells was investigated. From these studies, DPPO and BMPO appear to be more suitable spin traps than DMPO when one is interested in monitoring free radicals generated intracellularly. PMID- 3023613 TI - Synthesis and antiviral evaluation of 1,4-dioxane nucleoside analogues related to nucleoside dialdehydes. AB - Since biologically active nucleoside 2',3'-dialdehydes exist as six-membered cyclic acetals (1) in solution, we have investigated the antiviral activity of some structurally similar 1,4-dioxane nucleoside analogues. By reacting 2',3' seconucleoside tosylates with base, the guanine (10) and adenine (18) substituted (hydroxymethyl)dioxanes have been constructed. In addition, an unusual adenine substituted divinyl ether (22) was synthesized via a base-catalyzed, double elimination of a 2',3'-di-O-tosyl-2',3'-secoadenosine. None of these compounds showed significant antiviral activity. PMID- 3023614 TI - Stereoselectivity of a potent calcium antagonist, 1-benzyl-3-pyrrolidinyl methyl 2,6-dimethyl-4-(m-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate. AB - Four enantiomers (3a-d) of the title compound, YM-09730 (3), were synthesized by the reaction of (-)- or (+)-5-(methoxycarbonyl)-2, 6-dimethyl-4-(m-nitrophenyl) 1,4-dihydropyridine-3-carboxylic acid (1a or 1b) with (S)- or (R)-1-benzyl-3 pyrrolidinol (2a or 2b). [3H]Nitrendipine binding affinity and coronary vasodilating activity of these compounds were evaluated. The absolute configuration of the most potent enantiomer (3a) with the longest duration was unequivocally determined to be (S)-1,4-dihydropyridine-C4 and (S)-pyrrolidine-C3 (S,S) by X-ray crystallographic study on 3a X HBr as well as 3a X HCl. The configuration of 1a corresponds to R, and the other enantiomers of 3 were respectively determined by chemical correlation. The potency order of the four enantiomers was (S,S)-3a greater than (S,R)-3b greater than (R,R)-3d greater than (R,S)-3c. Latent chiral characters of nifedipine derivatives with the identical ester groups were assigned by comparison of their puckering modes of 1,4 dihydropyridine (DHP) rings with those found in 3a X HCl or 3a X HBr. On the basis of the assignment, it has been revealed that the (S)-DHP nifedipine derivatives possess the synperiplanar carbonyl group at C5. The conformational restriction may be a factor causing stereoselectivity of antagonism. PMID- 3023615 TI - Mutations linked to the pro alpha 2(I) collagen gene are responsible for several cases of osteogenesis imperfecta type I. AB - We have analysed six South African families with osteogenesis imperfecta type I using three DNA polymorphisms associated with the pro alpha 2(I) collagen gene. In four of these families linkage of the pro alpha 2(I) gene and the osteogenesis imperfecta phenotype was suggested, whereas in the remaining two families there was a lack of linkage. No distinct correlation could be made between the phenotypic features of the families studied and linkage or lack of linkage to the pro alpha 2(I) gene. Two different haplotypes were found to be associated with the mutant pro alpha 2(I) alleles. These findings suggest that molecular heterogeneity exists within osteogenesis imperfecta type I and that in a significant proportion of cases the defect is linked to the pro alpha 2(I) gene. PMID- 3023617 TI - Intrachromosomal insertion of chromosome 13 in a family with psychosis and mental subnormality. AB - A family is reported in which an apparently balanced intrachromosomal insertion of chromosome 13, inv ins(13)(q21.3q32q31), was detected in four members, three of whom also show psychiatric disorder including mental subnormality, personality defects and frank psychosis. The possible effects of the insertion are considered. PMID- 3023616 TI - A new strategy for mapping the human genome. AB - Recent advances in agarose gel electrophoresis of large DNA fragments raise the possibility of an entirely new approach to mapping mammalian genomes. In this article is discussed the potential of this technology for tackling problems such as construction of linkage maps, identifying chromosome translocation breakpoints, and moving from linked markers to genes causing diseases such as the muscular dystrophies and Huntington's chorea. PMID- 3023618 TI - Regulatory defects of a conditionally lethal nusAts mutant of Escherichia coli. Positive and negative modulator roles of NusA protein in vivo. AB - Previous studies have attributed two activities to the NusA protein of Escherichia coli; namely, termination and antitermination of transcription. To examine these activities, we isolated a temperature-sensitive mutant of the nusA gene (nusAts11). The mutant cells produce a thermolabile NusA protein and grow at 32 degrees C, but not at 42 degrees C. At 42 degrees C, nusAts11 is recessive to nusA+ and nusA1, indicating the absence of its active gene product at that temperature. In the mutant, the efficiency of termination at the lambda tR1 terminator decreases, resulting in an increased expression of distal gene(s). On the other hand, the synthesis of the beta-galactosidase and beta beta' subunits of RNA polymerase is reduced in the mutant. This mimics effects seen in vitro when NusA protein is removed from a coupled transcription-translation system. These results suggest that the NusA protein plays both negative and positive modulator roles in vivo. The mutation nusAts11, unlike nusA1, does not block lambda phage growth at non-permissive temperatures, suggesting that NusA protein is not required for N antitermination in the mutant. Besides, the nusAts11 allows lambda Nam7Nam53byp phage growth under sup0 conditions, indicating that the N antitermination function is dispensable (at least partly) in this mutant. PMID- 3023619 TI - Transcription termination and processing sites in the bacteriophage lambda pL operon. AB - S1 nuclease mapping was performed on transcripts from the major leftward operon of the bacteriophage lambda in order to locate the 3' ends of stable RNA species produced in vivo. The analysis was carried out on RNA purified from either an induced lambda prophage or bacteria carrying a plasmid containing a large segment of lambda including the intact PL operon through the bet gene. The S1 nuclease mapping was performed on transcripts produced in the presence and the absence of the N antitermination function, and in the presence and the absence of either the RNase III processing enzyme or the Rho factor. The results of this work indicate that the intercistronic region between the N and ral genes of lambda contains three sites at which transcripts end under N-Rho+ conditions (positions on the lambda sequence: 34,826, 34,558 and 34,393). The distal two correspond to the two sites previously described in this region as tL1 (on both sides of the BamHI site). In the region between ral and Ea10, we mapped the 3' ends of three species of RNA. The 3' end of one species was found to be located 90 nucleotides proximal to tL2a, at 34,000 in the lambda sequence. The terminator at this site may be partially N-resistant. In an RNase III deficient host, an additional RNA species is formed. The 3' end of this RNA species is located at tL2a (33,910 on the lambda sequence). In the presence of the antitermination N gene product, the readthrough transcripts are processed to form a 3' end at position 33,980 on the lambda sequence. These results suggest that elongation of transcription of the lambda PL operon is reduced gradually by clusters of termination located between genes and that the expression of the terminated products is further controlled by processing of the mRNA. PMID- 3023620 TI - Structure of replicating simian virus 40 minichromosomes. The replication fork, core histone segregation and terminal structures. AB - The structure of replicating simian virus 40 (SV40) minichromosomes was studied by DNA crosslinking with trimethyl-psoralen. The procedure was used both in vitro with extracted SV40 minichromosomes as well as in vivo with SV40-infected cells. Both procedures gave essentially the same results. Mature SV40 minichromosomes are estimated to contain about 27 nucleosomes (error +/- 2), except for those molecules with a nucleosome-free gap, which are interpreted to contain 25 nucleosomes (error +/- 2). In replicative intermediates, nucleosomes are present in the unreplicated parental stem with the replication fork possibly penetrating into the nucleosomal DNA before the histone octamer is removed. Nucleosomes reassociate on the newly replicated DNA branches at distances from the branch point of 225 ( +/- 145) nucleotides on the leading strand and of 285( +/- 120) nucleotides on the lagging strand. In the presence of cycloheximide, daughter duplexes contained unequal numbers of nucleosomes, supporting dispersive and random segregation of parental nucleosomes. These were arranged in clusters with normal nucleosome spacing. We detected a novel type of interlocked dimer comprising two fully replicated molecules connected by a single-stranded DNA bridge. We cannot decide whether these dimers represent hemicatenanes or whether the two circles are joined by a Holliday-type structure. The joining site maps within the replication terminus. We propose that these dimers represent molecules engaged in strand segregation. PMID- 3023621 TI - Fine analysis of the active H5 gene chromatin of chicken erythroid cells at different stages of differentiation. AB - We have analyzed the chromatin structure of a region that encompasses 14.4 X 10(3) base-pairs of the chicken histone H5 locus in adult erythroid cells at different stages of maturation. Seven of eight major lineage-specific DNase I hypersensitive sites, some of which show complex substructure, were found in the flanking regions of the gene. The hypersensitivity of some of these sites is modulated during erythrocyte maturation in a way that parallels the transcriptional activity of the gene. DNase I, micrococcal nuclease, and S1 nuclease recognize the same regions, which differ from those cleaved by S1 on supercoiled plasmid DNA. This suggests that hypersensitivity of DNA in chromatin reflects a greater accessibility of the DNA rather than its altered conformation. The DNA sequence of some of the DNase I target sites contains repeated motifs, (T C-C-C)2, (T-C-C)2, (T-G-G-G-G)2, which are found in the hypersensitive sites of other genes. Detailed analysis across sections of the H5 gene and flanking sequences revealed differences in the DNase I sensitivity of the different regions examined. Notably, the first one-third of the gene is more sensitive than the rest. The sequences downstream from the region where most RNA polymerases terminate transcription were found to be the most resistant. PMID- 3023622 TI - A new membrane-associated DNA replication protein, the gene 69 product of bacteriophage T4, shares a patch of homology with the Escherichia coli dnaA protein. AB - A new phage T4 DNA replication protein, gp69, is found to be associated with membrane fractions, as predicted by the translated base sequence of gene 69. In addition, gp69 shares a patch of homology with a segment of the Escherichia coli dnaA initiation protein. The patchy homology of dnaA protein and gp69 suggests that they may serve some similar functions, such as interactions with the same E. coli components in bacterial and viral DNA replication. We have shown before that gene 69 spans an origin of T4 DNA replication, and that this origin is preferentially associated with membrane fractions. We suggest the possibility that gp69 is involved in the attachment of this origin to the bacterial envelope. PMID- 3023623 TI - Molecular organization of the rudimentary gene of Drosophila melanogaster. AB - We have determined the molecular structure of the rudimentary gene of Drosophila melanogaster. The transcription unit, which extends over 14,000 bases, is split into seven exons and six introns. Sequences presenting homologies with a TATA box and a CAT box are located upstream from the transcription initiation site, and there is a consensus polyadenylation signal in the vicinity of the 3' end of the messenger RNA coding sequence. Short intronic sequences fit well with consensus signals found in other eukaryotic genes and involved in the splicing of the precursor messenger RNA. Partial genomic and complementary DNA sequencing has revealed stretches of amino acid sequence homologies with the corresponding enzymes in Saccharomyces cerevisiae and Escherichia coli. This is in good agreement with the genetic map of the locus, which indicates that the enzymic activities encoded by the gene are organized linearly along the DNA. PMID- 3023624 TI - Site-specific and illegitimate recombination in the oriV1 region of the F factor. DNA sequences involved in recombination and resolution. AB - We have defined some of the sequences involved in high frequency recA-independent recombination at the oriV1 region of the F factor. Using a mobilization assay, we determined that plasmid pMB080, a pBR322 derivative bearing the PvuII-BamHI (F factor co-ordinates 45.43 to 46.0) fragment from the oriV1 region of F, contained all sequences necessary to undergo efficient site-specific recombination with the F derivative pOX38, which retains the oriV1 region. We constructed a series of pMB080 deletions in vitro using exonucleases S1 and Bal31. Deletions removing a ten base-pair sequence, which forms part of an inverted repeat segment located 62 base-pairs to the left of the NcoI site (45.87) within the cloned fragment, totally eliminated the recA-independent recombination reaction. Other deletions differentially affected both the frequency and stability of cointegrate molecules formed by the site-specific recombination system. The F factor oriV1 region is involved also in low-frequency recombination with several sites on pBR322 and related plasmids. We have determined the precise location of these recombination sites within oriV1 by DNA sequencing. These studies revealed that recombination always took place within an eight base-pair spacer region between the ten base pair inverted repeats found to be important for oriV1-oriV1 interactions. We propose that the low-efficiency recombination between pBR322 and pOX38 results from the ability of the F site-specific recombination apparatus to weakly recognize and interact with sequences that bear some resemblance to the normal oriV1 recognition elements. Furthermore, we suggest, by analogy with the lambda paradigm, that the nucleotide sequences at the junctions of secondary site recombinants define at least one crossover site used during the normal site specific recombination process. PMID- 3023625 TI - Concatemer formation of ColE1-type plasmids in mutants of Escherichia coli lacking RNase H activity. AB - rnh mutants harboring pBR322 were found to contain several slowly migrating DNA species when examined by agarose gel electrophoresis. The plasmid DNA from rnh mutants included large molecules, i.e. plasmids two, three or four times the size of a single plasmid unit. That this DNA contained concatemeric plasmid joined in a head-to-tail fashion was determined by digestion with restriction endonucleases that cleaved the monomeric plasmid DNA at a unique site. This treatment resulted in migration of the plasmid DNA at a mobility identical to that of linearized monomeric plasmid by agarose gel electrophoresis. This was confirmed by electron microscopy. Plasmid concatemer formation was detected with several high-copy number (relaxed type) plasmids but not with low-copy-number (stringent) plasmids. Concatemer formation was dependent on RecA+ and RecF+ functions since several recA and recF mutations abolished concatemer formation. ColE1-type plasmids were previously shown to replicate in rnh mutants in the absence of DNA polymerase I (PolI) activity. This DNA PolI-independent plasmid replication was also examined for its dependence on the recF and recA gene products. rnh- polA(Ts) recF- strains were efficiently transformed with these plasmids at 30 degrees C and 42 degrees C, indicating the presence of DNA PolI-independent replication under recF conditions. The presence or absence of plasmid replication in rnh- polA- recA(Ts) strains was also examined by measuring the increase in total amounts of plasmid. The results indicated that DNA PolI-independent replication occurred in these triple mutants at 42 degrees C as well as at 30 degrees C. It was concluded that the recombination event giving rise to concatemer formation was not essential for DNA PolI-independent replication in rnh mutants. PMID- 3023626 TI - A recombinant Chinese hamster ovary cell line containing a 300-fold amplified tetramer of the hepatitis B genome together with a double selection marker expresses high levels of viral protein. AB - A new series of double-selection plasmids containing recombinant genes expressing the neomycin phosphotransferase (NEO) of transposon Tn5 and mouse dihydrofolate reductase (DHFR) in mammalian cells is described. Activity of the recombinant DHFR gene varied more than 50-fold, depending on the location of the simian virus 40 72 base-pair repeat or enhancer, which is part of the promoter of the NEO unit. A NEO-DHFR module with the enhancer located at the 3' end of the DHFR gene was inserted into a plasmid containing four tandem head-to-tail copies of the hepatitis B virus (HBV) genome and the new plasmid was used to transform DHFR- Chinese hamster ovary cells. In one of the cell lines obtained, an unrearranged copy of the HBV tetramer could be amplified 300-fold by increasing selective pressure with methotrexate, resulting in a proportional increase of the synthesis of HBV surface antigen. Four different mRNAs detected in the amplified cell line probably encode HBV core protein, pre-S and surface antigens, and the X protein. As a result of the DNA amplification, synthesis of HBV proteins is no longer restricted to resting cells. Integrated plasmid sequences appear to be stable during the amplification process. PMID- 3023627 TI - Control of Rous sarcoma virus RNA translation and packaging by the 5' and 3' untranslated sequences. AB - A cytopathic mutant of Rous sarcoma virus-PrB was isolated and shown to have two large deletions, which result in the junction of Gag sequences in P27 to the 5' end of Env, and in the loss of the Src gene. This replication-defective (rd) and transformation-defective (td) mutant can replicate in the presence of its helper, which is also td, but the viral particles produced are poorly infectious. Most of the virions do not contain viral RNA, and the mutant RNA accumulates in infected cells, where it is poorly translated and packaged. Molecular clones of the mutant, of its helper and of a PrBtd strain were obtained in lambda-EMBL3, characterized, shown to be biologically active by transfection assays and sequenced. Nucleotide sequence comparisons indicate that the strong ribosome binding site of Rous sarcoma virus RNA, responsible for the efficient RNA translation in vivo and in vitro, is mutated in PrB-(HM) mutant RNA; this causes the inhibition of RNA translation, as demonstrated by translation competition experiments using virus RNA made in vitro that carries the original or the mutated ribosome-binding site. In addition, an insertion present at the 3' end of both the mutant and the helper RNA, but absent in PrBtd RNA, is probably responsible for the inhibition of RNA packaging. Finally, these data are discussed in the light of a model of a 5'----3' Rous sarcoma virus RNA structure leading to a circular RNA molecule, which has implications in RNA translation, packaging and reverse transcription. PMID- 3023628 TI - Intermediates in the assembly of the TonA polypeptide into the outer membrane of Escherichia coli K12. AB - The tonA gene of Escherichia coli K12 was cloned into a multicopy plasmid, leading to substantial overproduction of the corresponding 78,000 Mr polypeptide in the outer membrane. The approximate size of the tonA gene and its direction of transcription were established by Tn1000 mutagenesis. A family of tonA deletions was constructed in vitro by Bal31 exonuclease digestion, followed by splicing of an "oligo stop" sequence to each 3' terminus in order to ensure prompt termination of translation of the truncated polypeptides in vivo. All these polypeptides proved to be extremely unstable in exponentially growing cultures but were relatively stable in maxicells. Under these conditions the truncated polypeptides, unlike wild-type TonA, fractionated with the Sarkosyl-soluble fraction of the cell envelope, indicating that these proteins are blocked in assembly as inner membrane (translocation) intermediates or as outer membrane (maturation) intermediates unable to form Sarkosyl-resistant complexes. We have also examined the kinetics of assembly of wild-type TonA into the outer membrane and the results indicate that, following cleavage of the N-terminal signal peptide, the protein passes through an apparently membrane-free intermediate form and only appears in the outer membrane after a delay of at least 50 seconds, following the completion of synthesis. From these results, we propose that the assembly of TonA involves translocation (with concomitant cleavage of the signal sequence) directly into the periplasm, followed by partitioning into the outer membrane. We further propose that the C terminus of TonA is essential for final maturation in the outer membrane in Sarkosyl-resistant form but that the C terminal half of the molecule probably does not contain any topogenic sequences required for partitioning to the outer membrane. PMID- 3023629 TI - Nuclear magnetic resonance study of the isotope exchange of the proximal histidyl ring labile protons in hemoglobin A. The exchange rates and mechanisms of individual subunits in deoxy and oxy-hemoglobin. AB - Proton nuclear magnetic resonance spectroscopy has been used to investigate the rates and mechanism of exchange with deuterium of the proximal histidyl imidazole labile ring proton in deoxy and oxy-hemoglobin A. The resolved signals for the two subunits indicate dynamic heterogeneity, with the exchange rate always faster in the alpha than the beta subunits, suggesting a lower dynamic stability for the alpha subunit. The activation energy for the exchange in both subunits (approximately 25 kcal; 1 cal = 4.184 J) indicates that exchange proceeds via an intermediate far from denaturation or global unfolding. The pH profiles for both hemoglobin states reflect the EX2 mechanism for both subunits. While the base catalysis expected for an iron-bound imidazole is observed in all cases, there are important differences in both rates and mechanisms between the subunits. In deoxy-hemoglobin, both base-catalyzed and water-assisted exchange contribute to the alpha subunit, but only the former to the beta subunit. For oxy-hemoglobin, the base-catalysis is retained for both subunits, but the slope is considerably less for the alpha relative to the beta subunit. Thus the two subunits in the two states of hemoglobin differ both in mechanisms and in the inherent dynamic stability reflected in any one mechanism. The relationships of the proximal histidyl ring NH exchange rates to previously characterized subsets of allosterically responsive protons in hemoglobin A is briefly discussed. PMID- 3023630 TI - Functional model of subcomponent C1 of human complement. AB - The domain organization of the zymogen subunits of the first component of human complement C1s, C1r2 and the complex C1s-C1r2-C1s was studied by electron microscopy. In the absence of Ca2+, monomeric C1s was visualized as a dumb-bell shaped molecule consisting of two globular domains (center-to-center distance 11 nm) connected by a rod. One of the globular domains is assigned to the light chain (B-chain) of the activated molecule, which is homologous to trypsin and other serine proteases. The second globular domain and the rod are assigned to the heavy chain (A-chain) of CIs. The subunit C1r is a stable dimer in the presence or absence of Ca2+. This dimer C1r2 was visualized as composed of two dumb-bells of dimensions similar to those observed for C1s. These are connected near the junctions between the rod and one of the globular domains. This leads to the structure of an asymmetrical X with two inner closely spaced globules (center to-center distance 7 nm) and two outer globules at a larger distance (14 nm). By comparison with fragment C1rII2, in which part of the A-chain is removed, the inner globular domains were assigned to the catalytic B-chains. This characteristic structure of C1r2 is readily recognized in the central portion of the thread-like 54 nm long C1s-C1r2-C1s complex formed in the presence of Ca2+. By affinity-labeling of C1s with biotin and visualization of avidin-ferritin conjugates in the reconstituted complex, it was demonstrated that C1s forms the outer portion of the complex. A detailed model of C1s-C1r2-C1s is proposed, according to which two C1s monomers bind to the outer globes of C1r2 by contacts between their heavy chains and those of C1r. According to this model the catalytic domains of C1r are located in the center and those of C1s at the very tips of the C1s-C1r2-C1s complex. On the basis of the structure of C1s-C1r2-C1s, we derived a detailed model of the C1 complex (composed of C1q and the tetrameric complex) and we discuss this model with a view to finding a possible activation mechanism of C1.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3023631 TI - Analysis of the ends of bacteriophage Mu using site-directed mutagenesis. AB - We showed previously that two regions at the left end (L1 and L3) and one at the right end (R2) of bacteriophage Mu are essential for transposition. These regions all contain a 22 base-pair sequence with the consensus YGtTTCAYtNNAARYRCGAAAR, where Y and R represent any pyrimidine and purine, respectively. The Mu A protein binds to these regions in vitro, and weakly to sequences between nucleotides 1 and 30 of the right end (R1) and between nucleotides 110 and 135 of the left end (L2). These weak A binding sites contain the sequence AARYRCGAAAR. Here we show, using site-directed mutagenesis, that the weak A binding sites are essential for transposition. Mutations in these weak A binding sites have a greater effect on transposition than mutations of corresponding base-pairs in the stronger A binding sites, located adjacent to these weak A binding sites. We confirm the importance of several of the conserved base-pairs in the consensus sequence YGtTTCAYtNNAARYRCGAAAR. The base-pairs in the A binding sites that are shown to be essential for transposition are all conserved in the ends of the related bacteriophage D108. Furthermore, it is shown that the distance of 90 base-pairs between the two regions at the left end (L1 and L2L3) is essential. PMID- 3023632 TI - Evidence for an intermediate in DNA synthesis involving pyrophosphate exchange. A possible role in fidelity. AB - The incorporation of exogenous deoxyribonucleotide monophates (dNMP) was measured under conditions of ongoing DNA synthesis, providing arguments for the existence of a [DNAn X dNMP X PPi] intermediate in the nucleotide incorporation step of DNA synthesis: (formula; see text). The existence of such an intermediate is suggested by an apparent exchange of both dNMP and pyrophosphate (PPi) moieties of the deoxyribonucleotide triphosphate (dNTP) substrate with exogenous molecules. Such exchange and the incorporation of exogenous dNMP into DNA, strictly require ongoing DNA synthesis, suggesting that the energy for exchange reactions is provided by the cleavage of dNTP substrate. We propose that nucleotide selection during ongoing DNA synthesis results largely from the different relative rates of forward (beta) and backward (-alpha) reactions involving the [DNAn X dNMP X PPi] intermediate: the forward (incorporation) reaction is expected to predominate for the correct nucleotide, whereas the backward (abortive) reaction is expected to predominate for incorrect nucleotides. PMID- 3023634 TI - DNA sequence and coding properties of mutD(dnaQ) a dominant Escherichia coli mutator gene. AB - The mutD(dnaQ) gene in Escherichia coli codes for the epsilon subunit of the DNA polymerase pol III holoenzyme. Previous work has shown that this gene lies adjacent to the gene coding for RNase H (rnh). The two products are translated from diverging promoters. Here we report on the 1.6 kb (1 kb = 10(3) bases or base-pairs) sequence of the region coding for both genes, and the transcripts encoded by them. mutD codes for two transcripts, one of whose origins lies within the rnh structural gene. Both transcripts overlap and are complementary to a region of the rnh transcript. Thus, they can potentially form double-stranded helices with rnh. Of the two possible double-stranded structures, the shorter does not interfere with a likely rnh ribosome binding site, while the longer one does. We suggest that this unique organization may regulate rnh translation rates. PMID- 3023633 TI - Modification enhancement by the restriction alleviation protein (Ral) of bacteriophage lambda. AB - The product of the lambda ral gene alleviates restriction and enhances modification by the Escherichia coli K-12 restriction and modification system. An open reading frame (orf) located between genes N and Ea10 has been assigned to the ral gene. We have cloned this orf in a plasmid where its transcription is controlled by a thermolabile lambda repressor. Inactivation of the lambda repressor caused a 1000-fold reduction in K-specific restriction of unmodified lambda phage and a 100-fold increase in modification. In minicells transformed with ral+ plasmids, derepression resulted in the appearance of a polypeptide with a lower mobility than that predicted for a protein encoded by the orf attributed to ral; in a transcription and translation system in vitro DNA from a ral+ plasmid encoded a polypeptide with the same mobility. This polypeptide was absent when the plasmid DNA carried a mutant ral gene. The nucleotide sequence of this mutant gene defined two base changes, one of which inactivates the initiation codon of the orf. The K restriction endonuclease, which is also a K-specific methylase, is encoded by three genes designated hsdR, hsdM and hsdS, although the hsdR polypeptide is not essential for the methylase activity. We show that Ral enhances modification in a host strain lacking the entire hsdR gene, and lambda phages carrying the hsdM and S genes modify their own DNA inefficiently in the absence of Ral, despite the fact that derivatives of these phages provide efficient amplification of the K-specific methylase. Our data support a model in which, as a consequence of the interaction of Ral with either the hsdM or the hsdS polypeptide, the conformation of the enzyme is changed and the efficiency of methylation of unmodified target sites is enhanced. It has been postulated that Ral counteracts Rho, but in our experiments Ral did not relieve transcriptional polarity. PMID- 3023635 TI - Gene regulation and evolution in the chorion locus of Bombyx mori. Structural and developmental characterization of four eggshell genes and their flanking DNA regions. AB - We report on the detailed structural and developmental characterization of four chorion genes and a truncated pseudogene located within a 9.5 X 10(3) base chromosomal segment. These genes belong to the A and B multigene families and, like previously characterized moth chorion genes, are arranged in tightly linked pairs, which are divergently transcribed (A/B.L11 and A/B.L12). On the basis of their high degree of sequence divergence, the A genes define two distinct subfamilies, while the more homologous B genes represent different copies of the same gene type. The A.L11 and B.L11 introns are much longer, in each case because of a single inserted DNA segment that is missing from A.L12 or B.L12. The 2.1 X 10(3) base insertion in A.L11 is the first retrovirus-like transposable element characterized in Bombyx mori. The very short 5' flanking sequences of A/B.L11 and A/B.L12 (277 and 276 base-pairs) are distinct as shown by hybridization but both recur in additional chorion gene pairs, forming two respective classes that are expressed during distinctly different developmental periods. The divergently transcribed genes of each pair, which border the same 5' flanking sequence, are expressed co-ordinately, during the same developmental period. Detailed comparisons of the 5' flanking regions, and of the corresponding region of the Drosophila s15-1 chorion gene, revealed numerous, very short sequence elements that are shared. One such element, T-C-A-C-G-T, is also associated with all five sequenced Drosophila chorion genes. Some elements are repeated in a dyad symmetrical pattern, i.e. are associated with each of the two genes in a pair, while others, including T-C-A-C-G-T, occur only once per 5' flanking region, and, if functionally important, would presumably act bi-directionally on both genes of the pair. PMID- 3023637 TI - Partitioning of plasmid R1. Structural and functional analysis of the parA locus. AB - The stability locus, parA+, of plasmid R1 is shown to be localized within a 1500 base-pair region of DNA on the largest EcoRI restriction fragment of plasmid R1. The nucleotide sequence of the region revealed the presence of two open reading frames, one of 320 codons, and another of 60 codons. The larger open reading frame encodes a polypeptide of 36,000 Mr. Deletions covering the promoter distal end of the 36,000 Mr reading frame give rise to synthesis of large amounts of truncated protein. Construction of promoter fusions between the parA+ promoter and the lacZ gene showed that the parA+ region encodes a factor that negatively regulates the expression of the 36,000 Mr protein. The locus exerting parA+ associated incompatibility, denoted incA+, was mapped to a 60 base-pair region covering the parA+ promoter. Most likely, this region is involved both in the negative regulation of the parA+ operon and in the parA+-associated incompatibility. Two explanations are suggested to explain this possible dual function of the parA+ promoter region. The parA+ region was cloned into an unstably inherited (par-) derivative of a mini-F derivative. The low copy number plasmid mini-F devoid of its own partition genes was stabilized more than 100 fold by carrying the parA+ genes. This observation is in accordance with the proposal that the parA+ locus specifies the true partition function of plasmid R1. PMID- 3023636 TI - Synthesis in vitro of a seven amino acid peptide encoded in the leader RNA of Rous sarcoma virus. AB - Sequences of avian retroviral RNAs suggest that short open reading frames in the putatively untranslated leader sequences might direct the synthesis of small peptides. Previous analyses indicate that translation of Rous sarcoma virus (RSV) RNA in vitro faithfully reflects translation of the viral RNA in the chick cell. Accordingly, we sought to determine if the heptapeptide LP1, encoded in the open reading frame closest to the 5' end of RSV RNA, could be synthesized in vitro since this would strongly suggest that it might also be synthesized in vivo. Here we confirm that RSV RNA directs the synthesis of LP1 in rabbit reticulocyte lysates. LP1 is rapidly degraded in the lysate by an aminopeptidase activity. On the basis of the following observations, we propose that the open reading frame encoding LP1 plays a role in the life cycle of avian retroviruses. The LP1 open reading frame is ubiquitous with respect to position and length in 12 strains of avian retrovirus. In the amino acid sequences of the 12 strains, only three of the seven residues are invariant. On the basis of the conservation of the -3 and +4 nucleotides flanking the AUG codon, the strengths of initiation for translation of LP1 are approximately the same in the different viruses. The LP1 open reading frame is positioned in front of sites on retrovirus RNA that are required for initiation of cDNA synthesis and for packaging of the RNA into mature virus. PMID- 3023638 TI - Transposable genetic element found in the 5'-flanking region of the fibroin H chain gene in a genomic clone from the silkworm Bombyx mori. AB - A transposable genetic element was found in the 5'-flanking region of the fibroin H-chain gene in one of the genomic clones from the silkworm Bombyx mori. This element, named K-1.4, is about 1 X 4 X 10(3) base-pairs long, contains an open reading frame of only 225 base-pairs and has inverted repeats of 12 base-pairs at both ends. Duplication of three base-pairs seems to have occurred when this element was integrated into the silkworm genome. About 15 copies of K-1.4 are present per haploid genome of various silkworm strains. Genomic loci of some of these elements are different among different strains or even among individual offspring of the same parents. K-1.4 is present also in the genome of Bombyx mandarina. The K-1.4-related sequences are present in some species belonging to the family Saturniidae. PMID- 3023639 TI - Topoisomerization of plasmid DNA in Escherichia coli infected with bacteriophage T4. AB - The degradation of host DNA, and the block to transcription of cytosine containing DNA, which are a part of the normal course of infection by bacteriophage T4, can be eliminated in an appropriate T4 genetic background (designated as our reference type, or r.t.), so that T4 late promoters carried on plasmid DNA can function. The changes of topoisomer distribution that ensue when phage T4 r.t. infect Escherichia coli carrying a plasmid containing a T4 late promoter were analyzed. The linking number of the covalently closed circular plasmid DNA increased (implying relaxation) at the same time as the distribution of topoisomers became much broader. The relaxation of plasmid DNA was primarily, but not exclusively, due to T4 DNA topoisomerase II. The bacterial DNA topoisomerase II (gyrase) continued to function after phage infection to maintain some degree of superhelicity in plasmid DNA. When the DNA gyrase was inhibited by coumermycin or oxolinic acid, the topoisomer distribution became distinctly bimodal, part of the DNA remaining highly negatively supercoiled. It is argued that the observed post-infection topological changes involve relaxation of torsional stress and changes of binding by proteins that topologically constrain the plasmid DNA. PMID- 3023640 TI - Functional analysis of the glucocorticoid regulatory elements present in the mouse mammary tumor virus long terminal repeat. A synthetic distal binding site can replace the proximal binding domain. AB - Transcription of mouse mammary tumor virus DNA is stimulated by steroid hormones. The DNA sequences involved in this regulation are located in the viral long terminal repeat between positions -200 and -50 with respect to the transcription initiation site. In this region four, one distal and three proximal, in vitro binding sites for the glucocorticoid hormone-receptor complexes have been identified. We have prepared a series of 5' and 3' deletions of this region, using the exonuclease ExoIII. Combination of suitable 5' and 3' fragments enabled us to reconstitute the entire long terminal repeat with small internal deletions. The mutated long terminal repeats linked to the coding region of the Herpes simplex virus thymidine kinase gene were introduced into LTK- aprt- cells by transfection. Transcription from the mouse mammary tumor virus promoter in the presence or absence of hormone was assayed by nuclease S1 mapping. Deletion of the proximal in vitro binding sites resulted in a decrease in hormonal inducibility. When a synthetic oligonucleotide harboring the sequence of the distal in vitro binding site was inserted at the site of the proximal ones, hormone response was restored. This indicated that the distal binding site can replace the proximal ones in their hormone-regulatory function. However, insertion at the same site of an oligonucleotide containing the sequence 5' TGTTCT 3' found in all four binding sites, did not restore the hormone response, indicating that sequences flanking the TGTTCT motif are required for hormone response. Insertion of an unrelated DNA fragment at the site of the proximal binding element deletion completely abolished the hormone response. Analyses of different proximal binding-site deletion and insertion mutants suggested the presence of a transcriptional element located downstream from the most proximal hormone-receptor binding site. PMID- 3023641 TI - Distinct sequence elements involved in the glucocorticoid regulation of the mouse mammary tumor virus promoter identified by linker scanning mutagenesis. AB - In the proviral DNA of mouse mammary tumor virus (MMTV), sequences up to approximately equal to 200 base-pairs from the RNA start site are required for stimulation of transcription by glucocorticoid hormones in cultured cells. A total of 26 mutant plasmids with clustered point mutations or small deletions in the hormone control region of the MMTV long terminal repeat were constructed, linked to the coding portion of the Herpes simplex virus thymidine kinase gene, and introduced by transfection into LTK- cells. Transcription from mutant DNA in the presence or absence of hormone was quantified by S1 nuclease protection assays. Our analysis revealed the presence of at least three control elements that affect the extent of transcription stimulation by glucocorticoid hormones: (1) a distal element, between -181 and -172 base-pairs from the RNA initiation site. Linker scanning mutants in this segment have a reduction of up to 20-fold in the hormone response with respect to wild type. (2) An element around position -120, defined by a mutation of 4 base-pairs between -121 and -117, which causes a fivefold reduction. (3) An element from approximately equal to -78 to -70, defined by a mutant with also a roughly fivefold lower stimulation. The first two are included in areas that have been shown by others to interact in vitro with hormone-receptor complexes; the last one overlaps the in vitro binding site of a nuclear protein factor. A mutant lacking all three elements (-193 to -70) is completely non-inducible by glucocorticoids. Together with earlier results obtained with 5' deletion mutants, the data show that the largest contribution to the stimulatory response is made by the distal element, which however does require the presence of both more-proximal ones for the response to be maximal. In the absence of the distal one, the two proximal elements together produce a residual stimulation in the order of 5 to 10% of wild type, while the -70 element alone is ineffective. In addition, we show that a functional TATA homology is required for maximum stimulation. It appears that transcriptional regulation of MMTV by glucocorticoid hormones is achieved by the concerted action of multiple sequence modules, not all of which correspond to receptor binding sites in vitro. PMID- 3023643 TI - Myocardial Na, K-ATPase in one-kidney, one-clip hypertensive rats. AB - Myocardial ventricular Na, K-ATPase activity of normotensive rats was compared with that of healthy rats with chronic benign one-kidney, one-clip hypertension. The yield of protein (mg/g wet wt left plus right ventricles) in microsomal and sarcolemmal membrane fractions was the same for both normotensive and hypertensive rat ventricles. However, the yield of protein (mg/ventricle) was 26% greater in the hypertensive relative to the normotensive animals, consistent with the presence of hypertrophy, as also indicated by an increase in the ratio of ventricular to body weight and a shift in the isomyosin composition. Na, K-ATPase activity, sodium-dependent phosphorylation and ouabain binding were significantly (P less than 0.05) decreased (by 20%, 40%, and 45%, respectively) in the hypertensive rat ventricles when the data were expressed in units/g tissue wet weight. However, when expressed in units per ventricle, values in normotensive and hypertensive animals were similar. The molecular activity or turnover number of ventricular (and also renal) Na, K-ATPase activity was the same in both groups of animals. These results suggest that the decrease in myocardial specific Na, K ATPase activity in the rat made hypertensive by removing one kidney and constricting the renal artery of the other kidney is related to the presence of cardiac hypertrophy. PMID- 3023642 TI - DNA engineering shows that nucleosome phasing on the African green monkey alpha satellite is the result of multiple additive histone-DNA interactions. AB - The mechanism underlying sequence-specific positioning of nucleosomes on DNA was investigated. African green monkey alpha-satellite DNA was reconstituted in vitro with histones. Histone octamers were found to adopt one major and several minor positions on the satellite repeat unit, very similar to those positions found previously in vitro, demonstrating that sequence-specific histone-DNA interactions are responsible for nucleosome positioning on this DNA. In order to understand the nature of these interactions in more detail, we have constructed a variant satellite fragment containing an insertion of half a helical DNA turn. The parent fragment directs histones to one major and two overlapping minor positions that are all affected by the insertion. All three frames respond in a unique fashion to the additional five base-pairs. From a quantitative analysis of the nucleosome positions on the engineered fragment, consensus "phasing boxes" as the basis for nucleosome positioning can be ruled out. Instead, our results argue very strongly that nucleosome positioning is due to the independent contribution of many different DNA-histone contacts along the entire core particle, in an apparently additive fashion. PMID- 3023645 TI - Regulation of intracellular sodium ions in acute reversible myocardial ischemia- a perspective. PMID- 3023644 TI - Ciba-Geigy award for outstanding research. Regulation of adrenergic receptor function by phosphorylation. AB - The various subtypes of adrenergic receptors represent distinct structural entities which are coupled in different ways to two major transmembrane signalling systems, the adenylate cyclase and phosphatidyl-inositol pathways. Recent evidence suggests that the functional linkage of both beta and alpha 1 adrenergic receptors to their respective effector systems is regulated by covalent modification of the receptors by phosphorylation-dephosphorylation reactions. Receptor phosphorylation appears to lead to desensitization of the biological response to receptor stimulation. Several kinases including protein kinase A, protein kinase C and a cAMP independent kinase appear to participate in these reactions. PMID- 3023646 TI - Permissive action of potassium on arrhythmogenesis in the rat heart. AB - The isolated perfused rat heart was used to assess the influence of extracellular potassium ([K+]0) on vulnerability of the heart to ventricular fibrillation (VF). The VF threshold is reduced when [K+]0 is lowered from 5.9 to 2.0 and 3.0 mmol/l. The vulnerable period is not only widened but VF can be obtained by stimuli on the R wave. The opposite effects are encountered when [K+]0 is increased to 9.0 mmol/l. These alterations in vulnerability to VF are accompanied by an increased incidence of tachyarrhythmias and spontaneous VF during coronary artery ligation and following reperfusion on reduction of [K+]0, with elimination of these arrhythmias on increasing [K+]0 to 9.0 mmol/l. The cellular biochemical responses that accompanied the altered electrical behaviour on alterations of [K+]0 involved the tissue levels of cyclic AMP in both non-ischaemic and ischaemic myocardium and tissue levels of Ca2+ whereas tissue levels of high energy phosphates, adenosine, inosine, hypoxanthine, lactate, Na+, K+ and Mg2+ did not discriminate between hearts vulnerable and hearts resistant to VF. In this model the extracellular K+ level is a determinant of vulnerability to ventricular fibrillation while ischaemic tissue levels of Ca2+ provide the best correlation with alterations in vulnerability. PMID- 3023647 TI - The influence of inhibitors of the ATP degradative pathway on recovery of function and high energy phosphate after transient ischemia in the rat heart. AB - The loss of the catabolic products of adenosine triphosphate in the form of purine nucleosides and oxypurines during ischemia and subsequent reperfusion may limit adenine nucleotide regeneration. This study compared the effects of infusion of inhibitors of the major reactions involved in the degradation of adenosine triphosphate to inosine on the postischemic recovery of high energy phosphate and myocardial function. Inhibitors of adenylate kinase, 5'nucleotidase, adenosine translocase and adenosine deaminase were studied. Following 30 minutes of ischemia, only hearts infused with alpha, beta, methylene adenosine diphosphate (5' nucleotidase inhibitor) recovered significantly better ventricular function than control (p less than 0.05), but all hearts had increased adenosine triphosphate regeneration (p less than 0.05). The formation and washout of greater than 30% of the total adenine pool metabolites was not prevented by any drug. Nevertheless all manipulations of adenine metabolism resulted in recruitment of high energy phosphate during preischemic infusion. PMID- 3023648 TI - Influence of adipocyte triglyceride on the partition of 2,2',4,4',5,5' hexabromobiphenyl between 3T3L1 adipocytes and surrounding pseudoblood. AB - Removal from adipose tissue is an important first step in ultimate removal of many lipophilic xenobiotics from the body. This study concerned the elucidation of mechanisms by which hexabromobiphenyl (HBB) was deposited in and removed from adipocytes. Adipocytes derived from the 3T3L1 cell line of mouse fibroblasts were used to conduct studies in vitro. Results support the idea that HBB enters the 3T3L1 adipocyte via passive diffusion. A plot of the velocity of uptake versus concentration was linear, the uptake of HBB does not appear to be energy dependent, and structurally similar biphenyls did not cause an inhibition of uptake. A linear relationship between the quantities of triglyceride and HBB in the cells was found during both uptake of HBB in lipogenesis and removal of HBB in lipolysis (r greater than 0.98). This supports the contention that the quantity of triglyceride in the cells has a strong influence on the movement of HBB between adipocytes and surrounding pseudoblood. Evidence has been presented that is consistent with the hypothesis that HBB moves freely across the adipocyte membrane and is sequestered in either cells or medium according to its relative solubility in these compartments. Methods to increase the removal of HBB from adipocytes have been proposed. PMID- 3023649 TI - Charge effect on binding, uptake and transport of ferritin through fenestrated endothelium. AB - The binding, uptake and transport of hydrosoluble macromolecules as a function of their electric charge have been investigated in mouse pancreatic exocrine fenestrated endothelium. After intravenous injection of ferritin derivatives with different isoelectric points (3.7, 4.5, 7.0 and 8.4) specimens of pancreas were collected at 5, 25, 45 and 60 min and processed for electron microscopy. Although the concentrations (10 mg/ml) and injected volumes (1 ml/100 g body weight) were identical, the tracers with similar molecular weight (Mr approximately 960,000), but with different net electric charge, followed different routes in endocytotic and transcytotic processes. The endothelial cell internalizes ferritins into multivesicular bodies and lysosomes. While anionic ferritins are endocytosed via plasmalemmal vesicles (fluid phase endocytosis), the cationic derivatives are taken up exclusively by coated pits (adsorptive endocytosis). The anionic and neutral macromolecules seem to be transcytosed via plasmalemmal vesicles. The circulating polycations are aggregated by plasma proteins and transcytosed as such either in fluid phase by large vacuoles, or by adsorption on the coated structures. The first mechanism is physiological, the latter probably appears in unusual conditions by which the endothelium participates in the plasma clearance. Our findings indicate that, for the transport of macromolecules, the capillary endothelium can be accounted not only as a size barrier but also as a selective differentiated charge barrier with complex and multiple mechanisms for the modulation of its transport systems. The endocytotic and transcytotic processes undergo the effect of net electric charge of the plasma components. PMID- 3023650 TI - Cytochemical localization of ouabain-sensitive, K+-dependent para nitrophenylphosphatase (transport ATPase) in the guinea pig sympathetic ganglia. AB - The cytochemical techniques, developed by Ernst in 1972 and Mayahara et al. in 1980 were used to study the localization of K+-pNPPase related Na+/K+-ATPase in the guinea pig sympathetic ganglia. Enzymatic activity was revealed at the cytoplasmic aspect of the plasma membranes. Both the neuronal and satellite cell membranes contained the enzyme. Most of the activity was seen in the membrane of the satellite cells, which was interpreted to indicate a membrane well equipped for conducting active transport of cations. The neuronal membrane also displayed enzymatic activity particularly at specialized areas such as synapses. PMID- 3023651 TI - Collagen changes in the ductal infiltrating (scirrhous) carcinoma of the human breast. A possible role played by type I trimer collagen on the invasive growth. AB - The tumour stroma in cases of ductal infiltrating carcinoma of the mammary gland contains substantial amounts of collagen type I trimer, besides the regular collagen types. Reconstituted collagen trimer consists of fibrils that are significantly thinner than reconstituted fibrils of type I collagen. The axial periodicity is somewhat longer due to widening of the c-d and d-e regions. Transmission EM of the tumours shows characteristic phenomena at the stromal tumour cell junctions: frequent absence of a basal lamina and thin disordered collagen fibrils that show frequent direct contacts with tumour cells. On the basis of literature data concerning the interaction between stroma and epithelia under physiological and pathological conditions it is hypothesized that the interaction of collagen type I trimer fibrils with tumour cells is instrumental in augmenting tumour cell progression and that the trimer may provide contact guidance for invasive growth. PMID- 3023652 TI - A physical map of the viral genome for infectious pancreatic necrosis virus Sp: analysis of cell-free translation products derived from viral cDNA clones. AB - The two segments of double-stranded RNA from infectious pancreatic necrosis virus Sp were cloned into the plasmid vector pUC8. Two sets of overlapping clones were identified by restriction enzyme and Southern blot analyses. Each of these sets was shown by Northern blot analysis to be exclusively related to either segment A or B of the genomic RNA. The entire lengths of the cloned segments were estimated to be 2.9 and 2.6 kilobases, respectively. Sequences from the two segments of viral cDNA were subcloned into the bacteriophage T7 RNA polymerase vectors pT71 and pT72. The activity of the single-stranded RNAs transcribed from these subclones in a rabbit reticulocyte lysate translation system provided information on the polarity of and the protein products coded for by each subclone. The four proteins encoded by the genome of infectious pancreatic necrosis virus were identified among the translation products of the individual cloned segments by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By constructing plasmids containing deletions in the sequences from either the 5' or 3' end of segment A, we were able to construct a physical map for the larger segment of double-stranded RNA. The proteins derived from these plasmids indicated that the linear gene order for viral proteins encoded in segment A is beta, gamma 2, and gamma 1. PMID- 3023653 TI - A kinase able to phosphorylate exogenous protein synthesis initiation factor eIF 2 alpha is present in lysates of mengovirus-infected L cells. AB - Infection of mouse L929 cells by mengovirus resulted in the expression of a kinase activity that selectively phosphorylated the small, 38,000-molecular weight subunit of eucaryotic initiation factor 2 and histone H2. This kinase activity was independent of host cell RNA synthesis and was located in the postribosomal supernatant (S-100 fraction) early after infection (up to 3 h). At later times after infection (5 h), kinase activity was also associated with the polysome fraction. The kinase present in the S-100 fraction bound strongly to DEAE-cellulose, its peak activity eluting at 0.5 M KCl. Kinase activity was independent of the presence of exogenous double-stranded RNA, and KCl at concentrations greater than 0.1 M inhibited eucaryotic initiation factor 2 phosphorylation. The 67,000-molecular-weight phosphoprotein activated in interferon-treated cells by double-stranded RNA was not detected by standard phosphorylation assays in lysates from mengovirus-infected cells. Labeling of this protein in vivo during 5 h of infection was also not detected. The DEAE cellulose-purified mengovirus kinase inhibited protein synthesis in reticulocyte lysates, and the inhibition was not reversible by high concentrations of poly(I).poly(C). PMID- 3023654 TI - Partition of E1A proteins between soluble and structural fractions of adenovirus infected and -transformed cells. AB - The partition of E1A proteins between soluble and structural framework fractions of human cells infected or transformed by subgroup C adenoviruses was investigated by using gentle cell fractionation conditions. A polyclonal antibody raised against a trpE-E1A fusion protein (K.R. Spindler, D.S.E. Rosser, and A. J. Berk, J. Virol. 132-141, 1984) synthesized in Escherichia coli was used to measure the steady-state levels of E1A proteins recovered in the various fractions by immunoblotting. The relative concentration of E1A proteins recovered in the soluble fraction of adenovirus type 2-infected cells was at least fivefold greater than the relative concentration in the corresponding fraction of transformed 293 cells. The observed distribution of E1A proteins was not altered by the sulfhydryl-blocking reagent N-ethylmaleimide. E1A proteins were recovered in nuclear matrix, chromatin, and cytoskeleton fractions after further fractionation of the structural framework fraction. However, the E1A protein species that could be identified by one-dimensional gel electrophoresis were not uniformly distributed among the subcellular fractions examined. The results obtained when fractionation was performed in the presence of the oxidation catalysts Cu2+ or (ortho-phenanthroline)2 Cu2+ indicate that E1A proteins can be efficiently cross-linked, via disulfide bonds, to the structural framework of both adenovirus-infected and adenovirus-transformed cells. PMID- 3023655 TI - Cellular mRNA translation is blocked at both initiation and elongation after infection by influenza virus or adenovirus. AB - During influenza virus infection, protein synthesis is maintained at high levels and a dramatic switch from cellular to viral protein synthesis occurs despite the presence of high levels of functional cellular mRNAs in the cytoplasm of infected cells (M. G. Katze and R. M. Krug, Mol. Cell. Biol. 4:2198-2206, 1984). To determine the step at which the block in cellular mRNA translation occurs, we compared the polysome association of several representative cellular mRNAs (actin, glyceraldehyde-3-phosphate dehydrogenase, and pHe7 mRNAs) in infected and uninfected HeLa cells. We showed that most of these cellular mRNAs remained polysome associated after influenza viral infection, indicating that the elongation of the proteins encoded by these cellular mRNAs was severely inhibited. Because the polysomes containing these cellular mRNAs did not increase in size but either remained the same size or decreased in size, the initiation step in cellular protein synthesis must also have been defective. Several control experiments established that the cellular mRNAs sedimenting in the polysome region of sucrose gradients were in fact associated with polyribosomes. Most definitively, puromycin treatment of infected cells caused the dissociation of polysomes and the release of cellular, as well as viral, mRNAs from the polysomes, indicating that the cellular mRNAs were associated with polysomes that were capable of forming at least a single peptide bond. A similar analysis was performed with HeLa cells infected by adenovirus, which also dramatically shuts down cellular protein synthesis. Again, it was found that most of the cellular mRNAs, which were translatable in reticulocyte extracts, remained associated with polysomes and that there was a combined initiation-elongation block to cellular protein synthesis. In cells infected by both adenovirus and influenza virus, influenza viral mRNAs were on larger polysomes than were several late adenoviral mRNAs with comparably sized coding regions. In addition, after influenza virus superinfection of cells infected by the adenovirus mutant dl331, a situation in which there is a limitation in the amount of functional initiation factor eIF-2 (M. G. Katze, B. M. Detjen, B. Safer, and R. M. Krug, Mol. Cell. Biol. 6:1741 1750, 1986), influenza viral mRNAs, but not late adenoviral mRNAs, were on polysomes. These results indicate that influenza viral mRNAs are better initiators of translation than are late adenoviral mRNAs. PMID- 3023656 TI - Genetic complementation among poliovirus mutants derived from an infectious cDNA clone. AB - We constructed several well-defined mutations in the nonstructural portion of the poliovirus type I (Mahoney strain) genome by making small insertions in an infectious cDNA clone. The derived viral strains carrying the mutations exhibited a variety of distinct plaque phenotypes. Thus, we were able to examine genetic complementation between different pairs of mutants by comparing the yields of progeny virus in mixed and single infections. Two mutants bearing lesions in the 2A and 3A regions of the genome, which are defective in the inhibition of host cell translation and the synthesis of viral RNA, respectively, could be rescued efficiently by genetic complementation; three replication-deficient mutants containing insertions in the 2B, 3D (replicase), and 3'-untranslated regions could not. Both the 2A and 3A mutants could be rescued by each other and by all of the other mutants tested. Because yield enhancement was apparent well before the completion of a single infectious cycle, it is likely that complementation of both mutants involved early diffusion of functional products. These data provide the first unambiguous evidence that the nonstructural portion of the poliovirus genome contains multiple complementation groups. The data also suggest that certain nonstructural functions act only in cis. PMID- 3023657 TI - Selective shedding and congenital transmission of endogenous avian leukosis viruses. AB - Shedding and congenital transmission of endogenous avian leukosis viruses were studied in viremic White Leghorn hens exogenously infected with viruses with endogenous long terminal repeats (LTRs) and in four semicongenic lines of hens that naturally express infectious endogenous viruses (EVs). Relatively high titers of infectious virus EV7 (encoded at locus ev7), Rous-associated virus-0 (RAV-0), and recombinant 882/-16 RAV-0 were detected in blood cells and sera from exogenously infected hens, but marked differences were noted in the incidence of congenitally infected progeny. In enzyme immunoassays that detect viral group specific antigen, little or no p27 was detected in albumens from dams infected with RAV-0. However, hatchmates infected with either EV7 or recombinant 882/-16 RAV-0, which was constructed with an RAV-0 LTR, shed high titers of p27. Similarly, semicongenic hens that expressed RAV-0 (EV2) (encoded at locus ev2) shed little or no p27 into albumens, but hens that harbored ev10, ev11, and ev12 shed high titers of p27. A slower electrophoretic mobility of p27, considered to be characteristic of EVs that are restricted in congenital transmission, was not associated with low levels of shedding or congenital transmission; p27 from other EVs and p27 from an avian leukosis virus field strain, all of which are shed at high levels, had mobilities identical to that of p27 from RAV-0. Although shedding and congenital transmission appear to be controlled by the viral genome, there was no correlation between low efficiency of shedding or congenital transmission and endogenous LTR or p27 sequences. PMID- 3023659 TI - Direct inactivation of viruses by human granulocyte defensins. AB - Human neutrophils contain a family of microbicidal peptides known as defensins. One of these defensins, human neutrophil peptide (HNP)-1, was purified, and its ability to directly inactivate several viruses was extensively tested. Herpes simplex virus (HSV) types 1 and 2, cytomegalovirus, vesicular stomatitis virus, and influenza virus A/WSN were inactivated by incubation with HNP-1. Two nonenveloped viruses, echovirus type 11 and reovirus type 3, were resistant to inactivation. Purified homologous peptides HNP-2 and HNP-3 were found to have HSV 1-neutralizing activities approximately equal to that of HNP-1. Inactivation of HSV-1 by HNP-1 depended on the time, temperature, and pH of incubation. Antiviral activity was abrogated by low temperature or prior reduction and alkylation of the defensins. Addition of serum or serum albumin to the incubation mixtures inhibited neutralization of HSV-1 by HNP-1. We used density gradient sedimentation techniques to demonstrate that HNP-1 bound to HSV-1 in a temperature-dependent manner. We speculate that binding of defensin peptides to certain viruses may impair their ability to infect cells. PMID- 3023658 TI - Simian virus 40 agnoprotein facilitates perinuclear-nuclear localization of VP1, the major capsid protein. AB - The agnoprotein of simian virus 40 (SV40) is a 61-amino-acid protein encoded in the leader of some late mRNAs. In indirect immunofluorescence studies with antisera against SV40 capsid proteins, we show that mutants which make no agnoprotein display abnormal perinuclear-nuclear localization of VP1, the major capsid protein, but not VP2 or VP3, the minor capsid proteins. In wild-type (WT) SV40-infected CV-1P cells, VP1 was found predominantly in the cytoplasm until 36 h postinfection (p.i.), approximately the time that high levels of agnoprotein became detectable under our infection conditions. Thereafter, VP1 localized rapidly to the perinuclear region and to the nucleus. In contrast, in agnoprotein minus mutant-infected CV-1P cells, perinuclear-nuclear accumulation of VP1 occurred much less efficiently; a significantly greater fraction of cells with predominantly cytoplasmic fluorescence was observed up to 48 h p.i. At 48 and 60 h p.i., more cells with largely perinuclear and little nuclear staining were seen than in WT-infected controls. In similar analyses with stably transfected cell lines constitutively expressing the agnoprotein, VP1 localized to the nucleus before 30 h p.i., regardless of the infecting virus. Delayed nuclear entry of VP1 in a mutant which makes no agnoprotein was also overcome in a revertant which has a second site point mutation in VP1. This suggests that an alteration of VP1 can partially overcome the defect of the agnogene mutation by enhancement of the rate of its own nuclear localization. Taken together, these results indicate that at least one function of the agnoprotein is to enhance the efficiency of perinuclear nuclear localization of VP1. PMID- 3023660 TI - Polyomavirus small t antigen: overproduction in bacteria, purification, and utilization for monoclonal and polyclonal antibody production. AB - Polyomavirus small t antigen was purified from genetically engineered Escherichia coli and used as the immunogen for the production of polyclonal and monoclonal antibodies. A new series of plasmids for increased expression of polyomavirus T antigens or a T antigen-beta-galactosidase fusion protein was constructed by replacing sequences coding for the ribosome-binding site of previously published plasmids with a chemically synthesized sequence that has a higher degree of complementarity to the 3' end of the 16S rRNA. Cells expressing the fusion protein from the plasmid with the synthetic sequence contained 5- to 10-fold more fusion protein after a 3-h induction than did control cells. Pulse-labeling of cells bearing the new plasmids revealed that the T antigens were synthesized at high levels after induction: 10% of total synthesis for small t; 15% for Py-1387T middle T, a truncated mutant of middle T; and probably 1 to 5% for middle T. Small t and Py-1387T middle T, but not wild-type middle T, were seen as minor bands in total cell protein analyzed on sodium dodecyl sulfate-polyacrylamide gels stained with Coomassie blue. A simple, rapid procedure for purification of bacterial small t from the pellet of sonicated bacteria yielded 1 to 2 mg of small t per liter of bacterial culture at 80 to 90% homogeneity. High-titer polyclonal rabbit antisera raised against purified small t recognized all three T antigens and were suitable for immunoaffinity purification of middle T. Mouse monoclonal antibodies raised against bacterial small t were of four classes, immunoprecipitating either all three polyomavirus T antigens, small t and middle T only, primarily small t, or middle T and large T in preference to small t. One of the latter monoclonal antibodies also immunoprecipitated large T but not small t of simian virus 40, suggesting that the site recognized by this antibody may be functionally important. None of the monoclonal antibodies yielded an immunoprecipitate active in phosphorylating middle T in vitro. PMID- 3023661 TI - Simian virus 40 agnoprotein facilitates normal nuclear location of the major capsid polypeptide and cell-to-cell spread of virus. AB - The simian virus 40 agnoprotein is a 61-amino-acid, highly basic polypeptide that is coded within the 5' leader of late 16S mRNAs. To better understand agnoprotein function and to more effectively differentiate cis-from trans-acting effects of an agnogene mutation, we constructed a mutant virus that carries a single-base pair substitution and fails to produce agnoprotein. pm 1493 contains a T/A to A/T transversion at sequence position 335. This mutation converts the agnoprotein initiation codon from ATG to TTG, preventing synthesis of the protein. The mutant displays only a modest growth defect in CV-1P and AGMK cells and no defect in BSC 1 cells. Early-gene expression, DNA replication, synthesis of late viral products, and the kinetics of virion assembly all appear normal in pm 1493 infected CV-1P cells. Immunofluorescent studies, however, indicate that localization of the major capsid polypeptide VP1 is different in mutant- than wild-type virus-infected cells. Furthermore, the lack of agnoprotein led to inefficient release of mature virus from the infected cell. Agnogene mutants could be severely compromised in their ability to propagate in monkeys given their reduced capacity for cell-to-cell spread. PMID- 3023662 TI - Diarrheal response of gnotobiotic pigs after fetal infection and neonatal challenge with homologous and heterologous human rotavirus strains. AB - Pigs exposed in utero to human rotavirus (HRV) strain Wa serotype 1 from 15 to 36 days prior to birth responded immunologically by modifying their clinical response to neonatal oral challenge with a pathogenic dose of homologous Wa or heterologous M serotype 3 HRV. In these cases, diarrhea was prevented in 12 of 14 pigs and greatly reduced in the other two. However, fecal virus shedding was not significantly modified, since it was detected in 12 of 14 pigs. These results suggest the existence of a closer antigenic relationship between these two different HRV serotypes which may only be expressed in an in vivo test system. Exposure of fetal pigs to HRV DS-1 serotype 2 failed to cause infection or to induce any protection when pigs were challenged at birth with HRV Wa. This model for cross-protection studies in gnotobiotic piglets offers good possibilities for the evaluation of potential HRV vaccine candidates, for the in vivo study of antigenic similarities between rotavirus serotypes, and for the understanding of protective immune responses against diarrhea and virus shedding. PMID- 3023663 TI - Characterization of West Nile virus RNA-dependent RNA polymerase and cellular terminal adenylyl and uridylyl transferases in cell-free extracts. AB - To facilitate further studies of flavivirus transcription, cell extraction methods and in vitro reaction conditions which increased West Nile virus (WNV) RNA-dependent RNA polymerase activity were determined. Subcellular fractions from WNV-infected BHK-21/W12 cells were characterized with regard to their protein and RNA content and in vitro polymerase activity. In both a cytoplasmic fraction, designated S1, and a fraction enriched for outer nuclear membranes, designated S2, seven virus-specific proteins, NS5 (96 kilodaltons [kDa]), NS3 (67 kDa), E (48 kDa), NS1 (47 kDa), ns4a (26 kDa), ns2a (17 kDa), and ns2b (14.5 kDa), were detected. The fractions also contained virus-specific RNA and cellular rRNA and mRNA. Polymerase activity in S1 and S2 fractions from WNV-infected cells was concentrated by pelleting and consisted of two types of enzyme activities: the WNV RNA-dependent RNA polymerase and terminal transferases of cellular origin. Enhanced levels of WNV polymerase activity were obtained from these cell fractions by altering several of the in vitro reaction conditions. Although Mg2+ was the divalent cation preferred by WNV polymerase, virus-specific in vitro transcription was detected at reduced levels when Mn2+ (0.05 or 0.5 mM) was present as the sole divalent cation. Product analysis revealed that the viral polymerase incorporated radiolabeled ribonucleotides into three distinct RNA species. Free single-stranded genome-sized RNA which was LiCl insoluble and RNase sensitive was found by fingerprint analysis to have an oligonucleotide pattern similar to that of WNV genomic RNA. RNA molecules which comigrated as a broad band near the top of the gel were separable into LiCl-insoluble, partially RNase sensitive replicative-intermediate RNA and LiCl-soluble, RNase-resistant replicative-form RNA. The cellular transferases added UMP or AMP residues to the 3'-termini of cellular mRNA, tRNA, and 18S and 28S rRNA. Although a cellular terminal transferase has been reported to function in initiation of poliovirus transcription, no labeling of the WNV RNA by either of these cellular enzymes was detected. Therefore, they appear to play no specific role in flavivirus RNA synthesis. PMID- 3023664 TI - Transcription initiation sites and nucleotide sequence of a herpes simplex virus 1 gene conserved in the Epstein-Barr virus genome and reported to affect the transport of viral glycoproteins. AB - Earlier reports have localized mutations which affect the processing and transport of herpes simplex virus 1 glycoproteins to a region located between the genes specifying glycoprotein B and the major viral DNA-binding protein (beta 8). The nucleotide sequence of this region contains a single long open reading frame encoding a 780-amino-acid protein with a predicted molecular weight of 83,845. To confirm the existence of this protein, rabbit polyclonal antibody was made against a synthetic peptide made according to the predicted sequence of a hydrophilic domain near the carboxy terminal of the protein. This antibody reacted with an infected cell protein of an apparent molecular weight of 95,500. We designated this protein infected cell protein 18.5 (ICP18.5). S1 nuclease analysis suggested that the 5.6-kilobase mRNA encoding ICP18.5 is initiated predominantly from one site, but three weaker initiation sites also seemed to occur within a 74-base-pair stretch of DNA. This gene appears to be conserved in the Epstein-Barr virus (EBV) genome, inasmuch as 174 of the 780 amino acids of ICP18.5 align with corresponding amino acids predicted by the EBV open reading frame BALF3. The EBV gene is located adjacent to the gene specifying a homolog of the herpes simplex virus 1 glycoprotein B. PMID- 3023665 TI - Increase of retroviral infection in vitro by the binding of antiretroviral antibodies. AB - Monoclonal antiretrovirus antibodies were assayed for their ability to influence retrovirus infection in vitro. Some antibodies increased murine cell infection by an ecotropic virus and both murine and human cell infection by an amphotropic virus. The stability of these viruses was not modified, suggesting that antibody virus complexes may be more infectious than free virus particles. PMID- 3023666 TI - p37mos-associated serine/threonine protein kinase activity correlates with the cellular transformation function of v-mos. AB - A serine/threonine-specific protein kinase activity is closely associated with v mos-encoded proteins. Experiments were conducted with several mutant forms of p37mos to determine whether or not the kinase function correlates with the biological activity of the mutant v-mos genes. Two mutants lacking cell transformation activity, one an arginine substitution for lysine-121 in the putative ATP-binding site and the other a 23-amino acid deletion from the C terminal end of p37mos, had no kinase activity associated with their mutant proteins. However, a third mutant with reduced biological activity had drastically less kinase activity than the wild-type protein. The latter mutant was able to phosphorylate the kinase-inactive p37mos(Arg-121) protein in vitro. These results indicate that even though p37mos(Arg-121) can be phosphorylated in trans by other kinase molecules, it lacks the ability to phosphorylate itself in vitro. This provides a compelling argument that the protein kinase function of p37mos is an intrinsic property of the protein. Moreover, since the kinase function correlates with the cellular transformation activity of the v-mos gene, we predict that it is required for the biological activity of the v-mos gene. PMID- 3023667 TI - Herpesvirus papio contains a plasmid origin of replication that acts in cis interspecies with an Epstein-Barr virus trans-acting function. AB - Herpesvirus papio (HVP) and Epstein-Barr virus (EBV) are closely related biologically and biochemically; lymphoblastoid cells infected with either virus contain episomal viral DNA. The putative origin of replication for EBV plasmids (oriP) has been assigned to a 1,790-base-pair fragment (cis) in the short unique region of the genome which requires a viral function supplied in trans from elsewhere in the genome (J. Yates, N. Warren, D. Reisman, and B. Sugden, Proc. Natl. Acad. Sci. USA 81:3806-3810, 1984). We report here the identification of the putative origin of replication (cis) in HVP; we assigned it to the HVP EcoRI K fragment. The results indicate that the HVP replication process requires both a cis and a trans-acting function, analogous to that found in EBV. PMID- 3023668 TI - Theiler's virus genome is closely related to that of encephalomyocarditis virus, the prototype cardiovirus. AB - Theiler's virus causes a persistent demyelinating infection of the mouse central nervous system. Our study of the molecular mechanism of persistence led us to sequence 1925 nucleotides located at the 3' end of the viral genome. We observed extensive homologies between this region and the corresponding region of encephalomyocarditis virus, the prototype cardiovirus, and only some homologies with the 3' ends of foot-and-mouth disease virus, rhinovirus, and poliovirus genomes. PMID- 3023669 TI - Deletions in vaccine strains of pseudorabies virus and their effect on synthesis of glycoprotein gp63. AB - The pseudorabies virus vaccine strains Norden and Bartha each have been reported to have deletions in the small unique component of the genome (B. Lomniczi, M. L. Blankenship, and T. Ben-Porat, J. Virol. 49:970-979, 1984). The deletion in Norden was shown to delete the entire coding region for gI but not any of the coding sequences for gp63. However, gp63 in Norden-infected cells was only 36 kilodaltons, and a 44-kilodalton form of gp63 was released into the medium. In Bartha, the deletion removed the coding region for all but 89 amino acids of gp63, and no gp63 was detected in either Bartha-infected cells or medium. PMID- 3023670 TI - Sequence of the genome ends and of the junction between the ends in concatemeric DNA of pseudorabies virus. AB - The nucleotide sequences of the termini of the mature pseudorabies virus genome and of the junction between these termini in concatemeric DNA were compared. To ensure conservation of unmodified 5' and 3' termini, the end fragments obtained directly (uncloned) from mature viral DNA were sequenced. The sequence obtained from 5' and 3' end labeling revealed that whereas the L terminus was blunt ended, the S terminus had a 2-base (GG) 3' overhang. The sequences spanning the junction between the termini present in concatemeric DNA was also determined and compared with that expected when the two ends of the mature DNA were juxtaposed. This comparison showed that in concatemeric DNA the ends of the mature genome had become joined by blunt-end ligation of one of the strands and that the 2 nucleotide gap on the other strand had been repaired. A significant degree of homology between the sequences spanning the junction between the ends of the varicella-zoster virus and pseudorabies virus genomes was found. PMID- 3023671 TI - Progressive reorganization of the host cell cytoskeleton during adenovirus infection. AB - Infection of cells with adenovirus lead to a characteristic reorganization of all cytoskeleton systems, starting with alterations at the microtubuli of the cells. During this progress, the flat, extended, and polar morphology of the cytoskeleton became nonpolar and rounder. These rearrangements were initiated before the appearance of adenovirus structural proteins hexon and fiber, as well as before the shutoff of host protein synthesis. We conclude that these alterations reflect a specific reorganization rather than an unorganized breakdown of the cell during adenovirus infection. PMID- 3023672 TI - Identification of the trans-acting Rep proteins of adeno-associated virus by antibodies to a synthetic oligopeptide. AB - Prior genetic analysis provided evidence for trans-acting regulatory proteins (Rep) coded by the left-hand open reading frame (orf-1) of adeno-associated virus (AAV). We have used immunoblotting analysis to identify four protein products of orf-1. Antibodies elicited against an oligopeptide encoded by orf-1 were reacted with extracts of cells that were infected with AAV or transfected with AAV recombinant vectors in the presence or absence of helper adenovirus. The antibody recognized four polypeptides with apparent molecular weights of 78,000, 68,000, 52,000, and 40,000. The 78,000-dalton (78K) (Rep78) and 68K (Rep68) proteins appear to be encoded by the unspliced 4.2-kilobase (kb) and spliced 3.9-kb mRNAs, respectively, transcribed from the p5 promoter. The 52K (Rep52) and 40K (Rep40) proteins appear to be the products of the unspliced 3.6-kb and the spliced 3.3-kb mRNAs, respectively, transcribed from the p19 promoter. Rigorous identification of Rep68 as an AAV-coded protein is compromised by a cross-reacting cellular protein of similar size. All four proteins were expressed in the human cell lines 293, HeLa, HT29, and A549 infected with AAV together with adenovirus. Rep78 and Rep52 were detected at lower levels in cells infected with AAV at high multiplicity in the absence of adenovirus. Human 293 cells transfected with a recombinant AAV vector (pAV2) also expressed Rep proteins in the presence or absence of adenovirus. Mutations introduced into the Rep region of pAV2 further identified the Rep proteins. The amount of each Rep protein varied between nuclear and cytoplasmic extracts, but all four proteins accumulated during the lytic cycle of the viral infection. Other studies have indicated that the Rep proteins have independent trans-acting functions in viral DNA replication and negative and positive regulation of gene expression. Correlation of each trans acting function with individual Rep proteins will be facilitated with the antibodies described herein. PMID- 3023673 TI - Genetic analysis of p60v-src domains involved in the induction of different cell transformation parameters. AB - The expression of p60v-src in chicken cells infected with Rous sarcoma virus causes stimulation of cell proliferation, morphological alteration, and anchorage independence. PA101 and PA104 are temperature-sensitive variants encoding mutant p60v-src proteins that are partially defective in the induction of these transformation parameters. To define the structural basis for the transformation defectiveness of the p60v-src mutants, the v-src genes of PA101 and PA104 were molecularly cloned and analyzed. Amino- and carboxy-terminal coding regions of the cloned mutant genes were exchanged with the corresponding regions of cloned wild-type v-src and chicken c-src genes, reconstructed into viral DNA, and expressed in infected cells maintained at various temperatures. This analysis revealed that lesions within the tyrosine kinase domains of the two mutant proteins confer temperature sensitivity on all three transformation functions of p60v-src. An amino-terminal region of the PA101 mutant protein, which coincides with the proposed modulatory domain and appears to interact with the kinase domain, affects morphological alteration in a temperature-independent manner. Our results suggest that the function of the kinase domain is essential to all three parameters examined, whereas the amino-terminal domain is important in determining cell morphology. PMID- 3023674 TI - Biochemical properties of p60v-src mutants that induce different cell transformation parameters. AB - PA101 and PA104 are Rous sarcoma virus variants that are differentially temperature sensitive in cell transformation parameters, including stimulation of cell proliferation, morphological alteration, and anchorage independence. To investigate the biochemical basis for the differential expression of these parameters, the tyrosine kinase activity and subcellular localization of the mutant p60v-src proteins encoded in the variants were examined. Analysis of chimeric src proteins derived from the mutant proteins revealed that lesions in the kinase domain inhibit in vitro kinase activity and confer temperature sensitivity on tyrosine phosphorylation of cellular protein p34 in vivo. The amino-terminal portions of the mutant src proteins also influence tyrosine phosphorylation in vivo and in vitro, which is consistent with an interaction between an amino-terminal region and the kinase domain. Large proportions of the mutant src proteins exist in soluble complexes with cellular proteins p50 and p90, even though the src proteins are myristylated. The formation of these soluble complexes segregates with lesions in the kinase domain and is independent of temperature. Our results demonstrate that the transformation parameters examined correlate to a limited extent with p34 phosphorylation but not with the levels of in vitro kinase activity or soluble complex formation. PMID- 3023676 TI - Rat embryo fibroblast cells expressing human papillomavirus 1a genes exhibit altered growth properties and tumorigenicity. AB - Human papillomavirus 1a (HPV1a) induces benign tumors (papillomas or warts) in humans under natural conditions of infection but has not been found to replicate significantly in cell culture or in experimental animals. To establish model systems to study the oncogenic properties and expression of HPV genes, we established cell lines by cotransfecting the 3Y1 rat fibroblast cell line with HPV1a DNA constructs containing an intact early gene region and the Tn5 neomycin resistance gene. Most cell lines selected for expression of the neomycin resistance gene by treatment with the antibiotic G-418 contained viral DNA in a high-molecular-weight form. The growth characteristics of several cell lines containing high copy numbers of HPV1a DNA were studied further. They were shown to differ from the parental cell line and from G-418-resistant cell lines that did not incorporate viral DNA in the following properties: morphological alteration, increased cell density at confluence, growth in 0.5% serum, efficient anchorage-independent growth in soft agar, and rapid formation of tumors in nude mice. Those cell lines that possessed altered growth properties and tumorigenicity were found to express abundant quantities of polyadenylated virus specific RNA species in the cytoplasm. PMID- 3023675 TI - Genetic lesions involved in temperature sensitivity of the src gene products of four Rous sarcoma virus mutants. AB - The src genes of four Rous sarcoma virus (RSV) mutants temperature-sensitive (ts) for cell transformation were analyzed. The mutant src genes were cloned into a replication-competent RSV expression vector, and the contribution of individual mutations to the ts phenotype was assessed by in vitro recombination with wild type src sequences. Three of the mutants, which were derived from the Schmidt Ruppin strain of RSV, each encoded two mutations within the conserved kinase domain. In all three cases, one of the two mutations was an identical valine to methionine change at amino acid position 461. Virus encoding recombinant src genes containing each of these mutations alone were not ts for transformation, demonstrating that two mutations are required for temperature sensitivity. The sequence of the src gene of the Bryan high-titer strain of RSV was determined and compared with that of the fourth ts mutant which was derived from it, again revealing two lesions in the kinase domain of the mutant. PMID- 3023677 TI - In vivo transcription of bacteriophage phi 29 DNA: transcription initiation sites. AB - The initiation sites of the RNA transcripts synthesized in vivo in Bacillus subtilis infected with bacteriophage phi 29 have been mapped by S1 protection experiments. Nine transcription initiation sites were localized along the entire phi 29 genome, close to previously reported B. subtilis and Escherichia coli RNA polymerase-binding sites. Eight of these sites corresponded to early transcription and only one corresponded to late transcription. By using 5'-end labeled RNA, four of the early sites and the late one were shown to be the main sites where initiation of transcription occurs in vivo in the phi 29 genome. PMID- 3023678 TI - Phosphorylation downregulates the DNA-binding activity of simian virus 40 T antigen. AB - Proteolytic fragments of simian virus 40 tumor (T) antigen and T antigen that was dephosphorylated with alkaline phosphatase bound between 1.5 to 2 times more origin-containing simian virus 40 DNA than did intact T antigen in DNA saturation experiments. Kinetic experiments showed that these treatments also enhanced the rate at which T antigen bound to the DNA. The enhanced binding of T-antigen fragments correlated with the generation of DNA-binding fragments that lacked the NH2-terminal region. Dephosphorylation of T antigen in vitro resulted in the removal of phosphate groups from the NH2-terminal region as well as from the COOH terminal region. To test the effects of dephosphorylation on the size of the protein, immunoaffinity-purified T antigen was subjected to sedimentation with and without prior treatment with alkaline phosphatase. Most of the purified protein sedimented as a monomer and no significant effect was observed after dephosphorylation, indicating that the enhanced DNA-binding activity was probably not due to the uncovering of additional binding sites buried specifically in oligomerized T antigen. Taken together, these results indicate that in vivo phosphorylation of the NH2-terminal region (residues 106 to 124) decreases the binding of the protein to the DNA origin. The effect is reversed by in vitro dephosphorylation or by proteolysis which removes the highly phosphorylated NH2 terminal arm of the polypeptide. We suggest that phosphorylation inactivates one of two distinct DNA-binding activities on the polypeptide chain perhaps corresponding to two separate regions in T antigen. PMID- 3023680 TI - Cas-Br-E murine leukemia virus: sequencing of the paralytogenic region of its genome and derivation of specific probes to study its origin and the structure of its recombinant genomes in leukemic tissues. AB - The ecotropic Cas-Br-E murine leukemia virus (MuLV) and its molecularly cloned derivative pBR-NE-8 MuLV are capable of inducing hind-limb paralysis and leukemia after inoculation into susceptible mice. T1 oligonucleotide fingerprinting, molecular hybridization, and restriction enzyme analysis previously showed that the env gene of Cas-Br-E MuLV diverged the most from that of other ecotropic MuLVs. To analyze proviruses in leukemic tissues, we derived DNA probes specific to Cas-Br-E sequences: two from the env region and one from the U3 long terminal repeat. With these probes, we found that this virus induced clonal (or oligoclonal) tumors and we documented the presence of typical mink cell focus forming-type proviruses in leukemic tissues and the possible presence of other recombinant MuLV proviruses. Since the region harboring the determinant of paralysis was mapped within the pol-env region of the virus (L. DesGroseillers, M. Barrette, and P. Jolicoeur, J. Virol. 52:356-363, 1984), we performed the complete nucleotide sequence of this region covering the 3' end of the genome. We compared the deduced amino acid sequences of the pol carboxy-terminal domain and of the env gene products with those of other nonparalytogenic, ecotropic, and mink cell focus-forming MuLVs. This amino acid comparison revealed that this part of the pol gene product and the p15E diverged very little from homologous proteins of other MuLVs. However, the Cas-Br-E gp70 sequence was found to be quite divergent from that of other MuLVs, and the amino acid changes were distributed all along the protein. Therefore, gp70 remains the best candidate for harboring the determinant of paralysis. PMID- 3023679 TI - Strain-specific transcription and translation of the BamHI Z area of Epstein-Barr Virus. AB - The expression of the 1,800-base-pair BamHI Z region of Epstein-Barr virus DNA was analyzed by hybrid-selected translation with several DNA subclones and RNA from different cell lines. Furthermore, large segments of the three reading frames extending in this area were expressed as fusion proteins into Escherichia coli. The fusion proteins were partially purified and used to immunize rabbits. These sera were used to confirm our mapping assignments and to identify the respective posttranslationally modified proteins in in vivo labeling experiments. The reading frame BRLF1 (the first reading frame starting in the BamHI R fragment in leftward orientation) encoded a 93- to 96-kilodalton (kDa) protein depending on the cell line. The molecular weight of in vivo-labeled proteins was increased relative to that of in vitro-translated proteins, indicating that a posttranslational modification had occurred. The BZLF1 reading frame encoded a 35 kDa protein. It was posttranslationally cleaved from a 38-kDa precursor in induced B95-8 and induced Raji cells and from a 40-kDa precursor in induced P3HR1 cells. In Raji cells superinfected with virus derived from P3HR1 cells, the protein seemed to be expressed both from endogenous Raji genomes and from infecting genomes. The transcripts for the 93- to 96-kDa and the 35-kDa protein overlapped partially. The serum against the expressed third reading frame BZLF2 specifically precipitated a 140-kDa protein. This reading frame contains only 650 nucleotides, and therefore further coding sequences were presumably spliced to BZLF2. The latter is deleted in the Raji cell line; therefore, the observed 140kDa protein in superinfected Raji cells was expressed from infecting P3HR1 genomes. PMID- 3023682 TI - Spacing between simian virus 40 early transcriptional control sequences is important for regulation of early RNA synthesis and gene expression. AB - We have analyzed the effect of insertion mutants between the simian virus 40 (SV40) 21-base pair (bp) repeats and the early-early (EE) TATA sequence. Insertion of 4, 42, or 90 bp of DNA at the SV40 NcoI site (map position 37) has been analyzed for its effect on expression of the SV40 early gene and positioning of the RNA 5' ends. Insertion of 4 bp reduced SV40 early promoter-dependent chloramphenicol acetyltransferase (CAT) expression by six- to eightfold. Increasing the size of the insertion to 42 or 90 bp resulted in a further drop in early gene expression to basal levels. At the RNA level, the 4-bp insertion reduced EE RNA synthesis approximately 10-fold. No concomitant increase in late early (LE) RNA synthesis was observed. Insertion of 42 or 90 bp of DNA resulted in a decrease of EE RNA synthesis and a stimulation of LE RNA synthesis. Deletion of the SV40 72-bp repeats from the insertion mutants demonstrated that some, but not all, of the LE RNA depends upon the presence of these sequences. These studies suggest that the ability of RNA polymerase II to utilize the EE (TATA directed) transcriptional control sequence requires an interaction with the upstream 21-bp repeats or the 72-bp repeats or both. That LE RNA levels in pJI1 in42 CAT and pJI1-in90 CAT were equivalent to the level of EE RNA in pJI1-CAT, yet the level of CAT gene expression was decreased greater than 10-fold, suggests that LE mRNA is under translational control and probably prefers a 5' initiation codon proximal to that of the CAT gene. PMID- 3023681 TI - Identification and nucleotide sequence of the thymidine kinase gene of Shope fibroma virus. AB - The thymidine kinase (TK) gene of Shope fibroma virus (SFV), a tumorigenic leporipoxvirus, was localized within the viral genome with degenerate oligonucleotide probes. These probes were constructed to two regions of high sequence conservation between the vaccinia virus TK gene and those of several known eucaryotic cellular TK genes, including human, mouse, hamster, and chicken TK genes. The oligonucleotide probes initially localized the SFV TK gene 50 kilobases (kb) from the right terminus of the 160-kb SFV genome within the 9.5-kb BamHI-HindIII fragment E. Fine-mapping analysis indicated that the TK gene was within a 1.2-kb AvaI-HaeIII fragment, and DNA sequencing of this region revealed an open reading frame capable of encoding a polypeptide of 176 amino acids possessing considerable homology to the TK genes of the vaccinia, variola, and monkeypox orthopoxviruses and also to a variety of cellular TK genes. Homology matrix analysis and homology scores suggest that the SFV TK gene has diverged significantly from its counterpart members in the orthopoxvirus genus. Nevertheless, the presence of conserved upstream open reading frames on the 5' side of all of the poxvirus TK genes indicates a similarity of functional organization between the orthopoxviruses and leporipoxviruses. These data suggest a common ancestral origin for at least some of the unique internal regions of the leporipoxviruses and orthopoxviruses as exemplified by SFV and vaccinia virus, respectively. PMID- 3023683 TI - Regulation of transcription in vitro from herpes simplex virus genes. AB - In vitro transcription assays were carried out by using as templates DNAs cut from the herpes simplex virus early glycoprotein D gene, the late glycoprotein C gene, the late VP5 gene, and the immediate-early ICP22 gene. Nuclear extracts from suspension cultures of uninfected HeLa cells effectively synthesized RNAs from genes of the immediate-early and delayed-early classes. To a lesser extent, the extracts also used DNAs cut from the late genes as templates. Transcription from the immediate-early gene was inhibited in extracts prepared from infected cells. Analysis of the proteins in infected-cell extracts by gel electrophoresis, transfer to nitrocellulose, and probing with specific antibody demonstrated the presence of the viral regulatory protein ICP4. Chromatographic fractionation of nuclear extract from infected cells yielded a mixture of proteins (fraction VIII) enriched in ICP4 (S.W. Faber and K.W. Wilcox, Nucleic Acids Res., 14:6067-6083, 1986). Addition of fraction VIII to the in vitro assay affected transcription. Depending on the DNA in the assay, an inhibitory or stimulatory effect was observed. Inhibition of RNA synthesis was found when DNA from the immediate-early gene was used as a template, and stimulation was found when DNA from the early or late gene was used. PMID- 3023686 TI - Nonecotropic murine leukemia viruses in BALB/c and NFS/N mice: characterization of the BALB/c Bxv-1 provirus and the single NFS endogenous xenotrope. AB - We used hybridization probes that react specifically with xenotropic and mink cell focus-forming virus envelope sequences to characterize the nonecotropic proviruses of BALB/c and NFS/N mice. Analysis of somatic cell hybrids with different BALB/c chromosomes showed that the 9 xenotropic and more than 20 MCF virus-related proviral sequences in this mouse were present on more than nine BALB/c chromosomes. Multiple copies were found on chromosomes 1, 4, 7, 12, and probably 11, and the copies found on a single chromosome were not identical by restriction enzyme mapping. We also identified and characterized the proviral sequences that give rise to infectious xenotropic virus in both BALB/c and NFS/N mice. BALB/c contains the major locus for induction of infectious virus in inbred mice, Bxv-1, which is on chromosome 1. We showed that this locus contains a single xenotropic provirus on an 18-kilobase HindIII fragment. Restriction enzyme analysis of a hybrid cell DNA that contains only the Bxv-1 xenotropic provirus showed that the Bxv-1 provirus contains restriction enzyme sites characteristic of the infectious virus induced from BALB/c fibroblasts. The Bxv-1 provirus and its flanking sequences also contain the same restriction sites as the provirus thought to contribute U3 long terminal repeat sequences to leukemogenic (class I) AKR MCF viruses. Analysis of cell hybrids made with the nonvirus-inducible strain NFS/N showed that the single xenotropic virus env gene of NFS mice, here termed Nfxv-1, is not on chromosome 1. Unlike that of Bxv-1, the restriction map of Nfxv 1 does not resemble that of any known infectious xenotropic virus including xenotropic viruses isolated from NFS mice. These data suggest that Bxv-1, but not Nfxv-1, is a full-length xenotropic provirus that can be transcribed directly to produce infectious virus. PMID- 3023685 TI - Conservation of the fourth gene among rotaviruses recovered from asymptomatic newborn infants and its possible role in attenuation. AB - RNA-RNA hybridization was performed to assess the extent of genetic relatedness among human rotaviruses isolated from children with gastroenteritis and from asymptomatic newborn infants. 32P-labeled single-stranded RNAs produced by in vitro transcription from viral cores of the different strains tested were used as probes in two different hybridization assays: undenatured genomic RNAs were resolved by polyacrylamide gel electrophoresis, denatured in situ, electrophoretically transferred to diazobenzyloxymethyl-paper (Northern blots), and then hybridized to the probes under two different conditions of stringency; and denatured genomic double-stranded RNAs were hybridized to the probes in solution and the hybrids which formed were identified by polyacrylamide gel electrophoresis. When analyzed by Northern blot hybridization at a low level of stringency, all genes from the strains tested cross-hybridized, providing evidence for some sequence homology in each of the corresponding genes. However, when hybridization stringency was increased, a difference in gene 4 sequence was detected between strains recovered from asymptomatic newborn infants ("nursery strains") and strains recovered from infants and young children with diarrhea. Although the nursery strains exhibited serotypic diversity (i.e., each of the four strains tested belonged to a different serotype), the fourth gene appeared to be highly conserved. Similarly, each of the virulent strains tested belonged to a different serotype; nonetheless, there was significant conservation of sequence among the fourth genes of three of these viruses. Significantly, the conserved fourth genes of the nursery strains were distinct from the fourth gene of each of the virulent viruses. These results were confirmed and extended during experiments in which the RNA-RNA hybridization was carried out in solution and the resulting hybrids were analyzed by polyacrylamide gel electrophoresis. Under these conditions, the fourth genes of the nursery strains were closely related to each other but not to the fourth genes of the virulent viruses. Full-length hybrids did not form between the fourth genes from the nursery strains and the corresponding genes from the strains recovered from symptomatic infants and young children. PMID- 3023687 TI - Amino-terminal sequence, synthesis, and membrane insertion of glycoprotein B of herpes simplex virus type 1. AB - Glycoprotein B (gB) was purified from cells infected with two strains (KOS and F) of herpes simplex virus type 1. Determination of amino acid sequence at the NH2 termini revealed, by comparison with amino acid sequence deduced from previously published nucleotide sequence, that gB is made with a cleavable signal sequence of 29 or 30 amino acids, depending on the virus strain. Analysis of gB translated in vitro in the presence and absence of membranes showed that gB is inserted into membranes and glycosylated cotranslationally; a large portion of the gB polypeptide made in vitro is protected from proteolysis by membranes; the large protected fragment carries N-linked carbohydrate and is probably the NH2 terminus based on locations of signals for the addition of N-linked carbohydrate; and the size of the protected fragment is 93 kilodaltons (kDa) for gB made in vitro and associated with dog pancreas membranes, whereas both 93- and 98-kDa protected fragments can be detected for gB made in vivo. These last results are consistent with a previous proposal that gB may traverse the membrane three times. PMID- 3023684 TI - Construction and characterization of hybrid polyomavirus genomes. AB - Several studies have suggested that certain unique features of the JC virus (JCV) regulatory region are responsible for the restricted lytic and transforming activities of this virus in vitro. To pursue this possibility, we have constructed hybrid polyomavirus genomes by exchanging the regulatory sequences of JCV, BK virus (BKV), and simian virus 40 (SV40). The host range of JCV was not expanded by the substitution of the BKV or SV40 regulatory signals; such hybrids were nonviable even in primary human fetal glial cells, the sole permissive cell for JCV. However, chimeric DNAs containing JCV regulatory sequences and BKV- or SV40-coding sequences were lytically active, indicating that the BKV and SV40 T proteins were capable of effectively interacting with the JCV replication and transcription signals to yield infectious hybrid viruses. Although JCV regulatory sequences and coding sequences both contributed to the restricted lytic activity of this virus, it appears that the latter sequences, most likely hose encoding the T protein, have a greater influence on this behavior. PMID- 3023688 TI - Kinetics of expression of herpes simplex virus type 1-specific glycoprotein species on the surfaces of infected murine, simian, and human cells: flow cytometric analysis. AB - The kinetics of expression of the herpes simplex virus type 1-encoded major glycoprotein species gB, gC, gD, and gE on the surfaces of cells of murine, simian, and human origins were studied. Viable cells were stained with monoclonal antibodies specific for each species, and the levels expressed were determined by fluorescence flow cytometry. Differences were observed in both the kinetics and the levels of expression of individual glycoprotein species, depending upon the origin of the host cells. Glycoprotein gC was expressed early and at high levels in cells of murine and human origins, but late and at relatively low levels in simian cells. In contrast, gE was expressed at high levels in simian cells, but was not detectable until late in the infectious cycle in murine and human cells. The kinetics and levels of expression of gB were similar for all cells investigated, whereas gD, with high levels of expression in all cells late in infection, appeared on the surfaces of murine cells very early postinfection. This approach has allowed a simple quantitative method for comparing levels of glycoprotein expression. PMID- 3023689 TI - Genomic localization, sequence analysis, and transcription of the putative human cytomegalovirus DNA polymerase gene. AB - The human cytomegalovirus (HCMV)-induced DNA polymerase has been well characterized biochemically and functionally, but its genomic location has not yet been assigned. To identify the coding sequence, cross-hybridization with the herpes simplex virus type 1 (HSV-1) polymerase gene was used, as suggested by the close similarity of the herpes group virus-induced DNA polymerases to the HCMV DNA polymerase. A cosmid and plasmid library of the entire HCMV genome was screened with the BamHI Q fragment of HSV-1 at different stringency conditions. One PstI-HincII restriction fragment of 850 base pairs mapping within the EcoRI M fragment of HCMV cross-hybridized at Tm - 25 degrees C. Sequence analysis revealed one open reading frame spanning the entire sequence. The amino acid sequence showed a highly conserved domain of 133 amino acids shared with the HSV and putative Epstein-Barr virus polymerase sequences. This domain maps within the C-terminal part of the HSV polymerase gene, which has been suggested to contain part of the catalytic center of the enzyme. Transcription analysis revealed one 5.4-kilobase early transcript in the sense orientation with respect to the open reading frame identified. This transcript appears to code for the 140-kilodalton HCMV polymerase protein. PMID- 3023690 TI - Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene. AB - DNA sequence analysis has revealed that the gene coding for the human cytomegalovirus (HCMV) DNA polymerase is present within the long unique region of the virus genome. Identification is based on extensive amino acid homology between the predicted HCMV open reading frame HFLF2 and the DNA polymerase of herpes simplex virus type 1. We present here a 5280-base-pair DNA sequence containing the HCMV pol gene, along with the analysis of transcripts encoded within this region. Since HCMV pol also shows homology to the predicted Epstein Barr virus pol, we were able to analyze the extent of homology between the DNA polymerases of three distantly related herpesviruses, HCMV, Epstein-Barr virus, and herpes simplex virus. The comparison shows that these DNA polymerases exhibit considerable amino acid homology and highlights a number of highly conserved regions; two such regions show homology to sequences within the adenovirus type 2 DNA polymerase. The HCMV pol gene is flanked by open reading frames with homology to those of other herpesviruses; upstream, there is a reading frame homologous to the glycoprotein B gene of herpes simplex virus type 1 and Epstein-Barr virus, and downstream there is a reading frame homologous to BFLF2 of Epstein-Barr virus. PMID- 3023691 TI - Characterization of a transcriptional promoter of human papillomavirus 18 and modulation of its expression by simian virus 40 and adenovirus early antigens. AB - RNA present in cells derived from cervical carcinoma that contained human papillomavirus 18 genomes was initiated in the 1.053-kilobase BamHI fragment that covered the complete noncoding region of this virus. When cloned upstream of the chloramphenicol acetyltransferase gene, this viral fragment directed the expression of the bacterial enzyme only in the sense orientation. Initiation sites were mapped around the ATG of open reading frame E6. This promoter was active in some human and simian cell lines, and its expression was modulated positively by simian virus 40 large T antigen and negatively by adenovirus type 5 E1a antigen. PMID- 3023692 TI - Physical mapping of two herpes simplex virus type 1 host shutoff loci: rescue of each ts mutation occurs with two unique cloned regions of the viral genome. AB - Two complementing temperature-sensitive (ts) herpes simplex virus type 1 (HSV-1) mutants, PAA1rts1 and ts199, were defective in viral DNA synthesis and in the shutoff of cellular macromolecular synthesis at 39.5 degrees C, the nonpermissive temperature. PAA1sts1 and PAA1rts1+ recombinants and PAA1rts1+ revertants were used to examine the contributions of the PAA1r mutation and the ts1 mutation of PAA1rts1 in affecting the levels of viral and cellular DNA synthesized at 34 and 39.5 degrees C. The results of this study suggests an interaction between the viral DNA polymerase and the ts1+ gene product during HSV-1 DNA replication and possibly in the inhibition of host DNA synthesis by HSV-1. Physical mapping of the ts mutations present in ts199 and the PAA1sts1 recombinant ts1-8 were performed by intratypic marker rescue experiments. Surprisingly, both the ts1-8 and ts199 mutations were rescued by two cloned fragments: ts1-8 by BglII-K (map coordinates 0.095 to 0.163) and BglII-I (map coordinates 0.314 to 0.417), while ts199 was rescued by BglII-K and BglII-O (map coordinates 0.163 to 0.197). In more refined mapping experiments, the regions between coordinates 0.347 to 0.378 and 0.126 to 0.163 were able to rescue the ts1-8 mutation. Southern hybridization analysis confirmed that the fragments that rescued ts1-8 and those that rescued ts199 had homology, as predicted by the physical mapping results. PMID- 3023693 TI - Preferential clustering of viral DNA sequences at or near the site of chromosomal rearrangement in fowl adenovirus type 1 DNA-transformed cell lines. AB - All six transformants obtained by inoculating fowl adenovirus type 1 (CELO virus) DNA or its fragments into a rat cell line of normal karyotype had more than 50 copy-equivalents of viral DNA sequences. Each of the transformants had almost all if not all of these viral DNA sequences clustered on a marker chromosome(s). Although the marker chromosome(s) differed from one cell line to another, viral DNA sequences preferentially clustered in or near the achromatic (or light stained) region of the G-banded marker chromosomes where chromosomal rearrangement or translocation occurred. These results indicate that no particular chromosome is required to act as the integration site of viral DNA for the transformation of cells, but chromosomal rearrangement at or near the cluster of viral DNA sequences might contribute to the transformation. PMID- 3023695 TI - Noncoding region between the env and src genes of Rous sarcoma virus influences splicing efficiency at the src gene 3' splice site. AB - Viral RNA and proteins in chicken embryo fibroblasts infected with different cloned variants of the Prague strain Rous sarcoma virus (RSV) were analyzed. The ratio of immunoprecipitated pp60src to the gag gene product p27 in Prague A (PrA) and Prague B (PrB) RSV-infected cells was two to three times that in Prague C (PrC) RSV-infected cells. A significant increase in the steady-state ratio of spliced 2.7-kilobase src gene mRNA to unspliced 9.3-kilobase genome-size RNA was observed in PrA- and PrB- compared with PrC-infected cells, consistent with the differences in the ratios of the gag to src gene protein products. Similar results were obtained when hybrid-selected RNA, which had been labeled for 3 h with [3H]uridine, was analyzed on formaldehyde-agarose gels, suggesting that the observed differences were due to splicing rather than RNA stability. Recombinant plasmids from infectious molecular clones of PrA and PrC were constructed to localize the regions responsible for the effects on src gene splicing. The substitution in place of the corresponding PrA region of the 262-base-pair region between the env gene and the src gene coding sequences from the PrC clone into the infectious PrA plasmid conferred the low src splicing efficiency of the PrC strain. The nucleotide sequence of this region of the PrA plasmid was determined and compared with the sequence of the PrC strain. Only four nucleotide differences were found; two changes were within the intron sequence, and two were in the exon sequence. The possible role of these differences in determining the extent of viral RNA splicing is discussed. PMID- 3023694 TI - Characterization of the simian virus 40 late promoter: relative importance of sequences within the 72-base-pair repeats differs before and after viral DNA replication. AB - We examined sequences involved in the simian virus 40 (SV40) late promoter in vivo, by using quantitative S1 nuclease analysis of a series of deletion mutants within the SV40 regulatory region. These mutants were constructed so as to place the altered promoter region in its normal position relative to the SV40 late genes. The effects of the deletions on late transcriptional activity were analyzed before and after viral DNA replication, by omitting or including SV40 large T antigen. The data show that (i) in the absence of large T antigen, the deletion of the 21-base-pair (bp) repeats results in a fourfold increase in late transcription, and (ii) the sequences within the 72-bp repeats are a component of the SV40 late promoter, acting not only before, but also after viral DNA replication. We identified two domains which contain sequences important for efficient late transcription. Domain I, at the late proximal end of each 72-bp repeat, was found to function before replication and was possibly also involved after replication. The contribution of domain II, at the late distal end of each 72-bp repeat, was much more significant after replication but only of minor importance before replication. PMID- 3023696 TI - Genetic resistance to mouse hepatitis virus correlates with absence of virus binding activity on target tissues. AB - The molecular mechanism of genetic resistance of inbred mouse strains to mouse hepatitis virus, a murine coronavirus, was studied by comparing virus binding to plasma membranes of intestinal epithelium or liver from susceptible BALB/c and resistant SJL/J mice with a new solid-phase assay for virus-binding activity. Virus bound to isolated membranes from susceptible mice, but not to membranes from resistant mice. F1 progeny of SJL/J X BALB/c mice had an intermediate level of virus-binding activity on their enterocyte and hepatocyte membranes. This correlated well with previous studies showing that susceptibility to mouse hepatitis virus strain A59 is controlled by a single autosomal dominant gene (M. S. Smith, R. E. Click, and P. G. W. Plagemann, J. Immunol. 133:428-432). Because virus binding was not prevented by treating membranes with sodium dodecyl sulfate, the virus-binding molecule could be identified by a virus overlay protein blot assay. Virus bound to a single broad band of Mr 100,000 to 110,000 in membranes from hepatocytes or enterocytes of susceptible BALB/c and semisusceptible C3H mice, but no virus-binding band was detected in comparable preparations of resistant SJL/J mouse membranes. Therefore, SJL/J mice may be resistant to mouse hepatitis virus A59 infection because they lack a specific virus receptor which is present on the plasma membranes of target cells from genetically susceptible BALB/c and semisusceptible C3H mice. PMID- 3023699 TI - A new common integration region (int-3) for mouse mammary tumor virus on mouse chromosome 17. AB - Mus musculus subsp. musculus (Czech II) mammary tumor DNA frequently contains an integrated proviral genome of the mouse mammary tumor virus (MMTV) within a specific 0.5-kilobase-pair region of the cellular genome (designated int-3). Viral integration at this site results in activation of expression of an adjacent cellular gene. We mapped int-3 to mouse chromosome 17 by analysis of PstI restricted cellular DNAs from mouse-hamster somatic cell hybrids. Restriction analysis of cellular DNA from (C3H/OuJ X Czech II) X Czech II backcross mice established the gene order T-H-2-int-3. These results demonstrated that the int-3 locus is distinct from two other common integration regions for mouse mammary tumor virus (designated int-1 and int-2) in mammary tumor DNA and suggest that several cellular genes may be at risk for virally induced activation during mammary tumor development. PMID- 3023697 TI - Comparison of upstream sequence requirements for positive and negative regulation of a herpes simplex virus immediate-early gene by three virus-encoded trans acting factors. AB - Using a short-term cotransfection system with recombinant chloramphenicol acetyltransferase (CAT) target genes and intact genes for regulatory proteins, we previously demonstrated that expression from the promoter-regulatory region of the gene for the immediate-early 175,000-molecular-weight (IE175K) protein of herpes simplex virus type 1 was subject to trans-acting effects by three different virus-encoded components. In the present work we have attempted to delineate the upstream cis-acting requirements within the IE175K promoter regulatory region for stimulation by the late structural protein Vmw65, stimulation by the IE110K protein, and repression by its own gene product, the IE175K protein. Our results augment previous reports of others by demonstrating that a construct containing only the single TAATGARAT consensus sequence, TAATGGAAT, between -115 and -106 was efficiently induced by Vmw65. Deletion to 108 effectively abolished the response to Vmw65. However, this latter construct remained responsive to IE110K stimulation and was induced as efficiently as the parental construct which contained sequences to -1900. Furthermore, not only basal levels of expression, but also Vmw65 activation of the parental construct and deletion mutants delta 380, delta 330, delta 300, and delta 160 and IE110K activated expression of the delta 108 construct were all subject to dominant repression by the IE175K protein. Finally, we show that expression from each of the deletions was open to stimulation by linkage to the simian virus 40 enhancer region. Enhancer-stimulated expression from each construct, including the -108 deletion, was efficiently repressed by the IE175K protein. In contrast, expression from the simian virus 40 enhancer when linked to its own promoter was unaffected by IE175K. These results place sequence requirements for both IE110K stimulation and IE175K autoregulation within the minimal promoter region -108 to +30, separate from the major requirements for Vmw65 activation located further upstream. PMID- 3023698 TI - Genome RNA terminus conservation and diversity among vesiculoviruses. AB - Sequence analysis of the RNA genome termini of various vesiculovirus standard and defective interfering (DI) particles demonstrated that some virus regulatory sequences and domains of virus N protein are highly conserved while others show considerable divergence. Clearly, distinct RNA signal sequences and protein coding regions of these virus genomes have quite different evolutionary pressures or constraints. Terminal regions of DI-particle RNA genomes of these viruses were found to possess self-complementary stems at the RNA termini, demonstrating the conservation of this DI-particle structural feature throughout the vesiculovirus group. A high degree of conservation of the 3'-terminal sequences of recent and historic isolates of vesicular stomatitis virus New Jersey was also demonstrated. PMID- 3023700 TI - Effects of S-adenosylhomocysteine and analogs on Epstein-Barr virus-induced transformation, expression of the Epstein-Barr virus capsid antigen, and methylation of Epstein-Barr virus DNA. AB - S-Adenosylhomocysteine was found to have no effect on Epstein-Barr virus-induced transformation of B-lymphocytes and to stimulate viral capsid antigen expression only slightly in the FF41-1 cell line. In contrast, the S-adenosylhomocysteine analogs sinefungin and S-isobutyladenosine inhibited Epstein-Barr virus transformation and induced a significant increase in the numbers of cells expressing the viral capsid antigen. An inverse relationship between levels of viral DNA methylation and gene expression was demonstrated. PMID- 3023701 TI - Varicella-zoster virus complements herpes simplex virus type 1 temperature sensitive mutants. AB - Varicella-zoster virus (VZV) can complement temperature-sensitive mutants of herpes simplex virus. Of seven mutants tested, two, carrying mutations in the immediate-early ICP4 and ICP27 proteins, were complemented. This complementation was not seen in coinfections with adenovirus type 5 or cytomegalovirus. Following transfection into CV-1 cells, a DNA fragment containing the VZV short repeat sequence complemented the ICP4 mutant. These data demonstrate a functional relationship between VZV and herpes simplex virus and have allowed localization of a putative VZV immediate-early gene. PMID- 3023702 TI - Replication and virulence of pseudorabies virus mutants lacking glycoprotein gX. AB - Pseudorabies virus (PRV) glycoprotein gX accumulates in the medium of infected cells. In an attempt to study the function of gX, two viruses were constructed that lacked a functional gX gene. One virus, PRV delta GX1, was derived by insertion of the herpes simplex virus thymidine kinase gene into the gX-coding region. The other virus, PRV delta GXTK-, was derived by subsequent deletion of the inserted herpes simplex virus thymidine kinase gene. Both viruses replicated in cell cultures but produced no gX. Furthermore, PRV delta GX1 was capable of killing mice with a 50% lethal dose of less than 100 PFU. PMID- 3023703 TI - Modification of acute murine cytomegalovirus adrenal gland infection by adoptive spleen cell transfer. AB - Homozygous (nu/nu), athymic nude mice, infected with murine cytomegalovirus (MCMV), develop unremitting and ablative virus infection involving both the adrenal cortex and medulla. During acute infection, adoptive transfer of MCMV immune, but not naive, spleen cells suppressed virus replication in the adrenal glands, but not the lungs or salivary gland. T lymphocytes, not macrophages or B cells, were responsible for limiting viral replication. The effect by donor cells was restricted by compatibility at the major histocompatibility locus. Restriction of MCMV replication in the adrenal gland was associated with T lymphocytes of the L3T4 phenotype. Thus, T-cell immunity is critical in regulating MCMV replication in the adrenal glands, and T lymphocytes restricted by class II major histocompatibility antigens mediate this effect. PMID- 3023704 TI - Ribavirin cures cells of a persistent infection with foot-and-mouth disease virus in vitro. AB - Ribavirin (1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) eliminates foot-and-mouth disease virus from persistently infected cell cultures. The latter are 10-fold more sensitive to ribavirin than lytically infected cells. In treated cells no viral RNA or proteins could be detected by dot-blot hybridization to cDNA probes, virus and RNA infectivity assays, or immunofluorescence. A potential application of the drug for the treatment of animals carrying the virus is suggested. PMID- 3023705 TI - Identification of two forms of an endogenous murine retroviral env gene linked to the Rmcf locus. AB - The Rmcf gene restricts the replication of recombinant murine mink cell focus inducing (MCF) viruses in cell cultures derived from mice carrying the resistance allele (Rmcfr) and may play a role in resistance to retrovirus-induced leukemias in vivo. We have characterized the endogenous gp70 expressed by Rmcfr and Rmcfs mice with a panel of type-specific monoclonal antibodies which discriminate xenotropic and MCF gp70. Embryo and tail skin cultures derived from Rmcfr mice (DBA/2 and CBA/N) expressed gp70 bearing a determinant unique to MCF viruses, whereas cultures from Rmcfs mice expressed either no detectable gp70 (NFS/N and IRW) or a gp70 serologically related to a subgroup of xenotropic viruses (C57BL/6, CBA/J, and A/WySn). Studies of progeny embryos derived from a (C57BL/6 X DBA/2) X C57BL/6 backcross established that the Rmcf resistance allele was linked to the expression of the MCF gp70 and that the gene encoding the xenotropic gp70 expressed by C57BL/6 Rmcfs mice was allelic with the MCF gp70 from Rmcfr mice. These data indicate that the Rmcf locus contains an endogenous gp70 gene having two allelic forms, one of which inhibits exogenous MCF infection in vitro by a mechanism of viral interference. PMID- 3023706 TI - Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses. AB - The complete nucleotide sequence of wild-type hepatitis A virus (HAV) HM-175 was determined. The sequence was compared with that of a cell culture-adapted HAV strain (R. Najarian, D. Caput, W. Gee, S.J. Potter, A. Renard, J. Merryweather, G.V. Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985). Both strains have a genome length of 7,478 nucleotides followed by a poly(A) tail, and both encode a polyprotein of 2,227 amino acids. Sequence comparison showed 624 nucleotide differences (91.7% identity) but only 34 amino acid differences (98.5% identity). All of the dipeptide cleavage sites mapped in this study were conserved between the two strains. The sequences of these two HAV strains were compared with the partial sequences of three other HAV strains. Most amino acid differences were located in the capsid region, especially in VP1. Whereas changes in amino acids were localized to certain portions of the genome, nucleotide differences occurred randomly throughout the genome. The most extensive nucleotide homology between the strains was in the 5' noncoding region (96% identity for cell culture-adapted strains versus wild type; greater than 99% identity among cell culture-adapted strains). HAV proteins are less homologous with those of any other picornavirus than the latter proteins are when compared with each other. When the sequences of wild-type and cell culture-adapted HAV strains are compared, the nucleotide differences in the 5' noncoding region and the amino acid differences in the capsid region suggest areas that may contain markers for cell culture adaptation and for attenuation. PMID- 3023707 TI - Integration of the DNA of filamentous bacteriophage Cflt into the chromosomal DNA of its host. AB - It was demonstrated for the first time that filamentous bacteriophage Cflt, which contains single-stranded DNA, can incorporate its genome into that of its host. Evidence in support of the incorporation was obtained from a Southern blot hybridization analysis of DNA isolated from Cflt-lysogenized cells. DNAs from different Cflt-lysogenized cells were purified, and the integration patterns were compared. Because all integration patterns were identical and only one fragment in Cflt replicative-form DNA was missing, it appears that the integration was site specific. Only one complement of viral DNA was integrated per host chromosome. To determine the attachment site on the viral DNA, the physical map of EcoRI, XhoI, SstII, and BglII on Cflt DNA was constructed. Based on this physical map and a Southern blot hybridization analysis of lysogen DNA with these restriction endonucleases, we demonstrated that DNA sequences from all regions of the Cflt genome were represented in the integrated viral sequences. The attachment site on the viral genome was located at 69.2 to 73.8 min on the Cflt DNA. PMID- 3023708 TI - Mammary tumorigenesis in feral mice: identification of a new int locus in mouse mammary tumor virus (Czech II)-induced mammary tumors. AB - A population of Mus musculus subsp. musculus (Czech II), recently isolated from the wild, lack endogenous mouse mammary tumor virus (MMTV) proviral genomes. Some of these mice carry an infectious MMTV [designated MMTV (Czech II)] that is transmitted in the milk and is associated with mammary tumor development. This virus is distinct from laboratory strains of MMTV present in inbred mice. An MMTV (Czech II) genome was found within a 0.5-kilobase region of the cellular genome in five of 16 Czech II mammary tumors. MMTV insertion at this site activates expression of a 2.4-kilobase species of RNA from a previously silent cellular gene. This region of the cellular genome was designated int-3 since it is unrelated to the int-1 and int-2 loci. The int-3 locus does not appear to correspond to other proto-oncogenes but is well conserved among mammalian species. PMID- 3023709 TI - Studies on the sequential development of acute interstitial pneumonia caused by Aleutian disease virus in mink kits. AB - We studied different parameters during the development of acute interstitial pneumonia in mink kits caused by neonatal infection with Aleutian disease virus (ADV). When histological lesions, presence of intranuclear inclusion bodies, and intranuclearly localized ADV antigen were correlated with levels of single stranded virion and duplex replicative forms of ADV DNA in the different tissues, it was concluded that the lung, probably alveolar type II cells, is the major primary target for viral replication and cytopathology. The presence of the duplex dimeric replicative-form DNA, a strong marker of parvovirus replication, was also observed in low amount in the mesenteric lymph node, suggesting replication of ADV in this organ, although no viral cytopathology could be demonstrated. Moreover, a few intranuclear inclusion bodies were demonstrated in kidney and liver from affected kits, but intranuclearly localized ADV antigen could not be demonstrated in liver sections, and neither could duplex dimer replicative-form DNA, suggesting that these organs are nevertheless not a major site of ADV replication. When the data were compared with results previously reported for ADV-infected adult mink and ADV-infected permissive cell cultures, the data suggested that the pattern of ADV replication in alveolar type II cells is similar to that seen in infected cell cultures but that the replication in the other kit organs resembles the restricted pattern seen in adult mink. PMID- 3023711 TI - Aplastic crisis or erythroid hypoplasia. PMID- 3023710 TI - Adult human endothelial cell enzymatic harvesting. Estimates of efficiency and comparison of crude and partially purified bacterial collagenase preparations by replicate microwell culture and fibronectin degradation measured by enzyme-linked immunosorbent assay. AB - Reliable enzymatic endothelial cell (EC) harvest methods are required for clinical EC seeding of vascular prostheses by methods analogous to those demonstrated in dogs. But crude collagenases used for EC harvest vary in efficacy and cytotoxicity, and purified collagenases reportedly give low EC yields. To compare different harvest methods, we studied growth curves of primary adult human saphenous vein EC (HSVEC) harvests plated in replicate microwell cultures. The EC yield, defined as attachment-capable ECs obtained per square centimeter of vein lumen, was estimated from the lowest number of ECs counted in lag phase before exponential growth began. With the use of morphometric studies of HSVs that were perfusion-fixed at their original dimensions, the baseline in situ density of ECs available for harvest from HSV was estimated at 1.3 X 10(5) EC/cm2. Crude (CBC) and partially purified bacterial collagenase (PBC) solutions at concentrations with equal levels of basement membrane lysis activity (BMLA) were compared by the replicate microwell method in a series of 21 harvests (six CBC, eight PBC, and seven enzyme-free control harvests). All 14 enzymatic harvests produced confluent EC cultures with no significant difference in mean harvest efficiency between CBC (12% of in situ EC number) and PBC (15%). However, PBC caused less degradation of human fibronectin (p less than 0.0001) as measured by an enzyme-linked immunosorbent assay employing a fibronectin-specific monoclonal antibody. These data suggest that chemically defined mixtures of pure enzymes with BMLA equal to the BMLA of crude collagenase might allow reliable EC harvesting without sacrifice in EC yield but with improved preservation of structures at the EC periphery. EC losses during initial vein dissection may have contributed to the low 12% to 15% efficiency we observed. PMID- 3023713 TI - [Effects of dopamine and dibutyryl cyclic AMP on coronary circulation in dogs]. PMID- 3023714 TI - [Diagnostic imaging of hepatic tumors]. AB - There are several imaging methods for the detection of small hepatocellular carcinomas (HCC). The combined use of computed tomography (CT) and angiography is an excellent method. CT following intraarterial injection of iodized oil is a sensitive and useful examination for the detection of small hypervascular HCCs. CT during arterial portography is superior in visualizing small hypovascular HCCs. These methods are complementary to each other. Magnetic resonance imaging (MRI) is a new examination. T1 and T2 of the HCC are usually longer than normal hepatic parenchyma. HCCs greater than 2 cm are almost detected by MRI. PMID- 3023712 TI - Cyclosporine improves psoriasis in a double-blind study. AB - In a double-blind trial, 21 patients with severe plaque psoriasis were randomly assigned to receive oral cyclosporine, 14 mg/kg/d, or its vehicle. After four weeks of therapy the 11 cyclosporine recipients had the following response to treatment: two had total clearing and six improved markedly, two moderately, and one minimally; whereas ten vehicle-treated patients showed no change or minimal improvement. Vehicle-treated patients, after a switch to cyclosporine for four weeks, demonstrated impressive improvement similar to that seen in patients who initially received only cyclosporine. Moderate or marked improvement or total clearing was noted in 17 (81%) of 21 and 20 (95%) of 21 after one and four weeks of therapy, respectively. Mitotic figures and leukotriene B4 levels in lesions decreased 86% and 64%, respectively, after seven days of cyclosporine therapy. Mononuclear (including activated T cells) and polymorphonuclear leukocyte infiltrates were markedly reduced in lesions of all patients after seven days of cyclosporine therapy. These results suggest that psoriasis may have an immunologic basis mediated by activated T cells and/or other immune cells; if a long-term regimen with a favorable efficacy-side effect ratio can be determined, cyclosporine would be a significant advance in the treatment of psoriasis. PMID- 3023715 TI - [Ultrasound diagnosis of hepatocellular carcinoma]. AB - Nowadays, ultrasound has been widely used in clinical practice of gastroenterology. A recently developed ultrasound apparatus with a higher resolution of image and less artifacts makes it possible to find out a small HCC sized about 1.0 cm. Moreover, a new convex type of probe with a wide field of view enables us to detect a small HCC locating in the upper part of the right lobe which has been done with difficulty before. Early diagnosis of HCC by detecting a small mass lesion will be carried out reliably by a modern ultrasound modality. PMID- 3023716 TI - [Surgical treatment of hepatocellular carcinoma]. AB - The result of surgical treatment for hepatocellular carcinoma in National Cancer Center Hospital is reported. Almost 80% of the cases in this series are cirrhotic. The cases were divided into two groups according to the period when hepatectomy was done, as the first half, [A], was from 1974 till 1981 when the treatment was only surgical removal and the latter half, [B], was from 1982 till the end of March, 1986 when pre- and post-operative embolization was combined with hepatectomy. The operative mortality rate was 10.1% (9/89) for [A] and 2.2% (3/121) for [B]. The cumulative survival rates of [A] for the 1st-5th year were 65.8%, 52.6%, 39.5%, 32.9% and 27.2%, respectively, and those of [B] were 86.1%, 78.1%, 65.1%, 50.6% and 50.6%. PMID- 3023717 TI - [Diagnosis and treatment of anal carcinoma]. AB - Because of several different histological types of epithelium in the anal canal, various kinds of carcinomas such as adenocarcinoma, squamous carcinoma, cloacogenic carcinoma arise from this region, and carcinoma related to fistula in ano is one of the characteristic malignancies. Though the fundamental treatment of anal carcinoma is rectal amputation, prophylactic gland dissection of inguinal lymph-node is not necessary except a case with definite metastasis. Numerous kinds of adjuvant chemotherapy to radical operation have been tried, however, selection of antitumor drugs, method of administration are not settled yet, and, at present, long term clinical effects are unknown. PMID- 3023718 TI - [Adenoid cystic carcinoma of the esophagus]. AB - A 56-year-old man was admitted with the complaint of dysphagia. X-ray studies and endoscopy revealed a protruding tumor at the middle third of esophagus. Under the diagnosis of esophageal cancer, subtotal esophagectomy was performed. A Borrmann type I like tumor measuring 6.7 X 3.8 X 2.2 cm was identified on the resected specimen. The surface of the tumor was irregular, nodular and covered with thin compressed esophageal mucosa. The histology of the tumor was consistent with adenoid cystic carcinoma. Incidentally, small foci of invasive squamous cell carcinoma were found adjacent to this tumor. There was no lymph node or remote organ metastasis. PMID- 3023720 TI - [Dynamic magnetic resonance imaging of hepatocellular carcinoma]. PMID- 3023719 TI - [Abnormalities of pyrimidine metabolism and hemolysis]. PMID- 3023722 TI - [Clinical, histopathological and immunohistochemical studies of myrmecia warts]. PMID- 3023721 TI - [Ruptured hepatocellular carcinoma following transcatheter arterial embolization: a case report]. PMID- 3023723 TI - [Case report of familial juvenile polyposis of the stomach associated with gastrointestinal malignancy]. PMID- 3023724 TI - [Clinical and pathological study of irradiation therapy in 24 patients with hepatocellular carcinoma]. PMID- 3023725 TI - [Quantification analysis of prognostic factors in patients with hepatocellular carcinoma treated by transcatheter arterial embolization]. PMID- 3023726 TI - [Effect of diethyldithiocarbamic acid on gastric mucosal potential difference and blood flow]. PMID- 3023727 TI - Evidence for human IgE antibodies specific for exogenous and endogenous cytochrome C in postmortem serum induced by bovine cytochrome C-allergy- detection by ELISA using avidin-biotin complex. PMID- 3023728 TI - Ascending fibers from the gigantocellular nucleus to the centromedian nucleus of the thalamus in cats. AB - Centrum medianum (CM) afferent pathways, which are involved in pain sensation, were analyzed using physiological techniques. Thirty-four neurons in the gigantocellular nucleus (nucleus gigantocellularis medullae oblongatae, GC) in cats were recorded intracellularly. Of these, 5 (15%) did not respond to electrical stimulation applied to any of the 4 limbs. Twenty-nine (85%) showed spike potentials that were superimposed on excitatory post-synaptic potentials (EPSPs) with an amplitude of 7.2 mV (n = 101) and a duration of 6.8 ms. The latencies from contra- and ipsilateral forelimb, contra- and ipsilateral hindlimb to the GC were 9.3 (n = 25), 7.2 (n = 23), 12.9 (n = 28), and 10.9 ms (n = 25), respectively. Of these responding neurons, 19 (66%) responded to stimuli to all 4 limbs, 7 (24%) to 3 limbs, 1 (3%) to 2 limbs, and 2 (7%) to 1 limb. These afferent neurons in the GC showed spike potentials without EPSPs after stimulation of the CM in the thalamus. Extracellular activities of 37 CM neurons were also tested. Of these, 4 neurons responded to GC stimulation with a short latency of 1.6 ms. Another 33 responded with a long latency of 6.7, and 11 of them were able to follow GC stimulation of over 200 Hz with a fixed long latency; 22 responded with varying long latencies but were not able to follow stimuli over 15 Hz. Three-quarters of the CM neurons received ipsilateral inputs from the GC, and the other contralateral inputs. Three CM neurons responded to stimulation of the GC in quasi-intracellular records. These findings suggest that some ascending fibers from the GC terminate on CM neurons and play a role in pain sensation. PMID- 3023730 TI - [Influence of ACTH on Kamin effect in passive avoidance learning in rats]. AB - The purpose of the present experiments was to compare the validity of the associative or "state dependent learning" hypothesis and the nonassociative ones, which had been proposed to explain the Kamin effect. In Experiment 1, rats were given one-trial passive avoidance trial with one of the three shock intensities (0.5-, 1.0- and 2.0-mA), and then the retention test was given after either one of the intervals of 0.05-, 2.5- and 24-hr. The clear Kamin effect (U-shaped retention function) was obtained for the animals treated with the 2.0-mA shock. In Experiment 2, the adrenocorticotropic hormone (ACTH) was injected two hours before the retention test to investigate the involvement of adreno-pituitary system in the retrieval of the memory. The retention performances at the intermediate (2.5-hr) interval were reinstated, whereas those at the 24-hr interval were impaired, as a function of dosage of ACTH. The results of these experiments favored the associative or state-dependent learning hypothesis on the Kamin effect. PMID- 3023729 TI - Focal perivascular alterations of white matter in herpes simplex encephalitis--a histological and immunocytochemical study. AB - Several cases of human herpes simplex encephalitis treated with Vidarabin have been investigated with the histological and immunocytochemical techniques. Cases with a subacute evolution revealed areas of focal perivascular myelin destruction in the white matter. The distribution of herpes simplex antigen did not show any preferential localization of the virus in perivascular oligoglial cells. In contrast, a spatial and temporal relationship has been found between the appearance of immunoglobulin-bearing cells around the vessels and that of areas of focal perivascular myelin damage. Therefore, it is postulated that the areas of focal destruction of myelin are not related to the cytotoxic effect of the virus but are rather dependent on the immune response of the host. PMID- 3023731 TI - Biological and physical comparison of mink enteritis virus feline panleukopenia virus and canine parvovirus. PMID- 3023732 TI - Enzyme-linked immunosorbent assay for the detection of antibodies to equid herpesvirus type 1 (EHV-1). PMID- 3023733 TI - [Features of the clinical picture and diagnosis of various types of myocardial infarct]. PMID- 3023734 TI - [Histochemical studies of neutrophils in myocardial infarct patients]. AB - Cytochemical assays of neutrophil acid phosphatase, beta-glucuronidase, N-acetyl beta-D-glycosamidase (NAG), myeloperoxidase activities, and the Sudan black B test, were carried out in 25 patients with myocardial infarction. Leucocytosis seen in the early days of infarction was associated with increased proportion of neutrophils characterized by high activities of the enzymes in question and enhanced reaction with Sudan black B. Neutrophilosis of the early myocardial infarction may result from the activity of the marginal cell fraction. The fact that increased myeloperoxidase activity and the enhanced response to Sudan black B persist through 14 days of the disease, coupled with a rise in neutrophil NAG activity on day 14, expands possibilities of myocardial infarction diagnosis. PMID- 3023735 TI - Responses of in vivo renal microvessels to dopamine. AB - The split hydronephrotic kidney preparation was used to directly observe the effects of locally applied dopamine on the in vivo diameters of renal vessels. Dopamine (1 X 10(-6) to 3 X 10(-5) M) produced a concentration-dependent dilation of the arcuate and interlobular arteries and afferent arterioles. Efferent arterioles near the glomeruli also dilated to dopamine but the dilation was less than that of the preglomerular vessels. Higher dopamine concentrations (3 X 10( 4) and 1 X 10(-3) M) produced more variable effects, with a tendency for the arcuate and interlobular arteries and the afferent and efferent arterioles away from the glomeruli to decrease in diameter. After pretreatment with haloperidol, dopamine (1 X 10(-6) to 1 X 10(-4) M) did not dilate any pre- or postglomerular vascular segment, but the tendency for pre- and postglomerular constrictions with higher dopamine concentrations were not abolished. Pretreatment with phentolamine and propranolol enhanced the dilator response of the pre- and postglomerular vessels (except the afferent arterioles near glomeruli and efferent arterioles near welling points) to dopamine (3 X 10(-5) and 1 X 10(-4) M), and abolished the reductions in diameter produced by the high dopamine levels. These data indicate that the dilator effect of dopamine is mediated by interactions with specific dopaminergic receptors, while alpha and beta adrenergic receptors appear to mediate a constrictor influence observed with high dopamine concentrations. The overall effect of dopamine on the renal vessel diameters thus appears to depend on the balance of dilator and constrictor stimuli mediated by multiple receptors. PMID- 3023736 TI - Endogenous digitalis-like factors in hypertension and chronic renal insufficiency. AB - Endogenous digitalis-like factors have been implicated in the adaptations that accompany renal insufficiency and in the pathogenesis of hypertension. We recently described several fractions of normal human plasma that inhibit NaK ATPase and exhibit apparent digoxin-like immunoreactivity. To determine if hypertension and/or renal insufficiency affect plasma levels of these factors, we examined four patient groups: normotensive controls; hypertensive subjects with normal renal function; hypertensives with moderate renal insufficiency; and chronic dialysis patients. Plasma levels of digoxin-like immunoreactivity and NaK ATPase inhibitory activity were significantly increased in hypertensive patients with mild renal failure (7.6 +/- 1.1 ouabain equivalents, mean +/- SEM, N = 21 vs 4.1 +/- 1.1 in normotensive controls, N = 20, P less than 0.05). NaK-ATPase inhibitory activity tended to be higher in patients with primary hypertension and normal renal function (5.5 +/- 0.7 ouabain equivalents, P less than 0.07); in dialysis patients, it was not different from controls. There was no correlation between NaK-ATPase inhibitory activity and blood pressure in any group. There was a significant rise in plasma NaK-ATPase inhibitory activity during dialysis (+ 1.8 +/- 0.7 ouabain equivalents, N = 22, P less than 0.03). As we have found that NaK-ATPase inhibitory activity in the plasma of normal humans can be separated into three distinct fractions, EI1, EI2, and EI3, we analyzed the plasma of 10 dialysis patients further. The increase in NaK-ATPase inhibitory activity could be attributed to fractions EI1 and EI3. These results suggest that plasma NaK ATPase inhibitors increase with chronic renal insufficiency, but not hypertension alone. Although hemodialysis may acutely raise plasma levels, long-term dialysis returns them to the normal range. PMID- 3023737 TI - [Carcinomatous invagination of the colon]. PMID- 3023738 TI - Role of the high density lipoprotein-receptor cycle in macrophage-cholesterol metabolism. AB - The intracellular catabolism of lipids and the blood cholesterol level are known to have an essential impact on the onset and progress of atherosclerosis. As lipoprotein receptors can influence the co-operation of macrophages, high density lipoproteins and enzymes of lipid metabolism, the contribution of these receptors to transport and intracellular processing of cholesterol are investigated by means of biochemical analysis and electron microscopy. Taking the effect of second messenger systems into consideration, a model for impaired lipoprotein receptor function affecting the cholesterol metabolism is developed. PMID- 3023740 TI - Susceptibility of Indonesia colonies of Aedes aegypti and Aedes albopictus mosquitoes to experimental infection with chikungunya virus. PMID- 3023739 TI - Rapid modifications of biophysical and biochemical parameters of red blood cell membrane from insulin dependent diabetics after insulin administration. AB - We have previously shown that red blood cell (RBC) filtrability in uncontrolled insulin dependent diabetics is abnormal compared to healthy subjects, but is corrected by insulin added in vivo or in vitro. We have now found biophysical abnormalities of the RBC membrane in such patients: Fluorescence polarization value (p) evaluated using DPH as probe is significantly lower in patients; insulin added in vitro increases and normalises the p value of diabetic RBC membrane, and has no effect on normal RBC membrane. As there is a relationship between the lipid bilayer and membrane cytoskeleton proteins, this abnormality of RBC membrane fluidity, correctable by insulin, may be an important determinant of the rheological behaviour of the RBC. In parallel, biochemical abnormalities of the RBC membrane are shown in the same patients: Higher cholesterol/phospholipid ratio, lower content in unsaturated fatty acids and higher content in saturated fatty acids. Decreased activity of ouabaine sensitive Na+K+ ATPase and increased activity of Mg++ ATPase. All of these RBC biochemical abnormalities are corrected after 24 h of normoglycaemia obtained by insulin treatment given in vivo with a biostator. Our results show the importance to check the degree of control of glycaemia when studying rheological parameters in diabetes mellitus. PMID- 3023742 TI - [Excitability of bronchial receptors in infectious allergic bronchial asthma]. PMID- 3023741 TI - [Endocardial angiofibroma simulating a mitral heart defect]. PMID- 3023743 TI - Hepatocellular adenoma related to oral contraceptive use--report of two cases. PMID- 3023744 TI - [The importance of bulk materials in enteral, formulated nutrition]. PMID- 3023745 TI - [Value and personal experiences with beverages and enteral foods rich in bulk]. PMID- 3023746 TI - Liver-specific glucose-6-phosphatase is not present in human placenta. AB - Type I glycogen storage disease (McKusick 23220), an inherited absence or deficiency of glucose-6-phosphatase (EC 3.1.3.9) activity in the liver, kidney and intestine, is associated with the accumulation of glycogen in those organs. Previous reports have shown that glucose-6-phosphatase exists in human placenta and that detection of a heterozygote for this disorder from placenta might be possible. Our finding of a normal glucose-6-phosphatase activity in a placenta from a patient at risk for type Ia glycogen storage disease prompted us to examine in more detail placental glucose-6-phosphatase. Unexpectedly, we found the properties of the placental enzyme differed from that in normal liver, and the placental enzyme hydrolyzed glucose-6-phosphate, mannose-6-phosphate, beta glycerol phosphate and glucose-1-phosphate equally well. Our data suggest the enzyme deficient in type I glycogen storage disease cannot be detected in placenta. PMID- 3023747 TI - Biochemical observations on a case of hepatic fructose-1,6-diphosphatase deficiency. AB - A case of hepatic fructose-1,6-diphosphatase deficiency is described. She presented with congenital bilateral cataracts, failure to thrive, hypoglycaemia and hyperlactacidaemia. A liver biopsy revealed normal levels of gluconeogenic enzymes except fructose-1,6-diphosphatase which was present at 30% of the level of the lower control values. The residual activity had a normal affinity for fructose-1,6-diphosphate, a decreased sensitivity for inhibition by fructose-2,6 diphosphate and an increased resistance toward conversion to the AMP-insensitive form of the enzyme. As a result of this mutation, the residual FDPase will always be maintained in the AMP-inhibited form. PMID- 3023749 TI - Introduction to recombinant DNA. AB - This paper describes the current state of knowledge of methods for analysing gene structure and localization. Illustrations are given of the preparation of complementary DNA libraries and their screening by positive-negative selection, the use of synthetic oligodeoxynucleotides and the use of antibodies. Analysis of the EGF precursor is used as an example to show its close relationship to plasma membrane receptor and its homology with the LDL receptor. Analysis of cloned genome DNA by use of bacteriophage lambda or cosmids gives useful information about gene regulation and evolution. Mutations by frame shift, point or missence mutations are discussed with reference to the LDL receptor and the apolipoproteins. The techniques of gene mapping by rat-human cell hybridization and hybridization in situ are illustrated, again with reference to genes coding for enzymes of cholesterol metabolism, the apolipoproteins and insulin-like growth factors. Finally the potential of in vitro mutagenesis and the injection of cloned DNA into the fertilized mouse ovum are discussed. PMID- 3023748 TI - Fructose-1,6-diphosphatase deficiency: diagnosis using leukocytes and detection of heterozygotes with radiochemical and spectrophotometric methods. AB - The first two cases of fructose-1,6-diphosphatase (FDPase) deficiency from the Middle East have been diagnosed on leukocytes using a spectrophotometric assay and a new radiochemical technique. The control mean for FDPase measured by the spectrophotometric assay was 178.2 nm mg-1 h-1 (n = 12), 66.8 nm mg-1 h-1 for obligate heterozygotes (n = 4) and non-detectable in the two patients. By the radiochemical assay the values were controls, 103.3; heterozygotes, 20.6; patients, 0.46 and 3.5 nm mg-1 h-1. Using both methods it was possible to identify two certain FDPase heterozygotes and three non-carriers in the family of one of the probands. The radiochemical method was found to be more effective in differentiating heterozygotes from controls than the spectrophotometric method. However, either technique may be conveniently used for the diagnosis of FDPase deficiency in leukocytes. PMID- 3023750 TI - Procollagen III peptide in bronchoalveolar lavage fluid. A potential marker of altered collagen synthesis reflecting pulmonary disease in sarcoidosis. AB - Type III procollagen N-terminal peptide was not detectable in bronchoalveolar lavage fluid from healthy volunteers but was present in fluid from the majority of patients with pulmonary sarcoidosis (N = 110); the mean concentration was 0.6 micrograms/liter returned fluid, range less than or equal to 0.2 to 19.5 or, expressed in relation to the amount of albumin recovered, 9.5 mg/gm albumin (range less than or equal to 1 to 45). The serum concentrations in the patients with sarcoidosis were normal. Significant inverse correlations were found between lavage fluid procollagen peptide and vital capacity (p less than 0.001), forced expiratory volume (p less than 0.01), and diffusion capacity (p less than 0.01). Lavage fluid procollagen peptide was also related to pulmonary radiological findings (p less than 0.001) and serum levels of angiotensin converting enzyme (p less than 0.001). These findings support the hypothesis that procollagen peptide in lavage fluid is a potential marker of activated pulmonary fibroblasts or an expanded fibroblast mass associated with interstitial lung fibrosis. PMID- 3023751 TI - Hepatocellular carcinoma cell line and peripheral blood lymphocytes from the same patient contain common chromosomal alterations. AB - A cell line derived from the biopsy of a human hepatocellular carcinoma which retains the differentiated phenotype of the liver parenchymal cell is described. Comparison of the integration sites of hepatitis B virus within the cellular genome of the biopsy specimen and within the genome of the multiply-passaged cell line reveals five stable sites of viral integration in the host cell genome. Multiple chromosome abnormalities are found in this cell line, some in the same area of the genome in which abnormalities were found in other human hepatomas. Chromosomes obtained from the peripheral blood cultures of the same patient manifest multiple signs of chromosome instability including breaks, minutes, chromosome pulverization, and acentric fragments, some of which localize to the chromosomal sites involved in the abnormalities in the tumor cell line. Chromosomal instability and/or virus-induced chromosome damage as factors in the etiology of human hepatocellular carcinoma are discussed. PMID- 3023752 TI - Postischemic alterations in ultrastructural cytochemistry of neuronal Golgi apparatus. AB - Functional activity of the Golgi apparatus in postischemic neurons was evaluated by using thiamine pyrophosphatase (TPPase) activity as an histochemical marker for the trans cisternae. Ischemia was produced in rats by permanent occlusion of vertebral arteries and 30-minute occlusion of the carotid arteries. This insult produces irreversible ischemic injury to neurons in the striatum and CA1 zone of hippocampus but only reversible injury to neurons in the paramedian cortex and CA3 hippocampus. The number of neurons with TPPase activity in controls correlated in part with neuronal size and was found in greater than 90% of neurons in cortex and CA3 hippocampus, 70% in CA1 hippocampus, and 40% in striatum. Ischemia plus recirculation for 30 minutes resulted in a decrease in the number of neurons with TPPase activity by 50% in CA1 hippocampus and by 80% in the three other areas. Resistant neurons in cortex and CA3 hippocampus showed partial recovery of TPPase activity by 2 hours after ischemia although the number of neurons was still less than that in controls (55% and 72%, respectively; p less than 0.01). At 24 and 48 hours, TPPase activity in cortical and CA3 neurons was similar to controls. In contrast, irreversibly injured neurons in striatum and CA1 hippocampus showed a persistent loss of TPPase activity during the entire postischemic period. Furthermore, TPPase activity remained significantly decreased in CA1 hippocampus even though previous studies in our laboratory indicated partial recovery of Golgi cisternae before subsequent cell death at 48 to 72 hours. Since TPPase activity has been correlated with functional activity within the Golgi apparatus these results suggest that glycosylation of glycoproteins and glycolipids may be markedly impaired in neurons after cerebral ischemia. The persistent abnormalities in Golgi function may contribute to the development of irreversible injury by interfering with the normal maintenance of plasma membranes and axonal transport. PMID- 3023753 TI - Inbred guinea pig aortic endothelial cell clones. Model for studying the vascular endothelium under totally isologous conditions. AB - Aortic endothelial cell clones were established from adult inbred strain 2 guinea pigs by using a simple method requiring neither feeder layers nor growth factors other than isologous serum. One clone has been maintained in continuous culture for 26 passages over a 9-month period. Cloned endothelial cells grew in a cobblestone pattern at confluence, formed intercellular junctions, and expressed factor-VIII-related antigen by immunofluorescence microscopy. The cloned cells also contained high levels of angiotensin-converting enzyme activity, although the level of enzyme activity expressed by different cloned cell lines varied. Cloned endothelial cells contained numerous Weibel-Palade bodies which appeared larger and less electron-dense than those of strain 2 guinea pig aortic endothelial cells in vivo. These clones may serve as an in vitro model to investigate the formation and function of this organelle. Cloned populations of strain 2 guinea pig vascular endothelial cells maintained in medium supplemented only with strain 2 guinea pig serum also provide an immunologically isologous system ideal for investigating interactions between the vascular endothelium and immunocompetent leukocytes in vitro. PMID- 3023755 TI - Significance of the "maturation" of metastases from Wilms' tumor after therapy. AB - Several metastatic nodules were discovered in the lungs of a 4-year-old boy two years after surgical excision of Wilms' tumor with subsequent chemotherapy and local irradiation. All these metastatic nodules were composed of differentiated mesenchymal elements similar to those encountered in the primary neoplasm. Similar "mature" metastases have been observed by pathologists in other tumors of embryonic origin. Review of available literature and clinical and experimental data supports the notion that "reversion" of malignant cells into phenotypically benign counterparts may take place spontaneously or under the influence of environmental factors. Anticancer agents (cytostatics, radiation) selectively destroy the more anaplastic cells present in a malignant tumor (or its metastatic implants), thus allowing the "benign" revertants to predominate. Although such lesions are benign-appearing, it is recommended that they be completely excised; however, but further chemo- or radiotherapy may not be necessary. PMID- 3023754 TI - The effect of enkephalins and prostaglandins on O-2 release by neutrophils. AB - Derivatives of superoxide (O-2), produced by phagocytic cells, are thought to play a role in the adult respiratory distress syndrome (ARDS) and other disease states. Control of the release of O-2 may prove beneficial. Using human neutrophils as a source of O-2, and an assay for O-2 based upon the reduction of cytochrome C, we found that prostaglandin D2 (PGD2), leucine enkephalin (LE), and methionine enkephalin (ME) inhibited O-2 release. The Escherichia coli product, N formyl methionyl leucyl phenylalanine (FMLP), was employed to stimulate O-2 release. PGD2 was most potent while there was no significant difference between LE and ME. Another peptide, thyrotropin releasing hormone (TRH), had no effect on O-2 release. There was no correlation between the potency of the inhibitory effect on O-2 release and the effect of these agents on the binding of [3H] FMLP to human neutrophils. Comparison of different but structurally related prostaglandins (PGD2, PGE2, and PGF2 alpha) revealed that PGD2 was more potent than PGE2 in inhibiting O-2 and that PGF2 alpha had no effect. This result suggested that the presence and position of the carbonyl group was an important determinant of the magnitude of inhibition. PMID- 3023756 TI - Properties of a 3H-adenine prelabeled vesicular preparation from guinea pig cerebral cortex. Optimization of incubation conditions for basal- and histamine stimulated 3H-cyclic AMP levels. AB - The effects of several variables related to incubation conditions were studied on both the basal and the histamine-stimulated 3H-cyclic AMP levels in a 3H-adenine prelabeled vesicular preparation of guinea pig cerebral cortex. Varying the preincubation time (i.e., the duration of incubation before labeling) caused pronounced differences in the postlabeling time course of 3H-cyclic AMP levels. A preincubation time of 45 min was found to minimize time-related postlabeling declines in 3H-cyclic AMP basal activity. Labeling of the homogenate for an entire experiment in one vessel ("bulk labeling") also contributed to time related declines in activity; these changes were minimized by labeling aliquots of homogenate individually. Rapid sample mixing during incubation was found to be necessary to ensure adequate suspension of the preparation and to achieve a stable basal activity. Such mixing altered the pH of the samples; maintenance of the pH at 7.4 required a decrease in the bicarbonate concentration and/or use of supplementary buffers. Several parameters related to histamine concentration response curves were found to be affected by various combinations of buffers and labeling methods. Individual labeling in Krebs-ringer-bicarbonate (15 mM) buffer gave the most stable and reproducible responses to histamine. This method permits detailed pharmacological characterization of transmitter-stimulated cyclic AMP changes in a cell-free system that retains many characteristics of brain slices. PMID- 3023758 TI - A synaptic model for the kindling effect. AB - In this work we present a model of the kindling effect based on the Hopf bifurcation for a system of ordinary differential equations. The model shows how quantitative changes in the physiological parameters at the microscopic, synaptic scale, produce the afterdischarge which is a macroscopic effect at the neuronal network scale. The presynaptic mechanisms are based on the vesicular hypothesis, or more generally on the quantal theory of synaptic transmission. The postsynaptic processes rely on Granit's law. This model gives a consistent framework which organizes and explains several experimental observations. PMID- 3023757 TI - Methodology for benzodiazepine receptor binding assays at physiological temperature. Rapid change in equilibrium with falling temperature. AB - Benzodiazepine receptors of rat cerebellum were assayed with [3H]-labeled flunitrazepam at 37 degrees C, and assays were terminated by filtration in a cold room according to one of three protocols: keeping each sample at 37 degrees C until ready for filtration, taking the batch of samples (30) into the cold room and filtering sequentially in the order 1-30, and taking the batch of 30 samples into the cold room and filtering sequentially in the order 30-1. the results for each protocol were substantially different from each other, indicating that rapid disruption of equilibrium occurred as the samples cooled in the cold room while waiting to be filtered. Positive or negative cooperativity of binding was apparent, and misleading effects of gamma-aminobutyric acid on the affinity of diazepam were observed, unless each sample was kept at 37 degrees C until just prior to filtration. PMID- 3023759 TI - Subunits in quantal transmission at the mouse neuromuscular junction: tests of peak intervals in amplitude distributions. AB - The regular spacing of peaks throughout the amplitude distribution of miniature end-plate potentials, quantal evoked end-plate potentials and quantal currents was demonstrated using autocorrelations and power density spectra calculated from the number of events in the successive bins of the histograms built by Matteson et al. (1979), Kriebel & Florey (1983) and Erxleben & Kriebel (1984). At the same mouse neuromuscular junction, the calculated interpeak was constant for evoked and spontaneous quantal releases, throughout sequential sampling and after change of bin size. The presence of regular peak intervals supports the hypothesis that quantal potentials are composed of potential subunits the size of the smallest subminiature potential. Challenging the hypothesis of an acetylcholine quantum composed of acetylcholine subunits, a postsynaptic origin of the subunit is proposed on the basis of the spatial arrangement in rows of the ACh receptors. The ACh-saturating patch evoked by a quantum release (Land et al., 1980, 1981) activates 10-20 rows of receptors, which is roughly the number of subunits composing a quantal event. Therefore the position of the ACh patch or the continuous variations in its size might cause stepwise variations in the total number of ACh receptors activated by an ACh quantum. PMID- 3023760 TI - Horseradish peroxidase histochemistry on mounted sections from embedded specimen: a simple method for serial reconstruction of neuronal projections. AB - A combination of two procedures, embedding of specimens and horseradish peroxidase (HRP) histochemistry on mounted serial sections, is proposed for three dimensional reconstruction of neuronal projections. To clarify the intrinsic organization of the peripheral nervous system, this simple method is more adequate than previously used techniques. PMID- 3023761 TI - Catecholamine-induced suppression of interleukin-1 production. AB - Recent evidence indicates that stress can suppress immune responses and thus increase the severity of viral and neoplastic diseases. Although, the mechanisms for stress-induced modulation of immunologic competence are unclear, neuroendocrine hormones are thought to be involved. A direct suppressive effect could result from the action of neuroendocrine hormones on lymphokine and monokine release. The central role of interleukin-1 (IL-1) in regulating cellular immune responses to infection and neoplasms stimulated the present study evaluating the effects of neuroendocrine hormones on IL-1 production. Norepinephrine and epinephrine inhibited the capacity of gamma interferon and lipopolysaccharide to stimulate IL-1 production from mouse peritoneal macrophages. Moreover, when intracellular and extracellular levels of IL-1 were quantitated, the studies demonstrated a catecholamine-mediated block in IL-1 synthesis without effect on its release. We also observed that exogenous cyclic AMP (cAMP) administered to mouse macrophages suppressed IL-1 production. This, coupled with the capacity of norepinephrine and epinephrine to enhance intracellular cAMP levels in macrophages, strongly suggested that the catecholamine-induced suppression of IL-1 production may be mediated by elevated intracellular cAMP levels. These findings demonstrate that selected stress related neuroendocrine hormones can modulate IL-1 production by macrophages and further support the hypothesis that alteration of macrophage function by neuropeptides and neurohormones is a significant feature of stress-induced enhancement of viral and neoplastic disease. PMID- 3023762 TI - Constitutive production of a non-interferon macrophage activating factor by HTLV transformed T cells. AB - Culture supernatants of several human T cell leukemia virus infected cell lines were assessed for the induction of tumoricidal activity against human melanoma target cells in a 72-hr assay. Two cell lines, MT-2 and C10/MJ2, were found to produce high levels of MAF activity constitutively. However, the MT-2 cell line, unlike C10/MJ2, produced little interferon. MT-2 MAF was distinct from interferon gamma by a number of biochemical and serological criteria. The MAF activity was associated with a non-glycosylated, acid stable protein which has an apparent molecular weight of 55,000 and an isoelectric point of 5.5. Furthermore, this molecule is not related to any of the known lymphokines or cytokines such as the interleukins or colony stimulating factors. PMID- 3023763 TI - In vitro synthesis of angiotensin-converting enzyme by alveolar macrophages is increased in disseminated sarcoidosis. PMID- 3023764 TI - Concerning the way to study the relative potencies of bronchodilator agents in vitro. PMID- 3023765 TI - Relationships between the age-dependent decay of glucose-1,6-bisphosphate synthesis, phosphoribomutase and phosphoglucomutase in human red cells. AB - In human red blood cells phosphoglucomutase exists in multiple molecular forms with different isoelectric points determined by two distinct loci called PGM1 and PGM2. With regard to the phosphoglucomutase PGM1 and PGM2 isoenzymes, the latter appear to be more important in erythrocyte metabolism owing to their ability to mutate ribose monophosphates and synthetize glucose-1,6-bisphosphate. In this paper we show that, beside undergoing age-related postranslational modifications, both phosphoglucomutase PGM1 and PGM2 forms decrease their activities as the mean cell age increases. Under the experimental conditions used to separate erythrocytes by age the comparison of the younger erythrocytes with the older shows that total phosphoglucomutase, phosphoribomutase and glucose-1,6 bisphosphate synthetic activities decay by 55%, 26% and 28%, respectively. We consider that these results substantiate the multifunctionality of PGM2 isoenzymes. Furthermore we discuss the role of these forms in the age-related decay of erythrocyte metabolism. PMID- 3023766 TI - Histochemistry and function of bombesin-like peptides. AB - Bombesin-like peptides are a group of brain-gut peptides found in several neuronal groups in the central nervous system and in peripheral intrinsic gut neurons and sensory neurons. The SIF cells (small intensely fluorescent cells) of the sympathetic ganglia also contain immunoreactivity for these peptides. These peptides are present in some pulmonary endocrine cells and tumors originating from these cells. Chromatographic studies suggest that several different peptides, possibly originating from at least two different precursors, are present in mammalian tissues. Authentic amphibian peptide bombesin does not appear to be found in mammalian tissues. Functional studies indicate that these peptides may be involved in many important functions, including sensory transmission, regulation of central autonomic pathways, thermoregulation, secretion of pituitary hormones, gastric and pancreatic secretion, food intake and satiety. PMID- 3023767 TI - Influence of neonatal steroid (diethylstilbestrol, allylestrenol) treatment on the sexual behaviour of the adult rat. AB - A single neonatal treatment with diethylstilbestrol (DES) or allylestrenol (AE) considerably depressed the sexual activity of male rats in adulthood. DES had a stronger depressive effect than AE. Though the adult sexual activity of intact female rats was also reduced by DES it was not influenced by AE. Ovariectomized females that had been hormone-treated before experimental mating showed reduced sexual activity under the influence of neonatal DES-treatment but increased sexual activity when treated neonatally with AE. PMID- 3023768 TI - Tofizopam selectively increases the action of anticonvulsants. AB - The effect of tofizopam, a 3,4-benzodiazepine (BZ) derivative, in modulating the anticonvulsive action of various drugs was investigated in mice. Electric shock and intravenous infusion of bicuculline were used as convulsive agents. Tofizopam increased the action of clonazepam, diazepam and flunitrazepam against bicuculline. The anticonvulsive effect of diazepam against electroshocks was augmented only slightly. Tofizopam failed to alter the actions of carbamazepine, phenobarbital, phenytoin, or sodium valproate against either of the convulsive stimuli. Both in vitro and in vivo, tofizopam has been shown to stimulate the binding of 1,4-BZs (e.g., flunitrazepam) to BZ receptors. Similarly, tofizopam enhances the binding of muscimol to GABA receptors. Although several anticonvulsants act on the GABA-BZ receptor complex, tofizopam seems to modify selectively the anticonvulsive action of 1,4-BZs, and this effect is seen better in bicuculline-induced seizures than in electroshocks. PMID- 3023769 TI - [Isolated deficit of adrenocorticotropin hormone (ACTH) associated with primary empty sella turcica]. PMID- 3023770 TI - [Temporal arteritis and peripheral neuropathy]. PMID- 3023771 TI - Angiofibroma: treatment trends in 150 patients during 40 years. AB - A series of 150 patients with histologically confirmed angiofibroma examined from 1945 through 1983 was studied to contrast treatment methods and surgical approaches. From 1945 to 1955, treatment consisted primarily of radiation. From 1955 through 1971, the primary method of treatment was surgical removal; the lateral rhinotomy approach was used to expose the tumor and its extensions in most cases. From 1971 through 1983, all tumors were removed surgically. Trends in diagnosis, treatment, and adjunctive therapy at a single institution were evaluated. Specifically, the trends considered were operative approaches, blood replacement with and without hypotensive anesthesia, adjunctive measures such as hormonal therapy or tumor embolization, mortality, and morbidity. Lateral rhinotomy provides wide exposure of and access to the nose, nasopharynx, paranasal sinuses, elements of the skull base, temporal fossa, and infratemporal fossa. Surgical treatment, specifically the lateral rhinotomy approach and its extensions, is recommended as the best method of managing angiofibroma in most patients. PMID- 3023772 TI - Preoperative parathyroid adenoma localization by the technetium-thallium subtraction scan. AB - The technetium-thallium subtraction scintigram was utilized preoperatively in 14 consecutive patients explored for primary hyperparathyroidism. The scintigram accurately identified the site of a parathyroid adenoma in 12 of 13 patients. PMID- 3023773 TI - Autoradiographic localization of subcomponents of the macromolecular GABA receptor complex. AB - The autoradiographic localization of subcomponents of the gamma-aminobutyric acid (GABA) receptor-chloride ionophore complex has provided insight into the distribution of this macromolecular system. GABA inhibits neurons by preferentially increasing the permeability of the affected membrane to chloride ions. This inhibition can be modified by the presence of other substances which bind to the GABA receptor complex. Autoradiographic localization of specific receptor subtypes associated with this complex has been accomplished in the central nervous system. This type of analysis has been performed on high and low affinity GABAA, benzodiazepine (BZ; both BZ1 and BZ2) and convulsant sites. These receptor sites are situated in distinct brain regions and co-exist in several areas. Other receptor subtypes, which may be influenced by the presence of GABA, can be analyzed for comparison in order to define regions of the brain where GABA may be exerting independent effects (i.e., those not associated with chloride channels). Microscopic localization of receptor sites indicates specific areas to investigate in further studies concerning the characterization of subcomponents of the macromolecular GABA complex associated with chloride ion channels. PMID- 3023774 TI - Conformational shifts at the benzodiazepine receptor related to the binding of agonists antagonists and inverse agonists. PMID- 3023775 TI - A dose-ratio comparison of mu and kappa agonists in formalin and thermal pain. AB - The effects of putative mu and kappa agonists, with and without naloxone, were compared in the formalin and tail flick tests in rats. The mu agonist sufentanil was more potent in the tail flick test than the formalin test while the opposite was true for the kappa agonist ethylketocyclazocine (EKC). MR2034 was equipotent in the two tests and in the tail flick test, analgesia decreased at high doses. The naloxone (0.1 mg/kg) dose-ratios (DR) for sufentanil and EKC were 3 to 7 times larger for the tail flick test than the formalin test. From this and other DR studies it is argued that in thermal pain tests, opioid analgesia is mediated primarily by mu receptors while in non thermal tests kappa effects predominate. PMID- 3023776 TI - Tissue slices in radioligand binding assays: studies in brain, pineal and muscle. AB - The use of tissue homogenates in receptor binding assays raises serious questions as to the physiological value of a preparation which examines receptors (binding sites) in disrupted tissue. In order to usefully study the regulatory properties of neurotransmitter receptors under physiological conditions, the necessity for tissue preparations which retain some degree of cellular integrity is clear. We review here the experiments which have utilized intact tissue - largely in the form of thick slices - to perform radioligand binding assays. There are many reports which note marked differences between studies in intact versus broken cell preparations. For example, significant discrepancies in KD and Bmax values are apparent for [3H] quinuclidinyl benzilate (muscarinic) and [3H] ouabain (Na+/K+-ATP ase, sodium pump) sites in brain and muscle respectively. A further example is the well-described stimulatory effect of GABA on benzodiazepine binding sites which is not seen in tissue slices. Other examples are highlighted. For all ligands so far examined, binding to slices is reversible, stereospecific, saturable, displaceable by appropriate drugs and of high affinity (nM). The method developed in our own laboratory is inexpensive, rapid and involves a minimum of tissue preparation. The technique is so simple as to allow many workers to enter this field who would not otherwise have done so. We suggest that metabolically active tissue slices offer the simplest approach to the study of cell-surface receptor regulation in living tissue. PMID- 3023777 TI - Fluidizing effects of centrophenoxine in vitro on brain and liver membranes from different age groups of mice. AB - This study examined the effects of different concentrations of centrophenoxine on physical properties of synaptic plasma membranes and liver microsomes using electron spin resonance procedures. Membranes of different age groups of mice were labeled with the 5-doxyl stearic acid spin-label and membrane fluidity determined in the presence and absence of different concentrations of centrophenoxine. Centrophenoxine had a direct effect on membranes as shown by a significant increase in membrane fluidity. This effect was greatest in liver microsomes as compared to synaptic plasma membranes. Age differences were not observed in centrophenoxine-induced fluidization. Effects of centrophenoxine in vivo may be due in part to the drug acting directly on the physical properties of the membrane lipid environment. PMID- 3023778 TI - Growth hormone responses to clonidine and GRF in spontaneously hypertensive rats: neuroendocrine evidence for an enhanced responsiveness of brain alpha 2 adrenoceptors in genetical hypertension. AB - Clonidine induces growth hormone (GH) release in rat. According to previous investigations this effect is mediated by postsynaptic alpha 2-adrenoceptors in the hypothalamus exerting a stimulatory influence on the recently discovered GH releasing factor (GRF). In the present study it is demonstrated that spontaneously hypertensive rats (SHR) of the Wistar-Kyoto strain display enhanced GH responses to clonidine as compared to normotensive Wistar-Kyoto control rats. In contrast, the GH responses to GRF are similar in hypertensive and normotensive animals. These findings indicate that brain alpha 2-adrenoceptors are more responsive in SHR than in normotensive controls. Since the enhanced GH responses to clonidine were observed also in young, prehypertensive SHR they are probably not secondary to the elevated blood pressure. The possible importance of an altered alpha 2-adrenergic neurotransmission for the development of elevated blood pressure in SHR is discussed. PMID- 3023779 TI - The effect of acute and chronic administration of timolol on cardiac sympathetic neural discharge, arrhythmia, and beta adrenergic receptor density associated with coronary occlusion in the cat. AB - The effect of timolol (5 mg/kg, p.o., b.i.d. 7 or 14 days) on cardiac beta adrenergic receptor density, the times to arrhythmia (AR) and death (D), heart rate, mean arterial blood pressure, and postganglionic cardiac sympathetic neural discharge after acute coronary occlusion in cats was examined. In the control animals, receptor densities in the left and right atria did not differ, but were lower than the right ventricle. Left ventricle and septum receptor densities were higher, with the left ventricle the highest. The importance of the gradation of beta receptors with increasing density from base to apex appears to be its relation to cardiac contractile function. Occlusion in cats not treated with timolol did not alter the cardiac beta receptor densities. After timolol for 7 or 14 days, no occlusion, receptor density increased in left ventricle and septum although the increase was only significant after 14 days. A comparison of the beta adrenergic receptor densities in cats pretreated with timolol for 7 or 14 days with or without occlusion revealed that, in general, a decrease (p greater than 0.05) occurred for the occlusion group. Timolol decreased heart rate and blood pressure prior to occlusion. The mean times to AR and D were not significantly increased by either dosing regimen of timolol, although the trend was for an increase in the time to D after 7 days of timolol and an increase in the time to AR and D after 14 days of timolol. When compared with data obtained in saline cats, chronic timolol produced minimal changes in postganglionic cardiac sympathetic neural discharge. Timolol given chronically (p.o.) or acutely (5 mg/kg, i.v. given 15 min prior to occlusion) also did not prevent the cardiac sympathetic discharge associated with the development of AR. The time to AR and D in the acutely treated cats was increased but not significantly. Since cardiac sympathetic neural discharge increased as blood pressure fell in the control period but did not increase after occlusion in the timolol treated animals, the combination of timolol and occlusion may have modified neural discharge via an action on the baroreceptor mechanism. That chronic administration of timolol produces an effect not present in cats in which only occlusion was done is supported by the observation that chronic treatment produced an occlusion-induced decrease in beta adrenergic receptor density.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3023780 TI - Inhibitory effects of calcitonin on adenylate cyclase activity in different rat brain areas. AB - We have investigated the effect of calcitonin (CT) on adenylate cyclase in membranes from different rat brain areas. Salmon calcitonin (sCT) dose dependently inhibited the enzyme activity in midbrain, hypothalamus, medulla, pons and caudate nucleus, but was ineffective in adenohypophysis. The inhibitory effect was enhanced by GTP. Comparison of calcitonins of different origin indicated that sCT was the most potent in inhibiting the enzyme in hypothalamic membranes, eel CT (eCT) was slightly less potent, and human CT (hCT) was ineffective. Chronic I.C.V. pretreatment with sCT did not modify the subsequent in vitro sensitivity of adenylate cyclase to sCT. It is concluded that some of CNS actions of CT might involve modulation of intracellular cAMP levels. PMID- 3023782 TI - [Interaction of d-tubocurarine and pancuronium on neuromuscular transmission in man]. PMID- 3023781 TI - Clinical, biochemical and histological features of primary haemochromatosis: a report of 67 cases. AB - In 67 patients (mean age 51 years, range 26-79), at diagnosis of primary haemochromatosis (PH), grade III or IV liver iron overload was present in all cases, cirrhosis in 85%, transferrin saturation greater than 80% in 75%, serum ferritin greater than 1000 micrograms/l in 84%, and overt diabetes in 48%. Alcohol intake was greater than 150 g/day in 11 patients; six were chronic hepatitis B surface antigen (HBsAg) carriers. HLA-A3 and B7 antigens were present in 64% and 23% versus respectively 22% (p less than 0.01) and 9% (p less than 0.025) in controls. Iron overload was found in the stomach, duodenum, skin and bone marrow in 57, 43, 45 and 59% of the patients studied. Sixty-three patients were followed for 1-260 months (median 24); 43 received regular iron-depleting treatment and 20 did not because of liver failure, cancer or refusal. Cumulative survival was 79%, 67% and 61% at 1, 4 and 10 years, respectively. Ten patients died from hepatocellular carcinoma and two from extrahepatic cancer. The early high mortality rate was due to some cases of advanced disease or cancer. Cumulative survival in the regularly treated group was 95% at 1 year and 91% at 4 and 10 years, which was higher than in the untreated group. PMID- 3023784 TI - Role of 2,3-dimercaptosuccinic acid in the treatment of heavy metal poisoning. PMID- 3023786 TI - Murine in vivo L-band ESR spin-label oximetry with a loop-gap resonator. AB - Small mice (approximately 20 g) were anesthetized and placed in a loop-gap resonator of diameter 25 mm resonating at 1.1 GHz. An oxygen-permeable capsule containing 2.5 X 10(-2) M perdeutero 15N TEMPONE (1-oxyl-2,2,6,6-tetramethyl-4 piperidone) in light paraffin oil was implanted in the peritoneal cavity. Electron spin resonance (ESR) spectra from the capsule are sensitive to the concentration of dissolved oxygen and calibration curves are given. The oxygen concentration was found to rise from a value close to zero when the animal breathes air to a level of 220 microM when the animal breaths pure oxygen. It is speculated that this surprisingly high level is related to the effect of the anesthetic on the cardiovascular system. Encapsulation provides a barrier to spin label reductants and to paramagnetic metal ions that might confound the spin label oximetric measurements. PMID- 3023785 TI - A proton relaxation enhancement investigation of the binding of fatty acid spin labels to human serum albumin. AB - Proton relaxation enhancement (PRE) values for fatty acid spin labels bound to human serum albumin have been investigated using the inversion-recovery method at 24 MHz. At 0.1 mM protein concentration and a label-to-protein ratio of one-to one, the PRE value for 12-Doxylsterate-albumin complex is 7.8 +/- 2.3, whereas the PRE values for 5-Doxylstearate and 16-Doxylstearate-albumin complexes are 1.5 +/- 0.6 and 1.7 +/- 0.7, respectively. Addition of 10-fold excess of stearic acid reduced the PRE values nearly to 1, indicating that the strong enhancements arise from direct binding of fatty acid spin labels to human serum albumin. PRE values for all three labels exhibit maxima as a function of the label-to-protein ratio, suggesting multiple binding sites for fatty acid spin labels with labels in the tightest binding sites not resulting in the most effective relaxation. Based on the rates of reduction of ESR signal amplitudes by sodium ascorbate, the difference in PRE values for the three fatty acid spin labels bound to albumin is attributed to the difference in water accessibility of the nitroxide moieties at various positions along the acyl chain, being greater at the C-12 position than at C-5 or C-16 position. The PRE value of 8 for 12-Doxylstearate bound to human serum albumin indicates that this complex may be a suitable paramagnetic contrast agent for in vivo NMR imaging. PMID- 3023787 TI - [Treatment of constipation with dietary fiber]. PMID- 3023783 TI - Adverse reactions with angiotensin converting enzyme (ACE) inhibitors. AB - Teprotide, a nonapeptide isolated from the venom of a Brazilian pit viper, Bothrops jararaca, was the first angiotensin converting enzyme (ACE) inhibitor to be discovered and tested. It was found to be an effective, non-toxic antihypertensive agent as well as an afterload-reducing agent for patients with congestive heart failure (CHF). The primary activity of teprotide resulted from blockade of the angiotensin I converting enzyme--the pivotal step in the renin angiotensin-aldosterone system (RAAS), and consequent reductions in angiotensin II levels. There was limited clinical testing for teprotide because of: its scarcity; the need for parenteral administration; and the subsequent discovery and synthesis of captopril, the first orally active angiotensin converting enzyme inhibitor. Captopril is the prototype oral angiotensin converting enzyme inhibitor and has been extensively studied since the initiation of formal studies in 1976. Perhaps one of the most closely researched drugs in modern times, the experience with captopril now includes more than 12,000 patients studied in formalized trials and over 4,000,000 patients treated world-wide by physicians for hypertension and congestive heart failure. Enalapril (MK421) is the first of what appears to be a growing number of analogues which are structurally and pharmacodynamically different from captopril; yet, they possess the same capacity for inhibiting the activity of angiotensin converting enzyme. The side effect profile of enalapril (and presumably future) angiotensin converting enzyme inhibitors appears to be similar to captopril, though clearly more experience is needed with newer agents. The initial use of captopril was troubled by a relatively high incidence of side effects which will form the focus of this discussion. Partially the result of incomplete pharmacokinetic information, captopril was administered in early studies at dosages now recognised to be far in excess of those necessary for drug action. In addition, dosages were given without regard for deficiencies of renal function, now known to be the main excretory route of captopril. The population of those patients studied frequently had chronic, treatment-resistant hypertension, often associated with concomitant end-organ disease (especially renal disease); and many additional factors further complicating the clinical setting, e.g. a relatively high incidence of collagen vascular disease and immunosuppressive treatments.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3023788 TI - [Mononuclear phagocytes in the resistance of mice to extraneural infection with Junin virus]. PMID- 3023790 TI - [Late neurologic syndrome in patients with Argentinian hemorrhagic fever treated with immune plasma]. PMID- 3023789 TI - [Immunohistochemical demonstration of neuron-specific enolase in nephroblastoma]. PMID- 3023791 TI - [Modification of the course of Junin virus infection in guinea pigs by administration of immune sera]. PMID- 3023792 TI - [Subtraction scintigraphy using Tc99m/Tl 201 in the preoperative localization of adenomas of the parathyroid]. PMID- 3023793 TI - Influence of repeated exposure to elevated environmental temperature on the activity of respiratory enzymes of rat liver mitochondria. AB - The paper presents studies of the activity of lipid-dependent enzymes of the respiratory chain of the liver of rats exposed to increased ambient temperature. The animals were heated in a chamber under controlled humidity (45-55% relative humidity), with forcer air flow and regulated temperature of 21 degrees +/- 1 degree C (control group) and 28 degrees +/- 1 degree C or 35 degrees +/- 1 degree C. They were affected by a relevant temperature for 7 or 14 consecutive days, 6 hrs daily. The enzymes activities were determined in a fraction of submitochondrial particles. The studies demonstrated that under the increased ambient temperature (7 X 6 hrs), the activity of the respiratory enzymes is changed. A statistically significant increase in the activity of NADH dehydrogenase, NADH cytochrome c reductase and cytochrome oxidase was found along with a decrease in the activity of succinate cytochrome c reductase and succinate dehydrogenase. On prolongation of thermal exposure (14 X 6 hrs) the activity of succinate dehydrogenase and succinate reductase: cytochrome c was further decreased. The activities of the other test enzymes did not exhibit any statistically significant differences as compared to controls. Kinetic tests of succinate dehydrogenase point to conformational changes of the enzyme when affected by an increased ambient temperature. This confirms the important role of this enzyme in the animals adaptation to thermally varying environmental conditions. PMID- 3023794 TI - [Evaluation of the biological effect of polishing powders]. AB - Polishing powders are applied as abrasives at the treatment of various metal surfaces prior to their electroplating. The polishing powder is composed of: technical paraffin, dibutyl phthalate, technical borax, ammonium sulfate, aloxite or corundum and, as a carrier, sawdust of decidous or coniferous trees. Evaluated were: exposure to the polishing powder dust of workers operating polishing machines, as well as fibrogenic properties of the powder in an experiment on animals polishing machines, and irritating and allergic effects upon human skin (determination of irritation halftime, rubbing, damping and epidermal tests). The results demonstrated that the mean geometric concentrations of the dust ranged from 3.1 to 6.4 mg/m3 of air, whereas the dust respirable fraction constituted approx. 20% of the total dust concentration. The content of free crystalline silica in the dust did not surpans 2%. In the experiment on animals the dust exhibited cytolytic effects and induced reactive lesions in form of granulomas. The content of hydroxyproline in rats' lungs when affected by the polishing powder approximated the values obtained with quartz of weak fibrogenic properties. Basing on dermatological studies, the irritating effects of the polishing powder were defined as medium, whereas the allergic effects as weak. PMID- 3023795 TI - Proinsulin radioimmunoassay in the evaluation of insulinomas and familial hyperproinsulinemia. AB - Two new radioimmunoassays for human proinsulin (hPI) have been developed and used to study patients with islet cell tumors and familial hyperproinsulinemia. Both antisera were adsorbed against human C-peptide conjugated to Sepharose, following which cross-reactivity to insulin and C-peptide was less than 0.001%. Antiserum 18D recognized the junction between the insulin B-chain and C-peptide and provided fivefold greater sensitivity than our previously reported hPI assay. Antiserum 11E recognized a determinant which includes or is adjacent to the A chain-C-peptide junction or which is specified by the tertiary structure. In all 20 patients studied with surgically confirmed islet cell tumors, fasting plasma proinsulinlike material (PLM) was abnormal (greater than 3 SD from the mean measured in either lean or obese subjects) in both assays. This provided better discrimination than has been reported for PLM measured by gel filtration (abnormal in 13 of 14 of the present samples) with a considerably less laborious procedure. Samples from two families in which a mutant proinsulin is present in the circulation have immunoreactivity in the two assays consistent with previous identification of the molecule as an A-chain-C-peptide-linked intermediate of proinsulin conversion. The immunoreactivity of a sample from another family in which large amounts of proinsulin circulate are consistent with an intact molecule being the predominant form. This assay will be useful for confirming the diagnosis of insulin-secreting tumor in patients suspected of recurrent fasting hypoglycemia and in physiologic studies of proinsulin secretion. PMID- 3023796 TI - The effect of chloroquine on the equilibrium density in a Percoll gradient of liver lysosomal populations. PMID- 3023797 TI - Modification of susceptibility to Klebsiella pneumoniae during murine cytomegalovirus infection. AB - The effect of murine cytomegalovirus (MCMV) infection on susceptibility to bacterial infection was studied in mice by a combination of intraperitoneal (ip) inoculation of a sublethal dose of MCMV with subsequent ip challenge of 2 X 10(3) cfu of a strain of Klebsiella pneumoniae (KP). When given alone, KP produced a mortality of 30-40%. Mortality was increased when KP was given 1 to 7 days after MCMV injection with the peak increase at the 4th to 5th day when 100% mortality occurred. Virus levels in various organs of mice infected with MCMV alone, or superinfected with KP did not differ. Bacterial counts on the other hand, showed that increased mortality in mixed MCMV and KP infected mice was due to an uncontrolled growth of bacteria at the site of primary lodgment, i.e., the peritoneum, and severe systemic infection. Neutrophil response to growth of KP during the first 3 days of bacterial infection was defective in MCMV infected mice. While the initial clearance of KP from the blood was more efficient in MCMV infected mice, probably due to activated reticuloendothelial function, it did not protect the mice against KP infection. Using the in vivo model, it was shown that poor neutrophil response and possibly other defective neutrophil functions were responsible for increased mortality to KP infection in MCMV infected mice. PMID- 3023798 TI - Effect of nalidixic acid on the conversion of Staphylococcus aureus cells to L forms in a liquid medium with 6-aminopenicillanic acid and lysozyme. PMID- 3023799 TI - Enzyme-linked immunosorbent assay for detection of antibodies to Epstein-Barr virus-associated nuclear antigen-1 using a synthetic oligopeptide. PMID- 3023800 TI - Effect of HTLV-III on the macromolecular synthesis in HTLV-I carrying cell line, MT-4. AB - Upon infection of human T-cell leukemia virus type I (HTLV-I)-carrying human T cell lines such as MT-4, HTLV-III, a probable etiologic agent of acquired immune deficiency syndrome (AIDS) caused fast and strong cytopathic effects leading ultimately to the death of the cells. Such effects were preceded by the rapid induction of HTLV-III antigens. Cell lines not infected with HTLV-I could, however, be subcultured after infection with HTLV-III, although they were also positive for HTLV-III antigens. To understand this cytopathogenicity of HTLV-III in HTLV-I bearing cells, macromolecular synthesis, including DNA synthesis and total protein synthesis, and also IL-2 receptor expression were investigated kinetically. In infected MT-4 cells DNA synthesis was markedly inhibited by HTLV III after the HTLV-III antigen synthesis became evident. This inhibition occurred before cell damage was detected in terms of viable cell-growth, but after induction of HTLV-III antigen. Puromycin, at 40 micrograms/ml, caused no toxic changes in MT-4 cells over 3 days but prevented viral antigen synthesis and virus induced cytopathic effect. Protein synthesis and IL-2 receptor expression were also inhibited at 4 and 5 days post infection. The degree of the effects and their kinetics suggest that they are the secondary effects of cytotoxicity by HTLV-III infection. PMID- 3023801 TI - Infections in patients with non-small-cell lung cancer treated with intensive induction chemotherapy. AB - The records of 65 consecutive patients with non-small-cell lung cancer (NSCLC) treated with intensive induction chemotherapy were reviewed to study the infectious complications during therapy and to analyze the relationship of the frequency of infections to various predisposing factors. A total of 44 infectious episodes were observed among 30 of the 65 patients. Of the 44 infections, 18 were microbiologically documented and 19 clinically. Seven (16%) infections were without microbiological or clinical documentation and were categorized as "possible" infections. Among the 18 microbiologically documented infections, fifteen (83%) were caused by bacteria and three by fungi. The most frequent bacteria identified in 11 (61%) of the 18 infections were gram-negative organisms, Escherichia coli and Klebsiella pneumoniae being the most frequent. Eight of the 44 infections were associated with bacteremia and three with microbiologically documented pneumonias. There were three drug-related infectious deaths, two associated with bacteremia and one with possible infections. All 44 infectious episodes presented with WBC counts of less than 1,000/microliter, and 34 (79%) had WBC counts of less than 500/microliter. We observed that during therapy, patients with poor performance status (less than 80%) are at a much higher risk to develop infectious complications than those with good performance status (greater than 80%; p less than .001). Although encouraging responses with intensive chemotherapy have been reported for NSCLC in several studies, a major impact of chemotherapy on the survival of patients with this disease has yet not been established. Thus, intensive chemotherapy for NSCLC should remain an experimental treatment modality and should be offered only to patients with good prognostic factors such as those defined by pretreatment performance status. PMID- 3023802 TI - Effect of transfusions on serologic testing for antibody to varicella. AB - Serologic testing of patients with acute lymphocytic leukemia for susceptibility to varicella was confounded by prior administration of blood products. Packed red cell transfusions produced fewer problems than plasma-rich transfusions. Seronegative individuals may be transiently seropositive for several weeks. Specimens submitted for serologic testing should be accompanied by information on recent receipt of blood products. PMID- 3023804 TI - [Granular cell tumor of the esophagus. Case report with a review of the literature]. PMID- 3023803 TI - Proton beam penumbra: effects of separation between patient and beam modifying devices. AB - The sharp lateral penumbra of a proton beam is often used to spare sensitive normal structures in treating clinical sites in which the target volume abuts, or even wraps around, these structures. Using Monte Carlo calculations and measurements, the factors which influence the penumbra of the proton beam at the Harvard Cyclotron Laboratory were investigated, with particular emphasis on the effects of separation between the patient and any beam modifying devices. Penumbra broadening, characterized by the distance over which the dose rises from 20% to 80% of the central dose, increases with greater amounts of scatterer introduced into the beam line. The broadening due to separation of the beam modifying devices and the patient is essentially linear with increasing air gap; the rate of increase depends on the details of these devices and on the depth of interest in the patient. For a particular portal, most of the parameters which affect the penumbra width are fixed by the patient's anatomy and the target volume. Only the thickness of the compensating bolus around the aperture edge and any air gap between the patient and the beam modifying devices can vary. Families of curves relating combinations of bolus thickness and air gap that maintain a constant penumbra width have been developed for guidelines during patient setup. PMID- 3023807 TI - [Fibrolamellar carcinoma of the liver: a case report]. AB - This is the first case report of fibrolamellar carcinoma of the liver (FCL) in Japan with reference to the relevant literature. A 56 year-old Korean male with positive HBsAg was complaining of generalized weakness. Alpha-fetoprotein level was elevated and a mass lesion in right lobe of the liver was detected. He underwent a wedge resection, of the liver and the follow-up study failed to show recurrence of the tumor in 15 months. The specimen showed a tumor consisting of a firm, grayish-white, circumscribed solitary mass measuring 4 cm in diameter. Microscopically the tumor was characterized by polygonal and eosinophilic tumor cells and abundant fibrous stroma around the tumor cells in lamellar fashion. FCL is a well-defined disease entirely with a distinct histologic pattern and a favorable prognosis. PMID- 3023806 TI - [Preoperative diagnosis in lung surgery interventions]. PMID- 3023805 TI - [Severe obstructive ileus caused by a linseed bezoar. Case report with critical comment on the use of dietary fiber]. PMID- 3023808 TI - Ni-coupled receptors in cultured neural hybrid cells: cell specificity for dibutyryl cyclic AMP-induced down-regulation but not morphological differentiation. AB - Opiate, muscarinic, and alpha 2-adrenergic receptors and the Ni-coupled response of adenylate cyclase (AC) inhibition were examined in neuroblastoma X glioma NG108-15 (108 CC15) and neuroblastoma X Chinese hamster brain NCB-20 clonal hybrid cells, induced to differentiate with 1.0 mM dibutyryl cAMP (dBcAMP). Scatchard analysis of binding of the opiate agonist 3H-(D-Ala2,D-Leu5)enkephalin (DADLE) and the antagonist [3H] diprenorphine to dBcAMP-treated NCB-20 cell membranes indicated an 80% reduction in opiate receptor density relative to untreated cells (Bmax = 47 +/- 11 fmol/mg of protein versus 220 +/- 48 fmol/mg of protein), with no change in ligand affinities. Binding of the muscarinic cholinergic antagonist [3H]quinuclidinyl benzilate and the alpha 2-adrenergic agonist [3H]-p-aminoclonidine to dBcAMP-treated NCB-20 membranes was also reduced by 50% and 28%, respectively. In contrast, treatment of NG108-15 cells with dBcAMP did not down-regulate opiate, muscarinic, or alpha 2-adrenergic receptor sites. Opiate and alpha 2-adrenergic receptor sites were not down-regulated in the N18TG2 neuroblastoma clone, the common parent of both the hybrid cells, and the apparent source of these receptors. The C6BU-1 parent of the NG108-15 hybrid showed poor specific binding of all ligands examined. dBcAMP was very potent in inducing opiate receptor site down-regulation of NCB-20 cells, with an ED50 after 4 days treatment of 8 microM. The time course of loss of [3H]DADLE and [3H]quinuclidinyl benzilate specific binding was similar, and maximum down regulation was achieved after 2 days. In contrast, neither higher concentrations of dBcAMP (5.0 mM) nor longer treatment times (7 days) resulted in down regulation of receptor sites on NG108-15 cells. Coupling of opiate receptors to AC was also selectively altered in differentiated NCB-20 cells. Prostaglandin E1 stimulated AC was maximally inhibited by 1 microM DADLE in membranes from undifferentiated cells to different degrees (30% in NCB-20 and 54% in NG108-15). dBcAMP treatment had no effect on opiate inhibition of AC in NG108-15 cells but reduced by 50% the maximum opiate inhibition of AC in NCB-20 cells. These data indicate that the signal for receptor down-regulation which was triggered by dBcAMP in the NCB-20 cell comes uniquely from the Chinese hamster brain cell NCB 20 parent. The differences between NCB-20 and NG108-15 cells in the regulation of Ni-coupled receptors provides an example of dBcAMP-induced heterologous down regulation with unique cell specificity, which is unrelated to the morphological differentiation process triggered by dBcAMP, which is common to both cells. PMID- 3023809 TI - Molecular characterization of the solubilized atrial natriuretic factor receptor from bovine adrenal zona glomerulosa. AB - The atrial natriuretic factor (ANF) receptor has been solubilized from bovine adrenal zona glomerulosa membranes with the nonionic detergent octyl-beta-D glucoside. Mathematical analysis of competition binding curves with solubilized receptor revealed the presence of two classes of binding sites with pK of 10.4 (Kd = 40 pM) and 8.2 (kd = 6000 pM), similar to the native receptor of intact membranes. The hydrodynamic properties of the ANF receptor were determined by prelabeling the membrane receptor with 125I-ANF prior to solubilization. The solubilized 125I-ANF-receptor complex eluted as a major peak with a Stokes radius of 50.8 A from a Superose 6 steric exclusion column. A partial specific volume of 0.770 ml/g and a sedimentation coefficient (S20,w) of 6.34 S were determined by sucrose density gradient centrifugation in H2O and D2O. These data were used to calculate a molecular weight of 158,000 and a frictional ratio of 1.25 for the labeled receptor-detergent complex. The amount of detergent bound to the receptor was estimated to be 0.45 g/g of protein, assuming a partial specific volume of 0.730 ml/g for the protein. Correction for the mass contributed by the bound detergent yielded a molecular weight of 109,000 for the receptor protein. Affinity cross-linking of 125I-ANF to its binding sites in zona glomerulosa membranes and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that one single band with apparent Mr 130,000 was specifically labeled. These results indicate that the ANF receptor from bovine adrenal cortex is a membrane protein with a total molecular weight of 110,000-130,000 and suggest that the native protein contains only one single polypeptide chain. PMID- 3023810 TI - Differences in muscarinic receptor reserve for inhibition of adenylate cyclase and stimulation of phosphoinositide hydrolysis in chick heart cells. AB - Carbachol is 100 times more potent for inhibiting cyclic AMP formation than for stimulating phosphoinositide (PI) hydrolysis in chick heart cells. To determine whether this reflects differences in agonist affinity of the receptor(s) coupled to the two responses, we measured these functional responses following removal of receptor reserve with propylbenzilycholine mustard (PrBCM). Conditions of PrBCM treatment that led to progressive loss of up to 95% of the [3H]-N methylscopolamine-binding sites decreased the potency but not the maximal capacity of carbachol to inhibit cyclic AMP formation. In contrast, there was a marked decrease in the maximal PI response to carbachol. The KA for carbachol, calculated by measuring functional responses following receptor inactivation, was similar whether the cyclic AMP or the PI response was examined. These KA values (approximately 40 microM) were similar to the KD calculated by examining carbachol competition for [3H]-N-methylscopolamine-binding sites on the intact cell. PrBCM treatment also decreased the maximal effect of oxotremorine on cyclic AMP formation under conditions in which carbachol remained a full agonist for this response. We interpret our data as indicating that: there is much greater receptor reserve in the coupling of muscarinic receptors to adenylate cyclase than to PI hydrolysis; this, rather than differences in receptor affinity underlies the disparate dose-response relationships for the two responses; and differences in the effects of weak agonist on the two responses may also reflect differences in receptor reserve. We suggest that muscarinic receptors with the same affinity for carbachol interact with different efficiency with the transducers (Gi and Gx) that regulate adenylate cyclase and phospholipase C. PMID- 3023812 TI - The glycine receptor: pharmacological studies and mathematical modeling of the allosteric interaction between the glycine- and strychnine-binding sites. AB - The displacement by glycine of 3H-strychnine binding to rat spinal cord membranes cannot be explained by a simple competitive interaction. Indeed, protein modifying reagents can completely abolish the inhibition of 3H-strychnine binding by glycine and other agonists, whereas the interaction of strychnine itself and other related compounds with the binding site is unimpaired. Moreover, glycine cannot inhibit completely saturable 3H-strychnine binding, the extent of its maximum inhibitory effect depending on the ionic composition of the medium. Hill coefficients less than 1 (whose magnitude also depends on the assay medium) were obtained from glycine displacement curves. These properties are consistent with a mathematical model of two different, but mutually interacting, binding sites for strychnine and glycine on the glycine receptor. The effect of ions and protein modifying reagents might be explained in this model as modifications of the mechanisms that mediate the allosteric interaction, and/or the affinity of glycine for the receptor. The agonists beta-alanine and taurine and the new antagonists, THAZ, iso-THAZ, and 4,5-TAZA, also seem to interact with a site different from the strychnine-binding site, probably the glycine-binding site. PMID- 3023811 TI - Relationships of the molecular structure of aldosterone derivatives with their binding affinity for mineralocorticoid receptor. AB - The molecular structures of 19-nor-11-deoxycorticosterone (III) and 21 hydroxypregna-4,11-diene-3,20-dione (IV) were determined by X-ray crystallographic analysis and the factors affecting the binding affinities for the mineralocorticoid receptor were examined with six aldosterone derivatives (I VI) containing these two compounds. The most important factor was found to be the steric one; affinity increased with increasing flatness of the structure. The electronic factor may be a minor influence although a good relationship was found between the affinity and the 13C-NMR chemical shift of the C(5) atom. The factor playing no role in the binding is the hydrophobic one. PMID- 3023814 TI - Vitamin D3 derivatives inhibit the differentiation of Friend erythroleukemia cells. AB - A number of vitamin D3 metabolites inhibit benzodiazepine- and dimethyl sulfoxide induced differentiation of Friend erythroleukemia cells. The inhibition is dose dependent and occurs at nM concentrations. The order of potency of these compounds is 1,25-dihydroxycholecalciferol greater than 1,25,26 trihydroxycholecalciferol greater than 1,24R,25-trihydroxycholecalciferol greater than 1 alpha-hydroxycholecalciferol greater than 24R,25-dihydroxycholecalciferol greater than 25S,26-dihydroxycholecalciferol. The inhibition is maximal when the vitamin D3 analogs are added together with the inducer, and becomes progressively decreased with delayed addition. These results suggest that the vitamin D3 metabolites may play a regulatory role in erythropoiesis. PMID- 3023813 TI - Activation and inhibition of 3H-strychnine binding to the glycine receptor by Eccles' anions: modulatory effects of cations. AB - The ammonium salts of Eccles' anions are inhibitors of 3H-strychnine binding. By contrast, the binding is activated by the sodium salts of these anions. This activation is due to an increase in the affinity for 3H-strychnine. Pretreatment of the membranes with acetic anhydride abolishes the effect of sodium salt, whereas ammonium salts remain unaffected. In the presence of chloride the inhibitory effect of ammonium predominates over the effect of sodium. These results suggest that the effect of Eccles' anions is profoundly modified by their counter-ions and that Na+ and ammonium are acting through distinct sites on the glycine receptor. PMID- 3023815 TI - Metabolic activation of phenol by human myeloperoxidase and horseradish peroxidase. AB - The oxidation of phenol catalyzed by human myeloperoxidase and horseradish peroxidase resulted in extensive binding of phenol-derived metabolites to boiled rat liver protein. This binding paralleled closely the removal of phenol from the incubations and was inhibited from 83 to 99% by the addition of the antioxidants, ascorbate and glutathione, suggesting that metabolism and binding were occurring via a one-electron oxidation pathway. Metabolic studies employing both human myeloperoxidase and horseradish peroxidase resulted in the identification of 4,4' biphenol and diphenoquinone as the principal identifiable metabolites. The addition of reduced glutathione to incubations containing horseradish peroxidase resulted in the formation of two conjugate species. These conjugate species were identified by fast atom bombardment mass spectrometry to be glutathione conjugates of diphenoquinone. The major gluthathione conjugate was identified as 3-(glutathion-S-yl)-4,4'-biphenol by NMR spectroscopy. These results suggest that the formation of highly reactive species through the peroxidase-mediated metabolism of phenol and other phenolic compounds could play an important role in the hematopoietic toxicity observed during chronic benzene exposure. PMID- 3023816 TI - Drosophila forked locus. AB - A 40-kilobase-pair region of the Drosophila X chromosome from band 15F was cloned, and DNA insertions were indentified for the forked alleles f1, f3, f3n, f5, f36a, fs, and fx. The positions of these insertions are consistent with the organization of the two pseudoallelic series present at the forked locus. Three RNAs of 0.8, 2.6, and 3.3 kilobases are transcribed from this chromosomal region. The 0.8-kilobase transcript(s), present at the larval and adult stages, and the 3.3-kilobase transcript, present at each developmental stage, are unaffected by the forked mutations examined. Only the 2.6-kilobase RNA, present exclusively at the pupal stage, was observed to be less abundant in each of the forked mutants analyzed, consistent with this transcript being the product of the forked gene. PMID- 3023817 TI - Direct identification of palmitic acid as the lipid attached to p21ras. AB - p21v-H-ras, the transforming protein of Harvey murine sarcoma virus, contains a covalently attached lipid. Using thin-layer chromatography, we identified the acyl group as the 16-carbon saturated fatty acid palmitic acid. No myristic acid was detected in fatty acids released from in vivo-labeled p21v-H-ras. The p21v-K ras protein encoded by Kirsten sarcoma virus was also palmitylated. The processing and acylation of p21v-K-ras however differed from that of p21v-H-ras. Three forms of [3H]palmitic acid-labeled p21ras proteins were detected in Kirsten sarcoma virus-transformed cells. This contrasted with Harvey sarcoma virus, in which two forms of p21v-H-ras contained palmitic acid. Analysis by partial proteolysis of p21v-H-ras labeled with [3H]palmitic acid suggested that all of the lipid found in intact p21v-H-ras was located in the C-terminal region. On sodium dodecyl sulfate-polyacrylamide gels, p21v-H-ras labeled with [3H]palmitic acid migrated slightly ahead of the majority of p21v-H-ras. Of the mature forms of p21v-H-ras, apparently only a subpopulation contains palmitic acid. PMID- 3023818 TI - Multiple C4/Slp genes distinguished by expression after transfection. AB - The S region of the murine major histocompatibility complex contains two closely related genes: C4, encoding the fourth component of complement, and Slp, encoding sex-limited protein. We cloned these genes from a cosmid library of the B10.W7R strain that does not show androgen regulation of the Slp protein. Restriction site polymorphisms revealed at least four C4-like genes within the Sw7 locus, indicating evolutionary amplification of this region. Transfection of these genes into L cells resulted in expression, processing, and secretion of immunologically correct C4 and Slp proteins. At least two different Slp genes and one C4 gene were capable, after transfection, of expressing C4 and Slp indistinguishable from macrophage-derived protein. A third Slp gene exists within this locus whose recombinant cognate did not express in L cells. Thus, the B10.W7R S region includes one C4 gene and at least three Slp-like genes. PMID- 3023819 TI - Integrative transformation of Candida albicans, using a cloned Candida ADE2 gene. AB - Candida albicans is a diploid dimorphic yeast with no known sexual cycle. The development of a DNA transformation system would greatly improve the prospects for genetic analyses of this yeast. Plasmids were isolated from a Candida Sau3A partial library which complements the ade2-1 and ade2-5 mutations in Saccharomyces cerevisiae. These plasmids contain a common region, part of which, when subcloned, produces ade2 complementation. Among the small number of auxotrophs previously isolated in C. albicans, red adenine-requiring mutants had been identified by several groups. In two of these strains, the cloned Candida DNA transformed the mutants to ADE+ at frequencies of 0.5 to 5 transformants per micrograms of DNA. In about 50% of the transformants, plasmid DNA sequences became stably integrated into the host genome and, in the several cases analyzed by Southern hybridization, the DNA was integrated at the site of the ADE2 gene in one of the chromosomal homologs. PMID- 3023820 TI - The complete sequence of the mouse skeletal alpha-actin gene reveals several conserved and inverted repeat sequences outside of the protein-coding region. AB - The complete nucleotide sequence of a genomic clone encoding the mouse skeletal alpha-actin gene has been determined. This single-copy gene codes for a protein identical in primary sequence to the rabbit skeletal alpha-actin. It has a large intron in the 5'-untranslated region 12 nucleotides upstream from the initiator ATG and five small introns in the coding region at codons specifying amino acids 41/42, 150, 204, 267, and 327/328. These intron positions are identical to those for the corresponding genes of chickens and rats. Similar to other skeletal alpha actin genes, the nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein. Comparison of the nucleotide sequences of rat, mouse, chicken, and human skeletal muscle alpha actin genes reveals conserved sequences (some not previously noted) outside of the protein-coding region. Furthermore, several inverted repeat sequences, partially within these conserved regions, have been identified. These sequences are not present in the vertebrate cytoskeletal beta-actin genes. The strong conservation of the inverted repeat sequences suggests that they may have a role in the tissue-specific expression of skeletal alpha-actin genes. PMID- 3023822 TI - Abelson virus potentiates long-term growth of mature B lymphocytes. AB - Abelson murine leukemia virus (A-MuLV) infection of mouse bone marrow cells usually leads to transformation of pre-B cells. However, when the environment is modified by the continuous presence of lipopolysaccharide (LPS), two novel types of membrane immunoglobulin (mIg)-positive B cell lines are generated. Because the cells which give rise to these cell lines copurify with mIg-positive bone marrow cells, the cell lines arise as a result of A-MuLV interaction with a new type of in vitro target cell. The cell lines generated fall into two groups which differ in several phenotypic characteristics. Group 1 cells are more differentiated than the typical pre-B cell transformant in that they synthesize mIgM and appear to resemble virgin B cells. The group 1 cells do not secrete immunoglobulin and are independent of LPS for growth. In addition, these cell lines synthesize the Abelson P160 protein, contain integrated abl proviral DNA, and are highly tumorigenic in syngeneic animals. The group 2 cell lines differ markedly from both the group 1 cells and from typical, pre-B cell A-MuLV transformants. These cells are mIgG positive and secrete large amounts of immunoglobulin into the culture medium. The cell lines are comprised of both adherent and nonadherent cells and do not synthesize P160 or contain integrated v-abl sequences. The group 2 cells are nontumorigenic in syngeneic animals and require LPS for growth and viability. Both types of cells have remained in culture for over 2 years with no changes in their phenotypic characteristics. This A-MuLV infection system and the novel mIg-positive cell lines may serve as useful models for studying biochemical and molecular properties of mature B cells. PMID- 3023821 TI - The sequence of a large L1Md element reveals a tandemly repeated 5' end and several features found in retrotransposons. AB - The complete nucleotide sequence of a 6,851-base pair (bp) member of the L1Md repetitive family from a selected random isolate of the BALB/c mouse genome is reported here. Five kilobases of the element contains two overlapping reading frames of 1,137 and 3,900 bp. The entire 3,900-bp frame and the 3' 600 bp of the 1,137-bp frame, when compared with a composite consensus primate L1 sequence, show a ratio of replacement to silent site differences characteristic of protein coding sequences. This more closely defines the protein coding capacity of this repetitive family, which was previously shown to possess a large open reading frame of undetermined extent. The relative organization of the 1,137- and 3,900 bp reading frames, which overlap by 14 bp, bears resemblance to protein-coding, mobile genetic elements. Homology can be found between the amino acid sequence of the 3,900-bp frame and selected domains of several reverse transcriptases. The 5' ends of the two L1Md elements described in this report have multiple copies, 4 2/3 copies and 1 2/3 copy, of a 208-bp direct tandem repeat. The sequence of this 208-bp element differs from the sequence of a previously defined 5' end for an L1Md element, indicating that there are at least two different 5' end motifs for L1Md. PMID- 3023823 TI - Polymorphism in an androgen-regulated mouse gene is the result of the insertion of a B1 repetitive element into the transcription unit. AB - The single-copy RP2 gene in mice produces three major mRNAs, the abundances of which are significantly increased in the kidneys by the administration of testosterone. S1 nuclease analysis of the kidney mRNAs indicated that they differ in the lengths of their 3' untranslated regions as a result of the use of different polyadenylation sites. When the mRNAs from different inbred mouse strains were examined by Northern blot analysis, it was observed that the largest mRNA varies in size, whereas the sizes of the other mRNAs remain the same. In DBA/LiHa and DBA/2J mice, the largest mRNA is approximately 2,150 nucleotides long, whereas the corresponding mRNA in C57BL/6J and BALB/cJ mice is only 1,950 nucleotides in length. All of these strains also have RP2 mRNAs that are 1,450 and 1,350 nucleotides long. By S1 nuclease mapping and comparison of the sequence of cDNA clones representing these mRNAs in DBA/LiHa and C57BL/6J mice, we determined that this size difference or polymorphism observed in the largest mRNA is the result of the insertion of a member of the B1 family of repeats into the 3' untranslated region of the RP2 gene in DBA mice. This particular B1 repeat is transcribed by RNA polymerase III in vitro, and its transcriptional orientation is opposite to that of the RP2 transcript. The polymorphism described here is evidence for the mobility of B1 repetitive elements within the genome. PMID- 3023825 TI - Duplicate upstream activating sequences in the promoter region of the Saccharomyces cerevisiae GAL7 gene. AB - We constructed a series of deletions in the 5' noncoding region of the Saccharomyces cerevisiae GAL7 gene, fused them to the Escherichia coli gene lacZ, and introduced them into yeasts by using a multicopy vector. We then studied the effect of the deletions on beta-galactosidase synthesis directed by the gene fusions in media with various carbon sources. This analysis identified a TATA box and two upstream activating sequences as necessary elements for galactose controlled GAL7 transcription. Two upstream activating sequences exhibiting 71% homology with each other were located 255 and 168 base pairs, respectively, upstream of the GAL7 transcription start point. Each sequence consists of 21 base pairs, displaying an approximate rotational symmetry with a core consensus sequence of GAA--AGCTGCTTC--CGCG. At least one of the two sequences is required for galactose induction and also for glucose repression of the GAL7'-lac'Z gene. Analysis with host regulatory mutants delta gal14 and delta gal180 suggests that these sequences are the site at which the GAL4 product exerts its action to activate the GAL7 gene. We also observed that a deletion lacking both upstream activation sequences allowed the gene fusion to be expressed in the absence of galactose at about 10% of the fully induced level of the intact fusion. This constitutive expression depended on the presence of the TATA box of GAL7 in cis but not on a functional GAL4 gene. The level of the uncontrolled expression was decreased by increasing the distance between the TATA box and the pBR322 sequence in the vector plasmid. PMID- 3023824 TI - Sequences from sea urchin TU transposons are conserved among multiple eucaryotic species, including humans. AB - Sequences homologous to various structural domains of the Strongylocentrotus purpuratus TU family of transposons are present in sea urchin species closely related to S. purpuratus and were found in close proximity to each other in linkage patterns that differed for different species. Sequence homologs of the inverted repeat outer domain (IVR-OD) segment were, in addition, present in a sea urchin related only distantly to S. purpuratus and in all other eucaryotic organisms surveyed. In humans, a polymorphic hybridization pattern was seen for genomic DNA obtained from different individuals. Sequence comparisons revealed that repeated sequence motifs similar to those making up the 15-base-pair direct repeat unit of the IVR-OD domain of the TU elements exist in the IVRs of transposons identified in Drosophila melanogaster and maize and in the transcription control regions of certain eucaryotic viral and cellular genes. The remarkable evolutionary conservation of IVR-OD homologs may reflect a biological role for these sequences in DNA transposition, the regulation of gene expression, or both. PMID- 3023826 TI - Biosynthesis and glycosylation of the epidermal growth factor receptor in human tumor-derived cell lines A431 and Hep 3B. AB - Biosynthesis of the receptor for epidermal growth factor was investigated in two human tumor-derived cell lines, Hep 3B and A431. When grown in the presence of tunicamycin, both cells expressed a receptor-related species p135, the presumptive aglycosylated form of the biosynthetic precursor, gp145, of the mature form of the receptor, gp165, expressed at the cell surface. Two additional receptor-related species, p115 and p70, were detected when A431, but not Hep 3B, cells were treated with tunicamycin. Furthermore, digestion of the A431 receptor related proteins with endoglycosidase F resulted in the detection of these three aglycosylated species. P70 appears to be the aglycosylated form of gp95, the presumptive intracellular precursor of the receptor-related species gp120 that is secreted by A431 but not Hep 3B cells; gp120 has a complex pattern of N-linked glycosylation, with consequent molecular weight and charge heterogeneity. P115 may be the aglycosylated form of a third biosynthetic intermediate, possibly a gp135 species detected in the early time points of pulse-chase labeling. Alternatively, p115 and gp135 may be derived co- or post-translationally by Ca2+ mediated proteolysis from p135 and gp145, respectively. The implications of the complexity of the biosynthesis of this molecule with regard to the multiple opportunities it affords the cell to modulate cell proliferation are discussed. PMID- 3023828 TI - DNA sequence homology between the terminal inverted repeats of Shope fibroma virus and an endogenous cellular plasmid species. AB - DNA hybridization experiments indicate that the genome of a tumorigenic poxvirus. Shope fibroma virus (SFV), possesses sequence homology with DNA isolated from uninfected rabbit cells. Southern blotting experiments, either with high complexity rabbit DNA as probe and SFV restriction fragments as targets or with high-specific activity, 32P-labeled, cloned SFV sequences as probes and rabbit DNA as target, indicate that the homologous sequences map at two locations within the viral genome, one in each copy of the terminal inverted repeat sequences. Unexpectedly, Southern blots revealed that the homologous host sequences reside in a rabbit extrachromosomal DNA element. This autonomous low-molecular-weight DNA species could be specifically amplified by cycloheximide treatment and was shown by isopycnic centrifugation in cesium chloride-ethidium bromide to consist predominantly of covalently closed circular DNA molecules. DNA sequencing of pSIC 9, a cloned 1.9-kilobase fragment of the rabbit plasmid species, indicated extensive homology at the nucleotide level over a 1.5-kilobase stretch of the viral terminal inverted repeat. Analysis of open reading frames in both the plasmid and SFV DNA revealed that (i) the N-terminal 157-amino acid sequence of a potential 514-amino acid SFV polypeptide is identical to the N-terminal 157 amino acids of one pSIC-9 open reading frame, and (ii) a second long pSIC-9 open reading frame of 361 amino acids, although significantly diverged from the comparable nucleotide sequence in the virus, possessed considerable homology to a family of cellular protease inhibitors, including alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III. The potential role of such cellular plasmid like DNA species as a mediator in the exchange of genetic information between the host cell and a cytoplasmically replicating poxvirus is discussed. PMID- 3023827 TI - Structure, organization, and regulation of human metallothionein IF gene: differential and cell-type-specific expression in response to heavy metals and glucocorticoids. AB - We describe a human genomic clone containing the metallothionein (MT) IF and MT IG genes. Southern blot analysis and partial DNA sequence determinations show that these genes are organized in a head-to-head fashion and are located approximately 7.0 kilobases apart from each other. Sequence analysis shows that the MT IF gene contains three exons separated by two introns. All of the intron exon junctions are defined by the GT-AG rule. The 5' flanking region shows the presence of a duplicated metal regulatory element (TGCGC CCGGCCC) important in heavy-metal induction of this gene and a sequence for its basal level expression (GCGGGGCGGGTGCAAAG). The 5' flanking region is also highly G + C rich (approximately 75%) and contains several GC boxes (GGGCGG), probably important in the binding of transcription factors. The TATAA box and the AATAAA sequence are represented by their variants, the TATCAA box and the AATTAA sequence, respectively. This gene is functional and inducible by heavy metals but not by dexamethasone in mouse LMTK- cells after its transfer on a plasmid containing the herpes simplex virus thymidine kinase gene. Further studies on various human cell lines show that this gene is not expressed in a splenic lymphoblastoid cell line (WI-L2) but is expressed in two hepatoma cell lines (Hep 3B2 and Hep G2) in response to cadmium, zinc, and copper. Dexamethasone appears to have no significant effect on its expression. The studies suggest that the MT IF gene shows cell-type-specific expression and is differentially regulated by heavy metals and glucocorticoids. PMID- 3023829 TI - Viral transfer, transcription, and rescue of a selectable myeloproliferative sarcoma virus in embryonal cell lines: expression of the mos oncogene. AB - A derivative of the myeloproliferative sarcoma virus (Neor-MPSV) carrying the mos oncogene and dominant selection marker for neomycin resistance (Neor) was introduced into embryonal carcinoma and embryo-derived cell lines by transfection and infection using pseudotypes with Friend helper virus (Friend murine leukemia virus [F-MuLV]). Cells resistant to G418 (a neomycin analog) were cloned and expanded. Transductants retained an undifferentiated phenotype as judged by morphology, tumorigenicity, and cell-surface antigen analyses. Nucleic acid analysis of infectants revealed both Neor-MPSV and F-MuLV proviruses, although no virus was released. G418-resistant transductants remained nonpermissive for the expression of other proviruses and for subsequent superinfection. Northern analysis showed expression of full-length Neor-MPSV, as well as mos-specific subgenomic RNA. mos sequences were deleted from Neor-MPSV (Neor mos-1), and pseudotypes were used to infect embryonal carcinoma cells. No morphological differences were observed in either mos+ or mos- transductants as compared with parental cell lines. However, mos+ transductants showed an enhanced anchorage independent growth compared with that of mos- transductants in agar cloning. PCC4 transductants were induced to differentiate with retinoic acid and superinfected with F-MuLV. Infection with viral supernatant in fibroblasts and in mice confirmed the rescue of biologically active Neor-MPSV. PMID- 3023831 TI - Liver-specific expression of a Qa-encoded class I gene is associated with DNA hypomethylation. AB - DNA methylation of two murine major histocompatibility complex (H-2) class I genes was examined in hybridizations to MspI and HpaII chromosomal DNA restriction digests. Q10, which exhibits liver-specific expression, and H-2Kb, a transplantation antigen gene, were examined in liver, spleen, thymus, and cell line DNAs. Unmethylated Q10 gene sequences were detected only in the liver, whereas the H-2Kb gene was unmethylated in all tissues examined. PMID- 3023830 TI - Rat metallothionein-1 structural gene and three pseudogenes, one of which contains 5'-regulatory sequences. AB - As shown by Southern blot analysis, the metallothionein-1 (MT-1) genes in rats comprise a multigene family. We present the sequence of the MT-1 structural gene and compare its features with other metallothionein genes. Three MT-1 pseudogenes which we sequenced apparently arose by reverse transcription of processed mRNA transcripts. Two of these, MT-1 psi a and MT-1 psi c, are retrogenes which derive from the MT-1 mRNA, having diverged from the MT-1 gene 6.9 and 2.6 million years ago, respectively. The third, MT-1 psi b, differs from the MT-1 cDNA by only three nucleotide alterations. Surprisingly, MT-1 psi b also preserves sequence homology for 142 base pairs 5' to the transcription initiation site of the parent gene; it contains a promoter sequence sufficient for specifying metal ion induction. We identified, by S1 nuclease mapping, an RNA polymerase II initiation site 432 base pairs 5' of the MT-1 transcription initiation site of the MT-1 structural gene which could explain the formation of the mRNA precursor to this pseudogene. We were unable to detect MT-1 psi b transcripts, either in liver tissue or after transfection. We conclude that the absence of detectable transcripts from this pseudogene is due to either a reduced level of transcription or the formation of unstable transcripts as a consequence of the lack of a consensus sequence normally found 3' of transcription termination in the MT-1 structural gene. PMID- 3023832 TI - Binding of alpha-factor pheromone to Saccharomyces cerevisiae a cells: dissociation constant and number of binding sites. AB - The number of alpha-factor binding sites on yeast MATa cells (8,000) and the equilibrium dissociation constant (6 X 10(-9) M) were determined from direct binding experiments. These values correct our previously reported estimates (D. D. Jennes, A. C. Burkholder, and L. H. Hartwell, Cell 35:521-529, 1983) that were based on indirect isotope dilution studies, and they lead to a revised rate constant for the association process (kon = 3 X 10(5) mol-1 s-1). PMID- 3023833 TI - Sensitive and specific DNA probe for detection of Plasmodium falciparum. AB - The isolation and some characteristics of a very sensitive DNA probe for the detection of Plasmodium falciparum are described. The probe is species specific and represents a large, albeit variable, fraction of the genome in all the strains tested. In addition to its immediate practical uses for the detection and quantitation of parasites, the probe defines an interesting family of repeated sequences. PMID- 3023835 TI - Expression of viral p21ras during acquisition of a transformed phenotype by rat adrenal cortex cells infected with Kirsten murine sarcoma virus. AB - Rat adrenal cortex cells infected with Kirsten murine sarcoma virus acquire a transformed phenotype in a progressive fashion. The expression of the viral p21ras does not appear to correlate with the degree of transformation of the adrenocortical cells but rather is produced at similar levels as the culture becomes transformed. This indicates that the expression of an oncogenic form of p21ras is not of itself sufficient to completely transform rat adrenal cortex cells. PMID- 3023836 TI - Interactions among the gypsy transposable element and the yellow and the suppressor of hairy-wing loci in Drosophila melanogaster. AB - We cloned and characterized the yellow locus of Drosophila melanogaster. We also studied its transcription pattern in the suppressible allele y2, which is caused by the insertion of the transposable element gypsy, and the effect of mutations at the unlinked suppressor of Hairy-wing locus on the transcription of yellow RNAs. The gypsy element is transcribed in a temporal fashion that correlates with the pattern of expression of the yellow locus. We propose that the mutational effect of the gypsy element is due to developmentally specific transcriptional interference on yellow transcription. Mutations at the su(Hw) locus reverse this effect by altering the quantitative expression of gypsy. PMID- 3023834 TI - Isolation of chicken cellular DNA sequences with homology to the region of viral oncogenes that encodes the tyrosine kinase domain. AB - A library of chicken genomic DNA was screened for sequences that could hybridize to a cloned DNA fragment containing the transforming gene (v-fps) of Fujinami sarcoma virus. In addition to c-fps, two unique chicken cellular DNA sequences were isolated that hybridized weakly to v-fps. These sequences hybridized with many other viral oncogenes encoding tyrosine kinases. Sequence analysis of the region where homology was detected revealed a region that is highly conserved among the tyrosine kinases both at the nucleotide and amino acid levels. Although we were unable to detect expression of either chicken cellular DNA sequence in a variety of avian tissues, the data suggest the existence of additional members of the tyrosine kinase gene family. Screening genomic libraries for sequences that hybridize weakly to functional regions of other genes may prove useful for the isolation and characterization of additional members of other gene families. PMID- 3023837 TI - Subnuclear localization of proteins encoded by the oncogene v-myb and its cellular homolog c-myb. AB - The retroviral transforming gene v-myb encodes a 45,000-Mr nuclear transforming protein (p45v-myb). p45v-myb is a truncated and mutated version of a 75,000-Mr protein encoded by the chicken c-myb gene (p75c-myb). Like its viral counterpart, p75c-myb is located in the cell nucleus. As a first step in identifying nuclear targets involved in cellular transformation by v-myb and in c-myb function, we determined the subnuclear locations of p45v-myb and p75c-myb. Approximately 80 to 90% of the total p45v-myb and p75c-myb present in nuclei was released from nuclei at low salt concentrations, exhibited DNA-binding activity, and was attached to nucleoprotein particles when released from the nuclei after digestion with nuclease. A minor portion of approximately 10 to 20% of the total p45v-myb and p75c-myb remained tightly associated with the nuclei even in the presence of 2 M NaCl. These observations suggest that both proteins are associated with two nuclear substructures tentatively identified as the chromatin and the nuclear matrix. The function of myb proteins may therefore depend on interactions with several nuclear targets. PMID- 3023838 TI - Ty insertions at two loci account for most of the spontaneous antimycin A resistance mutations during growth at 15 degrees C of Saccharomyces cerevisiae strains lacking ADH1. AB - The mutation rate to antimycin A resistance was determined for strains of Sacchromyces cerevisiae lacking a functional copy of the structural gene for alcohol dehydrogenase I (ADH1). One type of mutation that can cause antimycin A resistance in these strains is insertion of the transposable element Ty 5' to ADH2, the structural gene for the glucose-repressed isozyme of alcohol dehydrogenase, resulting in expression of this gene during growth on glucose. Here we show that after growth at 15 or 20 degrees C on glucose, 30% of the antimycin A resistance mutations are Ty insertions at ADH2 and another 65% of the mutations are Ty insertions at ADH4, a new locus identified and cloned as described in this paper. At 30 degrees C only 6% of the mutations are Ty insertions at either of these two loci. In addition, we show that the transposition rate is lower in mating-incompetent (a/alpha) cells than in either haploid or diploid mating-competent cells. Our results suggest that under certain conditions Ty transposition may be a major cause of spontaneous mutations in S. cerevisiae. PMID- 3023839 TI - Replicating plasmids in Schizosaccharomyces pombe: improvement of symmetric segregation by a new genetic element. AB - We characterized a number of widely used yeast-Escherichia coli shuttle vectors in the fission yeast Schizosaccharomyces pombe. The 2 micron vectors pDB248 and YEp13 showed high frequency of transformation, intermediate mitotic and low meiotic stability, and a low copy number in S. pombe, analogous to their behavior in [cir0] strains of Saccharomyces cerevisiae. The S. cerevisiae integration vectors pLEU2 and pURA3 transformed S. pombe at very low frequencies but, surprisingly, in a nonintegrative fashion. Instead, they replicated autonomously, and they showed very high copy numbers (up to 150 copies per plasmid-containing cell). This could reflect a lack of sequence specificity for replication of plasmid DNA in S. pombe. pFL20, an S. pombe ars vector, and a series of plasmids derived from it were studied to analyze the unusually high stability of this plasmid. Mitotic stability and partitioning of the plasmids was measured by pedigree analysis of transformed S. pombe cells. An S. pombe DNA fragment (stb) was identified that stabilizes pFL20 by improvement of plasmid partitioning in mitosis and meiosis. PMID- 3023841 TI - The nine amino-terminal residues of delta-aminolevulinate synthase direct beta galactosidase into the mitochondrial matrix. AB - delta-Aminolevulinate synthase, the first enzyme in the heme biosynthetic pathway, is encoded by the nuclear gene HEM1. The enzyme is synthesized as a precursor in the cytoplasm and imported into the matrix of the mitochondria, where it is processed to its mature form. Fusions of beta-galactosidase to various lengths of amino-terminal fragments of delta-aminolevulinate synthase were constructed and transformed into yeast cells. The subcellular location of the fusion proteins was determined by organelle fractionation. Fusion proteins were found to be associated with the mitochondria. Protease protection experiments involving the use of intact mitochondria or mitoplasts localized the fusion proteins to the mitochondrial matrix. This observation was confirmed by fractionation of the mitochondrial compartments and specific activity measurements of beta-galactosidase activity. The shortest fusion protein contains nine amino acid residues of delta-aminolevulinate synthase, indicating that nine amino-terminal residues are sufficient to localize beta-galactosidase to the mitochondrial matrix. The amino acid sequence deduced from the DNA sequence of HEM1 showed that the amino-terminal region of delta-aminolevulinate synthase was largely hydrophobic, with a few basic residues interspersed. PMID- 3023840 TI - DNA damage and heat shock dually regulate genes in Saccharomyces cerevisiae. AB - Two Saccharomyces cerevisiae genes isolated in a differential hybridization screening for DNA damage regulation (DDR genes) were also transcriptionally regulated by heat shock treatment. A 0.45-kilobase transcript homologous to the DDRA2 gene and a 1.25-kilobase transcript homologous to the DDR48 gene accumulated after exposure of cells to 4-nitroquinoline-1-oxide (NQO; 1 to 1.5 microgram/ml) or brief heat shock (20 min at 37 degrees C). The DDRA2 transcript, which was undetectable in untreated cells, was induced to high levels by these treatments, and the DDR48 transcript increased more than 10-fold as demonstrated by Northern hybridization analysis. Two findings argue that dual regulation of stress-responsive genes is not common in S. cerevisiae. First, two members of the heat shock-inducible hsp70 family of S. cerevisiae, YG100 and YG102, were not induced by exposure to NQO. Second, at least one other DNA-damage-inducible gene, DIN1, was not regulated by heat shock treatment. We examined the structure of the induced RNA homologous to DDRA2 after heat shock and NQO treatments by S1 nuclease protection experiments. Our results demonstrated that the DDRA2 transcript initiates equally frequently at two sites separated by 5 base pairs. Both transcriptional start sites were utilized when cells were exposed to either NQO or heat shock treatment. These results indicate that DDRA2 and DDR48 are members of a unique dually regulated stress-responsive family of genes in S. cerevisiae. PMID- 3023842 TI - Role of the avian retrovirus mRNA leader in expression: evidence for novel translational control. AB - Avian retroviral mRNAs contain a long 5' untranslated leader of approximately 380 nucleotides. The leader includes sequences required for viral replication and three AUG codons which precede the AUG codon used for translational initiation of the gag and env genes. We have used sensitive, quantitative assays of viral gene transcription and translation to analyze the role of this mRNA leader in viral gene expression. By substituting segments from related viruses, we had previously shown that the endogenous avian provirus ev-1 contained a defective leader segment (B. R. Cullen, A. M. Skalka, and G. Ju, Proc. Natl. Acad. Sci. USA 80:2946-2950, 1983). The sequence analysis presented here, followed by comparison with the nondefective ev-2 endogenous provirus segment, identified the critical changes at nucleotides 4 and 7 upstream of the initiator AUG. These differences do not alter the most conserved nucleotides within the consensus sequence which precedes eucaryotic initiation codons, but lie within a nine-nucleotide region that is otherwise highly conserved among avian retrovirus strains. Analysis of a series of deletion mutants indicated that other sequences within the leader are also required for efficient expression. Characterization of the altered transcripts demonstrated that the presence of the defective ev-1 segment or the deletion of a ca. 200-nucleotide leader segment did not affect the steady-state level or splicing efficiency of these mRNAs. Thus, we conclude that the reduced expression of these mRNAs is due to a translational deficiency. These results indicate that specific leader sequences, other than the previously identified consensus nucleotides which precede eucaryotic AUG initiator codons, can influence eucaryotic gene translation. PMID- 3023843 TI - Two modes of c-myb activation in virus-induced mouse myeloid tumors. AB - Two modes of disruption of the protooncogene c-myb by viral insertional mutagenesis in mouse myeloid tumor cells are described. The first mode was found in six tumors in which a Moloney murine leukemia virus component had inserted in the same transcriptional orientation upstream of the 5'-most exon with v-myb homology (vE1). cDNA sequence data indicate the presence of a truncated c-myb mRNA that is initiated in the upstream 5' long terminal repeat of the integrated provirus and processed via a cryptic splice donor sequence in the gag region to the splice acceptor site in vE1 of the c-myb gene, thus removing the remaining downstream viral and myb intronic sequences. Unlike most gag-onc transcripts, the gag and myb sequences in the hybrid transcript were not in the same reading frame. It is presumed that the gag sequence provides a cryptic translation initiation site for the novel amino-truncated c-myb protein. The second mode of disruption was by downstream virus insertion at the 3' side of the c-myb, which results in the synthesis of a small (approximately 2 kilobase) myb transcript. The 5' long terminal repeat of the inserted provirus provides a TGA termination codon that results in the elimination of 240 normal c-myb amino acid residues from the carboxyl terminus of the tumor-specific myb protein. These results suggest that truncated myb proteins play a role in neoplastic transformation of myeloid cells. PMID- 3023844 TI - Fine structure of the human hypoxanthine phosphoribosyltransferase gene. AB - The human hypoxanthine phosphoribosyltransferase (HPRT) gene has been characterized by molecular cloning, mapping, and DNA sequencing techniques. The entire gene, which is about 44 kilobases in length, is composed of nine exon elements. The positions of the introns within the coding sequence are identical to those of the previously-characterized mouse HPRT gene, although there are significant differences between intron sizes for the two genes. HPRT minigenes have been used in a transient expression assay involving microinjection into HPRT cells to demonstrate functional promoter activity within a 234-base-pair region upstream from the ATG codon. The promoter of this gene resembles those of other recently characterized "housekeeping" genes in that it lacks CAAT- and TATA-like sequences, but contains several copies of the sequence GGGCGG. Both RNase protection and primer extension analysis indicate that human HPRT mRNA is heterogeneous at the 5' terminus, with transcription initiation occurring at sites located congruent to 104 to congruent to 169 base pairs upstream from the ATG codon. Comparison of the mouse and human HPRT 5'-flanking sequences indicates that there are only limited stretches of conserved sequence, although there are other shared features, such as an extremely high density of potential methylation sites, that may have functional significance. PMID- 3023845 TI - Structure of the highly repeated, long interspersed DNA family (LINE or L1Rn) of the rat. AB - We present the DNA sequence of a 6.7-kilobase member of the rat long interspersed repeated DNA family (LINE or L1Rn). This member (LINE 3) is flanked by a perfect 14-base-pair (bp) direct repeat and is a full-length, or close-to-full-length, member of this family. LINE 3 contains an approximately 100-bp A-rich right end, a number of long (greater than 400-bp) open reading frames, and a ca. 200-bp G + C-rich (ca. 60%) cluster near each terminus. Comparison of the LINE 3 sequence with the sequence of about one-half of another member, which we also present, as well as restriction enzyme analysis of the genomic copies of this family, indicates that in length and overall structure LINE 3 is quite typical of the 40,000 or so other genomic members of this family which would account for as much as 10% of the rat genome. Therefore, the rat LINE family is relatively homogeneous, which contrasts with the heterogeneous LINE families in primates and mice. Transcripts corresponding to the entire LINE sequence are abundant in the nuclear RNA of rat liver. The characteristics of the rat LINE family are discussed with respect to the possible function and evolution of this family of DNA sequences. PMID- 3023846 TI - Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite strand exon in the 5' region of the CHO dhfr gene. AB - Transcription of the 26-kilobase (kb) dihydrofolate reductase (dhfr) gene in CHO cells is initiated at two sites: a major site (approximately 85% of the dhfr mRNA) at -63 relative to the translation start and a minor site (approximately 15%) at -107. Transcription also occurs from the opposite DNA strand in the dhfr 5' region, with a probable initiation site at approximately -195 relative to the dhfr translation start. A 4-kb polyadenylated RNA that is derived from the opposite-strand transcription increases threefold in abundance after serum starvation of CHO cells for 24 h. dhfr mRNA levels do not change during this time. The first dhfr exon lies within a 1-kb genomic region marked by exceptionally high G + C content and lack of DNA methylation. This region also includes a 214-base-pair (bp) exon for the opposite-strand transcript and five of the six DNase I-hypersensitive sites identified at the dhfr locus. Analysis of the DNA sequences of hamster, human (M. Chen, T. Shimada, A. D. Moulton, A. Cline, R. K. Humphries, J. Maizel, and A. W. Nienhuis, J. Biol. Chem. 259:3933 3943, 1984), and mouse (M. McGrogan, C. C. Simonsen, D. T. Smouse, P. J. Farnham, and R. T. Schimke, J. Biol. Chem. 260:2307-2314, 1985) dhfr genes reveals the presence of a 29-bp unit that is conserved 45 to 49 bp upstream of major and minor dhfr transcription start sites. This unit follows the consensus: GRGGCGGTGGCCTNNNNTGTCRCAARTRGGTR. The 5' part of the 29-bp unit contains a GC box that agrees with the GGGCGG consensus-binding site for the RNA polymerase II transcription factor Sp1 (D. Gidoni, W. A. Dynan, and R. Tjian, Nature (London) 312:409-413, 1984). Each of the three mammalian dhfr genes has several G-rich GC boxes proximal to the major dhfr transcription start site and several GC boxes of the opposite orientation (C rich) in a distal region about 500 bp upstream. PMID- 3023847 TI - Eucaryotic chromosome transfer: production of a murine-specific cosmid library from a neor-linked fragment of murine chromosome 17. AB - We recently developed a procedure for the molecular analysis of specific mammalian chromosomal fragments. This procedure allows for the transfer of contiguous chromosomal fragments, varying in size from a fraction to several centimorgans in length, from the donor cell of one species into a recipient cell of a different species. Specifically, we inserted a neor gene, encoded by a recombinant retrovirus, into the murine major histocompatibility complex (MHC). Metaphase chromosome transfers with this neor-tagged chromosome into recipient hamster, primate, and canine fibroblasts produced a panel of primary neor transferents, each containing a portion of, or all of, the murine MHC. A cosmid library was made from one such transferent, CHMD(D)B1. Cosmid clones were divided, using species-specific repeat probes, into those containing murine (donor) DNA sequences and those containing sequences derived from the recipient cell. The murine-specific cosmids were clustered into overlapping DNA segments by restriction enzyme digest analysis of the cosmid DNAs coupled with Southern blot analysis with, as probes, murine-specific repeat sequences and nick-translated murine genomic DNA. These cosmid clusters were analyzed for their position within or outside of the MHC, using recombinant mouse strains, and for the presence within them of known murine MHC genes. PMID- 3023849 TI - Regulation by copper of the expression of plastocyanin and cytochrome c552 in Chlamydomonas reinhardi. AB - Plastocyanin and cytochrome c552 are interchangeable electron carriers in the photosynthetic electron transfer chains of some cyanobacteria and green algae (P. M. Wood, Eur. J. Biochem. 87:9-19, 1978; G. Sandmann et al., Arch. Microbiol. 134:23-27, 1983). Chlamydomonas reinhardi cells respond to the availability of copper in the medium and accordingly accumulate either plastocyanin (if copper is available) or cytochrome c552 (if copper is not available). The response occurs in both heterotrophically and phototrophically grown cells. We have studied the molecular level at which this response occurs. No immunoreactive polypeptide is detectable under conditions where the mature protein is not spectroscopically detectable. Both plastocyanin and cytochrome c552 appear to be translated (in vitro) from polyadenylated mRNA as precursors of higher molecular weight. RNA was isolated from cells grown either under conditions favorable for the accumulation of plastocyanin (medium with Cu2+) or for the accumulation of cytochrome c552 (without Cu2+ added to the medium). Translatable mRNA for preapoplastocyanin was detected in both RNA preparations, although mature plastocyanin was detected in C. reinhardi cells only when copper was added to the culture. Translatable mRNA for preapocytochrome, on the other hand, was detected only in cells grown under conditions where cytochrome c552 accumulates (i.e., in the absence of copper). We conclude that copper-mediated regulation of plastocyanin and cytochrome c552 accumulation is effected at different levels, the former at the level of stable protein and the latter at the level of stable mRNA. PMID- 3023848 TI - Transcriptional regulation of the human cytomegalovirus major immediate-early gene is associated with induction of DNase I-hypersensitive sites. AB - Human teratocarcinoma cells were used to examine structural features associated with expression of the major immediate-early (IE) gene of human cytomegalovirus. By immunofluorescence, comparison of RNA levels, and in vitro transcription of nuclei, we showed that the major IE gene is inactive in undifferentiated but active in differentiated cells. Therefore, the block in human cytomegalovirus replication in teratocarcinoma cells appears to be at the transcriptional level, in one of the initial genes transcribed. In addition, the in vitro transcription experiments demonstrated that in permissive infections the gene was transcriptionally inactive late in infection. A comparison of the structural features of the promoter region with the active and inactive IE genes showed the presence of constitutive and inducible DNase I-hypersensitive sites. The majority of the constitutive sites existed at -175, -275, -375, -425, and -525 relative to the cap site in an area which has been shown to be capable of simian virus 40 enhancer function. In contrast, the inducible DNase I sites were located outside this region at -650, -775, -875, and -975. PMID- 3023850 TI - Extramitochondrial citrate synthase activity in bakers' yeast. AB - We isolated the gene for citrate synthase (citrate oxaloacetate lyase; EC 4.1.3.7) from Saccharomyces cerevisiae and ablated it by inserting the yeast LEU2 gene within its reading frame. This revealed a second, nonmitochondrial citrate synthase. Like the mitochondrial enzyme, this enzyme was sensitive to glucose repression. It did not react with antibodies against mitochondrial citrate synthase. Haploid cells lacking a gene for mitochondrial citrate synthase grew somewhat slower than wild-type yeast cells, but exhibited no auxotrophic growth requirements. PMID- 3023852 TI - Characterization and chromosome assignment of the human homolog of int-2, a potential proto-oncogene. AB - int-2 is one of two cellular genes (int-1 and int-2) currently implicated in the genesis of mammary carcinomas by mouse mammary tumor virus and may constitute a novel cellular proto-oncogene. Using low-stringency hybridization with mouse int 2 probes, we established that homologous genes exist in a variety of mammalian species, including humans, but failed to detect related sequences in other classes and phyla. Recombinant bacteriophage clones and a single cosmid encompassing the human int-2 gene were isolated and characterized by restriction enzyme mapping. A survey of nine primary human breast tumors, three breast tumor cell lines, and three normal individuals revealed no evidence for gross amplification or rearrangement of the int-2 locus. Three distinct restriction fragment length polymorphisms were observed which could prove useful in future linkage studies. By a combination of in situ hybridization of metaphase chromosomes and somatic cell genetics, the human int-2 gene was mapped to chromosome 11, band q13. PMID- 3023851 TI - A position effect on the expression of a tRNA gene mediated by the SIR genes in Saccharomyces cerevisiae. AB - The SIR genes of Saccharomyces cerevisiae are responsible for the position dependent regulation of the a and alpha mating-type genes. Previous work by others has shown that the products of the SIR genes prevent the accumulation of stable transcripts of the a and alpha genes at HML and HMR. Results of this study establish that this regulation is a region-specific effect rather than a gene specific effect since expression of a tRNA gene placed at HMR is repressed by the products of the SIR genes. PMID- 3023853 TI - Structure, expression, and chromosomal location of the human c-fgr gene. AB - The nucleotide sequence of seven exons of the human c-fgr gene, a cellular homolog of the oncogene of Gardner-Rasheed feline sarcoma virus, was determined. Twenty-six independent genomic clones were obtained from a human gene library with a DNA clone of Y73 avian sarcoma virus oncogene, v-yes, as a probe under relaxed hybridization conditions. Restriction mapping and partial sequence analyses revealed that two of these clones were derived from the c-fgr gene, distinct from the c-yes gene. Interestingly, the splicing points of the c-fgr gene were identical with those of the c-src gene throughout the seven exons, suggesting that the two proto-oncogenes were generated by gene duplication of an ancestral gene containing intervening sequences. On RNA blot hybridization the major transcript was found to be 2.6 kilobase long. Two additional transcripts of 3.5 and 4.7 kilobases were also detected. Furthermore, karyotype analysis of several human-mouse hybrid cells and Southern blot analyses of DNAs of the hybrids with a human c-fgr locus-specific probe showed that this gene is located on chromosome 1. PMID- 3023854 TI - The additional guanylate at the 5' terminus of Escherichia coli tRNAHis is the result of unusual processing by RNase P. AB - In eucaryotes the 5'-terminal guanylate moiety of mature tRNAHis is added posttranscriptionally. To determine whether the same mechanism occurs in procaryotes, we processed in vitro-derived Escherichia coli tRNAHis precursors to mature tRNA, either in E. coli extracts or by using pure M1-RNA, the catalytic component of RNase P. The results show that the extra guanylate at the 5' end of mature E. coli tRNAHis is encoded in the gene and is found in tRNA as the result of an unusual cleavage by RNase P. PMID- 3023855 TI - Upstream sequences required for efficient expression of a soybean heat shock gene. AB - A soybean gene (Gmhsp17.5-E) encoding a small heat shock protein was introduced into primary sunflower tumors via T-DNA-mediated transformation. RNA blot hybridizations and S1-nuclease hybrid protection studies indicated that the heat shock gene containing 3.25 kilobases of 5'-flanking sequences was strongly transcribed in a thermoinducible (40 degrees C) manner. Transcriptional induction also occurred to a lesser extent upon treatment of whole tumors with sodium arsenite and CdCl2. Basal (26 degrees C) transcription was not detected in soybean seedlings, but it was quite evident in transformed tumor tissue. A 5' deletion to -1,175 base pairs with respect to the CAP site had no effect on the levels of thermoinducible transcription, but it resulted in a large increase in basal transcription. Further removal of DNA sequences (including the TATA-distal heat shock consensus element) to -95 base pairs reduced thermoinducible transcription by 95% and also greatly decreased basal transcription. The termini of the Gmhsp17.5-E RNA in the tumor were generally the same as those present in soybean RNA, with the exception of several additional 3' termini. PMID- 3023856 TI - Structure and expression of ferritin genes in a human promyelocytic cell line that differentiates in vitro. AB - HL-60 is a human promyelocytic cell line with the capability of differentiating in vitro to give neutrophils, macrophages, or eosinophils. We screened libraries of HL-60 cDNA clones representing different time points during these differentiation processes to isolate clones corresponding to mRNAs whose expression is regulated during terminal differentiation. Upon sequencing this group of regulated clones, one clone encoding the heavy subunit and two clones encoding the light subunit of human ferritin were identified by reference to published amino acid sequences. Southern blot analyses showed that these clones are encoded by distinct multigene families. These clones identify two mRNAs whose ratios vary in a complex manner during both neutrophil and macrophage differentiation. PMID- 3023858 TI - Cellular response to DNA damage is enhanced by the pR plasmid in mouse cells and in Escherichia coli. AB - The pR plasmid, which enhances the survival of Escherichia coli C600 exposed to UV light by induction of the SOS regulatory mechanism, showed the same effect when it transformed mouse LTA cells (tk-, aprt-). With Tn5 insertion mutagenesis which inactivates UV functions in the pR plasmid, we recognized two different regions of the plasmid, uvp1 and uvp2. These pR UVR- mutants exhibited the same effect in LTA transformed cells, demonstrating that resistance to UV light, carried by the pR plasmid, was really due to the expression of these two regions, which were also in the mouse cells. Statistical analysis showed that the expression of the uvp1 and uvp2 regions significantly increased (P less than 0.01) the survival upon exposure to UV light in mouse cells and bacteria. These results might suggest the presence of an inducible repair response to DNA damage in mouse LTA cells. PMID- 3023857 TI - A distinct glucocorticoid hormone response regulates phosphoprotein maturation in rat hepatoma cells. AB - Glucocorticoid hormone-dependent maturation of the mouse mammary tumor virus (MMTV) phosphorylated polyprotein (Pr74) allows experimental access to certain posttranslational regulatory circuits under steroid control in M1.54 cells, an MMTV-infected rat hepatoma cell line. Pulse-chase experiments revealed that [35S]methionine-labeled Pr74 synthesized in uninduced cells could be converted posttranslationally into p24, a stable phosphorylated maturation product, only after 4 h of exposure to 1 microM dexamethasone, a synthetic glucocorticoid. This regulated processing could be prevented by prior exposure, during the chase period, to inhibitors of RNA (actinomycin D) or protein (cycloheximide or puromycin) synthesis. Moreover, half-maximal production of p24 occurred at 10 nM dexamethasone, a concentration that approximated half-maximal receptor binding and stimulation of MMTV transcript synthesis. Kinetic, hormonal, and genetic evidence suggest that p24 expression did not require or result from the overall glucocorticoid-dependent increase in polyprotein concentration. First, 20 h after dexamethasone withdrawal, Pr74 maturation was completely deinduced, whereas the absolute level of this MMTV precursor remained 10-fold over its basal level. Second, progesterone, which competes with dexamethasone for receptor binding, facilitated the regulated production of p24 but prevented the steroid-mediated accumulation of functional MMTV mRNA. Lastly, certain glucocorticoid-responsive variants, derived from M1.54 cells by resistance to complement cytolysis, expressed p24 in the presence or absence of glucocorticoid-induced levels of Pr74. Taken together, our results suggest that the glucocorticoid-regulated maturation of MMTV phosphopolyproteins resulted from an independent hormone response that required normal receptor function and de novo RNA and protein synthesis. PMID- 3023859 TI - The chronic myelocytic cell line K562 contains a breakpoint in bcr and produces a chimeric bcr/c-abl transcript. AB - In the DNAs of all Ph1-positive chronic myelocytic leukemia patients studied to date, a breakpoint on chromosome 22 (the Ph1 chromosome) can be demonstrated with a probe from the bcr (breakpoint cluster region). Although the K562 cell line was established from cells of a chronic myelocytic leukemia patient, we have been unable to detect the Ph1 chromosome by cytogenetic means. Employing a probe from the 5' region of bcr, we have cloned an amplified Ph1 breakpoint fragment from K562. This demonstrates that K562 contains multiple remnants of a Ph1 chromosome with a breakpoint within bcr and thus may serve as a model system for the study of Ph1-positive chronic myelocytic leukemia at a molecular level. The isolation of bcr cDNA sequences shows that parts of bcr encode a protein. Employing K562, we demonstrate the presence of an abnormally sized mRNA species hybridizing to c abl and to a bcr cDNA probe, indicating the possible consequence of the Ph1 translocation on a transcriptional level in chronic myelocytic leukemia. The isolation and sequencing of a cDNA containing the breakpoint area of this mRNA provide further evidence for its chimeric structure. Cloning of large stretches of chromosomal DNA flanking bcr and c-abl sequences in K562 and identification of the exons participating in the formation of the chimeric mRNA shows that a splice of at least 99 kilobases is made to fuse the 3' bcr exon to the 5' c-abl exon. Furthermore two chimeric cDNAs were isolated containing chromosome 9 sequences that map 43.5 kilobases downstream from the K562 breakpoint. These chromosome 9 sequences neither hybridize to the 8.5-kilobase chimeric c-abl mRNA nor to normal c-abl mRNAs in Hela cells and probably represent incorrect splicing products present in the K562 cell line. PMID- 3023860 TI - Sequences required for delivery and localization of the ADP/ATP translocator to the mitochondrial inner membrane. AB - The ADP/ATP translocator, a transmembrane protein of the mitochondrial inner membrane, is coded in Saccharomyces cerevisiae by the nuclear gene PET9. DNA sequence analysis of the PET9 gene showed that it encoded a protein of 309 amino acids which exhibited a high degree of homology with mitochondrial translocator proteins from other sources. This mitochondrial precursor, in contrast to many others, does not contain a transient presequence which has been shown to direct the posttranslational localization of proteins in the organelle. Gene fusions between the PET9 gene and the gene encoding beta-galactosidase (lacZ) were constructed to define the location of sequences necessary for the mitochondrial delivery of the ADP/ATP translocator protein in vivo. These studies reveal that the information to target the hybrid molecule to the mitochondria is present within the first 115 residues of the protein. In addition, these studies suggest that the "import information" of the amino-terminal region of the ADP/ATP translocator precursor is twofold. In addition to providing targeting function of the precursor to the organelle, these amino-terminal sequences act to prevent membrane-anchoring sequences located between residues 78 and 98 from stopping import at the outer mitochondrial membrane. These results are discussed in light of the function of distinct protein elements at the amino terminus of mitochondrially destined precursors in both organelle delivery and correct membrane localization. PMID- 3023861 TI - Interaction between Kb and Q4 gene sequences generates the Kbm6 mutation. AB - Genetic interaction as a mechanism for the generation of mutations is suggested by recurrent, multiple nucleotide substitutions that are identical to nucleotide sequences elsewhere in the genome. We have sequenced the mutant K gene from the bm6 mouse, which is one of a series of eight closely related, yet independently occurring mutants known collectively as the "bg series." Two changes from the Kb gene are found, positioned 15 nucleotides apart: an A-to-T change and a T-to-C change in the codons corresponding to amino acids 116 and 121, resulting in Tyr to-Phe and Cys-to-Arg substitutions, respectively. Hybridization analysis with an oligonucleotide specific for the altered Kbm6 sequence identifies one donor gene, Q4, located in the Qa region of the H-2 complex. The two altered nucleotides that differentiate Kbm6 and Kb are present in Q4 in a region where Kb and Q4 are otherwise identical for 95 nucleotides, delineating the maximum genetic transfer between the two genes. Because the Kbm6 mutation arose in an homozygous mouse these data indicate that the Q4 gene contains the only donor sequence and demonstrates that Q-region gene sequences can interact with the Kb gene to generate variant K molecules. PMID- 3023864 TI - The human hepatitis B virus enhancer requires trans-acting cellular factor(s) for activity. AB - The activity of the hepatitis B viral enhancer element was studied in various cell lines. This enhancer shows strict host and tissue specificity in that it is functional only in liver cells of human origin. Further, it requires trans-acting factor(s) present in liver cells for activity, and this activity is independent of hepatitis B virus gene products in the cell lines tested. PMID- 3023865 TI - Differential elimination of rDNA genes in bobbed mutants of Drosophila melanogaster. AB - In Drosophila melanogaster, the multiply repeated genes encoding 18S and 28S rRNA are located on the X and Y chromosomes. A large percentage of these repeats are interrupted in the 28S region by insertions of two types. We compared the restriction patterns from a subcloned wild-type Oregon R strain to those of spontaneous and ethyl methanesulfonate-induced bobbed mutants. Bobbed mutations were found to be deficiencies that modified the organization of the rDNA locus. Genes without insertions were deleted about twice as often as genes with type I insertions. Type II insertion genes were not decreased in number, except in the mutant having the most bobbed phenotype. Reversion to wild type was associated with an increase in gene copy number, affecting exclusively genes without insertions. One hypothesis which explains these results is the partial clustering of genes by type. The initial deletion could then be due either to an unequal crossover or to loss of material without exchange. Some of our findings indicated that deletion may be associated with an amplification phenomenon, the magnitude of which would be dependent on the amount of clustering of specific gene types at the locus. PMID- 3023862 TI - Tripartite upstream promoter element essential for expression of Saccharomyces cerevisiae ribosomal protein genes. AB - To initiate a genetic analysis of yeast ribosomal protein gene promoters, we have constructed a gene fusion between the yeast ribosomal protein gene RP39A and the Escherichia coli lacZ gene. This gene fusion contains approximately 1,030 nucleotides of the 5' flanking region and the first 49 1/3 codons of RP39A fused in frame to a large 3' end fragment of lacZ. Whether it is introduced into yeast cells on a moderately high-copy-number plasmid, or integrated into the yeast genome at the RP39A locus, this RP39A-lacZ gene directs the synthesis of a hybrid transcript which encodes beta-galactosidase activity. Deletions in the 5' flanking region of RP39A-lacZ were constructed by linker insertion and BAL 31 mutagenesis. The expression of the mutant genes in yeast cells was assayed by measuring RP39A-lacZ mRNA and beta-galactosidase levels. By these means we have shown that the sequences between nucleotides -256 and -170 upstream of RP39A are essential for expression of this gene. Three sequence motifs, HOMOL1, RPG, and a T-rich region, which were found in that order 5'----3' upstream of most yeast ribosomal protein genes, were present within this interval. We found that substitution of the CYC1-lacZ upstream activation site with the fragment from nucleotides -298 to -172 upstream of RP39A, containing the HOMOL1-RPG-T-rich motif in that 5'----3' orientation, fully restored expression of the CYC1-lacZ gene. The essentially of HOMOL1, the RPG sequence, and the T-rich region for wild type levels of expression of RP39A, the conserved location and order of these sequence motifs in yeast ribosomal protein genes, and the ability of a DNA fragment carrying these three sequence elements to substitute for the upstream activation site regions of CYC1 indicate that these three oligonucleotides may be essential to the transcription of yeast ribosomal protein genes. PMID- 3023863 TI - Cloning and characterization of four SIR genes of Saccharomyces cerevisiae. AB - Mating type in the yeast Saccharomyces cerevisiae is determined by the MAT (a or alpha) locus. HML and HMR, which usually contain copies of alpha and a mating type information, respectively, serve as donors in mating type interconversion and are under negative transcriptional control. Four trans-acting SIR (silent information regulator) loci are required for repression of transcription. A defect in any SIR gene results in expression of both HML and HMR. The four SIR genes were isolated from a genomic library by complementation of sir mutations in vivo. DNA blot analysis suggests that the four SIR genes share no sequence homology. RNA blots indicate that SIR2, SIR3, and SIR4 each encode one transcript and that SIR1 encodes two transcripts. Null mutations, made by replacement of the normal genomic allele with deletion-insertion mutations created in the cloned SIR genes, have a Sir- phenotype and are viable. Using the cloned genes, we showed that SIR3 at a high copy number is able to suppress mutations of SIR4. RNA blot analysis suggests that this suppression is not due to transcriptional regulation of SIR3 by SIR4; nor does any SIR4 gene transcriptionally regulate another SIR gene. Interestingly, a truncated SIR4 gene disrupts regulation of the silent mating type loci. We propose that interaction of at least the SIR3 and SIR4 gene products is involved in regulation of the silent mating type genes. PMID- 3023866 TI - Antipeptide antiserum identifies a widely distributed cellular tyrosine kinase related to but distinct from the c-fps/fes-encoded protein. AB - We raised antibodies directed against a synthetic peptide representing an amino acid sequence of the conserved kinase domain of the transforming protein of Fujinami sarcoma virus (FSV) (P140). The antiserum obtained specifically recognized FSV-P140 and its cellular homolog and in addition, it recognized a new cellular protein of 94,000 daltons (NCP94) in avian and mammalian cells. NCP94 was found to be associated with a cyclic nucleotide-independent protein kinase activity that was specific for tyrosine residues. Although NCP94 and FSV-P140 share antigenic determinants, NCP94 is not a cellular homolog of FSV-P140: NCP94 and the previously identified c-fps/fes product were different in their tryptic fingerprints and in their tissue specificities. Thus, the function of NCP94 in normal cells is probably different than that of the c-fps/fes product. NCP94 was expressed in every tissue and cell line that was examined. In chickens, NCP94 levels were highest during embryonic development and NCP94 expression was high in gizzard, brain, and spleen throughout embryonic and adult life. The universal expression of NCP94 suggests that this protein may be involved in an essential function of normal cells. NCP94 may be a new cellular tyrosine kinase of the src gene family. PMID- 3023867 TI - Transfer of cloned human class I major histocompatibility complex genes into HLA mutant human lymphoblastoid cells. AB - Three new kinds of recombinant DNA constructs were used to transfer cloned human class I HLA genes (A2 and B8) into unique HLA mutant lymphoblastoid cells: pHeBo(x): a class I gene, "x," in plasmid vector pHeBo, which contains a hygromycin resistance gene and Epstein-Barr virus oriP element that sustains extrachromosomal replication; pHPT(x): gene x in a vector with a hypoxanthine guanine phosphoribosyltransferase (HPRT) gene; pHPTe(x): gene x in a vector with the HPRT gene and oriP element. Cell surface class I antigen expression was strong in transferents made with class I-deficient lymphoblastoid cell line mutants .144 (A-null), .53 (B-null), and .184 (A-null, B-null). Transferents expressing HLA-A2 were recognized specifically by HLA-A2-specific cytotoxic T lymphocytes. When introduced on either of the vectors with the Epstein-Barr virus oriP element, the class I gene replicated extrachromosomally and was lost at rates of 0.2 to 0.3 per cell division. When introduced with vector pHPT (lacking Epstein-Barr virus oriP), the B8 gene was inserted at different chromosomal locations. Introduction of the HLA-B8 gene failed to restore antigen expression by HLA-B-null mutant .174, providing evidence that, unlike mutants exemplified by .53, .144, and .184, some HLA antigen loss mutants are deficient in a trans acting function needed for class I antigen expression. Of more general interest, the results obtained with HLA class I genes in vectors that replicate extrachromosomally suggest ways of relating genic expression to chromatin structure and function and of attempting to clone functional human centromeres. PMID- 3023868 TI - Transcription terminator-like element within a Saccharomyces cerevisiae promoter region. AB - We analyzed a cloned fragment of the yeast URA3 promoter region that contains a sequence of DNA capable of functioning as a highly efficient transcription terminator. BAL 31 deletions have shown the signal for the transcription termination activity is less than or equal to 110 base pairs and resides between bases 45 and 155 upstream of the URA3 primary ATG codon at base 227. In our in vivo assay system, the DNA fragment is able to terminate transcripts very efficiently in either orientation. The terminated transcripts bind to oligodeoxythymidylate cellulose columns and promote the synthesis of full-length cDNAs, suggesting that the transcripts are polyadenylated. The 110-base-pair region contains no sequence resembling terminator consensus sequences described by Zaret and Sherman (K.S. Zaret and F. Sherman, Cell, 28:563-573, 1982) or Henikoff and Cohen (S. Henikoff and E.H. Cohen, Mol. Cell. Biol., 4:1515-1520, 1984). We discuss the possible physiological relevance of this sequence to bona fide termination of transcription and to URA3 regulation in Saccharomyces cerevisiae. PMID- 3023869 TI - Enhanced mutagenesis of UV-irradiated simian virus 40 occurs in mitomycin C treated host cells only at a low multiplicity of infection. AB - Treatment of monkey kidney cells with mitomycin C (MMC) 24 h prior to infection with UV-irradiated simian virus 40 (SV40) enhanced both virus survival and virus mutagenesis. The use of SV40 as a biological probe has been taken as an easy method to analyse SOS response of mammalian cells to the stress caused by DNA damage or inhibition of DNA replication. The mutation assay we used was based on the reversion from a temperature-sensitive phenotype (tsA58 mutant) to a wild type phenotype. The optimal conditions for producing enhanced survival and mutagenesis in the virus progeny were determined with regard to the multiplicity of infection (MOI). Results showed that the level of enhanced mutagenesis observed for UV-irradiated virus grown in MMC-treated cells was an inverse function of the MOI, while enhanced survival was observed at nearly the same level regardless of the MOI. For the unirradiated virus, almost no increase in the mutation of virus progeny issued from MMC-treated cells was observed, while a small amount of enhanced virus survival was obtained. These results show that enhanced virus mutagenesis and enhanced virus survival can be dissociated under some experimental conditions. Enhanced virus mutagenesis, analogous to the error prone replication of phages in SOS-induced bacteria, was observed, at least for SV40, only when DNA of both virus and host cells was damaged and when infection occurred with a small number of viral particles. We therefore hypothesize that an error-prone replication mode of UV-damaged templates is observed in induced monkey kidney cells. PMID- 3023871 TI - The Drosophila melanogaster gypsy transposable element encodes putative gene products homologous to retroviral proteins. AB - We determined the complete nucleotide sequence of the gypsy element present at the forked locus of Drosophila melanogaster in the f1 allele. The gypsy element shares more homology with vertebrate retroviruses than with the copia element of D. melanogaster or the Ty element of Saccharomyces cerevisiae, both in overall organization and at the DNA sequence level. This transposable element is 7,469 base pairs long and encodes three putative protein products. The long terminal repeats are 482 nucleotides long and contain transcription initiation and termination signals; sequences homologous to the polypurine tract and tRNA primer binding site of retroviruses are located adjacent to the long terminal repeats. The central region of the element contains three different open reading frames. The second one encodes a putative protein which shows extensive amino acid homology to retroviral proteins, including gag-specific protease, reverse transcriptase, and DNA endonuclease. PMID- 3023872 TI - Primary DNA sequence determines sites of maintenance and de novo methylation by mammalian DNA methyltransferases. AB - Analysis of the enzymatic methylation of oligodeoxynucleotides containing multiple C-G groups showed that hemimethylated sites in duplex oligomers are not significantly methylated by human or murine DNA methyltransferase unless those sites are capable of being methylated de novo in the single- or double-stranded oligomers. Thus, the primary sequence of the target strand, rather than the methylation pattern of the complementary strand, determines maintenance methylation. This suggests that de novo and maintenance methylation are the same process catalyzed by the same enzyme. In addition, the study revealed that complementary strands of oligodeoxynucleotides are methylated at different rates and in different patterns. Both primary DNA sequence and the spacing between C-G groups seem important since in one case studied, maximal methylation required a specific spacing of 13 to 17 nucleotides between C-G pairs. PMID- 3023870 TI - Functional organization of the simian virus 40 origin of DNA replication. AB - To define the sequence elements involved in initiation of DNA synthesis at the simian virus 40 origin of replication, we determined the relative replication efficiencies in vitro and in vivo of templates containing a variety of mutations within the origin region. Replication of the mutants in vitro was assayed by the cell-free DNA replication system that we recently described (J.J. Li and T.J. Kelly, Proc. Natl. Acad. Sci. USA 81:6973-6977, 1984; J.J. Li and T.J. Kelly, Mol. Cell. Biol. 5:1238-1246, 1985), and replication in vivo was assayed after transfection of the mutant templates into COS-1 cells. The minimal origin of replication defined by both assays included a 15-base-pair (bp) imperfect inverted repeat, a 27-bp perfect inverted repeat, and a 17-bp A/T-rich region. T antigen binding site I was not required for DNA replication, but its presence increased replication efficiency severalfold both in vitro and in vivo. Although SP1 binding sites and enhancers had little or no effect on replication in vitro, the presence of either element markedly increased replication in vivo. Thus, the biological role of these elements is not restricted to stimulating transcription but may be more general. PMID- 3023875 TI - The t-unique coding domain is important to the transformation maintenance function of the simian virus 40 small t antigen. AB - The small t antigen (t) of simian virus 40, a 174-amino-acid-containing protein, when present together with the other early viral protein, large T antigen (T), plays an important role in the maintenance of simian virus 40-induced neoplastic phenotype in certain cells. Indeed, each protein functions in a complementary manner in this process. The t coding unit is composed of two segments, a 5' region of 246 nucleotides which is identical to that of the corresponding 5' region of the T coding unit and a 3' segment of 276 nucleotides which is unique. Two mutant, t-encoding genomes, one bearing a missense and the other a nonsense mutation at the same point in the t-unique coding region were constructed in vitro and found to be defective in their ability to dissolve the actin cytoskeleton of rat fibroblasts and to complement T in the growth of mouse fibroblasts in soft agar. Therefore, the unique segment of the t gene encodes a portion of the t molecule which is essential to its transformation maintenance function. PMID- 3023874 TI - Stable gene amplification and overexpression of sodium- and potassium-activated ATPase in HeLa cells. AB - Cell lines stably resistant to ouabain were isolated from an unstably resistant HeLa line after growth in nonselective medium. Stable resistant lines bound ouabain at levels 10-fold higher than did HeLa cells and at similar levels to those bound by the unstable C+ line previously described (J. F. Ash, R. M. Fineman, T. Kalka, M. Morgan, and B. Wire, J. Cell Biol. 99: 971-983). Expression and synthesis of the Na+, K+ -ATPase alpha chain showed a similar amplification over that for HeLa cells by Western blots and [35S]methionine pulse-labeling. In addition, a glycoprotein labeled with [3H]fucose and comigrating with the Na+, K+ -ATPase beta chain was eight- to ninefold amplified in stably resistant lines. Dot blots with a cDNA clone specific for Na+, K+ -ATPase alpha chain gene sequences confirmed the amplification of this gene. Karyotyping suggested that the amplification is associated with an expanded, abnormal banded region on the long (q) arm of one chromosome 17. PMID- 3023873 TI - Variable stability of a selectable provirus after retroviral vector gene transfer into human cells. AB - Human lymphoblasts deficient in the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) were infected with an amphotropic helper-free retroviral vector expressing human HPRT cDNA. The stability and expression of the HPRT provirus in five cell lines with different proviral integration sites were examined by determining HPRT mutation and reversion frequencies and by blot hybridization studies. Mutation to the HPRT-negative phenotype occurred at frequencies of approximately 4 X 10(-5) to 3 X 10(-6) per generation. Most mutations in each of the five cell lines were associated with partial or complete deletions or rearrangements of the provirus. Several mutants retained a grossly intact HPRT provirus, and in one such mutant HPRT shutdown resulted from a revertible epigenetic mechanism that was not associated with global changes in proviral methylation. Therefore, mutation and shutdown of the HPRT provirus in human lymphoblasts result from mechanisms similar to those reported for several other avian and mammalian replication-competent retroviruses. PMID- 3023876 TI - Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens. AB - We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa. PMID- 3023877 TI - Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis. AB - The RAD3 gene of Saccharomyces cerevisiae, which is involved in excision repair of DNA and is essential for cell viability, was mutagenized by site-specific and random mutagenesis. Site-specific mutagenesis was targeted to two regions near the 5' and 3' ends of the coding region, selected on the basis of amino acid sequence homology with known nucleotide binding and with known specific DNA binding proteins, respectively. Two mutations in the putative nucleotide-binding region and one in the putative DNA-binding region inactivate the excision repair function of the gene, but not the essential function. A gene encoding two tandem mutations in the putative DNA-binding region is defective in both excision repair and essential functions of RAD3. Seven plasmids were isolated following random mutagenesis with hydroxylamine. Mutations in six of these plasmids were identified by gap repair of mutant plasmids from the chromosome of strains with previously mapped rad3 mutations, followed by DNA sequencing. Three of these contain missense mutations which inactivate only the excision repair function. The other three carry nonsense mutations which inactivate both the excision repair and essential functions. Collectively our results indicate that the RAD3 excision repair function is more sensitive to inactivation than is the essential function. Overexpression of wild-type Rad3 protein and a number of rad3 mutant proteins did not affect the UV resistance of wild-type yeast cells. However, overexpression of Rad3-2 protein rendered wild-type cells partially UV sensitive, indicating that excess Rad3-2 protein is dominant to the wild-type form. These and other results suggest that Rad3-2 protein retains its affinity for damaged DNA or other substrates, but is not catalytically active in excision repair. PMID- 3023878 TI - A calcium ionophore-inducible cellular promoter is highly active and has enhancerlike properties. AB - We examined the regulatory/promoter sequence of a calcium ionophore-inducible gene isolated from the rat genome. Whereas the promoter of this ubiquitously expressed gene is active under noninduced conditions, after induction by calcium ionophore A23187 this promoter is 10- to 25-fold more active than the simian virus 40 early promoter, as measured by chloramphenicol acetyltransferase activities. Within this regulatory/promoter region, we have identified a DNA fragment with enhancer-like properties immediately 5' to the TATA sequence. This 291-nucleotide fragment acts in cis to enhance expression of the neomycin phosphotransferase (neo) gene driven by the herpes simplex virus thymidine kinase promoter in an orientation-independent manner. In addition, this fragment can confer A23187 inducibility to the neo gene and effectively compete for positive regulatory factors involved in A23187 induction. Sequence analysis of this promoter reveals homology with viral core enhancer sequences, and the apparent organization of direct repeat domains is similar to those observed in viral enhancers. PMID- 3023879 TI - Diverse long terminal repeats are associated with murine retroviruslike (VL30) elements. AB - The VL30 family is a retroviruslike gene family with no apparent nucleic acid homology to any known retrovirus. Over 100 copies of VL30 DNA elements are dispersed throughout the mouse genome. Sequence analysis of the VL30 long terminal repeat (LTR) units showed that, whereas the LTR units of a given VL30 DNA element were almost identical, the LTR units associated with distinct members of the family were very different from one another. Comparison of the LTR sequences possessed by two particular VL30 DNA elements revealed a pattern of extensively homologous DNA segments adjacent to only distantly related DNA sequences. With the aid of sub-LTR probes, it was shown that a certain LTR is composed of both U5 sequences that are abundantly present in all species of the genus Mus and a U3 region detected only in Mus musculus. In addition, we isolated a VL30 DNA element in which the LTR units were replaced by the LTR units of an apparently novel retroviruslike family. These findings suggest that recombinations have played a role in generating the diverse population of VL30 associated LTRs. PMID- 3023880 TI - trans Activation of the simian virus 40 enhancer. AB - We describe experiments which demonstrated that the simian virus 40 (SV40) enhancer affects certain transcriptional units differently. We also found that a specific enhancer-transcriptional unit interaction can be regulated by trans acting factors. Using transient assays, we examined the effects of the SV40 enhancer on herpesvirus thymidine kinase (tk) RNA levels when transcription was initiated either by the herpesvirus tk promoter or by an SV40 early promoter-tk fusion. We were unable to detect any effect of the enhancer on transcription from the tk promoter in CV-1 or HeLa cells. However, we found that the addition of T antigen in trans allowed the enhancer to stimulate expression from the tk promoter. This induction by T-antigen did not require T-antigen-binding sites in cis and appeared to be an indirect effect. In contrast, tk expression from the SV40 early promoter fusion was greatly stimulated by the enhancer in CV-1 cells. Furthermore, in 293 cells the SV40 enhancer had only a marginal effect on the SV40 promoter-tk fusion, whereas it strongly stimulated tk expression from the tk promoter. Our results raise the possibility that the enhancer function may not show cell specificity per se; rather, the interaction between the enhancer and a specific gene may be responsible for cell specificity. We discuss these observations in terms of the SV40 early gene-to-late gene switch that occurs during SV40 lytic growth. PMID- 3023882 TI - Orientation of the v-erb-B gene product in the plasma membrane. AB - The retroviral oncogene v-erb-B encodes a truncated version of the receptor for epidermal growth factor. To define the disposition of the v-erb-B protein within cells and across the plasma membrane, we raised antibodies against defined epitopes in the protein and used these in immunofluorescence to analyze cells transformed by v-erb-B. A small fraction of the v-erb-B protein was found on the plasma membrane in a clustered configuration. The bulk of the protein was located in the endoplasmic reticulum and Golgi apparatus. Epitopes near the amino terminus of the v-erb-B protein were displayed on the surface of the cell, whereas epitopes in the protein kinase domain were located exclusively within cells. We conclude that the v-erb-B protein spans the plasma membrane in a manner similar or identical to that of the epidermal growth factor receptor, even though the viral transforming protein does not possess the signal peptide that is thought to direct insertion of the receptor into the membrane. PMID- 3023881 TI - Rescue of chromosomal T-antigen sequences onto extrachromosomally replicating, defective simian virus 40 DNA by homologous recombination. AB - Recombination between chromosomal and extrachromosomal DNA sequences was analyzed by investigation of the recombinational rescue of a 1,018-base-pair (bp) segment of the T-antigen gene of simian virus 40 from the chromosome of monkey COS cells to two different, extrachromosomally replicating, simian virus 40 DNA molecules lacking this 1,018-bp sequence. The ratio of rescued to unrecombined virus was as high as 10(-3). The rescued molecules, detected optimally 5 to 9 days after transfection of COS cells, had completely recovered the 1,018-bp DNA segment from the chromosome. The recombination event is proposed to occur either by double reciprocal recombination or by gene conversion between the chromosomal T-antigen gene and the extrachromosomal molecules missing the 1,018-bp sequence. PMID- 3023884 TI - Structure of the c-Ki-ras gene in a rat fibrosarcoma induced by 1,8 dinitropyrene. AB - Restriction enzyme maps were made of the region around exons 1 and 2 of activated c-Ki-ras of a fibrosarcoma (1,8-DNP2) induced in a rat by 1,8-dinitropyrene. Nucleotide sequence analysis revealed that activated c-Ki-ras shows a G----T transversion in codon 12 and consequently encodes cysteine instead of glycine in normal rat c-Ki-ras. PMID- 3023883 TI - An anomalous Ty1 structure attributed to an error in reverse transcription. AB - We have determined the nucleotide sequence of both delta elements of a Ty1 transposon inserted near the CYC7 gene in the Saccharomyces cerevisiae CYC7-H2 mutant. The upstream delta element in this Ty1 has an unusual inverted repeat structure that may have been formed by an error during reverse transcription. PMID- 3023885 TI - An active chromatin structure acquired by translocated c-myc genes. AB - We used general sensitivity to DNase I digestion to analyze the chromatin structure of c-myc genes in seven murine plasmacytomas. In every case, the 3' portion of c-myc juxtaposed with C alpha displayed a much more DNase I-sensitive chromatin structure than untranslocated c-myc or, in one case analyzed, the reciprocally translocated 5' portion. Our data suggest the presence of regulatory sequences near the C alpha gene segment. PMID- 3023886 TI - In vivo localization of DNA topoisomerase II cleavage sites on Drosophila heat shock chromatin. AB - Similar to its inhibitory effect on mammalian DNA topoisomerase II, the cytotoxic drug VM26 (teniposide) also interferes with the breakage-reunion reaction of Drosophila melanogaster DNA topoisomerase II. VM26 induces topoisomerase II mediated DNA breakage in vitro and in cultured D. melanogaster cells presumably by stabilizing an enzyme-DNA cleavable complex. The drug-induced DNA breaks on D. melanogaster hsp70 genes were mapped in cultured cells using the indirect end labeling procedure. Multiple and specific cleavage sites occurred at both the 3' and 5' ends of the hsp70 genes. A number of these cellular topoisomerase II cleavage sites mapped close to the DNase I-hypersensitive regions of the hsp70 genes. The intensities of several topoisomerase II cleavage sites changed significantly on heat shock induction. Treatment of cultured D. melanogaster cells with VM26 at 25 degrees C resulted in the stimulation of transcription of the hsp70 genes. These results suggest that inhibition of DNA topoisomerase II may lead to heat shock transcription. PMID- 3023887 TI - Two different factors act separately or together to specify functionally distinct activities at a single transcriptional enhancer. AB - The expression of genes fused downstream of the Moloney murine sarcoma virus (MoMSV) long terminal repeat is stimulated by glucocorticoids. We mapped the glucocorticoid response element that conferred this hormonal regulation and found that it is a hormone-dependent transcriptional enhancer, designated Sg; it resides within DNA fragments that also carry a previously described enhancer element (B. Levinson, G. Khoury, G. Vande Woude, and P. Gruss, Nature [London] 295:568-572, 1982), here termed Sa, whose activity is independent of the hormone. Nuclease footprinting revealed that purified glucocorticoid receptor bound at multiple discrete sites within and at the borders of the tandemly repeated sequence motif that defines Sa. The Sa and Sg activities stimulated the apparent efficiency of cognate or heterologous promoter utilization, individually providing modest enhancement and in concert yielding higher levels of activity. A deletion mutant lacking most of the tandem repeat but retaining a single receptor footprint sequence lost Sa activity but still conferred Sg activity. The two enhancer components could also be distinguished physiologically: both were operative within cultured rat fibroblasts, but only Sg activity was detectable in rat exocrine pancreas cells. Therefore, the sequence determinants of Sa and Sg activity may be interdigitated, and when both components are active, the receptor and a putative Sa factor can apparently bind and act simultaneously. We concluded that MoMSV enhancer activity is effected by at least two distinct binding factors, suggesting that combinatorial regulation of promoter function can be mediated even from a single genetic element. PMID- 3023888 TI - Characterization of G1 transit induced by the mitogenic-oncogenic viral Ki-ras gene product. AB - NRK rat kidney cells infected with a temperature-sensitive mutant of the Kirsten sarcoma virus (ts371) were transformed at 36 degrees C but were phenotypically nontransformed at 41 degrees C because of the abnormal thermolability of the oncogenic 21-kilodalton product of the viral Ki-ras gene. Thus tsK-NRK cells were rendered quiescent in a G0-G1 state by a 48-h incubation in serum-free medium at the nonpermissive, p21-inactivating temperature of 41 degrees C. The serum starved cells could then be stimulated to transit G1 either as nontransformed cells by adding serum at 41 degrees C or as transformed cells by lowering the temperature to a p21-activating 36 degrees C. The viral p21 protein was as effective as serum in stimulating tsK-NRK cells to transit G1 and to start replicating DNA. While p21 effectively stimulated cells to transit G1 even in unconditioned, serum-free medium, they still needed cell-derived conditioning factors to subsequently divide. The p21 protein also enabled the cells to transit G1 in spite of an extracellular Ca2+ deficiency that inhibited the G1 transit of serum-stimulated cells. p21 activity was needed to stimulate both early and late G1 events. In contrast to serum, p21 did not stimulate total RNA or protein synthesis, but some RNA and protein synthesis must have been needed for the p21 driven G1 transit because it could be stopped by actinomycin D or cycloheximide. PMID- 3023889 TI - Interaction of distinct nuclear proteins with sequences controlling the expression of polyomavirus early genes. AB - The interaction between cellular factors and polyoma virus (Py) DNA was investigated by using a gel retention assay. Nuclear extracts from various cell lines (NIH 3T3, NIH 3T6, LTK-, F9) contained proteins that formed specific and distinct complexes with Py B enhancer fragments of either wild-type or F9-1 mutant origin. The presence of an excess amount of other well-characterized DNA sequences, including the Py A enhancer, the murine sarcoma virus enhancer, and the simian virus 40 enhancer-promoter region, did not interfere with this protein DNA interaction. However, a fragment previously defined as containing the lymphotropic papovavirus enhancer shares the binding of some common factor. This observation, in combination with the results of retention gel assays at different Mg2+ concentrations, indicates the interaction of several nuclear factors and Py DNA. The assay systems that were used allowed a distinction between some factors on the basis of their different biochemical and sequence requirements. The contact sites of these complexes were mapped to the B enhancer region of Py with Bal 31-derived mutant restriction fragments and ExoIII nuclease and are compatible with the functional domains determined in vivo. PMID- 3023890 TI - Restriction endonuclease activity induced by PBCV-1 virus infection of a Chlorella-like green alga. AB - An enzyme was isolated from a eucaryotic, Chlorella-like green alga infected with the virus PBCV-1 which exhibits type II restriction endonuclease activity. The enzyme recognized the sequence GATC and cleaved DNA 5' to the G. Methylation of deoxyadenosine in the GATC sequence inhibited enzyme activity. In vitro the enzyme cleaved host Chlorella nuclear DNA but not viral DNA because host DNA contains GATC and PBCV-1 DNA contains GmATC sequences. PBCV-1 DNA is probably methylated in vivo by the PBCV-1-induced methyltransferase described elsewhere (Y. Xia and J. L. Van Etten, Mol. Cell. Biol. 6:1440-1445). Restriction endonuclease activity was first detected 30 to 60 min after viral infection; the appearance of enzyme activity required de novo protein synthesis, and the enzyme is probably virus encoded. Appearance of enzyme activity coincided with the onset of host DNA degradation after PBCV-1 infection. We propose that the PBCV-1 induced restriction endonuclease participates in host DNA degradation and is part of a virus-induced restriction and modification system in PBCV-1-infected Chlorella cells. PMID- 3023891 TI - Hepatitis B surface antigen: an unusual secreted protein initially synthesized as a transmembrane polypeptide. AB - Hepatitis B surface antigen (HBsAg), the major coat protein of hepatitis B virus, is also secreted from cells as a subviral particle, without concomitant cleavage of N-terminal amino acid sequences. We examined this unusual export process in a cell-free system and showed that the initial product of HBsAg biosynthesis is an integral transmembrane protein, with most or all of its C-terminal half on the lumenal side of the endoplasmic reticulum membrane. To study the nature of its topogenic signals, we synthesized fusion proteins between HBsAg and the nonsecreted protein alpha-globin. Fusion proteins in which approximately 100 amino acids of globin preceded all HBsAg sequences were successfully translocated in vitro; the same domain as in the wild-type HBsAg was transported into the vesicle lumen. Fusions in which the entire globin domain was C terminal were able to translocate both the C-terminal region of HBsAg and its attached globin domain. Thus, uncleaved signal sequences in p24s function to direct portions of the molecule across the membrane and are able to perform this function even when positioned in an internal protein domain. PMID- 3023892 TI - Proto-oncogene c-ros codes for a molecule with structural features common to those of growth factor receptors and displays tissue specific and developmentally regulated expression. AB - A recombinant DNA clone containing cellular sequences homologous to the transforming sequence, v-ros, of avian sarcoma virus UR2 was isolated from a chicken genomic DNA library. Heteroduplex mapping and nucleotide sequencing reveal that the v-ros sequences are distributed in nine exons ranging from 65 to 204 nucleotides on cellular ros (c-ros) DNA over a range of 11 kilobases. Comparison of the deduced amino acid sequences of c-ros and v-ros shows two differences: v-ros contains a three-amino-acid insertion within the hydrophobic domain presumed to be involved in membrane association, and (ii) the carboxyl 12 amino acids of v-ros are completely different from those of the deduced c-ros sequence. The deduced amino acid sequence of c-ros bears striking structural features similar to those of insulin and epidermal growth factor receptors, including the presumed hydrophobic membrane binding domain, amino acids flanking the domain, and the distance between the domain and the catalytic region of the kinase activity. The expression of c-ros appears to be under a very stringent control. When tissues at various stages of chicken development were analyzed, only kidney was found to contain a significant level of c-ros RNA. The level of c ros RNA in kidney tissue is most abundant in 7- to 14-day-old chickens. Finally, nucleotide sequences of c-ros DNA and UR2-associated helper viral genome at regions corresponding to the gag ros recombination site suggest that the junction has been formed by RNA splicing. PMID- 3023893 TI - RAD7 gene of Saccharomyces cerevisiae: transcripts, nucleotide sequence analysis, and functional relationship between the RAD7 and RAD23 gene products. AB - The RAD7 gene of Saccharomyces cerevisiae was cloned on a 4.0-kilobase (kb) DNA fragment and shown to provide full complementation of a rad7-delta mutant strain. The nucleotide sequence of a 2.2-kb DNA fragment which contains the complete RAD7 gene was determined. Transcription of the RAD7 gene initiates at multiple sites in a region spanning positions -61 to -8 of the DNA sequence. The 1.8-kb RAD7 mRNA encodes a protein of 565 amino acids with a predicted size of 63.7 kilodaltons. The hydropathy profile of the RAD7 protein indicates a highly hydrophilic amino terminus and a very hydrophobic region toward the carboxyl terminus. A RAD7 subclone deleted for the first 99 codons complements the rad7 delta mutation, but not the rad7-delta rad23-delta double mutation, indicating that the RAD23 protein can compensate for the function that is missing in the amino-terminally deleted RAD7 protein. The RAD7 and RAD23 genes in multicopy plasmids do not complement the rad23-delta and rad7-delta mutations, respectively. These observations could mean that although the two proteins might share a common functional domain, they must also perform distinct functions. Alternatively, an interaction between the RAD7 and RAD23 proteins could also account for these observations. PMID- 3023894 TI - Molecular cloning of suppressor of sable, a Drosophila melanogaster transposon mediated suppressor. AB - A hybrid dysgenesis-induced allele [su(s)w20] associated with a P-element insertion was used to clone sequences from the su(s) region of Drosophila melanogaster by means of the transposon-tagging technique. Cloned sequences were used to probe restriction enzyme-digested DNAs from 22 other su(s) mutations. None of three X-ray-induced or six ethyl methanesulfonate-induced su(s) mutations possessed detectable variation. Seven spontaneous, four hybrid dysgenesis induced, and two DNA transformation-induced mutations were associated with insertions within 2.0 kilobases (kb) of the su(s)w20 P-element insertion site. When the region of DNA that included the mutational insertions was used to probe poly(A)+ RNAs, a 5-kb message was detected in wild-type RNA that was present in greatly reduced amounts in two su(s) mutations. By using strand-specific probes, the direction of transcription of the 5-kb message was determined. The mutational insertions lie in DNA sequences near the 5' end of the 5-kb message. Three of the seven spontaneous su(s) mutations are associated with gypsy insertions, but they are not suppressible by su(Hw). PMID- 3023895 TI - Transformation of chicken embryo fibroblasts and tumor induction by the middle T antigen of polyomavirus carried in an avian retroviral vector. AB - The middle T antigen of polyomavirus transformed primary chicken embryo fibroblasts when expressed from a replication-competent avian retrovirus. This in vitro-constructed retrovirus, SRMT1, is a variant of Rous sarcoma virus that encodes the middle T antigen in place of v-src. Inoculation of SRMT1 into 1-week old chickens rapidly induced hemangiomas and hemangiosarcomas. As shown with mammalian cells infected with polyomavirus, polyomavirus middle T antigen appears to be associated with p60c-src in chicken cells infected with SRMT1. When lysates of SRMT1-infected cells immunoprecipitated with either a monoclonal antibody against p60src or anti-T serum were assayed in an in vitro kinase reaction, the middle T antigen was heavily phosphorylated. To see whether an excess of p60c-src could alter the extent of phosphorylation of the middle T protein or the process of cell transformation by middle T, cells were doubly infected with SRMT1 and NY501, a virus which overexpresses p60c-src. Doubly infected chicken embryo fibroblasts transformed with the same kinetics and were morphologically indistinguishable from chicken embryo fibroblasts infected with SRMT1 alone. Phosphorylation of the middle T antigen was elevated two- to fivefold relative to cells infected only with SRMT1. PMID- 3023896 TI - Activation of a cryptic TACTAAC box in the Saccharomyces cerevisiae actin intron. AB - We constructed a translational fusion between the Saccharomyces cerevisiae actin gene and the Escherichia coli beta-galactosidase structural gene such that expression of beta-galactosidase activity required accurate splicing of the actin intron. Using this chimeric gene, we generated a series of internal deletions which removed the TACTAAC box or, in addition, TACTAAC-like sequences within the intron. Analysis of the fusion transcripts produced in these deletions allowed us to conclude that the TACTAAC-like sequence TACTAAG can substitute, albeit inefficiently, for the authentic TACTAAC box in the splicing process. These results indicate that the yeast splicing machinery can utilize a cryptic TACTAAC box, but there are requirements for primary sequence and proper position. PMID- 3023897 TI - Bidirectional transcription from a solo long terminal repeat of the retrotransposon TED: symmetrical RNA start sites. AB - A single copy of the retrotransposon TED was found integrated within the DNA genome of the insect baculovirus, Autographa californica nuclear polyhedrosis virus. After excision of the element from the viral genome, a single long terminal repeat (LTR) remained behind. We have examined the effect of this solo TED LTR on the local pattern of viral transcription. Most prominent was the transcription of two sets of abundant RNAs; both originated within the LTR but extended in opposite directions into flanking viral genes. By promoting symmetric transcription of adjacent genes, the solo LTR has the capacity to activate or repress gene expression in two directions. Primer extension analysis demonstrated that the divergent LTR transcripts were initiated near the same point within a 22 base-pair sequence having hyphenated twofold symmetry. Analogous symmetries at the initiation sites of other retrotransposon LTRs, including copia and Ty, suggested that these sequences serve to establish the precise start for transcription. PMID- 3023898 TI - Use of a novel S1 nuclease RNA-mapping technique to measure efficiency of transcription termination on polyomavirus DNA. AB - We devised a strategy to measure the efficiency of transcription termination in vivo by RNA polymerase on polyomavirus DNA. Pulse-labeled nuclear RNA was hybridized with a single-stranded polyomavirus DNA fragment which spans the transcription initiation region. Hybrids were treated with RNase, bound to nitrocellulose filters, eluted with S1 nuclease, and analyzed by gel electrophoresis. The ratio of full-length to less-than-full-length DNA-RNA hybrids was used to calculate transcription termination frequency. We found that 50% of the polymerases terminated per traverse of the L DNA strand during the late phase of infection. The method for mapping in vivo pulse-labeled RNAs which we developed is potentially useful for studying unstable cellular or viral RNAs. PMID- 3023899 TI - P transposons controlled by the heat shock promoter. AB - We have transformed Drosophila melanogaster with modified P-element transposons, which express the transposase function from the heat-inducible hsp70 heat shock promoter. The Icarus transposon, which contains a direct hsp70-P fusion gene, behaved like a very active autonomous P element even before heat shock induction. Although heat shock led to abundant somatic transcription, transposition of the Icarus element was confined to germ line cells. To reduce the constitutive transposase activity observed for the Icarus element, we attenuated the translational efficiency of the transposase RNA by inserting the transposon 5 neomycin resistance gene between the hsp70 promoter and the P-element sequences. The resulting construct, called Icarus-neo, conferred resistance to G418, and its transposition was significantly stimulated by heat shock. Heat shocks applied during the embryonic or third instar larval stage had similar effects, indicating that transposition of P elements is not restricted to a certain developmental stage. Both Icarus and Icarus-neo destabilized snw in a P-cytotype background and thus at least partially overcome the repression of transposition. Our results suggest that the regulation of P-element transposition occurs at both the transcriptional and posttranscriptional levels. PMID- 3023900 TI - Domain structure of the simian virus 40 core origin of replication. AB - The simian virus 40 core origin of replication consists of nucleotides 5211 through 31. These 64 base pairs contain three functional domains with strict sequence requirements and two spacer regions with relaxed sequence specificity but precise positional constraints. The early domain extends for 10 contiguous base pairs between nucleotides 5211 and 5220. A 9-base pair spacer from sequences 5221 through 5229 separates the early domain from the 23-base pair central palindrome that directs the binding of T antigen. The late end of the core between nucleotides 12 and 31 also contains spacer and sequence-specific functions that are not yet completely mapped. We propose that the sequence specific domains are interaction sites for viral and cellular proteins, determinants of DNA conformation, or both. The spacers would position these signals at required distances and rotations relative to one another. PMID- 3023901 TI - Nucleotide sequence of the two rat cellular rasH genes. AB - We present the nucleotide sequence of the coding region of the rat c-rasH-1 gene and a partial sequence analysis of the rat c-rasH-2 gene. By comparing these sequences with the Harvey murine sarcoma virus ras gene, we predict that the p21 protein encoded by the Harvey virus differs from the cellular c-rasH-1-encoded p21 at only two amino acids; those at positions 12 and 59. Alterations at each of these positions may play a role in activating the viral p21 protein. The c-rasH-2 gene is likely to be a nonfunctional pseudogene because it lacks introns, cannot be activated to transform NIH 3T3 cells, and differs in sequence from both c-rasH 1 and v-rasH at several base pair positions. PMID- 3023903 TI - Somatic excision of transposable element Tc1 from the Bristol genome of Caenorhabditis elegans. AB - We investigated the ability of the transposable element Tc1 to excise from the genome of the nematode Caenorhabditis elegans var. Bristol N2. Our results show that in the standard lab strain (Bristol), Tc1 excision occurred at a high frequency, comparable to that seen in the closely related Bergerac strain BO. We examined excision in the following way. We used a unique sequence flanking probe (pCeh29) to investigate the excision of Tc1s situated in the same location in both strains. Evidence of high-frequency excision from the genomes of both strains was observed. The Tc1s used in the first approach, although present in the same location in both genomes, were not known to be identical. Thus, a second approach was taken, which involved the genetic manipulation of a BO variant, Tc1(Hin). The ability of this BO Tc1(Hin) to excise was retained after its introduction into the N2 genome. Thus, we conclude that excision of Tc1 from the Bristol genome occurs at a high frequency and is comparable to that of Tc1 excision from the Bergerac genome. We showed that many Tc1 elements in N2 were apparently functionally intact and were capable of somatic excision. Even so, N2 Tc1s were prevented from exhibiting the high level of heritable transposition displayed by BO elements. We suggest that Bristol Tc1 elements have the ability to transpose but that transposition is heavily repressed in the gonadal tissue. PMID- 3023902 TI - Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae. AB - The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5 fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1). PMID- 3023904 TI - Foreign DNA introduced by calcium phosphate is integrated into repetitive DNA elements of the mouse L cell genome. AB - We investigated the sites of integration of exogenous DNA fragments introduced by DNA-mediated gene transfer. Mouse Ltk- cells were transformed with the herpes simplex virus thymidine kinase gene and pBR322 DNA by the calcium phosphate precipitation method. Some of the integrated exogenous DNA sequences were recovered from the stable tk+ transformants in the form of plasmids that were capable of propagation in bacteria. Four plasmids derived from two cloned cell lines were analyzed in detail by nucleotide sequencing and hybridization techniques. These plasmids contained a total of seven cellular-exogenous DNA junctions. In all cases, there was no sequence homology between the exogenous and cellular DNA sequences adjacent to the joining sites, and no specific exogenous or cellular sequences occurred at the junctions. Rearrangement or deletion of Ltk DNA was always associated with the integration of exogenous DNA. All of the assignable cellular sequences at the junctions were repetitive sequences. Two of these sequences were from the MIF-1 repetitive sequence family, and a third consisted of a 40-base pair simple copolymer of alternating deoxyadenosine deoxythymidine. Our results suggest that repetitive sequences are relatively favorable sites for the integration of exogenous DNA. PMID- 3023905 TI - Coordinate regulation of myelomonocytic phenotype by v-myb and v-myc. AB - Both avian myeloblastosis virus (by the action of v-myb) and avian myelocytomatosis virus MC29 (by the action of v-myc) transform cells of the myelomonocytic lineage. Whereas avian myeloblastosis virus elicits a relatively immature phenotype, cells transformed by MC29 resemble mature macrophages. When cells previously transformed by v-myb were superinfected with MC29, their phenotype was rapidly altered to that of a more mature cell. These superinfected cells expressed both v-myb (at a level similar to that found before superinfection) and v-myc. It therefore appears that the expression of v-myc can elicit certain properties of a more differentiated phenotype. In addition, unlike cells transformed by v-myb alone, the cells expressing both v-myb and v-myc could not be induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate to differentiate to fully mature macrophages. Cells with a morphology similar to that of the superinfected cells were elicited by simultaneously infecting yolk sac macrophages with avian myeloblastosis virus and MC29. Such cells expressed both v-myb and v-myc. These results indicate that expression of v-myb and v-myc in infected cells coordinately regulates myelomonocytic phenotype and that the two viral oncogenes vary in their ability to interfere with tumor promoter induced differentiation. Our findings also sustain previous suggestions that the oncogenes v-myb and v-myc may not transform target cells by simply blocking differentiation. PMID- 3023906 TI - Saccharomyces cerevisiae secretes and correctly processes human interferon hybrid proteins containing yeast invertase signal peptides. AB - Synthetic oligonucleotides coding for the yeast invertase secretion signal peptide were fused to the gene for the mature form of human interferon (huIFN alpha 2). Two plasmids (E3 and F2) were constructed. E3 contained the invertase signal codons in a reading frame with the mature huIFN-alpha 2 gene. F2 had a deletion of the codon for alanine at amino acid residue-5 in the invertase signal and an addition of a methionine codon located between the coding sequences for the invertase signal and mature huIFN-alpha 2. Both hybrid genes were located adjacent to the promoter from the 3-phosphoglycerate kinase gene on the multicopy yeast expression plasmid, YEp1PT. Yeast transformants containing these plasmids produced somewhat more IFN than did the same expression plasmid containing the IFN gene with its human secretion signal sequence. HuIFN-alpha 2, purified from the medium of yeast cells containing E3, was found to be processed at the correct site. The huIFN-alpha 2 made by plasmid F2 was found to be completely processed at the junction between the invertase signal (a variant) and the methionine of methionine-huIFN-alpha 2. These results strongly suggested that the invertase signal (or its variant) attached to huIFN was efficiently recognized by the presumed signal recognition particle and was cleaved by the signal peptidase in the yeast cells. These results also suggested that amino acid changes on the right side of the cleavage site did not necessarily prevent cleavage or secretion. PMID- 3023908 TI - Purified polyoma virus medium T antigen has tyrosine-specific protein kinase activity but no significant phosphatidylinositol kinase activity. AB - Medium T antigen, the transforming protein of polyoma virus, is associated with pp60c-src and strongly activates its tyrosine-specific protein kinase activity. We investigated whether the medium T-pp60c-src complex is also associated with an activity that phosphorylates the membrane phospholipid phosphatidylinositol, as shown for pp60v-src and p68v-ros, the transforming proteins of Rous sarcoma virus and avian sarcoma virus UR2, respectively. Medium T was purified by affinity chromatography from extracts of polyoma virus-infected mouse fibroblasts. It was bound to antibodies against a peptide corresponding to the carboxy terminus of medium T and released from the immune complex with an excess of the same peptide. In a second step, the partially purified medium T was bound to antibodies against another peptide corresponding to an internal region of medium T and released with excess peptide. Further purification was carried out with a monoclonal antibody against pp60c-src. Samples from each purification step were examined for protein kinase and phosphatidylinositol kinase activity. The highly purified preparations of the medium T-pp60c-src complex showed very low levels of phosphatidylinositol kinase activity, and no difference between medium T from transforming viruses and nontransforming hr-t mutants was detected. In contrast, protein kinase activity was associated with medium T purified from transforming viruses but not from hr-t mutants. PMID- 3023907 TI - Determination of DNA sequence changes induced by ethyl methanesulfonate in human cells, using a shuttle vector system. AB - The DNA sequence changes for 54 mutations induced in human cells by the alkylating agent ethyl methanesulfonate are reported. The mutations were obtained by using a shuttle vector system with the bacterial lacI gene as the target. Of the 54 mutations obtained, 53 were G:C to A:T transitions. PMID- 3023909 TI - Sequences involved in initiation of simian virus 40 late transcription in the absence of T antigen. AB - We analyzed the sequences involved in vivo in the initiation of simian virus 40 (SV40) late transcription occurring in the absence of both SV40 origin sequences and T antigen. The constituent elements of the SV40 late promoters have already been the subject of extensive studies. In vitro studies have resulted in the description of two putative domains of the late promoters. The first domain consists of an 11-base-pair (bp) sequence, 5'-GGTACCTAACC-3', located 25 nucleotides (nt) upstream of the SV40 major late initiation site (MLIS) (J. Brady, M. Radonovich, M. Vodkin, V. Natarajan, M. Thoren, G. Das, J. Janik, and N. P. Salzman, Cell 31:624-633, 1982). The second domain is located within the G C-rich region (J. Brady, M. Radonovich, M. Thoren, G. Das, and N. P. Salzman, Mol. Cell. Biol. 4:133-141; U. Hansen and P. A. Sharp, EMBO J. 2:2293-2303). Our previous in vivo studies permitted us to define a domain of the late promoter which extends from nt 332 to nt 113 and includes the 72-bp enhancer sequences. Here, by using transfection of the appropriate chimeric plasmids into HeLa cells in conjunction with quantitative S1 nuclease analysis, we analyzed in more detail the sequences required for the control of SV40 late-gene expression occurring before the onset of viral DNA replication. We showed that the major late promoter element is in fact the 72-bp repeat enhancer element. This element was able to drive efficient late transcription in the absence of T antigen. Under our experimental conditions, removal of the G-C-rich region (21-bp repeats) entailed a significant increase in the level of late-gene expression. Moreover, translocation of this element closer to the MLIS (53 nt upstream of the MLIS) enhanced the level of transcripts initiated at natural late initiation sites. Our results suggest that the G-C-rich regions have to be positioned between the enhancer element and the initiation sites to stimulate transcription from downstream sites. Thus, the relative arrangement of the various promoter elements is a critical factor contributing to the situation in which the early promoter is stronger than late promoters before viral DNA replication. PMID- 3023910 TI - Myeloma mutant with a novel 3' flanking region: loss of normal sequence and insertion of repetitive elements leads to decreased transcription but normal processing of the alpha heavy-chain gene products. AB - We isolated and characterized LP1.2, a mouse myeloma mutant with a deletion of at least 4 kilobases (kb) immediately 3' of the alpha gene and introduction of at least 5 kb of novel (nonimmunoglobulin) sequence in its place. A 6.2-kb genomic EcoRI fragment from the mutated allele was cloned, and a subfragment was sequenced. The deletion begins 11 base pairs (bp) beyond the normal site of cleavage and polyadenylation for the secreted form of alpha mRNA. A short direct repeat, eight copies of the 17-mer GCCT ATAGAAGTAAGGA, is located at the junction of the alpha and novel sequences. The first 4 bp of the 17-mer are identical to the last 4 bp of the alpha sequence. Novel sequences downstream of the direct repeats in LP1.2 include a low-copy-number sequence flanked by two distinct, highly repetitive elements. The low-copy-number portion of the novel sequence appears on a single 30-kb EcoRI fragment in several myelomas and in liver DNA; one copy of this fragment has rearranged in cell line W3129, and this allele has rearranged a second time in LP1.2. LP1.2 contains low levels of apparently normal alpha protein and mRNA. The S1 nuclease protection of nuclear and cytoplasmic RNAs shows that cleavage and polyadenylation are efficient and accurate and that they occur without the accumulation of aberrant transcripts. Alpha transcription in isolated nuclei is decreased sevenfold in LP1.2 relative to its parent, which accounts for the low steady-state levels of cytoplasmic alpha mRNA and protein in LP1.2. Decreased alpha transcription could result either from the deletion of a positive regulator in the 3' flanking region or from the introduction of novel sequences which exert a negative effect. PMID- 3023911 TI - Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells. AB - We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation. PMID- 3023912 TI - Saccharomyces cerevisiae contains two functional citrate synthase genes. AB - The tricarboxylic acid cycle occurs within the mitochondria of the yeast Saccharomyces cerevisiae. A nuclear gene encoding the tricarboxylic acid cycle enzyme citrate synthase has previously been isolated (M. Suissa, K. Suda, and G. Schatz, EMBO J. 3:1773-1781, 1984) and is referred to here as CIT1. We report here the isolation, by an immunological method, of a second nuclear gene encoding citrate synthase (CIT2). Disruption of both genes in the yeast genome was necessary to produce classical citrate synthase-deficient phenotypes: glutamate auxotrophy and poor growth on rich medium containing lactate, a nonfermentable carbon source. Therefore, the citrate synthase produced from either gene was sufficient for these metabolic roles. Transcription of both genes was maximally repressed in medium containing both glucose and glutamate. However, transcription of CIT1 but not of CIT2 was derepressed in medium containing a nonfermentable carbon source. The significance of the presence of two genes encoding citrate synthase in S. cerevisiae is discussed. PMID- 3023913 TI - Carcinogen-mediated methotrexate resistance and dihydrofolate reductase amplification in Chinese hamster cells. AB - We have investigated different parameters characterizing carcinogen-mediated enhancement of methotrexate resistance in Chinese hamster ovary (CHO) cells and in simian virus 40-transformed Chinese hamster embryo (C060) cells. We show that this enhancement reflects dihydrofolate reductase (dhfr) gene amplification. The carcinogens used in this work are alkylating agents and UV irradiation. Both types of carcinogens induce a transient enhancement of methotrexate resistance which increases gradually from the time of treatment to 72 to 96 h later and decreases thereafter. Increasing doses of carcinogens decrease cell survival and increase the enhancement of methotrexate resistance. Enhancement was observed when cells were treated at different stages in the cell cycle, and it was maximal when cells were treated during the early S phase. These studies of carcinogen mediated dhfr gene amplification coupled with our earlier studies on viral DNA amplification in simian virus 40-transformed cells demonstrate that the same parameters characterize the amplification of both genes. Possible cellular mechanisms responsible for the carcinogen-mediated gene amplification phenomenon are discussed. PMID- 3023914 TI - Establishment of two rabbit mammary epithelial cell lines with distinct oncogenic potential and differentiated phenotype after microinjection of transforming genes. AB - The goal of this work was to establish an assay for transformation of epithelial cells. Two epithelial cell lines were obtained after microinjecting transforming genes into primary rabbit mammary secretory cells. The cell lines were analyzed for their oncogenic potential and for the maintenance of a differentiated phenotype. A fully transformed cell line, which retained epithelial cell organization, was obtained by coinjecting simian virus 40 DNA and the activated human c-Ha-ras gene. The proliferation rate of these cells was high, with a doubling time of 16 h. Their growth was anchorage independent, and they had lost contact inhibition. The cells were tumorigenic in nude mice, but had no metastatic potential. Both microinjected DNAs were efficiently transcribed and translated, in contrast to the casein genes, which were expressed in primary cells but not in the transformed cell line. An immortalized cell line established after injection with simian virus 40 DNA alone was characterized by a moderate rate of proliferation with a doubling time of approximately 30 h. The growth of these cells was contact inhibited and anchorage dependent. The cells were not tumorigenic in nude mice. The viral DNA was expressed during early passages, as shown by the presence of the large T antigen in cell nuclei, but not at later passages. A high number of lactogenic hormone receptors were found associated with the cell surface. Despite the presence of these receptors, no induction of genes coding for milk proteins was observed after addition of prolactin. These data demonstrate that this assay system can be used to assess the immortalizing and transforming potential of candidate oncogenes in epithelial cells. PMID- 3023915 TI - Stimulation of the adenovirus E2 promoter by simian virus 40 T antigen or E1A occurs by different mechanisms. AB - We have examined the ability of simian virus 40 T antigen to stimulate transcription from the adenovirus E2 promoter. T antigen, produced from a cotransfected plasmid, stimulated chloramphenicol acetyltransferase enzyme and mRNA production from an E2 promoter-chloramphenicol acetyltransferase fusion plasmid (pEC113) in monkey kidney CV-1 cells. The level of stimulation of E2 transcription by simian virus 40 T antigen was equal to that observed in cotransfections of pEC113 and the adenovirus E1A gene product. Deletion mutations from the 5' end of the E2 promoter were examined for their ability to express basal, T-antigen, or E1A trans-activated promoter activity. In each case, deletion of upstream promoter sequences to -70 base pairs reduced chloramphenicol acetyltransferase expression to approximately 30% of the level observed with the intact E2 promoter. Deletion to -59 base pairs resulted in chloramphenicol acetyltransferase expression that was 3 to 5% of that observed with the intact E2 promoter. At saturating levels of the stimulatory proteins, the chloramphenicol acetyltransferase levels obtained in response to T antigen and adenovirus E1A were additive. COS-1 cells, which are derived from CV-1 cells and constitutively express simian virus 40 T antigen, do not support E2 promoter trans activation by T antigen. E1A trans activation of the E2 promoter is efficient in COS-1 cells. These results suggest that although promoter sequence requirements are similar, T antigen and E1A trans activate the E2 promoter by different mechanisms. PMID- 3023917 TI - Lymphoid and other tissue-specific phenotypes of polyomavirus enhancer recombinants: positive and negative combinational effects on enhancer specificity and activity. AB - Heterologous enhancer recombinants and deletions of the polyomavirus (Py) noncoding region were constructed and analyzed for tissue specificity of DNA replication and transcription in a number of lymphoid and other cell lines. The simian virus 40 72-base-pair repeat, mouse immunoglobulin heavy-chain enhancer, and Moloney murine leukemia virus enhancer were inserted into the PvuII-D locus (nucleotides 5128 through 5265) of Py. The ability of these recombinants and the parental PvuII-D deletion mutant to replicate in permissive 3T6 cells and MOP-6 cells as well as in nonpermissive mouse B lymphoid, T lymphoid, mastocyte, and embryonal carcinoma cells was determined. Wild-type Py DNA was not permissive for replication in most lymphoid cell lines, except one hybridoma line. Simply deleting the Py PvuII-D region, however, gave Py an expanded host range, allowing high-level replication in some T lymphoid and mastocytoma cell lines, indicating that this element can be a tissue-specific negative as well as positive element. Substitution of the murine leukemia virus enhancer for Py PvuII-D yielded a Py genome which retained the ability to replicate in 3T6 cells but also replicated well in B lymphoid cells. Substitution with the immunoglobulin heavy-chain enhancer allowed replication in B lymphoid cells but interfered with replication in 3T6 cells and mastocytomas. Surprisingly, substitution with the simian virus 40 72-base-pair enhancer repeat gave a recombinant which would not replicate in any cell line tried, including MOP-6 cells, even though other recombinants with this enhancer would replicate. Thus, we observed both cooperation and interference in these combinations between enhancer components and the Py genome and that these combined activities were cell specific. These results are presented as evidence that there may be a positional dependence, or syntax, for the recognition of genetic elements controlling Py tissue specificity. PMID- 3023916 TI - Competitive and cooperative functioning of the anterior and posterior promoter elements of an Alu family repeat. AB - Similar to tRNA genes and the VAI gene, the Alu family repeats are transcribed by RNA polymerase III and contain a split intragenic promoter. Results of our previous studies have shown that when the anterior, box A-containing promoter element (5'-Pu-Pu-Py-N-N-Pu-Pu-Py-G-G-3' in which Pu is any purine, Py is any pyrimidine, and N is any nucleotide) of a human Alu family repeat is deleted, the remaining box B-containing promoter element (5'-G-A/T-T-C-Pu-A-N-N-C-3') is still capable of directing weak transcriptional initiation at approximately 70 base pairs (bp) upstream from the box B sequence. This is different from the tRNA genes in which the box A-containing promoter element plays the major role in the positioning of the transcriptional initiation site(s). To account for this difference, we first carried out competition experiments in which we show that the posterior element of the Alu repeat competes with the VAI gene effectively for the transcription factor C in HeLa cell extracts. We then constructed a series of contraction and expansion mutants of the Alu repeat promoter in which the spacing between boxes A and B was systematically varied by molecular cloning. In vitro transcription of these clones in HeLa cell extracts was analyzed by RNA gel electrophoresis and primer extension mapping. We show that when the box A and box B promoter sequences are separated by 47 to 298 bp, the transcriptional initiation sites remain 4 to 5 bp upstream from box A. However, this positioning function by the box A-containing promoter element was lost when the spacing was shortened to only 26 bp or increased to longer than 600 bp. Instead, transcriptional initiation occurred approximately 70 bp upstream from box B, similar to that in the clones containing only the box B promoter element. All the mutant clones were transcribed less efficiently than was the wild type. An increase in the distance between boxes A and B also activated a second box A-like element within the Alu family repeat. We compare these results with the results of tRNA gene studies. We also discuss this comparison in terms of the positioning function of the split class III promoter elements and the evolutionary conservation of the spacing between the two promoter elements for optimum transcriptional efficiency. PMID- 3023918 TI - Specific protein binding to the simian virus 40 enhancer in vitro. AB - HeLa cell nuclear extracts and wild-type or mutated simian virus 40 enhancer DNA were used in DNase I footprinting experiments to study the interaction of putative trans-acting factors with the multiple enhancer motifs. We show that these nuclear extracts contain proteins that bind to these motifs. Because point mutations which are detrimental to the activity of a particular enhancer motif in vivo specifically prevent protection of that motif against DNase I digestion in vivo, we suggest that the bound proteins correspond to trans-acting factors involved in enhancement of transcription. Using mutants in which the two domains A and B of the simian virus 40 enhancer are either separated by insertion of DNA fragments or inverted with respect to their natural orientation, we also demonstrate that the trans-acting factors bind independently to the two domains. PMID- 3023920 TI - Inverted duplication-transposition event in mammalian cells at an illegitimate recombination join. AB - Illegitimate recombination events in mammalian cells often contain extraneous nucleotides or filler DNA at the recombinant joins. The polyomavirus-transformed cell line 7axB has previously been found to contain 37 base pairs (bp) of filler DNA at one virus-host join of the single insert of integrated viral DNA (A. Hayday, H. E. Ruley, and M. Fried, J. Virol. 44:67-77, 1982). By using a synthetic oligomer of these 37 bp as a probe, we demonstrated that this filler DNA is an inverted duplication of a single-copy rat sequence found 650 bp upstream from this virus-host join. The other virus-host join appears to be the result of a simple illegitimate recombination event between viral and host sequences. This is the first identification of filler DNA as a transposed copy of a chromosomal sequence. The relevance of the recombination events studied to cellular rearrangements and viral integration is discussed. PMID- 3023919 TI - Multiple regulation of STE2, a mating-type-specific gene of Saccharomyces cerevisiae. AB - The Saccharomyces cerevisiae STE2 gene, which is required for pheromone response and conjugation specifically in mating-type a cells, was cloned by complementation of the ste2 mutation. Transcription of STE2 is repressed by the MAT alpha 2 gene product, so that the 1.4-kilobase STE2 RNA is detected only in a or mat alpha 2 strains, not in alpha or a/alpha cells. However, STE2 RNA levels are also increased by the mating pheromone alpha-factor and decreased in strains bearing mutations in the nonspecific STE4 gene. Regulation of STE2 expression in a cells is therefore achieved by several mechanisms. PMID- 3023921 TI - Phenotypic change from transformed to normal induced by benzoquinonoid ansamycins accompanies inactivation of p60src in rat kidney cells infected with Rous sarcoma virus. AB - Three benzenoid ansamycin antibiotics (herbimycin, macbecin, and geldanamycin) were found to reduce the intracellular phosphorylation of p60src at a permissive temperature (33 degrees C) in a rat kidney cell line infected with a temperature sensitive mutant of Rous sarcoma virus. This effect was accompanied by morphological changes from the transformed to the normal phenotype. The filamentous staining pattern of actin fibers was observed in the cells treated with these antibiotics at 33 degrees C. Removal of the antibiotics allowed the cells to revert to the transformed morphology. Ansamitocin, another benzenoid ansamycin, and naphthalenoid ansamycins such as streptovaricin and rifamycins did not show this effect. Pulse-labeling of the antibiotic-treated cultures with 32Pi showed a marked reduction of 32P radioactivity incorporated into p60src. A parallel experiment with [35S]methionine showed that synthesis of p60src was slightly inhibited. The immune complex prepared by mixing the herbimycin-treated cell extracts with antibody against p60src was inactive in vitro in phosphorylating the complex itself. On the contrary, the immune complex derived from untreated cells was active in vitro even in the presence of the antibiotics. These results suggest that benzoquinonoid ansamycins have no direct effect on src kinase but destroy its intracellular environment, resulting in an irreversible alteration of p60src and loss of catalytic activity. PMID- 3023922 TI - A second domain of simian virus 40 T antigen in which mutations can alter the cellular localization of the antigen. AB - Previous studies have demonstrated that mutations at amino acid position 128 of the simian virus 40 large T antigen can alter the subcellular localization of the antigen. A second domain in which mutations can alter localization of the nuclear antigen has been identified by mutations at amino acid positions 185, 186, and 199. Mutations in this region cause the polypeptide to accumulate in both the nucleus and cytoplasm of monkey cells. These T-antigen variants accumulate to near normal levels, but they don't bind to the simian virus 40 origin of DNA replication and are unable to mediate DNA replication. Furthermore, the altered tumor antigens can no longer transform secondary rat cells at normal efficiency, but they retain the ability to transform established mouse and rat cell lines. PMID- 3023923 TI - Evidence for free and metabolically stable p53 protein in nuclear subfractions of simian virus 40-transformed cells. AB - To determine functional subcellular loci of p53, a cellular protein associated with cellular transformation, we analyzed the nucleoplasmic, chromatin, and nuclear matrix fractions from normal mouse 3T3 cells, from methylcholanthren transformed mouse (MethA) cells, and from various simian virus 40 (SV40) transformed cells for the presence of p53. In 3T3 and MethA cells, p53 was present in all nuclear subfractions, suggesting an association of p53 with different structural components of the nucleus. In 3T3 cells, p53 was rapidly turned over, whereas in MethA cells, p53 was metabolically stable. In SV40 transformed cells, p53 complexed to large tumor antigen (large T) was found in the nucleoplasmic and nuclear matrix fractions, as described previously (M. Staufenbiel and W. Deppert, Cell 33:173-181, 1983). In addition, however, metabolically stable p53 not complexed to large T (free p53) was also found in the chromatin and nuclear matrix fractions of these cells. This free p53 did not arise by dissociation of large T-p53 complexes, suggesting that stabilization of p53 in SV40-transformed cells can also occur by means other than formation of a complex with large T. PMID- 3023924 TI - Enhancer sequences responsible for DNase I hypersensitivity in polyomavirus chromatin. AB - DNase I preferentially cleaves polyomavirus minichromosomes at two sites in the enhancer, each of which comprises the sequence AAGCAPuPuAAG flanked by short inverted repeats. A tandem duplication of this sequence generates an additional hypersensitive locus. Mutations which alter either the AAGCAPuPuAAG or flanking repeats diminish hypersensitivity. This region must determine the chromatin conformation recognized by DNase I. PMID- 3023925 TI - Genetic determinants of growth phase-dependent and adenovirus 5-responsive expression of the Chinese hamster thymidine kinase gene are contained within thymidine kinase mRNA sequences. AB - We have constructed a chimeric thymidine kinase (TK) minigene, pHe delta 6Ha, which combines the complete coding and 3' noncoding regions of a Chinese hamster TK cDNA with the promoter region and 5' untranslated region of the TK gene of herpes simplex virus type 1. We have transformed rat 4 cells to Tk+ with this gene and analyzed the pattern of TK gene expression in these transformants under various conditions of in vitro cell culture. We find that TK gene expression in these Tk+ transformants is growth phase dependent, responsive to adenovirus 5 infection, and indistinguishable in character under a variety of cell culture conditions from the pattern of TK gene expression in rat 4 cells transformed to Tk+ with the genomic Chinese hamster TK gene clone lambda HaTK.5. We are led to the conclusion that the genetic elements which mediate growth phase-dependent TK gene expression are contained entirely within the sequences of the mature cytoplasmic hamster TK mRNA. PMID- 3023926 TI - Mutations at the suppressor of forked locus increase the accumulation of gypsy encoded transcripts in Drosophila melanogaster. AB - We studied the effect of mutations at the suppressor of forked [su(f)] locus in Drosophila melanogaster on the accumulation of transcripts encoded by the gypsy transposable element. Mutations at this locus do not affect the pattern of developmental expression of gypsy, but they cause an increase in the total amount of gypsy RNA present at different stages of development as compared with wild type or su(f)/+ flies. These results suggest that the su(f)-encoded products acts as a negative regulator of gypsy expression. PMID- 3023927 TI - A DNA fragment containing the upstream activator sequence determines nucleosome positioning of the transcriptionally repressed PHO5 gene of Saccharomyces cerevisiae. AB - The functional relationship of nucleosome positioning and gene expression is not known. Using high-copy plasmids, containing the yeast phosphate-repressible acid phosphatase gene (PHO5) and the TRP1/ARS1 vector system, I have determined the nucleosomal structure of the 5' region of the PHO5 gene and demonstrated that the nucleosomal positioning of this region is independent of orientation or position in the various plasmid constructions utilized. However, deletion of a 278-base pair BamHI-ClaI fragment from the 5'-flanking sequences of the PHO5 gene causes the nucleosome positioning to become dependent on orientation or position in the plasmids tested. Use of PHO5-CYC1-lACZ fusions have demonstrated that this DNA fragment contains the sequences responsible for the transcriptional regulation of the PHO5 gene in response to the level of phosphate in the growth media. The nucleosome positioning in the 5' region of PHO5 may be determined by an interaction with the sequences or machinery responsible for transcriptional regulation of the gene. PMID- 3023928 TI - The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro. AB - The sequence AAUAAA is found near the polyadenylation site of eucaryotic mRNAs. This sequence is required for accurate and efficient cleavage and polyadenylation of pre-mRNAs in vivo. In this study we show that synthetic simian virus 40 late pre-mRNAs are cleaved and polyadenylated in vitro in a HeLa cell nuclear extract, and that cleavage in vitro is abolished by each of four different single-base changes in AAUAAA. In this same extract, precleaved RNAs (RNAs with 3' termini at the polyadenylation site) are efficiently polyadenylated. This in vitro polyadenylation reaction also requires the AAUAAA sequence. PMID- 3023929 TI - Fine-structure analysis of the DNA sequence requirements for autonomous replication of Saccharomyces cerevisiae plasmids. AB - An autonomously replicating segment, ARS, is located 293 base pairs downstream from the histone H4 gene at the copy-I H3-H4 locus. The sequences needed for autonomous replication were defined by deletion analysis to include an ARS consensus sequence and an additional 3'-flanking region. External deletions into the 3'-flanking yeast sequences resulted in a loss of replication function. However, disruptions of the required 3'-flanking domain by either 10-base-pair linker-scanning substitutions or larger internal deletions did not impair autonomous replication. Thus, replication is dependent upon a flanking chromosome domain, but not an exact DNA sequence. The extent of the yeast sequences required in the 3'-flanking domain is variable depending on the nature of neighboring plasmid vector sequences. That is, there are certain vector sequences that prohibit replication when they are placed too close to the ARS consensus. These results suggest that the functional 3'-flanking domain of the H4 ARS is a specific DNA or chromatin structure or both. PMID- 3023930 TI - Site-specific methylation of adenine in the nuclear genome of a eucaryote, Tetrahymena thermophila. AB - DNA in the polyploid macronucleus of the ciliated protozoan Tetrahymena thermophila contains the modified base N6-methyladenine. We identified two GATC sites which are methylated in most or all of the 45 copies of the macronuclear genome. One site is 2 kilobases 5' to the histone H4-I gene, and the other is 5 kilobases 3' to the 73-kilodalton heat shock protein gene. These sites are de novo methylated between 10 and 16 h after initiation of conjugation, during macronuclear anlage development. The methylation states of these two GATC sites and four other unmethylated GATC sites do not change in the DNA of cells cultured under conditions which change the activity of the genes, including logarithmic growth, starvation, and heat shock. PMID- 3023931 TI - A mutant herpesvirus protein leads to a block in nuclear localization of other viral proteins. AB - The herpes simplex virus mutants KOS1.1 ts756 and HFEM tsLB2 express temperature sensitive ICP4 proteins that are not localized properly to the cell nucleus at the nonpermissive temperature. In these infected cells at the nonpermissive temperature, nuclear localization of at least two other viral proteins, ICP0 and ICP8, is impaired. Replacement of the mutated sequences in the ICP4 gene of tsLB2 restored proper nuclear localization of all of the proteins. The ICP0 and ICP8 proteins expressed in cells transfected with their individual genes were localized to the cell nucleus. Therefore, in infected cells, the mutant ICP4 gene product appears to be the primary defect which leads to the block in nuclear localization of the other proteins. One viral protein, ICP27, was not inhibited for nuclear localization in these cells. These data indicate that there are at least two pathways for nuclear localization of HSV proteins, one of which is inhibited by the mutant ICP4 protein. The mutant ICP4 protein may define a probe for one of the pathways of nuclear localization of proteins. PMID- 3023932 TI - Pharmacological characterization of cyclic AMP receptors mediating gene regulation in Dictyostelium discoideum. AB - Extracellular molecules regulate gene expression in eucaryotes. Exogenous cyclic AMP (cAMP) affects the expression of a large number of developmentally regulated genes in Dictyostelium discoideum. Here, we determine the specificity of the receptor(s) which mediates gene expression by using analogs of cAMP. The order of potency with which these analogs affect the expression of specific genes is consistent with the specificity of their binding to a cell surface receptor and is distinct from their affinity for intracellular cAMP-dependent protein kinase. Dose-response curves with cAMP and adenosine 3',5'-monophosphorothioate, a nonhydrolyzable analog, revealed that the requirement for high concentrations of exogenous cAMP for regulating gene expression is due to the rapid degradation of cAMP by phosphodiesterase. The addition of low concentrations of cAMP (100 nM) or analogs in pulses also regulates gene expression. Both the genes that are positively regulated by exogenous cAMP and the discoidin gene, which is negatively regulated, respond to cAMP analogs to the same degree. Genes expressed in prespore or prestalk cells are also similarly regulated. These data suggest that the effects are mediated through the same receptor. The specificity of this receptor is indistinguishable from that of the well-characterized cell surface cAMP receptor. PMID- 3023934 TI - Characterization and mutational analysis of a cluster of three genes expressed preferentially during sporulation of Saccharomyces cerevisiae. AB - A differential hybridization screen of a genomic yeast DNA library previously identified 14 genes of Saccharomyces cerevisiae that are expressed preferentially during sporulation. Three of these sporulation-specific genes, SPS1, SPS2, and SPS3, have been shown to be closely linked. A mutational analysis has demonstrated that expression of the SPS1 gene, but not the SPS2 gene, is essential for the completion of sporulation. A diploid MATa/MAT alpha strain homozygous for a disruption of the SPS1 gene failed to form asci when subjected to sporulation conditions. The 3' end of the transcript encoded by the SPS1 gene was found to map only 185 base pairs from the 5' end of the SPS2 gene. The SPS1 SPS2 intergenic region was shown to contain all of the regulatory sequences necessary for the sporulation-specific activation of the SPS2 gene as assessed by expression of a translational SPS2-lacZ fusion gene present on a replicating, centromere-containing plasmid. The fusion gene was found to be expressed at the same time during sporulation as the chromosomal wild-type SPS2 gene. PMID- 3023933 TI - Transcriptional regulation of ribosomal proteins during a nutritional upshift in Saccharomyces cerevisiae. AB - The relative rates of synthesis of Saccharomyces cerevisiae ribosomal proteins increase coordinately during a nutritional upshift. We constructed a gene fusion which contained 528 base pairs of sequence upstream from and including the TATA box of ribosomal protein gene rp55-1 (S16A-1) fused to a CYC1-lacZ fusion. This fusion was integrated in single copy at the rp55-1 locus in the yeast genome. During a nutritional upshift, in which glucose was added to cells growing in an ethanol-based medium, we found that the increase in the relative rate of synthesis of the beta-galactosidase protein product followed the same kinetics as the change in relative rates of synthesis of several ribosomal proteins measured in the same experiment. This demonstrates that the nontranscribed sequences upstream from the rp55-1 gene, which are present in the fusion, are sufficient to mediate the change in rates of synthesis characteristic of ribosomal proteins under these conditions. The results also suggest that a change in transcription rates is mainly responsible for the increase in relative rates of synthesis of ribosomal proteins during a nutritional upshift in S. cerevisiae. PMID- 3023935 TI - Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells. AB - We have studied the mechanism of cellular resistance to cardiac glycosides in C+ cells. C+ cells were resistant to ouabain and overproduced plasma membrane-bound Na,K-ATPase relative to parental HeLa cells. Overexpression of Na,K-ATPase in C+ cells correlated with increased ATPase mRNA levels and amplification (approximately 100 times) of the ATPase gene. Growth of C+ cells in ouabain-free medium resulted in a marked decline in ATPase mRNA and DNA levels. However, when cells were reexposed to ouabain, they proliferated and ATPase mRNA and DNA sequences were reamplified. Restriction analysis of C+ and other human DNA samples revealed the occurrence of rearrangements in the region of the Na,K ATPase gene in C+ cells. Furthermore, C+ cells expressed an ATPase mRNA species not found in HeLa cells. These results suggest that amplification of the gene coding for Na,K-ATPase results in overproduction of Na,K-ATPase polypeptides. Amplification of the ATPase gene or the expression of new ATPase mRNA sequences or both may also be responsible for acquisition of the ouabain-resistant phenotype. PMID- 3023937 TI - Intramolecular recombination between transfected repeated sequences in mammalian cells is nonconservative. AB - When plasmids carrying a fragmented gene with segments present as direct repeats are introduced into mammalian cells, recombination or gene conversion between the repeated sequences can reconstruct the gene. Intramolecular recombination leads to the deletion of the intervening sequences and the loss of one copy of the repeat. This process is known to be stimulated by double-strand breaks. Two current models for recombination in eucaryotic cells propose that the reaction is initiated by double-strand breaks, but differ in their predictions as to the fate of the intervening sequences. One model suggests that these sequences are always lost, while the other indicates that the reaction will be conservative as a function of the position of the double-strand break. We have constructed a plasmid in which two overlapping portions of the simian virus 40 early region, which contains the origin and T-antigen gene, are present as direct repeats separated by sequences containing a plasmid with a simian virus 40 origin of replication. Recombination across the repeated segments could produce a plasmid with an origin of replication and/or a plasmid with a gene for a functional T antigen which would drive the replication of both. Introduction of this construction into African green monkey kidney cells, without coinfection, establishes a condition in which the products of the recombination or gene conversion can be interpreted unambiguously. We find that the majority of the reconstruction reactions are nonconservative. PMID- 3023936 TI - PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors. AB - The proteinase A structural gene of Saccharomyces cerevisiae was cloned by using an immunological screening procedure that allows detection of yeast cells which are aberrantly secreting vacuolar proteins (J. H. Rothman, C. P. Hunter, L. A. Valls, and T. H. Stevens, Proc. Natl. Acad. Sci. USA, 83:3248-3252, 1986). A second cloned gene was obtained on a multicopy plasmid by complementation of a pep4-3 mutation. The nucleotide sequences of these two genes were determined independently and were found to be identical. The predicted amino acid sequence of the cloned gene suggests that proteinase A is synthesized as a 405-amino-acid precursor which is proteolytically converted to the 329-amino-acid mature enzyme. Proteinase A shows substantial homology to mammalian aspartyl proteases, such as pepsin, renin, and cathepsin D. The similarities may reflect not only analogous functions but also similar processing and intracellular targeting mechanisms for the two proteins. The cloned proteinase A structural gene, even when it is carried on a single-copy plasmid, complements the deficiency in several vacuolar hydrolase activities that is observed in a pep4 mutant. A strain carrying a deletion in the genomic copy of the gene fails to complement a pep4 mutant of the opposite mating type. Genetic linkage data demonstrate that integrated copies of the cloned proteinase A structural gene map to the PEP4 locus. Thus, the PEP4 gene encodes a vacuolar aspartyl protease, proteinase A, that is required for the in vivo processing of a number of vacuolar zymogens. PMID- 3023938 TI - Accurate in vitro transcription of Xenopus laevis mitochondrial DNA from two bidirectional promoters. AB - The mitochondrial RNA polymerase from Xenopus laevis oocytes was partially purified by heparin-Sepharose chromatography and phosphocellulose chromatography. This RNA polymerase preparation specifically initiated the transcription of X. laevis mitochondrial DNA (mtDNA) from two bidirectional promoters contained within a 123-base-pair segment of the mtDNA between the heavy-strand replication origin and the rRNA cistrons. Transcription in vitro initiated from precisely the same start sites previously mapped as initiation sites for transcription in vivo. At each of the four sites, initiation occurred within a conserved nucleotide sequence, ACPuTTATA. This consensus sequence is not related to promoters for transcription of human mtDNA. PMID- 3023939 TI - Localization of DNA sequences involved in dexamethasone-dependent expression of the rat alpha 1-acid glycoprotein gene. AB - Synthesis of rat alpha 1-acid glycoprotein (AGP), one of the major inflammation induced plasma proteins, is positively regulated by dexamethasone. To define the dexamethasone-responsive genetic element, we isolated and tested AGP gene sequences for the ability to confer glucocorticoid induction to the bacterial chloramphenicol acetyltransferase (CAT) gene in L cells. A 141-base-pair region of the AGP gene, including 120 base pairs of DNA upstream from the start site of transcription and 21 base pairs of the 5' untranslated region, was sufficient for maximal CAT gene induction by dexamethasone. To localize more precisely the AGP glucocorticoid-responsive element, parts of this 141-base-pair region were inserted 5' to either an AGP promoter-CAT gene or a human triosephosphate isomerase promoter-CAT gene, both of which lacked a response to the steroid. The AGP gene region between 120 and 42 base pairs upstream from the start site of transcription was found to mediate maximal dexamethasone induction of CAT enzyme levels. This result was unexpected because this region does not contain sequence homologies to known glucocorticoid receptor-binding sites and those AGP gene regions that lay further upstream and were homologous to other glucocorticoid receptor-binding sites were inactive in the CAT assay. The fact that the AGP gene region mediating dexamethasone regulation was distinct from the transcribed region indicates that glucocorticoids increase AGP gene expression primarily at the transcriptional rather than the posttranscriptional level. PMID- 3023940 TI - Effects of the position of the simian virus 40 enhancer on expression of multiple transcription units in a single plasmid. AB - We have examined the ability of the simian virus 40 72-base pair enhancer segment to simultaneously activate multiple transcription units with plasmids that contain one, two, or three simian virus 40-based transcription units in various arrangements. After transfection into CV1 cells, the expression of a marker gene, Ecogpt, was determined as a function of the position of that marker gene relative to the other transcription units and the position of the marker gene relative to enhancer elements on the plasmids. Two types of position effects were revealed by that analysis. The first, promoter occlusion, causes reduced transcription at a downstream promoter if transcription is initiated at a nearby upstream promoter. This effect does not involve enhancer elements directly, even though the effect is most pronounced when the downstream promoter lacks an enhancer element. The second effect stems from the ability of promoter sequences to reduce the effect of a single enhancer element on other promoters in the same plasmid. This latter effect is mediated by either promoters adjacent to the enhancer element or promoters interposed between the enhancer element and the other promoters on the plasmid. PMID- 3023941 TI - Saccharomyces cerevisiae PHO5 promoter region: location and function of the upstream activation site. AB - Saccharomyces cerevisiae repressible acid phosphatase (PHO5) is induced when inorganic phosphate in the culture medium is depleted. To study the mechanism of this regulation, we constructed various deletions in the 5'-flanking region of the PHO5 gene. Two elements were revealed by this analysis: an upstream activation site (UAS) and a downstream element, both playing parts in the expression of this gene. The UAS is located between -384 and -292 upstream of the initiation codon and activates expression of the gene when inorganic phosphate is depleted. It consists of two homologous regions (UAS I and UAS II) that contain CTGCACAAATG and an adenine-plus-thymine-rich sequence, either one of which suffices for the effect. The downstream element includes a putative TATA box at 100 from the ATG codon and is necessary for efficient transcription and expression of the normal-sized PHO5 transcript. The distance between the UAS and the downstream element can be altered without causing loss of expression efficiency, and the action of the UAS is not affected by its orientation. These results are consistent with a model wherein UAS acts as a site of activation for transcription by interaction with a protein factor(s) that becomes active when inorganic phosphate is depleted from the culture medium. PMID- 3023942 TI - Tissue-specific and developmentally regulated expression of a chimeric actin globin gene in transgenic mice. AB - A chimeric plasmid containing about 2/3 of the rat skeletal muscle actin gene plus 730 base pairs of its 5' flanking sequences fused to the 3' end of a human embryonic globin gene (D. Melloul, B. Aloni, J. Calvo, D. Yaffe, and U. Nudel, EMBO J. 3:983-990, 1984) was inserted into mice by microinjection into fertilized eggs. Eleven transgenic mice carrying the chimeric gene with or without plasmid pBR322 DNA sequences were identified. The majority of these mice transmitted the injected DNA to about 50% of their progeny. However, in transgenic mouse CV1, transmission to progeny was associated with amplification or deletion of the injected DNA sequences, while in transgenic mouse CV4 transmission was distorted, probably as a result of insertional mutagenesis. Tissue-specific expression was dependent on the removal of the vector DNA sequences from the chimeric gene sequences prior to microinjection. None of the transgenic mice carrying the chimeric gene together with plasmid pBR322 sequences expressed the introduced gene in striated muscles. In contrast, the six transgenic mice carrying the chimeric gene sequences alone expressed the inserted gene specifically in skeletal and cardiac muscles. Moreover, expression of the chimeric gene was not only tissue specific, but also developmentally regulated. Similar to the endogenous skeletal muscle actin gene, the chimeric gene was expressed at a relatively high level in cardiac muscle of neonatal mice and at a significantly lower level in adult cardiac muscle. These results indicate that the injected DNA included sufficient cis-acting control elements for its tissue-specific and developmentally regulated expression in transgenic mice. PMID- 3023943 TI - Mutational analysis of a ras catalytic domain. AB - We used linker insertion-deletion mutagenesis to study the catalytic domain of the Harvey murine sarcoma virus v-rasH transforming protein, which is closely related to the cellular rasH protein. The mutants displayed a wide range of in vitro biological activity, from those that induced focal transformation of NIH 3T3 cells with approximately the same efficiency as the wild-type v-rasH gene to those that failed to induce any detectable morphologic changes. Correlation of transforming activity with the location of the mutations enabled us to identify three nonoverlapping segments within the catalytic domain that were dispensable for transformation and six other segments that were required for transformation. Segments that were necessary for guanosine nucleotide (GDP) binding corresponded to three of the segments that were essential for transformation; two of the three segments share strong sequence homology with other purine nucleotide-binding proteins. Loss of GDP binding was associated with apparent instability of the protein. Lesions in two of the three other required regions significantly reduced GDP binding, while small lesions in the last required region did not impair GDP binding or membrane localization. We speculate that this latter region interacts with the putative cellular target of ras. The results suggest that transforming ras proteins require membrane localization, guanosine nucleotide binding, and an additional undefined function that may represent interaction with their target. PMID- 3023944 TI - Downstream sequences affect transcription initiation from the adenovirus major late promoter. AB - We analyzed a set of adenovirus-simian virus 40 (SV40) hybrids in which the SV40 T antigen coding sequences are inserted downstream from the adenovirus major late promoter within the first, second, and third segments of the tripartite leader. In infected cells, these viruses give rise to a matched set of hybrid SV40 mRNAs that differ only in the number of tripartite leader segments attached to the complete SV40 T antigen coding region. We found that the number of tripartite leader segments present at the 5' end of the hybrid SV40 mRNAs had little effect on the efficiency of T antigen translation. Surprisingly, insertion of SV40 sequences within the first leader segment, at +33 relative to the start of transcription, significantly reduced the frequency of transcription initiation from the major late promoter. The 3' boundary of this downstream transcriptional control element was mapped between +33 and +190 by showing that insertion of SV40 sequences within the intron after the first leader segment at +190 had very little effect on transcription initiation from the late promoter. A transient expression assay was used to show that the effect of downstream sequences on transcription initiation from the major late promoter is dependent on a trans acting factor encoded or induced by adenovirus. PMID- 3023945 TI - Termination-reinitiation occurs in the translation of mammalian cell mRNAs. AB - Many examples of internal translation initiation in eucaryotes have accumulated in recent years. In many cases terminators of upstream reading frames precede the internal initiation site, suggesting that translational reinitiation may be a mechanism for initiation at internal AUGs. To test this idea, a series of recombinants was constructed in the mammalian expression vector pSV2. Each contained a dicistronic transcription unit comprising the coding sequence for mouse dihydrofolate reductase (DHFR) followed by the gene for xanthine-guanine phosphoribosyl transferase (XGPRT) from Escherichia coli. Various versions of this pSV2dhfr-gpt recombinant plasmid altered the location at which the DHFR reading frame was terminated relative to the XGPRT initiation codon and demonstrated that this is a critical factor for the expression of XGPRT activity in transfected Cos-1 cells. Thus, when the DHFR frame terminated upstream or a very short distance downstream of the XGPRT initiator AUG, substantial levels of XGPRT activity were observed. When the DHFR frame terminated 50 nucleotides beyond the XGPRT initiator, activity was reduced about twofold. However, when the DHFR and XGPRT sequences were fused in-frame so that ribosomes which initiated at the DHFR AUG did not terminate until they encountered the XGPRT terminator, production of XGPRT activity was abolished. This dependence of internal translation initiation on the position of terminators of the upstream reading frame is consistent with the hypothesis that mammalian ribosomes are capable of translational reinitiation. PMID- 3023946 TI - Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants. AB - In a previous report (S. Subramani, R. Mulligan, and P. Berg, Mol. Cell. Biol. 1:854-864, 1981), it was shown that mouse dihydrofolate reductase (DHFR) could be efficiently expressed from simian virus 40 recombinant viruses containing the DHFR cDNA in different locations in the viral late region. This was true even in the case of the SVGT7dhfr26 recombinant, which had the DHFR coding sequence 700 to 800 nucleotides from the 5' end of the mRNA, where it was preceded by the VP2 and VP3 initiator AUGs and a number of other noninitiator AUGs. To investigate the process of internal translation initiation in mammalian cells, we constructed a series of SVGT7dhfr recombinants in which the upstream VP2 and VP3 reading frame was terminated in various positions relative to the DHFR initiation codon. The efficient production of DHFR in infected CV1 cells depended on having the terminators of the VP2-VP3 reading frame positioned upstream or nearby downstream from the DHFR initiation codon. These results reinforce the notion that mammalian ribosomes are capable of translational reinitiation. PMID- 3023947 TI - An excision event that may depend on patchy homology for site specificity. AB - In mouse cells transformed by a mutant polyomavirus genome, recombination between integrated viral DNA and flanking cellular DNA resulted in the excision of two readily amplifiable chimeras, designated RmI and RmII. The crossing-over that generated RmII was unique in that it involved a simple cellular sequence in which the triplet 5'-CTG-3' was repeated many times. We show that the sequence across the junction resulting from excision was identical in several molecules of RmII, as if the cross-over generating this junction always involved exactly the same two sites on the viral and cellular DNA. We also show that the cellular site mapped where the replacement of a G by an A in one of many successive 5'-CTG-3' triplets generated a homology of five nucleotides (5'-CTACT-3') with the viral site. Oligonucleotides on both sides of these sites are probably involved in matching the two DNAs prior to recombination. PMID- 3023948 TI - Transcriptional role for the nontranscribed spacer of rat ribosomal DNA. AB - In vitro transcription of the rat rRNA gene led to the identification of a region within a 3.4-kilobase fragment of the nontranscribed spacer (NTS) which significantly increased the transcription of rat ribosomal DNA. Promoter constructs containing this region were transcribed up to 17-fold more efficiently in vitro than templates with only 167 or 286 base pairs of NTS. This effect was also observed when the 3.4-kb fragment of the NTS was subcloned in the opposite orientation and 4 kb upstream of the promoter. The region responsible for the enhanced level of transcription was found between -286 and -1018. The results of order-of-addition experiments suggested that the enhanced level of transcription was the result of the formation of a stable complex between a trans-acting factor and the nontranscribed spacer. DNA-protein binding assays demonstrated that the same region of the NTS determined to have enhancer activity also specifically bound a proteinase K-sensitive factor present in nuclear extracts. The sequence of this region was not found to have any significant homology with the promoter of the rat rRNA gene. This is the first report to assign a transcriptional role to the NTS of a mammalian rRNA gene. PMID- 3023949 TI - Genetic manipulation of Saccharomyces cerevisiae by use of the LYS2 gene. AB - The structural gene for alpha-aminoadipate reductase (LYS2) was isolated from a Saccharomyces cerevisiae genomic DNA library by complementation of a lys2 mutant. Both genetic and biochemical criteria confirmed that the DNA obtained corresponds to the LYS2 locus on chromosome II. Subcloning and deletion analysis showed that a functional LYS2 gene is contained within a 4.6-kilobase (kb) EcoRI-HindIII fragment of the original insert, and the slightly larger EcoRI-ClaI segment (4.8 kb) was used to construct a series of cloning vehicles, including integrating, episomal, replicative, and centromeric vectors. The cloned DNA was also used to generate a genomic deletion that lacks all LYS2 coding sequences on chromosome II. The level of the LYS2 transcript (4.2 kb) was 10-fold higher in cells grown on minimal medium than in cells grown on complete medium and was not repressed by the presence of lysine alone. Gene disruption, gene replacement, and promoter analysis of the major alpha-factor structural gene (MF alpha 1) were performed to illustrate the utility of the LYS2 gene for the genetic manipulation of yeasts. Because all fungi synthesize lysine via the alpha-aminoadipate pathway, the techniques developed here for using the S. cerevisiae LYS2 gene should be directly applicable to other fungal systems. PMID- 3023951 TI - Multiple mechanisms are responsible for altered expression of ornithine decarboxylase in overproducing variant cells. AB - We selected and characterized a series of mouse S49 cell variants that overproduce ornithine decarboxylase (ODC). Previously, we described variants that have an amplified ODC gene and produce about 500-fold more ODC than the wild-type cells of origin (L. McConlogue and P. Coffino, J. Biol. Chem. 258:12083-12086, 1983). We examined a series of independent variants that overproduce ODC to a lesser degree and found that a number of mechanisms other than gene amplification are responsible for the increased ODC activity. Variants were selected for resistance to 0.1 mM difluoromethylornithine, an inhibitor of ODC, by either a single or a multistep process. All showed increased ODC activity and increased ODC mRNA steady-state levels. The half-life of the enzyme was not increased in any of the variants. In one class of variant the increase of ODC mRNA was sufficient to account for ODC overproduction. In a second class, the rate of synthesis of ODC polypeptide per ODC mRNA was at least four- to eightfold higher than that in wild-type cells. Therefore, these variants were altered in the translatability of ODC mRNA. Southern analysis showed that gene amplification does not account for the increased ODC mRNA levels in any of the variants. In both variant and wild-type cells, ODC activity was responsive to changes in polyamine pools; activity was reduced following augmentation of pool size. This change in activity was associated with modification of the rate of synthesis and degradation of ODC but no change in the level of ODC mRNA. PMID- 3023950 TI - Androgen regulation by the long terminal repeat of mouse mammary tumor virus. AB - Mouse mammary tumor virus (MMTV) has long been implicated in mouse mammary carcinogenesis, and it is now well established that the long terminal repeat (LTR) contains regulatory sequences responsible for glucocorticoid-mediated induction of viral RNA. However, we have demonstrated previously that androgens as well as glucocorticoids can regulate MMTV RNA in the S115 mouse mammary tumor cell line. To determine if androgens act directly on the LTR in these cells, plasmids were constructed with the MMTV LTR joined to the coding sequences of genes not normally expressed in the cells. Following transfection of these chimeric genes into S115 cells, we show that the expression of the genes is regulated by both androgens and glucocorticoids. Furthermore, hormonal regulation is also conferred by the LTR on the neighboring guanine phosphoribosyltransferase (gpt) gene. Thus, androgens can act on the LTR of MMTV when the appropriate receptors are present in the cells, and this interaction can influence the expression of additional adjacent genes. PMID- 3023952 TI - Construction of a helper-free recombinant adenovirus that expresses polyomavirus large T antigen. AB - Adenovirus-polyomavirus recombinant viruses were constructed in vitro by inserting a hybrid transcription unit composed of the adenovirus type 2 major late promoter and the early coding region of polyomavirus into the adenovirus type 5 vector Ad5 delta E1/dl309. The vector lacks the E1a and E1b transcription units and contains a unique restriction endonuclease cleavage site in their place. The polyomavirus genomic insert contained a small deletion which precluded the synthesis of functional small and middle T antigen but allowed for the synthesis of large T antigen. One recombinant virus, Ad5PyR39, which contained the hybrid transcription unit in the opposite transcriptional orientation from the overall direction of late-gene transcription, was studied in detail. Ad5PyR39 replicated efficiently without a helper virus in human 293 cells and expressed hybrid mRNAs of the expected size and composition that were translated to yield large T antigen. The large T antigen synthesized in 293 cells was the same size as that produced in mouse 3T6 cells lytically infected with polyomavirus, and this protein bound efficiently and specifically to the large-T-antigen-binding sites in polyomavirus DNA. Moreover, the large T antigen encoded by the recombinant virus proved capable of catalyzing the replication in mouse 3T6 cells of a plasmid containing the polyomavirus origin for DNA replication. Comparison of the amount of large T antigen produced in 3T6 cells infected with polyomavirus with that in 293 cells infected with Ad5PyR39, under optimal conditions for each system, revealed at least a fivefold greater yield of the protein on a per cell basis in the latter system compared with the former. Ad5PyR39 should prove to be useful to isolate large quantities of functional polyomavirus large T antigen for structural and biochemical studies. PMID- 3023953 TI - Nucleosome phasing on a DNA fragment from the replication origin of simian virus 40 and rephasing upon cruciform formation of the DNA. AB - Nucleosomes were reconstituted in vitro from a fragment of DNA spanning the simian virus 40 minimal replication origin. The fragment contains a 27-base-pair palindrome (perfect inverted repeat). DNA molecules with stable cruciform structures were generated by heteroduplexing this DNA fragment with mutants altered within the palindromic sequence (C. Nobile and R. G. Martin, Int. Virol., in press). Analyses of the structural features of the reconstituted nucleosomes by the DNase I footprint technique revealed two alternative DNA-histone arrangements, each one accurately phased with respect to the uniquely labeled DNA ends. As linear double-stranded DNA, a unique core particle was formed in which the histones strongly protected the regions to both sides of the palindrome. The cruciform structure seemed to be unable to associate with core histones and, therefore, an alternative phasing of the histone octamer along the DNA resulted. Thus, nucleosome positioning along a specific DNA sequence appears to be influenced in vitro by the secondary structure (linear or cruciform) of the 27 base-pair palindrome. The formation of cruciform structures in vivo, if they occur, might therefore represent a molecular mechanism by which nucleosomes are phased. PMID- 3023955 TI - Beta-adrenergic stimulation of c-fos gene expression in the mouse submandibular gland. AB - Isoproterenol (IPR), a beta-adrenergic agonist, induces division of acinar cells in the parotid and submandibular glands of adult rodents and produces hyperplastic and hypertrophic enlargements of these organs. We analyzed the effects of IPR on thymidine incorporation, c-fos mRNA levels, and the immunocytochemical localization of c-fos protein in the submandibular glands of adult and of 5- and 14-day-old mice. In the glands of untreated mice c-fos transcripts were not detectable. In all experimental groups, administration of IPR led to a rapid, transient increase in the c-fos mRNA level. Propranolol blocked the IPR effect, while treatment with IPR and cycloheximide led to superinduction. We observed no correlation between the effect of IPR on cell replication or organ growth and stimulation of c-fos expression, and conclude that the latter is the result of beta-adrenergic receptor-IPR interaction. The c fos protein was localized immunocytochemically in both the cytoplasm and the nuclei of acinar cells and in the nuclei of duct cells. PMID- 3023954 TI - Herpes simplex virus type 2 mutagenesis: characterization of mutants induced at the hprt locus of nonpermissive XC cells. AB - In a previous report, herpes simplex virus type 2 (HSV-2) was shown to increase the frequency of mutation at the hypoxanthine phosphoribosyltransferase (hprt) locus of nonpermissive rat XC cells (L. Pilon, A. Royal, and Y. Langelier, J. Gen. Virol. 66:259-265, 1985). A series of 17 independent mutants were isolated after viral infection together with 12 spontaneous noninfected mutants to characterize the nature of the mutations induced by the virus at the molecular level. The DNA of the mutants isolated after viral infection was probed with cloned HSV-2 fragments representing the entire genome. In these mutants, no authentic HSV-2 hybridization could be detected. This was indicative of a mechanism of mutagenesis which did not require the permanent integration of viral sequences in the host genome. The structure of the hprt gene was determined by the method of Southern (J. Mol. Biol. 98:503-517, 1975), and the level of hprt mRNA was analyzed by Northern blots. Except for the identification of one deletion mutant in each of the two groups, the HPRT- clones showed no evidence of alteration in their hprt gene. A total of 7 of 12 spontaneous mutants and 11 of 15 mutants isolated from the infected population transcribed an hprt mRNA of the same size and abundance as did the wild-type cells. Thus, the majority of the mutants seemed to have a point mutation in their hprt structural gene. Interestingly, the proportion of the different types of mutations was similar in the two groups of mutants. This analysis revealed that HSV-2 infection did not increase the frequency of rearrangements but rather that it probably induced a general increase of the level of mutations in the cells. This type of response is thought to be compatible with the biology of the virus, and the possible mechanisms by which HSV-2 induces somatic mutations in mammalian cells are discussed. PMID- 3023957 TI - Nonhomologous recombination in the parvovirus chromosome: role for a CTATTTCT motif. AB - The mechanism of nonhomologous recombination in murine cells infected with the parvovirus minute virus of mice (MVM) has been investigated by analysis of DNA sequences at recombination junctions in naturally occurring deletion variants of the virus. We report here that nonhomologous recombination in the MVM chromosome is characterized by short homologies, by insertion at recombination junctions of foreign DNA sequences that are enriched for preferred eucaryotic topoisomerase I cleavage sites, and by an association with a common DNA sequence motif of the type 5'-CTATTTCT-3'. Additional analyses of broken MVM chromosomes provided evidence for specific enzymatic cleavage within 5'-CTTATC-3' and 5'-CTATTC-3' sequences. The results indicate that the 5'-CTATTTCT-3' motif is an important genetic element for nonhomologous recombination in the parvovirus chromosome. PMID- 3023956 TI - Human c-ros-1 gene homologous to the v-ros sequence of UR2 sarcoma virus encodes for a transmembrane receptorlike molecule. AB - We isolated a human gene (designated c-ros-1) homologous to the v-ros sequence of UR2 sarcoma virus. Ten exons, 1,414 base pairs spanning 26 kilobases, contained a tyrosine kinase domain, a transmembrane domain, and a part of an extracellular domain carrying an N glycosylation site which was not acquired by UR2 sarcoma virus. The predicted structure of c-ros-1 is unique among the src family and clearly distinct from the human insulin receptor. PMID- 3023958 TI - Isolation and sequencing of a cDNA clone homologous to the v-sis oncogene from human endothelial cells. AB - A clone containing the 3' end of the mRNA for the human c-sis gene (homologous to the B chain of platelet-derived growth factor) was isolated from a cDNA library derived from human umbilical vein endothelial cells and then sequenced. The analysis of possible translation products in all three reading frames indicated that the A chain of platelet-derived growth factor was not coded for within the 3' end of the c-sis mRNA. The 3' end of the mRNA for c-sis is contained in or adjacent to exon 6. PMID- 3023959 TI - Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian cells. AB - We have used oligonucleotide-directed site-specific mutagenesis to convert serine codon 27 of the Escherichia coli chloramphenicol acetyltransferase (cat) gene to UAG, UAA, and UGA nonsense codons. The mutant cat genes, under transcriptional control of the Rous sarcoma virus long terminal repeat, were then introduced into mammalian cells by DNA transfection along with UAG, UAA, and UGA suppressor tRNA genes derived from a human serine tRNA. Assay for CAT enzymatic activity in extracts from such cells allowed us to detect and quantitate nonsense suppression in monkey CV-1 cells and mouse NIH3T3 cells. Using such an assay, we provide the first direct evidence that an opal suppressor tRNA gene is functional in mammalian cells. The pattern of suppression of the three cat nonsense mutations in bacteria suggests that the serine at position 27 of CAT can be replaced by a wide variety of amino acids without loss of enzymatic activity. Thus, these mutant cat genes should be generally useful for the quantitation of suppressor activity of suppressor tRNA genes introduced into cells and possibly for the detection of naturally occurring nonsense suppressors. PMID- 3023960 TI - Induction of specific transcription by RNA polymerase III in transformed cells. AB - RNA polymerase III (pol III) transcripts of the highly repeated mouse B2 gene family are increased in many oncogenically transformed murine cell lines. In cells transformed by simian virus 40, the small, cytoplasmic B2 RNAs are present at 20-fold-higher levels than in normal cells (M. R. D. Scott, K. Westphal, and P. W. J. Rigby, Cell 34:557-567, 1983; K. Singh, M. Carey, S. Saragosti, and M. Botchan, Nature [London] 314:553-556). We found that transcripts of the highly repeated B1 gene family are also increased 20-fold upon simian virus 40 transformation and showed that these RNAs result from pol III transcription. In contrast, transcripts from less highly repeated pol III templates such as the 5S, 7SL, 7SK, 4.5SI, tRNAMet, and tRNAPro genes are unaffected. The expression of the B2 RNAs in isolated nuclei shows that the augmentation is due mainly to an increased rate of transcription by pol III. There is thus specific transformation inducible pol III transcription. We developed an in vitro transcription assay which utilizes genomic DNA as a template to study the transcription of all members of a repetitive gene family in their native context. This assay reproduces the low cytoplasmic levels of B1 compared with B2 RNAs suggesting that this ratio is dictated by intrinsic signals in the DNA. PMID- 3023961 TI - Evolutionarily selected replication origins: functional aspects and structural organization. AB - A selective replicative pressure occurs during the evolution of simian virus 40 variants. When the replication origin is duplicated as an inverted repeat, there is a dramatic enhancement of replication. Having regulatory sequences located between the inverted repeat of ori magnifies their enhancing effect on replication. A passage 20 variant and a passage 45 variant containing three pairs of an inverted repeat of ori replicated more efficiently than a passage 13 variant containing nine copies of ori arranged in tandem. A 69-base-pair cellular sequence inserted between inverted repeats of ori of both passage 40 and 45 variants enhanced simian virus 40 DNA replication. Differences in replication efficiencies became greater as the total number of replicating species was increased in the transfection mixture, under conditions where T antigen is limiting. In a competitive environment, sequences flanking the replication origin may be inhibitory to replication. PMID- 3023962 TI - Simian virus 40 DNA replication: functional organization of regulatory elements. AB - The efficiency of simian virus 40 (SV40) DNA replication is dependent on the structural organization of the regulatory region. The enhancing effect of the G + C-rich 21-base-pair (bp) repeats on SV40 DNA replication is position and dose dependent and to some extent orientation dependent. The inverted orientation is about 50% as effective as the normal orientation of the 21-bp repeat region. Movement of the 21-bp repeat region 180 or 370 bp upstream of the ori sequence abolishes its enhancing effect, whereas no replication is detected if the 21-bp repeat region is placed downstream of the ori sequence. The dose-dependent enhancement of the 21-bp repeat of SV40 DNA replication as first described in single transfection by Bergsma et al. (D. J. Bergsma, D. M. Olive, S. W. Hartzell, and K. N. Subramanian, Proc. Natl. Acad. Sci. USA 79:381-385, 1982) is dramatically amplified in mixed transfection. In the presence of the 21-bp repeat region, the 72-bp repeat region can enhance SV40 DNA replication. In the presence of the 21-bp repeats and a competitive environment, the 72-bp repeat region exhibits a cis-acting inhibitory effect on SV40 DNA replication. PMID- 3023963 TI - c-erbB activation in avian leukosis virus-induced erythroblastosis: multiple epidermal growth factor receptor mRNAs are generated by alternative RNA processing. AB - Avian leukosis virus-induced erythroblastosis results from the specific interruption of the host oncogene, c-erbB, by the insertion of an intact provirus. This insertion results in the expression of two size classes (3.6 and 7.0 kilobases [kb]) of truncated c-erbB transcripts which are initiated in the 5' long terminal repeat of the integrated provirus. Through sequence analysis of erbB cDNA clones we have previously shown that the 3.6-kb activated erbB mRNA contains portions of viral gag and env genes fused to c-erbB sequences (T.W. Nilsen, P.A. Maroney, R.G. Goodwin, F.M. Rottman, L.B. Crittenden, M.A. Raines, and H.-J. Kung, Cell 41:719-726, 1985). In this report we show that the 7-kb mRNA differs from the shorter activated c-erbB mRNA in the length of its 3' untranslated sequence such that the longer mRNA has an extremely long (4.3 kb) 3' untranslated sequence. Additionally, we demonstrate that activated c-erbB mRNA precursors can be processed by alternative splicing to yield mRNAs with viral gag sequences fused directly to c-erbB sequences. Finally, blot hybridization evidence suggests that the two size classes of activated c-erbB mRNA in erythroblastic tissue represent truncated versions of the two c-erbB mRNAs present in normal tissue. PMID- 3023964 TI - Efficient transcription of a Caenorhabditis elegans heat shock gene pair in mouse fibroblasts is dependent on multiple promoter elements which can function bidirectionally. AB - A divergently transcribed pair of Caenorhabditis elegans hsp16 genes was introduced into mouse fibroblasts by stable transfection with vectors containing bovine papillomavirus plasmid maintenance sequences and a selectable gene. The hsp16 genes were transcriptionally inactive in the mouse cells under normal growth conditions and were strongly induced by heat shock or arsenite. In a cell line with 12 copies of the gene pair, there were estimated to be more than 10,000 hsp16 transcripts in each cell after 2 h of heat shock treatment. The hsp16 transcript levels were more than 100 times higher than those of a gene with a herpes simplex virus thymidine kinase gene promoter carried on the same vector. A single heat shock promoter element (HSE) could activate bidirectional transcription of the two hsp16 genes when placed between the two TATA elements, but the transcriptional efficiency was reduced 10-fold relative to that of the wild-type gene pair. Four overlapping HSEs positioned between the two TATA elements resulted in inducible bidirectional transcription at greater than wild type levels. The number of HSEs can therefore be a major determinant of the promoter strength of heat-inducible genes in mammalian cells. Partial disruption of an alternating purine-pyrimidine sequence between the two hsp16 genes had no significant effect on their transcriptional activity. PMID- 3023965 TI - Human growth hormone as a reporter gene in regulation studies employing transient gene expression. AB - The human growth hormone (hGH) transient assay system described here is based on the expression of hGH directed by cells transfected with hGH fusion genes. Levels of secreted hGH in the medium, measured by a simple radioimmunoassay, are proportional to both levels of cytoplasmic hGH mRNA and the amount of transfected DNA. The system is extremely sensitive, easy to perform, and is qualitatively different from other transient expression systems in that the medium is assayed and the cells themselves are not destroyed. The hGH transient assay system is appropriate for analyses of regulation of gene expression and was utilized here to investigate the effect of the simian virus 40 enhancer on the herpes simplex virus thymidine kinase promoter and the effect of zinc on the mouse metallothionein-I promoter. The expression of hGH can also be used as an internal control to monitor transfection efficiency along with any other transient expression system. All cell types tested thus far (including AtT-20, CV-1, GC, GH4, JEG, L, and primary pituitary cells) were able to secrete hGH into the medium. PMID- 3023967 TI - A consensus sequence polymer inhibits in vivo expression of heat shock genes. AB - Promoter function for hsp70 gene expression in Drosophila melanogaster was studied with an in vivo competition assay. A polymer of 40 tandem copies of the pair of regulatory elements of the hsp70 gene was constructed and cloned into a plasmid vector. Various marked genes were cotransfected with the polymer plasmid into Schneider line 2 cells, and their expression was determined by enzyme activity measurements. The polymer dramatically inhibited expression of cotransfected hsp70, hsp26, and hsp83 genes, but not cotransfected copia and histone genes. Our results indicate that in vivo, a trans-acting, positive regulatory factor, which can be titrated by heat shock consensus sequences, is required for activation of heat shock genes and is specific for these genes; the coordinate induction of different heat shock genes appears to be mediated by similar, but not identical, interactions of the trans-acting induction factor and the cis-acting heat shock consensus sequences; and the uninduced or basal level expression of the transfected hsp70 gene is also due to interaction of the consensus sequence with a positively acting factor. PMID- 3023968 TI - Organization of regionally expressed silkmoth chorion genes. AB - We described the organization of two silkmoth chorion genes, called E1 and E2, whose expression is largely restricted in time to the very late period of choriogenesis and in space to one of two major subpopulations of follicle cells. Using E1 and E2 clone cDNAs as probes, we showed that gene copy numbers per haploid genome remain constant throughout silkmoth development despite major changes in total DNA content per nucleus. Furthermore, gene copy numbers are the same in both cellular regions of the choriogenic follicle despite differences in nuclear size and levels of E gene expression. Southern analysis indicated between two and four copies each for E1 and E2 genes. Analysis of chromosomal clones showed that single copies of E1 and E2 are separated by about 7.5 kilobases and are transcribed from the same DNA strand. Two distinct pairs of cloned E1 and E2 genes were characterized. No other chorion genes were in their immediate vicinity. PMID- 3023966 TI - Binding of polyomavirus large T antigen to the human hsp70 promoter is not required for trans activation. AB - Polyomavirus large T antigen binds to two sites located between positions -110 and -170 of a human heat shock protein 70 (hsp70) promoter. Methylation interference studies show that binding for each site is determined by two GPuGGC pentanucleotide sequences. The specificity of this binding interaction is similar to that observed for large T binding to the viral genome. The existence of sequences that bind a viral protein in a cellular promoter raises the possibility that these sequences play a role in gene expression in an uninfected cell. We show that hsp70 large T antigen binding site 1 is capable of functioning as an upstream promoter element in cells that do not contain any viral T antigen. Genetic analysis of this effect suggests that a cellular factor exists that has a binding specificity that overlaps but is not identical to that of polyomavirus large T antigen. To determine whether binding of polyomavirus large T antigen can regulate expression of the intact human hsp70 promoter, we have introduced the promoter into mouse cells with plasmids that express the polyomavirus early proteins. These proteins stimulate the level of correctly initiated hsp70 transcripts, but surprisingly the degree of stimulation remains unchanged for promoter constructs in which the large T antigen binding sites have been deleted. These observations suggest that trans activation of the hsp70 promoter by the polyomavirus early proteins occurs through protein-protein interactions and not through sequence-specific DNA binding. PMID- 3023969 TI - Synergism of v-myc and v-Ha-ras in the in vitro neoplastic progression of murine lymphoid cells. AB - Murine bone marrow was either singly or doubly infected with retroviral vectors expressing v-myc (OK10) or v-Ha-ras. The infected bone marrow was cultured in a system that supports the long-term growth of B-lineage lymphoid cells. While the v-myc vector by itself had no apparent effect on lymphoid culture establishment and growth, infection with the v-Ha-ras vector or coinfection with both v-myc and v-Ha-ras vectors led to the appearance of growth-stimulated cell populations. Clonal pre-B-cell lines stably expressing v-Ha-ras alone or both v-myc and v-Ha ras grew out of these cultures. In comparison with cell lines expressing v-Ha-ras alone, cell lines expressing both v-myc and v-Ha-ras grew to higher densities, had reduced dependence on a feeder layer for growth, and had a marked increase in ability to grow in soft-agar medium. The cell lines expressing both oncogenes were highly tumorigenic in syngeneic animals. These experiments show that the v myc oncogene in synergy with v-Ha-ras can play a direct role in the in vitro transformation of murine B lymphoid cells. PMID- 3023971 TI - Recombination of homologous DNA fragments transfected into mammalian cells occurs predominantly by terminal pairing. AB - The mechanism by which double-strand cleavages stimulate the joining of plasmid DNA fragments introduced into cultured mammalian cells was investigated by cotransfecting pairs of plasmids encoding deletion mutations in a dominant selectable gene into LMtk- cells. Plasmid recombination substrates were produced by creating deletions of different sizes within the neo coding region of the pSV2neo plasmid. Complementing pairs of deleted plasmid DNAs were linearized at specific unique sites before cotransfection into mouse LMtk- cells by the calcium phosphate precipitation method. Cleaving one donor plasmid produced a 4- to 10 fold stimulation in the production of colonies able to survive in medium containing G-418. The linearization of the second plasmid further increased the efficiency by another factor of 6 to 15 when the cut was made on the opposite side of the homology, approximately equidistant from the center of the overlap. Fifty-seven individual G-418-resistant colonies representing the products of individual crosses were isolated, and the genomic DNAs containing the presumably integrated, functional recombinant neo genes were analyzed on Southern blots. A band consistent with the exchange of markers flanking the neo gene was present in 90% of the DNAs examined. In only one case was the pattern indicative of either a double crossover or a gene conversion event. These results support the idea that homologous extrachromosomal DNA fragments are joined through annealing of overlapping single-stranded ends. This DNA-joining phenomenon may represent the activity of cellular DNA repair enzymes; its relationship to genetic recombination occurring at the chromosomal level remains to be determined. PMID- 3023970 TI - Immunologically distinct p53 molecules generated by alternative splicing. AB - Transfection of a functional cloned p53 gene into an L12 p53 nonproducer cell line efficiently reconstituted p53 expression. The p53 protein synthesized in these clones was indistinguishable from that occurring naturally in tumor cells. When a p53 cDNA clone was used instead, we observed that the L12-derived clones exhibited a distinct immunological profile. In the present experiments we compared the immunological epitopes of p53 proteins encoded by several full length cDNA clones. Immunoprecipitation of p53 proteins generated by in vitro transcription and translation of the various cDNA clones indicated variations in the content of immunological epitopes. Basically, two p53 protein species were detected. Both species contained the same antigenic determinants except the PAb421-PAb122 site, which was present in proteins encoded by p53-M11 and pcD-p53, but not in the p53 protein encoded by the p53-M8 cDNA clone. Sequence analysis of the various cDNA clones indicated the existence of a 96-base-pair (bp) insert in clone p53-M8 as compared with clone p53-M11 or pCD-p53. The 96-bp insert contained a termination signal which caused the premature termination of the protein, leading to the generation of a p53 product 9 amino acids shorter than usual. The existence of this insert also accounted for the lack of the PAb421 PAb122 epitope which was mapped to the 3' end of the cDNA clone, following the 96 bp insert. This insert shared complete homology with the p53 intron 10 sequences mapping 96 bp upstream of the 5' acceptor splicing site of p53 exon 11. It was therefore concluded that the different cDNA clones represented p53 mRNA species which were generated by an alternative splicing mechanism. Differential hybridization of the mRNA population of transformed fibroblastic or lymphoid cells with either the 96-bp synthetic oligonucleotide or the p53-M11 cDNA indicated that the various mRNA species are expressed in vivo. PMID- 3023972 TI - Precise assignment of the light-strand promoter of mouse mitochondrial DNA: a functional promoter consists of multiple upstream domains. AB - Using deletion mutagenesis we localized the promoter for the light strand of mouse mitochondrial DNA to a 97-base-pair region, from -88 to +9 nucleotides of the transcriptional initiation site. Within this region the light-strand promoter could be dissected into at least three different functional domains. The specificity region, a maximum of 19 base pairs between -10 and +9 of the transcriptional initiation site, was essential and sufficient for accurate transcriptional initiation. A second region, extending to -29 nucleotides from the initiation site, facilitated the formation of a preinitiation complex between the template DNA and factor(s) present in the mitochondrial RNA polymerase fraction and was required for efficient transcription. A third, ill-defined upstream region, which extended up to -88 nucleotides from the initiation site, appeared to influence template transcriptional efficiencies in competition assays. Without the specificity domain, the upstream regions were incapable of supporting any transcription. The presence of multiple upstream domains was confirmed by disrupting nucleotide sequences in the upstream region by using linker insertion and linker replacement techniques. PMID- 3023973 TI - High-efficiency transformation of Saccharomyces cerevisiae cells by bacterial minicell protoplast fusion. AB - After a new transformation procedure, 10% of Saccharomyces cerevisiae cells were found to contain transforming DNA sequences. We used direct transfer of plasmid molecules by fusing bacterial minicell protoplasts to yeast protoplasts. Since the procedure significantly reduces the toxic effect of procaryotic protoplasm on the eucaryotic organism, it might be generally applicable in other systems in which transformation is inefficient or impossible. PMID- 3023974 TI - Polymyxin-B inhibition of LPS-induced interleukin-1 secretion by human monocytes is dependent upon the LPS origin. AB - Polymyxin-B (PMB) is an antibiotic known to inhibit various biological activities induced by lipopolysaccharides (LPS). We have investigated the ability of PMB to inhibit LPS-induced interleukin-1 (IL-1) secretion by human monocytes in vitro. Interleukin-1 was assayed by the conventional comitogenic assay using mice thymocytes. Our data demonstrate that PMB (1-2 micrograms/assay)-mediated inhibition of LPS-induced IL-1 secretion depends on the origin of the LPS. Interleukin-1 secretions induced by Escherichia coli and Acinetobacter calcoaceticus LPSs, when used at 1 microgram/assay were completely inhibited by PMB, whereas those induced by Neisseria gonorrheae, Neisseria meningitidis, Bordetella pertussis, and Salmonella enteritidis LPSs were unaffected. Neisseria meningitidis, the most potent IL-1 inducer tested could be inhibited by PMB only at concns below 5 ng/assay; when the assay was performed in the presence of serum (0.2%) PMB could not completely inhibit Neisseria meningitidis LPS-induced IL-1 secretion at LPS doses as low as 100 pg/assay. Polymyxin B itself, at doses greater than 50 micrograms/assay, stimulated IL-1 secretion and acted synergistically with LPS to induce IL-1 secretion when used at 10 micrograms/assay. Potential relevance of Lipid A-mediated IL-1 secretion and the use of PMB to detect endotoxin contamination is discussed. PMID- 3023976 TI - Liver cell cancer: insights into the pathogenesis of hepatocellular carcinoma in humans from experimental hepatocarcinogenesis in the rat. PMID- 3023977 TI - [Hepatoblastoma]. PMID- 3023979 TI - [The plasmid containing a fragment of Rous sarcoma virus genome transforms mouse cells and is expressed in Escherichia coli]. PMID- 3023975 TI - Molecular pathophysiology of persistent hepatitis B virus infection in relation to chronic liver disease and primary hepatocellular carcinoma. PMID- 3023978 TI - [Characteristics of the formation and properties of pLS plasmids resulting from deletions of bacteriophage lambda att80 (Tn9) genome]. PMID- 3023980 TI - [Construction and analysis of two-replicon hybrid plasmids containing the genome of simian papovavirus SV40]. PMID- 3023981 TI - [Possible common origin of poliovirus proteins responsible for different functions]. PMID- 3023982 TI - [Mapping the recognition sites for restrictase MluI on DNA from bacteriophage T4]. PMID- 3023983 TI - [The nature of RSI sequences flanking the vct gene encoding the synthesis of cholera toxin in Vibrio cholerae eltor]. PMID- 3023984 TI - [Migration of the IS5-like element in the plasmid R15]. PMID- 3023986 TI - [Transformation of mouse genome using microinjections of the thymidine kinase gene of herpes virus into fertilized ova]. PMID- 3023985 TI - [Cloning of the hly determinant from Pseudomonas aeruginosa strain PA-M7 in Escherichia coli cells]. PMID- 3023987 TI - [Effect of mutation in DNA unwinding protein on the viability, UV- and gamma sensitivity of Escherichia coli K12 mutants defective for DNA-polymerase I and DNA-methylase]. PMID- 3023988 TI - [Cleavage of the repeats in the liver DNA of rats during initial chromatin endonucleolysis]. PMID- 3023989 TI - [Genetic control of iron absorption systems in bacterial cells: mechanisms of the process and its role in pathogenicity]. PMID- 3023990 TI - [Molecular mechanism of phage DNA protection from the restriction endonucleases of Staphylococcus aureus cells]. PMID- 3023991 TI - [Effect of transposon insertions on the system regulating the gene transfer functions of plasmid pAP42]. PMID- 3023992 TI - Mutational alterations induced in yeast by ionizing radiation. AB - The cycl-9 ochre (UAA) mutant and the cycl-179 amber (UAG) mutant of the yeast Saccharomyces cerevisiae were reverted with X-rays and alpha-particles. The amino acid sequence changes of iso-1-cytochromes c from 36 of the intragenic revertants were determined by amino acid analysis and peptide mapping, aided by partial amino acid sequencing of 4 revertants. In addition, the DNA segments encompassing 3 unusual mutations with complex changes were cloned and sequenced. This study and previous studies of 16 other revertants of cycl-9 and cycl-179 revealed that ionizing radiation primarily induces single base-pair substitutions; 47 of the 52 revertants arose by transversions and transitions without any apparent preference. However, the A X T----T X A substitution at the first base pair for the cycl-179 UAG codon, leading to the normal protein, was not detected, nor was it found previously in 32 revertants of cycl-179 obtained spontaneously or induced with various other mutagens; apparently, there is a prohibition of certain base-pair substitutions at certain sites in DNA. In addition, 5 of the 52 revertants arose by multiple changes within a short region of 11 base pairs. These consisted of the deletion of 6 base pairs, the substitution of 3 base pairs, and 3 different kinds of substitutions of two base pairs. Compared to other mutagens previously tested with the cycl system, ionizing radiation produces the most random types of base-pair substitutions. PMID- 3023993 TI - The restriction endonuclease Alu I induces chromosomal aberrations in human peripheral lymphocytes in vitro. AB - The restriction endonuclease Alu I (recognition site AG/CT) produces chromosomal aberrations in isolated human peripheral lymphocytes in vitro. The aberrations are of the chromosome-type when the cells are treated in G1 and of the chromatid type when the cells are treated in late S, early G2. Additional treatment with ammonium sulphate leads to higher aberration frequencies than treatment with Alu I alone. PMID- 3023994 TI - Excessive chromosome fragility and abundance of sister-chromatid exchanges induced by UV in an Indian muntjac cell line defective in postreplication (daughter strand) repair. AB - Two UV-hypersensitive animal cell mutants defective in postreplication recovery (daughter strand synthesis) display quite different patterns of induced sister chromatid exchange (SCE). One, an SV40-transformed Indian muntjac cell (SVM), shows extremely high frequencies of SCE after UV; induced exchanges can be measured after UV doses as low as 0.01 J/m2. This cell also displays exaggerated levels of induced and spontaneous chromosome aberrations. By contrast SCE rates in the Chinese hamster cell mutant, UV-1, are essentially normal. In both SVM and UV-1, however, there is a clear correlation between the cell density and spontaneous frequencies of SCE, a feature which could be related to the observed density-dependent rate of DNA maturation. PMID- 3023995 TI - An SV40-transformed xeroderma pigmentosum group D cell line: establishment, ultraviolet sensitivity, transfection efficiency and plasmid mutation induction. AB - Fibroblasts from a patient with xeroderma pigmentosum complementation group D were treated with Simian virus 40 to establish a transformed cell line suitable for studies of DNA-mediated gene transfer. After progressing through 2 crises, a stable line, XP6Be(SV40), was established and cultured for more than 1 year. This line retains the characteristic xeroderma pigmentosum ultraviolet hypersensitivity and is able to complement a SV40-transformed group A line when fused and assayed for ultraviolet radiation inhibition of colony-forming ability. XP6Be(SV40) expressed high levels of transfected chloramphenicol acetyltransferase activity (0.1 nmole X mg-1 X min-1) in a transient expression assay, showed stable expression of transfected gpt or neo genes (frequency 1-20 X 10(-5)), and permitted replication of the mutagenesis shuttle vector plasmid, pZ189. Ultraviolet treatment (500 J X m-2) of pZ189 prior to replication in XP6Be(SV40) resulted in a large reduction in plasmid yield (5% survival) and a 60 fold increase in the mutation frequency, reflecting the reduced ability of these cells to repair ultraviolet-damaged transfecting DNA. This cell line provides the opportunity to utilize transfection studies in cells with the xeroderma pigmentosum group D defect in excision repair. PMID- 3023997 TI - AAEE case report #12: Common peroneal mononeuropathy at the fibular head. AB - Common peroneal mononeuropathies, usually located at the fibular head, are one of the many causes of foot-drop, a condition often evaluated in the electromyography laboratory. If appropriate nerve conduction studies are performed and particular muscles studied on needle myography, a satisfactory diagnosis can almost always be provided for what may be a perplexing problem clinically. With all peroneal mononeuropathies, the compound muscle action potential amplitude of the peroneal motor tibialis anterior nerve conduction studies, stimulating distal to the fibular head, is a semi-quantitative measure of the number of viable fibers supplying the tibialis anterior and allows for accurate prognostication regarding the foot-drop. PMID- 3023996 TI - Nuclear damage of colon epithelial cells by the food carcinogen 2-amino-3 methylimidazo[4,5-f]quinoline (IQ) is modulated by dietary lipids. AB - Intestinal damage in C57BL/6J female mice was quantified by measuring the frequency of nuclear aberrations in colonic crypts. The animals were maintained on the following diets: standard (5% lipids, 5% cellulose); low- and high cellulose (0-20% cellulose); high lipids (20% maize oil or 20% olive oil). All groups of animals were treated by gavage either with saline or 250 mg/kg of the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). After 24 h their colons were removed and stained and the nuclear aberrations scored under the microscope. The administration of IQ markedly increased the number of colon aberrations in all of the treated animals. Variations in dietary fiber did not modify the colon-damaging activity of this compound. Maize oil slightly increased the colon-damaging activity, whereas significant protection was observed in the animals on a high-lipid olive-oil diet. These results show that composition of the diet may vary the genotoxic effect of this dietary carcinogen. PMID- 3023998 TI - Thalidomide neuropathy: an electrophysiologic study. AB - Thalidomide is effective in the treatment of such disabling dermatologic diseases as aphthosis, discoid lupus erythematosus, and prurigo nodularis, in which other drugs fail. However, its use can induce neuropathy necessitating caution in its administration. It was found in this electrophysiologic study of 13 patients that the data best revealing neuropathy, even when clinical abnormalities were not apparent, were reduction of sensory nerve action potential amplitude on the sural nerve, increase of somatosensory evoked potential latency following sural nerve stimulation, and reduction of sensory action potential amplitude on stimulating the median nerve at the wrist. In two patients, electrophysiologic abnormalities had increased after withdrawal, suggesting a prolonged action of thalidomide. Timely reduction of dosage, after detection of changes indicating the onset of side effects, could reduce the risk of the sometimes rapid emergence of clinical symptoms. PMID- 3023999 TI - Repetitive DNA as a tool for the identification and comparison of nematode variants: application to Trichinella isolates. AB - DNA prepared from four isolates of Trichinella was compared by genomic DNA cross hybridisation, by electrophoresis following restriction endonuclease digestion and by hybridisation studies using a cloned repetitive DNA sequence from T. spiralis. The DNA from T. spiralis, T. nelsoni and T. pseudospiralis isolates was distinct and the interrelationships of these isolates were inferred. In contrast to previous work on T. nativa and T. spiralis, our work suggests that these two isolates are very similar. PMID- 3024000 TI - Homologous telomeric sequences are present in different species of the genus Plasmodium. AB - The telomeric sequence cloned from Plasmodium berghei (see M. Ponzi et al. (1985) EMBO J. 4, 2991-2995) was tested for species specificity. A telomeric and a subtelomeric fragment of the cloned insert served as separate, labelled probes on pulsed field gradient electrophoretical patterns and on genomic digests from the rodent malarias Plasmodium yoelii, Plasmodium chabaudi and from the human malaria Plasmodium falciparum. Results indicate that the subtelomeric fragment, abundantly represented in two chromosomes of P. berghei, is not present in the other DNA tested, while the telomeric fragment is present in every chromosome sized molecule in all the species tested. The telomeric location in the other genomes of the sequences homologous to the P. berghei telomeric probe is confirmed by experiments with Bal 31 exonuclease. In all cases, the TaqI site appears to delimit the common telomeric portion. PMID- 3024001 TI - Cloning and characterization of two Onchocerca volvulus repeated DNA sequences. AB - Two repeated sequences, plasmids pOV8 and pOV26, were cloned and characterized from the filarial parasite Onchocerca volvulus. Both clones can be used to distinguish O. volvulus DNA from other Onchocerca species or other nematodes by restriction fragment length polymorphisms, but neither clone can differentiate between DNA from savanna (Mali) or forest (Ivory Coast) O. volvulus isolates. DNA from one O. volvulus infective larva can be detected by both clones in dot blot hybridization assays. Neither clone cross hybridizes with DNA from host or vector species (human or simuliid, respectively). pOV26 is a member of an interspersed repeated DNA family composed of at least 100 members, and is only observed in the genus Onchocerca. Repeated DNA clone pOV8 cross reacts with DNA from other parasitic filarial nematodes, and is also present in at least 100 copies per O. volvulus genome. pOV26 is a potential tool in the diagnosis of human onchocerciasis, since it is specific for the genus Onchocerca. In the future, we plan to look for regions of these repeated sequences which may serve as a basis for the development of probes to discriminate among Onchocerca species and strains using a simple dot hybridization test. PMID- 3024003 TI - Carbohydrate antigen (CA 19.9) and malignant glucagonoma. PMID- 3024002 TI - Apolipoprotein B-gene DNA polymorphisms associated with myocardial infarction. AB - Levels of apolipoprotein B, the protein component of low-density lipoproteins, correlate with the risk of coronary heart disease. We examined whether genetic variation in apolipoprotein B is associated with myocardial infarction by studying apolipoprotein B-gene restriction-fragment-length polymorphisms in 84 patients with myocardial infarction and an equal number of matched controls. Southern blot analysis with apolipoprotein B-gene probes, performed after DNA was digested with the endonucleases XbaI and EcoRI, revealed alleles that we designated as X1, X2, and X3 and as R1 and R2, respectively. Similar studies with the endonuclease MspI revealed alleles of many different sizes (the difference was due to an insertion-deletion polymorphism), which we grouped as larger and smaller alleles and designated as ID1 and ID2, respectively. The frequencies of the X1, R1, and ID1 alleles were all significantly higher (P less than 0.01) in the cases than in the controls. None of the alleles, however, was significantly associated with variation in levels of low-density lipoprotein cholesterol or apolipoprotein B, and the functional importance of these alleles is therefore uncertain. Nonetheless, in addition to quantitative variation in apolipoprotein B levels in plasma, genetic variation at the apolipoprotein B locus may be a new and independent risk factor for myocardial infarction. PMID- 3024005 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 1-1987. A 58-year-old man with a malignant thymoma and confusion. PMID- 3024004 TI - Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 51-1986. A 44-year-old homosexual man with back pain and an interstitial pulmonary infiltrate. PMID- 3024006 TI - BK viruria and hemorrhagic cystitis. PMID- 3024007 TI - Predictors of the acquired immunodeficiency syndrome developing in a cohort of seropositive homosexual men. AB - In a cohort of 1835 homosexual men who were seropositive for human immunodeficiency virus (HIV) on entry into a prospective study, the acquired immunodeficiency syndrome (AIDS) developed in 59 during a median follow-up of 15 months. We matched 5 seropositive controls to each case according to study center and date of enrollment and performed a case-control analysis to determine factors predictive of AIDS. In a multivariate analysis, a decreased number of T helper lymphocytes, an increased number of T suppressor lymphocytes, a low level of antibody to HIV, a high titer of cytomegalovirus antibody, and a history of sex with someone in whom AIDS developed were independently associated with subsequent AIDS. Separate analyses of risk factors for Kaposi's sarcoma and opportunistic infections failed to support previously reported associations between the use of nitrites or an elevated cytomegalovirus-antibody titer and Kaposi's sarcoma. These variables may be markers rather than determinants of disease progression. A vigorous antibody response to HIV infection may confer at least temporary protection against the progression of immunodeficiency to AIDS, or a low level of antibody to HIV may reflect a later stage of infection. The increased risk associated with a history of sex with someone in whom AIDS developed may indicate earlier infection in cases or infection with a more virulent strain of HIV. These results may be useful in counseling HIV-seropositive persons and in designing studies of clinical interventions. PMID- 3024008 TI - A chimaeric receptor allows insulin to stimulate tyrosine kinase activity of epidermal growth factor receptor. AB - The cell surface receptors for insulin and epidermal growth factor (EGF) appear to share a common evolutionary origin, as suggested by structural similarity of cysteine-rich regions in their extracellular domains and a highly conserved tyrosine-specific protein kinase domain. Only minor similarity is found outside this catalytic domain, as expected for receptors that have different ligand specificities and generate different biological signals. The EGF receptor is a single polypeptide chain but the insulin receptor consists of distinct alpha and beta subunits that function as an alpha 2 beta 2 heterotetrameric receptor complex. Provoked by this major structural difference in two receptors that carry out parallel functions, we have designed a chimaeric receptor molecule comprising the extracellular portion of the insulin receptor joined to the transmembrane and intracellular domains of the EGF receptor to investigate whether one ligand will activate the tyrosine kinase domain of the receptor for the other ligand. We show here that the EGF receptor kinase domain of the chimaeric protein, expressed transiently in simian cells, is activated by insulin binding. This strongly suggests that insulin and EGF receptors employ closely related or identical mechanisms for signal transduction across the plasma membrane. PMID- 3024011 TI - Problems with AIDS vaccines. PMID- 3024009 TI - Cloning of complementary DNA encoding T-cell replacing factor and identity with B cell growth factor II. AB - Proliferation and maturation of antigen-stimulated B cells are regulated by several soluble factors derived from macrophages and T cells. These soluble factors are functionally divided into two groups: B-cell growth factor (BCGF), thought to be involved in B-cell proliferation; and B-cell differentiation factor (BCDF), responsible for maturation of activated B cells into immunoglobulin secreting cells. This classification needs to be re-examined in the light of the recent cloning of complementary DNA encoding IgG1 induction factor (interleukin 4, IL-4) from the 2.19 mouse T-cell line. Recombinant IL-4 has BCGF and BCDF activities and affects B cells, T cells and mast cells (refs 7, 8; our unpublished data). Another well-characterized B-cell factor is T-cell replacing factor (TRF), which, when secreted by the murine T-cell hybridoma B151K12, is defined by two activities: induction of IgM secretion by BCL1 leukaemic B-cell line; and induction of secondary anti-dinitrophenol (DNP) immunoglobulin G (IgG) synthesis in vitro by DNP-prime B cells. Although TRF from B151K12 was classified as BCDF, purified TRF has BCGF-II activity. To elucidate the molecular properties of TRF we isolated cDNA encoding TRF from the 2.19 T-cell line and report here the structure and multiple activities of this lymphokine. PMID- 3024010 TI - Rearrangement and enhanced expression of c-myc in hepatocellular carcinoma of hepatitis virus infected woodchucks. AB - Hepatocellularcarcinoma (HCC) that occur in woodchucks chronically infected with woodchuck hepatitis virus (WHV) were screened for activation of cellular oncogenes. Enhanced expression and allelic alterations of the c-myc oncogene were found in three HCC out of nine. Variations in the size of the c-myc transcripts, ranging from 2.0 kilobases (kb) to 5.6 kb, as well as in the level of c-myc gene expression, 5-50-fold higher than in adjacent liver tissues, were observed among the three HCC. Rearrangements of the c-myc locus were either upstream of the gene or within the first intron. Cloning and sequencing of the break-point region from one of the three tumours showed that the c-myc gene was truncated and joined to a unique cellular sequence of unknown function. WHV DNA was not integrated near the c-myc coding exons, excluding a direct role of the virus in c-myc activation. The novel type of rearrangement and activation of the c-myc gene, reported here in liver tumours of hepatitis virus infected animals, appears strikingly similar to those resulting from chromosomal translocations in human Burkitt's lymphomas, acute B- and T-cell leukaemias and mouse plasmacytomas. PMID- 3024012 TI - A complementary DNA clone for a macrophage-lymphocyte Fc receptor. AB - Macrophages, granulocytes and many lymphocytes express or secrete receptors for the Fc domain of immunoglobulins (Ig). These Fc receptors (FcRs) are heterogeneous and can be distinguished on the basis of their cellular distribution and specificities for different immunoglobulin isotypes. Although their functions are not completely understood, FcRs are known to be involved in triggering various effector cell functions and in regulating differentiation and development of B-cells. One of the best characterized is the mouse macrophage lymphocyte receptor for IgG1 and IgG2b (ref. 5). On macrophages, this FcR mediates the endocytosis of antibody-antigen complexes via coated pits and coated vesicles, the phagocytosis of Ig-coated particles, and the release of various inflammatory and cytotoxic agents. It is possible that the receptor possesses an intrinsic ligand-activated ion channel activity responsible for some of these functions. The IgG1/IgG2b FcR has been isolated and shown to be a transmembrane glycoprotein of relative molecular mass (Mr) 47,000-60,000 (47-60 K) containing four N-linked oligosaccharide chains and a large (greater than 10K) cytoplasmic domain. It is also immunologically indistinguishable from the murine Ly-17 alloantigen which, in turn, is tightly linked to the Mls lymphocyte activation locus. Here we describe the isolation and characterisation of a complementary DNA clone encoding the whole of the IgG1/IgG2b FcR expressed by the mouse macrophage like cell line P388D1. The receptor is a member of the immunoglobulin superfamily and, like Ly-17, maps to the distal portion of chromosome 1. cDNA probes detect one or two mRNA species in FcR+ macrophage and B-cell lines, but not in FcR- cells or a receptor-deficient variant derived from a FcR+ B-cell line. Finally, DNA hybridization analysis indicates the receptor gene is partially deleted or rearranged in the FcR- variant. PMID- 3024014 TI - Cyclic GMP is involved in the excitation of invertebrate photoreceptors. AB - The hyperpolarizing receptor potential in vertebrate rod photoreceptors appears to be mediated by the second messenger, cyclic GMP. Injection of cGMP into rods or application of cGMP to inside-out membrane patches activates a conductance resembling that produced by light. Light produces a rapid reduction of cGMP in living rods, leading to closure of sodium channels and membrane hyperpolarization. In most invertebrate photoreceptors the response to light is depolarizing. We have investigated whether cGMP is involved in controlling the increase in sodium conductance that underlies this depolarization. We show here that injection of cGMP into Limulus photoreceptors produces a depolarization that mimics the receptor potential. We also show that the cGMP concentration of the squid retina increases rapidly during exposure to light. These results support the hypothesis that cGMP mediates the light-induced depolarization in invertebrate photoreceptors and suggests that vertebrate and invertebrate phototransduction may be more similar than previously thought. PMID- 3024013 TI - The v-fms oncogene induces factor independence and tumorigenicity in CSF-1 dependent macrophage cell line. AB - The McDonough strain of feline sarcoma virus (SM-FeSV) transforms fibroblast cell lines in culture and produces fibrosarcomas in domestic cats. SM-FeSV does not induce haematopoietic malignancies in spite of the fact that its viral oncogene, v-fms, codes for a glycoprotein related to the receptor for the mononuclear phagocyte colony stimulating factor, CSF-1. The v-fms-coded polypeptide includes the complete extracellular domain of the c-fms proto-oncogene product and retains the ability to bind CSF-1 specifically. The two molecules have very similar sequences except at their extreme carboxyl terminal ends where 40 amino acids of the c-fms-coded glycoprotein are replaced by 11 unrelated residues in the v-fms product. Autophosphorylation of the c-fms gene product on tyrosine is enhanced by CSF-1 addition, whereas phosphorylation of the v-fms-coded glycoprotein appears to be constitutive. We now show that introduction of the v-fms gene into simian virus-40 (SV40)-immortalized, CSF-1 dependent macrophages renders them independent of CSF-1 for growth and tumourigenic in nude mice. These factor independent cell lines express unaltered levels of the c-fms product which is down-modulated in response to either CSF-1 or the tumour promoter 12-O tetradecanoyl-phorbol-13-acetate (TPA). The induction of factor independence by a non-autocrine mechanism suggests that the v-fms product is an unregulated kinase that provides growth stimulatory signals in the absence of ligand. PMID- 3024015 TI - Mature murine B lymphocytes immortalized by Kirsten sarcoma virus. AB - Clonal, antigen-specific, functionally responsive cell populations have proved critical for the analysis of the activation and regulation of lymphocytes. Such studies with B lymphocytes, the precursors of antibody-secreting cells, are hampered by the difficulty in generating phenotypically mature, antigen-reactive lines from defined cell populations. One method is to use acutely transforming retroviruses, which can transform B-lineage lymphocytes in vitro. However, Abelson murine leukaemia virus (A-MuLV) infection of murine bone marrow cells in vitro yields mostly immature B-cell lines, and infection of murine bone marrow cells with murine sarcoma viruses carrying ras related genes produces only immature lymphoid cell lines. Retroviruses which contain ras can immortalize nonlymphoid cells without causing loss of mature phenotypic characteristics. We used ras-containing Kirsten sarcoma virus (KiSV) pseudotyped with an amphotropic MuLV helper virus, to infect a purified population of mature, hapten-binding murine splenic B lymphocytes, aiming to generate mature B-cell lines to use as models for the study of B-cell growth and differentiation physiology. Immortalized B-cell lines which retain the same mature phenotype as the starting population, including hapten-specific binding, were produced. This is the first demonstration of a method for immortalizing selected antigen-binding B lymphocytes, and the first example of immortalization of mature B cells in vitro with an acutely transforming ras-containing retrovirus. PMID- 3024016 TI - AIDS research queried. Skulduggery at the lab bench. PMID- 3024017 TI - Lambda 5, a new light-chain-related locus selectively expressed in pre-B lymphocytes. AB - The development from stem cells to pre-B cells, B lymphocytes and, finally, plasma cells and memory cells proceeds through various stages which have been defined by the genomic context in which immunoglobulin (Ig) heavy (H) and light (L) chain gene segments are found, as well as by their state of expression. They have also been identified by surface marker analysis and susceptibility to various stimuli regulating growth and differentiation. We have searched for genes that are expressed at given stages in the B-lymphocyte development pathway and which might function to control this development at various stages. A complementary DNA sequence called pZ183 was found in a library constructed from messenger RNA of the murine pre-B lymphoma cell line 70Z/3 which is selectively expressed in pre-B cells. Here we report the nucleotide sequence of a cDNA clone (pZ183-1) containing 0.7 kilobases (kb) of the pZ183 gene. Part of this sequence shows strong homology to constant (C) and joining (J) region sequences of lambda 1 L chains. Our findings define a new immunoglobulin L-chain-related locus, which we call lambda 5, that is selectively transcribed in pre-B lymphocytes. PMID- 3024018 TI - Long-range restriction map around the Duchenne muscular dystrophy gene. AB - Duchenne muscular dystrophy is an X-linked recessive disease affecting about 1 in 4,000 newborn boys. As in many other inherited diseases, the biochemical basis of the condition is unknown, and as yet there is no effective treatment. Translocations, deletions and other mutations leading to the DMD phenotype are distributed over a chromosomal area of large, but unknown size. Using pulsed field gradient gel electrophoresis, we have now determined restriction maps of a major fraction of this area, covering two regions of three million basepairs in total, and used it to determine the position of several probes linked to DMD. The maps establish physical distances between structural changes associated with the DMD phenotype and provide evidence for a CpG-rich island proximal to the area containing translocations and deletions associated with the DMD phenotype. PMID- 3024019 TI - Effect of D2O on the transport of ascorbate across membranes of DPPC vesicles. PMID- 3024020 TI - Raising the ambient potassium ion concentration enhances carbachol stimulated phosphoinositide hydrolysis in rat brain hippocampal and cerebral cortical miniprisms. AB - The influence of the ambient potassium ion concentration ([K+]) upon agonist stimulated hydrolysis of phosphoinositides (PI) has been studied in isolated miniprisms of rat hippocampus and cerebral cortex. When the external [K+] was raised from 6 to 18 mmol/l, there was little or no increase in the hydrolysis of PI in the absence of agonist, however, carbachol (100 mumol/l) stimulated hydrolysis was greatly enhanced in both brain regions studied. Thus, carbachol stimulated the hydrolysis of PI to 146% and 386% of control levels at potassium concentrations of 5.88 and 18.2 mmol/l, respectively, in the rat hippocampus. A similar enhancement of muscarine (100 mumol/l) stimulation was observed in cortical miniprisms with 18 mmol/l [K+]. A further enhancement was seen at higher ambient [K+], although basal hydrolysis of PI was then also increased. The carbachol-stimulated hydrolysis of PI found at both 6 and raised [K+] was prevented by atropine (1 and 10 mumol/l) and tetraethylammonium (20 mmol/l), but not by 10 mmol/l Mg2+. Pirenzepine (50 nmol/l) also reduced this response. The ions Cs+ and Rb+ (but not Li+ or Tris+) produced a similar enhancement of the carbachol stimulation to that found with K+. At a buffer [K+] of 6 mmol/l, noradrenaline (100 mumol/l) produced a 2-fold increase in the hydrolysis of PI whereas 5-hydroxytryptamine (100 mumol/l) and histamine (500 mumol/l) had little or no effect. However, histamine and 5-hydroxytryptamine did stimulate the hydrolysis of PI when [K+] was increased. Miniprism ATP content was not changed by a rise in [K+] to 18 mmol/l. The significance of these results is discussed in terms of the postsynaptic cellular events following cholinergic stimulation. PMID- 3024022 TI - Characterization of the O-methylation of catechol oestrogens by intact rabbit thoracic aorta and subcellular fractions thereof. AB - In the present study we investigated the O-methylation of catechol oestrogens by intact rabbit thoracic aorta and subcellular fractions thereof. The O-methylation of 2-hydroxyoestradiol (2OHE2) and 2-hydroxyoestriol (2OHE3) displayed saturation kinetics in the intact tissue. The apparent Km and Vmax values for the O methylation of 2OHE2 were determined to be 0.91 mumol/l and 104 pmol g-1 min-1, respectively, when 2OHE2 was used as substrate; and 1.14 mumol/l and 188 pmol g-1 min-1 when 2OHE3 was used as substrate. The inhibitors of the extraneuronal uptake process (viz; phenoxybenzamine 33 mumol/l; normetanephrine, 46 mumol/l; and deoxycorticosterone acetate 27 mumol/l) failed to inhibit the O-methylation of either 2OHE2 (3.4 mumol/l) or 2OHE3 (3.4 mumol/l) in intact segments of the rabbit thoracic aorta. (-)-Isoprenaline (40 mumol/l) abolished the O-methylation of 2OHE2 (3.4 mumol/l) and markedly reduced that of 2OHE3 (3.4 mumol/l). Pretreatment of tissues with phenoxybenzamine (33 mumol/l) partially restored the O-methylation of 2OHE2 and 2OHE3 in the presence of (-)-isoprenaline (40 mumol/l). The O-methylation of 2OHE2 (5 mumol/l) was significantly reduced in segments of aorta in which the endothelium was removed. The latter reduction could not be attributed to damage to components of the vessel media. The O methylation of 2OHE2 and (-)-isoprenaline by subcellular fractions of the rabbit aorta also was examined. Both the microsomal and cytosolic fractions were shown to O-methylate 2OHE2 and (-)-isoprenaline, providing evidence for the existence of membrane-bound and soluble forms of COMT in the rabbit aorta. The O methylation of 2OHE2 by cytosolic and microsomal fractions of the aorta was determined and compared to that of (-)-isoprenaline. The kinetic constants for the O-methylation of 2OHE2 by cytosolic (Km: 0.27 mumol/l; V max: 112 pmol g-1 min-1) and microsomal (Km: 0.15 mumol/l; Vmax: 161 pmol g-1 min-1) fractions were similar. In contrast, the kinetic constants for the O-methylation of isoprenaline by cytosolic (Km: 121 mumol/l; Vmax: 174 pmol g-1 min-1) and membranal (Km: 0.91 mumol/l; Vmax: 105 pmol g-1 min-1) fractions were very different. It is concluded that catechol oestrogens are excellent substrates for catechol-O methyltransferase (COMT) in the rabbit aorta. Their O-methylation can occur in endothelial structures as well as in the smooth muscle-containing medial sections of the vessel.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3024023 TI - Cardiac ventricular beta 2-adrenoceptors in guinea-pigs and rats are localized on the coronary endothelium. AB - In mammalian heart tissue beta 2-adrenoceptors are known to coexist with beta 1 adrenoceptors. In the present study, evidence that beta 2-adrenoceptors in guinea pig and rat ventricles are primarily localized on the coronary endothelium is provided by competition binding studies with the subtype-selective beta adrenoceptor antagonists ICI 89.406 (beta 1-selective) and ICI 118.551 (beta 2 selective) on four different plasma membrane preparations. (1) Following density gradient centrifugation of cardiac ventricular microsomes from rats or guinea pigs, endothelial plasma membranes migrated at slightly higher density than the sarcolemmal membranes, as verified by endothelial (angiotensin converting enzyme) and sarcolemmal markers (adenylate cyclase, [3H]ouabain binding). At the activity peak of angiotensin converting enzyme, the relative amount of beta 2 adrenoceptors in guinea-pigs and rats was 25% and 65%, respectively. (2) On sarcolemmal membranes corresponding to the activity peak of adenylate cyclase, beta-adrenoceptors consisted of the beta 1-type exclusively (guinea-pig), or to at least 90% (rat). (3) Cultures of coronary endothelial cells derived from guinea-pigs revealed only beta 2-adrenoceptors. (4) Isolated guinea-pig cardiomyocytes contained only beta 1-adrenoceptors, a finding recently established in rat myocytes as well. PMID- 3024021 TI - Proabsorptive properties of forskolin: disposition of glycine, leucine and lysine in rat jejunum. AB - The effects of forskolin on mucosal cyclic AMP levels and active transport of glycine, L-lysine and L-leucine were studied in rat jejunum in vitro. Furthermore, the effects on lysine and glycine incorporation into mucosal protein and on mucosal cell volume were investigated. Elevation of intestinal mucosal cyclic AMP to threefold control levels by 10 mumol 1(-1) forskolin was accompanied by increased absorption of glycine (+33%), L-leucine (+72%) and L lysine (+188%), as determined in a three compartment model suitable to measure active transport. Increased intracellular accumulation could be demonstrated for lysine as a transport substrate. Accordingly, using a dual label method, calculated values for uphill transport of lysine at the site of the brush border membrane were markedly enhanced. Forskolin up to 10 mumol 1(-1) had no effects on the fraction of lysine or glycine incorporated into TCA-precipitable proteins of jejunal absorptive cells. Serosal to mucosal transfer, as well as basolateral entry into mucosal cells remained unchanged for all three amino acids. Likewise, intracellular fluid space, calculated from distribution spaces for 14C-inulin and 3H2O as well as the response of cellular volume to an osmotic gradient were not affected by forskolin. As comparable stimulatory effects of forskolin on active hexose transport were reported earlier, it is suggested that forskolin - known to inhibit sodium-coupled fluid absorption - may stimulate active transport by enhancing sodium availability for sodium dependent intestinal cotransporters in general. PMID- 3024024 TI - Effect of endothelium on basal and alpha-adrenoceptor stimulated calcium fluxes in rat aorta. AB - The rate of unstimulated influx of Ca2+ into rat aorta smooth muscle, measured as uptake of 45Ca, was inhibited in the presence of endothelium as compared to influx in the absence of endothelium. Efflux of 45Ca from unstimulated prelabelled tissues was also reduced in the presence of endothelium. In normal physiological solution the rate of influx and efflux of Ca2+ stimulated by B-HT 920 (1 and 10 microM), but not that stimulated by phenylephrine (30 nM and 1 microM), was also reduced in the presence of endothelium. In the presence of the calcium entry blocker flunarizine (3 microM), phenylephrine (1 microM) stimulated efflux of Ca2+ was inhibited by the presence of endothelium. A correlation between inhibition of Ca2+ influx and modulation of alpha-adrenoceptor agonist induced contractions by endothelium could not be demonstrated, and methylene blue, an antagonist of endothelium mediated inhibition of B-HT 920 contractions, did not affect Ca2+ influx stimulated by the agonist. The effects of endothelium on Ca2+ influx and efflux are unlikely to be due to alterations by endothelium of diffusion of 45Ca or the agonists in the vessel. The results demonstrate that an endothelial derived factor or factors can reduce calcium influx into smooth muscle cells and also modulate the release of calcium from cells, perhaps by affecting intracellular calcium pumping mechanisms. A reduction of calcium influx cannot be the sole explanation for the modulatory effect of endothelium on alpha adrenoceptor agonist-induced contractions but an effect on intracellular calcium metabolism may be important. PMID- 3024025 TI - Capsaicin sensitive afferent neurons from peripheral glucose receptors mediate the insulin-induced increase in adrenaline secretion. AB - The effect of insulin and of 2-deoxy-D-glucose (2-DG) on adrenaline secretion was compared in rats pretreated as neonates with capsaicin and in rats pretreated with the drug-vehicle. Capsaicin-pretreatment did not inhibit the fall in blood glucose concentrations induced by insulin or by fasting, nor did it affect the increase in blood glucose concentrations in response to 2-DG or restraint stress. Capsaicin greatly reduced the rise in urinary adrenaline excretion over 24 h and the fall in the adrenaline content of the adrenal glands normally induced by insulin. In contrast, capsaicin-pretreatment did not interfere with the rise in the adrenaline excretion and the fall in the adrenaline content of the adrenal glands normally induced by 2-DG. Insulin-induced hypoglycaemia as well as intracellular glucopenia in the brain caused by 2-DG activate hypothalamic centres which stimulate the nervous input to the adrenal medulla and adrenaline secretion. The fact that capsaicin interfered only with the adrenal effect of insulin suggests the involvement of afferent C-fibres in this insulin effect. Injection into the hepatic portal vein of a C-fibre stimulating dose of capsaicin increased arterial glucose concentrations in vehicle-pretreated rats but not in capsaicin-pretreated rats. The response was significantly diminished after bilateral vagotomy. From the present results it is concluded that glucose receptors in the hepatic portal vein transmit signals via afferent, capsaicin sensitive C-fibres to the brain and that activation of this pathway is essential for the increase in adrenaline secretion elicited by insulin-induced hypoglycaemia. PMID- 3024026 TI - Adenosine analogs mediating depressant effects on synaptic transmission in rat hippocampus: structure-activity relationships for the N6 subregion. AB - Previous studies have shown that analogs of adenosine with substituents upon the N6-nitrogen (e.g., N6-[1-phenyl-2(R)-propyl]adenosine; R-PIA) are often much more potent than the parent compound in activating adenosine receptors, particularly those of the A1 subtype. The present investigation characterized the potencies of a number of N6-substituted adenosine analogs in depressing excitatory synaptic transmission in slices of rat hippocampus, an electrophysiological response mediated by receptors of the A1 subtype. These potencies correlated well with previously reported affinities of these analogs for A1 receptor sites in brain, but not with coronary vasodilation in the dog heart, an A2 receptor mediated response. Analogs with alkyl or aryl substituents at the N6 position were generally more potent than adenosine, although analogs with a tertiary carbon attached directly to the N6-nitrogen were usually only weakly active. Although it has been suggested that there may be a subregion of the A1 receptor with some specificity for aryl groups, these experiments did not suggest that this was the case. Analogs with chiral centers attached to the N6-nitrogen usually displayed stereoselectivity, with R-isomers more potent than the S-isomers. The mechanism underlying this selectivity appeared to be both a facilitating effect of alkyl substituents in the propyl C1 position of R-PIA, and a hindering effect of substituents in the position normally occupied by the hydrogen attached to propyl C2 of R-PIA. These results indicate that although there are some similarities in terms of requirements for activity at A1 and A2 receptors, differences between the N6 sub-regions of these receptors are sufficient to permit the development of selective analogs for these two receptor sites. PMID- 3024027 TI - Qualitative differences in the effects of adenosine analogs on the cholinergic systems of rat striatum and hippocampus. AB - The effect of the purinergic agonist, 2-chloroadenosine (2-CADO), on central cholinergic parameters was studied in the rat. The drug (20 micrograms, i.c.v.) increased acetylcholine (ACh) content (approximately 30%) and inhibited sodium dependent high affinity choline uptake (30%) in the hippocampus. In striatum, the increase of ACh content was less marked (approximately 15%) and was not associated with inhibition of choline uptake. In both areas, ACh accumulation was prevented by theophylline but not by atropine or oxotremorine pretreatments. Differences were noted in the purinergic control of cholinergic function in the hippocampus and striatum. In hippocampus, the selective degeneration of noradrenergic, serotonergic and glutamatergic afferent pathways or the destructions of intrinsic neurons did not prevent the rise in ACh content induced by 2-CADO. Differently, in striatum, the action of 2-CADO was potentiated both by raphe deafferentation and by inhibition of serotonin synthesis and was completely prevented by chronic unilateral decortication. The cholinergic effect of 2-CADO was unchanged after impairment of the noradrenergic or dopaminergic systems. In addition, the D- and L-isomers of phenylisopropyladenosine, which have different affinities for A1 purinergic receptors but equal affinity for the A2 purinergic subtype, differed in their ability to affect acetylcholine content in these two brain regions, suggesting that A1 purinergic receptor activation mediates the effect of 2-CADO in the hippocampus and A2 receptor activation mediates the drug's action in the striatum. PMID- 3024028 TI - Diazepam attenuates the antagonism of haloperidol against apomorphine-induced stereotypic behavior after subchronic but not acute treatment in rats. AB - Apomorphine-induced stereotypic behavior was investigated in rats treated with diazepam or haloperidol and with the combination of both drugs in a one day trial or subchronically. The drugs were administered via the drinking water. Diazepam dose-dependently reduced apomorphine stereotypies after the subchronic (6 days) but not after the acute treatment. Haloperidol suppressed apomorphine-induced stereotypic behavior dose-dependently after acute as well as after subchronic administration apparently without the development of tolerance. This discrepancy to other studies may be explained by the concomitant increase in maximum number of D2-receptors in the striatum. The apomorphine antagonistic effect of haloperidol was attenuated when the neuroleptic was administered subchronically in combination with the benzodiazepine. This finding was unexpected since both drugs reduced apomorphine-induced stereotypic behavior when administered alone. The further increase in maximum number of D2-receptors due to combined treatment with low doses of diazepam, suggesting a sort of "over adaptation", possibly explains the haloperidol-antagonistic action of diazepam in the behavioral experiments. Binding studies on dopamine (D1), 5-hydroxytryptamine (5-HT2) and benzodiazepine receptors revealed that modification of the apomorphine-induced stereotypies by the combined treatment with haloperidol and diazepam cannot be explained by interactions of the drugs at the level of the D1, 5-HT2 or benzodiazepine-receptors. PMID- 3024029 TI - A unique pressor response to isoprenaline in the pithed rat during triiodo-L thyronine(T3)-induced hyperthyroidism. AB - Thyroid hormone appears to be involved in the regulation of beta-adrenoceptors affecting cardiovascular performance. In the present study, the influence of hyperthyroidism on beta-adrenoceptor-mediated response of the cardiovascular system was investigated in vivo using the pithed rat preparation. Hyperthyroidism was induced by triiodothyronine injections (500 micrograms/kg, i.p.) for 6 days. A markedly accelerated basal heart rate and a wider pulse pressure with a significantly elevated systolic blood pressure were observed in hyperthyroid pithed rats. Although the basal and the maximal heart rates were increased in hyperthyroid rats, EC50 of the heart rate response to isoprenaline did not significantly differ between euthyroid and hyperthyroid pithed animals. Markedly different responses of blood pressure to isoprenaline were obtained in the two groups; isoprenaline caused a dose-dependent decrease in diastolic pressure in euthyroid pithed rats, whereas it produced pressor response in hyperthyroid pithed rats. This unique pressor response to isoprenaline observed in hyperthyroid pithed rats was abolished by the beta 1-adrenoceptor selective antagonist metoprolol but not by the alpha-adrenoceptor antagonist phenoxybenzamine. The density of myocardial binding sites of the beta-type was markedly increased after T3 treatment (65%), whereas that of the mesenteric artery was not altered. The results indicate that thyroid hormone exerts different effects on cardiac and vascular beta-adrenoceptors, and this different susceptibility to thyroid hormone may in part be responsible for the altered response of blood pressure to isoprenaline seen in hyperthyroid pithed rats. PMID- 3024030 TI - Phenylethylamine-induced release of noradrenaline fails to stimulate alpha 1 adrenoceptors modulating [3H]-acetylcholine release in rat atria, but activates alpha 2-adrenoceptors modulating [3H]-serotonin release in the hippocampus. AB - The modulation of the depolarization induced release of [3H]-acetylcholine by agonists acting on alpha-adrenoceptors was studied in superfused rat atrial slices. In this model, noradrenaline and methoxamine, but not UK 14304 reduced the potassium evoked release of [3H]-acetylcholine. The inhibitory action of these drugs was antagonized by the alpha 1 selective adrenoceptor antagonist prazosin. Propranolol, idazoxan and sulpiride did not antagonize the inhibition by noradrenaline of the potassium-evoked release of [3H]-acetylcholine. Exposure to amphetamine, beta-phenylethylamine, m- or p-tyramine, increased in a concentration-dependent manner the spontaneous outflow of [3H]-noradrenaline from atrial slices. Yet, these concentrations of the indirectly acting sympathomimetic amines, tested in the presence of an inhibitor of monoamine oxidase (MAO), failed to modify the potassium evoked release of [3H]-acetylcholine. Desipramine 3 mumol/l or cocaine 10 mumol/l did not affect the release of [3H]-acetylcholine evoked by potassium stimulation. Under similar experimental conditions, beta phenylethylamine facilitated the spontaneous outflow of [3H]-noradrenaline, and inhibited the electrically-evoked release of [3H]-serotonin from the hippocampus by activation of alpha 2-adrenoceptors. It is concluded that the release of acetylcholine from atrial cholinergic neurons can be modulated through inhibitory alpha 1-adrenoceptors, which are not activated when the release of noradrenaline is induced by indirectly acting sympathomimetic amines. In addition, amphetamine or structurally related amines do not activate directly recognition sites in the cholinergic postganglionic parasympathetic neuron to modify the release of [3H] acetylcholine. PMID- 3024031 TI - Subendothelial beta 2-adrenoceptors in the rat vena cava: facilitation of noradrenaline release via local stimulation of angiotensin II synthesis. AB - Preparations of the cranial segment of the rat inferior vena cava preincubated with 3H-noradrenaline were superfused in the presence of desipramine and corticosterone. Tritium overflow was stimulated electrically (2 Hz). The experiments were carried out in spirally cut strips with or without intima or in segments ligated at both ends and superfused either on the adventitial side ("conventionally") or "inside out". In spirally cut strips electrically evoked 3H overflow was increased by isoprenaline and procaterol, but much less so by prenalterol. Adrenaline 1 nmol/l increased overflow, but at high concentrations it reduced it, just as noradrenaline did at all concentrations. The concentration response curve for isoprenaline was shifted to the right by propranolol (apparent pA2:8.29) and even more so by ICI 118-551, whereas atenolol was less potent (apparent pA2:6.42). Rauwolscine which, given alone, increased the evoked 3H overflow antagonized the inhibitory effect of noradrenaline (apparent pA2:7.58). These findings indicate that beta 2- and alpha 2-adrenoceptors mediating facilitation and inhibition of noradrenaline release, respectively, are present in the vena cava. The response to isoprenaline (at all concentrations) was considerably lower in segments superfused "conventionally" than in spirally cut strips, but no difference was observed with respect to the effects of noradrenaline, rauwolscine and angiotensin II. The effect of isoprenaline was clearly more pronounced in segments superfused "inside out" than in segments superfused "conventionally". In spirally cut strips angiotensin II increased 3H overflow. This effect was antagonized by saralasin, suggesting the involvement of facilitatory angiotensin receptors. In spirally cut strips or segments superfused "inside out", saralasin or captopril considerably attenuated the facilitatory effect of isoprenaline on 3H overflow. Conversely, in the presence of isoprenaline, captopril inhibited the electrically evoked 3H overflow in spirally cut strips, whereas in the absence of isoprenaline, captopril was ineffective. In conclusion, angiotensin receptors and alpha 2-adrenoceptors appear to be located on the sympathetic nerve endings, but a major part of the beta 2-adrenoceptors probably is subendothelial (most likely on smooth muscle cells). Angiotensin II, synthesized in response to beta 2-adrenoceptor activation, probably stimulates angiotensin receptors on the noradrenergic nerves, leading to an increase in noradrenaline release. PMID- 3024032 TI - Mutual interaction between presynaptic alpha 2-adrenoceptors and opioid kappa receptors at the noradrenergic axons of rabbit brain cortex. AB - The interaction between presynaptic, release-inhibiting alpha 2-adrenoceptors and opioid receptors was studied in slices of the parieto-occipital cortex of rabbits. The slices were preincubated with 3H-noradrenaline and then superfused with 3H-noradrenaline-free medium and stimulated electrically (3 or 7 Hz, 2 or 5 V/cm voltage drop between the electrodes). Clonidine and ethylketocyclazocine (EK) depressed, whereas yohimbine increased the electrically evoked overflow of tritium. When clonidine was administered first and retained in the medium for the rest of the experiment, the overflow-inhibiting effect of EK was reduced. When yohimbine was administered first and kept for the rest of the experiment, the effect of EK was enhanced. When, finally, EK was administered first and clonidine as the second drug, the overflow-inhibiting effect of clonidine was attenuated. The changes in the effect of EK (by clonidine or yohimbine) and clonidine (by EK) were not due to the changes in release per se produced by the drugs that were given first. Naloxone shifted the concentration-response curve of EK to the right; the dissociation constant of the naloxone-receptor complex, calculated from the shift, was 13 nmol/l. It is concluded that there is an interaction between presynaptic alpha 2-adrenoceptors and opioid kappa-receptors, either at the level of the receptors themselves or of the post-receptor reaction chains. Activation of one kind of receptor blunts the inhibition of release produced by activation of the other kind of receptor. PMID- 3024035 TI - [Chronic lymphatic leukemia with inclusion bodies, hypercalcemia and kidney infiltration]. PMID- 3024033 TI - The positive inotropic response to milrinone in isolated human and guinea pig myocardium. AB - The bipyridine derivative, milrinone, produced positive inotropic effects in isolated, contracting right ventricular papillary muscles and left atria from guinea pigs as well as in human papillary muscle strips. The inotropic effect was biphasic in guinea pig papillary muscles (EC50, high affinity, 1.5 X 10(-6) mol/l, about 35% of maximal effect; apparent EC50, 3 X 10(-5) mol/l with a maximal effect at 2 X 10(-4) mol/l) but monophasic in guinea pig left atria (EC50, 6 X 10(-5) mol/l) and in human papillary muscle strips (EC50, 5.8 X 10(-5) mol/l). In guinea pig papillary muscles, reserpine pretreatment or l-practolol preincubation reduced the low concentration effect only. In the presence of l practolol, carbachol reduced the low concentration effect only. In the presence of l-practolol, carbachol reduced but not abolished the inotropic effects of milrinone (3 X 10(-6) mol/l, 1 X 10(-4) mol/l) in both guinea pig and human myocardium. This antagonism was prevented by atropine preincubation. The maximum inotropic effect of milrinone was similar to that of ouabain and calcium in guinea pig myocardium but markedly less than either calcium or ouabain in human myocardium. Milrinone inhibited crude guinea pig and human cardiac phosphodiesterase activity in vitro but did not inhibit 3H-ouabain binding to partially purified human cardiac (Na+ + K+)-ATPase-containing membranes. We conclude that the primary mode of action of milrinone in both guinea pig and human myocardium is through inhibition of phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3024034 TI - Polymyxin B, a selective inhibitor of protein kinase C, diminishes the release of noradrenaline and the enhancement of release caused by phorbol 12,13-butyrate. AB - Slices of the rabbit hippocampus were labelled with 3H-noradrenaline, superfused continuously with a modified Krebs-Henseleit medium containing the uptake inhibitor cocaine and stimulated electrically (2 ms, 3 Hz, 24 mA, 5 V/cm). Phorbol 12,13-dibutyrate (PDB), a potent activator of protein kinase C (PKC), strongly enhanced the electrically-evoked overflow of tritium. In contrast, polymyxin B, a relatively selective inhibitor of PKC, diminished the evoked tritium overflow in a time- and concentration-dependent manner. The enhancement of the evoked overflow of tritium caused by PDB was strongly reduced in the presence of polymyxin B (100 mumol/l). These results suggest 1. that PKC may be involved in the physiological mechanism of action-potential-induced noradrenaline release from noradrenergic nerve terminals and 2. that the PDB-induced enhancement of noradrenaline release may be due to a direct activation of PKC. PMID- 3024036 TI - [Artificial insemination with donor semen and sexually transmissible disorders]. PMID- 3024037 TI - [Delayed neurotoxicity following poisoning with organophosphorous compounds]. PMID- 3024038 TI - Bovine leukosis virus: recloning of specific DNA fragments. AB - DNA fragments generated by Bam HI restriction endonuclease digestion of the provirus of bovine leukosis virus (BLV) was recloned in several plasmids. Recombinant plasmids containing X-region, env gene and a part of pol gene were prepared in pBR322, and in a plasmid containing promotor PR. Fragments env gene and a part of pol gene inserted were also into the pSV2-dhfr plasmid which has the both bacterial and eukaryotic promotors together with the gene for folic acid reductase. The expression possibility of these inserted BLV sequences either in mammalian cells after transfection or in bacteria is now tested. PMID- 3024039 TI - [Experiences with intradisk injection treatment with chymopapain and collagenase]. AB - In a comparative study 71 patients were treated by intradiscal injection of collagenase and 93 patients by chymopapain injections. Indication, technique of injection and post-injection treatment were based on uniform criteria and followed standardised procedure. In practically all cases, monosegmental injections were performed almost exclusively in the last two discs of the lumbar vertebral column; in cases where the x-ray and clinical findings were unequivocal, the injections were performed at one level of the lumbar vertebral column. After collagenase injection, patients suffered more from low back pain, needed higher doses of strong analgesics, and had a longer hospital stay. Results after one year were almost equal with success rates of 75% (chymopapain) and 72% (collagenase). In each group about three-quarters of the patients with unsatisfactory results were operated on. PMID- 3024041 TI - Behavioral effects of erythrosine following light exposure. AB - Three studies were conducted to examine the effects of erythrosine on the activity level of mice in a figure 8 maze. Results of the first study show that activity in a dark maze is not influenced by intraperitoneal doses as high as 1.25 mg/kg. In two additional studies, subjects were subjected to combinations of dye and pre-exposure to blue (Experiment II) or blue and green (Experiment III) light. Light exposure consistently produced increased activity levels. However, there was little evidence of a dye-light interaction effect. PMID- 3024040 TI - Prenatal diazepam: chronic anxiety and deficits in brain receptors in mature rat progeny. AB - Pregnant rats were treated with daily doses (5.0 to 7.5 mg/kg, SC) of the benzodiazepine (BDZ) receptor agonist, diazepam (DZ) between day 15 through day 20 of gestation, i.e., during ontogenesis of BDZ receptors in an attempt to alter their development. In the radial arm maze, 6 months old rat progeny showed behavioral anomalies and deficient exploratory behavior. One plausible interpretation of their poor performance is a deficit in short-term spatial "working memory." However, a more detailed evaluation of their behavior suggests a chronic state of hyperarousal/anxiety and thus, poor focus of attention on the task at hand. In addition, the number of brain BDZ receptors in the thalamus, at the age of one year, was significantly reduced. Hence, prenatal exposure to DZ has enduring and detrimental effects on brain function. PMID- 3024042 TI - The adrenocortical axis in the aged rat: impaired sensitivity to both fast and delayed feedback inhibition. AB - Aged rats secrete excessive amounts of the species-typical glucocorticoid, corticosterone, under basal conditions, following the end of stress and during habituation to mild stressors. Furthermore, the aged rat is resistant to the inhibitory effects of the synthetic glucocorticoid dexamethasone upon subsequent corticosterone secretion. These observations have led to the hypothesis that the aged adrenocortical axis is desensitized to the inhibitory effects of glucocorticoids. In the present study, we have defined this negative-feedback deficit more precisely. The aged adrenocortical axis is subject to both rate sensitive fast feedback regulation by corticosterone and to level-sensitive delayed feedback. Moreover, there is no age difference in the maximal extent of feedback inhibition which can be attained. However, the sensitivity to both forms of feedback regulation is diminished in aged rats, in that the aged adrenocortical axes are responsive under feedback conditions which completely inhibit corticosterone secretion in young animals. Such insensitivity is likely to underlie the incidences of hyperadrenocorticism apparent in the aged rat; we speculate that progressive degeneration in the aged hippocampus might be the cause of this dampened sensitivity to feedback inhibition. PMID- 3024043 TI - Aging does not alter the sensitivity of benzodiazepine receptors to GABA modulation. AB - The effects of GABA on benzodiazepine receptor binding in cerebral cortical, hippocampal, and cerebellar membranes from 2-3 months old and 28-32 months old rats were studied. GABA modulation of agonist, antagonist, and inverse agonist binding to the receptor was examined using the displacement of 3H-Ro15-1788 by diazepam, Ro15-1788, and beta-carboline-3-carboxylate methyl ester, respectively, in the absence and presence of 100 microM GABA and 150 mM sodium chloride. GABA modulation was alike in old and young rats, with respect to the particular ligand and brain region. The results support the hypothesis that, in the brain regions studied, the allosteric modulation of benzodiazepine receptor binding by GABA remains intact as a function of aging. PMID- 3024044 TI - Effects of pre-weaning undernutrition and post-weaning rehabilitation on polyphosphoinositide pools in rat brain regions. AB - In order to assess the effects of undernutrition during the pre-weaning period on polyphosphoinositide (PolyPI) pools in rat cerebral cortex, brain stem, and cerebellum, dams were fed 5% (L-) or 22% (L+) protein diets from birth to weaning and the pups were used at this age for analyses. To examine rehabilitation post weaning, L- and L+ pups were fed 22% protein diets (P+) for an additional six week period. Rats were decapitated and the dissection begun either immediately ("0 min" samples) or 10 min later (10 min samples). Body and tissue weights, and cerebroside levels were determined in addition ot PolyPI concentrations. In brain the extent of disappearance of PolyPI during the 10 min post-mortem period paralleled the content of gray matter: cerebral cortex greater than cerebellum greater than brain stem in all groups regardless of diet. Levels of PtdIns4P and PtdIns4,5P2 were decreased by 40% and 70% respectively in cerebral cortex of L- "0 min" samples. Deficits of both lipids in brain stem and cerebellum were 40 50%. In the L- 10 min samples, deficits were 20-30% in all three regions as compared with L+ 10 min levels, indicating the presence of a portion of both lipids affected only moderately by nutritional insufficiency. The effects on this relatively inert pool, much of it localized in myelin, were reversed on nutritional rehabilitation. The PolyPI pool lost post-mortem in L+ brain regions was practically absent in L- brain regions and was not restored in L-P+ animals. Thus, this study indicates that a metabolically labile pool, primarily located in gray matter structures, is more sensitive to nutritional deprivation during the pre-weaning period than the more stable pool. The precise role and function of these pools remain to be determined. PMID- 3024045 TI - Inactivation of kallikrein and kininases and stabilization of whole rat brain kinin levels following focused microwave irradiation. AB - Focused microwave irradiation was employed to stabilize endogenous whole rat brain bradykinin levels prior to a simple extraction procedure. Skull microwave exposure (2450 MHz, 3.8 kW., 2.45 sec) resulted in inactivation to less than 5% of control of whole brain kallikrein and kininase activity. Using this adequate exposure duration whole rat brain kinin levels as measured by a sensitive radioimmunoassay were approximately 0.6 pmol/g (wet weight). Further purification of irradiated brain extracts using HPLC revealed that immunoreactive kinin eluted as a single peak that co-chromatographed with authentic bradykinin. Microwave fixation duration of 1.25 sec yielded greatly increased levels of immunoreactive kinin which following HPLC purification eluted in two peaks, corresponding to authentic bradykinin and T-kinin, respectively. The tissue injury resulting from incomplete microwave fixation resulted in the release of kinins. This excess immunoreactive kinin may be derived from cerebral blood, since the predominant form of kinin-generating protein in plasma is T-kininogen. PMID- 3024047 TI - Effects of cannabinoids on the activities of mouse brain lipases. AB - Cannabinoids were found to augment phospholipase activities and modify lipid levels of mouse brain synaptosomes, myelin and mitochondria. Delta-1-tetra hydrocannabinol (delta 1-THC) and several of its metabolites induced a dose dependent (0.32-16 microM) stimulation of phospholipase A2 (PLA2) activity resulting in the increased release of free arachidonic acid from exogenous [1 14C]phosphatidylcholine (PC). The potencies of the cannabinoids in modulating PLA2 activity were approximately of the order: 7-OH-delta 1-THC greater than delta 1-THC greater than 7-oxo-delta 1-THC greater than delta 1-THC-7-oic acid = 6 alpha OH-delta 1-THC much greater than 6 beta-OH-delta 1-THC. The hydrolysis of phosphatidylinositol (PI) by synaptosomal phospholipase C (PLC) was enhanced significantly by delta 1-THC and promoted diacylglyceride levels by greater than 100 percent compared to control values. In contrast, arachidonate was the major product resulting from phospholipase activities of a 20,000 g pellet. Synaptosomal diacylglyceride lipase activity was inhibited by delta 1-THC. [1 14C]Arachidonic acid was readily incorporated into subcellular membrane phospholipids and after exposure to cannabinoids led to diminished phosphoglyceride levels and concomitant increases in released neutral lipid products. These data suggest that cannabinoids control phospholipid turnover and metabolism in mouse brain preparations by the activation of phospholipases and, through this mechanism, may exert some of their effects. PMID- 3024046 TI - Interindividual heterogeneity of molecular weight of human brain neutral sphingomyelinase determined by radiation inactivation method. AB - The molecular weight (Mr) of the membrane-bound neutral sphingomyelinase from human brain was determined using the radiation inactivation procedure. Previous studies on three human brains suggested a Mr of 165 +/- 25 kDa (J. Neurochem. 1985, 45:630-632). We now report that in another human brain the neutral sphingomyelinase had a Mr of 740 +/- 100 kDa; this higher Mr was not accompanied by differences in enzymatic properties nor heat-stability. PMID- 3024048 TI - [A case of primary multifocal glioblastoma multiforme of the brain]. AB - A 60-year-old patient is described with two independently located foci of glioblastoma multiforme in both frontal lobes. PMID- 3024049 TI - [Treatment of malignant brain tumors with slowly releasing anticancer drug polymer composites]. AB - The purpose of this study is to present the methodology and results of a clinical trial of local chemotherapy of malignant brain tumors based on slowly-releasing anticancer drug-polymer composites. The slowly releasing drugs were prepared by combining and mutually dispersing anticancer agents with glassified monomers containing 10% polymetacrylic methyl acid and then this compound was frozen at 78 degrees C and exposed to 1 X 10(6) rad of gamma rays from cobalt 60. Thus we prepared a compound of polymers and anticancer agents. We used needle-shaped capsules of this compound. These capsules release the drug very slowly over 40 days. We administered locally to the malignant brain tumors with either slowly releasing mitomycin, slowly releasing adriamycin, slowly releasing ACNU or slowly releasing 5 Fu drugs. The following techniques were employed in implantation these capsules. Implantation into the remaining tumor wall at the time of excision. Implantation into the tumor by CT-guided stereotactic method. We implanted these drugs into tumor of 55 cases, thereafter we conducted both radiation and chemotherapy with ACNU in most patients. This method has the following advantages: It is possible to be employed to different types of anticancer agents. Both dosage and releasing time can be adjusted. It is possible to administer these capsules postoperatively by the stereotactic method. The clinical study consists of 55 patients, 20 cases of anaplastic astrocytoma, 23 cases of glioblastoma multiforme, 5 cases of oligodendroglioma, 3 cases of medulloblastoma and 4 cases of others. Survival rate estimated by Kaplan-Meier method was 47% in glioblastoma at 12 months and 91% in anaplastic astrocytoma at 18 months.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3024050 TI - Stringhalt in horses: a distal axonopathy. AB - A detailed investigation of the neuropathology of a horse affected with stringhalt was performed. Qualitative and quantitative light and electron microscopy, and single teased fibre preparations of peripheral nerve demonstrated predominantly axonal degeneration, the stage of which was appropriate for the duration of clinical signs. There was selective involvement of large myelinated nerve fibres. A proximal to distal increase in the severity of pathological changes was present in the peripheral nerves. The long left recurrent laryngeal nerve was the most severely affected, followed in order by its right counterpart, the hindlimb and forelimb nerves. Neurogenic atrophy of muscles innervated by affected peripheral nerves also showed a distally graded increase in severity. No lesions were observed in the central nervous system. It was concluded that this disease should be classified as a distal axonopathy. PMID- 3024051 TI - Intranuclear vacuoles in Creutzfeldt-Jakob disease. AB - In an electron microscopic study of brain biopsy material from a case with Creutzfeldt-Jakob disease intranuclear vacuoles containing membrano-vesicular structures were found. To our knowledge this finding has not been previously reported in human spongiform encephalopathies. It may represent a specific alteration associated with Creutzfeldt-Jakob disease, and suggests the possibility of nuclear infection. PMID- 3024052 TI - Dopaminergic inhibition of anterior pituitary adenylate cyclase activity and prolactin release: the effects of perturbing calcium on catalytic adenylate cyclase activity. AB - The dopaminergic inhibition of anterior pituitary adenylate cyclase activity, cAMP accumulation, and prolactin release was studied in the presence of the Ca2+ channel activator, maitotoxin. In isobutylmethylxanthine (IBMX)-treated cells, maitotoxin stimulated prolactin secretion within 30 s and cAMP accumulation within 1 min. Although dopamine reduced cAMP accumulation and prolactin release, the effectiveness of the catecholamine was reduced in the presence of maitotoxin. When hemipituitary glands were exposed for 10 min to 100 ng maitotoxin/ml, their membranes showed increased adenylate cyclase activity. The hypothesis that maitotoxin stimulates adenylate cyclase activity by increasing Ca2+ availability was supported by the observation that, at concentrations up to 100 microM, Ca2+ stimulated anterior pituitary adenylate cyclase activity. Although dopamine decreased basal and maitotoxin-stimulated pituitary cAMP accumulation, via changes in adenylate cyclase activity, the decrement in cyclic nucleotide production, but not prolactin release, can be ascribed to the effect of the catecholamine on the basal activities of these parameters. These data provide additional evidence that an increased Ca2+ flux is stimulating to cAMP generation and prolactin release, whereas dopamine is inhibitory to these processes. PMID- 3024053 TI - Specific labelling of high-affinity vasoactive intestinal peptide receptors in rat liver membranes by a growth hormone-releasing factor analog. AB - (125I-His1, D-Ala2, Nleu27)-growth hormone-releasing factor (GRF) (1-29)-NH2, initially developed as a possible radioligand for identifying GRF receptors in the anterior pituitary, was found to bind to rat hepatic membranes. The tracer was stable, bound rapidly and reversibly, and its dissociation was accelerated by GTP. Radioligand binding was enhanced by the divalent cations Mg2+, Ca2+ and Mn2+ and inhibited by the chelating agent EDTA. Vasoactive intestinal peptide (VIP), PHI, secretin, GRF(1-29)-NH2, (His1, D-Ala2, Nleu27)-GRF(1-29)-NH2, and (D-Ala2, Nleu27)-GRF(1-29)-NH2 dose-dependently inhibited tracer binding. The order of potency of the unlabelled peptides tested suggested that (125I-His1, D-Ala2, Nleu27)-GRF(1-29)-NH2 specifically identified a high-affinity subclass of VIP receptors in rat liver membranes. PMID- 3024054 TI - Association of diurnal variations in hypothalamic but not cortical opiate ([3H] naloxone)-binding sites with the ability of naloxone to induce LH release in the prepubertal female rat. AB - We report the existence of a diurnal variation in the binding of the opiate antagonist [3H]-naloxone to slices of the mediobasal hypothalamus from prepubertal female rats. The binding is highest in the early morning and reaches a nadir in the late afternoon. Opiate binding in cortical slices from such animals is constant over the course of the day. Changes in receptor density, and not in receptor affinity, account for the diurnal variation in the amount of ligand bound. These diurnal variations in receptor numbers are associated with changes in the ability of naloxone to release LH and may be crucial in the transition from the juvenile state to one of competent reproductive functioning. PMID- 3024055 TI - Exercise-induced increases in plasma catecholamines and growth hormone are augmented by selective alpha 2-adrenoceptor blockade in man. AB - A specific alpha 2-adrenoceptor antagonist (idazoxan) was used in man to examine the neuroregulation of growth hormone (GH) release and the effect of alpha 2 adrenoceptors on noradrenaline release. In the first study, GH-releasing factor (GRF)-induced GH release was unaffected in 6 normal volunteers by the prior administration of idazoxan, suggesting that the alpha 2-receptors involved in GH release are not operative at pituitary level. In the second study, 6 normal volunteers performed a bicycle exercise test with and without prior treatment with idazoxan. The exercise-induced GH response (to 13 +/- 5 mU/l) was significantly augmented by idazoxan (to 20 +/- 7 mU/l) which contrasts with the current view of GH release being regulated by stimulatory hypothalamic alpha 2 adrenoceptors and suggests that these alpha 2-adrenoceptors are capable of exerting a dual effect on GH release. The exercise-induced increase in plasma noradrenaline (to 1.45 +/- 0.19 micrograms/l) and in heart rate (to 142 +/- 5 beats/min) were also augmented by idazoxan (to 2.24 +/- 0.23 micrograms/l and 152 +/- 6 beats/min). A similar augmentation of the plasma adrenaline response to exercise was also found, whereas the blood glucose, plasma insulin and potassium response to exercise was unaffected by idazoxan. These results suggest that during exercise in man, alpha 2-adrenoceptors exert a tonic inhibitory influence on noradrenaline release which also serves to limit the exercise-induced tachycardia. PMID- 3024056 TI - Pharmacological characterization of opioid receptors influencing the secretion of corticotrophin releasing factor in the rat. AB - The effects of selective agonists and antagonists of opioid receptors on the secretion of corticotrophin releasing factor (CRF) by isolated rat hypothalami in vitro were studied. Morphine (10(-8)-10(-6) M) and the mu-opioid receptor agonists, FK33-824CH (10(-8)-10(-6) M) and Tyr-D-Ala-Gly-MePhe-NH(CH2)2OH (10(-8) 10(-6) M), caused dose-related increases in the release of CRF from isolated hypothalami. The kappa-opioid receptor agonist, U50,488 (10(-8)-10(-6) M), was also weakly active in this respect but the delta-opioid receptor agonist, (D Pen2,D-Pen5)-enkephalin (2 X 10(-10)-2 X 10(-7) M), was not. The stimulatory actions of morphine and Tyr-D-Ala-Gly-MePhe-NH(CH2)2OH on CRF release were antagonized by naloxone (10(-8) M) and by the mu/delta-opioid receptor antagonist, beta-funaltrexamine (beta-FNA, 10(-9) M), but not by the delta-opioid receptor antagonist, ICI-154129 (5 X 10(-6) M). The effects of U50,488 on CRF release were unaffected by either beta-FNA or ICI-154129 but were antagonized by high doses of naloxone (10(-6) M). The results suggest that both mu- and kappa opioid receptors are involved in the stimulation of CRF secretion but that delta opioid receptors are not important in this respect. PMID- 3024057 TI - Evidence for multiple serotonergic influences on LH release in ovariectomized rats and for modulation of their relative effectiveness by estrogen. AB - Both opiates and serotonin (5HT) are known to inhibit LH release in ovariectomized rats, and estrogen has been shown to reverse certain serotonergic effects. Therefore studies were undertaken to compare the effects of morphine and the serotonin agonist 5-methoxy-N,N-dimethyltryptamine (5MEODMT) on LH release in ovariectomized rats with and without estrogen priming. Serial blood samples were collected via jugular cannulae before and 5, 15, 30 and 60 min after intravenous administration of morphine, 5MEODMT or both to rats receiving no pretreatment, or a serotonin receptor antagonist (methysergide, METH; or ketanserin, KET) 60 min earlier. In the absence of estrogen, morphine inhibited LH release, and the response was delayed by METH or abolished by KET, suggesting mediation by serotonin2 (5HT2) receptors. 5MEODMT alone failed to alter the release of LH significantly, but apparently activated both stimulatory and inhibitory serotonergic systems. Blockade of 5HT2 receptors with KET enabled an inhibitory system to prevail. No significant changes in LH concentrations were observed following combined administration of morphine and 5MEODMT. Similarly, in estrogen primed rats morphine appeared to activate both inhibitory (5HT2) and stimulatory (5HT1) systems, resulting in no net change unless the inhibitory system had been antagonized by KET. Administration of 5MEODMT alone or in combination with morphine resulted in a strong stimulatory effect which appeared to be mediated by 5HT1 receptors. These results suggest the existence of a multiplicity of serotonergic influences on the release of LH in the rat, not only in terms of particular species of 5HT receptors, but also in neuronal connectivity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3024058 TI - Hyperresponsiveness to the inhibitory action of dopamine agonists on luteinizing hormone secretion in the monosodium-L-glutamate-treated, orchidectomized rat. AB - The tuberoinfundibular (A12) dopaminergic pathway, which originates in the arcuate and periventricular nuclei, is thought to play an inhibitory role in the regulation of episodic luteinizing hormone (LH) secretion. Neonatal treatment of rats with the neurotoxin monosodium-L-glutamate (MSG) causes extensive damage to the arcuate nuclei and up to 60% depletion of dopamine (DA) in the mediobasal hypothalamus. We hypothesized that such DA depletion should result in a hyperresponsiveness to subsequent administration of a DA agonist. To test this hypothesis, male rats were treated neonatally with MSG. Control rats received injections of equiosmotic saline. As adults the rats were orchidectomized and fitted with indwelling venous catheters. Blood samples were taken from these unanesthetized, unrestrained rats at 5-min intervals for a 1-hour period, at which time the animals received an intraperitoneal injection of one of the following drugs: apomorphine (0.8 mg/kg, a DA receptor agonist), bromocriptine (8.0 mg/kg, a DA receptor agonist), 0.9% saline (vehicle for apomorphine) or 95% ethanol (vehicle for bromocriptine). Blood sampling was continued for a further 2 2.5 h. Plasma LH was measured by RIA. Both apomorphine and bromocriptine produced striking inhibition of circulating LH levels in MSG-treated rats. Neither of the control treatments altered pulsatile LH secretion patterns. Administration of exogenous gonadotropin-releasing hormone produced LH peaks in all animals so treated, including those whose endogenous LH secretion had been inhibited by the DA agonists. These findings suggest that the depletion of DA induced by neonatal MSG treatment results in a supersensitivity to DA agonists.(ABSTRACT TRUNCATED AT 250 WORDS) PMID- 3024060 TI - In vivo evidence for spinal delta-opiate receptor operated antinociception. PMID- 3024059 TI - Effect of chronic treatment of ethanol on benzodiazepine and picrotoxin sites on the GABA receptor complex in regions of the brain of the rat. AB - Ethanol has been shown to enhance gamma-aminobutyric acid (GABA)ergic transmission. In this study an examination was made of the effect of chronic treatment with ethanol and its withdrawal at 24 h on the binding of [3H]flunitrazepam and [35S]t-butylbicyclophosphorothionate (TBPS) to brain regions in rat. Rats were rendered tolerant to, and dependent on, ethanol by an intragastric intubation method. The affinity (KD) or the binding capacity (Bmax) of [3H]flunitrazepam or [35S]TBPS was not altered by chronic treatment with ethanol or during withdrawal from ethanol. Neither the enhancing effect of GABA on the binding of [3H]flunitrazepam nor its inhibitory effect on the binding of [35S]TBPS were affected by chronic treatment with ethanol or its withdrawal at 24 h. These results suggest that the sensitivity of benzodiazepine and picrotoxin sites on the oligomeric GABA receptor complex is not affected during tolerance to, or withdrawal from ethanol. It is suggested that the effects of ethanol on GABAergic transmission may be produced at the level of coupled chloride ion channels. PMID- 3024061 TI - Reactivation of herpesvirus in neurosurgical patients. AB - The authors are presenting seven patients who had operations between July 1984 and July 1985 and who developed herpes infections postoperatively. Four of the patients developed their infections in a dermatomal distribution that correlated with the nerve roots manipulated at operation. A spectrum of localized herpes reactivation is demonstrated in this series. The use of corticosteroids and other associated variables are discussed. Like reactivation of herpes simplex after trigeminal nerve operation, we believe reactivation of herpes simplex and herpes zoster can occur in operation of the cervical, thoracic, or lumbosacral spine. PMID- 3024062 TI - ACTH and attention in humans: a review. AB - In addition to the hormonal action of corticotropin (ACTH) on the adrenal cortex, this peptide and fragments of it may function as chemical signals in CNS synapses. This report reviews studies on behavioral and psychophysiological effects of ACTH-related neuropeptides. Experiments will be emphasized which applied EEG techniques for the measurement of peptide-induced changes on aspects of information processing in man. It is proposed to conceptualize the pattern of actions of ACTH-related neuropeptides as a blocking of suppressive functions occurring, for example, during habituation or selective attention. Disinhibitory effects mediated by structures of the limbic system may be responsible for repeatedly observed improvements of sustained attention, but impairments of selective attention following the administration of ACTH-related neuropeptides. Under the influence of these peptides the attention is more easily attracted by stimuli, irrespective of their relevance. PMID- 3024063 TI - Cholecystokinin-immunoreactive boutons in synaptic contact with hippocampal pyramidal neurons that project to the nucleus accumbens. AB - Neurons in the hippocampal formation of the rat that project to the medial nucleus accumbens were identified following the retrograde transport of a conjugate of horseradish peroxidase with wheat germ agglutinin. The great majority of such projecting neurons were located in the ventral subiculum and were pyramidal in shape; the pyramidal nature of 25 such retrogradely labelled neurons was established by Golgi impregnation. In material processed to reveal both retrogradely labelled cells and cholecystokinin-immunoreactivity, no immunoreactive projecting neurons were found. However, 48 identified projecting neurons, probably pyramidal, were found to receive input from cholecystokinin immunoreactive boutons that formed symmetrical synaptic contacts with the soma or proximal dendrites. It is suggested that one function of cholecystokinin immunoreactive neurons in the hippocampal formation might be to influence the output of the pyramidal neurons that project to the nucleus accumbens. Since this pathway is one of the main links between the limbic system and the basal ganglia, it is conceivable that changes in the cholecystokinin levels in the hippocampus, as found in schizophrenia, might influence behaviour through the pathway connecting the hippocampus with the nucleus accumbens. PMID- 3024064 TI - Increase in the number of presynaptic large intramembrane particles during synaptic transmission at the Torpedo nerve-electroplaque junction. AB - Small pieces of Torpedo electric organ were treated with 4-aminopyridine, a drug which greatly increases the duration of transmitter release in a single nerve impulse, transforming the normally brief electroplaque potential to a giant discharge. Specimens of tissue were cryofixed by rapid freezing using liquid coolants at precise time intervals during transmission of a single giant discharge, and then examined by freeze fracture. In each experiment, we monitored the electrical response of one specimen during the freezing run to check the physiological responsiveness of the tissue and to determine the precise time of contact with the cryogenic liquid. The general appearance of nerve terminals after cryofixation was similar to that of terminals from chemically fixed and cryoprotected tissue. The major morphological change observed during the time course of the giant discharge was a marked increase in the density of intramembrane particles larger than 10 nm on both the protoplasmic and external faces of the presynaptic membrane. This change appeared in specimens frozen within the first few milliseconds after the stimulus, that is, at a time corresponding to the onset of the rising phase of the potential (3 ms). At the end of the giant discharge, the particle density returned to control values with the same time course as the potential trace. Pits of 20 nm or larger, probably due to vesicle-membrane interaction, were found in a small proportion of nerve terminals. Their occurrence increased only at 120-150 ms after the stimulus, that is, a long time after the beginning of the giant potential and of the change in intramembrane particles. The size distribution of particles was also determined in the membrane of synaptic vesicles exposed by cross fracture of terminal boutons; it was found to be similar to that of the unstimulated presynaptic membrane and it did not change during the giant discharge. Stimulation experiments were also carried out in a modified solution containing no added calcium, 20 mM magnesium and 4-aminopyridine. The propagation of impulses along the nerves to the electric organ was not inhibited in the modified solution but acetylcholine release was prevented and no increase in particle density was found on the presynaptic membrane. These and previous biochemical experiments on this tissue suggest that the release of the neuro-transmitter acetylcholine is associated with a transient occurrence of large intramembrane particles on the two fracture faces of the presynaptic membrane.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3024065 TI - Functional organization of thalamic projections to the motor cortex. An anatomical and electrophysiological study in the rat. AB - In rats, horseradish peroxidase crystals were injected in motor cortical foci functionally identified by means of the motor effects evoked by electrical stimulations. The location in the thalamus of the neurons linked to different motor cortical foci was studied. Thalamic neurons were retrogradely labeled in both "motor" (ventralis lateralis and ventralis medialis) and "non-motor" nuclei: centralis lateralis, lateralis posterior, mediodorsalis and posterior thalamic nuclear group, as well as the ventrobasal complex. The ventrobasal complex was labeled after horseradish peroxidase injections in hindlimb and trunk motor areas. The ascending projections toward the motor cortex from both "motor" and "non-motor" thalamic nuclei are organized more precisely and more elaborately than previously reported. The motor cortical afferents from the nucleus ventralis lateralis are organized in three planes, rostrocaudally, dorsoventrally and mediolaterally. An inverted relation exists in the rostrocaudal plane between the nucleus ventralis lateralis and the motor cortex: the caudal motor cortex region (hindlimb) receives fiber inputs from the rostral region of the nucleus ventralis lateralis, whereas the caudal zone of the nucleus ventralis lateralis projects to the rostral motor cortex region (forelimb and vibrissae). A dorsoventral organization has also been observed in the rostral region of the nucleus ventralis lateralis: the ventral aspect is the source of fibers directed to the distal hindlimb region, whereas fibers originating from the dorsal aspect are directed to the proximal hindlimb area. A mediolateral relationship exists between medial and lateral sides of the nucleus ventralis lateralis and, respectively, proximal and distal forelimb cortical areas. There is some overlap between the various nuclear regions thus delineated. Four functional zones were found in the lateral half of the nucleus ventralis medialis and were classified according to their projection to the motor cortex; these are involved in motor control of the proximal and distal forelimb, vibrissae and ocular movements. The projection is topographically organized according to both an inverted rostrocaudal and a direct dorsoventral-mediolateral arrangement. Caudally, dorsal and ventral nuclear parts project to rostromedial (vibrissae) and rostrolateral (distal forelimb) regions of the motor cortex, respectively. More rostral nuclear zones project to more caudal (proximal forelimb, eye) cortical regions. There is little overlap between these four nuclear subdivisions. The nucleus centralis lateralis projects to vibrissae and proximal, as well as distal, forelimb areas.(ABSTRACT TRUNCATED AT 400 WORDS) PMID- 3024067 TI - [Neuropathology in acquired immune deficiency syndrome]. PMID- 3024066 TI - Nitrous oxide: clinical and electrophysiologic investigation of neurologic complications. AB - Prolonged exposure to nitrous oxide produces a recognized neurologic syndrome. We report clinical and electrophysiologic studies of nervous system involvement in a 25-year-old student who abused nitrous oxide. He developed signs of a sensorimotor polyneuropathy and of myelopathy. Routine blood studies, CSF examination, and myelogram were normal. Clinical electrophysiologic studies were performed serially. Nerve conduction studies demonstrated reduced amplitude and slowed sensory potentials, and mildly prolonged late responses. Sensory evoked potentials revealed prolonged latency of scalp-evoked potentials from tibial nerve stimulation with normal median nerve values. The foveal visual evoked potential was delayed in the right eye, with normal visual acuity, funduscopic examination, and spatial contrast sensitivity. Repeat electrophysiologic studies demonstrated improvement. Nitrous oxide produces multifocal reversible dysfunction within the nervous system similar to that described in patients with vitamin B12 deficiency. PMID- 3024068 TI - [Morphological characteristics of sudanophilic deposits appearing in abnormal myelination in pt rabbits]. PMID- 3024069 TI - Electron microscopic demonstration of luteinizing hormone-releasing hormone (LH RH) by the immunogold staining method. PMID- 3024070 TI - The glutamate analogue quisqualic acid is neurotoxic in striatum and hippocampus of immature rat brain. AB - To assess the neurotoxic properties of the glutamate agonist quisqualic acid (QA) in immature brain, we injected this compound (100 nmol QA/1 microliter) directly into the striatum of 7-day-old rats. QA produced neuronal necrosis and glial infiltration in 14 pups and reduced the size of the striatum and hippocampus on the side of injection. Intracerebral injection of QA provides a new method for producing neuronal lesions in the developing brain. PMID- 3024071 TI - The distribution pattern of adrenocorticotropin-like immunoreactivity in the cat central nervous system. AB - The distribution pattern of adrenocorticotropin-like immunoreactivity (ACTH-LI) in cats using the avidin-biotin modification of an immunocytochemical method shows cell bodies containing ACTH-LI in the medial basal hypothalamus, especially in the infundibular nucleus. The fibers from these neurons extended beyond the hypothalamus, into the paraventricular nucleus of the thalamus, rostral amygdala, periaqueductal gray, locus coeruleus, parabrachial nucleus and medial nucleus of the nucleus tractus solitarius. The distribution pattern of the cell bodies and fibers containing ACTH-LI bears several similarities to that seen in rats. The pattern differs from that of rats in the fact that the termination in the amygdala is more extensive and that ACTH-LI was not observed in cell bodies in any location other than the medial basal hypothalamus. PMID- 3024072 TI - Cerebellar benzodiazepine receptor distribution: an autoradiographic study of the normal C57BL/6J and Purkinje cell degeneration mutant mouse. AB - The distribution of [3H]flunitrazepam binding sites in the cerebella of normal mice and Purkinje cell degeneration mutant mice was studied by light microscopic autoradiography. In the cerebellar cortex of normal mice, a high density of [3H]flunitrazepam binding was observed over the molecular layer, an intermediate density over the Purkinje cell layer and a low density over the granule cell layer; the white matter was devoid of labeling. The deep cerebellar nuclei were labeled to an intermediate density. In the 54-day-old Purkinje cell degeneration mutant cerebellum, which is depleted of Purkinje cells, a 36% reduction in labeling density of the cerebellar cortex was observed. The density was reduced by approximately equal amounts in both the molecular and granule cell layers; labeling in the deep cerebellar nuclei was, however, substantially increased. PMID- 3024074 TI - Motoneurone activity in an isolated spinal cord preparation from the adult mouse. AB - This paper describes an isolated, hemisected preparation of adult mouse spinal cord, in which motoneurones remain viable. At 18-22 degrees C both orthodromic synaptic activation and antidromic invasion of populations of motoneurones could be demonstrated by extracellular recording of ventral root reflexes and ventral horn field potentials. Motoneurones had resting potentials of -55 to -65 mV and input resistances of 5-30 M omega, and, following ventral or dorsal root stimulation or during outward current injection, they generated action potentials which resembled those recorded from adult motoneurones in vivo. Recurrent inhibitory synaptic potentials followed antidromic spikes, demonstrating viability of the Renshaw cell pathway. PMID- 3024073 TI - Presence of neurons with GABA-like immunoreactivity in the superior cervical ganglion of the rat. AB - Using antibodies raised against gamma-aminobutyric acid (GABA)-glutaraldehyde complexes, we have found neurons with GABA-like immunoreactivity in the superior cervical ganglion of adult rats. The processes of these neurons formed pericellular networks around the principal ganglion cells. Electron microscopy revealed that the immunoreactive dendrites were innervated by non-reactive axon terminals which formed asymmetrical synapses and probably originated from the preganglionic nerve. Axons with GABA-like immunoreactivity, especially axonal varicosities filled with synaptic vesicles, were found in direct apposition to principal ganglion cells. The GABA-positive axons and axon varicosities persisted in experimentally decentralized (deafferented) ganglia, suggesting that the perikarya of the immunoreactive neurons were intrinsic to the superior cervical ganglion. Taken together with data on inhibitory effects of GABA in sympathetic ganglia, these findings suggest that the superior cervical ganglion of rats contains a subpopulation of inhibitory interneurons which is GABAergic. This would indicate that GABAergic neurons do not only occur in the central but also in the peripheral nervous system. PMID- 3024076 TI - Phosphatidylinositol turnover in neuroblastoma cells: regulation by bradykinin, acetylcholine, but not mu- and delta-opioid receptors. AB - The effect of opioids on phosphatidylinositol (PI) turnover to release inositol triphosphate (IP3) as second messenger was examined in mouse neuroblastoma X rat glioma hybrid cells NG108-15 (delta-receptors) and human neuroblastoma cells, SK N-SH (predominantly mu-receptors). PI turnover can be stimulated in both NG108-15 and SK-N-SH cells by bradykinin and acetylcholine, respectively. In contrast, etorphine, DADL ([D-Ala2,D-Leu5]-enkephalin) and DAGO ([D-Ala2,MePhe4,Gly-ol5] enkephalin), up to 1 microM concentrations failed to affect PI turnover in both cell lines. These results suggest that IP3 is not likely to serve as second messenger for both mu- and delta-opioid receptors. PMID- 3024077 TI - 'Choreic' movement induced by unilateral kainate lesion of the striatum and L DOPA administration in monkey. AB - A monkey whose unilateral striatum was extensively destroyed exhibited no choreic movement after administering L-DOPA, whereas another monkey whose unilateral striatum was partially (especially the dorsolateral part) destroyed showed 'choreic' movements exclusively on the contralateral extremities after the L-DOPA doses. Biochemical analysis disclosed a markedly increased activity of tyrosine hydroxylase in the unaffected (ventromedial) striatal area on the lesioned side when compared to the intact side of the choreic monkey, but not in the non choreic monkey. Therefore, choreic movements in a monkey were suggested to be generated by the hyperfunction of the nigrostriatal dopaminergic neurons that innervate the unaffected part of the striatum. PMID- 3024078 TI - Enduring effects of prenatal diazepam on the behavior, EEG, and brain receptors of the adult cat progeny. AB - Pregnant cats were treated with a benzodiazepine receptor agonist, diazepam (DZ; Valium; average dose of 0.4 mg/kg/day, i.m.) between day 20 through day 53 of gestation in an attempt to alter the ontogenesis of the benzodiazepine receptors. As tested in fully developed one-year-old progenies, prenatal exposure to DZ resulted in a behavioral syndrome of hyperactivity, aggressiveness, behavioral signs of chronic anxiety, inability to habituate to novel environment, suppression or absence of the reward-induced alpha-like (7 to 11 Hz) electroencephalographic patterns during operantly conditioned behavior (bar pressing for 1 ml of milk reward), and deficits in the numbers of benzodiazepine receptors in the hypothalamus (-47%), fronto-orbital cortex (-33%) and postcentral cortex (-19%). PMID- 3024079 TI - Early neurobehavioral and neurochemical alterations in rats prenatally exposed to imipramine. AB - Pregnant CD rats were treated subcutaneously with 0, 5 or 10 mg/kg/day of imipramine (IMI) on days 8-20 of gestation. Behavioral and neurochemical endpoints were measured at different postnatal days (PND). Three behavioral tests were conducted: negative geotaxis on PNDs 7-9; auditory startle habituation (ASH) on PNDs 14, 16 and 18; locomotor activity before and after intraperitoneal (i.p.) injection of saline or 0.5 mg/kg d-amphetamine on PND 21. Catecholamine levels, B adrenergic and muscarinic cholinergic binding were measured on PND 1 and in PND 21 rats 3 hours after challenge. Maternal weight gained during the dosing period was decreased in a dose-related manner, but there were no dose-related differences in offspring body weights. On PND 7, low-dose males turned significantly sooner in negative geotaxic testing, and more high-dose males successfully turned (94%) than did controls (61%). A significant reduction in ASH amplitude was found only in males from the low-dose group on PND 18. IMI-exposed males tended to be more active prior to and following amphetamine challenge. On PND 1, male offspring from the low-dose group showed a 65% reduction in B adrenergic receptor binding and a trend toward increased brain epinephrine (EPI) levels. On PND 21, no consistent dose-related receptor binding changes were observed. Cortical levels of EPI, however, tended to be higher in treated males and high-dose females challenged with d-amphetamine. These same rats also showed a marked elevation in locomotor activity following challenge with d-amphetamine. Thus, prenatal IMI exposure appeared to alter functional development of the central adrenergic systems in a complex manner, but one consistent with changes noted in both neurochemical and behavioral endpoints. PMID- 3024075 TI - Asymmetric distribution of purinergic and adrenergic neurotransmission cooperates in the motor activity along the rat vas deferens. AB - The distribution of purinergic and adrenergic responses in the epididymal and prostatic segment of the rat vas deferens were studied in vitro. Prazosin antagonizes the twitch elicited by electrical stimulation mainly in the epididymal segment while alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-mATP) preferentially inhibits the response of the prostatic segment. Using both prazosin plus alpha,beta-mATP, the response to field stimulation was completely inhibited. Concentration-response curves revealed that adrenergic compounds elicited a greater contraction in the epididymal portion than in the prostatic end of the ductus. Purinergic compounds caused a contraction of greater magnitude in the prostatic portion. The results suggest that adrenergic and purinergic mechanisms are asymmetrically distributed along the vas deferens reflecting a gradient of adrenergic and purinergic receptors along the ductus. PMID- 3024080 TI - Neurotoxicity of Bordetella pertussis. AB - Pertussis is a unique disease in which the harmful effects are mediated by an exotoxin that effects stimulation of the adrenergic system which is neuronally controlled. The interdependence of the growth of bacteria and toxin production, and the local colonization of the bacteria that precedes the clinical symptom of the disease reflect the nature of the disease. Pertussis toxin enzymatically alters the function of numerous regulatory cells that is demonstrable, after an interval of time, by a specific stimulus. The toxin also may act rapidly and effect action at a target tissue. The latter appears to be associated with the rapid adverse events after vaccination whereas both may occur in the disease. The pathophysiologic responses associated with specific clinical symptoms have not been clearly defined. Responses to be evaluated relative to encephalopathy are increased vascular permeability, hypoglycemia and enhanced activity of neuronal glutamate and aspartate. The intensity of responses is related to the amount of pertussis toxin available, genetic susceptibility, ethnic and allotype, and external factors. The reason for the non-linear dose response shown by the critical level between the sublethal and the lethal infection in mice is unclear. Bacterial adenylate cyclase may be a candidate. Much remains to be elucidated about the enzymatic pathways that effect the many disparate events, the identity of the neurons that effect the clinical symptoms and their CNS location, the identity of the neuronal transmitters and the pathoneuronal pharmacodynamics. PMID- 3024081 TI - Neurotoxicity of endosulfan in young and adult rats. PMID- 3024083 TI - Viral lesions of the oral cavity: a review. PMID- 3024082 TI - Effect of perinatal polybrominated biphenyl exposure on acquisition and performance of an autoshaping paradigm. AB - Pregnant Sprague-Dawley rats received oral doses of 0, 0.2 or 2 mg/kg/day polybrominated biphenyls (PBB) as fireMaster BP-6 (BP-6) from day 6 of gestation through day 24 postpartum. At approximately 6 months of age male and female offspring were food-deprived to 80% of their free-feeding weights and subjected to four phases of an autoshaping paradigm. Acquisition of Phase I, a VI-90 second schedule of responding, was significantly delayed for female offspring from dams administered 2 mg/kg/day BP-6; a trend toward delayed acquisition was observed in all other PBB-exposed animals. No BP-6-related difference in latency to respond during this phase was observed. Male offspring from dams administered BP-6 appeared to acquire Phase II responding (a FI-90 second contingency) at a faster rate than did control males. In contrast, BP-6-exposed females acquired Phase II responding at a somewhat slower rate than did control females. The sex-related difference in responding may involve a rate-dependent influence, as control females acquired Phase II responding much more quickly than did control males. Control males and females acquired Phase III (FR-20 responding) at approximately the same rate. No BP-6-related deficits were observed during the initial few days of acquisition of FR-20 responding. However, BP-6-exposed male offspring tended to respond more than did control males after this time. BP-6-exposed females tended to respond less than did control females only as the responding of controls approached an asymptote. Phase IV involved FR-20 responding following challenge with d-amphetamine or chloral hydrate; no significant BP-6-related changes in disruption of this behavior were observed. PMID- 3024084 TI - Central nervous system metastases in epithelial ovarian carcinoma. AB - With the advent of systemic chemotherapy capable of controlling metastases at most sites, central nervous system metastases are becoming more common in patients with epithelial ovarian carcinoma. A retrospective epidemiologic review at The University of Texas M. D. Anderson Hospital and Tumor Institute revealed central nervous system metastases in 13 of 4456 patients with epithelial ovarian carcinoma (0.29%) registered between 1944 and 1984. No patients were identified as having central nervous system metastases before 1968. The median survival overall was 29 months; following the diagnosis of brain metastases it was five months. Five of eight patients treated for central nervous system metastases lived ten months or longer. Patients with isolated metastases to the central nervous system lived longer than patients with accompanying systemic metastases. Patients treated with surgical resection lived longer than those who did not undergo surgery. With surgical resection, postoperative irradiation, and systemic chemotherapy, significant symptomatic improvement and long-term remission are possible. PMID- 3024086 TI - [X-ray diagnosis of malignant tumors of the lacrimal gland]. PMID- 3024085 TI - Radioimmunoassay of placental alkaline phosphatase in ovarian cancer sera and tissues. AB - A specific radioimmunoassay for human placental alkaline phosphatase has been developed using the 125I-labeled enzyme, highly purified with a fast protein liquid chromatography system and an absorbed rabbit antiserum. The sensitivity of this assay was 0.2 U/L. Serum levels of over 0.2 U/L were found in 27% of ovarian cancer patients, and most of these elevated enzyme levels occurred with more advanced stages of the disease. On the other hand, almost all ovarian cancer tissue contained detectable levels of the enzyme. Serous adenocarcinoma, endometrioid adenocarcinoma, and dysgerminoma had particularly large amounts. Placental alkaline phosphatase was more frequently detected in tissue than in the serum of ovarian cancer, and therefore may be a useful target in immunodetection and immunotherapy and in studying the histopathology of ovarian cancer. PMID- 3024087 TI - [Clinical x-ray and radionuclide diagnosis of tumors and tumor-like diseases of the ribs]. PMID- 3024088 TI - [Radionuclide diagnosis of tumors of the locomotor apparatus in children]. PMID- 3024091 TI - [Cylindroma of the nasal septum]. PMID- 3024090 TI - [Critical comments by the physician on so-called bulk-rich diet]. PMID- 3024089 TI - [Tumor antigens CA 125 and CA l9-9 in the serum of patients with gynecologic trophoblastic tumors and colorectal carcinoma]. PMID- 3024092 TI - Pain in early cancer of the lungs. AB - 164 patients with early cancer of the lungs, i.e., without extrathoracic spread or distant metastasis, were examined. Subjective and objective characteristics of the pain were studied. A correlation was found between the location of the neoplasm, the location of the pain and the characteristics of the sensory changes. These observations may be a useful contribution to the early diagnosis of primary carcinomas of the lungs. PMID- 3024094 TI - Evidence for genetic diversity in Trypanosoma (Nannomonas) congolense. AB - Genetic proximity between two karyotypic groups of Trypanosoma congolense was investigated using as hybridization probes: total genomic DNA, a 35 nucleotide long synthetic oligonucleotide, and non-variant antigen type (non-VAT) specific complementary DNAs. The phylogenetic relationship between Trypanosoma brucei and T. evansi, both of which are accepted species in the subgenus Trypanozoon, was used as a reference to assess the phylogenetic proximity of the two groups of T. congolense. Results indicate that some morphologically indistinguishable T. congolense populations differ in a variety of molecular and genetic properties: molecular karyotypes, majority of the DNA sequences, and the restriction enzyme sites in the genomic environments of various conserved genes. The implications of these findings for trypanosome evolution and T. congolense epidemiology are discussed. PMID- 3024095 TI - [Evaluation and comparative factors in the use of alumina ceramic (Cerestore) and vitreous silica (Dicor) in the preparation of single-unit jacket crowns]. PMID- 3024093 TI - [Experimental infection of ixodid ticks with Karshi virus]. AB - The ixodid ticks Hyalomma asiaticum, H. anatolicum, Dermacentor niveus were infected experimentally with Karsha virus. The virus replication has been proved to occur in the tick's organism. The titre of the virus grows gradually in infected ticks. Entering the tick's gut during its feeding virus particles penetrate into the gut walls where primary multiplication and accumulation of the virus take place.